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Chapter

New Advances in Fast Methods of

2D NMR Experiments
Abdul-Hamid Emwas, Mawadda Alghrably,
Samah Al-Harthi, Benjamin Gabriel Poulson,
Kacper Szczepski, Kousik Chandra and Mariusz Jaremko

Abstract

Although nuclear magnetic resonance spectroscopy is a potent analytical tool

for identification, quantification, and structural elucidation, it suffers from inher-
ently low sensitivity limitations. This chapter focuses on recently reported methods
that enable quick acquisition of NMR spectra, as well as new methods of faster,
efficient, and informative two-dimensional (2D) NMR methods. Fast and efficient
data acquisition has risen in response to an increasing need to investigate chemical
and biological processes in real time. Several new techniques have been successfully
introduced. One example of this is band-selective optimized-flip-angle short-
transient (SOFAST) NMR, which has opened the door to studying the kinetics of
biological processes such as the phosphorylation of proteins. The fast recording of
NMR spectra allows researchers to investigate time sensitive molecules that have
limited stability under experimental conditions. The increasing awareness that
molecular structures are dynamic, rather than static, has pushed some researchers
to find alternatives to standard, time-consuming methods of 15N relaxation observ-
ables acquisition.

Keywords: NMR, 2D NMR, ultrafast data processing, SOFAST, relaxation

1. Introduction

Nuclear magnetic resonance (NMR) spectroscopy is a powerful analytical tool

for identifying, quantifying, and elucidating structures of molecules. Moreover,
NMR offers several approaches to studying molecular dynamics and to probing the
interactions of inert molecules such as protein-protein, protein-DNA, and protein-
ligand. The main strength of NMR spectroscopy is its ability to discern or resolve
individual resonance frequencies of a wide range of different nuclei (e.g., 1H, 13C,
15
N, 29Si, 30Al, 31P to 235U). As the resonating frequency is unique for each type of
nuclei, one can envision an NMR for each nucleus as a separate spectroscopy such as
1
H NMR spectroscopy, 13C NMR spectroscopy, 235U NMR spectroscopy (Figure 1),
etc. More importantly, NMR has the ability to evaluate information about the envi-
ronment of each atom and their neighbor’s nuclei (both through space and through
bond), allowing researchers to differentiate the unique magnetic environments of
the same nuclei in different positions of a single molecule. Thus, NMR spectroscopy
is extensively used for the identification and in the structural elucidation in a wide

1
Nuclear Magnetic Resonance

Figure 1.
Frequency scale ranges and types of spectroscopies that correspond to them, respectively. The natural
abundances and chemical shifts of the most common (15N, 13C, 31P, 19F, and 1H nuclei) nuclei biomolecules
studies with respect to the scale of the 600 MHz proton frequency adapted from [1].

range of applications in gas [2, 3], liquid [4–16], and solid-state samples [17–28].
Nowadays, NMR spectroscopy is one of the most important analytical tools that has
been used in several fields. These fields include structural biology [29–38], organic
chemistry [39–52], polymer characterization [40, 46, 53–64], inorganic chemistry
[65–75], and physics [76–83].
Despite its significant advantages, NMR suffers from some limitations, of which
the relatively low sensitivity seems to be the most severe. An NMR sample can be
treated as a collection of many nuclear spins of magnetically active nuclei that act as
small bar magnets. These nuclear spins have two possible orientations with different
energy levels that adapt when placed within the strong magnetic field. The number
of nuclear spins occupying each energy level is determined by the Boltzmann
distribution equation:

NU / NL = e− ΔE/kT = e− hν/kT

where NU and NL are the nuclei (expressed in numbers) in the upper and
lower energy states, respectively, k is the Boltzmann constant, ΔE is the energy
gap between two energy states of the spins, higher (NU) and lower (NL) energy,
respectively, and T is the temperature expressed in Kelvin (K). Many of the nuclei

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New Advances in Fast Methods of 2D NMR Experiments
DOI: http://dx.doi.org/10.5772/intechopen.90263

