Uridine production

The product, uridine, was separated from cytidine and allopurinol (internal standard) by chromatography on a Hypersil ODS column (4.6 mm X 100 mm, 5 pun). Hie mobile phase consisted of 100 mM ammonium acetate (adjusted to pH 5.0 with 6 M HC1) containing 1% (v/v) methanol and 1mA/ 1-octanesulfonic acid. The effluent was monitored at 262 nm. Uridine production was calculated with reference to a uridine standard, and after correction based on the concentration of the internal standard. 

The residue crystallizes in ether and yields about 600 mg of (3-3, 5 -di-p-toluyl-2 -desoxy-5-iodo-uridine which is recrystallized from toluene. The product Is obtained as colorless crystals, soluble in chloroform and pyridine, sparingly soluble In acetone, benzene ether and alcohol. Insoluble in water, MP 193°C. 

Succinimide is readily silylated by HMDS 2 to the N-silylated product 201, which seems, however, to be in equilibrium with the O-silylated derivative 202 a (cf the closely related reactive center in persilylated uridine 3) and reacts after 6-10 days at 24 °C with one equivalent of primary or secondary amines such as morpholine to give the crystalline colorless cyclic acylamidine 203 and HMDSO 7, even in the absence of any protective gas [33] (Scheme 4.12). The reaction is much faster on heating to 120 °C under argon. At these temperatures 201 and 202 a, and possibly also the acylamidine 203, are apparently partially O-silylated by HMDS 2 to the very sensitive 2,5-bis(trimethylsilyloxy)pyrrole 202b or to 2-tri-... 

Online detection using 4H nuclear magnetic resonance (NMR) is a detection mode that has become increasingly practical. In a recent application, cell culture supernatant was monitored on-line with 1-dimensional NMR for trehalose, P-D-pyranose, P-D-furanose, succinate, acetate and uridine.33 In stopped-flow mode, column fractions can also be analyzed by 2-D NMR. Reaction products of the preparation of the neuromuscular blocking compound atracurium besylate were separated on chiral HPLC and detected by 4H NMR.34 Ten isomeric peaks were separated on a cellulose-based phase and identified by online NMR in stopped-flow mode. 

The thermal synthesis of nucleoside-5 -phosphite monoester using (NH HPCb was carried out under relatively mild conditions (60°C, reaction time about 24 h) by A. W. Schwartz s group in Nijmegen, Holland in the case of uridine, the yield was 20%. Ammonium phosphate, however, cannot be used it gave yields of only 0.15% after very long reaction times (46 days). This confirms earlier suggestions that nucleoside-H-phosphonates, and condensation products possibly derived from them, would have been formed more readily on the primeval Earth than nucleotides (de Graaf and Schwartz, 2005). 

A convenient synthesis of uridine 2, 3 -cyclic phosphite 15 (as a 3 4 mixture of diasteroisomers because of the chirality at phosphorus) was based on the reaction of the suitably protected uridine derivative 14 with ethyl dichlorophosphite carried out in the presence of triethylamine and anhydrous ethanol (Scheme 6) [21]. After adding ethanol into the reaction medium, an efficient chromatographic separation of the crude reaction product was achieved on silica gel with diethyl ether giving analytically pure phosphite 15 in 75% yield. It is interesting to note that without adding ethanol, 15 is very unstable. 

This is the first product of the action of water on phosphorus oxychloride,80 and might be expected to react with a 1,2-glycol to give a cyclic phosphate which is subsequently hydrolyzed to a mixture of 2- and 3-phosphates. No explanation can be given as yet for the behavior of uridine. 

Favored reaction at HO-5 in uridine 3 -phosphate through the formation of a mixed acetal of an aliphatic aldehyde with another alcohol has been claimed, but product isolation required chromatography on cellulose, and the yield reported343 was low ( 23%). 

Synthesis of sucrose 6 -phosphate by an enzymic method using uridine 5 -(a-D-glucopyranosyl diphosphate) plus D-glucose 6-phosphate has been reported.126-131 The first, unambiguous, chemical synthesis of sucrose 6 -phosphate was achieved by Buchanan and coworkers.18 The reaction of 2,3,4,6,l, 3, 4 -hepta-0-acetylsucrose, prepared by five steps of synthesis, with cyanoethyl phosphate in pyridine gave a crude product from which sucrose 6 -phosphate was isolated as the barium salt. 

Figure 8.19. Sequence reactions from aspartic acid (AA) and carbamoyl phosphate (CP) to the end product, cytidine triphosphate (CTP). The first reaction is catalyzed by ATCase. The intermediary compounds are N-carbamoyl aspartic acid (N-CAA), L-dihydroorotic acid (L-DHOA), orotic acid (OA), orotidine 5 -phosphate (0-5 -P), uridine 5 -phosphate (U-5 -P), uridine diphosphate (UDP), and uridine triphosphate (UTP).

The primary end product is uridine monophosphate (UMP). In the conversion of UMP to dTMP, three important enzymes are ribonucleotide reductase, thymidylate synthase, and dihy-drofoiate reductase. All three enzymes are targets of antineoplastic drugs and are summarized in Table I-18-1. 

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