Asymmetric oxidation catalyzed by myoglobin mutants

Article abstract of DOI:10.1016/S0957-4166(98)00498-4

The sperm whale myoglobin active site mutants (L29H/H64L and F43H/H64L Mb) have been shown to catalyze the asymmetric oxidation of sulfides and olefins. Thioanisole, ethyl phenyl sulfide, and cis-β-methylstyrene are oxidized by L29H/H64L Mb with more than

Full text of DOI:10.1016/S0957-4166(98)00498-4

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 10 (1999) 183–192
Asymmetric oxidation catalyzed by myoglobin mutants
Shin-ichi Ozaki,a Hui-Jun Yang,b Toshitaka Matsui,a Yoshio Goto b and
Yoshihito Watanabe a,b,∗
aInstitute for Molecular Science, Okazaki, Myodaiji, 444 Japan
bDepartment of Structural Molecular Science, The Graduate University for Advanced Studies, Okazaki, Myodaiji, 444 Japan
Received 11 November 1998; accepted 7 December 1998
Abstract
The sperm whale myoglobin active site mutants (L29H/H64L and F43H/H64L Mb) have been shown to catalyze
the asymmetric oxidation of sulfides and olefins. Thioanisole, ethyl phenyl sulfide, and cis-β-methylstyrene are
oxidized by L29H/H64L Mb with more than 95% enantiomeric excess (% ee). On the other hand, the F43H/H64L
mutant transforms trans-β-methylstyrene into the trans-epoxide with 96% ee. The dominant sulfoxide product in
the incubation of alkyl phenyl thioethers is the R isomer; however, the mutants afford dominantly the S isomer of
aromatic bicyclic sulfoxides. The results help us to rationalize the difference in the preferred stereochemistry of
the Mb mutant-catalyzed reactions. Furthermore, the Mb mutants exhibit an improvement in the oxidation rate up
to 300-fold with respect to wild type. © 1999 Elsevier Science Ltd. All rights reserved.
1. Introduction
Asymmetric biotransformations have been applied to organic synthesis as excellent alternatives to
chemical procedures.1 Chloroperoxidase from Cadariomyces fumago (CPO),2 a heme enzyme, is one
of the well studied enzymes for the enantioselective oxidation of sulfides and olefins.3 Recently, a
vanadium-containing non-heme bromoperoxidase from the alga C. officinalis (VBrPO)4 has been shown
to perform sulfoxidation of aromatic bicyclic sulfides in high enantiomeric excess (% ee).5 In order to
extend the biocatalytic methodology, we have examined the oxygenation by sperm whale myoglobin
(Mb) mutants.
Mb has protoporphyrin IX as a prosthetic group, and its major physiological role is the storage and
transfer of molecular oxygen.6 However, Mb can support the peroxide-dependent one- and two-electron
oxidation of a variety of substrates at a very slow rate.7 Recently, we reported as communications
that L29H/H64L and F43H/H64L Mb exhibit high catalytic turnover with high stereospecificity for the
sulfoxidation of thioanisole and the epoxidation of styrene.8 More importantly, a ferryl porphyrin radical
∗
Corresponding author. Tel: 81-564-55-7430; fax: 81-564-54-2254; e-mail: yoshi@ims.ac.jp
0957-4166/99/$ - see front matter © 1999 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(98)00498-4