are at 298 K, the most common temperature in which biomolecular NMR measure-
ments are performed. At this temperature, there is miniscule excess of the nuclei in
the lower energy state that could be excited to the higher energy state by absorbing
the energy given by the radio frequency (RF) pulse and later detected successfully
during the data acquisition process. Therefore, the relatively low sensitivity of the
NMR measurements seems to be the most severe limitation of this method. Another
limitation comes from each measured nuclei having a defined range of frequencies
at which it resonates (for practical reasons, frequencies are recalculated into the
“unit-less” ppm scale in order to make it independent of the applied external mag-
netic field, B0). This in turn depends on its magnetic neighborhood, and, therefore,
the whole range must be covered with good resolution.
In earlier days, most NMR experiments only recorded one-dimensional (1D)
spectra of which several were of 1H or 13C nuclei. For complex biomacromolecules,
such as proteins, nucleic acids and their complexes, and complex mixtures, this
technique has a particularly low spectral range of 1H resonance, what leads to the
low resolution of the spectra. This low resolution proves insufficient for analysis.
NMR signals depend on several nuclear properties such as the natural abun-
dance of investigated isotopes, nuclear spin (I), gyromagnetic ratio (γ), quadrupo-
lar moment (Q), and spin relaxation rates. Although most elements have an NMR
active isotope, NMR spectroscopy is still not a practical approach for detecting
many elements. For example, quadrupolar nuclei with nuclear spin > ½ possess a
quadrupolar moment that causes an effective spin relaxation mechanism, causing
dramatic line broadening in the NMR spectra. Unfortunately, the nuclear spin for
important elements such as carbon 12C isotope and 16O isotope is zero; thus, no
NMR signals can be detected for such nuclei. For example, the NMR active isotope
of oxygen, 17O has a low natural abundance (0.037%), low receptivity, quadrupolar
nuclear spin = 5/2, and very short spin relaxation T1 values. These factors make 17O
NMR spectroscopy an unfeasible approach. Thus, nuclei with nuclear spin = ½;
high natural abundance similar to those of 1H, 31P, and 19F; and high γ are the most
accessible nuclei for NMR studies in organic and biological samples. Even though
the 1D 1H NMR spectroscopy is the most common method, it suffers from the peak
overlap as the spectra is very narrow (about 10 ppm). To overcome the challenge
of spectra overlap, the multidimensional techniques which encode the indirect
dimensions were proposed (i.e., Ernst, 2D, Bax 3D 90s, Clore 4D, Michal, etc.).
Multidimensional NMR spectra have efficiently tackled the resolution problems
for the spectra of large molecules such functional biomolecules and their respec-
tive native complexes. Applying two-dimensional (2D) or multidimensional NMR
experiments are usually used to resolve overlapping peaks. Nevertheless, 2D experi-
ments require considerably larger amounts of time than the usual 1D experiments
do. The coincidence of simultaneous increase in measurement time with increase in
dimensionality is directly correlated with the exact mechanism of how exactly the
additional dimension is built in NMR. In an nD NMR experiment, n dimensions are
created by n independent time increments which encode chemical-shift informa-
tion. In 1D NMR, chemical shifts are encoded during acquisition time when data
is written referred to as direct dimension hereafter. There we have no restriction
in putting the number of complex points as it will not increase the measurement
time significantly. The scenario changes drastically when moving to multidimen-
sional NMR. In multidimensional NMR, first there is a prepare period and then a
mixing period where the magnetization is transferred to another dimension. Here
both homonuclear or heteronuclear magnetization can be generated which can be
frequency labeled. This frequency labeling is the evolutionary period, and here
the number of pints will determine the resolution of this dimension and the total
measurement time. Each time increment is equivalent to record one additional

3
Nuclear Magnetic Resonance

experiment. Hence, for nD experiment, we can construct n dimensions, and in

each dimension, some points have to be recorded to achieve the desired resolution
in that dimension. This is the main reason the measurement time increases in a
multiplicative manner with respect to the number of points in each dimension one
needs to acquire to achieve the desired resolution. In the simplest multidimensional
NMR, two-dimensional NMR thus uses two frequency axes. These are namely
direct as explained earlier and indirect axis which is created by time increments
after mixing. Now each time increment encodes one 1D spectra individually, and
the second frequency is generated by the second Fourier transformation. Signal
intensity is usually presented on the spectra as contours, bearing resemblance to
topographic maps, or as an intensity plot. Thus, some studies (such as untargeted
metabolomics) do not fully utilize 2D NMR because there are a large number of
samples that would require more time to complete. Several approaches have been
continuously developed to reduce the experimental time and to enhance the NMR
sensitivity. These approaches include hyperpolarization methods such as dynamic
nuclear polarization (DNP), cryoprobes, and new ultrahigh magnetic fields. These
methods, however, are costly and need substantial hardware additions.
Other methods such as cross-polarization, INEPT, DEPT, and fast methods
provide good enhancements without hardware additions and are available in most
existing NMR systems. Fast 2D NMR methods and band-selective optimized-flip-
angle short-transient (SOFAST) 2D NMR methods provide good NMR spectra
with a short experimental time, which in turn enables the researchers to carry
out powerful 2D NMR experiments in a few minutes. Increasing the number of
spectral dimensions increases the spectra acquisition time dramatically. In order
to overcome this issue and reduce the demand for the experimental time needed to
perform the spectra measurement, different methods of acquisition of the spectra
have been proposed. In this chapter, we introduce the relaxation mechanisms, and
then we briefly discuss a few methods of fast methods including SOFAST, ultrafast
(UF) 2D NMR, pure shift NMR, and post-processing methods. The pure shift NMR
method produces spectra that only contain chemical-shift data and is void of any
coupling information. This reduces the spectral complexities to a large extent as all
multiplets collapse in singlets. This saves a lot of analysis time. Pure shift as such is
not a fast method. However, we have incorporated this as it is indeed an important
milestone in achieving high spectral resolution and a very important step towards
minimizing the spectral analysis time.

