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Diazirines have been traditionally associated with chemical biology and materials chemistry, but their utility has been limited in organic chemistry. Recently in the Journal of the American Chemical Society, Arnold and co-workers employed diazirines as precursors to hemoprotein-derived metal carbenoid species.

The asymmetric reduction of ketones to chiral hydroxyl compounds by alcohol dehydrogenases (ADHs) is an established strategy for the provision of valuable precursors for fine chemicals and pharmaceutics. However, most ADHs favor linear aliphatic and aromatic carbonyl compounds, and suitable biocatalysts with preference for cyclic ketones and diketones are still scarce. Among the few candidates, the alcohol dehydrogenase from Thauera aromatica (ThaADH) stands out with a high activity for the reduction of the cyclic α-diketone 1,2-cyclohexanedione to the corresponding α-hydroxy ketone. This study elucidates catalytic and structural features of the enzyme. ThaADH showed a remarkable thermal and pH stability as well as stability in the presence of polar solvents. A thorough description of the substrate scope combined with the resolution and description of the crystal structure, demonstrated a strong preference of ThaADH for cyclic α-substituted cyclohexanones, and indicated structural determinants responsible for the unique substrate acceptance.

The synthesis of secondary and tertiary amines through the reductive amination of carbonyl compounds is one of the most significant reactions in synthetic chemistry. Asymmetric reductive amination for the formation of chiral amines, which are required for the synthesis of pharmaceuticals and other bioactive molecules, is often achieved through transition metal catalysis, but biocatalytic methods of chiral amine production have also been a focus of interest owing to their selectivity and sustainability. The discovery of asymmetric reductive amination by imine reductase (IRED) and reductive aminase (RedAm) enzymes has served as the starting point for a new industrial approach to the production of chiral amines, leading from laboratory-scale milligram transformations to ton-scale reactions that are now described in the public domain. In this perspective we trace the development of the IRED-catalyzed reductive amination reaction from its discovery to its industrial application on kg-ton scale. In addition to surveying examples of the synthetic chemistry that has been achieved with the enzymes, the contribution of structure and protein engineering to the understanding of IRED-catalyzed reductive amination is described, and the consequent benefits for activity, selectivity and stability in the design of process suitable catalysts.

Native Amine Dehydrogenases (nat-AmDHs) have recently emerged as a potentially valuable new reservoir of enzymes for the sustainable and selective synthesis of chiral amines, catalyzing the NAD(P)H-dependent ammoniation of carbonyl compounds with high activity and selectivity. MATOUAmDH2, recently identified from the Marine Atlas of Tara Oceans Unigenes (MATOUv1) database of eukaryotic genes, displays exceptional catalytic performance against its best identified substrate, isobutyraldehyde, as well as broader substrate scope than other nat-AmDHs. In the interests of providing a platform for the rational engineering of this and other nat-AmDHs, we have determined the structure of MATOUAmDH2 in complex with NADP+ and also with the cofactor and cyclohexylamine. Monomers within the structure are representative of more open and closed conformations of the enzyme and illustrate the profound changes undergone by nat-AmDHs during the catalytic cycle. An alanine screen of active site residues revealed that M215A and L180A are more active than the wild-type enzyme for the amination of cyclohexanone with ammonia and methylamine respectively, the latter suggesting that AmDHs have the potential to be engineered for the improved production of secondary amines.

Chiral amine diastereomers are ubiquitous in pharmaceuticals and agrochemicals,1 yet their preparation often relies on low-efficiency multi-step synthesis.2 These valuable compounds must be manufactured asymmetrically, as their biochemical properties can differ based on the chirality of the molecule. Herein, we report the discovery and characterisation of a multi-functional biocatalyst for amine synthesis, which operates using a previously unreported mechanism. This enzyme (EneIRED), identified within a metagenomic imine reductase (IRED) collection3 and originating from an unclassified Pseudomonas species, possesses an unusual active site architecture that facilitates amine-activated conjugate alkene reduction followed by reductive amination. This enzyme can couple a broad selection of α,β-unsaturated carbonyls with amines for the efficient preparation of chiral amine diastereomers baring up to three stereocentres. Mechanistic and structural studies have been carried out to delineate the order of individual steps catalysed by EneIRED 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.

