Absolute Minimal Sampling of Homonuclear 2D NMR TOCSY Spectra for High-Throughput Applications of Complex Mixtures

Modern applications of 2D NMR spectroscopy to diagnostic screening, metabolomics, quality control, and other high-throughput applications are often limited by the time-consuming sampling requirements along the indirect time domain t₁. 2D total correlation spectroscopy (TOCSY) provides unique spin connectivity information for the analysis of a large number of compounds in complex mixtures, but standard methods typically require >100 t₁ increments for an accurate spectral reconstruction, rendering these experiments ineffective for high-throughput applications. For a complex metabolite mixture it is demonstrated that absolute minimal sampling (AMS), based on direct fitting of resonance frequencies and amplitudes in the time domain, yields an accurate spectral reconstruction of TOCSY spectra using as few as 16 t₁ points. This permits the rapid collection of homonuclear 2D NMR experiments at high resolution with measurement times that previously were only the realm of 1D experiments.     

Advanced solvent signal suppression for the acquisition of 1D and 2D NMR spectra of Scotch Whisky

A simple and robust solvent suppression technique that enables acquisition of high‐quality 1D ¹H nuclear magnetic resonance (NMR) spectra of alcoholic beverages on cryoprobe instruments was developed and applied to acquire NMR spectra of Scotch Whisky. The method uses 3 channels to suppress signals of water and ethanol, including those of ¹³C satellites of ethanol. It is executed in automation allowing high throughput investigations of alcoholic beverages. On the basis of the well‐established 1D nuclear Overhauser spectroscopy (NOESY) solvent suppression technique, this method suppresses the solvent at the beginning of the pulse sequence, producing pure phase signals minimally affected by the relaxation. The developed solvent suppression procedure was integrated into several homocorrelated and heterocorrelated 2D NMR experiments, including 2D correlation spectroscopy (COSY), 2D total correlation spectroscopy (TOCSY), 2D band‐selective TOCSY, 2D J‐resolved spectroscopy, 2D ¹H, ¹³C heteronuclear single‐quantum correlation spectroscopy (HSQC), 2D ¹H, ¹³C HSQC‐TOCSY, and 2D ¹H, ¹³C heteronuclear multiple‐bond correlation spectroscopy (HMBC). A 1D chemical‐shift‐selective TOCSY experiments was also modified. The wealth of information obtained by these experiments will assist in NMR structure elucidation of Scotch Whisky congeners and generally the composition of alcoholic beverages at the molecular level.     

Study of near‐symmetric cyclodextrins by compressed sensing 2D NMR

The compressed sensing NMR (CS‐NMR) is an approach to processing of nonuniformly sampled NMR data. Its idea is to introduce minimal l_p‐norm (0 < p ≤ 1) constraint to a penalty function used in a reconstruction algorithm. Here, we demonstrate that 2D CS‐NMR spectra allow the full spectral assignment of near‐symmetric β‐cyclodextrin derivatives (mono‐modified at the C6 position). The application of CS‐NMR ensures experimental time saving and the resolution improvement, necessary because of very low chemical shift dispersion. In the overnight experimental time, the set of properly resolved 2D NMR spectra required for the unambiguous assignment of mono(6‐deoxy‐6‐(1‐1,2,3‐triazo‐4‐yl)‐1‐propane‐3‐O‐(phenyl)) β‐cyclodextrin was obtained. The highly resolved HSQC spectrum was reconstructed from 5.12% of the data. Moreover, reconstructed 2D HSQC–TOCSY spectrum yielded information about the correlations within one sugar unit, and 2D HSQC–NOESY technique allowed the sequential assignment of the glucosidic units. Copyright © 2013 John Wiley & Sons, Ltd.     

