Expanding the Substrate Specificity of Thermoanaerobacter pseudoethanolicus Secondary Alcohol Dehydrogenase by a Dual Site Mutation

Article abstract of DOI:10.1002/ejoc.201701351

Here, we report the asymmetric reduction of selected phenyl-ring-containing ketones by various single- and dual-site mutants of Thermoanaerobacter pseudoethanolicus secondary alcohol dehydrogenase (TeSADH). The further expansion of the size of the substrate binding pocket in the mutant W110A/I86A not only allowed the accommodation of substrates of the single mutants W110A and I86A within the expanded active site but also expanded the substrate range of the enzyme to ketones bearing two sterically demanding groups (bulky–bulky ketones), which are not substrates for the TeSADH single mutants. We also report the regio- and enantioselective reduction of diketones with W110A/I86A TeSADH and single TeSADH mutants. The double mutant exhibited dual stereopreference to generate the Prelog products most of the time and the anti-Prelog products in a few cases.

Full text of DOI:10.1002/ejoc.201701351

A Journal of
Accepted Article
Title: Expanding the Substrate Specificity of Thermoanaerobacter
pseudoethanolicus Secondary Alcohol Dehydrogenase by a Dual
Site Mutation
Authors: Musa M. Musa, Odey Bsharat, Ibrahim Karume, Claire Vieille,
Masateru Takahashi, and Samir Hamdan
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To be cited as: Eur. J. Org. Chem. 10.1002/ejoc.201701351
Link to VoR: http://dx.doi.org/10.1002/ejoc.201701351
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10.1002/ejoc.201701351
European Journal of Organic Chemistry
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Expanding the Substrate Specificity of Thermoanaerobacter
pseudoethanolicus Secondary Alcohol Dehydrogenase by a Dual
Site Mutation
Musa M. Musa,*[a] Odey Bsharat,[a] Ibrahim Karume,[a] Claire Vieille,[b] Masateru Takahashi,[c] and
Samir M. Hamdan[c]
Abstract: Here, we report the asymmetric reduction of selected
phenyl-ring-containing ketones by various single and dual site
mutants of Thermoanaerobacter pseudoethanolicus secondary
alcohol dehydrogenase (TeSADH). Further expanding the size of the
substrate binding pocket in the mutant W110A/I86A not only allowed
substrates of the single mutants W110A and I86A to be
accommodated within the expanded active site, but also expanded
the enzyme’s substrate range to ketones bearing two sterically
demanding groups (bulky-bulky ketones), which are not substrates for
TeSADH single mutants. We also report the regio- and
enantioselective reduction of diketones using W110A/I86A TeSADH
and single TeSADH mutants. The double mutant exhibited dual
stereopreference generating the Prelog products most of the time and
the anti-Prelog products in a few cases.
accumulate only in small concentrations, which complicates their
recovery.[2] Synthesis of optically-active alcohols from prochiral
ketones using enzymes has been extensively studied.[3] Alcohol
dehydrogenases (ADHs) are an important class of enzymes, in
which nicotinamide-adenine dinucleotide (NAD+) or its phosphate
derivative (NADP+) acts as a cofactor. ADHs, either in whole cells
or as purified enzymes, have been extensively used to produce
enantiopure alcohols. Among those are ADHs from
Thermoanaerobium brockii (TbSADH),[4] Rhodococcus ruber,[5]
Candida magnolia,[6] Lactobacillus kefir,[7] and horse liver,[8] to
name a few.
Thermoanaerobacter pseudoethanolicus secondary ADH
(TeSADH, EC 1.1.1.2) is identical in sequence to the well-known
and commercially available TbSADH,[9] Both enzymes are
NADP+-dependent, and have been successfully employed in the
reversible asymmetric reduction of ketones.[10,11] These enzymes
gained significant interest because of their high thermal stability
and tolerance to elevated concentrations of organic solvents.[12]
However, like other ADHs, TeSADH and TbSADH suffer from
high substrate specificity (i.e., limited substrate scope).[13]
TeSADH’s substrate binding site comprises two pockets of
different sizes and affinities.[10b,11] Phenyl-ring-containing ketones
and their corresponding alcohols are not substrates for wild-type
TeSADH.[13]
Introduction
Optically-active alcohols are important precursors in the
agrochemical and pharmaceutical industries. These building
blocks can be synthesized by chemical means using chiral
transition metal catalysts that are expensive and require
sophisticated reaction conditions. An alternative biocatalytic
approach is preferable because enzymes are not only highly
chemo-, regio-, and stereoselective, they are also economical and
environmentally benign.[1] Enzyme-catalyzed reactions are
carried out under mild conditions, which minimize by-products
formation by reducing the possibility of isomerization,
racemization, and epimerization.[2] Still, enzyme-based
approaches have disadvantages: enzymes may be insufficiently
stable for commercial applications, and reaction products might
Expanding TeSADH’s substrate scope would widen its
applicability as
a biocatalyst. Site-directed mutagenesis of
TeSADH has expanded its substrate range to a certain extent and
modified its stereoselectivity.[11] In particular, mutations of W110,
which lines the substrate binding site, allowed for the asymmetric
reduction of 4-phenyl-2-butanone and 1-phenyl-2-propanone and
their analogs, but not acetophenone (Scheme 1).[14] The
stereochemical outcome of redox reactions catalyzed by TeSADH
and its W110 mutants obeys Prelog’s rule,[15] in which the pro-R
hydride of NADPH is delivered to the Re face of a prochiral ketone,
producing S alcohols. Mutation I86A was designed to expand
what has been called the small pocket in the substrate binding
site.[16] I86A TeSADH reduced acetophenone analogs in anti-
Prelog mode, but could not reduce 4-phenyl-2-butanone and 1-
phenyl-2-propanone. None of these single TeSADH mutants
accommodated bulk-bulky ketones that are precursors to
pharmaceutically important analogs such as optically-active 1,2-
diarylethanols, which are analogs of the anti-cancer agent,
combretastatin (Figure 1).[17] Few reports have been published on
the asymmetric reduction of such bulky-bulky ketones using either
whole cells[18] or purified enzymes.[19] Developing a TeSADH
mutant that can accommodate a wide scope of substrates is of
interest to advance this robust enzyme.
