Modification of the Enantioselectivity of Biocatalytic meso-Desymmetrization for Synthesis of Both Enantiomers of cis-1,2-Disubstituted Cyclohexane by Amidase Engineering

Article abstract of DOI:10.1002/adsc.202100597

Both enantiomers of cis-1,2-disubstituted cyclohexane have been obtained enantioselectively through engineered amidase-catalyzed desymmetrization of meso carbocyclic 1,2-dicarboxamides under mild condition. Based on the enzyme-substrate binding model sugg

Full text of DOI:10.1002/adsc.202100597

COMMUNICATIONS
doi.org/10.1002/adsc.202100597
Modification of the Enantioselectivity of Biocatalytic meso-
Desymmetrization for Synthesis of Both Enantiomers of cis-1,2-
Disubstituted Cyclohexane by Amidase Engineering
Hui-Juan Hu,a, b Qi-Qiang Wang,a, c De-Xian Wang,a, c and Yu-Fei Aoa, c,*
a
Beijing National Laboratory for Molecular Sciences, CAS Key Laboratory of Molecular Recognition and Function, Institute of
Chemistry, Chinese Academy of Sciences, Beijing 100190 (People’s Republic of China)
E-mail: aoyufe@iccas.ac.cn
State Key Laboratory of NBC Protection for Civilian, Beijing 102205 (People’s Republic of China)
b
c
University of Chinese Academy of Sciences, Beijing 100049 (People’s Republic of China)
Manuscript received: May 18, 2021; Revised manuscript received: July 17, 2021;
Version of record online: ■■■, ■■■■
Supporting information for this article is available on the WWW under https://doi.org/10.1002/adsc.202100597
desymmetrization[5] of meso cyclic diesters.[6,7] Bioca-
Abstract: Both enantiomers of cis-1,2-disubstituted
talytic synthesis and in particular, esterase[6] or lipase-
cyclohexane have been obtained enantioselectively
catalyzed[7] hydrolysis, has demonstrated significant
through engineered amidase-catalyzed desymmetri-
advantages such as environmentally benign property,
zation of meso carbocyclic 1,2-dicarboxamides
mild reaction conditions, highly efficiency and enan-
under mild condition. Based on the enzyme-sub-
tioselectivity. However, one disadvantage of the bio-
strate binding model suggested by molecular dock-
catalytic approach is that only one enantiomer of the
ing, the amplification and reversal of enantioselec-
product can be obtained directly, whereas both the
tivity of amidase was quickly achieved through
enantiomers of products are frequently required for
generating and testing only 8 variants. The origin of
medicinal and synthesis applications. Protein engineer-
enantiopreference for amidase was revealed by MD
ing based on directed evolution could be a powerful
simulations and the reason for the reversal of
tool for addressing this challenge,[8] and the enantiose-
enantioselectivity towards amidase variant was
lectivity can be controlled, including improved and
disclosed. The synthetic application of biocatalytic
reversed, by reshaping the protein catalytic cavity.[9]
meso-desymmetrization was demonstrated by
straightforward transformation of the products to
As one of the most important hydrolases, amidase
[3.5.1.4] catalyzes the conversion of primary amides
enantiomerically pure heterocyclic compounds.
into carboxylic acids and releasing ammonia in the
process.[10] So far a large number of amidases and
microorganisms containing amidases have been
Keywords: Protein engineering; Biocatalysis; Ami-
dase; Desymmetrization; Enantioselectivity
identified;[11] Some of these amidases have been used
to investigate the desymmetrizations of meso or
prochiral diamides.[12] The only report on amidase-
catalyzed synthesis of enantiomerically enriched cis-
Enantiopure cis-1,2-disubstituted six-membered carbo- 1,2-disubstituted cyclohexene was shown in 1998 by
cyclic substrates occur as key intermediate for the T. Sugai,[13] who discovered that in the presence of
synthesis of many biologically active products such as Rhodococcus rhodochrous IFO 15564 cells, meso
anticapsin, (+)-aucantene, cis-carbapenems and vita- cyclohex-4-ene-1,2-dicarboxamide underwent enantio-
min D3.[1] As one most typical classes of these selective hydrolysis to yield corresponding monoacid
compounds, chiral 2-substituted cyclohex-4-ene-1-car- derivative with an unsatisfactory yield (39%).
