Biosynthesis of Alkaloids

Author(s): Edward Leete

Source: Science , Feb. 26, 1965, New Series, Vol. 147, No. 3661 (Feb. 26, 1965), pp. 1000-
1006
Published by: American Association for the Advancement of Science

Stable URL: https://www.jstor.org/stable/1715211

REFERENCES
Linked references are available on JSTOR for this article:
https://www.jstor.org/stable/1715211?seq=1&cid=pdf-
reference#references_tab_contents
You may need to log in to JSTOR to access the linked references.

JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide
range of content in a trusted digital archive. We use information technology and tools to increase productivity and
facilitate new forms of scholarship. For more information about JSTOR, please contact support@jstor.org.

Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at
https://about.jstor.org/terms

American Association for the Advancement of Science is collaborating with JSTOR to digitize,
preserve and extend access to Science

This content downloaded from

121.52.159.133 on Wed, 02 Jun 2021 09:58:24 UTC
All use subject to https://about.jstor.org/terms
the various lines of evidence described energy density of the plasma ap- 11. E. N. Parker, Astrophys. J. 133, 1014 (1961).
12. K. G. McCracken, J. Geophys. Res. 67, 423
here it is clear that an interplanetary proaches that of the galactic medium. (1962); ibid., p. 447.
13. - , ibid., p. 435.
magnetic field is always present, drawn Instabilities produce a disordered outer 14. K. I. Gringauz, V. V. Bezrukikh, V. D.
out from the sun by the radially stream- region of plasma and field. Direct ex- Ozerov, R. E. Rybchinskii, Dokl. Akad.
Nautk SSSR 131, 1301 (1960); S. Sh. Dol-
ing solar wind. The field is stretched perimental investigation awaits a deep ginov, E. G. Eroshenko, L. N. Zhuzov, N.
into a spiral pattern by the sun's rota- space probe capable of reaching and V. Pushkov, L. 0. Tyurmina, Proc. Intern.
Space Sci. Symp., Ist, Nice (1960), p. 863.
tion. The field appears to consist of operating at a distance of 100 astro- 15. C. P. Sonett, D. L. Judge, A. R. Sims, J. M.
relatively narrow filaments, the fields nomical
of units. Kelso, J. Geophys. Res. 65, 55 (1960).
16. P. J. Coleman, L. Davis, C. P. Sonett, Phys.
adjacent filaments having opposite direc- The weak interplanetary field exerts Rev. Letters 5, 43 (1960).
tions. At the earth's orbit the field considerable influence on the cosmic- 17. J. P. Heppner, N. F. Ness, C. S. Scearce, T.
L. Skillman, J. Geophys. Res. 68, 1 (1963).
points slightly below the eclipticray protons. Study of these particles
plane. 18. L. J. Cahill and P. G. Amazeen, ibid., p.
1835.
The magnitude of the field is steady has been a particularly valuable tool in 19. J. W. Dungey, Planetary Space Sci. 10,
and near 5 gammas in quiet times, but determining the large-scale properties 233 (1963).
20. T. Gold, Gas Dynamics of Cosmic Clouds,
it may rise to higher values at times of of the interplanetary field. Precise Van De Hulst, Ed. (North-Holland, Amster-
higher solar activity. A collision-free measurement of the magnitude and dam, 1955), p. 228; V. N. Zhigulev, Soviet
Phys. "Doklady" (English Transl.) 4, 514
shock front is formed in the plasma direction of the field at a point in space (1959); W. I. Axford, J. Geophys. Res. 67,
flow around the earth. In the transitionand study of small-scale fluctuations 3791 (1962); P. J. Kellogg, ibid., p. 3805.
21. M. Neugebauer and C. W. Snyder, Science
region between the shock front and thecan be accomplished only by means of 138, 1095 (1962); C. W. Snyder, M. Neuge-
bauer, U. R. Rao, J. Geophys. Res. 68,
magnetopause the magnitude of the a spacecraft magnetometer. The two
6361 (1963).
field is somewhat higher than it is in methods are complementary, and both 22. P. J. Coleman, Jr., L. Davis, Jr., E. J.
Smith, C. P. Sonett, Science 138, 1099 (1962).
the interplanetary region, and large will be used in further study of the in- 23. C. P. Sonett, D. S. Colburn, L. Davis, Jr.,
fluctuations in magnitude and direction terplanetary field during the increasing E. J. Smith, P. J. Coleman, Jr., Phys. Rev.
Letters 13, 153 (1964).
are common. A shock front has also solar activity of the next 5 years. 24. J. H. Wolfe and R. W. Silva, paper presented
been observed in space between a at the 45th Annual Meeting of the American
References and Notes Geophysical Union, Washington, D.C.,
slowly moving body of plasma and a 1964; H. Bridge, A. Egidi, A. Lazarus, E.
1. C. 0. Hines, Science 141, 130 (1963). Lyon, L. Jacobson, paper presented at the
faster, overtaking plasma stream. 2. H. W. Babcock and H. D. Babcock, Astro- 5th International Space Science Symposium,
Beyond the earth's orbit the solar phys. J. 121, 349 (1955). Florence, 1964.
3. S. Chapman and V. C. A. Ferraro, Terrest. 25. N. F. Ness, C. S. Scearce, J. B. Seek, J.
plasma must continue to expand with Magnetism Atmospheric Elec. 36, 77, (1931); Geophys. Res. 69, 3531 (1964).
the same velocity, carrying the inter- ibid., p. 177; ibid. 37, 147 (1932); ibid. 38, 79 26. L. Davis, J. Phys. Soc. Japan 17, suppl. 2,
(1933). 543 (1962); W. I. Axford, A. J. Dessler,
planetary field with it (26). The angle 4. H. Alfven, Cosmical Electrodynamics (Clar-
Gottlieb, Astrophys. J. 137, 1268 (1963);
endon, Oxford, 1950).
between the spiral field lines and the 5. L. Biermann, Observatory 77, 109 (1957).
-, ibid., p. 193.
27. The variance, a measure of the fluctuati
radial direction from the sun continues 6. S. Chapman, Smithsonian Contrib. Astrophys. field measurements in each 12-measurement
2, 1 (1957). average, is defined by Ness (25) as:
to decrease until, at great distances, the 7. E. N. Parker, Astrophys. J. 128, 664 (1958).
field is perpendicular to the direction of 8. T. Gold, J. Geophys. Res. 64, 1665 (1959).
9. J. A. Simpson, Annals International Geophysi-
plasma flow. The energy density of the cal Year (Pergamon, New York, 1956), vol. i = 1
4, p. 349; P. Meyer, E. N. Parker, J. A.
plasma decreases with plasma number Simpson, Phys. Rev. 104, 768 (1956); D. A.
density as the inverse square of the Bryant, T. L. Cline, U. D. Desai, F. B.
where N is 12, X, is an individua
McDonald, J. Geophys. Res. 67, 4983 (1962).
radial distance until ultimately, between10. D. K. Bailey, J. Geophys. Res. 62, 431 ment of the component X e, and
10 and 100 astronomical units, the (1957). average of 12 measurements.