Small optically active molecules, and more particularly short-chain chiral amines, are key 20 compounds in the chemical industry and precursors of various pharmaceuticals. Their chemo-21 biocatalytic production on a commercial scale is already established, mainly through lipase-22 catalyzed resolutions leading to ChiProsTM products among others. Nevertheless, their 23 biocatalytic synthesis still remains challenging for very short-chain C4 to C5 amines due to low 24 enantiomeric excess. To complement the possibilities recently offered by transaminases, this 25 work describes alternative biocatalytic access using amine dehydrogenases (AmDHs). Without 26 any protein engineering, some of the already described wild-type AmDHs (CfusAmDH, 27 MsmeAmDH, MicroAmDH and MATOUAmDH2) were shown to be efficient for the synthesis 28 of hydroxylated or unfunctionalized small 2-aminoalkanes. Conversions up to 97.1% were 29 reached at 50 mM, and moderate to high enantioselectivities were obtained, especially for (S)-30 1-methoxypropan-2-amine (98.1%), (S)-3-aminobutan-1-ol (99.5%), (3S)-3-aminobutan-2-ol 31 (99.4%) and the small (S)-butan-2-amine (93.6%) with MsmeAmDH. Semi-preparative scale 32 up experiments were successfully performed at 150 mM substrate concentrations for the 33 synthesis of (S)-butan-2-amine and (S)-1-methoxypropan-2-amine, the latter known as “(S)-34 MOIPA”. Modelling studies provided some preliminary results explaining the basis for the 35 challenging discrimination between similarly sized substituents in the active sites of these 36 enzymes.

Imine reductases (IREDs) offer biocatalytic routes to chiral amines and have a natural preference for the NADPH cofactor. In previous work, we reported enzyme engineering of the (R)-selective IRED from Myxococcus stipitatus (NADH-IRED-Ms) yielding a NADH-dependent variant with high catalytic efficiency. However, no IRED with NADH specificity and (S)-selectivity in asymmetric reductions has yet been reported. Herein, we applied semi-rational enzyme engineering to switch the selectivity of NADH-IRED-Ms. The quintuple variant A241V/H242Y/N243D/V244Y/A245L showed reverse stereopreference in the reduction of the cyclic imine 2- methylpyrroline compared to the wild-type and afforded the (S)- amine product with >99% conversion and 91% enantiomeric excess. We also report the crystal-structures of the NADPH-dependent (R)- IRED-Ms wild-type enzyme and the NADH-dependent NADH-IREDMs variant and molecular dynamics (MD) simulations to rationalize the inverted stereoselectivity of the quintuple variant.

The selective oxygenation of non-activated carbon atoms is an ongoing synthetic challenge and biocatalysts, particularly hemoprotein oxygenases, continue to be investigated for their potential, given both their sustainable chemistry credentials and also their superior selectivity. However, issues of stability, activity and complex reaction requirements often render these biocatalytic oxygenations problematic with respect to scalable industrial processes. A continuing focus on Cyto-chromes P450 (P450s), which require a reduced nicotinamide cofactor and redox protein partners for electron transport, has now led to better catalysts and processes with a greater understanding of process requirements and limitations for both in vitro and whole-cell systems. However, the discovery and development of unspecific peroxygenases (UPOs) has also recently provided valuable complementary technology to P450-catalyzed reactions. UPOs need only hydrogen peroxide to effect ox-ygenations but are hampered by their sensitivity to peroxide and also by limited selectivity. In this perspective we survey recent developments in the engineering of proteins, cells and processes for oxygenations by these two groups of hemopro-teins and evaluate their potential and relative merits for scalable reactions.

Cytochrome P450 CYP153AM.aq from Marinobacter aquaeolei serves as a model enzyme for the terminal (ω-) hy-droxylation of medium- to long-chain fatty acids. We have engineered this enzyme using different mutagenesis approaches based on structure-sequence-alignments within the 3DM database, crystal structures of CYP153M.aq. and a homolog CYP153AP.sp and site-directed saturation mutagenesis, to create a variant that ω-hydroxylates octanoic acid. The M.aqRLT variant exhibited 151-fold improved catalytic efficiency and showed strongly improved substrate binding, indicated by a 25-fold reduced Km compared to the wild type. Hence, we further investigated the variant computationally using MD simulations to gain deeper insights into the dynamics of the protein. We found the tunnel modifications and the two loop regions showing greatly reduced flexibility in the new variant were the main features responsible for stabilizing the ligand in the active site and enhancing catalytic efficiency. Additionally, we showed that a previously known fatty acid anchor (Q129R) interacts significantly with the ligand to hold it in the reactive position, thereby boosting the activity of the variant M.aq RLT towards octanoic acid. The study retrospectively demonstrates the drastic effects of substrate stabilization and simultaneously the impact of enzyme flexibility. These results could guide future engineering of enzymes with deeply buried active sites to increase or even establish new substrate activities.

