Cholesterol, a major lipid component of animal plasma membranes (PM) and a small component in some plants, has been associated with the development of various diseases and infections, like Alzheimer’s disease (AD), Parkinson’s diseases (PD), H. pylori infection, etc., through an imbalance in cholesterol homeostasis or cholesterol modification. In addition to the bioactivity of lipids like cholesterol, glycolipids (glycosylceramides and sterol glycosides) have been reported to influence the release of T-helper 1 (TH1) and T-helper 2 (TH2) cytokines in mammals. Also, cholesterol has been reported to undergo a sugar exchange with glucosyl ceramides. In previous studies, cholesterol analogues with intrinsic or extrinsic fluorescent groups were used as probes to monitor biodistribution of cholesterol and the sugar exchange with glucosyl ceramides. However, problems arose with these cholesterol probes from disrupting plasma membranes to difficult extraction for metabolite characterization. Recently, a dehydroepiandrosterone (DHEA) probe with a primary alkoxyl-azido group mimicked cholesterol well while providing efficient extraction via copper(I)-catalyzed azide/alkyne cycloaddition (CuAAC) with 4-N-methylamino-1,8-napthalimidopropyne (MAN) for metabolite characterization and minimal disruption of plasma membranes. Currently, there are no azido-glycolipid probes available to monitor the metabolism of glycolipids during TH1 and TH2 cytokine release in addition to the sugar exchange with ceramides. In this project, two secondary C17 azido-DHEA (5 and 8) probes were prepared and six azido-glycolipid (14, 15, 16, 17, 22, and 24) probes were proposed to study the metabolism of lipids and glycolipids in various environments. Chapter 1 will focus on the bioactivity of sterols/sterol glycosides and previous sterol probes. Chapter 2 will discuss the total synthesis of the two secondary C17 azido-DHEA probes (5 and 8). Chapter 3 will cover the NMR characterization of various DHEA derivatives while Chapter 4 will describe previous glycosylceramide derivatives, the bioactivity of glycosylceramides, and the future synthesis of six azido-glycolipid probes (14, 15, 16, 17, 22, and 24).

This dissertation delves into the contemporary significance of carbohydrates across diverse life forms and industries, emphasizing their pivotal role in shaping product quality, safety, and regulatory compliance within sectors like food and beverage, pharmaceuticals, and biofuels. The research capitalizes on significant advancements in nuclear magnetic resonance (NMR) spectroscopy, specifically silicon-29 NMR, extending its applications beyond protons to exert influence in fields such as materials science, biochemistry, and environmental science. A focal point of the study involves the synthesis of twelve per-O-silylated sugars using a microwave reactor, strategically reducing reaction time while preserving yield. Advanced NMR experiments identify silicon signals, providing valuable insights into spatial orientation and distinctive structural variations. The analysis of chemical shifts in per-O-silylated sugars contributes to the establishment of an extensive silicon NMR database for comprehensive carbohydrate characterization. Noteworthy patterns in per-O-silylated sugars, along with the discernible chemical shifts in diverse silylated compounds, further enhance the depth of the database. Recent strides in the 29Si NMR characterization technique are underscored, with a specific focus on per-O-silylated sugars. The characterization of twelve per-silylated sugars using 29Si NMR spectroscopy, complemented by a 2D graph incorporating 13C NMR chemical shifts, introduces an innovative approach that optimizes visualization and streamlines compound identification. Despite encountering challenges in a mixture synthesis experiment, where silicon chemical shifts undergo slight changes, the study contributes significantly to our understanding of per-O-silylated sugars using 29Si NMR spectroscopy. The dissertation concludes by addressing the limitations of 1H NMR spectroscopy, introducing the prominence of 1H quantitative nuclear magnetic resonance (qNMR). The Gervay-Hague research group employs 29Si NMR spectroscopy to quantify glucose, sucrose, and fructose within mixtures, which could extend the analysis to encompass coffee and tea leaves. Insights derived from 29Si NMR analysis affirm the reliability of integrated resonances, with the incorporation of a novel internal standard leading to a notable reduction in errors. The 29Si NMR signal corresponding to the 2-hydroxyl position emerges as a consistent and promising diagnostic tool for the quantitative assessment of sample mixtures, thereby showcasing the potential for accurate carbohydrate measurement across a spectrum of applications.

