Resonance Time Scale Research Articles

Studies on sulfenamides. X. Electron spin resonance spectra of radical cations electrochemically generated from N-(o-nirtrophenylthio)alicyclic amines.

The paramagnetic species formed by in situ electrochemical oxidation of eleven N-(o-nitrophenylthio) alicyclic amines in acetonitrile or propionitrile have been identified as the radical cations derived from the parent sulfenamides by one-electron transfer. The observed non-equivalence of the two N-methylene groups indicated the existence of restricted rotation around the S-N bond and three-electron π-bonded geometry. As regards the radical cations derived from six-membered alicyclic sulfenamides, the ring inversion was apparently conformationally frozen on the electron spin resonance (ESR) time scale even at 25°C. Based on the ESR and nuclear magnetic resonance data it is proposed that the C2N-S group in these radical cations is pyramidal and that the nitrogen inversion of these radicals is slow on the ESR time scale. The stability of the radical cations is discussed.

Conformational characterisation of valinomycin complexation with barium salts—A nuclear magnetic resonance spectroscopic study

Conformations of valinomycin and its complexes with Perchlorate and thiocyanate salts of barium, in a medium polar solvent acetonitrile, were studied using nuclear magnetic resonance spectroscopic techniques. Valinomycin was shown to have a bracelet conformation in acetonitrile. With the doubly charged barium ion, the molecule, at lower concentrations, predominantly formed a 1:1 complex. At higher concentrations, however, apart from the 1:1, peptide as well as ion sandwich complexes were formed in addition to a :final complex:. Unlike the standard 1:1 potassium complex, where the ion was centrally located in a bracelet conformation, the a 1:1 barium complex contained the barium ion at the periphery. The a :final complex: appeared to be an open conformation with no internal hydrogen bonds and has two bound barium ions. This complex was probably made of average of many closely related conformations that were exchanging very fast (on nuclear magnetic resonance time scale) among them. The conformation of the a:final complex a: resembled the conformation obtained in the solid state. Unlike the Perchlorate anion, the thiocyanate anion seemed to have a definite role in stabilising the various complexes. While the conformation of the 1:1 complex indicated a mechanism of ion capture at the membrane interface, the sandwich complexes might explain the transport process by a relay mechanism.

Torsional motion and elasticity of the deoxyribonucleic acid double helix and its nucleosomal complexes.

Torsional thermal oscillations of the DNA double helix within the electron paramagnetic resonance (EPR) time scale (10(-10)-10(-3) s) as indicated by a rigid, intercalating probe are much smaller in the spacer segment between nucleosomes in chromatin than in long, free DNA molecules. Still smaller DNA oscillation is indicated in intact nuclei and yet smaller if the nuclei have been treated with glutaraldehyde. The values of EPR measurements are not affected by the loading density of probe. If the probe were capable of substantial oscillations or movement different from that of the helix, those oscillations would be expected to dominate the spectra when movement of the helix is restrained. We conclude that the correlation time for torsional movement of free DNA inferred from EPR spectra is characteristic of the double helix and that there is no significant independent motion of the probe. The correlation time for the DNA double helix in molecules longer than approximately 500 base pairs is close to 30 ns, corresponding to an elastic constant of 1.5 X 10(-19) ergs cm for deformation by twisting. The motions observed in chromatin are consistent with a model in which spheres of 50-60-A radius are connected by simple elastic rods with the length of spacer DNA and the same elastic constant. The spin-labeled ethidium probe has been characterized in detail by nuclear magnetic resonance, infrared, fluorescence, and visible light spectroscopy. The binding equilibria are consistent with the hypothesis that strongly immobilized probe molecules are preferentially bound to spacer DNA.

Stochastically Perturbed Resonance

We analyze the motion of a periodically forced oscillator whose natural frequency is the superposition of a slowly varying component and a small stochastic perturbation. When the slowly varying component of natural frequency passes through the value corresponding to the forcing, there is resonance. The purpose of this study is to understand the effect of the resonance on the stochastic processes of the oscillator’s amplitude and phase. J. B. Kelley’s smoothing method is employed to derive a diffusion equation that governs the joint distribution of amplitude and phase. We study this equation in the limit where the time scale of resonance is much shorter than the time scale of diffusion. Far away from resonance, the distribution function satisfies to leading order the same diffusion equation as in the unforced case. The effect of the resonance is to create an internal layer in the solution at the time of resonance in which the distribution undergoes a small but abrupt change. A simple calculation gives the magnitude of the jump.

