NMR-spectroscopic analysis of mixtures: from structure to function

Abstract

NMR spectroscopy as a particularly information-rich method offers unique opportunities for improving the structural and functional characterization of metabolomes, which will be essential for advancing the understanding of many biological processes. Whereas traditionally NMR spectroscopy was mostly relegated to the characterization of pure compounds, the past few years have seen a surge of interest in using NMR-spectroscopic techniques for characterizing complex metabolite mixtures. Development of new methods was motivated partly by the realization that using NMR for the analysis of metabolite mixtures can help identify otherwise inaccessible small molecules, for example compounds that are prone to chemical decomposition and thus cannot be isolated. Furthermore, comparative metabolomics and statistical analyses of NMR spectra have proven highly effective at identifying novel and known metabolites that correlate with changes in genotype or phenotype. In this review, we provide an overview of the range of NMR-spectroscopic techniques recently developed for characterizing metabolite mixtures, including methods used in discovery-oriented natural product chemistry, in the study of metabolite biosynthesis and function, or for comparative analyses of entire metabolomes.

Figure 1

The range of applications for NMR spectroscopic analyses of metabolite mixtures can be categorized roughly into three areas. Screening for new natural products (green), structure determination of specific metabolites that are functionally connected with genotypic or phenotypic changes (yellow), and comparative profiling for the identification of biomarkers or assessment of general metabolic changes (red). The overlap of the ovals represents how NMR spectroscopic concepts and specific techniques find applicability in adjoining areas. The types of information sought by the three analytical vantages vary with regard to the emphasis placed on identifying new structures or associating compounds with biological activities, as indicated by placing them along a color-coded gradient. At the “structure” end of the spectrum (blue) fall purely discovery oriented natural product initiatives, whereas moving left across the gradient, emphasis moves away from procuring new structures towards associating structures with function.

Figure 2

Structures of new natural products identified via NMR-spectroscopic analyses of complex mixtures. Myrmicarin 430A (1) and bacillaene (6) represent members of a small but growing class of metabolites that have never been isolated in pure form.

Figure 3

Schematic representation of differential 2D NMR spectroscopy (DANS) applied to comparing C. elegans wildtype metabolome with that of daf-22 mutant worms [14]. Reprinted from [14] with permission from the National Academy of Sciences, USA.

Figure 4

Application of DemixC in the analysis of an unfractionated insect secretion [30]. (a) TOCSY spectrum of walking stick (A. buprestoides) defensive secretion. (b) Top red trace, representing the ¹H NMR spectrum of the secretion. (d, e, g, h, j, k) Black traces, representing one-dimensional subspectra obtained from DemixC analysis of the TOCSY, corresponding to the six different components of the secretion, including α-D- and β-D-glucose, dolichodial (2), peruphasmal (10), as well as the diol-derivatives 11 and 9 of 2 and 10. (c, f, i) Bottom three red traces, representing ¹H NMR reference spectra of purified components. Each reference spectrum contains two interconverting species, dialdehyde and diol forms of peruphasmal (trace c), dialdehyde and diol forms of dolichodial (trace f), as well as α-D- and β-D-glucose (trace i). Adapted with permission from [30]. Copyright 2007 American Chemical Society.