An efficient single-enzymatic cascade for asymmetric synthesis of chiral amines catalyzed by ω-transaminase

Article abstract of DOI:10.1039/c2cc37232k

An efficient single-enzymatic cascade approach for the asymmetric synthesis of chiral amines has been developed, which applies the amino donor 3-aminocyclohexa-1,5-dienecarboxylic acid spontaneously tautomerizing to reach reaction completion with excellen

Full text of DOI:10.1039/c2cc37232k

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An efficient single-enzymatic cascade for
asymmetric synthesis of chiral amines catalyzed by
x-transaminase†
Cite this: Chem. Commun.,
2013, 49, 161
Received 3rd October 2012,
Accepted 7th November 2012
Bo Wang, Henrik Land and Per Berglund*
DOI: 10.1039/c2cc37232k
www.rsc.org/chemcomm
An efficient single-enzymatic cascade approach for the asymmetric for the enzyme(s) applied in the secondary irreversible reactions,
synthesis of chiral amines has been developed, which applies the amino or are the optimum for one enzyme (either o-TA or other
donor 3-aminocyclohexa-1,5-dienecarboxylic acid spontaneously coexisting enzymes) and suboptimal for others. Therefore, under
tautomerizing to reach reaction completion with excellent ee values.
such conditions, the enzymes display only part of their optimum
performance (activity and enantioselectivity). Thus, even though
There is an increasing demand for enantiomerically pure chiral multi-enzyme cascades can successfully overcome problems
amines, which are pivotal building blocks for the development of relating to the reaction equilibrium, the major disadvantage
biologically active compounds including pharmaceuticals and remains that the majority of the enzymes in the reaction system
agrochemicals.1–8 The use of amine transaminases, also known cannot show their intrinsically full activities.2,20–27 Therefore,
as o-transaminases (o-TAs), for the purpose of producing chiral new and widely applicable protocols that can overcome the
amines has been shown to be both feasible and effective.2,9–14 deficiencies mentioned above are highly desirable.
Although o-TA catalyzed asymmetric synthesis theoretically gives
In this communication, we present a novel single-enzymatic
a 100% yield of the desired optically pure amines, there is a cascade approach for the preparation of chiral amines which
disadvantage that equilibrium usually favors the substrate over applies o-TA as the biocatalyst and a commercially available
the product during transamination,2 resulting in incomplete 3-aminocyclohexa-1,5-dienecarboxylic acid, 2, as the amino
transformation. The challenge in asymmetric synthesis with donor (Scheme 1). No additional enzymes, expensive cofactors
o-TAs is thus to shift the equilibrium to the product side, which or special measures are required for the purpose of pushing the
is usually achieved mainly by means of (i) overcoming problems equilibrium to the product side either by combining secondary
of substrate–product inhibition, and (ii) solving issues related to irreversible reactions or removing volatile substances. In trans-
poor conversion of ketones to amines.
amination, the generated ketone 3 (Scheme 1) is effectively
As described by a plethora of recent papers centered on o-TA removed by spontaneous tautomerization to 3-hydroxybenzoic
catalyzed transaminations, much effort has been devoted to solving acid 5,28,29 thus allowing a theoretical yield of 100%.
the issues relating to unfavored equilibrium.15–17 This unfavored
equilibrium has also been exploited to perform a complete
synthesis of pyridoxamine 50-phosphate.18 Special interest has
been shown in the area of multi-step enzyme cascade reactions
where the equilibrium is usually shifted towards the product
side by removing the formed ketone via one or several enzyme-
catalyzed reactions, thus allowing a theoretical yield of 100%.2,17,19–21
Nevertheless, multi-enzyme cascade reactions are complex systems
where the reaction conditions are usually a compromise to ensure
that all enzymes coexisting in the same system are active. Generally,
the compromised conditions are the optimum neither for o-TA, nor
Scheme 1 Effective synthesis of chiral amines through the transamination of
ketones by using an (S)- or (R)-selective o-TA. The employment of racemic
3-aminocyclohexa-1,5-dienecarboxylic acid drives the reaction to completion via
spontaneous and instant transformation from the product ketone to
3-hydroxybenzoic acid, pushing the equilibrium to the product side, allowing a
theoretical yield of 100%.
KTH Royal Institute of Technology, Division of Biochemistry, School of
Biotechnology, AlbaNova University Center, SE-106 91 Stockholm, Sweden.
E-mail: per.berglund@biotech.kth.se; Fax: +46 8 5537 8468; Tel: +46 8 5537 8366
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
c2cc37232k
c
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Chem. Commun., 2013, 49, 161--163 161

