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Sterols from the sponge Haliclona chilensis
Comp. Biochem. Physiol. Vol. 81B, No. 1, pp. 119-122, 1985 Printed in Great Britain
0305-0491/85 $3.00+0.00 © 1985 Pergamon Press Ltd
STEROLS F R O M THE SPONGE H A L I C L O N A C H I L E N S I S A. M. SELDES,* J. ROVIROSA,t A. SAN MARTiNt a n d E. G. GROS* *Departamento de Quimica Org~.nica y UMYMFOR, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Pab. 2, Ciudad Universitaria, 1428 Buenos Aires, Argentina and tDepartamento de Quimica, Facultad de Ciencias Bfisicas y Farmaceuticas, Universidad de Chile, Santiago, Chile
(Received 28 August 1984) A b s t r a c t - - 1 . The sterol composition of the sponge Haliclona chilensis (Thiele) has been analysed by argentic column- and thin-layer chromatography and reverse-phase high-performance liquid chromatography detecting twenty-eight components. 2. These were studied by gas chromatography-mass spectrometry and in some cases by proton and carbon-I 3 magnetic resonance spectroscopy. 3. It was established that the components were C26-, C27-, C28-, C29- and C30-A5 sterols and in minor amounts A° and A7 analogs with saturated and unsaturated side-chains. 4. 24-Keto-A 5 and -A° sterols with saturated and unsaturated side-chains were also found among the components of the mixture. 5. This is the first report on the presence of 24-keto-5~-cholestan-3fl-ol and 24-keto-5ct-cholest-25-en-3fl-ol in sponges.
INTRODUCTION M a r i n e organisms have been the source o f n u m e r o u s sterols, some o f t h e m h a v i n g unusual structures w i t h o u t terrestrial counterparts. A m o n g these organisms, sponges contain complex mixtures of sterols, in some cases with new a n d unique structures ( G o a d , 1978; Schmitz, 1978; Sica et al., 1978; Ballantine a n d Lavis, 1979; K a n a z a w a et al., 1979; Delseth et al., 1979; Djerassi, 1981; Li et al., 1981; Bohlin et al., 1981; I t o h et al., 1983; Di G i a c o m o et al., 1983). Since the earlier work o f B e r g m a n n a n d Feeney (1949) o n six species of Haliclona, a few papers on the same genus have a p p e a r e d reporting the presence o f mixtures o f mainly AS-sterols, a n d in some cases with A °- a n d AT-derivatives as m i n o r c o m p o n e n t s (Sheikh a n d Djerassi, 1974; Bergquist et al., 1980), epidioxy derivatives (Calder6n et al., 1982), t r i u n s a t u r a t e d sterols (Zielinski et al., 1982) a n d p r e g n a n e derivatives (Ballantine et al., 1977). In c o n t i n u i n g o u r current interest in sterols from aquatic organisms (Seldes a n d Gros, in press) we have investigated the constitution o f the sterol fraction derived from the sponge Haliclona chilensis, from which we have recently isolated a n d characterized two new dioxygenated steroid constituents (Seldes et al., in press). MATERIALS AND METHODS General Analytical thin-layer chromatography (TLC) was performed on silica gel 60-precoated (0.2 mm) aluminum sheets (Merck), developed with toluene-ethyl acetate (3:1) for free sterols and visualized by spraying with 50% sulfuric acid solution followed by heating. Argentic TLC for sterols acetates was carried out on precoated HPTLC silica gel 60 F254 plates (Merck) sprayed with a 10% silver nitrate hydroalcoholic solution and subsequently dried at 100°C. The plates were developed twice with petroleum ether (40-70°C fraction)-toluene (5:2 or 5:3) and spots were detected in the manner previously described. Column chromatography was carried out on silica gel 60 (70-230 mesh ASTM) (Merck) with petroleum ether-ethyl acetate
mixtures of increasing polarity as the eluant. Argentic column chromatography was performed on silica gel G (Merck) impregnated with 7% silver nitrate hydroalcoholic solution, taken to dryness and activated at 100°C. Chromatography of the sterol acetates was developed with petroleum ether-ethyl acetate (87: 3) as eluant and some fractions were recbromatographed in the same conditions. The progress of column chromatography was monitored by TLC and GC as indicated. Gas chromatography (GC) was performed on a Hewlett-Packard 5840A gas chromatograph equipped with a flame ionization detector and a fused silica capillary column (12 m x 0.2 ram) coated with methyl silicone fluid (HewlettPackard). The carrier gas was helium and analysis were performed between 200 and 280°C at 10°C/min. Relative retention times (RRT) are of the sterol acetates relative to cholesterol acetate. Combined gas chromatography-mass spectrometry (GC-MS) analysis of the free and acetylated sterols was performed on a Varian Mat CH7A mass spectrometer at 70 eV coupled to a Varian 1440 gas chromatograph using a 3% OV-17 GCQ glass column (1.8 m x 2 mm i.d.) and interfaced to a Varian Mat Data System 166 computer with magnetic libraries of standards. Quantification was made by automatic integration of the GC peaks and by single ion monitoring (SIM) of the sterols' base peaks in the mass chromatograms of the mixtures. High-performance liquid chromatography (HPLC) was used for analytical and preparative scale separations of the sterol mixtures obtained by argentic column chromatography, as well as for monitoring compounds purification. Analytical HPLC was performed with a Hewlett-Packard 1084 liquid chromatograph equipped with a refractive index detector and a variable volume injector with automatic sampling system. Separations were carried out with a R-Sil C-18HL 10 #m column (250 x 3.6 mm), packed in our laboratory with a Micromeritics 705 stirred slurry column packer, and with methanolwater (98:2) as the eluant at a flow rate of 3 ml/min. Preparative HPLC was carried out on a Micromeritics liquid chromatograph equipped with a 750 solvent delivery system, a 730 manual injector and a refractive index detector. Separations were performed using two different reversephase columns: Alltech R-Sil C-18HL 10 ,urn (500 x 10 mm, i.d.) and Altex Ultrasphere ODS 5 pm (250 x 10 mm, i.d.) using methanol or methanol-water mixtures at flow rates between 3 and 7 ml/min. Proton magnetic resonance spectra ([IH]NMR) at 100 MHz and carbon-13 magnetic resonance 119
120
A . M . SELDESet al.