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cation species (O_FeIV Por+·), equivalent to compound I of peroxidase, has recently been confirmed as
the catalytic species of a two-electron oxidation process by F43H/H64L, H64S, H64A, and H64L Mb.8
The mutants were designed to increase the life time of catalytic intermediate as well as the accessibility of
substrates. In this report, we have explored the scope of asymmetric oxygenation by the use of aromatic
bicyclic sulfides and methyl styrene. The results help us understand the substrate binding orientation on
the basis of the preferred stereochemistry for L29H/H64L and F43H/H64L Mb. Furthermore, we have
confirmed that compound I of L29H/H64L Mb is also a catalytic species for the oxidation of sulfides and
styrene.
2. Results and discussion
2.1. Asymmetric sulfoxidation of cyclic and acyclic sulfides
Among the Mbs studied here, L29H/H64L Mb is the best chiral catalyst (Table 1). The values of % ee
for the L29H/H64L mutant are greater than those of wild type and F43H/H64L Mb for the oxidation of
sulfides examined here. The largest improvement from 7.6% to 95% in enantioselectivity is observed
for the sulfoxidation of ethyl phenyl sulfide (2) by the L29H/H64L mutant. On the other hand, the
F43H/H64L mutant is the best catalyst in terms of the sulfoxidation rate. The Phe-43 -→ His and His-
64 -→ Leu mutation increases the rate of thioanisole (1) oxidation by 190-fold with respect to the wild
type, which is the largest enhancement achieved herein.
In order to elucidate the substrate recognition by the Mb mutants, we have determined the enantio-
selectivity for the oxidation of 2-hydroxyethyl phenyl sulfide (3). The absolute configuration for the
sulfoxide isomers of 3 was determined by comparison of their CD spectra with that of (R)-methyl
phenyl sulfoxide (Fig. 1). In the reaction with L29H/H64L and F43H/H64L Mb, the hydroxy group
of 3 decreases the value of % ee by approximately 25% with respect to ethyl phenyl sulfide (2), but
the R enantiomers are still the major sulfoxide products for 2 and 3 (Table 1). Thus, the orientation
of substrate binding does not appear to be controlled by hydrophilic properties of alkyl groups in the
case of sulfoxidation catalyzed by L29H/H64L and F43H/H64L Mb. Interestingly, cyclic sulfides give
different stereoselectivity from acyclic thioethers for the L29H/H64L mutant. The values of % ee in the
oxidation of 2,3-dihydrobenzothiophene (4) and 1-thiochroman (5) by L29H/H64L Mb are 67 and 66%
ee, respectively, and the dominant isomers are (S)-sulfoxides (Table 1). On the contrary, R is the major
enantiomer with 97% ee for thioanisole (1) oxidation by the L29H/H64L mutant. Since sulfide 6 bearing
an ortho substituent is oxidized to the corresponding (R)-sulfoxide with 82% ee by L29H/H64L Mb, the
cyclic moiety, not the ortho substituent of the benzene ring, appears to be important for the formation of
the S isomer. The similar changes in dominant isomers are also observed for F43H/H64L Mb.
In this study, hydrogen peroxide was added in one portion at the beginning of the reaction to determine
the initial rate, and the values can not be directly compared with the results from the continuous
addition of oxidants to the reaction mixture with CPO3g or VBrPO5 as previously reported. However,
the sulfoxidation system with F43H/H64L appears to be comparable to that of CPO in terms of the rate.9
2.2. Catalytic species for the sulfoxidation reaction
In order to confirm that compound I is the catalytic species for the oxidation of sulfides by the
L29H/H64L and F43H/H64L mutant, we have performed double mixing stopped-flow experiments.
Compound I was generated in the first mixing, and sulfides were added to compound I in the second

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185
Table 1
Enantiospecific sulfoxidation of cyclic and acyclic thioethersa
mixing. UV–visible spectra began to be collected just after the second mixing to monitor the spectral
changes.
The decrease in the absorbance of the Soret and the increase in the absorbance around 650 nm are
characteristic for the formation of ferryl porphyrin radical cation from the ferric state (Fig. 2). Compound
I (O_FeIV Por+·) of L29H/H64L Mb is reduced back to the ferric state in the presence of an excess
of thioanisole (1). Since compound II (O_FeIV Por), bearing one oxidation equivalent with respect to
the ferric state, is not observed, the sulfoxidation appears to proceed with a direct two-electron process.
Essentially the same spectral changes are observed in the presence of 2,3-dihydrobenzothiophene (4) with
L29H/H64L Mb. Therefore, the bicyclic sulfide does not change the oxidation mechanism. Thioanisole
and dihydrobenzothiophene also reduce compound I of the F43H/H64L mutant directly to the ferric state.
The similar UV–visible spectral changes were observed in the reaction of compound I of both mutants

