A New Generation of Smart Amine Donors for Transaminase-Mediated Biotransformations

Article abstract of DOI:10.1002/chem.201603188

The application of ω-transaminase biocatalysts for the synthesis of optically pure chiral amines presents a number of challenges, including difficulties associated with displacing the challenging reaction equilibria. Herein, we report a highly effective approach using low equivalents of the new diamine donor, cadaverine, which enables high conversions of challenging substrates to the corresponding chiral amines in excellent ee. This approach paves the way for the design of self-sufficient fermentation processes combining transaminase biotransformations with existing strategies for cadaverine production by decarboxylation of endogenous lysine.

Full text of DOI:10.1002/chem.201603188

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Accepted Article
Title: A new generation of smart amine donors for transaminase-mediated biotrans-
formations
Authors: Andrew Gomm; William Lewis; Anthony P Green; Elaine O’Reilly
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A new generation of smart amine donors for transaminase-
mediated biotransformations
Andrew Gomm[a], William Lewis[a], Anthony P. Green[b], and Elaine O’Reilly[a]*
a large excess of isopropylamine (IPA) as a low-cost donor
combined with in-situ evaporation of the acetone by-product to
displace reaction equilibria towards product formation.7a
However, to obtain the low concentrations of acetone required to
Abstract: The application of ω-transaminase biocatalysts for the
synthesis of optically pure chiral amines presents a number of
challenges, including difficulties associated with displacing the
challenging reaction equilibria. Herein, we report a highly effective
approach using low equivalents of the novel diamine donor,
cadaverine, which enables high conversions of challenging
substrates to the corresponding chiral amines in excellent ee. This
approach paves the way for the design of self-sufficient fermentation
processes combining transaminase biotransformations with existing
strategies for cadaverine production via decarboxylation of
endogenous lysine.
achieve high conversions presents
a significant technical
challenge. In addition, this methodology cannot be successfully
applied for the conversion of ketones with particularly
challenging positions of equilibria.9 Alternative methods
employed to displace reaction equilibria involve combinations of
expensive enzymes, which function to remove the by-product
and recycle costly co-factor.3d,7b-c,10 We have recently reported
an extremely efficient strategy, which allows high conversions to
be obtained using only 1.0 equivalent of the ‘smart’ diamine
donor, o-xylylenediamine.11 The success of this approach can
be attributed to spontaneous cyclization, tautomerization and
irreversible polymerization of the by-product, which prevents the
reverse reaction from compromising conversion. However, in
addition to the relatively high cost of o-xylylenediamine
(compared to IPA), the formation of coloured polymeric by-
products has the potential to complicate downstream processing
and product purification and therefore there remains
considerable demand for the discovery of alternative ‘smart’
amine donors. In conjunction with effectively displacing
challenging reaction equilibria, these new smart donors should
be derived from bulk chemicals in order to be suitable for
industrial scale chiral amine production.
Chiral amines are prevalent in active pharmaceutical ingredients
(APIs) and frequently feature as functional handles in widely
used chemical building blocks.1 However, cost effective and
sustainable access to optically pure derivatives continues to
present a significant challenge for the pharmaceutical and
chemical industries. In recent years, biocatalysis has emerged
as a competitive and sustainable technology for the production
of high-value chemicals.2 There has been a notable surge in the
discovery and development of novel biocatalysts and biocatalytic
processes for the preparation of optically pure amines, including
those involving amine oxidases,3 ammonia lyases,4 imine
reductases,5 amine dehydrogenases6 and ω-transaminases (ω-
TAs).3d,7
ω-TAs in particular are emerging as extremely promising
catalysts for chiral amine synthesis, starting from readily
available prochiral ketones. These pyridoxal-5’-phosphate
(PLP)-dependent proteins are part of the aminotransferase class
of enzyme (EC 2.6.1.18) and rely on a sacrificial amine donor to
mediate the reversible conversion of structurally diverse ketones
to the corresponding chiral primary amines.8 The development of
Diamines 1-6 (Scheme 1 (a)) were selected for initial evaluation,
as the aminoaldehyde by-products of these donors have the
potential to dimerize or cyclise in a similar manner to o-
xylylenediamine, but are not expected to lead to the formation of
insoluble polymers (Scheme 1 (b)). A key challenge was to
identify an ω-TA biocatalyst capable of accepting these
diamines to enable their suitability as amine donors to be
a
transaminase-catalyzed manufacturing route to the
evaluated.
