The neoclerodane furanoditerpenoid family of natural products is a compelling target for a divergent total synthesis due to the complexity around their spirolactone-containing decalin core, the similarities between their functional groups, and biological activities that indicate a potential for future medicinal application. While being a relatively small molecule, the most structurally complex compound of the family, teucrin A, contains six stereocenters, five of which are contiguous. Our divergent synthesis route involves the stereo-controlled formation of a common decalin intermediate in four concise steps and 32% overall yield from commercially available starting materials. Additionally, desmethyl montanin A - an analog of montanin A missing its methyl group - can be produced from this key intermediate in one step and 95% yield, for a total of 30% yield over five steps. The dual-key step of this route consists of a Lewis acid-catalyzed Diels-Alder cycloaddition which stereo-selectively forms the spirolactone, followed by a gold-catalysed 6-exo-dig cyclization of the Diels-Alder adduct to complete the decalin core. The complementarity of these two steps is the focus of the synthesis, with the remaining steps for the formation of select neoclerodane natural products consisting of simpler classical chemistry, highlighting the potential for the creation of a large "unnatural" product library for applications such as drug candidate screening. Progress has additionally been made toward teucrin A, setting a cornerstone for future advancements to be made on this project. This concise synthesis advocates for the Barriault group's Diels-Alder/gold cyclization method of forming complex structural cores and will be applied to the total syntheses of other natural products in the future.

3,5-Dimethylorsellinic acid (DMOA)-derived meroterpenoids are an extensive natural product family with significant structural complexity and diverse biological activity. Their structures feature various fused, bridged, spirocyclic skeletons and unconventional stereochemistries with significant strain, especially the trans-syn-trans fused drimane system, and extensive oxidative, skeletal rearrangements. Despite a long isolation history and extensive biosynthesis studies and encouraging biological activities, chemical synthesis of those natural products appeared only recently compared with other well-studied diterpene families such as the ent-kauranes, daphanes, and tiglianes. Thus, this thesis will present our work as an incremental advancement on the total syntheses of DMOA-derived meroterpenoids. In Chapter 1, a thorough biosynthetic relationship of DMOA-derived meroterpenoid subfamilies is reviewed and the most updated isolation and syntheses are covered with the purpose to provide the reader an understanding and appreciation of the origin of the exceptional diversity of DMOA-derived meroterpenoids. In Chapter 2, three generations of routes towards DMOA-derived spiromeroterpenoid are discussed in detail. The oxidative [3+2] cyclization approach was found to generate several complex dimers and the simplicission core. Lessons learned therein pointed to a successful strategy. In Chapter 3, a successful fragment coupling strategy culminated in a concise, modular, and collective synthesis of five spiromeroterpenoid natural products. The synthesis features a sterically hindered bis-neopentyl 1,2-addition coupling/oxidative Michael addition/MHAT reduction sequence to rapidly construct the conserved spirocycle with full stereo-control. The gateway natural product asnovolin A was secured in deca-milligram amounts which laid the foundation for chemoenzymatic synthesis of the highly oxidized spiromeroterpenoid novofumigatonin. In Chapter 4, significant progress was made towards the synthesis of the complex meroterpenoid andiconin. We developed a late stage de novo construction of the dearomatized biosynthetic precursor. A biomimetic radical fragmentation [4+2] cascade was designed to establish the highly congested [2.2.2] octane core of the natural product. / 2026-03-15T00:00:00Z

Enzymes are implicated in many diseases including neurodegenerative, cancer, immune deficiency, and inflammatory disorders. There is a constant need to develop novel drug compounds that target enzymes in order to modulate their function, thus treating the disease state. These compounds are typically small molecules with affinity to the enzyme active site or an allosteric site. In order to discover novel compounds for treating disease, the interaction between an enzyme and a small molecule must first be identified and then characterized. With the target enzyme known, it is beneficial to screen libraries of compounds against the target. Immobilizing the enzyme allows for pre-concentration of ligands on the surface and therefore increased signal enhancement, as well as permitting multiple wash steps and enzyme reuse. Immobilized enzyme columns are optimal for coupling to a variety of detection devices by way of liquid chromatography, including absorbance or mass spectrometric detection. Immobilized enzyme reactors (IMERs) were generated and optimized for two target molecules, acetylcholinesterase (AChE) and adenosine deaminase (ADA), for rapid function-based screening of enzyme inhibitors in mixtures. The IMER mode is useful for increasing throughput and facilitating the identification of hit mixtures, but it is slow and tedious to manually deconvolute hit compounds from mixtures and the IMER method is not amenable to natural product extracts, which are good sources of structurally diverse compounds that are more likely to result in a hit compound. Bio-selective solid-phase extraction (BioSPE) is an orthogonal method of isolating and identifying enzyme inhibitors in a single step, and was used to easily deconvolute complex mixtures, rapidly identifying to key compounds EHNA and MAC-0038732 out of mixtures using ADA columns. A data dependent acquisition MS method was developed and used to screen a set of fungal endophyte extracts, identifying two potentially novel inhibitors that were confirmed by IMER-MS/MS. / Thesis / Doctor of Philosophy (PhD) / The discovery of new drug compounds is crucial for the treatment of diseases. Enzymes are proteins that turn a substrate into a product; and in diseases they can often malfunction, overproducing the product. Small molecule compounds can sometimes inhibit enzyme function and can be further developed into therapeutic drugs. This thesis describes a method for detecting small molecule inhibitors that bind to an enzyme that is immobilized in a small column. Once the small molecule is bound to the immobilized enzyme, it can be detected by either showing that enzyme function is inhibited or by removing the compound from the enzyme and identifying the compound by mass spectrometry. These methods can quickly identify compounds at extremely low levels from complex mixtures, such as natural product extracts.

