Platinum, Gold, and Other Metal Chemotherapeutic Agents

Publication Date: January 26, 1983 | doi: 10.1021/bk-1983-0209.
fw001
Platinum, Gold, and Other

Metal Chemotherapeutic
Agents
Chemistry and Biochemistry
In Platinum, Gold, and Other Metal Chemotherapeutic Agents; Lippard, S.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1983.
Publication Date: January 26, 1983 | doi: 10.1021/bk-1983-0209.fw001

Platinum, Gold, and Other Metal
Chemotherapeutic Agents
Chemistry and Biochemistry
Stephen J. Lippard, EDITOR
Massachusetts Institute of Technology
Based on a symposium
sponsored by the ACS Division
of Inorganic Chemistry
at the 183rd Meeting of the
American Chemical Society,
Las Vegas, Nevada,
March 28-April 2, 1982
ACS SYMPOSIUM SERIES 209
AMERICAN CHEMICAL SOCIETY

WASHINGTON, D.C. 1983

Library of Congress Cataloging i n Publication Data
Platinum, gold, and other metal chemotherapeutic
agents.
(ACS symposium series, ISSN 0097-6156; 209)
"Based on a symposium sponsored by the Division
of Inorganic Chemistry of the American Chemical

Society at the ACS National Meeting, Las Vegas,
Nevada, March-April 1982."
Includes bibliographies and index.
1. Metals—Therapeutic use—Congresses. 2. Chem
istry, Pharmaceutical—Congresses. 3. Platinum
compounds—Therapeutic use—Congresses. 4. Anti
neoplastic agents—Congresses. I. Lippard, Stephen J.
II. American Chemical Society. Division of Inorganic
Chemistry. III. American Chemical Society. National
Meeting (1982: Las Vegas, Nev.) I V . Series.
RS431.M45P57 616.99'406l 82-24333
ISBN 0-8412-0758-5 ACSMC8 209 1-453
1983
Copyright © 1983
American Chemical Society
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The ACS S Y M P O S I U M SERIES was founded in 1974 to provide
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format of the Series parallels that of the continuing A D V A N C E S
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PREFACE
THE DISCOVERY OF THE REMARKABLE BIOLOGICAL ACTIVITIES of the anti

cancer platinum drug cw-diamminedichloroplatinum(II), cw-DDP, by
Barnett Rosenberg and his collaborators comprises an impressive chapter
Publication Date: January 26, 1983 | doi: 10.1021/bk-1983-0209.pr001
in the history of bioinorganic chemistry. Efforts are currently being made

by research groups throughout the world to understand the inorganic and
biological chemistry responsible for the antitumor properties of cfc-DDP,
and this symposium was held to promote an exchange of information
among many of the leading laboratories working on this problem. The aim
of the symposium was to stress chemical rather than clinical aspects of the
problem, which was consistent with the expectations of an audience of
chemists rather than physicians. During the planning stages for the meet
ing, several colleagues reminded us that the use of inorganic compounds
in chemotherapy was a broader subject than was embraced only by the
antitumor platinum compounds. Consequently, the scope of the symposium
was widened to include other metal chemotherapeutic agents, chiefly the
gold-based antiarthritic drugs.
The inorganic and the biological techniques being used to study the
mechanism of action of platinum antitumor drugs have become increas
195
ingly sophisticated. P t - N M R and other spectroscopic studies have re
vealed the complex composition of aqueous solutions of cw-DDP. D N A
molecules of varying complexity, including superhelical D N A molecules,
restriction fragments of known sequence, and Z - D N A , have been em
ployed in cw-DDP binding studies. Also, nucleosomes and chromatin are
being investigated; nuclease enzymes have been used to probe the platinum
binding sites; the alkaline elution technique is being applied to study in
vivo crosslinking; and antibodies raised against cw-DDP-modified D N A
have been used to probe the in vivo binding of the drug. N M R spectro
scopy has afforded a very powerful means of studying the stereochemistry
of platinum-oligonucleotide complexes in solution. Attempts to find more
powerful and less toxic metal-containing anticancer drugs have continued
through synthetic and biological studies of platinum group and other
transition elements.
Gold compounds have long been used for medicinal purposes, but a
recent flurry of interest in these complexes by the bioinorganic community
ix
has arisen because of the development of the first effective, orally admin
istered antiarthritic gold complex, RSAuPEt , where R is a thioglucose
3
derivative. As with the platinum antitumor drugs, the most powerful new
methods are beginning to be applied to studies of the mechanism of action
of this and other chrysotherapeutic agents. Two of the new methods are
multinuclear N M R and extended x-ray absorption fine structure (EXAFS)
spectroscopy.
The present volume discusses these topics through reports from most
of the laboratories represented at the Las Vegas meeting, including several
interesting papers presented in the poster sessions. I am grateful to the
authors for their prompt submission of manuscripts, assistance with refer-
eeing, and overall cooperation in helping to make the meeting and this
Publication Date: January 26, 1983 | doi: 10.1021/bk-1983-0209.pr001
volume a success. I also thank Dr. Barnett Rosenberg and Dr. Luigi Mar-
zilli for help in planning the symposium. Financial assistance from the
Donors of the Petroleum Research Fund administered by the American
Chemical Society; the Inorganic Division of the ACS; Engelhard Indus
tries; INCO Research and Development Center; Johnson Matthey, Inc.;
and Smith Kline and French Laboratories to support the symposium is
gratefully acknowledged. Through their help it was possible to bring many
foreign scientists to the meeting, the contributions of whom form an im
portant part of this book.
S T E P H E N J. L I P P A R D
Columbia University
October 1, 1982

1
DNA as a Target for Anticancer Coordination

Compounds
J. J. ROBERTS and M. F. PERA, JR.1

Institute of Cancer Research, Pollards Wood Research Station, Nightingales Lane, Chal-
font St. Giles, Bucks, HP8 4SP England
Publication Date: January 26, 1983 | doi: 10.1021/bk-1983-0209.ch001
The notion that DNA is the likely target for anti-

tumour platinum complexes will be discussed on the
basis of their interaction with nucleic acids in
vitro and of their interactions with the DNA of both
cells in culture and cells in whole animals.
Biochemical studies suggest that interactions with
cellular DNA result in an inactivation of the DNA
template for DNA replication. Support for these
views came from the demonstration that cells could
remove DNA bound platinum adducts by an excision
repair process that facilitated the recovery of
cells from toxic damage.
Since the first description of the biological properties of

platinum complexes there has been a considerable effort to
understand the molecular basis of their actions. The clear
demonstration of the usefulness of cisplatin in treatment of
human tumours has added impetus to this work, because ultimately
an understanding of the mechanism of drug action should provide
a more rational basis for improvement in therapy. Much evidence
is available now to support the view that platinum drugs exert
their cytotoxic effects through an interaction with DNA. The
main purpose of this review will be to assess critically the
evidence supporting this hypothesis. We will first briefly
summarise some of the biological properties of platinum compounds
other than antitumour activity that are probably a consequence
of their ability to react with DNA. Then we will discuss the
interaction of platinum compounds with nucleic acids before
considering in detail the evidence that indicates DNA as a major
target for drug action.
1
Current address: I C R F Laboratories, P O B 123, Lincoln's Inn Fields, London W C 2 A
3 P X England
0097-6156/83/0209-0003$06.75/0
© 1983 American Chemical Society

4 METAL CHEMOTHERAPEUTIC AGENTS
B i o l o g i c a l E f f e c t s o f Platinum Antitumour Drugs I n d i c a t i v e o f

I n t e r a c t i o n with DNA
Probably the f i r s t o b s e r v a t i o n o f an e f f e c t o f a platinum

c o o r d i n a t i o n complex i n a b i o l o g i c a l system and one which gave a
c l e a r i n d i c a t i o n o f i t s biochemical mode o f a c t i o n was the
o b s e r v a t i o n t h a t n e u t r a l platinum c o o r d i n a t i o n complexes i n h i b
i t e d b a c t e r i a l c e l l d i v i s i o n and induced the b a c t e r i a t o grow
i n t o long f i l a m e n t s . Filamentous growth i n b a c t e r i a may
be i n d i c a t i v e o f the a b i l i t y o f an agent t o r e a c t with DNA
l e a d i n g t o a s e l e c t i v e i n h i b i t i o n o f DNA s y n t h e s i s , with no
accompanying e f f e c t on other b i o s y n t h e t i c pathways such as RNA
or p r o t e i n s y n t h e s i s . A v a r i e t y o f agents, such as UV- and
X - i r r a d i a t i o n and c y t o t o x i c a l k y l a t i n g agents, can a l s o e l i c i t
t h i s response as a r e s u l t o f t h e i r common a b i l i t y t o damage DNA.

F u r t h e r important evidence f o r d i r e c t a t t a c k on DNA came
from the a b i l i t y o f platinum compounds t o induce the growth o f
phage from l y s o g e n i c s t r a i n s o f E . c o l i b a c t e r i a (2). The
r e l e a s e o f the phage DNA t o d i r e c t s y n t h e s i s o f new phage i s
normally a r a r e event. However, agents which can r e a c t with
DNA can cause the phage DNA t o be r e l e a s e d and phage p a r t i c l e s
t o be s y n t h e s i z e d with consequent c e l l l y s i s . Reslova (2)
was able t o show t h a t there e x i s t s an e x c e l l e n t c o r r e l a t i o n
between the antitumour a c t i v i t y of platinum compounds and t h e i r
a b i l i t y t o induce l y s o g e n i c Ε.coli t o enter the l y t i c c y c l e .
The i n t e r a c t i o n s o f platinum compounds with v i r u s e s have
f u r t h e r i n d i c a t e d the r e l a t i v e l y g r e a t e r importance o f r e a c t i o n s
with DNA as a g a i n s t those w i t h p r o t e i n i n producing b i o l o g i c a l
effects. Kutinova e t a l . (3) demonstrated the i n a c t i v a t i o n o f
the i n f e c t i o u s a c t i v i t y o f e x t r a c e l l u l a r papovavirus SV40 by
cis-ΡΡΡ. The i n a c t i v a t i o n o f B . s u b t i l i s transforming DNA by
platinum compounds, l i k e w i s e i n d i c a t e d the e f f e c t o f these
agents on the b i o l o g i c a l f u n c t i o n o f DNA (<4) .
The genotoxic nature o f platinum compounds and the import
ance o f the geometrical arrangement o f l i g a n d s f o r b i o l o g i c a l
e f f e c t , a l s o emerges from s t u d i e s on the mutagenic p r o p e r t i e s o f
these agents i n a number o f p r o k a r y o t i c and e u k a r y o t i c systems
(5-10). The c i s d e r i v a t i v e s were i n a l l cases a p p r e c i a b l y more
mutagenic than the corresponding t r a n s isomers. A comparison
o f c y t o t o x i c i t y and mutagenicity, e i t h e r per DNA i n t e r s t r a n d
c r o s s l i n k e d l e s i o n (9) or per t o t a l number o f l e s i o n s (10), f o r
c i s and trans-DPP showed t h a t these two b i o l o g i c a l e f f e c t s d i f f e r
i n t h e i r s e n s i t i v i t y to DNA b i n d i n g . Thus, i n a study o f mut
a t i o n a t the HGPRT locus i n Chinese hamster V79 c e l l s , e q u i t o x i c
doses o f trans-DPP were much l e s s mutagenic than cis-DDP, even
though i n t e r s t r a n d c r o s s l i n k i n g f o r the two isomers was comparable
(9). The study by Johnson e t a l . (10) i n CHO c e l l s r e v e a l e d
t h a t the mutagenicity per molecule bound t o DNA immediately a f t e r
16-24 h treatment was a t l e a s t 750 times l a r g e r f o r cis-DPP
than f o r the trans-isomer, while c y t o t o x i c i t y per DNA l e s i o n was
a f a c t o r o f 9 g r e a t e r f o r c i s than f o r trans-PPP.

1. ROBERTS AND PERA DNA and Anticancer Compounds 5
Another p r o p e r t y platinum antitumour drugs share with agents

t h a t i n t e r a c t with DNA i s i n d u c t i o n o f cancer. In v i t r o s t u d i e s
o f morphological t r a n s f o r m a t i o n , a p r o p e r t y o f t e n c o r r e l a t e d
with tumour i n d u c t i o n i n v i v o , showed cis-PPP was capable o f
transforming secondary S y r i a n hamster embryo c e l l s (11) . The
f i n d i n g t h a t trans-DPP transformed l O T i mouse c e l l s and 3T3 c e l l s
was o f i n t e r e s t i n l i g h t o f t h e low mutagenicity o f t h i s compound
(12). In c a r c i n o g e n e s i s s t u d i e s i n v i v o , cis-DDP produced lung
adenomas i n A/Jax mice, s k i n papillomas i n CD1 mice when adminis-
t e r e d i n combination with c r o t o n o i l , and sarcomas a t the s i t e o f
i n j e c t i o n i n F344 r a t s (13). When administered a t maximally
t o l e r a t e d doses, trans-DPP d i d not induce tumours i n the lung
adenoma o r s k i n papilloma systems, as expected from i t s low
mutagenicity (14).
Reaction o f Platinum Prugs w i t h PNA
Although other hypotheses have been proposed f o r the mechan-

ism o f a c t i o n o f platinum compounds (15-20), the bulk o f the
evidence a t present favour t h e theory t h a t PNA damage and synthe-
s i s o f PNA on a damaged template i s d i r e c t l y r e s p o n s i b l e f o r the
c y t o t o x i c a c t i v i t y o f platinum drugs. Before p r e s e n t i n g the
experimental evidence, i t i s u s e f u l f i r s t t o o u t l i n e the general
argument.
Platinum antitumour drugs are b i f u n c t i o n a l e l e c t r o p h i l i c
species which r e a c t i n a r e l a t i v e l y n o n - s e l e c t i v e f a s h i o n with
n u c l e o p h i l i c s i t e s i n c e l l u l a r macromolecules. On a p e r mole-
c u l e b a s i s , PNA i s the o n l y t a r g e t t h a t i s l a r g e enough t o under-
go one o r more r e a c t i o n s p e r molecule a t p h a r m a c o l o g i c a l l y
r e l e v a n t doses. Reaction w i t h PNA r e s u l t s i n disturbances i n
the r a t e o f PNA s y n t h e s i s , o r the q u a l i t y o f nascent PNA synthe-
s i z e d , which leads t o chromosome damage and c e l l death. Cells
possess t o v a r y i n g degrees the c a p a c i t y t o e x c i s e damage from
template DNA, and moreover, c e l l s vary i n t h e i r c a p a c i t y t o
synthesize PNA on a damaged template. Thus c y t o t o x i c i t y and
c e l l u l a r s e n s i t i v i t y t o platinum drugs w i l l be a f u n c t i o n o f the
extent o f r e a c t i o n with DNA, the c a p a c i t y t o remove damage from
the template, and the c a p a c i t y t o s y n t h e s i z e PNA on a template
c o n t a i n i n g damage.
Thus evidence f o r t h i s mechanism comes from s t u d i e s o f the
i n t e r a c t i o n o f the drugs with n u c l e i c a c i d s and n u c l e i c a c i d
components, from observations on the e f f e c t s o f the drugs on
DNA s y n t h e s i s , and from observations r e l a t i n g t o r e p a i r o f PNA
l e s i o n s and c y t o t o x i c i t y . F i r s t we w i l l consider the r e a c t i o n s
o f the drug with n u c l e i c a c i d s .
Reactions o f n e u t r a l platinum complexes with n u c l e i c a c i d

components i n v i t r o . Changes i n the u l t r a v i o l e t a b s o r p t i o n
spectrum o f salmon sperm PNA a f t e r r e a c t i o n with e i t h e r c i s o r

trans-DPP provided c o n c l u s i v e evidence t h a t both platinum com

pounds b i n d t o the organic bases o f DNA. Spectrophotometry
s t u d i e s f u r t h e r confirmed t h a t the three bases, guanine, adenine
and c y t o s i n e would a l l r e a c t with both isomers, the r a t e o f
r e a c t i o n with guanine being f a s t e r than with the other two bases
(22,23,24).
By b l o c k i n g the v a r i o u s p o s s i b l e b i n d i n g s i t e s i n the purine
bases by e i t h e r methylation o r p r o t o n a t i o n , Mansy e t a l . (22)
d e f i n e d the s i t e s most l i k e l y t o be i n v o l v e d i n r e a c t i o n w i t h
e i t h e r c i s o r trans-DPP. They concluded t h a t the c i s isomer
forms a bidentate c h e l a t e w i t h e i t h e r the 6-NH and N-7, o r t h e2
6-NH and N - l o f adenine, and the 4-NH and N-3 o f c y t o s i n e .

2 2
The t r a n s isomer, on the other hand, i n t e r a c t s monofunctionally

a t the N-7 and N - l o f adenine and the N-3 o f c y t o s i n e . Both
isomers r e a c t monofunctionally w i t h the N-7 o f guanine and hypox-

anthine. X-ray d i f f r a c t i o n s t u d i e s o f the complexes formed
between c i s P t ( N H 3 ) X and v a r i o u s bases have confirmed some o f
2 2
the c o n c l u s i o n s obtained from the e a r l y spectrophotometrie

studies. The product o f the r e a c t i o n between i n o s i n e and
P t ( N H 3 ) I c o n s i s t o f two hypoxanthine r i n g s bound t o the
2 2
platinum i o n v i a the N-7 p o s i t i o n (25). A similar structure

r e s u l t s from the i n t e r a c t i o n o f PtCl2 (en) with guanosine
(26,27); again the N-7 p o s i t i o n becomes occupied by the metal.
Other atoms which have been shown t o have an a f f i n i t y f o r
platinum i n c l u d e the deprotonated N ( l ) and Ν(3) p o s i t i o n s o f
guanine and thymine r e s p e c t i v e l y as w e l l as the deprotonated NH 2
group o f c y t o s i n e (28).
There i s no evidence y e t from c r y s t a l l o g r a p h i c s t u d i e s t h a t
the 0-6 p o s i t i o n o f guanine, the 6-NH group o f adenine o r the
2
4-NH group o f c y t o s i n e can be occupied by platinum(II) i o n s .

2
A bidentate b i n d i n g r e a c t i o n between the N-7 and the 0-6 p o s i t

ions o f guanine f o r the c i s b u t not the t r a n s platinum(II)
compounds was an a t t r a c t i v e p o s s i b i l i t y t o account f o r the d i f f
erence between the b i o l o g i c a l e f f e c t i v e n e s s o f the two isomers.
However, such bidentate b i n d i n g t o a s i n g l e base has not y e t been
i d e n t i f i e d unambiguously.
C i s - p l a t i n u m compounds can induce the formation o f i n t e r -
strand c r o s s l i n k s i n i s o l a t e d PNA (21,29-32) o r i n the PNA o f
whole c e l l s (33,34). There i s as y e t no d i r e c t evidence t o
i n d i c a t e which o f the many p o s s i b l e b i n d i n g s i t e s d i s c u s s e d above,
are i n v o l v e d i n such a r e a c t i o n . A p o s s i b i l i t y , suggested from
examination o f a PNA model, i s t h a t c r o s s l i n k i n g could occur
between the 6-amino groups i n adenines i n opposing strands o f
PNA i n a dA-dT sequence (35). These groups would be 3.5A apart
which approximates 3A; the d i s t a n c e between the c i s l e a v i n g
groups i n cis-PPP. There i s some evidence t o i n d i c a t e t h a t
cis-PPP can l i n k two NH groups i n t h i s way i n a simple nucleo
2
t i d e (36,37). A l t e r n a t i v e l y the amino groups o f guanines o r o f

c y t o s i n e s i n opposing strands o f PNA i n a dC-dG PNA sequence are
t h e o r e t i c a l l y amenable t o c r o s s l i n k i n g . The p r e f e r e n t i a l i n t e r -

strand c r o s s l i n k i n g o f DNAs r i c h i n guanine and c y t o s i n e was

noted from s t u d i e s o f the r e n a t u r a b i l i t y o f c r o s s l i n k e d DNAs o f
d i f f e r e n t G-C content (32), and from a study o f the i n h i b i t i o n o f
i n t e r c a l a t i o n o f 9-aminoacridine as a f u n c t i o n o f (G + C) content
o f p l a t i n u m - t r e a t e d DNA o r copolymers (38).
The frequency o f i n t e r s t r a n d c r o s s l i n k s was o r i g i n a l l y e s t i
mated from a combination o f measurements o f the amount o f p l a t i
num bound t o Hela c e l l c r o s s l i n k e d DNA molecules o f a presumed
s i z e (29,30): i t was shown t o be a r e l a t i v e l y r a r e event,
accounting f o r l e s s than one p e r cent o f the t o t a l number o f
r e a c t i o n s with c e l l u l a r DNA. Recent q u a n t i t a t i v e s t u d i e s o f
c r o s s l i n k i n g o f Chinese hamster c e l l DNA, u s i n g a v a r i e t y o f
techniques, confirmed the r a r i t y o f c r o s s l i n k s i n whole c e l l s a t
the time o f t h e i r maximum development (see below).
Despite the c l e a r evidence f o r the formation o f i n t e r s t r a n d

DNA c r o s s l i n k s both i n i s o l a t e d DNA and i n the DNA o f whole c e l l s ,
many r e c e n t observations i n d i c a t e the l i k e l y formation o f i n t r a -
s t r a n d c r o s s l i n k i n g between adjacent bases i n DNA by platinum
compounds. Moreover the importance o f t h i s r e a c t i o n as an
i n a c t i v a t i n g event i n bacteriophage was noted, f i r s t , by Shooter
e t a l . (39) f o r T7 bacteriophage and, more r e c e n t l y , by
F i l i p s k i e t a l . (40) f o r phage λ. F o r such a c r o s s l i n k i n g
r e a c t i o n t o occur, l o c a l p e r t u r b a t i o n o f the double h e l i x i s
r e q u i r e d , and indeed, some o b s e r v a t i o n s o f X-ray p h o t o e l e c t r o n
s p e c t r a l changes (41,42,43) o r CD s p e c t r a l changes (42);
Tamburio e t a l . (43) support such a m o d i f i c a t i o n . Marked enhan
cement o f the CD spectrum o f DNA was observed even a t low l e v e l s
o f r e a c t i o n and t h i s e f f e c t i n c r e a s e s w i t h i n c r e a s i n g GC content
o f s e v e r a l d i f f e r e n t DNAs (42). The b i n d i n g o f platinum t o the
N-7 p o s i t i o n o f guanine c o u l d weaken the G-C hydrogen bonding
which, i t i s suggested, makes the N - l p o s i t i o n o f guanine
a v a i l a b l e f o r f u r t h e r r e a c t i o n (44). Covalent b i n d i n g o f both
c i s and trans-DPP t o c l o s e d c i r c u l a r PM2 DNA a l t e r s the degree o f
s u p e r c o i l i n g and shortens the DNA, as r e v e a l e d by e l e c t r o n micro
scopy, presumably by d i s r u p t i n g and unwinding the complex (45,46).
Some recent elegant experiments by Cohen e t a l . (47) add f u r t h e r
strong support t o the a b i l i t y o f c i s but not trans-DPP t o form
i n t r a s t r a n d c r o s s l i n k s between adjacent guanines i n a dGn dCn
(n = 4) sequence i n c i r c u l a r pSMl PNA a t low l e v e l s o f Pt/P
ratios.
Reaction w i t h PNA o f c e l l s i n c u l t u r e . Studies w i t h

c u l t u r e d c e l l s have i n d i c a t e d the relevance o f platinum-PNA
binding t o c y t o t o x i c i t y . Pascoe and Roberts (29>,30) s t u d i e d
the i n t e r a c t i o n o f s e v e r a l platinum compounds w i t h macromolecules
a t measured l e v e l s o f c e l l k i l l .
To assess the p o s s i b l e importance o f PNA, RNA and p r o t e i n as
primary t a r g e t s f o r platinum(II) compounds, these b i n d i n g data
(expressed as moles/gm o f macromolecule) were used t o c o n s t r u c t
curves o f l o g s u r v i v a l a g a i n s t the amount o f drug bound t o each

type o f macromolecule. The r e s u l t i n g graphs were then

c h a r a c t e r i s e d i n a manner s i m i l a r t o those r e l a t i n g l o g c e l l
s u r v i v a l t o dose o f drug given t o the c e l l s . The shoulder
width o f the b i n d i n g curve was given the value Bq and the slope
o f the s t r a i g h t l i n e p o r t i o n B .
Q For both c i s and trans-DPP
the b i n d i n g c o e f f i c i e n t s were higher f o r RNA than DNA. However,
the t r u e s i g n i f i c a n c e o f these b i n d i n g c o e f f i c i e n t s can only be
appreciated i f account i s taken o f the molecular weights o f the
molecules concerned. I f one assumes no s e l e c t i v i t y i n the
b i n d i n g t o any p a r t i c u l a r RNA or p r o t e i n molecule, then i t i s
p o s s i b l e t o c a l c u l a t e the number o f platinum molecules bound t o
each macromolecule a t a given t o x i c dose. The r e s u l t s o f such
a c a l c u l a t i o n , performed a t the c o n c e n t r a t i o n o f cis-DDP which
reduced the s u r v i v i n g f r a c t i o n o f Hela c e l l s from f t o 0.37f
( t h i s i s t h e o r e t i c a l l y the c o n c e n t r a t i o n a t which one i n a c t i v a t -

i n g event occurs, on the average, i n each c e l l ) show the number
o f molecules bound t o DNA i s s t r i k i n g l y more than t h a t t o e i t h e r
RNA or p r o t e i n , c l e a r l y i n d i c a t i n g t h a t DNA i s the most suscep-
t i b l e c e l l u l a r t a r g e t f o r cis-DDP. The b i n d i n g data f u r t h e r
i n d i c a t e t h a t a t t h i s c o n c e n t r a t i o n o f cis-DDP approximately
only one molecule o f p r o t e i n out o f 1500 molecules w i l l have
r e c e i v e d one p l a t i n a t i o n r e a c t i o n . Unless there i s c o n s i d e r a b l e
s p e c i f i c i t y i n the r e a c t i o n o f platinum drugs with a p a r t i c u l a r
p r o t e i n enzyme molecule, then t h i s l e v e l o f r e a c t i o n would be
too low t o i n a c t i v a t e enzyme a c t i v i t y . Moreover, the l e v e l o f
r e a c t i o n with rRNA, tRNA or mRNA would not be expected, again,
i n the absence o f any s e l e c t i v i t y o f r e a c t i o n , t o i n a c t i v a t e a l l
such molecules and l e a d t o i n t e r f e r e n c e with p r o t e i n s y n t h e s i s .
S i m i l a r DNA b i n d i n g and c e l l s u r v i v a l s t u d i e s have been
c a r r i e d out i n Chinese hamster c e l l s i n c u l t u r e with a number o f
other platinum compounds t h a t have shown encouraging a c t i v i t y
against a number o f experimental animal tumours (48). Differ-
ences o f up t o t e n f o l d were found i n the molar concentrations
o f these agents t h a t were r e q u i r e d t o produce e q u i t o x i c e f f e c t s
on c e l l s i n c u l t u r e f o l l o w i n g one hour's i n c u b a t i o n .
The l e v e l s o f r e a c t i o n w i t h DNA a t e q u i t o x i c doses ( B Q
v a l u e s ) , on the other hançl, were, f o r most compounds, o f the

same order and d i f f e r e d by o n l y a few f o l d (Table I ) .
Reaction with the DNA o f c e l l s i n v i v o . I t i s c l e a r l y

e s s e n t i a l , f o r an understanding o f the mechanism o f the tumour-
i n h i b i t o r y a c t i o n o f platinum compounds, t o e s t a b l i s h that the
s e n s i t i v i t y o f tumour c e l l s i n v i v o i s r e l a t e d t o the extent o f
r e a c t i o n o f platinum with t h e i r DNA i n a manner s i m i l a r t o t h a t
f o r c e l l s t r e a t e d i n v i t r o w i t h these agents. In a p r e l i m i n a r y
study, mice b e a r i n g the t r a n s p l a n t e d ADJ/PC6 plasmacytoma were
t r e a t e d with cis-DDP and two other a c t i v e platinum congeners,
CHIP and cis-diammine(1:1-eyelobutanedicarboxylato)platinum(II)
at doses t h a t had an equal i n h i b i t o r y e f f e c t on the tumour
(IDgo) (49). Despite the d i f f e r e n c e i n the a c t u a l amounts o f

Table I sw
w
Comparison o f the t o x i c i t y o f v a r i o u s platinum complexes towards Η
Chinese hamster V79 379A c e l l s i n c u l t u r e and plasmacytoma ADJ/PC6
>
tumour c e l l s i n v i v o i n r e l a t i o n t o DNA b i n d i n g (Roberts, 1981)
α
ADJ/PC6A Chinese hamster
Mouse plasmacytoma V79 379A c e l l s >
Compound
L D I D
D Β
50 90 ο ο
(mg/kg) (mg/kg) (μΜ/l h) (nmoles/gm)
cis-Pt(II)Cl (NH )
2 3 2
13 1.6 15 8.5
ι
ι
cis-Pt(IV)C1 (iso-C H NH ) (OH)
2 3 ? 2 2 2
54 4.2 48 2.5
cis-Pt(II)(1,1-CBDCA)(NH ) 3 2
180 14.5 120 3.0
cis-Pt(II)(mal)(1,2-dac) N.D. N.D. 23 2.5 Î

cis-Pt(II)(SO )H 0(l,2-dac) 14 0.4 65 17.5
4 2
cis-Pt(II)Cl (C H NH ) 480 2.4 120 N.D.

2 5 g 2 2
N.D. Not determined

CBDCA Cyclobutanedicarboxylic acid; dac, 1,2-diaminocyclohexane

v©
the m a t e r i a l s administered t o the mice, the doses d i d not d i f f e r

by more than a f a c t o r o f two when expressed i n terms o f t h e i r
molar c o n c e n t r a t i o n s . I n t e r e s t i n g l y , the amounts o f the p l a t i -
num drugs bound t o the tumour DNA a t these e q u i t o x i c concentra-
t i o n s were a l l remarkably s i m i l a r .
Pera e t a l . (50) s t u d i e d the r e a c t i o n o f c i s p l a t i n and
hydroxymalonato diammine platinum(II) with DNA o f B16 melanoma
and bone marrow i n C57BL mice. I n h i b i t i o n o f tumour growth,
and colony formation assays f o r melanoma c e l l s as w e l l as bone
marrow stem c e l l s , were used t o q u a n t i t a t e t o x i c i t y and t o
i n d i c a t e antitumour s e l e c t i v i t y . Pt[OHmal(NH3)2] produced
g r e a t e r s e l e c t i v e i n h i b i t i o n o f tumour growth and more s e l e c t i v e
tumour c e l l k i l l i n g compared with c i s p l a t i n . In the case o f
c i s p l a t i n , b i n d i n g o f platinum t o DNA a t measured l e v e l s o f
s u r v i v a l i n v i v o was s i m i l a r t o values p r e v i o u s l y observed i n

c u l t u r e d c e l l s , a f i n d i n g t h a t again strengthens arguments
concerning the mechanism o f a c t i o n o f the drugs based on i n
v i t r o work. The g r e a t e r s e l e c t i v e t o x i c i t y o f Pt[OHmal(NH3)2)
towards the B16 melanoma was a s s o c i a t e d with an i n c r e a s e d
b i n d i n g o f platinum t o tumour DNA, r e l a t i v e t o c i s p l a t i n . The
enhanced DNA b i n d i n g i n the tumour seen with Pt[OHmal(NH^)2]
was not seen i n the marrow (Figure 1 A.B., Table I I ) . Thus
the i n c r e a s e d antitumour s p e c i f i c i t y o f the newer congener
probably r e s u l t s from pharmacologic f a c t o r s t h a t enhance d e l i v e r y
o f a c t i v e drug t o tumour c e l l s .
Role o f c r o s s l i n k i n g r e a c t i o n s . The s t r u c t u r a l requirement

f o r d i f u n c t i o n a l i t y and the p r i n c i p a l biochemical e f f e c t s o f the
platinum compounds, as d i s c u s s e d below, suggest a p a r a l l e l
between the platinum drugs and the c l a s s i c a l b i - f u n c t i o n a l a l k y l -
a t i n g agents such as the n i t r o g e n mustards. The l a t t e r compounds
have been thought f o r some time t o produce an i n h i b i t i o n o f DNA
s y n t h e s i s by t h e i r a b i l i t y t o introduce c r o s s l i n k s i n t o the
DNA o f mammalian c e l l s . I t has, however, been a matter o f
contention as t o whether the p r i n c i p a l l e s i o n i s a c r o s s l i n k
between strands o f the DNA h e l i x or c r o s s l i n k s between bases on
one s t r a n d o f DNA, o r p o s s i b l y , even between DNA and p r o t e i n .
C r o s s l i n k i n g was estimated by Roberts and Pascoe (33) from
the p r o p o r t i o n o f 'hybrid' DNA formed by l i n k i n g opposing strands
o f heavy (BudR l a b e l l e d ) and l i g h t (normal) DNA.
The r e l a t i v e t o x i c i t i e s o f the c i s and t r a n s isomers o f the
platinum(II) n e u t r a l complexes, can be d e f i n e d by the slopes o f
the s u r v i v a l curves (D ) obtained by t r e a t i n g Hela c e l l s i n
0
culture. Comparison o f these two s e t s o f values i n d i c a t e d t h a t

the r e l a t i v e a b i l i t i e s o f c i s and trans-DPP compounds t o c r o s s -
l i n k DNA i n v i v o (but not i n v i t r o ) were r e l a t e d t o t h e i r c y t o -
t o x i c a c t i o n (29,30). These s t u d i e s t h e r e f o r e suggest t h a t
i n t e r s t r a n d c r o s s l i n k i n g with both the platinum(II) compounds,
but not n e c e s s a r i l y platinum(IV) compounds, may be important i n
i n d u c i n g t h e i r c y t o t o x i c e f f e c t s and t h a t the c i s isomer i s most
e f f e c t i v e i n i n d u c i n g the r e a c t i o n .

35-1
Cisplatin dose (mg/kg)
80-,
70H
0 50 100 150
Pt [ OHMal(NH ) ] dose (mg/kg)
3 2
Figure L Binding of platinum to B16 melanoma DNA (Φ) or bone marrow DNA
(A) following treatment of C57B16 mice with cis-diamminedichloroplatinum(II)
(ch-DDP) (A) and hydroxymalonatodiammineplatinumfll) (B).

Table I I
Amount o f platinum bound t o marrow and tumour DNA

of C57BL mice a t doses o f c i s p l a t i n o r Pt[OHMal (NH3) ] 2
producing 37 per cent s u r v i v a l o f bone marrow stem

c e l l s (CFU-S) o r B16 lung colony forming c e l l s
B16
CFU-S
LCFC
Amount Amount
Bound Bound
Cisplatin 5 mg/Kg 4nmol/g 11 mg/Kg 22nmol/g

Pt[OHMal(NH ) ]3 2 40 mg/Kg 5nmol/g 20 mg/Kg 12.5nmol/g

A r e i n v e s t i g a t i o n o f c r o s s l i n k i n g o f DNA by platinum(II)
compounds u s i n g a d i f f e r e n t method from t h a t d e s c r i b e d above,
namely a l k a l i n e e l u t i o n (34) has confirmed the g r e a t e r a b i l i t y
of cis-DDP as compared with trans-DPP t o c r o s s l i n k c e l l u l a r DNA.
These i n v e s t i g a t o r s made the f u r t h e r i n t e r e s t i n g o b s e r v a t i o n t h a t
i n c u b a t i o n o f t r e a t e d mouse leukemic L1210 c e l l s i n a drug-free
medium r e s u l t e d i n an i n c r e a s e i n the number o f DNA c r o s s l i n k s .
C r o s s l i n k i n g e f f e c t s developed, f o l l o w i n g treatment w i t h concen-
t r a t i o n s as low as 1 ym f o r c i s and 5 ym f o r trans-DPP which
permitted over 80% s u r v i v a l o f colony-forming a b i l i t y . The
maximum c r o s s l i n k i n g e f f e c t by cis-PPP r e q u i r e d about 12 h post
treatment i n c u b a t i o n before i t was f u l l y developed by 6 h a f t e r
exposure t o the drug. The c r o s s l i n k i n g e f f e c t s o f both agents
were reversed upon f u r t h e r i n c u b a t i o n o f the c e l l s , presumably
due t o the o p e r a t i o n o f a PNA e x c i s i o n r e p a i r process.

A f u r t h e r study employing a l k a l i n e e l u t i o n showed t h a t the
c r o s s l i n k i n g e f f e c t produced by c i s and trans-PPP c o u l d be
separated i n t o two components, one p r o t e i n a s e - s e n s i t i v e and due
t o PNA-protein c r o s s l i n k i n g , another p r o t e i n a s e - r e s i s t a n t and due
to PNA i n t e r s t r a n d c r o s s l i n k i n g (51). PNA p r o t e i n c r o s s l i n k s
were a t maximum l e v e l s immediately a f t e r drug removal, while
PNA-PNA i n t e r s t r a n d c r o s s l i n k s reached maximum l e v e l s 6-12 hours
a f t e r drug removal. T o x i c i t y o f the two agents i n L1210
leukemia c e l l s , and V79 Chinese hamster c e l l s , c o r r e l a t e d w e l l
with PNA i n t e r s t r a n d c r o s s l i n k i n g , but not with PNA-protein
crosslinking (9,51).
Because the b i o p h y s i c a l b a s i s o f the e l u t i o n technique i s
p o o r l y understood, q u a n t i t a t i o n o f i n t e r s t r a n d c r o s s l i n k i n g with
t h i s technique i s based upon l a r g e l y unproven assumptions.
Therefore s t u d i e s were undertaken t o compare e l u t i o n r e s u l t s
with the a l k a l i n e caesium c h l o r i d e technique d e s c r i b e d above,
a l k a l i n e sucrose sedimentation, and e s t i m a t i o n o f renaturable
PNA i n c e l l l y s a t e s . D i r e c t q u a n t i t a t i o n o f c r o s s l i n k frequency
f o l l o w i n g c i s p l a t i n treatment was thus obtained over a wide dose
range u s i n g methods based upon known b i o p h y s i c a l p r o p e r t i e s o f
PNA. The r e s u l t s showed c l e a r l y t h a t a l k a l i n e e l u t i o n f o l l o w i n g
p r o t e i n a s e d i g e s t i o n gave an accurate measure o f i n t e r s t r a n d
c r o s s l i n k i n g , and showed a l s o t h a t c r o s s l i n k s were o n l y a small
f r a c t i o n o f the t o t a l drug-PNA r e a c t i o n products (52,53).
I t has been shown t h a t c e l l s can be p r o t e c t e d from the t o x i c
e f f e c t s o f cis-DDP by p r e v e n t i n g the formation o f PNA c r o s s l i n k s
by i n c u b a t i n g c e l l s i n the presence o f t h i o u r e a immediately a f t e r
treatment (54), a f i n d i n g which f u r t h e r supports a c y t o t o x i c r o l e
f o r PNA i n t e r s t r a n d c r o s s l i n k s . F u r t h e r i n v e s t i g a t i o n i n mouse
leukemia c e l l s and human f i b r o b l a s t s o f v a r y i n g s e n s i t i v i t y t o
c i s p l a t i n , have shown t h a t c e l l u l a r s e n s i t i v i t y o f t e n c o r r e l a t e s
with i n t e r s t r a n d c r o s s l i n k formation (55, 56, 57). However,
studies o f c e r t a i n mouse leukemia L1210 lines resistant to c i s -
platin (55,58), as w e l l as s t u d i e s o f Walker carcinoma c e l l s (59)

have i n d i c a t e d t h a t there i s not always a simple c o r r e l a t i o n

between c r o s s l i n k formation and c e l l k i l l .
I t i s e n t i r e l y p o s s i b l e t h a t i n t r a s t r a n d c r o s s l i n k i n g might
provide an even b e t t e r c o r r e l a t i o n , but i t i s not p o s s i b l e to
measure t h i s l e s i o n i n mammalian c e l l s at the present time.
Biochemical E f f e c t s of Drug-DNA Interaction
I n h i b i t i o n o f DNA s y n t h e s i s . The s i g n i f i c a n c e of the i n t e r

a c t i o n of platinum compounds with c e l l u l a r DNA i s apparent from
s t u d i e s of drug e f f e c t s on macromolecular s y n t h e s i s . Cis-DDP
s e l e c t i v e l y and p e r s i s t e n t l y i n h i b i t s the r a t e of DNA s y n t h e s i s
as compared with e f f e c t s on RNA and p r o t e i n s y n t h e s i s i n c e l l s i n
c u l t u r e (60-63) and c e l l s i n v i v o (64,65).
The l i k e l y b a s i s f o r t h i s s e l e c t i v e biochemical e f f e c t on
DNA s y n t h e s i s came from the o b s e r v a t i o n t h a t the i n h i b i t i o n of
DNA s y n t h e s i s was p e r s i s t e n t and p r o g r e s s i v e with time a f t e r
removal of the drug. I t i s now c l e a r , p a r t i c u l a r l y by com
p a r i s o n with analogous e f f e c t s produced by d i r e c t r e a c t i n g agents
such as mustard gas, t h a t both e f f e c t s are c o n s i s t e n t with the
view t h a t the primary chemical l e s i o n i s i n the DNA of the c e l l ,
which i s then i n h i b i t e d as a template f o r DNA r e p l i c a t i o n .
Thus m o d i f i c a t i o n s to the DNA template w i l l block DNA r e p l i c a t i o n
but not a f f e c t t r a n s c r i p t i o n or t r a n s l a t i o n . Under c o n d i t i o n s
of low c e l l k i l l i n g , the s e l e c t i v e i n h i b i t i o n by platinum com
pounds o f DNA s y n t h e s i s but not of RNA or p r o t e i n s y n t h e s i s ,
leads to the formation of g i a n t c e l l s , a feature observed i n
c e l l s t r e a t e d with a v a r i e t y o f agents a l s o known to block DNA
replication selectively.
Studies o f synchronized V79 Chinese hamster c e l l s t r e a t e d i n
Gl w i t h c i s p l a t i n showed t h a t depression of DNA s y n t h e s i s i n
these c e l l s was the r e s u l t of a decrease i n DNA s y n t h e t i c r a t e ,
r a t h e r than a decreased r a t e o f entry of c e l l s i n t o S (62).
As a r e s u l t , S phase was prolonged i n these c e l l s (Figure 2).
F o l l o w i n g Gl treatment with c i s p l a t i n , synchronized Hela c e l l s
a l s o showed a decrease i n the amount of thymidine i n c o r p o r a t i o n
i n t o DNA d u r i n g S phase, but the e f f e c t was not immediately
manifested i n these c e l l s (63) (Figure 3). Thus c e l l s d i f f e r i n
the way i n which t h e i r r e p l i c a t i o n machinery responds to c i s p l a -
t i n - i n d u c e d damage. Such d i f f e r e n c e s might account f o r v a r i a
t i o n s i n c e l l u l a r s e n s i t i v i t y (see below) and f u r t h e r s t u d i e s i n
t h i s area are warranted.
Johnson et a l . (66) used an i n v i t r o T7 DNA r e p l i c a t i o n
system which copies exogenous T7 DNA by a mechanism t h a t c l o s e l y
mimics i n v i v o DNA r e p l i c a t i o n to demonstrate the r e l a t i v e
i n h i b i t o r y e f f e c t of e i t h e r p y r i m i d i n e dimers or bound 195mpt:-
l a b e l l e d c i s or trans-DPP. I t c o u l d be shown t h a t cis-DDP
and pyrimidine dimers i n h i b i t e d DNA r e p l i c a t i o n to the same extent
per l e s i o n (63% i n h i b i t i o n per 3 χ 10"*^ l e s i o n s / n u c l e o t i d e phos
phate) and the trans-DPP was 5 - f o l d l e s s i n h i b i t o r y .

Τ
Time after harvest (hours)
Figure 2. Effects of cis-DDP treatment in Gl phase on DNA synthesis in synchro

nized Chinese hamster V79 cells. Key to cis-DDP concentration: · , 0; A> 1.0 μΜ;
5.0 /tM; and 10.0 μΜ. Τ indicates time of treatment.
Time after harvest (hours)
Figure 3. Effects of cis-DDP treatment in Gl phase on DNA synthesis in synchro

nized Hela cells. Key to cis-DDP concentration: · , 0; Δ , 0.1 μΜ; • , 0.25 μΜ; A ,
1.0 μΜ; and Ο , 2.0 Μ. μ

The a l t e r n a t i v e p o s s i b i l i t y t h a t DNA synthesis i s i n h i b i t e d

because o f the i n a c t i v a t i o n o f enzymes i n v o l v e d i n DNA r e p l i c a -
t i o n seems c o n t r a - i n d i c a t e d , not only by the f a i l u r e o f cis-DDP
to block p r o t e i n s y n t h e s i s , but a l s o by i t s f a i l u r e t o i n a c t i v a t e
DNA polymerase i n v i t r o except with very high concentrations (67).
R e l a t i o n between c a p a c i t y t o r e p l i c a t e on a damaged template

and c y t o t o x i c i t y . Further evidence r e l a t i n g drug induced
a l t e r a t i o n s i n DNA s y n t h e s i s t o c y t o t o x i c i t y i n b a c t e r i a and
mammalian c e l l s comes from s t u d i e s o f how c e l l s cope with DNA
template damage. Studies o f e x c i s i o n r e p a i r o f platinum induced
damage, discussed below, i n d i c a t e t h a t the m a j o r i t y o f DNA-
platinum products, i n v o l v i n g one strand o f a double h e l i x , are
chemically s t a b l e and are only slowly removed from DNA by an
enzymatic process. Thus i t could be supposed t h a t p e r s i s t e n t

l e s i o n s i n DNA are circumvented during DNA r e p l i c a t i o n by a
process which i s analogous t o t h a t which f a c i l i t a t e s the s u r v i v a l
of e x c i s i o n d e f e c t i v e b a c t e r i a a f t e r U V - i r r a d i a t i o n .
I t now seems c e r t a i n t h a t mammalian c e l l s a l s o possess
v a r y i n g c a p a c i t i e s t o r e p l i c a t e t h e i r DNA on a template c o n t a i n -
i n g unexcised damage. I t i s a l s o apparent, however, t h a t the
mechanism o f any such r e p a i r process d i f f e r s from t h a t which i s
thought t o occur i n b a c t e r i a . However, i r r e s p e c t i v e o f the
mechanism i n v o l v e d i n s y n t h e s i s i n g past r a d i a t i o n o r c h e m i c a l l y -
induced l e s i o n s i n DNA, i t has been found t h a t i n some c e l l s the
process i s amenable t o i n h i b i t i o n by the trimethylxanthine,
caffeine. Thus i t has been shown t h a t the r a t e o f elongation o f
newly synthesised DNA i n U V - i r r a d i a t e d o r c h e m i c a l l y - t r e a t e d
c e l l s , was d r a m a t i c a l l y impaired i n the presence o f c a f f e i n e .
As a consequence o f t h i s i n h i b i t i o n , many c e l l l i n e s , competent
i n t h i s r e p l i c a t i v e by-pass, are rendered extremely s e n s i t i v e t o
the l e t h a l e f f e c t s o f these agents by post treatment incubation
i n the presence o f non-toxic concentrations o f c a f f e i n e (68).
There i s now ample evidence t o i n d i c a t e t h a t UV- o r X - i r r a d -
i a t i o n o r chemically-induced c e l l death i s a f u n c t i o n o f the
amount o f chromosome damage which can be observed a t the f i r s t
or second m i t o s i s a f t e r treatment. Post treatment incubation i n
the presence o f c a f f e i n e enhances d r a m a t i c a l l y the chromosome
damaging e f f e c t s o f U V - i r r a d i a t i o n and chemicals i n both p l a n t
and animal c e l l s . The v a r i o u s c e l l u l a r e f f e c t s o f cis-DDP and
t h e i r m o d i f i c a t i o n s by c a f f e i n e , suggest t h a t l e s i o n s are
introduced onto DNA by platinum compounds which are a l s o circum-
vented by a c a f f e i n e s e n s i t i v e process (69). Post treatment
i n c u b a t i o n o f c e l l s i n medium c o n t a i n i n g 0.75 mM c a f f e i n e
d r a m a t i c a l l y p o t e n t i a t e d the t o x i c i t y o f c i s p l a t i n and increased
the number o f c e l l s c o n t a i n i n g chromosome damage. C a f f e i n e not
only increased the number o f cis-DDP t r e a t e d c e l l s c o n t a i n i n g
chromosomal aberrations but i t a l s o enhanced the s e v e r i t y o f the
damage observed. The delayed appearance o f chromosome abnormal-
i t i e s a f t e r cis-DDP treatment a l s o suggests t h a t DNA r e p l i c a t i o n

i s necessary f o r t h e i r formation, and i n t h i s r e s p e c t cis-DDP

resembles U V - i r r a d i a t i o n and a l k y l a t i n g agents r a t h e r than
X - i r r a d i a t i o n (69).
The proposal has t h e r e f o r e been made t h a t inadequate r e p l i -
c a t i o n o f DNA on a damaged template i s r e s p o n s i b l e f o r both c e l l
death and chromosome damage and t h a t post treatment i n c u b a t i o n
o f c e l l s i n medium c o n t a i n i n g c a f f e i n e , enhances these two e f f e c t s
o f DNA damage by i n h i b i t i n g a process which would permit r e p l i c a -
t i o n to proceed past l e s i o n s . Support f o r t h i s n o t i o n has come
from s t u d i e s on both the r a t e o f DNA s y n t h e s i s and the s i z e o f
DNA synthesised i n both asynchronous and synchronised populations
o f cis-DDP t r e a t e d c e l l s i n the presence and absence o f c a f f e i n e
(61,62). I t was found t h a t post treatment i n c u b a t i o n i n medium
c o n t a i n i n g a non-toxic c o n c e n t r a t i o n o f c a f f e i n e , reversed the
cis-DDP induced i n h i b i t i o n o f DNA s y n t h e s i s i n asynchronous popu-

l a t i o n s o f c e l l s (61). The s i z e of newly synthesised DNA i n
cis-DDP t r e a t e d c e l l s may be c o n t r a s t e d with the s i z e o f such
DNA i n c e l l s t r e a t e d s i m i l a r l y with cis-DDP and l a b e l l e d with.
(^H)TdR i n the presence o f non-toxic concentrations o f c a f f e i n e .
Under these c o n d i t i o n s the s i z e o f nascent DNA was markedly
reduced as compared with t h a t i n untreated c o n t r o l or cis-DDP
only t r e a t e d c e l l s . The s i z e o f the DNA synthesised d u r i n g 4
hours i n the presence o f c a f f e i n e i n cis-DDP t r e a t e d c e l l s was
dependent on the i n i t i a l dose o f DPP. I t thus appears t h a t
c a f f e i n e i n t e r f e r e s with the mechanism by which the c e l l r e p l i c -
ates i t s DNA past l e s i o n s on the DNA template. Some support f o r
t h i s n o t i o n was obtained from a comparison o f the d i s t a n c e
between platinum-induced l e s i o n s on the template strand o f DNA
and the s i z e o f the newly s y n t h e s i s e d DNA i n c e l l s t r e a t e d with
v a r i o u s doses o f cis-PPP and post incubated i n the presence o f
caffeine. The d i s t a n c e between platinum atoms on one strand o f
DNA was c a l c u l a t e d from atomic absorption measurements o f the
platinum bound to DNA i s o l a t e d from cis-DDP t r e a t e d c e l l s and
t h i s was found to correspond c l o s e l y to the s i z e o f the newly-
synthesised DNA. I t was concluded, t h e r e f o r e , t h a t i n Chinese
hamster c e l l s a l l unexcised p l a t i n a t i o n r e a c t i o n s are normally
circumvented d u r i n g DNA r e p l i c a t i o n by a c a f f e i n e - s e n s i t i v e
process.
R e l a t i o n s h i p Between E x c i s i o n Repair o f Platinum-induced PNA

Damage and C y t o t o x i c i t y
F r a v a l and Roberts (70) demonstrated removal o f platinum

adducts from DNA o f e x p o n e n t i a l l y growing Chinese hamster V79
cells. The h a l f - l i f e o f t o t a l drug-DNA r e a c t i o n products was
approximately 28 hours. As such products are s t a b l e chemically
under p h y s i o l o g i c a l c o n d i t i o n s , removal o f the DNA adducts could
be a t t r i b u t e d to r e p a i r .
Recent i n v e s t i g a t i o n s using a l k a l i n e e l u t i o n (51_) or a com-
b i n a t i o n o f a l k a l i n e e l u t i o n , a l k a l i n e sucrose sedimentation and

DNA r e n a t u r a t i o n , (53) have c l e a r l y demonstrated r e p a i r o f DNA-

p r o t e i n and DNA-DNA i n t e r s t r a n d c r o s s l i n k s i n c u l t u r e d mammalian
c e l l s f o l l o w i n g c i s p l a t i n treatment. Interstrand c r o s s l i n k i n g
i n Chinese hamster V79 c e l l s was demonstrated by a decrease i n
the r a t e o f f i l t e r e l u t i o n o f DNA from X - i r r a d i a t e d , t r e a t e d
c e l l s , a s h i f t i n a l k a l i n e sucrose g r a d i e n t s o f DNA molecules
towards the high molecular weight end o f the gradient, o r an
increase i n the r a p i d l y r e n a t u r i n g f r a c t i o n o f DNA i n c e l l l y s a -
tes. A l l o f these drug-induced phenomena reached a maximum
from 6-12 hours a f t e r drug treatment, then d e c l i n e d . The h a l f -
l i f e o f DNA i n t e r s t r a n d c r o s s l i n k s u s u a l l y appeared t o be between
12-24 hours. Some DNA degradation occurred, but i t was i n s u f f i -
c i e n t t o account f o r c r o s s l i n k r e v e r s a l . Because c r o s s l i n k s
induced by platinum are s t a b l e i n i s o l a t e d DNA under p h y s i o l o g -
i c a l c o n d i t i o n s , t h i s r e v e r s a l may a l s o be a t t r i b u t e d t o r e p a i r ,
though the mechanism remains unknown.
Although c o r r e l a t i o n s have been e s t a b l i s h e d i n some cases
between the extent o f drug-DNA i n t e r a c t i o n and c y t o t o x i c i t y , the
r e l a t i o n s h i p between e x c i s i o n r e p a i r o f DNA damage and t o x i c i t y
i s not always c l e a r .
The rare s k i n c o n d i t i o n , Xeroderma pigmentosum (XP), i s
c h a r a c t e r i s e d by extreme s e n s i t i v i t y t o s u n l i g h t and a p r e d i s p o -
s i t i o n t o s k i n cancer. C e l l s taken from persons s u f f e r i n g from
t h i s c o n d i t i o n are more s e n s i t i v e t o UV-irradiation than normal
c e l l s and are d e f i c i e n t i n e x c i s i o n r e p a i r o f UV-induced damage.
These same c e l l s are a l s o s e n s i t i v e t o other DNA damaging agents
such as hydrocarbon epoxides, 4-nitroquinoline-l-oxide and
7-bromomethylbenz(a)-anthracene and s e n s i t i v i t y i s again a s s o c i a -
ted with decreased l e v e l s o f v a r i o u s m a n i f e s t a t i o n s o f DNA
excision repair. (For review see (68). I t has now been found
(71) t h a t these r e p a i r d e f i c i e n t XP c e l l s are a l s o more s e n s i t i v e
than normal foetus lung c e l l s t o cis-DDP, when the l e t h a l e f f e c t s
o f the drug are expressed as a f u n c t i o n o f r e a c t i o n with DNA
r a t h e r than as a f u n c t i o n o f dose o f reagent. I t could t h e r e f o r e
be reasoned t h a t t h i s increased s e n s i t i v i t y o f XP c e l l s i s s i m i l -
a r l y due t o t h e i r decreased a b i l i t y t o excise cis-DDP induced
DNA damage.
C e l l s from p a t i e n t s with the genetic disease Fanconi's
anaemia, show unusual s e n s i t i v i t y t o the c y t o t o x i c and c l a s t o g e n i c
e f f e c t s o f d i f u n c t i o n a l a l k y l a t i n g agents. These c e l l s are
a l s o unusually s e n s i t i v e t o c i s p l a t i n (72). Such s e n s i t i v i t y i s
not the r e s u l t o f decreased binding o f platinum t o DNA but i t
remains t o be determined i f r e p a i r o f v a r i o u s l e s i o n s i n these
c e l l s i s abnormal.
I f DNA synthesis on a damaged template i s responsible f o r
t o x i c i t y , c e l l s t h a t are allowed t o r e p a i r DNA p r i o r t o S-phase
and c e l l d i v i s i o n should show l e s s t o x i c i t y than c e l l s e n t e r i n g
the p r o l i f e r a t i v e c y c l e immediately f o l l o w i n g treatment. In an
i n i t i a l study, F r a v e l and Roberts (70) t r e a t e d s t a t i o n a r y Chinese
hamster V79 c e l l s with c i s p l a t i n and measured t o x i c i t y and p l a t -

inum-DNA i n t e r a c t i o n a f t e r v a r i o u s h o l d i n g p e r i o d s i n the non-

dividing state. The c e l l s slowly excised platinum, and p l a t i n g
e f f i c i e n c y increased. There was a s i m i l a r r e l a t i o n s h i p between
the amount o f platinum bound t o DNA and c e l l s u r v i v a l whether one
compared the two immediately f o l l o w i n g treatment with s e v e r a l
drug doses, o r a f t e r v a r y i n g p e r i o d s o f recovery.
However, the c e l l s used i n these i n i t i a l s t u d i e s , Chinese
hamster V79 c e l l s , do not t o l e r a t e the n o n d i v i d i n g s t a t e w e l l ,
and i t was l a t e r found t h a t they do not recover from c i s p l a t i n
t o x i c i t y under some c o n d i t i o n s . Therefore, the experiment was
repeated using human f i b r o b l a s t s (73) under c o n d i t i o n s where
minimal DNA synthesis occurred. These c e l l s were healthy i n the
nondividing condition. The f i b r o b l a s t s recovered from c i s p l a t i n
t o x i c i t y i f h e l d i n the n o n d i v i d i n g s t a t e , and they excised
platinum l e s i o n s from DNA by a f i r s t order process with a h a l f -

l i f e o f 2.5 days (Figure 4 ) . DNA-DNA i n t e r s t r a n d c r o s s l i n k s ,
measured by a l k a l i n e e l u t i o n and by e s t i m a t i o n o f renaturable DNA
i n c e l l l y s a t e s , were found t o be r e p a i r e d with a h a l f - l i f e o f
about 36 hours (Figure 5). DNA p r o t e i n c r o s s l i n k s were removed
at a s i m i l a r r a t e and there was no evidence f o r any accompanying
degradation. Thus the r e v e r s a l o f c r o s s l i n k i n g was a t t r i b u t e d
to r e p a i r rather than i n t r o d u c t i o n o f DNA strand breaks. The
r e l a t i o n s h i p between c e l l s u r v i v a l and the amounts o f platinum
remaining bound t o DNA a t the time c e l l s were p l a t e d out f o r
e s t i m a t i o n o f c e l l s u r v i v a l , was s i m i l a r t o that observed i n c e l l s
t r e a t e d with s e v e r a l drug doses and p l a t e d immediately.
The r e s u l t s s t r o n g l y supported the hypothesis that damage present
on the DNA template a t the time o f entry i n t o the p r o l i f e r a t i v e
c y c l e s was r e s p o n s i b l e f o r c e l l u l a r t o x i c i t y . F u r t h e r , the
r e s u l t s showed t h a t the r e p a i r process was a c t u a l l y e f f e c t i v e i n
achieving b i o l o g i c a l recovery.
When, however, attempts have been made t o r e l a t e inherent
drug s e n s i t i v i t y t o r e p a i r c a p a c i t y , c l e a r c u t r e s u l t s have not
always emerged. Walker 256 carcinoma c e l l s d i s p l a y unusual
s e n s i t i v i t y t o d i f u n c t i o n a l a l k y l a t i n g agents and t o c i s p l a t i n .
There i s a s u b l i n e o f t h i s tumour t h a t shows a 30-fold increase
in resistance to c i s p l a t i n . Nevertheless, the two s u b l i n e s bind
the same amount o f platinum on DNA f o l l o w i n g treatment with a
given dosage, they excise platinum l e s i o n s from t h e i r DNA a t a
s i m i l a r r a t e , and they remove DNA-protein c r o s s l i n k s and DNA
i n t e r s t r a n d c r o s s l i n k s from DNA a t a s i m i l a r r a t e (74). Follow-
i n g sulphur mustard treatment, nascent DNA synthesis on a damaged
template appears t o be s i m i l a r i n both l i n e s (75). I t i s of
course p o s s i b l e t h a t these l i n e s might vary i n t h e i r c a p a c i t y t o
remove a s p e c i f i c l e s i o n t h a t was not measured - an i n t r a s t r a n d
c r o s s l i n k , f o r example - b u t i f t h i s i s not the case, then some
t a r g e t other than simply DNA e x i s t s i n c e r t a i n c e l l s that confers
unusual s e n s i t i v i t y t o b i f u n c t i o n a l agents. T h i s could conceiv-
ably be a t the l e v e l o f chromatin o r a DNA-nuclear matrix complex.

loo-.
•
Ο
Ο S
6S
i2 H
o.i-
2 4 6
Time post treatment (days)
Figure 4a. Recovery of nondividing human fibroblasts from cis-DDP toxicity.

Stationary-phase humanfibroblastswere treated with cis-DDP (A O , 32 μΜ; and
A • • · , 40 μΜ) and were either plated immediately or held in the nondividing
state for various time periods and recovered from drug toxicity.

Figure 4b. Excision of Pt from DNA of nondividing humanfibroblastsfollowing

cis-DDP treatment. The plot illustratesfirst-orderloss of the Pt reaction products.

METAL CHEMOTHERAPEUTIC AGENTS
Figure 5. Repair of cis-DDP-induced DNA-protein crosslinks and DNA inter-

strand crosslinks in humanfibroblastsduring holding in the nondividing state.
Stationary cultures were treated with 40 μτηοΐ cis-DDP, and after various periods
of holding in the nondividing state, crosslinking was measured by alkaline elution
(W, DNA-protein crosslinks; and O, DNA interstrand crosslinks) or by estimation
of renaturable DNA in cell lysates (Φ, DNA interstrand crosslinks).

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37. Kleinwachter, V.; Zaludova, R. Chem.-Biol. Interact. 1977,
16, 207.
38. Roos, I.A.G. Chem.-Biol. Interact. 1977, 16, 39.
39. Shooter, K.V.; Howse, R.; Merrifield, R.K.; Robbins, A.B.
Chem.-Biol. Interact. 1972, 5, 289.
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1980, 32, 321.
41. Millard, M.M.; Macquet, J-P,; Theophanides, T. Biochem.
Biophys. Acta 1975, 402, 166.
42. Macquet, J-P.; Butour, J-L. Eur. J. Biochem. 1978, 83, 375.
43. Tamburio, A.M.; Celotti, L.; Furcan, D.; Guantieri, V.
Chem.-Biol. Interact. 1977, 16, 1.
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and Oncology 1977, 7, 440.
45. Macquet, J-P.; Butour, J-L. Biochimie 1978, 60, 901.
46. Cohen, G.L.; Bauer, W.R.; Barton, J.K.; Lippard, S.J. Science
1979, 203, 1014.
47. Cohen, G.L.; Ledner, J . Α . ; Bauer, W.R.; Ushay, H.M.;
Caravana, C.; Lippard, S.J. J. Amer. Chem. Soc. 1980, 102,
2487.
48. Roberts, J.J.; Fraval, H.N.A. Biochimie 1978, 60, 869.
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Chemotherapeutic Agents", Academic Press, New York, p.17.
50. Pera, M.F.; Sessford, D.; Roberts, J.J. Biochem. Pharmacol.
1982 (in press).
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39, 365.
52. Roberts, J.J.; Friedlos, F. Biochim. Biophys. Acta 1981a,
655, 146.
53. Pera, M.F. Jr.; Rawlings, C.J.; Shackleton, J.; Roberts, J.J.
Biochim. Biophys. Acta 1981a, 655, 152.

54. Zwelling, L.A.; Filipski, J.; Kohn, K.W. Cancer Res. 1979c,
39, 4989.
55. Zwelling, L.A.; Michaels, S.; Schwartz, H.; Dobson, P.O.;
Kohn, K.W. Cancer Res. 1981, 41, 640.
56. Laurent, G.; Erickson, L.C.; Sharkey, N.A.; Kohn, K.W.
Cancer Res. 1981, 41, 3347.
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Kohn, K.W. Cancer Res. 1981, 41, 2791.
58. Strandberg, M.C.; Proc. Amer. Assoc. Cancer Res. 1981, 22,
202.
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61. van den Berg, H.W.; Roberts, J.J. Chem.-Biol. Interact. 1976,
12, 375.
62. Fraval, H.N.A.; Roberts, J.J. Chem.-Biol. Interact. 1978a,
23, 99.
63. Fraval, H.N.Α.; Roberts, J.J. Chem-Biol. Interact. 1978b,
23, 111.
64. Howie, J . Α . ; Gale, G.R. Biochem. Pharmacol. 1970, 19, 2757.
65. Taylor, D.M.; Tew, K.D.; Jones, J.D. Eur. J. Cancer 1976,
12, 249.
66. Johnson, N.P.; Hoeschele, J.D.; Kuemmerle, N.B.; Masker,
W.E.; Rahn, R.O. Chem.-Biol. Interact. 1978, 23, 267.
67. Harder, H.C.; Smith, R.G.; Leroy, A.F. Cancer Res. 1976, 36,
3821.
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69. van den Berg, H.W.; Roberts, J.J. Chem.-Biol. Interact.
1975b, 11, 493.
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71. Fraval, H.N.Α.; Rawlings, C.J.; Roberts, J.J. Mutation Res.
1978, 51, 121.
72. Pera, M.F., Jr.; Roberts, J.J. unpublished results.
73. Pera, M.F.; Rawlings, C.J.; Roberts, J.J. Chem.-Biol.
Interact. 1981b, 37, 245.
74. Rawlings, C.J.; Roberts, J.J. unpublished.
75. Roberts, J.J.; Friedlos, F. unpublished.
RECEIVED October 20, 1982

2
B i o l o g i c a l Consequences of Platinum-DNA
Crosslinks in M a m m a l i a n Cells
LEONARD A. ZWELLING
National Cancer Institute, Laboratory of Molecular Pharmacology, Developmental
Therapeutics Program, Division of Cancer Treatment, Bethesda, MD 20205
Bifunctional DNA adducts or crosslinks can be

formed by antineoplastic Pt-coordination complexes.
2 distinct DNA crosslinks can be produced in
mammalian cells by cis-Diamminedichloroplatinum(II)
(DDP) and its inactive isomer trans-DDP, DNA-protein
crosslinks (DPC) and DNA interstrand crosslinks
(ISC). Each can be quantified by using DNA alkaline
elution, a technique based upon the size-dependent
elution of DNA single strands from filters at pH 12.
Proteolytic enzymes can be used to distinguish DPC
from ISC. DPC form rapidly and are the major lesion
produced by trans-DDP. ISC form over 6-12 hr
following treatment and are more efficiently formed
by cis-DDP. ISC correlated more closely with cyto-
toxicity than did DPC. This was confirmed using
chemical blockade of ISC formation to enhance cell
survival following cis-DDP treatment. Additionally,
malignant cells resistant to cis-DDP in vitro and
in vivo showed lower levels of ISC than their
sensitive parent lines. Further studies in resis-
tant and sensitive cells have led to a model in
which the biological consequences of Pt-DNA damage
result from the temporal and quantitative relation-
ship between Pt-DNA damage and cellular repair at a
given functional DNA site. The kinetics of this
equilibrium betweenDNAdamage and repair of various
Pt-DNA adducts may determine the effect of cis-DDP
treatment on cytotoxicity, mutagenicity and sister
chromatid exchange frequency.
The consequences of damage to the PNA of living cells must

result from three related factors; (1) the number of damaged
sites, (2) the ability of the cell to repair the damage, and (3)
the temporal relationship between damage production, damage
repair and the normal cell functions with which unrepaired
This chapter not subject to U.S. copyright.

Published 1983, American Chemical Society

damage could i n t e r f e r e . This scheme (Figure 1) suggests an

e q u i l i b r i u m between DNA damage and r e p a i r superimposed upon
ongoing DNA f u n c t i o n s such as t r a n s c r i p t i o n and t r a n s l a t i o n .
The s t a t e of the e q u i l i b r i u m at the time and at the s i t e at
which a normal DNA f u n c t i o n i s to occur could determine the
consequences of the DNA damage. Therefore, d i f f e r e n c e s between
the s u s c e p t i b i l i t y of c e l l types to DNA damaging agents may
r e s u l t from d i f f e r e n c e s i n the i n i t i a l frequency of DNA damage,
d i f f e r e n c e s i n the c e l l s ' c a p a c i t y to r e p a i r damage or
d i f f e r e n c e s i n the a b i l i t y of the c e l l to t o l e r a t e damage e i t h e r
through by-passing damage or abating normal f u n c t i o n u n t i l damage
i s repaired.
The DNA damage produced by P t ( I I ) - c o o r d i n a t i o n complexes can
be i n c l u d e d i n such a schema. This d i s c u s s i o n w i l l be l i m i t e d
to damage produced by c i s - and trans-Diamminedichloroplatinum

( I I ) . These isomers (Figure 2) are f e l t to i n t e r a c t w i t h nucleo-
p h i l i c s i t e s i n i n t r a c e l l u l a r macromolecules through the
displacement by these moieties of the c h l o r i d e l i g a n d s ( l e a v i n g
groups) of the P t ( I I ) complexes. From stereochemical
c o n s i d e r a t i o n s the number and/or r a t e of macromolecular i n t e r
a c t i o n s would be d i f f e r e n t f o r Pt-complexes w i t h l e a v i n g groups
i n the c i s v s . trans c o n f i g u r a t i o n ( 1 ) . Each agent may e l i c i t
d i f f e r e n t c e l l u l a r responses w i t h correspondingly d i f f e r e n t
consequences. We w i l l present the r e s u l t s of work performed i n
the Laboratory of Molecular Pharmacology over the past 5 years
using various c e l l systems. These data e l u c i d a t e s e v e r a l Pt-DNA
i n t e r a c t i o n s , the c e l l u l a r DNA responses they e l i c i t and the
consequences of the e q u i l i b r i u m between DNA damage and r e p a i r i n
a c t i v e l y d i v i d i n g mammalian c e l l s .
M a t e r i a l s and Methods
I n V i t r o C e l l C u l t u r e . Mouse leukemia L1210 c e l l s ( d e s i g

nated K25) were grown i n RPMI 1630 medium supplemented w i t h 20%
h e a t - i n a c t i v a t e d f e t a l c a l f serum. V79 Chinese hamster lung
c e l l s were grown i n σ-ΜΕΜ medium w i t h 5% f e t a l c a l f serum i n
7.5% C02* The human c e l l l i n e s were grown i n Eagle's minimal
e s s e n t i a l medium w i t h 10% f e t a l c a l f serum.
Drug Treatments. Cis-Diamminedichloroplatinum(II) (NSC

119875) and trans-Diamminedichloroplatinum(II) (NSC 131558)
were obtained from the N a t i o n a l Cancer I n s t i t u t e and were always
c o n s t i t u t e d i n i s o t o n i c , aqueous s o l u t i o n j u s t p r i o r to use.
L-phenylalanine mustard (NSC 8806) was d i s s o l v e d i n 0.1 Ν HC1
and s t o r e d f r o z e n .
C e l l S u r v i v a l Measurements. L1210 c e l l s u r v i v a l was q u a n t i -

f i e d by colony forming a b i l i t y i n s o f t agar ( 2 ) . V79 colony-
forming a b i l i t y was measured using attached c e l l s on p l a t e s of
i n i t i a l p l a t i n g d e n s i t i e s of 300-3000/plate (3)· The cyto-

2. ZWELLING Biological Consequences of Pt-DNA Crosslinks 29
REPAIR
DAMAGE
FUNCTIONING CORRECT
ERROR DNA SITE FUNCTION
TIME
Figure 1. A schematic representation of the interaction between DNA damage

and repair of a random, functioning DNA site.
Figure 2. The structures of A, cis-DDP and B, trans-DDP.

t o x i c i t y of cis-DDP i n human c e l l s i s q u a n t i f i e d by drug

i n h i b i t i o n of c e l l growth d u r i n g 3 p o p u l a t i o n doublings i n
drug-free medium (4, 5)·
M u t a g e n i c i t y . V79 c e l l s were back-selected t o e l i m i n a t e a l l

p r e - e x i s t i n g HGPRT-mutants i n medium c o n t a i n i n g hypoxanthine,
5 6 6
10" M, methotrexate 3.2 χ 10" M, and thymidine, 5 χ 10" M (3)·
F o l l o w i n g drug treatments c e l l s were incubated i n f r e s h medium
f o r 2 days and then subcultured at 2-5 χ 10^ c e l l s / f l a s k f o r an
a d d i t i o n a l 3-5 days of growth to a l l o w mutant e x p r e s s i o n .
F o l l o w i n g t h i s , c e l l s were seeded on p l a t e s and incubated i n
6-thioguanine-containing medium. The f r a c t i o n of thioguanine-
res-istant c e l l s (mutation frequency) was determined from the
p l a t i n g e f f i c i e n c i e s i n the presence or absence of thioguanine.
Drug-Resistant L1210 C e l l s . The K25 L1210 c e l l s were used as

a parent l i n e t o develop a l i n e of c i s - D D P - r e s i s t a n t L1210 c e l l s .
The parent c e l l s were t r e a t e d w i t h 10"^ methylnitrosourea f o r 1
h r , allowed t o recover to e x p o n e n t i a l growth, t r e a t e d w i t h c i s -
DDP f o r 1 h r and seeded i n s o f t agar. A clone was s e l e c t e d from
the s u r v i v i n g c e l l s , r e t r e a t e d w i t h cis-DDP, reseeded i n s o f t
agar and again a clone was s e l e c t e d . An a d d i t i o n a l methyl
n i t r o s o u r e a treatment and 5 c y c l e s of cis-DDP treatment and
colony formation y i e l d e d the cis-DDP r e s i s t a n t l i n e (ZCR9).
These c e l l s were cryopreserved and f r e s h a l i q u o t s unfrozen
approximately every 6 weeks. By t h i s procedure r e s i s t a n c e was
maintained and r e p r o d u c i b i l i t y of experiments over many months
was assured ( 6 ) .
Murine L1210 tumor l i n e s i n v i v o were obtained from 2
sources. Dr. J . Burchenal, Memorial S l o a n - K e t t e r i n g I n s t i t u t e ,
provided the cis-DDP r e s i s t a n t l i n e (L1210/PDD) and i t s
s e n s i t i v e parent (L1210 (MSKI)). Dr. F. Schabel, Southern
Research I n s t i t u t e , d e r i v e d the L-PAM-resistant l i n e (L1210/
PAM). This tumor and i t s s e n s i t i v e parent (L1210 (NCI)) were
obtained from Dr. D. V i s t i c a , N a t i o n a l Cancer I n s t i t u t e . These
tumors were obtained i n e i t h e r C57BL X DBA/2Fi ( c a l l e d BD2Fi)
or DBA mice, but were maintained i n male BD2F^ mice o n l y .
Tumor l i n e s were passaged weekly by e x p l a n t i n g a s c i t e s f l u i d
6
from tumored animals and i n o c u l a t i n g 1 0 c e l l s i n t r a p e r i t o n e a l l y
( i . p . ) i n t o mice. To maintain drug r e s i s t a n c e , L1210/PDD and
L1210/PAM r e q u i r e d drug treatments f o l l o w i n g i n o c u l a t i o n ( 6 ) .
Mice bearing L1210 c e l l tumors to be used i n an experiment
were not t r e a t e d w i t h drugs t o maintain tumor r e s i s t a n c e during
the 7 days p r i o r to e x p l a n a t i o n . 6-7 days f o l l o w i n g t h i s tumor
i n o c u l a t i o n , a s c i t e s tumor c e l l s were explanted and e i t h e r
i n o c u l a t e d i n t o r e c i p i e n t animals upon whom s u r v i v a l s t u d i e s
would be performed or explanted i n t o RPMI 1630 medium + 20%
f e t a l c a l f serum, 50 μΜ 2-mercaptoethanol and e i t h e r [2-l^C]
thymidine (0.02 y C i ml-1) or [methyl-3H]thymidine (0.2 y C i ml-1)
and incubated f o r 20 h r at 37°. The f o l l o w i n g day explanted

c e l l s were washed f r e e of r a d i o a c t i v e l a b e l and 2-mercapto-

e t h a n o l . Equal numbers of o p p o s i t e l y l a b e l e d s e n s i t i v e and
r e s i s t a n t c e l l s were mixed, t r e a t e d w i t h 20 uM cis-DDP, 20
μΜ L-PAM o r no drug f o r 1 h r and then drug was removed.
C e l l s were resuspended i n drug-free medium and DNA damage assays
(see below) were performed a t v a r i o u s times t h e r e a f t e r .
The animals i n t o which these c e l l s were i n o c u l a t e d were
e i t h e r drug-treated o r untreated c o n t r o l s . They were checked
d a i l y f o r s u r v i v a l . I t i s c r i t i c a l t o note that c e l l s used f o r
DNA assays were a l i q u o t s of i d e n t i c a l c e l l s used f o r i n v i v o
s u r v i v a l measurements. F u r t h e r , by mixing o p p o s i t e l y l a b e l e d
c e l l s p r i o r t o drug treatment, uniform drug exposure was assured
(6).
The Assessment o f DNA C r o s s l i n k i n g by A l k a l i n e E l u t i o n . DNA

damage, that i s , i n t e r s t r a n d c r o s s l i n k s , DNA-protein c r o s s l i n k s ,
and s t r a n d breaks, was determined u s i n g the a l k a l i n e e l u t i o n
4
technique (_7, 8 ) . C e l l s l a b e l e d w i t h * C-thymidine f o r 20-24
hr were deposited on a membrane f i l t e r and l y s e d w i t h a
d e t e r g e n t - c o n t a i n i n g s o l u t i o n . An a l k a l i n e s o l u t i o n (pH 12.1-
12.2) was then slowly pumped through the f i l t e r , and f r a c t i o n s
were c o l l e c t e d t o determine the r a t e of r e l e a s e of DNA from the
f i l t e r . For assay of c r o s s l i n k s , the c e l l s were exposed t o
x-ray a t 0°C p r i o r t o d e p o s i t i o n on the f i l t e r . I n order t o
improve q u a n t i t a t i o n , c o n t r o l c e l l s l a b e l e d w i t h ^H-thymidine
and x - i r r a d i a t e d a t 0° were mixed w i t h the experimental Re
l a b e l e d c e l l s p r i o r t o d e p o s i t i o n on the f i l t e r s . The e l u t i o n
of ^H-DNA serves as an i n t e r n a l reference f o r n o r m a l i z a t i o n o f
14
the e l u t i o n of C-DNA.
DNA s t r a n d breaks are measured by the i n c r e a s e d e l u t i o n r a t e
of shortened s i n g l e strands. C r o s s l i n k s have the opposite
e f f e c t , and are measured by i n s e r t i n g a known frequency of s t r a n d
breaks by means of x-ray. I n t e r s t r a n d c r o s s l i n k s reduce e l u t i o n
r a t e by l i n k i n g together two o r more s i n g l e s t r a n d s . DNA-protein
c r o s s l i n k s reduce e l u t i o n because p r o t e i n s tend t o adsorb t o the
f i l t e r s under the a l k a l i n e conditions used. The e f f e c t s of
DNA-protein c r o s s l i n k s can be v i r t u a l l y e l i m i n a t e d by i n c l u d i n g
w i t h the detergent l y s i s , p r o t e o l y t i c enzymes ( 8 ) .
C r o s s l i n k i n g i s o f t e n expressed i n rad-equivalents· This
simply i n d i c a t e s that the degree of a l t e r a t i o n i n DNA e l u t i o n
r a t e [increase (strand breaks) or decrease ( c r o s s l i n k s ) ] produced
by the drug dose i n question i s equal i n q u a n t i t y t o that which
would have been produced by that dose of x - r a d i a t i o n . A d e t a i l e d
d i s c u s s i o n of the q u a n t i t a t i o n of e l u t i o n r e s u l t s can be found
i n references 7 and 8. (Also see F i g u r e 3 and legend).

ail—ι ι ι I ι I
0.9 0.8 0.7 0.6 0.5
3
FRACTION OF INTERNAL STANDARD H-DNA
RETAINED ON THE FILTER
Figure 3. The alkaline elution kinetics of DNA from L1210 cells treated with
cis-DDP (10 Μ) for 1 h followed by 12 h incubation in a drug-free medium.
μ
14
L1210 cells were labeled with C-thymidine for 20 h and were either cis-DDP
treated (A> A) or untreated (Φ, O). (Reproduced with permission from Ref. 20.
Copyright 1980, Academic Press.)
The two upper curves without irradiation indicate that cis-DDP produced no detectable DNA
breakage. The two lower curves quantify DNA crosslinking as the DNA from cells treated
with cis-DDP exhibiting slower elution kinetics than the DNA from control cells when both
had received prior irradiation (600 R). The abscissa is generated by the coelution of oppositely
labeled DNA from irradiated cells (internal standard cells). The point at which 60% of this
3 14
H-DNA remains on thefilteris the point at which the retention (R) of C-DNA is quantified
and compared with that of controls (R ).0

Results
The Nature of the DNA C r o s s l i n k i n g Produced by C i s - and Trans-
Diamminedichloroplatinum(II) and i t s R e l a t i o n t o C y t o t o x i c i t y .
I n i t i a l Observations w i t h L1210 C e l l s . Both compounds produced
p r o t e i n a s e - s e n s i t i v e and p r o t e i n a s e - r e s i s t a n t c r o s s l i n k s i n
L1210 mouse leukemia c e l l s as detected by a l k a l i n e e l u t i o n
(Figure 4 ) . Drug-induced c r o s s l i n k i n g which i s s e n s i t i v e t o
p r o t e o l y t i c d i g e s t i o n i s taken as r e p r e s e n t a t i v e of DNA-protein
c r o s s l i n k i n g (DPC). C r o s s l i n k i n g r e s i s t a n t t o p r o t e o l y t i c
d i g e s t i o n i s taken as i n t e r s t r a n d c r o s s l i n k i n g ( I S C ) . As can be
seen i n Figure 4, although both compounds produced DPC and ISC,
the c o n t r i b u t i o n of DPC t o the t o t a l c r o s s l i n k i n g produced by
trans-DPP was g r e a t e r than t o the t o t a l c r o s s l i n k i n g produced
by cis-DDP. F i g u r e 4 a l s o shows t h a t the time course of
formation and disppearance of DPC v s . ISC i s d i f f e r e n t w i t h

trans-DPP, but s i m i l a r w i t h cis-DDP. That i s both compounds
produced delayed ISC, but trans-DPP produced r a p i d PPC w h i l e the
m a j o r i t y of cis-PPP-PPC was delayed i n formation. I f the maximum
amount of the 2 types of c r o s s l i n k i n g i s compared w i t h the subse-
quent a b i l i t y of the c e l l s t o form c o l o n i e s ( F i g u r e 5 ) , PPC, the
major c o n t r i b u t o r t o t o t a l c r o s s l i n k i n g , was g r e a t e r f o r t r a n s -
than f o r cis-PPP at comparable t o x i c i t y . Comparing ISC and c e l l
s u r v i v a l b r i n g s the curves f o r the 2 drugs i n t o l i n e . This
r e s u l t suggests a mechanistic r e l a t i o n s h i p between ISC and c y t o -
t o x i c i t y f o r these 2 agents ( 9 ) .
This r e l a t i o n s h i p was f u r t h e r s u b s t a n t i a t e d by work i n which
i n t e r s t r a n d c r o s s l i n k formation could be prevented by the a v i d
Pt binder t h i o u r e a . Thiourea was capable of b l o c k i n g cis-PPP
c y t o t o x i c i t y and i n t e r s t r a n d c r o s s l i n k formation i n a s i m i l a r
dose- (Figure 6) and time- ( F i g u r e 7) dependent f a s h i o n ( 1 0 ) .
We have proposed a r e a c t i o n scheme f o r cis-PPP w i t h PNA bases
as a consequence of t h i s work (Figure 8) ( 1 1 ) . Cis-PPP could
undergo s e q u e n t i a l a c t i v a t i o n t o a p o s i t i v e l y charged species
capable of r e a c t i o n w i t h n u c l e o p h i l i c s i t e s on PNA (N). Thiourea
could compete w i t h PNA bases f o r P t - b i n d i n g and thus i n a c t i v a t e
cis-PPP d i r e c t l y (Reaction 5) o r f o l l o w i n g cis-PPP a c t i v a t i o n
1
(Reaction 5 ) . A d d i t i o n a l l y t h i o u r e a could prevent monoadduct
conversion t o ISC d i r e c t l y (Reaction 6) o r f o l l o w i n g a second
a c t i v a t i o n (Reaction 6'). Reversal of ISC (Reaction 7 ) , although
demonstrable i n i s o l a t e d chemical systems (12,13), was not
observed i n l i v i n g c e l l s .
The ISC produced by cift-PPP o r trans-PPP appears t o i n d i c a t e
the u l t i m a t e s u r v i v a l of t r e a t e d c e l l s .
I n V i t r o and I n Vivo s t u d i e s of s e n s i t i v e and r e s i s t a n t

murine c e l l s . A l i n e of L1210 c e l l s r e s i s t a n t t o cis-PPP was
developed from t h e parent K25 l i n e used i n the previous s t u d i e s .
M e t h y l n i t r o s o u r e a and cis-PPP treatments f o l l o w e d by s o f t - a g a r
colony formation were used t o o b t a i n t h i s l i n e designated ZCR9.
E q u i t o x i c concentrations of cis-PPP were 2.4 times h i g h e r i n
ZCR9 than i n K25. This d i f f e r e n c e was r e f l e c t e d i n the amount of
ISC but not PPC produced i n each c e l l type ( F i g u r e 9 ) . No d i f -

TIME AFTER DRUG TREATMENT (HOURS)
Figure 4. The kinetics of formation and disappearance of DNA-protein cross-

linking (DPC) and DNA interstrand crosslinking (ISC) in L1210 cells treated with
cis- or trans-DDP. Key to conditions: A, cis-DDP without proteinase (DPC and
ISC); B, trans-DDP without proteinase (DPC and ISC); C, cis-DDP with proteinase
(ISC only); and D, trans-DDP with proteinase (ISC only). (Reproduced with
permission from Ref. 9.)
All drug treatments were for 1 h at indicated doses followed by incubation in drug-free medium
for 0 to 24 h prior to crosslink quantification by alkaline elution.

SURVIVING FRACTION
Figure 5. Relation between DNA interstrand crosslinking and DNA-protein

crosslinking and survival of L1210 cells as measured by soft-agar colony formation
Key to conditions: top, without proteinase-K (DPC + ISC); and bottom, with
proteinase-K (ISC only). (Reproduced with permission from Ref. 9.)

THIOUREA CONCENTRATION (mM)
Figure 6. The dose dependence of thiourea's inhibition of cis-DDP cytotoxicity

(A) and inter strand crosslinking (B) in LI 210 cells. (Reproduced with permission
from Ref. 10.)
Cells were treated with 20 μΜ cis-DDP for 1 h, washed, and then treated for 1 h with various
concentrations of thiourea in medium after which colony formation in soft agar was quantified
immediately (A) or delayed 6-12 h before proteinase-resistant crosslinking (ISC) was quantified
(B).

T I M E A F T E R T R E A T M E N T (HR)
Figure 7. The effect of varying the time interval between cis-DDP treatment and
thiourea treatment on cytotoxicity (top) and ISC formation (bottom). (Reproduced
with permission from Ref. 10.)
L1210 cells were treated for 1 h with cis-DDP (20 μΜ), washed, and then treated immediately,
6 h, or 12 h later with thiourea (100 μΜ) for 1 h. Cytotoxicity and ISC were then quantified.
Key: · , no thiourea; O, 100 mM thiourea for 1 h just prior to inoculation into soft agar or
ISC measurement; and A, thiourea immediately following cis-DDP with ISC quantification
0, 6, or 12 h later.
HN CI H N OH H 3 H3N
3 Λ 3 N @ / 2
\e/® 3 Η 3
\ θ / ® 4
θ/
Pt
\
Pt
CI
—*
®/
Pt
\
-=H
®/
Pt
\Φ
HN
®/Pt ν([
HN 3 CI H3N
H3N CI
H3N 0H 2 3
6'
NH
H N^ S-C-NH
3 @ / 2
Pt Pt
®/ \ φ/ \
H3N CI
H3N S-C-NH 2
NH
Figure 8. Scheme for reactions of cis-DDP with nucleophilic site (N) or thiourea
at various points in the formation of a cis-DDP-DNA crosslink. (Reproduced with
permission from Ref. 11. Copyright 1981, Academic Press.)

CD
Ζ
(Λ
(/}
Ο
ο
<
I ' -l r~ ι ι I 1
7 I
/ ·
200
/
—
"κ25
-
160
•
•Jf
Ψ
• /
— —
120
80 m/
m /
Α
^CR9
40 - y -
-
•
0 I ι I ι ι • ι
10 20 30 40 50
Cis-DDP CONCENTRATION (μΜ)
Figure 9. Concentration dependence of cis-DDP DNA-protein crosslinking (top,

3000 R) and DNA interstrand crosslinking (bottom, 300 R) in sensitive (K25) and
resistant (ZCR9) LI210 cells. (Reproduced with permission from Ref. 6.)
All treatment times were 1 h. DPC was quantified immediately after treatment; ISC, 6 h later.
The ratios of the slopes of these lines (K25/ZCR9) are 3.0 for DPC and 2.5 for ISC. The ratio
for relative colony formation (survival) was 2.4 (ZCR9/K25). These ratios represent the ratios
of cis-DDP doses producing equal effects in each assay.

ferences were noted i n the k i n e t i c s of cis-DDP c r o s s l i n k

formation and r e p a i r i n these 2 l i n e s ( 6 ) . These s t u d i e s were
then extended t o tumors i n mice. S e n s i t i v e - r e s i s t a n t p a i r s of
LI210 c e l l s were obtained (see M a t e r i a l s and Methods) i n mice.
Animal s u r v i v a l i n tumor-bearing mice was compared w i t h DNA
c r o s s l i n k i n g i n expiants from a l i q u o t s of the i d e n t i c a l tumor
used t o i n o c u l a t e these mice. Lines were s e l e c t e d f o r r e s i s t a n c e
to the b i f u n c t i o n a l a l k y l a t i n g agent L-phenylalanine mustard
(L-PAM) as w e l l as t o cis-DDP. The r e s u l t s are summarized i n
Table I .
In the cis-DDP s e n s i t i v e - r e s i s t a n t p a i r (L1210 (MSKI) and
L1210/PDD), cis-DDP r e s i s t a n c e was confirmed i n L1210/PDD
and cis-DDP-induced ISC was almost absent. L1210/PDD was not
c r o s s - r e s i s t a n t t o L-PAM. Comparable ISC was produced by L-PAM
i n e x p i a n t s of both l i n e s . I n the L-PAM s e n s i t i v e - r e s i s t a n t

p a i r (L1210 (NCI) and L1210/PAM), L-PAM r e s i s t a n c e was confirmed
i n L1210/PAM and ISC was a p p r o p r i a t e l y depressed. However,
L1210/PAM was s t r i k i n g l y r e s i s t a n t t o cis-DDP, but cis-DDP-ISC
was only p a r t i a l l y depressed. Thus, although a l l our data t o
t h i s p o i n t i n d i c a t e d delayed, ISC would p r e d i c t f o r cis-DDP
c y t o t o x i c i t y , an e x c e p t i o n had been found ( 6 ) . This e x c e p t i o n
was then s t u d i e d i n f u r t h e r d e t a i l .
A Mechanism of Cis-DDP Resistance i n L1210/PAM. Malignant

a s c i t e s c e l l s were explanted from mice b e a r i n g L1210 (NCI) and
L1210/PAM i n t o s o f t - a g a r c l o n i n g tubes and c o l o n i e s allowed t o
form ( 2 ) . S i n g l e c o l o n i e s of each c e l l l i n e were s e l e c t e d ,
subcultured and t e s t e d f o r r e s i s t a n c e t o cis-DDP. The r e s i s t a n c e
of L1210/PAM t o cis-DDP i n v i v o was maintained i n v i t r o .
ISC assays revealed a peak of ISC 6-12 h r f o l l o w i n g a 1-hr
exposure t o cis-DDP i n both c e l l l i n e s . The magnitude of t h i s
c r o s s l i n k i n g i n L1210/PAM was 60% of that i n L1210 (NCI) f o r any
given drug dose. This d i f f e r e n c e was not s u f f i c i e n t t o e x p l a i n
the marked d i f f e r e n c e i n cis-DDP s e n s i t i v i t y between these c e l l
l i n e s (14).
F o l l o w i n g peak ISC, ISC d e c l i n e d i n both c e l l l i n e s , but the
r a t e was 1.7-fold f a s t e r i n L1210/PAM than L1210 (NCI). This
d i f f e r e n c e could a r i s e i f c r o s s l i n k s were more r a p i d l y removed
or more s l o w l y formed i n L1210/PAM. By p r e v e n t i n g f u r t h e r ISC
w i t h t h i o u r e a 5-6 h r f o l l o w i n g cis-DDP treatment ( 1 0 ) , the
removal of i n t e r s t r a n d c r o s s l i n k s could be examined w i t h no
c o n t r i b u t i o n from ongoing ISC f o r m a t i o n . The 2 l i n e s removed
formed c r o s s l i n k s at comparable r a t e s f o l l o w i n g blockade of
a d d i t i o n a l ISC formation by t h i o u r e a . The removal of ISC
appears normal i n L1210/PAM, but the conversion of monoadducts
to c r o s s l i n k s i s decreased. This could be due t o enhanced
monoadduct removal or i n a c t i v a t i o n . I n i t i a l Pt-DNA adducts are
l i k e l y t o be comparable i n these c e l l s as DPC was comparable
immediately f o l l o w i n g cis-DDP treatment ( 1 4 ) .

Table I . The S u r v i v a l of L1210-Bearing Mice v s . Q u a n t i f i e d

a
I n t e r s t r a n d C r o s s l i n k Formation i n I d e n t i c a l Explanted Tumors
Cis-DDP L-PAM
b C b C
Tumor L i n e %ILS ISC %ILS ISC
6 hr 12 h r 6 hr 12 h r
L1210 (MSKI) 151 0.20 0.21 94 0.23 0.20
L1210/PPD 11 0.01 0.01 76 0.18 0.17
L1210 (NCI) 66 0.30 0.26 96 0.17 0.18
L1210/PAM 6 0.19 0.16 25 0.07 0.08
a
S u r v i v a l compared w i t h u n t r e a t e d , tumor-bearing mice. Recipient
mice were i n o c u l a t e d w i t h 1-3 χ 10*> tumor c e l l s i . p . and t r e a t e d
w i t h e i t h e r cis-DDP, 4.5 mg k g ~ * i . p . on days 1 , 5 , 9 and 13
f o l l o w i n g tumor i n o c u l a t i o n or 13 mg k g ~ l L-PAM on day 1
f o l l o w i n g tumor i n o c u l a t i o n . C r o s s l i n k i n g measurements were
made on a l i q u o t s of the i d e n t i c a l c e l l s used f o r tumor
i n o c u l a t i o n which were explanted i n t o t i s s u e c u l t u r e r a t h e r
than i n o c u l a t e d i n t o r e c i p i e n t mice. (From Reference 6^ ) .
Increased l i f e span.
c
Interstrand WA)
c r o s s l i n k i n g : c r o s s l i n k i n g c o e f f i c i e n t • (- -1
where r and r a r e the f r a c t i o n of DNA r e t a i n e d on the f i l t e r

0
from drug-treated o r untreated c e l l s r e s p e c t i v e l y i n the

a l k a l i n e e l u t i o n assay w i t h proteinase. The time of e l u t i o n
at which r and r were measured was u s u a l l y 10 h r . 6 h r and
Q
12 h r i n d i c a t e the time f o l l o w i n g a 1 h r drug treatment at

which ISC was q u a n t i f i e d .
Reproduced with permission from Bet 6.

The r e s i s t a n c e of L1210/PAM to cis-DDP may r e s i d e i n a

combination of events which can be f i t i n t o the e q u i l i b r i u m
scheme i n Figure 1· I t appears that i n i t i a l Pt-DNA i n t e r a c t i o n s
are comparable i n these 2 c e l l l i n e s . ISC progresses to a higher
peak i n L1210 (NCI) than i n L1210/PAM because the e q u i l i b r i u m
between damage and r e p a i r (or i n a c t i v a t i o n ) i n Figure 1 i s
s h i f t e d f u r t h e r t o the r i g h t ( r e p a i r ) i n L1210/PAM. As c e l l
doubling times were comparable i n these c e l l s , the time a x i s o f
c r i t i c a l c e l l u l a r events i n v o l v i n g DNA i s probably s i m i l a r i n
L1210 (NCI) and L1210/PAM. Therefore, the increased r e s i s t a n c e
of L1210/PAM a r i s e s as a r e s u l t of a lower number of c r i t i c a l
l e s i o n s (ISC) which i n t u r n r e s u l t s from c e l l u l a r i n a c t i v a t i o n
or r e p a i r of the precursor (monoadduct) of that l e s i o n .
Pt-DNA Damage and Repair i n Human C e l l s . E r i c k s o n e£ a l .

have r e c e n t l y demonstrated a c o r r e l a t i o n between the c a p a b i l i t y
+
of s e v e r a l human c e l l types to r e p a i r m e t h y l a t i o n damage ( M e r
phenotype) and c e l l s u r v i v a l f o l l o w i n g treatment w i t h c h l o r o -
e t h y l n i t r o s o u r e a s (15). These c e l l l i n e s were a l s o examined
f o r t h e i r s e n s i t i v i t y or r e s i s t a n c e to cis-DDP. Although the
magnitude of cis-DDP ISC again c o r r e l a t e d w i t h c y t o t o x i c i t y , no
c o r r e l a t i o n was found between s u r v i v a l or c r o s s l i n k i n g and the
+
presence or absence of the M e r phenotype. This Mer repair
mechanism i s probably not i n v o l v e d i n the c e l l u l a r response to
Pt-DNA damage (4, 5 ) .
The R e l a t i o n s h i p between Pt-DNA C r o s s l i n k i n g and

Mutagenicity. V79 Chinese hamster c e l l s were used to compare
DNA damage w i t h 2 d i f f e r e n t consequences of that damage. Colony
forming a b i l i t y was again used to q u a n t i f y the l e t h a l i t y of c i s -
and trans-DPP. A d d i t i o n a l l y , mutagenicity was q u a n t i f i e d i n
these c e l l s at the hypôxanthine-guanine phosphoribosyl-
t r a n s f e r a s e locus (3) (Table I I ) .
Again, as had been seen i n L1210 c e l l s , ISC peaked 6-12 h r
f o l l o w i n g drug treatment, and DPC was a greater c o n t r i b u t o r t o
t o t a l c r o s s l i n k i n g f o l l o w i n g trans-DPP compared w i t h cis-DDP.
Once a g a i n , at e q u i t o x i c concentrations of each agent, ISC was
comparable whereas DPC was much greater i n trans_-DDP-treated
c e l l s . However, trans-DPP was v i r t u a l l y non-mutagenic (Figure
10); w h i l e cis-PPP was qui,te mutagenic. At doses of each agent
producing comparable c y t o t o x i c i t y and comparable ISC, trans-PPP
produced more PPC and was not mutagenic. Neither ISC nor PPC
are t h e r e f o r e n e c e s s a r i l y mutagenic ( 3 ) .
To f u r t h e r explore the r e l a t i o n s h i p between these PNA e f f e c t s
and t h e i r b i o l o g i c a l consequences i n V79 c e l l s , we expanded the
examined parameters to i n c l u d e measurement of Pt-induced s i s t e r
chromatid exchanges (SCE) (a v i s i b l y detectable i n d i c a t i o n of
r e c i p r o c a l exchanges of chromosomal PNA). Bradley et a l . showed
that both compounds can produce SCE, thus SCE producTion can be
d i s s o c i a t e d from mutagenicity (16).

Table I I . C y t o t o x i c i t y , M u t a g e n i c i t y and DNA C r o s s l i n k i n g i n V79
C e l l s Treated w i t h Cis-DDP or Trans-DDP
Concen- Treatment Post Crosslinking

tration time Incubation S u r v i v a l Mutation (rad equi-
0 5
(μΜ) (hr) time fraction frequency* valents)
(hr)
Cis-DDP -ProK +ProK

4
11 2 0 0.1 3 χ 10~ 124 6
6 270 46
12 311 94
187 41
18 101 33
Trans-DDP
5
320 2 0 0.19 <10~ 1303 40
6 2300 99
12 880 79
1200 116
18 522 106
Source: Reference 3.
a
S u r v i v a l of colony-forming a b i l i t y .
b
HGPRT l o c u s .

~ι Γ
4
3x10"
m 2x10"
Ξ
ce
4
I 1x10-
οί _L JL
ο 5 10 15 μΜ c/s-DDP
100 200 300 400 500 600 μΜ trans-DDP
DRUG CONCENTRATION
Figure 10. The mutation frequency produced by a 2-h treatment of V79 cells with
· , cis- or A trans-DDP. (Reproduced with permission from Ref. 3.)

F i n a l l y , t o understand the processes r e l a t i n g cis-DDP DNA

damage t o the v a r i o u s measured b i o l o g i c a l consequences, we
u t i l i z e d t h i o u r e a i n a f a s h i o n s i m i l a r t o t h a t used i n L1210
c e l l s ( v i d e supra - Figure 8 ) . As had been seen i n L1210 c e l l s ,
the treatment of V79 c e l l s w i t h t h i o u r e a immediately f o l l o w i n g
cis-DDP treatment, completely blocked the c y t o t o x i c e f f e c t even
at drug concentrations capable of producing 97% c e l l k i l l .
M u t a g e n i c i t y could a l s o be blocked wjien cis-DDP treatment was
immediately f o l l o w e d by t h i o u r e a however the t h i o u r e a i n h i b i t i o n
of mutagenicity diminished over the same cis-DDP dose range
wherein c y t o t o x i c i t y was t o t a l l y preventable by t h i o u r e a ( F i g u r e
11). A d d i t i o n a l l y i f the t h i o u r e a treatment was delayed f o r 4.5
h r a f t e r cis-DDP, some c y t o t o x i c i t y was s t i l l prevented, but
mutagenicity had escaped t h i o u r e a blockade. The mutagenic
process becomes f i x e d more r a p i d l y and at lower cis-DDP doses

than the c y t o t o x i c process ( 1 7 ) .
SCE p r o d u c t i o n by cis-DDP was a l s o a f f e c t e d by t h i o u r e a
(Figure 12). However over a dose range where immediate t h i o u r e a
completely blocked both c y t o t o x i c i t y and m u t a g e n i c i t y , SCE
formation could only p a r t i a l l y be prevented. This SCE formation
escaped t h i o u r e a blockade w i t h i n 4.5 h r . The order of suscept-
i b i l i t y of cis-DDP e f f e c t s to immediate t h i o u r e a blockade i s SCE
formation < mutagenicity < c y t o t o x i c i t y ( 1 7 ) .
A model connecting these v a r i o u s b i o l o g i c a l consequences of
cis-DDP i n V79 c e l l s w i t h p o t e n t i a l Pt-induced DNA l e s i o n s can
be constructed ( F i g u r e 13). S u b s t a n t i a t i n g the data accrued i n
L1210 c e l l s , V79 c y t o t o x i c i t y appeared t o be most c l o s e l y a s s o c i -
ated w i t h delayed i n t e r s t r a n d c r o s s l i n k f o r m a t i o n . E q u i t o x i c
doses of c i s - and trans-DPP produced comparable ISC and t h i o u r e a ,
a known b l o c k e r of delayed ISC formation i n L1210 c e l l s , blocked
cis-PPP c y t o t o x i c i t y i n a f a s h i o n c o n s i s t e n t w i t h the r e s u l t s of
the L1210 work.
Trans-PPP has l e a v i n g groups s t e r e o c h e m i c a l ^ disposed t o
make the formation of c r o s s l i n k s at a d j o i n i n g base p a i r s w i t h i n
one s t r a n d of PNA ( i n t r a s t r a n d c r o s s l i n k i n g ) u n l i k e l y (see F i g u r e
2 ) . I t i s not mutagenic. Cis-PPP, which can p o t e n t i a l l y form
i n t r a s t r a n d c r o s s l i n k s , i s mutagenic. I f cis-PPP i n t r a s t r a n d
c r o s s l i n k i n g forms t o a g r e a t e r magnitude or more r a p i d l y than
ISC, then the decreased a b i l i t y of t h i o u r e a t o b l o c k cis-PPP
mutagenicity compared w i t h i t s a b i l i t y t o b l o c k c y t o t o x i c i t y
suggests mutagenicity may r e s u l t from i n t r a s t r a n d c r o s s l i n k
formation.
SCE formation probably d e r i v e s from n e i t h e r the mutagenic
nor c y t o t o x i c cis-PPP l e s i o n . The non-mutagenic trans-PPP can
form SCE's. Immediate t h i o u r e a can only b l o c k SCE formation at
very low cis-PPP doses and d e l a y i n g t h i o u r e a a l l o w s almost t o t a l
escape of SCE formation from t h i o u r e a blockade. PNA monoadduct
formation may induce SCE formation. What l i t t l e e f f e c t t h i o u r e a
does have on SCE formation may d e r i v e from t h i o u r e a ' s b i n d i n g of
unreacted, i n t r a c e l l u l a r Pt p r i o r t o i t s b i n d i n g t o PNA at a l l .

c/s-DDP CONCENTRATION (μΜ)
Figure 11. The effect of thiourea on cis-DDP cytotoxicity and mutagenicity in V79
Chinese hamster cells. Cells were treated with various concentrations of cis-DDP for
1 h followed by a 1 h incubation in the presence (O) or absence (%) of 100 mM
thiourea. (See Ref. 17).
5 10
c/s-DDP CONCENTRATION (μΜ)
Figure 12. The effect of thiourea on cis-DDP sister-chromatid exchange production

in V79. Conditions are outlined in Fig. 11 legend.

Cytotoxicity
May Derive from
Interstrand Crosslinking
Mutagenicity SCE Formation

May Derive from May Derive from
Intrastrand Crosslinking Monoadduct Formation
Figure 13. The biological consequences of different DNA-Pt interactions in V79

cells. The appropriate DNA lesion is surrounded by the evidence relating it to the
particular biological response it produces.

Discussion
The scheme i n Figure 1 has i m p l i c i t w i t h i n i t a k i n e t i c

approach t o the study of DNA damage and r e p a i r and i t s
r e l a t i o n s h i p t o d e s i r a b l e and t o x i c consequences of a n t i n e o -
p l a s t i c , DNA r e a c t i v e drugs. I n t h i s paper the stepwise approach
to the study of t h i s scheme f o r one agent, cis-DDP, i s presented.
The i n i t i a l s e l e c t i o n of cis-DDP was made because of i t s c l i n i c a l
e f f i c a c y and the a v a i l a b i l i t y of an i n a c t i v e isomer ( 1 ) . We had
hoped that some c r i t i c a l DNA l e s i o n would be more e f f i c i e n t l y
produced by the a c t i v e compound. The development of the a l k a l i n e
e l u t i o n technique i n c o r p o r a t i n g enzymatic d e p r o t e i n i z a t i o n t o
d i s t i n g u i s h between i n t e r s t r a n d and DNA-protein c r o s s l i n k i n g was
c r i t i c a l f o r t h i s a n a l y s i s (7^, 8 ) .
The r e s u l t s of our i n i t i a l s t u d i e s i n L1210 c e l l s i n d i c a t e d

that ISC may be a key DNA l e s i o n produced by cis-DDP ( 9 ) . The
s t u d i e s w i t h t h i o u r e a (10) and r e s i s t a n t L1210 c e l l s (6) con-
firmed t h i s i d e a . However, the example of the r e s i s t a n c e o f
L1210/PAM t o cis-DDP d e s p i t e h i g h l e v e l s of c r o s s l i n k i n g
emphasized the n e c e s s i t y of studying the k i n e t i c s of l e s i o n
formation and r e p a i r (14) as w e l l as the magnitude of DNA damage
at any one p o i n t i n time. The s t u d i e s w i t h V79 confirmed t h i s
as the s u s c e p t i b i l i t y of these c e l l s t o v a r i o u s consequences of
cis-DDP was a l t e r e d i n d i f f e r e n t dose- and time-dependent
fashions by t h i o u r e a ( 3 , 17). These s t u d i e s demonstrate the
type of i n f o r m a t i o n o b t a i n a b l e i n w e l l - d e f i n e d c e l l u l a r systems
w i t h techniques which can q u a n t i f y c e r t a i n c e l l f u n c t i o n s and
drug-induced DNA l e s i o n s .
How can such an approach be extended i n the f u t u r e ? E r i c k s o n
et a l . have shown w i t h t h e i r work i n human c e l l s one important
s t e p . The r e s u l t s of s t u d i e s u s i n g human m a t e r i a l as a t a r g e t
f o r cis-DDP may r e v e a l d i f f e r e n c e s from r e s u l t s obtained i n
rodent systems. These r e s u l t s may have more bearing on c l i n i c a l
problems (4, 5)· E r i c k s o n e t a l . have a l s o developed a
f l u o r o m e t r i c a l k a l i n e e l u t i o n assay r e q u i r i n g no l a b e l e d DNA
(18). P a t i e n t m a t e r i a l explanted i n t o c u l t u r e o r obtained during
therapy could be analyzed f o r DNA damage w i t h t h i s assay and
conceivably could be c o r r e l a t e d w i t h plasma drug c o n c e n t r a t i o n s ,
other i n v i t r o assays, o r c l i n i c a l tumor response.
The i d e n t i f i c a t i o n of c r i t i c a l DNA l e s i o n s i n c e l l s could
a l s o a i d i n new drug development. Analogs of promise could be
compared w i t h parent compounds i n a manner s i m i l a r t o our
comparison of c i s - and trans-DPP as t o DNA damage and i t s
r e s u l t a n t e f f e c t s . An i n i t i a l t e s t of t h i s approach v s . animal
screening could be made t o see i f s i m i l a r r e s u l t s were obtained.
I f so, the i n v i t r o approach would be more r a p i d and l e s s c o s t l y .
Perhaps of greatest importance t o f u t u r e endeavors i s the
d i r e c t i o n s i n which t h i s current work moves us on a more b a s i c
l e v e l . I n p a r t i c u l a r , the r e s u l t s w i t h L1210/PAM and i t s c i s -
DDP-resistance r a i s e more questions than they answer. What
American Chemical
Society Library
In Platinum, Gold, and1155 164 Chemotherapeutic
Other Metal St. N. W. Agents; Lippard, S.;
ACS Symposium Series;
Washington, 0. C. Society:
American Chemical 20036Washington, DC, 1983.
kind of process would explain Pt-monoadduct inactivation? Could

it be a naturally-occuring substance which resembles thiourea in
its inactivating effect? Recent work suggests metallothionein
could play such a role (19). Or, is a specific platinum adduct
DNA repair mechanism extant? The human cell work would indicate
if so, it differs from that which responds to DNA methylation
(4, _5, 15). If such a repair process exists, is i t inducible,
and why would a cell possess a system which responds to metal-DNA
adduct formation as distinguished from organic adduct formation?
The answers to these questions should impact not only on the
field of antineoplastic drug development but also on normal and
malignant cell biology and physiology.
Abbreviations used: DDP, Diamminedichloroplatinum(II); L-PAM,

L-phenylalanine mustard; i . p . , intraperitoneal; RPMI 1630, Roswell

Park Memorial Institute medium 1630; DPC, DNA-protein crosslink
ing; ISC, DNA interstrand crosslinking; SCE, sister-chromatid
exchanges·
Acknowledgment s
Thanks to our numerous collaborators in this work: Drs.

Mattews 0. Bradley, Leonard C. Erickson, Kenneth Micetich,
Warren Ross, Tom Anderson, Guy Laurent and Jonathan Ducore; the
technical assistance of Stephen Michaels, Regina A. 6. Ewig,
Nancy Sharkey, Irene Clark, Suzanne Patterson, Patricia P. Dobson
and Howard Schwartz; and the secretarial efforts of Ms. Madie
Tyler. A special thanks to Dr. Kurt W. Kohn, Chief of the
Laboratory of Molecular Pharmacology, who has supported a l l of
this work with advice in experimental design and expert data
analysis and interpretation, as well as valuable critique of
this and a l l past manuscripts.
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Cancer Treatment; Chabner, Β. Α., Ed; W. B. Saunders:
Philadelphia, 1982; pp. 309-339.
2. Chu, M. Y.; Fisher, G. A. Biochemical Pharmacology, 1968, 17,
753-767.
3. Zwelling, L . Α.; Bradley, M. O.; Sharkey, Ν. Α.; Anderson,
T.; Kohn, K. W. Mutation Research 1979, 67, 271-280.
4. Erickson, L . C.; Zwelling, L. Α.; Ducore, J . M.; Sharkey, N.
Α.; Kohn, K. W. Cancer Research 1981, 41, 2791-2794.
5. Laurent, G.; Erickson, L . C.; Sharkey, Ν. Α.; Kohn, K. W.
Cancer Research 1981, 41, 3347-3351.
6. Zwelling, L . Α.; Michaels, S.; Schwartz, H . , ; Dobson, P. P.;
Kohn, K. W. Cancer Research 1981, 41, 640-649.

7. Kohn, K. W. "Methods in Cancer Research"; DeVita, V· T . ,

J r . ; Busch, H . , Ed.; Academic Press: New York, 1979; Vol.
XVI, pp. 291-345.
8. Kohn, K. W.; Ewig, R. A. G.; Erickson, L . C.; Zwelling, L . A.
"DNA Repair. A Laboratory Manual of Research Procedures";
Friedberg, E. C.; Hanawalt, P. C . , Ed.; Marcel Dekker: New
York, 198; pp. 379-401.
9. Zwelling, L . Α.; Anderson, T.; Kohn, K. W. Cancer Research
1979, 39, 365-369.
10. Zwelling, L . Α.; Filipski, J . and Kohn, K. W. Cancer Research
1979, 39, 4989-4995.
11. Kohn, K. W. "Molecular Actions and Targets for Cancer Chemo
therapeutic Agents", Academic Press: New York, 1981; pp. 3-
16.
12. Deutsch, W. Α.; Spierina, A. L . ; Newkome, G. R. Biochem.

Biophys. Res. Commun., 1980, 97, 1220-1226.
13. Filipski, J . ; Kohn, K. W.; Prather, R.; Bonner, W. M.
Science 1979, 204, 181-183.
14. Micetich, K.; Michaels, S.; Jude, G.; Kohn, K.; Zwelling, L .
Proc. Amer. Assoc. Cancer Research, 1981 22, 252.
15. Erickson, L . C.; Bradley, M. O.; Ducore, J . M.; Ewig, R. A.
G.; Kohn, K. W. Proc. Natl. Acad. Sci. USA, 1980, 77, 467-
471.
16. Bradley, M. O.; Hsu, I. C.; Harris, C. C. Nature, 1979, 282,
318-320.
17. Bradley, M. O.; Patterson, S.; Zwelling, L . A. Mutation
Research, in press.
18. Erickson, L . C.; Osieka, R.; Sharkey, Ν. Α.; Kohn, K. W.
Analytical Biochemistry, 1980, 106, 169-174.
19. Bakka, Α.; Endresen, L . ; Johnsen, A. B. S.; Edminson, P. D.;
Rugstad, Η. E. Toxicology and Applied Pharmacology, 1981
61, 215-226.
20. "Zwelling, L . Α.; Kohn, K. W. "Cisplatin. Current Status and
New Developments"; Prestayko, A. W.; Crooke, S. T.; Carter
S. K. Ed.; Academic Press: New York, 1980; pp. 21-35.

