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 A B S T R A C T

Keywords:
The state of the art and recent developments in application of nuclear magnetic resonance (NMR) for
CASE
structure elucidation and identification of small organic molecules are discussed. The recently sug-
Computer-assisted structure elucidation
Dereplication
gested new two-dimensional (2D)-NMR experiments combined with the advanced instrumentation allow
Expert system structure elucidation of new organic compounds at a sample amount of less than 10 μg. A pure shift ap-
Hydrogen-deficient molecule proach that provides 1H-decoupled proton spectra drastically simplified 1H and 2D NMR spectra
NMR interpretation. The structure elucidation of extremely hydrogen-deficient compounds was dramatically
Nuclear magnetic resonance facilitated due to the methodology based on combination of new 2D-NMR experiments providing long-
Structure elucidation range heteronuclear correlations with computer-assisted structure elucidation (CASE). The capabilities
Structure identification of CASE systems are discussed. The role of NMR-spectrum prediction in structure verification and NMR
Structure verification
approaches for qualitative mixture analysis are considered.
© 2015 Elsevier B.V. All rights reserved.

Contents

1. Introduction ........................................................................................................................................................................................................................................................... 88
2. Common set of 1D- and 2D-NMR experiments ........................................................................................................................................................................................ 89
3. Development of NMR experiments and instrumentation ..................................................................................................................................................................... 89
4. CASE expert systems .......................................................................................................................................................................................................................................... 93
5. NMR chemical-shift prediction ...................................................................................................................................................................................................................... 94
6. Structure verification ......................................................................................................................................................................................................................................... 94
7. Structure identification and dereplication in mixtures .......................................................................................................................................................................... 94
8. Is it possible to avoid an erroneous structure elucidation? ................................................................................................................................................................. 95
9. Conclusions ............................................................................................................................................................................................................................................................ 95
Acknowledgements ............................................................................................................................................................................................................................................. 95
References .............................................................................................................................................................................................................................................................. 95

1. Introduction To make clear the issues being discussed in this review, it is nec-
essary to consider some basic concepts. The first step in the structure
Molecular structure determination is a central theme of organic determination of an unknown is a spectral search against the rel-
and analytical chemistry. Nuclear magnetic resonance (NMR) spec- evant available databases using MS and NMR spectra. If the spectrum
troscopy in combination with high-resolution mass-spectrometry of the unknown fully coincides with a reference spectrum, it means
(HRMS) makes up a basic set of methods to solve this problem. Given that the structural formula of the unknown is identical to that of
the molecular formula of a complex organic molecule that has been the reference. This is termed as structure identification. Otherwise,
determined using HRMS, two-dimensional (2D)-NMR plays a crucial the problem of structure elucidation arises. Given the structure is
role in structure elucidation. In this review, we consider applica- elucidated, it is necessary to establish if the compound is new. The
tion of NMR to determine structures of small organic molecules. The structural search against corresponding databases {see review [10]}
results achieved in this area were discussed in monographs [1,2] to answer this question is called dereplication. This procedure is also
and reviews [3–9], including two comprehensive reviews pub- interpreted in literature as a structural identification of a known
lished recently [8,9]. chemical entity based on previously reported analytical and spec-
troscopic information [11].
Structure elucidation is obviously the most complicated task. It
is related to the class of inverse problems [12] for which solution am-
* Tel.: +7 495 438 2153; Fax: +4954382874. biguity is a distinctive peculiarity. A single solution is selected by
E-mail address: elyas@acdlabs.ru imposing additional constraints. On the whole, the problem of

http://dx.doi.org/10.1016/j.trac.2015.02.014
0165-9936/© 2015 Elsevier B.V. All rights reserved.
 M. Elyashberg/Trends in Analytical Chemistry 69 (2015) 88–97
structure elucidation from 2D-NMR data can be presented as a com-
bination of two inverse problems, which should be solved in
consecutive order [2]. The first problem is to determine all (if pos-
sible) 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 con-
straints coming from characteristic spectral features, NMR chemical-
shift 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 struc-
ture becomes problematic.
Analysis of spectroscopists’ reasoning during structure elucida-
tion 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 plau-
sible structures from the set of axioms. The axioms can be readily
formalized, and provide a theoretical basis for creation of algo-
rithms 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 qualita-
tive 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 corre-
lation (HSQC) and 1H-15N heteronuclear multi-bond correlation
(HMBC) 2D spectra (see below). The most frequently used set of 2D-
NMR experiments is presented in Table 1.
