Regioselective enzymatic acylation of vicinal diols of steroids

Article abstract of DOI:10.1016/j.tet.2005.01.104

Monoacylated derivatives of a complete set of 2,3- and 3,4-vicinal diols of steroids were prepared by regioselective lipase-catalysed transesterification reactions. The enzymes displayed different selectivities towards the vicinal diols depending on the configuration of the hydroxyl groups.

Full text of DOI:10.1016/j.tet.2005.01.104

Tetrahedron 61 (2005) 3065–3073
Regioselective enzymatic acylation of vicinal diols of steroids
a
M. Manuel Cruz Silva, Sergio Riva and M. Luisa S a´ e Melo
b
a,*
a
Centro de Estudos Farmac eˆ uticos, Lab. Qu ´ı mica Farmac eˆ utica, Faculdade de Farm a´ cia, Universidade de Coimbra,
Rua do Norte 3000-295 Coimbra, Portugal
b
Istituto di Chimica del Riconoscimento Molecolare, C.N.R., Via Mario Bianco 9, 20131 Milano, Italy
Received 28 October 2004; revised 29 November 2004; accepted 25 January 2005
Abstract—Monoacylated derivatives of a complete set of 2,3- and 3,4-vicinal diols of steroids were prepared by regioselective lipase-
catalysed transesterification reactions. The enzymes displayed different selectivities towards the vicinal diols depending on the configuration
of the hydroxyl groups.
q 2005 Elsevier Ltd. All rights reserved.
1
. Introduction
Studies on the transesterification of polyfunctionalyzed
steroids have shown that hydrolases can have access to
substituents either on the A-ring or on the D-ring and/or on
the side-chain of steroids. Several lipases showed a
Polyhydroxylated steroids bearing vicinal diols on the
A-ring are frequently found in Nature and some of them
have relevant biological activities. For instance, the trans-
diaxial 2b,3a-di-hydroxy pattern is present in natural
1
3,14
preference for C-3 hydroxyl groups,
whereas the
protease subtilisin Carlsberg catalysed the acylation of
1
4
1
2
C-17 OH. Moreover, stereoselective resolutions of
epimeric alcohols located on the steroid side-chain have
been carried out by lipase PS and, more recently, by
sulphated sterols with antiviral or anti-angiogenic action,
while 2a,3a-diols isolated from marine sources hold
1
5
3
cytotoxic activity. Steroidal saponins displaying trans-
1
6
subtilisin.
diequatorial 2a,3b-vicinal diols are quite frequent as
gitogenin derivatives with antitumor properties. In turn,
4
Concerning the modifications of A-ring substituents, the
selectivity of lipases for 3-hydroxysteroids has been applied
to the chemoenzymatic synthesis of pharmacologically
the 3b,4b-diol functionality is present in a variety of steroids
like in the agosterols, which induce reversal of multidrug
5
resistance and proteasome inhibition, as well as in
6
1
7
7
formestane metabolites and in volkendousins, which are
relevant tibolone metabolites. Quite recently, we have
reported a highly selective lipase-catalysed preparation of
epimerically pure 5a,6a- and 5b,6b-epoxysteroids through
8
potent antitumor agents. Finally, the 3a,4b-vicinal diol
pattern has been identified in contignasterol, a natural anti-
18
9
inflammatory compound and, recently, in a steroid
possessing chemotaxis activity.
acylation or deacylation reactions at the C-3 OH. Finally,
1
0
the ability to discriminate among different hydroxyl groups
on the A-ring has been demonstrated in the esterification of
ecdysteroids catalysed by Candida antarctica lipase, which
The discovery that lipases and proteases are able to act in
organic solvents opened the way to an intensive synthetic
exploitation of these biocatalysts, which, as shown in
hundreds of papers and several industrial applications,
19
afforded the 2b-monoacyl derivatives in good yields.
In this context, to further explore the enzymatic transform-
ations of steroids, we endeavoured a systematic study on the
selectivity of commercially available lipases towards a
complete set of stereoisomeric 2,3- and 3,4-vicinal diols, to
provide a new tool for the selective transformation of these
molecules and of related natural compounds.
1
1
display remarkable chemo-, regio- and stereoselectivity.
Specifically in the steroids field, enzyme catalysis can play
an important role for the mild and selective interconversion
1
2–19
of functional groups via regioselective transformations.
Keywords: Lipases; Steroids; Diols; Regioselectivity; Enzymatic
esterification.
2. Results and discussion
*
Corresponding author. Tel.: C351 239 859990; fax: C351 239 827030;
e-mail: samelo@ci.uc.pt
Different synthetic strategies were used to afford the
0040–4020/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2005.01.104

3066
M. M. Cruz Silva et al. / Tetrahedron 61 (2005) 3065–3073
a
Table 1. Lipase-catalysed monoacylation of vicinal diols
Substrate
Novozym
35
C. rugosa
lipase
Lipase
AY
Lipase
PS
Lipase
AK
C. viscosum
lipase
Porcine pancreatic
lipase
Lipozyme
IM 20
Lipase
CE
4
1
2
3
4
5
6
7
8
K
K
CC
CC
C
C
K
K
CC
C
C
C
K
C
CC
C
C
CC
K
CC
C
C
C
C
K
K
C
C
C
K
C
C
K
K
C
CC
C
K
K
K
K
K
K
K
K
K
K
K
K
K
K
K
K
K
K
K
K
K
K
K
K
C
b
b
CC
C
C
K
K
K
CC
K
K
a
Conversion and product(s) formation was evaluated by TLC.
The formation of two products was observed.
b
requested stereoisomeric vicinal diols 1–8. Woodward’s cis-
dihydroxylation method, which is known to be selective for
notable exception of compound 3 (2a,3a-diol) acylated only
by Novozym 435 (immobilized lipase B from Candida
antarctica). Enzymatic acylations were highly regioselec-
tive, showing the formation of only one product by TLC,
with the exception of Candida rugosa lipase (from Sigma or
Amano) acting on compound 4. Noteworthy, lipase PS and
Novozym 435 showed complementary regioselectivity
towards compound 4.
2
the more hindered b-face of 5a-steroids, was applied to
0
2
D - or D -unsaturated precursors affording the cis-b diols
3
1
7
and 5 (Scheme 2). Complementarily, the cis-a diols 3 and
were accessed through osmium tetroxide-mediated
2
1
dihydroxylation on the same olefins. Trans-diequatorial
diols 2 and 6 were obtained by two different approaches.
Starting from cholestan-3-one, a-acetoxylation by lead
tetracetate, followed by stereoselective reduction by
NaBH /CeCl and deacetylation rendered the diol 2. On
the other hand, the 3b,4a-diol 6 was directly accessed by
hydroboration of the D -3-one precursor. Finally, trans-
diaxial diols 4 and 8 were obtained by epoxide opening
reactions.