Sterols are part of an important class of molecules that modulate cellular signaling, recognition, and structure which make sterols an ideal probe to understand life processes. Dehydroepiandrosterone (DHEA) and sitosterol were used to synthesize novel chemical probes bearing an azide or alkyne as well as optimize reactions of known bioorthogonal sterols in Chapter 1. Routes to sterols with both bioorthogonal handles allows for a more comprehensive understanding of whether the type of or position of a bioorthogonal group on the sterol changes the metabolic outcomes when fed to an organism. The x-ray crystal structures of alkyne tagged DHEA and sitosterol analogs, as well as intermediates synthesized in effort to produce the bioorthogonal sterols, were collected and conformational analyses in Chapter 2 showed highly similar structures to that of endogenous sterols such as DHEA, cholesterol, and sitosterol. From the development of sterol probes arose the optimized synthesis of a fluorescent alkyne and invention of a novel fluorescent azide (Chapter 3). Routes to covalently attach the sterol and fluorophore by ‘click chemistry’ were developed for temperature-sensitive samples as well as for high atom economy with catalytic reagents. The culmination of this work was in the application of synthesized bioorthogonal sterols, fluorophores, and ‘click chemistry’ methods in Helicobacter pylori (Chapter 4). Target-based screening for the production of steryl glycoside metabolite analogs of bioorthogonal sterols by UPLC-fluorescence and characterization by UPLC-MS/MS was accomplished, showing the potential for this platform to be used in other living systems.

Tea has been enjoyed by many cultures world-wide for thousands of years. Recently, an expanded interest in tea drinking has occurred, which is attributed to the health benefits associated with tea consumption. This increase in demand is growing particularly quickly within the United States, where very few commercial tea fields and production outfits exist. Therefore, most tea that is consumed in the U.S. must travel internationally travel consumption. Unfortunately, tea consumed within the U.S. may undergo up to four rounds of international transportation: harvest to processing, processing to blending, blending to packaging, and packaging to retail. Given the reasonable international push towards sustainability, it then makes sense for tea fields and production facilities to be established within the U.S. to meet the growing demand. California boasts a rich agricultural history and UC Davis has continually provided cutting edge agricultural research. However, tea is historically grown in regions with higher levels of humidity, such as Taiwan, China, and Japan, and has only been established commercially in the Southern part of the United States previously. Therefore, the first task was to establish a test plot of Camellia sinensis in California. Such a field was established in conjunction with the University of California’s Agriculture and Natural Resources division at Kearney Agricultural Research and Extension Center in Parlier, CA. Two varieties (UC-K) were planted in December 2018 and sixteen varieties (UC-N) were planted in November 2019. A major reason for the increase in demand for tea in the U.S. stems from the reported health benefits. These health effects are generally credited to polyphenols, specifically the catechin (flavan-3-ol) subclass of flavonoids. However, only a few scientific endeavors have been attempted to determine the natural chemical profile of the flavan-3-ol compounds in tea plants. To overcome the lack of a more comprehensive survey, multiple methods were developed while waiting for the California test plot to mature enough for processing. Such methods include harvest, transportation, extraction, quantitative analysis, and qualitative analysis. In particular, the methods developed in this study limited the time between harvest and cryogenically freezing tea leaves to minimize the effects of oxidation on the flavan-3-ol profile. Tea samples were extracted with water to closely mimic the chemical profile ingested by a consumer, and compounds were identified and measured with a high-performance liquid chromatography (HPLC) instrument coupled with an ultraviolet-visible (UV-Vis) detector and a single quadrupole mass spectrometer (SQMS). The developed HPLC-UV-Vis-SQMS method was capable of independently analyzing nine phenol compounds and three alkaloid compounds. Once the California test field had matured, the natural flavan-3-ol profiles of six varieties was studied. The change in flavan-3-ol profile among black tea processing steps was also studied for a single variety. Both analyses were compared to previously reported values of flavan-3-ols, alkaloids, and select amino acids. Additionally, peaks not related to the twelve standards used were investigated and identified via standard and/or library comparison. Finally, one initially unknown metabolite was identified as theogallin and analyzed with the developed HPLC-UV-Vis-SQMS method. Theogallin is a conjugate of the well-studied quinic and gallic acids but is often not included in reported phenol profiles of tea. Present research into the potential health benefits of theogallin is therefore lacking. Furthermore, commercially available theogallin is extracted from tea leaves making the standard expensive to obtain and difficult to test for potential therapeutic effects. Such a deficiency makes the efficient synthesis of theogallin valuable for further studies. Given the potential benefits of theogallin, a four-step synthetic route utilizing previously developed regioselective silyl exchange technology (ReSET) was investigated. The current work presents the optimization, purification, and characterization of the first two steps, including a previously unreported crystal structure. The third step was attempted but not purified. With the first half of the synthesis available, further development of an efficient theogallin synthesis using ReSET is primed to continue.