J. Am. Chem. Soc. 10.1021/jacs.7b05125 (2017)

Chemoselectivity is a key consideration when selecting from the toolbox of bio-orthogonal chemical ('click') reactions for site-specific tagging of biological molecules, and new reactions are continually sought to enable precise, controllable labeling of multiple sites in tandem. Addy et al. have now added to this toolbox a chemoselective rapid azo-coupling reaction (CRACR) that uses an aromatic diazonium ion to conjugate a desired tag to a particular aromatic amino acid residue. In the optimized reaction, the 4-carboxybenzenediazonium (4CDz) ion reacts rapidly with the readily available noncanonical amino acid 5-hydroxytryptophan (5HTP) and, importantly, exhibits selectivity over canonical aromatic amino acids such as tyrosine. The use of this noncanonical amino acid, which can be site-specifically incorporated into a desired protein via nonsense suppression technology, also endows the reaction with an added degree of selectivity by limiting the number of sites within the proteome where the reactive handle is present. An added benefit of this reaction is that it is 'unclickable' as well: addition of dithionite reductively cleaves the azo-coupled conjugate, thus providing a means to remove the tag. Although this CRACR reaction has not yet been shown to be compatible with other click reactions, its selectivity and reversibility make it a potentially attractive choice for future labeling applications.