New DEFT sequences for the acquisition of one-dimensional carbon NMR spectra of small unlabelled molecules

The acquisition time and quality of 1D 13C{1H} spectra can be improved substantially by using a modified driven equilibrium Fourier transform (DEFT) sequence, which is specifically designed to compensate for the effects of B1 inhomogeneity, pulse miscalibration and frequency offsets. The new sequence, called uniform driven equilibrium Fourier transform (UDEFT), returns the carbon magnetization with a high accuracy along its equilibrium position after each transient is complete. Thus, the sequence allows the use of relaxation delays (RD), which are much shorter than the carbon T1 of the molecule, thereby speeding up the acquisition process of 1D 13C{1H} spectra. To achieve this level of performance, UDEFT employs a refocusing element constituted by a composite adiabatic carbon pulse surrounded by two 90 degrees carbon pulses whose phases are designed to compensate for 90 degrees pulse miscalibrations in an MLEV manner (90 degrees+x-tau(FID)-180+y(Adia)-tau-90 degrees+x-180 degrees+x(Adia)). A version of the UDEFT sequence allows recording 1D 13C{1H} spectra devoid of heteronuclear NOE by using a matched adiabatic 1H decoupling scheme where an even number of 180 degrees adiabatic pulses is applied during the UDEFT module. Spectra of a solution of 300 mM camphor that contains some carbon nuclei with very long T1 relaxation times (90 s and 78 s) were acquired with 128 scans in 10 min using a 5 s relaxation delay.     

13C-13C NOESY: an attractive alternative for studying large macromolecules

13C direct detection provides a valuable alternative to 1H detection to overcome fast relaxation because of its smaller magnetic moment. 13C-13C NOESY spectra were acquired for a dimeric protein of molecular mass 32 000 and for a monomeric analogue. With increasing molecular mass, the quality of 13C-13C NOESY spectra improves while the scalar-based experiments become less sensitive, as predicted by the increase in the molecular mass. 13C-13C NOESY spectra of the dimer were acquired with different mixing times. The mixing time can be tuned to detect mainly one-bond correlations, or it can be increased to also detect correlations between nuclei at longer distances. It is proposed that 13C-13C dipolar-based experiments provide a promising tool for signal detection and assignment in large macromolecules, such as multimeric species and macromolecular complexes, for which scalar-based experiments become less effective.     

1H-1H dipolar couplings provide a unique probe of RNA backbone structure

A NMR method is described that permits simultaneous measurement of the geminal 2JH1H2 + 2DH1H2 splitting and the sum of the 1JCH1 + 1DCH1 + 1JCH2 + 1DCH2 couplings for methylene groups, where 2DH1H2 and 1DCH are residual dipolar couplings, occurring when molecules are weakly oriented relative to the magnetic field. By suppressing either the upfield or downfield half of the 1H-1H geminal doublet, the experiment yields improved resolution relative to regular two-dimensional 1H-13C correlation spectra, making it applicable to systems of considerable complexity. The method is demonstrated for measurement of all 2DH5'H5' couplings in a 24-nucleotide 13C-enriched RNA stem loop structure, weakly aligned in liquid crystalline Pf1. The method is equally applicable to methylene groups in 13C-labeled proteins and to natural abundance samples of smaller molecules.     

Effect of atmosphere on collinear double-pulse laser-induced breakdown spectroscopy

