Trends in Analytical Chemistry 69 (2015) 88–97
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Trends in Analytical Chemistry j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / t r a c
Identification and structure elucidation by NMR spectroscopy Mikhail Elyashberg * Advanced Chemistry Development, Moscow Department, 6 Akademik Bakulev Street, Moscow 117513, Russian Federation
A R T I C L E
I N F O
Keywords: CASE Computer-assisted structure elucidation Dereplication Expert system Hydrogen-deficient molecule NMR Nuclear magnetic resonance Structure elucidation Structure identification Structure verification
A B S T R A C T
The state of the art and recent developments in application of nuclear magnetic resonance (NMR) for structure elucidation and identification of small organic molecules are discussed. The recently suggested new two-dimensional (2D)-NMR experiments combined with the advanced instrumentation allow structure elucidation of new organic compounds at a sample amount of less than 10 μg. A pure shift approach that provides 1H-decoupled proton spectra drastically simplified 1H and 2D NMR spectra interpretation. The structure elucidation of extremely hydrogen-deficient compounds was dramatically facilitated due to the methodology based on combination of new 2D-NMR experiments providing longrange heteronuclear correlations with computer-assisted structure elucidation (CASE). The capabilities of CASE systems are discussed. The role of NMR-spectrum prediction in structure verification and NMR approaches for qualitative mixture analysis are considered. © 2015 Elsevier B.V. All rights reserved.
Contents 1. 2. 3. 4. 5. 6. 7. 8. 9.
Introduction ........................................................................................................................................................................................................................................................... Common set of 1D- and 2D-NMR experiments ........................................................................................................................................................................................ Development of NMR experiments and instrumentation ..................................................................................................................................................................... CASE expert systems .......................................................................................................................................................................................................................................... NMR chemical-shift prediction ...................................................................................................................................................................................................................... Structure verification ......................................................................................................................................................................................................................................... Structure identification and dereplication in mixtures .......................................................................................................................................................................... Is it possible to avoid an erroneous structure elucidation? ................................................................................................................................................................. Conclusions ............................................................................................................................................................................................................................................................ Acknowledgements ............................................................................................................................................................................................................................................. References ..............................................................................................................................................................................................................................................................
1. Introduction Molecular structure determination is a central theme of organic and analytical chemistry. Nuclear magnetic resonance (NMR) spectroscopy in combination with high-resolution mass-spectrometry (HRMS) makes up a basic set of methods to solve this problem. Given the molecular formula of a complex organic molecule that has been determined using HRMS, two-dimensional (2D)-NMR plays a crucial role in structure elucidation. In this review, we consider application of NMR to determine structures of small organic molecules. The results achieved in this area were discussed in monographs [1,2] and reviews [3–9], including two comprehensive reviews published recently [8,9].
* Tel.: +7 495 438 2153; Fax: +4954382874. E-mail address:
[email protected] http://dx.doi.org/10.1016/j.trac.2015.02.014 0165-9936/© 2015 Elsevier B.V. All rights reserved.
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To make clear the issues being discussed in this review, it is necessary to consider some basic concepts. The first step in the structure determination of an unknown is a spectral search against the relevant available databases using MS and NMR spectra. If the spectrum of the unknown fully coincides with a reference spectrum, it means that the structural formula of the unknown is identical to that of the reference. This is termed as structure identification. Otherwise, the problem of structure elucidation arises. Given the structure is elucidated, it is necessary to establish if the compound is new. The structural search against corresponding databases {see review [10]} to answer this question is called dereplication. This procedure is also interpreted in literature as a structural identification of a known chemical entity based on previously reported analytical and spectroscopic information [11]. Structure elucidation is obviously the most complicated task. It is related to the class of inverse problems [12] for which solution ambiguity is a distinctive peculiarity. A single solution is selected by imposing additional constraints. On the whole, the problem of
M. Elyashberg/Trends in Analytical Chemistry 69 (2015) 88–97
structure elucidation from 2D-NMR data can be presented as a combination of two inverse problems, which should be solved in consecutive order [2]. The first problem is to determine all (if possible) pairs of atoms (nuclei) in the molecule for which there exist correlations observed in 1D and available 2D-NMR spectra. This goal is achieved as a result of interpretation of 1D- and 2D-NMR spectra, which may admit alternative solutions due to resonance overlap and other reasons. The second problem is to determine all structures that meet the revealed set of coupled nuclei and then to select the most probable structure by imposing additional constraints coming from characteristic spectral features, NMR chemicalshift prediction and chemical knowledge. It is evident that the solution of the second, main, problem strongly depends on the solution of the first problem. If erroneous spin couplings leak into the solution of the first problem, the possibility of a correct structure becomes problematic. Analysis of spectroscopists’ reasoning during structure elucidation led to the conclusion that initial NMR-based information used for this goal could be represented as a set of “axioms”, which make up a partial axiomatic theory formulated specifically for a given problem [1,2,13]. Hence, the problem reduces to inferring all plausible structures from the set of axioms. The axioms can be readily formalized, and provide a theoretical basis for creation of algorithms for computer-assisted structure elucidation (CASE). Note that both a human expert and a CASE program commonly use the same set of axioms, but the program is not governed by the chemical “prejudices” of the human mind and delivers all (without any exception) structures satisfying the given set of axioms adopted by the chemist. This task, as a rule, is impossible for a human expert. The program finds solution far more quickly and more reliably [13]. Generally speaking, progress in the reviewed area is going on in the following two directions:
•
•
suggestion of new NMR experiments and instrumentation for reliably acquiring as much structural information as possible from the smallest amounts of sample [8,14,15] in the shortest time (provides solution to the first inverse problem); and, enhancing the performance of the existing CASE programs and creating new ones (provides solution to the second inverse problem).
