Synthesis of protected 3-aminopiperidine and 3-aminoazepane derivatives using enzyme cascades

Article abstract of DOI:10.1039/d0cc02976a

Multi-enzyme cascades utilising variants of galactose oxidase and imine reductase led to the successful conversion of N-Cbz-protected l-ornithinol and l-lysinol to l-3-N-Cbz-aminopiperidine and l-3-N-Cbz-aminoazepane respectively, in up to 54% isolated yi

Full text of DOI:10.1039/d0cc02976a

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Synthesis of Protected 3-Aminopiperidine and 3-Aminoazepane
Derivatives Using Enzyme Cascades
Grayson J. Ford, a Nico Kress,a Ashley P. Mattey,a Lorna J. Hepworth,a Christopher R. Baldwin,a James
R. Marshall,a Lisa S. Seibt,a Min Huang,a William R. Birmingham,a Nicholas J. Turner,a Sabine L.
Flitsch*a
Received 24th April 2020,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
Multi-enzyme cascades utilising variants of galactose oxidase using
a transaminase-based biocatalytic synthesis of 3-
and imine reductase led to the successful conversion of N-Cbz- aminoazepane from ketone precursors.9 An attractive strategy
protected L-ornithinol and L-lysinol to L-3-N-Cbz- is the combination of biocatalysts in telescoped enzymatic
aminopiperidine and L-3-N-Cbz-aminoazepane respectively, in cascades resembling biosynthetic pathways to generate
up to 54% isolated yield. Streamlining the reactions into one- unnatural compounds.10–13
pot prevented potential racemisation of key labile
intermediates and led to products with high enantiopurity.
Chiral amine moieties are present in many of the most valuable
pharmaceutical compounds,1,2 with cyclic diamines in particular
often used as semi-rigid bifunctional linkers in medicinal
chemistry. Hence there is
a
demand for efficient,
stereoselective synthesis strategies of diamines from easily
accessible starting materials, preferentially in their semi-
protected form to allow direct application in the manufacture
of pharmaceutical intermediates. Recent biocatalytic
methodologies have proven to be very successful in the design
of alternative efficient and sustainable processes in the
synthesis of chiral amines, underpinned by an increasing
interest in their industrial application.3 In this regard, we were
interested in the chiral enzymatic synthesis of semi-protected
3-aminopiperidines 7 and 3-aminoazepanes 8, being core
structures in many valuable pharmaceutical drugs such as
alogliptin and besifloxacin.4,5
Current synthetic methods towards 3-aminopiperidines and
3-aminoazepanes encompass various approaches, including
Curtius and Hofmann rearrangements, hydrogenation of 3-
aminopyridine or the cyclisation of α-amino acids.6–8 Although
these routes are well established, they often lack chiral control
and the use of expensive and toxic reagents can hinder their
wider industrial application. The particular challenge of chiral 3-
aminoazepanes such as 8 was recently addressed by Feng et al.
Figure 1: Enzyme cascade for the synthesis of L-3-N-aminopiperidines and L-3-N-
aminoazepanes using a combination of galactose oxidase (GOase) and imine
reductase (IRED) enzymes.
When targeting 7 and 8, the challenge was to find enzymes that
could tolerate bulky unnatural protection groups such as
carboxybenzyl (Cbz) groups, which are needed for subsequent
coupling chemistries. Based on our previous expertise in the
application of galactose oxidase (GOase)14–17 and imine
reductase (IRED)18–22 biocatalysts, a multi-enzymatic cascade
towards amino-piperidine and amino azepanes was designed.
This streamlined approach uses readily available amino alcohols
such as 1a-c and 2a-c as substrates, (Figure 1), which were
directly accessible by chemical synthesis from natural amino
acids L-ornithine and L-lysine. (Supporting Information).
a. Department of Chemistry, Manchester Institute of Biotechnology, University of
Manchester, 131 Princess Street, Manchester, M1 7DN, UK
Electronic Supplementary Information (ESI) available: [details of any supplementary
information available should be included here]. See DOI: 10.1039/x0xx00000x
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The enzymatic cascade would then proceed by initial oxidation 102 and IR-110. AdRedAm was chosen becauseVioewf AitrtsiclebOronlainde
DOI: 10.1039/D0CC02976A
of amino alcohols 1 and 2 to the respective amino aldehydes 3 substrate range, whereas the other four IREDs have been
or 4 by GOase, followed by spontaneous formation of cyclic previously shown to accept the azepane scaffold.20,27
imine intermediates 5 or 6 that would finally be reduced by an
Previous studies directed the reaction design for the one-
IRED to generate the desired enantiopure products 7 and 8.17 pot in vitro GOase-IRED cascade, which included HRP for
Both individual enzymatic reactions have already shown activation of GOase, catalase for H2O2 decomposition, while
promise in industrial applications.23,24
NADP+ in combination with glucose dehydrogenase (GDH) and
The proposed strategy presented multiple challenges: the α- glucose was added as a cofactor recycling system to ensure
amino aldehyde intermediates from substrates 1a and 2a would electron supply for the IRED reaction.26,28 Considering
be predicted to be very unstable in a biotransformation. previously optimized reaction conditions for both GOase and
Considering the need for selective protection of the target IRED, initial analytical scale reactions were performed in
compounds for subsequent chemical coupling reactions in API phosphate buffer at pH 7.5 and 30 °C for 16 h.26,29
synthesis anyway, Boc- and Cbz-protected starting materials 1b-
c and 2b-c were investigated as leading to reagents that are
more valuable. However, the use of monoprotected substrates
would require the tolerance of the chosen biocatalysts towards
bulky hydrophobic protecting groups. To minimize side-
reactions, purified enzymes rather than whole cells or enzyme
lysates were used.
