title: Modified guanines representing O(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. 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.
authors.affiliation: Duke Comprehensive Cancer Center, Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710, USA.
authors.forename: Narayanan
authors.initials: N
authors.lastname: Balu
authors.forename: Michael P
authors.initials: MP
authors.lastname: Gamcsik
authors.forename: Michael E
authors.initials: ME
authors.lastname: Colvin
authors.forename: O Michael
authors.initials: OM
authors.lastname: Colvin
authors.forename: M Eileen
authors.initials: ME
authors.lastname: Dolan
authors.forename: Susan M
authors.initials: SM
authors.lastname: Ludeman
mesh_terms: D000079:Acetaldehyde / Q000031:analogs & derivatives* / Q000737:chemistry*; D000171:Acrolein / Q000737:chemistry*; D000478:Alkylation; D018906:Antineoplastic Agents, Alkylating / Q000009:adverse effects; D003520:Cyclophosphamide / Q000009:adverse effects / Q000737:chemistry*; D004249:DNA Damage; D006147:Guanine / Q000031:analogs & derivatives* / Q000138:chemical synthesis; D006801:Humans; D007938:Leukemia / Q000139:chemically induced
chemical_list: D018906:Antineoplastic Agents, Alkylating; D006147:Guanine; D000171:Acrolein; D003520:Cyclophosphamide; C004656:chloroacetaldehyde; D000079:Acetaldehyde