The correlation spectroscopy (COSY) and HMBC correlations
whose lengths most frequently do not exceed three bonds are re-
ferred to as standard correlations [1]. However, depending on the
spatial configuration of a molecule, correlations longer than stan-
dard 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.
89
Table 1
The most frequently used set of 2D NMR experiments [16]
Experiment Characteristics
HSQC, The HSQC spectrum shows resonances (heteronuclear
Heteronuclear correlations) which arise as a result of 1JCH couplings
Single Quantum between 13C nuclei and protons attached to the
Coherence 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.
COSY, Correlation The COSY spectrum usually reveals homonuclear
Spectroscopy 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, Total TOCSY allows one, in principle, to obtain sub-spectra for
Correlation different sequences of coupled protons in a molecule.
Spectroscopy In practice, investigators usually acquire only one of the
spectra.
HMBC, The HMBC spectrum reveals heteronuclear correlations
Heteronuclear between 1H and 13C (15N) nuclei separated by two or three
Multiple-Bond chemical bonds, allowing users to detect ”fuzzy”
Coherence 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.
NOESY, Nuclear The NOESY/ROESY [18] reveals couplings between
Overhauser hydrogen atoms separated in space by distance <5 Å, ,
Enhancement which is used for determining stereochemistry of an
Spectroscopy elucidated structure, as well as for clarifying positions of
ROESY, Rotated some substituents if HMBC and COSY data do not allow to
frame NOE do that. These spectra are usually not used during
Spectroscopy structure assembly.
3. Development of NMR experiments and instrumentation
The latest advances in development and improvements of 2D-
NMR experiments were extensively considered in recently published
reviews [6,8,19–24]. Significant efforts of researchers were focused
on creating methods for detecting correlations whose lengths could
(at least in principle) be unambiguously determined. For example,
distinguishing between correlations of coupling constants 2JCH and
3
JCH in the HMBC spectrum leads to obtaining crisper 2D-NMR struc-
tural information. Different methods were suggested to reduce the
time for spectral acquisition, to increase sensitivity and to simpli-
fy post-acquisition processing of 2D-NMR data. Some 2D-NMR
experiments that complement those shown in Table 1 are enumer-
ated in Table 2.
From the standpoint of a protocol for elucidating chemical struc-
ture, a 1H spectrum is almost always recorded first. The next step
is the acquisition of 2D-NMR data, and it is recommended to start
with a multiplicity-edited HSQC (ME-HSQC) spectrum [5]. The HSQC
data may provide some insight into the numbers of possible het-
eroatoms, as well as a partial carbon count. The next step is to acquire
a COSY spectrum. Having in hand 1H, COSY and ME-HSQC, it is pos-
sible to begin to assemble structural fragments comprising the
contiguous protonated carbon resonances.
Having the molecular formula from HRMS and HSQC, it is pos-
sible to establish the expected number of signals of quaternary
carbons in 13C-NMR spectrum and the minimum number of het-
eroatoms. Then, the HMBC spectrum is acquired, and in principle,
allows one to complete assembly of the structure.
If the molecule contains nitrogen atoms, employing the 1H-15N
HMBC spectrum gives valuable information that is crucial for the struc-
ture elucidation in many cases {see reviews [30–34]}. Low sensitivity
of the 1H-15N HMBC experiment is overcome using small-volume
90 M. Elyashberg/Trends in Analytical Chemistry 69 (2015) 88–97

Table 2 Hilton and Martin [35] investigated experimental performance

Experiments complementing the most frequently used 2D NMR techniques
Method Characteristics
HSQC-TOCSY The 1H-13C HSQC-TOCSY experiment combines 1H-13C HSQC
with a 1H-TOCSY experiments to give through-bond
correlations between a 13C-attached 1H to all other coupled
1
H. The resonances of coupled protons can be seen along a
line at the same 13C chemical shift from the carbon atom
attached to the primary 1H [16].
CIGAR-CHMBC Makes it much easier to interpret the spectrum, particularly
in the crowded regions. Is the best of the existing HMBC
sequences in terms of information content and ease of
interpretation [20].
