Dynamic NMR Studies Research Articles

Imaging of the intracellular topography of copper with a fluorescent sensor and by synchrotron x-ray fluorescence microscopy

Copper is an essential micronutrient that plays a central role for a broad range of biological processes. Although there is compelling evidence that the intracellular milieu does not contain any free copper ions, the rapid kinetics of copper uptake and release suggests the presence of a labile intracellular copper pool. To elucidate the subcellular localization of this pool, we have synthesized and characterized a membrane-permeable, copper-selective fluorescent sensor (CTAP-1). Upon addition of Cu(I), the sensor exhibits a 4.6-fold emission enhancement and reaches a quantum yield of 14%. The sensor exhibits excellent selectivity toward Cu(I), and its emission response is not compromised by the presence of millimolar concentrations of Ca(II) or Mg(II) ions. Variable temperature dynamic NMR studies revealed a rapid Cu(I) self-exchange equilibrium with a low activation barrier of deltaG++ = 44 kJ.mol(-1) and k(obs) approximately 10(5) s(-1) at room temperature. Mouse fibroblast cells (3T3) incubated with the sensor produced a copper-dependent perinuclear staining pattern, which colocalizes with the subcellular locations of mitochondria and the Golgi apparatus. To evaluate and confirm the sensor's copper-selectivity, we determined the subcellular topography of copper by synchrotron-based x-ray fluorescence microscopy. Furthermore, microprobe x-ray absorption measurements at various subcellular locations showed a near-edge feature that is characteristic for low-coordinate monovalent copper but does not resemble the published spectra for metallothionein or glutathione. The presented data provide a coherent picture with strong evidence for a kinetically labile copper pool, which is predominantly localized in the mitochondria and the Golgi apparatus.

Relating side-chain mobility in proteins to rotameric transitions: Insights from molecular dynamics simulations and NMR

The dynamic aspect of proteins is fundamental to understanding protein stability and function. One of the goals of NMR studies of side-chain dynamics in proteins is to relate spin relaxation rates to discrete conformational states and the timescales of interconversion between those states. Reported here is a physical analysis of side-chain dynamics that occur on a timescale commensurate with monitoring by 2H spin relaxation within methyl groups. Motivated by observations made from tens-of-nanoseconds long MD simulations on the small protein eglin c in explicit solvent, we propose a simple molecular mechanics-based model for the motions of side-chain methyl groups. By using a Boltzmann distribution within rotamers, and by considering the transitions between different rotamer states, the model semi-quantitatively correlates the population of rotamer states with 'model-free' order parameters typically fitted from NMR relaxation experiments. Two easy-to-use, analytical expressions are given for converting S2 (axis') values (order parameter for C-CH3 bond) into side-chain rotamer populations. These predict that S2 (axis') values below 0.8 result from population of more than one rotameric state. The relations are shown to predict rotameric sampling with reasonable accuracy on the ps-ns timescale for eglin c and are validated for longer timescales on ubiquitin, for which side-chain residual dipolar coupling (RDC) data have been collected.

A 13C NMR Study of the Molecular Dynamics and Phase Transition of Confined Benzene inside Titanate Nanotubes

This work investigated the nanoconfinement effect on the molecular dynamics and phase transition of confined benzene inside titanate nanotubes with a uniform inner diameter of approximately 5.3 nm. For 13C-enriched organics, the 13C nuclear spin-spin relaxation was demonstrated as a sensitive tool to differentiate molecular translational motion and reorientation and, thus, was shown to be advantageous over the commonly employed 1H and 2H NMR for studying complex phase diagram, specifically, for separating the phase behavior of translational motion and the phase behavior of molecular reorientation. In such an approach, the melting of translational motion of confined benzene was explicitly observed to take place in a broad temperature range below the bulk melting temperature. The abrupt change of the 13C nuclear spin-spin relaxation time of the confined liquid benzene at about 260 K suggested that nanoconfinement induced two topologically distinct liquid phases.