boxylic acid derivates have attracted continuous and
We recently demonstrated Rhodococcus erythrop-
intense attention.[2] While resolution processes using olis AJ270 cells can efficiently transform carbocyclic
stoichiometric amounts of chiral reagents have been 1,3-dicarboxamides to enantiopure functionalized car-
developed,[3] direct enantioselective synthetic methods boxylic acids in good yields,[14] and amidase from
are rare. Typical examples include desymmetrization Rhodococcus erythropolis AJ270 was rationally engi-
alcoholysis of meso cyclic anhydrides by enantiomeri- neered to modified its enantioselectivity toward hetero-
cally pure base or acid catalysts[4] and biocatalyzed cyclic 1,3-dicarboxamides.[15] Our continuous interest
Adv. Synth. Catal. 2021, 363, 1–7
1
© 2021 Wiley-VCH GmbH
��
These are not the final page numbers!

COMMUNICATIONS
asc.wiley-vch.de
in synthetic biocatalytic capability of amides led us to binding mode. This would be helpful in rationally
investigate the desymmetrization of meso carbocyclic selecting the mutation position and strategy, and reduce
1,2-dicarboxamides. Here we report the synthesis of the screening effort. The optimal outcome of molecular
both enantiomers of cis-1,2-disubstituted cyclohexane docking was illustrated in Figure 1: The carbonyl of R-
using engineered amidase-containing E. coli cells. configurated amide group (R-amide) of substrate can
Furthermore, the resultant biocatalytic products were be activated by forming multiple hydrogen bonds with
applied to synthesize a series of useful enantiomeri- the oxyanion hole composed of G192, G193, G194
cally pure heterocyclic compounds. In addition, the and S195 (the active site); The short distance between
origin of enantioselectivity reversal was revealed by γ-oxygen atom of S195 (OS195) and the carbonyl carbon
means of MD simulation.
atom of the R-amide (2.3 Å) suggested the formation
The starting meso carbocyclic dicarboxamides 1a of a covalent substrate-enzyme intermediate, which
and 1b were easily prepared from known compounds was consistent with the widely accepted Bi-Bi Ping-
by the literature methods (see Supporting Pong type acyl-transfer mechanism;[10] Through hydro-
Information).[16] We initially investigated the biotrans- phobic effect and H-bonds, the cyclic backbone and S-
formation of meso cyclohex-3-ene-1,2-dicarboxamide configurated amide group (S-amide) of substate could
1a. As illustrated in Scheme 1, amidase-containing E. nicely fit with the narrowly surrounding residues such
coli whole cells (E. coli-Ami) were able to catalyze the as F146, I198, W328 and I450 (the recognition site).
hydrolysis of 1a to mono-acid product under mild This binding mode was reasonable because it implied
°
conditions (neutral phosphate buffer at 37 C). The the preference of R-amide hydrolysis, which corre-
reaction was efficient and complete semi-hydrolysis sponded with the experimental results in Scheme 1.
was achieved in one hour monitored by TLC (all the
Judging from binding mode shown above, the S-
subsequent biocatalytic reactions were performed amide of substrate 1a was surrounded by residues I198
under the same conditions). To facilitate product and I450, and its steric hindrance and hydrophobic
isolation and detection, the carboxylic acid was effect may not be conducive to substrate-enzyme
alkylated by benzyl bromide under base conditions, binding, thereby affecting catalytic performance, par-
yielding the benzyl ester 2a in 66% yield and 94%ee ticularly enantioselectivity. Following this hypothesis,
(Scheme 1A), indicating the high enantioselectivity of we expected that site-directed mutagenesis of the large
amidase. When the carbon-carbon double bond in the residue Ile into the smaller one Ala would improve the
cyclic skeleton was changed to single bond, 2b was hydrolytic activity. To our delight, the I198A mutant-
produced in 72% yield and >99%ee (Scheme 1B). containing E. coli (variant M1) showed not only high
The improved enantioselectivity was probably due to efficiency but also high enantioselectivity, furnishing
the increased flexibility of the carbocyclic ring. To product 2a in 96% yield and >99%ee, and complete
assign the absolute configuration, high-quality single semi-hydrolysis of substrate 1a in 15 minutes (Table 1,
crystal[17] of 2b was obtained by slow evaporation of entry 1). Similar outstanding catalytic performance
the solution in a mixture of hexane and acetone. X-ray
diffraction analysis unambiguously revealed the 1R,2S-
configured stereogenic centers in 2b (Figure S1, SI),
indicating R-enantiopreference of amidase toward
meso carbocyclic dicarboxamide substrates.
