Biocatalytic Transamination for the Asymmetric Synthesis of Pyridylalkylamines. Structural and Activity Features in the Reactivity of Transaminases

Article abstract of DOI:10.1021/acscatal.6b00686

A set of transaminases has been investigated for the biocatalytic amination of 1-(4-chloropyridin-2-yl)alkan-1-ones. The influence of the chain length of the n-1-alkanone at the C-2 position of the pyridine has been studied in the reaction with different

Full text of DOI:10.1021/acscatal.6b00686

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Article
Biocatalytic Transamination for the Asymmetric
Synthesis of Pyridylalkylamines. Structural and
Activity Features in the Reactivity of Transaminases
María López-Iglesias, Daniel González-Martínez, Vicente Gotor,
Eduardo Busto, Wolfgang Kroutil, and Vicente Gotor-Fernández
ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.6b00686 • Publication Date (Web): 05 May 2016
Downloaded from http://pubs.acs.org on May 9, 2016
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Biocatalytic Transamination for the Asymmetric
Synthesis of Pyridylalkylamines. Structural and
Activity Features in the Reactivity of Transaminases
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María LópezꢀIglesias,†,§ Daniel GonzálezꢀMartínez,† Vicente Gotor,† Eduardo Busto, §,ǂ,*
Wolfgang Kroutil§,* and Vicente GotorꢀFernández†,*
† Departamento de Química Orgánica e Inorgánica, Instituto Universitario de Biotecnología de
Asturias, Universidad de Oviedo, Eꢀ 33071 Oviedo (Asturias), Spain.
§ Department of Chemistry, Organic and Bioorganic Chemistry, University of Graz, NAWI
Graz, Heinrichstrasse 28, 8010 Graz, Austria.
ǂ Departamento de Quımica Orgánica I, Facultad de Quımica, Universidad Complutense de
́
́
Madrid, 28040 Madrid, Spain.
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ABSTRACT. A set of transaminases has been investigated for the biocatalytic amination of 1ꢀ(4ꢀ
chloropyridinꢀ2ꢀyl)alkanꢀ1ꢀones. The influence of the chain length of the nꢀ1ꢀalkanone at the Cꢀ2
position of the pyridine has been studied in the reaction with different (R)ꢀ and (S)ꢀselective
transaminases. Thus, enantiopure amines were isolated with high purity starting from a wide
selection of prochiral ketones. On the one hand excellent yields (97ꢀ>99% conversion, up to 93%
isolated yield) and stereoselectivity values (>99% ee for both amine enantiomers) were found for
nꢀ1ꢀalkanone linear short chain substituents such as ethanone or propanone. On the other hand,
more hindered substrates were accepted only when using evolved enzymes such as an evolved
variant of (R)ꢀArthrobacter (ArRmut11ꢀTA). An initial common structural feature was the
presence of a chlorine atom on the Cꢀ4 position of the pyridine core, which was found to increase
the reactivity of the starting ketone, giving extra versatility for the introduction of other chemical
functionalities towards more complex and applicable organic molecules. In order to gain a
deeper understanding about the substrate specificity of different transaminases, additional
structural features were considered by variation of the acetyl group position on the pyridine ring
and the use of related acetophenone derivatives.
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KEYWORDS. Transaminases; amines; biocatalysis; enzymes; substrate specificity;
transamination
INTRODUCTION
The asymmetric synthesis of amines attracts significant attention due to their multiple immediate
applications and versatility as building blocks in the preparation of bioactive molecules.1 In
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addition, amines have been widely employed as chiral auxiliaries, asymmetric catalysts and
chiral resolving agents.2
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Enzymes offer highly sustainable and efficient synthetic alternatives to current existing chemical
methods,3 providing access to enantiopure amines and derivatives with excellent selectivity.4
Traditionally, hydrolases have been the biocatalysts of choice for the preparation of optically
active amines through classical or dynamic kinetic resolutions of the corresponding racemates,5
but the development of other classes of enzymes such as amine oxidases,6 transaminases,7
berberine bridge enzymes,8 amine dehydrogenases9 and more recently imine reductases10
presents biotransformations as versatile tools for the development of highly efficient asymmetric
transformations to prepare these compounds.