spectra ([I-~C]NMR) at 25.2 MHz were performed on a Varian XL-t00-15 spectrometer in the FT mode in deuterochloroform solution containing tetramethylsilane as internal standard. Extraction, isolation andfi'actionation of the sterol mixture Sponges were collected in Puyinqu~ (Chilo6, Chile) in March 1983 and immediately stored in acetone. They were identified by Ruth Desqueyroux-Faundez at the Museum d'Histoire Naturelle (Geneve, Switzerland) as Haliclona chilensis (Thiele) (order Haplosclerida). The material was blended and after keeping the extract at room temperature for 24 hr the resulting slurry was filtered. The solid residue was re-extracted twice more with the same solvent at room temperature. The air-dried sponge residue weighed 430 g. The combined organic extracts were concentrated at reduced pressure and the remaining aqueous residue was extracted twice with chloroform. The combined organic extracts were taken to dryness. The syrup obtained was chromatographed on a silica gel column to afford a crude sterol mixture (9.8 g) which was acetylated (acetic anhydride-pyridine 1 : 1, 25C, 18 he). The sterol acetates mixture was subjected to preliminary separation by argentic column chromatography. The fractions collected were monitored by argentic TLC and GC and those with approximately the same composition were combined to afford eight main fractions. These were subsequently chromatographed by reverse-phase HPLC and the subfractions were analyzed by GC MS as acetylated and free compounds, which were obtained by treatment of the ester derivatives with 53o potassium hydroxide in methanol at room temperature. Compounds were identified by their RRT in capillary GC, by comparison and co-injection with known standards, and by MS of the free and acetylated derivatives. The unknown sterols and those whose structures could not be unambiguously assigned by these methods were rechromatographed, leading in some cases to the isolation of pure compounds, which were studied by NMR spectroscopy.
RESULTS AND DISCUSSION Table 1 lists the twenty-eight sterols isolated from the sponge Haliclona chilensis, together with G C - R R T data, molecular weight, percentage of the sterol components in the mixture and main fragments of the mass spectra of their acetylated derivatives. As can be seen, besides a complex mixture of C26, C27, C28, C29 and C30-A5, and in minor amounts A°- and AT-sterols with saturated and unsaturated side-chains, the sponge contains 24-keto-A 5 and A°-derivatives also with saturated and unsaturated side-chains. 24-Methyl-cholesta-5,24(28)-dien-3/~-ol (13) was the principal component, accounting for 37~o of the total mixture, and cholest-5-en-3//-ol (7) (21%) and 22- trans-24-methyl-cholesta-5,22-dien-3/?-crl (9) (16~o) are also present as main components. These compounds are also found as major sterols in the other three species of Haliclona studied (Sheikh and Djerassi, 1974; Bergquist et al., 1980), differences resting upon percentage distribution of them. 3/3-Hydroxy-AS-derivatives (91%) with saturated and monounsaturated side-chains were characterized by their G C - R R T and comparison with authentic standards, and in some cases, when necessary, by [IH]and [~3C]NMR, and by typical peaks in their mass spectra as free and acetylated derivatives (Wyllie et al., 1977). Stanols (7%) were characterized, by the same procedures, as fully saturated and side-chain unsaturated derivatives. Mass spectra of the acetylated compounds
afforded molecular ions and fragmentation patterns characteristic for these type of sterols. Two AV-sterols were present in minor amounts (l~J~) as Cz6 and C28 compounds. The co-occurrence of compounds 2, 3 and 4 and of compounds 13, 15 and 16 has been reported in other sponges of the same genus (Zielinski et al., 1982). The presence of C26 sterols in marine invertebrates has been realised for some time (Idler et, al., 1970; Viala et al., 1972; G o a d et al., 1972; Erdman and Thomson, 1972) and their occurrence in plankton suggests that this may be the c o m m o n origin of all the C26 marine animal sterols. The presence of c o m p o u n d I may be regarded as an artifact of the extractive procedure, taking into account that 24-methylenecholesterol is the principal component and that initial work-up of the acetylated mixture may favor the deacylation. Its mass spectrum displays the same fragmentation