Asymmetric Synthesis of Chiral Halogenated Amines using Amine Transaminases

Article abstract of DOI:10.1002/cctc.201701962

Amine transaminases (ATAs) are versatile and industrially relevant biocatalysts that catalyze the transfer of an amine group from a donor to an acceptor molecule. Asymmetric synthesis from a prochiral ketone is the most preferred route to the desired amine product, as it is obtainable in a theoretical yield of 100 %. In addition to the requirement of active and enantioselective ATAs, the choice of a suitable amine donor is also important to save costs and to avoid additional enzymes to shift the equilibrium and/or to recycle the cofactors. In this work, we identified suitable (R)- and (S)-ATAs from Aspergillus fumigatus and Silicibacter pomeroyi, respectively, to afford a set of halogen-substituted derivatives of brominated or chlorinated 1-phenyl-2-propanamine, 4-phenylbutan-2-amine, and 1-(3-pyridinyl)ethanamine. Optimization of the donor–acceptor ratio enabled application of isopropylamine as an amine donor, which resulted in high conversions and amines with 73–99 % ee.

Full text of DOI:10.1002/cctc.201701962

Accepted Article
Title: Asymmetric synthesis of chiral halogenated amines using amine
transaminases
Authors: Ayad Dawood, Rodrigo de Souza, and Uwe Bornscheuer
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ChemCatChem
COMMUNICATION
utilizing the ATA from Vibrio fluvialis JS17 was reported by Shin
et al. applying several amine donors, e.g. 1-PEA, 1-aminoindane
or 1-aminotetralin.[5] Those amine donors are favored in a
thermodynamic manner since the corresponding ketones show a
drastically lower reactivity considering the reverse reaction,
which makes them interesting for a biocatalytic approach.[5–8]
However, the use of alanine – the natural amine donor – is one
of the most common strategies for the amination of the target
ketones.[9–11] The major drawback is the necessity of the removal
of the co-product pyruvate by additional enzymes, since a large
excess of alanine does not lead to a thermodynamic more
favorable situation.[12] For this purpose pyruvate decarboxylase[2],
alanine dehydrogenase or most commonly the combination of
lactate and glucose dehydrogenase (LDH/GDH-system)[13,14]
were applied, in order to drive the desired transamination
reaction to completeness. More recently, aliphatic diamines
were reported as effective amine donors.[15–20]
An interesting alternative as amine donor is isopropylamine
(IPA) since it is cheap, achiral and the by-product acetone can
be stripped off the reaction continuously as demonstrated on
industrial scale for the synthesis of (S)-sitagliptin using an
engineered (R)-ATA.[21] The only downside of this system is the
necessity of a large IPA excess to drive the equilibrium to the
desired product side, which might hamper the stability and
activity of the ATAs.[9,12,19,21]
Asymmetric synthesis of chiral
halogenated amines using
amine transaminases
Ayad W.H. Dawood,[a] Rodrigo O.M.A. de
Souza,[b] Uwe T. Bornscheuer*[a]
Abstract: Amine transaminases (ATA) are versatile and industrially
relevant biocatalysts, catalyzing the transfer of an amine group from
a donor to an acceptor molecule. Asymmetric synthesis from a
prochiral ketone is the most preferred route to the desired amine
product, since it is obtainable at a theoretical yield of 100 %. In
addition to the requirement of active and enantioselective ATAs, also
the choice of a suitable amine donor is important to save costs and
to avoid additional enzymes for shifting the equilibrium and/or
recycling of cofactors. In this work, we identified suitable (R)- and
(S)-ATAs from Aspergillus fumigatus and Silicibacter pomeroyi,
respectively to afford a set of halogen-substituted derivatives of
brominated or chlorinated 1-phenyl-2-propanamine, 4-phenylbutan-
2-amine and 1-(3-pyridinyl)ethanamine. Optimization of the donor-
acceptor-ratio enabled the application of isopropylamine as amine
donor resulting in high conversion and amines with 73-99 %ee.
