Enantioselective Synthesis of Chiral Vicinal Amino Alcohols Using Amine Dehydrogenases

Article abstract of DOI:10.1021/acscatal.9b03889

Chiral vicinal amino alcohols are an important motif found in many biologically active molecules. In this study, biocatalytic reductive amination of α-hydroxy ketones with ammonia was investigated using engineered amine dehydrogenases (AmDHs) derived from the leucine amino acid dehydrogenase (AADH) from Lysinibacillus fusiformis. The AmDHs thus identified enabled the synthesis of (S)-configured vicinal amino alcohols from the corresponding α-hydroxy ketones in up to 99% conversions and >99% ee. One of the AmDH variants was used to prepare a key intermediate for the antituberculosis pharmaceutical ethambutol.

Full text of DOI:10.1021/acscatal.9b03889

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Letter
Enantioselective Synthesis of Chiral Vicinal
Amino Alcohols Using Amine Dehydrogenases
Feifei Chen, Sebastian Cronin Cosgrove, William R. Birmingham, Juan Mangas-Sanchez,
Joan Citoler, Matthew Thompson, Gao-Wei Zheng, Jian-He Xu, and Nicholas J Turner
ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.9b03889 • Publication Date (Web): 18 Nov 2019
Downloaded from pubs.acs.org on November 18, 2019
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Enantioselective Synthesis of Chiral Vicinal Amino Alcohols
Using Amine Dehydrogenases
Fei-Fei Chen,†,‡ Sebastian C. Cosgrove,‡ William R. Birmingham,‡ Juan Mangas-Sanchez,‡ Joan
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Citoler,‡ Matthew P. Thompson,‡,∥ Gao-Wei Zheng,*,† Jian-He Xu,*,† and Nicholas J. Turner*,‡
†State Key Laboratory of Bioreactor Engineering, Shanghai Collaborative Innovation Center for Biomanufacturing,
East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, P.R. China
‡School of Chemistry, University of Manchester, Manchester Institute of Biotechnology, 131 Princess Street,
Manchester M1 7DN, UK
ABSTRACT: Chiral vicinal amino alcohols are an important motif found in many biologically active molecules. In this
study, biocatalytic reductive amination of α-hydroxy ketones with ammonia was investigated using engineered amine
dehydrogenases (AmDHs) derived from the leucine amino acid dehydrogenase (AADH) from Lysinibacillus fusiformis.
The AmDHs thus identified enabled the synthesis of (S)-configured vicinal amino alcohols from the corresponding α-
hydroxy ketones in up to 99% conversions and >99% ee. One of the AmDH variants was used to prepare a key
intermediate for the anti-tuberculosis pharmaceutical ethambutol.
KEYWORDS: amine dehydrogenase; biocatalysis; chiral amino alcohols; directed evolution; reductive amination
Chiral vicinal amino alcohols are important structural
moieties present in numerous synthetic and natural
the former route is also restricted to a maximum yield of
50% and the
bioactive molecules (Scheme 1).1
A representative
O
O
F
HO
Cl
example is (S)-2-amino-3-methyl-1-butanol (L-valinol),
used as a key intermediate in the synthesis of the HIV-1
integrase inhibitor Elvitegravir.1d,2 Chiral amino alcohols
also serve as important chiral auxiliaries or ligands in
various asymmetric syntheses.3 From a chemical synthesis
perspective, chiral vicinal amino alcohols can be accessed
via reduction of amino acids and α-amino carbonyl
compounds,4 cross-coupling of imines with carbonyl
compounds,5 opening of epoxides by nitrogen
nucleophiles,6 aminohydroxylation of alkenes,7 and
kinetic resolution of racemates.8 However, several
drawbacks remain, including harsh reaction conditions,
the use of expensive transition metal catalysts, and
insufficient regio- and enantioselectivity. Thus the
development of efficient, green synthetic routes for chiral
vicinal amino alcohol production remains a topic of great
interest.
HO
H
N
N
N
O
H
HO
OH
Ethambutol
(anti-tuberculosis)
Elvitegravir
(anti-HIV)
F
F
H
N
N
N
O
N
H
N
O
O
N
H
O
OH
N
O
NH
N
H
O
HO
A PDK1 inhibitor
Indinavir
(potential anticancer adjuvant)
(anti-HIV)
Compared to traditional chemical methods, biocatalysis
is an attractive alternative for the production of high-
value chemicals as enzymes are highly selective and
operate under mild reaction conditions, hence reducing
the environmental impact.9 Lipases and acylases have
been respectively harnessed for preparing optically pure
vicinal amino alcohols via classical kinetic resolution,
although the maximum yield is limited to 50%.10 Recently,
ω-transaminases (ω-TAs) have also proved to be useful
tools for the synthesis of chiral vicinal amino alcohols via
kinetic resolution of racemic amino alcohols and
asymmetric amination of α-hydroxy ketones.11 However,
Scheme 1. Biologically active molecules including
active pharmaceutical ingredients (APIs) containing
a chiral amino alcohol moiety.
latter requires the excessive addition of an effective amino
donor alongside in situ by-product removal11b,11c or in situ
product removal12 to overcome the unfavourable
thermodynamic equilibrium. On the other hand, by
combining ω-TAs with different enzymes such as lipases,13
epoxide
hydrolases,14
transketolases,15
alcohol
dehydrogenases,16 oxygenases,14,17 decarboxylases,17 and
synthases,18 elegant cascade reactions have been
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developed for the synthesis of chiral 1,2-amino alcohols.
