Modified Guanines Representing O 6 -Alkylation by the Cyclophosphamide Metabolites Acrolein and Chloroacetaldehyde: Synthesis, Stability, and ab Initio Studies Narayanan Balu, † Michael P. Gamcsik, † Michael E. Colvin, ‡ O. Michael Colvin, † M. Eileen Dolan, § and Susan M. Ludeman* ,† Duke Comprehensive Cancer Center, Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710, Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory, Livermore, California 94550, and Cancer Research Center and Department of Medicine, University of Chicago, Chicago, Illinois 60637 Received September 11, 2001 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 R,-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 °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 °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 °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 °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. Introduction The anticancer agent cyclophosphamide (1) undergoes a sequence of metabolic transformations leading to the formation of 4-hydroxycyclophosphamide (2), aldophos- phamide (3), and ultimately, phosphoramide mustard (4) and acrolein (5) (Scheme 1) (1). Phosphoramide mustard is responsible for cross-linking DNA (2) while acrolein is most commonly associated with side effects such as bladder cystitis (3). A competing, albeit minor (∼10%), metabolic fate of 1 results in dechloroethylation and the formation of 2-dechloroethylcyclophosphamide (6) and chloroacetaldehyde (7) (Scheme 1). The occurrence of neurotoxicity is attributed to this pathway (4, 5). Recently, we provided the first evidence that O 6 - alkylguanine-DNA alkyltransferase (AGT) plays an im- portant role in protecting against the toxicity and mu- tagenicity of 1 (6, 7). We also demonstrated that this relationship between AGT and 1 is specifically correlated with acrolein and not phosphoramide mustard (7). Con- sidering that AGT repair of DNA involves removal of an alkyl group from the O 6 position in a guanine residue, two pathways were presented by which acrolein might generate such a repairable adduct. Acting as a nucleo- phile, the guanylic oxygen could initiate a conjugate * To whom correspondence should be addressed. Phone: (919) 681- 2808. Fax: (919) 684-5653. Email: s.ludeman@duke.edu. † Duke Comprehensive Cancer Center and Department of Medicine, Duke University Medical Center. ‡ Lawrence Livermore National Laboratory. § University of Chicago. Scheme 1 380 Chem. Res. Toxicol. 2002, 15, 380-387 10.1021/tx0101503 CCC: $22.00 © 2002 American Chemical Society Published on Web 02/08/2002