been a rather random process, and

I consider that tens of thousands of
new alkaloids remain to be discovered
in the vast plant kingdom. Alkaloids
Biosynthesis
Biosynthesis of
of Alkaloids
Alkaloids may be defined as naturally occurring
organic compounds containing nitro-
gen, which is usually located in a het-
New and unexpected
unexpected routes
routes to
to the
the pyridine
pyridine and
and
erocyclic ring. The nitrogen in alkaloids
is present as an amino group, and this
piperidine rings
rings have
have been
been discovered.
discovered. group causes solutions of alkaloids in
water to be basic. Many nitrogen-con-
Edward Leete taining compounds (such as nocarda-
mine, gliotoxin) which are produced by
microorganisms could be regarded as
alkaloids, although one seldom finds
Some organic compounds, particu- known as alkaloids which to our knowl- such compounds discussed in treatises
larly the a-amino acids and carbohy- edge appear to have no biological role on alkaloids. The same alkaloid is some-
drates such as glucose and ribose, are in the plants which produce them. times found in quite unrelated species.
found in all plants, and it seems prob- About 3000 different alkaloids have Thus the fungus Claviceps produces a
able that these ubiquitous compounds been isolated from about 4000 plant group of compounds known as the
are formed by essentially the same species (1). Some typical alkaloids are ergot alkaloids which are derivatives
metabolic reactions in all plants. How- illustrated in Fig. 1. Until recently the of lysergic acid. Lysergic acid deriva-
ever certain plants contain compounds chemical investigation of plants has tives have also been isolated from the
SCIENCE VOL. 147
1000

This content downloaded from

121.52.159.133 on Wed, 02 Jun 2021 09:58:24 UTC
All use subject to https://about.jstor.org/terms
higher plant Rivea corymbosa (2). Basic Hypotheses of Biosynthesis labeled compound. The method which
heterocyclic compounds have also been we use most often in our current work
isolated from animals and are some- In general biochemists have ignored is that of wick feeding, where cotton
times referred to as animal alkaloids. the alkaloids, and it has been left to thread is passed through the stem of the
One such example is samandarine found organic chemists to suggest plausible plant by means of a fine sewing needle.
in the salamander, Salamandra macu- ways in which the alkaloids could be Both ends of the thread are then placed
losa (3). produced in the living plant. Robinson in a 5-ml beaker which contains a solu-
Over 150 years ago, morphine, pres- in 1917 (7) was one of the first to tion of the labeled compound. The
ent in the opium poppy Papaver somni- propose that many of the alkaloids solution is usually rapidly absorbed into
ferum, was the first alkaloid to be iso- could be derived from the common the plant in a few hours. Distilled water
lated in a crystalline state. Since that a-amino acids by relatively simple re- can then be added to the beaker to
time many organic chemists have been actions. The original ideas of Robinson ensure that all the labeled compound
busy elucidating the structures of al- have been amplified by many organic is taken into the plant. This procedure
kaloids. Eminent scientists spent many chemists over the years, and the chem-has the advantage that one does not
frustrating, but enjoyable, years carry- ical literature abounds in speculations have to disturb a plant which is grow-
ing out degradative reactions on strych- concerning the biogenesis of alkaloids ing in soil, and feeding experiments
nine (Fig. 1) before they were able to (8). However, until 1950 there was no have actually been carried out in the
ascertain its correct structure (4). The experimental evidence supporting any remote woods of northern Minnesota
newest generation of chemists can well of these hypotheses. Unsuccessful at- on Lycopodium annotinum (a club
marvel at the patience and ingenuity tempts were made to study alkaloid bio- moss), a species difficult to cultivate in
of earlier organic chemists who did not synthesis by feeding large amounts of a a greenhouse. Fine glass capillary tubes
possess the instrumentation, such as x- proposed precursor of an alkaloid to the have been used in a similar way to in-
ray crystallography and infrared and plant in the expectation that the yield of troduce solutions of labeled compounds
nuclear magnetic resonance spectros- alkaloid would be increased. Thus when into the stems of Ricinus communis
copy, now available for aiding in struc- lysine was fed to the opium poppy the plants (10). Labeled compounds have
ture determination. amount of morphine increased (9), also been injected directly into plants
Soon after alkaloids were isolated, at- whereas the actual precursors of mor- by means of a hypodermic syringe. A
tempts were made to synthesize them phine (tyrosine and methionine) are solution of radioactive tyrosine was in-
in the laboratory. Many will recall the quite unrelated to lysine. The lysine jected into the bulbous stem of the
efforts of W. H. Perkin who, in 1856, was in some way stimulating the en- peyote cactus Anhalonium lewinii, very
attempted to obtain quinine by oxidiz- zymes which catalyze the formation of little liquid exuding from the hole when
ing allyltoluidine with potassium di- morphine in the opium poppy. the needle was removed (11). Com-
chromate. The rationalization for his The general procedure that my col- pounds labeled with carbon-14 have
experiment was the following stoichio- leagues and I have used to study alka- also been injected into the seed capsule
metric equation: loid biosynthesis in vivo has been to of the opium poppy (12). A method
administer to the alkaloid-producing
2 C1oHi3N + 3 0 C.2,,H2,N20, + H2O plant a plausible precursor labeled with
allyltoluidine quinine
isotopic atoms. After a suitable period
The product from the reaction was of time, the alkaloid is isolated from
dark brown and unpromising. Lesser the plant and assayed for the isotope.
chemists would have probably aban- In most of our work we have used pre-
doned the experiment, but Perkin, at cursors labeled with carbon-14, al- Nicotblne Strychnine
the age of 18, repeated the oxidation (in Tobacco) (in Strychnos species)
though more recently we have carried
with crude aniline and, from the black out investigations with compounds la-
oxidation mixture, isolated not quinine beled with tritium and nitrogen-15. In HNo
but the first synthetic dye mauve. The some studies an intact plant growing ogN-CH HO <
synthesis of quinine was not achieved hydroponically has been used, and the
until 1944 (5). Although many alkaloids labeled organic compound has been CH3 1^-11-^^H N
have not yet been prepared in the added to the aerated nutrient solution Codeine Quinine
laboratory, it is possible to devise plau- (in Opium poppies) (in
in which the roots of the plant were
sible reaction sequences for their syn-
growing. One can never be sure that
0 OH
thesis. The varied methods used for the the nutrient solution or the plant roots
laboratory synthesis of alkaloids usual- are free of microorganisms, and there
ly depend on intermediates and reac- is always the possibility that the labeled H OH NCH3
tions which could not possibly occur in compound is modified or degraded by Nocardamine CH,OH
the living plant. But there have been microorganisms before it is absorbed (in Actinomyces buchanon) Gliotoxin
{in Trichoderma viride)
attempts to carry out alkaloid synthesis by the roots. Various techniques have
in aqueous solution at room tempera- been used to minimize this possibility.
ture, as a simulation of conditions which One method which has been successful
prevail in the living plant, and some of with tobacco plants is to cut off all the
these syntheses have been successful (6). old roots and allow new ones to grow
into a sterile solution of the labeled
The author is professor of organic chemistry compound. In other cases the roots Lysergic acid amide Somandarine
at the University of Minnesota, Minneapolis, and (in Rivea corymboso) (in Salamander)
is currently an Alfred P. Sloan Foundation have been washed with a germicide be-
fellow.
fore immersion in a solution of the Fig. 1. Typical alkaloids.
26 FEBRUARY 1965
1001

