Exploring the substrate scope of mutants derived from the robust alcohol dehydrogenase TbSADH

Article abstract of DOI:10.1016/j.tetlet.2016.06.134

Directed evolution of an enzyme as catalyst for a given stereoselective transformation provides a mutant for that particular reaction, but organic chemists need catalysts that are characterized by broad substrate acceptance. In a previous study we succeeded in evolving a set of variants of the thermally robust alcohol dehydrogenase TbSADH from Thermoanaerobacter brockii as a catalysts in the (R)- and (S)-selective reduction of tetrahydrofuran-3-one, this difficult-to-reduce compound being a sterically small substrate. These mutants were now tested in the asymmetric reduction of seven structurally unrelated and sterically more demanding substrates, including acetophenone, benzyl methyl ketone, 4-phenyl-2-butanone, and 2-oxo-4-phenyl-butanoic acid ethyl ester. The variants clearly out-perform WT TbSADH, but overly bulky substituted benzophenone derivatives are not accepted by WT or mutants.

Full text of DOI:10.1016/j.tetlet.2016.06.134

Accepted Manuscript
Exploring the substrate scope of mutants derived from the robust alcohol dehy-
drogenase TbSADH
Zhoutong Sun, Guangyue Li, Adriana Ilie, Manfred T. Reetz
PII:
S0040-4039(16)30820-6
DOI:
http://dx.doi.org/10.1016/j.tetlet.2016.06.134
TETL 47861
Reference:
To appear in:
Tetrahedron Letters
Received Date:
Accepted Date:
14 June 2016
30 June 2016
Please cite this article as: Sun, Z., Li, G., Ilie, A., Reetz, M.T., Exploring the substrate scope of mutants derived
from the robust alcohol dehydrogenase TbSADH, Tetrahedron Letters (2016), doi: http://dx.doi.org/10.1016/j.tetlet.
2
016.06.134
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Exploring the substrate scope of mutants derived from the robust alcohol
dehydrogenase TbSADH
Sun, Z.; Li, G.; Ilie, A.; and Reetz, M. T.
*

1
Tetrahedron Letters
journal homepage: www.elsevier.com
Exploring the substrate scope of mutants derived from the robust alcohol
dehydrogenase TbSADH
a,b,�
a,b,�
a,b,�
a,b
Zhoutong Sun
a
Guangyue Li
Adriana Ilie
and Manfred T. Reetz ∗
Max-Planck-Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 1, 45470, Mülheim an der Ruhr, Germany.
Fachbereich Chemie, Philipps-Universität Marburg, Hans-Meerwein-Strasse, 35032 Marburg, Germany.
b
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Directed evolution of an enzyme as catalyst for a given stereoselective transformation provides a
mutant for that particular reaction, but organic chemists need catalysts that are characterized by
broad substrate acceptance. In a previous study we succeeded in evolving a set of variants of the
thermally robust alcohol dehydrogenase TbSADH from Thermoanaerobacter brockii as
catalysts in the (R)- and (S)-selective reduction of tetrahydrofuran-3-one, this difficult-to-reduce
compound being a sterically small substrate. These mutants were now tested in the asymmetric
reduction of seven structurally unrelated and sterically more demanding substrates, including
acetophenone, benzyl methyl ketone, 4-phenyl-2-butanone and 2-oxo-4-phenyl-butanoic acid
ethyl ester. The variants clearly out-perform WT TbSADH, but overly bulky substituted
benzophenone derivatives are not accepted by WT or mutants.
Available online
Keywords:
Enantioselectivity
Alcohol dehydrogenase
Asymmetric ketone reduction
Biocatalysis
Directed evolution
2009 Elsevier Ltd. All rights reserved.
———
∗
Corresponding author. Tel.: +49-(0)6421-28-25500; fax: +49 -(0)6421-28-25620; e-mail: reetz@mpi-muelheim.mpg.de

