Redesign of enzyme for improving catalytic activity and enantioselectivity toward poor substrates: Manipulation of the transition state

Article abstract of DOI:10.1039/c2ob25614b

Secondary alcohols having bulky substituents on both sides of the hydroxy group are inherently poor substrates for most lipases. In view of this weakness, we redesigned a Burkholderia cepacia lipase to create a variant with improved enzymatic characteristics. The I287F/I290A double mutant showed a high conversion and a high E value (>200) for a poor substrate for which the wild-type enzyme showed a low conversion and a low E value (5). This enhancement of catalytic activity and enantioselectivity of the variant resulted from the cooperative action of two mutations: Phe287 contributed to both enhancement of the (R)-enantiomer reactivity and suppression of the (S)-enantiomer reactivity, while Ala290 created a space to facilitate the acylation of the (R)-enantiomer. The kinetic constants indicated that the mutations effectively altered the transition state. Substrate mapping analysis strongly suggested that the CH/π interaction partly enhanced the (R)-enantiomer reactivity, the estimated energy of the CH/π interaction being -0.4 kcal mol^-1. The substrate scope of the I287F/I290A double mutant was broad. This biocatalyst was useful for the dynamic kinetic resolution of a variety of bulky secondary alcohols for which the wild-type enzyme shows little or no activity. The Royal Society of Chemistry 2012.

Full text of DOI:10.1039/c2ob25614b

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Volume 10 | Number 31 | 21 August 2012 | Pages 6233–6444
ISSN 1477-0520
PAPER
Tadashi Ema et al.
Redesign of enzyme for improving catalytic activity and enantioselectivity
toward poor substrates: manipulation of the transition state
1477-0520(2012)10:31;1-D

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PAPER
Redesign of enzyme for improving catalytic activity and enantioselectivity
toward poor substrates: manipulation of the transition state†
Tadashi Ema,* Yasuko Nakano, Daiki Yoshida, Shusuke Kamata and Takashi Sakai
Received 24th March 2012, Accepted 30th May 2012
DOI: 10.1039/c2ob25614b
Secondary alcohols having bulky substituents on both sides of the hydroxy group are inherently poor
substrates for most lipases. In view of this weakness, we redesigned a Burkholderia cepacia lipase to
create a variant with improved enzymatic characteristics. The I287F/I290A double mutant showed a high
conversion and a high E value (>200) for a poor substrate for which the wild-type enzyme showed a low
conversion and a low E value (5). This enhancement of catalytic activity and enantioselectivity of the
variant resulted from the cooperative action of two mutations: Phe287 contributed to both enhancement
of the (R)-enantiomer reactivity and suppression of the (S)-enantiomer reactivity, while Ala290 created a
space to facilitate the acylation of the (R)-enantiomer. The kinetic constants indicated that the mutations
effectively altered the transition state. Substrate mapping analysis strongly suggested that the CH/π
interaction partly enhanced the (R)-enantiomer reactivity, the estimated energy of the CH/π interaction
being −0.4 kcal mol−1. The substrate scope of the I287F/I290A double mutant was broad. This
biocatalyst was useful for the dynamic kinetic resolution of a variety of bulky secondary alcohols
for which the wild-type enzyme shows little or no activity.
knowledge of the enzyme structure and reaction mechanism.5–7
In contrast, once the reaction mechanism has become clear,
Introduction
Enzymes are powerful biocatalysts that can accelerate chemical
reactions enormously.1 The kcat values, known as turnover
numbers, range from 102 to 106 s−1 for natural substrates.2,3
Considerable stabilization of transition states accounts for such
high turnover numbers.3 In contrast, enzymatic reactions for
unnatural substrates are much slower, and in some cases they are
poor substrates with little or no reactivity. If alteration of the
enzyme structure can stabilize the transition state, such a poor
substrate may become a good substrate. Another important
aspect of enzymatic reactions is high enantioselectivity for
natural substrates. However, enzymatic reactions do not necess-
arily attain high enantioselectivity for unnatural substrates. The
most straightforward way of solving this problem is to create a
mutant enzyme with an improved catalytic function.4
rational design, which uses site-directed mutagenesis, is also
useful.8–11 Comparison of the random and rational approaches
reveals that the latter seems to be more difficult and inefficient
than the former, and therefore directed evolution has recently
become the most popular method. Nevertheless, rational
approaches are also becoming more attractive as a mechanistic
understanding of biocatalysis is enhanced.8–11
Lipases are synthetically useful biocatalysts that can show
high enantioselectivity and broad substrate specificity in both
aqueous and nonaqueous media.1 However, the kinetic resolu-
tion of secondary alcohols bearing bulky substituents on both
sides of the hydroxy group remains difficult. Alteration of the
enzyme structure may overcome this drawback. We have pre-
viously used mechanistic knowledge to successfully control
(both increase and decrease) the enantioselectivity of a Burkhol-
deria cepacia lipase with a single mutation.10a In that study, we
controlled enantioselectivity by modulation of steric repulsion
between the enzyme and the slower-reacting (S)-enantiomer.
