One-Pot Cascade Synthesis of Mono- and Disubstituted Piperidines and Pyrrolidines using Carboxylic Acid Reductase (CAR), ω-Transaminase (ω-TA), and Imine Reductase (IRED) Biocatalysts

Article abstract of DOI:10.1021/acscatal.6b00855

Access to enantiomerically pure chiral mono- and disubstituted piperidines and pyrrolidines has been achieved using a biocatalytic cascade involving carboxylic acid reductase (CAR), ω-transaminase (ω-TA), and imine reductase (IRED) enzymes. Starting from keto acids or keto aldehydes, substituted piperidine or pyrrolidine frameworks can be generated in high conversion, ee, and de in one pot, with each biocatalyst exhibiting chemo-, regio-, and/or stereoselectivity during catalysis. The study also includes a systematic investigation of the effect of the position of a methyl group ring substituent on the IRED-catalyzed reduction of a chiral imine. Analysis of the selectivity observed in these reactions revealed an interesting balance between substrate versus enzyme control; the configurations of the products obtained were rationalized on the basis of minimizing 1,3- or 1,2-steric interactions with incoming NADPH.

Full text of DOI:10.1021/acscatal.6b00855

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Article
One Pot Cascade Synthesis of Mono- and Di-Substituted Piperidines
and Pyrrolidines using Carboxylic Acid Reductase (CAR), #-
Transaminase (#-TA) and Imine Reductase (IRED) Biocatalysts
Scott P. France, Shahed Hussain, Andrew M Hill, Lorna J Hepworth, Roger
M. Howard, Keith R. Mulholland, Sabine L. Flitsch, and Nicholas J Turner
ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.6b00855 • Publication Date (Web): 02 May 2016
Downloaded from http://pubs.acs.org on May 5, 2016
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One Pot Cascade Synthesis of Monoꢀ and Diꢀ
Substituted Piperidines and Pyrrolidines using
Carboxylic Acid Reductase (CAR), ωꢀTransaminase
(ωꢀTA) and Imine Reductase (IRED) Biocatalysts
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Scott P. France,a Shahed Hussain,a Andrew M. Hill,a Lorna J. Hepworth,a Roger M. Howard,b
Keith R. Mulholland,c Sabine L. Flitscha and Nicholas J. Turner a*.
a. School of Chemistry, University of Manchester, Manchester Institute of Biotechnology, 131
Princess Street, Manchester M1ꢁ7DN, United Kingdom.
b. Pfizer Chemical Research & Development, Discovery Park House, Ramsgate Road,
Sandwich, CT13 9NJ, United Kingdom.
c. Chemical Development, AstraZeneca, Silk Rd Business Park, Macclesfield, SK10 2NA,
United Kingdom.
ABSTRACT
Access to enantiomerically pure chiral monoꢀ and diꢀsubstituted piperidines and pyrrolidines has
been achieved using a biocatalytic cascade involving carboxylic acid reductase (CAR), ωꢀ
transaminase (ωꢀTA) and imine reductase (IRED) enzymes. Starting from keto acids or keto
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aldehydes, substituted piperidine or pyrrolidine frameworks can be generated in high conversion,
ee and de, in one pot, with each biocatalyst exhibiting chemoꢀ, regioꢀ and/or stereoselectivity
during catalysis. The study also includes a systematic investigation of the effect of the position of
a methyl group ring substituent on the IREDꢀcatalyzed reduction of a chiral imine. Analysis of
the selectivity observed in these reactions revealed an interesting balance between substrate
versus enzyme control; the configuration of the products obtained were rationalized based on
minimizing 1,3ꢀ or 1,2ꢀsteric interactions with incoming NADPH.
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KEYWORDS Biocatalysis, carboxylic acid reductase, ωꢀtransaminase, imine reductase,
piperidines, pyrrolidines
INTRODUCTION
Chiral substituted piperidines and pyrrolidines are important architectures that are found in
many biologically active natural products and pharmaceuticals. Further substitution of these
templates and concomitant addition of asymmetric centers gives rise to more structural
complexity, generating increasingly attractive scaffolds to explore chemical space in medicinal
chemistry.1 However, controlling the formation of two or more stereogenic centers, to access a
single desired piperidine/pyrrolidine stereoisomer, is often challenging.2–5 Current asymmetric
approaches that have been developed include those based upon diastereoselective
metalation/crossꢀcoupling,6–9 ringꢀclosing reactions,10–12 pyridine reductions13–16 and chemoꢀ
enzymatic synthesis.17–21 Alternative strategies involve the synthesis of the required
diastereomers as racemates followed by kinetic resolution to generate the single enantiomer
products.22,23 However, the recent emergence of new toolboxes of engineered biocatalysts, with
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broad substrate coverage, offers the prospect of constructing such targets via multiꢀenzyme
catalysis using simple and inexpensive precursors.
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Biocatalytic retrosynthetic analysis24 suggested that the substituted piperidine/pyrrolidine
skeleton could be prepared from the corresponding keto acid via a sequence involving carboxylic
acid reductase (CAR), ωꢀtransaminase (ωꢀTA) and imine reductase (IRED) (Scheme 1a).