2J,3J-HMBC
Affords the means of unequivocally differentiating between
2
JCH from 3JCH correlations. Peaks of 2JCH have skew
characteristics, while 2JCH signals for quaternary carbons are
missing. It also suffers from a severe lack of sensitivity
(almost 10 times lower than for HMBC) [20]
1
H-13C H2BC HMBC-type spectrum almost exclusively showing only
two-bond correlations and markedly also two-bond
correlations that are absent in HMBC spectra. As a rule,
correlations that are strong in an H2BC spectrum and weak
in an HMBC spectrum indicate two-bond correlations. H2BC
and HMBC are therefore quite complementary. Drawbacks
are that H2BC spectra only show peaks involving protonated
13
C carbons, they cannot identify adjacent quaternary
carbon resonances. In addition there is no absolute
guarantee that a peak in an H2BC spectrum represents a
two-bond correlation [20].
ADEQUATE 1,1-ADEQUATE exploits 1JCH and 1JCC couplings to allow the
identification of adjacent neighbor carbons. Both protonated
and non-protonated adjacent carbons are observed.
Correlations are equivalent to 2JCH correlations in
HMBC-type experiments. Method suffers from sensitivity
and sample limitations, which are overcome by employing
small volume high sensitivity and cryogenic NMR probes
[19].
1,n- ADEQUATE [19,25] provides predominantly 3JCC long-
range correlations that are analogous to 4JCH HMBC
correlations. Though it is less sensitive than 1,1-ADEQUATE
and 1JCC correlations “leak” into the spectrum, it has been
shown [26–28] that 1,n-ADEQUATE data acquired using
1.7 mm cryoprobe technology are very helpful for structure
characterization of proton-deficient molecules whose
structures are extremely difficult to elucidate by
conventional HMBC. Advantages of ADEQUATE over HMBC
were demonstrated experimentally in combination with
quantum-chemical coupling constant computations [28].
INADEQUATE A COSY-like experiment that yields 13C-13C correlations. It is
capable of establishing the identity of adjacent neighbor
carbons via 1JCC couplings. Major drawbacks of the
INADEQUATE experiment are extreme insensitivity and
prodigious sample requirements (>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].
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 (in-
cluding 1H -15N HMBC but not 1,1-ADEQUATE) that could, in principle,
be used to establish the full chemical structure and stereochemis-
try. 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 mol-
ecules 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 <2, it may be difficult and, in some cases, im-
possible to elucidate a structure unequivocally based solely on NMR
data and molecular formula information. This statement is known
as the “Crews rule” [42]. To circumvent this issue, several new ap-
proaches were suggested.
Gross and co-workers were the first to employ atomic force mi-
croscopy (AFM) [43] to determine a challenging structure of a small
molecule by making an image of its skeleton visible. Then, AFM com-
bined with 1D- and 2D-NMR, density functional theory (DFT) and
CASE system ACD/Structure Elucidator [44] (see Section 4) was used
[45] to determine the structure of natural product breitfussin A,
Structure 1(a “silent” fragment is highlighted in bold).
A new, high-sensitivity experiment complementary to HMBC, LR-
HSQMBC [46] extends the observation of long-range correlation data
to 4-, 5-, and even 6-bond long-range nJCH heteronuclear cou-
plings. The correlation lengths can be estimated from nJCH, for which
experiments were optimized. These correlations can reach quater-
nary carbons of “silent” fragments, allowing structure elucidation.
Proton-deficient model compound cervinomycin A2, Structure 2,
C29H21NO9, was used [27] to assess the benefits of including LR-
HSQMBC data as input for ACD/Structure Elucidator. Fig. 1 shows
the observed LR-HSQMBC correlations (optimized at 2 Hz).

inverse probes and/or cryogenic NMR probes, which allow acquisi-

tion of spectra on sub-micromole quantities of samples [35]. The
number of publications in which 1H-15N HMBC is utilized is con-
stantly growing. The very recently reported new experiments on long-
range heteronuclear single quantum multiple bond correlation
(LR-HSQMBC) optimized for 1H-15N long-range heteronuclear cou-
plings [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 eluci-
dation of nitrogen-containing molecules, particularly those belonging
to heterocyclic compounds and alkaloids.
In comparison with MS and optical spectroscopy, NMR pos-
sesses 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]. Structure 1. Natural product breitfussin A.
 M. Elyashberg/Trends in Analytical Chemistry 69 (2015) 88–97
Structure 2. Proton-deficient model compound cervinomycin A2.
