Enantioselective Synthesis of d- and l-α-Amino Acids by Enzymatic Transamination Using Glutamine as Smart Amine Donor

Article abstract of DOI:10.1002/adsc.201801278

Enzymatic transamination is a useful method for the green and highly enantioselective synthesis of chiral amines and non-canonical amino acids which are of major importance as intermediates in medicinal chemistry. However, transamination reactions are usually reversible and synthetic applications of transaminases often require the implementation of an equilibrium shift strategy. Herein, we report a highly effective approach using glutamine as smart amine donor. This amino acid is converted upon transamination into 2-oxoglutaramate which undergoes a fast cyclisation displacing the transamination equilibrium. We have developed a new activity assay in order to identify transaminases from biodiversity able to convert various α-keto acids into valuable amino acids of l- or d-series in the presence of glutamine as amine donor. Discovered transaminases were then used to prepare in high yield and with high enantioselectivity three amino acids of pharmaceutical importance, homophenylalanine, homoalanine and tert-leucine by simply using a nearly stoichiometric amount of glutamine as amine donor. (Figure presented.).

Full text of DOI:10.1002/adsc.201801278

Title: Enantioselective Synthesis of D- and L-α-Amino Acids by
Enzymatic Transamination Using Glutamine as Smart Amine
Donor
Authors: Egon Heuson, Franck Charmantray, Jean-Louis Petit,
Véronique de Berardinis, and Thierry Gefflaut
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To be cited as: Adv. Synth. Catal. 10.1002/adsc.201801278
Link to VoR: http://dx.doi.org/10.1002/adsc.201801278

10.1002/adsc.201801278
Advanced Synthesis & Catalysis
FULL PAPER
DOI: 10.1002/adsc.201((will be filled in by the editorial staff))
Enantioselective Synthesis of D- and L-α-Amino Acids by
Enzymatic Transamination Using Glutamine as Smart Amine
Donor
Egon Heuson,a Franck Charmantray,a Jean-Louis Petit,b Véronique de Berardinis,b and
Thierry Gefflaut a*
a
Université Clermont Auvergne, CNRS, SIGMA Clermont, ICCF, F-63000 Clermont-Ferrand, France.
Fax: (+33) 473407717; Phone: (+33) 473407866; E-mail: thierry.gefflaut@uca.fr
Génomique métabolique, Genoscope, Institut François Jacob, CEA, CNRS, Univ Evry, Univ Paris-Saclay, 91057 Evry,
b
France
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. Enzymatic transamination is a useful method for We have developed a new activity assay in order to identify
the green and highly enantioselective synthesis of chiral transaminases from biodiversity able to convert various α-
amines and non-canonical amino acids which are of major keto acids into valuable amino acids of L- or D-series in the
importance as intermediates in medicinal chemistry. presence of glutamine as amine donor. Discovered
However, transamination reactions are usually reversible and transaminases were then used to prepare in high yield and
synthetic applications of transaminases often require the with high enantioselectivity three amino acids of
implementation of an equilibrium shift strategy. Herein, we pharmaceutical
importance,
homophenylalanine,
report a highly effective approach using glutamine as smart homoalanine and tert-leucine by simply using a nearly
amine donor. This amino acid is converted upon stoichiometric amount of glutamine as amine donor.
transamination into 2-oxoglutaramate which undergoes a fast
cyclisation displacing the transamination equilibrium.
Keywords: Amination; Amino acids; Biocatalysis;
Transferases
are specific for amino acids of the D-series whereas
amine-TA (ATA also called ω-TA) are active with a
variety of amines lacking the α-carboxylic acid
moiety.[5] All L- or D-α-TA studied to date, proved to
be highly enantioselective catalysts and most of them
displayed relaxed substrate specificity. High
turnovers and no requirement for external cofactor
recycling are additional features making TA valuable
biocatalysts to access a variety of unusual amino
acids.
