Solvent Suppression in Pure Shift NMR.

Intense solvent signals in 1H solution-state NMR experiments typically cause severe distortion of spectra and mask nearby solute signals. It is often infeasible or undesirable to replace a solvent with its perdeuterated form, for example, when analyzing formulations in situ, when exchangeable protons are present, or for practical reasons. Solvent signal suppression techniques are therefore required. WATERGATE methods are well-known to provide good solvent suppression while enabling retention of signals undergoing chemical exchange with the solvent signal. Spectra of mixtures, such as pharmaceutical formulations, are often complicated by signal overlap, high dynamic range, the narrow spectral width of 1H NMR, and signal multiplicity. Here, we show that by combining WATERGATE solvent suppression with pure shift NMR, ultrahigh-resolution 1H NMR spectra can be acquired while suppressing intense solvent signals and retaining exchangeable 1H signals. The new method is demonstrated in the analysis of cyanocobalamin, a vitamin B12 supplement, and of an eye-drop formulation of atropine.

Rapid Measurement of Heteronuclear Coupling Constants in Complex NMR Spectra.

The NMR analysis of fluorine-containing molecules, increasingly widespread due to their importance in pharmaceuticals and biochemistry, poses significant challenges. Severe peak overlap in the proton spectrum often hinders the extraction of critical structural information in the form of heteronuclear scalar coupling constants, which are crucial for determining pharmaceutical properties and biological activity. Here, a new method, IPAP-FESTA, is reported that drastically simplifies measurements of the signs and magnitudes of proton-fluorine couplings. Its usefulness is demonstrated for the structural study of the steroidal drug fluticasone propionate extracted from a commercial formulation and for assessing solvent effects on the conformational equilibrium in a physically inseparable fluorohydrin mixture.

Pure shift FESTA: An ultra-high resolution NMR tool for the analysis of complex fluorine-containing spin systems.

NMR measurements of molecules containing sparse fluorine atoms are becoming increasingly common due to their prevalence in medicinal chemistry. However, the presence of both homonuclear and heteronuclear scalar couplings severely complicates their analysis by NMR. In complex systems, FESTA, a heteronuclear spectral editing method, allows simplified 1 H NMR spectra to be obtained containing only 1 H signals from the same spin system as a chosen 19 F. Despite spectral simplification, signal overlap due to the presence of scalar couplings is often a problem in FESTA spectra. Here, we report a new experiment that combines FESTA and pure shift methods to provide fully decoupled ultra-high resolution FESTA spectra showing a single signal for each 1 H chemical environment. The utility of the method is demonstrated for the analysis of two complex fluorine-containing mixtures of pharmaceutical and biochemical interest.

Giving Pure Shift NMR Spectroscopy a REST─Ultrahigh-Resolution Mixture Analysis.

Most interesting problems in chemistry, biology, and pharmacy involve mixtures. However, analysis of such mixtures by NMR remains a challenge, often requiring the mixture components to be physically separated before analysis. A variety of methods have been proposed that exploit species-specific properties such as diffusion and relaxation to distinguish between the signals of different components in a mixture without the need for laborious separation. However, these methods can struggle to distinguish between components when signals overlap. Here, we exploit the relaxation properties of selected nuclei to distinguish between different components of a mixture while using pure shift methods to increase spectral resolution by up to an order of magnitude, greatly reducing signal overlap. The advantages of the new method are demonstrated in a mixture of d-xylose and l-arabinose, distinguishing unambiguously between the five major species present.

Simultaneous Broadband Suppression of Homonuclear and Heteronuclear Couplings in 1 H†NMR Spectroscopy.

The 1 H NMR analysis of species containing NMR-active heteronuclei can be difficult due to signal overlap caused by the combined effects of homonuclear and heteronuclear scalar (J) couplings. Here, a general pure shift method is presented for obtaining ultra-high resolution 1 H NMR spectra where spectral overlap is drastically reduced by suppressing both homonuclear and heteronuclear J-couplings, giving one single signal per 1 H chemical environment. Its usefulness is demonstrated in the analysis of fluorine- and phosphorus-containing compounds of pharmaceutical and biochemical interest.

Improving the Sensitivity of FESTA Methods for the Analysis of Fluorinated Mixtures.

