Uridine phosphorylase and

Compound 25 (Fig. 18.9), a prodrug of 9-P-D-arabinofuranosyl guanine (26), was developed for the potential treatment of leukemia. Compound 24 is poorly soluble in water and its synthesis by conventional techniques is difficult. An enzymatic demethoxylation process was developed using adenosine deaminase (Mahmoudian et al., 1999, 2001). Compound 25 was enzymatically prepared from 6-methoxyguanine (27) and ara-uracil (28) using uridine phosphorylase and purine nucleotide phosphorylase. Each protein was cloned and overexpressed in independent Escherichia coli strains. Fermentation conditions were optimized for production of both enzymes and a co-immobilized enzyme preparation was used in the biotransformation process at 200 g/L substrate input. Enzyme was recovered at the end of the reaction by filtration and reused in several cycles. A more water soluble 5 -acetate ester of compound 26 was subsequently prepared by an enzymatic acylation process using immobilized Candida antarctica lipase in 1,4-dioxane (100 g/L substrate) with vinyl acetate as the acyl donor (Krenitsky et al., 1992). 

Two routes are known by which the free base, uracU, can enter the ribonucleotide pool. One proceeds by the sequential actions of uridine phosphorylase and uridine-cytidine kinase (reactions 5 and 6, Fig. 12-1) this route is discussed below. The other route is by way of a single-step phosphoribosyltransferase reaction specific for uracil (reaction 4, Fig. 12-1) ... 

For example, intact Ehrlich ascites tumor cells, or extracts therefrom, transfer the ribosyl group of uridine to hypoxanthine and thereby catalyze the net synthesis of inosine this reaction depends upon the coupled actions of uridine phosphorylase and purine nucleoside phosphorylase (89). Similar ribosyl transfers have been demonstrated with bacterial cells and extracts. Krenitsky has studied the kinetics of exchange between uracil-2- C and nonisotopic uridine catalyzed by highly purified uridine phosphorylase (30) ... 

The reversibility of uridine phosphorolysis suggests that uridine phosphorylase activity of the cell may be able to operate in the direction of synthesis, utilizing endogenous ribose 1-phosphate, and thereby contribute to the anabolism of uracil. Thus, uridine phosphorylase and uridine kinase acting in sequence would elevate uracil to the nucleotide level. 

In addition to participation in the deoxyribosyl transfer reactions described above, in which free deoxyribose 1-phosphate is formed as an intermediate, thymidine phosphorylase also catalyzes deoxyribosyl transfers involving thymine and uracil in which deoxyribosyl phosphate is an intermediate but is enzyme-bound (18-20). Such transfers require non-stoichiometric amounts of phosphate (19). The reaction mechanisms of uridine phosphorylase and purine nucleoside phosphorylase are not of this type and, accordingly, direct deoxyribosyl transfers occur only between substrates for thymidine phosphorylase, as exemplified by reaction (5) above and the following (15) (the asterisk indicates C-labeling) ... 

Felczak K, Drabikowska AK, Vilpo JA, Kuhkowski T, Shugar D (1996) 6-Substituted and 5,6-disubstituted derivatives of uridine stereoselective synthesis, interaction with uridine phosphorylase, and in vitro antitumor activity. J Med Chem 39 1720-1728... 

The key steps in Fluorouracil metabolism are shown in Scheme 1. Up to 80 % of 1 administered as injection is transformed to dihydrofluorouracil (DHFU, 13) by dihydropyrimidine dihydrogenase (mostly in liver tissues). However, this metabolite is not involved into antineoplastic activity instead, 13 itself and its further metabolites are responsible for most of the toxic effects of 1. The main mechanism of activation of Fluorouracil is conversion to fluorouridine monophosphate (FUMP, 14), either directly by orotate phosphoribosyltransferase, or via fluorouridine (FUR, 15) through the sequential action of uridine phosphorylase and uridine kinase. 14 is then phosphorylated to give fluorouridine diphosphate (FUDP, 16), which can be either phosphorylated again to the active metabolite fluorouridine triphosphate (ITJTP, 19), or reduced to fluorodeoxyuridine diphosphate (FdUDP, 18) by ribonucleotide reductase. In turn, 18 can either be dephosphorylated or phosphorylated to generate... 

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