Substituent Position-Controlled Stereoselectivity in Enzymatic Reduction of Diaryl- and Aryl(heteroaryl)methanones

Article abstract of DOI:10.1002/adsc.201801543

We report here the discovery of a novel ketoreductase (KRED), named KmCR2, with a broad substrate spectrum on bioreduction of sterically bulky diaryl- and aryl(heteroaryl)methanones. The position of the substituent on aromatic rings (meta versus para or ortho) was revealed to control the stereospecificity of KmCR2. The stereoselective preparation of both enantiomers of diaryl- or aryl(heteroaryl)methanols using strategically engineered substrates with a traceless directing group (bromo group) showcased the potential application of this substrate-controlled bioreduction reaction. The combined use of substrate engineering and protein engineering, was demonstrated to be a useful strategy in efficiently improving stereoselectivity or switching stereopreference of enzymatic processes. (Figure presented.).

Full text of DOI:10.1002/adsc.201801543

Title: Substituent Position-Controlled Stereoselectivity in Enzymatic
Reduction of Diaryl- and Aryl(heteroaryl)methanones
Authors: Zhining Li, Zexu Wang, Yuhan Wang, Xiaofan Wu, Hong Lu,
Zedu Huang, and Fener Chen
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10.1002/adsc.201801543
Advanced Synthesis & Catalysis
FULL PAPER
DOI: 10.1002/adsc.201501543
Substituent Position-Controlled Stereoselectivity in Enzymatic
Reduction of Diaryl- and Aryl(heteroaryl)methanones
Zhining Li,a, b Zexu Wang,a, b Yuhan Wang,a Xiaofan Wu,c Hong Lu,d, e Zedu Huang,*a,
b and Fener Chen*a, b
a
Engineering Center of Catalysis and Synthesis for Chiral Molecules, Department of Chemistry, Fudan University
220 Handan Road, Shanghai, 200433, P. R. China, E-mail: huangzedu@fudan.edu.cn, rfchen@fudan.edu.cn
Shanghai Engineering Research Center of Industrial Asymmetric Catalysis of Chiral Drugs, 220 Handan Road,
b
Shanghai, 200433, P. R. China
College of Chemical Engineering, Fuzhou University, 2 Xueyuan Road, Fuzhou, 350100, P. R. China
State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, 2005 Songhu Road,
c
d
Shanghai, 200438, P. R. China
Shanghai Engineering Research Center of Industrial Microorganisms, 2005 Songhu Road, Shanghai, 200438, P. R.
e
China
Received: ((will be filled in by the editorial staff))
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/adsc.201901543.
Abstract. We report here the discovery of a novel
ketoreductase (KRED), named KmCR2, with a broad
substrate spectrum on bioreduction of sterically bulky diaryl-
and aryl(heteroaryl)methanones. The position of the
substituent on aromatic rings (meta versus para or ortho)
was revealed to control the stereospecificity of KmCR2. The
stereoselective preparation of both enantiomers of diaryl- or
aryl(heteroaryl)methanols using strategically engineered
substrates with a traceless directing group (bromo group)
showcased the potential application of this substrate-
controlled bioreduction reaction. The combined use of
substrate engineering and protein engineering, was
demonstrated to be a useful strategy in efficiently improving
stereoselectivity or switching stereopreference of enzymatic
processes.
Keywords: Oxidoreductase; Asymmetric catalysis;
Substrate controlled stereoselectivity; Ketoreductase;
Diarylmethanol
substrate engineering strategy was also reported for
Introduction
hydroxynitrile
lyase,[2k]
lipase[2l-2o]
,
enoate
reductase[2p-2r] and glycosyltransferase[2s,2t] mediated
biotransformations. Finally, the combination of
protein engineering and substrate engineering
exhibits synergistic effect in some studies.[3]
Undoubtedly protein engineering is one major player
that transforms biocatalysis into an increasingly
sophisticated technology for organic synthesis of
high-value molecules.[1] On the other hand, it is worth
noting that substrate engineering is a useful
alternative approach to improve enzymatic processes,
the appropriate employment of which resulted in
tremendous successes previously.[2] For example,
Griengl and co-workers has introduced the
‘docking/protecting’ (d/p) group concept into
monooxygenase mediated biohydroxylation reactions,
achieving substantially improved reactivity and
selectivity.[2a,2b] Removable silyl groups were proved
as effective directing groups in ketoreductase
Figure 1. Select examples of pharmaceutical molecules
derived from diaryl- or aryl(heteroaryl)methanols or aryl
heteroarylmethylamines.
