Incredible Natural Abundance DoublE QUAntum Transfer Experiment Research Articles

PyINETA: Open-source platform for INADEQUATE-JRES integration in NMR metabolomics.

Annotating compounds with high confidence is a critical element in metabolomics. 13C-detection NMR experiment INADEQUATE (incredible natural abundance double-quantum transfer experiment) stands out as a powerful tool for structural elucidation, whereas this valuable experiment is not often included in metabolomics studies. This is partly due to the lack of community platform that provides structural information based INADEQUATE. Also, it is often the case that a single study uses various NMR experiments synergistically to improve the quality of information or balance total NMR experiment time, but there is no public platform that can integrate the outputs of INADEQUATE and other NMR experiments either. Here, we introduce PyINETA, Python-based INADEQUATE network analysis. PyINETA is an open-source platform that provides structural information of molecules using INADEQUATE, conducts database search, and integrates information of INADEQUATE and a complementary NMR experiment 13C J-resolved experiment (13C-JRES). Those steps are carried out automatically, and PyINETA keeps track of all the pipeline parameters and outputs, ensuring the transparency of annotation in metabolomics. Our evaluation of PyINETA using a model mouse study showed that our pipeline successfully integrated INADEQUATE and 13C-JRES. The results showed that 13C-labeled amino acids that were fed to mice were transferred to different tissues, and, also, they were transformed to other metabolites. The distribution of those compounds was tissue-specific, showing enrichment of particular metabolites in liver, spleen, pancreas, muscle, or lung. The value of PyINETA was not limited to those known compounds; PyINETA also provided fragment information for unknown compounds. PyINETA is available on NMRbox.

Revealing the roles of hydrocarbon pool mechanism in ethanol-to-hydrocarbons reaction

Ethanol-to-hydrocarbons (ETH) process, is investigated in this work due to the requirement of sustainable production route for light olefins such as ethene and propene. Majority of the mechanism insights of ETH process are principally proposed based on the methanol-to-hydrocarbons (MTH) process, which involves a homologous reaction system. The reaction intermediate species including cyclopentenyl cations and aromatics species, which are denoted as “hydrocarbon pool (HCP)” species in MTH process, were discovered and identified in the ETH process by in situ solid-state 13C NMR (ssNMR) experiments, advanced 2D 13C–13C INADEQUATE (Incredible Natural Abundance DoublE QUAntum Transfer Experiment) ssNMR experiment, 12C/13C-C2H5OH isotope switching experiments complemented by gas chromatography-mass spectroscopy (GC–MS). Especially, in the ETH reaction, ethylcyclopentenyl cations and their deprotonated form were first captured and exhibited relatively higher activity in the formation of ethene and propene. Multi-routes with the participation of these active intermediates were proposed and evaluated by density functional theory calculation (DFT), demonstrating that they can play important roles in the formation of olefins. Ethene formation are mainly formed from ethanol dehydration, whereas propene can be produced via multi-routes with the participation of the captured HCP species. Moreover, the detailed reaction routes may be modulated by the temperature. This work provides direct evidences of the critical function of HCP species in the ETH process and reveals the mechanism of olefin formation.

Application of INADEQUATE NMR techniques for directly tracing out the carbon skeleton of a natural product

Nuclear magnetic resonance (NMR) measurement of 1 JCC coupling by two-dimensional (2D) INADEQUATE (incredible natural abundance double quantum transfer experiment), which is a special case of double-quantum (DQ) spectroscopy that offers unambiguous determination of 13 C-13 C spin-spin connectivities through the DQ transitions of the spin system, is especially suited to solving structures rich in quaternary carbons and poor in hydrogen content (Crews rule). To review published literature on the application of NMR methods to determine structure in the liquid-state, which specifically considers the interaction of a pair of carbon-13 (13 C) nuclei adjacent to one another, to allow direct tracing out of contiguous carbon connectivity using 2D INADEQUATE. A comprehensive literature search was implemented with various databases: Web of Knowledge, PubMed and SciFinder, and other relevant published materials including published monographs. The keywords used, in various combinations, with INADEQUATE being present in all combinations, in the search were 2D NMR, 1 JCC coupling, natural product, structure elucidation, 13 C-13 C connectivity, cryoprobe and CASE (computer-assisted structure elucidation)/PANACEA (protons and nitrogen and carbon et alia). The 2D INADEQUATE continues to solve "intractable" problems in natural product chemistry, and using milligram quantities with cryoprobe techniques combined with CASE/PANACEA experiments can increase machine time efficiency. The 13 C-13 C-based structural elucidation by dissolution single-scan dynamic nuclear polarisation NMR can overcome disadvantages of 13 C insensitivity at natural abundance. Selected examples have demonstrated the trajectory of INADEQUATE spectroscopy from structural determination to clarification of metabolomics analysis and use of DFT (density functional theory) and coupling constants to clarify the connectivity, hybridisation and stereochemistry within natural products. Somewhat neglected over the years because of perceived lack of sensitivity, the 2D INADEQUATE NMR technique has re-emerged as a useful tool for solving natural products structures, which are rich in quaternary carbons and poor in hydrogen content.

