- Email: [email protected]
From the dinoflagellate Alexandrium hiranoi
~ )
Phytochemistt3,, Vol. 48, No. 1, pp. 85 88, 1998 ~) 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0031 9422/98 $19.00+0.00
Pergamon PII: S0031-9422(97)00756-5
F R O M THE D I N O F L A G E L L A T E
ALEXANDRIUM HIRANOI
MASAHIROMURAKAMI,* YuJ! OKITA,~rHISASHIMATSUDA,TATSUFUMIOKINO~ and KATSUM1YAMAGUCHI Laboratory of Marine Biochemistry, Graduate School of Agricultural Life Sciences, The University of Tokyo, Bunkyoku, Tokyo 113, Japan (Received in revisedJbrm 4 August 1997) Key Word Index--Alexandrium hiranoi; dinoflagellate; biosynthesis; goniodomin A; polyether macrolide; TCA cycle; polyketide.
Abstract--A biosynthetic study of polyether macrolide goniodomin A(1), isolated from the dinoflagellate Alexandrium hiranoi, was carried out by feeding sodium [1-~3C]-, [2-13C]- and [1,2-~3C2]-acetate, and [methyl13 " " C]-methlonme. Considering the labelling patterns in a few compounds such as brevetoxins or dinophysistoxins (DTX), it is suggested that the TCA cycle participates, or single carbon deletion processes occur in the biosynthesis of goniodomin A. © 1998 Elsevier Science Ltd. All rights reserved
INTRODUCTION
17/
Recently, it is known that some polyether metabolites of marine dinoflagellates, such as brevetoxins, okadaic acids and dinophysistoxins (DTX), are biosynthesized through a unique pathway [1-6]. Chou and Shimizu [1] proposed that this pathway is different from a normal polyketide pathway and is closely related to TCA cycle and terpenoid metabolism. Similar precursors with normal polyketide pathway are suggested for okadaic acid [3] and DTX-1 [4]. Wright et al. [6] proposed a different pathway for DTX-4, that is the normal polyketide pathway with a single carbon deletion process. In the course of our studies on biologically active substances from microalgae, we found that the dinoflagellate Alexandrium hiranoi K & F (formerly Goniodoma pseudogoniaulax) [7, 8] had a potent antifungal activity, and isolated the polyether macrolide goniodomin A(I) as the antifungal principle [9]. In this paper, we report the biosynthetic origin of goniodomin A.
RESULTS
~
19 \ 21 / " ~ 23
OH
II
Goniodomin A (1)
1 27
35
the biosynthetic pathway of goniodomin A. First, A. hiranoi was cultured with sodium [l-~3C]-acetate. The ~3C N M R spectra including DEPT of ~3C-labelled goniodomin A showed 13 carbons, noted as "c" in Fig. 1 and Table 1, were enriched with sodium [1-~3C]acetate (1,4, 6, 9, 12, 14, 19, 21, 23, 26, 28, 30 and 33). This fact suggests that these carbons were of acetate carbonyl origin. A signal at 6 27.5 was also enhanced but it was impossible to determine the position of enriched carbon in this experiment because two carbons, C 13 and C 17, had the same chemical shift at 6 27.5. A feeding experiment with sodium [ 2 - t 3 C ] acetate indicated that 1 had 24 carbons originating from acetate methyl (2, 3, 3=CH2, 5, 7, 8, 8=CH2, 10, 11, IZ-----CH4, 15, 16, 18, 20, 22, 24, 25, 25~-----CH2, 27, 29, 31, 32, 33-Me and 34). These carbons were noted as 'm' in Fig. 1 and Table 1. There were two enhanced signals, but those could not be assigned in
AND DISCUSSION
Some feeding experiments using A. hiranoi fed with ~3C-labelled compounds were carried out to examine
* Author to whom correspondence should be addressed. t Present address: Central Research Laboratory, Nippon Suisan Kaisha Ltd., Kitano-machi, Hachioji 192, Japan. Present address: Department of Applied Biochemistry, Utsunomiya University, 350 Mine-Machi, Utsunomiya, 321, Japan. 85
86
M. MURAKAMIel al.
I
__0/
m/ - - %
/
Table 1. Isotope incorporation results based on the J3C NMR data of goniodomin A
o.
