Modified Guanines Representing <i>O</i>⁶-Alkylation by the Cyclophosphamide Metabolites Acrolein and Chloroacetaldehyde: Synthesis, Stability, and ab Initio Studies

Abstract

Alkylation of DNA by acrolein and/or chloroacetaldehyde may result in the mutations that lead to the therapy-induced leukemia associated with cyclophosphamide (and ifosfamide) treatment. O6-(n-Propanalyl)guanine (O6-PAG) and O6-(ethanalyl)guanine (O6-EAG) were synthesized for use as authentic standards in investigations of DNA alkylation by acrolein and chloroacetaldehyde, respectively. Preparation of the O-methyl oximes of these aldehydes aided in confirming the structural assignments of O6-PAG and O6-EAG. HPLC was used to study the stability of O6-PAG under a variety of conditions. The decomposition of O6-PAG was attributed to an α,β-elimination reaction resulting in the formation of guanine and acrolein. In 0.1 M phosphate-DMSO (9:1), O6-PAG (1−10 mM) had a half-life of approximately 1 h (pH 7.4, 37 °C). In 0.05 M Tris-DMSO (9:1), the apparent half-life of O6-PAG (1−10 mM) was approximately 16 h (pH 7.4, 37 °C). The increased lifetime under the latter conditions was attributed to a reversible reaction between Tris and the aldehydic functionality of O6-PAG to give a more stable oxazolidine. Under conditions similar to those that would be used for hydrolysis of DNA [0.1 M HCl-DMSO (98:2), pH 1.3, 70 °C, 30 min], there was an estimated 10−35% loss of O6-PAG. Under the same conditions, O6-EAG had apparent half-lives of 6.6 h (phosphate-DMSO) and 2.5 days (Tris-DMSO) and the estimated loss at pH 1.3 over 30 min (70 °C) was 15−20%. Ab initio quantum chemical calculations were used to understand the energy factors that underlie the occurrence of O- versus N-alkylations as well as possible, subsequent intramolecular cyclizations. Simulations of the free energies of reactions between acrolein and guanine indicated that N-alkylation was favored over O6-alkylation and that cyclizations to tautomers were most favorable if they involved the N-1 or NH2 positions.