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.

Recovering sensitivity lost through convection in pure shift NMR.

Practical pure shift NMR experiments, especially on instruments equipped with cryoprobes, can sometimes give very disappointing results. Here we show for the first time that this is a consequence of signal loss due to sample convection, and demonstrate a simple adjustment to common pure shift NMR experiments that restores the lost signal.

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.

Lighting up spin systems: enhancing characteristic 1H signal patterns of fluorinated molecules.

Fluorine is becoming increasingly prevalent in medicinal chemistry, both in drug molecules and in molecular probes. The presence of fluorine allows convenient monitoring of such molecules in complex environments by NMR spectroscopy. However, sensitivity is a persistent limitation of NMR, especially when molecules are present at low concentrations. Here, sensitivity issues with 1H NMR are mitigated by sharing 19F photochemically-induced dynamic nuclear polarisation with 1H nuclei. Unlike direct 1H enhancement, this method enhances 1H signals without significantly distorting multiplet intensities, and has the potential to enable the use of suitable molecules as low-concentration probes.

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.

Ultra-selective 1D clean in-phase correlation spectroscopy.

Selective 1D COSY can unambiguously identify coupled spins but is often limited both by lack of selectivity, and by unfavourable multiplet lineshapes. Here, ultra-selective GEMSTONE excitation is employed with CLIP-COSY to provide through-bond correlations for nuclei whose NMR signals overlap. The new method is illustrated using the coccidiostat lasalocid and the immunosuppressant cyclosporin.

Resolving the complexity in human milk oligosaccharides using pure shift NMR methods and CASPER.

Human milk oligosaccharides belong to an important class of bioactive molecules with diverse effects on the development of infants. NMR is capable of providing vital structural information about oligosaccharides which can aid in determining structure-function relationships. However, this information is often concealed by signal overlap in 1H spectra, due to the narrow chemical shift range and signal multiplicity. Signal overlap in oligosaccharide spectra can be greatly reduced, and resolution improved, by utilising pure shift methods. Here the benefits of combining pure shift methods with the CASPER computational approach to resonance assignment in oligosaccharides are demonstrated.

Ultra-selective, ultra-clean 1D rotating-frame Overhauser effect spectroscopy.

An ultra-selective 1D NMR experiment - GEMSTONE-ROESY - enables clear, unambiguous assignment of ROE signals in the not uncommon situation that traditional selective methods fail. Its usefulness is demonstrated in the analysis of the natural products cyclosporin and lacto-N-difucohexaose I, providing detailed insight into the structures and conformations of these molecules.

Relaxational signal attenuation during soft refocusing pulses.

Quantitative NMR is widely used, but the systematic errors introduced when signals are excited by anything other than a single hard pulse are not always well understood. One important source of error in experiments using soft pulses is the spin relaxation that takes place during pulses, which contains contributions from both spin-spin and spin-lattice relaxation. Here it is shown that relaxation on resonance during shaped soft 180° refocusing pulses in practical experiments can be well represented by biexponential decay, with rate constants R2 and a shape-dependent linear combination of R1 and R2, where R1 and R2 are the inverses of the spin-lattice and spin-spin relaxation times T1 and T2. In principle this would allow correction for relaxational losses in experiments using on-resonance selective refocusing pulses.

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.

Improved Quantification by Nuclear Magnetic Resonance Spectroscopy of the Fatty Acid Ester Composition of Extra Virgin Olive Oils.

Analysis of foods, which are typically highly complex mixtures, by 1H NMR can be difficult because the prevalence of signal overlap complicates characterization and quantification. The various components of a food sample may have a wide range of concentrations, leading to a high dynamic range NMR spectrum and complicating the analysis of less concentrated species. One source of this complication is the presence of 13C satellites, peaks that appear either side of a parent peak with ∼0.56% of its intensity. Satellites of concentrated species can easily be comparable in intensity to the signals of minor components, and can partly or wholly obscure them. This is commonly seen in olive oil samples, leading to inaccurate calculation of the fatty acid ester composition of the oil, used for determining the quality of edible oils and for detecting adulteration. Here, we show that the recently introduced Destruction of Interfering Satellites by Perfect Echo Low-pass filtration (DISPEL) experiment is able to suppress 13C satellites and can substantially improve the accuracy of integration of minor signals. The DISPEL experiment does not require any complicated optimization, working "out of the box" with standard parameters, and incurs no significant loss of sensitivity. It has the potential to become the default experiment, replacing conventional 1D 1H NMR, for quantitative analysis of olive oil.

