(14)N NMR and two-dimensional suspension (1)H and (13)C HRMAS NMR spectroscopy of ionic liquids immobilized on silica

A variety of popular ionic liquids have been synthesized and characterized, including by optimized (14)N NMR spectroscopy of the neat and dissolved ionic liquids. Ionic liquids incorporating Si(OEt)(3) groups have been immobilized on silica in a well-defined manner with the imidazolium moiety remaining intact. This has been proved by optimized one- and two-dimensional (1)H and (13)C HRMAS NMR spectroscopy of the materials suspended in suitable solvents.     

Determination of long-range distances and dynamic order parameters by dipolar recoupling in high-resolution magic-angle spinning NMR spectroscopy

By introducing dipolar recoupling methods to high-resolution magic-angle spinning (HRMAS) NMR spectroscopy, a class of experiments has been delevoped that allows the measurement of residual dipole-dipole couplings of approximately 1 Hz in weakly immobilized molecules. Using homonuclear 1H-1H recoupling, distances of up to approximately 8 A can be selectively determined, while heteronuclear 1H-13C recoupling provides access to dynamic order parameters of individual molecular segments on the order of approximately 10-3. The experiments are demonstrated on functionalized oligopeptides that are attached to polymer resins.     

Linkers and catalysts immobilized on oxide supports: New insights by solid-state NMR spectroscopy

This review article describes classical and modern solid-state NMR methods that allow to gain insight into catalyst systems where one or two metal complexes are bound to oxide supports via bifunctional phosphine linkers, such as (EtO)₃Si(CH₂)₃PPh₂. Many aspects of the immobilized molecular catalysts can be elucidated with the corresponding NMR technique. The bulk of the support can be studied, as well as the interface of the support with the ethoxysilane. With respect to the linkers, their structural integrity and mobility are as easy to investigate by classical CP/MAS and high-resolution magic angle spinning (HRMAS) NMR techniques, as their adsorption behavior. Even electrostatic bonding to the support via phosphonium groups can be proven by solid-state NMR. For the immobilized catalysts, leaching, and even “horizontal” translational mobility effects, as probed by HRMAS NMR under “realistic conditions” in the presence of solvents, are described.     

Is high‐resolution magic angle spinning NMR a practical speciation tool for cheese samples? Parmigiano Reggiano as a case study

High-resolution magic angle spinning (HRMAS) NMR is probably the most apt NMR method to analyze complex materials involving a solid phase, e.g. foodstuffs. We present here an HRMAS analysis of grated cheese (Parmigiano Reggiano). A full NMR characterization of this cheese allows the identification of the presence of fatty acids (saturated and unsaturated), amino acids and other small organic molecules. Since the presence and relative concentration of these molecules have previously been shown to correlate with organoleptic, origin and age characterization, HRMAS NMR of cheese is likely to provide a good complimentary tool for the analysis of this food material.     

Inverse H-C ex situ HRMAS NMR experiments for solid-phase peptide synthesis

The growing importance of solid-phase peptide synthesis (SPPS) has necessitated the development of spectroscopic experiments that can be used to obtain structural and conformational information on resin-bound peptides. Despite the utility of two-dimensional high-resolution magic angle spinning (HRMAS) NMR experiments that provide homonuclear shift correlations, experiments that provide heteronuclear shift correlations are necessary for complex conformational and structural elucidatory problems. Here we report the optimization and implementation of non-gradient inverse NMR experiments for acquiring the 1H-13C shift correlations of resin-bound peptides. The use of non-gradient experiments is advantageous as many magic angle spinning (MAS) probes do not possess gradient coils. An HRMAS BIRD-HMQC experiment with a reduced 1JCH constant has proven very suitable for obtaining one-bond correlations. Long-range correlations can be interpolated by using a non-gradient HRMAS CT-HMBC-1 experiment where the resulting data is processed with forward linear prediction. It has been shown that removing the effects of 1H-1H J-modulation is crucial in order to view cross peaks that correspond to long-range correlations. Additionally, both experiments prove extremely useful over routine one-dimensional 13C HRMAS experiments for extracting carbon chemical shift data. The non-gradient HRMAS BIRD-HMQC and CT-HMBC-1 experiments can be used to assist in conformational analysis and to identify and deconvolute situations where accidental equivalence and seemingly correlated isochronous signals arise.     