The analysis of complex mixtures is an important but often intractable problem. When species contain sparse fluorine atoms, NMR spectra of fluorine-containing spin systems can be efficiently extracted from an intact mixture using the recently proposed FESTA (Fluorine-Edited Selective TOCSY Acquisition) methodology. Here an alternative approach to the existing selective reverse INEPT FESTA (SRI-FESTA) experiment is described, based on the use of a modulated spin echo for the initial excitation. MODO-FESTA (modulated echo FESTA) is simpler and has a significant sensitivity advantage over SRI-FESTA. Comparisons are presented of the relative sensitivity and spectral purity of the two types of methods.

Dissect and Divide: Putting NMR Spectra of Mixtures under the Knife.

Efficient, practical, and nondestructive analysis of complex mixtures is vital in many branches of chemistry. Here we present a new type of NMR experiment that allows the study of very challenging intact mixtures, in which subspectra of individual components can be extracted when other NMR means fail, for the case of a single, intact, static (constant composition) sample. We demonstrate the new approach, SCALPEL (Spectral Component Acquisition by Localized PARAFAC Extraction of Linear components), on a natural fermented beverage, beer, and other carbohydrate mixtures, obtaining individual carbohydrate component subspectra. This new class of NMR experiment is based on dissecting the spectrum rather than the sample, using pulse sequences tailored to generate data suitable for powerful tensor decomposition methods to allow highly complex spectra to be analyzed stepwise, one small section at a time. It has the clear potential to attack problems beyond the reach of current methods.

Unexploited Dimension: New Software for Mixture Analysis by 3D Diffusion-Ordered NMR Spectroscopy.

3D DOSY experiments have the potential to provide unique and valuable information, but they are underused, in part because of the lack of efficient processing software. Here, we illustrate the power of 3D DOSY and present MAGNATE, Multidimensional Analysis for the GNAT Environment, an open-source and free software package for the analysis of pulsed field gradient (PFG) 3D NMR diffusion data, distributed under the GNU General Public License. The new software makes it possible for the first time to efficiently analyze and visualize 3D diffusion (e.g., 3D HSQC-DOSY) data using both univariate (e.g., DOSY) and multivariate (e.g., OUTSCORE) methods in a user-friendly graphical interface. The software can be used either independently or as a module in the GNAT program.

FESTA: An Efficient Nuclear Magnetic Resonance Approach for the Structural Analysis of Mixtures Containing Fluorinated Species.

In complex mixtures, proton nuclear magnetic resonance (NMR) spectra are often very crowded, making spectral analysis complicated or even impossible, particularly when detailed structural information about the mixture components is needed. A new 1D NMR method (fluorine-edited selective TOCSY acquisition, FESTA) is introduced that facilitates the structural analysis of mixtures of species that contain fluorine. It allows simplified 1H spectra to be obtained that show only those protons that are in a spin system coupled to fluorine of interest. The new method is illustrated by factorizing a complex 1H spectrum into subspectra for individual spin systems involving different 19F sites.

The GNAT: A new tool for processing NMR data.

The GNAT (General NMR Analysis Toolbox) is a free and open-source software package for processing, visualising, and analysing NMR data. It supersedes the popular DOSY Toolbox, which has a narrower focus on diffusion NMR. Data import of most common formats from the major NMR platforms is supported, as well as a GNAT generic format. Key basic processing of NMR data (e.g., Fourier transformation, baseline correction, and phasing) is catered for within the program, as well as more advanced techniques (e.g., reference deconvolution and pure shift FID reconstruction). Analysis tools include DOSY and SCORE for diffusion data, ROSY T1 /T2 estimation for relaxation data, and PARAFAC for multilinear analysis. The GNAT is written for the MATLAB® language and comes with a user-friendly graphical user interface. The standard version is intended to run with a MATLAB installation, but completely free-standing compiled versions for Windows, Mac, and Linux are also freely available.

Pure shift 1 H NMR: what is next?

Currently, pure shift nuclear magnetic resonance (NMR) is an area of high interest. The aim of this contribution is to describe briefly how this technique has evolved, where it is now and what could be the next challenges in the amazing adventure of the development and application of pure shift NMR experiments. Copyright © 2016 John Wiley & Sons, Ltd.