3
Structural Chemistry of P l a t i n u m - D N A Adducts
THOMAS D. TULLIUS, H. MICHAEL USHAY, CAROLYN M. MERKEL, JOHN

P. CARADONNA, and STEPHEN J. LIPPARD
Columbia University, Department of Chemistry, New York, NY 10027
Aspects of the binding of the antitumor drug, cis-

-diamminedichloroplatinum(II) (cis-DDP), and other
platinum complexes to DNA are reviewed. This work
was undertaken because DNA is widely regarded as the
cellular target for cis-DDP and because of our
interest in heavy metal reagents as specific stains
for biological specimens. cis-DDP unwinds the
double helix when it binds to DNA through covalent
attachment to the bases. It also substantially
shortens the DNA. These structural effects are
revealed by gel electrophoretic study of closed
circular, superhelical plasmid DNAs and by electron
microscopy. When cis-DDP is allowed to react with
DNA in the presence of the intercalating drug ethid-
ium bromide, duplex unwinding still occurs but the
shortening effect is substantially diminished.
Ethidium can also switch the cis-DDP binding sites
that are sensitive to digestion by nucleases. In
the absence of ethidium, cis-DDP inhibits both
restriction enzymes and exonuclease III digestion of
DNA, and the latter has been used to demonstrate
that cis-DDP is especially effective at stopping the
enzyme when bound at oligo(dG) sequences. When
platinum is bound in the presence of ethidium,
however, different oligo(dG) regions are exposed.
These results are used to rationalize the synergism
observed when cis-DDP is used in combination chemo-
therapy with intercalating drugs. Using radio-
14
labeled [ C]methylamine it has been shown that
cis-[Pt(NH CH ) Cl ] does not release its amine
2 3 2 2
ligands upon binding to DNA. An intrastrand GpG

crosslink involving N7 atoms of guanine is currently
regarded as the most likely structure for the cyto-
toxic cis-DDP-DNA linkage. The compound [(dien)Pt-
Cl]Cl promotes theB->Zconformational transition of
poly(dG-dC)•poly(dG-dC).
0097-6156/83/0209-0051 $07.00/0

Heavy metals, especially the third row transition elements,

are useful reagents i n biology and biochemistry. The high
electron density of these elements (notably tungsten, osmium,
platinum and gold) makes them well suited for revealing aspects of
supramolecular structures i n electron microscopic images (j_).
More recently, simple platinum complexes have become widely used
in cancer chemotherapy ( 2 ) .
We wish to understand the interactions of heavy metal com-
pounds with biological materials at a molecular level. L i t t l e i s
known about the s t r u c t u r a l chemistry of heavy metal s t a i n s
attached to macromolecules, and the chemical basis of the tumor
k i l l i n g a b i l i t y of cis-diamminedichloroplatinum(II) (cis-DDP) i s
s t i l l obscure. By studying the chemistry of heavy metal complexes
of biological macromolecules we can design better, more specific
staining reagents. Finding out how the platinum antitumor drugs

bind to their cellular target, DNA, could help us design effective
drugs with fewer of the toxic side effects which limit the c l i n i -
cal efficacy of cis-DDP.
In this chapter we summarize some recent results from our
laboratory which bear on the question of the structural chemistry
of platinum-DNA complexes. We find the local structure of DNA, as
well as the nature of the platinum compound, to affect i n a pro-
found way the structure of the resulting platinum-DNA complex.
Levels of DNA Structure. A DNA molecule has several levels of

structure ranging from the primary structure of the sequence of
bases to the secondary structure of the Watson-Crick double helix
to the tertiary structure resulting from folding or supercoiling
the double helix to even higher order structures involved i n the
condensation of DNA i n the c e l l nucleus. To serve as a basis for
understanding the interaction of platinum complexes with DNA, we
f i r s t describe some of the more important features of DNA struc-
ture.
A supercoiled double helix apparently i s necessary for many
cellular processes involving DNA (3). DNA i s found to be super-
coiled i n the simplest procaryotes, as well as i n the c e l l s of
higher organisms. In cells with nuclei, the DNA double helix i s
wrapped i n shallow superhelices around complexes of eight histone
proteins. This structural unit i s termed the nucleosome (4). It
repeats along a chain of DNA to form the well known "beads on a
string" structure (5) seen i n electron micrographs of chromatin
spread on a grid.
From procaryotes, such as E. c o l i , much simpler supercoiled
DNA molecules can be isolated. These molecules, called plasmids
(6), consist of a few thousand DNA base pairs joined i n a closed
circle. The double helix of such a circular molecule i s super-
coiled upon i t s e l f , to form a condensed structure, called "form

3. TULLius ET A L . Structural Chemistry of Pt-DNA Adducts 53
I." The topological constraints inherent i n such a structure

result i n linking the number of superhelical turns to the winding
of the Watson-Crick double helix (7). For example, an intercala-
ting drug causes the DNA double helix to unwind when the drug
binds by insertion between base pairs. Ethidium bromide, a
commonly used intercalator, unwinds the DNA helix by 26° per bound
drug molecule (8). If such a molecule i s bound to closed circu-
x
lar, supercoiled DNA, the local unwinding of the double helix
results i n removal of negative superhelical turns, to maintain the
same total number of turns (Watson-Crick + supercoil) i n the DNA.
If, however, the closed circular DNA has a break i n one of i t s
chains, these topological constraints are relieved, there are no
supercoils, and the molecule exists i n a relaxed, open circular
form. This structure i s termed "form II." The advantage of
studying the binding of platinum complexes to such plasmid

molecules i s that very subtle changes i n the winding of the double
helix or i n the density of supercoiling translate to easily
observed shifts i n the mobility of the DNA on electrophoresis
gels. Thus structural changes that occur upon platinum binding
are amplified many times i n the experimentally observable event.
At the level of primary structure, several recent experiments
have shown the effect of base sequence on the local structure of
DNA. A dramatic example i s the crystal structure of d(CpG)~ as
determined by Rich and coworkers ( 9 ) . This molecule crystallizes
in a left-handed double h e l i c a l form called Z-DNA, which i s
radically different i n i t s structural properties from the familiar
right-handed B-DNA structure. Dickerson and Drew (10) showed i n
the crystal structure of the dodecanucleotide d(CGCGAATTCGCG) that
the local twist angle of a DNA double helix varies with sequence.
Deoxyribonuclease I cuts the phosphodiester backbone of the dode-
canucleotide preferentially at sites of high twist angle (11 ).
From these and other (12,13) experiments we see that the structure
of DNA varies with base sequence, and that enzymes are sensitive
to these details of structure.
Platinum Binding to DNA. How do simple platinum complexes

bind to DNA? What are the effects of the primary, secondary and
tertiary structures of DNA on this binding? We have approached
these questions experimentally over the last several years (14) ·
Our results and the results of others w i l l be summarized i n this
section.
An important early observation was that cis-DDP, the antitumor
drug, binds covalently and not by intercalation to DNA ( J_5 ) · We
present evidence to support this conclusion later i n the section
describing the effects of ethidium bromide on platinum binding.
Since platinum(ll), when bound to nitrogen ligands, i s rather
inert kinetically (16), platinum-DNA adducts may be isolated and
studied without appreciable platinum loss.
As a model for how platinum complexes might interact with the
DNA of higher organisms, we have studied the binding of the active

antitumor drug cis-DDP and the i n a c t i v e trans stereoisomer w i t h

the nucleosome core p a r t i c l e (17). We were i n t e r e s t e d to l e a r n
whether the platinum complexes would bind to the DNA, the p r o t e i n ,
or both, of the core p a r t i c l e . We found that cis-DDP, a t low
platinum to n u c l e o t i d e r a t i o s ( r ^ ) , i n t e r a c t e d almost e x c l u s i v e l y
w i t h nucleosomal DNA, w h i l e trans-DPP formed s p e c i f i c h i s t o n e -
h i s t o n e and DNA-histone c r o s s l i n k s . This experiment was one of
the f i r s t to demonstrate a c l e a r , q u a l i t a t i v e d i f f e r e n c e i n the
way i n which the two platinum complexes bind DNA i n an i n v i t r o
system.
We (18) and others (19,20,21 ) have s t u d i e d the e f f e c t of
platinum complexes on the conformation o f c o v a l e n t l y c l o s e d , c i r -
c u l a r s u p e r c o i l e d DNA u s i n g agarose g e l e l e c t r o p h o r e s i s . Both
c i s - and trans-DPP were found to unwind the Watson-Crick double
h e l i x , r e s u l t i n g i n removal of negative s u p e r c o i l s from these

t o p o l o g i c a l l y constrained PNA molecules (see above). An unwinding
angle of 22* per bound cis-PPP molecule was measured ( 2 2 ) . We
observed w i t h pSM1 (18) and pBR322 (22,25) plasmids that both c i s -
and trans-PPP could remove a l l s u p e r c o i l s , r e s u l t i n g i n a c l o s e d
c i r c u l a r PNA molecule which comigrated on g e l s w i t h r e l a x e d
(nicked) c i r c u l a r PNA. I n c o n t r a s t to subsequent r e s u l t s of
S c o v e l l and coworkers (20,21), we found (18) that e q u i v a l e n t
amounts of bound c i s - and trans-PPP caused e q u i v a l e n t changes i n
PNA conformation. For example, at comigration o f r e l a x e d - and
c l o s e d - c i r c u l a r p l a t i n a t e d pBR322, the r ^ was 0.050 0.005 f o r
both isomers (22). In a d d i t i o n , again i n c o n t r a s t to S c o v e l l ^ s
(20,21 ) work, we observed that both c i s - and trans-PPP cause
f u r t h e r p o s i t i v e winding of s u p e r c o i l e d PNA, r e s u l t i n g i n an
i n c r e a s e i n g e l m o b i l i t y of the PNA, at platinum b i n d i n g l e v e l s
g r e a t e r than that necessary to cause c o m i g r a t i o n of closed and
r e l a x e d c i r c u l a r PNA ( 1 8 ) .
A second e f f e c t of p l a t i n a t i o n was noted (18) on the e l e c t r o -
p h o r e t i c m o b i l i t y of relaxed c i r c u l a r PNA. The m o b i l i t y o f t h i s
form increased w i t h i n c r e a s i n g platinum b i n d i n g , as opposed to the
i n i t i a l decrease and subsequent increase i n m o b i l i t y o f super-
c o i l e d PNA. This behavior was a t t r i b u t e d to a shortening of the
PNA due to platinum induced c r o s s l i n k s . F u r t h e r evidence was
obtained from e l e c t r o n micrographs of the p l a t i n a t e d PNA samples
(18,24), which showed a dramatic decrease i n contour length o f
r e l a x e d plasmid molecules upon p l a t i n a t i o n (see F i g u r e 6 below).
E f f e c t o f Bound Platinum on PNA P r o c e s s i n g Enzymes. With some

of the e f f e c t s of p l a t i n a t i o n on PNA s t r u c t u r e now d e f i n e d , we can
ask whether and how these changes i n PNA conformation w i l l a f f e c t
enzymes which process PNA.
Type I I r e s t r i c t i o n endonucleases (25) are enzymes i s o l a t e d
from procaryotes which recognize and make double-stranded cuts a t
s p e c i f i c PNA sequences. We i n v e s t i g a t e d how p l a t i n a t i o n of p l a s -
mid pBR322 would a f f e c t the a b i l i t y of the enzyme Bam HI to cut a t
i t s s i n g l e r e c o g n i t i o n sequence i n t h i s plasmid (23.)· Bam HI

3. TULLius ET AL. Structural Chemistry of Pt-DNA Adducts 55
cleaves the sequence
-GGATCC-
-CCTAGG-
T
at the phosphodiester bonds between t h e p a i r s o f guanines as
shown. P r e v i o u s work on bacteriophage λ DNA had suggested (26)
that cis-DDP might c r o s s l i n k the adjacent guanine bases i n the
enzyme c u t t i n g s i t e , but s t i l l a l l o w the enzyme to cleave the DNA
backbone. The c r o s s l i n k e d , cut DNA would appear as uncut DNA on
g e l s , but i f the platinum were removed by cyanide treatment before
running the g e l , the cuts would be e v i d e n t . There are s e v e r a l Bam
HI s i t e s i n λ DNA, however, making the a n a l y s i s o f the r e s u l t s
d i f f i c u l t . Our experiment (25) u s i n g pBR322 DNA w i t h a s i n g l e Bam
HI s i t e showed c o n c l u s i v e l y that c r o s s l i n k i n g o f the guanines by

cis-DDP w i t h concomitant c u t t i n g o f the DNA backbone does not
occur. We found that Bam HI c u t t i n g was p r o g r e s s i v e l y i n h i b i t e d
by i n c r e a s i n g amounts o f bound cis-DDP, w i t h t o t a l i n h i b i t i o n
o c c u r r i n g a t r, = 0.050 0.005· Cyanide treatment d i d n o t
produce l i n e a r DNA from the samples which were apparently not cut
by Bam HI, as would have been p r e d i c t e d from the previous study
(26).
Some r e s t r i c t i o n endonucleases have s e v e r a l c u t t i n g s i t e s on a
p a r t i c u l a r DNA molecule. I n an experiment (27) w i t h such an
enzyme we used P s t I , which cleaves plasmid pSM1 a t f o u r s i t e s .
We asked whether cis-DDP b i n d i n g to any of one these s i t e s , which
n e c e s s a r i l y have i d e n t i c a l enzyme r e c o g n i t i o n sequences, would
render i t more d i f f i c u l t to cut than the o t h e r s . The c u t t i n g a t
one o f these s i t e s , the D-B j u n c t i o n , was indeed i n h i b i t e d a t a
lower platinum b i n d i n g l e v e l than the o t h e r s . Consideration o f
the sequences of bases f l a n k i n g the four s i t e s showed that the D-B
e #
j u n c t i o n has d(G) / d ( C ) . , d(GAG) d(CTC) and d ( G ) d ( C ) p sequences
2
surrounding the enzyme R e c o g n i t i o n sequence. None o r the o t h e r

three s i t e s has o l i g o ( d G ) * o l i g o ( d C ) s i t e s nearby. The c o n c l u s i o n
0
was made that cis-DDP bound a t o l i g o ( d G ) o l i g o ( d C ) ( o r perhaps
d(GAG)*d(CTC) (28)) s i t e s causes a more profound change i n DNA
s t r u c t u r e and i n h i b i t s the r e s t r i c t i o n enzyme. A g r e a t e r amount
of bound cis-DDP would then be necessary to i n h i b i t enzyme d i g e s
t i o n at other s i t e s l a c k i n g these sequences.
Sequence S p e c i f i c i t y o f cis-DDP B i n d i n g to DNA. Previous

e
experiments had a l s o i n d i c a t e d that o l i g o ( d G ) o l i g o ( d C ) sequences
might be p r e f e r r e d b i n d i n g s i t e s f o r cis-DDP. A g r e a t e r buoyant
d e n s i t y change was seen f o r p l a t i n a t e d poly(dG)*poly(dC) than f o r
p l a t i n a t e d poly(dG-dC) when compared to the u n p l a t i n a t e d polymers
(29) · To study i n more d e t a i l t h i s e f f e c t o f the primary s t r u c
ture o f DNA on platinum b i n d i n g , our group (30) as w e l l as that o f
H a s e l t i n e (31 ) developed a method f o r d e t e c t i n g platinum b i n d i n g
at s p e c i f i c sequences i n DNA.
This assay used the enzyme exonuclease I I I (32) which d i g e s t s

DNA e x o n u c l e o l y t i c a l l y from i t s 3^ ends- Bound cis-DDP stops t h i s

e x o n u c l e o l y t i c degradation. I f the p l a t i n a t e d DNA s u b s t r a t e i s
r a d i o a c t i v e l y l a b e l e d w i t h ^ Ρ a t one 5^ end (as f o r DNA sequenc
i n g (33))> d e n a t u r a t i o n of the exonuclease I l l - p r o d u c e d fragments,
s e p a r a t i o n by s i z e on a DNA sequencing e l e c t r o p h o r e s i s g e l , and
autoradiography w i l l r e v e a l the stopping p o i n t s of the enzyme.
The sequences at these stopping p o i n t s are determined by comparing
the fragment lengths w i t h the lengths of DNA molecules produced by
sequencing r e a c t i o n s . Since the bases at the 3^ ends of the
sequencing r e a c t i o n products are known, t h i s method a l l o w s the
determination of the base sequences at the s i t e s where p l a t i n a t i o n
stopped exonuclease I I I d i g e s t i o n . I t was found that cis-DDP
bound to oligo(dG) s i t e s stopped the enzyme; stopping at other
base sequences was not detected (30). We concluded that e i t h e r
cis-DDP binds p r e f e r e n t i a l l y to these oligo(dG) s i t e s to the

e x c l u s i o n of other sequences, or that the s t r u c t u r e r e s u l t i n g from
cis-DDP bound at oligo(dG) sequences i s s i n g u l a r l y e f f i c i e n t at
stopping exonuclease I I I .
Ethidium Bromide A l t e r s the Mode of B i n d i n g of cis-DDP to DNA
The antitumor a c t i v i t y of platinum drugs i s very s e n s i t i v e to

changes i n the stereochemistry of the platinum complex. For
example, w h i l e cis-DDP i s an e f f e c t i v e antitumor agent, the
corresponding t r a n s isomer i s not (2). We wanted to study how
changing the t e r t i a r y and secondary s t r u c t u r e of plasmid DNA would
a f f e c t the r e a c t i v i t y of cis-DDP toward the DNA. Several of the
drugs used i n a c l i n i c a l regimen w i t h cis-DDP to improve i t s
e f f i c a c y are a l s o able to a l t e r the s t r u c t u r e of DNA ( 2^). We
chose to study ethidium bromide ( E t d B r ) , a w e l l c h a r a c t e r i z e d DNA
i n t e r c a l a t i n g agent (8), as a model f o r other drugs which are used
w i t h cis-DDP.
Previous work has shown that DNA which had been p l a t i n a t e d
w i t h cis-DDP cannot bind as much EtdBr by i n t e r c a l a t i o n as u n p l a t -
i n a t e d DNA (15). Figure 1 i s a fluorescence Scatchard p l o t of the
b i n d i n g of EtdBr to c a l f thymus DNA to which v a r y i n g amounts of
cis-DDP were bound. Upon increased platinum b i n d i n g , the slope of
the fluorescence Scatchard p l o t remained constant. Therefore c i s -
DDP does not c o m p e t i t i v e l y i n h i b i t the b i n d i n g of ethidium. This
r e s u l t demonstrates that the platinum drug does not i n t e r c a l a t e
i n t o the DNA duplex, but binds c o v a l e n t l y . The change i n ab
s c i s s a , however, r e v e a l s that platinum bound to DNA reduces the
number of s i t e s a v a i l a b l e f o r ethidium b i n d i n g .
We then asked how b i n d i n g ethidium to DNA before p l a t i n a t i o n
would a f f e c t the a b i l i t y of cis-DDP to b i n d . Figure 2 shows the
r e s u l t s of atomic a b s o r p t i o n measurements of platinum b i n d i n g to
pBR322 DNA. The k i n e t i c s of b i n d i n g are unchanged when ethidium
i s present d u r i n g p l a t i n a t i o n . The a b i l i t y of cyanide to remove
bound platinum i s g r e a t l y enhanced, however. For example, w i t h no
ethidium present and more than approximately one platinum atom

60
Figure 1. Fluorescence Scatchard plot of the binding of EtdBr to calf-thymus

DNA in the absence (O) and the presence (·, 20 h; A, 66 h; and Δ , 139 h) of
cis-DDP in Tris-HCl (50 mM) and sodium chloride (0.2 M) at pH 7.5. Condi
tions: [DNA phosphate] = 1.05 χ 10~ M;[EtdBr] = 2.2 X 10' -1.7 X 10 M;
5 6 s
and T = 0.83. (Reproduced from Ref. 15. Copyright 1976, American Chemical
f
Society.)
Figure 2. Binding of cis-DDP to pBR322 DNA at 37 °C in sodium phosphate

(1 mM) and sodium chloride (3 mM) at pH 7.4. Conditions: [Pt] = 2.50 χ 10* M;
[DNA phosphate] = 2.50 X 10 M; and r, = 0.10. Key: O , no ethidium present;
A
and ·, 7.5 χ ΙΟ M ethidium present.

6

bound per f i v e base p a i r s , 8-10$ of the platinum cannot be removed

by cyanide treatment (23). When s a t u r a t i n g amounts of ethidium
are present during p l a t i n a t i o n , a l l bound platinum could be
removed from the DNA upon cyanide treatment.
The a b i l i t y of cis-DDP to unwind the DNA duplex i s u n a f f e c t e d
by the presence of ethidium d u r i n g p l a t i n a t i o n . F i g u r e 3 d e p i c t s
the changes i n g e l m o b i l i t i e s that occur when i n c r e a s i n g amounts
of platinum are bound to DNA forms I and I I . Note t h a t , whether
ethidium i s present or not, there i s at f i r s t a d i m i n u t i o n i n the
m o b i l i t y of the form I band upon platinum b i n d i n g due to duplex
unwinding (and concomitant i n t r o d u c t i o n of p o s i t i v e s u p o r c o i l s ) .
There i s then a subsequent i n c r e a s e i n m o b i l i t y i n the form I band
past the p o i n t where i t comigrates i n the g e l w i t h the form I I
band as platinum continues to bind and i n c r e a s e the number of
positive supercoils.
The a b i l i t y of cis-DDP to decrease the e f f e c t i v e l e n g t h of the
plasmid i s g r e a t l y reduced, however, when platinum i s bound i n the
presence of ethidium. Figure 4 shows that the presence of e t h i d -
ium d u r i n g p l a t i n a t i o n decreases the m o b i l i t y of p l a t i n a t e d form
I I DNA when compared w i t h the m o b i l i t y of c o n t r o l p l a t i n a t e d DNA.
This d i f f e r e n c e i n m o b i l i t y i s not due to a d i f f e r e n c e i n the
amount of bound platinum (see Figure 2 ) , and i t depends upon the
amount of ethidium present d u r i n g p l a t i n a t i o n .
A p l o t of g e l m o b i l i t y versus the amount of platinum bound,
Figure 5> demonstrates t h i s d i f f e r e n c e . We compare g e l m o b i l i t i e s
of samples p l a t i n a t e d w i t h a formal r a t i o of one cis-DDP per
n u c l e o t i d e , f o r samples c o n t a i n i n g e i t h e r no ethidium or three
ethidium molecules per ten n u c l e o t i d e s . The g e l m o b i l i t i e s are
normalized to 0 f o r form I I and 1.0 f o r form I DNA i n the c o n t r o l
sample. Note that there i s about a 35$ decrease i n m o b i l i t y where
forms I and I I comigrate i n the g e l when platinum i s bound i n the
presence of s a t u r a t i n g amounts of ethidium.
These changes i n g e l m o b i l i t y can be c o r r e l a t e d to changes i n
the p h y s i c a l s t r u c t u r e of the DNA, as seen i n Figure 6. The
uppermost e l e c t r o n micrograph of u n p l a t i n a t e d pBR322 DNA c l e a r l y
shows nicked and s u p e r c o i l e d molecules. The center micrograph i s
of pBR322 DNA p l a t i n a t e d w i t h cis-DDP to a l e v e l of 28 platinum
atoms per 100 n u c l e o t i d e s . The contour l e n g t h of the r e l a x e d
c i r c u l a r DNA i s g r e a t l y decreased, as has been seen p r e v i o u s l y
08).
The bottom micrograph i s of pBR322 p l a t i n a t e d to a l e v e l of 23
1
platinum atoms per 100 n u c l e o t i d e s i n the presence of s a t u r a t i n g
amounts of ethidium. I t i s c l e a r that t h i s DNA i s more s i m i l a r to
c o n t r o l DNA than are the samples p l a t i n a t e d w i t h no ethidium
present. There i s some compacting and " s h r i n k i n g " seen, but not
to the extent seen i n the sample p l a t i n a t e d i n the absence of
ethidium.
In summary, ethidium bromide does not a l t e r the k i n e t i c s of
platinum b i n d i n g . This r e s u l t i s not s u r p r i s i n g s i n c e these
b i n d i n g k i n e t i c s have been shown to be comparable to the pseudo-

Figure 3. Electrophoresis in a 1.5% agarose gel (Tris-acetate-EDTA buffer)

of approximately 0.5 ^g of forms I and II pBR322 DNA following incubation with
cis-DDP for the times indicated. Conditions: [Pt] = 4.25 X 10' M ; [DNA phos
5
phate] = 2.83 X 10 M ; and cis-DDP r, = 0.150. Key: left side, no EtdBr present;
A
and right side, 8.5 χ ΙΟ M EtdBr present.

6
Figure 4. Electrophoresis in a 1.5% agarose gel of forms I and II pBR322 DNA

following incubation with cis-DDP. Key to [EtdBr]: Ο M for lanes 1, 2,5, 8,11,
14, and 17; 2.7 X 10* M for lanes 3, 6, 9, 12, 15, and 18; and 5.4 χ 10« M for
lanes 4, 7, 10, 13, 16, 19, and 20. Key to incubation time: lanes 1-4, 0 h; lanes
5-7,1 h; lanes 8-10, 2 h; lanes 11-13,5 h; lanes 14-16, 8 h; and lanes 17-20,26 h.
Conditions: [Pt] = 2.70 χ 10 M; [DNA phosphate] = 1.80 χ 10' M ; and
s 4
cis-DDP r, = 0.150.

0.6 -1
i.oH
Figure 5. Gel mobilities for forms I and II pBR322 DNA as a function of the
amount of bound cis- or tmns-DDP. Distances traveled in the gel are normalized
so that in the control the mobility of form I DNA is 1.0 and the mobility of form 11
DNA is 0.0. Conditions: [Pt] = 2.34 χ 10' M ; [DNA phosphate] = 2.34 χ 10~
4 4
M ; and platinum r, = 1.0. Key: O, cis-DDP, no ethidium; · , cis-DDP, ethidium

r, = 0.3; Δ , trans-DDP, no ethidium; and A , trans-DDP, ethidium r, = 0.3.

Figure 6. Electron micrographs of pBR322 DNA. Key: a, untreated DNA; b,

no EtdBr and cis-DDP r = 0.28; c, EtdBr r, = 0.3 and cis-DDP r = 0.23.
6 6
Conditions in the platination mixture: [Pt] = 2.50 X 10~ M ; [DNA phosphate]

4
= 2.68 X 10~ M; cis-DDP r = 0.90; and an incubation time of 18 h (35).

4
f

f i r s t order r a t e of aquation of the platinum complex (34) » and

ethidium and cis-DDP do not react with each other i n s o l u t i o n
(35,)· The a b i l i t y o f the platinum complex to unwind the duplex
causing concomitant winding o f the s u p e r h e l i x i s a l s o unchanged.
The a b i l i t y of cyanide i o n to remove bound platinum i s g r e a t l y
enhanced when ethidium i s present during p l a t i n a t i o n . This
f i n d i n g i m p l i e s that the platinum i s more a c c e s s i b l e to cyanide
treatment e i t h e r through an e a s i l y a v a i l a b l e c o o r d i n a t i o n s i t e on
the platinum, or by the platinum being bound to DNA i n some
e n t i r e l y d i f f e r e n t manner. The DNA duplex shortening that i s
induced upon platinum b i n d i n g i s g r e a t l y reduced when ethidium i s
present, a l s o implying some d i f f e r e n t manner o f b i n d i n g . The
extent of r e d u c t i o n depends d i r e c t l y upon the amount of ethidium
present d u r i n g p l a t i n a t i o n .
We conclude that the presence of ethidium during p l a t i n a t i o n

b l o c k s a l o n g range i n t e r - o r i n t r a - s t r a n d c r o s s l i n k from
o c c u r r i n g , probably by s t a b i l i z i n g and s t i f f e n i n g the DNA duplex.
This e f f e c t would keep the duplex from c o l l a p s i n g and compacting
the DNA.
Ethidium Switches the Nuclease-Detectable Binding S i t e s o f cis-DDP

on DNA
In examining f u r t h e r t h i s e f f e c t of ethidium, we found that

the exonuclease I I I method described above detected d i f f e r e n t
cis-DDP b i n d i n g s i t e s on a DNA molecule of known sequence when
p l a t i n a t i o n was c a r r i e d out i n the presence of ethidium (36).
The o r i g i n a l exonuclease I I I experiment (30) had presented us
with an apparent anomaly. Although cis-DDP b i n d i n g to ( d G ) s i t e s n
(n = 2,3>5) i n the 165 base p a i r DNA molecule was detected t>y the
enzyme, a 5^-(dG),-dC-(dG) -3^ sequence seemed to have l i t t l e
9
bound platinum. Tnis s i t e i s l o c a t e d only 29 bases from the 3^

end, so i t s proximity to the o r i g i n o f exonuclease I I I d i g e s t i o n
coupled with i t s extensive run of adjacent guanines l e d us to
expect intense bands on sequencing g e l s corresponding to exo-
nuclease I I I stopping p o i n t s caused by p l a t i n a t i o n . Lanes 1 and 8
i n F i g u r e 7 show, however, that only weak bands appear i n t h i s
r e g i o n of the g e l . But i f ethidium i s present i n the p l a t i n a t i o n
r e a c t i o n mixture, a dramatic change i n the g e l p a t t e r n i s ob-
served. Lanes 2-5 and 9-12 i l l u s t r a t e the e f f e c t of i n c r e a s i n g
amounts of ethidium on the i n t e n s i t y of the bands i n the G^-C-Gp
r e g i o n . These bands become the most intense i n the autoradiograph
due to p l a t i n a t i o n .
A higher r e s o l u t i o n g e l , Figure 8, shows an i n t e r e s t i n g e f f e c t
of t h i s ethidium t i t r a t i o n . The f i r s t bands to appear are those
due to p l a t i n a t i o n of the 3^-(dG)p s i t e ; the (dG)g sequence shows
l i t t l e d e t e c t a b l e p l a t i n a t i o n at the lowest l e v e l o f ethidium. As
the amount of ethidium i n the r e a c t i o n mixture i s i n c r e a s e d , the
(dG)^ s i t e shows more p l a t i n a t i o n , and p l a t i n a t i o n at the ( d G ^
s i t e appears r e l a t i v e l y to decrease.

TULLIUS ET AL. Structural Chemistry of Pt-DNA Adducts
Figure 7. Autoradiograph of an 8% polyacrylamide/7 M urea electrophoresis gel

showing the effect of ethidium bromide on exonuclease Ill-detected cis-DDP binding
sites on a 165-base-pair DNA molecule.
Key to cis-DDP r : lanes 1-5,0.01; and lanes 8-12, 0.05. Key to ethidium bromide r : lanes 1 and
f f
8, 0; lanes 2 and 9, 0.012; lanes 3 and 10, 0.057; lanes 4 and 11, 0.12; and lanes 5 and 12, 0.
Lanes 6 and 13 show Maxam-Gilbert guanine-specific reaction products for sequencing. Lane
7 contains products of exonuclease III digestion of unplatinated, end-labeled 165-base-pair DNA.
Platination was carried out for 3 h at 37 °C. Electrophoresis was for 2 h at 1800 V (36).

Figure 8. Autoradiograph of an 8% polyacrylamide/7 M urea electrophoresis gel

showing the effect of ethidium bromide on exonuclease Ill-detected cis-DDP binding
sites on a 165-base-pair DNA molecule.
Key to cis-DDP r : lanes 1-5, 0.01; and lanes 8-12, 0.05. Key to ethidium bromide t : lanes
f t
1 and 8, 0; lanes 2 and 9, 0.012; lanes 3 and 10, 0.057; lanes 4 and 11, 0.12; and lanes 5 and
12, 0.23. Lane 6 shows Maxam-Gilbert guanine-specific reaction products for sequencing. Lane
7 contains products of exonuclease HI digestion of unplatinated, end-labeled 165-base-pair
DNA. Platination was carried out for 3 h at 37 °C. Electrophoresis was for 4 h at 1800 V (36).

What i s the reason for the effect of ethidium on the sites

where exonuclease III detects platination? It i s not simply due
to increased platinum binding in the presence of ethidium; the
results presented i n Figure 2 rule out this explanation. We think
that this experiment shows that cis-DDP binding i s sensitive to
the local structure of DNA. We consider two limiting cases. The
f i r s t i s that cis-DDP might bind only rarely to the Gg-C-G site 2
in the absence of ethidium, perhaps because the structure of the

helix i s unsuitable. Ethidium then would alter the DNA structure
such that this site becomes a good binding site for cis-DDP. The
second case i s that cis-DDP might bind to the G^-C-Gp sequence to
the same extent i n the presence or absence of ethidium, but the
mode of binding i s different i n these two circumstances. Without
ethidium, cis-DDP might bind in a manner to which exonuclease III
i s insensitive, so the enzyme detects no platinum binding. With

ethidium in the platination mixture, however, cis-DDP could now
bind i n a way which w i l l stop exonuclease III digestion, so that
intense bands are seen i n the sequencing gel. We are presently
attempting to determine which of these explanations i s correct.
More important, though, are the potential implications of
these results for the mechanism of synergism i n combination chemo-
therapy. We have shown that one drug can alter the biochemically
detectable binding site of another drug. This rationalization at
the molecular level i s i n contrast to the more common explanation
of synergism as resulting from action of two drugs at different
phases in the c e l l cycle.
Amine Ligands Are Not Lost From Platinum Upon Binding to DNA
There i s now considerable evidence from work described i n this

paper, others in this volume (57), and recent NMR papers (38,39,
40), that cis-DDP reacts i n a bifunctional manner with DNA. An
interesting question i s whether cis-DDP can interact with DNA
using more than two of i t s coordination sites. Hydrogen bonding
between the protons on the ammine ligands and oxygen atoms of the
DNA phosphate backbone i s one possibility. Another i s that, after
hydrolysis of one or both chloride ligands and subsequent binding
of platinum to a DNA base, the trans l a b i l i z i n g a b i l i t y of the
heterocyclic purine or pyrimidine ring nitrogen atom attached to
the PtiNH™)^ fragment would promote displacement of an ammine
ligand. As a result, a third and possibly a fourth coordination
site could be made available for platinum-DNA binding (41,42).
This mechanism, i n which two sites i n addition to the easily
hydrolyzable chloride sites are opened up for possible metal-DNA
coordination, i s not possible for trans-DPP and would explain i n
terms of ligand substitution kinetics the greater efficacy of c i s -
versus trans-DPP as an antitumor drug.
Evidence to support this mechanism comes from studies (45)
showing that [Pt(NIL) CA]CA can crosslink poly(A) bifunctionally.
Such a phenomenon couTLd only occur i f one or more of the ammine

l i g a n d s were l a b i l i z e d . P r e l i m i n a r y experiments c a r r i e d out i n

our l a b o r a t o r y (44) i n d i c a t e d the l o s s of two ammonia molecules
per molecule of cis-DDP bound to DNA at very low b i n d i n g l e v e l s .
Mass spectroscopic s t u d i e s a l s o suggested l o s s of ammonia but t h i s
r e s u l t could have been due to the c o n d i t i o n s under which the mass
spectrum was obtained ( 4 5 ) · I n d i r e c t support f o r an ammonia r e -
lease mechanism comes from the existence of cis-DDP bound to DNA
which i s r e s i s t a n t to removal by cyanide i o n ( 2 5 , 4 6 ) . The
i n a b i l i t y of cyanide to remove platinum may be explained by
c o o r d i n a t i o n of platinum to DNA through three or four Pt-DNA
bonds.
When d i c h ^ j ^ e t h y l e n e d i a m i n e p l a t i n u m i l l ) , [Pt(en)CFp]7 doubly
m 4
labeled with ^ P t and C , was allowed to react w i t h nucleo-
t i d e s , both l a b e l s remained together ( 4 7 ) · Subsequent work (48)
revealed t h a t , when doubly l a b e l e d [ P t t e n ) C £ J was i n j e c t e d i n t o

2
tumor bearing mice, the two l a b e l s became unequally d i s t r i b u t e d

w i t h respect to d i f f e r e n t biochemical f r a c t i o n s . The former r e -
s u l t does not preclude ammonia r e l e a s e from cis-DDP s i n c e e t h y l -
enediamine c o o r d i n a t i o n i s s t a b i l i z e d by the c h e l a t e e f f e c t .
Moreover, r e a c t i o n of cis-DDP w i t h DNA may d i f f e r from i t s
r e a c t i o n s w i t h n u c l e o t i d e s because a platinum bound to a guanine
base i n a region of l o c a l l y high guanine content i n DNA could
encounter a l o c a l l y very high c o n c e n t r a t i o n of other guanine bases
r e s u l t i n g i n a "DNA c h e l a t e e f f e c t . "
In order to determine whether or not monodentate amine l i g a n d s
could be released from a platinum complex upon b i n d i n g to DNA, we
studied the r e a c t i o n of ^C-labeled c i s - t P t C & ^ N l U C H - ^ ] w i t h T7
and M. l u t e u s DNAs. The compound cis-fPtCfc^NHpCE,)^] has a n t i -
tumor a c t i v i t y ( 4 9 , 5 0 ) w i t h a therapeutic index 15$ 1;hat of c i s -
DDP. A f t e r platinum i n c u b a t i o n followed by d i a l y s i s to remove
unbound reagents the amount of bound platinum was assayed by
atomic absorption spectroscopy (AAS) and the amount of methylamine
l i g a n d on the DNA was determined s e p a r a t e l y by l i q u i d s c i n t i l l a -
t i o n counting (LSC). From the l e v e l of platinum b i n d i n g d e t e r -
mined by AAS and the r a d i o a c t i v i t y a s s o c i a t e d w i t h the DNA i t i s
p o s s i b l e to determine whether platinum b i n d i n g followed by l o s s of
methylamine has occurred.
Figure 9 shows the r e s u l t of two such experiments. From the
AAS and LSC data i t i s c l e a r t h a t , w i t h i n experimental e r r o r , no
r e l e a s e of methylamine occurs upon b i n d i n g of cis-[PtC& (NHgCHL) ] 2 2
to DNA. N e i t h e r i n c r e a s i n g the amount of platinum complex added

nor i n c r e a s i n g the i n c u b a t i o n time at a f i x e d amount of added
platinum leads to l o s s of the amine l i g a n d s . Experiments c a r r i e d
out w i t h M. l u t e u s as w e l l as T7 DNA show the r e s u l t s to be unaf-
fected by i n c r e a s i n g the G+C content (75# vs 47$, r e s p e c t i v e l y ) of
the DNA. To determine whether DNA-bound platinum that i s r e s i s -
tant to cyanide treatment i s attached through more than two l i n k -
ages, we allowed [ C]cis-[PtC£ (NH CH ) ] to r e a c t w i t h T7
4
2 DNA.
The DNA was then t r e a t e d w i t h NauN as reported p r e v i o u s l y (25) and
b i n d i n g l e v e l s were determined by AAS and LSC. The data show very

TULLIUS ET AL. Structural Chemistry of Pt-DNA Adducts
Figure 9. Plots of r vs. time (top) and r vs. r, (bottom) for the binding of
5 b
14
[ CJcis-[PtCl (NH CH h] to T7 DNA (left) and M. luteus DNA (right). Key: O ,
g s s
r determined by AAS; and Δ , r determined by LSC. In the plots of r vs. r error

6 6 6 ff
bars are 2σ and the calculated best straight lines are drawn through the AAS (—)
and the LSC ( ; data.

c l e a r l y that two moles of amine l i g a n d per mole of platinum remain

w i t h the DNA. These r e s u l t s thus demonstrate that only two Pt-DNA
bonds are r e q u i r e d f o r cyanide r e s i s t a n t p l a t i n a t i o n and p o i n t to
b i f u n c t i o n a l c r o s s l i n k i n g as being most important.
Robins (48), however, has shown t h a t , i n v i v o , the ethylene-
anc w
diamine l i g a n d can be separated from [Ptten)C£p] * © now
address t h i s o b s e r v a t i o n . Although the trans e f f e c t of the purine
or py r i m i d i n e r i n g n i t r o g e n atoms i s not strong enough to l a b i l i z e
the amine l i g a n d trans to i t when c is-[PtCft (NH^CHL) ] and, by 2 2
analogy, cis-DDP and [ P t ( e n ) C j l ] bind to DNA, i t i s p o s s i b l e that

2
other compounds present i n the c e l l could bind to these platinum

complexes to d i s p l a c e the amine l i g a n d s . Sulfhydryl containing
compounds are prime candidates and g l u t a t h i o n e (51)> the most
abundant i n t r a c e l l u l a r s u l f h y d r y l c o n t a i n i n g molecule i n mammalian
c e l l s , i s a major contender. Binding s t u d i e s , as described above,

were c a r r i e d out i n the presence of g l u t a t h i o n e at the p h y s i o l o g i -
c a l l y r e l e v a n t c o n c e n t r a t i o n of 1 mM. This amount of g l u t a t h i o n e
does not d i m i n i s h the amount of platinum bound to DNA. Approxi-
mately 20-30$ of the r a d i o a c t i v i t y due to methlyaminé molecules i s
l o s t , however. This r e s u l t i s not s u r p r i s i n g i n view of. the
recent study of Sadler ( t h i s volume) and p r e v i o u s l y reported work
(16) showing that methionine and methionine c o n t a i n i n g peptides
can l a b i l i z e the ammine l i g a n d s upon b i n d i n g to cis-diamminedi-
chloroplatinum(ll). The i n f r a r e d spectrum and chemical a n a l y s i s
(52) of a 2:1 complex of g l u t a t h i o n e and cis-DDP are c o n s i s t e n t
w i t h the l o s s of the ammine l i g a n d s . Studies are c u r r e n t l y i n
progress to determine whether g l u t a t h i o n e remains bound to p l a t i -
num on the DNA. I n t e r e s t i n g l y , preliminary studies i n t h i s labor-
a t o r y point toward g l u t a t h i o n e enhancing the r a t e at which cis-DDP
a l t e r s the t e r t i a r y s t r u c t u r e of s u p e r h e l i c a l DNA (53).
F i n a l l y , as a word of c a u t i o n , i t should be remembered that
c i s - [ P t C J l ( N H C H - ) ] has only 15Î? of the antitumor a c t i v i t y of
2 2 2
cis-DDP. I t wou\Lcr be very i n t e r e s t i n g indeed i f t h i s d i f f e r e n c e

i n antitumor a c t i v i t y was due to the a b i l i t y of cis-DDP to l i b e r -
ate ammonia upon b i n d i n g to DNA.
B i n d i n g [(dien)PtC&]C& to poly(dG-dC)* poly(dG-dC) F a c i l i t a t e s the

B->Z Conformational T r a n s i t i o n
In the course of studying the e f f e c t s of cis-DDP on DNA

s t r u c t u r e and r e a c t i v i t y the compounds trans-DPP and [ ( d i e n ) P t -
CA]Cit are o f t e n used i n p a r a l l e l c o n t r o l experiments. The trans
isomer possesses the same charge, l i g a n d composition, and b i f u n c -
t i o n a l b i n d i n g a b i l i t y as cis-DDP but i s i n e f f e c t i v e as a drug
owing to i t s d i f f e r e n t stereochemistry. When hydrolyzed, the
compound [(dien)PtCil]Cjl has the same charge as the drug but can
only bind monofunctionally to DNA, s i n c e the other three s i t e s are
blocked by the t r i d e n t a t e d i e t h y l e n e t r i a m i n e (dien) l i g a n d . While
i n v e s t i g a t i n g changes i n the s p e c t r o s c o p i c p r o p e r t i e s of the syn-
t h e t i c polymers poly(dG)» poly(dC) and poly(dG-dC). poly(dG-dC) upon

platinum binding, we observed that the [(dien)PtC&]c& compound

could f a c i l i t a t e the right- to l e f t - handed, R*Z, conformational
transition i n poly(dG-dC)* poly(dG-dC). Interestingly, cis-DDP,
the primary subject of our investigation, was unable to induce or
f a c i l i t a t e the &>Z transition and actually prevented i t from
occurring even under very favorable conditions ( 5 4 ) . The same was
found to be true for trans-DPP.
The changes i n the circular dichroism (CD) spectrum of poly-
e
(dG-dC) poly(dG-dC) which occur upon cis-DDP binding are very
similar to those reported for calf thymus (24) , M. luteus (55.),
and salmon sperm (55) DNAs. Whereas [(dienTPtCfcJCfc has l i t t l e
effect on the CD spectrum of calf thymus DNA ( 2 4 ) , drastic changes
are observed upon the binding of this complex to poly(dG-dC)*poly-
(dG-dC), as shown i n Figure 10. At r^ < 0.1 there i s l i t t l e
change i n the long wavelength part of the spectrum but the 250 nm
band i s reduced in intensity. At r, = 0.1 and above, the positive
absorbance at 295 nm, which i s a snoulder i n the spectrum of the
unmodified polymer, increases dramatically i n intensity up to a
saturation binding level of approximately r^ = 0.33· A plot of
molar e l l i p t i c i t y at 295 nm versus r^ (not shown) reveals a s i g -
moidal shape characteristic of a cooperative transition. The
cooperative nature of the binding and the fact that the saturation
binding level of [(dien)Pt] i s one per three nucleotides i n d i -
cate the formation of a stable, regular structure. This structure
appears to be related to the monofunctional nature of [(dien)Pt]
since neither c i s - nor trans-DPP produce the effect.
The spectral changes noted above are not consistent with the
transition from right(B)- to left(Z)-handed PNA ( 5 6 ) , however.
Under the salt and buffer conditions used i n these experiments we
were unable to induce the B*Z transition i n the absence of ethanol
which lowers the water activity. MaLÇoy et_ a l . (57) could bring
about the transition with [(dien)Pt] alone by u s i M very low
salt buffers and excluding EPTA. When [(dien)Pt] binds to
poly(dG-dC).poly(dG-dC) at r = 0.054 and 0.093, for instance, the
amount of ethanol necessary to bring about the Ζ conformation i s
reduced from 55$ for unmodified polymer to 36$ and 25$, respect
ively (54). Interestingly, samples with r^ values greater than
0.1 and which showed the large positive CP absorption at 295 nm
could not be converted to Z-PNA, even at very high ethanol concen
trations. Moreover, when poly(dG-dC)»poly(dG-dC) i n the Ζ confor
mation (55$ ethanol) was allowed to react with saturating amounts
of [(dien)PtC£]Cil, the CP spectrum showed the large absorbance at
295 nm after removal of ethanol. Therefore, the polymer could not
be "locked into" the Ζ conformation by the platinum reagent.
Independent„jevidence supporting the occurrence of Z-PNA was
obtained by ^ Ρ NMR spectroscopy (56) as shown ^n Figure 11. The
conformational changes induced by [(dien)Pt] are completely
reversible as shown by experiments in which the platinated PNA was
treated with NaCN. Following removal of the platinum the normal
amount of ethanol (55$) was necessary to induce the Ζ confor
mation.

Figure 10. The circular dichroism spectra of poly(dG-dC) · poly(dG-dC) modified

at the indicated levels with [(dien)PtCl]Cl (54).

1» •» •» « » • I •' • I •« • I
2 3 4 5 6 7
ppm
31
Figure 11. P-NMR spectra (left) of a, poly(dG-dC) · poly(dG-dC) in buffer, b t
poly(dG-dC) · poly(dG-dC) modified to r = 0.10 with [(dien)PtCPJCl; and c,

b
sample in b after raising the ethanol concentration to 40%. The arrow designates
the resonance characteristic of Ζ -DNA. Circular dichroism spectra (right) of the
samples in b and c (54).

The ability of [(dien)PtC&]c& to promote the Ζ conformation

could be due to several factors. The addition of a bulky sub
stituent to the N7 atom of guanosine would help rotate the base
from the anti conformation, found in B-DNA, to the syn conforma
tion of Z-PNA. Studies with the carcinogen acetylaminofluorene,
which binds to C8 of guanine, showed i t to induce the Ζ confor
mation through a similar mechanism (58). Moreover, the addition
of a +2 charge to the guanine base would help stabilize the higher
charge density of the Z-DNA helix caused by the increased proxi
mity of the two phosphate chains. This effect would be similar to
that evoked to explain why methylation at N7 of guanine f a c i l i
tates the &>Z transition (59). Finally, examination of space
filling models of Z-DNA shows that [(dien)Pt] bound to N7 of
guanine forms good hydrogen bonding contacts between the amino
protons of the diethylenetriamine ligand and phosphate and cyto-

sine 02 oxygen atoms of the flanking nucleotides.
The N7 atom of guanine is located on the outside of the Z-DNA
helix and, as such, may be more available for platinum binding.
We attempted to test this point by comparing the amount of bound
[(dien)Pt] on poly(dG-dC)* poly(dG-dC) in both the Β and Ζ con
formations. After 24 h and at shorter times the binding levels
were approximately equal for both conformations. This result may
be due to the fact that the kinetics of platinum binding are
determined by the rate of chloride hydrolysis and therefore are
not a function of DNA structure.
Acknowledgment
The work described here was supported under a grant from
the National Cancer Institute, No. CA-,5826, PHS.
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4
P h y s i c o c h e m i c a l a n d S t r u c t u r a l S t u d i e s of the
I n V i t r o Interactions between P l a t i n u m ( I I )
C o m p o u n d s and DNA
J. P. MACQUET, J. L. BUTOUR, and N. P. JOHNSON

Laboratoire de Pharmacologie et de Toxicologie Fondamentales, CNRS,
205, Route de Narbonne, 31400 Toulouse, France
Perturbations of DNA secondary structure

and stability induced by c i s - [ P t ( Ν Η ) C l ] (cis-
3 2 2
DDP), trans-DDP and [Pt(dien)Cl]Cl at low levels

of DNA binding (r ) are summarized and preliminary
b
characterization of the corresponding platinum

-DNA adducts is presented. For r ≤0.01 both cis
b
-andtrans-DDPform interstrand crosslinks, shorten

the DNA and exclude stoichiometric amounts of
intercalating agents. However, cis-DDP destabilizes
DNA at this r while trans-DDP? stabilizes the
b
polymer and only the cis isomer causes an increase

in the circular dichroism of DNA. [Pt(dien)Cl]Cl
does not form crosslinks ; it changes neither the
length of DNA, the CD spectra nor the intercalation
of ethidium bromide. Fixation of [Pt(dien)Cl]Cl
stabilizes DNA at r ≤0.01. Two major products
b
of the reaction of cis-DDP with salmon sperm DNA

have been isolated and both contain guanine.
7
[Pt(dien)Cl]Cl fixes primarily at Ν of guanine
7
for r < 0.1 ; in addition, fixation at N of
b
1 7
adenine for r > 0.1 and at N and Ν of adenine
b
for 0.3 < r < 0.4 was observed.

b
Although cis-DDP binds covalently to proteins, RNA and

other nucleophiles in the cell (V), it is fixation on DNA which
seems to be responsible for the antitumor activity of this
compound (2). The toxicity of cis-DDP seems to be associated with
an inhibition of DNA synthesis rather than an inhibition of RNA
or protein synthesis (3, 4_, 5) ; similarly, mammalian cells (6)
and bacteria (_5, _7, 8) deficient in DNA repair are more sensitive
to killing by cis-DDP. This argument implies that inhibition of
DNA synthesis is responsible for the general cytotoxicity of cis-
DDP, but it does not explain why tumor cells are killed more read-
0097-6156/83/0209-0075$07.50/0

i l y than healthy ones without i n v o k i n g a s p e c i a l s e n s i t i v i t y of

tumor c e l l s , such as l a c k of DNA r e p a i r c a p a b i l i t y ( 9 ) . The
antitumor a c t i v i t y of a s e r i e s of platinum compounds i s g e n e r a l l y
c o r r e l a t e d w i t h t h e i r a b i l i t y to perturb v a r i o u s f u n c t i o n s of DNA
such as enhancement of mutagenesis (10, 11, 12), i n h i b i t i o n of DNA
s y n t h e s i s (8, 13, 14, 15) and i n d u c t i o n of l y s o g e n i c b a c t e r i a (10,
—
16).
Platinum compounds without antitumor a c t i v i t y (17) such as
trans-Wi? and [ P t ( d i e n ) C l ] C l (Figure 1) c o v a l e n t l y b i n d t o DNA
in vivo. Several s t u d i e s have compared the b i o l o g i c a l e f f e c t s
which r e s u l t when equal amounts of these three platinum compounds
are f i x e d on DNA ( t y p i c a l l y rb = 10~4-10""6). Cis-DDP i s 5-10 times
more t o x i c toward E. eoli (8) and mammalian c e l l s (J_, JJ_) than
trans-DDP. The r e l a t i v e t o x i c i t y i s c o r r e l a t e d w i t h the a b i l i t y
of these two isomers to i n h i b i t DNA r e p l i c a t i o n (<8, Γ3, J4_). The

cis isomer i s r e p a i r e d more e f f i c i e n t l y by E. coli (8) and i s a t
l e a s t 750 times more mutagenic i n mammalian c e l l s (11) than the
trans isomer. The compound [ P t ( d i e n ) C l ] C l binds c o v a l e n t l y to the
DNA of E. coli and seems not to be r e p a i r e d ; nevertheless t h i s
compound does not i n h i b i t DNA s y n t h e s i s or k i l l the b a c t e r i a ( 8 ) .
Repair of platinum compounds by E. coli may be under the c o n t r o l
of the SOS system ; cis-DDP induces 5-10 times more recA p r o t e i n
i n t r e a t e d E. Coli t h a n an e q u a l amount o f t r a n s - D P P o r
[ P t ( d i e n ) C l ] C l f i x e d on the DNA (18_). I t seems t h a t d i f f e r e n t
modes of f i x a t i o n on DNA are r e s p o n s i b l e f o r the d i f f e r e n t muta
g e n i c i t y , t o x i c i t y and DNA r e p a i r of these platinum complexes.
These r e s u l t s suggest t h a t the antitumor a c t i v i t y of p l a t i n u m ( I I )
compounds may a l s o depend on the formation of p a r t i c u l a r platinum-
DNA l e s i o n s .
To t e s t t h i s hypothesis we have measured the p e n e t r a t i o n and
DNA b i n d i n g i n LI210 leukemia c e l l s g r a f t e d i n mice which have
r e c e i v e d e q u i t o x i c doses of the drugs (Table I ) . [Pt(en)2]Cl2 has
f o u r platinum-nitrogen bonds and should not r e a c t w i t h DNA ; f o r
t h i s negative c o n t r o l , platinum f i x a t i o n on the DNA was not detec
t a b l e (rb < 5x10*6). R e s u l t s i n Table I show t h a t cis-DDP, trans-
DDP and [ P t ( d i e n ) C l ] C l enter the cancer c e l l and b i n d to i t s DNA,
but o n l y cis-DDP i s a n t i t u m o r a l . F i x a t i o n of 25 times more trans-
than cis-DDP on the DNA of LI210 leukemia c e l l s does not cause
antitumor a c t i v i t y f o r the trans isomer. From b a c t e r i a l s t u d i e s
mentioned above, a 25 f o l d excess of trans-Wi? on the DNA ought
to i n h i b i t DNA s y n t h e s i s more e f f i c i e n t l y than the smaller amount
of bound cis-DDP suggesting t h a t , f o r LI210 leukemia, i n h i b i t i o n
of DNA s y n t h e s i s may not be the mechanism of antitumor a c t i v i t y .
I f DNA i s the c e l l u l a r t a r g e t of these drugs, the antitumor a c t i v
i t y of cis-DDP nevertheless appears to r e q u i r e a platinum-DNA
lesion with a particular structure.

MACQUET ET AL. Pt(II) Compounds and DNA In Vitro
H o C _ CHo
H N
3 Cl H N Cl HN NH 2
3
\ / \ / HÇ
2 Pt
Pt
Pt
/ \ / \ Η 0
2 χ
Cl
Cl NH 3 HN 2
HN3 Cl
cis-|Pt(NH ) CI ]
3 2 2 trans-^t(NH ) CI ] 3 2 2
[pt(dien)Cl]d
Figure 1. Structures of the three platinum compounds.

TABLE I . PENETRATION AND DNA BINDING of P t ( I I )

COMPOUNDS IN L1210 LEUKEMIA CELLS (19).
10*> c e l l s were g r a f t e d i . p . i n DBA/2 mice on day 0 and animals
were t r e a t e d by i . p . i n j e c t i o n w i t h the maximum n o n - t o x i c dose
(LDo) of the drug on day 3. For the p e n e t r a t i o n and DNA b i n d i n g
experiments, the animals were s a c r i f i e d 2 h a f t e r treatment, the
leukemia c e l l s were removed and washed u n t i l no platinum was de
t e c t e d i n the supernatant a f t e r c e n t r i f u g a t i o n . The c e l l s were
counted and the p l a t i n u m c o v a l e n t l y f i x e d per c e l l was determined
by atomic a b s o r p t i o n . DNA was i s o l a t e d from these c e l l s by phenol
e x t r a c t i o n and the rb was determined. For the antitumor e x p e r i
ments, animals were g r a f t e d w i t h 10^ LI210 c e l l s and observed
f o r 30 days. The r e s u l t s are expressed as the mean s u r v i v a l time
of t r e a t e d animals (T) d i v i d e d by the mean s u r v i v a l time of non-
t r e a t e d animals (C). T/C ^ 125 % i s considered as s i g n i f i c a n t

antitumor a c t i v i t y .
Pt f i x e d
Compound LDo T/C to c e l l s rb
(ymol/kg) (%) (% treatment
dose)
c?is-DDP 30 200 0.4 1/30, 000

trans-ΌΏ? 167 116 1.5 1/1,200
[Pt(dien)Cl]Cl 203 100 0.1 1/10,000
[ P t ( e n ) 2 ] C l 2 :> 2,600 98 0,02 a
< 1/200,000
a f t e r 4 washings some f r e e compound i s s t i l l present due to the

b
l a r g e amount i n j e c t e d ; l i m i t of d e t e c t i o n .
I f the f i x a t i o n of P t ( I I ) compounds on DNA i s r e s p o n s i b l e

f o r the v a r i o u s b i o l o g i c a l a c t i v i t i e s of these compounds, then
the r e l a t i v e potencies of cis-DDP, trans-ΌΌ? and [ P t ( d i e n ) C l ] C l
must be a consequence of t h e i r modes of b i n d i n g on the DNA r a t h e r
than the s t o i c h i o m e t r y of f i x a t i o n . The o b j e c t i v e of t h i s paper
i s to determine whether or not the d i f f e r e n t b i o l o g i c a l a c t i v i t i e s
of these compounds are a s s o c i a t e d w i t h p a r t i c u l a r deformations of
DNA. To accomplish t h i s goal we w i l l f i r s t t r y to q u a n t i t a t e the
a l t e r a t i o n s of DNA conformation and s t a b i l i t y by these compounds
i n vitro at the lowest p o s s i b l e rb- The l i m i t of d e t e c t i o n f o r
most of the physicochemical techniques which w i l l be considered
i s rb = 0.005. The l e v e l of DNA b i n d i n g which produces most b i o
l o g i c a l e f f e c t s i s one or two orders of magnitude l e s s and so we
w i l l emphasize r e s u l t s from s t u d i e s w i t h 0.005 < r ^ < 0.05, Since
the k i n e t i c s of f i x a t i o n of these compounds are d i f f e r e n t and the
amount of p l a t i n u m bound to DNA w i l l change i f s i d e r e a c t i o n s are
p o s s i b l e , we w i l l l i m i t our d i s c u s s i o n t o physicochemical s t u d i e s
which have measured the platinum on the DNA. G e n e r a l l y p l a t i n u m
c o n c e n t r a t i o n has been determined by f l a m e l e s s atomic a b s o r p t i o n
or by u s i n g r a d i o a c t i v e 195mpt, both which have a l i m i t of detec
t i o n of rb = l-5xlO""6. Secondly, some p r e l i m i n a r y r e s u l t s from our

4. MACQUET ET AL. Pt(II) Compounds and DNA In Vitro 79
l a b o r a t o r y concerning the i s o l a t i o n and c h a r a c t e r i z a t i o n of the

platinum-DNA adducts formed by these compounds w i l l be presented.
F i n a l l y * we w i l l d i s c u s s b r i e f l y how the observed s t r u c t u r e s o f
the platinum-DNA l e s i o n s may be r e s p o n s i b l e f o r the a l t e r e d
s t r u c t u r e and s t a b i l i t y of DNA in vitro.
ALTERATIONS OF DNA SECONDARY STRUCTURE AND STABILITY BY P t ( I I )

COMPOUNDS, r < 0.05 b
Before examining, the conformation and s t a b i l i t y of the

platinum-DNA complex formed in vitro, i t i s worth b r i e f l y c o n s i d
e r i n g the mechanism of the r e a c t i o n between P t ( I I ) chloroammines
and DNA. C i s - [ P t ( N H ) 2 C l 2 l does not r e a c t w i t h DNA but the aquated
3
+ 2 +
forms, [Pt(NH3)2(H20)Cl] and [ P t ( N H ) ( H 2 0 ) l , bind c o v a l e n t l y
3 2 2
to the p o l y n u c l e o t i d e (20) and the o v e r a l l r e a c t i o n l i b e r a t e s

2 CI* (20.
[Pt(NH )2Cl ] 3 2
-Cl" I t +Cl"
[Pt (NH ) ( H 0 ) C l ]
3 2 2 [Pt (NH ) C1 (OH) ]
3 2
-ci JJ +C1~ -Cl" J J +C1

+
-H
[Pt ( N H ) (H 0) 1 " ~ ~ = ^ [Pt ( N H ) ( H 0 ) (OH) ] ^ ~ " = Ξ [Pt(NH ) (OH) 1
3 2 2 2
+
3 2 2
+
3 2 2
I f aqueous s o l u t i o n s of ois- o r trans-DDP are allowed t o e q u i l i

b r a t e , the k i n e t i c s of the r e a c t i o n s of these aquated species
w i t h DNA can be measured. At low r ^ the r e a c t i o n i s p s e u d o - f i r s t -
order w i t h respect t o platinum c o n c e n t r a t i o n . For cis-DDP, the
h a l f - l i f e of the r e a c t i o n of the diaquo species w i t h 10"4 M DNA
at 25°C, pH - 5-6 i s 0.8 min and the monoaquo species, c i s - and
t r a n s - [ P t ( N H ) ( H 2 0 ) C l ] J have h a l f - l i v e s of 6 h and 2 h r e s p e c t i
3 2
v e l y . The forms [Pt(NH )2Cl(OH)] and [ P t ( N H ) ( 0 H ) ] do not r e a c t

3 3 2 2
+
w i t h DNA and c i s - [ P t ( N H ) 2 ( H 2 0 ) ( O H ) ] r e a c t s w i t h the same k i n e t i c s
3
as the diaquo form (20). However, i f f r e s h l y d i s s o l v e d s o l u t i o n s

of platinum compounds are added t o the DNA, formation of the
monoaquo species i s the r a t e l i m i t i n g step (22). F i g u r e 2 shows
the r e a c t i o n s of f r e s h s o l u t i o n s of the three compounds w i t h DNA
at 37°C. Under these c o n d i t i o n s the h a l f - l i v e s of the r e a c t i o n s
were 3.9, 2.5 and 0.65 h f o r cis-DDP, trans-WV and [ P t ( d i e n ) C l ] C l
r e s p e c t i v e l y (19).
I t has been proposed that P t ( I I ) compounds b i n d t o DNA i n
three d i f f e r e n t ways, c i s - b i d e n t a t e , trarcs-bidentate and monoden-
t a t e , and that the compounds i n F i g u r e 1 are r e p r e s e n t a t i v e of
these three c l a s s e s of b i n d i n g (23). We w i l l now consider the
e f f e c t o f t h e f i x a t i o n o f t h e s e compounds in vitro,
0.005 Κ rt> < 0.05, on the s t r u c t u r e and the s t a b i l i t y of DNA.

TIME (hours)
Figure 2. Kinetics of the reaction between salmon-sperm DNA and cis-DDP (Φ),
trms-DDP (M), or [Pt(dien)Cl]Cl (A); U = 0.2.
Aliquots of cis- or trans-Z)DP were taken at different times and added to a solution of EtdBr.
Fluorescence measurements were performed as previously described (32), and platinum bound
to DNA was determined from standard curve of η vs. fluorescence. For [Pt(dien)Cl]Cl, aliquots
were taken at different times, and the reaction was stopped by increasing the Cl~ concentration
to 1 M. These samples were then passed through Sepharose-6B columns in order to remove the
unreacted platinum, and the concentration and the r» of the platinum-DNA complexes were
determined.

F i x a t i o n of P t ( I I ) compounds on DNA does not cause c h a i n

breaks. A s i n g l e c h a i n break i n s u p e r c o i l e d PM2 DNA converts the
polymer from a compact c l o s e d c i r c u l a r conformation t o a r e l a x e d
open c i r c u l a r form. This conformational change i s an extremely
s e n s i t i v e measure of c h a i n s c i s s i o n and i t has been used to d e t e c t
the n i c k i n g of DNA by P t ( I I ) compounds (24, 25, 26), Table I I
shows the percentage of n i c k e d PM2 DNA molecules which were
observed by e l e c t r o n microscopy a f t e r f i x a t i o n of v a r i o u s amounts
of the three p l a t i n u m compounds on the DNA (25), I t can be seen
that below rb = 0.1 none of the compounds cause c h a i n breaks i n
the DNA.
TABLE I I . RATIOS BETWEEN SUPERCOILED AND NICKED

PM2 DNA MOLECULES IN THE DIFFERENT PLATINUM-DNA
COMPLEXES VISUALIZED BY ELECTRON MICROSCOPY.
Supercoiled Nicked
Compound r molecules molecules
b
(%) (%)
DNA 0 81(78-84) 19
cis-Pt(NH ) -DNA
3 2 78(72-84) 22
10 73(69-77) 27
-2
78(77-79) 22
IS- 1
48(42-54) 52
trans-?t(NH^) ~DNA 2 77(75-79) 23

10
80(78-82) 20
-2 21
79(76-82)
IS-' 82(80-84) 18
Pt(dien)-DNA 82(79-85) 18
10 80(77-83) 20
-2
78(74-82) 22
10 1
69(68-70) 31
Values i n parentheses g i v e the range. 200 molecules of each

complex were v i s u a l i z e d at d i f f e r e n t p l a c e s on the g r i d .
Cis- and trans-DPP but not [ P t ( d i e n ) C l ] C l form i n t e r s t r a n d

c r o s s l i n k s . The best c h a r a c t e r i z e d of the c£s-Pt(NH3)2-DNÀ l e s i o n s
i s the i n t e r s t r a n d c r o s s l i n k which has been deduced from the
appearance of h i g h molecular weight DNA i n denaturing c o n d i t i o n s
(27), enhanced thermal r e n a t u r a t i o n (22) and a d i m i n i s h e d r a t e o f
a l k a l i n e e l u t i o n (28). I t i s evident from thermal r e n a t u r a t i o n

experiments (Figure 3) that c i s - and trans-DPP, but not

[ P t ( d i e n ) C l ] C l form i n t e r s t r a n d c r o s s l i n k s p e r m i t t i n g the reforma-
t i o n of the double h e l i c a l s t r u c t u r e of heat-denatured DNA which
i s subsequently cooled (25). Recently Roberts (29) has q u a n t i t a t e d
the number of i n t e r s t r a n d c r o s s l i n k s formed by "7s-DDP f o r
5
l x l O " < rb < 5xlO~4. Exposure of e i t h e r p u r i f i e d DNA or DNA i n
mammalian c e l l s to c i s - P P P f o r 2 hours produced about 1 c r o s s l i n k
per 150 bound p l a t i n u m molecules as determined by the percentage
of h i g h molecular weight DNA observed i n a l k a l i n e sucrose g r a d i -
ents. I n both cases the c r o s s l i n k i n g frequency increased t o about
1/30 when the platinum-DNA complex was s t o r e d i n b u f f e r f o r
24 hours a f t e r treatment. Assuming a c r o s s l i n k i n g frequency of
1/30, the data i n F i g u r e 3 i n d i c a t e s t h a t a s i n g l e c r o s s l i n k per
500-1000 base p a i r s i s necessary f o r complete r e n a t u r a t i o n of the
DNA.
Cis- and trans-DPP but not [ P t ( d i e n ) C l ] C l shorten DNA. The

length of PM2 DNA which has been t r e a t e d by the three compounds
has been measured u s i n g e l e c t r o n microscopy (23). This technique
appears to be more s e n s i t i v e than m i g r a t i o n d u r i n g g e l e l e c t r o -
phoresis which d i d not change a p p r e c i a b l y f o r r ^ < 0.05 (30). In
c o n t r a s t , f o r r ^ ^ 0.05 e l e c t r o n microscopy r e v e a l e d a l i n e a r
r e l a t i o n s h i p between the decrease i n the length of PM2 DNA and the
q u a n t i t y of bound cis- or trans-PPP. F i x a t i o n of [ P t ( d i e n ) C l ] C l
d i d not change the s i z e of the DNA (Figure 4 ) . Judging from the
slopes of the curves i n f i g u r e 4, f i x a t i o n of a s i n g l e cis-DDP
shortened the DNA by 17 A w h i l e each trans-PPP decreased the
o
l e n g t h by 10 A. A l t e r a t i o n s of the v i s c o s i t y (25) of DNA as a

f u n c t i o n of rb (Figure 5) are remarkably s i m i l a r t o the change i n
the l e n g t h of DNA observed u s i n g e l e c t r o n microscopy. The drop i n
v i s c o s i t y i s not caused by c h a i n breaks and i s c o n s i s t e n t w i t h the
hypothesis that these compounds shorten DNA,
F i x a t i o n of cis- and trqns-PPP, but not [ P t ( d i e n ) C l ] C l ,

d i s r u p t s DNA basestacking. Spectroscopic s t u d i e s such as UV
hyperchromism and c i r c u l a r d i c h r o i s m r e f l e c t the immediate
environment of the base and are s e n s i t i v e probes of conformational
changes i n v o l v i n g , at most, s e v e r a l n u c l e i c a c i d bases. Between
0.01 < r < 0.05 cis-DDP increased the p o s i t i v e CD band of DNA at
b
270 nm by about 40 % (Figure 6) u n l i k e trons-PPP and [ P t ( d i e n ) C l ] C l

which had no e f f e c t (31). UV hyperchromism was not observed a f t e r
f i x a t i o n of any of the three compounds i n t h i s range of rb (23),
A more d i r e c t measurement of the d i s r u p t i o n of base-base
i n t e r a c t i o n s by f i x a t i o n of these molecules i s t h e i r a b i l i t y t o
prevent the a s s o c i a t i o n of EtdBr w i t h DNA. EtdBr i n t e r c a l a t e s
between base p a i r s and the number of i n t e r c a l a t e d molecules can be
determined u s i n g f l u o r e s c e n c e spectroscopy (32). C i s - and trans-PPP
each prevented the i n t e r c a l a t i o n of one EtdBr molecule per
p l a t i n u m bound to DNA w h i l e [ P t ( d i e n ) C l ] C i had no e f f e c t (Figure 7),

MACQUET ET A L . Pt(H) Compounds and DNA In Vitro
ol , , , _
0 0.0125 0.025 0.050
r
b
Figure 3. The percentage of renaturation of cis-DDP (Φ), trans-DDP (M), and
[Pt(dien)Cl]Cl (A) at low r . 6
Platinum-T7 DNA complexes (25 ng/mL) in NaClO (10 mM) were denatured by raising the
t
temperature to 100°C and then renatured by decreasing the temperature at a rate of 1 °C/min
to 25 °C.
% renaturation = (A — Α) χ 100/(A — Ao)
f f
where A is the absorbance at 260 nm before denaturation, At is the absorbance at 260 nm corre
0
sponding to the maximum of hyperchromicity, and A is the absorbance at 260 nm after

renaturation.
LPNA-Pt
LDNA
50l ι ι ι
0 0.0125 0.025 0.050
r
b
Figure 4. Shortening of PM2 DNA as a function of r . Key: · , cis-DDP; M, 6
trans-DDP; and A , [Pt(dien)Cl]Cl.

Nicked circular PM2 DNA (0.02 mg/mL) was incubated in NaClO (10 mM) with platinum t
compounds at 20 °C in the dark for 8 d. After dialysis against NaClO (10 mM) and platinum t
determination by atomic absorption spectrophotometry, the complexes were absorbed on

carbon-coated copper grids and observed using a Philips 301 electron microscope. The average
length of control DNA (LDNA) and of platinum-DNA complexes (LDNA-PI) were determined
from an observation of at least 60 molecules.

Figure 6. Circular-dichroism spectra of salmon sperm DNA alone and complexée

with As-DDP {top), trms-DDP (middle), or [Pt(dien)Cl]Cl (bottom) for r = 0.05. 6

MACQUET ET AL. Pt(II) Compounds and DNA In Vitro
Figure 7. Number of EtdBr binding sites (η) in salmon sperm DNA complexed
with cis-DDP (Φ), trans-DDP (M), or [Pt(dien)Cl]Cl (A) at low r . Scatchard6
plots of EtdBr binding to different platinum-DNA complexes were used to deter

mine n.

For r b < 0.01, cis-ΌΌ? d e s t a b i l i z e s DNA w h i l e trans-W? and

[ P t ( d i e n ) C l ] C l s t a b i l i z e DNA. A l t e r a t i o n o f the m e l t i n g tempera-
t u r e o f DNA by f i x a t i o n of the three platinum compounds a t low r ^
i s shown i n F i g u r e 8. I n a l l cases t h e changes i n T were a l i n e a r
m
f u n c t i o n o f the q u a n t i t y o f bound platinum. For the f i x a t i o n of 5

p l a t i n u m per 1000 bases (30 platinum per T7 DNA molecule),
trans-ΌΌΈ and [ P t ( d i e n ) C l ] C l increased the m e l t i n g temperature
by 0.7°C and 1.7°C r e s p e c t i v e l y w h i l e e^s-DDP decreased the T by m
1.7°C (25).
S i n g l e stranded regions o f DNA are not detected f o r gis-DDP,

trans-ΌΌ? o r [ P t ( d i e n ) C l ] C l f o r r < 0.025. I n order t o see i f
K
s i n g l e stranded DNA i s formed by the f i x a t i o n of P t ( I I ) compounds

on DNA we have measured the nuclease SI d i g e s t i o n o f salmon sperm
DNA which had been t r e a t e d a t low r ^ by the three compounds i n

F i g u r e 1. The r e a c t i o n was f o l l o w e d by the r e l e a s e o f a c i d s o l u b l e
UV absorbing m a t e r i a l , l i m i t o f d e t e c t i o n 1 % d i g e s t i o n . Nuclease
SI from Aspergillus oryzae d i d not d i g e s t DNA which had been
t r e a t e d w i t h any of the compounds below r ^ = 0.025. At r ^ = 0,05
cùs-ΏΏ? but not trans-W? or [ P t ( d i e n ) C l ] C l induced s i n g l e
stranded regions which were s e n s i t i v e t o t h i s enzyme. At h i g h e r
rfc values SI nuclease was able t o p a r t i a l l y d i g e s t DNA which had
been t r e a t e d by a l l three compounds. I n a l l cases the extent o f
SI nuclease d i g e s t i o n increased n o n l i n e a r l y as a f u n c t i o n o f r ^ .
I t appears t h a t the formation o f s i n g l e stranded DNA may be a
cooperative phenomenon which r e q u i r e s the p a r t i c i p a t i o n of s e v e r a l
f i x e d platinum (33).
Summary. Physicochemical s t u d i e s of the a l t e r a t i o n o f DNA

conformation and s t a b i l i t y by the f i x a t i o n of <?£s-DDP, trans-WV
and [ P t ( d i e n ) C l ] C l a t 0.005 < r b < 0.05 r e v e a l t h a t each compound
a l t e r s the DNA i n a c h a r a c t e r i s t i c manner. Table I I I represents
β
an attempt t o q u a n t i f y these e f f e c t s f o r rb 0.01.
At t h i s r b , the f i x a t i o n of σ-ùs-OO? excludes one EtdBr
molecule per bound platinum, i m p l y i n g a change i n the conformation
which prevents i n t e r c a l a t i o n . Hydrogen bonding between the DNA
strands i s weakened, as i n d i c a t e d by the decrease i n the m e l t i n g
temperature o f DNA, but i t i s not weakened s u f f i c i e n t l y t o produce
s i n g l e stranded regions which a r e a s u b s t r a t e f o r SI nuclease. ©
F i x a t i o n of a s i n g l e eis-ΌΌΈ on DNA shortens the polymer by 17 A.
The s h o r t e n i n g o f DNA by f i x a t i o n o f cis-WP has been a t t r i b u t e d
to the formation o f microloops caused by a c r o s s l i n k between two
bases f a r apart from each other (23). Conceivably, DNA s h o r t e n i n g
could be the r e s u l t o f noncovalent charge-charge i n t e r a c t i o n s
between the p o s i t i v e platinum-base adduct and a negative phosphate,
but i n t h i s case [ P t ( d i e n ) C l ] C l should a l s o shorten the DNA, which
i s not observed.

4. MACQUET ET AL. Pt(H) Compounds and DNA In Vitro 87
TABLE I I I . CHANGES IN THE CONFORMATION

AND STABILITY OF DNA AFTER FIXATION OF
P t ( I I ) CHLOROAMINES IN VITRO, r - O.Ol. f e
Compound eis-ΌΌ? trans-WV [Pt(dien)Cl]Cl
a
DNA c h a i n b r e a k s 0 0 0
Interstrand crosslinks ,
per bound platinum 1/30 ND 0
a
ΔΤ -2.4°C 1.3°C 3,3°C
m
SI s e n s i t i v e s i n g l e
d
stranded DNA 0 0 0
DNA s h o r t e n i n g
e
(A/Pt) 17 10 0
f
EtdBr/Pt 1 1 0
a. r e f . 25 ; b. r e f . 29, 0.00005 < r b < 0.0004 ; c. r e f , 25,
0.001 < rb < 0.01 ; d. r e f . 33, r < 0.025 ; e. r e f . 23 ;
b
f. number of excluded EtdBr molecules per bound platinum, r e f . 32.
F i x a t i o n of trans-WV on DNA a t rb • 0,01 a l s o prevents the

i n t e r c a l a t i o n of an equimolar q u a n t i t y of EtdBr, As i n the case
of the eis isomer, b i d e n t a t e complexation a t two s i t e s on comple
mentary bases o r between w e l l separated bases c o u l d account f o r
the formation of i n t e r s t r a n d c r o s s l i n k s and microloops r e s p e c t i v e
l y . However, u n l i k e eis-DDP, the trans isomer s t a b i l i z e s r a t h e r
than d e s t a b i l i z e s the DNA at low r ^ . Trans-WV a l s o s t a b i l i z e s
P o l y l : PolyC and, i n t h i s case, the s t a b i l i z a t i o n was decreased
by i n c r e a s i n g the i o n i c s t r e n g t h (34) i m p l y i n g t h a t charge-charge
i n t e r a c t i o n s are l i k e l y r e s p o n s i b l e f o r the s t a b i l i z a t i o n . Trans-
P e 7
DDP binds t o yeast tRNA by a c o v a l e n t bond at N guanine and
hydrogen bonds between an ammine l i g a n d and a phosphate group (35).
Hence, charge-charge i n t e r a c t i o n s , hydrogen bonds o r water bridges
(34) may be r e s p o n s i b l e f o r the s t a b i l i t y of the trans-DDP-DNA
complex r e l a t i v e t o DNA a t low rb«
F i x a t i o n of [ P t ( d i e n ) C l ] C l does not cause c r o s s l i n k s o r micro-
loops because t h i s compound can not make a b i d e n t a t e complex w i t h
DNA. Below rb • 0.1 the Pt(dien)-DNA complex i s more s t a b l e than
DNA, probably because of charge-charge i n t e r a c t i o n s o r hydrogen
bonds between the platinum complex and adjacent n u c l e o t i d e s .
I t i s worth n o t i n g t h a t the a l t e r a t i o n s o f DNA conformation
and s t a b i l i t y f o r rb - 0.01 which have j u s t been d e s c r i b e d are

Figure 8. The melting-temperature variations of platinum-T7 DNA complexes

at low r (AT = T of the complex - T of DNA). Key: · , cis-DDP; M, trans-
b m m m
DDP; and A, [Pt(dien)Cl]CL Melting curves of platinum-T7 DNA complexes (25

μ-g/mL) in NaClO (10 mM) were recorded by measuring the hyperchromicity at
k
260 nm. The rate of temperature increase was 1 °C/min. In these conditions T7
DNA had a T of 73.0 ± 0.2 °C.
m

based on c e r t a i n physicochemical parameters, such as DNA l e n g t h ,

EtdBr i n t e r c a l a t i o n and m e l t i n g temperature, which appear to be
l i n e a r f u n c t i o n s of the amount of bound platinum from rb = 0.05 t o
^ = 0.005, the l i m i t of d e t e c t i o n . I n the next s e c t i o n we s h a l l
see that no new platinum-DNA adducts are formed between
0.0005 < rb < 0.1. Hence, i t seems l i k e l y that the changes i n DNA
s t r u c t u r e and s t a b i l i t y which have been observed f o r rb = 0.01
(Table I I I ) are a l s o present at lower l e v e l s of platinum b i n d i n g .
STRUCTURES OF THE PLATINUM-DNA ADDUCTS
Spectroscopic and c r y s t a l l o g r a p h i c s t u d i e s of platinum-base

complexes give some i n s i g h t i n t o the r e a c t i v i t y of the platinum
compounds and t h e i r p o s s i b l e b i n d i n g s i t e s on DNA. The r e a c t i o n s
of the d i f f e r e n t nucleosides or n u c l e o t i d e s w i t h the c h l o r o and

aquo d e r i v a t i v e s of ois- and trans-ΌΌΈ have been s t u d i e d by UV
spectroscopy (36), raman d i f f e r e n c e spectrophotometry (37) and
high pressure l i q u i d chromatography (38). For both c h l o r o isomers,
the r a t e s of the r e a c t i o n s w i t h v a r i o u s n u c l e i c a c i d monomers
show the f o l l o w i n g trend : GMP > AMP > CMP and dG > dA > dC » T,
The d i c h l o r o and diaquo d e r i v a t i v e s r e a c t s l o w l y w i t h thymidine
and UMP (37) or not at a l l (38, 39).
The s t o i c h i o m e t r y of the platinum-base complex depends on the
Pt:base r a t i o and a l s o on the pH. For i n s t a n c e , i t has been shown
t h a t the ois-diaqxxo species r e a c t s w i t h adenine, adenosine and AMP
to give 1:1, 1:2 and 2:1 (Pt:base) complexes. S i m i l a r r e s u l t s have
been observed f o r the trans isomer except that the 1:2 complexes
w i t h adenosine and AMP were not formed (40). The diaquo and
d i c h l o r o species of ois- and trans-ΌΏ? b i n d to GMP and dGMP t o
give 1:1 and 1:2 complexes as shown by raman spectroscopy (41) and
2+
NMR (42). ££s-[Pt(NH ) (H20)2] forms a 1:2 complex w i t h c y t i d i n e
3 2
or u r i d i n e at a c i d pH whereas under n e u t r a l c o n d i t i o n s the 1:1

complex i s formed (43). I t a l s o r e a c t s w i t h GpG and GpC forming
a b i d e n t a t e complex w i t h the two bases (44).
The platinum b i n d i n g s i t e s on n u c l e i c a c i d bases have been
e l u c i d a t e d by x-ray c r y s t a l l o g r a p h y (45, 46_, 47), NMR (37, 4^, 48,
49) and raman spectroscopy (37, 4J^, 43). The reported sTtes o f
f i x a t i o n of ois-WV are the N? of guanosine, AMP, GMP and GpG
3
(37, _4^, 44, 45, 49) and the N of CMP and c y t i d i n e (43, 47).
f
Trans-ΌΌΈ forms mono and b i s complexes w i t h 5 -GMP at N7 f o r
7
r ^ = 1 and at N* and N at pH > 9 f o r r ^ - 2 (41). I t a l s o r e a c t s
7 3
at N* and N of adenosine and at N of c y t i d i n e (39). The only
c r y s t a l s t r u c t u r e (35) reported f o r a trans-ΏΌ? base complex i s
7
the s t r u c t u r e of yeast tRNA i n which platinum i s bound to N o f
guanine 34. Hydrogen bonds were observed between one of the ammine
groups and 0^ of the same base and between the other ammine and
the three oxygen atoms belonging to the 5'-phosphate of t h i s nu
c l e o t i d e . The main b i n d i n g s i t e s of [ P t ( d i e n ) C l ] C l complexed w i t h
nucleosides and n u c l e o t i d e s as shown by x-ray c r y s t a l l o g r a p h y and
7 7 1
NMR are the N p o s i t i o n of guanine (46, 4J3), N and N of adenine

(48, 49, 51), N of c y t o s i n e (48, 5J_) and N of thymidine (49, 52),

3 3
No evidence f o r b i n d i n g at the phosphate group has yet been dem

o n s t r a t e d even w i t h a l a f g e excess of [ P t ( d i e n ) C l ] C l (48). Judging
from NMR r e s u l t s , [ P t ( d i e n ) C l ] C 1 gives three types of complexes
7
w i t h adenosine or 5'-AMP corresponding to b i n d i n g at N , at N^ or
7 7
at N and N* simultaneously ; N i s the k i n e t i c a l l y p r e f e r r e d
b i n d i n g s i t e (48, 51).
GC base p a i r s appear to be the p r e f e r r e d s i t e of f i x a t i o n of
p l a t i n u m ( I I ) compounds on DNA. This c o n c l u s i o n i s supported by
three types of arguments. F i r s t , the r a t e of f i x a t i o n of cis-DDP
to DNA i s c o r r e l a t e d w i t h i t s GC content (53). Hence, i f <?£s-DDP
i s mixed w i t h two DNAs of d i f f e r e n t GC content, i t w i l l f i x
p r e f e r e n t i a l l y to the DNA w i t h highest percentage of guanine (54,
55). Second, the q u a n t i t y of purine bases which have not reacted
w i t h platinum can be determined by means of paper chromatography

of the depurinated platinum-DNA complex. This experiment shows
that <?£s-DDP r e a c t s w i t h guanine before i t binds to adenine (56).
F i n a l l y , raman spectroscopy of platinum-DNA complexes r e v e a l s
t h a t f r e s h aqueous s o l u t i o n s of eis-DDP r e a c t p r e f e r e n t i a l l y
w i t h guanine at r ^ = 0.2 and a l s o w i t h adenine a t r ^ = 0.4 whereas
no r e a c t i o n w i t h c y t o s i n e or thymine was observed (37).
I s o l a t i o n and p r e l i m i n a r y c h a r a c t e r i z a t i o n of the platinum-

DNA adducts.Recently we have developped a method to separate the
platinum-DNA adducts (i.e. complexes formed on DNA between the
3
platinum compound and one or two bases) from the DNA (57). Since
the purpose of t h i s experiment was to determine the s t r u c t u r e of
these adducts, i t seemed important not to modify them d u r i n g the
i s o l a t i o n . Enzymatic d i g e s t i o n , such as has been used to cut
a l k y l a t e d bases from DNA (58) might be the g e n t l e s t technique, but
there i s i n c r e a s i n g evidence t h a t the platinum-DNA l e s i o n i s
r e s i s t a n t to nuclease d i g e s t i o n (59, 60, 61). A l k y l a t e d purines
detach spontaneously from damaged DNA (58, 62), but h e a t i n g the
platinum-DNA complex formed by the three complexes i n F i g u r e 1
d i d not r e l e a s e platinum. In f a c t , we have r e c e n t l y observed t h a t ,
7
u n l i k e a l k y l a t i n g agents, f i x a t i o n of a platinum on the N p o s i
t i o n of guanosine s t a b i l i z e s the bond between the sugar and the
base (63).
For these reasons we have used d e p u r i n a t i n g c o n d i t i o n s
(15 min, 100°C, 90 % HC00H) which separated adenine, guanine and
the platinum-DNA adducts from a p u r i n i c a c i d . These products were
r e s o l v e d by h i g h v o l t a g e paper e l e c t r o p h o r e s i s a t pH 2. The paper
was cut i n t o 1 cm s t r i p s which were e l u t e d i n water and the pro
f i l e of platinum c o n c e n t r a t i o n was determined by flameless atomic
a b s o r p t i o n . Under these c o n d i t i o n s , the a p u r i n i c a c i d migrated
toward the p o s i t i v e e l e c t r o d e w h i l e the purine bases and the
adducts migrated toward the negative e l e c t r o d e . For a l l three
compounds, l e s s than 10 % of the platinum was detected on the
a p u r i n i c a c i d , even at rf> = 0.4. The e l e c t r o p h o r e s i s p r o f i l e s of
hydrolysed platinum-DNA complexes formed by <?-£s-DDP, trans-ΌΏ? and

[ P t ( d i e n ) C l ] C l a t rb = 0.1 are shown i n F i g u r e 9. Subsequent paper

chromatography o f the major peaks gave a s i n g l e UV absorbing spot
which contained platinum. This evidence suggests that each major
peak i n Figure 9 represents a s i n g l e platinum-base(s) complex
(57). The s t r u c t u r e s o f s e v e r a l o f these complexes have been
determined.
[ P t ( d i e n ) C l ] C l . We have f i r s t i n v e s t i g a t e d the Pt(dien)-DNA

adducts because monofunctional f i x a t i o n of platinum on DNA was
expected to be simpler than b i d e n t a t e f i x a t i o n . In a d d i t i o n ,
complexes o f [ P t ( d i e n ) C l ] C l w i t h n u c l e i c a c i d bases which could
serve as model compounds f o r the i d e n t i f i c a t i o n of the Pt(dien)-DNA
adducts have been p r e v i o u s l y synthesized and w e l l c h a r a c t e r i z e d .
H y d r o l y s i s o f DNA which had been reacted w i t h [ P t ( d i e n ) C l ] C l
at 0.0005 < r ^ < 0.1 l i b e r a t e d a s i n g l e product which migrated

26 cm (Figure 9 ) . Above t h i s r b a second peak at 30 cm became
apparent and a s m a l l peak near 34 cm was observed at 0.3 < Γ^<0.4.
Paper chromatography revealed that each peak contained a s i n g l e
product. The m a t e r i a l i n each peak was e l u t e d i n water and i t s UV
a b s o r p t i o n was measured. Each adduct was then r e a c t e d w i t h t h i o u
rea to d i s p l a c e the platinum from the base which was subsequently
i d e n t i f i e d . The s i t e s o f platinum f i x a t i o n were determined by
comparison of the UV s p e c t r a , mass s p e c t r a and e l e c t r o p h o r e t i c
and chromatographic m o b i l i t y of the adducts w i t h hydrolysed
2 +
[ P t ( d i e n ) ( n u c l e o s i d e ) ] complexes of known s t r u c t u r e .
The r e s u l t s support the f o l l o w i n g mechanism f o r the r e a c t i o n
of [ P t ( d i e n ) C l ] C l w i t h DNA in vitro. The platinum compound binds
7
e x c l u s i v e l y t o Ν o f guanine below r ^ = 0.1. Above t h i s r ^ i t
7
a l s o f i x e s at Ν o f adenine. Above η> = 0.3 d e n a t u r a t i o n of the
DNA exposes the N* p o s i t i o n of adenine and [Pt(dien)] Α, w i t h 2
7
f i x a t i o n at N* and N , i s formed (63).
Cis-DDP. The e l e c t r o p h o r e s i s p r o f i l e of cr£s-DDP-DNA, η, = 0.1,

had p l a t i n u m - c o n t a i n i n g peaks which migrated 2, 14, 23 and 27 cm
toward the n e g a t i v e e l e c t r o d e (Figure 9 ) . A t rb = 0,4 a f i f t h peak
appeared at 32 cm. The r e l a t i v e i n t e n s i t y o f the m a t e r i a l at 14 cm
remained constant w i t h i n experimental e r r o r from rb = 0.006 to 0.4.
In c o n t r a s t , the r e l a t i v e s i z e s o f the peaks a t 23 cm(I) and
27 cm(II) v a r i e d from approximately equal i n t e n s i t i e s f o r
0.1 < rb < 0.4 t o a r a t i o of I / I I = 3.5 + 1 a t η, = 0.0005, t h e
l i m i t o f d e t e c t i o n f o r these peaks (57).
The two major peaks o f the platinum p r o f i l e each contained a
s i n g l e product judging by paper chromatography. R e a c t i o n of the
e l u t e d m a t e r i a l w i t h t h i o u r e a r e l e a s e d guanine from both adducts.
2+
H y d r o l y s i s of c i s - [ P t ( N H 3 ) 2 ( d G ) 2 ] i n which the platinum i s
7
complexed a t the N o f two guanine bases (45, 50), gave platinum-
c o n t a i n i n g peaks which migrated 14 and 23 cm (Figure 9 ) . Paper
chromatography revealed that the m a t e r i a l i n these peaks had the
sameRf as that found i n the corresponding cis-DDP-DNA h y d r o l y s i s
products. Hence, the m a t e r i a l a t 14 and 23 cm i n F i g u r e 9 appears

0 10 20 30
cm
Figure 9. Electrophoresis profiles of DNA treated with cis-DDP (top), trans-DDP

(middle), and [Pt(dien)Cl]Cl (bottom). Key: —, hydrolyzed platinum-DNA com
plex, r = 0.1; and
b , hydrolyzed platinum-DNA complex isolated from
treated E . coli, r = 0.0005 (intensity χ 2). The origin is at zero and the abscissa
&
is the distance migrated toward the negative electrode after 45 min of electro
phoresis at 100 mA (7000 V).

2+
to come from the a c i d h y d r o l y s i s o f <r£s-[Pt(NH3)2(dG>2] - The
peaks a t 27 and 32 cm have not yet been i d e n t i f i e d ,
Trans-ΏΏ?. For the hydrolysed trans-DDP-DNA complex, η, « 0,1,

the e l e c t r o p h o r e s i s p r o f i l e had peaks a t 3, 14, 19 and 23 cm
(Figure 9) whose r e l a t i v e i n t e n s i t i e s d i d not change between
rb = 0.003, the l i m i t o f d e t e c t i o n of the peak a t 23 cm, and
rb = 0.3. Above t h i s r b a peak near 26 cm appeared (57). E x p e r i
ments are i n progress t o determine the s t r u c t u r e s o f the adducts
which correspond t o the peaks o f the e l e c t r o p h o r e s i s p r o f i l e .
Platinum-DNA adducts in vivo. I n order t o compare the adducts

formed by these compounds w i t h DNA in vivo and in vitro, DNA was
i s o l a t e d from E. coli which had been t r e a t e d w i t h e i t h e r ois-W?
or [ P t ( d i e n ) C l ] C l . A f t e r h y d r o l y s i s and e l e c t r o p h o r e s i s , a l l o f
the p l a t i n u m from the DNA o f E. ooli which had been t r e a t e d w i t h
eis-DDP remained a t the o r i g i n w h i l e adducts from b a c t e r i a
t r e a t e d w i t h [ P t ( d i e n ) C l ] C l migrated about 10 cm (Figure 9 ) , S i m i
l a r r e s u l t s were obtained f o r DNA i s o l a t e d from LI210 leukemia
c e l l s which had been t r e a t e d w i t h [ P t ( d i e n ) C l ] C l i n c u l t u r e (57),
Recent immunochemical s t u d i e s have shown t h a t a n t i b o d i e s
against the ois-Vt(ΝΗβ^-ϋΝΑ complex formed in vitro do not recog~
n i z e the platinum-DNA complex i s o l a t e d from r a t s which have been
t r e a t e d w i t h cis-ΌΏ? (64). The present r e s u l t s show that the p l a t
inum-DNA adducts i s o l a t e d from t r e a t e d b a c t e r i a and mammalian
c e l l s do not give the same e l e c t r o p h o r e s i s p a t t e r n s a f t e r a c i d
h y d r o l y s i s as the corresponding adducts formed in vitro. I t seems
t h a t , in vivo, some o f the modes of f i x a t i o n o f p l a t i n u m compounds
to DNA may be d i f f e r e n t than those found in vitro.
I n summary, the p r e l i m i n a r y s t r u c t u r e s o f the platinum-DNA
adducts i n d i c a t e t h a t , i n DNA, [ P t ( d i e n ) C l ] C l and cis-OVP both
b i n d t o the Ν 7 p o s i t i o n o f guanine. I n the case o f [ P t ( d i e n ) C l ] C l ,
t h i s adduct i s the only d e t e c t a b l e mode of f i x a t i o n below
rb = 0.1. C^s-DDP forms two guanine-containing adducts w i t h DNA
2+
at rb = 0.0005. One o f these appears t o be cis-[Vt(NH3)2(dG)2]
which was present a t 3.5 ± 1 times the c o n c e n t r a t i o n o f the other
adduct a t t h i s rb« The s t r u c t u r e o f the second adduct has not y e t
been determined. I n the next s e c t i o n we w i l l b r i e f l y consider how
each of these l e s i o n s might cause the a l t e r a t i o n o f DNA s t r u c t u r e
and s t a b i l i t y which have been observed (Table I I I ) ,
HOW THE OBSERVED PLATINUM-DNA ADDUCTS MIGHT ALTER DNA CONFORMATION

AND STABILITY IN VITRO
F i x a t i o n o f p l a t i n u m compounds a t the N? p o s i t i o n o f guanine

l a b i l i z e s the proton a t N l . Table IV summarizes the changes i n pK
of the guanine Ν1 proton upon f i x a t i o n o f cis-Wi?, trans-DDP o r
[ P t ( d i e n ) C l ] C l a t the Ν 7 p o s i t i o n . The decrease i n the pK o f the
N* proton i s comparable f o r the f i x a t i o n o f [ P t ( d i e n ) C l ] C I , f o r
f f
the 5 -GMP : trans-DDP complexes and f o r the 2:1 5 -GMP : cis-ΌΌ?

complex, but o n l y ois-OOV d e s t a b i l i z e s the DNA (Table I I I ) , F i x a

7
t i o n o f p l a t i n u m a t Ν o f guanine may not s u f f i c i e n t l y change the
hydrogen bonding w i t h the complementary c y t o s i n e t o d e s t a b i l i z e
the DNA.
Unusual base p a i r i n g has been observed i n the c r y s t a l s t r u c
ture o f a platinum-guanosine complex and i t has been suggested
t h a t f i x a t i o n o f p l a t i n u m a t Ν 7 may l a b i l i z e the N l proton and
thereby cause m i s p a i r i n g d u r i n g DNA r e p l i c a t i o n (68) which would
l e a d t o mutations. This hypothesis seems u n l i k e l y s i n c e the pK of
+
[7-methy1guanosine] and [Pt(dien)G]2+ are s i g n i f i c a n t l y lower
+
than f o r guanosine, but [7-methy1guanosine] does not cause
m i s p a i r i n g (69) and [ P t ( d i e n ) C l ] C l i s , a t b e s t , a very weak muta
gen (70).
TABLE IV. VARIATION OF THE pK OF THE GUANINE

N* PROTON BY FIXATION OF PLATINUM(II)
COMPOUNDS AT THE Ν 7 POSITION.
Compound Δ pK Reference
il
, 2
cis-[Pt(NH ) (5 -GMP) ] ~3 2 2 - 0,3 a
+
c£s-[Pt(NH ) (GpG)] 3 2 - 1,3 C
44
ois-[Pt(NH ) (9-EtG)(1-MeC)]
3 2
2+
- l,6 b
66
IL
+
ois-[Pt(NH ) (5'-GMP)(H 0)]
3 2 2 - 2.8 ' a d
IL
1 2
trans-[Pt(ΝΗ ) (5 -GMP) ] β 2 " - 0.6 b
il
b
trans-[Pt(NH ) (5 -GMP)(H 0)]
3 2
f
2
+
- i,o
+
[7-methy1guanosine] - 2.I e
67
[Pt(dien)G] 2 +
- l,2 b
63
a. raman spectroscopy ; b. u l t r a v i o l e t a b s o r p t i o n ; c, NMR ;

d. p r e c i p i t a t i o n f o r 5 < pH < 7.5.
I t has been p r e v i o u s l y suggested that c h e l a t i o n of ois~WV

7 1
on p o s i t i o n s N and 0*> o f guanine might l a b i l i z e the N proton
s u f f i c i e n t l y t o induce m i s p a i r i n g and base p a i r s u b s t i t u t i o n
mutations ( 9 ) . Such a complex might a l s o be r e s p o n s i b l e f o r t h e
d e s t a b i l i z a t i o n of DNA a t low rb which was observed f o r ois-WP
but not f o r trans-W? o r [ P t ( d i e n ) C l ] C l .

F i x a t i o n of platinum compounds at N7 p o s i t i o n of guanine s t a

b i l i z e s the g l y c o s y l l i n k a g e . M e t h y l a t i o n of the N7 p o s i t i o n
of guanine l a b i l i z e s the g l y c o s y l l i n k a g e (62) and the spontane
ously created a p u r i n i c s i t e s on DNA in vivo can be recognized and
r e p a i r e d by a s p e c i f i c endonuclease (71). In a d d i t i o n , the l o s s
of a purine base could l e a d to a s i n g l e s t r a n d break i n the DNA,
For these reasons we have i n v e s t i g a t e d the i n f l u e n c e of platinum
7
at the N p o s i t i o n of guanosine on the s t a b i l i t y of the g l y c o s y l
linkage. ^ + . 2+
Guanosine, [7-methy1guanosine] and [ P t ( d i e n ) G ] were reac
ted at 37°C i n 1 M HC1 and the h y d r o l y s i s of the g l y c o s y l bond
was f o l l o w e d by changes i n the UV absorbance. The r e a c t i o n was
f i r s t order w i t h h a l f - l i v e s of 11.5 ± 1 h f o r guanosine, 8,5 ± 1 h
+
f o r [7-methylguanosine] and 135 ± 25 h f o r [ P t ( d i e n ) G ] C l 2 . S i m i
l a r l y , the h a l f — l i f e f o r the h y d r o l y s i s of the g l y c o s y l l i n k a g e of

dG and [Pt(dien)dG]Cl2 were about 4 min and 2,5 h r e s p e c t i v e l y .
These r e s u l t s i n d i c a t e t h a t , u n l i k e a l k y l a t i n g agents, f i x a t i o n of
platinum at the N? p o s i t i o n of guanine nucleosides s t a b i l i z e s the
bond between the sugar and the base (55),
There may be a simple e x p l a n a t i o n f o r the d i f f e r e n t behavior
of the a l k y l group and the platinum compound. In the case of
+
[7-methylguanosine] , the N' p o s i t i o n c a r r i e s a formal 1+ charge
which, judging from resonance s t r u c t u r e s , can be d e l o c a l i z e d t o
the N$ p o s i t i o n . In c o n t r a s t , the 2+ charge of the platinum com
p l e x i s shared by the four n i t r o g e n atoms which b i n d to the dsp2
o r b i t a l s of the platinum. Hence, i n the case of a l k y l a t i o n the
p o s i t i v e charge may r e s i d e on the imidazole r i n g and d e s t a b i l i z e
the g l y c o s y l l i n k a g e w h i l e f o r p l a t i n a t i o n , the charge would be
d e l o c a l i z e d on the platinum moiety.
Does f i x a t i o n of p l a t i n u m ( I I ) chloroamines on DNA in vitro

l a b i l i z e the amine l i g a n d s ? F i x a t i o n of ois-ΏΌ? on l-methylcyto-
s i n e rearranges the platinum complex to form a trans isomer which
i n d i c a t e s f i x a t i o n on a base may l a b i l i z e the NH3 l i g a n d (72), I t
has been proposed that f i x a t i o n of ois-ΌΏ? on a h e t e r o c y c l i c
n i t r o g e n atom of DNA may l i b e r a t e NH3 by means of the trans e f f e c t
(73, 74). Such an e v e n t u a l i t y would permit t h i s platinum compound
to b i n d t o DNA by 3 or 4 s i t e s and consequently introduce a number
of adducts i n DNA which might not be found i n r e a c t i o n s w i t h i s o
l a t e d n u c l e i c a c i d b a s e s . A l t h o u g h t h e r e a c t i o n o f ois-
[Pt[14c](en)Cl2] w i t h n u c l e i c a c i d bases does not l i b e r a t e
[14c]en (75), a d i f f e r e n t mode of b i n d i n g i n DNA might weaken the
platinum-nitrogen bond to a greater extent.
For t h i s reason we synthesized <r£s-[Pt[* χ ] ( e n ) C I 2 ] (76) and
measured the [ l ^ C ] d pt on the DNA, For rb = 0,1, 2091 ± 82 and
a n
2127 ± 76 cpm were found on the DNA before and a f t e r exhaustive

d i a l y s i s of the r e a c t i o n mixture, r e s p e c t i v e l y . Hence, f i x a t i o n
of [ P t ( e n ) C l 2 ] (which i s an a c t i v e antitumor agent (17)) on DNA
in vitro does not l a b i l i z e the ethylenediamine l i g a n d . In the
absence of published s t u d i e s on the l a b i l i t y of NH3 l i g a n d of

platinum chloroammines f i x e d on DNA, perhaps these r e s u l t s may be

c a u t i o u s l y e x t r a p o l a t e d to cis-ΌΌ?.
7
F i x a t i o n of [ P t ( d i e n ) C l ] C l at N G favors the Β — » Ζ conforma
t i o n a l t r a n s i t i o n . At rb = 0.05 [ P t ( d i e n ) C l ] C l , but not ois- o r
trarcs-DDP, f a v o r s the Β — » Ζ t r a n s i t i o n of poly(dG-dC)
poly(dG-dC). Monofunctional f i x a t i o n of platinum at the N? might
favor the syn conformation of guanine which i s found i n Ζ DNA (77) ·
Cis-DDP may form i n t r a s t r a n d c r o s s l i n k s and microloops. P r e

l i m i n a r y c h a r a c t e r i z a t i o n of the mode of f i x a t i o n of <^s-DDP on
2+
DNA i n d i c a t e s the formation of cis-[Vt(NH3)2(dG)2l which appears
to be the major adduct a t rb = 0.0005. For 0.1 < rb < 0.3 t h i s
adduct represents 47 ± 7 % of the platinum f i x e d on the DNA (57).
Such an adduct may be a r e s u l t of i n t r a s t r a n d c r o s s l i n k s between

adjacent guanine bases (54, 55). However, no more than 0.04 Pt/DNA
n u c l e o t i d e can be f i x e d on adjacent guanine bases (78) i n salmon
sperm DNA. Hence, ois-DOV l i k e l y forms bis-complexes w i t h non-
nearest neighbor guanine bases. Such complexes may be i m p l i c a t e d
i n the formation of microloops (23) o r guanine separated by one
or more bases such as i n GAG o r GCG sequences (79).
A HYPOTHESIS CONCERNING THE MECHANISM OF THE REACTION BETWEEN

CTS-DDP AND DNA.
Recent evidence suggests t h a t , in vitro and in vivo, the

formation of i n t e r s t r a n d c r o s s l i n k s proceeds by two steps i n which
the attachment of the two strands of DNA occurs much slower than
the i n i t i a l f i x a t i o n of platinum on DNA (29). S i m i l a r l y , the f o r
mation of i n t r a s t r a n d c r o s s l i n k s in vitro appears t o occur i n two
steps (80). This mechanism of r e a c t i o n i m p l i e s the i n i t i a l forma
t i o n of a r e a c t i v e intermediate which subsequently c o u l d make
e i t h e r i n t e r s t r a n d c r o s s l i n k s , i n t r a s t r a n d c r o s s l i n k s or perhaps
microloops (23) depending on whether i t reacted w i t h the base
complementary t o the i n i t i a l s i t e of f i x a t i o n , the nearest neighbor
base o r w i t h a base f a r t h e r away.
S e v e r a l arguments suggest t h a t c o v a l e n t b i n d i n g o f
cis-Vt(NH3)2 - a t the N? p o s i t i o n of guanine and f i x a t i o n at θ6
( e i t h e r through a water b r i d g e , a h y d r o x y l group or by d i r e c t
Pt-06 c o o r d i n a t i o n ) w o u l d be an a t t r a c t i v e candidate f o r
t h i s i n t e r m e d i a t e . The N? p o s i t i o n of guanine i s apparently the
i n i t i a l s i t e of f i x a t i o n f o r the c^s-DDP-DNA adduct whose s t r u c
ture has been determined. A second metastable bond between 0^ and
the p l a t i n u m moiety might reasonably delay the c r o s s l i n k i n g r e a c
t i o n of <r£s-DDP. L i b e r a t i o n of the N* p r o t o n i n the N^-O^ complex
has been p r e v i o u s l y suggested t o account f o r the mutagenicity of
cis-ΌΌ? ( 9 ) . Z w e l l i n g ( t h i s symposium) r e p o r t s t h a t t h i o u r e a
simultaneously i n h i b i t s mutagenicity and the formation of c r o s s
l i n k s i n mammalian c e l l s . I t may be that t h i o u r e a r e p l a c e s the
Pt-0^ l i n k a g e w i t h a p l a t i n u m - s u l f u r bond thereby d e p l e t i n g t h i s

metastable intermediate which is necessary for both mutagenicity

and crosslink formation.
Abbreviations. DDP = [Pt(NH3>2Cl2] ; dien = diethylenetriamine,

(NH2-CH2-CH2)2NH ; en « ethylenediamine, (NH2-CH2>2 5 EtdBr -
ethidium bromide, 3-8-diamino-6-phenyl-5-ethylphenanthridinium
bromide ; r^ = molar ratio Pt introduced/DNA nucleotide ; rb =
number of Pt covalently bound/DNA nucleotide ; T m = temperature
at which DNA is half denatured ; i.p^ - intraperitoneal.
Acknowledgments This work was supported by grants from D.G.R.S.T.

(décision d'aide n° 77.7.1348) and l'Association pour le Dévelop-
pement de la Recherche sur le Cancer. The comments of Drs. M. Leng,
B. Malfoy (Orléans) and of J.C. Chottard (Paris) are appreciated.
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5
Model for the Interaction of cis-Diamminedi-

chloroplatinum(II) with SimianVirus 40 DNA
1
WILLIAM M. SCOVELL, LEE R. KROOS, and VINCENT J. CAPPONI
Bowling Green State University, Department of Chemistry,
Bowling Green, OH 43403
Physico-chemical and nuclease probes which are

sensitive to modifications of DNA clearly indicate
that both cis- and trans-PtCl (NH ) bind to (G+C)
2 3 2
rich regions of DNA, although they exhibit different

modes of binding. Both electrophoretic profiles of
PtCl (NH ) treated SV40 DNA and the extent of S1
2 3 2
nuclease digestions on the modified DNAs emphasize

that the isomers exert different effects on DNA
structure. However, the isomers are comparable in
inhibiting site specific cleavage by a particular
restriction enzyme, with the relative inhibition for
a series of five restriction endonucleases paralleling
the number and relative position of guanines in and
adjacent to the recognition site. These data suggest
that the Bgl 1 site, which is in the regulatory region
of the SV40 genome, is a hyper-reactive site to
PtCl (NH ) binding. This may be directly related to
2 3 2
the impairment of biological functions of SV40 virus

in infected cells by cis-DDP treatment.
Perhaps the two most prominent differences between cis- and
+
trans-DDP are the (1) geometrically different structures and (2)
the therapeutic efficacy of cis-DDP as an anti-neoplastic agent,
while trans-DDP is inactive (1,2,3). The chemotherapeutic action
of cis-DDP is thought to result from a direct covalent binding to
DNA, which manifests selective inhibition of DNA synthesis, both
in vitro and in vivo (4,5,6). Notwithstanding that both isomers
may form a number and variety of different DNA or DNA-protein
crosslinked adducts, only one or more of the cis-DDP adducts is
responsible for the desired therapeutic effects. This signifi-
cant difference must be related, in part, to the different
1
Current address: Stanford University Medical School, Department of Biochem-
istry, Stanford, CA 94305
0097-6156/ 83/0209-0101 $06.25/0


stereochemical bonding c o n s t r a i n t s inherent i n the isomeric

s t r u c t u r e s . With t h i s i n mind, we have f e l t t h a t i t i s e s s e n t i a l
t o compare the e f f e c t t h a t both c i s - and trans-DDP b i n d i n g exerts
on DNA o r chromatin s t r u c t u r e and/or f u n c t i o n . Although both
isomers are expected t o b i n d p r e f e r e n t i a l l y t o (G+C) r i c h regions
m a
o f DNA (2'â'£) Y b i n d mono f u n c t i o n a l l y o r produce i n t e r -
s t r a n d c r o s s l i n k s (10,11 ), a mode o f b i n d i n g t o DNA which i s
s t e r e o c h e m i c a l l y unique t o cis-DDP i n v o l v e s an i n t r a s t r a n d c r o s s -
l i n k t o adjacent guanines on n a t i v e DNA o r t o other n u c l e o t i d e
bases (G,A o r C) i n denatured r e g i o n s . Stone e t a l (9) o r i g i -
n a l l y o f f e r e d the former proposal and a number of recent f i n d i n g s
are c o n s i s t e n t w i t h i t (12-16)·
As w i t h s i m i l a r b i o l o g i c a l problems, a major dichotomy sur-
f a c e s i n d e f i n i n g a b i o l o g i c a l system i n which one attempts t o
r e l a t e the chemical i n t e r a c t i o n s i n the genome t o the impairment

o f b i o l o g i c a l f u n c t i o n . I d e a l l y t h i s system would not o n l y be o f
s u f f i c i e n t s i m p l i c i t y t o make t h i s goal w i t h i n reach, but on the
other hand, complex enough t o be o f relevance i n human
(eukaryotic) e e l I s .
We have t h e r e f o r e focused much o f our e f f o r t s on comparing
the e f f e c t t h a t c i s - and trans-DDP e x e r t s on SV40 DNA and the
SV40 minichromosome. In t h i s communication, we s h a l l summarize
r e c e n t f i n d i n g s from our l a b o r a t o r y which help t o shape a working
model f o r d i s c e r n i n g how c i s - and trans-DDP b i n d i n g may modify
the DNA s t r u c t u r e and t h e r e f o r e d i s r u p t the normal b i o l o g i c a l
f u n c t i o n s o f SV40 v i r u s .
The SV40 Genome

As a r e s u l t o f an i n t e n s i v e e f f o r t i n the l a s t decade, the
oncogenic animal v i r u s , simian v i r u s 40 (SV40), i s perhaps one
o f the most w i d e l y used models f o r s t u d i e s on the molecular mech-
anisms o f gene expression and r e g u l a t i o n i n e u k a r y o t i c c e l l s (17,
J[8,JM3 ) · There i s good reason f o r i t s p o p u l a r i t y . The s m a l l DNA
genome, shown s c h e m a t i c a l l y i n Figure 1, i s e n t i r e l y sequenced
(5,243 b.p.) (20,21,22) contains o n l y f i v e known genes (denoted t ,
T, VP1, VP2, and VP3) and a r e g u l a t o r y r e g i o n (shown s t i p p l e d ) ,
the l o c a t i o n s o f which have been p r e c i s e l y mapped (17,18, 19 ) ·
The l o c a t i o n o f cleavage s i t e s ( i n map u n i t s ) f o r the r e s t r i c t i o n
endonucleases B g l 1, Kpn I , Hpa I I , Eco RI and Bam HI, a l l o f
which c u t SV40 DNA a t a s i n g l e , unique s i t e , are shown i n the
c i r c u l a r SV40 map. The temporal order o f v i r a l gene expression
i n a l y t i c i n f e c t i o n i s d i v i d e d i n t o the " e a r l y " and " l a t e "
r e g i o n s . R e p l i c a t i o n i s i n i t i a t e d a t the o r i g i n of r e p l i c a t i o n
(Ori) a t about 0.66 (23) and proceeds b i d i r e c t i o n a l l y t o t e r -
minate a t approximately 0.17. Τ a n t i g e n i s synthesized e a r l y and
then i t s s i t e s p e c i f i c b i n d i n g t o DNA a t 0.66 i s thought t o be
i n v o l v e d i n both c o n t r o l of f u r t h e r Τ antigen t r a n s c r i p t i o n and
the i n i t i a t i o n o f v i r a l DNA s y n t h e s i s (24)/ the l a t t e r of which
proceeds a t the same time t h a t the " l a t e " genes are expressed.

SCOVELL ET A L . cis-PtCltfNHsh and Simian Virus DNA 103
Figure 1. Schematic representation of SV40 génome indicating the locations of

principal features (19).
The genome is represented as a circle with the origin of replication (Ori) at the top (See Ref. 63
for details about the origin of DNA replication). Restriction endonuclease cutting sites are
shown for Bgl 1, Κρη I, Ηρα II, Eco RI, and Bam HI with Eco RI at 0/1.0. The genome is
divided into coding sequences for the early genes (large T, small t) and the late genes (VP/>
VP2, and VP3). Shaded arcs with arrowheads indicate coding positions of mRNA molecules with
arrowheads pointing in the 5' -+ 3' direction. The stippled area contains the regulatory region
of the genome.

Of p a r t i c u l a r note f o r our purposes, Kutinova e t a l (25)

have reported that i n SV40 i n f e c t e d A f r i c a n green monkey kidney
c e l l s , cis-DDP i n h i b i t s SV40 r e p l i c a t i o n and the production of
the l a t e p r o t e i n s , while e x h i b i t i n g no observable e f f e c t on Τ
antigen production.
Cis-DDP unwinds SV40 DNA Twice As E f f e c t i v e l y As Trans-DDP (15)

The binding of a v a r i e t y of molecules, i o n s , o r biomacromo-
l e c u l e s to DNA i s known to change the l o c a l s t r u c t u r e of DNA
(26,27,28) · Gel e l e c t r o p h o r e s i s provides a s e n s i t i v e method to
monitor such a l t e r a t i o n s i n t e r t i a r y s t r u c t u r e i n s u p e r c o i l e d
3
( c s ) DNA s i n c e the m o b i l i t y of the DNA i s p r o p o r t i o n a l to the
absolute number of s u p e r c o i l s , | f f (29). Figure 2 shows
g e n e r a l l y t h a t b i n d i n g to DNA may produce s t r u c t u r a l changes
which r e s u l t i n the unwinding of the DNA backbone. As i n c r e a s i n g

amounts of molecules or ions bind to the DNA, the negative super-
c o i l s are unwound u n t i l the i n t a c t relaxed form (ΔΟ* =0 ,f =0 ) i s
produced. On f u r t h e r b i n d i n g , the DNA u s u a l l y unwinds f u r t h e r
t o the p o s i t i v e l y s u p e r c o i l e d form.
F i g u r e 3 o u t l i n e s the experimental p r o t o c o l f o r t h i s aspect
o f the study, together with that f o r the subsequent i n v e s t i g a t i o n
with r e s t r i c t i o n endonucleases. The time dependent e f f e c t of
trans-DDP b i n d i n g on the e l e c t r o p h o r e t i c m o b i l i t y of s u p e r c o i l e d
and nicked SV40 DNA i s shown i n Figure 4a (JU>) · Four obser
v a t i o n s are of primary i n t e r e s t . The trans-DDP b i n d s to and
unwinds c^s DNA. At low l e v e l s of b i n d i n g , there i s l i t t l e or no
3
decrease i n the m o b i l i t y of c s DNA i n d i c a t i n g very l i t t l e e f f e c t
on unwinding of the DNA o r a l t e r i n g i t s t e r t i a r y s t r u c t u r e . A
decreased m o b i l i t y i s apparent at c a . 3 hours. The i n t a c t
r e l a x e d form of DNA i s observed at 24 hours, i t s m o b i l i t y being
c o i n c i d e n t with t h a t of nicked DNA. Under these r e a c t i o n con
d i t i o n s , and at elevated (trans-DDP/DNA(Ν)) mole r a t i o s 4 times
g r e a t e r , there was no evidence that trans-DDP unwound the DNA
f u r t h e r to a p o s i t i v e l y s u p e r c o i l e d form.
The e l e c t r o p h o r e t i c p r o f i l e f o r the cis-DDP r e a c t i o n under
i d e n t i c a l c o n d i t i o n s i s compared i n Figure 4b. Three aspects of
the p r o f i l e r e v e a l d i f f e r e n c e s between the e f f e c t t h a t c i s - and
trans-DDP e x e r t on the s t r u c t u r e of SV40 DNA. The cis-DDP b i n d s
t o and unwinds the s u p e r c o i l e d DNA t o the i n t a c t relaxed form i n
c a . 6 hours, a f a c t o r of 4 times f a s t e r than the trans-DDP. At
24 hours, the m o b i l i t y of the DNA has reversed and i s now greater
than t h a t of the i n t a c t relaxed form, i n d i c a t i v e of formation of
the p o s i t i v e l y s u p e r c o i l e d form. As noted above, the e l e c t r o
p h o r e t i c p a t t e r n i n d i c a t e s t h a t trans-DDP b i n d i n g cannot unwind
the DNA t o a s i m i l a r extent. A n a l y s i s of the r a t e of DDP b i n d i n g
t o DNA as measured by the atomic absorption of bound Pt i n d i c a t e s
t h a t cis-DDP b i n d s to DNA s l i g h t l y f a s t e r than trans-DDP. At the
p o i n t of the i n t a c t relaxed form, the (DDP/DNA(N) ) mole r a t i o s
f o r c i s - and trans-DDP a r e 0.08 and 0.15, r e s p e c t i v e l y . These

5. SCOVELL ET AL. Q\s-PtCU(NHsh and Simian Virus DNA 105
Figure 2. Schematic representation of the unwinding of negatively supercoiled

DNA caused by the binding of increasing amounts of drugs (dark circles). In the
unwinding process, the topological linking number (a) remains invariant while the
number of supercoils (τ) changes. The DNA is shaded also to denote different
degrees of superhelicity.

INTERACTION OF CIS- AND TRANS-DDP WITH SV40 DNA
E f f e c t o f the Covalent Binding on:
1· E f f e c t i v e Unwinding of DNA
2. Cleavage by R e s t r i c t i o n Endoviucleases
1.
(DDP + SV40 DNA) Gel Electrophoresis

o f DNA
(Figure 4 and 5)
2. t , Spot D i a l y s i s
x
(DDP-SV40 DNA)
t ,
x R e s t r i c t i o n Endonuclease
Gel E l e c t r o p h o r e s i s o f Linear DNA Fragments

(Figure 7)
Figure 3. Experimental protocols: Path 1 is used to compare the relative influence

of cis- and trans-DDP binding on unwinding Λ SV40 DNA (15). Path 2 is used
to determine the relative distribution of cis- and trans-DDP at specific sites in the
SV40 genome (16).

