Asymmetric Amination of α-Chiral Aliphatic Aldehydes via Dynamic Kinetic Resolution to Access Stereocomplementary Brivaracetam and Pregabalin Precursors

Article abstract of DOI:10.1002/adsc.201701449

Over the last decades biocatalysis has emerged as an indispensable and versatile tool for the asymmetric synthesis of active pharmaceutical ingredients (APIs). In this context, especially transaminases (TAs) have been successfully used for the preparation of numerous α-chiral, optically pure amines, serving as important building blocks for APIs. Here we elaborate on the development of transaminases recognizing the α-chiral centre adjacent to an aldehyde moiety with aliphatic residues, opening up concepts for novel synthetic routes to the antiepileptic drugs Brivaracetam and Pregabalin. The transformation proceeded via dynamic kinetic resolution (DKR) based on the bio-induced racemisation of the aldehyde enantiomers, enabling the amination of the racemic substrates with quantitative conversions. Medium, substrate as well as enzyme engineering gave access to both (R)- and (S)-enantiomers of the amine precursors of the stereocomplementary drugs in high optical purity, representing a short route to mentioned APIs. (Figure presented.).

Full text of DOI:10.1002/adsc.201701449

Title: Asymmetric Amination of α-Chiral Aliphatic Aldehydes via
Dynamic Kinetic Resolution to Access Stereocomplementary
Brivaracetam and Pregabalin Precursors
Authors: Christine Fuchs, Judith Farnberger, Georg Steinkellner,
Johann Sattler, Mathias Pickl, Robert Simon, Ferdinand
Zepeck, Karl Gruber, and Wolfgang Kroutil
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
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content of this Accepted Article.
To be cited as: Adv. Synth. Catal. 10.1002/adsc.201701449
Link to VoR: http://dx.doi.org/10.1002/adsc.201701449

10.1002/adsc.201701449
Advanced Synthesis & Catalysis
FULL PAPER
DOI: 10.1002/adsc.201((will be filled in by the editorial staff))
Asymmetric Amination of α-Chiral Aliphatic Aldehydes via
Dynamic Kinetic Resolution to Access Stereocomplementary
Brivaracetam and Pregabalin Precursors
Christine S. Fuchs,a Judith E. Farnberger,a Georg Steinkellner,a,b Johann H. Sattler,c
Mathias Pickl,c Robert C. Simon,c Ferdinand Zepeck,d Karl Gruber,b and Wolfgang
Kroutilc*
a
Austrian Centre of Industrial Biotechnology, ACIB GmbH, c/o University of Graz, Harrachgasse 21, 8010 Graz,
Austria
b
Institute of Molecular Biosciences, University of Graz, Humboldtstrasse 50/3, 8010 Graz, Austria
Institute of Chemistry, Organic and Bioorganic Chemistry, University of Graz, Harrachgasse 21, 8010 Graz, Austria
c
[Fax: (+43)-316-380-9840; phone: (+43)-316-380-5350; e-mail: wolfgang.kroutil@uni-graz.at]
Sandoz GmbH, Biocatalysis Lab, Biochemiestrasse 10, 6250 Kundl, Austria
d
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.201######.((Please
delete if not appropriate))
Abstract. Over the last decades biocatalysis has emerged as The transformation proceeded via dynamic kinetic
an indispensable and versatile tool for the asymmetric resolution (DKR) based on the bio-induced racemisation of
synthesis of active pharmaceutical ingredients (APIs). In this the aldehyde enantiomers, enabling the amination of the
context, especially transaminases (TAs) have been racemic substrates with quantitative conversions. Medium,
successfully used for the preparation of numerous α-chiral, substrate as well as enzyme engineering gave access to both
optically pure amines, serving as important building blocks (R)- and (S)-enantiomers of the amine precursors of the
for APIs. Here we elaborate on the development of stereocomplementary drugs in high optical purity,
transaminases recognizing the α-chiral centre adjacent to an representing a short route to mentioned APIs.
aldehyde moiety with aliphatic residues, opening up
concepts for novel synthetic routes to the antiepileptic drugs Keywords: amines; asymmetric catalysis; transaminases;
Brivaracetam and Pregabalin.
biotransformations; -chiral amines
Introduction
Both Brivaracetam 1 and Pregabalin 2 (Scheme 1) are
γ-amino carboxylic acids exhibiting remarkable
physiological activity on the central nervous system
(CNS).[1-2] Consequently, they represent highly
valuable pharmaceuticals for the treatment of various
neurological diseases. Pregabalin for instance, is
marketed by Pfizer under the trade name Lyrica® and
made it into the 20 top selling drugs worldwide in
2013.[3-4] Brivaracetam in turn is a relatively new and
promising anticonvulsant drug developed by UCB
pharma and has recently been approved as adjunctive
therapeutic for the treatment of epilepsy.[5] UCB
pharma has developed several synthetic routes for the
preparation of the 2-oxo-pyrrolidine derivative,[6-8]
however, these exclusively chemical methods come
along with poor stereoselectivity and hence elaborate
chromatography steps in order to obtain the far more
active (2S,4R)-stereoisomer in pure form. The
formation of side products and/or the unwanted
enantiomer is also an issue regarding the classical
Scheme 1. Brivaracetam 1 and Pregabalin 2: β-chiral
amines 3 and 4 were identified as precursors accessible by
reductive amination of corresponding α-chiral aldehydes 5
and 6.
manufacturing route towards Pregabalin developed
by Pfizer,[9] where the amine is prepared in racemic
form and the targeted (S)-enantiomer is obtained via
1
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10.1002/adsc.201701449
Advanced Synthesis & Catalysis
diastereomeric salt formation with mandelic acid.
This
process has been improved in terms of yield and
economical aspects by replacing chemical resolution
by a lipase-catalysed one.[10-11]
recycling system[60] to push the equilibrium towards
the product side (Scheme 2). Since γ-amino esters 3a-
d were converted to the corresponding lactam 7 by
ring closure due to basic
Table 1. Library of investigated wild-type TAs from
different organisms
Recently, biocatalysis has contributed significantly to
the synthesis of APIs[12-16] and a vast number of
methods involving ene-reductases,[17-19] nitrilases[20-22]
and tautomerases[23] were established serving as
alternative enzymatic routes towards Pregabalin via
asymmetric synthesis or resolution of enantiomers.
Interestingly, despite of their popularity for the
synthesis of chiral amines used for APIs and their
successful application also on industrial scale,[24-31]
transaminases (TA)[32-51] have not yet been considered
as the biocatalyst of choice to prepare key
intermediates of Pregabalin and Brivaracetam by
deracemisation of the corresponding aldehydes.
