Microcrystal Electron Diffraction-Guided Discovery of Fungal Metabolites

Abstract

Nature remains a vast repository of complex and functional metabolites whose structural characterization continues to drive innovations in pharmaceuticals, agrochemicals, and materials science. The cryogenic electron microscopy (cryoEM) method, microcrystal electron diffraction (microED, a 3D ED technique), has emerged as a powerful tool to structurally characterize small molecules. Despite this emerging role in structural chemistry, the cost and throughput of microED have limited its application in the discovery of natural products (NPs). While recent advances in sample preparation (e.g., arrayED) have provided a conceptual framework to address these challenges, they have remained unproven. Herein, we report the arrayED-driven discovery of a structurally unprecedented family of NPs (zopalides A-E), a muurolane-type sesquiterpene glycoside (rhytidoside A), aspergillicin analogs (aspergillicins H and I), and four crystal structures of previously reported fungal metabolites. Lastly, this the first time that the absolute stereo configuration of newly discovered NPs has been determined directly by microED alone without other methods using a small amount of sample.

1

Approaches to NP discovery. Traditional methods of NP discovery include bioassay-guided, structure-guided, and genome mining-guided isolation. In this work, we highlight the use of an electron diffraction-guided workflow (arrayED) to discover NPs.

2

ArrayED screening overview for Zopfiella sp. and the discovery of Zopalide NPs. (A) Chromatogram of crude fungal extract of Zopfiella sp. at a 254 nm wavelength with wells C7–C9 highlighted in green, blue, and red, respectively. Micrograph of the microarrayed extract with well C9 highlighted in red to show the presence of microcrystals and their associated diffraction pattern. (B) ab initio preliminary solution A1 (iso value = 1.085 eA^–1) resulted from the screening of wells C7–C9 as well as the final structure of zopalide A (1). (C) Chromatogram from an HRMS experiment on well C7 of Zopfiella sp. marked to distinguish the corresponding structures.

3

Reisolation from Zopfiella sp. and the discovery of zopalide analogs. (A) ab initio preliminary solution B1 (iso value = 0.772 eA^–1) in space group C ₂ provided by microED data collection on the final analyte isolated from the Zopalide family, followed by the final structure of zopalide E (5). (B) Statistics calculated from the dynamical refinement of both enantiomorphs of compound 5 in space group P2 ₁ 2 ₁ 2 ₁ . (C) Superposition of the structure provided by microED in space group P2 ₁ 2 ₁ 2 ₁ and the structure provided by SCXRD in space group P2 ₁ 2 ₁ 2 ₁ with an RSMD of 0.037 Å.

4

ArrayED screening overview of Rhytidhysteron hysterinum. (A) Chromatogram of mycelial extract from Rhytidhysteron hysterinum at a 254 nm wavelength with fraction D8 highlighted in red. Depiction of the 96 fractions generated for the arrayED workflow with wells containing crystalline particles highlighted in violet. Particles from well D8 are shown along with the corresponding diffraction. (B) ab initio preliminary solution C1 (iso value = 0.44 eA^–1) that resulted from the screening of well D8 (duplicate molecules found in the asymmetric unit omitted for clarity), followed by the final structure of rhytidoside A (6).

5

Overview of the isolation of metabolites from Aspergillus sp. (A) Chromatogram of mycelial extract from Aspergillus sp. at a 254 nm wavelength. (B) Chromatograms of purified fractions that exhibited crystallinity.

6

MicroED structures of fungal metabolites identified from the mycelial extract of Aspergillus sp. Depictions of the ab inito solution of compound 7 (iso value = 0.556 eA^–1), compound 9 (iso value = 1.4 eA^–1), compound 10 (iso value = 0.648 eA^–1), compound 11 (iso value = 0.955 eA^–1), compound 12 (iso value = 0.592 eA^–1), and compound 13 (0.571 eA^–1).