One-Shot Determination of Residual Dipolar Couplings: Application to the Structural Discrimination of Small Molecules Containing Multiple Stereocenters.

A novel approach for the fast and efficient structural discrimination of molecules containing multiple stereochemical centers is described. A robust J-resolved HSQC experiment affording highly resolved 1JCH/1TCH splittings along the indirect dimension and homodecoupled 1H signals in the detected dimension is proposed. The experiment enables in situ distinction of both isotropic and anisotropic components of molecules dissolved in compressed PMMA gels, allowing a rapid and direct one-shot determination of accurate residual dipolar coupling constants from a single NMR spectrum.

Ultrahigh-Resolution Diffusion-Ordered Spectroscopy.

Diffusion-ordered spectroscopy (DOSY) is an effective method for the analysis of intact mixtures, but the quality of results is critically limited by resolution in the NMR dimension. A new experiment integrating diffusion weighting into the PSYCHE method for pure shift NMR spectroscopy allows DOSY spectra to be measured with ultrahigh NMR resolution at improved sensitivity.

Disentangling Complex Mixtures of Compounds with Near-Identical (1) H and (13) C†NMR Spectra using Pure Shift NMR Spectroscopy.

The thorough analysis of highly complex NMR spectra using pure shift NMR experiments is described. The enhanced spectral resolution obtained from modern 2D HOBS experiments incorporating spectral aliasing in the (13) C indirect dimension enables the distinction of similar compounds exhibiting near-identical (1) H and (13) C NMR spectra. It is shown that a complete set of extremely small Δδ((1) H) and Δδ((13) C) values, even below the natural line width (1 and 5 ppb, respectively), can be simultaneously determined and assigned.

Broadband 1H homodecoupled NMR experiments: recent developments, methods and applications.

In recent years, a great interest in the development of new broadband 1H homonuclear decoupled techniques providing simplified JHH multiplet patterns has emerged again in the field of small molecule NMR. The resulting highly resolved 1H NMR spectra display resonances as collapsed singlets, therefore minimizing signal overlap and expediting spectral analysis. This review aims at presenting the most recent advances in pure shift NMR spectroscopy, with a particular emphasis to the Zangger-Sterk experiment. A detailed discussion about the most relevant practical aspects in terms of pulse sequence design, selectivity, sensitivity, spectral resolution and performance is provided. Finally, the implementation of the different reported strategies into traditional 1D and 2D NMR experiments is described while several practical applications are also reviewed.

Suppression of phase and amplitude J(HH) modulations in HSQC experiments.

The amplitude and the phase of cross peaks in conventional 2D HSQC experiments are modulated by both proton-proton, J(HH), and proton-carbon, (1)J(CH), coupling constants. It is shown by spectral simulation and experimentally that J(HH) interferences are suppressed in a novel perfect-HSQC pulse scheme that incorporates perfect-echo INEPT periods. The improved 2D spectra afford pure in-phase cross peaks with respect to (1)J(CH) and J(HH), irrespective of the experiment delay optimization. In addition, peak volumes are not attenuated by the influence of J(HH), rendering practical issues such as phase correction, multiplet analysis, and signal integration more appropriate.

Enantiodifferentiation through frequency-selective pure-shift (1)H nuclear magnetic resonance spectroscopy.

A frequency-selective 1D (1) H nuclear magnetic resonance (NMR) experiment for the fast and sensitive determination of chemical-shift differences between overlapped resonances is proposed. The resulting fully homodecoupled (1) H NMR resonances appear as resolved 1D singlets without their typical J(HH) coupling constant multiplet structures. The high signal dispersion that is achieved is then exploited in enantiodiscrimination studies by using chiral solvating agents.

Pure in-phase heteronuclear correlation NMR experiments.

A general NMR approach to provide pure in-phase (PIP) multiplets in heteronuclear correlation experiments is described. The implementation of a zero-quantum filter efficiently suppresses any unwanted anti-phase contributions that usually distort the multiplet pattern of cross-peaks and can hamper their analysis. The clean pattern obtained in PIP-HSQMBC experiments is suitable for a direct extraction of coupling constants in resolved signals, for a peak-fitting process from a reference signal, and for the application of the IPAP technique in non-resolved multiplets.