Aromatic hydroxylation reactions catalyzed by heme-thiolate enzymes proceed via an epoxide intermediate. These aromatic epoxides could be valuable building blocks for organic synthesis giving access to a range of chiral transdisubstituted cyclohexadiene synthons. Here we show that naphthalene epoxides generated by fungal peroxygenases can be subjected to nucleophilic ring opening yielding non-racemic trans-disubstituted cyclohexadiene derivates, which in turn can be used for further chemical transformations. Following the ring-opening reactions, the synthetic possibility of cyclohexadiene derivates also demonstrated by four examples yielding functional compounds. This novel approach may represent a promising shortcut for the synthesis of natural products and APIs.

Controlling the selectivity of a chemical reaction with external stimuli is common in thermal processes, but rare in visible-light photocatalysis. Here we show that the redox potential of a carbon nitride photocatalyst (CN-OA-m) can be tuned by changing the irradiation wavelength to generate electron holes with different oxidation potentials. This tuning was the key to realizing photo-chemo-enzymatic cascades that give either the (S)- or the (R)-enantiomer of phenylethanol. In combination with an unspecific peroxygenase from Agrocybe aegerita, green light irradiation of CN-OA-m led to the enantioselective hydroxylation of ethylbenzene to (R)-1-phenylethanol (99 % ee). In contrast, blue light irradiation triggered the photocatalytic oxidation of ethylbenzene to acetophenone, which in turn was enantioselectively reduced with an alcohol dehydrogenase from Rhodococcus ruber to form (S)-1-phenylethanol (93 % ee). Version of record

A chemical method for the treatment of harmful halogenated compounds that has recently become of interest is the reductive dehalogenation of carbon-halogen bonds. In the case of a fluorine atom, this process is called hydrodefluorination. While many transition metal-based approaches now exist to reductively defluorinate aromatic fluoroarenes, the cleavage of C−F bonds in aliphatic compounds is not so well-developed. Here we propose a biocatalytic approach exploiting a promiscuous activity exhibited by transaminases (TAs). Hence, a series of α-fluoroketones have been defluorinated with excellent conversions using Chromobacterium violaceum and Arthrobacter sp. TAs under mild conditions and in aqueous medium, using a stoichiometric amount of an amine (e. g. 2-propylamine) as reagent and formally releasing its oxidized form (e. g. acetone), with ammonia and hydrogen fluoride as by-products. It is also demonstrated that this process can be performed in a regio- or stereoselective fashion. Version of record

Biocatalytic imine reduction has been a topic of intense research by the artificial metalloenzyme community in recent years. Artificial constructs, together with natural enzymes, have been engineered to produce chiral amines with high enantioselectivity. This review examines the design of the main classes of artificial imine reductases reported thus far and summarises approaches to enhancing their catalytic performance using complementary methods. Examples of utilising these biocatalysts in vivo or in multi-enzyme cascades have demonstrated the potential that artIREDs can offer, however, at this time their use in biocatalysis remains limited. This review explores the current scope of artIREDs and the strategies used for catalyst improvement, and examines the potential for artIREDs in the future.

The biocatalytic asymmetric synthesis of amines from carbonyl compounds and amine precursors presents an important advance in sustainable synthetic chemistry. Oxidoreductases (ORs) that catalyze the NAD(P)H-dependent reductive amination of carbonyl compounds directly to amines using amine donors present advantages complementary to those of amine transaminases (ATAs) with respect to selectivity, stability and substrate scope. Indeed some ORs accept alkyl and aryl amines as reaction partners enabling access to chiral secondary amine products that are not directly accessible using ATAs. Moreover, superior atom economy can usually be achieved as no sacrificial amines are required as with ATAs. In recent years a number of ORs that apparently catalyze both imine formation and imine reduction in the reductive amination of carbonyls has been identified using structure informed protein engineering, sequence analysis from natural biodiversity and increasingly a mixture of both. In this review we summarize the development of such enzymes from the engineering of amino acid dehydrogenases (AADHs) and opine dehydrogenases (OpDHs) to become amine dehydrogenases (AmDHs), which are active toward ketones devoid of any requisite carboxylate and/or amine functions, through to the discovery of native AmDHs and reductive aminases (RedAms), and the engineering of all of these scaffolds for improved or altered activity. Structural and mechanistic studies have revealed similarities, but also differences in the determinants of substrate binding and mechanism in the enzymes. The survey reveals that a complementary approach to enzyme discovery that utilizes both natural genetic resources and engineering can be combined to deliver biocatalysts that have significant potential for the industrial synthesis of chiral amines.

