Asymmetric synthesis with the enzyme Coprinus peroxidase: Kinetic resolution of chiral hydroperoxides and enantioselective sulfoxidation

Article abstract of DOI:10.1021/jo990201p

The enzyme Coprinus peroxidase (CiP) was employed for the kinetic resolution of racemic hydroperoxides 1 and the asymmetric sulfoxidation of prochiral sulfides 4. Eleven hydroperoxides 1a-k were reduced by CiP and guaiacol as reductant under conditions of kinetic resolution with enantioselectivities of up to >98% for the (S)-hydroperoxide 1 and 90% for the (R)-alcohol 2. In the absence of a reductant, the hydroperoxide 1a afforded with CiP enantiomerically enriched hydroperoxide la (ee up to 54%) and alcohol 2a (ee up to 40%), as well as ketone 3a (which is also formed simultaneously in all other reactions) and molecular oxygen. Catalase activity was established for CiP with hydrogen peroxide. When aryl alkyl sulfides 4 were used as oxygen acceptors, three products, sulfoxides 5, alcohols 2, and hydroperoxides 1, were obtained, all in enantiomerically enriched form. The highest ee value (89%) was achieved for the sulfoxide derived from naphthyl methyl sulfide (4f). Thus, CiP may be utilized for the asymmetric synthesis of optically active hydroperoxides 1, alcohols 2, and sulfoxides 5.

Full text of DOI:10.1021/jo990201p

4834
J . Org. Chem. 1999, 64, 4834-4839
Asym m etr ic Syn th esis w ith th e En zym e Copr in u s P er oxid a se:
Kin etic Resolu tion of Ch ir a l Hyd r op er oxid es a n d En a n tioselective
Su lfoxid a tion
Waldemar Adam,* Cordula Mock-Knoblauch, and Chantu R. Saha-Mo¨ller
Institut fu¨r Organische Chemie der Universita¨t Wu¨rzburg, Am Hubland, D-97074 Wu¨rzburg, Germany
Received February 2, 1999
The enzyme Coprinus peroxidase (CiP) was employed for the kinetic resolution of racemic
hydroperoxides 1 and the asymmetric sulfoxidation of prochiral sulfides 4. Eleven hydroperoxides
1a -k were reduced by CiP and guaiacol as reductant under conditions of kinetic resolution with
enantioselectivities of up to >98% for the (S)-hydroperoxide 1 and 90% for the (R)-alcohol 2. In the
absence of a reductant, the hydroperoxide 1a afforded with CiP enantiomerically enriched
hydroperoxide 1a (ee up to 54%) and alcohol 2a (ee up to 40%), as well as ketone 3a (which is also
formed simultaneously in all other reactions) and molecular oxygen. Catalase activity was
established for CiP with hydrogen peroxide. When aryl alkyl sulfides 4 were used as oxygen
acceptors, three products, sulfoxides 5, alcohols 2, and hydroperoxides 1, were obtained, all in
enantiomerically enriched form. The highest ee value (89%) was achieved for the sulfoxide derived
from naphthyl methyl sulfide (4f). Thus, CiP may be utilized for the asymmetric synthesis of optically
active hydroperoxides 1, alcohols 2, and sulfoxides 5.
In tr od u ction
enzyme. Moreover, many enzymes accept only one of the
substrate enantiomers, such that the other antipode is
usually inaccessible. However, recently we have discov-
ered that the microorganism Bacillus subtilis possesses
peroxidase activity in which the opposite enantiomer of
the hydroperoxide is recognized as by the horseradish
peroxidase.7 This favorable finding encouraged us to
screen other peroxidases for their selectivity and sub-
strate acceptance to enable the biocatalytic synthesis of
both optically active enantiomers for a wide variety of
functionalized substrates.
The Coprinus peroxidase (CiP), which has been isolated
from the basidiomycete Coprinus cinereus,8 is known
from its X-ray crystal structure9 to have a rather large
substrate opening and should, therefore, be a favorable
candidate to accept a broad range of substrates, also
sterically encumbered ones. We have applied the enzyme
Coprinus peroxidase for the kinetic resolution of racemic
hydroperoxides and the enantioselective oxidation of
prochiral sulfides and report herein our results.
