Tricyclic Nucleobase Analogs and Their Ribosides as Substrates and Inhibitors of Purine-Nucleoside Phosphorylases III. Aminopurine Derivatives.

Etheno-derivatives of 2-aminopurine, 2-aminopurine riboside, and 7-deazaadenosine (tubercidine) were prepared and purified using standard methods. 2-Aminopurine reacted with aqueous chloroacetaldehyde to give two products, both exhibiting substrate activity towards bacterial (E. coli) purine-nucleoside phosphorylase (PNP) in the reverse (synthetic) pathway. The major product of the chemical synthesis, identified as 1,N2-etheno-2-aminopurine, reacted slowly, while the second, minor, but highly fluorescent product, reacted rapidly. NMR analysis allowed identification of the minor product as N2,3-etheno-2-aminopurine, and its ribosylation product as N2,3-etheno-2-aminopurine-N2--D-riboside. Ribosylation of 1,N2-etheno-2-aminopurine led to analogous N2--d-riboside of this base. Both enzymatically produced ribosides were readily phosphorolysed by bacterial PNP to the respective bases. The reaction of 2-aminopurine-N9- -D-riboside with chloroacetaldehyde gave one major product, clearly distinct from that obtained from the enzymatic synthesis, which was not a substrate for PNP. A tri-cyclic 7-deazaadenosine (tubercidine) derivative was prepared in an analogous way and shown to be an effective inhibitor of the E. coli, but not of the mammalian enzyme. Fluorescent complexes of amino-purine analogs with E. coli PNP were observed.

[Synthesis, conformation, and spectroscopy of nucleoside analogues concerning their antiviral activity]. / Synteza, konformacja i spektroskopia analogów nukleozydów oraz ich aktywnosc antywirusowa.

Chemically modified analogues of nucleosides and nucleotides, have been thoroughly investigated since the discovery of DNA double helix by Watson and Crick in 1953 (Nature 171: 737). Chemical structures, first of all tautomerism, of the nucleic acid bases, as well as the conformations of the nucleic acids constituents, determine the secondary and tertiary structures of DNA and RNA polymers. Similarly, structural and dynamic parameters of nucleoside derivatives determine their biological activity in mutagenesis, neoplastic transformation, as well as antiviral or anticancer properties. In this review, a multidisciplinary approach of Prof. David Shugar's group is presented in the studies on nucleosides and nucleotides. It consists in chemical syntheses of suitable analogues, measurements of physicochemical and spectral parameters, conformational analysis by means of nuclear magnetic resonance (NMR) and X-ray diffraction, as well as characteristics of the nucleoside analogues as inhibitors of some selected, target enzymes, crucial in respect to antiviral activity of the analogues. These long-lasting studies follows upon the line of the main paradigm of molecular biophysics, i. e. structure-activity relationship.

Structural basis for nematode eIF4E binding an m(2,2,7)G-Cap and its implications for translation initiation.

Metazoan spliced leader (SL) trans-splicing generates mRNAs with an m(2,2,7)G-cap and a common downstream SL RNA sequence. The mechanism for eIF4E binding an m²²7G-cap is unknown. Here, we describe the first structure of an eIF4E with an m(2,2,7)G-cap and compare it to the cognate m7G-eIF4E complex. These structures and Nuclear Magnetic Resonance (NMR) data indicate that the nematode Ascaris suum eIF4E binds the two different caps in a similar manner except for the loss of a single hydrogen bond on binding the m(2,2,7)G-cap. Nematode and mammalian eIF4E both have a low affinity for m(2,2,7)G-cap compared with the m7G-cap. Nematode eIF4E binding to the m7G-cap, m(2,2,7)G-cap and the m(2,2,7)G-SL 22-nt RNA leads to distinct eIF4E conformational changes. Additional interactions occur between Ascaris eIF4E and the SL on binding the m(2,2,7)G-SL. We propose interactions between Ascaris eIF4E and the SL impact eIF4G and contribute to translation initiation, whereas these interactions do not occur when only the m(2,2,7)G-cap is present. These data have implications for the contribution of 5'-UTRs in mRNA translation and the function of different eIF4E isoforms.

Synthesis of 3H and 13C labeled mRNA cap dinucleotides--useful tools for NMR, biochemical, and biological studies.

For deeper understanding the roles of the mRNA cap structure in cellular processes isotopically labeled dinucleotide cap analogues have been synthesized as tools for NMR and in vivo studies. Tritium or carbon C-13 labeled methyl iodide was used as a source of the isotope material. In order to minimize the number of steps during the radioisotopic synthesis the methylation with tritium labeled methyl iodide was performed with Gp(3)G as a substrate. The C-13 isotope was introduced into the cap dinucleotide by methylation of GDP with C-13 Methyl iodide, followed by coupling the product with guanosine 5'-phosphorimidazolide in DMF with zinc chloride as a catalyst.

Enzymatically stable 5' mRNA cap analogs: synthesis and binding studies with human DcpS decapping enzyme.

Four novel 5' mRNA cap analogs have been synthesized with one of the pyrophosphate bridge oxygen atoms of the triphosphate linkage replaced with a methylene group. The analogs were prepared via reaction of nucleoside phosphor/phosphon-1-imidazolidates with nucleoside phosphate/phosphonate in the presence of ZnCl2. Three of the new cap analogs are completely resistant to degradation by human DcpS, the enzyme responsible for hydrolysis of free cap resulting from 3' to 5' cellular mRNA decay. One of the new analogs has very high affinity for binding to human DcpS. Two of these analogs are Anti Reverse Cap Analogs which ensures that they are incorporated into mRNA chains exclusively in the correct orientation. These new cap analogs should be useful in a variety of biochemical studies, in the analysis of the cellular function of decapping enzymes, and as a basis for further development of modified cap analogs as potential anti-cancer and anti-parasite drugs.

