SNAr prep

(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
(19) World Intellectual Property Organization WO 2009/064471 A1

International Bureau
(43) International Publication Date ρ^πτ d*·) International Publication Number

22 M ay 2009 (22.05.2009) WO 2009/064471 A l
(51) International Patent Classification: (81) Designated States (unless otherwise indicated, fo r every
C07D 473/18 (2006.01) A 61K 31/70 (2006.01) kind o f national protection available)·. AE, AG, AL, AM,
AO, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ, CA,
(21) International Application Number: CH, CN, CO, CR, CU, CZ, DE, DK, DM, DO, DZ, EC, EE,
PCT/US2008/012804 EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID,
IL, IN, IS, JP, KE, KG, KM, KN, KP, KR, KZ, LA, LC, LK,
(22) International Filing Date:
LR, LS, LT, LU, LY, MA, MD, ME, MG, MK, MN, MW,
14 November 2008 (14.11.2008)
MX, MY, MZ, NA, NO, NI, NO, NZ, OM, PO, PH, PL, PT,
(25) Filing Language: English RO, RS, RU, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TJ,
TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM,
(26) Publication Language: English ZW.
(30) Priority Data: (84) Designated States (unless otherwise indicated, fo r every
60/988,200 15 November 2007 (15.11.2007) US kind o f regional protection available)·. ARIPO (BW, GH,
60/988,192 15 November 2007 (15.11.2007) US GM, KE, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, ZM,
ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM),
(71) Applicant (for all designated States except US)·. AVI
European (AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI,
BIOPHARMA, INC. [US/US]; 4575 S.W. Research Way,
WO 2009/064471 A1
#200, Corvallis, OR 97333 (US).
FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, MC, MT, NL,
NO, PL, PT, RO, SE, SI, SK, TR), OAPI (BF, BJ, CF, CO,
(72) Inventors; and CI, CM, GA, GN, GQ, GW, ML, MR, NE, SN, TD, TG).
(75) Inventors/Applicants (for US only)·. FOX, Christina,
Mary Josephine [GB/US]; 30587 Stout Lane, Corvallis, Published:
OR 97333 (US). REEVES, Matthew, Dale [US/US]; — with international search report
2325 Edgemont Street SE, Albany, OR 97322 (US). — before the expiration o f the time limit fo r amending the
WELLER, Dwight, D. [US/US]; 3323 N.W. Elmwood claims and to be republished in the event o f receipt o f
Drive, Corvallis, OR 97330 (US). amendments
— with sequence listing part o f description published sepa
(74) Agents: TODD, Stephen et al.; King & Spalding, P.O. rately in electronic form and available upon request from
Box 889, Belmont, CA 94002-0889 (US). the International Bureau
(54) Title: METHOD OF SYNTHESIS OF MORPHOLINO OLIGOMERS
O
WO 2009/064471 Al
(57) Abstract: Morpholino compounds are provided having the structure (I): where R1 is selected from the group consisting of
lower alkyl, di(lower alkyl)amino, and phenyl; R is selected from the group consisting of lower alkyl, monocyclic arylmethyl, and
monocyclic (aryloxy)methyl; R is selected from the group consisting of triarylmethyl and hydrogen; and Y is selected from the
group consisting of: a protected or unprotected hydroxyl or amino group; a chlorophosphoramidate group; and a phosphorodiami-
date linkage to the ring nitrogen of a further morpholino compound or a morpholino oligomer. Such compounds include doubly
protected morpholino guanine (MoG) monomers. Also described is their use in synthesis of morpholino oligomers, and further im
proved procedures for synthesis of morpholino oligomers, pertaining to deprotection of the protected morpholino ring nitrogen at
each monomer coupling step.
WO 2009/064471 PCT/US2008/012804
M ETH O D OF SY N TH ESIS O F M O R PH O LIN O O LIG O M ER S
Field o f the Invention

The invention relates to m ethods o f synthesizing phosphorodiam idate-linked
m orpholino oligom ers by coupling o f m orpholino subunit m onom ers, and in particular to
improved procedures for deprotection o f the protected m orpholino ring nitrogen at each
coupling step, and to the use o f guanine m orpholino (M oG) subunits with protection at
both the N2 and 06/N 1 groups o f the guanine base. M orpholino oligom ers synthesized
using these m odifications are obtained in higher purity and yield com pared to those
synthesized using m onoprotected guanine subunits and/or conventional ring nitrogen

deprotection procedures.
References
Albert, A., P hysical M ethods in H eterocyclic Chemistry, Vol. I, A.R. Katritzky, Ed.,
Academ ic Press, pp 44 (1963).

Fisher, A., Gallow ay, W .J., and Vaughan, J., J. Chem. Soc. 3591 (1964).
G arrison, A.W . and Boozer, C.E., J. A m . Chem. Soc. 90(13):3486-3494 (1968).
Gough et al. (1979) N ucleic Acids Research 7:1955-1964.
Hata et al. (1983) Tetrahedron Lett. 24:2775-2778.
Jones et al. (1982A) Tetrahedron Lett. 23:2253-2256.
Jones et al. (1982B) Tetrahedron Lett. 23:2257-2260.
M itsunobu, 0 . (1981) Synthesis 1:1-28.
Ravikum ar, V. et al., U.S. Patent No. 5,510,476.
Reese e t al. (1981) Tetrahedron Lett. 22:4755-4758.
Reese et al. (1984) J .C hem .Soc., Perkin Trans. 1 1263-1270.
Rogne, O., J. Chem. Soc. 727 (1970).
Sum m erton, J.E. and W eller, D.D. (1993) U.S. Patent No. 5,185,444.
Sum m erton, J.E. and W eller, D.D., Antisense Nucl. A c id D rug Dev. 7(3):187-195
(1997).
Sum m erton, J.E. and W eller, D.D., U.S. Patent N o. 5,185,444 (1993).
Background
Phosphorodiam idate-linked m orpholino oligomers, or PM O, are nucleic acid analogs
which bind tightly and sequence-specifically to com plem entary RNA and are useful in
m odulating protein synthesis and thus gene expression. These oligom ers are com posed o f
base-pairing recognition m oieties (heterocyclic bases) supported by a m orpholino
WO 2009/064471 PCT/US2008/012804
backbone system. M orpholino subunits for use in synthesizing such oligom ers can be
prepared easily from the corresponding ribonucleosides, which are readily available and
inexpensive precursors (see e.g. Sum m erton and W eller, 1993, 1997).
During such synthesis, as in conventional oligonucleotide synthesis, the functional
groups on the heterocyclic bases are typically m asked to prevent interference in the
synthetic transform ations. For exam ple, activation o f the N -tritylated m orpholino
m onom er (la -f; Figure I) entails reaction o f the 5 ’-hydroxyl with a suitable
phosphoram ido dichloridate to form the activated subunit 2a-f. At large scale (50-100
Gallon reactor), the crude activated subunit is generally contam inated with a high level o f
by-products. Follow ing chrom atographic purification, the activated subunit is isolated in
about 50% yield for A, C, I, T, U and their protected forms, but only in about 5% yield for
the activated singly protected G subunit, which is believed to be due to the presence o f the
unprotected 0 6 oxygen.
The 06-unprotected guanine subunit also gives rise to side reactions at the oligom er
stage. For exam ple, the 0 6 oxygen can react with activated subunit during coupling steps,
to form 06-phosphorylated or derivative species, and during final cleavage o f the base
protecting groups w ith am m onia, am m onia can react at C 6 to displace these species,
giving a diam inopurine derivative. Such impurities are difficult to rem ove by
chrom atography, and cause a large loss in yield.
Various protection schem es have been proposed in the art to reduce side reactions o f
unprotected guanine 0 6 positions in conventional oligonucleotide synthesis (see e.g.
Gough et al. 1979; Reese et al. 1981, 1984; Jones et al. 1982A, 1982B). However, these
protocols were largely unsuccessful when applied to PM O synthesis. Accordingly,
improved m ethods are sought to increase yield and purity in PM O synthesis, particularly
in the use o f G m orpholino subunits.
The m orpholino nitrogen o f a m orpholino subunit is also protected prior to use,
typically with a trityl or substituted trityl species. During oligom er synthesis, this group
must be removed during each cycle to allow incorporation o f the next subunit. Failure to
com pletely remove the protecting group leads to N -I deletion sequences that contam inate
the desired oligom er product.
Trityl groups are conventionally rem oved with acid, and deprotecting reagents used
for PM O synthesis have traditionally been carboxylic acids (Sum m erton et al. 1993,
1997). However, phosphorodiam idate groups are also sensitive to acid, and carboxylic
acids useful for detritylation are also capable o f prom oting hydrolysis o f
phosphorodiam idate linkages to am idate species, as shown in Fig. I, with the possibility o f
WO 2009/064471 PCT/US2008/012804
more extensive backbone degradation. For exam ple, cyanoacetic acid in 20%
acetonitrile/D CM is an effective deprotecting reagent, but it is found to cause substantial
(5-10% ) hydrolysis o f phosphorodiam idate linkages in the PM O product.
Carboxylic acids m ust also be com pletely removed from the synthesis support resin
prior to the coupling reaction; otherw ise, by-products are form ed that consist o f truncated
oligom ers containing a 3'-acylated species.
For these reasons, improved reagents are needed for m orpholino nitrogen deprotection
in PM O synthesis.
Sum m ary
In one aspect, the invention provides a m orpholino com pound com prising the
structure I:
I
wherein
R 1 is selected from the group consisting o f lower alkyl, di(low er alkyl)am ino, and
phenyl;
R 2 is selected from the group consisting o f lower alkyl, m onocyclic arylm ethyl, and
m onocyclic (aryloxy)m ethyl;
R 3 is selected from the group consisting o f triarylm ethyl and hydrogen; and
Y is selected from the group consisting of: a protected or unprotected hydroxyl or
am ino group; a chlorophosphoram idate group; and a phosphorodiam idate linkage to the
ring nitrogen o f a further m orpholino com pound or a m orpholino oligomer.
In selected em bodim ents, Y is selected from the group consisting o f a protected or
unprotected hydroxyl group and a chlorophosphoram idate group, e.g. a
chlorophosphoram idate group o f the form -0 -P(= 0 )-N (CH 3 ) 2 Cl. W hen Y is a protected
hydroxyl group, it is preferably a trialkyIsilyl-protected hydroxyl group.
The group R 3 is preferably selected from trityl (triphenylm ethyl), 4-m ethoxytrityl,
WO 2009/064471 PCT/US2008/012804
4-methyltrityl, 4,4'-dim ethyltrityl, and 4,4',4"-trim ethyltrityl. The group R 1 is preferably

lower alkyl, especially C1-C4 alkyl, and most particularly -C (C H 3 ) 3 (tert-butyl). The group
R 2 is preferably selected from benzyl and -CH (CH 3) 2 (isopropyl).
In a related aspect, the invention provides an improved m ethod o f synthesizing a
5 m orpholino oligom er, the m ethod comprising:
(a) reacting a solid-phase-supported m orpholino subunit, having an unprotected ring
nitrogen, with a base-protected m orpholino subunit m onom er, having a triarylm ethyl-
protected ring nitrogen and an activated phosphoram idate group on a 5 ’-exocyclic carbon,
thereby form ing a phosphorodiam idate linkage between the 5 ’-exocyclic carbon and
10 the unprotected ring nitrogen;
(b) deprotecting the protected ring nitrogen, to form an unprotected ring nitrogen; and
(c) repeating steps (a) and (b) one or more tim es with further base-protected
m orpholino subunit m onomers;
wherein at least one o f the base-protected m orpholino subunit m onom ers is a doubly
15 protected guanine m orpholino com pound having the structure I:
O
I
wherein
20 phenyl;
Y is a chlorophosphoram idate group.
25 Selected em bodim ents o f the variables represented in the above structure include
those described above.
In a further aspect, the invention provides an improved m ethod o f synthesizing a
morpholino oligom er, the m ethod comprising:
4
WO 2009/064471 PCT/US2008/012804