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Figure 1. CD spectra of (R)-methyl phenyl sulfoxide, (R)-sulfoxide of 3 (eluted at 20 min), and 6 (eluted at 19 min)
Figure 2. The UV–visible spectral changes from compound I of L29H/H64L Mb to the ferric state in the presence of thioanisole
in 50 mM NaOAc buffer (pH 5.3) at 5°C
with styrene (7). Thus, the results indicate that the ferryl porphyrin radical cation (O_FeIV Por+·) is the
reactive species for the peroxide dependent monooxygenation by the mutants.
2.3. Asymmetric epoxidation of styrene and β-methylstyrene
In order to gain an insight into the highly stereoselective oxidation by the L29H/H64L and F43H/H64L
mutant, we next studied epoxidation reactions. The epoxidation rate for styrene (7), trans- (8), and cis-

S. Ozaki et al. / Tetrahedron: Asymmetry 10 (1999) 183–192
187
β-methylstyrene (9) catalyzed by the L29H/H64L mutant is increased by 9-fold, 4-fold, and 46-fold,
respectively, compared with the rate of the wild type Mb (Table 2). The highest value of enantiomeric
excess was 99% ee for cis-β-methylstyrene epoxidation catalyzed by L29H/H64L Mb. On the other hand,
F43H/H64L Mb oxidized trans-β-methylstyrene to (1R,2S)-trans-epoxide with 96% ee. The F43H/H64L
mutant is the best enzyme in terms of epoxidation rate, and styrene, trans- and cis-β-methylstyrene are
found to be oxidized 300, 210, and 57 times faster by F43H/H64L than wild type Mb, respectively.
Synthetic iron porphyrins, hemoprotein model compounds, oxidize the cis- much faster than the trans-
isomer,10 but cis-β-methylstyrene is less reactive to Mbs than the trans isomer (Table 2), presumably due
to the limited accessibility of the cis isomer to the active site.
No more than a trace of trans-β-methylstyrene oxide is detected as a side product in the oxidation of
cis-β-methylstyrene. Thus, the epoxidation proceeds with the retention of olefin stereochemistry. Since
the trans-epoxide was generated in the oxidation of cis-β-methylstyrene by sterically unhindered Mn
porphyrin, the active site of the mutants might prevent the rotation of the two vinyl carbons of cis-β-
methylstyrene for steric reasons (Fig. 3).10
Only a trace of side product is observed in the oxidation of styrene and trans-β-methylstyrene;
however, a significant amount of phenylacetone is generated in the incubation of cis-β-methylstyrene
with F43H/H64L Mb. Compound I, an oxo-ferryl porphyrin π-cation radical species (O_FeIV Por+·),
is responsible for the epoxide production, but phenylacetone seems to be generated by the 1,2-hydrogen
rearrangement of the cationic species as previously proposed for the cytochrome c peroxidase-catalyzed
oxidation of olefin (Fig. 3).11 Since β-methylstyrene oxide is not transformed into phenylacetone in the
presence of F43H/H64L Mb and hydrogen peroxide, the carbocation at the benzylic position would be
formed from the reaction of compound I with β-methylstyrene.
2.4. Substrate binding orientation
The dominant isomers observed in the oxidation of thioanisole, dihydrobenzothiophene, and styrene by
L29H/H64L Mb are summarized in Fig. 4. We previously found that para substituents of thioanisole (1)
did not change the Mbs’ preference in enantioselectivity.8a In addition, a bulky substituent at the ortho-
position of 1 did not change the enantioselectivity (Table 1). Thus, the origin of enantioselectivity could
be rationalized by the conformational difference of sulfur or benzylic carbons in the putative reaction
intermediates. If we assume the aromatic group is bound in a fixed position, dihydrobenzothiophene (4)
and styrene seem to share a common binding orientation (Figs. 4 and 5). The vinyl group of styrene could
be in the same plane of the benzene ring to form a flat molecule like dihydrobenzothiophene (Fig. 5). On
the contrary, methyl phenyl sulfide approaches to the ferryl oxygen with the S–CH3 bond approximately
perpendicular to the plane of benzene ring. A similar substrate binding mode can be proposed for the
F43H/H64L mutant (Fig. 5).
In order to elucidate the origin of enantioselectivity for L29H/H64L Mb, we have crystallized the
mutant and performed the X-ray crystal structure analysis.12 Energy minimization followed by docking
either the substrates or products in the active site of L29H/H64L Mb did not immediately provide an
obvious rationale for the difference in enantiospecificity.13 Efforts to soak the substrate into the crystals
are underway to elucidate the reason for high stereospecificity of the Mb mutants.

