Deracemisation of mandelic acid to optically pure non-natural L-phenylglycine via a redox-neutral biocatalytic cascade

Article abstract of DOI:10.1002/adsc.200900891

A biocatalytic redox-neutral reaction cascade was designed for the deracemisation of racemic mandelic acid to yield optically pure L-phenylglycine employing three enzymes. The cascade consisted of three steps: a racemisation, an enantioselective oxidation and a stereoselective reductive amination. The enantioselective oxidation of Dmandelic acid to the corresponding oxo acid was coupled with the stereoselective reductive amination of the latter; thus the oxidation as well as the reduction reactions were performed simultaneously. The formal hydrogen abstracted in the first step the oxidation - was consumed in the reductive amination allowing a redox-neutral cascade due to a cascade-internal cofactor recycling. The enantiomers of the starting material were interconverted by a racemase (mandelate racemase) ensuring that in theory 100% of the starting material can be transformed. Using this set-up racemic mandelic acid was transformed to optically pure L-phenylglycine (ee >97%) at 94% conversion without the requirement of any additional redox reagents in stoichiometric amounts.

Full text of DOI:10.1002/adsc.200900891

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DOI: 10.1002/adsc.200900891
Deracemisation of Mandelic Acid to Optically Pure Non-Natural
l-Phenylglycine via a Redox-Neutral Biocatalytic Cascade
Verena Resch,a Walter M. F. Fabian,a and Wolfgang Kroutila,*
a
Department of Chemistry, Organic and Bioorganic Chemistry, University of Graz, Heinrichstrasse 28, A-8010 Graz,
Austria
Fax : (+43)-316-380-9840; e-mail: wolfgang.kroutil@uni-graz.at
Received: December 22, 2009; Published online: April 7, 2010
Abstract: A biocatalytic redox-neutral reaction cas-
cade was designed for the deracemisation of race-
mic mandelic acid to yield optically pure l-phenyl-
glycine employing three enzymes. The cascade con-
sisted of three steps: a racemisation, an enantiose-
lective oxidation and a stereoselective reductive
amination. The enantioselective oxidation of d-
mandelic acid to the corresponding oxo acid was
coupled with the stereoselective reductive amina-
tion of the latter; thus the oxidation as well as the
reduction reactions were performed simultaneously.
The formal hydrogen abstracted in the first step –
the oxidation – was consumed in the reductive ami-
nation allowing a redox-neutral cascade due to a
cascade-internal cofactor recycling. The enantio-
mers of the starting material were interconverted
by a racemase (mandelate racemase) ensuring that
in theory 100% of the starting material can be
transformed. Using this set-up racemic mandelic
acid was transformed to optically pure l-phenylgly-
cine (ee >97%) at 94% conversion without the re-
quirement of any additional redox reagents in stoi-
chiometric amounts.
enzymes might be the perfect catalysts to set up a
similar system for synthetic applications. Combina-
tions of simultaneous biocatalytic reduction and oxi-
dation have already been reported for the deracemi-
sation of sec-alcohols.[8,9]
Enantiomeric pure a-amino acids are becoming
more and more important as building blocks and
starting materials, especially in the pharmaceutical
and fine chemicals industry.[10] Particularly non-natu-
ral a-amino acids are in the focus of interest. For ex-
ample, d-phenylglycine or d-4-hydroxyphenylglycine
are used as building blocks for semi-synthetic broad-
spectrum antibiotics like Ampicillin and Amoxicil-
lin.[11] Other non-natural amino acids are needed as
templates in asymmetric synthesis.[12] Various methods
for the preparation of enantiopure non-natural a-
amino acids have been reported.[11]
The aim of the concept presented in this paper is to
transform the a-hydroxy acid mandelic acid 1 into the
corresponding optically pure non-natural a-amino
acid. In a first step the a-hydroxy acid should be oxi-
dised to the corresponding a-oxo acid (Scheme 1);
the latter should concurrently be transformed to the
corresponding a-amino acid via an asymmetric reduc-
tive amination, whereby the formal hydrogen ꢀab-
stractedꢁ in the oxidation is used in the reduction;
therefore both reactions – the enantioselective oxida-
tion as well as the stereoselective reduction – have to
run simultaneously.
Keywords: amino acids; deracemisation; domino re-
action; enzyme catalysis; oxidation; reduction
Such a concept of ꢀre-usingꢁ the hydrogen has been
Catalytic tandem reactions[1–5] are processes where referred to as ꢀborrowing hydrogenꢁ in metal cataly-
different catalysts operate concurrently in one pot to
overcome the limitations and disadvantages of a mul-
tistep synthesis such as time-consuming or yield-re-
ducing isolation and purification of the intermedi-
ates.[6] Although the concept of cascade reactions is
very elegant, there are still a lot of challenges that
need to be addressed. Especially a cascade involving
a concurrent reduction and an oxidation step renders
the system even more complex due to the diverging
Scheme 1. Concurrent redox-neutral cascade for the trans-
formation of an a-hydroxy acid to the corresponding a-
reaction conditions.[7] Since in living cells oxidation
and reduction processes are working simultaneously, amino acid. l-AADH: l-amino acid dehydrogenase.
Adv. Synth. Catal. 2010, 352, 993 – 997
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993

