Vol. 26, Issue 3, 246-256, March 1998

Mechanisms for Covalent Binding of Benoxaprofen Glucuronide to Human Serum Albumin
Studies by Tandem Mass Spectrometry

Y. Qiu, A. L. Burlingame, and L. Z. Benet

Departments of Biopharmaceutical Sciences (Y.Q., L.Z.B.) and Pharmaceutical Chemistry (Y.Q., A.L.B.) and the Liver Center (A.L.B., L.Z.B.), University of California, San Francisco

Tandem MS has been used to establish the structure and specific binding sites of covalent protein adducts formed upon incubation of the acyl glucuronide of the propionic acid nonsteroidal anti-inflammatory drug benoxaprofen with human serum albumin in vitro. Benoxaprofen 1-O--glucuronide was enzymatically synthesized in vitro and incubated with human serum albumin both in the presence and in the absence of NaCNBH₃. The modified human serum albumins were digested with trypsin and separated by HPLC. The modified peptides were detected using HPLC-electrospray MS (with selected-ion monitoring) and were structurally characterized by tandem MS using matrix-assisted laser desorption ionization in both the post-source decay and high-energy collision-induced dissociation modes. These studies established that benoxaprofen glucuronide forms covalent adducts with protein nucleophiles both by nucleophilic displacement of glucuronic acid at the anomeric center and by condensation of the rearranged acyl glucuronic acid isomers with -amino functions of lysine residues after acyl migration of the aglycone from the anomeric center. Lys-159 was identified as the major binding site. Thus, we have established that members of the less reactive propionic acid class of acyl glucuronides, such as the glucuronide of benoxaprofen, are also capable of reacting with protein nucleophiles to form covalent adducts analogous to those of tolmetin glucuronide (tolmetin is an acetic acid nonsteroidal anti-inflammatory drug), via the mechanisms previously reported from this laboratory, and that the specific covalent binding site profile appears to be drug dependent.