Inverse detection and heteronuclear editing in 1H-15N correlation and 1H-1H double-quantum NMR spectroscopy in the solid state under fast MAS

Signal enhancement in heteronuclear correlation spectra as well as signal selection in 1H experiments can be achieved through inverse, i.e., 1H, detection in the solid state under fast MAS conditions. Using recoupled polarization transfer (REPT), a heteronuclear 1H-15N single-quantum correlation (HSQC) experiment is presented whose symmetrical design allows the frequency dimensions to be easily interchanged. By observing the 15N dimension indirectly and detecting on 1H, the sensitivity is experimentally found to be increased by factors between 5 and 10 relative to conventional 15N detection. In addition, the inverse 1H-15N REPT-HSQC scheme can be readily used as a filter for the 1H signal. As an example, we present the combination of such a heteronuclear filter with a subsequent 1H-1H DQ experiment, yielding two-dimensional 15N-edited 1H-1H DQ MAS spectra. In this way, specific selection or suppression of 1H resonances is possible in solid-state MAS experiments, by use of which the resolution can be improved and information can be unravelled in 1H spectra.     

Observing in-phase single-quantum 15N multiplets for NH2/NH3+ groups with two-dimensional heteronuclear correlation spectroscopy

Two-dimensional (2D) F1-(1)H-coupled HSQC experiments provide 3:1:1:3 and 1:0:1 multiplets for AX(3) and AX(2) spin systems, respectively. These multiplets occur because, in addition to the 2S(y)H(z)(a)-->2S(y)H(z)(a) process, the coherence transfers such as 2S(y)H(z)(a)-->2S(y)H(z)(b) occurring in t(1) period provide detectable magnetization during the t(2) period. Here, we present a 2D F1-(1)H-coupled (1)H-(15)N heteronuclear correlation experiment that provides a 1:3:3:1 quartet for AX(3) spin system and a 1:2:1 triplet for AX(2). The experiment is a derivative of 2D HISQC experiment [J. Iwahara, Y.S. Jung, G.M. Clore, Heteronuclear NMR spectroscopy for lysine NH(3) groups in proteins: unique effect of water exchange on (15)N transverse relaxation. J. Am. Chem. Soc. 129 (2007) 2971-2980] and contains a scheme that kills anti-phase single-quantum terms generated in the t(1) period. The purge scheme is essential to observe in-phase single-quantum multiplets. Applications to the NH(2) and NH(3)(+) groups in proteins are demonstrated.     

A heteronuclear intermolecular single-quantum coherence scheme for high-resolution 2D J-resolved 1H NMR spectra in inhomogeneous magnetic fields

Based on heteronuclear intermolecular single-quantum coherences between proton (¹H) and quadrupolar nuclei (i.e. deuterium ²H), a three-dimensional nuclear magnetic resonance (NMR) pulse sequence is proposed for recovering high-resolution two-dimensional J-resolved NMR spectra from samples mixed with a deuterated solvent in the presence of large magnetic field inhomogeneities. Benefitting from excitation of spins via two different radio frequency (RF) transmit channels, this sequence is suitable for applications in randomly large inhomogeneous fields and the solvent suppression generally required in homonuclear intermolecular multiple-quantum coherence approaches is no longer necessary. Systematic theoretical analyses are given based on the distant dipolar field treatment. Experiment on a sample of corn oil in deuterated acetone and ethyl 3-bromopropionate and acetone dissolved in DMSO-d₆ in a deshimmed field with severe inhomogeneous broadening is performed to show the feasibility and applicability of this sequence.     

High resolution 2D 1H-13C correlation of cholesterol in model membrane

High resolution 2D NMR MAS spectra of liposomes, in particular 1H-13C chemical shifts correlations have been obtained on fluid lipid bilayers made of pure phospholipids for several years. We have investigated herein the possibility to obtain high resolution 2D MAS spectra of cholesterol embedded in membranes, i.e. on a rigid molecule whose dynamics is characterized mainly by axial diffusion without internal segmental mobility. The efficiency of various pulse sequences for heteronuclear HETCOR has been compared in terms of resolution, sensitivity and selectivity, using either cross polarization or INEPT for coherence transfer, and with or without MREV-8 homonuclear decoupling during t1. At moderately high spinning speed (9 kHz), a similar resolution is obtained in all cases (0.2 ppm for 1H(3,4), 0.15 ppm for 13C(3,4) cholesterol resonances), while sensitivity increases in the order: INEPT < CP(x4) < CP + MREV. At reduced spinning speed (5 kHz), the homonuclear dipolar coupling between the two geminal protons attached to C(4) gives rise to spinning sidebands from which one can estimate a H-H dipolar coupling of 10 kHz which is in good agreement with the known dynamics of cholesterol in membranes.     

