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Chirality plays a fundamental role in nature, but its detection and quantification still face many limitations. To date, the enantiospecific analysis of mixtures necessarily requires prior separation of the individual components. The simultaneous enantiospecific detection of multiple chiral molecules in a mixture represents a major challenge, which would lead to a significantly better understanding of the underlying biological processes; for example, via enantiospecifically analysing metabolites in their native environment. Here, we report on the first in situ enantiospecific detection of a thirty-nine-component mixture. As a proof of concept, eighteen essential amino acids at physiological concentrations were simultaneously enantiospecifically detected using NMR spectroscopy and a chiral solvating agent. This work represents a first step towards the simultaneous multicomponent enantiospecific analysis of complex mixtures, a capability that will have substantial impact on metabolism studies, metabolic phenotyping, chemical reaction monitoring, and many other fields where complex mixtures containing chiral molecules require efficient characterisation., (© 2020 Wiley-VCH GmbH.)

Homonuclear 13 C- 13 C couplings at natural abundance can be measured using the J-modulated Adequate DoublE QUAntumTransfer Experiment (ADEQUATE) experiment. To somewhat ameliorate F 1 digitization requirements, a scaling factor was incorporated into the original pulse sequence. Non-uniform sampling provides an obvious avenue to further facilitate the acquisition of 1 J CC and n J CC homonuclear coupling constant data. We introduce homonuclear decoupling (HD) analogous to that described for the 1,1-HD-ADEQUATE and 1,n-HD-ADEQUATE experiments and evaluate the combination of non-uniform sampling and HD on the acquisition of both 1 J CC and n J CC homonuclear 13 C- 13 C coupling constants using ibuprofen as a model compound. Copyright © 2015 John Wiley & Sons, Ltd., (Copyright © 2015 John Wiley & Sons, Ltd.)

Cryptospirolepine is the most structurally complex alkaloid discovered and characterized thus far from any Cryptolepis specie. Characterization of several degradants of the original, sealed NMR sample a decade after the initial report called the validity of the originally proposed structure in question. We now report the development of improved, homodecoupled variants of the 1,1- and 1,n-ADEQUATE (HD-ADEQUATE) NMR experiments; utilization of these techniques was critical to successfully resolving long-standing structural questions associated with crytospirolepine., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

NMR-aided enantiodiscrimination using chiral auxiliaries (CAs) is a recognized method for differentiating enantiomers and for measuring enantiomeric ratios (er). Up to the present, the study, optimization, and comparison of such methods have been performed based on the enantiodifferentiation of NMR signals via analyzing non-equivalent chemical-shift values (ΔΔδ) of the diastereoisomeric species formed. However, a poor and non-reliable comparison of results is often obtained via the analysis of ΔΔδ exclusively. In here, the concept of enantioresolution of an individual NMR signal and its importance for NMR-aided enantiodifferentiation studies is introduced and discussed. In addition, the enantioresolution quotient, E, is proposed as the parameter to describe its quantification. Complementary to measuring ΔΔδ, the experimental determination of E allows a more reliable interpretation of the results and opens up new possibilities for the study of enantiodifferentiation data derived from novel NMR experiments, setup improvements or new CAs. Finally, the different relationships between signal enantiodifferentiation, signal enantioresolution, and other main experimental issues of enantiodifferentiation experiments are addressed., (Copyright © 2015 Elsevier B.V. All rights reserved.)

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., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

NMR enantiodifferentiation studies are greatly improved by the simultaneous determination of (1)H and (13)C chemical shift differences through the analysis of highly resolved cross-peaks in spectral aliased pure shift (SAPS) HSQC spectra.

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., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The implementation of the HOmodecoupled Band-Selective (HOBS) technique in the conventional Inversion-Recovery and CPMG-based PROJECT experiments is described. The achievement of fully homodecoupled signals allows the distinction of overlapped (1)H resonances with small chemical shift differences. It is shown that the corresponding T1 and T2 relaxation times can be individually measured from the resulting singlet lines using conventional exponential curve-fitting methods., (Copyright © 2014 Elsevier Inc. All rights reserved.)

