Godwin A. Aleku

A reductive aminase from Aspergillus oryzae

Reductive amination is one of the most important methods for the synthesis of chiral amines. Here we report the discovery of an NADP(H)-dependent reductive aminase from Aspergillus oryzae (AspRedAm, Uniprot code Q2TW47) that can catalyse the reductive coupling of a broad set of carbonyl compounds with a variety of primary and secondary amines with up to >98% conversion and with up to >98% enantiomeric excess. In cases where both carbonyl and amine show high reactivity, it is possible to employ a 1:1 ratio of the substrates, forming amine products with up to 94% conversion. Steady-state kinetic studies establish that the enzyme is capable of catalysing imine formation as well as reduction. Crystal structures of AspRedAm in complex with NADP(H) and also with both NADP(H) and the pharmaceutical ingredient (R)-rasagiline are reported. We also demonstrate preparative scale reductive aminations with wild-type and Q240A variant biocatalysts displaying total turnover numbers of up to 32,000 and space time yields up to 3.73 g l−1 d−1. An enzyme (AspRedAm) capable of coupling carbonyls with a variety of amines in a reductive amination has now been discovered. Kinetic studies revealed that the enzyme catalysed both the imine formation step, as well as the reduction step. Structure and mutagenesis studies have highlighted essential catalytic residues and preparative scale examples have demonstrated total turnover numbers of up to 32,000.

Imine reductases (IREDs)

Imine reductases (IREDs) have emerged as a valuable new set of biocatalysts for the asymmetric synthesis of optically active amines. The development of bioinformatics tools and searchable databases has led to the identification of a diverse range of new IRED biocatalysts that have been characterised and employed in different synthetic processes. This review describes the latest developments in the structural and mechanistic aspects of IREDs, together with synthetic applications of these enzymes, and identifies ongoing and future challenges in the field.

Kinetic Resolution and Deracemization of Racemic Amines Using a Reductive Aminase

The NADP(H)‐dependent reductive aminase from Aspergillus oryzae (AspRedAm) was combined with an NADPH oxidase (NOX) to develop a redox system that recycles the co‐factor. The AspRedAm‐NOX system was applied initially for the kinetic resolution of a variety of racemic secondary and primary amines to yield S‐configured amines with enantiomeric excess (ee) values up to 99 %. The addition of ammonia borane to this system enabled the efficient deracemization of racemic amines, including the pharmaceutical drug rasagiline and the natural product salsolidine, with conversions up to >98 % and >99 % ee Furthermore, by using the AspRedAm W210A variant it was possible to generate the opposite R enantiomers with efficiency comparable to, or even better than, the wildtype AspRedAm.

Direct Alkylation of Amines with Primary and Secondary Alcohols through Biocatalytic Hydrogen Borrowing

The reductive aminase from Aspergillus oryzae (AspRedAm) was combined with a single alcohol dehydrogenase (either metagenomic ADH-150, an ADH from Sphingobium yanoikuyae (SyADH), or a variant of the ADH from Thermoanaerobacter ethanolicus (TeSADH W110A)) in a redox-neutral cascade for the biocatalytic alkylation of amines using primary and secondary alcohols. Aliphatic and aromatic secondary amines were obtained in up to 99 % conversion, as well as chiral amines directly from the racemic alcohol precursors in up to >97 % ee, releasing water as the only byproduct.

HIV point-of-care diagnostics: meeting the special needs of sub-Saharan Africa

Sub-Saharan Africa, accounting for 70% of the 35 million people living with HIV worldwide, obviously carries the heaviest burden of the HIV epidemic. Moreover, the regions poor health system occasioned by limited resources and inadequate skilled clinical personnel usually makes decentralization of HIV care difficult. Therefore, quality diagnostics that are easy to use, inexpensive, and amenable for use at point of care (POC) are a dire necessity. Clearly, such diagnostics will significantly lessen the pressure on the existing over-stretched centralized HIV laboratory services. Thankfully, some POC diagnostics are already being validated, while others are in the pipeline. As POC test kits emerge, implementation hurdles should be envisaged and planned for. This review examines emerging HIV diagnostic platforms, HIV POC product pipelines, gaps, perceived POC implementation challenges, and general recommendations for quality care.

Biocatalytic Routes to Enantiomerically Enriched Dibenz[c,e]azepines.

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.

Biocatalytic potential of enzymes involved in the biosynthesis of isoprenoid quinones

Naturally occurring isoprenoid quinones mediate electron transfer in respiratory or photosynthetic chains of living organisms. Tremendous progress has been made in the elucidation of biosynthetic pathways of prenylated quinones and a number of enzymes that catalyze specific transformation steps with remarkably high regio‐ and stereoselectivity have been characterized. Interestingly, some of these enzymes possess broad substrate scope towards synthetic analogues, thereby enabling their application as synthetic tools. The availability of mechanistic, structural and mutagenesis information for many of the pathway enzymes presents opportunities for protein engineering, a powerful approach that may improve the suitability of these biocatalysts for industrial processes.

Terminal Alkenes from Acrylic Acid Derivatives via Non-Oxidative Enzymatic Decarboxylation by Ferulic Acid Decarboxylases

Fungal ferulic acid decarboxylases (FDCs) belong to the UbiD‐family of enzymes and catalyse the reversible (de)carboxylation of cinnamic acid derivatives through the use of a prenylated flavin cofactor. The latter is synthesised by the flavin prenyltransferase UbiX. Herein, we demonstrate the applicability of FDC/UbiX expressing cells for both isolated enzyme and whole‐cell biocatalysis. FDCs exhibit high activity with total turnover numbers (TTN) of up to 55000 and turnover frequency (TOF) of up to 370 min−1. Co‐solvent compatibility studies revealed FDCs tolerance to some organic solvents up 20 % v/v. Using the in‐vitro (de)carboxylase activity of holo‐FDC as well as whole‐cell biocatalysts, we performed a substrate profiling study of three FDCs, providing insights into structural determinants of activity. FDCs display broad substrate tolerance towards a wide range of acrylic acid derivatives bearing (hetero)cyclic or olefinic substituents at C3 affording conversions of up to >99 %. The synthetic utility of FDCs was demonstrated by a preparative‐scale decarboxylation.