Hadamard Homonuclear Broadband Decoupled TOCSY NMR: Improved Efficacy in Detecting Long‐range Chemical Shift Correlations

The importance of Hadamard encoding pulses in one‐dimensional pure shift yielded by the chirp excitation version of selective total correlation spectroscopy (1D PSYCHE–TOCSY) experiments is discussed for chemical‐shift analysis of complex natural products at ultrahigh resolution. Herein, we adapted H_n Hadamard matrices to 1D PSYCHE–TOCSY and observed an overall circa square root of n‐fold enhancement in the signal‐to‐noise (S/N) ratio when compared to conventional 1D PSYCHE–TOCSY recorded by refocusing only one spin at a time. This enhancement in S/N facilitates the observation of very weak long‐range chemical‐shift correlations from Hadamard‐encoded PSYCHE–TOCSY (HE–PSYCHE–TOCSY). The proposed method will have a significant impact on structure determination of complex isolated/ synthetic natural products.     

Three New Pregnane Glycosides from Marsdenia tinctoria

Three new pregnane glycosides, tinctorosides A–C ( 1 – 3 , resp.), together with one known pregnane glycoside, stephanoside B ( 4 ), were isolated from the stems of Marsdenia tinctoria R. Br . (Asclepiadaceae). Their structures were elucidated by extensive spectral methods, especially 2D‐NMR experiments (¹H,¹H‐COSY, HSQC, HMBC, TOCSY, HSQC‐TOCSY, and ROESY), and chemical evidence.     

Keeping PASE with WEFT: SHWEFT–PASE pulse sequences for 1H NMR spectra of highly paramagnetic molecules

Metalloproteins are a category of biomolecules in which the metal site is usually the locus of activity or function. In many cases, the metal ions are paramagnetic or have accessible paramagnetic states, many of which can be studied using NMR spectroscopy. Extracting useful information from ¹H NMR spectra of highly paramagnetic proteins can be difficult because the paramagnetism leads to large resonance shifts (~400 ppm), extremely broad lines, extreme baseline nonlinearity, and peak shape distortion. It is demonstrated that employing polychromatic and adiabatic shaped pulses in simple pulse sequences, then combining existing sequences, leads to significant spectral improvement for highly paramagnetic proteins. These sequences employ existing technology, with available hardware, and are of short duration to accommodate short nuclear T₁ and T₂. They are shown to display uniform excitation over large spectral widths (~75 kHz), accommodate high repetition rates, produce flat baselines over 75 kHz while maintaining peak shape fidelity, and can be used to reduce spectral dynamic range. High‐spin (S = 5/2) metmyoglobin, a prototypical highly paramagnetic protein, was used as the test molecule. The resulting one‐dimensional (1D) pulse sequences combine shaped pulses with super‐water elimination Fourier transform, which can be further combined with paramagnetic spectroscopy to give shaped pulses with super‐water elimination Fourier transform–paramagnetic spectroscopy. These sequences require, at most, direct current offset correction and minimal phasing. The performance of these sequences in simple ¹H 1D, 1D NOE, and two‐dimensional NOESY experiments is demonstrated for metmyoglobin and Paracoccus denitrificans Co²⁺‐amicyanin (S = 3/2), and employed to make new heme hyperfine resonance assignments for high‐spin metBjFixLH_151–256, the heme sensing domain of Bradyrhizobium japonicum FixL. Copyright © 2013 John Wiley & Sons, Ltd.     

A double‐Fourier approach to enhance the efficiency of the indirect domain sampling in 2D NMR

The relatively long times that may be involved in high‐resolution two‐dimensional nuclear magnetic resonance (2D NMR) have stimulated the search for alternative schemes to collect these data. Particularly onerous situations arise when both high‐resolution and large spectral widths are sought along the indirect domain. Strategies proposed for dealing with such cases include folding‐over procedures, Hadamard encoding, and nonlinear data sampling. This communication discusses an alternative strategy, which exploits a partial prior knowledge regarding the position of the NMR resonances along the indirect domain together with customized excitations for every particular t₁ increment, to achieve an optimal sampling in terms of resolution and bandwidth. On the basis of such optimized encoding of the indirect‐domain evolution, which can easily be coped with by modern spectrometers, it becomes possible to maximize the resolution of fine structures without compromising on the spectral bandwidths. The processing of the resulting data along the indirect domain is based on the use of two serially applied discrete Fourier transforms; one to distinguish the main bands in the spectrum and the other to resolve the latter's fine features. A number of simple heteronuclear correlation experiments illustrating the significant acquisition time savings and simultaneous improvements in resolution that can be achieved with the resulting double‐Fourier encoding procedure are illustrated. Copyright © 2011 John Wiley & Sons, Ltd.     