More recently, we have created a variant that enhances the
reactivity of the (R)-enantiomer and that suppresses the reactivity
of the (S)-enantiomer.10b Here we report in detail the rational
creation of mutant lipases that display remarkably enhanced
catalytic activity and enantioselectivity for poor substrates
bearing bulky substituents on both sides of the hydroxy group.
Among several variants, the I287F/I290A double mutant was the
best biocatalyst, being useful not only for the kinetic resolution
There are two major ways of creating variants: directed
evolution and rational design. Directed evolution, which uses
random mutagenesis and high-throughput screening of a large
number of variants, can improve an enzymatic property without
Division of Chemistry and Biochemistry, Graduate School of Natural
Science and Technology, Okayama University, Tsushima, Okayama
700-8530, Japan. E-mail: ema@cc.okayama-u.ac.jp; Fax: +81-86-251-
8092; Tel: +81-86-251-8091
†Electronic supplementary information (ESI) available: Site-directed
mutagenesis, synthesis of 1, lipase-catalyzed reactions of 1, and copies
of NMR spectra. See DOI: 10.1039/c2ob25614b
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but also for the dynamic kinetic resolution (DKR) of bulky sub-
strates for which the wild-type enzyme showed little or no
activity. The kinetic study demonstrated that the mutations effec-
tively manipulated the transition state of the enzymatic reaction.
Results and discussion
Theoretical basis and working hypothesis
We have proposed a transition-state model to explain the enantio-
selectivity of lipases for secondary alcohols (Fig. 1a shows an
expanded version).12 Enantioselectivity results principally from
the conformational requirements and repulsive interactions in the
transition state, and no attractive interactions between the
enzyme’s pockets and the substrate’s substituents are involved.
In other words, the (R)-preference of lipases results from a
suppression mechanism working on the (S)-enantiomer in the
transition state. This mechanism has been supported by kinetic
and thermodynamic analyses,12a,c and the use of a large
secondary alcohol, 5-[4-(1-hydroxyethyl)phenyl]-10,15,20-
triphenylporphyrin.12b
Improvement of the catalytic activity and enantioselectivity of
an enzyme for a poor substrate may be achieved by a scenario
that involves careful alteration of the enzyme structure (Fig. 2).
Removal of steric hindrance or introduction of attractive inter-
actions may stabilize the (R)-enantiomer in the transition state,
while introduction of steric hindrance may destabilize the
(S)-enantiomer in the transition state. As the mutation sites, we
selected the following three amino acid residues, all of which
are in proximity to the catalytic residues: Ile287, Ile290, and
Gln292 (Fig. 1a). Although His86 was also a candidate for
mutation, we found this amino acid residue to be crucial for
enzymatic activity as predicted previously (data not shown).12a
We therefore left His86 unchanged in this study.
We employed 1-phenyl-1-hexanol (1a) as a poor substrate to
perform a docking experiment with respect to the transition-state
model (Fig. 1a). The wild-type enzyme has Ile287, which
appears to conflict with the alkyl chain of (R)-1a (Fig. 1b). We
hypothesized that the I287F mutation might realize the strategy
shown in Fig. 2 if Phe287 had the following two roles in the
transition state: an attractive interaction with (R)-1a and a
repulsive interaction with (S)-1a. More specifically, Phe287
appears to make favorable contact with the alkyl chain of (R)-1a
(Fig. 1c), which would accelerate the acylation of (R)-1a, while
Phe287 is likely to come into unfavorable contact with the
phenyl group of (S)-1a, which would hinder the acylation of
(S)-1a (not shown).10a On the other hand, Ile290 is the second
mutation site, which also seems to conflict with the alkyl chain
of (R)-1a (Fig. 1c). We hypothesized that the I290A mutation
would create a space to accommodate (R)-1a nicely in the tran-
sition state (Fig. 1d), leading to higher catalytic activity. Gln292
is the mutation site farthest from the active site. We hypothesized
that the Q292A mutation would also create a larger space for
(R)-1a (Fig. 1e).
