Selected NMR Works ...

Simultaneous Enantiospecific Detection of Multiple Compounds in Mixtures using NMR Spectroscopy

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; e.g. 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. 

LR-selHSQMBC: Simultaneous detection and quantification of very weak long-range heteronuclear NMR correlations   

The optimum detection and accurate measurement of longer‐range (4J and higher) heteronuclear NMR correlations is described. The magnitude and/or the sign of a wide range of large and small long‐range couplings can be simultaneously determined for protonated and non‐protonated 13C and 15N nuclei using the LR‐selHSQMBC experiment.  

Enhancing the utility of 1JCH coupling constants in structural studies through optimized DFT analysis 

Commonly used DFT methods for the calculation of 1JCH coupling constants have typically required the application of ad hoc correction factors, modification of functionals, or empirical scaling to improve the fit between predicted and experimental values. Here we demonstrate that highly accurate 1JCH coupling predictions can be obtained without such adjustments by careful selection of DFT methods for geometry optimization and J-coupling calculations (e.g. B3LYP/6-31G(d,p)(mixed)//mPW1PW/cc-pVTZ). The proposed method was cross-validated against a diverse set of 122 1JCH couplings and was successfully applied to the conformational and stereochemical analysis of strychnine and a previously unreported trachylobane diterpene natural product. 

Towards perfect NMR: Spin‐echo versus perfect‐echo building blocks 

The concept of “perfect NMR” includes the design of robust pulse sequences that allow an investigator to obtain undistorted pure in‐phase signals, with pure absorption lineshapes that are free of phase anomalies derived from undesired J modulations. Here, alternative NMR building blocks to the spin‐echo that are based on a general double SE module, known as a perfect‐echo, are reviewed. Several implementations to minimize/remove unwanted dispersive contributions in homonuclear and heteronuclear NMR experiments are described and illustrated with some examples of broad interest for small molecules 

Simultaneous acquisition of two 2D HSQC spectra with different 13C spectral widths

A time-efficient NMR strategy that involves the interleaved acquisition of two 2D HSQC spectra having different spectral widths in the indirect 13C dimension is presented. We show how the two equivalent coherence transfer pathways involved in sensitivity-enhanced HSQC experiments are managed selectively and detected separately in different FID periods within the same scan. The feasibility of this new SADA-HSQC (Spectral Aliasing in Dually Acquired HSQC) technique is demonstrated by recording simultaneously two complementary datasets, conventional and highly-resolved spectral-aliased 2D HSQC spectra, in a single NMR experiment. Combining the information from both datasets, accurate chemical shift determination and excellent signal dispersion is achieved in a unique measurement using only few t1 increments. 

Current developments in homonuclear and heteronuclear J‐resolved NMR experiments  

Two‐dimensional J‐resolved (Jres) NMR experiments offer a simple, user‐friendly spectral representation where the information of coupling constants and chemical shifts are separated into two orthogonal frequency axis. Since its initial proposal 40 years ago, Jres has been the focus of considerable interest both in improving the basic pulse sequence and in its successful application to a wide range of studies. Here, the latest developments in the design of novel Jres pulse schemes are reviewed, mainly focusing on obtaining pure absorption lineshapes, minimizing strong coupling artifacts, and also optimizing sensitivity and experimental measurements. A discussion of several Jres versions for the accurate measurement of a different number of homonuclear (JHH) and heteronuclear (JCH) coupling constants is presented, accompanied by some illustrative examples. 

Perfect 1JCH-resolved HSQC: Efficient measurement of one-bond proton-carbon coupling constants along the indirect dimension

A versatile 1JCH-resolved HSQC pulse scheme for the speedy, accurate and automated determination of one-bond proton-carbon coupling constants is reported. The implementation of a perfectBIRD element allows a straightforward measurement from the clean doublets obtained along the highly resolved F1 dimension, even for each individual 1JCHa and 1JCHb in diastereotopic HaCHb methylene groups. Real-time homodecoupling during acquisition and other alternatives to minimize accidental signal overlapping in overcrowded spectra are also discussed. 