Page 2 of 7
related variant K68C/N261L showed comparable activity
whereas K68T/N261L performed best. Kinetic parameters
towards 1a were subsequently determined for these three
variants (Table 1). Variant K68T/N261L (denoted AmDH-
Other biocatalysts including threonine aldolases,19
hydroxynitrile lyases,20 and halohydrin dehalogenases21
have also been recruited for the production of chiral
amino alcohols via multi-enzymatic cascades or chemo-
enzymatic approaches. Recently, Arnold and co-workers
reported the direct transformation of alkenes to chiral
amino alcohols with an engineered cytochrome c.22 These
reports demonstrate the potential of biocatalysis to use
readily available starting materials, simplify intermediate
operation steps for high atom efficiency, and avoid
environmentally hazardous waste.
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Table 1. Specific activities and kinetic parameters of
the selected top three AmDHs towards model
substrate 1a.
Spec. activity
(mU mg−1)a
kcat
Km
kcat/Km
Variant
9
(s−1)b
(mM)b (mM−1 s−1)b
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0.175 ±
0.009
9.7 ±
0.018
1.4
K68C/N261L
K68S/N261L
171 ± 5
Recently, amine dehydrogenases (AmDHs) have
emerged as a promising class of biocatalysts for accessing
optically active chiral amines.23 AmDHs catalyse the
asymmetric reductive amination of prochiral ketones
using inexpensive ammonia as the amino donor and
NAD(P)H as the reducing agent. The strategy of
engineering AmDHs from amino acid dehydrogenases
(AADHs) was first developed by Bommarius and co-
workers,23a,23b leading to the expansion of the substrate
scope of AmDHs by engineering new enzymes.23d-g,23m,23n
Recently, a family of native AmDHs with low amino acid
sequence identity to previously developed AmDHs was
discovered.24 Although a number of active and highly
stereoselective AmDHs have been reported, their
application is restricted to the amination of carbonyl
compounds without α-functionalization.25 Herein, we
describe the identification of AmDH variants with high
activity for the asymmetric amination of α-hydroxy
ketones allowing the preparation of optically pure vicinal
amino alcohols in good yield and ee (Scheme 2).
0.221 ±
0.005
14.6 ±
0.015
0.8
180 ± 2
K68T/N261L
(AmDH-M0)
0.227 ±
0.011
7.2 ±
0.031
1.0
233 ± 16
aActivity was measured in NH4Cl/NH4OH buffer (2 M, pH
9.5) containing 0.2 mM NADH and 20 mM 1a at 25°C. All
b
assays were performed in triplicate. Kinetic parameters (kcat
and Km) were determined by measuring the activities at
varied concentrations of substrate 1a.
M0) displayed the highest catalytic efficiency (kcat/KM
=
0.031 mM−1 s−1), due to a relatively high kcat and the lowest
KM. The K68S/N261L variant had previously been shown
to possess activity towards methyl ketone substrates,23m
and hence we compared the activity of all three variants
towards a panel of methyl ketones (Figure S2). As
expected the highest activity was observed with the
K68S/N261L variant. In contrast, AmDH-M0 displayed low
activity across the entire methyl ketone substrate panel
and hence this variant was selected for further
investigation due to its apparent preference for α-hydroxy
ketone substrates over methyl ketones.
NH3
H2O
O
NH2
R
AmDH
HO
HO
R
1
2
(S)-
NADH/H+
NAD+
CO2
Formate
FDH
Scheme 2. Synthesis of chiral vicinal amino alcohols
by amine dehydrogenase catalysed asymmetric
reductive amination of α-hydroxy ketones. AmDH,
amine dehydrogenase; FDH, formate dehydrogenase.
In our previous study,
successfully generated from
dehydrogenase from Lysinibacillus
(LfLeuAADH)26 showing extended catalytic scope,
satisfactory thermostability, and high
a
panel of AmDHs was
leucine amino acid
fusiformis
a
enantioselectivity.23m In this work the amino acid sites
K68 and N261 were found, as expected, to be two key
hotspots responsible for altering the substrate specificity
to afford AmDH activity.23a,23b We therefore initially
screened a library of K68/N261 variants for activity
towards hydroxy ketone 1a (See Table S2). To our delight
several variants showed high activity, including the
previously reported variant K68S/N261L23m (Table 1). The
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Further mutations were introduced into the substrate
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3
4
O
NH2
R
binding pocket of AmDH-M0 including A113G,
A113G/T134A, and A113G/T134G, as these sites had
previously been shown to enlarge the active-site of other
AmDHs to accept bulkier aliphatic ketones.23m The
specific activities of AmDH-M0 and the new variants
AmDH/FDH
HO
HO
R
NH4COOH buffer (1 M, pH 8.9)
NAD+ (1 mM)
1a 1h
-
2a 2h
(S)-
-
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AmDH-M1
(K68T/N261L/A113G/T134A),
(K68T/N261L/A113G),
and
AmDH-M2
AmDH-M3
NH2
NH2
HO
NH2
HO
HO
(K68T/N261L/A113G/T134G) were examined towards a
panel of α-hydroxy ketones with varying chain-lengths
(Figure 1). AmDH-M0 exhibited high activity towards
most of the substrates (1a1f), and, remarkably, the
activity observed towards the bulkier substrates 1b and
1d1f was higher than that for the model substrate 1a.
However, AmDH-M0 displayed almost no activity towards
the bulkiest α-hydroxy ketone 1g. In contrast, all the three
pocket-expanded variants exhibited marked activity
towards 1g, as well as higher activity towards 1f than
AmDH-M0. AmDH-M3, with the largest active site pocket,
exhibited the highest activity towards the two bulkiest α-
hydroxy ketones 1f and 1g.