To the best of our knowledge there are only a few reports
dealing with the acceptance of halogenated ketones by amine
transaminases.[15–17,19,20,22–31] For instance, Slabu et al.,[19]
Cassimjee et al.[22] and Paul et al.[24] investigated halogenated
acetophenone derivatives. López-Iglesias et al.[27] successfully
produced fluorinated 1-(3-pyridinyl)-ethanamine and Meadows
et al.[28] as well as Frodsham et al.[29] reported the synthesis of
(S)-1-(5-fluoropyrimidin-2-yl)-ethylamine. Gomm et al.,[15] Green
et al.[16] and Martinez-Montero et al.[17] subjected 4-fluoro-
phenylacetone to an asymmetric synthesis of the corresponding
amine. Finally Schmidt et al.[30] focused on halogenated
propargyl amies. However, the compounds described in this
work were not yet investigated. We thus herein report the
asymmetric synthesis of chlorinated and brominated 1-phenyl-2-
Optically pure amines are important and versatile building blocks
for the pharmaceutical and agrochemical industry. Chemical
synthesis strategies are usually waste intensive and the
formation of chiral amines requires the use of transition metals
and chiral auxiliaries.[1] Enzymatic routes to chiral compounds
are an environmentally benign alternative, because of their mild
operation conditions and intrinsic selectivity of the biocatalysts.
One strategy to access primary chiral amines is the utilization of
amine transaminases (ATA). ATAs are pyridoxal-5’-phosphate
(PLP)-dependent enzymes and belong to the PLP fold classes I
or IV. They catalyze the transfer of a primary amine group from
a donor molecule to an acceptor molecule via a Ping-Pong-Bi-
Bi-mechanism using PLP as co-factor. The substrate recognition
is ensured by two binding pockets: a small one only able to
accommodate a methyl group, and a large one with space for
e.g., a phenyl or a carboxyl moiety. An asymmetric synthesis is
the most convenient and economically favored route to a target
amine since it starts from the prochiral ketone and results in the
desired chiral product with a theoretical yield of 100 %.[2–4] The
first asymmetric synthesis of (S)-1-phenylethylamine (1-PEA)
propanamine,
4-phenylbutan-2-amine
or
1-(3-pyridinyl)-
ethanamine derivatives (Scheme 1) using different ATAs and
isopropylamine as amine donor.
The choice of suitable amine transaminases was mainly
guided by literature search, where similar, but usually non-
halogenated ketones served as substrates.[9,10,13,19,23,24,32–35] For
instance, ATA-117 was used to make (R)-1-(4-methoxy-
phenyl)propan-2-amine[13]
, the ATA from Chromobacterium
violaceum (Cvi-TA) catalyzed the conversion of 1-(4-
methoxyphenyl)propan-2-one and 3-acetylpyridine[33], the ATAs
from Silicibacter pomeroyi[32] (Spo-TA) and Aspergillus fumigatus
(Afu-TA)[35] were utilized for the synthesis of both enantiomers of
4-phenylbutane-2-amine. Hence, we chose for our investigations
the (S)-selective ATAs Spo-TA, Cvi-TA and due to structural
similarities to the enzymes mentioned above, the ATA from
Vibrio fluvialis (Vfl-TA) as well as the (R)-selective ATA from
Aspergillus fumigatus (Afu-TA).[32,36–38] Of particular interest was
the reported IPA acceptance by Spo-TA and Afu-TA.[39]
[a]
[b]
M.Sc. Biochem. A.W.H. Dawood, Prof. Dr. U.T. Bornscheuer
Dept. of Biotechnology & Enzyme Catalysis
Institute of Biochemistry, Greifswald University,
Felix-Hausdorff-Str. 4
17487 Greifswald (Germany)
E-mail: uwe.bornscheuer@uni-greifswald.de
Web: http://biotech.uni-greifswald.de
Prof. Dr. R.O.M.A. de Souza
Biocatalysis and Organic Synthesis Group
Institute of Chemistry
Federal University of Rio de Janeiro (Brazil)
In order to get additional indicators for successful catalysis,
docking experiments with representatives of the three substrate
groups (Scheme 1) were performed in silico to strengthen the
choice of focused ATAs. The quinonoid intermediates of 1b, 9b
and 12b were docked into the active sites of Spo-TA (PDB ID
Supporting information for this article is given via a link at the end of
the document.
