Phosphodiesterase 2

All nucleoside phosphoramidites including 5-methyl-dC, 5-hydroxymethyl-dC, 5-formyl-dC-III, 5-carboxy-dC, Ac-dC, dT, dA, dG, and dmf-dG, reagents, and controlled pore glass solid support for oligodeoxynucleotide synthesis were acquired from Glen Research Corporation (Sterling, VA). Human recombinant DNA methyltransferase 1 (DNMT1) and Δ580-DNMT1^{8 (link)} (missing the PCNA,^{23 (link)} DNMT3A/B interaction domains^{23 (link), 24 (link)}) were purchased from New England BioLabs (Ipswich, MA). Phosphodiesterase I, Phosphodiesterase II, and DNase I were acquired from Worthington Biochemical Corporation (Lakewood, NJ). Bovine intestinal alkaline phosphatase was procured from Sigma Aldrich Chemical Company (Milwaukee, WI). All remaining laboratory chemicals and solvents were purchased from ThermoFisher Scientific (Waltham, MA) and Sigma-Aldrich (Milwaukee, WI). The synthesis of ¹³C₁₀¹⁵N₂-5-Methyl-2′-deoxycytidine was described previously.^{22 (link)}

The AIN-93G and AIN-93M powdered mouse diets were purchased from Harlan Teklad (Cambridgeshire, UK). Natural DHM was isolated from a kava product supplied by Gaia Herbs, Inc. (Brevard, NC), following reported protocols.^{20 (link)} NNK, [¹³C₆]NNK, [¹³C₆]NNAL, [CD₃]O⁶-mG and [4-CD₂, CD₃]NNAL-O-gluc were purchased from Toronto Research Chemicals (Toronto, ON, Canada).^{22 (link)}O⁶-methylguanine (O⁶-mG) was purchased from Midwest Research Institute (Kansas City, MO). 7-[4-(3-Pyridyl)-4-oxobut-1-yl]guanine (7-pobG), O²-[4-(3-pyridyl)-4-oxobut-1yl]thymidine (O²-pobdT), O⁶-[4-(3-pyridyl)-4-oxobut-1-yl]-2′-deoxyguanosine (O⁶-pobdG), 7-[4-(3-pyridyl)-4-hydroxobut-1-yl]guanine (7-phbG), O²-[4–(3-pyridyl)-4-hydroxobut-1yl]thymidine (O²-phbdT), O⁶-[4-(3-pyridyl)-4-hydroxobut-1-yl]-2′-deoxyguanosine (O⁶-phbdG) and their corresponding deuterated analogs were synthetized following reported procedures.^{23 (link),24 (link)} Micrococcal nuclease and phosphodiesterase II were purchased from Worthington Biochemical Corporation (Lakewood Township, NJ). Alkaline phosphatase was from Roche Molecular Biochemicals (Pleasanton, CA).

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.

ɣ-OH-Acr-dGuo and [¹³C₁₀¹⁵N₅]ɣ-OH-Acr-dGuo were prepared by reaction of acrolein with dGuo or fully labeled dGuo (Toronto Research Chemicals). The resulting products were characterized and quantified by ¹H NMR, using toluene as an internal standard, as previously described;^{19 (link)} εdAdo and [¹³C₅]εdAdo (labeled with ¹³C in the five carbons of the deoxyribose ring) were purchased from Toronto Research Chemicals (North York, Ontario, Canada). Reagents for DNA isolation were obtained from Qiagen (Germantown, MD). Calf thymus DNA, micrococcal nuclease and phosphodiesterase II were purchased from Worthington Biochemical Co. (Lakewood, NJ). Alkaline phosphatase and other solvents were obtained from Sigma-Aldrich Chemical Co. (Milwaukee, WI).

Acr-dGuo and [¹³C₁₀¹⁵N₅]Acr-dGuo (comprising both adducts 1 and 2) were prepared as described^{29 (link)} by reaction of acrolein with fully labeled dGuo (Toronto Research Chemicals); εdAdo, [¹³C₅]εdAdo (labeled with ¹³C in the five carbons of the deoxyribose ring), εdCyd and [¹³C¹⁵N₂]εdCyd (labeled with ¹³C in position 5, and with ¹⁵N in positions 4 and 6) were purchased from Toronto Research Chemicals (North York, Ontario, Canada). Reagents for DNA isolation were obtained from Qiagen (Germantown, MD). Calf thymus DNA, micrococcal nuclease and phosphodiesterase II were purchased from Worthington Biochemical Co. (Lakewood, NJ). Alkaline phosphatase and other solvents were obtained from Sigma-Aldrich Chemical Co. (Milwaukee, WI).

ɣ-OH-Acr-dGuo (1) and [¹³C₁₀¹⁵N₅]ɣ-OH-Acr-dGuo were prepared by reaction of acrolein with dGuo or fully labeled dGuo (Toronto Research Chemicals, North York, Ontario, Canada).^{28 (link)} 1,N⁶-etheno-dAdo (4), [¹³C₅]1,N⁶-etheno-dAdo, 8-oxo-dGuo (5), and [¹³C₂¹⁵N]8-oxo-dGuo were purchased from Toronto Research Chemicals. (6S,8S)ɣ-OH-Cro-dGuo (2), (6R,8R)ɣ-OH-Cro-dGuo (3), and [¹⁵N₅](6S,8S;6R,8R)ɣ-OH-Cro-dGuo were prepared as described. ^{29 (link)} The purities of the standards were tested by LC-MS and were greater than 95%. Reagents for DNA isolation were procured from Qiagen (Germantown, MD). Calf thymus DNA, micrococcal nuclease, and phosphodiesterase II were purchased from Worthington Biochemical Co. (Lakewood, NJ). Alkaline phosphatase and other solvents were obtained from Sigma-Aldrich Chemical Co. (Milwaukee, WI).