Nucleoside Diphosphate Analogues

2 Nucleoside Pyrophosphates. - 2.2.1 Nucleoside Diphosphate Analogues. The enzymatic regeneration of 3 -phosphoadenosine-5 -phosphosulfate (76) catalysed by the rat liver sulfotransferase IV enzyme has been reported to provide useful quantities of the cofactor for enzyme kinetic experiments. The first thio-nucleotide analogues of adenosine 5 -phosphosulfate (77a) and of 3 -phosphoadenosine 5 -phosphosulfate (77b) have been synthesised in enantio-merically pure forms. Using these novel analogues, the sulfuryl transfer reaction to adenosine 5 -triphosphate was shown to proceed with inversion of configuration at the a-phosphorus.  

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Nucleoside Pyrophosphates. - 2.3.1 Nucleoside Diphosphate Analogues. A large variety of esters with different nucleoside and alkyl moieties (llla-j) have been synthesised in small amounts using different combinations of nucleoside triphosphate, alcohols and snake venom pyrophosphatase. Potato tube pyrophosphatase was also reported as being a possible practical biocatalyst to synthesise such nucleotide pyrophosphate-O-alkyl esters, but using more stringent reaction requirements than that of the snake venom enzyme. ... 

The Fe-protein has the protein fold and nucleotide-binding domain of the G-protein family of nucleotide-dependent switch proteins, which are able to change their conformation dependent on whether a nucleoside diphosphate (such as GDP or ADP) is bound instead of the corresponding triphosphate (GTP or ATP). However, nucleotide analogues, which induce the conformational switch of the Fe-protein, do not allow substrate reduction by the MoFe-protein, nor does reduction of the MoFe-protein by other electron-transfer reagents (whether small proteins or redox dyes) drive substrate reduction. Only the Fe-protein can reduce the MoFe-protein to a level that allows it to reduce substrates such as... 

Gonin, P. Xu, Y Milon, L. Dabernat, S. Morr, M. Kumar, R. Lacombe, M.L. Janin, J. Lascu, I. Catalytic mechanism of nucleoside diphosphate kinase investigated using nucleotide analogues, viscosity effects, and X-ray crystallography. Biochemistry, 38, 7265-7272 (1999)... 

E. I. Budowsky, T. N. Druzhinina, G. I. Eliseeva, N. D. Gabrielyan, N. K. Kochetkov, M. A. Novikova, V. N. Shibaev, and G. L. Zhdanov, Synthetic analogues of uridine diphosphate glucose Biochemical and chemical studies. The secondary structure of nucleoside diphosphate sugars, Biochim. Biophys. Acta, 122 (1966) 213-224. 

The method has the added advantage in that it provides a route not only to nucleoside diphosphates (upon reaction with pyrophosphate) but also allows the preparation of phosphonate analogues by the use of methylene or difluoromethylene bisphosphonates.10 The latter analogues are both stable to hydrolysis. 

The synthesis of nucleoside diphosphates is best achieved using the Poulter reaction,9 which involves reaction of the tris(tetra-n-butylammonium) salt of pyrophosphate with a nucleoside 5 -tosylate in acetonitrile. A general procedure for the synthesis of nucleoside tosylates of thymidine and 2 -deoxyadenosine is included (Protocol 15), whilst the syntheses of the other tosylates (including ribonucleosides) have been described using related procedures. Simple modification of the protocol, whereby the tetra-n-butylammonium salt of pyrophosphoric acid is replaced by methylene or difluomethylene bis phosphonate, allows the synthesis of hydrolytically stable dNTP analogues.10... 

Synthesis of glycopyranosylphosphonate analogues of certain natural nucleoside diphosphate sugars as potential inhibitors of gly-cosyltransferases. J. Med. Chem. 1987 30 1383-1391. 42. 

A series of myristoyl nucleoside di- and triphosphates (127) has been synthesised as membrane permeable prodrugs. Whilst the latter could be obtained by reaction of a suitable mixed anhydride of the acyl compound with the respective nucleotide in DMF, a novel and more efficient route to the diphosphate analogues involved treating myristoylimidazole with the diphosphate in the presence of methyl iodide. Methylation at the N-1 of the imidazolide presumably increases its reactivity toward the nucleotide. 

Some carbocyclic nucleoside phosphonates have been described. There has been a further report on compounds of type 208 (see Vol. 28, p. 291), with both purine and pyrimidine bases. Although initially made as racemates, a precursor diol could be resolved by enantioselective acetylation using a lipase and vinyl acetate.The triphosphate analogues 209 (B=Gua, Ade) have been made from the previously-described monophosphonates (Vol. 29, p. 285), and the cyclopropyl-fused diphosphate analogues 210 (X=CH2 and O) were also reported, along with their enantiomers. Trani-compounds of type 211 (n=l-3) have been made as racemates,and so have the related cw-isomers (n=l or 2). ... 