Synthesis of Dimethyl 1‐Aryl‐4‐ethoxy‐5‐oxo‐2,5‐dihydro‐1H‐pyrrole‐2,3‐dicarboxylates Mediated by Triphenylphosphine.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Rotational Isomerism Involving an Acetylenic Carbon VI. Syntheses, Structures, and Dynamic Stereochemistry of Bis(1-phenyl-9-anthryl)ethynes: Highly Restricted Rotation about Acetylenic Axis in Acyclic Diarylethynes

Abstract Some sterically congested di(9-anthryl)ethynes with substituted phenyl groups (4-Me, 4-iPr, 4-tBu, 3-iPr, 3,5-iPr2) at the 1-positions were synthesized by the Stille coupling of the corresponding triflate and bis(tributylstannyl)ethyne to realize the restricted rotation of the acetylenic axis in diarylethyne derivatives. In the X-ray structure of the 4-Me compound, the two 9-anthryl groups were staggered about the acetylenic axis by ca. 60°. The dynamic NMR study of the iPr substituted compounds afforded the kinetic data of the two independent processes: the faster and slower processes were assigned to the rotation about the acetylenic axis (ΔG≠ 45–51 kJ mol−1) and the rotation of the phenyl groups (ΔG≠ ca. 75 kJ mol−1), respectively, from the details of the lineshape changes. The observed barriers to the rotation about the acetylenic axis are rather high for acyclic diarylethynes, being attributed to the severe steric interactions between the 1-phenyl groups and the rigid di(9-anthryl)ethyne framework in the transition state. The mechanism of the dynamic stereochemistry and the substituent effect on the rotational barrier are discussed on the basis of the kinetic data and the molecular structures.

2H NMR Study of Aromatic Guest Dynamics in Clathrate Phases of Syndiotactic Polystyrene

AbstractSummary: 2H NMR solid‐state spectra of aromatic molecules (benzene, toluene, chlorobenzene, naphthalene) included in clathrate phases of syndiotactic polystyrene (s‐PS) or absorbed in the amorphous phase of s‐PS, have been recorded and analyzed for a broad range of temperature (230–360 K). The dynamic processes accessible to aromatic molecules absorbed in s‐PS clathrate phases are essentially temperature independent for the whole temperature range under investigation. Moreover, the molecular re‐orientations reflected in the NMR line‐shapes are similar to those accessible to the same molecules when absorbed in polystyrene amorphous phase at temperatures substantially lower than the polymer glass transition temperature. A simulation procedure for 2H NMR line‐shape analysis has been used to establish models for the dynamic processes of the different aromatic guest molecules.Solid‐state 2H NMR spectrum of naphtalene‐d8 in the amorphous phase of a semi‐crystalline s‐PS film (A), in the s‐PS clathrate phase (B) and the simulated spectrum for a static CD bond (C).magnified imageSolid‐state 2H NMR spectrum of naphtalene‐d8 in the amorphous phase of a semi‐crystalline s‐PS film (A), in the s‐PS clathrate phase (B) and the simulated spectrum for a static CD bond (C).

Structural Properties and Dissociative Fluxional Motion of 2,9-Dimethyl-1,10-Phenanthroline in Platinum(II) Complexes

A dynamic 1H NMR study has been carried out on the fluxional motion of the symmetric chelating ligand 2,9-dimethyl-1,10-phenanthroline (Me2-phen) between nonequivalent exchanging sites in a variety of square-planar complexes of the type [Pt(Me)(Me2-phen)(PR3)]BArf, 1-14, (BArf = B[3,5-(CF3)2C6H3]4). In these compounds, the P-donor ligands PR3 encompass a wide range of steric and electronic characteristics [PR3 = P(4-XC6H4)3, X = H 1, F, 2, Cl 3, CF3 4, MeO 5, Me 6; PR3 = PMe(C6H5)2 7, PMe2(C6H5) 8, PMe3 9, PEt3 10, P(i-Pr)3 11, PCy(C6H5)2 12, PCy2(C6H5) 13, PCy3 14]. All complexes have been synthesized and fully characterized through elemental analysis, 1H and 31P{1H} NMR. X-ray crystal structures are reported for the compounds 8, 11, 14, and for [Pt(Me)(phen)(P(C6H5)3)]PF6 (15), all but the last showing loss of planarity and a significant rotation of the Me2-phen moiety around the N1-N2 vector. Steric congestion brought about by the P-donor ligands is responsible for tetrahedral distortion of the coordination plane and significant lengthening of the Pt-N2 (cis to phosphane) bond distances. Application of standard quantitative analysis of ligand effects (QALE) methodology enabled a quantitative separation of steric and electronic contributions of P-donor ligands to the values of the platinum-phosphorus 1J(PtP) coupling constants and of the free activation energies DeltaG++ of the fluxional motion of Me2-phen in 1-14. The steric profiles for both 1J(PtP) and DeltaG++ show the onset of steric thresholds (at cone angle values of 150 degrees and 148 degrees , respectively), that are associated with an overload of steric congestion already evidenced by the crystal structures of 11 and 14. The sharp increase of the fluxional rate of Me2-phen can be assumed as a perceptive kinetic tool for revealing ground-state destabilization produced by the P-donor ligands. The mechanism involves initial breaking of a metal-nitrogen bond, fast interconversion between two 14-electron three-coordinate T-shaped intermediates containing eta1-coordinated Me2-phen, and final ring closure. By use of the results from QALE regression analysis, a free-energy surface has been constructed that represents the way in which any single P-donor ligand can affect the energy of the transition state in the absence of aryl or pi-acidity effects.

