Discovery of a multi-functional biocatalyst for asymmetric conjugate reduction-reductive amination

A major challenge in chemical synthesis is to develop catalytic systems that convert simple molecules to complex high-value products. Often these valuable compounds must be manufactured asymmetrically, as their biochemical properties can differ based on the chirality of the molecule. Of great interest are enantioenriched amine diastereomers, which are prevalent in pharmaceuticals and agrochemicals, 1 yet their preparation often relies on low-eciency multi-step synthesis. 2 Herein, we report the discovery and characterisation of a multi-functional biocatalyst, which operates using a previously unreported conjugate reduction-reductive amination mechanism. This enzyme (pIR-120), identied within a metagenomic imine reductase (IRED) collection 3 and originating from an unclassied Pseudomonas species, possesses an unusual active site architecture that facilitates an amine-activated conjugate alkene reduction followed by reductive amination. This enzyme enables the coupling of a broad selection of α,β-unsaturated carbonyls with amines for the ecient preparation of enantioenriched amine diastereomers. Moreover, employing a racemic substrate partner or conjugated dienyl-ketone provides a means of controlling additional stereocentres using the single catalyst. Mechanistic and structural studies have been carried out to delineate the order of individual steps catalysed by pIR-120 which have led to a proposal for the overall catalytic cycle. This work shows that the IRED family can serve as a platform for facilitating the discovery of further enzymatic activities for application in synthetic biology and organic synthesis. we report the discovery and characterisation of a multi-functional biocatalyst (pIR-120) that is able to catalyse CR, using a previously unreported amine activation mechanism, as well as imine reduction and RA. pIR-120 possesses broad substrate scope allowing for the stereoselective preparation of valuable amine diastereomers in a one-pot, one-catalyst reaction starting from simple prochiral starting materials. Mechanistic and structural studies reveal a multi-step process in which pIR-120, which possesses an unusual additional tyrosine residue (Y181) in its active site, rst catalyses amine-activated CR of α,β-unsaturated carbonyl via a previously undescribed enimine-NAD(P)H-enzyme complex, followed by RA. This new CR-RA reaction further expands the repertoire of imine reductases and emphasises their importance in the synthesis of stereochemically dened chiral amines.


Main
The prevalence of chiral amines in active pharmaceutical ingredients (APIs) and other high value chemicals 1 has led to an overarching goal in organic synthesis to develop e cient new catalytic methods for their preparation. 4 In this context, reductive amination (RA) is one of the most widely used and powerful methodologies in medicinal chemistry, enabling e cient formation of C-N bonds through the reductive coupling of carbonyls and amines. 2,5 The development of effective catalysts for asymmetric RA continues to be explored, including those based on metallo-, 6,7 organo-8 and biocatalysis. 3,[9][10][11] Furthermore, valuable amino-compounds often contain multiple stereogenic centres (Fig. 1a), however total control of their asymmetry is more challenging, resulting in less e cient multistep syntheses 12 or more complex tandem catalysis systems. 13,14 Whilst multi-enzyme systems are highly amenable to these biomimetic tandem processes (Fig. 1b), di culties in their assembly arise from incompatibilities of their reaction medium and reaction rates, which can lead to by-product formation and intricate reaction setup. 15 To address these issues and achieve the desired reaction metrics, signi cant protein engineering is often required on each enzyme component. 16 Discovery of a single enzyme that can control multiple stereocentres through a RA-like process would be highly desirable and enable e cient synthesis of valuable amine diastereomers using a one-pot, one-catalyst system (Fig. 1c).
Nicotinamide-dependent enzymes are versatile biocatalysts for both asymmetric conjugate reduction (CR) 17,18 and reductive amination (RA). 9,10,19,20 For RA, imine reductases (IREDs) have emerged as attractive catalysts since they possess broad substrate scope and can be engineered for industrial application. 21,22 IREDs are characterised as chemoselective for the reduction of C=N bonds [23][24][25] although under exceptional circumstances they can reduce C=O bonds of activated carbonyl species. 26 Furthermore, we recently demonstrated that IREDs could be combined with ene-reductases (EREDs) in a one-pot process to reduce both the C=C and C=N bonds of cyclic enimines (α,β-unsaturated imines). 27 We speculated that if an IRED could catalyse both of these steps, in a similar fashion to recently reported biosynthetic oxidoreductases, 28,29 this biocatalyst could be applied to the CR-RA of α,β-unsaturated carbonyls and allow access to enantioenriched amine diastereomers (Fig. 1c).