(KRED) catalyzed enantioselective synthesis of 3-
[2h,2i]
butyn-2-ol.
In addition, successful adoption of
1
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10.1002/adsc.201801543
Advanced Synthesis & Catalysis
Scheme 1. KmCR2-catalyzed synthesis of mono-substituted diaryl- or aryl(heteroaryl)methanols. [a] The analytical scale
reaction was carried out with 10 mM ketone substrate, 20 mM glucose, 0.2 mM NADP+, 12.3 M KmCR2 and 12.1 M
glucose dehydrogenase (GDH) in 1 mL of 50 mM NaPi, pH 7.0 at 30 ℃ and 180 rpm for 24 h. The reaction conversion
was determined using GC. [b] The preparative scale reaction was carried out with 250 mg ketone substrate, 1 g glucose, 50
mg NADP+, 35 mL 15% (w/v) cell-free extract (CFE) of KmCR2, 10 mL 15% (w/v) CFE of GDH and 5 mL DMSO at
30 ℃ for 24 h. The isolated yields are given in parentheses. [c] The ee from the analytical scale reaction was determined
using chiral HPLC. [d] The absolute configuration was determined by comparing the sign of the optical rotation of the
major enantiomer, the elution order in chiral HPLC with known data, or by 1H NMR spectroscopy using Mosher’s reagent.
[e]
The
ee
was
not
determined
(N.D.)
because
the
two
enantiomers
were
inseparable.
Chiral diaryl- and aryl(heteroaryl)methanols, as well
as their amine derivatives, are privileged structural
elements present in many pharmaceutical molecules
(Figure 1).[4] One direct approach to synthesize
diaryl- and aryl(heteroaryl)methanols is the reduction
of diaryl- and aryl(heteroaryl)methanones.[5] The
subtle structural differences between the two
aromatic rings of diarylmethanones render them
extremely challenging targets for asymmetric
named KmCR2, which shows a broad substrate
spectrum for the bioreduction of diaryl- and
aryl(heteroaryl)methanones. We disclose that the
position of the substituent on the substrate (meta
versus para or ortho) controls the stereospecificity of
KmCR2. Catalyzed by a single enzyme, both
enantiomers of diaryl- or aryl(heteroaryl)methanols
were prepared through reduction of strategically
designed substrates with a traceless directing group.
Moreover, the enantioselectivity of several
engineered substrates was improved further by using
protein engineering.
chemical
reduction.
Synthetically
useful
enantioselectivity has only been achieved for
diarylmethanone substrates that have either high
electronic dissymmetry or an ortho-substituent on
one of the aromatic rings.[6] Despite the enormous
progress made in enzyme-catalyzed ketone reduction,
Results and Discussion
efficient
bioreduction
of
diaryl-
and
Using KpADH,[7c]
a reported diarylmethanone
aryl(heteroaryl)methanones remains challenging due
to their sterically bulky nature.[5,6b,7] Ketoreductase
with ability to enantioselectively reduce a broad
range of diaryl- and aryl(heteroaryl)methanones is
highly wanted. Continuing our interests in
biocatalysis,[8] herein we report a novel ketoreductase
reductase from Kluyveromyces polysporus as the
query, a BLAST search against the genome of
Kluyveromyces marxianus CBS4857 uncovered the
presence of a putative diarylmethanone reductase
(49% amino acid identity), which we named KmCR2.
2
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10.1002/adsc.201801543
Advanced Synthesis & Catalysis
The corresponding gene was PCR amplified from
genomic DNA and ligated with linearized pET28a.
Transformation into E. coli BL21 (DE3) followed by
heterologous expression furnished a soluble N-
terminally His6-tagged protein of the expected size
(Figure S1). Using benzophenone as the substrate,
KmCR2’s catalytic ability was confirmed. Further,
we found that the enzyme prefers NADPH over
NADH, and the optimal reaction pH and temperature
are 7.0 and 45 ℃, respectively (Figure S2-S3).
position-dependent stereoselectivity was also
observed for the group II substrates. For ketones
consisting of a para-substituted phenyl and a meta-
substituted phenyl ring (3g and 3h), the reactions
afforded the corresponding alcohols in excellent
enantioselectivity. On the contrary, the combination
of a para-substituted phenyl and an ortho-substituted
phenyl ring led to the formation of alcohols 4j and
4k with unsatisfactory enantioselectivity.