Reports of transaminase mediated amination of α-
chiral aldehydes[52-55] prior to this work are scarce and
focus on substrates bearing the α-stereocentre in
benzylic position. These compounds allow
racemisation of the aldehyde in aqueous medium due
to the acid proton in benzylic position as well as in α-
position to the aldehyde, consequently enabling
dynamic kinetic resolution (DKR). In the most recent
example a transaminase mediated DKR of a cyclic α-
chiral aldehyde represented the key step in the
chemo-enzymatic synthesis of the heart-rate reducing
agent Ivabradine.[55]
In this study, we focused on the stereoselective
amination of racemic aldehydes 5 and 6 (Scheme 1),
bearing aliphatic residues at the chiral centre in α-
position to the aldehyde moiety.[56-57] The TA-
catalysed bioamination gives access to chiral amines
3 and 4, being precursors for the anticonvulsant
agents Brivaracetam 1 and Pegabalin 2. Since these
chiral compounds display an absolute configuration
opposite to each other, stereocomplementary
transaminases are required for their selective
preparation.
Source Organism
Abbr. Select. Ref.
[61]
Arthrobacter sp. KNK168
Aspergillus terreus
Giberella zeae
Hyphomonas neptunium
Neosartorya fischeri
Arthrobacter citreus
ArR
AT
GZ
HN
NF
(R)
(R)
(R)
(R)
(R)
(S)
(S)
(S)
(S)
(S)
(S)
(S)
(S)
(S)
(S)
[62]
[62]
[62]
[62]
[63]
[63]
[63]
[64]
[65]
[66]
[67]
[67]
[68]
[66]
ArS
BM
CV
OA
PD
PF
Bacillus megaterium
Chromobacterium violaceum
Ochrobactrum antropi
Paracoccus dentrificans
Pseudomonas fluorescens
Pseudomonas putida KT2440
Pseudomonas putida KT2440
Silicibacter pomeroyi
Vibrio fluvialis
PP1
PP2
SP
VF
work-up conditions, analytical methods were
established and optimised for product 7. Racemic
reference material was synthesised by chemical
means and the absolute configuration was assigned
by comparison of elution order on a chiral HPLC
phase with literature.[8] Most of the enzymes tested
successfully converted the chiral oxo esters 5a-d to
the corresponding amino esters in enantioenriched
form (Table 2). While eleven wild type enzymes
formed the (R)-amine (entry 1-11), only four
transaminases gave the (S)-enantiomer with mostly
poor stereoselectivity. In general, the highest ee
values of product 7 were reached when the t-butyl
ester 5c was applied as substrate, probably due to
steric reasons. In many cases the racemic substrates
were converted quantitatively to product 7, which
indicated that the reaction proceeded via a dynamic
kinetic resolution (DKR) involving racemisation of
Results and Discussion
the aliphatic aldehyde substrate. Actually,
a
racemisation of oxo esters 5a-d was not expected due
to the aliphatic residue at the chiral centre and a
proton being less acidic compared to benzylic
examples, which do not simply racemise in buffer.
Therefore, racemisation was investigated in more
detail later.
We started with the synthesis of oxo esters 5 serving
as precursors for Brivaracetam 1 and tested them in
the biocatalytic amination reaction. Then we intended
to employ the most promising biocatalysts and
reaction conditions for related oxo ester 6 leading to
Pregabalin 2. The ester form of precursor 5 and 6 was
preferred due to its easier handling; additionally, the
size of the ester moiety might allow tuning of chiral
recognition by the enzyme and therefore the optical
purity of the product obtained. Consequently, ethyl-,
i-propyl-, t-butyl and benzyl esters of Brivaracetam
precursor 5 were prepared.[58-59] The asymmetric
amination of aldehydes 5a-d was first tested with a
library of wild-type transaminases (TAs), showing
both (R)- and (S)-selectivity for the amination of
ketones (Table 1). In the first experiments, alanine
was used as amine donor together with the AlaDH-
2
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10.1002/adsc.201701449
Advanced Synthesis & Catalysis
Scheme 2. Transaminase-catalysed bioamination of
Brivaracetam precursors 5a-d and Pregabalin precursor 6..
Table 2. (R)- and (S)-selective bioamination of Brivaracetam precursors 5a-d.a)
rac-5a rac-5b
rac-5c
rac-5d
entry
TA
conv.b)
(%)
ee 7c)
(%)
conv.b)
(%)
ee 7c)
(%)
conv.b)
(%)
ee 7c)
(%)
conv.b)
(%)
ee 7c)
(%)
1
2
3
4
5
6
7
8
PP1
PP2
PD
ArS
CV
PF
VF
HN
SP
OA
NF
AT
ArR
BM
GZ
>99
99
rac
>99
86
4 (R)
14 (R)
rac
>99
78
>99
80
97
>99
99
73
98
80
15
22 (R)
42 (R)
56 (R)
52 (R)
17 (R)
54 (R)
62 (R)
92 (R)
14 (R)
52 (R)
11 (R)
16 (S)
28 (S)
rac
81
60
88
82
91
73
82
78
8 (R)
24 (R)
30 (R)
34 (R)
4 (R)
22 (R)
12 (R)
46 (R)
7 (R)
>99
>99
>99
>99
>99
75
>99
>99
>99
>99
>99
73
8 (R)
10 (R)
32 (R)
8 (R)
90 (R)
n.d.d)
n.d.d)
n.d.d)
30 (S)
20 (S)
14 (S)
n.d.d)
22 (R)
10 (R)
70 (R)
n.d.d)
20 (R)
26 (R)
80 (R)
n.d.d)
9
n.d.d)
n.d.d)
n.d.d)
>99
>99
50
n.d.d)
n.d.d)
n.d.d)
98
n.d.d)
n.d.d)
n.d.d)
62
10
11
12
13
14
15
n.d.d)
n.d.d)
n.d.d)
n.d.d)
14 (S)
20 (R)
10 (S)
n.d.d)
70
95
98
70
4 (S)
92
67
32 (S)
71
n.d.d)
n.d.d)
n.d.d)
n.d.d)
n.d.d)
55 (S)
n.d.d)
a) Reaction conditions: substrate 5 (50 mM), TA (lyophilised cells of E. coli, 20 mg/mL) in phosphate buffer (100 mM, pH
7, 1 mM PLP) at 30 °C, 120 rpm, 24 h, L-or D-alanine (250 mM), AlaDH (12 U), FDH (11 U), NAD+ (1 mM) and
b)
ammonium formate (150 mM). Conversion of 5 to 7 determined from area by GC-FID measurement on an achiral
stationary
phase.