pattern as the acetylated derivative of 13, considering that the last one does not present molecular ions. C o m p o u n d 27 presented G C - R R T and mass spectral data which are fully consistent with those of authentic aplysterol and its acetylated derivative used as standards. Aplystane derivatives had only been recorded previously as sterol components of sponges from the order Verongida (De Rosa et al., 1973), a fact that induced them to be considered as a chemotaxonomic feature restricted to the mentioned order, However, Bergquist et al. (1980) reported the presence of didehydroaplysterol as a major component of the sterol mixture of an Haliclona species (order Haplosclerida) in which, as in our case, the sterol profile was typical of most of the Ceractinomorpha except for the presence of the mentioned 26-methyl substituted derivative. Moreover, Li et al. (1981) reported from another sponge of the order Haplosclerida the presence of aplystane derivatives. In view of these findings, Bergquist et al. (1980) draw attention to the risks of diagnosing groups absolutely on the basis of the presence or absence of particular molecules. As we have already reported (Seldes et al., in press), we isolated from the sterol mixture two 24-keto derivatives namely, 24-keto-cholest-5-en-3/%ol (23) and 24-keto-cholesta-5,25-dien-3/3-ol (26). F r o m the same fraction (the last one from argentic column chromatography) we were able to isolate two minor components, present as traces in the sterol mixture, which were characterized as 24-keto-5e-cholestan-3/3-ol (24) and 24-keto-5~-cholest-25-en-3/3-ol (26) on the basis of their G C - R R T and spectroscopic properties. The mass spectra of their acetylated derivatives presented the characteristic fragmentation pattern of a saturated sterol nucleus. Besides the molecular ion, and the loss of C H 3 C O O H and CH3COOH plus CH3, they included major peaks at m/z 257 (M +-CH3COOH-side-chain) and 215 (M + CH3COOH-side-chainM2), which are diagnostic for saturated steroid nuclei, while the presence of an ion at m/z 255 was indicative of an unsaturated side-chain. Moreover, peaks at m/z 298 and 283 were formed from ( M + - C H 3 C O O H ) by a McLafferty type rearrangement on the C-24 carbonyl group (loss of part of the side-chain, C-23-C-27, plus 1H), while the latter resulted from the loss of an additional methyl group. Both ions have their counterpart (m/z 296 and m/z
Sterols from the sponge Haficlona chilensis
121
Table I. Composition, molecular weight (mol. wt), relative retention times (RRT) and main fragments of mass spectra (MS) ofsterol acetates of H. chilensis
Sterol No.
Sterol systematic nomenclature
Mol. wt
1
24-Methyl-cholesta-3,5,24(28)-triene
380
2
22-trans-24-Norcholesta-5,22-dien-3~-ol
412
3
22-trans-24-Nor-5~-cholest-22-en-3B-ol
414
4
22-trans-24-Norcholesta-7,22-dien-3fl-ol
412
5
22-trans-24-Methyl-27-norcholesta-5,22-dien-3fl-ol
426
6
22.trans-Cholesta-5,22-dien-3~-ol
426
7
Cholest-5-en-3/~-ol
428
8
5~-Cholestan-3/~-ol
430
9
22-trans-24-Methyl-cholesta-5,22-dien-313-ol
440
10
22-trans-24-Methyl-5~-cholest-22-en-3~-ol
442
I1
Cholesta-5,24-dien-3~-ol
426
12
24-Methyl-cholest-5-en-3/~-ol
442
13
24-Methyl-cholesta-5,24(28)-dien-3/~-ol
440
14
24-Methyl-5~-cholestan-3fl-ol
444
15
24-Methyl-5~-cholest-24(28)-en-3~-ol
442
16
24-Methyl-cholesta-7,24(28)-dien-3,0-ol
440
17
22.trans-24-Ethyl-cholesta-5,22-dien-3[~-ol
454
18
22.trans-24.Ethyl-5ot-cholest-22-en-3fl-ol
456
19
24-Ethyl-cholest-5-en-3/~-ol
456
20
24-Ethyl-5cc-cholestan-3/~-ol
458
21
24-Ethyl-cholesta-5,24(28)-dien-3/~-ol
454
22
A5,x-c30
468
23
24-Keto-cholest-5-en-3/]-ol
442
24
24-Keto-5~-cholestan-3/~-ol
444
25
24-Keto-cholesta-5,25-dien-3fl-ol
440
26
24-Keto-5~-cholesta-25-en-3/~-ol
442
27
24,26-Dimethyl-cholest-5-en-3/3-ol
456
28
A5-C30
470
281) in the AS-unsaturated analogues. The spectra of compounds 24 and 26 as well as those of 23 and 25 showed fragments at m/z 71 and 69, respectively, which came from the cleavage of 23,24-single bond (~ to C-24 carbonyl group). Two other compounds, 22 and 28, were isolated mainly from the same fraction as that previously described. The mass spectrum of the acetylated derivative of compound 22 showed no parent ion but an M +-CH3COOH peak at m/z 408 and main fragments at m/z 315 (M +-side-chain), 255 (M +-CH3COOHside-chain), 253 (M+-CH3COOH-side-chain-2H), 227 (M+-CH3COOH-side-chain-27), 213 (M +CH3COOH-side-chain-42) and 211 (M +-