protected ring nitrogen and an activated phosphoram idate group on a 5’-exocyclic carbon,
thereby form ing a phosphorodiam idate linkage between the 5’-exocyclic carbon and the
unprotected ring nitrogen;
(b) deprotecting the protected ring nitrogen, to form an unprotected ring nitrogen; and
m orpholino subunit m onom ers;
wherein said deprotecting com prises exposing the triarylm ethyl-protected ring
nitrogen to a reagent solution com prising a heterocyclic am ine salt in a trifluoroethanol-
containing solvent, the salt being a salt o f a heterocyclic amine, having a pK a in the range
o f 1-4 in its protonated form , w ith an acid selected from a sulfonic acid, trifluoroacetic
acid, and hydrochloric acid.
The heterocyclic am ine is preferably selected from the group consisting of: an
electron w ithdraw ing group-substituted pyridine, thiazole, pyridazine, pyrazole, triazole
and electron w ithdraw ing group-substituted substituted derivatives o f these. Such electron
w ithdraw ing groups (EW G) include halogen, cyano, aldehyde, keto, carboxyester, and
carboxamide.
Preferably, the heterocyclic amine is an electron w ithdraw ing group-substituted
pyridine, such as a chloro- or cyano-substituted pyridine. The am ine salt is preferably a
salt o f a sulfonic acid, such as an alkylsulfonate, (fluoroalkyl)sulfonate, or p-
toluenesulfonate, or a trifluoroacetate. In selected em bodim ents, the salt is selected from
3-chloropyridinium m ethanesulfonate (CPM) and 4-cyanopyridinium trifluoroacetate
(C Y TFA ).
The TFE-containing solvent preferably com prises dichlorom ethane and
trifluoroethanol in volum e ratio in the range o f about 90:10 to 25:75, and m ore preferably
in a volume ratio o f about 80:20 DCM :TFE.
The triarylm ethyl protecting group is selected from the group consisting o f trityl
(triphenylm ethyl), 4-m ethoxytrityl, 4-m ethyltrityl, 4,4'-dim ethyltrityl, and
4,4',4"-trim ethyltrityl.
The m odifications and improvem ents described herein m ay be com bined, such that
steps (a) - (c) above are carried out wherein:
(i) at least one o f the base-protected m orpholino subunit m onom ers is a doubly
protected guanine m orpholino compound having the structure I as set forth above; and
(ii) deprotecting the protected ring nitrogen com prises exposing the triarylm ethyl-
WO 2009/064471 PCT/US2008/012804
protected ring nitrogen to a reagent solution com prising a heterocyclic am ine salt in a
trifluoroethanol-containing solvent, the salt being a salt o f a heterocyclic am ine, having a
pKa in the range o f 1-4 in its protonated form, with an acid selected from a sulfonic acid,
trifluoroacetic acid, and hydrochloric acid.
5 Typically, the synthesis further com prises cleaving the m orpholino oligom er from the
solid phase and deprotecting the bases, in accordance with standard procedures.
These and other objects and features o f the invention will becom e m ore fully apparent
when the following detailed description o f the invention is read in conjunction with the
accom panying drawings.
10
B rief Description o f the Draw ings
Figure I illustrates the form ation o f an activated m orpholino subunit.
Figure 2 illustrates a route o f formation for a doubly protected m orpholino G subunit
(DPG) derivative in w hich the N2 position is phenylacetylated and the 0 6 position is
15 protected with the 4-nitrophenethyl (NPE) group.
Figures 3 illustrates an alternate route o f form ation for a doubly protected m orpholino
G subunit (DPG) derivative in which the N2 position is phenylacetylated and the 0 6
position is protected w ith the 4-nitrophenethyl (NPE) group.
Figure 4 illustrates the form ation o f a DPG derivative in w hich the N2 position is
20 phenylacetylated and the 0 6 position is protected w ith either the phenylsulfonylethyl
(PSE) or m ethylsulfonylethyl (M SE) group.
Figure 5 illustrates the form ation o f a DPG derivative in which the N2 position is
phenylacetylated and the 0 6 position is protected w ith the trim ethylsilylethyl (TM SE)
group.
25 Figure 6 illustrates the form ation o f a DPG derivative in which the N2 position is
phenylacetylated and the 0 6 position is protected w ith a series o f aryl derivatives.
Figure 7 illustrates the form ation o f a DPG derivative in which the N2 position is
phenylacetylated and the 0 6 position is protected with a series o f carbamoyl derivatives.
Figure 8 illustrates the form ation o f the DPG derivative in which the N2 position is
30 phenylacetylated and the 0 6 position is protected w ith the 4-(pivaloyloxy)benzyloxy
(POB) group.
Figure 9 shows conversion o f the phosphorodiam idate (PDA) linkage into the
phosphoram idate (am idate) linkages, in a side reaction that can occur upon treatm ent o f
phosphorodiam idate-linked m orpholino oligom ers (PM O) with carboxylic acids.
35 Figure 10 illustrates the preparation o f a disulfide anchor, for use in m odification o f a
6
WO 2009/064471 PCT/US2008/012804
synthesis resin used for stepw ise preparation o f a m orpholino oligom er, allow ing facile
release o f the oligom er by treatm ent with a thiol.
Figure 11 illustrates the preparation o f a triethylene glycol containing m oiety (“Tail”)
which increases aqueous solubility o f synthetic antisense oligom ers.
Figure 12 illustrates the preparation o f resins useful for the solid phase synthesis o f
m orpholino oligom ers.
D etailed D escription o f th e In v en tio n

I. Definitions
The term s below, as used herein, have the following meanings, unless indicated
otherwise:
A "morpholino oligomer" refers to a polymeric m olecule having a backbone which
supports bases capable o f hydrogen bonding to typical polynucleotides, wherein the polymer
lacks a pentose sugar backbone moiety, and more specifically a ribose backbone linked by
phosphodiester bonds which is typical o f nucleotides and nucleosides, but instead contains a
ring nitrogen with coupling through the ring nitrogen. A preferred m orpholino oligom er is
composed o f “morpholino subunit” structures, such as shown below, which in the oligomer
are preferably linked together by (thio)phosphorodiam idate linkages, joining the morpholino
nitrogen o f one subunit to the 5' exocyclic carbon o f an adjacent subunit. Each subunit
includes a purine or pyrim idine base-pairing m oiety Pi which is effective to bind, by base-
specific hydrogen bonding, to a base in a polynucleotide.
Morpholino oligom ers are detailed, for example, in co-owned U.S. Patent Nos.
5,698,685, 5,217,866, 5,142,047, 5,034,506, 5,166,315, 5,185,444, 5,521,063, and
5,506,337, all o f which are expressly incorporated by reference herein.
A "phosphorodiamidate" group comprises phosphorus having two attached oxygen
atoms and two attached nitrogen atoms, and herein may also refer to phosphorus having one
attached oxygen atom and three attached nitrogen atoms. In the intersubunit linkages o f the
oligomers described herein, one nitrogen is typically pendant to the backbone chain, and the
second nitrogen is the ring nitrogen in a morpholino ring structure, as shown in formula II
below. Alternatively or in addition, a nitrogen may be present at the 5’-exocyclic carbon, as
shown in formulas III and IV below.
WO 2009/064471 PCT/US2008/012804
? > >
N N N
O =P-N R 2 O = P-N R 2 O =P-O R "
I I I
N IN IN
Π III IV
In a thiophosphorodiamidate linkage, one oxygen atom, typically an oxygen pendant to

the backbone in the oligomers described herein, is replaced with sulfur.
A “solid-phase-supported m orpholino subunit” can be the first or any subsequent
m orpholino subunit m onom er incorporated into a m orpholino oligom er by solid-phase
stepw ise synthesis as described herein. The subunit is attached to the solid support, or to a
growing oligom er chain on the solid support, via its 5’ exocyclic carbon. “Base-
protected” refers to protection o f the base-pairing groups, e.g. purine or pyrim idine bases,
on the m orpholino subunits w ith protecting groups suitable to prevent reaction or
interference o f the base-pairing groups during stepw ise oligom er synthesis.
An “activated phosphoram idate group” is typically a chlorophosphoram idate group,
having substitution at nitrogen which is desired in the eventual phosphoram idate linkage
in the oligom er. An exam ple is (dim ethylam ino)chlorophosphoram idate, i.e.
-0 -P (= 0 )(N M e 2 )Cl.
The term s "charged", "uncharged", "cationic" and "anionic" as used herein refer to the
predominant state o f a ehemical moiety at near-neutral pH, e.g. about 6 to 8 . Preferably, the
term refers to the predominant state o f the chemical moiety at physiological pH, i.e. about
7.4.
"Lower alkyl" refers to an alkyl radical o f one to six carbon atoms, as exemplified by
methyl, ethyl, n-butyl, i-butyl, t-butyl, isoamyl, n-pentyl, and isopentyl. In selected
embodiments, a "lower alkyl" group has one to four carbon atoms, or 1 -2 carbon atoms; i.e.
methyl or ethyl. Analogously, "lower alkenyl" refers to an alkenyl radical o f two to six,
preferably three or four, carbon atoms, as exemplified by allyl and butenyl.
A "non-interfering" substituent is one that does not adversely affect the ability o f an
antisense oligom er as described herein to bind to its intended target. Such substituents
include small and preferably non-polar groups such as methyl, ethyl, methoxy, ethoxy,
hydroxy, or fluoro.
WO 2009/064471 PCT/US2008/012804
II. Base Protection in PM O Synthesis

Due to the specific challenges o f the m orpholino chem istry, a base protecting group
must fill several requirem ents. The protecting group should be readily introduced onto the
heterocyclic m oiety and thereafter be stable to subunit activation and purification
conditions, and solid phase synthesis. The protecting group should not be reactive with
the m orpholino am ine m oiety o f the grow ing chain, and should allow the activated
m orpholino subunit to couple cleanly with the growing oligom er chain. The protecting
group should be cleaved, preferably by am m onia, w ithout introducing new impurities.
Finally, it should result in crystalline subunit derivatives, in order to avoid the need for
chrom atographic purification prior to activation.
As described below and in the com parative Exam ples, protecting groups reported in
the literature for doubly protected guanosines, as used for nucleic acid synthesis, did not
adequately m eet these criteria. Thus, a new protecting strategy w as required for
m orpholino G subunits. A s described below, use o f the 4-(pivaloyloxy)benzyloxy group
at 0 6 was found to m eet al\ o f the above criteria.
A. 0 6 Protecting Groups: Com parative Data
A l. 4-nitrophenethyl ether fNPE)
This derivative was prepared as shown in Figure 2 (M itsunobu 1981) or Figure 3
(Jones et al. 1982B). W hile the crude 0 6 protected subunit could be prepared in
reasonable yield, the com pound was not readily crystalline and could be adequately
purified only by silica gel chrom atography, which is undesirable for large-scale
production. After testing an extensive range o f reslurrying and/or recrystallization
conditions, it was found that butoxyethanol-containing solvent com binations could, with
some difficulty, crystallize the material. However, excess butoxyethanol could not be
removed from the final product, as the com pound likely crystallized as a solvate. The
presence o f excess alcoholic solvent would not be acceptable in the activation reaction.
The NPE group is cleaved with strong base via a β-elim ination m echanism . These
conditions tend to generate the reactive by-product 4-nitrostyrene, which can then react
with reactive sites on the oligom er. W hile various scavenging agents (e.g. thiols and 1,3-
dicarbonyl com pounds) were introduced into the deprotection m ixture in an attem pt to
prevent trapping o f the by-product by the oligom er, none w ere com pletely successful in
elim inating this internal return problem . Even after purification, oligom ers prepared with
this subunit had a yellow tint.
A2. Phenvlsulfonvlethvl (PSEJ and M ethvlsulfonvlethvl (MSEJ
These groups were introduced via the corresponding 2-thioethanol derivatives (Jones
WO 2009/064471 PCT/US2008/012804
et al. 1982A, 1982B), as shown in Figure 4. H ow ever, no successful crystallization

