Department of Chemistry and Biochemistry

In recent decades, developments in glycobiology have enabled the use of glycopeptides as tools for studying complex diseases such as cancer. Mucin 1 (MUC1) is a heavily glycosylated transmembrane protein, altered in both expression and glycosylation pattern in human carcinomas of the epithelium. The presence of incomplete or truncated glycan structures, often capped by sialic acid, commonly known as tumor-associated carbohydrate antigens (TACAs), on the cell surface is a well-known cancer biomarker and therapeutic target for different types of cancer. Accumulating evidence suggests that TACAs are recognized by the endogenous carbohydrate binding proteins (lectins). These interactions frequently result in the development of a protumor microenvironment, favoring tumor initiation, progression, metastasis, and immune evasion. Macrophage galactose binding lectin (MGL) is a C-type lectin receptor found on antigen-presenting cells (APCs) which facilitates the uptake of carbohydrate antigens for antigen presentation, modulating the immune response in homeostasis, autoimmunity, and cancer. Considering the crucial role of tumor-associated forms of MUC1 and MGL in tumor immunology, a thorough understanding of this interaction is essential for it to be exploited for cancer vaccine strategies. The specific goal of this research is to synthesize structurally well-defined chemical probes, mono and multiple glycosylated MUC1 glycopeptide models bearing the Tn or sTn antigens, that provide control over the complexity of the chemical space of multivalent ligands. For this purpose, a concise scheme was developed for the large-scale synthesis of the Tn and sTn antigen building blocks in a relatively high yield with moderate stereoselectivity. Thiophenyl glycoside donors, in the presence TfOH/NIS or TMSOTf/NIS as promoter systems, were used for the galactosylation and sialylation steps of the amino acid building block synthesis, respectively. We explored the effect of the activator, temperature, solvent, and excess equivalent of sialic acid thioglycoside donor on the sialylation reaction.

Cyanobacteria are ancient prokaryotes that use photosynthesis and an accumulation of other adaptations to dominate aquatic ecosystems around the world. They are thus major contributors to biogeochemical cycling, a threat to human and environmental health, and an intriguing source for novel chemistry. We begin by providing an overview of bloom-forming cyanobacteria and their many toxic metabolites. We then discuss the characterization of some abundant extracellular pili of Microcystis aeruginosa, reporting a 2.4 Å cryoelectron microscopy pilus structure, revealing a novel class of pili that we have termed cyanobacterial tubular (CT) pili. The CT pili in M. aeruginosa were determined to be multi-functional, with a primary role in networking cells and enhancing colony formation, but also in controlling colony buoyancy, enriching iron, and accumulating toxins in the extracellular mucilage. Lastly, we explore the potential of heavy-labeling cyanobacterial cultures for the sake of isolating natural products that can be studied by vibrational spectroscopic imaging. The vibrational spectra of three classes of cyanopeptides along with their heavy-labeled counterparts are reported, and Density Functional Theory calculations are used to describe mode character, clarifying some unexpected changes in vibrational spectra upon heavy-labeling. As a whole, this work offers new insight into cyanobacterial physiology as well as a means to study cyanopeptides with imaging techniques and stable-isotope labeling.

Herein, we discuss a novel method for the synthesis of decorated 2,5-dihydrofurans. The base promoted 5-endo-dig cyclization of non-Conia-ene propargyl ethers produces 2,2 disubstituted dihydrofurans. Central to the reaction is the presence of an acidic C–H bond which is activated by an adjacent aromatic heterocycle. The transformation is viable with a wide range of substituents, including N, O, and S containing heterocycles, substituted phenyl rings, and alkyl groups. The cyclization proceeds within 30 seconds at room temperature under the action of potassium tert-butoxide. This work stands apart from the current literature due to the absence of transition metal catalysts and/or harsh reaction conditions. A thorough mechanistic investigation is undertaken to better understand the nature of this unprecedented reaction.

Cholesterol is a pivotal component of mammalian cell membranes and homeostasis. Due to its high concentration and heterogenous distribution in the brain, cholesterol is tightly regulated and its dyshomeostasis is frequently implicated in several neurodegenerative diseases. This dissertation reports the design, synthesis, and application of ten cholesterol naphthalimide probes (CND) to study cholesterol trafficking in live cells. The CND series was rationally designed by incorporating several of the structural features of endogenous cholesterol onto the naphthalimide (ND) scaffold conjugated via an ester bond. The modularity of the ND scaffold enabled all analogs to have the aliphatic tail of cholesterol, which is lacking in the most ubiquitously utilized probe, BODIPY-Cholesterol, a.k.a. Top-Fluor® Cholesterol (TFC). The CNDs were demonstrated to be optimal probes for cholesterol due to the ability to fluorescence exclusively in hydrophobic/membrane environments and exhibit low fluorescence in hydrophilic/aqueous environments. By incorporating a protonatable piperazine group on the C4 position of the ND scaffold, CND2 – CND4 possessed pH sensing capabilities, which were demonstrated to monitor intracellular vesicle turnover in neurons. The potential of the CNDs to bind to the lysosomal sterol transport protein NPC2 was investigated by molecular docking and molecular dynamics (MD) simulations. The docking pose with the CND’s aliphatic tail positioned inside the hydrophobic binding pocket was essential for mimicking endogenous cholesterol’s interactions and stabilizing the NPC2-ligand complex. Fluorescence confocal microscopy demonstrated a structure-dependent and cell-dependent intracellular distribution of the CND series in live cells.