Implicit in the design of this sequence is the expected exquisite chemoselectivity of each
individual biocatalyst, which could allow for a oneꢀpot reaction sequence owing to the
orthogonal reactivity of each enzyme. We envisaged employing an IRED as the key biocatalyst
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Scheme 1. Biocatalytic retrosynthesis and forward routes to (a) 2,xꢀdisubstituted piperidines
(x ≠ 6) and pyrrolidines (x ≠ 5) and (b) 2,6ꢀdisubstituted piperidines and 2,5ꢀdisubstituted
pyrrolidines.
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for establishing the CꢀN bond stereochemistry of the product (Scheme 1a). Adaptation of this
sequence (Scheme 1b) would also provide a route to the corresponding 2,6ꢀdiꢀsubstituted
piperidines/2,5ꢀdiꢀsubstituted pyrrolidines from the corresponding diketones. In this sequence,
there is a requirement for both regioselective and stereoselective transamination in order to
differentiate between two similar ketones and also establish the initial stereogenic center.
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CARs have recently gained interest as biocatalysts due to their ability to selectively and
efficiently reduce a broad range of carboxylic acids to aldehydes,25–27 a feat that is very difficult
to accomplish cleanly and in high yield using traditional chemical methods. CARs are both ATPꢀ
and NADPHꢀdependent and hence are best used as wholeꢀcell biocatalysts, with the addition of
glucose to allow for intracellular regeneration of cofactors by endogenous enzymes. For
maximum activity, coꢀexpression of a 4’ꢀphosphopantetheinyl transferase is required to postꢀ
translationally modify the native CAR apoꢀenzyme, by adding a 4’ꢀphosphopantetheine arm that
is required to transfer acylꢀAMP intermediates between adenylation and reduction domains
within the enzyme.25,28
ωꢀTransaminases are wellꢀestablished biocatalysts that convert ketones or aldehydes into
chiral primary amines, typically with high ee, at the expense of a suitable amine donor such as
alanine or isopropylamine.29–31 The ability of these enzymes to distinguish between two ketones,
with regioselective amination at the least hindered carbonyl, has been demonstrated by Kroutil
and coꢀworkers and ourselves previously.17–21 Furthermore, the synthetic utility and robustness
of these enzymes has already led to their incorporation as key biocatalysts in a number of
different cascade systems.31
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Imine reductases (IREDs) have been shown to be useful biocatalysts for generating chiral
monoꢀsubstituted piperidines and pyrrolidines from chemically synthesized cyclic imines.32–42
These NADPHꢀdependent enzymes typically accept a broad range of prochiral cyclic imines
which are reduced to the corresponding chiral amines, often with high conversion and ee.
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RESULTS AND DISCUSSION
In order to initially test the feasibility of the envisaged cascade, five simple keto acid substrates
1aꢀ1e were examined. The bacterial CAR from Mycobacterium marinum (MCAR), which has
been reported to reduce various medium to long chain fatty acids,27 was employed as a wholeꢀ
cell biocatalyst in E. coli. In addition, the gene for a 4’ꢀphosphopantetheinyl transferase (Sfp)
from Bacillus subtilis was coꢀexpressed within the cell as previously described.27
A
commercially available transaminase from Codexis, ATAꢀ113, was used with racemic DLꢀ
alanine supplied as the amine donor for the amination reaction. To ensure full conversion of the
intermediate aldehyde, a glucose dehydrogenase (GDH)/lactate dehydrogenase (LDH) system,
which has previously been reported,43,44 was also implemented to shift the transaminase
equilibrium. Finally, two enantiocomplementary wholeꢀcell IRED biocatalysts were employed in
the cascade reactions in order to access both enantiomers of the amine products: the (R)ꢀIRED
from Streptomyces sp GF358734,37,45 and the (S)ꢀIRED from Streptomyces sp GF3546.34,36,38,42
Table 1 shows the results of biotransformations carried out as oneꢀpot cascade processes.
Rapid and complete consumption of the starting keto acid was observed in all cases (24 h), with
subsequent reduction of the imine to the amine product with modest to excellent ee values that
are consistent with our previously reported work with these IREDs.37,38 No transamination of the
ketone carbonyl was observed, as predicted, which would have afforded the regioisomeric imine.
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Table 1. CARꢀTAꢀIRED Cascade Synthesis of 2ꢀSubstituted Piperidines
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Substrate
1a
Conv. to imine/%
IRED
Conv. to 2/%
ee/% (abs config)
17 (S)
>98
>98
>98
>98
>98
>98
>98
>98
>98
>98
(R)ꢀIRED
(S)ꢀIRED
(R)ꢀIRED
(S)ꢀIRED
(R)ꢀIRED
(S)ꢀIRED
(R)ꢀIRED
(S)ꢀIRED
(R)ꢀIRED
(S)ꢀIRED
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75 (R)
1a
>98
>98
97
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93 (R)
1b
90 (S)
1c
98
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>98
>98
>98
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>98 (S)
94 (R)
1d
1d
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74 (n.d.)a
1e
1e
Reaction conditions: 5 mM substrate , 75 mg/mL MCAR wet whole cells, 50 mg/mL IRED
wet whole cells, 2.5 mg/mL ATAꢀ113, 1 mg/mL GDH, 0.5 mg/mL LDH, 250 mM DLꢀalanine,
100 mM glucose, 1.5 mM NAD+, 1 mM PLP, 500 mM pH 7.0 NaPi buffer, 500 µL reaction
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volume, 30°C, 250 rpm, 24 h. Conversions and ee values were determined by GCꢀFID or HPLC
analysis on a chiral stationary phase with absolute configuration determined by comparison to
literature retention times.37 (a) Absolute configuration not determined, however, (R)ꢀ and (S)ꢀ
IRED afforded opposite enantiomers.