In order to improve amidase's enantioselectivity
toward substrate 1a, structure-guided engineering of
the protein[8] was undertaken. With the crystal structure
in hand,[18] we carried out the flexible molecular
docking of substrate 1a into the catalytic cavity of
amidase to better understand the substrate-enzyme
Figure 1. Molecular docking[19] of the substrate 1a into the
active site of amidase. Figures create by PyMOL[20] (A) and
LigPlus[21] (B). In figure 1A, the protein is drawn as a bluewhite
cartoon. The oxygen and nitrogen in the active pocket residues
and substrates are colored red and blue, respectively. The
carbon of substrates is colored green. The active site residues
are colored cyan. The recognition site residues are colored
Scheme 1. Biotransformations of substrates (A) 1a and (B) 1b.
brown.
Adv. Synth. Catal. 2021, 363, 1–7
2
© 2021 Wiley-VCH GmbH
��
These are not the final page numbers!

COMMUNICATIONS
asc.wiley-vch.de
Table 1. Variant-catalytic hydrolysis of substrate 1.[a]
Entry
1
Variant catalyst
Time
Product (yield %[b])
(ee %[c])
1
2
3
4
5
6
7
8
9
1a
1b
1a
M1 (I198A)
M1 (I198A)
M2 (I450A)
M3 (I450F)
M4 (I450S)
M5 (I450G)
15 min
10 min
45 min
90 min
2 h
2 h
3.5 h
2 h
2a (96) (>99)
2b (87) (>99)
ent-2a (87) (65)
2a (85) (70)
ent-2a (73) (10)
ent-2a (81) (16)
ent-2a (83) (98)
ent-2a (85) (98)
ent-2a (85) (>99)
ent-2b (76) (98)
M6 (I450A/F146A)
M7 (I450A/W328F)
M8[d]
1.5 h
9 h
10
1b
M8[d]
[a] Substrate (0.5 mmol) was incubated with variant-containing E. coli cells (0.5 g wet weight) in phosphate buffer (pH 7.0, 0.1 M)
°
at 37 C, and the acid product was alkylated by benzyl bromide (2 mmol) and K2CO3 (1 mmol) in 2 mL DMF solvent.
[b] Isolated yield.
[c] Determined by chiral HPLC analysis.
[d] I450A/F146A/W328F.
was demonstrated for substrate 1b, which could be
To further explore the origin of the biocatalytic
completely semi-hydrolyzed in only 10 min and enantiopreference toward substrate 1a, 100 ns molec-
afforded product 2b in 87% yield and >99%ee ular dynamic (MD) simulations were performed (Fig-
(Table 1, entry 2).
ure 2). Similar with the docking results in Figure 1, the
In contrast, the variant I450A (M2) reversed the MD simulation clearly showed that the R-amide of
enantioselectivity and yielded the enantiomer of 2a substrate rightly extended to the oxyanion hole of
(ent-2a) in 87% yield and 65%ee (Table 1, entry 3). It wild-type amidase (Figure 2A), and the carbon atom of
implied that I450A variant preferred of S-amide R-amide was closer to Oγ of S195 in distance than S-
hydrolysis, and this unexpected discovery prompted us amide during the simulation time (Figure 2C). While
to challenge the examination on thorough reversal of the binding mode was different when the variant M8
enantioselectivity, a critical subject to overcome the was used as biocatalyst: The S-amide was located in
limitation of its natural selectivity. However, switching the active site and closer to Oγ of S195 (Figure 2B and
I450 to residues Phe (M3), Ser (M4) and Gly (M5) did 2D), implying that the S-amide activation mode was
not obtain better results (Table 1, entries 4–6), imply- preferred, in line with the experimental results in
ing that a small and hydrophobic residue in this area Table 1. Interestingly, in both of the above binding
may be beneficial to enantioselectivity reversal. Based modes, the activated amide groups formed an equato-
on the above results, further decrease the bulkiness of rial bond with the cyclic backbone of substrates. On
residue in the recognition site (F146 and W328) was the other hand, the inactivated axial amide groups
performed and obtained variant M6 (I450A/F146A) demonstrated opposite orientation: The S-amide in
and M7 (I450A/W328F). To our delight, the variants Figure 2A extending to G193 and G194, while R-
M6 and M7 could both hydrolyze 1b and afforded amide in Figure 2B extending to A146. Based on the
corresponding product ent-2a in 98%ee (Table 1, above results, we proposed the reason for high
entry 7–8). Obviously, this result indicated the muta- enantioselectivity of amidase on desymmetrization.
