Real‐Time Band‐Selective Homonuclear Proton Decoupling for Improving Sensitivity and Resolution in Phase‐Sensitive J‐Resolved Spectroscopy

Real‐time band‐selective homonuclear ¹H decoupling during data acquisition of z‐filtered J‐resolved spectroscopy produces ¹H‐decoupled ¹H NMR spectra and leads to sensitivity enhancement and improved resolution, and thus aids the measurement of J couplings and residual dipolar couplings in crowded regions of ¹H NMR spectrum. High quality spectra from peptides, organic molecules, and also from enantiomers dissolved in weakly aligned chiral media are reported.     

PSYCHE CPMG–HSQMBC: An NMR Spectroscopic Method for Precise and Simple Measurement of Long‐Range Heteronuclear Coupling Constants

Among the NMR spectroscopic parameters, long‐range heteronuclear coupling constants convey invaluable information on torsion angles relevant to glycosidic linkages of carbohydrates. A broadband homonuclear decoupled PSYCHE CPMG–HSQMBC method for the precise and direct measurement of multiple‐bond heteronuclear couplings is presented. The PSYCHE scheme built into the pulse sequence efficiently eliminates unwanted proton–proton splittings from the heteronuclear multiplets so that the desired heteronuclear couplings can be determined simply by measuring frequency differences between peak maxima of pure antiphase doublets. Moreover, PSYCHE CPMG–HSQMBC can provide significant improvement in sensitivity as compared to an earlier Zangger–Sterk‐based method. Applications of the proposed pulse sequence are demonstrated for the extraction of ⁿJ(¹H,⁷⁷Se) and ⁿJ(¹H,¹³C) values, respectively, in carbohydrates; further extensions can be envisioned in any J‐based structural and conformational studies.     

Non‐Linear Signal Detection Improvement by Radiation Damping in Single‐Pulse NMR Spectra

When NMR lines overlap and at least one of them is affected by radiation damping, the resonance line shapes of all lines are no longer Lorentzian. We report the appearance of narrow signal distortions, which resemble hole‐burnt spectra. This new experimental phenomenon facilitates the detection of tiny signals hidden below the main resonance. Theoretical analysis based on modified Maxwell–Bloch equations shows that the presence of strong transverse magnetization creates a feedback through the coil, which influences the magnetization of all spins with overlapping resonance lines. In the time domain this leads to cross‐precession terms between magnetization densities, which ultimately cause non‐linear behavior. Numerical simulations corroborate this interpretation.     

Deuterium Quantification through Deuterium‐Induced Remote 1H and 13C NMR Shifts

Partial labeling by deuterium may be quantified through simple integrations of those ¹H (200 or 400 MHz ) and ¹³C (100.6 MHz ) NMR resonances that are split into pairs by chemical shifts ⁿΔ=δ(deuterated)?δ(nondeuterated) as induced by deuterium across n>2 chemical bonds. The relative intensities of the two components of a pair are shown to be influenced to practically equal degrees by relaxation effects, so that a deuterium fraction may be determined from ¹H and ¹³C integral pairs at more remote molecular positions under the routine conditions of fast accumulative spectral acquisition.     

Heteronuclear Cross‐Relaxation Effects in the NMR Spectroscopy of Hyperpolarized Targets

Dissolution dynamic nuclear polarization (DNP) enables high‐sensitivity solution‐phase NMR experiments on long‐lived nuclear spin species such as ¹⁵N and ¹³C. This report explores certain features arising in solution‐state ¹H NMR upon polarizing low‐γ nuclear species. Following solid‐state hyperpolarization of both ¹³C and ¹H, solution‐phase ¹H NMR experiments on dissolved samples revealed transient effects, whereby peaks arising from protons bonded to the naturally occurring ¹³C nuclei appeared larger than the typically dominant ¹²C‐bonded ¹H resonances. This enhancement of the satellite peaks was examined in detail with respect to a variety of mechanisms that could potentially explain this observation. Both two‐ and three‐spin phenomena active in the solid state could lead to this kind of effect; still, experimental observations revealed that the enhancement originates from ¹³C→¹H polarization‐transfer processes active in the liquid state. Kinetic equations based on modified heteronuclear cross‐relaxation models were examined, and found to well describe the distinct patterns of growth and decay shown by the ¹³C‐bound ¹H NMR satellite resonances. The dynamics of these novel cross‐relaxation phenomena were determined, and their potential usefulness as tools for investigating hyperpolarized ensembles and for obtaining enhanced‐sensitivity ¹H NMR traces was explored.     

