Determination of the syn-anti Equilibrium of Some Purine 3':5'-Nucleotides by Nuclear-Magnetic-Relaxation Perturbation in the Presence of a Lanthanide-Ion Probe

Abstract

The aqueous solution conformation of four purine 3' : 5'-nucleotides varying in their substituents at C-6 and C-8 has been studied using gadolinium(II1) to perturb the proton relaxation times. The ribose conformations are inferred. All the nucleotides are best described as being in a dynamic equilibrium between syn and anti conformations and the position of this equilibrium is not dramatically affected by changing the substituent at C-6. These nucleotides in their neutral base form slightly favour an anti conformation. In the presence of a bulky methylthio group at C-8 the equilibrium is shifted towards a dominance from the syn conformation due to steric repulsion factors. (Fig. 1). EXPERIMENTAL PROCEDUREThe cyclic nucleotides were the highest grade commercially available and were used without further purification. Lanthanide nitrates were prepared from the oxides (99.99%). The samples were 20 mM in nucleotide and 100 mM in praseodymium nitrate required to shift the proton resonances to allow accurate measurements of the relaxation times. The sodium salt of 3-trimethylsilyl propane sulphonic acid was used Abbreviations. Cyclic AMP, adenosine 3': 5'-monophosphate; cyclic IMP, inosine 3' : 5'-monophosphate; NMR, nuclear magnetic resonance.as an internal reference to monitor the effects of changes in bulk susceptibility and the data is corrected for this. The nucleotide solutions and a solution 1 mM in gadolinium nitrate were dissolved in 'H20 (99.8 %) and adjusted to pH 5.8 with 'HCl or NaO'H. They were then lyophilized three times with 'H20.The nucleotide solution containing 100 mM praseodymium nitrate was titrated with increasing volumes of the gadolinium nitrate solution. The change in the relaxation times, (TI p)-l or (Tz p)-l were measured after each addition and plotted against the metal ion/total substrate ratio. The observed slopes cfT~,)-' and cfT2p)-1 are equal to (TIM)-' and ( T~M )~' respectively. These results can be related to the internuclear distance between metal ion and observed proton, as described previously [12].Experiments were carried out at 28 "C on a Bruker WH 90DS spectrometer operating at 90 MHz in the Fourier transform mode. The Tl measurements were made using a standard 180 "-7-90 pulse sequence and the T2 measurements from line width studies taking care to avoid the effects of the removal of spin-spin coupling on titration with Gd(II1). RESULTS AND DISCUSSIONGadolinium(II1) induces no measurable shifts in the proton resonances of a molecule to which it is bound but markedly reduces the relaxation times. Under conditions of fast chemical exchange this reduction is related by the Bloembergen [I31 and Solomon

Molecular Recognition of cAMP by an RNA Aptamer

“…However, it is known that the glycosidic bond in nucleosides and nucleotides with C8 substitutions favor the syn conformation. (40,41) Since the two conformers are in an equilibrium state, we speculate that the aptamer might bind and trap the ligand in its anti conformation. A precedent for this effect is provided by crystallographic data of the complex between liver alcohol dehydrogenase and 8-bromoadenosine analogs of NAD + .…”

Co-Crystal Structures of PKG Iβ (92–227) with cGMP and cAMP Reveal the Molecular Details of Cyclic-Nucleotide Binding

“…Because cGMP and cAMP are structurally different at only the 2-, 6-, and N1-positions of their purine rings, different amino acid contacts at these positions were proposed to mediate the specificity. Due to rotation around their glycosidic bonds, cyclic nucleotides exist in equilibrium between syn and anti conformations, with cGMP and cAMP favoring syn and anti conformations respectively [18], [19]. The cGMP-binding site of PKG and CNG channels has a threonine residue distinct from the cAMP receptors, and previous models based on the known structures of PKA and HCN channels have predicted that the hydroxyl group of these threonine residues interacts with the guanine 2-NH 2 group of syn -cGMP through hydrogen bonds.…”

Proton nuclear magnetic resonance studies on cyclic nucleotide binding to the Escherichia coli adenosine cyclic 3',5'-phosphate receptor protein

“…We show that whereas cAMP and its analogous are bound in the syn conformation, cGMP and its analogues are bound in the anti conformation. This contrasts with their conformations in free solution where cyclic nucleotides exist in a syn/anti equilibrium (Davies, 1978), cAMP being predominantly anti and cGMP predominantly syn (Oida, 1977;Yathindra & Sundaralingam, 1974;Fazakerley et al, 1977). A preliminary report of part of this work has appeared (Gronenborn et al, 1981).…”