Nuclear-magnetic-resonance study of the conformation of a dinucleotide in solution using the lanthanide probe method.

The conformation of the dinucleotide adenylyl(3'-5')adenosine 2'-phosphate (ApA2'p) in aqueous solution at different pH values and temperatures has been studied using the lanthanide(III) ethylenodiaminetetraacetate(EDTA) 1:1 complexes as shift and relaxation probes. The conformational analysis, based on mixing different conformations in fast exchange within the nuclear magnetic resonance time scale, agrees well with the results from vicinal coupling constants and dimerization shifts obtained for the metal-free system. The dinucleotide exists in a temperature-dependent and pH-dependent conformational equilibrium between unstacked and base-stacked forms. At neutral pH and low temperature, the stacked form predominates, and it is a predominantly right helical structure, characterized as an anti, 3E, g-, g- g-, g'g', gg, 3E, anti conformation. This situation contrasts with adenylyl(3'-5')adenosine (ApA), where both right and left helices contribute to the stacked form. The nucleotidyl units of the unstacked form of ApA2'p have average conformations which are very similar to those of the corresponding mononucleotides in similar conditions.

Metal binding to pyridoxal derivatives. An NMR study of the interaction of Eu(III) with pyridoxal phosphate and pyridoxamine phosphate

The solution conformations of pyridoxal-5’-phosphate and pyridoxamine-5’-phosphate have been investigated using Eu(III) as a nuclear magnetic resonance shift probe. Binding of Eu(III) to pyridoxal phosphate results in the formation of two complexes, at the phosphate group and the \alpha-hydroxy-aldehyde moiety, which are in slow exchange on the nuclear magnetic resonance time-scale. The lanthanide-induced pseudo contact shifts calculated using the McConnell-Robertson equation. (J. Chem. Soc. (1950), 22, 1561) are in good agreement with the experimentally observed values for both pyridoxal phosphate and pyridoxamine phosphate and lead to a family of closely related conformations.

Nuclear-magnetic-resonance studies of 5'-ribonucleotide and 5'-deoxyribonucleotide conformations in solution using the lanthanide probe method.

The conformations of the metal-bound 5'-ribonucleotides and 5'-deoxyribonucleotides in aqueous solution at different pH values have been studied using the lanthanide probe method. The conformational analysis, based on mixing different conformations in fast exchange within the nuclear magnetic resonance time scale, agrees well with the results from coupling constants, nuclear Over-hauser effects and spin-lattice relaxation times, obtained for the metal-fixed systems. The equilibrium between the two basic conformational combinations for the 5'-nucleotides, anti-(N in equalibrium S)-gg-g'g' and syn-(N in equalibrium S)-gt-g'g' depends on the nature of the furanose ring, the base and also on the state of base protonation and phosphate ionization. The effect of base protonation is particularly strong for the guanine nucleotides.

Studies on the proton magnetic resonance spectra of aliphatic systems. IX. Complex shift and equilibrium constant of association between excess amount of aliphatic alcohol and tris(dipivalomethanato)europium.

From the two step equilibria of the complex formation between the shift reagent Eu (DPM)3 and a large excess of aliphatic alcohol, the two complex shifts Δ1, Δ2 and equilibrium constants K1, K2 are estimated. Δ1 and K1 are comparable with Δc and Kc obtained in an equimolar condition, and the magnitudes of K2 are included within the errors of K1. The infinite concentration shift Δic obtained in the presence of a large excess of the donor is also comparable with Δ1. These results suggest the role of the solvent molecule occupying an overwhelming majority in the sample solution, where the molecular interaction is a time-averaging 1 : 1 donor-acceptor collision in the nuclear magnetic resonance time scale.

Free acid, anion, alkali, and alkaline earth complexes of lasalocid a (X537A) in methanol: structural and kinetic studies at the monomer level.