Introduction
The tremendous structural diversity of biomolecules
notably relies on the combination of 20 standard
amino acids in proteins. Moreover, the chemists
ability of preparing an unlimited variety of non-
proteinogenic (also named “non-canonical” or
“unusual”) amino acids still expand the chemical
space of peptides and peptidomimetics which are of
increasing importance in modern drug discovery.[1]
Among the numerous synthetic routes towards
amino acids,[2] bioprocesses and biocatalytic methods
benefit from the high enantioselectivity of enzymes
while meeting the criteria of green and sustainable
chemistry.[3] Among various enzyme families used
for amino acids synthesis, transaminases (TA) have
been extensively studied in the large-scale
preparation of unusual L- or D-α-amino acids as well
as a variety of chiral amines.[4]
However, the major drawback of transamination
reactions lies in their equilibrium constants, often
close to unity and resulting in limited yields and
purification
issues.[6]
To
overcome
these
thermodynamic limitations, an equilibrium shift
strategy is needed. It usually consists in the removal
or transformation of the carbonyl coproduct formed
from the amine donor substrate. One striking example
is the removal under reduced pressure of acetone
formed from isopropylamine used as amine donor for
particular ATA.[7] This approach was notably adopted
in a synthetic process for the antidiabetic drug
sitagliptine.[8] However only few ATA are active with
isopropylamine and this strategy is not applicable to
α-TA which are highly specific for α-amino acids.
TA catalyze the pyridoxal 5’-phosphate-dependent
reversible transfer of the amino group from an amine
donor substrate onto an acceptor carbonyle substrate.
The TA family is highly diverse, comprising many
ubiquitous L-α-TA involved in proteinogenic L-amino
acids metabolism. Moreover, some microbial D-α-TA
1
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10.1002/adsc.201801278
Advanced Synthesis & Catalysis
Alternatively, equilibrium shift was often achieved
New TA activity assay with Gln as amine donor.
Assuming that Gln could be a substrate for various
TA from biodiversity,[20] we firstly developed a
continuous spectrophotometric assay in a 96-well
microplate format in order to precisely quantify
transamination activities with Gln as amine donor and
various keto acid precursors of amino acid targets. As
shown in Scheme 1, our assay was based on 2
auxiliary enzymes: an ω-amidase from Arthrobacter
nicotinovorans (ωA, Uniprot ID: Q93NG1)
catalyzing the hydrolysis of OGA amide into 2-
oxoglutaric acid (OG)[21] and a 2-oxoglutarate
reductase from Acidaminococcus fermentans (OGR,
Uniprot ID: D2RJU7) which catalyzes the reduction
through auxiliary enzymatic reactions as exemplified
by the lactic-dehydrogenase catalyzed reduction,[9]
acetolactate-synthase catalyzed autocondensation[10]
or
pyruvate-decarboxylase
catalyzed
decarboxylation[11] of pyruvic acid (Pyr) formed from
alanine (Ala) used as amine donor.
In a simpler way, a transamination reaction can be
displaced through the use of smart amine donors.
These so-called substrates are indeed converted upon
transamination into unstable carbonyl coproducts that
undergo a spontaneous transformation affording the
needed equilibrium shift. For example, aspartic acid
(Asp) or its analogue cysteine sulfinic acid (CSA)
used as amine donors are converted into β-keto
carboxylic or sulfinic acids which lose carbon or
sulfur dioxide to give pyruvic acid (Pyr). This
strategy was however limited to particular TA like
Aspartate–TA or Tyrosine-TA, that are active with
Asp or CSA as amine donor and unable to convert
Pyr, formed from these substrates, into Ala.[12]
Another class of smart amine donors is defined by
compounds bearing an extra amino group able to
react with the carbonyl generated upon
transamination to give a cyclic imine. Therefore, the
α,ε-diamino acid lysine was used as irreversible
amine donor with a genetically modified AspTA to
produce L-homophenylalanine (Hfe)[13] whereas
ornithine was used in an equilibrium shifted process
using an ornithine-ω-TA as auxiliary enzyme.[14]
More recently, several diamines proved efficient
irreversible amine donors with various ATA. Indeed,
the amino-carbonyl by-product formed from o-
xylylenediamine[15] and but-2-ene-1,4-diamine[16] are
readily cyclized and subjected to further
aromatization and polymerization. Interestingly,
several bio-based diamine including putrescine,
cadaverine and spermidine were efficiently used as
smart amine donors with ATA.[17]
of
OG
by
NADH,
thus
allowing its
spectrophotometric monitoring at 340 nm (ε = 6220
M-1.cm-1).[22] One NADH-based assay of OGA was
already described using ωA and glutamic-
dehydrogenase (GluDH).[23] However reductive
amination catalyzed by GluDH requires the addition
of ammonium ions which could impair TA activity.
