High resolution MAS-NMR in combinatorial chemistry

High-resolution magic angle spinning (hr-MAS) NMR is a powerful tool for characterizing organic reactions on solid support. Because magic angle spinning reduces the line-broadening due to dipolar coupling and variations in bulk magnetic susceptibility, line widths approaching those obtained in solution-phase NMR can be obtained. The magic angle spinning method is amenable for use in conjunction with a variety of NMR-pulse sequences, making it possible to perform full-structure determinations and conformational analysis on compounds attached to a polymer support. Diffusion-weighted MAS-NMR methods such as SPEEDY (Spin-Echo-Enhanced Diffusion-Filtered Spectroscopy) can be used to remove unwanted signals from the solvent, residual reactants, and the polymer support from the MAS-NMR spectrum, leaving only those signals arising from the resin-bound product. This review will present the applications of high-resolution magic angle spinning NMR for use in combinatorial chemistry research.     

Pre-analytical method for metabolic profiling of plant cell cultures of Passiflora garckei

Passiflora garckei cell cultures were used as a model to describe a reproducible sample preparation method. Solid phase extraction (SPE) was employed to isolate the plant metabolites for nuclear magnetic resonance (NMR) analysis and to subsequently detect the differences between yeast extract elicited and control cells. Compared with previous results obtained by using a Sephadex LH-20 column, SPE coupled with NMR spectroscopy improves the analysis of aromatic compounds e.g.: trans-feruloyl derivatives and trans-coumaroyl derivatives. Moreover, it decreases the concentration of sugars that usually overlap with many plant metabolite signals.     

High-Resolution 1H NMR Spectroscopy of Fish Muscle,Eggs and Small Whole Fish via Hadamard-Encoded Intermolecular Multiple-Quantum Coherence

Background and Purpose

Nuclear magnetic resonance (NMR) spectroscopy has become an important technique for tissue studies. Since tissues are in semisolid-state, their high-resolution (HR) spectra cannot be obtained by conventional NMR spectroscopy. Because of this restriction, extraction and high-resolution magic angle spinning (HR MAS) are widely applied for HR NMR spectra of tissues. However, both of the methods are subject to limitations. In this study, the feasibility of HR ¹H NMR spectroscopy based on intermolecular multiple-quantum coherence (iMQC) technique is explored using fish muscle, fish eggs, and a whole fish as examples.

Materials and Methods

Intact salmon muscle tissues, intact eggs from shishamo smelt and a whole fish (Siamese algae eater) are studied by using conventional 1D one-pulse sequence, Hadamard-encoded iMQC sequence, and HR MAS.

Results

When we use the conventional 1D one-pulse sequence, hardly any useful spectral information can be obtained due to the severe field inhomogeneity. By contrast, HR NMR spectra can be obtained in a short period of time by using the Hadamard-encoded iMQC method without shimming. Most signals from fatty acids and small metabolites can be observed. Compared to HR MAS, the iMQC method is non-invasive, but the resolution and the sensitivity of resulting spectra are not as high as those of HR MAS spectra.

Conclusion

Due to the immunity to field inhomogeneity, the iMQC technique can be a proper supplement to HR MAS, and it provides an alternative for the investigation in cases with field distortions and with samples unsuitable for spinning. The acquisition time of the proposed method is greatly reduced by introduction of the Hadamard-encoded technique, in comparison with that of conventional iMQC method.     

Complex glycosaminoglycans: profiling substitution patterns by two-dimensional nuclear magnetic resonance spectroscopy