The results of computational experiments (Table 3) clearly show
that complementing the standard 2D-NMR set by 1 H- 13 C LR-
HSQMBC data dramatically accelerates structure generation and
reduces the size of the output file.
Blinov et al. [27] performed analogous computational experi-
ments using staurosporine, C28H26N4O3, (a “Screw structure”), as a
model compound. In this case, the set of experiments shown in
Table 3 was complemented by IDR-HSQC-total correlation spec-
troscopy (TOCSY), 1H-15N HMBC, 1,1-ADEQUATE and 1,-n-ADEQUATE.
The results obtained were very interesting and highlighted the con-
tribution of the combination LR-HSQMBC and 1,-n-ADEQUATE.
It should be expected that the application of the described com-
bination of 2D-NMR experiments within CASE systems would allow
the problem of “silent fragments” to be overcome to a consider-
able degree. The methodology of the approach described should be
further developed on the basis of structure elucidation of other mol-
ecules subjected to the Crews rule.
Structure 3. An unusual proton-deficient molecule.
Fig. 1. Long-range heteronuclear single quantum multiple bond correlations (LR-
HSQMBCs) observed for cervinomycin A2. Note that numerous long-range correlations
reach carbon atoms of the “silent fragment” [27].
91
Table 3
Results obtained for cervinomycin A2 (2) with various 2D NMR aspects used as input.
Data that were provided as program input are denoted by (+), nJCH for which ex-
periments were optimized (in Hz), gt – time of structure generation, and k – number
of generated structures
1
COSY, HSQC H-13C 1
H-13C gt k
HMBC LR-HSQMBC
8 Hz 4 Hz 4 Hz 2 Hz
+ + + 49 h 314
+ + + + 37 h 4
+ + + + 2 m 30 s 7
+ + + + + 1m 44 s 1
Kummerlöwe et al. [47] concluded that 2D-NMR data, includ-
ing 1H -15N HMBC and 1,1-ADEQUATE, were insufficient to elucidate
the structure of an unusual proton-deficient molecule (3) manu-
ally, due to extremely contradictory 1H-13C HMBC data (presence
of nine NSCs, two of which had five bond lengths and were rather
strong).
Kummerlöwe et al. [47] were the first to utilize successfully the
NMR spectrum of a residual dipolar coupling (RDC) [48] to eluci-
date the structure of a small molecule.
Zangger and Sterk [49] developed a “pure shift” approach, pro-
viding 1H-decoupled proton spectra (i.e., all multiplets are transformed
into singlets). In recent years, efforts of researchers were directed
to overcome the major drawback of the approach – its low sensitivity
[50–58]. As a result, very recently, a new and very general pure shift
method, Pure Shift Yielded by CHirp Excitation (PSYCHE), was in-
troduced by Morris and co-workers [56]. PSYCHE has approximately
10-fold better sensitivity than competing “pure shift” methods.
Normal and PSYCHE spectra of estradiol in DMSO-d6 are shown in
Fig. 2as an example.
The same group applied PSYCHE to the TOCSY experiment [57].
In combination with covariance processing (see below), the result
is a high-quality, high-resolution TOCSY spectrum with singlets in
both dimensions (see Fig. 3). It is evident that the suggested ap-
proach dramatically facilitates interpretation of 1D- and 2D-NMR
spectrum and will significantly simplify spectroscopic data input
into CASE programs.
Analytical applications of the ultra-fast (UF) 2D-NMR tech-
nique were extensively discussed in a review [21]. This technique
acquires a 2D spectrum in a few seconds, but it suffers from low
sensitivity. For example, to use a single-scan HSQC spectra for re-
action monitoring, a 0.44 M solution was necessary [59]. Another
possibility to accelerate acquiring a spectrum appeared when NMR
spectrometers equipped with two or more independent receivers
became available, which allowed different types of 2D spectra to
be obtained simultaneously [60]. For example, the parallel-acquisition
NMR all-in-one combination of experimental applications (PANACEA)
Fig. 2. Spectra of estradiol obtained by (a) normal proton nuclear magnetic reso-
nance (1H-NMR) spectroscopy and (b) Pure Shift Yielded by CHirp Excitation (PSYCHE)
[56].
92 M. Elyashberg/Trends in Analytical Chemistry 69 (2015) 88–97

Fig. 3. Spectra of (a) normal Total Correlation Spectroscopy (TOCSY), (b) F1-Pure Shift Yielded by CHirp Excitation (PSYCHE)-TOCSY, and (c) double pure shift TOCSY using
PSYCHE in F1 and covariance processing in F2 of a sample of estradiol in DMSO-d6 [57].