Understanding J‐Modulation during Spatial Encoding for Sensitivity‐Optimized Ultrafast NMR Spectroscopy

Ultrafast (UF) NMR spectroscopy is an approach that yields 2D spectra in a single scan. This methodology has become a powerful analytical tool that is used in a large array of applications. However, UF NMR spectroscopy still suffers from an intrinsic low sensitivity, and from the need to compromise between sensitivity, spectral width, and resolution. In particular, the modulation of signal intensities by the spin–spin J‐coupling interaction (J‐modulation) impacts significantly on the intensities of the spectral peaks. This effect can lead to large sensitivity losses and even to missing spectral peaks, depending on the nature of the spin system. Herein, a general simulation package (Spinach) is used to describe J‐modulation effects in UF experiments. The results from simulations match with experimental data and the results of product operator calculations. Several methods are proposed to optimize the sensitivity in UF COSY spectra. The potential and drawbacks of the different strategies are also discussed. These approaches provide a way to adjust the sensitivity of UF experiments for a large range of applications.     

Single‐Scan Multidimensional NMR Analysis of Mixtures at Sub‐Millimolar Concentrations by using SABRE Hyperpolarization

Signal amplification by reversible exchange (SABRE) is a promising method to increase the sensitivity of nuclear magnetic resonance (NMR) experiments. However, SABRE‐enhanced ¹H NMR signals are short lived, and SABRE is often used to record 1D NMR spectra only. When the sample of interest is a complex mixture, this results in severe overlaps for ¹H spectra. In addition, the use of a co‐substrate, whose signals may obscure the ¹H spectra, is currently the most efficient way to lower the detection limit of SABRE experiments. Here, we describe an approach to obtain clean, SABRE‐hyperpolarized 2D ¹H NMR spectra of mixtures of small molecules at sub‐millimolar concentrations in a single scan. The method relies on the use of para‐hydrogen together with a deuterated co‐substrate for hyperpolarization and ultrafast 2D NMR for acquisition. It is applicable to all substrates that can be polarized with SABRE.     

On the Tuning of High‐Resolution NMR Probes

Three optimum conditions for the tuning of NMR probes are compared: the conventional tuning optimum, which is based on radio‐frequency pulse efficiency, the spin noise tuning optimum based on the line shape of the spin noise signal, and the newly introduced frequency shift tuning optimum, which minimizes the frequency pushing effect on strong signals. The latter results if the radiation damping feedback field is not in perfect quadrature to the precessing magnetization. According to the conventional RLC (resistor–inductor–capacitor) resonant circuit model, the optima should be identical, but significant deviations are found experimentally at low temperatures, in particular on cryogenically cooled probes. The existence of different optima with respect to frequency pushing and spin noise line shape has important consequences on the nonlinearity of spin dynamics at high polarization levels and the implementation of experiments on cold probes.     

A new technique for differentiating between 2J(C,H) and 3/4J(C,H) connectivities

We present a new pulse sequence that yields two simultaneously detected types of long‐range correlation spectra. The one spectrum is to show all ⁿJ(C,H) connectivities and the other is to show exclusively ²J(C,H) connectivities. The method is demonstrated by using strychnine as a test sample. A comparison with HMBC shows that the ²J(C,H)/ⁿJ(C,H) experiment supplies a ⁿJ(C,H) spectrum that is of equal standard with regard to sensitivity and spectral information. The additional ²J(C,H) spectrum allows the disentanglement of ²J(C,H) and ⁿJ(C,H) signals (n > 2) in HMBC type spectra, which greatly simplifies signal assignment and structure elucidation in general. Copyright © 2003 John Wiley & Sons, Ltd.     