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In the absence of the GDH/LDH system, conversion through to the final amine product was
achieved, however, some overꢀreduction of the keto acid to the keto alcohol was observed (see
Supporting Information). This suggests that, without a strong driving force for the transaminase
reaction, the keto aldehyde can be intercepted and reduced by endogenous reductases in the
wholeꢀcell before the desired transamination. Some keto alcohol byꢀproduct was also observed in
reactions with 1a despite employing the GDH/LDH system, however, for substrates 1bꢀe no keto
alcohol was detected.
The use of isopropylamine as an alternative amine donor for the transaminase was tested with
substrate 1b, however, lower conversions were obtained. Furthermore, a CAR orthologue from
Nocardia iowensis (NCAR)46,47 was tested in the cascade with substrates 1bꢀ1d, which led to
similar or slightly lower conversions in some cases and so was not pursued further. Substrate
loading (up to 50 mM) was also investigated which saw a decrease in conversion as substrate
concentration increased (see Supporting Information). Higher catalyst loading in this case may
be required to achieve greater conversion.
Having established the viability of the cascade for 2ꢀsubstituted piperidines, we turned our
attention to disubstituted analogues. Initially, the formation of 5ꢀmethylꢀ2ꢀphenylpiperidine 4
was investigated, since both enantiomers of the required keto aldehyde substrate 3 could be
prepared based on the report by Gellman et al..48 Aldehydes (R)ꢀ and (S)ꢀ3 were subjected to the
cascade conditions employing ATAꢀ113 and either (R)ꢀ or (S)ꢀIRED (Table 2). In all cases,
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conversion of the starting material to the imine intermediate was complete and subsequent
conversion to the product piperidine by the IRED was good to excellent. It was observed that the
chiral center alpha to the aldehyde functionality can racemize in buffer, however, this was
minimized in the cascade as upon transamination and formation of the imine, this stereocenter is
configurationally stable.
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Table 2. Synthesis of 2,5ꢀDisubstituted Piperidines
Substrate
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Product
Conv./%
de/%
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(R)ꢀIRED
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(S)ꢀIRED
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>98
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>98
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>98
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Reaction conditions: 5 mM substrate, 2.5 mg/mL ATAꢀ113, 1 mM PLP, 250 mM DLꢀalanine,
45 mg/mL IRED wet whole cells, 100 mM glucose, 100 mM pH 7.0 NaPi buffer, 500 µL
reaction volume, 30°C, 250 rpm, 24 h. Conversions, de and ee values were determined by GCꢀ
FID analysis on a chiral stationary phase.
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Interestingly, the preꢀexisting stereogenic center present in the imine intermediate did not
affect the stereoselectivity of the IRED reduction, i.e. the (R)ꢀIRED yielded the product with a Cꢀ
2ꢀ(S)ꢀstereocenter and the (S)ꢀIRED affording the Cꢀ2ꢀ(R)ꢀisomer.
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All four possible stereoisomers of 5ꢀmethylꢀ2ꢀphenylpiperidine 4 could thus be achieved using
this cascade, with the best result being the formation of (2R,5R)ꢀ4 in excellent conversion and de
by employing (S)ꢀIRED (Table 2, Entry 2). Compound (2R,5R)ꢀ4 is noteworthy as it is the minor
kinetic product upon reduction of the corresponding imine with NaBH4 and, therefore, the
cascade offers a convenient route to this diastereomer.
Access to the regioisomeric compounds 4ꢀmethylꢀ and 3ꢀmethylꢀ2ꢀphenylpiperidine (6 and 10
respectively) was then investigated. The corresponding keto acids were used as substrates and
the keto aldehydes generated in situ by means of the full CARꢀTAꢀIRED cascade (Table 3).
Complete consumption of the starting material was observed after 24 h, with only amine product
and unreacted imine intermediates detected. Interestingly, for these compounds, the inherent
selectivity of the IRED was overridden by the existing chirality of the imine, with only the cisꢀ
diastereomer observed in high conversion and de independent of whether the (R)ꢀ or (S)ꢀIRED
was used. This element of substrate control favored the more thermodynamically stable product
with both ring substituents equatorial.