SCOVELL ET A L . cis-PtCh(NHs)i and Simian Virus DNA
Figure 4. Electrophoretic profile of supercoiled (S) and nicked (N)

SV40 DNA incubated with trans-DDP (top) and cis-DDP (bottom)
both at [DDP/DNA(N)] atcd = 10 for various times at 25 °C.
incub
The outermost lanes contain control (C) SV40 DNA after incubation
for 0 min (left) and 24 h (right). Lanes 2-13 contain DNA reacted
with trans-DDP or cis-DDP for times (left to right) of 0, 3, 10, 20,
30, 50, and 85 min and 2, 3, 6, 12, and 24 h. (Reproduced with
permission from Ref. 15. Copyright 1982, Academic Press.)

r a t i o s i n d i c a t e t h a t the cis-DDP i s twice as e f f e c t i v e as

trans-DDP i n unwinding the DNA to the i n t a c t relaxed form.
P o s s i b l y the most s i g n i f i c a n t d i f f e r e n c e between the b i n d i n g
c h a r a c t e r i s t i c s o f the two isomers i s observed at very short
r e a c t i o n times, which corresponds to very low l e v e l s of DDP
b i n d i n g . We f i n d t h a t whereas the m o b i l i t y o f the trans-DDP
m o d i f i e d DNA e x h i b i t s very l i t t l e decrease i n m o b i l i t y a t t ^ O
( i . e . , mix DDP w i t h DNA and freeze immediately), the cis-DDP
modified DNA, on the other hand, has a comparatively l a r g e
decrease i n m o b i l i t y . T h i s l a t t e r e f f e c t i s more c l e a r l y evident
i n Figure 5 which compares the m o b i l i t i e s of the c i s - and
trans-DDP modified DNAs f o r the f i r s t 30 minutes of r e a c t i o n .
Although the cis-DDP b i n d i n g reduces the DNA m o b i l i t y a t t&0,
the m o b i l i t y o f the trans-DDP modified DNA i s not comparably
reduced u n t i l a t l e a s t 30 minutes. C l e a r l y the pronounced d i f

ference a t low l e v e l s o f b i n d i n g , perhaps a f a c t o r of 50 or more
as expressed by the d i f f e r e n t m o b i l i t i e s , i n d i c a t e s that the c i s -
and trans-DDP isomers e x h i b i t d i f f e r e n t modes o f b i n d i n g , while
the o v e r a l l e l e c t r o p h o r e t i c p r o f i l e f o r the r e a c t i o n i s con
s i s t e n t with a d i f f e r e n c e p e r s i s t i n g a t a l l r ^ l e v e l s . Although
we are unable to q u a n t i t a t e the amount of DDP bound at these very
s h o r t r e a c t i o n times, we estimate the (DDP/DNA(N) ) mole r a t i o to
4
be probably c a . 5 χ 10~ , which i s equivalent to only a few DDP
bound to the SV40 genome.
These f i n d i n g s do not agree with those of Cohen et a l (30)
who have reported that the b i n d i n g of c i s - or trans-DDP t o pSMI
DNA produced e s s e n t i a l l y i d e n t i c a l unwinding of the DNA. The
r e s u l t s presented here c l e a r l y demonstrate a number of w e l l
d e f i n e d d i f f e r e n c e s i n the manner i n which these isomers a l t e r
the t e r t i a r y s t r u c t u r e and unwind SV40 DNA. Although f u r t h e r
work i s r e q u i r e d to r e s o l v e the f a c t o r s r e s p o n s i b l e f o r the d i f
ferences i n the two s t u d i e s , we can r e p o r t t h a t r e s u l t s (not
shown) s i m i l a r to those with SV40 DNA are observed using 0X174RF
DNA.
Of the modes o f b i n d i n g which the cis-DDP may e x h i b i t , the
monodentate b i n d i n g would be expected to produce unwinding of the
DNA comparable to that of the trans-DDP. However, the
i n t r a s t r a n d c r o s s l i n k i n g of cis-DDP t o adjacent guanines, a mode
o f b i n d i n g s t e r e o c h e m i c a l l y unique to the cis-DDP isomer, and
which could e x p l a i n why cis-DDP unwinds the SV40 DNA twice as
e f f e c t i v e l y as does trans-DDP, should be regarded as a primary
candidate f o r producing t h i s e f f e c t . While t h i s cannot be
regarded as a unique i n t e r p r e t a t i o n of the data, a number of s t u
d i e s support i t (12-16)· Another b i n d i n g mode which cannot be
r i g o r o u s l y excluded, the cis-DDP c h e l a t i o n at 0-6 and N-7 on
guanine, i s a p o s s i b i l i t y , but there are no s t u d i e s which we f e e l
currently substantiate i t . The "cis-DDP p i n c h i n g " of the DNA
backbone, as a r e s u l t o f forming the i n t r a s t r a n d c r o s s l i n k to
adjacent guanines, may be expected to exert a pronounced

SCOVELL ET AL. cis-PtCl$(NHs)t and Simian Virus DNA
Figure 5. Comparative electrophoretic profile of supercoiled (S) and

nicked (Ν) SV40 DNA incubated with cis- and trans-DDP both at
[DDP/DNA(N)] = 1.0 for short reaction times at 25 ° C . The
incubated
outer lanes contain control (C) SV40 DNA at 0 min (left) and 30 min
(right). Lane 2 and even-numbered lanes contain trans-DDP modified
DNA, while lane 3 and odd-numbered lanes contain similarly cis-
DDP modified DNA for reaction times of 0, 3, 7,12, 20, and 30 min.
(Reproduced with permission from Ref. 15. Copyright 1982, Aca
demic Press.)

i n f l u e n c e on unwinding the phosphodiester backbone and a l s o i n

producing l o c a l l y denatured regions which would enhance the
e f f e c t i v e unwinding process* Since the trans-DDP i s expected to
e x h i b i t p r i m a r i l y monodentate or mono f u n c t i o n a l b i n d i n g , i t would
not be as e f f e c t i v e i n a l t e r i n g the DNA s t r u c t u r e .
Cis-DDP B i n d i n g t o DNA Stimulates Greater L e v e l s o f S1 Nuclease

D i g e s t i o n Than Does Trans-DDP (31 )
A number of endonucleases e x h i b i t a s p e c i f i c i t y f o r
d i g e s t i n g s i n g l e - s t r a n d e d regions i n DNA (32)· The u t i l i t y of
these nucleases, i n c l u d i n g S1 nuclease from A s p e r g i l l u s oryzae
(33), stems from t h e i r c a p a b i l i t y to recognize and excise m o d i f i -
c a t i o n s of DNA which induce l o c a l single-stranded segments.
Although s i n g l e base p a i r mismatches are not recognized (34,35 ),
p e r t u r b a t i o n s i n s t r u c t u r e produced by pyrimidine dimers (36,37 ),

carcinogens (38,39), cis-DDP (40), other drugs (41,42) or the
i n h e r e n t t o r s i o n a l s t r a i n i n s u p e r c o i l e d DNAs (44) stimulate S1
digestion*
F i g u r e 6 compares the e f f e c t of c i s - and trans-DDP modified
c a l f thymus DNA i n s t i m u l a t i n g S1 nuclease d i g e s t i o n (31^)· These
data, together with r e s u l t s with other DNAs at lower r ^ v a l u e s ,
c o n s i s t e n t l y show t h a t cis-DDP b i n d i n g produces a greater l e v e l
o f DNA d i g e s t i o n than does trans-DDP a t the same rj> values*
T h i s i s c o n s i s t e n t with the proposal t h a t cis-DDP i n t r a s t r a n d
w M
c r o s s l i n k s to guanines w i l l p i n c h the DNA strand and r e s u l t i n
d i s r u p t i o n of the complementary base p a i r i n g i n t e r a c t i o n . This
l o c a l l y denatured region i s then recognized and cut by S1
nuclease. Trans-DDP m o d i f i c a t i o n of DNA produces l e s s r e c o g n i -
zable m o d i f i c a t i o n s to DNA s t r u c t u r e , with the percent a c i d
s o l u b l e DNA produced being 3-5 times l e s s than t h a t f o r com-
parable l e v e l s of bound cis-DDP. Likewise, the data at r ^ l e v e l s
g r e a t e r than 0.05 i n d i c a t e t h a t the S1 d i g e s t i o n l e v e l s produced
on cis-DDP modified DNA are not obtained on trans-DDP modified
DNA u n t i l the r ^ values are twice as g r e a t .
Both probes used to monitor s t r u c t u r a l changes i n DDP-
3
modified DNA - (1) the m o b i l i t y of c s DNA i n gel e l e c t r o p h o r e s i s
and (2) S1 nuclease s e n s i t i v i t y - c l e a r l y and c o n s i s t e n t l y i n d i -
cate d i f f e r e n c e s i n the mode of binding f o r the c i s - and
trans-DDP isomers.
R e l a t i v e D i s t r i b u t i o n of C i s - and Trans-DDP B i n d i n g i n the SV40

Genome. ( 16)
Examination of SV40 DNA, which i s 58% (A+T), r e v e a l s t h a t
the most (G+C) r i c h region i n the genome, which i s l o c a l l y
greater than 55% (G+C), i s w i t h i n the region r e s p o n s i b l e f o r the
r e g u l a t i o n of gene expression (0.66 - 0.74 on the p h y s i c a l map)
(20,2V) · Since DDP e x h i b i t s a binding preference f o r (G+C) r i c h
regions and cis-DDP d i s r u p t s v i r a l r e p l i c a t i o n and the production
o f s e l e c t e d p r o t e i n s , i t was of i n t e r e s t to determine the r e l a -

5. SCOVELL ET AL. cis-PtCh(NH )$ and Simian Virus DNA
s 111
Figure 6. SI nuclease digestion of cis-DDP (O) and trans-DDP (A) modified

calf thymus DNA as a function of r . (Reproduced with permission from Réf. 16.
6
Copyright 1982, Academic Press.)

Digestions were carried out on DNA samples that were reacted with various levels of DDP
samples for 10 h at 37 °C, spin dialyzed, and then digested for 35 min at 37 °C with 90 u/mL
of SI nuclease. The assay procedure of Vogt (64) was used with the percent acid solubility
determined by the Ateo reading and assuming a 41% hyperchromicity (31).

t i v e d i s t r i b u t i o n o f bound DDP w i t h i n o r about s p e c i f i c l o c a t i o n s

i n the SV40 genome by monitoring the r e l a t i v e cleavage i n h i b i t i o n
o f s i t e s p e c i f i c r e s t r i c t i o n endonucleases, (R.E. ). The a n a l y s i s
assumes t h a t DDP b i n d i n g w i t h i n o r immediately adjacent t o the
r e c o g n i t i o n s i t e w i l l modulate the sequence s p e c i f i c protein-DNA
i n t e r a c t i o n and thereby i n h i b i t the subsequent cleavage o f the
DNA.
The DNA i s i n i t i a l l y reacted with DDP, spot d i a l y z e d and
then s u f f i c i e n t R.E. ( F i g u r e 3) i s added t o j u s t completely
c l e a v e an e q u i v a l e n t amount o f unmodified DNA. Figure 7 shows
the e l e c t r o p h o r e t i c p a t t e r n observed a f t e r r e a c t i o n o f the
modified DNA with B g l 1, Bam HI, Hpa I I , Kpn I and Eco RI,
r e s p e c t i v e l y . F i g u r e 1 i d e n t i f i e s the l o c a t i o n o f the cleavage
s i t e f o r each R.E. i n SV40 DNA. The r e c o g n i t i o n sequences f o r
each R. E. p l u s a 10 base p a i r sequence on each s i d e i s shown i n

F i g u r e 8. A comparison o f the band i n t e n s i t i e s i n each lane o f
the g e l (Figure 7) shows t h a t although the extents o f i n h i b i t i o n
produced by DDP b i n d i n g d i f f e r g r e a t l y f o r the f i v e R. E. s, t h e r e
i s l i t t l e o r no d i f f e r e n c e between the e f f e c t c i s - and trans-DDP
b i n d i n g t o DNA e x e r t s on any one R.E. S i m i l a r r e s u l t s were
obtained a t incubated [DDP/DNA(N)3 mole r a t i o s o f 0.2 and 0.05.
The r e l a t i v e band i n t e n s i t i e s i n d i c a t e t h a t the i n h i b i t i o n o f
cleavage produced by e i t h e r DDP isomer follows the order:
B g l 1&Bam HI > Hpa I I , K£n I > Eco RI
Previous s t u d i e s have l e d to p r e d i c t i o n s (jT) and have shown

experimentally (7^8) t h a t DDP binds p r e f e r e n t i a l l y t o guanines
and t h e r e f o r e t o (G+C) regions i n DNA. Since support f o r t h i s i s
d e r i v e d from both the determination o f c o n d i t i o n a l formation
constants and k i n e t i c measurements (2# 44 ), i t i s expected t h a t
t h i s i s the primary DNA m o d i f i c a t i o n r e s p o n s i b l e f o r the cleavage
i n h i b i t i o n . However, f u r t h e r s t u d i e s by Stone e t a l with
poly(dG) .poly( dC) and poly(dG-dC) showed there was a s t r i k i n g
base sequence e f f e c t on the binding r e a c t i o n ( 9 ) . In f a c t , i t
was t h i s unusually strong cis-DDP b i n d i n g i n poly(dG) .poly( dC)
t h a t stimulated the proposal that t h i s isomer may form
i n t r a s t r a n d c r o s s l i n k s to GpG u n i t s on the poly(dG) s t r a n d . T h i s
mode o f b i n d i n g , o f course, would be s t e r e o c h e m i c a l l y impossible
f o r the trans-DDP t o d u p l i c a t e . In l i g h t o f these arguments, and
a f t e r c o n s i d e r a t i o n o f the n u c l e o t i d e sequence w i t h i n and adja-
cent to the R.E. r e c o g n i t i o n sequence, a number o f a d d i t i o n a l
c o n c l u s i o n s may be drawn with regard to the nature o f DDP i n h i b i -
t i o n o f endonuclease a c t i v i t y .
The observed order o f cleavage i n h i b i t i o n f o r these R. E.s
does not p a r a l l e l the % (G+C) content w i t h i n the r e c o g n i t i o n
sequence as shown i n Table I . The most obvious d i s c r e p a n c i e s are
with Hpa I I and Bam HI. The Hpa I I R.E. i s o n l y moderately i n h i -
b i t e d although the r e c o g n i t i o n sequence i s 100% (G+C), while Bam

SCOVELL ET A L . cis-PtClt(NH )i and
s Simian Virus DNA 1
Figure 7. Cleavage of cis- and trans-DDP modified SV40 DNA by restriction

endonucleases that cut at a single unique site. (Reproduced with permission from
Réf. 16. Copyright 1982, Academic Press.)
Lane 1 shows the mobility of nicked (Ν) and supercoiled (S) forms of SV40 DNA. Lanes
2-6 contain SV40 DNA cleaved with Bam HI, Eco RI, Bgl 1, Ηρα II, and Kpn I, respectively.
These controls demonstrate complete cleavage to the linear (L) form. Lanes 7-11 contain
5
SV40 DNA [9.0 χ 70* M DNA(N)] that was reacted with cis-DDP (1.8 X 10~ M) for 3
h at 25 °C, spot dialyzed, and then cleaved with the restriction enzyme. The order is the
same as in the control series. Lanes 12-16 contain SV40 DNA reacted similarly with trans-DDP
followed by cleavage with restriction enzymes in the same order as controls.
AGCTTCCTGGGÂTCCAGACATGATA
BAM HI
TCG A A U G A C CCCTAGJGTCTG TAC Τ AT
TAGCTCAGAGGCCGAGG'CGGCCTCGGCCTCT
BGL I A T C G A G TC T C PGCC|TCCGCCGGAGCCGGAG A
AGTGT^CTAG'AATT^TTTQ^CTAA
ECO Rl TCACAiJGATCTTAAjGJAAAi&GATT
TiGiiGTGCTGCGdcGÇCTGTCACOSC
ΗΡΑ II AiiACGACGClilpGACAGTGiiG
CG|Q;(OTCAGAAS|GTAEBTAAigS)AAGTT
KPN I
G C We A G Τ C Τ Τ I p A J i i A T T U T TCA A
Figure 8. Cleavage sites in SV40 DNA for restriction endonucleases.

The recognition sequence plus 10 base pairs on each side is shown for Bam HI, Bgl 1, Eco RI,
Ηρα II, and Kpn I. The base pairs essential in the recognition sequence are underlined with a
solid line. Note that the centralfive-base-pairsequence for Bgl 1 underlined with a broken
line is not an essential sequence (22). Sequences of two or more adjacent guanines are shown
stippled. The vertical lines within the recognition sequence designate the cutting sites (16).

HI i s g r e a t l y i n h i b i t e d , yet with o n l y a 67% (G+C) content i n the

r e c o g n i t i o n sequence* We have t h e r e f o r e considered these sequen
ces p l u s 5-10 base p a i r s on e i t h e r side as important i n t h i s
sequence s p e c i f i c protein-DNA i n t e r a c t i o n . T h i s seems reasonable
i n t h a t , although the i n f l u e n c e of DDP b i n d i n g on DNA s t r u c t u r e
i s not expected to be long range, i t s i n f l u e n c e w i l l most l i k e l y
extend w e l l beyond i t s immediate s i t e of b i n d i n g . Therefore, DDP
b i n d i n g to n u c l e o t i d e s immediately adjacent to the r e c o g n i t i o n
sequence or cleavage s i t e s may modulate the extent of cleavage.
Focusing on these l a r g e r base p a i r segments, the cleavage i n h i b i
t i o n produced by DDP b i n d i n g to n u c l e o t i d e s p a r a l l e l s the number
and the r e l a t i v e p o s i t i o n of the guanines i n the sequence. We
f i n d t h a t adjacent guanines enhance the i n h i b i t i o n e f f e c t more so
than non-adjacent guanines, with the i n h i b i t i o n being s i g n i f i
c a n t l y greater i f the number of adjacent guanines i s more than

two. For example, Bam HI, which i s i n h i b i t e d comparably to Bgl 1,
has a sequence with fewer guanines (13 t o 21), fewer adj acent
guanine c l u s t e r s (3 t o 7 ), but has one n u c l e o t i d e t r a c t of four
adjacent guanines, while the l a r g e s t guanine t r a c t o c c u r r i n g i n
the Bgl 1 s i t e i s two. T h i s l a t t e r e f f e c t appears r e s p o n s i b l e
f o r DDP b i n d i n g producing a comparable i n h i b i t i o n o f Bgl 1 and
Bam HI cleavage, although the % (G+C) contents i n the n u c l e o t i d e
sequences d i f f e r s i g n i f i c a n t l y . Base p a i r s i n and about the
r e c o g n i t i o n sequences which have one or more adjacent guanines
are shown s t i p p l e d i n Figure 8. Our f i n d i n g s are c o n s i s t e n t with
those of Kelman and Buchbinder (45) and Ushay e t a l (46) to the
extent t h a t both previous r e p o r t s f i n d that cis-DDP b i n d i n g to λ
DNA and pBR322 DNA, r e s p e c t i v e l y , i n h i b i t s Bam HI c l e a v a g e . In
both s t u d i e s , however, no comparison of the cleavage i n h i b i t i o n
with trans-DDP was c a r r i e d out.
TABLE I. Percentage of (G+C) Content i n and Within 10 Base P a i r s

o f the R e s t r i c t i o n Endonuclease Recognition Sequence
Restric Location -%r (G+C) Content —^

t i o n Endo on Map Recognition Recognition
nuclease Sequence Sequence +
Bgl 1 • 660 91 71
Bam HI • 143 67 50
Hpa II .726 100 74
Kpn I .716 67 50
Eco RI 1.0/0 33 42

5. SCOVELL ET AL. cis-PtCh(NH$)2 and Simian Virus DNA 115
In a d d i t i o n , a s i m i l a r examination with the R.E. Hind I I I (6

cleavage s i t e s i n SV40 DNA) shows d i f f e r e n t i a l cleavage i n h i b i
t i o n a t the s i x s i t e s as a r e s u l t o f c i s - a n d trans-DDP b i n d i n g
(16)· T h i s a n a l y s i s adds f u r t h e r support t h a t the i n c l u s i o n o f
the 5-10 base p a i r s adjacent t o the r e c o g n i t i o n sequence i s
necessary t o e x p l a i n the i n h i b i t i o n e f f e c t . Our f i n d i n g t h a t
sequences o f adjacent guanines i n o r immediately adjacent t o the
recognition-sequence enhance the i n h i b i t i o n e f f e c t agrees with
Cohen e t a l (X2) who found that cis-DDP b i n d i n g on ρSMI DNA pro
duced p r e f e r e n t i a l i n h i b i t i o n a t o n l y one o f the four P s t I
s i t e s , presumably because the modified s i t e was the o n l y one with
a (dG)4(dC)4 c l u s t e r adjacent to i t . However, i n c o n t r a s t to
our f i n d i n g s t h a t both DDP isomers produce comparable i n h i b i t i o n
on a r e s t r i c t i o n enzyme, f o r a l l s i x r e s t r i c t i o n enzymes s t u d i e d ,
Cohen e t a l (Λ2) r e p o r t t h a t the p r e f e r e n t i a l i n h i b i t i o n a t one

o f the four P s t I s i t e s i s unique t o cis-DDP b i n d i n g and i s not
observed with trans-DDP.
The s t u d i e s presented i n t h i s work h i g h l i g h t the f a c t t h a t
d i f f e r e n t probes used to monitor DNA s t r u c t u r e may e x h i b i t very
d i f f e r e n t s e n s i t i v i t i e s t o the m o d i f i c a t i o n o f i n t e r e s t . For
3
example, both the e l e c t r o p h o r e t i c m o b i l i t y study with c s SV40
DNA and the S1 nuclease d i g e s t i o n on DNA c l e a r l y b r i n g out d i f
ferences i n the c i s - and trans-DDP modified DNA. Both these pro
bes are s e n s i t i v e t o , and, i n t h i s case, r e v e a l d i f f e r e n c e s i n
the mode o f b i n d i n g o f the two isomers. However, we f i n d that
f o r a p a r t i c u l a r r e s t r i c t i o n endonuclease, l i t t l e o r no d i f f e r e n
ces are observed between the cleavage i n h i b i t i o n on the modified
DNAs. The endonucleases used i n t h i s study, t h e r e f o r e , appear to
be l e s s s e n s i t i v e to the mode o f b i n d i n g used and are i n f l u e n c e d
predominantly by the l o c a l p h y s i c a l coverage o f DDP a t o r adja
cent to the r e c o g n i t i o n sequence. We have found t h i s same e f f e c t
t o occur with a more general nuclease, DNAase I , which d i g e s t s
both c i s - and trans-modified DNA with equal ease (47).
Segments o f t h e Regulatory Region o f t h e SV40 Genome A r e Hyper

r e a c t i v e Toward DDP B i n d i n g
The r e l a t i v e order o f cleavage i n h i b i t i o n f o r the f i v e
s i n g l e cut r e s t r i c t i o n endonucleases studied suggests the r e g u l a
t o r y r e g i o n , and s p e c i f i c a l l y the sequences i n and about the B g l
1 and perhaps the Bam HI r e c o g n i t i o n sequences, a r e hyper
r e a c t i v e s i t e s t o DDP b i n d i n g i n the genome. Further s t u d i e s ,
however, may r e v e a l t h a t these are but two o f a number o f such
s i t e s . These two sequences correspond to the s i t e a t o r near the
o r i g i n o f r e p l i c a t i o n and the general region a t which DNA r e p l i
c a t i o n terminates, r e s p e c t i v e l y (17,23,48). Beard e t a l . (49)
have r e c e n t l y reported t h a t the ultimate carcinogen, N-acetoxy-
acetylaminofluorene (AAAF), which binds almost e n t i r e l y t o guani
nes i n DNA, r e a c t s p r e f e r e n t i a l l y with the c o n t r o l r e g i o n i n
i n t r a c e l l u l a r SV40 minichromosome. Our f i n d i n g s with DDP, taken

together with t h a t f o r AAAF, would suggest t h a t agents which

b i n d p r e f e r e n t i a l l y to guanines may e x h i b i t a preference f o r
b i n d i n g i n the r e g u l a t o r y r e g i o n i n SV40 DNA and the minichromo-
some. I t i s of p a r t i c u l a r importance to consider of what r e l e
vance these f i n d i n g s on DNA are when extended to the minichromo-
some s i n c e t h i s would be the a c t u a l c e l l u l a r t a r g e t of i n t e r e s t .
In t h i s regard, a number of l a b o r a t o r i e s using e l e c t r o n
microscopy (50,51), d i g e s t i o n s with nucleases (52-55), i n c l u d i n g
B g l 1, have found that the region i n or about the o r i g i n of
r e p l i c a t i o n i s t o p o l o g i c a l l y exposed, nucleosome f r e e (56) and
encompasses sequences c r i t i c a l to gene expression and r e g u l a t i o n
(17,19,57,58,59)· T h i s gapped region of about 400 bps extends
from about the Bgl 1 s i t e to the Hpa I I s i t e and i s apparent a t
the top of Figure 9 which shows an e l e c t r o n micrograph of the
SV40 minichromosome. T h i s s t r u c t u r a l c h a r a c t e r i s t i c of a f r a c
t i o n of the minichromosomes may enhance the r e a c t i v i t y of t h i s
s i t e to exogenous small molecules, with the expectation being
t h a t t h i s would be e s p e c i a l l y true f o r many molecules e x h i b i t i n g
a preference f o r (G+C) r i c h r e g i o n s .
Since i t has been shown here that DDP b i n d i n g i n h i b i t s a
p a r t i c u l a r c l a s s of sequence s p e c i f i c protein-DNA i n t e r a c t i o n s
and that the SV40 DNA cleavage by Bgl 1 i s most i n h i b i t e d , i t
might be expected that other s i t e s p e c i f i c protein-DNA i n t e r a c
t i o n s i n or about the o r i g i n of r e p l i c a t i o n may be modulated by
DDP b i n d i n g . Relevant to t h i s p o i n t and p o s s i b l y of some connec
t i o n between the p r e f e r e n t i a l binding a f f i n i t y of DDP a t the Bgl
1 r e c o g n i t i o n sequence and the reported i n f l u e n c e of cis-DDP on
i n t r a c e l l u l a r SV40 v i r a l f u n c t i o n s (25), i t i s i n t r i g u i n g to spe
c u l a t e how cis-DDP b i n d i n g may i n f l u e n c e the b i n d i n g of T-antigen
t o i t s s p e c i f i c binding s i t e s a t or near the Bgl 1 r e c o g n i t i o n
sequence (60,61,62). As noted above, the s i t e s p e c i f i c binding
o f Τ antigen to DNA i s i n v o l v e d i n both the a u t o r e g u l a t i o n of Τ
antigen t r a n s c r i p t i o n and i s required f o r the i n i t i a t i o n of v i r a l
DNA s y n t h e s i s (V7,18,19). T j i a n (60) has shown t h a t the Τ a n t i
gen r e l a t e d p r o t e i n D2 binds s p e c i f i c a l l y to three d i s t i n c t and
c l o s e l y spaced r e c o g n i t i o n sequences a t and w i t h i n 60-70.base
p a i r s of the Bgl 1 s i t e . T-antigen b i n d i n g s i t e I I , which i s a t
the Bgl 1 r e c o g n i t i o n sequence, i s e n t i r e l y contained w i t h i n the
c u r r e n t l y known boundaries of the o r i g i n of r e p l i c a t i o n (23) · In
a d d i t i o n , methylation s t u d i e s on the D2 protein-DNA complex
revealed t h a t a m a j o r i t y of the guanines, many of which occur i n
c l u s t e r s of two or three i n the three b i n d i n g s i t e s , are s h i e l d e d
from methylation and t h e r e f o r e thought to be i n v o l v e d i n c r i t i
c a l l y important c o n t r a c t s i n the r e g u l a t o r y i n t e r a c t i o n . The
r e s u l t s of Shalloway (62), who used E. c o l i Exonuclease I I I t o
probe the T-antigen b i n d i n g s i t e , support T j i a n ' s c o n c l u s i o n s ,
but a l s o r e v e a l a d d i t i o n a l binding s i t e s i n the same general
r e g i o n which i n c l u d e the 6 times r e i t e r a t e d sequence 6 3 . 4 ^ 2 ? ^ ·
C o l l e c t i v e l y , these r e s u l t s emphasize the importance of t h i s

5. SCOVELL ET AL. cis-PtChfNHsh and Simian Virus DNA 117
Figure 9. Electron micrograph of an SV40 minichromosome that shows a stretch

of the génome devoid of nucleosomes. This gap has been mapped from the Bgl 1
site to about 0.74 on the physical map. The nucleosomes appear as white circular
clusters along the circular DNA. (Reproduced with permission from Ref. 50. Copy-
right 1980, Massachusetts Institute of Technology.)

(G+C) r i c h region o f the genome and the importance and i n v o l v e

ment o f c l o s e , w e l l - d e f i n e d contacts o f the guanines i n DNA i n
r e g u l a t o r y i n t e r a c t i o n s with Τ a n t i g e n . Therefore, any molecule
which d i s r u p t s or weakens t h i s sequence s p e c i f i c T-antigen-DNA
i n t e r a c t i o n may be expected to have an i n f l u e n c e on developmental
f u n c t i o n s o f the v i r u s . Although the d e t a i l e d nature of cis-DDP
and trans-DDP b i n d i n g to t h i s or other p a r t s of the genome and
the i n f l u e n c e t h a t the isomers may exert on SV40 v i r a l gene
expression i s now s p e c u l a t i v e , there are a number o f t e s t a b l e
p r e d i c t i o n s which should help t o understand the most s a l i e n t
f e a t u r e s which r e l a t e DDP b i n d i n g to the impairment o f s e l e c t e d
b i o l o g i c a l functions.
Conclusions
1. Cis-DDP unwinds SV40 DNA twice as e f f e c t i v e l y as trans-DDP

(15).
2. Cis-DDP d i s r u p t s the s t r u c t u r e of DNA t o a greater degree
than does trans-DDP a s measured by the greater s t i m u l a t i o n
o f S1 nuclease d i g e s t i o n on cis-DDP modified DNA ( 31_) ·
3. These data i n d i c a t e t h a t o f the modes o f binding that the
c i s - and trans-DDP e x h i b i t , the cis-DDP e x h i b i t s a mode o f
b i n d i n g which i s d i f f e r e n t than t h a t f o r trans-DDP and i s
r e s p o n s i b l e f o r the more e f f e c t i v e unwinding of the DNA
backbone and f o r the greater d i s r u p t i o n of DNA s t r u c t u r e
r e s u l t i n g i n S1 nuclease s e n s i t i v i t y . A bidentate mode o f
b i n d i n g by cis-DDP t o form e i t h e r ( 1 ) an i n t r a s t r a n d
c r o s s l i n k to adjacent guanines or p o s s i b l y (2) an N-7, 0-6
c h e l a t e complex with guanine may e x p l a i n our observations.
Current evidence more s t r o n g l y supports proposal ( 1 ) .
4. The region o f the B g l 1 s i t e on SV40 DNA i s a h y p e r - r e a c t i v e
s i t e f o r both c i s - and trans-DDP b i n d i n g (16) · This i s w i t h i n
the r e g u l a t o r y region o f SV40 v i r u s and may be one of the
primary l e s i o n s d i r e c t l y r e s p o n s i b l e f o r the impairment o f
v i r a l r e p l i c a t i o n and t r a n s c r i p t i o n i n SV40 i n f e c t e d A f r i c a n
green monkey kidney c e l l s .
3
^Legend o f Symbols: DDP, PtCl2(NH3)2; c s DNA, c o v a l e n t l y ,
c l o s e d , c i r c u l a r s u p e r c o i l e d DNA; b.p., base p a i r s ; DNA(N), DNA
concentration expressed i n m o l a r i t y o f n u c l e o t i d e s by using 1
O.D. » 50 ρ g DNA/ml; r ^ , mole r a t i o of DDP bound to DNA
n u c l e o t i d e ; Pu, purine base; V , the number of super c o i l s i n the
DNA;ot — the t o p o l o g i c a l l i n k i n g number f o r DNA.
Acknowledgments
We thank Mr. B i l l Butcher f o r a s s i s t a n c e with the pho
tographic work and to Ma they Bishop Co. f o r the loan o f cis-DDP
and the N a t i o n a l Cancer I n s t i t u t e f o r the g i f t of trans-DDP.
T h i s work was supported by the Ohio D i v i s i o n of the American
Cancer S o c i e t y , ttie Bowling Green State U n i v e r s i t y Biomedical
Research Support Grant, Alumni A s s o c i a t i o n and Parents Club,

5. SCOVELL ET AL. cis-PtCh(NH$h and Simian Virus DNA 119
Faculty Research Committee and The American Cancer Society

Support Grant number IN-130.
I would like this contribution dedicated to the memory of
Professor R. Stuart Tobias, a distinguished scholar and good
friend*
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6
Platinum-Oligonucleotide Structures and Their
Relevance to P l a t i n u m - D N A Interaction
JEAN-CLAUDE CHOTTARD, JEAN-PIERRE GIRAULT, and ERIC R.
GUITTET—Ecole Normale Supérieure, Laboratoire de Chimie, 75231 Cedex 05,
Paris, France
JEAN-YVES LALLEMAND—Laboratoire de RMN, CNRS, ICSN,
91190 Gif-sur-Yvette, France
GENEVIEVE CHOTTARD—Université Pierre et Marie Curie, Département de
Recherches Physiques, 75230 Cedex 05, Paris, France
The stoichiometric reactions of nine oxy and deoxy

guanine and/or cytosine containing dinucleotides with
-5 -4
cis - [Pt(NH ) (H O) ] (NO ) (10 - 5 x 10 M) in water
3 2 2 2 3 2
give monomeric platinum dinucleotide chelates in every case.

The complexes have been isolated by HPLC and characterized
1
by H NMR and CD analyses. GpG, d - GpG and d - pGpG
give a single N7 - N7 anti-anti complex. CpC and d-pCpC
give a single N3 - N3 syn - anti complex. CpG and d-pCpG
give a mixture of N3 - N7 C anti - G anti and C syn-G anti
isomers in equilibrium. GpC and d - pGpC give two couples
of N7 - N3 isomers: G syn - C anti, G syn - C syn(in equi-
librium) and G anti - C anti, G anti - C syn . The results
obtained point to a particular chelating aptitude of the
anti - anti GG sequence. Accordingly, the stoichiometric
reaction of the hexanucleotide d - TpGpGpCpCpA with the
platinum complex gives quantitatively the GN7 - GN7 chelate.
These results are in favor of the hypothesis of platinum
intrastrand cross-linking of adjacent guanines in DNA.
The mechanism of the cytotoxic action of the cis - diammine -

dichloroplatinum (II) complex (cis - DDP) is s t i l l unknown but there
is much evidence indicating that DNA is the main target in a
variety of biological systems (1). Three hypotheses are being
considered that imply a bifunctional coordination of the cis -
Pt I I (NH 3 ) 2 moiety to DNA: interstrand cross-linking (2, 3^, 4),
intrastrand cross-linking (_5, 6) and N(7) - 0(6) chelation of a
guanine (7_9 8) . The last two binding modes could be a first step
followed by further cross-linking reactions (6,jS). Intrastrand
cross-linking appears to be involved in the inactivating lesions
produced by cis - DDP in phage λDNA (9) . Data obtained from
platinum coordination to nucleotides (10, 11, 12) oligonucleotides
(13, 14, 15) and polynucleotides C5, 6) suggest that cross-linking
of two adjacent guanines could be a favored process. However,
platinum chelation by adjacent inosines seems to be a minor event
0097-6156/83/0209-0125$06.25/0

f o r the i n t e r a c t i o n o f c i s - DDP w i t h p o l y - I and p o l y - I . poly-C

(16). I t has been shown t h a t c i s - DDP , a t low platinum/nucleotide
r a t i o s , s e l e c t i v e l y i n h i b i t s the a c t i v i t y o f s e v e r a l r e s t r i c t i o n
endo-and exo - nucleases when t h e i r c u t t i n g s i t e s a r e adjacent t o
(dG) - (dC)
n n sequences w i t h η > 2 . This demonstrates the
sequence s p e c i f i c i t y o f the b i n d i n g o f c i s - DDP t o DNA (J7, J^,
19)· GAG and GCG sequences a l s o appear to be s e l e c t i v e l y
i n v o l v e d i n base - p a i r s u b s t i t u t i o n mutagenesis and t h i s suggests
the p o s s i b i l i t y o f platinum c h e l a t i o n by two guanines separated by
a t h i r d base (20).
The aim o f our work was t o i n v e s t i g a t e , from a chemical p o i n t
of view, the p o s s i b i l i t y o f i n t r a s t r a n d c r o s s - l i n k i n g of two
adjacent bases by the c i s - P t * * ( N H ) moiety. The s i m p l e s t models
3 2
to t e s t t h i s c h e l a t i o n are the d i n u c l e o t i d e s . I t i s known that

platinum b i n d i n g to DNA increases w i t h the %(G + C) content (21,22)

and we have t h e r e f o r e i n v e s t i g a t e d the f o l l o w i n g d i n u c l e o t i d e s :
GpG, d - GpG, d-pGpG (14, 23); CpG, d - pCpG ; GpC, d - pGpC and
CpC, d-pCpC. P r e l i m i n a r y r e s u l t s have a l s o been obtained w i t h the
hexanucleotide d - TpGpGpCpCpA .
ExpeAunzrvtaJL
The d i n u c l e o t i d e s were commercial ( e i t h e r ammonium s a l t o r

f r e e a c i d ) , the hexanucleotide was synthesized by Drs. J . I g o l e n
and T. Huynh-Dinh (Unité de Chimie Organique, I n s t i t u t Pasteur,
Paris).
The s t o i c h i o m e t r i c r e a c t i o n s , 1:1 c i s - [ P t ( N H ) ( H 0 ) ] ( N 0 ) /
3 2 2 2 3 2
5 -lf
o l i g o n u c l e o t i d e were run a t 10~ M t o 5 χ 10 M c o n c e n t r a t i o n
(^2 mg o f o l i g o n u c l e o t i d e ) a t pH *\J 5.5 and 37°C, i n doubly
d i s t i l l e d water. I n these c o n d i t i o n s the diaquo and monoaquomono-
hydroxy species a r e predominant (pKi - 5.51, p K = 7.37) and there 2
i s no s i g n i f i c a n t d i m e r i s a t i o n o f the l a t t e r (24). I n each case

comparative r e a c t i o n s have been r u n w i t h c i s - [ P t ( N H ) C l ] i n the 3 2 2
same c o n d i t i o n s . The r e a c t i o n s were monitored by UV and HPLC

and the r e a c t i o n products were analysed by Sephadex chromatography
(14), h i g h pressure g e l permeation chromatography and HPLC on a
Waters C 18 μ-Bondapak (reverse phase) column (14, 23).
The *H NMR s p e c t r a were recorded on a Cameca TSN 250
(250 MHz, N i c o l e t 1180 computer) o r on a WM 400 Bruker (400 MHz,
Aspect 2000) spectrometer (14, 23). The concentrations o f the
3 3
samples were 5 χ 10" t o ^ 10~ M i n D 0 . Chemical s h i f t s are 2
r e f e r r e d t o i n t e r n a l DSS (sodium 4,4 - dimethyl - 4 - s i l a p e n t a n e -

1 - s u l f o n a t e ) o r TSP (sodium 3 - t r i m e t h y l s i l y l - 2, 2, 3, 3 di» -
1 - p r o p i o n a t e ) . The CD s p e c t r a were recorded on a J o b i n Yvon
Mark I I I . The Ae(eL ~ £R> M" . cm" ) are given per n u c l e o t i d e
1 1
r e s i d u e . The molar e x t i n c t i o n c o e f f i c i e n t s o f the complexes were

determined from the r a t i o o f the o p t i c a l d e n s i t i e s o f the o l i g o
n u c l e o t i d e s o l u t i o n before and a f t e r i n c u b a t i o n w i t h the P t ( I I )
complex, assuming t h a t no c o n c e n t r a t i o n change had occurred. When a
mixture o f complexes i s formed, only a mean e x t i n c t i o n c o e f f i c i e n t

6. CHOTTARD ET AL. Pt-Oligonucleotide Structures 111
i s obtained by t h i s procedure; t h i s mean value has been used f o r

the d i f f e r e n t isomers separated by HPLC .
Analy&<u> th<L R&acXiorU
The r e a c t i o n s r u n e i t h e r w i t h c i s - [ P t ( N H ) ( H 0 ) ] ( N 0 ) o r
3 2 2 2 3 2
c i s - [ P t ( N H ) C l ] gave the same complexes o r mixtures o f

3 2 2
complexes. Those w i t h the d i c h l o r o complex were slower, e.g. the

second order r a t e constant f o r the disappearance o f GpC i s about
3 1 1
ten times smaller f o r the d i c h l o r o ('W χ 10 h " M" ) than f o r
the diaquo r e a c t i o n .
UV and HPLC monitoring o f the r e a c t i o n s r u n w i t h the diaquo
f
complex show that the presence of a f r e e 5 - phosphate increases
the r a t e of disappearance o f the d i n u c l e o t i d e (23). I n some cases
t h i s a c c e l e r a t i o n allowed us t o detect the formation o f an i n t e r

mediate by HPLC , that i s more slowly converted t o the f i n a l
complex(es). Such intermediates can a l s o be seen f o r r e a c t i o n s run
2
a t higher c o n c e n t r a t i o n (10~ M) but together w i t h the formation o f
new products ( f u r t h e r i n v e s t i g a t i o n s are i n p r o g r e s s ) . For a l l the
d i n u c l e o t i d e s s t u d i e d : GpG, d-GpG, d-pGpG, CpG, d - pCpG, GpC,
d-pGpC, CpC, d-pCpC , there were no unreacted s t a r t i n g m a t e r i a l
at the end o f the s t o i c h i o m e t r i c r e a c t i o n s w i t h
c i s - [ P t ( N H ) ( H 0 ) ] ( N 0 ) . We s t i l l have t o c o l l e c t p r e c i s e
3 2 2 2 3 2
comparative k i n e t i c data f o r the d i f f e r e n t cases, however we can

say that the diguanosine phosphates give the f a s t e s t o v e r a l l
r e a c t i o n s , whereas the d i c y t o s i n e phosphates give the slowest. For
example, f o r CpC 2.5 χ 10~**Μ, the f i n a l platinum c h e l a t e appears
a f t e r 6 hours and the r e a c t i o n i s complete a f t e r about one week,
_1
w h i l e f o r CpG 1.5 χ 10 *M the formation o f the c h e l a t e s i s over
a f t e r 17 hours.
HPLC a n a l y s i s o f the products r e s p e c t i v e l y e x h i b i t s one peak
f o r the GG d i n u c l e o t i d e s , two peaks f o r the CG d i n u c l e o t i d e s ,
three peaks f o r the GC d i n u c l e o t i d e s and one peak f o r the CC
d i n u c l e o t i d e s . I n each case these peaks represent a t l e a s t 95% o f
the o v e r a l l product. Sephadex and g e l permeation chromatographies
show that the complexes formed a r e monomeric. I n the CG cases
the two complexes e q u i l i b r a t e a f t e r HPLC s e p a r a t i o n , as do the
two complexes corresponding to peaks 1 and 3 ( e l u t i o n order) i n
the GC cases.
AàAÎgnm&rvt o& th<L Platinum Cootidincution Sit<tt>
The p o s s i b l e c o o r d i n a t i o n s i t e s of the s t u d i e d d i n u c l e o t i d e s
are guanine N7(G-N7), guanine N l and c y t o s i n e N3 (C - N3) (25).
I n our c o n d i t i o n s ( s t o i c h i o m e t r y o f the r e a c t i o n and pH) G - N l
T
c o o r d i n a t i o n i s very u n l i k e l y (11). For s e v e r a l 5 - GMP mono o r
b i s complexes, N7 c o o r d i n a t i o n induces a c h a r a c t e r i s t i c
0.4-0.7 ppm downfield s h i f t o f the H8 i n α p o s i t i o n (26, 27, 11).
I t i s very l i k e l y t h a t , i n s o l u t i o n the c i s - b i s (nucleotide)
P t H complexes have the two purines i n a h e a d - t o - t a i l arrangement

(12, 28). Platinum c o o r d i n a t i o n t o C - N3 o f Cyd and 5' - CMP

gives s m a l l e r downfield s h i f t s o f the H6 and H5 s i g n a l s ,
r e s p e c t i v e l y 0.11-0.28 and 0.12-0.26 ppm (27, 29). I n most
cases, f o r the platinum - n u c l e o t i d e complexes, one cannot observe,
1 9 5 X
a t high magnetic f i e l d s , the expected Pt - H c o u p l i n g constants
1 9 5
because o f a dominant Pt chemical s h i f t anisotropy r e l a x a t i o n
(30). ·* Platinum c o o r d i n a t i o n i s e a s i l y demonstrated by the d i s -
appearance o f the f r e e d i n u c l e o t i d e c h a r a c t e r i s t i c N7 and/or N3
t i t r a t i o n curves observed f o r the v a r i a t i o n o f the H8 and H5
f
chemical s h i f t s as a f u n c t i o n o f pH ( 5 - G M P - N 7 p K = 2.4 ,a
f
5 - C M P - N 3 p K - 4.3). F o r the f r e e d i n u c l e o t i d e s , the downfield
a
s h i f t s obtained upon a c i d i f i c a t i o n down t o pD2 a r e ^ 0 . 9 ppm f o r

G-H8 i n GpG (23) GpC and CpG and r e s p e c t i v e l y ^ 0 . 3 5 ppm
f o r C-H5 and ^ 0 . 2 5 ppm f o r C - H6 i n GpC and CpG). For
a l l the platinum d i n u c l e o t i d e complexes c o n t a i n i n g a guanine, the

curves o f the v a r i a t i o n o f the H8 chemical s h i f t s versus pD
e x h i b i t a t i t r a t i o n o f the NH1 ( p K 8-8.7) that r u l e s out a
a
platinum c o o r d i n a t i o n t o G - N l . Another consequence o f G-N7

c o o r d i n a t i o n i s u s u a l l y an a c c e l e r a t i o n o f the H8 deuterium
exchange w i t h D2O a t b a s i c pD (11). Such an a c c e l e r a t i o n occurs
f o r the GG complexes (23) but i s not observed f o r s e v e r a l isomers
of the CG and GC complexes a t 37°C, probably because o f neigh-
b o r i n g e f f e c t s . For a l l the platinum - d i n u c l e o t i d e complexes
s t u d i e d , the assignment o f the c o o r d i n a t i o n s i t e s leads t o the
c o n c l u s i o n that they are platinum c h e l a t e s , N7 - N7 f o r the GG
s e r i e s (14, 23), N3 - N7 f o r the CG s e r i e s , N7 - N3 f o r the GC
s e r i e s and N3 - N3 f o r the CC s e r i e s . The s t o i c h i o m e t r i c
r e a c t i o n o f [ P t ( d i e n ) B r ] Br w i t h CpG and GpC gives only the
G-N7 monocoordinated complex i n each case.
AàAÎgvumntà o& thz Bcu>z ConjiQUJiaXLovib and SugaA ConôûmZiorU
These assignments are made by *H NMR according to w e l l

documented analyses o f n u c l e o t i d e s (31) and o l i g o n u c l e o t i d e s
f
(32, 33, 34). The i d e n t i f i c a t i o n of the 5 - sugar i s based on the
3 1 f
Ρ - i H c o u p l i n g observed f o r i t s 3 - p r o t o n . The r e l a t i v e
r i g i d i t y o f the platinum d i n u c l e o t i d e chelates and the f a c t that we
have i s o l a t e d most o f the d i f f e r e n t isomers, f a c i l i t a t e the a s s i g n
ment o f the syn o r a n t i c o n f i g u r a t i o n t o the bases (syn and
a n t i r e s p e c t i v e l y correspond t o γ = 0 ± 90° and 0 ( - 180 ± 90°
f f
w i t h 9( = 0 ( 4 ) C ( D N(9) C(4) f o r purines and 0 ( 4 ) C ( l »)N(1)C(2)
f o r p y r i m i d i n e s (31)) and o f the conformation type t o the sugar
rings.
GpG, d - GpG and d - pGpG Platinum Complexes. Only one complex

i s obtained f o r each d i n u c l e o t i d e . The comparative *Η NMR data a r e
c o l l e c t e d i n Table I . The a n t i - a n t i c o n f i g u r a t i o n has been
p r e v i o u s l y assigned t o these three complexes (23) on the b a s i s o f
the f o l l o w i n g r e s u l t s : - a - The comparison o f the 3 - and 1
5'-H8 deuterium exchange r a t e s i n D 0 (pD 10) w i t h those o f the

2

3,b
Table I . *H NMR data for the GpG [Pt] , d - GpG [Pt] and d -PGPG [ Pt] complexes
GG GG [Pt]
M j δ J δ J Δδ
s 8.54 s + 0 . 5 5 , + 0.65
H8 Gp ,Î 7 . 9 7 *
GpG C + 0.35, + 0.4
pG k 7.90 s 8.32 s
(pD5.6)
GpG[Pt] d5 6.06 s + 0.2 , + 0.3
1
Gp ι 5.85
(pD5.5) HI 5.90 d7 + 0 . 0 5 , + 0.15
pG 1 5.76 d3
8 0 2 s 8.28 s + 0.25, + 0.5

d-GpG H8 i - r
C
pG [7.76 s 8.56 s + 0 . 5 5 , + 0.8
(pD 6 . 1 )
d-GpG[Pt] Î6.16 t
1 + 0.05, + 0.2
(pD5.5) Hi j 6.22
pG [6.01 t
8.51* s + 0.5
PGP d
H8 {8.02 + 0.75
d-pGpG pG 8.79 s
d-pGpG [Pt]
pGp /6.17 . t 6.19 d6 0, + 0.15
(pD 6 . 6 ) 1
HI d
6.28 q 6^ 10 + 0.1 , + 0.25
pG \6.02

a 3
2 5 0 MHz; samples 2
1 0 ~ M i n D 0 ; 1 7 ° C ; chemical s h i f t s (δ) i n ppm from DSS ;
* b
c o u p l i n g constants ( J ) i n Hz. a brace i s used f o r the δ values when the
c a
s i g n a l s have not been assigned t o each n u c l e o s i d e . s i g n a l broadening. broad
1
H8 o f c i s - b i s ( G u o ) and c i s - b i s ( 5 - GMP) p l a t i n u m complexes,
f
and the comparison o f the v a r i a t i o n of the 3 - and 5' -H8 chemical
s h i f t s vs.pD, show that both H8 protons o f d - pGpG [ P t ] experience
f 1
the p r o x i m i t y o f the f r e e 5 - phosphate. The 5 -G-H8 i s more
s l o w l y exchanged and much more s e n s i t i v e t o the 5'-phosphate
t i t r a t i o n (35). - fa - The absence o f a s i g n i f i c a n t i n c r e a s e o f the
1
Ti r e l a x a t i o n time o f the two H I protons o f GpG [ P t ] a f t e r the
complete deuterium exchange o f the two H8 p r o t o n s , excludes a
guanine syn c o n f i g u r a t i o n (36).
7 f
The absence of one H l " ^ H2 c o u p l i n g constant f o r the
GpG [ P t ] and d - pGpG [ P t ] complexes (as w e l l as f o r I p l [ P t ] and
ApA [ P t ] (14)) has a l s o been r e p o r t e d and taken as evidence f o r
an Ν - type conformation (31) of one furanose r i n g , supposed t o be
1
the 5 - one from examination o f the CPK models ( 2 3 ) .
CpG and d - pCpG P l a t i n u m Complexes. I n each case two isomers,

1 ( M 5 % ) and Ζ (^85%) i n the HPLC · e l u t i o n o r d e r , are obtained
that e q u i l i b r a t e a f t e r s e p a r a t i o n a t room temperature. The 400 MHz
*H NMR spectrum of t h e i r mixture i s presented on F i g u r e 1 f o r the
CpG case. The comparative *H NMR data o f the oxy and deoxy
isomers a r e c o l l e c t e d i n Table I I together w i t h those of the G-N7
monocoordinated complex [ P t ( d i e n ) ( C p G ) ] B r . L i k e i n the GG
2
s e r i e s , there i s an o v e r a l l s i m i l a r i t y between the CpG and the

d - pCpG complexes.
We a s s i g n the C a n t i - G a n t i c o n f i g u r a t i o n t o isomerà j, and
the C syn - G a n t i c o n f i g u r a t i o n t o the more abundant isomers 2
on the b a s i s o f the f o l l o w i n g r e s u l t s : - a - For the CpG [ P t ] - 2
isomer there i s a n u c l e a r Overhauser enhancement o f about 10%
f
between the C - H6 and C - H I protons. The T i r e l a x a t i o n time
of these protons i s 0.2 s a t 17°C and 250 MHz, w h i l e that o f the
C-H6 and C - Η Γ o f the CpG [ P t ] - 1 isomer i s 0.6s . - fa - For
CpG [ P t ] - 2 the C - Η Γ experiences a 0.4 ppm u p f i e l d s h i f t ,
compared t o i t s chemical s h i f t i n CpG , t h a t i s accompanied by a
f
0.8 ppm d o w n f i e l d s h i f t o f the corresponding H3 . Such s h i f t s
are not observed f o r CpG [ P t ] - 1 . These data r e s p e c t i v e l y
support C a n t i and C syn c o n f i g u r a t i o n s f o r the 1 and 2
isomers (38 - 41). - c - For the d - pCpG [ P t ] - 2 isomer, u p f i e l d
and downfield s h i f t s of C - HI ' and C-H3' are a l s o observed.
- d - For the two d-pCpG [ P t ] complexes o n l y the C - H6 chemical
s h i f t o f the I isomer i s s e n s i t i v e to the t i t r a t i o n o f the f r e e
f
5 - phosphate (Δ6 + 0.2 ppm w i t h pH i n c r e a s e ) (35). - e - For
both isomers, i n the oxy s e r i e s , t h e r e i s no NOE between G-H8
and G - Η Γ . The T i r e l a x a t i o n times, a t 17°C and 250 MHz, f o r
isomers 1 and 2 are r e s p e c t i v e l y 0.6 and 0.5 s f o r G-H8
1
and 0.6 s" f o r both G - H I . This i s evidence f o r a G- a n t i
c o n f i g u r a t i o n i n both 1 and 2 isomers. Examination o f CPK
models suggests that the s m a l l e r d o w n f i e l d s h i f t s o f G-H8 i n
the oxy and deoxy isomers I , compared to those i n isomers 2 ,
could be r e l a t e d t o the r i n g c u r r e n t e f f e c t of the adjacent a n t i
cytos i n e .

1
Figure L H-NMR spectrum (400 MHz) of the mixture of the CpG[Pt]-l and -2
3
isomers(15:85). Isomer 1 data are underlined. Conditions: ΙΟ M in D%0 at 15 °C.
Chemical shifts are in ppm from TSP.

to
l 5 3
Table II. H NMR data for the CpG[Pt1 and d-pCpG[Pt] complexes '*
CG CG [ Pt ] - 1 CG [ Pt ] - £
Δ6 Δ6
H8 8.02 s 8.22 + 0.2 8.44 + 0.4

H6 7.76 d 7.9 8.10 d 7.7 + 0.35 7.62 d 7.7 - 0.15
H5 5.87 d 7.9 6.03 d 7.7 + 0.15 5.99 d 7.7 + 0.1
1 6
CpG C - HI 5.73 d 3.2 5.78 s + 0.05 5.32 s - 0.4 w
H
CpG [ Pt ] 1 €
5.96 + 0.05 >
G - HI 5.90 d4.3 5.83 d 6 - 0.05 d 7.7
(pD7.4)
m m s
f
5.23 + 0.8
C - H3 4.44 f f
, I
(3 4 :6.7) (3 4 :9.8) §
H
f m 4.43 m - 0.1 Β
G - H3 4.51 f f
, I
(3 4 :5.4) (3 4 :2.5) m
>

M

C
H
Ο
%
M
H
8.1 ' 8.19 s + 0.1 8.42 s + 0.3

H8
7.83 d 7 8.13 d 7.5 + 0.3 7.52 d 7.5 - 0.3
H6
H5 * 6 6.01 d 7.5 5.91 d 7.5
d - pCpG
(pD5.5) 5.65 q - 0.5
ΗΓ 6.13 6.06 m 7;3.5
d-pCpG[Pt]
(pD 6.5) - 0.05,- 0.2
6.29 q 0
G - ΗΓ 6.26 6.09 m 10; 5
5.25 > + 0.3
C - H3»
a 3
400 MHz ; samples * 10" M i n D 0; 15°C ; chemical s h i f t s (δ) i n ppm from TSP ; c o u p l i n g contants (J) i n
2
b
Hz. a brace i s used f o r the δ values when the s i g n a l s have not been assigned to each n u c l e o s i d e .
c 2 +
For [Pt(dien)(CpG)] a t pD 7.5, 6(J) Δδ : H8, 8.51(e) + 0.5 ; H6, 7.83(d8) + 0.05; H5 * 5.9(d) 0 ;
f 1
C - H l / G - H I , 5.9/5.96 ( d 4 / d 4) 0,+ 0.15/0.05,+ 0.2 . °from r e f . 43. a s s i g n e d on the b a s i s
of the absence o f Ji» . c o u p l i n g t h a t has been found c h a r a c t e r i s t i c o f the 5» - n b o s e m a l l the studied
2
d i n u c l e o t i d e complexes (Tables I and I I I ) . 'broad.

For the J. and 2 isomers o f the oxy complex, the absence o f

, I
C - H 1 H 2 c o u p l i n g shows that the 5'-ribose adopts an Ν-type
1 f
conformation ( C 3 - endo) w h i l e the 3 - r i b o s e has a predominantly
S - type conformation (42). For the deoxy isomers the ( J i i + J i 2")
f
values show that the 5 - and 3'-sugar conformations a r e r e s p e c t
i v e l y predominantly o f the Ν - and S - type (42).
GpC and d - pGpC P l a t i n u m Complexes. I n each case a mixture of

isomers i s obtained which can be separated i n t o three f r a c t i o n s by
HPLC (numbered 1, 2, 3 i n the e l u t i o n o r d e r ) . F r a c t i o n s 1 and 3
c o n t a i n o n l y one complex each, r e s p e c t i v e l y I and 3 . These
complexes e q u i l i b r a t e a f t e r HPLC s e p a r a t i o n . This e q u i l i b r a t i o n
i s slow enough t o a l l o w independent s p e c t r o s c o p i c i n v e s t i g a t i o n s of
isomers I and 3 a f t e r c o l l e c t i o n a t low temperature. F r a c t i o n 2
contains two isomers 2a and 2 i · For GpC , the GpC [ Pt] - 2a

1
isomer represents about 95% o f f r a c t i o n 2 ( H NMR), and the three
main complexes GpC [ Pt ] - I , - 3 and - 2a are about i n 1:1:1
r a t i o s . F o r d - pGpC the f o u r isomers d - pGpC [ P t ] - \ , - 3, - 2a
and - 2J? a r e i n ca. 40:10:25:25 r a t i o s . T h e i r comparative
*H NMR data a r e c o l l e c t e d i n Table I I I , together w i t h those o f
the G-N7 monocoordinated complex [ P t (dien) (GpC) ] B r . L i k e i n 2
the GG and CG s e r i e s , there i s an o v e r a l l s i m i l a r i t y between

the GpC and the d - pGpC complexes i n s p i t e o f d i f f e r e n t
p r o p o r t i o n s o f the isomers.
We a s s i g n the G syn - C a n t i and G syn - C syn c o n f i g u r a t i o n s
to isomers \ and 3 on the b a s i s o f the f o l l o w i n g r e s u l t s :
- α - For the c y t o s i n e o f GpC [ P t ] - 3 , one observes a p o s i t i v e
1
NOE ( > 10%) between C - H6 and C - H i , a 0.6 ppm u p f i e l d s h i f t
1
of C - H l ' , and a 0.75 ppm d o w n f i e l d s h i f t o f C - H 2 . These
features a r e not observed f o r GpC [ P t ] - I and a r e c h a r a c t e r i s t i c
of a C syn c o n f i g u r a t i o n f o r the 3 isomer. - 6 - S i m i l a r
chemical s h i f t data a r e obtained f o r the c y t o s i n e s i n the c o r r e s
ponding complexes o f the deoxy s e r i e s . Moreover, w h i l e both H5
and H6 c y t o s i n e protons o f d - pGpC [ P t ] - 3 a r e i n s e n s i t i v e t o
the 5'- phosphate t i t r a t i o n , the C - H5 o f the I isomer e x h i b i t s
a s m a l l s e n s i t i v i t y i n agreement w i t h i t s C a n t i c o n f i g u r a t i o n .
- C - F o r GpC [ P t ] - 1 a p o s i t i v e NOE i s observed between
1
G-H8 and G - H I that supports a G syn c o n f i g u r a t i o n . A s m a l l
NOE f o r the 3 isomer r e q u i r e s f u r t h e r c o n f i r m a t i o n . Moreover
none o f the H8 o f d - pGpC [ P t ] - 1 and 3 i s d i r e c t l y
s e n s i t i v e t o the 5'- phosphate t i t r a t i o n , supporting a G syn
c o n f i g u r a t i o n f o r both isomers.
For the couple o f isomers o f f r a c t i o n 2 , we a s s i g n the
G a n t i - C a n t i c o n f i g u r a t i o n t o isomer 2§ and the G a n t i - C syn
c o n f i g u r a t i o n t o isomer 2b on the b a s i s o f the f o l l o w i n g data :
- a - For GpC [ P t ] - 2â~T there i s no NOE between G-H8 and
1
G- HI ' and between C - H6 and C - H l . This suggests a G a n t i
f f
c o n f i g u r a t i o n and, together w i t h the C - H l ' , - H2 and - H3
chemical s h i f t s , a C a n t i c o n f i g u r a t i o n . The l a t t e r should
c o n t r i b u t e t o the l a r g e d o w n f i e l d s h i f t o f C - H5 because i n the

6. CHOTTARD ET AL. Pt-Oligotiucleotide Structures 135
complex t h i s proton does not experience anymore the i n f l u e n c e o f

the guanine r i n g c u r r e n t . A s i m i l a r s h i f t i s observed f o r the
C-H5 o f d-pGpC [ P t ] - 2a , enhanced by the p r o x i m i t y o f the
1
5 - phosphate. I n the deoxy s e r i e s , the 2a/2b assignment i s based
X
on the s i m i l a r i t y between the H NMR data o f the oxy and deoxy
- 2a complexes. - b - For d - pGpC [ P t ] - 2a and - 2b t h e
f
G-H8 protons a r e s e n s i t i v e t o the 5 - phosphate t i t r a t i o n
(Δδ ^ 0.2 - 0.3 ppm) and t h i s supports an a n t i c o n f i g u r a t i o n o f the
guanines. Moreover the C - H5 o f 2a i s the o n l y other proton to
be s e n s i t i v e t o t h i s t i t r a t i o n (Δδ ^ 0.15 ppm) and t h i s confirms
the C a n t i c o n f i g u r a t i o n o f t h i s isomer. The CPK models show
that among a l l the p o s s i b l e c o n f i g u r a t i o n s o f d-pGpC [ P t ] ,
G a n t i - C a n t i i s the o n l y one t h a t can b r i n g the C - H5 i n the
v i c i n i t y o f the f r e e 5' - phosphate. - C - The complete c o n f i g u r a
t i o n assignment t o the 2b isomers i s more d i f f i c u l t because o f

the few data a v a i l a b l e f o r the oxy complex and because o f s i g n a l
o v e r l a p p i n g between the sugar protons o f the non separable 2a and
2b deoxy isomers. For d-pGpC[Pt] - 2b , the u p f i e l d s h i f t o f
C - H6 i s i n f a v o r o f a C syn c o n f i g u r a t i o n , but i s not accompanied
1
by the expected u p f i e l d s h i f t o f C - H l , however one sees a
1
s t r o n g l y deshielded H3 (5.07 ppm). We a s s i g n the G a n t i - C syn
c o n f i g u r a t i o n t o the GpC [ P t ] - and d - pGpC [ P t ] - 2b isomers,
because o f the previous assignments t o the \ , 3 and 2a isomers.
In the GpC [ Pt ] - 1 , - 3 and - 2a isomers, the 5 ' - r i b o s e s
adopt an Ν-type conformation (C3'- endo) c h a r a c t e r i s t i c o f the
1
platinum c h e l a t i o n . To the 3 - r i b o s e s corresponds an e q u i l i b r i u m
f
between type S- and type Ν - conformers (42). The 3 - r i b o s e s can
2 2
be considered as ^ 40% Ε f o r \ and 3 , and ^ 60% Ε f o r
2 3
2a i n the s i m p l i f i e d E ^ E d e s c r i p t i o n (32). I t i s note
worthy that even the d - pGpC [ P t ] - I and - 3 isomers present
f
an N - type conformation o f the 5 - sugar ( J , , + J n " 7 Hz) which
x 2 l t2
i s u s u a l l y l e s s favored i n the deoxy s e r i e s (41, 4 2 ) .
CpC and d - pCpC P l a t i n u m Complexes. Only one complex i s

obtained from each d i n u c l e o t i d e . T h e i r comparative *H NMR data
are c o l l e c t e d i n Table IV. Each complex contains both a syn and an
a n t i c y t o s i n e , the former being c h a r a c t e r i z e d by the u p f i e l d s h i f t s
?
of the r e l a t e d H I and H6 . From the sugar protons assignment,
f f
based on the 5 - c y t o s i n e - H3 c o u p l i n g w i t h the b r i d g i n g phos
phorus atom, the u p f i e l d s h i f t e d H I ' i s a 5 ' - C - H l ' . Therefore
the syn c y t o s i n e i s i d e n t i f i e d as Cp i n CpC [ P t ] and pCp i n
d-pCpC [ P t ] . For the oxy complex there i s no NOE between
f
pC-Hl and - H6 , however the e f f e c t between the corresponding
Cp protons i s not s i g n i f i c a n t enough to c o n f i r m the syn c o n f i g u r a
f
t i o n o f the 5 - c y t o s i n e . For the d - pCpC [ Pt ] complex o n l y the
3' - c y t o s i n e - H6 and - H5 protons a r e s e n s i t i v e t o the
1
5 - phosphate t i t r a t i o n (Δδ r e s p e c t i v e l y + 0.17 and 0.08) i n
agreement w i t h a pC - a n t i c o n f i g u r a t i o n . From these data we
t e n t a t i v e l y a s s i g n the 5* - C syn - 3' - C a n t i c o n f i g u r a t i o n t o
both CpC [ P t ] and d - pCpC [ Pt ] complexes .

9 1
Table III. *H NMR data o f the GpC[Pt] and d - pGpC [ Pt ] complexes '
GC GC [ P t ] - l f GC ί P t 1 - 3f GC ( P t l - ιέ GC l P t ] -
δ J δ J Δδ δ J Δδ δ J Δδ δ J Δδ
H8 8 03 s 8.05 s 0 8.13 s + 0.1 8.75 s + 0.7 8.37 s + 0.35

H6 7 79 d 7.7 7.76 d 8 -0.05 7.66 d 7.8 -0.15 7.67 d8 -0.1 7.37 d - 0.4
H5 5 65 d 7.7 6.2 d8 + 0.55 6.0 d 7.8 + 0.35 6.52 d 8 + 0.85
GpC
G - HI * 5 85 d 2.6 6.05 s + 0.2 6.0 s + 0.15 6.13 s + 0.3
(pD 7.4)
GpC [ Pt ] C - HP 5 88 d 2.2 5.88 d 3.7 0 5.25 d 4 -0.6 5.98 d 6 + 0.1
(pD 6.4)
m m m
G - H3« 4 61 ^4.3 4.78 + 0.15 4.71 + 0.1
(3' 4 :6.8) (3'4':9) (3'4 ':9.3)
m t t 1
C - H3' 4 28 4.49 + 0.2 4.6 + 0.3
(3» 4 Î7.5> (3'4':5.2) (3'4':6)
• %4.4
C - H2' 4 19 q 4.61 q + 0.4 4.95 q + 0.75
(2' 3 :5.2) (2·3':5.2) (2'3':6)
e
H8 8.11 7.82 s -0.3 8.05 s -0.05 8.92 s + 0.9 8.53 s + 0.4
e
d - pGpC H6 7.76 d 8 7.77 d 8 0 7.57 d 8 - 0.2 7.73 d8 0 7.4 d - 0.35
(pD 6.8) e
H5 5.81 d 8 6.33 d 8 + 0.5 5.90 d 8 + 0.1 6.98 d 8 + 1.2 6.39 + 0.6
d-pGpC [ P t ]
f f
(pD 6.4) G - HP 6.22 6.31 d 7 + 0.1 6.27 d 7 + 0.05 ' 6.31 6.31
f f
C - HP 6.27 6.37 q 8;4 + 0.1 5.72 q 8;5 -0.55 6.36 6.36
3 e
2
400 MHz ; samples Λ, 10~ M i n D 0 ; 15 C ; chemical s h i f t s (δ) i n ppm from TSP ; c o u p l i n g c o n s t a n t s ( J ) i n Hz. a brace
c 2 +
i s used f o r the δ v a l u e s when the s i g n a l s have not been a s s i g n e d t o each n u c l e o s i d e . f o r [Pt(dien)(GpC)] a t pD 6.4 ,

d

6U) Δ δ: H8 , 8.42 (s) + 0.4 ; H6, 7.87 (d 8) +0.1 ; H5 , 5.9 (d 8) + 0.35 ; G - Hl'/C - H P , 5.9 . f r o m réf. 43 .
e
broad. 'assigned by analogy w i t h the oxy isomers.
CHOTTARD ET AL. Pt-Oligonucleotide Structures 137
IX)
CO CVJ
ο Ο O ο
m i n IX) IX) IX)
co ο ο CVJ CO CO I
0\
1 1 +
ο ο ο ο ο ο IX) IX)
l-H i-H CVJ CO
+ I + I
Ο Ο ο ο
1 1 1 +
CO
•Η
ID IX) IX) LO CVJ
/-Ν
Ν Ν Ν Ν (Λ CM CO CO CO CO crco Ό
«* co Ν—/ Ο
Ό Ό Ό Ό Ό Ό Ό Ό Ο •
Ο 09
re
ft
Ο
°o
•Η
4d Φ ε
CO o
IX) IX) CO <x> IX) · cr» co •M
IX) Ο) Ο IX)
«3- vo o ο co IX) rH M-l
CO
ΙΟ IX) co m vo ϋ •H
•H
S Ό
ω eu •
CO ω
ο
σι •H
1^ 1^ CO CO
co CVJ co co •H Ο
ι— Ο <D
-ο - σ
-α Ό "Ο ο
m
eu i-4
Ο Ο
cti
a
ο • rt X )
ο •c
Τ3 Ο cd
ac
Ο IX) ΙΟ 00 co ca
σι §8 Οι ο CVJ r-1 Ο Χ ) eu
IX) IX) ιχ> CO o
•Η •
u
Ό
S Pd
W)
υ Ρ α CO •H
Ρ-. α pu I c CQ
ο • H CO
ce
/—\
e •-3 td
eu
νΟ νΟ m m —« νο νο m m ~-
SU CC pd pd ce pu pd pd Pd 33 Pd
U 00 PLI νο
υ m ο m
Pu ϋ
U Q ftQ α Ρ*
P-U α ,1 Ρ U Ω
pu α α
, ^

f f
In CpC [ P t ] both the 5 - and 3 - r i b o s e s adopt an Ν - type
conformation. For the deoxy complex the Ν-type predominates f o r
the 5'- sugar and the S - type f o r the 3'- sugar.
CP avuxlyAeA o& the. Olnactzotlde, ComplzxoA
The CD s p e c t r a o f the three diguanosine phosphate complexes

have been reported r e c e n t l y (23) and present a common c h a r a c t e r
i s t i c f e a t u r e . A t n e u t r a l and a c i d i c pHs there i s a broad p o s i
t i v e band i n the 275-290 nm r e g i o n i n s t e a d o f the negative band
that i s always present f o r the f r e e and protonated d i n u c l e o t i d e s .
This change appears c h a r a c t e r i s t i c o f the s t a c k i n g o f the a n t i
guanines w i t h i n the N7 - N7 c h e l a t e s . The observed spectra are
2 +
d i f f e r e n t from those reported f o r s o l u t i o n s o f GpG plus Zn
2 +
and C u (45) and from those o f the c i s - bis(nucleotide)complexes,
[ P t ( t n ) ( 5 - G M P ) ] 2 - and [ P t ( t n ) ( 5 - dGMP) ] " (46), which
f
2
f
2
2
possess a h e a d - t o - t a i l arrangement o f the purines (12,28). For the

CG- and GC - platinum complexes, the CD s p e c t r a o f the d i f f e r e n t
syn a n t i isomers are reported on F i g u r e 2 , together w i t h those o f
the corresponding f r e e d i n u c l e o t i d e s . The s p e c t r a o f the d i n u c l e o
t i d e s a l l show a p o s i t i v e composite band w i t h a maximum around
270-280 nm and a negative composite band w i t h a minimum around
230 nm. Upon platinum c h e l a t i o n an enhancement o f the CD s p e c t r a
i s n o t i c e a b l e f o r the CG complexes whereas i t i s accompanied by a
s t r i k i n g s i g n a l i n v e r s i o n f o r the GC complexes. The separated syn
and a n t i isomers do show d i s t i n c t CD s p e c t r a and these c h a r a c t e r
i s t i c s p e c t r a g i v e an e x c e l l e n t c o n f i r m a t i o n o f the correspondance
between the d i f f e r e n t oxy and deoxy isomers, f o r the CG and GC
X
s e r i e s , revealed by t h e i r E NMR s p e c t r a . The CD s p e c t r a o f the
CC- complexes are shown on F i g u r e 3. Platinum c h e l a t i o n leads t o a
red s h i f t and an enhancement o f the CD s p e c t r a .
Ptizlimina&y PqmxJÎU conceAviing d - TpGpGpCpCpA [d-TGGCCA)
The s t o i c h i o m e t r i c r e a c t i o n between d - TGGCCA and

c i s - [ P t ( N H ) 2 ( H 0 ) 2 ] ( N 0 ) ( 5 χ 10~ M) i n H 0 , a t pH 5.3 and
3 2 3 2
5
2
37°C, gives o n l y one complex as revealed by HPLC and by the

*H NMR spectrum of the crude r e a c t i o n product (Figure 4 ) . The
*Η NMR s p e c t r a o f the low f i e l d base protons o f the hexanucleotide,
at 70°C and pD 5.5 , and o f i t s platinum complex, a t 67°C and
pD 5.4 , are presented on F i g u r e 4 (at lower temperatures one
observes s i g n a l broadening). The 7.94 and 7.85 ppm s i g n a l s o f
d-TGGCCA are assigned t o the guanine H8 resonances, s i n c e these
are known t o occur a t higher f i e l d s than those o f adenine (47).
The 8.40 ppm resonance i s assigned t o the adenine - H8 , i n agree
1
ment w i t h t h a t o f a 3'- t e r m i n a l adenine ( presence o f a 5 - phos
phate bridge) (47). The other s t r a i g h t f o r w a r d assignments are
reported on F i g u r e 4 . Platinum c o o r d i n a t i o n leads t o downfield
s h i f t s o f the thymirte - H6 , o f one c y t o s i n e - H6 and o f the two
guanine - H8 protons. A c i d i f i c a t i o n o f the platinum complex, from

250 300
X nm
X nm Figure 2b. Circular dichroism spectra

4
of CG-platinum complexes (ca. 10~ M)
Figure 2a. Circular dichroism spectra in NaCl (0.05 M). Key: · · ·, d(pCpG)~
4
of CpG[Pt] (—, ca. ÎO M , pH 6.4) and [Pt]-1 at pH 5.6; —, d(pCpG)[Pt]-2 at
4
C G (-·-, ca. ΙΟ M , pH 6.3) in NaCl pH 6.5; and - · -, d(pCpG)[Pt] at pH
P
(0.05 M). 6.9.
250 300
X nm
Figure 2c. Circular dichroism spectra Figure 2d. Circular dichroism spectra
4
of GC-platinum complexes (ca. 10~ M) 4 of GC-platinum complexes (ca. 10~ M)

in NaCl (0.05 M). Key: --, GpC[Pt]-l in NaCl (0.05 M). Key: —, é(pGpC)-
at pH 5.5; · · ·, GpC[Pt]-2a at pH 6.1; fPtl-1 at pH 4.8; · · · , d(pGpC)[Pt]-2a
—, GpC[Pt]-3 at pH 6.7; and - · -, GpC + 2b) at pH 6.8; —, d(pGpC)[Pt]-3 at
at pH 6.8. pH 6.2; and - · -, d(pGpC) at pH 6.6.

I 1 ι
250 300
λ nm
Figure 3a. Circular dichroism spectra Figure 3b. Circular dichroism spectra
4
of CC-platinum complexes flO' M) in of CC-platinum complexes (10~ M) in 4
NaCl (0.05 M). Key: —, CpC[Pt] at pH NaCl (0.05 M). Key: —, d(pCpC)[Pt] at
6; - · -, CpC at pH 6.2; and · · ·, CpC pH 5; - · -, dfpCpC) at pH 6; and · · ;
atpH1.6. d(pCpC) at pH 1.5.
8.23 7.58
1
Figure 4. H-NMR spectra (400 MHz) of
the low-field base protons of dfTGGCCA)
and crude dfTGGCCA )[Pt]. Conditions
3
of dfTGGCCA: 2 χ ΙΟ M in D O, pD g
5.5, 70 °C. Conditions of dfTGGCCA)

3
[Pt]: 2 Χ ΙΟ M in 1 M KCl pD t
5.4, 67 °C. Chemical shifts in ppm from

TSP. The purine H8 protons of d-
(TGGCCA ) have been partially exchanged
for deuterium by previous heating of the
D O solution.
g

pD 7.6 t o pD 3 , a t 17°C, f i r s t r e v e a l s the p r o t o n a t i o n of the

two c y t o s i n e s (C - H6 doublets Δ6 + 0.3 and +0.4 ppm) f o l l o w e d
by the downfield s h i f t o f one of the 8.21-8.23 resonances. I n t h i s
pH range, the l a t t e r s h i f t (Δδ +0.36 ppm) can o n l y be assigned to
the H2 proton o f the unbound adenine. As thymine cannot be a
l i g a n d i n our c o n d i t i o n s , these data e s t a b l i s h G-N7 c o o r d i n a t i o n .
The i n f l u e n c e o f t h i s c o o r d i n a t i o n on the T-H6 and one of the
C-H6 protons suggests a GG - c h e l a t i o n . The G-H8 downfield
s h i f t s are i n agreement w i t h such a c h e l a t i o n when compared w i t h
those o f the diguanosine phosphate complexes.
V4J>cuAt>ion
Our r e s u l t s show t h a t , w i t h i n oxy o r deoxy d i n u c l e o t i d e s ,

adjacent guanines, c y t o s i n e s o r guanine and c y t o s i n e always s t o i -

I I
c h i o m e t r i c a l l y c h e l a t e the c i s - P t ( N H ) moiety upon r e a c t i o n
3 2
w i t h c i s - [ P t ( N H ) 2 ( H 0 ) 2 ] ( N 0 ) o r c i s - [ Pt (NH ) C 1 ] . GpG,
3 2 3 2 3 2 2
d - GpG and d - pGpG g i v e a s i n g l e N7 - N7 a n t i - a n t i complex.

CpC and d-pCpC give a s i n g l e N3 - N3 syn - a n t i complex. CpG
and d - pCpG g i v e a mixture o f N3 - N7 C a n t i - G a n t i (I) and
C syn - G a n t i (2) isomers. GpC and d - pGpC g i v e a mixture o f
two N7 - N3 couples of isomers G syn - C a n t i (V) , G syn - C syn
(3) and G a n t i - C a n t i (2a) , G a n t i - C syn (2b). P r e c i s e
comparative k i n e t i c data have t o be recorded but the formation o f
the platinum c h e l a t e s i s the f a s t e s t w i t h the diguanosine phosphates
and the slowest w i t h the d i c y t o s i n e phosphates.
When CpG o r GpC a r e r e a c t e d w i t h [ P t ( d i e n ) B r ] Br o n l y
the G - N7 monocoordinated complexes are formed and no other
complex could be detected by HPLC m o n i t o r i n g o f the r e a c t i o n .
This i s i n agreement w i t h the known b i n d i n g k i n e t i c s e l e c t i v i t y i n
favor of guanine - N7 (48). For the couples of CG o r GC e q u i
l i b r a t i n g complexes, the i s o m e r i s a t i o n o n l y a f f e c t s the c y t o s i n e
c o n f i g u r a t i o n . Examination o f the CPK models suggests t h a t t h i s
i s o m e r i s a t i o n r e q u i r e s a d i s s o c i a t i o n of the c y t o s i n e - N3 - Pt bond.
We have not y e t been a b l e t o trap the "open" i n t e r m e d i a t e . However
a d i s s o c i a t i o n - recombination process a f f e c t i n g o n l y the c y t o s i n e -
platinum bond might be r e l a t e d t o the l a r g e r s t a b i l i t y constants
i:[
r e c e n t l y r e p o r t e d f o r P d ( d i e n ) b i n d i n g to G-N7 than t o
C-N3 (49).
For a l l the d i n u c l e o t i d e s s t u d i e d , independently o f the nature
of the bases, platinum c h e l a t i o n induces an Ν-type conformation
1
f o r the 5 - sugar, w h i l e the 3'-sugar adopts a predominantly S-type
conformation (42, 32). A more d e t a i l e d a n a l y s i s o f the sugar
conformations i s undertaken.
There i s a s t r i k i n g d i f f e r e n c e between the CD s p e c t r a o f the
CG- and GC - complexes. While upon platinum c h e l a t i o n , the
o v e r a l l f e a t u r e s o f the CD are r e t a i n e d i n the CG s e r i e s , a
s i g n a l i n v e r s i o n occurs i n the GC s e r i e s . Moreover t h i s inversion
i s present f o r the d i f f e r e n t isomers which have been i s o l a t e d
(1, 3, 2a f o r GpC [ P t ] , 1, 3, 2a + 2b f o r d - pGpC [ Pt ] ) so

that we can r u l e out that i t i s c h a r a c t e r i s t i c of a guanine w i t h a

syn c o n f i g u r a t i o n (Isomers \ and 3 have a G syn - , isomers 2a
and 2b a G a n t i - c o n f i g u r a t i o n ) . This s i m i l a r i t y shows a l s o
that w i t h i n a s e r i e s a change of c o n f i g u r a t i o n from a n t i to syn
around one g l y c o s i d i c bond does not i n v e r t the CD . A c c o r d i n g l y
the CD of the GpC [ Pt ] - 1 syn - a n t i isomer i s not s i g n i f i c
a n t l y d i f f e r e n t from t h a t of the GpC [ Pt ] - 3 syn - syn isomer.
By c l o s e i n s p e c t i o n of the whole s e r i e s of s p e c t r a of the platinum
complexes, i t appears that f o r the oxy and deoxy s e r i e s the CD
of the GC [ P t ] - 3 syn - syn isomer i s almost the m i r r o r image of
that of the CG [ Pt ] - 2 syn - a n t i isomer. The use of CPK models
shows t h a t i n the case of CpG , the C syn - G a n t i c h e l a t i o n of
the platinum can be done w i t h a s l i g h t m o d i f i c a t i o n of the sugar -
phosphate backbone conformation, whereas f o r GpC the G syn -
C syn c h e l a t i o n i m p l i e s an important p e r t u r b a t i o n l e a d i n g to a
z i g z a g conformation of the backbone (50, 51). For the CG s e r i e s ,
the CPK models a l s o p o i n t to a backbone p e r t u r b a t i o n f o r the
c
a n t i - G a n t i c h e l a t i o n of platinum. However the CD of the
d-pCpG [ P t ] - 1 isomer i s of the r e g u l a r type w i t h a p o s i t i v e
maximum at 279 nm. So that i t appears that a change of the sugar
phosphate backbone conformation does not n e c e s s a r i l y i n v e r t the
CD . We t e n t a t i v e l y conclude t h a t the sequence i s p r i m a r i l y
r e s p o n s i b l e f o r the CD i n v e r s i o n observed i n the GC [ Pt ] s e r i e s
ΙΑ, that the z i g z a g h e l i c a l s t r u c t u r e leads to a reinforcement of
the negative long wavelength CD band c h a r a c t e r i s t i c of the
GC [ P t ] s e r i e s . Our conclusions are i n agreement w i t h the r e s u l t s
showing that the CD of s y n t h e t i c double stranded RNAs i s
p r i m a r i l y sequence dependent (52), i f we consider the conforma
t i o n a l r e s t r i c t i o n s brought by platinum c h e l a t i o n . They agree a l s o
w i t h c a l c u l a t i o n s on p o l y - I showing that a long wavelength negative
CD band i s not n e c e s s a r i l y r e l a t e d to a p a r t i c u l a r h e l i x sense (53).
When compared to the r e a c t i o n s of the other d i n u c l e o t i d e s
s t u d i e d the f a s t e r c o o r d i n a t i o n of the diguanosine phosphates, to
give a s i n g l e N7 - N7 a n t i - a n t i platinum complex, p o i n t s to a
p a r t i c u l a r c h e l a t i n g a p t i t u d e of the a n t i - a n t i GG sequence. A
p r e f e r r e d GG c h e l a t i o n has r e c e n t l y been reported f o r d - CCGG
(15) and our p r e l i m i n a r y r e s u l t s w i t h d-TGGCCA c l e a r l y demons
t r a t e the sequence s p e c i f i c i t y of platinum b i n d i n g . These r e s u l t s
are i n agreement w i t h those obtained from the r e s t r i c t i o n enzyme
s t u d i e s of p l a t i n a t e d DNAs (17, 18, 19) and support the hypothesis
of platinum i n t r a s t r a n d c r o s s - l i n k i n g of adjacent guanines.
Acknowledgment
We are deeply indebted to Drs. J . Igolen and T. Huynh - Dinh

f o r the synthesis of d-TGGCCA . We thank Miss V. Michon f o r NMR
t e c h n i c a l a s s i s t a n c e , Miss N. C l a q u i n f o r t y p i n g of the manuscript
and Engelhardt i n d u s t r i e s , France, f o r a l o a n of platinum.

Abbreviations tu&d
In dinucleoside monophosphates and oligonucleotides, A, C, G,

T respectively represent adenosine, cytidine, guanosine and thymi
dine, ρ to the left of a nucleoside symbol indicates a 5 f - phos
phate and to the right, i t indicates a 3 f - phosphate. In GpG the
5'- H8 is the H8 proton of the 5 f - guanine (Gp). The dinucleo
side monophosphates GpG* d - GpG, etc... will be occasionally
referred to as dinucleotides. Guo = guanosine, Cyd = cytidine,
5 1 - GMP - 5 f - guanosine monophosphate, 5 1 - CMP - 5 1 - cytosine mono
phosphate. HPLC — high - pressure liquid chromatography; NMR -
nuclear magnetic resonance, s - singlet, d = doublet, t • triplet,
q « quartet, m - multiplet, NOE = nuclear Overhauser effect, CD =
circular dichroism. tn = trimethylene diamine, dien = diethylene-
triamine. CPK models = Corey - Pauling - Koltun atomic models.
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7
Platinum(II) Complex Formation with Uracil
and Thymine
BERNHARD LIPPERT
Technische Universität München, Anorganisch-Chemisches Institut,
Lichtenbergstrasse 4, 8046 Garching, Federal Republic of Germany
Replacement of an amide proton in pyrimidine-2,4-

diones by less electron-attracting Pt(II) electro-
phile causes an increase in electron density at
the exocyclic oxygens, and allows easy formation
of Ν,Ο-bridged complexes with additional metal
ions, or proton binding at the oxygen(s). With
cis-Pt(NH ) L (L=monoanion of 1-methyluracil or
3 2 2
1-methylthymine) a number of dimeric and hetero

nuclear complexes of Pt,M, Pt ,M, and Pt ,M stoi2 4
chiometries have been isolated and studied using

crystallographic and spectroscopic methods.
With unsubstituted uracil and thymine the situa
tion is complicated due to the existence of two
tautomers of the monoanions. Pt binding occurs
through the Ν donors and via exocyclic oxygens.
In aqueous solution a slow mutual interconversion
of the various complexes takes place.
The i n t e r e s t i n the i n t e r a c t i o n between metal ions and

n u c l e i c a c i d s i n general (J_,2) and the f i n d i n g on the antitumor
a c t i v i t y of complexes formed between aquated c i s - K P t O J H ^ ^ C l o
and pyrimidine-2,4-dione l i g a n d s such as u r a c i l and thymine
i n p a r t i c u l a r (3,4) have l e d t o a c o n s i d e r a b l e a c t i v i t y i n t h i s
area of b i o i n o r g a n i c chemistry. With these l i g a n d s r e p r e s e n t i n g
t y p i c a l m u l t i s i t e l i g a n d s , f r e q u e n t l y no s t r a i g h t f o r w a r d i n t e r
p r e t a t i o n of s p e c t r o s c o p i c r e s u l t s i s p o s s i b l e . As w i l l subse
quently be shown, the combination of d i f f e r e n t s p e c t r o s c o p i c
techniques and the use o f X-ray c r y s t a l l o g r a p h y permits a
reasonable, although s t i l l incomplete, understanding of the co
o r d i n a t i n g behavior of c i s - P t ( N H )«(11), e n P t ( I I ) , and
P t ( N H ) ( I I ) w i t h these l i g a n d s .
3 3
0097-6156/83/0209-0125$06.25/0

P t ( I I ) Complexes of Unsubstituted U r a c i l and Thymine
The tautomerism of the monoanions of u n s u b s t i t u t e d u r a c i l

w i t h i t s N1 and N3 deprotonated forms i s w e l l e s t a b l i s h e d (5-9)
(Figure 1). With thymine, the c r y s t a l s t r u c t u r e of the N1 depro-
tonated tautomer has been performed (10), and IR and Raman spec-
t r o s c o p i c evidence has been presented f o r the e x i s t e n c e of the
second tautomer i n the s o l i d s t a t e as w e l l (11) . The high degree
of charge d e r e a l i z a t i o n i n both tautomers, deduced from theo-
r e t i c a l c a l c u l a t i o n s (12), suggests that metal b i n d i n g could
conceivably take place a t a number of s i t e s and combinations of
these, l e a d i n g t o a v a r i e t y of products. The s i t u a t i o n i s f u r t h e r
complicated by the f a c t that once a metal i s attached t o the
h e t e r o c y c l i c r i n g , the complex may be i n v o l v e d i n acid-base
e q u i l i b r i a , thus l e a d i n g t o d i a n i o n i c o r n e u t r a l l i g a n d s . With
the metal b i n d i n g s i t e being an e n d o c y c l i c n i t r o g e n , any n e u t r a l
l i g a n d i n a complex n e c e s s a r i l y occurs i n a r a r e tautomeric form
(Figure 2). Moreover, b i n d i n g of more than j u s t one metal t o a
s i n g l e u r a c i l or thymine l i g a n d i s p o s s i b l e .
I t may be a combination of these reasons why there appears
to be some u n c e r t a i n t y concerning metal b i n d i n g s i t e s of these
l i g a n d s i n s t u d i e s not supported by c r y s t a l s t r u c t u r e s (13-18).
Although the existence of tautomer complexes of u r a c i l and thy-
mine had been taken i n t o c o n s i d e r a t i o n (12.)* u n t i l r e c e n t l y ,
only i n a s i n g l e case, w i t h u r a c i l complexes o f t r i a m m i n e p l a t i -
num(II), i t has c l e a r l y been demonstrated by K i d a n i and Inagaki
(20).
Among the l i m i t e d number of c r y s t a l l o g r a p h i c a l l y confirmed
b i n d i n g s i t e s of metal ions w i t h the monoanions of u n s u b s t i t u t e d
u r a c i l (21,22,23) and thymine (21,24,25,26) one observes a s t r i k -
ing predominance of N1 coordination.Only i n one case, w i t h Cd
b i n d i n g t o u r a c i l , N3 b i n d i n g i s documented ( 2 3 ) .
Two more b i n d i n g p o s s i b i l i t i e s - v i a one of the e x o c y c l i c
oxygens (27,28) o r w i t h replacement of the proton a t C5 (29,30)
w i l l not be considered here, s i n c e they occur w i t h the n e u t r a l
l i g a n d s and do not i n v o l v e the N1,N3 tautomer e q u i l i b r i u m of the
monoanionic l i g a n d s , r e s p e c t i v e l y .
Methods of D i f f e r e n t i a t i o n . Apart from X-ray c r y s t a l l o -
graphy that had been a p p l i e d i n a few cases w i t h N1 p l a t i n a t e d
u r a c i l and thymine compounds (21,25,26), s p e c t r o s c o p i c methods
were used t o determine the s i t e s of platinum c o o r d i n a t i o n I n
sequence of t h e i r usefulness these were:
UV spectroscopy. I t proved t o be a p p l i c a b l e i n cases w i t h
i s o l a t e d tautomer complexes of u r a c i l (20) and thymine (31), but
i s of very l i m i t e d usefulness i n mixtures of v a r i o u s tautomer
and/or bridged complexes.
IR spectroscopy.The double bond s t r e t c h i n g r e g i o n i n a l l
cases permits a d i f f e r e n t i a t i o n of N1 and N3 tautomer complexes
of the thymine (31) and u r a c i l monoanion complexes. T y p i c a l l y ,
N1 c o o r d i n a t i o n gives r i s e t o an intense band around 1640 cm

7. LiPPERT Pt(U) Complex Formation with Uracil and Thymine 149
Figure 1. Two tautomeric forms of

0 ^ 0" "Ν' uracil (R = H) and thymine (R = CH ) 3
Η monoanions.
nT
C ^"MN/ H
nC^TM" N^ nC^TM^ ^
N' HCr^tvr
θ Η Η Η
Figure 2. Relevant acid-base equilibria of a Ν3 platinated uracil. Analogous

equilibria exist for Nl-platinated uracil as well (32).

w i t h shoulders both on the lower and higher frequency s i d e ,

whereas N3 c o o r d i n a t i o n i s recognized by a c h a r a c t e r i s t i c band
around 1550 cm w i t h a second one around 1650 cm , which f r e ^
quently i s s p l i t . These d i f f e r e n c e s r e s u l t from the d i f f e r e n c e s
i n charge d i s t r i b u t i o n i n both l i g a n d s and the i n a b i l i t y of the
metal e l e c t r o p h i l e t o r e s t o r e c o n d i t i o n s as w i t h the proton
attached. The s i t u a t i o n w i t h the u r a c i l tautomer complexes of
c i s - P t ( N H ) ( I I ) , c i s - P t ( N H ) ( H U ) C l . ( H 0 ) (HU= monoanion o f
3 3 2 x
u r a c i l ; x= Τ f o r HU-N1; x= ζ f o r HU-N3) expectedly i s r a t h e r

s i m i l a r t o the s i t u a t i o n w i t h the triammineplatinum(II) com
plexes described by K i d a n i and Inagaki (20), w i t h intense bands
around 1660sh, 1635vs, 1575sh, 1420s (HU-N1), and 1670s, 1625s,
1540vs, 1415s (HU-N3). However, the two c h l o r o complexes a r e
obtained q u i t e d i f f e r e n t l y from the corresponding (NH^)o com-^
pounds (32,33). On the other hand, w i t h mixtures of tautomer

complexes o r mixed tautomer complexes such as cijs-Pt (NH«) ~
(TH-N1)(TH-N3) (TH= thymine monoanion) these c h a r a c t e r i s t i c
bands a r e superimposed, and the i n t e r p r e t a t i o n of the i n f r a r e d
s p e c t r a i s l e s s s t r a i g h t f o r w a r d (31).
Raman spectroscopy. The fundementals of p l a n a r h e t e r o
c y c l i c r i n g s having C molecular symmetry are d i s t r i b u t e d i n t o
1
i n - p l a n e ( A ) and out-of-plane (A") v i b r a t i o n s . Both symmetry
species are IR and Raman a c t i v e , but o n l y i n - p l a n e v i b r a t i o n s
u s u a l l y are i n t e n s e i n the Raman. As has been shown, these modes
are q u i t e s u i t a b l e f o r a d i f f e r e n t i a t i o n of tautomers (9,11),
tautomer complexes (34) o r even mixtures of these (32), With
p y r i m i d i n e d e r i v a t i v e s , the most i n t e n s e Raman bands u s u a l l y a r e
those of r i n ^ - s t r e t c h i n g (1200-1300 cm ) and r i n g - b r e a t h i n g
(ca. 800 cm ) v i b r a t i o n s , w i t h the l a t t e r being p a r t i c u l a r l y
s e n s i t i v e t o changes i n metal c o o r d i n a t i o n s i t e s and t o the
charge of the l i g a n d . Due t o the h i g h i n t e n s i t i e s of these Raman
bands, i n many cases mixtures of complexes w i t h d i f f e r e n t metal
b i n d i n g s i t e s can be analyzed and s e m i - q u a n t i t a t i v e e s t i m a t i o n s
of the;j.r concentrations can be done (32) .
H NMR spectroscopy. D e s p i t e the disadvantage of a slow
time s c a l e which, i n c o n t r a s t t o v i b r a t i o n a l spectroscopy does
not permit a d i r e c t o b s e r v a t i o n of i n d i v i d u a l tautomers ( a t
l e a s t not of tautomers w i t h amide, i m i n o l s t r u c t u r e s ) , NMR spec
troscopy proved t o be a powerful technique f o r the i n v e s t i g a t i o n
of k i n e t i c a l l y i n e r t metal complexes o f tautomers. I t s u s e f u l
ness i s based on the f o l l o w i n g arguments:
(a) Chemical s h i f t . N1 and N3 p l a t i n a t e d u r a c i l e x h i b t s markedly
d i f f e r e n t s h i f t s f o r i t s H6 and H5 resonances, f o r example c a .
7.7. and 5.7 ppm, r e s p e c t i v e l y f o r the HU-N1 complexes (DÔ,
pD 8- 1.5), but ca. 7.4 and 5.7 ppm f o r the HU-N3 complexes
(DgO, pD 7- 3) (32). S i m i l a r l y , the corresponding thymine com
plexes have t h e i r H6 resonances i n Me^SO-d. around 7.3 ppm
(TH-N1) and 7.0 ppm (TH-N3) ( 3 j 0 . (b) A p a r t i c u l a r l y u s e f u l ^
property of P t complexes i s the n u c l e a r s p i n of 1/2 of the Pt
i s o t o p e ( n a t u r a l abundance 33.8 % ) , which gives r i s e t o s a t e l -

7. LiPPERT Pt(H) Complex Formation with Uracil and Thymine 151
l i t e s of H resonances i f c o u p l i n g occurs. For example, TH-N1

complexes show s a t e l l i t e s of the H6 resonance w i t h J= 36- 40 Hz,
whereas no c o u p l i n g i s observed w i t h N3 platinum b i n d i n g ( 3 1 ) .
The H NMR s p e c t r a of the u r a c i l tautomer complexes represent
textbook examples on the e f f e c t of bond s e p a r a t i o n between two
atoms having a nuclear s p i n on the magnitude of c o u p l i n g w i t h
5
J= 38.8 Hz, J= 15-4.3 Hz, and J * 0 Hz (Figure 3 ) . Taking
these d i f f e r e n c e s i n t o c o n s i d e r a t i o n , i t i s p o s s i b l e t o a s s i g n
b i n d i n g s i t e s on t h i s b a s i s . I t should be noted, however, that
a number of f a c t o r s , i n p a r t i c u l a r chem^gal s h i f t anisotropy
r e l a x a t i o n may prevent observation of P t - H c o u p l i n g (35).
(c) Under a c i d i c pH c o n d i t i o n s u r a c i l complexes w i t h N1 platinum
b i n d i n g r e a d i l y undergo i s o t o p i c exchange a t the C5 p o s i t i o n i n
w
DgO, thus s i m p l i f y i n g the ^ NMR spectrum ( t r i p l e t o f r e l a t i v e
i n t e n s i t i e s 1:4:1 f o r H6, H5 resonance disappeared) (32). I n
c o n t r a s t , u r a c i l complexes w i t h N3 platinum b i n d i n g do not show
t h i s phenomenon i n t h i s pH range. I n t e r e s t i n g l y , N3 p l a t i n a t e d
u r i d i n e does undergo i s o t o p i c exchange a t C5, but under a l k a l i n e
c o n d i t i o n s (36). (d) Another remarkable d i f f e r e n c e between N1
and N3 p l a t i n a t e d u r a c i l and thymine i n t h e i r response t o a c i d
treatment. While the Pt-N1 bond i s very s t a b l e , the Pt-N3 bond
i s r e a d i l y cleaved on a d d i t i o n of a c i d and s l i g h t warming, and
the n e u t r a l l i g a n d i s e x p e l l e d from the complex (31,32). This
mechanism can befollowed n i c e l y w i t h both NMR and Raman spec-
troscopy.
I s o l a t i o n of Tautomer Complexes.Until r e c e n t l y , there has

been l i t t l e o r no r a t i o n a l e behind the s e l e c t i v e s y n t h e s i s o f
u r a c i l and thymine tautomer complexes. With a b e t t e r understand-
i n g of the f a c t o r s that i n f l u e n c e metal b i n d i n g s i t e s - e.g.
s o l v e n t , s o l u b i l i t y , pH (with water being the s o l v e n t ) , r e a c t i o n
time, and hydrogen bonding p r o p e r t i e s o f adjacent l i g a n d s (31) -
i t now i s p o s s i b l e t o p r e d i c t w i t h a higher degree of c e r t a i n t y
the formation of a c e r t a i n tautomer complex. For example, i n
DMF,cis-Pt(NH ) (HU-N1)Cl i s formed p r e f e r e n t i a l l y due t o i t s
3 2
low s o l u b i l i t y i n t h i s s o l v e n t , whereas under moderately a c i d i c

c o n d i t i o n s i n water ,cis-Pt(NH ),» (HU-N3)CI * 2H^0 i s formed and no
3
N1 product (33). Formation of the u r a c i l tautomer complexes o f

t r i a m m i n e p l a t i n u m ( l l ) i n water i n a 1:1 r a t i o (20) appears t o be
the exception r a t h e r than the r u l e , and should be a t t r i b u t e d
mainly t o the s i m i l a r s o l u b i l i t i e s of both products, which
prevents e a r l y p r e c i p i t a t i o n of one s p e c i e s .
P r e p a r a t i v e High Pressure L i q u i d Chromatography, HPLC,
proved t o be a good technique f o r s e p a r a t i n g tautomer complexes.
For example, a l l three p o s s i b l e bis(thyminato) complexes o f
c i s - P t ( I I ) , c i s - P t ( N H ) ( T H - N 1 ) , cjÎs-Pt(NH«) (TH-N1)(TH-N3),
3 2 2 2
and c i s - P t ( N H ) ( T H - N 3 ) have been i s o l a t e d (3J_) .

3 2 2
Linkage I s o m e r i z a t i o n . Complexes o f c i s - P t ( I I ) and

e n P t ( I I ) c o n t a i n i n g a s i n g l e u r a c i l (32) o r thymine (31) l i g a n d
and an aquo l i g a n d , show a remarkable tendency t o undergo con-

Figure 3. *H-NMR spectra of [(NH ) Pt(HU-Nl)]+ (0.15 M , D 0, and pD 1-8)

3 3 2
and [(NH ) Pt(HU-N3)]+ (0.1 M , D O and pD 1) in the H5,H6 region. (Repro-

3 3 2 f
duced from Ref. 32. Copyright 1981, American Chemical Society.)

7. LiPPERT Pt(II) Complex Formation with Uracil and Thymine 153
densation r e a c t i o n s w i t h the p y r i m i d i n e l i g a n d a c t i n g as a
b r i d g e . As a r e s u l t , cleavage of the o r i g i n a l p l a t i n u m - l i g a n d
bond takes p l a c e , and formation of complexes w i t h the metal
l i n k e d t o another donor atom. This phenomenon i s a l s o observed
w i t h c o o r d i n a t i v e l y j a t u r a t e d complexes such as c i s - P t ( N H ^ ) ^ L ^ ,
e n P t L and Pt(NH > L (L= TH o r UH) i n the presence of aquo-
2 3 3
p l a t i n u m ( I I ) complexes and can be demonstrated p a r t i c u l a r l y w e l l

w i t h mixtures of the r e s p e c t i v e N1 and N3 complexes o f
+ Z
P t ( N H ) ( H U ) and Pt(NH > ( H 0 ) /as shown i n Figures 4 and 5:
3 3 3 2
a f t e r a w h i l e the proton NMR s p e c t r a of both mixtures are v i r

t u a l l y i d e n t i c a l w i t h a t l e a s t three d i s c e r n i b l e species present.
S i g n a l A represents the HU-N1 complex, s i g n a l C the HU-N3
complex, and s i g n a l Β a dimer which most l i k e l y i s the N j g l p
bridged complex. I n both s p e c t r a only H6 s i n g l e t s (with Pt
s a t e l l i t e s i n the case of HU-N1) are observed due t o i s o t o p i c

exchange a t C5. Since i t only proceeds v i a the N1 complex, y e t
a l s o i s observed f o r the mixture of the HU-N3/Pt(H 0) compounds, 2
an a d d i t i o n a l argument i n favour of the i n t e r c o n v e r s i o n of

b i n d i n g s i t e s i s posed.
. The very complicated Raman and, a f t e r p a r t i a l decomposition
H NMR s p e c t r a (32) of "Platinum P y r i m i d i n e B l u e s " 0 U) and 9
"Patinum P y r i m i d i n e Tans" (37) are not easy t o i n t e r p r e t . Never

t h e l e s s two important conclusions can be drawn from NMR and
Raman s p e c t r a : f i r s t , they c o n s i s t o f a mixture o f complexes
w i t h d i f f e r e n t b i n d i n g s i t e s o f platinum and d i f f e r i n g o l i -
gomeric s i z e . Second, i n s l i g h t l y t o moderately a c i d i c medium
an i n t e r c o n v e r s i o n of b i n d i n g s i t e s of the P t e l e c t r o p h i l e
occurs, as can be seen from the time dependence of the s p e c t r o -
skopic changes when the pH of the medium i s a l t e r e d (32).
P t ( I I ) Complexes of N1-Methylated U r a c i l and Thymine.
With the N1 p o s i t i o n f o r metal b i n d i n g b l o c k e d , the co

o r d i n a t i o n p r o p e r t i e s of u r a c i l and thymine are reduced
(Figure 6 ) . I n the course o f our systematic s t u d i e s of p o s s i b l e
r e a c t i o n products between c i s - P t ( I I ) and model nucleobases, the
2:1 complexes o f 1-MeT (monoanion o f 1-methylthymine, 1-MeTH)
and 1-MeU (monoanion o f 1 - m e t h y l u r a c i l , 1-MeUH) have been pre
pared.
The c r y s t a l s t r u c t u r e o f cis-Pt(NH ) (1-MeU)^'4H 0 has
3 2 2
been determined (38). The molecule has approximately C molecu 2
l a r symmetry w i t h the two h e t e r o c y c l i c l i g a n d s arranged i n

h e a d - t a i l f a s h i o n , s i m i l a r t o the r e s p e c t i v e complexes of
1-methylcytosine (39), v a r i o u s guanine d e r i v a t i v e s (40) and a l s o
Ct-hydroxopyridine (41 ).
Platinum b i n d i n g t o N3 of the u r a c i l and thymine l i g a n d s
f a c i l i t a t e s b i n d i n g of a d d i t i o n a l metals o r of a proton through
the e x o c y c l i c oxygens, and i s a consequence of an i n c r e a s e i n
e l e c t r o n d e n s i t y a t these s i t e s due t o the replacement of the
proton a t N3 by P t . Using t h i s property f o r s y n t h e t i c purposes,

Figure 4. *H-NMR spectra of a mixture

+
of [(NH ) Pt(HU-Nl)]
s s (0.1 M) and
2
[(NH ) Pt(D 0)] + (0.2 M) in D O in the
s 3 2 t
H5,H6 region. Key to conditions: a,

after 3 h at 40 °C and pD 2.6; b, after 43
h at 40 °C and pD 1.8; and c, after 10 d
at 40 °C and pD 1.7. Because of isotopic
exchange at C5, no H5 resonances are
observed in b and c. (Reproduced from
Ref. 32. Copyright 1981, American
Chemical Society.)

1
Figure 5. H-NMR spectra of a mixture
+
of [(NH ) Pt(HU-N3)]
s s (0.1 M) and
2
[(NH ) Pt(D 0)] + (0.2 M) in D O. Key
3 s 2 g
to conditions: a, after 6 h at 40 °C and

pD 2.3; b, after 4 d at 40 °C and pD 2.0;
and c, after 7 d at 40 °C and pD 1.9.
(Reproduced from Ref. 32. Copyright
1 ppm 1981, American Chemical Society.)
HN :
Figure 6. The structure of 1-methylura-

CH 3
cil (R = H) and 1-methylthymine (R =
CH ). S

i t i s p o s s i b l e t o o b t a i n a range of i n t e r e s t i n g compounds from

the 2:1 complex o f 1-HeU and 1-MeT, as i s o u t l i n e d i n the scheme
shown i n Figure 7.
Mono(1-MeT) and Mono(l-MeU)Complexes. A d d i t i o n o f a c i d t o

a s o l u t i o n of c i s - P t ( N I P ^ L ^ (L= 1-MeT o r 1-MeU) y i e l d s c r y s t -
a l l i n e products of composition c i s - [ P t ( N H ) L ( L H ) ] X·(H 0) ( 4 2 ) .
3 2 2
I n these compounds one o f t h e two l i g a n d s i s present i n i ? s neu-

t r a l form and, w i t h N3 being the s i t e of metal c o o r d i n a t i o n ,
consequently occurs i n a r a r e i m i n o l tautomeric s t r u c t u r e . The
a c i d i c proton has a pK - 2 i n the case o f the 1-MeT compound.
As w i t h the N3 p l a t i n a t e d u n s u b s t i t u t e d u r a c i l and thymine, the
Pt-N3(LH) bond i s e a s i l y cleaved on b r i e f warming and r e s u l t s i n
a s e l e c t i v e removal of the LH l i g a n d from the complex:
c i s - [Pt (NH ) L (LH)1 X

3 2 -A* cis-Pt (NH^LX + LH
N a t u r a l l y , the LH l i g a n d e x p e l l e d from the complex o r i g i n a l l y

has the r a r e tautomeric s t r u c t u r e . With water being the s o l v e n t ,
the i n t e r c o n v e r s i o n i n t o the normal d i k e t o tautomer i s an i n -
stantaneous one, but w i t h an a p r o t i c solvent i t i s f e a s i b l e
that an i m i n o l tautomer may have a measurable l i f e - s p a n . One
may f u r t h e r speculate that a metal c a t a l y z e d t a u t o m e r i z a t i o n o f
thymine o r u r a c i l under b i o l o g i c a l c o n d i t i o n s could take place
v i a such a mechanism (42).
With X= C l ~ , c i s - P t ( N H ^ L C l i s obtained i n good y i e l d .
cis-[Pt(NH )^L(HgO)]Y, prepared by r e a c t i o n o f the c h l o r o com-
3
p l e x w i t h Agi (Y= N 0 , C10^~), i s a s u i t a b l e s t a r t i n g m a t e r i a l

3
f o r a number of products.
Condensation Products. I n a condensation r e a c t i o n , the
aquo complex dimerizes t o g i v e the r e s p e c t i v e h e a d - t a i l P t d i -
mers w i t h N3,04 bridged u r a c i l (thymine) among other products.
The obtained dimers a r e i d e n t i c a l w i t h those prepared i n a
d i f f e r e n t way and s t u d i e d by Lock, Rosenberg and coworkers (43,
44), as evident from elemental a n a l y s i s , d e n s i t y measurements
and determination of the c e l l constants. With 1-MeT as l i g a n d ,
i n a d d i t i o n we i s o l a t e d an i n t e r e s t i n g second m o d i f i c a t i o n of
the h e a d - t a i l dimer, which has a r a t h e r d i f f e r e n t c r y s t a l s t r u c -
t u r e from the known one (43). This second m o d i f i c a t i o n c r y s t a l l -
i z e s i n stacks of dimers which are l i n k e d by strong hydrogen
bonds between the NH groups and the non-coordinating e x o c y c l i c
3
oxygen of adjacent dimers, l e a d i n g t o i n t e r - d i m e r separations of

about 3.9 Â (45).
Mixed Nucleobase Complexes. The p r e p a r a t i o n of p o s s i b l e
c r o s s l i n k i n g products of c i s - P t ( I I ) w i t h the a n i o n i c 1-MeU o r
1-MeT as one base, and n e u t r a l c y t o s i n e , guanine o r adenine as
second nucleobase proceeds without formation of undesired s i d e
products only i f the a n i o n i c l i g a n d i s attached t o Pt f i r s t . On
the other hand, r e a c t i o n of c i s - P t ( I I ) w i t h u r a c i l o r thymine i n
1:1 r a t i o r e s u l t s i n formation of the complicated platinum blues

cis-Pt(NH^)
1
+M + cis- [pt(NH ) (H 0) l 3 2 2 2
+ HX
L
HETERONUCLEAR COMPLEXES
crystallographically characterized
species: Pt M and Pt,M'
?
M- Mn(II), Ag(I); M'- Cu(II)
MONO-L-COMPLEXES HEAD-HEAD DIMER
9PtL(LH) X
a ,PtL( 2+
a P t L P t a
2 2 2
-LH
'4- r
+C1 +Mn+
a PtLCl (X= CI)
2
PtLC
decomposition heteronuclear +HN0„
+Ag+j -AgCl into complexes
a PtL(H Q)
2 2
cis-a PtCl
2 2
1 +Y +0.5 OH and
2+ cis-a PtL
2 2
a PtL Pta
2 2 2
a PtLY
2 a LPt(OH)PtLa
2 2
(differenti- 5+
head-tail mixed U-OH dimer ation from a Pt,L/
rt platinum blue
dimer nucleobase head-tail 8 4 4
complexes +OH" dimer)
oxidation
oxidation a PtL(0H)
2

Pt(III) dimers

products
Figure 7. Reaction scheme of compounds accessible via cis-Pt(NH hL (L = 1-MeU or 1-MeT).

s g
(3,4,37,46). The above o u t l i n e d s y n t h e s i s of 1:1 complexes of

1-MeU and 1-MeT enables formation of L c o n t a i n i n g mixed nucleo
base complexes according t o
+ +
ci£-[Pt(NH ) L(H 0)] 3 2 2 + Y • cis-[Pt(NH ) LY]3 2
With L= 1-MeT, the complexes w i t h Y= 1-methylcytosine, 9 - e t h y l -

guanine and 9-methyladenine have been prepared and s t u d i e d (47),
and s i m i l a r complexes w i t h L= 1-MeU a r e p r e s e n t l y being s t u d i e d
(48). I n another step the remaining L l i g a n d can be removed from
the compound v i a a c i d treatment and a new nucleobase Ζ i n t r o
duced, thus g i v i n g e v e n t u a l l y cis-Pt(ΝΗ^)^Ζ complexes. As an
example of such nucleobase complexes, the H NMR spectrum of
£Îsy^ (NH ) (1-MeT)(9-EtG)]Cl0 i n Me SO-d i s shown i n F i g u r e
t 3 2 4 2 6
8 3 P t c o u p l i n g of the H8 resonance of the guanine l i g a n d

( J 23.3 Hz) i n d i c a t e s N7 platinum b i n d i n g , and the chemical
s h i f t s of H8 and NH~ resonances are a l s o c o n s i s t e n t w i t h r e l a t e d
complexes w i t h N7 p l a t i n a t i o n of 9-ethylguanine (49).
Reaction w i t h Base. T i t r a t i o n of cis-[PtTNH^)(Η,,Ο))
w i t h one e q u i v a l e n t of base gives £is-Pt(NH^T^L(OH). T i t r a t i o n
w i t h 0.5 e q u i v a l e n t s y i e l d s a d i m e r i c complex of composition
[ ( N H ) P t L ( 0 H ) L P t ( N H ) ] w i t h a b r i d g i n g OH group and t e r m i n a l
3 2 3 2
L ligands:
+ +
2 cis-[pt(NH ) L(H 0)] 3 2 2 + 0H~-> [(NH ) PtL(OH)LPt ( N H ) ]
3 2 3 2 + H0
2
I n an analogous way a Jâ-OH dimer w i t h L= 1-methylcytosine has

been obtained (charge of the complex +3). From the pK o f the
aquo l i g a n d i n cis-ÇPt(NH^LQÔ)] * (ca 6- 6.5) i t i s evident
that the dimer can e x i s t a t n e u t r a l pH, and H NMR spectroscopy
confirms that i t i s the major component i n water of pH 7. Form-
a t i o n of t h i s dimer w i t h a s i n g l e hydroxo b r i d g e leads to an
a t t r a c t i v e hypothesis of how adjacent bases i n DNA might be
l i n k e d by a cis-Pt(OH)Pt moiety e i t h e r i n an i n t e r - o r i n t r a -
strand f a s h i o n . I n c o n t r a s t , a trans-Pt(OH)Pt u n i t could
accomplish i n t e r s t r a n d c r o s s l i n k i n g only (Figure 9 ) . I t had
p r e v i o u s l y been argued against the e x i s t e n c e of c l o s e l y ad-
j a c e n t P t centers i n DNA t r e a t e d w i t h c i s - P t ( I I ) on the b a s i s
of an Extended X-Ray Absorption Fine S t r u c t u r e Spectroscopy
study (EXAFS) (50). However, only P t - P t d i s t a n c e s s h o r t e r than
3.2 X could be r u l e d out whereas i n the present s i t u a t i o n , from
comparison w i t h c y c l i c hydroxo bridged complexes of composition
c i s - l ( N H ) P t ( O H ) ] ^ (n= 2, 3) (51), P t - P t separations of about
3 2
3.5 A might be expected. I t i s evident that a P t dimer w i t h a

s i n g l e OH b r i d g e can accomodate b i n d i n g of adjacent bases much
b e t t e r than a monomer can and that t h i s way of c r o s s l i n k i n g i s
by no means r e s t r i c t e d t o the p y r i m i d i n e beases. The r a t h e r un-
expected e x i s t e n c e of hydroxo complexes of c i s - P t ( I I ) , not only
i n the s o l i d s t a t e but a l s o over a wide pH range i n s o l u t i o n
(52,53), c e r t a i n l y makes the idea of t h i s type of c r o s s l i n k i n g
feasible.

TMS
Cis-[(NH ) PtTG]ClO^
3 2
Q ... 9 Τ
N\ C H
3 " ψ \ Λ
1
Figure 8. H-NMR spectrum of c\s-[(NH h(l-MeT)(9-EtG)]ClO
s k in Me SO-d .
g 6

Κ
m
H
>
ο
s
en
POR COMPARISON :
H o
H
\_^0> X
m
H >
H
309A 120-352 Â m

d

H
Figure 9. Possible arrangements of two nucleobases in a Pt-OH-Pt moiety. Key: cïs-Pt(II), left; O
and trans-P/(7/A right. >
O
m
H
Heteronùcléàr Complexes. L i k e p r o t o n a t i o n of c i s - P t
( N H ^ ^ I ^ , b i n d i n g of heterometals or of a second Pt (vide i n f r a )
occurs r e a d i l y . Guay and Beauchamp have reported s i m i l a r f i n d i n g s
w i t h Ag (54) and CH^Hg (55) coordinated to N3 o f 1-MeT. Among
the i s o l a t e d heteronuclear complexes o f c i s - P t ( N H ^ ^ I ^ two
types o f complexes have been e s t a b l i s h e d using X-ray c r y s t a l l o -
graphy: complexes of Pt,M and P t , M s t o i c h i o m e t r i e s .
?
For example, i n cis-[(NH-) Pt(1-MeU) Cu(P 0) 1

2 2 2 ?
S0^-4.5H 0 (38), P t i s coordinated by two NH« groups and two N3

2
donor atoms o f the 1-MeU l i g a n d s , Cu(II) by two aquo l i g a n d s and

two 04 donors o f the h e t e r o c y c l i c r i n g s . Both Pt and Cu have
roughly square p l a n a r . c o o r d i n a t i o n spheres w i t h the metals
s l i g h t l y out o f these planes and d i r e c t e d towards each o t h e r ,
g i v i n g Pt-Cu separations o f 2.765 X. Formation of the head-head
Pt,Cu dimer out of the h e a d - t a i l c i s - P t ( N H ^ ) L compound i n d i -

2 2
cates that r o t a t i o n of one of the two l i g a n d s i n the s t a r t i n g

compounds has preceded formation of the heteronuclear Pt,Cu
compound and may be considered a h i n t that 04 i s more b a s i c than
02. Nevertheless the p o s s i b i l i t y of N3,02 b r i d g i n g should not be
excluded.
Two c r y s t a l l o g r a p h i c a l l y confirmed examples of hetero-
n u c l e a r complexes of Pt~,M s t o i c h i o m e t r y e x i s t , j c i s - [ ( N H ~ ) ~
PtL MLJPt(NH ) ]
2 3 2X w i t h M= Ag (56) and Mn (57T"and L= T-MeT
Π
q
as b r i d g i n g l i g a n d (Figures 10 and 11). In both cases the two

Pt atoms have square planar c o o r d i n a t i o n , bonded to two NH^
groups and two N3 atoms o f the 1-MeT l i g a n d s , and the hetero-
metal M i s coordinated by f o u r oxygens of 1-MeT, most l i k e l y 04.
(An unambiquous d i f f e r e n t i a t i o n from 02 i s not p o s s i b l e due t o
the c r y s t a l l o g r a p h i c p e c u l i a r i t y of 1-MeT w i t h i t s pseudo-
twofold a x i s through N3 and C6.) The main d i f f e r e n c e between
the two compounds i s i n the c o o r d i n a t i o n sphere o f the h e t e r o -
metal: w h i l e Ag has a d i s t o r t e d t e t r a h e d r a l c o o r d i n a t i o n geo
metry, Mn has a square planar one. IR and Raman spectra of the
compounds suggest that only i n the Pt ,Mn compound the Mn-0 2
i n t e r a c t i o n has a marked covalent c h a r a c t e r , whereas the Ag-0

i n t e r a c t i o n appears to be e s s e n t i a l l y i o n i c , s i n c e only i n the
case .of the P t , Mn compound the h e t e r o c y c l i c modes o f the
2
1-MeT l i g a n d s are s t r o n g l y perturbed compared w i t h the b i s

(1-MeT)platinum complex. This c o n c l u s i o n i s i n agreement w i t h
the d i f f e r e n c e s i n M-0 distances as w e l l .
I f one compares the d i s t a n c e s between the r e s p e c t i v e
c o o r d i n a t i n g and non-coordinating oxygens i n the two compounds,
e.g. P t , A g : 014-024, 3.118 X; 034-044, 3.366 8; 012-022,
2
3.352 8; 032-042, 3.376 8, one f i n d s that they are r a t h e r

s i m i l a r . The same i s t r u e w i t h the Pt Mn compound, although the
2
distances are c o n s i d e r a b l y s h o r t e r , as expected. I f one t h i n k s

1
of b i n d i n g of another heterometal M to the a v a i l a b l e 02 atoms
f
of Pt ,ML^ t h i s i m p l i e s , that the c o o r d i n a t i o n sphere of M must
2

Figure 10. Molecular cation of [(NHshPtfl-MeThAgfl-MeThPtfNHshJNOs ·-

5H O. (Reproduced with permission from Ref. 56.)
t

LiPPERT Pt(H) Complex Formation with Uracil and Thymine
Figure 11. Molecular cation of [(NHshPtfl-MeThMnfl-MeThPtfNHshJCh

10H O. (Reproduced with permission from Ref. 57.)
2

be i d e n t i c a l w i t h that of the metal M. This i s , f o r example, con-

firmed w i t h the P t ^ j A g complex described below, where the square
p l a n a r c o o r d i n a t i o n geometry of the Pt bound t o 04 f o r c e s Ag to
take the unusual square p l a n a r c o o r d i n a t i o n as w e l l .
Head-Head Platinum Dimer. P r e p a r a t i o n and R e a c t i v i t y .

