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The authors have requested that this preprint be removed from Research Square.

Reductive amination of carbonyl compounds constitutes one of the most efficient ways to rapidly construct chiral and achiral amine frameworks. Imine reductase (IRED) biocatalysts represent a versatile family of enzymes for amine synthesis via NADPH mediated imine reduction. The reductive aminases (RedAms) are a sub-family of IREDs which have recently been shown to catalyse imine formation as well as imine reduction. Herein, a diverse library of novel enzymes were expressed and screened as cell-free lysates for their ability to facilitate reductive amination, in order to expand the known suite of biocatalysts for this transformation and to identify more enzymes with potential industrial applications. A range of ketones and amines were examined and enzymes were identified that were capable of accepting benzylamine, pyrrolidine, ammonia and aniline. Amine equivalents as low as 2.5 were employed to afford up to >99% conversion and for chiral products, up to >98% e.e. could be achieved. Preparative-scale reactions were conducted with low amine equivalents (1.5 or 2.0) of methylamine, allylamine and pyrrolidine, achieving up to >99% conversion and 76% isolated yield.

Biocatalytic retrosynthetic analysis of dibenz[c,e]azepines has highlighted the use of imine reductase (IRED) and ω-transaminase (ω-TA) biocatalysts to establish the key stereocentres of these molecules. Several enantiocomplementary IREDs were identified for the synthesis of (R)- and (S)-5-methyl-6,7-dihydro-5H-dibenz[c,e]azepine with excellent enantioselectivity, by reduction of the parent imines. Crystallographic evidence suggests that IREDs may be able to bind one conformer of the imine substrate such that, upon reduction, the major product conformer is generated directly. ω-TA biocatalysts were also successfully employed for the production of enantiopure 1-(2-bromophenyl)ethan-1-amine, thus enabling an orthogonal route for the installation of chirality into dibenz[c,e]azepine framework.

This publication describes the evaluation of four synthetic routes to the advanced pregabalin (Lyrica) intermediate 7. Asymmetric reduction of (E)-7 with an ene-reductase (OPR1 from Lycopersicon esculentum) gave a saturated cyanoester intermediate 5 with the desired S stereocenter in >99% ee. OPR1 also catalyzed the reduction of (Z)-7 to (S)-5, but with lower conversion and selectivity.

Neuronal hyperexcitability in both injured and adjacent uninjured neurons is associated with states of chronic injury and pain and is likely subject to neuroinflammatory processes. Chronic inflammatory responses are largely orchestrated by chemokines. One chemokine, monocyte chemoattractant protein-1 (MCP-1), in the presence of its cognate receptor, the β chemokine receptor 2 (CCR2), produces neural activity in dissociated neuronal cultures of neonatal dorsal root ganglion (DRG) neurons. Using a neuropathic pain model, chronic compression of the DRG (CCD), we compared anatomically separate populations of noncompressed lumbar DRG (L3/L6) with compressed lumbar DRG (L4/L5) for changes in the gene expression of CCR2.In situhybridization revealed that CCR2 mRNA was up-regulated in neurons and nonneuronal cells present in both compressed L4/L5 and ipsilateral noncompressed L3/L6 DRGs at postoperative day 5 (POD5). The total percentages of compressed and noncompressed neurons exhibiting CCR2 mRNA transcripts in L3, L5, and L6 DRG were 33 ± 3.5%, 49 ± 6.2%, and 41 ± 5.6%, respectively, and included cell bodies of small, medium, and large size. In addition, the preferred CCR2 ligand, MCP-1, was up-regulated by POD5 in both compressed L4/L5 and noncompressed L3/L6 DRG neurons. Application of MCP-1 to the cell bodies of the intact formerly compressed DRGin vitroproduced potent excitatory effects not observed in control ganglia. MCP-1/CCR2 signaling is directly involved with a chronic compression injury and may contribute to associated neuronal hyperexcitability and neuropathic pain.

Asymmetric bioreduction of an (E)-β-cyano-2,4-dienoic acid derivative by ene-reductases allowed a shortened access to a precursor of pregabalin [(S)-3-(aminomethyl)-5-methylhexanoic acid] possessing the desired configuration in up to 94% conversion and >99% ee. Deuterium labelling studies showed that the nitrile moiety was the preferred activating/anchor group in the active site of the enzyme over the carboxylic acid or the corresponding methyl ester.

The asymmetric bioreduction of a library of β-cyanoacrylate esters using ene-reductases was studied with the aim to provide a biocatalytic route to precursors for GABA analogues, such as pregabalin. The stereochemical outcome could be controlled by substrate-engineering through size-variation of the ester moiety and by employing stereochemically pure (E)- or (Z)-isomers, which allowed to access both enantiomers of each product in up to quantitative conversion in enantiomerically pure form. In addition, stereoselectivities and conversions could be improved by mutant variants of OPR1, and the utility of the system was demonstrated by preparative-scale applications.

Anaphylatoxin C5a is a potent inflammatory mediator associated with pathogenesis and progression of several inflammation-associated disorders. Small molecule C5a receptor (C5aR) antagonist development is hampered by species-specific receptor biology and the associated inability to use standard rat and mouse in vivo models. Gerbil is one rodent species reportedly responsive to small molecule C5aR antagonists with human C5aR affinity. We report the identification of the gerbil C5aR cDNA using a degenerate primer PCR cloning strategy. The nucleotide sequence revealed an open reading frame encoding a 347-amino acid protein. The cloned receptor (expressed in Sf9 cells) bound recombinant human C5a with nanomolar affinity. Alignment of the gerbil C5aR sequence with those from other species showed that a Trp residue in transmembrane domain V is the only transmembrane domain amino acid unique to small molecule C5aR antagonist-responsive species (i.e. gerbil, human, and non-human primate). Site-directed mutagenesis was used to generate human and mouse C5aRs with a residue exchange of this Trp residue. Mutation of Trp to Leu in human C5aR completely eliminated small molecule antagonist-receptor interaction. In contrast, mutation of Leu to Trp in mouse C5aR enabled small molecule antagonist-receptor interaction. This crucial Trp residue is located deeper within transmembrane domain V than residues reportedly involved in C5a- and cyclic peptide C5a antagonist-receptor interaction, suggesting a novel interaction site(s) for small molecule antagonists. These data provide insight into the basis for small molecule antagonist species selectivity and further define sites critical for C5aR activation and function.