Synthesis of endogenous sperm-activating and attracting factor isolated from ascidian Ciona intestinalis

Article abstract of DOI:10.1016/S0040-4039(03)01598-3

A chemoattractant candidate named sperm-activating and attracting factor (SAAF) from the eggs of ascidian Ciona intestinalis, was synthesized from chenodeoxycholic acid in 16 steps. The present synthesis led to the unambiguous structure determination of SAAF to be (3R,4R,7R,25S)-3,4,7,26-tetrahydroxycholestane-3,26-disulfate. The synthetic pure specimen was also used to confirm the dual sperm-activating and attracting activity.

Full text of DOI:10.1016/S0040-4039(03)01598-3

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 44 (2003) 6387–6389
Synthesis of endogenous sperm-activating and attracting factor
isolated from ascidian Ciona intestinalis
Tohru Oishi,a Hiroshi Tsuchikawa,a Michio Murata,a,* Manabu Yoshidab and Masaaki Morisawab
aDepartment of Chemistry, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan
bMisaki Marine Biological Station, Graduate School of Science, University of Tokyo, Miura, Kanagawa 238-0225, Japan
Received 30 May 2003; accepted 27 June 2003
Abstract—A chemoattractant candidate named sperm-activating and attracting factor (SAAF) from the eggs of ascidian Ciona
intestinalis, was synthesized from chenodeoxycholic acid in 16 steps. The present synthesis led to the unambiguous structure
determination of SAAF to be (3R,4R,7R,25S)-3,4,7,26-tetrahydroxycholestane-3,26-disulfate. The synthetic pure specimen was
also used to confirm the dual sperm-activating and attracting activity.
© 2003 Elsevier Ltd. All rights reserved.
Chemotaxis of sperm toward eggs during fertilization is
a crucial event for species conservation, particularly for
animals living in aquatic environments.1 Relevant
chemical attractants have been found from a few
marine organisms such as sea urchins and corals.2,3
Recently, we have reported a non-peptidic chemoat-
tractant candidate named sperm-activating and attract-
ing factor (SAAF) from the eggs of ascidian Ciona
intestinalis.4 SAAF is the first steroid possessing chemo-
taxis activity, as well as the first example of a single
agent concomitantly revealing both sperm activation
and attraction, which are reportedly elicited by differ-
ent mechanisms.5,6 Thus, SAAF may serve as a key
ligand for future biological studies on signal transduc-
tion pathways leading to sperm’s flagellum movement.
We proposed the structure of SAAF to be a novel
polyhydroxysterol sulfate (1) by means of 2D-1H NMR
and FAB-MS/MS analysis using approximately 4 mg of
sample.4 However, we could not rule out the possibility
that the biological activity might be attributed to a
minor constituent since spectral measurements were
carried out with a very limited amount of the specimen,
which was not completely pure as can be observed in
Figure 1. Synthesis is, therefore, essential for the
unequivocal identification of 1 as the active principle,
for the complete structure elucidation including the
stereochemistry at C-25, and for providing a specimen
for further biological studies. In this communication we
attempted to synthesize of SAAF and its C-25 epimer,
which allowed us not only to determine the complete
structure, but also to confirm the dual activity of
SAAF.
Synthesis of SAAF and its C-25 epimer commenced
with methyl 7a-hydroxy-3-oxo-5b-cholan-24-oate (2)7
as shown in Scheme 1. We planed a versatile route that
could provide both diastereomers by introducing the
side chain at the latest steps via a common intermedi-
ate. Oxidation of the ketone 2 with molecular oxygen in
the presence of t-BuOK proceeded regioselectively via
the C-4 enolate, and following esterification of the
concomitantly hydrolyzed product afforded 3-keto-4-
enol 3. Selective protection of the C-7 alcohol of 3 as its
benzyloxymethyl (BOM) ether in the presence of C-4
enol yielded 4. Although many attempts to reduce the
3-keto-4-enol
4 using conventional methods were
unsuccessful due to the formation of undesired
diastereomers preferentially with low reproducibility, a
solid-phase reduction of 4 with NaBH3CN on silica gel
was found to afford desired diol 5a as an inseparable
mixture with 5b-epimer 5b in a 3 to 1 ratio (44%);
Keywords: sperm-activating and attracting factor; sterol disulfate;
structure determination; synthesis.
* Corresponding author. Tel.: +81-6-6850-5774; fax: +81-6-6850-5774;
e-mail: murata@ch.wani.osaka-u.ac.jp
0040-4039/$ - see front matter © 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0040-4039(03)01598-3