The interconversion of non-activated alkenes and alcohols, catalyzed by (de)hydratases, has great potential in biotechnology for the generation of fine and bulk chemicals. LinD is a cofactor-independent enzyme that catalyzes the reversible (de)hydration of the tertiary alcohol (S)-linalool to the triene beta-myrcene, and also its isomerization to the primary alcohol geraniol. Structure-informed mutagenesis of LinD, followed by activity studies, confirmed essential roles for residues C171, C180 and H129 in water activation for the hydration of beta-myrcene to linalool. However, no evidence of covalent thioterpene intermediates was found using either X-ray crystallography, mass spectrometry, or QM/MM nudged elastic band simula-tions. Labelling and NMR experiments confirmed a role for residue D39 in (de)protonation of the linalool carbon C10 in the isomerization of linalool to geraniol and also the intermediacy of beta-myrcene in this isomerization reaction. X-ray, molecular dynamics and activity studies also suggested a significant role in catalysis for a mobile methionine residue M125, which exists in substantially altered orientations in different mutant structures.

The biocatalytic synthesis of amides from carboxylic acids and primary amines in aqueous media can be achieved using the ATP-dependent amide bond synthetase McbA, via an adenylate intermediate, using only 1.5 equivalents of the amine nucleophile. Following earlier studies that characterized the broad carboxylic acid specificity of McbA, we now show that, in addition to the natural amine substrate 2-phenylethylamine, a range of simple aliphatic amines, including methylamine, butylamine and hexylamine and propargylamine are coupled efficiently to the native carboxylic acid substrate 1-acetyl-9H-β-carboline-3-carboxylic acid by the enzyme, to give amide products with up to >99% conversion. The structure of wild-type McbA in its ami-dation conformation, coupled with modeling and mutational studies, reveal an amide access tunnel and a possible role for residue D201 in amide activation. Amide couplings were slower with anilines and alicyclic secondary amines such as pyrrolidine and piper-idine. The broader substrate specificity of McbA was exploited in the synthesis of the monoamine oxidase A inhibitor moclobe-mide, through the reaction of 4-chlorobenzoic acid with 1.5 equivalents of 4-(2-aminoethyl)morpholine, and utilizing polyphos-phate kinases SmPPK and AjPPK in the presence of polyphosphoric acid and 0.1 equivalents of ATP, required for recycling of the cofactor.

Cyclic ketones bearing alpha-quaternary stereocenters undergo efficient kinetic resolution using cyclohexanone monooxygenase (CHMO) from Acinetobacter calcoaceticus. Lactones possessing tetrasubstituted stereocenters are obtained with high enantioselectivity (up to >99% ee) and complete chemoselectivity. Preparative scale biotransformations were exploited in conjunction with a SmI2-mediated cyclization process to access complex, enantiomerically enriched cycloheptan- and cycloctan-1,4-diols. In a parallel approach to structurally distinct products, enantioenriched ketones from the resolution bearing an alpha- all carbon quaternary stereocenter were used in a SmI2-mediated cyclization process to give cyclobutanol products (up to >99% ee).

Cyclic ketones bearing α‐quaternary stereocenters underwent efficient kinetic resolution using cyclohexanone monooxygenase (CHMO) from Acinetobacter calcoaceticus. Lactones possessing tetrasubstituted stereocenters were obtained with high enantioselectivity (up to >99 % ee) and complete chemoselectivity. Preparative‐scale biotransformations were exploited in conjunction with a SmI2‐mediated cyclization process to access complex, enantiomerically enriched cycloheptan‐ and cycloctan‐1,4‐diols. In a parallel approach to structurally distinct products, enantiomerically enriched ketones from the resolution with an α‐quaternary stereocenter were used in a SmI2‐mediated cyclization process to give cyclobutanol products (up to >99 % ee).

