Catalytic Promiscuity of Transaminases: Preparation of Enantioenriched β-Fluoroamines by Formal Tandem Hydrodefluorination/Deamination

Article abstract of DOI:10.1002/anie.201510554

Transaminases are valuable enzymes for industrial biocatalysis and enable the preparation of optically pure amines. For these transformations they require either an amine donor (amination of ketones) or an amine acceptor (deamination of racemic amines). Herein transaminases are shown to react with aromatic β-fluoroamines, thus leading to simultaneous enantioselective dehalogenation and deamination to form the corresponding acetophenone derivatives in the absence of an amine acceptor. A series of racemic β-fluoroamines was resolved in a kinetic resolution by tandem hydrodefluorination/deamination, thus giving the corresponding amines with up to greater than 99 % ee. This protocol is the first example of exploiting the catalytic promiscuity of transaminases as a tool for novel transformations.

Full text of DOI:10.1002/anie.201510554

Angewandte
Communications
Chemie
International Edition: DOI: 10.1002/anie.201510554
German Edition: DOI: 10.1002/ange.201510554
Biocatalysis
Catalytic Promiscuity of Transaminases: Preparation of
Enantioenriched b-Fluoroamines by Formal Tandem
Hydrodefluorination/Deamination
Aníbal Cuetos+, Marina García-Ramos+, Eva-Maria Fischereder, Alba Díaz-Rodríguez,
Gideon Grogan, Vicente Gotor,* Wolfgang Kroutil,* and Ivµn Lavandera*
Abstract: Transaminases are valuable enzymes for industrial
biocatalysis and enable the preparation of optically pure
amines. For these transformations they require either an amine
donor (amination of ketones) or an amine acceptor (deami-
nation of racemic amines). Herein transaminases are shown to
react with aromatic b-fluoroamines, thus leading to simulta-
neous enantioselective dehalogenation and deamination to
form the corresponding acetophenone derivatives in the
absence of an amine acceptor. A series of racemic b-fluoro-
amines was resolved in a kinetic resolution by tandem hydro-
defluorination/deamination, thus giving the corresponding
amines with up to greater than 99% ee. This protocol is the
first example of exploiting the catalytic promiscuity of trans-
aminases as a tool for novel transformations.
I
n recent years, many enzymes have been shown to efficiently
catalyze transformations that appear far removed from their
natural activity. This catalytic promiscuity[1] can be useful for
synthetic purposes by broadening the applicability of biocat-
alysts. While many examples have recently been described for
hydrolases,[2] the application of these unconventional pro-
cesses for other enzyme classes has not been fully devel-
oped.[3] Among biocatalysts that have demonstrated broad
applicability, transaminases (TAs) can be highlighted, as they
perform the amination of an amine acceptor (ketone or
Scheme 1. Methodologies to obtain enantiopure amines using TAs by:
a) amination of a carbonyl compound using an amine donor, b) deam-
ination of a racemic amine employing an amine acceptor, and c) novel
tandem hydrodefluorination/deamination kinetic resolution of racemic
b-fluoroamines.
aldehyde) using an amine donor (e.g., alanine or isopropyl-
amine, Scheme 1a), mediated by the cofactor pyridoxal 5’-
phosphate (PLP).[4,5] Interestingly, transaminases can also
catalyze the reverse reaction, thus achieving the kinetic
resolution of racemic amines by amination of an amine
acceptor (e.g., pyruvate or acetone, Scheme 1b). This process
is hampered by the disadvantage of a maximum of 50% yield,
but it is thermodynamically favored in comparison to the
amination route.[4,6]
[*] Dr. A. Cuetos,[+] Prof. G. Grogan
York Structural Biology Laboratory, Department of Chemistry
University of York, Heslington, York, YO10 5DD (UK)
M. García-Ramos,[+] Dr. A. Díaz-Rodríguez, Prof. V. Gotor,
Dr. I. Lavandera
Departamento de Química Orgµnica e Inorgµnica
University of Oviedo
Instituto Universitario de Biotecnología de Asturias
C/Juliµn Clavería 8, 33006 Oviedo (Spain)
E-mail: vgs@uniovi.es
The introduction of fluorine atom(s) to an organic
derivative has a significant influence on, for example, their
physicochemical, conformational, and metabolic properties.[7]
lavanderaivan@uniovi.es
E.-M. Fischereder, Prof. W. Kroutil
À
À
The cleavage of a C F into a C H bond, hydrodefluorina-
tion,[8] has been performed in the case of aromatic derivatives
using transition-metal complexes under harsh reaction con-
ditions. For aliphatic fluorinated compounds, however, alter-
Department of Chemistry, Organic and Bioorganic Chemistry
University of Graz, Heinrichstrasse 28, 8010 Graz (Austria)
E-mail: Wolfgang.Kroutil@uni-graz.at
Dr. A. Díaz-Rodríguez
Current address: Medicines Research Centre, GlaxoSmithKline R&D
Ltd, Gunnels Wood Road, Stevenage, Hertfordshire, SG1 2NY (UK)
natives still remain to be found,[9] partly because of the
inertness of C F bonds. b-Fluoroamines are potent inhib-
itors of PLP-dependent enzymes such as aminotransferases
and decarboxylases.[11] These proteins are crucial in living
organisms and are therefore commonly envisaged as thera-
peutic targets.[12] Silverman and co-workers demonstrated
[10]
À
[+] These authors contributed equally to this work.