Complete 1H and 13C signal assignment of prenol‐10 with 3D NMR spectroscopy

The complete assignment of ¹H and ¹³C chemical shifts of natural abundance prenol‐10 is reported for the first time. It was achieved using 3D NMR experiments, which were based on random sampling of the evolution time space followed by multidimensional Fourier transform. This approach makes it possible to acquire 3D NMR spectra in a reasonable time and preserves high resolution in indirectly detected dimensions. It is shown that the interpretation of 3D COSY–HMBC and 3D TOCSY–HSQC spectra is crucial in the structural analysis of prenol‐10. Copyright © 2009 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.     

Spatial structure of heptapeptide Aβ16–22 (beta‐amyloid Aβ1–40 active fragment) in solution and in complex with a biological membrane model

The spatial structure of an active fragment of beta‐amyloid Aβ_1–40 heptapeptide Aβ_16–22 (Lys‐Leu‐Val‐Phe‐Phe‐Ala‐Glu) in aqueous buffer solution and in complex with sodium dodecyl sulfate micelles as a model membrane system was investigated by ¹H NMR spectroscopy and two‐dimensional NMR (TOCSY, HSQC‐HECADE (Heteronuclear Couplings from ASSCI‐domain experiments with E.COSY‐type crosspeaks), NOESY) spectroscopy. Complex formation was confirmed by the chemical shift changes of the heptapeptide's ¹H NMR spectra, as well as by the signs and values of the NOE effects in different environments. We compared the spatial structure of the heptapeptide in borate buffer solution and in complex with a model of the cell surface membrane. Copyright © 2012 John Wiley & Sons, Ltd.     

Identification of metabolites in coriander seeds (Coriandrum Sativum L.) aided by ultrahigh resolution total correlation NMR spectroscopy

NMR is a fast method for obtaining a holistic snapshot of the metabolome and also offers quantitative information without separating the compounds present in a complex mixture. Identification of the metabolites present in a plant extract sample is a crucial step for all plant metabolomics studies. In the present work, we used various two dimensional (2D) NMR methods such as J-resolved NMR, total correlation spectroscopy (TOCSY), and heteronuclear single quantum coherence sensitivity enhanced NMR spectroscopy for the identification of 36 common metabolites present in Coriandrum sativum L. seed extract. The identified metabolites belong to the following classes: organic acids, amino acids, and carbohydrates. ¹H NMR spectra of such complex mixtures in general display tremendous signal overlap due to the presence of a large number of metabolites with closely resonating multiplet signals. This signal overlapping leads to ambiguity in an assignment, and hence, identification of metabolites becomes tedious or impossible in many cases. Therefore, the utility of pure-shift proton spectrum along the indirect (F₁) dimension of the F₁-PSYCHE-TOCSY spectrum is demonstrated for overcoming ambiguity in assignment of metabolites in crowded spectral regions from Coriandrum sativum L. seed extract sample. Because pure-shift NMR methods yield ultrahigh resolution spectrum (i.e., a singlet peak per chemical site) along one or more dimensions, such spectra provide better identification of metabolites compared with regular 2D TOCSY where signal overlap and peak distortions lead to ambiguity in the assignment. Nine metabolites were unambiguously assigned by pure-shift F₁-PSYCHE-TOCSY spectrum, which was unresolved in regular TOCSY spectrum.     

Triterpenoidal Secondary Metabolites from Lantana Camara Linn.

Three new pentacyclic triterpenoids, camarin ( 1 ), lantacin ( 2 ), and camarinin ( 3 ) were isolated from the aerial parts of Lantana camara Linn ., together with seven known compounds. The structures of the new constituents were elucidated by chemical transformation, HR‐EI mass spectrometry, and NMR spectroscopy, including 1D (¹H‐ and ¹³C‐NMR) and 2D (¹H,¹H‐COSY, NOESY, ¹H,¹H‐TOCSY, J‐resolved, HMQC, and HMBC) experiments.     

