699
MINOR AND TRACE STEROLS IN MARINE INVERTEBRATES 1.
GENERAL METHODS OF ANALYSIS
Simeon Popov,+ Robert M. K. Carlson, Annemarie Wegrnann and Carl Djerassi Department of Chemistry, Stanford University, Stanford, California 94305 Received:
3/l/76 ABSTRACT
SS-Hydroxy sterols occurring at a concentration of at least 0.001% of the steroi mixtures of Pseudoplexaura porosa and Plexaura homomalla have been fractionated using a series of remtechniques and subsequently analyzed using combined gas chromatography-mass spectrometry (G&MS) in the development of a procedure for examining the minor and trace components of marine sterol mixtures. A total of 49 sterols were found4 which spanned a molecular weight range of 274 to 440. In addition A 3-keto analogs of cholesterol, 24-methylcholesterol and gorgosterol Initial separation of varwere found in the extracts of P. homomalla. ious natural sterol-containing-conjugates and free sterols was found to have a number of advantages. Fractional digitonin precipitation and alumina column chromatography were found to possess greater sterol separation abilities than previously recognized. Many of the minor sterols were found to possess novel structures including a series of short side chain sterols, 19-nor sterols, 5B-stanols and 4-monomethyl sterols for which structure elucidation work is continuing. INTRODUCTION 36-Monohydroxyl sterol extracts from marine invertebrates can be extremely complex mixtures.
Over 80 sterols have so far been identified
from marine sources Cl], and evidence has been presented for the occurrence of over 30 sterols in a single marine species C21.
The discovery
of novel sterols as major components of the extracts of marine animals is rare, although there have been a number of recent exceptions: dinosterol (4a,23,24~-trimethyl-5a-cholest-22-en-36-o1) a dinoflagellate t
the major sterol in
[3], calysterol (23,24-ethylidenecholesta-5,23-dien-
Visiting investigator on leave (1975-1976) from the Institute of Organic Chemistry, Bulgarian Academy of Sciences, Sofia, Bulgaria.
VoZwne 28, Number 5
S
T=EOXDI
November, 1976
36-01) the major sterol of a marine sponge C4], aplysterol 24,26-dimethylcholest-5-en-38-01)
((24k,25S)-
and 24,28-bisdehydroaplysterol,
the
major sterols of marine sponges of the genus Verongia [5], and a series of 19-nor-5a-stanols
again from a marine sponge [6].
The bulk of marine
invertebrates for which sterol analyses have been reported Cl], however, contain major sterols with well known structures. A number of workers have recently turned to characterizing the minor sterol components of marine extracts with very interesting results. Kobayashi, -et al., have recently characterized patinosterol nor-24-methyl-5a-cholest-22E-en-38-ol) 24-methylcholesta-5,22E-dien-38-01)
[7], occelasterol((24S)-27-nor-
[8] and amurasterol
24-methyl-5a-cholesta-7,22E-dien-3$-ol)
((24S)-27-
((24S)-27-nor-
[9] as minor components of a
scallop, marine annelid, and starfish respectively.
Kobayashi points
out [7] that these 27-nor sterols are possibly biosynthetic precursors to the 24-nor C
26
sterols which themselves are minor sterol components
of a great many marine organisms. isofucostanol in a jellyfish [lo].
Ballantine, et al., have detected 28Idler, et al., have also recently
discovered new sterols as minor components of marine invertebrates, the from a scallop Cl1 1.
most recent being (E)-24-propylidenecholest-5-en-38-01
Sterols possess very similar properties in conventional thin layer chromatography
(TLC) and column chromatography systems.
severe difficulties
This leads to
in isolation and analysis of minor sterol comoonents.
A number of reports have recently appeared which describe the use of new techniques in the fractionation of sterol mixtures. umn chromatography
over a hydroxyalkoxypropyl
LH-20 [12] and high pressure reversed-phase
These include col-
derivative of Sephadex
liquid chromatography
c13].
