Biosynthesis of Paralytic Shellfish Toxins - ACS Symposium Series

13 Biosynthesis of Paralytic Shellfish Toxins YUZURU SHIMIZU, MASARU KOBAYASHI, ALMOURSI GENENAH, and NAOSHI ICHIHARA

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Department of Pharmacognosy and Environmental Health Sciences, College of Pharmacy, University of Rhode Island, Kingston, RI 02881

Biosynthetic routes to saxitoxin and i t s analogs are discussed, and the current state of progress i n the feeding experiment using the d i n o f l a g e l l a t e s , Gonyaulax tamarensis, i s summarized. Also described are the biosynthetic and successf u l N-nmr assignment of neosaxitoxin and gonyautoxin-II. 15

In the past several years we have witnessed tremendous development i n the chemistry and biology of p a r a l y t i c s h e l l f i s h poisons (PSP). In addition to long-known saxitoxin, more than ten new toxins have been isolated and characterized, and t h e i r pharmacological propert i e s have been investigated (1,2,3). New toxin-producing organisms have been discovered, and we now have a better understanding of their l i f e - c y c l e . Despite a l l these discoveries, however, we have very l i t t l e knowledge regarding the biosynthesis of the toxins. P r i o r to our understanding, there was only one reported experiment on the biosynthesis of PSP, i n which l^C-amino acid precursors were fed to the culture of Gonyaulax catenella, and the incorporation of some r a d i o a c t i v i t y was observed i n the crude toxin f r a c t i o n (4). There are two major facets of the biosynthesis of the toxins: 1) the molecular o r i g i n of the t r i c y c l i c cyclopentanoperhydropurine skeleton, 2) the origins and biosynthetic sequence of the additional functional groups such as carbamate, 0-sulfate, and N-sulfate. The f i r s t question i s an i n t r i g u i n g one which arouses chemists imaginat i o n . One of the plausible pathways pointed out p r i v a t e l y by various researchers i s v i a a normal purine metabolite followed by the Michael type condensation of acrylate or an equivalent moiety (Scheme l a ) . Another possible pathway i s v i a a C7 sugar derivative (Scheme l b ) . In f a c t , some fungal metabolites are known to have amino sugars linked with guanido groups. The t h i r d p o s s i b i l i t y , which was stipulated by our group, i s the formation of a ninemembered intermediate by the condensation of an imidazole compound and a C2 unit (or two Ci units) followed by prototropic c y c l i z a t i o n (Scheme l c ) . The l a s t pathway seems to be the most a t t r a c t i v e because a sponge metabolite, phakellin, i s ostensibly synthesized through an analogous sequence ( 6 ) . 1

0097-6156/ 84/ 0262-0151 $06.00/ 0 © 1984 American Chemical Society

Ragelis; Seafood Toxins ACS Symposium Series; American Chemical Society: Washington, DC, 1984.

152

SEAFOOD TOXINS

14

Feeding Experiments using

C- and 13,C-Labeled Precursors (2)

Our f i r s t attempt was the feeding of commercially available [guanido-14c]-L-arginine to the cultures of Gonyaulax tamarensis (Ipswich s t r a i n ) . The toxin f r a c t i o n was isolated and further fractionated to the pure toxins (7). Figure 1 shows an example of the e l u t i o n pattern of the toxins from a Bio-Rex 70 column. A good c o r r e l a t i o n between the t o x i c i t y and r a d i o a c t i v i t y was observed. The major toxin, gonyautoxin-III was degraded to locate the radio a c t i v i t y i n the carbamoyl moiety. Thus gonyautoxin-III was con verted to saxitoxin by treating with zinc and hydrochloric acid (8), which was then hydrolyzed with 6.7 N HC1 to decarbamoylsaxitoxin and carbon dioxide (9). About one-third of the t o t a l a c t i v i t y (28%) was found to be associated with the released carbon dioxide and the rest with decarbamoylsaxitoxin (Scheme 2). The result seems to be i n Scheme 2 1

I

14,C=NH I NH

Η

I CH

Ν ν . NH,

9

2

Ν

γ 0

Zn-HCL

?2

CH„ HCNH

I

GTX-III

2

ι

OH

OH

OSOo

STX 6.7 N HC1

COOH L-Arginine

+

^= NH + C0 2

2

(28% a c t i v i t y )

Decarbamoyl STX (72% a c t i v i t y )

STX: GTX-III:

Saxitoxin Gonyautoxin-III


13.

