Hymenosulphate, a novel sterol sulphate with Ca-releasing activity from the cultured marine haptophyteHymenomonassp

摘要

J. CHEM. SOC. PERKIN TRANS. I 1989 Hymenosulphate, A Novel Sterol Sulphate with Ca-Releasing Activity from the Cultured Marine Haptophyte Hymenomonas sp. Jun'ichi Kobayashi," Masami Ishibashi, Hideshi Nakamura, and Yasushi Ohizumi Mitsubishi Kasei Institute of Life Sciences, Minamioo ya, Machida, Tokyo 794,Japan Yoshimasa Hirata Faculty of Pharmacy, Me40 University, Nag0 ya 468,Japan A novel sterol sulphate, hymenosulphate (I), with potent Ca-releasing activity in sarcoplasmic reticulum (SR), has been isolated from the cultured marine haptophyte Hymenomonas sp. and the structure elucidated on the basis of spectroscopic data and chemical reactions. This is the first isolation of a sterol sulphate from marine microalgae. The Haptophyceae are microscopic, unicellar algae, which are widely distributed in the oceans and often constitute a major proportion of marine phytoplankton.' Although symbiotic or ingested marine micro-organisms are of considerable current interest as a source of compounds with useful bioactivity,2 few studies have been performed on the chemistry and the biological activity of the metabolites of haptophycean algae.? During our research for bioactive substances from marine mi~roalgae,~.~ we have studied the chemical constituents of a laboratory-cultured haptophyte Hymenomonas sp.In this paper we report the isolation and structure elucidation of hymenosulphate (l),a new C2,-sterol sulphate with potent SR (sarcoplasmic reticulum) Ca-releasing activity from this microalga. (1) R s S0,Na (2) R = H Results and Discussion The haptophyte Hymenomonas sp.was isolated from an unidentified Okinawan stony coral. The microalga was grown unialgally in a sea water medium enriched with Provasoli's ES supplement2 and the cultured cells were harvested by centrifugation (ca. 400 g of cells from 1 000 1 of culture). The extracts of the harvested cells with methanol-toluene (3 :1)were partitioned between toluene and water. The aqueous layer was successively extracted with chloroform. The SR Ca-releasing activity was found in the toluene-soluble portion, which was chromatographed on a LH-20 column MeOH-CHCl, (1:l) followed by a silica gel column CCHC1,-1-BuOH-AcOH-H20 (1.5:6: 1:l) to give hymenosulphate (1) in 0.007 yield (wet ?The composition of sterols and fatty acids in four marine hapto- phycean algae has been examined by g.c.-m.s.analysis: J. K. Volkman, D. J. Smith, C.Eglinton, T. E. V. Forsberg, and E. D. S. Corner, J. Mar. Biol. Assoc. U. K., 1981, 61, 509. weight). The major components of the toluene layer proved to be glycolipids such as mono- and di-galactosyldiacylglycerols (3) and (4), respectively, while the chloroform layer was found to contain mainly octadecatetraenoic acid (5) together with a small amount of monogalactosylmonoacylglycerol (6).637 Hymenosulphate (l), m.p. 247-250deg;C, uk2 -23" (c 0.1 in MeOH), gave a quasi-molecular ion peak at m/z 491 (M--Na) in its negative fast-atom bombardment mass spectrum (f.a.b.m.s.).The 'H and 13C n.m.r. spectra of com- pound (1) indicated the presence of seven methyl groups and two trisubstituted double bonds. Compound (1) remained unchanged on reaction with Ac,O-pyridine, CH2N2, NaBH,, or KOH-MeOH. However, treatment in refluxing methanolic hydrochloric acid afforded a less polar product (2), m.p. 15amp; 152 "C.The molecular formula of compound (2) was established to be C2,H4,0 by high-resolution electron impact mass spectroscopy (e.i.m.s.) m/z412.3698 (M+),A -0.7 mmu). The difference in molecular weight between compounds (1) and (2), along with the observation of the remarkable change in the polarity RF values on silica gel t.1.c. developed with methanol- chloroform (2: 8); (1) 0.23 and (2) 