Site-specific bioorthogonal protein labelling by tetrazine ligation using endogenous β-amino acid dienophiles

Abstract

The tetrazine ligation is an inverse electron-demand Diels–Alder reaction widely used for bioorthogonal modifications due to its versatility, site specificity and fast reaction kinetics. A major limitation has been the incorporation of dienophiles in biomolecules and organisms, which relies on externally added reagents. Available methods require the incorporation of tetrazine-reactive groups by enzyme-mediated ligations or unnatural amino acid incorporation. Here we report a tetrazine ligation strategy, termed TyrEx (tyramine excision) cycloaddition, permitting autonomous dienophile generation in bacteria. It utilizes a unique aminopyruvate unit introduced by post-translational protein splicing at a short tag. Tetrazine conjugation occurs rapidly with a rate constant of 0.625 (15) M−1 s−1 and was applied to produce a radiolabel chelator-modified Her2-binding Affibody and intracellular, fluorescently labelled cell division protein FtsZ. We anticipate the labelling strategy to be useful for intracellular studies of proteins, as a stable conjugation method for protein therapeutics, as well as other applications.

Deep Learning-Driven Library Design for the De Novo Discovery of Bioactive Thiopeptides

“…In principle, analogous pipelines can be constructed for other RiPP biosynthetic pathways so long as the enzymatic reaction products can be chemically differentiated from the starting materials and intermediates. Many RiPP pathways amenable to combinatorial workflows (lanthipeptides, , lasso peptides, , cyanobactins, , graspetides, , and spliceotides, , among others) may benefit from comprehensive substrate profiling in an mRNA display format. Model-guided library design may be particularly useful for the pathways with enigmatic substrate preferences (e. g., prochlorosins , ) or highly cooperative biosynthesis (thiopeptides , and polytheonamides), where the substrate fitness landscapes become too complex for the manual construction of optimal library designs.…”

Genome Mining for New Enzyme Chemistry

“…94 This post-translational modification is widespread in nature and has been leveraged as a bioorthogonal labeling method and moiety for protease inhibition. 95,96 Ranthipeptides. Numerous RiPPs contain thioether-based macrocycles as the class-defining modification, including lanthipeptides, sactipeptides, and the more recently bioinformatically discovered ranthipeptides.…”

Novel Biocatalysts from Specialized Metabolism

“…The use of splicases offers a versatile and site‐selective method for introducing β‐residues into proteins [27] and generating diverse β‐α‐ketoamides for use in drug discovery programs [25] . These β‐α‐ketoamides have also been utilized as reactive handles for further derivatization with tetrazines to site‐specifically introduce fluorophores, for instance [28] …”