procedure could be found for the resulting subunits.
Like the N PE group, above, these groups are cleaved via a β-elim ination m echanism .
A fter incorporation into an oligom er, these derivatives gave the sam e problem s seen with
the NPE group; that is, internal return o f the reactive alkene by-product form ed during
deprotection.
A3. Trim ethvlsilvlethvl ether
As reported by Jones (Jones et al. 1982B), an 06-T M SE -m odified m orpholino
guanine subunit w as prepared as shown in Figure 5, but it was not stable during oligom er
synthesis. O ligom ers m ade with this subunit showed a range o f by-products sim ilar to
those made from 06-unprotected G subunits.
A4. Phenvl ether
M orpholino guanine subunits w ith 06-phenyl substitution (Figure 6 ) were prepared
according to the procedure o f Reese e t al. (1981, 1984). The derivatives included
unsubstituted phenyl, 2,5-dichlorophenyl, pentafluorophenyl, and 3-fluorophenyl. Such
subunits could be incorporated into PM O, but deprotection w ith the usual reagents, such as
2 -nitrobenzaldehyde oxim e and strong base, could not be carried to com pletion without
degradation o f the oligom er.
A5. Carbam ate
Several 0 6-carbam ate derivatives were synthesized, according to the procedure o f
Hata et al. 1983 (Figure 7). Use o f these derivatives in oligom er synthesis gave varying
results depending on the derivative used. For the more labile species, such as the diphenyl
carbam oyl analog, transfer o f the protecting group to the 3’-nitrogen o f the growing chain
was noted during the coupling step o f solid phase synthesis, resulting in truncated
oligom ers containing a 3'-diphenylcarbam oyl moiety. In addition, the 06-carbam ates
have two possible sites o f reaction with amm onia. W hile the m ore reactive m oieties such
as the diphenylcarbam oyl group gave relatively selective attack at the carbonyl, the more
stable dimethyl and pyrrolidinyl carbam ates showed significant com peting reaction o f
am m onia at the C 6 position, with conversion to diam inopurine.
B. 4-(Pivalovloxv')benzvloxv Protecting Group
4-(Pivaloyloxy)benzyloxy alcohol (4a, Figure 8 ) was introduced into the m orpholino
guanine subunit via an efficient, high-yielding synthesis. The subunit prior to activation
(com pound I f i n Figures I and 8 ) can be synthesized and reproducibly isolated at large
scale without chrom atographic purification, and it can be crystallized from a variety o f
solvents (e.g. TH F/w ater, TH F/heptane, acetonitrile, various ester/hydrocarbon mixtures).
WO 2009/064471 PCT/US2008/012804
Ten batches o f this subunit m ade at the 50-200 gallon scale (batch size: 8 - 27 kg o f
com pound lc ) gave an average yield o f 65% o f product, having a purity (by HPLC) o f
97.6% to 99.2% .
The subunit is converted to activated subunit (i.e., conversion to the 5’-
chlorophosphoram idate com pound) m uch more cleanly than m ono-protected G, and it can
be more easily purified by silica gel chrom atography. At scale, overall yield from
com pound I f t o com pound 2 f (Fig. I) is approxim ately 50%.
The POB protecting group m ay be employed w ith other com binations o f protecting
groups for the N2 and m orpholino ring nitrogens. Suitable N2 protecting groups include
phenylacetyl (as illustrated in Fig. 8 ) as well as acetyl, propionyl, isobutyryl, and
phenoxyacetyl. Trityl species suitable for m orpholino ring nitrogen protection between
coupling steps include unsubstituted trityl, 4-methyl-, 4,4'-dim ethyl-, and 4,4',4"-
trim ethyltrityl, and 4-m ethoxytrityl.
O ther acyl protecting groups can also be used in place o f pivaloyl for the phenol
m oiety o f the POB group. Suitable alternatives include Ν ,Ν -dim ethylcarbam oyl and
benzoyl.
During PM O synthesis, no products are seen wherein the pivaloyl group has become
attached to the 3'-term inus o f sm aller fragments o f the full length PM O, a side reaction
comm on to the 06-carbam ates discussed above. The only notable side product detected
was a PM O containing a phenolic residue, resulting from reaction with the deprotection
by-product quinone m ethide. H ow ever, this by-product could be reduced to trace levels
by sufficient dilution o f the am m oniacal deprotection solution. In addition, it is easily
rem oved by virtue o f strong binding o f the phenolic residue to the polym eric resins used
for strong anion exchange chrom atography. In general, the overall yield o f purified PM O
is greatly increased, as seen in Table I.
The im provem ent in PM O production fostered by the POB protected guanine group is
m ost evident in the purification following PM O solid phase synthesis, w here the difficulty
in rem oving diam inopurine and related byproducts can lead to severe loss during strong
anion exchange (SAX) chrom atography. For exam ple, crude purities for AVI-4126
prepared with CPM and M PG (m ono-protected guanine subunit, 2c) are in the 68-73%
range, which calculates to approxim ately 58% crude yield o f the PMO. During the Trityl-
On and T rityl-O ff purifications, significant material is lost to obtain pure product, and the
overall recovery from the chrom atography is 52%. For the AV I-4126 m ade using CYTFA
and DPG (di-protected guanine subunit), the crude purities are 70-75% , with com parable
N -I levels by m ass spectrom etry (indicating that detritylation efficiencies o f CYTFA and
WO 2009/064471 PCT/US2008/012804
CPM reagents are approxim ately equivalent) and crude yields o f about 61%. However,
application o f the usual purification m ethods recovers 80% o f the PM O from the crude
m ixture.
Table I.
PMO SEQ Sequence Detritylation Guanine Scale 2 Yield
AVI- ID reagent 1 M onom er
NO:
4126 I A C G TTG A G G G G C A TC G TC G C CAA 2c 54 gJ 18%

4557 2 CTG G G A TG A G A G CCA TCA CT CAA 2c 24 g 4 18%
Il Il Il
CAA 2c 48 g 5 15%
4126 I A C G TTG A G G G G C A TC G TC G C CPM 2c 25 $ 25%

M M Il
CPM 2c 25 g 27%
(I M Il
CPM 2c 25 g 30%
4020 3 CTT AGTC A TCG A G A TCTTCG TG CPM 2c 30 g 32%
4126 I A C G TTG A G G G G C A TC G TC G C CYTFA 2 f 25 g 49%

4065 4 G TG CTCA TG G TG CA CG G TC 6 CYTFA 2 f 120 g 46%
M Il Il
CYTFA 2 f 120 g 49%
M It
CYTFA 2f 120 g 50%
5
Syntheses were perform ed in accordance with m ethods described in co-ow ned US
application 11/801,885, using the m odifications indicated in the table; see Exam ples 3-6
below. AU PM O have a 5'-"tail" and are unsubstituted at the 3'-terminus.
1CAA = 11% Cyanoacetic acid (w/w) in a m ixture o f 20% acetonitrile/D CM (v/v);
10 CPM = 2% 3-Chloropyridinum m ethanesulfonate (w/v) and 0.9% ethanol (v/v) in 20%

trifluoroethanol/D C M (v/v); CY TFA = 2% 3-Cyanopyridinum trifluoroacetate (w/v) and
0.9% ethanol (v/v) in 20% trifluoroethanol/D C M (v/v).
7
Scale is weight o f starting resin in gram s. Resin loading is 480-520 m icrom oles/g
Com bined output o f 4x12 g and 1x8 g runs.
15 4Com bined output o f 2x12 g runs.
5Com bined output o f 4x12 g runs.
6Addition o f the final C subunit w as performed with an activated m orpholino C subunit
with 4-m ethoxytrityl protection on the m orpholino nitrogen.
20 Thus, use o f the doubly protected M oG m onom er o f the invention provides a method
o f synthesizing a m orpholino oligom er in increased purified yield relative to prior art
methods, and particularly in com parison to purified yields observed when a
12
WO 2009/064471 PCT/US2008/012804
m onoprotected M oG m onom er, or other protected M oG m onom er not o f the invention, is

em ployed. In particular, the m ethod preferably generates a reduced level o f
diam inopurine species than w ould be obtained using a M oG m onom er not o f the
invention.
5
IN. Doubly Protected G uanine M orpholino Subunits
The doubly protected guanine (DPG) m orpholino subunits o f the invention have the
structure
O
10 where
phenyl;
monocyclic (aryloxy)m ethyl;
15 R 3 is selected from the group consisting o f triarylm ethyl and hydrogen; and
amino group; a chlorophosphoram idate group; and a phosphorodiam idate linkage to the
ring nitrogen o f a further m orpholino compound or a m orpholino oligom er.
In selected em bodim ents, Y is a protected or unprotected hydroxyl group (as in the
20 pre-activated monomer) or a chlorophosphoram idate group (as in the activated m onomer).
Preferred protecting groups for the hydroxyl group include trialkylsilyl groups, such as
tert-butyldim ethylsilyl (TBDM S).
Em bodim ents in which Y is a phosphorodiam idate linkage to the ring nitrogen o f a
further m orpholino com pound, or a phosphorodiam idate linkage to a m orpholino
25 oligomer, refer to species form ed during the synthesis o f a m orpholino oligom er, prior to
base deprotection.
13
WO 2009/064471 PCT/US2008/012804
As discussed below, the substituents on the chlorophosphoram idate group (in the
activated monomer) can vary depending on the specific phosphorodiam idate linkage
desired.
The invention also provides, correspondingly, a m ethod o f synthesizing a m orpholino
5 oligom er, the m ethod com prising:
nitrogen, with a base-protected morpholino subunit m onom er, having a triarylm ethyl-
thereby form ing a phosphorodiam idate linkage between said 5 ’-exocyclic carbon and
10 said unprotected ring nitrogen;
(b) deprotecting said protected ring nitrogen, to form an unprotected ring nitrogen;
and
m orpholino subunit m onom ers;
15 w herein at least one o f said base-protected m orpholino subunit m onom ers is a doubly
protected guanine m orpholino com pound having the structure:
O
wherein
20 phenyl;
25 Preferred triarylmethyl protecting groups for the m orpholino ring nitrogen (R3)
include trityl (triphenylm ethyl), 4-methoxytrityl, 4-m ethyltrityl, 4,4'-dim ethyltrityl, and
4,4',4"-trim ethy ltrity I.
The R 1 substituent on the 0 6 protecting group is preferably Ci to C 4 alkyl, especially
14
WO 2009/064471 PCT/US2008/012804
-C(CH 3 )3 (tert-butyl), as in the 4-(pivaloyloxy)benzyloxy (POB) group. However, R 1can

also be di(lower alkyl)am ino, such as dim ethylam ino, or phenyl.
As noted above, substitution o f the chlorophosphoram idate group Y in “activated”
m onom ers varies depending on the structure o f the desired phosphorodiam idate linkage.
5 For preparation o f the “ standard” uncharged PM O linkage 5 '-O -P i= O X -N (C H 3)2) - 3 ’ (as
shown in Form ula II above w here R is methyl), the chlorophosphoram idate group Y is
5 ’-0 -P (= 0 )C l-N R 2 (see e.g. com pound 2f, Figure 8).
As described in co-ow ned application having USSN 11/801,885, filed M ay 10, 2007,
which is incorporated herein by reference, advantageous properties can be obtained by
10 preparing PM Os having cationic as well as neutral intersubunit linkages. In such
oligom ers, at least one intersubunit linkage between tw o consecutive m orpholino ring
structures contains a pendant cationic group. The pendant group bears a distal nitrogen
atom that can bear a positive charge at neutral or near-neutral (e.g. physiological) pH.
For preparation o f such linkages, the chlorophosphoram idate group Y in the subunit
15 m onom ers o f the invention m ay have one o f the following structures:
I? O Z O
, I Il
5 - O - P —N R - X 5 - O - P —N N -C (O )R f 5 - N — P -O R
I I \ / I
Cl Cl Cl
where R is lower alkyl, such as methyl or ethyl;

20 X = -R4-NHC(= 0)Rf, w here R4 is bivalent alkyl or oligo PEG, and Rf is fully or
partially fluorinated methyl, ethyl, or isopropyl; and
Z = X as defined above o r lower alkyl. Note that the Z-containing group results in a
5 ’-amine containing linkage.
The term “oligo PE G ” refers to a group such as -(C H 2-CH 2-O)n-CH2-CH2- , w here n
25 is typically I to 3, and “bivalent alkyl” is typically C 2 to Cg alkyl.
Follow ing preparation o f oligom ers using m onom ers having such activated
chlorophosphoram idate groups, the C(= 0)Rf protecting groups are rem oved from the
term inal nitrogen atoms, w hich may be further m odified, e.g. to form term inal guanidinyl
groups, as described in co-ow ned application USSN 11/801,885.
15
WO 2009/064471 PCT/US2008/012804
IV. Im proved Conditions for Deprotection o f the M orpholino Ring N itrogen in PM O

