LARGE-SCALE SYNTHESIS OF P-BENZOQUINONE-2'-DEOXYCYTIDINE AND P-BENZOQUINONE-2'-DEOXYADENOSINE ADDUCTS AND THEIR SITE-SPECIFIC INCORPORATIONINTO DNA OLIGONUCLEOTIDES

Benzene is a carcinogen in rodents and a cause of bone marrow toxicity and leukemia in humans. p-Benzoquinone (p-BQ) is one of the stable me tabolites of benzene, as well as of a number of drugs and other chemic als. 2'-Deoxycytidine (dC) and 2'-deoxyadenosine (dA) were allowed to react with p-BQ in aqueous solution at pH 7.4 and 4.5. The yields were considerably higher at pH 4.5 than at pH 7.4, as indicated by HPLC an alysis. The desired products were isolated by column chromatography on silica gel or cellulose. Identification was done by FAB-MS, H-1 NMR, and UV spectroscopy. The reaction of p-BQ with dC and dA at pH 4.5 pro duced the exocyclic compounds 3-hydroxy-1,N-4-benzetheno-2'-deoxycytid ine (p-BQ-dC), and 9-hydroxyl, N-6-benzetheno-2'-deoxyadenosine (p-BQ- dA), respectively, in a large scale and high yield. These adducts have been previously made in a microgram scale as the 3'-phosphate for P-3 2-postlabeling studies of their incidence in DNA. The p-BQ-dC and p-BQ -dA adducts have, in addition to the two hydroxyl groups of deoxyribos e, one newly formed hydroxyl group at the C-3 or C-9 of the exocyclic base of each product respectively. Incorporation of these adducts into oligonucleotides as the phosphoramidite requires the protection of al l three hydroxyl groups in these compounds. The exocyclic hydroxyl on the base has been successfully protected by acylation after protecting the 5'- and the 3'-hydroxyl groups of the sugar moiety with a 4,4'-di methoxytrityl group and a cyanoethyl N,N-diisopropylphosphoramidite gr oup, respectively. For the first time, to our knowledge, the fully pro tected phosphoramidites of p-BQ-dC and p-BQ-dA were prepared and incor porated site-specifically into a series of oligonucleotides. The coupl ing efficiency was very high (>98%). However, deprotection of the DNA oligomers with ammonia produced only 50% of the desired oligomers cont aining the adduct. In contrast, when 10% of 1,8-diazabicyclo[5.4.0]und ec-7-ene (DBU) in methanol at room temperature was used, only the desi red oligomers were detected by HPLC. Thus, by deprotecting the oligome rs with methoxide ions (DBU/methanol) and avoiding the use of ammonia, a high yield of modified DNA was obtained. After purification of thes e oligomers by HPLC, they were hydrolyzed enzymatically and analyzed b y HPLC, which confirmed the base composition and the incorporation of the adducts. The mass spectroscopic analysis of the DNA oligomers was confirmed by electrospray MS. These oligomers are now under investigat ion for their biochemical properties.