Applications of NMR Spectroscopy

Tautomerism is a chemical equilibrium which involves rapid transference of a hydrogen atom. Its importance in biochemistry, medicinal chemistry, pharmacology and organic synthesis, as well as the wide variety of molecules in which it occurs, makes it an interesting chemical issue to be studied. NMR bears the advantage of allowing equilibrium observation without shifting it. The aim of this work is to sum up a variety of experiments that can be carried out on tautomeric equilibria in order to obtain structural and mechanistic information. In every case, the two (or more) major tautomeric forms must have relatively low conversion rates, i.e., they must exist long enough to survive (in average) the NMR experiment and then show different but overlapped NMR spectra. Assignation of the peaks to their corresponding tautomeric form has to be done with regarding signal integration, multiplicity and chemical shift of the signals. Theoretical calculations might be carried out in order to do this assignation. Once found two (or more) independent and non-overlapped peaks corresponding to each tautomer, their integration permits tautomeric contents and tautomerization constants calculation. Herein, equilibrium shifts caused by the presence of substituents (causing electronic and steric effects), solvents (interacting in different ways with the tautomers), internal chemical interactions (such as hydrogen bonds), tautomer-tautomer interactions (producing the formation of dimers) and temperature variation are discussed using a variety of compounds, such as ketonitriles, ketoamides and salicylaldimines, among others. All these facts give information about the causes of the stabilization or destabilization of different tautomeric forms.

Living cells possess the ability to tune their response to various intra- and extracellular cues, e.g., oxidative stress, nutritional deprivation, etc., which in turn leads to an assortment of different metabolites in the cellular soup. Metabolomics – the science that deals with understanding the metabolic profile of a biological system under a set of conditions – is becoming a powerful tool that helps to comprehend the mechanistic information about cellular events. The information about the small molecules present in the biological soup is now being used in different facets of research in life sciences, including natural product identification; studying pharmacological responses of potential drugs; understanding the chemical diversity of a plant/microbial species based on geographical origin; translational metabolomics of bio-fluids to understand various diseases, and cellular pathway analysis; etc. The technological challenge in the identification as well as characterization of these small molecules has been answered in part by recent advances in the field of Mass spectrometry and NMR, coupled with high-throughput separation techniques. In this chapter, we will discuss the diversity of the metabolome; primary and secondary metabolites; overview of available techniques for metabolite identification and characterization; recent advances in NMR making it amenable for the study of metabolites; and the methods used for NMR data measurement and analysis.

In the field of diagnostic medicine, Nuclear Magnetic Resonance (NMR) spectroscopy has found wide applications. Simplicity of sample preparation, biological safety, non-invasiveness, and non-destructiveness are the advantages of NMR based metabolomic studies. One of the main drawbacks is lower sensitivity. This review summarises the current achievements of NMR spectroscopy in the diagnosis of human disorders. NMR spectroscopy has been useful in metabolomics-based diagnosis. NMRbased screening for inborn errors of metabolism is practised as more metabolites can be detected. Fast, simple and cost-effective screening is possible. Neuropsychiatric disorders like schizophrenia, panic disorders, major depression, bipolar disorders and autism-spectrum disorders have been investigated systematically using NMR spectroscopy. NMR metabolomics is used in the search for biomarkers of infectious diseases like tuberculosis, malaria and pneumonia, neurological disorders and Parkinson’s disease. NMR metabolomics has been used in the identification of biomarker for cardiovascular diseases and risk stratification. It has also been used in cancer diagnosis and therapy. NMR metabolomics has been used to investigate processes like transformation, progression, proliferation and metastasis in cancer cell lines. Other examples for applications of NMR metabolomics include gastro-intestinal disorders, endocrine and nutritional disorders, disorders of the nervous system and respiratory system disorders. In the coming years, it is expected that further developments to overcome the technical limitations will take place, making it one of the key diagnostic modalities of the future.

Nuclear magnetic resonance through the use of proton relaxometry measures the time needed by a population of spins once in the external magnetic field to recover from a resonant radiofrequency excitation. Generally, two relaxation parameters can be determined to describe the evolution of the spins system to the equilibrium state. Proton spin-lattice relaxation time with a time constant, T1H, and proton spin-spin relaxation time, with a time constant, T2H, are useful for elucidating molecular dynamics of complex solid materials, like food samples, for example. These parameters are of great interest because they are often used to obtain information on molecular mobility of molecules ranging from small molecular groups and domains size. Differences between values obtained for different samples can be used to evaluate changes in the molecular mobility in the food systems like the moisture absorption and water and oil distribution. Many studies can be done in food samples employing relaxation data, such as: cooking, food storage, storage processes, validity time of flours, major constituents and anti- oxidant in oils and so on.

The issues of climate change, land degradation, and sustainable agriculture are arguably three of the most significant scientific, political, and environmental challenges of the 21st century. However, one common denominator that is frequently overlooked when trying to understand and respond to key processes in the environment is the role of soil microbial biomass. As an example, soil microbial biomass controls the production of soil organic matter—the largest pool of terrestrial carbon—which is an important variable in studies of climate change and is also central to sustainable agriculture. Traditionally, the complex nature of this biomaterial renders it difficult to study at sufficiently fine resolution. At the molecular level, NMR spectroscopy is the single most powerful analytical technique available for the determination of structural components and their interactions in various environmental matrices, and can be performed on components of different physical phases [solids, semi-solids (gels), liquids, and gases]. NMR spectroscopy provides information about the chemical environment of nuclei within a molecule, and the three most frequently studied nuclei in microbial biomass are protons (1H), carbons (13C) and (15N). However, carbon and nitrogen NMR are generally considered relatively insensitive analytical techniques, because at natural abundance only 1% of the total carbon (13C) and 0.37% of the total nitrogen (15N) is NMR observable, while 99.9% of all protons are detectable. The complexity and multitude of components within a natural organic matter sample lead to extensive spectral overlap, especially in solid-state NMR. Therefore, the transformations of minor components are difficult to monitor unless we enhance NMR sensitivity by increasing the relative abundance of 13C and 15N through isotopic enrichment. This chapter provides an overview of the application of various NMR techniques used to provide comprehensive molecular and structural information, as well as some of the key considerations in the field of microbial biogeochemistry.