Synthesis and identification of an endogenous sperm activating and attracting factor isolated from eggs of the ascidian Ciona intestinalis; An example of nanomolar-level structure elucidation of novel natural compound

Article abstract of DOI:10.1016/j.tet.2004.02.075

A sperm-activating and attracting factor (SAAF) was isolated from the eggs of the ascidian Ciona intestinalis, and its structure was deduced with only approximately 4 μg of the specimen. Based upon the proposed structure, two epimers were synthesized from chenodeoxycholic acid in 16 steps. Comparison between synthetic and natural compounds 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 utilized to confirm that both sperm-activation and attraction were elicited by a single compound.

Full text of DOI:10.1016/j.tet.2004.02.075

Tetrahedron 60 (2004) 6971–6980
Synthesis and identification of an endogenous sperm activating
and attracting factor isolated from eggs of the ascidian
Ciona intestinalis; an example of nanomolar-level structure
elucidation of novel natural compound
b
b
Tohru Oishi,a Hiroshi Tsuchikawa,a Michio Murata,a Manabu Yoshida and Masaaki Morisawa
,
*
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 27 June 2003; revised 22 February 2004; accepted 22 February 2004
Available online 19 June 2004
Abstract—A sperm-activating and attracting factor (SAAF) was isolated from the eggs of the ascidian Ciona intestinalis, and its structure
was deduced with only approximately 4 mg of the specimen. Based upon the proposed structure, two epimers were synthesized from
chenodeoxycholic acid in 16 steps. Comparison between synthetic and natural compounds 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 utilized to confirm that
both sperm-activation and attraction were elicited by a single compound.
q 2004 Elsevier Ltd. All rights reserved.
1. Introduction
nism.8,9 In the present study, we report the structural
elucidation and synthesis of the sperm-activating and
attracting factor (SAAF) from a species of acidean.10,11
Sperm chemotractants, ubiquitously found in the animal
kingdom, play an essential role in fertilization.1,2 Chemo-
taxis of sperm toward an egg is a crucial event particularly
in aquatic environment, where sperm frequently travels long
distance to run into an egg. The relevant factors may control
the chance of intra- or inter-species mating, which may take
part in securing biodiversity, and thus have been a target
of biological research for a long time. To investigate the
molecular basis of fertilization, the pure specimen of a
sperm attractant is indispensable, particularly for tracking
signal transduction from chemoreception to flagellar move-
ment. Although numerous peptides and small organic
compounds have been so far proposed as a candidate of
chemoattractant,3,4 only a few of them have been unam-
biguously identified as an endogenous factor,5 such as those
for the sea urchin Arbacia punctulata,6 the coral Montipora
digitata,7 and the frog Xenopus laevis. The difficulty in
these studies lies in the quantification of chemotactic
activity of sperm. Two of the authors at Misaki Marine
Biological Station have extensively examined the chemo-
taxis in ascidian and clearly demonstrated that sperm
activation and attractant are caused by different mecha-
2. Results and discussion
2.1. Structure determination
SAAF was extracted and purified from the eggs of the
ascidian Ciona intestinalis as previously described.12,13
During the purification procedure, the sperm-activating and
attracting activities were always found in same fractions,
indicating that both activities were elicited by a single
compound. The chemical structure of SAAF was deduced
based on NMR and mass spectrometry. The molecular-
related ions were observed in negative ion FAB MS at m/z
617 and 595, which corresponded to (X–H)2 and (X–Na)2,
respectively. In addition, a prominent ion peak at m/z 515
corresponded to a fragment lost typical for a sulfate ester
(–SO3NaþH). These data indicate that SAAF has two
sulfate ester groups and, after one of them is lost, it still
possesses a negatively charged group (¼sulfate) to give rise
to a prominent ion at m/z 515. High-resolution ESI-TOF MS
further supported the presence of two sulfate esters by
giving rise to the exact mass of m/z 297.1273 for a divalent
anion, corresponding to C27H48O18S2 (calculated mass, m/z
297.1266). Two-dimensional 1H NMR (2D TOCSY)
spectrum in Figure 3 was successfully determined with
Keywords: Sperm-activating and attracting factor; Sterol disulfate;
Structure determination; Synthesis.
*
Corresponding author. Tel./fax: þ81-6-6850-5774;
e-mail address: murata@ch.wani.osaka-u.ac.jp
0040–4020/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2004.02.075

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T. Oishi et al. / Tetrahedron 60 (2004) 6971–6980
Figure 1. 1H NMR spectrum of SAAF (1, ca. 4 mg); D2O, 500 MHz. The spectrum was measured with using a special probe (Nanolac, Z-Spec-SMIDG500) for
1.7-mm diameter sample tube on a JEOL L-500 spectrometer. pSignals due to impurities. The broad peak at d 0.4 is due to silicon grease outside of the tube
used for sliding the tube into a spinner.
approximately 4 mg based upon an NMR spectrum in
comparison with that of a synthetic specimen. The spectra
revealed two singlet methyl signals at d 0.65 and 0.97
characteristic of steroid skeletons. Additional two methyl
doublets at d 0.90 and 0.92 and complex signals at 0.8–
2.0 ppm further support the presence of a steroid backbone.
The down-field signals at d 4.41, 3.96, 3.92, 3.83, and 3.72
correspond to five protons on four oxygen-bearing carbon
atoms (those at d 3.92/3.83 were apparently derived from a
methylene group). Among these, the connectivity of signals
at d 4.41, 3.92, and 3.72 was elucidated by 2D TOCSY (Fig.
3). This structural moiety fits the C3–C7 part of a steroid
skeleton. Signals at d 3.92 and 3.83, which revealed geminal
coupling, were assigned to a methylene group of C26 on the
basis of relayed coupling to one of the methyl doublets. The
location of sulfate groups was deduced from low-field
chemical shifts of signals at d 4.41 and 3.92/3.83 in
comparison with those of hydroxyl-bearing methine and
methylene groups. Moreover, their chemical shifts agreed
well with reported values for starfish sterol sulfates.12 Those
spectral data indicated that SAAF was a novel sulfated
steroid, 3,4,7,26-tetrahydroxy-cholestane-3,26-disulfate
(Fig. 1).
High-energy collision-induced dissociation (CID) MS/MS
is a versatile tool for structural elucidation of ionic
compounds.13 We adopted the tandem MS instrument of
EB–EB geometry with the negative-ion fast atom bombard-
ment (FAB) ionization method. The product ion spectrum
was recorded for precursor ions at m/z 515, which
corresponds to a desulfated product of SAAF presumably
generated through the first MS section. The structural basis
of the assignment for major product ions is shown in Figure
2. These fragmentations are reasonably explained by the
Figure 2. Normal FAB spectrum of SAAF (1) and fragment patterns for 3-O-defulfated ion of SAAF. Product ion spectra were obtained by CID MS/MS
experiments. See text for details.10

T. Oishi et al. / Tetrahedron 60 (2004) 6971–6980
6973
proposed structure, particularly for the part of three oxygen
functionalities, hence confirming the planner structure of
SAAF.
