A platform for HPLC-based natural product discovery

Numerous essential drugs have been developed on the basis of natural products: Antibiotics, anticancer drugs, immune suppressive or cholesterol lowering agents, just to name a few. In all these cases, the development started with the discovery of a natural product with a novel mode of action.

The search for bioactive natural products via the usual activity-directed preparative fractionation of extracts is a time consuming and tedious enterprise with a high probability of failure. We have, therefore, down-scaled and streamlined the process and use an HPLC-based platform operating in the 96-well microtiter format, with a high degree of standardization and automation.

Extracts are separated by gradient HPLC and analyzed on-line by various detectors. Via a T-split, the effluents are micro-fractionated in the 96-well format for bioassay. Activity profiles are matched with HPLC fingerprints and spectroscopic data. LC-MS with an ion trap mass spectrometer is used for efficient dereplication of active compounds in extracts. Accurate mass measurements are obtained by LC-TOF-MS, and NMR spectra are recorded on a dedicated NMR spectrometer equipped with a 1 mm microprobe and autosampler.

We have implemented a liquid storage concept for our libraries. Extract and compound libraries in 2D barcoded plates enable rapid and unambiguous tracking, and automated liquid handling. Sample related informations are managed via a customized database.

Automation of the extraction process: Plant extracts are generated by pressurized liquid extraction (PLE). Our group has been the first to implement this extraction technology in natural products screening and analysis.  

Instrumentation for microfractionation and rapid scale-up from analytical to semi-preparative HPLC.

Parallel evaporation of extracts and fractions is used.

A dedicated 500 MHz NMR spectrometer equipped with a 1mm microprobe and autosampler is used for structure elucidation. The active volume of the disposable 1mm tubes is 5µL. NMR spectra can be measured with µg-amounts of a compound which can be obtained from extracts by semipreparative HPLC.

Establishment of this platform was supported by a R'Equip grant from the Swiss National Science Foundation, the Steinegg Foundation, Herisau, the Fonds für Lehre und Forschung, Basel, the Hans und Wilma Stutz Foundation, and through gifts of equipment by Boehringer Ingelheim and Syngenta.