Synthesis and functional characterization of secondary metabolites from nematodes

Nematodes are the most abundant group of animals on Earth and include free-living and parasitic species, which have successfully adapted to nearly all ecosystems. Nematodes excrete several different molecules into their environment for inter- and intraspecies communication. Recent studies have found that a group of small molecules called ascarosides regulate many aspects of their life history such as mating, lifespan, and development. The ascarosides are glycolipids of the 3,6- dideoxysugar L-ascarylose linked to fatty acid-like aglycones derived from the peroxisomal β-oxidation cycle and are highly conserved in nematodes. Small structural alterations of the side chain or the attachment of additional building blocks downstream of the β-oxidation cycle can dramatically alter the biological activity of the ascarosides, which forms the molecular basis of the species-specific “chemical language” of nematodes.
Using HPLC-MS/MS analytical techniques along with one- and two-dimensional NMR spectroscopy, four families of species-specific modular ascarosides were identified in nematodes from the Elegans group: the ortho-aminobenzoate ascarosides (abas) in C. nigoni and C. tropicalis, the urocanate ascarosides (ucas) in C. nigoni, C. remanei and C. briggsae, the 4-epimeric ascaroside (caen) in C. nigoni, and the oligomeric ascarosides in C. tropicalis.
This PhD thesis focused on the total synthesis of these novel species-specific ascarosides, in order to confirm their structure assignment and to determine their biological functions in nematodes. For this purpose, this study first focused on the synthesis of two different types of ascaroside building blocks, prepared from commercially available L-rhamnose. First, the 2,4-di-O-benzoyl ascarylose commonly used for the synthesis of basic ascarosides that only differ by the length and the terminal function of their side chain was prepared. Then, the orthogonally protected ascarylose, first introduce by Zhang et al. with the 4-O-tert-butyldiphenylsilyl-2-O-benzoyl ascarylose and that allows the regioselective formation of 4- or 2-modified ascaroside, was synthesized.
Given the importance of the ascarylose building block for the synthesis of ascarosides, different synthetic strategies were studied to develop a short and selective synthesis to obtain the ascarylose building blocks. The six step synthesis of the dibenzoyl ascarylose from Jeong et al. based on regioselective elimination of the 2,3,4-tri-O-benzoyl rhamnose was successfully shortened to four steps via Barton McCombie deoxygenation of the methyl-3-O-henyloxythiocarbonyl rhamnoside while the seven steps synthesis of the orthogonally protected 4-O-tert-butyldiphenylsilyl-2-O-benzoyl ascarylose from Zhang et al. via regioselective sulfate ring opening of the methyl-2,3-(O-sulfate)-4-Otert-butyldiphenylsilyl rhamnoside was reduced to six steps via regioselective opening of methyl-2,3-O-benzylidène rhamnoside. Moreover, the synthesis also provided access to a wide range of 4-modified ascarylose building blocks while Zhang’s synthesis give only access to the 4-O-tert18 butyldiphenylsily-2-O-benzoyl ascarylose.
These building blocks were subsequently used to synthesize the 4-modified urocanate ascarosides and ortho-aminobenzoate ascarosides as well as the oligomeric ascarosides, while the 3,6-dideoxy-lyxohexose building block from the caenorhabdoside, was obtained via epoxide ring opening. In total ten novel species-specific ascaroside were synthesized. The (E)-configured urocanate ascarosides were synthesized in seven steps from the 4-O-tert-butyldiphenylsily-2-O-benzoyl ascarylose and there corresponding (Z)-isomers obtained by UV irradiation. The ortho-aminobenzoate ascarosides were also synthesized in seven steps from the 4-O-tert-butyldiphenylsilyl-2-O-benzoyl ascarylose. The oligomeric ascarosides were obtained by convergent synthesis from the 4-O-tert-butyldiphenylsily-2- O-benzoyl ascarylose and the 2,4-di-O-benzoyl ascarylose and the caenorhabdoside synthesized by glycosylation of the (7R,8R,2E)-threo-ethyl-7-benzoyloxy-8-hydroxy-2-nonenoate with the 3,6-dideooxy-lyxo-hexose.
Comparative analysis of HR-MS and NMR data of the synthetic and the natural compounds confirmed their structure assignment. Furthermore, the synthetic compounds were subsequently tested in behavioral assays. However, only the dimeric ascaroside showed significant retention activity in males of C. tropicalis. The identification of these novel species-specific ascarosides demonstrates that ascaroside signaling is more complex than previously anticipated and that nematodes have evolved to utilize the conserved basic ascarosides as scaffold from which they generate species-specific signaling molecules by the attachment of additional building blocks from diverse metabolic pathways, such as the amino acid metabolism, but also by oligomerization or epimerization of the ascarylose sugar.
The discovery of these modular ascarosides give some insights for the mechanism underlying the biosynthesis of species-specific ascaroside. Currently, limited information regarding the genes involved in the biosynthesis of these modular ascarosides is available. Therefore, additional investigation to decipher the mechanism of the biosynthesis of these ascarosides will be required in the future. Moreover, additional bioassays with maybe a focus on development or the formation of dauer worms will be required to find the biological functions of these highly specific modular ascaroside.

PhD thesis submitted to the Faculty of Sciences Institute of Chemistry University of Neuchatel

Dissertation Committee:
Prof. Stephan H. von Reuss, University of Neuchatel, Switzerland, Thesis director
Prof. Bruno Therrien, University of Neuchatel, Switzerland, Internal examinator
Prof. Frank Schroeder, BTI and Cornell University, USA, External examinator

Defended on the 7th of February 2023