Double-pulse laser-induced breakdown spectroscopy (DP-LIBS) has been shown to enhance LIBS spectra. Several researchers have reported significant increases in signal-to-noise and/or spectral intensity compared to single-pulse (SP) LIBS. In addition to DP-LIBS, atmospheric conditions can also increase sensitivity. Thus, in this study, a collinear DP-LIBS scheme was used along with manipulation of the atmospheric conditions. The DP-LIBS scheme consisted of an initial 45-mJ pulse at 1,064-nm fired into a sample contained in a controlled atmospheric/vacuum chamber. A second analytical 45-mJ pulse at 1,064-nm was then fired 0 to 200 μs after and along the same path of the first pulse. Ar, He, and air at pressures ranging from atmospheric pressure to 1 Torr are introduced during DP-LIBS and SP-LIBS experiments. For a brass sample, significant increases in the spectral intensities of Cu and Zn lines were observed in DP-LIBS under Ar compared to DP-LIBS in air. It was also found that Cu and Zn lines acquired with SP-LIBS in Ar are nearly as intense as DP-LIBS in air. While collinear DP-LIBS is effective for increasing the sensitivity for some reduced atmospheres (i.e., Ar and air at 630 to 100 Torr and He at 300 Torr), the enhanced spectral intensity ultimately dropped off as the pressure was reduced below 10 Torr for all atmospheric compositions in the experimental arrangement used in this study. At all pressures of air and Ar, the plasma temperature remained rather constant with increased inter-pulse delays; however, the plasma temperature was more variable for different He gas pressures and inter-pulse delays.     

Quantitative 2D HSQC (Q-HSQC) via suppression of J-dependence of polarization transfer in NMR spectroscopy: application to wood lignin

A quantitative method to record (1)H-(13)C correlation NMR spectra (Q-HSQC) is presented. The suppression of (1)J(CH)-dependence is achieved by modulating the polarization transfer delays of HSQC. In addition, the effect of homonuclear couplings, as well as relaxation during the pulse sequence are discussed. We developed the Q-HSQC approach for the quantitative analysis of wood lignin, a complex polymer where it has been difficult to obtain reliable data on the relative amounts of different structural units. The current method is applicable to a variety of complex mixtures, where normal 1D (1)H- and (13)C-NMR methods fail.     

Simultaneous quantification and identification of individual chemicals in metabolite mixtures by two-dimensional extrapolated time-zero (1)H-(13)C HSQC (HSQC(0))

Quantitative one-dimensional (1D) (1)H NMR spectroscopy is a useful tool for determining metabolite concentrations because of the direct proportionality of signal intensity to the quantity of analyte. However, severe signal overlap in 1D (1)H NMR spectra of complex metabolite mixtures hinders accurate quantification. Extension of 1D (1)H to 2D (1)H-(13)C HSQC leads to the dispersion of peaks along the (13)C dimension and greatly alleviates peak overlapping. Although peaks are better resolved in 2D (1)H-(13)C HSQC than in 1D (1)H NMR spectra, the simple proportionality of cross peaks to the quantity of individual metabolites is lost by resonance-specific signal attenuation during the coherence transfer periods. As a result, peaks for individual metabolites usually are quantified by reference to calibration data collected from samples of known concentration. We show here that data from a series of HSQC spectra acquired with incremented repetition times (the time between the end of the first (1)H excitation pulse to the beginning of data acquisition) can be extrapolated back to zero time to yield a time-zero 2D (1)H-(13)C HSQC spectrum (HSQC(0)) in which signal intensities are proportional to concentrations of individual metabolites. Relative concentrations determined from cross peak intensities can be converted to absolute concentrations by reference to an internal standard of known concentration. Clustering of the HSQC(0) cross peaks by their normalized intensities identifies those corresponding to metabolites present at a given concentration, and this information can assist in assigning these peaks to specific compounds. The concentration measurement for an individual metabolite can be improved by averaging the intensities of multiple, nonoverlapping cross peaks assigned to that metabolite.     

