Balance between photoreduction efficiency, cofactor affinity, and allosteric coupling of halogenase flavoenzymes.

Flavin-dependent halogenases (FDHs) are promising candidates for the sustainable production of halogenated organic molecules by biocatalysis. FDHs require only oxygen, halide and a fully reduced flavin adenine dinucleotide (FADH) cofactor to generate the reactive HOX that diffuses 10 Å to the substrate binding pocket and enables regioselective oxidative halogenation. A key challenge for the application of FDHs is the regeneration of the FADH.

The Impact of Metagenomics on Biocatalysis.

In the ever-growing demand for sustainable ways to produce high-value small molecules, biocatalysis has come to the forefront of greener routes to these chemicals. As such, the need to constantly find and optimise suitable biocatalysts for specific transformations has never been greater. Metagenome mining has been shown to rapidly expand the toolkit of promiscuous enzymes needed for new transformations, without requiring protein engineering steps.

Biocatalysis in Drug Design: Engineered Reductive Aminases (RedAms) Are Used to Access Chiral Building Blocks with Multiple Stereocenters.

Novel building blocks are in constant demand during the search for innovative bioactive small molecule therapeutics by enabling the construction of structure-activity-property-toxicology relationships. Complex chiral molecules containing multiple stereocenters are an important component in compound library expansion but can be difficult to access by traditional organic synthesis. Herein, we report a biocatalytic process to access a specific diastereomer of a chiral amine building block used in drug discovery.

Structure-Based Design of Small Imine Reductase Panels for Target Substrates.

Biocatalysis is important in the discovery, development, and manufacture of pharmaceuticals. However, the identification of enzymes for target transformations of interest requires major screening efforts. Here, we report a structure-based computational workflow to prioritize protein sequences by a score based on predicted activities on substrates, thereby reducing a resource-intensive laboratory-based biocatalyst screening.

Enzymatic Late-Stage Halogenation of Peptides.

The late-stage site-selective derivatisation of peptides has many potential applications in structure-activity relationship studies and postsynthetic modification or conjugation of bioactive compounds. The development of orthogonal methods for C-H functionalisation is crucial for such peptide derivatisation. Among them, biocatalytic methods are increasingly attracting attention.

One-Step Biocatalytic Synthesis of Sustainable Surfactants by Selective Amide Bond Formation.

N-alkanoyl-N-methylglucamides (MEGAs) are non-toxic surfactants widely used as commercial ingredients, but more sustainable syntheses towards these compounds are highly desirable. Here, we present a biocatalytic route towards MEGAs and analogues using a truncated carboxylic acid reductase construct tailored for amide bond formation (CARmm-A). CARmm-A is capable of selective amide bond formation without the competing esterification reaction observed in lipase catalysed reactions.

Novel Arylindigoids by Late-Stage Derivatization of Biocatalytically Synthesized Dibromoindigo.

Indigoids represent natural product-based compounds applicable as organic semiconductors and photoresponsive materials. Yet modified indigo derivatives are difficult to access by chemical synthesis. A biocatalytic approach applying several consecutive selective C-H functionalizations was developed that selectively provides access to various indigoids: Enzymatic halogenation of l-tryptophan followed by indole generation with tryptophanase yields 5-, 6- and 7-bromoindoles.

Enzymatic Late-Stage Modifications: Better Late Than Never.

Enzyme catalysis is gaining increasing importance in synthetic chemistry. Nowadays, the growing number of biocatalysts accessible by means of bioinformatics and enzyme engineering opens up an immense variety of selective reactions. Biocatalysis especially provides excellent opportunities for late-stage modification often superior to conventional de novo synthesis.

Cyclization of RGD Peptides by Suzuki-Miyaura Cross-Coupling.

Halogenated l- or d-tryptophan obtained by biocatalytic halogenation was incorporated into RGD peptides together with a variety of alkyl or aryl boronic acids. Suzuki-Miyaura cross-coupling either in solution or on-resin results in side chain-to-tail-cyclized RGD peptides, for example, with biaryl moieties, providing a new dimension of structure-activity relationships. An array of RGD peptides differing in macrocycle size, the presence of d-amino acid, -methylation, or connectivity between the indole moiety and the boronic acid showed that, in particular, connectivity exhibits a major impact on affinities toward integrins, for example, αβ.

Enzymatic Halogenation: A Timely Strategy for Regioselective C-H Activation.

Halogenating enzymes are increasingly attracting attention for biocatalytic C-H functionalization. Despite its importance for synthetic chemistry, selective introduction of halogens using conventional approaches often remains challenging, whereas biocatalysis offers excellent catalyst-controlled selectivity without requiring protecting groups or hazardous reagents. Owing to the high prevalence of halogenated secondary metabolites, a still growing repertoire of halogenases has been identified.

A High-Throughput Fluorescence Assay to Determine the Activity of Tryptophan Halogenases.

Biocatalytic halogenation with tryptophan halogenases is hampered by severe limitations such as low activity and stability. These drawbacks can be overcome by directed evolution, but for screening large mutant libraries, a facile high-throughput method is required. Therefore, we developed a quantitative halogenase assay based on a Suzuki-Miyaura cross-coupling towards the formation of a fluorescent aryltryptophan.