National Cancer Institute (NCI) Program for Natural Products Discovery: Rapid Isolation and Identification of Biologically Active Natural Products from the NCI Prefractionated Library

Abstract

An automated, high-capacity, and high-throughput procedure for the rapid isolation and identification of biologically active natural products from a prefractionated library is presented. The semipreparative HPLC method uses 1 mg of the primary hit fraction and produces 22 subfractions in an assay-ready format. Following screening, all active fractions are analyzed by NMR, LCMS, and FTIR, and the active principle structural classes are elucidated. In the proof-of-concept study, we show the processes involved in generating the subfractions, the throughput of the structural elucidation work, as well as the ability to rapidly isolate and identify new and biologically active natural products. Overall, the rapid second-stage purification conserves extract mass, requires much less chemist time, and introduces knowledge of structure early in the isolation workflow.

Chemical Elicitors Induce Rare Bioactive Secondary Metabolites in Deep-Sea Bacteria under Laboratory Conditions

“…The chemical samples were aliquoted (25 µl at 10 mg/mL) in 384-well plates, using half a row for each chromatographic series, containing the crude extract and the 9 fractions, totaling 18 crude extracts and 162 fractions. This layout was chosen to systematically evaluate the enrichment of metabolites by pre-fractionating the crude extracts, as previously suggested by Wagenaar [ 51 ] and Grkovic et al [ 54 ] Daughter plates were prepared at 1 mg/mL and 0.1 mg/mL concentrations for the bioassays.…”

Roseabol A, a New Peptaibol from the Fungus Clonostachys rosea

“…The NMR spectra were recorded in DMSO-d 6 as the signals showed well-redispersed resonances and unambiguous 2D-NMR correlations compared to other solvents that were examined (acetone-d 6 and CD 3 OH). The presence of a large number of amide NH protons (δ H 8.67-6.77) and carbonyl carbons (δ C 176.1-169.3) in the 1 H-and 13 C-NMR spectra of 1, respectively, indicated characteristic signals of a peptide (Table 1). Analysis of 2D-NMR (HSQC, COSY, HSQC-TOCSY, HMBC, and ROESY) data led to the assignment of seven common amino acids in 1 consisting of two serine (Ser), one glutamine (Glu), two leucine (Leu), and two valine (Val) residues, as well as one modified amino acid residue hydroxyproline (Hyp).…”

“…Despite relatively high response rates to these agents, less than half of patients achieve durable benefit; thus, alternative treatments are urgently needed. Concurrent with ongoing NCI natural product discovery efforts [12,13], an organic extract of a fungal isolate identified as Clonostachys rosea showed significant activity in a cell-based assay for inhibition of Merkel cell carcinoma cell growth and survival. Fractionation of the extract provided a new peptaibol that was named roseabol A (1) (Figure 1) and the linoleic acid oxidation product, 13-oxo-trans-9,10-epoxy-11(E)-octadecenoic acid (2) [14].…”

Screening open source traditional Chinese medicine extracts for growth-inhibiting properties towards Acinetobacter baumannii

“…These primary fractions, containing 0.5 mg of each fraction, were then reconstituted in DMSO and prepared for screening as noted above. Following identification of any active primary fractions meeting the 50% threshold, these hits were further subfractionated on semi-preparative high performance liquid chromatography (HPLC) where 1 mg of the active primary fraction was divided across 22 subfractions (that is, secondary fractions) collected into 96 deep-well plates followed by centrifugal evaporation to dry the HPLC solvent (Grkovic et al, 2020). As the secondary fractions resulting from this final step were of unknown exact mass, only the nominal assay mass of 45 µg per well (based on an equal split of HPLC input mass) can be reported.…”