2
2
For each substrate the best performing lipase (evaluated by
TLC) was chosen for scale-up reactions, allowing the
isolation of the corresponding monoester in good yields
(Scheme 1). Products identification was easily done by
NMR analysis (downfield shift of the signals due to the
proton geminal to the acylated OH) and, when possible, by
comparison with literature data.
4
3
4
23
As shown in the formula, the 2,3-diols were prepared
by modifying the cholestane skeleton, whereas the diffi-
culties found on separating the 5a- and 5b-epimers of
1
3,14
The general preference of lipases for the C-3 OH
was
observed with most of the substrates (Scheme 2). Specifi-
cally, the diequatorial vicinal diols 2 and 6 were converted
into the corresponding 3b-acetate, showing a common
preference of different lipases toward a 3b-equatorial OH
in the presence of 2a-equatorial OH (substrate 2) or of
4a-equatorial OH (substrate 6).
3
D -cholestane by sequential crystallizations led us to
prepare the set of 3,4-diols in the androstane series. The
cholestane derivative 9 was also synthesised to confirm that
lipases selectivity towards the A-ring OH was not affected
by different substituents on the D-ring.
The performances of a panel of 9 commercial lipases were
evaluated for the esterification of the vicinal diols 1–9, using
vinyl acetate as the acyl donor and toluene or acetone/THF
as solvents for the cholestane or the androstane derivatives,
respectively. TLC monitoring allowed the identification,
for each substrate, of the lipase(s) able to promote the
monoacylation of the substrates. As shown in Table 1, all
the stereoisomeric vicinal diols were accepted as substrates
by some of the enzymes tested with the exception of
compound 8.
Concerning the diaxial 2b,3a- and 3a,4b-diols (substrates 4
and 8), different outcomes were noticed. The diaxial 2b,3a-
diol was differently accepted by the lipases tested. Whereas
Novozym 435 converted this diol exclusively into the
3a-acetate 4a, lipase PS showed opposite selectivity
rendering the 2b-acyl derivative 4b as the only product.
Moreover, acylations catalysed by Candida rugosa lipases
were not regioselective with this substrate. Conversely, the
diaxial 3a,4b diol (8) was not accepted by any of the
enzymes tested.
Lipases from different sources (Candida antarctica, column
2
columns 5 and 6; Chromobacterium viscosum, column 7)
were able to acylate the target compounds. Usually more
than one enzyme was acting on the same substrate, with the
Finally, the equatorial/axial 2a,3a-diol (3), only accepted
by Novozym 435, was acylated at the axial 3a-position,
while, at variance, the 3a,4a-dihydroxy steroid (7) display-
ing axial/equatorial configuration, was acylated by different
lipases at its equatorial 4a-OH.
; Candida rugosa, columns 3 and 4; Pseudomonas strains,
Scheme 1.

M. M. Cruz Silva et al. / Tetrahedron 61 (2005) 3065–3073
3067
Scheme 2.
Moreover, in agreement with previous observations, the
lipases tested did not catalyse any acylation of the 17b-OH
(substrates 5–8), that was found unaffected in each of the
isolated products.
Specifically, this enzyme catalysed the acetylation of
substrates 1, 2, 5 and 6 at their 3b-OH, as was expectable
1
3b,c
from previous studies.
However, the 3a,4a-diol 7 was
also acylated by Candida rugosa lipase at the C-4 OH, and
the 2b,3a-diol 4 was acylated at both its hydroxy groups,
showing that this enzyme has a quite variable affinity and
selectivity pattern.
The ability of Novozym 435 to accept axial 3a-OH as a
nucleophile has been reported previously.
1
3d
Herein, we
noticed that this lipase catalyses the acylation of the 3a-OH
even when a second hydroxyl is located at C-2 (substrates 3
and 4), whereas the enzyme is inactive when the other OH is
located at C-4 (substrates 7 and 8).
Chromobacterium viscosum lipase, which is known to have
1
4
a strict selectivity for the 3b-hydroxyl of 5a-steroids,
showed the expected preference for diols carrying this OH
substrates 1, 2, 5 and 6). In addition, acylation of the
(
substrate 7 was also noticed, but at the 4a-OH.
Concerning lipase PS, the ability of this enzyme to esterify
3
3
-hydroxy steroids has been previously observed with
b-hydroxy-5,6-epoxy derivatives. In the present work,
1
8
Finally, the substrate 9, a cholestane displaying 3b,4a-
diequatorial configuration, was converted into the 3b-mono-
acetate 9a, a result which confirmed that the differences in the
steroid side chain do not influence the selectivity of lipases.
lipase PS was found able to acylate most of the substrates
Table 1), catalysing their regioselective modification, not
only at equatorial C-3 (product 2a), but also at equatorial C-4
product 7a) and axial C-2 (product 4a). Noteworthy, among
(
(
the panel of tested enzymes, lipase PS was the only one able
to acylate hydroxy groups located at the latter positions.
3. Conclusions
Candida rugosa lipase accepted most of the substrates.
The reported results clearly show that lipases are able to

3068
M. M. Cruz Silva et al. / Tetrahedron 61 (2005) 3065–3073
discriminate vicinal hydroxy groups located on the A-ring
of steroids, being sensitive to the configuration of the
different diols and affording the monoesters with high
regioselectivity and good yields.
35.61, 34.69, 31.82, 30.31, 28.77, 28.22, 28.00, 24.20,
23.82, 22.82, 22.56, 20.90, 18.68, 11.98, 11.67.
To a solution of cholest-2-ene (420 mg, 1.15 mmol) in
glacial acetic acid (35 ml), I2 (600 mg) and Cu(OAc)2
(500 mg) were added and the reaction mixture was refluxed
under magnetic stirring. After 6 h the reaction was complete
(TLC control). Then, toluene and NaCl were added and the
insoluble salts were filtered off. Diethyl ether was added to
the filtrate and the organic phase was washed with water and
saturated sodium bicarbonate solution. Evaporation of the
solvents under reduced pressure rendered the crude product,
which was dissolved in ethanol/chloroform (4:1) (20 ml),
and treated with NaOH 16% aqueous solution (1 ml) for 2 h.
Finally, upon addition of chloroform, the organic layers
were washed with water, HCl 5%, and brine and evaporated
to dryness. The residue was purified by flash chroma-
tography (petroleum ether/ethyl acetate 4:1), affording pure
cholestane-2b,3b-diol (1, 270 mg, 58%), which was crystal-
Considering the occurrence of steroidal vicinal diols in
Nature, some of which being mono-acylated, -glycosyl-
these findings can offer useful
3
,6
4
ated or -sulphated,
1,10
synthetic tools for the preparation of these compounds and
of related derivatives.
