Application of “Smart” Amine Donors for Rapid Screening and Scale-Up of Transaminase-Mediated Biotransformations

Article abstract of DOI:10.1002/ejoc.201800799

The “smart” amine donors o-xylylenediamine and cadaverine were employed for the rapid screening of a large ketone library and subsequent preparative-scale synthesis of selected compounds using a commercially available amine transaminase, ATA256. The methodology enables both screening and preparative-scale biotransformations to be performed with a single enzyme and simplifies the generation of sp3-rich small-molecule libraries.

Full text of DOI:10.1002/ejoc.201800799

A Journal of
Accepted Article
Title: Application of 'smart' amine donors for rapid screening and scale-
up of transaminase-mediated biotransformations
Authors: Andrew Gomm, Stylianos Grigoriou, Christopher Peel, James
Ryan, Nafees Mujtaba, Thomas Clarke, Evelina Evelina
Kulcinskaja, and Elaine O'Reilly
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To be cited as: Eur. J. Org. Chem. 10.1002/ejoc.201800799
Link to VoR: http://dx.doi.org/10.1002/ejoc.201800799
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European Journal of Organic Chemistry
10.1002/ejoc.201800799
COMMUNICATION
Application of ‘smart’ amine donors for rapid screening and
scale-up of transaminase-mediated biotransformations
Andrew Gomm,[a] [ǂ] Stylianos Grigoriou, [a] [ǂ] Christopher Peel,[a] James Ryan,[a] Nafees Mujtaba,[a]
Thomas Clarke,[a] Evelina Kulcinskaja[a] and Elaine O’Reilly* [a]
The ‘smart’ amine donors o-xylylenediamine and cadaverine
were employed for the rapid screening of a large ketone library
and subsequent preparative-scale synthesis of selected
compounds using the commercially available amine
transaminase, ATA256. The methodology enables both
screening and preparative-scale biotransformations to be
performed with a single enzyme and simplifies the generation of
sp3-rich small molecule libraries.
xylyenediamine can be used to effectively displace the
reaction equilibrium of some of the most challenging
substrates, using near stoichiometric equivalents of the
smart’ diamine donor.[17] Additionally, the co-products
‘
formed upon transamination with this donor are highly
coloured, suggesting that it could be employed for high-
throughput screening of both large enzyme and ketone
libraries. We have also extended this methodology to other
low-cost diamine donors, including cadaverine.[18] The
efficient conversion achieved using these diamines can be
attributed to the spontaneous cyclisation and subsequent
oligimerization/polymerization of the co-products, which
effectively displaces the reaction equilibrium. This
methodology has since been shown to be effective with other
ATAs and importantly, with other low-cost, bulk diamine
donors.[19] The development of biocatalytic approaches for
the synthesis of chiral amines that exploit ‘smart’ low-cost
diamine donors, in place of IPA coupled with complex co-
product removal, is of great interest.
Chiral amines are ubiquitous in pharmaceutical drugs,
bioactive natural products and privileged scaffolds.[1-2]
Asymmetric introduction of amines into small molecule
libraries represents an attractive way of increasing their
complexity.[3-5] It has been demonstrated that ‘sp -rich’
compounds possessing one or more stereocentres have
favourable properties for drug discovery processes.[6-7] In
order to produce diverse libraries of optically pure chiral
amines, robust strategies for their preparation must be
developed.
3
Current methods for asymmetric amine synthesis
typically rely on chiral auxiliaries or precious metal catalysts
and often require harsh reaction conditions (high
We have now demonstrated that o-xylylenediamine can
be employed for the rapid screening of a large library of
commercially available ketones and aldehydes, enabling
expedient identification of transaminase substrates. Based
on this high-throughput screening data, a selection of
ketones were converted to the corresponding chiral primary
amines on a preparative-scale, using cadaverine. The
approach demonstrates that these two smart donors can be
used in combination for efficient screening and subsequent
scale-up, using lower cost diamine donors.
[
8]
temperatures, pressures etc). The application of enzymes
for their synthesis represents a sustainable alternative, as
biotransformations are normally carried out under mild
conditions and often display superior regio- and
stereoselectivity to the traditional chemical approaches.