This working structure of SAAF was derived from limited
NMR and MS data, hence leaving some ambiguities to be
1
addressed. For the sterol skeleton, where H NMR signals
heavily overlap, no clear evidence was obtained regarding
stereochemistry. Chemical synthesis was thus necessary for
the complete structure identification. For this purpose, we
had to deduce the configuration of SAAF. First, the skeletal
stereostructure was deduced to be cholestan by the
following two reasons; most of sterols isolated from
tunicates possess a cholestan skeleton and a large coupling
constant between H-5 and H-6 suggested the trans fusion of
rings A and B. The stereochemistry of oxygenated carbon
atoms was deduced from the small coupling constants for
the signals at d 4.41, 3.96, and 3.72 as one can observe in the
attached 1D spectrum of Figure 3. The data clearly reveal
that no diaxial coupling is involved in these spin systems,
which indicates that all the three oxygen atoms were axially
oriented. The stereochemistry at C25, which could not be
assigned by NMR, remained to be elucidated by synthesiz-
ing both epimers at this positions.
2.2. Synthesis of SAAF and 25-epi-SAAF
Synthesis plan of SAAF and its C-25 epimer is shown in
Scheme 1. We planed a versatile route that could provide
both diastereomers by introducing the side chain using 4 and
5 at the latest steps via a common intermediate 6, which
would be synthesized from chenodeoxycholic acid 7.
Figure 3. TOCSY spectrum of SAAF (1) of an extremely limited amound
(ca. 4 mg). The spectrum was measured as the same conditions as those in
Figure 1.
Synthesis of SAAF commenced with 7a-hydroxy-3-oxo-
5b-cholan-24-oate (8), prepared from 7 in two steps
(Scheme 2).14 Oxidation of 8 with molecular oxygen in
the presence of t-BuOK proceeded regioselectively via the
C-4 enolate, and following esterification of the concomi-
tantly hydrolyzed product afforded 3-keto-4-enol 9.
Selective protection of the C-7 alcohol of 9 as its
benzyloxymethyl (BOM) ether in the presence of C-4 enol
yielded 10. Reduction of 10 with NaBH4 proceeded
stereoselectively at C-3 to give cyclic enol borate 11, and
hydrolysis of 11 with 1 M HCl yielded 4-keto-3-ol 12a
concomitant with its C-5 epimer 12b as an inseparable
mixture in a 1:1.8 ratio. Treatment of the mixture with
NaBH4 gave the desired diol 13a from 12a and diastereomer
13b from 12b in 20 and 36%, respectively. The stereo-
1
chemistry of 12a was unambiguously determined by H
NMR analysis. Isomerization of 12b to 12a under the basic
conditions failed to give 3-keto-4-ol 12c as a single isomer.
Attempts to reduce 9 using other conventional methods
were unsuccessful due to the formation of undesired
diastereomers preferentially with low reproducibility.
Finally, a solid-phase reduction of 10 with NaBH3CN on
Scheme 1. Synthesis plan of SAAF and 25-epi-SAAF.

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T. Oishi et al. / Tetrahedron 60 (2004) 6971–6980
to give 13a, whereas 12b remained intact and easily
separated from 13a. However, partial isomerization of 12b
occurred to give 12c, which was further reduced to 13c
resulting in the formation of inseparable mixture of 13a and
13c in a ratio of 3:1 (44%). Even though the yield of 13a
should be improved, the contiguous 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 13a and 13c with thionyl
chloride followed by oxidation with RuO2–NaIO4 gave
cyclic sulfate 14a in 65% yield for two steps,15 which was
easily separated from 5b-isomer 14b (11%) by silica gel
chromatography (Scheme 3). Regioselective opening of the
cyclic sulfate at C-3 position was successfully achieved by
treating with benzoic acid in the presence of Cs2CO3 in
DMF (75%) followed by hydrolysis of the resulting
sulfate.16 Protection of the resulting C-4 alcohol (15) as
BOM ether afforded benzoate 16 as a single isomer (71%).
Exposure of 16 to t-BuOK in t-BuOH resulted in the
selective hydrolysis of the methyl ester in the presence of
benzoate ester to yield carboxylic acid 17 (95%), which was
further converted to iodide 18 through the decarboxylation
by treating with Pb(OAc)4 in the presence of iodine under
irradiation using a tungsten lamp (84%).17Oxidation of 18
with DMSO in the presence of 2,4,6-collidine gave an
aldehyde 6 (94%), a common precursor of the two
diastereomers at C-25. Elongation of the side chain was
achieved through a Wittig reaction between the aldehyde 6,
and an ylide generated from (R)-phosphonium salt 418 and
n-BuLi–TMSCl19 to give olefin 19 (E/Z¼1:8) in 69% yield.
Reductive removal of the benzoyl group of 19 with LiAlH4
gave diol 20, which was converted to the corresponding
sodium bis-sulfate through the successive treatment with
SO3·Py and ion-exchange resin (IR-120B, Naþ form).
Hydrogenation of the double bond and concomitant removal
of the BOM groups afforded 25S-isomer (2), and the
resulting polar material was purified by reverse-phase
HPLC. The C-25 epimer 3 was synthesized by the identical
procedure as 2 except for the use of (S)-phosphonium salt 5
in place of 4.
2.3. Comparison with the natural product
The 1H NMR spectrum of the natural product was compared
with those of the synthetic samples, 2 (25S) and 3 (25R), as
shown in Figure 4.10 The areas other than those in Figure 4
were indistinguishable among the natural SAAF and the
synthetic pair. While the chemical shifts of the 25-methyl
group and H-26a in 3 do not match those of the natural
product (Dd values for H-26a and 25-methyl are 20.014 and
20.007 ppm, respectively), those of 2 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.
Scheme 2. Reagents and conditions: (a) t-BuOK, O2, t-BuOH, then CH2N2,
Et2O, MeOH, CHCl3 (71%); (b) BOMCl, i-Pr2NEt, CH2Cl2 (60%);
(c) NaBH4; CHCl3, MeOH, 0 8C; (d) 1 M HCl; (e) DBU, THF, 60 8C;
(f) NaBH3CN, SiO2, CHCl3, MeOH, then evaporated.
silica gel was found to afford desired diol 13a in moderate
yield. A solution of 10 in chloroform and methanol was
added NaBH3CN (5 equiv.) and silica gel (10–silica
gel¼1:3, w/w). After stirring for 10 min the solvent was
removed under reduced pressure, and the residue was stirred
without solvent. Under the reaction conditions, hydrolysis
of the borate 11 proceeded smoothly to give a mixture of
12a and 12b. Further reduction of 12a proceeded smoothly
Both the sperm-activating and attracting activity of
synthetic SAAF were bioassayed using methods previously
reported.4 Synthetic SAAF (2) activated sperm of the
ascidian C. intestinalis at 3.7 nM and concurrently exhibited

T. Oishi et al. / Tetrahedron 60 (2004) 6971–6980
6975
comparative activities as those with SAAF (activated at
,3.7 nM and attracted at ,10 nM).