Protein─protein interactions (PPIs) are essential for cell─cell interactions and cellular signal transduction, which play a crucial role in various human diseases. PPIs involved in cancer immunology pathways, known as immune checkpoints, have been intensely studied, leading to a new approach to cancer therapy. The PD1:PDL1 interaction is one of the most essential immune checkpoints. Studies on PD1:PDL1 showed over ten clinical monoclonal antibodies (mAb) used to treat cancer patients. Unfortunately, antibodies do not penetrate the tumor microenvironment well, and clearance from the body is slow, leading to unwanted side effects. There is a significant gap in the drug market between the typical Rule of 5 (Ro5) small-molecule drugs (MW<0.5 kDa, SASA ~150 Å) and large antibodies with molecular weights greater than 40 kDa (SASA >2,000 Å). PPIs remain challenging to modulate by small molecules due to their large, shallow, often dynamic, and water-exposed surfaces lacking well-defined binding pockets. Thus, our lab was drawn to work on large β-hairpin peptides (2-3 kDa) that can potentially mimic the CDR-H3 loops of some of the most potent and clinical anti-PD1 antibodies. Exploration of these β-hairpin peptides provided valuable insights into their folding stability, conformational flexibility, passive membrane permeability, and protein-protein interaction (PPI) blocking activities. Additionally, the rational design of TCIs against PD1, specifically targeting a lysine residue, emerged as a strategy to irreversibly obstruct the PD1:PDL1 protein-protein interaction enhancing potency of the non-covalent inhibitors by taking advantage of their specificity. Meticulous structural analysis, peptide synthesis, and biological evaluations are presented contributing comprehensions into covalent inhibitor development of drugs fbRo5.

Misfolding and aggregation of Cellular Prion Protein (PrPc) is a major molecular process involved in the pathogenesis of Prion diseases. An N-terminal portion of the Prion protein, PrP106-128, is a 23-residue peptide fragment characterized by an amphipathic structure with two domains: a hydrophilic N-terminal domain and a hydrophobic C-terminal domain. Here, we studied the aggregation properties of the prion fragment peptide PrP106-128. The results show that the peptide aggregates in a concentration-dependent manner in an aqueous solution and that the aggregation is sensitive to pH and the preformed amyloid seeds.Furthermore, we show that the zwitterionic POPC liposomes moderately inhibit the aggregation of PrP(106–128), whereas POPC/cholesterol (8:2) vesicles facilitate peptide aggregation likely due to the increase of the lipid packing order and membrane rigidity in the presence of cholesterol. In addition, anionic lipid vesicles of POPG and POPG/cholesterol above a certain concentration accelerate the aggregation of the peptide remarkably. The strong electrostatic interactions between the N-terminal region of the peptide and POPG may constrain the conformational plasticity of the peptide, preventing insertion of the peptide into the inner side of the membrane and thus promoting fibrillation on the membrane surface. The results suggest that the charge properties of the membrane, the composition of the liposomes, and the rigidity of lipid packing are critical in determining peptide adsorption on the membrane surface and the efficiency of the membrane in catalyzing peptide oligomeric nucleation and amyloid formation.

Alzheimer’s disease (AD) is a common neurodegenerative disorder. The most recognized disease pathology is the Amyloid-β (Aβ) cascade hypothesis which states that the accumulation of Aβ plaques might be the cause of AD. In the AD brain, Aβ plaques stockpile a variety of molecular components including metals, lipids, nucleic acids, carbohydrates, and peptides, indicating Aβ aggregation might be influenced by these modulators. In this dissertation, we investigated the effects of Zn2+ and carnosine, phospholipids, and β-hairpins on Aβ aggregation to dissect their mechanistic roles in the amyloidogenesis of Aβ. We first systematically studied the kinetic impact of Zn2+ on the aggregation of Aβ40 and Aβ40-M. Our results show that the presence of Zn2+ transforms the Aβ40 aggregation kinetics from a single sigmoidal to a biphasic process, while the aggregation of Aβ40-M is significantly suppressed by Zn2+. We also found that a nature dipeptide, carnosine, remarkably decreases the activity of Zn2+ on modulating Aβ aggregation, although it has a weak direct effect on the peptide aggregation kinetics. Second, we investigated the activities of Aβ40 and Aβ42 in inducing membrane damage and the effects of lipid membranes on the aggregation of these peptides using liposome models containing mitochondrial-specific phospholipid–cardiolipin (CL).