I
%...."m~m/C%m/OH "
HO.m**C.m~m
m/_-o
I
m
Intensity ratio (labelled/unlabelled)a Jcc(Hz)
I
m
c%m _
// ---~ km~C~.O/ ~c~U'.m ~ m I/OH ! m
m M
Fig. 1. The labeling pattern of goniodomin A. m: acetate methyl, c: acetate carbonyl, M: methionine methyl, bold bond: acetate unit.
this experiment. The signal at 6 27.5 was also e n h a n c e d with [2-t3C]-acetate, as in the case o f the feeding experiment with [1-13C]- acetate. T h e 9-Me a n d 34-Me c a r b o n s which h a d a same chemical shift at ~ 20.0 were enriched with sodium [2-13C]-acetate. F u r t h e r m o r e , A . hiranoi was fed with sodium [1, 2-13C2]-acetate to determine the positions o f C2 units t h a t originate f r o m the same acetate unit. T h e result o f the 1D- a n d 2DI N A D E Q U A T E [10, 11] spectra of isolated labelled g o n i o d o m i n A confirmed the positions o f 14 intact acetate units n o t e d as . . . . . in Fig. 1 a n d Table 1. Since the signal for C13, which was n o t assigned by the feeding experiment o f [1J3C]-acetate, showed a cross-peak due to coupling with C 12 originating f r o m acetate carbonyl, C13 was s h o w n to originate from acetate methyl. C 17 was d e t e r m i n e d to originate from acetate c a r b o n y l because the signal for C17 showed a cross-peak due to coupling with C18. C h o u a n d Shimizu [1] p r o p o s e d t h a t the biosynthesis o f brevetoxins is closely related to the T C A cycle a n d terpenoid metabolism. Y a s u m o t o a n d Torigoe [3] a n d N o r t e et al. [4] suggested a similar i n t e r v e n t i o n o f such precursors in a n o r m a l polyketide p a t h w a y to explain the biosynthesis o f okadaic acid a n d D T X - 1 , respectively. F r o m this p o i n t o f view, the biosynthetic origin o f g o n i o d o m i n A could be explained as follows. T w o moieties (C17-C22, C 2 6 - C 3 1 ) are p r e s u m e d to be biosynthesized by a n o r m a l polyketide process because the labelling p a t t e r n s are constructed f r o m a succession o f acetate units. C h o u a n d Shimizu [1] suggested t h a t two " m - m " moieties a n d three " m - m m " moieties are p r e s u m e d to be derived from the intermediates in the T C A cycle. Acetate is c o n d e n s e d with oxaloacetate to form citrate in the cycle. In the first cycle, C2 or C3 o f the newly formed succinate will be originated f r o m acetate methyl (Scheme 1). As a result o f the " n e w " succinate, the " n e w " citrate formed by c o n d e n s a t i o n o f acetate with oxaloacetate in the second cycle will possess " m - m - m " labelling patterns. T h u s the two " c - - m - m " moieties ( C 9 ~ 1 1 , C 1 4 - C 1 6 ) could be derived from succinate a n d the
Position
[I-'3C]acetate
[2-'3C]acetate
1
4.1
0.9
68
c
2 3 3--------CH2 4 5 6 7 8 8~------CH2 9 9-Me 10 11 12 12------CH2 13 14 15 16 17 18 19 20 21 22 23 24 25 25-----CH2 26 27 28 29 30 31 32 33 33-Me 34 34-Me 35 36
1.2 1.0 1.0 3.5 0.6 3.3 0.6 1.3 1.4 3.3 (1.1) c
2.2 1.9 2.3 1.3 2.1 0.9 2.0 1.8 2.5 1.0 (2.0) c 2.0 1.8 1.0 2.1 (1.8)d 1 2.2 3.0 (1.8) a 2.2 1.1 2.4 0.9 2.2 0.9 1.9 2.1 2.2 1.0 2.4 1.0 2.2 1.2 1.7 2.4 1.1 2.6 2.4 (2.0) c 1.1 1.4
68
m m m c m c m m m c m
1.3
1.3 3.0 1.2 (2.3)a 2.8 0,9 1,1 (2.3) d 1.1 3.5 0.9 4.4 1.3 35 0.7 1,4 0,8 3.2 1.0 3.5 1.5 3.3 0.9 1.1 3.4 1 1.3 (1.1)¢ 1.2 1.2
[1,2-'3C2]acetate Assignmentb
-
-
-37 36 48 48 --32 -
-
32 -41 -(41) d 35 35 -(41) d 40 44 44 34 34 35 35 --38 38 43 43 50 49 -33 -33 ----
m
m c m m c m m c m c m c m c m m m c m c m c m m c m m M ---
Intensity of each peak in the labelled 1 divided by that of the corresponding signal in the unlabelled 1, normalized to give a ratio of 1 for an unenriched peak (33-Me for [l'3C]acetate labelling and C 14 for [233C]acetate labelling), bm: acetate methyl, c: acetate carbonyl, M: methionine methyl. C9-Me and 34-Me completely overlapped each other. These values are calculated from the peak at ~ 20.1. dC 13 and C 17 completely overlapped each other. These values are calculated from the peak at 6 27.5.