The Pictet-Spengler reaction is a valuable route to 1,2,3,4-tetrahydro-Β-carboline (THBC) and isoquinoline scaffolds found in many important pharmaceuticals. Strictosidine synthase (STR), catalyzes the Pictet-Spengler condensation of tryptamine and the aldehyde secologanin to give (S)-strictosidine as a key intermediate in indole alkaloid biosynthesis. STRs also accept shortchain aliphatic aldehydes to give enantioenriched alkaloid products with up to 99% e.e. STRs are thus valuable asymmetric organocatalysts for applications in organic synthesis. The STR catalysis of reactions of small aldehydes gives an unexpected switch in stereopreference, leading to formation of the (R)-products. Here we report a rationale for the formation of the (R)-configured products by the STR enzyme from Ophiorrhiza pumila (OpSTR) using a combination of X-ray crystallography, mutational and molecular dynamics (MD) studies. We discovered that short chain aldehydes bind in an inverted fashion compared to secologanin leading to the inverted stereopreference for the observed (R)-product in those cases. The study demonstrates that the same catalyst can have two different productive binding modes for one substrate – but give different absolute configuration of the products by binding the aldehyde substrate differently. These results will guide future engineering of STRs and related enzymes for biocatalytic applications.

A tandem enzymatic strategy to enhance the scope of Calkylation of small molecules via the in situ formation of S-adenosyl methionine (SAM) cofactor analogues is described. A solventexposed channel present in the SAM-forming enzyme SalL tolerates 5'-chloro-5’-deoxyadenosine (ClDA) analogues modified at the 2position of the adenine nucleobase. Coupling SalL-catalyzed cofactor production with C-(m)ethyl transfer to coumarin substrates catalyzed by the methyltransferase (MTase) NovO forms C(m)ethylated coumarins in superior yield and greater substrate scope relative to that obtained using cofactors lacking nucleobase modifications. Establishing the molecular determinants which influence C-alkylation provides the basis to develop a late-stage enzymatic platform for the preparation of high value small molecules

The synthesis of the amide bond between an amine and a carboxylic acid is one of the most significant reactions in industrial pharmaceutical synthesis. Despite the apparent simplicity of synthetic methods for amide bond formation, many of these are disadvantaged by their requirement for toxic or hazardous reagents for the activation of the acid component, or poor atom economy resulting from the need for stoichiometric amounts of coupling reagents. In this context, biocatalysis has emerged as an alternative catalytic method for amide bond formation, presenting the advantages of both environmentally benign reagents and conditions and also atom economy. In this review we detail developments in the enzyme-catalyzed preparation of secondary and tertiary amides for the synthesis of pharmaceutical-type molecules and review the applications of hydrolases, such as lipases and penicillin acylases, to these reactions. We also summarise the activity of ATP-dependent enzymes for amide bond formation and assess their potential for the preparative synthesis of amides from carboxylic acids and amines in aqueous media. Keywords: Biocatalysis, Amides, Lipase, Penicillin Acylase, NRPS, Amide Bond Synthetase

Having identified inconsistencies when repeating literature examples of photochemical transformations and difficulties recreating experimental setups, we devised several criteria that an ideal labscale reactor should achieve. Herein, we introduce a versatile photoreactor for high throughput screening, preparative scale batch reactions and continuous processing, all with a single light source. The reactor utilizes interchangeable arrays of pseudo-monochromatic high-power LEDs in a range of synthetically useful wavelengths, combined with excellent temperature control. Moreover, light intensity can be modulated in an accurate and straightforward manner. This system has subsequently been tested on a range of literature methodologies.

One of the major challenges in chemical synthesis is the selective oxyfunctionalization of non-activated C-H bonds, which can be enabled by biocatalysis using cytochrome P450 monooxygenases. In this study, we report on the characterization of the versatile CYP109Q5 from Chondromyces apiculatus DSM436, which is able to functionalize a wide range of substrates (terpenes, steroids and drugs), including the ring of β-ionone in non-allylic positions. The crystal structure of CYP109Q5 revealed flexibility within the active site pocket that permitted the accommodation of bulky substrates, and enabled a structure-guided approach to engineering the enzyme. Some variants of CYP109Q5 displayed a switch in selectivity towards the non-allylic positions of β-ionone, allowing the simultaneous production of 2- and 3-hydroxy-β-ionone, which are chemically challenging to synthesize and are important precursors for carotenoid synthesis. An efficient whole-cell system finally enabled the production of up to 0.5 g L-1 hydroxylated products of β-ionone; this system can be applied to product identification in further biotransformations. Overall, CYP109Q5 proved to be highly evolvable and active. The studies in this work demonstrate that, using rational mutagenesis, the highly versatile CYP109Q5 generalist can be progressively evolved to be an industrially valuable specialist for the synthesis of specific products