This content downloaded from

121.52.159.133 on Wed, 02 Jun 2021 09:58:24 UTC
All use subject to https://about.jstor.org/terms
 synthesis. Often alkaloids are synthe- the nicotine formed in Nicotiana plants
HO-e CHzCHzNH ~-HOO CHCH2NHCH3
sized only in the aerial parts of a plant. by methods which have proven success-
Tyramine N-Methyltyramine
Quite large organic molecules such ful in the study of photosynthesis. This
H0 f\cH2NH CH30 CH2 NH as tryptophan and nicotine can enter a is probably the ideal way to study alka-
HOOC HO plant by way of its root system, and, loid biosynthesis, since there is no inter-
in the examples reported, the size of a ference with the normal physiology of
HOt73CH,CHC HOfCH2 H2
labeled compound has not prevented the plant if the amount of carbon-14
Norbelladine 0-Methylnorbelladine
its absorption by a plant. However, we fed does not cause radiation damage
Fig. 2. Methylations catalyzed by isolated can say very little about translocation to the plant (15).
enzymes. The incorporation of carbon-14 or
of labeled compounds inside plants.
The lack of incorporation of a given other isotope into an alkaloid after
compound into an alkaloid may be due administration of a labeled organic
which has received little attention is the to the fact that the administered labeled compound to a plant does not necessari-
direct absorption of organic compounds compound was unable to reach the site ly mean that the administered com-
through the leaves (13). I have sprayed of alkaloid synthesis. In this kind of pound is a precursor of the alkaloid.
the leaves of Digitalis purpurea plants experiment alkaloid synthesis should It is always possible that the labeled
with an equeous solution of mevalonic be occurring at the time when the la- compound is degraded into smaller
acid-2-CT' and have observed excellent beled compound is being fed to the fragments which are then utilized for
translocation of the mevalonic acid into plant. To determine whether alkaloid alkaloid synthesis. For this reason the
the plants. synthesis is taking place at a particular use of uniformly or randomly labeled
In the extensive work with sterile cul- stage in development, a plant may be compounds in the study of metabolic
tures of excised roots (14), the environ-grown in an atmosphere containing pathways has its limitations. Investiga-
ment in which the roots are growing radioactive carbon dioxide, and alka- tions with such compounds can provide
can be very well controlled, and one loids produced during growth may be preliminary information which should
can be sure that the nutrient solution examined for radioactivity. Rapoport is then be followed by work with specif-
is free of microorganisms. However, ambitiously attempting to determine ically labeled precursors. Even when it
such experiments can be successful the radioactive intermediates between is shown that a specifically labeled com-
only if the roots are the site of alkaloid such atmospheric carbon dioxide and pound results in the formation of an

SPECIES PRECURSOR ALKALOID SPECIES PRECURSOR ALKALOID

COOH
Anhalonium lewinii
CH30,1
(11) CH30 l NH2 Hordeum distichum
.......CHa
OCH3 fN77jflNHN KN, H3
(54)
Tyrosine
Mescoline
ONH H H
COOH 3-Aminome
Cotha edulis
(47) Nicotiona tobocum COOH
d-nor- -Ephedrine
Phenylotanine (55) oNHa

f-O NH2
Chelidonium majus Ni CH3
(48)
NOIr- COOH 0,, CH3 Nicotine
Hof NH2 N I N
Popover somniferum /-r.-. COOH
(56)
(49)
HOp mine OH N'0> Ho NHz
0

Dopomine Chel i donine HO/>NN

(49)
HO0 NHz
Cinchona succirubro COOH HO , Morphine
OH
(50) Rauwolfia serpentina
Hy CH
NHz N^C^O^H
ON (57)

Tryptophan Quinine
H CN3 CH3
Ajmoline
Colchicum byzantinum COOH
O ICH3z * ' NHCOCH3 Tobernanthe iboga CH3O s--I
(51)
(58)
Jr CH30 (0 N H H
N"H

