Trichothecenes such as T2-toxin are toxic natural products produced by several species of fungi that grow on grain crops. This class of sesquiterpenes comprises over 200 family members that feature a tricyclic core with varying degrees of oxidation and further cyclization, and has received attention for their toxicity as well as their anticancer, antifungal, and immunomodulatory effects. Cellular and animal model studies support the hypothesis that their manifold biological effects arise at least in part due to inhibition of protein synthesis. Here we report a modular synthesis of the minimal trichothecene pharmacophore verrucarol, enabling straightforward structural modifications to investigate toxicity and molecular mechanisms of action. We found that while verrucarol inhibits human cytosolic protein synthesis in vitro, functionalization of the C4 and C15 alcohols is required for potent cellular toxicity in cancer cell lines and fibroblasts. We characterized the binding of verrucarol and diacetylverrucarol to the 50S subunit of the human cytosolic ribosome at 2.7 A resolution, revealing binding determinants that could not be resolved from the previous structures in yeast ribosomes. This work will enable new studies of trichothecenes required for food safety and for exploration of their use as therapeutics.

Macrolide antibiotics are a structurally diverse class of polyketide natural products, produced by bacteria, that exhibit antimicrobial activity through inhibition of bacterial protein synthesis. Macrolides and their semisynthetic derivatives are critical for the treatment of infectious disease, many of which are first-line therapeutics. Ketolides are a subclass of the macrolides which bear a ketone at the C3 position of the macrocycle. While predominantly semisynthetic, there are some ketolide natural products, like narbomycin, that have marked structural simplicity compared to other macrolides yet maintain good antibiotic activity. Here we report a modular, convergent synthesis of narbomycin, which we propose to be a minimal pharmacophore for macrolide antibiotics. This platform enables the facile incorporation of desired structural modifications to this minimal pharmacophore to generate novel unnatural products that can increase potency and overcome clinically relevant resistance mechanisms. In chapter 1, I describe our first attempts to synthesize narbomycin through a critical macrocyclization step using the thermolytic properties of a dioxinone moiety. A vinylogous Mukaiyama aldol was used to incorporate this dioxinone and set the C4/C5 stereochemical configuration. Despite screening for reaction conditions, the desired syn aldol products were unable to be obtained, but a method for anti vinylogous Mukaiyama dioxinone aldol products was successfully developed. Some mechanistic rationale for our findings is discussed. In chapter 2, I describe the successful total synthesis of narbomycin. This route uses a robust Liebeskind-Srogl coupling to join the two halves of the molecule together and employs an unprecedented macrocyclization strategy via direct intramolecular displacement of an oxazolidinone chiral auxiliary. Some of the challenges faced in the final steps of the synthesis involving a difficult reduction of an exocyclic alkene to a methyl group are also outlined.

(+)-Pleuromutilin is a fungal natural product that binds to the peptidyl transferase center of bacterial ribosomes, inhibiting protein synthesis as its mechanism of action. This tricyclic diterpene has been successfully modified through semi-synthesis to access more potent analogs such as the recently FDA-approved lefamulin, which is used for treating adults with community –acquired bacterial pneumonia. However, semi-synthesis provides an inherently restricted approach to incorporating variations in the structure of chemical matter. Thus, pleuromutilins remain limited in their spectrum of activity and susceptible to modifications in the ribosomal binding site that confer resistance. Previous studies have provided evidence that modifications of the core are well-tolerated in that fully synthetic analogs may retain biological activity. Here, we will describe our efforts in developing an efficient synthesis of pleuromutilin that may not strictly follow its natural substitution pattern, but will allow for the construction of a diverse library of simplified pleuromutilin analogs.In Chapter 1, we report a synthetic route towards simplified pleuromutilin antibiotic candidates that uses a key photochemical reaction to access the six-, eight-membered bridged ring system. We describe our efforts in optimizing steps in this route, including the conditions necessary for the [2+2] cycloaddition in the de Mayo-type reaction sequence. In Chapter 2, we report the synthesis of antibody-drug conjugates for the targeted degradation of cell surface proteins.

Cell-penetrating peptides (CPPs) have been widely explored as a means to transport molecular cargo across cellular membranes. Their applications range from delivering small molecules, such as fluorophores and chemotherapeutic agents, to larger, typically cell-impermeable macromolecules, such as peptides, nucleic acids, and proteins. To enhance selectivity, numerous strategies have been developed to mask the peptides' positive charges until their release is desired. Without these charges, CPPs cannot adopt the conformation necessary to interact with negatively charged cell membranes, rendering them inactive in membrane penetration.

One effective masking strategy relies on protease-triggered cleavage. In this approach, the CPP is linked to a masking domain via a protease-cleavable sequence. Proteolytic cleavage releases the CPP from the masking domain, restoring its activity. Disease-associated proteases, such as cathepsins and matrix metalloproteinases, have been extensively investigated for this purpose due to their upregulation in pathological tissues, including cancers. However, these proteases are also present and active at basal levels in healthy tissues, posing challenges for the selective delivery of cytotoxic agents.

To address this limitation, we present a side chain dual-masking strategy for CPPs. Positive charges on key residues, such as lysines, are covalently conjugated to protease-cleavable sequences, effectively neutralizing the charges until proteolytic cleavage occurs. By masking two or more lysines on the CPP, an AND gate system is created, enabling CPP activation only in the presence of multiple active proteases. This approach enhances selectivity, ensuring CPP activation primarily in target disease tissues where the relevant proteases are simultaneously upregulated.