4. Experimental
4
.1. General
All commercially available chemicals were used as supplied
by the manufacturers. Steroids, porcine pancreatic lipase
(
13.3 U/mg) and crude Candida rugosa lipase (665 U/mg)
21a
lized from n-hexane, mp 173–175 8C (lit. 174–177 and
1
were from Sigma. Novozym 435 (8600 PLU/g) and
Lipozyme IM 20 (24 BIU/g) were from Novozymes. Lipase
PS (30.8 U/mg, from Pseudomonas cepacia) was purchased
from Amano and adsorbed on celite as described else-
26
75–176 8C).
1
Selected data: H NMR (CDCl ) d: 0.65 (3H, s, CH -18),
3
3
0
.86 (6H, two d, JZ6.6 Hz, CH -26 and CH -27), 0.89 (3H,
1
6
3 3
where. Lipases AK (20 U/mg, from Pseudomonas
fluorescens), CE (5.5 U/mg, from Humicola lanuginosa)
and AY (30 U/mg, from Candida rugosa) were from
Amano. Chromobacterium viscosum lipase (100 U/mg)
d, JZ6.5 Hz, CH -21), 1.00 (3H, s, CH -19), 3.63 (1H, dt,
3
3
13
JZ4.6, 10.8 Hz, H-3a), 4.02 (1H, brq, H-2a). C NMR d:
7
4
2
1
2
2.42, 70.22, 56.41, 56.26, 55.24, 45.33, 43.17, 42.62,
0.03, 39.50, 36.16, 35.78, 35.23, 34.83, 32.54, 31.96,
8.34, 28.22, 27.99, 24.17, 23.81, 22.80, 22.55, 21.29,
1
13
was supplied by Finnsugar. H and C NMR spectra were
recorded on a Bruker AC-300 at 300 M Hz and at
8.64, 14.56, 12.09. FTIR (ATR): n
931.3, 3311.2 cm
1049.1, 2850.3–
max
7
5.47 M Hz, respectively, using CDCl3 and CD OD as
3
K1
.
solvents. Chemical shifts are reported on the d (ppm) scale
and are relative to tetramethylsilane as internal standard.
Infrared spectra were recorder on a Jasco FT/IR-420
spectrometer. Mass spectra were recorded on a GCT
Micromass spectrometer. Melting points were measured in
a B u¨ chi B-540. Flash chromatography was performed using
silica gel (230–400 Mesh) from Merck and petroleum ether/
ethyl acetate mixtures as eluents. TLC monitoring was done
in petroleum ether/ethyl acetate as eluent and detected with
4
(
1
.2.2. Cholestane-2a,3b-diol (2). Glacial acetic acid
60 ml) was refluxed with acetic anhydride (5 ml) for
0 min, then cholestan-3-one (769 mg, 2 mmol) was added
and, finally, lead tetracetate (1.5 g, 3.4 mmol) was slowly
added. The reaction was heated under reflux, with magnetic
stirring until the consumption of the starting material (24 h).
After cooling, diethyl ether was added and the organic
solution was washed with HCl 5%, saturated bicarbonate
solution and brine. The crude product was purified by
flash chromatography (petroleum ether/ethyl acetate 5:1)
affording pure 3-oxocholestan-2a-yl acetate (710 mg, 80%),
2
4
the Komarowsky’s reagent.
4
4
.2. Chemical synthesis of the diols
2
7
as a white amorphous powder, mp 120–122 8C (lit. 124.7–
125.2 8C).
.2.1. Cholestane-2b,3b-diol (1). According to a known
2
5
procedure, silica gel (70–230 Mesh, 100 g) was added to a
solution of p-toluenesulfonic acid (3 g) in acetone (20 ml).
The mixture was stirred, then evaporated under reduced
pressure and left in a vacuum oven for 2 days. A mixture of
cholestan-3b-ol (1.17 g, 3 mmol), p-toluenesulfonic acid/
silica (15 g) and anhydrous toluene (200 ml) was stirred
under reflux, until the consumption of the starting material
1
Selected data: H NMR (CDCl ) d: 0.67 (3H, s, CH -18),
3
3
0.85 0.91 (9H, CH -21, CH -26 and CH -27), 1.12 (3H, s,
3
3
3
CH -19), 2.15 (3H, s, CH CO), 5.29 (1H, dd, JZ6.6,
3
3
1
3
12.9 Hz, H-2b). C NMR d: 204.3, 170.15, 74.46, 56.16,
56.06, 53.78, 47.84, 44.82, 43.57, 42.56, 39.71, 39.47,
37.18, 36.10, 35.74, 34.67, 31.56, 28.38, 28.20, 27.99,
(
24 h). After cooling, diethyl ether was added and the silica
24.16, 23.78, 22.80, 22.54, 21.60, 20.79, 18.63, 12.75,
12.04. FTIR (ATR): nmax 1222.7, 1727.9, 1750.1 cm
K1
was removed by filtration. The organic solution was washed
with water, saturated bicarbonate solution and brine. Upon
evaporation, cholest-2-ene (1.0 g, 90%) was recovered as a
single product.
.
To a solution of 3-oxocholestan-2a-yl acetate (700 mg,
1.6 mmol) in THF/methanol 2:1, CeCl $7H O (745 mg,
3
2
2
at room temperature before NaBH (120 mg, 3.2 mmol,
mmol) was added and the mixture was stirred for 10 min
1
Selected data: H NMR (CDCl ) d: 0.66 (3H, s, CH -18),
3
3
4
0
.75 (3H, s, CH -19), 0.86 (6H, two d, JZ6.6 Hz, CH -26
8 equiv) was slowly added. After 30 min, the reaction was
complete (TLC monitoring) and HCl 5% was added
dropwise. The mixture was poured into water and extracted
with diethyl ether. The organic solution was washed with
3
3
and CH -27), 0.90 (3H, d, JZ6.5 Hz, CH -21), 5.59 (2H, m,
3
3
1
H-2 and H-3). C NMR d: 125.96, 125.85, 56.49, 56.27,
3
5
4.07, 42.47, 41.45, 40.03, 39.78, 39.51, 36.17, 35.79,

M. M. Cruz Silva et al. / Tetrahedron 61 (2005) 3065–3073
3069
HCl 5%, saturated bicarbonate solution and brine, and
evaporated to dryness, yielding 3b-hydroxycholestan-2a-yl
acetate (641 mg, 91%), as a white powder, mp 73–75 8C.
evaporated at reduced pressure, affording 2a,3a-epoxy-
cholestane (395 mg, 93%).
1
Selected data: H NMR (CDCl ) d: 0.64 (3H, s, CH -18),
3
3
1
Selected data: H NMR (CDCl ) d: 0.64 (3H, s, CH -18),
3
3
0.75 (3H, s, CH -19), 0.86 (6H, two d, JZ6.6 Hz, CH -26
3 3
0
.84–0.90 (12H, CH -19, CH -21, CH -26, CH -27), 2.08
3
and CH -27), 0.90 (3H, d, JZ6.5 Hz, CH -21), 3.14 (2H, m,
3
3
3
3
3
(
3H, s, CH CO), 3.59 (1H, ddd, JZ5.4, 9.5, 11.1 Hz, H-3a),
H-2b and H-3b).