ZH3
CH3COONa 3 O
lbogaine
Colchicine OCH3
Vinca rosea COOH
COOH
Datura stramoniunm (59)
(52) NHz N Hz CH3
NHZ
Ornithine CH 0 ^N / O^CO
3 CH COOCH3
c,OOH Vindoline
(53)

c^ H Hyascyamine

Fig. 3. Some alkaloids and their established precursors [location of isotopic atoms indicated by *, ::, o, ]. (Included in parentheses
are the reference numbers of the sources of the data.)
SCIENCE, VOL. 147
1002

This content downloaded from

121.52.159.133 on Wed, 02 Jun 2021 09:58:24 UTC
All use subject to https://about.jstor.org/terms
alkaloid labeled at specific positions, in XCOOH sCONH2 investigations the on
agreement with a preconceived bio- ical sourcce of nicoti
genetic scheme, one must proceed with Nicotinic acid Nicotinamide tophan. I The acid is
caution in interpreting the data. Sup- sequence of reactions
/
pose that an alkaloid F is formed from 5. This pa athway oper
OCH3
carbon dioxide by way of the inter- organism, s, such as Neurospora, and in
mediates A, B, C, D, and E. A com-
N 2 CH3 K-%Nz?
H CHN
animals ( (21). Nicotinic acid labeled at
NHN
pound X could be incorporated into the CH3 C-2 is obi tained from tryptophan labeled
alkaloid if the plant is able to convert Nicotine Anobosine Ricinine at C-7a. If the same sequence of reac-
it to any of the intermediates in this Fig. 4. Alkaloids derived from nicotinic tions occ urs in the tobacco plant we
biosynthetic sequence, whereas com- acid. would ex pect nicotine isolated from a
pound X may not play any role in the ich had been fed whi
plant tryptophan-
normal biosynthesis of the alkaloid. 7a-CT"
be labeled also at C-2to
in the
We actually know very little about the o COOH pyridine i ring. In actual fact no activity
capacity of plants to carry out chem- ^V-^Y00 f ~NHH ^NCOOH
cted in thewas
nicotinedetec
(22). The
ical reactions which do not take place NH 2 NHCHO "NHz failure of f tryptophan to serve as a pre-
H OH
in a normal plant. nicotinic acid or related com-
Tryptophon Formylkynurenine 3-Hydroxy- sor
n other
anthranilic acid pounds ir higher plants has also
been obsc erved (23).
Isolation of Enzymes C OCOOO H CCOOH COSix ye ars ago I (24) and Griffith
" ,CHO, NCOH NH2 and Byei rrum (25) showed that the
Very little work has yet been re- Qui'nolinic Nicot inicstr ation of acetic acid-2-C11 to
ported on the isolation of enzymes acid acid Nicotitan t plants led to the formation
which control alkaloid synthesis in
Fig. 5. The biosynthesis of nicotinic acid of radioa .ctive nicotine and anabasine.
plants. An enzyme has been isolated from tryptophan. An apprm eciable amount of the radio-
from the roots of germinating barley activity v was located in their pyridine
Hordeurn vulgare which catalyzes the rings. On the other hand, there was no
transfer of a methyl group from S-ade- Equisetum arvense (horse tails), and activity ir n the pyridine ring of nicotine
nosyl-L-methionine to tyramine afford- Lycopodium clavatum (club moss). when ace -tate-l1-CT1 was fed to tobacco
ing N-methyltyramine (16). Mudd and There has been much work on the bio- plants. O1 ther investigators studying the
co-workers have also obtained from synthesis of nicotine and its isomer biosynthe sis of ricinine found that ace-
Neriine bowdenii a cell-free enzyme anabasine (Fig. 4), the main alkaloid tate-2-C14, , succinate-2,3-C"4, glycerol-
(17) which catalyzes the O-methyla- of Nicotiana glauca (wild tobacco). 1,3-CT4, and glycerol-2-C14 were also
tion of norbelladine. These two reac- Dawson and his co-workers established excellent precursors of the pyridone
tions are illustrated in Fig. 2. Mothes that nicotinic acid is a precursor of the ring of lis th
alkaloid (10, 26). However,
(18) was able to show that radioactive pyridine ring of these two alkaloids, the the admi inistration of acetate-l-C14 or
morphine was obtained when uniform- carboxyl group being lost at some step succinate- -1,4-C14 to the castor bean
ly labeled tyrosine-C" was added to the in the biosynthesis (14, 19). Nicotinic yielded ri icinine which had most of its
aqueous latex obtained from the opium acid was also found to be a precursor activity 1 located on the cyano group.
poppy. of ricinine, an alkaloid found in Ricinius The ificance signi of these results could
Some of the recent results obtained commnunis (castor bean). In this case not be e valuated until methods were
in my laboratory by feeding specifically the carboxyl group of nicotinic acid developed I for the systematic degrada-
labeled compounds to alkaloid-produc- ultimately becomes the cyano group of tion ofacid
cotinic niand ricinine to de-
ing plants are summarized in Fig. 3. ricinine, nicotinamide apparently being termine tctivity on a each of the carbon
A relatively small number of a-amino an intermediate (20). Thus by study- atoms.
acids-ornithine, lysine, phenylalanine. ing the origin of the pyridine or pyri- Radioa ctive anabasine isolated from
tyrosine, and tryptophan-serve as di- done rings of these alkaloids, important Nicotiana t glauca plants which had been
rect precursors of many alkaloids. information has been obtained concern- fed aceta te-2-C"4 or glycerol-2-C'4 was
Peripheral 0- and N-methyl groups ing are the origin of nicotinic acid in degraded by oxidation of the anabasine
derived from the S-methyl group of higher plants. When we commenced our with pot :assium permanganate (27).
methionine. Acetic acid takes part in
the biosynthesis of some alkaloids.
The structures of the alkaloids are 4 7 L.SOCiz

so varied that it is not possible to COOH Ci oH20H rOCHH Q CH3 i. CH-31 CH3
5:iCOOO1 2H H. H30OH QCH3 PhLi
formulate a single hypothesis concern- N 3. LiAIH4 2. H2Pt NPh
ing the biogenesis of all of them. In
this review I will illustrate several ways ^^~CHs O