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10.1002/cctc.201701962
ChemCatChem
COMMUNICATION
3HMU), Afu-TA (PDB ID 4CHI), Cvi-TA (PDB ID 4A6T) and Vfl-
TA (PDB ID 4E3Q). The result showed a plausible coordination
of all docked intermediate complexes for every considered ATA
(Figure S1, Supporting Information).
phenyl-2-propanamine,
pyridinyl)-ethanamine with isopropylamine as
4-phenylbutan-2-amine
or
1-(3-
a
cheap and
convenient amine donor. The use of the ATAs from Silicibacter
pomeroyi (Spo-TA) and Aspergillus fumigatus (Afu-TA) enabled
mostly quantitative conversion of compounds 1a–12a yielding
the desired amines with excellent optical purity (mostly
>99 %ee).
ATA
NH2
O
buffer, PLP, pH 7.5, 30 °C
R
R
∗
Table 1. Conversion and enantiomeric excess values of the amine products
1b–12b obtained by asymmetric synthesis using IPA as donor and crude cell
lysate containing overexpressed Spo-TA (0.75 U) or Afu-TA (0.5 U).
n
n
1a 12a
-
1b-12b
NH2
O
Spo-TA
c [%][a,b]
Afu-TA
c [%][a,b]
excess
eeP
eeP
Substrate
[%][b]
[%][b]
NH2
NH2
1a
2a
95.6
96.6
91.4
97.6
96.3
97.1
93.3
95.9
88.0
88.8
93.9
90.7
± 3.0
± 0.8
± 0.4
± 1.3
± 2.9
± 2.2
± 2.8
± 1.9
± 6.0
± 5.0
± 4.7
± 8.3
>99
>99
98
96.2
96.1
95.0
95.9
93.4
91.1
93.9
84.5
95.6
78.4
91.4
86.4
± 0.3
± 0.4
± 0.3
± 0.5
± 1.1
± 0.4
± 0.4
± 4.1
± 0.7
± 2.3
± 0.4
± 2.9
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
R
R
R
NH2
N
1b R= 4-Br
R= 4-Cl
R= 3-Br
4b R= 3-Cl
R= 3,4-Cl
R= 2,4-Cl
7b R= 4-Br
10b R= 4-Br
11b
12b
3a
2b
3b
8b
R= 3-Br
R= 3-Br
R= 3,4-Cl
9b
R= 3,4-Cl
4a
>99
98
5b
6b
5a
Scheme 1. Amine products (bottom) obtained by the asymmetric synthesis
(top) from the corresponding prochiral ketones (1a-12a) using isopropylamine
as amine donor and different amine transaminases.
6a
>99
80
7a
8a
95
For preliminary test purposes, we decided to make the reaction
with 1-(4-bromophenyl)propan-2-one 1a as a model reaction to
investigate several reaction conditions. Initially, we tested the
ATAs for activity towards 1a using various amine donors. By the
application of the alanine/LDH/GDH system or providing 1-PEA
in a slight excess (5-fold), very good conversion of 1a to 1b was
observed using Cvi-TA (LDH/GDH system: 99 %, no conversion
with 1-PEA), Afu-TA (LDH/GDH system: 99 % and 1-PEA: 98 %)
and Spo-TA (1-PEA: 95 %; Table S1, Supporting Information).
Vfl-TA did not convert 1a at all. However, as 1-PEA is rather
cost-intensive[6] and the alanine/LDH/GDH system requires the
involvement of additional enzymes, isopropylamine as donor
was investigated (especially the donor-acceptor-ratio) using the
model substrate 1-(4-bromophenyl)propan-2-one 1a with Spo-
TA and Afu-TA. In case of Spo-TA a relative low 10-fold excess
of IPA was already sufficient to observe high conversion (>80 %).