With the enzyme activity confirmed, we employed
14 mono-substituted benzophenones (left box,
Scheme
1)
and
12
mono-substituted
phenyl(heteroaryl)methanones (right box, Scheme 1)
to explore the substrate spectrum of KmCR2. In
analytical scale reactions, most substrates were
completely consumed, and the desired alcohol
products were produced with varying levels of
enantioselectivity (up to 99% ee). Interestingly, we
noticed a striking correlation between the position of
the substituent and the facial selectivity. For instance,
meta-methyl benzophenone (1b) was reduced to
generate the corresponding (R)-alcohol (2b) with
74% ee. In contrast, substrates with a methyl group
on the para- (1a) or ortho- (1c) positions both gave
(S)-alcohols stereoselectively. Apparently this
substituent position-dependent stereoselectivity is
general, irrespective of the type of substituent
(fluorine, chlorine or methoxy) or the type of ketone
(benzophenone or phenyl(heteroaryl)methanone).
Previously, a similar substituent position dependent
stereopreference was observed for mutant enzymes
of thiamine diphosphate (ThDP)-dependent enzymes
EcMenD, BsMenD and ApPDC, and the reason
behind this phenomenon is still unknown.[9] To the
best of our knowledge, this kind of substrate
(substituent position) dependent stereoselectivity has
not been reported for enzyme-catalyzed diaryl- or
aryl(heteroaryl)methanone reduction reactions. The
developed bioreduction was performed in preparative
scale (250 mg ketone substrate) for select substrates,
affording the desired alcohols in good isolated yields
(up to 92%) and with similar enantiomeric purities as
those observed in analytical scale reactions (see SI
for details).
We next studied the bioreduction of bis-substituted
diarylmethanones, which can be divided into two
groups based on whether both two aromatic rings
contain a substituent (Scheme 2). When the two
substituents were attached to the same ring (group I),
the substituent positions strongly influenced
enantioselectivity. For instance, substrates bearing at
least one substituent at the meta-position (3a, 3b, 3c
and 3d) were reduced in poor to moderate
enantioselectivity (25% to 69% ee). In contrast,
ketone 3e with two chlorines at ortho- and para-
positions was biotransformed into alcohol 4e in 99%
ee. Probably due to the steric hinderance, KmCR2
was unable to reduce ketone 3f, containing a chloro
group at both ortho- positions. The substituent-
Scheme 2. KmCR2-catalyzed synthesis of bis-substituted
diarylmethanols. [a] The analytical scale reaction was
carried out with 10 mM ketone substrate, 20 mM glucose,
0.2 mM NADP+, 12.3 M KmCR2 and 12.1 M glucose
dehydrogenase in 1 mL of 50 mM NaPi, pH 7.0 at 30 ℃
and 180 rpm for 24 h. The reaction conversion was
determined using GC. [b] The ee was determined using
chiral HPLC. [c] The absolute configuration was
determined by comparing the elution order in chiral HPLC
with known data.
Collectively, the above studies suggest that an
intrinsic property of the substrates, the substituent
position, governs the observed stereoselectivity of
the KmCR2-catalyzed bioreduction of diaryl- and
aryl(heteroaryl)methanones. Given that such
substrate-dependent
stereoselective
reduction
reactions possess great potential in organic
synthesis,[2j,10] we next devoted our efforts to
examining the synthetic utility of KmCR2 in this
regard. To better guide the experimental design, we
first constructed a model to aid in predicting the
stereochemical outcomes of KmCR2-catalyzed
reactions (Figure 2). According to the data shown in
Scheme 1 and 2, we concluded the priority of aryl- or
heteroaryl units as follows: ortho- or para-
substituted phenyl> pyridinyl> phenyl> thiophenyl>
meta-substituted phenyl. Under this definition, the
attack on the carbonyl group by hydride from
NADPH would occur from the back face,
3
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10.1002/adsc.201801543
Advanced Synthesis & Catalysis
affording alcohol products with the indicated
stereochemistry (Figure 2).
meta-position, substrates engineered at the para-
position
were
generally
reduced
more
enantioselectively, regardless of retention or
inversion of configuration. For example, bromo-
incorporation at the para-position of ketone 1t
generated the engineered substrate 5e. The sequential
bioreduction and debromination reactions yielded
alcohol 2t with inverted configuration as expected
and with remarkably high enantioselectivity (99%
Figure 2. Model for predicting the stereochemical
outcomes of KmCR2-catalyzed reduction of diaryl-
and aryl(heteroaryl)methanones. Aromatic rings of
higher (A ring) and lower (B ring) priorities were
colored with blue and red, respectively.