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Table 3. Synthesis of 2,4ꢀDisubstituted Piperidines
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Substrate
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Product
Conv./%
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ee/%
(R)ꢀ5
(R)ꢀIRED
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>98
(R)ꢀ5
(S)ꢀ5
(S)ꢀ5
(S)ꢀIRED
(R)ꢀIRED
(S)ꢀIRED
>98
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>98
>98
>98
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Reaction conditions: 5 mM substrate , 75 mg/mL MCAR wet whole cells, 50 mg/mL IRED
wet whole cells, 2.5 mg/mL ATAꢀ113, 1 mg/mL GDH, 0.5 mg/mL LDH, 250 mM DLꢀalanine,
100 mM glucose, 1.5 mM NAD+, 1 mM PLP, pH 500 mM pH 7.0 NaPi buffer, 500 µL reaction
volume, 30°C, 250 rpm, 24 h. Conversions, de and ee values were determined by GCꢀFID
analysis on a chiral stationary phase.
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For keto acid 7 we observed racemization of the preꢀexisting stereogenic center at Cꢀ4 once it
had entered the cascade, likely due to the equilibrium of the imine intermediate with its enamine
tautomer (Scheme 2). As a consequence, supplying either a single enantiomer or the racemic
keto acid into the cascade furnished an identical product distribution. Under these conditions a
chiral imine intermediate is generated that can equilibrate its enantiomers, allowing the IREDs to
catalyze a dynamic kinetic resolution (DKR) process (Table 4).
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Scheme 2. Racemization of (S) or (R)ꢀ7 by imineꢀenamine tautomerization
Table 4. Synthesis of 2,3ꢀDisubstituted Piperidines
Substrate
IRED
Conv./%
de/%
ee/%
(±)ꢀ7
(R)ꢀIRED
89
95
18
(±)ꢀ7
(S)ꢀIRED
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81
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Reaction conditions: 5 mM substrate, 75 mg/mL MCAR wet whole cells, 50 mg/mL IRED
wet whole cells, 2.5 mg/mL ATAꢀ113, 1 mg/mL GDH, 0.5 mg/mL LDH, 250 mM DLꢀalanine,
100 mM glucose, 1.5 mM NAD+, 1 mM PLP, 500 mM pH 7.0 NaPi buffer, 500 µL reaction
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volume, 30°C, 250 rpm, 24 h. Conversions, de and ee values were determined by GCꢀFID
analysis on a chiral stationary phase.
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Remarkably, in all cases the cisꢀdiastereomer was the major product and was obtained in high
conversion and in good to high de. Starting with a single enantiomer of 7 afforded comparable
results (see Supporting Information).
For the synthesis of 2,6ꢀdisubstituted piperidines, a panel of 1,5ꢀdiketones (Table 5) were
treated with either the (S)ꢀselective ATAꢀ113 or the enantiocomplementary (R)ꢀselective ATAꢀ
117.49 In these cases, the transaminases were used to establish the first stereogenic center by
transformation of an achiral starting material. As previously described,18–20 the transaminases
catalyzed the amination of the lessꢀhindered carbonyl exclusively, producing the disubstituted
chiral imine in very high ee. Initial results suggested that the presence of IREDꢀcontaining
wholeꢀcells in a oneꢀpot reaction cascade inhibited conversion of the starting diketone by the
transaminase enzymes. Therefore, the procedure was optimized to allow complete consumption
of the diketone starting material by the transaminase, before sequential addition of the IRED
wholeꢀcell biocatalysts after 24 h in the same pot.
The 2,6ꢀdisubstituted piperideines proved to be challenging substrates for imine reduction,
therefore the IRED cell loading was increased to 200 mg/mL in order to achieve higher
conversions. Substrate control was once again apparent, as seen for the 2,4ꢀdisubstituted
piperidines; the more stable 1,3ꢀdiequatorial product was formed regardless of the IRED used.
This outcome was particularly pronounced with bulky aromaticꢀsubstituted piperideines, which
were converted to their corresponding amines in high de. In cases where the inherent
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Table 5. Synthesis of 2,6ꢀDisubstituted Piperidines
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Substrate
ATA
Conv.
IRED
Amine
Conv./%
de/%
ee/%
to 12/%
product
113
>98
>98
>98
>98
(R)ꢀIRED
81
>98
>98
11a
117
113
117
(S)ꢀIRED
(R)ꢀIRED
(S)ꢀIRED
32
71
61
>98
>98
>98
>98
>98
>98
11a
11b
11b
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7
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9
113
117
113
117
>98
>98
>98
>98
(R)ꢀIRED
42
>98
>98
95
>98
11c
11c
11d
11d
10
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60
(S)ꢀIRED
(R)ꢀIRED
(R)ꢀIRED
63
>98
>98
>98
>98
21
13
Reaction conditions: 5 mM substrate, 2.5 mg/mL ATAꢀ113 or ATAꢀ117, 1 mg/mL GDH, 0.5
mg/mL LDH, 250 mM Lꢀalanine (with ATAꢀ113) or Dꢀalanine (with ATAꢀ117), 100 mM
glucose, 1.5 mM NAD+, 1 mM PLP, 100 mM pH 7.0 NaPi buffer, 500 µL reaction volume,
30°C, 250 rpm, 24 h followed by addition of 200 mg/mL IRED wet whole cells and incubation at
30°C, 250 rpm for 24 h. Conversions, de and ee values were determined by GCꢀFID analysis on
a chiral stationary phase.