2a
(S)-
2b
(S)-
2c
(S)-
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AmDH-M0
91% conv., >99% ee
AmDH-M3
AmDH-M0
94% conv., >99% ee
AmDH-M3
AmDH-M0
75% conv., >99% ee
AmDH-M3
84% conv., >99% ee
96% conv., >99% ee
27% conv., >99% ee
NH2
NH2
NH2
HO
HO
HO
2d
(S)-
2e
(S)-
2f
(S)-
AmDH-M0
92% conv., >99% ee
AmDH-M3
AmDH-M0
>99% conv., >99% ee
AmDH-M3
AmDH-M0
90% conv., >99% ee
AmDH-M3
To gain further insight into the reactivity of the AmDH
variants, molecular docking analyses were then
performed for AmDH-M0 and AmDH-M3, respectively
(See the Supporting Information). The docking result
shows that the initial mutation K68T/N261L around the
90% conv., >99% ee
>99% conv., >99% ee
92% conv., >99% ee
NH2
NH2
HO
HO
2g
(S)-
2h
(S)-
active site of AmDH-M0 may provide
a suitable
interaction between the hydroxyl group of substrate and
the mutated residues for the proper coordination of
substrate (Figure S3 (A)), which is in correspondence
with the previous report that the two key residues K68
and N261 of the wild-type AADH directly interact with
the carboxyl group of the amino acid substrate.23a,27
However, the binding of bulkier substrates could be
impeded due to the limited substrate-binding space. By
introducing the pocket-expanding mutations (Figure S3
(B)), additional space could be generated in the distal end
of the active site to facilitate the accommodation of the
bulkier α-hydroxy ketone substrates in a rather stretched
conformation, thus leading to enhanced reactivity.
AmDH-M0
42% conv., >99% ee
AmDH-M3
AmDH-M0
37% conv., >99% ee
AmDH-M3
38% conv., >99% ee
99% conv., >99% ee
aReaction conditions: 0.75 mg AmDH (purified protein), 3 mg
FDH (crude cell-free extract), 50 mM substrate, 2% (v/v)
DMSO, 1 mM NAD+, NH4COOH/NH4OH buffer (1 M, pH 8.9),
0.5 mL total volume, 30ºC, 250 rpm, 24 h. Conversions
determined by HPLC analysis after derivatisation or GC-FID
analysis. ee determined by HPLC analysis after derivatisation.
Scheme 3. Synthesis of chiral amino alcohols using
the engineered AmDHs.a
Biotransformations of the α-hydroxy ketones to the
corresponding chiral vicinal amino alcohols were then
performed on an analytical scale by employing the
engineered AmDH-M0 and the best pocket-expanded
variant AmDH-M3 (Scheme 3). All α-hydroxy ketone
substrates tested, including an aryl-containing α-hydroxy
ketone (1h), were efficiently aminated yielding the (S)-
configured 1,2-amino alcohol products 2a2h with high ee
(>99%) and ≥75% conversion for all but the aromatic
substrate. For 1b and 1d1f, both AmDH-M0 and AmDH-
M3 afforded >90% conversion. However, for the long-
chain aliphatic α-hydroxyl ketone 1g, AmDH-M3 gave a
significantly higher conversion (99%) than AmDH-M0
(42%), whereas AmDH-M0 appeared to be more active
towards shorter chain substrates 1a and 1c.
To examine the performance of these new AmDHs for
the preparative synthesis of chiral vicinal amino alcohols,
the amination of 1f was scaled up (10 mmol), using
AmDH-M3 in the form of a crude cell-free extract,
affording 662 mg of (S)-2-amino-1-hexanol (2f) in 56%
isolated yield and >99% ee (Scheme 4 (A)). A preparative
scale reaction for the synthesis of (S)-2a was also
performed using AmDH-M0. This afforded the desired
product in 74% isolated yield with >99% ee, which thus
achieved a formal synthesis of the active (S,S)-form of the
tuberculosis drug ethambutol (Scheme 4 (B)).28 This
route to (S)-2a could be more advantageous than those
currently employed, as it avoids the need for chiral pool
starting materials or precious-metal catalysis, and also
exclusively generates the (S)-enantiomer required for the
active form of the drug.29
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(A)
Gao-Wei Zheng: 0000-0001-5918-6047
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Jian-He Xu: 0000-0003-4301-7630
Nicholas J. Turner: 0000-0002-8708-0781
AmDH-M3 crude CFE
FDH crude CFE
O
NH2
HO
HO
NH4COOH buffer (1 M, pH 8.9)
NAD+ (1mM)
Present Addresses
∥Present address: EnginZyme AB, Teknikringen 38A, 114 28,
Stockholm, Sweden.
88% conv.
>99% ee
56% yield
1f
2f
(S)-
30oC, 200 rpm, 24 h
100 mM
(B)
Notes
The authors declare no competing financial interest.
AmDH-M0 crude CFE
FDH crude CFE
O
NH2
HO
HO
9
NH4COOH buffer (1 M, pH 8.9)
NAD+ (1mM)
ASSOCIATED CONTENT
Supporting Information
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1a
2a
(S)-
95% conv.
>99% ee
74% yield
30oC, 200 rpm, 24 h
100 mM
The Supporting Information is available free of charge on the
ACS Publications website.