Ile, Leu, and Val methyl assignments of the 723-residue malate synthase G using a new labeling strategy and novel NMR methods

New NMR experiments are presented for the assignment of methyl (13)C and (1)H chemical shifts from Ile, Leu, and Val residues in high molecular weight proteins. The first class of pulse schemes transfers magnetization from the methyl group to the backbone amide spins for detection, while the second more sensitive class uses an "out-and-back" transfer scheme in which side-chain carbons or backbone carbonyls are correlated with methyl (13)C and (1)H spins. Both groups of experiments benefit from a new isotopic labeling scheme for protonation of Leu and Val methyl groups in large deuterated proteins. The approach makes use of alpha-ketoisovalerate that is (13)C-labeled and protonated in one of its methyl groups ((13)CH(3)), while the other methyl is (12)CD(3). The use of this biosynthetic precursor leads to production of Leu and Val residues that are (13)CH(3)-labeled at only a single methyl position. Although this labeling pattern effectively reduces by 2-fold the concentration of Leu and Val methyls in NMR samples, it ensures linearity of Val and Leu side-chain (13)C spin-systems, leading to higher sensitivity and, for certain classes of experiments, substantial simplification of NMR spectra. Very near complete assignments of the 276 Ile (delta 1 only), Leu, and Val methyl groups in the single-chain 723-residue enzyme malate synthase G (MSG, molecular tumbling time 37 +/- 2 ns at 37 degrees C) have been obtained using the proposed isotopic labeling strategy in combination with the new NMR experiments.     

Simultaneous measurement of N?H and Cα?Hα coupling constants in proteins

We present a pulse sequence for the simultaneous measurement of N? H and Cα? Hα couplings in double‐labeled proteins from 2D spectra. The proposed sequence, a modification of the HN(CO)CA experiment, combines the J‐modulation method and the IPAP scheme. The couplings can be readily retrieved from a series of 2D ¹⁵N? ¹H correlation spectra, differing in the time point at which a ¹H 180° pulse is applied. This induces an intensity modulation of the ¹⁵N? ¹H correlation peaks with the Cα? Hα coupling. The Cα? Hα coupling is then obtained by fitting the observed intensities to the modulation equation. The N? H coupling is measured in each member of the set from peak‐to‐peak separations in the IPAP subspectra. The pulse sequence is experimentally verified with a sample of ¹⁵N/¹³C‐enriched ubiquitin. Copyright © 2009 John Wiley & Sons, Ltd.     

Identification of histidine tautomers in proteins by 2D 1H/13C(delta2) one-bond correlated NMR

If the 13Cdelta2 chemical shift of neutral ("high pH") histidine is >122 ppm, primarily Ndelta1-H tautomer (2) is indicated; if it is <122 ppm, primarily Nepsilon2-H tautomer (1) is indicated. His resonances from the catalytic triad of active serine proteases, for example, are readily distinguished from those of denatured enzyme. The 13Cdelta2 chemical shifts increased by 6.2 ppm for the catalytic histidines in both alpha-lytic protease and subtilisin BPN' in raising the pH from that of imidazolium cation to that of tautomer 2. This tautomer identification method is easy to implement, requiring only bioincorporation of [U-13C] (or the more readily available [U-13C,15N])-histidine. Standard 1H/13C correlation HMQC or HSQC NMR pulse programs then yield the 13Cdelta2 chemical shifts with the benefit of high 1H sensitivity. Because of large one-bond spin-couplings (1JCH approximately 200 Hz), the method should extend to proteins having large 1H and 13C line widths, including very high molecular weights.     

Supercycled homonuclear dipolar decoupling in solid-state NMR: toward cleaner 1H spectrum and higher spinning rates

A homonuclear dipolar decoupling scheme based on windowed phase-modulated Lee-Goldburg (wPMLG) pulse sequences that causes a"z-rotation" of the spins for high-resolution proton NMR spectroscopy of solids is described and analyzed. This supercycled scheme suppresses the effect of pulse imperfections on the spectra and significantly relaxes the off-resonance dependence of the line-narrowing efficiency and scale factor. This leads to a broad spectral window that is free of artifacts such as zero lines, image peaks, and localized rotor-radio-frequency resonances. High-resolution (1)H spectra and two-dimensional homonuclear (1)H-(1)H correlation spectra of standard amino acids, obtained by a combination of this supercycled scheme with magic angle spinning frequencies up to 25 kHz, are demonstrated.     