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be achieved (Fig. 1a). Reaction times were longer compared with
those employing excess amino donor 2, and when a bit more than
one equivalent of amino donor 2 was introduced, for instance, 1.05
equivalent, conversions of up to >99% could be achieved (Table 1).
In addition, the stoichiometric amount of amino donor will also
relieve inhibition towards o-TA, possibly caused by the excess amino
donor. In the following studies, all reactions were performed with a
ratio of 1 : 1.05 (prochiral ketone/amino donor) to make the
prochiral ketones undergo complete conversions.
The inhibition caused by the generated co-product 3 was also
evaluated, by continuously adding 5, which tautomerizes to ketone
3, to the reaction solution. The concentration of ketone 3 was
increased and initial reaction rates versus varied concentrations of
3 + 5 were measured by following the formation of 1-phenylethyla-
mine at a wavelength of 270 nm (Fig. 1b). It was observed that
within the range of 0–20 mM, only a slight decline in the initial rate
could be observed, and when the concentration reached 20 mM, the
transaminase still maintained 80% of its highest activity. Never-
theless, when the concentration exceeded 20 mM (Fig. 1b), an
obvious decline in the initial reaction rate was thus observed. As a
stoichiometric amount of amino donor will be sufficient for a nearly
full conversion, a maximum of 5 mM (product 5) will be formed no
matter how much amino donor 2 is used. This is far less than the
upper limit (20 mM) of ketone 3 + product 5 above which inhibition
occurs. The possible pH shift, which would probably result in the
loss of transaminase activity, was also tested. The result showed that
no detectable pH change was found, indicating that in the designed
system, no deactivation caused by the pH shift exists.
The possible reverse reaction was also investigated by employing
the generated co-product 5, which tautomerizes to ketone 3, as the
amino acceptor, (S)-1-phenylethylamine as the amino donor
(Scheme 2a and b) and Cv-o-TA W60C as the biocatalyst. Data
collected showed that even after reaction for 36 h, no acetophenone
was detected, indicating that no detectable reverse reaction occurred
during transamination in the system. This might be the reason for
why a full conversion can be achieved even when amino donor 2
was employed at only a bit more than one equivalent excess. In
addition, it also indicates that there seems to be no equilibrium
existing in the assay due to the tautomerization reaction.
Fig. 1 (a) Profile of conversion of acetophenone vs. concentration of 2
(equivalents); (b) effect of the concentration of a mixture of 3 + 5 on the initial
rate of Cv-o-TA W60C obtained by following the formation of
1-phenylethylamine at 270 nm.
During transamination, prochiral ketone 1 and amino donor
2 are treated with o-TA and pyridoxal 50-phosphate (PLP).
Successful substrates are converted to the corresponding chiral
amines and ketone 3, which is unstable and spontaneously
transforms to phenol 5. The tautomerization of 3 to 5 serves
the triple purpose of (i) introducing secondary irreversible
reactions, (ii) removing ketone inhibition towards o-TA, and
(iii) shifting equilibrium to the product side.
To establish the basis for the designed single-enzymatic cascade
approach, initial studies were performed in HEPES buffer (50 mM,
pH 7.0) using acetophenone as the model amino acceptor and the