Ref. 28b
Materials, experimental procedures, analytic methods,
HO
GC spectra, 1H and 13C NMR spectra (PDF)
H
N
N
H
ACKNOWLEDGMENT
OH
ethambutol
The work was financially supported by the National Natural
Science Foundation of China (Nos. 21472045, 21878085,
21536004, and 21871085). Fei-Fei Chen gratefully
acknowledges the China Scholarship Council for providing a
scholarship to support his one year’s overseas study in the
UK. Sebastian Cosgrove acknowledges the UK Catalysis Hub
(EPSRC) for funding. William Birmingham, Juan Mangas-
Sanchez and Nicholas Turner acknowledge an ERC Advanced
Grant (Grant Number 742987). We are also grateful to Dr
Reynard Spiess, Dr Fabio Parmeggiani, and Dr Kun Huang
for their valuable assistance with MS, NMR, and HPLC
analyses, respectively.
Scheme 4. (A) Preparative biocatalytic synthesis of
chiral vicinal amino alcohol (S)-2f using AmDH-M3;
(B) Preparative synthesis of chiral vicinal amino
alcohol (S)-2a using AmDH-M0 for formal synthesis
of ethambutol. CFE, cell-free extract.
In summary, conversion of α-hydroxy ketones to the
corresponding vicinal amino alcohols has been achieved
using AmDH variants derived from the AADH from
Lysinibacillus fusiformis (LfLeuDH). Using ammonia as
the amino donor, two new AmDH variants (LfAmDH-M0
& LfAmDH-M3) showed high activity and complementary
substrate spectrum towards a panel of structurally diverse
α-hydroxy ketones to afford the corresponding chiral
vicinal amino alcohols in good conversion (up to 99%)
and high ee (>99%). These variants also proved to be
efficient in preparative scale reactions including the
synthesis of (S)-2a which is a key intermediate in the
preparation of the anti-tuberculosis drug ethambutol. The
methodology described herein expands the catalytic
scope of AmDHs and presents a potentially green and
sustainable approach to vicinal amino alcohols. We
envisage performing further laboratory evolution for the
AmDH variants with different techniques (e.g., error-
prone PCR, cassette mutagenesis, DNA shuffling),30 which
could provide the opportunity of generating new variants
with elevated catalytic efficiency.
REFERENCES
(1) (a) Wilkinson, R.; Thomas, J. P.; Shepherd, R. G.; Baughn,
C. Stereospecificity in a New Type of Synthetic Antituberculous
Agent. J. Am. Chem. Soc. 1961, 83, 2212-2213. (b) Erlanson, D. A.;
Arndt, J. W.; Cancilla, M. T.; Cao, K.; Elling, R. A.; English, N.;
Friedman, J.; Hansen, S. K.; Hession, C.; Joseph, I.; Kumaravel,
G.; Lee, W. C.; Lind, K. E.; McDowell, R. S.; Miatkowski, K.;
Nguyen, C.; Nguyen, T. B.; Park, S.; Pathan, N.; Penny, D. M.;
Romanowski, M. J.; Scott, D.; Silvian, L.; Simmons, R. L.;
Tangonan, B. T.; Yang, W. J.; Sun, L. H. Discovery of a Potent
and Highly Selective PDK1 Inhibitor via Fragment-Based Drug
Discovery. Bioorg. Med. Chem. Lett. 2011, 21, 3078-3083. (c)
Ekegren, J. K.; Ginman, N.; Johansson, A.; Wallberg, H.; Larhed,
M.; Samuelsson, B.; Unge, T.; Hallberg, A. Microwave-
Accelerated Synthesis of P1'-Extended HIV-1 Protease Inhibitors
Encompassing
a Tertiary Alcohol in the Transition-State
Mimicking Scaffold. J. Med. Chem. 2006, 49, 1828-1832. (d) Sato,
M.; Kawakami, H.; Motomura, T.; Aramaki, H.; Matsuda, T.;
Yamashita, M.; Ito, Y.; Matsuzaki, Y.; Yamataka, K.; Ikeda, S.;
Shinkai, H. Quinolone Carboxylic Acids as a Novel Monoketo
Acid Class of Human Immunodeficiency Virus Type 1 Integrase
Inhibitors. J. Med. Chem. 2009, 52, 4869-4882. (e) Bergmeier, S.
C. The Synthesis of Vicinal Amino Alcohols. Tetrahedron 2000,
56, 2561-2576. (f) Blakemore, D. C.; Castro, L.; Churcher, I.; Rees,
D. C.; Thomas, A. W.; Wilson, D. M.; Wood, A. Organic
Synthesis Provides Opportunities to Transform Drug Discovery.
Nat. Chem. 2018, 10, 383-394.
AUTHOR INFORMATION
Corresponding Author
*Email: gaoweizheng@ecust.edu.cn
*Email: jianhexu@ecust.edu.cn
*Email: nicholas.turner@manchester.ac.uk
(2) (a) Ramanathan, S.; Mathias, A. A.; German, P.; Kearney, B.
P. Clinical Pharmacokinetic and Pharmacodynamic Profile of the
HIV Integrase Inhibitor Elvitegravir. Clin. Pharmacokinet. 2011,
50, 229-244. (b) Radl, S.; Stach, J.; Pisa, O.; Cinibulk, J.; Havlicek,
ORCID
Fei-Fei Chen: 0000-0002-8567-2054
Sebastian C. Cosgrove: 0000-0001-9541-7201
William R. Birmingham: 0000-0002-8880-5502
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J.; Zajicova, M.; Pekareka, T. An Improved Synthesis of
Elvitegravir. J. Heterocycl. Chem. 2016, 53, 1738-1749.
