He Synthesis And Study Of Spectral Properties Of BPQ -DNA Adducts

Benzo[a]pyrene (B[a]P) is a classical one of the Polycyclicaromatic hydrocarbons (PAHs), which are ubiquitous environmental pollutants and suspect human carcinogens. B[a]P is an product of incomplete combustion and is emitted into the air we breathe, and it is carcinogenic and mutagenic. B[a]P itself is biologically inert, and its carcinogenic effects requires metabolic activation to biologically reactive intermediates by many enzymes. In one of the pathways, benzo[a]pyrene-7,8-dione (BPQ) is produced by the oxidation of (±)-B[a]P-7,8-trans-dihydrodiol, w hich is catalyzed by aldo-keto reductases(AKRs). B[a]P-7,8-dione is both electrophilic and redox-active. The electrophilic BPQ can undergo 1,4-or 1,6-Michael addition with DNA to form BPQ-DNA adducts. DNA adduct is an important biological monitoring indicator, and is the most important and common form of DNA chemical injuries. If the adducts avoide their own repair system of the biological cells, they could become the minimum factor of mutagenicity and carcinogenicity.In this study, BPQ-dC, BPQ-dA, BPQ-dG adduct are synthesized, and the best chromatographic separation conditions of the three kinds of adduct are discussed. BPQ-dC, BPQ-dA, BPQ-dG adduct are prepared, and their spectral properties are studied.The synthesis of BPQ-DNA adducts:BPQ-dC:A 4500 μL solution of BPQ (7 mg) and dC (80 mg) in 2:1 DMF-sodium phosphate buffer (0.25 M, pH=7.0) was stirred at 65 ℃ for 16 h. The reaction mixture was filtered, and the solution was subjected to reverse phase (RP)-HPLC on a YMC-ODS-A C18 column (10×250 mm,5μm, Waters, USA)BPQ-dA:A 3000 μL, solution of BPQ (4 mg) and dA(50 mg) in 1:1 DMF-sodium phosphate buffer(0.25 M, pH=7.0) was stirred at 60 ℃ for 20 h. The reaction mixture was filtered, and the solution was subjected to reverse phase (RP)-HPLC on a YMC-ODS-A C18 column (10× 250 mm,5μm, Waters, USA)BPO-dG:A 3000 μL solution of BPQ (5 mg) and dG(40 mg) in 2:1 DMF-sodium phosphate buffer(0.25 M, pH=7.0) was stirred at 60 ℃ for 6 h. The reaction mixture was filtered, and the solution was subjected to reverse phase (RP)-HPLC on a YMC-ODS-A C18 column (10 ×250 mm,5 μm, Waters, USA)Chromatographic separation conditions of BPQ-DNA adducts:BPQ-dC:At room temperature, using a mobile phase of 35% solvent A (HAC-NH4AC buffer,10 mM, pH=5.0) and 65% solvent B (3:1methanol-acetonitrile) for 5 min, increasing linearly to 20% solvent A and 80% solvent B withi n 60 min at a flow rate of 1.0 ml/min. UV detector wavelength is 260 ran.BPQ-dC:At room temperature, using a mobile phase of 35% solvent A (TEAA buffer,50mM, pH=7.0) and 65% solvent B (3:lmethanol-acetonitrile) for 5 min, increasing linearly to 100% solvent B within 45 min at a flow rate of 1 ml/min. UV detector wavelength is 260 nm.BPQ-dG:At room temperature, using a mobile phase of 40% solvent A (TEAA buffer,50mM, pH=7.0) and 60% solvent B (3:1methanol-acetonitrile) for 5 min, increasing linearly to 10% solvent A and 90% solvent B within 40 min at a flow rate of 1 ml/min. UV detector wavelength is 260 nm.Preparation and spectral properties of BPQ-DNA adducts:BPQ-dC:Under the selected conditions, each individual peak of BPQ-dC was pooled and reduced in volume by rotary evaporation in vacuo, the residues were subjected to UV, CD, MS and so on. Finally four major products:BPQ-dC2, BPQ-dC3-3,BPQ-dC6, BPQ-dC7 were obtained.Their molecular weights were all 526.BPQ-dA:Under the selected conditions, each individual peak of BPQ-dA was pooled and reduced in volume by rotary evaporation in vacuo, the residues were subjected to UV, CD, MS and so on. Finally two major products:BPQ-dAl, BPQ-dA2 were obtained.Their molecular weights were both 550.BPQ-dG:Under the selected conditions, each individual peak of BPQ-dG was pooled and reduced in volume by rotary evaporation in vacuo, the residues were subjected to UV, CD, MS and so on. Finally four major products:BPQ-dGI, BPQ-dG2, BPQ-dG3 and BPQ-dG4 were obtained. Their molecular weignis were an 566.