determined
c)
d)
Optical
purity
was
measured
by
GC-FID
on
a
chiral
phase.
not
The most promising amination results concerning
conversion were obtained with HN-TA, affording the
product with 73% conversion and 92% ee (R). While
the (R)-amine is required for the production of
Brivaracetam, the identification of enantio-
complementary transaminases leading to the (S)-
enantiomer was of importance to access also
Pregabalin intermediates in due course. For this
purpose, the most selective TA was the one from
Giberella zeae forming the amine (S)-3c with 70%
conversion and 55% ee (Table 2, entry 15).
upon co-solvent addition. In order to identify further
efficient transaminases yielding the (S)-product in
higher optical purity, bioamination of substrates 5a,
5c and 5d was tested with a variant of ArR-TA,
namely ArRmut11, which has been evolved for the
synthesis of Sitagliptin.[24] Furthermore, three variants
of ArR-TA and six ones of ArRmut11-TA were
designed to achieve higher activity and improved
optical purity of the amine (Table 3). With these
enzymes 2-propylamine (IPA) was employed as
amine donor and DMSO (20 vol%) was added as co-
solvent. Using mentioned reaction conditions, imine
formation between aldehyde 5 and either IPA or
product amine 3, respectively, was observed in
Interestingly, ArR-TA showed
a
switch in
enantiopreference depending on the ester moiety of
the substrate. Thus, it preferred the (R)-enantiomer of
5a and the (S)-enantiomer of 5b-d (Table 2, entry 13). several samples. Nevertheless, many reactions went
It may also be noted, that the stereopreference for the
amination of aldehydes does not correlate with the
one observed for the amination of ketones, as
indicated before.[54] Since it has been shown in
previous work[54] that reaction parameters such as
temperature, pH and especially organic co-solvents
can have a significant effect on the biocatalytic
amination of α-chiral aldehydes, Brivaracetam
precursor 5c was chosen for a detailed investigation,
employing selected enzymes (ArR, ArS, PD, PF, VF)
under varying reaction conditions (for detailed data
see SI). While reaction temperature did not
significantly affect the stereoselectivity of the
to completion. The variants of ArR-TA were
designed based on the presented approach in previous
studies[24] in order to raise the activity of the enzyme.
From the three tested ArR-TA variants only the
double variant (ArR-G132F/S218P, entry 4)
transformed the tested aldehydes to the corresponding
(S)-amines with high conversion (90 to >99%), albeit
with moderately improved ee’s (up to 34%)
compared to the wild-type (ArR-WT). The results
also showed that the enantiopreference can be easily
inverted at low ee, since with variant ArR-G132F the
(R)-enantiomer was obtained, whereas the double
variant led to the (S)-enantiomer. Since ArRmut11
only led to racemic amine for two of the oxo esters
and 20% ee in the best case (entry 5), further variants
were designed. Originally, ArRmut11 has been
evolved to enable the transformation of sterically
demanding ketones as well as to increase enzyme
stability and activity.[24] To ideally maintain the high
enzymes with exception of
a
switch in
enantiopreference of ArR, the addition of organic co-
solvents such as DMSO, DMF and DME positively
influenced ee-values obtained. However, (S)-selective
enzymes like GZ, which would be of special interest
for the preparation of Pregabalin, lost their activity
3
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10.1002/adsc.201701449
Advanced Synthesis & Catalysis
stability of the peptide backbone, ArRmut11 was
taken as starting point for further variations with the
idea to reduce the size of one of the two binding
pockets. The mutations M117F and G279A were
expected to make the binding pocket tighter, however
only
variant
ArRmut11-G279A
led
to
Table 3. Bioamination of Brivaracetam precursors 5a, 5c and 5d employing variants of ArR-TA and ArRmut11.a)
rac-5a
rac-5c
rac-5d
entry
TA
7b) (%) ee 7c) (%)
7b) (%) ee 7c) (%)
7b) (%) ee 7c) (%)
1
2
3
4
5
6
7
8
ArR-WT
ArR-S218P
ArR-G131F
ArR-G131F/S218P
ArRmut11
>99
<1
51
20 (R)
n.d.e)
26 (R)
34 (S)
rac
95
<1
<1
>99
90
>99
>99
<1
97
98
75
55
20 (S)
n.d.e)
62
8
4 (S)
n.d.e)
40 (R)
33 (S)
rac
n.d.e)
25
90
81
80
65
91
93
68
52
n.d.f)
>99
>99
>99
>99
56
>99
>99
>99
n.d.f)
20 (S)
20 (S)
6 (S)
ArRmut11-M117F
ArRmut11-G279A
rac
rac
35 (S)
12 (R)
rac
40 (S)
50 (S)
n.d.f)
20 (S)
30 (S)
10 (R)
rac
32 (S)
50 (S)
n.d.f)
ArRmut11-M117F/G279A
ArRmut11-A60V/M117F
ArRmut11-A60V/M117F/G279A
ArRmut11-A60V/M117F/G279V
ArRmut11-A60V/M117F/G279Vd)
n.d.e)
9
8 (S)
10
11
12
23 (S)
74 (S)
80 (S)
a) Reaction conditions: substrate 5 (50 mM), TA (lyophilised cells of E. coli, 20 mg/mL) in phosphate buffer (100 mM, pH
b)
7, 1 mM PLP), 20 vol% DMSO if not indicated otherwise, 2-propylamine (500 mM) at 45 °C, 120 rpm, 24 h.
Determined from area by GC-FID measurement on an achiral stationary phase. The value for 7 represents the percentage
c)
d)
e)
of 7 in the mixture of 5/7/imines. Optical purity was measured by GC-FID on a chiral phase. 20 vol% DMF. not
determined due to low conversion. f) not determined.
an improvement, allowing transformation of the three
esters 5a,c,d with low stereo-recognition (entry 7, ee
20-35%). Interestingly, the variant bearing both
mutations (ArRmut11-M117F/G279A) turned out to
be useless due to the decrease of both conversion and
product ee. Comparing the wild type (ArR) and the
new variants, the additional amino acid exchange
A60V was suspected to improve the compactness of
corresponding alcohol, catalysed by the alcohol
dehydrogenase ADH-A from Rhodococcus ruber[71-73]
(overexpressed in E. coli and partially purified by
heat precipitation, please note that here DKR was not
observed).
Actually, no significant racemisation of optically
enriched 5 (within 2-4 hours) was observed neither in
buffer (phosphate, pH 7, 100 mM), nor in buffer
the structure, especially at the dimer interface. Indeed, supplemented with pyridoxamine phosphate (PMP,
variant ArRmut11-A60V/M117F/G279A led to
>99% conversion and slightly improved ee (entry 9).