2. Fast 2D NMR approaches

The power of NMR is well realized because it is the only noninvasive tech-
nique which has an atomic level resolution and can work well for both solution
and solid-state samples. Although one-dimensional NMR in principle contains a
wealth of information, the situation becomes severely limited due to signal overlap
for small organic molecules onwards. This requires spreading the signals in other
dimensions which can be the same nuclei or different NMR active nuclei. Here,
concepts of 2D or multidimensional NMR arise, and with the development of 2D,
3D, and even higher-dimensional NMR, the overlap problem is solved even for large
biomolecules. The price we pay is an increase in measurement time. Any higher-
dimensional spectra require certain time increments to achieve certain spectral
resolution, and as a result, the measurement time increases enormously as the
number of dimensions increases. Generally, for the same sample moving from 1D
NMR to 2D, the measurement time increases by at least two orders of magnitudes.
Moving from 2D to 3D the experimental time increases further by more than one

4
New Advances in Fast Methods of 2D NMR Experiments
DOI: http://dx.doi.org/10.5772/intechopen.90263

order of magnitude. Similarly, 4D and 5D become so time-consuming that they are

hardly used in practice. This is the main reason for steady research in the direction
to overcome the time limitation so that speedy acquisition can be feasible. Here we
will discuss some of these advancements and will limit the discussion to 2D. The
basic building block of the 2D or any multidimensional experiment consists of a
preparation period, evolution period that incorporates incremental time delay,
mixing period, and one recycle delay which is required for getting longitudinal
magnetization back to equilibrium. There have been numerous methodologi-
cal developments focusing on each aspect of these building blocks. Nonuniform
sampling (NUS) reduces the number of incremental delays. FAST or SOFAST
methods reduce recycle delay which is on the order of seconds. Ultrafast works on
preparation and mixing components, and with the help of slice selection gradient,
single-scan experiments are designed. We will go through each aspect separately.
Lastly, there is pure shift NMR which focuses on enhancement of resolution.

2.1 SOFAST 2D NMR

We first focus on SOFAST method that aims to reduce the inter-scan delay
and eventually manages to lower the 2D acquisition times [84]. This technique is
optimized in order to obtain fast and sensitive 2D NMR HMQC with 1H-15N and
1
H-13C correlation of biomolecules with a different size range [84]. The SOFAST-
HMQC experiment makes use of selective 1H pulses affecting only a portion of
proton spins while keeping a large pool of proton spins unperturbed at Z axis.
These unperturbed spins enhance the spin-lattice relaxation (T1) rate via dipo-
lar interactions with the perturbed spins and effective recycle delay decreases.
Additionally, selective excitation is achieved with Ernst angle excitation which helps
in acquiring more sensitivity [85] at lower recycle delay. Therefore, the concerted
use of Ernst angle excitation and higher repetition rates of the pulse sequence yield
high signal-to-noise ratio compared to conventional experiments. This strongly is
recommended in biomolecular NMR, to study protein modifications and real-time
molecular kinetics [85]. Numerous versions of SOFAST-HMQC experiments have
been proposed for different purposes depending on the experimental conditions
[86, 87]. As an example, 13C methyl SOFAST experiment has been developed, allow-
ing the recording of high-quality methyl 1H-13C correlation spectra of protein with

Figure 2.
Profits of the high-field NMR magnetic fields on spectral resolution and sensitivity. High-resolution 1D
1H NMR spectral of a representative small well-folded protein (A) U-15 N human ubiquitin of 1 mM
concentration in 20 mM phosphate buffer, 10% D2O, and 0.02% NaN3 recorded with the same parameter set at
three spectrometers working at 500, 700, and 950 MHz proton frequencies at 24°C.

