2H[19F] REDOR for distance measurements in biological solids using a double resonance spectrometer

A new approach for distance measurements in biological solids employing 2H[19F] rotational echo double resonance was developed and validated on 2H,19F-D-alanine and an imidazopyridine based inhibitor of the gastric H+/K+-ATPase. The 2H-19F double resonance experiments presented here were performed without 1H decoupling using a double resonance NMR spectrometer. In this way, it was possible to benefit from the relatively longer distance range of fluorine without the need of specialized fluorine equipment. A distance of 2.5 +/- 0.3 A was measured in the alanine derivative, indicating a gauche conformation of the two labels. In the case of the imidazopyridine compound a lower distance limit of 5.2 A was determined and is in agreement with an extended conformation of the inhibitor. Several REDOR variants were compared, and their advantages and limitations discussed. Composite fluorine dephasing pulses were found to enhance the frequency bandwidth significantly, and to reduce the dependence of the performance of the experiment on the exact choice of the transmitter frequency.     

29Si and 19F MAS NMR spectra of isolated 29Si(19F)2 and 29Si(19F)3 spin systems: experiments and simulations

We consider ²⁹Si and ¹⁹F MAS NMR spectra of isolated ²⁹Si(¹⁹F)₂ and ²⁹Si(¹⁹F)₃ spin systems in two organosilicon compounds of the type RR’SiF₂ and RSiF₃(R,R′=organic ligands). Experimental spectra are analysed by means of numerical simulations. It is found that the SiF₃ group in RSiF₃ is reorienting rapidly around the molecular Si–C bond direction in the solid state. The two ¹⁹F shielding tensors in RR’SiF₂ have strongly differing orientations relative to the two Si–F bond directions in the molecule. Possibilities and limitations of straightforward MAS NMR approaches for the full characterisation of ²⁹Si(¹⁹F)₂ and ²⁹Si(¹⁹F)₃ spin systems and other dipolar coupled two and three-spin systems are discussed.     

A 4-mm probe for (13)C CP/MAS NMR of solids at 21.15 T

The design of a broadband 4-mm magic-angle spinning (MAS) X-(1)H/(19)F double resonance probe for cross-polarization (CP)/MAS NMR studies at 21.15 T ((1)H at 900 MHz) is described. The high-frequency (1)H/(19)F channel employs a new and efficient transmission line tuning design. The first (13)C CP/MAS NMR spectra recorded at 21.15 T have been obtained with this probe and exhibit the best S/N per milligram sample of hexamethylbenzene achieved so far for a 4-mm rotor.     

A 1H/19F minicoil NMR probe for solid-state NMR: application to 5-fluoroindoles

We show that it is feasible to use a minicoil for solid-state 19F 1H NMR experiments that has short pulse widths, good RF homogeneity, and excellent signal-to-noise for small samples while using low power amplifiers typical to liquid-state NMR. The closely spaced resonant frequencies of 1H and 19F and the ubiquitous use of fluorine in modern plastics and electronic components present two major challenges in the design of a high-sensitivity, high-field 1H/19F probe. Through the selection of specific components, circuit design, and pulse sequence, we were able to build a probe that has low 19F background and excellent separation of 1H and 19F signals. We determine the principle components of the chemical shift anisotropy tensor of 5-fluoroindole-3-acetic acid (5FIAA) and 5-fluorotryptophan. We also solve the crystal structure of 5FIAA, determine the orientation dependence of the chemical shift of a single crystal of 5FIAA, and predict the 19F chemical shift based on the orientation of the fluorine in the crystal. The results show that this 1H/19F probe is suitable for solid-state NMR experiments with low amounts of biological molecules that have been labeled with 19F.     

Carbon-13 and fluorine-19 NMR spectroscopy of the supramolecular solid p-tert-butylcalix(4)arene.alpha,alpha,alpha-trifluorotoluene

