The activation energies for the pseudorotation of the furanose ring in adenosine, guanosine, inosine and xanthosine dissolved in liquid deuteroammonia have been determined by analysis of the longitudinal relaxation rates of the single tertiary carbons between + 40 "C and -60 "C. For the purine ribosides the average activation energy was found to be 4.7 f-0.5 kcal . mol-I (20 f 2 kJ . mol-').For the pyrimidine nucleosides cytidine and uridine the respective activation energy should be higher since it could not be determined by I3C relaxation measurements. This result can be explained by the formation of a hydrogen bond between the 5'-hydroxymethyl group and the base. In adenosine, guanosine, inosine and xanthosine the relaxation rates of C(5') are smaller than all others thus excluding the formation of a hydrogen bond between the purine base and the 5'-hydroxymethyl group of a strength comparable to the one suggested for cytidine and uridine.From the analysis of the high-resolution proton magnetic resonance spectra it is well known that the conformation of the common nucleosides can only be described by assuming equilibria between several conformers [l-31. The transitions between the different conformations are rapid on the time scale of a high-resolution nuclear magnetic resonance experiment. Two controversial ways of describing a dissolved nucleoside molecule in solution exist, the first one explaining the experimental results by a "continuously changing envelope of conformations" [4], the other arguing that the molecule exists in the time average predominantly in a few fixed or rigid conformers, occasionally interconverting into each other [5]. It is obvious that this controversy can be settled by determination of the different energies of activation for the internal modes of motion. Except for the rotation of hydrogens in amino and hydroxy side groups, three internal modes of motion exist ( around the exocyclic C(4')-C(5') bond in the ribose moiety; (c) the pseudorotation of the furanose ring of the pentose. The energy of activation for the rotation around the glycosidic bond has been determined for adenosine by Rhodes and Schimmel [6] to 6.2 kcal . mol-I (26 kJ . mol-I). A lower limit of 3.5 to 4 kcal . mol-' (15-17 kJ . mol-l) can be gained for the rotation around C(4') -C(5') from results obtained on substituted ethane fragments [7].The insertion of activation energies between 3 and 6 kcal . rno1-l (12.5-25 kJ . mol-') into the Boltzmann equation yields the result that at room temperature and below that vast majority of the molecules are locked in a fixed conformation. At any given instant only very few molecules possess sufficient energy to perform conformational transitions between the syn anti and gg gt tg states. To our knowledge the activation energy of pseudorotation of the furanose ring or any comparably substituted tetrahydrofuran derivative has not been determined experimentally. Values between 2.0 and 4.0 kcal . mol-' (8.5-17 kJ . mol-I) are favored in the literature [7-91. The energy barriers of...
With the use of PMR the ribose conform ations have been studied in the tem perature range - 60 to + 40 °C in ND3 solutions of adenosine (A), guanosine (G), inosine (I), xanthosine (X), purineriboside (P R), 2-am inopurineriboside (2am P R), N6-isopentenyladenosine (N6ipA), 8-bromo-adenosine (8-BrA), 8-bromoguanosine (8-BrG), formycin B (F), tubercidin (T), isopropylidene-adenosine (iA), and isopropylideneguanosine (iG). The analysis is based on the two-state S ⇄ N model of the ribose m oiety proposed by Altona and Sundaralingam . The compounds studied can be classified into two groups: 1. A, I, G, X, PR , 2am PR, N6ipA, and T show a small tem perature dependence of the S ⇄ N equilibrium and [ S ] ~ 0 .6 ; 2. 8-BrA, 8-BrG, and F have a stronger tem perature dependence and [S] ~ 0 .8 . W ithin these two groups the sim ilarities observed are greater than observed in the solid state. Some therm odynam ic conclusions about the S ⇄ N and the syn ⇄ anti equilibria are presented. The results support the previously proposed correlation of the S state of the ribose with the syn conform ation of the base and of the N state of the ribose with the anti conform ation of the base. Furtherm ore, it is derived that the gg rotam er is correlated with the S state of the ribose and therefore stabilizes the syn conformation of the base.
The solution conformations of adenosine, guanosine and inosine in liquid ND3 have been determined by NMR. Comparison of the Karplus analysis of the proton HR spectra of the ribose moiety obtained in this solvent with the data from aqueous solutions of A and I proves that the conformations of the nucleosides are very similar in both liquids. From the analysis of the vicinal coupling constants of the ring protons it has been deduced that the S state C(2')-endo is slightly preferred. The mole fraction in S approximates 0.6 for all three nucleosides. C-13 relaxation measurements have been applied in the determination of the correlation times for rotational diffusion. Only at temperatures below - 40 degrees C is the pseudo-rotation of the furanoside ring slowed down sufficiently for it not to contribute to the measured relaxation rates. From NOE studies and T1 measurements on the individual protons it is derived that the N, C(3')-endo, form of the ribose is correlated with an anti conformation of the base (Y approximately 210 degrees to 220 degrees) and the S, C(2')-endo, form of the ribose with a syn conformation of the base (Y approximately 30 degrees to 50 degrees). The glycosyl torsion angles derived for the two conformations of A, G, and I are equal within the limits of accuracy.
The solution conformation of xanthosine in liquid N2H3 has been determined by nuclear magnetic resonance. The correlation times for rotational diffusion were derived from 13C relaxation measurements. A Karplus analysis of the high-resolution spectra of the ribose moiety yields the conformation of the sugar from the H -H coupling constants. The mole fraction of the sugar in the N conformation is 0.4 at room temperature and increases slightly as the temperature decreases. From nuclear Overhauser enhancement studies, and T1 measurements of the various protons it is deduced that the N ribose is correlated with an anti conformation of the base (Y= 210 ") while the S form of the sugar is coupled to a glycosyl torsion angle in the syz range ( Y z 50 O -90 ").At the molecular level, the explanation for a wide range of biological phenomena which involve nucleic acids or coenzymes containing nucleosides, relies critically on the conformation of the monomeric constituents in solution [1,2]. The flexibility of dissolved nucleosides and nucleotides, the preferred conformation, and the modes of internal motion are controversial topics in the current literature [3 -61. The application of magnetic resonance techniques to the study of this problem is well established [7-111. Most of the results published so far deal with the properties of pyrimidine nucleosides [4,12,13], though some purine nucleotides have been investigated [ 14 -161. The study of purine nucleosides has been hampered by the low solubility of most of these compounds in water. Furthermore, the purine nucleosides associate strongly in aqueous solution. Hence, since it is nearly impossible to separate the intramolecular interactions from the intermolecular contributions, quantitative analysis of nuclear magnetic resonance data becomes dubious. It thus appeared desirable to investigate the behaviour of the purine nucleosides in a polar solvent with molecular properties similar Abbreviations. NMR, nuclear magnetic resonance; NOE, nuclear Overhauser enhancement.to those of water. In this context the ability of solvent particles to participate in hydrogen bonds equally well either as donor or as acceptor molecules is of special importance. Polar solvents lacking this property are not likely to yield the same conformation as that found in aqueous solution, since in the former the formation of intramolecular hydrogen bonds produces pronounced minima of energy. These are lacking, or are at least, less prominent, if the solvent molecules participate in the hydrogen bonding. However, the solvent should preferably have a lower tendency to support solvophobic interactions than water. Among the solvents fulfilling these requirements, liquid ammonia appears to be the most suitable for our applications. All purine nucleosides tested to date have a solubility of at least 0.6 molal in this solvent. The low melting point and viscosity of ammonia allows highresolution nuclear magnetic resonance (NMR) investigations at temperatures low enough to partially "freeze" the conformational ...
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