S
TEEOIDI
Such new preparative techniques when used in conjunction with refined pre-existing techniques offer new opportunities for studying minor sterol components. A study of the minor sterols of marine organisms is pertinent for a number of important reasons. It is reasonable to assume that some of the minor sterol components are active metabolites and therefore are of major relevance to the study of sterol biosynthesis in marine organisms a promising field which is only just being opened. Other minor components may be sterols carried through the marine food chain and are, therefore, of ecological interest. Analysis of minor sterols is particularly important with respect to biosynthetic studies by radioisotope labeling because a large radiolabel incorporation in an unresolved minor component could lead to ambiguous results and misleading conclusions. As part of a search for new biosynthetically important marine sterols, and as a prelude to a sterol biosynthetic study in gorgonians, a number of different methods of sterol separations were investigated by us using the extracts of two Caribbean gorgonians, Pseudoplexaura porosa and Plexaura homomalla. Our procedure represents a refinement and synthesis of several new and older methods of sterol fractionation. Particularly noteworthy is the finding that several conventional fractionation procedures including chromatography over alumina (Activity III) and digitonin fractionation possess previously unrecognized potential in separating 3-monohydroxy sterol mixtures and that organisms from which only eight sterols have been identified previously by conventional gas chromatography-massspectrometer (GC-MS) Cl41 analysis
S
TEIROXDN
can be shown to contain over 30 sterols (many of which possess novel structures) using the fractionation and analytical procedures described below.
Further structural elucidation
and syntheses of many of these
novel sterols- is underway in this laboratory and will be the subject of forthcoming communications. RESULTS AND DISCUSSION The purpose of the procedure presented below is to create fractions of a sterol mixture with enrichments of various sterols sufficient for the production of a good quality GC-MS derived mass spectrum of each sterol component.
This requires in some cases a several hundredfold
enrichment of a particular minor sterol relative to the more abundant sterols.
Ps :e
1); however, ture.
porosa can be shown to contain over 40 sterols (see Table nine
of these sterols make up 97% of the mass of the mix-
Many of the minor and trace sterols are only present in micro-
gram quantities even if gram quantities of the sterol mixture are available.
At present GC-MS is the only viable tool for obtaining strue
tural information concerning these minor sterol components.
Present
knowledge of sterol mass spectrometric behavior has advanced so that many times GC-MS data are sufficient to suggest a structure for a newly detected sterol [15], which then warrants confirmation by synthesis. When quantities of the minor sterols are sufficient, production of fractions significantly enriched in the minor sterols also allow preparative GLC or HPLC collection of these minor components for Nuclear Magnetic Resonance
(NMR) or other analysis.
A number of chromatographic proceed-
ures were used by us to effect sufficient enrichments of the various minor sterol components.
These procedures include:
S Table
stem1 Reference Number
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
i”7 18 19 20 21 22 23 24 25 26 27 28
Structure
* * * * * * * * * * * * * * 1
* 15 7 1 I z 10 Y 5 li x
29
ii 5
30 31
T z
32 33 34 35 36 37 38 39 40 41
< G * : * * G
1.
TXIEOXDb
703
4-Demethyl' Sterols Present in ps. porosaand P. homoma.l.la M+
274 288 290 300 302 312 314 314 316 316 318 318 330 344 358 360 372 384 384 386 396 386 389 398 398 398 400 402 412 412 414 4J.4 414 416 416 424 426 426 426 426 428 428
Number of Carbon Atoms
19 20 20 21 21 21 22 22 22 21 22 21 23 24 25 25 26 27 27 27 27 27 27 28 28 28 28 28 29 29 29 29 29 29 29 30 30 30 30 30 30 30
Relative Retention Time 3% ov-17 276Y
Fraction Used in GC-MS+ Analysis
T5-6 0.13 TS-6 0.25 T5-6 0.25, b c 0.53 ' TS-6 0.23 T5-6 0.57 T5-6 0.57 T5-6 0.31 T4-lt2 0.27 TS-6,T6-12 0.57 T5-6 0.27 TS-6 0.57 TS-6 0.33 0.6-0.8 T5-6 0.6-0.8 T5-6 0.6-0.6 T5-6 0.92b T8-20t21 g 1.17 0.93 T8-19 T6-7 l.OOb 1.15 Tg8-20+21 1.21 T6-(l-4jd 1.00 TB-15t16 1.13 c,Tt?-22+23 1.2gb 1.35 T8-20+21 1.27 T6-7 1.27 T8-(l-4)d 1.36 T8-13t14 1.65 TS-3 1.54 T6-7 1.65 T8-(l-4jd 1.62 T7-5 T7-5 1.33 1.53 T8-(l-4jd 2.25 T8-22t23.c 2.20 T6-10tllj 3.38a Tgs-(1-4) 1.53a 2.15 TB-22 2.20_ T;-(l-4)d 1.53.=
Percent of Sterol Mixture Mixture II.phomomalla Ps. porosa Percent of Sterol
0.3
MINOR AND TRACE STEROLS IN MARINE INVERTEBRATES 1.