The explosive xenobiotic 2,4,6-trinitrotoluene (TNT) is a major worldwide environmental pollutant and its persistence in the environment presents health and environmental concerns. The chemical structure of TNT dictates that biological detoxification pathways follow predominantly reductive transformation of the nitro groups, and as a result, TNT is notoriously recalcitrant to mineralization in the environment. Plant-based technologies to remediate this toxic pollutant rely on a solid understanding of the biochemical detoxification pathways involved. Towards this, two Arabidopsis Tau class glutathione transferases, GSTU24 and GSTU25, have been identified that catalyze the formation of three TNT-glutathionylated conjugates. These two GSTs share 79 % identity yet only GSTU25 catalyzes the substitution of a nitro group for sulfur to form 2-glutathionyl-4,6-dinitrotoluene. The production of this compound is of interest because substitution of a nitro group could lead to destabilization of the aromatic ring, enabling subsequent biodegradation. To identify target amino acids within GSTU25 that might be involved in the formation of 2-glutathionyl-4,6-dinitrotoluene, the structure for GSTU25 was determined, in complex with oxidized glutathione, and used to inform site-directed mutagenesis studies. Replacement of five amino acids in GSTU24 established a conjugate profile and activity similar to that found in GSTU25. These findings contribute to the development of plant-based remediation strategies for the detoxification of TNT in the environment.

Reductive Aminases (RedAms) catalyze the asymmetric reductive amination of ketones with primary amines to give secondary amine products. RedAms have great potential for the synthesis of bioactive chiral amines, however, insights into their mechanism are currently limited. Comparative studies on reductive amination of cyclohexanone with allylamine in the presence of RedAms, imine reductases (IREDs) or NaBH3CN support the distinctive activity of RedAms in catalyzing both imine formation and reduction in the reaction. Structures of AtRedAm from Aspergillus terreus, in complex with NADPH and ketone and amine substrates, along with kinetic analysis of active-site mutants, reveal modes of substrate binding, the basis for the specificity of RedAms for reduction of imines over ketones, and the importance of domain flexibility in bringing the reactive participants together for the reaction. This information is used to propose a mechanism for their action and also to expand the substrate specificity of RedAms using protein engineering.

Chiral amines are valuable constituents of many important pharmaceutical compounds and their intermediates. It is estimated that nearly 40-45% small molecule pharmaceuticals contain chiral amine scaffolds in their structures. The major challenges encountered in the chemical synthesis of enantiopure amines are the use of toxic chemicals, formation of a large number of by-products, and multi-step syntheses. To address these limitations, cost-effective biocatalytic methods are maturing and proving to be credible alternatives for the synthesis of chiral amines in enantiomerically pure forms. Herein, we report the recent progress achieved and current perspectives in the enzymatic synthesis of chiral amines using four important enzymes i.e. imine reductases, amine dehydrogenases, monoamine oxidases and cytochrome P450s. Applications to the industrial synthesis of chiral amines are highlighted. Protein engineering approaches, which play a critical role in improving or altering enzyme activity and substrate scope, are also addressed along with the discovery of pioneering enzymatic activities from nature. This survey of recent work demonstrates that enzymatic approaches to the synthesis of chiral amines will continue to be a major focus of research in biocatalytic chemistry in the years to come.

Amide bond formation is one of the most important reactions in pharmaceutical synthetic chemistry. The development of sustainable methods for amide bond formation, including those that are catalyzed by enzymes, is therefore of significant interest. The ATP‐dependent amide bond synthetase (ABS) enzyme McbA, from Marinactinospora thermotolerans, catalyzes the formation of amides as part of the biosynthetic pathway towards the marinacarboline secondary metabolites. The reaction proceeds via an adenylate intermediate, with both adenylation and amidation steps catalyzed within one active site. In this study, McbA was applied to the synthesis of pharmaceutical‐type amides from a range of aryl carboxylic acids with partner amines provided at 1–5 molar equivalents. The structure of McbA revealed the structural determinants of aryl acid substrate tolerance and differences in conformation associated with the two half reactions catalyzed. The catalytic performance of McbA, coupled with the structure, suggest that this and other ABS enzymes may be engineered for applications in the sustainable synthesis of pharmaceutically relevant (chiral) amides.