Fig. 1 (a) The transition-state model to rationalize the enantioselectivity
in the lipase-catalyzed kinetic resolution of secondary alcohols (residues
287, 290, and 292 are added to the original version). (i) The C–O bond
of the substrate takes the gauche conformation with respect to the break-
ing C–O bond, which is due to the stereoelectronic effect. (ii) The
hydrogen atom attached to the stereocenter in the substrate is syn-
oriented toward the carbonyl oxygen atom to minimize the torsional
strain. Enantioselectivity is explained by the conformational require-
ments and repulsive interactions and/or strains caused in the transition
state. The catalytic triad residues, the ester being produced, residue 287,
residue 290, and residue 292 are shown in green, blue, red, magenta,
and orange, respectively. Typically, the (R)-enantiomer reacts faster
because, in this favorable conformation shown in blue, the larger substi-
tuent (R1) can be directed toward external solvent without severe strain
and/or steric hindrance. (b)–(e) The active sites of (b) the wild-type
enzyme, (c) the I287F mutant, (d) the I287F/I290A double mutant, and
(e) the I287F/I290A/Q292A triple mutant. (R)-1a connected to Ser87 in
the tetrahedral intermediate is shown in light blue. Gln292 is omitted for
clarity in (b)–(d).
(Scheme 1) and evaluated enantioselectivity on the basis of
E values.13 The results are summarized in Table 1. Because of
the low activity of the wild-type enzyme for 1a, high catalyst
loading and a long reaction time were required (entry 1). To our
delight, the conversion and E value of the I287F mutant for 1a
were twice and six times, respectively, the corresponding values
of the wild-type enzyme (entry 2). Homolog 1b exhibited the
same trend (entries 10 and 11). In contrast, the I287A, I287W,
and I287Y mutations resulted in lower conversions of 1a (entries
3–5). These results suggest that only the I287F mutant had a
Rational creation of mutant enzymes
We conducted the recombinant lipase-catalyzed kinetic resolu-
tions of 1a and 1b with vinyl acetate in dry i-Pr2O at 30 °C
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Table 1 Kinetic resolution of 1 with the wild-type and mutant
enzymesa
Entry
1
Lipase
Time (h)
cb (%)
Ec
1
2
3
4
5
6
7
8
1a
1a
1a
1a
1a
1a
1a
1a
1a
1b
1b
1b
1c
1c
1c
1d
1e
1e
1e
Wild-type
I287F
I287A
I287W
I287Y
41
41
41
41
41
2.5
41
41
6
41
22
4
41
22
4
23
46
20
17
10
50
10
41
45
39
47
47
34
47
50
19
45
42
43
5
32
1.4
5
13
>200
4
79
52
9
71
>200
14
55
>200
10
5
16
I287F/I290A
I287F/I290F
I290A
I287F/I290A/Q292A
Wild-type
I287F
I287F/I290A
Wild-type
I287F
I287F/I290A
I287F/I290A
Wild-type
I287F
9
Fig. 2 (a) Energy diagram for an enantioselective enzymatic reaction
toward a poor substrate. (b) Improvement of catalytic activity and enantio-
selectivity by stabilization of the (R)-enantiomer and destabilization of
the (S)-enantiomer in the transition state.
10
11
12
13
14
15
16
17
18
19
75
41
41
3
I287F/I290A
>200
a Conditions: immobilized lipase (700 mg, 0.5% (w/w) enzyme/
Toyonite-200M), 1 (0.50 mmol), vinyl acetate (1.0 mmol), molecular
sieves 3A (three pieces), dry i-Pr2O (5 mL), 30 °C. b Conversion
calculated from c = ee(1)/(ee(1) + ee(2)). c Calculated from E = ln[1 − c
(1 + ee(2))]/ln[1 − c(1 − ee(2))].
entries 6 and 12). In both cases the conversions reached almost
50% within a few hours, and the E values exceeded 200. These
results, which were beyond our expectation, strongly suggest that
the effects of the double mutations were cooperative. In sharp
contrast with the I287F/I290A double mutant, the I287F/I290F
double mutant showed very poor activity and enantioselectivity
for 1a (entry 7). The I290A mutation had a positive effect on
catalytic activity and enantioselectivity (entry 8). These results
strongly suggest that the second mutation (I290A) enhanced the
reactivity of (R)-1a and (R)-1b by eliminating steric hindrance.