[a]
Prof. M. M. Musa, O. Bsharat, Dr. I. Karume
Chemistry Department
King Fahd University of Petroleum and Minerals
Dhahran, 31261, KSA
E-mail: musam@kfupm.edu.sa
http://faculty.kfupm.edu.sa/CHEM/musam/index.html
Prof. C. Vieille
Department of Microbiology and Molecular Genetics and
Department of Biochemistry and Molecular Biology
Michigan State University
[b]
[c]
East Lansing, MI 48824, USA
Dr. M. Takahashi, Prof. S. M. Hamdan
Division of Biological and Environmental Sciences and Engineering
King Abdullah University of Science and Technology
Thuwal, 23955-6900, KSA
Supporting information for this article is given via a link at the end of
the document.
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10.1002/ejoc.201701351
European Journal of Organic Chemistry
FULL PAPER
tris(hydroxymethyl)aminomethane–HCl (Tris–HCl) containing 2-
propanol (30%, v/v), which works as a co-solvent and a co-
substrate. Although the asymmetric reductions of 4-phenyl-2-
butanone (1a), 4-(4'-methoxyphenyl)-2-butanone (1b), 4-phenyl-
3-buten-2-one (1d), 1-phenyl-2-propanone (1f), and 1-phenyl-2-
butanone (1j) using W110A TeSADH, as well as those of 1a and
1f using W110G and W110V TeSADHs, have already been
reported,[14] we screened the asymmetric reduction of these
substrates using the aforementioned mutants under the
conditions used in the current study to allow proper comparison.
The discrepancies in enantioselectivities between the results
reported here and earlier are due to the different reaction
conditions used.[20]
The four mutants tested showed similar catalytic properties
in the asymmetric reduction of 1a, producing (S)-4-phenyl-2-
Scheme 1. Ketones used in this study.
butanol
[(S)-2a]
with
high
conversion
yields
and
enantioselectivities (Table 1). Optically-active 2a is a synthon in
protease inhibitors such as VX-478 and SC-52151 (Figure 1),
which are potential therapeutic agents for the treatment of
acquired immunodeficiency syndrome (AIDS).[21] Single TeSADH
mutants W110A, W110G, and W110V showed a slight edge in
conversion yield over the double mutant (W110A/I86A) in the
asymmetric reductions of 1b and 4-(4'-hydroxyphenyl)-2-
butanone (1c). However, W110A/I86A TeSADH showed a higher
stereoselectivity than the single mutants did in the asymmetric
reduction of 1b (Table 1). W110G TeSADH showed the least
stereoselectivity in the reduction of 1b, which is consistent with
previously reported results for asymmetric reduction of 1a.[14a] It is
well noted that the conversion yield and stereoselectivity of
TeSADH-catalyzed reduction reactions are highly dependent on
the structures of both the enzyme’s active site and the substrate.
The high percent ee (>94%) registered in the asymmetric
reduction of 1c using all TeSADH mutants when compared with
1b could be due to higher binding affinity of 1c’s hydroxyl group
for the enzyme active site compared to 1b’s methoxy group, which
results in fewer selectivity mistakes in 1c reduction. (S)-1c, known
as (S)-rhododendrol, has been used to prevent hepatotoxicity[22]
and as melanin-inhibitor in skin-lightening cosmetics.[23] A low
conversion yield was observed in the W110A/I86A TeSADH-
catalyzed reduction of 1d compared to those obtained with single
mutants (Table 1). However, the asymmetric reduction of 4-
phenyl-3-butyn-2-one (1e) resulted in high conversion yield and
high ee with all mutants. The asymmetric reduction of 1e with high
enantioselectivity is of great interest as this substrate, known as
To expand substrate specificity of TeSADH and thus make
it a more attractive biocatalyst, we built the W110A/I86A TeSADH
double mutant. W110 and I86 line the large and the small pockets,
respectively, of TeSADH’s substrate binding site, and thus
mutation of these two residues to Ala is expected to expand both
binding pockets. This double mutation not only expanded the
substrate scope of TeSADH to accommodate diaryl ketone
substrates, but it also altered the enzyme’s stereoselectivity for a
few substrates. We also compared the performance of this double
mutant with those of single TeSADH W110 mutants and
performed docking studies of 1,2-diphenylethanone in the
W110A/I86A TeSADH active site.
a
difficult substrate, is not easily reduced with high
enantioselectivity.[24,25] During optimization of the co-substrate
concentration, we observed an increase in stereoselectivity in the
asymmetric reduction of 1b and 1c when using 10% (v/v) instead
of 30% (v/v) 2-propanol, as used for 1a, 1d and 1e; this effect
could be due to differences in TeSADH’s active site solvation
affecting substrate binding affinity.[26]
Figure 1. Chemical structures of (R)-combretastatin, VX-478, and SC-52151.
Results and Discussion
To check the efficiency of W110A/I86A TeSADH in the
asymmetric reduction of phenyl-ring-containing ketones that are
substrates for W110A TeSADH,[14b] we conducted
a
comprehensive study of the activity and stereoselectivity of
various W110 mutants (W110A, W110G, and W110V) as well as
W110A/I86A TeSADH in asymmetric reduction of these ketones.
