Biosynthesis of Red Tide Toxins - American Chemical Society

Chapter 2 Biosynthesis of Red Tide Toxins Yuzuru Shimizu, Sandeep Gupta, and Hong-Nong Chou

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Department of Pharmacognosy and Environmental Health Sciences, College of Pharmacy, University of Rhode Island, Kingston, RI 02881-0809 T h e biosynthesis o f two major classes o f red tide toxins, saxitoxin analogs and brevetoxins, have been studied. It was shown that saxitoxin is biosynthesized from arginine, acetate, and methionine methyl group. Brevetoxins were shown to be unique polyketides, w h i c h are probably biosynthesized from dicarboxylic acids. S o m e details o f the biosynthetic mechanism have been elucidated. T h e elucidation o f the biosynthetic mechanism o f red tide toxins is an important step to understand the toxigenesis o f the deleterious red tide organisms. Questions have been raised about what triggers the production o f the toxins i n the organisms, where the regulating step is, o r h o w the organisms acquire the ability to produce the toxins. M o r e than a decade ago when we started to study the biosynthesis o f saxitoxin analogs and brevetoxins, very little was k n o w n about the molecular origins o f the unique classes o f compounds. W e n o w have the knowledge o f a l l the building blocks o f saxitoxin molecule, and some details o f the biosynthetic process. Biosynthesis of Saxitoxin Analogs Saxitoxin was first isolated from A l a s k a n butter clam Scuddomus giganteus, but is actually produced by the dinoflagellate, Gonyaulax spp (1,2). M o r e than a dozen related toxins have been isolated by several groups including ours (3). A l l o f the toxins have the same unique tricyclic perhydropurine skeleton, and there were various speculations about the origin o f the perhydropurine ring (I). In the early stage o f o u r investigation, we obtained experimental results which indicated that the major p o r t i o n o f the ring system is derived from arginine as shown i n Scheme 1 (4). However, o u r repeated attempts to incorporate [1C ] a r g i n i n e into the toxin molecule d i d not result i n any specific labeling. In Scheme 1, two C . units o r o n e C unit w o u l d become C - 5 and C-13 o f the toxin molecule. Therefore, as an alternate approach, we fed [ 1 , 2 - C ] acetate to a toxin-producing strain o f the blue-green alga, Aphanizomenon flos-aquae (5). T w o intact units o f acetate were incorporated into neosaxitoxin as evidenced by the appearance o f two sets o f A B type signals i n the C - N M R spectrum. T h e orientation o f the incorporated acetate units was determined by feeding [2C ] acetate as shown i n Scheme 2. This result clearly excludes the original pathway i n which C - 5 should come from C - l o f arginine. T o accommodate this new finding and the previous results, w e considered a new pathway (Scheme 3), i n which acetate o r its derivative condenses with arginine followed by decarboxylation. Such Claisen-type condensation o n alpha-amino acid has some precedent i n biochemical systems (6). T o prove this hypothesis, we synthesized [ 2 - C , 2 - N ] a r g i n i n e and ornithine and fed to A. flos-aquae (5). 13

2

13

2

1 3

1 3

1 3

15

0097-6156/90/0418-0021$06.00/0 o 1990 American Chemical Society

Hall and Strichartz; Marine Toxins ACS Symposium Series; American Chemical Society: Washington, DC, 1990.

MARINE TOXINS: ORIGIN, STRUCTURE, AND MOLECULAR PHARMACOLOGY

H N 2

TT"" HONT I

/=NH

N

\ - ^

-

2

'CH *CH

Saxitoxin

CH

Neosaxitoxin

H2N ,

o

H

\S H

H

)=NH2


Combinations of X=H,OH

Y=H, SO3H

z

Gonyautoxms, B - , C-1.4 toxms

Z=H, O S 0 H a . b 3

I

13

II O

COOH

Hr,J

H NH + 2

H N J V HN

:

?

Arginine

Scheme 1

ll

>

O

m c H3C"""COOH

A. flos-aquae

H Lr'

HON-^m ^

H

H

N ^>=NH

2

OH C

^

OH

Neosaxitoxin Scheme 2


2.

SHIMIZU ET AL.