That is why OGR was preferred to GluDH for OG
titration.
Besides cyclic imine formation from diamino
substrates, other transamination by-product formed
from polyfunctional amine donors are prone to
cyclisation. Noteworthy, reactions catalyzed by
glutamine-TA (GlnTA, EC 2.6.1.15) yield 2-
oxoglutaramate (OGA) which was shown to exist
almost
exclusively (99,7%)
in
a
cyclic
hydroxylactame form[18] and some natural
transaminations involving Gln as donor were reported
to be displaced towards OGA formation.[18], [19]
Herein, we report the design of a new activity
assay for TA using L- or D-Gln as amine donor, the
implementation of this assay to identify L- and D-α-
TA from biodiversity active with Gln, and application
examples of these newly discovered TA for the
efficient synthesis of valuable unusual amino acids.
Hence, we demonstrate that Gln can truly be
considered as a smart amine donor for equilibrium-
shifted enzymatic transaminations.
Scheme 1. Enzymatic assay for transaminase with Gln as
amine donor.
Both auxiliary enzymes, ωA and OGR, were
cloned and overexpressed in E. coli with addition of
an His-Tag for easy purification (see table S1).
Approximately 150,000 U of purified OGR were
obtained from 1 L culture of the over-expressing
strain. OGR proved to be highly active with a specific
Results and Discussion
activity close to 1600 U.mg-1 . In the case of ωA, a
.
2
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10.1002/adsc.201801278
Advanced Synthesis & Catalysis
total activity of approximately 500 U was obtained
from 1 L microbial culture, corresponding to a
modest specific activity of 2.4 U.mg-1. However, it
should be pointed out that ωA activity was measured
with OGR as auxiliary enzyme in non-optimal
conditions, using a sample of OGA that was shown
that were identified in previous experiments for their
synthetic potential with various keto acid acceptors
and Glu as standard amine donor.[20] A series of 9
keto acids were used as acceptor substrates, including
precursors of canonical amino acids (glyoxylic acid
(GL), Pyr, MOP, hydroxypyruvic acid (HPA) and
phenylpyruvic acid (PhP)) as well as unusual amino
acids (2-oxobutyric acid (OB), trimethylpyruvic acid
(TMP), phenylglyoxylic acid (PhG) and 2-oxo-4-
phenylbutyric acid (OPB)). Recombinant E. coli
strains overexpressing each of the 24 TA were
cultured in 96-well microplates and cell-lysates were
directly used in the Gln-TA assay. For each substrate,
a cell lysate from host E coli without overexpressed
TA was used as control in order to discard false
positives coming from side reactions catalysed by the
host-cell enzymes. The average protein content of
cell lysates was 1 mg.mL-1 and 0.5-5 μg TA were
used in each assay. In these conditions, the 2 D-TA
and 5 L-TA among 22 showed activities > 0.1 U/mL
of lysate with at least one keto acid substrate (table
1). The best activities in D- and L-series were
measured with D-TAEi and L-TA from Megasphaera
elsdenii DSM 20460 (L-TAMe, Uniprot ID: G0VQA2)
respectively. Although L-TAMe displayed lower
activity levels than those of D-TAEi, a broad substrate
spectrum was evidenced for both enzymes.