GENERAL METHODS OF ANALYSIS
Simeon Popov,+ Robert M. K. Carlson, Annemarie Wegrnann and Carl Djerassi Department of Chemistry, Stanford University, Stanford, California 94305 Received:
3/l/76 ABSTRACT
SS-Hydroxy sterols occurring at a concentration of at least 0.001% of the steroi mixtures of Pseudoplexaura porosa and Plexaura homomalla have been fractionated using a series of remtechniques and subsequently analyzed using combined gas chromatography-mass spectrometry (G&MS) in the development of a procedure for examining the minor and trace components of marine sterol mixtures. A total of 49 sterols were found4 which spanned a molecular weight range of 274 to 440. In addition A 3-keto analogs of cholesterol, 24-methylcholesterol and gorgosterol Initial separation of varwere found in the extracts of P. homomalla. ious natural sterol-containing-conjugates and free sterols was found to have a number of advantages. Fractional digitonin precipitation and alumina column chromatography were found to possess greater sterol separation abilities than previously recognized. Many of the minor sterols were found to possess novel structures including a series of short side chain sterols, 19-nor sterols, 5B-stanols and 4-monomethyl sterols for which structure elucidation work is continuing. INTRODUCTION 36-Monohydroxyl sterol extracts from marine invertebrates can be extremely complex mixtures.
Over 80 sterols have so far been identified
from marine sources Cl], and evidence has been presented for the occurrence of over 30 sterols in a single marine species C21.
The discovery
of novel sterols as major components of the extracts of marine animals is rare, although there have been a number of recent exceptions: dinosterol (4a,23,24~-trimethyl-5a-cholest-22-en-36-o1) a dinoflagellate t
the major sterol in
[3], calysterol (23,24-ethylidenecholesta-5,23-dien-
Visiting investigator on leave (1975-1976) from the Institute of Organic Chemistry, Bulgarian Academy of Sciences, Sofia, Bulgaria.
VoZwne 28, Number 5
S
T=EOXDI
November, 1976
36-01) the major sterol of a marine sponge C4], aplysterol 24,26-dimethylcholest-5-en-38-01)
((24k,25S)-
and 24,28-bisdehydroaplysterol,
the
major sterols of marine sponges of the genus Verongia [5], and a series of 19-nor-5a-stanols
again from a marine sponge [6].
The bulk of marine
invertebrates for which sterol analyses have been reported Cl], however, contain major sterols with well known structures. A number of workers have recently turned to characterizing the minor sterol components of marine extracts with very interesting results. Kobayashi, -et al., have recently characterized patinosterol nor-24-methyl-5a-cholest-22E-en-38-ol) 24-methylcholesta-5,22E-dien-38-01)
[7], occelasterol((24S)-27-nor-
[8] and amurasterol
24-methyl-5a-cholesta-7,22E-dien-3$-ol)
((24S)-27-
((24S)-27-nor-
[9] as minor components of a
scallop, marine annelid, and starfish respectively.
Kobayashi points
out [7] that these 27-nor sterols are possibly biosynthetic precursors to the 24-nor C
26
sterols which themselves are minor sterol components
of a great many marine organisms. isofucostanol in a jellyfish [lo].
Ballantine, et al., have detected 28Idler, et al., have also recently
discovered new sterols as minor components of marine invertebrates, the from a scallop Cl1 1.
most recent being (E)-24-propylidenecholest-5-en-38-01
Sterols possess very similar properties in conventional thin layer chromatography
(TLC) and column chromatography systems.
severe difficulties
This leads to
in isolation and analysis of minor sterol comoonents.
A number of reports have recently appeared which describe the use of new techniques in the fractionation of sterol mixtures. umn chromatography
over a hydroxyalkoxypropyl
LH-20 [12] and high pressure reversed-phase
These include col-
derivative of Sephadex
liquid chromatography
c13].