We succeeded in elucidating the structure of SAAF with
only ca. 4 mg of a specimen. This may be partly due to the
sensitivity improvement of modern NMR instruments as
demonstrated by the special probe for a capillary tube. We
rather think that the high purity of the specimen is a key
point. Usually, it is very difficult to prepare the microgram
amount of an NMR sample without contaminations, which
may come from both HPLC and NMR solvents or from
containers. In this study, the sample was mostly treated as a
aqueous solution in plastic vessels, which helped reducing
the contamination with detergents or plasticizers; the only
prominent contaminant seen in Figure 1 is lactic acid, one of
common impurities under aqueous conditions. Another
problem encountered in 2D NMR measurements is that an
enormous solvent peak often results in an uneven base
plane. In this study, the volume of the solution was only
20 mL, which effectively reduced the contamination and
secure the flatness of the base line or base plane in NMR
Scheme 3. Reagents and conditions: (a) SOCl2, Et3N, THF, 94%;
(b) RuCl3·nH2O, NaIO4, 14a (65%), 14b (11%); (c) PhCO2H, Cs2CO3,
DMF; then conc. H2SO4, THF, 75%; (d) BOMCl, i-Pr2NEt, CH2Cl2 (84%);
(e) t-BuOK, t-BuOH, 95%; (f) Pb(OAc)4, I2, hn, CCl4, hn (300 W tungsten
lamp), 84%; (g) DMSO, 2,4,6-collidine, 94%; (h) n-BuLi, TMSCl, THF,
then 0.5 M HCl, 69%; (i) LiAlH4, THF, 80%; (j) SO3·Py, Py, then Amberite
IR-120B Naþ form; (k) Pd/C, H2, MeOH.
the attracting activity at ,10 nM;11 these quantitative
evaluations were first accomplished with the synthetic
specimen. The concentration of SAAF (10 nM) is for an
aqueous gel in a capillary from which SAAF diffuses to a
sperm-containing medium. Thus, the minimum active
concentration is thought to be the subnano–pico molar
range. It is noteworthy that 25-epi-SAAF (3) possesses
Figure 4. Partial 1H NMR spectra of SAAF, 2(25S), and 3(25R). Asterisks
indicate the signals attributed to contaminants.

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T. Oishi et al. / Tetrahedron 60 (2004) 6971–6980
measurements. The other methodology useful for the
structure analysis in nanomolar scale is MS/MS; in
particular, high energy collision experiments are helpful
since C–C bond cleavages provides the information about
skeletal structures. Key steps in this study was to obtain the
reliable working structures and to synthesize these
effectively.
at 23 8C on a JEOL L-500 spectrometer (500 MHz) with a
1.7-mm probe (Nanolac, Z-Spec-SMIDG500). 2D TOCSY
spectra were measured for a spin locking time of 20 ms with
data matrices of 2k£512 (a total of 82,000 acquisitions for
35 h) while a peak of solvent-derived deuterium being
presaturated. Fast atom bombardment (FAB) MS/MS
experiments were carried out in the negative-ion mode
using a HX-110/HX-110 tandem mass spectrometer (JEOL)
equipped with a variable dispersion array detector (MS-
ADS11). Collision-induced dissociation (CID) was effected
by introducing helium at a pressure that reduced the
intensity of precursor ions to 30%.
3. Conclusion
In conclusion, the sperm-activating and attracting factor
(SAAF) was synthesized from 7 in 17 steps, which led to the
unambiguous structure determination of SAAF to be
(3R,4R,7R,25S)-3,4,7,26-tetrahydroxycholestane-3,26-di-
sulfate (2). The synthetic pure specimen 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 pathway(s).
4.2. Synthesis of SAAF and 25-epi-SAAF
4.2.1. Methyl 7a-hydroxy-3,4-dioxo- cholan-24-oate 9.
t
To a stirred solution of 8 (2.57 g, 6.35 mmol) in BuOH
t
(15 mL) was added BuOK (2.10 g, 19.1 mmol) and the
resulting suspension was stirred for 1.5 h at rt under oxygen
atmosphere (under the reaction conditions, the methyl ester
was hydrolyzed to give the corresponding carboxylate). The
reaction was carefully quenched with 1.87 M NH4Cl
(130 mL). The reaction mixture was extracted with ethyl
acetate, and the combined organic layer was concentrated.
The residue was dissolved in chloroform (10 mL) and
methanol (10 mL), which was treated with a solution of
diazomethane in ether at 0 8C and the mixture stirred for 1 h.
Evaporation followed by silica gel column chromatography
(hexane/ethyl acetate¼8:1–2:1) gave 9 (1.81 g, 68% from
8) as colorless solid.
4. Experimental
All reactions sensitive to air or moisture were carried out
under argon atmosphere in dry, freshly distilled solvents
under anhydrous conditions, unless otherwise noted. THF
was distilled sodium/benzophenone, diethyl ether (Et2O)
from LiAlH4, diisopropylamine, diisopropylethylamine
(i-Pr2NEt), pyridine, from calcium hydride, and DMF,
DMSO and HMPA from calcium hydride under reduced
pressure. Dichloromethane (CH2Cl2) and toluene was dried
over activated MS4A. All other reagents were used as
supplied unless otherwise stated. Analytical thin-layer
chromatography (TLC) was performed using E. Merck
Silica gel 60 F254 pre-coated plates. Column chromato-
graphy was performed using 100–210 mm Silica Gel 60N
(Kanto Chemical), and for flash column chromatography
Compound 9. Rf 0.20 (hexane/ethyl acetate¼2:1); IR (KBr)
3448, 2940, 1736, 1670, 1636, 1381, 1169 cm21; 1H NMR
(500 MHz, CDCl3) d 6.11 (1H, s, 4-OH), 3.96 (1H, s, 7-H),
3.64 (3H, s, CO2CH3), 3.14 (1H, dd, J¼15.5, 3.5 Hz, 6-Ha),
2.16 (1H, dd, J¼15.5, 3.5 Hz, 6-Hb), 1.16 (3H, s, 10-Me),
0.90 (3H, d, J¼7.0 Hz, 20-Me), 0.68 (3H, s, 13-Me); 13C
NMR (500 MHz, CDCl3) d 192.9, 174.5, 143.3, 135.9, 67.8,
55.7, 51.5, 50.3, 45.6, 42.4, 39.4, 39.2, 37.6, 35.4, 34.4,
31.8, 31.6, 31.0, 31.0, 28.1, 23.6, 20.8, 18.3, 16.8, 11.9.