5
Nuclear Magnetic Resonance

high molecular weight in a few second acquisition time [88]. Methyl groups are used
as spectroscopic probes of protein structure, dynamics, and kinetics because they
are dispersed naturally throughout regions of folded protein. It has been shown that
the use of methyl groups as a probe is very useful, particularly for drug binding and
molecular interactions studies [88].
For biomolecular NMR applications, the use of longitudinal relaxation opti-
mized experiments permits the acceleration of 2D NMR data acquisition which
provides a tool for high-throughput screening for macromolecules like proteins
or nucleic acids. Such experiments allow one to obtain a high signal-to-noise ratio
compared to its counterpart, as mentioned previously. Theillet et al. present an
example of the use of SOFAST-HMQC experiment in order to monitor protein phos-
phorylation reactions [89]. In this study, the N-terminal transactivation domain of
human p53 15N-labeled protein was phosphorylated by either recombinant enzyme
or endogenous kinases present in cell extracts. The protein phosphorylation reac-
tions then were monitored by SOFAST-HMQC NMR experiments in real time to
obtain a high atomic-resolution understating of phosphorylations level of serine
and threonine residues in kinase reactions [89].

2.2 Ultrafast 2D NMR

Secondly, we focus on ultrafast 2D NMR that is able to deliver any kind

of 2D NMR spectra involving homo- or heteronuclear correlation in a single
scan and, therefore, in a fraction of a second. The basis of this approach relies
on the concept of revealing the spin interactions measured by spatiotemporal
encoding [90]. The strategy is essentially that, instead of measuring N1 succes-
sive experiments multiple times on the sample with an independent increase
of time, a better way to do it, as in the case of UF spectroscopy, is the one that
“divides” the sample into N1 fractions and records all of them simultaneously
within one scan (Figure 3) [90]. The application of slice-selective gradients
encodes different phases in different slices similarly to incremental decay. As a
result, the entire spectrum can be built from a single scan. However, the cost is
reduced sensitivity.

Figure 3.
Comparison between the conventional and UF 2D NMR approaches. (a) Conventional approach of 2D NMR
is done on the sample by measuring N1 successive experiments multiple times with an independent increase of
time. (b) UF 2D NMR approach can be conceptualized as “dividing” the sample to fractions and recording all
of them simultaneously within one scan [adapted and modified from ref.[90]].

6
New Advances in Fast Methods of 2D NMR Experiments
DOI: http://dx.doi.org/10.5772/intechopen.90263

The so-called fast 2D NMR spectroscopy has found multiple applications in

the field of metabolomics [86, 91, 92], as well as in the analysis of natural products
[92–94]. The ultrafast quantitative 2D NMR spectroscopy is also an appropriate
choice for measuring 13C enrichments in a single scan (with ultrafast COSY and
zTOCSY experiments). The authors of this study have reported an accuracy of
1–2%, an average precision of 3%, which is believed to be better than those previ-
ously achieved with different approaches [95]. This approach is also used in acquir-
ing R1 longitudinal relaxation rates [4].

2.3 Nonuniform sampling and processing

Multidimensional experiments have two significant limitations: sensitivity and

low resolution, especially for large molecules. For obtaining good sensitivity, we need
a very high number of scans for 13C, 15N-labeled samples, and J-coupling transfer,
and dipole-dipole interactions were utilized. For instance, metabolomics research
is based on the analysis of the natural abundance of carbon and hydrogen nuclei;
therefore, the sensitivity remains the main issue because isotope labeling cannot be
applied. Enhancing the sensitivity and reducing the acquisition time may require
the concerted use of methods such as SOFAST method and nonuniform sampling
while decreasing the number of recorded points for indirect dimensions. Sometimes
the single-scan approach can be used [96–98]. The main limitation of single-scan
methods, however, is recording only 1H plane, even for 2D experiments [99].
Some studies demonstrated that a combination of fast methods with reducing
the number of data points can benefit multidimensional NMR spectra like BEST-
Trosy, which provide magnification of sensitivity and resolution for structural and
kinetic investigation of biomolecular and complex systems [100].
Normal 2D experiments yield 2D spectra, while during data acquisition in the
indirect dimension (marked as t1), the second “vertical” dimension has incremental
time delay. This time delay is linear, and the gap between the time points is the
same. In contrast, NUS approaches the t1 change in a semi-random way. In NUS,
only some of the time increments are recorded, and then the complete spectra are
constructed after reconstructing the missing data points. Various algorithms are
designed to select which data points to acquire and what will lead to successful
reconstruction. The advantage of NUS is that the same level of resolution can be
achieved with significantly fewer t1 increments. Thus, overall measurement time
decreases. This is practical for NMR spectroscopist because this means one can
obtain well-resolved 2D NMR spectra in less time.
There has been extensive research on the optimization of finding suitable com-
binations for sampling schemes and reconstruction algorithms [98]. NUS sampling
conventionally selects random acquisition points in the indirect dimensions. The
“randomness” of NUS is restricted by factors such as the transverse relaxation rate.
Exponentially weighted random sampling is designed to overcome the T2 decay as
more signals decay incrementally in the initial phase. Poisson gap sampling has also
been used to yield excellent results as described later. Processing the recorded data
can be classified in several ways [101]. For example, the sparse time domain data
may be directly transformed with Fourier transform (FT) algorithms, and subse-
quently the missing data are added by interpolation and then followed by conven-
tional Fourier transform [101] in the second dimension. This can also be reversed
for reconstruction, followed by FT in both dimensions.
NUS can be a good tool, especially for triple resonance experiments, which have
a low dynamic range. An almost identical intensity and a small number of peaks
allow the use of low-density sampling. For a highly dynamic range of signals in
experiments like NOESY (which provides information on the restraint through