%
RRT
MS
0.10 0.73 380 (M +), 365, 296, 281,255,253, 228, 213, 211 1.37 0.85 352 (M + CH3COOH)~ 337, 282, 281, 255,253,213,211 0.42 0.86 414 (M +), 399, 344, 329, 315, 255, 215 0.56 0.90 412 (M +), 397, 352, 337, 313,299, 288, 273,255, 253,213 0.28 0.94 366 (M+-CH3COOH), 351,337, 282, 255,253, 213 5.60 0.96 366 (M+-CH3COOH), 351,282, 255, 253, 228, 213 2 1 . 0 0 1.00 368 (M+-CH3COOH), 353, 275, 260, 255,247, 228,213 1.83 1.01 430 (M +), 415, 370, 355, 328, 316, 275, 262, 257, 230, 215 16.41 1.03 380 (M+-CH3COOH), 365, 337, 282, 255, 253, 213 0.82 1.04 442 ~,M+), 427, 382, 344, 329, 315, 257, 229, 215 2.20 1.05 366 (M+-CH3COOH), 351,281,255 253,228,213, 211 1.05 1.07 382 (M + CH3COOH), 367,289, 274, 261,255, 228, 213 37.02 1.07 380 (M+-CH3COOH), 365, 296, 281, 255, 253, 228, 213,211 0.37 1.08 444 (M +), 429, 384, 369, 276, 275,257, 230, 215 2.84 1.09 442 (M+), 427, 367, 358, 343, 315, 298, 255, 229,215 0.96 1.10 440 (M +), 425,401,380, 365, 356, 341,327, 313, 255, 253,229, 227, 213 0.90 1.13 394 (M+-CH3COOH), 379, 351,296, 282, 255,253,228,213,211 0.22 1.14 456 (M +), 413, 396, 353,344, 315, 257, 215 0.92 1.16 396 (M + CH3COOH), 381,303, 288, 275, 255,228,213 0.32 1.17 458 (M +), 443, 398, 383, 290, 275,257, 230, 215 2.30 1.17 394 (M+-CH3COOH), 379, 352, 296, 281,255, 253, 213,211 0.10 1.18 408 (M+-CH3COOH), 315, 296, 281, 255, 253,227, 213,211, 125 0.57 1.20 382 (M + CH3COOH), 367, 315, 296, 281,255,253, 228,213,211, 71 traces 1.21 444 (M +), 384, 369,298, 283, 257,255, 215, 71 1.62 1.23 380 (M+-CH3COOH), 365, 315, 296, 281,255, 253, 228, 2ll, 69 traces 1.24 442 (M+), 382, 367, 298, 283, 257,255, 215, 213, 69 0.10 1.26 396 (M+-CH3COOH), 381,315, 288, 275, 255, 213 0.12 1.30 410 (M+-CH3COOH), 395, 380, 315, 296, 281,255, 253, 213, 211
CH3COOH-side-chain-42-2H), which gave indications ofa A 5-monounsaturated steroid nucleus having 'extra' groups at the side-chain that should weight 153 (CzlH21 or Cl0HlTO). In addition, the mass spectrum presented fragments at m/z 296 (408-112), 283 (408-125) and 281 (408-127) and the base peak at m/z 125, which are indicative that the side-chain should have an unsaturated group at C-24; the absence of a peak at m/z 310 would rule out structures with a A25-double bond, as was found for stelliferasterol, strongylosterol, xestospongesterol, verongurasterol and 24-isopropenylcholesterol (Theobald et al., 1978; Kokke et al., 1978, 1979; Li and Djerassi, 1981). Hence this hypothesis tentatively points to a 'fucosterol'
A. M. SELDES et al.
122
structure plus an additional methyl group or a 24-methylene sterol plus two additional methyl groups. The possibility o f having a keto group at the side-chain could not be discarded from the available data. The failure to isolate a large enough a m o u n t o f this c o m p o u n d , present as 0.1% o f the total sterol mixture, did not allow a definitive determination o f the side-chain structure by [ I H ] N M R spectroscopy. The other c o m p o u n d isolated from the same fraction, c o m p o u n d 28, showed in its mass spectrum as an acetylated derivative the absence o f a molecular ion but an M + CH3COOH fragment, as a base peak, at m / z 410 instead. Main fragments at m / z 315 ( M + side-chain), 255 ( M + - C H 3 C O O H side-chain), 253 ( M + - C H 3 C O O H - s i d e - c h a i n - 2 H ) , 213 (255 42) and 211 (253-42) pointed to a AS-monounsaturated steroid nucleus. The presence o f ions at m / z 296 (410 112) and 281 (196 15) would indicate a A24-unsa turation at the side-chain, As the side-chain should weight 155 (C11H23 or Ct0HI90) the presence o f an unsaturation would point to the latter possibility, giving indirect evidence o f an oxygenated function such as a keto group plus two extra methyl groups. A saturated side-chain, C~lH23, would not produce the McLafferty rearrangement peaks that were observed in the spectrum. As in the previous case, the extremely low a m o u n t o f the c o m p o u n d , 0.1% o f the total sterol mixture, inhibited the side-chain structural elucidation by [~H]NMR spectroscopy. It is, however, our intention to reinvestigate these c o m p o n e n t s to a t t e m p t a complete characterization. Acknowledgments--We thank the Departamento de Desarrollo de Investigaci6n de Universidad dc Chile, CONICET (Argentina), and the Organization of the American States for financial support.