three "c~rn-m-m" moieties (1/2/3/3~-------CH2, 6/7/8/8--------CH2, 23/24/25/25---------CH2) from a tricarboxylic acid like citrate. T w o intact acetate units (4/5,
Biosynthesis of polyether macrolide goniodomin A #
CO2H CH3~CO2H I #r2C--O I _ ~#/2CH 2 I CO2 H
\
#CH2CO2 H
#D~H 2 I CO2H
#CH2CO2H I CH2 I
#/2CH2CO2H 41 I #12CH2CO2H
C--O
I CO2 H Scheme 1.
12/13) are derived from acetate directly. A methyl and a methylene group noted as "m" in Fig. i (9-Me, 12------CH2)are presumed to be of acetate methyl origin. The methylations at C9 and C 12 are by methyl groups of acetate units with subsequent loss of one C~ unit. This type of methylation was observed in other polyethers from dinofiagellates [2, 4, 12] and bacterial metabolite [13]. Feeding of [methylJ3C]-methionine showed enhancement of one carbon signal at 6 20.0.* The signal at 6 20.0 was also enhanced with sodium [2'3C]-acetate. This fact indicated that one of two methyl carbons (9-Me and 34-Me) was of acetate origin and the other of methionine origin. The ~H selective decoupling experiment (irradiation at 6 0.740; H34Me) revealed that C34-Me was originated from the methionine methyl. Consequently, the origin of C9Me was the acetate methyl. Recently, Wright et al. [6] proposed that backbone carbons of DTX-4 are derived from polyketide without participation of other precursors. This pathway is also applicable to biosynthesis of goniodomin A. Six backbone carbons from the methyl groups of cleaved acetate units (3, 8, 11, 16, 25, 32) are considered to be derived from a single carbon deletion process [6]. The advantage of this proposal is that the isotope enrichment would be expected to be uniform, which is suggested from the enrichment data (Table 1), whereas this is unlikely when TCA intermediates are involved (Scheme 1). In the biosynthesis of DTX-1 and 4 [5, 14], the starter unit is shown to be glycolate. Although there is no data to explain the biosynthetic origin of the remaining two methylene carbons (35 and 36), these carbons are likely of glycolate origin.
* Labelled/unlabelled ratio of signal at 6 20.0 to that of 6 25.4 (C14) was 5 up.
87
EXPERIMENTAL
Culture. Alexandrium hiranoi was isolated from the rock pool at Jogashima, Kanagawa Prefecture (Japan), and cultured in 10 1 glass bottles containing Provasoli medium [nutritive salts mixture (NaNO3350 mg, fl-Na2glycerophosphate 50 mg, Fe (as EDTA; 1 : 1 molar) 2.5 mg, PII metals 25 ml, vitamin B~2 10 #g, vitamin BI 500 #g, biotin 5 #g, Tris 500 mg, HzO 100 ml) 2 ml, 75% sea water 100 ml] [15] under illumination of 500/~E-: s -~ on a 12L-12D cycle at 25 ° for about 20 days. It was made axenic with an antibiotic mixt. (penicillin-G 50 mg -], polymixin-B 50 mg 1-~, neomycin 2.5 mg 1-~, streptomycin 50 mg 1-~, chloramphenicol 0.5 mg 1-~). All ~3C-labelled compounds (NaOAc, methionine) were added 3 days before harvesting and those concentrations were 50 mg 1 ~. Extraction and isolation of goniodomin A. The organism was collected by filtration and extracted with MeOH × 3. The extract was combined, concd to remove MeOH, and partitioned between H20 and Et20. The organic layer was subjected to silica gel column chromatography with C6H6-EtOAc (4:1, 3:2). The C6H6-EtOAc (3:2) fr. was subjected to ODS cc with aq. MeOH. The 83% MeOH fr. was subjected to reversed-phase HPLC with 83% MeOH to yield goniodomin A(1); column CAPCELL P A K Cts, 250 x 10 mm; flow-rate 1.5 ml min-t: detection UV (210 nm) and refractive index. The yield was about 2 mg from 10 1 culture in each feeding experiment. lHand 13CNMR data. The ~H and J3C N M R spectra assignments of 1 were previously described [9]. The J3C N M R data used in this study were acquired in CDC13 at 75 MHz. Chemical shifts are referenced to internal CDC13 at 6 77.0 for carbon. Acknowledgements--This work was partly supported by a Grant-in-aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan. H.M. is financially supported by Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists.
