Nicolas Giraud

Nuclear Magnetic Resonance Using a Spatial Frequency Encoding: Application to J-Edited Spectroscopy along the Sample

Nuclear magnetic resonance provides chemists with a unique insight into molecular structure and dynamics over very wide spatial and time ranges. Unfortunately, in most of the recent applications of NMR, the size of the spin networks that are investigated often remain challenging, even for spectrometers that operate at very high fields, since the overlap of signals, as well as the complexity of their multiplet structure, overwhelm the resolution of the NMR spectra. In this context, considerable methodological developments have been devoted to the design of multidimensional experiments that aim at simplifying the analytical process. For instance, COSY and J-resolved experiments illustrate the extent to which chemical shift and scalar coupling information can be separated, thus allowing a rapid assignment of each proton site, and then a measurement of H–H couplings. Unfortunately, as hard pulses are used in such experiments, they often give overcrowded spectra as soon as the size of the molecular system increases. One way to reduce the number of interactions that contribute to the structure of NMR spectra involves the use of semiselective pulses, which allow use of coherences that involve a single spin nucleus; for instance, selective refocusing experiments have opened the way to a site-specific measurement of each interaction from unresolved spectra. However, in this latter case, up to n(n 1)/2 selective experiments need to be recorded in order to extract all the couplings out of a network of n fully coupled spins, thus requiring a significantly longer experimental time. In order to overcome the above-mentioned difficulty, we proposed to run different selective experiments on different parts of the sample, that is, we carried out a parallel acquisition of different experiments using a single-receivercoil system. For this purpose, we created a spatial frequency encoding of the sample, in a manner similar to magnetic resonance imaging techniques. Application of the principle to menthol is shown in Figure 1. We simultaneously applied a semiselective p/2 pulse and a pulsed field gradient along the NMR tube (i.e., a z gradient). This irradiation scheme can be described as an rf field with a position-dependent offset, and results in the polarization of spin nuclei with different resonance frequencies in different cross sections of the sample. Although the same signals are observed on the gradient-encoded (Figure 1a) and on the broadband excitation spectra of menthol (Figure 1b), the gradient-encoded experiment should actually be compared to the semiselective spectrum (Figure 1c), where one single proton nucleus is excited by the application of a soft pulse over the whole sample. Indeed, the spatial encoding allows this selective experiment to be carried out on each of the proton spins in separate “slices”. We note that each line of the resulting spatially encoded spectrum arises from a restricted cross section of the sample: if the spatial frequency sweep that is induced by the pulsedfield gradient is adjusted so that the width of the spectrum (ca. 3 ppm on menthol) exactly matches the height of the sample, then each signal (whose width can be roughly Figure 1. a) Proton spectrum of l-( )-menthol. The spectrum was acquired using a semiselective pulse (H channel) applied together with a pulsed z-field gradient (PFG channel). An NMR tube is drawn along the spectrum in order to illustrate the spatial encoding of proton lines according to their resonance frequencies. b) Broadband excitation spectrum, recorded on the same compound. c) Spectrum that results from the application of the semiselective pulse used in (a), at the resonance frequency of proton H. The corresponding pulse sequence is shown for each spectrum).

Application of a 1H δ‐resolved 2D NMR experiment to the visualization of enantiomers in chiral environment, using sample spatial encoding and selective echoes

We present the application of a 2D broadband homodecoupled proton NMR experiment to the visualization of enantiomers. In a chiral environment, the existence of diastereoisomeric intermolecular interactions can yield—generally slight—variations of proton chemical shifts from one enantiomer to another. We show that this approach, which relies on a spatial encoding of the NMR sample, is particularly well suited to the analysis of enantiomeric mixtures, since it allows, within one single 2D experiment, to detect subtle chemical shift differences between enantiomers, even in the presence of several couplings. This sequence, which uses semiselective radio‐frequency (rf) pulses combined to a z‐field gradient pulse, produces different selective echoes in various parts of the sample. The resulting homonuclear decoupling provides an original δ‐resolved spectrum along the diagonal of the 2D map where it becomes possible to probe the chiral differentiation process through every proton site where the resulting variation in the chemical shift is detectable. We discuss the advantages and drawbacks of this approach, regarding other experiments which provide homodecoupled proton spectra. This methodology is applied to the observation of enantiomers of (1) ( ± )2‐methyl‐isoborneol coordinated to europium (III) tris[3‐(trifluoromethyl‐hydroxymethylene)‐(+)‐camphorate] in isotropic solution, and (2) ( ± )3‐butyn‐2‐ol dissolved in a chiral liquid‐crystal solvent, in order to show the robustness of this pulse sequence for a wide range of chiral samples. Copyright

Spin–spin coupling edition in chiral liquid crystal NMR solvent

The application of the G-SERFph pulse sequence is presented on enantiomeric mixtures dissolved in a chiral liquid crystal. It aims at editing, within one single 2D spectrum, every proton coupling which is experienced by a given proton site in the molecule, and leads to real phased T-edited spectroscopy (T=J+2D). This NMR experiment is based on the combination of homonuclear semi-selective refocusing techniques with a spatial frequency encoding of the sample. This approach, which consists in handling selectively each coupling in separate cross sections of the sample, is applied to the visualization of enantiomers dissolved in a chiral liquid crystalline phase. Advantages and limits of this methodology are widely discussed.

Simultaneous determination of all forms of biopterin and neopterin in cerebrospinal fluid.