Traditional methods of chemical synthesis of alkaloids exhibit various problems such as lack of enantioselectivity, the use of toxic chemical and intermediates, and multiple numbers of synthetic steps. Consequently, various enzymatic methods for the formation of C-C bonds in the alkaloid skeleton have been developed. Herein, we report advances achieved in the enzymatic or chemo-enzymatic synthesis of pharmaceutically important alkaloids that employ three C-C bond forming enzymes: two Pictet-Spenglerases and the oxidative C-C bond forming flavoenzyme Berberine Bridge Enzyme. Protein engineering studies, improving the substrate scope of these enzymes, and thereby leading to the synthesis of non-natural alkaloids possessing higher or newer pharmacological activities, are also discussed. Furthermore, the integration of these biocatalysts with other enzymes, in multi-enzymatic cascades for the enantioselective synthesis of alkaloids, is also reviewed. Current results suggest that these enzymes hold great promise for the generation of C-C bonds in the selective synthesis of alkaloid compounds possessing diverse pharmacological properties.

The kinetic resolution of amino acid esters (AAEs) is a useful synthetic strategy for the preparation of single enantiomer amino acids. The development of an enzymatic dynamic kinetic resolution (DKR) process for AAEs, which would give a theoretical yield of 100% of the enantiopure product, would require an amino acid ester racemase (AAER), however, no such enzyme has been described. We have identified low AAER activity of 15 U mg-1 in a homolog of a PLP-dependent α-amino ε-caprolactam racemase (ACLR) from Ochrobactrum anthropi. We have determined the structure of this enzyme, OaACLR, to a resolution of 1.87 Å and using structure-guided saturation mutagenesis, in combination with a colorimetric screen for AAER activity, we have identified a mutant, L293C, in which the promiscuous AAER activity of this enzyme towards L-phenylalanine methyl ester is improved 3.7 fold.

Chiral amines are important components of 40-45% of small molecule pharmaceuticals and many other industrially important fine chemicals and agrochemicals. Recent advances in synthetic applications of ω-transaminases for the production of chiral amines are reviewed herein. Although a new pool of potential ω-transaminases is being continuously screened and characterized from various microbial strains, their industrial application is limited by factors such as disfavored reaction equilibrium, poor substrate scope and product inhibition. We present a closer look at recent developments in overcoming these challenges by various reaction engineering approaches. Furthermore, protein engineering techniques, which play a crucial role in improving the substrate scope of these biocatalysts and their operational stability, are also presented. Last, the incorporation of ω-transaminases in multi-enzymatic cascades, which significantly improves their synthetic applicability in the synthesis of complex chemical compounds, is detailed. This analysis of recent advances shows that ω-transaminases will continue to provide an efficient alternative to conventional catalysis for the synthesis of enantiomerically pure amines.

We report the exploration of the evolutionary relationship between imine reductases (IREDs) and other dehydrogenases. This approach is informed by the sequence similarity between these enzyme families and the recently described promiscuous activity of IREDs for the highly reactive carbonyl compound 2,2,2-trifluoroacetophenone. Using the structure of the R-selective IRED from Streptosporangium roseum (R-IRED-Sr) as a model, β-hydroxyacid dehydrogenases (βHADs) were identified as the dehydrogenases most similar to IREDs. To understand how active site differences in IREDs and βHADs enable the reduction of predominantly C = N or C = O bonds respectively, we substituted amino acid residues in βHADs with the corresponding residues from the R-IRED-Sr and were able to increase the promiscuous activity of βHADs for C = N functions by a single amino acid substitution. Variants βHADAt-K170D and βHADAt-K170F lost mainly their keto acid reduction activity and gained the ability to catalyze the reduction of imines. Moreover, the product enantiomeric purity for a bulky imine substrate could be increased from 23% ee (R-IRED-Sr) to 97% ee (βHADAt-K170D/F-F231A) outcompeting already described IRED selectivity.

Cyclic ketones bearing α-quaternary stereocenters undergo efficient kinetic resolution using cyclohexanone monooxygenase (CHMO) from Acinetobacter calcoaceticus. Lactones possessing tetrasubstituted stereocenters are obtained with high enantioselectivity (up to >99% ee) and complete chemoselectivity. Preparative scale biotransformations were exploited in conjunction with a SmI2-mediated cyclization process to access complex, enantiomerically enriched cycloheptan- and cycloctan-1,4-diols. In a parallel approach to structurally distinct products, enantioenriched ketones from the resolution bearing an α- all carbon quaternary stereocenter were used in a SmI2-mediated cyclization process to give cyclobutanol products (up to >99% ee).