Unless otherwise mentioned, reactions were performed in
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10.1002/ejoc.201701351
European Journal of Organic Chemistry
FULL PAPER
2f with excellent conversion yields and moderate ees, and that
using W110V TeSADH resulted in a significant improvement in ee
(>99%). One possible explanation is that 1f can fit in two
orientations in the large binding pocket of W110A and W110G
TeSADHs; but a single orientation is allowed in W110V, whose
large pocket is smaller than that of the W110A and W110G
mutants. Interestingly, in the current report, the W110A/I86A
Table 1. Asymmetric reduction of phenyl-ring-containing ketones by the
TeSADH mutants.[a]
TeSADH-catalyzed reduction of 1f resulted in
a reverse
stereochemical outcome, producing (R)-2f with high conversion
yield and good enantioselectivity (Table 2). Both substrate-
binding pockets are enlarged in the double mutant, likely allowing
the benzyl group of 1f to fit in either pocket.
Asymmetric reduction of 2-chlorophenylacetone (1g) by the
single mutants produced (S)-2g with high enantioselectivity. In
contrast, W110A/I86A TeSADH produced the opposite
enantiomer (R)-2g with high ee (99%). Reduction of 4-
chlorophenylacetone (1h) resulted in (S)-2h irrespective of the
enzyme used, which demonstrates the importance of the
substituent location on the phenyl ring in controlling
stereoselectivity. The presence of a trifluoromethyl group in the
meta position in 3-(trifluoromethyl)phenylacetone (1i) resulted in
reduction to (S)-2i with high conversion yield and high
enantioselectivity using all mutants, which indicates that the CF3
in meta position does not allow 1i to fit within the small pocket of
W110A/I86A TeSADH as 1f and 1g do. Reduction of 1j
predominantly produced the S-alcohol in high ees using the single
mutants and a moderate ee using the double mutant. The
moderate enantioselectivity of the W110A/I86A TeSADH-
catalyzed asymmetric reduction of 1j could be due to the ability of
the benzyl group to fit in the small pocket, leading to noticeable
selectivity mistakes. Extending the alkyl moiety to propyl in 1-
phenyl-2-pentanone (1k) resulted in a reversed fit of this substrate
in the active site of W110A/I86A TeSADH producing (R)-2k in
high ee. This result indicates that the propyl group forces this
substrate to fit in an orientation that allows for a Si-face attack of
the ketone by the hydride of NADPH in anti-Prelog mode.
Surprisingly, we also observed this reversed stereochemistry in
1k reduction by W110V TeSADH with low conversion yield. 1k
was not a substrate for W110G and W110A TeSADHs.
R
Substrate
X
Conv.
(%)[b]
ee
(%)[c]
PhCH2CH2
1a
W110G
W110A
W110V
>99
99
95
96
99
>99
98
W110A/I86A
91
p-MeO-C6H4-CH2CH2
p-HO-C6H4-CH2CH2
(E)-Ph-HC=CH
Ph-C≡C
1b[d]
1c[d]
1d
W110G
W110A
W110V
93
98
94
40
20
60
79
84
W110A/I86A
W110G
W110A
W110V
95
97
92
80
99
>99
94
W110A/I86A
>99
W110G
W110A
W110V
75
73
67
21
>99
92
92
W110A/I86A
90
1e
W110G
W110A
W110V
>99
99
97
97
99
>99
98
W110A/I86A
>99
[a] Unless otherwise stated, reactions were performed using 0.0191 mmol
ketone 1a–e, 100 μL enzyme [0.2 mg])], 1.0 mg NADP+, 600 μL buffer A,
and 300 μL 2-propanol; for W110A/I86A TeSADH-catalyzed reactions,
ZnCl₂ and DTT were added at 1.4 μM and 6.5 mM final concentrations,
respectively; the W110V TeSADH-catalyzed reactions contained 0.0038
mmol 1a–e , 950 μL buffer A, and 50 μL 2-propanol. [b] Percent conversion
was determined by Gas chromatography (GC). [c] The % ee of the acetate
ester derivative of the alcohol produced was determined by GC using a chiral
stationary phase. [d] Reactions were performed using 10% 2-propanol.
Table 2. Asymmetric reduction of phenyl ring-containing ketones by the
TeSADH mutants.[a]
Reduction of substrates 1a-e using W110A/I86A TeSADH
followed Prelog mode, consistent with the results obtained with
W110 single mutants. The ability of W110A/I86A TeSADH to
reduce these substrates with efficiencies similar to those obtained
with the single mutants is exciting. Note that W110A/I86A
TeSADH was first designed to enlarge the two substrate-binding
pockets to increase the efficiency of enantiopure alcohol
racemization by increasing selectivity mistakes.[27] Yet,
W110A/I86A TeSADH shows enantioselectivity comparable to
the single mutants, which indicates that the size of the substrate-
binding site is not the only factor that controls stereoselectivity of
TeSADH-catalyzed reactions.
