- Email: [email protected]
Marine sterols IV. C21 sterols from marine sources. Identification of pregnane derivatives in extracts of the sponge Haliclona rubens .
Tetrahedron Letters No. 18, pp 1547 - 1550, 1977.
MARINE
STEROLS
OF PREGNANE
IV.
’
Czl
STEROLS
DERIVATIVES
IN
FROM
EXTRACTS
Pergamon Ress.
MARINE OF
THE
SOURCES.
Printed in Great Britain.
IDENTIFICATION
SPONGE HALICLONA RUBENS.
James A. Ballantine' and Kevin Williams Department of Chemistry, Institute of Marine Studies, University College of Swansea, U.K. Basil A. Burke Department of Chemistry, University of the West Indies, Mona, Kingston 7, Jamaica. (Received in UK 9 March 1977; accepted
for publioation
21 March 1977)
As part of our investigations into marine Invertebrate sterol profiles and their relation to the marine
food
web,
we
wish to report the occurrence of a number of pregnane
derivatives in the sterol mixture isolated from a sponge Haliclona rubens.
This animal
has been found to contain 3g-hydroxy-17g-pregn-5-ene-20-one (11, 3B,20f3-dihydroxy17g-pregn-5-ene (111 and
3fi,20a-dihydroxy-17fi-pregn-%ene [III1 In addition to other,
as yet unidentified, keto sterols. This is the first report of free pregnane derived sterols in the marine environment. In fact the only other C21 steroid which has been isolated from marine sources is the aglycone IIVl from the hydrolysis of a complex saponln obtained by a number of workers2.3.4 from three species of starfish.
“o&H
Ho&
H mu
OH
(IV)
The sponge was collected off the north coast of Jamaica and the chopped material was extracted with methanol and the residue from the chloroform soluble portion of the The star01 fraction was crystallised A very from methanol and examined by GC-MS techniques as previously described. 1.5 methanol extract was chromatographedon silica.
complex sterol g.1.c. profile was observed [Fig. 11 and temperature programming was employed over the lower end of the chromatogram to increase the resolution.
The
retention data for each of the peaks is given in Table 1 , accompanied by the masses 1547
No. 18
23
w
210°
250°
b
FIGURE 1.
Chromatogran of Halicona rubens Sterol TMS Ethers on 1% SE-30 Ultraphase
TABLE 1.
Data for Haliclona rubens Star01 TMS Ethers.
Peak No.
RRT
M+'
Identification
1 2 3 4 5 6 7 6 9 10 11 12 13 14 15 16 17 16 19 20 21 22
0.10 0.17 0.18 0.23 0.30 0.34 0.39 0.41 0.43 0.45 0.51 0.54 0.59 0.61 0.79 0.65 0.88 1.00 1.10 1.17 1.26 1.36
346 372,374
l
23 24 25 26
1.54 1.64 1.60 1.94
t l
344 366 374
l
21c 5
36-hydroxy-17$-pregn-5-ene-ZO-one
l l
402,462 402,416 462
l/ 21c 5 l/* 21c 5
36,206-dihydroxy-176-pregn-5-ene 36,20a-dihydroxy-176-pragn-5-ene
l l
402 402,442 490 456 456 456 470 504 472 464 466 466 472 546.464.472 486,456
Footnote * requires further
l
*/ 26C 5,22[El *
24-norcholesta-5,22-dien-36-01
27C 27C 27C 28C * 28C 29C 29C 29c
5,22(El 5.22tEl 5 5,22(El
occelasterol cholesta-5,22-dien-36-01 cholesterol brassica- or crinosterol
5 5,22(E) 22(E) 5
dihydrobrassica-or campesterol stigma- or poriferasterol stigma- or poriferastanol @site- or clionasterol
l
l/*/* l/* for identification.
lo.
18
1549
of the molecular ions of the sterol trlmethylsilyl ethers which have been Identified within each peak. The comnon marine sterols were Identified by comparison of g.1.c. retention data and mass spectra with those of trimethylsilyl ethers of standard sterols and by multiple mass spectrometrlc scanning of each g.1.c. peak to identify all overlapping constituents. The new marine sterols were identified as follows : A molecular formula of
PEAK 5:
was established for the
C21H3102~SiMe31
accurate mass measurement of the molecular ion during the GC-MS scan.