in which pyridine and piperidine rings ~"~CH3 . CH3 ! r-, CH3

are produced in nature. HCHO 0S041
-H 2.AgO IH N. a NH3 [cH3 PhCOOH fC
rc-iJ / NoIO 1 Z Ph -- N Ph
3 heat / \
---NPh +
Nicotine is the best known alkaloid
CH3CH3 1 CH
containing a pyridine ring, and it oc-
3
curs in most Nicotiana species. It is,
however, found in quite unrelated spe- CH3NH2 CO2
OHCN C3 Cr03 HOOC-"' CH 2 C g[c-
cies: Asclepias syriaca (milk weed), Ph ' Ph
Atropa belladonna (deadly nightshade), Fig. 6. Degradation of
ity on each of the carbon atoms. nicot
26 FEBRUARY 1965
1003

This content downloaded from

121.52.159.133 on Wed, 02 Jun 2021 09:58:24 UTC
All use subject to https://about.jstor.org/terms
 Krebs
lated to glycerol. The other carbons Robinson's Hypothesis:
CH3COOH - - CH2COOH CH2 COOH
cycle I were derived from succinate or a closely
O=C-COOH
0 r0'"
Acetic acid CH2COOH related compound. A tentative scheme
C H ",_ CHa-- - C H c'CH` N
Succinic
acid
Oxaloacetic
acid for the biosynthesis of nicotinic acid OH3 CH2 +, ,.+--- H
COOH H COOH
based on these tracer results is illus- Acetoccetic A-Piperideine
trated in Fig. 7. It is suggested that the acid I
CH0OH H-C -- - 3CH COO.
1 I COOH heterocyclic ring is formed by a con-
CHOH - CHOH 20CH-COOH OCH3 N CH; CH
densation between glyceraldehyde-3- Pnt-, a ~ : : HA
lH20H -'O-CH - NH2 phosphate and aspartic acid. The re- 0
Aspartic acid N.w HyUpthesis:
Glycerol Glyceraldehyde sultant piperidine derivative then under-
3-phosphate
goes dehydration and dehydrogenation 4 CH3COOH - - -. -
CH3
OH i to yield quinolinic acid, which is then NH3
HO .COOH _OOH 40..C,OOFt
decarboxylated affording nicotinic acid.
K COOH -NT "'COOH N Quinolinic acid is indeed a precursor
0
H
Quinolinic Nicotinic of nicotinic acid in corn and castor
ocid acid CH-, N
bean plants (30). Therefore the admin- H 3 OH
Fig. 7. Biogenetic scheme for the forma- istration of aspartic acid-3-C'4 to tobac-
tion of nicotinic acid in higher plants and Fig. 9. Old and new biogenetic schemes
co should result in the formation of
some microorganisms. for coniine.
nicotine labeled at C-3 of the pyridine
ring. In actual fact only about 50 per-
cent of the activity of the pyridine ring
The nicotinic acid so obtained was then I showed that the administration of
was located at this position (31). This
degraded by the scheme illustrated in lysine-2-CT" to Nicotiana glauca plants
result can be rationalized by postulating
afforded anabasine which was labeled
Fig. 6. Activity at each of the carbons that the administered aspartic acid-3-C"
except C-4 was determined directly. solely at C-2' of the piperidine ring
is metabolized by way of the Krebs
Activity at C-4 was determined by dif- (34). Recently we have carried out
cycle to the symmetrical succinic acid.
ference. A quite different degradative feeding experiments with lysine-2-C11
Aspartic acid which could then be re-
scheme for nicotinic acid has been pub- labeled with N1' on the a- or e-nitrogen
synthesized from this succinic acid
lished by Christman et al. (28). Com- (35). The results indicated that the
would have radioactivity equally dis-
plete degradations have also been car- piperidine nitrogen was derived only
tributed between C-2 and C-3. In
ried out on radioactive ricinine derived from the e-amino group of lysine. This
Escherichia coli glycerol and succinic
from acetate, succinate, and glycerol result is consistent with the hypothesis
acid are also precursors of nicotinic
(10, 26, 29). The results obtained from that the piperidine ring of anabasine is
acid (32). Exciting results were recent-
these degradations are all consistent. It ly obtained by Mothes et al. studying formed from lysine by way of a-keto-
was found that acetate-2-C14 leads to the e-aminocaproic acid (Fig. 8). I con-
the biosynthesis of nicotinic acid by
same pattern of labeling as succinate- sider that this keto acid then cyclizes
Mycobacterium tuberculosis (33). By
2,3-C'4, and it is suggested that the to Az-piperideine-2-carboxylic acid. De-
feeding aspartic acid-l,4-C1'-N-', they
acetate is converted to succinate or a carboxylation affords A'-piperideine
were able to show that the pyridine
closely related metabolite prior to its which could condense with 1,6-dihydro-
nitrogen was derived from the amino
incorporation into nicotinic acid. The nicotinic acid to yield ultimately ana-
group of aspartic acid and the carboxyl
results indicated that carbons-4, -5, and group was derived from the 7-carboxyl basine as illustrated in Fig. 7. Other
-6 of nicotinic acid were derived from piperidines which are derived from
of aspartic acid. Glycerol also served
a three-carbon compound closely re- lysine are pipecolic acid (36) found in
as a precursor of nicotinic acid in this
various plants and microorganisms, and
same organism. Thus it seems quite
homostachydrine (37) found in Medi-
likely that nicotinic acid is produced
cago sativa (alfalfa).
by the same sequence of reactions in
.HOOC NH2 HOOC NH2 HOOC^( E. coli, M. tuberculosis, Ricinus com-
munis, and Nicotiana species. Since the
Lysine ca-Keto-c-amino- L-Piperideine- 0
caproic acid 2-carboxylic proposed precursors of nicotinic acid,
acid
glyceraldehyde-3-phosphate and aspart- 2-Methylpiperidine
OH COOH
CH3 CH3 (in Pinus sabiniana
ic acid, are such active metabolites, it
H-J?O -O
seems unlikely that the actual mecha- 0

nism of the formation of nicotinic acid

H H
by this new route will be determined CH COOH CH N c H

Z'(a hydride until the enzymes responsible for its Pinidine

a cceptor) 1,6-Dihydro-
nicotinic acid
synthesis are isolated. (in Pinus torreyana)