R1
R2
Substrate
X
Conv. ee
(%)[b] (%)[c]
PhCH2
CH3
1f
W110G
W110A
W110V
>99
95
79(S)
84(S)
>99(S)
80(R)
Previously,[14] we observed that the asymmetric reduction of
1f using the W110G and W110A TeSADH mutants produced (S)-
>99
W110A/I86A >99
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10.1002/ejoc.201701351
European Journal of Organic Chemistry
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binds in the small pocket. Reduction of such bulky-bulky substrate
with high enantioselectivity is of great interest because the similar
sizes of the phenyl and benzyl moieties do not guide the binding
based on pocket size only. The conversion yield for the reductions
of the bulky-bulky substrate 1m by W110A/I86A TeSADH was
clearly about 2-fold higher than that for 1l, which is a typical
example of a small–bulky ketone; this result could be due to a
good fit of bulky-bulky substrates within the active site of
W110A/I86A TeSADH that allows for maximum utilization of the
active site.
o-Cl-C6H4-CH2
CH3
1g
1h
1i
W110G
W110A
W110V
13
84
99
89(S)
98(S)
>99(S)
99(R)
W110A/I86A >99
p-Cl-C6H4-CH2
CH3
W110G
W110A
W110V
70
>99
>99
89(S)
97(S)
98(S)
80(S)
W110A/I86A 99
m-CF3-C6H4-
CH2
CH3
W110G
W110A
W110V
>99
>99
99
96(S)
>99(S)
99(S)
95(S)
W110A/I86A >99
Table 3. Asymmetric reduction of 1,2-diphenylethanone by W110A/I86A
TeSADH.[a]
PhCH2
PhCH2
Ph
CH3CH2
1j
W110G
W110A
W110V
96
98
99
92(S)
92(S)
>99(S)
71(S)
W110A/I86A 72
CH3CH2
CH2
1k
1l
W110G
W110A
W110V
n.r.
3
32
-
n.d.
92(R)
98(R)
W110A/I86A 95
X
Conv. (%)[b]
ee (%)[c]
CH3
W110G
W110A
W110V
n.r.
n.r.
n.r.
-
-
-
W110G
W110A
W110V
n.r.
n.r.
n.r.
87
-
-
-
W110A/I86A 46
98(R)
W110A/I86A
95
[a] Reactions were performed using 0.0191 mmol ketones 1f–l, 0.2 mg
enzyme, 1.0 mg NADP+, 700 μL buffer A, and 300 μL 2-propanol; for
W110A/I86A TeSADH-catalyzed reactions, ZnCl₂ and DTT were added at
1.4 μM and 6.5 mM final concentrations, respectively; the W110V TeSADH-
catalyzed reactions contained 0.0038 mmol 1f–l, 950 μL buffer A, and 50 μL
2-propanol. [b] Percent conversion was determined by GC. [c] The % ee of
the acetate ester derivative of the alcohol produced was determined by GC
using a chiral stationary phase. [n.r.: no reaction, n.d.: not determined].
[a] Reaction mixtures contained 0.0191 mmol 1m, 0.4 mg enzyme, 1.0 mg
NADP+, 700 μL buffer A, 300 μL 2-propanol, and ZnCl₂ and DTT to final
concentrations of 1.4 μM and 6.5 mM, respectively. [b] Percent conversion
was determined by GC. [c] The % ee for (R)-2m was determined by high-
performance liquid chromatography (HPLC) using hexane/methanol (v/v =
95: 5). [n.r.: no reaction].
The binding orientation of 1m in W110A/I86A TeSADH was
confirmed by computational docking (Figure 2A) using the crystal
structure of TbSADH (identical in sequence to TeSADH[9]) in
complex with NADP as the template. The lowest binding energy
configuration (-5.9 kJ/mol) placed 1m’s phenyl ring in the small
pocket and the carbonyl group at 3.7 Å from His59. The carbonyl
is unusually far from His59 for catalysis, but this distance likely
explains why the 1m reduction reaction required double the
amount of enzyme compared to most other conversions in this
study, and why, even with extra enzyme, the 1m reduction
reaction did not reach full completion (Table 2). In comparison,
the lowest energy docking of 1m in W110A TeSADH (-5.1 kJ/mol)
placed the carbonyl 4.0 Å away from His59 (Figure 2B), and the
lowest energy docking of 1m in W110V TeSADH (-1.8 kJ/mol)
created a steric clash with F112 (not shown).
Acetophenone (1l) is not a substrate for wild-type TeSADH
or for any of the W110 mutants. 1l was previously shown to be a
substrate for I86A TeSADH.[16] Its reduction by W110A/I86A
TeSADH resulted in (R)-1-phenylethanol [(R)-2l] in high
enantioselectivity and moderate conversion, as shown in Table 2.
This outcome is consistent with that observed with I86A TeSADH,
which reduced 1l in anti-Prelog mode.[16] 1a-j are substrates for
W110 TeSADH mutants, but not for I86A TeSADH. In contrast, 1l
is a substrate for I86A TeSADH but not for the W110 TeSADH
mutants.[28] The W110A/I86A double TeSADH mutant, though,
can reduce ketones that are substrates for all individual mutants.
This striking finding prompted us to look for other valuable
substrates like bulky-bulky ketones to expand TeSADH’s
substrate range.
The enlarged substrate binding site in W110A/I86A
TeSADH makes this enzyme mutant a potential catalyst to reduce
bulky-bulky ketones such as 1,2-diphenylethanone (1m) (Table 3),
which is not a substrate for any previously reported mutant of
TeSADH including W110 mutants. Reduction of 1m using
W110A/I86A TeSADH resulted in (R)-2m in high
enantioselectivity and high conversion yield. The ability of
W110A/I86A TeSADH to accommodate 1m is attributed to
extensions of both its small and large substrate-binding pockets.
