Two-dimensional Exchange Spectroscopy Research Articles

DOTA-bis(amide)]lanthanide complexes: NMR evidence for differences in water-molecule exchange rates for coordination isomers.

Two derivatives of 1,4,7,10-tetraazacyclododecane with trans-acetate and trans-amide side-chain ligating groups have been prepared and their complexes with lanthanide cations examined by multinuclear NMR spectroscopy. These lanthanide complexes exist in aqueous solution as a mixture of slowly interconverting coordination isomers with 1H chemical shifts similar to those reported previously for the major (M) and minor (m) forms of the tetraacetate ([Ln(dota)]-) and tetraamide ([Ln(dtma)]3+) complexes. As in the [Ln(dota)]- and [Ln(dtma)]3+ complexes, the m/M ratio proved to be a sensitive function of lanthanide size and temperature. An analysis of 1H hyperfine shifts in spectra of the Yb3+ complexes revealed significant differences between the axial (D1) and non-axial (D2) components of the magnetic susceptibility tensor anisotropy in the m and M coordination isomers and the energetics of ring inversion and m <==> M isomerization as determined by two-dimensional exchange spectroscopy (EXSY). (17)O shift data for the Dy3+ complexes showed that both have one inner-sphere water molecule. A temperature-dependent (17)O NMR study of bulk water linewidths for solutions of the Gd3+ complexes provided direct evidence for differences in water exchange rates for the two coordination isomers. The bound-water lifetimes (tauM298) in the M and m isomers of the Gd3+ complexes ranged from 1.4-2.4 micros and 3-14 ns, respectively. This indicates that 1) the inner-sphere water lifetimes for the complexes with a single positive charge reported here are considerably shorter for both coordination isomers than the corresponding values for the [Gd(dtma)]3+ complex with three positive charges, and 2) the difference in water lifetimes for M and m isomers in these two series is magnified in the [Gd[dota-bis(amide)]] complexes. This feature highlights the remarkable role of both charge and molecular geometry in determining the exchange rate of the coordinated water.

Investigation of Polymorphism in Aspartame and Neotame Using Solid-State NMR Spectroscopy

We have been studying the artificial sweeteners aspartame (l-aspartyl-l-phenylalanine methyl ester) and neotame (N-(3,3-dimethylbutyl)-l-aspartyl-l-phenylalanine methyl ester) as compounds which exhibit polymorphism. 13C CP/MAS NMR shows that aspartame exists in three distinct forms at room temperature, depending on preparation conditions. For two of the forms, there exists three resonances for each carbon, indicating three crystallographically inequivalent sites and therefore three distinct conformations and/or arrangements of aspartame molecules within the unit cell. Two-dimensional exchange spectroscopy using high-speed MAS and very high-power 1H decoupling on uniformly 13C labeled aspartame is a very powerful tool for unambiguously assigning each resonance in the NMR spectrum of aspartame. Even for forms of aspartame that possesses multiple crystallographically inequivalent sites, it is possible to identify connectivities between the nuclei of each conformation and/or arrangement of molecules using two-dimensional NMR techniques. 13C CP/MAS NMR also shows that neotame exists in multiple solid forms. The most stable form of neotame under ambient conditions is a monohydrate. However, other forms can be prepared by heating or using reduced pressures. We have found that high-speed magic-angle spinning can cause a change in polymorphic forms. Three different forms were produced upon spinning at 29kHz for several days. The monohydrate was identified as the second form produced. Also, altering the crystallization and drying conditions can generate mixtures of the solid forms of neotame. When the monohydrate form of neotame was heated under vacuum, a mixture of anhydrate forms was produced. In the reconversion to the monohydrate upon exposure to moisture under ambient conditions no significant changes were observed in the powder X-ray diffraction patterns during part of the reconversion process. This suggests that no change in form had occurred. The 13C CP/MAS NMR spectra, however, indicated the presence of many forms of neotame during the reconversion. One possible reason that solid-state NMR spectroscopy detected the changes in forms and powder X-ray diffraction did not is that the conformation of the neotame molecules changes between forms but the unit cell parameters do not change significantly.