Amine transaminase (ATA) enzymes have emerged as
powerful catalysts for the production of chiral amines, with
many impressive examples of their application present in the
scientific literature.[9-12] ATAs are pyridoxal-5-phosphate-
dependent (PLP-dependent) and can catalyse the reversible
formation of chiral amines from pro-chiral ketones using a
suitable sacrificial amine donor. The reversibility of this
transformation means that efficient methods to displace the
often challenging reaction equilibrium are necessary to
achieve high conversion of substrate to product.
A library containing 400 ketones/aldehydes was supplied
by our industrial partner, Key Organics. Compounds within
this library were structurally diverse and many of the
corresponding amines were of commercial interest.
Screening was conducted in 96-well plates and results
analysed after 1 and 24 hours (figure 1). The formation of a
dark precipitate in the wells enabled qualitative identification
of enzyme substrates.
Commonly employed strategies include the use of a large
excess of amine donor coupled with in situ co-product
removal or enzymatic degradation of the reaction by-
product.[9,10, 13-16] We previously demonstrated that ortho-
[
a]
School of Chemistry
University of Nottingham
University Park, Nottingham, NG7 2RD.
E-mail: elaine.oreilly@nottingham.ac.uk
These authors contributed equally to this manuscript
[ǂ]
Figure 1. Representative assay plate showing the screening of a ketone
library using o-xylylenediamine. Wells containing dark precipitate indicate
ATA activity.
Supporting information for this article is given via a link at the end of
the document.
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European Journal of Organic Chemistry
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Comparison between Cadaverine and IPA
Analysis of the assay data (figure. 2) revealed that
smaller, low molecular weight compounds (<225 Da) are
readily accepted by commercially available ATA 256; an
enzyme developed by Codexis.[20] This data demonstrates
that in a library of diverse compounds, larger molecules are
less likely to be accepted. However, molecular weight and
size are not the only important factors and electronic effects,
as well as the nature of the substituents on the ketone are
also extremely relevant. As expected, the data showed that
alpha aryl-substituted methyl ketones were less amenable to
transamination compared to those with an alkyl substituent
100
9
8
7
6
5
4
3
2
1
0
0
0
0
0
0
0
0
0
0
(
figure. 3). Cyclic ketones were well tolerated whereas α,β
unsaturated ketones were poorer substrates.[21]
1
2
3
4
5
6
7
8
9
10 11 12 13
Compound
375
325
275
225
175
125
Analysis of ketone substrates- Molecular Weight vs
Calculated Volume
Conversion with 1eq Cadaverine
Conversion with 3 eq cadaverine
Conversion with 1 eq IPA
Conversion with 3 eq IPA
Negative
Positive
Molecular Weight
75
Figure 3: Analytical scale biotransformations with a selection of ketone
substrates 13, utilising either 1 or 3 equivalents of the amine donors,
cadaverine or IPA. Conversions of compounds 13 (20 mM) to the
50
100
150
200
250
300
350
400
1-
1
-
corresponding amine were carried out using ATA256 (2.5 mg/ml), PLP (1
mM) and cadaverine or IPA (20mM). Conversions are an average of three
replicates.
Figure 2. Characterisation of ketones used in the library screening and a
plot of their molecular weight vs volume.[22, 22]
preparative-scale biotransformations with ketones
and volatile ketones
1 and 12
Several ketones were then selected for quantitative
analysis (Figure 3) based on our interest in producing the
corresponding chiral amines on a preparative scale. The
amines selected were either not commercially available,
were extremely expensive or were difficult to produce using
traditional chemical approaches. Analytical scale reactions
were performed using our recently reported diamine donor
cadaverine[17] and compared to transformations using
isopropylamine (the current industry standard amine donor).
As expected, reactions employing cadaverine outperformed
isopropylamine in almost all cases (figure 3). Using 3
equivalents of cadaverine was sufficient to achieve a 98%
6-8.
Initial studies showed that using DMSO as a co-solvent was
likely to complicate chromatographic purification when
reactions were performed on a preparative scale. A variety
of other co-solvents were screened and biotransformations
containing methanol and ethanol reached similar
conversions to those with DMSO, indicating that these were
good alternative solvents. Acetone was the only co-solvent
that gave poor conversions, with some of the
biotransformations being completely inhibited (see
supplementary data).
