Reagents and methods for the solid-phase synthesis of protein-EDTA for use in affinity cleaving

Synthetic procedures for the introduction of the metal chelator ethylenediaminetetraacetic acid (EDTA) at unique amino acid positions of proteins by solid-phase methods are described. Two protected derivatives of EDTA compatible with Merrifield solid-phase protein synthesis employing N-tert-butyloxycarbonyl- (Boc-) protected amino acids were developed. The first reagent is a dipeptide with three of four carboxyl groups of EDTA protected as benzyl esters and the fourth coupled to a γ-aminobutanoic acid linker, referred to as tribenzyl-EDTA-CABA (BEG). A second reagent is the tricyclohexyl ester of EDTA, TCE. BEG and TCE allow the modification of the NH_2 terminus and/or lysine side chains of resin-bound peptides and proteins. Upon deprotection and cleavage from the resin, a protein is produced with EDTA at a defined amino acid position. The availability of protein-EDTA conjugates extends the affinity cleaving method to the study of protein-DNA complexes in solution.