J‐Edited Pure Shift NMR for the Facile Measurement of nJHH for Specific Protons

We report a novel 1D J‐edited pure shift NMR experiment (J‐PSHIFT) that was constructed from a pseudo 2D experiment for the direct measurement of proton–proton scalar couplings. The experiment gives homonuclear broad‐band ¹H‐decoupled ¹H NMR spectra, which provide a single peak for chemically distinct protons, and only retain the homonuclear‐scalar‐coupled doublet pattern at the chemical‐shift positions of the protons in the coupled network of a specific proton. This permits the direct and unambiguous measurement of the magnitudes of the couplings. The incorporation of a 1D selective correlation spectroscopy (COSY)/ total correlation spectroscopy (TOCSY) block in lieu of the initial selective pulse, results in the exclusive detection of the correlated spectrum of a specific proton.     

Spatially Selective Heteronuclear Multiple‐Quantum Coherence Spectroscopy for Biomolecular NMR Studies

Spatially selective heteronuclear multiple‐quantum coherence (SS HMQC) NMR spectroscopy is developed for solution studies of proteins. Due to “time‐staggered” acquisitioning of free induction decays (FIDs) in different slices, SS HMQC allows one to use long delays for longitudinal nuclear spin relaxation at high repetition rates. To also achieve high intrinsic sensitivity, SS HMQC is implemented by combining a single spatially selective ¹H excitation pulse with nonselective ¹H 180° pulses. High‐quality spectra were obtained within 66 s for a 7.6 kDa uniformly ¹³C,¹⁵N‐labeled protein, and within 45 and 90 s for, respectively, two proteins with molecular weights of 7.5 and 43 kDa, which were uniformly ²H,¹³C,¹⁵N‐labeled, except for having protonated methyl groups of isoleucine, leucine and valine residues.     

Non‐Uniform Sampling and J‐UNIO Automation for Efficient Protein NMR Structure Determination

High‐resolution structure determination of small proteins in solution is one of the big assets of NMR spectroscopy in structural biology. Improvements in the efficiency of NMR structure determination by advances in NMR experiments and automation of data handling therefore attracts continued interest. Here, non‐uniform sampling (NUS) of 3D heteronuclear‐resolved [¹H,¹H]‐NOESY data yielded two‐ to three‐fold savings of instrument time for structure determinations of soluble proteins. With the 152‐residue protein NP_372339.1 from Staphylococcus aureus and the 71‐residue protein NP_346341.1 from Streptococcus pneumonia we show that high‐quality structures can be obtained with NUS NMR data, which are equally well amenable to robust automated analysis as the corresponding uniformly sampled data.     

Improved excitation uniformity in multiple‐quantum NMR experiments of mixtures

Multiple‐quantum ¹H NMR spectroscopy has been finding a renewed interest for its possible applications in the analysis of mixtures of small molecules, due to its simplification properties. A crucial aspect of this application of multiple‐quantum NMR is the sensitivity of the spectrum intensity to the molecular structure and to the parameterization of the experiment, which could result in the missing of some components. We demonstrate that a general scheme to overcome this drawback consists in varying the experiment parameterizations over a small number of values, selected according the values of the couplings and the relaxation rates. Copyright © 2013 John Wiley & Sons, Ltd.     

Isoquinoline alkaloids: a 15N NMR and x‐ray study. Part 2

Continuing our systematic ¹⁵N NMR study of isoquinoline alkaloids, we report a contribution extending our previous paper. The ¹⁵N NMR chemical shifts and ¹⁵N,¹H long‐range coupling pathways of tertiary and quaternary isoquinoline alkaloids of several constitutional types are presented. The selected compounds belong to the protoberberine, proaporphine, pavinane, rhoeadine and phtalideisoquinoline classes of alkaloids and were investigated by gradient‐selected inverse‐detected multiple bond correlation experiments (GHMBC and GSQMBC). In addition, x‐ray data and the principal geometric parameters of stylopine, mecambridine, norchelerythrine, isothebaine and mecambrine are reported and discussed. Copyright © 2002 John Wiley & Sons, Ltd.