The reaction with Afu-TA reached the same value only at 1 M
IPA (a 200-fold excess; Figure S5, Supporting Information).
Consequently, Spo-TA and Afu-TA were used for the
asymmetric synthesis of all substrates using IPA as amine donor
(Scheme 1, Table 1). Both, Spo-TA and Afu-TA showed
excellent conversions (>90 %) with minor exceptions (8a–10a
and 12a, ~78–88 % conv.). The optical purity of the chiral amine
products were predominantly excellent (>95 %ee, with two
exceptions: amines 7b and 9b, 73–80 %ee, Table 1) as
determined by chiral gas chromatography (GC) analysis (see
Supporting Information for chromatograms and retention times,
Table S4). Preparative scale reactions (50 or 100 mg 1a) using
Spo-TA or Afu-TA confirmed the identity of 1-(4-bromophenyl)-2-
propanamine 1b after GCMS analysis, 1H- and 13C-NMR
9a
73
10a
11a
12a
>99
>99
>99
[a] Reaction conditions: HEPES buffer (50 mM) pH 7.5,
isopropylamine (0.25 M for Spo-TA, 1 M for Afu-TA) , 10 % DMF, 30°C, 18 h.
[b] Conversion and enantiomeric excess were determined via chiral GC
analysis using
measurements were performed in triplicates.
5 mM ketone,
a Hydrodex-ß-TBDAc column (Macherey & Nagel). All
Experimental Section
All chemicals were purchased either from Sigma Aldrich (Darmstadt,
Germany), Roth (Karlsruhe, Germany), Acros/Thermofisher Scientific
(Waltham, USA) or Enamine (Monmouth, USA) in analytical grade.
Enzyme expression and cell lysis
Genes encoding the ATAs from Silicibacter pomeroyi and Aspergillus
fumigatus were subcloned into the pET22b vector with a C-terminal His-
tag and transformed into E. coli BL21 (DE3) as described previously.[32]
ATAs from Chromobacterium violaceum and Vibrio fluvialis were
available in a pET24b and pET28a vector containing a C-terminal or N-
terminal His-tag, respectively. The protein expression was done in TB
media with 100 µg/mL ampicillin or 50 µg/mL kanamycin at 160 rpm and
20°C. After the optical density at 600 nm (OD600) reached 0.7, expression
was induced by adding 0.2 mM isopropyl β-D-1-thiogalactopyranoside
(final concentration). After 18 h the cultures were centrifuged (4,000 x g,
15 min, 4 °C) and washed with lysis buffer (HEPES (50 mM pH 7.5), 0.1
mM PLP, 300 mM NaCl). Cell disruption was performed via sonication
using the Bandelin Sonoplus HD 2070 (8 min, 50 % pulsed cycle, 50 %
power) on ice followed by centrifugation in order to remove cell debris
(12,000 x g, 45 min, 4 °C, Sorvall centrifuge). The supernatant containing
the crude ATA was stored at 4 °C until use.
spectroscopy
(Supporting
Information).
The
absolute
configuration of each enantiomer was verified by literature data
using optical rotation values.[40]
Thus, we demonstrated the enzymatic synthesis of a set of
halogen-substituted chiral benzylic amines derived from 1-
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10.1002/cctc.201701962
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COMMUNICATION
Determination of transaminase activity
The combined organic layers were washed with 2x 15 mL saturated brine
solution, dried over anhydrous MgSO4 and evaporated under vacuum.
The consistency of the amine product was a yellow oil. Each product was
confirmed via GCMS and 10-12 mg each were subjected to 1H- and 13C-
NMR spectroscopy. A racemic standard was synthesized for comparison
reasons according to literature[42] (1H- and 13C-NMR spectra, see
Supporting Information).