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Transaminases (TAs), also known as aminotransferases (EC 2.6.1.X) or amine transaminases, are
pyridoxal 5’ꢀphosphate (PLP) dependent enzymes11 that catalyze the kinetic resolution of
racemic amines and, conversely, asymmetric synthesis of amines from the corresponding
prochiral ketones.7,12 The latter reaction is preferred over the first as it culminates in a 100%
theoretical maximum yield. In this sense and taking into account thermodynamic considerations,
the main limitation of an unfavorable equilibrium shift towards the formation of the amine has
been broadly studied and overcome by diverse strategies.13
The pyridine core constitutes an interesting nitrogenꢀbased scaffold, both from a biological and
synthetic point of view.14 It can participate in chemical transformations either via the nitrogen or
via the aromatic ring, which makes pyridines attractive compounds for many different
applications.15 Particularly, the presence of a chlorine atom on the aromatic ring opens up a
variety of synthetic possibilities for increasing molecular complexity, allowing for instance the
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preparation of enantiopure 4ꢀ(N,Nꢀdimethylamino)ꢀ2ꢀ(1ꢀhydroxyalkyl)pyridines using lipases16
or alcohol dehydrogenases,17 compounds that offer interesting applications in asymmetric
organocatalysis as ligands or chiral catalysts.17
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Herein, we describe the enzymatic transamination of 1ꢀ(4ꢀchloropyridinꢀ2ꢀyl)alkanꢀ1ꢀones
bearing different size alkyl chains on the Cꢀ2 position of the aromatic ring (1aꢀd, Figure 1 top).
A range of transaminases overexpressed in E. coli was investigated to establish the optimal
reaction conditions and study the influence of the substitution pattern on the reactivity of the
prochiral ketones. Furthermore, additional structural parameters such as the type of aromatic
ring, the presence of chlorine atoms or the position of the ketone functionality on the aromatic
ring will be considered (structures 1eꢀi, Figure 1 bottom), in order to provide an insight in the
interaction between selected transaminases and aromatic substrates with different structural
cores. Correlations between conversion of the enzymatic transamination processes and the
infrared carbonyl stretch of the starting ketones will be done, discussing the best structural motifs
to allow the displacement of the equilibrium towards the amine formation.
Figure 1. Chemical structure of 1ꢀ(4ꢀchloropyridinꢀ2ꢀyl)alkanꢀ1ꢀones (top) and structural
modifications considered for the study of transaminase reactivity (bottom).
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RESULTS AND DISCUSSION
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Prochiral ketones 1aꢀd were chemically synthesized starting from commercially available 4ꢀ
chloropyridine Nꢀoxide in a twoꢀstep sequence previously developed in our research group.16a
The biocatalytic transamination of ketones 1aꢀd was initially performed at a 50 mM substrate
concentration using a set of TAs overexpressed in E. coli (Table 1): the (S)ꢀselective TAs from
Vibrio fluvialis (Vf)18 and Chromobacterium violaceum (Cv),19 or the (R)ꢀselective TAs from
(R)ꢀArthrobacter species (ArR)20 and Aspergillus terreus (At).21
respectively, were initially used as amine source depending on the enzyme’s selectivity, and the
equilibrium was shifted towards amine formation by removing the pyruvate to ꢀalanine with
Lꢀ or Dꢀalanine (L/DꢀAla),
L
alanine dehydrogenase (AlaDH) at the expense of ammonia and formate generating only CO2
and H2O as byproducts.22
As shown in Table 1, quantitative conversions and excellent stereoselectivities were observed for
the methyl ketone 1a with all tested TAs (entries 1ꢀ4). Similarly, access to both enantiomers of
amine 2b was accomplished by selecting the appropriate TA (entries 5ꢀ8). Thus, the enantiopure
amine (S)ꢀ2b was obtained with quantitative conversion (entries 5 and 6) and the complementary
(R)ꢀantipode with very high conversions (up to 97%, entries 7 and 8).