Reduction of 1m by W110A/I86A TeSADH followed Prelog’s rule
with an inverted Cahn-Ingold-Prelog priority, which indicates that
the benzyl group binds in the large pocket and the phenyl group
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10.1002/ejoc.201701351
European Journal of Organic Chemistry
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Figure 2. Lowest energy dockings of 1m in the catalytic sites of W110A/I86A
TbSADH (A) and W110A TbSADH (B). The substrate is viewed from the face
occupied by NADP in the TbSADH crystal structure. The residues lining up the
binding pocket and those in close proximity to the reactive ketone are shown in
stick representation. Colors are based on atoms (green or white: carbon; blue:
nitrogen; red: oxygen; and yellow: sulfur). Mutated residues are in white. The
surface of the binding pocket is shown when it does not hide the substrate.
Conclusions
We showed that the W110A/I86A double mutation expanded the
substrate scope of TeSADH. This double mutant accommodates
and reduces ketones that are substrates for W110 mutants only
and others that are substrates for I86A TeSADH only, with
conversion yields and enantioselectivities comparable to those
obtained with the single mutants. W110A/I86A TeSADH
accommodates 1m, whcih bears bulky groups on each side of the
carbonyl, and reduces it with high conversion yield and
enantioselectivity. W110A/I86A TeSADH showed a reverse
enantiopreference for a few substrates, compared to the TeSADH
single W110 mutants. We also succeeded in conducting
regioselective reductions of diketones using both the TeSADH
double and single mutants, a feature that is tough to accomplish
by non-enzymatic approaches. The ability of W110A/I86A
TeSADH to accommodate a wider scope of substrates than those
accommodated by wild-type TeSADH and other previously
reported single mutants is of great interest to organic chemists.
A highly advantageous characteristic of enzymes, besides
their high enantioselectivity, is their high regioselectivity. This very
important feature, which is not easy to accomplish in
organometallic or organic catalysis, was observed in the
asymmetric reduction of 1-phenyl-1,2-propanedione (1n) and 1-
phenyl-1,3-butanedione (1o). While 1n was not a substrate for
TeSADH single W110 mutants, W110A/I86A TeSADH activity
with 1n resulted in a regioselective reduction of the homobenzylic
carbonyl to produce (R)-2-hydroxy-1-phenyl-1-propanone [(R)-
2n] with excellent conversion yield and enantioselectivity (Table
4). Production of the R alcohol indicates that 1n fits in an
orientation that allows for a Si-facial attack of the carbonyl (i.e.,
anti-Prelog mode). This stereochemical outcome is similar to that
obtained in the W110A/I86A TeSADH-catalyzed reduction of 1f
and 1g (Table 2), which reflects the similarity of the three
structures. In contrast, excellent performance was observed for
all enzyme mutants in the regioselective asymmetric reduction of
1o in Prelog mode, predominantly yielding (S)-3-hydroxy-1-
phenyl-1-butanone [(S)-2o] and leaving the benzylic carbonyl
intact.
Experimental Section
General: NADP+, NADPH, ketones 1a-e and 1g-o, (rac)-2a, (rac)-2f, (rac)-
2l, (R)-2a, (S)-2a, (R)-2f, and (S)-2f were purchased from Sigma-Aldrich
and used without further purification. (rac)-2b-e, (rac)-2g-k, and (rac)-2m
were produced by NaBH4 reduction of their corresponding ketones as
reported.[29a] All pHs were adjusted at room temperature. Nuclear
Magnetic Resonance (NMR) spectra were recorded on a 500 MHz
spectrometer at 500 MHz (1H) and at 125 MHz (13C) at room temperature
using either Tetramethylsilane (TMS) or the solvent peak as an internal
standard. Optical rotation measurements were performed by using MCP
200 polarimeter.
Table 4. Asymmetric reduction of phenyl ring-containing ketones by the
TeSADH mutants.[a]
Gene expression and purification of TeSADH mutants: W110A,
W110G, W110V, and W110A/I86A mutants of TeSADH with a C-terminal
His-tag were expressed and purified as previously reported.[27] In brief,
proteins were expressed in Escherichia coli BL21 (DE3) (Novagen) grown
in LB containing 100 µg/mL ampicillin. Two-L liquid cultures at an OD600 of
1.0 were induced with isopropyl β-D-1-thiogalactopyranoside for three
hours. Once resuspended in lysis buffer, the cell pellet was lysed by high
pressure differential lysis (Cell disruptor, Constant Systems), followed by
centrifugation. The supernatant was heat-treated at 85 °C for 15 min to
denature proteins from E. coli and centrifuged at 96,000 g for 40 min.
Finally, the proteins were purified by Ni-affinity chromatography ( HisTrap
FF 5 mL, GE Healthcare). The purified enzymes were dialyzed against 20
mM Tris-HCl (pH 8.0) containing 10 mM 2-mercaptoethanol and 50 %
glycerol) and stored at -80 °C until used. Protein concentrations were
determined by OD280 using the extinction coefficient and molecular weight
calculated based on the amino acid sequence composition of TeSADH.
R1
R2
Substrate
X
Product Conv.
(%)[b]
ee
(%)[c]
PhCO
CH3
1n
W110G
W110A
W110V
(R)-2n
n.r.
n.r.
n.r.