tions of I450A, F146A and W328F were all beneficial For the wild-type amidase, the narrow catalytic cavity
to the reversal of enantioselectivity. Through accumu- forced the substrate to adjust its conformation and
lating the mutations, a triple variant M8 (I450A/ location to adopt the R-amide activation mode (Fig-
F146A/W328F) was obtained, which demonstrated not ure 2A), in spite of steric hindrance between axial S-
only high efficiency but also complete reversal of amide group and residue G194 (the shortest distance is
enantioselectivity, furnishing ent-2a in 85% yield and 3.0 Å). In terms of the variant M8, the mutation
>99%ee (Table 1, entry 9). The variant M8 was also enlarged the catalytic cavity and allowed the substrate
applied to substrate 1b, and ent-2b was achieved in to fit in the cavity with a more comfortable orientation
76% yield and 98%ee (Table 1, entry 10).
owing to the absence of steric hindrance with G194.
Adv. Synth. Catal. 2021, 363, 1–7
3
© 2021 Wiley-VCH GmbH
��
These are not the final page numbers!

COMMUNICATIONS
asc.wiley-vch.de
Scheme 2. Synthesis of enantiomerically pure heterocyclic
compounds 5, 8 and 9.
Figure 2. MD simulation (GROMACS 2018.8)[22] results of 1a
into the active site of wild-type amidase (Figure A and C) and
variant M8 (I450A/F146A/W328F) (Figure B and D). The
representative binding modes were shown in Figure A and B;
The fluctuation of the distance between carbonyl carbon atom
of 1a and the Oγ atom of Ser195 during the MD simulations
were shown in Figure C and D.
In conclusion, we demonstrated the versatile and
efficient synthesis of both enantiomers of vicinally
functionalized cyclohexane compounds under mild
condition using engineered amidase. Through protein
engineering, the catalytic enantioselectivity has been
improved or completely reversed. The origin of
enantiopreference of amidase has also been revealed
by MD simulations. As a result, this study not only
sheds light on the regulation of stereoselectivity by
To illustrate the synthetic application of biocatalytic engineered proteins, but also inspires the construction
method, the biocatalyzed meso-desymmetrization of of valuable enantiomerically pure building blocks
substrate 1a was conducted in the gram scale and led through biocatalysis.
to the formation of enantiopure mono-acid product 3a
(1.22 g, 90%) or ent-3a (1.15 g, 85%) (Scheme S3,
SI), which is an important building block for the
Experimental Section
synthesis of enantiomerically pure heterocyclic com- Materials
pounds. Heterocyclic compounds 5, 8 and 9 are
All commercial chemicals were used without further purifica-
tion. DNA manipulations including plasmid DNA extraction,
restriction endonuclease digestion, agarose gel electrophoresis,
valuable core structure of some natural products with
antibacterial and antitumor activity.[23] In the presence
of NaClO, enantiopure 3a smoothly underwent Hof-
mann Rearrangement to corresponding amino acid 4,
followed by cyclization with ethyl benzimidate afford-
ing tetrahydroquinazoline derivate 5 in 70% yield and
>99%ee as shown in Scheme 2. The reduction of 4
led to amino alcohol 6, which could be transformed
into compound 7 in a good yield, with the subsequent
intramolecular cyclization yielding tetrahydro-benzox-
azinone derivate 8 in 87% yield and >99%ee.
