Effect of Solvent Polarity on Enantioselectivity in Candida Antarctica Lipase B Catalyzed Kinetic Resolution of Primary and Secondary Alcohols

Article abstract of DOI:10.1021/jo502521e

The Candida antarctica lipase B (CAL-B) catalyzed kinetic resolution of primary and secondary alcohols via acetylation is dependent on the permittivity (ε) of the reaction solvent. For example, the enantiomeric ratio (E) vs ε plot for the acetylation of 1-(naphth-2-yl)ethanol (1) exhibits a convex shape, taking the maximum E value at a medium ε value (11.2), whereas the same plot for the acetylation of benzyl 3-hydroxybutylate (3) exhibits a concave shape, taking the minimum E value at a similar ε value (11.6). Kinetic studies reveal that the difference in shape of the E vs ε plots originates from the relative reaction rate between the enantiomers with different Michaelis constants (K_m). Thus, when the enantiomer with a larger K_m value in the middle ε region reacts more slowly than its antipode, the ε dependence of E exhibits a convex shape. On the other hand, when the enantiomer reacts more quickly, it exhibits a concave shape. The E vs ε plot for the acetylation of 2-methoxy-2-phenylethanol (7) exhibits a convex shape with the maximum E value (20) at ε = 14.1. The E value can be further improved to almost reach the efficiency required for industrial applications (E ≈ 30) by the addition of a nitro compound.

Full text of DOI:10.1021/jo502521e

Article
pubs.acs.org/joc
Effect of Solvent Polarity on Enantioselectivity in Candida Antarctica
Lipase B Catalyzed Kinetic Resolution of Primary and Secondary
Alcohols
Yuichi Kitamoto,* Yosuke Kuruma, Kazumi Suzuki, and Tetsutaro Hattori*
Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, 6-6-11 Aramaki-Aoba, Aoba-ku,
Sendai 980-8579, Japan
*
S Supporting Information
ABSTRACT: The Candida antarctica lipase B (CAL-B)
catalyzed kinetic resolution of primary and secondary alcohols
via acetylation is dependent on the permittivity (ε) of the
reaction solvent. For example, the enantiomeric ratio (E) vs ε
plot for the acetylation of 1-(naphth-2-yl)ethanol (1) exhibits
a convex shape, taking the maximum E value at a medium ε
value (11.2), whereas the same plot for the acetylation of
benzyl 3-hydroxybutylate (3) exhibits a concave shape, taking
the minimum E value at a similar ε value (11.6). Kinetic
studies reveal that the difference in shape of the E vs ε plots
originates from the relative reaction rate between the
enantiomers with different Michaelis constants (K ). Thus, when the enantiomer with a larger Km value in the middle ε
m
region reacts more slowly than its antipode, the ε dependence of E exhibits a convex shape. On the other hand, when the
enantiomer reacts more quickly, it exhibits a concave shape. The E vs ε plot for the acetylation of 2-methoxy-2-phenylethanol (7)
exhibits a convex shape with the maximum E value (20) at ε = 14.1. The E value can be further improved to almost reach the
efficiency required for industrial applications (E ≈ 30) by the addition of a nitro compound.
9
INTRODUCTION
transition state. On the other hand, Cainelli and co-workers
pointed out the importance of solvent effects on substrate
molecules. They reported that in the Candida antarctica lipase
■
Enzymatic kinetic resolution is a method widely employed in
the drug industry, as well as in the laboratory, to optically
resolve a racemic compound by an enzyme-catalyzed reaction,
taking advantage of the difference in reaction rates between the
10
(
CAL) catalyzed acetylation of 1-(naphth-2-yl)ethanol (1),1
1
the ln E vs 1/T plot (herein, E denotes the enantiomeric ratio
1
,2
(eq 1)) comprises two half-segments with different slopes,
enantiomers. Resolution reactions have often been con-
ducted using enzymes suspended in organic solvents in order to
resolve water-insoluble compounds, suppress undesired side
reactions, and shift the thermodynamic equilibrium to
kcat,R/Km,R
E =
kcat,S/Km,S
3
products. For example, enantiopure carbocylic nucleosides,
ln[1 − c(1 + ee )]
ln[1 − c(1 − ee )]
ln[(1 − c)(1 − ee )]
ln[(1 − c)(1 + ee )]
p
which are key intermediates for the synthesis of antiviral agents,
were prepared by the pancreatin-catalyzed acetylation of their
=
p
3e
precursory alcohols. Synthesis of the C16−C20 segment of an
endogenous lipid mediator, resolvin E1, was achieved by the
lipase-catalyzed acetylation of its precursory secondary
s
=
s
3
f
v
alcohol. Dynamic kinetic resolution, which allows us to
obtain an enantiopure product quantitatively from a racemic
substrate by racemizing the substrate during kinetic resolution,
0,R
=
v0,S
(1)
4
has also been successfully conducted in organic solvents. In the
resolution reaction of alcohols, amines, and carboxylic acids via
acylation or transesterification, it is often the case that the
stereoselectivity varies depending on the solvent employed. In
some reports, the solvent effects are correlated with the polarity
or hydrophobicity of the solvents.