An NMR method to enhance the sensitivity and resolution in band-selective long-range heteronuclear correlation spectra is proposed. The excellent in-phase nature of the selHSQMBC experiment allows that homonuclear and/or heteronuclear decoupling can be achieved in the detected dimension of a 2D multiple-bond correlation map, obtaining simplified cross-peaks without their characteristic fine J multiplet structure. The experimental result is a resolution improvement while the highest sensitivity is also achieved. Specifically, it is shown that the (1)H-homodecoupled band-selective (HOBS) HSQMBC experiment represents a new way to measure heteronuclear coupling constants from the simplified in-phase doublets generated along the detected dimension., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The utility of (13)C NMR spectroscopy for the differentiation of enantiomers using chiral solvating agents (CSA) is stated. Three examples involving the enantiodifferentiation of a drug, a metabolite and a reactant in aqueous and organic solutions have been chosen to show it. The intrinsic high dispersion of (13)C nucleus, as well as the singlet nature of the signals in standard experiments makes (13)C NMR experiments a powerful alternative or complement to (1)H NMR experiments; specially, when studying pure compounds with complex proton spectra or mixtures of compounds, as in chiral metabonomics, where severe overlapping exists in proton spectrum. To evaluate and compare the quality of the enantioresolution of a NMR signal we introduce the enantiodifferentiation quotient, E, that considers the complexity of (1)H multiplets (and in general the width) of the original signal. It has been observed that the error in the measurement of the enantiomeric molar ratio can be related to the E value. The sensitivity and experimental time of a wide range of chiral analyte concentrations were also assessed.

The effects of phase modulation due to homonuclear proton-proton coupling constants in HSQMBC-IPAP and HMBC-IPAP experiments are experimentally evaluated. We show that accurate values of small proton-carbon coupling constants, (n)J(CH), can be extracted even for phase-distorted cross-peaks obtained from a selHSQMBC experiment applied simultaneously on two mutually J-coupled protons. On the other hand, an assessment of the reliability of (n)J(CH) measurement from distorted cross-peaks obtained in broadband IPAP versions of equivalent HMBC and HSQMBC experiments is also presented. Finally, we show that HMBC-COSY experiments could be an excellent complement to HMBC for the measurement of small (n)J(CH) values., (Copyright © 2013 John Wiley & Sons, Ltd.)

A general review of novel NMR methods to measure heteronuclear long-range proton-carbon coupling constants ((n)JCH; n>1) in small molecules is made. NMR experiments are classified in terms of NMR pulse scheme and cross-peak nature. A discussion about simplicity, general applicability and accuracy for each particular NMR experiment is presented and exemplified. Important aspects such as the sign determination and measurement of very small coupling values involving protonated and non-protonated carbons as well as the complementarity between different experiments are also discussed. Finally, a compilation of applications in structural and conformational analysis of different types of molecules since 2000 is surveyed., (Copyright © 2013 Elsevier B.V. All rights reserved.)

A user-friendly 2D NMR approach denoted as CLIP-HSQMBC is proposed for the very easy, direct and accurate measurement of long-range proton-carbon coupling constants in organic molecules and natural products. The J value can be extracted directly from the analysis of resolved in-phase (1)H multiplets that show an additional splitting arising from the proton-carbon coupling. In cases of unresolved peaks, a simple fitting analysis using the internal satellite lines as a reference is performed. Addition of a spin-lock period results in a CLIP-HSQMBC-TOCSY experiment that is suitable for the measurement of very small coupling values or to observe correlations from overlapped resonances.