Dynamic NMR study on the trans-fused eight-membered ether ring model representing G ring of brevetoxin A

Conformational analysis of the trans-fused eight-membered ether ring model ( 2 ) representing G ring of brevetoxin A ( 1 ) was carried out by dynamic NMR study in combination with molecular mechanics calculations.

Study of gas-fluidization dynamics with laser-polarized 129Xe

We report initial NMR studies of gas dynamics in a particle bed fluidized by laser-polarized xenon (129Xe) gas. We have made preliminary measurements of two important characteristics: gas exchange between the bubble and emulsion phases and the gas velocity distribution in the bed. We used T2* contrast to differentiate the bubble and emulsion phases by choosing solid particles with large magnetic susceptibility. Experimental tests demonstrated that this method was successful in eliminating 129Xe magnetization in the emulsion phase, which enabled us to observe the time dependence of the bubble magnetization. By employing the pulsed field gradient method, we also measured the gas velocity distribution within the bed. These results clearly show the onset of bubbling and can be used to deduce information about gas and particle motion in the fluidized bed.

Synthesis of Dimethyl 1-Aryl-4-ethoxy-5-oxo-2,5-dihydro-1H-pyrrole-2,3-dicarboxylates Mediated by Triphenylphosphine

Ethyl 2-arylamino-2-oxo-acetates undergo a complex reaction with dimethyl acetylendicarboxylate in the presence of triphenylphosphine to produce dimethyl 1-aryl-4-ethoxy-5-oxo-2,5-dihydro-1H-pyrrole-2,3-dicarboxylates in good yields. Dynamic NMR study of dimethyl 1-(2-methyl-6-nitrophenyl)-4-ethoxy-5-oxo-2,5-dihydro-1H-pyrrole-2,3-dicarboxylate shows a fairly high energy barrier (ΔG # = 95 ± 2 kJ mol− 1) for rotation around the N-aryl single bond, which leads to an observable atropisomerism.

Synthesis of Fluorinated Dialkyl 1‐Aryl‐4‐alkoxy‐5‐oxo‐2,5‐dihydro‐1H‐pyrrole‐2,3‐dicarboxylates.

AbstractFor Abstract see ChemInform Abstract in Full Text.

CP/MAS,13C and17O NMR studies of proton transfer and dynamics of cyanopyridine hydrogen-bond complex with trichloroacetic acid in crystal and in solution

The hydrogen bond (H-bond) in the complex of cyanopyridine (4-pyridincarbonitrile, C6H4N2, further CyPy) with trichloroacetic acid (TCA) was investigated in the solid state and in the solution (1 M in CH3CN). The 13 C CP/ MAS results as well as X-ray and neutron diffraction reveal a complete proton transfer (CPT) for the CyPyTCA complex. An experimental criterion of the threshold of CPT is proposed. Reorientational dynamics of 'free' and 'bonded' CyPy molecules in solution were investigated by 17 O and 13 C NMR relaxation time and nuclear Overhauser effect (NOE) factor measurements. The ro- tational diffusion even of 'free' CyPy molecules is anisotropic, with a corresponding correlation time of 3 ps for rotation and that of 6 ps for tumbling at 293 K. The formation of the CyPyTCA H-bond complex causes a general slowdown of the overall rotational motion with a very slight increase in its anisotropy (7 ps for rotation and 17 ps for tumbling, respectively). The results are compared with similar data on H-bonding in pyridine N-oxide/acid systems.

