Lipase-catalyzed resolution of stereogenic centers in steroid side chains by transesterification in organic solvents: The case of a 26- hydroxycholesterol

Article abstract of DOI:10.1016/S0957-4166(98)00222-5

The Pseudomonas cepacia (PCL) lipase selectively catalyzes the acylation of the (25S)-isomer of the (25R,S)-26-hydroxycholesterol 1a when the transesterification is irreversibly carried out with vinyl acetate in a mixture of organic solvents (chloroform/t

Full text of DOI:10.1016/S0957-4166(98)00222-5

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 9 (1998) 2193–2196
Lipase-catalyzed resolution of stereogenic centers in steroid side
chains by transesterification in organic solvents: the case of a
26-hydroxycholesterol
Patrizia Ferraboschi, Shahrzad Rezaelahi, Elisa Verza and Enzo Santaniello †,∗
Dipartimento di Chimica e Biochimica Medica, Università degli Studi di Milano, Milano, Italy
Received 24 March 1998; accepted 29 May 1998
Abstract
The Pseudomonas cepacia (PCL) lipase selectively catalyzes the acylation of the (25S)-isomer of the (25R,S)-
26-hydroxycholesterol 1a when the transesterification is irreversibly carried out with vinyl acetate in a mixture of
organic solvents (chloroform/tetrahydrofuran). © 1998 Elsevier Science Ltd. All rights reserved.
The lipase-catalyzed transesterification in organic solvents of hydroxylated substrates is now a well
established method extensively applied to the synthesis of enantiomerically pure compounds1 and seems
especially useful when applied to sterols that are highly insoluble in water. A few lipases have already
shown the capability of catalyzing the regioselective acylation of hydroxy groups in the steroid rings
and deacylation of the corresponding esters has already been described.2,3 We have recently reported4
that the Pseudomonas cepacia lipase5 (PCL or PFL from the previous name Pseudomonas fluorescens)
catalyzes the stereoselective acylation of a hydroxy group in the steroid side chain, under the conditions of
irreversible transesterification in an organic solvent,6 a method that we have used for the enantioselective
resolution of a variety of 2-substituted alkanols.7 We have now extended the above observation to another
primary alcohol in the steroid side chain bearing the stereogenic center at position 25, namely 26-
hydroxycholesterol 1a.
∗
†
Corresponding author.
Centro Interdipartimentale LITA Vialba, Via G. B. Grassi, 74-20128 Milano.
0957-4166/98/$ - see front matter © 1998 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(98)00222-5

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P. Ferraboschi et al. / Tetrahedron: Asymmetry 9 (1998) 2193–2196
We had already prepared the (25S)-epimer of 1a by a different biocatalytic approach, i.e. preparing a C-
5 chiral synthon necessary for the construction of the side chain by baker’s yeast-mediated bioreduction.8
An alternative approach could be constituted by the use of other enantiomerically pure chiral building
blocks such as phenylsulfones prepared by PFL-catalyzed resolution.9
In order to study the resolution of the stereogenic center present in the side chain, the synthesis of
(25R,S)-1a was required and we started from the 22-iodo derivative 2 obtained as previously described.8
The phenylsulfone 3 was easily prepared from the corresponding phenylsulfonyl ketone10 and reacted
with the intermediate compound 2.11 The conversion of the steroidal phenylsulfone 4 to the desired 1a
required the removal of the phenylsulfonyl moiety followed by the deprotection to the 25-ketosteroid
5.12 The 3β-hydroxy group of this intermediate was silylated and the 25-keto group transformed into the
corresponding methyl enol ether (compound 6).13 The enol ether was hydrolyzed to the corresponding
aldehyde with simultaneous removal of the silyl protection and reduction of the intermediate 25-aldehyde
afforded the final compound 1a.14
The (2R,S)-3,26-diol 1a prepared as above underwent a reaction with the lipase and vinyl acetate in
chloroform/tetrahydrofuran15 and after 1 h, 30% of the 26-acetate 1b was formed (as established by GLC
analysis)16 thus showing that the enzymatic reaction was highly regioselective (no trace of the 3-acetate
1
was observed).17 A 70% conversion to 1b was reached in 3 h and the 500 MHz H-NMR of the 26-
MTPA esters of the unreacted 1a (the enzymatic product at 70% conversion) and of the alcohol from the
acetate 1b (at 30% conversion) showed that the enzymatic reaction may be carried out to produce pure
epimers.18