7
Nuclear Magnetic Resonance

the space between atoms), reconstruction will be better with less mixing time.
The positive results of reconstructed spectra were demonstrated with 20% and
more sparse incremental time [102]. NUS methods have also been widely used for
resonance backbone and side-chain assignments of macromolecules, determina-
tion of cross-correlated relaxation rates (CCR), kinetic processes, posttranslational
modifications, and metabolomics [103].
The determinative criteria of spectra quality have a big gap in the random
sampling schedule, especially at the beginning and at the end of dataset points. To
overcome this limitation, the Poisson distribution [104] was used with sinusoidal
variation of average gap length. In particular, sinusoidal variation of gap length
keeps a degree of order in the sampling schedule, and it condenses points at the
beginning of the time domain data. This provides significant benefits for time
domain data with exponential decay [104].
The approach for the reconstruction of traditional uniform sampling is FT
[105]. The processing spectra from NUS acquisition experiments, however, demand
different types of reconstruction methods because regular discrete FT leads to
artifacts as several data points are missing. Among the reconstruction algorithms,
multidimensional decomposition (MDD) is the model where the full matrix of
different dimensions can be identified as a sum of one-dimensional vectors (com-
ponents), in which each of the components is coherent to a peak or group of peaks.
MDD essentially breaks multidimensional data into sets of one-dimensional data,
which are much easier to analyze and solve. The overlap in multidimensional NMR
is common, and since MDD is able to resolve the overlapping resonances, MDD
is well equipped for resolution enhancement of crowded datasets. MDD typically
only needs a subset (20–30%) of the full dataset in order to reconstruct the full
NMR signal [106, 107]. Figure 4 gives a visual demonstration of the sampling of
the full dataset to make a smaller dataset and create the spectra. One paper reports
a method demonstrated on nonuniformly sampled [16]N-NOESY-HSQC datasets
recorded for the 14 kDa protein azurin [108]. MDD typically only needs a subset
(20–30%) of the full dataset in order to reconstruct the full NMR signal [106, 107].
Another algorithm is compressed sensing (CS) which is one of the best recon-
struction methods available. Maximum entropy method (Max Ent) is another
algorithm. CS is very useful in under sampled multidimensional experiments for
solid-state NMR, fast metabolomics studies, and chemical-shift imaging [109, 110].
CS was demonstrated to give better reconstruction of weaker peaks, compared to
an existing Max Ent implementation [110]. In one paper, two time-equivalent 3D

Figure 4.
Visual demonstration of NUS for obtaining NMR spectra. NUS takes a small subset of the total dataset
(crowded blue dots) and uses that comparatively small dataset (20–30% of the total dataset) to create an
accurate NMR spectra of the molecule of interest.

8
New Advances in Fast Methods of 2D NMR Experiments
DOI: http://dx.doi.org/10.5772/intechopen.90263

NUS TROSY-HNCA semi-constant time experiments were recorded, with CS giving

better overall peak resolution [111].
Iterative soft threshold (IST) is another type of reconstruction algorithm. Like
other methods, IST is able to “clean up” the NMR data by filtering the spectra. It
does this by setting a threshold value and then, as its name suggests, runs through
a fixed number of iterations until the procedure is complete. The best approach to
IST is to use it in sync with an optimized NUS schedule. IST computational methods
only take a reasonable amount of time and greatly reduce the analysis time required
to obtain NMR spectra [112, 113]. One paper reports successful application of the
IST approach to the ubiquitin protein. On a dual core, 2.2GHz CPU, the overall
time required to process the sampled NMR dataset took under 60 seconds [114], a
significant contribution to decreasing total analysis time.