The supramolecular 1:1 host-guest inclusion compound, p-tert-butylcalix[4]arene x alpha,alpha,alpha-trifluorotoluene, 1, is characterized by 19F and 13C solid-state NMR spectroscopy. Whereas the 13C NMR spectra are easily interpreted in the context of earlier work on similar host-guest compounds, the 15F NMR spectra of solid 1 are, initially, more difficult to understand. The 19F[1H] NMR spectrum obtained under cross-polarization and magic-angle spinning conditions shows a single isotropic resonance with a significant spinning sideband manifold. The static 19F[1H] CP NMR spectrum consists of a powder pattern dominated by the contributions of the anisotropic chemical shift and the homonuclear dipolar interactions. The 19F MREV-8 experiment, which minimizes the 19F-19F dipolar contribution, helps to identify the chemical shift contribution as an axial lineshape. The full static 19F[1H] CP NMR spectrum is analysed using subspectral analysis and subsequently simulated as a function of the 19F-19F internuclear distance (D(FF) = 2.25 +/- 0.01 A) of the rapidly rotating CF3 group without including contributions from additional libration motions and the anisotropy in the scalar tensor. The shielding span is found to be 56 ppm. The width of the centerband in the 19F[1H] sample-spinning CP NMR spectrum is very sensitive to the angle between the rotor and the magnetic field. Compound 1 is thus an attractive standard for setting the magic angle for NMR probes containing a fluorine channel with a proton-decoupling facility.     

Solid state 19F NMR parameters of fluorine-labeled amino acids. Part I: aromatic substituents

Structural parameters of peptides and proteins in biomembranes can be directly measured by solid state NMR of selectively labeled amino acids. The 19F nucleus is a promising label to overcome the low sensitivity of 2H, 13C or 15N, and to serve as a background-free reporter group in biological compounds. To make the advantages of solid state 19F NMR fully available for structural studies of polypeptides, we have systematically measured the chemical shift anisotropies and relaxation properties of the most relevant aromatic and aliphatic 19F-labeled amino acids. In this first part of two consecutive contributions, six different 19F-substituents on representative aromatic side chains were characterized as polycrystalline powders by static and MAS experiments. The data are also compared with results on the same amino acids incorporated in synthetic peptides. The spectra show a wide variety of lineshapes, from which the principal values of the CSA tensors were extracted. In addition, temperature-dependent T(1) and T(2) relaxation times were determined by 19F NMR in the solid state, and isotropic chemical shifts and scalar couplings were obtained in solution.     

Study of molecular dynamics and cross relaxation in tetramethylammonium hexafluorophosphate (CH(3))(4)NPF(6) by (1)H and (19)F NMR

(CH(3))(4)NPF(6) is studied by NMR measurements to understand the internal motions and cross relaxation mechanism between the heterogeneous nuclei. The spin lattice relaxation times (T(1)) are measured for (1)H and (19)F nuclei, at three (11.4, 16.1 and 21.34 MHz) Larmor frequencies in the temperature range 350-50K and (1)H NMR second moment measurements at 7 MHz in the temperature range 300-100K employing home made pulsed and wide-line NMR spectrometers. (1)H NMR results are attributed to the simultaneous reorientations of both methyl and tetramethylammonium groups and motional parameters are evaluated. (19)F NMR results are attributed to cross relaxation between proton and fluorine and motional parameters for the PF(6) group reorientation are evaluated.     

2,3-二羟基萘钼多酸有机衍生物的合成及1H NMR和IR谱学研究

本文对二种新合成的2，3－二羟基萘二钼和四钼多酸有机衍生物[n-Bu)4N]2[Mo2O5(OC10H6O)2](Ⅰ）和[n-Bu)4N]2[Mo4O10(OC10H6O)2(OCH3)2](Ⅱ)进行了红外光谱与核磁共振波谱研究，发现[Mo2O5]^2 中钼氧多桥键的红外振动频率较[Mo4O10(OCH3)2]^2 中钼氧多桥键的红外振动频率红移，而在配合物Ⅱ中2，3－二羟基中芳环的^1H化学位移较配合物Ⅰ中向低场移动。同时还发现含二钼配位中心[Mo2O5]^2 的[Mo2O5(OC10H6O)2]^2-与含四钼配位中心[Mo4O10(OCH3)2]^2 的[Mo4O10(OC10H6O)2(OCH3)2]^2-生成条件的差异仅仅只在反应体系的pH值的微小变化，说明钼多酸有机衍生物阴离子是对体系酸碱度极为敏感的物质。     

1H and 13C NMR Spectra of Alditolyl Derivatives of 3-Hydrazino-5-Methyl[1,2,4]triazino[5,6-b]indole and Their Cyclized Products.

The ¹H and ¹³C NMR spectra of sugar (5-methyl [1, 2, 4]-triazino [5, 6-b] indol-3-yl) hydrazones (1), per-0-acetyl aldehydo sugar 1-acetyl-1-(5-methyl [1, 2, 4] triazino [5, 6-b]-indol-3-yl) hydrazones (2), l- (penta-0-acetyl-pentitol-1-yl)-10-methyl [l, 2, 4] triazolo [3′, 4′:3, 4] [l, 2, 4] triazino [5, 6-b]-indoles (3) have been investigated. The 2 D NMR (H, C COSY) spectrum of 2a has been studied.     