Determination ofsyn/anti Isomerism in DCNQI Derivatives by 2D Exchange Spectroscopy: Theoretical Underpinning

The dynamic syn/anti isomerism resulting from the inversion of the cyano group at the C=N double bond in a series of substituted DCNQIs has been investigated in solution by two-dimensional exchange spectroscopy (2D EXSY). The isomers formed were characterised by 1H NMR at 223 K to slow down the inversion process of the cyanoimine group as well as by H,H COSY spectra. Whereas compounds 6, 7, 9 and 10 show only one isomer, compounds 8 and 11 show two isomers with two inversion processes and compounds 12 and 13 show three isomers and four inversion processes. The energy barrier for the syn/anti isomerization of the NCN groups has been estimated from the rate constants determined from the 2D EXSY spectra, and very close values (13.16−13.93 kcal/mol for a single inversion) were found for all compounds. Isomerizations involving two NCN groups (compounds 12 and 13) exhibited higher activation free energy values (13.41−14.40 kcal/mol). Theoretical calculations of these free energy barriers are in excellent agreement with the experimental values, especially when solvent effects are taken into account. Theoretical calculations at B3LYP/6−31G* level predict planar geometries for the DCNQI derivatives studied (6 and 8), in particular when solvent effects are considered. This is in perfect agreement with the experimental data. Two-electron stabilizing interactions, as well as solvent effects, are related to the relative energies of the different stereoisomers. The relative equilibrium populations of the different isomers have been calculated using semiempirical energies and Boltzmann’s distribution. Although the most stable isomer as determined by AM1 method is in qualitative agreement with that deduced from NMR experiments in all DCNQI derivatives studied, better quantitative correlations are obtained at the B3LYP(L1A1)/6−31G* calculation level. Comparison of both semiempirical and ab initio calculations reveal that in these DCNQI systems AM1 is the method of choice for those larger systems whose size prevents energy calculations at higher levels.

Structure and Dynamic Exchange in Rhodium η2-Naphthalene and Rhodium η2-Phenanthrene Complexes: Quantitative NOESY and EXSY Studies

Complexes of naphthalene and phenanthrene with rhodium(η5-cyclopentadienyl)(trimethylphosphine) have been studied by quantitative two-dimensional nuclear Overhauser (NOESY) and exchange spectroscopy (EXSY). Naphthalene coordinates in the η2-1,2-mode as (η5-C5H5)Rh(PMe3)(η2-C10H8). At 260 K, NOESY peaks establish that the solution conformer has the hydrogen atoms on the coordinated double bond bent out of the arene plane toward the PMe3 ligand. The effective average distance, reff, of these hydrogen atoms from those in the PMe3 ligand is calculated as 3.52 Å by matrix analysis of the NOESY spectrum. At room temperature, (η5-C5H5)Rh(PMe3)(η2-C10H8) undergoes an intramolecular [1,3]-metallotropic shift within the coordinated ring with ΔG⧧300 of 74.4 kJ mol-1 detected by EXSY spectroscopy. This species is in equilibrium (a) with the C−H activated isomer (η5-C5H5)Rh(PMe3)(C10H7)H and (b) with the dinuclear complex [(η5-C5H5)Rh(PMe3)]2(μ-1,2-η2-3,4-η2-C10H8) and free naphthalene. The free energy change at 300 K for conversion of (η5-C5H5)Rh(PMe3)(η2-C10H8) to (η5-C5H5)Rh(PMe3)(C10H7)H is +11.5 kJ mol-1 compared to +2.2 kJ mol-1 for the (η5-C5Me5) analogue. The crystal structure of the dinuclear complex, [(η5-C5H5)Rh(PMe3)]2(μ-1,2-η2-3,4-η2-C10H8), shows that this molecule adopts the structure with the two rhodium centers coordinated antifacially to the same ring of the naphthalene ligand. The C−C bond lengths of the coordinated ring show conspicuous alternation, while those of the uncoordinated ring differ less than those in free naphthalene. The mean separation of the hydrogen atoms attached to the coordinated CC bond from the PMe3 hydrogen atoms, averaged from the crystal structure as 1/〈r-3〉1/3 for each independent rhodium center, is 3.56 Å, compared to 3.48 Å for reff measured in solution by NOESY spectroscopy. The phenanthrene complex (η5-C5H5)Rh(PMe3)(η2-9,10-C14H10) adopts a conformation similar to the naphthalene complex; the value of reff is estimated to be reduced to 3.43 Å.

