MP17:Natural Product Isolation

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Natural product isolation for chemical ecology: rapid screening, analysis, and characterization

Kayla A. Kaiser, Christian Paetz, Cynthia K. Larive, Bernd Schneider

Department of Chemistry, University of California, Riverside, USA

Biosynthesis/NMR Group, Max Planck Institute for Chemical Ecology, Jena, Germany

Bottlenecks in the field of chemical ecology arise in isolation and structure elucidation of molecules responsible for biological interactions. The utilization of on-line high-pressure liquid chromatography, solid-phase extraction, and nuclear magnetic resonance with a cryoprobe (HPLC-SPE-NMR) can overcome challenges of sample complexity and sensitivity while seeking new chemical and biological knowledge. In this research, culture medium of Xiphidium caeruleum root was found to contain a set of phenylpropanoid glycosides, of interest because this class of compounds possesses an extensive bioactivity profile. Liquid growth medium was first treated by solid-phase extraction (SPE) to remove hydrophilic constituents of the sample matrix, reducing sample complexity. Rapid screening for molecular identity was carried out using high-pressure liquid chromatography (HPLC) coupled to electrospray ionization mass spectrometry (ESI-MS). This powerful combination of high-resolution separation and information-rich detection allowed us to focus our further studies only on molecules containing functional groups of interest. Automated peak-trapping of novel phenylpropanoid glycosides on SPE cartridges following multiple injections of growth media accumulated a sufficient quantity of material of reproducible chemical content for generating structural hypotheses by heteronuclear two-dimensional nuclear magnetic resonance (NMR). Automated sample handling from SPE cartridges to a cryogenically-cooled flow probe reduced volume of deuterated solvent necessary and hence cost of analysis, in addition to providing structural information from a single root culture flask (80 mL). Without having to expose the compounds to light, oxygen or acidic conditions, we could be more confident that the structure we observed reflected the bioactive molecules excreted by the plant. By fractionating the molecules contained in the medium into single components, the problem of dynamic range can be addressed. Dereplication of identified compounds was assisted by in-house (ACD/Labs) and publicly available (NMRShiftDB) databases as well as comparison with literature. For complete structure elucidation, precision isolation of milligram quantities of pure material was achieved by preparative HPLC and computerized fraction collection. Large scale SPE (10g sorbent) was applied to post-column effluent to prevent sample degradation prior to concentration. Finally, isolates were partitioned and a portion (5%) was reconstituted in ultrapure protonated solvent for high-resolution mass measurement using double sector double focusing MS while the remainder (95%) was reconstituted in deuterated solvent for full NMR characterization. This approach has utilized efficient handling of limited quantities of bioactive molecules excreted by roots of Xiphidium caeruleum, ostensibly for the purposes of chemical communication, perhaps to manipulate partners, competitors, and ecosystems. Automation has contributed at various points throughout this discovery approach to the speed at which identification of chemical substances which carry biological information, ultimately allowing further studies of the developmental, behavioral, and ecological consequences of these previously unexplored chemical signals.

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