832
J . Org. Chem. 1999, 64, 832-835
Sem isyn th etic En zym es in Asym m etr ic Syn th esis:
En a n tioselective Red u ction of Ra cem ic Hyd r op er oxid es Ca ta lyzed
by Selen o-Su btilisin †
Dietmar Ha¨ring,‡ Ellen Schu¨ler,‡ Waldemar Adam,§ Chantu R. Saha-Mo¨ller,§ and
Peter Schreier*,‡
Institutes of Organic Chemistry and of Pharmacy and Food Chemistry, University of Wu¨rzburg,
Am Hubland, D-97074 Wu¨rzburg, Germany
Received August 17, 1998
The serine protease subtilisin was chemically converted into the peroxidase-active seleno-subtilisin.
This semisynthetic enzyme catalyzes the enantioselective reduction of racemic hydroperoxides in
the presence of thiophenols to yield optically active hydroperoxides and alcohols on the semi-
preparative scale. The kinetic parameters and enantioselectivities of seleno-subtilisin-catalyzed
reduction of various chiral hydroperoxides were determined. The catalytic efficiency of this
semisynthetic enzyme is comparable to that of the native horseradish peroxidase. The sense in the
enantioselectivity of the seleno-subtilisin is opposite to the natural enzymes previously used in the
synthesis of optically active hydroperoxides. Consequently, the semisynthetic enzyme seleno-
subtilisin complements the naturally available peroxidases for the asymmetric synthesis of both
enantiomers.
In tr od u ction
As a challenging proposition to overcome these short-
comings, we focused our interest on semisynthetic en-
zymes with designed properties. In this strategy of
biocatalyst design a known enzyme is chemically modi-
fied at a specific site to introduce the desired catalytic
activity with conservation of the selectivity of the peptide
framework.4 To date, the preparative application of
semisynthetic enzymes in asymmetric catalysis has been
limited to only few examples, either at the lower micro-
molar scale or under noncatalytic conditions.5
Enantiomerically pure hydroperoxides have a high
potential as chiral oxidants;1 however, their general and
convenient preparation still demands intensive research
efforts.2,3 The enantioselective synthesis by purely chemi-
cal means has so far been restricted to sugar1a and furyl
hydroperoxides.1d Best results have been achieved by
enzymatic methods with lipase,2a lipoxygenase,2b chlo-
roperoxidase (CPO),2c and horseradish peroxidase (HRP).3
Especially our efficient kinetic resolution with HRP has
made available a broad structural variety of optically
active hydroperoxides. Despite the success, all these
enzymes prefer the same absolute configuration of the
chiral hydroperoxides, and their application is limited to
sterically unencumbered substrates.
Previously, it has been reported6 that the specific
chemical modification of serine 221 into selenocystein at
the active site of subtilisin imparts this protease with
seleno-peroxidase activity. The semisynthetic peroxidase
seleno-subtilisin with the peptide framework of the serine
protease subtilisin catalyzes the reduction of hydrogen
peroxide, tert-butyl hydroperoxide, and cumol hydroper-
oxide in the presence of 5-mercapto-2-nitrobenzoic acid
(Figure 1).6 Our preliminary study7a with chiral organic
hydroperoxides revealed that seleno-subtilisin reduces
racemic hydroperoxides enantioselectively and estab-
lished this semisynthetic enzyme as the first one with
catalytic efficiency and enantioselectivity comparable to
the native enzymes. From the well-known regio- and
stereoselectivity of the subtilisin template,8 the affinity
and enantioselectivity of seleno-subtilisin for chiral hy-
droperoxides may be rationalized and even predicted.7b
* To whom correspondence should be addressed. Phone: +49-931-
888-5481. Fax: +49-931-888-5484. E-Mail: schreier@pzlc.uni-
wuerzburg.de.
† Dedicated to Professor C. -H. Brieskorn on the occasion of his 85th
birthday.
‡ Institute of Pharmacy and Food Chemistry.
§ Institute of Organic Chemistry.
(1) (a) Hamann, H.-J .; Ho¨ft, E.; Mostowicz, D.; Mishnev, A.; Ur-
banczyk-Lipkowska, Z.; Chmielewski, M. Tetrahedron 1997, 53, 185.