Amino 1-Alcohols. J. Org. Chem. 1987, 52, 5079-5085. (b) Honig,
H.; Seuferwasserthal, P.; Fulop, F. Enzymatic Resolutions of
Cyclic Amino Alcohol Precursors. J. Chem. Soc., Perkin Trans. 1
1989, 2341-2345. (c) Fadnavis, N. W.; Sharfuddin, M.; Vadivel, S.
K. Resolution of Racemic 2-Amino-1-butanol with Immobilised
Penicillin G Acylase. Tetrahedron: Asymmetry 1999, 10, 4495-
4500. (d) Lundell, K.; Kanerva, L. T. Enantiomers of Ring-
Substituted 2-Amino-1-phenylethanols by Pseudomonas cepacia
Lipase. Tetrahedron: Asymmetry 1995, 6, 2281-2286. (e)
Lourenco, N. M. T.; Barreiros, S.; Afonso, C. A. M. Enzymatic
Resolution of Indinavir Precursor in Ionic Liquids with Reuse of
Biocatalyst and Media by Product Sublimation. Green Chem.
2007, 9, 734-736.
(11) (a) Malik, M. S.; Park, E. S.; Shin, J. S. ω-Transaminase-
Catalyzed Kinetic Resolution of Chiral Amines Using L-
Threonine as an Amino Acceptor Precursor. Green Chem. 2012,
14, 2137-2140. (b) Fuchs, C. S.; Simon, R. C.; Riethorst, W.;
Zepeck, F.; Kroutil, W. Synthesis of (R)- or (S)-Valinol Using ω-
Transaminases in Aqueous and Organic Media. Biorg. Med.
Chem. 2014, 22, 5558-5562. (c) Nobili, A.; Steffen-Munsberg, F.;
Kohls, H.; Trentin, I.; Schulzke, C.; Höhne, M.; Bornscheuer, U.
T. Engineering the Active Site of the Amine Transaminase from
Vibrio fluvialis for the Asymmetric Synthesis of Aryl-Alkyl
Amines and Amino Alcohols. ChemCatChem 2015, 7, 757-760. (d)
Zhang, J. D.; Wu, H. L.; Meng, T.; Zhang, C. F.; Fan, X. J.; Chang,
H. H.; Wei, W. L. A High-Throughput Microtiter Plate Assay for
the Discovery of Active and Enantioselective Amino Alcohol-
Specific Transaminases. Anal. Biochem. 2017, 518, 94-101. (e)
Zhao, J. W.; Wu, H. L.; Zhang, J. D.; Gao, W. C.; Fan, X. J.; Chang,
H. H.; Wei, W. L.; Xu, J. H. One Pot Simultaneous Preparation of
Both Enantiomer of β-Amino Alcohol and Vicinal Diol Via
Cascade Biocatalysis. Biotechnol. Lett. 2018, 40, 349-358. (f)
Smithies, K.; Smith, M. E. B.; Kaulmann, U.; Galman, J. L.; Ward,
J. M.; Hailes, H. C. Stereoselectivity of an ω-Transaminase-
Mediated Amination of 1,3-Dihydroxy-1-phenylpropane-2-one.
Tetrahedron: Asymmetry 2009, 20, 570-574. (g) Rios-Solis, L.;
Bayir, N.; Halim, M.; Du, C.; Ward, J. M.; Baganz, F.; Lye, G. J.
Non-Linear Kinetic Modelling of Reversible Bioconversions:
Application to the Transaminase Catalyzed Synthesis of Chiral
Amino-Alcohols. Biochem. Eng. J. 2013, 73, 38-48. (h) Kaulmann,
U.; Smithies, K.; Smith, M. E. B.; Hailes, H. C.; Ward, J. M.
Substrate Spectrum of ω-Transaminase from Chromobacterium
violaceum DSM30191 and Its Potential for Biocatalysis. Enzyme
Microb. Technol. 2007, 41, 628-637.
(12) (a) Rehn, G.; Adlercreutz, P.; Grey, C. Supported Liquid
Membrane as a Novel Tool for Driving the Equilibrium of ω-
Transaminase Catalyzed Asymmetric Synthesis. J. Biotechnol.
2014, 179, 50-55. (b) Hülsewede, D.; Tänzler, M.; Süss, P.;
Mildner, A.; Menyes, U.; von Langermann, J. Development of an
in situ-Product Crystallization (ISPC)-Concept to Shift the
Reaction Equilibria of Selected Amine Transaminase-Catalyzed
Reactions. Eur. J. Org. Chem. 2018, 2018, 2130-2133.
(13) Yun, H.; Kim, J.; Kinnera, K.; Kim, B. G. Synthesis of
Enantiomerically Pure trans-(1R,2R)- and cis-(1S,2R)-1-Amino-2-
Indanol by Lipase and ω-Transaminase. Biotechnol. Bioeng.
2006, 93, 391-395.
(14) Wu, S. K.; Zhou, Y.; Wang, T. W.; Too, H. P.; Wang, D. I.
C.; Li, Z. Highly Regio- and Enantioselective Multiple Oxy- and
Amino-Functionalizations of Alkenes by Modular Cascade
Biocatalysis. Nat. Commun. 2016, 7, 11917.
(15) (a) Ingram, C. U.; Bommer, M.; Smith, M. E. B.; Dalby, P.
A.; Ward, J. M.; Hailes, H. C.; Lye, G. J. One-Pot Synthesis of
Amino-Alcohols Using a De-Novo Transketolase and β-Alanine:
Pyruvate Transaminase Pathway in Escherichia coli. Biotechnol.
