Deoxynucleoside 5 -diphosphate

Since 1976 a number of 2 -substituted 2 -deoxynucleoside 5 -diphosphates have been shown to be mechanism-based inactivators of the ribonucleotide reductases from a variety of sources (Table I). This work was extended by Stubbe and Kozarich 50, 51), who studied the reaction of several 2 -halo-substituted 2 -deoxynucleoside 5 -diphosphates with RDPR. Incubation of RDPR with CIUDP, 2 -deoxy-2 -fluoroadenosine 5 -diphosphate (FADP), or 2 -deoxy-2 -fluorocytidine 5 -diphosphate (FCDP) resulted in time-dependent enzyme inactivation, concomitant and stoichiometric loss of all substituents from the ribose moiety, increase in the UV-Vis absorbance of the protein near 320 nm, and... 

This enzyme [EC 2.7.4.13] catalyzes the reaction of ATP with a deoxynucleoside phosphate to produce ADP and a deoxynucleoside diphosphate. The enzyme can also utilize dATP as the phosphorylating substrate as well. 

Sj"herg, B.-M., Gr%oslund, A., and Eckstein, E., 1983, A substrate radical intermediate in the reaction between ribonucleotide reductase from Escherichia coli and 2 -azido-2 -deoxynucleoside diphosphates. J. Biol. Chem. 258 8060n8067. 

NRTIs are phosphorylated and converted into diphosphate forms by nucleoside kinases. These acdvated forms have high levels of afdnity for HTV-1 reverse d anscriptase and compete with the natural deoxynucleoside diphosphates. Once incorporated into the growing chain of DNA, lack of a 3 -hydroxyl group that can form a phosphodiester bond with the incoming nucleoside causes chain terminadon. Tenofovir is an exception in this group as it is a nucleotide analogue rather than nucleoside and, as such, requires only tw o phosphorylation steps instead of three to become the active form. Pharmacological characteristics of FDA approved NRTIs are presented in Table 41.3. 

Negative feedback. A ribonucleoside diphosphate, with 2 -OH shown in purple, is converted to the corresponding deoxynucleoside diphosphate in the reaction shown across the top. The deoxynucleoside diphosphate is then phosphorylated to the corresponding deoxynucleoside triphosphate with the "g-phosphate shown in purple. This is the final product of the pathway and it acts as a negative allosteric inhibitor of the initial enzyme, as shown by the large purple arrow. 

All NRTIs, as exemplified for AZT (Fig. 7), act in a similar fashion following their uptake by the cells, they are phosphorylated successively to their 5 -monophosphate, 5 -diphosphate, and 5 -triphosphate form (De Clercq 2002). Unlike the first phosphorylation step in the metabolic pathway of the acyclic guanosine analogues (see above), which is carried out by a virus-encoded enzyme (thymidine kinase), the first as well as the subsequent phosphorylations of the 2, 3 -dideoxynucleosides are carried out by cellular enzymes, that is, a 2 -deoxynucleoside (e.g., dThd) kinase, a 2 -deoxynucleotide (e.g., dTMP) kinase, and a (2 -deoxy)nucleoside 5 -diphosphate (NDP) kinase. 

DNA-directed DNA polymerases [EC 2.7.7.7], also called DNA nucleotidyltransferases (DNA-directed), are enzymes that catalyze the DNA template-directed extension of the 3 -end of a nucleic acid strand one nucleotide at a time. Thus, n deoxynucleoside triphosphates produce n pyrophosphate (or, diphosphate) ions and DNA . This enzyme cannot initiate the synthesis of a polymeric chain de novo it requires a primer which may be DNA or RNA. RNA-directed DNA polymerases [EC 2.7.7.49], also referred to as reverse transcriptases, DNA nucleotidyltransferases (RNA-directed), and revertases, are enzymes that catalyze the RNA template-directed extension of the 3 -end of a nucleic acid strand one nucleotide at a time. Thus, n deoxynucleoside triphosphates produce n pyrophosphate (or, diphosphate) ions and DNA . As was the case above, this enzyme cannot initiate the synthesis of a polymeric chain de novo it requires a primer which may be DNA or RNA. 

This enzyme [EC 2.7.7.49], also known as RNA-directed DNA polymerase, DNA nucleotidyltransferase (RNA-directed), and revertase, catalyzes the RNA-template-directed extension of the 3 -end of a DNA strand by one deoxynucleotide at a time n deoxynucleoside triphosphate to produce n pyrophosphate (or, diphosphate) and DNA . The enzyme cannot initiate a DNA chain de novo and requires a DNA or RNA primer. See also Viral Polymerases... 

Krishnan, P. Fu, Q. Lam, W. Lion, J.-Y. Dutschman, G. Cheng, Y.-C. Phosphorylation of pyrimidine deoxynucleoside analog diphosphates selective phosphorylation of L-nucleoside analog diphosphates by 3-phos-phoglycerate kinase. J. Biol. Chem., 277, 5453-5459 (2002)... 