>99
-
-
-
W110A/I86A
>99
PhCOCH2 CH3
1o
W110G
W110A
W110V
(S)-2o
92
99
89
92
>99
>99
>99
92
W110A/I86A
General procedure for asymmetric reduction of ketones: Enzyme
reaction mixtures contained 0.2 mg enzyme and 1.0 mg NADP+ in 100 μL
Tris-HCl (50 mM, pH 8.0, buffer A), 600 μL buffer A, 300 μL 2-propanol,
and 0.0191 mmol ketone (1a-o). Reactions were started by adding the
ketone. The mixture was incubated in a thermostated shaker maintained
at 50 °C and 180 rpm. W110V TeSADH-catalyzed reactions were carried
out using 0.00382 mmol ketones in reaction mixtures containing 5% 2-
propanol (v/v). W110A/I86A TeSADH-catalyzed reactions were performed
in the presence of 1.4 μM ZnCl2 and 6.5 mM DDT. In a few cases, enzyme
loading and 2-propanol concentrations were varied to achieve maximum
[a] Reactions were performed using 0.0191 mmol ketones (1n, 1o), 0.2 mg
enzyme, 1.0 mg NADP+, 700 μL buffer A, and 300 μL 2-propanol; for
W110A/I86A TeSADH-catalyzed reactions, ZnCl₂ and DTT were added at
1.4 μM and 6.5 mM final concentrations, respectively; the W110V TeSADH-
catalyzed reactions contained 0.0038 mmol ketone, 950 μL buffer A, and 50
μL 2-propanol. [b] Percent conversion was determined by GC. [c] The % ee
of the acetate ester derivative of the alcohol produced was determined by
GC using a chiral stationary phase. [n.r.: no reaction].
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10.1002/ejoc.201701351
European Journal of Organic Chemistry
FULL PAPER
conversions. Upon achieving maximum conversion to alcohol products
(monitored by GC), the reaction mixture was extracted with ethyl acetate
(2 × 500 µL). The combined organic layers were dried with sodium sulfate,
and then concentrated. The remaining residue was treated with acetic
anhydride (one drop) and pyridine (two drops) to obtain the acetate ester
derivative prior to percent ee determination by chiral GC.[29b] The percent
ee of (R)-2m was determined by a chiral HPLC.
(S)-4-Phenyl-2-butanol [(S)-2a]: 1H NMR (CDCl3): δ = 1.23 (d, J = 6.1 Hz,
3 H), 1.59 (s, 1 H), 1.74–1.85 (m, 2 H), 2.67–2.81 (m, 2 H), 3.79–3.86 (m,
1 H), 7.17–7.31 (m, 5 H) ppm. 13C NMR (CDCl3): δ = 23.8, 32.3, 41.1, 67.8,
126.1, 128.6, 128.7, 142.4 ppm. 1H and 13C NMR data were in accordance
with those previously reported.[33]
(S)-4-(4'-Methoxyphenyl)-2-butanol [(S)-2b]: 1H NMR (CDCl3): δ = 1.21
(d, j = 6.1 Hz, 3 H), 1.46 (br s, 1 H), 1.69–1.78 (m, 2 H), 2.57–2.70 (m, 2
H), 3.76 (s, 3 H), 3.77–3.81 (m, 1 H), 6.81 (d, J = 8.3 Hz, 2 H), 7.10 (d, J =
8.3 Hz, 2 H) ppm. 13C NMR (CDCl3): δ = 23.6, 31.2, 41.0, 55.2, 67.5, 113.8,
129.2, 134.1, 157.7 ppm. 1H and 13C NMR data were in accordance with
those previously reported.[14b]
General procedure for large-scale asymmetric reduction of ketones:
A mixture of 0.191 mmol ketone, 7.0 mL buffer A, 3.0 mL 2-propanol, 100
μL enzyme [0.2-0.4 mg in buffer A] and 2 mg NADP+ was placed in a 50-
mL round-bottomed flask equipped with a condenser and a magnetic
stirrer. The reaction mixture was stirred at 50 °C for 24 h. It was then
extracted with ethyl acetate (2 × 7 mL). The combined organic layers were
dried with sodium sulfate and then concentrated. A portion of the remaining
residue was derivatized as described above prior to GC analysis. (R)-1,2-
Diphenylethanol [(R)-2m] was purified by flash chromatography using
diethyl ether:hexane (1:3, v/v).
(S)-4-(4'-Hydroxyphenyl)-2-butanol [(S)-2c]: 1H NMR (CD3OD): δ = 1.18
(d, J = 6.1 Hz, 3 H), 1.60–1.75 (m, 2 H), 2.50–2.59 (m, 1 H), 2.60–2.71 (m,
1 H), 3.32 (s, 1 H), 3.68–3.76 (m, 1 H), 6.70 (d, J = 8.3 Hz, 2 H), 7.01 (d, J
= 8.3 Hz, 2 H) ppm. 13C NMR (CD3OD): δ = 23.5, 32.3, 42.4, 67.9, 116.1,
130.2, 134.4, 156.3 ppm.1H and 13C NMR data were in accordance with
those previously reported.[34]
GC Analysis (chiral): The percent ee values of the acetate ester
derivatives of alcohol products were determined using an Agilent 7890A
capillary GC equipped with a flame ionization detector and an HP chiral-
20B column (30 m, 0.32 mm [i.d.], 0.25 μm film thickness) using He as the
carrier gas. The temperature program for 2a-d, 2f, 2h, 2i, 2j, 2n, and 2o
was set as: oven 70 °C (initial, hold time 10 min) to 180 °C (final, hold time
20 min) at 5 °C/min rate; injector 220 °C, detector 230 °C; and 15 mL/min
He flow. For 2e, 2g, and 2k, the rate of the oven temperature change was
1.5 °C/min. The injection volume was 1.0 µL with split ratio of 10:1.