This work encompasses the synthesis, analysis, and optimization of [3.2.1] all-carbon bridged bicyclic compounds, known as resveramorphs (RVM), Studies were conducted using a Caenorhabditis elegans model, where RVMs were tested for antiseizure capabilities. In both applications, RVMs proved potent with activities in the sub-nanomolar level in one case. A structure-activity relationship (SAR) was hypothesized for the identification of the pharmacophore. The six to seven step synthesis route towards the RVM analogues is discussed in further detail. The bicyclization of the RVMs is achieved through a reductive aldol reaction. The reaction suffers from selectivity issues leading to multiple bicyclic products. By following a one-factor-at-a-time (OFAT) methodology, attempts at optimization for this reaction were made, however, despite important gains, the overall yields of the bicyclic product remain low. Other products from this reaction have been used to understand the reaction mechanism, which will be the basis for future efforts to further optimize this key step.

The amyloid beta (Aβ) peptide has been linked to Alzheimer’s Disease (AD) since the early 1990s. Since then, many studies have characterized the peptide and examined its aggregation process. Aβ is a 40 or 42-residue peptide, composed of a charged N-terminal and hydrophobic C-terminal, that aggregates into characteristic β-sheets forming insoluble plaques in the brains of (AD) patients. In recent years an intermediate oligomeric species has been shown to interact with lipid membranes, largely resulting in the etiology of AD. In this study, two fragments are used, the 23-residue N-terminal fragment, Aβ23 and the 30-residue C-terminal fragment, Aβ11-40, to better understand the role of the N and C-terminus in the aggregation of Aβ peptide. Aβ11-40 has also been found in the brains of AD patients, playing a biological role in the disease. This study used analytical and biophysical techniques to systematically synthesize, purify, characterize, and study these fragments' aggregation in different conditions. We investigated the effects of lipid membranes on the aggregation of Aβ23 and Aβ11-40 and the activities of these peptides in inducing membrane damage. The results show that the aggregation of Aβ23 was increased in the presence of lipid membranes, likely due to favorable electrostatic interactions. However, the aggregation of Aβ11-40 was not influenced by lipid membranes. A dye leakage study was carried out to study the membrane damage occurring as a result of fragments' interaction with lipid membranes. The results showed that neither fragment had a profound effect on membrane destruction, although the charge of the lipid head seemed to play a role. This work's second study focused on the effect of three specific polysaccharides, heparin, chitosan (CHT), and trimethyl chitosan (TMC), on the aggregation of Aβ23 and Aβ11-40. The results showed that for Aβ23, heparin increased aggregation, while both CHT and TMC decreased aggregation. However, for Aβ11-40, both heparin and CHT did not affect aggregation, while TMC decreased aggregation.

The mechanisms of larval fish transport have been rigorously studied in the past several decades, building foundational knowledge of key biological and environmental factors with which to inform decisions about species management. This study has been built upon information gained from previous studies to further elucidate the processes involved at the recruitment stage of larval fishes. Vertical swimming behaviors of larval fishes enable deliberate orientation within the water column to allow organisms of limited mobility greater control over their horizontal movements. Vertical accumulation patterns of larvae are found to be tightly dependent on the strength of stratification within the water column at nursery entrances, such as estuaries. Onshore currents, such as upwelling and surface intrusions, are found to be conduits for entry into these systems. This study observed and analyzed the influence of intrusions by the Gulf Stream into the Fort Pierce Inlet and the vertical accumulation patterns of late-stage larvae associated with those events. This study incorporated a well-established zooplanktonic abundance sampling technique to achieve two primary goals: (1) to analyze the vertical abundances of larval fishes in stratified flow during Gulf Stream intrusions and (2) to assess the correlation between larval influx and intrusion events. The results of this study show a significant and positive correlation between propagule pressure of larval fishes and incidence of Gulf Stream intrusion events. Whereas previous studies have primarily described the spatiotemporal aspects of larval transport in a broader sense, our findings revealed a greater layer of complexity in the mechanisms of transport by incorporating localized hydrographic features. The information gleaned from these results can inform the ecological considerations of future fisheries management and study efforts via additional understanding about the role of physical oceanographic events in a critical life stage.