Biological and pharmacological interactions of heparin and structurally related glycosaminoglycans (GAGs) such as heparan sulfate (HS) involve complex sequences of variously sulfated uronic acid and aminosugar residues. Due to their structural microheterogeneity, these sequences are usually characterized in statistical terms, by high-performance liquid chromatographic analysis of fragments obtained by enzymatic or chemical degradation. Nuclear magnetic resonance (NMR) spectroscopy is also currently used for structural characterization of GAGs. However, the use of monodimensional NMR analysis of complex GAGs is often limited by severe signal overlap that does not allow reliable quantitative measurements. Using magnetically equivalent signals, the higher resolution achieved by two-dimensional NMR methods could be also exploited for quantitative applications. In this work, heteronuclear single quantum coherence (HSQC) spectroscopy has been evaluated to determine variously substituted monosaccharide components of HS and HS mimics obtained by chemical modification of the Escherichia coli K5 polysaccharide (K5-PS) structurally related to the common biosynthetic precursor of heparin and HS. Heparin was used as a model for assessing the influence of 1H-13C spin-spin couplings on "volumes" of the corresponding signals. For major signals, the HSQC approach permitted quantification of additional structural features both in heparins and in a typical HS. The method was applied to profile the substitution patterns of K5-PS derivatives involving different degrees of N,O-sulfation and N-acetylation, including O-sulfated heparosans bearing free amino groups.     

Bimodal quantitative monitoring for enzymatic activity with simultaneous signal increases in 19F NMR and fluorescence using silica nanoparticle-based molecular probes

We describe the bimodal quantitative assay for enzymatic activity in (19)F NMR spectroscopy and fluorescence spectroscopy using a nanoparticle-based molecular probe. Perfluorinated dendrimers were tethered on silica nanoparticles with a phosphate-caged fluorescein as a linker. Before enzymatic reaction, the molecular rotation of the perfluorinated dendrimers should be highly restricted, and the (19)F NMR signals from the perfluorinated dendrimers were too broad to be detected relative to the noise level. Fluorescence signals of fluorescein were suppressed by the presence of the diphosphate groups. Following the enzymatic reaction with an alkaline phosphatase, perfluorinated dendrimers and fluorescein were released, and the NMR signals of perfluorinated dendrimers and strong fluorescence from fluorescein were correspondingly observed. The enzymatic activity and reaction rates of the hydrolysis of alkaline phosphatase were detected from the increases of fluorescence and (19)F NMR signals. Finally, the feasibility of the probe in the presence of miscellaneous molecules under biomimetic conditions was demonstrated by determining of the enzymatic activity in cell lysate. Quantitative analysis using both (19)F NMR spectroscopy and fluorescence spectroscopy can be accomplished.     

2D selective-TOCSY-DQFCOSY and HSQC-TOCSY NMR experiments for assignment of a homogeneous asparagine-linked triantennary complex type undecasaccharide

The assignment of ¹H and ¹³C NMR signals of a complex type triantennary asialooligosaccharide was examined using 2D selective-TOCSY–DQFCOSY and HSQC–TOCSY experiments. The 2D selective-TOCSY–DQFCOSY experiment exhibits a 2D DQFCOSY spectrum of an individual monosaccharide in the undecasaccharide, although the NMR signals of several monosaccharides in the triantennary undecasaccharide are heavily overlapped. Selective excitation of each anomeric proton signal and subsequent TOCSY experiment afforded transverse magnetization corresponding to all of the proton signals of the monosaccharide. This magnetization was then developed with the corresponding DQFCOSY pulse sequence to afford the DQFCOSY spectrum of the individual monosugars. In this case, four GlcNAc-b, -e, -j, and -h residues were excited as a mixture. In order to assign ¹³C signals, a conventional 2D HSQC–TOCSY spectrum was examined and compared with an unambiguous assignment of 2D selective-TOCSY–DQFCOSY thus obtained. This systematic analysis made it possible to obtain an assignment of the ¹H and ¹³C NMR signals of the triantennary undecasaccharide. In addition, these experiments also revealed all of the glycosyl positions in the triantennary undecasaccharide.     

Nuclear magnetic resonance chromatography: applications of pulse field gradient diffusion NMR to mixture analysis and ligand–receptor interactions

Pulse field gradient (PFG) diffusion NMR spectroscopy is a non-invasive method for the spectroscopic separation and identification of compounds of interest from a mixture. Because it relies on differences in translational diffusion rates to resolve NMR signals from individual components, pulse field gradient NMR is a unique method for analyzing complex mixtures and for detecting intermolecular interactions. A number of multidimensional pulse field gradient NMR experiments have been developed to alleviate the overlap of NMR signals arising from a complex mixture and facilitate component identification. The applications of pulse field gradient NMR for mixture analysis and for the direct identification of high affinity ligands are reviewed.     