HOOC .
Kll O O0 /
Piperidines from Lysine
? H yCooH ^CoC CHo O CHiCH N
H
H CH3 CH3
Anabasine Pipecolic Homostachydrine The amino acid lysine serves as a Carpaine
acid precursor of the reduced pyridine ring (in Carico papaya)

Fig. 8. Biosynthesis of the piperidine ring (piperidine) in several alkaloids and Fig. 10. Some piperidine alkaloids which
of anabasine. other natural products. Some time ago are plausibly derived from acetate.
1004 SCIENCE, VOL. 147

This content downloaded from

121.52.159.133 on Wed, 02 Jun 2021 09:58:24 UTC
All use subject to https://about.jstor.org/terms
Piperidines from Acetate carbon fragment (represented here as
formaldehyde), and malonic acid.
Until recently it has been generally In this review the methods which are
accepted that the piperidine ring of CH3 being used for studying the biosynthesis
Actinidine Motatabiloctone Skytonthine of alkaloids in plants have been de-
coniine, one of the main alkaloids of
Conium maculatum (hemlock) was also CH2OH - / scribed. Many of the biogenetic hy-
derived from lysine. Robinson (7) had potheses proposed almost 50 years ago
suggested that coniine and related alka- 2 1 N by Sir Robert Robinson have now been
O00H
loids are produced by a condensation shown to be correct. However, new and
COOH
between Az-piperideine and acetoacetic unexpected biosynthetic routes to the
Mevalonic acid
acid (Fig. 9). Decarboxylation fol- pyridine and piperidine rings have
lowed by reduction of the side-chain Fig. 1 1. Terpenoid pyridine and piperidine been discovered. Many problems re-
alkaloids.
then yields coniine. Initial tracer experi- main to be solved and we are only
ments (38) were apparently consistent just beginning to understand the rela-
with this hypothesis. Uniformly labeled found in nature do not fit readily into tion of alkaloid biosynthesis to other
lysine-C'4 was fed to hemlock plants. any of the previously mentioned metabolic reactions which occur in the
and radioactive coniine was formed. schemes. 3-Methoxypyridine is found living plant.
However the alkaloid was not degraded in Equisetum arvense (43), and I sug- References and Notes
to determine the distribution of radio- gest that it is formed by the cyclization1. H.-G. Bolit, Ergebnisse der Alkaloid-Chemie
activity. On the other hand, 1 admin- and dehydration of an amino pentose his 1960 (Akademie-Verlag, Berlin, 1961).
2. A. Hofmann and H. Tscherter, Experientia
istered lysine-2-C"4 to hemlock by va- such as 5-amino-5-deoxy-D-xylose as 16, 414 (1960).
rious methods and observed but little illustrated in Fig. 12. The cyclization of 3. G. WVeitz and E. Wolfel, Acta Cryst. 15, 484
(1962); E. W1olfel, C. Sch6pf, G. Weitz, G.
incorporation of activity into the crude such amino sugars to pyridine deriva- Habermehl, Chem. Ber. 94, 2361 (1961).
hemlock alkaloids. I thus considered a tives has been observed in vitro (44). 4. L. H. Briggs, H. T. Openshaw, R. Robinson,
J. Chem. Soc. 1946, 903 (1946); R. B. Wood-
new scheme for the biogenesis (Fig. The 9) important vitamin pyridoxine (Fig. ward, W. J. Brehm, A. L. Nelson, J. Am.
Chem. Soc. 69, 2250 (1947).
of coniine whereby both the side-chain 12) is a pyridine derivative, but little
5. R. 13. Woodward and W. von E. Doering,
and the piperidine ring are formed is known of its precursors. Another J. A ns. Chem. Soc. 66, 849 (1944).
6. E. E. Van Tamelen, Fortschr. Chem. Org.
from a poly-f-ketoacid derived from pyridine derivative whose origin appears Naturstoffe 19, 242 (1961).
four acetate units. Tracer experiments to be unknown is 3-hydroxypicolinic 7. R. Robinson, J. Chei. Soc. 111, 876 (1917);
The Structural Relations of Natural Products
confirmed this hypothesis (39). When acid (Fig. 12), part of the cyclic poly- (Clarendon Press, Oxford, 1955).