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0305-0491/85 $3.00+0.00 © 1985 Pergamon Press Ltd
STEROLS F R O M THE SPONGE H A L I C L O N A C H I L E N S I S A. M. SELDES,* J. ROVIROSA,t A. SAN MARTiNt a n d E. G. GROS* *Departamento de Quimica Org~.nica y UMYMFOR, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Pab. 2, Ciudad Universitaria, 1428 Buenos Aires, Argentina and tDepartamento de Quimica, Facultad de Ciencias Bfisicas y Farmaceuticas, Universidad de Chile, Santiago, Chile
(Received 28 August 1984) A b s t r a c t - - 1 . The sterol composition of the sponge Haliclona chilensis (Thiele) has been analysed by argentic column- and thin-layer chromatography and reverse-phase high-performance liquid chromatography detecting twenty-eight components. 2. These were studied by gas chromatography-mass spectrometry and in some cases by proton and carbon-I 3 magnetic resonance spectroscopy. 3. It was established that the components were C26-, C27-, C28-, C29- and C30-A5 sterols and in minor amounts A° and A7 analogs with saturated and unsaturated side-chains. 4. 24-Keto-A 5 and -A° sterols with saturated and unsaturated side-chains were also found among the components of the mixture. 5. This is the first report on the presence of 24-keto-5~-cholestan-3fl-ol and 24-keto-5ct-cholest-25-en-3fl-ol in sponges.
INTRODUCTION M a r i n e organisms have been the source o f n u m e r o u s sterols, some o f t h e m h a v i n g unusual structures w i t h o u t terrestrial counterparts. A m o n g these organisms, sponges contain complex mixtures of sterols, in some cases with new a n d unique structures ( G o a d , 1978; Schmitz, 1978; Sica et al., 1978; Ballantine a n d Lavis, 1979; K a n a z a w a et al., 1979; Delseth et al., 1979; Djerassi, 1981; Li et al., 1981; Bohlin et al., 1981; I t o h et al., 1983; Di G i a c o m o et al., 1983). Since the earlier work o f B e r g m a n n a n d Feeney (1949) o n six species of Haliclona, a few papers on the same genus have a p p e a r e d reporting the presence o f mixtures o f mainly AS-sterols, a n d in some cases with A °- a n d AT-derivatives as m i n o r c o m p o n e n t s (Sheikh a n d Djerassi, 1974; Bergquist et al., 1980), epidioxy derivatives (Calder6n et al., 1982), t r i u n s a t u r a t e d sterols (Zielinski et al., 1982) a n d p r e g n a n e derivatives (Ballantine et al., 1977). In c o n t i n u i n g o u r current interest in sterols from aquatic organisms (Seldes a n d Gros, in press) we have investigated the constitution o f the sterol fraction derived from the sponge Haliclona chilensis, from which we have recently isolated a n d characterized two new dioxygenated steroid constituents (Seldes et al., in press). MATERIALS AND METHODS General Analytical thin-layer chromatography (TLC) was performed on silica gel 60-precoated (0.2 mm) aluminum sheets (Merck), developed with toluene-ethyl acetate (3:1) for free sterols and visualized by spraying with 50% sulfuric acid solution followed by heating. Argentic TLC for sterols acetates was carried out on precoated HPTLC silica gel 60 F254 plates (Merck) sprayed with a 10% silver nitrate hydroalcoholic solution and subsequently dried at 100°C. The plates were developed twice with petroleum ether (40-70°C fraction)-toluene (5:2 or 5:3) and spots were detected in the manner previously described. Column chromatography was carried out on silica gel 60 (70-230 mesh ASTM) (Merck) with petroleum ether-ethyl acetate
mixtures of increasing polarity as the eluant. Argentic column chromatography was performed on silica gel G (Merck) impregnated with 7% silver nitrate hydroalcoholic solution, taken to dryness and activated at 100°C. Chromatography of the sterol acetates was developed with petroleum ether-ethyl acetate (87: 3) as eluant and some fractions were recbromatographed in the same conditions. The progress of column chromatography was monitored by TLC and GC as indicated. Gas chromatography (GC) was performed on a Hewlett-Packard 5840A gas chromatograph equipped with a flame ionization detector and a fused silica capillary column (12 m x 0.2 ram) coated with methyl silicone fluid (HewlettPackard). The carrier gas was helium and analysis were performed between 200 and 280°C at 10°C/min. Relative retention times (RRT) are of the sterol acetates relative to cholesterol acetate. Combined gas chromatography-mass spectrometry (GC-MS) analysis of the free and acetylated sterols was performed on a Varian Mat CH7A mass spectrometer at 70 eV coupled to a Varian 1440 gas chromatograph using a 3% OV-17 GCQ glass column (1.8 m x 2 mm i.d.) and interfaced to a Varian Mat Data System 166 computer with magnetic libraries of standards. Quantification was made by automatic integration of the GC peaks and by single ion monitoring (SIM) of the sterols' base peaks in the mass chromatograms of the mixtures. High-performance liquid chromatography (HPLC) was used for analytical and preparative scale separations of the sterol mixtures obtained by argentic column chromatography, as well as for monitoring compounds purification. Analytical HPLC was performed with a Hewlett-Packard 1084 liquid chromatograph equipped with a refractive index detector and a variable volume injector with automatic sampling system. Separations were carried out with a R-Sil C-18HL 10 #m column (250 x 3.6 mm), packed in our laboratory with a Micromeritics 705 stirred slurry column packer, and with methanolwater (98:2) as the eluant at a flow rate of 3 ml/min. Preparative HPLC was carried out on a Micromeritics liquid chromatograph equipped with a 750 solvent delivery system, a 730 manual injector and a refractive index detector. Separations were performed using two different reversephase columns: Alltech R-Sil C-18HL 10 ,urn (500 x 10 mm, i.d.) and Altex Ultrasphere ODS 5 pm (250 x 10 mm, i.d.) using methanol or methanol-water mixtures at flow rates between 3 and 7 ml/min. Proton magnetic resonance spectra ([IH]NMR) at 100 MHz and carbon-13 magnetic resonance 119
120
A . M . SELDESet al.