[61] technique can in principle obtain all data [including indirect
ADEQUATE (INADEQUATE)] needed for small molecule structure elu-
cidation in a single experiment, but low sensitivity prevents its wide
application. More sensitive timeshared (TS) versions of 2D-NMR ex-
periments (TS-HMBC, TS-HSQC, TS-HSQC-TOCSY, and TS-HSQMBC)
were suggested by Parella and Nollis [62] who demonstrated uti-
lization of this approach for simultaneous acquisition of 1H/13C and
1
H/15N-NMR spectra.
New methods for 2D-NMR data processing, which facilitate spec-
trum interpretation, were developed and reviewed [8]. Covariance
processing involves reprocessing 2D data sets, singly or in pairs, to
improve resolution or to present the existing information in a dif-
ferent, clearer fashion. Unsymmetric Covariance Processing (UCP),
developed by Martin and co-workers [63], and the closely-related
Generalized Covariance Processing (GCP), suggested by Snyder and
Bruschweiler [64], are the most useful. These methods allow one
 M. Elyashberg/Trends in Analytical Chemistry 69 (2015) 88–97 93

to combine data from two kinds of 2D spectra that have a common
axis (usually 1H). The resultant spectrum does not include any new
information that was not present in the two original 2D spectra. Nev-
ertheless, the data are presented in a clearer manner, which is easier
to interpret {see review [65]}. The software for covariance process-
ing is available from Bruker, ACD Labs and Mestranova.
4. CASE expert systems
CASE expert systems mimic the reasoning of a hum-
an expert during the process of structure elucidation. The
following main advantages of CASE systems should be noted
[1]:
1) all statements about interrelation between a spectrum and
a structure (“axioms”) are expressed explicitly;
2) all logical consequences (structures) following from the system
of “axioms” are deduced completely, without any exclusions;
3) if the initial data are complete and consistent, the process of
computer-based structure elucidation is usually fast, signifi-
cantly saving time and labor of the scientist; and,
4) if the chemist has several sets of axioms related to a given
structural problem, an expert system allows rapid genera-
tion of all consequences from each of the sets and identifies
the most probable structure by comparing the solutions
obtained.
System performance can be illustrated by the following example:
with its assistance, 1500 unknown molecules were elucidated by
two spectroscopists in 6 months [77]. More than 50 examples of
structure elucidation for natural products with unprecedented or
unique skeletons were described [2].
Significant improvements [78] were introduced in the LSD
program [69], which is now capable of treating 2D-NMR data con-
taining NSCs, and selection of the most probable structure is
performed by NMR spectrum prediction. LSD is the first version of
CASE program freely available from the Internet [79].
The SESAMI system [68] was combined with a 13C-NMR inter-
pretive library-search system (INFERCNMR) [80], capable of searching
substructures in a database containing assigned 13C-NMR spectra.
The search resulted in a set of substructures predicted to be present
in the unknown, each of which is assigned an estimated predic-
tion accuracy. Involving the best substructures in the structure-
generation procedure allows a significant reduction in the generation
time and the size of the output file. The suggested approach is prom-
ising for the further development of CASE methodology, as it does
not require application of the fragment database incorporated into
the program.
As for the Bruker CMC-se program, the absence of any publica-
tions utilizing the program does not allow a comparative evaluation
to be made.
New expert system CAST/CNMR Structure Elucidator is based on
13
C-NMR and a database containing structures with chemical-
shift assignment [71]. A series of advanced graph-theory algorithms

The state of the art in this area was extensively reviewed

[1,5,66,67]. The contemporary CASE programs include SESAMI [68],
LSD [69], COCON [70], ACD/Structure Elucidator [1,2], and Bruker
CMC-se, which are based on 2D-NMR, and CAST\CNMR Structure
Elucidator [71], based on 13C spectra.
ACD\Structure Elucidator (StrucEluc) [1,2,44,72] is the most ad-
vanced expert system. The 1D- and 2D-NMR spectra can be imported
to the program from a spectrometer or input manually from a table
prepared by the user. The imported data must be thoroughly checked
and edited by the spectroscopist. The strategy of the system rests
upon a series of databases containing factual and axiomatic knowl-
edge. All axioms are explicitly presented on the Molecular Connectivity
Diagram (MCD) for visual analysis (e.g., Fig. 4).