stereoselectivity of the IREDs and the imine enantiomer matched to afford a cisꢀdiastereomer
product, higher conversions and de of the amine were achieved. In this respect, the (R)ꢀIRED
was best matched with ATAꢀ113 in order to furnish the highest conversions of the starting imine
whereas the (S)ꢀIRED was paired more effectively with ATAꢀ117 (Table 5). For further
information on all ATAꢀIRED combinations see Supporting Information. Substrate 11d with
ATAꢀ113 and the (R)ꢀIRED afforded the natural product (ꢀ)-dihydropinidine (2S,6R)ꢀ13d in
excellent conversion and de. Unusually, following transamination of diketone 11d by ATAꢀ117,
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the (R)ꢀIRED showed low conversion to the product amine (2R,6S)ꢀ13d in only 13% de, giving
rise to a larger proportion of the transꢀdiastereomer product in this case.
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To test the synthetic utility of these cascade reactions, preparativeꢀscale reactions were carried
out (Table 6). Substrates were chosen to exemplify the formation of monoꢀ and diꢀsubstituted
piperidines starting from a keto acid, keto aldehyde or diketone via the CARꢀTAꢀIRED or TAꢀ
IRED cascades. The isolated product yields demonstrate that these cascades can be successfully
employed on synthetically relevant scale to afford compounds that are difficult to prepare by
traditional chemical means.
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Table 6. PreparativeꢀScale Synthesis Employing the CARꢀTAꢀIRED or TAꢀIRED Cascade
Substrate
Enzyme Cascade
MCAR, ATAꢀ113,
(S)ꢀIRED
Product
Isolated yield/% (Quantity)
(R)ꢀ2b
83 (70 mg)
1b
(R)ꢀ3
(±)ꢀ7
ATAꢀ113,
(2R,5R)ꢀ4
(2R,3S)ꢀ10
79 (73 mg)
76 (65 mg)
(S)ꢀIRED
MCAR, ATAꢀ113,
(S)ꢀIRED
(S)ꢀ15a
(R)ꢀIRED
(2S,6S)ꢀ13a
(2S,6R)ꢀ13d
92 (46 mg)
90 (51 mg)b
ATAꢀ113, (R)ꢀIRED
11d
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Reactions were carried out as per the analyticalꢀscale reactions as described above. Crude
product des and ees matched those obtained in the analyticalꢀscale reactions. (a) (S)ꢀ15 was
prepared from 11a and ATAꢀ113 and isolated (93% yield). (b) Isolated as hydrochloride salt.
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The observed stereoselectivities of the IREDꢀcatalysed reduction of the chiral piperideine
substrate panel can be tentatively rationalized as shown in Figure 1. For monoꢀsubstituted 2ꢀ
phenylpiperideine, we suggest that there is a preferred binding orientation that allows hydride
delivery from NADPH to one face of the prochiral imine, resulting in the observed
stereochemistry of the product. Assuming that the lowest energy pathway operates, the hydride is
predicted to attack antiperiplanar with respect to the σꢀCꢀH bond vicinal to the imino functional
group, as described by Yamamoto et al.50,51 and based on the theoretical predictions by Houk et
al.52 and Cieplak.53 For all of the disubstituted compounds, the corresponding chiral imines
would also presumably prefer to bind in the same orientation at the enzyme activeꢀsite. However,
if this binding mode places the methyl group on the ring in a pseudoꢀaxial position, such that
there is a steric 1,3ꢀinteraction with the incoming hydride from NADPH, it is presumably more
favorable for the substrate to enter the active site in a 180° rotated orientation. An associated
ringꢀflip of the imine would also be expected assuming hydride delivery via the lowest energy
nucleophilic attack trajectory. In this new binding mode, hydride is delivered to the opposite face
of the imine as there is no longer an unfavorable 1,3ꢀ steric interaction. This situation applies for
one enantiomer of the 2,6ꢀ and 2,4ꢀpiperideines with each IRED and explains why, in these
cases, the inherent selectivity of the enzyme is overridden. For the 2,3ꢀpiperideine 8, which can
tautomerize via the enamine 9, the lowest energy route minimizes 1,2ꢀsteric interactions between
the methyl group and the incoming hydride, resulting in the cisꢀdiastereomer as the major
product.
