Total Synthesis of Okadaic Acid

Till now, two total syntheses of okadaic acid (82) have been reported.244 Isobe et al published first total synthesis of (82) using D-glucose derived synthon (85) through the coupling of three segments A, B, C in 28 steps (8590). The coupling is principally based on the strategy utilizing sulfonyl-carbanion as key reactions. The high acyclic stereoselectivity is attributed to a successful development of switching the syn/anti-diastereoselectivity in the heterocunjugate addition.

The compound (82) was made through a series of steps from (85) in good yield. Treatment of (89) with NaClO2 at room temperature for 1 hr in aq. t-BuOH (containing NaHPO4 and 2-methyl-2-butene) followed by treatment of (88) with lithium metal in liquid ammonia afforded okadaic acid (82) in 80% yield.

5.3.1 Mode of Action of Okadaic Acid and its Congeners Okadaic acid (82) has been found to act as an inhibitor of the protein phosphatases.245 Numerous biochemical and pharmacological studies have been carried out using (82) as a probe. The biological activities of (82) are now considered to be due to its inhibitory action against protein phosphatases. Okadaic acid (82) inhibited PP2A at the lowest concentration (Ki of 30 pm) PPI inhibited at the next lowest concentration, and PP2B at the highest concentration. Okadaic acid (82) showed no effect on PP2C. Okadaic acid and DTX1 had been reported to be non-phorbol ester type cancer promoter.246 In contrast to phorbol esters, which activate protein kinase C, okadaic acid inhibited dephosphorylation of proteins, predominantly serine/threonine residues. Okadaic acid and phorbol both cause the accumulation of essentially the same phosphorylated proteins, some of which are involved in tumor promotion.247 Several groups have studied the structure activity relationship of (82). Alteration of the C1 carboxylic acid or 24-OH greatly reduced the activity. Hydrogenation of C14 and C15 double bond or deoxidation at C2

82, R = Bz 90, R = H

which affect the pseudo cyclic conformation formed by interaction between Q carboxylic acid and C24 hydroxyl group also reduced the potency.

5.4 Pectenotoxins

Pectenotoxin-l (PTXb 91 )248254 was isolated as one of the diarrheic shellfish toxins from the digestive glands of the scallop, Patinopecten yessoensis found Northeastern Japan.242 The toxins were isolated by repeated chromatography on silicic acid and gel permeation through Sephadex LH-20. DTX1, DTX2 and DTX3 which had bulky fatty acid moiety were separated by this procedure, HPLC on reversed phase column and HPLC on a silicic


acid column were used to separate PTX1 from PTX3 and PTX2 from PTX4. 200 kg of digestive glands of scallop yielded pectenotoxin-l (PTX1) (20 mg) m.p. 208-209°C; [a]D +17.1° (c, 0.41 MeOH); pectenotoxin-2 (PTX2) (40 mg) as white amorphous solid, [a]D +16.2° (c, 0.015 MeOH); pectenotoxin-3 (PTX3)10 mg; pectenotoxin-4 (PTX4) (7 mg); and pectenotoxin-5 (PTX5) (0.5 mg) as colorless solids.

Minimum lethal dose of PTX1 to mouse was 250 ^g/kg (ip); PTX2 60 ^g/kg (ip). Structure (91) for pectenotoxin-l (PTX1) was assigned by X-ray crystallographic analysis.242 The structures of four pectenotoxin homolog have been elucidated so far.255 They all have essentially the same structure except at C43 where various stages of oxidation from methyl to carboxylic acid are found (PTX2, CH3; PTX1, CH2OH; PTX3, CHO, and PTX6, CO2H). PTX1 was also found in dinoflagellate Dinophysis fortii along with dinophysistoxin-l.233

Histopathological investigations have shown that PTX1 is hepatotoxic and induces rapid necrosis of hepatocytes. The pathological action of PTX! resembles that of phalloidin.256 PTXs are substantially different from other dinoflagellate toxins, especially in a longer carbon backbone (C40), a C33 lactones ring rather than an acyclo structure, and a novel dioxabicyclo moiety. The large oxygen-rich internal cavity is grossly similar to cavities found in the polyether ionophores from terrestrial microorganisms.

5.5 Yessotoxin

Yessotoxin (92), a novel polyether toxin, had been isolated from scallops Patinopecten yessoensis implicated in diarrheic shellfish poisoning.242,257261 Digestive glands of scallops (84 kg) collected at Mutsu Bay, Japan, yielded 60 mg yessotoxin (92) as an amorphous solid. The toxin was isolated from the toxin fractions by initial chromatography on an alumina column, and subsequently by successive chromatography on Fujgel (DDS-Q3), Develosil and Toyopearl. It had [a]D + 3.01 (c, 0.45, MeOH); UV ^max (MeOH) 230 nm (e 10,600), IR (KBr), 3400, 1240, 1220 cm- ; FABMs (negative), m/z 1163 (M+-Na).

The toxin killed mice at a dose of 100 ^g/kg (ip), but caused no fluid accumulation in suckling mice intestines even at the fatal dose. Yessotoxin (C55H80Na2O21S2) in 13C NMR revealed the presence of 55 carbons consisting of six methyls, 18 methylenes, 24 methines and seven quaternary carbons. The connectivities of the protons were established by detailed analysis of 1H-1H COSY, 1H-1H-RELAY and 13C-1H COSY. Positions of ether bonds were determined mainly by COLOC (7 Hz) and NOE measurements with use of phase sensitive NOESY and ROESY experiments. The presence of sulphate ester(s) suggested by IR bands at 1240, 1220 and 820 cm- was confirmed by elemental analysis for sulphur by ion chromatography of sulfate ions liberated by solvolysis. The position of the esters was determined by comparison between 1H NMR spectra of desulphated YTX and intact toxin. The data suggested that yessotoxin structure (92)262 partly resembles those of brevetoxins, yet YTX is distinct from brevetoxins in having a longer backbone of 42 carbons or a terminal side chain of nine carbons, two sulphate esters, and in lacking carbonyl groups.