Two-dimensional exchange spectroscopy revisited: Accounting for the number of participating spins

The magnetization exchange between two groups of equivalent spins each having different populations and overall relaxation rates has been analyzed. The results suggest that either the spin population difference or the overall relaxation rate difference in an exchange spectrum can produce cross-peaks with volumes larger than that of the corresponding diagonal line. This is important for interpretation of the magnetization exchange between water and macromolecular protons where both the population differences and auto-relaxation rate differences can be very large. Theoretical predictions of peak volume evolution were experimentally verified in the intermolecular magnetization exchange between the water and labile amide proton in a model system N-acetylglycine/water.

Ordering of Silicate Layers by Hydrogen-Bonding Networks: Solid State NMR Investigation of the Perfect Three-Dimensional Registration in the Layer Silicate RUB-18

Solid state 1H and 29Si NMR spectroscopies were applied to the layer silicate RUB-18 in order to investigate the hydrogen bonding network providing the three-dimensional ordering of the silicate layers. Two 1H NMR signals corresponding to water molecules and strongly hydrogen-bonded silanol groups were resolved, the relative intensities of which are in good agreement with the recently proposed structure. By means of two-dimensional heteronuclear correlation NMR, the protons in hydroxyl groups are clearly identified as the source of polarization transfer to the 29Si nuclei of the silicate layer. The dynamics of the exchange process between the protons in hydroxyl groups and those in the water molecules were investigated by two-dimensional 1H exchange spectroscopy, giving insight into the nature of the exchange processchemical exchange or spin diffusion. In addition, the ratio of exchanging protons of the two species has been found to differ from the stoichiometric ratio. 29Si cross-polarization/magic angle...

Chain Flips in Polyethylene Crystallites and Fibers Characterized by Dipolar13C NMR

The occurrence and rate of 180° chain-flip motions in the crystalline regions of two polyethylenes were studied by 13C NMR. In high-density polyethylene (HDPE) and in ultradrawn ultrahigh molecular weight polyethylene (UHMWPE) fibers, the changes in the 13C−13C dipolar couplings brought about by the reorientations of the 13C−13C internuclear vectors in the crystallites were observed. In the HDPE sample, which was labeled with 4% of 13C−13C pairs, the rotational motion was observed directly via two-dimensional exchange spectroscopy, stimulated-echo decays, and 1D line shape changes monitoring the 13C−13C dipolar coupling. The data show that the jumps occur between two sites, with a rotation angle of 180° and with a jump rate of ∼10/s at ambient temperature. The correlation function of the motion was found to be slightly nonexponential, with a stretched-exponential β parameter of 0.8 ± 0.1. The data yield an activation energy of 93 ± 10 kJ/mol for the 180° chain flips. In the fibers, the narrowing of natura...

Protonation of β-Cyanoenamines: NMR Spectroscopic and Calculational Studies of Ketenimine Amidinium Ion Formation

NMR spectroscopy and quantum mechanical calculations have been used to investigate the protonation of β-cyanoenamines. One-dimensional variable-temperature spectroscopy, two-dimensional exchange spectroscopy (2D EXSY), and density functional calculations provide convincing evidence that the favored site of protonation in 1,1-bis(dimethylamino)-2-cyano-2-p-X-phenyl-substituted ethylenes (X = NO2, CF3, Br, F, or CH3) is the nitrogen of the cyano group. This results in the formation of a ketenimine amidinium ion in solution. When X is less electron withdrawing than −NO2, an equilibrium mixture is formed consisting of 60−93% CN-protonated enamine with the remainder protonated at the β-carbon. Calculational results also support predominantly CN-protonation in these systems. Both experimental and calculational results show no evidence of protonation at the amino nitrogen.

