Thymidine 3 -phosphate, preparation

In an investigation of a possible way of delivering nucleoside drugs across cell membranes, the glucosyl phospholipid (115) of thymidine was prepared. This was found to interact with large unilamellar vesicles so as to place the nucleotide derivative Inside the vesicles, suggesting transport across the lipid bilayer. i Similar mannose phosphate derivatives of AZT, ddT and FDU were... 

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. 

Manson and Lampen243 reported that they obtained the phosphorolysis and arsenolysis of hypoxanthine desoxyriboside by enzyme preparations from calf-thymus gland and rat liver. An acid-stable phosphate ester was isolated as a product of phosphorolysis. Results to be outlined suggested that this ester was 2-desoxy-D-ribose 5-phosphate and evidence was obtained for its formation by a mutase type reaction from 2-desoxy-D-ribose 1-phosphate. This evidence was extended and reinforced when Manson and Lampen244 obtained indications for the formation of desoxy-D-ribose 1-phosphate during the phosphorolysis of thymidine. Consequently the conversions outlined may be depicted as shown. 

Epithelial cells of small intestine were prepared in a fractional way (4), the older, less adherent villus tip cells being washed out by EDTA-containing phosphate buffer first, while mitotic crypt cells appeared in the final fractions. The enzyme characteristics of the series of fractions obtained (Fig. 13) followed conventional criteria for differentiated (villus) and less differentiated (crypt) cells (3, 4). The thymidine kinase activity decreased from crypt to villus while the activity of alkaline phosphatase increased (Fig. 13). 

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. 

Figure 1.6 Antigen presentation by lung DCs in a primary MLR effect of prior depletion of alveolar macrophages in DC donors. Semi-purified lung DCs from groups of control or treated BN rats (n = 3) were titrated into cultures of WAG lymph node cells, and resulting DNA synthesis determined as Incorporation of [ H] thymidine at the 120 h time point. Panel A lung DC pools were from untouched controls ( ), and animals Intratracheally inoculated 48 h previously with either phosphate-buffered saline (PBS) (O) or liposomes containing PBS ( ) or DMDP ( ). Panel B MLR stimulatory activity of lung DCs prepared from BN rats at varying periods after intratracheal administration of DMDP liposomes zero time control ( a ) 24 h post-administration ( ) 48 h (O) 72 h ( ). Reproduced with permission from Holt et af. (1993).

When 5 -0-tritylthymidine-3 -phosphate is treated with excess tri-isopropyl benzenesulphonylchloride (TPS) and thymidine, and then deprotected, the trinucleoside monophosphate (7a) is obtained. The 5-bromo- and 5-fluoro-deoxyuridine analogues (7b) and (7c) are prepared similarly. All are resistant to snake venom and spleen phosphodiesterases, and hydrolyse too slowly under physiological conditions for the cytotoxic moiety to be effective. When protected UpU is treated with bis-(4-nitrophenyl) phosphorochloridate, and subsequently with an amine or amino-acid ester, the dinucleoside phosphor-amidates (8) are formed. Although the compounds investigated split the P—N bond under the conditions required for protecting-group removal, the method has potential for the preparation of easily fissionable neutral phospho-triesters. 

Preparation of Crude Extracts The parasite larvae or adult forms were homogenized by grinding with sand in a mortar (placed on ice) with three to five volumes of ice-cold 0.05 M phosphate buffer, pH 7.5, containing 0.1 M KCl and 0.01 M 2-mercaptoethanol, except for assays of thymidine kinase activity, when phosphate buffer was substituted by Tris-HCl buffer at the same concentration and pH. The suspension was sonicated and centrifuged to obtain crude extract. 

Scheme 6 illustrates the use of these protecting groups in the phosphorylation of 2 -deoxynucleosides by Michelson and Todd. 5 -0-Trityl-thymidine (110) was phosphorylated with dibenzyl phosphorochloridate (44) to (111), which, after treatment with 80% acetic acid, afforded thymidine 3-(benzyl phosphate) (112). Catalytic hydrogenolysis of (112) gave thymidine 3 -phosphate. Acetylation of (110) yielded the 3 -acetate (114) which, on detritylation to (115), followed by phosphorylation, catalytic reduction, and deacetylation, gave thymidine 5 -phosphate (116), identical with the thymidylic acid obtained by enzymic hydrolysis of 2 -deoxyribonueleic acid. A rather similar sequence was applied to the preparation of the 2 -deoxycytidine analogs of (113) and (116). 

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