Reaction of cis-Pt(NH^X^L^ w i t h one equivalent of the "diaquo"
species of C i s p l a t i n gives i n high y i e l d the platinum dimer w i t h
head-head arranged L l i g a n d s :
2 +
cis-Pt(NH ) L 3 2 2 + cis-[Pt(NH ) (H 0) ]
3 2 2 2
2 +
cis-[Pt(NH ) L Pt(NH ) ]
3 2 2 3 2
With L= 1-MeT the s t r u c t u r e of the head-head dimer has been

determined (58). P t c o o r d i n a t i o n occurs through N3 and (most
l i k e l y ) 04. The compound bears some s i m i l a r i t y w i t h one h a l f of
the OC-pyridone dimer-of-dimers d e s c r i b e d by H o l l i s and L i p p a r d
(41) which i s the precursor of the t e t r a m e r i c OC-pyridone blue
described by Barton et a l . (59), but the i n t e r m o l e c u l a r P t - P t
s e p a r a t i o n i n the 1-MeT compound (5.62 8) i s almost twice as
long as w i t h the OC-pyridone dimer-of-dimers (3.13 8 ) .
I n an attempt t o a t t a c h yet another metal t o the
a v a i l a b l e second e x o c y c l i c oxygen of L, the 1-MeU head-head dimer
was reacted w i t h Ag , and a compound of composition [ ( N H ^ g P t ^
(1-MeU)Âg](N0 )^ has been i s o l a t e d (60). The pentanuclear
3
complex i s composed of two head-head dimers, each w i t h Pt bonded

to N3 and 04, and the two dimers are l i n k e d by Ag through the
a v a i l a b l e 02 atoms ( F i g u r e 13). Ag has square planar c o o r d i n a t i o n
and i s s i t u a t e d on the i n v e r s i o n centre of the c a t i o n , having
Ag-0 d i s t a n c e s of 2.34- 2.45 8. Adjacent pentanuclear c a t i o n s
are r e l a t e d by an i n v e r s i o n c e n t e r , thus l e a d i n g t o stacks of Pt^,
u n i t s i n t e r r u p t e d by Ag (Figure 14). The i n t e r m o l e c u l a r d i s t a n c e
between neighboring Pt atoms i s 3.25 8 o n l y , and the Pt dimers
are o r i e n t e d i n a way i d e n t i c a l w i t h that of the P t ( I I ) dimer-of-
dimers of 0L-pyridone and that of the p a r t i a l l y o x i d i z e d s p e z i e s .
The c r y s t a l s t r u c t u r e of t h i s Pt,,Ag complex o f f e r s a
r a t i o n a l e as to how formation of a p a r t i a l l y o x i d i z e d Pt complex
could conceivably take p l a c e . I f one assumes t h a t i n a redox
r e a c t i o n Ag o x i d i z e s a P t ^ u n i t and i s reduced to Ag , then the
o x i d a t i o n s t a t e of P t i s increased from +2 to +2.25, as i n the
ClL-pyridone blue. Indeed, s l i g h t warming of the Pt^,Ag compound
i n water gives a c r y s t a l l i n e blue product of composition
[ ( N H ) g P t ( 1 - M e U ) ] ( N 0 ) - 5 H 0 (60). Despite the l a c k of a
3 4 4 3 5 2
c r y s t a l l o g r a p h i c c o n f i r m a t i o n due to i n s u f f i c i e n t c r y s t a l s i z e ,
a l l the other evidence - elemental a n a l y s i s , r e d u c t i v e t i t r a t i o n ,
EPR spectrum - suggests that i t i s the analogue of the (X-pyridone
b l u e . T h i s assumption i s f u r t h e r supported by an a l t e r n a t i v e
s y n t h e s i s of the 1-methyluracil blue through HN0« o x i d a t i o n of
the head-head dimer analogous to a method of H o l l i s and L i p p a r d

Figure 12. Molecular cation of the head-head dimer cis-[(NH hPt(l-MeT) Pt-
s t
(NH ) ](NO )g. (Reproduced with permission from Ref. 58.)

s 2 s

1^6 METAL CHEMOTHERAPEUTIC AGENTS
I
ι
I
Is
"δ g
S3
δ
ί
.r
s
ϋ
ι
ι
Si
.s

7. LIPPERT Ρΐ(ΙΙ) Complex Formation with Uracil and Thymine 167
Figure 14. Arrangement of adjacent cations of the Pt Ag compound. Only two

k
halves of pentanuclear units are shown. (Reproduced from Ref. 60. Copyright 1982,
American Chemical Society.)

to obtain the ot-pyridone blue, and by the fact that the 1-MeU
blue, like the OC-pyridone blue (61), can be further oxidized to
yellow-orange, diamagnetic, 1-MeU bridged complexes which most
likely are the analogues of the Pt(III) dimers obtained by
Hollis and Lippard (61).
Acknowledgments
The work presented here has been made possible by the

joint efforts of Dr. Dietmar Neugebauer, Dr. Ulrich Schubert,
Jurgen Riede (crystal structures of 1-MeU and 1-MeT compounds),
Ruth Beyerle and Gabi Raudaschl (mixed nucleobase complexes),
Dr. Rudolf Pfab (complexes of unsubstituted thymine), Dr. Petr
Jandik (HPLC), Professor Colin J. L. Lock and Romolo Faggiani of
McMaster University, Hamilton, Canada (crystal structures of

complexes of unsubstituted uracil and thymine) and Dr. H. Howard-
Lock for corrections of the text. The generous financial support
by the Deutsche Forschungsgemeinschaft, DFG, and the Technische
Universitat MUnchen is gratefully acknowledged.
literature Cited
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2. Eichhorn, G. L. "Metal Ions in Biological Systems",
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3. Davidson, J. P.; Faber, P. J.; Fischer, R. G.; Mansy, S.;
Peresie, H. J.; Rosenberg, B.; Van Camp, L. Cancer
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Howe, Κ. E.; Liebermann, D. Z.; Newman, A. D.; Hill, J. M.
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Soc. 1974, 96, 6832.
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11. Lippert, B. J. Raman Spectrosc. 1980, 9, 324.
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15. Weiss, R.; Venner, H. Hoppe-Seyler Z. Physiol. Chem. 1969,
350, 396.

7. LIPPERT Pt(II) Complex Formation with Uracil and Thymine 169
16. Srivastava, R. C.; Srivastava, M. N. J. Inorg. Nucl. Chem.

1978, 40, 1439.
17. Taqui Khan, M. M.; Krishnamoorthy, C. R. J. Inorg. Nucl. Chem.
1974, 36, 711.
18. Bhandari, A. M.; Solanki, A. K.; Wadhwa, S. J. Inorg. Nucl.
Chem. 1981, 43, 2995.
19. Beck, W.; Kottmair, N. Chem. Ber. 1976, 109, 970.
20. Inagaki, K.; Kidani, Y. Bioinorg. Chem. 1978, 9, 157.
21. Faggiani, R.; Lippert, B.; Lock, C. J. L. Inorg. Chem. 1980,
19, 295.
22. Lumme, P.; Mutikainen, I. Acta Crystallogr., Sect. B. 1980,
B36, 2251.
23. Mutikainen, I.; Lumme, P. Acta Crystallogr., Sect. B. 1980,
B36, 2237.
24. Kistenmacher, T. J.; Sorrell, T.; Marzilli, L. G. Inorg.
Chem. 1975, 14, 2479.

25. Lippert, B.; Pfab, R.; Neugebauer, D. Inorg. Chim. Acta 1979,
37, L 495.
26. Faggiani, R.; Lippert, B; Lock, C. J. L . ; Pfab, R.
Inorg. Chem. 1981, 20, 2381.
27. Carrabine, J. Α.; Sunderalingam, M. Biochemistry 1971, 10,
292.
28. Goodgame, M.; Johns, K. W. J. Chem. Soc., Dalton 1977, 17,
1680.
29. Dale, R. M. K.; Martin, E . ; Livingston, D. C.; Ward, D. C.
Biochemistry 1975, 14, 2447.
30. Mansy, S.; Tobias, R. S. Inorg. Chem. 1975, 14, 287.
31. Pfab, R.; Jandik, P.; Lippert, B. Inorg. Chim. Acta, in
press.
32. Lippert, B. Inorg. Chem. 1981, 20, 4326.
33. Lippert, B.; unpublished results.
34. Lippert, B. Proc. Int. Conf. Raman Spectrosc. 7th 1980, 582.
35. Lallemand, J. Y.; Soulie, J.; Chottard, J. C. J. Chem. Soc.,
Chem. Commun. 1980, 436.
36. Lim, M. C.; Martin, R. B. J. Inorg. Nucl. Chem. 1976, 38,
1915.
37. Flynn, C. M.; Viswanathan, T. S.; Martin, R. B. J. Inorg.
Nucl. Chem. 1977, 39, 437.
38. Neugebauer, D.; Lippert, B.; submitted.
39. (a) Orbell, J. D.; Marzilli, L. G.; Kistenmacher, T. J.
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Lippert, B.; Lock, C. J. L. Inorg. Chem., in press.
40. (a) Gellert, R. W.; Bau, R. J. Am. Chem. Soc. 1975, 97,
7379. (b) Cramer, R. E . ; Dahlstrom, P. L . ; Seu, M. J. T . ;
Norton, T.; Kashiwaga, M. Inorg. Chem. 1980, 19, 148.
(c) Bau, R.; Gellert, R. W. Biochimie 1978, 60, 1040.
(d) Marzilli, L. G.; Chalilpoyil, P.; Chiang, C. C.;
Kistenmacher, T. J. J. Am. Chem. Soc. 1980, 102, 2480.
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103, 1230.

42. Lippert, B. Inorg. Chim. Acta 1981 , 55, 5.

43. Lock, C. J. L . ; Peresie, H. J.; Rosenberg, B.; Turner, G.
J. Am. Chem. Soc. 1978, 100, 3371.
44. Faggiani, R.; Lock, C. J. L . ; Pollock, R. J.; Rosenberg, B.;
Turner, G. Inorg. Chem. 1981, 20, 804.
45. Neugebauer, D.; Lippert, B.; submitted.
46. Lippert, B. J. Clin. Hematol. Oncol. 1977, 7, 26.
47. Beyerle, R.; Lippert, B. Inorg. Chim. Acta 1982, 66, 141.
48. Raudaschl, G.; Lippert, B.; unpublished results.
49. Lippert, B. J. Am. Chem. Soc. 1981, 103, 5691.
50. Teo, B.-K.; Eisenberger, P.; Reed, J.; Barton, J. K.;
Lippard, S. J. J. Am. Chem. Soc. 1978, 100, 3225.
51. Lippert, B.; Lock, C. J. L . ; Rosenberg, B.; Zvagulis, M.
Inorg. Chem. 1978, 17, 2971 and references.
52. Rosenberg, B. Biochimie 1978, 60, 859.

53. Boreham, C. J.; Broomhead, J. Α.; Fairlie, D. P.
Aust. J. Chem. 1981, 34, 659.
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55. Guay, F.; Beauchamp, A. L. Inorg. Chim. Acta 1982, 66, 57.
56. Lippert, B.; Neugebauer, D. Inorg. Chim. Acta. 1980. 46. 171.
57. Lippert, B.; Schubert, U. Ibid. 1981, 56, 15.
58. Lippert, B.; Neugebauer, D.; Schubert, U. Ibid. 1980, 46,L11.
59. (a) Barton, J.K.; Rabinowitz, Η. N.; Szalda, D. J.;
Lippard, S. J. J. Am. Chem. Soc. 1977, 99, 2827.
(b) Barton, J. K.; Szalda, D. J.; Rabinowitz, Η. N.;
Waszczak, J. V.; Lippard, S. J. Ibid. 1979, 101, 1434.
60. Lippert, B.; Neugebauer, D. Inorg. Chem. 1982, 21, 451.
61. (a) Hollis, L. S.; Lippard, S. J. J. Am. Chem. Soc.
1981, 103, 6761.
(b) Hollis, L. S.; Lippard, S. J.; personal communication.

8
195 15
Pt- and N-NMR Studies of Antitumor
Complexes
ISMAIL M. ISMAIL and PETER J. SADLER
Birkbeck College, Department of Chemistry, University of London,

Malet Street, London WC1E 7HX England
195 15
Pt and N NMR studies at 4.7 and 9.4 Τ are used
to characterise new Pt(II) and Pt(IV) anti-tumour
drugs, to study the interaction of inosine (I) and
1
5 -adenosine monophosphate (AMP) with cisplatin, and
release of amines induced by binding to methionine
sulphur of peptides and proteins. Drug characteri
195
sation is aided by the predictability of Pt
195 15
shifts and Pt - N coupling constants. Broaden
14
ing through scalar coupling to N can sometimes be
overcome by raising the temperature. Contributions
195
to Pt relaxation from chemical shift anisotropy,
not only make it difficult to observe signals from
Pt bound to macromolecules, but can also lead to
195 1 13 15
broadening of Pt satellites of H, C, and N
resonances. N7-06 chelation was not detected for I
15
bound to cis-Pt( NH )Cl at acidic pH levels, but
3 2
may occur for the diaquo complex. Several previously

undetected I and AMP species are reported. Finally,
facile release of both NH and a chelated diamine
3
(ethylenediamine) was observed on reaction with

N-Ac-Met, Gly-Met and RNase A in solution near
neutral pH and it is suggested that such reactions
may play an important part in the mechanism of
action of Pt anti-tumour drugs.
0097-6156/83/0209-0171 $06.00/0

In t h i s paper we study three problems:

( i ) the c h a r a c t e r i s a t i o n o f P t ( I I ) and P t ( I V ) anti-tumour drugs i n
1 9 5
s o l u t i o n by P t NMR,
( i i ) t h e i n t e r a c t i o n o f two n u c l e i c a c i d bases i n o s i n e ( I ) and
1 5
adenosine 5^monophosphate (AMP) w i t h N - l a b e l l e d c i s - P t ( N H ) C l 3 a 2
2 + 1 5 1 9 5
and c i s - P t ( N H ) ( H 0 ) 3 by N and P t NMR, and
2 2 2
( i i i ) the r e l e a s e o f amines from c i s - P t ( N H ) C l and P t ( e n ) C l on 3 2 2
b i n d i n g t o methionine-containing peptides and p r o t e i n s , u s i n g

1 5
p r i m a r i l y N NMR.
M u l t i n u c l e a r NMR i s p o t e n t i a l l y a very powerful technique f o r
d e f i n i n g f u l l y the c o o r d i n a t i o n sphere of platinum anti-tumour
drugs i n s o l u t i o n and upon i n t e r a c t i o n w i t h biomolecules. The
p r o p e r t i e s of some u s e f u l n u c l e i a r e l i s t e d i n Table I . I n our
s t u d i e s we seek i n f o r m a t i o n about the o x i d a t i o n s t a t e o f platinum
usually Pt(II) or Pt(IV),
Table I
P r o p e r t i e s o f some NMR n u c l e i u s e f u l i n the study o f

Pt anti-tumour drugs
Isotope I Abund.(%) Freq.(MHz) n.O.e. Recept.
H 1
h 99.98 200 _ 1.0
13 C h 1.11 50.29 +3.0 1
Ι.βχΙΟ" *
1 5
N h 0.37 20.27 -3.9 3.9xl0- 6
35 C1 3/2 75.53 39.19 - 3.6x10- 3
19 5 p t h 33.8 42.82 +3.3 3.4x10- 3
the number and types of coordinated l i g a n d s , t h e i r stereochemical

arrangements, and dynamic processes i n c l u d i n g the r a t e s o f l i g a n d
f
s u b s t i t u t i o n . Pt-induced l i g a n d a c t i v a t i o n s (e.g. lowered pK s )
are sometimes a l s o d e t e c t a b l e . A l l o f these f e a t u r e s appear to
c o n t r i b u t e t o the b i o l o g i c a l a c t i v i t y o f platinum complexes as
e x e m p l i f i e d by the f o l l o w i n g : c i s but not t r a n s P t ( I I ) diamine
2 - 2
complexes a r e a c t i v e anti-tumour drugs ( 1 ) , P t C l * and P t C l ~ 6
l f +
are potent a l l e r g e n s ( 2 ) , whereas P t ( e n ) i s a neurotoxin (3).
3

8. ISMAIL AND SADLER 5
Pt- and N-NMR Studies 15
173
*H NMR has the advantage o f h i g h s e n s i t i v i t y , and P t b i n d i n g

can o f t e n be detected v i a s p i n - s p i n couplings t o l i g a n d n u c l e i up
to 4 bonds away from P t . These couplings can provide stereochem
1 9 5 X 1 3
i c a l i n f o r m a t i o n ( 4 ) . However, P t s a t e l l i t e s o f H, C and
15
N resonances can be very broad and d i f f i c u l t t o a s s i g n a t h i g h
f i e l d s (5., 6) where s e n s i t i v i t y i s g r e a t e s t f o r work on biomolecules.
35
C 1 NMR i s u s e f u l f o r studying C I " r e l e a s e (7), but s i g n a l s f o r
Pt-bound c h l o r i d e are too broad t o observe.
1 9 5
We were a t t r a c t e d t o P t NMR by i t s l a r g e , p r e d i c t a b l e chem
1 9 5 5
i c a l s h i f t range o f ca. 15,000 ppm. (8). P t - * N one-bond
c o u p l i n g constants are p a r t i c u l a r l y s e n s i t i v e t o the nature o f
t h e 1 9 5
t r a n s l i g a n d C9,10,11,12). Sometimes P t c o u p l i n g t o quad-
llf
r u p o l a r N i s r e s o l v a b l e but, i n g e n e r a l , we have r e s o r t e d t o
1 5
enrichment o f P t diamines w i t h N . This has the advantage o f
1 5
making d i r e c t o b s e r v a t i o n o f N NMR s i g n a l s p o s s i b l e . Most o f
the work d e s c r i b e d here has only become p o s s i b l e i n the l a s t few
years w i t h the r o u t i n e a v a i l a b i l i t y o f high f i e l d (4.7 - 9.4 T)
F o u r i e r transform spectrometers w i t h l a r g e sample tubes (10 - 15
mm diameter).
Experimental
1 9 5 1 5
P t and N NMR s p e c t r a were recorded on V a r i a n XL 200
(4.7 T ) , Bruker WM200 (4.7 T) o r Bruker WH400 (9.4 T) spectrometers
i n 10 o r 15 mm diameter tubes. H 0 was used as s o l v e n t , w i t h 5- 2
10% D 0 added o r D 0 i n a c o n c e n t r i c tube f o r l o c k . This avoided

2 2
s i g n i f i c a n t d e u t e r a t i o n o f amine l i g a n d s . References were 1 M

1 9 5 15 1 5
Na PtCl i n D 0 for
2 6 2 P t , and 2.4 M NH*C1 i n 1 M HC1 f o r N .
1 5
Most N s p e c t r a were obtained from overnight accumulations u s i n g
30°-60° p u l s e s , 3.6s pulse r e p e t i t i o n r a t e s and a data p o i n t
r e s o l u t i o n o f 0.6 Hz/point. I n view o f p o s s i b l e negative n.O.e.'s
s e v e r a l s p e c t r a were checked a g a i n s t a phase r e f e r e n c e . The h i g h
frequency - p o s i t i v e s h i f t s i g n convention i s used.
1 9 5
P t Chemical S h i f t Range
Most P t complexes have l o w - l y i n g e x c i t e d e l e c t r o n i c s t a t e s ,

1 9 5
and P t NMR s h i f t s are dominated by the paramagnetic term i n
Ramsey's equation ( 8 ) . The P t ( I V ) h a l i d e s alone span some 12,500
ppm ( 8 ) :
2 2 2 2
PtF ~ 6 PtCl 6 " PtBr " 6 Ptl "*"
6
δ/ppm 7326 0 -1870 -5121
The c a l c u l a t e d s h i f t o f P t ( 0 ) i s -10,427 ppm ( 8 ) , and removal o f

e l e c t r o n s t o g i v e P t ( I I ) leads t o d e s h i e l d i n g and high-frequency
s h i f t s . P t ( I V ) resonances tend t o be f u r t h e s t t o h i g h frequency
(low f i e l d ) , but there i s c o n s i d e r a b l e o v e r l a p .

Ligand exchange r e a c t i o n s of P t ( I I ) and (even more so) P t ( I V )

are u s u a l l y slow enough on the NMR t i m e s c a l e f o r separate reson
ances to be observable f o r each d i s t i n c t Pt complex i n s o l u t i o n .
I n t e r e s t i n g exceptions are P t ( I I ) aquo-hydroxo complexes which
t i t r a t e as a s i n g l e , averaged resonance (13), and exchange of
amine w i t h t r a n s - P t ( e t h e n e ) ( a m i n e ) C l which i s r a p i d i n CDC1 (14).
2 3
The l a t t e r may be r e l e v a n t to the behavior of anti-tumour drugs i n

membranes·
S u b s t i t u t i o n s h i f t s u s u a l l y show r e g u l a r , incremental p a t t e r n s
z z
(15). For s u b s t i t u t i o n of B r " i n t o P t C l " and P t C l ~ , the s h i f t 4 6
increments (Δ6) vary w i t h s u b s t i t u t i o n s t e p , n, according to (16):
- Δ ό ( Ι Ι ) - 203 + 23.5n ppm

- Δ δ ( ΐ ν ) - 275 + 11.7n ppm
Even c i s - t r a n s and mer-fac isomers are u s u a l l y w e l l - s e p a r a t e d i n

2
the spectrum: 10 ppm f o r the isomers of P t C l B r " " , 208 ppm f o r 2 2
P t ( N H ) ( H 0 ) , and 310 ppm f o r P t ( N 0 ) i , C l - (12). S u b s t i t u t

3 2 2 2
2 +
2 2
2
i o n s h i f t s are s m a l l e s t f o r s u b s t i t u t i o n s t r a n s to l i g a n d s w i t h
h i g h t r a n s i n f l u e n c e s . Thus, i n P t ( I V ) n i t r i t e complexes, a Br"*
f o r CI"" s u b s t i t u t i o n i s ca. 50 ppm l a r g e r when t r a n s to C I "
compared to t r a n s to N0 ~ (12). The term "trans i n f l u e n c e " i s used
2
to denote the dependence of NMR (ground-state) parameters on the

nature of the t r a n s l i g a n d . This i s d i s t i n c t from the "trans
e f f e c t " which i s r e l a t e d to bond l a b i l i z a t i o n as measured by
ligand substitution rates.
3 5 3 7 7 9 8 1
At very h i g h f i e l d s (> 4.7 Τ ) , C 1 / C 1 and B r / B r i s o t o p -
omers are r e s o l v a b l e (17) and, i n p r i n c i p l e , the number of h a l i d e
ions bound to Pt can be deduced from the isotopomer s p l i t t i n g
p a t t e r n . In p r a c t i c e , the s h i f t s (0.167 ppm f o r C I , 0.028 ppm f o r
Br) are too s m a l l i n comparison w i t h the temperature dependence of
1 9 5
P t s h i f t s (0.3-1 ppm per °C r i s e i n temp.) and n a t u r a l l i n e -
widths of many complexes. Temperature g r a d i e n t s across samples
X
are p a r t i c u l a r l y marked when l a r g e sample tubes and H decoupling
are used.
1 9 5
Representative P t s h i f t s f o r anti-tumour and r e l a t e d com
plexes are given i n Table I I . Figure 1 i l l u s t r a t e s the r e g u l a r
p a t t e r n of NH - H 0 s u b s t i t u t i o n s h i f t s i n P t ( I I ) complexes. A
3 2
s i m i l a r graph i s obtained f o r the NH - C l ~ system, g i v i n g the3
f o l l o w i n g incremental s h i f t s :
Δδ(NH by 0H ) = + 590
3 2 ppm
Δ6(ΝΗ by CI") = + 270
3 ppm
1 9 5
The s e n s i t i v i t y of P t NMR s h i f t s to s m a l l changes i n the types
of bound l i g a n d s i s very u s e f u l f o r the c h a r a c t e r i s a t i o n of new
drugs. For example, the c o o r d i n a t i o n of b i s - c a r b o x y l a t e s i s o f t e n
X 13
not c o n v i n c i n g l y d e t e c t a b l e by H or C NMR, but Table I I shows
t h a t replacement of two H 0 l i g a n d s by c y c l o b u t a n e d i c a r b o x y l a t e
2
g i v e s a low frequency s h i f t of 133 ppm (18).

Table I I
1 5 1 9 5
N and P t NMR s h i f t s and one-bond c o u p l i n g constants f o r P t ( I I ) and P t ( I V ) diamines
195 15 195 χ5
Complex Ô( Pt)/ppm 6( N)/ppm ^( Ρί- N)/Hz Solvent
Pt(IV)
jc, t-Pt(NH ) (mal) (OH) 3 2 2
+1570 D 0/H 0
2 2 2
c-Pt(C H NH ) (0H)
3 7 2 2 4
+1521 249 D0
2
c , c , t - P t (C H NH ) C 1 (OH) 3 7 2 2 2 2
+881 266 D0
2
ç, ç,t-?t(NH ) C1 (OH)2 3 2 2
+860 -37.9 275 H 0/H 0
2 2 2
c-Pt(NH ) CU 3 2
-145 -30.5 247 H0
2
Pt(II)
2 H0
-81.7 342 2
[Pt(NH ) (OH)] + 3 2 2
3 H0
-1499 -79.1 339 2
[Pt(NH ) (0H)] + 3 2 3
2 + H0
-1590 -89.0 387 2
c-Pt(NH ) (H 0) 3 2 2 2
Pt(NH ) (Etmal)
3 2
-1694 -83.7 366 H0
2
Pt(NH ) (CBDCA)
3 2
-1723 360 H0
2
2 + H0
-1914 -51.9 411 2
Pt(en)(H 0) 2 2

c_-Pt(NH ) Cl 3 2 2
-2048 302 DMF
-2097 312 DMSO
-2168 312 H0
2
Pt(H)(NH ) ( H / » _
3 x 4 x
1 9 5 1 9 5 1 5
P t Chemical shift P t - N Coupling
m m 15
Figure 1. Plots of the variation in Pt chemical shift (left) and Pt- N one-bond
coupling constants with the number of NH ligands in the complexes [Pt(NH ) -
S s x
2 2
(H 0) . ] * (right). The values for tiam-Pt(NH ) (H O) + are those reported in
% k x s g g 2
Ref. 12.

m 15
8. ISMAIL AND SADLER Pt- and N-NMR Studies 177
19 5 p t _ ltf N > 15 N C o u p l l n g s
1 9 5 15
One bond P t - N c o u p l i n g constants depend on the s
c h a r a c t e r of the Pt o r b i t a l s used i n bonding to N, and are
expected to be s m a l l e r f o r a Ν l i g a n d t r a n s to a l i g a n d which has
a l a r g e t r a n s i n f l u e n c e s i n c e t h i s tends to weaken the t r a n s bond
(9,10,11). The remarkable r e g u l a r i t y of c o u p l i n g s i n P t ( I I ) N H / 3
H 0 complexes i s shown i n F i g u r e 1, where i t can be seen t h a t

2
s m a l l c i s e f f e c t s a l s o occur.
For c i s - P t ( N H ) 2 complexes, the order of t r a n s i n f l u e n c e s
a
l
based on J i s :
ligand : H0 2 <C0 " 2 <0H~ <C1" <NH 3 <S-Met

1
J/Uz : 390 360 340 310 285 265
1 9 5 15
The r a t i o of P t ( I I ) : P t ( I V ) P t - N couplings would be
2
expected to be 1.5, based on a h y b r i d i z a t i o n change from d s p t o
2 3
d s p ( 9 ) . From our l i m i t e d data, Table I I , i t i s apparent t h a t
c i s e f f e c t s from the a d d i t i o n a l a x i a l l i g a n d s i n P t ( I V ) p l a y a
r o l e . Many of the P t ( I V ) c o u p l i n g s are h i g h e r than p r e d i c t e d ,
g i v i n g r a t i o s of c a . 1.2.
X X 1 9 5
*N and *N c o u p l i n g s to P t are r e l a t e d by t h e i r gyro-
magnetic r a t i o s :
1 9 5 1 5 1 9 5 χΐ|
^( Ρί- Ν)/^( Ρϋ - Ν) = 2.711/1.932 = 1.40
llf
However d i r e c t l y bonded l i g a n d N n u c l e i o f t e n cause severe
1 9 5
broadening of P t resonances and hence couplings are unresolved.
This leads us to a c o n s i d e r a t i o n of some important r e l a x a t i o n
1 9 5
mechanisms f o r Pt.
1 9 5
P t Relaxation
S c a l a r R e l a x a t i o n of the Second K i n d . The l i n e w i d t h s of

1 9 5 1If
P t resonances from complexes w i t h N l i g a n d s are determined
χι 1
by the quadrupolar r e l a x a t i o n r a t e of *Ν, ( T i q ) " where
1 ι 2 2 1 2
(Txq)- » < Τ « Γ α = 3π (2Ι + 3) χ (1 + /3η )τ 0
101*(21-1)
- 1
At h i g h temperatures ( T i ) decreases as the motional c o r r e l a t i o n q
time x decreases. Provided t h a t f i e l d g r a d i e n t s are s m a l l ,

c
( u s u a l l y the case f o r coordinated primary amines), then T . may i(

1 9 5 XI
become long enough to a l l o w r e s o l u t i o n of P t - *N c o u p l i n g s .
24
T h i s i s i l l u s t r a t e d i n F i g u r e 2. S i m i l a r l y , [cis-Pt(NH ) (Η 0) ] 3 2 2 2
g i v e s a broad resonance a t 300 K, but a t 343 Κ c o u p l i n g s are

resolved (6). Conversely, a t low temperatures, or i n l a r g e mole
XI 1 9 5
c u l e s , e f f e c t i v e decoupling of *N from P t may occur.

l
Figure 2. { H}-**Pt-NMR spectra (43
MHz) of cis,cis,trans-Pt(isopropylamine) - g
Cl (OH)
g in D O at 333 Κ
g g (top)
and 298 Κ (bottom) showing resolution
195 14
of Pt- N coupling at high temperature.
The shift is temperature dependent. (Re
produced with permission from Ref. 6.) 8/ppm 900 880 860

195 15
Chemical S h i f t Anisotropy (CSA). CSA r e l a x a t i o n i n c r e a s e s

2
w i t h the square o f the a p p l i e d f i e l d ( B ) n u c l e a r screening 0 f
2
a n i s o t r o p y ( Δ σ ) , and w i t h molecular weight and lowering o f t h e
temperature ( T i n c r e a s e s ) : c
1
C T ( P t ) . r ( C S A ) - 6/7 C T a i P O r ^ C S A )
1
2
= (2/15) y Bo Δσ T P t
2 2
C
1 5
This has r e s t r i c t e d many o f our N s t u d i e s o f P t ( I I ) complexes t o
intermediate f i e l d s (e.g. 4.7 T). P t ( I V ) complexes are l e s s a n i
s o t r o p i c and the e f f e c t i s l e s s marked. I t i s probable that CSA
1 9 5
r e l a x a t i o n i s p a r t l y r e s p o n s i b l e f o r our f a i l u r e t o observe Pt
s i g n a l s d i r e c t l y from P t bound to macromolecules. CSA r e l a x a t i o n
1 9 5 1 9 5
of P t can a l s o l e a d t o the disappearance o f P t s a t e l l i t e s
X 1 3 1 5
from H, C o r N s p e c t r a . These are normally used as i n d i c a t
1 9 5
ions o f b i n d i n g s i t e s . P t couplings appear i n the s p e c t r a o f
coupled l i g a n d n u c l e i as 1:4:1 m u l t i p l e t s only i f r e l a x a t i o n times
1 9 5
are the same i n Pt(I-O) and P t s p e c i e s . I n d i c a t i o n s t h a t
X
d i f f e r e n c e s could e x i s t were noted i n H NMR s t u d i e s by E r i c k s o n
et a l (4) on 1,2-diaminoethane complexes, and Lallemand e t a l (5)
f o r n u c l e o s i d e complexes. We have r e c e n t l y shown (6) f o r t r a n s -
X
P t ( e t h e n e ) ( 2 - c a r b o x y - p y r i d i n e ) C l t h a t the broadening o f H 2
1 9 5
s a t e l l i t e s a t h i g h f i e l d a r i s e s from P t r e l a x a t i o n v i a the CSA
mechanism. The e f f e c t on the s a t e l l i t e l i n e w i d t h s i s p r o p o r t i o n a l
1 9 5
to the s p i n - l a t t i c e r e l a x a t i o n r a t e o f P t , [2ττΤι (Pt) , and
2
therefore to B as shown above.
0
S p i n - r o t a t i o n . This r e l a x a t i o n mechanism i s important f o r

s m a l l complexes and u n l i k e the o t h e r s , i n c r e a s e s w i t h temperature.
2 2
I t appears t o be dominant f o r P t C l z , " and P t C l " (19). 6
15
N Chemical S h i f t Range
15
N NMR s h i f t s o f amines bonded t o P t ( I I ) appear t o be d e t e r
mined l a r g e l y by the nature o f the trans l i g a n d . We f i n d the
15
f o l l o w i n g approximate ranges f o r l i g a n d s t r a n s t o N H : 3
6/ppm : -40 t o -50 ; -50 t o -70 ; -80 t o -90

ligand : S (Met, DMSO) NH , C I "
3 H 0, C 0 ~
2 2
I t i s notable t h a t t h i s order p a r a l l e l s t r a n s i n f l u e n c e s i n a
1 1 9 5 1 5
s i m i l a r manner t o J ( P t - N ) . Motschi and Pregosin (11a) have
1 5 x 1 9 5
p r e v i o u s l y noted a strong c o r r e l a t i o n between 6 ( N ) and J ( Pt-
15
N) f o r a range o f P t ( I I ) S c h i f f ' s base complexes.
1 5
A l e i e t a l (20) c l e a r l y demonstrated that changes i n N
1 9 5 T 5
s h i f t s and P t ^ N couplings can be used t o f o l l o w r e a c t i o n s o f
15
N - e n r i c h e d c i s - d i a m i n e P t ( I I ) diaquo complexes w i t h 1-methyl-
imidazole ( a l s o e n r i c h e d ) . The s h i f t ranges f o r ethylenediamine
complexes are s i m i l a r t o those above f o r NH except that a l l peaks 3
are s h i f t e d to h i g h frequency by about 30 ppm.

Hydroxide-bridged Dimers and Trimers

1 9 5
An elegant use of P t NMR has been i n k i n e t i c s t u d i e s of the
1 9 5
o l i g o m e r i z a t i o n of P t ( I I ) diamines around n e u t r a l pH. Pt
15
s p e c t r a of N - e n r i c h e d [ c i s - P t ( N H ) ( H 0 ) ( O H ) ] + show w e l l 3 2 2
r e s o l v e d resonances f o r monomer, dimer and t r i m e r (hydroxide-

bridged) s p e c i e s (21a). From these s t u d i e s the h a l f - l i f e f o r d i s -
appearance of the monomer a t pH 7 was c a l c u l a t e d t o be about 7
•min. T h i s system has a l s o been s t u d i e d by Boreham et a l (22).
G i l l and Rosenberg have r e c e n t l y r e p o r t e d (21b) a s i m i l a r
1 9 5
P t study f o r unenriched 1,2-diaminocyclohexane complexes.
llf
Although N s c a l a r c o u p l i n g broadens the resonances, T i s now x
s h o r t enough t o enable r a p i d p u l s e r a t e s t o be used without

s a t u r a t i o n . Spectra w i t h good s i g n a l - t o - n o i s e r a t i o s were acqu-
i r e d from 50 mM s o l u t i o n s i n j u s t a few minutes. The s h i f t of the

dimer i s about 436 ppm to h i g h frequency of the diaquo monomer.
This i s probably due to the s h o r t Pt - P t c o n t a c t (3.1 Â) s i n c e
the resonance f o r the t r i m e r (Pt - P t , 3.7 A) i s s h i f t e d back t o
w i t h i n 141 ppm of the diaquo complex. The stronger t r a n s i n f l u -
ence of b r i d g i n g OH" compared to H 0 i s r e f l e c t e d i n a 40 Hz 2
T T 9 5 1 5
decrease i n J ( P t - N ) , Table I I . Terminal OH" l i g a n d s appar-
e n t l y have comparable t r a n s i n f l u e n c e s to NH (22). 3
2 +
I n t e r a c t i o n of c i s - P t ( N H ) 3 2 w i t h N u c l e i c A c i d Bases
1 9 5 15
Using a combination of P t and N NMR, we have r e c e n t l y
2+
shown (23) t h a t ç i s - P t ( N H ) ( H 0 ) and i t s ethylenediamine (en)
3 2 2 2
analogue r e a c t r a p i d l y w i t h acetamide to g i v e a product i n which

the C O group i s bound to P t through 0 w i t h H 0 displacement. A 2
15
2 ppm s h i f t of the N NH or en resonance t o h i g h frequency was
3
1 9 5
observed i n a d d i t i o n to a 47 ppm P t s h i f t (23). We t h e r e f o r e
sought to use s i m i l a r methods to d e t e c t p o s s i b l e N7-06 c h e l a t i o n
w i t h n u c l e i c a c i d bases, the s u b j e c t of much s p e c u l a t i o n .
For our i n i t i a l s t u d i e s , we chose i n o s i n e ( I ) and adenosine
monophosphate (AMP), F i g u r e 3. Only the former has the p o s s i b i l -
i t y of N7-06 c h e l a t i o n . We chose to work a t low pH's of c a . 2
where a l l the l i t e r a t u r e evidence seemed to p o i n t t o N7 as the
o n l y b i n d i n g s i t e on both I and AMP (24,25). A c i d i c s o l u t i o n s
a l s o a v o i d any competing o l i g o m e r i z a t i o n r e a c t i o n s through OH"
1 9 5
bridging. A P t NMR spectrum of a 1:1 mixture of I and
1 5
c i s - P t ( N H ) C l which had r e a c t e d f o r 7 days a t pH 2.3 i n the
3 2 2
dark i s shown i n F i g u r e 4. The long r e a c t i o n time was chosen

merely to ensure the attainment of e q u i l i b r i u m . Unreacted complex
( t r i p l e t , J = 312 Hz) as w e l l as two s e t s of s i g n a l s w i t h s h i f t s
near those expected f o r P t N ( H 0 ) (-2330 ppm) and PtN* (-2440 ppm) 3 2
species are seen. The broadening of these suggests c o o r d i n a t i o n

llf
of I v i a N. C u r i o u s l y t h e r e appear t o be two d i f f e r e n t
P t N ( H 0 ) complexes p o s s i b l y due to c o o r d i n a t i o n of both N7 and Nl.
3 2
The s h i f t of the proposed 1:2 complex compares w e l l w i t h the p r o -

duct of a s i m i l a r r e a c t i o n we have c a r r i e d out w i t h 2 e q u i v a l e n t s
of GMP.

8. ISMAIL AND SADLER
m
Pt- 15
and N-NMR Studies 181
0 NH 2
N
toi 2° H H N
V°^ C H 2 0 P
° 3 H
" Figure 3. Molecular structures of ino-
^--^ VrXu sine (left) and adenosine 5'-monophos-
0 H 0 H 0 H 0 H
phate (right). At acidic pH values, the
Inosine AMP only Pt binding site expected is N7.
-2000 -2100 -2300 S/ppm -2400 -2500
Figure 4. {^-^Pt-NMR spectrum (43 MHz) of a 1:1 solution of cis-Pi-

15
( NH ) Cl and inosine (50 mM in H O/10% D 0). Some of the Pt complex was
s 2 g 2 2
present in suspension at the start of the reaction but almost all of it had dissolved
after 7 d at 40 °C when this spectrum was recorded (pH 2.3).

A On} 15
- N NMR spectrum of the same i n o s i n e s o l u t i o n i s
shown i n Figure 5. There i s no observable s i g n a l i n the 80-90
ppm range (not shown). This i s the r e g i o n expected f o r NH t r a n s 3
t o C=0 i f c h e l a t i o n occurs. The resonance of 64.7 ppm (J=312 Hz)

confirms the presence of unreacted cis-Pt(NH ) Cl · A l l the r e s 3 2 2
onances i n the 61-69 ppm r e g i o n are assigned to NH l i g a n d s t r a n s 3
to Ν or CI". There seems to be a t l e a s t 7 r e s o l v e d peaks, 5 of

which have v i s i b l e s a t e l l i t e s . Resonance 5^ has a c o u p l i n g most
a p p r o p r i a t e to a PtN* complex (288 Hz) the other couplings l i e
+
between 304 and 335 Hz. No l i b e r a t i o n of N H i s observed. I f 4
2
[ c i s - P t ( N H ) ( N 7 - I ) C I 3 + and [ c i s - P t ( N H ) ( N 7 - I ] + were the o n l y
3 2 3 2 2
products, o n l y three resonances would be expected. The presence

of f o u r a d d i t i o n a l peaks suggests e i t h e r t h a t there are a d d i t i o n a l
b i n d i n g s i t e s ( N l or N3, not 0 ) , or t h a t species w i t h d i f f e r e n t
base o r i e n t a t i o n s or Η-bonding i n t e r a c t i o n s i n v o l v i n g NH are be 3
i n g detected. The sharpness of the s a t e l l i t e peaks suggests t h a t

a l l species are of low molecular weight.
Chu and Tobias (24) were unable to detect N7-06 c h e l a t i o n f o r
i n o s i n e by Raman spectroscopy, and suggested t h a t a s p e c i a l s t a b i l
i t y was a s s o c i a t e d w i t h c i s - P t ( N H ) ( I ) perhaps through s t a c k i n g
3 2 2
of coordinated i n o s i n e s . T h e i r work on a c i d i c s o l u t i o n s of
2 +
cis-Pt(NH ) (H 0)
3 2 2 suggested that only N7- coordinated 1:1 and
2
15
1:2 complexes were formed. Our N s p e c t r a of s i m i l a r s o l u t i o n s
are shown i n F i g u r e 6 where i n o s i n e i s compared to AMP. Peaks are
15
now present i n the 85-95 ppm range as expected f o r N H t r a n s to 3
H 0.
2 I f we ignore n.O.e. e f f e c t s on i n t e n s i t i e s (although there
i s l i t t l e j u s t i f i c a t i o n f o r t h i s ) then there i s a s i m i l a r amount
of unreacted diaquo complex i n both cases. This i s c u r i o u s i n
view of the second b i n d i n g s i t e a v a i l a b l e on AMP, although Pt has
to d i s p l a c e a proton (pK of N l ca. 4 ) . Indeed, although peaks 1
a
and 3 are common to both s p e c t r a , peak 5 i s unique to the AMP

r e a c t i o n and maybe due to the NH t r a n s to H 0 i n [ P t ( N H )
3 2 3 2
2+
(Nl-AMP)(H 0)] . 2 Again i n the 60-70 ppm r e g i o n there are more
resonances than can be simply accounted f o r by N7 c o o r d i n a t i o n
alone f o r I , unless Η-bonding, b a s e - o r i e n t a t i o n or inter-molecular
a s s o c a t i o n are a l s o i n v o l v e d .
We p r e d i c t e d t h a t c l o s u r e of the N7-06 c h e l a t e r i n g would
l e a d to a ca. 2 ppm s h i f t of the t r a n s NH to h i g h frequency. 3
P o s s i b l e candidates are resonances 1 ( r i n g open) and 2 (closed)

f o r I (Figure 6 ) . Resonance 2 i s absent f o r the AMP system as
would be expected.
15
N NMR i s t h e r e f o r e a powerful method f o r studying the i n t e r
a c t i o n between n u c l e i c a c i d bases and platinum drugs i n s o l u t i o n .
l
When combined w i t h P t and U NMR, more d e t a i l s of the i n t e r
1 9 5
a c t i o n s i n s o l u t i o n should be revealed. Further work along these

l i n e s i s i n progress.

8. ISMAIL AND SADLER m
Pt- and 15
Ν-NMR Studies 183
Figure 5. ^Hy^N-NMR spectrum (20.3 MHz) of the same 1:1 solution of cis-
15 195
Pt( NH ) Cl and inosine used in Figure 4. Main peaks and their Pt satellites
s t t
are numbered.

ι ι ι ι » I > r r r ι ι ι ι ι j f > r ι r ι ι ι ι j ι ι ι ι ι ι ι ι ι j ι ι ι ι ι ι ι ι ι ι
-60 -70 -60 -90 ppm
10
•
15 2
Figure 6. {'Hy^N-NMR spectra (40.6 MHz) of 0.32 M cis-Pt( NH h(H O) + s t 2
in the presence of 0.5 molar equivalents of AMP (top, pH 2.2) and inosine (bottom,
pH 1.9) 1 d after mixing. Resonances from ca. —65 to —75 ppm and —85 to —95
15
ppm are assigned to NH ligands trans to Ν or CI' and O, respectively. A resonance
S
due to unreacted Pt complex appears in both spectra at —89 ppm.

Pt- and 15
N-NMR Studies 185
Drug Binding to Methionine i n Peptides and P r o t e i n s : Release of

Coordinated Amines
Our i n t e r e s t i n Pt b i n d i n g to methionine r e s i d u e s of p r o t e i n s
was f i r s t aroused by the observations of c r y s t a l l o g r a p h e r s (26)
2
t h a t PtCli» " and cis-Pt(NH )2CI2 bind s p e c i f i c a l l y to the Met S
3
atoms i n p r o t e i n c r y s t a l s , sometimes w i t h secondary b i n d i n g to

His i f the pH i s h i g h enough (pH 6-7). However, the r e s o l u t i o n i n
e l e c t r o n d e n s i t y maps of p r o t e i n s i s r a r e l y high enough to a l l o w
complete d e f i n i t i o n of the c o o r d i n a t i o n sphere of P t . This l e a d
2
Dickerson to propose that on b i n d i n g to cytochrome c, P t C l * "
becomes 6-coordinate: a P t ( I V ) C U ( S M e t ) L adduct (26). Chemically
t h i s seemed very u n l i k e l y , as discussed by Petsko e t a l (27), and
1 9 5 15
we sought to use a combination of P t NMR s h i f t s and N coup
l i n g s t o d e f i n e the b i n d i n g s i t e s .
1 9 5
A l l our attempts to observe P t NMR s i g n a l s from e i t h e r
P t C l z , " or c i s - P t ( N H ) 2 C I 2 bound to reduced cytochrome c or r i b o -
2 15
3
nuclease A (RNase) have so f a r f a i l e d . These platinum complexes

are known to b i n d to the sulphur atoms of exposed methionines
(residues 65 and 29 of Cyt c and RNase r e s p e c t i v e l y ) as shown by
our previous *H NMR s t u d i e s on RNase (28) and those of Boswell et
a l on Cyt c (29) and x-ray c r y s t a l l o g r a p h y . We assume t h e r e f o r e
t h a t the resonances are broadened beyond d e t e c t i o n v i a chemical
s h i f t a n i s o t r o p y r e l a x a t i o n . The r e s t r i c t i o n of Pt m o b i l i t y on
the p r o t e i n w i l l l e a d to a l a r g e i n c r e a s e i n T (see CSA equation c
above). The a n i s o t r o p y term would a l s o be expected to i n c r e a s e .

llf
S c a l a r c o u p l i n g to N w i l l a l s o c o n t r i b u t e to the i n c r e a s e i n
l i n e w i d t h s i f n i t r o g e n b i n d i n g s i t e s are i n v o l v e d .
1 9 5
To make sure t h a t our search of the P t chemical s h i f t
range was concentrated i n the c o r r e c t r e g i o n s , we s t u d i e d a number
of model r e a c t i o n s . Displacement of one C I " by S of Met leads toa
high f i e l d s h i f t of ca. 1000 ppm, whereas i n t r o d u c t i o n of f u r t h e r
S to give P t N S , P t N S and PtS* gives f u r t h e r incremental s h i f t s
2 2 3
of 600, 300 and 0 ppm r e s p e c t i v e l y . The most i n t e r e s t i n g f e a t u r e

15
of the N NMR spectrum of a s o l u t i o n c o n t a i n i n g cis-Pt(NH )2CI2 3
and N-acetyl-L-Met (1:2) pH 2.2, a f t e r 3.5 hr r e a c t i o n a t 298 K,

i s the l a r g e peak f o r ΝΗ<Λ. A l l the other peaks are e x p l i c a b l e
by S c o o r d i n a t i o n : c i s - P t ( N H ) ( S - N A c M e t ) C l and c i s - P t ( N H )
3 2 3
(S-NAcMet)Cl . 2
Reactions of c i s - P t ( N H ) C l w i t h RNase A a t pH 6.5 a l s o l e a d

3 2 2
to the r e l e a s e of coordinated NH , Figure 7. U n l i k e the N-Ac-Met 3

1 5
system above, no s i g n a l s are observed f o r N H t r a n s to S. I t 3
+
seems t h a t N H production induced by the h i g h t r a n s i n f l u e n c e
4
of S i s a general r e a c t i o n and, i n p a r t i c u l a r , does not r e q u i r e

a low pH. Once S d i s p l a c e s CI", NH r e l e a s e appears to be more 3
favourable w i t h the p r o t e i n , although i n analogous r e a c t i o n s of

2 +
c i s - P t ( N H ) 2 ( H 0 ) 2 added to RNase A a t pH 6, intermediates w i t h
3 2
NH t r a n s to S are d e t e c t a b l e .
3
Our r e p o r t seems to be the f i r s t d i r e c t demonstration of NH 3
r e l e a s e on r e a c t i o n of Pt drugs w i t h peptides and p r o t e i n s ,

although V o l s t e i n et a l (30,31) have i s o l a t e d analogous products
from r e a c t i o n of methionine i t s e l f (which can c h e l a t e v i a S and
NH ) w i t h c i s - P t ( N H ) C l .
2 3 2 2

Figure 7. {W^'N-NMR spectrum (40.6 MHz) of a 6 mM solution of RNase A

15
that had reacted with 5 molar equivalents of cis-Pt( NH ) Cl for 8 d (final pH 6.5).
s t t

m 15
This r a i s e s the question of whether the d i f f e r i n g b i o l o g i c a l

a c t i v i t i e s of c i s and trans P t ( I I ) diamines could be r e l a t e d to
S-induced amine r e l e a s e . This would be a k i n d of b i o l o g i c a l
Kurnakov t e s t ! Kurnakov d i s t i n g u i s h e d (32) between c i s and
t r a n s - P t ( N H ) C l 2 by t h e i r r e a c t i o n s w i t h t h i o u r e a : the c i s
3 2
isomer g i v e s a y e l l o w t e t r a k i s t h i o u r e a s p e c i e s , whereas no NH 3
i s r e l e a s e d from the t r a n s complex g i v i n g the white product,

[trans-Pt(NH )2(thiourea)23 .
3
2+
However, f o r t h i s to be an import
ant b i o l o g i c a l event, i t would need to apply to chelated diamine
complexes a l s o , and t h i s seemed l e s s l i k e l y . Indeed, Romeo e t a l
reported (33) t h a t s i g n i f i c a n t amounts of the ring-opened form of
+ 4
[Pt(en)(DMS0)C1] are present i n s o l u t i o n o n l y a t h i g h H * (molar)
and CI"" c o n c e n t r a t i o n s , based on u v - v i s i b l e s p e c t r a .
Thus we were somewhat s u r p r i s e d to f i n d t h a t r e a t i o n s of
c i s - P t ( e n ) C l w i t h both N-acetyl-L-methionine
2 (as a model system)
and w i t h RNase A l e a d to s i g n i f i c a n t r e l e a s e of f r e e ethylene-
diamine, Figures 8 and 9. The greater d i f f i c u l t y i n d i s p l a c i n g
1
en r e l a t i v e to NH i s r e f l e c t e d i n the presence of peaks f o r
3 Ν
trans to S i n the spectrum. A c u r i o u s f e a t u r e i n Figure 9 i s the
2 +
a d d i t i o n a l peak ca. 3 ppm to low f i e l d of e n H . Only f u r t h e r 2
experiments w i l l determine whether t h i s i s monodentate ( d a n g l i n g -

arm) en, or perhaps en bound to the p r o t e i n .
F i n a l l y , we d e s c r i b e a model r e a c t i o n which suggests t h a t some
b i n d i n g s i t e s i n p r o t e i n s could l e a d to very e f f i c i e n t amine r e
l e a s e . Reaction of one e q u i v a l e n t of the d i p e p t i d e Gly-L-Met
w i t h çis-Pt(NH ) C1 at pH 5.5 leads to a very e f f i c i e n t r e l e a s e
3 2 2
of NH . 3 This i s a t t r i b u t a b l e to the t r i d e n t a t e b i n d i n g of the

peptide v i a S, deprotonated NH, and NH groups: a mode of c o o r d i n - 2
a t i o n found by Freeman and Golomb (34) i n the c r y s t a l s t r u c t u r e

of Pt(Gly-L-Met)CI w i t h deprotonation of NH even a t pH 2.5. This
r a i s e s the question as to whether b i n d i n g of Pt to Met residues
of p r o t e i n s can be followed by b i n d i n g to N" (deprotonated NH) of
the neighbouring peptide l i n k a g e , although i t seems l i k e l y t h a t
t h i s r e a c t i o n needs to be d r i v e n by a d d i t i o n a l NH c o o r d i n a t i o n .
2
2 +
I t i s p e r t i n e n t to note t h a t the SCH *H NMR s h i f t of F e 3 cyto-
chrome c on r e a c t i o n w i t h Pt complexes d i f f e r s markedly from t h a t
observed i n a model system of N-Ac-Met where c o o r d i n a t i o n i s to
S only (29).
B i o l o g i c a l S i g n i f i c a n c e of Amine Release. Our suggestion

that amine r e l e a s e may be an important i n v i v o event f o r Pt a n t i -
tumour drugs was not supported by animal s t u d i e s , u n t i l r e c e n t l y .
The e a r l y work of Talyor et a l (35) appeared to e s t a b l i s h t h a t
1 9 5 ll
P t and * C - l a b e l l e d P t ( e n ) C l were metabolised s i m i l a r l y i n
2
r a t s . However t h i s study, which was based on a s i n g l e 24h time

p o i n t , has r e c e n t l y been extended. Robins and Leach now f i n d
1 9 1 1
(36), using P t and " C - l a b e l l e d P t ( e n ) C l and c i s - P t ( N H ) C 1 ,
2 3 2 2
t h a t d i s s o c i a t i o n of l i g a n d from Pt occurs as soon as 1 hr a f t e r

a d m i n i s t r a t i o n to mice bearing ADJ/PC6 plasma c e l l tumours.
llf
P r o l i f e r a t i n g t i s s u e s of the gut and tumour take up C i n t o DNA

30 20 10 -10 -20 -30 -40 -50 Î I ppm

_ 1 ! 1 j !
292 _ ·
X
CI S
295 Hz
15
Figure 8. {*H)->*N-NMR spectrum (20.3 MHz) of a 0.1 M solution of Pt( N-
en)Cl containing two molar equivalents of N-acetyl-L-methionine at pH 2.8 and
t
reaction time of 1 wk, although equilibrium may be reached earlier than this.
, s
ppm 20 -20 -40 -60 -80
?
βηΗΓ
Pt-N-VN Nl'- /N
v
as
HtenCl2
15
Figure 9. ^Hy^N-NMR spectrum (40.6 MHz) of Pt( N-en)Cl after reaction g
with RNase A (6 mU) in a 5:1 molar ratio for 7 d (3 d at 37 °C and 4 d at 20 °C

in darkness). Initially much of the Pt complex was present as a suspension. Peaks
for unreacted complex appear in the spectrum. The phases of peaks were checked
against a phase reference. The loss of n.O.e. on the signals from the proposed
Pt(en)Cl(S-Met-Protein) species is consistent with a lowered mobility (longer r ) e
15 X
for these N nuclei. Relaxation via mechanisms other than dipolar coupling to H
may also be occurring.

Pt- and 15N-NMR Studies 189
from day 4 in parallel with the rise in synthetic activity, but

this is not paralleled by an uptake of 1 9 1 P t . Levels of1 9 1 P t
in the gut and tumour from cis-Pt(NH3)2CI2 are much greater than
those from PtenCl 2 , perhaps a reflection of the ease of release
of NH3 compared to en. We therefore conclude that studies of the
mechanism of action of Pt anti-tumour complexes should include
consideration of the consequences of protein as well as nucleotide
binding. The active metabolites may well be different from those
currently considered. A controlled transfer of Pt from proteins
to DNA could occur, and i t is pertinent to note that methionine
is thought to play a key role in the initiation of protein syn
thesis. The fate of released amines also requires further atten
tion. Taken together, these new approaches may lead to a better
understanding of structure-activity relationships.
Acknowledgments
We thank the SERC, MRC, Johnson Matthey Ltd and the University
of London Intercollegiate Research Service for support. We are
also grateful to Mr. P. Hydes and Dr. C. Barnard (Johnson Matthey)
for discussions and loan of Pt compounds, Mr. M. Buckingham and
Dr. G. Hawkes (ULIRS High Field NMR Service), Dr. J. Feeney and
Dr. P. Morris (MRC Biomedical NMR Centre) for assistance with some
NMR measurements, and Dr. A.B. Robins (Royal Marsden) for dis
cussion of his unpublished results.
PJS is a Nuffield Foundation Science Research Fellow 1981-82.
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12. (a) Kerrison, S.J.S. Ph.D. Thesis, London University,

London, 1982.
(b) Kerrison, S.J.S., Sadler, P.J. J. Chem. Soc. Dalton,
1982, in press.
13. Freeman, W., Pregosin, P.S., Sze, S.N., Venanzi, L.M. J.
Magn. Res.,1976,22,473.
14. Al-Najjar, I.M., Green, Μ., Kerrison S.J.S., Sadler, P.J.
J. Chem. Res. (S),1979,206.
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Marshall, I.R.H. J.C.S. Dalton,1976,459.
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Commun.,1980,1175.
18. Neidle, S., Ismail, I.M., Sadler, P.J. J. Inorg. Biochem.,
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19. Pesek, J.J., Mason, W.R. J. Magn. Res.,1977,25,519.
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(b) G i l l , G.S., Rosenberg, B. J. Amer. Chem. Soc., 1982,in
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Interactions", Spiro, T.G. Ed, Wiley Inter. S c i . , New York,
1980,1,(Metal Ions in Biology Series, p.32).
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9
C o n f o r m a t i o n a l P r o p e r t i e s of P u r i n e and
P y r i m i d i n e C o m p l e x e s of c i s - P l a t i n u m
Implications for Platinum(II)-DNA Crosslinking Modes
THOMAS J. KISTENMACHER and JOHN D. ORBELL

The Johns Hopkins University, Department of Chemistry, Baltimore, MD 21218
LUIGI G. MARZILLI
Emory University, Department of Chemistry, Atlanta, GA 30322
The stereochemical properties of a variety of cis

complexes of Pt(II) containing purine or pyrimidine
ligands are examined. The c r i t i c a l intramolecular
conformational parameters [the interbase dihedral
angle and the base/coordination plane dihedral
angles] are systematically studied and trends sought.
Where intramolecular interactions are determinative
of the adopted molecular conformation, the nature of
the steric demands imposed by increasing numbers of
exocyclic functional groups contiguous to the Pt
binding site are clearly of major import.
Within the past several years, there has been l i t e r a l l y an

explosion of chemical and biochemical studies (1) directed toward
elucidating the mode of action of platinum(II) anti-tumor drugs,
typified by cis-Pt(NH3) 2Cl2 (2)· It is perhaps not inappropriate
to suggest, however, that while significant progress has been
made, the mode (or modes) of action of such Pt(II) reagents at
the molecular level remains elusive. A leading hypothesis is
that the primary molecular targets for active Pt(II) complexes
are the nucleobases of cellular DNA. In the event that DNA is
the "active" macromolecular target, several experimental results
can be cited (3-6) which implicate metal-base binding at regions
rich i n guanosine-cytidine (G-C) residues as both prevalent and
possibly of mechanistic importance.
The various results accumulated thus far have led to
numerous proposals for ways i n which Pt(II) agents may bind to
the nucleobases of DNA so as to cause mutagenesis (by inducing
base-mispairing) or c e l l death (by introducing one or more
"defects" which cancer cells may find d i f f i c u l t or impossible to
repair). Both the cis and trans isomers of a reagent such as
Pt(NH 3 ) 2 Cl 2 are mutagenic, and to some extent carcinogenic, but
only the cis isomer has significant anti-tumor activity (1). One
of the requirements for anti-tumor activity in these systems,
0097-6156/83/0209-0191$06.00/0

then, centers about the a v a i l a b i l i t y of two adjacent s i t e s a t the

P t ( I I ) center. The p o s s i b i l i t y has n a t u r a l l y a r i s e n that each of
these s i t e s i s u t i l i z e d to form a c o v a l e n t bond to a nucleobase -
l e a d i n g to the formation of an i n t e r s t r a n d or an i n t r a s t r a n d
metal-mediated c r o s s - l i n k .
One chemical approach toward a c h i e v i n g r e s o l u t i o n of the
mode of a c t i o n of c i s - P t ( I I ) complexes a t the molecular l e v e l has
been to s y n t h e s i z e v a r i o u s model complexes of the g e n e r a l formu
l a t i o n cis-A2PtB2> where A i s NH3 or A2 i s a b i d e n t a t e c h e l a t e
such as ethylenediamine (en) or trimethylenediamine (tn) and the
Β l i g a n d s are nucleobases, n u c l e o s i d e s or n u c l e o t i d e s . The p r i n
c i p a l advantage of t h i s approach i s that these complexes are
u s u a l l y i s o l a b l e ( o f t e n i n c r y s t a l l i n e form) and r e a d i l y s t u d i e d
both i n s o l u t i o n and the s o l i d s t a t e by a v a r i e t y of s p e c t r o
s c o p i c techniques (e.g., NMR, IR, RAMAN, ORD/CD, and X-ray

s c a t t e r i n g - both EXAFS and s i n g l e c r y s t a l ) . The major disadvan
tage of these model systems l i e s i n deducing to what degree
c o r r e l a t i o n s that a r i s e from such s y s t e m a t i c s t u d i e s can be
c a r r i e d over w i t h f i d e l i t y to a Ρt(II)-DNA complex.
I n t h i s r e p o r t , we b r i e f l y summarize some of the major c o r
r e l a t i o n s , emphasizing molecular c o n f o r m a t i o n a l p r o p e r t i e s , f o r
cis-A2PtB2 complexes which have become a v a i l a b l e through s i n g l e -
c r y s t a l X-ray d i f f r a c t i o n s t u d i e s . An attempt i s a l s o made to
suggest which of these deductions are most l i k e l y to be worthy of
c o n s i d e r a t i o n as p o s s i b l e elements of any d e t a i l e d d e s c r i p t i o n of
an " i n v i v o " Pt(II)-DNA complex and i t s p o s s i b l e r o l e i n a n t i
tumor a c t i v i t y .
Nucleobase B i n d i n g S i t e s
I n any s y s t e m a t i c a n a l y s i s of the b i n d i n g of a metal to the

nucleobases of DNA, a c a r e f u l s c r u t i n y of the v a r i e t y of endo-
c y c l i c n i t r o g e n atom s i t e s a v a i l a b l e or p o t e n t i a l l y a v a i l a b l e a t
p h y s i o l o g i c a l pH i s of c e n t r a l importance. I n t h i s s e c t i o n , we
enumerate f o r each nucleobase the v a r i o u s metal b i n d i n g s i t e s ,
paying p a r t i c u l a r a t t e n t i o n to the number and the nature of the
e x o c y c l i c f u n c t i o n a l groups near to these s i t e s . To t h i s end, we
present i n F i g u r e 1 i l l u s t r a t i o n s of four commonly employed
nucleobases (9-methylguanine, 9-methyladenine, 1-methylcytosine
and the thymine monoanion). A l s o presented i n F i g u r e 1 are i n -
plane e l e c t r o s t a t i c p o t e n t i a l energy maps f o r these bases. These
maps are ready v i s u a l i z a t i o n s of the a v a i l a b i l i t y of the lone
p a i r d e n s i t y f o r each base and the p o s s i b l e r o l e of e l e c t r o
s t a t i c s i n any p a r t i c u l a r metal b i n d i n g mode.
Guanosine. Near pH 7, the N(7) s i t e ( F i g u r e 1) on a guanine

base has an a v a i l a b l e p a i r of e l e c t r o n s and i s known to b i n d
metals (notably P t ( I I ) and P d ( I I ) ) to produce s t a b l e complexes
(Z> iL> Î)· T n e
Presence of the nearby 0(6) c a r b o n y l oxygen atom
has l e d to a c o n t i n u i n g s p e c u l a t i o n (10-13) that t h i s e x o c y c l i c

KISTENMACHER ET A L . Purine and Pyrimidine cis-Pt Complexes 193
Figure 1. Molecular topology, atomic numbering, and in-plane molecular electro

static potential energy maps for a, 9-methylguanine (9-MeG); b, 9-methyladenine
(9-MeA); c, 1-methylcytosine (1-MeC); and d, the Ν1-deprotonated thymine mono
anion (Thy-). Contour levels for the electrostatic potential energy maps are given in
kcal/mol as in Ref. 21 and 26.

o x y g e n atom may p l a y an i m p o r t a n t r o l e i n M - N ( 7 ) - b o u n d g u a n o s i n e
systems ( e . g . , the p o s s i b l e p r e s e n c e under c e r t a i n c o n d i t i o n s o f
a M-N(7),0(6) c h e l a t i o n b i n d i n g mode).
The N ( l ) s i t e on g u a n o s i n e ( F i g u r e 1) i s p r o t o n a t e d a t pH 7.
I t i s k n o w n , h o w e v e r , t h a t b o t h a l k y l a t i o n (14) a n d P t ( I I ) b i n d
i n g ( 1 4 , 1 5 , 16) a t N(7) c.an r e d u c e t h e pKa o f t h e N ( l ) - H p r o t o n
by c a . 2 l o g u n i t s , l e a d i n g to s i g n i f i c a n t N ( l ) d e p r o t o n a t i o n a t
pH 7 . A s f o r t h e c a s e o f N(7) m e t a l b i n d i n g , t h e p o s s i b l e r o l e
of 0(6) f o r m e t a l a t t a c k a t N ( l ) has been c o n s i d e r e d ( 1 7 - 2 2 ) .
(It i s w o r t h w h i l e t o n o t e t h a t t h e r e i s one e x a m p l e i n t h e l i t e r
a t u r e o f a C u ( I I ) complex w h i c h e x h i b i t s a d i r e c t M-0(6) b i n d i n g
mode (23) and r e c e n t s p e c t r o s c o p i c e v i d e n c e w h i c h s u g g e s t s 0 ( 6 )
b i n d i n g by N ( l ) - d e p r o t o n a t e d g u a n i n e d e r i v a t i v e s i n s o l u t i o n
(22)). A p o i n t o f p a r t i c u l a r i n t e r e s t t o us h e r e , h o w e v e r , i s
t h a t the deprotonated N ( l ) s i t e of guanosine i s expected to be a

strong nucleophile. I n a d d i t i o n , we n o t e t h a t t h i s s i t e i s
f l a n k e d b y an e x o c y c l i c amino g r o u p a s w e l l a s an e x o c y c l i c o x o
group ( F i g u r e 1 ) . ( F o r t h e l e s s common n u c l e o s i d e i n o s i n e , t h e
e x o c y c l i c amino g r o u p a t C ( 2 ) i s m i s s i n g , and t h e d e p r o t o n a t e d
N ( l ) b i n d i n g s i t e h a s o n l y t h e a d j a c e n t oxo g r o u p n e a r b y ) .
Adenosine. U n l i k e g u a n o s i n e , b o t h the Ν ( 7 ) and N ( l ) b a s e

b i n d i n g s i t e s of adenosine have l o n e p a i r d e n s i t y a v a i l a b l e f o r
m e t a l b i n d i n g a t pH 7 ( F i g u r e 1 ) . T h e s e two s i t e s a r e , f o r N(9)-
s u b s t i t u t e d 6 - a m i n o p u r i n e s , of about e q u a l metal b i n d i n g s t r e n g t h
( 9 , 2 4 ) , and t h e r e i s r e a s o n t o s p e c u l a t e ( 2 5 , 26) t h a t t h e
a v a i l a b i l i t y o f t h e s e two m e t a l b i n d i n g s i t e s l e a d s t o m u l t i p l e
m e t a l b i n d i n g and polymer f o r m a t i o n i n m e t a l - a d e n o s i n e s y s t e m s .
The major s t e r e o c h e m i c a l d i f f e r e n c e i n the e n v i r o n m e n t s
a b o u t t h e N(7) a n d N ( l ) s i t e s o f a d e n o s i n e i s t h a t , f o r t h e l a t
t e r s i t e , t h e e x o c y c l i c amino g r o u p i s a d j a c e n t t o t h e s i t e o f
p o t e n t i a l m e t a l a t t a c k , w h i l e f o r M-N(7) b i n d i n g t h i s amino g r o u p
l i e s o f f a 3 - c a r b o n atom ( F i g u r e 1 ) .
Cytidine. N e a r n e u t r a l p H , o n l y t h e Ν ( 3 ) s i t e on a c y t o s i n e
base has lone p a i r d e n s i t y a v a i l a b l e f o r metal b i n d i n g ( F i g u r e
1). We w i s h t o e m p h a s i z e t h a t t h e N(3) s i t e o n a c y t o s i n e b a s e
i s d i r e c t l y a n a l o g o u s t o t h e Ν ( 1 ) - d e p r o t o n a t e d s i t e on g u a n o s i n e ,
i n t h a t b o t h h a v e a n e x o c y c l i c amino and a n e x o c y c l i c oxo g r o u p
on t h e i r r e s p e c t i v e α - c a r b o n atoms. ( I t h a s a l s o b e e n shown t h a t
u n d e r r e l a t i v e l y m i l d c o n d i t i o n s ( 2 7 , 28) d e p r o t o n a t i o n o f t h e
e x o c y c l i c amino g r o u p o f c y t o s i n e c a n o c c u r f o l l o w e d b y m e t a l
binding at this group).
Thymidine. Under b a s i c c o n d i t i o n s , thymine e x i s t s as a

t a u t o m e r i c m i x t u r e o f m o n o a n i o n s ( 2 9 ) , w i t h e i t h e r N ( l ) o r N(3)
d e p r o t o n a t e d ( t h e N ( l ) d e p r o t o n a t e d tautomer and i t s e l e c t r o
s t a t i c p o t e n t i a l map a r e i l l u s t r a t e d i n F i g u r e 1 ) . Deprotonation
of a thymidine r e s i d u e i n a p o l y n u c l e o t i d e a l l o w s the p o t e n t i a l
for metal attack at N(3). T h i s s i t e i s c o n t i g u o u s t o two oxo

9. KISTENMACHER ET AL. Purine and Pyrimidine cis-Pt Complexes 195
f u n c t i o n a l groups; the p o s s i b l e u t i l i z a t i o n of these carbonyl

oxygen atoms i n the formation of dimeric and polymeric métallo
species has been an a c t i v e area of i n v e s t i g a t i o n (17, 18, 30-32
and references t h e r e i n ) .
I n Table I , we summarize the e n d o c y c l i c base b i n d i n g s i t e s
f o r the common n u c l e o s i d e s discussed above and r e i t e r a t e the
nature of the e x o c y c l i c f u n c t i o n a l groups near these s i t e s .
Table I
Nucleobase M e t a l B i n d i n g S i t e s and the Nature o f the

S u b s t i t u e n t s Contiguous to the B i n d i n g S i t e
Nucleos ide Binding S i t e Adjacent S u b s t i t u e n t s

Adenosine N(7) -H;

N(l) -H; -N(6)H 2
Guanosine N(7) -H;

N(l)* -N(2)H ; =0(6)
2
Inosine N(7) —H*

N(l)* -H; =0(6)
Cytidine N(3) -N(4)H ; =0(2)
2
Thymidine N(3)* =0(2); =0(4)
Requires deprotonation p r i o r to o r concomitant w i t h metal b i n d i n g
Stereochemical P r o p e r t i e s of cis-A2PtB2 Complexes
For the c l a s s of complexes symbolized by cis-A2PtBB' (where

1
Β and B a r e p o t e n t i a l l y d i f f e r e n t nucleobases, nucleosides o r
n u c l e o t i d e s ) , the p r i n c i p a l i n t r a m o l e c u l a r c o n f o r m a t i o n a l param
e t e r s a r e the f o l l o w i n g : (1) the i n t e r b a s e d i h e d r a l angle (B/B*);
and, (2) the base/PtN^ c o o r d i n a t i o n plane d i h e d r a l angles (B/PtN^
f
and B /PtN^). I t became c l e a r to us i n the course of attempting
to compare these conformational parameters over a wide v a r i e t y of
complexes s t u d i e d by s i n g l e - c r y s t a l X-ray d i f f r a c t i o n methods
that some convention needed to be advanced (33). For example,
1
the B/B d i h e d r a l angle, as w i t h other d i h e d r a l a n g l e s , may be
defined as an angle or i t s supplement, l e a d i n g to p o t e n t i a l con
f u s i o n and i n a p p r o p r i a t e comparisons.
In order to achieve a meaningful comparison among d i f f e r e n t
complexes, i t was decided t h a t a v i s u a l as w e l l as an a n a l y t i c a l
d e s c r i p t i o n of these d i h e d r a l angles was e s s e n t i a l . As our pro
posed convention has been d e t a i l e d elsewhere (33), we o f f e r here
a s i m p l i f i e d i l l u s t r a t i o n s u f f i c i e n t f o r the a p p r e c i a t i o n of the
1
comparisons and trends presented l a t e r . I n F i g u r e 2, the B/B ,
B / P t N C l and B ' / P t ^ C ^ d i h e d r a l angles are i l l u s t r a t e d f o r the
2 2
complex c i s - d i c h l o r o b i s p y r i d i n e p l a t i n u m ( I I ) (34). The B/B' draw

i n g , f o r example, i s achieved by c o n f i n i n g one of the two c i s
p y r i d i n e bases (the one c o n t a i n i n g atom N(2) has been chosen) to

BASE/BASE DIHEDRAL ANGLE
(1) PLACE ONE BASE IN-PLANE

SO N^PT IS LEFTWARD
(2) SECOND BASE OUTWARD
(3) DEDUCE IF ANGLE IS ^90°
BASE/PTN 4 DIHEDRAL ANGLE
(1) ORIENT ONE Ν-Ρτ NORMAL
TO THE VIEWING PLANE
(2) SECOND PT-N LEFTWARD
(3) SENSE OF A N G L E ( * 9 0 ° ) AS
INDICATED
Figure 2. Systematic molecular iformational

co drawings for cis-dichlorobispyri-
dineplatinum(II) and the essence
ή the proposed conformational convention.

the plane of the paper and to the r i g h t o f the second base (con
t a i n i n g atom N ( l ) ) , which p r o j e c t s outward toward the viewer.
f
The convention (33) y i e l d s a B/B i n t e r b a s e d i h e d r a l angle
of 72° f o r the present complex as the p y r i d i n e r i n g c o n t a i n i n g
N(l) i s o b v i o u s l y t i l t e d toward that c o n t a i n i n g N(2). I t i s
c l e a r t o us that the d e f i n i t i o n of the i n t e r b a s e d i h e d r a l angle
at 72° i s s u p e r i o r t o that of i t s supplement 108°. By a s i m i l a r
procedure (see Figure 2 ) , one obtains b a s e / P t N C l d i h e d r a l 2 2
angles of 124° ( N ( l ) - r i n g ) and 118° ( N ( 2 ) - r i n g ) . I n t h i s case

(and a l l others considered subsequently), we have employed l e a s t -
squares planes o b t a i n a b l e from p u b l i s h e d f r a c t i o n a l coordinates
to d e r i v e numerical values f o r the d i h e d r a l angles.
A p p l i c a t i o n s t o c i s - B i s ( p u r i n e ) P t ( I I ) Complexes. There a r e
p r e s e n t l y a v a i l a b l e a l a r g e number of c i s - b i s ( N ( 7 ) - b o u n d purine
b a s e ) P t ( I I ) complexes whose molecular s t r u c t u r e s have been d e t e r
mined by X-ray d i f f r a c t i o n techniques. For the most p a r t , the
nucleobase i n these systems i s a 6-oxopurine d e r i v a t i v e ( 7 ) .
Figure 3 presents conformational drawings f o r three of these com
2 +
p l e x e s : the c i s - [ P t ( e n ) ( 1 , 3 , 9 - t r i m e t h y l x a n t h i n e ) ] c a t i o n (35);
2
2 + f
the [ P t ( e n ) ( g u a n o s i n e ) ] c a t i o n (36); and [Pt(tn)(Me-5 -GMP )]°
2 2
(37), where Me-5'-GMP i s the phosphate methyl e s t e r of guanosine

5'-monophosphate. Since each of these three complexes i s r e
q u i r e d to possess C molecular symmetry i n the s o l i d s t a t e , only
2
one of the B/PtN^ d i h e d r a l angles i s i l l u s t r a t e d i n F i g u r e 3.

1
From Figure 3, i t i s c l e a r that the B/B i n t e r b a s e conforma
t i o n a l angle (as w e l l as the B/PtN^ d i h e d r a l angle) decreases
s i g n i f i c a n t l y on going from the bis(nucleobase) complex, through
the b i s ( n u c l e o s i d e ) complex, t o the b i s ( n u c l e o t i d e ) complex.
This trend has been r a t i o n a l i z e d by suggesting t h a t across the
s e r i e s there i s an i n c r e a s i n g amount o f a t t r a c t i v e intracomplex
base/base i n t e r a c t i o n which culminates w i t h the n u c l e o t i d e com
p l e x . S p e c i f i c i n t e r b a s e i n t e r a c t i o n s i n v o l v i n g the carbonyl
oxygen atom 0(6) of one base and the Pt-bound i m i d a z o l e r i n g of
the two-fold r e l a t e d n u c l e o t i d e have been c i t e d (37) as p o s s i b l y
of p a r t i c u l a r importance. Such i n t e r b a s e i n t e r a c t i o n s are a l s o
of notable s t r e n g t h i n s e v e r a l P t ( I I ) complexes c o n t a i n i n g two
cis-bound i n o s i n e 5'-monophosphate l i g a n d s (12, 38, 39). A f a c
tor which may a l s o be o f some importance w i t h regard to these
b i s ( n u c l e o t i d e ) complexes i s the common occurrence (35, 37) of a
0(phosphate)··-Η-0-Η···0(6) intracomplex hydrogen bonding scheme
which i s favored by ( f a v o r a b l e to) a s m a l l i n t e r b a s e d i h e d r a l
angle.
Nonetheless, i t has been suggested (35) that i t i s u n l i k e l y
that these intracomplex i n t e r a c t i o n s a r e of s u f f i c i e n t s t r e n g t h
to be determinative of t h e adopted molecular conformation under a
v a r i e t y of c o n d i t i o n s . I f so, i t i s l i k e l y that the molecular
conformation of such N(7)-bound 6-oxopurine complexes (and N(7)-
bound 6-aminopurine systems (26)) w i l l be d i c t a t e d by a competi
t i o n between intracomplex (base/base, hydrogen bonding) and


9. KiSTENMACHER ET AL. Purine and Pyrimidine cis-Pt Complexes 199
intercomplex (base/base s t a c k i n g , base/counterion, hydrogen bond

ing) i n t e r a c t i o n s .
A p p l i c a t i o n s to cis-bis(pyrimidine)Ρt(II) Complexes. C o n s i
derably l e s s stereochemical data are p r e s e n t l y a v a i l a b l e f o r c i s -
bis(pyrimidine)Ρt(II) complexes from X-ray d i f f r a c t i o n s t u d i e s
(20, 33, 40-42). Of these, we have chosen to present conforma
t i o n a l drawings i n Figure 4 f o r the c i s - [ P t ( N H ^ ) 2 ( l - m e t h y l c y t o -
2 , _
s i n e ) ] " c a t i o n (33), the mixed base c a t i o n c i s - f P ^ N H o K i l -
2
+
methylcytosine) (N(l)-deprotonated thymine monoanion)] (41) and
the [Pt(en)(7,9-dimethylhypoxanthine) J 2 c a t i o n (20). I n the
l a t t e r complex, the nucleobases are a c t u a l l y m o d i f i e d p u r i n e s ;
however, s i n c e the p y r i m i d i n e r i n g s i t e N ( l ) o f the purine base
i s bound to the P t ( I I ) c e n t e r , t h i s complex i s i n c l u d e d i n the
present s e r i e s .
I t i s evident from a study of F i g u r e 4 that there a r e exact
1
l y opposite trends observed f o r the B/B and the B/PtN^ d i h e d r a l
angles. The i n t e r b a s e d i h e d r a l angle i n c r e a s e s smoothly and the
b a s e / c o o r d i n a t i o n plane d i h e d r a l angle decreases p r o g r e s s i v e l y as
the number of e x o c y c l i c f u n c t i o n a l groups adjacent to the P t atom
b i n d i n g s i t e increases from (1 + 1) f o r the 7,9-dimethylhypoxan
t h i n e c a t i o n to ( 2 + 1 ) f o r the mixed 1-methyIcytosine/thymine
monoanion complex to (2 + 2) f o r the bis(1-methyIcytosine) com
p l e x c a t i o n . These trends suggest t h a t as the number of exocy
c l i c s u b s t i t u e n t s ortho to the P t atom b i n d i n g s i t e i n c r e a s e s ,
intracomplex s t e r i c f a c t o r s become i n c r e a s i n g l y more determina
t i v e of the adopted molecular conformation.
These trends culminate i n the s t e r i c a l l y crowded c i s -
2 +
[ P t ( N H ) ( l - m e t h y l c y t o s i n e ) ] c a t i o n (33). I t can be contended
3 2 2
that the conformational parameters i n t h i s c a t i o n are p r i m a r i l y

d i c t a t e d by intracomplex s t e r i c e f f e c t s . I n c o n j u n c t i o n w i t h
e a r l i e r remarks (vide s u p r a ) , t h i s deduction i s expected to c a r r y
over d i r e c t l y t o cis-bis(N(1)-bound)Ρt(II) complexes of deproton-
ated guanine as the nature and number of f u n c t i o n a l groups ortho
to t h e N ( l ) b i n d i n g s i t e are e x a c t l y those f o r the N(3) b i n d i n g
s i t e of cytosine.
Mixed P u r i n e / P y r i m i d i n e Complexes. U n f o r t u n a t e l y , only very

l i m i t e d s t r u c t u r a l i n f o r m a t i o n i s a v a i l a b l e (15, 16) f o r mixed
p u r i n e / p y r i m i d i n e complexes of P t ( I I ) . I n f a c t , so f a r only com
plexes c o n t a i n i n g N(7)-bound 9-ethylguanine (or i t s N ( l ) - d e p r o -
tonated monoanion) and Ν(3)-bonded 1-methyIcytosine have been
s t r u c t u r a l l y c h a r a c t e r i z e d . Conformational drawings f o r these
complexes are presented i n Figure 5.
F o l l o w i n g the e m p i r i c a l trends o f f e r e d i n the previous two
s e c t i o n s , i t might be expected that the r o l e of intracomplex
s t e r i c f a c t o r s f o r such complexes would be intermediate between
those f o r a cis-bis(N(7)-bound guanine) complex and those f o r a
cis-bis(N(3)-bound c y t o s i n e ) complex. The conformational
parameters d i s p l a y e d i n F i g u r e 5 seem to be i n accord w i t h t h i s


Figure 4. Base/base and base/PtN dihedral angles for three bis(pyrimidine-ring-bound)Pt(II)
k
complexes. 1-MeC is 1-methyIcy tosine; Thy is the Nl-deprotonated monoanion of thymine; and
7,9-Dmhyp is 7,9-dimethylhypoxanthine.
9. KiSTENMACHER ET AL. Purine and Pyrimidine cis-Pf Complexes 201
S*
t :
.11
II·
ΊΟ
«*•»-
•G I
L-s .
-Si c£
§ 1-5
g§ε
•11-
«I
«•ο "a
ftj
3 "S
3*!
CO
S?
3 ft*

f
n o t i o n . The B/B i n t e r b a s e d i h e d r a l angles are l a r g e f o r a l l
three complexes. There i s , however, c l e a r l y some degree of v a r i -
a b i l i t y i n the i n t e r b a s e d i h e d r a l angle, probably owing to the
r e l a t i v e l y d i f f e r e n t environment ( e s p e c i a l l y the presence of 9-
ethylguanine:9-ethylguanine monoanion i n t e r b a s e hydrogen bonding
i n some cases) about each of the species i n t h e i r r e s p e c t i v e
c r y s t a l l i n e s o l i d s (15, 16).
Environmental E f f e c t s . To f u r t h e r expose the v a r y i n g e f f e c t s

of c r y s t a l l i n e environment on molecular conformation, we present
i n F i g u r e 6 two complexes where only the charge-balancing counter-
ion has been changed (35) or the degree of h y d r a t i o n m o d i f i e d
(41, 42).
In £he f i r s t example of the [ P t ( e n ) ( 1 , 3 , 9 - t r i m e t h y l x a n -
thine^] c a t i o n , the change i n c o u n t e r i o n from PF5"" to N0^~

1
causes a reasonably dramatic r e d u c t i o n i n the B/B d i h e d r a l angle
by ca. 16°. S i m i l a r l y , a d d i t i o n to the c r y s t a l l i n e s t r u c t u r e of
three water molecules per complex of the cis-[Pt(NH3)2(1-methyl-
+
c y t o s i n e ) ( N ( l ) - d e p r o t o n a t e d thymine monoanion)] (as the per-
c h l o r a t e s a l t i n each case) causes a decrease i n the i n t e r b a s e
1
d i h e d r a l angle by some 11°. That the a l t e r a t i o n i n the B/B
d i h e d r a l angle owing to environmental e f f e c t s i s l e s s f o r the
mixed p y r i m i d i n e complex than f o r the b i s ( p u r i n e ) complex would
c e r t a i n l y be c o n s i s t e n t w i t h the s t e r e o c h e m i c a l i n f l u e n c e of the
number of e x o c y c l i c f u n c t i o n a l groups adjacent to the metal b i n d -
ing s i t e f o r each of the nucleobases. However, the p a u c i t y of
the data p r e s e n t l y a v a i l a b l e l i m i t s our confidence i n such a
proposal.
I m p l i c a t i o n s f o r Pt-DNA C r o s s - l i n k i n g Modes
In the f o r e g o i n g s e c t i o n s , we have d e s c r i b e d a convention

f o r the s y s t e m a t i c a p p r a i s a l of the conformational ( n o t a b l y , the
i n t e r b a s e d i h e d r a l angle) p r o p e r t i e s of c i s complexes of P t ( I I )
c o n t a i n i n g purine or p y r i m i d i n e bases or t h e i r n u c l e o s i d e s or
n u c l e o t i d e s as l i g a n d s . From an a n a l y s i s of these c o n f o r m a t i o n a l
parameters, the p r i n c i p a l e m p i r i c a l deduction i s that the r o l e of
i n t r a m o l e c u l a r f o r c e s i n determining the adopted molecular con-
formation i s most s t r o n g l y i n e f f e c t when there are two ortho
s u b s t i t u e n t s adjacent to the P t b i n d i n g s i t e on each of the c i s -
bound nucleobase l i g a n d s .
In t h i s s e c t i o n , we w i l l attempt to apply t h i s deduction to
an " i n v i v o " Pt(II)-DNA complex. I n p a r t i c u l a r , we w i l l concen-
t r a t e our a t t e n t i o n on regions of a DNA duplex which are r i c h i n
(G-C) r e s i d u e s . These regions are k i n e t i c a l l y f a v o r a b l e to
P t ( I I ) b i n d i n g , and, t h e r e f o r e , most l i k e l y i n i t i a l s i t e s f o r
a t t a c k by a P t ( I I ) anti-tumor drug. Our a t t e n t i o n w i l l be f u r -
ther focused onto p o t e n t i a l i n t r a s t r a n d or i n t e r s t r a n d m e t a l -
mediated c r o s s - l i n k s i n these (G-C) r i c h r e g i o n s .
In Table I I , the s i x d i f f e r e n t types of c r o s s - l i n k i n g modes

9. KISTENMACHER E T A L . Purine and Pyrimidine cis-Pt Complexes 203
BEN>PT(|.3,9-TMX) ] 2
N 0 " SALT
3
+
0NH ) PT(1-MECKTHY)]
3 2
ANHYDROUS
TRIHYDRATE
Figure 6. Base/base and base/PtN conformational drawings for the PF " and
k 6
2
NOf salts of [Pt(en)(l,3,9-trimethylxanthine) ] + and the anhydrate and trihydrate
2
of cis-[Pt(NH ) (l-methylcytosine)(Nl-deprotonated thymine anion)]\

s t

Table I I
C r o s s - l i n k i n g Modes i n Regions of High (G-C) Content
(1) N ( 7 ) - P t - N ( 7 )
G G (Periphery-Periphery)
(2) N ( 7 ) - P t - N ( 1 )
G G (Periphery-Core)
(3) N ( 1 ) - P t - N ( 1 )
G G (Core-Core)
(4) N ( 7 ) - P t - N ( 3 )
G c (Periphery-Core)
(5) N ( 1 ) - P t - N ( 3 )
G c (Core-Core)
(6) N ( 3 ) - P t - N ( 3 )
c c (Core-Core)
which can be e n v i s i o n e d i n a r e g i o n o f h i g h (G-C) content a r e

enumerated. I n each case, the e n d o c y c l i c N-atom b i n d i n g s i t e

u t i l i z e d f o r each nucleobase i s i n d i c a t e d , as w e l l as the r e l a -
t i v e p o s i t i o n o f t h i s b i n d i n g s i t e a t the " p e r i p h e r y " o r " c o r e "
of a DNA duplex (these l a t t e r l a b e l s are most a p p r o p r i a t e f o r a
B-DNA h e l i x (43) and l e a s t a p p r o p r i a t e f o r a Z-DNA h e l i x (44,
45)). I n t h i s c o n t e x t , only the N(7) s i t e on a guanine base i s
considered to be near the periphery o f the h e l i x (these s i t e s are
o f t e n exposed to the surrounding medium i n the major grove o f a
B-DNA h e l i x ) ; the core s i t e s (N(3) of c y t o s i n e and N ( l ) of
guanine), a r e , o f course, i n v o l v e d i n the G-C t r i p l e hydrogen-
bond scheme. C l e a r l y , the u t i l i z a t i o n o f e i t h e r o f these core
s i t e s by a P t ( I I ) reagent w i l l d i s r u p t , a t l e a s t l o c a l l y , the
c o n t i n u i t y of the DNA h e l i x .
A p a r t i c u l a r p o i n t we wish to make i s t h a t the core-core
c r o s s - l i n k i n g modes of Table I I w i l l be r e s t r i c t e d , i n the
absence o f s t e r i c s t r a i n , by i n t r a m o l e c u l a r f o r c e s t o l a r g e
(>100°) i n t e r b a s e d i h e d r a l a n g l e s , owing to the presence o f the
e x o c y c l i c amino and oxo groups adjacent t o t h e metal b i n d i n g s i t e
on each base. The i n t r a m o l e c u l a r s t e r i c demands f o r the core-
core c r o s s - l i n k i n g modes are expected to exceed those f o r the two
s t e r e o c h e m i c a l l y e q u i v a l e n t periphery-core modes and almost c e r -
t a i n l y exceed those f o r the lone p e r i p h e r y - p e r i p h e r y c r o s s - l i n k
of Table I I . I t i s d i f f i c u l t to p r e d i c t , a t t h i s time, how the
o v e r a l l s t r u c t u r e of the DNA polymer would i n f l u e n c e the i n t e r -
base d i h e d r a l angle.
The formation o f a core-core c r o s s - l i n k i n g mode r e q u i r e s the
p e n e t r a t i o n of the DNA duplex by the P t ( I I ) reagent; i n a d d i t i o n ,
f o r those modes i n v o l v i n g N ( l ) of guanine, proton r e l e a s e must
take p l a c e p r i o r to or concomitant w i t h metal a t t a c k . One p o s s i -
b l e means by which proton r e l e a s e a t N ( l ) of a guanine base can
be f a c i l i t a t e d i s through b i n d i n g o f the P t ( I I ) reagent a t N(7)
of the same base (14, 15, 16). I n such a model, the P t ( I I ) agent
would a c t i n an a u t o - s y n e r g i s t i c mode, i n which i t s r o l e would be
twofold - t o f a c i l i t a t e proton r e l e a s e a t N ( l ) of G through b i n d -
ing a t N(7) and to form a c r i t i c a l i n t r a s t r a n d o r i n t e r s t r a n d

core-core c r o s s - l i n k which i s expected to cause a severe l o c a l

deformation of a DNA duplex. I n a s i m i l a r v e i n , i n a s y n e r g i s t i c
combination of a P t ( I I ) agent w i t h a n i t r o g e n mustard ( 1 ) , the
r o l e of the l a t t e r component could a l s o be construed as f a c i l i -
t a t i n g N ( l ) deprotonation through covalent c r o s s - l i n k s i n v o l v i n g
N(7) of G. However, the a t t a c k of two agents at a s i m i l a r l o c a -
t i o n may be s t a t i s t i c a l l y improbable.
I n summary, i t i s a t l e a s t a p e r m i s s i b l e p o s s i b i l i t y t h a t
the c r i t i c a l l e s i o n s i n a Pt(II)-DNA complex are those i n v o l v i n g
P t b i n d i n g a t core s i t e s of a DNA h e l i x . I n regions of high
(G-C) content, i n t r a s t r a n d or i n t e r s t r a n d core-core c r o s s - l i n k i n g
modes are expected to place severe demands on the l o c a l c o n t i n u -
i t y ( v i s - a - v i s , the normal base stacked c o n f i g u r a t i o n ) of a DNA
h e l i x , owing to the l a r g e i n t e r b a s e d i h e d r a l angle favored by
i n t r a m o l e c u l a r s t e r i c e f f e c t s . I t i s a l s o important to note that

a l l p o s s i b l e core-core c r o s s - l i n k s i n regions of h i g h (G-C) con-
tent are e q u i v a l e n t l y demanding i n the v i c i n i t y of the Pt center.
C e r t a i n l y f o r these c r o s s - l i n k s , the i n t e r b a s e d i h e d r a l angle
w i l l be modified by f o r c e s present i n the polymer. Since the
polymer backbone i s attached to the bases i n a d i f f e r e n t manner
for C and G, s p e c i f i c d i f f e r e n c e s i n the d i s r u p t i o n of the DNA
s t r u c t u r e i s expected. Inadequate i n f o r m a t i o n i s p r e s e n t l y
a v a i l a b l e to f u l l y assess these p e r t u r b a t i o n s , but i t i s h i g h l y
u n l i k e l y that d i h e d r a l angles approaching 0°, as found i n n a t i v e
DNA, w i l l e x i s t at m e t a l l a t e d c r o s s - l i n k i n g s i t e s . I t i s reason-
able to b e l i e v e that the degree of p e r t u r b a t i o n i n the Pt-DNA
complex w i l l p a r a l l e l the trend found f o r s m a l l molecules. This
i n t u r n could mean that the i n i t i a l a t t a c k of the Pt agent w i l l
be s e l e c t i v e . That i s , i f the s t e r i c s t r a i n i n f l u e n c e of a d j a -
cent e x o c y c l i c groups near the p o t e n t i a l metal b i n d i n g s i t e i s
too g r e a t , that p a r t i c u l a r l e s i o n might not form.
F i n a l l y , i f one accepts the p r e v a l e n t n o t i o n (13) that
cancer c e l l s are d e f i c i e n t i n t h e i r a b i l i t y to e x c i s e defects
from strands of DNA, then such c e l l s may f i n d defects imposed by
core-core or even periphery-periphery c r o s s - l i n k s i n regions of
high (G-C) content d i f f i c u l t to r e p a i r .
Acknowledgments
This i n v e s t i g a t i o n was supported by the N a t i o n a l I n s t i t u t e s

of H e a l t h , P u b l i c Health S e r v i c e Grant No. GM 29222. We are
indebted to Drs. C. J . L. Lock and B. L i p p e r t f o r making many of
t h e i r r e s u l t s known to us p r i o r to p u b l i c a t i o n .

Literature Cited
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platin - Current Status and New Developments"; Academic Press:
New York, 1980.
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J. J. In "Metal Ions in Genetic Information Transfer", Adv.
Inorg. Biochem. 1981, 3, 274.
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251, 736.
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Eds.; Agents and Actions: Basel, 1981, Chapter 21.
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12. Goodgame, D. M. L.; Jeeves, I.; Phillips, F. L.; Skapski,
A. C. Biochim. Biophys. Acta 1975, 378, 153.
14. See discussion in Chu, G. Y. H.; Mansy, S.; Duncan, R. E.;
Tobias, R. S. J. Am. Chem. Soc. 1978, 100, 593.
15. Faggiani, R.; Lock, C. J. L.; Lippert, B. J. Am. Chem. Soc.
1980, 102, 5419.
16. Faggiani, R.; Lippert, B.; Lock, C. J. L.; Speranzini, R. A.
Inorg. Chem., in press.
17. Barton, J. K.; Lippard, S. J. Ann. N. Y. Acad. Sci. 1978,
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18. Barton, J. K.; Szalda, D. J.; Rabinowitz, H. N.; Waszczak,
J. V.; Lippard, S. J. J. Am. Chem. Soc. 1979, 101, 1434.
19. Marzilli, L. G.; Wilkowski, K.; Chiang, C. C.; Kistenmacher,
T. J. J. Am. Chem. Soc. 1979, 101, 7504.
20. Kistenmacher, T. J.; Wilkowski, K.; de Castro, B.; Chiang,
C. C.; Marzilli, L. G. Biochem. Biophys. Res. Commun. 1979,
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21. Orbell, J. D.; Wilkowski, K.; Marzilli, L. G.; Kistenmacher,
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9. KISTENMACHER ET AL. Purine and Pyrimidinecis-PtComplexes 207
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Soc. 1982, 104, 461.
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10
Platinum Complexes with DNA Bases,
Nucleotides, and DNA
A. P. HITCHCOCK, C. J. L. LOCK, and W. M. C. PRATT

McMaster University, Institute for Materials Research, Hamilton, Ontario, L8S 4M1 Canada
B. LIPPERT
Technische Universität München, Anorganisch-Chemisches Institut,
Lichtenbergstrasse 4, 8046 Garching, Federal Republic of Germany
Models for the interaction of cis-platin and its

hydrolysis products with DNA are discussed. The
models are the interstrand cross-link, intrastrand
cross-link, N7-06 clip, platinum dimer interaction,
Eichhorn costacking, and N1 pK shift. Interactions
observed in model compounds have been observed to
occur in DNA for most of the models, but i t is not
possible to say which, i f any, is the important
lesion in the anti-cancer action of cis-platin.
In this paper we intend to discuss various ways in which

platinum complexes may interact with DNA bases and perhaps with
DNA. In previous papers in this symposium much has been said
about interstrand and intrastrand cross-links involving one
platinum atom. We shall consider these only briefly and concen-
trate on other methods of interaction. Whether these other modes
of binding are relevant to the anti-cancer action of platinum
complexes cannot be said at this point, but we have concentrated
on interactions which are peculiar to the cis-form of Pt(NH3)2Cl2,
and which do not occur with the trans form.
Platinum complexes have been shown to bind to either the
neutral or anionic forms of adenine at N(l) and N(7)(1), thymine
at 0(2), N(3), 0(4), (2, 3), cytosine at N(3), N(4) (4) and
guanine at N(7) (5), N(l) (6) and possibly 0(6) (7) when the
deoxyribose positions are blocked. Platinum complexes can be
considered to bind to these bases in DNA in four ways. Binding
can occur to one base, either monofunctionally or bifunctionally
when the base acts as a chelating agent, and to two bases, in
which case a base from each DNA strand may be bound forming an
interstrand cross-link or the two bases may be on the same DNA
strand forming an intrastrand clip. Considering a l l the
possibilities, six possible models of interaction have been
proposed. These are:
(i) Interstrand cross-link
(ii) Intrastrand cross-link
0097-6156/83/0209-0209$06.00/0

(iii) N7-06 c l i p
(iv) Dimer-DNA i n t e r a c t i o n
(v) Eichhorn c o - s t a c k i n g
(vi) N l pK s h i f t
and below we s h a l l consider them s e p a r a t e l y .
Interstrand Cross-Link
The i n t e r s t r a n d c r o s s - l i n k can occur f o r both c i s and trans
complexes and recent work has confirmed our p o s t u l a t e t h a t t r a n s
complexes can be accomodated i n the DNA double h e l i x much more
e a s i l y than c i s (X). I f we consider the complex [cis-Pt(NH3>-
2 +
GC] (G, C e t c . a r e used t o i n d i c a t e the bases guanine,
c y t o s i n e , e t c . blocked a t the deoxyribose p o s i t i o n w i t h e i t h e r a
methyl o r e t h y l group), as shown i n F i g u r e 1, the bases are

roughly a t r i g h t angles to each other and to the l i g a n d square
plane ( 8 ) . Large d i h e d r a l angles a r e found i n a l l complexes o f
t h i s type. The e f f e c t on the DNA s t r u c t u r e i s shown i n F i g u r e 2.
E i t h e r the bases w i l l have to "bend down" (2b) t o coordinate the
platinum o r they w i l l have t o " t u r n on edge" ( 2 c ) . E i t h e r w i l l
cause marked d i s t o r t i o n o f the DNA s t r u c t u r e . Such d i s t o r t i o n i s
c o n s i s t e n t w i t h the d e s t a b i l i z a t i o n o f the DNA s t r u c t u r e and
lowering of the m e l t i n g p o i n t ( 9 ) . Our argument t h a t the t r a n s -
complex would pack b e t t e r i n t o the DNA s t r u c t u r e was based on the
model shown i n F i g u r e 3. Binding o f the type shown w i l l i n c r e a s e 0
the Ν$1).··Ν(3) d i s t a n c e i n a GC p a i r from 2.8 A t o 4.1 A. The

p i c t u r e was based on the s t r u c t u r e o f [ t r a n s - P t i N H ^ ^ i C ^ ] ( ^ 3 ) 2
(10), i l l u s t r a t e d i n F i g u r e 4, which £as the two bases co-planar,
w i t h an N(3)...N(3)' d i s t a n c e o f 4.1 A. The n i t r a t e groups (ghes£
would be other bases i n DNA) l i e p a r a l l e l t o the C bases 3.4 A
above and below. The ammonia groups, which l i e above and below
the plane o f the bases, hydrogen bond t o the n i t r a t e groups (these
would be bases i n DNA). I n p r a c t i c e , the packing o f a t r a n s
complex w i l l l i k e l y be even more e f f i c i e n t , as we have shown i n
Figure 5 (11). The deoxyribose p o s i t i o n s a r e i n s i m i l a r p o s i t i o n s
to those i n a Watson-Crick GC p a i r but the guanine molecule has
been r o t a t e d 180° about the deoxyribose-N9 bond, a l l o w i n g both
N7 of guanine and N3 of c y t o s i n e to bond to platinum. The
s t r u c t u r e i s f u r t h e r s t a b i l i z e d by a weak i n t e r n a l 06(G)...N4(C)
hydrogen bond. The d i s t a n c e between the deoxyribose p o s i t i o n s i s
10.38 A, v e r y c l o s e t o the d i s t a n c e of 10.85 1 f o r a normal GC
p a i r . The group w i l l t h e r e f o r e pack n e a t l y i n t o the DNA double
h e l i x . Any d e s t a b i l i z i n g e f f e c t w i l l come o n l y from the ammonia
groups l y i n g above and below the base plane, but s i n c e these a r e
almost c e r t a i n l y i n v o l v e d i n hydrogen bonding t o the base p a i r s
above and below the platinum atom, t h i s d e s t a b i l i z a t i o n may be
n e g l i g i b l e . The whole packing may indeed s t a b i l i z e the DNA and
e x p l a i n why t r a n s - P t C N I ^ ^ C ^ r a i s e s the m e l t i n g p o i n t of DNA (13).
I n t r a s t r a n d Cross-Link
The i n t r a s t r a n d c r o s s - l i n k has been discussed a t l e n g t h ,
p a r t i c u l a r l y the one i n v o l v i n g an i n t e r a c t i o n w i t h the N7

HITCHCOCK ET A L . Pt Complexes with Nucleotides and DNA 21
2+
Figure L The (NH ) PtGC cation showing the large dihedral angles between the
s t
bases and the ligating atom square plane.
RtprtMRtotlon of Crooo-IM oyds-olotli

DNA bests bood éom or turn oasdfs
α b c
Figure 2. The interaction of the cis-platinum complex with DNA. Key: a, DNA;
b crosslink with bases bent down to accommodate the large dihedral angles; and
f
c, crosslink with bases turned on edge to accommodate the large dihedral angles.
2
Figure 3. The packing of the \izn$-Pt(NH ) * moiety in a GC pair lengthening
s t
the N3-N1 distance. N3,N1 binding is assumed.

2+
Figure 5. The trans-/Pt(TVH )*GC]
S cation. The two bases and Pt are coplanar.

10. HITCHCOCK ET AL. Pt Complexes with Nucleotides and DNA 213
p o s i t i o n s of two adjacent guanine molecules on a s i n g l e s t r a n d .

We s h a l l o n l y remind you t h a t i n such b i n d i n g the requirements o f
the l a r g e d i h e d r a l angles cause marked d i s t o r t i o n o f the DNA
s t r u c t u r e because the bases can no l o n g e r l i e p a r a l l e l . I t has
been suggested (14) t h a t t h i s b i n d i n g causes the bases t o " f o l d -
out" o f the double h e l i x . The a t t r a c t i o n o f such an i n t e r a c t i o n
as the primary l e s i o n i n DNA i s that i t can o n l y occur f o r the
c i s form o f ΡίίΝΗβ^Ο^, and not f o r t h e t r a n s form.
The N7-06 C l i p
Another model which can o n l y occur f o r cis-complexes i s the
N7-06 c l i p o f guanine (15). I t i s proposed t h a t bonding o f 06 t o
p l a t i n u m i n t e r f e r e s w i t h hydrogen bonding t o 06 i n a manner
s i m i l a r t o t h a t achieved by m e t h y l a t i o n o f 06 i n DNA. T h i s
l a t t e r i s a d e f e c t f o r which r e p a i r i s v e r y d i f f i c u l t i n DNA (15).

Evidence f o r such an i n t e r a c t i o n i s sparse. C e r t a i n l y such a
c l i p does occur when 06 i s r e p l a c e d by a sulphur atom (16,17).
An N7-06 c l i p i s suggested i n the s t r u c t u r e o f a copper complex
determined by Kistenmacher e t a l . (18). Nevertheless the Cu-06
d i s t a n c e i s long (see F i g u r e 6) and one i s faced w i t h the
q u e s t i o n o f whether the i n t e r a c t i o n i s r e a l o r whether the oxygen
cannot "get away" from the copper atom because o f the geometric
r i g i d i t y o f the l i g a n d . One t e s t would be t o compare the M-N7-C8
and M-N7-C5 angles w i t h those i n compounds i n which no i n t e r a c t i o n
i s p r e s e n t . The former should be l a r g e r w i t h Cu-06 b i n d i n g than
the standard (no i n t e r a c t i o n ) and the l a t t e r s m a l l e r . Comparison
of v a l u e s f o r the copper complex (18) w i t h those f o r two p l a t i n u m
s t r u c t u r e s we have determined (19) suggests t h a t the i n t e r a c t i o n
is real.
Cu(18) Pt(19) Pt(19) Δ
M-N7-C8 138.9(3)° 124(1)° 124(1)° +15°
M-N7-C5 117.8(3)° 129(1)° 126(1) -9
C5-N7-C8 102.9(3)° 107(1)° 109(2) -5
359.6 360 359
D e s p i t e t h i s , there i s no evidence o f a Pt-06 i n t e r a c t i o n i n any
of the dozen o r so platinum-guanine compounds s t u d i e d so f a r by
c r y s t a l l o g r a p h y , even i f the guanine i s a n i o n i c , where one would
expect 06 to be a b e t t e r donor. I t can be argued t h a t we have
not y e t looked a t the i d e a l compound; namely, [cis-Pt(NHo)2«
G i O H ^ ) ] ^ * On making guanine a n i o n i c one might expect Oo t o b i n d
to p l a t i n u m and water t o be e l i m i n a t e d , i f an N7-06 c l i p i s
p o s s i b l e . So f a r we and o t h e r s have been unable t o i s o l a t e the
c a t i o n i n a s t a b l e form.
We r e j e c t the argument t h a t experiments i n the s o l i d s t a t e do
not apply t o s o l u t i o n s . We have shown t h a t v i b r a t i o n a l s p e c t r a o f
s o l i d s and s o l u t i o n s are s i m i l a r , and s h i f t s i n v(C=0) o f 20-30
cm~l, supposed t o g i v e evidence o f the N7-06 c l i p , are caused by
changing hydrogen bonding p a t t e r n s . Deprotonation a t N l causes
1
even b i g g e r s h i f t s i n v(C=0) o f up t o 70 cm" . The i s s u e i s s t i l l


i n doubt but i t i s our f e e l i n g t h a t the N7-06 c l i p i s not a

v i a b l e model.
The Dimer-DNA I n t e r a c t i o n
A model we have spent some time examining i s based on the
i n t e r a c t i o n of the dimeric c a t i o n ( N H ) P t ( 0 H ) 9 P t ( N H ) ^ w i t h
3 2 3
p y r i m i d i n e s a t N3 and a t the e x o c y c l i c 04 o r N4 p o s i t i o n w i t h the

consequent formation of an anion (2^ 4^ 20). Reaction a l s o occurs
with, guanine but the r e a c t i o n product has not been c h a r a c t e r i z e d
so f a r . The formation o f the complex w i t h cy t o s i n e suggests how
r e a c t i o n occurs. The pK of the NH group which i s deprotonated
2
i s > 12, yet the complex i s formed a t near pH 7. This suggests

t h a t a marked change i n pK of the NH group occurs upon i n i t i a l
2
b i n d i n g of platinum a t the p r e f e r r e d N3 s i t e . I t should be

p o s s i b l e to detect such a pK s h i f t by NMR. A downfield s h i f t i n

the NMR of the NH group on c o o r d i n a t i o n of platinum a t N3 i s
2
shown i n F i g u r e 7, where the s p e c t r a of 1-methylcytosine i n

dg-dimethylsulphoxide, and of the same system w i t h K-^PtCl^ added,
are compared. The s h i f t i s n e a r l y 2 p.p.m. The platinum i s
known to bond a t N3 under the c o n d i t i o n s of the experiment (21).
The s p l i t t i n g of the NH protons i n t o two peaks on platinum
2
c o o r d i n a t i o n i s not s u r p r i s i n g ; there i s c o n s i d e r a b l e m u l t i p l e
bonding between the NH group and the r i n g , and i t i s best
2
represented as I , w i t h the two protons l y i n g i n the

plane of the ^ ^ ^ ^ ^ ^ H r i n g , and thus i n g e o m e t r i c a l l y
different N3 γ p o s i t i o n s w i t h respect to a platinum
atom bonded a t Η N3. The probable r e a c t i o n mechanism
i s i l l u s t r a t e d i n F i g u r e 8; i n a d d i t i o n to the pK s h i f t of the
N ^ group the p o s i t i o n of the proton-accepting hydroxide groups
probably a i d s the r e a c t i o n .
The platinum-platinum d i s t a n c e i n the three compounds
i s o l a t e d so f a r i s 2.95 Î . Both L i p p a r d (22) and Mansy (23) have
suggested t h a t a s i m i l a r r e a c t i o n c o u l d occur w i t h guanine, i n
the manner shown i n Figure 9. I t seemed t h a t i t should be
p o s s i b l e to d e t e c t such a r e a c t i o n w i t h bases i n DNA by l o o k i n g
f o r the P t - P t d i s t a n c e of 2.95 A w i t h extended x-ray a b s o r p t i o n
f i n e s t r u c t u r e (EXAFS) techniques. We have performed such
experiments on the C o r n e l l High Energy Synchrotron Source.
We s h a l l not d i s c u s s the theory of EXAFS (24), which has been
covered i n other l e c t u r e s , but b r i e f l y i t i s p o s s i b l e to s u b t r a c t
a smooth background curve from the a c t u a l P t L3 a b s o r p t i o n
spectrum (Figure 10) to g i v e an o s c i l l a t i n g f u n c t i o n (Figure 11).
The energy s c a l e of the f i r s t spectrum has been converted to
p h o t o e l e c t r o n wave number (k s c a l e , k = (0.263 ( E - E ) ) % where E
Q Q
i s the midpoint of the a b s o r p t i o n jump). I t i s then p o s s i b l e t o

F o u r i e r transform the EXAFS o s c i l l a t i o n s to g i v e i n f o r m a t i o n about
d i s t a n c e s to atoms surrounding the t a r g e t platinum atom.
The i n f o r m a t i o n one can g a i n , however, i s very dependent on
how much of the a b s o r p t i o n spectrum i s analysed. Three p o i n t s
are marked on the P t L^ a b s o r p t i o n spectrum of the dimer c a t i o n ,

(a)
I
1 1 1 1
900 750 600 450
Hz
Figure 7. Part of the NMR spectra in d -DMSO of a, 1-methy Icy tosine; and b,
6
1-methylcy tosine and K PtCl . 2 k
2
Figure 8. Binding of [(NH ) Pt(OH) Pt(NH ) ] + at N3 of 1-methylcy tosine and
3 2 g s 2
subsequent reaction to form the 1-methylcytosinate anion bound to two platinum

atoms at Ν3 and N4.
Figure 9. Binding of two platinum atoms to guanine at Nl,06 (22) and N7,06
(23).

HITCHCOCK ET AL. Pt Complexes with Nucleotides and DNA 217

z t
( N H ) P t ( O H ) P t ( N H ) " ' , (Figure 10) and these are marked
3 2 2 3 2
s i m i l a r l y on the o s c i l l a t i n g f u n c t i o n (Figure 11). The

o s c i l l a t i o n s a t low v a l u e s of k a r i s e p r i m a r i l y from b a c k s c a t t e r -
i n g of the i o n i z e d Pt L3 e l e c t r o n s from the nearest neighbour
atoms Ν and 0, whereas o s c i l l a t i o n s from the P t - P t i n t e r a c t i o n s ,
i n which we are i n t e r e s t e d , a r i s e p r i m a r i l y i n the h i g h k range,
where s i g n a l to n o i s e e f f e c t s become s i g n i f i c a n t . Thus the
i n f o r m a t i o n i n the F o u r i e r transform i s dependent on the k range
and k-weighting used. This e f f e c t i s i l l u s t r a t e d i n Figure 12,
where a s e r i e s gf transforms over the wavenumber ranges 5 - l g ,
5-14 and 5-18 ( A ~ l ) are p l o t t e d . The Pt-(N,0) peak a t 1.8 A i s
e a s i l y v i s i b l e i n a j l p l o t s but the P t - P t peak a t ^ 3 1 i s o n l y
1
marked i n the 5-18 A" p l o t and i s not observed i n the 5-10 l " *
p l o t . This provides a p o t e n t i a l problem i n the DNA experiments
s i n c e the EXAFS data f o r the Pt-DNA complexes became e x c e s s i v e l y

n o i s y above 14 1-1. I t should be noted (Figure 12) t h a t the peak
maxima do not occur e x a c t l y a t the known i n t e r a t o m i c d i s t a n c e s f o r
the c a t i o n (marked by v e r t i c a l l i n e s ) . This i s because of phase
s h i f t s i n the b a c k s e a t t e r i n g which have the p r i n c i p a l e f f e c t of a
s h i f t i n the F o u r i e r transform spectrum of the P t dimer of 0.31 1
f o r Pt-(0,N) and 0.04 A f o r P t - P t d i s t a n c e s . We have assumed these
s h i f t s are t r a n s f e r a b l e from the dimer c a t i o n to the Pt-DNA complex
(25).
The DNA experiments (26) i n v o l v e d a comparison o f the EXAFS
s p e c t r a of [ P t ( N H ) C ] ( N 0 ) ( C ) , C = 1-methyl c y t o s i n e , which
3 2 2 3 2
does not c o n t a i n a short (<4 A) P t - P t d i s t a n c e (27), [(NHo) Pt(OH) - 2 2
Pt(NH ) ](N0 )3 2 3and the r e a c t i o n product of the dimer c a t i o n

2
2 +
[ ( N H ) P t ( O H ) P t ( N H ) ] w i t h DNA.
3 2 2 3 This l a s t sample was made by
2
r e a c t i n g a s o l u t i o n or c a l f thymus DNA (buffered w i t h lOmM NaN0 , 3
5mM t r i s - N 0 ) w i t h [ ( N H ) P t ( O H ) P t ( N H ) ] ( N 0 ) f o r 1 and 8 days.

3 3 2 2 3 2 3 2
The dimer to PO," r a t i o was 1:10. A f t e r i n c u b a t i o n the s o l u t i o n s

were c e n t r i f u g e a a t 190,000g f o r twenty hours, the supernatant
decanted, and the DNA p e l l e t washed w i t h more b u f f e r to remove any
excess platinum dimer. I t was c e n t r i f u g e d again f o r twenty hours
a t 190,000g, the supernatant removed and the DNA p e l l e t d r i e d under
n i t r o g e n gas.
F o u r i e r transform s p e c t r a of the P t L EXAFS o f the c y t o s i n e 3
and dimer standards and the one-day DNA sample are shown i n Fig.13.
For these r e s u l t s the data, over a k-range of 4-14 1~1, was
3 5
m u l t i p l i e d by k , k* or k before F o u r i e r a n a l y s i s . A peak appears c
i n each gf the s p e c t r a of the DNA sample a t about 2.9 A as compared

to 3.08 A i n the dimer standard, whereas none i s observed i n Jhe
c y t o s i n e standard. The i n c r e a s e i n the i n t e n s i t y of the ^ 3 A peak
w i t h increased k-weighting supports i d e n t i f i c a t i o n of t h i s f e a t u r e
as a P t - P t d i s t a n c e s i n c e higher k-weighting emphasizes the l a r g e
k-regions whgre the EXAFS amplitude f o r a h i g h Ζ atom i s l a r g e r .
The 2.87(8) A P t - P t d i s t a n c e observed i s c o n s i s t e n t w i t h values
found i n the model compounds (2, 4^ 20), suggesting a s i m i l a r
r e a c t i o n takes p l a c e i n DNA i t s e l f . ο
1
Comparison of the data (analysed over 4<k<14 A" w i t h weight-

Figure 12. Fourier transform of the EXAFS spectrum of [(NH ) Pt(OH) Pt- s 2 2
1
(NH ) ](NO ) over various k ranges. Key to k range in A' : top, 5-10; middle,
s 2 s 2
5-14; and bottom, 5-18.
(a) (b) A (c)
Pt-cyto*int Pt-ONA
1
J1 Pt-OH-dim*r
ι • 1
a.
-V.
Ε
si
1 r—1—ι Γ—·—1 "
î -#X\ 5
1
Figure 13. Comparison of the EXAFS results transformed over 4 < k < 14 A"
with a weighting of k , η = 3, 4, 5. Key: a, [(NH ) PtC ](ClO ) *C,C = 1-methyl
n
s 2 2 k 2
cy tosine; b, [(NH ) Pt(OH) Pt(NH ) ](NO )

s 2 2 + DNA; and c, [(NH ) Pt(OH) Pt-
s 2 s 2 s t t
(NH ) ](NO ) . 2 2 s 2

3 D
ing o f k and k ) from the one day and e i g h t day DNA samples,
however, (Figure 14) r e v e a l s t h a t the P t - P t v e c t o r i s not present
i n the second sample, suggesting that the i n i t i a l i n t e r a c t i o n i s
unstable and that the platinum atoms e v e n t u a l l y move away from
each other.
Although there i s a d i f f e r e n c e i n P t - P t d i s t a n c e i n the dimer
and the DNA r e a c t i o n product, one would l i k e to make sure that the
peak does n o t a r i s e from unreacted dimer. A d d i t i o n a l i n f o r m a t i o n
i s obtained from t h e a b s o r p t i o n edges o f the v a r i o u s compounds
(Figure 15). I t i s c l e a r t h a t the edge p o s i t i o n f o r the dimer i s
q u i t e d i f f e r e n t from that f o r the r e a c t i o n product w i t h DNA,
suggesting a d i f f e r e n t chemical environment of the P t atom. EXAFS
s t u d i e s o f other P t standards and of Pt-dimer-DNA samples prepared
under a v a r i e t y o f c o n d i t i o n s are i n progress.
Eichhorn Co-stacking
Another model we have examined i s based on Eichhorn's
o b s e r v a t i o n that c i s - P t i N I L j ^ C ^ and enPtCl« pack i n the s o l i d so
t h a t the square planes a r e ^ 3.4 Î a p a r t , whereas t h i s i s n o t
true f o r trans-Pt(NH3) Cl . 2 2 Because the d i s t a n c e between base
p a i r s along the double h e l i x i n DNA i s a l s o 3.4 Â , Eichhorn
suggested the çis-Pt(NH3)Cl molecules might "costack" (28).
2
Since t h a t time we have examined other complexes o f c i s - d i h a l o -

p l a t i n u m ( I I ) and have found t h i s type o f packing i s v e r y common,
whereas i t has n o t been found i i j trans-complexes (Figure 1 6 ) .
We do n o t suggest that the 3.4 A packing d i s t a n c e i s found i n a l l
c i s - d i h a l o p l a t i n u m ( I I ) complexes; i t i s not (Figure 17). Now i f
platinum b i n d i n g t o two p a r a l l e l adjacent bases i s t o occur i n
DNA i t cannot be t o the same platinum atom. I t i s true that a
complex, w i t h platinum bound t o two guanosine monophosphate
molecules, i s known i n which the d i h e d r a l angle between the bases
i s s m a l l , but as the authors p o i n t out (29) t h i s s t r u c t u r e cannot
occur i n DNA. The platinum atom i s bound t o two GMP n u c l e o t i d e s
such t h a t the phosphate groups l i e on opposite s i d e s o f the
molecule. The t r u e analogue t o the DNA s i t u a t i o n would be
b i n d i n g t o a d i n u c l e o t i d e which contains o n l y one phosphate group.
Thus i n DNA t h e two phosphate groups would condense t o one and
would thus have to be on the same s i d e of the molecule, prevent-
ing the type of s t r u c t u r e observed.
1
I f one examines E i c h h o r n s model i n more d e t a i l t o see how
b i n d i n g might occur, we see a d i f f e r e n c e between t h e a c t i o n of
c i s and t r a n s - P t ( N H 3 ) C l .
2 2 The reason f o r the 3.4 1 d i s t a n c e
i s t h a t the molecules are h e l d together by N-H...CI o r N-H...Br
hydrogen bonds which are ^ 3.4 Â. I n F i g u r e 18 we show how c i s -
and t r a n s - P t ( N H 3 ) C l ((a) and (b) r e s p e c t i v e l y ) might be bonded
2 2
to two adjacent bases on DNA. Hydrogen bonding can s t i l l occur

for t h e c i s complex but cannot occur f o r the t r a n s . Even a l l o w i n g
for the t w i s t i n DNA, when the bases are not one above the other
we can s t i l l get one hydrogen bond f o r the cis-complex. One
may reasonably ask whether the energy o f a s i n g l e hydrogen bond i s

Figure 15. Pt L absorption edge spectra for

s [(NH hPt(OH) Pt(NH ) ](NO h
s t s 2 s
and the reaction product with DNA after 1- and 8-d reaction time.

