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One new (1) and four known (2-5) ursene triterpenes with potent inhibition of the formation of the bacterial biofilm Pseudomonas aeruginosa PA01 were obtained from Diospyros dendo using a high-throughput natural products chemistry procedure. These compounds were isolated as mass-limited samples. The miniaturization of the structure elucidation and dereplication was performed primarily utilizing a capillary-scale NMR probe.

Two 2,7'-cyclolignans, ocholignans A and B, were obtained as mass-limited samples from Scyphocephalium ochocoa via high-throughput natural products chemistry methods. The rapid structure elucidation of each compound was primarily facilitated by NMR data acquisition using a capillary-scale NMR probe, CapNMR probe. Ocholignan A was found to possess significant in vitro antibacterial activity against Gram-positive bacteria methicillin-resistant Staphylococcus aureus ATCC 33591 and S. aureus 78-13607A with a MIC of 16 microg/mL, respectively.

Employing a capillary-scale NMR probe enables the miniaturisation of structure determination and de-replication of purified natural products from plants using only 5-100 microg of material. Approximately 5 microg are required to perform one-dimensional proton and two-dimensional homonuclear (COSY and NOESY) NMR experiments; some 30 microg are needed to acquire HMQC- or HSQC-NMR spectra; ca. 75-100 microg are necessary to measure HMBC-NMR spectra; and around 200 microg of a compound are needed to perform 13C- and DEPT-NMR experiments. In order to illustrate the integration of the outputs from high-throughput natural product chemistry methods with the capabilities of the state-of-the-art CapNMR technology, the preparation of a natural product library from the extract of Penstemon centranthifolius, and the subsequent isolation, purification and structure determination of six known iridoid glycosides with 25-300 microg of material are presented.

High-throughput isolation, purification and analysis methods applied to natural products libraries from plants gave rise to the discovery of two novel acylated caprylic alcohol glycosides (1, 2) produced by Arctostaphylos pumila. The NMR spectra were acquired using the CapNMR probe and performed on mass-limited samples, which enabled us to elucidate the structures of 2,6-diacetyl-3,4-diisobutyl-1- O-octylglucopyranoside (1, 200 microg) and 2,6-diacetyl-3,4-dimethylbutyl-1- O-octylglucopyranosid (2, 70 microg). Compounds 1 and 2 exhibited antibacterial activity against Gram-positive methicillin-resistant Staphylococcus aureus with an MIC of 128 microg/mL and 64 microg/mL, respectively.

Utilizing high-throughput isolation, purification, and analysis methods applied to a natural products library, a new mass-limited antibacterial indolosesquiterpene, suaveolindole (1), was obtained from Greenwayodendron suaveolens. The miniaturization of the structure elucidation of 1 was performed primarily using the CapNMR probe. Compound 1 was found to possess significant in vitro antibacterial activity against the Gram-positive bacteria Bacillus subtilis (ATCC 43223), Staphylococcus aureus (ATTC 6538P), and methicillin-resistant Staphylococcus aureus (ATTC 33591), with MIC values of 4, 8, and 8 microg/mL, respectively.

The application of an 8-way fully automated parallel LC-MS-ELSD system to the analysis of a library of 96 structurally diverse natural products is described. A 10-min separation incorporating a universal gradient allowed elution of 86 of these 96 compounds, all of which were detected by positive or negative mode electrospray ionization in conjunction with ELSD. This method is demonstrated to be one of the most universal means of detection for polar, nonvolatile, thermally labile natural products. It also allows an 8-fold increase in throughput. The analysis and profiling of constituents present in a library derived from plant material of Sarcostemma hirtellum, in terms of their retention times and mass spectra, are shown. This rapid characterization of plant constituents in terms of compound libraries is important in searching for new biologically active compounds. As a high-throughput tool to support our natural product discovery program, this method has been successfully used to analyze a library of 36,000 partially purified fractions derived from plant materials.

High-throughput methods were applied to the production, analysis, and characterization of libraries of natural products in order to accelerate the drug discovery process for high-throughput screening in the pharmaceutical and biotechnology industries. Library production integrates automated flash chromatography, solid-phase extraction, filtration, and high-throughput parallel four-channel preparative high-performance liquid chromatography to obtain the libraries in 96- or 384-well plates. Libraries consist of purified fractions with approximately one to five compounds per well. Libraries are analyzed prior to biological screening by a high-throughput parallel eight-channel liquid chromatography-evaporative light scattering detection-mass spectrometry system to determine the molecular weight, number, and quantity of compounds in a fraction. After biological screening, active fractions are rapidly purified at the microgram level and individual compounds are rescreened for confirmation of activity. Structures of active compounds are elucidated by NMR spectroscopy and mass spectrometry. Utilization of a novel microcoil probe allows NMR data to be gathered on 50 microg. As a demonstration, a library was made from the stem bark of Taxus brevifolia. Biological screening in the National Cancer Institute's in vitro panel of three cancer cell lines demonstrates that the process enables the discovery of active anticancer compounds not detected in the flash fractions from which the library originates.