In recent years, enzymes have increasingly been used
for the synthesis of optically active compounds.1 In
particular, peroxidases have proven to be versatile bio-
catalysts for a number of redox transformations.2 Among
the best-known peroxidases is chloroperoxidase from
Caldariomyces fumago (CPO), which, for example, cata-
lyzes enantioselective epoxidations,3 sulfoxidations,4 and
benzylic hydroxylations.5 Also, horseradish peroxidase
(HRP) has been intensively studied for the kinetic
resolution of hydroperoxides.6 In many of these reactions,
good to excellent enantioselectivities have been obtained;
unfortunately, this selectivity advantage comes at the
dear price of limited scope of substrate acceptance by the
* To whom correspondence should be addressed. E-mail: adam@
chemie.uni-wuerzburg.de. Fax: +49-931-8884756.
(1) (a) Enzymes in Synthetic Organic Chemistry; Wong, C. H.,
Whitesides, G. M., Eds.; Pergamon Press: Oxford, 1994. (b) Drautz,
K.; Waldmann, H. Enzyme Catalysis in Organic Synthesis; VCH:
Weinheim, 1995.
(2) (a) van Deurzen, M. P. J .; van Rantwijk, F.; Sheldon, R. A.
Tetrahedron 1997, 39, 13183-13220. (b) Adam, W.; Lazarus, M.; Saha-
Mo¨ller, C. R.; Weichold, O.; Hoch, U.; Ha¨ring, D.; Schreier, P. In
Advances in Biochemical Engineering/ Biotechnology; Faber, K., Ed.;
Springer-Verlag: Heidelberg, 1999; Vol. 63, p 73.
(3) (a) Colonna, S.; Gaggero, N.; Casella, L.; Carrea, G.; Pasta, P.
Tetrahedron: Asymmetry 1993, 4, 1325-1330. (b) Allain, E. J .; Hager,
L. P.; Deng, L.; J acobsen, E. N. J . Am. Chem. Soc. 1993, 115, 4415-
4416. (c) Lakner, F. J .; Hager, L. P. J . Org. Chem. 1996, 61, 3923-
3925. (d) Dexter, A. F.; Lakner, F. J .; Campbell, R. A.; Hager, L. P. J .
Am. Chem. Soc. 1995, 117, 6412-6413.
(4) (a) Colonna, S.; Gaggero, N.; Casella, L.; Carrea, G.; Pasta, P.
Tetrahedron: Asymmetry 1992, 3, 95-106. (b) Fu, H.; Kondo, H.;
Ichikawa, Y.; Look, G. C.; Wong, C. H. J . Org. Chem. 1992, 57, 7265-
7270. (c) van Deurzen, M. P. J .; Remkes, I. J .; van Rantwijk, F.;
Sheldon, R. A. J . Mol. Catal. A: Chemical 1997, 117, 329-337.
(5) (a) Miller, V. P.; Tschirret-Guth, R. A.; Ortiz de Montellano, P.
R. Arch. Biochem. Biophys. 1995, 319, 333. (b) Zaks, A.; Dodds, D. R.
J . Am. Chem. Soc. 1995, 117, 10419-10424.
Resu lts a n d Discu ssion
Kin etic Resolu tion of Hyd r op er oxid es. In our
efforts to provide optically enriched hydroperoxides,
which may be used as oxidants in asymmetric synthe-
sis,10 we have shown that the kinetic resolution of racemic
hydroperoxides with horseradish peroxidase (HRP) con-
stitutes a useful way to achieve this goal.6 Unfortunately,
(7) Adam, W.; Boss, B.; Harmsen, D.; Lukacs, Z.; Saha-Mo¨ller, C.
R.; Schreier, P. J . Org. Chem. 1998, 63, 7598-7599.
(8) (a) Shinmen, Y.; Asami, S.; Amachi, T.; Shimizu, S.; Yamada,
H. Agric. Biol. Chem. 1986, 50, 247-249. (b) Morita, Y.; Yamashita,
H.; Mikami, B.; Iwamoto, H.; Aibara, S.; Terada, M.; Minami, J . J .
Biochem. 1988, 103, 693-699.
(6) (a) Adam, W.; Hoch, U.; Lazarus, M.; Saha-Mo¨ller, C. R.;
Schreier, P. J . Am. Chem. Soc. 1995, 117, 11898-11901. (b) Adam,
W.; Hoch, U.; Humpf, H.-U.; Saha-Mo¨ller, C. R.; Schreier, P. J . Chem.