Synthesis
A s noted above, deprotection o f the m orpholino ring nitrogen, w hich is typically
protected by a triarylm ethyl group such as trityl, in PM O synthesis, m ust be com plete
enough at each step to m inim ize N -I deletion species. However, studies in support o f the
invention showed that reagents used in the prior art for this purpose caused an undesirable
am ount o f backbone hydrolysis (see Fig. I) and degradation. Therefore, efficient
deprotecting reagents w hich at the sam e time m inim ized such hydrolysis were sought.
A sim ple assay was used to test the efficiency o f various reagents in deprotection
(typically detritylation) o f N -protected m orpholino subunits. A m odel com pound, the
tritylated m oCBz(i.e. benzoyl-protected cytosine m orpholino) subunit shown below, is
dissolved in the detritylation solution to be investigated. A t various tim epoints (e.g. I, 2, 4
min), an aliquot was quenched and analyzed by TLC or HPLC for com pletion o f
m orpholino nitrogen deprotection. Generally, for prediction o f effective detritylation
during solid phase PM O synthesis, this model reaction should be com plete within about 2
m inutes at room tem perature.
Tr m o(T r)C Bz
Using this assay and further experim entation, it was determ ined that various
pyridinium salts o f strong acids in m ixtures o f trifluoroethanol (TFE) and dichlorom ethane
(DCM ) are excellent catalysts for rem oving the triarylm ethyl protecting group, e.g. a trityl
group, from the m orpholino nitrogen during solid phase PM O synthesis.
A m inimum am ount o f TFE (~10% v/v or greater) is preferred for reasonable reaction
rates and solubilization o f the pyridinium salts. Because TFE alone does not swell naked
polystyrene, m ixtures with DCM (dichlorom ethane) are preferred, especially in the early
cycles o f PM O synthesis. Preferred solvent com positions include 10 to 75% TFE.
The use o f the TFE solvent is believed to enhance the selectivity o f the detritylation
reaction over amidate form ation (hydrolysis) and phosphorodiam idate (PDA) cleavage,
described above, by addressing the differing m echanism s o f PDA cleavage and
WO 2009/064471 PCT/US2008/012804
detritylation. TFE is a potent hydrogen bonding solvent and decreases the reactivity o f
nucleophiles in solution; therefore, it is believed to slow the attack on phosphorus
necessary for P-N bond cleavage. TFE also prom otes SN l type solvolysis reactions. The
solvolytic character o f am ine detritylation reactions with TFE is evidenced by the yellow
color o f detritylation reaction m ixtures and the orangish color o f dem ethoxytritylation
reaction mixtures. Therefore, increasing TFE concentration is believed to both suppress
nucleophilic attack on the PDA linkage and promote detritylation.
U nsubstituted pyridinium salts are not sufficiently acidic for optim al deprotection, but
the use o f pyridinium species containing electron w ithdraw ing groups (EW G) (e.g.
halogen, carbonyl, cyano) allows rapid cleavage o f the protecting group. G enerally at
least 2% (w/v) o f such a salt in the TFE:D CM solvent is sufficient for rapid detritylation.
Preferred levels o f the pyridinium salts are 2 to 10% (w/v).
A cids useful in form ing the pyridinium salts include sulfonic acids, such as
m ethanesulfonic, trifluorom ethanesulfonic, and p-toluenesulfonic acid, trifluoroacetic
acid, and hydrochloric acid. Although a carboxylic acid, trifluoroacetic acid does not cap
the grow ing PM O chain if present during the coupling reaction, and its carboxylate is not
sufficiently nucleophilic to prom ote amidate formation. Particularly preferred are
trifluoroacetic and especially m ethanesulfonic acid.
The pyridines useful in form ing the pyridinium salts include halogen substituted
pyridines, especially the less expensive chloropyridines, o f w hich 3-chloropyridine is
preferred, and cyanopyridines, for which 4-cyanopyridine is preferred. The 3- and 4-
cyanopyridines are readily available, inexpensive bulk chem icals. In general, the efficacy
o f the salts correlates inversely with the pKa o f the pyridinium species. Pyridines with
electron w ithdraw ing groups range in pKa from about I to 4 (Fisher et al. 1964, Rogne
1970).
Also useful are nicotinic acid derivatives (i.e. esters, such as ethyl nicotinate, and
nicotinam ide), as well as their ketone and aldehyde congeners. G enerally, however, these
are less potent reagents than the cyanopyridinium salts.
It will be appreciated that salts o f heterocycles other than pyridines can function as
selective detritylation reagents under the conditions described, provided the pKa o f the
protonated form is sim ilar to that o f substituted pyridines o f the invention. Exam ples may
be found in the m any tables o f pKa for heterocycles found in the literature (e.g. Albert
1963). Exam ples include thiazole (pKa 2.53), pyridazine (pKa 2.33), pyrazole (pKa 2.47),
triazole (pKa 2.30), and substituted derivatives thereof, especially derivatives substituted
with EW G as described above..
WO 2009/064471 PCT/US2008/012804
Tw o particularly preferred salts are 3-chloropyridinium m ethanesulfonate (CPM ) and

4-cyanopyridinium trifluoroacetate (CYTFA), and particularly preferred em bodim ents o f
detritylation reagents include solutions o f 2% (w/v) o f CPM or CY TFA in 20%
trifluoroethanol/D C M (v/v) containing 0.9% ethanol (v/v). As shown in Table I above,
use o f these reagents resulted in a significant increase in yield over the conventional
cyanoacetic acid reagents.
The m ore acidic CY TFA is found to be slightly m ore efficient than CPM . However,
much o f the increase in yield betw een the CPM and CY TFA reagents in the Table can be
attributed to the use o f a doubly protected guanine m onom er (DPG) in w hich the 0 6
position is protected with a 4-(pivaloyloxy)benzyloxy group, as disclosed in the co-owned
and concurrently filed provisional application entitled “ Improved Synthesis o f M orpholino
Oligom ers using Doubly Protected Guanine M orpholino Subunits” . In general, use o f the
DPG m onom er reduces the am ount o f diam inopurine-containing side products, while the
improved detritylation reagents reduce the am ount o f backbone hydrolyzed or truncated
side products.
Thus, this m odification provides a m ethod o f synthesizing a m orpholino oligom er
with reduced hydrolysis o f phosphorodiam idate linkages in the backbone, and preferably a
reduced or equivalent level o f N -I deletion species, relative to prior art m ethods. In
another aspect, the invention provides a m ethod o f deprotecting a triarylm ethyl-protected
m orpholino ring nitrogen during synthesis o f a m orpholino oligom er, with reduced
hydrolysis o f phosphorodiam idate linkages in the backbone o f the m orpholino oligom er
relative to that observed when cyanoacetic acid is used as the deprotecting reagent.
Preferably, the m ethod also provides a reduced or equivalent level o f N -T deletion species
than would be observed when cyanoacetic acid is used as the deprotecting reagent.
A useful further m odification is the use o f a trityl trapping agent, such as a thiol, to
shift the reaction equilibrium tow ards products. The use o f thiol trapping agents has been
employed for nucleic acid synthesis (Ravikum ar et al., U.S. Patent N o. 5,510,476).
M ercaptoethanol is a readily available, inexpensive agent useful for this purpose. The
presence o f the hydroxyl group is not critical for trapping, because sim ple thiols such as
benzylm ercaptan perform equally well. Alcohols, such as ethanol and butanol, and even
water also serve as trapping agents o f the trityl cation.
WO 2009/064471 PCT/US2008/012804
E xam ples
Exam ple I: Synthesis ofN 2 -P h A c. 06-P Q B D oubly Protected M orpholino G ( Ό Ρ Ο
Subunit (See Fig 8)
Preparation o f 3 (Starting with 35 kg o f lc): A 100 G reactor is charged with Ic (35
5 kg; 1.0 eq), im idazole (5.0 kg; 1.3 eq) and dichlorom ethane (279 kg). The batch is cooled
to 3°C. A 50 G reactor is cooled to 3°C and charged with t-butylchlorodim ethylsilane
(10.1 kg; 1.2 eq) and dichlorom ethane (93 kg). The solution in the 50 G reactor is
transferred to the 100 G reactor, and the batch is adjusted to 20°C. Upon reaction
com pletion (1-3 hours), m ethanol (1.8 kg; 1.0 eq) is charged to the 100 G reactor. A fter
10 30 minutes, the solution in the 100 G reactor is charged to a 200 G reactor containing pH 3
citrate buffer (376 kg o f I M citric acid adjusted to pH 3 with solid NaO H ). The batch is
agitated for 30 m inutes, and the layers are separated. The lower organic layer is washed
once m ore with pH 3 citrate buffer, and once with brine solution (287 kg o f 2.5%
N aC l/w ater (w:w)). The resulting organic solution is distilled at <35°C until Karl Fischer
15 analysis o f the batch shows < 0.05% water. This solution is cooled to 3°C in the 100 G
reactor and is used directly in the preparation o f compound 4.
Preparation o f 4 \ The 100 G reactor containing the solution o f com pound 3 is charged
with triethylam ine (6.8 kg; 1.2 eq), 4-dim ethylam inopyridine (0.68 kg; 0.1 eq), and
triisopropylbenzenesulfonyl chloride (18.6 kg; 1.1 eq). The batch is warmed to 20°C.
20 Upon reaction com pletion (3-9 hours), the solution is charged to a 200 G reactor
containing pH 4.5 phosphate buffer (228 kg o f I M KH2PO4). The batch is agitated for 30
minutes, and the layers are separated. The lower organic layer is w ashed with brine (212
kg o f 2.5% NaC 1/water (w:w)). The resulting organic solution is distilled at <35°C until
Karl Fischer analysis o f the batch shows < 0.01% water. This solution is cooled to 3°C in
25 the 100 G reactor and is used directly in the preparation o f com pound 5.
Preparation o f 4a (Starting with 60 kg o f 4-hydroxybenzaldehyde): A 750 G reactor
is charged with 4-hydroxybenzaldehyde (60 kg; 1.0 eq), toluene (260 kg), and 1-
methyl imidazole (8.1 kg; 0.2 eq). To this solution is charged a solution o f potassium
bicarbonate (100 kg; 2.0 eq) in w ater (400 kg), followed by trim ethylacetyI chloride (83
30 k g ;1 .4 e q ). This tw o-phase m ixture is agitated at 20°C. Upon reaction com pletion (1-5
hours), m ethanol (15.7 kg; 1.0 eq) is charged to the batch. The batch is agitated at 20°C
for I hour. The layers are separated. To the upper organic layer is charged w ater (200
kg). The batch is agitated for 30 m inutes, and the layers are separated. To the upper
organic layer is charged pH 4.5 phosphate buffer (16.5 kg KH2PO4 in 242 kg water). The
35 batch is agitated for 30 m inutes, and the layers are separated. To the upper organic layer is
19
WO 2009/064471 PCT/US2008/012804
charged water (200 kg). The batch is agitated for 30 m inutes, and the layers are separated.
The upper organic layer is distilled under vacuum at <30°C to achieve a batch volum e o f
200 L. TH F (70 kg) is charged to the batch, and the batch is transferred to a 500 G reactor
containing Pd/C (9.6 kg; 0.004 eq; 5% Pd/C, 50% wet Johnson M atthey Type A405028-5
or A570129-5). The reactor is initially pressurized to 5 psi H 2 with the agitation set at 50
rpm. Both the pressure and agitation rate are slowly increased as the reaction proceeds, to
a m axim um o f 25 psi H 2 and 90 rpm. Upon reaction com pletion (8 -4 8 hours), the batch is
filtered through a pad o f Celite followed by a 0.1 micron inline filter. The Celite is rinsed
with toluene (20 kg). To the batch is charged pH 6.5 phosphate buffer solution (2.7 kg
KH2PO4 and 2.3 kg potassium phosphate, dibasic, trihydrate in 200 kg w ater). The batch
is agitated for 30 m inutes, and the layers are separated. The upper organic layer is
distilled under vacuum at <30°C to achieve a batch volum e o f 140 L. Toluene (126 kg) is
charged to the batch, and the batch is distilled under vacuum at <30°C to achieve a batch
volume o f 140 L. The batch is adjusted to 20°C, and transferred to a 500 G reactor
containing n-heptane (821 kg) and seed crystals o f com pound 4a (100 gram s) held at 0°C.
The batch is held at 0°C for 1-2 hours. A second portion o f seed crystals (100 grams) is
added, and the batch is held at 0°C for 1-2 hours. Com pound 4a is isolated by filtration.
Yield = 70 - 80% from 4-hydroxybenzaldehyde.
The derivative in which the phenol m oiety is protected as its N ,N -dim ethylcarbam ate
instead o f the pivalate ester is m ade under conditions sim ilar to 4a. In order to push to
com pletion the reaction betw een 4-hydroxybenzaldehyde and dim ethylcarbam oyl
chloride, the reaction is perform ed in refluxing dichlorom ethane in the presence o f N-
m ethylim idazole as base and 0.2 eq DM AP as catalyst. . ....................
Preparation o f 5 : A 100 G reactor containing the solution o f com pound 4 is charged
with N -m ethylpyrrolidine (9.5 kg; 2.0 eq dissolved in 23 kg o f dichlorom ethane). A fter 10
m inutes, com pound 4a (14.0 kg; 1.2 eq) is added, followed by 1,8-
diazabicyclo[5.4.0]undec-7-ene (10.2 kg; 1.2 eq in 23 kg dichlorom ethane). T he batch is
warmed to 20°C. Upon reaction com pletion (1-9 hours), the solution is diluted with 327
kg o f dichlorom ethane and charged to a 200 G reactor containing pH 4.5 phosphate buffer
(334 kg o f I M KH2PO4). The batch is agitated for 30 m inutes, and the layers are
separated. The lower organic layer is washed once more w ith pH 4.5 phosphate buffer
(111 kg o f I M KH2PO4), then once with brine (212 kg o f 2.5% N aC l/w ater (w:w)). The
resulting organic solution is distilled at < 35°C until Karl Fischer analysis o f the batch
shows < 0.05% water. This solution is used directly in the preparation o f com pound If.
Preparation o f I f : A 100 G reactor containing the solution o f com pound 5 is charged
WO 2009/064471 PCT/US2008/012804
with triethylam ine trihydrofluoride (18.0 kg; 2.0 eq). The batch is agitated at 20°C. Upon
reaction com pletion (4-20 hours), the batch is charged to a 200 G reactor. The 200 G
reactor is charged with NaH COs solution (230 kg o f a 5% (w:w) solution). The batch is
agitated for 30 m inutes, and the layers are separated. The low er organic layer is washed
5 once m ore with NaHCOs solution (230 kg o f a 5% (w:w) solution), then once with pH 6.5
phosphate buffer (9.3 kg K H 2 PO 4 and 14.0 kg K 2 HPO 4 in 215 kg water). The resulting
organic solution undergoes solvent exchange to TH F (to achieve <1% DCM by w eight in
the batch). The solution is diluted with THF (124 kg) and heated to 60°C. W ater (8 kg
per kg o f com pound I f in solution based on LOE analysis; pre-heated to 60°C) is charged
10 slow ly to the THF solution. The solution is slowly cooled to 3°C and held for >4 hours.
Crude com pound I f is isolated by filtration. The crude m aterial is re-dissolved in THF
(342 kg) and heated to 60°C. W ater (315 kg; pre-heated to 60°C) is charged slowly to the
THF solution. The solution is cooled to 3°C and held for >4 hours. Com pound I f is
isolated by filtration. A second recrystallization can be perform ed to further purify
15 com pound I f if desired. Y ield = 53 - 73% from lc .
Preparation o/~2/TStarting with 12 kg o f If): A 50 G reactor is charged with
com pound I f (12 kg; 1.0 eq), dichlorom ethane (159 kg), 2,6-lutidine (2.5 kg; 1.6 eq) and
1-methylimidazole (0.36 kg; 0.3 eq). This solution is distilled to achieve a batch volume
o f 69 L, and cooled to 5°C. N , N -D im ethylphosphoram idodichloridate (3.8 kg; 1.6 eq) is
20 charged to the batch. The batch is adjusted to 20°C. Upon reaction com pletion (6-16
hours), toluene (78 kg) is charged to the batch. The resulting m ixture is distilled at 25°C
to achieve a batch volum e o f 126 L (GC analysis o f the batch m ust show 30-45% DCM
by weight), and transferred to a 100 G reactor containing pH 3 citrate buffer (15.4 kg citric
acid m onohydrate, 1.4 kg N aO H , 80 kg water). The batch is agitated for 10 m inutes, and
25 the layers are separated. The lower aqueous layer is sent to waste. T h e u p p e ro rg a n ic
layer is transferred to the 50 G reactor containing sodium sulfate (8.0 kg). The batch is
agitated for 30 minutes, and the sodium sulfate waste cake is removed by filtration. The
sodium sulfate cake is rinsed with dichlorom ethane (16 kg). The resulting product
solution is distilled in the 50 G reactor to achieve a batch volum e o f 53 L (GC analysis o f
30 the batch must show 11-15% DCM by weight). The 100 G reactor is charged with
heptane (238 kg). The batch in the 50 G reactor is transferred to the 100 G reactor over 2
hours. At the end o f the transfer, the batch is held at 20°C for 4 -1 6 hours. The crude
compound 6 is collected by filtration. The crude m aterial is charged to the 100 G reactor.
To the crude solids is added a solution o f toluene (16 kg) and heptane (50 kg). This
21
WO 2009/064471 PCT/US2008/012804
m ixture is agitated for 3 hours and filtered. The reslurry is repeated one or m ore tim es.
Yield o f crude 2 f = 80% from If.
Purification o f C om pound 2 f \sy Silica Gel Chrom atography (Starting with ~ 6.5 kg o f
crude com pound 2f): The “ strength” o f crude com pound 2 f is calculated by correcting the
5 w eight o f crude material for HPLC purity and volatiles. For this purification step, 5.75 kg
o f m aterial (corrected for strength) is used per injection on a 50 cm chrom atography
colum n. The 50 cm chrom atography colum n is packed with a slurry o f heptane/silica gel
(51.8 kg o f silica gel). The crude material is loaded onto the colum n as a solution in
dichlorom ethane/2,6-lutidine (15 kg dichlorom ethane, 0.16 kg 2,6-lutidine). The product
10 is eluted with a tw o-step gradient o f 4-m ethyl-2-pentanone (M IBK )/heptane/2,6-lutidine
(first step is 827 L o f 39:61 M IB K :heptane (w:w) with 0.06% 2,6-lutidine (w:w); second
step is 1343 L o f 73:27 M IB K :heptane (w:w) with 0.06% 2,6-lutidine (w:w)). The
approved fraction pool is concentrated via thin-film evaporation to a concentration o f 150
g/L. This concentrated pool is precipitated onto 6 volum es o f heptane. The purified 2 f is
15 isolated by filtration. Yield o f purified 2 f = 50% from If; 65% from crude 2f.
Exam ple 2. Preparation o f CY TFA Pyridinium Salt Detritylation Solution