SHIMIZU ET AL.

cpm

153

Biosynthesis of Paralytic Shellfish Toxins

GTX-IHI

UK C 29269 cpm)

MU

6000 10000-1

4000 ί ο < ο χ ο

< 5000H

•2000

GTX-V-fV NEOSTX

STX

Fr 60 80 J00 120 140 F i g u r e 1. E l u t i o n p a t t e r n o f t o x i n s from Bio-Rex 70 column i n C - l a b e l e d a r g i n i n e f e e d i n g , ( F r . : f r a c t i o n number; cpm: counts p e r minute; and MU: mouse u n i t s )


154

SEAFOOD TOXINS

agreement with the general understanding that carbamate groups are derived from the ornithine-urea cycle v i a carbamoyl phosphate. The next experiment was the feeding of [l-13c]-d,l-arginine, which was synthesized by the following route. Both gonyautoxin-II and neosaxitoxin were isolated from the culture, and their 13c-nmr S

G

h

e

m

e

3

C H.C0C1 N NCH CH CH CH(OC H ) — * 2

2

2

2

2

5

I

2

1)

1

3

5

2

2

2

2

5

2

O

2

2

-—»·

o

2

"*

C,H C0NHCH CH CH CH0 O

c

6 5

2

O

2

n

2

C N , " N H -

ο)

6

,

C,H-C0NCH.CH CH CH0H

6 5

C H CONHCH CH CH CH(OC H )

H

+

H NCH CH CH_CH 0

2

0

2

2

d,1-ornithine

>

2

H N-C-NH(CH ) CH II I NH NH 0

2

I NH

9

COOH

0

2

0

2

COOH

0

3

2

d,l-arginine

spectra were examined. The r e s u l t s , however, indicated that the carbon-13 was incorporated only randomly i n both compounds. In view of an apparent d i f f i c u l t y for large organic molecules to penetrate into the organism and reach p a r t i c u l a r biosynthetic s i t e s i n intact forms, we have t r i e d the feeding of small basic metabolic u n i t s . The feedings of [l-13c]-acetate resulted i n random incorporations of 13c atoms. S i m i l a r l y , the feeding of doubly labeled [l,2-13c]acetate afforded the toxin molecules whose C-C spin-spin coupling pattern i n the cmr spectra showed no s p e c i f i c groups pattern (Table I and H). Feeding experiments were also t r i e d using [l-13c]and [2-13c]-glycine, both of which were commercially a v a i l a b l e . The main purpose of this experiment was to prove or to disprove a possible involvement of the ordinary purine metabolism i n the toxin biosynthesis (Scheme l a ) . In such a scheme, C-4, C-5, and N-7 of the toxin should be derived from a glycine molecule. Disappointingly the feedings also resulted i n general enrichment of a l l the carbons i n both neosaxitoxin and gonyautoxin-II. However, i n one feeding experiment with [2-13c]-glycine, we observed extra-enrichment of C - l l and C-12 i n gonyautoxin-II, while a l l the other carbons were also enriched (about 10 times that of the natural abundance) (Scheme 4). This unexpected enrichment of two neighboring carbons from a single-labeled precursor can be explained by assuming that labeled glycine was introduced into TCA cycle v i a malate. In the cycle, the molecular asymmetricity w i l l be lost at the succinate step, and the labeling w i l l appear on both C-2 and C-3 of succinate. The r e s u l t seems to support Scheme l c , since C-4 and C-3 of g l u t a mate correspond to C - l l and C-12 of the toxin molecule i n such a scheme. Despite e f f o r t s to pinpoint the molecular o r i g i n of PSP, the results have been far from conclusive. The major obstacle i s the reluctance of the organism to u t i l i z e exogenous organic compounds. Generally prototrophic d i n o f l a g e l l a t e s such as Gonyaulax tamarensis are known to be very s e l e c t i v e i n u t i l i z a t i o n of organic compounds,



