Expanding the synthetic scope of biocatalysis by enzyme discovery and protein engineering

This paper is dedicated to the memory of Jon Williams who was a pioneer of chemo-enzymatic catalysis, a field which has grown significantly thanks to his vision and ideas.Despite the growing impact of enzyme catalysis in industrial chemistry, the full potential of this technology is yet to be unlocked. Accessing new chemistries and expanding the scope of existing reactions is necessary in order to make biocatalysis a pivotal technology in the manufacturing of chemicals across the whole industrial spectrum. This review highlights how the biocatalytic toolbox for synthetic chemistry has recently been expanded by extending the scope of industrially relevant reactions, and the addition of new reactions via enzyme discovery or protein engineering.We would like to thank the Industrial Biotechnology Innovation Centre (IBioIC) and Biotechnology and Biological Sciences Research Council (BBSRC) for the awarding the CASE studentship to J.R.M from Prozomix Ltd. J.M − S thanks the ARAID Foundation for personal funding and the Research Group E07_20R for scientific support. N.J.T is grateful to the ERC for the award of an Advanced Grant (Grant number 742987).Peer reviewe

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) which 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

Cascade processes merging chemical and enzyme catalysis

Cascade processes are an attractive strategy to rapidly build molecular complexity and circumvent the need to isolate reaction intermediates, providing higher efficiencies into synthetic routes with lower environmental toll. We have recently developed a new method to synthesise chiral 1,4-nitro alcohols by sequentially combining three transformations in the same reaction vessel via asymmetric C–C bond formation using a chiral thiourea catalyst and a bioreduction process as key steps.We would like to thank the Agencia Estatal de Investigación (AEI), the Ministerio de Ciencia e Innovación (Ministry of Science and Innovation, MCIN), and the EU for the financial support (PID2020-113351RA-100/AEI/10.13039/501100011033 and TED2021-130803B-I00 MCIN/AEI /10.13039/501100011033 NextGenerationEU/PRTR). J.M-S also thanks the Fundación Agencia Aragonesa para la Investigación y el Desarrollo (Aragonese Foundation for Research & Development, ARAID) for personal funding.Peer reviewe

Construction of chemoenzymatic linear cascades for the synthesis of chiral compounds

Inspired by nature, synthetic chemists try to mimic the efficient metabolic networks in living organisms to build complex molecules by combining different types of catalysts in the same reaction vessel. These multistep cascade processes provide many advantages to synthetic procedures, resulting in higher productivities with lower waste generation and cost. However, combining different chemo- and biocatalysts can be challenging as reaction conditions might differ greatly. As a highly multidisciplinary field that benefits from advances in chemical catalysis, molecular biology and reaction engineering, this area of study is rapidly progressing. In this Review, we highlight recent trends and advances in the construction of multistep chemoenzymatic one-pot cascades to access chiral compounds as well as the different strategies to solve current challenges in the field.We would like to thank the Agencia Estatal de Investigación for financial support (PID2020-113351RA-100/AEI/10.13039/ 501100011033), and the Research Group E07_20R for scientific support. J.M-S also thanks the ARAID Foundation for personal funding.Peer reviewe

Asymmetric (Transfer) Hydrogenation of Imines

Peer reviewe

Application of engineered biocatalysts for the synthesis of active pharmaceutical ingredients (APIs)

Genetic engineering of enzymes has played a significant role in multiple pharmaceutical synthetic processes. The need for selective, robust catalysts that can operate under process chemistry conditions has driven the need for mutagenesis approaches, which have successfully delivered a variety of process-optimized biocatalysts. In this chapter we give an overview of different directed evolution methods that have been applied to pharmaceutical intermediates during the last decade. The exciting progress that has been made since the landmark sitagliptin study in 2010 demonstrates the role that engineered biocatalysts can play in the manufacture of active pharmaceutical ingredients (APIs).Peer reviewe

Chemoenzymatic one-pot cascade for the construction of asymmetric C-C and C-P bonds via formal C-H activation

The integration of organocatalysis and enzyme catalysis in one-pot cascade processes allows for the efficient construction of complex molecular architectures with high levels of stereocontrol. However, challenges related to reaction compatibility between both processes are often a limitation for the development of efficient synthetic routes. In this study, we describe the combination of an enzymatic aerobic oxidation followed by the squaramide-mediated asymmetric formation of C-P and C-C bonds to access important building blocks such as chiral α-hydroxy phosphonates and β-nitro alcohols in good yields and enantiomeric ratios. This sequential process is conducted in a one-pot fashion within a biphasic system and represents a pioneering example of a chemoenzymatic cascade involving aerobic biooxidation and an organocatalytic step operating under hydrogen-bond activation mode

Combinación de procesos bio y organocatalíticos para la generación de nuevos enlaces C-C

Resumen del póster presentado a la 9ª Jornada de Jóvenes Investigadores de Química y Física de Aragón, celebrada el 16 de diciembre de 2021 en la Universidad de Zaragoza.Peer reviewe

NAD(P)H-Dependent Dehydrogenases for the Asymmetric Reductive Amination of Ketones : Structure, Mechanism, Evolution and Application

Abstract. Asymmetric reductive aminations are some of the most important reactions in the preparation of active pharmaceuticals, as chiral amines feature in many of the world’s most important drugs. Although many enzymes have been applied to the synthesis of chiral amines, the development of reductive amination reactions that use enzymes is attractive, as it would permit the one-step transformation of readily available prochiral ketones into chiral amines of high optical purity. However, as most natural 'reductive aminase' activities operate on keto acids, and many are able to use only ammonia as the amine donor, there is considerable scope for the engineering of natural enzymes for the reductive amination of ketones, and also for the preparation of secondary amines using alkylamines as donors. This review summarises research into the development of NAD(P)H-dependent dehydrogenases for the reductive amination of ketones, including amino acid dehydrogenases (AADHs), natural amine dehydrogenases (AmDHs), opine dehydrogenases (OpDHs) and imine reductases (IREDs). In each case knowledge of the structure and mechanism of the enzyme class is addressed, with a further description of the engineering of those enzymes for the reductive amination of ketones towards primary and also secondary amine products