Ex-vivo NMR of unprocessed tissue in water: a simplified procedure for studying intracranial neoplasms

Ex-vivo and in-vitro nuclear magnetic resonance (NMR) spectroscopy techniques have been used for studying chemical metabolites in surgically resected specimens of human neoplasms, and may provide complementary information to in-vivo whole-body magnetic-resonance spectroscopy (MRS). We describe an ex-vivo NMR in water method for measurement of water-soluble metabolites in unprocessed normal rat brain tissue and human intracranial neoplasms. The NMR spectra obtained using the method described here were comparable to those obtained using high-resolution magic-angle spinning (HRMAS) NMR methods, with good correlation in metabolite concentrations relative to creatine (r ² = 0.7635). Improved spectral resolution and baseline were noted compared to HRMAS, but macromolecule resonances were not detected. Ex-vivo NMR of unprocessed tissue in water is rapid and technically simple to perform, and has the potential to be used for direct assessment of intracranial neoplasms.     

Multidimensional HRMAS NMR: a platform for in vivo studies using intact bacterial cells

In vivo analysis in whole cell bacteria, especially the native tertiary structures of the bacterial cell wall, remains an unconquered frontier. The current understanding of bacterial cell wall structures has been based on destructive analysis of individual components. These in vitro results may not faithfully reflect the native structural and conformational information. Multidimensional High Resolution Magic Angle Spinning NMR (HRMAS NMR) has evolved to be a powerful technique in a variety of in vivo studies, including live bacterial cells. Existing studies of HRMAS NMR in bacteria, technical consideration of its successful application, and current limitations in studying true human pathogens are briefly reviewed in this report.     

Conformational analysis by HRMAS NMR spectroscopy of resin-bound homo-peptides from C(alpha)-methyl-leucine

A series of [L-(alphaMe)Leu]n (n = 1-5) homo-peptides have been covalently linked to Tentagel and POEPOP resins and submitted to a conformational study using HRMAS NMR spectroscopy. Whereas the mono- and dipeptide are mainly fully-extended, stable 3(10)-helical structures are formed beginning from the trimer.     

DEUSS: a perdeuterated poly(oxyethylene)-based resin for improving HRMAS NMR studies of solid-supported molecules

A novel resin called DEUSS (perdeuterated poly(oxyethylene)-based solid support) has been prepared by anionic polymerization of deuterated [D4]ethylene oxide, followed by cross-linking with deuterated epichlorohydrin. DEUSS can be suspended in a wide range of solvents including organic and aqueous solutions, in which it displays a high swelling capacity. As measured by proton HRMAS of the swollen polymer, the signal intensity of the oxyethylene protons is reduced by a factor of 110 relative to the corresponding nondeuterated poly(oxyethylene)poly(oxypropylene) (POEPOP) resin, thus facilitating detailed HRMAS NMR studies of covalently linked molecules. This 1H NMR invisible matrix was used for the solid-phase synthesis of peptides, oligoureas, and a series of amides as well as their characterization by HRMAS NMR spectroscopy. On-bead NMR spectra of high quality and with resolution comparable to that of liquid samples were obtained and readily interpreted. The complete absence of the parasite resin signals will be of great advantage, for example, for the optimization of multistep solid-phase stereoselective reactions, and for the conformational study of resin-bound molecules in a large variety of solvents.     

Resolving the structure of ligands bound to the surface of superparamagnetic iron oxide nanoparticles by high-resolution magic-angle spinning NMR spectroscopy

A major challenge in magnetic nanoparticle synthesis and (bio)functionalization concerns the precise characterization of the nanoparticle surface ligands. We report the first analytical NMR investigation of organic ligands stably anchored on the surface of superparamagnetic nanoparticles (MNPs) through the development of a new experimental application of high-resolution magic-angle spinning (HRMAS). The conceptual advance here is that the HRMAS technique, already being used for MAS NMR analysis of gels and semisolid matrixes, enables the fine-structure-resolved characterization of even complex organic molecules bound to paramagnetic nanocrystals, such as nanosized iron oxides, by strongly decreasing the effects of paramagnetic disturbances. This method led to detail-rich, well-resolved (1)H NMR spectra, often with highly structured first-order couplings, essential in the interpretation of the data. This HRMAS application was first evaluated and optimized using simple ligands widely used as surfactants in MNP synthesis and conjugation. Next, the methodology was assessed through the structure determination of complex molecular architectures, such as those involved in MNP3 and MNP4. The comparison with conventional probes evidences that HRMAS makes it possible to work with considerably higher concentrations, thus avoiding the loss of structural information. Consistent 2D homonuclear (1)H- (1)H and (1)H- (13)C heteronuclear single-quantum coherence correlation spectra were also obtained, providing reliable elements on proton signal assignments and carbon characterization and opening the way to (13)C NMR determination. Notably, combining the experimental evidence from HRMAS (1)H NMR and diffusion-ordered spectroscopy performed on the hybrid nanoparticle dispersion confirmed that the ligands were tightly bound to the particle surface when they were dispersed in a ligand-free solvent, while they rapidly exchanged when an excess of free ligand was present in solution. In addition to HRMAS NMR, matrix-assisted laser desorption ionization time-of-flight MS analysis of modified MNPs proved very valuable in ligand mass identification, thus giving a sound support to NMR characterization achievements.     