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Table 1. Enzymeꢀcatalyzed asymmetric transamination of ketones 1a-d.a
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Entry
1
R
TA
c (%)b
>99
>99
>99
>99
>99
>99
95
ee (%)c
>99 (S)
>99 (S)
>99 (R)
>99 (R)
>99 (S)
>99 (S)
>99 (R)
>99 (R)
>99 (S)
>99 (S)
n.m.
Me (1a) Vf
Me (1a) Cv
Me (1a) ArR
Me (1a) At
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Et (1b)
Vf
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Et (1b) Cv
Et (1b) ArR
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Et (1b)
At
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nPr (1c) Vf
nPr (1c) Cv
nPr (1c) ArR
nPr (1c) At
nBu (1d) Vf
nBu (1d) Cv
nBu (1d) ArR
nBu (1d) At
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3
<1
n.m.
15
81 (S)
<1
19
<1
n.m.
<1
n.m.
a Reaction conditions: phosphate buffer (100 mM, pH 7), ketone 1a-d (50 mM), lyophilized cells
E. coli/TA (20 mg), PLP (1 mM), NAD+ (1 mM),
Lꢀ or Dꢀalanine (250 mM), AlaDH (10 µL, 11
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U), formate dehydrogenase (FDH, 2.6 mg, 11 U), ammonium formate (150 mM), 24 h at 30 ºC
and 700 rpm.
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b Conversion values were determined by GC analysis after isolation of products by extraction
from basified media (See experimental section).
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c Enantiomeric excess values were measured by GC or HPLC (See Supporting Information) on a
chiral phase. n.m.: not measured. The stereochemistry of the products, which appear in brackets,
was confirmed by comparing the corresponding optical rotations with the values reported in
literature (See experimental section).
The exquisite preliminary results encouraged us to carry out representative examples on
preparative scale (50 mg of substrate). Consequently, transamination reactions of methyl ketone
1a with CvꢀTA, allowed the isolation of (S)ꢀ2a (ee >99%) in 82% isolated yield. The synthetic
potential of the VfꢀTA was also demonstrated obtaining the same enantiopure amine in 93%
isolated yield (see Experimental Section). Similarly, VfꢀTA led to quantitative transformation
and 93% isolated yield of (S)ꢀ2b after a simple extraction protocol. The isolated amines were
analyzed by GC and NMR techniques, 2a and 2b being obtained with very high purity without
no additional purification.
A dramatic decrease of conversion was observed when bulkier ketones were considered
containing longer alkyl rests (1c and 1d, propyl and butyl, respectively), which is in agreement
with previous TAꢀcatalyzed biotransamination examples with structurally similar acetophenone
derivatives.23 Nevertheless, amine (S)ꢀ2c was still obtained with good conversion and in
enantiopure form when Vfꢀ TA was used (entry 9), while low conversions were attained for the
(S)ꢀselective CvꢀTA or the (R)ꢀselective enzymes (entries 10ꢀ12). Other TAs (Arthrobacter
citreus, Bacillus megaterium, Paracoccus denitrificans, Pseudomonas fluorescens or
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Hyphomonas neptunium) proved to be not suitable. Low or no detectable conversion was
obtained for the pentanone 1d in all cases (entries 13ꢀ16).
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Since none of the enzymes tested provided an efficient access to the more hindered amines, an
evolved variant of (R)ꢀArthrobacter (ArRmut11ꢀTA) was explored, also displaying (R)ꢀ
selectivity as the wildꢀtype enzyme. This enzyme has been previously successfully applied in the
transamination of sterically demanding substrates,24 and presents additional advantages as its
high thermal stability and tolerance to 2ꢀpropylamine as amine source. Thus, the reaction was
run at elevated temperature (45 ºC) circumventing a recycling system by adding an excess of the
amine donor. The results are summarized in Table 2.
