Strategies for Profiling and Discovery of Acylsugar Specialized Metabolites

Plant metabolic processes have evolved to produce an enormous array of chemically diverse metabolites that serve important roles in growth, development and resistance, while offering nutritional, medicinal, and economic value for humans. In the post-genome era, comprehensive measurements of plant chemistries, termed plant metabolomics, have experienced rapid growth as a field of research, with a broader goal being to provide the foundation for uncovering the relationship between the functions of genes, proteins and enzymes responsible for the biosynthesis of metabolites. However, researchers in this field face formidable challenges because unlike proteins and oligonucleotides which are constructed from a limited set of precursors, the building blocks of metabolites are far more varied. As a result, annotation and identification of novel metabolites remains the greatest obstacle to understanding the mechanisms responsible for metabolite accumulation and the functional significance of new metabolites. To address these challenges, the goals of this research have been to discover novel specialized metabolites (those that are taxonomically restricted and not involved in central metabolism) to facilitate the discovery of enzymatic processes responsible for assembly of plant chemical defenses, and to develop improved methods and technologies for annotation, identification and dereplication of metabolite discovery.Chapters 2 and 3 of this dissertation demonstrate approaches for untargeted metabolite profiling of specialized metabolites extracted from leaf surface glandular trichomes (hair-like epidermal cells) from two species of the family Solanaceae, Salpiglossis sinuata and Solanum quitoense. Liquid chromatography/mass spectrometry (LC/MS) profiling revealed diverse multiply esterified sugar metabolites, known as acylsugars, a family of metabolites known for anti-insect activity. Acylsugar metabolites were purified and their structures were elucidated using one- and two-dimensional (1D and 2D) nuclear magnetic resonance spectroscopy (NMR). These efforts established structures of 16 (of more than 400) new acylsucrose metabolites extracted from S. sinuata and established a novel group of acylated myo-inositols and myo-inositol glycosides (N-acetylglucosaminyl, glucopyranosyl and xylopyranosyl) from S. quitoense (9 structures). These results guided the discovery of previously unidentified acylsugar biosynthetic enzymes operating in S. sinuata and S. quitoense acylsugar biosynthetic pathways and extended our understanding of the evolution of specialized metabolism in the Solanaceae, while providing new analytical approaches for defining acylsugar composition and biodiversity.Mass spectrometry-based platforms are extremely effective tools for metabolite detection and investigations of metabolomes. However, for many metabolites, mass spectrometry alone does not provide unambiguous metabolite identification. While NMR spectroscopy provides more detailed structural information than MS, some researchers avoid its use because it commonly involves time-consuming metabolite purifications. In addition, as more metabolite structures are determined, there is a growing chance that researchers will purify and identify compounds that are already known but thought to be novel isomers. To aid dereplication of metabolite discovery, a homologous set of S-alkyl glutathione (GS-n-alkyl) standards featuring normal saturated chain lengths (1-24 carbons) was synthesized and exploited as liquid chromatographic (LC) retention index standards (Chapter 4). These standards encompass a wide reversed phase-LC retention range, are easily ionized by electrospray ionization (ESI) in positive- and negative-ion modes, and show improved capacity for standardizing chromatographic retention. A thorough investigation of the dependence of acylsucrose retention index values using GS-n-alkyl standards was performed while altering several important chromatographic experiment parameters, including a comparison of columns, solvent delivery systems, aqueous mobile phase pH, column temperature, LC gradient slope, and organic solvent component. GS-n-alkyl standard LC/MS analysis also shows promise for evaluating RP-HPLC column performance, batch-to-batch column reproducibility, and degradation of column performance with use. This research has potential to improve interspecies SM metabolite discovery and dereplication.