Modified Guanines Representing O⁶-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.

C‐terminal lysine processing of human immunoglobulin G2 heavy chain in vivo

“…Acrolein and crotonaldehyde are continuously produced by lipid peroxidation of polyunsaturated fatty acids 16, 17. Acrolein is also a product of myeloperoxidase‐catalyzed amino acid oxidation17 and anticancer drug metabolism, such as cyclophosphamide 29. Living cells have developed several lines of defense mechanisms to eliminate these intracellular cytotoxins.…”

Aldo–keto reductase family 1 B10 gene silencing results in growth inhibition of colorectal cancer cells: Implication for cancer intervention

“…16,17 Acrolein is also a product of myeloperoxidase-catalyzed amino acid oxidation 17 and anticancer drug metabolism, such as cyclophosphamide. 29 Living cells have developed several lines of defense mechanisms to eliminate these intracellular cytotoxins. Aldehyde dehydrogenases (ALDH) mediate the oxidation of aldehyde carbonyls, forming carbonic acids; 30 glutathione-Stransferases (GST) catalyze the conjugation of carbonyls with glutathione; 31,32 and aldo-keto reductases such as AR participate in the reduction of carbonyl groups to alcohol forms.…”

Lack of Mutagenicity of Acrolein-Induced DNA Adducts in Mouse and Human Cells

“…[8][9][10]. Acrolein-induced DNA adducts have been characterized in vitro (11)(12)(13)(14) and quantified in vivo in various organs of experimental animals and humans (9,10,15,16). A recent in vitro study has shown preferential formation of acrolein-DNA adducts at lung cancer mutational hotspots in the p53 tumor suppressor gene in normal human bronchial epithelial cells and lung fibroblasts (17).…”