T o a solution o f 4-cyanopyridine (10.1 g; 1.055 eq) in dichlorom ethane (790 mL) is
added trifluoroacetic acid (10.5 g; 1.0 eq) followed by 2,2,2-trifluoroethanol (198 mL) and
ethanol (10 mL) and the solution is stirred for 10-30 min.
20
Exam ple 3: P re p ara tio n o fD isu lfid e A n c h o r (See Fig. 10)
Preparation o fN -tritv l piperazine, succinate salt (NTP): To a cooled solution o f
piperazine (.10.eq) in toluene/m ethanol-(5:l toluene/m ethanol (v:v); 5 m L/g piperazine)
was added slowly a solution o f triphenylm ethyl (trityl) chloride (1.0 eq) in toluene (5
25 mL/g trityl chloride). Upon reaction completion ( 1 - 2 hr), this solution was w ashed four
tim es with water. To the resulting organic solution was added an aqueous solution o f
succinic acid (L I eq; 13 m L w ater/g succinic acid). This m ixture w as stirred for 90 min,
and the solid product was collected by filtration. The crude NTP was purified by two
reslurries in acetone. Yield = 70%.
30 Preparation o f sym m etrical disulfide 7: I, Γ -C arbonyldiim idazole (CDI) (12.402 g;
2.2 eq.) was suspended in dichlorom ethane (5.25 m L/g) and cooled on an ice bath.
Hydroxyethyl disulfide 6 (5.36 g; I eq.) was dissolved in dichlorom ethane (10 m L/g) and
tetrahydrofuran (I mL/g). The diol solution was added to the CDI slow ly such that the
tem perature o f the m ixture stayed below 4 °C for the duration o f the reaction. Upon
35 reaction com pletion (once addition was com plete), de-ionized w ater (93.8 pL, 0.15 eq.)
22
WO 2009/064471 PCT/US2008/012804
was added to quench the reaction. Independently, N-trityl piperazine, succinate salt (NTP)
(32.59 g; 2.1 eq.) was dissolved in toluene (8 m L/g NTP), dichlorom ethane (2 m L/g NTP),
and m ethanol (2 m L/g N TP). K2CO3 (22.09 g; 4.6 eq.) was dissolved in de-ionized w ater
(10 mL/g). The K2CO3 solution added to the solution o f NTP; the m ixture was stirred and
5 then separated into two layers. The cloudy organic layer was distilled to remove 90
grams; the resulting w ater droplets w ere separated and acetone (8 m L/g N TP) was added
to the organic layer. The solution o f CDI activated disulfide diol was added to the solution
o f the free base and concentrated to 225 mL. Acetone (10 m L/g NTP) w as added and the
m ixture was concentrated to 225 mL. The m ixture was heated to reflux and solid began
10 crystallizing out o f solution. Upon com pletion, the reaction m ixture was cooled and the
solid (7) was isolated by filtration. Yield: 27.92 g; 93.1% (based on w eight-based assay).
Preparation o f disulfide alcohol 8 : I (36.00 g; 32.1 m mol; I eq.) w as suspended in
acetone (2.8 mL/g 7). H ydroxyethyl disulfide (78.51 mL; 20 eq.) was added followed by
acetone (1.7 mL/g 7). 5% N aO H /m ethanol (2.85 mL; 0.1 eq.) was added; the pH o f the
15 m ixture was 10 by pH paper. Triphenylphosphine (8.42 g; I eq.) w as added followed by
acetone (1.1 mL/g 7). AU solids went into solution and then product began to crystallize
out. A fter sixteen hr, the reaction m ixture was neutralized w ith acetic acid (2.4 g; 0.2 eq.).
The crude product was isolated by filtration. The crude solid 8 was subjected to two
refluxing acetone reslurries (5 mL/g 7).
20 A fter filtration the crude product was suspended in dichlorom ethane (7.25 m L/g 7).
The m ixture was heated until a clear solution formed (35 °C). The solution was extracted
five tim es with an equal volume o f de-ionized water and the final organic layer was
concentrated to 155 mL. D ichlorom ethane was added (4.3 m L/g 7), and the solution was
again concentrated to 155 mL. CDI (9.17 g; 1.1 eq.) was added and the m ixture was
25 stirred at room tem perature. Upon reaction com pletion (~20 min) the reaction mixture
was washed twice with an equal volum e o f de-ionized water, then ethylbenzene (2.1 m L/g
7) was added. The solution was concentrated to 65.2 g, reducing the dichlorom ethane in
the solution to 0.17% , and stirred on an ice bath to crystallize the product. The product 9
was isolated by filtration. Yield: 44%.
30
Exam ple 4: T rieth v len eg lv co lT ail /See Fig. 11)
Preparation o f tritvl piperazine phenyl carbamate 10: To a cooled suspension o f N TP
in dichlorom ethane (6 mL/g NTP) was added a solution o f potassium carbonate (3.2 eq) in
w ater (4 mL/g potassium carbonate). To this tw o-phase m ixture was slow ly added a
35 solution o f phenyl chloroform ate (1.03 eq) in dichlorom ethane (2 g/g phenyl
23
WO 2009/064471 PCT/US2008/012804
chloroform ate). The reaction m ixture was warmed to 20 °C. Upon reaction com pletion
(1-2 hr), the layers were separated. The organic layer was w ashed with water, and dried
over anhydrous potassium carbonate. The product 10 w as isolated by crystallization from
acetonitrile. Yield = 80%
5 Preparation o f carbamate alcohol 11: Sodium hydride (1.2 eq) was suspended in 1-
m ethyl-2-pyrrolidinone (32 m L/g sodium hydride). To this suspension were added
triethylene glycol (10.0 eq) and com pound 10 (1.0 eq). The resulting slurry was heated to
95 °C. Upon reaction com pletion (1-2 hr), the m ixture was cooled to 20 °C. To this
m ixture was added 30% dichlorom ethane/m ethyl tert-butyl ether (v:v) and water. The
10 product-containing organic layer was w ashed successively with aqueous NaOH, aqueous
succinic acid, and saturated aqueous sodium chloride. The product 11 was isolated by
crystallization from dichlorom ethane/m ethyl tert-butyl ether/heptane. Yield = 90%.
Preparation o f Tail acid 12: To a solution o f com pound 11 in tetrahydrofuran (7 m L/g
11) was added succinic anhydride (2.0 eq) and DM AP (0.5 eq). The m ixture was heated
15 to 50 0C. Upon reaction com pletion (5 hr), the m ixture w as cooled to 20 0C and adjusted
to pH 8.5 with aqueous NaHCC>3 . M ethyl tert-butyl ether w as added, and the product was
extracted into the aqueous layer. D ichlorom ethane was added, and the m ixture was
adjusted to pH 3 with aqueous citric acid. The product-containing organic layer was
washed with a m ixture o f pH=3 citrate buffer and saturated aqueous sodium chloride.
20 This DCM solution o f 12 was used without isolation in the preparation o f com pound 13.
Preparation o f 13 : To the solution o f compound 12 was added N -hydroxy-5-
norbom ene-2,3-dicarboxylic acid imide (HONB) (1.02 eq), 4-dim ethylam inopyridine
(DM AP) (0.34 eq), and then l-(3-dim ethylam inoprqpyl)-N '-ethylcarbodiim ide
hydrochloride (EDC) (1.1 eq). The m ixture was heated to 55 0C. Upon reaction
25 completion (4-5 hr), the m ixture was cooled to 20 0C and w ashed successively with 1:1 0.2
M citric acid/brine and brine. The dichlorom ethane solution underw ent solvent exchange
to acetone and then to Ν ,Ν -dim ethylform am ide, and the product was isolated by
precipitation from a c eto n e /Ν ,Ν -dim ethylform am ide into saturated aqueous sodium
chloride. The crude product was reslurried several tim es in w ater to rem ove residual N,N -
30 dim ethylform am ide and salts. Yield = 70% o f 13 from com pound 11. Introduction o f the
activated “Tail” onto the disulfide anchor-resin was perform ed in N M P by the procedure
used for incorporation o f the subunits during solid phase synthesis.
24
WO 2009/064471 PCT/US2008/012804
Exam ple 5: Preparation o f the Solid Support for Synthesis o f M orpholino Oligom ers
Exam ple 5a: Preparation o f Am inom ethvlpolvstvrene-disulfide resin
This procedure was perform ed in a silanized, jacketed peptide vessel (custom made
by Chem Glass, NJ, USA) with a coarse porosity (40-60 pm ) glass frit, overhead stirrer,
5 and 3-way Teflon stopcock to allow N 2 to bubble up through the frit or a vacuum
extraction. Tem perature control w as achieved in the reaction vessel by a circulating water
bath.
The resin treatm ent/w ash steps in the following procedure consist o f two basic
operations: resin fluidization and solvent/solution extraction. For resin fluidization, the
10 stopcock was positioned to allow N 2 flow up through the frit and the specified resin
treatm ent/w ash was added to the reactor and allowed to perm eate and com pletely w et the
resin. M ixing was then started and the resin slurry mixed for the specified time. For
solvent/solution extraction, m ixing and N 2 flow were stopped and the vacuum pum p was
started and then the stopcock was positioned to allow evacuation o f resin treatm ent/w ash
15 to waste. AU resin treatm ent/w ash volum es were 15 m L/g o f resin unless noted otherwise.
To am inom ethylpolystyrene resin (100-200 mesh; -1 .0 m m ol/g N 2 substitution; 75 g,
I eq, Polym er Labs, UK, part #1464-X 799) in a silanized, jacketed peptide vessel was
added l-m ethyl-2-pyrrolidinone (N M P; 20 m l/g resin) and the resin was allow ed to swell
with m ixing for 1-2 hr. Follow ing evacuation o f the swell solvent, the resin w as washed
20 with dichlorom ethane (2 x 1-2 m in), 5% diisopropylethylam ine in 25%
isopropanol/dichlorom ethane (2 x 3-4 min) and dichlorom ethane (2 x 1-2 min). A fter
evacuation o f the final wash, the resin was fluidized with a solution o f disulfide anchor 9
in l-m ethyl-2-pyrrolidinone (0.17 M ; 15 mL/g resin, -2 .5 eq) and the resin/reagent
m ixture was heated at 45°C for 60 hr. On reaction completion, heating was discontinued
25 and the anchor solution was evacuated and the resin washed with l-m ethyl-2-
pyrrolidinone (4 x 3-4 min) and dichlorom ethane (6 x 1-2 m in). The resin was treated
with a solution o f 10% (v/v) diethyl dicarbonate in dichlorom ethane (16 mL/g; 2 x 5-6
min) and then washed with dichlorom ethane (6 x 1-2 min). The resin 14 w as dried under
a N 2 stream for 1-3 hr and then under vacuum to constant weight (± 2%). Yield: 110-
30 150% o f the original resin weight.
Example 5b: D eterm ination o f the L oadins o f Am inom ethvlpolvstvrene-clisulfide
resin
The loading o f the resin (num ber o f potentially available reactive sites) is determined
by a spectrom etric assay for the num ber o f triphenylm ethyl (trityl) groups per gram o f
35 resin.
25
WO 2009/064471 PCT/US2008/012804
A known weight o f dried resin (25 ± 3 mg) is transferred to a silanized 25 ml