(b) Adam, W.; Korb, M. N. Tetrahedron: Asymmetry 1997, 8, 1131. (c)
Adam, W.; Peters, K.; Renz, M. J . Org. Chem. 1997, 62, 3183. (d)
Lattanzi, A.; Bonadies, F.; Scettri, A. Tetrahedron: Asymmetry 1997,
8, 2141. (e) Adam, W.; Korb, M. N.; Roschmann, K. J .; Saha-Mo¨ller,
C. R. J . Org. Chem. 1998, 63, 3423. (f) Shum, W. P.-S.; Saxton, R. J .;
Zajacek, J . G. US Patent 5,663,384, 1997.
(2) (a) Baba, N.; Mimura, M.; Hiratake, J .; Uchida, K.; Oda, J . Agric.
Biol. Chem. 1988, 52, 2685. (b) Dussault, P.; Porter, N. A. J . Am. Chem.
Soc. 1988, 110, 6276. (c) Fu, H.; Kondo, H.; Ichikawa, Y.; Look, G. C.;
Wong, C.-H. J . Org. Chem. 1992, 57, 7265. (d) Adam, W.; Lazarus,
M.; Saha-Mo¨ller, C. R.; Weichold, O.; Hoch, U.; Ha¨ring, D.; Schreier,
P. Adv. Biochem. Eng. Biotech. 1998, 63, 73.
(3) (a) Adam, W.; Hoch, U.; Saha-Mo¨ller, C. R.; Schreier, P. Angew.
Chem., Int. Ed. Engl. 1993, 105, 1737. (b) Adam, W., Hoch, U.; Lazarus,
M.; Saha-Mo¨ller, C. R.; Schreier, P. J . Am. Chem. Soc. 1995, 117,
11898. (c) Adam, W.; Fell, R. T.; Hoch, U.; Saha-Mo¨ller, C. R.; Schreier,
P. Tetrahedron: Asymmetry 1995, 6, 1047. (d) Adam, W.; Hoch, U.;
Humpf, H. U.; Saha-Mo¨ller, C. R.; Schreier, P. J . Chem. Soc. Chem.
Commun. 1996, 2701. (e) Hoch, U.; Adam, W.; Fell, R.; Saha-Mo¨ller,
C. R.; Schreier, P. J . Mol. Catal. A: Chem. 1997, 117, 321.
(4) Kaiser, E. T. Angew. Chem., Int. Ed. Engl. 1988, 27, 913.
(5) (a) Kuang, H.; Davies, R. R.; Distefano, M. D. Bioorg. Med. Chem.
Lett. 1997, 7, 2055. (b) Kuang, H.; Brown, M. L.; Davies, R. R.; Young,
E. C.; Distefano, M. D. J . Am. Chem. Soc. 1996, 118, 10702. (c) Davies,
R. R.; Distefano, M. D. J . Am. Chem. Soc. 1997, 119, 11643.
(6) (a) Wu, Z.-P.; Hilvert, D. J . Am. Chem. Soc. 1990, 112, 5647. (b)
Bell, I. M.; Hilvert, D. Biochemistry 1993, 32, 13969.
(7) (a) Ha¨ring, D.; Herderich, M.; Schu¨ler, E.; Withopf, B.; Schreier,
P. Tetrahedron: Asymmetry 1997, 8, 853. (b) Ha¨ring, D.; Hubert, B.;
Schu¨ler, E.; Schreier, P. Arch. Biochim. Biophys. 1998, 354, 263. (c)
Ha¨ring, D.; Schreier, P. Biotech. Bioeng. 1998, 59, 786.
(8) (a) Takagi, H. Int. J . Biochem. 1993, 25, 307. (b) Kazlauskas, R.
J .; Weissfloch, A. N. E. J . Mol. Catal. B: Enzymol. 1997, 3, 65. (c)
Moree, W. J .; Sears, P.; Kawashiro, K.; Witte, K.; Wong, C.-H. J . Am.
Chem. Soc. 1997, 119, 3942. (d) Fitzpatrick, P. A.; Klibanov, A. M. J .
Am. Chem. Soc. 1991, 113, 3166.
10.1021/jo981665a CCC: $18.00 © 1999 American Chemical Society
Published on Web 01/15/1999