2

2

2

(co

(co

(co

3

2-^" C-glycine

6

C-glycme

(2.2 χ 10 dpm)

1-

(3.3 χ 10 dpm)

7

guanido-"^C-L-arginine

7

14 guanidoC-L-arginine (2.2 χ 10 dpm)

( r a d i o a c t i v i t y i n dpm*)

Precursors

**calculated

from the r a d i o a c t i v i t y of co-fed

d i s i n t e g r a t i o n per minute

meager)

4

meager)

3

sufficient)

2

meager)

*dpm:

2

(co

1

Feeding Experiments

14, C-glycine.

(0.5%**)

71,250 mu

(1 .2 χ 10"* dpm, 0,.56%)

35,600 mu

21,900 mu 4 0,.19%) (6 .1 χ 10 dpm,

(2 .4 χ 10"* dpm, 1,,1%)

50,000 mu

Total Toxins

4

dpm, 0.24%)

3

dpm, 0.09%)

4

dpm, 0.2%)

(2,.2 atom excess %**]

43,500 mu

(4.8 X 10

15,500 mu

(9.9 X 10

6,940 mu

(5.3 X 10

21,200 mu

GTX-III

Amounts of toxins i s o l a t e d i n mouse units (mu) and r a d i o a c t i v i t y

Table I. Incorporation r a t i o s of precursors into the toxins and gonyautoxin-III (GTX-III) produced by Gonyaulax tamarensis


13 C

Histidine-C -(U) (1.1 χ 10? dpm)

U

Arginine-1,2,3,4,5(1.45 χ 107 dpm)

Arginine-1-

14 C

46,000 mu 122,857 dpm

48,000 mu 47,143 dpm

35,000 mu

56,000 mu

Ornithine-l- C

13

75,000 mu

13

Toxin isolated and amount

1.7% of RA i n C02. 98.3% i n decarbamoylSTX r a t i o (1:57.8)

GTX-VIII, 8,35/dpm

1

7.9% of RA i n C0 . 92.1% i n decarbamoylSTX r a t i o (1:12.7) 2

0.27% incorporation, no s p e c i f i c incorporation from ^ C . J ^ J R

13 C-NMR showed no s p e c i f i c incorporation

13 C-NMR showed no s p e c i f i c incorporation

Comments

GTX-III, 8,285 dpm

GTX-II, 12,000 mu

GTX-II, 11,570 mu

GTX-II, 12,000 mu

(in mouse units (mu) and r a d i o a c t i v i t y (dpm))

Amount of Crude Toxins Obtained

Acetate-l,2- C

Precursor

Table I I . Results of feedings of various precursors to G. tamarensis culture

13.

SHIMIZU ET AL.

157

Biosynthesis of Paralytic Shellfish Toxins

Scheme 4

TCA

Cycle

1.70 H

2

N

Η

V

9

Ν ^

Ν

^ · Ν

(1.70) GTX-II 4.50"" SO 13. ^C-Enrichment pattern of gonyautoxin-II (GTX-II) from the [ 2 - C ] glycine fed Gonyaulax tamarensis culture, and a possible pathway leading to the enrichment at C - l l and C-12. The numbers depict atom excess % calculated from the r e l a t i v e peak i n t e n s i t i e s i n the spectra of the enriched and unenriched samples based on an average enrich ment of 2.2 atom excess %. 13