1
40–63 mm Silica Gel 60N (Merck) was used. H and 13C
NMR spectra were recorded on a JEOL EX-270 (270 MHz),
a JEOL GSX-500 (500 MHz), or JEOL JMN-LA500
(500 MHz) spectrometer. Chemical shifts are reported in d
(ppm) using residual CHCl3 as an internal standard of d 7.26
and d 77.00 for 1H and 13C NMR, respectively. Signal
patterns are indicated as s, singlet; d, doublet; t, triplet; q,
quartet; m, multiplet; br, broad peak. IR spectra were
recorded on a Jasco FT/IR-300E spectrometer. Mass spectra
(ESI) were recorded on a ThermoQuest LCQ DECA
instrument. Optical rotations were recorded on a Perkin–
Elmer 241 polarimeter.
4.2.2. Methyl 7a-benzyloxymethoxy-3,4-dioxocholan-24-
oate 10. To a solution of 9 (2.63 g, 6.29 mmol) in
dichloromethane (18.9 mL) was added diisopropylethyl-
amine (2.19 mL, 12.6 mmol) and BOMCl (1.31 mL,
9.44 mmol), and the mixture was stirred for 18 h at rt. The
reaction was quenched with water and extracted with
dichloromethane. The combined organic layer was washed
with saturated NaCl, dried over anhydrous MgSO4, and
filtered. Concentration followed by purification by flash
column chromatography (hexane/ethyl acetate¼10:1–5:1)
gave 10 (2.03 g, 60%) as colorless amorphous, and 17% of 9
(464 mg) was recovered.
4.1. Structure determination
Materials. The ascidians, C. intestinalis was collected at
the Onagawa Bay (Miyagi prefecture), Aburatsubo Bay
(Kanagawa prefecture), and Ootsuchi Bay (Iwate prefec-
ture) in Japan. SAAF was extracted and purified as reported
previously.10
Compound 10. Colorless amorphous; Rf 0.55 (hexane/ethyl
acetate¼2:1); [a]2D2 22.48 (c 1.58, CHCl3); IR (KBr) 3456,
2960, 1740, 1664, 1636, 1380, 1172, 1052 cm21; 1H NMR
(500 MHz, CDCl3) d 7.36–7.24 (5H, m, Ph), 6.03 (1H, s,
4-OH), 4.88 (1H, d, J¼7.0 Hz, OCH2OBn), 4.74 (1H, d,
J¼7.0 Hz, OCH2OBn), 4.65 (1H, d, J¼11.5 Hz, OCH2Ph),
4.57 (1H, d, J¼11.5 Hz, OCH2Ph), 3.88 (1H, s, 7-H), 3.65
(3H, s, CO2CH3), 3.39 (1H, dd, J¼16.0, 3.0 Hz, 6-Ha), 2.00
(1H, dd, J¼16.0, 3.0 Hz, 6-Hb), 1.16 (3H, s, 10-Me), 0.91
Structure analysis of SAAF. Electrospray-ionization time-
of-flight (ESI-TOF) MS was performed in the negative-ion
1
mode with a Mariner (ABI) mass spectrometer. H NMR
spectra of SAAF in 20 mL of a D2O solution were recorded

T. Oishi et al. / Tetrahedron 60 (2004) 6971–6980
6977
(3H, d, J¼6.5 Hz, 20-Me), 0.68 (3H, s, 13-Me); 13C NMR
(500 MHz, CDCl3) d 193.1, 174.5, 142.8, 137.9, 136.7,
128.2, 127.6, 127.5, 93.7, 73.7, 70.0, 55.6, 51.5, 49.9, 45.9,
42.4, 39.6, 39.1, 37.6, 35.4, 34.5, 31.9, 31.1, 31.0, 28.5,
28.1, 24.0, 20.8, 18.4, 16.9, 11.8; MS (ESI) m/z 561
(MþNa)þ.
(241 mg, 1.13 mmol) in a mixed solvent of acetonitrile
(2.3 mL), CCl4 (1.5 mL), and 50 mM phosphate buffer
(1.5 mL). The reaction mixture was stirred for 20 min, then
quenched with saturated Na2S2O3 and extracted with ethyl
acetate. The organic layers were combined, washed with
saturated NaCl, dried over dried over MgSO4, filtered.
Concentration followed by florisil column chromatography
(hexane/ethyl acetate¼30:1–6:1) gave cyclic sulfate 14a
(367 mg, 65%) as colorless amorphous, and side product
14b (62 mg, 11%).
4.2.3. Methyl 7a-benzyloxymethoxy-3b,4b-dihydroxy-
5a-cholan-24-oate 13a. To a stirred solution of 10 (1.01 g,
1.88 mmol) in chloroform (4 mL) and methanol (4 mL) was
added NaBH3CN (589 mg, 9.37 mmol) and silica gel
(3.00 g), and the reaction mixture was stirred for 10 min
at rt. The solvent was evaporated and the residue was stirred
for 1 h at rt. The residue was added chloroform (4 mL) and
methanol (4 mL) again and stirred for 10 min. The solvent
was removed by evaporation and the resulting residue was
stirred for 3 h at rt. The same procedure was repeated again
and the residue was stirred for 13 h at rt. The residue was
washed with a mixed solvent of chloroform and methanol
(chloroform/methanol¼2:1), and the precipitates were
removed by filtration. The residue was washed with the
same solvent three times. The combined filtrate was
concentrated, and the residue was dissolved in ethyl acetate.
The organic layer was washed with saturated NaCl, dried
over MgSO4, and concentrated under reduced pressure. The
residue was purified by flash column chromatography
(hexane/ethyl acetate¼8:1–2:1) to give a mixture of 13a
and 13c (443 mg, 44% 13a/13c¼3:1), which was separated
from 12b (220 mg, 22%).
Compound 14a. Colorless amorphous; Rf 0.32 (hexane/ethyl
acetate¼5:1, 3 times development); [a]2D3 þ6.48 (c 0.12,
CHCl3); IR (thin film) 2945, 2869, 1736, 1381, 1208,
;
1039 cm21 1H NMR (500 MHz, CDCl3) d 7.36–7.27
(5H, m, Ph), 4.82 (1H, d, J¼7.0 Hz, OCH2OBn), 4.74
(1H, d, J¼7.0 Hz, OCH2OBn), 4.63–4.56 (4H, m, OCH2Ph,
3,4-H), 3.74 (1H, d, J¼2.0 Hz, 7-H), 3.64 (3H, s, CO2CH3),
1.03 (3H, s, 10-Me), 0.90 (3H, d, J¼7.0 Hz, 20-Me), 0.63
(3H, s, 13-Me); 13C NMR (500 MHz, CDCl3) d 174.5,
137.7, 128.4, 127.8, 127.3, 94.7, 85.8, 83.0, 75.2, 70.3, 55.6,
51.5, 49.9, 46.5, 42.5, 40.0, 39.6, 39.0, 35.4, 34.9, 34.1,
31.1, 31.0, 30.6, 28.0, 23.7, 20.4, 18.3, 12.9, 11.9; MS (ESI)
m/z 627 (MþNa)þ.
