Deoxyuridine diphosphate

Deoxyribose Deoxyuridine Deoxyuridylic acid deoxyuridine monophosphate (dUMP) Deoxyuridine diphosphate (dUDP) Deoxyuridine triphosphate (dUTP)... 

Scheme 14.11. A proposal for the use of ribonucleoside diphosphate reductase (ribonucleotide diphosphate reductase, EC 1.17.4.1) to convert adenosine diphosphate (ADP), uridine diphosphate (UDP), and cytidine diphosphate (CDP) into their respective 2 -deoxy analogues deoxyadenosine diphosphate (dADP),deoxyuridine diphosphate (dUDP),and deoxy-cytidine diphosphate (dCDP), or, in general, ribonucleic acids (RNAs) into the corresponding deoxyribonucleic acids (DNAs) (after Stubbe, J. 7. Biol. Chem., 1990,265, 5330).

Ribonucleotide reductase works on ribo-A, -U, -G, -C diphosphates to give the deoxynucleotide. The deoxyuridine, which is useless for RNA synthesis, is converted to deoxythymidine by the enzyme thymidylate synthase, which uses methylene tetrahydrofolate as a one-carbon donor. The odd thing here is that ribonucleotide reductase uses the UDP as a substrate to give the dUDP. This must then be hydrolyzed to the dUMP before thymidylate synthase will use it to make dTMP. Then the dTMP has to be kinased (phosphorylated) up to dTTP before DNA can be made. 

Fig. 3. Metabolism of the fluoropyrimidines dTMP = deoxythymidine monophosphate, dUMP = deoxyuridine monophosphate, FdUDP = fluorodeoxyuridine diphosphate, FdUMP - fluoro-deoxyuridine monophosphate, FdUTP = fluorodeoxyuridine triphosphate, FU-DNA= fluorouracil-deoxyribonucleic acid, FUDP = fluorouracil diphosphate, FUMP = fluorouracil monophosphate, FU-RNA = fluorouracil-ribonucleic acid, FUTP = fluorouracil triphosphate.

A polynucleotide chain such as that in Chart 10 would then be indicated in the trivial system as in the following example UpCpTpApGp. This system differentiates simply among the various dinucleotides. UpU is deoxyuridylyl-3, 5 -deoxyuridine, dUpU is de-oxyuridylyl-(3, 5)-deoxyuridine, pUp is uridine-3, 5 -diphosphate. 

Another route to dUMP is the reduction of UDP to dUDP, followed by phosphorylation of dUDP to dUTP (or direct reduction of UTP to dUTP in some microorganisms). The dUTP is then hydrolyzed to dUMP. This circuitous route to dUMP is dictated by two considerations. First, the ribonucleotide reductase in most cells acts only on ribonu-cleoside diphosphates, probably because this permits better regulation of its activity. Second, cells contain a highly active deoxyuridine triphosphate diphosphohydrolase (dUT-Pase). It prevents the incorporation of dUTP into DNA by keeping intracellular levels of dUTP low by means of the reaction... 

The thymidylate synthase reaction involves the methylation of deoxy-UMP to deoxy-TMP (thymidylate). Deoxy-UMP is the result of dephosphorylation of the product of the ribonucleotide reductase reaction, dUDP. The conversion of the diphosphate nucleotide to the monophosphate nucleotides helps channel deoxyuridine to thymidylate synthase rather than directly to DNA. N5,N10-methylene tetrahy-drofolate donates the methyl. 

EPR investigations of the inactivation of E. coU ribonucleotide reductase with 2 -azido-2 -deoxyuridine 5 -diphosphate Evidence... 

Fig. 2.1. Examples from The Energy Hall of Fame. These molecules not only deliver energy, but transfer special groups in the process. Acyl, RCO— ADP, adenosine diphosphate ATP, adenosine triphosphate dUMP deoxyuridine monophosphate FAD, flavin adenine dinucleotide GTP, guanosine triphosphate NADH, nicotinamide adenine dinucleotide NADP, nicotinamide adenine dinucleotide phosphate P, phosphate TMP, thymidine monophosphate UDP, uridine diphosphate UTP, uridine triphosphate.

