Modified guanines representing O(6)-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 O(6)-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 degrees C). 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 degrees C). 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 degrees C, 30 min], there was an estimated 10-35% loss of O(6)-PAG. Under the same conditions, O(6)-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 degrees 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 O(6)()-alkylation and that cyclizations to tautomers were most favorable if they involved the N-1 or NH(2) positions.