Compound 14b. Amorphous material; Rf 0.43 (hexane/
EtOAc¼5:1, 3 times development); [a]2D3 þ32.18 (c 1.44,
CHCl3); IR (thin film) 2945, 2870, 1736, 1382, 1208,
1035 cm21; 1H NMR (500 MHz, CDCl3) d 7.35–7.26 (5H,
m, Ph), 5.76 (1H, dd, J¼10.0, 4.5 Hz, 4-H), 5.12 (1H, q,
J¼4.5 Hz, 3-H), 4.81 (1H, d, J¼6.5 Hz, OCH2OBn), 4.74
(1H, d, J¼6.5 Hz, OCH2OBn), 4.62 (1H, d, J¼12.0 Hz,
OCH2Ph), 4.59 (1H, d, J¼12.0 Hz, OCH2Ph), 3.76 (1H, d,
J¼2.5 Hz, 7-H), 3.64 (3H, s, CO2CH3), 1.03 (3H, s, 10-Me),
0.90 (3H, d, J¼7.0 Hz, 20-Me), 0.63 (3H, s, 13-Me); 13C
NMR (500 MHz, CDCl3) d 174.4, 137.5, 128.3, 127.6,
127.5, 94.2, 86.4, 81.5, 74.7, 70.6, 55.6, 51.5, 49.7, 42.7,
42.6, 39.1, 39.0, 36.2, 35.4, 35.0, 31.1, 31.0, 29.3, 28.1,
25.6, 23.9, 23.0, 21.6, 20.7, 18.4, 11.8; MS (ESI) m/z 627
(MþNa)þ.
Compound 13a: Rf 0.15 (hexane/ethyl acetate¼2:1); 1H
NMR (500 MHz, CDCl3) d 7.35–7.24 (5H, m, Ph), 4.83
(1H, d, J¼7.5 Hz, OCH2OBn), 4.76 (1H, d, J¼7.5 Hz,
OCH2OBn), 4.60 (1H, d, J¼12.0 Hz, OCH2Ph), 4.57 (1H, d,
J¼12.0 Hz, OCH2Ph), 3.75 (1H, d, J¼1.5 Hz, 7-H), 3.64
(3H, s, CO2Me), 3.59 (1H, s, 4-H), 3.54 (1H, m, 3-H), 1.59
(1H, d, J¼13.0 Hz, 5-H), 1.01 (3H, s, 10-Me), 0.90 (3H, d,
J¼7.0 Hz, 20-Me), 0.62 (3H, s, 13-Me).
1
Compound 13c. H NMR (500 MHz, CDCl3) d 7.36–7.24
(5H, m, Ph), 4.93 (1H, d, J¼5.5 Hz, OCH2OBn), 4.63 (1H,
d, J¼5.5 Hz, OCH2OBn), 4.62 (1H, d, J¼12.0 Hz,
OCH2Ph), 4.57 (1H, d, J¼12.0 Hz, OCH2Ph), 4.26 (1H,
m, 4-H), 3.95 (1H, d, J¼2.5 Hz, 3-H), 3.64 (3H, s, CO2Me),
3.57 (1H, d, J¼2.5 Hz, 7-H), 3.35 (1H, d, J¼6.0 Hz, 4-OH),
0.94 (3H, s, 10-Me), 0.90 (3H, d, J¼7.0 Hz, 20-Me), 0.62
(3H, s, 13-Me).
4.2.5. Methyl 3a-benzoyloxy-4b,7a-dibenzyloxy- meth-
oxy-5a- cholan-24-oate 15. A mixture of 13a (109 mg,
0.180 mmol), benzoic acid (55 mg, 0.450 mmol), and
Cs2CO3 (147 mg, 0.450 mmol) in a 10 mL flask was dried
under the reduced pressure. To the mixture was added DMF
(2 mL) and stirred for 6 h at rt. The solvent was removed
under reduced pressure, and the residue was purified
by silica gel column chromatography (hexane/ethyl
acetate¼5:1–2:1, then chloroform/methanol¼1:0–1:2) to
give 3-O-benzoyl-4-O-sulfate (Rf 0.30, 5:1—chloroform/
methanol) (110 mg) as a mixture of benzoic acid, and 21 mg
(19%) of 14a was recovered. To the solution of 3-O-
benzoyl-4-O-sulfate (110 mg) in THF (2.4 mL) was added a
solution of conc. sulfuric acid (54 mL) in THF (0.1 mL).
The mixture was stirred for 15 min at rt, then quenched with
saturated NaHCO3 and extracted with ethyl acetate. The
combined organic layer was washed with saturated
NaHCO3, dried over MgSO4, filtered, and concentrated
under reduced pressure to give 15 (71 mg, 75% from 14a).
4.2.4. Methyl 7a-benzyloxymethoxy-3b,4b-cyclic sul-
fate-5a-cholan-24-oate 14a. To a stirred solution of 13a
and 13c (437 mg, 0.801 mmol) and triethylamine (446 mL,
3.22 mmol) in THF (3 mL) was added a solution of thionyl
chloride (94.6 mL, 1.21 mmol) in THF (1 mL) and stirred
for 10 min at 0 8C. The reaction mixture was quenched with
water and extracted with ethyl acetate. The combined
organic layer was washed with saturated NaCl, dried over
anhydrous MgSO4. Concentration followed by flash column
chromatography (hexane/ethyl acetate¼50:1–10:1) gave a
mixture of cyclic sulfites (441 mg, 94%) as colorless oil.
The mixture of cyclic sulfites (552 mg, 0.938 mmol) was
dissolved in a mixed solvent of acetonitrile (4.6 mL), CCl4
(3.0 mL), and 50 mM phosphate buffer (3.0 mL). To the
solution was added RuCl3 (19.5 mg, 94 mmol), NaIO4
Compound 15. Amorphous material; Rf 0.43 (hexane/ethyl
acetate¼2:1); IR (thin film) 3487, 2940, 1734, 1717, 1270,

6978
T. Oishi et al. / Tetrahedron 60 (2004) 6971–6980
1111, 1041 cm21; 1H NMR (500 MHz, CDCl3) d 8.01 (2H,
d, J¼8.0 Hz, PhCO2), 7.53–7.06 (8H, m, PhCO2, Ph), 5.13
(1H, d, J¼2.5 Hz 3-H), 4.81 (1H, d, J¼7.0 Hz, OCH2OBn),
4.72 (1H, d, J¼7.0 Hz, OCH2OBn), 4.47 (1H, d, J¼12.0 Hz,
OCH2Ph), 4.43 (1H, d, J¼12.0 Hz, OCH2Ph), 3.74 (1H, s,
7-H), 3.67 (1H, s, 4-H), 3.64 (3H, s, CO2CH3), 2.08 (1H, dt,
J¼13.5, 3.0 Hz, 5-H), 1.08 (3H, s, 10-Me), 0.91 (3H, d, J¼
7.0 Hz, 20-Me), 0.64 (3H, s, 13-Me); 13C NMR (500 MHz,
CDCl3) d 174.6, 165.5, 137.6, 132.9, 130.3, 129.5, 128.3,
128.2, 127.4, 127.1, 95.0, 73.2, 73.2, 69.5, 55.7, 51.5, 50.2,
47.3, 42.6, 39.8, 39.2, 38.2, 35.9, 35.4, 32.9, 31.1, 31.1,
30.8, 28.1, 23.8, 22.1, 20.1, 18.4, 13.5, 11.9; MS (ESI) m/z
669 (MþNa)þ.