Enantiopure compound 9 was readily obtained from
compound 6, CS2 and Pb(NO3)2 under base condition.
Notably, no racemization was observed in any of
transformations and all of the products were enantio-
pure.
and the transformation were performed according to standard
protocols. All the restriction enzymes were purchased from
Thermo Fisher Scientific. High fidelity PCR DNA-polymerase,
and dNTPs were purchased from Vazyme Biotech Co., Ltd.
PCR primers were synthesized and DNA sequencing was
conducted by TsingKe Biotech Co., Ltd. Other common
biochemical and media components were obtained from
standard commercial sources and used directly. The plasmid
pET22b for amidase (from Rhodococcus erythropolis AJ270
strain) expression is gifted from Yapeng Chao and Shijun Qian
from Institute of Microbiology, Chinese Academy of
Sciences.[15]
Adv. Synth. Catal. 2021, 363, 1–7
4
© 2021 Wiley-VCH GmbH
��
These are not the final page numbers!

COMMUNICATIONS
asc.wiley-vch.de
vacuum, the residue was purified by silica gel chromatography
using ethyl acetate as mobile phase to give product 2a and 2b.
General procedure for the variant construction and
protein expression
E. Coli BL21 (DE3) strains were applied for heterologous
protein expression. The plasmid of wild-type amidase served as
a PCR template for generating variants I198A (M1), I450A
(M2), I450F (M3), I450S (M4) and I450G (M5); double
variants I450A/F146A (M6) and I450A/W328F (M7) were
obtained by using I450A as a template; triple variants I450A/
F146A/W328F (M8) were respectively obtained by using
I450A/F146A as template. The PCR mixture (50 μL) contained
25 μL 2× Phanta Max Master Mix, 17 μL H2O, 2 μL DMSO,
2 μL (about 50 ng) template DNA and 2 μL (about 10 μM) each
Supporting Information
For the detailed description about general information,
synthesis procedure, variants construction, MD simu-
lations, characterization data, copies of H, 13C NMR
1
spectra, HPLC, and crystallographic data, please see
the Supporting Information.
°
primer mix. The PCR program used was 5 min at 95 C
Acknowledgements
°
°
followed by 30 cycles of 0.5 min at 95 C, 0.5 min at 60 C, and
°
°
6 min at 72 C and a final extension step for 7 min at 72 C and
Financial supports from National Natural Science Foundation
of China (21977098 and 21502202) are gratefully acknowl-
edged.
°
5 min at 15 C. After confirmation by 0.9% agarose gel
electrophoresis, the PCR products were incubated with DpnI
(1 μL) at 37 C for 8 h to digest the template plasmid, and the
reaction mixtures were transformed into competent E. coli
BL21 (DE3) PlysS cells and then plated on Luria-Bertani
medium agar plates containing 100 μg/mL of Ampicillin.
°
References
[1] a) R. M. Borzilleri, S. M. Weinreb, M. Parvez, J. Am.
Chem. Soc. 1995, 117, 10905–10913; b) J. E. Baldwin,
R. M. Adlington, M. B. Mitchell, J. Chem. Soc. Chem.
Commun. 1993, 1332–1335; c) S. Kobayashi, J. Shibata,
M. Shimada, M. Ohno, Tetrahedron Lett. 1990, 31,
1577–1580; d) S. Kobayashi, Y. Eguchi, M. Shimada, M.
Ohno, Chem. Pharm. Bull. 1990, 38, 1479–1484; e) H.-J.
Gais, K. L. Lukas, W. A. Ball, S. Braun, H. J. Lindner,
Liebigs Ann. Chem. 1986, 1986, 687–716; f) W. Boland,
U. Niedermeyer, L. Jaenicke, H. Görisch, Helv. Chim.
Acta. 1985, 68, 2062–2073.
[2] a) K. T. Tran, J. S. Pallesen, S. M. Solbak, D. Narayanan,
A. Baig, J. Zang, A. Aguayo-Orozco, R. Carmona, A.
Garcia, A. Bach, J. Med. Chem. 2019, 62, 8028–8052;
b) L. Turbanti, G. Cerbai, C. D. Bugno, R. Giorgi, G.