connecting at a point identified as an inversion temperature
(T ). Similar observations have been made in other enzymatic
and nonenzymatic reactions. Variable-temperature (VT)
NMR analysis of the substrate strongly suggested that the
breakpoint at Tinv represents a transition between two different
5
inv
12
6
7
−9
These reports assert that
solvents affect the enzymes or enzyme−substrate complexes by7
producing changes in the conformational rigidity of enzymes,
Received: November 4, 2014
8
invading the active site, or altering the solvation of the
©
XXXX American Chemical Society
A
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solute−solvent clusters, which serve as distinct supramolecules
with different reactivities and enantioselectivities. During the
optical resolution of racemic 1,1′-binaphthalene-2,2′-dicarbox-
ylic acid by crystallization after conversion into diastereomeric
amides with (S)-1-phenylethylamine, we recently found that
the deposited diastereomer can be switched by the permittivity
relationship with that of the whole system, because the initial
concentrations of solutes were kept constant throughout a
series of runs. The E value in the reaction of racemic alcohol
rac-1 was too large to be accurately determined from the
conversion (c) and the enantiomeric excess of either the
product (ee ) or substrate (ee ) (eq 1). Consequently, each
p
s
13
(ε) of the crystallization solvent, which provides an example
enantiomer was separately subjected to the acetylation, and the
E value was calculated as the ratio of the initial rate of the faster
reacting R isomer to that of the S isomer, v /v . Figure 1a
of dielectrically controlled resolution (DCR) proposed by
1
4,15
Sakai, Sakurai, and co-workers.
Mechanistic studies
0,R 0,S
revealed that this phenomenon was caused by ε-dependent
changes in the aggregation state of the amide molecules in
solution and the ease with which solvent molecules are
incorporated into the crystals. On the basis of our findings
combined with those of Cainelli’s group, we reasoned that
enzymatic kinetic resolution should be controllable by
principles similar to those of DCR. Herein, we report that
the enantioselectivity for the lipase-catalyzed kinetic resolution
of primary and secondary alcohols is controllable by altering
solvent polarity, using solvent permittivity as a measure
(Scheme 1).
Scheme 1. CAL-B Catalyzed Acetylation of Alcohols 1 and 3
Figure 1. Dependence of enantiomeric ratio (E) on solvent
permittivity (ε) for the CAL-B-catalyzed acetylation of (a) alcohol 1
and (b) alcohol 3.
shows the plot of E vs ε. Analogous to the temperature
10
dependence of ln E reported by Cainelli (vide supra), the plot
comprises two half-segments with different slopes, connecting
at a point with a medium ε value (ε = 11.2). The racemic
alcohol rac-3 was also acetylated under similar conditions
(
Scheme 1b); in this case, the E value could be calculated from
c and ee (eq 1). The E vs ε plot for alcohol 3 also comprises
p
two half-segments with different slopes, connecting at a point
with a medium ε value (ε = 11.6) (Figure 1b). These results
indicate that the enantioselectivity of enzymatic kinetic
resolution may be controlled by altering the polarity of the
reaction media.
RESULTS AND DISCUSSION
■
Acetylation of Alcohols 1 and 3 in Mixed Solvents
with Different Polarities. First, alcohol 1 was acetylated with
vinyl acetate in toluene−acetonitrile using CAL-B as a catalyst
1
(
Scheme 1a). It has been reported that CAL-B maintains a rigid
Kinetic Studies and H NMR Analysis. In order to clarify
16
conformation in organic solvents and its acyl transfer reaction
the origin of the ε dependence of the enantiomeric ratio, the
two reactions were examined further. The Michaelis constants
for the individual enantiomers, Km,R and Km,S, were determined
by Lineweaver−Burk plots in toluene−acetonitrile with differ-
follows Michaelis−Menten kinetics: in particular, the bibi ping-
1
7
pong mechanism. The polarity of the reaction solvent was
varied by changing the volumetric ratio of the two components
(
v/v), instead of employing a series of solvents with different ε
ent composition ratios. The K vs ε plot for alcohol 1 exhibited
m
values. Therefore, the structural effect of the solvents, e.g.
