InnovatIve tools In organIc / organometallIc chemIstry
CHIMIA 2020, 74, No. 11 891
tary technologies that have enabled the intensification of indus- PfTA, however the ee was again low in both cases, at 66% and
trially relevant biocatalytic processes. In particular, the transfer 20% respectively. Interestingly, the use of compounds with a me-
of enzyme-based processes into continuous flow is currently thyl group at the 4-position (3a, 4a) gave significantly higher ee
the focus of growing attention.[9] While flow biocatalysis can be values, which suggests that the methyl group may help orientate
performed using immobilised whole-cells or cell-free enzymes, the molecule in the active site in such a way that the formation of
some excellent examples of cell-free reactions in continuous flow the (S)-enantiomer is favoured. To further probe this hypothesis,
have appeared in the literature in recent years.[10–12] For example, the crystal structure of HeWT
(PDB: 6GWI) was used to dock
our group were the first to report on the use of ω-transaminase selected compounds (1a and 4a) from our panel into its active
(ω-TA) in continuous flow for the synthesis of a panel of aromatic
site.[14]
amines.[13] The enzyme, which was covalently immobilised onto
a commercially available metal-derivatised epoxy resin proved to Docking Analysis of Ketones 1a and 4a in the Active
be highly stable in a packed bed reactor (PBR) at high flow rates. Site of HeWT
An in-line purification strategy was used, in which basification
HeWT is a homodimer, with an active site positioned at the
of the reaction stream and extraction with ethyl acetate, afforded interface of the monomers and a substrate binding region that is
the desired amine product. Traces of the unreacted aldehyde sub-
composed of a small and a large binding pocket. This character-
strate in the organic phase were effectively trapped using a ben- istic architecture typically limits the substrate scope of wild-type
zylamine functionalised resin. In addition excellent conversions ω-TAs to ketones bearing at least one small substituent, not ex-
and reaction times were achieved compared to those obtained in ceeding that of a methyl group. Our molecular docking studies
batch. Bearing this information in mind, we report herein on an have confirmed that small cyclic ketones are particularly challeng-
enzyme-based system for the synthesis of a series of small cyclic ing for HeWT
as they evidently do not display the well-defined
molecules that are inherently difficult to produce using tradition- small and large substituents that are typically a prerequisite for the
al chemical methods. Furthermore, the application of flow bio- transaminase biocatalytic mechanism.[19] Fig. 1(left) shows the
catalysis in this case effectively demonstrates how biocatalytic docking of tetrahydrofuran-3-one (1a) in the active site of HeWT.
processes can easily be scaled-up, allowing for the direct, one-pot In this case, the tetrahydrofuranyl ring oxygen appears to form
conversion of inexpensive and readily available compounds to a hydrogen bond with the side chain of W56. This interaction
high-value molecules, while also maintaining a very low envi- anchors the substrate in the active site such that the reductive am-
ronmental impact.
ination of the carbonyl is favoured. In the case of 1-methyl-3-pip-
eridone (4a) the docking displaying the most favourable score
places the substrate in a position that is further away from the PLP
cofactor (3.23Å, Fig. 1(right)) when compared to 1a (2.70Å, Fig.
1(left)). Interestingly, the substrate is positioned in the vicinity of
Results and Discussion
Substrate Screening of Structurally Different ω-TAs
We started our studies on the sustainable formation of small the large binding pocket. The high stereoselectivity achieved by
cyclic amines by selecting two structurally different ω-TAs from
our toolbox of enzymes, a dimeric ω-TA from Halomonas elonga- mode favours the formation of the (S)-enantiomer.
ta HeWT and a tetrameric ω-TA from Pseudomonas fluorescens
PfTA.[14,15]
The previously reported screening for HeWT with this Continuous Flow Synthesis of Small Cyclic Amines
set of substrates[14] is given in Table 1 for comparison. 1a was
The promising results obtained with HeWT in batch mode led
HeWT in the case of 4b, suggests that this orientation and binding
first employed on a 10 mM scale using 1 equivalent of (S)-ω- us to explore the possibility of carrying out continuous reactions
methylbenzylamine, (S)-MBA, as the amine donor. Remarkably, as an alternative strategy for the scale-up synthesis of the target
complete conversion to the corresponding amine 1b was achieved amines. Compared to batch processes, performing reactions in
when HeWT
was employed as the biocatalyst, whereas PfTA continuous flow mode offer several benefits including better pa-
reached a final conversion of ∼48.6%, possibly due to thermody- rameter control, reduced reaction times, improved yields and re-
namic limitations. Initial efforts to shift the equilibrium towards cycling of unreacted reagents.[20] Specifically with respect to bio-
product formation, by increasing the concentration of the amine catalytic transformations, continuous flow can also offer addition-
donor to 20 mM were successful, resulting in a final conversion al benefits such as reduced enzyme inhibition through continuous
of 66.5% (Supplementary Information Table 3). However, fur-
ther improvements could not be achieved as precipitation of the therefore covalently immobilized onto a commercially available
product removal and facile downstream processing.[21] HeWT was
biocatalyst was observed at higher concentrations of (S)-MBA epoxy support as previously reported.[13] With an enzyme loading
(concentrations >20 mM). In terms of stereoselectivity, (S)-1b of 5mg/g resin, the resulting biocatalyst displayed an activity of
was preferred in both cases, however the enantiomeric excess 38 U/g resin. A continuous flow reactor, packed with immobi-
was low for both TAs (HeWT, 70% (S); PfTA, 42% (S)). Given
lized-HeWT, was subsequently set up and optimized for the prepa-
the widespread occurrence of amino-substituted tetrahydrothio- ration of 1b. The substrate loading in this case was increased to 50
phenes, pyrrolidines and piperidines in pharmaceutically active mM, while the amine donor concentration was kept constant at 1
compounds, the substrate scope of HeWT
probed with compounds 2a–4a.[16,17] Using the same reaction with a residence time of just 5 min. Furthermore, a downstream
conditions as outlined above, HeWT was able to transform the purification step to remove the acetophenone by-product, fol-
selected compounds into the corresponding amines (2b–4b) with lowed by liquid–liquid extraction facilitated the isolation of the
excellent molar conversions. PfTA, on the other hand, afforded final product with excellent yield (91%). While full conversion of
moderate conversions in the case of compounds 2a and 4a, while the starting material 1a was observed, the transamination reaction
no conversion at all was detected in the case of 3a (Table 1). did not take place selectively. In fact, a significant loss in ee was
and PfTA was further equivalent. Remarkably, complete molar conversion was achieved
Optimisation of the reaction conditions again proved challenging observed with respect to the batch reactions (Table 2). A similar
due to the instability of the biocatalyst in the presence of high phenomenon was also observed in the case of 2b. Although high
concentrations of (S)-MBA. Regarding the stereoselectivity, com- conversion of 2a was achieved after 10 mins of residence time,
pound 2b was initially assumed to be S, in the absence of stand- the ee of the final product was 26% (S). Interestingly, these results
ards, by comparison with the other substrates.[14] This has been
are much more comparable to those obtained in batch mode when
recently re-evaluated by synthetic preparation of standards, to be, the tetrameric TA from Pseudomonas fluorescence was employed
in fact, R.[18] The same enantiomer was produced by HeWT
and as the biocatalyst. While the active sites of PfTA and HeWT were