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Table 2. Asymmetric transamination of ketones 1a-d using the ArRmut11ꢀTA.a
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ee
Entry
R
TA preparation
(%)b (%)c
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nBu (1d)
nBu (1d)
nPr (1c)
nPr (1c)
Et (1b)
crude
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>99
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>99
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semipurified
crude
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semipurified
semipurified
Me (1a)
a Reaction conditions: phosphate buffer (100 mM, pH 7), ketone 1aꢀd (50 mM), ArRmut11ꢀTA
(20 mg of crude enzyme or 500 µL of semipurified version), PLP (0.5 mM), 2ꢀpropylamine (1
M), 24 h at 45 ºC and 700 rpm.
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b Conversion values were determined by GC analysis after isolation of products by extraction
from basified media (See experimental section).
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c Enantiomeric excess values were measured by GC or HPLC (See Supporting Information) on a
chiral phase.
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Good conversions and excellent enantiomeric excesses were achieved with the ArRmut11ꢀTA
crude preparation for the formation of (R)ꢀamines 2d and 2c (entries 1 and 3). Next, a
semipurified preparation of the enzyme was tested aiming for an improvement of the conversion
values. Indeed, very high conversions were detected (84ꢀ98%) without affecting the selectivity of
the reaction. As a proof of concept, scale up of the process was performed using 50 mg of
substrate 1c, recovering the enantiopure amine (R)ꢀ2c in 68% isolated yield after an extraction
step. The biocatalytic system was also successfully used for the preparation of amines bearing
short alkyl chains (2a-b, R=Me, Et) reaching completion within 24 h (entries 6 and 5,
respectively).
In order to get a deeper insight about the structureꢀactivity relationship for TAs, additional
enzymatic studies were performed by considering structural parameters such as: (i) chain length
of the alkyl group, (ii) presence of nitrogen and chlorine atoms in/on the aromatic ring; (iii) the
position of substitution of the acyl group on the pyridine ring.
The transamination of ketones 1aꢀd was followed over time catalyzed by the (S)ꢀselective VfꢀTA
(Figure 2), which displayed a good activity even for the butanone 1c (72% conversion, entry 9 in
Table 1). The ethanone 1a (R=Me) was converted very quickly under the reaction conditions
employed reaching 74% conversion in 1 hour, and almost complete consumption of the ketone
after 7 hours. On the other hand, propanone 1b (R=Et) was transformed more slowly. Reaction
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progress was similar for substrates 1b and 1c at 1 and 3 hours (5 vs. 3% and 14 vs. 12%
conversion, respectively), the difference between these two ketones became more significant
after 5 hours (34 vs. 18% conversion) and 7 hours (44 vs. 26% conversion). In line with the
conversions achieved before (entry 13 in Table 1), the pentanone 1d (R=Bu) only reached 4% of
conversion at 7 hours, which clearly illustrates the decrease of reactivity for substrates bearing
mediumꢀlarge aliphatic chains.
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t (h)
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Et
nPr
nBu
Figure 2. Graphical representation of VfꢀTA reactivity as function of the chain size of ketones
1aꢀd after 1, 3, 5 and 7 hours.
Following our aim to establish a structureꢀactivity relationship for TAs, we focused next on other
structural aspects related to the aromatic ring, including the presence of heteroatoms (nitrogen
and chlorine) and the position of the acetyl group on the pyridine ring. Therefore, ethanone 1a
was chosen as model substrate and methyl ketones 1e-i were included in this study (Figure 1).
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Amination experiments were carried out employing transaminases of complementary
stereopreference, under previously optimized reaction conditions (temperature, substrate
concentrations, amine donors, recycling systems, reaction time, pH and type of buffer) for an
accurate comparison between substrates (Scheme 1).
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Scheme 1. Enzymatic transamination of 1a and 1e-i using alanine or 2ꢀpropylamine as amino
donor.