Solid‐State NMR Correlation Experiments and Distance Measurements in Paramagnetic Metalorganics Exemplified by Cu‐Cyclam

We show how to record and analyze solid‐state NMR spectra of organic paramagnetic complexes with moderate hyperfine interactions using the Cu‐cyclam complex as an example. Assignment of the ¹³C signals was performed with the help of density functional theory (DFT) calculations. An initial assignment of the ¹H signals was done by means of ¹H–¹³C correlation spectra. The possibility of recording a dipolar HSQC spectrum with the advantage of direct ¹H acquisition is discussed. Owing to the paramagnetic shifting the resolution of such paramagnetic ¹H spectra is generally better than for diamagnetic solid samples, and we exploit this advantage by recording ¹H–¹H correlation spectra with a simple and short pulse sequence. This experiment, along with a Karplus relation, allowed for the completion of the ¹H signal assignment. On the basis of these data, we measured the distances of the carbon atoms to the copper center in Cu‐cyclam by means of ¹³C R₂ relaxation experiments combined with the electronic relaxation determined by EPR.     

PASADENA hyperpolarization of succinic acid for MRI and NMR spectroscopy

We use the PASADENA (parahydrogen and synthesis allow dramatically enhanced nuclear alignment) method to achieve 13C polarization of approximately 20% in seconds in 1-13C-succinic-d2 acid. The high-field 13C multiplets are observed as a function of pH, and the line broadening of C1 is pronounced in the region of the pK values. The 2JCH, 3JCH, and 3JHH couplings needed for spin order transfer vary with pH and are best resolved at low pH leading to our use of pH approximately 3 for both the molecular addition of parahydrogen to 1-13C-fumaric acid-d2 and the subsequent transfer of spin order from the nascent protons to C1 of the succinic acid product. The methods described here may generalize to hyperpolarization of other carboxylic acids. The C1 spin-lattice relaxation time at neutral pH and 4.7 T is measured as 27 s in H2O and 56 s in D2O. Together with known rates of succinate uptake in kidneys, this allows an estimate of the prospects for the molecular spectroscopy of metabolism.     

Electronic structure and solvatochromism of merocyanines NMR spectroscopic point of view

(1)H and (13)C NMR spectra of two series of malononitrile-based merocyanines, which possess positive and negative solvatochromism have been in detail investigated in low polar chloroform and polar dimethyl sulfoxide (DMSO). Careful attribution of signals in spectra has been made with the help of two-dimensional NMR experiments (COSY, NOESY, HMBC, and HMQC). Hence, the dependence of merocyanines electronic structure on their chemical structure and solvent nature has been studied by this powerful method. It has been shown that there exists a good correlation between the calculated charges on carbon atoms of a polymethine chain and their chemical shifts in (13)C NMR spectra. The influence of solvent polarity on bond orders for dyes with positive and negative solvatochromism is also observed. The comparison of (13)C NMR spectra of merocyanines and corresponding parent ionic dyes allows to determine their sign of solvatochromism irrespectively of electronic spectra, and also to find the key atoms of chromophore whose signals in (13)C NMR spectra are most informative.     

Order parameters based on (13)C(1)H, (13)C(1)H(2) and (13)C(1)H(3) heteronuclear dipolar powder patterns: a comparison of MAS-based solid-state NMR sequences

Order parameters describing conformational exchange processes on the nanosecond to microsecond timescale can be obtained from powder patterns in solid-state NMR (SSNMR) experiments. Extensions of these experiments to magic-angle spinning (MAS) based high-resolution experiments have been demonstrated, which show a great promise for site-specific probes of biopolymers. In this study, we present a detailed comparison of two pulse sequences, transverse Manfield-Rhim-Elleman-Vaughn (T-MREV) and Lee-Goldburg cross-polarization (LGCP), using experimental and simulation tools to explore their utility in the study of order parameters. We discuss systematic errors due to passively coupled (13)C or (1)H nuclei, as well as due to B(1) inhomogeneity. Both pulse sequences can provide quantitative measurements of the order parameter, but the LGCP experiment is capable of greater accuracy provided that the B(1) field is highly homogeneous. The T-MREV experiment is far better compensated for B(1) inhomogeneity, and it also performs better in situations with limited signal.     