No close contact (4A)
trans-dichlorodiammineplatinum(II)
trans-dichlorobis(ethyleneimine)platinum(II)
trans-dichlorobis(cylco-butylamine)platinum(II)
trans-dichlorobis(eyelo-heptylamine)platinum(II)
trans-dichlorobis(eyelo-hexylamine)platinum(II)
trans-dibromobis(cyclo-hexylamine)plat inum(II)
Figure 16. List of known trans-dihalodiammineplatinum(II) structures. Not one

of these has a Pt-Pt contact less than 4 A.
No close contact (48)
cis-dichlorodi(cyclopropylamine)platinum(II)
cis-dichlorodi(cyclobutylamine)platinum(II)
cis-dichloro(1-methylamino-2(S)-aminopropane)piat inum(II)
cis-dichloro4i(N-methylimidazole)platinum(II)
cis-dichlorodi(ethyleneimine)platinum(II)
Figure 17. List of cis-dichlorodiammineplatinum(II) structures with no Pt-Pt

contact less than 4 A.
Figure 18. Pairjs of cis- and trans-/PtfRiNH^Ch] molecules bound to adjacent

bases on a DNA chain.

enough to s t a b i l i z e the system. We t h i n k not but L i p p e r t , i n a

previous paper, has d e s c r i b e d a mono-hydroxide b r i d g e d two
platinum atom complex which could a c t as a s u i t a b l e model
(Figure 19). Again, such an i n t e r a c t i o n , which would keep the
bases p a r a l l e l a t 3.4 1 s e p a r a t i o n , can o n l y occur f o r the
a n ( n o t o rt ie t r a n s
h y d r o l y s i s products of cis=Pt (NH3) * ^ *
complex.
N l pK S h i f t Model
The q u e s t i o n that a r i s e s i s : How can b i n d i n g a t N7 on the
o u t s i d e of the DNA h e l i x i n t e r f e r e w i t h the hydrogen bonding
r e g i o n and cause m i s p a i r i n g ? Work we have done on a s e r i e s of
2 +
complexes c o n t a i n i n g the c a t i o n s and molecules [ P t i N ^ ^ G C ] ,
[Pt(NH3) (G-H)C]+,
2 [(Pt(NH3) GC)(Pt(NH3) (G-H)C]3+, t P t ( N H 3 ) ~
2 2 2
(G-H) J, [ P t ( N H ) ( G - H ) ] G has suggested Kow t h i s might occur

2 3 2 2
(8, 19, 30). B i n d i n g of p l a t i n u m a t N7 of guanine causes a

marked change i n the pK of N l of guanine from about 9.4 to 8.0.
Thus a t p h y s i o l o g i c a l pH (^ 7.6) about 15% of a l l p l a t i n a t e d
guanine molecules are deprotonated a t N l . I t has thus proved
p o s s i b l e to form complexes c o n t a i n i n g G-G" p a i r w i t h three
hydrogen bonds l i k e the Watson-Crick G-C p a i r (Figure 20a). A
comparison of hydrogen bond d i s t a n c e s f o r v a r i o u s base p a i r s
(Figure 21) shows the G-G"" p a i r i s s t r o n g l y hydrogen-bonded and
t h i s i s borne out by v i b r a t i o n a l s p e c t r a l s t u d i e s i n s o l u t i o n
which suggest i t i s bonded much more s t r o n g l y than the Watson-
C r i c k base p a i r . The G-G" p a i r cannot f i t i n t o the DNA double
h e l i x because the deoxyribose p o s i t i o n s are on the opposite s i d e
of thg base p a i r (Figure 20) and the sugar-sugar d i s t a n c e i s
* 14 A. N e v e r t h e l e s s , i t i s p o s s i b l e to see how p l a t i n u m induced
base m i s - p a i r i n g could occur. Using the example i n Figure 20
deprotonated guanine should bond to thymine and i n s o l u t i o n we
have shown t h i s occurs. I s o l a t i o n of a s o l i d has proved
d i f f i c u l t , s i n c e thymine i s much l e s s s o l u b l e than any complex
and separates f i r s t . What we have induced, however, i s G~-T
m i s p a i r i n g by c o o r d i n a t i n g p l a t i n u m to the N7 p o s i t i o n of guanine
Because of the s t r e n g t h of the G"-G bond we should examine
how much bonding might occur i n DNA. I f we bond two guanine
molecules to one p l a t i n u m atom, the bases w i l l " f o l d out" of the
h e l i x . Deprotonation of one guanine molecule might a l l o w us to
a f f e c t the DNA t e r t i a r y s t r u c t u r e by a G-G" bond between two
f o l d e d out p o s i t i o n s . T h i s might occur i n a simple bond of the
type shown i n F i g u r e 22(a) or a l t e r n a t i v e l y i n v o l v e a c r u c i f o r m
segment of DNA as shown i n F i g u r e 22(b). Such c r u c i f o r m
s t r u c t u r e s , although common i n RNA, are o n l y normal i n DNA where
one f i n d s i n v e r t e d sequence repeats. These u s u a l l y occur i n the
c o n t r o l r e g i o n s of DNA (Figure 23). I n t h i s case such b i n d i n g
would not cause base m i s p a i r i n g i n the DNA but would prevent
r e p l i c a t i o n completely.
In summary, we have d i s c u s s e d some ways i n which c i s - p l a t i n ,
cis-Pt(NH«j) Cl , and i t s aquation products r e a c t w i t h DNA bases.
2 2

Figure 19. A hydroxide-bridged platinum dimer bound to two guanine molecules

co-stacked as in a dinucleotide.
Τ " 'Ï
R A
c
Figure 20. Structural formulas of a, Watson-Crick GC base pair; b, N7-platinated

G-G~ base pair; c, methylated G-T base pair.

HITCHCOCK ET A L . Pt Complexes with Nucleotides and DNA 225
Hydrogen bond distances in Â

N2 06, NI Nl, 06- -N2,
N4 02or N3 N3or 02 N4or
N2 06 N3 Nl 02 N2
(NH ) Pt(G-H) G
3 2 2
2880 2 95(2) 2 87(2)
(NH^PtGC^
2 99(1) 273(1) 2-99(1)
(NH3)Pt(G-H)C
2
GC 2 81(1) 2 92(1) 2-94(1)

GFC 2 82(1) 2 94(1) 2 96(1)

C2H+ 276 283 293
Figure 21. Hydrogen-bond distances in A for various triply hydrogen-bonded base

pairs (FC = 5-fluorocytosine).
Figure 22. Possible ways in which G - G " pairs could occur.
I I I I U U U U I 1 L
I 2 3 4 4 3 2 I
Symmetrically related regions of the lac operator
Figure 23. An inverted sequence repair in the control region of the lac gene.

Because of the complexity of the aquation and hydrolysis reaction

the possible modes of interaction are more numerous and complex
than had previously been realized. In some cases i t has been
shown that the interaction of cis-platin with DNA is accurately
described by the model compounds. It has not been established
which, i f any, of the above models describes the primary lesion
in the anti-cancer activity of cis-platin.
Acknowledgments
We acknowledge with thanks the contributions of our
co-workers. Currently at McMaster these are J. Britten, B. Brown,
R. Faggiani, Dr. Ή.Ε. Howard-Lock, P. Pilon and M.A. Turner and
those who have graduated are Dr. J. Bradford, Dr. R.A. Speranzini,
Dr. G. Turner and Dr. M. Zvagulis. Other cooperation has taken
place with the research groups of Profs. B. Rosenberg, F.
Ghadially, J. Powell and T. Theophanides.
Literature Cited
1. Lock, C.J.L.; Speranzini, R.A.; Turner, G.; Powell, J.

J. Amer. Chem. Soc. 1976, 98, 7865.
2. Lock, C.J.L.; Peresie, H.J.; Rosenberg, B.; Turner, G.
J. Amer. Chem. Soc. 1978, 100, 3371.
3. Faggiani, R.; Lippert, B.; Lock, C.J.L. Unpublished work.
4. Faggiani, R.; Lippert, B.; Lock, C.J.L.; Speranzini, R.A.
J. Amer. Chem. Soc. 1981, 103, 1111.
5. Gellert, R.W.; Bau, R. J.. Amer. Chem. Soc. 1975, 97, 7379
J. Clinical Hemat. Oncol. 1977, 7, 51.
6. DeCastro, B. ; Chiang, C.C.; Wilkowski, K.; Marzilli, L.G.;
Kistenmacher, T.J. Inorg. Chem. 1981, 20, 1835.
7. Gellert, R.W.; Fischer, B.E.; Bau, R. J. Amer. Chem. Soc.
1980, 102, 7812.
8. Faggiani, R.; Lock, C.J.L.; Lippert, B. J. Amer. Chem. Soc.
1980, 102, 5418.
9. Harder, H.C. Chem.-Biol. Interactions 1975, 10, 27.
10. Lippert, B.; Lock, C.J.L.; Speranzini, R.A. Inorg. Chem.
1981, 20, 808.
11. Britten, J.; Lippert, B.; Lock, C.J.L. Manuscript in
preparation.
12. Guschlbauer, W. "Nucleic Acid Structure" Springer-Verlag.
New York 1976 p. 35.
13. Macquet, J.-P.; Butour, J.-L.; Biochimie, 1978, 60, 901.
14. Lippard, S.J. in "Inorganic Chemistry in Biology and
Medicine", Martell, A.E. Ed.; ACS Symposium Series
No. 140, 1980.

15. Rosenberg, Β. Biochimie 1978, 60, 859.

16. Sleiten, Ε.; Apeland, A. Acta Crystallogr. 1975, B31, 2019.
17. Heitner, H . I . ; Lippard, S.J. Inorg. Chem. 1974, 13, 815.
18. Szalda, D.; Kistenmacher, T . J . ; M a r z i l l i , L.G. J . Amer.
Chem. Soc. 1976, 98, 8371-7.
19. Faggiani, R.; Lippert, B . ; Lock, C.J.L.; Speranzini, R.A.
Inorg. Chem. In press.
20. Lock, C.J.L.; Pollock, R . J . ; Rosenberg, B . ; Turner, G.
Inorg. Chem. 1981, 20, 804.
21. Lock, C.J.L.; Speranzini, R.A.; Powell, J. Can. J. Chem.
1976, 54, 53.
22. Barton, J.K.; Szalda, D . J . ; Rabinowitz, H.N.; Waszczack,

J.V.; Lippard, S.J. J . Amer. Chem. Soc. 1979, 101, 1434.
23. Chu, G.Y.H.; Mansy, S.; Duncan, R.E.; Tobias, R.S. J. Amer.
Chem. Soc. 1978, 100, 593.
24. Lee, P.Α.; Citrin, P.H.; Eisenberger, P.; Kincaid, B.M.
Rev. Mod. Phys. 1981, 53, 769.
25. Citrin, P.H.; Eisenberger, P.; Kincaid, B.M. Phys. Rev. Lett.
1976, 36, 1346.
26. Hitchcock, A.P.; Lock, C.J.L.; Pratt, W.M.C. Inorg. Chim.
Acta, 1982, 66, L45.
27. Faggiani, R.; Lippert, B . ; Lock, C.J.L. Inorg. Chem. 1982.
In press.
28. Srivastava, R.C.; Froehlich, J.; Eichhorn, G.L. Biochimie,
1978, 60, 879.
29. M a r z i l l i , L.G.; Chalilpoyil, P.; Chiang, C.C.; Kistenmacher,
T.J. J . Amer. Chem. Soc. 1980, 102, 2480.
30. Faggiani, R.; Lippert, B . ; Lock, C.J.L. Submitted to
Inorg. Chem.

11
Hydrolytic Equilibria and N 7 Versus N1
Binding in Purine Nucleosides of
cis-Diamminedichloroplatinum(II)
Palladium(II) as a Guide to Platinum(II) Reactions at Equilibrium
R. BRUCE MARTIN
University of Virginia, Department of Chemistry, Charlottesville, VA 22901
Antitumor Pt(II) amines are usually administered

as cis dichloro complexes. This form persists in
-
human blood plasma with its high 103 mM C1 con
tent. The net zero charge on the complex fosters
its passage through cell walls. Within many cells
-
the C1 concentration is much lower, only ~4 mM.
-
Substitution of C1 by H O then occurs. Depending
2
upon pH deprotonation may also yield hydroxo con

taining complexes. In neutral solutions of low
-
C1 content the chloro-hydroxo complex predomi
nates but aquo-hydroxo, chloro-chloro, aquo-chloro,
and hydroxo-hydroxo complexes also appear. Of the
three leaving groups H O is by far the best,
2
- -
followed by C1 and OH , which is inert. Most
likely the aquo complexes react with cell consti
tuents. At sufficient Pt(II) concentrations
complexes with hydroxo bridges form at the ex
pense of a water ligand, reducing the Pt(II)
complex reactivity. Dihydroxo bridges were first
observed on analogous Pd(II) complexes in solution
and found to form more slowly with Pt(II).
In purine nucleosides metal ion binding may
occur at either N(7) or N(1). In guanosine and
inosine the ratio of binding at N(1) to N(7) is
pH dependent. At pH 7 the stability order for
dienPd(II) binding at the common 5'-nucleotides
is G7>I7>I1>G1>U3>T3>C3>A1>A7. This series
represents a considerable promotion of guanosine
+
and inosine N(7) stabilities over that found for H
+
and CH Hg . Pt(II) appears to favor N(7) to N(1)
3
even more than Pd(II). Even when binding at N(1) is

favored, Pt(II) may become kinetically fixed at Ν(7)
in the 6-oxopurines. Of the several common nucleoside
binding sites both thermodynamic and kinetic factors
favor Pt(II) binding at guanosine N(7).
0097-6156/83/0209-0231$06.00/0
© 1983 A m e r i c a n Chemical Society

Cis (NH3)2PtCl2 and other P t ( I I ) complexes r e a c t only s l o w l y

w i t h t h e n u c l e i c bases. The slowness may be e s s e n t i a l t o t h e i r
e f f i c a c y as tumor i n h i b i t o r s , f o r i t provides i n t e g r i t y and
n e u t r a l i t y during c i r c u l a t i o n and passage i n t o c e l l s . Equili-
brium constants f o r a s s o c i a t i o n of c i s (NH3>2Pt(II) complexes
w i t h n u c l e i c bases remain unknown. I t has been argued that
p u b l i s h e d constants f a i l t o r e f l e c t systems a t e q u i l i b r i u m ( 1 ) .
The aqueous chemistry of P t ( I I ) r e l e v a n t t o b i o l o g i c a l molecules
has r e c e i v e d review ( 2 ) .
As an i n d i c a t o r of e q u i l i b r i u m tendencies and constants f o r
P t ( I I ) complexes, we have employed analogous P d ( I I ) complexes.
Both metal ions form diamagnetic, planar complexes and p r e f e r
n i t r o g e n t o oxygen dgnor atoms. They e x h i b i t s i m i l a r e f f e c t i v e
i o n i c r a d i i of 0.60 A f o r P t ( I I ) and 0.64 A f o r P d ( I I ) ( 3 ) .
However, complexes of d i e n P d ( I I ) r e a c t about 10^ times f a s t e r

than those of d i e n P t ( I I ) ( 4 ) . At t h e same time P d ( I I ) exchanges
s l o w l y among n u c l e i c base l i g a n d s i t e s so that i n -4î NMR s p e c t r a
i n d i v i d u a l l i g a n d resonances appear f o r each k i n d of P d ( I I )
b i n d i n g s i t e . Because of i t s convenient r a t e of attainment of
e q u i l i b r i u m c o n c e n t r a t i o n s , as w e l l as i t s diamagnetism and slow
r a t e o f exchange on t h e NMR time s c a l e , P d ( I I ) o f f e r s one of
the most a t t r a c t i v e metal ions f o r i n v e s t i g a t i o n o f i t s complexes.
This a r t i c l e deals w i t h two separate aspects of P t ( I I )
b i n d i n g t o n u c l e i c bases. The f i r s t s e c t i o n d e s c r i b e s t h e com-
plexes present i n aqueous s o l u t i o n s c o n t a i n i n g c i s (NH3>2Pt(II)
over a range of pH i n media of h i g h and low c h l o r i d e i o n content.
The second s e c t i o n considers aspects of t h e N ( l ) versus N(7)
dichotomy f o r b i n d i n g a t purine n u c l e o s i d e s . To e l u c i d a t e t h e
b i n d i n g of P t ( I I ) a t e q u i l i b r i u m , both s e c t i o n s r e l y on r e -
search performed on analogous P d ( I I ) complexes.
S o l u t i o n P r o p e r t i e s of c i s (NH-Q2Pt(II) Complexes
Antitumor P t ( I I ) complexes e x i s t i n human blood plasma i n

an ambient c h l o r i d e i o n c o n c e n t r a t i o n of 103 mM. A much lower
v a l u e of o n l y "4 mM CI"" occurs w i t h i n many c e l l s . E q u i l i b r i u m
and r a t e constants have been reported f o r s u b s t i t u t i o n of CI"" by
H2O i n c i s (NH3>2PtCl2. The s u c c e s s i v e e q u i l i b r i u m constants
f o r formation of aquo and c i s diaquo complexes a r e 3.3 and 0.4mM,
r e s p e c t i v e l y ( 5 ) . S i m i l a r values a r e found f o r enPtCl2 ( 6 ) . The
above values a r e used h e r e i n as prototypes f o r c i s P t ( I I ) amines.
+
The complex c i s (NH3>2Pt(H20>2^ undergoes two deprotona-
t i o n s t o g i v e f i r s t t h e aquo-hydroxo, and f i n a l l y t h e dihydroxo
complexes. From t h e t i t r a t i o n data f u r n i s h e d by Jensen (7) a t
20°, I c a l c u l a t e from a n o n - l i n e a r l e a s t squares program of pH
versus equiv base, ρΚχ = 5.5 and pK2 = 7.1. These values come
out s l i g h t l y lower than those c a l c u l a t e d by Jensen. However,
the newly c a l c u l a t e d values a r e i d e n t i c a l t o those reported by
the Russian s c h o o l f o r s o l u t i o n s a t 25° w i t h 0.1 - 0.3 M
NaN0 ( 8 ) .
3

11. MARTIN Equilibria and Binding of cis-PtCh(NHs)B 233
The a c i d i t y constant f o r water deprotonation i n c i s

(NH3>2Pt(Cl)(H2O) i s unknown and needs t o be estimated. For
t h r e e examples (9) I note that t h e p K v a l u e f o r deprotonation
a
from an aquo-chloro complex i s about t h e average o f t h e succes

s i v e ρΚχ and pK2 values from the corresponding diaquo complex.
Thus from t h e average o f t h e ρΚχ and pK2 v a l u e s i n the preceding
paragraph I estimate f o r c i s (NH3>2Pt(Cl)(H2O) that p K = 6.3. a
T h i s v a l u e s i s i d e n t i c a l t o that reached i n r e f e r e n c e 9 by a
s i m i l a r argument.
Combination o f the above e q u i l i b r i u m constant values w i t h
the ambient C I " c o n c e n t r a t i o n s y i e l d s the P t ( I I ) complex d i s
t r i b u t i o n curves shown i n F i g u r e s 1-3.
F i g u r e 1 shows mole f r a c t i o n P t ( I I ) versus pH f o r c i s
(NH3>2PtX,Y i n t h e presence o f 103 mM C I " , as occurs i n blood
plasma. From low pH t o pH 7.8 t h e c i s (NH3)2PtCl2 complex pre

dominates. From 7.8<pH<8.7 t h e c i s (NH3)2Pt(CI)(OH) complex
dominates. At pH>8.7 the dihydroxo complex becomes predomi
nant.
F i g u r e 2 shows a s i m i l a r p l o t but i n t h e presence o f o n l y
4 mM C l ~ , as might occur w i t h i n a c e l l . The d i s t r i b u t i o n curves
vary more than a t t h e higher c h l o r i d e c o n c e n t r a t i o n . At low pH
the d i c h l o r o and aquo-chloro complexes appear i n comparable
amounts and account f o r most o f t h e P t ( I I ) . I n n e u t r a l s o l u t i o n s
the chloro-hydroxo complex becomes dominant and accounts f o r
about 38% o f the P t ( I I ) , w h i l e t h e aquo-hydroxo complex accounts
f o r about 24%. At pH >^ 7.3 the dihydroxo complex becomes i n
c r e a s i n g l y predominant.
F i g u r e 3 shows mole f r a c t i o n P t ( I I ) versus mM c h l o r i d e
c o n c e n t r a t i o n a t pH 7,0. The predominant complexes present a r e
s t r o n g l y dependent upon c h l o r i d e c o n c e n t r a t i o n a t low concentra
t i o n s . At zero c h l o r i d e c o n c e n t r a t i o n t h e aquo-hydroxo and
hydroxo-hydroxo complexes account f o r >98% o f t h e P t ( I I ) ; t h e
aquo-aquo complex occurs a t l e s s than 2%. A l l three complexes
decrease i n amount as C I " i s added. Mole f r a c t i o n s o f t h e
chloro-hydroxo and chloro-aquo complexes peak a t 9 mM C l ~ . Above
17mM C I " t h e c h l o r o - c h l o r o complex predominates. T h i s complex
a l s o predominates i n c l i n i c a l f o r m u l a t i o n s c o n t a i n i n g 0.15 M
NaCl.
From the second order r a t e constants f o r s u b s t i t u t i o n by
p y r i d i n e i n d i e n P t ( I I ) complexes (4-, 10), I estimate the r e l a t i v e
l e a v i n g group a b i l i t i e s o f H2O t o C I " t o be 70 t o 1. Bound
hydroxide i o n appears i n e r t and a p p a r e n t l y undergoes s u b s t i t u t i o n
o n l y a f t e r conversion t o water i n a r a p i d p r o t o n i c e q u i l i b r i a .
D i s t r i b u t i o n curves i n Figures 1-3 r e p r e s e n t i n g complexes
w i t h t h e good l e a v i n g group H2O appear as s o l i d curves. Com
plexes w i t h o n l y the poor l e a v i n g groups C I " and OH" a r e shown
as dashed curves. At 103 mM C I " i n F i g u r e 1 o n l y t r a c e s o f
complexes c o n t a i n the good water l e a v i n g group. Under c o n d i
t i o n s such as occurs i n plasma, t h e P t ( I I ) complexes remain
r e l a t i v e l y i n e r t . At t h e i n t r a c e l l u l a r 4 mM C I " shown i n

1.0I J J—
1
cr, c r ^ OH", OH"/
Ζ (NH ) 3 2 Pt X,Y
g
μ- [CI"] = 103 mM
Ï0.5h V
y
UJ
-I
ο cr.oH -
χ
\ /
A \
\ / \
7
* \
/ \ ^
• S
CI", H 0
2
~r
6 8 9 10
PH
Figure 1. Mole fraction of Pt(II) vs. pH for cis-(NH ) Pt(H) complexes in presence
s 2
of 103 mM ambient Ct~. Solid curve represents complex with a water ligand. The
mole fractions of the diaquohydroxo and aquohydroxo complexes never exceed
0.015.
PH
Figure 2. Mole fraction of Pt(U) vs. pH for cis-(Ν H ) Pt(II) complexes in presence
3 2
of 3.5 mM ambient Cl~. Solid curves represent complexes with a water ligand.
Polymerization of the aquohydroxo complex to give hydroxo-bridged dimers and
turners is not considered.

11. MARTIN Equilibria and Binding of as~PtCh(NH )2 3
235
CI" CI"
(NH ) 3 2 Pt X,Y
pH = 7.0
y H f i O . O H - " ^ ^ c r , OH"
OH", OH"
CI", H 0
2
20 40 60 80 100 120 140 160

mM CI"
Figure 3. Mole fraction of Pt(II) vs. [Cl~] for cis-(TVH ) Pt(11) complexes at pH
3 2
7.0. Solid curves represent complexes with a water ligand.

F i g u r e 2 t h e s o l i d c u r v e s i n d i c a t e a p p r e c i a b l e mole f r a c t i o n s o f
c o m p l e x e s w i t h g o o d w a t e r l e a v i n g g r o u p s f r o m pH 4 « - 8 . In n e u t r a l
s o l u t i o n s a b o u t 33% o f t h e P t ( I I ) c o m p l e x e s c o n t a i n good w a t e r
l e a v i n g g r o u p s a t 4 mM C l ~ w h i l e o n l y a b o u t 3% do s o a t 103 mM
Cl~. When e q u i l i b r i u m i s a t t a i n e d , a much g r e a t e r f r a c t i o n o f
P t ( I I ) c o m p l e x e s c o n t a i n good w a t e r l e a v i n g g r o u p s w i t h i n c e l l s
than i n t h e plasma.
C o m b i n a t i o n o f t h e r a t e a n d d i s t r i b u t i o n r e s u l t s a t pH 7
i n d i c a t e s t h a t t h e P t ( I I ) complexes w i t h i n a c e l l a r e about 7
t i m e s more r e a c t i v e t h a n t h o s e i n t h e p l a s m a . Aquo c o m p l e x e s
a r e almost t h e o n l y r e a c t i v e s p e c i e s w i t h i n a c e l l and t h e major
r e a c t i v e species i n the plasma. Due t o t h e i r g e n e r a l p r e d o m i -
n a n c e , c i s d i c h l o r o c o m p l e x e s p r o v i d e a b o u t 1/3 o f t h e r e -
a c t i v i t y i n t h e plasma.
Upon t i t r a t i o n o f 10 mM enPd(0H2)2 w i t h s t a n d a r d b a s e a n
e n d p o i n t i s r e a c h e d a t pH 7 . 5 a f t e r a d d i t i o n o f o n e e q u i v b a s e .
We d i s c o v e r e d , h o w e v e r , t h a t t h e r e v e r s i b l e t i t r a t i o n c u r v e
f l a t t e n s o n t h e pH a x i s a n d c a n n o t b e f i t t e d w i t h a n e q u i l i b r i u m
e x p r e s s i o n f o r a s i m p l e d e p r o t o n a t i o n ( 1 1 ) . We s u c c e e d e d i n
f i t t i n g t h e t i t r a t i o n curve t o a combination deprotonation and
dimerization process that y i e l d s a b i n u c l e a r , dihydroxo bridged
dimer. F o r t h i s r e a c t i o n t h e o v e r a l l e q u i l i b r i u m c o n s t a n t was
f o u n d t o b e Kd = 1 0 · M. - 8 3
enPd(OH ) 2 2
2 +
J 2 H 0
3
+
+ (en)Pd^ J>>d(en) 2+
^ 0 ^
H
F o r t h e c o r r e s p o n d i n g P t ( I I ) c o m p l e x , enPt(OH2)2 > 2+ a n
e n d p o i n t i s r e a c h e d a f t e r a d d i t i o n o f two e q u i v s b a s e . T h e
d i m e r i z a t i o n proceeds s l o w l y , and i t i s p o s s i b l e t o r e s o l v e
t h e t i t r a t i o n c u r v e o b t a i n e d a t 2 3 ° w i t h 0.2 M KNO3 i n t o two
s u c c e s s i v e d e p r o t o n a t i o n s (11) w i t h p % = 5 . 8 f o r f o r m a t i o n o f
t h e a q u o - h y d r o x o c o m p l e x a n d pK2 = 7 . 6 f o r f o r m a t i o n o f t h e
dihydroxo complex. I f o n l y o n e e q u i v b a s e i s added t o
enPt(OH2)2 2 +
and t h e s o l u t i o n c o n t a i n i n g predominantly t h e aquo-
hydroxo complex i s a l l o w e d t o s t a n d , no t i t r a b l e groups r e -
m a i n ( 1 1 ) . Thus d i m e r i z a t i o n d o e s o c c u r , b u t more s l o w l y t h a n
w i t h t h e corresponding Pd(II) complex.
Presence o f b i n u c l e a r , dihydroxo b r i d g e d P d ( I I ) and P t ( I I )
complexes i n s o l u t i o n r e c e i v e s support from c r y s t a l s t r u c t u r e
d e t e r m i n a t i o n s o f c i s diamine P t ( I I ) complexes (12,13). T r i -
n u c l e a r complexes w i t h t h r e e P t ( I I ) and t h r e e hydroxo b r i d g e s
also occur i n c r y s t a l s (14,15).
R e d u c i n g t h e c o n c e n t r a t i o n o f enPd(0H2)2 t o 1 mM makes i t
p o s s i b l e t o r e s o l v e t h e p r e v i o u s r e a c t i o n i n t o component d e -
p r o t o n a t i o n and d i m e r i z a t i o n s t e p s .

11. MARTIN Equilibria and Binding of as-PtCh(NH k 3 237
H
K D
+
2+
2 enPd(H 0)(OH)2 t 2 H 0 + 2 (en)Pd :Pd(en)
Consistent with the c r y s t a l structure r e s u l t s we a l s o w r i t e a

reaction f o r formation o f t r i n u c l e a r , trihydroxo bridged trimer.
+ -> 3+
3 enPd(H 0)(0H)
2 «- 3 H 0 + 2 trimer
A new n o n - l i n e a r l e a s t s q u a r e s a n a l y s i s o f pH v s . e q u i v b a s e
f r o m t h e 1976 t i t r a t i o n d a t a a t 2 3 ° w i t h 0 . 2 M K N 0 (11) y i e l d s3
1 2
p K = 6 . 2 , l o g K = 3.7 ( M " ) , and l o g K = 6 . 5 ( M ~ ) .
x D T At t o t a l
P d ( I I ) c o n c e n t r a t i o n s g r e a t e r t h a n 0 . 2 mM more P d ( I I ) o c c u r s a s
d i h y d r o x o b r i d g e d dimer than a s t h e aquo-hydroxo complex.
I t i s p o s s i b l e t o make a n a p p r o x i m a t e c o m p a r i s o n o f t h e
t r i m e r i z a t i o n and d i m e r i z a t i o n c o n s t a n t s o f e n P d ( I I ) w i t h t h o s e
for c i s (NH3)2Pt(II). From t h e c o n s t a n t s g i v e n above i n t h e
3 2
e n P d ( I I ) s y s t e m , Κχ = 9 K ' . D From t h e p a i r o f p o i n t s f o r d i m e r
( 0 . 6 0 ) a n d t r i m e r ( 0 . 4 0 ) P t ( I I ) m o l e f r a c t i o n s a t t h e end o f
3 2
F i g u r e 7 i n r e f e r e n c e ( 1 6 ) I c a l c u l a t e t h a t Κχ = 4 Κ ρ / . (If
the system i s not a t e q u i l i b r i u m , t h e numerical 4 f a c t o r should
be g r e a t e r . ) T h e c o m p a r i s o n s u g g e s t s t h a t t h e two m e t a l i o n
systems have comparable t e n d e n c i e s t o form hydroxo b r i d g e d
dimers and t r i m e r s .
The d i s t r i b u t i o n c u r v e s i n F i g u r e 2 a r e i n c o m p l e t e i n
n e u t r a l s o l u t i o n s where t h e a q u o - h y d r o x o complex p o l y m e r i z e s t o
y i e l d hydroxo b r i d g e d dimer and t r i m e r . The extent o f t h e
d i m e r i z a t i o n depends upon t h e t o t a l P t ( I I ) c o n c e n t r a t i o n , and
t h e d i m e r i z a t i o n e q u i l i b r i u m c o n s t a n t i s unknown. If the value
+ 3 , 7
f o r enPd(H20) ( 0 H ) o f Krj = 1 0 i s taken as r e p r e s e n t a t i v e , at
a l l c o n c e n t r a t i o n s g r e a t e r t h a n 0 . 2 mM more P t ( I I ) o c c u r s a s
dimer than a s t h e aquo-hydroxo complex. The d i h y d r o x o b r i d g e d
dimer i s expected t o b e v i r t u a l l y i n e r t t o s u b s t i t u t i o n . B e
cause i t i s l e s s s t r a i n e d , t h e hydroxo bridged t r i m e r promises
t o b e e v e n more i n e r t t h a n t h e d i m e r . Thus t h e r e a c t i v i t y o f
P t ( I I ) complexes i n n e u t r a l s o l u t i o n s i n t h e p r e s e n c e o f a low
C l ~ b a c k g r o u n d d r o p s m a r k e d l y a t g r e a t e r t h a n 0 . 1 mM t o t a l
Pt(II) concentrations. C o n c e n t r a t i o n s g r e a t e r t h a n 0 . 1 mM
t o t a l P t ( I I ) a r e u n l i k e l y w i t h i n a c e l l , b u t t h e y a r e common i n
many i n v i t r o e x p e r i m e n t s .

2+
E f f o r t s t o use c i s (NH3)2Pt(H20>2 and other c i s diaquo
complexes t o provide g r e a t e r r e a c t i v i t y than t h e c i s d i c h l o r o
complexes may be s e l f - d e f e a t i n g under some circumstances. I n
n e u t r a l s o l u t i o n s w i t h a low C l ~ background, s u b s t a n t i a l f r a c -
t i o n s o f i n e r t hydroxo bridged dimer and t r i m e r form depending
upon t h e t o t a l P t ( I I ) c o n c e n t r a t i o n . Dimer and t r i m e r formation
occurs v i a t h e aquo-hydroxo monomer which occurs i n maximum con-
c e n t r a t i o n i n n e u t r a l s o l u t i o n s . The r a t e o f monomer disappear-
ance e x h i b i t s a maximum i n n e u t r a l s o l u t i o n s (16). At
c o n c e n t r a t i o n s g r e a t e r than 0.1 mM t o t a l P t ( I I ) , t h e r a t e o f
i n e r t dihydroxo bridged dimer formation may exceed that o f water
s u b s t i t u t i o n by another l i g a n d . Thus c o n c l u s i o n s based on t h e
r a t i o o f r e a c t i v e P t ( I I ) complex t o other l i g a n d s such as
n u c l e i c bases may have t o be reconsidered. This c o n c l u s i o n
appears e s p e c i a l l y r e l e v a n t t o r e a c t i o n o f supposed c i s diaquo

P t ( I I ) complexes w i t h h e l i c a l n u c l e i c a c i d s , where t h e secondary
s t r u c t u r e slows f u r t h e r t h e r a t e o f complex formation w i t h t h e ^
l i g a n d . An otherwise i n e x p l i c a b l e r e p o r t that c i s (NH3)2Pt(1^0)2
r e a c t s more s l o w l y a t pH 7 w i t h DNA than does c i s (NH3)2PtCl (17X 2
i s probably due t o formation of i n e r t hydroxo complexes and hydroxo

bridged dimers and t r i m e r s .
+
A dimer w i t h a s i n g l e hydroxo b r i d g e forms w i t h dienPd(H20)? .
The i r r e g u l a r nature of t h e t i t r a t i o n curve had been noted (11).
A recent n o n - l i n e a r l e a s t squares a n a l y s i s r e s o l v e d t h e t i t r a t i o n
curve obtained i n 0.5 M KNO3 and 21° i n t o a deprotonation w i t h
pK = 7.74 + 0 - 0 1 and a d i m e r i z a t i o n r e a c t i o n (18). We can
a
reformulate t h e d i m e r i z a t i o n r e a c t i o n as
K6
dienPd(H 0) 2
2 +
+ dienPd(0H) +
t (dien)Pd-OH-Pd(dien) + H 0 2
and from t h e constants a l r e a d y given i n r e f e r e n c e 18 c a l c u l a t e

1
K$ = 132 M" . At pH 7.74 t h e c o n c e n t r a t i o n s of t h e two r e a c t -
ants a t t h e l e f t appear i n equal c o n c e n t r a t i o n s , and a t 60 mM
P d ( I I ) i s d i v i d e d evenly between t h e two s i d e s o f t h e r e a c t i o n .
Higher t o t a l P d ( I I ) c o n c e n t r a t i o n s favor t h e dimer. The ana-
2 +
logous compound d i e n P t ( H 2 0 ) exhibits a regular t i t r a t i o n
curve w i t h p K = 6.13 a t 25° i n 0.1 M NaC104 (19). The slow
a
d i m e r i z a t i o n r e a c t i o n permits easy e v a l u a t i o n of t h e a c i d i t y
2+
constant. The d i m e r i z a t i o n r e a c t i o n i n d i e n P t ( H 2 O )
awaits study.
Hydroxo groups on P t ( I I ) complexes might a l s o r e a c t as
2
n u c l e o p h i l e s . Though i t i s 1 0 ^ · times l e s s b a s i c than unbound
4
hydroxide, t h e hydroxide bound t o dienPtOH * i s only 450 times
l e s s r e a c t i v e i n promoting h y d r o l y s i s o f p - n i t r o p h e n y l a c e t a t e (13).
Metal i o n bound hydroxide o f g r e a t l y decreased b a s i c i t y but only
modestly reduced n u c l e o p h i l i c i t y occurs commonly w i t h metal ions
and provides a r e l a t i v e l y h i g h c o n c e n t r a t i o n o f potent hydroxide
n u c l e o p h i l e s i n n e u t r a l and even a c i d i c s o l u t i o n s (20).

11. MARTIN Equilibria and Binding of cis-PtCh(NH )i 3 239
N ( l ) versus N(7) Dichotomy i n P u r i n e Nucleosides (21)
When s u b s t i t u t e d a t N(9) as i n n u c l e o s i d e s and d e r i v a t i v e s ,

the purine bases o f f e r two p o t e n t i a l metal i o n b i n d i n g s i t e s . I n
n e u t r a l s o l u t i o n s o f adenosine both N ( l ) and N(7) deprotonate
w h i l e w i t h the 6-oxopurines i n o s i n e and guanosine only N(7)
deprotonates. N(1)H deprotonations occur w i t h p K values o f 3.6,
a
8.7, and 9.2 i n adenosine, i n o s i n e , and guanosine, r e s p e c t i v e l y

(21). The r e l a t i v e extent o f metal i o n b i n d i n g t o N(7) and N ( l )
i n n e u t r a l s o l u t i o n s o f i n o s i n e and guanosine depends on pH.
Despite r e p o r t s on s t u d i e s o f many metal i o n s , the N(7)/N(l)
metal i o n b i n d i n g r a t i o s a t e q u i l i b r i u m i n purine nucleosides are
known q u a n t i t a t i v e l y f o r only two cases, owing t o an e a r l y study
+
on CH3Hg (22) and a more recent one on d i e n P d ( I I ) (18). The
i n t r i n s i c N(l)/N(7) metal i o n b i n d i n g r a t i o s o f C^Hg"*" approach

more c l o s e l y those o f the proton than do the b i n d i n g r a t i o s o f
d i e n P d ( I I ) , which e x h i b i t a s t r o n g b i a s toward the l e s s b a s i c
N(7) (18).
This research seeks t o c l a r i f y the i n t r i n s i c N(l)/N(7)
b i n d i n g r a t i o s f o r c i s diamine P t ( I I ) . Owing t o the d i f f i c u l t y
of a t t a i n i n g and j u d g i n g attainment of e q u i l i b r i u m w i t h P t ( I I ) (1),
we performed experiments w i t h P d ( I I ) . The enPd(II) binds a t
both N ( l ) and N(7) t o produce extensive polymer formation (23).
We f i n d no evidence f o r any N(7)-0(6) c h e l a t i o n o f enPd(II) i n
6-oxopurines. With o n l y a s i n g l e s i t e f o r s u b s t i t u t i o n ,
2+ 2+
d i e n P d H 2 0 avoids the c o m p l e x i t i e s o f f e r e d by e n P d ( H 2 0 ) 2 and
provides an i n t r i n s i c measure o f P d ( I I ) b i n d i n g a t i n d i v i d u a l
n u c l e i c base s i t e s . Studies w i t h t r i d e n t a t e d i p e p t i d e s i n s t e a d
of d i e n c h e l a t e d about P d ( I I ) y i e l d s i m i l a r r e s u l t s (24,25).
2
A comprehensive i n v e s t i g a t i o n o f dienPdH20 + b i n d i n g t o
nucleosides and 5-nucleotides has been reported (18); t h i s de-
s c r i p t i o n w i l l h i g h l i g h t o n l y a few p o i n t s . From the separate
peaks i n *H NMR s p e c t r a i t i s p o s s i b l e t o f o l l o w the chemical
s h i f t s of each k i n d o f d i e n P d ( I I ) complex as a f u n c t i o n o f pH.
F i g u r e 4 shows a p l o t o f H(8) chemical s h i f t versus pH f o r
f
s e v e r a l species i n a s o l u t i o n o f 5 -GMP and d i e n P d ( I I ) . Upon
p r o t o n a t i o n o f the phosphate group, (designated by Hp) the H(8)
chemical s h i f t moves u p f i e l d , r e v e r s i n g the u s u a l d i r e c t i o n .
For i n o s i n e and adenosine and t h e i r 5'-nucleotides, the H(2)
chemical s h i f t has a l s o been i d e n t i f i e d over a wide pH range (18).
Chemical s h i f t data such as t h a t shown i n F i g u r e 4 a l l o w a c a l -
c u l a t i o n o f a c i d i t y constants f o r deprotonations from f r e e l i g a n d
and complexes. ^
Comparison o f areas under i n d i v i d u a l peaks i n the H NMR
spectrum of d i e n P d ( I I ) - n u c l e o s i d e and n u c l e o t i d e complexes per-
mits e v a l u a t i o n o f e q u i l i b r i u m s t a b i l i t y constants f o r complex
formation. F i g u r e 5 shows the species d i s t r i b u t i o n i n a n e a r l y
T
equimolar s o l u t i o n o f d i e n P d ( I I ) and 5 -GMP and F i g u r e 6 does
f
the same f o r 5 -AMP. The d i f f e r e n c e between the d i s t r i b u t i o n s
i n F i g u r e 5 and 6 r e s u l t s l a r g e l y from the d i f f e r e n t absolute


11. MARTIN Equilibria and Binding of cis-PtCl (NH )2 2 3 241
1 I 1 1 1 ι
BN^Hp
BHjHp /Â MB 7
L 0.4
M
*Β"Μ7_
M BH7 P zr
A
\ / ' Δ ^?
π π Β
•
MyBM^p 9 §
,^'ΒΜ,Ηρ M BMf
7
1 I I I ι
1 2 3 4 5 6 7 8 9
PH
2
Figure 5. Species distribution of 5'-GMP binding by dienPd * presented as ligand
2
mole fraction (LMF) vs. pH at 0.10 M dienPd * and 0.11 M GMP. Individual points
1
are calculated from H-NMR intensities. Curves are derived from equilibrium
constants given in Ref. 18. (Reproduced from Ref. 18. Copyright 1981, American
Chemical Society.)
1.0 ι 1 1 1 1 1 1 Γ
2
Figure 6. Species distribution of 5'-AMP binding by dienPd * presented as ligand
mole fraction (LMF) vs. pH with both total Pd(II) and total AMP at 0.10 M .
1
Individual points are calculated from H-NMR intensities. Curves are derived from
equilibrium constants given in Ref. 18. (Reproduced from Ref. 18. Copyright 1981,
American Chemical Society.)

b a s i c i t i e s o f N ( l ) ; the two 5'-nucleotides possess a s i m i l a r

i n t r i n s i c P d ( I I ) N(7)/N(l) b i n d i n g r a t i o o f -1.4. This r a t i o
d i f f e r s r a d i c a l l y from t h a t f o r the proton, which amounts t o
4 6 f 2
1 0 · f o r 5 -GMP and 10 .7 f o r 5'-AMP (18). Thus the extent o f
d i e n P d ( I I ) b i n d i n g t o N(7) o f GMP and guanosine g r e a t l y exceeds
what might be expected from N(7) b a s i c i t y .
I n n e u t r a l s o l u t i o n s our r e s u l t s show t h a t the d i e n P d ( I I )
f
complex d i s t r i b u t i o n favors MyBH^ f o r 5 -GMP and BMi f o r i n o s i n e
and adenosine w h i l e f o r 5'-IMP and 5'-AMP both N(7) and N ( l )
complexed s p e c i e s occur i n comparable amounts (18). Presence o f
the 5'-phosphate group f a v o r s formation o f the N(7) complexes.
The s t a b i l i t y order o f decreasing d i e n P d ( I I ) b i n d i n g strengths a t
the common 5'-nucleotides a t pH 7 i s G7>I7>I1>G1>U3>T3>C3>A1>A7
(18). Thus o f the common n u c l e o s i d e s , N(7) o f a guanine base
provides the strongest d i e n P d ( I I ) b i n d i n g s i t e i n n e u t r a l

solutions.
How do the i n t r i n s i c N(l)/N(7) b i n d i n g r a t i o s f o r d i e n P t ( I I )
compare w i t h those now known f o r d i e n P d ( I I ) ? Because i t i s
d i f f i c u l t t o achieve e q u i l i b r i u m w i t h P t ( I I ) complexes o f t h e
N ( l ) protonated 6-oxopurines, we approached e q u i l i b r i u m w i t h an
2+
equimolar s o l u t i o n o f d i e n P t H 2 0 and 5'-AMP a t pH 5.1 (26). The
s o l u t i o n sat f o r two months, during which time i t was heated a t
50° f o r 13 days. L e v e l i n g o f f i n t h e slow growth o f the ! H NMR
peaks due t o the ΒΜχ complex suggests t h a t e q u i l i b r i u m may have
been obtained. The l a s t r a t i o of M7BHP t o BMiHp complexes i s
1.9 (26). This v a l u e i s 4.4 times g r e a t e r than t h a t found f o r
d i e n P d ( I I ) t o AMP (18). I f we g e n e r a l i z e the r e s u l t f o r AMP t o
the other n u c l e i c bases, we conclude t h a t P t ( I I ) favors N(7)
over N ( l ) b i n d i n g t o an even g r e a t e r extent than P d ( I I ) . Thus
the N(7) b i n d i n g s i t e o f guanosine a l r e a d y atop the s t a b i l i t y
order f o r P d ( I I ) becomes r e l a t i v e l y s t r o n g e r f o r P t ( I I ) compared
to s i t e s on the other bases. We conclude t h a t the predominant
P t ( I I ) b i n d i n g s i t e i n n u c l e o s i d e s and n u c l e o t i d e s i s N(7) o f
guanosine.
During a study o f the H(8) promoted exchange by P t ( I I )
bound t o N(7) i n p u r i n e s , we noted a t pH 5-6 a m i g r a t i o n o f
+
d i e n P t ( I I ) from i n o s i n e N(7) t o N ( l ) (27). F i r s t d i e n P t H 0 2
r e a c t s a t N(7).
Μ + ΒΗχ + MyBH-L
The metal i o n a t N(7) promotes the a c i d i t y o f the proton a t N ( l )

by up t o 2 l o g u n i t s (18).
+
M BH! +
7 H + M B~
7
The g r e a t e r f r a c t i o n o f N ( l ) deprotonated s p e c i e s w i t h M7BH1 than

w i t h ΒΗ^ provides a f a c i l e pathway f o r metal i o n c o o r d i n a t i o n a t
N ( l ) a t lower pH than occurs w i t h unbound l i g a n d

11. MARTIN Equilibria and Binding of c\s-PtCU(NHn)z 243
M + M B~
7 Μ ΒΜ? 1
2+
Our experiments w i t h equimolar i n o s i n e and d i e n P t H 2 0 produced a
complete r e a c t i o n t o g i v e M7BH1, f o l l o w e d by appearance o f t h e
b i n u c l e a r complex M7BM1 and reappearance o f f r e e l i g a n d ΒΗχ.
F i n a l l y the metal i o n bound a t N(7) may be r e l e a s e d .
M BM7 1 + Μ + ΒΜχ
Even w i t h d i e n P d ( I I ) and i n o s i n e the o v e r a l l t r a n s f e r o f m e t a l

i o n from N(7) t o N(.l) i s slow (18).
With N ( l ) protonated n u c l e i c bases, metal i o n c o o r d i n a t i o n
at N ( l ) occurs v i a the b i n u c l e a r M7BM1 complex because o f t h e
g r e a t e r a c i d i t y o f M7BH1 compared t o ΒΗχ, For a l l f i v e l i g a n d s
s t u d i e d w i t h d i e n P d ( I I ) , the greater f r a c t i o n o f N(.l) depro

tonated species when the l i g a n d i s metalated a t N(7) more than
o f f s e t s the decrease i n s t a b i l i t y f o r metal i o n b i n d i n g a t N ( l )
K K V S e K K n
i n M7B compared t o B, ( 7 l / a 7 B l / a B * r e f e r e n c e 18).
The above mechanism provides the main pathway f o r metal i o n
c o o r d i n a t i o n a t N ( l ) i n a c i d i c and n e u t r a l s o l u t i o n s o f i n o s i n e ,
guanosine, and d e r i v a t i v e s c o n t a i n i n g comparable amounts o f
metal i o n .
P t ( I I ) does d i s p l a c e the b a s i c N(3) proton from u r i d i n e
(pK = 9.2); the s t a b i l i t y constant f o r d i e n P t ( I I ) b i n d i n g t o
a
u r i d i n e has been estimated as a t l e a s t 10 times greater than

t h a t f o r d i e n P d ( I I ) (28). For d i e n P d ( I I ) and u r i d i n e i n H 0 the 2
s t a b i l i t y constant i s l o g Κ = 8,1 (29), Since d i e n P t ( I I ) y i e l d s

a g r e a t e r adenosine N(7)/N(l) s t a b i l i t y constant r a t i o than
d i e n P d ( I I ) , the s t a b i l i t y constant f o r P t ( I I ) a t N(7) o f purines
should be about 50-100 times greater than t h a t f o r P d ( I I ) , D i s
placement o f the N ( l ) proton o f guanosine and i n o s i n e and
d e r i v a t i v e s by P t ( I I ) i s d i v e r t e d by p r i o r c o o r d i n a t i o n and
k i n e t i c f i x a t i o n at N(7), Of the s e v e r a l common n u c l e o s i d e
b i n d i n g s i t e s , both e q u i l i b r i u m and k i n e t i c f a c t o r s favor P t ( I I )
b i n d i n g a t guanosine N(7), Reactions o f P d ( I I ) compounds w i t h
n u c l e i c bases serve as a thermodynamic r e f e r e n c e f o r r e a c t i o n s o f
analogous P t ( I I ) compounds a t e q u i l i b r i u m . Large d i f f e r e n c e s
between r e a c t i o n products o f analogous P d ( I I ) and P t ( I l ) com
pounds may be a t t r i b u t e d t o i n t r o d u c t i o n o f k i n e t i c e f f e c t s i n
the l a t t e r .
Acknowledgments
This research was supported by a research grant from the

N a t i o n a l Science Foundation.

Literature Cited
1. Vestues, P.; Martin, R.B. J. Am. Chem. Soc. 1981, 103, 806.
2. Howe-Grant, M.E.; Lippard, S.J. Metal Ions Biol. Syst. 1980,
11, 63.
3. Shannon, R.D. Acta Crystallogr. 1976, A32, 751.
4. Basoĺo, F.; Gray, H.B.; Pearson, R.G. J. Am. Chem. Soc. 1960,
82, 4200.
5. Reishus, J.W.; Martin, Jr., D.S. J. Am. Chem. Soc. 1961, 83,
2457.
6. Coley, R.F.; Martin, Jr., D.S. Inorg. Chim. Acta 1973, 7, 573·
7. Jensen, K.A. Z. Anorg. Allg. Chem. 1939, 242, 87.
8. Grinberg, Α.Α.; Stetsenko, A . I . ; Mitkinova, N.D.; Tikhonova,
L.S. Russ. J. Inorg. Chem. 1971, 16, 137.
9. LeRoy, A.F. Cancer Treat. Rep. 1979, 63, 231.

10. Gray, H.B.; Olcott, R.J. Inorg. Chem. 1962, 1, 481.
11. Lim, M.C.; Martin, R.B. J. Inorg. Nucl. Chem. 1976, 38, 1911.
12. Faggiani, R.; Lippert, B.; Lock, C.J.L.; Rosenberg, B. J. Am.
Chem. Soc. 1977, 99, 777.
13. Lippert, B.; Lock, C.J.L., Rosenberg, B.; Zvagulis, M. Inorg.
Chem. 1978, 17, 2971.
14. Faggiani, R.; Lippert, B.; Lock, C.J.L.; Rosenberg, B. Inorg.
Chem. 1977, 16, 1192.
15. Faggiani, R.; Lippert, B.; Lock, C.J.L.; Rosenberg, B. Inorg.
Chem. 1978, 17, 1941.
17. Mansy, .S.; Chu, G.Y.H.; Duncan, R.E.; Tobias, R.S. J. Am.
Chem. Soc. 1978, 100, 607.
18. Scheller, K.; Scheller-Krattiger, V.; Martin, R.B. J. Am.
Chem. Soc. 1981, 103, 6833.
19. Alcock, R.M.; Hartley, F.R.; Rogers, D.E. J. Chem. Soc.
Dalton 1973, 1070.
20. Martin, R.B. J. Inorg. Nucl. Chem. 1976, 38, 511.
21. Martin, R.B.; Mariam, Y.H. Metal Ions Biol. Syst. 1979, 8, 57.
22. Simpson, R.B. J. Am. Chem. Soc. 1964, 86, 2059.
23. Sovago, I.; Martin, R.B. Inorg. Chem. 1980, 19, 2868.
24. Vestues, P.I.; Martin, R.B. Inorg. Chim. Acta 1981, 55, 99.
25. Scheller-Krattiger, V.; Scheller, K.H.; Martin, R.B. Inorg.
Chim. Acta 1982, 59, 281.
26. Kim, S-H.; Martin, R.B. 1981, unpublished results.
27. Noszal, B.; Scheller-Krattiger, V.; Martin, R.B. J. Am. Chem.
Soc. 1982, 104, 1078.
28. Lim, M.C.; Martin, R.B. J. Inorg. Nucl. Chem. 1976, 38, 1915.
29. Lim, M.C. J. Inorg. Nucl. Chem. 1981, 43, 221.

12
Extended X-Ray Absorption Fine Structure
(EXAFS) Spectroscopic Analysis of
6-Mercaptopurine Riboside Complexes of
Platinum(II) and Palladium(II)
MICHAEL A. BRUCK, HANS-JÜRGEN KORTE, and ROBERT BAU—
University of Southern California, Department of Chemistry,

Los Angeles, CA 90089
NICK HADJILIADIS—University of Ioannina, Department of Chemistry,
Domboli 31, Ioannina, Greece
BOON-KENG TEO—Bell Laboratories, Murray Hill, NJ 07974
EXAFS evidence is presented to support the existence of N(7)-S(6)

chelates in the structures of Pt(6—mercaptopurineriboside), 2
Pt(2—amino—6—mercaptopurineriboside), 2
Pd(6—mercaptopurineriboside) 2 and
Pd(2—amino—6—mercaptopuri neriboside) . Relevant bond distances
2
are as follows: for Pt(6—mercaptopurineriboside) ,Pt—Ν = 2.02(2)Å,

2
Pt—S = 2.31(1)Å; for Pt(2-amino—6—mercaptopurineriboside), 2
Pt-N = 2.02(2)Å, Pt-S = 2.31 (1)Å.
The possible existence of an N(7)-O(6) chelate from guanine to platinum remains

one of the most actively-debated issues among researchers studying the mode of action
of anti-tumor platinum drugs (1-13)· Even though many compounds have been
prepared which are believed to contain such chelates (3-7), there are as yet no reported
crystal structure determinations which unambiguously prove the existence of this mode
(14-17). In contrast, the related question of an N(7)-S(6) chelate involving the sulfur
analog of guanine, 6-mercaptopuri ne, is much less controversial: there is general
agreement that such chelates are readily formed. Crystal structures have been reported
showing the existence of such chelates in complexes of Cu(II) (18), Cd(II) (19), and,
most notably, Pd(II) (20,21).
The syntheses of several Pt complexes with 6-mercaptopurines have been
reported (22,21). Spectroscopic evidence, conductivity measurements and chemical
analyses suggest that these compounds have the formula Pt(6—mercaptopurine) 2 with
0097-6156/83/0209-0245$06.00/0

the N(7)-S(6) chelate binding mode. Since Pd complexes are often considered to be
good structural models for Pt complexes, the known existence of an N(7)-S(6) chelate
in Pd(6—mercapto—9—benzylpurine) (2&2L) provided further support for the chelate
2
model in analogous Pt complexes. In the absence of an X-ray crystallographic

determination, we report here the determination of the structure in the vicinity of the
Pt atom for Pt(6—mercaptopurine riboside) [Pt(6—MPR) ]
2 and 2
Pt(2—amino—6—mercaptopurine riboside) [Pt(2—A—6—MPR) ], using Extended X-ray

2 2
Aborption Fine Structure ( E X A F S ) spectroscopy. The E X A F S technique (24-M)

allows an accurate determination of interatomic distances between an X-ray absorbing
atom and its near-neighbors as well as the coordination numbers.
The abbreviations for ligands used in this paper are shown in Figure 1. It should
be noted that for the complexes described in this paper, the ligands become
deprotonated at the N ( l ) position when complexed to a metal and hence acquire a
formal charge of —1.
Experimental $ ç ç t i o n
The compounds studied in this work are cis—Pt(NH ) Cl ( A ) ,

3 2 2
Pt(2-A-6-MPR) (B), P t ( 6 - M P R )
2 2(C), P d ( 6 - M P R ) (D),
2 Pd(2-A-6-MPR) 2
(E), and P d ( G u o ) C l (F). Compounds A and D were used as models and compound F
2 2
was used as a check for the model compound approach used in this study. The
synthesis and characterization of the Pt-6-mercaptopurineribosides has been previously
reported (23). The Pd complexes were prepared in an analogous manner.
C i s — P t ( N H ) C l QS) and P d ( G u o ) C l (2) were prepared according to literature
3 2 2 2 2
methods. The E X A F S experiments were conducted on the C2 beam-line at the Cornell

High Energy Synchrotron Source (CHESS) (22)· The synchrotron radiation was
monochromated by a channel-cut silicon < 2 2 0 > crystal.
Samples were prepared by first grinding the solids into homogenous fine powders
and then pressing the powder into a pellet (cross-section of pellet: 3mm X 19mm;
cross-section of synchrotron beam: ~ 1mm X 14mm). For some samples it was
necessary to dilute the powder with boron nitride. The sample concentration and the
pellet thickness were adjusted such that the change in μ Χ across the absorption-edge
was approximately equal to one (where μ is the linear absorption coefficient and X is
the pellet thickness).
A l l data were collected in the transmission mode using gas-filled ionization
chambers to measure the X-ray intensity before (I ) and after (I) the sample. For the
0
Pt samples, the Pt L - e d g e absorption spectra ( E ~ 11560 eV) were collected using

m 0
N in both ion chambers. For Pd samples, the Pd K-edge spectra ( E ~ 24350 eV)
2 c
were collected using N in the I chamber and A r in the I chamber.

2 0
Each spectrum was measured at a series of discrete energy values, starting at a

point approximately 100 eV below the absorption edge and continuing up in energy to a
point approximately 900 e V above the absorption edge. The actual increments were in
steps of constant k and therefore ranged from 2 to 9 eV from the beginning to the end
of a scan. The average dwell time per data point was about 1 sec., the actual time per
data point was varied so that a constant count was obtained for IQ. Plots of the raw data
in the form In ( V I ) vs. Ε are shown in Figure 2.

12. BRUCKET A L . EXAFS Analysis of Purine Complexes 247
American Chemical
Society Library
1155 16th St. n. w.
Washington,
ACS Symposium Series; 0. C. Society:
American Chemical 20036 Washington, DC, 1983.
11800 12000 24200 24400 24800 25000

E, eV E, eV
1400 11600 Π800 12000

24200 24400 24600 24800 25000 25200 25400
E, eV E. eV
E. eV E > e V
Figure 2. X-Ray absorption spectra, χ vs. E , eV. Key: A, c\s-Pt(NH ) Cl ; B,

μ 3 2 2
Pt(2-A-6-MPR) ; C, Pt(6-MPR) ; D, Pd(6-MPR) ; E, Pd(2-A-6-MPR) ; and F,

2 2 S 2
Pd(Guo) Cl . 2 2

12. BRUCKET AL. EXAFS Analysis of Purine Complexes 249
Data Reduction
The raw data were processed as described in the literature (2SL22.M.22). The
procedure involves (a) transformation of the absorption data from energy space into
momentum or k-space according to the equation,
1/2
2
k= [(2m/!î )(E-E ) 0 (1)
3
followed by (b) background subtraction using a cubic spline, (c) k -weighting,
(d) normalization of the edge-jump, and (e) corrections for the fall-off in absorption
3
cross-section according to Victoreen's equation (40). The resultant k X ( k ) vs. k plots
are shown as solid curves in Figure 3.
Fourier transforms of the k-space data are depicted as solid curves in Figure 4.
T o obtain the actual distances, r, a phase-shift factor must be added to the apparent
distance, r*, in the Fourier transform. This phase-shift may be estimated from model
compounds. However, more accurate values for r can be obtained from curve-fitting as
described below.
For the purposes of this study, the first two major maxima in the Fourier
transform, corresponding to the M - N (M=Pt or Pd) and M - S or M - C l distances, were
Fourier filtered and back-transformed into k-space. T h e filtering windows are shown as
dashed curves in Figure 4 and the back-transformed, filtered data in k-space are shown
as clashed curves in Figure 3.
A non-linear least-squares minimization technique was used to fit the filtered
spectrum with a semi-empirical expression for the E X A F S as follows:
(2)
Subscript 1 refers to M - N parameters and subscript 2 refers to M - S or M - C l parameters.

The functions Fj(kj) and 0j(kj) used in this study are the theoretically calculated
t h
amplitude and phase functions for the j back-scatterer (41). Four parameters are
least-squares refined for each term: the scale factor, B; the Debye-Waller thermal
parameter, σ ; the interatomic distance, r; and the energy threshold, Δ Ε . 0
A set of best-fit values (denoted by subscript bf) were obtaind by curve-fitting the
filtered k-space data. Plots of the filtered data (solid curve) and associated best-fit
values (dashed curve) are shown in Figure 5. These results, which are based upon
theory, and hence totally independent of "model" compounds, are listed in the left-hand
portion of Table I, under the heading "BFBT results" (best-fit based upon theory).
The P d - N distances are between 1 . 9 7 À and 2 . 0 4 Â for the compounds studied.
The P t - N distances range from 2 . 0 4 Â to 2 . 0 6 Â . The Pd-S and Pt-S B F B T distances, as
well as the Pd-Cl and Pt-Cl distances, are all around 2 . 3 3 À . These distances are within
the ranges of expected values. For comparison, the average value for the Pt-Cl
distance in cis — P t ( N H ) C l is 2 . 3 3 1 Â . However, the coordination numbers (N) were
3 2 2
not well determined by the B F B T technique.

In order to improve the accuracy of the B F B T results, as well as to determine
reliable coordination numbers, a newly developed fine adjustment procedure (42) was
applied to the best-fit results ( F A B M : fine adjustment based upon model). This
technique helps to alleviate parameter correlation problems and provides a simple
graphical technique to distinguish a good "model" from a "bad" model. A "good" model
has been operationally defined as one which has parameters and parameter correlation

Figure 3. Background-subtracted (solid curve) and Fourier-filtered (dashed curve)

3 3 1
EXAFS data {k (k), [A~ ] vs. k, [A' ]}. Key: A, às-Pt(NH ) Ch;
x B, Pt(2-A-6-
s g
MPRh; C, Pt(6-MPR) ; D, Pd(6-MPR) ; E, Pd(2-A-6-MPR) ; and F, Pd(Guo) Cl .

g 2 2 t t

BRUCKET A L . EXAFS Analysis of Purine Complexes 251
150
140 -
ί
130
120 -
Ε
110
100
90
1
8 0
!
I 70
60
50
40
30
20
10
0 û 2
r\ Â
3
Figure 4. Fourier transforms (solid curves) [φ (ΐ) vs. r,A (before phase shift
3
correction)] of the background-subtracted EXAFS spectra in Figure 3 and Fourier-

filtering windows (dashed curves). Key: A, cis-Pt(NH ) Cl ; B, Pt(2-A-6-MPR) ;
s 2 2 t
C, Pt(6-MPR) ; D, Pd(6-MPR) ; E, Pd(2-A-6-MPR) ; and F, Pd(Guo) Cl .

2 2 2 t 9

3
Figure 5. Fourier-filtered EXAFS spectra, k (k) vs. k, (solid curve) and BFBT
x
nonlinear least-squares curve fit (dashed curve). Key: A, cis-Pt(NH ) Cl ; B, Pt(2-

s 2 2
A-6-MPR) ; C, Pt(6-MPR) ; D, Pd(6-MPR) ; E, Pd(2-A-6-MPR) ; and F, Pd-

2 2 2 2
(Guo) Cl .
2 g

TABLE I. Results of the EXAFS Analysis
BFBT Results FABM Results

p p
Compound Term r,Â E ,eV
0 AE ,eV
0 r,Â σ,Α r,À Ν
a e e
(A) cis-Pt(NH ) Cl
3 2 2 Pt-N 2.000 11560.3 9.60 2.06(3) 0.075(12) 2.000 2.0
a e e
Pt-Cl 2.331 11560.3 17.11 2.34(1) 0.032(20) 2.331 2.0
(B) Pt(2-A»6-MPR) 2 Pt-N 11560.8 5.25 2.04(3) 0.063(48) 2.02(2) 2.0(5)

_ 2.33(2) 0.040(14) 2.31(1) 1.9(6)
Pt-S 11560.8 19.77
(C) Pt(6-MPR) 2
Pt-N - 11560.8 4.62 2.04(4) 0.058(48) 2.02(2) 1.9(4)
Pt-S 11560.8 18.13 2.32(1) 0.040(16) 2.31(1) 2.0(7)
e e
(D) Pd(6-MPR) 2
Pd-N 2.064^ 24349.0 -10.36 2.03(4) 0.057(48) 2.064 2.0
e e
Pd-S 2.308° 24349.0 12.39 2.33(2) 0.055(26) 2.308 2.0
(E) Pd(2-A-6-MPR) 2
Pd-N 24350.0 -10.75 2.04(5) 0.059(50) 2.08(4) 2.2(19)
Pd-S 24350.0 12.49 2.33(2) 0.047(20) 2.31(2) 2.1(13)
(F) Pd(Guo) Cl
2 2
Pd-N - 24350.0 -15.56 1.97(5) 0.025(32) 2.02(4) 1.9(14)
Pd-Cl 24350 12.18 2.33(2) 0.051(20) 2.31(2) 2.1(13)
a) Reference 57
b) Assuming that Pd-N and Pd-S distances in Pd(6—mercaptopurineriboside) 2
(Compound D) are the same as those in Pd(6—mercapto—9—benzylpurine) 2

(reference 20).

c) Known from model compounds, (refs. 20,57).
=
d) Calculated with σ^-κ 0.075Â and σ^_ = 0.032Â from the model compound A.
χ
e) Calculated with a£ _ = 0.057Â and σ^-χ — 0.055Â from the model compound D.
d N
behavior similar to that of the unknown. This procedure is described in detail

elsewhere (421. and details of our results are given in the Appendix.
The final results of the E X A F S analysis ( F A B M results) are summarized in the
right-hand portion of Table I. The F A B M values for all M - N distances in this study are
between 2 . 0 0 À and 2 . 0 8 À . A l l of the values are reasonable, though they do show a
wider range than the B F B T results. The range for M - S and M - C l distances is 2 . 3 1 Â to
2 . 3 3 Â . The F A B M values for coordination numbers, N , fall within ± 1 0 % of the
expected values of 2 nitrogen and 2 chlorine or sulfur atoms per metal.
Discussion
Transition metal complexes of 6-mercaptopurines have been characterized with
three binding modes: N-only, S(6)-only and [N(7)-S(6)] chelation. Two additional
binding modes, the [N(l)-S(6)] chelate and the [N(3)-N(9)] chelate, have also been
proposed, but not characterized.

S(6)-only binding has been shown crystallographically to occur in the complexes
H g ( 6 - M P ) C l (42), and [ C u ( 6 - M P ) C l ] (44,45,46); and also is believed to exist
2 2 2 2
(based upon IR evidence) in W ( C O ) ( 6 — M P ) (41,4&). Nitrogen-only binding has been

5
implied from E S R studies on iron-nitrosyl/mercaptopurine complexes (49).

The N(7)-S(6) chelation mode appears to be the dominant mode of bonding in
metal complexes of 6-mercaptopurines. It has been crystallographically shown to exist
in [Cu(9—methyl—6—mercaptopurine) C l ] (IS), 2 [Cd(6—mercaptopurine) ( H 0 ) C 1 ]
2 2 2
(12), and Pd(9—benzyl—6—mercaptopurine) (2Q,21); and also indicated in some

2
tungsten and iron complexes (4L4L42).

Various other spectroscopic studies have been reported on metal-mercaptopurine
complexes (50-56). In these studies, however the specific binding sites are not
discussed. These studies have included the metals A g , A u , Pd, Pt, Pb, Z n , N i , C o , C u ,
C d , M g , C a , Be, A l , C r , and U .
Spectroscopic evidence (2Î) indicates that P t ( 6 - M P R ) and P t ( 2 - A - 6 - M P R )
2 2
have the [N(7)-S(6)] chelate structure. A comparison of the Fourier-transformed

spectra for the Pt and Pd complexes of 6 - M P R in this study (Figure 4) shows that they
all contain two distinguishable peaks. The assignment of these peaks to P t - N and Pt-S
back-scatterings, in increasing distance, was subsequently supported by curve-fitting.
In this paper we have left unresolved the actual geometrical configuration (i.e., çj&
or trans) of the Pt and Pd mercaptopurine complexes. The original paper by Grinberg
and co-workers (22), which favored a trans configuration, cited the strong trans-
influence of sulfur as being a factor that would support a trans-geometrv. This
argument was also cited by Hadjiliadis and Theophanides (23.36). who presented infra-
red evidence consistent with the trans-configuration in their platinum/mercaptopurine
complexes. On the other hand, the crystal structure of
Pd(6—mercapto—9—benzylpurine) by Heitner and Lippard (2fi,21) unambiguously
2
shows the cis configuration and these authors argued that the cj& geometry allows
maximum x-backbonding to sulfur (in other words, that a soft ligand like sulfur prefers
to be opposite a hard ligand like nitrogen). Our present E X A F S data do not allow us to
distinguish the and trans, isomers.
Finally, we should point out that the existence of N(7)-S(6) chelates to platinum
does not mean that N(7)-0(6) chelates to platinum are also present in the
corresponding platinum-purine complexes. In fact, Heitner and Lippard have pointed
out that the steric requirements for a [N—C—C—S—M] five-membered ring are
significantly different from those for a [ N - C - C - O - M ] five-member ring, so that the
existence of the former does not necessarily imply the existence of the latter.

Conclusion
The purpose of these experiments was to determine the binding mode of 6-
mercaptopurineriboside to platinum using EXAFS spectroscopy. The Pt-EXAFS data
presented here establish the presence of two nitrogen and two sulfur atoms at normal
bonding distances from the platinum in Pt(6—mercaptopurineriboside)2 and
Pt(2—amino—6—mercaptopurine—riboside)2 . For Pt(6—MPR)2 and
Pt(2-NH2-6-MPR)2, wefindPt-N - 2.02(2)À, Pt-S - 2.31(1)Â. The results from
the Pt complexes in conjunction with the Pd-EXAFS, also presented here, [of
Pd(6-MPR)2 and Pd(2-A-6-MPR)2 which are chemical analogs of the known N(7)-
S(6) chelate Pd(9—benzyl—6—mercaptopurine)2 (2Q,21)1, define the binding mode in
the Pt-complexes of this study to be of the [N(7)-S(6)]-chelate type.
Acknowledgments
This research was supported by NIH Grant #CA-17367 to R.B., and NATO Grant
#1809 awarded jointly to R.B. and N.H. We thank the administrative and technical
staff of the Cornell High Energy Synchrotron Source (CHESS) for their enthusiastic
assistance. We would like to thank Dr. Douglas M. Ho for helpful discussions. Finally,
we would like to express our gratitude for the technical and computer software
assistance provided to us by M. R. Antonio (M.S.U.) and V. Bakirtzis (Bell Labs).
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Acta 1975, 378, 153.
3. Millard, M. M.; Macquet, J. P.; Theophanides, T. Biochim. Biophys. Acta 1975,
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Appendix: the FABM technique as applied to the title compounds.

The structurally known compound cis—Pt(NH3)2Cl2 (57) was chosen as the Pt
model compound for use in thefineadjustment based on model (FABM) technique
(for a full discussion of the method, see ref. 42). Pd(9-ribose-6-mercaptopurine) (21)
was used as the Pd-model, assuming the average values for the Pd-N and Pd-S
distances from Pd(9—benzyl—6—mercaptopurine)2 (2&21)·
The choice of a model compound with two nitrogen and two chlorine neighbors
(hereafter designated as Pt—N2C12) [i.e., cis—Pt(NH3)2Cl2; compound A] for an
unknown with two nitrogen and two sulfur (Pt-N^) neighbors is not unreasonable.
Small differences in atomic number are not distinguishable using EXAFS. To
demonstrate this point, the cis—Pt(NH3)2Cl2 data wasfitusing the theory for (Pt-N,S)
and then again with (Pt-N,Cl) theory. The resulting best-fit values are shown in
Table Π. Note that the a and r values are very similar.
Correlations between the parameters Bj and σ} and ΔΕ£. and η within each term
can affect the accuracy of the best-fit results. An even worse problem for the present
system is that the phase and amplitude functions for the two types of neighbors
(nitrogen and sulfur or chlorine) are not sufficiently different to prevent "trade offs"
between the two terms. Both of these problems can be circumvented by the FABM
method.
To explore and characterize the Bj vs. σ} correlation, a series offitsare made for
each absorber-backscatt ere r pair. For thesefitsthe parameter σ} isfixedat various
values in the vicinity of the best fit value, abf. While least-squares refining the
parameters IJ, ΔΕ£., Bj, and Bj the parameters σ{, τ·χ and ΔΕ£. are held at their best-fit
values, here i and j denote two different terms. The Bj values are then plotted against
σ} to give a graphical representation of the correlation. This correlation is fit with a
quadratic least-squares curve (goodness-of-fit R > 0.998). The correlation of ΔΕ£. and
η is probed in a similar manner. A series offitsis performed byfixingη at values
around its best-fit value and allowing ΔΕ£., σν Bj and Bj to refine while holding AEg., σχ
and η at their best-fit values. The ΔΕ|! values are then plotted against Δη = IJ — (rbf)j.
The plots of ΔΕξ vs. Δη generally exhibit linear behavior. However, if a compound
shows significant deviation from linearity, a quadratic term is added to reflect the
curvature. In either case R > 0.998.

T A B L E II. Comparison of an (N,C1) Fit with an (N,S) Fit
Pt(NH ) Cl
3 2 2 Pt(NH ) Cl
3 2 2
Parameter (N,C1 fit) (N,S fit)
Bi 1.136 0.994
σ
ι 0.0753 0.0727
Γι 2.059 2.050
B 2 0.693 0.775
*2 0.0365 0.0335
Γ2 2.337 2.344
ΔΕ 0 ι 0.20 -1.24
ΔΕ 0 2 7.71 8.10
The correlation curves for the platinum and palladium compounds are plotted in
Figure 6 and Figure 7 respectively: (a) Δ Ε £ vs. ΔΓ plots for M - N ; (b) Δ Ε | vs. ΔΓ plots
for M - X ; (c) Β vs. σ plots for M - N ; (d) Β vs. σ plots for M - X (M=Pt or Pd; X=C1 or
S). T h e regression coefficients corresponding to the curves are tabulated in Table III.
The fact that the curvatures and slopes of the curves within each figure are quite similar
is a strong indication that we have chosen reasonable model compounds for our
unknown systems.
The F A B M procedure makes use of these curves as described below. The results
are collected into Table IV with the best-fit values and relevant intermediate variables.
The Β values and σ values are now related as Bj — b + b σ + ο σ / . The ΔΕ£. values
0 x } 2
are related to the η values by Δ Ε ξ — ao 4- aÂrj) + a ( A r j ) and Δ η — η — r .

2
2
bf
The distance correction Δ η is determined for each term of each unknown from
the characteristic ΔΕΟ.. The ΔΕΟ. is obtained using a Ar m for the model compound
calculated as Ar m = r ^ — r . A t Ar , ΔΕΟ is found using the Δ Ε £ vs. ΔΓ correlation
bf m
curve of the model compound. The values of A r m and Δ Ε ο thus determined are:


Figure 6. Parameter correlation curves for Pt complexes A, B, and C. Key: α, ΔΓ vs. Δ Ε / for Pt-
N terms; b, ΔΓ vs. Δ Ε / for Pt-X terms; c, σ vs. Β for Pt-N terms; and d, σ vs. Β for Pt-X terms.
(See Appendix and Ref. 42 for explanation.;

12. BRUCKET A L . EXAFS Analysis of Purine Complexes 261
T A B L E III Least-Squares Regression Coefficients
Compound Term an ai a<> bn bi b,
(A) Pt(NH ) Cl 3 2 2 PtN 9.585 143.187 0.515 0.767 98.696

PtCl 16.733 229.349 1126.518 0.563 0.055 129.196
(B) Pt(2-A-6-MPR) 2 PtN

PtS
5.263
19.914
185.393
201.866
— 0.522
0.542
1.070
0.523
93.536
114.464
(Q Pt(6-MPR) 2 PtN
PtS
4.617
18.258
189.777
210.133
— 0.515
0.574
0.955
0.315
90.518
126.000
(D) Pd(6-MPR) 2 PdN -10.348 224.389 -334.344 0.421 0.973 76.786

PdS 12.40 200.934 0.476 0.152 122.018
(E) Pd(2-A-6-MPR) 2 PdN

PdS
-10.651
12.49
193.634
217.974
— 0.459
0.510
0.852
0.082
88.857
120.089
(F) Pd(Guo) Cl 2 2 PdN -15.094 314.474 -1110.490 0.440 1.421 49.054

PdCl 12.104 198.006 0.506 0.491 114.714
Δ Γ
» ΡΙ-Ν)
(
=
-°- 0 5 9
' A E
ô ( P t - N ) = 1-14; Ar m ( p t _ X ) - -0.0062, AE O ( P T _ X ) = 15.35;
Δ Γ
» ( Μ -Ν) = 0 0 3 8 4
' ΔΕ
ο*(Μ-Ν) - - 2 . 2 2 ; Δ Γ Η ΐ ( Μ _ Χ ) = -0.0205, A E V _ 0 d X ) = 8.28.
The correction, ΔΓ, for each bond-type is then determined from the AEg vs. ΔΓ
curve of the unknown at Δ Ε * . In this way the Δ Ε * from the model is transferred to the
unknown and a small adjustment, ΔΓ, is determined and applied to r b f
R
(FABM β r
bf + ΔΓ). The final distances are given in Table I and repeated in Table I V ,
along with the starting values and intermediate results.
To determine the coordination numbers, the best-fit Debye-Waller factors
σ^_ Ν — 0.075Â; σ ^ _ — 0.032À;
χ σ ^ _ — 0.057Â
Ν and σ ^ χ — 0 . 0 5 5 Â ; (where Χ
indicates S or Cl) of the models ( A and D ) were transferred to the corresponding terms
of the unknowns (B, C and E , F ) .
For each type of bond, the Β values of the model compounds, B , and the m
unknowns, B , are determined from the Β vs. σ correlation curves (subscripts m and u
m
refer to model and unknown respectively). The B m values of the model compounds
((A) Bpt_ == 1.132, B p ^ = 0.694; (D)
N x B P d _ N - 0.722, B P d _ x = 0.848) are used to
determine a amplitude reduction factor, S*. Which is just a proportionality factor such
that S* = Bn/Nm where N m is the known coordination number of the model.
The coordination numbers for the unknowns, N , are
u then calculated as
N u — BJS*. The final values of the unknown coordination numbers are listed in
Table I and also in Table IV with the values of intermediate variables B , S* and B . m u

T A B L E IV Summary of the F A B M Method
Parameters Used for Calculating Parameters Used for Calculating

a
F A B M Distances FABM
Coordination Numbers**
ΔΓ ΔΕ; ΔΓ + r •
Tbf = FABM B m
S* B„ N U
σ
(À) (eV) (Â) (À) (À) (Â) (BM/NM) (Bu/S')
(A) cis-Pt(NH ) Cl
3 2 2
Pt-N -0.059 1.14 -0.059 2.059 2.000 0.075 1.132 0.566 2.00
Pt-Cl -0.006 15.35 -0.006 2.337 2.331 0.032 0.694 0.347 - 2.00
(B) Pt(2-A-6-MPR) 2 Pt-N - - -0.022 2.037 2.015 _
1.133 2.00
Pt-S - - -0.023 2.333 2.310 - - - 0.673 1.94
(Q Pt(6-MPR) 2 Pt-N - - -0.018 2.035 2.017 _ _
1.100 1.94
Pt-S - - -0.014 2.322 2.308 - - - 0.710 2.05
(D) Pd(6-MPR) 2 Pd-N 0.038 -2.22 0.038 2.025 2.063 0.057 0.722 0.361 2.00
Pd-S -0.021 8.28 -0.021 2.329 2.308 0.055 0.848 0.424 - 2.00
(E) Pd(2-A-6-MPR) 2 Pd-N - - 0.044 2.036 2.079 _ _
0.791 2.19
Pd-S - - -0.019 2.330 2.301 - - - 0.872 2.06
(F) Pd(Guo) Cl
2 2 Pd-N - - 0.050 1.966 2.016 _
0.677 1.88
Pd-Cl - - -0.019 2.331 2.312 - - - 0.847 2.06
a) Δ Ε = a + a ^ r 4- a Ar* where an, a , a are given in Table III.

0 0 2 t 2

2
b) B = b + b i σ + ο σ
0 2 where b , b
0 l f b are given in Table III.
2
13
Synthesis and Biological Studies of a New Class
of Antitumor Platinum Complexes
DAVID B. BROWN, ABDUL R. KHOKHAR, MILES P. HACKER,
JOHN J. McCORMACK, and ROBERT A. NEWMAN
University of Vermont and the Vermont Regional Cancer Center,
Departments of Chemistry and Pharmacology, Burlington, VT 05405
A series of platinum complexes having the general

formula LPtCl , where L is a ligand bearing a formal
3
positive charge, have been prepared and have been dem-

onstrated to exhibit significant antitumor activity.
Complexes with this formulation satisfy the generally
accepted criteria for structural features required in
an antitumor-active platinum complex, but are expected
to possess kinetic advantages when compared to the
prototype antitumor platinum complex, cis-dichloro-
diamineplatinum (II). Complexes have been prepared
where L is a monoprotonated diamine and also where L
is a protonated amino-olefin. All complexes have been
examined for cytotoxicity against L1210 leukemia in
cell culture, and selected compounds have been tested
for antitumor activity in vivo (L1210 leukemia in BDF 1
mice). One compound, where L is protonated 3-amino-

-quinuclidine, has significant in vivo activity, with
T/C approaching 150%.
More than a decade has elapsed since Rosenberg first reported
(1) the antitumor activity of cis-dichlorodiamineplatinum(II),
DDP. It would be fortuitous indeed if the first platinum complex
reported proved to be the most effective compound of its type.
Furthermore, although DDP is one of the most effective antitumor
drugs available, its use is accompanied by severe nausea and a
range of toxic effects Ç2). These factors, combined with the
rather limited range of clinical activity for DDP, have led to
the search for new antitumor platinum compounds having either
increased efficacy or decreased toxicity, or both.
A vast number of platinum compounds have now been screened
for antitumor activity, and the results of these investigations
have led to a set of empirical guidelines for the minimum struc-
tural features required for activity. Although they may be
stated in various forms, the key structure-activity relationships
(3) are:
0097-6156/83/0209-0265$06.00/0

1. The complex must have a p a i r of c i s - a n i o n i c l i g a n d s (the

l e a v i n g groups), as the trans analogs are i n v a r i a b l y much
less active.
2. The a n i o n i c l i g a n d s must be moderately hard Lewis bases,
such as c h l o r i d e i o n s or oxygen donors.
3. The Pt complexes must be water s o l u b l e , but must a l s o be
capable of e n t e r i n g the c e l l i n t a c t . For a l l p r a c t i c a l
purposes, t h i s l i m i t s compounds to those bearing no net
charge.
4. The i d e n t i t y of the n e u t r a l l i g a n d s (the amines) i s
c r u c i a l to a c t i v i t y i n that there i s suggestion that only
complexes of primary (and p o s s i b l y secondary) amines are
e f f e c t i v e o n c o l y t i c agents.
Compliance w i t h these g u i d e l i n e s means that v i r t u a l l y a l l

compounds which have been examined have had the general s t r u c t u r e
I.
(Y)
In I , X i s an a n i o n i c l i g a n d , t y p i c a l l y c h l o r i d e , and A i s an
amine (or A^ a c h e l a t i n g diamine.) O x i d a t i o n to the f r e q u e n t l y
more s o l u b l e platinum (IV) analogs r e q u i r e s the i n c o r p o r a t i o n of
two a d d i t i o n a l a n i o n i c l i g a n d s , Y.
The general f a t e of DDP analogs i n the body seems c l e a r
(equation 1). I t i s thought that DDP enters the c e l l i n t a c t .
+2
CI /0H 2
Ptr DNA^ A 2 Pt/DNA Complex

«2?
A/ CI
In the c e l l , an area of low c h l o r i d e i o n c o n c e n t r a t i o n , e q u i l i b -

rium favors l o s s of the a n i o n i c l i g a n d s w i t h the formation of
v a r i o u s hydrated d e r i v a t i v e s . I t i s these very r e a c t i v e com-
p l e x e s , w i t h t h e i r l a b i l e aquo l i g a n d s , which coordinate to DNA
and lead t o the observed c y t o t o x i c i t y . Excepting c o n s i d e r a t i o n s
of s o l u b i l i t y , the broad s t r u c t u r e / a c t i v i t y observations of the
past decade are probably a r e f l e c t i o n of v a r i a t i o n s i n only the
end of t h i s process. In p a r t i c u l a r , v a r i a t i o n s i n the amine
i d e n t i t y are l i k e l y to have only minor e f f e c t s on the k i n e t i c s

13. BROWN ET AL. New Class of Antitumor Pt Complexes 267
and theraodynamics o f the h y d r o l y s i s e q u i l i b r i u m . The amine

i d e n t i t y w i l l however have a s i g n i f i c a n t e f f e c t (due t o s t e r i c
f a c t o r s , hydrogen bonding, etc.) on the thermodynamics o f p l a t i
num b i n d i n g t o DNA, and hence on o n c o l y t i c a c t i v i t y .
In p r i n c i p l e , i t should be p o s s i b l e t o vary the a c t i v i t y of
platinum complexes not only by a l t e r i n g the thermodynamics o f
platinum b i n d i n g t o DNA, but a l s o by a l t e r i n g the r a t e at which
the e q u i l i b r i u m of equation 1 i s a t t a i n e d . The k i n e t i c s of t h i s
h y d r o l y s i s r e a c t i o n w i l l be d i c t a t e d by the t r a n s - e f f e c t , that i s ,
by the a b i l i t y of the l i g a n d l y i n g trans t o the c h l o r i d e t o i n
crease the r a t e of the c h l o r i d e i o n s u b s t i t u t i o n r e a c t i o n s .
Amines l i e low i n the t r a n s - e f f e c t s e r i e s , and consequently the
h y d r o l y s i s of a c h l o r i d e i o n l y i n g trans t o an amine i s a r a t h e r
s l u g g i s h r e a c t i o n . Our work centers on e f f o r t s t o improve a n t i
tumor a c t i v i t y by v a r y i n g the s u b s t i t u e n t s on platinum i n such a

f a s h i o n that the r a t e of the h y d r o l y s i s r e a c t i o n i n equation (1)
i s enhanced. We have chosen t o do t h i s by r e p l a c i n g one of the
amines by a l i g a n d l y i n g higher i n the t r a n s - e f f e c t s e r i e s .
P r a c t i c a l c o n s i d e r a t i o n s g e n e r a l l y r e s t r i c t the choice of l i g a n d
to a n i o n i c s p e c i e s , and we have focused on the use of the c h l o r
i d e i o n . Replacement of a n e u t r a l amine by an a n i o n i c c h l o r i d e
would lead t o a net negative charge on the complex. I n order t o
m a i n t a i n o v e r a l l complex n e u t r a l i t y (which i s presumably neces
sary f o r c e l l u l a r p e n e t r a t i o n ) , the remaining amine must be r e
placed by a l i g a n d bearing a formal p o s i t i v e charge. This leads
to a complex having the general formula I I :
CI
Θ I
L Pt
I
_ CI
II
Such complexes can i n f a c t , as w e l l as i n p r i n c i p l e , e x h i b i t

s i g n i f i c a n t antitumor a c t i v i t y . We report here the s y n t h e s i s ,
c h a r a c t e r i z a t i o n , and antitumor a c t i v i t y o f two broad c l a s s e s of
complex having s t r u c t u r e I I , those where the p o s i t i v e l y charged
l i g a n d i s a monoprotonated (or monoalkylated) diamine and those
where i t i s a monoprotonated amino-olefin.
Complexes o f Protonated Diamines
P r i o r t o our work, there had been few r e p o r t s o f platinum

complexes bearing monoprotonated (or monoalkylated) diamines as
l i g a n d s . To our knowledge, only three complexes having t h i s
s t r u c t u r e have been c h a r a c t e r i z e d . Terζis (4) prepared and
determined the c r y s t a l s t r u c t u r e of trichloro(9-methyladeninium)

p l a t i n u m ( I I ) , Maresca, et a l . (5) prepared t r i c h l o r o ( t e t r a -

methylethylenediammonium)platinijm(II), and Adeyemo, et a l . (6)
prepared t r i c h l o r o ( 4 - a m i n o - 2 , 6 - d i m e t h y l p y r i d i n e ) p l a t i n u m ( I I ) . We
have found a l a r g e number of such complexes can be prepared using
the two general r e a c t i o n s o u t l i n e d below.
N~N + K PtCl,
0 + HC1 CI
+ I
HN~N Pt CI
N-N · 2HC1 + K PtCl.

0
2 4
+ NaOH-JJ CI
Under these p r e p a r a t i v e c o n d i t i o n s , a l a r g e number of a l t e r n a t i v e

products (e.g., ( N ~ N ) P t C l , (Ν ~ N H ) P t C l ^ , etc.) are p l a u s i b l e ,

2 2 2
and indeed i n c e r t a i n cases were found. For the compounds r e

ported here, the combination of C, H, N, and CI m i c r o a n a l y t i c a l
data was adequate to demonstrate u n e q u i v o c a l l y a r a t i o of 3C1/
diamine/Pt.
Although t h i s i s supportive of the c o o r d i n a t i o n sphere of
platinum being P t C l ^ N , i t i s a more d i f f i c u l t problem to determine
which of the two n i t r o g e n s i t e s of the diamine i s coordinated to
platinum and, consequently, which n i t r o g e n s i t e i s protonated. It
i s probable that the s p e c i f i c isomeric form which i s produced i s a
consequence of some complex i n t e r p l a y of both k i n e t i c and thermo
dynamic f a c t o r s . In p a r t i c u l a r , i t s i d e n t i t y w i l l depend upon the
Lewis b a s i c i t y of each n i t r o g e n donor s i t e toward the Lewis a c i d
platinum i n the form P t C l ^ . Since knowledge of that Lewis b a s i c
i t y does not e x i s t , we have no p r e d i c t i v e a b i l i t y .
In the case of the compound d e r i v e d from 3-aminoquinuclidine
( t r i c h l o r o ( 3 - a m i n o q u i n u c l i d i n i u m ) p l a t i n u m ( I I ) , or QTP) we have
t r i e d to r e s o l v e the s t r u c t u r a l question by examination of i n f r a
red s p e c t r a i n the N-H s t r e t c h i n g r e g i o n . Comparisons of s p e c t r a
f o r 3-aminoquinuclidine, i t s mono- and di-protonated h y d r o c h l o r
i d e s , and QTP suggest that i n the platinum complex i t i s the -NH 2
group which i s ^ p r o t o n a t e d . S p e c i f i c a l l y , a broad and i n t e n s e band

at ca 2800 cm which can be assigned to the protonated t e r t i a r y
amine group i n the mono- and d i - h y d r o c h l o r i d e i s absent from QTP.
I n f r a r e d s p e c t r a thus suggest s t r u c t u r e I I I f o r QTP.
CI
I
Pt CI
III

B i o l o g i c a l Studies of Protonated Diamine Complexes
A l l of the compounds we have prepared have been evaluated as

i n h i b i t o r s of the growth of murine leukemic c e l l s (L1210) and a
DDP-resistant s u b l i n e (L1210/DDP) i n v i t r o . We have chosen a 50%
i n h i b i t o r y dose ( I D ^ Q ) of 10 jag/ml as our u p p e r - l i m i t c r i t e r i o n
f o r a s i g n i f i c a n t l e v e l of a c t i v i t y . Compounds t h a t s a t i s f y the
c r i t e r i o n are evaluated f u r t h e r f o r t h e i r a b i l i t y t o prolong the
l i f e span of mice i n o c u l a t e d w i t h L1210 c e l l s . Although we have
prepared a l a r g e number of complexes having the g e n e r a l formu-
l a t i o n I I , most e x h i b i t r e l a t i v e l y l i t t l e a c t i v i t y a g a i n s t L1210
i n the c e l l c u l t u r e screen (Table I g i v e s s e l e c t e d I D ^ Q v a l u e s ) .
Table I . +
Compounds (L )PtCl« Which S a t i s f y the C r i t e r i o n
f o r A c t i v i t y i n C e i l C u l t u r e Studies
I D
L sn'
L1210 L1210/DDP
* S o l u b i l i z e d i n DMSO.

The most i n t e r e s t i n g compound i n terms of b i o l o g i c a l a c t i v i t y

i s QTP, the compound w i t h 3-aminoquinuclidine as the l i g a n d , espe-
c i a l l y i n view of i t s a b i l i t y t o produce roughly e q u i v a l e n t i n h i -
b i t i o n of the growth of both L1210 and L1210/DDP c e l l s . QTP i s ,
u n f o r t u n a t e l y , very p o o r l y s o l u b l e i n water. I n an attempt to
f i n d w a t e r - s o l u b l e analogs of QTP, s e v e r a l d e r i v a t i v e s were p r e -
pared by the routes o u t l i n e d below:
LPtCl 3 + Ag S0 2 4 *LPtCl(S0 ) · H 0 4 2 + 2AgCl

I V
LPtCl 3 + 2H 0 2 2 >LPt Cl (OH) 3 2 + 2^0
I V
LPtCl 3 + H 0
2 2 + 2HC1 > LPt Cl 5 + 21^0
LPtCl(S0 ) 4 + Ba C H ( C 0 0 )2 2 > L P t C l CH (COO> 2 2 + BaS0 4
These compounds, s e v e r a l having enhanced aqueous s o l u b i l i t y , were

evaluated f o r i n v i t r o a c t i v i t y . Each of the compounds (Table I I )
has a c t i v i t y a g a i n s t DDP-sensitive L1210 c e l l s comparable to that
of QTP, but i s l e s s e f f e c t i v e against L1210 c e l l s r e s i s t a n t t o
DDP.
Table I I . A c t i v i t y of 3-Aminoquinuclidinium D e r i v a t i v e s
Against L1210 C e l l s I n V i t r o
ID 5 Q Oig/ml)
Compound L1210/0 L1210/DDP
LPtCl 3 2 5
LPtCl 5 4.4 >10
LPtCl (OH)3 2 4.5 >10
LPtCl (S0.)
o 4.6 >10
3 4
LPtCl(CH (C00) ) 2 2 2.7

To evaluate the a c t i v i t y of compounds against L1210 i n v i v o ,

the f o l l o w i n g procedure was employed. L1210 c e l l s (1 χ 106
suspended i n 0.1 ml of p h y s i o l o g i c a l s a l i n e s o l u t i o n ) were i n o c u
l a t e d i n t r a p e r i t o n e a l l y ( i . p . ) i n t o BDF^ mice (20-22 gm) and drug
treatment ( i . p . ) was i n i t i a t e d 24 hours l a t e r . Animals that r e
ceived no drug treatment d i e d between 7 and 9 days a f t e r i n o c u l a
t i o n of L1210 c e l l s (mean s u r v i v a l time ~8.5 days). R e s u l t s ex
press the r a t i o of the mean s u r v i v a l time of t r e a t e d animals t o
the mean s u r v i v a l time of c o n t r o l animals (%T/C).
Studies i n our l a b o r a t o r y r e v e a l that QTP has s i g n i f i c a n t
antileukemic a c t i v i t y . For example, when given as a s i n g l e dose
(10 mg/kg; day 1) the compound gave a T/C value of 125% and the
%T/C value was increased somewhat (to 138) when a more i n t e n s i v e
schedule of a d m i n i s t r a t i o n was employed (5 mg/kg; days 1, 2, 5,
6, 9). Our r e s u l t s were confirmed by independent s t u d i e s c a r r i e d

out under the auspices of the Developmental Therapeutics Program,
N a t i o n a l Cancer I n s t i t u t e (Table I I I ) .
The water s o l u b l e malonato d e r i v a t i v e of QTP e x h i b i t e d
l i m i t e d i n v i v o a c t i v i t y . Thus, at a s i n g l e dose (day 1) o f
50 mg/kg the compound gave a %T/C value of 126%. We a l s o e v a l u
ated the s o l u b l e s u l f a t o d e r i v a t i v e of QTP i n v i v o but found i t
to be more t o x i c than QTP and to be i n e f f e c t i v e against murine
leukemia at a non-toxic dose (5 mg/kg). The b i o l o g i c a l a c t i v i t y
of QTP i s not shared by the l i g a n d per se s i n c e 3-aminoquinucli
dine hydrochloride was found to e x h i b i t no c y t o t o x i c i t y i n v i t r o
under c o n d i t i o n s i n which the complex was q u i t e a c t i v e .
The a c t i v i t y of QTP i n the experimental leukemia system
prompted us to c a r r y out t o x i c o l o g i c a l s t u d i e s on t h i s complex.
When administered as a s i n g l e i . p . i n j e c t i o n , QTP caused no immed
i a t e signs of t o x i c i t y but w i t h i n 48 hours mice became l e t h a r g i c ,
developed p i l o e r e c t i o n and d i s p l a y e d signs of tetany. The t o x i c
e f f e c t s progressed u n t i l p a r a l y s i s of the hind limbs was observed.
Deaths occurred 3 to 8 days f o l l o w i n g treatment. Gross necropsy
revealed signs of r e n a l t o x i c i t y i n c l u d i n g pale r e n a l c o r t e x ,
f r i a b i l i t y and exaggerated c o r t i c o m e d u l l a r y border. Other g r o s s l y
observable s i g n s of t o x i c i t y were s h a r p l y reduced spleen s i z e and
d i s t e n t i o n of s m a l l i n t e s t i n e . The c a l c u l a t e d L D ^ Q , L D ^ Q and
LD^Q values were 22, 42 and 83 mg/kg r e s p e c t i v e l y .
These s t u d i e s of the b i o l o g i c a l a c t i v i t y of QTP i n d i c a t e that
complexes of the type L P t C l ^ represent a new c l a s s of antitumor
platinum c o o r d i n a t i o n compounds. We are c u r r e n t l y engaged i n
s t u d i e s of the p r e p a r a t i o n and e v a l u a t i o n of new examples of t h i s
c l a s s of compound that may be more e f f e c t i v e and l e s s t o x i c than
QTP.
Complexes of Protonated Amino-Olefins
Although monoprotonated diamines are a convenient choice f o r

+
the p o s i t i v e l y charged l i g a n d i n s t r u c t u r e I I , other l i g a n d s L
may be considered. We have consequently examined complexes where

Table I I I . A c t i v i t y of Trichloro(3-Aminoquinuclidinium)
a
P l a t i n u m ( I I ) Against L1210 C e l l s I n V i v o
(mg/kg) Schedule %T/C
5 1 123
10 1 125
50 1 Toxic
5 1,2,5,6,9 138
2.50 1,5,9 126
5 1,5,9 120
10 1,5,9 130
20 1,5,9 133
2.5 1,5,9 119
5 1,5,9 122
10 1,5,9 139
20 1,5,9 140
40 1,5,9 148
a. ) Because of the low s o l u b i l i t y of t h i s compound, data

from d i f f e r e n t l a b o r a t o r i e s may not be d i r e c t l y com-
parable due t o different sonification/suspension/
solubilization procedures.
b. ) Data obtained a t the U n i v e r s i t y of Vermont.
c. ) Experiments 2 and 3 represent data obtained from the

NCI u t i l i z i n g two d i f f e r e n t screening c o n t r a c t o r s .

the l i g a n d L+ i s a protonated a m i n o - o l e f i n . In p a r t i c u l a r , we
have prepared a s e r i e s of compounds having s t r u c t u r e IV, i n which
a s u b s t i t u t e d a l l y l amine i s coordinated to platinum through the
H Cl
IV
o l e f i n i c i r - s y s t e m . This s t r u c t u r e possesses the k i n e t i c advantages

discussed above f o r a l l compounds of type I I , but i n a d d i t i o n the
t h i r d c h l o r i d e i s h i g h l y a c t i v a t e d toward s u b s t i t u t i o n because of
the l a r g e t r a n s - e f f e c t of the coordinated o l e f i n .
S e v e r a l compounds analogous to I V have been r e p o r t e d , and
X-ray c r y s t a l l o g r a p h i c s t u d i e s have e s t a b l i s h e d t h a t i n each case
the c o o r d i n a t i o n environment a t platinum i s s i m i l a r to t h a t i n
Zeise's s a l t , e.g., square-planar platinum c o o r d i n a t i o n by three
c h l o r i d e s and the o l e f i n ff-system ( 7 ) . We have prepared complexes
having s t r u c t u r e I V by the d i r e c t r e a c t i o n of the N - s u b s t i t u t e d
a l l y l amine w i t h potassium t e t r a c h l o r o p l a t i n i t e i n a c i d i c aqueous
s o l u t i o n . A n a l y t i c a l data e s t a b l i s h the f o r m u l a t i o n , and i n f r a r e d
s p e c t r a demonstrate that the o l e f i n i s coordinated to platinum.
The s t r u c t u r e i s r e t a i n e d i n dimethylformamide s o l u t i o n , as demon-
s t r a t e d by e x t e n s i v e nmr measurements.
B i o l o g i c a l Studies of Amino-Olefin Complexes
The compounds prepared i n t h i s study have been evaluated as

i n h i b i t o r s of L1210 growth u s i n g the c e l l c u l t u r e screen. Using
our c r i t e r i o n f o r a s i g n i f i c a n t c y t o t o x i c i t y , many of the com-
pounds having s t r u c t u r e I V are found to be a c t i v e i n v i t r o
(Table I V ) . S e v e r a l trends are apparent from these data. F i r s t ,
most of the protonated a m i n o - o l e f i n complexes have comparable
c y t o t o x i c i t y (the exceptions w i l l be discussed s h o r t l y ) . F u r t h e r -
more, most are approximately e q u i t o x i c to the s e n s i t i v e and r e s i s -
tant c e l l l i n e s . This suggests that there may be a s i g n i f i c a n t
d i f f e r e n c e i n the mechanism of a c t i o n of these compounds compared
to DDP, s i n c e i n DDP and many of i t s analogs there i s a ca 2 0 - f o l d
d i f f e r e n c e i n a c t i v i t y toward these c e l l l i n e s . The three proton-
a r e
ated a m i n o - o l e f i n compounds w i t h l a r g e values of I D C Q equiva-
l e n t , and d i s t i n c t from the other complexes, i n that they are
d e r i v e d from t e r t i a r y amines. For simple DDP analogs there i s
s i g n i f i c a n t evidence t h a t complexes of primary, and p o s s i b l y
secondary, amines are much more a c t i v e than complexes of t e r t i a r y
amines (8). T h i s trend seems to be repeated here, but i n t h i s
case the amine i s not coordinated to platinum. Consequently,
s u b s t i t u t i o n a t the amine would not be expected to have any

Table IV. C e l l C u l t u r e A c t i v i t y f o r Protonated and

N e u t r a l Complexes o f A l l y l a m i n e s
Protonated Ligand N e u t r a l Ligand

A l l y l Amine, L Complex, (LH+)PtCl 3 Complex, L P t C l 2
Compound No. I D f a g / m l ) Compound No.

5Q ID 5 Q
L1210 L1210/DDP L1210

la 3.0 5.0 lb 5
2a >10
3a 2.5 4.0 3b >10
4a 6 4.3 4b 7
5a 8 27 5b >10
6a >10
7a >10

s i g n i f i c a n t e f f e c t upon a c t i v i t y . The o b s e r v a t i o n that the e f f e c t

p e r s i s t s here should be accounted f o r i n any d i s c u s s i o n of the
o r i g i n of the e f f e c t of the amine s u b s t i t u t i o n upon antitumor
activity.
In the absence of p r o t o n a t i o n , the n e u t r a l a m i n o - o l e f i n i s ,
i n p r i n c i p l e , capable of c o o r d i n a t i o n as a c h e l a t e l i g a n d , u t i l z -
ing both the o l e f i n fr-system and the n i t r o g e n lone p a i r . This
would produce a n e u t r a l complex of type V, which should a l s o be
a c t i v a t e d toward the s u b s t i t u t i o n r e a c t i o n of equation ( 1 ) , and,
consequently, have p o t e n t i a l as an antitumor agent, Yellow s o l i d s

having the composition ( a m i n o - o l e f i n ) P t C l can be prepared by
?
n e u t r a l i z a t i o n of the (protonated a m i n o - o l e f i n ) P t C l ^ complexes

w i t h aqueous hydroxide, f o l l o w e d by e x t r a c t i o n i n t o chloroform.
A l t e r n a t i v e l y , at l e a s t i n c e r t a i n cases, i d e n t i c a l m a t e r i a l s can
be prepared by the d i r e c t r e a c t i o n of the a m i n o - o l e f i n w i t h
aqueous K ^ P t C l ^ . Once formed, these compounds are o n l y p o o r l y
s o l u b l e i n s o l v e n t s which do not cause decomposition, and t h i s
f a c t has made t h e i r c h a r a c t e r i z a t i o n e l u s i v e . Denning and Venanzi
(9) have reported complexes of t h i s form w i t h N - e t h y l a l l y l a m i n e
and N - o c t y l a l l y l a m i n e , which they formulate as polymeric and
d i m e r i c , r e s p e c t i v e l y . Apparently, a monomeric c h e l a t e complex
having S t r u c t u r e V cannot be formed because of the s t e r i c
problems inherent i n the formation of the 4%-memb«red c h e l a t e
r i n g . Based on t h e i r low s o l u b i l i t y , we b e l i e v e that the com-
plexes we have prepared have o l i g o m e r i c s t r u c t u r e s as w e l l . I n -
f r a r e d s p e c t r a do, however, demonstrate c o n c l u s i v e l y that both
the amine and the o l e f i n are coordinated to platinum.
Although the n e u t r a l l i g a n d complexes corresponding to each
of the protonated l i g a n d complexes could not be prepared, b i o l o g i -
c a l data i s a v a i l a b l e f o r four such complexes. In g e n e r a l , c e l l
c u l t u r e a c t i v i t y i s comparable f o r both the protonated l i g a n d and
n e u t r a l l i g a n d complexes. Although t h i s may be f o r t u i t o u s , i t i s
a l s o p o s s i b l e that under b i o l o g i c a l c o n d i t i o n s the two types o f
compound are i n t e r c o n v e r t i b l e . Since the n e u t r a l l i g a n d complexes
are t y p i c a l l y prepared by deprotonation of the protonated l i g a n d
complexes, i t i s conceivable that t h i s deprotonation r e a c t i o n
occurs at p h y s i o l o g i c a l pH.
As r e p r e s e n t a t i v e samples, the complexes (protonated N - e t h y l -
a l l y l a m i n e ) P t C l ^ and ( N - e t h y l a l l y l a m i n e ) P t C ^ have been given a
p r e l i m i n a r y screening f o r antitumor a c t i v i t y i n v i v o . The com-
plexes were administered i n t r a p e r i t o n e a l l y to mice w i t h L1210

Leukemia, At the dosages used, neither compound exhibited signif-
icant antitumor activity at less than toxic dose.
None of these compounds are particularly water soluble.
Consequently, we considered that the poor in vivo activity might
reflect poor water solubility, rather than inherent molecular in-
activity. In order to test this hypothesis, we prepared the sul-
fate derivative of (protonated allylamine)PtCl« by reaction 4.
This sulfate complex is readily water soluble but, like its insol-
uble chloride analogs, appears to be inactive in vivo.
We are forced to conclude that although these compounds are
indeed cytotoxic, they do not have significant antitumor effects

at acceptable levels of host toxicity.
Conclusion
No single compound among those reported here exhibits the
combination of increased activity, decreased toxicity and desir-
able physical properties which would provide for a likely clinical
improvement over cis-dichlorodiamineplatinum(II). Nonetheless,
this work demonstrates clearly that activity is not restricted to
simple analogs of DDP, but may be found as well in significantly
different structural classes. In particular, at least one com-
pound having a structure (L^PtCl^-trichloroquinuclidiniumplatin-
um(II)—has exhibited activity comparable to that of DDP.
Acknowledgment : This work was sponsored by the National Cancer
Institute through grants 24543 and 22435.
Literature Cited
1. Rosenberg, B.; Van Camp, L; Trosko, J.E.; Mansour, V.H.
Nature 1969, 222, 385-386.
2. Schaeppi, U.; Hoyman, L.A.; Fleischman, R.W.; Rosenkrantz, H.;
Ilievski, V.; Phelan, R.; Cooney, D.; Davis, R.D. Toxicol-
Appl. Pharmacol. 1973, 25, 230.
3. Rosenberg, B. "Cisplatin: Current Status and New Develop-
ments"; A.W. Prestayko, S.T. Crooke, and S.K. Carter, eds.
Academic Press, Inc., 1980, pp. 9-20.
4. Terzis, A. Inorg. Chem., 1976, 15, 793.
5. Maresca, L.; Natile, G.; Rizzardi, G. Inorg. Chim. Acta,
1980, 38, 137.

13. BROWN ET AL. New Class of Antitumor Pt Complexes111
6. Adeyemo, Α.; Teklu, Y.; Williams, T. Inorg. Chim. Acta, 1981,

51, 19.
7. Mura, P.; Spagna, R.; Zambonelli, L. J. Organometal. Chem.
1977, 142, 403.
8. Roberts, J.J. "Metal Ions in Genetic Information Transfer";
Vol.'3, G.L. Eichlorn and L.G. Marzilli, eds., 273-332
(1981).
9. Denning, R.G.; Venanzi, L.M. J. Chem. Soc. 1963, 3241.

14
Chemical and Biological Activity of Metal
Complexes Containing Dimethyl Sulfoxide
1
NICHOLAS FARRELL
Simon Fraser University, Department of Chemistry,
Burnaby, British Columbia V5A 1S6 Canada
The requirement for inert amine-type ligands in

the coordination sphere is a major feature of the
structure-activity relationships so far delineated
for antineoplastic complexes such as cis-
-[PtCl (NH ) ].
2 3 In the ensuing study of the b i o l o g i -
2
cal activity of other heavy metal complexes, much

emphasis has therefore been placed on amine com-
plexes which are structurally analogous to the o r i -
ginal platinum species. There are few systematic
studies using other ligands or mixed ligand sys-
tems. This contribution summarises the biological
and related chemical activity of complexes contain-
ing dimethylsulfoxide (DMSO).
The scope of the article requires, f i r s t , a
brief summary of the nature of DMSO as a ligand i n
coordination compounds. The discussion is then
divided into simple complexes containing DMSO as the
only donor ligand and the recently-developed mixed
amine-sulfoxide complexes, where DMSO plays the role
of the leaving group.
Nature of DMSO as a Ligand

The alkyl sulfoxides of general formula, R2SO, are examples
of ambidentate ligands which may bind to metal ions through either
the sulfur or oxygen donor atom, the canonical forms being repre-
sented by:
1
Current address: Departamento de Quimica, Universidade Federal de Minas Gérais,
Belo Horizonte MG 30.000, Brazil
0097-6156/83/0209-0279$06.00/0

R R R.
\ \ \
+ +
j;s"=o - s=o - s-o~
R R' R^
Complexes f o r most d-block elements have been prepared and a

recent review (1) updating that of Reynolds ( 2 ) , summarises
these. C o n s i d e r a t i o n s of the hard and s o f t p r o p e r t i e s of the
donor atoms a l l o w us to p r e d i c t t h a t , i n g e n e r a l , s u l f o x i d e s
should bind v i a oxygen to the hard f i r s t - r o w t r a n s i t i o n metals and
v i a s u l f u r to s o f t metals, such as those of Group V I I I . Both
s t e r i c and e l e c t r o n i c e f f e c t s have been shown to d i c t a t e the
choice of donor atom and these e f f e c t s may be summarized: -
S t e r i c E f f e c t s . These may be manifested by the s u l f o x i d e ,

other ligands i n the c o o r d i n a t i o n sphere and the c e n t r a l metal
ion.
e f f e c t of sulfoxide ligand
Whereas [PdCl (DMSO) ] and [PdCl (MBSO) ] (MBSO * 3-methyl-
2 2 2 2
b u t y l s u l f o x i d e ) have both s u l f o x i d e s bound through s u l f u r , the

s t e r i c a l l y more demanding MBSO l i g a n d r e s u l t s i n a trans con
+
f i g u r a t i o n . S i m i l a r l y , [Pd(DMS0)iJ has been shown to have two
sulfur-boung trans to two oxygen-bound s u l f o x i d e s , w h i l e
+
[Pd(MBSO)^] r e s u l t s i n a complex w i t h a l l oxygen-bound l i
gands ( 3 ) .
other l i g a n d s
T h i s i s demonstrated i n complexes of the type
[M(dppe)(DMSO)Cl](A) and [Pd(dppe)(DMSO) ](A) (dppe - l , 2 ( d i -2 2
β β
phenylphosphino)ethane, Μ Pd,Pt, Α non-coordinating anion)
(4) . Bonding through s u l f u r i s d i s f a v o u r e d because of i n t e r a c
t i o n of the methyl groups w i t h the phenyl groups of the bulky
phosphine l i g a n d and, thus, bonding v i a the s t e r i c a l l y - u n r e s t r i c t -
ed oxygen i s p r e f e r r e d .
s i z e of c e n t r a l metal ion
The e f f e c t of the metal i o n may be gauged by comparison of
trans-[FeCl (DMSO)u]+, a l l oxygen-bound ( 5 ) , and c i s -
2
[RuCl (DMSO)i ], w i t h three s u l f u r and one oxygen-bound l i g a n d

2 t
(5) . W i t h i n the same row, the [RuCl3(DMSO)3]" anion contains

three sulfur-bound s u l f o x i d e s i n the fac c o n f i g u r a t i o n ( 7 ) , where
as, i n the i s o e l e c t r o n i c [RhCl3(DMSO)^T7 the s m a l l e r s i z e of the
R h ( l l l ) a l l o w s f o r only two S-bonded ligands (8,9), although minor
amounts of linkage and geometric isomers have been observed i n
s o l u t i o n (10).
E l e c t r o n i c E f f e c t s . A graphic demonstration of how p u r e l y -

e l e c t r o n i c e f f e c t s c o n t r o l the mode of c o o r d i n a t i o n of the
s u l f o x i d e l i g a n d i s demonstrated i n DMSO adducts of rhodium c a r -
boxy l a t e s , [Rh (0 CR)i (DMSO) ] (11,12). The dimeric s t r u c t u r e of
2 2 + 2
the rhodium c a r b o x y l a t e s w i t h b r i d g i n g c a r b o x y l a t e groups has been

confirmed (3) and these r e a d i l y add donor ligands L i n the a x i a l

14. FARRELL Metal Complexes Containing Dimethyl Sulfoxide 281
s
p o s i t i o n s . Where R CH3 or C2H5 s u l f u r c o o r d i n a t i o n i n DMSO
occurs but, upon the replacement of a l k y l by CF3 groups, the h i g h
l y - e l e c t r o n e g a t i v e CF3 s u b s t i t u e n t s force preference o f oxygen-
atom bonding.
Solvent E f f e c t s
The s t e r i c and e l e c t r o n i c e f f e c t s i l l u s t r a t e d are not the

unique determining f a c t o r s of b i n d i n g s i t e s i n s u l f o x i d e complexes
and, as noted, linkage and geometric isomers may e x i s t i n s o l u
t i o n . A f u r t h e r relevant e f f e c t i s that o f s o l v e n t .
The X-ray c r y s t a l s t r u c t u r e of [Ru(DMSO)g ] (BFi# ) shows three 2
sulfur-bound fac t o three oxygen-bound ligands (14), confirming

the s t r u c t u r e proposed from H N.M.R. studies i n CD2CI2. However,
i n D2O, the *H N.M.R. measurements show four S-bound ligands and

two free DMSO molecules a r i s i n g from s u b s t i t u t i o n o f the
e a s i l y - d i s p l a c e d O-bound s u l f o x i d e s by s o l v e n t :
2 + 2 +
[Ru(DMSO)i+ (DMS0)2 ] [Ru(DMSO\ ( H 0 ) ] 2 2 (1)
Thus, an aqueous medium favours S-bonding and that t h i s i s a

general c h a r a c t e r i s t i c was confirmed i n the case o f platinum. The
2+
[ P t i D M S O ) ^ ] c a t i o n i s considered to have two S- and two O-bound
ligands i n a c i s arrangement, as judged by I.R. and H N.M.R.
s t u d i e s ( 3 ) , and analogy with the Pd analog whose s t r u c t u r e i s now
known (15). In D2O, only a trace of O-bonded or free DMSO i s ob
served, i n d i c a t i n g that the predominant species i n aqueous s o l u
t i o n contains a l l S-bound s u l f o x i d e s , the only example so f a r o f a
t o t a l l y S-bound homoleptic s u l f o x i d e complex. A r a t i o n a l e f o r
t h i s behaviour may be found by c o n s i d e r i n g that S-bound species
w i l l allow f o r continued Η-bonding i n t e r a c t i o n with H2O and, i n
deed, that the Η-bonding i n aqueous s o l u t i o n may reduce the
m e t a l - a f f i n i t y o f the oxygen atom.
A f u r t h e r feature of the s o l u t i o n chemistry observed i n
these systems i s t h e i r s o l v o l y s i s behaviour. The O-bound s u l f o x
ides are e a s i l y d i s p l a c e d by solvent molecules; h y d r o l y s i s pro
ducts are immediately formed i n water or even i n wet non-aqueous
s o l v e n t s . Recently, slower h y d r o l y s i s o f the sulfur-bound l i g a n d
has a l s o been observed (16). The H N.M.R. spectrum o f
2+
[Ru(DMSO)g] i n D2O shows a t h r e e - l i n e p a t t e r n at 6.51, 6.58 and
6.62τ and a s i n g l e t a 7.4τ, w i t h an i n t e n s i t y r a t i o o f 2:1. Upon
e
r a i s i n g the temperature, the r a t i o v a r i e s u n t i l , at 82 C, the ob
served i n t e g r a t i o n i s 1:1, the basic t h r e e - l i n e p a t t e r n f o r the
methyl resonances of the S-bound ligands being u n a l t e r e d . The
spectrum i s not reversed upon c o o l i n g t o room temperature. The
data are r e a d i l y explained:
2 + R Te 2 +
[Ru(DMSO)i (DMSO)i ]
t f 4- [Ru(DMS0)i, ( H 0 ) ]
2 2
2+
[Ru(DMSO) (H 0)3]
3 2

A f u r t h e r , unusual base l a b i l i s a t i o n was observed at 32° i n 2%

0D"/D20, where only two 1:1 s i n g l e t s at 6.8τ and 7.4τ are found.
Thus, both temperature and b a s i c i t y may l a b i l i s e the S-bound l i
gand .
2+
For the [ P t i D M S O ) ^ ] c a t i o n , s i m i l a r behaviour i s also
e
shown; slow h y d r o l y s i s of one S-bound l i g a n d a f t e r 24 h at 32 C
e
being observed o r , upon h e a t i n g , to 82 C. The interconversions
may be summarised:

Thus, a p i c t u r e emerges of s u l f o x i d e complexes i n aqueous

s o l u t i o n , where i n i t i a l f a s t h y d r o l y s i s o f O-bound l i g a n d o c c u r s ,
followed by a slow displacement of S-bound groups, the exact
species formed being under i n v e s t i g a t i o n . (At time of w r i t i n g , a
complex corresponding t o [Pt2 (DMSO)g ] (BFi^ has been i s o l a t e d
from the h y d r o l y s i s of the monomeric species.) That t h i s behav
i o u r i s not r e s t r i c t e d t o homoleptic complexes i s evidenced by r e
s u l t s on the more l a b i l e Rh and Pd systems. Thus, [PdCl2(DMSO)2]
i n D2O immediately shows 1:1 s i n g l e t s at 6.45 and 7.4τ and a s o l u
t i o n of mer-[RhCl3(DMSO)3] shows i n i t i a l loss of the unique 0-
bound l i g a n d , followed by l o s s o f one S-bound s u l f o x i d e w i t h i n
hours:
trans-PdCl (DMSO) 2 2 [PdCl (DMSO)] + 2DMS0

2 2 (2)
t R 1 !
[RhCl (DMSO) (DMSO)]
3 2 [RhCl (DMS0) (H 0] ^ ' '
3 2 2
[RhCl (DMSO)(H 0) ]
3 2 2 (3)
Displacement o f S-bound s u l f o x i d e has been observed i n non-aqueous

solvents f o r these metals (17,18).
K i n e t i c and Thermodynamic Studies
The k i n e t i c c i s and trans e f f e c t s and the thermodynamic c i s

and trans i n f l u e n c e s of DMSO i n platinum c o o r d i n a t i o n chemistry
have been e x t e n s i v e l y i n v e s t i g a t e d .
From the study of a c i d h y d r o l y s i s o f the complex
K[PtCl3(DMSO)], formed r e a d i l y by the r e a c t i o n o f one equivalent
of DMSO w i t h K2PtCli i n water, E l d i n g (19) concluded that the c i s
f
and trans i n f l u e n c e s were o f the magnitude of 02Η^, a point made

by Kukushkin i n comparison of the DMSO complex w i t h Zeise's s a l t ,
K[Pt(C2Hi )Cl3] ( 2 0 ) . These i n f l u e n c e s are small compared to the
+
k i n e t i c e f f e c t s which are i n the order:
trans e f f f e c t C HV > DMSO

2 > C I " > NH > H 0 3 2
11 6
(10 : 2 x 1 0 : 330 : 200 : 1)
cis effect DMSO > H 0 « NH > C I " > 0 Η%
2 3 2
(5 : 1 : 1 : 0.4 : 0.05)
Thus, DMSO i s considered to have an intermediate trans e f f e c t

and a r e l a t i v e l y large c i s e f f e c t , compared t o other l i g a n d s . The
approximate order o f magnitude d i f f e r e n c e i n the c i s e f f e c t s o f
DMSO and NH3 has a l s o been confirmed i n the f o l l o w i n g system ( 2 1 ) :

L - DMSO, NH ; Nuc = NH , N R% , X", SC(NH )2

3 3 2 2
This r e l e v a n t f i n d i n g i s a t t r i b u t e d t o the greater n u c l e o p h i l i c

d i s c r i m i n a t i o n f a c t o r of DMSO i n i t s complex, whereas the trans
effect i s attributed to t r a n s i t i o n - s t a t e s t a b i l i s a t i o n a r i s i n g
from the π-acceptor p r o p e r t i e s of the l i g a n d , S-bound DMSO being
considered a b e t t e r σ-donor and π-acceptor than ethylene (22).
The l a b i l i t y o f S-bound DMSO was studied by measuring the
+
k i n e t i c s o f l i g a n d replacement i n [ P t ( d i e n ) L ] ( d i e n • 1,5-
s
diamino-3-aza-pentane; L DMSO, H 0 ) . 2 The H 0 l i g a n d i s ap
2
proximately three orders o f magnitude more l a b i l e than DMSO and a

l a b i l i t y sequence, derived by c o m p i l a t i o n of the data from the r e
action:
n + 2 +
[Pt(dien)L] + SC(NH )2 2 [Pt(dien)SC(NH ) ] 2 2 + L (or L") (4)
(n • 1,2) was suggested (23):
H 0 > I " > Br > CI > DMSO « SCN~ > N ~ > N 0 "
2 3 2
B i o l o g i c a l A c t i v i t y o f M e t a l - D i m e t h y l s u l f o x i d e Complexes
The most comprehensively studied complex i s c i s -

[ R u C l (DMSO)i* ]. I n a s e r i e s o f s t u d i e s comparing i t s b i o l o g i c a l
2
a c t i v i t y w i t h that o f c i s - [ P t C l ( N H ) ] , the ruthenium species

2 3 2
was shown t o produce filamentous growth i n E. c o l i and induce λ

prophage (24). I t i s mutagenic (25) and has i n v i v o anti-tumour
a c t i v i t y (26).
The comparisons w i t h the platinum complex showed t h a t , a t
10 μg/mL, filamentous growth was an order o f magnitude l e s s f o r
[RuCl (DMSO)iJ, whereas, at 100 μg/mL, 100% f i l a m e n t a t i o n was
2
found, the platinum complex i n h i b i t i n g growth at t h i s concentra

+
t i o n . Both complexes are mutagenic f o r uvr Β and u v r s t r a i n s o f
T. thypimurium c a r r y i n g the His G46 missense mutation, s p e c i f i c
for base p a i r s u b s t i t u t i o n mutagens. However, some d i f f e r e n c e s

i n response were noted, which may r e f l e c t a d i f f e r e n c e i n mode o f

a c t i o n at the DNA l e v e l .
The s i m i l a r i t y i n a c t i v i t y i s a l s o demonstrated by i n h i b i -
t i o n of the i n v i t r o b l a s t o g e n i c response of human p e r i p h e r a l
lymphocytes, stimulated by mixed c u l t u r e of phytohaemaglutinin
(PHA) (27).
These s t u d i e s culminated i n a comparison of the anti-tumour
a c t i v i t y of the complexes. The ruthenium compound was shown to
be a c t i v e at doses of 400 mg/kg. The animal numbers involved
are, however, small and i t would be unwise to e x t r a p o l a t e f u r -
t h e r . The dose l e v e l s p o s s i b l e f o r [RuCl2(DMSO)^] are noteworthy
and i n d i c a t e a h i g h LD50 f o r t h i s complex.
These f i n d i n g s prompted us to undertake a survey of the b i o -
l o g i c a l a c t i v i t y of the other M-DMSO complexes (28). The method
used was c y t o t o x i c assay, using R a j i B u r k i t t lymphoma c e l l s and L

5178Y c e l l s . These p a r t i c u l a r assay r e s u l t s , i n g e n e r a l , c o r r e -
l a t e w e l l w i t h observed i n v i v o anti-tumour a c t i v i t y and c i s -
[PtCl2(NH3)2l was used f o r comparison.
The r e s u l t s f o r the Pt and Ru complexes are shown i n F i g u r e s
1 and 2. For the L 5178Y case, the approximate order of magni-
tude d i f f e r e n c e i n a c t i v i t y between cis-[RuCl2 (DMS0)i+ ] and c i s -
[PtCl2(NH3)2l was confirmed from counts a f t e r 24 h. Using R a j i
B u r k i t t lymphoma c e l l s , at 24 and 48 h, some d i f f e r e n c e s were
noted. Increased time d i d not improve c e l l k i l l f o r the r u t h e n i -
um complex, a f a c t which may be a t t r i b u t e d to inherent lack of
c y t o t o x i c i t y or a f a i l u r e to enter the c e l l s i n the f i r s t p l a c e .
+
S i m i l a r behaviour was found f o r [Ru(DMS0)g] , [RhCl3(DMS0)3],
2+ 2+
[RhCl(DMSO) ] 5 and [ P t i D M S O ) ^ ] ( c a t i o n i c species as t h e i r BF^""
s a l t s ) , none of the complexes being more a c t i v e than the o r i g i n a l
ruthenium species and the complexes do not appear to be promising
candidates f o r anti-tumour a c t i v i t y . A rhodium complex mer-
[RhCl3(DMSO)py2l has been reported as having remarkable a c t i v i t y
i n v i t r o a g a i n s t P388 leukemia and KB carcinoma. The r o l e o f
DMSO i n the complex i s not obvious but the s i m i l a r i t y i n s t r u c -
ture to the b a c t e r i o s t a t i c s e r i e s (30) [ R h A ^ C ^ l C l (A • aromatic
h e t e r o c y c l i c ; e.g., p y r i d i n e ) should be noted.
I n t e r a c t i o n of Metal-DMSO Complexes w i t h N u c l e i c Acids and

N u c l e i c A c i d Bases
Apart from the understanding of the mechanism of anti-tumour

a c t i o n of c i s - [ P t C l 2 ( N H ) ] , a major i n t e r e s t i n the area o f
3 2
n u c l e i c acid-metal i o n i n t e r a c t i o n s i s the p o s s i b i l i t y of se-

quence d e t e r m i n a t i o n , using s p e c i f i c b i n d i n g of metal complexes.
The s p e c i f i c i n t e r a c t i o n of ^ P t C l i * w i t h DNA bases was f i r s t
reported i n 1967 (31). The n e c e s s i t y f o r a monofunctional agent
to dominate s i d e r e a c t i o n s , such as c r o s s - l i n k i n g , prompted the
use of [PtCl3(DMS0)]~ u t i l i s i n g the trans i n f l u e n c e of the s u l f o x -
ide l i g a n d . Reaction c o n d i t i o n s w i t h DNA and RNA were found, i n
which the complex could be used as a d i f f e r e n t i a l s t a i n i n g agent



for adenine and guanine residues i n both p o l y n u c l e o t i d e s (32).

