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Scholarly Works (5 results)

  • Thesis
  • Peer Reviewed
UC San Diego Electronic Theses and Dissertations (2021)

Organisms across all domains of life have developed elegant strategies to produce specialized metabolites, also referred to as natural products, for defense, structure, and communication. Natural products provide novel inspiration for pharmaceutical development given their potent biological activities and structural diversity. Recent advancements in DNA sequencing technology and the development of predictive bioinformatics tools have unveiled a vast collection of biosynthetic machinery responsible for generating this structural diversity that may be harnessed by synthetic chemists. Many of the enzymes encoded by biosynthetic genes catalyze chemical reactions that are difficult to replicate with the same selectivity by traditional synthetic methods. An example of this discrepancy is illustrated by the current enzymatic and chemical methods known to generate a highly reactive ortho-quinone methide (o-QM) intermediate. The utility of o-QMs in total synthesis has been hampered by complications that surround the preparation of their precursors, the harsh generation methods, and poor chemo-, regio-, and stereoselective control. In contrast, multiple enzymes have been reported to catalyze o-QM formation under mild conditions with remarkable selectivity. Chapter 1 of this dissertation examines several different enzymatic strategies developed to generate o-QMs. Chapter 2 describes progress towards the biomimetic total synthesis of (–)-chlorizidine A that aims to synthetically replicate the final biosynthetic intramolecular cyclization via an o-QM intermediate. Chapter 3 reports the discovery of tetrachlorizine, a novel tetrachlorinated marine natural product, and the identification of its associated biosynthetic gene cluster. Biochemical characterization of a pivotal flavin-dependent oxidase revealed a dehydrogenation reaction that proceeds via an unprecedented o-QM mechanism. This oxidase was then repurposed to generate an isolable o-QM, an extremely rare chemical motif. Chapter 4 investigates the structure-function relationships of two homologous flavin-dependent oxidases with divergent reaction selectivities and highlights their favorable biocatalytic properties. This chapter also discloses the first reported bacterial enzymes capable of performing oxidative cyclization reactions to produce cannabinoids.