165.4, 137.7, 137.6, 132.8, 130.4, 129.4, 128.3, 128.2,
127.7, 127.5, 127.4, 127.2, 94.6, 94.5, 79.2, 76.4, 70.5, 69.7,
69.5, 55.7, 50.2, 47.2, 42.6, 39.8, 39.2, 38.1, 35.9, 35.4,
32.8, 31.0, 30.8, 28.1, 23.8, 22.4, 20.3, 18.3, 13.3, 11.9; MS
(ESI) m/z 776 (MþNa)þ.
4.2.8. 24-Nor-5a-cholane-4b,7a-dibenzyloxymethoxy-
23-iodo-3a-ol, benzoate 18. A solution of 17 (43 mg,
57.1 mmol), Pb(OAc)4 (38 mg, 86 mmol), and iodine
(43 mg, 171 mmol) in CCl4 (2 mL) was refluxed for
20 min under irradiation of tungsten lamp (300 W). The
reaction mixture was quenched with saturated Na2S2O3, and
extracted with dichloromethane. The combined organic
layer was washed with saturated NaCl, and dried over
MgSO4. Concentration followed by silica gel column
chromatography (hexane/ethyl acetate¼20:1–2:1) gave 18
(40 mg, 84%) as colorless oil.
4.2.6. Methyl 3a-benzoyloxy-4b,7a-dibenzyloxy-meth-
oxy-5a-cholan-24-oate 16. To a solution of 15 (71 mg,
0.110 mmol) in dichloromethane (1 mL) was added diiso-
propylethylamine (345 mL, 1.98 mmol) and BOMCl
(122 mL, 0.880 mmol) and the mixture was stirred for
22 h at rt. The reaction mixture was quenched with water
and extracted with dichloromethane. The combined organic
layer was washed with saturated NaCl, dried over MgSO4,
and concentrated under reduced pressure. The residue was
purified by silica gel column chromatography (hexane/ethyl
acetate¼15:1–4:1) to give 16 (56 mg, 67%) as colorless oil
and 11 mg (15%) of 15 was recovered.
Compound 18. Colorless oil; Rf 0.43 (hexane/ethyl
acetate¼5:1); IR (thin film) 3424, 1715, 1452, 1270,
1109, 1039 cm21 1H NMR (500 MHz, CDCl3) d 8.00
;
(2H, d, J¼7.5 Hz, PhCO2), 7.53–7.09 (13H, m, PhCO2, Ph),
5.27 (1H, q, J¼2.5 Hz 3-H), 4.82, 4.79 and 4.70£2 (4H,
d£4, J¼7.0 Hz, OCH2OBn), 4.64, 4.60, 4.49 and 4.47
(4H, d£4, J¼12.0 Hz, OCH2Ph), 3.73 (1H, d, J¼1.5 Hz
7-H), 3.57 (1H, s, 4-H), 3.29 (1H, td, J¼9.0, 5.5 Hz,
23-Ha), 3.08 (1H, q, J¼9.0 Hz, 23-Hb), 1.07 (3H, s, 10-Me),
0.91 (3H, d, J¼6.5 Hz, 20-Me), 0.66 (3H, s, 13-Me);
13C NMR (500 MHz, CDCl3) d 165.4, 137.7, 137.6,
132.8, 130.4, 129.4, 128.3, 128.2, 127.7, 127.5, 127.4,
127.2, 94.6, 94.5, 79.2, 76.4, 70.5, 69.7, 69.5, 55.6, 50.2,
47.2, 42.7, 39.7, 39.2, 38.1, 37.3, 35.9, 32.8, 31.0, 28.1,
23.8, 22.4, 20.2, 17.9, 13.3, 11.9, 5.4; MS (ESI) m/z 857
(MþNa)þ.
Compound 16. Colorless oil; Rf 0.63 (hexane/ethyl
acetate¼2:1); [a]2D2 26.48 (c 0.64, CHCl3); IR (thin film)
2943, 1734, 1717, 1270, 1099, 1038, 1026 cm21; 1H NMR
(500 MHz, CDCl3) d 8.00 (2H, d, J¼8.0 Hz, PhCO2), 7.52–
7.09 (13H, m, PhCO2, Ph), 5.28 (1H, d, J¼3.0 Hz 3-H),
4.82, 4.80, 4.71 and 4.70 (4H, d£4, J¼7.0 Hz, OCH2O),
4.64, 4.60, 4.49 and 4.47 (4H, d£4, J¼12.0 Hz, OCH2Ph),
3.74 (1H, s, 7-H), 3.65 (3H, s, CO2CH3), 3.57 (1H, s, 4-H),
1.07 (3H, s, 10-Me), 0.91 (3H, d, J¼7.0 Hz, 20-Me), 0.65
(3H, s, 13-Me); 13C NMR (500 MHz, CDCl3) d 174.6,
165.4, 137.7, 137.6, 132.8, 130.4, 129.4, 128.3, 128.2,
127.7, 127.5, 127.4, 127.2, 94.7, 94.5, 79.2, 76.4, 70.5, 69.7,
69.5, 55.7, 51.5, 50.1, 47.2, 42.6, 39.8, 39.2, 38.1, 35.9,
35.4, 32.9, 31.1, 28.1, 23.8, 22.4, 20.3, 18.4, 13.3, 11.9; MS
(ESI) m/z 789 (MþNa)þ.
4.2.9. 3a-Benzoyloxy-4b,7a-dibenzyloxymethoxy-24-
nor-5a-cholan-23-al 6. To a solution of iodide 18
(14 mg, 17 mmol) in DMSO (0.5 mL) was added 2,4,6-
collidine (11 mL, 81 mmol). After stirring for 10 min at
150 8C, the reaction mixture was cooled to rt, then quenched
with water, and extracted with dichloromethane. The
combined organic layer was washed with 0.5 M HCl and
saturated NaCl, dried over MgSO4, filtered, and concen-
trated under reduced pressure. The residue was purified
by silica gel column chromatography (hexane/ethyl
acetate¼15:1–8:1) to give 6 (11 mg, 94%) as colorless oil.