1
by H NMR to be almost exclusively cyclized in
aqueous solution. Considering that the acyclic form
of OGA is the true substrate of ωA,[18] it is likely that
the activity was underestimated in this assay and it
was expected that ωA should display a much higher
specific activity in the multienzymatic assay
described in scheme 1 where OGA hydrolysis can
compete efficiently with cyclisation. Moreover Both
enzymes showed good stability and could be stored
for few months without noticeable loss of activity.
In order to adjust the parameters and evaluate the
performances of the new Gln-TA assay, we used as
model enzyme,
a
D-amino acid-TA from
Enterococcus italicus DSM 15952 (D-TAEi, Uniprot
ID : E6LHY8) which was previously identified as a
highly active broad spectrum TA.[20], [24] The activity
of D-TAEi towards D-Gln (40 mM) was first evaluated
in the presence of Pyr (10 mM) as acceptor substrate
by monitoring Pyr consumption using lactic
dehydrogenase (LDH). In these conditions, a specific
activity of 45 U.mg-1 was measured. D-TAEi (20 mU)
was then combined with various amounts of ωA and
OGR in the Gln-TA assay. We could observe that 0.4
U OGR and 40 mU ωA are sufficient to observe non-
limiting auxiliary reactions and these conditions were
fixed for the following experiments. The apparently
low needed amount of ωA confirmed the assumption
that ωA specific activity measured with cyclic OGA
was largely underestimated.
Table 1. Transamination activities (U.mL-1) of 22 L-TA
and 2 D-TAa) measured in cell lysates with L- or D-Gln as
amine donor and various acceptor substrates b).
Uniprot ID. GL
Pyr OB MOP TMP HPA PhG PhP OPB
G0VQA2
L-TA1
0,10 0,12 0,15 0,19 0,03 0,01 0,18 0,09 0,06
B2JE86
L-TA2
L-TA3
L-TA4
L-TA5
L-TA6
L-TA7
L-TA8
L-TA9
L-TA10
L-TA11
L-TA12
L-TA13
L-TA14
L-TA15
L-TA16
L-TA17
L-TA18
L-TA19
L-TA20
L-TA21
L-TA22
0,04 0,15 0,13 0,15
0,03 0,02
0,01 0,15 0,06 0,03
0,03
B7WY27
DOMDHO
G8R8R4
E1VWL8
C1CZ11
Q1J263
Q9RRM7
C0GI63
0,02 0,07 0,04 0,03
0,11
0,02
0,03
As shown in figure S1, the calibration curve using
various amounts of D-TAEi showed a broad dynamic
range with a good linearity up to 20 mU/well and a
calculated limit of detection of 0.3 mU/well. Finally,
we considered that possible hydrolytic activity of ωA
towards Gln could generate a false positive signal.
Indeed, hydrolysis of Gln would give Glu which is a
standard amine donor for most L-α-TA. In order to
0,03 0,01
0,02 0,05
0,02
0,03 0,01 0,01
0,02 0,04 0,06 0,02
0,01
0,03
0,02
0,02
0,03
0,03
0,02 0,07 0,04 0,03
0,05 0,06 0,01
0,02 0,03
0,05
0,04
0,01
0,01
C4LDS0
Q0AV15
Q0AT08
Q3SH06
B7X3V3
Q72ES5
E1Q1V7
A0A0H3K2P4
C5CIR2
0,01
0,08 0,08 0,01
0,03 0,04
0,01 0,06
0,05 0,01
rule
out
this
possibility,
Branched-Chain-
aminotransferase from Escherichia coli (BCATEc)
was used as a negative control enzyme. BCATEc
activity was measured in our assay with L-Gln as
donor and its natural substrate 4-methyl-2-
oxopentanoic acid (MOP) as acceptor. At the same
time, BCATEc activity was also measured in the same
conditions with L-Glu as amine donor. Specific
activities of 0.02 and 11.5 mg.mL-1 were measured
with Gln and Glu respectively, confirming the very
low activity of BCAT towards Gln and indicating that
no false positive signal comes from Gln hydrolysis.