Garzelli, M. Criscuoli, A. R. Renzetti, A. Subissi, G.
Bramanti, J. Med. Chem. 1993, 36, 699–707.
The pre-cultures of 5 mL Luria-Bertani broth (1% Tryptone, 1%
NaCl, and 0.5% yeast extract) containing 100 μg/mL ampicillin
were inoculated by selecting one single colony of E. coli BL21
°
(DE3). After incubation overnight at 37 C and 220 rpm, the
precultures were diluted 1:100 into 300 mL LB medium
containing Ampicillin and grown to an optical density of
°
600 nm reach around 0.6~0.8. After cooling at 4 C for 0.5 h,
Protein expression was induced by the addition of 300 μM
isopropyl-β-D-thiogalactopyranoside (IPTG), and growth con-
°
tinued for an additional 6 h at 25 C. The cells were harvested
°
by centrifugation at 8000 rpm and 4 C for 5 min and the
supernatants were discarded. Then they were re-suspended in
°
phosphate buffer (pH 7.0, 0.1 M) and stored in À 20 C. All the
resulting variants sequences were confirmed by DNA sequenc-
ing.
General procedure for the biotransformations
[3] a) C. Bolm, A. Gerlach, C. L. Dinter, Synlett 1999, 1999,
195–196; b) I. N. Houpis, A. Molina, R. A. Reamer,
Tetrahedron Lett. 1993, 34, 2593–2596; c) N. Tamura, Y.
Kawano, Y. Matsushita, K. Yoshioka, M. Ochiai,
Tetrahedron Lett. 1986, 27, 3749–3752.
In an Erlenmeyer flask (150 mL) with a screw cap a suspension
of E. coli cells (0.5 g wet weight) in aqueous phosphate buffer
°
(pH 7.0, 0.1 M, 25 mL) was activated at 37 C for 0.5 h.
Substrates 1a or 1b (0.5 mmol) was dissolved in aqueous
phosphate buffer (pH 7.0, 0.1 M, 5 mL) and added in one
[4] a) E. Schmitt, I. Schiffers, C. Bolm, Tetrahedron 2010,
66, 6349–6357; b) Y.-M. Song, J. S. Choi, J. W. Yang, H.
Han, Tetrahedron Lett. 2004, 45, 3301–3304; c) J.
Woltinger, H.-P. Krimmer, K. Drauz, Tetrahedron Lett.
2002, 43, 8531–8533; d) Y. Uozumi, K. Yasoshima, T.
Miyachi, S.-i. Nagai, Tetrahedron Lett. 2001, 42, 411–
414; e) Y. Chen, S.-K. Tian, L. Deng, J. Am. Chem. Soc.
2000, 122, 9542–9543; f) C. Bolm, I. Schiffers, C. L.
Dinter, A. Gerlach, J. Org. Chem. 2000, 65, 6984–6991;
g) D. Seebach, G. Jaeschke, Y. M. Wang, Angew. Chem.
Int. Ed. 1995, 34, 2395–2396; Angew. Chem. 1995, 107,
2605–2606.
°
portion, and the resulting mixture was incubated at 37 C with
orbital shaking (220 rpm). The reaction process was monitored
using TLC method. When TLC showed the complete disappear-
ance of the substrate, the reaction was quenched immediately
by removing microbial cells through a celite pad filtration. The
filtration cake was washed consecutively with water (10 mL×
°
3). The combined filtrate was heated at 50 C under vacuum to
remove solvent, giving a waxy solid which is a mixture of acid
product and salt. The mixture was dissolved in DMF (2 mL)
followed by the addition of K2CO3 (138 mg, 1 mmol) and
benzyl bromide (0.25 mL, 2 mmol). The mixture was stirred
overnight, and the reaction was then quenched by adding water
(20 mL). Extraction with ethyl acetate (30 mL×3) and dried
over anhydrous MgSO4. After removing the solvent under
[5] For reviews, see: a) J. M. Palomo, Z. Cabrera, Curr. Org.
Synth. 2012, 9, 791–805; b) E. García-Urdiales, I.
