title: Large scale synthesis of p-benzoquinone-2'-deoxycytidine and p-benzoquinone-2'-deoxyadenosine adducts and their site-specific incorporation into DNA oligonucleotides.
abstract: 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 metabolites of benzene, as well as of a number of drugs and other chemicals. 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 analysis. The desired products were isolated by column chromatography on silica gel or cellulose. Identification was done by FAB-MS, 1H NMR, and UV spectroscopy. The reaction of p-BQ with dC and dA at pH 4.5 produced the exocyclic compounds 3-hydroxy-1,N4-benzetheno-2'-deoxycytidine (p-BQ-dC), and 9-hydroxy-1,N6-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 32P-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 deoxyribose, 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 all 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'-dimethoxytrityl group and a cyanoethyl N,N-diisopropylphosphoramidite group, respectively. For the first time, to our knowledge, the fully protected phosphoramidites of p-BQ-dC and p-BQ-dA were prepared and incorporated site-specifically into a series of oligonucleotides. The coupling efficiency was very high (> 98%). However, deprotection of the DNA oligomers with ammonia produced only 50% of the desired oligomers containing the adduct. In contrast, when 10% of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) in methanol at room temperature was used, only the desired oligomers were detected by HPLC. Thus, by deprotecting the oligomers with methoxide ions (DBU/methanol) and avoiding the use of ammonia, a high yield of modified DNA was obtained. After purification of these oligomers by HPLC, they were hydrolyzed enzymatically and analyzed by 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 investigation for their biochemical properties.
mesh_terms: D001483:Base Sequence; D001562:Benzimidazoles / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism; D016227:Benzoquinones / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D018736:DNA Adducts / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D003839:Deoxyadenosines / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D003841:Deoxycytidine / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism*
chemical_list: C096961:3-hydroxy-1,N(4)-benzetheno-2'-deoxycytidine; C096962:9-hydroxy-1,N(6)-benzetheno-2'-deoxyadenosine; D001562:Benzimidazoles; D016227:Benzoquinones; D018736:DNA Adducts; D003839:Deoxyadenosines; D009838:Oligodeoxyribonucleotides; D003841:Deoxycytidine; C004532:quinone