Since position 279 seemed to have significant impact
on the ee, a more demanding amino acid (valine) was
introduced at this position instead of alanine. This
triple variant (ArRmut11-A60V/M117F/G279, entry
11) enabled the highest ee (up to 74%) obtained so
far for the (S)-enantiomer. Addition of 20 vol% DMF
further improved the optical purity to 80% (entry 12),
making this variant a promising candidate for the
preparation of Pregabalin.
0.5 mM), the aminated form of the cofactor pyridoxal
phosphate (PLP) used by TAs. Racemisation was not
promoted by amino acids such as L-proline or L-
lysine (5 mM) either. However, when the aldehyde
was incubated in buffer together with elevated
concentration of amine donor rac-alanine (250 mM)
clearly detectable racemisation was observed within 4
hours (but too slow for a DKR). While racemisation
was not induced by the TA (ArRmut11, partially
purified by heat treatment) itself in buffer, it was
slightly increased by addition of PMP as well as the
presence of L-alanine (250 mM). It turned out that
amines, e.g. IPA efficiently caused racemisation,
which was even doubled when enzyme and PLP (1
mM) were present in addition to IPA (50 mM).
Nevertheless, also crude E. coli extract significantly
provoked racemisation, most likely due to the amine
compounds present. Therefore, it was assumed that
the substrate bearing aliphatic moieties at the chiral
centre in α-position to the aldehyde moiety were
racemised via Schiff-base formation (Scheme 3).[74]
Since in the amination of 5a-d the unreacted substrate
was detected in racemic form in every experiment
and several transformations went to completion with
high ee for the product, it became obvious that the
reaction proceeded via
a
dynamic kinetic
resolution[69-70] involving racemisation of the
substrate. Notably, substrates 5 do not racemise if
only applied in buffer at pH 7-9, as it would be the
case for substrates bearing a benzylic proton.[52-54]
Investigating the racemisation of enantioenriched
precursor 5c in the presence of various components
of the transaminase reaction system allowed to
elucidate the reason for the observed racemisation
and subsequent DKR. Enantioenriched 5c was
prepared by enantioselective reduction of rac-5c in a
kinetic resolution, transforming one enantiomer to the
4
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10.1002/adsc.201701449
Advanced Synthesis & Catalysis
NAD+ (1 mM) and ammonium formate (150 mM);
2-propylamine: substrate (50 mM), TA (lyophilised cells
of E. coli, 20 mg/mL) in phosphate buffer (100 mM, pH 7,
1 mM PLP), 2-propylamine (500 mM) at 45 °C, 120 rpm,
b)
24 h (1 mM).
Determined from area by GC-FID
measurement on an achiral stationary phase. The value for
8 represents the percentage of 8 in the mixture of
6/8/imines. c) Optical purity was measured by GC-FID on a
chiral phase. d) in presence of 20 vol% DME. e) in presence
of 20 vol% DMSO. f) in presence of 20 vol% DMF. g) not
Scheme 3. Proposed mechanism for the racemisation of
substrate 5c via imine/enamine formation exemplified with
2-propylamine (IPA).
h)
determined due to quantitative imine formation.
determined due to low conversion.
not
The anticipated effect of increased stereoselectivity
caused by the additional methyl functionality of 6
was not found, except when ArRmut11-A60V/M11F/
G279A was used as biocatalyst (entry 9). In this case,
the ee of product 8 was doubled compared to lactam
7 obtained from Brivaracetam precursor rac-5c
(Table 3, entry 10). In general, the ee values obtained
for product 8 were comparable to the ones of product
7, albeit conversions to 8 were significantly lower,
exceeding 50% only in few examples. In case of the
desired (S)-product an increase of optical purity was
again achieved by employing ArRmut11 variants.
The smaller the binding pocket by substitution of
glycine at position 279 with a bigger amino acid
residue, the better the ee, however going in hand with
In a next step, the focus was set on Pregabalin. Since
in the case of Brivaracetam precursors the tert-butyl
ester 5c gave the best results with respect to optical
purity in most experiments, tert-butyl ester 6 was
chosen as precursor substrate for Pregabalin. It was
prepared similarly as described in literature,[58-59]
however, since the starting material was not
commercially available, the route was expanded by
one additional step (for further information check SI).
The optical purity to be obtained with used TAs was
assumed to be higher than for Brivaracetam
precursors 5 due to the additional methyl group and
consequently, the higher steric demand. The
asymmetric bioamination of aldehyde 6 (Scheme 2)
was performed using the most promising biocatalysts
and reaction conditions identified previously for
aldehyde 5c. Consistent with the behaviour of amino
esters 3a-d the formed Pregabalin intermediate 4 was
transformed to the corresponding lactam 8 during
basic work-up. Racemic reference material was
synthesised by transformation of Pregabalin to its
corresponding methyl ester, followed by subsequent
spontaneous cyclisation. Two of the tested TAs did
not convert aldehyde 6 at all (Table 4) and in
accordance with the results obtained for Brivaracetam
precursor 3, the (R)-enantiomer of 8 was rather easily
accessible in high optical purity (entry 2-3).
a
loss of conversion. Thus, ArRmut11-
A60V/M117F/G279V transformed rac-6 with
moderate conversion (27%) and 76% ee (entry 11)
into the (S)-amine.
To further improve the stereoselectivity of the most
promising enzymes and to increase the optical purity
of aminated product (S)-4, more variants were
constructed. The fact that only a slightly reduced size
of the binding pocket of ArRmut11 had significant
impact on the selectivity, encouraged the study of
further mutations at this position. However, replacing
glycine 279 by bulkier amino acid residues such as
leucine, isoleucine and phenylalanine led to variants
with decreased efficiency and negligibly improved
stereoselectivity (see SI). Thus, a different approach
for developing variants was pursued, involving the
generation of a homology model based on the
structure of the wild type enzyme ArR-TA. At first
we aimed for understanding why an enlargement of
the amino acid residue at position 279 from glycine to
alanine and then to valine successively increased the
stereoselectivity of ArRmut11-A60V/M117F, but a
substitution to even bigger residues such as Leu, Ile
or Phe did not have beneficial effects. Based on the
homology model (see SI), we envisioned that these
residues were rather flexible and thereby able to
adapt conformations which did not result in
interaction with the substrate molecule. Since the TA
from Giberella zeae (GZ) allowed the formation of
(S)-3c with 70% conversion and 55% ee, this enzyme
was chosen as second candidate to be engineered.