In pursuit of this activity, we screened both reported 9,25,30-32 and our recently established (meta)genomic IREDs 3,33 for the complete reduction of cyclic enimine I to amine II ( Fig. 1d and Supplementary Table. ST1). Amongst the 389 IREDs screened, we observed that 262 catalysed reduction of I, with the majority behaving conventionally, i.e. reducing the C=N bond only (206 enzymes, 53%). Gratifyingly, a smaller subset of IREDs were able to catalyse the reduction of both the C=C and C=N bonds of I to the diastereomerically enriched product II (44 enzymes, 11%). Furthermore, in a complementary fashion, some IREDs reduced solely the C=C bond (12 enzymes, 3%). Mapping the reaction pro les against genetic sequence indicated localised sequence-activity correlation only (Extended Data Fig. ED1). One particular metagenomic enzyme, likely originating from an unclassi ed Pseudomonas species (pIR-120, Fig. 1d), exhibited excellent full reduction of I to II, and hence this enzyme was selected for further study.
pIR-120 was next examined for the ability to catalyse CR-RA of cyclohex-2-enone 1 with allylamine a, monitoring for potential reduced and coupled products 1a, 1', 1'a including aza-conjugate addition. The reaction proceeded with high conversion, forming predominately CR-RA product 1'a and CR product 1' without concomitant generation of direct RA product 1a. In the absence of pIR-120, or the nicotinamide cosubstrate regeneration system, no reduction products could be detected. Optimisation of the buffer type, pH as well as co-solvent (Extended Data Fig. ED2) increased the conversion to 1'a and could be scaled-up using 20 eq. of a yielding the hydrochloride salt in 69% yield (Fig. 1e).
We explored the substrate scope of pIR-120 using the optimised reaction conditions (Fig. 2). Preliminary experiments revealed that cyclopropylamine b displayed high activity with α,β-unsaturated carbonyls and hence was screened against a large panel of substrate partners 1-23 (for a full list of substrates see Supplementary Fig. SF4). pIR-120 exhibited a broad substrate scope, accepting enals as well as acyclic and cyclic enones, yielding CR and mono-RA products. Generally, unhindered enals and enones could be transformed with high chemoselectivity to the corresponding saturated amines. This trend is observed by comparing the reaction pro les of increasingly hindered C 2 -substituted but-2-enals 3-5 or decreasingly hindered amine donors a-c with cinnamaldehyde 6.
A broad selection of amine partners was explored using 3-methyl-cyclohexenone 15 as substrate partner.
Excellent conversion, chemo-, enantio-and diastereoselectivities were observed for small linear primary amines a-c, e-h. Notably, functionalised products from amines a, e, g, h could be formed e ciently as well as the secondary amine pyrrolidine i.
We were also keen to see if CR-RA products with additional stereocentres could be synthesised. (R) or (S)-3-uoropyrrolidine j could be coupled e ciently with 3-methylcyclohexenone 15, affording (cis)-15'j with high chemo-and diastereoselectivity (see Supplementary Discussion for details on the use of rac-j). Note that the introduction of an F-substituent on the pyrrolidine ring led to a change in relative stereochemistry across the cyclohexylamine ring. Furthermore, CR-RA of cyclopropylamine b with racemic enone 23 demonstrated that the single catalyst could control three stereocentres on cyclohexylamine ring 23'b, offering excellent enantioselectivity as well as good chemo-and diastereoselectivity.