REFERENCES
1. Chou, H. N. and Shimizu, Y., Journal of the American Chemical Society, 1987, 109, 2184. 2. Lee, M. S., Qin, G., Nakanishi, K. and Zagorski, M. G., Journal of the American Chemical Society, 1989, 111, 6234. 3. Yasumoto, T. and Torigoe, K., Journal of Natural Products, 1991, 54, 1486. 4. Norte, M., Padilla, A. and Fern~mdez, J. J., Tetrahedron Letters, 1994, 35, 1441. 5. Needham, J., Hu, T., McLachlan, J. L., Walter, J. A. and Wright, J. L. C., Journal of the Chemical Society, Chemical Communications, 1995, 1623. 6. Wright, J. L. C., Hu, T., McLachlan, J. L., Needham, J. and Walter, J. A., Journal of the American Chemical Society, 1996, 118, 8757.
88
M. MURAKAMIet al.
7. Kita, T., Fukuyo, Y., Tokuda, H. and Hirano, R., Bulletin of Marine Science, 1985, 37, 643. 8. Kita, T. and Fukuyo, Y., Bulletin of the Plankton Society of Japan, 1988, 35, 1. 9. Murakami, M., Makabe, K., Yamaguchi, K., Konosu, S. and W~lchli, M. R., Tetrahedron Letters, 1988, 29, 1149. 10. Bax, A., Freeman, R. and Kempsell, S. P., Journal of the American Chemical Society, 1980, 102, 4849. 11. Mareci, T. H. and Freeman, R., Journal of Magnetic Resonance, 1982, 48, 158. 12. Kobayashi, J., Takahashi, M. and Ishibashi, M.,
Journal of the Chemical Society, Chemical Communications, 1995, 1639. 13. Needham, J., Andersen, R. J. and Kelly, T., Journal of the Chemical Society, Chemical Communications, 1992, 1367. 14. Needham, J., McLachlan, J. L., Walter, J. A. and Wright, J. L. C., Journal of the Chemical Society, Chemical Communications, 1994, 2599. 15. Watanabe, M. M. and Nozaki, H., ed., NIESCollection List of Strains, 4th edn, Microalyae and Protozoa. National Institute for Environmental Studies, Tsukuba, 1994, p. 37.
Phytochemistt3,, Vol. 48, No. 1, pp. 85 88, 1998 ~) 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0031 9422/98 $19.00+0.00
Pergamon PII: S0031-9422(97)00756-5
F R O M THE D I N O F L A G E L L A T E
ALEXANDRIUM HIRANOI
MASAHIROMURAKAMI,* YuJ! OKITA,~rHISASHIMATSUDA,TATSUFUMIOKINO~ and KATSUM1YAMAGUCHI Laboratory of Marine Biochemistry, Graduate School of Agricultural Life Sciences, The University of Tokyo, Bunkyoku, Tokyo 113, Japan (Received in revisedJbrm 4 August 1997) Key Word Index--Alexandrium hiranoi; dinoflagellate; biosynthesis; goniodomin A; polyether macrolide; TCA cycle; polyketide.