2
9
a
The asymmetric reduction of prochiral ketones with structurally different ketones.
Since screening is the
8
formation of chiral alcohols can be performed either with chiral bottleneck of directed evolution, methods and strategies for
transition metal catalysts or enzymes of the type alcohol evolving small but “smart” mutant libraries requiring a
dehydrogenases (ADHs), often in a complementary manner. minimum of screening are needed. In this endeavor we recently
1
2
Numerous ADHs are commercially available, but not all are proposed triple code saturation mutagenesis (TCSM) as a
robust enough for industrial applications. We were attracted by particularly efficient approach, and applied it successfully to
10
the pronounced thermostability of the ADH from limonene epoxide hydrolase and to the alcohol dehydrogenase
3,4
Thermoanaerobacter brockii (TbSADH), while realizing that TbSADH, in the latter case as catalyst in the reduction of
5
its substrate scope with high stereoselectivity is not as broad as ketone 1. It was possible to enhance (R)-selectivity to 99% ee,
organic chemists would like it to be. For example, the and to invert stereoselectivity up to 95% ee (S) without
5
asymmetric reduction of tetrahydrofuran-3-one (1) catalyzed by significant tradeoff in thermostability (see also Table 2). The
wildtype (WT) TbSADH leads to the formation of (R)-2 with best variants were also successful in the asymmetric reduction
5
5
low enantioselectivity (23% ee) (Scheme 1), which is of three structurally related ketones.
unfortunate because its enantiomer (S)-2 is a building block in In the present study we report the performance of some of
5
the synthesis6 of the HIV inhibitors amprenavir and the previously evolved TbSADH variants as catalysts in the
fosamprenavir. Indeed, ketone 1 is a challenging substrate asymmetric reduction of prochiral ketones which are not
because the α- and α’-substituents flanking the carbonyl structurally related to 1, namely 3a-g (Scheme 2). Rather than
function are isosteric. Whenever prochiral ketones are screening the previous libraries or resorting to additional
characterized by sterically and electronically similar α- and α’- genetic optimization, a select set of mutants (Table S1) was
2
substituents, WT TbSADH and many other ADHs as well as assayed. The results are summarized in Table 1.
1
modern Ru-based synthetic catalysts fail to deliver sufficiently
high enantioselectivity.
O
HO
HO
ADH
O
O +
(R)-2
O
NAD(P)H
1
(S)-2
Scheme 1. ADH-catalyzed asymmetric reduction of prochiral
ketone 1.
Protein engineering based on rational site-specific
7
5,8
mutagenesis or directed evolution has been applied for
enhancing and sometimes inverting the enantioselectivity of
ADH-catalyzed ketone reductions. For example, Phillips has
engineered several active and enantioselective mutants of
TbSADH which catalyze the asymmetric reduction of several
Scheme 2. Asymmetric reduction of ketones 3a-g catalyzed by
TbSADH mutants previously evolved as catalysts for the reduction
of substrate 1.
9
Table 1. Biocatalytic reduction of ketones 3a-g by TbSADH mutants evolved for substrate 1.
Reactions
3d→4d
a
b
Entry
ADH
code
3a→4a
ee%
3b→4b
ee%
3c→4c
ee%
3e→4e
ee%
90(S)
99(S)
99(S)
99(S)
nd
nd
nd
nd
nd
3f→4f
ee%
>99(S)
71(S)
>99(S)
>99(S)
94(S)
>99(S)
nd
>99(S)
>99(S)
>99(S)
>99(S)
90(S)
>99(S)
>99(S)
>99(S)
>99(S)
>99(S)
>99(S)
>99(S)
>99(S)
>99(S)
>99(S)
3g→4g
ee%
92(R)
27(R)
71(R)
9(S)
74(R)
44(R)
5(R)
39(R)
64(R)
96(R)
22(R)
8(R)
21(R)
97(R)
>99(R)
91(R)
>99(R)
50(R)
86(R)
17(S)
59(R)
4(S)
c%
12
25
98
97
90
<5
<5
<5
<5
96
97
26
99
98
71
97
98
6
c%
46
99
>99
>99
8
7
7
7
c%
36
16
82
65
86
<5
<5
<5
7
97
77
20
98
98
97
97
99
8
ee%
>99(S)
97(S)
>99(S)
>99(S)
>99(S)
95(S)
c%
>99
>99
>99
>99
75
c%
37
94
94
95
<5
<5
<5
<5
<5
<5
92
95
95
8
c%
>99
>99
>99
>99
34
29
<5
13
24
18
36
99
>99
97
84
79
98
98
99
22
32
c%
92
77
76
55
81
50
21
41
59
99
28
69
43
99
99
95
>99
55
97
25
59
32
1
2
3
4
5
6
7
8
9
WT
18(S)
91(S)
>99(S)
>99(S)
97(R)
72(R)
nd
50(R)
28(R)
99(R)
99(R)
92(S)
>99(S)
98(R)
93(S)
96(R)
99(R)
56(R)
96(S)
5(R)
78(S)
51(S)
91(S)
96(S)
63(S)
81(S)
89(S)
87(S)
24(S)
nd
41(S)
87(S)
>99(S)
96(R)
95(S)
54(R)
77(R)
>99(S)
>99(S)
73(S)
85(S)
>99(S)
45(S)
95(S)
98(S)
97(S)
93(R)
nd
SZ2006
SZ2007
SZ2012
SZ2052
SZ2055
SZ2056
SZ2057
SZ2058
SZ2063
SZ2074
SZ2110
SZ2111
SZ2114
SZ2172
SZ2176
SZ2205
SZ2218
SZ2219
SZ2224
SZ2225
SZ2257
15
8
7
84
nd
nd
>99(S)
92(S)
10
<5
9
36(S)
98(R)
91(S)
98(S)
>99(S)
97(R)
97(R)
97(R)
98(R)
51(R)
98(S)
90(R)
15(S)
82(S)
>99(S)
>99(S)
>99(S)
>99(S)
>99(S)
>99(S)
>99(S)
>99(S)
>99(S)
>99(S)
>99(S)
>99(S)
>99(S)
>99(S)
10
11
12
13
14
15
16
17
18
19
20
21
22
34
nd
>99
>99
>99
99
99(S)
99(S)
99(S)
92(S)
nd
87(S)
93(S)
93(S)
99(S)
95(S)
86(S)
99(S)
>99
99
95
99
13
41
99
>99
19
19
97
64
94
98
91
99
97
69
98
<5
<5
7
9
95
<5
11
<5
98
59
24
10
97
13
<5
64
10(R)
69(S)
96

