Efficient kinetic resolution of racemic amines using a transaminase in combination with an amino acid oxidase

Article abstract of DOI:10.1039/b902995h

A range of enantiomerically pure (R)- and (S)-configured chiral amines has been prepared in excellent e.e. (99%) by combining a transaminase enzyme with an amino acid oxidase and catalytic quantities of pyruvate.

Full text of DOI:10.1039/b902995h

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Efficient kinetic resolution of racemic amines using a transaminase
in combination with an amino acid oxidasew
Matthew D. Truppo,*a Nicholas J. Turner*a and J. David Rozzellb
Received (in Cambridge, UK) 12th February 2009, Accepted 5th March 2009
First published as an Advance Article on the web 16th March 2009
DOI: 10.1039/b902995h
A range of enantiomerically pure (R)- and (S)-configured chiral
amines has been prepared in excellent e.e. (99%) by combining a
transaminase enzyme with an amino acid oxidase and catalytic
quantities of pyruvate.
Enantiomerically pure chiral amines are key intermediates in a
number of pharmaceutical compounds that possess a wide
range of biological activities.1 Biocatalytic approaches to these
building blocks have traditionally relied upon kinetic
resolution of racemic substrates using hydrolytic enzymes2
Scheme 1 Resolution of a racemic amine using a transaminase with
such as lipases, acylases and proteases and indeed some of
catalytic quantities of pyruvate and an amino acid oxidase.
these processes have been operated successfully at large scale.3
to alanine which undergoes in situ oxidation back to pyruvate
catalysed by AAO in the presence of molecular oxygen. In
addition to significantly reducing the quantities of pyruvate
required, we reasoned that this system should also overcome
the unfavourable equilibrium, since one of the components
(pyruvate) is being removed. Reduction of pyruvate concen-
tration should in addition lead to reduced inhibition of the
transaminase enzyme.
In certain cases it is possible to racemise the unreactive
enantiomer by addition of a racemisation catalyst leading to
a dynamic kinetic resolution (DKR) process.4 Alternative
approaches based upon deracemisation have also been
developed in which enantioselective amine oxidases are com-
bined with non-selective reducing agents resulting in yields
of 450% and high enantioselectivities.5 Recently trans-
aminases have emerged as viable biocatalysts for chiral amine
production.6 Transaminases can be used in two complemen-
tary ways, either (i) in the asymmetric synthesis7 of
chiral amines from ketones using a suitable amine donor
(e.g. L-alanine) or (ii) in the kinetic resolution8 of racemic
amines using a suitable amine acceptor (e.g. pyruvate).
Although the former process is attractive in terms of the
potential for high yields of product, it often suffers from poor
conversion due to the equilibrium favouring the ketone and
also product inhibition of the transaminase by the amine
product.9,10 Thus the kinetic resolution of amines using trans-
aminases remains an option in many instances and has been
applied in some cases on a large scale.11 However, it is
necessary to add stoichiometric quantities of pyruvate which
in addition to the cost can also result in inhibition of the
reaction. In this communication, we report a new approach in
which catalytic quantities of pyruvate are used in combination
with an amino acid oxidase.
To establish the basis for this approach, the kinetic
resolution of (Æ)-a-methylbenzylamine (MBA 1) at 25 mM
concentration was carried out using the (R)-selective trans-
aminase ATA-117 together with D-amino acid oxidase
(D-AAO) and a catalytic amount of pyruvate (2 mM; 8 mol%).12
The reaction proceeded to 50% conversion after 1 h yielding
(S)-1 (99% e.e.) (Fig. 1). A control reaction was also run in
which the D-AAO was omitted. The reaction proceeded to
only 7.5% conversion after 1 h affording (S)-1 in 8% e.e. This
result corresponds well to a maximum theoretical conversion
of 8% based on a 25 mM substrate charge and a 2 mM amine
acceptor concentration.
The resolution of (Æ)-1 was then attempted at concentra-
tions up to 100 mM using ATA-117, D-amino acid oxidase,
and a catalytic amount of pyruvate (8 mol%). All reactions
proceeded to 50% conversion and yielded (S)-1 in 99% e.e.
(Fig. 2).
To improve the efficiency of the reaction we decided to
explore the effect of reducing the concentration of pyruvate.
Kinetic resolutions were run with (Æ)-1 (100 mM) and
pyruvate loadings from 2 mM (2 mol%) to 0.05 mM
(0.05 mol%). As expected the reaction rate decreased with
decreasing concentrations of pyruvate, although even with
0.1 mM pyruvate (0.1 mol%) the reaction proceeded to
completion in 12 h (Fig. 3).