3
1
3
4
1
4
3
2
.82 (1H, ddd, JZ4.8, 9.4, 11.6 Hz, H-2b). C NMR d:
71.59, 76.49, 73.53, 56.28, 56.17, 54.17, 44.43, 42.52,
2.12, 39.81, 39.47, 37.27, 36.11, 35.89, 35.73, 34.70,
1.79, 28.19, 27.97, 27.77, 24.15, 23.77, 22.79, 22.53,
1.38, 18.63, 13.09, 12.02.
2
8
According to a known procedure, to a solution of 2a,3a-
epoxycholestane (374 mg, 0.9 mmol) in acetone (30 ml),
periodic acid (350 mg) in acetone/water (1:1.5 ml) was
added and the mixture was heated to reflux for 5 min, then
concentrated until 1/3 of the initial volume and kept at room
temperature for 30 min. This mixture was refluxed again
while water (2 ml) was added dropwise for 30 min. Finally,
the mixture was cooled, concentrated under vacuum and
purified by flash chromatography (petroleum ether/ethyl
acetate 4:1), yielding cholestane-2b,3a-diol (4, 309 mg,
3
b-Hydroxycholestan-2a-yl acetate (500 mg, 1.12 mmol)
was treated with NaOH as described in Section 4.2.1. The
crude product was purified by flash chromatography
(
2
petroleum ether/ethyl acetate 4:1), rendering cholestane-
a,3b-diol (2, 370 mg, 82%), which was crystallized from
methanol, mp 196.0–197.5 8C (lit. 204 8C).
2
2b
8
1
5%), which was crystallized from methanol, mp 183–
85 8C (lit. 178–180 and 197–200 8C).
1
b
21a
1
Selected data: H NMR (CDCl ) d: 0.64 (3H, s, CH -18),
3
3
1
Selected data: H NMR (CDCl ) d: 0.65 (3H, s, CH -18),
0
.84–0.91 (12H, CH -19, CH -21, CH -26, CH -27), 3.40
3 3 3 3
3
3
(
1H, ddd, JZ5.1, 8.9, 10.9 Hz, H-3a), 3.59 (1H, ddd, JZ
0
.86 (6H, two d, JZ6.6 Hz, CH -26 and CH -27), 0.90 (3H,
3 3
13
4
5
3
2
.7, 9.0, 11.5 Hz, H-2b). C NMR d: 73.11, 72.3, 56.30,
6.20, 54.26, 45.03, 44.83, 39.89, 39.48, 38.14, 37.46,
6.13, 35.77, 35.57, 34.73, 31.88, 28.23, 28.00, 27.91,
4.17, 23.80, 22.81, 22.55, 21.36, 18.64, 13.50, 12.04. FTIR
.
d, JZ6.5 Hz, CH -21), 0.99 (3H, s, CH -19), 3.87 (2H, m,
3
3
1
H-2a and H-3b). C NMR d: 71.82, 70.61, 56.41, 56.19,
3
5
3
5.10, 42.60, 40.54, 40.01, 39.49, 38.93, 36.14, 35.78,
5.73, 34.88, 31.90, 31.71, 28.20, 28.01, 24.13, 23.80,
K1
(
ATR): nmax 1051.0, 2850.1–2930.3, 3312.9 cm
22.82, 22.55, 20.86, 18.64, 14.59, 12.10. FTIR (ATR): n
1047.9, 2843.2–2930.9, 3310.1 cm
max
K1
.
4
.2.3. Cholestane-2a,3a-diol (3). A solution of K Fe(CN)6
3
(
mide (96 mg) in 20 ml of t-BuOH/H O (3:2) was prepared.
1 g), Et N (7 ml), K CO (420 mg) and methanesulphona-
3 2 3
4
.2.5. Androstane-3b,4b,17b-triol (5). A mixture of
2
testosterone acetate (661 mg, 2 mmol), glacial acetic acid
30 ml) and zinc dust (4 g) was stirred at room temperature.
Cholest-2-ene (370 mg, 1 mmol), (Section 4.2.1) was
dissolved in 30 ml THF/t-BuOH/H O (10:3:2) and added
(
2
After disappearance of the starting material (5 h), diethyl
ether was added and the suspension was filtered through a
celite pad. The filtrate was evaporated under reduced
pressure, then dissolved in diethyl ether and washed with
water, saturated sodium bicarbonate solution and brine.
After drying with MgSO , the organic phase was evapo-
4
rated, affording an epimeric mixture of 5a- and 5b-androst-
3-en-17b-yl acetate (595 mg, 94%).
to the previous solution. Then, 30 ml of OsO4 solution
(
0.1 mg/ml in CH CN) was added and the reaction was
3
stirred at room temperature until the total consumption of
the starting material (48 h). Sodium sulphite 5% was added
and the mixture was stirred for 5 h. Upon addition of diethyl
ether, the organic layer was washed with HCl 5%, saturated
bicarbonate solution and brine, and then, evaporated. The
residue was purified by flash chromatography (petroleum
ether/ethyl acetate 4:1), rendering cholestane-2a,3a-diol
1
(
2
3, 242 mg), which was crystallized from n-hexane, mp
Selected data: H NMR (CDCl
3
) d: 0.78 (3H, s, 18-CH
), 2.03 (3H, s, CH CO),
.56 (1H, m, H-17a), 5.29 (1H, dq, H-4), 5.54 and 5.66 (1H,
two m, H-3). The 5a- and 5b- epimeric ratio, evaluated by
3
),
22b
17–218 8C (lit. 216–219 8C).
0.80 and 0.96 (3H, two s, 19-CH
3
3
4
1
Selected data: H NMR (CDCl ) d: 0.65 (3H, s, CH -18),
3
3
2
9
0
.80 (3H, s, CH -19), 0.86 (6H, two d, JZ6.6 Hz, CH -26
the integration of the H-3 multiplets, was 0.8:1. Epimeri-
cally pure 5a-androst-3-en-17b-yl acetate was obtained
after 3 sequential crystallizations in n-hexane.
3
3
and CH -27), 0.90 (3H, d, JZ6.6 Hz, CH -21), 3.77 (1H, br
3
3
1
3
d, JZ11.0 Hz, 2b-H), 3.96 (1H, br s, 3b-H). C NMR d:
6
3
2
1
2
9.27, 69.12, 56.35, 56.16, 54.16, 42.55, 40.93, 39.90,
9.49, 38.14, 36.90, 36.13, 35.78, 34.76, 34.22, 31.82,
8.22, 28.00, 27.65, 24.17, 23.80, 22.81, 22.55, 20.90,
5a-Androst-3-en-17b-yl acetate (200 mg, 0.63 mmol)
reacted with I /Cu(OAc) , the product obtained was treated
2 2
8.65, 12.40, 12.06. FTIR (ATR): n
930.0, 3315.0 cm
1048.7, 2852.2–
with NaOH (Section 4.2.1) and purified by flash chroma-
tography (petroleum ether/ethyl acetate 1:2), rendering
androstane-3b,4b,17b-triol (5, 95 mg), as a white amor-
max
K1
.
