Gyula Sági

Base-modified oligodeoxynucleotides. I. effect of 5-alkyl, 5-(1-alkenyl) and 5-(1-alkynyl) substitution of the pyrimidines on duplex stability and hydrophobicity

Abstract Properties of oligodeoxynucleotides essential for the antisense effect can be favourably modified by substitution of the heterocyclic base moiety. This is shown here for duplex stability by 5 alkyl, 5-(1-alkenyl) and 5-(1-alkynyl) substitution of the pyrimidines in the self-complementary (dT-dA) 10 and (dC-dG) 6 oligomers as well as for the hydrophobic character of the (dT-dA) 10 series.

Characterisation of an uridine-specific binding site in rat cerebrocortical homogenates

Parameters of [3H]uridine binding to synaptic membranes isolated from rat brain cortex (K(D)=71+/-4 nM, B(max)=1.37+/-0.13 pmol/mg protein) were obtained. Pyrimidine and purine analogues displayed different rank order of potency in displacement of specifically bound [3H]uridine (uridine>5-F-uridine>5-Br-uridine approximately adenosine>>5-ethyl-uridine approximately suramin>theophylline) and in the inhibition of [14C]uridine uptake (adenosine>uridine>5-Br-uridine approximately 5-F-uridine approximately 5-ethyl-uridine) into purified cerebrocortical synaptosomes. Furthermore, the effective ligand concentration for the inhibition of [14C]uridine uptake was about two order of magnitude higher than that for the displacement of specifically bound [3H]uridine. Adenosine evoked the transmembrane Na(+) ion influx, whereas uridine the transmembrane Ca(2+) ion influx much more effectively. Also, uridine was shown to increase free intracellular Ca(2+) ion levels in hippocampal slices by measuring Calcium-Green fluorescence. Uridine analogues were found to be ineffective in displacing radioligands that were bound to various glutamate and adenosine-recognition and modulatory-binding sites, however, increased [35S]GTPgammaS binding to membranes isolated from the rat cerebral cortex. These findings provide evidence for a rather specific, G-protein-coupled site of excitatory action for uridine in the brain.

Synthesis and NMR characterization of diastereomeric CPSMeG derivatives.

Abstract Synthesis and stereochemical characterization of enantiomerically pure CPSMeG derivatives by NMR methods are reported. The effect of 5′-dimethoxytrityl on the conformational properties is described. It was found that in P-diastereomers the conformational differences about the C3′-O3′ bond, as discernible from the J values, are enhanced by the presence of this protecting group.

Crystal and molecular structure of (+)-carba-thymidine, C11H16N2O6

Abstract The molecular structure of (+)-carba-thymidine possessing notable anti-HSV activity has been determined by single crystal X-ray diffraction. It crystallizes in the monoclinic space group P2 with unit cell dimensions a = 4.810(2), b = 11.560(1)1, c = 10.014(1) A, β = 92.34(2)°, Z = 2. The structure was solved by direct methods and refined by least squares to a final R = 0.038 for 1027 reflections (I < 36(I)). The torsion angle x around the glycosidic N1-C1′ bond agrees with that of thymidine (37.5°vs 39.1°) whereas the C3′-exo pucker of the five-membered ring is shifted to an even less common C1′-eao form.

PNA-DNA chimeras containing 5-alkynyl-pyrimidine PNA units. Synthesis, binding properties, and enzymatic stability.

Abstract Three chimeric dimer synthons (oeg_tNHT, oeg_upNHT and oeg_uhNHT) containing thymine (t), 5-(l-propynyl)-uracil (up) and 5-(1-hexyn-1-yl)-uracil (uh) PNA units with N-(2-hydroxyethyl)glycine (oeg) backbone were synthesized in solution and incorporated into T20 oligonucleotide analogues, using standard P-amidite chemistry. Insertion of dimer blocks led to destabilization of duplexes with dA20 target. The smallest T m drops were found for chimeras containing oeg_upNHT dimers. Incorporation of the chimeric synthons into the 3′-end of T20 brought about growing resistance to 3′-exonucleolytic (SV PDE) cleavage in the order of oeg_tNHT < oeg_upNHT < oeg_uhNHT. Due to different endonuclease activities of 3′- and 5′-exonucleases applied, placing of five consecutive dimers at the 5′-terminus resulted in a relatively smaller, but also side-chain dependent, stabilization towards the hydrolysis by 5′-exonuclease (BS PDE). Neither exonucleases (SV and BS PDE) nor an endonuclease (Nuclease P1) could hydrolyse the unnatural phosphodiester bond linking the 3′-OH of thymidine to the terminal OH of N-(2-hydroxyethyl)glycine PNA backbone.

