Synthesis, characterization, and immunochemical detection of O6-(carboxymethyl)-2'-deoxyguanosine: a DNA adduct formed by nitrosated glycine derivatives.
Chem Res Toxicol
; 10(6): 652-9, 1997 Jun.
Article
en En
| MEDLINE
| ID: mdl-9208171
O6-(Carboxymethyl)-2'-deoxyguanosine (O6-CMdG) is formed in DNA by nitrosated glycine derivatives and appears to be nonrepairable by O6-alkylguanine transferases. O6-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 O6-CMdG in good yield. A similar route was used to synthesize O6-(carboxymethyl)guanosine (O6-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 O6-CMdG/ well. O6-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 O6-CMdG. The binding of O6-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 O6-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 O6-CMdG at levels of 7.3, 393.9, and 496 mumol of O6-CMdG/mol of dG. The antiserum also recognized O6-CMdG in intact DNA.
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Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Carcinógenos
/
Aductos de ADN
/
Desoxiguanosina
/
Glicina
Tipo de estudio:
Diagnostic_studies
Límite:
Animals
Idioma:
En
Revista:
Chem Res Toxicol
Asunto de la revista:
TOXICOLOGIA
Año:
1997
Tipo del documento:
Article
Pais de publicación:
Estados Unidos