3
0
4
.2.4. Cholestane-2b,3a-diol (4). A mixture of cholest-2-
phous powder, mp 257–260 8C (lit. 263–265 8C).
ene (400 mg, 1.1 mmol), CHCl3 (15 ml) and 3-chloro-
peroxybenzoic acid (400 mg, 2.3 mmol) was stirred at room
temperature. After the disappearance of the starting material
1
Selected data: H NMR (CDCl
/CD
-19), 3.50 (1H, m, H-3a), 3.57
(1H, t, JZ8.6 Hz, H-17a), 3.68 (1H, br t, JZ2.30 Hz,
OD) d: 0.72 (3H, s,
3
3
CH -18), 1.04 (3H, s, CH
3 3
(
extracted with CH Cl . The organic phase was washed with
24 h), the reaction mixture was poured into water and
1
3
H-4a). C NMR d: 81.89, 75.12, 72.67, 56.03, 51.64, 43.42,
37.66, 37.17, 36.12, 36.05, 32.54, 30.09, 26.34, 25.80,
2
2
sodium sulphite 5%, water and brine, dried with MgSO and
4

3070
M. M. Cruz Silva et al. / Tetrahedron 61 (2005) 3065–3073
2
2
3.79, 20.69, 14.98, 11.42. FTIR (ATR): nmax 1064.6,
845.5–2934.2, 3308.3 cm
4.2.9. Cholestane-3b,4a-diol (9). Hydroboration of
cholest-4-en-3-one (384 mg, 1 mmol) under the conditions
described in Section 4.2.8 rendered cholestane-3b,4a-diol
K1
.
(9, 352 mg, 87%), which was crystallized from n-hexane,
mp 235–237 8C (lit. 237 8C).
4
.2.6. Androstane-3b,4a,17b-triol (6). Testosterone
288.4 mg, 1 mmol) was dissolved in THF (10 ml) and
3
1
(
cooled to 0 8C. Then, BH in THF (1.0 M, 5 ml) was added
slowly. The mixture was stirred for 3 h, then warmed to
room temperature over 2.5 h and cautiously quenched with a
10 N NaOH aqueous solution (2 ml), followed by slow
addition of a 30% H O aqueous solution (2 ml). This
mixture was stirred overnight, then poured into water and
extracted with diethyl ether. The organic layers were
washed with HCl 5%, saturated bicarbonate solution and
3
1
Selected data: H NMR (CDCl ) d: 0.62 (3H, s, CH -18),
3
3
0
.79–0.87 (12H, CH -19, CH -21, CH -26, CH -27), 3.18
3 3 3 3
1
3
(1H, t, JZ10.2 Hz, H-4b), 3.29 (1H, m, H-3a). C NMR d:
7
3
2
1
3
6.24, 75.25, 56.54, 56.37, 54.6, 50.86, 42.63, 40.08, 39.62,
7.28, 36.36, 36.27, 35.92, 35.16, 31.74, 28.37, 28.26,
8.11, 24.28, 23.93, 22.83, 22.78, 22.58, 21.08, 18.70,
2
2
3.58, 12.11. FTIR (ATR): n
315.0 cm
1047.1, 2851.2–2930.2,
max
K1
.
brine. After drying with MgSO , the solvent was evaporated
4
at reduced pressure and the residue purified by flash
chromatography (petroleum ether/ethyl acetate 1:2), render-
ing androstane-3b,4a,17b-triol (6, 246 mg, 80%), which
was crystallized from methanol, mp 253.3–255.8 8C (lit.
4.3. Enzymatic acylation of 2,3- and 3,4-vicinal diols
In a typical screening assay, a solution of the substrate
(2 mg), in 0.9 ml of solvent (toluene for the cholestane diols
or acetone/THF for the androstane diols) and vinyl acetate
(0.1 ml) was prepared. This solution was added to the
enzyme (30 mg of crude enzymes, 10 mg of Lipozyme IM
20 or 5 mg of Novozym 435) in 3 ml vials, which were
stopped with a cap and shaken at 250 rpm at 45 8C. The
reactions were monitored by TLC (see Table 1).
2
3a
30
2
48–250 and 258–259 8C).
1
Selected data: H NMR (CDCl /CD OD) d: 0.72 (3H, s,
3
3
CH -18), 0.85 (3H, s, CH -19), 3.20 (1H, t, JZ8.7 Hz,
3
3
H-4b), 3.29 (1H, m, H-3a), 3.59 (1H, t, JZ8.5 Hz, H-17a).
C NMR d: 81.87, 76.52, 75.53, 55.24, 51.53, 51.48, 43.38,
1
3
3
2
2
7.75, 37.23, 36.85, 35.68, 31.76, 30.13, 28.78, 23.77,
3.10, 21.10, 13.89, 11.47. FTIR (ATR): n
1066.0,
max
K1
847.5–2932.0, 3309.1 cm
.
4
.3.1. 2b-Hydroxycholestan-3b-yl acetate (1a). To a
solution of cholestane-2b, 3b-diol (1, 25 mg, 0.062 mmol)
in toluene (8 ml) and vinyl acetate (2 ml), Candida rugosa
lipase (100 mg) was added and the reaction was shaken at
250 rpm, at 45 8C. After 24 h the reaction was complete.
The enzyme was filtered off and the solvent was evaporated,
the residue was purified by flash chromatography (petro-
leum ether/ethyl acetate 5:1) yielding 2b-hydroxycholestan-
4
1
.2.7. Androstane-3a,4a,17b-triol (7). 5a-Androst-3-en-
7b-yl acetate (200 mg, 0.63 mmol) (Section 4.2.5) was
oxidized by OsO (Section 4.2.3). The product was treated
4
with NaOH (Section 4.2.1) and purified by flash chroma-
tography (petroleum ether/ethyl acetate 1:2), affording
androstane-3a,4a,17b-triol (7, 110.6 mg, 57%).
3b-yl acetate (1a, 22 mg, 80%), which was crystallized from
methanol, mp 147–148 8C (lit. 154 8C).
1
Selected data: H NMR (CDCl /CD OD) d: 0.72 (3H, s,
26
3
3
CH -18), 0.85 (3H, s, CH -19), 3.45 (1H, d, JZ10.2 Hz,
3
3
1
Selected data: H NMR (CDCl ) d: 0.65 (3H, s, CH -18),
H-4b), 3.6 (1H, t, JZ8.5 Hz, H-17a), 3.97 (1H, s, H-3b).