1H‐HRMAS NMR study of smoked Atlantic salmon (Salmo salar)

High‐resolution magic angle spinning (HRMAS) NMR spectroscopic data of smoked Atlantic salmon (Salmo salar) were fully assigned by combination of one‐ and two‐dimensional‐HRMAS experiments. Complete representative spectra, obtained after few minutes of analysis time, revealed a large number of minor and major compounds in the sample. The methodology is limited by the low sensitivity of NMR, and therefore HRMAS only enables the determination of the most relevant components. These were fatty acids (FAs), carbohydrates, nucleoside derivatives, osmolytes, amino acids, dipeptides and organic acids. For the first time, spectra were resolved sufficiently to allow semiquantitative determination in intact muscle of the highly polyunsaturated FA 22:6 ω‐3. Additionally, the feasibility of ¹H‐HRMAS NMR metabolite profiling was tested to identify some bioactive compounds during storage. This profiling was carried out by the non‐destructive and direct analysis (i.e. without requiring sample preparation and multiple step procedures) of intact salmon muscle. The proposed procedure can be applied to a large number of samples with high throughput due to the short time of analysis and quick evaluation of the data. Copyright © 2010 John Wiley & Sons, Ltd.     

Discrimination of allied species within the genus Turbinaria (Fucales, Phaeophyceae) using HRMAS NMR spectroscopy

A novel chemotaxonomical method based on 1D (1)H HRMAS NMR spectroscopy is being tested for taxonomical purposes. This powerful technique allowed us to discriminate between specimens belonging to two sister species of Turbinaria, which are difficult to tell apart using only morphological characters. Based on spectra analysis, the results allowed us to successfully group the specimens according to their species. Thus, the efficiency of HRMAS NMR spectroscopy for the discrimination of algal species and for the pre-screening of potential chemomarkers is demonstrated.     

Study of metabonomic profiles of human esophageal carcinoma by use of high-resolution magic-angle spinning 1H NMR spectroscopy and multivariate data analysis

Esophageal carcinoma (EC) is one of the most common malignant tumors. EC survival has remained disappointingly low because of the high malignancy of esophageal cancer and the lack of obvious clinical symptoms at an early stage. Early diagnosis is often difficult because the small tumor nodules are frequently missed. Metabonomics based on high-resolution magic-angle spinning (HRMAS) NMR has been popular for tumor detection because it is highly sensitive, provides rich biochemical information and requires no sample pretreatment. ¹H HRMAS spectra of non-involved adjacent esophageal tissues and of well differentiated and moderately differentiated esophageal carcinoma tumors were recorded and analyzed by use of multivariate and statistical analysis techniques. Moderately differentiated EC tumors were found to have increased total choline, alanine, and glutamate and reduced creatine, myo-inositol, and taurine compared with non-involved adjacent tissues. Moreover, clear differences between the metabonomic profiles of EC tissues enabled tumor differentiation. Furthermore, the integral Gly/MI ratio for samples of different tissue types were statistically significantly different; this was sufficient both for distinguishing non-involved tissues from esophageal carcinoma and for classification of well differentiated and moderately differentiated EC tumors.

Multistep synthesis of 2,5-diketopiperazines on different solid supports monitored by high resolution magic angle spinning NMR spectroscopy

The solid-phase synthesis of 2,5-diketopiperazines containing the trans-4-hydroxy-L-proline amino acid residue (Hyp) was performed on Ellman polystyrene, polyoxyethylene-polyoxypropylene (POEPOP), polystyrene-polyoxyethylene NovaSyn, and Wang resins, respectively. The reaction pathway allowed the introduction of different functional groups around the bicyclic scaffold in a combinatorial approach, and it generated mixtures of isomers. A detailed characterization of the single reaction steps by high resolution magic angle spinning (HRMAS) NMR spectroscopy was performed. The NMR spectral resolution of the resin-bound intermediates and final products was greatly influenced by the polymer matrix. The POEPOP resin permitted to obtain HRMAS NMR spectra with a resolution comparable with that of the spectra of the molecules in solution. Moreover, configurational and conformational isomers formed during the solid-phase reaction steps could be detected and easily assigned. Therefore, the combination of the HRMAS NMR technique with the use of nonaromatic resins may become an extremely powerful tool in solid-phase organic synthesis. This approach will allow the monitoring of multistep reactions and the conception of on-bead structural studies either on small molecules or on natural and/or synthetic oligomers.     