We next tested a third-generation biocatalyst, the I287F/
I290A/Q292A triple mutant. We unexpectedly found this variant
to be less active and enantioselective than the I287F/I290A
double mutant (entry 9). This result, however, suggests an
important role of Gln292, which is located at the outer side of
Phe287 and Ala290 (Fig. 1a). The Q292A mutation, which
locally creates a space to facilitate the acylation of (R)-1a
(Fig. 1e), may bring about fluctuation of the adjacent Phe287,
which may decrease catalytic activity and enantioselectivity. Of
all the variants that we prepared (Table 1), the I287F/I290A
double mutant was the best biocatalyst for the bulky substrates
examined.
Scheme 1 Lipase-catalyzed kinetic resolution of 1. Only 2e has the
(S)-configuration according to the Cahn–Ingold–Prelog (CIP) priority
system for nomenclature.
The total turnover number (TTN) and the turnover frequency
(TOF) provide a rough estimate of the extent of catalytic activity.
TTN is defined as the number of substrate molecules converted
by one enzyme molecule, while TOF is the turnover number per
unit time. These values can be calculated from the data in
Table 1. For example, the TTN of the wild-type enzyme for 1a
specific mechanism to enhance the reactivity of (R)-1a, such as
an attractive interaction between Phe287 and the alkyl chain of
(R)-1a, which was supported by kinetic measurements and sub-
strate mapping analysis (vide infra). Steric bulkiness of Trp287
and Tyr287 seems to hinder the acylation, although we expected
the same enhancement effect as that produced by Phe287.
We next examined a second-generation biocatalyst, the I287F/
I290A double mutant. Surprisingly, this variant exhibited much
higher activity and enantioselectivity for 1a and 1b (Table 1,
(entry 1) is 1080 in 41 h, and the TOF is 26 h−1 14
In contrast,
.
the TTN of the I287F/I290A double mutant for 1a (entry 6) is
2350 in 2.5 h, and the TOF is 940 h−1. Clearly, the double
mutation has remarkably improved enzymatic activity for this
substrate, although the values of the I287F/I290A double mutant
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for 1a are still smaller than the corresponding values of the wild-
type enzyme for an inherently good substrate, 1-phenylethanol
(TTN = 7800 in 4.5 h; TOF = 1700 h−1).10a
transition state, while the Km value is related to the substrate-
binding step. Fig. 4 indicates that enantioselectivity originated
from the difference in Vmax values (in other words, from differ-
ences in the transition state), as demonstrated previously.12a,c
Importantly, the mutations caused the following changes in the
Vmax values: the I287F mutant had a Vmax value for (R)-1a that
was approximately three times that of the wild-type enzyme,
whereas the I287F mutant had a Vmax value for (S)-1a that was
half that of the wild-type enzyme. These results clearly support
our hypothesis concerning the two roles of Phe287: enhance-
ment of the reactivity of (R)-1a and suppression of the reactivity
of (S)-1a. Furthermore, the I287F/I290A double mutant had a
Vmax value for (R)-1a that was approximately five times that of
the I287F mutant. Obviously the second mutation (I290A)
enhanced the reactivity of (R)-1a. These kinetic constants are
consistent with our expectation that the mutations would
successfully manipulate the transition state.
Kinetic measurements
We measured time courses to compare the relative activities of
recombinant lipases for each enantiomer of 1a. A mixture of
enantiomerically pure alcohol 1a (50 mM), immobilized lipase
(300 mg), and molecular sieves 3A (one piece) in dry i-Pr2O
(1.0 mL) was stirred at 30 °C for 30 min. We started the reaction
by addition of vinyl acetate (1.0 M). At appropriate time inter-
vals, we withdrew aliquots (10 μL) and added them to EtOAc
(0.5 mL). After centrifuging the diluted solution, we filtered
the supernatant through a syringe filter (pore size 0.45 μm). The
filtrate was then analyzed by gas chromatography to determine
the conversion rates (calibrated). The time courses are shown in
Fig. 3. The use of enantiomerically pure alcohol 1a was helpful
for understanding the effect of each mutation on the enzymatic
activity. Fig. 3 clearly showed the relative activity of the recom-
binant lipases for 1a because all the reactions were conducted
under the same conditions. The I287F mutant showed higher
activity for (R)-1a and lower activity for (S)-1a than the wild-
type enzyme. Furthermore, the I287F/I290A double mutant
exhibited much higher activity for (R)-1a than the wild-type
enzyme or the I287F mutant. These results are consistent with
our working hypothesis described above.
