Methylene analogues of adenosine 5'-tetraphosphate. Their chemical synthesis and recognition by human and plant mononucleoside tetraphosphatases and dinucleoside tetraphosphatases.

Adenosine 5'-polyphosphates have been identified in vitro, as products of certain enzymatic reactions, and in vivo. Although the biological role of these compounds is not known, there exist highly specific hydrolases that degrade nucleoside 5'-polyphosphates into the corresponding nucleoside 5'-triphosphates. One approach to understanding the mechanism and function of these enzymes is through the use of specifically designed phosphonate analogues. We synthesized novel nucleotides: alpha,beta-methylene-adenosine 5'-tetraphosphate (pppCH2pA), beta,gamma-methylene-adenosine 5'-tetraphosphate (ppCH2ppA), gamma,delta-methylene-adenosine 5'-tetraphosphate (pCH2pppA), alphabeta,gammadelta-bismethylene-adenosine 5'-tetraphosphate (pCH2ppCH2pA), alphabeta, betagamma-bismethylene-adenosine 5'-tetraphosphate (ppCH2pCH2pA) and betagamma, gammadelta-bis(dichloro)methylene-adenosine 5'-tetraphosphate (pCCl2pCCl2ppA), and tested them as potential substrates and/or inhibitors of three specific nucleoside tetraphosphatases. In addition, we employed these p4A analogues with two asymmetrically and one symmetrically acting dinucleoside tetraphosphatases. Of the six analogues, only pppCH2pA is a substrate of the two nucleoside tetraphosphatases (EC 3.6.1.14), from yellow lupin seeds and human placenta, and also of the yeast exopolyphosphatase (EC 3.6.1.11). Surprisingly, none of the six analogues inhibited these p4A-hydrolysing enzymes. By contrast, the analogues strongly inhibit the (asymmetrical) dinucleoside tetraphosphatases (EC 3.6.1.17) from human and the narrow-leafed lupin. ppCH2ppA and pCH2pppA, inhibited the human enzyme with Ki values of 1.6 and 2.3 nm, respectively, and the lupin enzyme with Ki values of 30 and 34 nm, respectively. They are thereby identified as being the strongest inhibitors ever reported for the (asymmetrical) dinucleoside tetraphosphatases. The three analogues having two halo/methylene bridges are much less potent inhibitors for these enzymes. These novel nucleotides should prove valuable tools for further studies on the cellular functions of mono- and dinucleoside polyphosphates and on the enzymes involved in their metabolism.

Calcium-stimulated guanosine--inosine nucleosidase from yellow lupin (Lupinus luteus).

Guanosine-inosine-preferring nucleoside N-ribohydrolase has been purified to homogeneity from yellow lupin (Lupinus luteus) seeds by ammonium sulfate fractionation, ion-exchange chromatography and gel filtration. The enzyme functions as a monomeric, 80kDa polypeptide, most effectively between pH 4.7 and 5.5. Of various mono- and divalent cations tested, Ca(2+) appeared to stimulate enzyme activity. The nucleosidase was activated 6-fold by 2mM exogenous CaCl(2) or Ca(NO(3))(2), with K(a)=0.5mM (estimated for CaCl(2)). The K(m) values estimated for guanosine and inosine were 2.7+/-0.3 microM. Guanosine was hydrolyzed 12% faster than inosine while adenosine and xanthosine were poor substrates. 2'-Deoxyguanosine, 2'-deoxyinosine, 2'-methylguanosine, pyrimidine nucleosides and 5'-GMP were not hydrolyzed. However, the enzyme efficiently liberated the corresponding bases from synthetic nucleosides, such as 1-methylguanosine, 7-methylguanosine, 1-N(2)-ethenoguanosine and 1-N(2)-isopropenoguanosine, but hydrolyzed poorly the ribosides of 6-methylaminopurine and 2,6-diaminopurine. MnCl(2) or ZnCl(2) inhibited the hydrolysis of guanosine with I(50) approximately 60 microM. Whereas 2'-deoxyguanosine, 2'-methylguanosine, adenosine, as well as guanine were competitive inhibitors of this reaction (K(i) values were 1.5, 3.6, 21 and 9.7 microM, respectively), hypoxanthine was a weaker inhibitor (K(i)=64 microM). Adenine, ribose, 2-deoxyribose, 5'-GMP and pyrimidine nucleosides did not inhibit the enzyme. The guanosine-inosine hydrolase activity occurred in all parts of lupin seedlings and in cotyledons it increased up to 5-fold during seed germination, reaching maximum in the third/fourth day. The lupin nucleosidase has been compared with other nucleosidases.

Synthesis and enzymatic characterization of methylene analogs of adenosine 5'-tetraphosphate (P4A).

A new methodology for synthesis of biologically important nucleoside tri- and tetraphosphates containing a bisphosphonate moiety instead of the terminal pyrophosphate bond is described. The series consists of tri- and tetraphosphate analogs of adenosine, guanosine and 7-methylguanosine (characteristic for mRNA cap). We have adopted a two-step procedure that allowed us to insert a methylene bridge into the phosphate chain. Nucleoside mono- or diphosphates were first activated (as imidazole derivatives) and then used in coupling reactions with organic salts of bisphosphonate. The resulting synthetic method enabled us to obtain the desired compounds with high yields and does not require any protective groups. This makes it very useful for the synthesis of labile compounds such as those containing the 7-methylguanosine ring. The structures of the synthesized compounds were confirmed by NMR spectroscopy. They were tested as potential substrates and inhibitors of several hydrolases.

