Evidence of secondary structure by high-resolution magic angle spinning NMR spectroscopy of a bioactive peptide bound to different solid supports.

The structure of the 19-amino acid peptide epitope, corresponding to the 141-159 sequence of capsid viral protein VP1 of foot-and-mouth disease virus (FMDV), bound to three different resins, namely, polystyrene-MBHA, PEGA, and POEPOP, has been determined by high-resolution magic angle spinning (HRMAS) NMR spectroscopy. A combination of homonuclear and heteronuclear bidimensional experiments was used for the complete peptide resonance assignment and the qualitative characterization of the peptide folding. The influence of the chemicophysical nature of the different polymers on the secondary structure of the covalently attached FMDV peptide was studied in detail. In the case of polystyrene-MBHA and polyacrylamide-PEGA resins, the analysis of the 2D spectra was hampered by missing signals and extensive overlaps, and only a propensity toward a peptide secondary structure could be derived from the assigned NOE correlations. When the FMDV peptide was linked to the polyoxyethylene-based POEPOP resin, it was found to adopt in dimethylformamide a helical conformation encompassing the C-terminal domain from residues 152 to 159. This conformation is very close to that of the free peptide previously analyzed in 2,2,2-trifluoroethanol. Our study clearly demonstrates that a regular helical structure can be adopted by a resin-bound bioactive peptide. Moreover, a change in the folding was observed when the same peptide-POEPOP conjugate was swollen in aqueous solution, displaying the same conformational features as the free peptide in water. The possibility of studying solid-supported ordered secondary structures by the HRMAS NMR technique in a wide range of solvents can be extended either to other biologically relevant peptides and proteins or to new synthetic oligomers.     

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.     

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.     

CSA-enabled spin diffusion leads to MAS rate-dependent T1's at high field

A surprisingly strong spin rate dependence of (15)N and (13)C NMR T(1) times in magic angle spinning experiments on solid peptides is demonstrated. Using a variety of isotopomers, the phenomenon is shown to be the result of chemical shift anisotropy-mediated spin diffusion. This effect has the potential to be used to detect long-range distance constraints in macromolecular systems.     

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.     

High-resolution characterization of liquid-crystalline [60]fullerenes using solid-state nuclear magnetic resonance spectroscopy

Liquid-crystalline materials containing fullerenes are valuable in the development of supramolecular switches and in solar cell technology. In this study, we characterize the liquid-crystalline and dynamic properties of fullerene-containing thermotropic compounds using solid-state natural abundance (13)C NMR experiments under stationary and magic angle spinning sample conditions. Chemical shifts spectra were measured in isotropic, liquid-crystalline nematic and smectic A and crystalline phases using one-dimensional (13)C experiments, while two-dimensional separated local-field experiments were used to measure the (1)H- (13)C dipolar couplings in mesophases. Chemical shift and dipolar coupling parameters were used to characterize the structure and dynamics of the liquid-crystalline dyads. NMR data of fullerene-containing thermotropic liquid crystals are compared to that of basic mesogenic unit and mesomorphic promoter compounds. Our NMR results suggest that the fullerene-ferrocene dyads form highly dynamic liquid-crystalline phases in which molecules rotate fast around the symmetry axis on the characteristic NMR time scale of approximately 10 (-4) s.     

Characterization of the 310-helix in model peptides by HRMAS NMR spectroscopy

A tetra- and a hepta-homopeptide from the C(alpha)-tetrasubstituted Aib (alpha-aminoisobutyric acid) residue were covalently linked to the POEPOP resin by the fragment-condensation approach. The conformational preferences of the two model peptides were determined for the first time on a solid support by means of high-resolution magic angle spinning NMR spectroscopy. The results obtained indicate that the Aib homopeptides adopt a regular 3(10)-helical structure even when they are covalently bound to a polymeric matrix, and thus confirm the remarkable conformational stability of the peptides rich in this amino acid. An ATR-FTIR spectroscopic investigation, performed in parallel, also confirmed that these polymer-bound peptides do indeed adopt a helical conformation. The results of this study open the possibility to exploit the peptide-resin conjugates based on C(alpha)-tetrasubstituted alpha-amino acids as helpful, structurally organized templates in molecular recognition studies or as catalysts in asymmetric synthesis.     