Bioeng. 2007, 96, 559-569. (b) Smith, M. E. B.; Chen, B. H.;
Hibbert, E. G.; Kaulmann, U.; Smithies, K.; Galman, J. L.; Baganz,
F.; Dalby, P. A.; Hailes, H. C.; Lye, G. J.; Ward, J. M.; Woodley, J.
1
2
3
4
5
6
7
8
(3) Ager, D. J.; Prakash, I.; Schaad, D. R. 1,2-Amino Alcohols
and Their Heterocyclic Derivatives as Chiral Auxiliaries in
Asymmetric Synthesis. Chem. Rev. 1996, 96, 835-875.
(4) (a) Reetz, M. T.; Drewes, M. W.; Schmitz, A.
Stereoselective Synthesis of β-Amino Alcohols from Optically
Active α-Amino Acids. Angew. Chem. Int. Ed. Engl. 1987, 26, 1141-
1143. (b) Abiko, A.; Masamune, S. An Improved, Convenient
Procedure for Reduction of Amino Acids to Aminoalcohols: Use
of NaBH4-H2SO4. Tetrahedron Lett. 1992, 33, 5517-5518.
(5) Fan, G. Q.; Liu, Y. H. Titanium-Mediated Cross-Coupling
Reactions of Imines with Ketones or Aldehydes: An Efficient
Route for the Synthesis of 1,2-Amino Alcohols. Tetrahedron Lett.
2012, 53, 5084-5087.
(6) (a) Harris, C. E.; Fisher, G. B.; Beardsley, D.; Lee, L.;
Goralski, C. T.; Nicholson, L. W.; Singaram, B. Boranes in
Synthesis .6. A New Synthesis of β-Amino Alcohols from
Epoxides. Use of Lithium Amides and Aminoborane Catalysts to
Synthesize β-Amino Alcohols from Terminal and Internal
Epoxides in High Yield. J. Org. Chem. 1994, 59, 7746-7751. (b)
Posner, G. H.; Rogers, D. Z. Organic Reactions at Alumina
Surfaces. Mild and Selective Opening of Epoxides by Alcohols,
Thiols, Benzeneselenol, Amines, and Acetic Acid. J. Am. Chem.
Soc. 1977, 99, 8208-8214. (c) Posner, G. H.; Rogers, D. Z. Organic
Reactions at Alumina Surfaces. Mild and Selective Opening of
Arene and Related Oxides by Weak Oxygen and Nitrogen
Nucleophiles. J. Am. Chem. Soc. 1977, 99, 8214-8218.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(7) (a) Li, G. G.; Chang, H. T.; Sharpless, K. B. Catalytic
Asymmetric Aminohydroxylation (AA) of Olefins. Angew. Chem.
Int. Ed. Engl. 1996, 35, 451-454. (b) Nilov, D.; Reiser, O. The
Sharpless
Limitation. Adv. Synth. Catal. 2002, 344, 1169-1173. (c) Bodkin, J.
A.; McLeod, M. D. The Sharpless Asymmetric
Asymmetric
Aminohydroxylation–Scope
and
Aminohydroxylation. J. Chem. Soc., Perkin Trans. 1 2002, 2733-
2746.
(8) (a) Sakurai, R.; Sakai, K. Resolution of Racemic cis-1-
Amino-2-indanol by Diastereomeric Salt Formation with (S)-2-
Phenylpropionic Acid. Tetrahedron: Asymmetry 2003, 14, 411-413.
(b) Larrow, J. F.; Schaus, S. E.; Jacobsen, E. N. Kinetic Resolution
of Terminal Epoxides via Highly Regioselective and
Enantioselective Ring Opening with TMSN3. An Efficient,
Catalytic Route to 1,2-Amino Alcohols. J. Am. Chem. Soc. 1996,
118, 7420-7421. (c) Kawabata, T.; Yamamoto, K.; Momose, Y.;
Yoshida, H.; Nagaoka, Y.; Fuji, K. Kinetic Resolution of Amino
Alcohol Derivatives with a Chiral Nucleophilic Catalyst: Access
to Enantiopure Cyclic cis-Amino Alcohols. Chem. Commun.
2001, 2700-2701.
(9) (a) Huisman, G. W.; Collier, S. J. On the Development of
New Biocatalytic Processes for Practical Pharmaceutical
Synthesis. Curr. Opin. Chem. Biol. 2013, 17, 284-292. (b) Zheng, G.
W.; Xu, J. H. New Opportunities for Biocatalysis: Driving the
Synthesis of Chiral Chemicals. Curr. Opin. Biotechnol. 2011, 22,
784-792. (c) Hollmann, F.; Arends, I. W. C. E.; Holtmann, D.
Enzymatic Reductions for the Chemist. Green Chem. 2011, 13,
2285-2314. (d) Höhne, M.; Bornscheuer, U. T. Biocatalytic Routes
to Optically Active Amines. ChemCatChem 2009, 1, 42-51. (e)
Kroutil, W.; Mang, H.; Edegger, K.; Faber, K. Recent Advances in
the Biocatalytic Reduction of Ketones and Oxidation of sec-
Alcohols. Curr. Opin. Chem. Biol. 2004, 8, 120-126. (f) Reetz, M.
T. Biocatalysis in Organic Chemistry and Biotechnology: Past,
Present, and Future. J. Am. Chem. Soc. 2013, 135, 12480-12496. (g)
Schrittwieser, J. H.; Velikogne, S.; Hall, M.; Kroutil, W. Artificial
Biocatalytic Linear Cascades for Preparation of Organic
Molecules. Chem. Rev. 2017, 118, 270-348.