TRS
ether by
The presence of
prominent ions at m/e 129 and (M-1291+ were indicative of a AS-3G-trimethylsllylether grouping' and the ion at m/e 43 suggested the presence of an acetyl function. The mass spectrum was Identical with the published GC-I'IS data for the TMS ether' of the mamnalian hormone 3l3-hydroxy-17~-pregn-5-ene-ZO-one and was slightly different from particularly in the lower abundance of the m/e 213 ion. of the 17cr-analogue8. g.1.c. retention data for the 17G-isomer were also in agreement with
those
that
The published for
observed
Peak 5. Accordingly the sterol in Peak 5 was compared with an authentic sample of 3@hydroxy17G-pregn-5-ene-ZO-one'by GC-MS techniques. Is
given
in
Table
for
2
both
the
The
comparison
of
g.1.c.
free sterols and their TNS ethers.
retention
times
The GC-mass spectra
of the two materials were identical and Peak 5 sterol Is formulated as 3G-hydroxy-17Bpregn-5-ene-20-one [Il. PEAKS 8 and 10 :
A
molecular formula of C21H3202~SiMe312was established for these
sterols by accurate mass measurements and corresponds to that required for pregnene dials. 10 The major ion at m/e 117 supported the presence of a 20-hydroxy pregnane system and the ion at m/e 129 was indicative of a A5-3G-TNS ether structure6. In addition the g.1.c. 10 retention times were comparable with the published data for A5-3G-pregnen-3,20-diols. The compounds were therefore compared with authentic samples of 38,20G-dihydroxyJ epimers which are not distinguish17G-pregn-5-ene'and 3$,20a-dihydroxy-17$-pregn-5-ene' able by mass spectrometry.
The g.1.c. retention times are given in Table 2 for both the
free sterols and the di-TMS ethers. the
The mass spectra of Peaks 8 and 10 were similar to
published spectra of the TMS ethers of the dlols and are identical with those of
authantic samples when run under identical conditions. formulated as
Peak 8 star01 is therefore
3B,20B-dihydroxy-17g-pragn-5-ene (III and Peak 10 sterol is formulated as
3G,20cL-dihydroxy-178_pregn-5-ene [III]. MINOR CONSTITUENTS
in these extracts.
:
There
are
Indications
In particular Peaks
that
15 and
resolution and correspond to a C2203-di-TMS
20
ether
some have and
other keto-sterols may be present been
mass
a C2303-dl-TMS
measured ether
at
high
respectively.
These constituents and other minor sterols in this extract are being further Investigated following large scale extractions of the sponge.
1550
No.
Table 2.
Relative Retention Times on SE-30 at 210'
18
( cholastane = 1.001
36-hydroxy-176-pragn-5-ene-ZO-one'
b.62
0.76
Peak 5 star01
0.63
0.79
3B-hydroxy-17a-prsgn-S-ene-ZO-one6
0.56
0.70
9.10 36,20B-dihydroxy-176-pregn-5-ene
0.62
1.09
Peak 6 sterol
0.62
1.06
9.10 38,20cr-dihydroxy-176-pregn-5-ene
0.66
1.16
Peak 10 star01
0.67
1.16
The presence of these ZO-keto and ZO-hydroxy sterols containing the usual A5-36-OH system is presumably due to oxidative cleavage of the normal marine star01 sidechains at C20 perhaps via a 20,22-diol intermediate metabolite. Acknowledgements : (NERC
F60/81/61
A
research contract from the Institute of Oceanographic Sciences
and an SRC studentship [to
KWI
are gratefully acknowledged.
REFERENCES. 1.
Part III. J.A. Ballantine, J.C. Roberts and R.J. Norris, Biomed. Mass Spectrom.. 3. 14 (19761.
2.
Y.N. Shaikh, B.N. Tursch and C. Djerassi. J. Amer. Chem. Sot., 94. 3276 (19721.
3.
Y. Shimizu. J. Amer. Chem. Sot.. 94, 4051 [19721.
4.
S. Ikegami, Y. Kamiya and S. Tamura. Tetrahedron Letters, -16, 1601 (19721.
5.
J.A. Ballantine, J.C. Roberts and R.J. Norris, J. Chromatogr..-103. 269 (19751.
6.
B.A. Knights, J. Gas Chromatogr.,5_, 273 (19671.
7. 6.
J. Sjovall and R. Vihko, Acta endocr.. -57. 247 (19661. H. Eriksson, J.A. Gustafsson and J. Sjovall, Europ. J. Biochem., 2, 219 [19661.
9.
Sigma London, Chemical Co. Ltd.
10.