spectra ([I-~C]NMR) at 25.2 MHz were performed on a Varian XL-t00-15 spectrometer in the FT mode in deuterochloroform solution containing tetramethylsilane as internal standard. Extraction, isolation andfi'actionation of the sterol mixture Sponges were collected in Puyinqu~ (Chilo6, Chile) in March 1983 and immediately stored in acetone. They were identified by Ruth Desqueyroux-Faundez at the Museum d'Histoire Naturelle (Geneve, Switzerland) as Haliclona chilensis (Thiele) (order Haplosclerida). The material was blended and after keeping the extract at room temperature for 24 hr the resulting slurry was filtered. The solid residue was re-extracted twice more with the same solvent at room temperature. The air-dried sponge residue weighed 430 g. The combined organic extracts were concentrated at reduced pressure and the remaining aqueous residue was extracted twice with chloroform. The combined organic extracts were taken to dryness. The syrup obtained was chromatographed on a silica gel column to afford a crude sterol mixture (9.8 g) which was acetylated (acetic anhydride-pyridine 1 : 1, 25C, 18 he). The sterol acetates mixture was subjected to preliminary separation by argentic column chromatography. The fractions collected were monitored by argentic TLC and GC and those with approximately the same composition were combined to afford eight main fractions. These were subsequently chromatographed by reverse-phase HPLC and the subfractions were analyzed by GC MS as acetylated and free compounds, which were obtained by treatment of the ester derivatives with 53o potassium hydroxide in methanol at room temperature. Compounds were identified by their RRT in capillary GC, by comparison and co-injection with known standards, and by MS of the free and acetylated derivatives. The unknown sterols and those whose structures could not be unambiguously assigned by these methods were rechromatographed, leading in some cases to the isolation of pure compounds, which were studied by NMR spectroscopy.
RESULTS AND DISCUSSION Table 1 lists the twenty-eight sterols isolated from the sponge Haliclona chilensis, together with G C - R R T data, molecular weight, percentage of the sterol components in the mixture and main fragments of the mass spectra of their acetylated derivatives. As can be seen, besides a complex mixture of C26, C27, C28, C29 and C30-A5, and in minor amounts A°- and AT-sterols with saturated and unsaturated side-chains, the sponge contains 24-keto-A 5 and A°-derivatives also with saturated and unsaturated side-chains. 24-Methyl-cholesta-5,24(28)-dien-3/~-ol (13) was the principal component, accounting for 37~o of the total mixture, and cholest-5-en-3//-ol (7) (21%) and 22- trans-24-methyl-cholesta-5,22-dien-3/?-crl (9) (16~o) are also present as main components. These compounds are also found as major sterols in the other three species of Haliclona studied (Sheikh and Djerassi, 1974; Bergquist et al., 1980), differences resting upon percentage distribution of them. 3/3-Hydroxy-AS-derivatives (91%) with saturated and monounsaturated side-chains were characterized by their G C - R R T and comparison with authentic standards, and in some cases, when necessary, by [IH]and [~3C]NMR, and by typical peaks in their mass spectra as free and acetylated derivatives (Wyllie et al., 1977). Stanols (7%) were characterized, by the same procedures, as fully saturated and side-chain unsaturated derivatives. Mass spectra of the acetylated compounds
afforded molecular ions and fragmentation patterns characteristic for these type of sterols. Two AV-sterols were present in minor amounts (l~J~) as Cz6 and C28 compounds. The co-occurrence of compounds 2, 3 and 4 and of compounds 13, 15 and 16 has been reported in other sponges of the same genus (Zielinski et al., 1982). The presence of C26 sterols in marine invertebrates has been realised for some time (Idler et, al., 1970; Viala et al., 1972; G o a d et al., 1972; Erdman and Thomson, 1972) and their occurrence in plankton suggests that this may be the c o m m o n origin of all the C26 marine animal sterols. The presence of c o m p o u n d I may be regarded as an artifact of the extractive procedure, taking into account that 24-methylenecholesterol is the principal component and that initial work-up of the acetylated mixture may favor the deacylation. Its mass spectrum displays the same fragmentation pattern as the