Attractive interaction in the transition state
We considered the possibility of a CH/π interaction as an attrac-
tive interaction between the phenyl group of Phe287 and the
alkyl chain of (R)-1a (Fig. 1c and d).15–17 We decided to use
substrate mapping analysis to specify and characterize the attrac-
tive interaction. Alcohols 1c–e (Scheme 1) were selected
To investigate the mutational effect more quantitatively, we
used previously determined kinetic constants (Fig. 4).10b This
reaction is heterogeneous because the immobilized enzyme
powder is insoluble. The Vmax value is normalized by the weight
of the enzyme powder and corresponds to the kcat value in a
homogeneous system. The Vmax value is therefore related to the
Fig. 3 Time courses of the lipase-catalyzed acylations of 1a. The same
data are plotted in (a) and (b), but the scales of the x- and y-axes are
adjusted to clarify the difference in the reaction rate. Conditions:
immobilized lipase (300 mg, 0.5% (w/w) enzyme/Toyonite-200M), 1a
(0.050 mmol), vinyl acetate (1.0 mmol), molecular sieves 3A (one
piece), dry i-Pr2O (1.0 mL), 30 °C. Orange square: wild-type enzyme
toward (R)-1a; red diamond: I287F mutant toward (R)-1a; magenta
circle: I287F/I290A double mutant toward (R)-1a; black square: wild-
type enzyme toward (S)-1a; blue diamond: I287F mutant toward (S)-1a;
green circle: I287F/I290A double mutant toward (S)-1a.
Fig. 4 Kinetic constants for the lipase-catalyzed acylations of 1a.
(a) Vmax values. (b) Km values. Because of the heterogeneous reaction,
the nonlinear least-squares method was applied to the Michaelis–Menten
type of equation: v0 = Vmax(E)mg[S]0/(Km + [S]0), where Vmax is normal-
ized by the weight of the immobilized enzyme powder (E)mg
.
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because they are almost isosteric to 1a and because a CH/π inter-
action cannot take place at the fluorine or oxygen atoms in 1c–e.
As shown in Table 1, the enzymatic activity and enantioselec-
tivity for 1c decreased in the following order: the I287F/I290A
double mutant (entry 15) > the I287F mutant (entry 14) > the
wild-type enzyme (entry 13). This trend is quite similar to that
for 1a, which rules out the possibility that the terminal methyl
group of 1a participated in attractive interactions. In sharp con-
trast, the fact that the I287F/I290A double mutant showed poor
activity and enantioselectivity for 1d (entry 16) strongly suggests
that the fluorinated methylene moiety of (R)-1d was subject to
severe steric repulsion (Fig. 5b). The conformation shown
in Fig. 1c suggests that the hydrogen atoms at the C5 and C3
positions of (R)-1a participate in the CH/π interaction with the
phenyl group of Phe287 (Fig. 5a).
On the basis of this model (Fig. 5a), we considered that 1e
could be used to quantify the energy of the CH/π interaction
stabilizing (R)-1a in the transition state. Because (S)-1e cannot
participate in a CH/π interaction (Fig. 5c), the increased activity
and enantioselectivity of the enzyme variant for 1a compared to
1e can be ascribed to the additional CH/π interactions. The
results of the lipase-catalyzed kinetic resolution of 1e shown in
Table 1 (entries 17–19) reveal that the I287F mutant converted
1e a little more slowly than the wild-type enzyme (entries 17
and 18), whereas the I287F/I290A double mutant converted 1e
much faster than the wild-type enzyme (entries 17 and 19). This
strongly suggests that the I290A mutation but not the I287F
mutation enhanced the catalytic activity of the I287F/I290A
double mutant toward (S)-1e by reducing steric hindrance. The
E value for the I287F mutant toward 1e was ∼3 times that for
the wild-type enzyme (entries 17 and 18), whereas that for the
I287F/I290A double mutant toward 1e was more than 40 times
that for the wild-type enzyme (entries 17 and 19). Although 1a
and 1e exhibited a similar trend, a clear difference between 1a
and 1e was apparent in the I287F mutation: the conversion and
E value of the I287F mutant for 1a were twice and six times,
respectively, the corresponding values of the wild-type enzyme
(entries 1 and 2), whereas the conversion and E value of the
I287F mutant for 1e were comparable and three times, respecti-
vely, the corresponding values of the wild-type enzyme (entries
17 and 18). On the basis of the E values for the wild-type and
I287F mutant enzymes toward 1a and 1e, we estimated the free
energy of the CH/π interaction to be −0.4 kcal mol−1 according
to the following equation: Δ1a–1eΔF–IΔR–SΔG‡ = −RTln(EF/EI)1a
+ RTln(EF/EI)1e = −RTln[(32/5)/(16/5)].18
This value (−0.4 kcal mol−1) is reasonable when compared
with the data in the literature.15–18 For example, Hunter and co-
workers have determined the energy of the CH/π interaction in
synthetic host–guest complexes to range from −0.3 to −1.1 kcal
mol−1 18a,b
depending on aromatic–aromatic combinations.
