Angewandte
Communications
Chemie
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
Angew. Chem. Int. Ed. 2016, 55, 3144 –3147
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
3145