23

Biosynthesis of Red Tide Toxins

1 3

T h e C - N M R spectrum o f neosaxitoxin obtained from the feeding experiment showed an enhanced signal for C-4, which was split into a doublet by the spin-spin c o u p l i n g w i t h the neighboring N (J=9.3 H z ) (Scheme 4). T h e result clearly i n d i cated that the connectivity C - 2 - N - 2 o f arginine was incorporated intact into the toxin molecule, supporting the pathway i n Scheme 3. T h e next question was the origin o f the side-chain carbon C-13. W e first fed formate as a general precursor o f C« units without success. T h e failure made us consider C 0 as a possible source. T h e idea seemed attractive, since C C L can be fixed as malonyl C o A , which c o u l d undergo condensation w i t h arginine (Scheme 5). V i g o r o u s efforts were made to effect the selective enrichment o f C-13 by pulsefeeding o f C 0 , but only some random incorporation was observed after prolonged incubation. Meanwhile, ^-alanine isolated from the feeding experiments showed enrichment i n C - 3 , suggesting the pulse feeding was operating. [ 3 - C ] - 3 Hydroxypropionate was also prepared and was fed as a closer putative intermediate i n such a pathway. A g a i n no significant incorporation was observed. T h e negative results made us reexamine C precursors. Despite o u r earlier failure i n formate feeding experiments, [3- C]serine, [1,2C ] g l y c i n e , and [ M e - C ] m e t h i o n i n e were found to enrich C-13 i n neosaxitoxin effectively (7). T h e best incorporation was observed w i t h methionine, indicating it is the direct precursor via S-adenosylmethionine. G l y c i n e C - 2 and serine C-3 must have been incorporated through tetrahydrofolate system as methyl donors i n methionine biosynthesis. In order to obtain more insights into the biosynthetic mechanism, [ M e - C M e - d ] double-labeled methionine was fed to the organism. T h e C - N M R spectrum o f resulting neosaxitoxin showed a clean triplet for C-13 beside the natural abundance singlet. T h e result indicated that only one deuterium was left o n the methylene carbon. In another experiment, [ l , 2 - C 2 - 2 - d ] double-labeled acetate was fed. First we observed a complete loss o f deuterium atoms. In a short incubation, however, we obtained neosaxitoxin partially retaining a deuterium atom (40% equivalent o f incorporated acetate molecule). T h e location o f the deuterium atom was o n C - 5 , which was originally the carboxyl carbon o f acetate, suggesting that it migrated from the adjacent methyl-derived carbon C-6. T h e result is not totally surprising, because hydride i o n shifts are k n o w n i n many methylations. Thus, it was proposed that the methyl carbinol is formed by the sequence: methylation o f a double bond - hydride shift - formation o f terminal methylene - epoxidation - opening o f the epoxide to aldehyde - reduction to carbinol (Scheme 6). T h e pathway can explain well the loss o f two original hydrogens i n methionine methyl group. 1

5

2

1 3

2


13

1

13

13

13

1 3

1 3

3

13

3

Biosynthesis of Brevetoxins Brevetoxins are the toxic principles o f the F l o r i d a red tide organism, Gymnodinium breve (Ptychodiscus brevis). T h e compounds are potent ichthyotoxins. Several toxins have been isolated from the organisms and their structures elucidated. They can be divided into two types o f toxins having different ring systems, represented by brevetoxin A and brevetoxin B (II). Brevetoxin B was the first brought to a pure state, and its structure was determined by X - r a y crystallography (8). Brevetoxin A is the most toxic principle i n the organism, and its structure was also determined by X - r a y crystallography (9). B o t h compounds have unique a\\-trans polyether ring systems, w h i c h made us believe that the compounds are products o f successive opening o f trans-epoxides. T h e precursors o f the epoxides should be polyketidal trans polyenes. 13

W e started feeding experiments using C - l a b e l e d acetate l o n g before the establishment o f the toxin structures w i t h a hope that the analysis o f the C-NMR 1 3


24


CI *, ? •

HN

^COOH

2


^

1

~

Scheme 3 .0^13

X-NHCH CH CH 2

2

CHCOOH I

2

1 5

X=

NH

2

H NC(NH)— 2

Scheme 4

CH OH 2

CH3COOH

CH NH 2

2

COOH P-Alanine

Scheme 5


SHIMIZU ET AL.

Biosynthesis ofRed Tide Toxins


.