Asymmetric (13)C-(13)C polarization transfer under dipolar-assisted rotational resonance in magic-angle spinning NMR

A two-dimensional (2D) homonuclear exchange NMR spectrum in solids often shows an asymmetric cross-peak pattern, which disturbs a quantitative analysis of peak intensities. When magnetization is prepared using cross polarization (CP), the asymmetry can naively be ascribed to nonequilibrium initial magnetization. We show, however, that the CP effect cannot fully explain the observed mixing-time dependence of the peak intensities in 2D (13)C-(13)C exchange spectra of [2,3-(13)C] l-alanine (2,3-Ala) under (13)C-(1)H dipolar-assisted rotational resonance (DARR) recoupling, which has recently been proposed for a broadband recoupling method under magic-angle spinning. We develop a theory to describe polarization transfer in a two-spin system under DARR recoupling. By taking into account the effects of the partial spectral overlap among (13)C signals, which is a unique feature of DARR recoupling, and (1)H-(1)H flip-flop exchange, we can successfully explain the observed mixing-time dependence of the peak intensities of 2D (13)C-(13)C DARR exchange spectra of 2,3-Ala. A simple initial-rate analysis is also examined.     

Synthesis and testing of a p-H2 hyperpolarized 13C probe based on the pyrazolo[1,5-a]pyrimidineacetamide DPA-713, an MRI vector to target the peripheral benzodiazepine receptors

DPA-713 is the lead compound of a recently developed 2-phenylpyrazolo[1,5-a]pyrimidineacetamide series that has been shown to display a good targeting capability toward peripheral benzodiazepine receptors, recently renamed translocator protein (18 kDa) or in short TSPO. On the basis of this structure, a novel derivative bearing a [(13)C]butynoate moiety has been designed and synthesized (three steps-42% overall yield) providing, upon rapid and quantitative para-hydrogenation, the corresponding hyperpolarized [(13)C]alkene. Para-hydrogen-induced polarization effects have been detected in both (1)H and (13)C-NMR spectra. Upon applying a field cycling procedure, the spin order of para-H(2) added hydrogens is transferred on the (13)C carboxylate moiety yielding a signal enhancement of approximately 4500 times. T(1) of the carboxylate carbon atom is approximately 21.9 s (at 9.37 T). A (13)C-MR image has been acquired by using the (13)C RARE (Rapid Acquisition by Relaxation Enhancement) acquisition protocol on a 10-mM solution. The main limitation to the in vivo use of this novel para-hydrogenated [(13)C]derivative is its relatively low solubility in aqueous systems.     

Investigation of structural transition of regenerated silk fibroin aqueous solution by Rheo-NMR spectroscopy

In this study we applied Rheo-NMR to investigate the structural change of Bombyx mori silk fibroin in aqueous solution under shear. Monitoring the time dependence of 1H solution NMR spectra of silk fibroin subjected to constant shear strain, signal intensities of random coil decreased suddenly during shear while peaks from beta-sheet structure did not arise in the solution spectra. After these experiments, an aggregate of silk was found in the Couette flow cell and its secondary structure was determined as beta-sheet by 13C solid-state NMR. In conclusion the moderate shear applied here triggered the change in the secondary structure.     

