15N-15N spin-spin coupling constants through intermolecular hydrogen bonds in the solid state.

A 2hJNN intermolecular spin-spin coupling constant (SSCC) of 10.2±0.4 Hz has been measured for the powdered tetrachlorogallate salt of pyridinium solvated by pyridine (pyridine-H+⋯pyridine cation 3). Density Functional Theory (DFT) calculations at the B3LYP/6-311++G(d,p) level reproduced this value and two others reported in the literature for 2hJ intermolecular SSCCs, which were measured for complexes in solution.

Complete (1)H resonance assignment of beta-maltose from (1)H-(1)H DQ-SQ CRAMPS and (1)H (DQ-DUMBO)-(13)C SQ refocused INEPT 2D solid-state NMR spectra and first principles GIPAW calculations.

A disaccharide is a challenging case for high-resolution (1)H solid-state NMR because of the 24 distinct protons (14 aliphatic and 10 OH) having (1)H chemical shifts that all fall within a narrow range of approximately 3 to 7 ppm. High-resolution (1)H (500 MHz) double-quantum (DQ) combined rotation and multiple pulse sequence (CRAMPS) solid-state NMR spectra of beta-maltose monohydrate are presented. (1)H-(1)H DQ-SQ CRAMPS spectra are presented together with (1)H (DQ)-(13)C correlation spectra obtained with a new pulse sequence that correlates a high-resolution (1)H DQ dimension with a (13)C single quantum (SQ) dimension using the refocused INEPT pulse-sequence element to transfer magnetization via one-bond (13)C-(1)H J couplings. Compared to the observation of only a single broad peak in a (1)H DQ spectrum recorded at 30 kHz magic-angle spinning (MAS), the use of DUMBO (1)H homonuclear decoupling in the (1)H DQ CRAMPS experiment allows the resolution of distinct DQ correlation peaks which, in combination with first-principles chemical shift calculations based on the GIPAW (Gauge Including Projector Augmented Waves) plane-wave pseudopotential approach, enables the assignment of the (1)H resonances to the 24 distinct protons. We believe this to be the first experimental solid-state NMR determination of the hydroxyl OH (1)H chemical shifts for a simple sugar. Variable-temperature (1)H-(1)H DQ CRAMPS spectra reveal small increases in the (1)H chemical shifts of the OH resonances upon decreasing the temperature from 348 K to 248 K.

Powder crystallography by combined crystal structure prediction and high-resolution 1H solid-state NMR spectroscopy.

A fast method for crystal structure determination using crystal structure prediction and solid-state (1)H NMR is presented. This technique does not need any prior knowledge except the chemical formula; resonance assignment is not necessary. Starting from an ensemble of predicted crystal structures for powdered thymol, comparison between experimental and calculated (1)H solid-state isotropic NMR chemical shifts is sufficient to determine which predicted structure corresponds to the powder under study. The same approach using proton-proton spin-diffusion data is successful and can be used for cross-validation.

Statistical recoupling prior to significance testing in nuclear magnetic resonance based metabonomics.

Significance testing is a crucial step in metabolic biomarker recovery from the metabolome-wide latent variables computed by multivariate statistical analysis. In this study we propose an algorithm based on the landscape of the covariance/correlation ratio of consecutive variables along the chemical shift axis to restore, prior to significance testing, the spectral dependency and recouple variables in clusters which correspond to physical, chemical, and biological entities: statistical recoupling of variables (SRV). Variables are associated into a series of clusters, which are then considered as individual objects for the control of the false discovery rate. Compared to classical procedures, it is found that SRV allows efficient recovery of statistically significant metabolic variables. The proposed SRV method when associated with the Benjamini-Yekutieli correction retains a low level of significant variables in the noise areas of the nuclear magnetic resonance (NMR) spectrum, close to that observed using the conservative Bonferroni correction (false positive rate), while also allowing successful identification of statistically significant metabolic NMR signals in cases where the classical procedures of Benjamini-Yekutieli and Benjamini-Hochberg (false discovery rate) fail. This procedure improves the interpretability of latent variables for metabolic biomarker recovery.

Powder NMR crystallography of thymol.

A protocol for the structure determination of powdered solids at natural abundance by NMR is presented and illustrated for the case of the small drug molecule thymol. The procedure uses proton spin-diffusion data from two-dimensional NMR experiments in combination with periodic DFT refinements incorporating (1)H and (13)C NMR chemical shifts. For thymol, the method yields a crystal structure for the powdered sample, which differs by an atomic root-mean-square-deviation (all atoms except methyl group protons) of only 0.07 A from the single crystal X-ray diffraction structure with DFT-optimized proton positions.

Metabolic profiling strategy of Caenorhabditis elegans by whole-organism nuclear magnetic resonance.