J. Sjovall and R. Vihko, Stemids, 7. 447 (19661 R. Vihko, Acta endocr. Suppl. -109 (19661.
MARINE
STEROLS
OF PREGNANE
IV.
’
Czl
STEROLS
DERIVATIVES
IN
FROM
EXTRACTS
Pergamon Ress.
MARINE OF
THE
SOURCES.
Printed in Great Britain.
IDENTIFICATION
SPONGE HALICLONA RUBENS.
James A. Ballantine' and Kevin Williams Department of Chemistry, Institute of Marine Studies, University College of Swansea, U.K. Basil A. Burke Department of Chemistry, University of the West Indies, Mona, Kingston 7, Jamaica. (Received in UK 9 March 1977; accepted
for publioation
21 March 1977)
As part of our investigations into marine Invertebrate sterol profiles and their relation to the marine
food
web,
we
wish to report the occurrence of a number of pregnane
derivatives in the sterol mixture isolated from a sponge Haliclona rubens.
This animal
has been found to contain 3g-hydroxy-17g-pregn-5-ene-20-one (11, 3B,20f3-dihydroxy17g-pregn-5-ene (111 and
3fi,20a-dihydroxy-17fi-pregn-%ene [III1 In addition to other,
as yet unidentified, keto sterols. This is the first report of free pregnane derived sterols in the marine environment. In fact the only other C21 steroid which has been isolated from marine sources is the aglycone IIVl from the hydrolysis of a complex saponln obtained by a number of workers2.3.4 from three species of starfish.
“o&H
Ho&
H mu
OH
(IV)
The sponge was collected off the north coast of Jamaica and the chopped material was extracted with methanol and the residue from the chloroform soluble portion of the The star01 fraction was crystallised A very from methanol and examined by GC-MS techniques as previously described. 1.5 methanol extract was chromatographedon silica.
complex sterol g.1.c. profile was observed [Fig. 11 and temperature programming was employed over the lower end of the chromatogram to increase the resolution.
The
retention data for each of the peaks is given in Table 1 , accompanied by the masses 1547
No. 18
23
w
210°
250°
b
FIGURE 1.
Chromatogran of Halicona rubens Sterol TMS Ethers on 1% SE-30 Ultraphase
TABLE 1.
Data for Haliclona rubens Star01 TMS Ethers.
Peak No.
RRT
M+'
Identification
1 2 3 4 5 6 7 6 9 10 11 12 13 14 15 16 17 16 19 20 21 22
0.10 0.17 0.18 0.23 0.30 0.34 0.39 0.41 0.43 0.45 0.51 0.54 0.59 0.61 0.79 0.65 0.88 1.00 1.10 1.17 1.26 1.36
346 372,374
l
23 24 25 26
1.54 1.64 1.60 1.94
t l
344 366 374
l
21c 5
36-hydroxy-17$-pregn-5-ene-ZO-one
l l
402,462 402,416 462
l/ 21c 5 l/* 21c 5
36,206-dihydroxy-176-pregn-5-ene 36,20a-dihydroxy-176-pragn-5-ene
l l
402 402,442 490 456 456 456 470 504 472 464 466 466 472 546.464.472 486,456
Footnote * requires further
l
*/ 26C 5,22[El *
24-norcholesta-5,22-dien-36-01
27C 27C 27C 28C * 28C 29C 29C 29c
5,22(El 5.22tEl 5 5,22(El
occelasterol cholesta-5,22-dien-36-01 cholesterol brassica- or crinosterol
5 5,22(E) 22(E) 5
dihydrobrassica-or campesterol stigma- or poriferasterol stigma- or poriferastanol @site- or clionasterol
l
l/*/* l/* for identification.
lo.
18
1549
of the molecular ions of the sterol trlmethylsilyl ethers which have been Identified within each peak. The comnon marine sterols were Identified by comparison of g.1.c. retention data and mass spectra with those of trimethylsilyl ethers of standard sterols and by multiple mass spectrometrlc scanning of each g.1.c. peak to identify all overlapping constituents. The new marine sterols were identified as follows : A molecular formula of
PEAK 5:
was established for the
C21H3102~SiMe31
accurate mass measurement of the molecular ion during the GC-MS scan.