acetylated derivative of 13, considering that the last one does not present molecular ions. C o m p o u n d 27 presented G C - R R T and mass spectral data which are fully consistent with those of authentic aplysterol and its acetylated derivative used as standards. Aplystane derivatives had only been recorded previously as sterol components of sponges from the order Verongida (De Rosa et al., 1973), a fact that induced them to be considered as a chemotaxonomic feature restricted to the mentioned order, However, Bergquist et al. (1980) reported the presence of didehydroaplysterol as a major component of the sterol mixture of an Haliclona species (order Haplosclerida) in which, as in our case, the sterol profile was typical of most of the Ceractinomorpha except for the presence of the mentioned 26-methyl substituted derivative. Moreover, Li et al. (1981) reported from another sponge of the order Haplosclerida the presence of aplystane derivatives. In view of these findings, Bergquist et al. (1980) draw attention to the risks of diagnosing groups absolutely on the basis of the presence or absence of particular molecules. As we have already reported (Seldes et al., in press), we isolated from the sterol mixture two 24-keto derivatives namely, 24-keto-cholest-5-en-3/%ol (23) and 24-keto-cholesta-5,25-dien-3/3-ol (26). F r o m the same fraction (the last one from argentic column chromatography) we were able to isolate two minor components, present as traces in the sterol mixture, which were characterized as 24-keto-5e-cholestan-3/3-ol (24) and 24-keto-5~-cholest-25-en-3/3-ol (26) on the basis of their G C - R R T and spectroscopic properties. The mass spectra of their acetylated derivatives presented the characteristic fragmentation pattern of a saturated sterol nucleus. Besides the molecular ion, and the loss of C H 3 C O O H and CH3COOH plus CH3, they included major peaks at m/z 257 (M +-CH3COOH-side-chain) and 215 (M + CH3COOH-side-chainM2), which are diagnostic for saturated steroid nuclei, while the presence of an ion at m/z 255 was indicative of an unsaturated side-chain. Moreover, peaks at m/z 298 and 283 were formed from ( M + - C H 3 C O O H ) by a McLafferty type rearrangement on the C-24 carbonyl group (loss of part of the side-chain, C-23-C-27, plus 1H), while the latter resulted from the loss of an additional methyl group. Both ions have their counterpart (m/z 296 and m/z
Sterols from the sponge Haficlona chilensis
121
Table I. Composition, molecular weight (mol. wt), relative retention times (RRT) and main fragments of mass spectra (MS) ofsterol acetates of H. chilensis
Sterol No.
Sterol systematic nomenclature
Mol. wt
1
24-Methyl-cholesta-3,5,24(28)-triene
380
2
22-trans-24-Norcholesta-5,22-dien-3~-ol
412
3
22-trans-24-Nor-5~-cholest-22-en-3B-ol
414
4
22-trans-24-Norcholesta-7,22-dien-3fl-ol
412
5
22-trans-24-Methyl-27-norcholesta-5,22-dien-3fl-ol
426
6
22.trans-Cholesta-5,22-dien-3~-ol
426
7
Cholest-5-en-3/~-ol
428
8
5~-Cholestan-3/~-ol
430
9
22-trans-24-Methyl-cholesta-5,22-dien-313-ol
440
10
22-trans-24-Methyl-5~-cholest-22-en-3~-ol
442
I1
Cholesta-5,24-dien-3~-ol
426
12
24-Methyl-cholest-5-en-3/~-ol
442
13
24-Methyl-cholesta-5,24(28)-dien-3/~-ol
440
14
24-Methyl-5~-cholestan-3fl-ol
444
15
24-Methyl-5~-cholest-24(28)-en-3~-ol
442
16
24-Methyl-cholesta-7,24(28)-dien-3,0-ol
440
17
22.trans-24-Ethyl-cholesta-5,22-dien-3[~-ol
454
18
22.trans-24.Ethyl-5ot-cholest-22-en-3fl-ol
456
19
24-Ethyl-cholest-5-en-3/~-ol
456
20
24-Ethyl-5cc-cholestan-3/~-ol
458
21
24-Ethyl-cholesta-5,24(28)-dien-3/~-ol
454
22
A5,x-c30
468
23
24-Keto-cholest-5-en-3/]-ol
442
24
24-Keto-5~-cholestan-3/~-ol
444
25
24-Keto-cholesta-5,25-dien-3fl-ol
440
26
24-Keto-5~-cholesta-25-en-3/~-ol
442
27
24,26-Dimethyl-cholest-5-en-3/3-ol
456
28
A5-C30
470
281) in the AS-unsaturated analogues. The spectra of compounds 24 and 26 as well as those of 23 and 25 showed fragments at m/z 71 and 69, respectively, which came from the cleavage of 23,24-single bond (~ to C-24 carbonyl group). Two other compounds, 22 and 28, were isolated mainly from the same fraction as that previously described. The mass spectrum of the acetylated derivative of compound 22 showed no parent ion but an M +-CH3COOH peak at m/z 408 and main fragments at m/z 315 (M +-side-chain), 255 (M +-CH3COOHside-chain), 253 (M+-CH3COOH-side-chain-2H), 227 (M+-CH3COOH-side-chain-27), 213 (M +CH3COOH-side-chain-42) and 211 (M +-
