Chemoenzymatic syntheses of (25R)- and (25S)-25-hydroxy-27-nor- cholesterol, a steroid bearing a secondary hydroxy group in the side chain

Article abstract of DOI:10.1016/S0957-4166(99)00257-8

(25R)- and (25S)-25-hydroxy-27-nor-cholesterol 1c were prepared by enzymatic resolution of the stereogenic center in the side chain of compound 4a or by synthesis of the side chain using, as a chiral synthon, the enantiomerically pure phenylsulfonyl alkan

Full text of DOI:10.1016/S0957-4166(99)00257-8

Pergamon
Tetrahedron: Asymmetry 10 (1999) 2497–2500
Chemoenzymatic syntheses of (25R)- and (25S)-25-hydroxy-27-
nor-cholesterol, a steroid bearing a secondary hydroxy group in
the side chain
Patrizia Ferraboschi,a Fabio Pecora,a Shahrzad Reza-Elahi a and Enzo Santaniello b,∗
aDipartimento di Chimica e Biochimica Medica, Università degli Studi di Milano, Italy
bDipartimento di Scienze Precliniche LITA Vialba, Università degli Studi di Milano, Italy
Received 6 May 1999; accepted 21 June 1999
Abstract
(25R)- and (25S)-25-hydroxy-27-nor-cholesterol 1c were prepared by enzymatic resolution of the stereogenic
center in the side chain of compound 4a or by synthesis of the side chain using, as a chiral synthon, the
enantiomerically pure phenylsulfonyl alkanol 6a prepared by different biocatalytic approaches. © 1999 Elsevier
Science Ltd. All rights reserved.
We have recently reported that the Pseudomonas cepacia lipase (PCL) catalyzes the stereoselective
acylation of the primary hydroxy group of a steroid side chain, as shown for the compound 1a1 and for
26-hydroxycholesterol 1b.2
We present here our results on the biocatalytic approach to the preparation of diastereomerically pure
25-hydroxy-27-nor-cholesterol 1c, a cholesterol metabolite that has been used as the (25RS)-epimeric
mixture in studies concerning the inhibitory effects of oxysterols on the hydroxymethylglutaryl coenzyme
(HMGCoA) reductase activity.3 The resolution of the C-25 stereogenic center was of special interest,
because the stereochemical outcome of the process might be foreseen according to the model that
has been proposed to explain the enantiopreference of a few lipases on secondary alcohols.4 Further-
more, (25R)- and (25S)-1c could, in principle, be transformed into the corresponding stereoisomers
∗
Corresponding author. E-mail: enzo.santaniello@unimi.it
0957-4166/99/$ - see front matter © 1999 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(99)00257-8

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of 26-aminocholesterol,5 that has been reported to interfere with myocite proliferation and cholesterol
synthesis.6
In order to study the resolution of the C-25 stereogenic center, we prepared the 3β-O-silyl ether of
(25RS)-1c (compound 4a) as substrate of the enzymatic reaction7 from the 25-ketosteroid 3,8 that was
in turn obtained from the 22-iodo derivative 2, an intermediate of the synthesis of 26-hydroxycholesterol
1b (Scheme 1).2
Scheme 1.
The compound (25RS)-4a was subjected to the enzymatic reaction (PCL/vinyl acetate)9 in chloroform
and the 25-acetate 4b at 30% conversion (24 h) and the unreacted alcohol 4a at 70% conversion (48 h)
were isolated (Scheme 2).10 The 500 MHz 1H NMR spectra of the (R)-MTPA ester11 of the alcohol 4a
and of the alcohol obtained from the acetate 4b showed that the enzymatic reaction afforded the pure
epimers12 that could be prepared in nearly 30% yield after silica gel column chromatography.
Scheme 2.
In order to assign the configuration of the enzymatic products, samples of the required (25R)-
or (25S)-alcohol 4a or deprotected 1c could be prepared from the previous 22-iodo steroid 2 and
a chiral phenylsulfonyl intermediate, by an approach that we had already applied to the synthesis
of marine sterols13 and 26-hydroxycholesterol.14 (S)-4-Phenylsulfonyl-2-butanol 6a was prepared in
an enantiomerically pure form (>98% ee) by a baker’s yeast-mediated reduction15 of the known 4-
phenylsulfonyl-2-butanone 5.16 The compound 6a was converted to the corresponding THP ether 7 that