In this study, we present a methodology for metabotyping of C. elegans using 1H high resolution magic angle spinning (HRMAS) whole-organism nuclear magnetic resonance (NMR). We demonstrate and characterize the robustness of our metabolic phenotyping method, discriminating wild-type N2 from mutant sod-1(tm776) animals, with the latter being an otherwise silent mutation, and we identify and quantify several confounding effects to establish guidelines to ensure optimal quality of the raw data across time and space. We monitor the sample stability under experimental conditions and examine variations arising from effects that can potentially confuse the biological interpretation or prevent the automation of the protocol, including sample culture (breeding of the worms by two biologists), sample preparation (freezing), NMR acquisition (acquisition by different spectroscopists, acquisition in different facilities), and the effect of the age of the animals. When working with intact model organisms, some of these exogenous effects are shown to be significant and therefore require control through experimental design and sample randomization.

Methyl proton contacts obtained using heteronuclear through-bond transfers in solid-state NMR spectroscopy.

A two-dimensional proton-mediated carbon-carbon correlation experiment that relies on through-bond heteronuclear magnetization transfers is demonstrated in the context of solid-state NMR of proteins. This new experiment, dubbed J-CHHC by analogy to the previously developed dipolar CHHC techniques, is shown to provide selective and sensitive correlations in the methyl region of 2D spectra of crystalline organic compounds. The method is then demonstrated on a microcrystalline sample of the dimeric protein Crh (2 x 10.4 kDa). A total of 34 new proton-proton contacts involving side-chain methyl groups were observed in the J-CHHC spectrum, which had not been observed with the conventional experiment. The contacts were then used as additional distance restraints for the 3D structure determination of this microcrystalline protein. Upon addition of these new distance restraints, which are in large part located in the hydrophobic core of the protein, the root-mean-square deviation with respect to the X-ray structure of the backbone atom coordinates of the 10 best conformers of the new ensemble of structures is reduced from 1.8 to 1.1 A.

Metabotyping of Caenorhabditis elegans reveals latent phenotypes.

Assigning functions to every gene in a living organism is the next challenge for functional genomics. In fact, 85-90% of the 19,000 genes of the nematode Caenorhabditis elegans genome do not produce any visible phenotype when inactivated, which hampers determining their function, especially when they do not belong to previously characterized gene families. We used (1)H high-resolution magic angle spinning NMR spectroscopy ((1)H HRMAS-NMR) to reveal the latent phenotype associated to superoxide dismutase (sod-1) and catalase (ctl-1) C. elegans mutations, both involved in the elimination of radical oxidative species. These two silent mutations are significantly discriminated from the wild-type strain and from each other. We identify a metabotype significantly associated with these mutations involving a general reduction of fatty acyl resonances from triglycerides, unsaturated lipids being known targets of free radicals. This work opens up perspectives for the use of (1)H HRMAS-NMR as a molecular phenotyping device for model organisms. Because it is amenable to high throughput and is shown to be highly informative, this approach may rapidly lead to a functional and integrated metabonomic mapping of the C. elegans genome at the systems biology level.

On the orientational dependence of resolution in 1H solid-state NMR, and its role in MAS, CRAMPS and delayed-acquisition experiments.

Numerical simulations and experiments are used to show that the spin dynamics of the dipolar-coupled networks in solids is often strongly dependent on crystallite orientation. In particular, different rates of dephasing of the magnetisation mean that NMR signals obtained at longer dephasing times are dominated by orientations in which the local dipolar coupling strength is relatively weak. This often leads to a distinct improvement in spectral resolution as the dephasing time is increased. The effects are particularly noticeable under magic-angle spinning (MAS), but are also observed when homonuclear decoupling is used to reduce the rate of dipolar dephasing. Numerical simulation is seen to be a powerful and easily used tool for understanding the behaviour of solid-state NMR experiments involving dipolar-coupled networks. The implications for solid-state NMR spectra of abundant spins acquired under MAS and homonuclear decoupling are discussed, as well as insights provided into the performance of 'delayed-acquisition' and 'constant-time' experiments.

Two-photon microscopy and spectroscopy of lanthanide bioprobes.

The linear and non-linear photophysical properties of tris-dipicolinate europium and terbium complexes (absorption, emission, lifetime, luminescence induced by two-photon absorption) are studied in the crystalline state as well as in protein derivative crystals and compared to those in solution. Upon laser irradiation at 532 nm, luminescence of terbium is induced by a two-photon antenna effect, whereas luminescence of europium results from one-photon absorption in forbidden f-f transitions. Finally, linear and two-photon microscopy imaging experiments on biological and bio-inspired crystals are performed. These first proof-of-concept experiments open the way for the development of time-resolved non-linear microscopy that should combine the advantages of lanthanide luminescence (long lifetime, sharp emission bands, insensitivity to oxygen) with those of confocal biphotonic excitation (near-IR excitation, 3D resolution and reduced photodamage).

Assigning powders to crystal structures by high-resolution (1)H-(1)H double quantum and (1)H-(13)C J-INEPT solid-state NMR spectroscopy and first principles computation. A case study of penicillin G.

We show how powder samples at natural isotopic abundance can be assigned to crystal structures by using high-resolution proton and carbon-13 solid-state NMR spectra in combination with first principles calculations. Homonuclear proton double-quantum spectra in combination with through-bond proton-carbon HSQC spectra are used to assign the NMR spectra. We then show that the proton chemical shifts can be included in the process of assigning the spectra to a crystal structure using first principles calculations. The method is demonstrated on the K salt of penicillin G.