Importantly, this ω-TA must also have broad
blockbuster antidiabetic drug sitagliptin, using heavily
a
substrate scope towards structurally diverse ketones.
engineered (R)-selective ω-TA, highlights the high level of
interest in this enzyme family.7a However, despite the enormous
synthetic potential of these biocatalysts, there remain limited
examples of their application in industry. Challenges associated
with overcoming unfavourable reaction equilibria as well as a
restricted substrate scope has contributed to the relatively slow
uptake of these biocatalysts in both academia and industry.
Biotransformations employing ω-TAs typically involve the use of
[a]
[b]
A. Gomm, Dr. W. Lewis, Dr. E. O’Reilly
School of Chemistry, University of Nottingham
University Park, NG7 2RD, UK
Scheme 1. (a) Amine donors 1-6 screened with commercial enzymes and (b)
predicted dimerization or cyclization of the by-products. (i) The aminoaldehyde
by-products of 1 and 2 have the potential to undergo dimerization followed by
spontaneous and irreversible oxidation to the pyrazine. (ii) The by-products of
diamines 3-5 are expected to cyclize to the corresponding pyrroline or
tetrahydropyridine and undergo further trimerization.
E-mail: elaine.oreilly@nottingham.ac.uk
Dr. A. P. Green
Manchester Institute of Biotechnology, University of Manchester
131 Princess Street, Manchester, M1 7DN
Supporting information for this article is given via a link at the end of
the document.
While there are examples of diamino- and poly
aminotransferases that accept similar amine donors,12 these

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10.1002/chem.201603188
COMMUNICATION
enzymes suffer from an extremely narrow ketone substrate
scope, which is thus far limited to alpha-keto acids. In contrast,
commercially available ω-TAs from Codexis offer an extremely
broad substrate scope and have been engineered for increased
stability, making them the biocatalysts of choice in both
academic and industrial laboratories. Therefore, our initial
screen focused on evaluating twelve optimized Codexis
biocatalysts13 for their ability to utilize diamines 1-6. This
preliminary screen, with 1.0 equivalent of donors 1-6 and
acetophenone as the acceptor, revealed donors 4 and 5 allowed
significant conversion of the ketone to the corresponding chiral
amine using (S)-selective ATA256 at pH 8.5 (preliminary data
not shown). Having identified ATA256 as a suitable biocatalyst,
the conversion of acetophenone to MBA was monitored at a
range of pH values using both diamine 4 and IPA as the amine
donor (Figure 1). Despite this commercial enzyme being
specifically engineered to accept high concentrations of IPA,
cadaverine (also referred to as 1,5-DAP) outperformed this
donor across all three pH ranges tested. With both donors,
increasing the reaction pH resulted in higher conversions.
Significantly, only modest enhancements in conversions with
increasing pH were obtained using the standard IPA donor. In
contrast, conversions with cadaverine increased dramatically
upon variation of pH from 9 to 10.9 (a more detailed discussion
of the effect of pH is given below).
fluoroamphetamine was achieved. Under the same conditions, a
modest conversion of 69% was observed using IPA.
Acetophenone is routinely used as a benchmark substrate in ω-
TA biotransformations. An impressive conversion of 94% was
achieved for this aromatic ketone with only 3.0 equivalents of
diamine 4, while under the same conditions, IPA enabled only a
modest conversion of 44%.
Table 1. Conversion of 7-9 to the corresponding amines using ATA256 and
diamines 4, 5 and IPA.
Entry
Acceptor
Amine
donor
Equiv.
Conv.
ee
[%]
1
2
7
7
8
9
7
7
8
9
7
8
9
IPA
IPA
IPA
IPA
4
3.0
1.5
3.0
3.0
3.0
1.5
3.0
3.0
3.0
3.0
3.0
79%
69%
44%
15%
98%
96%
94%
57%
81%
91%
57%
>99 (S)
>99 (S)
>99 (S)
>99 (S)
>99 (S)
>99 (S)
>99 (S)
>99 (S)
>99 (S)
>99 (S)
>99 (S)
3
4
5
6
4
7
4
8
4
9
5
10
11
5
5
The ketone concentration was 20mM. Conversions are calculated from the
mean of 3-5 replicates. ee values were measured by GC-FID using a chiral
column (see supporting information).