Supporting information and ORCID(s) from the author(s) for this
article are available on the WWW under http://dx.doi.org/10.1002/
anie.201510554.
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that 4-amino-5-fluoropentanoic acid was an irreversible
inhibitor of g-aminobutyrate aminotransferase (GABA-
AT).[13]
Herein we show that the promiscuous reactivity of
transaminases can be applied to obtain a series of enantiopure
b-fluoroamines by an unprecedented formal tandem hydro-
defluorination/deamination kinetic resolution of racemic
b-fluoroamines. The reaction proceeds in the absence of an
amine acceptor and under simple and mild reaction con-
ditions in aqueous medium (Scheme 1c). Thus, it was
exploited that the inhibition of certain transaminases by
b-fluoroamines is negligible under the reaction conditions
employed.
To study the reactivity of TAs with fluorinated com-
pounds,[5c,14] we focused on the transformation of 2-fluoro-1-
phenylethan-1-amine (1a) with acetone as the amine acceptor
to obtain the remaining enantioenriched amine 1a, together
with transformed 2-fluoro-1-phenylethan-1-one (2a) under
typical kinetic resolution conditions as generalized in Sche-
me 1b. Several commercially available TAs did not afford this
product but acetophenone (3a) was produced (data not
shown). Furthermore, similar conversions were attained in
the absence of the amine acceptor (Table 1), with only
Scheme 2. Proposed dehalogenation (route a) and inhibition (route b)
mechanisms as catalyzed by TAs employing the b-fluoroamine 1a.
mechanistic studies,[15,16] we propose a dual mechanism for
defluorination and inhibition (Scheme 2). As a first step, PLP,
which is linked to the catalytic lysine of the TA,[17] is
transferred to the reacting 1a, thus forming the external
aldimine intermediate. Then, it rearranges into the ketimine
intermediate, which is converted into the dehalogenated
aldimine I as a result of the fluorine atom serving as a leaving
group. Then, the catalytic lysine can attack the imine bond to
generate the internal aldimine together with an enamine
derivative. At this stage, two different pathways can occur. If
the enamine quickly diffuses into the reaction medium
(route a), it will be hydrolyzed, thus affording the defluori-
nated ketone (3a), and the enzyme will be able to start a new
catalytic cycle as it is not inactivated. However, if the enamine
remains in the active site of the enzyme for a sufficient time
(route b), it will attack the internal aldimine to provide an
imine which, after hydrolysis, will furnish a final covalent
adduct (II), thus inactivating the transaminase irreversibly.
Moreover, the proposed mechanism explains why an amine
acceptor is not necessary, that is, the transfer of an amine
group does not occur. Instead, it involves the hydrolysis of an
imine bond to release a molecule of ammonia.
Then, it was investigated if this reaction could be
performed enantioselectively. Ideally, a kinetic resolution by
tandem hydrodefluorination/deamination could be achieved
(Scheme 1c). A series of transaminases overexpressed in
E. coli was employed: the S-selective TA from Chromobac-
terium violaceum (CV-TA),[18] S- and R-selective TAs from
Arthrobacter sp. (ArS-TA[19] and ArR-TA,[20] respectively),
and a variant of the ArR-TA with mutations in 27 positions
(ArRmut11-TA),[5c] which is also R-selective. These TAs have
displayed excellent activities for aromatic substrates, thus
providing the corresponding amines with high selectivities.[21]
Racemic aromatic b-fluoroamines (1a–f; Table 2) were
either purchased or synthesized as described in the Support-
ing Information.[22] We used lyophilized preparations of
Table 1: Enzymatic transformations on b-fluoroamine 1a.[a]
Entry
Enzymatic preparation
Conversion [%][b]
1a
2a
3a
1
2
3
4
5
6
7
–
>99
41
53
<1
<1
<1
<1
<1
<1
<1
<1
59
47
ATA-231[c]
TA-P1-F03[c]
TA-P1-G06[c]
BSA
28
72
>99
>99
>99
<1
<1
<1
CAL-B
Lyo. E. coli
[a] For reaction conditions, see the Supporting Information. [b] Mea-
sured by GC analysis. [c] Transaminase commercially available from
Codexis company. DMSO=dimethylsulfoxide.
acetophenone detected as a product (entries 2–4). Since the
substrate was stable in the blank reaction (entry 1), and in the
presence of other enzymatic or protein preparations such as
bovine serine albumin (BSA), lipase B from Candida
antarctica (CAL-B), and lyophilized cells of E. coli
(entries 5–7), it was clear that this amine was transformed
by the tested TAs, thus resulting in both hydrodefluorination
and deamination reactions.