Synthesis and Stereochemistry of N-Phenyl-N-9-triptycylhydroxylamine Derivatives and Related Compounds

Abstract During the course of attempts at synthesis of N-phenyl-N-9-triptycylhydroxylamine (1), we found that the reaction of N-9-triptycylhydroxylamine (5) with benzenediazonium-2-carboxylate afforded 3-(2-carboxyphenyl)-1-(9-triptycyl)triazene 1-oxide (6), the structure of which was confirmed by X-ray crystallography. Oxidation of N-(9-triptycyl)aniline (4) with mCPBA gave phenyl 9-triptycyl nitroxide (7) and N-(9-triptycyl)-1,4-benzoquinonimine (8) in small yields. Reduction of 7 with phenylhydrazine gave 1, and O-ethylation of 1 gave O-ethyl-N-phenyl-N-9-triptycylhydroxylamine (2). Results of X-ray crystallography for compounds 2, 4, 7, and 8 and results of dynamic NMR studies for 1, 2, and 8 are presented. In compound 8, Tp–N rotation and C=N topomerization took place on the NMR timescale at high temperatures above 60 °C, and the energy barriers to both processes were obtained. In compounds 1 and 2, dynamic NMR behavior was observed at very low temperatures below ca. −50 °C, and the energy barriers to the respective processes could not be determined except for those related to “Ph-passing”.

NMR and molecular dynamics studies of the interaction of melatonin with calmodulin

Pineal hormone melatonin (N-acetyl-5-methoxytryptamine) is thought to modulate the calcium/calmodulin signaling pathway either by changing intracellular Ca(2+) concentration via activation of its G-protein-coupled membrane receptors, or through a direct interaction with calmodulin (CaM). The present work studies the direct interaction of melatonin with intact calcium-saturated CaM both experimentally, by fluorescence and nuclear magnetic resonance spectroscopies, and theoretically, by molecular dynamics simulations. The analysis of the experimental data shows that the interaction is calcium-dependent. The affinity, as obtained from monitoring (15)N and (1)H chemical shift changes for a melatonin titration, is weak (in the millimolar range) and comparable for the N- and C-terminal domains. Partial replacement of diamagnetic Ca(2+) by paramagnetic Tb(3+) allowed the measurement of interdomain NMR pseudocontact shifts and residual dipolar couplings, indicating that each domain movement in the complex is not correlated with the other one. Molecular dynamics simulations allow us to follow the dynamics of melatonin in the binding pocket of CaM. Overall, this study provides an example of how a combination of experimental and theoretical approaches can shed light on a weakly interacting system of biological and pharmacological significance.

42(P-3) 有機合成モデルの動的NMRおよび分子力場計算によるポリ環状エーテルの立体配座解析(ポスター発表の部)

42(P-3) 有機合成モデルの動的NMRおよび分子力場計算によるポリ環状エーテルの立体配座解析(ポスター発表の部)

63,65Cu NMR study of low-frequency spin dynamics in the infinite-layer antiferromagnetic compound SrCuO2

The spin-lattice and spin-spin relaxation times have been measured for 63,65Cu NMR in the infinite-layer anti-ferromagnet SrCuO2 in the ordered state for temperatures from 4.2 to 361 K. In the region of low temperatures (T≤250 K), both relaxation processes are of the same nature and the main contribution to the relaxation rate is associated with the diffusion of a small number of holes with an activation energy of ∼42 meV. In the high-temperature range (T > 250 K), contributions to the transverse relaxation rate exhibit redistribution and this relaxation process is determined predominantly by indirect interactions.

1H NMR study of molecular dynamics of acetylcholine chloride

The longitudinal relaxation timeT1 and the second momentM2 of1H nuclear magnetic resonance line in a wide temperature range have been measured for acetylcholine chloride. Two different types of the methyl groups reorientation occurred. The first type was the hindered rotation of the methyl group denoted as C(1)H3 about the threefold symmetry axis. The second type was the reorientation of the trimethyl group-N(CH3)3 around the pseudo C3′ axis of C(6)-N(7) bond, which accompanied the standard C3 motion of the methyl group. The Dunn-McDowell model was applied to analyze the dynamics observed.

Backbone assignment of proteins with known structure using residual dipolar couplings.