The amination of 1a,eꢀi was performed using various TAs (10 mg E. coli/TA, 1 h) at 30 ºC for
Vf, Cv, ArR, while 45 ºC was selected for the more thermostable ArRmut11ꢀTA used as
semipurified preparation (500 ꢁL, 8 U, Figure 3). From the results obtained some general facts
concerning their reactivity can be concluded. A decrease in conversion values was detected when
comparing the model substrate 1ꢀ(4ꢀchloropyridinꢀ2ꢀyl)ethanꢀ1ꢀone (1a, conversion up to 87%)
with 2ꢀacetylpyridine (1f, conversion up to 50%), which lacks the chlorine atom on the Cꢀ4
position. This drop in the conversion became more significant when investigating aromatic
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derivatives such as 3’ꢀchloroacetophenone (1e, conversion up to 17%) and acetophenone (1g),
which reached the highest conversion (21%) using the (R)ꢀselective ArRꢀTA. A similar tendency
was observed with 3ꢀacetylpyridine (1h, conversion up to 19%), while no reaction was found
with semipurified ArRmut11ꢀTA. Finally, the amination of 4ꢀacetylpyridine (1i) was studied,
finding a similar trend with respect to 2ꢀacetylpyridine (1f), reaching the best conversion (42%
and 57%) with the Cvꢀ and ArRꢀTAs, respectively. In any case, there are major differences
compared to 1ꢀ(4ꢀchloropyridinꢀ2ꢀyl)ethanꢀ1ꢀone (1a) and in fact conversions for 4ꢀ
acetylpyridine (1i) are just comparable to those for 2ꢀacetylpyridine (1f).
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Figure 3. Reactivity comparison for the amination of the methyl ketones 1a,e-i with (a) Cv, (b)
Vf and (c) ArRꢀTAs (30 ºC, 1 h), and (d) ArRmut11ꢀTA (45 ºC, 1 h).
Interestingly, an analysis of the spectroscopical data revealed a positive correlation between
conversions and vibrational frequencies of the carbonyl band in the infrared spectra for ketones
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1a,eꢀi, and therefore with the C=O bond length.25 The conversions achieved with CvꢀTA for
various substrates presented in Figure 4 shows this correlation with the electronic effect of the
chlorine atom and the pyridine nitrogen, the last being the predominant one. The pyridine
derivatives 1a, 1f and 1i bearing the acetyl group at Cꢀ2 or Cꢀ4 positions, in which the electron
withdrawing effect of the nitrogen is stronger, displayed frequencies higher than 1690 cmꢀ1,
those compounds leading to higher conversion values. Among them, the pyridine 1a is the more
reactive one as it has the additional electron withdrawing effect of the chlorine. On the contrary,
the larger C=O bonds fit to the acetophenone derivatives 1g and 1e, and to the pyridine 1h, in
which the conversion of the reaction reached a maximum 10% after 1 hour. The acetophenone 1e
with a chlorine atom and the pyridine 1h displayed a slight higher reactivity than acetophenone
(1g), which does not present the electron withdrawing effects of 1e and 1h.
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Figure 4. Graphical representation of vibrational frequencies of the carbonyl IR band (bars)
towards conversion values (line) in the enzymeꢀcatalyzed transamination of 1a and 1e-i using
CvꢀTA (700 rpm, 30 ºC and 1 hour).
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Finally, the high conversions observed for 1a within 1 hour using semipurified ArRmut11ꢀTA
(85% conversion, Figure 3d) might indicate a higher thermodynamic stability of the product
amine 2a, which may facilitate a shift of the equilibrium towards its formation. In this case, less
amount of the amine donor (2ꢀpropylamine) would be required for a practical biotransformation.
Accordingly, the concentration of 2ꢀpropylamine was reduced from 1 M to 100 mM (Table 3). In
addition, the same experiment was made employing 3’ꢀchloroacetophenone (1e).
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Table 3. Asymmetric enzymeꢀcatalyzed transamination of 1a and 1e using semipurified
ArRmut11ꢀTA.a
[2ꢀpropylamine]
(M)
t
c
Entry
X
(h) (%)b
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N (1a)
N (1a)
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N (1a)
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CH (1e)
CH (1e)
CH (1e)
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0.1
a Reaction conditions: phosphate buffer (100 mM, pH 7), ketone 1a and 1e (50 mM), ArRmut11ꢀ
TA (500 µL), PLP (0.5 mM), 2ꢀpropylamine (0.1ꢀ1 M), 1ꢀ24 h at 45 ºC and 700 rpm.
b Conversion values were determined by GC analysis after isolation of products by extraction
from basified media (See experimental section).
