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. Tryptophan halogenases emerged as valuable catalysts to functionalise tryptophan (Trp), while direct enzyme‐catalysed halogenation of synthetic peptides is yet unprecedented. Here, it is reported that the Trp 6‐halogenase Thal accepts a wide range of amides and peptides containing a Trp moiety. Increasing the sequence length and reaction optimisation made bromination of pentapeptides feasible with good turnovers and a broad sequence scope, while regioselectivity turned out to be sequence dependent. Comparison of X‐ray single crystal structures of Thal in complex with d‐Trp and a dipeptide revealed a significantly altered binding mode for the peptide. The viability of this bioorthogonal approach was exemplified by halogenation of a cyclic RGD peptide.

Biocatalytic syntheses often require unfavorable conditions, which can adversely affect enzyme stability. Consequently, improving the stability of biocatalysts is needed, and this is often achieved by immobilization. In this study, we aimed to compare the stability of soluble and immobilized transaminases from different species. A cysteine in a consensus sequence was converted to a single aldehyde by the formylglycine-generating enzyme for directed single-point attachment to amine beads. This immobilization was compared to cross-linked enzyme aggregates (CLEAs) and multipoint attachments to glutaraldehyde-functionalized amine- and epoxy-beads. Subsequently, the reactivity and stability (i.e., thermal, storage, and solvent stability) of all soluble and immobilized transaminases were analyzed and compared under different conditions. The effect of immobilization was highly dependent on the type of enzyme, the immobilization strategy, and the application itself, with no superior immobilization technique identified. Immobilization of HAGA-beads often resulted in the highest activities of up to 62 U/g beads, and amine beads were best for the hexameric transaminase from Luminiphilus syltensis. Furthermore, the immobilization of transaminases enabled its reusability for at least 10 cycles, while maintaining full or high activity. Upscaled kinetic resolutions (partially performed in a SpinChemTM reactor) resulted in a high conversion, maintained enantioselectivity, and high product yields, demonstrating their applicability.

An enzyme cascade was established previously consisting of a recycling system with an l‐amino acid oxidase (hcLAAO4) and a catalase (hCAT) for different α‐keto acid co‐substrates of (S)‐selective amine transaminases (ATAs) in kinetic resolutions of racemic amines. Only 1 mol % of the co‐substrate was required and l‐amino acids instead of α‐keto acids could be applied. However, soluble enzymes cannot be reused easily. Immobilization of hcLAAO4, hCAT and the (S)‐selective ATA from Vibrio fluvialis (ATA‐Vfl) was addressed here. Immobilization of the enzymes together rather than on separate beads showed higher reaction rates most likely due to fast co‐substrate channeling between ATA‐Vfl and hcLAAO4 due to their close proximity. Co‐immobilization allowed further reduction of the co‐substrate amount to 0.1 mol % most likely due to a more efficient H2O2‐removal caused by the stabilized hCAT and its proximity to hcLAAO4. Finally, the co‐immobilized enzyme cascade was reused in 3 cycles of preparative kinetic resolutions to produce (R)‐1‐PEA with high enantiomeric purity (97.3 %ee). Further recycling was inefficient due to the instability of ATA‐Vfl, while hcLAAO4 and hCAT revealed high stability. An engineered ATA‐Vfl‐8M was used in the co‐immobilized enzyme cascade to produce (R)‐1‐(3‐ethoxy‐4‐methoxyphenyl)‐2‐(methylsulfonyl)ethanamine, an apremilast‐intermediate, with a 1,000 fold lower input of the co‐substrate.