FIGURE 13-12 Ping-Pong mechanism of nucleoside diphosphate kinase. The enzyme binds its first substrate (ATP in our example), and a phosphoryl group is transferred to the side chain of a His residue. ADP departs, and another nucleoside (or deoxynucleoside) diphos-... 

FYotein B1 (Mr 160,000), a dimer of general structure aa (25), binds the ribonucleotide substrates (29) and contains active thiol groups that directly participate in the catalytic process (30). The B1 subunit also contains the binding site for the nucleoside diphosphate substrates and the deoxynucleoside triphosphates and ATP, which act as allosteric effectors. 

Scheme 6. Mechanism of inhibition of RDPR by 2 -halo-substituted deoxynucleoside S -diphosphates.

When the nitrate ester 277 was treated with BuaSnD and AIBN, the deuteriated alcohol 278 was formed, by 1,5-radical translocation. However, the 2 -chloro-2 -deoxy-system 279 gave the furanone 280 under the same conditions. These results suggest that the inhibition of ribonucleoside diphosphate reductase by 2 -chloro-2 -deoxynucleosides involves loss of Cl from a radical at C-3. Some related studies from the same team, also supporting this conclusion, were mentioned earlier. ... 

McKenna reported the efiicient synthesis of the alpha, P-difluoromethylene deoxynucleoside 5 -triphosphate of adenosine and cytosine (89a,b), obtained enzymatically from catalytic ATP, phosphoenol pyruvate, nucleoside diphosphate kinase and pyruvate kinase using the nucleoside difluor-omethylene bisphosphonates as substrate for the kinase. The bispho-sphonate was prepared by nucleophilic displacement of the 5 -0-tosyl nucleoside precursor with tris(tetrabutylammonium) difluoromethylenebi-sphosphonate. Finally Mackman synthesised the diphosphate esters of two cyclopentyl based nucleoside phosphonates (90a,b). While (90b) was unstable following reduction of (90a) and therefore unsuitable for biological evaluation, (90a) exhibited potent inhibition against HIV reverse transcriptase. ... 

Ribonucleotide reductase of Escherichia coli, which catalyzes the reduction of ribonucleoside 5 -diphosphates to 2 -deoxynucleoside 5 -diphos-phates, consists of two nonidentical subunits, proteins B1 and B2. In the presence of Mg , the two subunits form a 1 1 complex of active enzyme. When separated, neither subunit has any known biological activity. Protein B1 has a molecular weight of 160,000, contains the active dithiols, is capable of interacting with thioredoxin, and contains... 

The number of bound phosphates can vary in a nucleotide. When phosphate is absent, the molecule is referred to as a nucleoside or a deoxynucleoside, depending on the hydroxylation-state of the sugar. Up to three phosphate groups can be present. When one or more phosphates are present, the compound is commonly referred to by the base present, how many phosphates are present, and whether the deoxy-form of the sugar is used. For example, when the base adenine is present and there are two phosphates, the corresponding deoxyribonucleotide and ribonucleotide are typically referred to by the abbreviations, dADP and ADR The former and latter abbreviations indicate deoxy-adenine diphosphate and... 

The enzyme purified from bull semen dephosphorylates the 5 -phosphate esters of adenosine, uridine, cytidine, guanosine, nicotinamide riboside, and the 5 -phosphate esters of deoxynucleosides, but is inactive with adenosine pyrophosphates and substrates which bear other substituents on the ribose, e.g., adenosine 2, 5 -diphosphate (124). However 5 -nucleo-tidase from potato can hydrolyze the 5 -phosphate of this diphosphate... 

It has been shown that purine-pyrimidine dinucleotides are considerably more add-labile than pyrimidine-pyrimidine dinudeotides and that the cytosine deox3uibose bond is more labile than the thymine deoxyribose bond. Thus, when thymidylic-uridylic add dinucleotide is hydrolyzed more thymidine 3, 5 -diphosphate is formed lhan uridine 3, 5 -diphos-phate [Eq. 56]. On the basis of these data an attempt was made to gain information on the distribution of pyrimidine deoxynucleotides in different preparations of DNA (255). Differences were detected in the rate of appearance of the pyrimidine deoxynucleoside dipho hates, i.e., early appearance from isolated pyrimidine deoxynucleotides and later appearance from adjoining pjuimidine deoxynucleotide sequences. This indicates that the distribution of pyrimidines in DNA is not random and differs from one preparation to another, even in samples of DNA with the same base composition. 

Only the triphosphates can function as substrates and these have a high affinity for polymerase. The diphosphates and monophosphates are inactive in the presence of purified enzyme. The synthesis of deoxynucleo-tide triphosphates from deoxynucleosides and their mono- and diphosphate derivatives has been discussed previously (see Section II, C, 1). 

Treatment of nucleoside 5 -phosphates with sodium cyclotriphosphate gives 2 ,5 - and 3 ,5 -diphosphates and 2 ,3 -cyclic-5 -diphosphates, with the product distribution dependent on pH, reaction temperature and reaction time. 191 3, 5 -Diphosphates of labile deoxynucleosides can be prepared by the action of phosphoryl tetrakis-triazole, followed by hydrolysis.192... 

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