volum etric flask and ~5 mL o f 2% (v/v) trifluoroacetic acid in dichlorom ethane is added.
The contents are m ixed by gentle swirling and then allowed to stand for 30 min. The
volume is brought up to 25 mL with additional 2% (v/v) trifluoroacetic acid in
5 dichlorom ethane and the contents thoroughly m ixed. Using a positive displacem ent
pipette, an aliquot o f the trityl-containing solution (500 pL) is transferred to a 10 mL
volum etric flask and the volum e brought up to 10 mL with m ethanesulfonic acid.
The trityl cation content in the final solution is m easured by UV absorbance at 431.7
nm and the resin loading calculated in trityl groups per gram resin (pm ol/g) using the
10 appropriate volumes, dilutions, extinction coefficient (ε: 41 p m o f'c m '1) and resin weight.
The assay is perform ed in triplicate and an average loading calculated.
The resin loading procedure in this exam ple will provide resin w ith a loading o f
approxim ately 500 pm ol/g. A loading o f 300-400 in pm ol/g w as obtained if the disulfide
anchor incorporation step is perform ed for 24 hr at room tem perature.
15
Example 5c: Tail loading (See Fig. 121
Using the same setup and volum es as for the preparation o f am inom ethylpolystyrene-
disulfide resin, the Tail can be introduced into the m olecule. For the coupling step, a
solution o f 13 (0.2 M) in N M P containing 4-ethylm orpholine (NEM , 0.4 M ) was used
20 instead o f the disulfide anchor solution. After 2 hr at 45 0C, the resin 15 was washed
tw ice with 5% diisopropylethylam ine in 25% isopropanol/dichlorom ethane and once with
DCM . To the resin was added a solution o f benzoic anhydride (0.4 M ) and NEM (0.4 M).
A fter 25 min, the reactor jack et was cooled to room tem perature, and the resin w ashed
tw ice with 5% diisOpropyrethylamine in 25% isopropanol/dichlorom ethane and eight
25 tim es with DCM . The resin 15 was filtered and dried under high vacuum . The loading for
resin 15 is defined to be the loading o f the original am inom ethylpolystyrene-disulfide
resin 14 used in the Tail loading.
Exam ple 6: Synthesis o f M orpholino Oligom ers

30 Example 6a: S o lid Phase Synthesis
Protected oligom ers were prepared m anually by solid phase oligom er synthesis on
am inom ethylpolystyrene-disulfide resin (~500 μιτιοΐ/g loading) at 10 g scale (starting resin

weight). Solutions used were as follows:
Detritylation solutions: CA A = 11% Cyanoacetic acid (w/w) in a m ixture o f 20%
35 acetonitrile/D CM (v/v);
CPM = 2% 3-Chloropyridinum m ethanesulfonate (w/v) and 0.9% ethanol (v/v) in
26
WO 2009/064471 PCT/US2008/012804
20% trifluoroethanol/D C M (v/v);

CYTFA = 2% 3-Cyanopyridinum trifluoroacetate (w/v) and 0.9% ethanol (v/v) in
20% trifluoroethanol/D C M (v/v).
Neutralization solution: 5% diisopropylethylam ine in 25%
isopropanol/dichlorom ethane;
Coupling solutions: 0.165 M (for 2 f (DPG), 2c, and 2d or other T subunits) or 0.18 M
(for 2a and 2b or other A/C subunits) activated M orpholino Subunit and 0.4 M N-
ethylm orpholine in 1,3-dim ethylim idazolidinone (DMI).
Activated M PG (2c) w as prepared as in Sum merton et al. (1993).
A fter transfer o f the resin to the synthesis reactor and prior to initiating synthesis
cycles, l-m ethyl-2-pyrrolidinone (NMP, 20 m L/g resin) was added and allow ed to sit for
1-2 hrs. A fter w ashing 2 tim es with dichlorom ethane (10 m L/g resin), the following
synthesis cycle was used w ith addition o f the appropriate coupling solution o f activated
M orpholino Subunit o f the desired base and desired linkage type at each cycle to give the
proper sequence.
Step V olum e (m L /g o f s ta rtin g resin)* T im e (m in)

DCM 10-30 1-2
DCM 10-30 1-2
Detritylation A 10-30 2-3
Neutralization A 10-30 3-4
N eutralization A 10-30 3-4
DCM 10-30 1-2
DCM 10-30 1-2
Coupling 7-1 2 " 90
DCM 10-30 1-2
* W ash volum es are incremented to account for resin swelling; volum e is 10
m L/g o f actual resin volum e at each cycle
** Coupling volum es are sufficient to m aintain good m ixing and are
incremented to account for resin swelling
After incorporation o f the final subunit, a final cycle (m ethoxytritylation) was

performed with 0.32 M 4-m ethoxytriphenylm ethyl chloride and 0.4 M N-ethylm orpholine
WO 2009/064471 PCT/US2008/012804
in D M I. A fter m ethoxytritylation, the resin was washed 8 tim es with N M P and then
treated w ith cleavage solution consisting o f 0.1 M 1,4-dithiothreitol (DTT) and 0.73 M
triethylam ine in N M P (27 m L/g starting resin) for 30 m in. A fter collection o f the
protected oligom er solution, the resin (significantly reduced in volum e) was w ashed with
two additional portions o f cleavage solution (13 mL/g starting resin for 15 min each) and
the w ashes were com bined w ith the bulk solution. To the protected oligom er solution in
an appropriately sized pressure bottle with Teflon plug (Ace Glass, N J, USA) was added
concentrated aqueous am m onia (106 mL/g starting resin, previously cooled to -20°C), the
bottle sealed, and the contents m ixed by swirling. The bottle was placed in a 45°C oven
for 16-20 hr to rem ove base and backbone protecting groups.
Follow ing am m onolysis, the crude oligom er solution is cooled to room tem perature
and then diafiltered against 0.28% aqueous am m onia using a PLBC 3kd Regenerated
Cellulose m em brane (M illipore) to remove solvents and small m olecules prior to ion
exchange chrom atography.
Exam ple 6b: Purification o f M orpholino O lisom ers by Anion E xchanse
Chrom atozraphv
The crude oligom er solution obtained from diafiltration is adjusted to pH 11-11.5 and
loaded onto a colum n o f ToyoPearl Super-Q 650S anion exchange resin (Tosoh
Bioscience). The m ethoxytritylated oligom er is eluted with a gradient o f 5-35% B over 17
column volum e (Buffer A: 10 mM sodium hydroxide; Buffer B: I M sodium chloride in
10 mM sodium hydroxide) and fractions o f acceptable purity (anion exchange HPLC and
mass spec) pooled.

Exam ple 6c: D em ethoxvtritvlation o f M orpholino O lisom ers
To the pooled fractions from anion exchange chrom atography is added acetonitrile
(10% by volum e) followed by 2 M H3PO4 to adjust the pH to 3. The solution is m ixed for
45 min and then neutralized w ith concentrated aqueous am m onia to pH 7. The oligom er
solution is diafiltered against 20 m M sodium acetate using a PLBC 3kd Regenerated
Cellulose m em brane (M illipore) to exchange buffers prior to cation exchange
chrom atography.
Exam ple 6d: Purification o f M orpholino O lisom ers by Cation Exchanse
C hrom atosraphv
The oligom er solution is adjusted to pH 4.5 with acetic acid and loaded onto a column
o f Source 30S cation exchange resin (GE Healthcare). The oligom er is eluted with a
gradient o f 0-35% B over 17 colum n volum es (Buffer A: 20 mM sodium acetate, 25%
acetonitrile, pH 4.5; Buffer B: 0.5 M sodium chloride, 20 mM sodium acetate, 25%
WO 2009/064471 PCT/US2008/012804
acetonitrile, pH 4.5) and fractions o f acceptable purity (cation exchange HPLC and mass
spec) pooled.
29
WO 2009/064471 PCT/US2008/012804
CLAIM S
I. A m orpholino com pound com prising the structure

O
5 wherein
phenyl;
R2 is selected from the group consisting o f lower alkyl, m onocyclic arylm ethyl, and
10 R3 is selected from the group consisting o f triarylm ethyl and hydrogen; and
amino group; a chlorophosphoram idate group; and a phosphorodiam idate linkage to the
ring nitrogen o f a further m orpholino com pound or a m orpholino oligom er.
15 2. The com pound o f claim I, w herein Y is selected from the group consisting o f a
protected or unprotected hydroxyl group and a chlorophosphoram idate group.
3. The com pound o f claim 2, w herein Y is a trialky lsily 1-protected hydroxyl or

unprotected hydroxyl group.
20
4. The com pound o f claim 2, w herein Y is a chlorophosphoram idate group o f the form
-0 -P (= 0 )-N (C H 3)2Cl.
5. The com pound o f claim I, wherein R3 is selected from trityl (triphenylmethyl),