An unprecedented example of the application of an organic azide as an enzyme inhibitor derives from the elegant studies of Stubbe and coworkers at MIT, who have investigated the mechanism of action of ribonucleotide reductase (RNR) using several mechanism-based inhibitors including 2 -azido-2 -deoxyuridine-5 -diphosphate (NjUDP) (71) [82]. RNR plays a... 

The earliest mechanistic studies utilizing alternate substrates were first reported in 1976 by Thelander and co-workers on the inactivation of RDPR by 2 -chloro-2 -deoxyuridine 5 -diphosphate (CIUDP) and the cytidine analog, CICDP (9). 

FIGURE 48-2 Action of flucytosine in fungi. 5-Flucytosine is transported by cytosine permease into the fungal cell, where it is deaminated to 5-fluorouracil (5-FU). The 5-FU is then converted to 5-fluorouracil-ribose monophosphate (5-FUMP) and then is either converted to 5-fluorouridine triphosphate (5-FUTP) and incorporated into RNA or converted by ribonucleotide reductase to 5-fluoro-2 -deoxyuridine-5 -monophosphate (5-FdUMP), which is a potent inhibitor of thymidylate synthase. 5-FUDP, 5-fluorouridine-5 -diphosphate dUMP, deoxyuridine-5 -monophosphate dTMP, deoxyuridine-5 -monophosphate UPRTase, uracil phosphoribosyl transferase. 

Fig. 11.1.4. Separation of uracil and 5-fluorouracil bases, nucleosides and nucleotides by reversed phase ion-pair HPLC. Chromatographic conditions column, Bondapak Cig (300 x 4 mm) mobile phase, (from 0-30 min) 0.1 mM tetrabutylammonium hydrogen sulphate (Cjg), 2.5 mM tetraethylammonium bromide (Cg) and 2% methanol in 2 mM sodium acetate, 1.5 mM phosphate buffer, pH 6.0 (Buffer A) (from 30-50 min) Buffer A-i-30 mM phosphate detection, UV at 254 nm. Peaks FU, fluorouracil FUR, fluorouracU riboside/ FUdR, fluorouracil deoxyriboside FUMP, fluorouridine 5 -monophosphate 5 dFUR, 5 -deoxyfluorouracil riboside FdUMP, deoxyfluorouri-dine monophosphate UDPG, uridine diphosphoglucose UDP, uridine diphosphate dUDP, deoxyuridine monophosphate UTP, uridine triphosphate. Reproduced from Au et al. (1982), with permission.

The de novo pathway to 2 -deoxythymidine monophosphate (dTMP) synthesis first requires the use of dUMP (2 -deoxyuridine-5 -monophosphate) from the metabolism of either UDP or CDP (cytidine diphosphate). The hydrolysis of dUTP (2 -deoxyuridine-5 -triphosphate) to dUMP and subsequent methylation at C-5 by the action of thymidylate synthase, using A, A i°-methylenetetrahydrofolate (THF) as the methyl donor, generate dTMP (Figure 6.54). The latter is subsequently phosphorylated to deoxy-thymidine triphosphate (dTTP) used in DNA synthesis and repair. 

As we have already noted, the deoxyribonucleoside phosphates occur in animal tissues in small amounts and their concentrations are increased in cells engaged in DNA synthesis. The triphosphates of the four deoxyri-bonucleosides represented in DNA have been demonstrated in cell extracts, as have the mono- and diphosphates of thymidine and deoxycytidine. The mono-, di-, and triphosphates of deoxyuridine are known as intermediary compounds in the metabolism of the pyrimidine deoxyribonucleotides. 

These results are consistent with anabolism studies with cell cultures. A close analog of BrVdUrd, (E)-5-(2-iodovinyl)-2 -deoxyuridine, was efficiently converted to its monophosphate derivative in cells infected with both HSV-1 and HSV-2. However, a diphosphate (or triphosphate) could only be detected with extracts of cells infected with HSV-1 . The inefficient phosphorylation of BrVdUMP by the dThd-dTMP kinase from HSV-2 correlates well with the observation that replication of HSV-2 is at least 100-fold less sensitive to BrVdUrd than is that of HSV-1 and suggests a cause-and-effect relationship. The sensitivities of the two viruses to acyclovir are about equal. ... 

---