The r e a c t i o n s found are worthy of note i n that b i n d i n g t o both py
r i m i d i n e and purine bases bases was found, the d i f f e r e n t i a l s t a i n
ing being made p o s s i b l e by b i n d i n g of two platinum atoms per aden
ine residue a t both pH 6.0 and pH 7.5, while at the lower pH one
platinum and a t high pH two platinum atoms were bound t o guanine,
a l b e i t i n a much slower r e a c t i o n .
Neither t h i s complex nor i t s n e u t r a l analogue,
cis-[PtCl2(DMS0)21 » are a c t i v e against tumours and t h i s fact has
prompted s t u d i e s w i t h simple bases i n attempts to explore the d i f
ference between the amine and s u l f o x i d e l i g a n d . Both complexes
react w i t h a v a r i e t y o f purines and p y r i m i d i n e s , the a n i o n i c com
plex r e a c t i n g more r e a d i l y . A g e n e r a l i z e d scheme may be w r i t t e n :
JPtCl (R2SO)]-
3
Ad
[{PtCl R2SO} Ad]
2 2 [PtCl2(DMS0)Nuc]
Ad ^Nuc β
Gua, Xan
Cytd
The b i n d i n g o f two Pt atoms per adenine moiety v i a an Νχ, N7 dimer

was confirmed by an X-ray c r y s t a l s t r u c t u r e determination o f
X
[{PtCl (DIPSO)} (9-MeAde)] (33) and by H N.M.R. observations o f
2 2
the r e a c t i o n between £Îs-[PtCl2(DMSO)2] and adenosine (34). The

other b i n d i n g s i t e s are summarized i n Table I , along with r e l e v a n t
s t u d i e s on the octahedral ruthenium and rhodium systems.
The anti-tumour a c t i v i t y of cis-[RuCl2(DMS0)i»] prompted us
to study the r e a c t i v i t y o f t h i s complex w i t h simple bases (35).
An i n t e r e s t i n g d i f f e r e n c e emerges between the octahedral and
square-planar cases; f o r adenine or adenosine only monodentate
b i n d i n g v i a N7 i s found f o r both Ru and i t s Rh analogue (Table
I ) , w i t h a f u r t h e r d i s t i n c t preference f o r adenine over guanine
b i n d i n g . Hydrogen-bonding i n t e r a c t i o n s between the e x o c y c l i c
groups of purine and pyrimidine bases and l i g a n d atoms may
d i c t a t e s p e c i f i c i t y and b i n d i n g s i t e s i n metal complex-base i n t e r -
a c t i o n s (36). By analogy w i t h the f a c t o r s d e l i n e a t e d , H-bonding
between the e x o c y c l i c 6-NH2 group of adenine and the DMSO oxygen
atoms or c h l o r i n e atoms (both Η-bond acceptors) i n the octahedral
sphere may d i c t a t e s p e c i f i c i t y , these i n t e r a c t i o n s being minimised
i n a square-planar complex upon removal o f the a x i a l l i g a n d s .
These i n t e r p r e t a t i o n s await d e f i n i t i v e s t r u c t u r a l confirma
t i o n . C e r t a i n l y , DMSO as l i g a n d i n the ruthenium complexes d i c
tates a d i f f e r e n t behaviour than that observed by C l a r k and
2+
Taube i n t h e i r study o f the [ R u ( N H ) s C l ]3 system ( 7 ) . F u r t h e r ,
depending on the s t r u c t u r e , displacement of c h l o r i d e and both S-
and O-bound s u l f o x i d e has been observed i n these r e a c t i o n s w i t h
the simple n u c l e i c a c i d bases.

Table I
Reaction Products and Binding S i t e s of M-DMSO Complexes
w i t h Nucleobases
Site
çis-[PtCl (DMS) ]
2 2 Adenosine trans-ffPtCl-,ίΟΜςη^Ι AA ι
N L 7 3 4
. P t C l DIPSO)]-
3 9-Me Adenie ïftnT- Pt"dllllÀ î î°l Λ, , ' *
(D )( Cy0]
7 8 1 1 1 6
cis- « 1 2S5! Adfni e " S) îcT 2 7 3 4 3 5
3 3 Adenine fîf_
i£ç- RuCl (DMSO) ]-
l^â^ (S)
A^HÎM^
** »·
-- Cl3( M 0)
[ R h D S 3 ] Adenosine J £ 35

Chemical and B i o l o g i c a l A c t i v i t y o f c i s - [ P t ( a m i n e ) ( D M S 0 ) 1 ( A )
2 2 2
A major o b s t a c l e t o the more widespread c l i n i c a l use o f c i s -

[PtCl (NH3> ] i s i t s low s o l u b i l i t y and high t o x i c i t y . Many "se-
2 2
cond-generation" analogues have s u b s t a n t i a l l y improved t o x i c i t i e s

but s o l u b i l i t y s t i l l remains a handicap.
A novel approach t o t h i s problem suggested i t s e l f from our
work on h y d r o l y s i s of S-bound s u l f o x i d e . The unique p r o p e r t i e s
of DMSO i n i t s pharmacological a c t i o n s , such as membrane t r a n s
port and membrane p e n e t r a t i o n , are w e l l known (41) and the water-
s o l u b i l i s i n g p r o p e r t i e s i n i t s metal complexes was r e a d i l y appar
ent from the good aqueous s o l u b i l i t y of the M-DMSO s p e c i e s .
F u r t h e r , the l a b i l i t y seemed q u a l i t a t i v e l y of the order o f h a l i d e
(a point confirmed q u a n t i t a t i v e l y during the course of these ex
periments (23)) and these observations prompted us t o probe the

following structure-activity relationship:
2 +
c i s - [ P t C l (amine) ]
2 2 ·* [Pt(H 0) (amine) ]
2 2 2
2
H 0 2
cis-[Pt(DMSO)2(amine) ] 2 [Pt(H 0) (amine) ]

2 2 2
to study the s o l u b i l i t y of these l a t t e r species and e s p e c i a l l y i f

h y d r o l y s i s o f DMSO would occur. C l e a r l y , the paramount r e q u i r e
ment i s f o r a S-bonded l i g a n d . F u r t h e r , weak non-coordinating
anions, t o avoid formation of c h l o r o s p e c i e s , are deemed neces
sary.
The r e s u l t s obtained t o date w i l l be i l l u s t r a t e d using com
plexes o f 1,2-diaminocyclohexane (dac). A d d i t i o n o f t h i s c h e l a t
+
ing diamine t o a methanolic s o l u t i o n of [Pt(DMS0)iJ readily
g i v e s , upon r e d u c t i o n to half-volume and a d d i t i o n o f e t h e r , the
+
[Pt(dac)(DMSO) ] 2 c a t i o n w i t h both DMSO l i g a n d s bound throueh
1
s u l f u r (42) (v(NH) = 3280, 3220 cm" ; v(S0) = 1150, 1070 cni ;
x{(CH ) SO} - 6.4τ i n D 0; J(Pt-H) - 26.0 Hz). The formation o f
3 2 2
t h i s c a t i o n was confirmed by r e a c t i o n of [ P t C l ( d a c ) ] w i t h two

2
e q u i v a l e n t s of a s i l v e r s a l t i n DMSO and by r e a c t i o n of c i s -
[ P t C l ( D M S 0 ) ] w i t h two e q u i v a l e n t s of a s i l v e r s a l t i n methanol,
2 2
followed by a d d i t i o n of a methanolic s o l u t i o n of the diamine.

2+
The [Pt(DMS0)i|] r e a c t i o n i s , however, the c l e a n e s t and i s
e a s i l y extendable t o other amines f o r a range of counter-anions:
The complexes are a l l remarkably s o l u b l e i n water and, because the

aqueous medium i s avoided i n t h e i r p r e p a r a t i o n , are e a s i l y

obtained as pure c r y s t a l l i n e s o l i d s . T o x i c i t y s t u d i e s show an

LD50 s i g n i f i c a n t l y greater than the parent d i c h l o r o d e r i v a -
t i v e s . The s o l u b i l i t y and LD50 values f o r some complexes o f
1,2-diaminocyclohexane are shown i n Table I I .
Table I I . S o l u b i l i t y and T o x i c i t y o f [ P t ( l , 2 - d a c ) X ]2
X S o l u b i l i t y (mg/mL) LD (mg/kg i p ) Reference

50
CI <0.5 28 42
S0i*(=X ) 10 22.5 43
malonato <0.5 36.0 43

,
4 carboxyphthalato <0.1 76.5 43
25 (1.5% NaHC0 ) 3
DMSO ( B F ) H >200 140 42
Of the complexes shown, three ( s u l f a t o , malonato and c a r -

boxy l p h t h a l a t o ) are deemed t o be promising candidates f o r f u l l
c l i n i c a l use as anti-tumour agents (44), p a r t l y because o f t h e i r
low incidence of r e n a l t o x i c i t y (the l i m i t i n g s i d e - e f f e c t o f the
o r i g i n a l c i s - [ P t C l ( N H 3 ) ] ) and because of t h e i r a c t i v i t y against
2 2
c i s - p l a t i n - r e s i s t a n t tumours (45). Only the malonato d e r i v a t i v e

presents a higher LD50 than the DMSO complex; the s o l u b i l i t y i s ,
however, 10 -10 times l e s s .
The remarkable s o l u b i l i t y of these mixed amine-sulfoxide c a -
t i o n s i s a general property. Thus, f o r the NH3 d e r i v a t i v e (coun-
t e r - a n i o n NO3"), s o l u b i l i t i e s of 75-100 mg/mL are e a s i l y o b t a i n -
able. This i s p a r t i c u l a r l y u s e f u l i n the case o f the a l i c y c l i c
amines developed by Tobe (46). The complex c i s - [ P t C l ( c h a ) ] has 2 2
one o f the highest t h e r a p e u t i c i n d i c e s o f any o f the platinum com-

plexes but i s one of the most i n s o l u b l e of a l l d e r i v a t i v e s . The
complex [Pt(cha) (DMSO) ](NO3) has an aqueous s o l u b i l i t y o f
2 2 2
«5 mg/mL which now compares favourably w i t h £Îs-[PtCl (NH3) ] 2 2
(1 mg/mL).
The behaviour of the [Pt(amine) (DMSO) ] species i n aqueous
2 2
s o l u t i o n p a r a l l e l s that o f [Pt(DMS0)i*] . Slow d i s s o c i a t i o n

e
(18 h, 25 C) of one DMSO l i g a n d occurs. S i m i l a r d i s s o c i a t i o n oc-
e
curs upon heating t o 82 C, a l s o v e r i f i e d f o r c i s -
[Pt(NH3) (DMSO) ](N03) . Thus, the k i n e t i c i n e r t n e s s of the
2 2 2
amine l i g a n d s , and perhaps the mutual c i s e f f e c t s of the DMSO l i -

gands, favour DMSO h y d r o l y s i s , even though t h i s S-bonded l i g a n d
would be considered to have a greater t r a n s - i n f l u e n c e . I t i s es-
p e c i a l l y i n t e r e s t i n g t o note that use of DMSO as a solvent f o r
c i s - [ P t C l ( N H 3 ) ] r e s u l t s i n a complex mixture (47), whereas i n -
2 2
c o r p o r a t i o n of DMSO i n a w e l l - d e f i n e d molecular species g r e a t l y

enhances the w a t e r - s o l u b i l i t y of the Pt(amine)2 moiety which r e -

mains i n t a c t even upon i n i t i a l h y d r o l y s i s . The
+
[Pt(amine)2(DMSO>2] c a t i o n s may be considered t o be molecular
slow-release systems.
In the quest f o r improved analogues of cis-[PtCl2(NH3>2l,
the major c o n s i d e r a t i o n s i n the choice of s u i t a b l e analogs are
s o l u b i l i t y , t o x i c i t y and a c t i v i t y . R e s u l t s are not yet a v a i l a b l e
on anti-tumour t e s t s of t h i s s e r i e s ; however, r e s u l t s have been
obtained on other systems and, indeed, the general a c t i v i t y of
1,2-dac complexes (44) bodes w e l l i n t h i s p a r t i c u l a r case.
Platinum-Metal Complexes i n P a r a s i t i c Diseases
In a p a r a l l e l study, we have i n v e s t i g a t e d the a c t i v i t y o f

metal complexes i n p a r a s i t i c diseases and, e s p e c i a l l y , trypano-

s o m i a s i s , r e s p o n s i b l e f o r s l e e p i n g s i c k n e s s i n man. Of the p a r a -
s i t i c d i s e a s e s , t h i s does not now present a major problem,
compared to m a l a r i a , f i l a r i a s i s and s c h i s t o s o m i a s i s , but the p r e -
valence of the disease has increased i n l a t t e r years due t o drama-
t i c changes i n the p o l i t i c a l and economic c l i m a t e i n the t r o p i c a l
areas (48). A number of platinum d e r i v a t i v e s has been found t o be
a c t i v e _in v i t r o and i n v i v o against T^ rhodesiense and the p l a t i n -
um complexes, as a f a m i l y , may be considered a c t i v e trypanocides
(49). The i n v i t r o r e s u l t s of s e l e c t e d complexes (Table I I I )
shows that the DMSO and c h l o r o d e r i v a t i v e s of the Pt(dac) complex
show e s s e n t i a l l y the same a c t i v i t y .
Table I I I . B i o l o g i c a l A c t i v i t y of Trypanocidal Platinum Complexes
Trypanocidal LD50 (mg/kg)

Complex titre* (single
in v i t r o intraperitone-
aldose; mice)
Motility Abolition
of
infectivity
Total Partial
cis-[PtCl (NH ) ]*
2 3 2
3 <3 5 13
ÎPtCl2(dac)] 3 3 5 28 (17.5-44.8)
[Pt(dac)(DMSO) ](BFi ) 2 t 2
3 3 5 140 (107-183)
s
Titre logio[molarity]" ( i . e . , 3 • mM). Values <3 have no
significance.
A r e p r e s e n t a t i v e trypanocide (Ethidium bromide) gives values of 4,

4 and 5.
The s i m i l a r i t y of the metabolism of A f r i c a n typanosomes t o
that of some tumour c e l l s has prompted the c o r r e l a t i o n between

anti-trypanosomal and anti-tumour properties of drugs and, indeed,

a good correspondence exists (51,52). These results place the
platinum complexes in this category, also demonstrated by Steck et
al. (63) for cis-[PtCl2(NH3)2], a further demonstration of their
general pharmacological utility. The advantages of trypanosomes
as tumour cell models and biochemical probes has been emphasised
(54,55) and should not be under-emphasised in the case of heavy
metal complexes. Indeed, ruthenium complexes, such as
[RuCl(NH3)5] + and Ruthenium Red, also show activity with roughly
the order of magnitude difference observed in tumour systems
(56). A series of complexes is now being tested further under
the auspices of the World Health Organisation.
Interaction of [Pt(amine)2(DMSO)2]2+ with Nucleic Acid Bases

Despite the extensive data accumulated on the anti-tumour ac

tivity of Pt-amine complexes, few model studies with simple bases
and dinucleotides have been reported with amines, other than NH3
and ethylene-diamine. The complexes cis-[Pt(amine)2(DMS0)2] +
readily react with bases such as guanosine, displacing a DMSO
molecule. Thus, the series is useful for studying the generality
of intra-ligand Η-bonding interactions delineated in earlier
studies but for a much wider range of amines.
Summary
Dimethylsulfoxide, as a ligand, has been shown to possess

many of the properties desirable for metal-based chemotherapeutic
agents. Complexes containing DMSO, in place of amine, do not gen
erally exhibit much activity but consideration of DMSO as a leav
ing group and substitution for CI in the cis-[Pt(amine)2X2l unit
results in more water-soluble, less toxic derivatives. Activity
has been demonstrated in trypanosomiasis for a series of complex
es. In chemical studies, DMSO, as a ligand, may be displaced by
nucleobases, the products and binding sites being dictated by the
nature of the starting complex.
Legend of Symbols
DMSO β dimethylsulfoxide
DIPSO β diisopropylsulfoxide
DMSO or S a S-bound sulfoxide in a metal complex
DMS£ or 0 - O-bound sulfoxide
dac β 1,2-diaminocyclohexane (as a mixture of isomers)
en. = 1,2-diaminoethane
cha - eyelohexylamine
Ad • substituted adenine

294
Acknowledgments
I thank Johnson-Matthey and Co. Ltd. for a loan of precious
metal salts. The financial support of CNPq, Brasil and Simon
Fraser University is acknowledged. Collaboration with
Prof J; Williamson on the trypanocidic properties of platinum com
plexes is especially acknowledged as is that of Prof. J.M. Goldie
for the crytotoxicity studies.
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22. Braddock, P.D.; Romeo, R.; Tobe, M.L. Inorg. Chem. 1974, 13,
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23. Romeo, R.; Cusuraano, M. Inorg. Chim. Acta 1981, 49, 167.

14. FARRELL Metal Complexes Containing Dimethyl Sulfoxide
24. Monti-Bragadin, C.; Ramani, L . ; Samer, L . : Mestroni, G.;

Zassinorich, G. Antimicr. Ag. Chemother. 1975, 7, 825.
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28. Farrell, N . ; Goldie, J.M. unpublished results.
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Gylys, G.A. "Cisplatin, Curent Status and New Developments",
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49. Farrell, N . ; Vargas, M.D.; McLaren, D.J.; Williamson, J.

Abstract A19, International Conference on the Chemistry of
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Relationships", (Van der Bossche, H., Ed.), North-Holland,
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15
D e t e r m i n a t i o n a n d L o c a t i o n of P l a t i n u m M e t a l s
in Biological Samples by N e u t r o n Activation and
Microprobe Analyses
M. E. FARAGO and P. J. PARSONS

Bedford College, Chemistry Department, Regent's Park, London NW1 4NS England
Instrumental Neutron Activation Analysis (INAA) was

applied to the determination of the platinum metals
as part of a study of their uptake, accumulation and
toxicity in plants. Long irradiations are described
for iridium, osmium and ruthenium determinations in
specially grown plant materials. An interference in
the determination of platinum in plant matrices by
this method is reported also. Short irradiations,
utilising thermal and epithermal fluxes are
investigated for rhodium and palladium determinations,
with further studies using cyclic activation analysis.
Water hyacinths (Eichhornia crassipes MART Solms)
were grown in nutrient solutions containing platinum
2-
as PtCl6 . The roots were examined using electron
microscopes equipped with X-ray microanalysis
f a c i l i t i e s . Platinum deposits were detected and
located within the cortical layers of the root.
Several studies have demonstrated already the effects of the
platinum group metals on plants and that the metals can be taken
up into the tissues either from soil or from solution in hydro-
ponic experiments (1-5). The principal aims of this study are
summarised:
(1) to investigate whether aquatic plants, such as the water
hyacinth, Eichhornia erassïpes, are able to assimilate
soluble complexes of the platinum group metals from a
nutrient solution;
(2) to develop sensitive methods of analysis for the platinum
metals in bio-materials;
(3) to characterise the sites of storage and the mode of chemical
binding of the metals within the tissues;
(4) to examine the biological effects of platinum group metals
on the plants.
In this paper we report on the results of an investigation
into the use of (a) Instrumental Neutron Activation Analysis (INAA)
for the determination of platinum group metals in plants, and
0097-6156/83/0209-0297$06.00/0

(b) E l e c t r o n Microscopy w i t h X-ray Microprobe A n a l y s i s f o r the

l o c a t i o n o f platinum d e p o s i t s i n p l a n t r o o t s .
A n a l y t i c a l Techniques
A wide range of techniques i s a v a i l a b l e f o r the q u a n t i t a t i v e

determination of the platinum group metals (Table I ) . However,
there i s no NBS B i o l o g i c a l Standard Reference m a t e r i a l c o n t a i n i n g
c e r t i f i e d l e v e l s of the platinum group metals f o r the comparison
of a n a l y t i c a l techniques. For t h i s reason, p l a n t m a t e r i a l s were
s p e c i a l l y prepared, each o f which contained one of the platinum
group metals. The f r e e f l o a t i n g a q u a t i c p l a n t , water h y a c i n t h
(Eiehhovnia erassipes MART Solms) was grown i n h a l f s t r e n g t h
n u t r i e n t s o l u t i o n c o n t a i n i n g one o f the platinum group metals as
a s o l u b l e complex. Each metal was s u p p l i e d a t two c o n c e n t r a t i o n s ,

f f f f
designated as H l and L 0 .
A f t e r two week's growth, the p l a n t s were harvested, washed,
d r i e d and homogenised. I n t h i s way a number of r e p r e s e n t a t i v e
samples were obtained.
Non-destructive methods such as INAA o r PIXE remove the need
to o x i d i s e organic matter p r i o r to metal determination. I n some
f f
cases i t i s p r e f e r a b l e t o a s h before a n a l y s i s , s i n c e t h i s
concentrates the metals of i n t e r e s t and may remove an i n t e r f e r i n g
m a t r i x . I n other cases dry ashing can concentrate an i n t e r f e r i n g
element.
Other methods i n Table I have been i n v e s t i g a t e d and w i l l be
reported elsewhere ( 6 ) . F o r INAA i n t h i s work standards were
prepared by s p i k i n g Bowen's k a l e w i t h platinum metals.
Neutron A c t i v a t i o n A n a l y s i s
The use of a c t i v a t i o n methods f o r the a n a l y s i s of b i o l o g i c a l

n
m a t e r i a l has been reviewed by Bowen 07)· ^ Instrumental Neutron
A c t i v a t i o n A n a l y s i s , the d r i e d sample i s placed i n the core of a
nuclear r e a c t o r where i t i s bombarded w i t h neutrons. Many of the
elements present i n the sample undergo n u c l e a r r e a c t i o n s of which
the most common are the (η, γ) type. The products of these
r e a c t i o n s are r a d i o a c t i v e and decay w i t h the emission of gamma
photons of c h a r a c t e r i s t i c energy.
Although determination of the platinum metals by t h i s method
i s w e l l documented (8-10), those concerning b i o l o g i c a l m a t e r i a l s
are few (11,12). The i r r a d i a t i o n time i n the r e a c t o r i s set
according t o the h a l f - l i f e , t | , of the daughter product (Figure 1)
and the v a r i o u s c h a r a c t e r i s t i c s f o r the r e a c t i o n being monitored,
jLe_. thermal neutron cross s e c t i o n , i s o t o p e abundance and type of
backgrounds. (Table I I ) . I n t h i s work the platinum metals can be
d i v i d e d i n t o the long i r r a d i a t i o n s : P t , I r , Ru, Os: and the short
i r r a d i a t i o n s : Rh, Pd.

Table I . Modern Methods f o r the A n a l y s i s of B i o l o g i c a l M a t e r i a l s

>
Proton Induced X-Ray Emission ^ Materials dried Neutron A c t i v a t i o n A n a l y s i s
Ο
and homogenised (INAA) Ο
(PIXE)
>
Ό
Wet ashing Dry ashing ι

00
X-Ray Spectrometry Nuclear Methods

Atomic Spectroscopy (INAA) §
SX
3
Thermal NAA
Atomic A b s o r p t i o n Atomic Emission X-Ray Fluorescence PIXE
(XRF) Epithermal NAA 3
Flame (FAAS) Inductively Coupled
Plasmas
Flameless (ETA AAS) Cyclic (CAS)
(ICP) 9"

DC Argon Plasma
Atom Emission
AC/DC A r c , Spark (AES) vo
TIME
ACTIVATION DECAY
Figure 1. A typical plot of radioactivity vs. time showing both activation and
decay.

Table I I . Nuclear Data f o r the P l a t i n u m Group Metals
Isotopic Thermal Neutron A c t i v a t e d Isotope

Nuclear R e a c t i o n
Element Nuclide Abundance Cross S e c t i o n
Utilised Half Decay
% σ (barns) Nuclide γ keV
Life Mode
1 0 8 1 0 8
Pd 26.7 Pd (η,γ) mPd 0.2 m Pd 4.69 m IT 188.9
Pd
1 0 6 1 0 6
Pd 27.3 Pd (η,γ) mPd 0.013 m Pd 21.3 s 215.0
1 0 3
R h (η,γ) mRh 11 mRh 4.34 m IT 51.4
1 0 3
Rh Rh 100.0 1 0 3
104 104
R h (η,Ύ) Rh 139 Rh 42.3 s 555.8
1 9 1 1 9 1
192 T 192 T 316.5
Ir lr 38.5 l r (η,Ύ) Ir 300 Ir 74.4 d 3 " EC
468.1
190. 190.
Os Os 26.4 Os (η,γ) Os 3.9 0s 14.6 d β~ 129.4
1 0 2 1 0 2 1 0 3 1 0 3
Ru Ru 31.6 R u (η,γ) Ru 1.4 Ru 38.9 d β~ 497.0
198 p t 199 p t 199 p t

30.8 m (47.960)
198 p t (η,γ)
Pt 7.2 4 β~
199 p t 1 9 9 1 9 9
Au Au 3.15 d 158.37

208.20
Long I r r a d i a t i o n s
Table I I I gives the c o n d i t i o n s , data and r e a c t i o n s f o r the

long i r r a d i a t i o n s of i r i d i u m , osmium and ruthenium. Samples and
standards were i r r a d i a t e d i n the U n i v e r s i t y of London Reactor
12 2 1
under a maximum thermal neutron f l u x of 1.4 χ Ι Ο η cm" s" .
The samples were counted u s i n g a l i t h i u m d r i f t e d germanium
d e t e c t o r (Ortec Inc) l i n k e d to a computer based gamma r a y
spectrometer (Nuclear Data Inc. 6620 M u l t i c h a n n e l A n a l y s e r ) . A
general Neutron A c t i v a t i o n Package w r i t t e n i n FORTRAN IV was
employed t o run a peak search and c a l c u l a t e PGM concentrations
i n the p l a n t samples. The i r r a d i a t i o n of platinum i s a s p e c i a l
case 'and d e t a i l s are given i n Table IV. Various n u c l i d e s emit
γ-rays of s i m i l a r energy, and i n INAA these become a s e r i o u s
i n t e r f e r e n c e i n the determination of platinum i n b i o l o g i c a l

samples.
Table I I I . Neutron A c t i v a t i o n A n a l y s i s : Long I r r a d i a t i o n s
I r , Os and Ru I r r a d i a t e d f o r 1 week; allowed to decay over

2-3 days and counted f o r 30 m (long l i v e d
r a d i o a c t i v e products)
(n> γ) ^ Y 316 keV, 406 keV ^
191, 1 9 2 m
"Tr Ir Pt
37.3% t j - 74d stable
109 in^D , 1 0 3 ^497keV ^

K u
Ru m Rh
31.6% tj-39.8d
»°0. ^ - »lo. Y l 2 9 k 6 V
* »li r
26.4% t^ - 14.6d
Short I r r a d i a t i o n s
Both rhodium and p a l l a d i u m undergo (η, γ) r e a c t i o n s which

r e s u l t i n short l i v e d decay products. Short i r r a d i a t i o n s and
short counting times are thus r e q u i r e d . In b i o l o g i c a l samples
some m a t r i x elements g i v e r i s e to a background of s h o r t l i v e d
decay products. These are not a s e r i o u s problem w i t h long
i r r a d i a t i o n s , where long c o o l i n g periods can reduce t h e i r a c t i v i t y ,
but w i t h short l i v e d products they produce an unfavourable back
ground. Various refinements of Instrumental Neutron A c t i v a t i o n
A n a l y s i s can be used to reduce t h i s i n t e r f e r e n c e i n the
determination of short l i v e d n u c l i d e s .

15. FARAGO A N D PARSONS INAA and Microprobe Analysis 303
Table IV. Determination of Platinum by Neutron A c t i v a t i o n
198 (η, γ) 199 199

p t y p t
Au
7.21% • 30 m γ 158.37 keV, 208.20 keV
t^ = 3.15d
(Long i r r a d i a t i o n ) 198
Au
Interferences :
Nuclide keV
117 14.Od 158.40

m Sn c
4 7 4 7
4 7
Sc 3.43d 159.40 ( v i a T i ( n , p ) S c )
117 _ 158.40
m In 1.9h
1 "mHg
u„ 42 m 158.30
177 6.74d 208.20

Lu
Epithermal I r r a d i a t i o n
Figure 2 gives the general features of a r e a c t o r neutron

spectrum. Most of the a v a i l a b l e neutrons from f i s s i o n are thermal,
w i t h fewer i n the epithermal r e g i o n , and even fewer i n the f a s t
r e g i o n . I n thermal neutron a c t i v a t i o n a n a l y s i s , the whole
r e a c t o r spectrum c o n t r i b u t e s t o the a c t i v a t i o n process. Some
n u c l i d e s have a neutron spectrum which i s s u i t e d t o resonance
absorbance i n the epithermal r e g i o n . By screening out thermal
neutrons, a c t i v a t i o n o f the m a t r i x i s reduced r e l a t i v e t o the
n u c l i d e of i n t e r e s t . Such a f l u x i s p o s s i b l e under a cadmium
f i l t e r . I n t h i s study epithermal i r r a d i a t i o n s were c a r r i e d out
1 2 2 1
under a neutron f l u x of 0.5 χ 1 0 η cm" s " . The samples were
then t r a n s f e r r e d by a f a s t pneumatic system t o a Ge(Li) detector
s i t u a t e d c l o s e t o the r e a c t o r . Gamma s p e c t r a were accumulated
u s i n g a Laben 8000 MCA of 4096 channels. Peak areas and thus
concentrations were c a l c u l a t e d using C o v e l l ' s method (13).

NEUTRON ENERGY/eV
Figure 2. The general features of a reactor neutron spectrum. Key: A, thermal
region; B, epithermal region; and C, fast region.

Cyclic Activation Analysis
C y c l i c a c t i v a t i o n a n a l y s i s i s a recent improvement i n NAA i n

which optimum use i s made of the experimental time a v a i l a b l e f o r
the counting of short l i v e d i s o t o p e s . The s i g n a l to noise r a t i o
f o r the r a d i o n u c l i d e of i n t e r e s t i s enhanced i f the sample i s
i r r a d i a t e d f o r a short p e r i o d of time, allowed to decay, and then
counted f o r a short p e r i o d of time; the sample i s then
f f
r e - i r r a d i a t e d and the process repeated f o r n c y c l e s . F i g u r e 3
gives the v a r i a t i o n i n i s o t o p e a c t i v i t y w i t h time and c y c l e number.
The p r i n c i p l e s and a p p l i c a t i o n s of c y c l i c a c t i v a t i o n a n a l y s i s
have been reviewed by Spyrou (14). C y c l i c a c t i v a t i o n was c a r r i e d
out u s i n g the CAS system of the U n i v e r s i t y of London Reactor
1 2 2 1
where a thermal f l u x of 1.3 χ Ι Ο η cm" s " i s o b t a i n a b l e .
Epithermal f l u x e s i n the r e g i o n are a v a i l a b l e w i t h CAS a l s o . I n

t h i s study the CAS programme i n v o l v e d f i v e c y c l e s , each of 6 0 s
epithermal i r r a d i a t i o n , 1 s decay and f o l l o w e d by 60 s counting
u s i n g a Ge(Li) d e t e c t o r coupled to a Laben 8000 MCA of 4096
channels. Table 5 gives the r e s u l t s of the i n v e s t i g a t i o n of the
determination of rhodium and p a l l a d i u m by v a r i o u s refinements o f
INAA, and Table VI compares the determination of platinum metals
i n p l a n t s by INAA and v a r i o u s techniques.
Table V. Short I r r a d i a t i o n of P l a n t M a t e r i a l c o n t a i n i n g
e i t h e r Rhodium or P a l l a d i u m
Sample Nuclide γ, keV Method Metal Concentration

(yg/g dry wt)
Rh L0 1 0 4
Rh 555.8 A 5.25 (±0.54)
Rh L 0 1 0 4
Rh 555.8 C 4.43 (±0.14)
104 _ 5.24 (±0.03)
Rh L0 mRh 51.4 C
Pd L0 mPd 188.8 Β 4.13 (±1.43)
Pd L0
1 0 9
m T>A
Pd 188.8 C 4.62 (±1.26)
Key: Method A = S i n g l e Thermal I r r a d i a t i o n
Β = S i n g l e Epithermal Irradiation
C = C y c l i c A c t i v a t i o n Epithermal

to
Ο
ACTIVITY ON
SATURATION
3
tfl
H
>
r
ο
X
TIME w
I
Η
>
Μ

Figure 3. Cyclic activation analysis. Variation of induced activity with time and cycle number.
Key: T , cycle period; U, time of irradiation; U» time between end of irradiation and start of count;
t , time of counting; and tj, time between end of counting and start of irradiation. The detected
c >
radiations at each counting period are summed to give a cumulative detector response, D , over
c
Ο
m
the total experimental time. D is maximized when Uv = t«/ and U = t = T/2 (See reference 17
c c
Η
for details). CO
Table V I . A Comparison between INAA and other

Determination Methods
P l a n t Grown i n ETA
Sample INAA PIXE DC A r c
a solution of: AAS
Rh LO Na [RhCl ]
3 6 4.43(±0.14) 3.8 4.22 8
Pd LO [Pd(NH ) Cl ] 3 2 2 4.62(±1.26) 5.6 3.40 1
I r LO Na [IrCl ]
3 6 1.28(±0.02) nd nd nd
Os LO Na [OsCl ]
2 6 1.79(±0.07) nd - -
Ru LO (NH ) [RuCl ]
4 3 6 nd 1.1 nd 0.5
P t LO (NH ) [PtCl ]
4 2 6
5.6 14.7 8
E l e c t r o n Microscopy w i t h X-ray M i c r o a n a l y s i s
When a specimen i s examined w i t h an e l e c t r o n microscope, i t i s

bombarded w i t h a beam o f h i g h v e l o c i t y e l e c t r o n s . As the beams
of e l e c t r o n s bombards the specimen many events take p l a c e , some
e l e c t r o n s are absorbed by the specimen, some pass through, some
are d e f l e c t e d o r s c a t t e r e d . I f one of the i n n e r e l e c t r o n s i s
removed by the e l e c t r o n beam, then an e l e c t r o n f a l l s from a
h i g h e r energy s h e l l i n t o the h o l e t o s t a b i l i s e the atom. When
t h i s happens energy i s emitted as X-rays. Each element has i t s
own c h a r a c t e r i s t i c X-ray spectrum.
Energy D i s p e r s i v e A n a l y s i s
The recent development of s o l i d s t a t e d e t e c t o r s has enabled

h i g h l y e f f i c i e n t X-ray a n a l y s i s t o be c a r r i e d out by energy
d i s p e r s i v e methods. The d e t e c t o r u n i t c o n s i s t s of a s i l i c o n -
l i t h i u m doped c r y s t a l cooled t o l i q u i d n i t r o g e n temperature.
X-rays of a l l energies are processed by a microcomputer
simultaneously and the energy spectrum i s d i s p l a y e d on a CRT
screen.
Wavelength D i s p e r s i v e A n a l y s i s
This method of a n a l y s i s uses a r o t a t i n g c r y s t a l t o d i f f r a c t

the X-rays. The c r y s t a l i s p r e s e t t o a p a r t i c u l a r angle
corresponding t o the d i f f r a c t i o n of s p e c i f i c X-ray wavelengths.
A l l other wavelengths are e l i m i n a t e d from the d e t e c t o r ; the
r e s u l t i s much b e t t e r s p e c t r a l r e s o l u t i o n and lower d e t e c t i o n

l i m i t s , when compared w i t h the energy d i s p e r s i v e systems. The

l a t t e r has the advantage of a r a p i d multi-element determination.
With the wavelength d i s p e r s i v e system, the l o c a t i o n of i n d i v i d u a l
elements may be mapped, e i t h e r on the surface of a sample or i n a
t i s s u e s e c t i o n (15).
3
The roots of water h y a c i n t h t r e a t e d w i t h 10 yg cm" of Pt
a p p l i e d as (NH4)2[PtClô] were i n v e s t i g a t e d u s i n g scanning e l e c t r o n
microscopy (SEM) . The specimens were prepared f o r i n v e s t i g a t i o n
by conventional methods; f i x e d i n g l u t a r a l d e h y d e , and dehydrated
i n a l c o h o l . The cut specimens were d r i e d and mounted on
aluminium stubs, then s p u t t e r coated w i t h e i t h e r carbon or a metal.
Energy d i s p e r s i v e a n a l y s i s (EDXA) of a t y p i c a l r o o t surface of a
t r e a t e d p l a n t was performed, the r e s u l t s are presented i n
F i g u r e 4. EDXA of a s i m i l a r s e c t i o n of a c o n t r o l root grown i n
n u t r i e n t s o l u t i o n without platinum added was a l s o performed. The

s p e c t r a of both samples show A l from the stubs. The c o n t r o l sample
shows a h i g h c o n c e n t r a t i o n of Ca, and s m a l l amounts of Ρ and S.
The platinum-treated root shows Ca a l s o , but a d i s t i n c t l y l a r g e
peak at the platinum Μα p o s i t i o n , 2.05 keV i s seen; t h i s
completely masks the Ρ and S l i n e s seen i n the c o n t r o l sample.
The platinum l i n e at 9.44 keV (La) can a l s o be seen.
A s e c t i o n of root was s t u d i e d u s i n g wavelength d i s p e r s i v e
a n a l y s i s . The d i s t r i b u t i o n of P t , as s t u d i e d u s i n g both the Μα
and La l i n e s show t h a t the platinum i s concentrated i n the c o r t i c a l
l a y e r s of the r o o t , w i t h very l i t t l e passing i n t o the v a s c u l a r
system.
Discussion
INAA of rhodium and p a l l a d i u m by the epithermal c y c l i c

a c t i v a t i o n method i s very s a t i s f a c t o r y . Osmium and i r i d i u m appear
to be s a t i s f a c t o r i l y determined by the long i r r a d i a t i o n method.
However, the most i n t e r e s t i n g element, platinum, appears to
s u f f e r from an i n t e r f e r e n c e which i s s t i l l under i n v e s t i g a t i o n .
Determination on the Pt LO sample f o r platinum using v a r i o u s
techniques are given i n Table VI f o r INAA, the f i g u r e f o r the
d r i e d p l a n t sample was u n u s u a l l y high ( ^ 44 ppm P t ) , and no
agreement was obtained between the 158 keV peak and 208 keV peak.
C o n t r o l water h y a c i n t h s , grown under the same c o n t r o l l e d
hydroponic c o n d i t i o n s , but without platinum added were i r r a d i a t e d
under s i m i l a r c o n d i t i o n s . A l a r g e photopeak at 158 keV r e s u l t e d ,
whereas the 208 keV peak was not v i s i b l e . Thus some i n t e r f e r i n g
n u c l i d e w i t h a photopeak around 158 keV i s present even i n the
c o n t r o l m a t e r i a l . The i n t e r f e r i n g elements are under f u r t h e r
i n v e s t i g a t i o n . This i n t e r f e r e n c e seems to be present both i n
animal and i n p l a n t t i s s u e . LeRoy (16) has compared the r e s u l t s
of the a n a l y s i s of canine h e a r t , l i v e r and muscle by both
e l e c t r o t h e r m a l a t o m i s a t i o n atomic absorption spectroscopy (ETA AAS)
and INAA. I t was found t h a t the r e s u l t s f o r Pt u s i n g INAA

were higher i n a l l cases. A t present i t appears that r e s u l t s of

the a n a l y s i s of b i o l o g i c a l t i s s u e f o r platinum by INAA must be
treated with caution.
Scanning e l e c t r o n microscope s t u d i e s (SEM) have shown that the
energy d i s p e r s i v e system (EDXA) i s s u c c e s s f u l i n d e t e c t i n g h i g h
concentrations (<*v 10,000 ppm Pt) of platinum i n b i o l o g i c a l
t i s s u e s , both the Μα and L a l i n e s being v i s i b l e . The wavelength
d i s p e r s i v e system can be used s u c c e s s f u l l y to map platinum
l o c a t e d i n b i o l o g i c a l samples when l o c a l concentrations are h i g h
enough.
This work has a l s o given an i n d i c a t i o n of the r e l a t i v e
t o x i c i t y of the platinum metals towards the water h y a c i n t h , these
are shown i n Table V I I .
Table V I I . R e l a t i v e T o x i c i t y of the Platinum Group Metals

to the Water Hyacinth (Eiohhomia evassipes)
3
(Applied at a c o n c e n t r a t i o n of 10 mg dm" )
2 2
Pt: c i s [ P t ( N H ) C l ] ^ i [ P t C l ] " > trans[PtOïH^Cl^ »
3 2 2 4 [PtCl ] " 6
2
Pd: cis[Pd(NH ) Cl ] > [PdC^] '
3 2 2
2 +
Ru: [Ru(phen) J > [RuClJ " 3
1 1 1 1 1 I V 1 1
PGM's: Pd * * P t " > Ru > 0s ** Ir" ~ Pt™ » R h "
I t can be seen t h a t as f a r as P t i s concerned, P t ( I I ) i s

f a r more t o x i c than P t ( I V ) . R h ( I I I ) i s r e l a t i v e l y n o n - t o x i c
compared w i t h the other platinum metals. In f a c t low
concentrations of R h ( I I I ) appear to have a t o n i c e f f e c t ( 5 ) . A t
low concentrations t h i s metals i s not p h y t o t o x i c to corn and
tomato ( 5 ) . The growth of Kentucky bluegrass i s s t i m u l a t e d by
s m a l l amounts of p a l l a d i u m ( I I ) c h l o r i d e , whereas at higher
concentrations i t i s p h y t o t o x i c (3). We have found t h a t the
e f f e c t of K2[PtCl4] on the grass Setavia vertioillata (L) P. Beauv.
i s very s i m i l a r , low l e v e l s s t i m u l a t e growth and higher l e v e l s
are p h y t o t o x i c . (Figure 5 ) . We have found a l s o t h a t p l a t i n u m
and rhodium complexes s t i m u l a t e v e g e t a t i v e r e p r o d u c t i o n i n the
water h y a c i n t h (17). Thus i n the case of higher p l a n t s , the
b i - p h a s i c r e a c t i o n , s t i m u l a t i o n at low l e v e l s and p h y t o t o x i c i t y
at h i g h l e v e l s , has been demonstrated f o r compounds of platinum,
p a l l a d i u m and rhodium.

KeV
Figure 4. Energy dispersive x-ray analysis (EDXA ) of water hyacinth root treated
with (NH ) [PtCl ]. This method gives the x-ray lines of all elements in the whole
k 2 s
field of the microscope (X625). Conditions: Pt, Ma = 2.05 keV, La = 9.44 keV;
and Ca,Ka = 3.69 keV.
Figure 5. Effects of platinum on a grass species compared with control. Key: B,

0.5 ppm of K [PtClJ; and C, 2.5 ppm of K [PtClJ.
g t

Admowledgmeiits
We thank Johnson Matthey Research Centre f o r the loan o f

platinum group metals and f o r p r o v i s i o n of EDXA and microprobe
f a c i l i t i e s . We thank Miss M. M i n s k i , U n i v e r s i t y of London
Reactor C e n t r e , A s c o t , f o r co-operation and h e l p f u l d i s c u s s i o n s .
P.J.P. thanks SERC f o r the award f o r a studentship.
Literature Cited
1. Brenchley, W.E. Ann. App. Biol. 1934, 21, 389.
2. Hamner, C.H. Bot. Gaz. 1942, 104, 161.
3. Sarwar, M.; Thirbert, R . J . ; Benedict, W.G. Can. J. Plant Sci.
1970, 50, 91.
4. Pallas, J . E . ; Jones, J.B. Plant and Soil, 1978, 50, 207.

5. Farago, M.E.; Mullen, W.A.; Payne, J.B. Inorg. Chim. Acta,
1979, 34, 151.
6. Farago, M.E.; Parsons, P . J . Analyst, in the press, 1982.
7. Bowen, H.J.M. CRC Crit. Revs. Amalyt. Chem. Dec. 1980, 127.
8. Caramella-Crespi, V . ; Pisani, U . ; Ganzerli-Valentini, M.T.;
Meloni, S.; Maxia, V. J. Radioanalyt. Chem. 1974, 23.
9. Nadkarni, R.A.; Morrison, G.H. J. Radioanalyt. Chem. 1977,
38, 435.
10. Parry, S.J. Analysts, 1980, 105, 1157.
11. Farago, M.E.; Parsons, P . J . Royal Society of Chemistry
Inorganic Biochemistry Discussion Group Meeting, London,
December 1980.
12. Valente, I.M.; Minski, M.J.; Peterson, P . J . ; personal
communication 1981.
13. Covell, D.F. Anal. Chem. 1959, 31, 1785.
14. Spyrou, N.M. J. Radioanalyt. Chem. 1981, 61, 211.
15. Lauchli, Α.; Spurr, A.R.; Epstein, E. Plant Physiol. 1971,
48, 118.
16. LeRoy, A.F.; Whehling, M.L.; Sponseller, H.L.; Friaf, W.S.;
Solomon, R.E.; Dedrick, R.L.; Litterst, C.L.; Gram, T.E.;
Guarino, A.M.; Becker, D.A. Biochem. Med. 1977, 18, 194.
17. Parsons, P . J . Ph.D. Thesis, University of London, 1982.

16
T u m o r I n h i b i t i o n b y M e t a l l o c e n e D i h a l i d e s of
Early Transition Metals
Chemical and Biological Aspects
H. KÖPF
Technische Universität Berlin, Institut für Anorganische und Analytische Chemie,
1 Berlin 12, Federal Republic of Germany
P. KÖPF-MAIER
Freie Universität Berlin, Institut für Anatomie, 1 Berlin 33, Federal Republic of Germany
5
Metallocene dihalides, (η -C H ) MX , represent the
5 5 2 2
first organometallic antitumor agents. Their anti

tumor activity has been established in vitro and in
vivo, e.g. against the Ehrlich ascites tumor, the
lymphoid leukemia L1210, or the lymphocytic leuke
mia P388. Considering the structure-activity rela
tion three statements can be made: (i) There ex
ists a strong dependence of the cytostatic activity
of the metallocene dichlorides (C H ) MCl upon the 5 5 2 2
central metal atom M. ( i i ) Within the titanocene

dihalide system (C H ) TiX the Cl ligands can be
5 5 2 2
replaced by other halide or pseudohalide ligands

without reduction of the tumor-inhibiting potency,
(iii) Chemical modification of the cyclopentadienyl
rings, e.g. mono- or 1,1'-disubstitution with orga
nic residues, leads to a diminution of the cytosta
tic activity in dependence on the degree of substi
tution. With regard to the mechanism of action of
the metallocene dihalides, precursor incorporation
studies indicate an attack within the nucleic acid
metabolism. This is confirmed by cytokinetic and by
microanalytical (electron energy loss-spectrosco
pic) investigations as well as by the light-micro
scopic and ultrastructural alteration patterns of
treated tumor cells.
The development of the inorganic compound cis-diamminedi-

chloroplatinum(II) (DDP) to a potent antitumor drug during the
last twelve years, leading to a valuable enrichment of the thera
peutic arsenal of cytostatic drugs (J^, 2), has strongly stimula
ted the search for other metal-containing tumor-inhibiting spe
cies. Thus, the antitumor activity of certain representatives of
the metallocene dihalides was detected three years ago (3). Since
then, the cytostatic behavior of the metallocene dihalides has
0097-6156/83/0209-0315$06.00/0

been demonstrated at the experimental stage a g a i n s t v a r i o u s a n i -

mal tumor systems as w e l l as a g a i n s t i n v i t r o c u l t u r e d c e l l s .
The metallocene d i h a l i d e s represent the f i r s t example of a
group of o r g a n o m e t a l l i c compounds which e x h i b i t cancero-
s t a t i c p r o p e r t i e s by t h e i r own. Both c o n t a i n i n g a c i s - d i h a l o m e t a l
moiety, the metallocene d i h a l i d e s a r e , i n c o n t r a s t to the tumor-
i n h i b i t i n g i n o r g a n i c noble metal complexes of the p l a n a r L2MX2
type, t e t r a h e d r a l l y coordinated and are c h a r a c t e r i z e d by e a r l y
t r a n s i t i o n metals f e a t u r i n g as the c e n t r a l atoms M and r r - c y c l o -
p e n t a d i e n y l r i n g s as the l i g a n d s L.
Structure-Activity Relation
To study the s t r u c t u r e - a c t i v i t y r e l a t i o n of the metallocene
d i h a l i d e s , the i n f l u e n c e of chemical v a r i a t i o n upon the tumor-in-
h i b i t i n g a c t i v i t y was i n v e s t i g a t e d . P r i n c i p a l l y , a metallocene
d i h a l i d e molecule o f f e r s t h f e e d i f f e r e n t p o s i t i o n s to be m o d i f i e d
(Figure 1): ( i ) At the p o s i t i o n of the c e n t r a l metal atom M, one
of e i g h t d i f f e r e n t e a r l y t r a n s i t i o n metals of the t i t a n i u m , vana-
dium, and chromium subgroups of the P e r i o d i c Table can be i n t r o -
duced, ( i i ) The p o s i t i o n s of the acido l i g a n d s X can be occupied
by v a r i o u s h a l i d e as w e l l as pseudohalide l i g a n d s . ( i i i ) S i n g l e
hydrogen atoms of the two c y c l o p e n t a d i e n y l r i n g s may be r e p l a c e d
by other groups i n d i f f e r e n t s u b s t i t u t i o n modes such as monosub-
s t i t u t i o n , 1 , 1 ' - d i s u b s t i t u t i o n , or b r i d g i n g of the two r i n g s by
a b i f u n c t i o n a l group.
Studies on the s t r u c t u r e - a c t i v i t y r e l a t i o n of the m e t a l l o c e -
ne d i h a l i d e s were g e n e r a l l y performed u s i n g as experimental sy-
stem the E h r l i c h a s c i t e s tumor (EAT) growing i n female CF1 mice.
The t h e r a p e u t i c e f f e c t was judged by determining the mean s u r v i -
v a l time (MST) and by c a l c u l a t i n g the i n c r e a s e i n l i f e span (ILS)
compared to the untreated c o n t r o l s . The key-date f o r the s u r v i v a l
s t u d i e s was day 180 a f t e r tumor t r a n s p l a n t a t i o n (Table I , I I I ) or
day 60 (Table V I I I ) .
V a r i a t i o n of the Metal Atom. C o n s i d e r i n g the i n f l u e n c e of

the c e n t r a l metal M w i t h i n the metallocene d i c h l o r i d e system
^ I ^ ^ M C ^ upon the t u m o r - i n h i b i t i n g a c t i v i t y , there i s a s t r o n g
dependence of the antitumor a c t i o n upon the metal atom present
C 3 - 7 ) . Whereas the complexes c o n t a i n i n g the f i r s t and second row
t r a n s i t i o n metals T i , V, Nb, or Mo e f f e c t marked tumor i n h i b i t i o n
w i t h d i s t i n c t d o s e - a c t i v i t y r e l a t i o n s and w i t h cure r a t e s of
100 % at optimum dose (Table I ) , the metallocene d i c h l o r i d e s of
the h i g h e r homologues Ta or W only s p o r a d i c l y induce the s u r v i v a l
of t r e a t e d tumor-bearing mice. The extreme case of t h i s tendency
i s reached at the metallocene d i c h l o r i d e s of Zr and Hf, f o r which
no t u m o r - i n h i b i t i n g p r o p e r t i e s could be observed a g a i n s t EAT.
Regarding the p o s i t i o n of the c e n t r a l metal atoms i n the Pe-
r i o d i c Table, a d i a g o n a l r e l a t i o n T i - Nb and V - Mo i s obvious of
those metals which d i s p l a y as c e n t r a l atoms i n metallocene d i -
c h l o r i d e s marked c a n c e r o s t a t i c a c t i v i t y ( 6 ) . This corresponds to

16. KOPF AND KÔPF-MAIER Metallocene Dihalides 317
Figure 1. Molecular structure of the

5
metallocene dihalides, fa -C H ) MX . s s 2 t
The arrows indicate the three positions

that can be chemically modified.
Table I
Pharmacological data of metallocene d i c h l o r i d e s
b)
Optimum Cure r a t e ILS L D
T I
Réf.
50
dose at o p t i - at o p t i - ν
mum dose mum dose
(mg/kg) (%) (%) (mg/kg)
M = Ti 30-60 80-90 c)
842-951 100 3.3 (3,4)
d
M - Zr 75 -> (4)
M = Hf .d) 220 J*> (4)
M =V 80-90 100 984 110 1.4
M = Nb 20-25 1Q0 1076 35 3.5 (6)
M - Ta 80-220 e)
570 f )
220 .e) -
M - Mo 75-100 100 1039 175 2.9
M =W 100-525 e)
360 f )
500 (6)
a) I L S (increase i n l i f e span) values are defined as p e r c e n t u a l

increase of the mean s u r v i v a l time (MST) of a dose group r e l a t e d
to the untreated tumor-bearing c o n t r o l group (key-date: day 180).
With T i , V, Nb, Mo i n optimum doses, the MST values are s i g n i f i -
c a n t l y d i f f e r e n t from c o n t r o l values (Wilcoxon-Mann-Whitney U-
s t a t i s t i c s ) . b) Τ I (therapeutic i n d e x ) , defined as the r e l a t i o n
LD50/ED75. c) 100 % a f t e r a p p l i c a t i o n i n buffered s o l u t i o n (17).
d) No cures o r any therapeutic e f f e c t observed, e) Only sporadic
cures w i t h i n the i n d i c a t e d dose range, f ) Maximum v a l u e .

the s i m i l a r i t y of atomic r a d i i w i t h i n a diagonal p a i r of e l e -

ments, e.g. T i (1.32 Â) and Nb (1.34 Â ) , and p o i n t s to the s i g n i -
f i c a n c e of e f f e c t s of s i z e f o r the mechanism of a c t i o n .
Thus, the s i z e of M i n f l u e n c e s the C l - M - C l bonding angle,
the M-CI bonding d i s t a n c e and the non-bonding Cl...CI d i s t a n c e
( b i t e ) w i t h i n the d i c h l o r o m e t a l moiety of the complexes. These
s t r u c t u r a l parameters are given i n Table I I f o r metallocene d i -
c h l o r i d e s (8 - 11) and f o r the i n o r g a n i c c y t o s t a t i c drug DDP (12).
For DDP the C l . . . C I b i t e amounts to 3.35 Â and corresponds
to the d i s t a n c e between two appropriate DNA-base donor atoms of
a DNA h e l i x (13) i n a manner that c r o s s - l i n k s can be b u i l t up
a f t e r d i s s o c i a t i o n of the CI atoms (13 - 16). I f the s i m p l i f i c a -
t i o n i s admitted that the b i t e between the CI atoms as the l e a -
v i n g groups i s d i r e c t l y c o r r e l a t e d to the d i s t a n c e of adjacent
c o o r d i n a t i o n s i t e s at the metal atom against another given p a i r

of entrant l i g a n d s , the p o s s i b i l i t y of a s i m i l a r molecular a t t a c k
f o r complexes e x h i b i t i n g s i m i l a r b i t e values has to be taken i n
account. I t i s remarkable that w i t h a l l t u m o r - i n h i b i t i n g m e t a l l o -
cene d i c h l o r i d e s t h i s b i t e i s of a s i m i l a r s i z e as w i t h DDP, i . e .
the value i s at the most by 0.12 Â higher or 0.11 Â lower than
the b i t e of DDP. In c o n t r a s t , the zirconocene and hafnocene d i -
c h l o r i d e s , which do not induce a n t i p r o l i f e r a t i v e a c t i v i t y , are
c h a r a c t e r i z e d by a b i t e which i s markedly (by 0.25 or 0.31 Â)
l a r g e r than that of DDP. These two C l . . . C I d i s t a n c e s exceed a
c r i t i c a l value l y i n g between 3.50 and 3.60 Â which seems to be an
upper l i m i t f o r the formation of DNA-metal c r o s s - l i n k s , and are
t h e r e f o r e p o s s i b l y r e s p o n s i b l e f o r the t h e r a p e u t i c i n e f f e c t i v i t y
of zirconocene and hafnocene d i c h l o r i d e s .
From these c o n s i d e r a t i o n s i t can be deduced that the c i s - d i -
chlorometal moiety w i t h i n the metallocene d i h a l i d e s i s apparently
of c r i t i c a l s i g n i f i c a n c e f o r the mechanism of a c t i o n . The forma-
t i o n of adjacent, perhaps c h e l a t i n g bonds to b i o l o g i c a l macromo-
l e c u l e s a f t e r d i s s o c i a t i o n of the h a l i d e l i g a n d s seems to be an
important step.
V a r i a t i o n of the Acido Ligands. In c o n t r a s t to the v a r i a -

t i o n of the c e n t r a l metal atoms, the exchange of the CI l i g a n d s
i n the titanocene model system (05115)2^X2 by the h a l i d e or pseu-
dohalide l i g a n d s X • F, Br, I , NCS, N3 does not a f f e c t the a n t i -
tumor a c t i v i t y (17). Table I I I shows that a l l i n v e s t i g a t e d com-
pounds l e a d to cure r a t e s of 100 % when a p p l i e d i n optimum doses.
This means that the antitumor a c t i v i t y of the metallocene species
i s independent of the h a l i d e l i g a n d X; provided that a c e n t r a l
metal i s present which causes marked antitumor a c t i v i t y , the a c i -
do l i g a n d s can apparently be widely v a r i e d without l o s s or d i m i -
n u t i o n of the c a n c e r o s t a t i c e f f e c t i v i t y . In some cases, e s p e c i a l -
l y i n the case of X - Br, the t h e r a p e u t i c range i s even enlarged
l e a d i n g to an increase of the t h e r a p e u t i c index (Τ I) to 4.5,
compared to 3.3 a f t e r treatment w i t h titanocene d i c h l o r i d e .
Moreover, the a p p l i c a t i o n of the titanocene d i h a l i d e s i n so-

16. KÔPF A N D KÔPF-MAIER Metallocene Dihalides 319
Table II
S t r u c t u r a l parameters
of DDP and some metallocene d i c h l o r i d e s
Compound M M-Cl a )
Cl-M-Cl Cl...Cl b )
Réf.
(Â) (°) (Â)
(C H ) MoCl
5 5 2 2
Mo 2.471(5) 82.0(2) 3.242 (8)
(C H CH ) VC1
5 4 3 2 2 V 2.398(2) 87.06(9) 3.303 (9)
cis-(NH ) PtCl 3 2 2 Pt 2.330(1) 91.9(3) 3.349 (12)
(C H ) NbCl
5 5 2 2
Nb 2.470(4) 85.6(1) 3.356 (8)
(C H ) TiCl
5 5 2 2
Ti 2.364(3) 94.43(6) 3.470 (10)
<CH ) (C H ) HfCl
2 3 5 4 2 2
Hf 2.423(3) 95.87(8) 3.598 <JJ)
(C H ) ZrCl
5 5 2 2
Zr 2.441(5) 97.1(2) 3.660 (8)
a) Mean M - C l d i s t a n c e , b) C a l c u l a t e d non-bonding C l . . . C l
distance ( b i t e ) .

Table I I I
Pharmacological data of titanocene dihalides
Optimum Cure r a t e I L S LD^Q TI Ref.

a
^\ x dose ) at o p t i - at o p t i -
">»v mum dose mum dose**)
(mg/kg) (%) (%) (mg/kg)
X = F 40 100 1039 60 2.0 (J7)

c)
X - Cl 30-60 7 5
960-971 100 3.3 (il)
Χ - Br 40-80 100 1162 135 4.5 07)

Χ - I 60-100 100 1011 145 2.6 <J7)
Χ = NCS 80 100 1081 135 3.4 (17)
Χ = N 3 50-70 100 1084 95 2.4 07)
a) A f t e r a p p l i c a t i o n i n b u f f e r e d s o l u t i o n s , t o x i c l e v e l s are
s h i f t e d to higher doses so that the optimum dose ranges are broa
dened and the Τ I values are enlarged ( f o r d e t a i l s see r e f . 17).
b) The b a s i c MST values are i n a l l cases s i g n i f i c a n t l y d i f f e r e n t
from the c o n t r o l v a l u e s , c) 100 % a f t e r a p p l i c a t i o n i n L u f f e r e d
solution.

16. KÔPF AND KÔPF-MAIER Metallocene Dihalides 321
l u t i o n s b u f f e r e d to pH 5.1-5.7 leads to a f u r t h e r increase o f

the Τ I values and to a r e d u c t i o n of the substance-induced s i d e
e f f e c t s ; e s p e c i a l l y the appearance of p o s t p e r i t o n i t i c symptoms
s e v e r a l weeks a f t e r i n t r a p e r i t o n e a l a p p l i c a t i o n of high doses o f
titanocene d i h a l i d e s i s s t r i k i n g l y reduced by pH e l e v a t i o n i n the
drug s o l u t i o n s before i n j e c t i o n (17).
This phenomenon may be explained by the tendency of a l l t i
tanocene d i h a l i d e s to p a r t i a l l y cleave o f f t h e i r acido l i g a n d s X
w i t h i n a h y d r o l y s i s e q u i l i b r i u m i n aqueous systems (18). This
leads to a c i d p r o p e r t i e s o f the drug s o l u t i o n s which are respon
s i b l e f o r the s i d e e f f e c t s and which can be reduced by b u f f e r i n g .
On the other hand, the reason f o r the s i m i l a r t u m o r - i n h i b i
t i n g potencies o f the titanocene d i h a l i d e s w i t h X • F, C l , Br, I ,
NCS, N3 can be seen i n t h e i r s i m i l a r a b i l i t y to d i s s o c i a t e the
l i g a n d s X i n aqueous s o l u t i o n s , which apparently means a s i m i l a r

a b i l i t y to r e l e a s e the metals' c o o r d i n a t i o n s i t e s f o r i n t e r a c t i o n
w i t h the t a r g e t molecule i n the b i o l o g i c a l system. In consequen
ce, titanocene d e r i v a t i v e s w i t h s t r o n g l y bonded l i g a n d s X such as
( C H j ) T i ( S R ) 2 (19) o r (C5H )2TiS5 (20) do not show any cancero-
5 2 5
s t a t i c a c t i v i t i e s against EAT. Thus, the l e a v i n g a b i l i t y of the X

l i g a n d s w i t h i n the dihalometal moiety p l a y s apparently an impor
tant r o l e f o r the t u m o r - i n h i b i t i n g p r o p e r t i e s of the metallocene
d i h a l i d e s , as was s i m i l a r l y p o s t u l a t e d f o r the D D P - t y p e complexes
(21).
M o d i f i c a t i o n of the Cyclopentadienyl Ligands. The r e s u l t s

described so f a r suggest that the MX2 moiety w i t h i n the m e t a l l o
cene d i h a l i d e s represents the a c t i v e center of the molecules.
However, m o d i f i c a t i o n of the c y c l o p e n t a d i e n y l r i n g s i n
(C5H5)2TiCl2 as the model system a l s o has a s i g n i f i c a n t i n f l u e n c e
on the t u m o r - i n h i b i t i n g potency of titanocene d i c h l o r i d e .
The v a r i a t i o n of one of the two r i n g s by e t h y l o r t r i m e t h y l -
s i l y l monosubstitution reduces the cure r a t e s to 60 o r 80 % (Ta
b l e IV, t o p ) . Whereas these values are only moderately diminished
i n comparison to the u n s u b s t i t u t e d titanocene d i c h l o r i d e , the t u
m o r - i n h i b i t i n g a c t i v i t y of complexes c o n t a i n i n g two modified
r i n g s i s much more reduced. Thus, f o r 1 , 1 ' - d i s u b s t i t u t e d or 1,1'-
bridged compounds (Table IV, middle and bottom) a t the best spo
r a d i c cures can be observed. For (C5H5)(C5H4Me)TiCl2>
( C H M e ) T i C l 2 , ( C ^ ^ ^ H ^ T i C ^ ( L D Q = 35 mg/kg), and
5 4 2 5
SiMe2(C5H4)2TiCl2 c a n c e r o s t a t i c i n a c t i v i t y i s noted; because o f

the outstanding t o x i c i t y of the f i r s t three compounds i t may be
argued t h a t here the t o x i c e f f e c t i n t e r f e r e s w i t h a p o s s i b l e cy
t o s t a t i c a c t i o n (22).
In c o n t i n u a t i o n of the m o d i f i c a t i o n of the c y c l o p e n t a d i e n y l
l i g a n d s , one or two of these can be replaced by the r e l a t e d r e
bound systems i n d e n y l o r t e t r a h y d r o i n d e n y l . The t e s t e d compounds
of t h i s type i n c l u d e ( C g H y ^ T i C ^ , (C9H7) (CoJHj j ) T i C l 2 , and
(C H )(0 Η )ΤΐΟΐ2 (Table V). Whereas f o r the mixed c y c l o p e n t a d i
9 7 5 5
e n y l complex, which r e v e a l s a maximum cure r a t e of 100 %, no d i -

Table IV
Pharmacological data of monosubstituted,
1 , 1 ' - d i s u b s t i t u t e d , and 1,1'-bridged t i t a n o c e n e d i c h l o r i d e s (22)
a ) b
0D Maximum 0C > L D
50
cure r a t e
Ι ^Cl w i t h i n OD
(mg/kg) (%) (mg/kg)
R = Me -c) -c) .c) 45

R - Et 50-70 60 9/15 100
R = SiMe 3 40-70 80 16/20 100
d ) e)
SD SC L D
50
(mg/kg) (mg/kg)
R - Me -c) -c) 30
R - SiMe 3 280-300 2/10 360
n
R = SiMe Bu 2 320-360 3/15 420
R = GeMe„ 240-320 7/25 400
d) e)
.ci SD SC LD,
50
Cl
(mg/kg) (mg/kg)
Ζ = CH 2 220-260 2/15 360

Ζ = CHMe 200-260 2/20 320
Ζ = SiHMe 160-220 6/20 300
_c)
Ζ - SiMe 2 220
Ζ - SiEt 2 140-200 3/20 220
Ζ - GeMe 2 220-260 2/15 280
a) 0 D = Dose range w i t h optimum t u m o r - i n h i b i t i n g a c t i v i t y (cure

r a t e s > 50 % ) . b) 0 C = Number of cures i n the OD , r e l a t e d t o
the t o t a l number of animals i n the OD. c) No cures or any t h e
r a p e u t i c e f f e c t observed, d) SD = Dose range of sporadic c u r e s ,
e) SC = Number of sporadic c u r e s , r e l a t e d t o the t o t a l number of
animals w i t h i n S D .

Table V
Pharmacological data of
i n d e n y l and t e t r a h y d r o i n d e n y l d e r i v a t i v e s L L ' T i C ^ (22)
\ ^Cl \ ri ^ \ ^Cl
Ti
C Ti
C Ti
C
I ^Cl / ^Cl / ^Cl
b )
L L' SD a )
sc L D
50
(mg/kg) (mg/kg)
C H C H
280-320 2/15 360
9 7 9 7
C H C H
260-300 2/15 320
9 11 9 11
C H
C H 5 5 80-120 11/15 c)
120
9 11
a) and b) see Table IV. c ) 100 % cures a t optimum dose (100

mg/kg).
Table V I
Pharmacological data of
monocyclopentadienyl complexes C^H^TiX2Y
.X
Ti^
x
/
a ) b )
SD SC LD 5 Q
(mg/kg) (mg/kg)
Cl Cl 50-130 10/45 120
Cl SPh 60-100 6/25 100
NCS NCS 70-110 6/25 130
a) and b) see Table IV.

minution of the c a n c e r o s t a t i c a c t i v i t y can be observed i n compa

r i s o n to the u n a l t e r e d titanocene d i c h l o r i d e , again the tumor-in
h i b i t i n g p r o p e r t i e s of the complexes c o n t a i n i n g two modified r i n g
l i g a n d s are s t r o n g l y reduced (22).
On extending the m o d i f i c a t i o n of c y c l o p e n t a d i e n y l l i g a n d s to
the t o t a l replacement of one C5H5 r i n g by an a d d i t i o n a l acido l i
gand Y, an exhaustion of the c a n c e r o s t a t i c a c t i v i t y i s to be ob
served i n no case (Table V I ) . Instead, sporadic cures occur w i t h
a l l t e s t e d compounds l e a d i n g to a reduced antitumor e f f e c t i v i t y
i n a range which i s comparable to that of the m a j o r i t y of the
compounds i n Tables IV and V.
From the r e s u l t s of t h i s chapter the hypothesis i s deduced
that the c y c l o p e n t a d i e n y l r i n g s act as c a r r i e r l i g a n d s which en
able the t r a n s p o r t of the a c t i v e centers of the molecules to the
i n t r a c e l l u l a r t a r g e t s . M o d i f i c a t i o n of the r i n g s apparently i n
j u r e s the t u m o r - i n h i b i t i n g a c t i v i t y by a f f e c t i n g the t r a n s p o r t a
t i o n p r o p e r t i e s of the molecules. P o s s i b l e reasons f o r t h i s be
h a v i o r are s t e r i c e f f e c t s , changes i n the s o l u b i l i t y p r o p e r t i e s ,
or a l t e r a t i o n s of r i n g ligand-metal bond s t a b i l i t i e s of the modi
f i e d metallocene d i h a l i d e s (22).
E f f e c t of H y d r o l y t i c a l l y Formed Species. As the metallocene

d i h a l i d e s are a p p l i e d under aqueous c o n d i t i o n s , p o s s i b l e e f f e c t s
of the h y d r o l y t i c a l l y formed species are of i n t e r e s t . Some d i s s o
c i a t i o n , aquation, and h y d r o l y s i s r e a c t i o n s of titanocene d i h a l i
des have r e c e n t l y been e l u c i d a t e d i n more d e t a i l (23 - 30). In
aqueous s o l u t i o n s , the parent compounds (05115)2^X2 do not only
form the n e u t r a l oxo-bridged d i n u c l e a r complexes
(C5H )2(X)Ti-0-Ti(X)(C H5)2 0 8 ) , but a d d i t i o n a l l y seem to co
5 5
e x i s t i n pH-dependent r e v e r s i b l e e q u i l i b r i a w i t h the c a t i o n i c
2+ +
aquo species [ ( C H ) 2 T i ( O H 2 ) 2 ] » [(C H )2Ti(OH2)OH] , and
5 5 5 5
2+
[(C5H5)2(H20)Ti-0-Ti(OH2)(C5H5) ] (23). X-ray s t r u c t u r e d e t e r
2
minations of the n e u t r a l complex (05115)2(Cl)Ti-O-Ti(CI) ( 0 ^ 5 ) 2

(24) as w e l l as of c r y s t a l l i n e s a l t s of the mononuclear diaquo
(25, 26) and the d i n u c l e a r μ-οχο (27, 28) c a t i o n s show them to
have the suggested s t r u c t u r e s w i t h s t i l l v a l i d b i s ( c y c l o p e n t a d i
enyl) titanium skeletons.
On the other hand, on r e a c t i o n of titanocene d i c h l o r i d e w i t h
equimolar amounts of water and diethylamine i n t e t r a h y d r o f u r a n
the t r i n u c l e a r monocyclopentadienyl h y d r o l y s i s product
[C5H5Ti(Cl)0]3 together w i t h unchanged ^ I ^ ^ T i C ^ could be i s o
l a t e d (29). Thus, the p o s s i b i l i t y of h y d r o l y t i c cleavage of cyc
l o p e n t a d i e n y l r i n g s during the b i o l o g i c a l a c t i o n has a l s o to be
taken i n account.
As t y p i c a l examples of a (05115)2^012 and a 0511511013 hydro
l y s i s product, we have t e s t e d against EAT the d i n u c l e a r
[(C5H5)2Ti(Cl)] 0 and the t e t r a n u c l e a r ^ ^ T i i C O O ] ^ , r e s p e c t i
2
v e l y , which are e a s i l y a c c e s s i b l e i n pure form (24_; 29, 30). Com

pared to t h e i r parent compounds, both μ-οχο-complexes show redu
ced antitumor a c t i v i t y . Thus, h y d r o l y s i s seems not to be a step
producing the i n t r i n s i c l y a c t i v e s p e c i e s .

Tumor Systems Tested i n v i v o
The c a n c e r o s t a t i c a c t i v i t i e s of titanocene d i c h l o r i d e and

vanadocene d i c h l o r i d e as t y p i c a l r e p r e s e n t a t i v e s of the m e t a l l o -
cene d i h a l i d e s were t e s t e d against v a r i o u s experimental tumor s y -
stems (Table V I I ) . The two complexes e x h i b i t c a n c e r o s t a t i c p r o -
p e r t i e s against f l u i d a s c i t i c tumors such as EAT i n e a r l y and ad-
vanced stages, lymphoid leukemia L1210, and lymphocytic leukemia
P388 (31). The growth of s o l i d tumors, e.g. of a subcutaneously
implanted s o l i d EAT, i s i n h i b i t e d i n a h i g h l y s i g n i f i c a n t manner
by i n t r a p e r i t o n e a l a p p l i c a t i o n of the metallocene d i c h l o r i d e s
(31); t h i s r e s u l t demonstrates that the substances a r e capable t o
achieve a n t i p r o l i f e r a t i o n by a systemic e f f e c t . Test s e r i e s
against other experimental tumor systems a r e p r e s e n t l y under i n -
vestigation.
C e l l Growth I n h i b i t i o n i n v i t r o
For determination of the g r o w t h - i n h i b i t i n g potencies i n

v i t r o , the i n f l u e n c e of the metallocene d i c h l o r i d e s (C5H5)2MCl2
(M = T i , Zr, Hf, V, Mo) upon the p r o l i f e r a t i o n of i n v i t r o c u l t u -
red tumor and embryonic c e l l s both of animal and of human o r i g i n ,
was i n v e s t i g a t e d . Three main c o n c l u s i o n s can be drawn: ( i ) There
are no h i n t s t o a c e l l s p e c i f i t y of the g r o w t h - i n h i b i t i n g a c t i o n ,
( i i ) Vanadocene d i c h l o r i d e i s the most e f f e c t i v e g r o w t h - i n h i b i -
t i n g r e p r e s e n t a t i v e i n v i t r o and accomplishes h i g h l y s i g n i f i c a n t
d i m i n u t i o n of c e l l p r o l i f e r a t i o n i n a c o n c e n t r a t i o n as low as
5 x 1 0 ~ 6 mol/1. Titanocene and molybdocene d i c h l o r i d e s i n h i b i t
c e l l u l a r growth only i n c o n c e n t r a t i o n s of 5 x 1 0 " ^ o r 10~3 mol/1,
r e s p e c t i v e l y , whereas zirconocene and hafnocene d i c h l o r i d e s ,
which a r e i n e f f e c t i v e a g a i n s t EAT i n v i v o , r e q u i r e even higher
c o n c e n t r a t i o n s (32). ( i i i ) The growth i n h i b i t i o n i s apparently
caused by a c y t o t o x i c a c t i o n of the compounds, as i s demonstrated
by a p p l i c a t i o n of the dye l i s s a m i n e green (32).
Investigations Concerning the Mechanism of A c t i o n
The i n v e s t i g a t i o n s concerning the mode of a c t i o n of the me-

t a l l o c e n e d i h a l i d e s represented by titanocene d i c h l o r i d e and v a -
nadocene d i c h l o r i d e are based on four experimental p i l l a r s :
3
( i ) I n c o r p o r a t i o n studies wi t h H - l a b e l l e d , s p e c i f i c precursors
of the DNA, RNA, or p r o t e i n synthesis were performed t o r e v e a l
d i f f e r e n t i n f l u e n c e s of the substances upon these synthesis
pathways, ( i i ) The c y t o k i n e t i c behavior of EAT c e l l s a f t e r
treatment was pursued t o recognize a l t e r a t i o n s i n the c e l l
t r a n s i t through the c e l l c y c l e , ( i i i ) The l i g h t - m i c r o s c o p i c
and u l t r a s t r u c t u r a l a l t e r a t i o n p a t t e r n s were observed t o gain i n -
formations about the c e l l u l a r events d u r i n g the i n h i b i t i o n of

Table V I I
T u m o r - i n h i b i t i n g p r o p e r t i e s of (C5H5)2MCl2
i n optimum doses against v a r i o u s experimental tumor systems
M Fluid Fluid L1210 P388 Solid

EAT EAT EAT
a ) a ) a ) a ) a)
day 1 day 5 day 1 day 1 days 1 , 3 , ( 5 )
Ti 70 mg/kg: 70 mg/kg: 70-90 40-70 2 χ (30-50)

mg/kg: mg/kg: mg/kg:
100 % 90 % ILS c )
I L S c) T/C d)
sur- sur 21-26 % 26-30 14-25 %

•b) v i v o r s b)
80 mg/kg: 80 mg/kg: 50-60 30-50 3 x 5 0 mg/kg:

mg/kg: mg/kg:
100 % 20 % ILS c )
I L S c) T/Cd)
(
sur- sur 24-29 % 18-24 % 31 %

,b) v i v o r s b)
a) Day a f t e r tumor t r a n s p l a n t a t i o n on which the animals were

t r e a t e d w i t h a s i n g l e dose, b) On key-date (day 180 a f t e r t r a n s
p l a n t a t i o n ) , c) For d e f i n i t i o n see Table I . d) T/C = mean tumor
weight of a dose group r e l a t e d as percentage t o the mean tumor
weight of the c o n t r o l s (key-date: day 8 a f t e r t r a n s p l a n t a t i o n ) .

proliferation. ( i v ) Using the e l e c t r o n energy l o s s spectroscopy

(EELS), a new m i c r o a n a l y t i c a l method which combines the u l t r a -
s t r u c t u r a l r e p r e s e n t a t i o n of b i o l o g i c a l o b j e c t s w i t h the a n a l y s i s
of metals l i k e t i t a n i u m o r vanadium i n low c o n c e n t r a t i o n s , i t was
intended t o determine the l o c a l i z a t i o n of .the c e n t r a l metal atoms
of (C H5)2MCl2 (M = T i , V) w i t h i n the tumor c e l l s a f t e r t r e a t -
5
ment.
Precursor I n c o r p o r a t i o n S t u d i e s . Studies of the i n c o r p o r a -

t i o n of t r i t i a t e d precursors of the DNA, RNA, or p r o t e i n synthe-
s i s , i . e . of thymidine, u r i d i n e , or l e u c i n e , i n t o the a c i d - i n s o -
l u b l e f r a c t i o n of EAT c e l l s were accomplished a f t e r i n v i v o as
w e l l as i n v i t r o treatment w i t h (C5H5)2MCl2 (M = T i , V ) . I n a l l
experiments, the n u c l e i c a c i d metabolism turns out t o be much more
s e n s i t i v e t o the metallocene d i h a l i d e a c t i o n than the p r o t e i n me-

t a b o l i s m (33, 34). I n p a r t i c u l a r , treatment w i t h vanadocene d i -
c h l o r i d e induces an extended and p e r s i s t e n t suppression of the DNA
s y n t h e s i s without signs of r e g e n e r a t i o n processes (Figure 2 ) .
In t h i s connection i t i s worth mentioning t h a t an i n t e r -
a c t i o n between metallocene d i c h l o r i d e s and n u c l e i c a c i d s can be
demonstrated i n v i t r o by the f a c t t h a t , dependent on the sub-
stance c o n c e n t r a t i o n , the UV absorptions of both c a l f thymus DNA
and c a l f l i v e r RNA are i n f l u e n c e d . The maximum a t 258 nm i s s h i f -
ted t o lower wave l e n g t h s , w h i l e the absorbance of the band i n -
creases which i s i n d i c a t i v e of an a l t e r a t i o n of the secondary
s t r u c t u r e of the n u c l e i c a c i d s .
C y t o k i n e t i c S t u d i e s . Marked m i t o t i c depressions and c e l l

accumulations i n the l a t e S and i n the p r e m i t o t i c G2 phases are
the main r e s u l t s a f t e r i n v i v o treatment of EAT c e l l s w i t h
optimum t u m o r - i n h i b i t i n g doses of metallocene d i c h l o r i d e s (35).
Whereas the G2 b l o c k a f t e r (C5H5)2VCl2 a p p l i c a t i o n i s r e v e r s i b l e
and gives r i s e t o slow p a r t i a l l y synchronized waves and, l a t e r -
on, t o the désintégration of the c e l l s , the (C5H5)2TiCl2~treated
EAT c e l l populations are removed s e v e r a l days a f t e r substance ap-
p l i c a t i o n by c e l l s belonging to the defensive system of the host
animals.
Considering the c y t o k i n e t i c behavior of EAT a f t e r i n v i t -
r o treatment w i t h metallocene d i h a l i d e s , the appearance of c e l l
a r r e s t s i n the p r e m i t o t i c G2 phase i s again the dominant r e s u l t .
The r e v e r s i b i l i t y of t h i s G2 b l o c k depends on the a p p l i e d concen-
t r a t i o n s . A d d i t i o n a l l y , c e l l accumulations a t the G<|/S boundary
can be recognized d u r i n g the exposure t o the substances i n low
c o n c e n t r a t i o n s . F o l l o w i n g short exposure p e r i o d s , these c e l l s e s -
cape the a r r e s t a t Gj/S and continue t h e i r t r a n s i t through the
c e l l c y c l e as synchronized populations which can be pursued du-
r i n g one f o l l o w i n g c y c l e (36).
The comparison w i t h the c y t o k i n e t i c behavior a f t e r treatment
w i t h other c y t o s t a t i c drugs shows t h a t many substances f o r which
the molecular a t t a c k upon DNA molecules i s g e n e r a l l y assumed p r o -

CO
Κ)
00
DNA RNA Protein
3
M
24 ' 48 [h] 8 16 24 ^ 48 [h]

s
• Time after treatment m
s
F/gwre 2. Incorporation rates related as percentage to the controls (- · -) of [methyl- H]thy
3 3
midine (left), [5- H]uridine (middle), and L-[4,5- H]leucine (right) as a measure of the DNA, RNA,
and protein synthesis activities respectively after a 90-min treatment of EAT cells with vanadocene
M

dichloride in vitro. C

H
M
H
16. KOPF A N D KÔPF-MAIER Metallocene Dihalides 329
voke s i m i l a r f i n d i n g s : A l k y l a t i n g agents such as cyclophosphamide

(37) and melphalan (38), the a n t h r a c y c l i n e s C39, 4 0 ) , and DDP
(41) cause c e l l a r r e s t s i n the G phase, whereas c e l l accumula-
2
t i o n s a t the G<|/S boundary are observable under the i n f l u e n c e of

a n t i m e t a b o l i t e s (40, 42) and DDP (41).
M o r p h o l o g i c a l S t u d i e s . I n analogy t o other c y t o s t a t i c drugs

such as a l k y l a t i n g agents, bleomycin, o r DDP, the treatment w i t h
the metallocene d i c h l o r i d e s of t i t a n i u m and vanadium induces the
formation of g i a n t c e l l s w i t h one o r s e v e r a l n u c l e i as w e l l as
the appearance of abnormal m i t o t i c f i g u r e s (43).
At the u l t r a s t r u c t u r a l l e v e l (Figure 3) the f i r s t observable
a l t e r a t i o n s a f t e r i n v i v o and i n v i t r o treatment c o n s i s t i n chro-
matin condensation and i n n u c l e a r segmentation. The f u r t h e r - o n
developing morphological f e a t u r e s show many s i m i l a r i t i e s t o the

r e s u l t s a f t e r treatment w i t h a l k y l a t i n g agents (44, 45) or w i t h
DDP (46) and can be d e s c r i b e d as the development of the morpholo-
g i c a l signs of an unbalanced c e l l u l a r growth and, l a t e r - o n , as
the formation of necroses of the tumor c e l l s (43).
A s u r p r i s i n g event a f t e r i n v i v o and i n v i t r o treatment w i t h
titanocene d i c h l o r i d e , but not w i t h vanadocene d i c h l o r i d e , i s the
appearance of complete type-Α v i r u s p a r t i c l e s w i t h i n the c y t o
plasm some days a f t e r treatment as w e l l as of probable e a r l y s t a
ges of the v i r u s e s (Figure 3 ) . Thus, t i t a n o c e n e d i c h l o r i d e i s ap
p a r e n t l y able t o a c t i v a t e the formation of p r e v i o u s l y unexpressed
endogenous v i r u s e s . I n animals, a massive immigration of c e l l s
such as macrophages, l e u k o c y t e s , or lymphocytes which belong t o
the defensive system of the host animals, can be observed d u r i n g
the f o l l o w i n g days. I t might be supposed that t h i s process i s the
consequence of the v i r u s a c t i v a t i o n and a c t s as a f o r t i f y i n g f a c
tor i n the course of tumor i n h i b i t i o n by t i t a n o c e n e d i c h l o r i d e
(43)·
The f i n a l events a f t e r metallocene d i c h l o r i d e a p p l i c a t i o n
are the d e s t r u c t i o n of the tumor c e l l s and/or t h e i r phagocytosis
by macrophages i n v i v o (Figure 3 ) .
M i c r o a n a l y t i c a l S t u d i e s . The EELS i n v e s t i g a t i o n s a f t e r i n
v i v o as w e l l as a f t e r i n v i t r o treatment of EAT w i t h the a n t i t u
mor agents t i t a n o c e n e d i c h l o r i d e and vanadocene d i c h l o r i d e r e v e a l
r e p r o d u c i b l y the main enrichment of the c e n t r a l metals T i and V
w i t h i n the n u c l e a r heterochromatin. To a minor e x t e n t , the metals
are a s s o c i a t e d w i t h the euchromatin, the n u c l e o l u s , and the c y t o
plasmic ribosomes. I n most cases, no metal atoms can be detected
i n other c e l l u l a r regions by EELS. This means that the treatment
w i t h titanocene or vanadocene d i c h l o r i d e r e s u l t s apparently i n an
accumulation of the c e n t r a l metals T i and V w i t h i n those c e l l u l a r
regions which a r e r i c h i n n u c l e i c a c i d s .
In connection w i t h the s t r u c t u r e - a c t i v i t y r e l a t i o n and the
other experimental r e s u l t s , the d e s c r i b e d i n t r a c e l l u l a r d i s t r i b u
t i o n p a t t e r n of the c e n t r a l metal atoms which apparently r e p r e -

Figure 3. Schematic representation of ultrastructural alterations of ΕA Τ cells after

treatment with titanocene dichloride or DDP in vivo. Key to treatment (time after
treatment): O, untreated; A, titanocene dichloride (2-6 h); B, titanocene dichloride
(8-10 h) or DDP (4-12 h); C, titanocene dichloride (12-24 h) or DDP (24-36 h);
and D, titanocene dichloride (36-120 h) or DDP (48-120 h).

16. KÔPF A N D KÔPF-MAIER Metallocene Dihalides 331
Table V I I I
a)
I L S values determined on day 60
a f t e r t r a n s p l a n t a t i o n of the f l u i d EAT
(C H ) MC1
5 5 2 2 I L S (C H ) TiX
5 5 2 2 I L S
(%) (%)
M Ti 217-244 X = F 280
M = V 261 X - Cl 237-241
M s Nb 292 X = Br 323
M = Ta 164 X I 270
M = Mo 280 X = NCS 294
M = W 97 X N 295
3
a) This Table was i n c l u d e d t o f a c i l i t a t e comparison w i t h I L S

values of other tumor systems.

sent the a c t i v e centers of the molecules i s i n d i c a t i v e of a mole-

c u l a r a t t a c k of the metal-containing species upon the n u c l e i c
a c i d s w i t h i n the t r e a t e d c e l l s .
Acknowledgments
The authors are indebted t o the Deutsche Forschungsgemein-

s c h a f t , the Deutsche K r e b s h i l f e , and the Fonds der Chemischen
I n d u s t r i e f o r f i n a n c i a l grants.
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21, 1656.
40. Büchner, T.; Barlogie, B.; Göhde, W.; Schumann, J.; in
"Pulse-Cytophotometry", Haanen, C. A. M.; Hillen, H. F. P.;
Wessels, J. M. C., Eds.; European Press Medikon: Ghent,
1975, p. 293.
41. Bergerat, J.-P.; Barlogie, B.; Göhde, W.; Johnston, D. Α.;
Drewinko, B. Cancer Res. 1979, 39, 4356.
42. Ernst, P.; F a i l l e , Α.; Killmann, S.-A. Scand. J. Haema.
1973, 10, 209.
43. Köpf-Maier P. J. Cancer Res. Clin. Oncol. 1982, in press.
44. Bienengräber, Α.; Putzke, H.-P.; Tessmann, D. Z. Krebs-
forsch. 1965, 66, 438.
45. Brandes, D.; Anton, E . ; Lam, K. W. J. Nat. Cancer Inst.
1967, 39, 385.
46. Aggarwal, S. K. J. Clin. Hematol. Oncol. 1977, 7, 760.

17
R u t h e n i u m A n t i c a n c e r A g e n t s and R e l e v a n t
R e a c t i o n s of R u t h e n i u m — P u r i n e C o m p l e x e s
MICHAEL J. CLARKE
Boston College, Department of Chemistry, Chestnut Hill, MA 02167
Attempts to incorporate ruthenium into pharmaceutical

agents have typically followed two avenues of approach.
Early studies were directed toward ruthenium-containing
1
chemotherapeutic agents and more recently workers have begun
97
to explore the possibility of using Ru for diagnostic organ
2,3
imaging. While ruthenium complexes have shown promise
along both directions, there has been only one clinical trial
103
of a ruthenium compound, which involved RuCl for 3
4
radio-imaging tumors. At present, the only commonly used
ruthenium compound for biomedical purposes is the cytological
5
stain, ruthenium red. The interactions of ruthenium
complexes with biologically important molecules have recently
been reviewed and the early literature dealing with the
metabolic fate of different types of ruthenium compounds has
1,3,6
been summarized elsewhere.
Antitumor Aâatt
7
With few exceptions, approaches to ruthenium
antineoplastic agents have related to the putative mechanism 1 8
of action of the clinically employed c i s - C ^ N ^ K P t . ' The
binding of ammine complexes of both Ru(II) and Ru(III) to
nucleotides and nucleic acids is, in many ways, analogous to
9
that of cisplatin. ' On the other hand, ruthenium(II)
complexes usually undergo ligand substitution somewhat more
rapidly and the tripositive oxidation state is much more
accessible under mild conditions. Advantage can be taken
from the ready availability of both the Ru(II) and Ru(III)
oxidation states under physiological conditions and the
general inertness of these ions toward substitution, when
coordinated to nitrogen ligands.
Studies reported in an earlier symposium revealed the
general anticancer activity of compounds containing the
0097-6156/83/0209-0335$06.00/0

tetraamminerutheniumdll) ion or its congeners. Due to the

hypoxic conditions often holding in tumor cells, the
dipositive oxidation state is more likely to occur. Rapid
coordination to nitrogen or sulfur ligands can then fix the
metal in its environment. A corollary of this hypothesis is
that these complexes should tend to accumulate in tumors
relative to more aerobic normal tissue. While the exact
mechanism of action is difficult to prove with certainty, the
hypothesis accomodates most of the available data and such
complexes tend to concentrate in tumors as predicted.
In harmony with the cis-platinum drugs, biological
studies have also indicated that cellular DNA is the target
site for this class of ruthenium complexes. * * In
contrast to cis-Cl^NHg^Pt, ruthenium compounds screened in
the Ames test were able to cause mutations by frame shift as

well as by base pair substitution; however, these compounds
were approximately a hundredfold less active in the
generation of mutations. The platinum compound also shows
greater activity in inducing the bacterial SOS DNA repair
mechanism in the B. subtilis Comptest. In general, the
ruthenium complexes are an order of magnitude less toxic than
cisplatin; however, they must also be administered at higher
doses to achieve the same therapeutic effect. * . Thus the
therapeutic indices of these agents are no better than that
of cisplatin.
The compound, cis-Cl (DMSO) Ru, exhibits marginal
2 4
antitumor activity, inhibits nucleic acid synthesis and

exerts a pattern of cell damage and mutagenicity similar to
that of c i s - C l ( N H o )
2 2 Some purine and pyrimidine
Pt
adducts of the DMSO compound have recently been reported.

In contrast to the ammine complexes, the dimethylsulfoxide
complexes appear to coordinate at the N-7 of adenine owing to
hydrogen bonding between DMSO and the exocyclic amine protons
0
on adenine.
+
Ruthenium red, [(NH ) Ru-0-Ru(NH ) -0-Ru(NH ) ]^ ,
3 5 3 4 ? 5
selectively stains cell membrane mucopolysaccharides",

2+ 17
specifically inhibits cell membrane Ca -ATPase and
inhibits calcium ion transport across the mitochondrial
18
membrane } however, impurities may be responsible for at
least some of these biological effects. It also exhibits
fairly good antitumor activity against Lewis lung carcinoma
in mice (T/C - 141 at a dose of 2.5 mg/kg*day) and caused a
39% reduction in tumor volume at 5 mg/kg*day. '
While adenine containing coenzymes are easily complexed
by ammineruthenium ions, studies with the NAD-requiring
enzymes alcohol dehydrogenase, lactate dehydrogenase and
glucose-6-phosphate dehydrogenase, the NADH-requiring malate
dehydrogenase, and isocitrate dehydrogenase, which utilizes
NADP, revealed no inhibition or utilization when these

17. CLARKE Ruthenium Anticancer Agents 337
coenzymes were coordinated to (NH3) Ru(III) at the N-6 site

5
21
of the adenine residue. Therefore, it is unlikely that
complexation of these coenzymes plays a significant metabolic
role, except at very high ruthenium concentrations. Recent
work by Matthews has shown that ribonuclease is mildly
inhibited by a mixture of 2,3'-CMP coordinated to
( N H « ) c R u ( I I I ) at the exocyclic nitrogen.
Radiodiaanostic Agent»
07
7
The isotope 'Ru emits an essentially monoenergetic
gamma ray at 216 keV, which is well suited for use in present
radioscintigraphic equipment. Moreover the half-life of the

isotope (2.9 days) is sufficiently long to allow for chemical
synthesis and quality control of these radio-imaging agents.
Ruthenium radiopharmaceuticals would be especially useful
when periodic or delayed scans are necessary in order to
diagnose or monitor the progress of disease. Owing to the
relatively scant availability of this isotope at the present
1
time, the longer-lived isotope, Ru, is often used for
initial tissue-distribution studies. Refinement of earlier
1
studies has now verified that Ru introduced into animals
as cis-iCîMB^^Ru]* or related complexes exhibits a
concentration in tumors comparable to that of the widely used
tumor-imaging agent, ° Ga-citrate ' . Unfortunately, these
complexes do not provide high tumor to blood contrast ratios,
so that improvement is necessary for medically useful agents.
Earlier studies with rat-liver microsomes indicated that
NADH-dehydrogenase systems readily reduced
ammineruthenium(III) complexes. Crane has since discovered
plasma membrane NADH-dehydrogenase enzymes on a number of
4
different types of cells. Experiments with pig
erythrocytes showed that ammineruthenium(III) ions are fairly
readily reduced by the plasma membrane NADH-dehydrogenase.
As expected, the reduction rates are a function of the metal
ion's reduction potential and its intrinsic electron-transfer
rate. Comparison of the hexammineruthenium(III) reductase
activities of subcellular fractions of mouse liver revealed
the plasma membrane and Golgi apparatus to be about
equivalent (when measured per mg of protein) and the
2 4
endoplasmic reticulum to be 3-4 times more effective . It
is possible that erythrocyte plasma membrane dehydrogenases
may be partially responsible for the reduction of ruthenium
complexes and their subsequent binding to blood proteins.
Surface NADH-dehydrogenase activity has also been shown to
25
occur with at least one tumor cell l i n e , so that these
systems may also be indirectly responsible for the binding of
ruthenium complexes to tumor sites. This is in harmony with

Pitha's earlier suggestion that cellular toxicity of

2 3 4
2
cis-[Cl (NH ) Ru] occurred th through initial binding of the
metal to the cell surface and subsequent uptake into the cell
by pinocytosis.
An important finding by Srivastaya et al. is that a
103
portion of the metal administered as RuCl *31^0 is 3
complexed by the glycoprotein transferrin, and so mimics iron

metabolism to some extent. Since rapidly growing tumor
cells require substantial quantities of iron, a
Ru-transferrin complex should be readily received by the
tumor. Substantiation of this is the higher uptake of the
purified Ru-transferrin relative to RuCl alone. Assuming3
the presence of transferrin receptor sites on the tumor cell

surface, initial accumulation by this mechanism should also
occur on the cell membrane followed by subsequent transport

to the cell interior. The recent report that Ru
introduced as RuCl specifically binds to glycoproteins on
3
the surface of rat acites hepatoma (AH-130) cells in vitro

may correlate with this . Subcellular distribution of
Ru in tumor and liver cells isolated from Ehrlich's solid
tumor-bearing mice, which had been injected with RuCl , 3
are similar to those of Ga and, by implication, to those of

iron. Fractionation of these and AH-130 cells innoculated in
tissue culture revealed particular accumulation of the metal
ion in the mitochondria followed by the cell debris plus
nuclear portion. '
The antitumor antibiotic bleomycin has long been of
interest in radiopharmacy since it selectively localizes in
tumors. In vitro, the drug requires the presence of Fe(II)
and oxygen to exhibit its full biochemical activity, which
involves introducing both single- and double-strand breaks
into cellular DNA through an as yet unidentified radical
2
intermediate . Stern and coworkers have recently reported
a remarkable Ru-bleomycin complex, which, it is claimed,
retains 100% of native bleomycin's cytotoxic activity,
remains stable through injection and excretion, and exhibits
the same tissue distribution and tumor accumulation as
9
fr-labelled bleomycin. A drug of this type may have the
highly desirable advantage of simultaneous chemotherapeutic
and diagnostic activities. Independent work involving direct
combination of [(HÔ^Ru] with bleomycin also yielded
stable products, which exhibited Sephadex chromatographic
behavior similar to those of native bleomycin and the complex
cited above . However, given the multiple coordination
sites available on this antibiotic and the normal
substitut ion-inert character of ammine ruthenium ions
coordinated to these types of sites, it is unlikely that
either mode of preparation yields a single structural
product. Further characterization of these complexes will be
necessary before mechanistic conclusions can be drawn.

Ruthenium red has shown some tendency to localize in

tumors, with the likely receptor sites being
mucopolysaccharides on cell surfaces Ruthenocenes have
provided a versatile ogranometallic approach to the synthesis
of radioruthenium agents, however, no high specificity has
been shown for organs which are not already well-imaged by
3 2 - 3 9
existing agents . A number of ruthenium complexes with
chelating ligands either similar or identical to the those
99m
employed with clinically used T c radiopharmaceuticals have
been tested with similar results. The DTPA complex shows
4 0
promise for cerebrospinal fluid imaging ,
8-hydroxyquinoline, 3,4,7,8-tetramethyl-l,10-phenanthroline
and iminodiacetate complexes may be useful for delayed
4
hepatobiliary studies , complexes of
dimercaptopropanesulfonic acid and dimercaptosuccinic acid

4 2 4 3
are potential long-term renal imaging agents * , phosphate
44
and phosphonate derivatives yield bone-imaging agents .
Reaction Mechanisms of Purine and Pvrimidinc Complexes with

1Μ^1 *»<ΙΙΙ)
5
While a substantial amount of structural information has

been collected on metal complexes of purine and
4 5 5 0
pyrimidines, * " much less is known about their
reactivities. Of particular interest are the effects of the
metal ion on the organic chemistry of nucleosides and the
reactions of the metal ion when coordinated to the
heterocyclic base, since these may contribute to either
oncogenesis or anticancer activity.
Since the most common coordination position for
relatively "soft" transition metal ions in nucleic acids is*
t!<e N-7 of guanine, the reaction chemistry of N-7 bound
guanosine and deoxyguanosine complexes is of special
importance. It has long been speculated that such complexes
may behave analogously to N-7 protonated or alkylated
5
nucleosides, which undergo fairly rapid sugar hydrolysis ·
However, early studies at low pH showed that the effect of
the metal ion was to hinder the proton assisted reaction by
blocking protonation at the more catalytically effective N-7
site and making the formation of the conjugate acid of the
nucleoside more difficult by virtue of the cationic charge on
52
the metal. HFLC analysis of the decomposition of
(dGuo)(NH ) Ru(III) at neutral pH reveals a complicated
3 5
3
product distribution. Assuming the general type of
reaction scheme outlined in Figure 1, it is possible to
determine: the net rate of disappearance for the dGuo complex
( k • k^ + *2 + £ 3 ) , the net rate of formation of the Gua
dl

dGuo—deoxyguanosine
Gua — guanine
dR — deoxyribose
1B
^ k4-*iGuaHNH3) _xLxRu
M 5
(Gua)(NH ) Ru*dR^C k ^(NH ) (L)Ru* Gua

3 5 5 3 5 +
K
(dGuo)(NH ) R *
3 5 u _J<2^(NH )5(L)Ru dGuo
3 +
( d G u o ) ( N H ) . L x R uk " c (NH
J 0 3 : ") -
5 x
k6 (Gua)(NH3)5
-
L , Ru% dGuo
xLxRue+dR
7 3 5 X x+
Figure 1. Proposed reaction scheme for the decomposition of (dGuo)(NH ) - s s
Ru(III) in neutral solution. dGuo is deoxyguanosine; Gua is guanine; and dR is

deoxyribose. (Reproduced with permission from Ref. 53.)

CLARKE Ruthenium Anticancer Agents 341
complex (kj), and the net disappearance of this species (k^

= k + k ) . At pH 7.0 plots of the yield of
4 5
(Gua)(NH ),Ru(III) as a function of time give rate constants

3
5 1 6 1 s
of: k j = 8.2 χ 10~ sec"" , k * 5.8 χ 10"" sec" and k
d x d 2
5
4.8 χ I0~" sec . In harmony with studies on the hydrolysis
of N-7 protonated and alkylated nucleosides, , the value
of k distinctly decreases in the range of the pK (7.6) for
1 ft
5 8
proton loss from the N-l site , so that this pathyway
becomes negligible above pH 8. However, over the same pH
range (6-8.5) a second pathway becomes dominant and yields an
as yet unidentified product (see below)·
Under physiological conditions the half-life for the
decomposition of (dGuo)(NH3) Ru(III) is approximately 4.5
5
3
days. Since both free guanine and deoxyguanosine are
observed to form, ligand dissociation is a competetive

reaction. Ammine ligand loss is known to be catalyzed by
base, so that it is likely that at least one of the as yet
unidentified products involves substitution of ammonia.
Another possibility is imidazole ring opening of the purine,
which isfcnownto occur with N-7 alkylated nucleosides in
54
basic media . Since comparable rates for the hydrolysis of
free deoxyguanosine are not available under the same
conditions, it is difficult to assess the effect of metal ion
coordination in depurinating nucleic acids; however, the
sugar hydrolysis rates probably represent an acceleration of
2 3
10 to 10 over those for the free ligand below pH 7.0.
Since the half-life of these reactions are relatively long
under physiological conditions, immediate metal ion induced
genetic effects are probably the result of the presence of
the metal ion on the nucleic acid rather than metal-catalyzed
depurination. This should be particularly true of ions of
lower charge such as Pt(II). However, in a slightly basic
environment at somewhat elevated temperatures a major side
reaction takes place, which may be a significant process for
metal ions of tripositive or higher charge.
Free Energy Correlations
A substantial amount of structural and acidity data on

pentaammineruthenium(III) complexes with purines and similar
ligands has been correlated through the modification of the
59
Kirkwood-Westheimer equation given in Eq.(l). Such a
correlation can only be expected to hold when dealing with
the same metal ion similarly coordinated to the identical
tautomeric form of a rigid ligand for which the pK value is ft
known. In addition, this relationship is valid only over a

fairly narrow range of r (between 3.3 and 5.6 A).

Nevertheless, these restrictions obtain for most purine and

pyrimidine complexes with (NH3) Ru(III), so that this and
5
related equations have considerable predictive power (as will

be seen below.)
2
ApK * 1 . 4 / r - 2.6
a (1)
In this approach ion-dipole interactions are assumed to

predominate and a l l other terms are simply collected into the
intercept value. An analogous correlation can be extracted
from reduction potential data and relates ΔΕ, the change in
reduction potential between the complex and its conjugate
base, and r. This relation has the distinct advantage of
lifting the requirement that identical tautomeric forms be
compared, which allows a significantly larger number of

5
complexes to be accomodated.
2
ΔΕ - 0.056/r - 0.1
Using these two equations it is easily possible to

derive additional relationships to predict the difference in
pK values between isostructural complexes of Ru(II) and
Ru(III).
Metal-Ion Movement on Purines and Pyrimidine s
Even though metal ions such as Ru(II), Ru(III) and

Pt(II) are usually fairly inert to substitution with the type
of nitrogen ligands found on nucleic acids, the close
juxtaposition of intrabase, intrastrand and interstrand sites
may facilitate metal ion movement so as to modulate mutagenic
or anticancer effects. Two clear examples of this have now
been studied with ruthenium ammine complexes which point out
the effects of both pH and electrochemical environments on
these linkage isomerization reactions.
Pentaammineruthenium(III)-hypoxanthine complexes undergo
a facile linkage isomerization from N-3 to N-9. Figure 2
shows that the rate of this reaction is substantially
decreased in the pH range above the pK for the N-3 ft
coordinated complex, owing to the residual anionic charge on

the pyrimidine. The negative activation entropy is in the
range predicted for a mechanism in which the metal ion is
simultaneously coordinated to both the N-3 and N-9 sites in
the activated complex leading to the loss of five degrees of
rotational freedom (four for the cis ammonias and one for the
metal center). The decrease in the linkage isomerization
rate between pH 1 and 2 (see*Figure 2) may be due to the
partial protonation of the N-9 site (pK - - 0 . 4 ) . The ft

2. Plot of k o68 vs. pH for the N3 to N9 linkage isomerization of (7-

MeHyp)(NH ) Ru(III).
3 5

subsequent increase in the rate in the pH range 0 - 1 is

surprising and may be due to a change in the ionic media from
+ + 4 5 6 2
predominately L i to H ' .
Despite the generally high stability of
pentaammineruthenium(II) complexes with aromatic nitrogen
heterocycles, complexes of this ion with adenosine and
cytidine ligands have proven to be somewhat unstable due to
the presence of the adjacent exocyclic amine. However, owing
to its greater pi-acceptor ability, the ligand
2-aminopyrimidine (2-AmPta) affords a complex suitable for
model studies. The effect of the exocyclic amine on the
stability of these complexes is apparent from a comparison of
(Pm)(NH|) Ru(II) (Pm - pyrimidine), which remains stable in
5
acid solution for extended periods, and

l-[(2-AmPm)(NH ) Ru(II)], which undergoes an acid catalyzed
3 5
dissociation. Even under neutral conditions the latter ·

complex persists with a half-life of only about an hour.
Figure 3 indicates that it is possible to add two
protons to this complex and that both catalyze the
hydrolysis. The first protonation undoubtedly occurs at the
free ring nitrogen, whereas the second may be at the
exocyclic nitrogen or directly on the metal. Space-filling
models suggest that a proton of the exocyclic amine impinges
on an octahedral face of the metal ion coordinated at the
adjacent ring nitrogen. Such a juxtaposition should
facilitate protonation of the metal center to form a labile
seven-coordinate intermediate^ · Interestingly,
l-[(4-aminopyridine)(NH ) Ru] has also been shown to
3 5
undergo a rapid acid catalyzed dissociation.

The formation of exocyclically coordinated
ruthenium(III) complexes of adenine and cytidine derivatives
has been proposed to proceed through initial coordination of
65
Ru(II) to the adjacent ring nitrogen (see Figure 4 ) . An
analogous mechanism, but not necessarily requiring oxidation
of the metal ion, has recently been suggested for the
synthesis of exocyclically coordinated platinum complexes
with these ligands , so that this type of linkage
isomerization may well be pertinent to the activity of
platinum group anticancer agents .
Use of Eq.(l), with an estimate of the pK for the ft
exocyclic nitrogen on adenosine to be in the range of 18,

allows prediction of the pE of the N-l coordinated Ru(III)
ft
complex to be approximately 8. This can be verified through

a determination of the pH dependence of the reduction
potential of this complex which is predicted to be:
1 1 1
[H+1K 11
+ Λ
E^ - Ε - .059 log 1 1
[H+]2 + [ f f ^ K

CLARKE Ruthenium Anticancer Agents 345
Figure 3. Plot of k vs. pH for the dissociation of 2'(AmPm)(NH,) Ru(IU).

0b8 5
pKi has been determined to be 1.79 by independent spectrophotometric titration.

pK is derived from the kinetic data.
2

Figure 4. Reaction mechanism for the formation of exocyclically coordinated

complexes of adenosine and cytidine. Step 4 is taken to be rate-limiting.

where Κ is the acid dissociation constant for the conjugate

acid of the Ru(II) form (with the proton presumably at N-3 or
N-7) and Κ is the equilibrium constant for the loss of a
proton from the exocyclic amine of the Ru(III) complex.
Electrochemical data fitted to this equation (see Figure 5)
determines the actual pK for l-CAdoiN^) Ru(III)] to be 8.2.
ft 5
The corresponding potential-pH curve for the model complex,

l-I(2-AmPm)(NH ) Ru(III)l, is similar with ρΚ^-Ι.β and
3 5
III
p K « 8 . 9 . The higher E° value of the 2-AmPm complex (363
mV) is consistent with stronger retrodative bonding between
Ru(II) and 2-AmPm than with Ado (342 mV).
Confirmation of the reaction mechanism is provided by
kinetic data dependent upon the same pK for the N-l ft
coordinated ruthenium(III) complex (see Figure 6). Owing to

the instability of l-[(Ado)(NH ) Ru(II)] and severe 3 5
restrictions required of the oxidizing partner, isomerization

kinetic rates were derived from cyclic voltammetrie data
68
using the method of Nicholson and Shain " , after forming
the N-l coordinated Ru(II) complex at the electrode surface
by electrolytic reduction of the N-6 bound Ru(III) species.
Since the specific rates estimated by this method were
independent of concentration, the rate law is taken to be
first order in the complex.
The reverse (N-6 to N-l) linkage isomerization has also
been studied and requires the metal to be in the dipositive
oxidation state on the protonated (neutral) ligand. Previous
electrochemical work determined the pK of a
6-[(Ado)(NH ) Ru(II)] to be 11.3, with deprotonation probably

3 5
occuring at N-l. A plot of specific rate constants for the

exo- to endocyclic movement of Ru(II) (as determined by the
cyclic voltammetrie method) versus pH shows that the kinetic
pK value of 10.6 falls within the range of the thermodynamic
ft
value. The limiting rate of the N-6 linkage isomerization of

,
6-[(5 -AMP)(NH ) Ru(II)l has been spectrophotometrically
3 5
1
determined to be 1.6 sec" following reduction of the
corresponding Ru(III) complex by either Cr(II) or Eu(II) over
1
the pH range 1 to 5. The electrochemical results indicate
that this rate becomes insignificant in the pH range
substantially above the pK . The activation parameters for
this reaction when measured at pH 3 are AH « 86 kJ/mol and
AS = 43.5 J/mol'K. Since both forward and reverse
isomer!ζations can occur at neutral pH, the adenine
coordination site on cellular nucleic acids should be
determined by the environmental electrochemical potential and
the relative efficiencies of the electron-transfer agents
present.
American Chemical
Society Library
1155 16ft St. N. w.
Washington,
ACS Symposium Series; 0. C.
American Chemical 20036
Society: Washington, DC, 1983.
500
Figure 5. Plot of reduction potential vs. pH for HAdo)(NH ) Ru(Ul).

s s


μη
Figure 6. 0 e
Plot of k & as determined from cyclic voltammetric data for the Nl to N6 linkage
isomerization of (Ado)(NH ) Ru(lll).
3 s
Acknowledgments This work was supported by PHS grant

GM26390. Particular acknowledgement is made of the
substantial assistance provided by P.H. Fackler, A. Abelleira
and J . Lacava in the preparation of the manuscript.
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and references therein.
2. Srivastava, S. C., Richards, P., Meinken, G. Ε., Som, P.,
Atkins, H. L . , Larson, M. S., Grunbaum, Z., Rasey, J. S.,
Dowling, M., Clarke, M. J., "Radiopharm. 2, Proc. Int.
Symp., 2nd", Sorenson, J. Α., Ed., Soc. Nucl. Med., Inc.,

New York, 1979, pp. 265-74.
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D.C. 1980, pp. 157-80.
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Kelman, A. D., J. Inorg. Biochem., 1980, 12, 79-87.
10. Taube, Η., Comments in Inorg. Chem., 1981, 1, 17-31.
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J., J. Clin. Hematol. Oncol., 1977, 7, 274.
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13. Giraldi, T., Sava, G., Bertoli, G., Mestroni, G.,
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16. Farrell, Ν., de Oliveira, N.G., Inorg. Chim. Acta., 1982,
in press.
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20. Matsunaga, K., Okayama Igakkai Zasshi, 1979, 91, 141-52.
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23. Srivastava, S. C., Richards, P., Meinken, G. Ε., Larson,

S. Μ., Grunbaum, Ζ., "Radiopharm.: Stuct.-Act. Relat.",
Spencer, R. P., Ed., Grune and Stratton, New York, 1981, pp.
207-23.
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Jacobsen, L. B., Schirrmacher, V., Biochim. Biophys. Acta,
1981, 634, 11-18.
25. Laliberte, J.-F., Crane, F. L . , Clarke, M. J., submitted
for publication, 1982.
26. See citations in reference 1.
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and 135-40.
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73-100.
29. Stern, P. Ε., Halpern, S. E . , Hagan, P. L . , Howell, S.

