Synthesis of 8-carbon-14-labeled O6-methyldeoxyguanosine and its deoxynucleotide copolymers

Abstract

To study the nature and repair of the promutagenic DNA lesion O6-methylguanine, we have synthesized 8-14C-labeled O6-methyldeoxyguanosine triphosphate and investigated the kinetics of its incorporation into the synthetic copolymers poly(dC'm6dG) and poly(dT,m6dG). Deoxy[8-14C]guanosine was methylated with ethereal diazomethane and the products were separated by high-pressure liquid chromatography. O6-Methyldeoxy[14C]guanosine was converted to the 5'-monophosphate with carrot phosphotransferase and then to the 5'-triphosphate via the phosphorimidazolidate formed by the action of N,N'-carbonyldiimidazole. Although m6dGTP was a poor substrate for Escherichia coli DNA polymerase I, copolymers could be synthesized from dCTP or dTTP and m6dGTP with terminal deoxynucleotidyl transferase. The percent of m6dG in the polymer increased linearly as the percentage of m6dGTP in the polymerization mixture was increased to 20% of the total. The percent incorporation of m6dGTP into poly(dT,m6dG) was, however, higher than into poly(dC,m6dG). Good yields of both polymers were readily obtained. The stability of O6-methyldeoxyguanosine in poly(dT,m6dG) was found to be pH dependent, and the half-life has been measured at four different pH values.

The synthesis of oligoribonucleotides containing O6-methylguanosine: the role of conserved guanosine residues in hammerhead ribozyme cleavage

“…The latter compounds require further conversion into the 6-trimethylamino-(21, 29) or the 6-Nmethylpyrrolo-(30, 31) derivatives in order to effect displacement by methoxide ion. With the exception of the diazomethane treatment of (deoxy)nucleosides which gives rise to extensive side products that include N7-methyland N'-methyl-(deoxy)guanosines (24,25), no direct route to the synthesis of 06alkyl guanosine or deoxyguanosine has been reported.…”

Escherichia coli polymerase I can use O2-methyldeoxythymidine or O4-methyldeoxythymidine in place of deoxythymidine in primed poly(dA-dT).poly(dA-dT) synthesis.

“…Therefore, the fiact that 06-methyl dGTP and 06-ethyl dGTP could be incorporated into polymers by terminal deoxynucleotidyltransferase but not by pol I (8,10) indicates that either no incorporation occurred or that once incorporated the nucleotide was excised. Abbott et aL (10) have evidence that 06-methyl dGTP is not incorporated by pol I. Copolymers containing 06-methyl or 06-ethyldeoxyguanosine have been prepared but only with terminal deoxynucleotidyltransferase, although both derivatives inhibit polymerization of unmodified dNTPs (7,8,10).…”

“…The derivatives studied in this way include 06-methyldeoxyguanosine (7), 06-ethyldeoxyguanosine (8), and 3-methyl-and 1,N6-ethenodeoxycytidine (9). In the case of the best-studied derivatives, 06-alkyldeoxyguanosine, both Abbott et aL (10) and Miller et al (8) report that the triphosphate was not incorporated by pol I and, in fact, appeared to be an inhibitor of normal synthesis.…”

3H‐labelled alkyl‐nucleotides, ‐nucleosides and ‐bases for the immunoanalytical quantification of DNA damage and repair

“…-Aminopentyl-7-hydro-8-oxo-9-ethyl-guanine (24). Twenty-nine micromoles (23) were dissolved in CH 3 COOH (conc.)…”