Natural Abundance, Single-Scan 13C-13C-Based Structural Elucidations by Dissolution DNP NMR.

While 13C-based Incredible Natural Abundance DoublE QUAntum Transfer Experiment (INADEQUATE) experiments offer an attractive alternative for establishing molecular structures, they suffer from low sensitivities arising from the scarcity of spin pairs present at natural abundance. Herein we demonstrate that dissolution dynamic nuclear polarization (dDNP) provides sufficient sensitivity to acquire 1D 13C INADEQUATE spectra in a single scan and at natural abundance. Moreover, if steps are adopted to endow sub-Hertz precision to these measurements, they allow one to measure carbon-carbon J couplings over both one and multiple bonds for each chemical site. As these JCC-couplings are usually sufficiently distinct to enable univocal pairing of the nuclei involved, essentially the same information as in 2D INADEQUATE can be obtained. The feasibility of the method is demonstrated for a range of compounds, including natural products such as α-pinene, menthol and limonene. Features and extensions of this approach are briefly discussed.

Correlation Spectroscopy (COSY), Exclusive Correlation Spectroscopy (ECOSY), Total Correlation Spectroscopy (TOCSY), Incredible Natural-Abundance Double-Quantum Transfer Experiment (INADEQUATE), Heteronuclear Single-Quantum Correlation Spectroscopy (HSQC), Heteronuclear Multiple-Bond Correlation Spectroscopy (HMBC), Nuclear Overhauser Effect Spectroscopy (NOESY) and Rotating Frame Nuclear Overhauser Effect Spectroscopy (ROESY) Comparative Study on Malignant and Benign Human Cancer Cells and Tissues Under Synchrotron Radiation

In the current study, we have experimentally and comparatively investigated and compared malignant human cancer cells and tissues before and after irradiating of synchrotron radiation using Correlation Spectroscopy (COSY), Exclusive Correlation Spectroscopy (ECOSY), Total Correlation Spectroscopy (TOCSY), Incredible Natural-Abundance Double-Quantum Transfer Experiment (INADEQUATE), Heteronuclear Single-Quantum Correlation Spectroscopy (HSQC), Heteronuclear Multiple-Bond Correlation Spectroscopy (HMBC), Nuclear Overhauser Effect Spectroscopy (NOESY) and Rotating Frame Nuclear Overhauser Effect Spectroscopy (ROESY).

Preliminary Quality Evaluation and Characterization of Phenolic Constituents in Cynanchi Wilfordii Radix.

A new phenolic compound, 2-O-β-laminaribiosyl-4-hydroxyacetophenone (1), was isolated from Cynanchi Wilfordii Radix (CWR, the root of Cynanchum wilfordii Hemsley), along with 10 known aromatic compounds, including cynandione A (2), bungeisides-C (7) and –D (8), p-hydroxyacetophenone (9), 2′,5′-dihydroxyacetophenone (10), and 2′,4′-dihydroxyacetophenone (11). The structure of the new compound (1) was elucidated using spectroscopic methods and chemical methods. The structure of cynandione A (2), including a linkage mode of the biphenyl parts that remained uncertain, was unambiguously confirmed using the 2D 13C–13C incredible natural abundance double quantum transfer experiment (INADEQUATE) spectrum. Additionally, health issues related to the use of Cynanchi Auriculati Radix (CAR, the root of Cynanchum auriculatum Royle ex Wight) instead of CWR have emerged. Therefore, constituents present in methanolic extracts of commercially available CWRs and CARs were examined using UV-sensitive high-performance liquid chromatography (HPLC), resulting in common detection of three major peaks ascribed to cynandione A (2), p-hydroxyacetophenone (9), and 2′,4′-dihydroxyacetophenone (11). Thus, to distinguish between these ingredients, a thin-layer chromatography (TLC) method, combined with only UV irradiation detection, focusing on wilfosides C1N (12) and K1N (13) as marker compounds characteristic of CAR, was performed. Furthermore, we propose this method as a simple and convenient strategy for the preliminary distinction of CWR and CAR to ensure the quality and safety of their crude drugs.

