Second-generation synthesis of endogenous sperm-activating and attracting factor (SAAF) isolated from the ascidian Ciona intestinalis

Article abstract of DOI:10.1016/j.tetlet.2010.03.011

Stereoselective synthesis of the chemoattractant sperm-activating and attracting factor (SAAF), isolated from the eggs of the ascidian Ciona intestinalis, was achieved via reductive 1,3-transposition of an allylic alcohol and the axial opening of an epoxide as key steps. This second-generation synthesis improved the total yield of SAAF over that of the first-generation synthesis and provided a key intermediate for synthesizing molecular probes of SAAF.

Full text of DOI:10.1016/j.tetlet.2010.03.011

Tetrahedron Letters 51 (2010) 2600–2602
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Second-generation synthesis of endogenous sperm-activating and attracting
factor (SAAF) isolated from the ascidian Ciona intestinalis
*
Tohru Oishi , Kouichiro Ootou, Hajime Shibata, Michio Murata
Department of Chemistry, Graduate School of Science, Osaka University, 1-1 Machikaneyama, Toyonaka, Osaka 560-0043, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Stereoselective synthesis of the chemoattractant sperm-activating and attracting factor (SAAF), isolated
from the eggs of the ascidian Ciona intestinalis, was achieved via reductive 1,3-transposition of an allylic
alcohol and the axial opening of an epoxide as key steps. This second-generation synthesis improved the
total yield of SAAF over that of the first-generation synthesis and provided a key intermediate for synthe-
sizing molecular probes of SAAF.
Received 19 January 2010
Revised 26 February 2010
Accepted 4 March 2010
Available online 7 March 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Chemoattractant
Sperm-activating and attracting factor
Sterol sulfate
Stereoselective synthesis
26
OSO3-Na+
Chemotaxis of sperm toward an egg is a critical step in repro-
duction, particularly in aquatic environments, where sperm fre-
quently travel long distances to contact an egg.1 Yoshida et al.
isolated a non-peptidic chemoattractant called sperm-activating
and attracting factor (SAAF) from eggs of the ascidian Ciona intesti-
nalis. The structure of SAAF was determined to be a novel poly-
hydroxysterol sulfate (Fig. 1, 1).2,3 SAAF is the first steroid
possessing chemotactic activity, and the first example of a single
agent that possesses both sperm activation and attraction activi-
ties, which are reportedly elicited through different mechanisms.4
Thus, SAAF may serve as a key compound for biological studies
on signal transduction pathways leading to sperm flagellum move-
ment. Because of the limited availability of SAAF from natural
sources, the ability to produce the compound by chemical synthe-
sis is needed for further biological investigations. The first synthe-
sis of 1 by our group indicated that construction of the A ring was
problematic due to the low stereoselectivity and/or poor reproduc-
ibility of reduction of the 3-keto-4-enol intermediate.3 Herein, a
highly stereoselective synthesis of 1 is described in which con-
struction of the contiguous stereogenic centers at C3, C4, and C5
is completely controlled. The present synthesis also provides a
key intermediate for synthesizing molecular probes of SAAF
(Fig. 1, 2) for identification of the target proteins.5
3
+Na-O3SO
7
OH
4
H
OH
SAAF (1)
OSO3-Na+
+Na-O3SO
OH
H
H
O
O
O
Molecular probes (2)
N
R
O
S
HN
4
NH
O
2a: R =
2b: R =
N
4
H
O
CF3
O
S
Stereoselective synthesis of the steroid core 11 is shown in
Scheme 1. The known alcohol 4, prepared from chenodeoxycholic
acid 3 in two steps (93%),6 was protected as BOM ether 5. Dehydro-
N
N
HN
NH
H
N
O
O
O
3
2
O
* Corresponding author. Tel.: +81 6 6850 5775; fax: +81 6 6850 5785.
E-mail address: oishi@chem.sci.osaka-u.ac.jp (T. Oishi).
Figure 1. Structures of SAAF (1) and its molecular probes (2a–b).