,
Shimizu and co-workers have used molecular balances to deter-
mine that the energy of the Me–arene CH/π interaction is
−0.95 kcal mol−1 16f
.
Fersht and co-workers have reported that
the energetic contribution of the Tyr–Tyr CH/π interaction to
protein stabilization is −1.3 kcal mol−1 18c
The energy of the
CH/π interaction between carbohydrate and the aromatic side
chain of a protein has been determined to be −0.8 kcal mol−1 18d
.
.
Substrate scope of the I287F/I290A double mutant
We next investigated the substrate scope of the I287F/I290A
double mutant (Scheme 1). The results of the kinetic resolutions
of 1f–q are summarized in Table 2. Clearly, this variant showed
high activity and enantioselectivity for various bulky alcohols.
For example, alcohols 1f and 1i, which contain an olefin and a
branched chain, respectively, in the aliphatic substituent, reacted
smoothly with high E values (entries 1 and 4). Although the
reaction of 1g, bearing a methoxymethyl ether group at the ter-
minus of the alkyl group, took a long time, the E value was high
(entry 2). In contrast, the acylation of 1h, which has a phenyl
group at the terminus of the alkyl group, proceeded rapidly with
a high E value (entry 3). The fact that the reactivity of 1j was
only modest (entry 5) was probably due to a decrease of the
nucleophilicity of the hydroxy group that was caused by the tri-
fluoromethyl group or due to the steric bulkiness of the trifluoro-
methyl group, whereas 1k and 1l, both of which contain an ether
group in the aromatic substituent, were very reactive (entries 6
and 7). Alcohol 1m, which contains a naphthyl group, also
showed good reactivity (entry 8). Interestingly, the reaction of
1n, with a shorter alkyl chain, took more time (entry 9). The
TTN values for the I287F/I290A double mutant toward 1f–n
(entries 1–9) reached a practical level, 5770–7590.14 In sharp
Fig. 5 Proposed interactions in the transition state of the I287F/I290A
double mutant-catalyzed reactions. (a) The CH/π interactions for (R)-1a.
(b) The steric repulsion for (R)-1d. (c) No attractive interactions for
(S)-1e (absolute configuration based on the CIP priority system for
nomenclature).
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Table 2 Substrate scope of the I287F/I290A double mutant and wild-type enzymea
I287F/I290A
cb (%)
Wild-type
Entry
1
Substrate 1
Time (h)
7
Ec
cd (%)
Ec
39
>200
0
—
2
3
22
46
41
143
6
3
—
—
6.5
>200
4
5
9
35
45
147
0
4
—
—
54
>200
6
7
41
>200
2
—
7
7
7
42
41
41
42
36
50
>200
>200
199
3
0
—
—
8
9
24
50
50
3
5
—
10
11
12
>200
>200
>200
18b
38b
45b
19
113
68
a Conditions: immobilized lipase (200 mg, 0.5% (w/w) enzyme/Toyonite-200M), 1 (0.50 mmol), vinyl acetate (1.0 mmol), molecular sieves 3A (three
pieces), dry i-Pr2O (5 mL), 30 °C. b Conversion calculated from c = ee(1)/(ee(1) + ee(2)). c Calculated from E = ln[1 − c(1 + ee(2))]/ln[1 − c(1 − ee
(2))]. d Conversion calculated from 1H NMR.
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contrast, the conversions of these bulky alcohols 1f–n were
much lower (<6%) when catalyzed by the wild-type enzyme.
The reactivity of 1n–q for the wild-type enzyme decreased in
the following order: 1q > 1p > 1o > 1n (Table 2, entries 9–12).