Incredible natural–abundance double–quantum transfer experiment (INADEQUATE), nuclear overhauser effect spectroscopy (NOESY) and rotating frame nuclear overhauser effect spectroscopy (ROESY) comparative study on malignant and benign human cancer cells and tissues under synchrotron radiation

Incredible natural–abundance double–quantum transfer experiment (INADEQUATE), nuclear overhauser effect spectroscopy (NOESY) and rotating frame nuclear overhauser effect spectroscopy (ROESY) comparative study on malignant and benign human cancer cells and tissues under synchrotron radiation

A novel approach to correlation spectroscopy (COSY), exclusive correlation spectroscopy (ECOSY), total correlation spectroscopy (TOCSY), incredible natural–abundance double–quantum transfer experiment (INADEQUATE), heteronuclear single–quantum correlation spectroscopy (HSQC), heteronuclear multiple–bond correlation spectroscopy (HMBC), nuclear overhauser effect spectroscopy (NOESY) and rotating frame nuclear overhauser effect spectroscopy (ROESY) comparative study on malignant and benign human cancer cells and tissues under synchrotron radiation

A novel approach to correlation spectroscopy (COSY), exclusive correlation spectroscopy (ECOSY), total correlation spectroscopy (TOCSY), incredible natural–abundance double–quantum transfer experiment (INADEQUATE), heteronuclear single–quantum correlation spectroscopy (HSQC), heteronuclear multiple–bond correlation spectroscopy (HMBC), nuclear overhauser effect spectroscopy (NOESY) and rotating frame nuclear overhauser effect spectroscopy (ROESY) comparative study on malignant and benign human cancer cells and tissues under synchrotron radiation

(13)C NMR Metabolomics: INADEQUATE Network Analysis.

The many advantages of (13)C NMR are often overshadowed by its intrinsically low sensitivity. Given that carbon makes up the backbone of most biologically relevant molecules, (13)C NMR offers a straightforward measurement of these compounds. Two-dimensional (13)C-(13)C correlation experiments like INADEQUATE (incredible natural abundance double quantum transfer experiment) are ideal for the structural elucidation of natural products and have great but untapped potential for metabolomics analysis. We demonstrate a new and semiautomated approach called INETA (INADEQUATE network analysis) for the untargeted analysis of INADEQUATE data sets using an in silico INADEQUATE database. We demonstrate this approach using isotopically labeled Caenorhabditis elegans mixtures.

Structures of Storage‐Induced Transformation Products of the Beer’s Bitter Principles, Revealed by Sophisticated NMR Spectroscopic and LC–MS Techniques

Besides undesirable changes in the attractive aroma, a significant decrease in the intensity of the bitterness as well as a change of the taste into a lingering, harsh bitterness has long been known as a shelf-life limiting factor of beer. Multiple studies have demonstrated that the aging of beer induces a decrease of the total amount of cis- and trans-iso-alpha-acids, the well-known bitter principles of beer. Although the trans-iso-alpha-acids exclusively, not the cis-iso-alpha-acids, were found to be degraded upon storage of beer, the key transformation products formed exclusively from the trans isomers in beer are not known. In the present study, suitable model experiments followed by LC-MS/MS and sophisticated NMR spectroscopic experiments, including the measurement of residual dipolar couplings (RDCs) in gel-based alignment media as well as a novel broadband and B(1)-field-compensated incredible natural abundance double-quantum transfer experiment (INADEQUATE) pulse sequence, enabled the identification of a series of previously unknown trans-specific iso-alpha-acid transformation products, namely, tricyclocohumol, tricyclocohumene, isotricyclocohumene, tetracyclocohumol, and epitetracyclocohumol, respectively. HPLC-MS/MS analysis of these compounds, which exhibit the aforementioned harsh lingering bitter taste and have threshold concentrations ranging from 5 to 70 micromol L(-1), confirmed their generation during aging of beer and, for the first time, explained the storage-induced changes of the beer's bitter taste on a molecular level.