acetate-l-C1 was fed to hemlock radio- peptide etamycin which is produced by 8. T accept with enthusiasm the suggestion of
F. R. Stermitz and H. Rapoport, J. Am.
active coniine was obtained, the activitycertain Streptomyces species (45). Chem. Soc. 83, 4045 (1961) that the word
being equally distributed between the Three different biogenetic schemes (46) biosynthesis should describe in vivo experi-
mental studies on the mode of formation of
four positions indicated with heavy have been suggested for gentianine, an natural products, while the word biogenesis
should be applied to the hypothetical schemes
dots in the formula of coniine (Fig. 10).alkaloid found in various Gentiana
suggested to rationalize the formation and
Several other piperidine alkaloids are species. I favor a fourth scheme (Fig. structures of natural products.
9. 0. V. Bogdashevskaya, Fiziol. Rast. 5, 185
also plausibly formed from acetate de- 12). The carbon skeleton is built up (1958).
rived poly-,f-ketoacids (Fig. 10). The from a six-carbon poly-/l-ketoacid de-
10. P. F. Juby and L. Marion, Biochem. Biophys.
Res. Commun. 5, 461 (1961); Can. J. Chem.
fatty acids are of course formed by the rived from three acetate units, a one- 41, 117 (1963).
linear combination of acetate units, and 11. E. Leete, Chem. Ind. London 1959, 604
(1959).
it seems that these piperidine deriva- CHO 12. A. M. Kuzin and V. I. Merenova, Bio-
tives arise by some deviation in the chimiya 19, 616 (1954).
40HNH -3N HO 13. K. Mothes and G. Trefftz, Naturwissen-
normal biosynthesis of fatty acids. HOi- 2 schaften 41, 382 (1954); E. C. Humphries,
-fOH 0 ?HOX OHOH
Proc. Fertiliser Soc. Soc. Anal. Chem. 48, 1
CH2NH2 OH I OH (1948).
14. R. F. Dawson, D. R. Christman,. R. C. An-
5-Amino-5-deoxy-
derson, J. Am. Chem. Soc. 75, 5114 (1953).
Pyridines from Mevalonic Acid D-xylose 15. W. L. Alworth, R. C. DeSelms, H. Rapoport,
ibid. 86, 1608 (1964); W. L. Alworth, A. A.
CH2OH Liebman, H. Rapoport, ibid. 86, 3375 (1964).
The pyridine ring of the alkaloid OCH3 16. J. D. Mann, C. E. Steinhart, S. H. Mudd,
HO, CHHOH 3-Methoxy- J. Biol. Chem. 238, 676 (1963).
actinidine (Fig. 11) is probably formed r-OH pyridine 17. H. M. Fales, J. D. Mann, S. H. Mudd, J.
by yet another biosynthetic route. This CH3-NJ N COOH Am. Chemr. Soc. 85, 2025 (1963).
18. G. Kleinschmidt and K. Mothes, Z. Natur-
alkaloid occurs in the plant Actinidia Pyridoxine 3-Hydroxy forsch. 14b, 52 (1959), Arch. Pharm. 293, 948
polygama, along with the nitrogen-free picolinic acid (1960); L. Meissner and K. Mothes. Phyto-
chemistry 3, 1 (1964); J. W. Fairbairn and
compound matatabilactone (40). Skyt- G. Wassel, ibid. 3, 583 (1964).
anthine, having the same carbon skele- 0 CH3
19. R. F. Dawson, D. R. Christman, A.
D'Adamo, M. L. Solt, A. P. Wolf, J. Am.
ton as actinidine, but with the pyridine Chemn. Soc. 82, 2628 (1960); M. L. Solt, R.
ring reduced, is found in Skytanthus HOOC J CH2OH
F. Dawson, D. R. Christman, Plant Physiol.
35, 887 (1960).
acutus (41). These compounds are ap- HOOC; COOH 20. E. Leete and F. H. B. Leitz, Chem. Ind. Lon-
parently terpenes being readily con- )OH
don 1957, 1572 (1957); G. R. Waller and L.
M. Henderson, J. Biol. Chem. 236, 1186
structed from two isoprene units (Fig. (1961).
11). The biological precursor of the 21. W. A. Krehl, L. J. Teply, P. S. Sarma, C. A.
Gentianine L Elvehjem, Science 101, 489 (1945); G. W.
isoprene unit is mevalonic acid, and Beadle, H. K. Mitchell, J. F. Nyc, Proc.
this compound is an excellent precursor 0 Natl. Acad. Sci. U.S. 33, 155 (1947); Y.
Nishizuka and 0. Hayaishi, J. Biol. Chem.
of skytanthine in S. acutus (42). Fig. 12. Miscellaneous pyridine deriva- 238, 3369 (1963); R. K. Gholson, I. Ueda,
N. Ogasawara, L. M. Henderson, ibid. 239,
Several other pyridine compoundstives of unknown origin. 1208 (1964).
26 FEBRUARY 1965
1005

