A “Tag-and-Modify” Approach to Site-Selective Protein Modification

• Published in: Accounts of chemical research 2011-09, Vol.44 (9), p.730-741
• Main Authors: Chalker, Justin M, Bernardes, Gonçalo J. L, Davis, Benjamin G
• Format: Article
• Language: English
• Subjects: Alanine - analogs & derivatives; Alanine - chemistry; Alkynes - chemistry; Amino acids; Azides - chemistry; Catalysis; Chemists; Copper - chemistry; Covalence; Cysteine - chemistry; Cysteine - metabolism; Disulfides - chemistry; Free Radicals - chemistry; Genetics; Glycosylation; Marking; Palladium - chemistry; Protein Prenylation; Proteins; Proteins - chemistry; Proteins - metabolism; Ruthenium - chemistry; Strategy; Transformations
• ISSN: 0001-4842; 1520-4898
• DOI: 10.1021/ar200056q

Covalent modification can expand a protein’s functional capacity. Fluorescent or radioactive labeling, for instance, allows imaging of a protein in real time. Labeling with an affinity probe enables isolation of target proteins and other interacting molecules. At the other end of this functional spectrum, protein structures can be naturally altered by enzymatic action. Protein–protein interactions, genetic regulation, and a range of cellular processes are under the purview of these post-translational modifications. The ability of protein chemists to install these covalent additions selectively has been critical for elucidating their roles in biology. Frequently the transformations must be applied in a site-specific manner, which demands the most selective chemistry. In this Account, we discuss the development and application of such chemistry in our laboratory. A centerpiece of our strategy is a “tag-and-modify” approach, which entails sequential installation of a uniquely reactive chemical group into the protein (the “tag”) and the selective or specific modification of this group. The chemical tag can be a natural or unnatural amino acid residue. Of the natural residues, cysteine is the most widely used as a tag. Early work in our program focused on selective disulfide formation in the synthesis of glycoproteins. For certain applications, the susceptibility of disulfides to reduction was a limitation and prompted the development of several methods for the synthesis of more stable thioether modifications. The desulfurization of disulfides and conjugate addition to dehydroalanine are two routes to these modifications. The dehydroalanine tag has since proven useful as a general precursor to many modifications after conjugate addition of various nucleophiles; phosphorylated, glycosylated, peptidylated, prenylated, and even mimics of methylated and acetylated lysine-containing proteins are all accessible from dehydroalanine. While cysteine is a useful tag for selective modification, unnatural residues present the opportunity for bio-orthogonal chemistry. Azide-, arylhalide-, alkyne-, and alkene-containing amino acids can be incorporated into proteins genetically and can be specifically modified through various transformations. These transformations often rely on metal catalysis. The Cu-catalyzed azide–alkyne addition, Ru-catalyzed olefin metathesis, and Pd-catalyzed cross-coupling are examples of such transformations. In the course of adapting these reactions