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.