Nucleotide promiscuity of 3-phosphoglycerate kinase is in focus: implications for the design of better anti-HIV analogues

The wide specificity of 3-phosphoglycerate kinase (PGK) towards its nucleotide substrate is a property that allows contribution of this enzyme to the effective phosphorylation (i.e. activation) of nucleotide-based pro-drugs against HIV. Here, the structural basis of the nucleotide-PGK interaction is characterised in comparison to other kinases, namely pyruvate kinase (PK) and creatine kinase (CK), by enzyme kinetic analysis and structural modelling (docking) studies. The results provided evidence for favouring the purine vs. pyrimidine base containing nucleotides for PGK rather than for PK or CK. This is due to the exceptional ability of PGK in forming the hydrophobic contacts of the nucleotide rings that assures the appropriate positioning of the connected phosphate-chain for catalysis. As for the D-/L-configurations of the nucleotides, the L-forms (both purine and pyrimidine) are well accepted by PGK rather than either by PK or CK. Here again the dominance of the hydrophobic interactions of the L-form of pyrimidines with PGK is underlined in comparison with those of PK or CK. Furthermore, for the l-forms, the absence of the ribose OH-groups with PGK is better tolerated for the purine than for the pyrimidine containing compounds. On the other hand, the positioning of the phosphate-chain is an even more important term for PGK in the case of both purines and pyrimidines with an L-configuration, as deduced from the present kinetic studies with various nucleotide-site mutants of PGK. These characteristics of the kinase-nucleotide interactions can provide a guideline for designing new drugs.

Synthesis and cytotoxic activity of novel 5-substituted-1-(β-L- arabinofuranosyl) pyrimidine nucleosides

A series of new 5-halogeno-1-(ß-L-arabinofuranosyl)uracils and their cytosine analogues were synthesized by halogenation of ara-L-uridine and ara-L-cytidine, respectively. The 5-(2-thienyl) and 5-halogenothienyl derivatives of both series were also prepared in excellent yields by Stille coupling followed by halogenation. All of these syntheses were based on benzoyl-protected derivatives. In vitro cytotoxicity experiments carried out using L1210 mouse leukemia cells showed that 5-(2-thienyl)-ara-L-uridine was the most potent compound of the new compounds; the majority of the analogues were not effective up to 200 μM concentrations.

Synthesis, biophysical, and biochemical properties of PNA-DNA chimeras.

Abstract Three PNA-DNA chimeric dimer synthons (tT, upT and uhT, see Sch. 1) have been synthesized in solution and used to make T20-analogue chimeras applying standard solid-phase DNA synthesis protocol. Duplex forming ability of chimeras with dA20 and their hydrolyses by 3′- and 5′-exonucleases (snake venom and bovine spleen phosphodiesterase, respectively) have been investigated.

SYNTHESIS AND ENZYMATIC CHARACTERIZATION OF P1-THIO-P2-OXO TRIDEOXYNUCLEOSIDE DIPHOSPHATES HAVING AZT, FdU, OR dT AT THE 3′-POSITION

ABSTRACT Model compounds for oligonucleotide-prodrugs, P1-thio-P2-oxo-trideoxyribonucleoside diphosphates: d[GsCoX] and d[TsAoX] (X=AZT, FdU or dT) have been prepared, and their hydrolyses by snake venom phosphodiesterase and nuclease S1 are described.

13C-31P couplings in the study of conformational properties of some diastereomeric nucleoside-3'-thiophosphate derivatives.

Synthesis and stereochemical characterization of enantiomerically pure nucleoside-3′-phosphorothioate esters and salts are reported. Vicinal carbon–phosphorus couplings reflect different predominance of the ϵ conformation in the isomeric (Rp and Sp) esters, while for the salts the ϵt conformation prevails in both stereoisomers. The influence of solvent and temperature on the conformational preferences is also described.