Carbon-13 and Fluorine-19 NMR Spectroscopy of the Supramolecular Solid p-tert-Butylcalix[4]arene ·α,α,α-trifluorotoluene

The supramolecular 1 : 1 host–guest inclusion compound, p-tert-butylcalix[4]arene ·α,α,α-trifluorotoluene, 1, is characterized by ¹⁹F and ¹³C solid-state NMR spectroscopy. Whereas the ¹³C NMR spectra are easily interpreted in the context of earlier work on similar host–guest compounds, the ¹⁹F NMR spectra of solid 1 are, initially, more difficult to understand. The ¹⁹F{¹H} NMR spectrum obtained under cross-polarization and magic-angle spinning conditions shows a single isotropic resonance with a significant spinning sideband manifold. The static ¹⁹F{¹H} CP NMR spectrum consists of a powder pattern dominated by the contributions of the anisotropic chemical shift and the homonuclear dipolar interactions. The ¹⁹F MREV-8 experiment, which minimizes the ¹⁹F–¹⁹F dipolar contribution, helps to identify the chemical shift contribution as an axial lineshape. The full static ¹⁹F{¹H} CP NMR spectrum is analysed using subspectral analysis and subsequently simulated as a function of the ¹⁹F–¹⁹F internuclear distance (D_FF = 2.25 ± 0.01 Å) of the rapidly rotating CF₃ group without including contributions from additional libration motions and the anisotropy in the scalar tensor. The shielding span is found to be 56 ppm. The width of the centerband in the ¹⁹F{¹H} sample-spinning CP NMR spectrum is very sensitive to the angle between the rotor and the magnetic field. Compound 1 is thus an attractive standard for setting the magic angle for NMR probes containing a fluorine channel with a proton-decoupling facility.     

Low-E probe for (19)F-(1)H NMR of dilute biological solids

Sample heating induced by radio frequency (RF) irradiation presents a significant challenge to solid state NMR experiments in proteins and other biological systems, causing the sample to dehydrate which may result in distorted spectra and a damaged sample. In this work we describe a large volume, low-E (19)F-(1)H solid state NMR probe, which we developed for the 2D (19)F CPMG studies of dilute membrane proteins in a static and electrically lossy environment at 600MHz field. In (19)FCPMG and related multi-pulse (19)F-(1)H experiments the sample is heated by the conservative electric fields E produced in the sample coil at both (19)F and (1)H frequencies. Instead of using a traditional sample solenoid, our low-E (19)F-(1)H probe utilizes two orthogonal loop-gap resonators in order to minimize the conservative electric fields responsible for sample heating. Absence of the wavelength effects in loop-gap resonators results in homogeneous RF fields and enables the study of large sample volumes, an important feature for the dilute protein preparations. The orthogonal resonators also provide intrinsic isolation between the (19)F and (1)H channels, which is another major challenge for the (19)F-(1)H circuits where Larmor frequencies are only 6% apart. We detail steps to reduce (19)F background signals from the probe, which included careful choice of capacitor lubricants and manufacture of custom non-fluorinated coaxial cables. Application of the probe for two-dimensional (19)F CPMG spectroscopy in oriented lipid membranes is demonstrated with Flufenamic acid (FFA), a non-steroidal anti-inflammatory drug.     

Conformational Analysis of (S)-4,5,6,7-Tetrahydro-5-Methylimidazo [4,5,1-jk][1,4]-Benzodiazepin-2(1H)-one (R78362)

The solution and solid state conformation of (S)-4,5,6,7-tetrahydro-5-methylimidazo [4,5,1-jk][1,4]-benzodiazepin-2(1H)-one (R78362) have been investigated by low temperature NMR and x-ray diffraction studies. The ¹H NMR spectrum of R78362 shows no evidence of the presence of multiple conformers in the temperature range 340K - 177K. Molecular mechanisms and semiempirical molecular orbital calculations suggest that the nitrogen and ring inversion barriers of R78362 are small and thus a time-averaged ¹H NMR spectrum is probably occurring at 298K. The x-ray diffraction data indicated that there were two independent molecules in the asymmetric unit of the crystal. The two molecules had similar conformations with the benzoimidazole ring being planar and the diazepine ring in a “half-chair” conformation.     