P. Ferraboschi et al. / Tetrahedron: Asymmetry 9 (1998) 2193–2196
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The configuration of the enzymatic products was assigned by comparison of the published
resonances19 and from the results it was clear that the 25S-acetate 1b is produced by the enzymatic
transesterification. The fact that the 25S-alcohol 1a is the substrate accepted by the enzyme in the
conditions of the transesterification reaction to yield the 25S-acetate 1b confirms the configurational
outcome of the enzymatic reaction when 2-methyl alkanols are the substrates20 and include the side
chain of 1a in this class of compounds.21 This reaction is faster than the formation of the (20S)-acetate
from the (20R,S)-22-hydroxy steroid reported by us4 (30 h for a 30% conversion). However, it should
be remembered that, due to different steric hindrance, the C-26 alcohol is more accessible than the
C-22 analogue. In conclusion, this result offers an additional example of the regio- and enantioselective
control of the enzymatic reaction on a polyfunctional steroid as a substrate and from this and our
previous work4 a new approach is opened to the stereoselective construction of steroid side chains.
Acknowledgements
This work has been financially supported by Ministero dell’Università e della Ricerca Scientifica
(MURST, fondi 60%).
References
1. (a) Faber, K.; Riva, S. Synthesis 1992, 895. (b) Santaniello, E.; Ferraboschi, P.; Grisenti, P. Enzyme Microb. Technol. 1993,
15, 367. (c) Fang, J.-M.; Wong, C.-H. Synlett 1994, 393. (d) Carrea, G.; Ottolina, G.; Riva, S. Trends Biotechnol. 1995,
13, 63.
2. For a review, see: Riva, S. in Applied Biocatalysis, Blanch, H. W., Clark, D. S. Eds, M. Dekker, New York 1991, Vol. 1,
179–219.
3. (a) Njar, V. C. O.; Caspi, E. Tetrahedron Lett. 1987, 28, 6549. (b) Riva, S.; Klibanov, A. M. J. Am. Chem. Soc. 1988,
110, 3291. For more recent examples, see: (c) Adamczyk, M.; Chen, Y.-Y.; Fishpaugh, J. R.; Gebler, J. C. Tetrahedron:
Asymmetry 1993, 4, 1467. (d) Bertinotti, A.; Carrea, G.; Ottolina, G.; Riva, S. Tetrahedron 1994, 50, 13165. (e) Baldessari,
A.; Maier, M. S.; Gros, E. G. Tetrahedron Lett. 1995, 36, 4349. (f) Baldessari, A.; Maier, M. S.; Gros, E. G. Helv. Chim.
Acta 1997, 36, 4349.
4. Ferraboschi, P.; Molatore, A.; Verza, E.; Santaniello, E. Tetrahedron: Asymmetry 1996, 7, 1551
5. The lipase was a generous gift from the italian subsidiary of Amano (Japan) through the kind interest of Mr. I. Gaudio
(Mitsubishi Italia, Milano).
6. (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.
7. Santaniello, E.; Ferraboschi, P.; Casati, S.; Manzocchi, Tetrahedron: Asymmetry 1995, 6, 1521 and references cited therein.
8. (a) Ferraboschi, P.; Grisenti, P.; Casati, R.; Fiecchi, A.; Santaniello, E. J. Chem. Soc., Perkins Trans. 1 1987, 1743. (b)
Ferraboschi, P.; Fiecchi, A.; Grisenti, P.; Santaniello, E. ibidem 1 1987, 1749.
9. Santaniello, E.; Ferraboschi, P.; Fiecchi, A.; Grisenti, P.; Manzocchi, A. J. Org. Chem. 1987, 117, 701.
10. Reaction of sodium phenylsulfinate with methyl vinyl ketone quantitatively afforded 4-phenylsulfonyl-2-butanone (Julia,
M.; Badet, B. Bull. Soc. Chem. 1975, 1363). Ketalization (ethylene glycol in the presence of p-toluenesulfonic acid, 90%
yield) afforded the required intermediate 3.

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11. The 22-iododerivative 2 prepared starting from stigmasterol as described in Ref. 8b (50% yield) was treated with
the phenylsulfone 3 and LDA (from butyl lithium and diisopropylamine) affording the intermediate 4 (87%). The
phenylsulfonyl moiety was quantitatively removed by reaction with sodium amalgam (see Ref. 8b).
12. Treatment of compound 4 with H2SO4 in water/tetrahydrofuran (1/1) hydrolyzed both protecting groups (the i-steroid
moiety and the ketal function) affording the 25-keto-27 norcholesterol 5 (95%).
13. Compound 5 was silylated (89%) with t-butyldimethyl silyl chloride and imidazole (Corey, E. J.; Venkateswarlu, A. J. Am.
Chem. Soc. 1972, 94, 6190) and the 25-keto group was reacted with methoxy methyl triphenyl phosphonium chloride and
LDA to afford the intermediate 6 (85%).
14. Treatment of compound 6 with perchloric acid removed the silyl and the methyl enol ether groups affording the
intermediate 25-aldehyde that was directly reduced to the required 1a (NaBH4 in methanol, 79%).
15. A solution of 25R,S-1a (0.4 g, 1 mmol) in chloroform/tetrahydrofuran 1/2 (5.5 ml) and vinyl acetate (0.32 ml, 3.46 mmol)
was added to the solid lipase (14 mg, 31.5 U/mg) with stirring at room temperature.
16. GLC analysis (Hewlett Packard, mod. 5890/II, HP-5 capillary column, T 280°C) showed two peaks for the products at TR
15.0 (alcohol) and 17.0 min (acetate).
17. The structure of the product from the enzymatic reaction was determined by 1H-NMR (500 MHz): the proton at position
3 showed a multiplet centered at 3.50 ppm and the protons at positon 26 a multiplet centered at 3.87 ppm.
18. Although the enzymatic products were epimers, the optical purity could not be established directly by 1H-NMR (500 MHz)
analysis and the corresponding (R)-MTPA-esters were prepared. The derivative from (25R,S)-26-hydroxycholesterol
showed a signal constituted by three groups of peaks: a pair of double doublets between 4.00–4.08 ppm and 4.18–4.25
ppm and a doublet at 4.13 ppm. In the case of MTPA ester of unreacted 1a the doublet at 4.13 ppm was not detectable and
the same derivative prepared from the alcohol obtained by the hydrolysis of acetate 1b showed only the signal centered at
4.13 ppm.
19. Uomori, A.; Seo, S.; Sato, T.; Yoshimura, Y.; Takeda, K. J. Chem. Soc., Perkins Trans. 1 1987, 1713.
20. Ferraboschi, P.; Casati, S.; De Grandi, S.; Grisenti, P.; Santaniello, E. Biocatalysis 1994, 10, 279.
21. A recent interpretation of the enantiopreference of Pseudomonas cepacia lipase toward primary alcohols has been
proposed; see: Weissfloch, A. N. E.; Kazlauskas, R. J. J. Org. Chem. 1995, 60, 6959.