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T. Oishi et al. / Tetrahedron Letters 44 (2003) 6387–6389
converted to the corresponding sodium bis-sulfate
through the successive treatment with SO3·Py and ion-
exchange resin (IR-120B, Na+ form). Hydrogenation of
major side product was 4-keto-3-ol 6 (20%). Even
though the yield of 5a should be improved, the contigu-
ous stereogenic centers at C-3, C-4, and C-5 on the
steroid framework were properly installed for the next
operations. Treatment of the mixture of diols 5a and 5b
with thionyl chloride followed by oxidation with RuO2–
NaIO4 gave cyclic sulfate 7a in 65% yield for two
steps,8 which was easily separated from 5b-isomer 7b
(11%) by silica gel chromatography. Regioselective
opening of the cyclic sulfate at C-3 position was suc-
cessfully achieved by treating with benzoic acid in the
presence of Cs2CO3 in DMF (75%),9 and successive
protection of the resulting C-4 alcohol as BOM ether
afforded benzoate 8 as a single isomer (71%). Exposure
of 8 to t-BuOK in t-BuOH resulted in the selective
hydrolysis of the methyl ester in the presence of ben-
zoate ester to yield carboxylic acid 9 (95%), which was
further converted to iodide 10 through the decarboxyla-
tion by treating with Pb(OAc)4 in the presence of iodine
under irradiation using a tungsten lamp (84%).10 Oxida-
tion of 10 with DMSO in the presence of collidine gave
an aldehyde 11 (94%), a common precursor of the two
diastereomers at C-25. Elongation of the side chain was
achieved through a Wittig reaction between the alde-
hyde 11, and an ylide generated from (R)-phosphonium
salt 1211 and n-BuLi–TMSCl12 to give olefin 13 (E:Z=
1:8) in 69% yield. Reductive removal of the benzoyl
group of 13 with LiAlH4 gave diol 14, which was
Scheme 1. Reagents and conditions: (a) t-BuOK, O2, t-BuOH,
then CH2N2, Et2O, MeOH, CHCl3 (71%); (b) BOMCl, i-
Pr2NEt, CH2Cl2 (83%); (c) NaBH3CN; CHCl3, MeOH, then
evaporated; (d) SOCl2, Et3N, THF, 93%; (e) RuCl3·nH2O,
NaIO4; (f) PhCO2H, Cs2CO3, DMF, 75%; (g) BOMCl, i-
Pr2NEt, CH2Cl2 (84%); (h) t-BuOK, t-BuOH, 95%; (i)
Pb(OAc)4, I2, hw, CCl4, 84%; (j) DMSO, 2,4,6-collidine, 94%;
(k) n-BuLi, TMSCl, THF, 69%; (l) LiAlH4, THF, 87%; (m)
SO3·Py, Py, then Amberite IR-120B Na+ form; (n) Pd/C, H2,
MeOH.
Figure 1. Partial 1H NMR spectra of SAAF, 15 (25S), and 16
(25R). Asterisks indicate the signals attributed to contami-
nants.

