2.1. One Dimensional NMR Spectroscopy
The one-dimensional (1D) experiment is by far the most common NMR experiment used for drug studies. The 1D acquisition takes the least amount of time, has one of the simplest hardware requirements, and therefore, in most cases, 1D-NMR is more attractive for high throughput studies. One dimensional NMR spectroscopy normally incorporates a preparation period, some form of induced excitation to form coherence, and lastly, a signal “read” detection period. The preparation period can be modified according to the needs of the experiment or the specifics of the sample. Simple 1D NMR is capable of rapidly producing high-quality spectra of drugs and their targets while revealing how the drugs and targets may interact at the atomic level. 1D 1H-NMR is extremely effective in drug design studies because it has a (relatively) high sensitivity, it is non-destructive, and because hydrogen atoms are extremely abundant in most molecules of interest. Therefore the resulting spectra usually contains a large amount of relevant information and this wealth of data can be acquired in a relatively short period of time. The basic 1D 1H-NMR, along with 1D 13C-NMR, 1D 15N-NMR, and 1D 31P-NMR, and their respective uses in drug design/discovery are briefly discussed below.

2.1.1. 1D 1H-NMR
The 1H hydrogen isotope is NMR visible, has the highest gyromagnetic ratio (apart from tritium) of all of NMR active nuclei, and is combined with a vast natural abundance in organic chemical compounds. This makes the 1D-1H-NMR experiment the most commonly applied NMR approach. Moreover, many software databases [99,100,101,102] are well established for 1H-NMR spectra therefore assisting with processing, analyzing, and identifying the detected molecules automatically. Since almost all drug discovery and drug development studies are performed on samples dissolved in water, many different solvent suppression methods have been applied. The most common is presaturation [103,104]. The key point of this method is to use a low power induced field at the specific frequency of water. This effectively averages out any coherence of the water resonance. The experiment is simple for common hardware to perform and easy to set up; however, presaturation has a substantial disadvantage in that signals resonating close to the solvent signal will show decreased intensity [103,104] or may be lost entirely. This is due to the fact the even selective pulses or very low power pulses also excite some area around the water signal. Also suppressed hydrogen from H2O in solution can exchange with atoms of interest in the molecule and effectively bleed the suppressive spin state to any neighboring atoms. The water signal itself is usually broad, so a wider area of suppression is not necessarily undesirable but affects more of the molecule(s) of interest. More recent water suppression techniques have been developed such as those based on a scheme known as excitation sculpting [105,106]. The basic pulse sequence consists of a double pulsed field gradient echo (DPFGE) in each of which a selective component pulse is flanked by two pulsed field gradients [107]. The particular elements differ for different applications. In the case of water suppression known as WATER suppression by GrAdient Tailored Excitation (WATERGATE), this involves an initial encoding gradient along with the middle element; a combination of two selective 90° rotations on the water along with a central non-selective 180° excitation of all resonances [108]. This is predicated in that water experiences a 360° rotation (effectively nothing) while all other spins experience 180° rotation. The application of the second refocusing gradient does not rephase the water and therefore removes the signal. The reader is referred to the detailed literature [103,104,109] for further information. In principle, a water suppression element (or many elements combined) can be incorporated in any existing pulse sequence to enhance the performance, and it has been implemented in various 1D, 2D, and triple resonance 3D/4D experiments. Although 1H is the most sensitive nucleus for NMR yielding strong, sharp signals within a few minutes [110], chemical shift dispersion of 1H is quite narrow (only around 10 ppm). This has prompted the consideration of other nuclei such as 13C, 15N, or 31P for resolution improvements.