Preparative biotransformations were carried out with
ketones 6-8 in HEPES buffer at pH 10.9, using DMSO as a
co-solvent and were purified by distillation (see ESI for
further details). All reactions proceeded with excellent ee
conversion of challenging ketone
1 to the corresponding
amine. A conversion of only 70% was achieved using IPA at
the same concentration. The diamine donor also significantly
outperformed IPA in the biotransformations involving the low
(
>99%), which was established by the synthesis and NMR
analysis of the corresponding Mosher’s acid chlorides (see
and 12
were extracted and purified via column chromatography in
0% and 39% yield respectively. In all cases, the compounds
molecular weight ketones,
5-8. These ketones (5-8) have
particularly low boiling points and therefore using high
concentrations of IPA in combination with in situ acetone co-
product removal is likely to be challenging, as the volatile
ketone starting material would also be evaporated under
these conditions. For this reason, we choose to demonstrate
the utility of the ‘smart’ amine donor, cadaverine, and by
extension similar low-cost diamine donors, by preforming
supporting information for details).[24] Compounds
1
7
were of sufficient purity for commercialisation.
3
The synthesis and application of ‘sp -rich’ fragments
remains highly important in modern drug-discovery
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European Journal of Organic Chemistry
10.1002/ejoc.201800799
COMMUNICATION
processes and it is expected that this methodology can
provide synthetic and medicinal chemists with a facile route
for the synthesis of chiral amine fragments. There is
increasing interest from industry in the development of
sustainable, bio-derived chiral molecules and the use of
commercially available enzymes and mild reaction
conditions make this approach attractive for accessing these
synthetically challenging compounds. Work is on-going to
directly evolve new enzymes that possess a broader
substrate scope and that can utilise ‘smart’ amine donors.
This will expand the use of the biocatalytic toolbox in modern
synthesis.
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P. A. Clemons, N. E. Bodycombe, H. A. Carrinski, J. A. Wilson, A. F.
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Acknowledgements
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We thank our industrial partners, Key Organics, for supplying the
compound library and BIOCATNET,
BB/L013649/1) for proof-of-concept funding. Research leading to
these results has also received funding from the BBSRC
BB/M021947/1) and Royal Society Research Grant (RG150134).
a
BBSRC NIBB
[17]
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The authors gratefully acknowledge the BBSRC/EPSRC
Synthetic Biology Research Centre, Nottingham and the
University of Nottingham for the financial support provided to SG.
We also acknowledge funding from the UoN to support AG,
funding from the UK Catalysis Hub for supporting JR and funding
from the EPSRC Centre for Doctoral Training in Sustainable
Chemistry for supporting CP and TC.
J. L. Galman, I. Slabu, N. J. Weise, C. Iglesias, F. Parmeggiani, R.
C. Lloyd, N. J. Turner, Green Chem., 2017 9, 285–288.
The sequence for ATA256 can be found in the following patent.
Crowe et. al. Chemical processes for preparing spiroindolones and
intermediates thereof. US Patent US 2015/0045562 A1, filed 22
March 2013 and issued 12 February 2015.
[21]
Minor modification of the o-xylylenediamine assay to use 0.15
mg/mL of commercially available enzyme enzyme and leaving for 3
hours instead of 24 hours gave enabled accurate prediction of
substrates which underwent facile transamination. (see
Supplementary Data for more information).
Keywords: amine transaminase • biocatalysis • chiral amine •
high-throughput screening
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European Journal of Organic Chemistry
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COMMUNICATION
Entry for the Table of Contents
COMMUNICATION
[
a] [ǂ]
Andrew Gomm,
Stylianos Grigoriou,
[
a] [ǂ]
Christopher Peel,[a] James Ryan,
Nafees Mujtaba,[a] Thomas Clarke,
[a]
[a]
[
a]
Evelina Kulcinskaja and Elaine
O’Reilly* [
a]
Page No. – Page No.
The ‘smart’ amine donors o-xylylenediamine and cadaverine have been employed
for the rapid screening of a large ketone library and subsequent preparative-scale
synthesis of a selection of optically pure chiral amines, using a commercially
available amine transaminase biocatalyst.
Application of ‘smart’ amine donors
for rapid screening and scale-up of
transaminase-mediated
biotransformations
This article is protected by copyright. All rights reserved.