A general procedure for assigning the 31P spectra of drug-oligonucleotide complexes

Taking advantage of the slow exchange at the NMR time scale occurring in drug oligonucleotides complexes the 31P signals in the bound forms are assigned by using 31P NMR two dimensional chemical exchange. This technique was applied to complexes between Actinomycin D and d[CpGpCpG] or d[m5CpGpm5CpG]. As compared to the labelled 17O, 18O this method proved to be a powerful and unique way to assign 31P in broad spectrum or with long oligonucleotides.     

Observation of arginyl-deoxyoligonucleotide interactions in Taq I endonuclease by detection of specific 1H NMR signals from 140kD [N eta 1, N eta 2, 15N Arg]Taq I/oligomer complexes

Proton and nitrogen signals of the guanidinium amines in [N eta 1, N eta 2 15N Arg]Taq I endonuclease were observed using isotope filtered experiments and proton detected 1H[15N] heterocorrelated two dimensional NMR spectroscopy. These rapidly exchanging protons could be detected in the free enzyme only at pH 4.5; at pH 8.5, no signals were measured after extensive signal averaging. Addition of deoxyribonucleotide oligomers resulted in the appearance of two groups of signals at about 6.8 and 7.5 ppm. Since these signals are independent of the presence of cognate sequence or Mg2+, it is assumed they represent nonspecific arginyl-DNA interactions. This labeling/NMR approach provides a new method for investigating the role of arginine in protein-DNA interactions.     

31P NMR analysis of red blood cell UDPGlucose and UDPGalactose: comparison with HPLC and enzymatic methods.

The levels of uridine diphosphogalactose (UDPGal) and uridine diphosphoglucose (UDPGlu) in trichloroacetic acid extracts of human red blood cells (RBC) were measured by 31P NMR spectroscopy. Individual determinations were compared to results obtained by enzymatic and high-pressure liquid chromatographic (HPLC) methods. The characteristic doublet of the P beta resonance signals of both UDPGal and UDPGlu were detected in proton-decoupled spectra of extracts. Quantitative analyses were obtained by employing a standard, methylene diphosphonate, in an external capillary tube during data acquisition for periods of 14 to 24 h using an "inverse-gated" pulse sequence. The ratio of the integrated area of each of the uridine sugar nucleotide doublets to the area of the external reference peak was linear with concentrations between 0.03 and 0.50 mM. There was no difference between the mean value obtained by 31P NMR of 6.6 +/- 1.4 mumol UDPGlu/100 g Hgb or 2.1 +/- 0.6 mumol UDPGal/100 gHgb and the corresponding levels determined enzymatically or by HPLC in identical RBC extracts. When analyzed as paired data, only UDPGlu by NMR was found to be lower than the value obtained by HPLC. As a quantitative analytical tool, NMR spectrometry validated both the enzymatic and HPLC methods used for measurement of uridine sugar nucleotides in our laboratories.     

Measurement of a wide range of intracellular sodium concentrations in erythrocytes by 23Na nuclear magnetic resonance.

The accuracy of the 23Na nuclear magnetic resonance (NMR) method for measuring the sodium concentration in erythrocytes was tested by comparing the NMR results to those obtained by emission-flame photometry. Comparisons were made on aqueous solutions, hemolysates, gels, ghosts, and intact erythrocytes. The intra- and extracellular 23Na NMR signals were distinguished by addition of the dysprosium tripolyphosphate [Dy(PPP)7-2] shift reagent to the extracellular fluid. The intra- and extracellular volumes of ghosts and cells were determined by the isotope dilution method. Our results indicate that greater than 20% of the intracellular signal remains undetected by NMR in ghosts and cells. When the cells are hemolyzed, the amount of NMR-detectable sodium varies depending on the importance of gel formation. In hemolysates prepared by water addition, the NMR and flame photometry results are identical. The loss of signal in ghosts, cells, and undiluted hemolysates is attributed to partial binding of the Na+ ion to intracellular components, this binding being operative only when these components exist in a gel state. In a second part, 31P NMR was used to monitor the penetration of the shift reagent into the cells during incubation. Our data demonstrate that free Dy3+ can slowly accumulate inside the red cell.     

