2 deoxyuridine

The following tritium radiolabeled compounds were used during the project: [³H]-Uracil (40 Ci/mmol), [³H]-Thymidine (71.7 Ci/mmol), [³H]-Uridine (60 Ci/mmol) were obtained from PerkinElmer (Waltham, MA, USA). [³H]-Cytidine (20 Ci/mmol) was purchased from American Radiolabeled Chemicals Incorporated (St Louis, MO, USA). Uridine, Uracil, Thymidine, 5-FlourUracil, 5-FlouroUridine, 2’-DeoxyUridine, 3’-deoxyThymidine, Sulfadiazine, NBMPR, Adenosine, Inosine, resazurin sodium salt and Phenyl Arsine Oxide were bought from Sigma-Aldrich (Poole, UK). 5-Flouro 2’DeoxyUridine was from VWR; 5-MethylUridine was from Alfa Aesar; 2’,3’-dideoxyUridine was from Carbosynth; Pyrimethamine was from Fluka; Ara-A was from ICN Biomedicals.

Thymidine, 2’-deoxyuridine and formic acid (LC-MS grade) were obtained from Sigma Aldrich (Poole, UK). Internal standards (IS) Thymidine-1’,2’,3’,4’,5’-¹³C₅ and 2’-deoxyuridine-1’,2’,3’,4’,5’-¹³C₅ were from Toronto Research Chemicals (Toronto ON, Canada) and 70% perchloric acid (PCA) was obtained from VWR (Lutterworth, UK). Methanol (HPLC grade) was supplied by Rathburn (Walkerburn, Scotland). Deionized water was prepared in-house (<1.0 µS/cm). Analyte-free dialyzed human plasma (with K₂EDTA) was obtained from TCS Biosciences Ltd (Buckingham, UK). Analyte-free human plasma containing K₃EDTA, lithium heparin, analyte-free human serum from individual donors and whole blood (for the preparation of hemolyzed plasma) were obtained from Biological Speciality Corporation (Colmar, PA, USA). Hyperlipidaemic plasma containing 247 mg/dL cholesterol was supplied by Seralab (Haywards Heath, UK). Urine was obtained from in-house healthy volunteers.

The enzymatic reaction was carried out using 99% pure uridine, citidine, adenosine, guanosine, inosine, xanthosine, 2′–deoxyuridine, thymidine, vidarabine or 5–methyluridine from Sigma–Aldrich (Burlington, MA, USA) and water purified using a Milli-Q unit (Merck, Darmstadt, Germany). The reaction was carried out as follows: the desired stock concentration of nucleoside was prepared by weighing it and dissolving it in Tris–HCl buffer pH 7.5, then 500 μL of said nucleoside at the required concentration (diluted with water when needed) was added to plastic test tubes (total volume 1.5 mL), and five replicates were made for each final concentration of nucleoside. The number of points on the curve (i.e., the number of nucleoside samples) depends on the further accuracy of plotting the reaction rate versus substrate concentration curve; in this work, it was 15 points for each substrate where applicable. Next, 5 μL of purified RihC solution at a concentration of approximately 200 μg mL⁻¹ was added to each sample (the final concentration in the solution is 2 μg mL⁻¹) and stirred. At certain time intervals, the enzymatic reaction was stopped by adding 5 µL of concentrated HCl (time differed for different nucleosides). The prepared samples were then used for analysis.