Identification of fluorine sites at the surface of fluorinated gamma-alumina by two-dimensional MAS NMR

By means of 27Al triple quantum Magic-Angle Spinning Nuclear Magnetic Resonance (3QMAS NMR) and 27Al[19F] WISE MAS NMR, we were able to detect three different Al-F sites on the surface of fluorinated gamma-alumina. Three 19F resonances at 9, 20, and 33 ppm (from C6F6) correlated to 27Al resonances in the octahedral range. While the positions of the maxima in the 27Al dimension were ill-defined due to the inherently low efficiency of the 27Al[19F] CPMAS process, the center of gravity of the lines shifted significantly upfield in that dimension with increasing wt.% F. Tentatively, these three resonances were assigned to (VI)Al(O(6-n)Fn) (n = 1, 2, 3) environments on the F/gamma-Al2O3 surface. At F contents above levels corresponding to the full fluorination of the gamma-Al2O3 surface, neoformation of an AlF3 x 3H2O phase was also evidenced with an 19F resonance at -8 ppm and with an 27Al resonance at -17 ppm.     

Cross-correlated (19)F relaxation measurements for the study of fluorinated ligand-receptor interactions.

Fluorine is often used in drug-design efforts to enhance the pharmacokinetic properties of biologically active compounds. Additionally fluorine nuclei ((19)F) have properties that are well suited to current pharmaceutical NMR screening programs. Together, these considerations have motivated our interest in the utility of fluorine relaxation parameters to study ligand-receptor interactions. Here, we investigate the potential for cross-correlated relaxation effects between the (19)F anisotropic chemical-shift and (19)F-(1)H dipole-dipole relaxation mechanisms to help pinpoint and quantify exchange processes. Methods are proposed and demonstrated in which the magnitude ratio of the transverse cross-correlation rate constant eta(xy) and the fluorine transverse relaxation rate constant, R(2), help estimate the exchange rate constant for ligand-binding equilibria. These exchange rate constants provide estimates of the ligand dissociation rate constants k(off) and can thus provide a means for rank-ordering the binding affinities of ligands identified in pharmaceutical screens.     

H MAS and H[Na] double resonance NMR studies on the modification of surface hydroxyl groups of γ-alumina by sodium

The modification of surface hydroxyl groups with sodium in a series of Na₂CO₃-γ-Al₂O₃ catalysts was investigated as a function of both the Na₂CO₃ loading and the calcination temperature by means of ¹H magic angle spinning (MAS) and ¹H[²³Na] spin-echo double resonance NMR techniques. The ¹H NMR experiments revealed that sodium ions are homogeneously distributed over the alumina surface and closely coordinated with the surface hydroxyl groups. In the catalysts calcined at 250 °C, the acidic hydroxyl groups (with a chemical shift of 2.0 ppm) are preferentially associated with sodium ions at low Na₂CO₃ coverages (5 and 10%), while both the acidic and the basic (0 ppm) hydroxyl groups are accessible for sodium ions at high coverages (15 and 20%). The coordination causes a low-field shift of about 2 ppm in the ¹H MAS spectra, and a broad signal at 4.5 ppm appears. It is interesting that the 4.5 ppm signal is completely suppressed in the ¹H[²³Na] MAS experiments, providing direct evidence that a strong interaction exists between adsorbed sodium ions and the surface hydroxyl groups. Increasing the calcination temperature to 450 °C results in preferential removal of the acidic hydroxyl groups, and only the most basic hydroxyl groups remain when the calcination temperature is raised to 600 °C. This is attributed to the formation of the coordinated species which enhances the acidity of the surface hydroxyl groups and prompts their dehydroxylation, especially at high calcination temperature. Correlation of the ¹H MAS NMR results and catalytic activity measurements indicates that the basic hydroxyl groups are essential for the carbonyl sulfide hydrolysis reaction.     

Very high-resolution 1H MAS NMR of a natural polymeric material

The use of ultrafast magic angle spinning (> 30 kHz) in tandem with delayed echo acquisition is shown to yield very high-resolution lH MAS NMR spectra of complex natural organic materials. For the first time, very high-resolution 1H MAS NMR spectra are reported for cork and wood components, two natural materials with great economic importance. The effect of the spinning rate on the 1H NMR spectra was evaluated with single-pulse acquisition and delayed-echo acquisition. The delayed-echo acquisition spectra presented linewidths as sharp as 67 and 25 Hz. The narrow peaks, characterised by proton spin-spin and spin-lattice relaxation, were assigned to the isotropic chemical shifts and the general spectral features were shown to correlate with the sample chemical structure. The tentative assignments of cork 1H MAS NMR signals were presented.     