P. Ferraboschi et al. / Tetrahedron: Asymmetry 10 (1999) 2497–2500
2499
was coupled to the 22-iodo derivative 2 and, after a few additional steps, a sample of (25S)-compound 1c
was prepared in 58% yield (Scheme 3).
Scheme 3. (i) Baker’s yeast, 30°C, 96 h (35%); (ii) DHP, pTSA, rt, 24 h (quant.); (iii) LDA, −78°C, 5 h (60%); (iv) Hg/Na,
EtOH, 25°C, 6 h (quant); (v) H2SO4, H2O/THF (1/1), 4 h (96%)
For a synthesis of both C-25 stereoisomers, the racemic phenylsulfonyl alcohol 6a was also resolved by
the PCL-catalyzed irreversible transesterification procedure9 in chloroform, obtaining both (S)-alcohol
6a and (R)-acetate 6b enantiomerically pure (>98% ee, E=189) (Scheme 4).17
Scheme 4.
1
In any event, the H NMR spectrum of the 3,25-diMTPA ester of 25S-1c prepared using (S)-
phenylsulfonyl alcohol 6a was identical to that obtained from the same diMTPA-ester of the desilylated
product of the enzymatically prepared 4a.18 This result demonstrated that the stereochemical outcome
of the enzymatic acylation of the alcohol 4a is in agreement with the configurational preference of the
enzyme when a secondary alcohol is the substrate.4 Finally, the results presented here offer additional
examples of the stereoselective control of the enzymatic reaction on a hydroxy group present in a
steroid chain and propose a few biocatalytic approaches to the synthesis of chiral synthons such as
phenylsulfonyl alcohols, useful intermediates for the construction of steroid side chains.
Acknowledgements
This work has been financially supported by the Ministero dell’Università e della Ricerca Scientifica e
Tecnologica (Fondi ex-MURST 60% to Università degli Studi di Milano and MURST PRIN Biocatalisi
e Bioconversioni) and Consiglio Nazionale delle Ricerche (CNR, Target Project in Biotechnology).
References
1. Ferraboschi, P.; Molatore, A.; Verza, E.; Santaniello, E. Tetrahedron: Asymmetry 1996, 7, 1551.
2. Ferraboschi, P.; Rezaelahi, S.; Verza, E.; Santaniello, E. Tetrahedron: Asymmetry 1998, 9, 2193.
3. (a) Defay, R.; Astruc, M. E.; Roussillon, S.; Descomps, B.; Crastes de Paulet, A. Biochem. Biophys. Res. Commun. 1982,
106, 362. (b) Taylor, F. R.; Saucier, S. E.; Shown, E. P.; Parish, E. J.; Kandutsch, A. A. J. Biol. Chem. 1984, 259, 12382.