This easily allows users to investigate the dependence of the
structural problem solution on any change in the initial set of axioms.
The system is capable of inferring all plausible structures from a com-
bination of a molecular formula and 1D- and 2D-NMR data (e.g.,
HSQC, HMBC, and COSY), even in those cases when the spectrum-
structural information is very fuzzy, incomplete and contradictory.
Selection of the most probable structure is performed on the basis
of the 13C chemical-shift prediction using three algorithms imple-
mented into the system – HOSE code based [73], neural networks
and additivity rules [1,5]. The program is capable of elucidating a
structure of an unknown in the presence of an unknown number
of non-standard correlations of unknown length. The software is
commercially available and was used for solving many complex an-
alytical problems. For example, with its aid, the structure of complex
alkaloid quindolinocryptotackieine was determined [74] in an in-
teractive mode, allowing for step-by-step resolution of many
ambiguous correlations, which took a spectroscopist a week of work.
It was the first time that the program solved the structure that had
been unsolvable by experienced spectroscopists. Structure 3 (see Fig. 4. The molecular connectivity diagram created from 1H, 13C, heteronuclear single-
Section 3) declared as manually unsolvable from the full set of 2D- quantum correlation (HSQC), heteronuclear multi-bond correlation (HMBC) and
NMR data was unambiguously elucidated by StrucEluc in a fraction correlation spectroscopy (COSY) spectra of gymnopalyne (upper segment of the figure).
of a second [75]. The software was recently applied for structure Atom properties are adjusted to the structure of gymnopalyne to illustrate differ-
ent conventional signs: atom hybridization (sp3 – blue, sp2 – violet, sp – green, not
elucidation of armeniaspirols A–C [76] . Fig. 5illustrates a typical sp – light blue, not defined – black), connectivities (COSY – blue, HMBC – green),
representation of the most probable structures suggested by the and labels (ob – obligatory neighbor heteroatom, fb – forbidden neighbor heteroa-
program. tom). Information about 1H chemical shifts can also be visualized [2].
94 M. Elyashberg/Trends in Analytical Chemistry 69 (2015) 88–97
Fig. 5. The three top-ranked structures according to the dA deviation. dA(13C), dI(13C) and dN(13C) – average deviations calculated by Hierarchical Organization of Spherical
Environments (HOSE) code-based algorithm, incremental approach and neural networks correspondingly. The correct structure #1 (armeniaspirol B) is reliably selected as
the most probable [76] in accordance with the criteria suggested [1].
are used for selecting appropriate substructures, then the most prob-
able structures are formed by merging the substructures that have
common parts. The program produces a correct structure if all the
fragments are included in the database. As only one example
illustrates the suggested approach, no conclusions regarding its ef-
ficacy can be made.
It should be noted that the first open source structure genera-
tor, OMG [81], recently became available. It can be used for solving
structural problems as a stand-alone program and as a block of a
CASE system. The CASE program COCON is also freely accessible from
the WEBCOCON server [70].
As discussed in Section 3, utilization of new 2D-NMR experi-
ments within the CASE systems leads to rapid structure elucidation
of complex proton-deficient molecules.
5. NMR chemical-shift prediction
As mentioned above, NMR chemical-shift prediction plays an in-
valuable role in the estimation of suggested structures [1,2,5].
Chemical shifts of 13C, 1H, 15N, 19F and 31P nuclei can be calculated
using empirical and quantum-mechanical (QM) methods. NMR spec-
trum predictors based on HOSE codes [73], neural nets, and an
incremental approach are used most frequently and were re-
viewed [1,5]. The predictors can be incorporated in expert systems
(as described for ACD/Structure Elucidator and LSD) or used sep-
arately as commercial programs [82–85] or free [86]. The programs
provide an accuracy of 13 C chemical-shift prediction about
1.5–1.8 ppm, which almost always allows for selecting the most prob-
able structure(s) among candidates. The prediction is very fast. For
example, the incremental program incorporated into ACD/Structure
Elucidator calculates ~10, 000 shifts per second on a standard PC.
A new program, NMRscape [87] for 13C chemical-shift calcula-
tion based on new principles {OptiChem theory [88]} was recently
developed and tested on several families of structures. The results
seem rather promising, as the accuracy provided by this approach
is expected to be higher than that inherent to existing empirical
methods.