Alfonso, V. Gotor, Chem. Rev. 2011, 111, PR110–PR180.
Adv. Synth. Catal. 2021, 363, 1–7
5
© 2021 Wiley-VCH GmbH
��
These are not the final page numbers!

COMMUNICATIONS
asc.wiley-vch.de
[6] a) M. P. Meissner, P. Süss, H. Brundiek, J. M. Woodley, [11] a) M.-X. Wang, Acc. Chem. Res. 2015, 48, 602–611;
J. v Langermann, Org. Process Res. Dev. 2018, 22,
1518–1523; b) P. Süss, S. Borchert, J. Hinze, S. Illner, J.
b) T. Sugai, T. Yamazaki, M. Ykoyama, H. Ohta, Biosci.
Biotechnol. Biochem. 1997, 61, 1419–1427.
v Langermann, U. Kragl, U. T. Bornscheuer, R. Warden- [12] Y.-F. Ao, Q.-Q. Wang, D.-X. Wang, Chin. J. Org. Chem.
ga, Org. Process Res. Dev. 2015, 19, 2034–2038; c) P. 2016, 36, 2333–2343.
Süss, S. Illner, J. v Langermann, S. Borchert, U. T. [13] K. Matoishi, A. Sano, N. Imai, T. Yamazaki, M.
Bornscheuer, R. Wardenga, U. Kragl, Org. Process Res.
Dev. 2014, 18, 897–903; d) H. A. Sousa, C. A. M.
Yokoyama, T. Sugai, H. Ohta, Tetrahedron: Asymmetry
1998, 9, 1097–1102.
Afonso, J. P. B. Mota, J. G. Crespo, Ind. Eng. Chem. Res. [14] H.-J. Hu, P. Chen, Y.-F. Ao, Q.-Q. Wang, D.-X. Wang,
2003, 42, 5516–5525; e) L. Heiss, H.-J. Gais, Tetrahe- M.-X. Wang, Org. Chem. Front. 2019, 6, 808–812.
dron Lett. 1995, 36, 3833–3836; f) S. Kobayashi, K. [15] a) Y.-F. Ao, H.-J. Hu, C.-X. Zhao, P. Chen, T. Huang, H.
Kamiyama, T. Iimori, M. Ohno, Tetrahedron Lett. 1984,
25, 2557–2560; g) G. Sabbioni, M. L. Shea, J. B. Jones,
J. Chem. Soc. Chem. Commun. 1984, 236–238.
[7] A. Goswami, T. P. Kissick, Org. Process Res. Dev. 2009,
13, 483–488.
Chen, Q.-Q. Wang, D.-X. Wang, M.-X. Wang, ACS
Catal. 2021, 11, 6900–6907; b) Z. Xue, Y. Chao, D.
Wang, M. Wang, S. Qian, J. Ind. Microbiol. Biotechnol.
2011, 38, 1931–1938.
[16] G. E. Ficken, H. Ficken, R. P. Linstea, J. Am. Chem. Soc.
1954, 76, 3730–3734.
[8] For recent reviews, see: a) S. Wu, R. Snajdrova, J. C.
Moore, K. Baldenius, U. T. Bornscheuer, Angew. Chem. [17] CCDC-2074238 (2b) contains the supplementary crystal-
Int. Ed. 2021, 60, 88–119; Angew. Chem. 2021, 133, 89–
123; b) K. Chen, F. H. Arnold, Nat. Catal. 2020, 3, 203–
213; c) G. Qu, A. Li, C. G. Acevedo-Rocha, Z. Sun,
lographic data for this paper. These data can be obtained
free of charge from The Cambridge Crystallographic
Data Centre via www.ccdc.cam.ac.uk/data_request/cif.
M. T. Reetz, Angew. Chem. Int. Ed. 2020, 59, 2–30; [18] The crystal structure for amidase from Rh. sp. N-771 is
Angew. Chem. 2020, 132, 2–2; d) U. T. Bornscheuer, B.
Hauer, K. E. Jaeger, U. Schwaneberg, Angew. Chem. Int.
Ed. 2019, 58, 36–40; Angew. Chem. 2019, 131, 36–41;
e) R. B. Leveson-Gower, C. Mayer, G. Roelfes, Nat.