B., Dabbs, J. Ε., Gordon, R. M., J. Nat. Can. Inst., 1981,
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Compd. Radiopharm., 1980, 17, 421-30.
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Radiat. Isot., 1981, 32, 797-802.
38. Wenzel, Μ., Hoffmann, Κ., Naturwissenschaften. 1979, 66,
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40. Oster, Ζ. H., Som, P., Gil, M. C., Fairchild, R. G.,
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L., Meinken, G. Ε., Srivastava, S. C., Richards, P., Brill,
A. B., J. Nucl. Med.. 1981, 22, 269-73.
41. Schachner, E. R., Gil, M. C., Atkins, H. L . , Som, P.,
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Eur. J. Nucl. Med., 1981, 6, 403-5.
43. Oster, Ζ. Η., Som, P., Gil, M. C., Goldman, A. G.,
Fairchild, R. G., Meinken, G. E . , Srivastava, S. C., Atkins,
H. L . , Richards, P., Brill, Α. Β., Radiology. 1981, 141,
185-90.
44. Srivastava, S. C., Som, P., Meinken, G. Ε., Sewatkar, Α.,
Ku, T. H., 1981,
45. Kastner, M. E . , Coffey, K. F., Clarke, M. J., Edmonds, S.

Ε., Eriks, Κ., J. Am. Chem. Soc., 1981, 103, 5747-52.

46. Graves, B. J., Hodgson, D. J., J. Am. Chem. Soc., 1979,
101, 5608.
47. Marzilli, L. G., Kistenmacher, T. J., Eichhorn, G. L . ,
Met. Ions in Biol., 1980, 1, 179-250.
48. Marzilli, L. G., Prog. Inorg. Chem., 1977, 23, 255-378.
49. Hodgson, D. J., Prog. Inorg. Chem., 1977, 23, 211-254.
50. Gellert, R. W., Ban, R., Met. Ions Biol. Syst., 1979, 8,
1-49.
51. Thomson, A. J., Williams, R. J. P., Resolva, S., Struct.
Bonding. 1972, 11, 1.
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5413-18.
53. Clarke, M. J., Perpall, Η., Coffey, Κ. Ε., Anal.
Biochem., 1982, in press.

54. Zoltewicz, J. Α., Clark, D. F . , Sharpless, T. W., Grahe,
G., J. Am. Chem. Soc., 1970, 92, 1741-50.
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1193-97.
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Chem. Soc., 1978, 100, 7620-24.
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Β., Brnylants, Α., J. Am. Chem. Soc., 1972, 94, 4715-20.
58. Morrissey, P. Ε., Clarke, M. J., unpublished results,
1982
59. Perrin, D. D., Dempsey, B., Sergeant, E. P., "pKa
Prediction for Organic Acids and Bases", Chapman and Hall,
New York, 1981.
60. Clarke, M. J., Inorg. Chem., 1977, 16, 738.
61. Coffey, K. F . , M. S. Thesis, Boston College, 1981.
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4021-23.
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66. Faggiani, R., Lippert, Β., Lock, C. J. L . , Speranzini, R.
Α., J. Am. Chem. Soc., 1981, 103, 1111.
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results, 1979.

18
O v e r v i e w and C u r r e n t Status of G o l d - C o n t a i n i n g
Antiarthritic D r u g s
BLAINE M. SUTTON
Smith Kline and French Laboratories, Philadelphia, PA 19101
Auranofin, (2,3,4,6-tetra-O-acetyl-1-thio-β-
D-glucopyranosato-S)(triethylphosphine)gold is a new
compound for treating rheumatoid arthritis (RA).
Gold has a long medical history. Gold compounds
were first investigated for RA treatment because of
the reported relief of joint pain in tubercular
patients being treated unsuccessfully with sodium
aurothiosulfate. Use of gold therapy and its
mechanism of therapeutic benefit have long been
controversial. However, clinical studies reported
in 1960 confirmed its antiarthritic value.
Auranofin has several potential advantages over gold
compounds in use today, most important is its route
of administration. It is clinically effective when
administered orally. Other gold compounds must be
given by injection. Auranofin's unique therapeutic
properties may be due in part to physical and
chemical characteristics which differ markedly from
those of currently used agents.
Excellent studies of the interaction of platinum

compounds with biologically important small and
macromolecular species have been reported both in this
volume and elsewhere (1). Consequences of these
observations have been associated with proposed modes of
antineoplastic action of cis-platinum (II) compounds* The
relatively recent discovery of this new class of antineo
plastic agents (2) has led to an intensification of research
into the therapeutic potential of transition metal
compounds. It is surprising that the intensity of
scientific zeal directed towards this class of
antineoplastic agents has not focused on gold compounds,
probably one of the first metals to be used in treatment of
0097-6156/83/0209-0355$06.00/0

disease and one that s t i l l holds a prominent p o s i t i o n as a

s p e c i f i c therapeutic form f o r treatment of a c t i v e rheumatoid
a r t h r i t i s (RA).
History
Gold u n l i k e platinum i s an ancient metal and has a long

medical h i s t o r y . I t was probably one of the f i r s t to
a t t r a c t the a t t e n t i o n of man, because i t i s one of the few
that e x i s t e d i n the elemental s t a t e i n nature. Being
r e l a t i v e l y i n e r t i t r e t a i n s i t s l u s t e r and does not t a r n i s h
on exposure to a i r . Since pure gold was too s o f t to form
u s e f u l implements, p r i m i t i v e man valued gold f o r ornamental
purposes. The ancient Egyptians obtained t h e i r gold i n

a l l u v i a l diggings from the sand and g r a v e l i n s e v e r a l
d i s t r i c t s between the N i l e and the Red Sea, the Nubian
1
area. The Rameside papyrus', the o l d e s t map i n the world,
l o c a t e s these d i s t r i c t s ( 3 ) . Ornamental gold became a s i g n
o f c h i e f t a i n s h i p and remains today as evidence of personal
wealth* I t s a c q u i s i t i o n has enslaved unknown numbers of
people from ancient to modern times and has motivated
unbelievable a c t s of v i o l e n c e and crime: these l i n e s from
the Greek w r i t e r Phyoclides ( 4 ) .
"Gold and s i l v e r are i n j u r i o u s to mortals: gold i s the
source of crime, the plague o f l i f e and r u i n of a l l
things* Would be that thou were not such an a t t r a c t i v e
scourge: because of these a r i s e r o b b e r i e s , homicide,
brothers are maddened against brothers and c h i l d r e n
against parents"·
The b i o l o g i c a l mystique a t t r i b u t e d to gold i n h i s t o r y
equaled and p o s s i b l y surpassed i t s monetary a t t r a c t i o n (5>)*
The Egyptian Sun God Ra i n the Pyramid Age was b e l i e v e d to
be the procréator o f kings* The l i q u i d of Ra, the gold o f
the gods and goddesses, flowed i n t h e i r veins and gave them
strength and endurance* Search f o r the golden l i v e r of l i f e
was prevalent i n ancient India and P e r s i a * Thus there i s
l i t t l e wonder that gold e a s i l y occupied a m a g i c a l l y honored
place when i t was considered f o r therapeutic purposes*
Older races respected i t s c u r a t i v e power when l a i d upon
parts of the body with s u i t a b l e i n c a n t a t i o n s or worn as
r i n g s or amulets* I t s golden c o l o r i d e n t i f i e d i t as a
remedy f o r jaundice "the Golden Disease" among the Hindus
and l a t e r the Greeks, southern Persians and Germans (60.
As e a r l y as 2500 BC the Chinese employed gold f o r
b i o l o g i c a l b e n e f i t (7)· Ancient Arabic physicians a l s o
record i t s b e n e f i t * Gold was revered i n medieval medicine
and h e l d an honored place i n pharmacopoeias of those days*
P l i n e y describes manifold e f f i c a c i o u s uses of gold i n
wounded persons, to ward o f f sorcerous curses; i n ashen form

18. SUTTON Gold-Containing Antiarthritic Drugs 357
to cure f i s t u l a s and discharges, p u t r i d u l c e r s and sores;

b o i l e d i n honey as a purgative l i n i m e n t (50· Gold i n some
form has been recommended as a panacea f o r a l l diseases from
the 8th Century by Abn Moussa the Wise t o as r e c e n t l y as
1500 AD by Paracelsus ( 7 ) . Homeopathic medicine i n the 18th
and 19th century valued the b e n e f i t o f gold therapy ( 8 ) .
Hahnemann p r a i s e d the v i r t u e o f g o l d i n homeopathy,
published a pharmacodynamic study o f powdered g o l d and i n
a s s o c i a t i o n w i t h ten other p h y s i c i a n s experienced and
d e s c r i b e d b i z a r r e b i o l o g i c a l responses a f t e r the i n g e s t i o n
o f t r i t u r a t e d gold l e a f (aurum f o l i a t i u r a ) . He a l s o reported
i n 1879 treatment o f a 55 year o l d women w i t h b a c t e r i a l
e n d o c a r d i t i s , n o t i n g improvement i n the heart as w e l l as the
j o i n t s , freed o f rheumatism.
Robert Koch reported the f i r s t experimental

a n t i b a c t e r i a l a c t i v i t y o f gold s a l t s i n 1890 ( 9 ) . He
described i n v i t r o a n t i t u b e r c u l a r a c t i v i t y o f gold cyanide
at a d i l u t i o n o f 1 t o 2 m i l l i o n , a c o n c e n t r a t i o n much too
d i l u t e t o invoke cyanide i o n as the a c t i v e agent. When he
l a t e r f a i l e d t o demonstrate a n t i t u b e r c u l a r a c t i v i t y w i t h
gold s a l t s i n experimental animals, Koch terminated h i s work
w i t h gold compounds. However h i s observations d i d not go
unnoticed. D i f f e r e n t p h y s i c i a n s administered i n o r g a n i c gold
s a l t s o f t e n i n t r a v e n o u s l y i n l a r g e doses and reported
b e n e f i c i a l r e s u l t s i n s k i n t u b e r c u l o s i s , s y p h i l i s (10),
lupus v u l g a r i s (11) and pulmonary t u b e r c u l o s i s (12). As one
might suspect i n many o f these cases the treatment was
probably more t e r m i n a l than the d i s e a s e . The i n t r o d u c t i o n
o f g o l d sodium t h i o s u l f a t e (Sanochrysin) by Mollgaard i n
1924 r e k i n d l e d use o f gold compounds f o r t u b e r c u l o s i s
therapy (13) and sparked what P. D'Arcy Hart termed the
"Gold Decade" (1925-1935) (14). The mystique o f gold
therapy again caught the p u b l i c imagination* D i f f e r e n t
chemical forms (15) and s m a l l e r dosages reduced f a t a l i t i e s
due t o the therapy. However t o x i c i t y and i n c o n c l u s i v e
t h e r a p e u t i c b e n e f i t c o n t r i b u t e d t o a d e c l i n e i n the use o f
g o l d compounds f o r treatment o f t u b e r c u l o s i s . Although the
r e a l value o f gold therapy f o r t u b e r c u l o s i s may never be
answered, the c l i n i c a l b e n e f i t , h i s t o r i c a l l y , d i d not
outweigh the p e r s i s t e n t r i s k a s s o c i a t e d w i t h i t s t o x i c i t y .
Development o f Chrysotherapy f o r Rheumatoid A r t h r i t i s
Lande f i r s t used aurothioglucose ( S o l g a n a l , Schering) t o

t r e a t non-tuberculosis i n f e c t i o n s (16). He t r e a t e d a group
of 39 p a t i e n t s w i t h b a c t e r i a l e n d o c a r d i t i s and v a r i o u s
p o o r l y defined diseases i n c l u d i n g rheumatic f e v e r . H i s
o b s e r v a t i o n o f the s i g n i f i c a n t l o s s o f j o i n t p a i n i n these
p a t i e n t s and recommendations f o r t r i a l s t u d i e s i n chronic
a r t h r i t i s l e d J . F o r e s t i e r t o i n v e s t i g a t e the use o f gold

compounds f o r treatment of rheumatoid a r t h r i t i s (17)· Six

years l a t e r (1935) F o r e s t i e r reported evidence of the
b e n e f i c i a l a c t i o n of gold s a l t s on the e v o l u t i o n of chronic
rheumatoid a r t h r i t i s (18). In h i s i n i t i a l s t u d i e s he used
sodium aurothiopropanolsulfonate ( A l l o c h r y s i n e ) . Later he
used other preparations sodium aurothiomalate (Myochrysine),
aurothioglucose (Solganal) and o t h e r s . He recommended that
the preparations be administered i n t r a m u s c u l a r l y and noted
that c o l l o i d a l g o l d , gold c h l o r i d e and gold cyanide were not
useful.
Chrysotherapy was not r e a d i l y accepted i n the United
S t a t e s . As l a t e as 1934, C e c i l reported that drugs played a
r e l a t i v e l y minor r o l e i n treatment of rheumatoid a r t h r i t i s
and that h i s experience w i t h gold compounds had never shown
s t r i k i n g r e s u l t s (19).
The f i r s t report of the s u c c e s s f u l use of gold compounds
i n t r e a t i n g a r t h r i t i s induced i n experimental animals was
that of Sabin and Warren i n 1940 (20). Intravenous
i n j e c t i o n of a pleuropneumonia-like microorganism (Type Β
s t r a i n ) w i t h a p a r t i c u l a r a f f i n i t y f o r the j o i n t s of the
animals (mice) produced a migratory and progressive
p o l y a r t h r i t i s l e a d i n g to a n k y l o s i s i n two to four months. A
v a r i e t y of g o l d ( l ) t h i o l a t e s e x h i b i t e d p r o p h y l a c t i c
prevention as w e l l as t h e r a p e u t i c r e v e r s a l of the a r t h r i t i c
syndrome i n the i n f e c t e d mice. I t was noted that the
i n f e c t i o u s agent was c l e a r e d q u i c k l y from the v a s c u l a r
system and l o c a l i z e d i n the j o i n t areas. G o l d ( l ) t h i o l a t e s
d i d not i n h i b i t growth o f the pleuropneumonia-like
microorganism i n v i t r o l e a d i n g the authors to hypothesize an
i n v i v o a c t i v a t i o n of the r e t i c u l o e n d o t h e l i a l system as a
mechanism whereby the gold s a l t s produced t h e i r e f f e c t s .
G o l d ( l l l ) i n the form of sodium t e t r a c h l o r o a u r a t e was
somewhat b e n e f i c i a l at a near t o x i c dose l e v e l . C o l l o i d a l
g o l d was i n e f f e c t i v e . Freyberg and co-workers l a t e r
reproduced t h i s a r t h r i t i c mouse model and demonstrated that
the b e n e f i c i a l e f f e c t s of gold c o o r d i n a t i o n compounds
r e s i d e d i n the gold c o n t a i n i n g complexes and not i n the
t h i o l l i g a n d (21). Sodium aurothiomalate was 100% p r o t e c t i v e
when administered i n t r a v e n o u s l y at a dose of 2 or 4 mg/mouse
on a l t e r n a t e days u n t i l 9 or 10 i n j e c t i o n s had been given.
Sodium thiomalate was i n e f f e c t i v e at doses as h i g h as 15.4
mg administered i n the same regimen. Sodium succinimide
aurate, a compound not c o n t a i n i n g a t h i o l l i g a n d was e q u a l l y
e f f e c t i v e as sodium aurothiomalate, r a i s i n g f u r t h e r question
as to the r o l e of the t h i o l l i g a n d i n the t h e r a p e u t i c
response.
C o n f l i c t i n g r e p o r t s of the b e n e f i t of chrysotherapy i n
rheumatoid a r t h r i t i s motivated S i r Stanley Davidson of the
Empire Rheumatism C o u n c i l (Great B r i t a i n ) to p l a n f o r a

18. SUTTON Gold-Containing Antiarthritic Drugs
m u l t i c e n t e r , double b l i n d study o f the treatment i n 1938.

The Second World War took p r i o r i t y so that the study was not
i n i t i a t e d u n t i l 1957. The f i r s t report o f t h i s study i n
1960 s t a t e d that "by a l l c r i t e r i a except r a d i o l o g i c a l ,
p a t i e n t s w i t h acute rheumatoid a r t h r i t i s t r e a t e d , over a
p e r i o d o f f i v e months, w i t h a t o t a l treatment dose o f l g o f
sodium aurothiomalate fared b e t t e r than those t r e a t e d w i t h a
t o t a l dose o f 0.01g o f the same substance given i n a double
b l i n d t r i a l over the same p e r i o d " (22). The f i n a l r e p o r t as
recent as 1961 compared the r e s u l t s o f 77 p a t i e n t s t r e a t e d
w i t h a f i v e months course o f twenty weekly gold i n j e c t i o n s
w i t h 81 c o n t r o l s followed f o r a t o t a l o f 30 months. I n
general "gold t r e a t e d p a t i e n t s fared b e t t e r than c o n t r o l s
from the t h i r d t o s i x t h month and on the whole up t o month
18, one f u l l year a f t e r completion o f the 5 month course o f

treatment. Thereafter gold t r e a t e d p a t i e n t s d e t e r i o r a t e d t o
an appreciable extent although they r e t a i n e d some s m a l l
advantage over c o n t r o l s a t 30 months". No s i g n i f i c a n t
r a d i o l o g i c a l d i f f e r e n c e was found between the gold t r e a t e d
and c o n t r o l groups (23). Data from t h i s study supported the
e f f i c a c y o f chrysotherapy i n p a t i e n t s w i t h rheumatoid
a r t h r i t i s but d i d not d e s c r i b e a dosage regimen that was
c o n s i s t e n t w i t h a r e l i a b l e c l i n i c a l response.
Current A p p l i c a t i o n s
Although the report o f the Empire Rheumatism C o u n c i l

e s t a b l i s h e d the b e n e f i t o f gold therapy, i t was o n l y
subsequent t o the r i s e and f a l l o f the c o r t i c o s t e r o i d
a r t h r i t i c panacea p e r i o d (24) that rheumatology reevaluated
gold preparations as major t h e r a p e u t i c m o d a l i t i e s f o r RA
(25). Recent s t u d i e s have presented r a d i o l o g i c a l evidence
t h a t gold therapy slows and o f t e n terminates the progress o f
degenerative j o i n t changes a s s o c i a t e d w i t h the disease
process (26). However there i s yet no u n i v e r s a l agreement
upon the p a t i e n t p o p u l a t i o n that should r e c e i v e
chrysotherapy, the most appropriate time t o i n i t i a t e therapy
nor the most appropriate dosage regimen. There a l s o does
not appear t o be a c o r r e l a t i o n between blood gold l e v e l s and
a t h e r a p e u t i c response. The degree o f a l l o w a b l e s i d e e f f e c t
s e v e r i t y f o r continued therapy i s unresolved and the
mechanisms o f the a n t i a r t h r i t i c a c t i v i t y o f gold compounds
are r e c e i v i n g continued but not n e c e s s a r i l y c o n c l u s i v e
a t t e n t i o n . However, i t i s now accepted that b e n e f i t o f gold
therapy outweighs the r i s k a s s o c i a t e d w i t h i t s use and that
low maintenance therapy i s appropriate i n those p a t i e n t s who
have responded t o treatment (27).
Since the Report o f the B r i t i s h Rheumatism C o u n c i l ,
there has been l i t t l e change i n the chemical forms o f gold

used to t r e a t RA (sodium aurothiomalate and a u r o t h i o g l u c o s e )

i n the United S t a t e s . However, there have been notable
m o d i f i c a t i o n s of the dosage regimens employed w i t h the
i n t e n t o f decreasing t o x i c r e a c t i o n s and i n c r e a s i n g
t h e r a p e u t i c responses* The present conventional treatment
procedure i n v o l v e s an i n i t i a l intramuscular i n j e c t i o n of a
gold compound c o n t a i n i n g 10 mg of gold* This i n i t i a l small
dose i d e n t i f i e s those p a t i e n t s that might be h y p e r s e n s i t i v e
to g o l d therapy and should not continue treatment*
Thereafter the dose i s increased stepwise i n the next two to
three weeks to 25 mg and 50 mg (based on Au content of the
compound). A f t e r a dose l e v e l of compound c o n t a i n i n g 50 mg
o f g o l d has been reached, i t i s continued f o r twenty weeks
u n t i l a t o t a l of l g of gold has been administered. I f a
c l i n i c a l response occurs, weekly dosing i s g r a d u a l l y changed

to a maintenance regimen c o n s i s t i n g o f a s i n g l e monthly
i n j e c t i o n of compound c o n t a i n i n g 50 mg of gold (28) ·
Serum gold l e v e l s i n p a t i e n t s on t h i s t h e r a p e u t i c
regimen are q u i t e v a r i a b l e reaching 4 to 8 ug/ml a f t e r the
i n j e c t i o n and f a l l i n g o f f to 0.75 to 1.25 ug/ml when the
gold compounds are administered on a monthly bases. Lorber
(29) has claimed that a more enhanced and prolonged c l i n i c a l
response can be obtained i f the gold compounds are
administered at weekly doses s u f f i c i e n t to m a i n t a i n a steady
s t a t e serum gold l e v e l of 3 ug/ml, however, c o r r e l a t i o n of
serum gold l e v e l s w i t h c l i n i c a l response has been challenged
repeatedly (28). Gold t h i o l a t e s comprise the major c l a s s o f
gold compounds used today t o t r e a t RA, sodium
aurothiomalate, Myochrisine (I), and a u r o t h i o g l u c o s e ,
Solganal (£), being the drugs of choice i n the United
S t a t e s . The former i s administered as an i n j e c t a b l e aqueous
s o l u t i o n and the l a t t e r as an i n j e c t a b l e suspension i n o i l .
lAu
HO
1 2

18. SUTTON Gold-Containing Antiarthritic Drugs
New Developments
The mechanisms whereby g o l d ( l ) compounds a r r e s t

progression o f the disease syndrome i n RA are not w e l l
understood. L e i b f a r t h and P e r s e l l i n i n a recent review
h i g h l i g h t t h i s f a c t (30). They evaluated the a n t i m i c r o b i a l ,
antiinflammatory, antienzyme and immunosuppressive
a c t i v i t i e s r e p o r t e d l y observed w i t h d i f f e r e n t i n j e c t a b l e
gold products. Their conclusions favored antiinflammatory
and antienzyme a c t i v i t i e s of g o l d ( l ) compounds as being the
best documented mechanisms i n v o l v e d i n a r r e s t i n g the
progressive c r i p p l i n g processes a s s o c i a t e d w i t h RA (Table
I)· I n t e r f e r e n c e w i t h other proposed mediators o f
inflammation (macrophage a c t i v a t i o n , r e l e a s e of mast c e l l
r e a c t o r s , chemotactic f a c t o r s , p r o s t a g l a n d i n p a r t i c i p a t i o n )
were considered i n need o f f u r t h e r study.
We reported p r e v i o u s l y on a s e r i e s of phosphine g o l d
c o o r d i n a t i o n compounds that produced a n t i a r t h r i t i c a c t i v i t y
i n adjuvant induced a r t h r i t i s i n r a t s when administered by
the o r a l route (32). A l l other gold compounds are e f f e c t i v e
o n l y when administered p a r e n t e r a l l y . One member from t h i s
group, (2,3,4,6-tetra-0-acetyl-l-thio-3-D-glucopyranosato-S_)
( t r i e t h y l p h o s p h i n e ) g o l d , a u r a n o f i n (jj}, e x h i b i t e d potent
a c t i v i t y o f t h i s type and i s now undergoing c l i n i c a l t r i a l .
CHoOAc
S-Au-P-Et
This compound d i f f e r s from c u r r e n t l y used gold t h i o l a t e s

i n that the g o l d ( l ) i n the molecule i s two coordinated
through t h i o l and phosphine l i g a n d s . This novel molecular
environment appears to endow a u r a n o f i n w i t h p h y s i c a l and
b i o l o g i c a l p r o p e r t i e s unique to g o l d compounds i n c u r r e n t
t h e r a p e u t i c use. Auranofin e x i s t s as a monomolecular
species i n the s o l i d s t a t e and i n s o l u t i o n as determined by
x-ray c r y s t a l l o g r a p h i c s t u d i e s and osmometric molecular
weight determinations (33, 34). Mossbauer s p e c t r o s c o p i c
s t u d i e s confirm both the monovalent s t a t e of gold i n the
molecule as w e l l as the monomolecular nature of the compound
(35). E a r l i e r Mossbauer Spectroscopic s t u d i e s have shown
t h a t there i s a l i n e a r r e l a t i o n s h i p between isomer s h i f t
(IS) and quadrupole s p l i t t i n g (QS) f o r two c o o r d i n a t e ,
monovalent gold compounds which d i f f e r s s i g n i f i c a n t l y from
that of t r i v a l e n t gold (36). The p l o t i n Figure 1 l o c a t e s
a u r o t h i o l a t e s , aurothioglucose and sodium aurothiomalate i n
the lower r e g i o n of that l i n e a r p l o t i n an area where gold
i s coordinated to two s u l f u r atoms, i . e . b i s - ( e t h y l e n e t h i o -

Table I
I n h i b i t i o n of Enzyme Activity
b y G o l d Compounds, i n v i t r o (30)
Enzyme Enzyme S o u r c e Compound Molar Inh. Cone.

-3
guinea pig peritoneal macrophage 5x10
-4
Acid Phosphatase human s y n o v i a l f l u i d Sodium Aurothiomalate 2x10
-3
guinea p i g p e r i t o n e a l macrophage 5x10
B-Glucuronidase* -5
human s y n o v i a l . f l u i d Sodium Aurothiomalate 5x10
$-Glucosaminidase human s y n o v i a l f l u i d
-4
plasma l e u k o c y t e s Sodium Aurothiomalate
1x10
3 4 w
Elastase human leukocytes Sodium Aurothiomalate 5xl0" -10" H
>
Cathepsin human s y n o v i a l fluid 4x10
s
Collagenase human leukocyte Sodium Aurothiosulfate m
1x10
ι
w
* L e w i s et a l . r e c e n t l y r e p o r t e d t h a t sodium a u r o t h i o m a l a t e ( 1 χ 1 0 ~ M) failed to inhibit 3-glucuroni-
d a s e and 3 - g l u c o s a m i n i d a s e d e r i v e d f r o m mouse m a c r o p h a g e s (31). w

Reproduced with permission from Ref. 30.
>
o
w
Η

1 2 3 4 * neii/ sec
F/gwre i . P/oi sfowmg relationship between isomer shifts (IS) and quadrupole splitting (QS) for S
go/d compounds.
u r o n i u m ) g o l d ( l ) c h l o r i d e , e t c . The c l o s e f i t to the
two-coordinate l i n e i n d i c a t e s t h a t i n the s o l i d s t a t e these
compounds are two-coordinate through two s u l f u r l i g a n d s * In
c o n t r a s t a u r a n o f i n i s l o c a t e d i n upper r e g i o n of the p l o t , a
l o c a t i o n occupied by monomeric molecular species*
These data as w e l l as nmr, EXAFS and g e l f i l t r a t i o n
s t u d i e s i n d i c a t e t h a t g o l d t h i o l a t e s , sodium aurothiomalate
and a u r o t h i o g l u c o s e , e x i s t i n polymeric s t a t e s (37, 38,
39)* S u i t a b l e c r y s t a l l i n e forms have not been prepared t o
i n v e s t i g a t e these molecules by x-ray c r y s t a l l o g r a p h y * In
f a c t the s t r u c t u r e of g o l d t h i o l a t e s i s b e t t e r represented
by 4 r a t h e r than those s t r u c t u r e s d e s c r i b e d e a r l i e r *
Auranofin i s a l i p o p h i l i c , n o n i o n i c compound whereas g o l d
t h i o l a t e s i n general are h y d r o p h i l i c (40, 41)* The

characteristic physical
2 2 +
R= (SCHCO2CH2CO2) ~" Na , sodium aurothiomalate
s
R SC6H12O5, a u r o t h i o g l u c o s e
p r o p e r t i e s o f a u r a n o f i n ( l i p o p h i l i c i t y , monomolecular,
n o n - i o n i c ) are d i f f e r e n t from those o f g o l d t h i o l a t e s i n
c l i n i c a l use and may c o n t r i b u t e t o i t s novel b i o l o g i c a l
properties*
No exact experimental model of rheumatoid a r t h r i t i s has
been developed* However i n numerous b i o l o g i c a l assays
designed t o i n t e r p r e t drug e f f e c t on models of or events
a s s o c i a t e d w i t h RA, a u r a n o f i n produced d i s t i n c t l y d i f f e r e n t
responses than g o l d t h i o l a t e s * Auranofin i n h i b i t e d the
development of e x p e r i m e n t a l l y induced adjuvant a r t h r i t i s i n
r a t s when administered o r a l l y * Sodium aurothiomalate
i n h i b i t e d only when administered i n t r a m u s c u l a r l y (31, 42)*
Serum g o l d l e v e l s i n animals t r e a t e d e f f e c t i v e l y w i t h
a u r a n o f i n were approximately one t h i r d of those t r e a t e d w i t h
sodium aurothiomalate* Kidney r e t e n t i o n of gold i n the
sodium aurothiomalate animals was ten o r more times t h a t o f
a u r a n o f i n t r e a t e d animals* Thus a u r a n o f i n produced
a n t i a r t h r i t i c a c t i v i t y i n t h i s a r t h r i t i c animal model a f t e r
o r a l a d m i n i s t r a t i o n a t lower serum concentrations than those
r e q u i r e d by sodium aurothiomalate w i t h much l e s s kidney g o l d
retention*

Gold i n the blood f o l l o w i n g the a d m i n i s t r a t i o n of sodium

aurothiomalate i s a s s o c i a t e d p r i m a r i l y w i t h p r o t e i n s i n the
serum. In c o n t r a s t gold from o r a l a d m i n i s t r a t i o n o f
a u r a n o f i n i s e q u a l l y d i s t r i b u t e d between the c e l l u l a r
components and serum p r o t e i n s o f the blood during the 24-48
h r . p e r i o d a f t e r a d m i n i s t r a t i o n . A f t e r seventy hours
a u r a n o f i n gold i s a l s o l o c a l i z e d i n the serum p r o t e i n s
(43). The b i o l o g i c a l s i g n i f i c a n c e o f the property has not
been i n t e r p r e t e d .
Tissue damage present i n RA i s a t t r i b u t e d i n part t o
a c t i o n of l y t i c lysosomal enzymes released by phagocytic
c e l l s i n inflammed t i s s u e . Auranofin i n h i b i t s the r e l e a s e
o f lysosomal enzymes from phagocytizing leukocytes a t
micromolar concentrations (1-10 uM) (44, 45). I t d i d not
produce leukocyte c y t o t o x i c i t y nor i n h i b i t i o n o f c e l l f r e e

lysosomal enzyme a c t i v i t y at these e f f e c t i v e
c o n c e n t r a t i o n s . Sodium aurothiomalate, aurothioglucose and
sodium a u r o t h i o s u l f a t e d i d not e x h i b i t t h i s potent a c t i v i t y .
The e t i o l o g y and pathogenesis o f RA are unknown, however
both humoral and c e l l mediated immune responsiveness are
thought to p l a y a r o l e i n the pathogenesis o f chronic
inflammation (46, 47). Auranofin has been shown to suppress
humoral immunity (antibody production) i n assay systems i n
which sodium aurothiomalate was e i t h e r i n e f f e c t i v e o r
s t i m u l a t e d antibody production (48). In other s t u d i e s both
a u r a n o f i n and sodium aurothiomalate were shown to be capable
of s t i m u l a t i n g l o w - l e v e l delayed h y p e r s e n s i t i v i t y i n mice
but o n l y a u r a n o f i n was capable o f r e s t o r i n g iramunosuppressed
(methotrexate) c e l l mediated immunity (49). These
c o n t r a s t i n g immunopharmacological p r o p e r t i e s o f a u r a n o f i n ,
suppression o f humoral immunity and enhancement of c e l l
mediated immunity, appear i d e a l l y s u i t e d to c o r r e c t
immunological abnormalites observed i n RA (hyperactive
humoral immunity, suppressed c e l l mediated immunity).
U n l i k e the t r a n s i t i o n elements, copper and i r o n , which
are normal body c o n s t i u e n t s , gold does not have a w e l l
defined t r a n s p o r t , storage or enzyme f u n c t i o n w i t h i n the
body. Gold t h i o l a t e compounds used c l i n i c a l l y to t r e a t RA
have l i m i t i n g s i d e e f f e c t l i a b i l i t y , i . e . m i l d d e r m a t i t i s ,
p r o t e i n u r e a , nephrosis, e x f o l i a t i v e d e r m a t i t i s , leukopenia,
thrombocytopenia. Auranofin, f i r s t studied c l i n i c a l l y by
F i n k e l s t e i n (50) and B e r g l o f (51), i s now undergoing
extensive c l i n i c a l e v a l u a t i o n . The r e s u l t s o f i t s use i n
treatment o f 1200 RA p a t i e n t s f o r periods up to 3 years have
been reported t o the Food and Drug A d m i n i s t r a t i o n . Both
symptomatic and s e r o l o g i c a l improvement was seen i n p a t i e n t s
a f t e r s i x months treatment. A dose o f 6mg/day o f drug was
observed to be safe and e f f e c t i v e . Side e f f e c t s r e q u i r i n g

discontinuance of therapy have been minimal, about 10%· The

most prevalent of these were m i l d d i a r r h e a and s k i n rash*
The m a j o r i t y of p a t i e n t s showing these symptoms continued
treatment without f u r t h e r c o m p l i c a t i o n s * Data c o l l e c t e d t o
date i n d i c a t e that a u r a n o f i n i s a safe and e f f e c t i v e form o f
chrysotherapy f o r treatment of RA. I t has s e v e r a l p o t e n t i a l
advantages: o r a l dose form, no p a i n from i n j e c t i o n , no
n i t r i t o i d r e a c t i o n , p o t e n t i a l low t i s s u e accumulation o f
gold (52).
Future A p p l i c a t i o n s
As a r e s u l t of a u r a n o f i n , chrysotherapy promises to f i n d
a new p o s i t i o n i n the treatment of RA* Continued research
i n t o i t s mechanism of a c t i o n i s necessary* Development o f

more e f f e c t i v e gold agents should be the o b j e c t of continued
a t t e n t i o n which w i l l r e q u i r e extensive comparative
l a b o r a t o r y and c l i n i c a l study w i t h heavy research investment*
Metal c o n t a i n i n g compounds are a t t r a c t i n g i n c r e a s i n g
a t t e n t i o n as a n t i n e o p l a s t i c agents* Cisplatin i s
e s t a b l i s h e d as u s e f u l therapy f o r treatment of t e s t i c u l a r
and o v a r i a n tumors, although a s s o c i a t e d severe s i d e e f f e c t s
l i m i t the doses that may be given to p a t i e n t s * Work i s
underway to develop l e s s t o x i c , e q u a l l y e f f i c a c i o u s agents
(53)* Palladium ( I I ) complexes r e c e n t l y have been reported
to e x h i b i t a n t i n e o p l a s t i c p o t e n t i a l i n i n v i t r o s t u d i e s , i n
v i v o a c t i v i t y has not been reported (54, 55)* Simon et a l
observed i n v i t r o i n h i b i t i o n of DNA synthesis and
a n t i p r o l i f e r a t i v e a c t i v i t y w i t h a u r a n o f i n (56, 57)· In
l i g h t of the low l e v e l of s e r i o u s s i d e e f f e c t s observed i n
c l i n i c a l s t u d i e s , these authors speculated on i t s
a n t i n e o p l a s t i c p o t e n t i a l * Other workers have observed
i n h i b i t i o n of lymphocyte DNA synthesis w i t h a u r a n o f i n ,
however t h e i r data i n d i c a t e that the i n h i b i t i o n was a r e s u l t
of i n t e r f e r e n c e w i t h membrane transport a c t i v i t y r a t h e r than
d i r e c t a c t i o n on DNA synthesis (58)*
Simon et a l a l s o reported antitumor a c t i v i t y w i t h
a u r a n o f i n u s i n g mouse lymphocytic leukemia P388 (59)*
Studies c a r r i e d out at the Southern Research I n s t i t u t e
i n d i c a t e d t h a t a u r a n o f i n at maximum non-toxic doses
e x h i b i t e d minimal a c t i v i t y i n the P388 system and that
continued treatment f a i l e d to reduce the tumor burden or
prolong animal s u r v i v a l time* In c o n t r a s t c i s p l a t i n both
reduced tumor burden and prolonged animal s u r v i v a l (60).
Although a u r a n o f i n , i t s e l f , does not appear to have
potent a n t i n e o p l a s t i c a c t i v i t y , r e s u l t s obtained are
encouraging. I n v e s t i g a t i o n of the a n t i n e o p l a s t i c a c t i v i t y
of other g o l d ( l ) as w e l l as g o l d ( l l l ) compounds appears
j u s t i f i e d . G o I d ( i l l ) compounds may be more a t t r a c t i v e i n
l i g h t of the s i m i l a r s t r u c t u r a l c h a r a c t e r i s t i c s o f

gold(lll), platinum(ll) and palladiumillj coordination

compounds.
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J. Am. Chem. Soc. 1981, 103, 3423.
55. Graff, G.M. Chem. Week 1982, 130, 35.
56. Simon, T.M.; Kunishima, D.H.; Vibert, G.J.; Lorber Α.,
J. Rheumatol. 1979, 6 (S5), 91.
57. Simon, T.M.; Kunishima, D.H.; Vibert, G.J.; Lorber, A.
Cancer 1979, 44, 1975.
58. Finkelstein, A . E . ; Burrone, O.R.; Walz, D.T.; Misher, A.
J. Rheumatol. 1977, 4, 245.
59. Simon, T.M.; Kunishima, D.H.; Vibert, G.J.; Lorber, A.
Cancer Res. 1981, 41, 94.
60. Shabel, F . ; Laster, R.; Corbett, T. personal

communication.

19
L i g a n d E x c h a n g e R e a c t i o n s of G o l d D r u g s i n
M o d e l S y s t e m s and i n R e d C e l l s
M. TAHIR RAZI, GABRIEL OTIKO, and PETER J. SADLER

Birkbeck College, Department of Chemistry, Malet Street, London, WC1E 7HX England
31 1
80 MHz P NMR and 400 MHz H spin-echo NMR are
used to compare and contrast reactions of auro-
thiomalate (Myocrisin) Et PAuCl, and Et PAu(TATG),
3 3
where TATG is 2,3,4,6-tetra-0-acetyl-1-thio-B-D-

-glucopyranosato-S, (auranofin) with intact red
blood cells, glutathione in model reactions, and
dimercaptopropanol (dmp), an agent thought to be
useful for reversal of gold toxicity. The hydro-
phobic phosphine complexes readily penetrate cells,
and their exchange reactions are more rapid on the
NMR timescale than those of aurothiomalate. In red
cells, Et PAuCl is partitioned between GSH and a
3
+
second site, and (Et P) Au is also formed at high
3 2
Au levels. Significant Et P release and oxidation

3
to OPEt is observed inside cells only when dmp is

3
+
added. (Et P) Au is not auranofin-treated red
3 2
cells. We also show that auranofin, when added to

whole blood, is partitioned between plasma and
cells. It is suggested that a key feature in the
molecular pharmacology of gold(I) is its ability
to transport, store, and release reducing equiva-
lents (thiols and phosphines).
Gold compounds were originally brought into clinical use

accidentally for the treatment of rheumatoid arthritis ÇL,2_,_3) ·
However, controlled clinical trials (4) confirmed their effective-
ness, and gold therapy ("chrysotherapy") is now considered to be
as good as that using any other drug for the difficult cases, and
one of the few that can lead to repair of joint tissue.
Currently, the most widely used gold drugs are gold(I) thiolate
complexes such as aurothiomalate ("Myocrisin") and aurothioglucose
("Solganal"), administered by intramuscular injection. These have
now been supplemented (5) by the orally-active complex (2,3,4,6-
tetra-0-acetyl-l-thio-3-D-glucopyranosato-S)(triethylphosphine)
gold(I) (auranofin), see Figure 1. The initial clinical pictures
0097-6156/83/0209-0371 $06.00/0

Au-S-CH-COo
Δ
I
CH2-C0" 2
Myocrisin "•OJC-CK-CH^CHJC-N-CH-C N - C H o2^2

CO
NH 3
+
H ÇH 2
SH
CH OAc fGlu Cys Gly

2
PEt 3
AcOÂ- 0 Glutathione
CH -CH-CH
2 2
I I I
SH SH OH
+
Et P-Au-CI
3 [Et P-Au-PEt ]
3 3 Dimercaptopropanoi
CI"
Figure 1. Molecular structures.
emerging from use of t h i o l a t e and phosphine complexes appear to be

s i m i l a r , although responses seem to be e a r l i e r w i t h a u r a n o f i n and
s i d e - e f f e c t s fewer. The aim of chrysotherapy i s u s u a l l y to estab-
l i s h a constant serum gold l e v e l (ca. 15 yM w i t h aurothiomalate
a d m i n i s t r a t i o n ) and to m a i n t a i n i t w i t h f u r t h e r doses, although
c l i n i c a l responsiveness i s not n e c e s s a r i l y c o r r e l a t e d w i t h serum
gold l e v e l s .
The r e s u l t s described here suggest t h a t the e f f e c t i v e n e s s of
gold i n therapy may be r e l a t e d to i t s very strong preference f o r
t h i o l a t e sulphur as a l i g a n d compared to others t h a t are a v a i l a b l e
i n the b i o l o g i c a l system. D e p l e t i o n of p r o t e i n t h i o l groups i n
serum through o x i d a t i o n i s thought to be a c o n t r i b u t i n g f a c t o r to
connective t i s s u e diseases such as rheumatoid a r t h r i t i s (6,7).
Polymeric Au(I) t h i o l a t e s can bind f u r t h e r t h i o l a t e s r e v e r s i b l y to
1 - n
form [ A u ( S R ) ] n species (l<n^2) w i t h favourable exchange
energies (8,9). Au(I) avoids c h e l a t i o n s i t e s , t h a t might t r a p
other metal ions w i t h s i m i l a r l i g a n d preferences, through i t s
strong tendency toward l i n e a r two-coordination. However, there
seems to be a c e r t a i n " f l o p p i n e s s " i n i t s c o o r d i n a t i o n sphere.
The i n t r i g u i n g p o s s i b i l i t y a r i s e s t h a t gold phosphine b i o -
chemistry may d i f f e r markedly from t h a t of gold t h i o l a t e s . Many
gold phosphines are hydrophobic and too l i t t l e a t t e n t i o n has been
p a i d i n the past to d i f f e r e n c e s i n metal i o n a f f i n i t y f o r l i g a n d s

19. RAZI ET AL. Ligand Exchange Reactions of Gold Drugs 373
i n aqueous compared to non-aqueous media. Since c e l l s are "mixed

s o l v e n t s " the q u e s t i o n a r i s e s as t o the c o r r e c t c o n d i t i o n s under
which t o c a r r y out model r e a c t i o n s . L i t t l e i s known, too, about
phosphine b i o c h e m i s t r y . I n t h i s paper we d e s c r i b e the development
of NMR methods f o r the o b s e r v a t i o n o f l i g a n d exchange r e a c t i o n s on
g o l d ( I ) and phosphine b i o c h e m i s t r y d i r e c t l y i n c e l l s , and i n
comparison w i t h analogous r e a c t i o n s i n model systems.
Experimental
S o l i d M y o c r i s i n , as an o f f - w h i t e powder, Au(I) thiomalate

0.3 g l y c e r o l .2H 0, was the g i f t o f May and Baker Ltd.(Dagenham).
2
1
A u r a n o f i n , trademark ' R i d a u r a , was the g i f t o f S,K and F Labora
tories (Philadelphia).
Blood was drawn from male v o l u n t e e r s and placed i n S t e r i l i n

p l a s t i c v i a l s c o n t a i n i n g K EDTA as a n t i - c o a g u l a n t . NMR e x p e r i
2
ments were u s u a l l y c a r r i e d out w i t h i n the next few hours.

X
H NMR s p e c t r a a t 400 MHz were obtained on the Bruker WH400
instrument o f the ULIRS s e r v i c e , w i t h the help o f Mr. M. Bucking
ham, u s i n g 5 mm tubes. For SEFT s p e c t r a , 300 r e p e t i t i o n s o f a
(90°-τ-180°-τ)-collect sequence w i t h τ = 60 msec was used (10).
The r e f e r e n c e was DSS o r TSS.
31
P NMR s p e c t r a a t 80 MHz were recorded on the Bruker WM200
spectrometer a t the MRC Biomedical NMR Centre, M i l l H i l l , u s i n g
15 mm tubes and ca. 4 ml o f sample. T y p i c a l l y , 3 ml o f packed
red c e l l s , p r e v i o u s l y washed twice w i t h s a l i n e s o l u t i o n , were
resuspended i n 1 ml o f D 0 - s a l i n e . Compounds which were i n s o l u b l e
2
i n water were added i n c a . 5-20 μΐ o f MeOH. Spectra are u s u a l l y

the r e s u l t o f c a . 1 hour accumulations : 2000 p u l s e s , 1.8 sec
p u l s e r e p e t i t i o n time, 16 Κ computer p o i n t s , 45° p u l s e , and peaks
are referenced t o e x t e r n a l H P0z» (85% i n H 0)/15% D 0.
3 2 2
T h i o l Uptake and Exchange Reactions o f Aurothiomalate ("Myocrisin")
Model systems. Aurothiomalate i s a 1:1 complex o f Au(I) and

disodium t h i o m a l a t e . Many commercial samples are hydrated and
a l s o c o n t a i n g l y c e r o l ( 8 ) . EXAFS, Mossbauer spectroscopy and g e l
permeation chromatography a l l i n d i c a t e t h a t the complex i s p o l y
meric both i n the s o l i d s t a t e and i n s o l u t i o n (11,12,13). L i n e a r
AuS 2 c o o r d i n a t i o n i s a t t a i n e d through b r i d g i n g thiomalate S atoms.
I t i s p o s s i b l e t h a t Au(I)-Au(I) bonds s t a b i l i s e r i n g o r c h a i n
s t r u c t u r e s , and there i s evidence t h a t such polymeric s t r u c t u r e s
are f l e x i b l e , changing i n response, f o r example, t o i o n i c s t r e n g t h
changes (14,15). We have r e c e n t l y obtained evidence t h a t up t o
23% o f gold i n s o l i d samples i s s t r u c t u r a l l y d i f f e r e n t t o t h e
remainder (14).
Further t h i o m a l a t e , added t o aqueous s o l u t i o n s o f the 1:1
complex a t pH 7, binds to Au(I) and a l l thiomalate i s i n f a s t
3 x
exchange (ca. 1 0 sec ) u n t i l a r a t i o o f A u ( I ) : t h i o m a l a t e o f 4:7
i s reached, then resonances f o r f r e e thiomalate appear. I t i s

s t i l l not c l e a r whether [ A u ( t m ) ] ~ monomers are a c t u a l l y formed,

2
3
or whether c l u s t e r species of the type [ A u ( t m ) ] " " e x i s t . There
4 7
are no r e p o r t s of c r y s t a l s t r u c t u r e s of b i s ( t h i o l a t o ) A u ( I ) com
plexes from aqueous s o l u t i o n , although a few w e l l - c h a r a c t e r i s e d
complexes have been i s o l a t e d from non-aqueous media (16). C l u s t e r s
may c o n t a i n 3-coordinate A u ( I ) , although a recent attempt to p r e
2
pare such species l e a d to [Au(I) M (II) SR<»] ~ heteronuclear
2 2 2
e
c l u s t e r s (M=Ni or Pd, S R D - p e n i c i l l a m i n e ) c o n t a i n i n g two c o o r d i n
ate AuS and s t a b i l i s e d by short Au-Au contacts of 2.9A (17). I t
2
i s now known t h a t t h r e e - and f o u r - c o o r d i n a t e Au(I) phosphines

e x i s t and t h a t there i s a degree of " f l o p p i n e s s " i n t h e i r c o o r d i n
a t i o n bonds (18). However Ρ cannot b r i d g e gold ions and so s t a b i l
i z e Au(I)-Au(I) bonds as S can.
1 n
T h i o l s w i t h pK 's > 8 e x h i b i t slow exchange w i t h [ A u ( S R ) ] "
a n
13
on the C NMR time s c a l e and we have determined exchange para
meters f o r t h i o m a l a t e , N - a c e t y l c y s t e i n e and mercaptoacetate ( 8 ) .
1
The a c t i v a t i o n f r e e energies are ca. 16 K c a l mole" and a c t i v a t i o n
e n t r o p i e s are n e g a t i v e , c o n s i s t e n t w i t h a s s o c i a t i v e i n t e r m e d i a t e s .
T h i o l displacement orders tend to f o l l o w the pK (SH) order: those a
w i t h the lowest pK 's b i n d most s t r o n g l y a t pH 7.

a
Using h i g h f i e l d *H NMR we have now observed f r e e and bound

g l u t a t h i o n e (GSH) when added to aurothiomalate s o l u t i o n s . This we
13
were unable to do i n previous C experiments. Most of the t h i o
malate i s d i s p l a c e d by two e q u i v a l e n t s of GSH a t pH 7, F i g u r e 2,
and i n t e g r a t i o n of the Cys aCH resonance of GSH suggests t h a t a
species of s t o i c h i o m e t r y [ A u ( S G ) ] i s formed as i n d i c a t e d by our
1 # 5
13
previous measurements on other t h i o l a t e s by C NMR. Again, t h i s
emphasizes the i n s t a b i l i t y of monomers r e l a t i v e to polymers and
c l u s t e r s . I t i s i n t e r e s t i n g to note t h a t a t a GSH:Au(tm) r a t i o of
1:2, peaks f o r bound tm are broadened but not those of f r e e . T h i s
suggests t h a t exchange r e a c t i o n s are o c c u r r i n g w i t h i n c l u s t e r
species.
Using SEFT NMR we can observe resonances from GSH i n i n t a c t
red c e l l s and we t h e r e f o r e s t u d i e d the entry of Au(tm) i n t o c e l l s .
These s t u d i e s w i l l be reported i n more d e t a i l elsewhere (19).
Entry of aurothiomalate i n t o red c e l l s . F i g u r e 3 shows 400

MHz *H SEFT NMR s p e c t r a of GSH (ca. 2 mM) and other s m a l l molecules
i n red c e l l s . Resonances f o r l a r g e r molecules such as haemoglobin
which have short T values disappear during the r e f o c u s s i n g p e r i o d
2
(10). Upon a d d i t i o n of Au(tm), the GSH resonances f o r Cys aCH,

3CH and Gly CH a l l decrease i n i n t e n s i t y . We i n t e r p r e t t h i s to
2 2
mean t h a t some gold enters c e l l s forming A u ( S G ) complexes, asl e 5
above, i n slow exchange w i t h f r e e GSH. I n c r e a s i n g the Au(tm) con

c e n t r a t i o n from 0.5 mM to 1 mM causes l i t t l e f u r t h e r change i n the
spectrum. I t i s p o s s i b l e t h a t polymeric Au(tm) coats the s u r f a c e
of the c e l l s preventing entry of more g o l d . This could be an
important f u n c t i o n of polymeric gold t h i o l a t e s i n v i v o and could
a l t e r the a n t i g e n i c determinants on lymphocyte c e l l s u r f a c e s . Red
c e l l s from p a t i e n t s t r e a t e d w i t h M y o c r i s i n u s u a l l y have a low gold

3 2 S/ppm
Figure 2. *H-NMR spectra (400 MHz) of glutathione (20 mM) in the presence
of 2 equivalents (top) and 0.5 equivalents (bottom) of aurothiomalate w D 0 ej f
pH* 7 and 298 Κ showing the displacement of coordinated thiomalate by GSH

and the slow exchange of the latter between free (f) and bound (b) forms. Thio
malate CH and CH resonances are circled. Key to the glutathione assignments:
2
, Gly CH ; g , Cys CH ; g , Glu y-CH ; g , Glu β-CH,; g , Cys CH.

g l 2 2 2 3 2 h 5
erg ere
I • ' »
k 3 S/ppm 2 1
1
Figure 3. SEFT H-NMR spectra (400 MHz). Key: top, 500 Μ auranofin B; μ
middle, 500 Μ aurothiomalate (note the decrease in intensity of the inverted multi
μ
plet for Cys 8-CH relative to ergothioneine or lactate); and bottom, packed red cells
2
0.45 mL plus 0.05 mL D Osaline. The singlet near 2 ppm is from auranofin
2
acetyls; other assignments are g Gly CH ; g , Glu y-CH ; g , Glu β-CH, (all GSH);
lt 2 3 2 h
erg, ergothioneine; ere, creatine; ala, alanine; and lac, lactate.

content, except f o r smokers (20)! Most gold i n blood i s c a r r i e d

on albumin. We have yet to study Au(tm) i n whole blood, but have
done so w i t h a u r a n o f i n , v i d e i n f r a .
Reactions of Gold Phosphines w i t h T h i o l s
These are r e l e v a n t to both the o r a l a b s o r p t i o n of g o l d and

e n t r y i n t o c e l l s . We compare a u r a n o f i n w i t h E t P A u C l s i n c e r e a c t
3
ions w i t h t h i o l s could l e a d to the same products i f the E t P - A u 3
bond stays i n t a c t . E t P A u C l could a l s o be a product of a u r a n o f i n

3
r e a c t i o n s w i t h HC1 media i n the stomach, although NMR i n v e s t i g a t

ions (21,22) suggest t h a t slow d e a c e t y l a t i o n of the sugar i s the
major r e a c t i o n under such c o n d i t i o n s . We have a l s o s t u d i e d the
bis(phosphine) species ( E t P ) A u C l . Not only i s t h i s o r a l l y -
3 2
a c t i v e (23), but c u r i o u s l y a l s o appears as an intermediate v i a

E t P t r a n s f e r r e a c t i o n s both i n some model systems and i n red
3
cells.
Model r e a c t i o n s of gold phosphines. Figure 4 shows *H NMR

s p e c t r a of E t P A u C l i n the presence of v a r y i n g amounts of GSH a t
3
pH 7. I t i s c l e a r from the s h i f t and s e p a r a t i o n of resonances f o r

the two Cys £CH protons t h a t GS" d i s p l a c e s CI", as deduced from
2
31
our previous P NMR s t u d i e s (24):
+
E t P A u C l + GSH -* Et PAuSG + C l " +
3 3 H
+
A s m a l l amount of ( E t P ) A u 3
i s a l s o produced presumably v i a d i s p r o p o r t i o n a t i o n of Et PAuSG. 3
I t i s i n t e r e s t i n g to note t h a t the Gly CH protons a l s o become

2
non-equivalent suggesting t h a t secondary i n t e r a c t i o n s occur w i t h i n

the Et PAuSG complex. The presence of E t P bound to Au a l s o
3 3
X
appears to enhance t h i o l exchange r a t e s . The Cys aCH H resonance
i s a s i n g l e peak even w i t h two e q u i v a l e n t s of GSH present (f$CH i s 2
+
more complex). We a l s o observe (25) f a s t exchange of E t P A u w i t h 3
13
N - a c e t y l c y s t e i n e on the C NMR t i m e - s c a l e , i n c o n t r a s t to Au(tm).
The h i g h t r a n s i n f l u e n c e of phosphines (weakening of the t r a n s
bond) has a p a r a l l e l i n P t ( I I ) chemistry.
The b i s complex ( E t P ) A u C l , which i s i o n i z e d i n H 0 (24),
3 2 2
does not r e a c t r a p i d l y w i t h t h i o l s , but i s a good reducing agent

toward d i s u l p h i d e s (24,25), due to the l a b i l i t y of one of the
phosphines which a c t s as the reducing agent. This l a b i l i t y i s
+
n e a t l y i l l u s t r a t e d by the NMR s p e c t r a of ( E t P ) A u .3 2 In H 0, E t P 2 3
exchange i s slow and e f f e c t s due to v i r t u a l c o u p l i n g are observed

13
i n *H and C s p e c t r a , whereas i n CDC1 or MeOD exchange i s slow
3
only a t low temperatures (25).

The choice of s o l v e n t presents a problem i n the study of
t h i o l exchange r e a c t i o n s of a u r a n o f i n . The c l i n i c a l l y - u s e d
c r y s t a l l i n e polymorph, a u r a n o f i n A, i s s o l u b l e to only 20 μΜ i n
H 0 and does not d i s s o l v e to the mM l e v e l on shaking w i t h GSH
2
s o l u t i o n s , as does E t P A u C l .
3 This suggests t h a t GSH does not

RAZI ET AL. Ligand Exchange Reactions of Gold Drugs 377
ppm
1
Figure 4. H-NMR spectra (400 MHz) of Eu?A uCl reacted with 20 mM solutions
of GSH. Key to GSH: Et PAuCl molar ratios: top, 1:1; middle, 1:0.5; and bottom,
s
1:0. Final pH values were adjusted to pH* 7. Note the large shift of the GSH Cys
β-CH, protons and AB coupling pattern for Gly CH (g ) at 1:1 ratio. GSH is in
2 t
fast exchange between free and bound forms at 1:0.5 molar ratio, but the Cys
fi-CH resonances are complex and have yet to be analyzed.
2

r e a d i l y d i s p l a c e TATG. However, TATG i s hydrophobic and t h e r e f o r e

not a good l e a v i n g group i n H 0! Using the polymorph a u r a n o f i n Β
2
(H 0 s o l u b i l i t y 0.7 mM), we have observed some broadening but

2
l i t t l e s h i f t o f the Cys &CH resonance o f GSH a t a u r a n o f i n : GSH

2
r a t i o s o f 1:1 t o 1:4 i n D 0. This suggests t h a t GSH exchange

2
occurs a t i n t e r m e d i a t e r a t e s , but t h a t there i s l i t t l e d i s p l a c e

ment o f TATG. S i m i l a r r e s u l t s were obtained f o r GSH-auranofin A
i n MeOD-D 0 s o l u t i o n s .
2
The exchange behaviour o f TATG w i t h a u r a n o f i n i s c u r i o u s . A t

1 3
0.2M c o n c e n t r a t i o n s the C NMR spectrum i n MeOH i s a s u p e r p o s i t
ion o f resonances f o r a u r a n o f i n and added TATG, whereas the peak
for the anomeric ΟχΈ. proton i s broadened beyond d e t e c t i o n i n 400
X
MHz H s p e c t r a o f s i m i l a r s o l u t i o n s a t 1:1 (10 mM) t o 1:4 r a t i o s
and a s i n g l e s e t o f averaged resonances i s observed f o r t h e
remaining protons. I t would appear t h e r e f o r e t h a t the TATG

exchange r a t e i n c r e a s e s w i t h decrease i n the c o n c e n t r a t i o n s o f
a u r a n o f i n and TATG. This i s being f u r t h e r i n v e s t i g a t e d .
Reactions o f g o l d phosphines w i t h r e d c e l l s . (Et P) AuCl 3 2
does not appear t o penetrate r e d c e l l s , but when added t o whole

blood, r e a c t s r e a d i l y w i t h albumin, reducing d i s u l p h i d e c r o s s
l i n k s , so denaturing i t (24).
E t P A u C l , on the other hand, r a p i d l y enters c e l l s and occu
3
p i e s two s i t e s (24). We have now been a b l e t o i n v e s t i g a t e t h e

t i t r a t i o n curve i n more d e t a i l . I t can be seen (Figure 5) t h a t
two resonances A and Β f o r Au-bound phosphine a r e seen a t low
E t P A u C l c o n c e n t r a t i o n s . Above 2 mM, a t h i r d resonance a s s i g n a b l e
3
+
to ( E t P ) A u appears. We had p r e v i o u s l y assigned resonance A a t
3 2
40.1 ppm t o Et PAu-SG s i n c e i t s chemical s h i f t i s s i m i l a r t o t h a t

3
i n model r e a c t i o n s . However, s i n c e t h e p o p u l a t i o n o f S i t e Β (42.1

ppm) s a t u r a t e s , t h i s i s more l i k e l y t o a r i s e from the GSH complex.
I t i s notable t h a t s i t e A i s populated a t E t P A u C l l e v e l s w e l l
3
below t h e GSH c o n c e n t r a t i o n (ca. 1.5 mM i n the c e l l suspension

used). We considered t h a t s i t e A might be due t o E t P A u C l i n t h e 3
membrane but when c e l l s a r e l y s e d and the ghosts removed, both

resonances A and Β a r e r e t a i n e d . Thus we observe a marked d i f f e r
ence between c e l l and model r e a c t i o n s .
Confirmation o f i n t r a c e l l u l a r GSH b i n d i n g was obtained by *H
SEFT s t u d i e s , F i g u r e 3: a t 0.5 mM Au the Cys 3CH resonance d i s 2
appears from the spectrum and the marked decrease i n i n t e n s i t y o f

the G l y CH resonance i n d i c a t e s non-equivalence, as seen f o r model
2
r e a c t i o n s . I t remains t o be seen whether GSH p r o t e c t s oxyhaemo-

g l o b i n from Et PAuCl-induced s p i n - s t a t e changes (26) s i m i l a r t o
3
those we have observed w i t h cytochrome c (27).

Auranofin a l s o leads t o the disappearance o f the GSH Cys 3CH 2
resonance on r e a c t i o n w i t h r e d c e l l s , F i g u r e 3. A l l the GSH i n

the c e l l i s e v i d e n t l y i n intermediate exchange on the NMR time-
s c a l e , thus the peak i s broadened ( s h o r t e r T ) and i t disappears
2
from the SEFT spectrum.

RAZI ET AL. Ligand Exchange Reactions of Gold Drugs 379
peak
height
2 4 6 Δυ/mM β
Β Α ι
F i W 5. {W-^P-NMR spectra (80 MHz) of EhPAuCl (left, 1 m M ; middle,

4 m M ; am/ Wgfo, 6 mM) <m wee/ion wi/Λ red cells (3 mL of packed cells) in
D 0-saline solution (1 mL) at 297 K. The inset shows the variation in peak heights
2
with Au concentration.

Reaction of Auranofin w i t h Whole Blood

3 1
P NMR s p e c t r a of whole blood c o n t a i n i n g 1 mM a u r a n o f i n (20
μΐ of an MeOH s o l u t i o n added to 5 ml of blood) at average r e a c t i o n
times of 2, 3.25 and 4.25 hours, showed a s i n g l e peak a t 42.8 ppm.
A f t e r 7 h r , the red c e l l s were separated, washed three times w i t h
s a l i n e , resuspended i n s a l i n e and NMR s p e c t r a recorded a t 39.5,
40.5 and 41.5 h r . Spectra taken from both separated plasma and
resuspended red c e l l s showed s i n g l e peaks at 42.8 ppm. From the
peak i n t e n s i t i e s i t could be concluded t h a t a u r a n o f i n was p a r t i t
3 1
ioned between plasma (45%) and red c e l l s (55%). The P NMR s h i f t
suggests that the species c o u l d be i n t a c t a u r a n o f i n although other
Et PAuSR species i n c e l l s may have s i m i l a r s h i f t s .
3
Previous s t u d i e s , u s i n g r a d i o - l a b e l l e d a u r a n o f i n (μΜ) incub

ated w i t h r a t and dog blood f o r 20 min, have a l s o i n d i c a t e d p a r t
i t i o n i n g between plasma (60%) and c e l l s (40%) (28). The r e p o r t e d
data appears to show t h a t when Au i s t r a n s f e r r e d to c e l l s i t
leaves behind the TATG l i g a n d (mostly bound to p r o t e i n ) and h a l f
of the E t P (as 0 P E t ) . We d i d not observe formation of 0 P E t and,
3 3 3
as y e t , have no NMR i n f o r m a t i o n about TATG t r a n s f e r , but f u r t h e r

s t u d i e s are i n progress.
Dimercaptopropanol as an I n t r a c e l l u l a r Au-binding Ligand
2,3-Dimercaptopropanol (dmp, F i g u r e 1, a l s o c a l l e d B r i t i s h
A n t i - L e w i s i t e ) i s thought to be u s e f u l f o r the treatment of t o x i c
s i d e e f f e c t s a r i s i n g from g o l d therapy (29). We r e p o r t here
s t u d i e s on r e a c t i o n s of dmp w i t h red c e l l s t r e a t e d w i t h E t P A u C l 3
or a u r a n o f i n .
3 1
P NMR s p e c t r a of these r e a c t i o n s are shown i n Figures 6 and
7. I t seems l i k e l y t h a t dmp promotes m i g r a t i o n of gold i n
E t P A u C l - t r e a t e d c e l l s i n t o hydrophobic regions of the c e l l , prob
3
a b l y as the complex CH (SAuPEt )CH(SAuPEt )CH 0H. In a hydro

2 3 3 2
phobic environment, E t P t r a n s f e r would probably be f a c i l e , g i v i n g

3
+
( E t P ) A u . This may d i s s o c i a t e or a c t as a reducing agent
3 2
(although there i s no obvious i n t r a c e l l u l a r candidate f o r r e d u c t

+
ion) l e a d i n g to 0 P E t and E t P A u , which i n t u r n binds to t h i o l
3 3
forming Et PAuSR. Hence we see an i n c r e a s e i n i n t e n s i t y of reson

3
ance Β a f t e r an i n i t i a l f a l l and the intermediate formation of

+
(Et P) Au .3 2 I n the presence of one e q u i v a l e n t of dmp, e q u i l i b r i u m
was reached a f t e r ca. 9 h r , by which time j u s t under h a l f of the
phosphine had been converted to the o x i d e . A d d i t i o n of a second
e q u i v a l e n t of dmp causes f u r t h e r conversion, F i g u r e 6.
No intermediate formation of ( E t P ) A u + i s seen i n the r e
3 2
a c t i o n of dmp w i t h a u r a n o f i n - t r e a t e d red c e l l s , perhaps another

i n d i c a t i o n t h a t l i t t l e TATG i s d i s p l a c e d i n s i d e the c e l l s under
these c o n d i t i o n s although i t i s p o s s i b l e that the b i s species i s
s h o r t e r - l i v e d . A f t e r ca. 2.5 hr i n the presence of one e q u i v a l e n t
of dmp about h a l f o f the coordinated E t P has been converted to 3
E t P 0 , Figure 7.
3

, , , j
70 60 ppm 50 40
Figure 6α. {W^P-NMR spectrum (80 MHz) of red blood cells that had reacted
with 1 mM Et PAuCl for 3 h (a). One molar equivalent dimercaptopropanol (dmp)
s
was then added and spectra were taken after 0.5 h (b) and 4.5 h (c). The reaction
appeared to reach equilibrium after ca. 9 hat 297 K. After 24 ha second equivalent
of dmp was added and 2 h later another spectrum (d) was obtained.
dmp dmp
Figure 6b. Plot of peak heights from spectra shown in Figure 6a vs. time after dmp
addition showing the formation of intermediates. Because the peak heights do not
appear to account for all the phosphine present during the reaction, they may be
subject to relaxation effects.

OPEt 3
Figure 7. {Wy^P-NMR spectrum (80 MHz) of red cells (4 mL packed cells +

1 mL D O/saline) that had reacted with 1 mM auranofin (added in 20 /*L of MeOH)
x
for 9 h (a). One molar equivalent of dimercaptopropanol (dmp) was then added
and spectra were recorded after 1.5 h (b), 2.5 h (c), and 3.5 h (d) at 297 K. The
insert shows the variation of peak heights with time after dmp addition.

Dimercaptopropanol is insoluble in H20, and attempts to model

the above reactions by addition of a MeOH solution of dmp to an
aqueous solution of Et3PAuSG (Et3PAuCl + GSH, 1:1, pH 7) gives a
yellow precipitate, probably the dmp-(AuPEt3)2 complex. We have
previously studied a mixed Au(I), Au(III)-dmp complex by Mossbauer
spectroscopy (30).
The ability of dimercaptopropanol to induce the formation of
0PEt3 is of biological interest since rapid degradation of aurano
fin to produce the oxide has been observed in rats and dogs (28).
Once the phosphine is released, Au molecular pharmacology could
resemble that of gold thiolate drugs, although there are two
important differences. Firstly, gold may have been delivered to
hydrophobic intracellular sites which gold thiolates could not
reach, and secondly the phosphine, on release, may carry out bio
chemically significant reductions.

Finally, we note that it may be possible for strong oxidants
to remove thiolate ligands even from 1:1 Au(I) thiolates. This
may generate a highly reactive Au(I) ion. Experiments to explore
this area are in progress.
Acknowledgments We thank the MRC, SERC and ULIRS for the

provision of NMR facilities. We also thank the Arthritis and
Rheumatism Council and Smith Kline and French Laboratories (Phila
delphia) for support. We are grateful to Drs. D. Hill and B.
Sutton (S, Κ and F Labs.) for fruitful discussions.
Literature Cited
1. Sadler, P.J. Structure and Bonding (Berlin) 1976, 29, 171.

2. Shaw, C.F. Inorg. Perspec. Biol. Med. 1979, 2, 287.
3. Brown, D.M., Smith W.E. Chem. Soc. Revs. 1980, 9, 217.
4. Empire Rheumatism Council, Ann. Rheum. Dis. 1960, 19, 95.
5. Walz, D.T., DeMartin, M.J., Chakrin, L.W., Sutton, B.M.,
Misher, A. J. Pharm. Exp. Therap. 1976, 197, 1.
6. Haafaja, M. Scan. J. Rheum. 1975, 4 (Suppl. 7) 1.
7. Lorber, Α., Simon, T.M. Gold Bull., 1979, 12, 149.
8. Isab, Α.Α., Sadler, P.J. J.C.S. Dalton, 1982, 135.
9. Isab, Α.Α., Sadler, P.J. J.C.S. Chem. Comm. 1976, 1051.
10. Brown, F.F., Campbell, I.D., Kuchel, P.W., Rabenstein, D.L.
FEBS Lett. 1977, 82, 12.
11. Mazid, M.A., Razi, M.T., Sadler, P.J., Greaves, G.N., Gurman,
S.J., Koch, M.H.J., Phillips, J.C. J.C.S. Chem. Comm. 1980,
1261.
12. Hill, D.T., Sutton, B.M., Isab, A.A., Razi, M.T., Sadler, P.J.,
Trooster, J.M., Calis, G.H.M. Submitted for publication.
13. Shaw, C.F., Schmitz, G., Thompson, H.O. Witkiewics, P.
J. Inorg. Biochem. 1979, 10, 317.
14. Grootveld, M.C., Sadler, P.J. Submitted for publication.
15. Isab, Α.Α., Sadler, P.J. J.C.S. Dalton, 1981, 1657.

16. Bowmaker, G.A., Dobson, B.C. J.C.S. Dalton Trans, 1981, 267.
17. Birker, P.J.M.W.L., Verschoor, G.C. Inorg. Chem. 1982, in
press.
18. Jones, P.G. J.C.S. Chem. Comm., 1980, 1031.
19. Otiko, G., Razi, M.T., Sadler, P.J., Isab, Α.Α., Rabenstein,
D.L. Manuscript in preparation.
20. Graham, G. Personal communication.
21. Malik, N.A., Otiko, G., Razi, M.T., Sadler, P.J. Symposium on
"Bioinorganic Chemistry of Gold Coordination Compounds"
Philadelphia Nov. 16-17, 1981.
22. Smith, I.C.P. Symposium on "Bioinorganic Chemistry of Gold
Coordination Compounds" Philadephia Nov. 16-17, 1981.
23. Hill, D.T. US Patent 4,057,630, Nov. 8, 1977.
24. Malik, N.A., Otiko, G., Sadler, P.J. J. Inorg. Biochem. 1980,
12, 317.
25. Malik, N.A. Ph.D Thesis, University of London, 1980.
26. Otiko, G. Ph.D Thesis, University of London, 1981.
27. Otiko, G., Sadler, P.J. FEBS Lett. 1980, 116, 227.
28. Intoccia, A.P., Flanagan, T.L., Walz, D.T., Gutzait, L.,
Swagzdis, J . E . , Flagieollo, J., Hwang, B.Y-H., Dewey, R.H.
"Symposium on Bioinorganic Chemistry of Gold Coordination
Compounds", Pennsylvania Nov. 16-17, 1981.
29. Margolis, H.M., Kaplan, P.S. Ann. Intern. Med. 1947, 27, 353.
30. Calis, G.H.M., Trooster, J.M., Razi, M.T., Sadler, P.J.
J. Inorg. Biochem. 1982, in press.

20
G o l d - B a s e d A n t i a r t h r i t i c D r u g s and M e t a b o l i t e s
Extended X-Ray Absorption Fine Structure (EXAFS)
Spectroscopy and X-Ray Absorption Near Edge
Spectroscopy (XANES)
R. C. ELDER, M. K. EIDSNESS, and M. J. HEEG—University of Cincinnati,
Department of Chemistry, Cincinnati, OH 45221
K. G. TEPPERMAN—University of Cincinnati, Department of Biological Sciences,
Cincinnati, OH 45221
C. F. SHAW III and NANCY SCHAEFFER—University of Wisconsin-
-Milwaukee, Department of Chemistry, Milwaukee, WI 53201
Gold oxidation state and local structure in bio-

logically relevant samples have been probed by X-ray ab-
sorption spectroscopy. XANES, X-ray absorption near edge
spectroscopy, distinguishes gold(III) from gold(I) and gold(0),
due to a 2p 5d transition which occurs only for gold(III).
Also XANES apparently indicates the presence of any Au-P
bonds in a sample. EXAFS, extended X-ray absorption fine
structure, spectroscopy has been used with curve fitting
techniques to show that the gold atoms in solutions of
sodium gold(I)thiomalate (GTM) are coordinated to two
sulfur atoms at 2.29 Å. Aurosomes, gold-containing lyso-
somal bodies formed in the kidneys of rats given chronic
doses of GTM, have two sulfur atoms bound to gold(I) at 2.30
Å. Administration of gold(III) to rats results in rapid reduc-
tion and renal deposition of gold(I) containing aurosomes.
Considering that tens of millions of people suffer from rheuma-
toid arthritis (1) and that gold-based anti-arthritis pharmaceuticals
have been proven to induce remission of this frequently crippling
disease (2), it is surprising how little is known about gold biochemistry
(3,4,5). Significant medical progress has been made in fine-tuning
protocols for drug administration (chrysotherapy) (6) and a new orally-
administered, gold-based drug, auranofin, has undergone clinical trials
(7), yet the metabolism, mode of action, site of action, cause of
toxicity, etc. of these drugs are not understood. A partial explanation
may lie in the fact that gold is spectroscopically "quiet," that is gold
has no useful NMR or ESR spectra (excepting rare gold(II) complexes
(8)) and its visible-UV spectrum is either nonexistent or difficult to
interpret in terms of structural implications. Another explanation
might be that gold chemistry has been considered uninteresting. Two
oxidation states, III and I, dominate the aqueous chemistry. Gold(I)
complexes are frequently unstable with respect to disproportionation to
gold(0) metal and gold(III). "Soft" ligands binding through sulfur and
phosphorus stabilize gold(I). The structural chemistry of gold(III) is
largely that of four coordinate square planar species, whereas gold(I)
chemistry is dominated by two coordinate linear complexes. Recently,
0097-6156/83/0209-0385$06.00/0

however, an increasing number of three (trigonal) and four coordinate

(tetrahedral) gold(I) complexes have been characterized (9,10). Finally,
gold has no known biological function in the body (11). This simple
observation has had two results: first, it has reduced interest in gold
biochemistry and, second, it makes gold biochemistry difficult. There
are no evolved storage systems or transport mechanisms for gold. As a
result, once introduced, gold is found throughout the body, involved in
seemingly non-specific interactions.
For whatever reasons, gold biochemistry is not well understood.
Any attempt to improve the situation seemed to require the intro
duction of a new, more sensitive technique for the characterization of
gold complexes in a biological milieu. X-ray absorption spectroscopy,
XAS, (12,13,14) offered such a tool. This type of spectroscopy has
enjoyed a recent revitalization due to the advent of intense new sources
(13) and to the suggestion of a new theoretical basis for interpretation

( ï | ) . XAS may be divided into two categories: XANES (X-Ray
Absorption Near Edge Spectroscopy) and EXAFS (Extended X-Ray
Absorption Fine Structure). From XANES it may be possible to
discover oxidation state and specific ligands bound to the absorbing
atom (16) and from EXAFS one determines the types of atom bound,
numbers of neighbors and coordination distances. XAS has several
extremely attractive features. First, it is element specific, that is
absorption edges for each element are well separated and few direct
interferences occur. Second, the sample may be in any state. We have
obtained spectra from amorphous solids, solutions, gums, oil suspensions
and animal tissues. Third, the sample size is relatively small, typically
less than 50 mg of material is required. Fourth, the technique is
relatively sensitive, permitting examination of samples containing as
little as 1 ppm of the atom of interest. Fifth, the technique is
relatively non-destructive, causing much less radiation damage to
samples than might be anticipated (vide infra). Disadvantages are that
XAS does have limited sensitivity, that the information discovered is
limited in scope (vide infra) and that an extremely intense source of x-
rays is required.
Experimental Considerations for X-Ray Absorption Spectroscopy
A typical spectrum, that of a 0.015 M solution of sodium

gold(I)thiomalate, is shown in Figure 1 and a diagram of the experi
mental setup is given as Figure 2. The spectrum which is a plot of
log(I /I) versus x-ray energy may be discussed most easily in terms of
three regions. At the lowest energy is the pre-edge region. Here there
is general background absorption by all the atoms in the sample with
absorption decreasing as the energy of the incident x-rays increases.
This region is of no interest other than it provides a measure of the
background to subtract. At higher energy there is a sudden rise in
absorption known as the absorption edge. For gold compounds this
feature occurs at ca. 11.9 Kev. The absorption increase results once
the incident x-ray photons have sufficient energy to promote the gold
2p electrons into some available excited state or to ionize them. If a
2p electron is excited into a bound state having significant ligand

ELDER ET AL. EXAFS and XANES Studies of Gold Drugs 387
Gold(l)thiomalate
0.015M
11.40 11.80 12.20 12.60 13.00 KEV
Figure 1. X-Ray absorption spectrum at the L edge of a 0.015 M solution of

IU
the drug Myocrisin (sodium gold(I)thiomalate).
Double
Crystal B e a m
Transmission
Monochromator M o n i t o r
Monitor
X-Ray
Sample I
Beam Lu-
Ion Chamber 2 -H 1
Fluorescence
Detector
Figure 2. Schematic diagram of experimental setup to measure x-ray absorption

spectra showing both transmission andfluorescencemodes.

character, it may give a signal characteristic for the particular ligating

Tt
atom. For example, vanadyl complexes give a signal typical of the yl"
oxygen (16). If, on the other hand the excited state is largely centered
on the metal atom there may be an indication of the metal oxidation
state. Both types of features occur in the near edge region for gold
compounds. Beyond the L edge the 2p electron is ionized and excess
m
energy is carried off by the ejected photo-electron. In this, the EXAFS

region, which extends for roughly 1000 ev beyond the edge, there is
again a gradual decrease in absorption with increase in x-ray energy.
The decrease is not smooth, however. There is a series of wiggles,
some of which can be seen in Figure 1. It is this wiggling modulation of
the x-ray absorption which is known as EXAFS and which can be
interpreted in terms of local structure around the absorbing atom.
Most simply, it is due to interference phenomena arising from back-
scattering of the ejected photo-electron by the neighbor atoms.
Apparatus Used to Measure X-Ray Absorption Spectroscopy. To

obtain a sufficiently intense source of x-rays, we and most others use
synchrotron radiation from an electron storage ring (13). A l l of the
experiments described here have been performed at the Stanford
Synchrotron Radiation Laboratories. Electrons are injected into the
storage ring and held at high energy (typically 3.0 Gev). Since the
electron path must be bent around the ring there is necessarily an
acceleration applied and this results in the emission of synchrotron
radiation. The spectrum of this radiation is continuous with the
intensity peaking in the x-ray region. The x-ray beam is extracted from
the storage ring through a series of beryllium windows and brought
down a beam pipe where it is available for experimenters. The x-ray
beam is extremely intense (several orders of magnitude more intense
than the continuous spectrum from a high intensity x-ray tube) and
intensity decreases slowly with time as electrons are lost from the
storage ring.
The x-ray beam first is diffracted by a double crystal mono-
chromator which passes a narrow energy band (typically a few ev wide)
as indicated on the left of Figure 2. Next the beam intensity, I . is
monitored by an ion chamber which absorbs a few percent of the beam
to ionize a gas such as nitrogen or argon. The ion current which flows
between charged plates is then a measure of beam intensity. Sub
sequently the beam passes through the sample where it is partially
absorbed and, in transmission mode, on to a second ion chamber to
measure I, the remaining intensity. In a fluorescence mode, a detector
placed at 90° to the beam is used to measure the fluorescent L α x-ray
which may be emitted in the relaxation of the 2p hole. Fluorescence
yields are acceptably high (ca. 30% for gold); however, the emission is
nearly isotropic, so a large area fluorescence detector is desirable. The
fluorescence technique is more sensitive and so preferred for dilute
samples.
Sample preparation is relatively simple. Solids are packed into a
slot cut into aluminum or plastic sheet of 1 mm or less thickness. The
area of the sample is larger than the beam which is normally 3 χ 25
mm. The samples are covered front and back with a thin adhesive tape,

20. ELDER ET AL. EXAFS and XANES Studies of Gold Drugs 389
typically Kapton. Liquid holders follow an equally simple design with

injection ports on the top. For fluorescence detection the sample is
placed at 45° to the beam.
Problems of Gold-Arthritis to be Considered

The questions which arise concerning the gold-based, anti-arthritis
drugs may be categorized as arising from chemical or medical points of
view. Chemical questions concerning the interactions of gold at the
molecular level include: what are the structures of the drugs them
selves (sodium gold(I)thiomalate - Myochrisin and gold(I)thioglucose -
Solganol are both amorphous solids), what happens at the molecular
level to these and other drugs in the body, do they proceed to a common
metabolite and, most generally, what is involved in gold biochemistry?
More medical questions include: what is the mode of action of the gold
drugs, what is the site of action, what causes the toxic side effects such
as renal failure and how can side effects be prevented or reduced?
We have attempted to answer several of these questions and will
dwell here on our results concerning drug structure in solid and solution
and on a somewhat detailed knowledge of the form of gold deposits
accumulated in the kidneys.
Kidney Deposits - The Separation of Aurosomes. That gold

concentrates in pre-existing lysosomal bodies called aurosomes has been
known for some time (17). These electron-rich areas are seen on
electron micrographs and considerable morphological characterization
of them has taken place. However, the oxidation state of the gold was
not known, nor were the chemical identities of the ligands (if any)
bound to gold known. We have developed a method of separating
aurosomes from bulk kidney tissue (18) in order to subsequently examine
the aurosomes by XAS. The samples used here were generated by two
different procedures. In one case 12 male Sprague-Dawley strain rats
(200-225 g initially) were given weekly interperitoneal injections of 0.4
mg Au/Kg body weight as sodium gold(I)thiomalate in 0.9% saline
solution for a period of 20 weeks. In another, similar rats were given an
acute dose, 35 mg/Kg of body weight, of sodium gold(HI)tetrachloride
and killed by suffocation with C 0 18 hours later. In both cases, the
2
kidneys were immediately excised and chilled in ice cold homogenizing

medium (0.25 M sucrose, 1 mM EDTA, 5 mM MgCl«, 25 mM KC1 and 50
mM Tris/HCl, pH 8.6). During all subsequent steps the tissues and
resulting samples were maintained at 0-4°C. The renal capsules were
removed and connective tissue was cut away from the collected
kidneys. The renal cortex was diced, washed, and patted dry to yield
tissue, which was then homogenized in 4 ml homogenizing medium per
gram of tissue using a Thomas glass/Teflon tissue grinder.
The homogenates were centrifuged at 600 g for 10 minutes to
pellet the nuclear/aurosomal fraction (18). Because of their anoma
lously high density, aurosomes pellet with nuclei as shown by electron
microscopy. To remove cell debris, the pellet was resuspended in 2.3 M
sucrose/3.3 mM calcium acetate to two-thirds of the original volume
and centrifuged at 40,000 g for 60 minutes. The pellet was resuspended
in 1.0 M sucrose/1 mM calcium acetate to one-half the original volume,

then centrifuged at 3000 g for 5 minutes. The pellet was twice

resuspended in 0.25 M sœrose/50 m M Tris/HCl, pH 8.6 and pelleted by
centrifugation at 3000 g for 5 minutes. The final washed pellet was
resuspended in water, frozen at -78° and lyophilized. The lyophilized
sample contained ca. 200 y g gold/g of sample. This technique provided
two aurosomal samples from the kidneys of normal rats, one from rats
given normal pharmacological doses of a currently used drug over an
extended time and the other from rats given a single acute dose of a
gold(III) compound and relatively quickly thereafter sacrificed.
XANES - Results and Interpretation

Figure 3 shows x-ray absorption near edge spectra, XANES, taken
at the L edge for Au{m) and Au(I) complexes as well as for gold(0)
foil. The plots are typical in that all the gold(III) complexes we have
examined are characterized by a sharp spike at 11.920 (1) Kev, whereas
the spike is missing from the edge spectra of gold foil and also of all
the gold(I) complexes we fiave examined. These results may be
interpreted most simply as follows: the L™, edge results from the
excitation of a 2p electron. Above the edge energy, this electron is
excited to the continuum. However, the 2p electron can also be
promoted to a bound state subject to the selection rule, ai = ±1. For a
2p electron this means transitions to s and d ..slates are allowed (19).
The electron configuration for Au(0) is Xe 4f *5dT 6s , that for Au(I)
A 4 l u 14
is Xe 4 f 5 d and that for Au(III) is Xe 4f 5 d * . Thus, the gold(III)
complexes have a vacancy in the 5d orbitals and the spike in the
absorption edge may be ascribed to the 2p-> 5d transition. Since both
gold(0) and gold(I) have a filled 5d subshell, this transition is not
possible and the spike is not seen. Gold(0) metal has an extremely
characteristic absorption spectrum above the edge as may be seen in
Figure 3. These two sharp peaks at 11.945 and 11.967 Kev serve to
differentiate gold(0) from gold(I). We have also obtained spectra from
colloidal gold(0), generated in such a way to give particles of gold metal
with a mean diameter of 64 nm (20), much smaller than the size of
aurosomes seen in electron micrographs. The sol spectra are identical
to that shown from gold foil in Figure 3. XANES for gold foil and the
chronic dose sodium gold(I)thiomalate aurosomes are compared in
Figure 4. Clearly the spike which characterizes gold(III) complexes is
missing in the aurosome spectrum and thus the gold in these aurosomes
has not been oxidized to gold(III). Similarly, comparison with the
gold(0) foil spectrum indicates that aurosomal gold has not been
reduced to colloidal gold(0). Thus, these aurosomes contain gold(I).
(Gold(n) may be ruled out on several bases. First, it is extremely rare.
Second, the gold(II) cytidine complex (8) which we have examined and
which might be of biological relevance shows a spike similar to that for
gold(III), a finding not too surprising in view of the Xe 4f 5d
electron configuration expected for gold(II).)
XANES for the gokkni) tetrachloride induced aurosomes and for
gold foil are compared in Figure 5. Once again, it is clearly evident
that the gold in these aurosomes is gold(I). Thus, the administration of
gold(III) tetrachloride has been followed by rapid reduction to gold(I). It

ELDER ET AL. EXAFS and XANES Studies of Gold Drugs 391
Figure 4. XANES spectra for chronic dose gold(I) thiomalate aurosomes (Π)
showing that the gold is still in the + 1 oxidation state as compared with spectra of
gold(0) (O); and gold(III) (—).

© 11.88 11.92 11.96 12.00 KEV
Figure 5. XANES spectrum for aurosomes (O) produced by injecting gold(III)

tetrachloride showing that reduction of gold(III) to gold(I) has taken place. Spectra
forgold(O) (O) and gold(III) (—) are given for comparison.
11.904 11.920 11.936 11.952 KEV
Figure 6. XANES spectra for a series of gold(I) complexes with zero, one, two,
and four phosphorus atoms coordinated to gold. The absorption peak at 11.927 keV
increases as the number of bound phosphorus atoms increases. Compound identifi-
cation given in text.

appears that gold(I) is the prédominent oxidation state in a biological

milieu.
Interestingly, these samples show little if any evidence of radia
tion damage. A spectrum taken during the first 10 minutes exposure to
the beam is identical with one taken after several hours of x-ray
irradiation. Also aurosomal samples reexamined one year after their
original irradiation show no changes.
XANES as an Indicator of Ligating Atoms
If the spectral transition which causes a XANES peak is to an
unoccupied molecular orbital which contains substantial ligand charac
ter, then that peak may be used to identify ligation by a particular
atom. This situation is illustrated in Figure 6. XANES for four
compounds are shown. In order of increasing absorbance at 11.927 Kev

the compounds are sodium bis(thiosulfato)gold(I), triethylphosphine-
gold(I)tetraacetylthioglucose (auranofin-Ridaura), bis(diphenylmethyl-
phosphine)gold(I)hexafluorophosphate and tetrakis(diphenylmethylphos-
phine)gold(I)hexafluorophosphate. Thus, the series progresses through
zero, one, two and four bound phosphorus atoms and the peak absorb
ance increases accordingly. It appears that this peak which occurs at
significantly higher energy than the gold(m) spike may be diagnostic of
an Au-P interaction. AU of the complexes containing phosphorus which
we have examined show this peak. Auranofin can be recognized from
its XANES Au-P peak, and since this orally administered drug is known
to lose triethylphosphine during metabolism (21), the XANES peak may
prove quite useful in determining at which stage the Au-P bond is
broken.
EXAFS Results and Structural Interpretation

In order to extract useful information from the EXAFS region of
an x-ray absorption spectrum considerable data manipulation is neces
sary. We have chosen to follow the methodology of Keith Hodgson and
coworkers (22). First, the general background absorption due to non-
gold atoms in the sample may be subtracted by extrapolation of the
pre-edge absorption. Second, a smooth curve through the EXAFS
portion of the spectrum is subtracted and the EXAFS normalized. This
curve, shown for sodium gold(I)thiomalate solution data in Figure 7, is
usually a cubic spline of two or three segments. The smooth curve
represents the absorption which would occur if the absorbing atom was
isolated from all neighbors. The resultant wiggles are then multiplied
by κ ί and plotted versus Κ as shown in Figure 8. Κ is proportional to
( E) where Ε is the difference between the energy of a particular
point and the energy chosen as the start of the EXAFS region (usually
20-40 ev above the edge inflection point of some standard material such
as gold foil). The advantage to working in Κ space is that the EXAFS
now appears as a damped sine wave with a period related to the
distance between absorber and back scattering neighbor plus a phase
shift characteristic of the absorber-scatterer pair. The amplitude is
related to the number of neighbors and the damping factor arises from

3J- • ,
Γΐ.40 11.80 12.20 12.60 13.00 KEV
Figure 7. The pre-edge subtracted absorption data for a solution of 0.14 M
sodium gold(I)thiomalate (—). The smooth curve ([J) is the three-part cubic spline
which is subtracted to give the EXAFS.
0 6 12 18 Κ
Figure 8. EXAFS times K? vs. Κ for a solution of 0.14 M sodium gold(I)thio-
malate.

thermal and positional disorder, that is, all neighbors in all sites are not
at exactly the same distances. If there are several types of neighbor
atoms (or the same kinds of atoms but at different distances) then
interferences or a beat pattern results in the EXAFS which most simply
arises, from the sum of the individual sine waves. Thus, the EXAFS data
in this form can show whether the absorber has only one type of
neighbor. As shown in Figure 9, the maximum in the amplitude
envelope occurs at different Κ values for different absorber-scatterer
pairs. Three scatterers, widely different in atomic number, are used.
In general, the peak in the amplitude envelope moves to higher Κ for
higher atomic number scatterers, occurring ca. K=2 for nitrogen, ca.
K=5 for chlorine and ca. K=9 for bromine. For a simple EXAFS pattern
it is relatively easy to guess the approximate atomic number of the
scattering partner. A comparison of Figures 9 and 8 will show there is
relatively little difference between the EXAFS when the neighbors are
changed from sulfur to chlorine. (The position of the maximum in the
amplitude envelope will depend on the power of Κ used to weight the
EXAFS. Κ 2 is used here.)
Fourier Transforms, Filtering and Curve Fitting. The EXAFS data

can be Fourier transformed into R space. The resulting function which
is plotted versus distance is not a true radial distribution function since
the phase shift now results in a distance shift from true gold-ligand
distance. A Fourier transform for sodium gold(I)thiomalate is shown in
Figure 10. In practice, the phase shifts are evaluated from model
compounds with known crystal structures. From the peak shape(s),
positionfe) and height(s) reasonable estimates may be made of the
number and kinds of scatters and their distance(s) from the absorber.
Finally, the data may be filtered by back transforming a selected range
of data from R into Κ space. It is this Fourier filtered data to which
curve fitting techniques are applied. The curve is determined by six
parameters (22) of which four describe the phase shift, amplitude and
disorder for a particular absorber-scatterer pair. A l l six parameters
are varied for a model compound of known structure. These parameters
are transferred to an unknown compound and only the two parameters
which may be interpreted in terms of coordination number and bond
distance are varied to give the best fit.
Aurosome Results
The results of fitting the chronic dose sodium gold(I)thiomalate
aurosomes based on a sodium bis(thiosulfato)gold(I) model (23) are
presented in Figure 11 and the values derived from the fit are listed in
Table I. A first attempt to fit to a gold-nitrogen environment is
disasterous, the gold coordination number becomes negative and the
bond distance unrealistically short. A fit with sulfur neighbors is much
better giving a reasonable coordination numbr of 1.8 and bond distance
of 2.30 R. An attempt to fit two sets of neighbor atoms, nitrogen and
sulfur results in an apparently better fit value, however, there are now
four variable parameters and the nitrogen coordination number is
unreasonably low at 0.4 with an extremely short distance of 1.90 A .

18 Κ
2
Figure 9. EXAFS times K vs. K . Key: top, tetraamminegoldflll) nitrate (maxi
mum amplitude at ca. Κ = 2); middle, potassium gold(lll)chloride (maximum
amplitude at ca. Κ = 6); and bottom, potassium gold(III)bromide (maximum
amplitude at ca. Κ = 9).

b
0.0 0.8 1.6 2.4 3.2 4.0 R
Figure 10. Fourier transform for a solution of 0.14 M sodium gold(I)thiomalate

showing the gold-sulfur peak.
Chronic Thiomalate Aurosomes
4.00 8.00 Κ
Figure 11. The Fourier-filtered data (boxes) fit to a gold-sulfur scattered pair
with coordination number of 1.84 and bond distance of 2.30 A, (line).

Table I
Chronic Dose Gold Thiomalate Aurosomes
EXAFS Curve Fitting: Single Shell
Atom type Coord # R(£) Fit
Nitrogen negative 1.7 1.95 1.61
Sulfur 1.84 2.30 0.33
Phosphorus 2.07 2.34 0.29
EXAFS Curve Fitting: Double Shell
Nitrogen and 0.41 1.90 n 9 f i
U , Z 0
Sulfur 1.89 2.27
This result merely indicates that nitrogen is not in the gold coordination
sphere. A phosphorus fit is included to show that we are unable to
distinguish between sulfur and phosphorus neighbors on the basis of
curve fitting. However, both on the basis of the XANES data which
shows no evidence of phosphorus coordination and chemical sense we
are able to rule out phosphorus coordination here. Based on our
experience in fitting known compounds and the results of Hodgson's
group (22) for an extensive series of molybdenum complexes, the errors
which we expect in these calculations are ca. 0.02 R in distance and ca.
20% in coordination number. Thus, we feel confident that the gold in
these aurosomes is bound to two sulfur atoms at a distance of 2.30 R.
Gold-Based Drug Results

These are shown in Table II.
Table II
EXAFS Results Sulfur Ligands
Compound Coord # Au-S (R) Fit
Chronic aurosomes 1.84 2.304 .33
Au thiomalate solid 1.81 2.298 .17
Au thiomalate soin .14 M 1.86 2.292 .17
Au thiomalate soin .015 M 1.81 2.292 .36
Au thioglucose solid 1.72 2.304 .23
Au (cysteine) solid 1.87 2.309 .17
Au (glutathione) solid 1.72 2.304 .23
Sodium gold(I)thiomalate has been fit successfully with sulfur neighbors
giving coordination numbers ca. two and bond distances of 2.29 R in
solutions of 0.14 and 0.015 M concentration. This requires the
compounds to be polymeric with bridging sulfur atoms since thiomalic
acid has a single thiolate function. From the minor differences
between the aurosome fit and the fit to the drug Myochrisin it is not
possible to exclude that aurosomes might be mere storehouses of the
drug. On the basis of rather low circulating concentrations of gold in

the blood (24), it seems unlikely that the polymer would remain intact.
Also, the liïmilarity of the fit aurosome data to that from the
gold(III)tetraehloride aurosomes (which we have yet to fit) would
suggest that aurosomes contain a drug metabolite.
Possible metabolites previously suggested include gold complexes
of cysteine and glutathione (25). We have examined solid samples of
each of these and find them to fit satisfactorily to a model in which
gold is coordinated to two bridging sulfur atoms with bond lengths
identical to those found in the chronic sodium gold(I)thiomalate auro-
somes.
Conclusions
X-ray absorption spectroscopy shows considerable promise as a
powerful tool to aid in the elucidation of the role of gold-based
pharmaceuticals in the management of rheumatoid arthritis. This

technique has already given considerable new insight into gold oxidation
state and binding in aurosomal samples prepared from rat kidneys.
Acknowledgments
We thank the Research Corporation, the National Science Founda-
tion and Smith Kline and French Laboratories for support of this work.
XAS experiments were performed at SSRL which is supported by the
NSF through the Division of Materials Research and the NIH through
the Biotechnology Resource Program in the Division of Research
Resources. We thank Professors K.O. Hodgson and R.A. Scott for
immense help in introducing us to these techniques.
Literature Cited
1. "Primer on the Rheumatic Diseases" 7th Edtion, Rodman, G.P.,

Ed., The Arthritis Foundation, New York, NY, 1973.
2. Empire Rheumatism Council, Ann. Rheum. Dis. 1961, 20, 315-354.
3. Shaw, III, C.F., Inorganic Perspectives in Biology and Medicine
1979, 2, 287-355.
4. Sadler,P.J.,Struct. Bonding 1976, 29, 171-214.
5. Brown, D.H.; Smith, W.E., Chem. Soc. Rev. 1980, 9, 217-240.
6. Jessop,J.D.,J.Rheumatol. Suppl. 5, 1979, 6, 12-17.
7. Sutton, B.L., this volume.
8. Hadjiliadis, N.; Pneumatikakis, G.; Basosi, R., J. Inorg. Biochem.,
1981, 14, 115-126.
9. Jones, P., Gold Bull. 1981, 14, 102-118.
10. Elder, R.C.; Zeiher, E.H.K.; Onady, M.; Whittle, R.R., J. Chem.
Soc. Chem. Commun. 1981, 900-901.
11. Shroeder, H.A., in "Metal Binding in Medicine," Seven, H.A., ed.,
J.P. Lippincott, Philadelphia, PA, 1960, 59-67.
12. Cramer, S.P.; Hodgson, K.O., Prog. Inorg. Chem., 1979, 25, 1-39.
13. Winick, H., in "Synchrotron Radiation Research," Winick, H. and
Doniach, S., eds., Plenum, New York, NY, 1980, 27-58.
14. Teo, B., in "EXAFS Spectroscopy," Teo, B. and Joy, D., eds.,
Plenum, New York, NY, 1981, 13-58.

15.
Sayers, D.E.; Stern, E.A.; Lytle, F.W., Phys. Rev. Lett., 1971, 27
1204-1207.
16. Tullius, T.D.; Gillium, W.O.; Carlson, R.M.K.; Hodgson, K.O., J.
Am. Chem. Soc., 1980, 102, 5670-5676.
17. Ghadially, F.N., J. Rheumatol. Suppl. 5, 1979, 45-50.
18. Shaw, III, C.F.; Thompson, H.O; Witkiewicz, P.; Satre, R.W.;
Siegesmund, K., Toxicol. Appl. Pharmacol., 1981, 61, 349-357.
19. Lytle, F.W.; Via, G.H.; Sinfelt, J.H., in "Synchrotron Radiation
Research," Winick, H. and Doniach, S., eds. Plenum, New York,
NY, 1980, 401-423.
20. Horisberger, Marc, Biol. Cell., 1979, 36, 253-258.
21. Walz, D.T., private communication.
22. Cramer, S.P.; Hodgson, K.O.; Stiefel, E.I. and Newton, W.E., J.
Am. Chem. Soc., 1978, 100, 2748-2761.
23. Ruben, H.; Zalkin, Α.; Felkens, M.O. and Templeton, D.H., Inorg.
Chem., 1974, 13, 1836-1839.
24. Lorber, Α.; Wilcox,J.A.;Vibert,G.J.;Simon, T.M.,J.Rheumatol.,
Suppl. 5, 1979, 6, 31-39.
25. Shaw, III, C.F., in "Inorganic Chemistry in Biolgy and Medicine,"
Martell, Α., ed., ACS Symposium Series, No. 140. Washington,
D.C., 1980, 349-372.

21
G o l d Thiolate Complexes In V i t r o and In V i v o
D. H. BROWN and W. E. SMITH

University of Strathclyde, Department of Pure and Applied Chemistry,
GlasgowG11XL,Scotland
Naturally occurring thiols and other thiol

ligands used in the complexes administered
during gold therapy react with gold(O), gold
(I) and gold(III). The products formed i n
each case are dependent on the ligand, the
solvent and the presence or absence of oxy-
gen. Polymers, clusters, co-precipitates or
mixed crystals and monomeric complexes have
all been identified, as have gold(I), gold
(III) and mixed valence complexes. In vivo
gold distributes widely and reacts readily.
Despite this, the reactivity of serum gold
is dependent on the compound originally ad-
ministered. It is suggested that the thera-
peutic effect of gold compounds is dependent
on the a b i l i t y of gold to bind to the thiol
system on c e l l membrane.
Gold compounds have been used for many years in
the treatment of rheumatoid a r t h r i t i s . They have
proved to be effective therapeutic agents in some pa-
tients but they are also potentially toxic(1-5). As a
result, enthusiasm for these compounds has waxed and
waned over the years but no drug has yet been developed
to replace them completely, although compounds such as
d-penicillamine have become viable alternatives (6).
They differ from standard anti-inflammatory agents i n
that they affect chronic rather than acute inflammation
and they may cause a remission of the disease activity.
Gold compounds and most other compounds claimed to pro-
duce remission in rheumatoid arthritis affect the im-
mune system i n some way and i t seems l i k e l y that this
action is responsible for the antirheumatic effect
(4,6,7). A l l these agents can produce serious toxic
side reactions in some patients and it is hoped that
0097-6156/83/0209-0401$06.00/0

t h e i r mode o f a c t i o n m i g h t make i t p o s s i b l e t o d i f f e r e n
t i a t e b e t w e e n the t h e r a p e u t i c and t o x i c a c t i o n o f g o l d
complexes i n c o n v e n t i o n a l t h e r a p e u t i c regimes.
M o s t a u t h o r s a g r e e t h a t g o l d compounds r e a c t w i t h
the t h i o l / d i s u l p h i d e system i n v i v o . T h i s system pro
v i d e s t h e l a r g e s t s o u r c e o f s o f t l i g a n d s and g o l d has
been i d e n t i f i e d bound to p r o t e i n s i n a l m o s t e v e r y
t i s s u e f r a c t i o n so f a r i n v e s t i g a t e d , i n c l u d i n g b r a i n
t i s s u e (θ). G o l d compounds a f f e c t t h e r e s u l t s o f a s s a y s
f o r t h e t h i o l c o n t e n t o f p r o t e i n s (β) a n d t h e y a l t e r t h e
t h i o l b a l a n c e across the c e l l w a l l s of c i r c u l a t i n g
b l o o d c e l l s (2). T h i s paper d e s c r i b e s the r e a c t i o n of
g o l d compounds w i t h t h i o l l i g a n d s i n v i t r o and u s e s
t h i s i n f o r m a t i o n i n i n t e r p r e t i n g the l e s s d i r e c t e v i
dence f o r t h i o l / g o l d r e a c t i o n s i n v i v o . A possible
mechanism f o r a c t i o n o f g o l d compounds i n t h e therapy
of rheumatoid a r t h r i t i s i s suggested.
In V i t r o Gold Chemistry
The l i g a n d s c o n s i d e r e d i n t h e i n v i t r o s t u d i e s a r e
s h o w n i n F i g u r e 1. C y s t e i n e and t h e t r i p e p t i d e g l u t a
t h i o n e a r e n a t u r a l l y o c c u r r i n g t h i o l c o n t a i n i n g com
pounds. P e n i c i l l a m i n e i s an e f f e c t i v e d r u g i n the
t r e a t m e n t o f r h e u m a t o i d a r t h r i t i s and c o n t a i n s a n -SH
g r o u p on a t e r t i a r y c a r b o n . T h i o m a l i c a c i d i s the
l i g a n d used i n the drug M y o c r i s i n . I t does not c o n t a i n
a n a m i n e g r o u p a n d , s i n c e c a r b o x y l a t e g r o u p s do n o t
complex r e a d i l y w i t h g o l d , i t i s a b e t t e r model f o r
comparison with p r o t e i n sulphydryl groups. The dithiol
2,3-dimercaptopropanol ( B r i t i s h A n t i - L e w i s i t e or BAL)
has b e e n u s e d t o t r e a t c a s e s where an o v e r d o s e o f g o l d
has been a d m i n i s t e r e d ( l O ) .
Spectroscopic Studies. Many o f t h e s o l i d s i s o l a t e d

f r o m t h e r e a c t i o n s o f t h i o l s w i t h g o l d compounds a r e
n o n c r y s t a l l i n e and p o l y m e r i c . They can, t h e r e f o r e , not
be r e c r y s t a l l i z e d and a r e o f t e n n o t s u i t a b l e f o r X - r a y
s t r u c t u r a l a n a l y s i s , a l t h o u g h a u r a n o f i n c a n be c r y s t a l
l i z e d and i t s s t r u c t u r e has b e e n d e t e r m i n e d ( l l . ) . As
a c o n s e q u e n c e , s p e c t r o s c o p i c p r o b e s o f s t r u c t u r e and
e l e c t r o n i c c o n f i g u r a t i o n are w i d e l y used i n the s o l i d
s t a t e as w e l l as i n s o l u t i o n .
The o x i d a t i o n s t a t e o f t h e g o l d p r e s e n t i n t h e s e
compounds i s u s u a l l y d e t e r m i n e d by M O s s b a u e r s p e c t r o
s c o p y (12) but X-ray p h o t o e l e c t r o n spectroscopy has
a l s o b e e n u s e d s u c c e s s f u l l y ( l ^ . ) . The m a j o r a d v a n t a g e s
o f the l a t t e r technique are s i m p l e r sample h a n d l i n g
p r o c e d u r e s and the e l i m i n a t i o n o f a n e e d f o r l i q u i d
helium c o o l a n t , but i t r e q u i r e s very c a r e f u l i n t e r p r e -

21. BROWN AND SMITH Gold Thiolate Complexes 403
H COJP CH 3 CO^H
H- -H CH3 C C H
SH NHg SH NHg
a b
H H H
H- H- -H
SH H OH SH SH
CCfeH H H H COjH
N H g H H O H Ç H g O H H
SH
Figure 1. The thiol-containing ligands used in most of the reactions considered.

Key: a, cysteine; b, penicillamine; c, thiomalic acid; d, 2,3-dimercaptopropanol
(BAL); and e, glutathione.

t a t i o n of the r e s u l t s . S u r f a c e c h a r g i n g of the samples

i s overcome by u s i n g the C lg s i g n a l from the l i g a n d .
B o t h t e c h n i q u e s are i n s u f f i c i e n t l y s e n s i t i v e f o r routine
use i n b i o l o g i c a l s y s t e m s and c a n c a u s e c h e m i c a l r e a c
t i o n s t o o c c u r i n the s a m p l e . An example o f t h i s w i t h
XPS a l s o i l l u s t r a t e s t h e d e t e c t i o n o f g o l d f l ) and
g o l d f l l l ) b y t h i s t e c h n i q u e f F i g u r e z) and a s i m i l a r
r e s u l t h a s b e e n r e p o r t e d f o r g o l d f l l l ) dithiοcarbamate
f14), C o n s i d e r a b l e e x p o s u r e t o t h e X - r a y beam was re
q u i r e d to a c h i e v e the complete removal of the g o l d f l l l )
s i g n a l and t h e c o m p o u n d s u s e d a r e p a r t i c u l a r l y s u i t a b l e
f o r t h i s experiment. EXAFS and r e l a t e d t e c h n i q u e s a r e
a l s o p r o v i n g t o be v a l u a b l e i n u n d e r s t a n d i n g t h e s e
structures. Sodium g o l d f l ) t h i o m a l a t e c o n t a i n s an ap
p r o x i m a t e l y l i n e a r S-Au-S g r o u p i n g f 1 ^ ) , a n d t h i s a n d
r e l a t e d s t u d i e s are r e v i e w e d i n another a r t i c l e i n t h i s
volume f 1 6 ) .
I f a n Au-S bond i s p r e s e n t i n the sample, a combi
n a t i o n o f u v - v i s i b l e s p e c t r o m e t r y and c i r c u l a r d i c h r o -
i s m fC.D.) p r o d u c e s a s p e c t r u m i n t h e v i s i b l e and n e a r
uv r e g i o n w h i c h i s c h a r a c t e r i s t i c o f t h i s bond f F i g u r e
3) a n d w h i c h i s p r e s e n t i n b o t h g o l d f l ) and goldflll)
compounds. I n g o l d f l l l ) compounds, however, the bands
a r e s h i f t e d b y a b o u t 60 nm t o w a r d s t h e i n f r a r e d and,
c o n s e q u e n t l y , an e s t i m a t i o n o f t h e g o l d o x i d a t i o n state
i s p o s s i b l e by t h i s method i n b o t h s o l i d and solution
4
s a m p l e s and a t c o n c e n t r a t i o n s down t o a b o u t 1 0 ~ molar
f17). C.D. s p e c t r a are v e r y s e n s i t i v e to environment
a n d so p r o v i d e a s i m p l e m e t h o d o f f o l l o w i n g g e o m e t r y
changes i n s o l u t i o n . C.D. i s c o m p l e m e n t a r y t o NMR in
t h a t i t i s s i m p l e r t o c a r r y o u t and i s r e a d i l y a p p l i c
a b l e i n a w i d e r r a n g e o f s o l v e n t s and i s more s u i t a b l e
for kinetic studies. NMR m e t h o d s p r o v i d e a more d e
t a i l e d a n a l y s i s of the s t r u c t u r e of s o l u t i o n s p e c i e s
(18).
A C.N.D.O. c a l c u l a t i o n o n P - A u f r ) - S e n t i t i e s was u s e d
to a i d i n a s s i g n i n g the s p e c t r a . S i n c e an e r r o r o f
l e s s t h a n 1% i s s u f f i c i e n t t o p r e v e n t a n a s s i g n m e n t o f
i n d i v i d u a l b a n d s , t h e c a l c u l a t i o n was u s e d o n l y t o p r o
v i d e a d e s c r i p t i o n of the o r b i t a l s expected to c o n t r i
b u t e t o t r a n s i t i o n s i n t h i s e n e r g y r e g i o n and f o r t h i s
p u r p o s e , i t s h o u l d be a c c e p t a b l e . I n d i v i d u a l band
a s s i g n m e n t s w e r e made e m p i r i c a l l y . The b o n d f o r m e d
b e t w e e n g o l d a n d s u l f u r h a s b o t h a <T a n d π c o n t r i b u t i o n .
The s t r o n g e r 6 o r b i t a l c o n t a i n s a l a r g e r p r o p o r t i o n o f
A u f s ) t h a n A u f p ) , p r o b a b l y because the energy s e p a r a
t i o n o f the s and ρ i s q u i t e l a r g e compared t o b o n d i n g
i n a n e q u i v a l e n t b o n d w i t h a 3d m e t a l . Thus, g o l d f l )
with sulfur halide ligands prefers linear coordination.
W i t h p h o s p h o r u s , t h e Ρ f p ) o r b i t a l s m i x more r e a d i l y

BROWN AND SMITH Gold Thiolate Complexes 405
90 95
B i n d i n g energy / e V
Figure 2. Decomposition of Et NAuBr in the x-ray beam of an x-ray photo-

k k
electron spectrometer. Peaks 1 and 2 refer to goldflll) and peaks 3 and 4 refer to
goldfl). Key to exposure time before spectra were recorded: a, 15 min; b, 3 h; c,
24 h; and d, control spectrum of Et NAuBr . fReproduced with permission from
h t
Ref. 13.)

Figure 3. Near-UV electronic (top) and circular dichroism (CD) (bottom) spectra
of goldfl) cysteine attributable to transitions centered on the gold-sulfur bond.
The changes in sign in the CD spectrum can be helpful in separating more complex
spectra.

w i t h the g o l d , p r o d u c i n g a d e c r e a s e d energy s e p a r a t i o n
of* the g o l d s and ρ o r b i t a l s and p r e d i s p o s i n g the phos
p h i n e g o l d e n t i t y to f u r t h e r s u b s t i t u t i o n . Thus,
h i g h e r c o o r d i n a t i o n numbers are to be e x p e c t e d w i t h
phosphorus-containing l i g a n d s .
Chemical Reactions. The r e a c t i o n s o f a l l o f the

l i g a n d s w i t h g o l d compounds are v e r y dependent on the
s o l v e n t u s e d . F o r example, sodium t e t r a c h l o r o a u r a t e
r e a c t s w i t h t h i o m a l i c a c i d i n water to g i v e a g o l d f l )
t h i o m a l a t e complex i n s o l u t i o n . I n t h o r o u g h l y d r i e d
e t h a n o l , sodium c h l o r i d e i s p r e c i p i t a t e d and g o l d f l l l )
complexes o f t h i o m a l i c a c i d and c h l o r i d e are o b t a i n e d
i n s o l u t i o n , and w i t h a c e t o n i t r i l e a g o l d f l ) complex,
g o l d f l ) t h i o m a l a t e . N a C l , i s p r e c i p i t a t e d and a g o l d f l l l )
s o l u t i o n remains f1ft). F u r t h e r , a l t h o u g h the u s u a l
form o f g o l d f l ) complexes i s AuL, i n g l a c i a l a c e t i c
a c i d , g o l d f l ) compounds o f f o r m u l a AuL have been i s o
3
l a t e d f20). I t i s presumed t h a t t h e y c o n t a i n a d i s u l
fide. However, the i n f r a r e d spectrum o f t h e s e com
pounds i s not c o n s i s t e n t w i t h a m i x t u r e o f the
component p a r t s , i r r e s p e c t i v e o f the form o f d i s u l f i d e
which i s p o s t u l a t e d to e x i s t and, t h e r e f o r e , some form
o f complexing i s b e l i e v e d to e x i s t .
The s o l u t i o n s p e c i e s appear to be q u i t e l a b i l e .
The r e s u l t s o f c o n d u c t i o m e t r i c t i t r a t i o n were dependent
on the speed w i t h which t h e y were c a r r i e d out f19).
T h u s , the s o l i d s o b t a i n e d are o f t e n s i m p l y the l e a s t
s o l u b l e component a r i s i n g from a s e r i e s o f c o m p e t i t i v e
r e a c t i o n s between c l u s t e r s . Depending on c o n d i t i o n s ,
a series of stoichiometric products, AuftmH ), 2
N a ^ A u f t m ) ] , CaJAuftm)]21Lp, BaJAuf tmX^HgO, and m i x e d l i

gand complexes c o n t a i n i n g t h i o m a l i c a c i d ftm) and
g l u t a t h i o n e o r c y s t e i n e have been p r e p a r e d f2l).
The s t a b i l i t y o f the o x i d a t i o n s t a t e i s a l s o de
pendent on the l i g a n d u s e d . F o r example, the compound
goldflll)fpenicillamine) 2 can be p r e p a r e d by d i s s o l v i n g
p e n i c i l l a m i n e and sodium t e t r a c h l o r o a u r a t e i n e t h a n o l
but under the same c o n d i t i o n s , c y s t e i n e produced g o l d f l )
c y s t e i n e f 11.) . R e a c t i o n o f BAL w i t h sodium t e t r a c h l o r o
a u r a t e i n aqueous e t h a n o l produced a range o f polymers
o f d e f i n i t e s t o i c h i o m e t r y Au* f B A L ) C l which i n c l u d e
x v
examples o f g o l d f l ) and goldîlll) compounds and mixed

v a l e n c e compoundsf22). Further, s t a r t i n g with a solu-
t i o n o f N a A u en^feÔ^, s u b s t i t u t i o n w i t h d i f f e r e n t
3
l i g a n d s produced g o l d f l ) complexes and w i t h d-

p e n i c i l l a m i n e , a g o l d f l l l ) complex f20) fFigure 4).
The g o l d o x i d a t i o n s t a t e s i n t h i s l a t t e r s t u d y were
d e t e r m i n e d by XPS.
Many o f the compounds formed are p o l y m e r i c .

Figure 4. Products identified in the reaction of Na Au en (SO3)2 with sulfur-

s t
containing ligands. Reactions were carried out at 1:1 stoichiometry in aqueous

ethanol. Key to abbreviations: cys, 1-cysteine; en, diaminoethane; pen, à-penicilla-
mine; and tu, thiourea.