This result is quite reasonable because steric repulsion between
the alkyl substituent of (R)-1 and the wild-type enzyme
decreases in the following order: 1n > 1o > 1p > 1q (Fig. 1a). In
contrast, the reactivity of 1n–q for the I287F/I290A double
mutant varied as follows: 1q > 1n > 1o > 1p (entries 9–12). The
reactivity of 1p was much lower than that of 1q (entries 11 and
12) because of steric hindrance around the ethyl group of (R)-1p
(Fig. 1a). Further elongation of the alkyl chain of 1 led to an
acceleration of the reaction catalyzed by the I287F/I290A double
mutant (entries 9 and 10), a result that can be explained by an
attractive interaction as described above. The I287F/I290A
double mutant exhibited higher E values for 1n–q than the wild-
type enzyme (entries 9–12), which was partly due to the I287F
mutation. Because phenylalanine is more bulky than isoleucine,
the former hindered the acylation of (S)-1n–q more efficiently.
Hult and co-workers have created an (S)-selective variant of
Candida antarctica lipase B that shows activity for bulky
alcohols such as 1a.9f In that study they examined only simple
secondary alcohols having phenyl or alkyl groups, and their
variant showed low enantioselectivity for 1q. They proposed that
their variant has a cavity to accommodate the (unsubstituted)
phenyl group, which is responsible for the inversion of the enantio-
preference. In contrast, the results in Table 2 demonstrate a
wide range of applicability of the I287F/I290A double mutant.
To the best of our knowledge, this is the first example of the suc-
cessful enzyme-catalyzed kinetic resolution of bulky alcohols
1f–m.
Scheme 2 Dynamic kinetic resolution of 1 using the I287F/I290A
double mutant and Ru catalyst 3.
(69–88%) with high enantiomeric purities (95–98% ee). In the
DKR of 1g and 1k, we obtained considerable amounts of the
corresponding ketones, which decreased the yields of 2g and 2k
(entries 6 and 9). Bulky alcohols having an ether group (1e, 1g,
and 1k), except for 1l, reacted sluggishly. Even when the reac-
tion required a very long time, TLC indicated that the reaction
proceeded gradually, an indication that both the biocatalyst and
the racemization catalyst were active for a long time. In contrast
to the DKR of the bulky substrates, which required a larger
amount of 3 (10 mol%) and longer reaction times (72–145 h)
(entries 1–11), the DKR of the least bulky substrate 1q with a
smaller amount of 3 (4 mol%) was completed within 24 h
without producing the corresponding ketone (entry 12). The
TTN values for the I287F/I290A double mutant toward 1
reached 9070–14500 (entries 1–12).14 These results clearly
demonstrate that this variant is robust and can be used in the
DKR of a wide range of bulky secondary alcohols.
Dynamic kinetic resolution
DKR is a fascinating technology because a racemic substrate
can be converted into an optically pure product in 100% yield
at most.19–23 Among various racemization catalysts reported so
far, chlorodicarbonyl(1,2,3,4,5-pentaphenylcyclopentadienyl)-
ruthenium(II) (3) is highly active and commercially available.20
Although most DKRs with native lipases are successful only for
secondary alcohols with a small substituent at the stereocenter,
Bäckvall and co-workers have recently reported the DKR
of bulky secondary alcohols by using an (S)-selective variant of
Candida antarctica lipase B.20j We expected the I287F/I290A
double mutant to be an excellent biocatalyst for the (R)-selective
DKR of bulky secondary alcohols.