bulkiness and functional groups, on E can be reduced. The ε
a convex shape, taking the maximum value at the same ε value
as that of the node observed in the E vs ε plot (Figure 2a
compared with Figure 1a). For most ε ranges, the S enantiomer
exhibited a larger Km value than the R enantiomer. The
18
value of the mixed solvent, which was calculated as a weighted
19
average of the component ε values, was employed as a
measure of the polarity of the reaction system; although effects
of solutes on ε were not taken into account, the ε value
calculated from the solvent ε values should be in a linear
magnitude of change in K was also larger for the S enantiomer.
m
Similar observations were made for alcohol 3, except that the
K value and its magnitude of change were larger for the R
m
B
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Figure 2. Dependence of Michaelis constant (K ) on solvent
m
permittivity (ε) for the acetylation of (a) alcohol 1 and (b) alcohol
3
. The solid line with ● and broken line with ○ represent Michaelis
constants Km,S and Km,R, respectively.
enantiomer than for the S enantiomer (Figure 2b). On the
other hand, the ε-dependent change of maximum velocity, Vmax
,
was very small (Figure 3). This indicates that the catalytic
activity of the enzyme is almost constant regardless of the
solvent polarity. Therefore, since the enantiomeric ratio (E) is
expressed as the product of the ratio of catalytic constants,
kcat,R/kcat,S, and the ratio of Michaelis constants, Km.S/Km,R, the ε
dependence of E is mostly attributable to the latter quotient,
the difference in the ε dependence of Km between the
enantiomers. The K vs ε plot for each enantiomer of alcohols
m
1
and 3 exhibited a convex shape (Figure 2). This indicates
that, in a medium-permittivity solvent, the substrate molecules
do not easily diffuse into the substrate-binding pocket of the
enzyme.
Figure 3. Dependence of maximum velocity (Vmax) on solvent
permittivity (ε) for the CAL-B-catalyzed acetylation of (a) alcohol 1
and (b) alcohol 3.
We then analyzed the behavior of the alcohol molecules by
1
H NMR spectroscopy in toluene-d −acetonitrile-d3 with
8
varying compositions. The chemical shift vs ε plot for the
methyl protons of each racemic alcohol comprised two half-
segments with different slopes, connecting at a point with a
medium ε value (Figure 4). The ε values of the nodes (ε = 10.9
and 11.3 for alcohols 1 and 3, respectively) are in reasonable
are stabilized by inclusion into the pocket, which decreases the
K value. In a solvent with an ε value similar to that of the
m
node, alcohol molecules will have a polarity similar to that of
the solvent and be stabilized by solvation (Figure 5b). In such a
solvent, the polarity of the substrate-binding pocket will also be
similar to that of the solvent. Under these conditions, the
inclusion of the alcohol molecules into the pocket comes at the
expense of the stabilization energy of solvation. As a result, the
Km value becomes high. In a mixed solvent with an ε value
lower than that of the node, alcohol molecules are more polar
than the solvent and are associated with each other by
intermolecular hydrogen bonds (Figure 5c). In such a solvent,
the polarity of the substrate-binding pocket will also be higher
than that of the solvent. As a result, alcohol molecules are
stabilized by inclusion into the pocket, which decreases the Km
value. As two enantiomeric substrate molecules form
diastereomeric enzyme−substrate complexes, the magnitude
agreement with those of the nodes of the K vs ε plots (vide
m
supra). Similar ε-dependent changes were observed for the
other protons of alcohols 1 and 3 (Figure S1 in the Supporting
Information). It is conceivable that the two half-segments in the
δ vs ε plots correspond to different changes in the aggregation
state of the alcohol molecules.
Mechanistic Considerations. With these observations in
mind, a feasible mechanism for the ε-dependent convex change
of K is proposed as follows (Figure 5). In a solvent with an ε
m
value higher than that of the node, the alcohol molecules are
less polar than the solvent and are associated with each other by
hydrophobic interactions (Figure 5a). In such a solvent, the
polarity of the substrate-binding pocket of the enzyme will also
be lower than that of the solvent. As a result, alcohol molecules
C
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Figure 4. Changes in the chemical shifts of (a) alcohol 1 and (b)
alcohol 3 depending on solvent permittivity (ε) for the methyl
protons. Conditions: alcohol 1 (0.200 M) or alcohol 3 (0.141 M),
toluene-d −acetonitrile-d , 30 °C.