(S)-4-Phenyl-3-buten-2-ol [(S)-2d]: 1H NMR (CDCl3): δ = 1.31 (d, J = 6.4,
2 H), 1.63 (br s, 1 H), 4.39–4.47 (m, 1 H), 6.20 (dd, J = 15.8, 6.4 Hz, 1 H)
6.51 (d, J = 15.9 Hz, 1 H), 7.18–7.33 (m, 5 H) ppm. 13C NMR (CDCl3): δ =
23.4, 68.9, 126.4, 127.6, 128.6, 129.4, 133.5, 136.7 ppm. 1H and 13C NMR
data were in accordance with those previously reported.[35]
(S)-4-Phenyl-3-butyn-2-ol [(S)-2e]: 1H NMR (CDCl3): δ = 1.56 (d, J = 6.7
Hz, 3 H), 2.11 (br s, 1 H), 4.76 (q, J = 6.7 Hz, 1 H), 7.29–7.43 (m, 5 H). 13
C
NMR (CDCl3): δ = 24.4, 58.8, 84.0, 90.9, 122.6, 128.2, 128.3, 131.6 ppm.
1H and 13C NMR data were in accordance with those previously
reported.[36]
GC-mass spectrometry (MS) analysis (non-chiral): GC-MS analysis
was conducted using Agilent GC model 7890A combined with Agilent
5975C Inert MSD with Triple-Axis Detector using capillary column [(5%
phenyl)–methylpolysiloxane, 30 m × 320 µm × 0.25 µm]. The following
temperature program was used for GC: oven 70 °C (initial, hold time 10
min) to 180 °C (final, hold time 20 min) at 5 °C/min rate; injector 220 °C;
detector 230 °C, and 15 mL/min He flow. The injection volume was 1.0 µL
with split ratio 10:1.
1
(S)-1-Phenyl-2-propanol [(S)-2f]: H NMR (CDCl3): δ = 1.27 (d, J = 6.1
Hz, 3 H), 1.61 (br s, 1 H), 2.71 (dd, J = 13.6, 8.1 Hz, 1 H), 2.81 (dd, J =
13.4, 4.9 Hz, 1 H), 4.01–4.08 (m, 1 H), 7.22–7.35 (m, 5 H) ppm. 13C NMR
(CDCl3): δ = 23.0, 46.0, 69.1, 126.8, 128.9, 129.7, 138.8 ppm. 1H and 13
NMR data were in accordance with those previously reported.[33]
C
HPLC Analysis: HPLC measurements were performed on a Dionex-
UltiMate 3000 system equipped with a Chiralcel OD–H column (Daicel).
The percent ee of (S)-1m was determined using hexane/methanol (95:5,
v/v) as the mobile phase.
(R)-1-(2'-Chlorophenyl)-2-propanol [(R)-2g]: [훼]2퐷1 –20° (c 0.011,
CH2Cl2) 99% ee, lit.[37] [훼]퐷20 +40.3 (c 1.0, CH2Cl2), 95% ee for S isomer.
1H NMR (CDCl3): δ = 1.27 (d, J = 6.1 Hz, 3 H), 2.83 (dd, J = 13.7, 7.9 Hz,
1 H), 2.96 (dd, J = 13.6, 4.6 Hz, 1 H), 4.12–4.16 (m, 1 H), 7.17–7.38 (m, 4
H) ppm. 13C NMR (CDCl3): δ = 23.0, 43.2, 67.5, 126.8, 127.9, 129.6, 131.7,
134.3, 136.4 ppm. 1H and 13C NMR data were in accordance with those
previously reported.[37]
Determination of absolute configuration: The absolute configurations
of (S)-2a, (S)-2f, and (R)-2f were determined by co-injection on a chiral
GC with their commercially available enantiopure forms. The absolute
configurations of (S)-2b,[14b] (S)-2c,[30] (S)-2d,[14b] (S)-2e,[31] (S)-2i,[27] (S)-
2j,[31] and (S)-2o,[14b] were demonstrated by co-injection with enantiopure
alcohols produced by W110A TeSADH-catalyzed reduction of their
corresponding ketones, which were reported to have (S)-configuration.
The absolute configuration of (R)-l was determined by co-injection with
enantiopure alcohol produced by I86A TeSADH-catalyzed reduction of
acetophenone.[16] The absolute configurations of (R)-2g, (S)-2g, (S)-2h,
(R)-2k, (S)-2m, and (R)-2n were determined by comparing the sign of
optical rotation with those reported in literature.
(S)-1-(4'-Chlorophenyl)-2-propanol [(S)-2h]: [훼]2퐷1 +7.7° (c 0.011,
CHCl3) >99% ee, lit.[38] [훼]퐷25 +31 (c 1.0, CHCl3), >99% for S isomer. 1H
NMR (CDCl3): δ = 1.17 (d, J = 6.4 Hz, 3 H), 2.6 (dd, J = 13.6, 7.6 Hz, 1 H),
2.68 (dd, J = 13.4, 4.9 Hz, 1 H), 3.92–3.95 (m, 1 H), 7.08 (d, J = 8.2 Hz, 2
H), 7.20 (d, J = 8.2 Hz, 2 H) ppm. 13C NMR (CDCl3): δ = 22.9, 44.9, 68.7,
128.6, 130.7, 132.3, 137.0 ppm. 1H and 13C NMR data were in accordance
with those previously reported.[38]
(S)-1-[(3'-Trifluoromethyl)-phenyl)]-2-propanol [(S)-2i]: 1H NMR
(CDCl3): δ = 1.26 (d, J = 6.1 Hz, 3 H), 1.70 (br s, 1 H), 2.78 (dd, J = 13.7,
7.9 Hz, 1 H), 2.84 (dd, J = 13.4, 4.6 Hz, 1 H), 4.03–4.09 (m, 1 H), 7.40–
Docking experiment: The 3D coordinates of 1m were generated with
CORINA-Gasteiger Research. Mutations were introduced in the structure
of TbSADH complexed with NADP (PDB no. 1YKF) using PyMOL
Molecular Graphics System, Version 1.8 (Schrödinger, LLC). The 3D
structure of 1m was docked in the 3D structures of the mutant enzymes
with AutoDock Vina[32] using a search box of 12 Å x 12 Å x 12 Å centered
on the TbSADH catalytic Zn atom. Structures were visualized using
PyMOL.