0040-4039/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2010.03.011

T. Oishi et al. / Tetrahedron Letters 51 (2010) 2600–2602
2601
CO2Me
CO2H
ref. 6
IBX, TFA,
DMSO, rt, 48 h
5
4
93% (2 steps)
OR1
O
H
HO
OH
BOMCl, i-Pr2NEt,
4: R1 = H
H
5: R1 = BOM
NaI, TBAI, CH2Cl2,
3
rt, 11.5 h, 98%
CO2Me
+
+
O
O
H
OBOM
O
6c (10%)
6a (68%)
6b (6%)
CO2Me
CO2Me
NBSH, DEAD,
PPh3, NMM,
-30 to 0 ºC,
87%
CeCl3·7H2O,
NaBH4, MeOH,
0ºC, 1 h, 96%
6a
5
3
OBOM
HO
OBOM
H
8
7
CO2Me
CO2Me
MCPBA, NaHCO3,
CH2Cl2, 0 ºC, 6 h,
94%
AcONa, AcOH,
45 ºC, 48 h, 67%
5
3
3
4
4
R2O
OBOM
OBOM
H
O
H
OAc
9
10: R2 = H
TBSOTf, 2,6-lutidine,
CH2Cl2, 0 ºC, 30 min,
99%
11: R2 = TBS
Scheme 1. Synthesis of the steroid core of SAAF (11).
lectively to furnish
a
-epoxide 9 as a single isomer.10 Regioselective
CH3
Hax
H
opening of epoxide 9 was achieved by treatment with sodium ace-
tate in acetic acid at 45 °C for two days to yield 10 (67%) via diaxial
opening (Fürst-Plattner rule)11 with recovery of 9 (20%). The struc-
ture of 10 was confirmed by 1H NMR analysis as shown in Figure 2.
The resulting secondary alcohol was protected as the TBS ether 11.
Thus, stereoselective construction of the contiguous stereogenic
centers of the steroid core at C3, C4, and C5 was achieved. The
overall yield from chenodeoxycholic acid for nine steps was 32%,
a dramatic improvement over that of the corresponding intermedi-
ate in the first-generation synthesis (9.2%, nine steps).3
OAc
H
6
H
5
3
4
H
Heq
OH
H
OBOM
10
J
(H3,H4) = 3 Hz
J (H4,H5) = 3 Hz
J (H5,H6ax) = 13 Hz
Figure 2. Selected 1H NMR coupling constants of 10.
Next, side chain elongation was accomplished in a sequence
analogous to that in the first-generation synthesis (Scheme 2).3
Selective hydrolysis of the methyl ester in the presence of acetate
was achieved by treatment with t-BuOK in t-BuOH to yield carbox-
ylic acid 12 (72%) with recovery of 11 (20%). Conversion of carbox-
ylic acid 12 to aldehyde 14 was achieved through decarboxylative
iodination (86%),12 followed by treatment of the resulting iodide 13
with DMSO in the presence of collidine (80%).13 Wittig olefination
using phosphonium salt 1514,15 gave a Z-olefin (Z:E = 20:1) in 84%
yield, with protection of the resulting primary alcohol as a TBS
ether (16) in 90% yield. Removal of the acetyl group with LiAlH4
provided alcohol 17 (98%),16 a common intermediate of SAAF (1)
and its molecular probes (2).5 Protection of the secondary alcohol
as a BOM ether provided 18 (86%) followed by removal of the
TBS groups with TBAF to give diol 19 (73%) which was converted
to sulfate 20 by successive treatment with sulfur trioxide–pyridine
complex and ion exchange resin (Amberlite IR-120B). Finally, re-
moval of the BOM groups using palladium black as a catalyst under
genation of ketone 5 proceeded in a regioselective manner by
treatment with o-iodoxybenzoic acid (IBX)7 in the presence of tri-
fluoroacetic acid (TFA) to afford the C4–C5 unsaturated ketone 6a
in 68% yield with concomitant formation of regioisomer 6b (C1–
C2 unsaturated, 6%) and dienone 6c (C1–C2 and C4–C5 unsatu-
rated, 10%). Luche reduction8 of enone 6a resulted in the formation
of b-alcohol 7 as a single isomer. Reductive 1,3-transposition of
allylic alcohol 7 was achieved successfully according to the Myers
protocol9 [i.e., 7 was subjected to Mitsunobu reaction conditions
using diethyl azodicarboxylate (DEAD), triphenylphosphine, and
o-nitrobenzenesulfonylhydrazine (NBSH) in the presence of N-
methylmorphorine (NMM) in toluene at ꢀ30 to 0 °C] to afford ole-
fin 8 in 87% yield as a single isomer. Since stereocontrol at the C5
position was complete, the stereoselective introduction of a 3,4-
diol unit in trans-diaxial orientation was conducted. Epoxidation
of 8 with m-chloroperbenzoic acid (MCPBA) proceeded stereose-

2602
T. Oishi et al. / Tetrahedron Letters 51 (2010) 2600–2602
R1
date signal transduction pathways induced by SAAF are currently
in progress.