Solid state 19F NMR parameters of fluorine-labeled amino acids. Part II: aliphatic substituents

A representative set of amino acids with aliphatic 19F-labels has been characterized here, following up our previous compilation of NMR parameters for single 19F-substituents on aromatic side chains. Their isotropic chemical shifts, chemical shift tensor parameters, intra-molecular 19F dipole-dipole couplings and temperature-dependent T1 and T2 relaxation times were determined by solid state NMR on twelve polycrystalline amino acid samples, and the corresponding isotropic 19F chemical shifts and scalar couplings were obtained in solution. Of particular interest are amino acids carrying a trifluoromethyl-group, because not only the 19F chemical shift but also the intra-CF3 homonuclear dipolar coupling can be used for structural studies of 19F-labeled peptides and proteins. The CF3-groups are further compared with CH2F-, CD2F-, and CD3-groups, using both 19F and 2H NMR to describe their motional behavior and to examine the respective linebroadening effects of the protonated and deuterated neighbors. We have also characterized two unnatural amino acids in which a CF3-label is rigidly connected to the backbone by a phenyl or bicyclopentyl moiety, and which are particularly well suited for structure analysis of membrane-bound polypeptides. The 19F NMR parameters of the polycrystalline amino acids are compared with data from the correspondingly labeled side chains in synthetic peptides.     

1H, 13C and 19F NMR Studies of the Diels-Alder Adduct of p-Fluoranil with Phencyclone. II. Hindered Phenyl Rotations and Anisotropic Effects in a Model Compound for Drugs

Abstract

The Diels-Alder adduct of phencyclone, compound 1, with p-fluoranil, compound 3, has been prepared in refluxing toluene. The adduct, compound 2, has been examined by ¹H, ¹³C and ¹⁹F NMR spectroscopy at 300, 75 and 282 MHz, respectively. At ambient temperature, the unsubstituted bridgehead phenyl groups in adduct 2 are found to exhibit hindered rotation, resulting in slow exchange limit (SEL) ¹H NMR spectra. Full aryl proton assignments are made based on 1D and 2D (COSY45) NMR. The ¹⁹F NMR (proton coupled) reveals one of the two ¹⁹F signals to be a triplet. This resonance collapses to a singlet in the proton decoupled ¹⁹F spectrum, implying an unexpected long range ¹H-¹⁹F coupling. For the ¹³C NMR spectrum, tentative assignments are presented. Data for compound 2 as a model compound for drugs are discussed in terms of the hindered aryl rotation and evidence of magnetic anisotrppic effects.     

Intermolecular (1)H[(19)F] NOEs in studies of fluoroalcohol-induced conformations of peptides and proteins

Mixtures of fluorinated alcohols and water can selectively stabilize certain secondary structures of peptides and proteins. Such mixtures may also be of use in solubilizing hydrophobic or membrane-bound proteins. We show that intermolecular dipolar interactions between the fluorine nuclei of such solvents and the protons of a dissolved protein lead to readily detected (1)H[(19)F] nuclear Overhauser effects. These NOEs can potentially provide information about solvent exposure of particular groups as well as indicate the formation of long-lived fluoroalcohol-solute complexes. Results obtained with HEW lysozyme in solutions containing trifluoroethanol illustrate these possibilities.     

F/Si distance determination in fluoride-containing octadecasil by Hartmann–Hahn cross-polarization under fast magic-angle spinning

¹⁹F/²⁹Si Hartmann–Hahn continuous wave cross-polarization (CP) has been applied under fast magic-angle spinning (MAS) to a powder sample of octadecasil. Strong oscillations occur during CP on a sideband matching condition between the isolated ²⁹Si–¹⁹F spin pairs formed by the silicons in the D4R units and the fluoride anions. The magnitude of the dipolar coupling constant was deduced directly from the line-splitting between the intense singularities of the Pake-like patterns obtained by Fourier transformation of the oscillatory polarization transfer. The corresponding Si–F internuclear distance, r=2.62±0.05 Å, is found to be in very good agreement with the X-ray crystal structure and the value of 2.69±0.04 Å recently reported from rotational echo double resonance (REDOR) and transferred echo double resonance (TEDOR) nuclear magnetic resonance (NMR) experiments. Furthermore, the CP technique is still reliable under fast MAS where both REDOR and TEDOR sequences suffer from severe artefacts due to finite pulse lengths. In octadecasil, a spinning frequency of 14 kHz is shown to be necessary for an effective suppression of ¹⁹F–¹⁹F spin diffusion. The influences of experimental missettings and radiofrequency (RF) field inhomogeneity are taken into account.