Characterization of three saponins from a fraction using 1D DOSY as a solvent signal suppression tool. Agabrittonosides E–F. Furostane Saponins from Agave brittoniana Trel. spp. Brachypus

A careful NMR analysis, especially by 1D TOCSY and 1D ROESY, of a refined saponin fraction allowed us to determine the structure of three saponins from a polar extract of Agave brittoniana Trel. spp. Brachypus leaves. The use of 1D DOSY for the suppression of the solvent signal was useful to obtain the chemical shifts of anomeric signals. A full assignment of the ¹H and ¹³C spectral data for the new saponins, agabrittonosides E–F (1–2) and the well‐known Karatavioside C (3) and their methoxyl derivatives, is reported. The structures were established using a combination of 1D and 2D (¹H, ¹H‐COSY, TOCSY, ROESY, g‐HSQC, g‐HMBC and g‐HSQC‐TOCSY) NMR techniques and ESI–MS. In addition, the methoxylation of these furostane saponins in the presence of MeOH was studied. Copyright © 2010 John Wiley & Sons, Ltd.     

Spectroscopic separation of 13C NMR spectra of complex isomeric mixtures by the CSSF‐TOCSY‐INEPT experiment

Isomeric mixtures from synthetic or natural origins can pose fundamental challenges for their chromatographic separation and spectroscopic identification. A novel 1D selective NMR experiment, chemical shift selective filter (CSSF)‐TOCSY‐INEPT, is presented that allows the extraction of ¹³C NMR subspectra of discrete isomers in complex mixtures without physical separation. This is achieved via CSS excitation of proton signals in the ¹H NMR mixture spectrum, propagation of the selectivity by polarization transfer within coupled ¹H spins, and subsequent relaying of the magnetization from ¹H to ¹³C by direct INEPT transfer to generate ¹³C NMR subspectra. Simple consolidation of the subspectra yields ¹³C NMR spectra for individual isomers. Alternatively, CSSF‐INEPT with heteronuclear long‐range transfer can correlate the isolated networks of coupled spins and therefore facilitate the reconstruction of the ¹³C NMR spectra for isomers containing multiple spin systems. A proof‐of‐principle validation of the CSSF‐TOCSY‐INEPT experiment is demonstrated on three mixtures with different spectral and structural complexities. The results show that CSSF‐TOCSY‐INEPT is a versatile, powerful tool for deconvoluting isomeric mixtures within the NMR tube with unprecedented resolution and offers unique, unambiguous spectral information for structure elucidation. Copyright © 2014 John Wiley & Sons, Ltd.     

Application of Pulsed Field Gradients in High-Resolution NMR Spectroscopy

In this paper the applicability of pulsed field gradients in high-resolution NMR spectroscopy are considered. Applications include solvent suppression, spectral editing of one- and two-dimensional (2D) NMR spectra and the induction of quadrature detection in the w₁, direction of many 2D experiments. The applications are analysed using simple product operator algebra. Particular attention is directed to discussing the experimental requirements of this technology in order that high-resolution NMR conditions are maintained throughout the experiment.     

A conformational NMR analysis of methymycin aglycones: complete and unambiguous assignments of stereochemically diverse glycosylated methymycin analogs by 1D and 2D NMR techniques and molecular modeling

