ABSTRACT This study is responsive to the Notice of Special Interest (NOSI) NOT-AT-21-006 “Fundamental Science Research on Complementary and Integrative Health Approaches, Including Natural Products or Mind and Body Interventions” objectives to “Develop targeted and untargeted bioinformatic approaches to identify active components in a natural product mixture.” Structural elucidation of natural products (NPs) remains a critical rate-limiting step in NP discovery campaigns. Difficulties in structural elucidation can arise from i) the lack of sufficient quantities of material for traditional analytical methods (e.g. nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography); ii) intrinsic physical properties of the NP, and iii) limitations of NMR capabilities in determining relative configuration. X-ray crystallography remains the gold-standard for unambiguous structural determination, including the assignment of stereochemistry. However, X-ray crystallographic analysis of newly-isolated NPs is often thwarted by insufficient quantities to provide crystals large enough for single-crystal diffraction or poor solid-state properties that preclude the formation of large, pristine crystals even when sufficient material is available. Given these challenges, we envision that application of the recently reported cryo-electron microscopy (CryoEM) modality micro-crystal electron diffraction (MicroED) could lead to vertical advances in the field of NP discovery directly responsive to this NOSI, as MicroED has recently been demonstrated to provide unambiguous structures from sub-micron-sized crystals of structurally complex chemical compounds that had failed to yield large crystals suitable for X-ray analysis. In this proposal, we aim to leverage a CryoEM/MicroED approach to resolving major bottlenecks in the structure elucidation of (partially) purified NPs and chemically complex NP mixtures. We hypothesize that we can advance the field of NP research through development and optimization of a high-throughput platform technology to identify NPs in complex mixtures and yield a novel diffractomics signature of molecules for integration into bioinformatics approaches. To evaluate this hypothesis, we will carry out three specific aims: 1) Use MicroED to solve structures of recalcitrant (partially) purified NPs; 2) Develop a high-throughput MicroED-based platform for compound discovery; and 3) Resolve major bottlenecks in structure determination of complex NP mixtures. For all aims, we will leverage a one-of-a-kind and expansive group of three NP collections (chemical libraries of extracts and partially purified fractions) derived from plants, marine organisms, and filamentous fungi. We anticipate advancement in the speed and accuracy of NP structural identification as a result of these studies, accelerating the rate of discovery of pharmacologically relevant NPs key to the improvement of human health.

NARRATIVE Regardless of the biological source (bacterial, fungal, plant, animal, or engineered organism), natural product scientists face a common bottleneck: structure elucidation of compounds found in complex mixtures. Here, we propose development and optimization of a micro-crystal electron diffraction platform technology for high- throughput compound identification with nanogram quantities of analytes found in natural mixtures.