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.

Synthesis and properties of mRNA cap analogs containing phosphorothioate moiety in 5',5'-triphosphate chain.

Nucleosides and oligonucleotides with an oxygen replaced by sulfur atom are an interesting class of compounds because of their improved stability toward enzymatic cleavage by nucleases. We have synthesized several dinucleotide mRNA cap analogs containing a phosphorothioate moiety in the alpha, beta, or gamma position of 5',5'-triphosphate chain [m7Gp(s)ppG, m7Gpp(s)pG, and m7Gppp(s)G]. These are the first examples of the biologically important 5'mRNA cap analogs containing a phosphorothioate moiety, and these compounds may be useful in a variety of biochemical and biotechnological applications. Incorporation of a sulfur atom in the alpha or gamma position within the dinucleotide cap analog was achieved using PSCl3 in a nucleoside phosphorylation reaction followed by coupling the phosphorothioate of nucleoside with a second nucleotide. Synthesis of cap analogs with the phosphorothioate moiety in beta position was performed using an organic phosphorothioate salt in a coupling reaction with an activated nucleotide. The structures of newly synthesized compounds was confirmed using MS and 1H and 31P NMR spectroscopy. We present here the results of preliminary studies on their interaction with translation initiation factor eIF4E and enzymatic hydrolysis with human and nematode DcpS scavengers.

Novel dinucleoside 5',5'-triphosphate cap analogues. Synthesis and affinity for murine translation factor eIF4E.

Chemical synthesis of a series of novel dinucleoside cap analogues, m7GpppN, where N is formycin A, 3'-O-methylguanosine, 9-beta-D-arabinofuranosyladenine, and isoguanosine, has been performed using our new methodology. The key reactions of pyrophosphate bonds formation were achieved in anhydrous dimethylformamide solutions employing the catalytic properties of zinc salts. Structures of the new cap analogues were confirmed by 1H NMR and 31p NMR spectra. The binding affinity of the new cap analogues for murine eIF4E(28-217) were determined spectroscopically showing the highest association constant for the analogue that contains formycin A.

The antiviral drug ribavirin does not mimic the 7-methylguanosine moiety of the mRNA cap structure in vitro.

The eukaryotic initiation factor eIF4E binds the mRNA 5' cap structure and has a central role during translational initiation. eIF4E and the mechanisms to control its activity have oncogenic properties and thus have become targets for anticancer drug development. A recent study (Kentsis et al. 2004) presented evidence that the antiviral nucleoside ribavirin and its phosphorylated derivatives were structural mimics of the mRNA cap, high-affinity ligands for eIF4E, and potent repressors of eIF4E-mediated cell transformation and tumor growth. Based on these findings, we tested ribavirin, ribavirin triphosphate (RTP), and the dinucleotide RpppG for their ability to inhibit translation in vitro. Surprisingly, the ribavirin-based compounds did not affect translation at concentrations where canonical cap analogs efficiently block cap-dependent translation. Using a set of reporter mRNAs that are translated via either cap-dependent or viral internal ribosome entry sites (IRES)-dependent initiation, we found that these ribavirin-containing compounds did inhibit translation at high (millimolar) concentrations, but there was no correlation of this inhibition with an eIF4E requirement for translation. The addition of a ribavirin-containing cap to mRNA did not stimulate translation. Fluorescence titration experiments with eIF4E and the nuclear cap-binding complex CBC indicated affinities for RTP and RpppG that were two to four orders of magnitude lower than those of m(7)GTP and m(7)GpppG. We conclude that, at least with respect to translation, ribavirin does not act in vitro as a functional mimic of the mRNA cap.

Synthesis of novel mRNA 5' cap-analogues: dinucleoside P1, P3-tri-, P1, P4-tetra-, and P1, P5-pentaphosphates.

A series of new mRNA anti reverse cap analogues (ARCA) was designed to obtain a tool for studying the mechanism of protein translation. Dinucleoside P1, P3-tri-, P1, P4-tetra- and P1, P5-pentaphosphates, linked by a 5'-to-5' phosphate bridge and composed of modified 7-methylguanosine and guanosine, have been synthesized. The hydroxyl group (2'OH or 3'OH) in 7-metylguanosine moiety was replaced by -OCH3 or -H in order to obtain the cap analogues capable to be correctly incorporated into synthetic mRNA transcripts. Tri-, tetra-, and pentaphosphates were prepared by ZnCl2 catalyzed condensation in DMF of derivatives of the 7-methylguanosine diphosphates with the guanosine mono-, di- and triphosphate P-imidazolides, respectively. The structures of the novel compounds were established by means of 1H and 31P NMR spectra.

Binding studies of eukaryotic initiation factor eIF4E with novel mRNA dinucleotide cap analogues.

Studies on the interaction of the murine translation initiation factor 4E with two new-synthesized cap-analogues, modified at C2' of 7-methylguanosine, have been performed by means of the fluorescence titration method. No difference in the binding affinity for eIF4E was observed compared with the "anti reversed" cap analogues, possessing the analogous modifications at C3'. Potential significance of the novel caps as research tools for examination of the nuclear cap binding complex CBC80/20 has been discussed.