TRS
ether by
The presence of
prominent ions at m/e 129 and (M-1291+ were indicative of a AS-3G-trimethylsllylether grouping' and the ion at m/e 43 suggested the presence of an acetyl function. The mass spectrum was Identical with the published GC-I'IS data for the TMS ether' of the mamnalian hormone 3l3-hydroxy-17~-pregn-5-ene-ZO-one and was slightly different from particularly in the lower abundance of the m/e 213 ion. of the 17cr-analogue8. g.1.c. retention data for the 17G-isomer were also in agreement with
those
that
The published for
observed
Peak 5. Accordingly the sterol in Peak 5 was compared with an authentic sample of 3@hydroxy17G-pregn-5-ene-ZO-one'by GC-MS techniques. Is
given
in
Table
for
2
both
the
The
comparison
of
g.1.c.
free sterols and their TNS ethers.
retention
times
The GC-mass spectra
of the two materials were identical and Peak 5 sterol Is formulated as 3G-hydroxy-17Bpregn-5-ene-20-one [Il. PEAKS 8 and 10 :
A
molecular formula of C21H3202~SiMe312was established for these
sterols by accurate mass measurements and corresponds to that required for pregnene dials. 10 The major ion at m/e 117 supported the presence of a 20-hydroxy pregnane system and the ion at m/e 129 was indicative of a A5-3G-TNS ether structure6. In addition the g.1.c. 10 retention times were comparable with the published data for A5-3G-pregnen-3,20-diols. The compounds were therefore compared with authentic samples of 38,20G-dihydroxyJ epimers which are not distinguish17G-pregn-5-ene'and 3$,20a-dihydroxy-17$-pregn-5-ene' able by mass spectrometry.
The g.1.c. retention times are given in Table 2 for both the
free sterols and the di-TMS ethers. the
The mass spectra of Peaks 8 and 10 were similar to
published spectra of the TMS ethers of the dlols and are identical with those of
authantic samples when run under identical conditions. formulated as
Peak 8 star01 is therefore
3B,20B-dihydroxy-17g-pragn-5-ene (III and Peak 10 sterol is formulated as
3G,20cL-dihydroxy-178_pregn-5-ene [III]. MINOR CONSTITUENTS
in these extracts.
:
There
are
Indications
In particular Peaks
that
15 and
resolution and correspond to a C2203-di-TMS
20
ether
some have and
other keto-sterols may be present been
mass
a C2303-dl-TMS
measured ether
at
high
respectively.
These constituents and other minor sterols in this extract are being further Investigated following large scale extractions of the sponge.
1550
No.
Table 2.
Relative Retention Times on SE-30 at 210'
18
( cholastane = 1.001
36-hydroxy-176-pragn-5-ene-ZO-one'
b.62
0.76
Peak 5 star01
0.63
0.79
3B-hydroxy-17a-prsgn-S-ene-ZO-one6
0.56
0.70
9.10 36,20B-dihydroxy-176-pregn-5-ene
0.62
1.09
Peak 6 sterol
0.62
1.06
9.10 38,20cr-dihydroxy-176-pregn-5-ene
0.66
1.16
Peak 10 star01
0.67
1.16
The presence of these ZO-keto and ZO-hydroxy sterols containing the usual A5-36-OH system is presumably due to oxidative cleavage of the normal marine star01 sidechains at C20 perhaps via a 20,22-diol intermediate metabolite. Acknowledgements : (NERC
F60/81/61
A
research contract from the Institute of Oceanographic Sciences
and an SRC studentship [to
KWI
are gratefully acknowledged.
REFERENCES. 1.
Part III. J.A. Ballantine, J.C. Roberts and R.J. Norris, Biomed. Mass Spectrom.. 3. 14 (19761.
2.
Y.N. Shaikh, B.N. Tursch and C. Djerassi. J. Amer. Chem. Sot., 94. 3276 (19721.
3.
Y. Shimizu. J. Amer. Chem. Sot.. 94, 4051 [19721.
4.
S. Ikegami, Y. Kamiya and S. Tamura. Tetrahedron Letters, -16, 1601 (19721.
5.
J.A. Ballantine, J.C. Roberts and R.J. Norris, J. Chromatogr..-103. 269 (19751.
6.
B.A. Knights, J. Gas Chromatogr.,5_, 273 (19671.
7. 6.
J. Sjovall and R. Vihko, Acta endocr.. -57. 247 (19661. H. Eriksson, J.A. Gustafsson and J. Sjovall, Europ. J. Biochem., 2, 219 [19661.
9.
Sigma London, Chemical Co. Ltd.
10.
J. Sjovall and R. Vihko, Stemids, 7. 447 (19661 R. Vihko, Acta endocr. Suppl. -109 (19661.