Methods and reagents for site-selective functionalization of peptides and proteins. The methods most generally involve the reaction of a
thioester with hydrazine. Reagents include
bifunctional reagents of formula:H2N—NH—CH2-M-L-FGand salts thereof where M is a
single bond or a chemical group carrying a non-bonding
electron pair, such as —C(O)NR′—, where R′ is H, or an
alkyl or
aryl group; L is an optional
linker group as described above; and FG is a functional group having reactivity that is orthongonal to that of the hydrazine group. FG can, among others, be an
azide, alkenyl, alkynyl,
nitrile (—CN) or
triazole group and is preferably an
azide group (—N3). Methods and reagents can, for example, be combined with
intein-mediated
protein splicing to link proteins or fragments thereof to various
chemical species or to a surface. Surface immobilization of proteins via the methods herein results in
immobilized proteins which substantially retain
biological activity and is thus useful for the generation of
peptide or
protein microarrays. Kits for functionalization and / or immobilization of peptides and proteins are provided as well as microarrays of peptides, proteins or both.