Broadband measurement of true transverse relaxation rates in systems with coupled protons: application to the study of conformational exchange.

Accurate measurement of transverse relaxation rates in coupled spin systems is important in the study of molecular dynamics, but is severely complicated by the signal modulations caused by scalar couplings in spin echo experiments. The most widely used experiments for measuring transverse relaxation in coupled systems, CPMG and PROJECT, can suppress such modulations, but they also both suppress some relaxation contributions, and average relaxation rates between coupled spins. Here we introduce a new experiment which for the first time allows accurate broadband measurement of transverse relaxation rates of coupled protons, and hence the determination of exchange rate constants in slow exchange from relaxation measurements. The problems encountered with existing methods are illustrated, and the use of the new method is demonstrated for the classic case of hindered amide rotation and for the more challenging problem of exchange between helical enantiomers of a gold(i) complex.

Single-scan ultra-selective 1D total correlation spectroscopy.

Selective 1D TOCSY is a powerful tool in the assignment of NMR spectra of organic molecules. Here an order of magnitude improvement in selectivity, allowing overlapping multiplets to be excited separately, is achieved in a single scan using the very recent GEMSTONE method. The new experiment is illustrated using an antibiotic and a mixture of diastereomers.

Signal-to-noise ratio in diffusion-ordered spectroscopy: how good is good enough?

Diffusion-ordered NMR spectroscopy (DOSY) constructs multidimensional spectra displaying signal strength as a function of Larmor frequency and of diffusion coefficient from experimental measurements using pulsed field gradient spin or stimulated echoes. Peak positions in the diffusion domain are determined by diffusion coefficients estimated by fitting experimental data to some variant of the Stejskal-Tanner equation, with the peak widths determined by the standard error estimated in the fitting process. The accuracy and reliability of the diffusion domain in DOSY spectra are therefore determined by the uncertainties in the experimental data and thus in part by the signal-to-noise ratio of the experimental spectra measured. Here the Cramér-Rao lower bound, Monte Carlo methods, and experimental data are used to investigate the relationship between signal-to-noise ratio, experimental parameters, and diffusion domain accuracy in 2D DOSY experiments. Experimental results confirm that sources of error other than noise put an upper limit on the improvement in diffusion domain accuracy obtainable by time averaging.

Single-Scan Selective Excitation of Individual NMR Signals in Overlapping Multiplets.

2D NMR is an immensely powerful structural tool but it is time-consuming. Targeting individual chemical groups by selective excitation in a 1D experiment can give the information required far more quickly. A major problem, however, is that proton NMR spectra are often extensively overlapped, so that in practice only a minority of sites can be selectively excited. Here we overcome that problem using a fast, single-scan method that allows selective excitation of the signals of a single proton multiplet even where it is severely overlapped by other multiplets. The advantages of the method are illustrated in a selective 1D NOESY experiment, the most efficient way to determine relative configuration unambiguously by NMR. The new approach presented here has the potential to broaden significantly the applicability of selective excitation and unlock its real potential for many other experiments.

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.

Varian Associates and the birth of commercial NMR spectroscopy.

The birth and early growth of commercial NMR instrumentation, and the parts played by the brothers Russell and Sigurd Varian and their colleagues, are described.

Improved ultra-broadband chirp excitation.

The design and application of ultra-broadband excitation pulses have been among the most interesting and timely areas in NMR and EPR methodology in recent years, due especially to advances in hardware design in EPR, the advent and popularity of high- and ultrahigh-field NMR, and the application of numerical methods like optimal control theory to the design and optimization of radiofrequency pulses and pulse sequences. In this communication, we present a short, robust, and flexible version of the CHORUS family of constant-phase, very broadband excitation sequences. We demonstrate that more than 0.5 MHz excitation with uniform amplitudes and phases can be achieved with this excitation sequence.

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.