Structural studies on aryl‐substituted enaminoketones and their thio analogues. Part I. Analysis of high‐resolution 1H, 13C NMR and 13C CP MAS spectra combined with GIAO‐DFT calculations

A series of aryl-substituted enaminoketones and their thio analogues in CDCl(3) solution and in the solid state were studied by the use of high-resolution (1)H and (13)C as well as (13)C cross polarization magic angle spinning (CP MAS) NMR spectra in combination with gauge including atomic orbitals-density functional theory (GIAO-DFT) calculations performed at the B3PW91/6-311 + + G(d,p) level of theory using the B3PW91/6-311 + + G(d,p)-optimized geometries. The analysis of the (13)C NMR spectra in solution was done by using the Incredible Natural Abundance DoublE QUAntum Transfer Experiment (INADEQUATE) technique, whereas trends observed in the (13)C shielding constants, calculated for the compounds studied, were a great help in assigning most of the signals in the (13)C CP MAS NMR spectra. It was established on the basis of the experimental and theoretical NMR data that both groups of compounds exist in the form of Z-s-Z-s-E isomers in CDCl(3) solution as well as in the solid state, with the NH hydrogen atom involved in intramolecular hydrogen bonding. This conclusion is in agreement with the fact that some of the compounds studied reveal liquid-crystalline properties. Three-bond H, H and C, H coupling constants measured in solution played a crucial role in the structure elucidation.

13C‐detected IPAP‐INADEQUATE for simultaneous measurement of one‐bond and long‐range scalar or residual dipolar coupling constants

The sensitivity of cryoprobes, which are rapidly becoming available, means that the measurement of coupling constants involving 13C, 13C pairs at the natural abundance of 13C can now, in principle, be done by using tens rather then hundreds of milligrams of compounds. However, a robust method that would yield reliable values of small long-range carbon--carbon coupling constants is still missing. In this Communication, we describe a novel 13C-detected incredible natural-abundance double-quantum transfer experiment (INADEQUATE) experiment for simultaneous correlation of one-bond and long-range 13C- 13C pairs and the measurement of both types of coupling constants in 13C natural abundance samples. This method yields accurate values of one-bond and long-range coupling constants by manipulation of pure phase in-phase (IP) and antiphase (AP) doublets, and is referred to as 13C-detected IPAP-INADEQUATE. It is illustrated by the measurement of interglycosidic (3)J(CCOC) coupling constants in a disaccharide molecule providing important information about the conformation of the glycosidic linkage. Owing to the simplicity of INADEQUATE spectra the carbon-carbon coupling constants are particularly suitable for studies of partially oriented molecules through the measurement of carbon-carbon residual dipolar couplings (RDCs). An example of this approach is presented. We expect the method to find a variety of applications in the conformational analysis of small molecules, determination of diastereoisomers and enantiomers, and studies of molecules in aligned media.

Through-space contributions to two-dimensional double-quantum J correlation NMR spectra of magic-angle-spinning solids

A routinely used assumption when interpreting two-dimensional NMR spectra obtained with a commonly used double-quantum (DQ) magic-angle-spining (MAS) pulse sequence referred to as the refocused incredible natural abundance double-quantum transfer experiment (INADEQUATE) [A. Lesage, M. Bardet, and L. Emsley, J. Am. Chem. Soc. 121, 10987 (1999)] has been that correlation peaks are only observed for pairs of nuclei with a through-bond connectivity. The validity of this assumption is addressed here by theory, experiment, and computer simulations. If the isotropic chemical shifts of the two nuclei are different and the MAS frequency is far from rotational resonance, the theoretical description demonstrates that DQ correlation peaks are indeed indicative of a J coupling. However, if the isotropic chemical shifts are the same, it is shown that DQ peaks can appear for pairs of nuclei even in the absence of a through-bond J coupling. These peaks appear in the specific case of a pair of nuclei with a nonzero through-space dipole-dipole coupling and chemical shift anisotropy tensors having different principal magnitudes or orientations, provided that the MAS frequency is comparable to or smaller than the chemical shift anisotropies. Experimental 31P spectra recorded on a sample of TiP2O7 and computer simulations show that the magnitude of these anomalous peaks increases with increasing B0 magnetic field and that they decrease with increasing MAS frequency. This behavior is explained theoretically.