8
3
of ε-dependent change in Km may be different between the
enantiomers (Figure 2).
Interestingly, the ε dependence of enantiomeric ratio (E)
exhibited a convex shape for alcohol 1 (Figure 1a), whereas it
exhibited a concave shape for alcohol 3 (Figure 1b). As shown
in Figure 2, as the solvent permittivity approaches the ε value of
the node, it becomes difficult for the enantiomer with a larger
K value to form an enzyme−substrate complex to a greater
m
extent than its antipode. Therefore, the reaction rate of this
enantiomer is reduced by a larger magnitude in comparison to
that of its antipode, as evidenced by the initial rate v vs ε plot
0
(
Figure S2 in the Supporting Information). The relevant
Figure 5. Feasible mechanism for the ε dependence of K : the order
m
enantiomer is the S enantiomer for alcohol 1 and the R
enantiomer for alcohol 3, while the R enantiomer reacts more
quickly than the S enantiomer in both cases. Thus, we
of the polarities of the mixed solvent, the alcohol molecule, and the
substrate-binding pocket of the enzyme, the aggregation state of
molecules in solution, and the ease for alcohol molecules to be
included into the pocket in (a) high-, (b) medium-, and (c) low-
polarity solvents. The ellipsoids and blue and red circles represent
alcohol molecules and solvent molecules with low and high ε values,
respectively. The dotted lines indicate intermolecular hydrogen bonds
between alcohol molecules.
20
concluded that, when the enantiomer with a larger K value
m
reacts more slowly than its antipode in the middle ε region, the
ε dependence of E exhibits a convex shape. On the other hand,
when the enantiomer reacts more quickly, it exhibits a concave
shape.
Control of Enantioselectivity in Other Kinetic Reso-
lution Systems. The mechanistic considerations strongly
suggest that CAL-B-catalyzed acetylation exhibits the maximum
or minimum E value in a solvent with polarity similar to that of
the substrate regardless of the substrate and solvent system
employed, unless the solvent does not affect the reaction other
than by solvating the substrate. We then tested the applicability
of the present polarity-controlled method to other kinetic
resolution systems (Scheme 2). The E vs ε plot for the CAL-B-
catalyzed acetylation of alcohol 5 in toluene−acetonitrile
exhibited a concave shape (Figure 6a). On the other hand,
the acetylation of alcohol 7 conducted under the same
conditions exhibited a convex shape (Figure 6b). The latter
reaction was also carried out in 1,4-dioxane−acetonitrile, which
exhibited a slightly different change from that observed in
toluene−acetonitrile. This indicates that the enantiomeric ratio
(E) is not solely determined by the solvent permittivity but is
also affected by the structures of solvent molecules.
We reasoned that, when the E vs ε plot exhibits a convex
shape, the resolution efficiency at the node can be improved by
using an additive which increases K by retarding the diffusion
m
of a substrate into the binding pocket of the enzyme. We found
that nitro compounds are useful for this purpose (Table 1).
Interestingly, the maximum E value (20) obtained for alcohol 7
in toluene−acetonitrile (2/1, v/v) at ε = 14.1 (Figure 6b and
entry 1 in Table 1) increased to 28 with the addition of an
equimolar amount of 4-nitrophenyl N-hexylcarbamate (12)
(entry 5 in Table 1); the improved E value almost reaches the
1
,2
1
efficiency required for industrial applications. In the H NMR
D
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Scheme 2. CAL-B-Catalyzed Acetylation of Alcohols 5 and 7
Table 1. Addition Effect of Nitro Compounds in the CAL-B-
Catalyzed Acetylation of Alcohol 7
a
a
Conditions: rac-7 (0.700 mmol), vinyl acetate (1.40 mmol), CAL-B
(
10.0 mg), additive (0.700 mmol), toluene−acetonitrile (2/1, v/v; 5.0
b c
mL), 30 °C, 4 h. Determined by GC analysis. Calculated by ee c/(1
−
P
d
c). Calculated by ln[1 − c(1 + ee )]/ln[1 − c(1 − ee )].
P
P
decreases to a greater extent than that of the R enantiomer
to improve the E value.