Scheme 1 outlines the process in which a catalytic amount of
amine acceptor (pyruvate) is added to the reaction together
with an amino acid oxidase (AAO). Transaminase catalysed
kinetic resolution of the amine leads to conversion of pyruvate
a School of Chemistry, University of Manchester, Manchester
Interdisciplinary Biocentre, 131 Princess Street, Manchester,
UK M1 7DN. E-mail: Nicholas.turner@manchester.ac.uk;
Fax: (+44)161 306 5173
To examine the generality of the process, two transaminases
ATA-113 [(S)-selective] and ATA-117 [(R)-selective] were
applied to the resolution of a variety of racemic amine sub-
strates 1–6. Reactions were run with amine substrate (25 mM)
b Codexis Inc., 129 N. Hill Avenue, Pasadena, CA 91106, USA
w Electronic supplementary information (ESI) available: HPLC assay
conditions; resolution conditions; HPLC chromatograms. See DOI:
10.1039/b902995h
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Fig. 1 Transaminase (ATA-117) catalyzed resolution of a-methyl-
benzylamine 1 (25 mM) using a catalytic amount (2 mM) of amine
acceptor (pyruvate), with and without D-amino acid oxidase (D-AAO).
Fig. 3 Resolution of a-methylbenzylamine 1 (100 mM) using a
catalytic amount of amine acceptor (pyruvate). The molar ratio of
amine acceptor to substrate is shown in bold.
Table 1 Synthesis of (R)- and (S)-amines 1–6 using transaminases
ATA-113 and ATA-117
#
Compound Structure Product (R)-amine
Product (S)-amine
1
499% ee (ATA113) 499% ee (ATA117)
499% ee (ATA113) 499% ee (ATA117)
2
3
499% ee (ATA113) 499% ee (ATA117)
499% ee (ATA113) 499% ee (ATA117)
Fig. 2 Resolution of various concentrations of a-methylbenzylamine
1 using a catalytic amount (8 mol%) of amine acceptor (pyruvate).
Closed lines and symbols show enantiomeric excess (e.e.). Open lines
and symbols show conversion.
4
and a catalytic amount of pyruvate (2 mM; 8 mol%). L-Amino
acid oxidase and D-amino acid oxidase were used in the
ATA-113 and ATA-117 catalysed reactions in view of their
known specificities for L- and D-alanine respectively. Reactions
were run for 3 h and monitored by HPLC to determine %
conversion and e.e. (Table 1). All six amines were successfully
resolved by transaminase ATA-113, giving the corresponding
(R)-amine products with 99% e.e. at 50% conversion. Trans-
aminase ATA-117 successfully resolved amines 1–4, giving the
(S)-amine product with 499% e.e. at 50% conversion.
However, ATA-117 showed no activity towards the more
sterically demanding amines 5 and 6 and hence we sought to
prepare the (S)-enantiomers of these two amines from the
corresponding ketones according to the procedure that we
have previously reported.13
5
6
499% ee (ATA113) 499% ee (ATA113)
synthesis direction
499% ee (ATA113) 499% ee (ATA113)
synthesis direction
amine donor (Scheme 2). Lactate dehydrogenase (LDH) was
added to drive the reaction to completion by reduction of the
pyruvate formed to lactate and the recycling of NADH was
achieved by using glucose dehydrogenase (GDH). It is inter-
esting to note that a single broad specificity transaminase
ATA-113 can be used to prepare both (R)-(kinetic resolution)
and (S)-amines (asymmetric synthesis) in high enantiomeric
excess illustrating the benefit of the two complementary
approaches.
Amines (S)-5 and (S)-6 were prepared in 99% e.e. from the
corresponding ketones using ATA-113 and L-alanine as the
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Fl., 2007, pp. 743–755; S. Brase, T. Baumann, S. Dahmen and
H. Vogt, Chem. Commun., 2007, 1881.
2 H. Ismail, R. M. Lau, F. van Rantwijk and R. A. Sheldon, Adv.
Synth. Catal., 2008, 350, 1511.
3 M. Breuer, K. Ditrich, T. Habicher, B. Hauer, M. Keßeler,
R. Sturmer and T. Zelinski, Angew. Chem., Int. Ed., 2004, 43, 788.
4 C. Roengpithya, D. A. Patterson, A. G. Livingston, P. C. Taylor,
J. L. Irwin and M. R. Parrett, Chem. Commun., 2007, 3462;
A. N. Parvulescu, P. A. Jacobs and D. E. De Vos, Adv. Synth.
Catal., 2008, 350, 113; C. E. Hoben, L. Kanupp and J.-E. Backvall,
Tetrahedron Lett., 2008, 49, 977; M.-J. Kim, W.-H. Kim, K. Han,
Y. K. Choi and J. Park, Org. Lett., 2007, 9, 1157.
5 C. J. Dunsmore, R. Carr, T. Fleming and N. J. Turner, J. Am.
Chem. Soc., 2006, 128, 2224.