C NMR d: 82.40, 73.56, 70.14, 54.19, 50.76, 42.52, 37.50,
3
3
1
3
0.86 (6H, two d, JZ6.5 Hz, CH -26 and CH -27), 0.89 (3H,
3 3
3
2
2
7.11, 36.89, 34.90, 31.76, 31.10, 23.47, 22.58, 21.20,
0.30, 12.70, 11.49. FTIR (ATR): n
d, JZ6.5 Hz, CH -21), 1.03 (3H, s, CH -19), 2.08 (3H, s,
3 3
1065.0, 2846.1–
CH CO), 4.09 (1H, br q, JZ2.6 Hz, H-2a), 4.78 (1H, ddd,
max
3
K1
13
933.3, 3307.8 cm
.
JZ3.34, 4.71, 11.75 Hz, H-3a). C NMR d: 170.13, 75.58,
6
3
2
1
1
3
8.72, 56.30, 56.19, 55.15, 45.40, 42.90, 42.59, 39.95,
9.47, 36.12, 35.76, 35.30, 34.77, 31.86, 28.58, 28.20,
8.13, 27.98, 24.13, 23.79, 22.80, 22.54, 21.34, 21.22,
4
1
.2.8. Androstane-3a,4b,17b-triol (8). 5a-Androst-3-en-
7b-yl acetate (290 mg, 0.92 mmol) obtained as described
8.62, 14.51, 12.07. FTIR (ATR): n
1025.1, 1259.3,
max
K1
above (2.5) reacted with 3-chloroperoxybenzoic (Section
.2.4). The product obtained was treated with periodic acid
Section 4.2.4), deacetylated with NaOH (Section 4.2.1)
721.8, 2858.0–2950.2, 3510.8 cm . FD-MS m/zZ
4
(
C
86.3462 (100%, M KCH COOH), 447.3828 (83%,
3
C
C
M C1), 404.3776 (67%, M C1KCH CO), 448.3870
3
and purified by flash chromatography (petroleum ether/
ethyl acetate 1:2), affording androstane-3a,4b,17b-triol
C
25%, M C2), 61.0324 (8%, CH COOHC1).
(
3
(
2
8, 142 mg), which was crystallized from methanol, mp
63–264.3 8C.
4.3.2. 2a-Hydroxycholestane-3b-yl acetate (2a). To a
solution of cholestane-2a, 3b-diol (2, 50 mg, 0.124 mmol)
in toluene (8 ml), vinyl acetate (1 ml) and lipase PS
(100 mg) were added and the reaction mixture was shaken
for 24 h, under the conditions described above. After usual
work-up and purification by flash chromatography, 2a-
hydroxycholestane-3b-yl acetate was recovered (2a, 48 mg,
1
Selected data: H NMR (CDCl /CD OD) d: 0.71 (3H, s,
3
3
CH -18), 1.03 (3H, s, CH -19), 3.49 (1H, br s, H-4a), 3.55
3
3
(
1H, t, JZ8.6 Hz, H-17a), 3.75 (1H, br q, JZ2.6 Hz, H-3b).
C NMR d: 82.54, 76.56, 71.24, 56.94, 52.51, 45.24, 44.08,
1
3
3
2
2
7.99, 37.09, 36.96, 33.32, 33.21, 30.61, 26.60, 25.11,
1064.9,
4.31, 20.97, 14.85, 11.67. FTIR (ATR): n
87%) as a white powder, mp 157–159 8C. The TLC R of 2a
f
max
K1
849.8–2935.1, 3309.5 cm . FD-MS m/zZ308.2386
and of 3b-hydroxycholestan-2a-yl acetate (synthesised in
Section 4.2.2) using petroleum ether/ethyl acetate (2:1) as
eluent were 0.47, and 0.39, respectively.
C
C
(100%, M ), 309.2424 (27%, M C1), 290.2237 (17%,
M KH O).
C
2

M. M. Cruz Silva et al. / Tetrahedron 61 (2005) 3065–3073
3071
1
Selected data: H NMR (CDCl ) d: 0.65 (3H, s, CH -18),
1b
(4b, 37 mg, 67%), mp 115.7–116.2 8C (lit. 85–86 and
27
113 8C).
3
3
0
.84–0.90 (12H, CH -19, CH -21, CH -26, CH -27), 2.08
3
3
3
3
(
3H, s, CH CO), 3.77 (1H, ddd, JZ4.8, 9.4, 11.5 Hz, H-2b),
3
13
1
Selected data: H NMR (CDCl ) d: 0.64 (3H, s, CH -18),
4
1
4
3
2
1
3
3
.59 (1H, ddd, JZ5.4, 9.5, 10.9 Hz, H-3a). C NMR d:
71.37, 78.99, 69.85, 56.13, 56.09, 54.03, 45.28, 44.34,
2.46, 39.75, 39.39, 36.81, 36.03, 35.68, 34.62, 32.53,
1.69, 30.82, 28.13, 27.90, 27.63, 24.08, 23.70, 22.70,
2.44, 21.25, 18.54, 13.17, 11.94. FTIR (ATR): nmax 1025.9,
3
3
0.86 (6H, two d, JZ6.6 Hz, CH -26 and CH -27), 0.89 (3H,
3
3
d, JZ5.3 Hz, CH -21), 0.90 (3H, s , CH -19), 2.04 (3H, s,
3
3
CH CO), 3.84 (1H, br q, JZ2.3 Hz, H-3b), 4.87 (1H, br q,
3
1
3
H-2a). C NMR d: 170.30, 73.15, 67.59, 56.35, 56.17,
54.85, 42.55, 39.92, 39.48, 38.52, 37.10, 36.12, 35.77, 35.56
34.94, 31.84, 31.74, 28.19, 28.11, 27.99, 24.12, 23.81,
22.80, 22.54, 21.44, 20.20, 18.63, 13.64, 12.06. FTIR
K1
259.8, 1718.8, 2857.0–2951.3, 3512.1 cm . FI-MS m/zZ
C C
86.3553 (100%, M KCH COOH), 446.3842 (54%, M ),
3
C
87.3591 (35%, M C1KCH COOH), 447.3853 (33%,
3
C
M C1), 61.0251 (1%, CH COOHC1).
3
(ATR): n
3481.8 cm
1034.5, 1263.2, 1716.4, 2862.8–2928.4,
max
K1
.
4.3.3. 2a-Hydroxycholestane-3a-yl acetate (3a). To a
solution of cholestane-2a,3a-diol (3, 40 mg, 0.1 mmol) in
4.3.6. 4b,17b-Dihydroxyandrostane-3b-yl acetate (5a).
To a solution of androstane-3b,4b,17b-triol (5, 35 mg,
0.114 mmol) in toluene (5 ml), THF (5 ml) and vinyl acetate
(1 ml), Candida rugosa lipase (100 mg) was added and the
reaction mixture was shaken for 3 days, under the conditions
described above. After usual work-up and purification by
flash chromatography (petroleum ether/ethyl acetate 2:1),
toluene (8 ml), vinyl acetate (1 ml) and Novozym 435
(
80 mg) were added and the reaction mixture was shaken
for 2 days, under the conditions described above. After
usual work-up and purification by flash chromatography,
2
3
a-hydroxycholestane-3a-yl acetate was recovered (3a,
3 mg, 75%)andcrystallizedfrommethanol, mp163–164 8C.