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Differences between substrate 1a and 1e are for instance the higher conversion for 1a (85% vs.
39%) after 1 hour at 1 M of 2ꢀpropylamine (entries 1 and 4), something already discussed.
Furthermore, at 100 mM 2ꢀpropylamine concentration, ketone 1e reached 24% conversion after
24 h (entry 6), while the reaction with 1a achieved 76% (entry 3). The values obtained for 1e are
in agreement with the fact that the deamination reaction is favored for acetophenone derivatives
compared to the reverse amination reaction.26 On the other hand, the excellent results obtained
for the amination of 1a even with low excesses of amine donor are consistent with
thermodynamic preference for the formation of amine (R)ꢀ2a. This inclination was analyzed
through different perspectives. The first attempt to explain it focuses on an intramolecular
hydrogen bond between the amino group and the pyridine nitrogen which would provide an
additional stabilization to the amine and therefore drive the reaction to the amination direction.27
Computational studies28 on the model substrate 2a confirmed the presence of the hydrogen bond
(Figure S4, for further details see the Supporting Information).
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Alternatively, energy calculations supported the thermodynamic stabilization by the difference
observed in the ꢂG of the reaction for the amination of both ketones 1a and 1e, the pyridine
transformation being 4.3 kJ/mol more favorable (see Table S1 and Figure S3).
CONCLUSIONS
The asymmetric synthesis of 1ꢀ(4ꢀchloropyridinꢀ2ꢀyl)alkanꢀ1ꢀamines has been performed
through transaminaseꢀcatalyzed reactions starting from their corresponding prochiral ketones.
Hence, ethanꢀ and propanone derivatives were successfully transformed with a variety of (R)ꢀ
and (S)ꢀselective transaminases. After finding adequate reaction conditions, both amine
antipodes have been obtained with excellent conversion values and high to excellent isolated
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yields in preparative biotransformations, recovering the desired amines with high purity after a
simple extraction protocol. To achieve full stereoselectivity and high conversion for the most
hindered pyridine substrates, the use of an evolved variant from (R)ꢀArthrobacter species was
required. The 1ꢀ(4ꢀchloropyridineꢀ2ꢀyl)ethanꢀ1ꢀone 1a was transformed very efficiently even at
reduced concentration of amine donor most likely due to thermodynamic reasons as a result of
the formation of an internal hydrogen bond. Different structural parameters including pyridine
and acetophenone derivatives, some bearing chlorine atoms at the aromatic ring, and with the
alkanone rest at variable positions in the aromatic ring were examined, which enabled to identify
ideal structural motives for the substrate to be converted by the enzymes investigated.
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EXPERIMENTAL SECTION
1ꢀ(4ꢀChloropyridineꢀ2ꢀyl)alkanꢀ1ꢀones 1aꢀd and racemic amines 2aꢀd were prepared according
to previously reported procedures from 4ꢀchloropyridine Nꢀoxide.16 Chemical reagents and
solvents were used as received from commercial sources.
TLC analyses were carried out with precoated aluminium sheets (TLC silica gel 60 F254,
Merck) with detection by UV or staining with potassium permanganate or ninhydrin. Preparative
chromatographic separations were performed by flash chromatography on Merck silica gel (230ꢀ
400 mesh). Optical rotation was measured at 20 °C with a PerkinꢀElmer 241 polarimeter at the
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sodium Dꢀline. H and C NMR spectra were recorded at 20 °C with a Bruker 300 MHz unit;
chemical shifts are given in ppm relative to the resonance of the solvent. Analytical data and
retention times for the measurement of conversion and enantiomeric excess values are given in
the Supporting Information section. The purity of the resulting optically active amines obtained
through transaminaseꢀcatalyzed processes was determined by GC and NMR analyses.