Magic angle spinning NMR experiments for structural studies of differentially enriched protein interfaces and protein assemblies

Protein-protein interactions play vital roles in numerous biological processes. These interactions often result in formation of insoluble and noncrystalline protein assemblies. Solid-state NMR spectroscopy is rapidly emerging as a premier method for structural analysis of such systems. We introduce a family of two-dimensional magic angle spinning (MAS) NMR experiments for structural studies of differentially isotopically enriched protein assemblies. Using 1-73((13)C,(15)N)/74-108((15)N) labeled thioredoxin reassembly, we demonstrate that dipolar dephasing followed by proton-assisted heteronuclear magnetization transfer yields long-range (15)N-(13)C correlations arising exclusively from the interfaces formed by the pair of differentially enriched complementary fragments of thioredoxin. Incorporation of dipolar dephasing into the (15)N proton-driven spin diffusion and into the (1)H-(15)N FSLG-HETCOR sequences permits (1)H and (15)N resonance assignments of the 74-108((15)N) enriched C-terminal fragment of thioredoxin alone. The differential isotopic labeling scheme and the NMR experiments demonstrated here allow for structural analysis of both the interface and each interacting protein. Isotope editing of the magnetization transfers results in spectral simplification, and therefore larger protein assemblies are expected to be amenable to these experiments.     

Selective interface detection: mapping binding site contacts in membrane proteins by NMR spectroscopy

Intermolecular contact surfaces are important regions where specific interactions mediate biological function. We introduce a new magic angle spinning solid state NMR technique, dubbed "selective interface detection spectroscopy" (SIDY). In this technique, 13C-attached protons (1Hlig) are dephased by 1H-13C REDOR. A spin diffusion period is then used to enhance long distance 1H-1H correlations, and the results are detected by a short period of cross polarization to the 13C isotope labels. This SIDY approach allows selective observation of the interface between 13C-labeled and unlabeled moieties. We have used SIDY to probe the ligand-protein binding surface between a uniformly isotopically labeled ligand cofactor, U-13C20-11-cis-retinal, and its binding site in rhodopsin (Rho), an unlabeled, membrane-embedded G-protein coupled receptor (GPCR). The observed 1HGPCR-13Clig correlations indicate multiple close contacts between the protein and the ionone ring of the ligand, in agreement with binding studies. The polyene tail of the ligand displays fewer strong correlations in the SIDY spectrum. Some correlations can be assigned to the protein side chains lining the ligand binding site, in agreement with the crystal structure.     

NMR analysis of surfaces and interfaces in 2-nm CdSe

Solid-state NMR analysis on wurtzite 2-nm hexadecylamine-capped CdSe nanocrystals (CdSe-HDA) provides evidence of discrete nanoparticle reconstruction within the Se sublattice of the nanomaterial. The cadmium and selenium atoms are probed with (1)H-(113)Cd and (1)H-(77)Se cross-polarization magic angle spinning (MAS) experiments, which demonstrate five ordered selenium sites in the nanoparticle that can be assigned to contributions arising from different surface sites and a selenium site one layer down from the surface. Intriguingly, in these materials both HDA and thiophenol are observed to selectively bind to specific sites on the nanoparticle surface. 2D heteronuclear chemical shift correlation (HETCOR) experiments provide evidence for thiophenol selectively binding at surface vacancies. Analysis of the NMR provides a model of a 2-nm CdSe-HDA molecular surface.     

High magnetic field solid‐state NMR analyses by combining MAS,MQ‐MAS,homo‐nuclear and hetero‐nuclear correlation experiments

A general strategy of structural analysis of alumina silicate by combining various solid‐state NMR measurements such as single pulse, multi‐quantum magic angle spinning, double‐quantum homo‐nuclear correlation under magic angle spinning (DQ‐MAS), and cross‐polarization hetero‐nuclear correlation (CP‐HETCOR) was evaluated with the aid of high magnetic field NMR (800 MHz for ¹H Larmor frequency) by using anorthite as a model material. The high magnetic field greatly enhanced resolution of ²⁷Al in single pulse, DQ‐MAS, and even in triple‐quantum magic angle spinning NMR spectra. The spatial proximities through dipolar couplings were probed by the DQ‐MAS methods for homo‐nuclear correlations between both ²⁷Al–²⁷Al and ²⁹Si–²⁹Si and by CP‐HETCOR for hetero‐nuclear correlations between ²⁷Al–²⁹Si in the anorthite framework. By combining various NMR methodologies, we elucidated detailed spatial correlations among various aluminum and silicon species in anorthite that was hard to be determined using conventional analytical methods at low magnetic field. Moreover, the presented approach is applicable to analyze other alumina‐silicate minerals. Copyright © 2012 John Wiley & Sons, Ltd.     