4b,17b-dihydroxyandrostan-3b-yl acetate was recovered
(5a, 29.3 mg, 74%).
1
Selected data: H NMR (CDCl ) d: 0.65 (3H, s, CH -18),
3
3
0
.82 (3H, s, CH -19), 0.87 (6H, two d, JZ6.5 Hz, CH -26
3
3
1
Selected data: H NMR (CDCl ) d: 0.73 (3H, s, CH -18),
and CH -27), 0.90 (3H, d, JZ6.5 Hz, CH -21), 2.12 (3H, s,
3
3
3
3
CH CO) 3.84 (1H, dt, JZ4.2, 11.4 Hz, H-2b), 5.12 (1H, br
q, JZ2.6 Hz, H-3b). C NMR d: 171.58, 73.03, 68.11,
1.06 (3H, s, CH -19), 2.09 (3H, s, CH CO), 3.63 (1H, t, JZ
8.7 Hz, H-17a), 3.83 (1H, br t, H-4a), 4.72 (1H, ddd, JZ
13
3
3 3
1
3
5
3
2
1
2
6.33, 56.23, 54.14, 42.57, 41.70, 39.88, 39.47, 39.29,
6.74, 36.13, 35.78, 34.71, 32.21, 31.77, 28.23, 27.99,
7.45, 24.16, 23.83, 22.81, 22.54, 21.38, 20.89, 18.63,
2.55, 12.04. FTIR (ATR): nmax 1027.4, 1259.8, 1714.7,
3.2, 4.8, 8.0 Hz, H-3a). C NMR d: 170.27, 81.87, 75.53,
72.88, 55.29, 50.98, 48.71, 42.93, 36.85, 36.54, 35.61,
35.44, 31.82, 30.45, 25.55, 23.34, 22.13, 21.34, 20.11,
14.71, 11.11. FTIR (ATR): nmax 1041.5, 1258.2,
K1
K1
859.0 2952.5, 3516.3 cm . FI-MS m/zZ446.3828
1709.3, 2840.9–2943.0, 3442.0 and 3530.1 cm . FD-MS
C
C
C
C
(
100%, M ), 386.3293 (91%, M KCH COOH),
m/zZ351.2470 (100%, M C1), 290.2104 (78%, M K
3
C C
47.3842 (29%, M C1), 387.3349 (23%, M C1K
C
CH COOH), 350.2468 (37%, M ).
4
CH COOH).
3
3
4.3.7. 4a,17b-Dihydroxyandrostane-3b-yl acetate (6a).
4.3.4. 2b-Hydroxycholestane-3a-yl acetate (4a). To a
solution of cholestane-2b,3a-diol (4, 50 mg, 0.124 mmol) in
To a solution of androstane-3b,4a,17b-triol (6, 30 mg,
0.1 mmol) in acetone (6 ml), THF (3 ml) and vinyl acetate
(1 ml), Chromobacterium viscosum lipase (200 mg) was
added and the reaction mixture was shaken for 3 days, under
the conditions described above. After usual work-up and
purification by flash chromatography, 4a,17b-dihydroxy-
androstan-3b-yl acetate was recovered (6a, 20.2 mg, 60%),
and crystallized from methanol, mp 182–184 8C.
toluene (10 ml), vinyl acetate (1 ml) and Novozym 435
80 mg) were added and the reaction mixture was shaken
(
for 3 days, under the conditions described above. After
usual work-up and purification by flash chromatography,
2
single product (4a, 47.6 mg, 86%), mp 110–111 8C (lit.
b-hydroxycholestane-3a-yl acetate was recovered as a
1b
1
06–107 8C).
1
Selected data: H NMR (CDCl ) d: 0.73 (3H, s, CH -18),
3
3
1
Selected data: H NMR (CDCl ) d: 0.65 (3H, s, CH -18),
0.86 (3H, s, CH -19), 2.09 (3H, s, CH CO), 3.46 (1H, t, JZ
3
3
3 3
0
.86 (6H, two d, JZ6.6 Hz, CH -26 and CH -27), 0.90 (3H,
9.5 Hz, H-4b), 3.64 (1H, t, JZ8.5 Hz, H-17a), 4.58 (1H,
13
ddd, JZ5.4, 9.2, 11.6 Hz, H-3a). C NMR d: 171.51,
81.85, 79.23, 72.48, 54.32, 51.29, 50.83, 42.88, 36.58,
36.01, 35.05, 31.00, 30.50, 25.62, 23.32, 22.48, 21.37,
3
3
d, JZ6.5 Hz, CH -21), 0.99 (3H, s, CH -19), 2.07 (3H, s,
CH CO), 3.88 (1H, br s, H-2a), 4.82 (1H, br s, H-3b). C
NMR d: 170.52, 72.92, 68.65, 56.43, 56.25, 54.97, 42.58,
3
3
1
3
3
4
3
2
1
0.46, 39.99, 39.89, 39.46, 36.13, 35.77, 35.34, 34.81,
1.85, 28.66, 28.21, 27.97, 24.11, 23.83, 22.80, 22.53,
1.40, 20.81, 18.62, 14.18, 12.06. FTIR (ATR): n 1033.1,
20.54, 15.26, 13.51, 11.11. FTIR (ATR): nmax 1035.6,
1262.2, 1707.7, 2841.6–2940.9, 3443.3 and 3522.3 cm
FI-MS m/zZ290.2061 (100%, M KCH COOH),
3
C C
350.2541 (39%, M ), 291.2042 (12%, M C1K
K1
.
C
max
K1
261.2, 1714.2, 2860.1–2953.8, 3500.3 cm
.
CH COOH).
3
4
.3.5. 3a-Hydroxycholestane-2b-yl acetate (4b). To a
solution of cholestane-2b,3a-diol (4, 50 mg, 0.124 mmol) in
toluene (9 ml), vinyl acetate (1 ml) and lipase PS (200 mg)
were added and the reaction mixture was shaken for 3 days,
under the conditions described above. After usual work-up
and purification by flash chromatography, 3a-hydroxy-
cholestane-2b-yl acetate was recovered as a single product
4.3.8. 3a,17b-Dihydroxyandrostan-4a-yl acetate (7a).
To a solution of 3a,4a,17b-trihydroxyandrostane (7,
118 mg, 0.38 mmol) in acetone (18 ml), THF (4 ml) and
vinyl acetate (2 ml), lipase PS (500 mg) was added and the
reaction mixture was shaken for 4 days, under the conditions
described above. After usual work-up and purification by

3072
M. M. Cruz Silva et al. / Tetrahedron 61 (2005) 3065–3073
flash chromatography, 3a,17b-dihydroxyandrostan-4a-yl
acetate was recovered (7a, 102.3 mg, 76%) as a white
powder.
