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The ability to programme new modes of catalysis into proteins would allow the development of enzyme families with functions beyond those found in nature. To this end, genetic code expansion methodology holds particular promise, as it allows the site-selective introduction of new functional elements into proteins as non-canonical amino acid side chains.1-4 Here, we exploit an expanded genetic code to develop a photoenzyme that operates via triplet energy transfer catalysis, a versatile mode of reactivity in organic synthesis that is currently not accessible to biocatalysis.5-12 Installation of a genetically encoded photosensitiser into the beta-propeller scaffold of DA_20_0013 converts a de novo Diels-Alderase into a photoenzyme for [2+2]-cycloadditions (EnT1.0). Subsequent development and implementation of a platform for photoenzyme evolution afforded an efficient and enantioselective enzyme (EnT1.3, up to 99% e.e.) that can promote intramolecular and bimolecular cycloadditions, including transformations that have proven challenging to achieve selectively with small molecule catalysts. EnT1.3 performs >300 turnovers and, in contrast to small molecule photocatalysts, can operate effectively under aerobic conditions and at ambient temperatures. An X-ray crystal structure of an EnT1.3-product complex shows how multiple functional components work in synergy to promote efficient and selective photocatalysis. This study opens up a wealth of new excited-state chemistry in protein active sites and establishes the framework for developing a new generation of enantioselective photocatalysts.

Imine reductases catalyse the reductive amination of aldehydes or ketones with amines to produce chiral amines—a key transformation in the preparation of fine chemicals and active pharmaceutical ingredients. Although significant progress has been recently made in the field, their industrial application has not been demonstrated. Herein, we describe a wild-type imine reductase that was engineered to perform reductive amination with concomitant substrate amine resolution to give a commercially relevant manufacturing process to lysine-specific demethylase-1 inhibitor GSK2879552. Three rounds of evolution resulted in an enzyme variant showing a >38,000-fold improvement over wild type. The engineering of a more stable and active enzyme variant enabled process optimization to an economic, high quality and sustainable operating space. Using the evolved enzyme, kilogram quantities of a key intermediate to GSK2879552 were produced in 84% yield, at 99.9% purity and >99.7% enantiomeric excess, with improved process mass intensity. Imine reductases have been regarded as one of the most promising enzymes by the pharmaceutical industry—but their industrial application is still lacking. This work reports the successful industrial application of an imine reductase, enabled by directed evolution.

The ability to quickly and easily assess the activity of large collections of enzymes for a desired substrate holds great promise in the field of biocatalysis. Cell-free synthesis, although not practically amenable for large-scale enzyme production, provides a way to accelerate the timeline for screening enzyme candidates using small-scale reactions. However, because cell-free enzyme synthesis requires a considerable amount of template DNA, the preparation of high-quality DNA “parts” in large quantities represents a costly and rate-limiting prerequisite for high throughput screening. Based on time-cost analysis and comparative activity data, a cell-free workflow using synthetic DNA minicircles and rolling circle amplification enables comparable biocatalytic activity to cell-based workflows in almost half the time. We demonstrate this capability using a panel of sequences from the carbon-nitrogen hydrolase superfamily that represent possible green catalysts for synthesizing small molecules with less waste compared to traditional industrial chemistry. This method provides a new alternative to more cumbersome plasmid- or PCR-based protein expression workflows and should be amenable to automation for accelerating enzyme screening in industrial applications.

Several biocatalytic approaches for the preparation of optically pure methyl 3-oxocyclohexanecarboxylates (S)-, (R)-1 and 3-oxocyclohexanecarbonitriles (S)-, (R)-2 have been successfully demonstrated. Screening of reaction-focused enzyme collections was used to identify initial hits using three enzymatic strategies. Reaction optimization and scale-up enabled the production of chiral intermediates for route scouting efforts on scales of up to 100 g. The enzymes applied in these processes (lipases, enoate reductases, and nitrilases) have been shown to be robust catalysts for drug manufacturing and represent a green alternative to conventional methods to access these chiral cyclohexanone building blocks.

An epoxide resolution process was rapidly developed that allowed access to multigram scale quantities of (R)-2-butyl-2-ethyloxirane 2 at greater than 300 g/L reaction concentration using an easy-to-handle and store lyophilized powder of epoxide hydrolase from Agromyces mediolanus. The enzyme was successfully fermented on a 35 L scale and stability increased by downstream processing. Halohydrin dehalogenases also gave highly enantioselective resolution but were shown to favor hydrolysis of the (R)-2 epoxide, whereas epoxide hydrolase from Aspergillus niger instead provided (R)-7 via an unoptimized, enantioconvergent process.

[Image: see text] The use of biocatalysis in the manufacture of small molecule active pharmaceutical ingredients has seen a marked increase over the past decade. Driven by academic and industrial interest in the application of enzymes as catalysts for transforming chemical routes, the biocatalytic toolbox available to a chemist has continued to expand. Despite this, the application of biocatalysis in early discovery chemistry has trailed in comparison to its use in manufacturing routes. The authors offer their perspective on the adoption of biocatalysis in the early discovery space: highlighting challenges including enzyme supply and the biocatalysis business model, as well as recent trends that could spur more collaboration and access to enzymes for early discovery R&D activities.

Synthesis of 1,2- and 1,4-bis-thiohydantoins and hydantoins employing ethylenediamine and 1,4-diaminobutane as spacers is described. Compounds containing a two carbon bridge were synthesized by alkylation of ethylenediamine with two equivalents of N-t-butyl-α-(p-toluenesulfonyloxy)phenylacetamide 3. The phenyl isothiocyanate adduct of 3 cyclized in refluxing toluene to form 1a. Other isothiocyanate or isocyanate adducts derived from alkylation product 4 required hydrolysis to induce cyclization. Compounds 1b-1f were obtained in this way. Compounds with a four carbon bridge were obtained by reaction of two equivalents of methyl α-bromophenyl acetate and 1,4-diaminobutane to produce N,N'-bis-[(α-phenyl-α-methoxycarbonyl)methyl]butylenediamine 6. The isothiocyanate or isocyanate adducts from 6 cyclized, without hydrolysis, to form compounds 2a-2e.

A two step synthesis of a C(3) disubstituted oxindoles via the rhodium(II)-catalyzed coupling of diazoketone (6) and 3-methyloxindole (9) is reported.

The new chiral amino thiourea catalyst 3d promotes the highly enantioselective cyanosilylation of a wide variety of ketones. The hindered tertiary amine substituent plays a crucial role with regard to both stereoinduction and reactivity, suggesting a cooperative mechanism involving electrophile activation by thiourea and nucleophile activation by the amine.

[reaction: see text] Two complementary strategies for the synthesis of the diazonamide A bisaryl quaternary center are described. The first strategy relies upon an extremely facile tandem cyclopropanation/ring-opening sequence, which has proven amenable to chiral catalysis to provide enantioenriched material. The second strategy relies upon a more concise alkylation route ideal for material advancement.