First isolated in 1948 from the Trichothecium roseum fungus, trichothecenes are a diverse class of sesquiterpene natural products with the structures of over 250 congeners established to date. Their general structure consists of the tricyclic “trichothecene” core, marked by the C12,13 exocyclic epoxide and C9,10 olefin, with further structural complexity generated through esterification of macrolide chains to afford macrocyclic trichothecenes including verrucarin A. Owing to their complex structures and potent anticancer activity, trichothecenes have captured the attention of numerous academic groups since the 1970s, resulting in total syntheses of both non-macrocyclic and macrocyclic trichothecenes. The macrocyclic compounds exhibit increased potency, with recent biological studies suggesting involvement of novel molecular targets. The dissertation research described herein is focused on efforts toward the total synthesis of (–)-verrucarol, the flagship trichothecene natural product, and related trichothecene congeners. (–)-Verrucarol was the subject of six total syntheses throughout the 1980s, serving as an entry point for macrocyclic trichothecenes including verrucarin A. Compelled by the recent literature on insight into their therapeutic activity, we have established a concise route to the trichothecene core of verrucarol in eight steps. The construction of the tricyclic system is enabled by a novel samarium (II) iodide (SmI2)-mediated radical cyclization to an enol sulfonate to generate a complex oxabicyclo[3.2.1]octanone ring system. Significant synthetic efforts focused on a key late-stage olefin isomerization, which was ultimately accomplished through Mukaiyama-hydration and elimination. Furthermore, while significant synthetic efforts have gone into constructing the tricyclic trichothecene core, only a single successful, albeit lengthy, enantioselective synthesis of (–)-verrucarol has been reported. To evaluate the full therapeutic potential of trichothecenes, a concise asymmetric synthesis to access the prerequisite tricyclic core is needed. Herein, we report a Cr (III)-salen complex-mediated Diels-Alder cycloaddition of 4-pyrones and the simple diene, isoprene, for rapid access of an enantioenriched precursor for the trichothecene core. Future efforts and synthetic strategies to generate macrocyclic analogues for structure-activity-relationship studies are provided, as focused synthetic efforts to evaluate their clinical potential are sorely needed. / 2025-09-20T00:00:00Z