We report below on nuclear magnetic resonance investigations of the structure and exchange kinetics for the free acid, anion, sodium complex, and barium complex of the ionophore lasalocid A (X537A) in methanol solution. A comparison between the proton and carbon longitudinal relaxation times of lasalocid in nonpolar and polar solvents demonstrates that the free acid (HX) is a monomer in methanol solution. Parallel proton and carbon relaxation measurements demonstrate that the anion (X-), sodium complex (NaX), and barium complex (BaX+) are also monomeric in methanol solution. These results are in contrast to the Na2X2 dimer and the BaX2-H20 dimer observed in crystals and in nonpolar (cyclohexane and methylene chloride) solutions. Large downfield shifts on complex formation (X- to NaX and BaX+) are detected for protons located on the polar face of the ionophore with their C-H bonds directed towards and proximal to the metal ion. The exchange of lasalocid anion between free (X-) and complexed (BaX+) states in methanol can be monitored from the temperature-dependent line shapes of the proton resonances at superconducting fields. The exchange rates are independent of the reactant concentrations and are characteristic of a rate-determining dissociation of BaX+ in methanol solution with activation parameters delta H++ = 6.5 kcal mol-1 (25 degrees) and delta S++ = -20.0 cal mol-1 degree -1 (1 cal = 4.184 J). The rate constants for dissociation and formation of BaX+ complex in methanol, 25 degrees, are 5.2 X 10(3) sec-1 and 1.5 X 10(10) M-1 sec-1, respectively. These studies were extended to derive the activation parameters for the exchange of lasalocid anion between BaX+ and NaX and between BaX+ and HX in methanol, while the exchange among HX, X-, and NaX is too rapid to be monitored on the time scale of nuclear magnetic resonance.

Dimensionality of spin fluctuations in highly anisotropic TCNQ salts

An anisotropic three-dimensional random walk model is developed and used to describe the frequency dependence of proton spin–lattice relaxation rates in several TCNQ salts. The triplet exciton system (φ3AsCH3) (TCNQ)2 at 145 °K is shown to display two-dimensional diffusive behavior. Measurements of the proton relaxation rate in NMP–TCNQ at room temperature are reported and interpreted as displaying two-dimensional diffusive behavior with a transition to three-dimensional behavior. These results suggest that the nuclear resonance time scale is so long as to preclude observing one-dimensional behavior even in highly anisotropic one-dimensional systems. The random walk model is extended to display the dependence of relaxation rate upon charge transfer and thus explain the unusually rapid relaxation in NMP–TCNQ.

The charge-relay system of serine proteinases: Proton magnetic resonance titration studies of the four histidines of porcine trypsin

Peaks corresponding to the C (2)-protons of all four histidine residues of porcine β-trypsin were resolved in 250 MHz nuclear magnetic resonance spectra after deuteration of the slowly exchangeable N- H groups (whose resonances obscure the histidine peaks) by reversible unfolding of the protein in D 2O. One of the four peaks was assigned to the charge-relay histidine in the active site of trypsin (His(57) in the bovine chymotrypsinogen numbering system). Whereas the three other histidine C (2)-peaks exhibited normal titration curves with single p K′ values of 7.20, 6.71 and 6.67, the peak assigned to His(57) had an abnormal titration curve showing two protonation steps in the pH range from 1 to 9. The first protonation with a pH′ mid of 5.0 is rapid on the nuclear magnetic resonance time-scale; the second with a pH′ mid of 4.5 is slow and apparently involves conformational transitions between two states having lifetimes of approximately 18 ms. In the complex between porcine β-trypsin and bovine pancreatic trypsin inhibitor (Kunitz) His(57) was found to be insensitive to pH over the range from 4 to 9 and its chemical shift resembles that of His(57) in the singly protonated charge relay of free trypsin. This result provides direct evidence that the trypsin charge relay acts as a proton acceptor in the initial catalytic step which leads to the formation of a tetrahedral complex. In the presence of equimolar bovine pancreatic trypsin inhibitor (Kunitz) the pH' mid of the conformational transition that affects the charge-relay histidine is lowered from 4.5 to approximately 3.5.