Quantitative determination of aliphatic hydrocarbon compounds by 2D NMR

Quantitative analysis of complex mixtures by NMR is often hampered by heavily overlapping signals in 1D ¹H or ¹³C spectra. To resolve the overlap problem, we have been looking at the possibilities of using heteronuclear correlated 2D NMR methods for quantification. In this work, we applied 2D INEPT to analyze mixtures of tetradecane and squalane, which represent typical substructures of lube oil fractions. The factors affecting correlation peak volumes, namely the polarization transfer delays within pulse sequence, multiplicity of CH_n group and the magnitude of ¹J_{(C, H)} couplings were taken into account by product operator formalism calculations. The results indicate that if absolute precision in quantification is not essential, the current approach can be used for the quantitative analysis of the molecular composition of complex mixtures when conventional 1D NMR methods fail. Copyright © 2002 John Wiley & Sons, Ltd.     

A new approach in 1D and 2D 13C high-resolution solid-state NMR spectroscopy of paramagnetic organometallic complexes by very fast magic-angle spinning

Novel 1D and multidimensional solid-state NMR (SSNMR) methods using very fast magic-angle spinning (VFMAS) (spinning speed > 20 kHz) for performing 13C high-resolution SSNMR of paramagnetic organometallic complexes are discussed. VFMAS removes a majority of 13C-1H and 1H-1H dipolar couplings, which are often difficult to remove by RF pulse techniques in paramagnetic complexes because of large paramagnetic shifts. In the first systematic approach using the unique feature of VFMAS for paramagnetic complexes, we demonstrate a means of obtaining well-resolved 1D and multidimensional 13C SSNMR spectra, sensitivity enhancements via cross polarization, and signal assignments, and applications of dipolar recoupling methods for nonlabeled paramagnetic organometallic complexes of moderate paramagnetic shifts ( approximately 800 ppm). Experimental results for powder samples of small nonlabeled coordination complexes at 1H frequencies of 400.2-400.3 MHz show that highly resolved 13C SSNMR spectra can be obtained under VFMAS, without requirements of 1H decoupling. Sensitivity enhancement in 13C SSNMR via cross polarization from 1H spins was demonstrated with an amplitude-sweep high-power CP sequence using strong RF fields ( approximately 100 kHz) available in the VFMAS probe. 13C CPMAS spectra of nonlabeled Cu(II)(dl-alanine)2.(H2O) and V(III)(acetylacetonate)3 (V(acac)3) show that it is possible to obtain high-resolution spectra for a small quantity ( approximately 15 mg) of nonlabeled paramagnetic organometal complexes within a few minutes under VFMAS. Experiments on Cu(II)(dl-alanine)2.(H2O) demonstrated that 1H-13C dipolar recoupling for paramagnetic organometal complexes can be performed under VFMAS by application of rotor-synchronous pi-pulses to 1H and 13C spins. The results also showed that signal assignments for 13CH, 13CH3, and 13CO groups in paramagnetic complexes are possible on the basis of the amount of 13C-1H dipolar dephasing induced by dipolar recoupling. Furthermore, the experimental 2D 13C/1H chemical-shift correlation NMR spectrum obtained for nonlabeled V(acac)3 exhibits well-resolved lines, which overlap in 1D 13C and 1H spectra. Signals for different chemical groups in the 2D spectrum are distinguished by the 13C-1H dipolar dephasing method combined with the 2D 13C/1H correlation NMR. The assignments offer information on the existence of nonequivalent ligands in the coordination complex in solids, without requiring a single-crystal sample.     

Evaluation of NMR spectroscopy for the quantitative characterization of urea-formaldehyde resins

A comparative evaluation has been made of both proton and (13)C nuclear magnetic resonance techniques in the quantitative characterization of commercial urea-formaldehyde resins. There is good agreement between data derived from (13)C NMR spectra and from (1)H high-field continuous wave, or low-field (Fourier transform) NMR spectra. Low-field continuous wave proton spectra exhibit inferior resolution and provide inaccurate quantitative data. Combination of (13)C and proton NMR with nitrogen analysis gives a quantitative characterization technique for these resins.