For the first step of the enzyme cascade, suitable
recombinant GOase variants F2 and M3-5 were tested for the
oxidation of amino alcohols 1a-c and 2a-c (Supporting
Information).14,17 An initial activity screen showed that the Cbz-
protected amino alcohol derivatives 1c and 2c gave highest
initial activity, whilst unprotected and Boc-protected
derivatives 1a-b and 2a-b were not well accepted as substrates
(Supporting Information). The poor results for the unprotected
substrates 1a and 2a might be due to instability of the amino
aldehyde intermediates and problems with amino alcohol
functionality, which can result in potential chelation of the
active copper center in GOase.25
Given that Cbz-protected substrates 1c and 2c
Figure 2: Result of activity screening for the synthesis of 3-N-Cbz-aminopiperidine
7 and 3-N-Cbz-aminoazepane 8 comparing 2 GOases and 5 IREDs. Reactions were
demonstrated good activity, a colorimetric HRP-ABTS assay was
performed with 3 mM substrate in NaPi buffer (pH 7.5) at 30°C and 200 rpm for
16 h. The resulting analytical yields were determined by GC-FID and are given as
used to determine kinetic constants comparing the two GOase
mutants F2 and M3-5 (Table 1).26 Both mutants showed overall
colour-coded ranges.
comparable activity, L-lysinol derivative 2c appeared to be
preferred over L-ornithinol 1c, with significant higher catalytic
efficiency, particularly for the M3-5 variant.
Chromatographic analysis of the reaction mixtures showed only
trace amounts of products for 1a and 2a, and so were not
investigated further (Supporting Information). The Boc-
protected substrates 1b and 2b showed no detectable activity,
but we were pleased to observe a range of potential GOase-
IRED combinations to be active towards the Cbz-protected
substrates 1c and 2c (Figure 2 & Supporting Information). We
were surprised to find a marked preference for the L-lysinol
substrate 2c over the L-ornithinol substrate 1c, considering that
the azepane imine intermediate would be expected to be less
readily formed than the corresponding piperidine imine. For
both substrates, the GOase M3-5 variant performed
considerably better than F2, which is in agreement with
previously reported amino alcohol conversions.17 The IRED
preference was found to be strongly substrate dependent, with
AdRedAm performing best in the case of the piperidine product
7, while IR-49 was optimal for azepane 8 synthesis.
Table 1. Kinetic constants Km and kcat for oxidations of N-α-Cbz-ornithinol 1c and
N-α-Cbz-lysinol 2c using GOase variants M3-5 and F2.
Substrate -
GOase
Km
[mM]
Vmax
[U mg-1]
kcat
[s-1]
Kcat/Km
[mM s-1]
1c – F2
1c – M3-5
2c – F2
70.7  7.2
47  15
70.5  2.2
40.5  7.3
0.83  0.09
0.77  0.22
1.74  0.05
2.29  0.34
0.9  0.1
0.013
0.019
0.028
0.063
0.87  0.25
1.97  0.06
2.57  0.42
2c – M3-5
Conditions: ABTS-HRP coupled assay in NaPi (100 mM, pH 7.4) with substrates 1c
and 2c at various concentrations, λ = 420 nm, 25 oC (Supporting Information)
Encouraged by these results, the GOase variants were tested in
combination with an IRED panel in a one-pot reaction, thus
avoiding the need to isolate potentially unstable
aldehyde/imine intermediates. Five IREDs were selected from
available libraries for the first screen: AdRedAm, IR-23, IR-49, IR-
A set of reaction optimizations were performed for both
cascades to give products 7 and 8, including pH, reaction
temperature, enzyme loading and substrate concentration
(Supporting Information). Each parameter was altered
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individually while other parameters stayed constant for both. While the phenyl group binds atop P463 in M3-5, it is bound
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DOI: 10.1039/D0CC02976A
The cascade was screened over a range of reaction pHs (6-8). above F290 in F2 allowing for stronger π-π stacking. Two of the
An optimal pH of 8 was determined for the enzyme cascades, mutational differences between M3-5 and F2 seem to be mainly
resulting in higher product yields for both when compared to a responsible for the altered interaction networks in substrate
lower reaction pH, following general previous trends for both binding being M/K330 and T/E406. K330 in F2 seems to cause a
enzymes used.26,29 Increasing reaction temperatures above 30 displaced hydroxy orientation, while E406 is a potential salt
oC resulted in lower conversions, suggesting deactivation of the bridge partner for the terminal charged amino group.