This review discusses the state of progress in solving these problems.
2. Common set of 1D- and 2D-NMR experiments 1
H- and 13C-NMR spectra carry information about the qualitative and quantitative composition of an unknown and they are used first for the determination of the molecular formula. Along with 1D 1 H- and 13C (15N if available)-NMR spectra, many two-dimensional NMR experiments were developed for structure elucidation [16,17]. For molecules containing nitrogen atoms, resonances of 15N nuclei are determined from 1H-15N heteronuclear single-quantum correlation (HSQC) and 1H-15N heteronuclear multi-bond correlation (HMBC) 2D spectra (see below). The most frequently used set of 2DNMR experiments is presented in Table 1. The correlation spectroscopy (COSY) and HMBC correlations whose lengths most frequently do not exceed three bonds are referred to as standard correlations [1]. However, depending on the spatial configuration of a molecule, correlations longer than standard correlations can also be observed. These correlations are referred to as non-standard correlations (NSCs). The presence of NSCs, their number and lengths in HMBC and COSY spectra are difficult to detect, and this issue can make the initial information not only fuzzy but also contradictory.
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Table 1 The most frequently used set of 2D NMR experiments [16] Experiment
Characteristics
HSQC, Heteronuclear Single Quantum Coherence
The HSQC spectrum shows resonances (heteronuclear correlations) which arise as a result of 1JCH couplings between 13C nuclei and protons attached to the corresponding atoms. This allows one to detect all CH, CH2 and CH3 groups with chemical shift assignment. ME-HSQC (Multiplicity Edited HSQC) alleviates distinguishing responses from CH3, CH2 and CH groups in HSQC spectrum obviating the acquisition of DEPT data. The COSY spectrum usually reveals homonuclear correlations (spin couplings) between vicinal hydrogens separated by three bonds (3JHH). This makes it possible to identify the neighbor carbon atoms connected by a chemical bond. TOCSY allows one, in principle, to obtain sub-spectra for different sequences of coupled protons in a molecule. In practice, investigators usually acquire only one of the spectra. The HMBC spectrum reveals heteronuclear correlations between 1H and 13C (15N) nuclei separated by two or three chemical bonds, allowing users to detect ”fuzzy” fragments around a given C or N atom. There is no routine approach that would allow determining which intervening 1 H-13C pairs are separated by two bonds and which – by three. Therefore the information carried by HMBC is fuzzy by nature. 1H-13C HMBC data are made even more “fuzzy” by the occasional observation of 4JCH correlations. In contrast, for conventional 1H -15N HMBC, 4JNH correlations are almost never observed. The NOESY/ROESY [18] reveals couplings between hydrogen atoms separated in space by distance 10 mg). To an extent, the limitations of the experiment have been overcome by the development of small volume high sensitivity and cryogenic NMR probes [29].
CIGAR-CHMBC
2J,3J-HMBC
1
H-13C H2BC
ADEQUATE
INADEQUATE
inverse probes and/or cryogenic NMR probes, which allow acquisition of spectra on sub-micromole quantities of samples [35]. The number of publications in which 1H-15N HMBC is utilized is constantly growing. The very recently reported new experiments on longrange heteronuclear single quantum multiple bond correlation (LR-HSQMBC) optimized for 1H-15N long-range heteronuclear couplings [36] and H-C-N multiple-bond correlation (HCNMBC) [37,38] produced data complementary to 1H-15N HMBC-type correlations. It is expected that such experiments can facilitate the structure elucidation of nitrogen-containing molecules, particularly those belonging to heterocyclic compounds and alkaloids. In comparison with MS and optical spectroscopy, NMR possesses significantly less sensitivity, which becomes especially notable regarding the sample size and the acquisition time of 2D-NMR. As a result of technical progress, cooled microprobes became available [39,40].
Hilton and Martin [35] investigated experimental performance limits for an ensemble of 2D-NMR experiments using a 600 MHz spectrometer with 1.7 mm Bruker TCI MicroCryoProbe. A solution containing 870 μg (2.6 μmol) of a model compound – strychnine in 30 μL of CDCl3 – was used. The following acquisition times to obtain adequate signal-to-noise ratios were determined: COSY – 7 min; rotating-frame Overhauser-effect spectroscopy (ROESY) – 1 h 11 min; 13C reference – 25 min; ME-HSQC – 7 min; 1H-13C HMBC – 33 min; 1H-13C heteronuclear 2-bond correlation (H2BC) – 3 h 11 min; 1H-15N HMBC – 1 h 22 min; adequate sensitivity double-quantum spectroscopy (1,1-ADEQUATE) – 14 h 40 min. Further dilutions have shown that, with samples of 45 μg (150 nmol) even 1 H- 15 N HMBC remains accessible experimentally over a weekend. The authors [35] concluded that, with a 1 mg sample of strychnine (~3 μmol), it is now possible to acquire the full set of homonuclear and heteronuclear 2D-NMR experiments in 4 h (including 1H -15N HMBC but not 1,1-ADEQUATE) that could, in principle, be used to establish the full chemical structure and stereochemistry. Using the same equipment as in [35], high signal-to-noise pure shift (see below) HSQC data from a 7.4 μg metabolite sample were acquired in just over 30 min [41]. Structure elucidation becomes especially challenging for molecules for which a severe deficit of protons is inherent. These molecules contain “silent” (deprived of hydrogen) fragments, which prevent structure assembly using HMBC correlations. If the ratio of the number of protons in a molecule to the sum of the heavy atoms (e.g., C, N, O, and S) is
