NMR studies of the conjugation of mechlorethamine with glutathione

Many cancer cells are resistant to chemotherapeutic treatment with mechlorethamine and other alkylating agents. These drug-resistant cells often show an increase in the intracellular concentration of glutathione and an increase in the activity of glutathione-S-transferase when compared to the sensitive cells. Both of these components are thought to be involved with inactivation of the drug either through conjugation with glutathione or by hydrolysis. NMR spectroscopy was used to monitor the nonenzymatic conjugation of mechlorethamine with glutathione. Several intermediates along the pathway to the doubly glutathione substituted mustard, including both mustard-aziridinium adducts, can be observed. The assignment of the 1H NMR spectrum of these adducts are presented. At 30 degrees C, pH 7.0, no hydrolyzed mustard was detectable. With the use of 13C-labeled mustard, the conjugation reaction can be shown to proceed through an aziridinium intermediate rather than by direct nucleophilic substitution.