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Table 2
Enantioselective epoxidation of olefins
3. Experimental section
3.1. Materials
All substrates except for sulfide 4, 5, and 6 are commercially available. 4, 5, and H218O2 were
chemically synthesized as previously reported.3g,7a Sulfoxides and epoxides were synthesized from the

S. Ozaki et al. / Tetrahedron: Asymmetry 10 (1999) 183–192
189
Figure 3. Proposed mechanism for the oxidation of cis-β-methylstyrene by Mbs11
Figure 4. The dominant isomers observed for sulfoxidation and epoxidation
Figure 5. Proposed conformational rationalization for the asymmetric oxidation by the Mb mutants

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corresponding starting substrates by the oxidation with m-chloroperbenzoic acid (mCPBA) or H2O2 and
used to make standard curves.
The L29H/H64L and F43H/H64L Mb were constructed utilizing the polymerase chain reaction based
method.14 The Mbs were expressed and purified as previously reported.15 The protein concentration was
determined spectrometrically at 408 nm (ε=1.8×105 M−1 cm−1).
GC–MS analysis of sulfide 6 and its sulfoxide was performed on a Shimadzu QP5000 equipped with
a Shimadzu CBP1 capillary column. The column temperature was kept at 150°C for 2 min, then rose to
250°C at 20°C/min.
3.2. Synthesis of sulfide 6 and its sulfoxide
A mixture of thiosalicylic acid (4.0 g), methyl iodide (36 mL), and 28% ammonium hydroxide (24
mL) was stirred at 25°C for 48 h. The products (a mixture of 6 and its free acid) were extracted
with dichloromethane, and purified by silica gel 60 (2.1 cm×8 cm). The column was washed with
dichloromethane to elute 6 and then with 20% isopropanol containing hexane to recover the free acid.
NMR: δ (CDCl3) 2.49 (s, 3H), 3.95 (s, 3H), 7.18 (t, 1H, J=6.9 Hz), 7.31 (d, 1H, J=7.9 Hz), 7.50 (t,
1H, J=7.2 Hz) 8.03 (d, 1H, J=7.9 Hz). GC–MS: retention time 4.3 min, MS found 182.1 (calculated for
C9H10O2S 182.23).
The sulfoxide of 6 was synthesized in a water–methanol mixture (2.0 mL) containing 6 (10 mg) and
H2O2 (0.01 mol) at 25°C for 4.5 h. The sulfoxide product was purified by silica gel 60 (2.1 cm×8 cm)
with the eluent system of isopropanol:hexane, 20:80. NMR: δ (CDCl3) 2.85 (s, 3H), 3.96 (s, 3H), 7.57 (t,
1H, J=7.6 Hz), 7.83 (t, 1H, J=6.6 Hz), 8.09 (d, 1H, J=7.6 Hz), 8.32 (d, 1H, J=7.9 Hz). GC–MS: retention
time 5.6 min, MS found 198.1 (calculated for C9H10O3S 198.24). The S and R isomers were separated and
collected by isocratic HPLC on a Daicel chiral column OD (0.46 cm×25 cm) at a flow rate of 0.5 mL/min
(isopropanol:hexane=20:80). The CD spectra were obtained on a JASCO J-40 spectropolarimeter, and the
absolute configuration was assigned by comparison of the CD spectra of (R)-methyl phenyl sulfoxide and
the synthetic sulfoxide of 6 (Fig. 1).
3.3. Enzymatic sulfoxidation
H2O2 (1 mM) was added to a solution of either Mb or the Mb mutants (5 µM) and 1 mM sulfide in
0.5 mL of 50 mM sodium phosphate buffer (pH 7.0) at 25°C. A selected internal standard was added, the
mixture was extracted with dichloromethane, and analyzed by isocratic HPLC on Daicel chiral column
OD (0.46 cm×25 cm) at a flow rate of 0.5 mL/min.16 The reaction time varies from 1 to 30 min, and the
rates were determined from the linear portion of the product versus time plot. Since the enzymes were
inactivated during the reaction, we normally observed up to 2500 turnovers. Standard curves prepared
using synthetic sulfoxides were used for quantitative analysis, and the values of % enantiomeric excess
were determined on a basis of peak area of HPLC traces. It was previously reported that the (S)-sulfoxides
of 1 and 2 eluted from the column.16 The absolute configurations for sulfoxides of 3 and 6 were confirmed
by the comparison of the CD spectra of (R)-methyl phenyl sulfoxide and those of sulfoxide isomers for
3 and 6 collected from the chiral column. To determine the stereochemistry for sulfoxides of 4 and 5,
authentic (R)-sulfoxides were synthesized by CPO as described previously,3g and the retention times
were determined as 21 (4) and 36 min (5), respectively. The sulfoxide formed in control incubations
without enzyme was subtracted when necessary.