T. Oishi et al. / Tetrahedron Letters 44 (2003) 6387–6389
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Education, Sciences, Sports, Culture, and Technology,
Japan.
the double bond and concomitant removal of the BOM
groups afforded 25S-isomer (15), and the resulting
polar material was purified by reverse-phase HPLC.
The C-25 epimer 16 was synthesized by the identical
procedure as 15 except for the use of (S)-phosphonium
salt in place of 12.
References
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The 1H NMR spectrum of the natural product was
compared with those of the synthetic samples, 15 (25S)
and 16 (25R), as shown in Figure 1.13 While the chem-
ical shifts of the 25-methyl group and H-26a in 16 do
not match those of the natural product (Dl values for
H-26a and 25-methyl are −0.014 and −0.007 ppm,
respectively), those of 15 are identical with SAAF as
are other resonances of the steroid framework.4 Thus,
the configuration of the side chain was confirmed as
25S, resulting in the first synthesis of SAAF. When the
bioactivity and biosynthetic rationality are taken into
account, the absolute stereochemistry is assigned to be
3R,4R,7R, and 25S.
Both the sperm-activating and attracting activity of
synthetic SAAF were bioassayed based on methods
previously reported.4 Synthetic SAAF (15) activated
sperm of ascidian Ciona intestinalis at 3.7 nM and
concurrently exhibited the attracting activity at <10
nM;14 these quantitative evaluations were first accom-
plished with the synthetic specimen. It is noteworthy
that 25-epi-SAAF (16) possesses comparative activities
as those with SAAF (activated at ꢀ3.7 nM and
attracted at <10 nM).
10. Sheldon, R. A.; Kochi, J. K. Org. React. 1972, 19, 279.
11. (a) Kozikowski, A. P.; Chen, Y. Y. J. Org. Chem. 1981,
46, 5248; (b) Bergmann, J.; Lo¨fstedt, C.; Ivanov, V. D.;
Francke, W. Eur. J. Org. Chem. 2001, 16, 3175.
12. (a) White, J. D.; Jeffrey, S. C. J. Org. Chem. 1996, 61,
2600; (b) Wang, X.; Erickson, S. D.; Iimori, T.; Still, W.
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1
13. 15: H NMR (500 MHz, D2O) l 4.41 (1H, d, J=2.5 Hz,
In conclusion, the sperm-activating and attracting fac-
tor (SAAF) was synthesized from 2 in 16 steps, which
led to the unambiguous structure determination of
SAAF to be (3R,4R,7R,25S)-3,4,7,26-tetrahydroxy-
cholestane-3,26-disulfate (15). The synthetic pure speci-
men was also used to confirm the dual sperm-activating
and attracting activity. Currently, we are preparing
molecular probes to be used for identification of the
receptor and the relevant signal transduction path-
way(s).
3-H), 3.96 (1H, d, J=2.0 Hz 7-H), 3.92 (1H, dd, J=9.0,
5.5 Hz, 26-Ha), 3.83 (1H, dd, J=9.0, 7.0 Hz, 26-Hb),
3.72 (1H, s, 4-H), 1.81 (1H, d, J=15.0 Hz, 5-H), 0.97
(3H, s, 10-Me), 0.92 (3H, d, J=6.5 Hz, 25-Me), 0.90 (3H,
d, J=6.5 Hz, 20-Me), 0.65 (3H, s, 13-Me); MS (ESI) m/z
297 (M−2Na)2−; 16: 1H NMR (500 MHz, D2O) l 4.41
(1H, d, J=2.5 Hz, 3-H), 3.96 (1H, d, J=2.5 Hz 7-H),
3.91 (1H, dd, J=9.0, 5.5 Hz, 26-Ha), 3.83 (1H, dd,
J=9.0, 7.0 Hz, 26-Hb), 3.72 (1H, s, 4-H), 1.81 (1H, d,
J=15.0 Hz, 5-H), 0.97 (3H, s, 10-Me), 0.91 (3H, d,
J=7.0 Hz, 25-Me), 0.90 (3H, d, J=6.0 Hz, 20-Me), 0.65
(3H, s, 13-Me); MS (ESI) m/z 297 (M−2Na)2−
.
Acknowledgements
14. The concentration of SAAF (10 nM) is for an aqueous
gel in a capirally from which SAAF diffuses to a sperm-
containing medium. Thus, the minimum active concentra-
tion is thought to be the subnano-pico molar range.
This work was supported by a Grant-in-Aid for Scien-
tific Research on Priority Area (A) from the Ministry of