The conversion of 1-indanone 9 to aminoindane is known to be
particularly challenging due to the extremely unfavourable
position of equilibria associated with this transformation. We
recently reported that 1.5 eq of o-xylylenedimaine was sufficient
to achieve a 73% conversion to the corresponding chiral
amine.11 In the same study we reported that a modest
conversion (21%) was achieved using 10 eq of alanine and the
widely used lactate dehydrogenase/glucose dehydrogenase
pyruvate removal system. Using only 3.0 eq of 1,5-DAP, a 54%
Figure 1. pH-profile for the conversion of acetophenone to (S)-MBA using 3
equivalents of IPA and 1,5-DAP as the amine donors. Conversions are
reported after 48 hours. The values given are the mean of 2-3 repeats and the
error bars represent the upper and lower limits of conversions obtained for
individual reactions.
Having established the optimum pH for performing these
reactions, ATA256 was screened with ketones 7-9 and amine
donors 4 and 5 (Table 1). In each case, the conversions were
compared to those achieved using IPA as the amine donor. The
ketone substrates (7-9) were rationally selected to allow the
efficiency of these diamine donors to be evaluated in
combination with progressively challenging substrates.
Biocatalytic amination of 7-9 becomes successively more
challenging due to increasingly unfavourable positions of
thermodynamic equilibria, as determined previously with
alternative amine donors.11
conversion of
9
to (S)-(+)-1-aminoindane is achieved
demonstrating the ability of this diamine donor to displace the
equilibria of extremely challenging substrates. Under the same
conditions, IPA performed poorly as expected, reaching only
15% conversion after 48 hours. The conversions with substrates
7-9 obtained with the related diamine
5 were broadly
comparable to those achieved with 1,5-DAP (Entries 9-11).
The conversion of acetophenone 8 to MBA was subsequently
carried out on
a preparative scale (Scheme 2 (b)) to
demonstrate the suitability of this system for performing
preparative scale transformations and to allow by-product
characterization. We anticipated that the fate of the initially
formed cyclic imine by-product may provide a rationale for the
Using only 1.5 equivalents of diamine 4, essentially complete
conversion (96%) of 4-fluorophenylacetone
7
to (S)-4-

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COMMUNICATION
dramatic increase in conversions with increasing pH. To aid by-
the displacement of the thermodynamic equilibrium towards the
desired amine products.
product characterization, imine 12 was prepared synthetically
following
a
literature procedure14 (Scheme
2 (a)). The
Table 2. Conversion of amine acceptor 14 to cadaverine 4 using ATA256 and
(S)-aminoindane over pH range 8-10.9.
symmetrical product 13 was initially isolated exclusively and
subsequently converted to isotripiperidine 14.14 The
unsymmetrical trimer was characterised by mass spectrometry
and NMR14,15 and the x-ray crystal structure of the molecule was
solved (Scheme 2 (c)). Only trimer 14 was observed in the
preparative scale biotransformation (Scheme 2 (b)) by both GC
and NMR (see supporting information Figure S1 for NMR
overlay) and could be seperated from the chiral amine product
by filtering through silica. This result is in line with literature
reports that this trimer is stable at pH1016 and may rationalise
why a higher reaction pH leads to increased conversions.
Entry
ATA
Amine donor
Acceptor
pH
Conv.
1
2
3
4
256
256
256
256
15
15
15
15
14
14
14
14
8.0
9.0
68%
57%
28%
14%
10
10.9
Conversions are based on the formation of ketone 9 from (S)-aminoindane 15,
monitored by GC-FID.
In summary, we have identified an ω-TA that has broad
acceptor substrate scope and is capable of using the bulk
diamine cadaverine as an amine donor. This new
generation ‘smart’ diamine is highly effective at displacing
challenging reaction equilibria, enabling high conversions to
the desired optically pure chiral amine products in the
absence of by-product removal systems. The success of
this donor can be attributed to a spontaneous dehydrative
cyclization/trimerization of the reaction by-product following
transamination. In all cases, cadaverine significantly
outperforms the industry standard amine donor,
isopropylamine, and therefore this work represents an
important proof-of-concept study. Future efforts will focus
on the identification of ω-TAs that accept alternative bulk
diamines (including 1,4-diaminobutane or putrescine),
performing reactions at significantly higher substrate
concerntrations and exploring the potential of self-sufficient
fermentation processes whereby the cadaverine donor is
produced biosynthetically via lysine decarboxylation.