  • 1 supplemental video
Cover page: Synthetic and Biocatalytic Strategies for Natural Product Synthesis via ortho-Quinone Methide Intermediates
  • Article
  • Peer Reviewed
UC San Diego Previously Published Works (2021)
Ortho-quinone methides (o-QMs) are reactive intermediates in biosynthesis that give rise to a variety of intra- and intermolecular cyclization/addition products in bacteria, fungi, and plants. Herein, we report a new metabolic deviation of an o-QM intermediate in a benzylic dehydrogenation reaction that links the newly described marine bacterial natural products dihydrotetrachlorizine and tetrachlorizine. We discovered these novel dichloropyrrole-containing compounds from actinomycete strain AJS-327 that unexpectedly harbors in its genome a biosynthetic gene cluster (BGC) of striking similarity to that of chlorizidine, another marine alkaloid bearing a different carbon skeleton. Heterologous expression of the homologous flavin-dependent oxidoreductase enzymes Tcz9 and Clz9 revealed their native functions in tetrachlorizine and chlorizidine biosynthesis, respectively, supporting divergent oxidative dehydrogenation and pyrrolizine-forming reactions. Swapping these berberine bridge enzyme-like oxidoreductases, we produced cyclized and dehydrogenated analogs of tetrachlorizine and chlorizidine, including a dearomatized chlorizidine analog that stabilizes an o-QM via conjugation with a 3H-pyrrolizine ring.
    Cover page: Discovery and Biosynthesis of Tetrachlorizine Reveals Enzymatic Benzylic Dehydrogenation via an ortho-Quinone Methide
    • Article
    • Peer Reviewed
    UC Davis Previously Published Works (2021)
    Bromopyrroles (BrPyr) are synthesized naturally by marine sponge symbionts and produced anthropogenically as byproducts of wastewater treatment. BrPyr interact with ryanodine receptors (RYRs) and sarco/endoplasmic reticulum (SR/ER) Ca2+-ATPase (SERCA). Influences of BrPyr on the neuronal network activity remain uncharted. BrPyr analogues with differing spectra of RYR/SERCA activities were tested using RYR-null or RYR1-expressing HEK293 and murine cortical neuronal/glial cocultures (NGCs) loaded with Fluo-4 to elucidate their mechanisms altering Ca2+ dynamics. The NGC electrical spike activity (ESA) was measured from NGCs plated on multielectrode arrays. Nanomolar tetrabromopyrrole (TBP, 1) potentiated caffeine-triggered Ca2+ release independent of extracellular [Ca2+] in RYR1-HEK293, whereas higher concentrations produce slow and sustained rise in cytoplasmic [Ca2+] independent of RYR1 expression. TBP, 2,3,5-tribromopyrrole (2), pyrrole (3), 2,3,4-tribromopyrrole (4), and ethyl 4-bromopyrrole-2-carboxylate (5) added acutely to NGC showed differential potency; rank order TBP (IC50 ≈ 220 nM) > 2 ≫ 5, whereas 3 and 4 were inactive at 10 μM. TBP >2 μM elicited sustained elevation of cytoplasmic [Ca2+] and loss of neuronal viability. TBP did not alter network ESA. BrPyr from marine and anthropogenic sources are ecological signaling molecules and emerging anthropogenic pollutants of concern to environmental and human health that potently alter ER Ca2+ dynamics and warrant further investigation in vivo.
      Cover page: Marine and Anthropogenic Bromopyrroles Alter Cellular Ca2+ Dynamics of Murine Cortical Neuronal Networks by Targeting the Ryanodine Receptor and Sarco/Endoplasmic Reticulum Ca2+-ATPase
      • Article
      • Peer Reviewed
      UCLA Previously Published Works (2019)
      Enzymatic dehalogenation is an important and well-studied biological process in both the detoxification and catabolism of small molecules, many of which are anthropogenic in origin. However, dedicated dehalogenation reactions that replace a halogen atom with a hydrogen are rare in the biosynthesis of natural products. In fact, the debrominase Bmp8 is the only known example. It catalyzes the reductive debromination of the coral settlement cue and the potential human toxin 2,3,4,5-tetrabromopyrrole as part of the biosynthesis of the antibiotic pentabromopseudilin. Using a combination of protein crystallography, mutagenesis, and computational modeling, we propose a catalytic mechanism for Bmp8 that is reminiscent of that catalyzed by human deiodinases in the maintenance of thyroid hormones. The identification of the key catalytic residues enabled us to recognize divergent functional homologues of Bmp8. Characterization of one of these homologues demonstrated its debromination activity even though it is found in a completely distinct genomic context. This observation suggests that additional enzymes outside those associated with the tetrabromopyrrole biosynthetic pathway may be able to alter the lifetime of this compound in the environment.
        Cover page: Bacterial Tetrabromopyrrole Debrominase Shares a Reductive Dehalogenation Strategy with Human Thyroid Deiodinase
        • Article
        • Peer Reviewed
        UC San Diego Previously Published Works (2016)
        The cars we drive, the homes we live in, the restaurants we visit, and the laboratories and offices we work in are all a part of the modern human habitat. Remarkably, little is known about the diversity of chemicals present in these environments and to what degree molecules from our bodies influence the built environment that surrounds us and vice versa. We therefore set out to visualize the chemical diversity of five built human habitats together with their occupants, to provide a snapshot of the various molecules to which humans are exposed on a daily basis. The molecular inventory was obtained through untargeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of samples from each human habitat and from the people that occupy those habitats. Mapping MS-derived data onto 3D models of the environments showed that frequently touched surfaces, such as handles (e.g., door, bicycle), resemble the molecular fingerprint of the human skin more closely than other surfaces that are less frequently in direct contact with humans (e.g., wall, bicycle frame). Approximately 50% of the MS/MS spectra detected were shared between people and the environment. Personal care products, plasticizers, cleaning supplies, food, food additives, and even medications that were found to be a part of the human habitat. The annotations indicate that significant transfer of chemicals takes place between us and our built environment. The workflows applied here will lay the foundation for future studies of molecular distributions in medical, forensic, architectural, space exploration, and environmental applications.
          Cover page: Mass Spectrometry-Based Visualization of Molecules Associated with Human Habitats
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