1H‐HRMAS NMR study of cold smoked Atlantic salmon (Salmo salar) treated with E‐beam

¹H High resolution magic angle spinning (HRMAS) NMR spectroscopic data in combination with principal components analysis and analysis of variance were used to differentiate between irradiated and non‐irradiated cold‐smoked Atlantic salmon (Salmo salar). NMR profiling was obtained, with a spectral acquisition time of less than 8 min, from a small sample size of intact white salmon muscle, by non‐destructive analysis that includes a very simple and rapid sample preparation step. Results obtained enable the use of creatine, trimethylamine oxide and the sum of phosphorylcholine and glycerophosphorylcholine as diagnostic compounds to detect irradiation treatment. This study shows the potential of ¹H‐HRMAS to be a rapid method for investigating compositional changes due to food processing as well as to confirm the presence or absence of some bioactive compounds in irradiated samples. Copyright © 2013 John Wiley & Sons, Ltd.     

Structural characterization and quantitation of compound loading of disubstituted benzoates bound to Wang resin through high-resolution magic angle spinning NMR spectroscopy

The combination of 1D and 2D high-resolution magic angle spinning NMR experiments led to the assignment of the proton and carbon resonances for several disubstituted benzoates bound to a polystyrene resin through a Wang linker. It is shown that the signal corresponding to the methylene protons of the linker can be utilized to monitor the solid-phase reactions and determine the loading of the compounds on the resin.     

Quantitative monitoring of solid phase organic reactions by high-resolution magic angle spinning NMR spectroscopy

Three possible high-resolution magic angle spinning (HR MAS) NMR experiments to quantitatively monitor a solid phase supported Horner-Emmons reaction are presented. In the first experiment we follow the solid phase reaction in deuterated solvent directly in the NMR rotor. The second quantification is done by reconditioning of a few milligrams of resin from an undefined reaction vessel by washing, drying, and reswelling in deuterated solvent, and the evaluation of the amount of resin bound structures by comparing to an external standard. The third experiment represents the first analytical quantification of resin-bound structures without any sample preparation, except the transfer of resin-solvent suspension (large excess of reagents in protonated dimethylformamide) from the reaction vessel to the NMR rotor.     

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.     

Multinuclear solid state NMR investigation of two polymorphic forms of ciprofloxacin-saccharinate

Two polymorphic forms of a novel pharmaceutical compound, ciprofloxacin-saccharinate (CIP-SAC), are analyzed using one dimensional (1D) and two dimensional (2D) (1)H nuclear magnetic resonance (NMR) at fast magic angle spinning (MAS). Additionally (15)N spectroscopy and (1)H-(13)C correlation experiments were performed to complement our conclusions. The 1D (1)H NMR spectra of CIP and complexes reveal valuable information about the ionic bonding between ciprofloxacin and saccharine. Additionally, these spectra allow us to perform a clear characterization of each solid form, giving the number of molecules per unit cell in one of the polymorphs. From 2D (1)H-(1)H spectra obtained through double quantum correlations we can arrive at important conclusions about the hydrogen bonding, conformation, and intra and inter-molecular interactions present in these compounds. Comparing and contrasting the (1)H-(1)H correlation data obtained for both polymorphic forms and taking into account the single crystal structure data existing for the solid form CIP-SAC (II) was possible to extract some conclusions on the polymorph CIP-SAC (I) where no single crystal information is available. (1)H MAS NMR is shown to be an important tool in the field of polymorphism and for the characterization of multicomponent pharmaceutical compounds.     

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.     

Solid‐state NMR characterization of oxygen sites in organically modified aluminosilicate xerogels

Solid‐state NMR characterization of hybrid aluminosilicate xerogels, by ¹⁷O magic angle spinning (MAS) and triple quantum magic angle spinning (MQMAS) techniques, evidences Si—O—Si and Si—O—Al oxygen sites, spectrally separated in MQMAS experiments. Inversion of the MQMAS spectra allows the measurement of quadrupolar parameters, isotropic chemical shifts, distribution of chemical shift and discussion of the mobility of the structural units. Copyright © 2003 John Wiley & Sons, Ltd.     

Two-dimensional (13)C-(13)C correlation spectroscopy with magic angle spinning and dynamic nuclear polarization

The sensitivity of solid-state NMR experiments can be enhanced with dynamic nuclear polarization (DNP), a technique that transfers the high Boltzmann polarization of unpaired electrons to nuclei. Signal enhancements of up to 23 have been obtained for magic angle spinning (MAS) experiments at 5 T and 85-90 K using a custom-designed high-power gyrotron. The extended stability of MAS/DNP experiments at low temperature is demonstrated with (1)H-driven (13)C spin-diffusion experiments on the amino acid proline. These (13)C-(13)C chemical shift correlation spectra are the first two-dimensional MAS/DNP experiments performed at high field (>1.4 T).     

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.