2.1.2. 1D 13C-NMR
Compared with 1H, 13C has a much higher chemical shift dispersion (~200 ppm), however the natural abundance of 13C is low (1.1%). Additionally, the gyromagnetic ratio is ~4 times weaker than 1H and therefore 13C spectra are far more difficult to obtain especially for less concentrated samples. There are some polarization transfer techniques such as Distortionless Enhancement by Polarization Transfer (DEPT) or Insensitive Nuclei Enhanced by Polarization Transfer (INEPT), which can enhance signal intensity by starting the magnetization on a higher sensitivity and abundance proton and then transferring magnetization to the less sensitive carbon nuclei for subsequent direct detection [111], but this requires additional hardware and acquisition times. The use of 1D 13C in drug design studies was illustrated by Tsujimoto et al. [112]. The goal of the study was to examine if a metabolomics approach based on 1H and 13C offers significant improvements when comparing potential drugs. The authors prepared a total of 40 samples with five different citrus-type crude drugs (kijitsu, tohi, chimpi, kippi and seihi) and measured 1D 1H and 1D 13C for each sample. While 1H-NMR spectra allowed the identification of three compounds (naringin, sucrose, and β-glucose), using 13C-NMR allowed unambiguous identification of eight additional compounds (naringin, neohesperidin, α- and β-glucose, sucrose, limonene, narirutin, and synephrine). The added signal resolution from 13C-NMR spectra allowed researchers to obtain better structural information about the compounds than from 1H-NMR spectra alone.

2.1.3. 1D 15N-NMR
In comparison to the previous example, 15N has a lower shift dispersion (~100ppm) than 13C, but higher than that of 1H. Here, the situation is unfortunately severely limited due to an even lower natural abundance (0.37%) and a gyromagnetic ratio ~10 times smaller than 1H. This means that 15N’s combined sensitivity is around 260,000 times lower than 1H. As a result, isotopic enrichment of 15N combined with 1H-mediated enhancement via indirect detection is often needed in order to obtain a satisfactory 1D 15N spectra. Similar to 13C, a few methods are available to overcome such low sensitivity. One of them focuses on tagging molecules with carboxyl groups using 15N-ethanolamine and later detecting the signal using a 2D heteronuclear correlation NMR experiment [113]. Currently, novel approaches such as “smart isotope labeling” have been developed [114]. Also, the SOFAST (Band-Selective Optimized Flip Angle Short Transient) technique can help but results in substantial hardware considerations/drawbacks and often increased concentrations, and/or dramatically longer experiments are still required [115,116,117].
Promising methods are on the horizon. These methods include 15N heteronuclear signal enhancement via Signal Amplification by Reversible Exchange in SHield Enables Alignment Transfer to Heteronuclei (SABRE-SHEATH); however, more work and research are required before such methods can be applied for biomedical purposes [118].

2.1.4. 1D 31P-NMR
With a natural abundance of 100% and a gyromagnetic ratio of about 2.5 times smaller than 1H, one may think that phosphorus could be broadly used for NMR experiments regarding the drug discovery and development. However, the application of 31P is limited due to the fact that most of the molecules of interest simply do not contain a phosphorus atom. Therefore 31P-NMR is usually applicable for studies related to energy, phospholipid metabolism (ATP, NADP), and/or characterization of changes in DNA [94,119,120]. For example, Overall et al. conducted an experiment in which they showed that 31P solid-state NMR can be used for quantitative analysis of DNA dynamics within live bacteria [94]. For that, the researchers first prepared untreated cultures of E. coli, and measured them using a Hartmann-Hahn 1H to 31P cross-polarization (31P CP) experiment. Afterwards, they measured E. coli treated with ampicillin and maculatin 1.1 (Mac1.1) in a similar manner. Spectra obtained from treated bacteria compared to those obtained from untreated bacteria showed alterations in the lineshape, reduced signal intensity at the spectrum’s edges, and a shift in spectral density towards 0 ppm which indicated the increased dynamics of the phosphorus from nucleic acids [94].
Over time, several innovations have been applied to expand the usage of 31P. Like in 13C and 15N labeling of specific biological compounds, incorporation of 31P can also be used. In order to achieve that, 2-chloro-4,4,5,5-tetramethyldioxaphospholane (CTMDP) can be used for tagging lipids containing hydroxyl, aldehyde, and carboxyl groups that can later be detected with better resolution [121]. Another fairly recent method enables toxicological screening of 31P in living cells for several hours without affecting cell viability [122]. This specific method can be used to observe the changes in energy metabolism in real-time while enabling the evaluation of the effects of administered drugs.