Abstract--A biosynthetic study of polyether macrolide goniodomin A(1), isolated from the dinoflagellate Alexandrium hiranoi, was carried out by feeding sodium [1-~3C]-, [2-13C]- and [1,2-~3C2]-acetate, and [methyl13 " " C]-methlonme. Considering the labelling patterns in a few compounds such as brevetoxins or dinophysistoxins (DTX), it is suggested that the TCA cycle participates, or single carbon deletion processes occur in the biosynthesis of goniodomin A. © 1998 Elsevier Science Ltd. All rights reserved
INTRODUCTION
17/
Recently, it is known that some polyether metabolites of marine dinoflagellates, such as brevetoxins, okadaic acids and dinophysistoxins (DTX), are biosynthesized through a unique pathway [1-6]. Chou and Shimizu [1] proposed that this pathway is different from a normal polyketide pathway and is closely related to TCA cycle and terpenoid metabolism. Similar precursors with normal polyketide pathway are suggested for okadaic acid [3] and DTX-1 [4]. Wright et al. [6] proposed a different pathway for DTX-4, that is the normal polyketide pathway with a single carbon deletion process. In the course of our studies on biologically active substances from microalgae, we found that the dinoflagellate Alexandrium hiranoi K & F (formerly Goniodoma pseudogoniaulax) [7, 8] had a potent antifungal activity, and isolated the polyether macrolide goniodomin A(I) as the antifungal principle [9]. In this paper, we report the biosynthetic origin of goniodomin A.
RESULTS
~
19 \ 21 / " ~ 23
OH
II
Goniodomin A (1)
1 27
35
the biosynthetic pathway of goniodomin A. First, A. hiranoi was cultured with sodium [l-~3C]-acetate. The ~3C N M R spectra including DEPT of ~3C-labelled goniodomin A showed 13 carbons, noted as "c" in Fig. 1 and Table 1, were enriched with sodium [1-~3C]acetate (1,4, 6, 9, 12, 14, 19, 21, 23, 26, 28, 30 and 33). This fact suggests that these carbons were of acetate carbonyl origin. A signal at 6 27.5 was also enhanced but it was impossible to determine the position of enriched carbon in this experiment because two carbons, C 13 and C 17, had the same chemical shift at 6 27.5. A feeding experiment with sodium [ 2 - t 3 C ] acetate indicated that 1 had 24 carbons originating from acetate methyl (2, 3, 3=CH2, 5, 7, 8, 8=CH2, 10, 11, IZ-----CH4, 15, 16, 18, 20, 22, 24, 25, 25~-----CH2, 27, 29, 31, 32, 33-Me and 34). These carbons were noted as 'm' in Fig. 1 and Table 1. There were two enhanced signals, but those could not be assigned in
AND DISCUSSION
Some feeding experiments using A. hiranoi fed with ~3C-labelled compounds were carried out to examine
* Author to whom correspondence should be addressed. t Present address: Central Research Laboratory, Nippon Suisan Kaisha Ltd., Kitano-machi, Hachioji 192, Japan. Present address: Department of Applied Biochemistry, Utsunomiya University, 350 Mine-Machi, Utsunomiya, 321, Japan. 85
86
M. MURAKAMIel al.
I
__0/
m/ - - %
/
Table 1. Isotope incorporation results based on the J3C NMR data of goniodomin A
o.
I
%...."m~m/C%m/OH "
HO.m**C.m~m
m/_-o
I
m
Intensity ratio (labelled/unlabelled)a Jcc(Hz)
I
m
c%m _
// ---~ km~C~.O/ ~c~U'.m ~ m I/OH ! m
m M
Fig. 1. The labeling pattern of goniodomin A. m: acetate methyl, c: acetate carbonyl, M: methionine methyl, bold bond: acetate unit.