The characterization of the ATAs was done via the acetophenone assay
according to Schätzle et al.[41] with slight modifications. As all used ATAs
showed their optimum at basic pH values, the assay was performed in
CHES buffer (50 mM pH 9). In the reaction solution the concentrations of
the amine donor ((R)- or (S)-1-PEA) and the acceptor pyruvate was set
to 1.25 mM in 0.25 % (v/v) DMSO. Briefly, 10 µL of a pre-diluted ATA
solution was mixed with buffer and the reaction was initiated by the
addition of the reaction solution. The formation of acetophenone was
quantified at 245 nm using the Tecan Infinite M200 Pro (Crailsheim,
Germany) at 30 °C. One unit (U) of ATA activity was defined as the
formation of 1 µmol acetophenone per minute (ε= 12 mM-1cm-1). All
measurements were performed in triplicates.
The absolute configuration was determined via optical rotation data using
the Polar-L polarimeter from IBZ Messtechnik.
A solution of each
enantiomer in chloroform was prepared (concentration as indicated). The
specific optical rotation data of each enantiomer were as followed and
could be verified with literature.[40]
(S)-1-(4-bromophenyl)-2-propanamine (S)-1b, 25 % isolated yield (not
optimized), >99 %ee, [α]20D = +20.8 (c =0.9, CHCl3).
(R)-1-(4-bromophenyl)-2-propanamine (R)-1b, 27 % isolated yield (not
optimized), >99 %ee, [α]20D = -25.9 (c =1.2, CHCl3).
Asymmetric synthesis of the chiral amines 1b–12b
Biotransformations of ketones 1a–12a were performed in triplicates and
in 0.25 mL scale using 1.5 mL glass vials at 30 °C and 950 rpm shaking.
The reaction mixtures contained 50 µL ATA crude lysate (9–14 U/mL),
5 mM ketone, 10 % DMF as co-solvent, 250 mM IPA in HEPES 50 mM
pH 7.5–8. After 24 h incubation, the reaction was quenched by adding
3 M NaOH (resulting in pH ≥13). Samples for gas chromatography (GC)
analysis were taken immediately after this quenching.
Bioinformatic analyses
Docking studies were done using YASARA Structure (v.17.1.28).[43] The
quinonoid intermediate (the reactive intermediate in the ATA reaction)
consisting of PLP and 1b/9b/12b was modelled by a combination of
ChemDraw® (v.11) and YASARA followed by energy minimization. Prior
to the docking experiments a refinement of the lowest energy ligand
conformation and all used crystal structures from the PDB database was
performed with the built in macro (YAMBER3 force field at 298 K for
500 ps, standard settings). Since Spo-TA (PDB ID 3HMU) was
crystallized in the apo-form, the position of the PLP co-factor had to be
determined first as its position was crucial for the visual evaluation of the
docking result (mainly by superposing the docked ligand-receptor
complex with the refined, PLP containing crystal structure and checking
the coverage of the pyridine ring of the PLP). A PDB-BLAST search was
done, revealing the best result in sequence identity of 53 % with the ATA
from Chromobacterium violaceum (PDB ID 4A6T, for BLAST result see
Supporting Information). Both structures were superimposed and the
position of the PLP was transferred from one structure to another.
Because of plausibility reasons the surrounding binding residues of the
phosphate group and the nitrogen of the PLP pyridine ring were
confirmed with literature data.[44] The applied docking method was the
implemented AutoDock VINA algorithm with standard settings, which
means 100 runs in total and subsequent clustering to give distinct
complex conformations. The output was evaluated visually. Usually the
best conformation revealed by this way was one of those with the highest
binding energy and lowest dissociation constant according to the docking
log file. All illustrations were made with Pymol (v.1.7).
