The Natural Products Discovery Core’s five-member team has more than 50 years of cumulative experience in natural products-related research, including natural product extract preparation, isolation, small molecule characterization, genome mining and mutasynthesis. Projects in these areas can be completed by the NPDC following our standard operating procedure (SOP), which can be broadly divided into five areas:
Chemical Enrichment and Profiling
- Re-growth of top 30 bioactive natural product extracts to 1-L culture.
- Chemical class enrichment using the Biotage Selekt fractionation technique, creating eight fractions from each natural product extract.
- Chemical profiling of active crude extracts and fractions. The post prioritization analysis will provide vital information regarding abundance of individual chemical entities within high-interest extracts. This effort also will enable sorting of molecules with identical/common masses and establish that the molecules with similar molecular weights are the same (or different) molecules. Most importantly, this process will establish the method development of downstream chromatographic processes to isolate active entities in a data-informed manner.
- Assessment and prioritization of obtained active natural product extracts to create a dataset comprising the biosynthetic gene clusters (BGCs) for each of the microbial strains and produce the top 30 active natural product extracts. Based on our recently developed screening campaigns prototype, we expect a high-quality BGC map will provide judicious snapshots of the similar genetic scaffolds and unique biosynthetic systems as a primary tool for prioritization of active natural product extracts and identification of novel chemical entities.
- Biological activity assessment of each fraction.
Large Scale Culture and Optimization
Once the top five strains/NPEs are identified from Chemical Enrichment and Profiling, those strains are up-scaled to 80 to 100 L of microbial culture and organically extracted, through the following processes:
- Microbial growth: Large-scale growth.
- C18 fractionation: Fractionation using Biotage Selekt.
- Mass spectrometry: Compare the enriched fraction with the bioactive C18 fraction.
- Informatics and bioassay guided fractionation: The purification will be based on the acquired genomic and chemical profiling data.
E-PuRE Platform-Guided Isolations
This step involves the pure chemical entity isolation using our proprietary Extracts to Pure Entity (E-PuRE) purification methodology and sophisticated chromatography methods.
Dereplication, Structure Elucidation, Chemical Derivatization
- Chemical structure determination:The complete chemical structure of the bioactive pure chemical entity will be confirmed by 1H and 13C 1D and 2D 600 MHz/ 800 MHz with cryoprobe nuclear magnetic resonance and high-resolution mass spectrometry in the Natural Products Discovery Core.
- Stereochemical determination: The isolated new chemical entities are likely to possess multiple stereocenters, which will be determined using various methodologies (nuclear magnetic resonance, chemical derivatization, etc.).
Yield Optimization, Mutasynthesis
- Perform media optimization for high-quantity production of an active chemical entity.
- Perform strain improvement/mutasynthesis to obtain non-natural analogs for securing IP.