The ¹H and ¹³C NMR spectra of 10‐deoxymethynolide (1), 8.9‐dihydro‐10‐deoxymethynolide (2) and its glycosylated derivatives (3–9) were analyzed using gradient‐selected NMR techniques, including 1D TOCSY, gCOSY, 1D NOESY (DPFGSENOE), NOESY, gHMBC, gHSQC and gHSQC‐TOCSY. The NMR spectral parameters (chemical shifts and coupling constants) of 1–9 were determined by iterative analysis. For the first time, complete and unambiguous assignment of the ¹H NMR spectrum of 10‐deoxymethynolide (1) has been achieved in CDCl₃, CD₃OD and C₆D₆ solvents. The ¹H NMR spectrum of 8,9‐dihydro‐10‐deoxymethynolide (2) was recorded in CDCl₃, (CD₃)₂CO and CD₃OD solutions to determine the conformation. NMR‐based conformational analysis of 1 and 2 in conjugation with molecular modeling concluded that the 12‐membered ring of the macrolactones may predominantly exist in a single stable conformation in all solvents examined. In all cases, a change in solvent caused only small changes in chemical shifts and coupling constants, suggesting that all glycosylated methymycin analogs exist with similar conformations of the aglycone ring in solution. Copyright © 2013 John Wiley & Sons, Ltd.     

Petrorhagiosides A – D,New γ‐Pyrone Derivatives from Petrorhagia saxifraga Link

Four novel γ‐pyrone (=4H‐pyran‐4‐one) metabolites, petrorhagiosides A–D, along with four known analogs, have been isolated from the MeOH extract of Petrorhagia saxifraga, a perennial herbaceous plant typical of Mediterranean vegetation. The structures of the new compounds were established on the basis of extensive spectroscopic analyses including 1D‐ an 2D‐NMR (¹H,¹H‐DQ‐COSY, TOCSY, HSQC, CIGAR‐HMBC, and HSQC‐TOCSY) experiments.     

Ultrafast Single-Scan 2D NMR Spectroscopic Detection of a PHIP-Hyperpolarized Protease Inhibitor

Two-dimensional NMR spectroscopy is one of the most important spectroscopic tools for the investigation of biological macromolecules. However, due to the low sensitivity of NMR spectroscopy, it takes usually from several minutes to many hours to record such spectra. Here, the possibility of detecting a bioactive derivative of the sunflower trypsin inhibitor-1 (SFTI-1), a tetradecapeptide, by combining parahydrogen-induced polarization (PHIP) and ultrafast 2D NMR spectroscopy is shown. The PHIP activity of the inhibitor was achieved by labeling with O-propargyl-l -tyrosine. In 1D PHIP experiments a signal enhancement of a factor of approximately 1200 compared to standard NMR was found. This enhancement permits measurement of 2D NMR correlation spectra of low-concentrated SFTI-1 in less than 10 seconds, employing ultrafast single-scan 2D NMR detection. As experimental examples PHIP-assisted ultrafast single-scan TOCSY spectra of SFTI-1 are shown.     

Positional Enrichment by Proton Analysis (PEPA): A One‐Dimensional 1H‐NMR Approach for 13C Stable Isotope Tracer Studies in Metabolomics

A novel metabolomics approach for NMR‐based stable isotope tracer studies called PEPA is presented, and its performance validated using human cancer cells. PEPA detects the position of carbon label in isotopically enriched metabolites and quantifies fractional enrichment by indirect determination of ¹³C‐satellite peaks using 1D‐¹H‐NMR spectra. In comparison with ¹³C‐NMR, TOCSY and HSQC, PEPA improves sensitivity, accelerates the elucidation of ¹³C positions in labeled metabolites and the quantification of the percentage of stable isotope enrichment. Altogether, PEPA provides a novel framework for extending the high‐throughput of ¹H‐NMR metabolic profiling to stable isotope tracing in metabolomics, facilitating and complementing the information derived from 2D‐NMR experiments and expanding the range of isotopically enriched metabolites detected in cellular extracts.     

NOAH: NMR Supersequences for Small Molecule Analysis and Structure Elucidation

Nested NMR experiments combining up to five conventional NMR pulse sequences into one supersequence are introduced. The core 2D NMR techniques routinely employed in small molecule NMR spectroscopy, such as HSQC, HMQC, HMBC, COSY, NOESY, TOCSY, and similar, can be recorded in a single measurement. In this way the data collection time may be dramatically reduced and sample throughput increased for basic NMR applications, such as structure elucidation and verification in synthetic, medicinal, and natural product chemistry.