Scheme 2. a) Synthesis of tripiperidine 13 and unsymmetrical isotripiperidine
14.14 (i) N-Chlorosuccinimide, Et2O, 0ºC, 2 h; (ii) EtOH, KOH, reflux, 2 h, 68%
over two steps; (iii) recrystallized in acetone, -20 ºC, 5 days. b) Preparative-
scale biotransformation of acetophenone 8 to (S)-MBA using ATA256 and 1,5-
DAP 4. c) Crystal structure of isotripiperidine 14.
Acknowledgements
To determine whether the formation of trimer 14 is reversible
under biotransformation conditions and to further investigate the
influence of pH on reaction equilibria, purified trimer 14 was
evaluated as an amine acceptor in combination with (S)-
aminoindane 15 (i.e. the reverse process of entry 8, Table 1), at
a range of pH values (pH 8-10.9) (Table 2). Table 2 shows that
at pH 8, (S)-aminoindane 15 is readily converted to indanone 9
(68%) while at higher pH values, this conversion is much less
efficient. At pH 10.9 (the optimal reaction conditions employed in
this study) a modest conversion of 14% was achieved. These
results suggest that formation of unsymmetrical trimer 14 is
favoured at higher pH and that trimer formation contributes to
Research leading to these results has received funding from the
BBSRC (BB/M021947/1), EPSRC (EP/K038869/1) and RSC
Research Fund. We also gratefully acknowledge a Royal
Society Research Grant (RG150134) and Codexis for supplying
enzymes used in this study.
Keywords: asymmetric catalysis • biocatalysis • chiral amine •
diamine• transaminase

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[1]
[2]
[3]
a) N. M. Cassiano in Alkaloids: Properties, Applications and
Pharmacological Effect, Nova Science Pub. Inc.; New York, 2010; b) M.
E. Welsch, S. A. Snyder, B. R. Stockwell, Curr. Opin. Chem. Biol. 2010,
14, 347; c) D. J. Newman, G. M. J. Cragg, Nat. Prod. 2012, 75, 311.
a) N. J. Turner, M. D. Truppo in Biocatalytic routes to nonracemic chiral
amines, Wiley_VCH Verlag GmbH, 2010, pp 431-459; b) M. Höhne, U.
T. Bornscheuer, ChemCatChem 2009, 1, 42; c) C. M Clouthier, J. N.
Pelletier, Chem. Soc. Rev., 2012, 41, 1585-1605.
a) C. J. Dunsmore, R. Carr, T. Fleming, N. J. Turner, J. Am.
Chem. Soc. 2006, 128, 2224; b) D. Ghislieri, A. P. Green, M. Pontini, S.
C. Willies, I. Rowles, A. Frank, G. Grogan, N. J. Turner, J. Am. Chem.
Soc. 2013, 135, 10863; c) D. Ghislieri, D. Houghton, A. P. Green, S. C.
Willies, N. J. Turner, ACS Catal. 2013, 3, 2869; d) E. O’Reilly, C.
Iglesias, N. J. Turner, ChemCatChem 2014, 6, 992.
[4]
[5]
a) M. M. Heberling, B. Wu, S. Bartsch, D. B. Janssen, Curr.
Opin.Chem. Biol. 2013, 17, 250; b) S. L. Lovelock, R. C. Lloyd, N. J.
Turner, Angew. Chem. Int. Ed. 2014, 53, 4652; Angew. Chem. 2014,
126, 4740; c) F. Parmeggiani, S. L. Lovelock, N. J. Weise, S. T. Ahmed,
N. J. Turner, Angew. Chem. Int. Ed., 2015, 54, 4608–4611; Angew.
Chem., 2015, 127, 4691–4694.
a) F. Leipold, S. Hussain, D. Ghislieri, N. J. Turner, ChemCatChem
2013, 5, 3505; b) M. Gand, H. Müller, R. Wardenga , M. Höhne, J. Mol.
Cat: B Enzym., 2014, 110, 126-132; c) P. N. Scheller, S. Fademrecht, S.