Proton resonance assignments and ligand exchange kinetics in high-spin and mixed-spin myoglobin complexes using two-dimensional exchange spectroscopy

The task of assigning resonances in proton nuclear magnetic resonance spectra of paramagnetic heme proteins can be an arduous process, but with the development of multi-dimensional NMR methods the situation has improved. It is demonstrated here that two-dimensional exchange spectroscopic experiments can be used to obtain to assignment correlations for the heme protons of methydroxy-, metthiocyano-, metaquo-, and metimidazole-myoglobin forms. All the assignments are unambiguous and straightforward when the temperature and mixing times are adjusted to minimize nuclear Overhauser cross-peaks from each complex. Moreover, saturation transfer experiments allow the study of ligand binding kinetics. The exchange rates between metaquo- and metimidazole- (or methyl substituted imidazole) myoglobin complexes are estimated. The differences between the exchange rates reflect differences in the hydrophobic and steric interactions between the ligands and the protein moiety.

Unidirectional Steady State Rates of Central Metabolism Enzymes Measured Simultaneously in a Living Plant Tissue

The unidirectional steady state reaction rates of several enzymes and metabolic fluxes of distinct processes were measured simultaneously in hypoxic maize root tips using two-dimensional phosphorus NMR exchange spectroscopy. A single spectrum monitors ATP synthesis and hydrolysis as well as the activities of four enzymes involved in key pathways of central metabolism: UDP-glucose pyrophosphorylase, phosphoglucomutase, hexose-phosphate isomerase, and enolase. The corresponding unidirectional reaction rates and net metabolic fluxes were calculated from spectral intensities. This method provides a unique picture, at enzyme resolution, of how metabolism reacts in a concerted fashion to changes in external parameters such as temperature and oxygen concentration. By increasing hypoxia via an increase in temperature, we measured the expected increase in glycolysis through enolase activity while total ATP synthesis settled. At the same time, we observed a net flux through phosphoglucomutase and UDP-glucose pyrophosphorylase toward carbohydrate synthesis. This result is discussed in relation to the current hypothesis on the turnover of cell walls and sucrose. This reaction also produces a net flux of pyrophosphate, which is needed by pyrophosphate:fructose-6-phosphate 1-phosphotransferase to work as a glycolytic enzyme.

NMR Spectroscopic Studies of β-Methyl-Pyridine Binding to Cytochrome C

The binding of β-methyl-pyridine (β-MePy) to horse heart ferricytochorme c (cyt c) was studied by two-Dimensional exchange spectroscopy. Kinetic and equilibrium data for β-MePy binding to cyt c have been determined. Some hyperfine shifted resonances of β-MePy-cyt c have been assigned. Differences between β-MePy-cyt c and pyridine bound cyt c (py-cyt c) have been disscussed.

Effect of pyridine on the alkaline isomerization of cytochrome c

The effect of pyridine on the isomerization of cytochrome c between the native and the alkaline form was studied by 1H NMR. The different effects of the concentration of pyridine, the pH and temperature on the ratio of two alkaline cytochrome c isomers were investigated. The results of the investigation showed that pyridine above a certain concentration and high temperature can change the relative concentration of the two alkaline isomers and that it was more efficient than the temperature effect. This phenomenon was further investigated by two-dimensional exchange spectroscopy (2D-EXSY), which, for the first time, was used to measure both the three species exchange rate constants between the native form and two individual alkaline isomers — with or without the presence of pyridine — and the multiple species exchange rate constants between native cytochrome c, pyridine = bound cytochrome c and two alkaline forms.

1H and 11B NMR evidence for specific binding of borate ion to cytochrome c

Cytochrome c possesses a positively charged surface which binds specifically to mitochondria enzymes and metabolites. The affinity of the cytochrome c surface to charged ligands is demonstrated by specific binding to borate ions. The effect of the ionic bond formed between the borate and the cytochrome is observed in both 1H and 11B NMR spectra. New narrow peaks with prolonged T 1 values appear in the 11B spectrum and are assigned to the bound tetrahedral borate ion. In the 1H spectrum changes are observed in the peaks of the iron ligand histidine 18β and the heme methyls 3 and 8. These changes are affected by the ionic strength of the solution. The exchange process between the free and borate-bound forms of the protein is established by a two-dimensional exchange spectroscopy experiment. The k diss for the process is 24 ± 2 s −1, as measured at 5°C by an inversion transfer experiment. K D values were calculated at several temperatures and range from 67 ± 7 mM at 2.7°C to 143 ± 14 mM at 14°C.

