Modified guanines representing O6-alkylation by the cyclophosphamide metabolites acrolein and chloroacetaldehyde:: Synthesis, stability, and ab initio studies

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. O-6-(n-Propanalyl)guanine (O-6-PAG) and O-6-(ethanalyl)guanine (O-6-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 O-6-PAG and 06-EAG. HPLC was used to study the stability of O-6-PAG under a variety of conditions. The decomposition of O-6-PAG was attributed to an alpha,beta-elimination reaction resulting in the formation of guanine and acrolein. In 0.1 M phosphate-DMSO (9:1), O-6-PAG (1-10 mM) had a half-life of approximately 1 h (pH 7.4, 37 degreesC). In 0.05 M Tris-DMSO (9:1), the apparent half-life of O-6-PAG (1-10 mM) was approximately 16 h (pH 7.4, 37 degreesC). The increased lifetime under the latter conditions was attributed to a reversible reaction between Tris and the aldehydic functionality of O-6-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 degreesC, 30 min], there was an estimated 10-35% loss of O-6-PAG. Under the same conditions, 06-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 degreesC) 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 O-6-alkylation and that cyclizations to tautomers were most favorable if they involved the N-1 or NH2 positions.