Supplementary data
TBSO
OBOM
H
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.tetlet.2010.03.011.
OAc
11: R1 = CH2CO2Me
12: R1 = CH2CO2H
13: R1 = CH2I
t-BuOK, t-BuOH, 72%
Pb(OAc)4, I2, hν, CCl4, 86%
collidine, DMSO, 150 ºC, 80%
References and notes
14: R1 = CHO
1. Miller, R. L. In Biology of Fertilization; Metz, C. B., Monoray, A., Eds.; Academic:
New York, 1985; Vol. 2, p 275.
2. Yoshida, M.; Murata, M.; Inaba, K.; Morisawa, M. Proc. Natl. Acad. Sci. U.S.A.
2002, 99, 14831.
3. (a) Oishi, T.; Tsuchikawa, H.; Murata, M.; Yoshida, M.; Morisawa, M.
Tetrahedron Lett. 2003, 44, 6387; (b) Oishi, T.; Tsuchikawa, H.; Murata, M.;
Yoshida, M.; Morisawa, M. Tetrahedron 2004, 60, 6971; (c) Murata, M.; Oishi, T.;
Yoshida, M. In Progress in Morecular and Subcellular Biology, Subseries Marine
Molecular Biotechnology; Fusetani, N., Clare, A. S., Eds.; Springer: Berlin, 2006; p
203.
Br-
1) 15, n-BuLi, THF, -78ºC;
TMSCl, 0 ºC,
Ph3P+
OH
then 13, -78 ºC to rt;
0.5 M HCl, 84%
15
2) TBSCl, imidazole, 90%
OR3
4. (a) Yoshida, M.; Inaba, K.; Morisawa, M. Dev. Biol. 1993, 157, 497; (b) Yoshida,
M.; Inaba, K.; Ishida, K.; Morisawa, M. Dev. Growth Differ. 1994, 36, 589.
5. (a) Kondoh, E.; Konno, A.; Inaba, K.; Oishi, T.; Murata, M.; Yoshida, M. Dev.
Growth Differ. 2008, 50, 665; (b) Yoshida, M.; Shiba, K.; Yoshida, K.; Tsuchikawa,
H.; Ootou, K.; Oishi, T.; Murata, M. FEBS Lett. 2008, 582, 3429.
6. Tserng, K.-Y. J. Lipid Res. 1978, 19, 501.
7. Zhang, D.-H.; Cai, F.; Zhou, X.-D.; Zhou, W.-S. Org. Lett. 2003, 5, 3257.
8. Gemal, A. L.; Luche, J.-L. J. Am. Chem. Soc. 1981, 103, 5454.
9. Myers, A. G.; Zheng, B. Tetrahedron Lett. 1996, 37, 4841.
10. Silva, E. J.; Melo, M. L.; Neves, A. S. J. Chem. Soc., Perkin Trans. 1 1996, 1649.
11. Royals, E. E.; Leffingwell, J. C. J. Org. Chem. 1966, 31, 1937.
12. Sheldon, R. A.; Kochi, J. K. Org. React. 1972, 19, 279.
7
4
R3O
OBOM
H
R2O
16: R2 = Ac, R3 = TBS
17: R2 = H, R3 = TBS
LiAlH4, THF, 98%
BOMCl, i-Pr2NEt, 86%
TBAF, THF, 73%
18: R2 = BOM, R3 = TBS
19: R2 = BOM, R3 = H
20: R2 = BOM, R3 = SO3Na
SO3·Py, Py, 60 ºC;
Amberlite IR-120B
H2, Pd black, MeOH,
78% (for two steps).