Soc., Chem. Commun. 1996, 2701-2702. (c) Hoch, U.; Adam, W.; Fell,
R. T.; Saha-Mo¨ller, C. R.; Schreier, P. J . Mol. Catal. A, Chem. 1997,
117, 321-328.
(9) (a) Petersen, J . F. W.; Kadziola, A.; Larsen, S. FEBS Lett. 1994,
339, 291-296. (b) Abelskov, A. K.; Smith, A. T.; Rasmussen, C. B.;
Dunford, H. B.; Welinder, K. G. Biochemistry 1997, 36, 9453-9463.
(10) (a) Adam, W.; Korb, M. N.; Roschmann, K. J .; Saha-Mo¨ller, C.
R. J . Org. Chem. 1998, 63, 3423-3428. (b) Adam, W.; Korb, M. N.
Tetrahedron: Asymmetry 1997, 8, 1131-1142.
10.1021/jo990201p CCC: $18.00 © 1999 American Chemical Society
Published on Web 06/06/1999

Asymmetric Synthesis with Coprinus Peroxidase
J . Org. Chem., Vol. 64, No. 13, 1999 4835
Ta ble 1. CiP -Ca ta lyzed Kin etic Resolu tion of Hyd r op er oxid es 1 in th e P r esen ce of Gu a ia cola
HRP does not catalyze the kinetic resolution of sterically
hindered and, particularly, tertiary hydroperoxides.6a For
this purpose, we have employed CiP, since, as already
mentioned, its X-ray crystal structure exposed a rather
large opening for substrate entry.9a To establish the
catalytic efficiency and the substrate selectivity of CiP,
we examined a series of sterically and electronically
varied racemic hydroperoxides 1, with emphasis on those
substrates that are not satisfactorily converted by HRP.
The results (Table 1) show that the hydroperoxide
structure dramatically influences the enantioselectivity
and the enantiomeric preference of the CiP enzyme. An
increase of the chain length of the alkyl group in the case
of the alkyl aryl hydroperoxide leads to a drastic reduc-
tion of the reactivity and selectivity. Whereas for the
hydroperoxides 1a -c with a methyl group good to
excellent enantioselectivities were obtained at low en-
zyme concentration in relatively short reaction times
(entries 1-3), for the substrates with sterically more
demanding alkyl groups, e.g., 1d -f, low or even no
selectivity was observed (entries 4-6). By comparison,
the variation of the aryl group does bring about signifi-
cant changes in the efficiency of enantiomeric control, as
exemplified by the hydroperoxides 1a -c (entries 1-3),
but not as pronounced as does the alkyl group in the
derivatives 1d -f (entries 4-6). Interestingly, the enzyme
CiP shows better reactivity and selectivity toward the
cyclic hydroperoxide 1g than toward the corresponding
acyclic hydroperoxide 1d (entries 7 and 4, respectively).
Apparently, the conformationally more rigid cyclic struc-
ture fits well into the enzyme pocket. Sterically more
demanding hydroperoxides such as 1i,j show almost no
conversion to the corresponding alcohols even at large
enzyme concentrations and long reaction times (entries
9 and 10). Instead, the transformation of the hydroper-
oxide to the corresponding ketone was observed. Consid-
erable amounts of ketone were also obtained for sub-
strates 1d -f with alkyl groups larger than methyl
(entries 4-6). Unfortunate for our purposes, the tertiary
hydroperoxide 1k (entry 11) was only ineffectively con-
verted to the corresponding alcohol 2k .

4836 J . Org. Chem., Vol. 64, No. 13, 1999
Ta ble 2. CiP -Ca ta lyzed Su lfoxid a tion w ith Ra cem ic (1-P h en yl)eth yl Hyd r op er oxid ea
Adam et al.
Interestingly, a â-hydroxy group in the hydroperoxide
1h does not decrease the reactivity and selectivity as
much as prolongation of the alkyl chain in the ethyl
derivative 1d (entries 8 and 4, respectively). Thus,
although steric effects play an important role, electronic
factors are as well significant for enantiodifferentiation
by the enzyme.