i f they accept them at a l l . The second problem may be the compartmentalization of biochemical reactions involved i n the formation of the toxins. Under such a circumstance, a precursor, i f exogenously fed, reaches the biosynthetic s i t e only after cleaved to basic fragments. The t h i r d problem i s a technical one. Unlike other microorganisms, the growth of d i n o f l a g e l l a t e s i s very slow, and the maximum attainable population i s also low. As a r e s u l t , the feeding experiment had to be carried out t y p i c a l l y i n 50 L of culture media to obtain a few milligrams of pure toxins. Although s t e r i l e tech niques were used throughout the experiment, there was a p o s s i b i l i t y that small amounts of precursors added to the vast amounts of culture media were quickly metabolized by contaminated or symbiotic bacteria before the d i n o f l a g e l l a t e u t i l i z e d them. Attempts to con centrate the organisms before feeding the precursors or to use the c e l l homogenate have so far given negative r e s u l t s . Biochemical Conversion of PSP When the homogenates of toxic scallops were incubated, other d r a s t i c changes i n the toxin p r o f i l e were observed (10); propor t i o n a l l y gonyautoxin-I - IV and neosaxitoxin decreased and saxitoxin increased. In another instance, the analysis of Mytilus exposed to the 1980 red tide at Sonoma County, C a l i f o r n i a , showed the almost exclusive presence of neosaxitoxin i n mussels collected just after the red tide and a gradual increase of saxitoxin (Krueger, Meyer and Shimizu, unpublished). These observations suggested the possible


158

SEAFOOD TOXINS

Scheme 5 z

T-Labeled neoSTX

H

» L H STX: , ^ C-Labeled STX RN' ^4-' \ . R=H,X=H C-Labeled GTX-II V N H neoSTX: „ xt^at^VN/ R=OH,X=H In Scallop and Clam Homogenates 2 ) \ Η GTX-II· X

N

4

2

h

N

\ / O H

R=H,X=OSO "

H

3

transformation of neosaxitoxin and gonyautoxins to saxitoxin i n the s h e l l f i s h bodies. The available C - l a b e l e d toxins obtained by C labeled arginine feeding made i t possible to study the conversion i n a more d e f i n i t i v e way. Experiments using the homogenates of Placopecten magellanicus , Mya arenaria, and Mercenaria mercenaria showed the d e f i n i t e bioconversion of neosaxitoxin to saxitoxin. The conversion of gonyautoxins with 11-0-sulfate moieties to saxitoxin was also recognized. However, the same experiments using boiled homogenates also afforded a small amount of saxitoxin, indicating that the conversion may also undergo non-enzymatically or microb i a l l y by contaminated bacteria. 14

1 4

15

Biosynthetic N-Enrichment of PSP Toxins (11) The *"*N-enrichment of PSP toxins i s important to obtain the ^N-nmr data of the toxins. Such data w i l l be useful i n future biosynthetic studies using 15 -labeled precursors. T y p i c a l l y only a few m i l l i grams of i n d i v i d u a l p u r i f i e d toxins can be obtained out of 50 L of culture medium, and i t i s simply p r o h i b i t i v e to obtain enough of the toxin for the natural abundance ^N-nmr spectroscopy. The purpose of this experiment was to check the f e a s i b i l i t y of biosynthesizing highly enriched toxins by giving this photosynthetic organism an inorganic l^N-precursor. Gonyaulax tamarensis was grown i n enriched sea water. The f u l l - s t r e n g t h G u i l l a r d medium (77 L) added with N a N 0 (99% enrich ment, 0.1 g/L). After 33 days, the c e l l s (2 χ 10^) were harvested and processed according to the previously reported method (6). Chromatography of the crude toxic f r a c t i o n on Bio-Rex 70 afforded gonyautoxin-II (14 mg) and neosaxitoxin (6 mg). For l^N-iimr measure ments, the samples were further p u r i f i e d by Bio-Rex 70 and Chelex 100 r e s i n . The spectra were obtained i n two modes: protondecoupled with NOE and proton-coupled with NOE. The observed chemical s h i f t s , coupling constants and parameters used were sum marized i n Table I I I . These r e s u l t s are i n good agreement with the f u l l y protonated forms of the proposed structures i n both compounds excluding alternative structures such as one having a hydroxyl group on N^. The experiment also opened a way to use l^N-nmr for structure study and biosynthetic work of the toxins even i f they are produced only i n minute quantities. N