1H assisted 13C/15N heteronuclear correlation spectroscopy in oriented sample solid-state NMR of single crystal and magnetically aligned samples

(1)H-irradiation under mismatched Hartmann-Hahn conditions provides an alternative mechanism for carrying out (15)N/(13)C transfers in triple-resonance heteronuclear correlation spectroscopy (HETCOR) on stationary samples of single crystals and aligned samples of biopolymers, which improve the efficiency especially when the direct (15)N-(13)C dipolar couplings are small. In many cases, the sensitivity is improved by taking advantage of the (13)C(α) labeled sites in peptides and proteins with (13)C detection. The similarities between experimental and simulated spectra demonstrate the validity of the recoupling mechanism and identify the potential for applying these experiments to virus particles or membrane proteins in phospholipid bilayers; however, further development is needed in order to derive quantitative distance and angular constraints from these measurements.     

High spatial resolution in vivo 2D (1)H magnetic resonance spectroscopic imaging of human muscles with a band-selective technique.

This report demonstrates a 2D (1)H magnetic resonance spectroscopic imaging (MRSI) technique that can address some technical difficulties often encountered in MRS studies of human muscles. A preliminary application of this whole-slice technique in human skeletal muscles demonstrates clearly noticeable differences in (1)H metabolite spectra between different human muscles. This observation illustrates the importance of multi-voxel and high spatial resolution in a heterogeneous environment. This technique is robust, can be easily implemented on a commercial MR scanner, and should prove useful for investigators in both basic and clinical (1)H MRS studies.     

Computer optimized spectral aliasing in the indirect dimension of (1)H-(13)C heteronuclear 2D NMR experiments. A new algorithm and examples of applications to small molecules

A new algorithm for optimizing spectral width in the indirect dimension of heteronuclear 2D experiments is introduced. It takes a list of carbon chemical shifts as input and calculates the optimal spectral width and number of time increments to use in the carbon dimension of 2D experiments such as HSQC, HMBC, etc. When using optimized conditions where signals are better distributed along the carbon dimension, the number of time increments needed to resolve all of the signals is reduced by one to two orders of magnitude. This decreases the experimental time by the same factors and makes the acquisition of spectra such as HSQC-TOCSY, HSQC-NOESY, etc. more practical. The new algorithm allows users to limit the maximal t(1) evolution time when relaxation is a concern, and can take lists of carbons that do not need to be resolved. For any carbon, insights regarding the position of signals in the proton dimension increase the efficiency of the optimization by allowing the overlap of pairs of carbons with incompatible proton dispersions. The application of a second optimization using a fully-resolved spectrum as a source of proton dispersion for the carbons allows the number of time increments to be reduced further. Application to cyclosporine A shows that the time taken to acquire fully resolved HSQC spectra can be 126 times less than would be required in a full-width spectrum with the same resolution. The most interesting applications concern experiments where series of HSQC-based experiments have to be acquired, for example in relaxation time measurements. It is shown that the acquisition of quickly acquired series of selective-TOCSY-HSQC can facilitate assignment in carbohydrates. Computer-optimized spectral aliasing (COSA) generally requires no modification of the pulse sequence and can therefore be easily applied by non specialists.     

Very fast magic angle spinning (1)H-(14)N 2D solid-state NMR: sub-micro-liter sample data collection in a few minutes

Substantial resolution and sensitivity enhancements of solid-state (1)H detected (14)N HMQC NMR spectra at very fast MAS rates up to 80 kHz, in a 1mm MAS rotor, are presented. Very fast MAS enhances the (1)H transverse relaxation time and efficiently decouples the (1)H-(14)N interactions, both effects leading to resolution enhancement. The micro-coil contributes to the sensitivity increase via strong (14)N rf fields and high sensitivity per unit volume. (1)H-(14)N HMQC 2D spectra of glycine and glycyl-L-alanine at 70 kHz MAS at 11.7 T are observed in a few minutes with a sample volume of 0.8 μL.     

New (15)N NMR exchange experiments for the unambiguous assignment of (1)H(N)/(15)N resonances of proteins in complexes in slow chemical exchange with free form

The potentialities of a 2D proton-detected heteronuclear exchange experiment to assign the nitrogen and amide proton resonances in a uniformly (15)N-enriched macromolecule involved in a complex, starting from the free form assignments, are demonstrated on a protein-DNA complex. This 2D experiment is further extended to a 3D experiment in the case of severe superpositions.     

PFG-omega1-filtered TOCSY experiments for the determination of long-range heteronuclear and homonuclear coupling constants and estimation of J-coupling "crosstalk" artifacts in 2-D omega1-filtered "E. COSY-style" spectra.