6. Miscellaneous Toxins

Screening of micro algae for toxin production led to the isolation of a wide variety of bioactive metabolites from dinoflagellates. Some of these compounds were possibly implicated in poisonings.

6.1 Amphidinolides

Three groups of macrolides named amphidinolides had been isolated from the dinoflagellate Amphidinium spp. symbiotic to flatworm Amphiscolops breviviridis263 The cultivated dinoflagellate Amphidinium spp. from an Okinawan flatworm Amphiscolops spp. furnished amphidinolide-A,264 and amphidinolide-B265 and amphidinolide-C (0.0015% wet weight).263 Amphidinolide-C (93), obtained as colorless amorphous solid, [a]D-106° (c, 1.0 CHCl3) had molecular formula C41H62O10. 1H and 13C NMR studies of the toxin revealed the presence of two isolated ketones, an ester carbonyl, five olefins, 12 methines (nine of them bearing oxygen atoms), 10 methylenes, and six methyl groups. Extensive 400 and/or 500 MHz NMR analyses, 2D NMR techniques, in particular, conventional COSY coupled with double relayed coherence transfer (RCT2) experiments were very effective for deducing the partial structures. The assignments of the carbons bearing hydrogen were established by 1H and 13C COSY via one bond coupling. The phase sensitive 2D COSY facilitated by one-dimensional difference NOE experiments provided useful information to determine the geometries of double bonds. Three segments (A-C) were separated by three carbonyls. The connectivities of three segments (A-C) were clearly established by HMBC spectrum, and finally structure (93) was assigned to amphidinolide-C. A number of macrolides with structures similarities, have been isolated from sponges,266 nudibranchs267 and cyanophytes.268,269 The question remains as to whether these macrolides were produced by host animals or symbiotic microorganisms.


6.2 Amphidinol

Amphidinol 3 (94) (12 mg)270,271 was isolated from cultured cells (440 L) of the dinoflagellate Amphidinium klebsii, collected at Ishigaki island, Japan.272 Recently, Murata et al273 reported the absolute configuration of amphidinol 3 (94) by using newly developed configurational analysis based on carbon-hydrogen spin coupling constant. In order to facilitate measurements of

2.3 JCH authors prepared a 13C-enriched sample of (94) (8 mg) by making another culture (200 L) in the presence of 12 mM NaH13CO3. Stereochemistry of (94) was accomplished as below; (a) the /-based method was used for the determination of stereochemistry at the acyclic parts with 1,2- and 1,3-chiral


centers, C20-C27, C32-C34, C38-C39, C44-C45, and C50-C51; (b) the NOE analysis combined with J analysis was used for two ether cycles and their linkage C39-C44; (c) the modified Mosher method and chromatographic/ NMR comparison were used for degradation products to determine the absolute stereochemistry at C2, C6, C10, C14, C23, and C39. 3JH,H and 2,3 JCH values of intact (94) were measured by E. COSY and hetero half-filtered ' TOCSY (HETLOC), respectively, phase sensitive HMBC was also used for parts where the small magnetization transfer by TOCSY hampered the accurate measurement of 2,3 JCH by HETLOC. The values for 2J(C32, H-33) and 3J(C34, H-32) indicate that H-33 is anti to C32-OH8 and H-32 is gauche to C34, respectively. These interactions unambiguously establish the threo configuration for C32-C33. For C38-C39, 3J(H-38, H-39), which is intermediate between anti and gauche, suggests that this bond undergoes a conformational change. The two small values for 3J(C37, H-39) and 3J(C40, H-38) indicate gauche C37/H-39 and gauche C40/H-38 interactions in both conformers. Of the six possible pairs of alternating rotamers arising from erythro and threo configurations, only one pair in satisfies all of these requirements. The relative configurations of the consecutive stereogenic center in C20-C27 was determined using this method. The diastereomeric relationships of C44-C45 and C50-C51 were assigned in the same manner on the basis of 3JH,H and 2,3JC,H. The configurations of rings A/B and their linkage (C39-C44) were elucidated using NOEs in combination with 3JH,H and 2'3JC,H data. These NMR-based analyses using intact (94) have revealed the relative configurations of C20-C27 and C32-C51. The absolute configurations of (94) was determined by the analysis of the degradation products of Amphidinol 3 (94). Treatment of (94) with HIO4/NaBH4, followed by esterification with (R)- and (S )-MTPA (2-methoxy-2-trifluoromethyl-2-phenylacetic acid) and separation by HPLC, furnished MTPA esters of fragments corresponding to C2-C20, C21-C24 and C33-C50. Based on this elegant study amphidinol 3 (94) was assigned the structure with the absolute stereochemistry of 2S, 6R, 10R, 14R, 20S, 21S, 23S, 24R, 25S, 27S, 32R, 33S, 34R, 35R, 36R, 38R, 39R, 43R, 44R, 45R, 47R, 48R, 49R, 50S, and 51R. Amphidinol (94) is the first representative of a new class of polypeptide metabolites exhibiting potent antifungal and hemolytic activities. Growth inhibiting activity (6 ^g/disk) of 94 against

Aspergillus niger was three times that of amphotericin-B; the hemolytic activity was 120 times that of standard saponin.

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