Verena Resch et al.
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sis.[13] In biocatalysis only few related examples of
redox-neutral cascades with internal cofactor recy-
cling have been mentioned,[14–16] where the nicotin-
Table 1. Reductive amination of phenyl glyoxylate 2 yielding
3.
Entry
AADH[a]
Conv.[b,c] [%]
ee[d] [%]
ACHTUNGTRENNUNG
amide cofactors NAD(P)+ are reduced in the first
1
2
3
4
5
6
7
8
l-AADH-101
l-AADH-102
l-AADH-103
l-AADH-104
l-AADH-105
l-AADH-106
l-AADH-107
l-AADH-108
l-AADH-109
l-AADH-110
l-AADH-111
l-AADH-112
l-AADH-113
l-AADH-114
l-AADH-115
l-AADH-116
l-AADH-117
l-AADH-118
d-AADH-101
d-AADH-102
d-AADH-103
d-AADH-104
d-AADH-105
d-AADH-106
>97
<1
5
>97
>97
>97
>97
>97
4
>97 (l)
n.d.[e]
52 (l)
>97 (l)
>97 (l)
>97 (l)
>97 (l)
>97 (l)
51 (l)
step of a cascade and the NAD(P)H formed is then
consumed in a second step, whereby oxidation and re-
duction mediated by the NAD(P)-cofactor occur at
the same molecule. This approach of internal cofactor
recycling avoids the additional use of other enzymes
as well as reagents for the recycling of the cofactors,
therefore leading to a redox-neutral cascade.
Since in such a sequence all transformations are re-
versible the favoured product formed depends on
thermodynamics. Calculation[17] of the Gibbs free
energy DG for the interconversion of lactic acid and
alanine showed that the thermodynamic equilibrium
is on the side of the amino acid (À5.7 kcalmolÀ1);
thus an a-hydroxy acid should be preferentially con-
verted to the amino acid, which encouraged us to per-
form further investigations on this subject.
Since the enantioselective oxidation of d-mandelic
acid by mandelate dehydrogenase is well described[18]
we tested first various commercial amino acid dehy-
drogenases (AADHs) exclusively for the reductive
amination of phenyl glyoxylate 2 employing glucose
and glucose dehydrogenase for the recycling of the
cofactor NADH (Table 1).
Interestingly l- as well as d-AADHs led to the for-
mation of the l-amino acid, although in case of d-
AADHs at rather low conversion. Probably these d-
AADHs are only stereospecific for aliphatic amino
acids as stated by the provider.[19]
Before the d-mandelate dehydrogenase (d-MDH)
was coupled with an AADH the activity of the d-
MDH as well l-AADH (l-AADH-101) was deter-
mined by a UV assay, following the formation/con-
sumption of NADH. The units of the enzymes em-
ployed for the cascade were chosen in such a way that
the reductive amination step is six times faster than
the oxidation step.
