Analysis of oxidative DNA damage, Part II: Synthesis of the internal standard 8-[18O]hydroxy-2'-deoxyguanosine

Abstract

In the project 'Oxidative DNA Damage' the first aim is to develop a mass spectrometric method for the quantification of 8-hydroxy-2'- deoxyguanosine (oh8dG). The required precision of the method requires the application of a labeled analogue as an internal standard. This report describes the syntheses of 8-[18O]hydroxy-2'-deoxyguanosine (18Oh8dG), which is to be used as internal standard in the quantitative GC-MS analysis of 8-hydroxy-2'deoxyguanosine. 8-[18O]Hydroxy-2'- deoxyguanosine was synthesised starting from 2'-deoxyguanosine (dG) in three steps. In the first step dG was brominated at the C-8 position, resulting in 8-bromo-2'-deoxyguanosine (8-Br-dG). 8-Br-dG was converted into 8-[18O]benzyloxy-2'-deoxyguanosine (8-[18O]OBz-dG) by treatment of 8-Br-dG with the sodium salt of [18O]benzyl alcohol. Finally 8-[18O] OBz-dG was hydrogenated resulting in 18Oh8dG. The sodium salt of [18O]benzyl alcohol was synthesised in three steps starting from ethyl benzimidate hydrochloride. In the first step ethyl benzimidate hydrochloride was converted in ethyl [18O]carboxy benzoate by treatment with H218O (95%). In the second step ethyl benzoate was reduced by lithium aluminium hydride to [18O]benzyl alcohol. In the last step [18O]benzyl alcohol was converted into the sodium salt of [18O] benzyl alcohol by treatment with sodium hydride. Starting with dG (5 g, 17 mmol), the overall yield of the total procedure was approximately 18 mg (0.11 mmol, 4%). The chemical purity of [18O]oh8dG was estimated to be 89%, based on HPLC analysis. The isotopic purity of 18Oh8dG was determined to be 93.4% based on GC-MS analysis. The mentioned chemical and isotopic purity comply with the planned application.<br>