Hence, this new Gln-TA assay appeared well
adapted to perform the screening of TA collections in
order to highlight transamination activities with L- or
D-Gln as amine donor.
0,03 0,06
0,05 0,01 0,02
0,01 0,01 0,01
0,02
0,06 0,04 0,07
0,02
0,09 0,01 0,04 0,07
A1HUC4
F6B3V8
F6CII1
0,09 0,02
0,11 0,01
0,03 0,17 0,01
0,01 0,08
0,09 0,01 0,01
0,03
0,02
P54694
E6LHY8
D-TA1
D-TA2
0,50 0,78 0,60 0,04 0,01 0,25
0,07 0,18
2,43 13,78 14,45 4,36 0,01 0,98 0,08 14,10 16,27
a)
Activities of L-TA and D-TA are highlighted in blue and
red respectively and are given after subtraction of the
control activities measured for each substrate without
overexpressed TA.b) Glyoxylic acid (GL), pyruvic acid
(Pyr), 2-oxobutyric acid (OB), 4-methyl-2-oxopentanoic
acid (MOP), trimethylpyruvic acid (TMP), hydroxypyruvic
acid (HPA), phenylglyoxylic acid (PhG), phenylpyruvic
acid (PhP), 4-phenyl-2-oxobutanoic acid (OPB).
Screening of a TA collection. Screening experiments
were performed on a set of 22 L-TA from various
classes and 2 D-TA including D-TAEi (see table S1)
3
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10.1002/adsc.201801278
Advanced Synthesis & Catalysis
Table 2. Apparent kinetic parameters of transaminations catalysed by D-TAEi and L-TAMe
TA
Substrate
KM (mM)
kcat (min-1)
kcat/ KM (min-1.mM-1)
Specific activity (U.mg-1)
D-TAEi
D-Gln a)
Pyr b)
OB b)
9.0 ± 0.1
0.9 ± 0.1
0.6 ± 0.1
0.5 ± 0.1
1904 ± 28
1631 ± 16
1266 ± 14
836 ± 31
211 ± 5
57 ± 1
49 ± 1
38 ± 1
25 ± 1
1812 ± 219
2110 ± 375
1672 ± 396
OPB b)
L-TAMe
L-Gln a)
Pyr b)
OB b)
TMP b)
OPB b)
29.9 ± 1.8
0.3 ± 0.1
0.9 ± 0.2
1.4 ± 0.1
1.1 ± 0.1
39 ± 2
22 ± 1
21 ± 1
5.6 ± 0.1
83 ± 8
1.3 ± 0.1
73 ± 28
23 ± 6
0.9 ± 0.1
0.5 ± 0.1
0.5 ± 0.1
0.12±0.01
1.8 ± 0.2
4.0 ± 0.4
75 ± 14
a) variable concentrations of D- or L-Gln were used in combination with 5 mM Pyr. b) variable concentrations of keto
acid were used in combination with 40 mM D- or L-Gln.
In the case of D-TAEi, activities ≥ 1 U.mL-1 were
measured with all substrates except TMP and PhG. In
the case of L-TAMe activities were always ≥ 0.1 U.mL-
1
except with TMP and HPA. However L-TAMe
activity with TMP was significantly higher than the
control indicating that L-TAMe could be used for L-
tert-leucine (Tle) synthesis from TMP and Gln.
Kinetic parameters. In order to better characterize
both enzymes and to confirm screening results, the
Gln-TA assay was used with purified D-TAEi and L-
TAMe, to determine kinetic parameters of
Figure 1. Activities of D-TAEi and L-TAMe with OPB (1
mM) and various amine donors (40 mM): OGR or LDH
were used as auxiliary enzymes for the reduction by
NADH of OG or Pyr formed from D/L-Glu and D/L-Ala
transamination reactions with Pyr, KB, TMP or OPB
as acceptor and L- or D-Gln as amine donor (table 2).
