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Allylic amines are a versatile class of synthetic precursors of many valuable nitrogen-containing organic compounds, including pharmaceuticals. Enzymatic allylic amination methods provide a sustainable route to these compounds but are often restricted to allylic primary amines. We report a biocatalytic system for the reductive N-allylation of primary and secondary amines, using biomass-derivable cinnamic acids. The two-step one-pot system comprises an initial carboxylate reduction step catalyzed by a carboxylic acid reductase to generate the corresponding α,β-unsaturated aldehyde in situ. This is followed by reductive amination of the aldehyde catalyzed by a bacterial reductive aminase pIR23 or BacRedAm to yield the corresponding allylic amine. We exploited pIR23, a prototype bacterial reductive aminase, self-sufficient in catalyzing formal reductive amination of α,β-unsaturated aldehydes with various amines, generating a broad range of secondary and tertiary amines accessed in up to 94% conversion under mild reaction conditions. Analysis of products isolated from preparative reactions demonstrated that only selective hydrogenation of the C=N bond had occurred, preserving the adjacent alkene moiety. This process represents an environmentally benign and sustainable approach for the synthesis of secondary and tertiary allylic amine frameworks, using renewable allylating reagents and avoiding harsh reaction conditions. The selectivity of the system ensures that bis-allylation of the alkylamines and (over)reduction of the alkene moiety are avoided.

Monoacylated diamines are common motifs present in pharmaceuticals, agrochemicals, and natural products. However, the synthesis of these compounds often requires selective protection/deprotection s...

Allylic amines are a versatile class of synthetic precursors of many valuable nitrogen-containing organic compounds, including pharmaceuticals. Enzymatic allylic amination methods provide a sustainable route to these compounds but are often restricted to allylic primary amines. We report a biocatalytic system for the reductive

Sustainable production of fine chemicals and biofuels from renewable biomass offers a potential alternative to the continued use of finite geological oil reserves. However, in order to compete with current petrochemical refinery processes, alternative biorefinery processes must overcome significant costs and productivity barriers. Herein, we demonstrate the biocatalytic production of the versatile chemical building block, coniferol, for the first time, directly from lignocellulosic biomass. Following the biocatalytic treatment of lignocellulose to release and convert ferulic acid with feruloyl esterase (XynZ), carboxylic acid reductase (CAR) and aldo-keto reductase (AKR), this whole cell catalytic cascade not only achieved equivalent release of ferulic acid from lignocellulose compared to alkaline hydrolysis, but also displayed efficient conversion of ferulic acid to coniferol. This system represents a consolidated biodegradation–biotransformation strategy for the production of high value fine chemicals from waste plant biomass, offering the potential to minimize environmental waste and add value to agro-industrial residues.\ud \ud

The alkylation of amines with either alcohols or carboxylic acids represents a mild and safe alternative to the use of genotoxic alkyl halides and sulfonate esters. Here we report two complementary one-pot systems in which the reductive aminase (RedAm) from Aspergillus oryzae is combined with either (i) a 1° alcohol/alcohol oxidase (AO) or (ii) carboxylic acid/carboxylic acid reductase (CAR) to affect N-alkylation reactions. The application of both approaches has been exemplified with respect to substrate scope and also preparative scale synthesis. These new biocatalytic methods address issues facing alternative traditional synthetic protocols such as harsh conditions, overalkylation and complicated workup procedures.

The direct C-H carboxylation of aromatic compounds is an attractive route to the corresponding carboxylic acids, but remains challenging under mild conditions. It has been proposed that the first step in anaerobic microbial degradation of recalcitrant aromatic compounds is a UbiD-mediated carboxylation. In this study, we use the UbiD enzyme ferulic acid decarboxylase (Fdc) in combination with a carboxylic acid reductase to create aromatic degradation-inspired cascade reactions, leading to efficient functionalization of styrene through CO

Carboxylic acid reductases (CARs) are an emerging biocatalyst platform for the synthesis of a diverse array of aldehydes from carboxylic acids, operating chemoselectively and under mild aqueous conditions. As such, there is growing interest in the industrial application of these enzymes, both for the synthesis of aldehyde end-products, which are particularly prevalent in the flavor and fragrance industries, and aldehyde intermediates in multi-enzyme cascades. This perspective aims to review recent developments in the applications of CARs with a focus on the challenges and considerations involved in their implementation, as well as potential solutions with a view to increased industrial utility.