A common drawback of all empirical methods is that predic-
tion accuracy depends on the composition of the databases used
for program training. In contrast, the DFT GIAO quantum-chemical
method does not suffer from this limitation, but it is very time-
consuming and there is the problem of selecting appropriate
functional and basic sets for calculations {see reviews [89,90]}. The
following optimal strategy of jointly utilizing empirical and QM
methods for structure elucidation was suggested [91]: all candidate
structures must be ranked first by empirical 13C spectrum predic-
tion, and, if the average deviations between the experimental and
predicted spectra calculated for two to three top-ranked struc-
tures are large or very close, only then should QM predictions be
performed for those questionable structures. The possibility was
demonstrated of successful application of JHH, JHC, and JCC coupling-
constant calculations by QM methods for distinguishing isomers
having very similar structures [92].
6. Structure verification
Structure verification based on 13C chemical-shift prediction
allows selection of the correct structure among thousands of plau-
sible hypotheses produced by expert systems [1,2]. For verification
of structures suggested for large sets of synthesized molecules, 1H
chemical-shift prediction is frequently used due to the possibility
of quickly acquiring a spectrum with a small sample size. Proton
chemical-shift dependence on, e.g., solvent, temperature, and pH,
limits the utility of this approach. Utilization of PSYCHE 1H-NMR
coupled with pure shift ME-HSQC acquired using the 1.7-mm
MicroCryoProbe would make the approach more robust.
Verification and identification of organic molecules from a struc-
ture database using both 1H and 13C-NMR spectra were reported [93].
Keyes et al. [93] examined a method of structure validation using
a set of 500 compounds supplied with 1H, HSQC, LC-MS and HPLC
data. 1H and HSQC spectra were predicted using the ACD/NMR Pre-
dictor [82] and compared with experimental spectra. It was
concluded that the approach was very practical for application in
a pharmaceutical company. As current implementations of auto-
mated structure-verification systems allow false-positive results, an
approach based on 1H and HSQC spectra was suggested [94] that
greatly reduces the probability of an automated validation system
passing incorrect structures (i.e., false positives). The novel method
was examined by automatic validation of 127 compounds, which
showed a reduction in the false-positive rate from 20% to 5%.
Plainchont and Nuzillard [95] proposed to verify a molecular struc-
ture using a combination of 1D and 2D HSQC, COSY and HMBC-
NMR spectra. This approach is not suitable for rapid structure
verification, but it rather fits analysis of doubtful structures passed
for verification.
7. Structure identification and dereplication in mixtures
Dereplication of organic compounds (identification in a mixture)
is a challenging problem for NMR spectroscopy [96]. If the sample
 M. Elyashberg/Trends in Analytical Chemistry 69 (2015) 88–97
amount is enough for separation of individual components, they can
be determined by a spectral search against an NMR database [97],
or structures of unknowns are elucidated using MS and a common
set of 1D- and 2D-NMR spectra. For example, Thummala et al. [98]
isolated an unknown impurity of ambroxol using preparative HPLC
and established its structure by combined application of 2D-NMR,
FTIR and LC-MS/MS.
Codina et al. [99] isolated five impurities from a pharmaceuti-
cal matrix (samples of 20–40 μg) and identified them from MS and
2D-NMR spectra with the aid of the ACD/Structure Elucidator. An
effective dereplication strategy for identification of natural me-
tabolites directly within mixtures was suggested [100,101]. A
multigram quantity of a crude extract was rapidly fractionated by
centrifugal partition extraction (CPE). The fractions of simplified
chemical composition were subsequently analyzed by 13C-NMR, and
hierarchical clustering analysis (HCA) suggested by the authors for
pattern recognition. A locally-built 13C-NMR chemical-shift data-
base was used for identification. This methodology resulted in the
direct identification (without time-consuming isolation proce-
dures) of seven major compounds of a bark extract [100] and six
out of eight prominent constituents of the crude extract of lichen
[101].
Pauli and co-workers [11] comprehensively investigated the pre-
cision necessary for measuring spectral parameters of 1H-NMR
spectra for their tabulation to be used for structure dereplication
and identification. It was shown that δ and J values of 1H-NMR should
be routinely reported with Δδ = 0.1–1 ppb and ΔJ = 10 mHz preci-
sion, respectively.