this experiment. The signal at 6 27.5 was also e n h a n c e d with [2-t3C]-acetate, as in the case o f the feeding experiment with [1-13C]- acetate. T h e 9-Me a n d 34-Me c a r b o n s which h a d a same chemical shift at ~ 20.0 were enriched with sodium [2-13C]-acetate. F u r t h e r m o r e , A . hiranoi was fed with sodium [1, 2-13C2]-acetate to determine the positions o f C2 units t h a t originate f r o m the same acetate unit. T h e result o f the 1D- a n d 2DI N A D E Q U A T E [10, 11] spectra of isolated labelled g o n i o d o m i n A confirmed the positions o f 14 intact acetate units n o t e d as . . . . . in Fig. 1 a n d Table 1. Since the signal for C13, which was n o t assigned by the feeding experiment o f [1J3C]-acetate, showed a cross-peak due to coupling with C 12 originating f r o m acetate carbonyl, C13 was s h o w n to originate from acetate methyl. C 17 was d e t e r m i n e d to originate from acetate c a r b o n y l because the signal for C17 showed a cross-peak due to coupling with C18. C h o u a n d Shimizu [1] p r o p o s e d t h a t the biosynthesis o f brevetoxins is closely related to the T C A cycle a n d terpenoid metabolism. Y a s u m o t o a n d Torigoe [3] a n d N o r t e et al. [4] suggested a similar i n t e r v e n t i o n o f such precursors in a n o r m a l polyketide p a t h w a y to explain the biosynthesis o f okadaic acid a n d D T X - 1 , respectively. F r o m this p o i n t o f view, the biosynthetic origin o f g o n i o d o m i n A could be explained as follows. T w o moieties (C17-C22, C 2 6 - C 3 1 ) are p r e s u m e d to be biosynthesized by a n o r m a l polyketide process because the labelling p a t t e r n s are constructed f r o m a succession o f acetate units. C h o u a n d Shimizu [1] suggested t h a t two " m - m " moieties a n d three " m - m m " moieties are p r e s u m e d to be derived from the intermediates in the T C A cycle. Acetate is c o n d e n s e d with oxaloacetate to form citrate in the cycle. In the first cycle, C2 or C3 o f the newly formed succinate will be originated f r o m acetate methyl (Scheme 1). As a result o f the " n e w " succinate, the " n e w " citrate formed by c o n d e n s a t i o n o f acetate with oxaloacetate in the second cycle will possess " m - m - m " labelling patterns. T h u s the two " c - - m - m " moieties ( C 9 ~ 1 1 , C 1 4 - C 1 6 ) could be derived from succinate a n d the
Position
[I-'3C]acetate
[2-'3C]acetate
1
4.1
0.9
68
c
2 3 3--------CH2 4 5 6 7 8 8~------CH2 9 9-Me 10 11 12 12------CH2 13 14 15 16 17 18 19 20 21 22 23 24 25 25-----CH2 26 27 28 29 30 31 32 33 33-Me 34 34-Me 35 36
1.2 1.0 1.0 3.5 0.6 3.3 0.6 1.3 1.4 3.3 (1.1) c
2.2 1.9 2.3 1.3 2.1 0.9 2.0 1.8 2.5 1.0 (2.0) c 2.0 1.8 1.0 2.1 (1.8)d 1 2.2 3.0 (1.8) a 2.2 1.1 2.4 0.9 2.2 0.9 1.9 2.1 2.2 1.0 2.4 1.0 2.2 1.2 1.7 2.4 1.1 2.6 2.4 (2.0) c 1.1 1.4
68
m m m c m c m m m c m
1.3
1.3 3.0 1.2 (2.3)a 2.8 0,9 1,1 (2.3) d 1.1 3.5 0.9 4.4 1.3 35 0.7 1,4 0,8 3.2 1.0 3.5 1.5 3.3 0.9 1.1 3.4 1 1.3 (1.1)¢ 1.2 1.2
[1,2-'3C2]acetate Assignmentb
-
-
-37 36 48 48 --32 -
-
32 -41 -(41) d 35 35 -(41) d 40 44 44 34 34 35 35 --38 38 43 43 50 49 -33 -33 ----
m
m c m m c m m c m c m c m c m m m c m c m c m m c m m M ---
Intensity of each peak in the labelled 1 divided by that of the corresponding signal in the unlabelled 1, normalized to give a ratio of 1 for an unenriched peak (33-Me for [l'3C]acetate labelling and C 14 for [233C]acetate labelling), bm: acetate methyl, c: acetate carbonyl, M: methionine methyl. C9-Me and 34-Me completely overlapped each other. These values are calculated from the peak at ~ 20.1. dC 13 and C 17 completely overlapped each other. These values are calculated from the peak at 6 27.5.
three "c~rn-m-m" moieties (1/2/3/3~-------CH2, 6/7/8/8--------CH2, 23/24/25/25---------CH2) from a tricarboxylic acid like citrate. T w o intact acetate units (4/5,
Biosynthesis of polyether macrolide goniodomin A #
CO2H CH3~CO2H I #r2C--O I _ ~#/2CH 2 I CO2 H
\
#CH2CO2 H
#D~H 2 I CO2H
#CH2CO2H I CH2 I
#/2CH2CO2H 41 I #12CH2CO2H
C--O
I CO2 H Scheme 1.
