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Click chemistry has found wide application in bioconjugation, enabling control over the site of modification in biomolecules. Demonstrations of this chemistry to construct chemically defined antibody-drug conjugates (ADCs) have increased in recent years, following studies that support benefits of homogeneity and site-specificity of drug placement on the antibody. In this chapter, a brief history of early applications of this chemistry in ADCs is presented. Examples of click chemistries that are utilized for ADC synthesis, including those currently undergoing clinical investigations, are enumerated. Protocols for two common conjugation methods based on carbonyl-aminooxy coupling and strain-promoted azide-alkyne cycloaddition are described.

The substrate promiscuity of microbial transglutaminase (mTG) has been exploited in various applications in biotechnology, in particular for the attachment of alkyl amines to glutamine‐containing peptides and proteins. Here, we expand the substrate repertoire to include hydrazines, hydrazides, and alkoxyamines, resulting in the formation of isopeptide bonds with varied susceptibilities to hydrolysis or exchange by mTG. Furthermore, we demonstrate that simple unsubstituted hydrazine and dihydrazides can be used to install reactive hydrazide handles onto the side chain of internal glutamine residues. The distinct hydrazide handles can be further coupled with carbonyls, including ortho‐carbonylphenylboronic acids, to form site‐specific and functional bioconjugates with tunable hydrolytic stability. The extension of the substrate scope of mTG beyond canonical amines thus substantially broadens the versatility of the enzyme, providing a new approach to facilitate novel applications. [ABSTRACT FROM AUTHOR]