Fatty amine synthesis from renewable sources is an energetically-demanding process involving toxic metal catalysts and harsh reaction conditions as well as selectivity problems. Herein we present a mild, biocatalytic alternative to the conventional amination of fatty acids through a one-pot tandem cascade performed by a carboxylic acid reductase (CAR) and a transaminase (ω-TA). Saturated and unsaturated fatty acids, with carbon chain lengths ranging from C6 to C18, were successfully aminated obtaining conversions of up to 96%.

Carboxylic acid reductases (CARs) catalyze the reduction of a broad range of carboxylic acids to aldehydes using the cofactors adenosine triphosphate and nicotinamide adenine dinucleotide phosphate, and have become attractive biocatalysts for organic synthesis. Mechanistic understanding of CARs was used to expand reaction scope, generating biocatalysts for amide bond formation from carboxylic acid and amine. CARs demonstrated amidation activity for various acids and amines. Optimization of reaction conditions, with respect to pH and temperature, allowed for the synthesis of the anticonvulsant ilepcimide with up to 96 % conversion. Mechanistic studies using site-directed mutagenesis suggest that, following initial enzymatic adenylation of substrates, amidation of the carboxylic acid proceeds by direct reaction of the acyl adenylate with amine nucleophiles.

Carboxylic acid reductase (CAR) catalyzes the ATP- and NADPH-dependent reduction of carboxylic acids to the corresponding aldehydes. The enzyme is related to the nonribosomal peptide synthetases, consisting of an adenylation domain fused via a peptidyl carrier protein (PCP) to a reductase termination domain. Crystal structures of the CAR adenylation-PCP didomain demonstrate that large-scale domain motions occur between the adenylation and thiolation states. Crystal structures of the PCP-reductase didomain reveal that phosphopantetheine binding alters the orientation of a key Asp, resulting in a productive orientation of the bound nicotinamide. This ensures that further reduction of the aldehyde product does not occur. Combining crystallography with small-angle X-ray scattering (SAXS), we propose that molecular interactions between initiation and termination domains are limited to competing PCP docking sites. This theory is supported by the fact that (R)-pantetheine can support CAR activity for mixtures of the isolated domains. Our model suggests directions for further development of CAR as a biocatalyst.

Correction for ‘A biocatalytic cascade for the conversion of fatty acids to fatty amines’ by Joan Citoler, et al., Green Chem., 2019, 21, 4932–4935.

Colicin E9 is a nuclease antibiotic produced by E.coli to target and kill competing bacteria in times of stress. Immunity protein 9 (Im9) is expressed co-translationally and binds strongly to E9, inactivating the nuclease to protect the producing cell from the cytotoxic effects of E9. The affinity between E9:Im9 is highly avid with an off-rate of the order of days. However, upon binding to an outer membrane protein of a competing cell, a complex set of protein:protein interactions coupled to protein remodelling results in rapid Im9 release allowing E9 translocation into the cytoplasm and cell death on a timescale of minutes.In order to investigate the molecular basis for such an extreme switch in complex avidity we have used an atomic force microscope to perform dynamic force spectroscopy (DFS) measurements on E9:Im9. We observe a force-induced switching mechanism from the long lived complex (koff ∼ 1x10−5 s−1) in the absence of force to a much shorter lived state (koff ∼ 5 s−1) under the application of small biologically relevant forces. The decrease in complex lifetime under force can be abrogated by introduction of disulfide cross cross-links. This suggests that protein dynamics lie at the heart of the bipartite function of the complex switching from a highly stable complex protective to host, to one where rapid dissociation facilitates competitor death.