Enzyme kinetics clearly confirmed the greater
activity of D-TAEi over L-TAMe due to a better affinity
respectively.
towards the amine donor D-Gln as well as a 10 to 75
fold increased kcat with the various substrates. The
inactivity of D-TAEi with TMP was confirmed
Residual Pyr was titrated over time with LDH and
NADH until equilibration (stabilization of Pyr
concentration). In order to highlight the interest of
Gln as amine donor, the same experiment was run
using L-Glu as amine donor. Final Pyr concentrations
measured at various initial donor/Pyr ratios are
reported in Figure 2. As expected, we could observe a
linear correlation between residual [Pyr] at
equilibrium and the initial donor/Pyr ratio indicating
an apparently complete reaction and therefore a very
efficient equilibrium shift afforded by OGA
cyclisation. Considering that the conversion obtained
with one molar equivalent of Gln was above 98%,
indicated an equilibrium constant over 2000 for the
transamination reaction between Pyr and Gln.
Conversely, when using a stoichiometric amount of
Glu as amine donor, the conversion was less than
60%, thus indicating an equilibrium constant lower
than 2.25. A larger-scale reaction between Pyr (50
mM, 0.7 mmol) and Gln (60 mM) was then run using
L-TAMe (9.3 μM) as catalyst. In those conditions,
whereas a modest activity was evidenced for L-TAMe
with this substrate. Moreover, L-TAMe showed lower
KM and higher kcat with all other acceptor substrates,
and the best specific activity of this enzyme was
measured with OPB.
In order to better characterize the substrate
spectrum of both TA, activities with OPB and D/L-
Gln were compared to those measured with D/L-Glu
and D/L-Ala as standard amine donors. Results
reported in figure 1 indicated that although D-TAEi
showed comparable activities with the 3 tested
donors, L-TAMe displayed a marked preference for
Glu with a ten-time activity reduction when Gln was
used as amine donor. Nevertheless, L-TAMe was
considered as a suitable enzyme to demonstrate the
relevance of the equilibrium shift strategy using Gln
as amine donor.
Synthetic experiments. As a first model reaction,
the transamination reaction between Pyr and L-Gln
was studied using L-TAMe as catalyst. In a first
analytical experiment, Pyr (50 mM) was reacted with
L-Gln at various concentrations (0-60 mM).
4
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10.1002/adsc.201801278
Advanced Synthesis & Catalysis
was assayed for the synthesis of L-homo-alanine
(Hal), L-tert-leucine (Tle) and L-homophenylalanine
(Hfe). These three amino acids are included in the
peptidomimetic structures of widely used drugs[1] and
their stereoselective synthesis has been the subject of
many studies.[2], [26] Furthermore, the potential of D-
TAEi was assessed through the synthesis of D-Hal and
D-Hfe which also constitute important building
blocks for biologically active compounds.[27]
Synthesis experiments were run on 0.1 mmol scale
and 100 mM Gln and 120 mM OB or TMP were first
chosen as standard substrate concentrations. A slight
excess of acceptor was used in order to achieve a
complete conversion of Gln allowing the isolation of
pure amino acid products by selective adsorption on a
cation exchange resin. Enzymes specific activities
were measured in these conditions in order to address
possible inhibition at high substrate concentrations. In
the case of D-TAEi, the results were very close from
those reported in table 1 (36 and 28 U.mg-1 for OB
and OPB respectively). Specific activities of L-TAMe
with 120 mM OB or TMP and 100 mM L-Gln (1,8
and 0.7 U.mg-1 respectively) were found higher than
those measured previously at 40 mM L-Gln (table 1).