With this model, we envisioned that we could
enzymatically synthesize both enantiomers of diaryl
or aryl(heteroaryl)methanols using
a
traceless
directing group (substrate engineering strategy). For
instance, assume that KmCR2 reduces the
unmodified ketone I to the corresponding alcohol II
with low ee (Scheme 3). By strategically
incorporating a bromo group onto the meta-position
of aromatic ring B of lower priority (substrate III),
the priority difference between the two aromatic
rings becomes more significant, and the bioreduction
is expected to proceed with greater enantioselectivity.
Upon debromination, the same alcohol II would be
generated with improved ee. On the other hand, the
attachment of a bromo group onto the para-position
of aromatic ring B is expected to reverse the
priorities of the two aromatic rings (substrate V). As
a result, KmCR2-catalyzed reduction followed by
debromination would furnish alcohol II with inverted
configuration. Not only the aromatic ring of lower
priority, but the one of higher priority could be
engineered in a similar fashion to afford the same
pair of enantiomeric products (Scheme S8). We
decided to use the bromo group mainly because of its
ease of removal, rendering it as a traceless directing
group.
Six diaryl- or aryl(heteroaryl)methanones (1b, 1o, 1p,
1g, 1t and 1u, Figure 3) were selected to validate our
substrate engineering strategy. The enantioselectivity
of KmCR2 towards those substrates was in the range
of 42% to 84% ee. For all six substrates, by
selectively incorporating a bromo group to the
designated position, alcohol products were obtained
either with significantly improved ee or with an
inverted configuration. In particular, both
enantiomers of alcohol 2t were synthesized in
excellent enantiomeric purity (>90% ee), which in
principal can be further transformed to the
synthetically useful phosphite-pyridine ligands.[11]
Compared to their counterparts engineered at the
ee).
Scheme 3. Design principle for KmCR2-catalyzed
synthesis of both enantiomers of diaryl or
aryl(heteroaryl)methanols using a traceless directing
group (substrate engineering strategy). Aromatic rings
of higher and lower priorities were colored with blue
and red, respectively.
Given the fact that substrates engineered at the meta-
position (e.g. 5b and 5c, Figure 3) were reduced less
stereoselectively by KmCR2, we sought to test if the
stereoselectivity could be improved using protein
engineering. Thus, a homology model of KmCR2
was built and 14 amino acid residues were found to
be within 5 Å of the docked substrate phenyl(m-
tolyl)methanone 1b (Figure S5). Residues C85, F87,
F162, S197, Y198, F215 and S216 were found to be
close to the m-tolyl ring, and we hypothesized that
the stereoselectivity could be improved by
engineering those residues. In this regard, the
mutation of one residue to a smaller amino acid (e.g.
Ala) might increase space to better accommodate
meta-substituted substrates therefore resulting in
higher stereoselectivity. To test this hypothesis, we
first individually mutagenized each of the above 7
residues. Indeed, compared with wild-type KmCR2,
2 such mutants (C85A and F162A) exhibited
improved stereoselectivity towards meta-substituted
substrates 1b, 1e and 1s (Table S3). On the other
hand, by substituting Cys85 with the branched amino
acid Val, this generated mutant showed diminished
stereoselectivity relative to the wild-type enzyme,
indirectly supporting our hypothesis that increasing
4
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10.1002/adsc.201801543
Advanced Synthesis & Catalysis
Figure 3. KmCR2-catalyzed preparative-scale synthesis of both enantiomers of diaryl or aryl(heteroaryl)methanols using a
substrate engineering strategy. Aromatic rings of higher and lower priorities were colored with blue and red, respectively.
the room around the meta-substituted phenyl ring
improves stereoselectivity.