As a manual 1H spectrum assignment is a time consuming,
tedious and error-prone procedure, an expert system for the auto-
matic atom-to-peak or multiplet assignment of 1H-NMR spectra of
small molecules has been developed [102]. It was found with the
test set of 90 compounds that 94% of assignments were correct, so
confirming the efficiency of the program.
Different NMR experimental approaches were developed for qual-
itative analysis of mixtures without any preliminary fractionation
or separation. The NMR diffusion-ordered spectroscopy (DOSY) ex-
periment is based on molecules of different sizes and shapes
frequently having different diffusion coefficients [8]. DOSY is very
powerful for studying complex mixtures, but its applicability is still
limited, particularly when dynamic or unstable systems are studied.
A UF version of DOSY was recently described as overcoming this
drawback [21]. Morris and co-workers [58] developed a pure shift
DOSY experiment, which greatly extended the range of applicabil-
ity of DOSY. Simplified (pure shift) PSYCHE 1H-NMR [56] spectra (see
Fig. 2) are obviously very promising for analyzing complex mix-
tures by 1H-NMR. A method of mixture analysis based on 13C-
NMR [100,101] can probably be extended to PSYCHE 1H-NMR spectra
of mixtures.
Although the sensitivity of NMR is significantly lower than MS,
LC-NMR is now available as a coupled method for mixture analy-
sis [8,103–105]. Only 1 H spectra can usually be obtained in
continuous flow mode. The low sensitivity of the approach pre-
vents its wide application. However, very recently, Foley and co-
workers [106,107] described application of on-line reaction
monitoring using continuous flow NMR. A standard 5 mm NMR
probe enables the researcher to conduct experiments on flowing
reaction mixtures using a range of spectrometers of varying mag-
netic field strengths [107]. It was reported [59] that UF COSY spectra
were utilized for reaction monitoring using a commercial HPLC-
NMR set-up.
A combination of LC with solid-phase extraction (SPE) car-
tridges led to the possibility of obtaining MS and NMR spectra of
components from a single experiment [108]. After LC separation,
the sample was split, and a small portion sent for MS analysis, while
the remainder was directed to SPE cartridges for collection.
95
Application of a cryogenically-cooled probe reduced the time and
the sample amount necessary for structure identification (elucida-
tion) [8].
8. Is it possible to avoid an erroneous structure elucidation?
As spectroscopic structure elucidation is a complex logical-
combinatorial process, it is not surprising that different scientists
may come to different structures from the same initial data. A series
of reviews [13,109–112] discussed many structural misassignments.
The following most typical reasons of obtaining erroneous struc-
tures can be noted:
a) severe resonance overlap in NMR spectra;
b) erroneous user suggestions (”axioms”);
c) mistaken logical conclusions inferred from the presence or
the absence of characteristic spectral features in 1D-NMR and
2D-NMR spectra;
d) complexity, inconsistency and entanglement of initial infor-
mation {see [75]}.
Expert system-based analysis of many cases, when erroneous
structures were inferred, shows [1,13,75,113] that application of CASE
allows the determination of the correct structure and causes of the
human error to be detected. Figuratively speaking, a CASE system
can be used as a “polygraph detector”. To avoid upsetting errors, it
is recommended to predict 13C-NMR and 1H-NMR chemical shifts
for any structural hypothesis in the process of chemical research.
9. Conclusions
Contemporary NMR spectroscopy is the most powerful routine
analytical tool for molecular structure elucidation and identifica-
tion, given that the molecular formula is determined using HRMS
and all available spectroscopic data. 2D-NMR plays a crucial role in
establishing structures of new organic molecules, and, to date, a
plethora of different kinds of 2D-NMR experiments has been elabo-
rated. These methods are extensively developed to provide the
possibility of acquiring as much as possible structural information
from a minimum amount of sample (<10 μg [41]) in the shortest
instrument time. NMR is successfully used for structure dereplication
and analysis of mixtures, including usage of LC-NMR. Many studies
have shown that CASE is a powerful amplifier of human intelli-
gence. It should be expected that application of pure shift spectra
as input to artificial intelligence programs developed ad hoc for data
import to CASE systems will automate this procedure signifi-
cantly, so facilitating and accelerating the stage of data preparation.
The author believes that, in the near future, CASE will become
a routine analytical tool, which will serve as an integral part of any
NMR spectrometer similar to the software used in X-ray
crystallography.
Acknowledgements
The author thanks Gary Martin for constructive comments and
pieces of valuable advice.
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