0.91 suggested the presence of a sulphate group in (l),which was supported by the i.r.absorption' of (1) at 1 250 and 1 220 cm-' and confirmed by a sodium rhodizonate test as well as ion chromatography of sulphate ions liberated by solvolysis.lo The e.i.m.s. analysis of compound (2) revealed that it was a C2,-sterol with an unusual C,, side-chain m/z 271 (M+ -C10H21); base peak. By comparison of the 'H n.m.r. data with those in the literature this sterol (2) was identified as (24R)-23, 24-dimethylcholesta- (22E)-5,22-dien-3P-o1, which was previously obtained from some marine organisms.?.' ',I2 Thus the structure of hymeno- sulphate (C,,H,,NaO,S) was established as (1). Hymenosulphate (1) is the first sterol sulphate from marine microalgae, although several sterol sulphates have been isolated from marine sponges.13 In the SR,4 the Ca-releasing activity of hymenosulphate (1) was ten times more potent than that of caffeine, a well known Ca-releaser.The glycolipids (3), (4), and (6)exhibited inhibition of Naf, K+-ATPase activity l4 with an IC,, value of 2 x lOP5~each. Experimental M.p.s were determined with a Yanagimoto micro melting point apparatus and are uncorrected. Optical rotations were measured on a JASCO DIP-360 polarimeter. 1.r. spectra were obtained with a Hitachi 260-50 spectrometer. 'H and 13C N.m.r. spectra were recorded on a Bruker AM-500 spectrometer. Mass spectra were obtained with a JEOL HX-100 spectrometer. Wako C-300silica gel (Wako Pure Chemical) was used for glass 102 J.CHEM. SOC. PERKIN TRANS. I 1989 (3)R' = R2= X (6)R1= H,R2= X R2 HO OH HOh HO ? X column chromatography. T.1.c. was carried out on Merck silica gel GF254* Isolation.-The haptophyte Hymenomonas sp. was obtained from an unidentified cylindrical stony coral collected at Sesoko Island, Okinawa. The isolation of the microalga was performed by shaking the coral after a rinse with sterilized sea water. The alga was mass cultured in the laboratory by the procedure previously described.2 The alga (408 g, wet weight) harvested from the culture (1 040 1) by centrifugation was extracted with methanol-toluene (3:l; 500 ml x 3). After addition of IM aqueous NaCl(750 ml), the mixture was extracted with toluene (250 ml x 4) and the aqueous layer was then extracted with chloroform (250 ml x 4).The toluene-soluble fraction was evaporated under reduced pressure to give a crude residue (9.7 g), part of which (1.8 g) was subjected to gel filtration on Sephadex LH-20 (2.1 x 110 cm) eluted with chloroform-methanol (1 : 1). Evaporation of the fraction eluting between 13amp;160 ml gave a residue (620 mg), part of which (130 mg) was further separated by silica gel column chromatography (1.7 x 34 cm) with methanol-chloro- form (5:95; 300 ml, 10:90; 100 ml, and 25: 75,200 ml) as eluant to afford monogalactosyldiacylglycerol (3) (27 mg), f.a.b.m.s. m/z 771 (M' + H) in the 160-220 ml fraction and digalacto- syldiacylglycerol(4) (22 mg), f.a.b.m.s. mjz 933 (M+ +H) in the 37W20 ml fraction.The acid part of both glycolipids was identified as being mainly octadecatetraenoic acid after alkaline hydrolysis. Galactose was also identified by g.1.c. after methanolysis and trimethylsilylation. The fraction (33 mg) eluting between 430480 ml of the Sephadex LH-20 column described above was purified by flash chromatography on a silica gel column (1.8 x 36 cm) eluted with chloroform-butan- 1-01-acetic acid-water (1.5:6: 1: 1) to give, in the 120-150 ml fraction, hymenosulphate (1) (6 mg), m.p. 247-250 "C (from MeOH); cti2 -23" (c 0.1 in MeOH); i.r. (KBr) 1 575, 1410, 1 250,l 220,l 065,995,860, and 805 cm-'; 6, CDCl,-CD,OD (1: l) 5.39 (1 H, d, J4.9 Hz, 6-H), 4.91 (1 H, d, J9.6 Hz, 22-H), 4.23 (1 H, m, 3-H), 2.41 (1 H, m, 20-H), 1.53 (3 H, s, 29-H,), 1.04 (3 H, s, 19-H3), 0.96 (6 H, d, J6.7 Hz, 21-and 28-H3), 0.86 (3 H, d, J6.6 Hz, 26-H,), 0.80 (3 H, d, J6.6 Hz, 27-H3), and 0.74 (3 H, s, 18-H3); 6, CCDC1,-CD,OD (1: l) 140.70, 135.86, 132.40, 123.00,79.76,57.52,57.46,50.92, 50.90,42.81,40.34, 39.76,37.82, 37.12, 35.15, 32.54, 32.51, 31.39, 29.38,28.47,24.81, 22.02, 21.62, 20.98, 20.39, 19.62, 17.26, 13.45, and 12.57; f.a.b.m.s.