Promiscuous Enzymes for Residue‐Specific Peptide and Protein Late‐Stage Functionalization

Abstract

The late‐stage functionalization of peptides and proteins holds significant promise for drug discovery and facilitates bioorthogonal chemistry. This selective functionalization leads to innovative advances in in vitro and in vivo biological research. However, it is a challenging endeavor to selectively target a certain amino acid or position in the presence of other residues containing reactive groups. Biocatalysis has emerged as a powerful tool for selective, efficient, and economical modifications of molecules. Enzymes that have the ability to modify multiple complex substrates or selectively install nonnative handles have wide applications. Herein, we highlight enzymes with broad substrate tolerance that have been demonstrated to modify a specific amino acid residue in simple or complex peptides and/or proteins at late‐stage. The different substrates accepted by these enzymes are mentioned together with the reported downstream bioorthogonal reactions that have benefited from the enzymatic selective modifications.

Selective Late-Stage Functionalization of Tryptophan-Containing Peptides To Facilitate Bioorthogonal Tetrazine Ligation

“…LSF offers a cost- and time-effective approach to functionalize C–H bonds without the need for numerous synthetic steps and labor-intensive workup and purification procedures . Additionally, LSF allows for the introduction of reactive handles amenable to bioorthogonal chemical reactions. , However, site-selective LSF of peptides is challenging as a result of the presence of several reactive groups, stereocenters that are prone to racemization, multiple C–H bonds, or the need for physiological conditions. While chemical, , enzymatic, and photocatalytic methods have been developed for peptide LSF, they have faced challenges including focus on residues carrying nucleophilic functional groups, selectivity concerns, or the use of toxic metals or harsh conditions that affects the structural integrity of delicate macromolecules.…”

“…Additionally, LSF allows for the introduction of reactive handles amenable to bioorthogonal chemical reactions. , However, site-selective LSF of peptides is challenging as a result of the presence of several reactive groups, stereocenters that are prone to racemization, multiple C–H bonds, or the need for physiological conditions. While chemical, , enzymatic, and photocatalytic methods have been developed for peptide LSF, they have faced challenges including focus on residues carrying nucleophilic functional groups, selectivity concerns, or the use of toxic metals or harsh conditions that affects the structural integrity of delicate macromolecules.…”

“…The work described herein adds to the growing role of biocatalysis in the selective modification of peptides. ,, Our approach is distinctive compared to other reported methods used to chemoenzymatically label Trp in peptides. First, we demonstrate the ability to selectively target Trp in the presence of other residues and reactive functionalities on broad complex cyclic , and linear peptides with diverse handles.…”