S. Ozaki et al. / Tetrahedron: Asymmetry 10 (1999) 183–192
191
3.4. Reactions of compound I with sulfides or styrene
Rapid scan spectra were collected on a Hi-Tech SF-43 stopped-flow apparatus equipped with an MG
6000 diode array spectrometer. Single mixing experiments (i.e. mixing of ferric Mb and mCPBA) were
performed to determine the rate of compound I formation. mCPBA was used as an oxidant because it was
better than H2O2 at generating compound I on the time scale of stopped-flow experiments. The reaction
of L29H/H64L Mb (10 µM) with mCPBA (250 µM) was performed in 50 mM sodium acetate buffer (pH
5.3) at 5°C. Since the F43H/H64L mutant (10 µM) does not require a large excess of mCPBA to generate
compound I, the mCPBA concentration was reduced to 100 µM. Based on the results of single mixing
experiments, the delay times, defined as the interval between the first and the second mixing, were set
as 10 and 0.3 s for L29H/H64L and F43H/H64L Mb, respectively. Sulfides (100 µM) or styrene (100
µM) were added to compound I by the second mixing after the appropriate delay time to collect spectral
changes.
The rates of compound I reduction for L29H/H64L Mb by thioanisole, 2,3-dihydrobenzothiophene,
and styrene were 5.9ꢀ0.1 s−1, 4.5ꢀ0.1 s−1, and 0.92ꢀ0.02s−1, respectively. The values for the
F43H/H64L mutant were 66ꢀ3 s−1, 44ꢀ1 s−1, and 44ꢀ2 s−1, respectively.
3.5. Enzymatic epoxidation
The wild type or mutants (10 µM) in 0.5 mL of 50 mM sodium phosphate buffer (pH 7.0) were
incubated with 0.5 µL of neat styrene, cis-, or trans-β-methylstyrene and 1 mM H2O2 at 25°C. A selected
internal standard was added, and the dichloromethane extracts were analyzed by GC equipped with a
Chiraldex G-TA capillary column at 80°C. The reaction time varies from 2 to 20 min, and the rates were
determined from the linear portion of the product versus time plot. Since the enzymes were inactivated
during the reaction, we normally observed up to 100 turnovers. The standard curve was prepared for
quantitative analysis, and the absolute stereochemistry was determined based on a retention time of the
authentic epoxide.
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