These results were in accordance with the high KM
value determined for L-Gln and L-TAMe (table 1) and
indicated that no significant substrate inhibition was
observed for L-TAMe with these 2 substrates. In the
case of the lithium salt of OPB, a maximum solubility
of 14 mM was determined in water at pH 7.6.
Consequently, reactions were run in heterogeneous
conditions with a suspension of OPB (18.4 g.L-1,
theoretically 100 mM). As Hfe is even less soluble in
water (maximum solubility < 3 mM), it was simply
isolated by filtration at the end of the reaction. For
that reason, a slight excess of Gln (120 mM) was
used in that case to ensure a complete conversion of
OPB. Syntheses results are summarized in table 3.
Amino acids of L- or D-series were isolated with
excellent enantiopurity, confirming again that TA are
highly enantioselective catalysts. All reactions
showed conversions over 90%, clearly demonstrating
that using Gln as amine donor is an effective and
Figure 2. Final concentrations of Pyr measured in
transaminations with various initial [donor]/[Pyr] ratios.
the conversion rate reached 95% in less than 1.5 h
and Pyr titration after 3 h indicated a completely
displaced reaction (> 99% conversion). Amino acids
were removed from the reaction mixture by selective
adsorption on a sulfonic acid containing ion exchange
resin and the lithium salt of OGA was quantitatively
isolated after neutralization with lithium hydroxide
and lyophilisation. NMR analyses showed that OGA
was the only keto acid present in the reaction mixture
and confirmed that it exists almost exclusively in its
cyclic form in aqueous solution (see SI). It should be
pointed out that this transamination reaction between
Pyr and Gln thus constitutes a highly efficient method
to prepare OGA, which is an important biomarker of
hyperammonemia.[25]
In light of these first encouraging results, L-TAMe
and D-TAEi were evaluated as catalysts for the
synthesis of valuable unusual L- or D-amino acids
using L- or D-Gln as amine donor (scheme 2). In
accordance with the substrate spectra evaluated
through screening and kinetic experiments, L-TAMe
Scheme 2. Synthesis of unsual L- and D- amino acids using L- or D-Gln as amine donor.
5
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10.1002/adsc.201801278
Advanced Synthesis & Catalysis
Table 3. Synthesis of unsual amino acids catalysed by L-TAMe and D-TAEi with L- or D-Gln as amine donor
TA (μM)
Acceptor (mM)
Donor (mM)
Time (h)
Conversiona) (%)
eeb) (%)
Product
L-TAMe (65)
L-TAMe (130)
L-TAMe (90)
D-TAEi (8.4)
D-TAEi (8.4)
OB (120)
TMP (120)
OPB (100)
OB (120)
OPB (100)
L-Gln (100)
L-Gln (100)
L-Gln (120)
D-Gln (100)
D-Gln (120)
24
48
24
7
L-Hal
L-Tle
L-Hfe
D-Hal
D-Hfe
98
92
93
99
96
> 99
> 99
> 99
> 99
> 99
24
a) Conversions of the limiting substrate were calculated by NMR analysis of reaction media and were always inferior or
equal to yields calculated from isolated products. b) Enantiomeric excess determined by HPLC.
with Gln or Glu using Pyr as acceptor (0.6 or 1.1
U.mg-1 respectively). The process of scheme 3 was
implemented on 0.1 mmol scale using TMP (110
mM), Gln (100 mM), Glu (10 mM), and 100 μM of
each TA. Under these conditions, a conversion of
94% was reached in 48 h, thus demonstrating the
applicability of this Gln-TA coupled approach.