Connectivities in molecules by INADEQUATE: recent developments

AbstractThe INADEQUATE (Incredible Natural Abundance DoublE QUAntum Transfer Experiment) consists of a pulse sequence which eliminates the NMR signals from isolated spins (spin system A) displaying signals from coupled spins (spin system AX or higher spin systems). It is of great importance when applied to molecules with skeleton elements such as carbon, silicon or tungsten, all of which contain a small percentage of spin‐½ isotopes embedded in magnetically inactive isotopes. Analysis of the AX type spectra gives one‐bond and long‐range coupling constants (see compound 6) and, most important, the connectivity pattern of the skeleton atoms in molecules of unknown structure such as the carbon compounds 1 or 2, the silicon compound 3 or the lithium compound 4b. Unfortunately, INADEQUATE is rather insensitive, in the case of carbon only one out of 104 molecules gives the desired response. Efforts to reduce this drawback are described; recently, a remarkable step forward has been made by concentrating the four lines of an AX spin system to just two signals (see Fig. 2). Copyright © 2001 John Wiley & Sons, Ltd.

Cyclopolymerization: XXVI. Repeating unit structure of cyclopolymers derived from N-substituted-N-allyl-2-(methoxycarbonyl)allylamines and mechanism of intramolecular cyclization

NMR studies on completely cyclized polymers derived from N-substituted-N-allyl-2-(methoxycarbonyl)allylamines (1) were undertaken to determine the repeat cyclic unit. NMR techniques employed are two-dimensional INADEQUATE (Incredible Natural Abundance DoublE QUAntum Transfer Experiment), DEPT (Distortion Enhancement by Polarization Transfer) measurements, and those under suppressed nuclear Overhauser effect. These studies along with the comparison of the spectra of poly(1) with those of related compounds revealed that the poly(1) consists of two sets of five-membered rings. The main chain methylene carbons attached to the ring has a trans configuration for one of them and a cis arrangement for the other. The former was found to be a main component. The value 4.5kJ/mol as the difference of the activation energy for the formation of the respective configuration, Ecis−Etrans, was obtained by the investigation of temperature effect. Comparison of these results with those reported so far for polymers with similar structures led to the conclusion that five-membered ring formation could be interpreted by a kinetic mechanism as in the radical cyclization reactions in low molecular weight compounds.

Clean Selective Spin-Locking Spectra Using Pulsed Field Gradients

This chapter presents the introduction of two-dimensional (2D) methods, specifically in the context of determining homonuclear correlations. This is so that the 2D concept is introduced with a realistically useful experiment in mind, although it also becomes apparent that the general principles involved are common to any 2D experiment. Following this, a variety of correlation techniques are presented for identifying scalar (J) couplings among homonuclear spins, which for the most part means protons although methods for correlating spins of low abundance nuclides such as carbon are also discussed. Thes chapter introduces the operation and use of pulsed field gradients. The techniques themselves begin with basic correlation spectroscopy (COSY), which maps nuclei sharing a mutual scalar coupling within a molecule, making the proton–proton COSY one of the workhorse techniques in organic structure elucidation. In the chapter, some variants of the basic COSY experiment that display a number of beneficial characteristics are presented. The chapter discusses techniques such as total correlation spectroscopy (TOCSY), which provides an alternative to the COSY approach for establishing correlations within a molecule. This provides efficient transfer of information along a network of coupled spins, a feature that can be extremely powerful in the analysis of more complex spectra. The final method—incredible natural-abundance double-quantum transfer experiment (INADEQUATE)—establishes correlations between like spins of low natural abundance.

INADEQUATE NMR spectra of extremely strongly coupled spin systems

A novel product operator formalism for strongly coupled two-spin systems was developed which allows the time evolution for such systems to be estimated much more easily than using the density matrix. The newly developed theory was applied to describe the INADEQUATE (Incredible Natural Abundance DoublE QUAntum Transfer Experiment) experiments, and the condition for the efficiency of the creation of the double-quantum coherences was formulated, which is useful for setting the experimental conditions. The refocused INADEQUATE spectra of benzyl bromide, in which two one-bond 13C—13C pairs have extremely strong couplings compared with the chemical shift differences, were observed under several experimental conditions. The signals attributable to these strongly coupled spin pairs were detected with reasonably large intensities, and their coupling constants were determined by simulation of the spectral intensities; the weak and the long-range couplings in the compound were also obtained.