4.2.7. 3a-Benzoyloxy-4b,7a-dibenzyloxymethoxy-5a-
cholan-24-oic acid 17. To a solution of 16 (4.9 mg,
t
t
6.39 mmol) in BuOH (0.1 mL) was added BuOK (2.2 mg,
19.2 mmol), and the mixture was stirred for 2 h at rt. The
reaction mixture was quenched with saturated NH4Cl, and
extracted with ethyl acetate. The combined organic layer
was washed with saturated NaCl. Concentration followed
by silica gel column chromatography (hexane/ethyl
acetate¼8:1–4:1) gave 17 (4.6 mg, 95%) as colorless
amorphous.
Compound 6. Colorless oil; Rf 0.25 (hexane: ethyl
acetate¼5:1); [a]2D3 211.38 (c 0.23, CHCl3); IR (thin film)
2933, 2719, 1718, 1270, 1108, 1038, 1026 cm21; 1H NMR
(500 MHz, CDCl3) d 9.73 (1H, dd, J¼3.5, 1.0 Hz, CHO),
8.00 (2H, d, J¼8.5 Hz, PhCO2), 7.56–7.09 (13H, m,
PhCO2, Ph), 5.27 (1H, q, J¼3.5 Hz 3-H), 4.82, 4.78, 4.70
and 4.69 (4H, d£4, J¼7.0 Hz, OCH2O), 4.64, 4.60, 4.49 and
4.46 (4H, d£4, J¼12.0 Hz, OCH2Ph), 3.72 (1H, s, 7-H),
3.57 (1H, s, 4-H), 1.07 (3H, s, 10-Me), 1.01 (3H, d,
J¼6.0 Hz, 20-Me), 0.70 (3H, s, 13-Me); 13C NMR
(500 MHz, CDCl3) d 203.3, 165.4, 160.7, 137.7, 137.6,
132.8, 130.4, 129.4, 128.3, 128.2, 127.7, 127.5, 127.4,
127.2, 94.6, 94.6, 79.2, 76.4, 70.5, 69.8, 69.5, 55.8, 50.9,
50.2, 47.2, 42.7, 39.8, 39.1, 38.1, 35.9, 32.9, 31.7, 31.0,
28.4, 23.8, 22.4, 20.2, 20.1, 13.3, 11.9; MS (ESI) m/z 777
(MþMeOHþNa)þ.
Compound 17. Amorphous material; Rf 0.27 (hexane/
ethylacetate¼2:1); IR (thin film) 1714, 1451, 1270, 1101,
1
1038 cm21; H NMR (500 MHz, CDCl3) d 8.00 (2H, d,
J¼8.0 Hz, PhCO2), 7.53–7.09 (13H, m, PhCO2, Ph), 5.27
(1H, d, J¼3.0 Hz 3-H), 4.82, 4.79, 4.71 and 4.70 (4H, d£4,
J¼7.0 Hz, OCH2O), 4.64, 4.60, 4.49 and 4.47 (4H, d£4,
J¼12.0 Hz, OCH2Ph), 3.73 (1H, s, 7-H), 3.56 (1H, s, 4-H),
1.06 (3H, s, 10-Me), 0.92 (3H, d, J¼6.5 Hz, 20-Me), 0.65
(3H, s, 13-Me); 13C NMR (500 MHz, CDCl3) d 179.9,

T. Oishi et al. / Tetrahedron 60 (2004) 6971–6980
6979
4.2.10. 104b,7a-Dibenzyloxymethoxy-5a-cholest-23-
ene-3a,26-diol,3a-benzoate, (25S) 19. To a stirred suspen-
sion of phosphonium salt 4 (101 mg, 0.243 mmol) in (1 mL)
was added in dropwise a solution of 1.6 M n-BuLi in hexane
(304 ml, 0.486 mmol) at 278 8C, then stirred for 10 min at
0 8C. TMSCl (31 ml, 0.243 mmol) was added to the reaction
mixture at 0 8C, and stirred for 30 min at rt. A portion of the
resulting solution of the ylide (324 mL, 60.9 mmol) was
added to the solution of 6 (11.0 mg, 15.2 mmol) in THF
(0.54 mL) at 278 8C, and stirred for 15 min, then stirred at
0 8C for 1 h, and stirred for 2 h at rt. The reaction mixture
was quenched with 0.5 M HCl (0.2 mL) and stirred for
10 min at rt, and extracted with ether. Combined organic
layer was washed with saturated NaCl. Concentration
followed by silica gel column chromatography (hexane/
ethylacetate¼10:1–5:1) gave 19 (8.2 mg, E/Z¼1:8, 69%) as
colorless oil.
the reduced pressure to give the pyridinium salt. To the
solution of the residue in methanol (2.0 mL) was added
Amberite IR-120B (1.7 g), and stirred for 3 h at rt. The resin
was removed by filtration, and the filtrate was concentrated.
The residue was dissolved in methanol (2.0 mL) again, and
the solution was added Amberite IR-120B (1.7 g), and
stirred for 4.5 h at rt. The resin was removed by filtration,
and the filtrate was concentrated. The residue was dissolved
in methanol (2.0 mL) and added 10% Pd on charcoal
(52 mg, 61 mmol). The mixture was stirred under the
hydrogen atmosphere for 2.5 h at rt. The catalyst was
removed by filtration through Celite pad, and the filtrate was
concentrated in vacuo. The residue was purified by HPLC to
give 2 as colorless solid.
Compound 2. Colorless solid; Rf 0.13 (chloroform/
methanol¼2:1); HPLC RT 5.2 min; HPLC conditions
{column, TSK-GEL ODS-120T (f 4.6£250 mm); flow
rate, 1.0 mL/min; solvent A, H2O; solvent B, CH3CN;
gradient, solvent B 21% (0–20 min)}; 1H NMR (500 MHz,
D2O) d 4.41 (1H, d, J¼2.5 Hz, 3-H), 3.96 (1H, d, J¼2.0 Hz
7-H), 3.92 (1H, dd, J¼9.0, 5.5 Hz, 26-Ha), 3.83 (1H, dd,
J¼9.0, 7.0 Hz, 26-Hb), 3.72 (1H, s, 4-H), 1.81 (1H, d, J¼
15.0 Hz, 5-H), 0.97 (3H, s, 10-Me), 0.92 (3H, d, J¼6.5 Hz,
25-Me), 0.90 (3H, d, J¼6.5 Hz, 20-Me), 0.65 (3H, s,
Compound 19. Colorless oil; Rf 0.45 (1:2—ethyl acetate/
hexane); 1H NMR (500 MHz, CDCl3) d 8.00 (2H, d,
J¼7.5 Hz, PhCO2), 7.52–7.09 (13H, m, PhCO2, Ph), 5.53–
5.44 (1H, m, 23-H), 5.27 (1H, d, J¼2.5 Hz 3-H), 5.22 (1H,
dd, J¼15.5, 8.0 Hz, 24-H,E), 5.15 (1H, t, J¼10.5 Hz,
24-H,Z), 4.82, 4.80, 4.71 and 4.70 (4H, d£4, J¼6.5 Hz,
OCH2O), 4.64, 4.60, 4.49 and 4.47 (4H, d£4, J¼12.0 Hz,
OCH2Ph), 3.73 (1H, s, 7-H), 3.57 (1H, s, 4-H), 3.46 (1H, dd,
J¼10.0, 6.0 Hz 26-Ha), 3.36–3.31 (1H, m, 26-Hb), 1.07
(3H, s, 10-Me), 0.93 (3H, d, J¼6.0 Hz, 20-Me or 25-Me),
0.92 (3H, d, J¼6.0 Hz, 20-Me or 25-Me), 0.66 (3H, s,
13-Me).