(10) (a) Francalanci, F.; Cesti, P.; Cabri, W.; Bianchi, D.;
Martinengo, T.; Foa, M. Lipase-Catalyzed Resolution of Chiral 2-
ACS Paragon Plus Environment

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Page 6 of 7
M.; Micheletti, M. A Multidisciplinary Approach toward the
D.; Schell, U. E. Understanding and Overcoming the Limitations
of Bacillus badius and Caldalkalibacillus thermarum Amine
Dehydrogenases for Biocatalytic Reductive Amination. ACS
Catal. 2017, 7, 3204-3209. (g) Chen, F. F.; Liu, Y. Y.; Zheng, G. W.;
Xu, J. H. Asymmetric Amination of Secondary Alcohols by Using
a Redox-Neutral Two-Enzyme Cascade. ChemCatChem 2015, 7,
3838-3841. (h) Mutti, F. G.; Knaus, T.; Scrutton, N. S.; Breuer, M.;
Turner, N. J. Conversion of Alcohols to Enantiopure Amines
through Dual-Enzyme Hydrogen-Borrowing Cascades. Science
2015, 349, 1525-1529. (i) Mayol, O.; David, S.; Darii, E.; Debard, A.;
Mariage, A.; Pellouin, V.; Petit, J. L.; Salanoubat, M.; de
Berardinis, V.; Zaparucha, A.; Vergne-Vaxelaire, C. Asymmetric
Reductive Amination by a Wild-Type Amine Dehydrogenase
from the Thermophilic Bacteria Petrotoga mobilis. Catal. Sci.
Technol. 2016, 6, 7421-7428. (j) Liu, J.; Pang, B. Q. W.; Adams, J.
P.; Snajdrova, R.; Li, Z. Coupled Immobilized Amine
Dehydrogenase and Glucose Dehydrogenase for Asymmetric
Synthesis of Amines by Reductive Amination with Cofactor
Recycling. ChemCatChem 2017, 9, 425-431. (k) Thompson, M. P.;
Turner, N. J. Two-Enzyme Hydrogen-Borrowing Amination of
Rapid and Preparative-Scale Biocatalytic Synthesis of Chiral
Amino Alcohols: A Concise Transketolase-/ω-Transaminase-
Mediated Synthesis of (2S,3S)-2-Aminopentane-1,3-diol. Org.
Process Res. Dev. 2010, 14, 99-107. (c) Rios-Solis, L.; Morris, P.;
Grant, C.; Odeleye, A. O. O.; Hailes, H. C.; Ward, J. M.; Dalby, P.
A.; Baganz, F.; Lye, G. J. Modelling and Optimisation of the One-
Pot, Multi-Enzymatic Synthesis of Chiral Amino-Alcohols Based
on Microscale Kinetic Parameter Determination. Chem. Eng. Sci.
2015, 122, 360-372. (d) Villegas-Torres, M. F.; Martinez-Torres, R.
J.; Cazares-Korner, A.; Hailes, H.; Baganz, F.; Ward, J. Multi-Step
Biocatalytic Strategies for Chiral Amino Alcohol Synthesis.
Enzyme Microb. Technol. 2015, 81, 23-30.
(16) Sehl, T.; Hailes, H. C.; Ward, J. M.; Menyes, U.; Pohl, M.;
Rother, D. Efficient 2-Step Biocatalytic Strategies for the
Synthesis of All Nor(pseudo)ephedrine Isomers. Green Chem.
2014, 16, 3341-3348.
(17) Baud, D.; Peruch, O.; Saaidi, P. L.; Fossey, A.; Mariage, A.;
Petit, J. L.; Salanoubat, M.; Vergne-Vaxelaire, C.; de Berardinis,
V.; Zaparucha, A. Biocatalytic Approaches Towards the Synthesis
of Chiral Amino Alcohols from Lysine: Cascade Reactions
Combining α-Keto Acid Oxygenase Hydroxylation with
Pyridoxal Phosphate-Dependent Decarboxylation. Adv. Synth.
Catal. 2017, 359, 1563-1569.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Alcohols
Enabled
by
a
Cofactor-Switched
Alcohol
Dehydrogenase. ChemCatChem 2017, 9, 3833-3836. (l) Bohmer,
W.; Knaus, T.; Mutti, F. G. Hydrogen-Borrowing Alcohol
Bioamination
with
Coimmobilized
Dehydrogenases.
(18) (a) Sehl, T.; Hailes, H. C.; Ward, J. M.; Wardenga, R.; von
Lieres, E.; Offermann, H.; Westphal, R.; Pohl, M.; Rother, D. Two
Steps in One Pot: Enzyme Cascade for the Synthesis of
Nor(pseudo)ephedrine from Inexpensive Starting Materials.
Angew. Chem. Int. Ed. 2013, 52, 6772-6775. (b) Erdmann, V.; Sehl,
T.; Frindi-Wosch, I.; Simon, R. C.; Kroutil, W.; Rother, D.
Methoxamine Synthesis in a Biocatalytic 1-Pot 2-Step Cascade
Approach. ACS Catal. 2019, 9, 7380-7388.
(19) Steinreiber, J.; Schurmann, M.; Wolberg, M.; van Assema,
F.; Reisinger, C.; Tesko, K.; Mink, D.; Griengl, H. Overcoming
Thermodynamic and Kinetic Limitations of Aldolase-Catalyzed
Reactions by Applying Multienzymatic Dynamic Kinetic
Asymmetric Transformations. Angew. Chem. Int. Ed. 2007, 46,
1624-1626.