An NMR investigation of the incoherent tunnelling exchange in a niobium trihydride

The incoherent contribution to the tunnelling exchange of hydrogen pairs in NbH3cp′2 (cp′ = C5 H3(Si(CH3)3)2) was investigated by 1H-NMR line-shape analysis and two-dimensional 1H-NMR exchange spectroscopy. The experimental data were analysed by fitting them with simulated one- and two-dimensional spectra, taking into account the coherent and incoherent tunnelling contributions and nuclear cross relaxation. The dissipative exchange process shows an Arrhenius-like temperature dependence with an activation energy E a = 69(2) kJ mol-1 over the temperature range 246–341 K.

Chemical exchange and conductivity processes in betaine phosphate and betaine phosphite

Chemical exchange processes of deuterons in partially deuterated betaine phosphate (DBP) and betaine phosphite (DBPI) crystals are investigated by means of NMR experiments over a wide temperature range. The lineshape analysis of one-dimensional (1D) NMR experiments on DBP reveals a behaviour which is characteristic for exchange processes between all deuterons in the hydrogen bonds. In the temperature range between 230 and 310 K two-dimensional (2D) NMR exchange spectroscopy has been applied, revealing a uniform rate of exchange between all sites. The exchange rates for both crystals are found to show an Arrhenius-like behaviour with activation energies of 83 and , respectively, where for temperatures above 360 K the rates are obtained from the lineshape analysis of 1D NMR spectra. In addition, conductivity measurements for DBP and DBPI are reported. The dc conductivity in the direction of the quasi-one-dimensional chains, formed essentially by the phosphate and phosphite groups in DBP and DBPI, is characterized by activation energies of 115 and , respectively. For DBPI this value is close to that obtained for the exchange processes by means of NMR, whereas a difference of about is found in the case of DBP. Quantitative relations between the exchange and conductivity processes are established and, on the basis of simplifying assumptions, an estimation of effective charge-carrier densities is discussed.

Use of cross-links to study the conformational dynamics of triplex DNA.

The conformational dynamics of a 34-base-long pyrimidine purine-pyrimidine motif intramolecular DNA triple helix possessing three cytosine residues in the Hoogsteen strand (1) and a disulfide cross-linked analog (2) were studied by two-dimensional exchange and NOE spectroscopy and by measuring base-catalyzed imino proton exchange rates. Under acidic conditions that stabilize triplexes containing Hoogsteen strand cytosines (pH 6.0 and 1 degree C), sequences 1 and 2 exhibit a small and identical degree of conformational heterogeneity. However, at a higher temperature (pH 6.0 and 37 degrees C), 1 exhibits much more extensive conformational heterogeneity than 2. The exchange times for Watson-Crick imino protons are approximately 1 h for both triplexes. However, the Hoogsteen base-pair lifetimes of 1 could not be measured because this sequence is conformationally labile under the alkaline conditions necessary to conduct these experiments. Because of the extraordinary pH stability conferred by the cross-link, it is possible to estimate the base-pair lifetimes for 2. The lifetimes of the Hoogsteen base pairs range from about 3 to 370 ms, and in all cases are shorter than that of the Watson-Crick base pair contained in the same triplet. These experiments represent the first measurement of base-pair lifetimes within Hoogsteen triplets. The ability to measure individual base-pair lifetimes may prove useful in studies that attempt to modulate triplex properties through rational design.

