p.m. Different nucleosides

were assayed at 30 °C and pH 7. Reaction mixtures contained 1 × 1010 CFU, 2.5 mM 5-fluorouracil and 10 mM uridine, thymidine, 2′-deoxyuridine, 2′-deoxycytidine or 2′,3′-dideoxyuridine. Reactions were performed at different 5-fluorouracil and thymidine ratios (1 : 1, 4 : 1 and 1 : 4) at 30 °C, pH 7, and 200 r.p.m. All assays were performed selleck inhibitor three times in 1 mL of reaction medium. Subsequent to system characterization with 5-fluorouracil, the incorporation of other halogenated pyrimidine bases was tested using 5-chlorouracil and 5-bromouracil. Reactions were performed in a 1 : 4 ratio (2.5 mM halogenated base and 10 mM thymidine) in potassium phosphate buffer (30 mM, pH 7) at 30 °C. 1 × 1010 CFU were mixed with 3 mL of 1, 2, and 3% (w/v) agar or agarose. The mixture was then added dropwise to stirred sunflower oil (20 mL) at 25 °C. The resulting gel beads were cooled, filtered, washed with hexane and then with physiological solution to obtain solvent-free beads. 1 × 1010 CFU were mixed with 3 mL of phosphate buffer (30 mM, pH 7) containing 15, 20, and 25% (w/v) acrylamide/bis-acrylamide,

subsequently 50 μL of 10% (w/v) ammonium persulfate (APS) and 14 μL of N, N, N′, N′tetramethylethylenediamine (TEMED). The resulting gel was cut into small cubic pieces (1.0 × 1.0 × 0.2 cm). The biosynthesis of nucleoside analogues selleck chemicals llc was qualitatively evaluated by TLC Merck Silica gel 60 F254 in chloroform/methanol (80 : 20, v/v) as mobile phase. The quantitative analysis was performed by HPLC (Gilson) equipped with a UV detector (254 nm) using a Nucleodur 100-5 C18 column (5 μm, 125 × 5 mm). The isocratic mobile phase used was water/methanol (95 : 5, v/v) at room temperature and at a flow rate of 1.2 mL min−1. Retention times of substrates and products were as follows: Floxuridine biosynthesis: (1) uracil (1.0 min), 5-fluorouracil (1.4 min), 2′-deoxyuridine (2.0 min), floxuridine (3.0 min); (2) cytosine (1.1 min), 5-fluorouracil (1.4 min),

2′-deoxycytidine (2.2 min), floxuridine (3.0 min); (3) 5-fluorouracil (1.4 min), thymine (2.6 min), U0126 cell line floxuridine (3.0 min), thymidine (4.2 min). 5-fluorouridine biosynthesis: uracil (1.0 min), 5-fluorouracil (1.4 min), uridine (1.8 min), 5-fluorouridine (2.8 min). 5-chloro-2′-deoxyuridine biosynthesis: (1) uracil (1.0 min), 2′-deoxyuridine (2.0 min), 5-chlorouracil (4.8 min), 5-chloro-2′-deoxyuridine (6.0 min); (2) cytosine (1.1 min), 2′-deoxycytidine (2.2 min), 5-chlorouracil (4.8 min), 5-chloro-2′-deoxyuridine (6.0 min); (3) thymine (2.6 min), thymidine (4.2 min), 5-chlorouracil (4.8 min), 5-chloro-2′-deoxyuridine (6.0 min). Product identification was performed by MS-HPLC (See Supporting Information, Data S1). 5-fluoro-2′-deoxyuridine biosynthesis from thymidine and 5-fluorouracil was used as reaction test for the screening (Fig. 1).