Sharing a sequence identity of 35% with ArRmut11,
it was also comprised in the homology modelling
approach, leading to active site models of both
enzymes (Figure 1). To identify amino acid residues
Table 4. Bioamination of Pregabalin precursor 6.a)
rac-6
entry TA
8b) (%) ee 8c) (%)
1
2
3
4
5
6
7
8
PFd)
30
70
50
<1
40
<1
85
65
n.d.g)
90 (R)
94 (R)
n.d.h)
VFd)
HN
AT
GZ
ArR-WT
n.d.g)
n.d.h)
12 (S)
20 (S)
ArRmut11e)
ArRmut11f)
ArRmut11-
A60V/M117F/G279Af)
ArRmut11-
A60V/M117F/G279Ve)
ArRmut11-
A60V/M117F/G279Vf)
9
48
46
27
50 (S)
64 (S)
76 (S)
10
11
a) Reaction conditions: AlaDH-system: substrate (50 mM),
TA (lyophilised cells of E. coli, 20 mg/mL) in phosphate
buffer (100 mM, pH 7, 1 mM PLP) at 30 °C, 120 rpm, 24 h,
L-or D-alanine (250 mM), AlaDH (12 U), FDH (11 U),
5
This article is protected by copyright. All rights reserved.

10.1002/adsc.201701449
Advanced Synthesis & Catalysis
which are important for the stereoselective
transformation of substrates investigated, residues
from the active site of GZ-TA were replaced with the
ones in equivalent position of ArRmut11-
A60V/M117F/G279V (for sake of easier reading
designated as ArRmut*) and vice versa (for more
detailed information see SI). Hence, five mutants of
tested for the amination of substrates rac-5c and rac-
6. Two of the prepared ArRmut* variants converted
Brivaracetam precursor 5c quantitatively with 77% ee
and 56% ee (S), respectively (Table 5), representing a
significant improvement of conversion, while
preserving the optical purity achieved by the template
ArRmut* and six of GZ-TA were constructed and
Figure 1. Active site models of ArRmut11-A60V/M117F/G279V (A) and GZ-TA (B).
Table 5. Bioamination of precursors 5c and 6 using variants of ArRmut*-TAa) and GZ-TAb).
rac-5c
rac-6
7c) (%)
ee 7d) (%)
8c) (%)
ee 8d) (%)
analogy of mutation
number for ArRmut*
entry
TA
1
2
3
4
5
6
7
8
ArRmut*
54
<1
<1
>99
>99
<1
70
62
48
86
<1
94
80 (S)
n.d.e)
n.d.e)
77 (S)
56 (S)
n.d.e)
55 (S)
48 (S)
rac
27
<1
<1
<1
15
10
40
63
3
60
<1
65
25
92
76 (S)
n.d.e)
n.d.e)
n.d.e)
58 (S)
n.d.f)
ArRmut*-T64V
ArRmut*-T119E
ArRmut*-I152V
ArRmut*-S277T
ArRmut*-S277N
GZ
GZ-L56V
GZ-V60T
GZ-E115T
GZ-V148I
n.d.f)
L60V (red)
V64T
80 (S)
42 (S)
15 (S)
n.d.e)
72 (S)
n.d.f)
9
10
11
12
13
E119T (green)
V152I (yellow)
T277S (orange)
T277N (orange)
L60V-T277S
6 (S)
n.d.e)
34 (S)
n.d.e)
25 (S)
GZ-T273S
GZ-T273N
GZ-L56V-T273S
<1
97
14
60 (S)
) Reaction conditions: substrate (50 mM), ArRmut*-TA=ArRmut11-A60V/M117F/G279V (lyophilised cells, 20 mg mL-1)
in phosphate buffer (100 mM, pH 7, 1 mM PLP) containing 20 vol% DMF and 2-propylamine (500 mM) at 30 °C, 120
rpm, 24 h. b) Reaction conditions: substrate (50 mM), GZ-TA (lyophilised cells, 20 mg mL-1) in phosphate buffer (100 mM,
pH 7, 1 mM PLP) at 30 °C, 120 rpm, 24 h; D-alanine (250 mM), AlaDH (12 U), FDH (11 U), NAD+ (1 mM) and
ammonium formate (150 mM). c) Determined from area by GC-FID measurement on an achiral stationary phase. The value
for 7 represents the percentage of 7 in the mixture of 5/7/imines. The value for 8 represents the percentage of 8 in the
a
d)
e)
mixture of 6/8/imines. Optical purity was measured by GC-FID on a chiral phase. not determined due to low
conversion. f) not determined due to quantitative imine formation.
enzyme. In case of Pregabalin precursor 6 only the Even though most of the developed GZ-TA variants
ArRmut*-S277T variant gave the (S)-amine with low either produced the desired products with lower or
conversion and mediocre optical purity (58% ee). equal optical purity as the template enzyme or did not
6
This article is protected by copyright. All rights reserved.

10.1002/adsc.201701449
Advanced Synthesis & Catalysis
convert the substrates at all, two variants attracted was prepared with 94% ee (R) and 80% ee (S). Finally,
attention. Pregabalin precursor rac-6 was transformed in order to confirm the results obtained on an
to the desired (S)-product with high optical purity and analytical scale, the biotransformation was
conversions of up to 65% using GZ-L56V (80% ee) successfully carried out on a preparative scale,
and GZ-T273S (72% ee), respectively. Mutation leading to isolated GABA derivatives 3c and 4 which
L56V in GZ relates to mutation A60V in ArRmut*, were further transformed to the targeted
which has already been introduced before and anticonvulsant APIs. The herein developed process
improved
the
stereoselectivity
significantly. represents a short method for the preparation of
Brivaracetam precursor by
According to the homology model of GZ (Figure 1B, Pregabalin and
a
red residue) this position is not located directly in the amination via dynamic kinetic resolution of the
active site, but close to the dimer interface. A further aldehyde moiety.
amino acid that has been identified to have an impact
both on the stereoselectivity and activity of the
enzymes was the one in position 273 of GZ-TA and
Experimental Section
the equivalent position 277 of ArRmut* (see Figure 1,
General Information
orange). When the serine in ArRmut* was replaced
by a threonine, being the respective amino acid in GZ,
stereoselectivity dropped. In contrast, substituting
T273 in GZ to serine led to an increased optical purity
of (S)-4, while an asparagine at the same position
implied complete loss of activity. As a final attempt
to further increase the optical purity of (S)-4, a variant
was prepared bearing both beneficial mutations L56V
and T273S. This constructed enzyme aminated
substrates 5c and 6 with high conversions, however,
formed (S)-4 with mediocre 60% ee.
All chemicals/reagents were purchased from commercial
suppliers and used as received unless stated otherwise.