Employing this set-up it is ensured that NADH is
quickly consumed in the second step providing NAD+
for the thermodynamically unfavoured first step, the
asymmetric oxidation.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
5
48 (l)
>97
4
<1
>97
>97
4
>97 (l)
>97 (l)
n.d.
>97 (l)
>97 (l)
>97 (l)
>97 (l)
>97 (l)
54 (l)
5
>97
4
5
3
4
51 (l)
>97 (l)
>97 (l)
>97 (l)
>97 (l)
4
3
[a]
Amino acid dehydrogenase, the numbers refer to the
enzyme kit of Codexis.
Reaction conditions: phenylglyoxylic acid
0.07 mmol), amino acid dehydrogenase (2 mg, l-AADH-
101–118, d-AADH-101–105), glucose (30 mg,
0.17 mmol), glucose dehydrogenase (10 mL, 5 U), NADH
(1 mg, 0.002 mmol), buffer (1 mL, pH 9.5, sodium bicar-
bonate 100 mM, NH4Cl 200 mM), shaking at 308C with
120 rpm for 22 h.
Conversions were measured by HPLC-DAD analysis on
an achiral phase. The measured values were corrected
using a calibration curve.
[b]
2 (10 mg,
[c]
[d]
[e]
Enantiomeric excess was determined by HPLC analysis
on a chiral phase. In brackets the isomer obtained in
excess is given.
n.d. not determined due to too low conversion.
itial idea for internal cofactor recycling to set up a
Having now chosen possible suitable reaction con- redox-neutral cascade. Since the substrate possessed
ditions, enantiopure d-mandelic acid was transformed the d-configuration and the product showed the l-
in the presence of d-MDH, l-AADH, cofactor and configuration an inversion of absolute configuration
ammonium to yield indeed the desired l-phenylgy- was achieved resembling a Mitsunobu stereoinver-
cine. At a substrate concentration of 10 gLÀ1 a con- sion.[20–23] In contrast to the reagent intensive Mitsu-
version of up to 76% was reached, whereby the nobu reaction (stoichiometric amount of, for example,
amount of oxo acid intermediate was below 0.3%. In- PPh3 and DEAD), the here presented system requires
terestingly, longer reaction times did not lead to just two biocatalysts, catalytic amounts of cofactor
higher conversion. Additional experiments showed and ammonium ions.
that the achieved concentration of l-phenylglycine
In the first experiments the substrate was optically
caused inhibition of d-MDH. Reducing the substrate pure d-mandelic acid. As a further challenge our aim
concentration to 5 gLÀ1 circumvented inhibition. Nev- was to start from racemic mandelic acid and to con-
ertheless, the successful transformation proved the in- vert the racemate to the optically pure l-amino acid,
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Adv. Synth. Catal. 2010, 352, 993 – 997