In humans, genetic defects of the synthesis or regeneration of tetrahydrobiopterin (BH4), an essential cofactor in hydroxylation reactions, are associated with severe neurological disorders. The diagnosis of these conditions relies on the determination of BH4, dihydrobiopterin (BH2), and dihydroneopterin (NH2) in cerebrospinal fluid (CSF). As MS/MS is less sensitive than fluorescence detection (FD) for this purpose, the most widely used method since 1980 involves two HPLC runs including two differential off-line chemical oxidation procedures aiming to transform the reduced pterins into their fully oxidized fluorescent counterparts, biopterin (B) and neopterin (N). However, this tedious and time-consuming two-step indirect method underestimates BH4, BH2, and NH2 concentrations. Direct quantification of BH4 is essential for studying its metabolism and for monitoring the efficacy of BH4 supplementation in patients with genetic defects. Here we describe a single step method to simultaneously measure BH4, BH2, B, NH2, and N in CSF by HPLC coupled to FD after postcolumn coulometric oxidation. All target pterins were quantified in CSF with a small volume (100 μL), and a single filtration step for sample preparation and analysis. As compared to the most widely used method in more than 100 CSF samples, this new assay is the easiest route for accurately determining in a single run BH4, BH2, and NH2 in CSF in deficit situations as well as for monitoring the efficacy of the treatment.

Improvements to selective refocusing phased (SERFph) experiments

Selective refocusing experiments are very powerful for extracting proton-proton couplings one by one. However we demonstrate in the present work that various spectral artefacts are produced by the initial sequence and we show that the combined addition of a refocusing pi pulse and a zero-quantum filter greatly improves the experimental sensitivity, and moreover leads to observation of pure absorption lineshapes in the resulting phased 2D spectrum. These developments are applied to the differentiation of enantiomers dissolved in a chiral liquid crystal.

Magnetic field dependence of spatial frequency encoding NMR as probed on an oligosaccharide

The magnetic field dependence of spatial frequency encoding NMR techniques is addressed through a detailed analysis of 1H NMR spectra acquired under spatial frequency encoding on an oligomeric saccharide sample. In particular, the influence of the strength of the static magnetic field on spectral and spatial resolutions that are key features of this method is investigated. For this purpose, we report the acquisition of correlation experiments implementing broadband homodecoupling or J‐edited spin evolutions, and we discuss the resolution enhancements that are provided by these techniques at two different magnetic fields. We show that performing these experiments at higher field improves the performance of high resolution NMR techniques based on a spatial frequency encoding. The significant resolution enhancements observed on the correlation spectra acquired at very high field make them valuable analytical tools that are suitable for the assignment of 1H chemical shifts and scalar couplings in molecules with highly crowded spectrum such as carbohydrates. Copyright

Exploration of the supramolecular interactions involving tris-dipicolinate lanthanide complexes in protein crystals by a combined biostructural, computational and NMR study

Incorporating in a non-covalent manner lanthanide derivatives into protein crystals has shown to be of prime interest for X-ray crystallography, insofar as these versatile compounds can co-crystallize with proteins through supramolecular interactions, in addition to being strong anomalous scatterers for anomalous-based diffraction techniques. In this paper, the selective affinity of tris-dipicolinate lanthanide complexes for cationic amino-acid residues is explored, using a panel of experimental (X-ray diffraction, NMR titration) and theoretical methods that provides access to an accurate description of the interaction process.

A multidimensional approach to the analysis of chemical shift titration experiments in the frame of a multiple reaction scheme

We present a method for fitting curves acquired by chemical shift titration experiments, in the frame of a three‐step complexation mechanism. To that end, we have implemented a fitting procedure, based on a nonlinear least squares fitting method, that determines the best fitting curve using a “coarse grid search” approach and provides distributions for the different parameters of the complexation model that are compatible with the experimental precision. The resulting analysis protocol is first described and validated on a theoretical data set. We show its ability to converge to the true parameter values of the simulated reaction scheme and to evaluate complexation constants together with multidimensional uncertainties. Then, we apply this protocol to the study of the supramolecular interactions, in aqueous solution, between a lanthanide complex and three different model molecules, using NMR titration experiments. We show that within the uncertainty that can be evaluated from the parameter distributions generated during our analysis, the affinities between the lanthanide derivative and each model molecule can be discriminated, and we propose values for the corresponding thermodynamic constants. Copyright

Combining pure shift and J-edited spectroscopies: A strategy for extracting chemical shifts and scalar couplings from highly crowded proton spectra of oligomeric saccharides

We report the application of pure shift and J‐edited nuclear magnetic resonance spectroscopies to the structural analysis of a protected maltotrioside synthetic intermediate whose crowded 1H spectrum displays highly crowded regions. The analytical strategy is based on the implementation of J‐edited and TOCSY experiments whose resolution is optimized by the use of broadband homonuclear decoupling and selective refocusing techniques, to assign and measure chemical shifts and homonuclear scalar couplings with high accuracy. The resulting data show a high level of complementarity, providing a detailed insight into each subunit of this oligomeric saccharide, even for proton sites whose nuclear magnetic resonance signals strongly overlap. This approach allowed for fully assigning proton chemical shifts and extracting 80% of the 3JHH couplings that are in excellent agreement with those expected for D‐gluco‐pyranosyl units in 4C1 conformations.

Paramagnetic DOSY: An Accurate Tool for the Analysis of the Supramolecular Interactions between Lanthanide Complexes and Proteins

Diffusion ordered NMR is implemented to determine accurately the mobility of paramagnetic tris-dipicolinate lanthanide complexes that are versatile probes of protein structure. It is shown that diffusion coefficient ratios can be measured with an accuracy of 1 % using a standard BPPLED pulse sequence, which allows for observing significant, though weak, variations when different species are interacting with the paramagnetic compound. We demonstrate that this approach is complementary to classical chemical shift titration experiments, and that it can be applied successfully to probe the supramolecular dynamic interactions between lanthanide complexes and small molecules on the one hand, or to determine rapidly their affinity for a targeted protein.