Sodium g o l d f l ) t h i o m a l a t e has been shown to be a p o l y -

mer i n s o l u t i o n by NMR and t h e r e i s more than one
polymer p r e s e n t i n the s o l u t i o n o f t h i s compound u s e d
as the d r u g M y o c r i s i n f 2 3 ) . I n aqueous s o l u t i o n ,
c y s t e i n e produces an i n s o l u b l e p o l y m e r i c g o l d f l ) c y s -
t e i n e i n many r e a c t i o n s s t u d i e d . When t h i s r e a c t i o n i s
s t a r t e d from t r i e t h y l p h o s p h i n e g o l d f l ) c h l o r i d e , a t
l e a s t f o u r i n t e r m e d i a t e s p e c i e s can bè o b s e r v e d i n the
c i r c u l a r d i c h r o i s m fz). However, s t a r t i n g w i t h t r i -
p h e n y l p h o s p h i n e g o l d T l ) c h l o r i d e , the r e a c t i o n s t o p s a t
the f i r s t s t a g e , p r o d u c i n g monomeric triphenylphosphine
g o l d f l ) c y s t e i n e f11) Λ The k i n e t i c s o f s u b s t i t u t i o n
can be q u i t e r a p i d but some o f the more complex r e a c
t i o n s , such as the f o r m a t i o n o f p o l y m e r i c g o l d f l )
c y s t e i n a t e , can be slow, t a k i n g from 24 h o u r s to one

week to complete. In i n vivo r e a c t i o n s , s o l u b i l i t y i s
also important. Triethylphosphine g o l d f l ) c h l o r i d e
r e a c t s r e a d i l y i n e t h a n o l , i n which i t i s s o l u b l e and
w i t h d i f f i c u l t y i n water, i n which i t i s not a p p r e c i a b
l y s o l u b l e , whereas the r e v e r s e i s t r u e o f sodium g o l d
f l ) t h i o m a l a t e f24). I n s o l u b l e g o l d f l ) polymers,
a l t h o u g h t h e y can be made to r e d i s s o l v e w i t h s u i t a b l e
l i g a n d s , are r e l a t i v e l y u n r e a c t i v e and i n the c o r r e c t
medium i n v i v o , they are the most l i k e l y s t o r a g e forms
of gold.
G o l d f o ) , g o l d f l ) and g o l d f l l l ) o x i d a t i o n s t a t e s
can r e a d i l y be i n t e r c h a n g e d , the f i n a l p r o d u c t depend
i n g on the l i g a n d and on the c o n d i t i o n s u s e d . In
a d d i t i o n to the p r o d u c t i o n o f g o l d f l ) from g o l d f l l l ) as
a l r e a d y d i s c u s s e d , r e a c t i o n o f KAufCN) w i t h a f i v e
2
f o l d e x c e s s o f p e n i c i l l a m i n e i n a i r i n aqueous e t h a n o l
produced g o l d ( l l l ) f p e n i c i l l a m i n e ) 2 fll). However,
r e a c t i o n o f sodium t e t r a c h l o r o a u r a t e w i t h i n s u f f i c i e n t
p e n i c i l l a m i n e produces g o l d f θ ) . G o l d f o ) c o l l o i d can be
o x i d i z e d to g o l d f l ) o r g o l d f l l l ) w i t h p e n i c i l l a m i n e i n
the p r e s e n c e o f oxygen and g o l d f l l l ) can be r e d u c e d to
g o l d f l ) w i t h d i s u l f i d e s fZ6). This l a t t e r reaction i s
u n u s u a l i n t h a t a d i s u l f i d e i s b e i n g u s e d as a r e d u c i n g
agent. The e x p l a n a t i o n o f t h i s e f f e c t i s t h a t oxygen
causes f u r t h e r o x i d a t i o n o f the d i s u l f i d e to form, i n
s t e p w i s e f a s h i o n , s u l f e n i c , s u l f i n i c and s u l f o n i c a c i d s
f26;. F u r t h e r , i n the r e d u c t i o n o f g o l d f l l l ) w i t h c y s
t e i n e the c o n v e n t i o n a l r e a c t i o n ,
NaAuCl 4 + RSH > RSAu + RSSR + 3HC1 + NaCl
r e q u i r e s 3 moles o f c y s t e i n e p e r mole o f g o l d compound

and t h i s appears to be the r e a c t i o n which o c c u r s under
nitrogen. I n the p r e s e n c e o f oxygen, however, much
l e s s l i g a n d i s r e q u i r e d , oxygen i s consumed i n the

r e a c t i o n fll) and c y s t e i c a c i d has been i d e n t i f i e d i n

the p r o d u c t s f 2 7 ) . I n a study o f the r e d u c t i o n o f g o l d
f i l l ) to g o l d f l ; w i t h t h i o e t h e r s , a key s t e p i n the
r e d u c t i o n p r o c e s s i s postulated as a r e a c t i o n a t the co-
o r d i n a t e d l i g a n d r a t h e r t h a n a t the m e t a l i o n f.28) and
i t i s p o s s i b l e t h a t oxygen complexes to the c o o r d i n a t e d
s u l f u r atom i n the r e d u c t i o n o f g o l d f l l l ) by t h i o l s o r
disulfides.
In Vivo Chemistry
The approach r e q u i r e d f o r i n v i v o c h e m i s t r y i s
q u i t e d i f f e r e n t from t h a t employed i n v i t r o i n t h a t not
a l l the t e c h n i q u e s employed i n v i t r o are a p p l i c a b l e and
the b a s i c c h e m i s t r y i s more complex. The a d m i n i s t r a -

t i o n o f a g o l d compound e v e n t u a l l y l e a d s to i t s metabo-
l i s m and the f i n a l s t o r a g e o r e x c r e t i o n o f the g o l d i n
a time s c a l e comparable to those o b s e r v e d f o r i n v i t r o
k i n e t i c s and, hence, t h e r e i s an e s s e n t i a l l y non-
e q u i l i b r i u m s i t u a t i o n c h e m i c a l l y as w e l l as m e t a b o l i c -
ally. F u r t h e r , t h e r e i s no guarantee t h a t the l a r g e s t
f r a c t i o n s o f the g o l d f o u n d i n v i v o a r e , i n f a c t , the
therapeutic or t o x i c f r a c t i o n s j i t i s p o s s i b l e that
v e r y s m a l l amounts o f g o l d p r e s e n t i n the c o r r e c t en-
v i r o n m e n t s are r e s p o n s i b l e f o r the drug a c t i o n . During
the p r o c e s s o f metabolism, the g o l d w i l l pass t h r o u g h
b i o l o g i c a l compartments i n which the d i e l e c t r i c cons-
t a n t w i l l be such as to produce a c h e m i s t r y more a k i n
to t h a t i n non-aqueous s o l v e n t s than to t h a t i n water.
I n s p i t e o f these d i f f i c u l t i e s , s u f f i c i e n t i s known
about the r e a c t i v i t y o f i n v i v o g o l d to e n a b l e some
c o n c l u s i o n s to be drawn on the b a s i s o f a comparison o f
the a v a i l a b l e i n v i v o d a t a and the i n v i t r o c h e m i s t r y .
I n t h i s r e s p e c t , the development o f a u r a n o f i n f t r i e t h y l -
phosphinef2,3,4,6-tetra-o-acetyl-l-thio-p-D-glucopyra-
n o s a t o ) g o l d f l ) f1.) , which c o n t a i n s the t r i e t h y l p h o s -
p h i n e g o l d f l ) moiety, i s h e l p f u l i n t h a t a comparison
can be made between i t and the more c o n v e n t i o n a l g o l d -
s u l f u r drugs such as M y o c r i s i n fsodium g o l d f l ) t h i o -
malate) .
M y o c r i s i n and a u r a n o f i n are w i d e l y d i s t r i b u t e d
among the p r o t e i n s i n b l o o d serum (2£_) # For Myocrisin,
the g o l d c o n t e n t o f each p r o t e i n f r a c t i o n i s o l a t e d by
e l e c t r o p h o r e s i s has been shown to v a r y from p a t i e n t to
p a t i e n t but to remain c o n s t a n t f o r any one p a t i e n t as
the g o l d l e v e l o f the serum i n c r e a s e s d u r i n g t h e r a p y
f30). Thus, t h e r e seems to be a number o f g o l d b i n d i n g
s i t e s i n serum which are o f comparable s t r e n g t h and
which are not s a t u r a t e d a t the g o l d l e v e l s a c h i e v e d
d u r i n g t h e r a p y . These s i t e s are p r o b a b l y t h i o l groups

or d i s u l f i d e s . The l a r g e s t s i n g l e f r a c t i o n i s on a l b u
m i n , w i t h r e p o r t s o f b e t w e e n 50 a n d 90% o f t h e g o l d
being present on t h i s p r o t e i n . T h i s wide s c a t t e r i s
due t o d i f f e r e n c e s i n a n a l y s i s m e t h o d s a n d s e p a r a t i o n
techniques used, i n the time o f sampling a f t e r i n j e c
t i o n andi n the v a r i a t i o n i n b i o c h e m i s t r y o f p a t i e n t s
with rheumatoid a r t h r i t i s . I n a d d i t i o n t o the t i g h t l y
b o u n d g o l d , some g o l d i s a b s o r b e d o n a l b u m i n , p r o b a b l y
as t h e u n c h a n g e d o r p a r t l y m e t a b o l i s e d d r u g . There are
fewer r e s u l t s a v a i l a b l e f o r a u r a n o f i n , but there i s a
f r a c t i o n o f the g o l d on albumin as w e l l as a f r a c t i o n
bound t o i t a n d t h e r e i s a d i f f e r e n t d i s t r i b u t i o n o f
t i g h t l y bound g o l d w i t h h i g h e r c o n c e n t r a t i o n s o n the β
a n d y g l o b u l i n f r a c t i o n s (29 %2θ). F u r t h e r , the d i s t r i
b u t i o n o f g o l d between the c i r c u l a t i n g b l o o d c e l l s and

p l a s m a i s d i f f e r e n t w i t h t h e d i f f e r e n t compounds ( F i
g u r e 5). Up t o 50% o f t h e b l o o d g o l d i n a u r a n o f i n i s
present i n the c e l l s . W i t h M y o c r i s i n , u p t o a b o u t 30%
o f t h e g o l d may b e p r e s e n t i n some c a s e s b u t n o g o l d a t
a l l has been d e t e c t e d i n o t h e r s . The d i f f e r e n c e a p
p e a r s t o b e t h a t c i g a r e t t e s m o k i n g i n c r e a s e s t h e amount
of i n t r a c e l l u l a r g o l d , p o s s i b l y because the a d d i t i o n a l
cyanide present i n blood a c t s as a t r a n s p o r t i n g agent
but a u r a n o f i n p r o d u c e s a t l e a s t one f o r m o f serum g o l d
w h i c h c a n c r o s s t h e c e l l membrane i n a n y c a s e (2.>22Û ·
The d i f f e r e n c e i n r e a c t i v i t y o f t h e s e r u m g o l d
p r o d u c e d b y the two d r u g s can be d e m o n s t r a t e d c l e a r l y
by i n c u b a t i n g the serum f r o m M y o c r i s i n a n d a u r a n o f i n
t r e a t e d p a t i e n t s w i t h blood c e l l s from a placebo
t r e a t e d p a t i e n t . Most o f the a u r a n o f i n serum g o l d i s
t r a n s f e r r e d (2)· T h i s c o n c l u s i o n i s r e i n f o r c e d b y a n i -
mal e x p e r i m e n t s w i t h t r i e t h y l p h o s p h i n e g o l d c h l o r i d e
and M y o c r i s i n . T r i e t h y l p h o s p h i n e g o l d c h l o r i d e i s v e r y
e f f i c i e n t l y a b s o r b e d o r a l l y (j52^.H). A v e r y h i g h c o n -
c e n t r a t i o n appears t o adhere t o the stomach w a l l i n i t i -
a l l y , s u g g e s t i n g t h a t t h e a b s o r p t i o n may o c c u r b y
chemical attack with n a t u r a l l y occurring t h i o l ligands
i n t h e l i p i d l a y e r s o f t h e s t o m a c h w a l l (β) . M y o c r i s i n
reacts readily with zinc metallothionen i n vitro with
t h e g o l d r e p l a c i n g z i n c (34). T h e w a y i n w h i c h d i f f e
r e n t compounds o r m e t a b o l i t e s r e a c t w i t h t h i s p r o t e i n
i n the gut c o u l d be a n important step i n o r a l absorp
tion. Both M y o c r i s i n andauranofin d i s t r i b u t e widely
but the r e s i d e n c e time o f g o l d i n t i s s u e s such as k i d
ney a n d l i v e r i s a p p r e c i a b l y s h o r t e r f o r o r a l l y admi
n i s t e r e d auranofin than f o r i n j e c t e d M y o c r i s i n , again
s u g g e s t i n g a d i f f e r e n t r e a c t i v i t y f o r the i n v i v o g o l d
and d e m o n s t r a t i n g t h a t t h e change i n r e a c t i v i t y a p p l i e s
t o more b i o c h e m i c a l c o m p a r t m e n t s t h a n b l o o d . Thus, i t
seems l i k e l y t h a t b o t h M y o c r i s i n a n d a u r a n o f i n a r e

s
bo
w 71
1
.Ζ. η . Tri
2
ο
Ο 1.2-
S"
/ /
/
Π
/
/ / /
.4
/
/ / /
/
/ /
/
/ /
/ 12
A 24 week
β
Figure 5. Distribution of gold between the cells and the plasma in patients treated
with Myocrisin (top) and auranofin (bottom).

21. BROWN A N D SMITH Gold Thiolate Complexes 413
metabolised quite q u i c k l y i n v i v o but the metabolites

f o r m e d r e t a i n a memory o f t h e p a r e n t c o m p o u n d i n some
way a n d p r o d u c e b i o c h e m i c a l f r a c t i o n s of quite d i f f e -
rent reactivity.
T h e r e h a v e b e e n many more s t u d i e s i n w h i c h gold
d i s t r i b u t i o n and the r e a c t i o n s of gold with proteins
have been i n v e s t i g a t e d . The general conclusion of
these ex v i v o s t u d i e s i s that the g o l d i s coordinated
to the t h i o l group. A g o l d - s u l f u r bond i n albumin has
been demonstrated by Mô'ssbauer measurements (3j) . A
major problem with such studies is that the separation
and subsequent reaction of these biochemical fractions
may w e l l c a u s e c h a n g e s i n r e a c t i v i t y as t h e m a t r i x is
removed d u r i n g s e p a r a t i o n . Using N.M.R. methods, i t
has been p o s s i b l e to demonstrate the e x i s t e n c e o f a

gold glutathione complex i n the i n t a c t r e d c e l l and i t
may b e t h a t t h i s m e t h o d o f a p p r o a c h may y e t p r o v i d e a
more d e t a i l e d u n d e r s t a n d i n g o f the i n v i v o processes
Mechanism of A c t i o n o f Gold Drugs. In only a few

cases is the a c t i o n o f a drug understood i n sufficient
d e t a i l to be o f use to the chemist i n p o s t u l a t i n g a me-
chanism at the molecular l e v e l . In the case o f p l a t i -
num c o m p o u n d s , t h e e f f e c t o n c e l l r e p l i c a t i o n i s regar-
ded as a k e y s t e p w h i c h l e a d s to the p o s t u l a t e that the
mechanism o f a c t i o n i s due to f o r m a t i o n o f complexes
o f p l a t i n u m w i t h DNA, as d i s c u s s e d elsewhere i n this
volume. The equivalent o b s e r v a t i o n i n the case of gold
compounds and o f o t h e r agents b e l i e v e d to be c a p a b l e of
p r o d u c i n g a r e m i s s i o n o f the symptoms o f r h e u m a t o i d a r -
t h r i t i s may be t h a t t h e y a l l m o d u l a t e t h e immune res-
p o n s e i n some w a y . To date, a l l the m a j o r compounds
c l a i m e d to have r e m i s s i o n i n d u c i n g p r o p e r t i e s and which
are not either cytotoxic agents or steroids, are thiols
or h y d r o l y s e to t h i o l s i n v i v o , o r are compounds such
as the g o l d compounds o r c h l o r o q u i n e w h i c h w o u l d be ex-
pected to r e a c t r e a d i l y w i t h t h i o l s i n vivo. However,
t h e way i n w h i c h t h e immune s y s t e m i s a f f e c t e d varies
f r o m compound to compound and i s d i f f e r e n t f o r each gold
compound, as might have been i n f e r r e d f r o m t h e i r diffe-
rent i n vivo reactivity. Further, t h e r e i s no evidence
of a common f a c t o r s u c h a s t h e s u p p r e s s i o n o f activity
o f one p a r t i c u l a r enzyme (^fifà) t h e r e i s no general
c o n s e n s u s t h a t the amount o f any g o l d f r a c t i o n s u c h as
serum g o l d , small molecule gold or i n t r a c e l l u l a r gold,
i s r e l a t e d to t h e r a p e u t i c response.
One way c o m p o u n d s w i t h s u c h d i s p a r a t e p r o f i l e s of
a c t i v i t y might affect t h e immune s y s t e m i s b y reaction
w i t h s u l f h y d r i l and d i s u l f i d e groups on c e l l membrane.

These membranes r e q u i r e m e t a l i o n s f o r t h e i r s t a b i l i t y
f 37 ) and the -SH groups on and i n them a f f e c t the che-
m i c a l t r a n s p o r t o f v i t a l n u t r i e n t s such as a l a n i n e (^J?),
the s y n t h e s i s o f components such as g l u t a t h i o n e (39)
and c e l l adherence.
The t h i o l groups on the membranes a r e i n many d i f -
f e r e n t environments w i t h d i f f e r e n t degrees o f p r o t e c -
t i o n by s t e r i c o r d i e l e c t r i c c o n s t a n t f a c t o r s (40).
Thus, they w i l l r e a c t d i f f e r e n t l y w i t h d i f f e r e n t com-
pounds. A r e l a t e d example which has been w i d e l y studied
i s t h e d e t e r m i n a t i o n o f the t h i o l c o n t e n t o f hemoglobin
(4l). Due t o the p r o t e c t i o n o f t h e t h i o l groups by the
p r o t e i n s t r u c t u r e , from 1 t o 8 t h i o l groups p e r mole-
c u l e have been r e p o r t e d , depending on t h e e s t i m a t i o n
u s e d and one s t a n d a r d r e a g e n t , E l l m a n ' s r e a g e n t , does

not appear t o r e a c t a t a l l . Thus, each p o t e n t i a l t h e -
r a p e u t i c agent would be e x p e c t e d t o r e a c t w i t h a d i f f e -
r e n t c o m b i n a t i o n o f t h i o l groups and, hence, t o a f f e c t
c e l l u l a r f u n c t i o n s i n a unique way.
The t h i o l system i t s e l f i s i n a complex and l i t t l e
u n d e r s t o o d e q u i l i b r i u m i n v i v o . The main t h i o l contain-
i n g s p e c i e s i n serum i s albumin and i t has between 0.3
and 0.7 t h i o l groups p e r m o l e c u l e , depending on d i s e a s e
s t a t e , w i t h the r e m a i n i n g t h i o l e i t h e r b l o c k e d by c y s -
teine or other t h i o l ligands or missing altogether.
The k i n e t i c s o f r e a c t i o n o f albumin t h i o l a r e complex
and t h e r e may be more than one microenvironment round
the t h i o l group. T h i s group i s l i k e l y t o be i n e q u i -
l i b r i u m w i t h o t h e r t h i o l s and the way i n which g o l d
adds p r o p o r t i o n a t e l y t o each serum f r a c t i o n as the l e v e l
i n c r e a s e s i s an i n d i c a t i o n t h a t g o l d c e r t a i n l y e q u i l i -
b r a t e s between d i f f e r e n t serum p r o t e i n s . A soluble
small molecule d i s u l f i d e could e q u a l l y w e l l accomplish
this i n vivo. I t would be e x p e c t e d t h a t some form o f
e q u i l i b r i u m w i t h each p r o t e i n and w i t h the membrane
s u r f a c e would e x i s t . On the i n s i d e o f the membrane, an
11 1 1
analogous e q u i l i b r i u m would a l s o e x i s t . To a f f e c t
t h i s e q u i l i b r i u m , the drugs must s u r v i v e i n v i v o and
be t o l e r a t e d w e l l by the p a t i e n t . Thus, a l t h o u g h c y s -
t e i n e would be o x i d i z e d t o c y s t i n e and p r e c i p i t a t e a t
c e l l membrane and t i s s u e s u r f a c e s a t b l o o d pH, p e n i c i l -
lamine w i l l form a mixed d i s u l f i d e and remain i n
c i r c u l a t i o n and p o t e n t i a t e the t h i o l - d i s u l f i d e e q u i l i -
b r i u m f43). Thus, g o l d would be e x p e c t e d t o modulate
the t h i o l system i n an analogous but n o t i d e n t i c a l way
to p e n i c i l l a m i n e o r c h l o r o q u i n e ( F i g u r e 6).
I n c h r o n i c i n f l a m m a t i o n , the i n f l a m m a t o r y r e s p o n s e
which b e g i n s as a b e n e f i c i a l e f f e c t o f the immune s y s -
tem remains i n a c t i o n s u f f i c i e n t l y l o n g t o c r e a t e
t i s s u e d e s t r u c t i o n and so produce more a n t i b o d i e s

BROWN A N D SMITH Gold Thiolate Complexes 415
HaemoglobinSH
Albumin SH
ι
! -SH Glutathione
RSSR HS-j
-SH
ιι
._ J '
- <|
- > - >
it t <
Synthesis pathway
1
Other Protein SH
Plasma Membrane Intracellular fluid
Figure 6. Schematic diagram of the thiol/disulfide equilibrium inside and outside

cells. Gold interacts with different compounds of this system and is expected to
modify the equilibrium.

r e q u i r i n g a further inflammatory response. This sus-

tained inflammation i s achieved i n a s l i g h t l y different
way than the i n i t i a l inflammation. One effect i s that
c i r c u l a t i n g monocytes adhere to the polymorphonuclear
c e l l s i n the j o i n t and enhance t h e i r phagocytic a c t i o n .
Myocrisin i s known to reduce t h i s adherence and may
affect the immune response i n that way (44). This
change could well be caused by the i n t e r a c t i o n of gold
with the t h i o l group on c e l l membranes. Thus, the
mechanism of action of gold drugs may not be known with
c e r t a i n t y but a reasonable postulate as to t h e i r chemi-
c a l a c t i v i t y can be given. Much remains to be done to
test t h i s hypothesis and to define the precise nature
of the effect on the membrane of c i r c u l a t i n g blood cells
and the synovium.
AcknowledgmentB
We thank Dr. H . A . Capell and the s t a f f at the

Centre for Rheumatic Diseases i n Glasgow for t h e i r
encouragement of our work on gold over a number of
years.
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inflammatory Agents", Ed.: Scherrer, R.A.; White-
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1980; 42, 551.
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McNeillie, A.; Dunlop, J.; Smith, W.E.; Brown, D.H.;
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Inorg. Chim.Acta 1982; 67, 27.
26. Shaw III, C . F . ; Canero, M.P.; Witkiewicz, P.L.;
Eldridge, J.F.; Inorg.Chim. 19, 1980.
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G . , J . C . S . Dalton,1980, 1017.
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Dis. 1980; 31.
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J.Rheumatol. Supp. 5 1980, 30.
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W.E.; in press.
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Cottney, J.; Lewis, A.J.; Arth. and Rheum. 1978,
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Cottney, J.; Lewis, A.J.; Agents and Actions 1978;
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37. Ludwig, J.S.; Chapvil, M.; in "Trace Elements in

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S.S.; in "Sulphur in Biology", Pg. 135, CIBA Sym
posium 72, Exerpta Medica, 1980, Amsterdam.
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and Biophys. Acta 1979; 557, 363.
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Group in Aminoacids, Peptides and Proteins,
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C.J.; Smith, W.E.; Sturrock, R.D.; Rheumatology

International 1982; in press.
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1979, 22, 1352.

22
O s m i u m C a r b o h y d r a t e P o l y m e r s as P o t e n t i a l
Antiarthritic D r u g s
C. C. HINCKLEY and J. N. BEMILLER—Southern Illinois University at

Carbondale, Department of Chemistry and Biochemistry, Carbondale, IL 62901
L. E. STRACK—Southern Illinois University at Carbondale, Department of
Animal Industries, Carbondale, IL 62901
L. D. RUSSELL—Southern Illinois University at Carbondale, Department of
Physiology, Carbondale, IL 62901
Osmium carbohydrate polymers, termed osmarins,

have been investigated as potential antiarthritic
agents. They are prepared in three steps from
OsO , and have been characterized by elemental
4
analysis, gel filtration, gel electrophoresis,

solution density and viscosity measurements, and
ultracentrifugation. The polymers are of variable
composition dependent upon the details of prepara-
tion. They are polydisperse anionic polyelectro-
lytes, and preparations containing 30%-40% osmium
have average molecular weights in the range,
50-100K daltons. The polymers react in strong
base with the oxygen species O , O , and H O .2 2 2 2
They form complexes in solution with proteins.

In antiarthritic application, dilute solutions of
the compounds are injected directly into the
synovial spaces of effected joints. Though some
evidence of microscopic toxicity has been found,
the materials have very low toxicity. In mice,
IP injection at dose levels of 1g/Kg body weight
produce no mortality. In pigs and rabbits,
osmarins of high molecular weight stain the joint
capsules and articular surfaces of injected
joints irreversibly. In limited experimental
treatments of arthritic dogs, improvements have
been noted in five of six cases. It is proposed
that, in vivo, osmarins may react with superoxide
ion and remove this damaging species.
New osmium compounds may prove to be antiinflammatory
agents useful in the treatment of some forms of arthritis. The
materials are osmium-carbohydrate polymers (1), termed osmarins.
These compounds bind irreversibly with living tissue and exhibit
other properties which suggest their use.
0097-6156/83/0209-0421$06.00/0

There i s a long standing connection between a r t h r i t i s

treatment and osmium ( 2 ) . Osmium t e t r o x i d e has been used on a
l i m i t e d b a s i s f o r the treatment of a r t h r i t i s i n humans, p r i n c i -
p a l l y i n Europe, f o r about 30 years. Osmium t e t r o x i d e treatment
i s not used i n the United States and i s c o n t r o v e r s i a l everywhere.
In theory, the treatment makes use of the t o x i c i t y of osmium
t e t r o x i d e to achieve a "chemical synovectomy". D e t r a c t o r s
b e l i e v e t h a t the damage t o j o i n t t i s s u e s inherent i n the proce-
dure exacts a p r i c e too high f o r the method to be g e n e r a l l y
acceptable. S t i l l , the s m a l l but p e r s i s t e n t l i t e r a t u r e which
has developed over the years provides a growing l i s t of apparent
successes.
In 1976 a group of researchers i n S w i t z e r l a n d reported a
study of over seventy people who had been helped by the t r e a t -
ment ( 3 ) . They found t h a t there were osmium c o n t a i n i n g d e p o s i t s

i n the j o i n t s of these people long a f t e r the osmium t e t r o x i d e
treatment. They i n c l u d e d i n t h e i r paper a suggestion, upon
which our work i s p a r t l y based, t h a t the osmium c o n t a i n i n g
deposits may c o n t r i b u t e to the long term e f f i c a c y of the pro-
cedure .
Recent work provides s u p p o r t _ f o r t h e i r suggestion ( 4 ) .
Involvement of superoxide i o n , 0^ , i n inflammation i s w e l l
e s t a b l i s h e d (5,6). In a r t h r i t i s , superoxide may be present i n
an e f f e c t e d j o i n t as p a r t of a response to the disease ( 4 ) .
There i t may a t t a c k the s y n o v i a l f l u i d , d e s t r o y i n g i t s c a p a c i t y
to l u b r i c a t e the j o i n t surfaces (7,8). Metal complexes have
been shown to c a t a l y t i c a l l y remove superoxide ( 9 ) , and osmium
c o n t a i n i n g d e p o s i t s could perform a s i m i l a r f u n c t i o n . Osmium
e x h i b i t s many of the c h a r a c t e r i s t i c s b e l i e v e d necessary f o r
these c a t a l y t i c p r o p e r t i e s . The f a c t t h a t the enzyme superoxide
dismutase has been s u c c e s s f u l l y used to t r e a t a r t h r i t i s i n both
animals (10) and humans (11,12) i s f u r t h e r support f o r the
suggestion.
In our proposed a p p l i c a t i o n , s o l u t i o n s of osmarins are t o
be i n j e c t e d d i r e c t l y i n t o the s y n o v i a l spaces of a r t h r i t i c
j o i n t s . The aim i s to provide osmium deposits s i m i l a r t o those
which Bousinna, e t . a l . , (3) have suggested may be b e n e f i c i a l ,
w h i l e at the same time, a v o i d i n g the damage t h a t accompanies
OsO^ i n j e c t i o n s .
In t h i s paper, we f i r s t present the p r e p a r a t i o n and
c h a r a c t e r i z a t i o n of osmium-carbohydrate polymers. This d i s -
c u s s i o n i s f o l l o w e d by a review of our s t u d i e s i n v o l v i n g animals.
Mice have been used f o r t o x i c i t y s c r e e n i n g , and we have examined
the e f f e c t of osmarins upon the t i s s u e s of the s y n o v i a l space
i n both p i g s and r a b b i t s . F i n a l l y , we r e p o r t the r e s u l t s of a
s m a l l number of experimental treatments of a r t h r i t i c dogs.
Our f i n d i n g s are t h a t osmarins of high average molecular
weight appear t o have the p r o p e r t i e s we seek, t h a t i s , they
have minimal t o x i c i t y , no evident p e r s i s t e n t s i d e e f f e c t s , are
r e t a i n e d f o r long periods w i t h i n the j o i n t , and are e f f e c t i v e

22. HINCKLEY E T AL. Osmium Carbohydrate Polymers 423
i n some cases i n r e s t o r i n g freedom of movement of a r t h r i t i c

j o i n t s and a l l o w i n g recovery of the j o i n t s u r f a c e s . Comments
concerning apparent e f f i c a c y are not presented as a s s e r t i o n s o f
proven f a c t , but r a t h e r as i n d i c a t i o n s which prompt our con
t i n u i n g study.
Osmarins
P r e p a r a t i o n and c h a r a c t e r i z a t i o n . Osmarins are prepared

i n three s t e p s , the f i r s t of which begins w i t h osmium t e t r o x i d e .
OsO^ (0.5 g ) , d i s s o l v e d i n a s m a l l amount (15 mL) of methanol
c o n t a i n i n g 0.1M KOH, r e a c t s w i t h the a l c o h o l t o produce d i -
potassium tetramethylosmate(VI), K [ 0 s ( 0 C H ^ ) ^ 0 ] , which
2 2
Table I . Elemental compositions f o r s e v e r a l osmarin pre

parations.
Preparation % Os % c % H % κ
1 9.7 26.7 4.5 11.7
2 13.6 26.0 4.3 10.0
3 15.0 14.5 3.6 0.7
4 23.0 32.4 5.6 6.9
5 35.0 14.6 5.8 4.9
p r e c i p i t a t e s as a green s o l i d (13). When t h i s product i s

d i s s o l v e d i n a c e t i c a c i d (100 mL), blue potassium t r i a c e t a t o d i -
oxoosmate(VI) i s formed. Glucose (1.0 g ) , d i s s o l v e d f i r s t i n a
small amount of water (20 ml) and then mixed w i t h a l a r g e r
q u a n t i t y of a c e t i c a c i d (100 mL) i s added t o the blue s o l u t i o n
and allowed t o r e a c t overnight a t room temperature t o form the
osmarin. P u r i f i c a t i o n i s e f f e c t e d by g e l f i l t r a t i o n of an
aqueous s o l u t i o n through Sephadex G-25 a f t e r the a c e t i c a c i d
has been removed by evaporation under reduced pressure.
Osmarins of w i d e l y d i f f e r i n g composition (Table I ) have
been prepared by v a r y i n g the r e a c t i o n temperature, the concen
t r a t i o n s of osmium and carbohydrate i n the p r e p a r a t i v e m i x t u r e ,
the nature of the carbohydrate, and the time of r e a c t i o n . F o r
i n s t a n c e , when the q u a n t i t i e s of r e a c t a n t s i n d i c a t e d above are
allowed t o r e a c t a t room temperature f o r twenty-four hours, the
f i n a l p r e p a r a t i o n w i l l c o n t a i n 30 t o 40% osmium. Longer r e a c t i o n
times (1 week) w i t h m i l d h e a t i n g on a steam bath y i e l d prepara
t i o n s having 20 t o 30% osmiums. I n c r e a s i n g the q u a n t i t y of
glucose (2-3 g) and h e a t i n g y i e l d s p r e p a r a t i o n s having l e s s
than 20% osmium. The p r e p a r a t i o n s are brown or b l a c k depending
upon t h e i r composition. P r e p a r a t i o n s c o n t a i n i n g l e s s than 20%
osmium are brown, those c o n t a i n i n g more than 20% are b l a c k .
The v e r y i n t e n s e c o l o r s are the consequence of broad a b s o r p t i o n

throughout the v i s i b l e r e g i o n . E x t i n c t i o n c o e f f i c i e n t s are on

the order of 2000 L/cm. mole o f Os. These values compare
f a v o r a b l y t o those found f o r some charge t r a n s f e r complexes
(14) .
The r e l a t i v e l y h i g h percentages of carbon i n the polymers
and t h e i r h i g h aqueous s o l u b i l i t y suggest the presence of bound
carbohydrate. I n f r a r e d s p e c t r a o f - t h e polymers e x h i b i t broad
bands i n the^regions 1610-1550 cm" , 1400-1300 cm" , and
1200-950 cm , c o n s i s t e n t w i t h the presence of gluconate and
glucose. Permanganate t i t r a t i o n s of the polymers i n a c i d
s o l u t i o n i n d i c a t e an o x i d a t i o n s t a t e f o r osmium of +4, but do
not e l i m i n a t e the p o s s i b i l i t y of mixed o x i d a t i o n s t a t e s . These
f a c t o r s are c o n s i s t e n t w i t h the e m p i r i c a l formula:
K 0 s 0 ( g l u c o s e ) ( g l u c o n a t e ) OÔ) . This formula embodies an
u v x
i n t e r p r e t a t i o n of the materYals as glucose and/or gluconate

s o l u b i l i z e d osmium d i o x i d e . In t h i s i n t e r p r e t a t i o n , d i r e c t
Os-Os bonds are not assumed. B r i d g i n g between osmium ions
through oxo and/or carbohydrate groups i s thought t o be most
l i k e l y . R e c e n t l y , a new c l a s s of osmium(IV) complexes con
t a i n i n g both oxide and c a r b o x y l a t e b r i d g e s has been reported
(15) . The compounds, 0 β ( μ - 0 ) ( M - O ^ C H ^ X ^ P R ^ where X=Cl,Br,
2
e x h i b i t b r i d g i n g between the osmium atoms of tne k i n d suggested

f o r osmarins.
Gel f i l t r a t i o n has i n d i c a t e d t h a t the components have
r e l a t i v e l y l a r g e molecular volumes. They e l u t e as broad bands
at or near the v o i d volume of a Sepharose £-B column. T h i s g e l
excludes g l o b u l a r p r o t e i n s which exceed 10 d a l t o n s i n molecular
weight. Since the g e l i s not c a l i b r a t e d f o r osmarins, g e l
f i l t r a t i o n does not provide estimates of molecular weight.
In g e l e l e c t r o p h o r e s i s , the p r e p a r a t i o n s migrate as s i n g l e
broad bands toward the p o s i t i v e e l e c t r o d e . P l o t s of m i g r a t i o n
d i s t a n c e versus g e l c o n c e n t r a t i o n (Ferguson p l o t s ) , measured
f o r the same c o n d i t i o n s of temperature and e l e c t r i c f i e l d , show
t h a t molecules i n the t r a i l i n g edge of the bands are l a r g e r
than those i n the l e a d i n g edge. T h i s demonstrates t h a t the
p r e p a r a t i o n s are p o l y d i s p e r s e , an i n d i c a t i o n which i s confirmed
i n sedimentation v e l o c i t y experiments.
Osmarins are extremely s o l u b l e ; aqueous s o l u t i o n s con
t a i n i n g up to 10% osmarin were used f o r s o l u t i o n d e n s i t y ,
p a r t i a l s p e c i f i c volume, and v i s c o s i t y measurements. The
e f f e c t of the d i s s o l v e d polymer upon the v i s c o s i t y o f the
l i q u i d i s s l i g h t , even a t the extreme c o n c e n t r a t i o n s t u d i e d ,
i n d i c a t i n g t h a t , i n aqueous s o l u t i o n , the polymers are g e n e r a l l y
s p h e r i c a l i n shape.
Sedimentation v e l o c i t y experiments, supplemented by p a r t i a l
s p e c i f i c volume and v i s c o s i t y measurements, p r o v i d e estimates
of molecular weights ( F i g u r e 1). M o l e c u l a r weights may v a r y
from 10,000 to 100,000 d a l t o n s or more i n a s i n g l e p r e p a r a t i o n .
There i s a general r e l a t i o n s h i p between the composition of the
osmarin p r e p a r a t i o n and the range of molecular weights of the
components of the mixture. The molecular weights of osmarin

22. HINCKLEY ET AL. Osmium Carbohydrate Polymers 425
2Π
5 10 15 20 25
SEDIMENTATION COEFFICEIENT
Figure 1. Sedimentation profiles for several osmarins plotted as absorbance vs.

sedimentation coefficient.

p r e p a r a t i o n s c o n t a i n i n g as l i t t l e as 15% osmium average 10K

d a l t o n s , w h i l e the average molecular weight of those c o n t a i n i n g
35% i s 80K daltons or more.
The combined r e s u l t s a l l o w us to draw the o u t l i n e s of
s t r u c t u r e of b l a c k osmarins. The p r e p a r a t i o n s are p o l y d i p e r s e
mixtures of osmium-carbohydrate polymers. The number of osmarin
atoms v a r i e s from 10 to 50 per molecule most commonly, w i t h
some having as many as 100 or more. W i t h i n the molecules,
osmium atoms are l i n k e d together w i t h oxygen or carbohydrate
bridges (gluconate or g l u c o s e ) . Osmium e x h i b i t s an average +4
o x i d a t i o n s t a t e , and the polymers are a n i o n i c . In s o l u t i o n ,
the molecules are d i s t o r t e d spheres. A suggested o r g a n i z a t i o n a l
geometry i s t h a t of c o i l e d chains of v a r y i n g l e n g t h . The
chains may be branched and/or c r o s s l i n k e d , but not l i n e a r l y
extended.
Chemical P r o p e r t i e s . Chemical p r o p e r t i e s of the polymers

are c o n s i s t e n t w i t h those expected f o r the c o n s t i t u e n t s of the
m a t e r i a l s , and w i t h those expected of a n i o n i c polymers. Osmarins
are o x i d i z e d by a wide v a r i e t y of common o x i d i z i n g agents
i n c l u d i n g potassium permanganate and e e r i e ammonium n i t r a t e .
In these r e a c t i o n s , both the osmium and the bound carbohydrate
are o x i d i z e d .
Studies of osmarin-oxygen species chemistry are i n progress
and w i l l be reported l a t e r . P r e l i m i n a r y experiments i n d i c a t e
an extensive oxygen chemistry. The polymers r e a c t i n v i t r o
w i t h the oxygen species HÔ^, 0^ , and 0^, i n the presence of
strong base. The r e a c t i o n w i t h hydrogen peroxide i s accompanied
by vigorous gas e v o l u t i o n i n d i c a t i n g t h a t the peroxide has
decomposed. Osmium i s o x i d i z e d to 0s0^ which may be removed by
e x t r a c t i o n i n t o chloroform. In the r e a c t i o n of osmarins w i t h
K 0 i n DMSO, no gas i s evolved i n s p i t e of the presence of
2
water i n the s o l u t i o n . Gas i s evolved when osmarins are not

present.
F i g u r e 2 i s a p l o t of absorbance a t 480 αιμ as a f u n c t i o n
of time, f o r the r e a c t i o n of an osmarin w i t h the oxygen i n a i r .
The r e a c t i o n w i t h oxygen i s complicated by the f a c t t h a t both
the osmium i n the compound and the attached carbohydrate r e a c t
w i t h the oxygen. Product mixtures are complex, and c o n t a i n
s e v e r a l osmium c o n t a i n i n g components as w e l l as a carbohydrate
mix t h a t i n c l u d e s hexoses and evidence f o r pentoses.
Osmarins r e a c t w i t h p r o t e i n s . Evidence f o r r e a c t i o n i s
the formation of p r e c i p i t a t e s and the presence of p r o t e i n
dependent bands i n e l e c t r o p h o r e s i s . P r o t e i n s s t u d i e d are
albumin, cytochrome C, myglobin, and lysozyme. In these e x p e r i
ments, s o l u t i o n s c o n t a i n i n g an osmarin p r e p a r a t i o n and the
p r o t e i n are mixed and allowed t o stand f o r some time. Reaction
i s not immediate, and r e a c t i o n time i s dependent upon the
osmium content of the osmarin p r e p a r a t i o n . When the osmium
percentage i s h i g h (25%), p r e c i p i t a t e s form i n two or three

HINCKLEY ET AL. Osmium Carbohydrate Polymers 427
-l.O
-1.2
ο
-1.3
-1.4
TIME (min.)
Figure 2. Logarithms of absorbance (480 nm) are plotted vs. time for basic
solution of an osmarin reacting with air. Key: I, no pretreatment; II, helium bubbled
through prior to beginning reaction by adding strong base; and III, O gas pre t
treatment.

hours, i f the osmium percentage i s low (15%) p r e c i p i t a t e s may

not form a t a l l . I n every case, however, e l e c t r o p h o r e s i s
demonstrates the f o r m a t i o n of complexes which appear as d i s t i n c t
and narrow bands, present o n l y when p r o t e i n i s p r e s e n t .
P r o t e i n b i n d i n g i s a reasonable e x p e c t a t i o n f o r osmarins.
I t i s c o n s i s t e n t w i t h the known s t r u c t u r e of the polymers and
the p r o p e r t i e s of osmium ( I V ) . Osmarins are p o l y a n i o n i c , a
s t r u c t u r a l f e a t u r e which p r o v i d e s the means t o form m u l t i p l e
i o n i c l i n k a g e s w i t h p r o t e i n s , and a d d i t i o n a l l y , Os(IV) i s known
to form complexes w i t h p r o t e i n sidegroups (16).
Animal s t u d i e s
T o x i c i t y i n mice. Osmarin p r e p a r a t i o n s t o be used i n

animal experiments are screened f o r acute t o x i c i t y . IP i n -

j e c t i o n s i n mice a t dose l e v e l s of l g osmarin/kg body weight
produce no m o r t a l i t y . As y e t , no l e t h a l dose has been d e t e r -
mined. At the 1 g/kg.b.w. dose l e v e l the s k i n and eye c o l o r of
the mice are darkened. No other e f f e c t s are observed. A f t e r
about an hour, the u r i n e of i n j e c t e d mice i s darkened by osmarin,
but s i g n i f i c a n t amounts are r e t a i n e d i n the animal. Gross
examination of both l i v e r and kidney shows t h a t they are s l i g h t l y
darkened. I n m i c r o s c o p i c examination of these t i s s u e s , dense
deposits not seen i n u n i n j e c t e d mice are found i n the Kuppfer
c e l l s of the l i v e r and i n the kidney p r o x i m a l convoluted t u b u l e
c e l l s . These d e p o s i t s are presumed t o be osmarin w a l l e d o f f
w i t h i n lysosomes. A f t e r about a month, mice which are not
s a c r i f i c e d r e g a i n t h e i r normal c o l o r .
S t a i n i n g and R e t e n t i o n i n p r o c i n e synovia. I f the Boussina,

et a l . , (3) suggestion t h a t osmium c o n t a i n i n g d e p o s i t s have a
long term b e n e f i c i a l e f f e c t i s c o r r e c t , then to be u s e f u l ,
osmarins should s t a i n t i s s u e w i t h i n s y n o v i a l spaces and be
r e t a i n e d there f o r long p e r i o d s . I n i t i a l experiments w i t h p i g s
and r a b b i t s have been d i r e c t e d toward examining t h i s and r e l a t e d
i s s u e s . I n these experiments, osmarin s o l u t i o n s are i n j e c t e d
i n t o the s y n o v i a l spaces of the l i v i n g animal, and some time
l a t e r , the t i s s u e s of the j o i n t are examined a f t e r s a c r i f i c e of
the animal. Our gross and m i c r o s c o p i c f i n d i n g s are t h a t osmarins
w i l l s t a i n t i s s u e i n j o i n t s and be r e t a i n e d there. The
e f f e c t i v e n e s s of both s t a i n i n g and r e t e n t i o n , however, i s de-
pendent upon the composition of the osmarin.
P i g s used i n t h i s study have been, f o r the most p a r t ,
a r t h r i t i c . Mycoplasmal a r t h r i t i s i s common i n l a r g e herds, and
i s e n z o o t i c i n the SIU herd.
I n i t i a l experiments w i t h p i g s were short term and i n v o l v e d
four a r t h r i t i c p i g s which r e c e i v e d d i f f e r e n t osmarin prepara-
t i o n s . J o i n t t i s s u e s were examined f o r r e t a i n e d osmarin and
photographed one t o three days a f t e r i n j e c t i o n . A second group
of p i g s i n c l u d i n g n o n a r t h r i t i c c o n t r o l s , were i n j e c t e d and

examined one month a f t e r the i n j e c t i o n . Tissue samples f o r

microscopic study were obtained from these animals.
Osmarin s t a i n i n g was observed through the c o l o r imparted
to the t i s s u e . In most cases, the s t a i n i n g was obvious as a
grey or b l a c k c o l o r when the s t a i n i n g was due t o an osmarin
p r e p a r a t i o n c o n t a i n i n g more than 30% osmium, or a brown c o l o r
f o r l e s s osmium r i c h osmarins. The c o l o r of the s t a i n e d t i s s u e
was c l e a r l y r e l a t e d to t h a t of the i n j e c t e d osmarin, which
suggests t h a t the b i n d i n g i s a s s o c i a t i v e , and not accompanied
by changes i n osmium o x i d a t i o n s t a t e or the c o n n e c t i v i t y of the
osmium i o n s .
F i g u r e 3 i s a photograph of a s t a i n e d j o i n t . The osmarin
used i n t h i s case contained 35% osmium as a dry s o l i d . A l l
surfaces w i t h i n the s y n o v i a l space were s t a i n e d , a r t i c u l a r
c a r t i l a g e on the j o i n t s u r f a c e s and the j o i n t capsule which

encloses the space. The most proximal lympth node was s t a i n e d
(not shown).
Except f o r the c o l o r , the s t a i n e d t i s s u e appears normal.
There was no gross evidence of t o x i c i t y . In the month-long
study from which F i g u r e 3 i s drawn, o n l y one t a r s a l and c a r p a l
j o i n t of the p i g s were i n j e c t e d w i t h osmarin s o l u t i o n . The
other j o i n t s were untreated and served as c o n t r o l s on the same
animal. In the a r t h r i t i c animals, the untreated t a r s a l j o i n t s
e x h i b i t e d l e s i o n s , c o n f i r m i n g the o r i g i n a l d i a g n o s i s . The
t r e a t e d j o i n t s e i t h e r e x h i b i t e d no l e s i o n (one case) or s t r u c -
tures which are i n t e r p r e t e d as h e a l i n g l e s i o n s (two c a s e s ) .
This l a s t assessment i s t e n t a t i v e and i s based upon m i c r o s c o p i c
evidence of c a r t i l a g e growth i n t o the l e s i o n areas ( f o l l o w i n g
section).
In a f o l l o w i n g experiment, two a r t h r i t i c p i g s and one
normal c o n t r o l were i n j e c t e d i n r i g h t t a r s a l and c a r p a l j o i n t s
and s a c r i f i c e d one month l a t e r . The osmarin p r e p a r a t i o n used
i n t h i s case contained o n l y 17% osmium as a dry s o l i d . On
examination, the t r e a t e d j o i n t s were found t o be unstained.
There was no d i f f e r e n c e between i n j e c t e d and u n i n j e c t e d j o i n t s .
The e a r l i e r s h o r t term experiments (discussed above) i n d i c a t e d
t h a t s t a i n i n g from low osmium percentage polymers was l i g h t .
These f i n d i n g s i n d i c a t e a r a p i d c l e a r i n g time as w e l l , and i n
these cases, no evidence of b e n e f i c i a l e f f e c t could be found.
In subsequent experiments i n v o l v i n g r a b b i t s and dogs, o n l y
osmarin p r e p a r a t i o n s c o n t a i n i n g more than 30% osmium i n the dry
s o l i d were used.
An i n t e r e s t i n g secondary f i n d i n g was made i n the experiment
i n which one month separated i n j e c t i o n and s a c r i f i c e of the
animal. In these cases, the s t a i n e d a r t i c u l a r s u r f a c e was
marked w i t h a lacework p a t t e r n of unstained c a r t i l a g e (Figure
3). The p a t t e r n i s a t t r i b u t e d t o growth s i n c e p i g s used i n
these s t u d i e s are young (3 mo) animals and double t h e i r weight
i n the one month i n t e r v a l p r i o r to s a c r i f i c e .

Figure 3. The osmarin-stained surface of a pig tarsal joint. The pattern of white
lines (unstained cartilage) is attributed to growth.

The white l i n e d p a t t e r n observed appears t o be due t o the

new c a r t i l a g e formed as the p i g s grew. Osmarin bound t o the
c a r t i l a g e surface d i d not s t a i n the new c a r t i l a g e . The p a t t e r n
i s an i n d i c a t i o n o f the s t r e n g t h and s t a b i l i t y o f the b i n d i n g .
I t serves t o demonstrate t h a t once the osmium polymers have
bound t o the c a r t i l a g e surface they do not move. The p a t t e r n i s
not a r e f l e c t i o n o f normal growth and i s an e f f e c t o f the
osmarin.
H i s t o l o g y of s t a i n e d t i s s u e i n p i g s . H i s t o l o g i c a l s t u d i e s
have had s e v e r a l o b j e c t i v e s . F i r s t i s the determination o f
what the osmarin s t a i n s . Secondly, these s t u d i e s r e v e a l the
extent o f microscopic t o x i c i t y . Stained t i s s u e appears h e a l t h y
and e n t i r e l y undamaged. M i c r o s c o p i c a l l y , some dose r e l a t e d
c e l l death i s observed. F i n a l l y , microscopic examination o f

the p i g c a r t i l a g e confirmed t h a t the unusual l i n e d p a t t e r n i s
due t o growth of c a r t i l a g e . Evidence o f c a r t i l a g e growth has
been found, a s s o c i a t e d w i t h l e s i o n s i n osmarin t r e a t e d j o i n t s ,
which suggests regeneration.
Tissue samples f o r microscopic study were obtained from
p i g s s a c r i f i c e d one month a f t e r i n j e c t i o n . Samples were obtained
from the j o i n t capsules and a r t i c u l a r s u r f a c e s . When a r t h r i t i c
l e s i o n s were found, samples from these areas were obtained.
The experiment i n c l u d e s two l e v e l s o f c o n t r o l . F i r s t , a l l o f
the p i g s r e c e i v e d i n j e c t i o n s o f osmarin s o l u t i o n o n l y i n the
r i g h t t a r s a l and c a r p a l j o i n t s . Thus, s t a i n e d and unstained
t i s s u e was obtained from the same animal by examining both
t a r s a l and c a r p a l j o i n t s . Secondly, three o f the p i g s were
a r t h r i t i c and one was h e a l t h y . Assessments o f the a f f e c t s
accompanying osmarin s t a i n i n g are made on the b a s i s o f com-
parisons o f s t a i n e d and unstained t i s s u e from both a r t h r i t i c
and n o n - a r t h r i t i c animals.
The d i s t r i b u t i o n o f osmarin i n a s t a i n e d s y n o v i a l space
i s , f o r the most p a r t , independent o f a r t h r i t i c c o n d i t i o n s .
J o i n t capsule and c a r t i l a g e o f a r t h r i t i c and h e a l t h y animals
are s i m i l a r l y s t a i n e d . The growth r e l a t e d l i n e d p a t t e r n i s
observed i n both a r t h r i t i c and h e a l t h y animals. Disease r e l a t e d
d i f f e r e n c e s are found o n l y i n t i s s u e s r e l a t i n g t o a r t h r i t i c
lesions.
H i s t o l o g i c a l examination o f the s y n o v i a l membrane revealed
osmarin t o be taken up by s u b s y n o v i a l macrophages (Figure 4 ) .
Accumulations o f t h i s type are not seen i n c o n t r o l t i s s u e s . I n
both osmarin t r e a t e d and u n i n j e c t e d j o i n t s the s y n o v i a l membrane
appeared i n t a c t .
Stained a r t i c u l a r c a r t i l a g e appears h e a l t h y and undamaged.
M i c r o s c o p i c a l l y , s t a i n i n g o f the surface l a y e r o f the p i g
c a r t i l a g e was confirmed by a strong a f f i n i t y o f t o l u i d i n e blue
f o r t h i s r e g i o n which was not seen i n c o n t r o l t i s s u e s (Figure
5 ) . Most surface c e l l s o f the a r t i c u l a r c a r t i l a g e appeared
healthy and were present as s i n g l e c e l l s . I n white l i n e r e g i o n s ,

Figure 4. A tissue section taken through the synovial membrane and underlying
connective tissue from a pig that received a single osmarin injection 30 d previously.
Synovial cells (SC) appear healthy and line the joint cavity. Numerous sybsynovial
macrophages (M) show dense osmarin deposits.
Figure 5. A section of the articular cartilage from a pig injected 30 d previously

with osmarin. The surface zone (length of double-headed arrow) shows dense
staining with toluidine blue to a depth of about 0.1 mm. Chrondrocyte nests (arrows)
usually display one or two cells on the surface and one to ten cells in the deeper
zones. At the right is a region that corresponds to a white line (arc) seen in Figure
3. Surface staining is not apparent at the white line region.

a l l c e l l s appeared h e a l t h y and numerous m u l t i c e l l u l a r lacunae

were present. C a r t i l a g e i n t h i s r e g i o n appears p i n k w i t h the
metachromatic t o l u i d i n e blue s t a i n . The m a t r i x was l e s s dense
w i t h the e l e c t r o n microscope.
U l t r a s t r u c t u r a l l y the osmarin-treated c a r t i l a g e appeared
normal except f o r evidence f o r an o c c a s i o n a l dead chondrocyte.
These chondrocytes i n e v i t a b l y d i s p l a y e d e l e c t r o n dense i n -
c l u s i o n s which were most l i k e l y osmarin. For the most p a r t the
c e l l u l a r r e a c t i o n t o osmarin appears to be phagocytosis. The
compound i s absorbed and w a l l e d o f f i n s p e c i f i c i n c l u s i o n s .
I n regions of a r t h r i t i c l e s i o n s the c a r t i l a g e was l e s s
darkened by the osmarin. This i n d i c a t e d a c t i v e growth of
c a r t i l a g e . This was confirmed by l i g h t microscope observations
(Figure 6 ) . C a r t i l a g e nests contained numerous c e l l s , and the
matrix s t a i n e d p i n k w i t h t o l u i d i n e b l u e .
Rabbit s t u d i e s . An on going companion study i n v o l v i n g

t h i r t y - f i v e r a b b i t s y i e l d e d p r e l i m a r y r e s u l t s somewhat s i m i l a r
to those of the p i g s t u d i e s . Osmarin s o l u t i o n s i n j e c t e d i n t o
the knee j o i n t s of the r a b b i t s s t a i n a r t i c u l a r c a r t i l a g e and
the j o i n t capsule, as i n the p i g . In t h i s study, a measure o f
osmarin r e t e n t i o n has been developed based upon the standard
grey s c a l e . S e c t i o n s of s t a i n e d a r t i c u l a r c a r t i l a g e were
removed and compared v i s u a l l y w i t h standard grey s c a l e s . I n
t h i s study groups of ten r a b b i t s were i n j e c t e d a t the same
time, and then animals were s a c r i f i c e d and the j o i n t s examined
at i n t e r v a l s from one day to seven weeks post i n j e c t i o n . There
appears to be v e r y l i t t l e change i n c a r t i l a g e d i s c o l o r a t i o n
w i t h time, i n d i c a t i n g s t r o n g b i n d i n g of osmarin. G r o s s l y , the
c a r t i l a g e and synovium of the j o i n t s , through s t a i n e d , appeared
otherwise normal a t a l l time p e r i o d s . The i n t e r l a c i n g white
l i n e s seen i n long term i n j e c t e d p i g s were not p r e s e n t , even
when young growing r a b b i t s were examined. The darkening of the
s y n o v i a l membrane appeared upon microscopic examinations t o be
due to accumulation of osmarin i n s u b s y n o v i a l macrophages,
s i m i l a r to those found i n p i g s . The a r t i c u l a r c a r t i l a g e was
m i n i m a l l y d i s r u p t e d w i t h osmarin. L i t t l e evidence of c e l l
death i n the c a r t i l a g e was noted. S y n o v i a l t i s s u e examined one
day a f t e r osmarin i n j e c t i o n was d i s r u p t e d and contained numerous
dead c e l l s . S y n o v i a l t i s s u e examined three weeks or l a t e r
a f t e r i n j e c t i o n was normal. This i s a p o t e n t i a l l y important
o b s e r v a t i o n . An e f f e c t upon the synovium suggests p o s s i b l e
short term b e n e f i t s .
In order t o provide comparisons w i t h osmarin t r e a t e d j o i n t
t i s s u e s , a number of r a b b i t s were i n j e c t e d w i t h e q u i v a l e n t
amounts (based upon osmium content) of osmium t e t r o x i d e s o l u t i o n s .
The r a b b i t s were s a c r i f i c e d and the j o i n t s examined according
to the same time i n t e r v a l s t h a t were employed i n the osmarin
s t u d i e s . The r e s u l t s of these s t u d i e s were s i m i l a r t o those
reported by others (17). H i s t o l o g i c a l l y , the osmium t e t r o x i d e

Figure 6. Articular cartilage taken from a region less than 2 mm from an arthritic
lesion. The surface is not differentially stained as shown in Figure 5. Chrondrocyte
nests (arrows) contain from one to eight cells and suggest active growth in this region.

treatments, were harsher on j o i n t t i s s u e s than were osmarin

treatments and t h e i r d e l e t e r i o u s e f f e c t s were longer l a s t i n g .
A f t e r one day the s y n o v i a l membranes of both groups appeared
n e c r o t i c . However, the osmarin t r e a t e d j o i n t s regenerated a
healthy appearing s y n o v i a l membrane by three weeks. The s y n o v i a l
membrane of the osmium t e t r o x i d e group regenerated a f t e r seven
weeks, although there remained much subsynovial f i b r o s i s and
many f o r e i g n body g i a n t c e l l s . For the OsO^ t r e a t e d r a b b i t s ,
the e n t i r e c a r t i l a g e surface (about 2mm deep) showed n e c r o t i c
c e l l s . In a d d i t i o n , i n the osmium t e t r o x i d e i n j e c t e d r a b b i t s ,
there was a surface exudate upon the c a r t i l a g e and a h i g h l y
granulated c a r t i l a g e matrix.
C l i n i c a l data i n dogs. Osmarins have been used i n a

l i m i t e d number of c l i n i c a l s i t u a t i o n s i n dogs. S i x dogs have

been t r e a t e d so f a r . With s p e c i a l p e r m i s s i o n from the owners
of the dogs, i n t r a a r t i c u l a r i n j e c t i o n s of osmarin s o l u t i o n s
were made i n t o j o i n t s a f f l i c t e d w i t h a r t h r i t i s . The e t i o l o g y
ranged from trauma to probable genetic to o l d age. The t r e a t -
ments and c l i n i c a l e v a l u a t i o n s of the e f f e c t s of treatments
were made by l i c e n s e d v e t e r i n a r i a n s i n p r i v a t e p r a c t i c e . I n
some cases, pre- and post-treatment roentgenograms were used i n
the c l i n i c a l e v a l u a t i o n s . There was no opportunity f o r necropsy
and h i s t o l o g i c a l e v o l u t i o n of the t r e a t e d j o i n t s . No c l i n i c a l l y
normal j o i n t s were i n j e c t e d . C r i t e r i a f o r improvement i n c l u d e d
increased locomotor a c t i v i t y , increased freedom of movement o f
the a f f e c t e d j o i n t , decreased l i m p i n g , and diminished evidence
of p a i n upon p a l p a t i o n of the j o i n t . Where roentgenograms were
made, a smoother, more c l e a r l y defined a r t i c u l a r surface and
decreased r a d i o o p a c i t y i n the s o f t t i s s u e s immediately surround-
i n g the j o i n t were i n t e r p r e t e d as post-treatment signs o f
improvement.
For a p e r i o d of 1 to 3 days f o l l o w i n g osmarin i n j e c t i o n
the dogs e x h i b i t e d evidence of increased discomfort i n the
t r e a t e d j o i n t . A s i m i l a r p e r i o d of increased discomfort was
noted when normal r a b b i t j o i n t s were t r e a t e d w i t h osmarin, but
was not seen i n e i t h e r normal or a r t h r i t i c osmarin t r e a t e d
j o i n t s of p i g s . The authors do not a t t i r b u t e t h i s short p e r i o d
of discomfort t o the p h y s i c a l trauma of the i n j e c t i o n . More
l i k e l y explanations could be t h a t osmarins cause a temporary
decrease i n s y n o v i a l f l u i d v i s c o s i t y , or t h a t the discomfort i s
due to a type of chemical synovectomy which was p r e v i o u s l y
noted i n the r a b b i t s .
In f i v e of the s i x dogs, the temporary increase i n discom-
f o r t was followed by a p e r i o d of gradual improvement over the
o r i g i n a l a r t h r i t i c c o n d i t i o n s . Two of the dogs have recovered
normal f u n c t i o n i n the e f f e c t e d j o i n t s . Three of the dogs have
improved, but s t i l l r e t a i n some l o s s of f u n c t i o n i n the a r t h r i t i c
j o i n t s a t t h i s time. I n the dog which f a i l e d to show any im-
provement, the knee j o i n t was markedly deformed by e x o s t o s i s
due to the long standing a r t h r i t i s .

Conclusions
The study of osmarins as potential arthritic agents follows
directly, in this case, from the Boussina, et a l . , suggestion
that osmium containing deposits may have long term beneficial
effects in arthritis treatment. Osmarins exhibit properties,
e.g. low toxicity and tissue staining and retention, which are
thought necessary for such an application. In preliminary
experiments involving dogs and pigs, favorable responses to
treatment have been observed. We propose that osmarins in a
joint, may react with superoxide ion and thereby protect the
synovial fluid from damage by this species.
A secondary finding in the study is that osmarins have a
short term effect upon the synovial membrane. This effect may
also contribute to the utility of these substances.

It is important to emphasize the preliminary character of
the research discussed in this paper. No claims of cures are
implied. At the same time, i t is also important to recognize
the potentialities that the research findings suggest. If the
several uncertainties in the proposed application of osmarins
can be successfully resolved, then new alternatives in arthritis
treatment will be available.
Acknowledgments
This research has been supported by the departments listed
and by the office of Research Development and Administration of
Southern Illinois University. Drs. T. 0. Miller and D. M. Lane
of Murphysboro, Illinois and L. F. Striegel of Carbondale,
Illinois are veterinarians in private practice and have treated
the dogs included in the study. Dr. G. H. Gass guided early
studies of toxicity. Dr. W. J. Roth characterized osmarins.
P. S. Ostenburg prepared many of the materials used in the
study. A. M. Islam and P. A. Kibala are currently studying
osmarin reactivities. Several undergraduate research assistants
have contributed to the project.
Literature Cited
1. Hinckley, C. C.; Ostenburg, P. S.; Roth, W. J. Polyhedron

1982, in press.
2. Nissila, M. Scand. J. Rheumatology 1979, Suppl. 29, 1.
3. Bonssina, I; Lagier, R.; Ott, H.; Fallet, G. H. Scand. J.
Rheumatology 1976, 5, 53.
4. Fridovitch, Irwin. Science 1978, 201, 875.
5. Bannister, W. H.; Bannister, J. V., Eds,; "Biological and
Clinical Aspects of Superoxide and Superoxide Dismutase",
Elsevier/North Holland, New York, 1980, pp. 147-153.
6. Bannister, W. H.; Bannister, J. V., Eds.; "Biological and

7. McCord, J. M. Science 1974, 185, 530.

8. Greenwald, R. A.; Moy, W. W. Arthritis Rheum. 1980, 23,
455-463.
9. Spiro, T. G., Ed.; "Metals Ion Activation of Dioxygen",
John Wiley and Sons, New York, 1980, pp. 209-237.
10. Michelson, A. M.; McCord, J. M.; Fridovitch, I., Eds.;
"Superoxide and Superoxide Dismutases", Academic Press,
New York, 1977, pp. 517-536.
11. Michelson, A. M.; McCord, J. M.; Fridovitch, I., Eds.;
"Superoxide and Superoxide Dismutases", Academic Press,
New York, 1977, pp. 537-550.
12. Bannister, W. H.; Bannister, J. V., Eds.; "Biological and
13. Criegee, R.; Marchand, B.; Wannowius, Η.; Ann. der Chem.
1942, 550, 99-133.
14. Cotton, F. A.; Wilkinson, G.; "Advanced Inorganic Chemistry",
4th Ed., "John Wiley and Sons, New York, 1980.
15. Armstrong, J. E . ; Robinson, W. R.; Walton, R.A. J. C. S.
Chem. Comm. 1981, 1120-1121.
16. Nielson, A. J.; Griffith, W. P.; J. Chem. Soc. Dalton
1979, 1084.
17. Mitchell, N; Laurin, C.; Shepart, N.; J. Bone Joint Surgery
(British) 1973, 55B, 814-821.

INDEX
A Agar colony formation, soft, mouse
leukemia cells 35/
Absorber-backscatterer pair, Alkaline cesium chloride
best-fit results 251 technique 13-14,17-18
Absorption edge spectra for Alkaline elution studies, D N A cross-
[(NH ) Pt(OH) Pt(NH ) ]-
3 2 2 3 2
linking by Pt(II) 13,31
( N 0 ) and D N A reaction
3 2
Alkaline sucrose
product 221/ sedimentation 13-14,17-18
Acid-base equilibria of a N 3 - Alkyl sulfoxides, dimethylsulfoxide-
platinated uracil 149/ containing metal complexes ...279-93
Acid dissociation constant Alkylating agents, difunctional, and
N l shift model 223 cw-DDP, similarities 10,13-14, 17-19
R u complexes 344-47 Allylamines, cell culture activity for
Publication Date: January 26, 1983 | doi: 10.1021/bk-1983-0209.ix001
Acid hydrolysis of K[PtCl (Me SO)] 283

3 2
protonated and neutral Pt
Acid phosphatase, inhibition of complexes 274*
activity by gold compounds 362* Ambidentate ligands, dimethylsulf-
Acid treatment of platinated uracil oxide-containing metal
and thymine tautomers 151,153 complexes 279-93
Acidity constant for water deprotona- (2-Amino-6-mercaptopurine ribo-
tion in cw-(NH ) Pt(Cl)(H 0) .... 233
3 2 2
side)^ Pt and Pd complexes,
Acido ligands 318 E X A F S analysis 245-62
Activity Amino olefin complexes,
allylamine Pt complexes, protonated 271,273-76
protonated and neutral 274* 3-Aminoquinuclidinium derivatives,
3-aminoquinuclidinium derivatives activity against murine leukemia
against murine leukemia cells cells in vitro 269-71
in vitro 269-71 Ammine ligand loss, (dGuo)-
ethidium bromide 292* (NH ) Ru(III) 341
3 5
gold compounds against enzymes Ammine ligands and Pt binding

in vitro 362* to D N A 65-68
trichloro(3-aminoquinuclidinium)- to D N A in vitro by chloroamines .95-96
platinum(II) against murine to peptides and proteins 185-89
leukemia cells in vivo 272* Ammineruthenium(III) complexes .. .335-38
trypanocidal Pt complexes 292* AMP—See Adenosine 5'-mono-
Adducts, P t - D N A phosphate
D N A conformation and stability Analysis, energy and wavelength
in vitro and in vivo 93-94 dispersive 307-8
structures 89-93 Analysis of biological materials,
Adenine modern methods 299*
monodentate binding 288 Anhydrate of c w - [ P t ( N H ) ( l -
3 2
reaction with [ P t C l ( R S O ) ] -
3 2 288 methylcytosine) (Nl-deproto-
Adenine derivatives, ruthenium (III) nated thymine anion)]*, base/
complexes 344-45 base and base/PtN conforma-
4
Adenosine tional drawing 197,198/

molecular topology, atomic num- Animal studies, osmarins 428-36
bering, potential energy maps, Antiarthritic drugs, gold 355-66
and metal binding sites 192-95 Antibiotic bleomycin 338-39
monodentate binding 288
Antitumor agents, R u 335
pKa, exocyclic nitrogen 344
Antitumor complexes, analysis by
Adenosine 5'-monophosphate
N M R spectroscopy 172-89
interaction with Pt(II)
diamines 180-82 Apparatus, x-ray absorption
structures 181/ spectroscopy 388-89
Adenosine 5'-nucleotides, chemical Applications, gold drugs 359-60
shift and p H range 239-40 Aquo-hydroxo Pt complexes,
African typanosomes 292 equilibria 232-43
439

Argon plasma, in summary of Base/PtN coordination plane

4
analytical methods for biological dihedral angles of cis-Vi com

materials 299* plexes with nucleobases, nucleo
Arthritis, rheumatoid sides, and nucleotides 195-202
development of chrysotherapy . ...357-59 N-3 ^-Benzosalicylidene-NSN - 1
gold-based anti-arthritis dimethylethylenediamine ( theo-

pharmaceuticals 385-99 phyllinato)copper(II), C u - 0 6
Articular cartilage, pig, and osmarin .. 432/ interaction v 214/
Ashing, wet and dry, and atom emis Binding of m - D D P to D N A 53-54, 57/
sion, in summary of analytical amine ligands 65-68
methods for biological materials 299* mouse leukemia cells 76-78
Atomic absorption spectroscopy, sequence specificity 55-56
,4
binding of [ C]cw-[PtCl - 2 vs. trans-DO? 4-7, 110
(NH CH )2]
3 3 67/ Binding of ethidium bromide to calf
Atomic numbering for nucleobases ... 193/ thymus D N A , fluorescence
Atomic spectroscopy, in summary of Scatchard plot 57/
analytical methods for biological Binding of Pt
materials 299* on guanine at N l , 0 6 215, 216/

Auranofin 364-67 on unsubstituted uracil and
ligand exchange reactions 371-83 thymine 148-51
reaction with whole blood 380 Binding mode
and red cells, proton N M R cis-ΌΌΡ to D N A and effect of
spectra 382/ ethidium bromide 56-62
Aurosome transition metal complexes of
E X A F S spectroscopy 395-99 6-mercaptopurines 254-62
separation, kidney deposits 389 Binding ratios for c/s-diammine
Aurothioglucose and arthritis 357-67 Pt(II) complexes 239
Aurothiomalate 371 Binding sites
and arthritis 357-67 dimethylsulfoxide complexes on
proton N M R spectra 375/ nucleobases 289*
thiol uptake and exchange exonuclease-III-detected cis-DOP
reactions 373-76 on D N A and effect of
Autoradiograph of electrophoresis gel, ethidium 62-65
ethidium bromide effect on exo- nucleobase, platinated purine and
nuclease-III-detected cw-DDP pyrimidine complexes 192
binding sites 63/, 64/ Binding studies, M . luteus and calf
Β thymus D N A 66-69
Bacteria, E. coli, induction of phage Biochemical effects of Pt d r u g - D N A
growth 4 interaction 14-17
Base, nucleic acid Biological activity
interaction with metal-dimethyl- of ethidium bromide 292*
sulfoxide complexes ...285,288-89 of metal-dimethylsulfoxide
interaction with Pt(II) complexes 284-85
diammines 180-82 of ris-[Pt(amine) (Me SO) ](A) .290-92
2 2 2 2
interaction with [Pt(NH ) - 3 2

trypanocidal Pt complexes 292*
(Me SO) ]
2 2
2+
293 Biological consequences
Base configurations of P t - D N A crosslinks in
assignments, of Pt dinucleotide mammalian cells 27-48
chelates 128 of Pt antitumor drugs on D N A 4-5
nucleic acids and Pt(II) diammines 182 Biological materials, summary of
Base pair modern methods for analysis 299*
triply hydrogen-bonded, hydrogen Biological studies
bond distances 225/ of amino-olefin complexes 273-76
Watson-Crick G C and methylated of antitumor Pt complexes,
GT 224/ LPtCl 3 265-76
Base reaction with rij-[Pt(NH ) L- 3 2 Bis (purine )Pt( II) complexes and
(H 0)]
2
+
158 bis(pyrimidine-ring-bound)Pt(II)
Base stacking, D N A and fixation of complexes, base/base and base/
cis- and trans-ΏΌΡ 82 P t N dihedral angles
4 100,197

INDEX 441
Bis(thyminato) complexes of Pt(II), Cells, kinetics—Continued

separation of tautomers 151,153 sensitive and resistant, in vivo
Bleomycin 338-39 and in vitro studies 33, 39
Blood, whole, reaction with auranofin 380 thiourea inhibition of cis-DDP
Bonding angles, chloride, structure- cytotoxicity and interstrand
activity relation 318-21 crosslinking 36/, 37/
Bone marrow, mice, cw-diammine- mouse plasmacytoma, Pt(II) and
dichloroplatinum(II) and P t ( I V ) complexes, toxicity ... 9*
hydroxymalonatodiammine- Cells, red—See Red blood cells
platinum(II) 10-12 Cesium chloride technique,
alkaline 13-14,17-18
C Characterization
of osmarins 423-26
Caffeine and inhibition of D N A of P t - D N A adducts 90-91
repair 16-17 Chelation ( N 7 - 0 6 ) for inosine by
Calf thymus D N A Raman spectroscopy 182
binding to ethidium bromide, Chemical activity of cw-[Pt ( amine ) -
fluorescence Scatchard plot .... 57/ (Me SO) ](A)

2 2 2 290-92
binding studies 66-69 Chemical properties, osmarins 426-28
circular dichroism spectra 69-70 Chemical shift anisotropy 179
SI nuclease digestion of trans- and Chemical shift of inosine and adeno
cw-DDP 111/ sine and their 5'-nucleotides ...239-40
Carbohydrate polymers, osmium, as 1 5
Chemical shift range, N N M R 179
antiarthritic drugs 421 195
Chemical shift range, P t N M R ...173-77
Carboxyphthalato complexes, Chinese hamster cells
solubility and toxicity 29It effect on D N A synthesis of
Cathepsin, inhibition of activity by C / J - D D P treatment 15/
gold compounds 362* growth 28, 30
Cell culture mutation frequency by cis- or
activity for protonated and neutral trans-ΌΌΡ 43/
Pt complexes of allylamines . . . 274* Pt(II) and P t ( I V ) complexes,
mouse leukemia cells in vitro 30-31 toxicity 9*
P t - D N A reactions 7-10 Chloride bonding angles, structure-
Cell lysates, estimation of renaturable activity relation 318-21
DNA 13-14,17-18 Chloroamine-Pt(II) complexes, fixa
Cell replication and caffeine 16-17 tion in vitro, conformational
Cell survival measurements 28, 30 changes and stability of
Cells DNA 88*, 95-96
Chinese hamster Chromatography, high pressure gel
growth 28, 30 permeation 126
mutation frequency by cis- or Chromatography, high pressure
trans-ΌΌ? 43/ liquid
toxicity of Pt(II) and P t ( I V ) reactions of diaquo Pt complex
complexes 9*, 15/ with dinucleotides 127
human, P t - D N A damage and
separation of tautomers 151,153
repair 41
Chrondrocyte nests, osmarin 434/
malignant ascites, mechanism of
cw-DDP resistance 39,41 Chronic dose gold thiomalate auro
mammalian, biological conse somes, E X A F S curve fitting 398*
quences of P t - D N A cross Chrysotherapy 371
links 27-48 Chrysotherapy for rheumatoid
mouse leukemia arthritis, development 357-59
activity of 3-aminoquinuclidi- Circular dichroism spectra
nium derivatives in vitro .269-72 M. luteus and calf thymus D N A ...69-70
kinetics monomeric Pt dinucleotide
alkaline elution 32/ chelates 138-40
formation and disappearance poly(dG-dC) · poly(dG-dC)
of DNA-protein and with [(dien)PtCl]Cl 70/
D N A interstrand cross- salmon sperm D N A and Pt
linking 34/, 35/ complexes 84/

Cisplatin—See cw-Diamminedichloro- CSA—See Chemical shift anisotropy

platinum(II) Curve fitting, Fourier transforms 395
Collagenase, inhibition of activity by Cyclic activation analysis 305
gold compounds 362* Cyclic voltammetric data, linkage
Colony formation, soft-agar, mouse isomerization of [ ( A d o ) -
leukemia cells 35/ (NH ) Ru(III)]
3 5 349/
Concentration dependence of d s - D D P Cyclobutanedicarboxylate replace
DNA-protein crosslinking and 1 9 5
ment and P t N M R shifts 174-76
D N A interstrand crosslinking ... 38/ Cyclopentadienyl ligands, modification 321
Condensation products of Ν 1 - m e t h y l Cytidine, molecular topology, atomic
ated thymine and uracil 156 numbering, potential energy
Configurations maps, and metal binding sites .192-95
base, of Pt dinucleotide chelates ... 128 Cytidine derivatives, ruthenium (III)
base/PtN coordination plane
4 complexes 344-45
dihedral angles of « > P t com Cytokinetic studies 327
plexes with nucleobases, nucle Cytosine
otides, and nucleosides 195-202 coordination sites with Pt
cis- vs. trans-ΌΌΡ binding to D N A complexes 127-28

and nuclease digestion 110 oxy and deoxy, containing dinucleo
cis and trans influences of dimethyl- tides with cw-Pt(II)
sulfoxide in Pt coordination complexes 125-42
chemistry, kinetic and thermo in restriction endonuclease recogni
dynamic studies 283-84 tion sequence 114*
Conformation Cytosine-guanine residues, high
P t - D N A adducts in vitro 93-94 regions, D N A - P t crosslinking
sugar and Pt dinucleotide chelates .. 128 modes 202-5
Conformation parameters of cis-Pt Cytotoxicity, cw-DDP
complexes with nucleobases, capacity to replicate on a damaged
nucleosides, and nucleotides .195-202 template 16-22
Conformational changes of D N A D N A crosslinking produced 31-33
after fixation of Pt(II) chloro- inhibition by thiourea, mouse
amines in vitro 88* leukemia cells 36/, 37/
Conformational drawings 195,196/
Conformational transition ( B - Z ) and
fixation of diethylenetriammine-
platinum compounds 68-72,96 Data reduction, E X A F S 249-54
Coordination sites of Pt(II) complex Debye-Waller thermal parameter,
formation EXAFS 249-62
with dinucleotides 127-28 Deoxyguanine- and deoxycytosine-
with uracil and thymine 148-51 containing dinucleotides with
Copper, N-3,4-benzosalicylidene- cis-Pt(II) complexes 125-42
NS^-dimethylethylenediamine- Detector, lithium-drifted
(theophyllinato)(II), C u - 0 6 germanium 302
interaction 214/ 1,2-Diaminocyclohexane com
Corticosteroid arthritic panacea 195
plexes, P t study 180
period and gold therapy 359 1,2-Diaminocyclohexane and
195
Coupling constants, one-bond, Pt P t - M e S O complexes
2 290-92
1 5
and N N M R , for Pt(II) and ds-Diamminedichloroplatinum(II)
Pt(IV) diammines 173-77 binding to D N A 4-12,57/
Crosslinking reactions 10-14 calf thymus, SI nuclease
D N A - P t interstrand 6-7,81-82, digestion 111/
191,210 ethidium bromide effect 56-62
alkaline elution studies 13, 31 sequence specificity 55-56
biological consequences in simian virus 40, electro-
mammalian cells 27-48 phoretic profiles 104,107-10
repair in human fibroblasts 96 synthesis 15/
intrastrand, cis- and trans-Pt comparison of an (N,C1) fit with
complexes 96, 210 an(N,S)fit 258*
kinetics of formation and disappear and difunctional alkylating agents,
ance in mouse leukemia cells .34-37 similarities 10,13-14,17-19

INDEX 443
cw-Diamminedichloroplatinum(II)—Cont'd. Dinucleotide chelates—Continued

E X A F S analysis 245-62 Pt, assignments of base con
mole fraction Pt(II) vs. p H 234/ figurations and sugar
N M R spectrum 181/, 183/ conformations 128
nucleophilic site in c w - D D P - Dinucleotides
D N A crosslink 37/ and cw-Pt complexes, syn- and
and plasmid D N A , electro anti-isomers of reaction
phoresis 59/ products 125-42
resistance in malignant ascites U V and H P L C monitoring of
cells, mechanism 39,41 the reactions of diaquo Pt
structural parameters 319* complex 127
structures of cis- and trans- 29/, 222/ Discrimination factor, nucleophilic,
toxicity of dimethylsulfoxide 284
inhibition by thiourea 36/, 37/ Diseases, parasitic, Pt-metal
recovery of nondividing human complexes 292
fibroblasts 20/ Distribution curves for cw-diammine-
/ranf-Diammineplatinum cation, dichloroplatinum(II) 233-35

packing 211/, 212/ Ι,Γ-Disubstituted titanocene dichlor
c/s-Diammineplatinum complexes, ides, pharmacological data 322*
solution properties 232-38 DNA
Diaquo-hydroxo Pt complexes, amine ligands and Pt binding 65-68
equilibria 232-43 base stacking and fixation of cis-
Diaquo Pt complex with dinucleo and trans-ΌΌΡ 82
tides, U V and H P L C moni binding and penetration of Pt(II)
toring of reactions 127 complexes in mouse
Dichlorides leukemia cells 76-78
metallocene, structural biological effects of Pt antitumor
parameters, bond angles, drugs 4-5
and distance of D D P 319* calf thymus
titanocene, pharmacological data .. 322* binding studies 66-69
cw-Dichlorobispyridineplatinum(II), circular dichroism spectra 69-70
conformational drawings 195, 196/ ethidium bromide, fluorescence
Difunctional alkylating agents and Scatchard plot 57/
cw-diamminedichloroplatinum, conformational changes and
similarities 10,13-14,17-19 stability 88*
Dihalides crosslinking
metallocene, of transition metals by cis- and trans-ΌΌΡ 31-33
structures 222/ by Pt(II), alkaline elution
tumor inhibition 315-31 studies 13, 31
damage, Pt-induced excision
titanocene, pharmacological data .. 320*
repair and cytotoxicity,
Dihedral angle, interbase, of cw-Pt relationship 17-22
complexes 195-202, 211/ damage and repair of a random,
Dimer functioning D N A site,
D N A interaction 215 interaction 29/
head-head Pt, preparation and and D D P
reactivity 164-68 binding, cis vs. trans 4-7
hydroxide-bridged 180 electrophoresis profile 91-93
Dimercaptopropanol in intracellular hypothetical mechanism of
Au-binding ligand 380 reaction 96-97
Dimethylsulfoxide and nuclease digestion 110
as ligand in metal complexes, dimer interaction 215
interaction with nucleic acids drug interaction, biochemical
and biological activity 279-93 effects 14-17
with R u 281, 335-37 and enzyme exonuclease III 55-56
Dinucleotide chelates ethidium bromide effect on
monomelic Pt, proton N M R cw-DDP binding 56-62
and C D spectra 125-42 fixation of Pt(II) complexes 81

DNA—Continued DNA—Continued
interstrand crosslinking, structures 52-53,89-93
kinetics of formation and supercoiled and nicked
disappearance 34/, 35/, 38/ ratios by electron microscopy .... 81/
M. luteus simian virus 40, incubated with
14
binding of [ C]c/s- cis- and trans-DDP electro-
PtCl (NH CH )
2 3 3 2 67/ phoretic profile 104, 107-10
binding studies and circular synthesis, effect of c/s-DDP treat
dichroism spectra 66-69 ment in synchronized Chinese
nuclease digestion (SI) of trans- hamster cells and inhibition ....14-16
and c/s-DDP 111/ tumor, c/s-DDP and hydroxymalo-
Pt chloroamines after fixation natodiammineplatinum ( II ) .... 10-12
in vitro 88/ Dogs, clinical data, osmarins 435
P t - D N A adducts in vitro 93-94 Dose-activity relation, transition
nuclease-detectable binding sites metals 316-18
on c/s-DDP and effect of Double helix structure, supercoiled
ethidium 62-65 DNA 52-53
plasmid D r u g - D N A interaction, biochemical

and c/s-DDP, electrophoresis .... 59/ effects 14-17
electron micrographs 61/ Drugs, gold
enzyme effect and recognition antiarthritic 355-66
sequence 54-55 ligand exchange reactions 371-83
gel mobilities, as a function of Dry ashing, in summary of analytical
the amount of bound cis- methods for biological materials 299/
or trans-ΌΌΡ 60/
and protein crosslinking
concentration dependence 38/ ε
in human fibroblasts, repair 22/
in mouse leukemia cells, Ε. coli bacteria, induction of phage
kinetics of formation and growth 4
disappearance 34/, 35/ Eichhorn co-stacking 220,223
and Pt complexes Eichhornia crassipes, study of biologi
binding 53-54 cal effects of Pt complexes 298, 308-10
crosslinking Elastase, inhibition of activity by
guanine-cytosine residues 202-5 gold compounds 362/
interstrand 6-7, 191 Electron micrograph
in mammalian cells, biological plasmid D N A 61/
consequences 27-48 simian virus 40 minichromosome .. 117/
and mutagenicity, Electron microscopy
relationship 41-44 supercoiled and nicked D N A ratios 81/
damage and repair in human cells 41 with x-ray microanalysis 307
physicochemical and structural Electronic effects, dimethylsulfoxide
studies, in vitro inter as ligand 281-83
actions 75-96 Electrophoresis gel, autoradiograph,
reactions, cells in culture and ethidium bromide effect on exo-
in vivo 7-10 nuclease-III-detected c/s-DDP
renaturation 83/ binding sites 63/, 64/
renaturable, estimation in cell Electrophoresis profile
lysates 13-14,17-18 of trans- and c/s-DDP-DNA 91-93
repair, inhibition, and caffeine 16-17 of plasmid D N A and c/s-DDP 59/
salmon sperm, viscosity and circular of supercoiled and nicked simian
dichroism spectra 84/ virus 40 D N A incubated with
secondary structure and stability by cis- and trans-OD? 104, 107-10
Pt(II) complexes, alterations 79-89 Electrostatic potential energy maps
sequence specificity of c/s-DDP for nucleobases 192
binding 55-56 Endonuclease, restriction, recognition
simian virus 40, model for inter sequence of guanine and cytosine 114/
action with c/s-DDP 101-18 Endonuclease, Type II restriction 54

INDEX 445
Energy correlations, free, Fixation of Pt(II) compounds on

( N H ) R u ( I I I ) complexes with
3 5
DNA 81
purines 341-42 Fixation of Pt(II) compounds on
Energy dispersive analysis 307 guanine 93-95
Energy threshold, E X A F S Flame and flameless atomic
spectroscopy 249-62 spectroscopy, in summary of
Enzyme activity inhibition by gold analytical methods for biological
compounds in vitro 362/ materials 299/
Enzyme effect on plasmid D N A 54-56 Fluorescence modes, x-ray absorption
Enzymes, D N A processing and bound spectrum 387/
Pt effects 54-55 Fluorescence Scatchard plot of the
Epithermal irradiation 303 binding of ethidium bromide to
Equilibria, acid-base, of N 3 calf thymus D N A 57/
platinated uracil 149/ Fourier transform
Equilibria, hydrolytic, c/s-DDP 236-38 E X A F S spectra of [(NH ) Pt(OH) -
3 2 2
Equilibrium constant of guanine Pt(NH ) ](N0 )

3 2 3 2 218,219/
filtered data 395
during fixation 94/

Equilibrium, Pd(II) as guide to gold-sulfur scatter pair 397/
Pt(II) reactions 236-38 Pt and Pd mercaptopurine
Ethidium bromide, biological activity 292/ riboside complexes 249-54
Ethidium bromide and calf thymus sodium gold(I) thiomalate 397/
D N A , fluorescence Scatchard Free energy correlations,
plot 57/ ( N H ) R u ( I I I ) complexes with
3 5
Ethidium bromide and purines 341-42

c/s-DDP-DNA 56-62
Ethyl monosubstitution 321 G
Ethylenediamine Pt(II) complexes
and amine release 187-89 Gamma ray spectrometer 302
shift ranges 179 Gel mobilities for plasmid D N A as a
Exchange reactions of function of the amount of bound
aurothiomalate 373-76 cis- or /r<ws-DDP 60/
Excision repair of Pt-induced D N A Gel permeation chromatography,
damage and cytotoxicity 17-22 high pressure 126
Exonuclease-III-detected c/s-DDP Genome, simian virus 40 102
binding sites, autoradiograph of Germanium detector, lithium drifted .. 302
electrophoresis gel 63/, 64/ β-Glucosaminidase and ^-glucu
Exonuclease III enzyme and D N A ...55-56 ronidase, inhibition of activity by
Extended x-ray absorption fine gold compounds 362/
structure ( E X A F S ) spectroscopy Glutathione ( G S H ) and auro
gold-based anti-arthritis drugs and thiomalate solutions, proton
metabolites 385-90, 393-99 NMR 374-75
Pt and Pd mercaptopurine riboside Glycosyl linkage and guanine, fixation
complexes 245-62 of Pt compounds at N 7 position 95
spectrum, Fourier transform, of Gold, X A N E S spectra 391/
[(NH ) Pt(OH) Pt(NH ) ]- Gold antiarthritic drugs 355-66
3 2 2 3 2
(N0 ) 215-21 Gold complexes

3 2
inhibition of enzyme activity
in vitro 362/
ligand exchange reactions 371-83
Gold phosphines
Fibroblasts, human, recovery from reactions with red cells 378
cw-DDP(II) toxicity 20/, 22/ reactions with thiols 376
Filtering, Fourier transforms 395 Gold-sulfur scatter pair, Fourier-
Fixation of cis- and trans-OOP filtered data fit 397/
conformational transition ( B - Z ) 96 Gold therapy, applications 359-60
and D N A base stacking 82 Gold thiomalate aurosomes, chronic
Fixation of Pt(II) chloroamines on dose, E X A F S curve fitting 398/
D N A in vitro and amine Gold (I) complexes
ligands 88/, 95-96 with coordinated phosphorus atoms 392/

Gold(I) complexes—Continued Hydrogen bond

with thiolate 374,381-83 distances in triply hydrogen-bonded
Gold(I) thiomalate base pairs 225/
Fourier transform 397/ nucleic acid and Pt(II) diammine
x-ray absorption spectrum 387/ interaction 182
Gold(III)bromide and gold(III)- Hydrolysis of dimethylsulfoxide-
chloride, E X A F S spectra 396/ metal complexes 281
Grass species, effects of Pt complexes 310/ Hydrolytic equilibria, c/s-diammine-
Guanine dichloroplatinum(II) 236-38
coordination sites with Pt Hydroxide-bridged dimers and
complexes 127-28 trimers 180
and cytosine residues, D N A - P t Hydroxide-bridged Pt dimer bound
crosslinking modes 202-5 to guanine 224/
fixation of Pt compounds at the
N7 position 93-95
hydroxide-bridged Pt-dimer 224/
N l , 0 6 binding of Pt .215, 216/
N 7 - 0 6 clip 213, 215 Indenyl derivatives, pharmacological

oxy- and deoxy-containing dinucleo data 323/
tides with ds-Pt(II) Inductively coupled plasmas, in
complexes 125-42 summary of analytical methods
and restriction endonuclease for biological materials 299/
recognition sequence 114* Inhibition
Guanosine, molecular topology, of D N A repair and caffeine 16-17
atomic numbering, potential of D N A synthesis 14-16
energy maps, and metal binding of enzyme activity by gold
sites 192-95,202-5 complexes in vitro 362/
Guanosine and R u complexes 339-41 by metallocene dihalides of
transition metals 315-31
by thiourea of c/s-DDP cytotoxicity,
H mouse leukemia cells 36/, 37/
Inosine
Ή N M R spectroscopy—See N M R 5'-monophosphate, structures 181/
spectroscopy, proton 5'-nucleotides, chemical shift and
Hafnium and structure-activity p H range 239-40
relation 316-18 andPt(II) diammines 180-82
Halide isotopomers 174 Instrumental neutron activation
Halide ligands 318 analysis ( I N A A ) 297-310
195
Halides and P t ( I V ) , P t N M R 173-77 Interatomic distance, E X A F S 249-62
Head-head Pt dimer, preparation and Interbase dihedral angle of c/s-Pt
reactivity 164-68 complexes with nucleobases,
Heteronuclear complexes, P t , M
2
nucleosides, and nucleotides .195-202
stoichiometry 161-64 Interferences in determination of Pt
High pressure gel permeation by neutron activation 303/
chromatography 126 Interstrand crosslinking, D N A 38/
High pressure liquid chromatography with guanine-cytosine residues 202-5
monitoring of reactions of diaquo in human fibroblasts, repair 22/
Pt complex with dinucleotides 127 kinetics of formation and disappear
separation of tautomers 151,153 ance in mouse leukemia
Histology of stained tissue in pigs, cells 34-37
osmarin studies 431-33 cis- and trans-Pt complexes
History of gold 356-57 6-7, 81-82, 210
Homoleptic complexes, hydrolysis ...282-83 Intracellular Au-binding ligand,
HPLC—See High pressure liquid dimercaptopropanol 380
chromatography In-plane molecular electrostatic
Human cells, P t - D N A damage potential energy maps for
and repair 41 nucleobases 193/
Human fibroblasts, recovery from cis- Ion-metal movement on purines and
D D P toxicity 20/, 22/ pyrimidines 342-49

INDEX 447
IR spectroscopy, differentiation of Linkage isomerization

platinated thymine and uracil of [(Ado)(NH ) Ru(III)], cyclic
3 5
tautomers 148, 150 voltammetric data 349/

Irradiation by iridium, osmium, and hypoxanthineruthenium(III)
ruthenium 302 complexes 342
Isolation of P t - D N A adduots 90-91 platinated uracil and thymine ...151,153
Isolation of tautomer complexes of Liquid chromatography, high pressure,
platinated uracil and thymine. 151, 153 separation of tautomers 151,153
Isomers, syn- and anti-, of reaction Liquid scintillation counting, binding
14
products of dinucleotides and of [ C ] c / s - [ P t C l ( N H C H )
2 3 3 67/
2
c/s-Pt complexes 125-42 Lithium-drifted germanium detector .. 302

Isotopic abundance, Pt group metals .. 301/ Lung cell growth, Chinese hamster ....28, 30
Isotopomers, halide 174 Lysates, estimation of renaturable
DNA 13-14, 17-18
Κ M
Kidney deposits and separation of Malonato Pt complexes, solubility and

aurosomes 389 toxicity 29It
Kinetic studies Mammalian cells, biological conse
formation and disappearance of quences of P t - D N A cross-
DNA-protein and D N A inter linking 27-48
strand crosslinking 34/, 35/ Measurements, cell survival 28, 30
cis and trans influences of dimethyl Mechanism
sulfoxide in Pt coordination of c/s-DDP resistance in malignant
chemistry 283-84 ascites cells 39,41
Kinetics, alkaline elution 32/ hypothetical, of the reaction between
Kirkwood-Westheimer equation 341-42 c/s-DDP and D N A 96-97
of purine complexes with
L (NH ) Ru(III)
3 5 339-41
Mechanistic study of Pt antitumor
195 1 5
Leukemia cell D N A , mouse, alkaline drugs using P t and N
elution kinetics .: 32/ NMR 172-89
Leukemia cells, mouse Melting temperature variations 87/
activity of aminoquinuclidinium 6-Mercaptopurines, binding modes of
derivatives in vivo 269-72 transition metal complexes 254-62
kinetics of formation and Metallocene dichlorides
disappearance of DNA-protein structural parameters, bond angles,
and D N A interstrand cross- and distance of D D P 319/
linking 34/, 35/ of transition metals, tumor
penetration and D N A binding of inhibition 315-31
Pt(II) compounds 76-78 Metals
thiourea inhibition of cw-DDP binding sites on unsubstituted uracil
cytotoxicity and interstrand and thymine 148-51
crosslinking 36/, 37/ complexes, dimethylsulfoxide as
Ligand ligand 279-93
cyclopentadienyl, modification 321 in biological samples by neutron
dimercaptopropanol, Au-binding.... 380 activation and microprobe
dimethylsulfoxide 279-93 analyses, determination and
in gold complexes, X A N E S as location 297-310
indicator 393 transition, tumor inhibition by metal
sulfoxide, in [PtCl (Me SO)]", trans
3 2
locene dihalides 315-31
influence 285,288 Metal-ion movement on purines and
sulfur-bound [Pt(Me SO) ]
2 4
2+
282 pyrimidines 342-49
in trichloro Pt antitumor Metal-Pt complexes in parasitic
complexes 265-76 diseases 292
Ligand exchange reactions Methionine in peptides and proteins,
of gold drugs and red cells 371-83 binding and release of coordi
of Pt(II) complexes 172-89 nated amines 185-89
American Chemical
Society Library
115$Metal
In Platinum, Gold, and Other 1 · *Chemotherapeutic
i t . n. w. Agents; Lippard, S.;
ACS Symposium Series; American Chemical
WuMiMMl, 0. C.Society:
20036Washington, DC, 1983.
9-Methyladenine, metal binding Myocrisin 371

sites 192-95 See also Aurothiomalate
Methylated G T base pair and Watson- and arthritis 357-67
Crick G C base pair 224/ x-ray absorption spectrum 387/
x
N -Methylated uracil and thymine,
Pt(II) complexes 153,156-67
1-Methylcytosinate anion 216/
Ν
1-Methylcytosine
metal binding sites 192-95 Neutral Pt complexes of allylamines,
N M R spectra 216/ cell culture activity 274/
9-Methylguanine, metal binding Neutron activation analysis, determi
sites 192-95 nation and location of Pt metals
1-Methylthymine and 1-methyluracil, in biological samples 297-310
structure 155/ Nicked simian virus 40 D N A incubated
Mice, osmarin toxicity 428-36 with c/s- and /ra/js-DDP, electro-
Mice bone marrow, d s - D D P and phoretic profile 104,107-10
hydroxymalonatodiammine- Nicked and supercoiled D N A ratios
platinum(II) 10-12 by electron microscopy 81/

Microloops of c/s-DDP 96 Niobium and structure-activity
Microprobe analyses, determination and relation 316-18
location of Pt metals in biological Nitrogen N l p K shift model 223
samples 297-310 N M R nuclei, properties 172/
Minichromosome, simian virus 40, N M R spectra
electron micrograph 117/ carbon-13 and phosphorus-31, of
Model for interaction of c/s-DDP gold drugs 373, 374, 378-82
with simian virus 40 D N A 101-18 of 1-methylcytosine 216/
Mole fraction Pt(II) vs. p H for c/s- of p o l y ( d G » d C ) modified with
diammineplatinum complexes .... 234/ [(dien)PtCP] CI 71/
Molecular topology for nucleobases .. 193/ proton
Molybdenum and structure-activity of gold drugs 373-87
relation 316-18 of c/s-[(NH ) (l-MeT)(9-EtG)]-
3 2
Monocyclopentadienyl complexes, C10 4 159/

pharmacological data 323/ of Pd(II) nucleoside and nucleotide
Monomeric Pt dinucleotide chelates, complexes 239-41
proton N M R and C D spectra ...125-42 of platinated uracil tautomers
Monosubstitution 152/, 154/, 155/
ethyl and trimethylsilyl 321 of c/s, c/s, trans-Pt (isopropyl-
titanocene dichlorides, pharmaco amine) Cl (OH)
2 2 2 178/
logical data 322/ of [Ru(Me SO) ] 2 6
2+
281-82
Mouse leukemia cells proton and platinum-195 of c/s-
activity of 3-aminoquinuclidinium DDP 181/, 183/
derivatives 269-72 ofRNaseA 186/, 188/
alkaline elution kinetics 32/ N M R spectroscopy
cell culture in vitro 30-31 antitumor complexes 172-89
kinetics of formation and disap proton, dichlorodimethylsulfoxide
pearance of DNA-protein and complexes 283
D N A interstrand cross- Nuclear data for the Pt group metals .. 301 /
linking 34/, 35/ Nuclear methods, in summary of
penetration and D N A binding of analytical methods for biological
Pt(II) complexes 76-78 materials 299/
thiourea inhibition of c/s-DDP cyto Nuclear reaction of R h and Pd 302-6
toxicity and interstrand cross- Nuclease-detectable binding sites of
linking 36/, 37/ c/s-DDP on D N A and effect of
Mouse plasmacytoma tumor cells, ethidium 62-65
Pt(II) and Pt(IV) complexes, Nuclease digestion and c/s- vs. trans-
toxicity 9/ D D P binding to D N A 110
Mutagenicity of Chinese hamster cells 30 Nucleic acid bases
Mutagenicity and P t - D N A cross- interaction with metal-dimethyl-
linking, relationship 41-44 sulfoxide complexes ...285,288-89

INDEX 449
Nucleic acid bases—Continued Palladium(II)

interaction with Pt(II) diam dimethylsulfoxide 283
mines . . A 180-82 E X A F S spectroscopy of 6-mercapto-
interaction with purine riboside complexes 245-62
[Pt(NH ) (Me SO) ]
3 2 2 2
2+
293 guide to Pt(II) reactions at
Nucleic acid components in vitro, equilibrium 236-38
reactions of neutral Pt complexes 5-7 titration 236-38
Nucleic acids Parameter correlation curves for
and anticancer Pt complexes, in vitro 6-mercaptopurine riboside Pt
studies 5-7 complexes 256-62
interaction with metal dimethyl Parasitic diseases, Pt-metal
sulfoxide complexes 285,288-89 complexes 292
Nucleobase binding sites, platinated Penetration and D N A binding of
purine and pyrimidine complexes 192 Pt(II) complexes in mouse
Nucleobase Pt complexes, mixed ...156,158 leukemia cells 76-78
Nucleobases, reaction products and Peptides, methionine, binding of
binding sites of dimethylsulfoxide Pt(II) and release of coordi
complexes 289/ nated amines 185-89

Nucleophilic discrimination factor of p H range of inosine and adenosine
dimethylsulfoxide 284 and their 5'-nucleotides 239-40
Nucleophilic site of c/s-DDP in c/s- Phage growth, E. coli bacteria 4
D D P - D N A crosslink 37/ Pharmacological data of indenyl and
Nucleosides, purine, N l vs. N 7 tetrahydroindenyl derivatives,
dichotomy 239-43 metallocene dichlorides, mono-
Nucleotides cyclopentadienyl complexes, and
conformational transition ( B - Z ) of titranocene dihalides 317-23
diethylenetriammineplatinum Phosphines, gold, reactions with thiols 376
complex binding 68-72 Phosphorus atoms and gold(I)
coordination sites with Pt 127-28 complexes 392/
Nucleotides, reaction with dichloro- Physicochemical studies of in vitro
ethylenediammineplatinum(II) .. 66 interactions between Pt(II)
5'-Nucleotides of inosine and adenosine, complexes and D N A 75-96
chemical shift, and p H range ...239-40 Pig synovia, histology of stained tissue,
osmarin studies 428-33
Ο Plasmacytoma tumor cells, mouse,
Octahedral complexes and antitumor Pt(II) and P t ( I V ) complexes,
activity 288 toxicity 9t
Olefin amino complexes, protonated Plasmid D N A
271, 273-76 and c/s-DDP, electrophoresis 59/
Oligomerization of Pt(II) diammines, electron micrographs 61/
195
P t studies 180 enzyme effect and recognition
Oligonucleotide-Pt structures, relevance sequence 54-55
to P t - D N A interaction 125-42 gel mobilities, as a function of the
One-bond coupling constants, P t 195 amount of bound c/s- or
1 5
and N N M R , for Pt(II) and trans-ΌΌΡ 60/
Pt(IV) diammines 173-77 Platinated uracil, acid-base equilibria 149/
Osmarins, sedimentation profiles 425/ Platinated uracil tautomers, proton
Osmium, irradiation 302 N M R spectra 152/, 154/, 155/
Osmium carbohydrate polymers as anti Platinated uracil and thymine, reaction
arthritic drugs 421 with base 158
Oxy guanine and cytosine containing Platinum—See c/s-diamminedichloro-
dinucleotides with c/s-Pt(II) platinum(II) and more specific
complexes 125-42 entries
Polymers, osmium carbohydrate, as
Ρ antiarthritic drugs 421
Palladium, nuclear reactions 302-6 Potassium gold (III) bromide and
Palladium(2-amino-6-mercaptopurine gold(III)chloride, E X A F S
riboside)2, E X A F S analysis ...245-62 spectra 396/

Potassium trichlorodimethylsulfoxide Radiodiagnostic agents, R u 337

platinum complex, acid Raman spectroscopy, differentiation of
hydrolysis 283-84 platinated uracil and thymine
Potential energy maps for nucleobases 192 tautomers 150,153
Preparation Rate constants, dGuo-Ru complex .... 339
of head-head Pt dimer 164-68 Reactivity of head-head Pt dimer ...164-68
of osmarins 423-26 Reactor neutron spectrum 303, 304/
sample, x-ray absorption Recognition sequence, plasmid D N A .54-55
spectroscopy 388 Recovery of nondividing human fibro
Properties blasts from c/s-DDP toxicity 20/
N M R nuclei 172* Red blood cells
of ( 0 H ) M C 1 , tumor-inhibiting 326*
5 5 2 2 ligand exchange reactions 371-83
solution, of c/s-diammineplatinum proton N M R spectra of auranofin
complexes 232-38 and aurothiomalate 375/, 382/
stereochemical, of cw-Pt complexes reactions with gold phosphines 378
with nucleobases, nucleosides, Reduction potential vs. p H for
and nucleotides 195-202 l-[(Ado)(NH ) Ru(III)] 348/
3 5
Protein-DNA crosslinking Relaxation, P t 195

177
concentration dependence 38/ Replication on a damaged template,
in human fibroblasts, repair 22/ relation between capacity and
kinetics of formation and disappear cytotoxicity 16-17
ance in mouse leukemia Resistance, c/s-DDP, mechanism in
cells 34/, 35/ malignant ascites cells 39, 41
Proteins, methionine, binding of Rheumatoid arthritis, chryso
Pt(II) release of coordinated therapy 357-59, 371-83, 385-99
amines 185-89 Rhodium, nuclear reactions 302-6
Proton-induced x-ray emission, in Rhodium dimethylsulfoxide complex .. 283
summary of analytical methods R N A , target for Pt(II) complexes .... 7-8
for biological materials 299* RNase A , N M R spectrum 186/, 188/
Protonated amino-olefin Ruthenium
complexes 271,273-76 irradiation 302
Protonated diamine complexes 267-71 reaction mechanisms of purine and
Protonated Pt complexes of allyl- pyrimidine complexes 339-41
amines, cell culture activity 274* Ruthenium-97 and -103 337-38
Purine Ruthenium dimethylsulfoxide complex 281
free-energy correlations, Ruthenium red 336-37
( N H ) R u ( I I I ) complexes .341-42
3 5
metal-ion movement 342-49

nucleosides, N l vs. N 7
S
dichotomy 239-43 Salmon sperm D N A
reaction with [ P t C l ( R S O ) ] - 3 2 288 circular dichroism spectra, and
Purine-pyrimidine complexes of Pt complexes 84/
Pt(II), base/base and base/ viscosity, complexed with c/s-
P t N dihedral angles
4 197-202 and trans-ΌΌ? 84/
Pyrimidine, reaction with Sample preparation, x-ray absorption
[PtCl (R SO)r
3 2 288 spectroscopy 388
Pyrimidine complexes 19S
Satellites, P t N M R 173, 179,183/
with ( N H ) R u ( I I I )
3 5 339-42 195
Scalar relaxation, P t N M R 177
of Pt(II), base/base and base/ Scale factor, E X A F S 249-62
P t N dihedral angles
4 199,201-2 Scanning electron microscopy
Pyrimidines, metal-ion movement .342-49 studies ( S E M ) 309
Scatchard plot, fluorescence, of the
Q binding of ethidium bromide to
Quadrupolar relaxation rate 177 calf thymus D N A 57/
Sedimentation profiles, osmarins 425/
R Sequence specificity of c/s-DDP
Rabbit studies, osmarin 433-35 binding to D N A 55-56
Radiation of indium, osmium, and Simian virus 40 D N A , model for
ruthenium 302 interaction with cw-DDP 101-18

INDEX 451
Single shell, E X A F S curve fitting 398* Spectra—Continued

Sister-chromatid exchange production N M R —Continued
by c/s-DDP in Chinese hamster of c/s,c/s,*raws-Pt(isopropyl-
cells 45/ amine) Cl (OH) 2 2 178/ 2
Sodium aurothiomalate, arthritis 357-67 X A N E S of gold(III), gold(I),

Sodium gold ( I ) thiomalate and gold(0) 391/
Fourier transform 397/ x-ray absorption
x-ray absorption spectrum 387/ of myocrisin and gold(I)-
Solganal and arthritis 357-67 thiomalate 387/
Solubility of [(NH )oPt(OH) Pt(NH ) ]-
3 2 3 2
of carboxyphthalato Pt complexes .. 291* (N0 ) 3 2 217/

of diaminocyclohexane Pt Spectrometer, gamma ray 302
complexes 291* Spectrophotometric titration,
of malonato Pt complexes 291* [2-(AmPm)(NH ) Ru(III)] 345/
3 5
Solution properties of cw-diamminô-
Spectroscopy
platinum complexes 232-38
atomic, in summary of analytical
Solvent effects, dimethylsulfoxide
methods for biological

as ligand 281-83
materials 299*
Species distribution of adenosine
atomic absorption, binding of
5'-phosphate binding by a 14
[ C]c/s-[PtCl (NH CH ) 67/
diethylene Pd cation 241/ 2 3 3 2
extended x-ray absorption fine

Specificity, sequence, of c/s-DDP
structure ( E X A F S )
binding to D N A 55-56
of gold-based anti-arthritis drugs
Spectra
and metabolites .385-90, 393-95
circular dichroism
of 6-mercaptopurine riboside
of monomeric Pt dinucleotide
complexes of Pt(II)
chelates 125-42
and Pd(II) 245-62
of poly(dG-dC) · poly(dG-dC)
IR, differentiation of platinated
with [(dien)PtCl]Cl 70/
thymine and uracil
of salmon sperm D N A and Pt
tautomers 148,150
complexes 84/
NMR
extended x-ray absorption fine
antitumor complexes 172-89
structure ( E X A F S )
proton
of [ ( N H ) P t ( O H ) P t ( N H ) ] -
3 2 2 3 2
dichlorodimethylsulfoxide
( N 0 ) and D N A reaction
3 2
complexes 283
product 215-21
differentiation of platinated
of potassium gold (III)bromide,
uracil and thymine
chloride, and nitrate 396/
tautomers 150-52,153,158
NMR
Raman, differentiation of platinated
of 1-methylcytosine 216/
uracil and thymine
phosphorus-31
tautomers 150,153
of gold drugs 373, 378-82
x-ray, in summary of analytical
of poly(dG-dC) · poly(dG-
methods for biological
dC) modified with
materials 299*
[(dien)PtCP]Cl 71/
x-ray absorption near edge, gold-
proton
based anti-arthritis drugs and
of gold drugs 373-87
metabolites 391/
of monomeric Pt dinucleotide 195
Spin-rotation, P t N M R 179
chelates 138-40 195 1 5
Spin-spin couplings, P t and N
of Pd( II)-nucleoside and N M R , for Pt(II) and P t ( I V )
nucleotide complexes .239-41 diammines 173-77
of platinated uracil Square-planar complexes and anti
tautomers 151-55 tumor activity 288
of [ R u ( M e S O ) F 2 6 281-82 Stability of D N A - P t (II) complexes .79-94
proton and nitrogen-15 Staining and retention in pig
of ethylenediammine complex 188/ synovia 428-31
of RNase A 186/, 188/ Stereochemical properties
proton and platinum-195 c/s- vs. trans-ΌΌΡ binding to D N A
of c/s-DDP 181/, 183/ and nuclease digestion 195-202

Stereochemical properties—Continued Supercoiled and nicked D N A ratios

trans influence of sulfoxide ligand by electron microscopy 81/
in [ P t C l ( M e S O ) r
3 2 285-88 Supercoiled simian virus 40 D N A
cis vs. trans influences of dimethyl incubated with cis- and trans-
sulfoxide in Pt coordination OOP, electrophoretic
chemistry, kinetic and thermo profile 104,107-10
dynamic studies 283-84 Symmetry of c / s - P t ( N H ) ( l -
3 2
cis and trans isomers, P t N M R .. 195

174 MeU) -4H 0
2 2 153
cis-Pt complexes with nucleobases, Synchronized Chinese hamster cells,
nucleosides, and nucleo effect on D N A synthesis of
tides 195-202 c/s-DDP treatment 15/
Steric effects, dimethylsulfoxide as Synovial cells ( S C ) , pig, and
ligand 281-83 osmarin 432/
Structural and acidity data, ( N H ) - 3 5 Synthesis of D N A , inhibition 14-16
Ru(III) complexes with Synthesis of L P t C l antitumor Pt
3
purines 341-42 complexes 265-76

Structural interpretation, and E X A F S
results, gold complexes 393 Τ

Structural parameters of D D P and Tantalum and structure-activity
some metallocene dichlorides .... 319/ relation 316-18
Structural studies of in vitro inter Tautomers
actions between Pt(II) com of platinated thymine and uracil,
plexes and D N A 75-96 differentiation using spec
Structure of 1-methyluracil and troscopy 148-51
1-methylthymine 155/ of uracil and thymine mono-
Structure-activity relationship anions 149-53
dimethylsulfoxide species 290 Temperature variations, melting, of
L P t C l antitumor Pt complexes . .265-66
3 P t - D N A complexes 87/
metallocene dihalides 316-25 Template, capacity to replicate on
Structures and cytotoxicity 16-17
adenosine 5'-monophosphate and Tetra-O-acetyl-1 -thio-£-D-(gluco-
inosine 181/ pyranosato-S) (triethylphos
aurothioglucose 357-67 phine) gold (I)—See Auranofin
cis- and trans-OOP 29/ Tetraamminegold ( III ) nitrate,
DNA E X A F S spectra 396/
levels 52-53 Tetrahydroindenyl derivatives,
P t - D N A adducts 79-83 pharmacological data 323/
1-methylthymine 155/ Therapeutic index and acido ligands .. 318
myocrisin 357-67 Therapy, gold, applications 359-60
Substitution, mono, ethyl, and Thermal neutron activation analysis .. 303
trimethylsilyl 321 Thermal neutron cross-section, Pt
Substitution, in O-bound sulfoxides group metals 301/
2
in [ R u ( M e S O ) ] \
2 e 281-82 Thermodynamic studies of cis and
Substitution shifts, halide, in Pt trans influences of dimethylsulf
complexes 174 oxide in Pt coordination
Sucrose sedimentation, chemistry 283-84
alkaline 13-14,17-18 Thiol ligand
Sugar conformations, assignments, of reaction with gold phosphines 376
Pt dinucleotide chelates 128 role in the therapeutic response to
Sulfoxide sodium aurothiomalate 358
complexes, hydrolysis of O-bound Thiol uptake of aurothiomalate 373-76
ligand 282-83 Thiomalate S atoms, coordination 373
_
ligand in [ P t C l ( M e S O ) ] , trans
3 2 Thiourea inhibition of c/s-DDP cyto
influence 285,288 toxicity in mouse leukemia
S-bound hydrolysis 290 cells 36/, 37/
Sulfur ligands in gold drugs, Thymidine, molecular topology,
E X A F S results 398/ atomic numbering, potential
2+
Sulfur-bound ligands, [Pt(Me SO) ] 282 2 4 energy maps, and metal binding
Supercoiled double helix structure ...52-53 sites 192-95

INDEX 453
Thymine, 1-methyl-, structure 155/ Tumor-inhibiting properties—Continued

Thymine, Pt(II) complex of metallocene dihalides of
formation 147-68 transition metals 315-31
Thymine monoanion, metal binding Tungsten and structure-activity
sites 192-95 relation 316-18
Thymus D N A , calf, bound to ethidium
bromide, fluorescence Scatchard U
plot 57/ Uracil
Tissue, pig, histology and osmarin Nl-methylated Pt(II)
studies 431-33 complexes 153,156-67
Titanium and structure-activity Pt(II) complex formation 147-68
relation 316-18 tautomers, platinated, proton
Titanocene dihalides, pharmacological N M R spectra 152/, 154/, 155/
data 320*, 322* U V monitoring of reactions of diaquo
Titanocene dihalides and acido ligands 318 Pt complex with dinucleotides .... 127
Titration
[2-(AmPm)(NH ) Ru(III)] 345/
3 5 V
Pd(II) complex 236-38
Topology, molecular, for nucleo Vanadium and structure-activity
bases 193/ relation 316-18
Toxicity Virus, simian 40 D N A , model for
of carboxyphthalato complexes 291* interaction with cw-DDP 101-18
of diaminocyclohexane Pt Viscosity of sonicated salmon sperm
complexes 291* D N A complexed with cis- and
of cw-DDP, recovery of nondivid- trans-ΌΌ? 84/
ing human fibroblasts 20/
of malonato Pt complexes 291* W
of osmarins in mice 428-38 Water deprotonation in C I J - ( N H ) - 3 2
of Pt(II) and Pt(IV) complexes to Pt(Cl) ( H 0 ) , acidity constant .. 233

2
Chinese hamster and mouse Water hyacinth, study of biological

plasmacytoma tumor cells 9* effects of Pt complexes ... 298, 308-10
Transmission modes, x-ray absorp Watson-Crick G C base pair and
tion spectrum 387/ methylated G T base pair 224/
Trichloro ( 4-amino-2,6-dimethylpyri- Wavelength dispersive analysis 307-8
dine) platinum (II) preparation .. 268 Wet ashing, in summary of analytical
Trichloro ( 3-aminoquinuclidinium ) - methods for biological materials 299*
platinum (II) against murine
leukemia cells in vivo, activity .... 272* X
Trichlorodimethylsulfoxide rhodium
and platinum complexes, X-Ray absorption near edge spectros
hydrolysis 283-84 copy, gold-based anti-arthritis
Trichloro ( 9-methyladeninium ) plati drugs and metabolites 385-93
num (II) and trichloro (tetra- X-Ray absorption spectra
methylethylenediammonium ) - of [ ( N H ) P t ( O H ) P t ( N H 3 ) ] -
3 2 2 2
platinum(II), preparation 267-68 (N0 )

3 2 217/
Trihydrate of ci.y-[Pt(NH ) (l- 3 2
of Pt and Pd mercaptopurine
methylcytosine) (Nl-deproton- riboside complexes 248/
ated thymine anion)]*, base/ X - R a y absorption spectroscopy,
base and base/PtN conforma EXAFS 215
4
tional drawings 195-202 X - R a y microanalysis, electron

Trimers, hydroxide-bridged 180 microscopy 307
Trimethylsilyl monosubstitution 321 X - R a y spectrometry, in summary of
analytical methods for biological
Trypanosomiasis, Pt complexes 292
materials 299*
Tumor D N A , cw-diamminedichloro-
platinum(II) and hydroxymalo- Ζ
natodiammineplatinum(II) 10-12
Tumor-inhibiting properties Zirconium and structure-activity
of ( C H ) M C 1
5 5 2 2 326* relation 316-18


Platinum, Gold, and Other Metal Chemotherapeutic Agents

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Publication Date: January 26, 1983 | doi: 10.1021/bk-1983-0209.

Platinum, Gold, and Other

In Platinum, Gold, and Other Metal Chemotherapeutic Agents; Lippard, S.;

In Platinum, Gold, and Other Metal Chemotherapeutic Agents; Lippard, S.;

Massachusetts Institute of Technology

sponsored by the ACS Division

at the 183rd Meeting of the

American Chemical Society,

Las Vegas, Nevada,

March 28-April 2, 1982

ACS SYMPOSIUM SERIES 209

AMERICAN CHEMICAL SOCIETY

In Platinum, Gold, and Other Metal Chemotherapeutic Agents; Lippard, S.;

of Inorganic Chemistry of the American Chemical

In Platinum, Gold, and Other Metal Chemotherapeutic Agents; Lippard, S.;

M. Joan Comstock, Series Editor

David L. Allara Robert Ory

In Platinum, Gold, and Other Metal Chemotherapeutic Agents; Lippard, S.;

In Platinum, Gold, and Other Metal Chemotherapeutic Agents; Lippard, S.;

THE DISCOVERY OF THE REMARKABLE BIOLOGICAL ACTIVITIES of the anti­

in the history of bioinorganic chemistry. Efforts are currently being made

In Platinum, Gold, and Other Metal Chemotherapeutic Agents; Lippard, S.;

DNA as a Target for Anticancer Coordination

J. J. ROBERTS and M. F. PERA, JR.1

The notion that DNA is the likely target for anti-

Since the first description of the biological properties of

In Platinum, Gold, and Other Metal Chemotherapeutic Agents; Lippard, S.;

B i o l o g i c a l E f f e c t s o f Platinum Antitumour Drugs I n d i c a t i v e o f

Probably the f i r s t o b s e r v a t i o n o f an e f f e c t o f a platinum

t h i s response as a r e s u l t o f t h e i r common a b i l i t y t o damage DNA.

In Platinum, Gold, and Other Metal Chemotherapeutic Agents; Lippard, S.;

Another p r o p e r t y platinum antitumour drugs share with agents

Reaction o f Platinum Prugs w i t h PNA

Although other hypotheses have been proposed f o r the mechan-

Reactions o f n e u t r a l platinum complexes with n u c l e i c a c i d

In Platinum, Gold, and Other Metal Chemotherapeutic Agents; Lippard, S.;

trans-DPP provided c o n c l u s i v e evidence t h a t both platinum com­

6-NH and N - l o f adenine, and the 4-NH and N-3 o f c y t o s i n e .

The t r a n s isomer, on the other hand, i n t e r a c t s monofunctionally

isomers r e a c t monofunctionally w i t h the N-7 o f guanine and hypox-

the c o n c l u s i o n s obtained from the e a r l y spectrophotometrie

platinum i o n v i a the N-7 p o s i t i o n (25). A similar structure

4-NH group o f c y t o s i n e can be occupied by platinum(II) i o n s .

A bidentate b i n d i n g r e a c t i o n between the N-7 and the 0-6 p o s i t ­

t i d e (36,37). A l t e r n a t i v e l y the amino groups o f guanines o r o f

In Platinum, Gold, and Other Metal Chemotherapeutic Agents; Lippard, S.;

strand c r o s s l i n k i n g o f DNAs r i c h i n guanine and c y t o s i n e was

Despite the c l e a r evidence f o r the formation o f i n t e r s t r a n d

Reaction w i t h PNA o f c e l l s i n c u l t u r e . Studies w i t h

In Platinum, Gold, and Other Metal Chemotherapeutic Agents; Lippard, S.;

type o f macromolecule. The r e s u l t i n g graphs were then

( t h i s i s t h e o r e t i c a l l y the c o n c e n t r a t i o n a t which one i n a c t i v a t -

v a l u e s ) , on the other hançl, were, f o r most compounds, o f the

Reaction with the DNA o f c e l l s i n v i v o . I t i s c l e a r l y

In Platinum, Gold, and Other Metal Chemotherapeutic Agents; Lippard, S.;

cis-Pt(II)(mal)(1,2-dac) N.D. N.D. 23 2.5 Î

cis-Pt(II)Cl (C H NH ) 480 2.4 120 N.D.

N.D. Not determined

In Platinum, Gold, and Other Metal Chemotherapeutic Agents; Lippard, S.;

the m a t e r i a l s administered t o the mice, the doses d i d not d i f f e r

s u r v i v a l i n v i v o was s i m i l a r t o values p r e v i o u s l y observed i n

Role o f c r o s s l i n k i n g r e a c t i o n s . The s t r u c t u r a l requirement

culture. Comparison o f these two s e t s o f values i n d i c a t e d t h a t

THE DISCOVERY OF THE REMARKABLE BIOLOGICAL ACTIVITIES of the anti

trans-DPP provided c o n c l u s i v e evidence t h a t both platinum com

A bidentate b i n d i n g r e a c t i o n between the N-7 and the 0-6 p o s i t

I n h i b i t i o n o f DNA s y n t h e s i s . The s i g n i f i c a n c e of the i n t e r

Figure 2. Effects of cis-DDP treatment in Gl phase on DNA synthesis in synchro

Figure 3. Effects of cis-DDP treatment in Gl phase on DNA synthesis in synchro

I n V i t r o C e l l C u l t u r e . Mouse leukemia L1210 c e l l s ( d e s i g