Nuclear magnetic resonance spectroscopic studies of pyridine methyl derivatives binding to cytochrome c

The binding of pyridine methyl derivatives (2-, 3- and 4-methylpyridine) to horse heart ferricytochrome c (cyt c) by displacing methionine-80 was studied by 1H NMR spectroscopy to elucidate the effects of the different methyl substitution positions on the affinity and kinetics of binding to cytochrome c and the hyperfine-shifted NMR signals of the ligand–cytochrome c complex. Two-dimensional exchange spectroscopy (2D-EXSY) showed that except for 2-methylpyridine (2-mpy) these pyridine derivatives can form stable complexes with cytochrome c. The complexes 3-mpy–cyt c and 4-mpy–cyt c exhibit different hyperfine shift patterns compared to that of py–cyt c. The temperature dependence of the methyl resonances of 3-mpy–cyt c differs from those of 4-mpy– and py–cyt c. Kinetic and equilibrium data for the binding of 3- and 4-mpy to cyt c have been obtained by 2D-EXSY. Based on these data a comprehensive comparison between the binding properties of these pyridine derivatives and those of pyridine towards cyt c was made. The 1H NMR resonances of 3-mpy–cyt c have also been assigned including the heme peripheral protons and some aliphatic and aromatic side chain protons.

Structural investigations of C-nitrosobenzenes. Part 2.1 NMR studies of monomer–dimer equilibria including restricted nitroso group rotation in monomers

Energy barriers associated with rotation of nitroso groups in monomeric nitrosobenzene and eleven monomeric substituted nitrosobenzenes in solution have been measured by total 1H NMR bandshape analysis. ΔG ‡ (298.15 K) values for the rotations are in the range 31–41 kJ mol–1. The values for 4-substituted compounds correlate well with the Hammett σp+ parameter for the substituent. The experimental energy barrier for nitrosobenzene is compared with theoretical calculations. Monomer–dimer equilibria of these compounds in solution have been investigated, with ΔH, ΔS and ΔG data calculated for the dissociation of both Z- and E- azodioxy dimers to monomers. Kinetic data, based on time-dependent and two-dimensional exchange spectroscopy (2D-EXSY) NMR measurements, have been obtained for the dissociation of 3-methylnitrosobenzene and 3,5-dimethylnitrosobenzene dimers. Energy profiles for the ground and transition states of both dimers and monomers (including exchange between both their rotameric forms) are discussed.

Fluxional rearrangements in molybdenum(&gt;0&gt;), tungsten(&gt;0&gt;) and rhenium(I) carbonyl complexes of 2,6-bis[(4S )-isopropyloxazolin-2-yl]pyridine (L). Crystal structure of [Mo(CO)4L] ‡

Treatment of the [ReX(CO)5] (X = Cl, Br or I) and [M(CO)4(pip)2] (M = Mo or W, pip = piperidine) compounds with the C2-symmetric ligand 2,6-bis[(4S)-isopropyloxazolin-2-yl]pyridine (L) yielded complexes of general formulae fac-[ReX(CO)3L] and cis-[M(CO)4L], in which the ligand is co-ordinated in a bidentate fashion. In solution these complexes undergo a fluxional process that exchanges the co-ordinated and pendant oxazoline rings. In the case of the M(CO)4 complexes all permutational isomers are equivalent. However, in the rhenium(I) complexes the lower symmetry of the metal moiety leads to the formation of chemically distinct species. The different exchange pathways between these species gives rise to different magnetisation transfers, providing a spectroscopic handle on the mechanism of the ligand rearrangement. The activation parameters have been evaluated by standard one-dimensional band shape analysis and by two-dimensional exchange spectroscopy; ΔG ‡ (298 K) ≈ 52.0 and 62.6 kJ mol–1, respectively for the complexes of Mo0 and W0 and is in the range 78.5–80.5 kJ mol–1 for the rhenium(I) complexes.

Determination of fast proton exchange rates of biomolecules by NMR using water selective diffusion experiments

We present a new water selective pulse sequence allowing rapid determination of exchange rates of labile protons on the millisecond time scale. Using diffusion measurements, exchange rates of resolved protons can be determined without prior knowledge of relaxation parameters in a short overnight experiment. The use of a sensitive, highly selective and easy to implement water excitation scheme allows for its straightforward application to a wide range of biomolecules. The results obtained for the imino proton exchange rates of a 16 bp DNA are in strong agreement with values obtained by the classical approach of two-dimensional exchange spectroscopy.