Use of 1H and 31P HRMAS to evaluate the relationship between quantitative alterations in metabolite concentrations and tissue features in human brain tumour biopsies

Quantitative multinuclear high-resolution magic angle spinning was performed in order to determine the tissue pH values of and the absolute metabolite concentrations in 33 samples of human brain tumour tissue. Metabolite concentrations were quantified by 1D (1)H and (31)P HRMAS using the electronic reference to in vivo concentrations (ERETIC) synthetic signal. (1)H-(1)H homonuclear and (1)H-(31)P heteronuclear correlation experiments enabled the direct assessment of the (1)H-(31)P spin systems for signals that suffered from overlapping in the 1D (1)H spectra, and linked the information present in the 1D (1)H and (31)P spectra. Afterwards, the main histological features were determined, and high heterogeneity in the tumour content, necrotic content and nonaffected tissue content was observed. The metabolite profiles obtained by HRMAS showed characteristics typical of tumour tissues: rather low levels of energetic molecules and increased concentrations of protective metabolites. Nevertheless, these characteristics were more strongly correlated with the total amount of living tissue than with the tumour cell contents of the samples alone, which could indicate that the sampling conditions make a significant contribution aside from the effect of tumour development in vivo. The use of methylene diphosphonic acid as a chemical shift and concentration reference for the (31)P HRMAS spectra of tissues presented important drawbacks due to its interaction with the tissue. Moreover, the pH data obtained from (31)P HRMAS enabled us to establish a correlation between the pH and the distance between the N(CH(3))(3) signals of phosphocholine and choline in (1)H spectra of the tissue in these tumour samples.     

Preparation and sorption properties of a β‐cyclodextrin‐linked chitosan derivative

β‐Cyclodextrin (β‐CD) was coupled to chitosan by the intermediate of its monochlorotriazinyl derivative, called βW7MCT, so that a chitosan derivative bearing cyclodextrin was obtained. Because the average degree of substitution of the cyclodextrin derivative was 2.8, the reaction yielded crosslinked insoluble products. The structure of these materials has been investigated by high‐resolution magic‐angle spinning (HRMAS) with gradients. For the first time, HRMAS spectra of chitosan polymers containing β‐CD were obtained. This NMR technique produced one‐ and two‐dimensional well‐resolved solid‐state spectra. These data confirm the proposed structure. Decontamination of waters containing textile dyes were carried out with the crosslinked derivatives. These tests showed that the new chitosan derivatives are characterized by a rate of sorption and a global efficiency superior to that of the parent chitosan polymer and of the well‐known cyclodextrin‐epichlorohydrin gels. © 2000 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 39: 169–176, 2001     

High resolution magic angle spinning NMR in combinatorial chemistry

Solid phase organic chemistry coupled with combinatorial methods promises to increase dramatically the diversity and number of small molecules available for medical and biological applications. However, optimizing the reaction conditions can be a time consuming step, especially since analytical tools to monitor reaction progress and detect impurities for solid phase chemistry are less developed than for solution chemistry. The use of high resolution magic angle spinning (HRMAS) NMR is described here as such an analytical tool. Whereas initial applications of molecular identification using deuterated organic solvents to swell the resins presented a significant gain in time over the cleave-and-analysis methods, the introduction of a differential diffusion filter has made immediate recording of spectra possible without any sample treatment. The applications of HRMAS NMR to different solid supports that are used in combinatorial chemistry will be described in terms of rapidity, robustness and sensitivity.     

High-resolution magic angle spinning NMR of the neuronal tau protein integrated in Alzheimer's-like paired helical fragments

HRMAS NMR of tau paired helical fragments assembled with heparin show an intensity decrease for those amino acids that are incorporated into the rigid core region, whereas the N-terminal amino acids maintain their full mobility.     

High-resolution magic-angle spinning NMR for the identification of reaction products directly from thin-layer chromatography spots

We have investigated the prospect of identifying organic reaction products directly from separated thin-layer chromatography (TLC) spots with high-resolution magic-angle spinning (HRMAS) NMR. The concept is to use the TLC spots for NMR analysis so that spectra can be obtained before the reaction is worked up, but without having to elute the product from the TLC stationary phase. Thus, the separated spot is scraped from the plate, transferred to an HRMAS sample rotor, and suspended with a deuterated solvent. Herein, we describe the effects of having the stationary phase present during NMR acquisition. Using a Varian 4 mm gHX Nanoprobe and rotenone as a test compound, we found that the presence of the stationary phase during NMR acquisition resulted in (i) a large, broad 'background' signal near 4.6 ppm and (ii) a decrease in the signal-to-noise ratio due to the adsorption of the product molecules to the adsorbent. However, both effects could be adequately and conveniently eliminated. The background signal was removed by using either a CPMG pulse sequence or chemical exchange. The adsorption was avoided by using a more polar solvent system. Finally, we found that spectra with good signal-to-noise ratio and resolution could be acquired in a matter of minutes even for cases of limited product concentration. Therefore, we believe the technique has value and provides the organic chemist with another option to obtain NMR data critical for structural elucidation or verification.