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Preferred binding
orientation of parent imine
in IRED active site
Substrate
Product
5
6
7
8
H
H
H
H
N
N
Ph
N
H
Ph
HN
HN
14
Ph
Ph
Ph
Ph
(R)-2b
H
9
Me
10
11
12
13
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Me
N
H
Ph
Me
Me
N
Ph
Ph
N
Me
Ph
(R)-
15
(2R,6R)-
13a
Me
HN
H
1,3-steric
clash
Me
Me
N
Ph
N
N
H
(S)-15
Ph
180°
(2S,6R)-13a
and ring flip
H
Ph
H
N
Me
N
Me
N
H
Ph
Ph
Me
(2S,6S)-13a
H
Me
Me
Me
H
Ph
H
H
N
N
H
Ph
N
Ph
Ph
HN
Ph
Me
(R)-16
(2R,5R)-4
Me
Me
H
Me
HN
N
H
Ph
N
N
N
Me
(S)-16
Ph
(2R,5S)-4
Ph
Me
Me
H
H
H
Me
Me
HN
HN
N
Ph
N
(S)-17
Ph
Ph
Ph
H
Ph
(2R,4S)-6
Me
Me
Me
Me
H
1,3-steric
clash
N
N
Ph
Ph
180°
N
(R)-17
Ph
H
(2R,4R)-6
and ring flip
Me
H
Me
H
Ph
H
Me
N
N
Ph
N
H
Ph
(2S,4R)-6
Me
Ph
Me
H
H
N
N
Ph
N
N
HN
HN
H
8
Ph
Ph
Me
(2R,3S)-10
Me
Me
Ph
Me
H
N
H
H
Me
N
H
Ph
9
Me
1,2,steric
interaction
Ph
Ph
10
(2S,3S)-
Figure 1. Proposed rationale for selectivity of IREDꢀcatalysed reduction of chiral imines.
Hydride (blue) represents NADPH as hydride source. Lowest energy routes represented by green
arrows and higher energy routes indicted by red arrows. Major products are in green boxes and
minor products are in red boxes.
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In contrast to the piperideine ring systems, investigations into the effect of a preꢀexisting
stereogenic center on the reduction of 2,5ꢀdisubstituted pyrrolines by IREDs revealed a less
predictable pattern (Table 7).
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Table 7. Synthesis of 2,5ꢀDisubstituted Pyrrolidines
Entry
ATA
IRED
Conv. to
15/%
Amine
Conv./%
de/%
ee/%
product
1
113
(R)ꢀIRED
>98
>98
>98
>98
11
63
>98
2
3
4
113
117
117
(S)ꢀIRED
(R)ꢀIRED
(S)ꢀIRED
17
95
68
65
>98
>98
>98
97
>98
Reaction conditions: 5 mM substrate, 2.5 mg/mL ATAꢀ113 or ATAꢀ117, 1 mg/mL GDH, 0.5
mg/mL LDH, 250 mM Lꢀalanine (with ATAꢀ113) or Dꢀalanine (with ATAꢀ117), 100 mM
glucose, 1.5 mM NAD+, 1 mM PLP, 100 mM pH 7.0 NaPi buffer, 500 µL reaction volume,
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30°C, 250 rpm, 24 h followed by addition of 200 mg/mL IRED wet whole cells and incubation at
30°C, 250 rpm for 24 h. Conversions, de and ee values were determined by GCꢀFID analysis on
a chiral stationary phase.
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Taking the ATAꢀ113 product (S)ꢀ15 as a substrate, the IREDs displayed modest conversion of
the chiral imine, giving the corresponding amines in moderate de (63–65%). When compared to
the IRED reductions of monoꢀsubstituted 2ꢀphenylpyrroline, substrate (S)ꢀ15 appeared to
override the enzyme’s inherent selectivity, producing pyrrolidines of the opposite configuration.
When the opposite enantiomer (R)ꢀ15 was treated with the IREDs, higher levels of conversion
were obtained and remarkably both enzymes furnished amines of the same configuration in high
de (97ꢀ>98%) (Table 7, entries 3 and 4). It is interesting to note that although there appears to be
a preference for the (R)ꢀenantiomer as a substrate for both enzymes, it is clear that more subtle
and complex binding interactions are in play.
CONCLUSIONS
In summary, a versatile and highly efficient oneꢀpot cascade route to chiral piperidines and
pyrrolidines has been successfully demonstrated utilizing CAR, ωꢀTA and IRED biocatalysts to
afford valuable chiral amines from simpler, acyclic starting materials. Disubstituted compounds
can also be accessed using this approach, with one stereogenic center being set by an IRED and
the other derived from the substrate or generated using an ωꢀTA. In certain cases, chiral imine
substrates can override inherent IRED selectivity, providing an insight into substrate binding and
substituent effects with these enzymes which has not been investigated previously.
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ASSOCIATED CONTENT
AUTHOR INFORMATION
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Corresponding Author
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*Email nicholas.turner@manchester.ac.uk
Funding Sources
S.P.F acknowledges a CASE award from Biotechnology and Biological Sciences Research
Council (BBSRC) and Pfizer. S.H acknowledges a CASE award from Engineering and Physical
Sciences Research Council (EPSRC) and AstraZeneca. A.M.H received funding from the Centre
of Excellence for Biocatalysis, Biotransformations and Biocatalytic Manufacture (CoEBio3) and
L.J.H from the BBSRC sLoLa programme (BB/L502005/1). N.J.T thanks the Royal Society for a
Wolfson Research Merit Award.
Supporting Information. This material is available free of charge via the Internet at
http://pubs.acs.org. Experimental procedures and methods, substrate synthesis and isolated
product characterization, analytical data.
ACKNOWLEDGMENT
Thanks go to Nicholas J. Weise for help with rationalization of stereochemical outcomes.
