pH-Dependent mismatch discrimination of oligonucleotide duplexes containing 2′-deoxytubercidin and 2- or 7-substituted derivatives: protonated base pairs formed between 7-deazapurines and cytosine

Oligonucleotides incorporating 2′-deoxytubercidin (1a), its 2-amino derivative 2a and related 2-, or 7-substituted analogs (1d, 2b–d, 3 and 4) are synthesized. For this purpose, a series of novel phosphoramidites are prepared and employed in solid-phase synthesis. Hybridization experiments performed with 12mer duplexes indicate that 7-halogenated nucleosides enhance the duplex stability both in antiparallel and parallel DNA, whereas 2-fluorinated 7-deaza-2′-deoxyadenosine residues destabilize the duplex structure. The 7-deazaadenine nucleosides 1a, 1d and their 2-amino derivatives 2a–d form stable base pairs with dT but also with dC and dG. The mispairing with dC is pH-dependent. Ambiguous base pairing is observed at pH 7 or under acid conditions, whereas base discrimination occurs in alkaline medium (pH 8.0). This results from protonated base pairs formed between 1a or 2a and dC under neutral or acid condition, which are destroyed in alkaline medium. It is underlined by the increased basicity of the pyrrolo[2,3-d]pyrimidine nucleosides over that of the parent purine compounds (pK(a) values: 1a = 5.30; 2a = 5.71; dA = 3.50)

Cyclic nucleotide specificity of the activator and catalytic sites of a cGMP-stimulated cGMP phosphodiesterase from Dictyostelium discoideum

The cellular slime mold Dictyostelium discoideum has an intracellular phosphodiesterase which specifically hydrolyzes cGMP. The enzyme is activated by low cGMP concentrations, and is involved in the reduction of chemoattractant-mediated elevations of cGMP levels. The interaction of 20 cGMP derivatives with the activator site and with the catalytic site of the enzyme has been investigated. Binding of cGMP to the activator site is strongly reduced (more than 80-fold) if cGMP is no longer able to form a hydrogen bond at N2H2 or O2’H. Modifications at N7, C8, O3’ and O5’ induce only a small reduction of binding affinity. A cyclic phosphate structure, as well as a negatively charged oxygen atom at phosphorus, are essential to obtain activation of the enzyme. Substitution of the axial exocyclic oxygen atom by sulphur is tolerated; modification of the equatorial oxygen atom reduces the binding activity of cGMP to the activator site by 90-fold. Binding of cGMP to the catalytic site is strongly reduced if cGMP is modified at N1H, C6O, C8 and O3’, while modifications at N2H2, N3, N7, O2’H, and O5’ have minor effects. Both exocyclic oxygen atoms are important to obtain binding of cGMP to the catalytic site. The results indicate that activation of the enzyme by cGMP and hydrolysis of cGMP occur at different sites of the enzyme. cGMP is recognized at these sites by different types of molecular interaction between cGMP and the protein. cGMP derivatives at concentrations which saturate the activator site do not induce the same degree of activation of the enzyme (activation 2.3-6.6-fold). The binding affinities of the analogues for the activator site and their maximal activation are not correlated. Our results suggest that the enzyme is activated because cGMP bound to the activator site stabilizes a state of the enzyme which has a higher affinity for cGMP at the catalytic site.

The base pairing properties of 8-aza-7-deaza-2′-deoxyisoguanosine and 7-halogenated derivatives in oligonucleotide duplexes with parallel and antiparallel chain orientation

The base pairing properties of oligonucleotide duplexes containing 8-aza-7-deaza-2′-deoxyisoguanosine, its 7-bromo or its 7-iodo derivative are described. The nucleosides were synthesized on a convergent route, protected and converted into phosphoramidites. Oligonucleotides were prepared on a solid-phase and were hybridized to yield duplexes with parallel (ps) or antiparallel (aps) chain orientation. The 8-aza-7-deaza-2′-deoxyisoguanosine-containing duplexes show almost identical base pairing stability as those containing 2′-deoxyisoguanosine, while the 7-substituted derivatives induce a significant duplex stabilization both in ps and aps DNA. Self-complementary duplexes with parallel chain orientation are exceptionally stable due to the presence of 5′-overhangs. The bulky halogen substituents were found to be well accommodated in the grooves both of aps and ps DNA