Limitations of Azide-Alkyne Chemistry in Genetic Code Expansion

Abstract

Genetic code expansion (GCE) has enabled the incorporation of non-canonical amino acids (ncAAs) into proteins, offering powerful opportunities for site-specific labelling and functionalization. Among these, azide- and alkyne-containing ncAAs are particularly attractive due to their compatibility with bioorthogonal “click” reactions, such as strain-promoted and copper-catalysed azide–alkyne cycloaddition (SPAAC and CuAAC). This review outlines the principles of azide–alkyne chemistry and its integration with GCE, highlighting key applications in live-cell imaging, proteomics, and targeted drug conjugation. At the same time, it discusses major limitations, including copper-associated cytotoxicity, steric and stability issues of reactive groups, and challenges with incorporation efficiency across different host systems. By summarizing significant advances across bacterial, yeast, and mammalian models, this review provides a critical perspective on the current state and future potential of azide–alkyne GCE systems.