25 4-m ethoxytrityl, 4-m ethyltrityl, 4,4'-dim ethyltrityI, and 4,4',4"-trim ethyltrityl.
6. The com pound o f claim I, wherein R 1 is lower alkyl.
30
WO 2009/064471 PCT/US2008/012804
7. The com pound o f claim 6, w herein R 1 is -C(CHs)3 (tert-butyl).
8. The com pound o f claim I, w herein R2 is benzyl or -C H (C H 3)2.
5 9. A m ethod o f synthesizing a m orpholino oligom er, the m ethod comprising:

thereby form ing a phosphorodiam idate linkage between said 5’-exocyclic carbon and said
10 unprotected ring nitrogen;
(b) deprotecting said protected ring nitrogen, to form an unprotected ring nitrogen;
and
15 w herein at least one o f said base-protected m orpholino subunit m onom ers is a doubly
protected guanine m orpholino com pound having the structure:
O
wherein
R 1 is selected from the group consisting o f lower alkyl, di(Iower alkyl)am ino, and
20 phenyl;
R2 is selected from the group consisting o f lower alkyl, m onocyclic arylmethyl, and
R3 is selected from the group consisting o f triarylm ethyl and hydrogen; and
25
10. The method o f claim 9, w herein Y is a chlorophosphoram idate group o f the form
-O-P(sO )-N (C H 3)2Cl.
31
WO 2009/064471 PCT/US2008/012804
11. The method o f claim 9, w herein R3 is selected from trityl (triphenylm ethyl),
4-methoxytrityl, 4-m ethyltrityl, 4,4'-dim ethyltrityl, and 4,4',4"-trim ethyltrityl.
12. The method o f claim 9, w herein R 1 is lower alkyl.
5
13. The method o f claim 12, w herein R 1 is -C(CH 3 )3 (tert-butyl).
14. The method o f claim 9, w herein R2 is benzyl or -CH (CH 3)2.
10 15. The method o f claim 9, w herein said deprotecting o f step (b) com prises exposing said
triarylm ethyl-protected ring nitrogen to a reagent solution com prising a heterocyclic amine
salt in a trifluoroethanol-containing solvent, said salt being a salt o f a heterocyclic amine,
having a pKa in the range o f 1-4 in its protonated form , with an acid selected from a
sulfonic acid, trifluoroacetic acid, and hydrochloric acid.
15
16. The method o f claim 15, w herein said salt is selected from 3-chloropyridinium
m ethanesulfonate (CPM ) and 4-cyanopyridinium trifluoroacetate (CYTFA).
17. The method o f claim 15, w herein said solvent com prises dichlorom ethane and
20 trifluoroethanol in volum e ratio in the range o f about 90:10 to 25:75.
18. The method o f claim 17, w herein said volum e ratio is about 80:20.
19. A method o f synthesizing a m orpholino oligom er, the method comprising:

25 (a) reacting a solid-phase-supported m orpholino subunit, having an unprotected ring
thereby form ing a phosphorodiam idate linkage between said 5 ’-exocyclic carbon and said
unprotected ring nitrogen;
30 (b) deprotecting said protected ring nitrogen, to form an unprotected ring nitrogen;
and
wherein said deprotecting com prises exposing said triarylm ethyl-protected ring
35 nitrogen to a reagent solution com prising a heterocyclic am ine salt in a trifluoroethanol-
32
WO 2009/064471 PCT/US2008/012804
containing solvent, said salt being a salt o f a heterocyclic amine, having a pKa in the range
o f 1-4 in its protonated form, with an acid selected from a sulfonic acid, trifluoroacetic
acid, and hydrochloric acid.
5 20. The m ethod o f claim 19, w herein said heterocyclic am ine is selected from the group
consisting o f an electron w ithdraw ing group-substituted pyridine, thiazole, pyridazine,
pyrazole, triazole and electron w ithdraw ing group-substituted substituted derivatives
thereof.
10 21. The method o f claim 20, w herein said heterocyclic am ine is an electron w ithdraw ing
group-substituted pyridine.
22. The m ethod o f claim 20, w herein said electron withdrawing group is selected from the
group consisting o f halogen, cyano, aldehyde, keto, carboxyester, and carboxam ide.
15
23. The method o f claim 21, w herein said heterocyclic am ine is a chloro- or cyano-
substituted pyridine.
24. The method o f claim 19, w herein said salt is a salt o f a sulfonic acid, selected from an
20 alkylsulfonate, a (fluoroalkyl)sulfonate, and a p-toluenesulfonate, or a trifluoroacetate.
25. The method o f claim 23, w herein said salt is selected from 3-chloropyridinium
m ethanesulfonate (CPM ) and 4-cyanopyridinjum trifluoroacetate (CYTFA).
25 26. The method o f claim 19, w herein said solvent com prises dichlorom ethane and
trifluoroethanol in volum e ratio in the range o f about 90:10 to 25:75.
27. The method o f claim 26, w herein said volum e ratio is about 80:20.
30 28. The method o f claim 19, w herein said triarylm ethyl is selected from the group
consisting o f trityl (triphenylm ethyl), 4-m ethoxytrityl, 4-methyltrityl, 4,4'-dim ethyltrityl,
and 4,4',4"-trim ethyltrityl.
33
WO 2009/064471 PCT/US2008/012804
Sheet I of 12
HO Me2N - P - O
1. 2,6-lutidine/DCM
Cl
2. N -methylim idazole
3. N ,N -dim eth ylph osp ho ram ido -
/ \ dichloridate
/ \
\ / W
la - f, where Pi: 2a- f
a = f=
HN
NH
O
b=
HN
α λ λ Ι α /v
c=
NH
N
H
I ^ nh
N ^O
yw nJvruv
e =
(
N 'N
^Λ/vK/V'
Figure I
WO 2009/064471 PCT/US2008/012804
Sheet 2 o f 12
NH
Si-O NH
Im id a z o l e
TBDMS-CI
D ichlorom ethane
NO;
T riphenylphosphine
DIAD
4 - n i t r o p h e n e t h y l a lc o h o l
Si-O NH
I
Tr
NO;
T r ie th y la m in e - 3 HF
HO NH
I
Tr
D ich lo ro m eth an e
2 ,6 - lu ti d in e
N - m e th y lim id a z o le
N ,N -d im e th y lp h o sp h o ra m id o d ic h lo rid a te
Figure 2
WO 2009/064471 PCT/US2008/012804
Sheet 3 of 12
I
NH O NH O
< <
HO N NH -Si-O N NH
I m id a z o le
N TBDMS-CI N
I
I
Tr Ic D ic h l o r o m e t h a n e Tr
TIPS-CI N -m e th y lp y rr o lid in e ; DBU
T r ie th y la m in e 4 - n i t r o p h e n e t h y l alcohol
DMAP
Si-O NH
I
Tr
NO2 NO·
Si-O NH HO NH
NO·
D ic h lo r o m e t h a n e
2 ,6 - lu t id in e
N ,l\ l- d i m e th y lp h o s p h o r a m id o d i c h lo r id a t e
C l-P -O NH
Me2N
I
Tr
Figure 3
WO 2009/064471 PCT/US2008/012804
Sheet 4 of 12
I
NH O
<
-Si-O- N '^ N H "
t ip s - ci
Triethylamine
DMAP
-Si-O N 'NH
O
Oxone™
N-methylpyrrolidine; DBU <
-Si-O N ^ 'NH
HO 'R
w h e re R = m ethyl; phenyl
I w h e re R = methyl;
Tr phenyl
O
Il
O
^ Il5—R
/x ^ S -R O
O
r
N O
s^N O </N
<f A H0^
N N '^ N H '
-Si-O N NH
Triethylamine-3HF Iv w h e re R = m ethyl; phenyl
N
N I
Tr
I
Tr
w h e re R = m ethyl; phenyl
O
O ^ S -R
D ichlorom ethane I’
2,6-lutidine ^ n 0 O
N-methylimidazole O
Ii
< A
N,N-dim ethylphosphoram idodichloridate C l-P -O N NH
Me2N
^ w h e re R = methyl; phenyl
Tr
Figure 4
WO 2009/064471 PCT/US2008/012804
Sheet 5 o f 12
NH NH
HO NH PhAc— O NH
N -m ethylim idazole/D CM
P h e nylacetyl chloride
TIPS-CI N -m ethylpy rro lidine; DBU
T rie th y la m in e 2-(T rim ethy lsilyl)eth ano l

DMAP
PhAc— O NH
,Si(CH3)3 ,Si(CH3)3
PhA c-O NH HO NH
T e tr a m e th y lg u a n id in e
Ethanol/DCM
Tr Tr
,Si(CH3)3
D ic h lo r o m e th a n e
2,6 -lu tid in e
N -m eth ylim id azole
N ,N - d im e th y lp h o sp h o ra m id o d ic h lo rid a te
C l-P -O - NH
Me2N
I
Tr
Figure 5
WO 2009/064471 PCT/US2008/012804
Sheet 6 of 12
NH NH
HO NH Si-O NH
I m id a z o le
TBDMS-CI
D ic h l o r o m e t h a n e 3
N - m e th y lp y r r o lid i n e ; DBU
TIPS-C l
T riethylam ine
DMAP
HO
Si-O NH
Where H1- H5 can be substituted
with various electron-withdrawing
groups
Tr
N 'NH
-Si-O N 'NH
T r i e t h y l a m i n e - 3 HF
D ich lo ro m eth an e
2 ,6 - lu ti d i n e
N ,N -d im eth y lp h o sp h o ram id o d ich lo rid ate
C l-P -O N 'NH
Figure 6
WO 2009/064471 PCT/US2008/012804
Sheet 7 of 12
NH
Si-O NH
Si-O NH
DIEA/Pyridine/DMF
N
O
'N
Ph
O
O"^ NR2
N O
< Λ
HO N NH
N
I
Tr
D ichlorom ethane
2 ,6 - lu ti d in e C l-P -O NH
Me2N
N - m e th y l im id a z o le
N ,N -dim ethylphosphoram idodichloridate
Figure 7
WO 2009/064471 PCT/US2008/012804
Sheet 8 of 12
Γ I
NH O NH O
< <
HO N NH -S i-O N^ 'N H '
x
Imidazole
TBDMS-CI
Tr Ic Dichloromethane
N-methylpyrrolidine; DBU
TIPS-CI
Triethylamine
DMAP
HO
S i-O NH
4a
I
Tr
o \ /r°
-S i-O
Cfo S N NH
HO
r
<
^N
Λ
N NH
O
Triethylamine - 3 HF
If
Dichloromethane
2,6-lutidine
N-methylimidazole
N.N-dimethvlDhosphoramidodichloridate
M e2N
Tr = \ //
Figure 8
WO 2009/064471 PCT/US2008/012804
Sheet 9 o f 12
I !-I Is-I
Vr" N
I^NMe2
1X NT '
| .,NIVIe2
O ^ pS O ^ pS
Carboxylic Acid
y r <
S PS Detritylation
P4
M e2N ' ' 0 H O ^^P
PDA
/ V
gro up
Amidate
/ 1
g ro u p
V
[•■I [*·]
Figure 9
WO 2009/064471 PCT/US2008/012804
Sheet IO of 12
O
1. Carbonyldiimidazole .A, „s.
6 2. NTP Free Base
NaOH, 6
Triphenylphosphine
S •OH
I , I - C a r b o n y ld iim id a z o le
O A '\
N
"Λ
N
JJ
T)
Zx y S ^ O v
T Nx /
V / O
Figure 10
WO 2009/064471 PCT/US2008/012804
S h e e t ll o f 12
Phenyl Chloroformate
K2CO3 w
\ /
-N
®
NH2
DCM/H20
-N A,
N O
\ /
10
HO.
&
NaH/NMP
O Triethylene glycol
95 C
r\ -N N Po
.OH
ll
Succinic anhydride/DMAP
THF; 55 C
HO-N
EDC
DMAP
DCM; reflux
O— N
Figure 11
WO 2009/064471 PCT/US2008/012804
Sheet 12 of 12
o
Il
o
Il
n< ^ n^ o^ ss^ o^ n^ ss ^
NH2 k __ _ NTrityl N
NTrityI
NH2 NH C O O E t 14
Aminomethyl
polystyrene resin
O k NTrityI
13
O O
N ^ O ^ — SS— N ^ 1 o
H I I, U
NH COOEt
is ° r^ °vO
J V ^ , N Trityl
Figure 12
5399260vl
INTERNATIONAL SEARCH REPORT International a p p lica tio n N o
PCT/US2008/012804
A. CLASSIFICATION OF SUBJEC T MATTEFi
INV. C07D473/18 A61K31/70
A ccording Io International P a te n t C lassification (IPC) o r Io both national classification a n d IPC
B. FIELDS SEARCHED_________________________________________________________________
M inimum docum entation s e a r c h e d (classification s y s te m followed by classification sym bols)
C07D A61K
D ocum entation s e a rc h e d o th e r th a n m inim um d o cu m en tatio n to th e ex ten t th a t s u c h d o c u m e n ts a r e in clu d ed In th e fields s e a r c h e d
E lectronic d a ta b a s e co n su lted during th e in tern atio n al s e a r c h (n am e of d a ta b a s e a n d , w h e re practical, s e a r c h te rm s u se d )
E P O - I n t e r n a I , EMBASE, CHEM ABS D a ta , BEILSTEIN D a ta
C. DOCUMENTS CONSIDERED TO BE RELEVANT
C ategory* Citation of d o cu m en t, with indication, w h e re ap p ro p riate, of th e relev an t p a s s a g e s R elev an t to claim No.
US 5 185 444 A (SUMMERTON JAMES E [US] ET 1 ,9 ,1 9