3. Pure shift NMR

Proton NMR suffers from extensive signals splitting due to the presence of many
weak and strong couplings, leading to significant spectral overlap, even for small
molecules. The problem is severe at low magnetic fields. Although the coupling
constant gives useful structural information, difficulty in obtaining them due to
strong overlap often becomes a major roadblock. Ideally, a spectrum, where all mul-
tiplets collapse in singlets, gives the most straightforward information which can be
achieved by pure shift. Subsequently, the analysis of couplings can be introduced in
a systematic manner. The problem is simple for 13C NMR as natural isotopic abun-
dance is low and only singlets are present. However, the situation becomes complex
for 13C isotope-enriched samples, especially for large biomolecules. In those cases,
spin-state-selective decoupling are most commonly used. Both components of the
splitting due to 13C-13C coupling are recorded in a linear combination of inphase
mode and antiphase mode. This is known as IPAP (inphase-antiphase) [115] or
direct excitation of single-quantum coherences [116]. This method mainly works
because of clear 13C signals in different spectral shift regions and different and
distinct spin-spin coupling values between those different classes of spins. This
is clearly not the case for proton NMR where coupling values spread much wider
ranges and also spectral overlap is more severe.
Pure shift NMR focuses on chemical shift and removes homonuclear couplings
which lead to complex multiplet structure of individual peaks. This results in
severe overlap even for small molecules and particularly for 1H spectra since the
chemical-shift dispersion of the proton is normally lower (~10 ppm) compared
to other heteronuclei like 13C or 15N. Having the highest gyromagnetic ratio and
natural abundance, 1H NMR is the most obvious choice for almost all routine and
conventional small and large molecule analysis. However, the spectral overlap
due to homonuclear coupling limiting the resolution presents a major limitation
of 1D proton NMR spectroscopy. Pure shift NMR was developed to simplify the
overlapped proton NMR spectra by keeping only chemical-shift information. There
have been great contributions coming first from Zangger [117–120] and subse-
quently by Morris and others [117, 121–127] in this direction. Some of the recent
advances are presented here.
First experimental demonstration of homonuclear broadband decoupling in
proton NMR was reported by Ernst and coworkers [128]. The basic pulse program
consists of a 90° excitation pulse followed by spin echo. So, in direction dimen-
sion, homonuclear coupling is active, and chemical-shift evolution refocuses at the
end of t1, while both chemical-shift and homonuclear couplings are active during
acquisition. So, the resultant spectrum shows tilted multiplets where the indirect