Scheme 6


26



Me

4

5

45 Brevetoxin-B

II spectra might help the structure elucidation. It was assumed then that the c o m pounds were n o r m a l polyketides. In fact, the elucidated structures have all the appearance o f the ordinary polyketides with some methyl branches, which could come from methionine o r propionate. However, the labeling pattern o f brevetoxin B obtained from the acetate feedings was totally inexplicable i n terms o f the k n o w n polyketide biosynthesis (10). Similar observations were made by Nakanishi's group (11). W e n o w believe that we have evidence to explain the apparent randomization. W h e n the organism was exposed to a high concentration o f [ 2 - C ] acetate for a brief period, certain rows o f carbons (mostly three carbons i n a row) i n brevetoxin B had C - C spin-spin couplings (10) (Scheme 7 A ) . These couplings clearly indicate rather "fresh" condensations between actate methyl-derived carbons. Similar spin-spin couplings were not observed when [ l - C ] a c e t a t e was fed under the same c o n d i t i o n . T h e most plausible explanation o f this very unusual labeling pattern is to assume the involvement o f succinate, which is synthesized from acetate as shown i n Scheme 8a. 13

13



2.

27

Biosynthesis ofRedTide Toxins

SHIMIZU ET AL.

Scheme 7 •CH3COOH

COOH * CHjCOOH I C(OH)COOH I

+

a)

COOH | C=0 | r>CH

*

(*)CH2COOH 2

2

| 'CHjCOOH — — * CH — - . | | CHoCOOH

^

2

(*) 0 I

COOH

COOH

C)

C =

w

y/

Scheme 8


28


Three other portions o f the molecule which do not fit the polyketide combination pattern (Scheme 7 B a,f,n) can be also explained by assuming they are derived from a dicarboxylic acid such as 3-hydroxy-3-methylglutaric acid (Scheme 8b). If that is the case, brevetoxins are a new type o f mixed polyketides which are formed by condensation o n b o t h ends o f dicarboxylic acids (Scheme 8c). In order to prove further the hypothesis, feeding experiments with such putative precursors as succinate, acetoacetate, and propionate are i n progress.


Literature Cited 1. Schantz, E. J.; Mold, J. D.; Stanger, D. W.; Shave, J.; Riel, F. J.; Bowden, J. P.; Lynch, J. M.; Wyler, R. S.; Riegel, B. R.; Sommer, H. J. Am. Chem. Soc. 1957, 79, 5230. 2. Schantz, E. J.; Lynch, J. M.; Vayada, G.; Matsumoto, K.; Rapoport, H. Biochemistry 1966, 5, 1191. 3. Shimizu, Y. In Progress in the Chemistry of Organic Natural Products; Springer-Verlag: New York, 1984; Vol 45, p 235. 4. Shimizu, Y.; Kobayashi, M.; Genenah, A.; Ichihara, N. In Seafood Toxins ACS Symposium Series 262; Ragelis, E. R., Ed.; American Chemical Society: Washington, DC, 1984; p 151. 5. Shimizu, Y.; Norte, M.; Hori, A .; Genenah, A ; Kobayashi, M. J. Am. Chem. Soc. 1984, 106, 6433. 6. Ohuchi, S.; Okuyama, A .; Nakagawa, H.; Aoyagi, T.; Umezawa, H. J. Antibiot. 1984, 37, 518 7. Shimizu, Y.; Gupta, S.; Norte, M.; Hori, A .; Genenah, A In Toxic Dinoflagellates; Anderson, D. M.; White, A ; Baden, G. D., Eds.; Elsevier: New York, 1985; p 271. 8. Lin, Y. Y.; Risk, M. M.; Ray, S. M.; Van Engen, D.; Clardy, J.; Golik, J.; James, C.; Nakanishi, K. J. Am. Chem. Soc. 1981, 103, 6773. 9. Shimizu, Y.; Chou, H-N.; Bando, H.; Van Dyne, G.; Clardy, J. J. Am. Chem. Soc. 1986, 108, 514. 10. Chou, H-N.; Shimizu, Y. J. Am. Chem. Soc. 1987, 109, 2184. 11. Lee, M. S.; Repeta, D. J.; Nakanishi, K.; Zagorski, M. G. J. Am. Chem. Soc. 1986, 108, 7855. RECEIVED May 22, 1989


Biosynthesis of Red Tide Toxins - American Chemical Society

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