Chromobacterium violaceum o-transaminase variant W60C (Cv-o-TA
W60C) as the biocatalyst. In our previous work we successfully
obtained the Cv-o-TA W60C mutant with enhanced substrate
specificity, however its optimum pH value was shifted from the
original 8.2 of the wild type to 7.0.9 In transamination, the concen-
tration of acetophenone was set at 5 mM and varied concentrations
of amino donor 2 were explored to find the suitable amount of
amino donor for a full conversion of acetophenone. The collected
data showed that even when amino donor 2 was used at an
equivalent amount, a nearly ideal conversion of up to 98% could
To further explore the versatility of the approach, a group of
transaminases listed in Table 1 were tested. After 12 hours, samples
were taken and analyzed by GC to calculate the conversions. Similar
Table 1 Investigation of the generality of amino donor 2 when subjected to
various transaminasesa
Entry Transaminase
Ratio (2/1) Abs. Configb Convc (%)
1
2
3
4
5
6
7
8
Cv-o-TA wild type
Cv-o-TA wild type
Cv-o-TA W60C
Cv-o-TA W60C
Cv-o-TA F88A/A231F 1.05 : 1
ATA-113
ATA-117
o-TA-001
1 : 1
1.05 : 1
1 : 1
S
S
S
S
S
S
R
S
97
>99
98
>99
>99
>99
>99
>99
1.05 : 1
1.05 : 1
1.05 : 1
1.05 : 1
a
Reactions were performed on a 1 mL scale, conditions: ketone, 5 mM;
Scheme 2 (a) Asymmetric synthesis of (S)-1-phenylethylamine with an (S)-
selective o-transaminase by applying acetophenone and 3-aminocyclohexa-
1,5-dienecarboxylic acid as the model amino acceptor and donor, respectively;
(b) reverse reaction using 3-hydroxybenzoic acid as the amino acceptor and
(S)-1-phenylethylamine as the amino donor.
temperature, 37 1C; the pH value for all enzymes was 8.2 except for
Cv-o-TA W60C for which the pH value was 7.0; reaction time, 12 h.
b
Absolute configuration was determined by comparison with
c
standard samples. Conversions were determined by GC-analysis
with an internal standard.
c
162 Chem. Commun., 2013, 49, 161--163
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Table 2 Asymmetric syntheses by the designed single-enzymatic cascade
o-TA caused by an excess amino donor. It is expected that the
assay will be useful for many applications, especially for
medical research, in pharmaceutical and green chemistry.
This research was financially supported by KTH Royal
Institute of Technology. The authors would like to acknowledge
Prof. Wolfgang Kroutil (Graz University) for supplying the gene
for Cv-o-TA. The gift of the enzymes ATA 113, ATA 117 and o-TA
001 from Jeffrey Lutje Spelberg (Codexis) is gratefully
acknowledged.
approacha
Abs.
ee
Convc
Entry Transaminase
Product Configb pH (%) (%)
1
2
3
4
Cv-o-TA W60C
Cv-o-TA wild type
Cv-o-TA F88A/A231F
ATA-113
ATA-117
o-TA-001
S
S
S
S
R
S
S
S
7.0 >99 >99
8.2 >99 >99
8.2 >99 >99
8.2 >99 >99
8.2 >99 >99
5
6
8.2
62 >99
7d
Cv-o-TA wild type
Cv-o-TA W60C
8.2 >99 >99
7.0 >99 >99
8d
Notes and references
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9d
Cv-o-TA wild type
Cv-o-TA W60C
S
S
8.2
7.0
26 >99
41 98
10d
11
12
Cv-o-TA wild type
Cv-o-TA W60C
S
S
8.2 >99 >99
7.0 >99 >99
13
14
Cv-o-TA wild type
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S
S
8.2 >99 >99
7.0 >99 >99
a
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c
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d
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c
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Chem. Commun., 2013, 49, 161--163 163