9
Nuclear Magnetic Resonance

dimension spreads out the J-coupling. Now, 45° projection of the 2D spectrum
yields pure shift spectra where all the scalar couplings have been removed. The
main problem of J-resolved spectra is the twisted phase accumulation during t1
which leads to both absorptive and dispersive phase components in the indirect
dimension. This results in partial or complete cancelation after taking the projec-
tion. To avoid this, normally absolute value mode calculation or power spectrum is
taken before the calculation 45° projection. This generates broad peaks with long
tails in the final decoupled spectrum.
The most prominent contribution came from Zangger and Sterk [120] who
employed selective pulses and gradient encoding simultaneously. In this approach,
first a selective 90° pulse was applied, followed by an incremental delay and com-
bination of hard and soft selective 180° pulse with the same incremental delay
and subsequent acquisition. The pulses were applied with weak gradient encoding
implying spatial resolution in the sample. Hence, the whole sample is now divided
in different slices, and the first frequency-selective pulse excites signals of different
frequencies in different slices of the sample tube. Most importantly all signals are
excited at once, but the price we pay is loss in sensitivity as the effective signal is com-
ing from a single slice. Normally, the first experiment proposed was ~2% sensitive
compared to conventional 1D NMR. In each slice, selective signals get excited by the
selective first excitation pulse. Subsequently, coupling with all other spins evolves in
the incremental delay. Next the selective spins undergo complete 360° rotation, while
all other spins undergo 180° rotation initiated by hard 180° pulse. Therefore, after
the same incremental delay, the J-coupling between the selected spin with all other
spins gets refocused and homonuclear decoupling is achieved. The initially proposed
scheme recorded FID with different incremental time delays. Then, from each FID,
the first few points (tc/DW) determined by the incremental delay (tc) and the dwell
time (DW) were concatenated to generate the final FID. The excitation bandwidth
is determined by the relative proximity of the scalar-coupled protons. If coupled
protons with very similar chemical shifts need to be resolved, then the selective
pulses must be highly selective. This also implies the application of higher gradient
strengths to generate more slices to cover the full spectral width which decreases sen-
sitivity immensely as the effective slice thickness reduces. There have been consider-
able developments to overcome the limitations in the first proposed scheme.
First the two-dimensional mode of data acquisition was omitted, and the
homonuclear broadband decoupling was performed during acquisition [119]. This
generates a single-scan decoupling sequence like normal 1D NMR. This simplified the
whole thing as these signals can be stored as regular 1D or as acquisition dimension
of multidimensional spectra. Most importantly, the processing of the data becomes
a routine, and there is a huge reduction of total measurement time. In this modi-
fied scheme shown in Figure 5, the acquisition is interrupted approximately every
1/3(3JHH) to incorporate decoupling block. Additionally the decoupling of the signals
should be placed right in the middle of the data chunk. So the first and the last acqui-
sition block length is only half as long of all other blocks sandwiched between two.
Since relaxation is always there, some chunking artifacts arise after Fourier
transform as FID gets interrupted in between. Further, to reduce the overall
measurement time, this slice-selective excitation was combined with fast pulsing
by shifting the frequency of the selective pulse between individual scans [117]. In
slice-selective decoupling, in each slice only the excited magnetization is used for
the acquisition. So, by shifting the offset of the 90° excitation pulse and also the
selective 180° refocusing pulse after every scan, the unused equilibrium magnetiza-
tion can be used without any recycle delay. The next scan or the subsequent scans
with different offset act as recycle delay for the first set of spins which were excited
in the first scan. This makes repetition much faster, and overall signal-to-noise ratio

10
New Advances in Fast Methods of 2D NMR Experiments
DOI: http://dx.doi.org/10.5772/intechopen.90263

Figure 5.
(a) Pulse sequence of first Zangger-Sterk experiment. The delay τ c was incremented by several milliseconds.
The first of each FID was concatenated to produce the final FID as outlined in the text. (b) the real-time
slice-selective broadband homodecoupling approach. The data chunking times are variable which is represented
by number n. frequency shifting is achieved for all selective pulses in parallel after each transient. The slice-
selective gradients are represented by g1 and g2; G2 and G3 clear any imperfection of the refocusing pulse.

increases per unit time as shown in Figure 6. Apart from varying offset in between
different transients, FID chunk size also varied to suppress the decoupling side-
bands. The decoupling sidebands arise due to truncation of FID, and the longer the
FID chunk, the more dominant are the artifacts as more antiphase magnetization
can build up. On the contrary, if keeping the individual FID blocks, short leads to
broad signals as more relaxation losses occur during the more frequent acquisition
interruptions. The variation of chunking time between subsequent scans leads to
smearing out of the decoupling sidebands which otherwise add up if they are kept
the same. The slice-selective decoupling has been employed for two-dimensional
homonuclear TOCSY [127, 129] and NOESY [122] spectra. The enhanced resolution
in the direct dimension can be propagated to the indirect dimension by applying
covariance processing which yield pure shift spectra in both the dimension for
TOCSY spectrum. Additionally for heteronuclear HSQC, the slice-selective excita-
tion was employed for intrinsically disordered proteins (IDPs) [130] where proton
spectral dispersion is severely low. Additionally slice-selective excitation has been
used for fast data acquisition [131] and was able to monitor chemical reaction from
different spatial location of the sample tube [132].
Recently a new method named as pure shift yielded by chirp excitation
(PSYCHE) experiment has been proposed by Morris et al. [125] which is based on
anti Z-COSY experiment. This experiment employs two small flip angle chirp pulses
which selectively refocus a small proportion of spins, namely, active spins, while
leaving the majority ones undisturbed, namely, passive spins. Now this selection of
active and passive spins are purely statistical which leads to an obvious advantage
over ZS method as there the selection was based on selective pulse. The sequence is
closely related to anti Z-COSY except to the fact that instead of using two hard small

11
Nuclear Magnetic Resonance

Figure 6.
(a) A regular 1D spectrum, (b) pure shift with fixed chunking time of 25 ms, (c) pure shift with variable
chunking time with real-time acquired spectrum of a mixture of strychnine with unknown degradation
products in CDCl3. Gray circles indicate artifacts resulting from decoupling sidebands. Weak peaks visible in
(b) result from additional minor compounds in the mixture, which are way below the level of artifacts in the
fixed chunking time spectrum. The figure presented is from [117].