13-Me); MS (ESI) m/z 297 (M22Na)22
.
4.2.13. (3a,4b,7a,25R)-3,4,7,26-Tetrahydroxy-5a-cho-
lestane-3,26-disulfate 3. Compound 3. Colorless solid;
Rf 0.13 (chloroform/methanol¼2:1); HPLC conditions
{column, TSK-GEL ODS-120T (f 4.6£250 mm); flow
rate, 1.0 mL/min; solvent A, H2O; solvent B, CH3CN;
gradient, solvent B 21% (0–20 min)}; 1H NMR (500 MHz,
D2O) d 4.41 (1H, d, J¼2.5 Hz, 3-H), 3.96 (1H, d, J¼2.5 Hz
7-H), 3.91 (1H, dd, J¼9.0, 5.5 Hz, 26-Ha), 3.83 (1H, dd,
J¼9.0, 7.0 Hz, 26-Hb), 3.72 (1H, s, 4-H), 1.81 (1H, d, J¼
15.0 Hz, 5-H), 0.97 (3H, s, 10-Me), 0.91 (3H, d, J¼7.0 Hz,
25-Me), 0.90 (3H, d, J¼6.0 Hz, 20-Me), 0.65 (3H, s,
4.2.11. 4b,7a-Dibenzyloxymethoxy-5a-cholest-23-ene-
3a,26-diol, (25S) 20. To a solution of 19 (8.2 mg,
10.5 mmol) in THF (0.6 mL) was added LiAlH4 (4.0 mg,
105 mmol) at 0 8C. After stirring for 15 min, the
reaction was quenched with water and diluted with ether,
and the resulting precipitates were removed by filtration
through the Celite pad. The filtrate was concentrated
and purified by silica gel column chromatography (hexane/
ethyl acetate¼5:1–2:1) to give 20 (6.2 mg, 87%) as
colorless oil.
13-Me); MS (ESI) m/z 297 (M22Na)22
.
4.3. Estimation of the amount of natural SAAF
Compound 20. Colorless oil; Rf 0.20 (1:2—hexane/ethyl
acetate¼2:1); 1H NMR (500 MHz, CDCl3) d 7.35–7.24
(10H, m, Ph), 5.53–5.44 (1H, m, 23-H), 5.21 (1H, dd,
J¼15.5, 8.0 Hz, 24-H,E), 5.16 (1H, t, J¼10.5 Hz, 24-H,Z),
4.83, 4.75 and 4.72 (3H, d£3, J¼7.5 Hz, OCH2O), 4.65–
4.55 (5H, m, OCH2O£1 and OCH2Ph£4), 3.93 (1H, d,
J¼2.5 Hz, 3-H), 3.74 (1H, s, 7-H), 3.48–3.43 (1H, m,
26-Ha), 3.36–3.31 (2H, m, 26-Hb and 4-H), 0.99 (3H, s,
10-Me), 0.92 (3H, d, J¼7.0 Hz, 20-Me or 25-Me), 0.91 (3H,
d, J¼5.0 Hz, 20-Me or 25-Me), 0.64 (3H, s, 13-Me).
A standard solution of synthetic SAAF in D2O was
1
prepared and its concentration was estimated by H NMR
using DMF as internal standard (1.26 mM). 1H NMR
spectra of natural SAAF (x mg) and synthetic SAAF (4 mg
and 40 mg) in 40 mL of D2O solution were recorded on a
JEOL L-500 spectrometer (500 MHz) with a 1.7-mm probe
(Nanolac, Z-Spec-SMIDG500) under the same conditions
(receiver gain: 28, acquisition times: 6176, temperature,
29.9 8C). The signal intensity of the natural SAAF was
identical with that of the synthetic SAAF containing 4 mg
of sample, and approximately tenth part of 40 mg of
sample.
4.2.12. (3a,4b,7a,25S)-3,4,7,26-Tetrahydroxy-5a-cho-
lestane-3,26-disulfate 2. A mixture of 20 (4.1 mg,
6.07 mmol) and SO3·Py (19.3 mg, 0.121 mmol) in a 10 mL
flask was dried under the reduced pressure. To the mixture
was added pyridine (0.6 mL) and the resulting mixture was
stirred for 50 min at 60 8C. The pyridine was removed and
dried in vacuo for 5 h. To the residue was added ether, and
decanted to remove excess SO3·Py. The precipitates were
dissolved in chloroform, and insoluble impurities were
removed by decantation. The solvent was removed under
4.4. Biological activities
A sample in an aqueous solution was mixed with the same
volume of 2% agar, and the mixture was enclosed in the
tip of glass capillary with a diameter of ca. 50 mm. The
capillary was inserted in sea water including an appropriate
number of sperm, and images of sperm around the capillary
tip were recorded onto a personal computer every 20 msec

6980
T. Oishi et al. / Tetrahedron 60 (2004) 6971–6980
´
3. Cosson, J.; Carre, D.; Cosson, M. P. Cell Motil. Cytoskeleton
1986, 6, 225.
using a high-speed CCD camera (HAS-200, Ditect) and a
video card (HAS-PCI, Ditect). The position of each
sperm was analyzed using the image analyzing program
(Dip-motion 2D, Ditect), and parameters (the chemotaxis
index, D, dD/dt, velocity, u) were calculated from the
position of the data. See a previous report for details of the
bioassay.10
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Acknowledgements
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We thank Drs. M. Ikeda and H. Ohtake (Dokkyo University
School of Medicine) for their help in the measurement of
ESI/TOF-MS; Dr. H. Naoki (Suntory Institute for Bio-
organic Research) for measurement of MS/MS; to Mr.
S. Adachi (Osaka University) for NMR measurements;
Dr. Tsutsui (Univ. of Tokyo) for his advice; and to
Ms M. Ishikawa, Mr. H. Iryu, and Mr. M. Sekifuji (Univ.
of Tokyo) for their technical work. We also thank the
directors and staff of the Education and Research Center
of Marine Bioresources (Tohoku University) and Otsuchi
Marine Research Center (University of Tokyo) for
supplying materials. This work was supported in part by
a Grant-In-Aid for Scientific Research on Priority Area
(A) (No. 12045235) from MEXT, Japan to M. Yoshida,
M. Murata, and by Grant-In-Aids from the Ministry of
Education, Culture, Sports, Science and Technology, Japan
to M. Morisawa.
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