We present novel one- and two-dimensional versions of the omega1-filtered TOCSY experiment. These experiments utilize pulsed-field gradient techniques and INEPT-reverse INEPT magnetization transfer to generate heteronuclear filtering by means of coherence pathway selection. The major advantages of this approach are twofold: first, each experiment requires a reasonable number of transmitter pulses, gradient pulses, and delays to implement. Second, the use of z-axis gradients at the beginning and termination of the pulse sequences prevents the recovery of dephased magnetization prior to FID detection. This technique was incorporated into 1-D and 2-D omega1-filtered JXH- and JHH-TOCSY-style experiments. As demonstrated on 15N-enriched peptide samples, the use of the pulsed-field-gradient coherence selection scheme effectively filters out unwanted magnetization components, thereby improving the overall sensitivity of the experiments. In addition to this suite of pulse sequences, we also present a method for correcting the reduction in J-coupling that results from crosspeak shifting in 2-D omega1-filtered E. COSY-style spectra. This correction is applicable to both Lorentzian and Gaussian 2-D crosspeak lineshapes.     

Absorption spectra and isotope shifts of the(2, 0),(3, 1), and(8, 5) bands of the A~2Π_u-X~2Σ_g~+ system of ~(15)N_2~+ in near infrared

The high-resolution absorption spectra of the(2, 0),(3, 1), and(8, 5) bands of the A~2Π_u–X~2Σ_g~+system of~(15) N_2~+ have been recorded by using velocity modulation spectroscopy technique in the near infrared region. The rotational constants of the X~2Σ_g~+and A~2Π_u states of~(15) N_2~+ were derived from the spectroscopic data. The isotope shifts of these bands of the A~2Π_u–X~2Σ_g~+system of~(14) N_2~+ and~(15) N_2~+ were also analyzed and discussed.     

Methods for the measurement of (1)J(NCalpha) and (2)J(NCalpha) from a simplified 2D (13)C(alpha)-coupled (15)N SE-HSQC spectrum

Two methods for the measurement of (2)J(NCalpha) and (1)J(NCalpha) in (15)N/(13)C-labeled small and medium-size proteins are described. The current approach is based on simplified (13)C(alpha)-coupled (15)N HSQC spectra, where the two (2)J(NCalpha) doublets are separated into two subspectra corresponding to the alpha and beta spin states of the residue's own alpha carbon. The displacement of the two (2)J(NCalpha) doublets between the two subspectra provides an accurate value for (1)J(NCalpha). The alpha/beta filtration is achieved by taking the sum and difference of the recorded complementary in-phase and antiphase J-coupled spectra. J-multiplication is utilized in one of the proposed methods. In this method, an additional coupling evolution period, which is incremented in concert with t(1), is included in the pulse sequence making it possible to scale the peak-to-peak separation.     

2D(13)C-(13)C MAS NMR correlation spectroscopy with mixing by true (1)H spin diffusion reveals long-range intermolecular distance restraints in ultra high magnetic field

An improved 2D (13)C-(13)C CP(3) MAS NMR correlation experiment with mixing by true (1)H spin diffusion is presented. With CP(3), correlations can be detected over a much longer range than with direct (1)H-(13)C or (13)C-(13)C dipolar recoupling. The experiment employs a (1)H spin diffusion mixing period tau(m) sandwiched between two cross-polarization periods. An optimized CP(3) sequence for measuring polarization transfer on a length scale between 0.3 and 1.0 nm using short mixing times of 0.1 ms < tau(m) < 1 ms is presented. For such a short tau(m), cross talk from residual transverse magnetization of the donating nuclear species after a CP can be suppressed by extended phase cycling. The utility of the experiment for genuine structure determination is demonstrated using a self-aggregated Chl a/H(2)O sample. The number of intramolecular cross-peaks increases for longer mixing times and this obscures the intermolecular transfer events. Hence, the experiment will be useful for short mixing times only. For a short tau(m) = 0.1 ms, intermolecular correlations are detected between the ends of phytyl tails and ring carbons of neighboring Chl a molecules in the aggregate. In this way the model for the structure, with stacks of Chl a that are arranged back to back with interdigitating phytyl chains stretched between two bilayers, is validated.     

(2 + 1) Resonance enhanced multiphoton ionization of hydrogen chloride in a pulsed supersonic jet: Vacuum wavenumbers of rotational lines with detailed band analysis for excited electronic states of HCl

A comprehensive study of the excited electronic states of HCl is reported. Using resonance enhanced multiphoton ionization ((2 + 1) REMPI) and time-of-flight mass spectrometry (TOFMS) over 120 band systems are analyzed. Supersonic jet techniques are used to prepare rotationally cold species for laser spectroscopy in the 77 000 to 96 000 cm⁻¹ region. At least 50 new electronic states are characterized as well as several features only tentatively assigned previously. A long vibrational progression consisting of 29 vibrational levels of the deeply bound V¹Σ⁺(0⁺) valence/ion-pair state is studied. We also extend the identification and analysis to high vibrational levels of low-lying Rydberg states. The assignments of [²Π_i] Rydberg state complexes are evaluated in terms of spin-orbit coupling and united-atom calculations. In several band systems, the spectra exhibit anomalous rotational line intensities and broadened linewidths which are consistent with predissociation. Multiphoton ionization with mass spectrometry permits the investigation of isotope effects as well as the appearance of fragment species associated with repulsive potential curves.