Synthesis, characterization, and immunochemical detection of O6-(carboxymethyl)-2'-deoxyguanosine: a DNA adduct formed by nitrosated glycine derivatives.
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.
Harrison
K L
KL
MRC Toxicology Unit, University of Leicester, U.K.
Fairhurst
N
N
Challis
B C
BC
Shuker
D E
DE
D000818:Animals; D002273:Carcinogens / Q000138:chemical synthesis* / Q000737:chemistry; D002417:Cattle; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D003429:Cross Reactions / Q000276:immunology; D018736:DNA Adducts / Q000138:chemical synthesis* / Q000737:chemistry / Q000276:immunology; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000032:analysis / Q000138:chemical synthesis; D004797:Enzyme-Linked Immunosorbent Assay; D005998:Glycine / Q000031:analogs & derivatives / Q000737:chemistry*; D007074:Immunoglobulin G / Q000032:analysis / Q000302:isolation & purification; D015538:Nitrosation; D011817:Rabbits
D002273:Carcinogens; D018736:DNA Adducts; D007074:Immunoglobulin G; C107252:O(6)-(carboxymethyl)-2'-deoxyguanosine; D003849:Deoxyguanosine; D005998:Glycine