CONCLUSION
■
In conclusion, we have shown that the enantioselectivity of
enzymatic kinetic resolution may be controlled by adjusting the
polarity of the reaction solvent, by using solvent permittivity as
a measure. The results presented in this paper clearly indicate
that the solvent permittivity is a useful parameter to optimize
the conditions for enzymatic kinetic resolution.
Figure 6. Dependence of enantiomeric ratio (E) on solvent
permittivity (ε) for the acetylation of (a) alcohol 5 in toluene−
acetonitrile and (b) alcohol 7 in toluene−acetonitrile (solid line with
EXPERIMENTAL SECTION
General Considerations. H NMR spectra were measured with
tetramethylsilane as an internal standard. Candida antarctica lipase B
CAL-B) powder was prepared by freeze-drying of Lypozyme CALB-L
■
(
1
●) and 1,4-dioxane−acetonitrile (broken line with ○).
−1
solution (Novozymes, Lot No. LCN02106, 5000 Units/g, 1.2 g mL ),
after dialysis with regenerated cellulose tubing (Spectra/Por 1 dialysis
tubing, MWCO 6000−8000 Da). The activity of the freeze-dried
spectrum, the hydroxy signal of alcohol 7 was shifted downfield
by the addition of compound 12, suggesting the formation of
intermolecular hydrogen bonds with compound 12 (Figure S3
5
−1
CAL-B powder was determined to be 1.9 × 10 Units g , following
2
1
22
the literature procedure. 1-(Naphth-2-yl)ethanol (1), benzyl 3-
2
3
24
in the Supporting Information). In addition, the initial rate (v )
hydroxybutyate (3), 1-phenyl-2-propanol (5), 2-methoxy-2-
25 26
0
−4
−1
−1
of the acetylation of alcohol 7 (7.98 × 10 M h (mg-Enz) )
phenylethanol (7), and 4-nitrophenyl N-hexylcarbamate (12)
−4
−1
−1
were prepared according to the literature procedures. Reaction
solvents and vinyl acetate were freshly distilled before use. Other
reagents were used as purchased.
Preparation of 1-Nitro-4-nonylbenzene (13). To a stirred
solution of nonylbenzene (d = 0.858; 1.0 mL, 5.71 mmol) in acetic
anhydride (8.0 mL) was added 69% nitric acid (d = 1.42; 256 μL, 5.71
mmol) dropwise over a period of 20 min at 0 °C, and the mixture was
stirred at room temperature for 2 h. The mixture was poured onto ice-
cold water and extracted with diethyl ether. The extract was washed
was diminished (6.03 × 10 M h (mg-Enz) ) by the
addition of compound 12. These observations indicate that the
nitro compound serves as a hydrogen-bond acceptor and forms
aggregates with the alcohol molecules, which retards the
formation of an enzyme−substrate complex. This should affect
the S enantiomer with a larger Km value to a greater extent
similarly to the solvent effects described above. As a result, the
reaction rate of the more slowly reacting S enantiomer
E
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successively with saturated aqueous NaHCO and water, dried over
3
AUTHOR INFORMATION
■
MgSO , and evaporated. The residue was purified by column
4
Corresponding Authors
*E-mail for Y.K.: kitamoto@orgsynth.che.tohoku.ac.jp.
*E-mail for T.H.: hattori@orgsynth.che.tohoku.ac.jp.
chromatography with hexane−ethyl acetate (3/1) as an eluent to
give nitro compound 13 (608 mg, 43%) as a pale yellow oil: IR (neat)
−1 1
2
926, 2855, 1601, 1519, 1346, 1110, 854, 747, 697 cm ; H NMR
(
(
2
2
400 MHz) δ 0.86 (t, 3H, J = 6.9 Hz), 1.26−1.32 (m, 12H), 1.60−1.68
Notes
m, 2H), 2.71 (t, 2H, J = 7.7 Hz), 7.32 (d, 2H, J = 8.8 Hz), 8.13 (d,
H, J = 8.8 Hz); 13C NMR (100 MHz) δ 14.2, 22.8, 29.3, 29.4, 29.5,
The authors declare no competing financial interest.
9.6, 31.1, 32.0, 36.0, 123.7, 129.3, 146.3, 151.0; HRMS (ESI) calcd
+
for C H NNaO (M + Na) 272.1626, found 272.1621.