A long-range optimized P.E.HSQC experiment, named P.E.HSQMBC, is proposed for the simultaneous measurement of a complete set of homonuclear and heteronuclear coupling constants from a single 2D cross-peak. The sign and the magnitude of proton-proton coupling constants are measured along the direct dimension from the relative E.COSY-type multiplet pattern displacement due to the passive one-bond coupling constant splitting generated in the indirect dimension. On the other hand, long-range proton-carbon coupling constants are independently determined in the detected dimension from a traditional fitting analysis of antiphase multiplet patterns or, more conveniently, from the IPAP multiplet displacement obtained from extended HSQMBC experiments., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Differences in molecular chirality remain an important issue in drug metabolism and pharmacokinetics for the pharmaceutical industry and regulatory authorities, and chirality is an important feature of many endogenous metabolites. We present a method for the rapid, direct differentiation and identification of chiral drug enantiomers in human urine without pretreatment of any kind. Using the well-known anti-inflammatory chemical ibuprofen as one example, we demonstrate that the enantiomers of ibuprofen and the diastereoisomers of one of its main metabolites, the glucuronidated carboxylate derivative, can be resolved by (1)H NMR spectroscopy as a consequence of direct addition of the chiral cosolvating agent (CSA) β-cyclodextrin (βCD). This approach is simple, rapid, and robust, involves minimal sample manipulation, and does not require derivatization or purification of the sample. In addition, the method should allow the enantiodifferentiation of endogenous chiral metabolites, and this has potential value for differentiating metabolites from mammalian and microbial sources in biofluids. From these initial findings, we propose that more extensive and detailed enantiospecific metabolic profiling could be possible using CSA-NMR spectroscopy than has been previously reported.

A simple proton-selective α/β-HSQMBC experiment is proposed for the accurate measurement of long-range proton-carbon coupling constants (nJCH) in small molecules without need for an individualized and time-consuming post-processing fitting procedure. The method acquires two pure-phase In-phase (IP) and Anti-phase (AP) multiplets completely free of any phase distortion due to the absence of JHH evolution. Accurate nJCH values can be directly measured analyzing the relative displacement of the resulting IPAP cross-peaks. Discussion about signal intensity dependence and cross-talk is made for a range of experimental conditions. The robustness of the method is evaluated by comparing the nJCH value measured from the analysis of the three available IP, AP and IPAP multiplet patterns. Multiple-frequency and region-selective versions of the method can also be efficiently recorded provided that excited protons are not mutually coupled., (Copyright © 2011 Elsevier Inc. All rights reserved.)

A new NMR approach is proposed for the measurement of long-range heteronuclear coupling constants ((n)J(XH), n>1) in natural abundance molecules. Two complementary in-phase (IP) and anti-phase (AP) data are separately recorded from a modified HSQMBC experiment and then added/subtracted to provide spin-state-selective α/β-HSQMBC spectra. The magnitude of (n)J(XH) can be directly determined by simple analysis of the relative displacement between α- and β-cross-peaks. The robustness of this IPAP-HSQMBC experiment is evaluated experimentally and by simulation using a variety of different conditions. Important aspects such as signal intensity dependence and presence of unwanted cross-talk effects are discussed and examples on the measurement of small proton-carbon ((n)J(CH)) and proton-nitrogen ((n)J(NH)) coupling constants are provided., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Improved pulsed-field gradient echo methods are presented and discussed for the direct selective excitation of the (13)C-satellite lines in overcrowded (1)H NMR spectra of small molecules. Sensitivity enhancements in (13)C spin-state selection can be achieved by combining multiple-proton-frequency excitation and Hadamard phase encoding. Several satellite-selective (SATSEL) NMR experiments are proposed and exemplified by measuring the sign and the magnitude of small, long-range proton-carbon coupling constants for (1)H resonances showing several levels of signal overlapping., (2008 John Wiley & Sons, Ltd.)