<p dir="ltr">Phosphatidylinositol phosphates (PIPs) and soluble inositol phosphates (IPs) serve as critical secondary messenger molecules that regulate cellular processes. The INPP5 family of phosphatases play an essential role in regulating levels of PIP-5’ and IP-5’ molecules. Src homology 2-containing-inositol phosphatases (SHIP), are a subgroup of the INPP5 family that consists of two members, SHIP1 and SHIP2. Both SHIP proteins have been identified to hydrolyze PI(3,4,5)P3 into PI(3,4)P2. Interestingly, the dysregulation of PI(3,4,5)P3 and SHIP proteins have been observed in multiple diseases, such as cancer, diabetes, and neurodegenerative disease. Recently, SHIP1 was identified as a potential risk factor for the development of Late-onset Alzheimer’s Disease (LOAD). Furthermore, knockdown and inhibition of SHIP1 using small-molecule inhibitors were shown to reduce phenotypes associated with LOAD. Taking these studies together suggests SHIP1 to be a potential therapeutic target for the treatment of LOAD.</p><p dir="ltr"><br></p><p dir="ltr">Despite SHIP1’s therapeutic potential, the development of specific small-molecule inhibitors that target SHIP1 has been challenging. One explanation for this challenge is that very little is known about the overall structure and function of SHIP1. In this thesis I will discuss in detail how we generated multiple SHIP1 constructs to improve our understanding of SHIP1’s overall structure and function in an <i>in vitro </i>setting.</p><p><br></p><p dir="ltr">Efficient protein production is essential for studying enzyme structure and function. The choice of expression system can impact protein yield and stability. The E. coli (BL21) and Baculovirus expression systems are two commonly used systems for protein production. While E. coli is cost-effective and can yield a large amount of protein, the Baculovirus system offers advantages in terms of protein folding and post-translational modifications. Using both systems to generate SHIP1 protein, we demonstrate that the Baculovirus system significantly enhances SHIP1 solubility for all generated constructs, making it the preferable choice for investigating the structure and function of SHIP1.</p><p><br></p><p dir="ltr">SHIP1, a 133 kDa protein, which comprises five established domains: an N-terminal Src Homolgy 2 (SH2) domain, 2.) a pleckstrin homology-related (PH) domain, 3.) an inositol phosphatase catalytic (Ptase) domain, 4.) a C2 domain, and 5.) a C-terminal domain containing proline-rich regions (PXXP) and tyrosine phosphorylated (NPXY) motifs. Despite their regulatory roles in phosphatase activity, protein-protein interactions, and membrane association, limited information is available about their structures and how they contribute SHIP1’s biochemical functions. In this study, we utilized baculovirus-expressed SHIP1 constructs to investigate the impact of each domain on macromolecular structure. Interestingly, a previously unrecognized domain within SHIP1 that directly impacts the enzyme's oligomeric state was identified. This work highlights that SHIP1's individual domains can significantly impact its overall structure and function, providing valuable insights for the development of potential therapeutics in the treatment of LOAD.</p><p><br></p><p dir="ltr">Accurate determination of phosphatase kinetics is vital for understanding the enzymatic activity and its potential involvement in disease. Using our baculovirus generated SHIP1 constructs, we employed in-vitro assays, including the malachite green (MG) and the 2-amino-6-mercapto-7-methylpurine riboside (MESG) coupled enzyme assays, to gain insight into SHIP1 kinetics. Results from the MG assay shows that SHIP1 can hydrolyze the PI(3,4,5)P3 diC8 substrate more efficiently than I(1,3,4,5)P4. Additionally, SHIP1’s PH domain was observed to increase the turnover of PI(3,4,5)P3 diC8. Furthermore, dimerization of SHIP1 was not observed to alter SHIP1 kinetics in any way. Lastly, no major differences in I(1,3,4,5)P4 kinetics were observed with the addition of SHIP1’s N-terminus. These results offer the first comprehensive biochemical characterization of SHIP1 across its substrates and N-terminal domains.</p><p><br></p><p dir="ltr">The development of potent and specific small-molecule inhibitors that target SHIP1 remains challenging. One potential cause for this challenge is that no structures of SHIP1 have been solved in complex with active compounds, making structure-based drug design impossible. In this study, we developed a covalent compound, <b>TAD-58547</b>, from a previously published fragment-based screen that was conducted on SHIP1’s Ptase and C2 domain. <b>TAD-58547 </b>was shown to effectively inhibited SHIP1's Ptase and C2 domains at modest potency. Using X-ray crystallography, this compound was observed to form a covalent interaction with a cysteine residue near the Phosphatase-C2 domain interface. Intriguingly, the inhibitor's potency was observed to be reduced in the presence of the SH2 domain. In addition to testing <b>TAD-58547</b> against our SHIP1 constructs, we investigated the effect of SHIP1’s N-terminus on the potency of a literature compound, <b>TAD-58616</b>. This compound was shown to inhibit all our tested constructs at low µM concentrations. Furthermore, using x-ray crystallography <b>TAD-58616 </b>was solved in complex with SHIP1’s Ptase and C2 domain. Intriguingly, density for <b>TAD-58616 </b>was shown to interact with a site previously identified from the fragment-based screen. While we initially determined this site to be a result of crystal packing, fragments bound to this site may have the potential to inhibit SHIP1. The work presented in this study reinforced the importance of testing inhibitors against physiological relevant forms of SHIP1, when developing potential therapeutics.</p><p><br></p><p dir="ltr">Lastly, new evidence has suggested that the binding of phosphorylated immunoreceptor tyrosine-based activation motifs (p-ITAM) and immunoreceptor tyrosine-based inhibitory motifs (p-ITIM) to SHIP1’s N-terminal SH2 domain is essential for its “Anchorage and Activation” at the plasma membrane (PM). With this model it is believed that SHIP1’s SH2 domain, places the phosphatase into an auto-inhibited state. Upon binding to immune receptor proteins and adaptor proteins that contain ITAM/ITIM sequences, SHIP1 becomes un-auto-inhibited, allowing it to efficiently hydrolyze PI(3,4,5)P3 embedded in the PM. While this model does support the notion that SHIP1 activity is mediated by its PM localization, our biophysical and biochemical characterization add another level of complexity to this regulatory event. Taking all these results together, we propose a novel model for SHIP1 called “Anchorage and Assist” and suggest innovative therapeutic strategies for targeting SHIP1.</p><p><br></p><p dir="ltr">In conclusion, this thesis highlights the importance of choosing suitable expression systems for efficient protein production. Additionally, it offers insight into SHIP1's regulatory mechanisms through the discovery of a novel domain impacting its oligomeric state. Furthermore, the accurate determination of SHIP1 kinetics enhances our understanding of this phosphatase and its potential implications in disease. Also, the identification and crystallization of a novel and previously determined inhibitor scaffolds in complex with SHIP1 increases our ongoing efforts to develop a small-molecule inhibitor that specifically targets SHIP1. Lastly, using recently published data, detailing SHIP1 PM localization and activation, we proposed a new model for SHIP1 activity and suggest novel therapeutic strategies for targeting SHIP1.</p>

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