15

3



99.0 (s)

101.0 ( d )

N-3

N-7

,]

,Hz

Hz

d

J

NC(5)=9.4

NC(10)=8.4

NC(6)=7.8

NC

r

S

1

·ς

|

î

^

2

(d): doublet; and ( t ) : t r i p l e t .

f) Assignment may be exchangeable

e) Assignment may be exchangeable

from Carbon-13 NMR Spectra

c) ( s ) : s i n g l e t ;

d) Obtained

2

^

90 .9

99 .1

93 .2

99 .6

lj

J

c m

I

χ

S

2

6

76.6 ( t )

91.1

f

)

86.2 ( d )

s

94.7

(

8

4

70.4 ( t )

6

96.4 (s) ·

93.1

9

J

N NH H'

92 .8

e

J

87.0 ( t )

m

95 .5

p

ppm

80.4 (d)

P

f

2

d

NC(10)=6.4

NC

101.0 ( d )

90.7

lj

J

Gonyautoxin-II^ __——,

;

a) Solute concentration 1.8 mM, 5% D 0/H 0, pH 4.0

e

NH

94.2

J

Neosaxitoxin

3

—

Nitrogen-15 chemical s h i f t s of Neosaxitoxin and Gonyautoxin-II

b) Solute concentration 3.3 mM, 5% D 0/H 0, pH 4.0

N

76.8 ( t )

f

87.4 ( d )

N-9

1 4

6

N

70.7 ( t )

f

e

8

N

88.7 ( t )

140.1 (s)

ppm

2

N-l

Position

Table I I I .

SEAFOOD

160

TOXINS

Acknowledgments The work was supported by USPHS grants GM24425 and GM28754. L i t e r a t u r e Cited 1. 2. 3.

4. 5. 6. 7. 8. 9. 10. 11.

Taylor, D. L.; Seliger, H. H. "Toxic Dinoflagellate Blooms"; Elsevier/North Holland: New York, 1979. Shimizu, Y. Pure and Appl. Chem. 1982, 54, 1973. Shimizu, Y. "Paralytic S h e l l f i s h Poisons"; In "Progress i n the Chemistry of Organic Natural Products"; Herz, W.; Griesbach, H.; Kirby, G. W., Eds.; Springer-Verlag: Wien-New York, 1983, i n press. Proctor, Ν. H.; Chan, S. L.; Travor, A. J. Toxicon 1975, 13, 1. Sharma, G.; Magdoff-Fairchild, B. J . Org. Chem. 1977, 42, 4118. Foley, L. H.; Buchi, G. J . Am. Chem. Soc. 1982, 104, 1776. Oshima, Y.; Buckley, L. J.; Alam, M.; Shimizu, Y. Comp. Biochem. Physiol. 1977, 57c, 31. Shimizu, Y.; Hsu, C. P. Chem. Commun. 1981, 314. Ghazarossian, V. E.; Schantz, E. J.; Schnoes, H. K.; Strong, F. M. Biochem. Biophys. Res. Comm. 1976, 68, 776. Shimizu, Y.; Yoshioka, M. Science 1981, 212, 546. Hori, Α.; Shimizu, Y. Chem. Commun. 1983, 790-792.

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February

6, 1984


Biosynthesis of Paralytic Shellfish Toxins - ACS Symposium Series

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