Reference compounds (S)- and rac-Pregabalin were
obtained from Sandoz. NMR spectra were recorded on a
300 MHz Bruker NMR unit at 293 K; chemical shifts are
given in ppm relative to Me4Si or the corresponding
solvent signal (1H: CDCl3 = 7.26 ppm, CD3OD = 3.31
ppm; 13C: CDCl3 = 77.0 ppm, CD3OD = 49.00 ppm).
Plasmids for overexpression of transaminases were
obtained from GeneArt AG (Regensburg, Germany),
cloned and transformed into E. coli BL21(DE3) as reported
previously.[63] Purified recombinant L-alanine dehydro-
genase was prepared as described before.[61] Biocatalytic
small scale reactions were carried out in a thermo-shaker
Eppendorf Comfort. Preparative scale reactions were
performed in an Infors Unitron shaker. For analytical
methods, NMR-spectra, DNA- and amino acid sequences
Finally, the TA-mediated bioamination of aldehyde
substrates being precursors for Brivaracetam and
Pregabalin was performed on preparative scale (200
mg, 20 mL), employing the enzymes which gave
highest conversions on an analytical scale. Thus, PD- of enzymes see electronic Supporting Information.
TA was used to aminate Brivaracetam precursor 5c
and Pregabalin precursor 6 was converted by VF-TA.
By replacing the aqueous NaOH solution (10 N) in
the basic workup procedure by aqueous saturated
NaHCO3 the formation of lactam was prevented and
the tert-butyl-ester 3c was obtained in 65% isolated
yield with a very good ee of 90% (R). Similarly,
Pregabalin precursor 4 was isolated in 55% yield,
however, with 60% ee (R). Subsequently, the tert-
butyl ester moiety of obtained GABA derivatives was
cleaved under acidic conditions resulting in the
Brivaracetam acid intermediate (55% yield) and
Pregabalin 2 (63% yield) as HCl salt.
Preparation of Oxo Esters 5a-d
Conclusion
A
novel
Brivaracetam precursors and Pregabalin has been
developed, involving transaminase-catalysed
chemo-enzymatic
route
towards
Scheme 4. Synthesis of Brivaracetam precursors rac-5a-d.
a
Valeraldehyde (30 mM) and diisobutylamine (30 mM)
were dissolved in toluene (120 mL) and refluxed for 8
hours in a Dean Stark apparatus. Molecular sieve was
added and the mixture was stirred at 21 °C overnight.
Molecular sieve was removed by filtration over Celite® and
the appropriate bromo acetate ester (45 mM) was added
and the reaction was refluxed for 8 h. Acetate buffer (70
mL; 35 g acetic acid, 35 g sodium acetate, 70 mL water)
was added, the suspension was stirred for 1 hour at 21 °C
and the organic phase was washed with Na2CO3 saturated
solution. The organic phase was dried over Na2SO4 and the
solvent was evaporated.[59] The product was purified by
amination of an α-chiral aldehyde moiety as the
biocatalytic asymmetric key step. This stereoselective
reaction was run via DKR, enabled by efficient
racemisation of the substrate induced by the
transaminase reaction system. The application of (R)-
and (S)-selective enzymes provided access to both
enantiomers of the desired chiral amines in optically
enriched form. By substrate and medium engineering
as well as the design of transaminase variants, the
Brivaracetam precursor 3c was formed with 92% ee column chromatography (silica, eluent: hexane/EtOAc
97:3). For a more detailed procedure, obtained yields and
NMR data see SI.
(R) and 80% ee (S) and the Pregabalin precursor 4
7
This article is protected by copyright. All rights reserved.

10.1002/adsc.201701449
Advanced Synthesis & Catalysis
Preparation of Oxo Ester 6
and aldehyde rac-5c (9 µL, 50 mM) were added to sodium
phosphate buffer (1 mL, 100 mM, pH 7.0, 1mM NAD free
acid) containing 2-propanol (30 µL). The reduction was
performed at 30 °C and 750 rpm for 15-20 min. After
extraction with EtOAc (2x 500 µL) the combined organic
phases were dried over Na2SO4 and analysed via gas
chromatography.
Racemisation: The organic solvent was removed under air
flow and enantioenriched aldehyde 5c was incubated in
phosphate buffer containing lyophilised E. coli BL21
(DE3) cells (20 mg), 2-propylamine (50 mM), ArRmut11-
TA (100 µL, 1.5 U, purified by heat treatment), PMP (1
mM) or PLP (1 mM) or appropriate mixtures of these
compounds. Racemisation was performed at 30 °C and 750
rpm. Samples were taken after 1 min, 10 min, 30 min, 1 h,
2 h and 4 h. After extraction with EtOAc (2x 500 µL) the
combined organic phases were dried over Na2SO4 and
analysed via chiral gas chromatography.
Isolation of γ-Amino Esters 3c and 4 and cleavage
of tert.-Butyl-Group
Lyophilised cells of E. coli containing overexpressed TA
(400 mg) and FDH (100 mg, 220 U) were rehydrated in
phosphate buffer (18 mL, pH 7, 100 mM) containing PLP
(1 mM) and NADH free acid (1 mM) at 30 °C and 120 rpm
for 30 min. AlaDH (300 µL, 240 U), ammonium formate
(180 mg, 150 mM), alanine (446 mg, 250 mM) and co-
solvent 1,2-dimethoxyethane (200 mL) as well as the
substrate (1 mmol) were added. Transamination was
carried out at 30 °C in an orbital shaker (120 rpm) for 24 h.
Cell material was removed by centrifugation and the
supernatant was basified by addition of aqueous saturated
NaHCO3 solution (3.5 mL) and extracted with t-butyl
methyl ether (7 × 10 mL). The combined organic phases
were dried over Na2SO4 and the solvent was evaporated to
yield the product.
Scheme 5. Synthesis of Pregabalin precursors rac-6.
The starting material 4-methyl-pentanal 9 was synthesised
according to three different methods. One the one hand
aldehyde 9 was formed by PCC- or ADH-mediated
oxidation of 4-methyl-1-pentanol. On the other hand it was
synthesised via hydrogenation of 4-methyl-2-pentenal.
Regardless of which procedure was used, intermediate 9
was isolated as the corresponding enamine 10 after addition
of diisobutylamine. The following reaction steps were
continued as described before for Brivaracetam precursors,
yielding in oxo ester rac-6. For detailed procedures of all
steps, related yields and NMR data see SI.
General Procedure for Biotransformations
t
tBu-3-(aminomethyl)hexanoate 3c: Obtainend from Bu-3-
All experiments were performed in duplicates on a one
millilitre scale using 2 mL Eppendorf tubes as reaction
vessels.
formylhexanoate 5c (201 mg, 1 mmol), using lyophilised
cells of E. coli containing overexpressed PD-TA (400 mg).