12/13) are derived from acetate directly. A methyl and a methylene group noted as "m" in Fig. i (9-Me, 12------CH2)are presumed to be of acetate methyl origin. The methylations at C9 and C 12 are by methyl groups of acetate units with subsequent loss of one C~ unit. This type of methylation was observed in other polyethers from dinofiagellates [2, 4, 12] and bacterial metabolite [13]. Feeding of [methylJ3C]-methionine showed enhancement of one carbon signal at 6 20.0.* The signal at 6 20.0 was also enhanced with sodium [2'3C]-acetate. This fact indicated that one of two methyl carbons (9-Me and 34-Me) was of acetate origin and the other of methionine origin. The ~H selective decoupling experiment (irradiation at 6 0.740; H34Me) revealed that C34-Me was originated from the methionine methyl. Consequently, the origin of C9Me was the acetate methyl. Recently, Wright et al. [6] proposed that backbone carbons of DTX-4 are derived from polyketide without participation of other precursors. This pathway is also applicable to biosynthesis of goniodomin A. Six backbone carbons from the methyl groups of cleaved acetate units (3, 8, 11, 16, 25, 32) are considered to be derived from a single carbon deletion process [6]. The advantage of this proposal is that the isotope enrichment would be expected to be uniform, which is suggested from the enrichment data (Table 1), whereas this is unlikely when TCA intermediates are involved (Scheme 1). In the biosynthesis of DTX-1 and 4 [5, 14], the starter unit is shown to be glycolate. Although there is no data to explain the biosynthetic origin of the remaining two methylene carbons (35 and 36), these carbons are likely of glycolate origin.
* Labelled/unlabelled ratio of signal at 6 20.0 to that of 6 25.4 (C14) was 5 up.
87
EXPERIMENTAL
Culture. Alexandrium hiranoi was isolated from the rock pool at Jogashima, Kanagawa Prefecture (Japan), and cultured in 10 1 glass bottles containing Provasoli medium [nutritive salts mixture (NaNO3350 mg, fl-Na2glycerophosphate 50 mg, Fe (as EDTA; 1 : 1 molar) 2.5 mg, PII metals 25 ml, vitamin B~2 10 #g, vitamin BI 500 #g, biotin 5 #g, Tris 500 mg, HzO 100 ml) 2 ml, 75% sea water 100 ml] [15] under illumination of 500/~E-: s -~ on a 12L-12D cycle at 25 ° for about 20 days. It was made axenic with an antibiotic mixt. (penicillin-G 50 mg -], polymixin-B 50 mg 1-~, neomycin 2.5 mg 1-~, streptomycin 50 mg 1-~, chloramphenicol 0.5 mg 1-~). All ~3C-labelled compounds (NaOAc, methionine) were added 3 days before harvesting and those concentrations were 50 mg 1 ~. Extraction and isolation of goniodomin A. The organism was collected by filtration and extracted with MeOH × 3. The extract was combined, concd to remove MeOH, and partitioned between H20 and Et20. The organic layer was subjected to silica gel column chromatography with C6H6-EtOAc (4:1, 3:2). The C6H6-EtOAc (3:2) fr. was subjected to ODS cc with aq. MeOH. The 83% MeOH fr. was subjected to reversed-phase HPLC with 83% MeOH to yield goniodomin A(1); column CAPCELL P A K Cts, 250 x 10 mm; flow-rate 1.5 ml min-t: detection UV (210 nm) and refractive index. The yield was about 2 mg from 10 1 culture in each feeding experiment. lHand 13CNMR data. The ~H and J3C N M R spectra assignments of 1 were previously described [9]. The J3C N M R data used in this study were acquired in CDC13 at 75 MHz. Chemical shifts are referenced to internal CDC13 at 6 77.0 for carbon. Acknowledgements--This work was partly supported by a Grant-in-aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan. H.M. is financially supported by Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists.
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