We conducted the DKR of 1 with the I287F/I290A double
mutant, isopropenyl acetate, Ru complex 3, KOt-Bu, and
Na2CO3 in dry i-Pr2O at 30 °C (Scheme 2). During optimization
of the reaction conditions for 1a, we observed the formation of a
considerable amount of the corresponding ketone (data not
shown), which was released from the catalytic cycle of the
Ru-catalyzed racemization, probably because of the bulkiness of
1a.20a–c,j The results obtained under optimized conditions are
shown in Table 3. The simple bulky alcohols 1a, 1b, 1h, 1i, and
1m and functionalized bulky alcohols 1c and 1f, which have
trifluoromethyl and alkenyl groups, respectively, were converted
successfully into the corresponding (R)-esters in good yields
Conclusions
Improvement of the catalytic function of enzymes is important
from scientific and industrial viewpoints. Despite remarkable
advances in enzyme science and technology, rational control of
the enzymatic function remains difficult because of the complex-
ity of reaction mechanisms. The fact that secondary alcohols
having bulky substituents on both sides of the hydroxy group are
inherently poor substrates for most lipases has been an unre-
solved weakness that has limited the usefulness of the biocata-
lysts. In this study the synergic effects of only two mutations
(I287F/I290A) dramatically enhanced both catalytic activity and
enantioselectivity for poor substrates. Phe287 contributed to
both enhancement of the reactivity of the (R)-enantiomer and
suppression of the reactivity of the (S)-enantiomer, and
Ala290 made space to facilitate the acylation of the (R)-enantio-
mer. Kinetic constants indicated that the mutations effectively
manipulated the transition state. Substrate mapping analysis
strongly suggested that the reactivity of the (R)-enantiomer was
partly enhanced by a CH/π interaction, of which the estimated
energy was −0.4 kcal mol−1. To the best of our knowledge, this
is the first example of an introduction of a CH/π interaction in
the transition state to promote a more enantioselective enzymatic
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Table 3 Dynamic kinetic resolution of 1 with I287F/I290A double
mutant and 3a
reaction. The substrate scope of the I287F/I290A double mutant
was broad, and we were able to use this variant in the DKR of
various bulky secondary alcohols for which the wild-type
enzyme showed little or no activity. The I287F/I290A double
mutant achieved a TTN up to 14500. We expect that this variant
will be useful for the synthesis of chiral intermediates that
cannot be prepared with other lipases. Although the structures of
the variants that we have prepared and presented here have not
yet been fully optimized, for example, by directed evolution, the
impact of the mutations was nevertheless great. The present
results clearly demonstrate the efficiency and power of rational
design for the creation of excellent biocatalysts.
Entry Product (R)-2
% Yieldb (% ee) Ketone
1
88 (95)
69 (97)
87 (98)
70 (95)c
88 (95)
55 (96)
87 (95)
2
9
2
3
4
5
6
7
2
Experimental
6
General
1H and 13C NMR spectra were measured on Varian 600 or
400 MHz NMR spectrometers. IR spectra were recorded on a
Shimadzu FTIR-8900 spectrometer. Silica gel column chromato-
graphy was performed using Fuji Silysia BW-127 ZH
(100–270 mesh), and thin layer chromatography (TLC) was per-
1
formed on Merck silica gel 60 F254
.
29
9
General procedure for dynamic kinetic resolution
Immobilized lipase (0.5% (w/w) enzyme/Toyonite-200M) was
dried under reduced pressure overnight. A solution of KOt-Bu
(0.5 M in THF, 250 μL) was added to a Schlenk flask, and THF
was removed under reduced pressure. Immobilized lipase
(400 mg), Ru catalyst 3 (chlorodicarbonyl(1,2,3,4,5-pentaphenyl-
cyclopentadienyl)ruthenium(II)) (63.8 mg, 0.100 mmol),
Na2CO3 (106 mg, 1.00 mmol), and molecular sieves 4A
(20 pieces) were added to the flask. The flask was evacuated and
filled with Ar. Dry i-Pr2O (2 mL) was added, and the mixture
was stirred at 30 °C for 15 min. Alcohol 1 (1.0 mmol) was
added, and the mixture was stirred for 15 min. The reaction was
started by addition of isopropenyl acetate (165 μL, 1.5 mmol).
The mixture was stirred at 30 °C for 24–145 h. The reaction was
stopped by filtration, and the mixture was concentrated. Purifi-
cation by silica gel column chromatography gave ester 2. In the
case of 2g and 2k, the corresponding ketone was removed by
silica gel column chromatography after conversion of 2g and 2k
to the corresponding alcohols.
8
9
80 (95)
67 (94)
6
16
10
11
12
85 (97)
78 (98)
87 (96)
4
5
0
Acknowledgements
This work was supported by a Grant-in-Aid for Scientific
Research from Japan Society for the Promotion of Science
(JSPS) and by The Sumitomo Foundation. We are grateful to the
SC-NMR Laboratory of Okayama University for the measure-
ment of NMR spectra.
a Conditions: immobilized lipase (400 mg, 0.5% (w/w) enzyme/
Toyonite-200M), 1 (1.0 mmol), isopropenyl acetate (1.5 mmol), 3
(10 mol% except for 1q (4 mol%)), KOt-Bu (0.13 mmol except for 1q
(0.05 mmol)), Na2CO3 (1.0 mmol), molecular sieves 4A (20 pieces),
dry i-Pr2O (2 mL), 30 °C, 72 h except for 1g (120 h), 1i (145 h), and 1q
(24 h). b Isolated yield. c Only 2e has the (S)-configuration according to
the CIP priority system for nomenclature.
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