Kinetics and mechanism of the acid-catalyzed hydrolysis of O6-benzylguanine

Abstract

A variety of analytical techniques were used to elucidate the kinetics, reaction pathway and mechanism of hydrolysis of O 6-benzylguanine, an O 6-alkylguanine-DNA alkyltransferase depleting agent, as a function of pH, buffer concentration, temperature, substituents and halide nucleophiles. The reaction was also carried out in H 2 18O in order to determine the site of cleavage. The pH-rate profile indicated that the hydrolysis of O 6-benzylguanine was acid-catalyzed, with the neutral O 6-benzylguanine having greater intrinsic susceptibility to undergo acid catalyzed hydrolysis compared to its protonated form. Alternatively, the kinetics could be described by the kinetically indistinguishable process of spontaneous degradation of fully di- and mono-protonated O 6-benzylguanine. In this case, the di-protonated species is more susceptible than the mono-protonated species. Based on the 18O incorporation data, the site of the bond cleavage for hydrolysis of O 6-benzylguanine was unambiguously assigned to the benzylic carbon-oxygen bond leading to the formation of benzyl alcohol and guanine as the predominant products. Benzyl chloride was also detected as a degradation product when the ionic strength of the solution was adjusted with sodium chloride. The rate of hydrolysis of p-substituted O 6-benzylguanines increased with increasing electron donating capability of p-substituents, consistent with a mechanism involving positive charge formation on the benzylic carbon in the transition state. An Eyring plot resulted in a value for the observed entropy of activation, Δ S ≠, of −2.4 e.u. which was consistent with a unimolecular, S n 1 , reaction. Although the rate of hydrolysis of O 6-benzylguanine was well correlated with the nucleophilicity of various halide nucleophiles, the magnitude of the catalysis was less than anticipated for S n 2 type reactions. The results suggested that in the presence of bromide and iodide, the transition state had some S n 2 character. Based on the above observations, a late transition state for this reaction was suggested, where positive charge development at the benzylic carbon atom was quite advanced.