3
a
b
The mutations of the ADH codes are summarized in Table S1, Reaction time is 2h. The others are 14~16h; nd: not determined.
Several results are noteworthy, beginning with the
WT TbSADH and the best (R)- and (S)-selective mutants
performance of WT TbSADH. It proved to be a poor catalyst in SZ2074 and SZ2172, respectively, were characterized by
the reduction of ketones 3a-c, both enantioselectivity and kinetic experiments (Table 2). Relative to WT, both mutants
conversion under the reaction conditions being unacceptable. In show notably higher catalytic efficiency as reflected by the
contrast, several mutants are excellent catalysts. For example,
respective
kcat/K
m
values. There is some tradeoff in
the poor (S)-selectivity (18% ee) and low conversion (12%) of thermostability (Table 2), but the mutants are still robust and
WT TbSADH in the reaction of acetophenone (3a) was boosted likely to be acceptable candidates of industrial applications.
by several variants to >99% ee at 97-98% conversion. One of the best mutants, SZ2074, was tested under preparative
Moreover, it was possible to invert stereoselectivity with scale conditions using 30 mM (129 mg) of ketone 3a. The
preferential formation of (R)-4 using several mutants (99% ee; conversion reached 94% within
7-98% conversion). Similar results were observed for remaining at 99% ee with an isolated yield of 75%.
substrates 3b-c. It is interesting to note that single mutants
SZ2006 (W110A) and SZ1114 (I86A) have been prepared Table 2. Kinetic parameters of WT TbSADH and best
6 h, enantioselectivity
9
9a
previously by directed evolution based on a different strategy.
mutants in the reaction of substrate 3a
W110A is a poor catalyst for substrates 3a-c, and I86A
9a
-1
5
provides only to (R)-enantiomeric products. The advantage of
the present new mutants is the possibility of obtaining both (R)-
Enzymes
K
m
(mM)
kcat (min )
k
cat/K
m
Thermostability
15
-
1
-1
(
min M )
94.69
171.92
144.63
T
50 (℃)
5
WT
SZ2074
SZ2172
19.01±1.68
15.82±0.81
18.53±1.18
1.80±0.07
2.72±0.06
2.68±0.08
86
70
75
and (S)-products (see also Table S1).
In the case of substrates 3d-f, WT TbSADH shows good to
excellent enantioselectivity in the range 90-99% ee in favor of
the (S)-alcohols, although conversion in the reaction of 3e
turned out to be moderate (37% with 90% ee). In this case
several mutants led to values of 99% ee and 94-95% conversion.
However, for the keto-ester 3g, WT TbSADH leads to (R)- 4g
In order to explain the effect of mutations on
stereoselectivity at the molecular level, induced fit docking
calculations using substrate 3a were performed. The
configuration of reduced product is determined by attack of the
nicotinamide hydride of NADPH from either the Si- or Re-face
of the ketone, which leads to the (R)- or (S)-alcohol,
respectively. Upon docking substrate 3a into WT TbSADH,
both Si- and Re-face additions of hydride to the carbonyl group
appeared to be possible in the least-energy conformations (Fig.
S1). This model explains the low enantioselectivity (18% S)
observed for the WT-catalyzed reduction of 3a. The situation in
the case of the best mutants is quite different. According to the
docking results using variant SZ2074, the nicotinamide ring of
cofactor NADPH is located at the Si-face of 3a (Fig. 1a),
consistent with the observed (R)-selectivity. In the case of the
(
92% ee), which is the key precursor for the production of
11
angiotensin-converting enzyme (ACE) inhibitors. Fortunately,
several mutants show improved enantioselectivity reaching 99%
ee with full conversion. Although all of the ketones are already
structurally very different from the original substrate 1, we
decided to go one step further by subjecting the very bulky
benzophenone derivatives 5a-b to reduction using the same
TbSADH variants (Scheme 3). None of the mutants accepted
these ketones, probably due to steric reasons. In a previous
directed evolution study of TbSADH reported by Phillips and
coworkers, mutants for the stereoselective reduction of
structurally similar benzophenone substrates were in fact
9a
evolved.
Thus, a toolbox of different robust TbSADH
(
S)-selective variant SZ2172, the Re-face of 3a is exposed to
mutants now exists for a variety of different substrates.
hydride attack by NADPH (Figure 1b). Additionally, the
distances between NADPH cofactor and the carbonyl C-atom
of the substrate are shorter in mutants SZ2074 and SZ2172 than
WT TbSADH, in line with the observation that both mutants
show higher catalytic efficiency relative to WT TbSADH.
Scheme 3. Attempted asymmetric reduction of ketones 5a-b
catalyzed by TbSADH mutants previously evolved for the
reduction of substrate 1.