New Tripodal, "Supercharged" Analogues of Adenosine Nucleotides: Inhibitors for the Fhit Ap3 A Hydrolase.

Methanetrisphosphonic acids provide a branch point for synthetic nucleotide analogues which can be exploited either to generate novel tripodal nucleotides or to incorporate additional negative charge into linear analogues relative to the parent nucleotide, as exemplified in the picture for ATP and diadenosine tetraphosphate (Ap4 A). These compounds show valuable discriminatory behavior as competitive inhibitors for the tumor suppressor protein Fhit and a second Apn A pyrophosphohydrolase. X=H, Cl, F.

Novel diadenosine polyphosphate analogs with oxymethylene bridges replacing oxygen in the polyphosphate chain: potential substrates and/or inhibitors of Ap4A hydrolases.

Dinucleoside polyphosphates (Np(n)N's; where N and N' are nucleosides and n = 3-6 phosphate residues) are naturally occurring compounds that may act as signaling molecules. One of the most successful approaches to understand their biological functions has been through the use of Np(n)N' analogs. Here, we present the results of studies using novel diadenosine polyphosphate analogs, with an oxymethylene group replacing one or two bridging oxygen(s) in the polyphosphate chain. These have been tested as potential substrates and/or inhibitors of the symmetrically acting Ap(4)A hydrolase [bis(5'-nucleosyl)-tetraphosphatase (symmetrical); EC 3.6.1.41] from E. coli and of two asymmetrically acting Ap(4)A hydrolases [bis(5'-nucleosyl)-tetraphosphatase (asymmetrical); EC 3.6.1.17] from humans and narrow-leaved lupin. The six chemically synthesized analogs were: ApCH(2)OpOCH(2)pA (1), ApOCH(2)pCH(2)OpA (2), ApOpCH(2)OpOpA (3), ApCH(2)OpOpOCH(2)pA (4), ApOCH(2)pOpCH(2)OpA (5) and ApOpOCH(2)pCH(2)OpOpA (6). The eukaryotic asymmetrical Ap(4)A hydrolases degrade two compounds, 3 and 5, as anticipated in their design. Analog 3 was cleaved to AMP (pA) and beta,gamma-methyleneoxy-ATP (pOCH(2)pOpA), whereas hydrolysis of analog 5 gave two molecules of alpha,beta-oxymethylene ADP (pCH(2)OpA). The relative rates of hydrolysis of these analogs were estimated. Some of the novel nucleotides were moderately good inhibitors of the asymmetrical hydrolases, having K(i) values within the range of the K(m) for Ap(4)A. By contrast, none of the six analogs were good substrates or inhibitors of the bacterial symmetrical Ap(4)A hydrolase.

Adenosine-5'-O-phosphorylated and adenosine-5'-O-phosphorothioylated polyols as strong inhibitors of (symmetrical) and (asymmetrical) dinucleoside tetraphosphatases.

Dinucleoside 5',5"'- P (1), P ( n )-polyphosphates, and particularly the diadenosine compounds, have been implicated in extracellular purinergic signalling and in various intracellular processes, including DNA metabolism, tumour suppression and stress responses. If permitted to accumulate, they may also be toxic. One approach to understanding their function is through the various specific degradative enzymes that regulate their levels. Eight adenosine-5'- O -phosphorylated polyols (derivatives of glycerol, erythritol and pentaerythritol) and 11 adenosine-5'- O -phosphorothioylated polyols (derivatives of glycerol, erythritol, pentaerythritol, butanediol and pentanediol) have been tested as inhibitors of specific diadenosine tetraphosphate (Ap(4)A) hydrolases. Of these two groups of novel nucleotides, the adenosine-5'- O -phosphorothioylated polyols were generally stronger inhibitors than their adenosine-5'- O -phosphorylated counterparts. 1,4-Di(adenosine-5'- O -phosphorothio) erythritol appeared to be the strongest inhibitor of ( asymmetrical ) Ap(4)A hydrolases (EC 3.6.1.17) from both lupin and human, with K (i) values of 0.15 microM and 1.5 microM respectively. Of eight adenosine-5'- O -phosphorylated polyols, 1,4-di(adenosine-5'- O -phospho) erythritol was the only compound that inhibited the lupin enzyme. Two derivatives of pentaerythritol, di(adenosine-5'- O -phosphorothio)-di(phosphorothio) pentaerythritol and tri(adenosine-5'- O -phosphorothio)-phosphorothio-pentaerythritol, proved to be the strongest inhibitors of the prokaryotic ( symmetrical ) Ap(4)A hydrolase (EC 3.6.1.41) so far reported. The estimated K (i) values were 0.04 microM and 0.08 microM respectively. All of these inhibitors were competitive with respect to Ap(4)A. These new selectively acting Ap(4)A analogues should prove to be valuable tools for further studies of Ap(4)A function and of the enzymes involved in its metabolism.