7.51 (m, 4 H) ppm. 13C NMR (CDCl3): δ = 22.9, 45.3, 68.6, 123.3 (q, JC-F
=
3.1 Hz), 124.1 (q, JC-F = 270.3 Hz), 126.0 (q, JC-F = 3.1 Hz), 128.9, 130.7
(q, JC-F = 32 Hz), 132.8, 139.5 ppm. 1H and 13C NMR data were in
accordance with those previously reported.[33]
This article is protected by copyright. All rights reserved.

10.1002/ejoc.201701351
European Journal of Organic Chemistry
FULL PAPER
(S)-1-Phenyl-2-butanol [(S)-2j]: 1H NMR (CDCl3): δ = 1.01 (t, J = 7.3 Hz,
3 H), 1.49-1.60 (m, 3 H), 2.60 (dd, J = 13.5, 8.4 Hz, 1 H), 2.85 (dd, J = 13.5,
4.3 Hz, 1 H), 3.71-3.78 (m, 1 H), 7.21–7.63 (m, 5 H) ppm. 13C NMR
(CDCl3): δ = 10.0, 29.6, 43.6, 74.0, 126.4, 128.5, 129.4, 138.6 ppm.1H and
13C NMR data were in accordance with those previously reported.[35]
Seisser, K. Faber, S. F. Mayer, R. Oehlein, A. Hafner, W. Kroutil, Eur. J.
Org. Chem. 2006, 1904–1909.
[6]
[7]
D. Zhu, Y. Yang, L. Hua, J. Org. Chem. 2007, 71, 4202–4205.
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(R)-1-Phenyl-2-pentanol [(R)-2k]: [훼]2퐷1 –1.01° (c 0.015, EtOH) 98 % ee,
lit.[39] [훼]퐷20 +3.7° (c 1.1, EtOH), 99% ee for S isomer. 1H NMR (CDCl3): δ =
0.94 (t, J = 7.0 Hz, 3 H), 1.41–1.55 (m, 4 H), 2.65 (dd, J = 13.6 Hz, 8.5 Hz,
1 H), 2.83 (dd, J = 13.6 Hz, 4.0 Hz, 1 H), 3.67 (br s, OH), 3.81-3.86 (m,
1H), 7.21-7.35 (m, 5H) ppm.13C NMR (CDCl3): δ = 14.0, 18.9, 39.1, 44.1,
72.4, 126.4, 128.5, 129.4, 138.6 ppm. 1H and 13C NMR data were in
accordance with those previously reported.[40]
[8]
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lit.[41] [훼]퐷22 –13.0 (c 1.05, CHCl3), 90% ee for S isomer. 1H NMR (CDCl3):
δ = 1.93 (br s, 1 H), 2.99 (dd, J =13.7, 8.6 Hz, 1 H), 3.05 (dd, J = 13.6, 4.6
Hz, 1 H), 4.88–4.92 (m, 1 H), 7.19–7.37 (m, 10 H) ppm. 13C NMR (CDCl3):
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1H and 13C NMR data were in accordance with those previously
reported.[41,42]
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1H NMR (CDCl3): δ = 1.46 (d, J = 7.0 Hz, 3 H), 3.7 (s, 1 H), 5.17 (q, J = 7.6
Hz, 1 H), 7.48–7.53 (m, 2 H), 7.62–7.65 (m, 1 H), 7.92–7.95 (m, 2 H) ppm.
13C NMR (CDCl3): δ = 22.3, 69.3, 128.7, 128.9, 133.4, 134.0, 202.4 ppm.
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Acknowledgements
The authors acknowledge the support provided by the Deanship
of Scientific Research (DSR) at King Fahd University of Petroleum
and Minerals (KFUPM) for funding this work under project number
IN151032. They also acknowledge the supported by baseline
research fund to S.M.H. by King Abdullah University of Science
and Technology.
[20] Reaction conditions used in ref. 14b for asymmetric reductions of 1a, 1b,
1d, and 1f: 0.34 mmol substrate, 2.0 mg NADP+, 0.75 mg W110A
TeSADH, 7.0 mL Tris-HCl (50 mM, pH 8.0), and 3.0 mL 2-propanol.
Reaction conditions used in ref. 14a for asymmetric reductions of 1a and
1f: 0.04 mmol ketone, 1.0 mg NADP+, 0.35 mg mutant TeSADH, 9.5 mL
Tris-HCl (50 mM, pH 8.0), and 0.5 mL 2-propanol.
Keywords: active site • alcohol dehydrogenases • asymmetric
reduction • site-directed mutagenesis • substrate specificity
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10.1002/ejoc.201701351
European Journal of Organic Chemistry
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Author(s), Corresponding Author(s)*
Musa M. Musa,* Odey Bsharat, Ibrahim
Karume, Claire Vieille, Masateru
Takahashi, and Samir M. Hamdan
Page No. – Page No.
Expanding the Substrate Specificity
of Thermoanaerobacter
pseudoethanolicus Secondary
Alcohol Dehydrogenase by a Dual
Site Mutation
The enlarged substrate binding pocket of Thermoanaerobacter pseudoethanolicus
secondary alcohol dehydrogenase W110A/I86A double mutant (W110A/I86A
TeSADH) enabled the asymmetric reduction of ketones bearing two sterically
demanding groups. W110A/I86A TeSADH also reduced ketones that are substrates
for W110A TeSADH single mutant in high enantioselectivity.
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