This content downloaded from

121.52.159.133 on Wed, 02 Jun 2021 09:58:24 UTC
All use subject to https://about.jstor.org/terms
22. E. Leete, Chem. Ind. London 1957, 1.270
(1957); L. M. Henderson, J. F. Someroski,
D. R. Rao, P. L. Wu, T. Griffith, R. U.
Byerrum, J. Biol. Chem. 234, 93 (1959).
23. E. Leete, L. Marion, I. D. Spenser, Can. J.
Chem. 33, 405 (1955).
24. E. Leete, Chem. Ind. London 1958, 1477
(1958).
25. T. Griffith and R. U. Byerrum, Science 129,
1485 (1959).
26. G. R. Waller and L. M. Henderson, Biochem.
Biophys. Res. Commun. 5, 5 (1961); J. M.
Essery, P. F. Juby, L. Marion, E. Trumbull,
Can. J. Chem. 41, 1142 (1963).
27. A. R. Friedman and E. Leete, J. Am. Chem.
Soc. 85, 2141 (1963); 86, 1224 (1964).
28. D. R. Christman, R. F. Dawson, K. I. C.
Karlstrom, J. Org. Chenm. 29, 2394 (1964).
29. U. Schiedt, G. Boeckh-Behrens, A. M. Del-
luva, Z. Physiol. Chem. 330, 46, 58 (1962).
30. L. A. Hadwiger, S. E. Bodiei, G. R. Waller,
R. K. Gholson, Biochem. Biophys. Res.
Commun. 13, 466 (1963).
31. T. Griffith, K. P. Hellman, R. U. Byerrum,
Biochemistry 1, 336 (1962).
32. M. V. Ortega and G. M. Brown, J. Biol.
Chem. 235, 2939 (1960).
33. D. Gross, H. R. Schiitte, G. Hubner, K.
Mothes, Tetrahedron Letters 1963, 541 (1963).
34. E. Leete, J. Am. Chem. Soc. 78, 3520 (1956).
35. E. Leete, E. G. Gros, T. J. Gilbertson, ibid.
86, 3907 (19,64).
36. N. Grobbelaar and F. C. Steward, ibid. 75,
4341 (1953); P. H. Lowy, Arch. Biochem.
Biophys. 47, 228 (1953); A. Meister and S.
D. Buckley, Biochim. Biophys. Acta 23, 202
(1957); L. Fowden, J. Exptl. Botany 11, 302
(1960).
37. A. V. Robertson and L. Marion, Can. J.
Chem. 37, 1043 (1959).
38. U. Schiedt and H. G. H6ss, Z. Naturforsch.
13b, 691 (1958); Z. Physiol. Chem. 330, 74
(1962).
39. E. Leete, J. Am. Chem. Soc. 85, 3523 (1963);
86, 2509 (1964).
40. T. Sakan, A. Fujino, F. Murai, Y. Butsugan,
A. Sazui, Bull. Chem. Soc. Japan 32, 315
(1959).
41. C. Djerassi, J. P. Kutney, M. Shamma, J. N.
Shoolery, L. F. Johnson, Chemn. Ind. London
1961, 210 (1961); G. C. Casinovi, J. A. Gar-
barino, G. B. Marini-Bettolo, ibid. 1961,
(1961).
42. G. C. Casinovi and G. B. Marini-Bettolo,
Abstracts A, meeting of International Union
Pure and Applied Chemistry, London (1963),
p. 285.
43. R. H. F. Manske and L. Marion, Can. J.
Res. B20, 87 (1942).
44. H. Paulsen, Angew. Chem. Intern. Ed. Engl.
2, 555 (1963).
45. J. C. Sheehan, H. G. Zachau, W. B. Lawson,
J. Am. Chem. Soc. 79, 3933 (1957).
46. R. Thomas, Tetrahedron Letters 1961, 544
(1961); T. R. Govindachari, K. Nagarajan,
S. Rajappa, Experientia 14, 55 (1958); E.
Wenkert, J. Am. Chem. Soc. 81, 1474 (1959).
47. E. Leete, Chem. Ind. London 1958, 1088
(1958).
48. -, J. Am. Chem. Soc. 85, 473 (1963).
49. and J. B. Murrill, Tetrahedron Letters
1964, 167 (1964); A. R. Battersby and R. J.
Francis, J. Chem. Soc. 1964, 4078 (1964).
50. N. Kowanko and E. Leete, J. Am. Chem.
Soc. 84, 4919 (1962).
51. E. Leete and P. E. N6meth, ibid. 82, 6055
(1960); E. Leete, ibid. 85, 3666 (1963); E.
Leete, Tetrahedron Letters 1965, 333 (1965);
A. R. Battersby, R. Binks, D. A. Yeowell,
Proc. Chem. Soc. 1964, 86 (1964); A. R. Bat-
tersby, R. Binks, J. J. Reynolds, D. A. Yeo-
well, J. Chem. Soc. 1964, 4257 (1964).
52. E. Leete, J. Am. Chem. Soc. 84, 55 (1962);
Tetrahedro,n Letters 1964, 1619 (1964).
53. M. L. Louden and E. Leete, J. Am. Chem.
Soc. 84, 1510, 4507 (1962).
253
54. B. G. Gower and E. Leete, ibid. 85, 3683
(1963).
55. E. Leete, E. G. Gros, T. J. Gilbertson,
Tetrahedron Letters 1964, 587 (1964).
56. E. Leete, Chemn. Ind. London 1958, 977
(1958); J. Am. Chem. Soc. 81, 3948 (1959);
A. R. Battersby and B. J. T. Harper, Chem.
Ind. London 1953, 364 (1958); A. R. Bat-
tersby, R. Binks, B. J. T. Harper, J. Chem.
Soc. 1962, 3534 (1962).
57. E. Leete, J. Am. Chem. Soc. 82, 6338 (1960).
58. - and M. Yamazaki, Tetrahedron Let-
ters 1964, 1499 (1964).
59. E. Leete, A. Ahmad, I. Kompis, unpublished
work.
60. I thank the National Science Foundation and
the U.S. Public Health Service for grants
(NSF-GB-363, NIH-MH-02662) which have
supported my work on alkaloid biosynthesis.

which these are organized in transac-

tional systems involving all of the
senses (1). "Zoosemiotics" was then
coined (2) to identify a very rapidly
Animal Communication expanding discipline within the be-
havioral sciences, one which has crys-
tallized at the intersection of semiotics,

A communication network model for languages the science of signs, and ethology, a
field which Niko Tinbergen char-
is applied to signaling behavior in animals. acterized, in the first book ever written
on the subject, as "the objective study
of behavior," but which he more re-
Thomas A. Sebeok
cently-and more fairly-redefined as
"the biological study of behaviour"
(3). Zoosemiotics has not only emerged
as a dominant theme in ethology, but
The term "semiotic," in its earliest to absorb logic, mathematics, and "data on animal communication have
sense equivalent to symptomatology, linguistics entirely within semiotic. contributed a thread of continuity that,
was introduced into philosophical dis- "The whole science of language," the in some ways and at some times, has
course at the end of the 1 7th century logician Rudolf Carnap then reaffirmed seemed to be the principal axis of
by John Locke to label one of the in 1942, "is called semiotic," and, in synthesis in the entire field of animal
three branches of contemporary sci- 1946, Morris introduced further refine- behavior" (4).
ence, to wit, the doctrine of signs. The ments when he distinguished among Modern developments in the study of
real founder and first systematic investi-pure semiotic, which elaborates dis- animal communication stem largely
gator of the field, however, was the course about signs; descriptive semiotic, from Charles Darwin (5). They re-
subtle and profound American philos- which focuses on actual signs; and ap- ceived substantial impetus from the
opher, Charles Sanders Pierce. The plied semiotic, which utilizes knowl- classic investigations of K. von Frisch,
unique place of semiotic among the edge about signs for the accomplish- and were placed in their present aca-
sciences-not merely one among the ment of various purposes. In 1962, the demic frame by K. Z. Lorenz, Tinber-
others, "but an organon or instrument anthropologist Margaret Mead proposed gen, W. H. Thorpe, and many others.
of all the sciences"-was insisted on by a variant, "semiotics," as a term which The period from Darwin until the end
Charles Morris who, in 1938, proposed might aptly cover "patterned communi- of the last decade has been conveniently
cations in all modalities," that is, for summarized by Kainz (6), whose book
The author is professor of linguistics and the global study of the interactional may be complemented by a series of
chairman of the Research Center in Anthro- and communicational context of the easily accessible review articles and a
pology, Folklore, and Linguistics at Indiana
University, Bloomington. human use of signs and the way in recent, semi-popular, survey of the field

1006 SCIENCE, VOL. 147

This content downloaded from

121.52.159.133 on Wed, 02 Jun 2021 09:58:24 UTC
All use subject to https://about.jstor.org/terms