Further experiments also demonstrated that this trans-
formation may occur along with enzymatic deactivation
because in the presence of fluorinated substrates, selected
TAs lost their activity, at least partially (see the Supporting
Information for more details). Our next objective was the
proposal of a plausible mechanism which could explain these
results. Based on the work by Silverman and co-workers on
GABA-AT inhibitors,[13] and with reference to his previous
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Table 2: Tandem hydrodefluorination/deamination kinetic resolution of
the b-fluoroamines 1a–f.[a]
Entry Substrate
CV-TA
Conv. 3 [%][b] ee 1 [%][c,d]
ArR-TA
Conv. 3 [%][b] ee 1 [%][c,d]
Figure 1. Active site at the dimer interface of ArRmut11-TA inhibited by
complexation with 1d and showing PLP covalently bonded to both
K188 and the residual ligand, thus representing complex II as shown
in Scheme 2. The PLP adduct is shown in ball-and-stick format.
Electron density maps correspond to the FoÀFc omit map and the
2FoÀFc map in black and gray meshing, respectively, and at the
corresponding levels of 3s and 1s. The omit map is that obtained
prior to modelling and refinement of the ligand.
1
2
3
4
5
6
1a
1b
1c
1d
1e
1 f
53
50
52
51
60
55
99 (S)
99 (S)
>99 (S)
>99 (S)
>99 (S)
99 (S)
22
67
51
28
54
55
23 (R)
>99 (R)
>99 (R)
40 (R)
>99 (R)
>99 (R)
[a] For reaction conditions, see the Supporting Information. [b] Measured
by GC analysis. [c] Measured by GC analysis using a chiral stationary
phase. [d] Change in Cahn–Ingold–Prelog (CIP) priority.
conditions. This process was demonstrated for the kinetic
resolution of racemic b-fluoroamines to obtain the non-
converted amines with excellent ee values. Remarkably no
amine acceptor, such as acetone, was necessary. Depending
on the biocatalyst of choice, both enantiomers could be
obtained. Hydrodefluorination is an interesting reaction that
has been studied during recent years.[8] The use of metal
E. coli cells because of their easy handling and high stabili-
ty.[21c,d] After screening the enzymes (see the Supporting
Information), we obtained the best results for CV-TA and
ArR-TA, thus resulting in excellent resolutions for most of
the substrates. Therefore, different derivatives, including
those with either electron-donating or electron-withdrawing
groups at the phenyl moiety, were obtained with high
enantiomeric excess. Moreover, depending on the transami-
nase used, both enantiomers were attained either in optically
pure form or at least optically enriched. Thus, CV-TA reacted
preferentially with the R antipode and ArR-TA with the
S enantiomer.
À
complexes has allowed the cleavage of aromatic C F bonds,
but methods to cleave aliphatic bonds are not accessible. The
proposed biocatalytic concept expands the toolkit for asym-
metric synthesis complementing previous chemical processes,
and illustrates the potential of catalytic promiscuity as a tool
for designing unprecedented reactions.
Finally, we applied this transformation concept on a prep-
arative scale. Thus, 100 mg each of the racemic amines 1a and
1d were resolved using CV-TA into the corresponding
S-configured derivatives (> 99% ee) within 32 h, giving 75–
80% isolated yield (referring to the single enantiomer) after
a simple extraction protocol (see the Supporting Informa-
tion).
Acknowledgments
A.C. thanks the Principado de Asturias for his postdoctoral
fellowship Clarín. E.-M.F. acknowledges the European
Unionꢀs 7th framework program FP7/2007-2013 under grant
agreement #289646 (KyroBio). Financial support from
MICINN (Project CTQ2013-44153-P) is gratefully acknowl-
edged.
To obtain evidence of the mechanism of inhibition
proposed in Scheme 2, the X-ray crystal structure of
ArRmut11-TA was obtained from a protein that had been
pre-incubated with 1d. Details of the structure can be found
in the Supporting Information. After building and refinement
of the protein and water atoms, clear electron density was
observed in the omit map at the interface of six dimer pairs for
PLP (corresponding to twelve PLP sites). Two regions of
continuous electron density projected from the electrophilic
carbon atom of PLP (Figure 1): the first region connected the
PLP to the side-chain of Lys188, and the second extended
from the PLP into the active site. This density was successfully
modelled as the adduct resulting from inhibition of the
enzyme by 1d, thus representing the structure II in Scheme 2.
In summary, we have reported a novel enantioselective
transformation based on the promiscuous activity of trans-
aminases, namely a tandem hydrodefluorination/deamination
reaction in aqueous medium under very mild reaction
Keywords: amines · biocatalysis · enzymes · fluorine ·
kinetic resolution
How to cite: Angew. Chem. Int. Ed. 2016, 55, 3144–3147
Angew. Chem. 2016, 128, 3196–3199
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Received: November 13, 2015
Published online: February 2, 2016
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