enzyme at these temperatures. Increasing IRED concentration Comparing 1c and 2c, the extended length of the lysinol allows
showed minimal variation in the yield, but increasing the GOase for a strong salt bridge formation with E195 and E406 in M3-5
concentration improved product yield, confirming that the IRED and F2 respectively; which is less pronounced for the shorter
does not appear to be rate limiting. It was also noted that ornithinol, serving as a potential structural explanation for the
conversions for the transformation did not increase further observed preference for lysinol.
when more than 0.5 mg ml-1 GOase was used for the
Using optimized reaction conditions of higher GOase
transformation of 2c to product 8, but did increase up to 1 mg concentrations and a reaction pH of 8, the biotransformations
ml-1 for the transformation of 1c to product 7. Combined with of 1c and 2c were scaled up to a final volume of 90 mL. For
the initial kinetic screening results, this further confirms the substrate 1c (3 mM), GOase M3-5 and AdRedAm were used
higher affinity GOase M3-5 has for substrate 2c over 1c.
(1 mg ml-1 and 0.25 mg ml-1 respectively). Protein precipitation
Given that the GOase is the gatekeeper for the cascade, of AdRedAm was observed after 1–3 h of the reaction in
molecular docking studies were performed to better contrast to the analytical scale reactions (Supporting
understand substrate specificity. F2 and M3-5 models were information). Nonetheless, we were able to extract and isolate
created based on the reported E1 variant structure (PDB 2WQ8). product 7 in 16% yield (10 mg) using a simple acid-base wash.
1c and 2c were used as ligands for the docking simulation into For substrate 2c, we were able to use less enzyme with
the two GOase mutants. The displayed binding modes in Figure 0.5 mg ml-1 M3-5 and 0.25 mg ml-1 IR-49. Under these reaction
3 represent the catalytic binding modes with the highest conditions, no precipitate formed within the 48 h of reaction
calculated binding affinity. For catalytic binding, the free copper time and following the same workup protocol as before, a yield
binding site needs to be occupied by the hydroxy group that is of 54% (36 mg) product 8 was isolated and characterized from
to be oxidized in the radical-based GOase mechanism.30 In the the reaction.
E1 crystal structure, a co-crystallized acetate binds the copper in
The enantiopurity of 7 and 8 was determined using chiral
2.3 Å distance, which is closely mimicked by the calculated normal phase HPLC analysis, comparing the products from the
binding of 1c and 2c to M3-5. In contrast, more than 1 Å greater scaled-up biotransformations, with authentic and synthesized
distances are calculated for binding to F2, supporting the kinetic standards (Supporting Information). Chromatograms showed
data. Interestingly, the ligands adopt two opposing binding the presence of only one enantiomer per reaction, confirming
modes with respect to the Cbz phenyl group.
preservation of the initial amino acid-derived L-configuration.
In conclusion, we showed that industrially relevant
enzymes, GOase and IRED can be combined in a novel one-pot
enzymatic cascade to synthesize enantiopure Cbz-protected L-
3-aminopiperidine 7 and L-3-aminoazepane 8 products from
amino alcohols derived from bio-renewable amino acids as a
green feedstock under ambient conditions. The strategy allows
for directly generating selectively Cbz protected targets, that
are compatible for subsequent incorporation into chemical
processes. The lack of racemization demonstrates the
advantage of using one-pot streamlined cascades.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
We kindly acknowledge financial support from the European
Research Council (788231-ProgrES-ERC-2017-ADG to SLF; BIO-
H-BORROW: Grant No. 742987 to NJT); EU (CarboMet 737395)
BBSRC (BB/M027791/1; BB/M02903411; BB/M028836/1). JRM.
acknowledges the Industrial Biotechnology Innovation Centre
Figure 1: Molecular docking (AutoDock VINA as implemented in YASARA) of N-α-
Cbz-ornithinol 1c and N-α-Cbz-lysinol 2c in GOase variants M3-5 and F2 modelled
based on the E1 variant crystal structure (PDB 2WQ8). Substrate (cyan)
conformations positioning the target hydroxyl group towards the copper centre
(dark orange) are depicted with the respective O-Cu distance in red. Substrate-
interacting active site amino acids are highlighted (purple) indicating residues
altered in between the GOase variants (green). Selected substrate-receptor
interactions are highlighted (yellow).
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(IBioIC) and BBSRC funding through a CASE studentship with
Prozomix Ltd. W.R.B. gratefully acknowledges the 21
Biotechnology and Biological Sciences Research Council (BBSRC)
2018, 140, 17872–17877.
View Article Online
P. Matzel, M. Gand and M. Höhne, DGOreI:e1n0C.1h0e39m/D.,02C0C1072,91796,A
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for the award of a Discovery Fellowship (BB/S010459/1).
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