D. V.; Fahy, E.; Faulkner, D. J. J. Nat. Prod. 1988, 51,
954–958.
4. (a) Mimaki, Y.; Kuroda, M.; Kameyama, A.; Yokosuka, A.;
Sashida, Y. Phytochemistry 1998, 48, 1361–1369. (b) Mimaki,
Y.; Kuroda, M.; Fukasawa, T.; Sashida, Y. Chem. Pharm. Bull.
1999, 47, 738–743. (c) Candra, E.; Matsunaga, K.; Fujiwara,
H.; Mimaki, Y.; Kuroda, M.; Sashida, Y.; Ohizumi, Y.
J. Pharm. Pharmacol. 2002, 54, 257–262.
1
Selected data: H NMR (CDCl ) d: 0.72 (3H, s, CH -18),
3
3
0
8
.87 (3H, s, CH -19), 2.09 (3H, s, CH CO), 3.64 (1H, t, JZ
3 3
.5 Hz, H-17a), 4.01 (1H, q, JZ2.9, 2.7 Hz, H-3b). 4.85
1
3
(
1H, dd, JZ11.8, 2.9 Hz, H-4b). C NMR d: 171.24, 82.76,
7
3
2
1
3
5.20, 67.58, 54.11, 50.67, 42.83, 42.48, 37.68, 36.80,
4.79, 31.14, 30.84, 27.48, 26.75, 23.41, 22.42, 21.13,
0.27, 12.82, 11.51. FTIR (ATR): nmax 1039.2, 1265.1,
5. Aoki, S.; Yoshioka, Y.; Miyamoto, Y.; Higushi, K.; Setiawan,
A.; Murakami, N.; Chen, Z.-S.; Sumizawa, T.; Akiyama, S.-I.;
Kobayashi, M. Tetrahedron Lett. 1998, 39, 6303–6306.
6. Tsukamoto, S.; Tatsuno, M.; van Soest, R. W. M.; Yokosawa,
H.; Ohta, T. J. Nat. Prod. 2003, 66, 1181–1185.
K1
710.7, 2841.6, 3445.7 and 3528.1 cm . FI-MS m/zZ
C
08.2328 (100%, M C1KCH CO), 290.2052 (71%,
3
C
C
M KCH COOH), 350.2441 (62%, M ), 332.2306 (26%,
3
7. Lønning, P. E.; Geisler, J.; Johannessen, D. C.; Gschwind,
H.-P.; Waldmeier, F.; Schneider, W.; Galli, B.; Winkler, T.;
Blum, W.; Kriemler, H.-P.; Miller, W. R.; Faigle, J. W.
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C C
M KH O), 351.2468 (12%, M C1).
2
4
.3.9. 4a-Hydroxycholestane-3b-yl acetate (9a). To a
8
. Rogers, L. L.; Zeng, L.; McLaughlin, J. L. J. Org. Chem. 1998,
3, 3781–3785.
solution of cholestane-3b,4a-diol (9, 50 mg, 0.124 mmol) in
acetone (9 ml), vinyl acetate (1 ml) and Chromobacterium
viscosum lipase (500 mg) were added and the reaction
mixture was shaken under the conditions described above
for 6 days. The usual work-up and flash chromatography
yielded 4a-hydroxycholestane-3b-yl acetate (9a, 42.3 mg,
6
9
. (a) Burgoyne, D. L.; Andersen, R. J.; Allen, T. M. J. Org.
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Acad. Sci. 2002, 99(23), 14831–14836.
11. (a) Carrea, G.; Riva, S. Angew. Chem., Int. Ed. 2000, 39,
7
6%), mp 169–170 8C.
1
Selected data: H NMR (CDCl ) d: 0.65 (3H, s, CH -18),
3
3
2
226–2254. (b) Gotor, V. Org. Process Res. Dev. 2002, 6,
20–426. (c) Schulze, B.; Wubbolts, M. G. Curr. Opin.
0
.84–0.91 (12H, CH -19, CH -21, CH -26, CH -27), 2.08
3 3 3 3
4
Biotechnol. 1999, 10, 609–615.
(
3H, s, CH CO), 3.45 (1H, t, JZ9.7 Hz, H-4b), 4.57 (1H,
3
13
ddd, JZ5.3, 9.1, 11.5 Hz, H-3a). C NMR d: 171.49,
7
3
2
2
1
3
3
9.24, 72.47, 56.30, 56.18, 54.21, 51.23, 42.45, 39.85,
9.46, 36.83, 36.10, 35.96, 35.76, 34.97, 31.44, 28.22,
7.97, 25.65, 24.12, 23.80, 22.79, 22.60, 21.53, 21.36,
0.94, 18.61, 13.46, 12.01. FTIR (ATR): nmax 1036.6,
12. (a) Secundo, F.; Carrea, G.; De Amici, M.; Joppolo di
Ventimiglia, S.; Dordick, J. S. Biotechnol. Bioeng. 2003, 81,
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62, 564–577.
K1
263.2, 1715.4, 2860.9–2930.2, 3549.3 cm . FI-MS m/zZ
13. (a) Njar, V. C. O.; Caspi, E. Tetrahedron Lett. 1987, 28,
6549–6552. (b) Riva, S.; Bovara, R.; Ottolina, G.; Secundo, F.;
Carrea, G. J. Org. Chem. 1989, 54, 3161–3164. (c) Ottolina,
G.; Carrea, G.; Riva, S. Biocatalysis 1991, 5, 131–136.
C C
86.3400 (100%, M KCH COOH), 446.3787 (33%, M ),
3
C
87.3417 (27%, M C1KCH COOH), 447.3893 (13%,
3
C
M C1).
(d) Bertinotti, A.; Carrea, G.; Ottolina, G.; Riva, S.
Tetrahedron 1994, 50, 13165–13172.
1
1
4. Riva, S.; Klibanov, A. M. J. Am. Chem. Soc. 1988, 110,
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Acknowledgements
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5. (a) Ferraboschi, P.; Molatore, A.; Verza, E.; Santaniello, E.
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Thanks are due to Funda c¸ a˜ o para a Ci eˆ ncia e Tecnologia
FCT) through POCTI (FEDER) and to the CNR-GRICES
bilateral exchange program (2003–2004) for financial
support. M. M. C. S. wishes to thank Funda c¸ a˜ o Calouste
Gulbenkian for a Ph.D. grant.
(
1
1
6. Cruz Silva, M. M.; S a´ e Melo, M. L.; Parolin, M.; Tessaro, D.;
Riva, S.; Danieli, B. Tetrahedron: Asymmetry 2004, 15,
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1–27.
7. Ferraboschi, P.; Colombo, D.; Reza-Elahi, S. Tetrahedron:
Asymmetry 2002, 13, 2583–2586.
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