%
RRT
MS
0.10 0.73 380 (M +), 365, 296, 281,255,253, 228, 213, 211 1.37 0.85 352 (M + CH3COOH)~ 337, 282, 281, 255,253,213,211 0.42 0.86 414 (M +), 399, 344, 329, 315, 255, 215 0.56 0.90 412 (M +), 397, 352, 337, 313,299, 288, 273,255, 253,213 0.28 0.94 366 (M+-CH3COOH), 351,337, 282, 255,253, 213 5.60 0.96 366 (M+-CH3COOH), 351,282, 255, 253, 228, 213 2 1 . 0 0 1.00 368 (M+-CH3COOH), 353, 275, 260, 255,247, 228,213 1.83 1.01 430 (M +), 415, 370, 355, 328, 316, 275, 262, 257, 230, 215 16.41 1.03 380 (M+-CH3COOH), 365, 337, 282, 255, 253, 213 0.82 1.04 442 ~,M+), 427, 382, 344, 329, 315, 257, 229, 215 2.20 1.05 366 (M+-CH3COOH), 351,281,255 253,228,213, 211 1.05 1.07 382 (M + CH3COOH), 367,289, 274, 261,255, 228, 213 37.02 1.07 380 (M+-CH3COOH), 365, 296, 281, 255, 253, 228, 213,211 0.37 1.08 444 (M +), 429, 384, 369, 276, 275,257, 230, 215 2.84 1.09 442 (M+), 427, 367, 358, 343, 315, 298, 255, 229,215 0.96 1.10 440 (M +), 425,401,380, 365, 356, 341,327, 313, 255, 253,229, 227, 213 0.90 1.13 394 (M+-CH3COOH), 379, 351,296, 282, 255,253,228,213,211 0.22 1.14 456 (M +), 413, 396, 353,344, 315, 257, 215 0.92 1.16 396 (M + CH3COOH), 381,303, 288, 275, 255,228,213 0.32 1.17 458 (M +), 443, 398, 383, 290, 275,257, 230, 215 2.30 1.17 394 (M+-CH3COOH), 379, 352, 296, 281,255, 253, 213,211 0.10 1.18 408 (M+-CH3COOH), 315, 296, 281, 255, 253,227, 213,211, 125 0.57 1.20 382 (M + CH3COOH), 367, 315, 296, 281,255,253, 228,213,211, 71 traces 1.21 444 (M +), 384, 369,298, 283, 257,255, 215, 71 1.62 1.23 380 (M+-CH3COOH), 365, 315, 296, 281,255, 253, 228, 2ll, 69 traces 1.24 442 (M+), 382, 367, 298, 283, 257,255, 215, 213, 69 0.10 1.26 396 (M+-CH3COOH), 381,315, 288, 275, 255, 213 0.12 1.30 410 (M+-CH3COOH), 395, 380, 315, 296, 281,255, 253, 213, 211
CH3COOH-side-chain-42-2H), which gave indications ofa A 5-monounsaturated steroid nucleus having 'extra' groups at the side-chain that should weight 153 (CzlH21 or Cl0HlTO). In addition, the mass spectrum presented fragments at m/z 296 (408-112), 283 (408-125) and 281 (408-127) and the base peak at m/z 125, which are indicative that the side-chain should have an unsaturated group at C-24; the absence of a peak at m/z 310 would rule out structures with a A25-double bond, as was found for stelliferasterol, strongylosterol, xestospongesterol, verongurasterol and 24-isopropenylcholesterol (Theobald et al., 1978; Kokke et al., 1978, 1979; Li and Djerassi, 1981). Hence this hypothesis tentatively points to a 'fucosterol'
A. M. SELDES et al.
122
structure plus an additional methyl group or a 24-methylene sterol plus two additional methyl groups. The possibility o f having a keto group at the side-chain could not be discarded from the available data. The failure to isolate a large enough a m o u n t o f this c o m p o u n d , present as 0.1% o f the total sterol mixture, did not allow a definitive determination o f the side-chain structure by [ I H ] N M R spectroscopy. The other c o m p o u n d isolated from the same fraction, c o m p o u n d 28, showed in its mass spectrum as an acetylated derivative the absence o f a molecular ion but an M + CH3COOH fragment, as a base peak, at m / z 410 instead. Main fragments at m / z 315 ( M + side-chain), 255 ( M + - C H 3 C O O H side-chain), 253 ( M + - C H 3 C O O H - s i d e - c h a i n - 2 H ) , 213 (255 42) and 211 (253-42) pointed to a AS-monounsaturated steroid nucleus. The presence o f ions at m / z 296 (410 112) and 281 (196 15) would indicate a A24-unsa turation at the side-chain, As the side-chain should weight 155 (C11H23 or Ct0HI90) the presence o f an unsaturation would point to the latter possibility, giving indirect evidence o f an oxygenated function such as a keto group plus two extra methyl groups. A saturated side-chain, C~lH23, would not produce the McLafferty rearrangement peaks that were observed in the spectrum. As in the previous case, the extremely low a m o u n t o f the c o m p o u n d , 0.1% o f the total sterol mixture, inhibited the side-chain structural elucidation by [~H]NMR spectroscopy. It is, however, our intention to reinvestigate these c o m p o n e n t s to a t t e m p t a complete characterization. Acknowledgments--We thank the Departamento de Desarrollo de Investigaci6n de Universidad dc Chile, CONICET (Argentina), and the Organization of the American States for financial support.
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