Chem. Rev. 2019, 3, 687–705; f) P. N. Devine, R. M.
Howard, R. Kumar, M. P. Thompson, M. D. Truppo,
known (PDB code: 3 A1 K) and 100% identities in
amino acid sequences with the amidase in Rhodococcus
erythropolis AJ270. See: A. Ohtaki, K. Murata, Y. Sato,
K. Noguchi, H. Miyatake, N. Dohmae, K. Yamada, M.
Yohda, M. Odaka, BBA-Proteins Proteom. 2010, 1804,
184–192.
N. J. Turner, Nat. Chem. Rev. 2018, 2, 409–421; g) R. A. [19] G. M. Morris, R. Huey, W. Lindstrom, M. F. Sanner,
Sheldon, P. C. Pereira, Chem. Soc. Rev. 2017, 46, 2678–
2691.
R. K. Belew, D. S. Goodsell, A. J. Olson, J. Comput.
Chem. 2009, 30, 2785–2791.
[9] For some recent examples, see: a) S. Yu, J. Li, P. Yao, J. [20] Schrodinger, LLC. The PyMOL molecular graphics
Feng, Y. Cui, J. Li, X. Liu, Q. Wu, J. Lin, D. Zhu, system, version 2.3.0. New York, NY.
Angew. Chem. Int. Ed. 2021, 60, 3679–3684; Angew. [21] R. A. Laskowski, M. B. Swindells, J. Chem. Inf. Model.
Chem. 2021, 133, 3723–3728; b) J. Li, G. Qu, N. Shang, 2011, 51, 2778–2786.
P. Chen, Y. Men, W. Liu, Z. Mei, Y. Sun, Z. Sun, Green [22] a) B. Hess, C. Kutzner, D. van der Spoel, E. Lindahl, J.
Synth. Catal. 2021, 2, 45–53; c) X. Ren, N. Liu, A. L.
Chandgude, R. Fasan, Angew. Chem. Int. Ed. 2020, 59,
21634–21639; Angew. Chem. 2020, 132, 21818–21823;
d) S. P. Godehard, C. P. S. Badenhorst, H. Müller, U. T.
Chem. Theory Comput. 2008, 4, 435–447; b) J. A. Maier,
C. Martinez, K. Kasavajhala, L. Wickstrom, K. E.
Hauser, C. Simmerling, J. Chem. Theory Comput. 2015,
11, 3696–3713.
Bornscheuer, ACS Catal. 2020, 10, 7552–7562; e) Y.-C. [23] a) J. Wang, V. Lee, H. O. Sintim, Chem. Eur. J. 2009, 15,
Zheng, F.-L. Li, Z. Lin, G.-Q. Lin, R. Hong, H.-L. Yu, J.-
H. Xu, ACS Catal. 2020, 10, 5757–5763; f) B.-M. Su,
Z.-H. Shao, A.-P. Li, M. Naeem, J. Lin, L.-D. Ye, H.-W.
Yu, ACS Catal. 2020, 10, 864–876.
2747–2750; b) S. Höck, H.-J. Borschberg, Helv. Chim.
Acta 2003, 86, 1397–1409; c) G. Bernáth, G. Stájer,
A. E. Szabó, F. Folöp, Tetrahedron 1985, 41, 1353–1365;
d) W. L. F. Armarego, T. Kobayashi, J. Chem. Soc. C
1971, 238–245.
[10] For a recent review, see: Z. Wu, C. Liu, Z. Zhang, R.
Zheng, Y. Zheng, Biotechnol. Adv. 2020, 43, 107574.
Adv. Synth. Catal. 2021, 363, 1–7
6
© 2021 Wiley-VCH GmbH
��
These are not the final page numbers!

COMMUNICATIONS
Modification of the Enantioselectivity of Biocatalytic meso-
Desymmetrization for Synthesis of Both Enantiomers of cis-
1,2-Disubstituted Cyclohexane by Amidase Engineering
Adv. Synth. Catal. 2021, 363, 1–7
H.-J. Hu, Q.-Q. Wang, D.-X. Wang, Y.-F. Ao*