ABBREVIATIONS
PLP (pyridoxal 5'ꢀphosphate)
REFERENCES
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(1)
(2)
(3)
(4)
Lovering, F.; Bikker, J.; Humblet, C. J. Med. Chem. 2009, 52, 6752–6756.
Buffat, M. G. P. Tetrahedron 2004, 60, 1701–1729.
Vo, C.ꢀV. T.; Bode, J. W. J. Org. Chem. 2014, 79, 2809–2815.
Mitchell, E. A.; Peschiulli, A.; Lefevre, N.; Meerpoel, L.; Maes, B. U. W. Chem. Eur. J.
2012, 18, 10092–10142.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(5)
(6)
Campos, K. R. Chem. Soc. Rev. 2007, 36, 1069–1084.
Seel, S.; Thaler, T.; Takatsu, K.; Zhang, C.; Zipse, H.; Straub, B. F.; Mayer, P.; Knochel,
P. J. Am. Chem. Soc. 2011, 133, 4774–4777.
(7)
(8)
(9)
Coldham, I.; Leonori, D. Org. Lett. 2008, 10, 3923–3925.
Beak, P.; Lee, W.ꢀK. J. Org. Chem. 1993, 58, 1109–1117.
Millet, A.; Larini, P.; Clot, E.; Baudoin, O. Chem. Sci. 2013, 4, 2241.
(10) Mix, S.; Blechert, S. Adv. Synth. Catal. 2007, 349, 157–160.
(11) Yadav, J. S.; Reddy, B. V. S.; Chaya, D. N.; Kumar, G. G. K. S. N.; Naresh, P.;
Jagadeesh, B. Tetrahedron Lett. 2009, 50, 1799–1802.
(12) Launay, G. G.; Slawin, A. M. Z.; O’Hagan, D. Beilstein J. Org. Chem. 2010, 6, 4–9.
(13) Lu, S. M.; Wang, Y. Q.; Han, X. W.; Zhou, Y. G. Angew. Chem. Int. Ed. 2006, 45, 2260–
2263.
(14) Glorius, F.; Spielkamp, N.; Holle, S.; Goddard, R.; Lehmann, C. W. Angew. Chem. Int.
Ed. 2004, 43, 2850–2852.
(15) Ye, Z. S.; Chen, M. W.; Chen, Q. A.; Shi, L.; Duan, Y.; Zhou, Y. G. Angew. Chem. Int.
Ed. 2012, 51, 10181–10184.
(16) Cai, W.; Colony, J. L.; Frost, H.; Hudspeth, J. P.; Kendall, P. M.; Krishnan, A. M.;
Makowski, T.; Mazur, D. J.; Phillips, J.; Brown Ripin, D. H.; Ruggeri, S. G.; Stearns, J.
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Page 22 of 25
1
2
3
4
F.; White, T. D. Org. Process Res. Dev. 2005, 9, 51–56.
5
6
7
8
(17) O’Reilly, E.; Iglesias, C.; Ghislieri, D.; Hopwood, J.; Galman, J. L.; Lloyd, R. C.; Turner,
N. J. Angew. Chem. Int. Ed. 2014, 53, 2447–2450.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(18) Simon, R. C.; Grischek, B.; Zepeck, F.; Steinreiber, A.; Belaj, F.; Kroutil, W. Angew.
Chem. Int. Ed. 2012, 51, 6713–6716.
(19) Simon, R. C.; Zepeck, F.; Kroutil, W. Chem. Eur. J. 2013, 19, 2859–2865.
(20) Simon, R. C.; Fuchs, C. S.; Lechner, H.; Zepeck, F.; Kroutil, W. Eur. J. Org Chem. 2013,
3397–3402.
(21) Payer, S. E.; Schrittwieser, J. H.; Grischek, B.; Simon, R. C.; Kroutil, W. Adv. Synth.
Catal. 2016, 358, 444–451.
(22) Wanner, B.; Kreituss, I.; Gutierrez, O.; Kozlowski, M. C.; Bode, J. W. J. Am. Chem. Soc.
2015, 137, 11491–11497.
(23) Binanzer, M.; Hsieh, S. Y.; Bode, J. W. J. Am. Chem. Soc. 2011, 133, 19698–19701.
(24) Turner, N. J.; O’Reilly, E. Nat. Chem. Biol. 2013, 9, 285–288.
(25) NaporaꢀWijata, K.; Strohmeier, G. A.; Winkler, M. Biotechnol. J. 2014, 9, 822–843.
(26) Lamm, A. S.; Venkitasubramanian, P.; Rosazza, J. P. N. in: Science of Synthesis:
Biocatalysis in Organic Synthesis 2; (Eds.: K. Faber, W.ꢀD. Fessner, N. J. Turner), Georg
Thieme Verlag, Stuttgart, 2015; pp 459–478.
(27) Akhtar, M. K.; Turner, N. J.; Jones, P. R. Proc. Natl. Acad. Sci. U. S. A. 2013, 110, 87–92.
(28) Venkitasubramanian, P.; Daniels, L.; Rosazza, J. P. N. J. Biol. Chem. 2007, 282, 478–485.
