Thymidine solution preparation

This method is also used for the analysis of the drug in tablets after employing a resolution solution prepared by dissolving accurately weighed quantities of thymine and thymidine in water, diluted with water to obtain a solution having a known concentration of 0.1 JLg/ml of each component. The resolution, R, between thymine and thymidine is not less than 2.0, and thymine is resolved from the void volume... 

Preparation of Radioactive FdUMP. [ H]- and [ CJFUdR are commercially available and conveniently converted to the 5 -monophosphate using thymidine kinase found in the 20-40% (NH4) 2SO4 fraction of the cell-free supernatant from Escherichia coli B. A solution (1.0 ml) containing about 1 mAf [ H]- or [ CJFUdR, 7.5 mAf ATP, 7.5 mAf MgCU, 30 mAf KF, 0.5 mg of bovine serum albumin, 70 mAf Tris-HCl (pH 7.8), and approximately 0.1 mg of the crude thymidine kinase preparation is incubated at 37°. The reaction is monitored by application of aliquotes to a micro DEAE-cellulose column (0.5 X0 8 cm) and stepwise elution with 3 ml of 5 mAf ammonium formate (pH 4.5) to elute unreacted nucleoside and 3 ml of 100 mAf of the same buffer to elute the nucleotide radioactivity is determined in each fraction to ascertain the extent of conversion. After the reaction is 80-90% complete (usually 60 min), the mixture is diluted to 20 ml with water, and purified on a DEAE-cellulose column (1X5 cm) by the method of Rustum and Schwartz- ... 

An amount of enzyme preparation equivalent to 900 mg of wet cells was made up to 25 ml with the above potassium phosphate buffer solution. 150 mg (1.15 mmol) of 5-fluorouracil and 1.0 gram of thymidine (4.12 mmol) were dissolved in 15 ml of the above potassium phosphate buffer solution. The mixture was incubated at 37°C for 18 hours. After this time, enzyme action was stopped by the addition of four volumes of acetone and one volume of peroxide-free diethyl ether. The precipitated solids were removed by filtration, and the filtrate was evaporated under nitrogen at reduced pressure until substantially all volatile organic solvent had been removed. About 20 ml of aqueous solution, essentially free of organic solvent, remained. This solution was diluted to 100 ml with distilled water. 

Esters of /3-D-mannopyranosyl pyrophosphates have not yet been prepared, and consequently their behavior in alkaline solution is unknown. However, thymidine 5 -(/3-L-rhamnopyranosyl pyrophosphate), in which the steric disposition of groups should be similar in the normal (1C) conformation of the glycosyl group (85), is cleaved readily under alkaline conditions.103... 

An amount of enzyme preparation equivalent to 900 mg of wet cells was made up to 25 ml with the above potassium phosphate buffer solution. 150 mg (1.15 mmol) of 5-fluorouracil and 1.0 gram of thymidine (4.12 mmol) were dissolved in 15 ml of the above potassium phosphate buffer solution. 

Thymidine (85.4 g 0.353 mol) was dissolved in 500 mL dry DMF (dimethyl formamide) and added to N-(2-chloro-l,l,2-trifluoroethyl)diethylamine (100.3 g 0.529 mol) [prepared according to the method of D. E. Ayer, J. Med. Chem. 6, 608 (1963)]. This solution was heated at 70°C for 30 minutes then poured into 950 mL ethanol with vigorous stirring. The product precipitated from this solution and was filtered. The ethanol supernatant was refrigerated then filtered to yield a total of 47.75 g (0.213 mol 60.3%) of 2,3 -anhydrothymidine melting point 228°-230°C. 

Bone marrow is placed into a balanced salt solution containing preservative-free heparin and a single cell suspension prepared by passing the marrow through a fine wire mesh. Alternatively, a small Potter-Elvehjem tissue homogenizer (Hll) may be used. The nucleated cell count is adjusted to 5-10 X 106 ml of balanced salt solution and deoxyuridine added. Following incubation, 5 p,Ci of tritiated thymidine is added and the mixture incubated for an additional hour. The cells are then washed and the nucleated cell count determined. A 0.1 ml aliquot is then placed on a filter paper disk and dried. The activity of the dried disks is then measured in a scintillation... 

Liver cells are then exposed in vitro to the test compound and incubated with tritium-labeled thymidine for about 18 hours. At the end of the incubation, the cells are fixed on slides and prepared for autoradiography. For that the slides are first exposed to liquid photographic emulsion, air-dried and following a 7-day exposure in the dark, exposed to developing solution. 

Among the first cell viability assays developed for HTS was the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] tetrazolium reduction assay (Mosmann 1983) that served as a milestone for this type of study. The assay offered a non-radioactive alternative to tritiated thymidine incorporation into DNA as a method of measuring cell proliferation. In many cases, the MTT assay can directly substitute for the tritiated thymidine incorporation assay (Figure 6.3). The MTT tetrazolium compound is prepared in a physiologically balanced solution, added to cells in culture, and incubated for approximately 4 hr. Viable cells convert MTT into an intensely colored formazan product that can be quantitated by recording changes in absorbance at specific wavelengths. 

The mixed lymphocyte reaction (MLR) test was performed in 96-well flat-bottomed microtiter plates. Selected experimental agents were prepared as 10 mM stock solution in DMSO and a 50-fold dilution of this was prepared in RPMI. Serial dilutions were prepared from this subsequent solution and 10 xl of the diluted stock was added to the well to give concentrations in the assay starting at 9.5 xm. In each well was placed 1.5 x 105 donor cells to give a final volume of 0.2 ml RPMI 1640 medium supplemented with 10% human serum, 2mM L-glutamine, and penicillin/streptomycin. Cells were incubated at 37°C in a humidified atmosphere containing 5% carbon dioxide for 120 hours. 3H-Thymidine (0.5 xCi) was added in the final 6 hours of incubation and cell radioactivity levels determined, which were indicative of T-cell proliferation. 

Shaw has reported the first syntheses of thymidine- and 2 -deoxy-5-fluorouridine - 3, 5 - cyclic boranophosphorothioate (103a,b). These analogues, displaying increased lipophilicity, were prepared from a key intermediate, a cyclic phosphoramidite obtained by heating a thoroughly degassed HMPA solution of the nucleoside. This phosphoramidite was then converted to a cyclic phosphite triester in the presence of 4-nitrophenol and 5-ethylthio-lH-tetrazole, and converted to the boranated complex in situ. The cyclic boranophosphite was then converted to the 3, 5 -cyclic boranophosphorothioate. ... 

Uridine diphosphate galactose (109) was prepared using seven enzymes involved in three biosynthetic pathways, immobilised on super-bead columns. This method, which converted 50% of uracyl monophosphate into UDP-galactose (109), was superior to the solution approach as enzyme stability was improved. To study the biosynthesis of the pseudosaccaharide acarbose, thymidine 5 -diphospho-4-amino-4,6-dideoxy-a-D-glucopyranose(110) was synthesised from galactose in sixteen steps. 

CldU, IdU, BrdU, and thymidine (Sigma, St Louis, MO cat. 19250). Prepare 0.20 mAf stock solutions of CldU, IdU, or BrdU and a 10 mM (100-fold concentrated) stock solution of thymidine. Store at 4°C. 

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