Deracemisation of Mandelic Acid to Optically Pure Non-Natural l-Phenylglycine
Table 2. Transformation of racemic mandelic acid to optical-
ly pure l-phenylglycine via a redox-neutral cascade (see
Scheme 2).[a]
Entry AADH[b]
1[c] [%] 2[c] [%] 3[c] [%] eeP[d] [%]
1
2
3
4
5
6
7
8
l-AADH-101
14.0
15.7
14.9
16.1
16.7
19.4
14.8
14.6
19.7
21.0
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.3
0.3
0.1
85.8
84.1
84.9
83.7
83.1
80.3
85.0
85.2
80.0
78.7
93.6
>97
>97
>97
>97
>97
>97
>97
>97
>97
>97
>97
l-AADH-104
l-AADH-105
l-AADH-106
l-AADH-107
l-AADH-108
l-AADH-111
l-AADH-114
l-AADH-115
l-AADH-118
Scheme 2. Deracemistaion of mandelic acid via a concurrent
redox-neutral cascade to yield l-phenylgycine.
9
10
11
l-AADH-101[e] 6.30
[a]
Reaction conditions: rac-mandelic acid
1
(5 mg,
thus performing a deracemisation. For this purpose
mandelate racemase[24] as a third enzyme was em-
ployed in an extended system (Scheme 2) for the in-
terconversion/racemisation of mandelic acid. Mande-
late racemase has been shown to racemise a broad
spectrum of substituted mandelic acid derivatives as
well as heteroaromatic analogues.[24,25] The racemase
was required since d-mandelate dehydrogenase is a
highly stereoselective enzyme exclusively transform-
ing d-mandelic acid and leaving the l-enantiomer un-
touched. Fortunately, mandelate racemase racemises
only the a-hydroxy acid 1 but not the corresponding
amino acid 3.[24]
Consequently, racemic mandelic acid was trans-
formed in the presence of mandelate racemase, d-
MDH and various AADHs at a substrate concentra-
tion of 5 gLÀ1 (Table 2, entries 1–10) on an analytical
scale to yield l-phenylglycine l-3 in up to 86% con-
version (entry 1). Performing the redox-neutral cas-
cade at a 10-fold larger scale led to 94% conversion
of l-3 (entry 11). Considering in an approximation
the Gibbs free energy for the interconversion of lac-
tate/alanine (À5.7 kcalmolÀ1, see above), a maximum
conversion of 98.6% to the l-amino acid is possible at
the equilibrium under the conditions employed.
0.03 mmol), amino acid dehydrogenase (2 mg, l-AADH-
101, 104, 105, 106, 107, 108, 111, 114, 115, 118), mande-
late dehydrogenase (5 mL, 2.9 U), mandelate racemase
(20 mg), NAD+ (1 mg, 0.002 mmol), buffer (1 mL,
pH 9.5, sodium bicarbonate 100 mM, ammonium chloride
200 mM), shaking at 308C with 120 rpm for 22 h.
Amino acid dehydrogenase, the numbers refer to the
enzyme kit of Codexis.
Composition was measured by HPLC-DAD analysis on
an achiral phase. The measured values were corrected
using a calibration curve.
Enantiomeric excess of l-3 was determined by HPLC
analysis on a chiral phase.
[b]
[c]
[d]
[e]
Cascade performed on 50-mg scale. For details see Ex-
perimental Section.
Scheme 3. Dynamic kinetic asymmetric transformation
(DYKAT): a) type II as introduced by Trost; b) the general
concept applied in this paper for deracemisation. A,B: sub-
strate enantiomers, I: prochiral intermediate P,Q: product
Therefore, the obtained conversion of 94% of l- enantiomers.
amino acid is rather close to the theoretical maxi-
mum. The concept for the deracemisation employed
in this paper (Scheme 2) is definitely different from a
dynamic kinetic resolution,[26,27] which encompasses a
In summary, a biocatalytic, redox-neutral cascade
racemisation step and a kinetic resolution. Scheme 2 was designed for the deracemisation of rac-mandelic
resembles more a dynamic kinetic asymmetric trans- acid to l-phenylglycine by employing three enzymes.
formation (DYKAT) of type II via a prochiral inter- The substrate enantiomers were interconverted by the
mediate I as introduced by Trost (see Scheme 3, enzyme mandelate racemase. In the first redox step
a).[28–32]
d-mandelic acid was oxidised by the d-selective man-
However, the difference is that the here presented delate dehydrogenase to give the corresponding a-
concept involved an additional racemisation step of oxo acid consuming NAD+ and giving NADH. In the
the substrate enantiomers (Scheme 3, b) since the second step, the reductive amination, an l-selective
transformation of the substrate enantiomers is highly amino acid dehydrogenase (EC 1.4.1.5) converted the
stereoselective due to the usage of intrinsic chiral en- oxo acid to the corresponding a-amino acids. For this
zymes.
step NADH is required, which is provided by the first
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995

Verena Resch et al.
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Experimental Procedure
The reactions were carried out in 1.5-mL Eppendorf tubes.
Each reaction mixture contained the crude commercial
preparation of amino acid dehydrogenase (2 mg), d-mande-
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11.2 min.
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asc.wiley-vch.de
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