In all the hydroperoxides in Table 1, CiP preferentially
accepts the R enantiomer as substrate with concurrent
formation of the (R)-alcohol, while the (S)-hydroperoxide
is left behind (entries 1-6, 8). An exception is the cyclic
hydroperoxide 1g, for which the S enantiomer is con-
verted to the alcohol and the (R)-hydroperoxide remains
(entry 7).11 Evidently, the structural arrangement of the
CiP enzyme pocket, which preferentially accommodates
the short alkyl chain in the hydroperoxides 1a -d , also
accepts well the larger but flat phenyl group.
hydroperoxides with short alkyl chains. Sterically more
encumbered and particularly tertiary hydroperoxides,
however, are not or only unselectively reduced. The sense
of the enantioselectivity is also the same for CiP as for
HRP, except for the indanyl hydroperoxide 1g. The larger
substrate opening of the CiP enzyme, which had prompted
us to examine sterically more demanding hydroperoxides,
has not held up to our expectations.
En a n tioselective Su lfoxid a tion . Peroxidases also
possess the ability to catalyze oxygen-transfer reactions.
Whereas CPO also catalyzes epoxidations,3 most other
peroxidases only oxidize sulfides.4,12 We have investigated
herein the oxygen-transfer propensity of CiP by using the
enantioselective sulfoxidation as the model reaction. For
this purpose, seven electronically and sterically varied
prochiral sulfides 4 were oxidized by CiP (Table 2).
As oxidant, racemic (1-phenyl)ethyl hydroperoxide (1a )
was used, which had been shown to be best in the CiP-
These results clearly establish the limitations of CiP-
catalyzed kinetic resolution of hydroperoxides. Like HRP,
CiP also shows very good enantioselectivity for aryl alkyl
(12) (a) Colonna, S.; Gaggero, N.; Carrea, G.; Pasta, P. J . Chem. Soc.,
Chem. Commun. 1992, 357-358. (b) Ozaki, S.-I.; Ortiz de Montellano,
P. R. J . Am. Chem. Soc. 1994, 116, 4487-4488. (c) Ozaki, S.-I.; Ortiz
de Montellano, P. R. J . Am. Chem. Soc. 1995, 117, 7056-7064. (d) Ortiz
de Montellano, P. R. Annu. Rev. Pharmacol. Toxicol. 1992, 32, 89-
107.
(11) It should be noted that for the hydroxy-functionalized hydro-
peroxide 1h a change in the sequence priority applies, and thus, the
stereoselection is the same.

Asymmetric Synthesis with Coprinus Peroxidase
J . Org. Chem., Vol. 64, No. 13, 1999 4837
catalyzed kinetic resolution experiments. If successful,
three optically active compounds would be prepared
simultaneously, namely, the sulfoxide 5, the alcohol 2,
and the hydroperoxide 1. That this is feasible was nicely
demonstrated by Wong et al. for the CPO enzyme.4b
In a preliminary experiment, the same reaction condi-
tions were used for the enantioselective sulfoxidation of
methyl phenyl sulfide (4a ) as for the kinetic resolution
of hydroperoxides (Table 2, entry 1). At complete sulfide
conversion, only racemic sulfoxide 5a was obtained. A
control experiment without enzyme showed that under
these conditions the sulfide was almost completely oxi-
dized by the hydroperoxide (entry 2). This undesirable
direct chemical sulfoxidation was minimized by employ-
ing low sulfide concentrations and a higher amount of
enzyme (entry 3). Under these conditions, the CiP-
catalyzed enantioselective sulfoxidation of methyl phenyl
sulfide (4a ) was achieved in moderate (39%) enantio-
selectivities for the sulfoxide 5a . In the case of the alkyl
aryl sulfides 4b and 4c, in which the phenyl rings are
substituted with para electron-donating substituents,
among the best (79%) enantioselectivities were obtained
for the (S)-(-)-sulfoxides 5b and 5c (entries 4 and 5).
Most pleasingly, high ee values were also found for the
(S)-(-)-hydroperoxide 1a (>95%) and for the (R)-(+)-
alcohol 2a (ca. 65%). In contrast, methyl p-nitrophenyl
sulfide (4d ) was not converted at all to the corresponding
sulfoxide (entry 6), which may be ascribed to the deac-
tivation of the sulfide toward oxidation due to the
electron-withdrawing para substituent.4a,c
As was the case for the kinetic resolution of the
hydroperoxides 1, prolongation of the alkyl chain in the
ethyl tosyl sulfide (4e) strongly reduced the reactivity and
selectivity (entry 7). Benzyl methyl sulfide (4g) also
proved to be a poor substrate for the CiP-catalyzed
sulfoxidation (entry 9). Only low conversion and almost
no enantioselectivity were observed. The best substrate
was methyl naphthyl sulfide (4f), which was oxidized
with the highest ee value of 89% (entry 8). The large
steric difference between the naphthyl and the methyl
groups in this prochiral sulfide is clearly advantageous
for effective stereodifferentiation.
