NMR in Metabolomics and Natural Products Research: Two Sides of the Same Coin

Abstract

Small molecules are central to biology, mediating critical phenomena such as metabolism, signal transduction, mating attraction, and chemical defense. The traditional categories that define small molecules, such as metabolite, secondary metabolite, pheromone, hormone, and so forth, often overlap, and a single compound can appear under more than one functional heading. Therefore, we favor a unifying term, biogenic small molecules (BSMs), to describe any small molecule from a biological source.In a similar vein, two major fields of chemical research,natural products chemistry and metabolomics, have as their goal the identification of BSMs, either as a purified active compound (natural products chemistry) or as a biomarker of a particular biological state (metabolomics). Natural products chemistry has a long tradition of sophisticated techniques that allow identification of complex BSMs, but it often fails when dealing with complex mixtures. Metabolomics thrives with mixtures and uses the power of statistical analysis to isolate the proverbial “needle from a haystack”, but it is often limited in the identification of active BSMs. We argue that the two fields of natural products chemistry and metabolomics have largely overlapping objectives: the identification of structures and functions of BSMs, which in nature almost inevitably occur as complex mixtures.Nuclear magnetic resonance (NMR) spectroscopy is a central analytical technique common to most areas of BSM research. In this Account, we highlight several different NMR approaches to mixture analysis that illustrate the commonalities between traditional natural products chemistry and metabolomics. The primary focus here is two-dimensional (2D) NMR; because of space limitations, we do not discuss several other important techniques, including hyphenated methods that combine NMR with mass spectrometry and chromatography.We first describe the simplest approach of analyzing 2D NMR spectra of unfractionated mixtures to identify BSMs that are unstable to chemical isolation. We then show how the statistical method of covariance can be used to enhance the resolution of 2D NMR spectra and facilitate the semi-automated identification of individual components in a complex mixture. Comparative studies can be used with two or more samples, such as active vs inactive, diseased vs healthy, treated vs untreated, wild type vs mutant, and so on. We present two overall approaches to comparative studies: a simple but powerful method for comparing two 2D NMR spectra and a full statistical approach using multiple samples. The major bottleneck in all of these techniques is the rapid and reliable identification of unknown BSMs; the solution will require all the traditional approaches of both natural products chemistry and metabolomics as well as improved analytical methods, databases, and statistical tools.

The smell of moulting:N-acetylglucosamino-1,5-lactone is a premoult biomarker and candidate component of the courtship pheromone in the urine of the blue crab,Callinectes sapidus

“…Biomarker targeting has been used to discover bioactive molecules associated with a variety of specific conditions (Robinette et al, 2012). An example in mice is DB, which was first identified as a testosterone-level dependent urinal biomarker (Novotny et al, 1984) and then later confirmed as a pheromone through behavioural bioassays (Novotny et al, 1985).…”

<i>Aspergillus flavus</i> Secondary Metabolites: More than Just Aflatoxins

“…Purification of metabolites is usually required for structure elucidation, however, recently, several metabolites have been identified and structurally characterized using NMR without having been isolated in pure form 135) . 2D-NMR analysis of unpurified extracts can also be used to investigate an organisms' metabolome 136) . Differential analysis by 2D-NMR spectroscopy (DANS) 137,138) was employed to examine the gliotoxin biosynthesis pathway (gli) in A. fumigatus, which revealed many new gli-dependent metabolites 139) .…”

Homo- and Hetero-Covariance NMR Spectroscopy and Applications to Process Analytical Technology

“…While the concept of homo-and hetero-covariance spectroscopy was developed nearly three decades ago, there are relatively few reports on the use of synchronous and asynchronous spectra involving NMR spectroscopy [3,14,55,58,59]. In contrast, an abundant number of investigations have applied so-called statistical hetero-spectroscopy (STOCSY) that has delivered important contributions to the field of metabolomics and whose variants have recently been depicted like a phylogenetic tree [22,32,60]. In the current report, the focus is however laid on examples from chemical processes rather than metabolomics.…”