Colicins are antimicrobial proteins produced by bacteria. Their highly active modes of killing make colicins of interest as a potential new class of antibiotics. However, the precise mechanism of cytotoxicity is not understood thus limiting their translation to biotechnological applications.It is thought that colicin intoxication occurs via a series of protein-protein interactions (PPIs) that span the periplasm (Housden et al. Science, 2013). Mechanical force may play a role in this process by an inside-out energy transduction mechanism. Periplasmic proteins that are subverted by colicins have either a known role in applying force during their normal function in-vivo or are highly homologous to proteins that do.In this work we study the colicin E9 system and the PPIs that remodel during its translocation across the outer and inner membranes of E. coli. Through a series of in-vitro single molecule force spectroscopy experiments performed using an atomic force microscope, we identify a mechanism by which dissociation of the highly avid colicin E9-immunity protein interaction (fM affinity, half-life days) can take place via the application of small forces (

A single-molecule force study shows that rapid dissociation of a high-affinity protein interaction can be triggered by site-specific remodelling of one protein partner, and that prevention of remodelling maintains avidity., Colicins are protein antibiotics synthesised by Escherichia coli strains to target and kill related bacteria. To prevent host suicide, colicins are inactivated by binding to immunity proteins. Despite their high avidity (Kd≈fM, lifetime ≈4 days), immunity protein release is a pre-requisite of colicin intoxication, which occurs on a timescale of minutes. Here, by measuring the dynamic force spectrum of the dissociation of the DNase domain of colicin E9 (E9) and immunity protein 9 (Im9) complex using an atomic force microscope we show that application of low forces (, Author Summary Many proteins interact with other proteins as part of their function. One method of modulating the activity of protein complexes is to break them apart. Some complexes, however, are extremely kinetically stable and it is unclear how these can dissociate on a biologically relevant timescale. In this study we address this question using protein complexes between colicin E9 (a bacterial toxin) and its immunity protein Im9. These highly avid complexes (with a lifetime of days) must be broken apart for colicin to be activated. By using single-molecule force methods we show that pulling on one end of colicin E9 drastically destabilises the complex so that it dissociates a million-fold faster than its intrinsic rate. We then show that preventing this destabilisation (by the insertion of cross-links that pin the N-terminus of E9 in place) yields a kinetically stable complex. It has previously been postulated that force can destabilise a protein complex by partially unfolding one or more binding partners. Our work provides new experimental evidence that shows this is the case and provides a mechanism for this phenomenon, which we term a trip bond. For the E9:Im9 complex, trip bond behaviour allows a stable complex to be rapidly dissociated by application of a surprisingly small force.

The application of biocatalysis in the pharmaceutical industry is rapidly growing as a result of increased access to enzymes that meet the demands of industrial processes. This expansion of activity has led to a corresponding increase in the demand for immobilised enzymes. EziG™ (EnginZyme AB, Sweden) is marketed as a general matrix for enzyme immobilisation on controlled porosity glass. In this work we identified criteria for a “general” enzyme immobilisation technology in the context of the requirements of the pharmaceutical industry. We subsequently evaluated EziG™ for generality in a series of case studies for the application of immobilised biocatalysts. In this study we have focussed on the challenges facing both academic and industrial applications such as enzyme stability, multistep reactions and reactions in continuous flow.

The direct C–H carboxylation of aromatic compounds is an attractive route to the corresponding carboxylic acids, but remains challenging under mild conditions. It has been proposed that the first step in anaerobic microbial degradation of recalcitrant aromatic compounds is a UbiD-mediated carboxylation. In this study, we use the UbiD enzyme ferulic acid decarboxylase (Fdc) in combination with a carboxylic acid reductase to create aromatic degradation-inspired cascade reactions, leading to efficient functionalization of styrene through CO2 fixation. We reveal that rational structure-guided laboratory evolution can expand the substrate scope of Fdc, resulting in activity on a range of mono- and bicyclic aromatic compounds through a single mutation. Selected variants demonstrated 150-fold improvement in the conversion of coumarillic acid to benzofuran + CO2 and unlocked reactivity towards naphthoic acid. Our data demonstrate that UbiD-mediated C–H activation is a versatile tool for the transformation of aryl/alkene compounds and CO2 into commodity chemicals. Biocatalytic cascade reactions using engineered variants of ferulic acid decarboxylase coupled to carboxylic acid reductase utilize carbon dioxide fixation to enable the carboxylation and functionalization of styrene and other aromatic compounds.