Conclusion
In summary, we have developed an efficient
spectrophotometric assay, which is suitable not only
for screening enzyme collections but also for kinetic
studies of TA which are active with L- or D-Gln as
amine donor. The screening of a small collection of
Scheme 3. Synthesis of L-Tle by coupling BCATEc with L-
TATc using Gln as smart amine donor
24 TA highlighted several L- or D-TA showing
significant activity with L- or D-Gln. The 2 most
active enzymes in L- and D-series were used for high
yielding and enantioselective synthesis of three
general method to shift the equilibrium of
valuable unusual amino acids, thus demonstrating
transamination reactions.
that Gln, a readily available and bio-based substrate,
It might be argued however that Gln is maybe not
can be used as a smart amine donor in order to shift
commonly accepted as a good substrate by L-α-TA
and that finding one enzyme showing a good activity
the equilibrium of transamination reactions. Further
large scale screening experiments will undoubtedly
for both Gln and the keto acid precursor of a valuable
allow the discovery of highly active and robust new
target might be a cumbersome task. Nevertheless, a
Gln-TA for use as catalyst or auxiliary enzyme in
Gln-TA could also be used as an auxiliary enzyme to
large scale and highly enantioselective syntheses of a
shift the equilibrium of another transamination
variety of chiral amines.
reaction catalyzed by an α-TA or an Amine-TA
devoid of activity towards Gln. In this bienzymatic
system, Ala or Glu could be used as standard primary
Experimental Section
amine donor ensuring the coupling between both
transamination reactions. In order to illustrate this
approach, we designed the new synthetic process of
L-Tle described in scheme 3, by coupling BCATEc
and a previously assayed serine-glyoxylate-TA from
Thermosinus carboxydivorans Nor1 (L-TATc, Uniprot
General procedure for amino acid synthesis using
L-TAMe or D-TAEi. Reactions were carried out in
Eppendorf tubes (1,5 mL). To a 1 mL aqueous
solution containing L- or D-Gln (0.1 or 0.12 mmol),
OPB (0.1 mmol), OB (0.12 mmol) or TMP (0.12
mmol) and PLP (0.1 μmol) at pH 8 was added L-
TAMe (6.5-13 nmol) or D-TAEi (0.84 nmol): a
suspension of the TA in 3 M (NH4)2SO4 was
centrifuged (14000 rpm, 5 min.), the supernatant was
discarded and the pellet diluted with the solution of
substrates and cofactor. The reaction mixture was
orbitally stirred at room temperature and controlled
by thin layer chromatography. After 7 to 48 h, L- or
ID: A1HUC4). BCATEc was previously used for Tle
[28]
synthesis[14],
whereas thermostable L-TATc was
recently combined with a transketolase for in situ
production of HPA from L-Serine.[29] This TA couple
was chosen because although neither of the two TA
showed any activity with TMP and Gln (< 0.02 U.mg-
1), BCATEc proved active with TMP and Glu (1.1
U.mg-1) whereas L-TATc showed significant activity
6
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10.1002/adsc.201801278
Advanced Synthesis & Catalysis
D-Hfe was isolated by filtration on a 0.22 μm
membrane. The white solid was washed with H2O
(4x0.5 mL) and dried under vacuum (0.5 mm Hg) for
24 h at 40 °C. In the case of Hal and Tle, the reaction
mixture was poured on a small column of dowex®
50WX8 (H+ form, 1 mL). The column was washed
with H2O (10 mL) before elution with 1 M NH3.
Ninhydrin positive fractions were pooled and
concentrated under reduced pressure. Amino acids
were isolated as white solids. Conversions, chemical
and enantiomeric purity were assessed by NMR and
HPLC analyses (See SI).
Meier, M. T. Gundersen, J. M. Woodley, M.
Schürmann, ChemCatChem 2015, 7 (17), 2594–2597.
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Acknowledgements
We thank the French National Center for Scientific
Research (CNRS) and University Clermont-
Auvergne for financial support. We also thank
Martine Sancelme for the enzyme production and
Aurélie Job for HPLC analyses.
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8
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FULL PAPER
Enantioselective Synthesis of D- and L-α-Amino
Acids by Enzymatic Transamination Using
Glutamine as Smart Amine Donor
Adv. Synth. Catal. Year, Volume, Page – Page
Egon Heuson, Franck Charmantray, Jean-Louis Petit,
Véronique de Berardinis, and Thierry Gefflaut a*
9
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