To assess the synthetic applicability of the pIR-120-catalysed CR-RA, preparative-scale syntheses were performed using 15 partnered with a, b or i as well as 16 combined with b forming 15'a, 15'b, 15'i, and 16'b as the hydrochloride salts in 81%, 77%, 60% and 72% isolated yield respectively (Extended Data We next carried out mechanistic investigations to further characterise pIR-120 and identify any intermediates formed during CR-RA. Isotopic labelling experiments, using the in situ generated deuterated nicotinamide cosubstrate from D-glucose-1-d 1 , yielded 1,3-d 2 -15'b from 15 and b as the hydrochloride salt (Fig. 3a, 75% isolated yield, 91% 2D incorporation). This isotopic labelling pattern suggests pIR-120 mediates asymmetric hydride transfer at both C1 and C3 of the unsaturated carbonyl substrate via a nicotinamide cosubstrate. We were also keen to probe the enzyme-substrate complex formed during the CR step. Omitting any amine donor from the reaction yielded no product of either CR or RA (Fig. 3d), suggesting that the IREDcatalysed reaction explicitly requires the presence of an amine in the catalytic cycle. Furthermore, no activity was observed when combining either enone 15 with tertiary amine donor triethylamine k or unsaturated ester methyl cyclohex-1-ene-1-carboxylate and cyclopropylamine b, suggesting that pIR-120catalysed CR likely occurs via an enimine-NAD(P)H-enzyme complex, reminiscent of organocatalytic CR systems. 34 To the best of our knowledge this is the rst example of an enzyme that achieves CR by this type of enimine intermediate.
The multiple activities of pIR-120 prompted us to study its structure using X-ray crystallography. Crystals of pIR-120 in complex with NADP + were obtained in the P2 1 space group with two molecules in the asymmetric unit, forming the now familiar domain swapping dimeric fold observed in for the IRED family ( Fig. 3e). 9,35 A comparison of the monomer structure with others in the Protein DataBank using the DALI server 36 revealed that the closest existing IRED structures in the database were those from Streptosporangium roseum (PDB code 5OCM; 30% seq id; rmsd 1.6 Å over 286 Ca atoms), 37 Aspergillus oryzae (5G6S; 30%; 1.6 Å) 9 and Stackebrandtia nassauensis (6JIT, 30%; 2.0 Å). The most striking differences with other IRED folds having structures in the database were observed in the active site (Fig.   3f).
pIR-120 possesses a tyrosine residue, Y177 at the top of the ceiling of the active site as drawn, in common with other IREDs, such as those from Streptomyces sp. GF3546 (4OQY), 38 Bacillus cereus (4D3F), 39 and Nocardopsis halophila (4D3S) 38 , which have been shown to display (S)-stereoselectivity for the reduction of the model imine compound 2-methyl pyrroline. In common with those enzymes, in pIR-120 Y177 forms a hydrogen bond with the hydroxyl group of a side-chain, in this case threonine T101, which in turn H-bonds to the 2'-hydroxyl of the ribose in NADP + . However, pIR-120 also possesses an additional tyrosine residue Y181 that also points into the active site towards the cosubstrate binding cleft, which is a hydrophobic leucine in both 5OCM and 6JIT. The active site also features a number of cyclic and hydrophobic amino acid side-chains F185, Y269, H245 and A240 with Y129 at the rear and V244 at the front, that form a closed cavity which has been previously observed to be suitable for binding, especially of planar cyclic imines in IRED structures. 9,40 A model of the enzyme active site in complex with the enimine formed by condensation of 15 with b was constructed using AutoDock Vina (Fig. 3f). 41 In the top pose, the model suggests that the closest atom to the C4 of the pyridinium ring of the cosubstrate, suitable for acceptance of a hydride, is the prochiral carbon atom of the C=C bond. Delivery of a hydride to this atom as shown in the model would give the experimentally observed (R)-con guration at this centre.
Based on our structural and mechanistic investigation the following dual pIR-120 catalytic cycle is proposed for productive CR-RA (Fig. 4a). First, the nicotinamide cosubstrate and condensation product of α,β-unsaturated carbonyl V and amine form an active-site enimine-NAD(P)H-pIR-120 complex VI. Where substrate orientation kinetically favours the C4 of the enimine orientated toward the nicotinamide hydride, CR yields the stereoenriched 1-enamine-NAD(P) + -pIR-120 complex VII. Following this, the oxidised cosubstrate and prochiral 1-enamine are expelled from the enzyme, with the latter being hydrolysed in solution to form the stereoenriched carbonyl VIII. A further NAD(P)H cosubstrate binds to the enzyme together with the condensation product of the previously released carbonyl VIII and amine to form complex IX which undergoes the expected IRED-catalysed RA, 9 yielding the stereoenriched nal product X.
