Conversion of Proteins to Diazeniumdiolate-Based Nitric Oxide Donors

Michael reaction of the methoxymethyl-protected monodiazeniumdiolate of piperazine (MOM-PIPERAZI/NO) with 4-maleimidobutyric acid followed by its conversion to the N-hydroxy-succinimido ester produces a reagent capable of transferring the nitric oxide (NO)-donating diazeniumdiolate group to the terminal amines of the lysine residues contained in proteins. The reagent has been used to produce diazeniumdiolated bovine serum albumin (D-BSA) and diazeniumdiolated human serum albumin (D-HSA) containing 22 and 19 modified lysyl groups, respectively. Upon dissolution in pH 7.4 phosphate buffer at 37 °C, these albumin derivatives gradually released all of their contained NO (approximately 40 mol/mol of protein) with initial rates of about 30−40 pmol/min/mg and half-lives on the order of 3 weeks. This methodology is now available for use in exploiting the unique specific metabolic interactions of proteins to target NO therapy to specific physiological processes in vivo.