(negative; glycerol as matrix) mjz 491 (M--Na); f.a.b.m.s. (positive; (4)R'= R2= X (5)X-OH diethanolamine as matrix) m/z 725 (M+ + diethanolamine x 2 + H). The chloroform-soluble fraction, after evaporation, gave a residue (0.25 g), part of which (68 mg) was separated by silica gel column chromatography (1.7 x 25 cm) with methanol-chloro- form (1 :9) as eluant to give octadecatetraenoic acid (5)(34 mg) in the 40-60 ml fraction and monogalactosylmonoacylgly-cero16 (6) (3 mg), f.a.b.m.s.mjz 513 (Mf + H) in the 19amp;270 ml fraction. Acid Hydrolysis of Hymenosulphate (l).-Hymenosulphate (1) (3 mg) was heated in 2~ HCl(2 ml) and MeOH (2 ml) under reflux for 1 h. After cooling, the reaction mixture was extracted with chloroform (5 ml x 3). The combined chloroform layers were purified by silica gel column chromatography (0.7 x 7 cm) with methanol-chloroform (4: 96) as eluant to give (24R)-23,24-dimethylcholesta-(22E)-5,22-dien-3~-ol(2) (1 mg), m.p. 1-152 "C (lit.," 156-158.5 "C); -50" (c 0.5 in CHC1,) -53.7"); 6,(CDC13) 5.35 (1 H, m, 6-H), 4.89 (1 H, d, J9.6 Hz, 22-H), 3.53 (1 H, m, 3-H), 1.51 (3 H, s, 29-H,), 1.02 (3 H, s, 19-H3), 0.94 (6 H, d, J6.8 Hz, 21-and 28-H,), 0.85 (3 H, d, J 6.5 Hz, 26-H,), 0.79 (3 H, d, J6.7 Hz, 27-H3), and 0.72 (3 H, s, 18-H3);e.i.m.s.m/z (relative intensity, ) 412 (M+),(94), 394 (95), 369 (18), 351 (22), 341 (14), 323 (13), 300 (83), 282 (25), 271 (loo), 255 (82), and 253 (36) (Found: M+, 412.3698.Calc. for C2,H4,0: M,412.3705). Detection of Sulphate Ions by Ion Chromut0gruphy.-Hymenosulphate (1) (1.1 mg) was dissolved in pyridine-dioxane (1 :1) (1 ml) and the solution was heated at 120 "C for 4 h. The solution was evaporated, the residue was suspended in water, and the suspension was loaded on a SEP-PAK C,, cartridge column (Waters Associates). The cartridge column was first washed with water which eluted S042-, then with MeOH which yielded desulphated sterol.Quantitative deter- mination of S042-was carried out by h.p.1.c. on a YMC-Pack AM-314 (ODS) Yamamura Chemical, 7 x 300 mm with an aqueous solution containing 1.0 mM-tetrabutylammonium hydroxide and 0.8 m~-benzene-l,3,5-tricarboxylicacid (flow rate 1.O ml min-I). Elution of the ions was monitored at 280 nm. A calibration curve prepared with Na2S04 was used for quantitative determination. The sulphate ion was detected at R, 4.4 min. This h.p.1.c. analysis showed that 0.73 mol of SO,2-was liberated from one mole of compound (1) by solvolysis. J. CHEM. SOC. PERKIN TRANS. I 1989 Biological Assay.-The extravesicular Ca2 + concentration in sarcoplasmic reticulum was monitored with a Ca2 -+ electrode prepared by the method of Tsien and Rink with modification^.^ Na+, K +-ATPase assay was carried out as previously described.Acknowledgements We thank Professors M. Chihara and I. Inoue (Institute of Biological Sciences, University of Tsukuba) for their kind gift of the haptophyte, and Ms. M. Hamashima and Ms. A. Muroyama of this Institute for their technical assistance. References 1 M. Parke and P. S. Dixon, J. Mar. Bid. Assoc. U. K., 1976,56, 527. 2 J. Kobayashi, M. Ishibashi, M. R. Walichli, H. Nakamura, Y. Hirata, T. Sasaki, and Y. Ohizumi, J. Am. Chem. Soc., 1988, 110, 490 and references cited therein. 3 J. Kobayashi, M. Ishibashi, H. Nakamura, Y.Ohizumi, T. Yamasu, T. Sasaki, and Y. Hirata, Tetrahedron Lett., 1986, 27, 5755; M. Ishibashi, Y. Ohizumi, M. Hamashima, H. Nakamura, Y. Hirata, T. Sasaki, and J. Kobayashi, J. Chem. Soc., Chem. 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