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Formate dehydrogenase was purchased from Evocatal GmbH (lyophilized powder 4.1 U/mg) and
NAD+ free acid were purchased from Codexis Inc. Lyophilized E. coli cells containing
overexpressed transaminases were prepared as previously reported.20a,29 Lyophilized cells applied
as catalyst displayed the following activities (determined for the deamination of αꢀ
methylbenzylamine with pyruvate): ArR: 2.2 U/mg, ArRmut11: 0.3 U/mg, At: 0.5 U/mg, Vf: 1.3
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U/mg, Cv: 2.1 U/mg, purified Lꢀalanine dehydrogenase: 1.1 U/µL. Small scale reactions were
shaken in 2 mL thermo shaker Eppendorf® Comfort. Preparative scale reactions were performed
in an Infors Unitron shaker.
Heat purification protocol for ArRmut11
Lyophilized E. coli cells containing overexpressed enzyme (2 g) were suspended in a 100 mM
phosphate buffer pH 7 (20 mL) and the suspension was sonicated with the following protocol: 1
s pulse; 4 s pause; 2.5 min; 40% amplitude. The mixture was centrifuged for 20 min at 45000 g
and 4 ºC and the supernatant was treated for 30 min at 50 ºC. After this time the suspension was
centrifuged again for 20 min at 45000 g and 4 ºC and the supernatant containing the active
enzyme was stored at ꢀ18 ºC. Protein concentration: 26.7 mg/mL according to Bradford’s assay.
Activity (determined for the deamination of αꢀmethylbenzylamine with pyruvate): 16 U/mL.
Typical procedure for asymmetric transamination of ketones 1a-d employing alanine
dehydrogenase as regeneration system
To a suspension of ketones 1a-d (0.05 mmol, 50 mM) in a 100 mM phosphate buffer pH 7 (440
µL) were successively added ammonium formate (100 µL of 1.5 M solution in a 100 mM
phosphate buffer pH 7; final concentration 150 mM), alanine (250 µL of 1 M solution in
phosphate buffer 100 mM pH 7; final concentration 250 mM), NAD+ (100 µL of 10 mM solution
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in a 100 mM phosphate buffer pH 7; final concentration 1 mM), PLP (100 µL of 10 mM solution
in a 100 mM phosphate buffer pH 7; final concentration 1 mM), lyophilized cells of E. coli
containing overexpressed transaminases (20 mg), formate dehydrogenase (FDH, 2.6 mg, 11 U)
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and alanine dehydrogenase (AlaDH, 10 µL, 11 U).
Dꢀ or Lꢀ alanine were used as amine donor
depending on the (R) or (S)ꢀtransaminase selectivity, respectively.
The resulting mixture was shaken at 30 °C and 700 rpm for 24 h. After this time the reaction was
quenched by adding aqueous NaOH 4 M (400 µL), extracted with ethyl acetate (3 x 500 µL), and
organic phases were combined and dried with Na2SO4. Reaction crude was analyzed through GC
to determine conversion values and, after an in situ derivatization, enantiomeric excess.
Typical experimental procedure for asymmetric transamination of ketones 3a-d catalyzed
by semipurified ArRmut11-TA
Ketones 1a-d (0.05 mmol, 50 mM) were suspended in a 100 mM phosphate buffer pH 7 (500
µL) containing PLP (1 mM; final concentration 0.5 mM) and isopropylamine (2 M; final
concentration 1 M). Then, semipurified ArRmut11 (500 µL) was added and the mixture was
shaken at 45 °C and 700 rpm for 24 h. The reaction was quenched by adding aqueous NaOH 4 M
(400 µL), extracted with ethyl acetate (3 x 500 µL), and organic phases were combined and dried
with Na2SO4. Reaction crude was analyzed through GC to determine conversion values and,
after an in situ derivatization, enantiomeric excess.
