Are natural deep eutectic solvents the missing link in understanding cellular metabolism and physiology?

Over the past decade, metabolomics has developed into a major tool for studying the metabolism of organisms and cells, and through this approach much has been learned about metabolic networks and the reactions of organisms to various external conditions (Lay et al., 2006). Most of this work involves a number of chemometric methods to identify markers in the metabolomic data for various events. But in fact, little work has been done on understanding the meaning of the metabolomic data itself and the role of the total of the compounds observed. Is there any logic in the studying the combination of compounds itself, instead of looking at the correlations between the compounds observed and any disease or applied experimental conditions? NMR-based metabolomics in particular give a clear view of the major compounds present in an organism or cell and enable the direct quantitative comparison of all major compounds. Considering all the information we have collected in recent years using our protocol for NMR-based metabolomics (Kim et al., 2010), we asked ourselves why a few very simple molecules are always present in considerable amounts in all microbial, mammalian, and plant cells. It seems that these compounds must serve some basic function in living cells and organisms. These compounds include sugars, some amino acids, choline, and some organic acids such as malic acid, citric acid, lactic acid, and succinic acid. With the exception of sugars, which may serve as storage products and a source of energy, the other compounds are present in such large amounts that it does not make sense to consider them as only intermediates in metabolic pathways. Here, we develop a novel theory about the role of these compounds, which may explain many questions in the biochemistry of cells and organisms. The theory is based on analogy with green chemistry, where in past years various synthetic ionic liquids (ILs) have been developed for chemical and enzymatic reactions as well as for the extraction of natural products. The field of ILs began in 1914, when Paul Walden (Plechkova and Seddon, 2008) reported on the physical properties of ethylammonium nitrate. But it is only in recent years that ILs and deep eutectic solvents (DES) have been revisited by chemical engineering, because such solvents can replace conventional organic solvents. Mixing salts and/or organic compounds may cause a considerable reduction of the melting point, turning them into liquids even at very low temperatures. Using the liquids made from synthetic chemicals, ILs and DES now have many different applications such as dissolving polymers and metals and as media for biotransformation (Welton, 1999; Wasserscheid and Keim, 2000; Abbott et al., 2004; Gorke et al., 2008). In fact, many of the synthetic ILs contain choline and in some cases also natural organic acids. In analogy with the synthetic ILs, we hypothesized that themetabolites that occur in large amounts in cells may form a third type of liquid, one separate from water and lipids. Taking the plant metabolomics data we have collected over recent years into consideration, we saw a clear parallel with the synthetic ILs. The above-mentioned major cellular constituents seemed perfect candidates for making ILs and DES. As the first step, we made various combinations of these candidates, thereby discovering more than 30 combinations that form viscous liquids (Table I). Here, we will use “natural deep eutectic solvents” (NADES) as a common term for these mixtures. The preparation of NADES and NMRmeasurements are described in Supplemental Materials and Methods S1. In a H-H-nuclear Overhauser enhancement spectroscopy spectrum of the Suc and malic acid mixture, some of the protons showed intermolecular interaction, implying that the molecules of these compounds in the liquid are aggregated into larger structures, as in liquid crystals (Fig. 1). In some mixtures, such as different sugars with choline chloride, water can be present as part of the solvent (1:1:1 molar ratio, corresponding to approximately 6% water; Table I). This water is strongly retained in the liquid and cannot be evaporated. 1 These authors contributed equally to the article. * Corresponding author; e-mail [email protected]. [W] The online version of this article contains Web-only data. www.plantphysiol.org/cgi/doi/10.1104/pp.111.178426