S
TEEOIDI
Such new preparative techniques when used in conjunction with refined pre-existing techniques offer new opportunities for studying minor sterol components. A study of the minor sterols of marine organisms is pertinent for a number of important reasons. It is reasonable to assume that some of the minor sterol components are active metabolites and therefore are of major relevance to the study of sterol biosynthesis in marine organisms a promising field which is only just being opened. Other minor components may be sterols carried through the marine food chain and are, therefore, of ecological interest. Analysis of minor sterols is particularly important with respect to biosynthetic studies by radioisotope labeling because a large radiolabel incorporation in an unresolved minor component could lead to ambiguous results and misleading conclusions. As part of a search for new biosynthetically important marine sterols, and as a prelude to a sterol biosynthetic study in gorgonians, a number of different methods of sterol separations were investigated by us using the extracts of two Caribbean gorgonians, Pseudoplexaura porosa and Plexaura homomalla. Our procedure represents a refinement and synthesis of several new and older methods of sterol fractionation. Particularly noteworthy is the finding that several conventional fractionation procedures including chromatography over alumina (Activity III) and digitonin fractionation possess previously unrecognized potential in separating 3-monohydroxy sterol mixtures and that organisms from which only eight sterols have been identified previously by conventional gas chromatography-massspectrometer (GC-MS) Cl41 analysis
S
TEIROXDN
can be shown to contain over 30 sterols (many of which possess novel structures) using the fractionation and analytical procedures described below.
Further structural elucidation
and syntheses of many of these
novel sterols- is underway in this laboratory and will be the subject of forthcoming communications. RESULTS AND DISCUSSION The purpose of the procedure presented below is to create fractions of a sterol mixture with enrichments of various sterols sufficient for the production of a good quality GC-MS derived mass spectrum of each sterol component.
This requires in some cases a several hundredfold
enrichment of a particular minor sterol relative to the more abundant sterols.
Ps :e
1); however, ture.
porosa can be shown to contain over 40 sterols (see Table nine
of these sterols make up 97% of the mass of the mix-
Many of the minor and trace sterols are only present in micro-
gram quantities even if gram quantities of the sterol mixture are available.
At present GC-MS is the only viable tool for obtaining strue
tural information concerning these minor sterol components.
Present
knowledge of sterol mass spectrometric behavior has advanced so that many times GC-MS data are sufficient to suggest a structure for a newly detected sterol [15], which then warrants confirmation by synthesis. When quantities of the minor sterols are sufficient, production of fractions significantly enriched in the minor sterols also allow preparative GLC or HPLC collection of these minor components for Nuclear Magnetic Resonance
(NMR) or other analysis.
A number of chromatographic proceed-
ures were used by us to effect sufficient enrichments of the various minor sterol components.
These procedures include:
S Table
stem1 Reference Number
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
i”7 18 19 20 21 22 23 24 25 26 27 28
Structure
* * * * * * * * * * * * * * 1
* 15 7 1 I z 10 Y 5 li x
29
ii 5
30 31
T z
32 33 34 35 36 37 38 39 40 41
< G * : * * G
1.
TXIEOXDb
703
4-Demethyl' Sterols Present in ps. porosaand P. homoma.l.la M+
274 288 290 300 302 312 314 314 316 316 318 318 330 344 358 360 372 384 384 386 396 386 389 398 398 398 400 402 412 412 414 4J.4 414 416 416 424 426 426 426 426 428 428
Number of Carbon Atoms
19 20 20 21 21 21 22 22 22 21 22 21 23 24 25 25 26 27 27 27 27 27 27 28 28 28 28 28 29 29 29 29 29 29 29 30 30 30 30 30 30 30
Relative Retention Time 3% ov-17 276Y
Fraction Used in GC-MS+ Analysis
T5-6 0.13 TS-6 0.25 T5-6 0.25, b c 0.53 ' TS-6 0.23 T5-6 0.57 T5-6 0.57 T5-6 0.31 T4-lt2 0.27 TS-6,T6-12 0.57 T5-6 0.27 TS-6 0.57 TS-6 0.33 0.6-0.8 T5-6 0.6-0.8 T5-6 0.6-0.6 T5-6 0.92b T8-20t21 g 1.17 0.93 T8-19 T6-7 l.OOb 1.15 Tg8-20+21 1.21 T6-(l-4jd 1.00 TB-15t16 1.13 c,Tt?-22+23 1.2gb 1.35 T8-20+21 1.27 T6-7 1.27 T8-(l-4)d 1.36 T8-13t14 1.65 TS-3 1.54 T6-7 1.65 T8-(l-4jd 1.62 T7-5 T7-5 1.33 1.53 T8-(l-4jd 2.25 T8-22t23.c 2.20 T6-10tllj 3.38a Tgs-(1-4) 1.53a 2.15 TB-22 2.20_ T;-(l-4)d 1.53.=
Percent of Sterol Mixture Mixture II.phomomalla Ps. porosa Percent of Sterol
0.3