flip angle pulses, it uses two symmetric, low power, frequency-swept chirp pulses
along with weak pulsed-field gradient. This gradient employs spatial resolution and
suppresses the cross-peak terms as related to anti Z-COSY since they experience dif-
ferent chemical shifts as they are excited at different times during the chirp pulses.
The major advantage of PSYCHE is that it is most sensitive among the existing pure
shift methods. Several further developments are achieved to obtain ultraclean arti-
fact-free spectra and applied in 2D NMR as well [133, 134]. Very recently one such
modification named SAPPHIRE-PSYCHE methodology is applied for plant metabo-
lomics study. In that report, single-pulse, PSYCHE, and SAPPHIRE-PSYCHE spectra
were compared from aqueous extracts of Physalis peruviana fruits. It was found using
pure shift methodology; many proton NMR signals can be cleanly observed which

12
New Advances in Fast Methods of 2D NMR Experiments
DOI: http://dx.doi.org/10.5772/intechopen.90263

were not possible in conventional NMR. These signals belong to amino acids, organic
acids, and sugars which are critical components of plant mixture. One example of
such metabolite was the identification of glutamic acid from Cape gooseberry which
was not possible to isolate due to heavy overlap. Thus, ultra-clean pure shift spectra
look very promising and provide new benchmark for metabolomics studies.

4. Summary and future perspectives

High-resolution NMR with high sensitivity is the desired aim for any NMR
application. There have been significant hardwire advances in achieving high
sensitivity. This includes the development of high magnetic field, cryogenically
cooled probes [135], recent advent of high-mass-sensitive probe for small volume
[136], etc. A combination of these advances results in an increase of sensitivity by
more than an order of magnitude. These advancements fueled the developments of
fast methodology. Additionally due to the sample instability, which results in the
relatively fast decomposition of the biomolecule being an object of the study, the
quick methods of 2D spectra acquisition needed to be developed. In this regard,
previously mentioned SOFAST, NUS, etc. methodologies were developed and
subsequently applied in many different scenarios [90, 93, 94, 137]. Above that,
these fast methods can be also used for studying either chemical or biochemical
processes that are characterized by the relatively fast kinetics and also carry some
structural information, like hydrogen/deuterium (H/D) exchange [138, 139]. With
NUS approach, one problem is the generation of very large file size, especially for
4D and 5D experiments. Artifacts are also produced, which generates “unwanted”
signals that do not accurately represent the studied molecule. However, there is
considerable effort in the scientific community to reduce the amount of produced
artifacts. For example, newly proposed MUNIN uses three-way decomposition
and a simplified model for NMR spectra based on generally accepted assumptions.
This method achieves high-resolution and good sensitivity while avoiding artifacts
[106]. Also, a signal separation algorithm is shown to suppress sampling artifacts
in high-resolution four-dimensional NMR spectra [140]. It is likely that NUS-based
methods will continue to be used in the future, mainly because they will greatly
reduce experimental time, and still provide satisfactory results with increased
resolution of the spectra what facilitates their analysis. However, with the increase
in magnetic field, resolution has been improved only two times and not more than
that. Even with the availability of very high-field magnet such as 1.1 GHz and
upwards which is limited, resolution was still a problem. Hence, the development of
pure shift methods became necessary, and with the techniques great resolution can
be achieved uniformly for all scenarios. In this regard, both the methodologies and
developments in the direction of reducing measurement time and increasing resolu-
tion remain of utmost importance and have become an area of research itself.

Acknowledgements

We would like to thank King Abdullah University of Science and Technology for
financial support.

Conflict of interest

The authors declare no conflict of interest.

13
Nuclear Magnetic Resonance

Author details

Abdul-Hamid Emwas1, Mawadda Alghrably2, Samah Al-Harthi2,

Benjamin Gabriel Poulson2, Kacper Szczepski2, Kousik Chandra2
and Mariusz Jaremko2*

1 Core Labs, King Abdullah University of Science and Technology (KAUST),

Thuwal, Saudi Arabia

2 Biological and Environmental Science and Engineering (BESE), King Abdullah

University of Science and Technology (KAUST), Thuwal, Saudi Arabia

*Address all correspondence to: mariusz.jaremko@kaust.edu.sa

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms
of the Creative Commons Attribution License (http://creativecommons.org/licenses/
by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,
provided the original work is properly cited.

14
New Advances in Fast Methods of 2D NMR Experiments
DOI: http://dx.doi.org/10.5772/intechopen.90263

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