Hofelzer, J. Pleiss, F. Leipold, N. J. Turner, B. N. Nestl, B. Hauer,
Chembiochem. 2014,15, 2201-4; d) S. Hussain, F. Leipold, H. Man, E.
Wells, S.P. France, K. R. Mulholland, G. Grogan, N. J. Turner,
ChemCatChem, 2015, 7, 579–583.
[6]
[7]
M. J. Abrahamson, E. Vazquez-Figueroa, N. B. Woodall, A. S.
Bommarius, Angew. Chem. Int. Ed. 2012, 51, 3969; Angew. Chem.
2012, 124, 4036; b) M. J. Abrahamson, J. W. Wong, A. S. Bommarius,
2013, Adv. Synth. Catal., 355, 1780–1786; c) L. J. Ye, H. H. Toh, Y.
Yang, J. P. Adams, R. Snajdrova, Z. Li, ACS Catalysis, 2015, 5, 1119-
1122.
a) C. K. Savile et al., Science 2010, 329, 305; b) E. O’Reilly, C.
Iglesias, D. Ghislieri, J. Hopwood, J. L. Galman, R. C. Lloyd, N. J.
Turner, Angew. Chem. Int. Ed. 2014, 53, 2447; Angew, Chem. 2014,
126, 2479; c) E. O’Reilly, N. J. Turner, Perspectives in Science 2015,
4, 55– 61; d) T. Sehl, H. C. Hailes, J. M. Ward, R. Wardenga, E. von
Lieres, H. Offermann, R. Westphal, M. Pohl, D. Rother, Angew. Chem.
Int. Ed., 2013, 52, 6772–6775; Angew. Chem., 2013, 125, 6904–6908;
e) A. Cuetos, M. García-Ramos, E.-M. Fischereder, A. Díaz-Rodríguez,
G. Grogan, V. Gotor, W. Kroutil, I. Lavandera, Angew. Chem. Int. Ed.
2016, 55, 3144; Angew. Chem. 2016, 128, 3196.
[8]
[9]
a) S. Mathew, H. Yun, ACS Catal. 2012, 2, 993; b) D. Koszelewski, K.
Tauber, K. Faber, W. Kroutil, Trends Biotechnol. 2010, 28, 324.
a) P. Tufvesson, C. Bach, J. M. Woodley, Biotechnol. Bioeng. 2014,
111, 309–319.
[10] a) B. Wang, H. Lan, P. Berglund, Chem. Commun., 2013, 49, 161-163;
b) L. Martínez-Montero, V. Gotor, V. Gotor-Fernández, I. Lavandera,
Adv. Synth. Catal., 2016, 358, 1618-1624; c) D. Koszelewski, I.
Lavandera, D. Clay, G. M. Guebitz, D. Rozzell, W. Kroutil, Angew.
Chem., Int. Ed. 2008, 47, 9337; Angew. Chem. 2008, 120, 9477; d) R.
Abu, J. M. Woodley, ChemCatChem, 2015, 7, 3094.
[11] A. P. Green, N. J. Turner, E. O'Reilly, Angew. Chem. Int. Ed. 2014, 53,
10714; Angew. Chem. 2014, 126, 10890.
[12] I. Slabu, J. L. Galman, N. J. Weise, R. C. Lloyd, N. J. Turner,
ChemCatChem, 2016, 8, 1038-1042.
[13] The following TA biocatalysts were screened: ATA254, ATAG05,
ATA260, ATA256, ATA234, ATA113; (R)-selective ATA025, ATA303,
ATA013, ATA301, ATA415, ATA117.
[14] E. Gravel, E. Poupon, R. Hocquemiller, Tetrahedron 2006, 62, 5248–
5253. It is worth noting that our 1H MNR data for compound 14 is not
fully consistent with the data reported in this manuscript.
[15] H. Keske, H. Möhrle, G. Zimmermann, J. Org. Chem. 1977, 42, 66–72.
[16] A. Rouchaud, A., J. –C. Braekman, Eur. J. Org. Chem., 2009, 2666–
2674.

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A new generation of smart amine
donors for transaminase-mediated
biotransformations
ω-Transaminase (ω-TA) biocatalysts have been used in combination with a ‘smart’
diamine donor, enabling the highly efficient preparation of optically pure chiral
amines. This new generation donor is highly effective at displacing the challenging
reaction equilibrium and significantly outperforms the widely used donor,
isopropylamine.