Importantly, the use of these beneficial single
mutants and the corresponding double mutant
(C85A/F162A) improved the stereoselectivity of the
difficult substrates 5b and 5c encountered in the
substrate engineering study (Table S4). For example,
phenyl(thiophen-3-yl)methanone (1p) was reduced
selectively to the R-enantiomer of phenyl(thiophen-
3-yl)methanol (2p) with only 75% ee by wild-type
KmCR2 (Figure 4). Through our substrate
engineering approach, we were able to increase the
enantioselectivity of the reduction of the engineered
substrate (6c with a para-bromo group) to the R-
alcohol to 99% ee. However, the S-alcohol was
prepared with only 39% ee from the engineered
substrate 5c with a meta-bromo group. Gratifyingly,
our engineered enzyme, C85A/F162A increased the
enantiomeric purity of the S-alcohol to 83% ee for
the reduction of 5c. The efficiency of our
engineering strategy is noteworthy: we were able to
enhance the enantioselectivity of our reaction by
screening only 9 mutant enzymes.
Conclusion
In
stereoselectivity (meta versus para or ortho) was
revealed for KmCR2, novel diaryl- or
aryl(heteroaryl)methanone reductase discovered
summary,
substituent
position-dependent
Figure 4. Improved stereoselective synthesis of both
enantiomers of phenyl(thiophen-3-yl)methanol (2p
with the combined use of substrate engineering and
protein engineering.
)
a
through genome mining. Using
a
substrate
5
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10.1002/adsc.201801543
Advanced Synthesis & Catalysis
engineering approach, both enantiomers of diaryl- or
aryl(heteroaryl)methanols were chemo-enzymatically
synthesized from designed substrates with a traceless
directing group, showcasing the potential application
of such substrate-controlled stereoselective enzyme
processes. The combined use of substrate
engineering and protein engineering, was further
demonstrated to be a useful strategy in efficiently
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improving
stereoselectivity
or
switching
stereopreference of enzymatic reactions.
Experimental Section
KmCR2-catalyzed reduction in analytical scale
To a solution of 10 mM ketone substrate, 20 mM glucose
and 0.2 mM NADP+ in 50 mM NaPi buffer (pH 7.0), were
added KmCR2 and glucose dehydrogenase (GDH) (0.5
mg/mL final concentration for each enzyme). The 1.5 mL
Eppendorf tube containing 1 mL of the above mixture was
shaken at 180 rpm and 30 ℃. After 24 h, the reaction
mixture was extracted with EtOAc (5 mL) and the organic
layer was subjected to GC-MS and chiral HPLC analyses.
The reaction conversion was determined using GC-MS on
Agilent 5975 equipped with HP-5MS column (30 m x
0.25 mm, 0.25 mm film). The temperature program is as
follows: 80 ℃ for 2 min, 20 ℃/min from 80 to 280 ℃,
280 ℃ for 7 min.The ee of the products was determined
using chiral HPLC, and the detailed data is shown in Table
S1. The absolute configuration of certain products was
determined by comparing the elution order in chiral HPLC
with known data.
KmCR2-catalyzed reduction in preparative scale
The preparative scale reaction was carried out with 250
mg ketone substrate, 1 g glucose, 50 mg NADP+, 35 mL
15% (w/v) cell-free extract (CFE) of KmCR2, 10 mL 15%
(w/v) CFE of GDH and 5 mL DMSO (for substrates 1o
and 1p, 0.5 mL DMSO was used) at 30 ℃and 600 rpm for
24 h. The reaction mixture was extracted with EtOAc. The
organic layer was then washed with brine and dried with
anhydrous Na2SO4, then filtered, and the filtrate was
evaporated to dryness. The product was purified by flash
chromatography (silica gel, petroleum ether: EtOAc = 4:1).
The ee of the products was determined using chiral HPLC.
The absolute configuration was determined by comparing
either the sign of the optical rotation of the major
enantiomer or the elution order in chiral HPLC with
known data.
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Acknowledgements
We thank Prof. Qi Zhang (Fudan University), Dr. Weixin Tang
(Harvard University) and Mr. Abraham Wang (UIUC) for
critical proofreading of this manuscript. Prof. Wei Li and Prof.
Xiaojian Hu (Fudan University) are acknowledged for the help
of docking study. Financial support from Shanghai Sailing
Program (18YF1402100) is greatly appreciated.
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10.1002/adsc.201801543
Advanced Synthesis & Catalysis
FULL PAPER
Substituent Position-Controlled Stereoselectivity in
Enzymatic Reduction of Diaryl- and
Aryl(heteroaryl)methanones
Adv. Synth. Catal. Year, Volume, Page – Page
Zhining Li, Zexu Wang, Yuhan Wang, Xiaofan
Wu, Hong Lu, Zedu Huang,* and Fener Chen*
TOC Graphic
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