(29) Koszelewski, D.; Tauber, K.; Faber, K.; Kroutil, W. Trends Biotechnol. 2010, 28, 324–
332.
(30) Mathew, S.; Yun, H. ACS Catal. 2012, 2, 993–1001.
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2
3
4
5
6
7
8
9
(31) Simon, R. C.; Richter, N.; Busto, E.; Kroutil, W. ACS Catal. 2014, 4, 129–143.
(32) Schrittwieser, J. H.; Velikogne, S.; Kroutil, W. Adv. Synth. Catal. 2015, 357, 1655–1685.
(33) Leipold, F.; Hussain, S.; France, S. P.; Turner, N. J. in: Science of Synthesis: Biocatalysis
in Organic Synthesis 2; (Eds.: K. Faber, W.ꢀD. Fessner, N. J. Turner), Georg Thieme
Verlag, Stuttgart, 2015; pp 359–382.
10
11
12
13
14
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19
20
21
22
23
24
25
26
27
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29
30
31
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41
42
43
44
45
46
47
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49
50
51
52
53
54
55
56
57
58
59
60
(34) Mitsukura, K.; Suzuki, M.; Tada, K.; Yoshida, T.; Nagasawa, T. Org. Biomol. Chem.
2010, 8, 4533–4535.
(35) Mitsukura, K.; Suzuki, M.; Shinoda, S.; Kuramoto, T.; Yoshida, T.; Nagasawa, T. Biosci.
Biotechnol. Biochem. 2011, 75, 1778–1782.
(36) Mitsukura, K.; Kuramoto, T.; Yoshida, T.; Kimoto, N.; Yamamoto, H.; Nagasawa, T.
Appl. Microbiol. Biotechnol. 2013, 97, 8079–8086.
(37) Hussain, S.; Leipold, F.; Man, H.; Wells, E.; France, S. P.; Mulholland, K. R.; Grogan, G.;
Turner, N. J. ChemCatChem 2015, 7, 579–583.
(38) Leipold, F.; Hussain, S.; Ghislieri, D.; Turner, N. J. ChemCatChem 2013, 5, 3505–3508.
(39) RodriguezꢀMata, M.; Frank, A.; Wells, E.; Leipold, F.; Turner, N. J.; Hart, S.;
Turkenburg, J. P.; Grogan, G. ChemBioChem 2013, 14, 1372–1379.
(40) Scheller, P. N.; Fademrecht, S.; Hofelzer, S.; Pleiss, J.; Leipold, F.; Turner, N. J.; Nestl, B.
M.; Hauer, B. ChemBioChem 2014, 15, 2201–2204.
(41) Wetzl, D.; Berrera, M.; Sandon, N.; Fishlock, D.; Ebeling, M.; Müller, M.; Hanlon, S.;
Wirz, B.; Iding, H. ChemBioChem 2015, 16, 1749–1756.
(42) Huber, T.; Schneider, L.; Präg, A.; Gerhardt, S.; Einsle, O.; Müller, M. ChemCatChem
2014, 6, 2248–2252.
(43) Shin, J. S.; Kim, B. G. Biotechnol. Bioeng. 1999, 65, 206–211.
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3
4
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6
(44) Koszelewski, D.; Lavandera, I.; Clay, D.; Rozzell, D.; Kroutil, W. Adv. Synth. Catal.
2008, 350, 2761–2766.
7
8
9
(45) Mitsukura, K.; Suzuki, M.; Shinoda, S.; Kuramoto, T.; Yoshida, T.; Nagasawa, T. Biosci.
Biotechnol. Biochem. 2011, 75, 1778–1782.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
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46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(46) Li, T.; Rosazza, J. P. N. Appl. Environ. Microbiol. 2000, 66, 684–687.
(47) NaporaꢀWijata, K.; Robins, K.; OsorioꢀLozada, A.; Winkler, M. ChemCatChem 2014, 6,
1089–1095.
(48) Chi, Y. G.; Gellman, S. H. Org. Lett. 2005, 7, 4253–4256.
(49) Savile, C. K.; Janey, J. M.; Mundorff, E. C.; Moore, J. C.; Tam, S.; Jarvis, W. R.;
Colbeck, J. C.; Krebber, A.; Fleitz, F. J.; Brands, J.; Devine, P. N.; Huisman, G. W.;
Hughes, G. J. Science. 2010, 329, 305–309.
(50) Maruoka, K.; Miyazaki, T.; Ando, M.; Matsumura, Y.; Sakane, S.; Hattori, K.;
Yamamoto, H. J. Am. Chem. Soc. 1983, 105, 2831–2843.
(51) Maruoka, K.; Yamamoto, H. Angew. Chem. Int. Ed. 1983, 55, 668–682.
(52) Mazzocchi, P. H.; Pople, J. A.; Jeffrey, A.; Wipff, G.; Caramella, P.; Houk, N. J. Am.
Chem. Soc. 1981, 103, 2438–2440.
(53) Cieplak, A. S. J. Am. Chem. Soc. 1981, 103, 4540–4552.
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