ACKNOWLEDGMENTS
15
23
2
■
Typical Procedure for the Analysis of ε Dependence of
Enantiomeric Ratio (E) for CAL-B-Catalyzed Acetylation. To a
solution of alcohol (R)-1 (344 mg, 2.00 mmol) and vinyl acetate (d =
We wish to thank Novozymes Japan Ltd. of Japan for a
generous gift of Lypozyme CALB-L solution. We also wish to
thank Prof. S. Shoda, Assist. Prof. A. Kobayashi, Assist. Prof. M.
Noguchi, and Dr. M. Ishihara (Tohoku University) for
courteous permission to use their instruments and technical
guidance.
0
.93; 740 μL, 8.0 mmol) in toluene−acetonitrile (3/1, v/v (ε = 11.2),
2
.0 mL) in a 30 mL screw-cap vial was added CAL-B powder (15.0
mg), and the suspension was shaken at 30 °C. A small portion (20 μL)
of the mixture was taken out at 20 min intervals, quenched with 5%
(
w/v) aqueous trichloroacetic acid, and extracted with dichloro-
methane. The organic extract was dried over MgSO , concentrated,
4
REFERENCES
■
and submitted to GC analysis (column, Quadrex MPS-10 (0.32 mm
i.d. × 25 m); oven temperature, 150 °C; detector, FID) to determine
the conversion (c). The initial rate (v ) was determined to be 4.93 ×
(
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,R
−
3
−1
−1
1
0
M h (mg-Enz) from the slope of the c vs t plot at t = 0. The
acetylation of antipode (S)-1 was carried out by the same procedure,
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0
,S
−7
−1
−1
4
.03 × 10 M h (mg-Enz) . The E value was calculated from these
4
initial rates to be 1.22 × 10 at ε = 11.2. The E vs ε plot (Figure 1) was
obtained by repeating this procedure using toluene−acetonitrile
mixtures with varying composition ratios as reaction solvents.
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procedure similar to that mentioned above. Racemic alcohol was used
as a substrate, and the enantiomeric excess of the resulting ester (eep)
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(
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acetylation are essentially identical with those reported in the
literature.
(
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H, J = 6.3 Hz), 1.95 (s, 3H), 2.55 (dd, 1H, J = 15.5, 5.5 Hz), 2.68
−1 1
1
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(
1
6
dd, 1H, J = 15.5, 7.7 Hz), 5.13 (d, 1H, J = 15.0 Hz), 5.25−5.33 (m,
13
H), 7.32−7.37 (m, 5H); C NMR (100 MHz) δ 20.0, 21.2, 41.0,
6.6, 67.4, 128.4, 128.5, 128.7, 135.9, 170.2, 170.4; HRMS (FAB)
+
calcd for C H O (M + H) 237.1127, found 237.1126.
13
17
4
Typical Procedure for the Analysis of ε Dependence of
Michaelis Constant (K ) and Maximum Velocity (V ) for CAL-
m
max
B-Catalyzed Acetylation. The initial rate of the acetylation of
alcohol 1 was determined for each enantiomer by the same procedure
as that mentioned for the analysis of the ε dependence of E with
varying concentrations of alcohol 1 (1.0, 1.5, 2.0, and 2.5 M) in
toluene−acetonitrile (3/1, v/v (ε = 11.2)). Using the relation between
the concentration of alcohol 1 and the initial rate, K and V were
(4) (a) Pam
(b) Pallissier, H. Tetrahedron 2003, 59, 8291. (c) Martín-Matute, B.;
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ies, O.; Backvall, J.-E. Chem. Rev. 2003, 103, 3427.
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m
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determined by Lineweaver−Burk plots. The K vs ε plot (Figure 2)
m
and V
vs ε plot (Figure 3) were obtained by repeating this
max
procedure using toluene−acetonitrile mixtures with varying composi-
tion ratios as reaction solvents.
ASSOCIATED CONTENT
■
̂
Tetrahedron: Asymmetry 2004, 15, 3331. (f) Chenevert, R.; Pelchat, N.;
*
S
Supporting Information
Jacques, F. Curr. Org. Chem. 2006, 10, 1067. (g) Hydrolases in Organic
Synthesis, 2nd ed.; Bornscheuer, U. T., Kazlauskas. R. J., Eds.; Wiley-
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Biophys. Acta 1994, 1206, 1. (b) Carrea, G.; Ottolina, G.; Riva, S.
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1
of nitro compound 12, data for Figures 1−3 and 6, and H and
1
3
C spectra of the compounds synthesized. This material is
available free of charge via the Internet at http://pubs.acs.org.
F
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dx.doi.org/10.1021/jo502521e | J. Org. Chem. XXXX, XXX, XXX−XXX