The product was isolated as a yellow oil (130 mg, 65%
Biotransformations using alanine as amine donor: D-
alanine was used as amine donor together with ArR-TA,
AT-TA, NF-TA, GZ-TA and HN-TA because these
transaminases show (R)-selectivity for the amination of
ketones. When using other TAs, L-alanine was applied as
donor. Lyophilised cells of E. coli containing
overexpressed transaminase (20 mg) were rehydrated in
phosphate buffer (1 mL, pH 7, 100 mM) containing PLP (1
mM) and NADH free acid (1 mM) at 30 °C and 120 rpm
for 30 min. AlaDH (15 µL, 12 U), FDH (5 mg, 11 U),
ammonium formate (9.5 mg, 150 mM) and alanine (22.3
mg, 250mM) as well as the substrate (50 mM) were added.
Transamination was carried out at 30 °C in a thermo shaker
(750 rpm) for 24 h. The reaction was quenched by addition
of aqueous NaOH 10 N solution (200 µL). After extraction
with EtOAc (2x 500 µL) the combined organic phases were
dried over Na2SO4 and analysed via gas chromatography.
For medium engineering respective parameters were
changed.
1
yield) and was used without further purification. NMR: H-
NMR (300 MHz, CDCl3): H [ppm] = 0.92 (t, 3J6,5 = 6.9 Hz,
3 H, Me), 1.20-1.48 (m, 6 H, H-4, H-5, NH23), 1.45 (s, 9 H,
tBu), 1.86 (mc, 1 H, 3-C), 2.14-2.22 (dd, J2a,3= 6.3 Hz,
2J2a,2b= 14.6 Hz, 1 H, 2-Ha), 2.20-2.30 (dd, J2b,3= 7.1 Hz,
3
2J2b,2a= 14.6 Hz, 1 H, 2-Hb), 2.56-2.65 (dd, 3JNCHa,3= 6.3 Hz,
2J NCHa, NCHb= 12.9 Hz, 1 H, NCH2), 2.65-2.75 (dd, J3,NCHb
3
= 5.6 Hz, 2J NCHb, NCHa= 12.9 Hz, 1 H, NCH2). 13C-NMR
(75MHz, CDCl3): C [ppm] = 14.43(-CH3), 19.99 (5-C),
28.23 (-CH(CH3)3), 34.22 (3-C, 4-C), 38.44 (2-C), 45.52
(NCH2), 80.31 (CO), 172.95 (OCO).
tButyl-3-(aminomethyl)-5-methylhexanoate 4: Prepared
from tBu-3-formyl-5-methylhexanoate 6 (220 mg, 1 mmol),
using lyophilised cells of E. coli containing overexpressed
VF-TA (400 mg). The product was isolated as a yellow oil,
purified by column chromatography (silica, CH2Cl2
:
MeOH 100/0 to 94/6) and isolated as an orange oil (108 mg,
Biotransformations using 2-propylamine as amine
donor: Lyophilised cells of E. coli overexpressing
transaminase (20 mg) were rehydrated in phosphate buffer
(800 µL, pH 7, 100 mM) containing PLP (1 mM) at 30 °C
and 750 rpm for 30 min. DMSO (200 µL) and 2-
propylamine (50 µL, 500mM) as well as the substrate (9
µL, 50 mM) were added. Transamination was carried out at
30 °C in a thermo shaker (750 rpm) for 24 h. The reaction
was quenched by addition of aqueous NaOH 10 N solution
(200 µL). After extraction with EtOAc (2x 500 µL) the
combined organic phases were dried over Na2SO4 and
analysed via gas chromatography. For medium engineering
respective parameters were changed.
1
0.4 mM, 50% yield). H-NMR (300 MHz, CD3OD): H
[ppm] = 0.89 (d, 3J6,5 = 6.5 Hz, 3 H, Me), 0.90 (d, 3J6,5 = 6.5
t
Hz, 3 H, Me), 1.14 (mc, 2H, 4-H), 1.44 (s, 9H, Bu), 1.64
(mc, 1H, 5-H), 1.69 (bs, 2H, NH2), 1.93 (mc, 1H, 3-H),
2.13-2.21 (dd, J2a,3 = 6.1 Hz, 2J 2a,b= 14.6 Hz, 1 H, 2-Ha),
3
2.21-2.30 (dd, J2b,3 = 7.3 Hz, 2J 2b,a= 14.6 Hz, 1 H, 2-Hb),
3
2.55-2.65 (dd, JNCHa,3= 6.5 Hz, 2J NCHa, NCHb= 12.9 Hz, 1 H,
3
NCH2), 2.66-2.76 (dd, JNCHb,3= 5.1 Hz, 2J NCHb, NCHa= 12.9
3
Hz, 1 H, NCH2).13C-NMR (75MHz, CD3OD): C [ppm] =
22.82(Me), 23.06 (5-C), 25.30 (tBu), 28.40 (tBu), 36.25 (3-
C), 38.61 (2-C), 41.52 (4-C), 45.71 (NCH2), 80.40 (CO),
172.91 (OCO).
t
General procedure for ester hydrolysis: Bu-ester 3c and
Racemisation of -Chiral Aldehydes
4, respectively, were diluted with aqueous HCl (0.3 M), the
suspension was heated to 65 °C for 5-12 h and after cooling
to room temperature the solvent was evaporated. The
remaining brown oil was dissolved in a small amount of
Preparation of enantioenriched t-butyl-3-formyl-
hexanoate 5c: Purified (heat pulse) ADH-A (20 µL, 0.3 U)
8
This article is protected by copyright. All rights reserved.

10.1002/adsc.201701449
Advanced Synthesis & Catalysis
water and extracted with EtOAc (3x 0.5-1 mL). The
aqueous phase was removed under vacuum to give the
product as a yellow oil.
[3]
[4]
[5]
[6]
http://www.firstwordpharma.com/node/1194000#
axzz3BTtPfUfJ, (Oct. 17, 2017)
http://www.drugs.com/stats/top100/sales, (Oct. 17,
2017)
G. Coppola, G. Iapadre, F. F. Operto, A. Verrotti,
Drug. Des. Dev. Ther. 2017, 11.
3-(Aminomethyl)hexanoic acid hydrochloride: Prepared
t
from Bu-3-(aminomethyl)hexanoate (40 mg, 0.20 mmol),
using aqueous HCl solution (4 mL, 0.3 M). The product
was isolated as brown oil (20 mg, 0.11 mmol, 55% yield).
