Synthesis and Characterization of Tobacco Metabolites

NNK and NNKOAc were purchased from Toronto Research Chemicals. (S)-NNAL, (R)-NNAL, N⁶-PHB-dAdo, 2-(3-pyridyl)-1,3-dithiane, tert-butyl 3-(2-(3-pyridyl)-1,3-dithianyl)-1-propylcarbamate, and [pyridine-D₄]tert-butyl 3-(2-(3-pyridyl)-1,3-dithianyl)-1-propylcarbamate were synthesized as previously described.^{15 (link),33 (link),34 (link)} [¹⁵N₅]2′-deoxyadenosine (>99% ¹⁵N₅ incorporation) was obtained from Cambridge Isotope Laboratories (Tewksbury, MA). 6-Chloropurine-2′-deoxyriboside was obtained from Carbosynth (Compton, United Kingdom). Reagents for DNA isolation were purchased from Qiagen (Hilden, Germany). Calf thymus DNA, phosphodiesterase II, and micrococcal nuclease were obtained from Worthington Biochemical Co. (Lakewood, NJ). Alkaline phosphatase was procured from Roche Diagnostics GmbH (Mannheim, Germany). Porcine liver esterase and all other chemicals and solvents were obtained from either Sigma Aldrich (Milwaukee, WI) or Thermo Scientific (Waltham, MA) in reagent grade or higher and used without further purification.