In all cases, the (S)-sulfoxide was formed, in contrast
to the reactions catalyzed by CPO, which displays enan-
tioselectivities up to >98% for a number of aryl sulfides4c
in favor of the (R)-sulfoxide.4 This makes the enzyme CiP
a valuable native biocatalyst for the asymmetric synthe-
sis of sulfoxides since the “opposite” enantiomer is made
available. However, compared to CPO, the turnover
number of CiP in the asymmetric sulfoxidation is lower
and the substrate acceptance is more limited. The major
advantage of the CiP-catalyzed sulfoxidation is that the
hydroperoxide, sulfide, and enzyme are all mixed at once,
while with CPO the cumbersome slow continuous addi-
tion of the oxygen donor is required.4 Thus, the present
CiP-catalyzed sulfoxidation contributes a biocatalytic
process for the synthesis of selected (S) sulfoxides, which
should serve as valuable synthons for the preparation of
natural products and as powerful stereodirecting groups
in asymmetric synthesis.13
F igu r e 1. (a) CiP-catalyzed disproportionation of hydrogen
peroxide [UV absorption at 240 nm, 200 µmol of H2O2, 60 µmol
of CiP in 3 mL of 1 M phosphate buffer (pH 7.0)]. (b) Oxygen-
gas evolution during the CiP-catalyzed disproportionation of
(1-phenyl)ethyl hydroperoxide (1a ) [18 µmol 1a , 300 nmol CiP
in 2 mL of 1 M phosphate buffer (pH 7.0)].
hydroperoxide 1a used in the sulfoxidation reactions is
converted to the corresponding ketone 3a , whereby the
oxidation of the sulfide to sulfoxide is substantially
reduced. When, however, 1 equiv of hydrogen peroxide
was used as oxidant instead of hydroperoxide 1a in the
CiP-catalyzed sulfoxidation, no conversion of the sulfide
was observed although the H2O2 was completely con-
sumed.14 This prompted us to test whether the peroxidase
CiP also possesses catalase activity, i.e., catalyzes the
disproportionation of hydrogen peroxide to water and
molecular oxygen. Indeed, the UV absorption of hydrogen
peroxide at 240 nm decreased upon addition of CiP to a
solution of hydrogen peroxide in phosphate buffer
(pH 7.0), as displayed in Figure 1a.
Clearly, with hydrogen peroxide as oxygen source, the
resulting compound I of the enzyme prefers to react with
another molecule of hydrogen peroxide to produce O2
through paths A and D rather than oxidize the sulfide
to its sulfoxide along paths A and C in Scheme 1.
Ca ta la se Activity a n d Keton e F or m a tion fr om th e
Alk yl Hyd r op er oxid es. Unfortunately, up to 40% of the
(14) While this manuscript was in preparation, the following paper
appeared: Tuynman, A.; Vink, M. K. S.; Dekker, H. L.; Schoemaker,
H. E.; Wever, R. Eur. J . Biochem. 1998, 258, 906-913. The authors
describe the CiP-catalyzed formation of (S)-methyl phenyl sulfoxide
with a yield of 84% and an ee of 73%, when an excess of H2O2 is added
slowly, as catalase activity accounts for 85% of the H2O2 consumption.
(13) (a) Mata, E. G. Phosphorus, Sulfur Silicon 1996, 117, 231-
286. (b) Kagan, H. B.; Rebiere, F.; Samuel, O. Phosphorus, Sulfur
Silicon 1991, 58, 89-110. (c) Carren˜o, M. C. Chem. Rev. 1995, 95,
1717-1760.

4838 J . Org. Chem., Vol. 64, No. 13, 1999
Adam et al.