A general Monte Carlo/simulated annealing algorithm for resonance assignment in NMR of uniformly labeled biopolymers

We describe a general computational approach to site-specific resonance assignments in multidimensional NMR studies of uniformly ¹⁵N,¹³C-labeled biopolymers, based on a simple Monte Carlo/simulated annealing (MCSA) algorithm contained in the program MCASSIGN2. Input to MCASSIGN2 includes lists of multidimensional signals in the NMR spectra with their possible residue-type assignments (which need not be unique), the biopolymer sequence, and a table that describes the connections that relate one signal list to another. As output, MCASSIGN2 produces a high-scoring sequential assignment of the multidimensional signals, using a score function that rewards good connections (i.e., agreement between relevant sets of chemical shifts in different signal lists) and penalizes bad connections, unassigned signals, and assignment gaps. Examination of a set of high-scoring assignments from a large number of independent runs allows one to determine whether a unique assignment exists for the entire sequence or parts thereof. We demonstrate the MCSA algorithm using two-dimensional (2D) and three-dimensional (3D) solid state NMR spectra of several model protein samples (α-spectrin SH3 domain and protein G/B1 microcrystals, HET-s_218–289 fibrils), obtained with magic-angle spinning and standard polarization transfer techniques. The MCSA algorithm and MCASSIGN2 program can accommodate arbitrary combinations of NMR spectra with arbitrary dimensionality, and can therefore be applied in many areas of solid state and solution NMR.     

Structural studies of Desulfovibrio vulgaris ferrocytochrome c3 by two-dimensional NMR.

Two-dimensional NMR has been used to make specific assignments for the four haems in Desulfovibrio vulgaris (Hildenborough) ferrocytochrome c3 and to determine their haem core architecture. The NMR signals from the haem protons were assigned according to type using two-dimensional NMR experiments which led to four sets of signals, one for each of the haems. Specific assignments were obtained by calculating the ring current shifts which arise from other haems and aromatic residues. Observation of interhaem NOEs confirmed the assignments and established that the relative orientation of the haems is identical to that found in the crystal structure of D. vulgaris (Miyazaki F.) ferricytochrome c3. Assignments were also made for all the aromatic residues except for the haem ligands and F20, which is shifted under the main envelope of signals. The NOEs observed between these aromatic protons and haem protons confirm the similarity between the structures in solution and in the crystal. The assignments reported here are the basis for the cross-assignments of the four microscopic haem redox potentials to specific haems in the protein structure [Salgueiro, C. A., Turner, D. L., Santos, H., LeGall, J. and Xavier, A. V. (1992) FEBS Lett., in the press]     

Characterization of insoluble calcium alginates by solid-state NMR

Three series of 9 insoluble calcium alginate powders with different average calcium contents (1.5, 3.5 and 8%, w/w) are investigated by means of ¹³C solid-state NMR spectroscopy. The effect of the increased calcium content on the determination of the mannuronate (M) to guluronate (G) ratio from spectral deconvolution of the ¹³C CP/MAS spectra is discussed, and the variations observed are commented in function of possible structural modifications related to the interaction with the divalent cations. The possibility of using solid-state NMR spectroscopy for the quantification of the calcium content in unknown alginate samples is explored performing principal component analysis (PCA) of the spectra. The results obtained show that a clear separation of alginates with slightly different calcium content is possible. The proposed method relies on the sole use of the chemical shifts of the signals corresponding to pyranose carbons, suggesting that PCA of solid-state NMR data holds promises as a rapid and undestructive method for screening the calcium content of alginate-based materials with biomedical uses.     