GC analysis
Samples of 30–40 µL were taken for chiral GC analysis and extracted
with 250 µL ethyl acetate containing 1 mM 4’-iodoacetophenone as
internal standard for quantification. The organic layers were dried over
anhydrous MgSO4 and derivatized with N-Methyl-bis-trifluoroacetamide
(MBTFA) by adding 7.5 µL of the commercial stock solution to 100 µL of
the organic layer and incubation at 60 °C for 30 min. Afterwards, the
samples were analyzed immediately using the chiral Hydrodex-ß-TBDAc
column (Macherey & Nagel). For the analysis of all ketones the following
temperature gradient program was established: initial temperature 80 °C,
kept for 10 min, linear gradient to 175 °C with a slope of 4 °C min-1, kept
for 13 min, linear gradient to 220 °C with a slope of 20 °C min-1, kept for
5 min. The conversion for each compound was determined by
quantification of substrate consumption via calculation of the response
factor. Each sample included an internal standard and was set in relation
to control experiments in each batch experiment with known substrate
concentration. The linearity of the response factor over a certain range
was verified via a calibration curve. The chiral analysis of the amine
products was either done as described above or with the following
temperature profile: 60 °C, kept for 35 min, linear gradient to 165 °C with
a slope of 2 °C min-1, kept for 20 min, linear gradient to 220 °C with a
slope of 5 °C min-1, kept for 10 min.
Acknowledgements
Preparative scale synthesis of 1b
We thank the Bundesministerium für Bildung und Forschung
(Grant 01DN14016) for financial support. Prof. Dr. de Souza
thanks the Alexander-von-Humboldt foundation for a Capes-
Humboldt research fellowship. We are also grateful to Prof. Dr.
Bednarski and Patrick Werth (Pharmazeutische/Medizinische
Chemie, Institut für Pharmazie, Greifswald) for assisting with
some equipment.
The conversion of 1-(4-bromophenyl)propan-2-one 1a to 1-(4-
bromophenyl)-2-propanamine 1b was performed in preparative scale
with crude cell lysate containing Spo-TA or Afu-TA, respectively. The
following conditions were applied: 50 mg 1a (using Spo-TA) or 100 mg
1a (using Afu-TA) were added to an Erlenmeyer flask and mixed with
DMF (12 % final concentration), HEPES buffer (50 mM final, pH 7.5) and
isopropylamine (850 mM final concentration). The pH was adjusted with
aqueous HCl. In the end 15 vol% of crude cell lysate was added, which
led to a final working volume of 30 mL in case of Spo-TA and 50 mL in
case of Afu-TA. The reaction mixture was incubated for 72 h at 30 °C
under agitation. For the quantification of the conversion, samples were
taken and extracted as described above. The reaction was stopped when
no further conversion was observed during reaction monitoring (for Spo-
TA 98 %, for Afu-TA 62 %). The following reaction workup was done as
followed: After acidification with 3 M aq. HCl to pH 1, an extraction with
2x 30 mL ethyl acetate was performed in a separation funnel. The
aqueous layer was basified afterwards with 3 M aq. NaOH solution to a
pH of 11–12 and extracted 5 times with 15 mL methyl tert-butyl ether.
Keywords: Amine transaminases, biocatalysis, chiral amines,
isopropylamine, halogenated ketones, molecular docking
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This article is protected by copyright. All rights reserved.

10.1002/cctc.201701962
ChemCatChem
COMMUNICATION
COMMUNICATION
The asymmetric synthesis of chiral
brominated and chlorinated 1-phenyl-
Ayad W.H. Dawood, Rodrigo O.M.A. de
Souza, Uwe T. Bornscheuer*
2-propanamine,
4-phenyl-butan-2-
amine and 1-(3-pyridinyl)-ethanamine
derivatives is presented. Using
suitable (R)- and (S)-selective ATAs
from Aspergillus fumigatus and
Silicibacter pomeroyi a set of halogen-
substituted amines was obtained at
high conversion (74–99%) with mostly
excellent optical purity (73–99%ee)
using isopropylamine as amine donor.
Page No. – Page No.
Asymmetric synthesis of chiral
halogenated amines using amine
transaminases
This article is protected by copyright. All rights reserved.