Sch em e 1. Ca ta lytic Cycles of Copr in u s
P er oxid a se (CiP ): P er oxid a se (A + B), Ca ta la se (A
+ D), a n d Mon ooxygen a se (A + C) Activity
electron oxidation of the ethanol, which in turn derives
from the ethyl hydroperoxide through reduction.15 This,
indeed, is the known mechanism for the reaction of alkyl
hydroperoxides with catalases18 and has also been re-
ported to work enantioselectively with soybean lipoxy-
genase (SBLO).19
More recently, the mechanism of oxygen production
was clarified. EPR-spectral evidence demonstrated the
generation of peroxyl radicals in the reaction of peroxi-
dases with hydroperoxides.20 Moreover, it was shown that
about 12% of the released oxygen is electronically excited
singlet oxygen.21 It was, thus, suggested that the peroxyl
radicals, generated by enzymatic one-electron oxidation
of the hydroperoxide, disproportionate according to the
Russell mechanism22 into ketone, alcohol, and O2 in a
ratio of 1:1:1 (Scheme 2).
This reaction, however, only constitutes a minor path
for the formation of acetaldehyde; the major one involves
the oxidation of ethanol, as suggested by Hager.21
In our experiments with CiP, however, the possibility
that the ketone 3a is formed by the oxidation of the
alcohol 2a 15,21,23 was discounted by the fact that the
alcohol resisted CiP-catalyzed treatment with hydrogen
peroxide or alkyl hydroperoxides 1b and 1e as oxygen
sources. Thus, a plausible pathway for the formation of
O2 and ketone (Scheme 2) is through the intervention of
peroxyl radicals, enzymatically generated by the CiP,
which disproportionate into alcohol 2, ketone 3, and O2
in a 1:1:1 ratio according to the Russell mechanism.22
However, if the overall reaction in Scheme 2 were the
only pathway for the ketone formation, an alcohol/ketone
ratio of 2:1 would be expected, i.e., 1 mol of alcohol results
from the reduction of the hydroperoxide for the formation
of compound I and another mol from the enzymatically
produced peroxyl radicals through the Russell cleavage,
together with 1 mol of ketone. Instead, an alcohol/ketone
ratio of 1:1 was obtained for hydroperoxide 1a and even
1:4 for 1f. This implicates that, besides the Russell
cleavage, some of the ketone must be formed from the
hydroperoxide by an additional pathway. That this
pathway is also an enzyme-catalyzed reaction and not
merely base-catalyzed dehydration of the hydroperoxide
(Kornblum-DeLaMare reaction24), has been confirmed
by means of a control experiment, in which each hydro-
peroxide 1a and 1f was stirred under the usual reaction
conditions [phosphate buffer (pH 7.0)] without the CiP
enzyme; after 4 h (1a ) and even 3 days (1f), the hydro-
peroxides were recovered unchanged.
In contrast, with the hydroperoxide 1a as oxygen
source, some asymmetric sulfoxidation takes place (Table
2); however, when CiP was allowed to react with hydro-
peroxide 1a in the absence of a reductant, oxygen-gas
evolution, which was monitored by means of an oxygen
electrode, was observed (Figure 1b). To understand the
origin of the oxygen evolution, a careful product study of
the reaction between hydroperoxide 1a and CiP was
conducted (Table 3, entry 1).
Mechanistically significant, in addition to alcohol 2a
substantial amounts of ketone 3a were also observed,
even when the isolated pure enzyme was employed (entry
2), accompanied by oygen-gas release. The ratio of alcohol
to ketone was 1:1, independent of the extent of conversion
(entry 3). Also the sterically more hindered hydroperoxide
1f gave the corresponding ketone 3f in addition to the
alcohol 2f, but in an alcohol-to-ketone ratio of about 1:4
(entry 4). The appreciable ee values for the hydroperoxide
1a and alcohol 2a (entries 1-3) show clearly that an
enzymatic stereodifferentiating process is involved.
In this context, Hager et al. were the first who reported
the formation of molecular oxygen in the reaction be-
tween CPO and ethyl hydroperoxide.15 They attributed
the liberation of oxygen to the catalase activity of CPO
toward alkyl hydroperoxides, as has been documented
for hydrogen peroxide.16 However, this seems question-
able because early work claims that catalase itself does
not catalyze the disproportionation of alkyl hydroperox-
ides into alcohol and O2.17 Also acetaldehyde was ob-
served, presumed to be formed by CPO-catalyzed two-
With the experimental data on hand, it is difficult to
suggest which additional enzymatic pathway is respon-
sible for the ketone formation. What may be stated
definitely (see control experiment) is that the ketone is
(18) Fita, I.; Rossmann, M. G. J . Mol. Biol. 1985, 185, 21-37.
(19) Scettri, A.; Bonadies, F.; Lattanzi, A.; Palombi, L.; Pesci, S.