An out-and-stay 2D proton-proton NMR correlation experiment is proposed to detect long-range proton-proton connectivities up to six and seven bonds away. The magnetization flow pathway is based on a consecutive, dual-step J(CH)-transfer mechanism and it allows one to trace out (1)H-(1)H connectivities between protons belonging to different spin systems. This novel experimental scheme will be particularly useful in cases when carbon resonances overlap, providing connectivity information that could not be obtained in a HMBC experiment. The success of the experiment is demonstrated in the structural studies of a wide variety of chemical compounds., (2008 John Wiley & Sons, Ltd.)

This study presents a simple method for measuring long-range heteronuclear coupling constants between protons and proton-bearing carbons. The approach involves recording two conventional 1D-TOCSY experiments in which the offset of the selective proton pulse is set on the low- and high-frequency 13C satellites of an isolated proton signal, Hi. Long-range heteronuclear coupling values between the 13Ci bonded to Hi and the protons Hj,k...n that belong to the same spin system were easily determined from the relative displacement of the relayed Hj,k...n signals in the satellite-selective TOCSY spectra. The sense of the displacement indicated the sign of the coupling constants.

New spin-state-selective (S3) NMR pulse sequences exclusively applying cross-polarization schemes to achieve optimum homonuclear and heteronuclear 1H-X coherence transfer are reported for the simple and accurate measurement of the magnitude and sign of heteronuclear coupling constants for samples at natural abundance. The proposed spin-edited HCP-TOCSY experiments are based on clean heteronuclear S3 excitation, generated by simultaneous co-addition of two independent in-phase and anti-phase components created during the mixing heteronuclear J-cross-polarization (HCP) step, which is finally transferred to other protons by a conventional homonuclear TOCSY mechanism. Selective 1D and non-selective 2D approaches for the easy determination of long-range proton-carbon and proton-nitrogen coupling constants on any protonated and non-protonated heteronuclei are presented and discussed for several organic molecules., (Copyright 2004 John Wiley & Sons, Ltd.)

A suite of multiple-purpose sensitivity-enhanced 2D correlation NMR experiments based on heteronuclear J-cross polarization (HCP) techniques are introduced for isotropic liquid samples. Several pulse sequences using an adaptable heteronuclear TOCSY mixing building block are proposed for different types of effective coherence-order-selective (COS) heteronuclear coherence-transfer mechanisms. They are based on the anisotropic behaviour of the involved HCP process that is easily described and analysed in terms of cartesian product-operator formalism. A number of different versions are given for in-phase to in-phase (II-COS: S (-) \\ to I (-)), in-phase to anti-phase (IA-COS: S (-) \\ to 2 I (-) S (z)), in-phase to spin-state-selective (IS(3)-COS: S (-) \\ to 2 I (-) S (alpha /beta)), anti-phase to in-phase (AI-COS: 2 I (z) S (-) \\ to I (-)), anti-phase to anti-phase (AA-COS: 2 I (z) S (-) \\ to 2 I (-) S (z)), anti-phase to spin-state-selective (AS(3)-COS: 2 I (z) S (-) \\ to 2 I (-) S (alpha /beta)) and spin-state-selective to spin-state-selective (S(3)S(3)-COS: 2 I (alpha /beta) S (-) \\ to 2 I (-) S (alpha /beta )) coherence transfers. The combination of the echo/anti-echo approach, heteronuclear gradient echoes and the preservation of equivalent pathways (PEP) methodology affords a general approach to obtain sensitivity-enhanced pure-absorption 2D spectra that can be used as interesting alternatives to conventional pulse-interrupted free-precession INEPT-based pulse schemes, such as HSQC-type and TROSY-type experiments.

A very simple strategy is proposed to extract carbon multiplicity information along with the classic knowledge of carbon-carbon connectivities in ADEQUATE experiments without affecting the sensitivity ratios of the original pulse schemes. These new multiplicity-edited ADEQUATE experiments prove to be highly helpful for complete 1H and 13C resonance assignment and also for automated and easy spin system characterization of samples at natural abundance, using a single NMR experiment.