Synthesis, Characterization, and Immunochemical Detection of O 6-(Carboxymethyl)-2‘-deoxyguanosine:  A DNA Adduct Formed by Nitrosated Glycine Derivatives

• Published in: Chemical research in toxicology 1997-06, Vol.10 (6), p.652-659
• Main Authors: Harrison, Kathryn L, Fairhurst, Neil, Challis, Brian C, Shuker, David E. G
• Format: Article
• Language: English
• ISSN: 0893-228X; 1520-5010
• DOI: 10.1021/tx960203u

O 6-(Carboxymethyl)-2‘-deoxyguanosine (O 6-CMdG) is formed in DNA by nitrosated glycine derivatives and appears to be nonrepairable by O 6-alkylguanine transferases. O 6-CMdG has been synthesized by an unambiguous route involving the introduction of a methyl glycolate moiety at C6 of a 3‘,5‘-bis-O-(methoxyacetyl)dGuo derivative by displacement of a quinuclidinium ion. Methanolysis of the methoxyacetyl groups and calcium hydroxide-mediated hydrolysis of the methyl ester afforded the calcium salt of O 6-CMdG in good yield. A similar route was used to synthesize O 6-(carboxymethyl)guanosine (O 6-CMGuo), which was used to prepare protein conjugates for immunization. Rabbit antisera were prepared, and a quantitative competitive ELISA was developed which showed 50% inhibition at 2 pmol of O 6-CMdG/well. O 6-CMGuo was 30 times less cross-reactive (50% inhibition at 60 pmol/well), and normal nucleosides and carboxymethylated purines did not cross-react to any significant extent. The antiserum was also used to prepare reusable immunoaffinity columns which retained O 6-CMdG. The binding of O 6-CMdG was so strong that conditions used to elute the adduct (1 M trifluoroacetic acid) resulted in partial hydrolysis (becoming quantitative on heating) of the glycosidic bond to give O 6-CMguanine which was detected by HPLC with fluorescence detection. DNA treated with azaserine (5 mmol), N-(N‘-acetyl-l-prolyl)-N-nitrosoglycine (5 mmol), and potassium diazoacetate (5 mmol) afforded O 6-CMdG at levels of 7.3, 393.9, and 496 μmol of O 6-CMdG/mol of dG. The antiserum also recognized O 6-CMdG in intact DNA.