(20) Purkarthofer, T.; Pabst, T.; van den Broek, C.; Griengl, H.;
Maurer, O.; Skranc, W. Large-Scale Synthesis of (R)-2-Amino-1-
(2-furyl)ethanol via a Chemoenzymatic Approach. Org. Process
Res. Dev. 2006, 10, 618-621.
(21) Molinaro, C.; Guilbault, A. A.; Kosjek, B. Resolution of 2,2-
Disubstituted Epoxides via Biocatalytic Azidolysis. Org. Lett.
2010, 12, 3772-3775.
(22) Cho, I.; Prier, C. K.; Jia, Z. J.; Zhang, R. K.; Gorbe, T.;
Arnold, F. H. Enantioselective Aminohydroxylation of Styrenyl
Olefins Catalyzed by an Engineered Hemoprotein. Angew. Chem.
Int. Ed. 2019, 58, 3138-3142.
(23) (a) Abrahamson, M. J.; Vazquez-Figueroa, E.; Woodall, N.
B.; Moore, J. C.; Bommarius, A. S. Development of an Amine
Dehydrogenase for Synthesis of Chiral Amines. Angew. Chem.
Int. Ed. 2012, 51, 3969-3972. (b) Abrahamson, M. J.; Wong, J. W.;
Bommarius, A. S. The Evolution of an Amine Dehydrogenase
Biocatalyst for the Asymmetric Production of Chiral Amines.
Adv. Synth. Catal. 2013, 355, 1780-1786. (c) Au, S. K.; Bommarius,
B. R.; Bommarius, A. S. Biphasic Reaction System Allows for
ChemCatChem 2018, 10, 731-735. (m) Chen, F. F.; Zheng, G. W.;
Liu, L.; Li, H.; Chen, Q.; Li, F. L.; Li, C. X.; Xu, J. H. Reshaping the
Active Pocket of Amine Dehydrogenases for Asymmetric
Synthesis of Bulky Aliphatic Amines. ACS Catal. 2018, 8, 2622-
2628. (n) Tseliou, V.; Knaus, T.; Masman, M. F.; Corrado, M. L.;
Mutti, F. G. Generation of Amine Dehydrogenases with
Increased Catalytic Performance and Substrate Scope from ε-
Deaminating L-Lysine Dehydrogenase. Nat. Commun. 2019, 10,
3717.
(24) Mayol, O.; Bastard, K.; Beloti, L.; Frese, A.; Turkenburg, J.
P.; Petit, J. L.; Mariage, A.; Debard, A.; Pellouin, V.; Perret, A.; de
Berardinis, V.; Zaparucha, A.; Grogan, G.; Vergne-Vaxelaire, C. A
Family of Native Amine Dehydrogenases for the Asymmetric
Reductive Amination of Ketones. Nat. Catal. 2019, 2, 324-333.
(25) Knaus, T.; Bohmer, W.; Mutti, F. G. Amine
Dehydrogenases: Efficient Biocatalysts for the Reductive
Amination of Carbonyl Compounds. Green Chem. 2017, 19, 453-
463.
(26) Li, J.; Pan, J.; Zhang, J.; Xu, J. H. Stereoselective Synthesis
of L-tert-Leucine by a Newly Cloned Leucine Dehydrogenase
from Exiguobacterium sibiricum. J. Mol. Catal. B: Enzym. 2014,
105, 11-17.
(27) Sekimoto, T.; Fukui, T.; Tanizawa, K. Involvement of
Conserved Lysine 68 of Bacillus stearothermophilus Leucine
Dehydrogenase in Substrate Binding. J. Biol. Chem. 1994, 269,
7262-6.
(28) (a) Smith, M.; Accinelli, A.; Tejada, F. R.; Kharel, M. K.
Chapter 28–Drugs Used in Tuberculosis and Leprosy. Side Eff.
Drugs Annu. 2016, 38, 283-293. (b) Pi, J.; Zhao, T.; Zhang, W.;
Guo, D.; Zhang, Q.; Xie, G. Ethambutol Hydrochloride Synthesis
Method. CN108218724 (A).
(29) (a) Kumar, J. K.; Narsaiah, A. V. Stereoselective Synthesis
of Tuberculostatic Agent (S,S)-Ethambutol. Org. Commun. 2014,
7, 28-33. (b) Aguiar, R. M.; Leao, R. A. C.; Mata, A.; Cantillo, D.;
Kappe, C. O.; Miranda, L. S. M.; de Souza, R. O. M. A.
Continuous-Flow Protocol for the Synthesis of Enantiomerically
Pure Intermediates of Anti Epilepsy and Anti Tuberculosis
Active Pharmaceutical Ingredients. Org. Biomol. Chem. 2019, 17,
1552-1557.
Conversion
of
Hydrophobic
Substrates
by
Amine
Dehydrogenases. ACS Catal. 2014, 4, 4021-4026. (d) Bommarius,
B. R.; Schurmann, M.; Bommarius, A. S. A Novel Chimeric
Amine Dehydrogenase Shows Altered Substrate Specificity
Compared to Its Parent Enzymes. Chem. Commun. 2014, 50,
14953-14955. (e) Ye, L. J.; Toh, H. H.; Yang, Y.; Adams, J. P.;
Snajdrova, R.; Li, Z. Engineering of Amine Dehydrogenase for
Asymmetric Reductive Amination of Ketone by Evolving
Rhodococcus Phenylalanine Dehydrogenase. ACS Catal. 2015, 5,
1119-1122. (f) Pushpanath, A.; Siirola, E.; Bornadel, A.; Woodlock,
(30) Zeymer, C.; Hilvert, D. Directed Evolution of Protein
Catalysts. Annu. Rev. Biochem. 2018, 87, 131-157.
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