Glutathione transferases (GSTs) are involved in many processes in plant biochemistry, with their best characterised role being the detoxification of xenobiotics through their conjugation with glutathione. GSTs have also been implicated in noncatalytic roles, including the binding and transport of small heterocyclic ligands such as indole hormones, phytoalexins and flavonoids. Although evidence for ligand binding and transport has been obtained using gene deletions and ligand binding studies on purified GSTs, there has been no structural evidence for the binding of relevant ligands in noncatalytic sites. Here we provide evidence of noncatalytic ligand-binding sites in the phi class GST from the model plant Arabidopsis thaliana, AtGSTF2, revealed by X-ray crystallography. Complexes of the AtGSTF2 dimer were obtained with indole-3-aldehyde, camalexin, the flavonoid quercetrin and its non-rhamnosylated analogue quercetin, at resolutions of 2.00, 2.77, 2.25 and 2.38 Å respectively. Two symmetry-equivalent-binding sites (L1) were identified at the periphery of the dimer, and one more (L2) at the dimer interface. In the complexes, indole-3-aldehyde and quercetrin were found at both L1 and L2 sites, but camalexin was found only at the L1 sites and quercetin only at the L2 site. Ligand binding at each site appeared to be largely determined through hydrophobic interactions. The crystallographic studies support previous conclusions made on ligand binding in noncatalytic sites by AtGSTF2 based on isothermal calorimetry experiments (Dixon et al. (2011) Biochem J438, 63-70) and suggest a mode of ligand binding in GSTs commensurate with a possible role in ligand transport.

The structure of a P450 ω-hydroxylase bound to its fatty acid product was determined, which revealed a narrow substrate tunnel that leads to the heme. The introduction of an arginine side chain in proximity to the carboxyl group of the fatty acid led to a reduced KM value for dodecanoic acid, which suggests the importance of an anchoring point in the active site. An increase in the flexibility of the substrate recognition region was also engineered, which resulted in a threefold improved product formation.

Chiral primary amines are important intermediates in the synthesis of pharmaceutical compounds. Fungal reductive aminases (RedAms) are NADPH-dependent dehydrogenases that catalyse reductive amination of a range of ketones with short-chain primary amines supplied in an equimolar ratio to give corresponding secondary amines. Herein we describe structural and biochemical characterisation as well as synthetic applications of two RedAms from Neosartorya spp. (NfRedAm and NfisRedAm) that display a distinctive activity amongst fungal RedAms, namely a superior ability to use ammonia as the amine partner. Using these enzymes, we demonstrate the synthesis of a broad range of primary amines, with conversions up to >97% and excellent enantiomeric excess. Temperature dependent studies showed that these homologues also possess greater thermal stability compared to other enzymes within this family. Their synthetic applicability is further demonstrated by the production of several primary and secondary amines with turnover numbers (TN) up to 14000 as well as continuous flow reactions, obtaining chiral amines such as (R)-2-aminohexane in space time yields up to 8.1 g L-1 h-1. The remarkable features of NfRedAm and NfisRedAm highlight their potential for wider synthetic application as well as expanding the biocatalytic toolbox available for chiral amine synthesis.

Artificial metalloenzymes can be defined as the fusion of chemo- and enzymatic catalysts by insertion of non-specific achiral catalytic moieties into the chiral environment of a protein cavity, thereby broadening the scope of both fields. Presently, the field of artificial metalloenzymes is at an exciting stage, with interesting features already demonstrated and a growing reaction repertoire, but has not yet reached maturity. To exploit the full potential of catalytic reactions using hybrid catalysts is far from trivial and as the ability to screen for novel properties remains limited. Albeit computational simulations are advanced, directed/designed evolution of such selective catalysts remains challenging, hence their development relies to a great extent on trial-and-error. Hitherto, screening and evaluation of ligands for activity or of proteins for selectivity still remains an elaborate process, as all library members have to be evaluated individually. High-throughput analysis to simultaneously evaluate large hybrid catalyst libraries within a short time-frame has therefore become increasingly important. The key focuses of this research were the development of (i) novel purification strategies to simplify the challenge of producing functional protein (streptavidin), in sufficient quantity and appropriate purity for high-throughput screening of organometallic moieties; (ii) a reiterative approach to protein expression: re-design of streptavidin construct, moving to a more complex expression system, Pichia pastoris, to increase the production of streptavidin and simplify the purification process; and (iii) a novel biomolecular scaffold, human carbonic anhydrase II, for the creation of a new artificial metalloenzyme. This thesis addresses the issues and feasibility of purification and expression processes and, highlights bottlenecks that arose using these technologies, key learning's and where to go from now.