4
Figure 1. Docking poses of 3a in (a) SZ2074 mutant of TbSADH and in (b) SZ2172 mutant. The distance between NADPH cofactor and
the carbonyl C-atom of the substrate are highlighted by green dashed lines.
Tetrahedron: Asymmetry 2009, 20, 513-557; (e) Musa, M. M.;
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In conclusion, several robust mutants of TbSADH, previously
3
.
evolved for the asymmetric reduction of the sterically small
5
prochiral ketone 1 with little tradeoff in thermostability, proved
to be active and highly stereoselective in the reduction of a set of
structurally unrelated and sterically more demanding ketones 3a-
g, but not of the extremely bulky benzophenone derivatives 5a-b.
In most cases these variants clearly out-performed WT TbSADH
in terms of conversion and enantioselectivity. Thus, the substrate
scope of this robust ADH has been expanded, while extending
4
.
5.
6
.
1
983, 48, 2767-2769; (b) Nobili, A.; Gall, M. G.; Pavlidis, I. V.;
5
,9a,9b,9h
the list of useful TbSADH mutants.
If stereoselectivity of
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the present mutants utilizing other substrates in future studies
should prove to be insufficient, they can be employed as
templates for further genetic optimization.
7
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Supplementary Material
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Supplementary data associated with this article can be found, in
the online version, at www.elsevier.com.
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Highlights



Wide substrate scope of alcohol dehydrogenase mutants in asymmetric ketone reduction.
Thermostable alcohol dehydrogenase mutants in enantioselective ketone reduction.
Mild stereoselective biocatalysts as an alternative to chiral man-made Ru-catalysts.