NMR Assignment of the mTOR Domain Responsible for Rapamycin Binding

A fully automated, NOE-based NMR structure determination of a uniformly ¹³C,¹⁵N-labeled protein was achieved in crude cell-extract, without purification of the overexpressed protein. Essentially complete sequence-specific assignments were obtained using triple resonance experiments, based on the high intensity of the resonances from the overexpressed protein relative to those of the background. For the collection of NOE distance constraints, efficient discrimination between NOE cross peaks from the target protein and background signals was achieved using the programs ATNOS and CANDID. In the iterative ATNOS/CANDID procedure, the identification of the desired protein NOEs is initially guided by the self-consistency of the protein NOE-network. Although the intensities of the signals in this network vary over a wide range, and are in many instances comparable to or smaller than those of the background, the first cycle of calculations resulted in the correct global polypeptide fold, and the structure was then refined in six subsequent cycles using the intermediate NMR structures for additional guidance. The experience gained with this work demonstrates that the ATNOS/CANDID procedure for automatic protein structure determination is highly robust and reliable in the presence of intense background signals, and might thus also represent a platform for future protein structure determinations in physiological fluids.     

NMR study of seed hydration with deuterated water: dependence of proton signals on hydration level.

Proton NMR signals in seeds are shown to depend on hydration level. In fact at low water amount, as it occurs in many native seeds, protons can have a restricted mobility and are not detectable. A NMR method for measuring the dependence of proton signals on hydration is reported. The method also allows the separation of the contributions of water and non-water protons in a low-resolution NMR experiment. It is based on successive hydrations (with deuterated water) - desiccation steps and on the analysis of the transverse magnetization decay curves.     

Automated SPE-RP-HPLC fractionation of biofluids combined to off-line NMR spectroscopy for biomarker identification in metabonomics

NMR-based metabonomics is a valuable and straightforward approach to measuring hundreds of metabolites in complex biofluids. However, metabolite identification is sometimes limited by overlapped signals in NMR spectra. We describe a new methodology using an automated hyphenation of solid phase extraction (SPE) with RP-HPLC combined to NMR spectroscopy, which allowed identification of 72 metabolites of various molecular classes in human urine. This methodology was also successfully applied to the fractionation of a cat urine sample to aid identification of aromatic compounds and felinine. The SPE-RP-HPLC method appears to be a reliable tool to support biomarker discovery in metabonomic studies.     

Direct in vivo measurement of absolute metabolite concentrations using 31P nuclear magnetic resonance spectroscopy

Whilst in vivo NMR spectroscopy provides much useful biochemical information, a limitation to such studies has been the difficulty in quantitating the results to obtain absolute metabolite concentrations. We report here a simple direct method to obtain absolute metabolite concentrations when using in vivo NMR with radiofrequency surface coils. The method has been validated for nucleoside triphosphates in two tissues; rat brain and skeletal muscle. The results obtained are in close agreement with nucleoside triphosphate concentrations obtained using other methods. Precautions for the accurate application of the method are discussed. This method can be applied to other metabolites, coils and NMR nuclei.     

Rapid assignment of solution 31P NMR spectra of large proteins by solid-state spectroscopy

The application of the (31)P NMR spectroscopy to large proteins or protein complexes in solution is hampered by a relatively low intrinsic sensitivity coupled with large line widths. Therefore, the assignment of the phosphorus signals by two-dimensional NMR methods in solution is often extremely time consuming. In contrast, the quality of solid-state NMR spectra is not dependent on the molecular mass and the solubility of the protein. For the complex of Ras with the GTP-analogue GppCH(2)p we show solid-state (31)P NMR methods to be more sensitive by almost one order of magnitude than liquid-state NMR. Thus, solid-state NMR seems to be the method of choice for obtaining the resonance assignment of the phosphorus signals of protein complexes in solution. Experiments on Ras.GDP complexes show that the microcrystalline sample can be substituted by a precipitate of the sample and that unexpectedly the two structural states observed earlier in solution are present in crystals as well.