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P. Ferraboschi et al. / Tetrahedron: Asymmetry 10 (1999) 2497–2500
4. Kazlauskas, R. J.; Weissfloch, A. N. E.; Rappaport, A. T.; Cuccia, L. A. J. Org. Chem. 1991, 56, 2656. The
enantiopreference for primary alcohols has been also studied; see: Weissfloch, A. N. E.; Kazlauskas, R. J. J. Org. Chem.
1995, 60, 6959.
5. We have outlined the main steps to transform compound 4a into 26-aminocholesterol (conversion of the 25-hydroxy into
a 25-nitrile group, followed by lithium hydride reduction, 54% overall yields).
6. Corsini, A.; Verri, D.; Raiteri, P.; Quarato, P.; Paoletti, R.; Fumagalli, R. Arterioscler. Thromb. Vasc. Biol. 1995, 15, 420.
7. We also prepared (25RS)-1c but it was insoluble in chloroform/tetrahydrofuran; we therefore prepared a less polar
substrate such as the silyl derivative 4a, so that we could carry on the enzymatic reaction in a solvent like chloroform
or dichloromethane, that we have used for all our transesterifications.
8. The ketone 3 (83% from 2) was silylated (89%) and reduced (NaBH4/MeOH, 85%).
9. (a) Degueil-Castaing, M.; De Jeso, B.; Drouillard, S.; Maillard, B. Tetrahedron. Lett. 1987, 28, 953. (b) Wang, Y.-F.;
Lalonde, J. J.; Momongan, M.; Bergbreiter, D. E.; Wong, C.-H. J. Am. Chem. Soc. 1988, 110, 7200. The lipase (31.5
U/mg) was a generous gift from the Italian subsidiary of Amano (Japan) and was used for reactions at 30°C with vinyl
acetate (4:1 molar ratio with respect to the substrate).
10. A solution of (25RS)-4a (0.5 g, 1 mmol) in chloroform (3 mL) was treated with vinyl acetate and the lipase (14 mg)
under stirring for the time required for the desired conversion. The enzymatic reactions were monitored by GLC analysis
(Hewlett Packard, mod. 5890/II, HP-5 capillary column, T 280°C, TR 21 min for the alcohol and 25 min for the acetate).
11. Dale, J. A.; Mosher, H. S. J. Am. Chem. Soc. 1973, 95, 512. The (R)-MTPA esters are quantitatively prepared by reaction
of the alcohol with (S)-α-methoxy-α-trifluoromethylphenylacetic acid (MTPA) chloride (JPS, Switzerland, >99% ee). For
the experimental protocol, see, for instance: Ferraboschi, P.; Grisenti, P.; Manzocchi, A.; Santaniello, E. Tetrahedron 1994,
50, 10539.
12. In the spectrum of (25RS)-derivative the signal due to the C-26 methyl group showed two doublets at 1.24 and 1.32 ppm.
The signal at 1.24 ppm was not detectable in the spectrum of the (R)-MTPA ester of the enzymatically prepared alcohol
4a. In the spectrum of the (R)-MTPA ester of the alcohol obtained after removal (LiAlH4 in THF) of the acetate 4b only
the signal at 1.24 ppm was present.
13. Santaniello, E.; Ferraboschi, P. Synth. Commun. 1984, 14, 1199.
14. Ferraboschi, P.; Fiecchi, A.; Grisenti, P.; Santaniello, E. J. Chem. Soc., Perkin Trans. 1 1987, 1749. Ferraboschi, P.; Grisenti,
P.; Casati, R.; Fiecchi, A.; Santaniello, E. ibid. 1987, 1743.
15. Robin, S.; Huet, F.; Fauve, A.; Veschambre, H., Tetrahedron: Asymmetry, 1995, 4, 239. Using a 1/20 ratio (mmol substrate
per gram yeast) instead of the described 1/6 ratio we reached a 75% conversion of the substrate and incubation time was
lowered (from 192 h to 96 h). A 35% yield of the product was obtained after purification by silica gel chromatography.
16. Julia, M.; Badet, B. Bull. Soc. Chim. France 1975, 1363.
17. A solution of (RS)-6a (0.3 g, 1.4 mmol) in chloroform (2.6 mL) was treated with vinyl acetate and the lipase (19.5 mg)
1
under stirring. The ees were determined by H NMR analysis of the (R)-MTPA esters of the alcohol 6a and the alcohol
from the acetate 6b after treatment with LiAlH4 in THF. The signals at 1.24 and 1.32 ppm present in the derivative from
(RS)-6a were absent respectively in the derivative from (S)- and (R)-6a. The value of E was calculated according to Sih and
Wu (Sih, C. J.; Wu, S.-H. Topics in Stereochemistry 1989, 19, 63). The S-configuration of 6a ([α]D +19, c 1 in chloroform)
was established by comparison with the specific rotation reported in the literature (Ref. 15).
1
18. H NMR of 3β,25S-diMTPA ester of 1c: δ 0.63 (s, 3H, CH3-18), 0.80 (d, 3H, CH3-21), 0.97 (s, 3H, CH3-19), 1.30 (d,
3H, CH3-26), 3.377 (s, 3H, OCH3), 3.379 (s, 3H, OCH3), 4.81–4.89 (m, 1H, CHOCO), 5.09–5.15 (m, 1H, CHOCO),
5.39–5.41 (m, 1H, CH_C). The significant signals for a stereochemical analysis were those due to the following methyl
groups [from the 1H NMR spectrum of (3β,25RS)-diMTPA ester of 1c): 0.63 (s, 3H, CH3-18), 0.65 (s, 3H, CH3-18), 0.80
(d, 3H, CH3-21), 0.87 (d, 3H, CH3-21), 1.23 (d, 3H, CH3-26), 1.30 (d, 3H, CH3-26)]. The C-19 methyl group was a singlet
at 0.97 ppm for both (25R)- and (25S)-isomers.