Finally, we sought to further extend the enzyme catalysed CR-RA by employing a conjugated dienylketone. Whilst 24 was susceptible to aza-conjugate addition, pIR-120-catalysed 4-and 6-electron CR-RA of α,β,γ,δ-unsaturated enone 24 in combination with cyclopropylamine b, affording 24'b and (trans)-24"b=16'b respectively (Fig. 4b). Interestingly, 6-electron CR-RA product 24"b=16'b possessed analogous diastereo-and enantioselectivity to the CR-RA of the corresponding ethyl substituted α,βunsaturated enone 16, suggesting that reduction of 24 proceeds by a similar pathway to that of 16. This experiment suggests that pIR-120 could be used to establish additional stereogenic centres during the CR-RA process.
In summary, we report the discovery and characterisation of a multi-functional biocatalyst (pIR-120) that is able to catalyse CR, using a previously unreported amine activation mechanism, as well as imine reduction and RA. pIR-120 possesses broad substrate scope allowing for the stereoselective preparation of valuable amine diastereomers in a one-pot, one-catalyst reaction starting from simple prochiral starting materials. Mechanistic and structural studies reveal a multi-step process in which pIR-120, which possesses an unusual additional tyrosine residue (Y181) in its active site, rst catalyses amine-activated CR of α,β-unsaturated carbonyl via a previously undescribed enimine-NAD(P)H-enzyme complex, followed by RA. This new CR-RA reaction further expands the repertoire of imine reductases and emphasises their importance in the synthesis of stereochemically de ned chiral amines.

Methods
Cloning, expression and protein puri cation The codon-optimised pIR-120 gene sequence (TWIST Biosciences, US, GenBank accession number MW854365) was cloned into pET-28a-(+) (Supplementary Fig. SF1) and used to transform chemically competent E. coli BL21 (DE3). Cultivation was performed in 400 mL Terri c Broth (TB) media (Formedium, Hunstanton, Norfolk, UK) supplemented with 35 µg·mL -1 kanamycin in 2 L Erlenmeyer ba ed asks. Cultures were incubated at 37 °C and shaken at 200 rpm until an optical density (OD 600nm ) of 0.6-0.8, before gene expression was induced with isopropyl β-D-1-thiogalactopyranoside (IPTG) at a nal concentration of 100 µM. Incubation was continued at 23 °C and 200 rpm for 25 h before harvesting the biomass using centrifugation. Disruption of the cells using sonication in an ice bath, then clari cation by centrifugation and lyophilisation afforded powdered pIR-120 cell-free extract (CFE). To obtain puri ed enzyme, powdered pIR-120 CFE was resuspended, clari ed by centrifugation and immobilised metal a nity chromatography (IMAC) was performed. Further puri cation of the protein for crystallography and control and mechanistic experiments was realised using gel ltration (GF) chromatography.   Substrate scope of pIR-120-catalysed CR-RA. Major RA product is drawn, product distribution is illustrated by the donut chart, % conversion = ∑(area% of CR and RA products). a non-enzymatic aza-conjugate addition product observed, b certain components determined by analogy based on the GC-MS spectrum, see Supplementary Table S2 for further details. Mechanistic and structural studies. a: deuterium labelling experiment, b and c: isolated reactions of potential CR-RA pathway intermediates, d: amine donor control experiment, e: Structure of pIR-120 dimer in ribbon format with monomers A and B shown in green and blue respectively, f: Active site of pIR-120 in complex with NADP+. Side chains of monomers A and B are shown in cylinder format with carbon atoms in green and blue respectively. The structure has been used to model the enimine ligand (the condensation product of 15' and b), shown with carbon atoms in yellow. The distance between these atoms is given in Ångstroms.

Figure 4
Proposed catalytic cycle of productive pIR-120 conjugate reduction-reductive amination (CR-RA) and extension to 6-electron CR-RA. a: schematic of the proposed catalytic cycles for pIR-120-catalysed CR and RA demonstrating the productive CR-RA reaction of α,β-unsaturated carbonyls and amines, b: example of 6-electron enzymatic CR-RA catalysed by pIR-120 only, by-product formation observed, see Supplementary Information for further details.

Supplementary Files
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