Scheme 2 Asymmetric synthesis of amines from ketones using
transaminase and lactate dehydrogenase to drive the reaction to
completion.
6 K. Faber and W. Kroutil, Curr. Opin. Chem. Biol., 2005, 9, 181;
J. D. Stewart, Curr. Opin. Chem. Biol., 2001, 5, 120;
D. Koszelewski, I. Lavandera, D. Clay, G. M. Guebitz,
D. Rozzell and W. Kroutil, Angew. Chem., Int. Ed., 2008, 47,
9337; U. Kaulmann, K. Smithies, M. E. B. Smith, H. C. Hailes and
J. M Ward, Enzyme Microb. Technol., 2007, 41, 628.
7 D. Koszelewski, I. Lavandera, D. Clay, D. Rozzell and W. Kroutil,
Adv. Synth. Catal., 2008, 350, 2761; H. Yun and B.-G. Kim,
Biosci., Biotechnol., Biochem., 2008, 72, 3030; A. Iwasaki,
Y. Yamada, N. Kizaki, Y. Ikenaka and J. Hasegawa, Appl.
Microbiol. Biotechnol., 2006, 69, 499; J.-S. Shin and B.-G. Kim,
Biotechnol. Bioeng., 1999, 65, 206.
8 M. Hohne, K. Robins and U. T. Bornscheuer, Adv. Synth. Catal.,
2008, 350, 807; J.-S. Shin and B.-G. Kim, Biotechnol. Bioeng.,
1997, 55, 348; J.-S. Shin, B.-G. Kim and D.-H. Shin, Enzyme
Microb. Technol., 2001, 29, 232; J.-S. Shin and B.-G. Kim,
Biotechnol. Bioeng., 1998, 60, 534; J.-S. Shin, B.-G. Kim,
A. Liese and C. Wandrey, Biotechnol. Bioeng., 2001, 73, 179.
9 Y. B. Tawari, R. N. Goldberg and J. D. Rozzell, J. Chem.
Thermodyn., 2000, 32, 1381.
10 For approaches to overcome the problems associated with equili-
brium see: M. Hohne, S. Kuhl, K. Robins and U. T. Bornscheuer,
ChemBioChem, 2008, 9, 363.
11 R. L. Hanson, B. L. Davis, Y. Chen, S. L. Goldberg, W. L. Parker,
T. P. Tully, M. A. Montana and R. N. Patel, Adv. Synth. Catal.,
2008, 350, 1367.
12 All enzymes, including transaminases (ATA-113 and ATA-117),
L-aminoacid oxidase (L-AAO) and D-amino acid oxidase (D-AAO),
glucose dehydrogenase (GDH CDX-901) and lactate dehydro-
genase (LDH-102), were generously supplied by Codexis
(Redwood City, CA).
In summary, we have developed a novel system for the
resolution of racemic amines using a transaminase (ATA)
coupled with an amino acid oxidase (AAO). In contrast to
previously reported approaches that use a stoichiometric
amount of amine acceptor, the system described here employs
a catalytic amount of amine acceptor (pyruvate) that is
continuously recycled in situ by an amino acid oxidase and
molecular oxygen. The effective replacement of a molar
quantity of amine acceptor with oxygen will result in signifi-
cant cost savings when this process is applied on a large scale.
Additionally, the AAO system drives the reaction equilibrium
and significantly reduces transaminase inhibition by the
co-substrate pyruvate. Finally, we have demonstrated two
complementary systems for the synthesis of chiral amines. Given
a single transaminase with broad substrate specificity and high
enantioselectivity, both the (R)- or (S)-amine product can be
obtained via either resolution of racemic amine substrates or
asymmetric synthesis of the chiral amine through transamina-
tion of the corresponding prochiral ketone substrate.
Notes and references
1 N. J. Turner and R. Carr, in Biocatalysis in the Pharmaceutical and
Biotechnology Industries, ed. R. N. Patel, CRC Press, Boca Raton,
13 M. D. Truppo, D. J. Rozzell, J. C. Moore and N. J. Turner, Org.
Biomol. Chem., 2009, 7, 395.
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Chem. Commun., 2009, 2127–2129 | 2129