A prerequisite for NMR studies of protein-ligand interactions or protein dynamics is the assignment of backbone resonances. Here we demonstrate that protein assignment can significantly be enhanced when experimental dipolar couplings (RDCs) are matched to values back-calculated from a known three-dimensional structure. In case of small proteins, the program MARS allows assignment of more than 90% of backbone resonances without the need for sequential connectivity information. For bigger proteins, we show that the combination of sequential connectivity information with RDC-matching enables more residues to be assigned reliably and backbone assignment to be more robust against missing data. Structural or dynamic deviations from the employed 3D coordinates do not lead to an increased error rate in RDC-supported assignment. RDC-enhanced assignment is particularly useful when chemical shifts and sequential connectivity only provide a few reliable assignments.

Dynamic NMR study of the mechanisms of double, triple, and quadruple proton and deuteron transfer in cyclic hydrogen bonded solids of pyrazole derivatives.

Using dynamic solid state (15)N CPMAS NMR spectroscopy (CP = cross polarization, MAS = magic angle spinning), the kinetics of the degenerate intermolecular double and quadruple proton and deuteron transfers in the cyclic dimer of (15)N labeled polycrystalline 3,5-diphenyl-4-bromopyrazole (DPBrP) and in the cyclic tetramer of (15)N labeled polycrystalline 3,5-diphenylpyrazole (DPP) have been studied in a wide temperature range at different deuterium fractions in the mobile proton sites. Rate constants were measured on a millisecond time scale by line shape analysis of the doubly (15)N labeled compounds, and by magnetization transfer experiments on a second timescale of the singly (15)N labeled compounds in order to minimize the effects of proton-driven (15)N spin diffusion. For DPBrP the multiple kinetic HH/HD/DD isotope effects could be directly obtained. By contrast, four rate constants k(1) to k(4) were obtained for DPP at different deuterium fractions. Whereas k(1) corresponds to the rate constant k(HHHH) of the HHHH isotopolog, an appropriate kinetic reaction model was needed for the kinetic assignment of the other rate constants. Using the model described by Limbach, H. H.; Klein, O.; Lopez Del Amo, J. M.; Elguero, J. Z. Phys. Chem. 2004,218, 17, a concerted quadruple proton-transfer mechanism as well as a stepwise consecutive single transfer mechanism could be excluded. By contrast, using the kinetic assignment k(2) approximately k(3) approximately k(HHHD) approximately k(HDHD) and k(3) approximately k(HDDD) approximately k(DDDD), the results could be explained in terms of a two-step process involving a zwitterionic intermediate. In this mechanism, each reaction step involves the concerted transfer of two hydrons, giving rise to primary kinetic HH/HD/DD isotope effects, whereas the nontransferred hydrons only contribute small secondary effects, which are not resolved experimentally. By contrast, the multiple kinetic isotope effects of the double proton transfer in DPBrP and of the triple proton proton transfer in cyclic pyrazole trimers studied previously indicate concerted transfer processes. Thus, between n = 3 and 4 a switch of the reaction mechanism takes place. This switch is rationalized in terms of hydrogen bond compression effects associated with the multiple proton transfers. The Arrhenius curves of all processes are nonlinear and indicate tunneling processes at low temperatures. In a preliminary analysis, they are modeled in terms of the Bell-Limbach tunneling model.

Gated Electron Transfers and Electron Pathways in Azurin: A NMR Dynamic Study at Multiple Fields and Temperatures

Dynamic properties of electron transfer pathways in a small blue copper cupredoxin are explored using an extensive 15N NMR relaxation study of reduced Pseudomonas aeruginosa azurin at four magnetic fields (500-900 MHz) and at two temperatures chosen well below the melting point of the protein. Following a careful model-free analysis, several protein regions with different dynamic regimes are identified. Nanosecond time-scale mobility characterizes various residues of the hydrophobic surface patch believed to mark the natural entry point for electrons, notably the surface-exposed copper-ligand His117. These findings are consistent with a gated electron transfer process according to the "dynamic docking" model. Residues 47-49 along intramolecular pathways of electrons show rigidity that is remarkably conserved when increasing the temperature. Three different conformational exchange processes were observed in the millisecond range, one near the only disulfide bridge in the molecule and two near the copper ion. The latter two processes are consistent with previous data such as crystal structures at various pH values and NMR relaxation dispersion experiments; they may indicate an additional gated electron transfer mechanism at slower time-scales.