Modification of proteins by 1 (2 chloroethyl) 3 cyclohexyl 1 nitrosourea (NSC 79037) in vitro

The binding of 1 (2 chloroethyl) 3 cyclohexyl 1 nitrosourea (CCNU), labeled with ¹⁴C in the carbonyl moiety, to albumin, poly L lysine, calf thymus DNA, and Escherichia coli tRNA was studied. Binding was extensive to proteins and negligible to nucleic acids. The most active interaction was found with poly L lysine. These results parallel those of cyclohexyl labeled CCNU and suggest that the carbonyl moiety is also part of the protein bound adduct. The radioactive products obtained from enzymatic hydrolysates of proteins previously reacted in vitro with cyclohexyl ¹⁴C labeled CCNU and from proteins isolated from L1210 leukemia cells incubated with the labeled drug were characterized by comparison with an authentic sample of N ⁶ cyclohexylcarbamoyllysine. The latter was synthesized by the reaction of cyclohexylisocyanate with lysine. The derivatives obtained from protein hydrolysates in all cases had the same R(F), in four different paper chromatographic solvent systems, as the authentic sample. These studies demonstrate that CCNU binds to proteins by cyclohexylcarbamoylation of their lysine residues.