Incidence and distance matrices

It is now recognised that many aspects of chemistry are, in some considerable measure, topologically determined. For example, graph theory has been applied to problems in Valence Theory an aspect which has been explored by several authors.1,2,3*4 Perhaps the earliest application of formal graph theory to a chemistry problem was Cayley’s work in determining the isomer counts of the paraffinic hydrocarbons.’ A much more recent application also concerns isomers and is the subject of the present communication. Fluxional (stereochemically non-rigid) molecules, undergo rapid molecular rearrangements. All the isomers have the same equilibrium geometry and differ only in atomic permutations, but on a nuclear magnetic resonance time scale the properties of individual atoms may be a time average of those associated with mote than one position. Although is not always possible to associate a unique permutation with a unique permutational mechanism each species has a definite number of permutational isomer?j’ each permutational mechanism leading to a characteristic pattern of interconversions between these isomers. The various mechanisms are conveniently distinguished by the different shortest path sequences by which any given isomer is converted into another. The shortest paths are detailed in a so-called distance matrix of the system, examples of which have been given by several authors. However there appears to be in the chemical literature no details of the derivation of such matrices. It is the purpose of the present communication to remedy this ommision and to propose some innovations that will increase the information content of them.

13C high-resolution nuclear magnetic resonance studies of enzyme-substrate reactions at equilibrium. Substrate studies of chymotrypsin-N-acetyltyrosine semicarbazide complexes.

N-Acetyl-L-tyrosine semicarbazide is hydrolyzed by chymotrypsin (EC 3.4.21.1) to N-acetyl-L-tyrosine and semicarbazide. If a high concentration of semicarbazide is present, the equilibrium for the reaction can be shifted from hydrolysis to synthesis. Using N-acetyl-L-[(13)C]tyrosine enriched at the carboxyl carbon and high concentrations of semicarbazide hydrochloride, we have studied the enzyme-substrate complex of N-acetyl-L-[(13)C]tyrosine semicarbazide and chymotrypsin A(delta) by (13)C nuclear magnetic resonance. We observe no shift within the experimental accuracy of +/-0.05 ppm as the fraction of substrate bound is changed from 0.17 to 0.70. Since E + S right arrow over left arrow ES is in fast exchange on the nuclear magnetic resonance time scale, it is possible to show that when the substrate is bound to the enzyme in the Michaelis complex, the (13)C resonance is shifted less than 0.1 ppm, indicating that negligible substrate strain occurs in this complex at the site of enzymatic attack. These experiments demonstrate the application of nuclear magnetic resonance to the study of particular states along the reaction pathway for enzyme-substrate reactions at equilibrium.

Nuclear magnetic resonance studies of lanthanide complexes. Part I. Solvation numbers and kinetics of substrate exchange in lanthanide shift reagent systems

Proton resonance spectra are reported for a variety of lanthanide shift reagents with four different substrates, dimethyl sulphoxide, hexamethylphosphoramide, tetramethylurea, and triethylamine. At low temperatures substrate exchange is slow on a proton resonance time-scale. Solvation numbers and bound chemical shifts were derived. In some cases the solvation numbers are solvent dependent. For many systems, quantitative kinetic data were obtained.

Fluidity of phospholipid bilayers and membranes after exposure to osmium tetroxide and gluteraldehyde

The response of fluid bilayer regions to osmium tetroxide and glutaraldehyde fixation was examined in phospholipid multilayers and in nerve bundles from the walking legs of the lobster Homarus americanus. The samples were spinlabeled either with 5-doxylstearic acid (the 4′4′-dimethyloxazolidine- N-ozyl derivative of 5-ketostearic acid) or the maleimide spin label, 4-maleimido-2,2,6,6-tetramethylpiperidine-1-oxyl. Osmium tetroxide fixation abolishes the characteristic orientation of the spin-labeled lipid bilayer regions and virtually eliminates motion on the electron spin resonance time scale. Glutaraldehyde treatment reduces the motion of maleimide spin labels covalently attached to proteins. However, in contrast to osmium tetroxide fixation, glutaraldehyde has essentially no effect on the orientation and mobility in the fluid bilayer regions, and hence probably does not restrict directly the potential for translational motion in membrane phospholipid bilayer regions.