General procedure for preparative reductive amination of 1a and 1b employing alanine
dehydrogenase as regeneration system
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To a suspension of ketones 1a or 1b (0.32 mmol, 50 mM) in a 100 mM phosphate buffer pH 7
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(2.8 mL) were successively added ammonium formate (643 µL of 1.5 M solution in a 100 mM
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phosphate buffer pH 7; final concentration 150 mM), Lꢀalanine (1.6 mL of 1 M solution in a 100
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mM phosphate buffer pH 7; final concentration 250 mM), NAD+ (643 µL of 10 mM solution in a
100 mM phosphate buffer pH 7; final concentration 1 mM), PLP (643 µL of 10 mM solution in a
100 mM phosphate buffer pH 7; final concentration 1 mM), lyophilized cells of E. coli
containing overexpressed transaminases (32 mg for 1a, 96 mg for 1b), formate dehydrogenase
(16.4 mg, 69 U) and AlaDH (63 µL, 69 U). The resulting mixture was shaken at 30 °C and 120
rpm for 24 h. The reaction was quenched by adding aqueous NaOH 4 M until pH ≈ 10 and
extracted with ethyl acetate (3 x 10 mL). The organic phases were combined, dried with Na2SO4
and the solvent was removed under reduced pressure. Enantiopure amines (S)ꢀ2a and (S)ꢀ2b
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were isolated as colorless liquids in 82ꢀ93% yield. For (S)ꢀ2a in >99% ee: [α]D = ꢀ23.4 (c 0.5,
CHCl3); lit. [α]D20 = ꢀ20.4 (c 0.5, CHCl3).16b For (S)ꢀ2b in >99% ee: [α]D20 = ꢀ5.8 (c 0.6, CHCl3);
lit. [α]D20 = ꢀ5.1 (c 0.6, CHCl3).16b
Preparative reductive amination of 1c catalyzed by semipurified ArRmut11-TA
In a Falcon tube, ketone 1c (50 mg, 0.27 mmol, 50 mM) was suspended in a 100 mM phosphate
buffer pH 7 (2.7 mL) containing PLP (1 mM; final concentration 0.5 mM) and isopropylamine (2
M; final concentration 1 M). Then, semipurified ArRmut11ꢀTA (2.7 mL) was added and the
mixture was shaken at 45 °C and 120 rpm for 24 h. The reaction was quenched by adding
aqueous NaOH 4 M until pH ≈ 10 and extracted with ethyl acetate (3 x 10 mL). The organic
phases were combined, dried with Na2SO4 and the solvent was removed under reduced pressure.
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Enantiopure amine (R)ꢀ2c was isolated as colorless liquid in 68% yield. For (R)ꢀ2c in >99% ee:
[α]D20 = +10.8 (c 0.5, CHCl3); lit. [α]D20 = ꢀ9.1 (c 0.5, CHCl3) for (S)ꢀ2c in 98% ee].16b
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ASSOCIATED CONTENT
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Supporting Information.
Information about the transaminases employed in this study, details of the computational
calculations, the chemical synthesis of the starting and reference materials including the NMR
data of the products, the specific data of the conversions depicted in Figures 2 and 3, the
analytics including the chromatograms, as well as the NMR spectra for described organic
compounds. This material is available free of charge via the Internet at http://pubs.acs.org
AUTHOR INFORMATION
Corresponding Author
* Eꢀmail: vicgotfer@uniovi.es
* Eꢀmail: wolfgang.kroutil@uniꢀgraz.at
* Eꢀmail: bbusto@ucm.es
Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval
to the final version of the manuscript.
ACKNOWLEDGMENT
Financial support of this work by the Spanish Ministerio de Economía y Competitividad (CTQꢀ
2013ꢀ44153ꢀP project) and the Asturian Regional Government (FCꢀ15ꢀGRUPIN14ꢀ002) are
gratefully acknowledged. M.L.ꢀI. and D.G.ꢀM. thank FICYT for a predoctoral fellowship. E. B.
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thanks the European Union for a Marie Curie IEF grant (Project IEFꢀ2011ꢀBIOCASCADE). We
thank R. SoriaꢀMartínez for the assistance in computational studies.
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ABBREVIATIONS
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TA, transaminase; E. coli, Escherichia coli; Vf, Vibrio fluvialis; Cv, Chromobacterium
violaceum; ArR, Arthorbacter species; At, Aspergillus terreus; AlaDH, alanine dehydrogenase;
FDH, formate dehydrogenase; TLC, thin layer chromatography.
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