Tetrahedron 1997, 53, 17139-17150.
(20) (a) Davies, M. J . Biochim. Biophys. Acta 1988, 964, 28-35. (b)
Chamulitrat, W.; Takahashi, N.; Mason, R. P. J . Biol. Chem. 1989,
264, 7889-7899.
(21) Hall, R. D.; Chamulitrat, W.; Takahashi, N.; Chignell, C. F.;
Mason, R. P. J . Biol. Chem. 1989, 264, 7900-7906.
(22) (a) Russell, G. A. J . Am. Chem. Soc. 1957, 79, 3871-3877. (b)
Horward, J . A.; Ingold, K. U. J . Am. Chem. Soc. 1968, 90, 1058-1059.
(c) Nakano, M.; Takayama, K.; Shimizu, Y.; Tsuji, Y.; Inaba, H.; Migita,
T. J . Am. Chem. Soc. 1976, 98, 1974-1975.
(23) Geigert, J .; Dalietos, D. J .; Neidleman, S. L.; Lee, T. D.;
Wadsworth, J . Biochem. Biophys. Res. Commun. 1983, 114, 1104-
1108.
(15) Thomas, J . A.; Morris, D. R.; Hager, L. P. J . Biol. Chem. 1970,
245, 3129-3134.
(16) (a) Hager, L. P.; Hollenberg, P. F.; Rand-Meir, T.; Chiang, R.;
Doubek, D. Ann. N. Y. Acad. Sci. 1975, 244, 80-93. (b) Araiso, T.;
Rutter, R.; Palcic, M. M.; Hager, L. P.; Dunford, H. B. Can. J . Biochem.
1981, 59, 233-236.
(24) Kornblum, N.; DeLaMare, H. E. J . Am. Chem. Soc. 1951, 73,
880-881.
(17) Chance, B. J . Biol. Chem. 1949, 180, 947-959.

Asymmetric Synthesis with Coprinus Peroxidase
J . Org. Chem., Vol. 64, No. 13, 1999 4839
Ta ble 3. CiP -Ca ta lyzed Decom p osition of th e Hyd r op er oxid es 1 to Alcoh ols 2 a n d Keton es 3 in th e Absen ce of
Gu a ia cola
Sch em e 2. F or m a tion of P er oxyl Ra d ica ls by
CiP -Ca ta lyzed Oxid a tion of Hyd r op er oxid es a n d
Th eir Disp r op or tion a tion to Keton e, Alcoh ol, a n d
Molecu la r Oxygen (Ru ssell Mech a n ism )
toward hydrogen peroxide. In its reaction cycle (Scheme
2), CiP uses alkyl hydroperoxides, on one hand, as
substrates for the formation of compound I and, on the
other hand, for the generation of peroxyl radicals by the
reduction of compound I back to the native enzyme
through compound II. The peroxidase activity of CiP may
be used for the synthesis of optically active products by
the kinetic resolution of hydroperoxides and enantio-
selective sulfoxidation, which provides (S)-(-)-hydroper-
oxides, (R)-(+)-alcohols, and (S)-(-)-sulfoxides in enan-
tiomeric excess up to >90%. Clearly, the CiP peroxidase
constitutes a promising enzyme for large-scale synthetic
applications.
Ack n ow led gm en t. We thank NovoNordisk for the
generous gift of CiP samples and the Deutsche Fors-
chungsgemeinschaft (SFB-347 “Selektive Reaktionen
Metall-aktivierter Moleku¨le”) and the Fonds der Che-
mischen Industrie for financial support. We also thank
Prof. P. Schreier, University of Wu¨rzburg, for help with
the oxygen measurements.
not formed by enzymatic oxidation of the alcohol, which
is known for the CPO-catalyzed transformation of pri-
mary alcohols to aldehydes.15,23 Be this as it may, the
enantioselective degradation of a chiral secondary hy-
droperoxide by a peroxidase in the absence of a reductant
is unprecedented for such enzymes.
Su p p or tin g In for m a tion Ava ila ble: Experimental pro-
cedures with the tabulated HPLC conditions for the determi-
nation of the ee values of the hydroperoxides 1, alcohols 2,
and sulfoxides 5 on chiral columns. This material is available
free of charge via the Internet at http://pubs.acs.org.
In summary, we have demonstrated that, besides the
usual peroxidase activity, CiP also acts as catalase
J O990201P