Molecular structure determination in powders by NMR crystallography from proton spin diffusion.

The inability to determine molecular structures from powdered samples is a key barrier to progress in many areas of molecular and materials science. We report an approach to structure determination that combines molecular modeling with experimental spin diffusion data obtained from the high-resolution solid-state nuclear magnetic resonance of protons, and which allows the determination of the three-dimensional structure of an organic compound, in powder form and at natural isotopic abundance.

Proton to carbon-13 INEPT in solid-state NMR spectroscopy.

A refocused INEPT through-bond coherence transfer technique is demonstrated for NMR of rigid organic solids and is shown to provide a valuable building block for the development of NMR correlation experiments in biological solids. The use of efficient proton homonuclear dipolar decoupling in combination with a direct spectral optimization procedure provides minimization of the transverse dephasing of coherences and leads to very efficient through-bond (1)H-(13)C INEPT transfer for crystalline organic compounds. Application of this technique to 2D heteronuclear correlation spectroscopy leads to up to a factor of 3 increase in sensitivity for a carbon-13 enriched sample in comparison to standard through-bond experiments and provides excellent selectivity for one-bond transfer. The method is demonstrated on a microcrystalline sample of the protein Crh (2 x 10.4 kDa).

Powder crystallography by proton solid-state NMR spectroscopy.

The investigation of 1H-1H spin-diffusion build-up curves using a rate matrix analysis approach shows that high-resolution magic angle spinning NMR of protons, applied to powdered organic compounds, provides a method to probe crystalline arrangements. The comparison between experimental 1H data and simulation is shown to depend strongly on the parameters of the crystal structure, for example on the unit cell parameters or the orientation of the molecule in the unit cell, and those parameters are experimentally determined for a model organic compound.

Probing proton-proton proximities in the solid state: high-resolution two-dimensional 1H-1H double-quantum CRAMPS NMR spectroscopy.

A new 1H DQ (double-quantum) CRAMPS (combined rotation and multiple-pulse sequence) solid-state nuclear magnetic resonance experiment incorporating DUMBO homonuclear 1H dipolar decoupling is presented. The major resolution enhancement enables DQ peaks corresponding to all 22 close (<3.5 A) proton-proton proximities in the dipeptide beta-AspAla to be observed. In particular, the DQ CRAMPS spectrum provides access to the alkyl region of the spectrum and yields a clear assignment of the two CH and two diastereotopic CH2 proton resonances.

Characterization of heteronuclear decoupling through proton spin dynamics in solid-state nuclear magnetic resonance spectroscopy.

The work presented here aims at understanding the performance of phase modulated heteronuclear decoupling sequences such as Cosine Modulation or Two Pulse Phase Modulation. To that end we provide an analytical description of the intrinsic behavior of Cosine Modulation decoupling with respect to radio-frequency-inhomogeneity and the proton-proton dipolar coupling network. We discover through a Modulation Frame average Hamiltonian analysis that best decoupling is obtained under conditions where the heteronuclear interactions are removed but notably where homonuclear couplings are recoupled at a homonuclear Rotary Resonance (HORROR) condition in the Modulation Frame. These conclusions are supported by extensive experimental investigations, and notably through the introduction of proton nutation experiments to characterize spin dynamics in solids under decoupling conditions. The theoretical framework presented in this paper allows the prediction of the optimum parameters for a given set of experimental conditions.

Experimental aspects of proton NMR spectroscopy in solids using phase-modulated homonuclear dipolar decoupling.

In this paper we demonstrate experimentally that the continuously phase-modulated homonuclear decoupling sequence DUMBO-1 is suitable for high-resolution proton NMR spectroscopy of rigid solids. Over a wide range of experimental conditions, we show on the model sample L-alanine as well as on small peptides that proton linewidths of less than 0.5 ppm can be obtained under DUMBO-1 decoupling. In particular the DUMBO-1 sequence yields well resolved proton spectra both at slow and fast MAS. The DUMBO-1 decoupling scheme can in principle be inserted in any multi-nuclear or multi-dimensional solid-state NMR experiment which requires a high-resolution 1H dimension. An example is provided with the 13C-1H MAS-J-HMQC experiment.

Correlation of fast and slow chemical shift spinning sideband patterns under fast magic-angle spinning.

A new two-dimensional solid-state NMR experiment, which correlates slow and fast chemical shift anisotropy sideband patterns is proposed. The experiment, dubbed ROSES, is performed under fast magic-angle spinning and leads to an isotropic spectrum in the directly detected omega(2) dimension. In the evolution dimension omega(1), the isotropic chemical shift is reduced by a factor S, and spinning sidebands are observed spaced by a scaled effective spinning speed omega(R)/S. These spinning sidebands patterns are not identical to those observed with standard slow magic-angle spinning experiments. Chemical shift anisotropy parameters can be accurately extracted with standard methods from these spinning sideband patterns. The experiment is demonstrated with carbon-13 experiments on powdered samples of a dipeptide and a cyclic undecapeptide, cyclosporin-A.