Direct Monitoring of Exogenous γ‑Hydroxybutyric Acid in Body Fluids by NMR Spectroscopy

γ-Hydroxybutyric acid (GHB) is a popular drug increasingly associated with cases of drug-facilitated sexual assault (DFSA). Currently, expanding procedures of analysis and having forensic evidence of GHB intake in a long term are mandatory. Up to now, most studies have been performed using GC/MS and LC-MS as analytical platforms, which involve significant manipulation of the sample and, often, indirect measurements. In this work, procedures used in NMR-based metabolomics were applied to a GHB clinical trial on urine and serum. Detection, identification, and quantification of the drug by NMR methods were surveyed, as well as the use of NMR-based metabolomics for the search of potential surrogate biomarkers of GHB consumption. Results demonstrated the suitability of NMR spectroscopy, as a robust nondestructive technique, to fast and directly monitor (detect, identify, and quantify) exogenous GHB in almost intact body fluids and its high potential in the search for metabolites associated with GHB intake. 

Homodecoupled 1,1- and 1,n-ADEQUATE: Pivotal NMR Experiments for the Structure Revision of Cryptospirolepine

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.

Disentangling complex mixtures of compounds having near-identical 1H and 13C NMR spectra by pure shift NMR 

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 13C indirect dimension enables the distinction of similar compounds exhibiting near‐identical 1H and 13C NMR spectra. It is shown that a complete set of extremely small Δδ(1H) and Δδ(13C) values, even below the natural line width (1 and 5 ppb, respectively), can be simultaneously determined and assigned. 

Simultaneous 1H and 13C NMR enantiodifferentiation from highly resolved pure shift HSQC spectra 

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

LR-HSQMBC: A sensitivity NMR technique to probe very long-range heteronuclear coupling pathways

HMBC is one of the most often used and vital NMR experiments for the structure elucidation of organic and inorganic molecules. We have developed a new, high sensitivity NMR pulse sequence that overcomes the typical 2,3JCH limitation of HMBC by extending the visualization of long-range correlation data to 4-, 5-, and even 6-bond long-range nJCH heteronuclear couplings. This technique should prove to be an effective experiment to complement HMBC for probing the structure of proton-deficient molecules. The LR-HSQMBC NMR experiment can, in effect, extend the range of HMBC to provide data similar to that afforded by 1,n-ADEQUATE even in sample-limited situations. This is accomplished by optimizing responses for very small nJCH coupings as opposed to relying on the markedly less sensitive detection of long-range coupled 13C–13C homonuclear pairs at natural abundance. DFT calculations were employed to determine whether the very long-range correlations observed for cervinomycin A2 were reasonable on the basis of the calculated long-range couplings. 

Enantiodifferentiation through Frequency-Selective Pure-Shift 1H Nuclear Magnetic Resonance Spectroscopy

A frequency-selective 1D 1H nuclear magnetic resonance (NMR) experiment for the fast and sensitive determination of chemical-shift differences between overlapped resonances is proposed. The resulting fully homodecoupled 1H 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.

13C NMR spectroscopy for the differentiation of enantiomerics using chiral solvating agents

The utility of 13C 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 13C nucleus, as well as the singlet nature of the signals in standard experiments makes 13C NMR experiments a powerful alternative or complement to 1H 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 1H 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. 

Long-Range Proton-Carbon Coupling Constants: NMR Methods and applications

A general review of novel NMR methods to measure heteronuclear long-range proton–carbon coupling constants (nJCH; 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. 

A definitive NMR solution for the simple and accurate measurement of the magnitude and the sign of small heteronuclear coupling constants on protonated and non-protonated carbons.

A proton‐selective HSQMBC‐TOCSY experiment can be used to measure small proton–carbon (nJCH; n>1) coupling constants on both protonated and non‐protonated carbon atoms (see spectrum). The method combines in a single pulse scheme all the benefits of the widely used HSQMBC and HSQC‐TOCSY experiments. The magnitude and the sign of nJCH can be determined simply with excellent accuracy.