NMR: 1H-NMR (300 MHz, CD3OD): H [ppm] = 0.95 (mc,
3H, Me), 1.29-1.51 (m, 4H, 4-H, 5-H), 2.17 (mc, 1H, CH),
2.46 (d, 3J2,3= 6.4 Hz, 2 H, 2-H), 2.97 (d, 3JNCH,3= 6.5 Hz, 2
H, NCH2). 13C-NMR (75MHz, CD3OD): C [ppm] = 14.32
(Me), 20.51 (5-C), 34.61(4-C), 34.74 (3-C), 36.71 (2-C),
44.04 (NCH2), 174.33 (OCOH).
E. Differding, B. Kenda, B. Lallemand, A.
Matagne, P. Michel, P. Pasau, P. Talaga,
WO2001062726 A2 (UCB S.A.), Belgium, 2001.
J. Surtees, F. Lurquin, O. Diouf, WO2005028435
A1 (UCB S.A.), Belgium, 2005.
C. Ates, F. Lurquin, Y. Quesnel, A. Schule,
WO2007031263 A1 (UCB Pharma, S.A.),
Belgium, 2007.
M. S. Hoekstra, D. M. Sobieray, M. A. Schwindt,
T. A. Mulhern, T. M. Grote, B. K. Huckabee, V. S.
Hendrickson, L. C. Franklin, E. J. Granger, G. L.
Karrick, Org. Process Res. Dev. 1997, 1, 26-38.
C. A. Martinez, S. Hu, Y. Dumond, J. Tao, P.
Kelleher, L. Tully, Org. Process Res. Dev. 2008,
12, 392-398.
P. J. Dunn, K. Hettenbach, P. Kelleher, C. A.
Martinez, in Green Chemistry in the
Pharmaceutical Industry, Wiley-VCH Verlag
GmbH & Co. KG, 2010, pp. 161-177.
R. N. Patel, ACS Catal. 2011, 1, 1056-1074.
M. D. Truppo, ACS Med. Chem. Lett. 2017, 8,
476-480.
R. N. Patel, Bioorg. Med. Chem. 2017, doi:
10.1016/j.bmc.2017.05.023.
A. S. Wells, J. W. Wong, P. C. Michels, D. A.
Entwistle, K. Fandrick, G. L. Finch, A. Goswami,
H. Lee, S. Mix, T. S. Moody, L. Pang, R. K. Sato,
N. J. Turner, T. J. Watson, Org. Process Res. Dev.
2016, 20, 594-601.
M. López-Iglesias, D. Mendez-Sanchez, V. Gotor-
Fernández, Curr. Org. Chem. 2016, 20, 1204-
1221.
C. K. Winkler, D. Clay, S. Davies, P. O’Neill, P.
McDaid, S. Debarge, J. Steflik, M. Karmilowicz, J.
W. Wong, K. Faber, J. Org. Chem., 2013, 78,
1525-1533.
S. Debarge, P. McDaid, P. O’Neill, J. Frahill, J. W.
Wong, D. Carr, A. Burrell, S. Davies, M.
Karmilowicz, J. Steflik, Org. Process Res. Dev.
2014, 18, 109-121.
A. J. M. Burrell, C. A. Martinez, P. O. Mcdaid, P.
M. O'neill, J. W. Wong, WO 2012/025861 A1
(Pfizer Manufacturing), Ireland, 2012.
Z. Xie, J. Feng, E. Garcia, M. Bernett, D. Yazbeck,
J. Tao, J. Mol. Catal. B: Enzym. 2006, 41, 75-80.
M. P. Burns, J. K. Weaver, J. W. Wong, WO
2005/100580 A1 (Pfizer Products Inc.), USA,
2005.
[7]
[8]
3-(Aminomethyl)-5-methylhexanoic acid 1 hydrochloride:
t
Prepared from Bu-3-(aminomethyl)-5-hexanoate (140 mg,
0.65 mmol), using aqueous HCl solution (5 mL, 0.3 M).
The product was isolated as brown oil (104 mg, 0.41 mmol,
[9]
1
3
63% yield). H-NMR (300 MHz, CD3OD): 0.93 (d, J6,5
=
3
6.7 Hz, 3 H, Me), 0.95 (d, J6,5 = 6.7 Hz, 3 H, Me), 1.27 (t,
3J = 7.2 Hz, 2 H, 4-H), 1.70 (mc, 1H, 5-H), 2.22 (mc, 1H, 3-
H), 2.45 (d, 3J2,3 = 6.2 Hz, 2 H, 2-H), 2.97 (d, 3J2,3 = 6.3 Hz,
2 H, NCH2). 13C-NMR (75MHz, CDCl3): C [ppm] = 22.52
(Me), 23.18 (Me), 26.14 (5-C), 32.60 (3-C), 37.11 (2-C),
42.03 (4-C), 44.50 (NCH2), 175.79 (OCO). NMR spectra
are in agreement with literature.[75-77]
[10]
[11]
Molecularbiological and microbiological methods
Construction of TA variants: Models of the enzyme
variants were generated using YASARA 13.6.16 and the
dimeric structure of ArR-TA as template. Primers were
designed using the Agilent QickChange primer design tool
and mutagenesis was performed using Agilent QickChange
Mutagenesis Kit. For detailed information about used
plasmids, primers and conditions see supporting
information.
[12]
[13]
[14]
[15]
Overexpression of enzymes: To one shot of E. coli BL21
(DE3) competent cells plasmid solution (10-100 ng) was
added and the cells were incubated on ice for 30 min. Then
cells were heat-shocked at 42 °C for 30 sec, preheated SOC
medium (250 µL) was added and incubated at 37 °C and
350 rpm for 1 h. The cells were streaked out on LB agar
and incubated at 37 °C overnight. For the preculture 10 mL
of LB medium containing ampicillin (100 mg/mL) were
inoculated with one colony and incubated at 30 °C and 120
rpm overnight. Main cultures (330 mL LB medium)
containing the antibiotic ampicillin were inoculated with 2-
5 mL of the preculture. After growing the cells at 30 °C
and 120 rpm until an OD600 = 0.6 – 0.8 was reached,
expression was induced and shaking was continued at 120
rpm and 20 °C overnight. Cells were harvested by
centrifugation (20 min, 8000 rpm), washed with phosphate
buffer (100 mM, pH 7, 0.5 mM PLP), resuspended in
phosphate buffer (100 mM, pH 7, 0.5 mM PLP) and
lyophilised. For detailed information about plasmids,
inducer concentration, and enzyme activities see SI.
[16]
[17]
[18]
[19]
Acknowledgements
[20]
[21]
This study was financed by the Austrian FFG, BMWFJ, BMVIT,
SFG, Standortagentur Tirol and ZIT through the Austrian FFG-
COMET- Funding Program.
[22]
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