With the advent of an adequate nucleotide technology and the development of widespread interest in nucleotide metabolism, many free nucleotides have been found in cells however, no comprehensive survey has appeared since the 1958 report of Henderson and LePage (17), which lists over 100 nucleotides, and the list has probably more than doubled since. A large number of free nucleoside diphosphate conjugates have been isolated from cells and characterized many such compounds serve as coenzymes in group-transfer reactions, for example, CDP-X and UDP-X derivatives participate, respectively, in phospholipid and polysaccharide synthesis (see Chapter 3). As well, many new nucleotides have been recognized as metabolites of nucleoside antibiotics and of synthetic purine and pyrimidine analogues. 

S.-K. (2009) Chemoenzymatic syntheses of carbasugar analogues of nucleoside diphosphate sugars UDP-carba-Gal, UDP-carba-GlcNAc, UDP-carba-Glc, and GDP-carba-Man. Chem. Commun. (Cambridge, U.K.), 1733-1735. 

Whitesides and coworkers have carried out a comparison of enzymic and chemical routes to CTP, GTP and UTP on a 10-gram scale. They concluded that CTP and GTP were best made enzymically, and UTP by reaction of CTP with nitrous acid. The triphosphates were then employed for the enzymic synthesis of UDP-Glucose, UDP-Glucuronic acid, and GDP-Mannose.i94 Cytidine diphosphate sugars have been prepared from the 3,6-dideoxyhexoses paratose and abequose,193 and all four nucleoside diphosphate sugars of 6-sulpho-a-D-quinovose have been synthesized for studies of sulpholipid biosynthesis in chloroplasts.196 The stable analogue (138) of CMP-KDO has been prepared by a triester approach, but was only a weak inhibitor of KDO incorporation into lipopolysaccharides.197 A reference to acetylated forms of UDPGlc is mentioned in Chapter 7. 

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. 

Entecavir, telbivudine, clevudine, and the other nucleoside analogues (Fig. 4aa) need to be phosphorylated to their 5 -triphosphate form to be antivirally active (Fig. 8). This again implies three phosphorylation steps based successively on a nucleoside kinase, nucleoside 5 -monophosphate kinase, and nucleoside 5 -diphosphate kinase. These reactions have been characterized only in a few cases, that is, thymidylate kinase in the metabolism of clevudine (Hu et al. 2005). 

Acyclovir Zovirax) is a guanine nucleoside analogue most effective against HSV-1 and HSV-2, but it has some activity against VCV, CMV, and EBV. Valacyclovir (Valtrex) is the L-valine ester prodrug of acyclovir. Acyclovir is converted to its active metabolite via three phosphorylation steps. First, viral thymidine kinase converts acyclovir to acyclovir monophosphate. Next, host cell enzymes convert the monophosphate to the diphosphate and then to the active compound, acyclovir triphosphate. Because viral thymidine kinase has a much greater affinity for acyclovir triphosphate than does mammalian thymidine kinase, acyclovir triphosphate accumulates only in virus-infected cells. 

Tenofovir disoproxil fumarate (Viread) is a prodrug of tenofovir, a phosphorylated adenosine nucleoside analogue, and is the only available agent of its class. It is converted by cellular enzymes to tenofovir diphosphate, which competes with deoxyadenosine triphosphate (dATP) for access to reverse transcriptase and causes chain termination following its incorporation. Tenofovir was approved as part of a combination therapy for HIV in adults who failed treatment with other regimens it appears to be effective against HIV strains that are resistant to NRTIs. The pharmacokinetic properties of tenofovir are provided in Table 51.2. 

Fig. 4. The use of arsonomethyl phosphonate as an analogue of diphosphate by RNA polymerase. The enzyme accepts the analogue in the reverse of polymerase action. Since the product hydrolyzes, the overall effect is that of an exonuclease, producing nucleoside 5 -phosphates (59).

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. 

Subtypes of adenosine receptors exist - A, Aj and A3 -which have differential sensitivities to adenosine nucleoside analogues, including 2-methylthio-AMP, 2-thioadenosine, DPMA. IB-MECA, NECA, CPA. CCPA and DPCPX. These receptors, and subtypes within A2, have all been cloned. They have structures typical of the seven-transmembrane G-protein-coupled superfamily of receptors, but have amongst the shortest sequences known (A3 has only 318 amino acids), and a lack of sequence similarity with any other receptors appears to put them in a class of their own. Adenosine receptors are not sensitive to nucleotides such as ADP (adenosine diphosphate) and ATP (adenosine triphosphate), which instead act as P2 receptor agonists that are nucleotide-... 

Nucleoside Cyclic Pyrophosphates. Extensive work has been carried out by Matsuda et al. in their efforts to synthesise chemically stable cyclic adenosine diphosphate ribose (cADPR) analogues. The carbocyclic inosine analogue (83) was first prepared through an efficient cyclisation of an 8-bromo-A-1 -[5"-(phosphoryl)carbocyclic-ribosyl]inosine 5 -phenylthiophos-... 

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