AL) 9 F e b r u a r y 1993 ( 1 9 9 3 - 0 2 - 0 9 )
c i t e d in t h e a p p l i c a t i o n
c l a i m 10; e x a m p le s 1 2 ,1 3
SUMMERTON ET AL: " M o rp h o lin o a n t i s e n s e

o l i g o m e r s : d e s i g n , p r e p a r a t i o n , and
p ro p e rtie s"
SENSE & NUCLEIC ACID DRUG DEVELOPMENT,
MARY ANN LIEBERT, IN C ., NEW YORK, US,
v o l . 7 , I J a n u a r y 1997 ( 1 9 9 7 - 0 1 - 0 1 ) , p a g e s
1 8 7 -1 9 5 , XP002136176
ISSN: 1 0 8 7-29 06
c i t e d in t h e a p p l i c a t i o n
p ag e 189; f i g u r e 5
- / -
F u rth er do c u m e n ts a r e listed in th e continuation of Box C. S e e p a te n t family an n e x .
* S p e c ia l ca te g o rie s of cited d o c u m e n ts :
la ter d o c u m e n t p u b lish e d afte r th e in ternational filing d a te
o r priority d a te a n d not in conflict with th e application but
'A ' d o c u m e n t defining th e g e n e ra l s ta te of th e art w hich is not
cited to u n d e rs ta n d th e principle o r th e o ry underlying th e
co n s id e re d to b e of particular re le v a n c e invention
'E ' ea rlie r d o cum ent but pu b lish ed on o r a fte r th e international d o c u m e n t o f particu lar relev an ce ; th e claim ed invention
filing d ate ca n n o t b e c o n s id e re d novel o r c a n n o t b e co n s id e re d to
■L‘ d o c u m e n t which m a y th ro w d o u b ts o n priority claim (s) or involve a n inventive s te p w h en th e d o c u m e n t is ta k en alo n e
w hich is cited to esta b lish th e publication d a te of a n o th e r d o c u m e n t o f particu lar relev an ce ; th e claim ed invention
citation o r o th e r sp e c ia l re a s o n (a s sp ecified )
ca n n o t b e c o n s id e re d to involve a n inventive s te p w h en th e
■O' d o c u m e n t referring to an oral d isclo su re , u s e , exhibition o r d o c u m e n t is co m b in ed with o n e o r m o re o th er s u c h d o c u
o th e r m e a n s m en ts, s u c h com b in atio n b ein g o b v io u s to a p erso n skilled
■p* d o c u m e n t published p rio rto th e in tern atio n al filing d a te but in th e art.
la te r th a n th e priority d a te claim ed '&■ d o c u m e n t m e m b e r of th e s a m e p a te n t family
D a te of th e a c tu a l com pletion of th e international s e a rc h D ate of m ailing of th e in tern atio n al s e a r c h report
5 F e b r u a r y 2009 0 7 /0 4 /2 0 0 9
N am e a n d mailing a d d r e s s of th e ISA/ A u thorized officer
E u ro p ean P a te n t Office, P .B . 5 8 1 8 P a te n tla a n 2
N L - 2 2 8 0 HV RiJswijk
Tel. (+ 3 1 -7 0 ) 3 4 0 -2 0 4 0 ,
Fax: (+ 3 1 -7 0 ) 3 4 0 -3 0 1 6 S k u lj , P rim oz
Fomn PCT/lSA/210 (second sh e et) (April 20Q5)
INTERNATIONAL SEARCH REPORT
International a p p lica tio n N o
PCT/US2008/012804
C (C on tin u atlon ). DOCUM ENTS CONSIDERED TO BE RELEVANT
C ategory* Citation of d o cu m en t, with indication, w h e re a p p ro p riate, of th e relev an t p a s s a g e s R elev an t to claim No.
US 2 0 0 7 /1 3 5 3 3 3 Al (GELLER BRUCE L [US] ET

AL) 14 J u n e 2007 ( 2 0 0 7 - 0 6 - 1 4 )
p a ra g ra p h [0034]
CASTRO M J L ET AL: "Gemini s u r f a c t a n t s 19

from a l k y l g l u c o s i d e s "
TETRAHEDRON LETTERS 19970609 GB,
v o l . 3 8 , n o . 2 3 , 9 J u n e 1997 ( 1 9 9 7 - 0 6 - 0 9 ) ,
p a g e s 3 9 9 5 - 3 9 9 8 , XP002513601
ISSN: 0 0 4 0 -4 0 3 9
ABRAMOVA T V ET AL: "S y n th esis of

m o rp h o lin e n u c le o s id e t r i p h o s p h a t e s "
TETRAHEDRON LETTERS, ELSEVIER, AMSTERDAM,
v o l . 4 5 , n o . 2 2 , 24 May 2004 ( 2 0 0 4 - 0 5 - 2 4 ) ,
p a g e s 4 3 6 1 - 4 3 6 4 , XP004506361
ISSN: 0 0 4 0 -4 0 3 9
ABRAMOVA TATIANA V ET AL: "New

o l i g o n u c l e o t i d e a n a l o g u e s b a s e d on
m o r p h o l i n e s u b u n i t s j o i n e d by o x a l y l
d i am ide t e t h e r . "
BI00RGANIC CHEMISTRY JUN 200 7,
v o l . 35, n o . 3 , J u n e 2007 ( 2 0 0 7 - 0 6 ) , p a g e s
2 5 8 -2 7 5 , XP002513602
ISSN: 0 0 4 5 -2 0 6 8
P,x WO 2 0 0 8 /0 0 8 1 1 3 A (AVI BIOPHARMA INC [U S ]; 1 -3 ,5 -8

WELLER DWIGHT D [U S ]; GELLER BRUCE L [U S ];
IVE) 17 J a n u a r y 2008 ( 2 0 0 8 - 0 1 - 1 7 )
t h e w h ole docum ent
f i g u r e s 2 K ,(1 2 /3 7 )
p,x WO 2 0 0 8 /0 3 6 1 2 7 A (AVI BIOPHARMA INC [U S ]; 1 9 -2 8

WELLER DWIGHT D [U S ]; HASSINGER JED N
[U S ]) 27 March 2008 ( 2 0 0 8 - 0 3 - 2 7 )
t h e w h o le docum ent
ex a m p le 13
Form PCT/ISA/210 (continuation of s e c o n d sh e et) (April 2005)

INTERNATIONAL SEARCH REPORT
International a p p lica tio n N o
Inform ation o n p aten t fam ily m em b ers
PCT/US2008/012804
P a ten t d o c u m en t Publication P a ten t fam ily Publication
cited in s e a r c h report date m em b er(s) date
US 5185444 A 0 9 -0 2 -1 9 9 3 NONE
US 200 7135333 Al 14 -0 6 -2 0 0 7 AU 2006267051 Al 18-01-2007

CA 2614191 Al 18-01-2007
EP 1915161 A2 30-04 -2 0 0 8
WO 2007009094 A2 18-01-2007
WO 20 08008113 A 1 7 -0 1 -2 0 0 8 US 2008194463 Al 14-08-2008
WO 20 08036127 A 27-03-2008 NONE
Form PCT/ISA/210 (patent family annex) (April 20D5)

SNAr prep

Uploaded by

Copyright:

Available Formats

You might also like

SNAr prep

Uploaded by

Document Information

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

SNAr prep

Uploaded by

Copyright:

Available Formats

(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)

(19) World Intellectual Property Organization WO 2009/064471 A1

(43) International Publication Date ρ^πτ d*·) International Publication Number

(54) Title: METHOD OF SYNTHESIS OF MORPHOLINO OLIGOMERS

M ETH O D OF SY N TH ESIS O F M O R PH O LIN O O LIG O M ER S

Field o f the Invention

synthesized using m onoprotected guanine subunits and/or conventional ring nitrogen

Academ ic Press, pp 44 (1963).

4-methyltrityl, 4,4'-dim ethyltrityl, and 4,4',4"-trim ethyltrityl. The group R 1 is preferably

(a) reacting a solid-phase-supported m orpholino subunit, having an unprotected ring

D etailed D escription o f th e In v en tio n

In a thiophosphorodiamidate linkage, one oxygen atom, typically an oxygen pendant to

II. Base Protection in PM O Synthesis

et al. 1982A, 1982B), as shown in Figure 4. H ow ever, no successful crystallization

4126 I A C G TTG A G G G G C A TC G TC G C CAA 2c 54 gJ 18%

4126 I A C G TTG A G G G G C A TC G TC G C CPM 2c 25 $ 25%

4126 I A C G TTG A G G G G C A TC G TC G C CYTFA 2 f 25 g 49%

1CAA = 11% Cyanoacetic acid (w/w) in a m ixture o f 20% acetonitrile/D CM (v/v);

10 CPM = 2% 3-Chloropyridinum m ethanesulfonate (w/v) and 0.9% ethanol (v/v) in 20%

Com bined output o f 4x12 g and 1x8 g runs.

15 4Com bined output o f 2x12 g runs.

5Com bined output o f 4x12 g runs.

6Addition o f the final C subunit w as performed with an activated m orpholino C subunit

with 4-m ethoxytrityl protection on the m orpholino nitrogen.

m onoprotected M oG m onom er, or other protected M oG m onom er not o f the invention, is

-C(CH 3 )3 (tert-butyl), as in the 4-(pivaloyloxy)benzyloxy (POB) group. However, R 1can

where R is lower alkyl, such as methyl or ethyl;

IV. Im proved Conditions for Deprotection o f the M orpholino Ring N itrogen in PM O

Tw o particularly preferred salts are 3-chloropyridinium m ethanesulfonate (CPM ) and

Exam ple 2. Preparation o f CY TFA Pyridinium Salt Detritylation Solution

A known weight o f dried resin (25 ± 3 mg) is transferred to a silanized 25 ml

Exam ple 6: Synthesis o f M orpholino Oligom ers

am inom ethylpolystyrene-disulfide resin (~500 μιτιοΐ/g loading) at 10 g scale (starting resin

20% trifluoroethanol/D C M (v/v);

Step V olum e (m L /g o f s ta rtin g resin)* T im e (m in)

After incorporation o f the final subunit, a final cycle (m ethoxytritylation) was

mass spec) pooled.

I. A m orpholino com pound com prising the structure

3. The com pound o f claim 2, w herein Y is a trialky lsily 1-protected hydroxyl or

5. The com pound o f claim I, wherein R3 is selected from trityl (triphenylmethyl),

6. The com pound o f claim I, wherein R 1 is lower alkyl.

7. The com pound o f claim 6, w herein R 1 is -C(CHs)3 (tert-butyl).

8. The com pound o f claim I, w herein R2 is benzyl or -C H (C H 3)2.

5 9. A m ethod o f synthesizing a m orpholino oligom er, the m ethod comprising:

12. The method o f claim 9, w herein R 1 is lower alkyl.

14. The method o f claim 9, w herein R2 is benzyl or -CH (CH 3)2.

19. A method o f synthesizing a m orpholino oligom er, the method comprising:

TIPS-CI N -m e th y lp y rr o lid in e ; DBU

TIPS-CI N -m ethylpy rro lidine; DBU

T rie th y la m in e 2-(T rim ethy lsilyl)eth ano l

6 2. NTP Free Base

A ccording Io International P a te n t C lassification (IPC) o r Io both national classification a n d IPC

D ocum entation s e a rc h e d o th e r th a n m inim um d o cu m en tatio n to th e ex ten t th a t s u c h d o c u m e n ts a r e in clu d ed In th e fields s e a r c h e d

E lectronic d a ta b a s e co n su lted during th e in tern atio n al s e a r c h (n am e of d a ta b a s e a n d , w h e re practical, s e a r c h te rm s u se d )

E P O - I n t e r n a I , EMBASE, CHEM ABS D a ta , BEILSTEIN D a ta

C. DOCUMENTS CONSIDERED TO BE RELEVANT

C ategory* Citation of d o cu m en t, with indication, w h e re ap p ro p riate, of th e relev an t p a s s a g e s R elev an t to claim No.

US 5 185 444 A (SUMMERTON JAMES E [US] ET 1 ,9 ,1 9

SUMMERTON ET AL: " M o rp h o lin o a n t i s e n s e