13. (a) Kornblum, N.; Jones, W. J.; Anderson, G. J. J. Am. Chem. Soc. 1959, 81, 4113;
(b) Van Gool, M.; Zhao, X.-Y.; Sabbe, K.; Vandewalle, M. Eur. J. Org. Chem. 1999,
2241.
26
OSO3-Na+
14. (a) Kozikowski, A. P.; Chen, Y. Y. J. Org. Chem. 1981, 46, 5248; (b) Bergmann, J.;
Löfstedt, C.; Ivanov, V. D.; Francke, W. Eur. J. Org. Chem. 2001, 16, 3175.
15. (a) White, J. D.; Jeffrey, S. C. J. Org. Chem. 1996, 61, 2600; (b) Wang, X.; Erickson,
S. D.; Iimori, T.; Still, W. C. J. Am. Chem. Soc. 1992, 114, 4128.
16. Physical data of 17: Colorless amorphous; ½a D28
ꢁ
+11.3 (c 0.91, CHCl3); Rf = 0.47
3
(hexane/EtOAc = 5/1); IR (film) 3501, 2886, 2857, 1471, 1460, 1361, 1255,
+Na-O3SO
4
7
OH
1149, 1083, 1046, 1027, 1004, 836, 774, 734, 697, 673 cmꢀ1
;
1H NMR
H
OH
(500 MHz, CDCl3) d 7.32–7.24 (5H, m, Ph), 5.37–5.32 (1H, m, H23), 5.17 (1H,
t, J = 9.5 Hz, H24), 4.85 (1H, d, J = 7.5 Hz, –OCH2O–), 4.75 (1H, d, J = 7.5 Hz, –
OCH2O–), 4.63–4.53 (2H, m, PhCH2–), 3.78 (1H, d, J = 2.5 Hz, H4), 3.76 (1H, d,
J = 2.5 Hz, H3), 3.47–3.45 (1H, m, H26), 3.41 (1H, d, J = 1.5 Hz, H7), 3.34–3.30
(1H, m, H26), 2.57–1.02 (23H, m, H1, 2, 5, 6, 8, 9, 11, 12, 14, 15, 16, 17, 20, 22,
25), 1.00 (3H, s, H19), 0.93 (3H, d, J = 7.5 Hz, H27), 0.91 (3H, d, J = 7.5 Hz, H21),
0.84 (18H, s, –Sit-Bu), 0.63 (3H, s, H18), 0.02 (6H, s, –SiCH3), 0.01 (6H, s, –
SiCH3); 13C NMR (125 MHz, CDCl3) d 137.95, 133.18, 128.55, 128.26, 127.63,
127.47, 94.74, 77.26, 76.42, 69.73, 67.97, 55.99, 50.14, 47.11, 42.61, 42.58,
39.94, 39.14, 36.67, 36.53, 35.91, 34.85, 34.03, 31.74, 30.85, 28.32, 26.06, 26.03,
25.89, 25.73, 25.03, 24.00, 20.05, 18.89, 18.48, 18.11, 17.58, 17.02, 13.58, 11.89,
ꢀ4.67, ꢀ4.87, ꢀ5.14, ꢀ5.17; HRMS (ESI-TOF) calcd for C47H82O5Si2 [M+Na+]
805.5593, found: 805.5601.
SAAF (1)
Scheme 2. Synthesis of SAAF (1).
hydrogen and concomitant hydrogenation of the double bond
afforded SAAF (1) in 78% yield over two steps.
In conclusion, stereocontrolled synthesis of SAAF (1) was
achieved via an improved route that completely controlled the
contiguous stereogenic centers at C3, C4, and C5. The present syn-
thesis also provided a key intermediate (17) for synthesizing
molecular probes of SAAF. Syntheses of molecular probes to eluci-