Synthesis, Characterization, and Immunochemical Detection of O⁶-(Carboxymethyl)-2‘-deoxyguanosine:  A DNA Adduct Formed by Nitrosated Glycine Derivatives

Abstract

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.

Synthesis of deuterium and C‐13‐labelled ethyl glycolate and their subsequent use in the synthesis of labelled analogues of the DNA adduct O6‐carboxymethyl‐2′‐deoxyguanosine

“…The synthesis of 1 was carried out with methyl glycolate and gave a lower overall yield (6.2%) than previously reported [6] despite several attempts to improve yields at each stage. We then investigated the hydrolysis of ethyl diazoacetate to give ethyl glycolate in both aqueous and deuterated solvents (Scheme 2, step (iii)).…”

“…In terms of cost, 2 was the preferred option but proved to be problematic for LC-MS analysis. Thus, 5, the C-13 labelled analogue of O 6 CMdG was obtained in sufficient yield for future analyses of biological samples by LC-MS . 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 …”

“…Hence, we pursued the use of LC-MS as a more selective, and potentially sensitive, technique to analyse DNA adducts in biological samples. The synthesis of unlabelled O 6 CMdG, 1 (Scheme 1), has been reported by other researchers [6] where methyl glycolate was used to furnish the carboxymethyl group at the O6 position of 2'-deoxyguanosine. However, we utilized ethyl glycolate due to the availability of starting materials to insert the labelled atoms for [ 2 …”

“…Previous research has examined this adduct using an immunoslot blot assay which is very sensitive but only a single adduct can be analysed at a time and difficulties can occur due to cross-reactivity of the antibodies [3,4] . A limited supply of a polyclonal antibody does exist for O 6 CMdG but attempts to produce a monoclonal antibody have been unsuccessful and a different technique will therefore be needed in the future. There has been considerable progress in the area of DNA adduct analysis by LC-MS and good sensitivity can be achieved (e.g.…”

Optimizing immunoslot blot assays and application to low DNA adduct levels using an amplification approach

“…The ISB has since been used to detect the M 1 dG adducts in mice livers (17) and human colorectal mucosa (18) and more recently we applied this methodology to the measurement of O 6 -carboxymethyl-deoxyguanosine adducts (O 6 CMdG, Fig. 1), using a polyclonal antibody (19), derived from nitrosated amines, for in vitro studies and in human blood samples of volunteers consuming a high red meat diet (20). The latter was confirmed by alternative qualitative methods of measuring O 6 CMdG (21).…”