Conformations of diphosphopyridine coenzymes upon binding to dehydrogenases.

The binding of oxidized as well as reduced coenzyme to some dehydrogenases has been studied under different concentration ratios and temperatures by nuclear magnetic resonance spectroscopy. A significant difference in the spectral behavior between DPN(+) and DPNH upon binding is interpreted in terms of fast and slow on-off rates relative to the nuclear magnetic resonance time scale in the binding of these two coenzymes. Significant downfield shifts of DPN(+) were observed upon binding, comparable in magnitude to those expected upon opening (destacking) of the coenzymes in the case of chicken-muscle and lobster-tail lactate dehydrogenase (EC 1.1.1.27) and yeast alchol dehydrogenase (EC 1.1.1.1.). A preliminary survey of several other dehydrogenases is consistent with these findings. In the case of 3-phosphoglyceraldehyde dehydrogenase, there is a possibility that the coenzyme exists in the folded form.

A Spin Label Study of the Influence of Cholesterol on Egg Lecithin Multibilayers

A detailed electron spin resonance study of a cholestane spin label in hydrated egg lecithin multibilayers of variable cholesterol content is presented. Several theoretical models are proposed in an attempt to explain the observed electron spin resonance spectra for the egg lecithin–cholesterol multibilayer system; we conclude that the most probable model is that of restricted random walk of individual spin labels where the amplitude of the random walk decreases from about 46° at 0 mol% cholesterol content to a minimum of 17° at 55 mol% cholesterol. Also, as the cholesterol content of the multibilayers increases, so the rate of random walk decreases from rapid to intermediate on the electron spin resonance time scale. The results clearly indicate that cholesterol is able to order egg lecithin films, orientating all molecules towards a normal to the surface of the film and decreasing their mobility. The condensing and stiffening influence of cholesterol in phospholipids is undoubtedly one of its major roles in biological membranes.

Method for correlation of proton magnetic resonance assignments in different solvents: conformational transition of oxytocin and lysine vasopressin from dimethylsulfoxide to water.

The peptide amide, primary carboxamide, and aromatic proton resonances were assigned to specific hydrogens of oxytocin and lysine vasopressin (Lys-VP) in water at 23 degrees at pH 2.5 and 4.2, respectively. We started with the spectral assignments of oxytocin and Lys-VP determined in deuterated dimethylsulfoxide (Me(2)SO) and monitored the course of each of these resonances as the proportion of water to Me(2)SO was gradually increased. Changes in each of the two hormones in chemical shifts and in some coupling constants indicate that conformational alterations occur in both oxytocin and Lys-VP during the solvent transition from Me(2)SO to water. This study is a specific application of a general method for correlating spectral assignments in different solvents and for monitoring conformational changes accompanying solvent transitions. Application of this technique requires only that the solvent components be miscible over the entire transitional range, that spectral changes of the solute be simple enough to follow, and that the associated structural changes of the solute be "rapid on the proton magnetic resonance time-scale."

Proton resonance studies of the Schlenk equilibria for methyl- and ethyl-zinc iodide in tetrahydrofuran

Proton resonance spectra are reported for mixtures of methyl- and ethyl-zinc iodide with the corresponding bisalkylzincs in tetrahydrofuran solution. At low temperatures, alkyl exchange is slow on a proton resonance timescale. For the Schlenk equilibria R2Zn + Znl2⇌ 2RZnl K is 500 at –70 °C, in agreement with previous results.

Studies on Grignard reagents. Part III. Proton resonance spectra of alkyl and aryl Grignard reagents

Proton resonance spectra are reported for a variety of Grignard reagents in a number of different solvents, and also for mixtures with the corresponding bis-alkyl-and-aryl-magnesiums. At low temperatures chemical exchange of alkyl or aryl groups is slow on a proton resonance time-scale. The position of the Schlenk equilibrium, for which quantitative data are presented, depends very markedly on the alkyl or aryl group and on the solvent. Alkyl or aryl exchange is slower in strongly co-ordinating solvents.