Yellow Fever (YF) caused by yellow fever virus (YFV) has historically been a threat to global health. This risk posed by YFV was highly mitigated with the development of the vaccine strain YFV-17D, but challenges caused by this virus linger despite global vaccination attempts. With outbreaks occurring in urban areas in the past 20 years and the lack of an antiviral for YFV, there is demand for expanding the arsenal of global health tools to fight against Flaviviruses like YFV.

To address this gap, a high-throughput screen was done on the ~75,000 molecules in the Princeton University Small Molecule Library to find a compound that exhibited antiviral activity against YFV while also maintaining low cytotoxicity. The initial screen identified 626 molecules with antiviral activity, of which 43 were determined to be noncytotoxic. While these initial stages of the screen seemed promising, we were unable to identify a molecule that consistently exhibited antiviral activity. Of the 43 molecules that continued to the second round of screening, 5 exhibited a titratable effect where an increase in concentration led to a decrease in viral activity. Those 5 were preliminarily retested but did not validate the original antiviral activity exhibited in the initial screening process.

Due to time constraints, more thorough workup could not be completed to establish firmly whether any of the compounds are suitable for further refinement in structure activity relationship analysis. It is also conceivable that some of these compounds may exhibit antiviral activity against other related flaviviruses. Moreover, the search for an antiviral that acts on YFV can continue by screening other molecule libraries or identifying targets in the YFV replication cycle.

Rising atmospheric carbon dioxide levels have led to increasingly severe environmental and economic consequences. To mitigate these effects, there is an urgent need to develop technologies that utilize waste and atmospheric carbon dioxide in order to stabilize its atmospheric concentration and prevent further emissions. Thus far, the Bocrasly group has developed, optimized, and characterized a chromium–gallium oxide (CrGaOx) catalyst capable of producing 1-butanol via the electrochemical CO2 reduction reaction. This study builds on that foundation by synthesizing and investigating three additional mixed transition metal oxide catalysts: chromium–aluminum, manganese–aluminum, and manganese–gallium. Each combination tested was found to form metal oxides. In addition, metal composition was found to play a significant role in determining catalyst structure and morphology. Chromium was identified as the source of the characteristic “puff” of the CrGaOx catalyst. Manganese-based systems were shown to form large chunks composed of MnO and an intermetallic oxide. Importantly, both Mn-Al and Mn-Ga catalysts were found to be electrochemically active, producing formate at low Faradaic efficiencies. These findings advance our understanding of pore formation and catalyst morphology, features believed to be critical for the effectiveness of CrGaOx. This work contributes to the broader effort to design and develop more efficient electrocatalysts for CO2 reduction, which in turn will help to close the carbon loop.

Soil in Hawaii has been extensively studied to better understand the properties that make it effective in capturing and storing organic carbon for long periods of time. In particular, the age and rainfall gradients found naturally occurring on the Hawaiian Islands make it salient for soil research, thus far yielding insights into how mineral composition, age, and vegetation influence the abundance of soil carbon. However, the character of organic carbon along these gradients has remained virtually unknown, holding space for this study to explore how the soil profile changes with respect to the functional groups present in organic carbon molecules. This study focuses first on how previously identified properties of soil important for carbon sequestration (i.e. mineral content) independently influence sorption and collection of organic matter, and then explore how organic carbon molecule change by experimenting on whole soil samples, via experimentation on both the solid and mobile phases of the molecules. In doing so, this determination of the type of carbon present in soil and how it changes as a function of age and climate will further the understanding of soil carbon dynamics in carbon turnover and sequestration.

This study aims to develop software for modelling electrodes covered in a porous insulating layer for applications to carbon dioxide reduction. The project focuses on modular design and efficiency in its construction of these modules. It successfully produces a library for cyclic voltammogram analysis including visualization and baseline correction. It also develops a data structure for partitioning 3D space, allowing efficient collision checking. Finally, it presents designs for electrode modules that allow clients to specify the geometry and conductivity of the electrode surface.

Carbonate rocks record information about the environmental conditions under which they formed, offering a window into Earth history. Among carbonate rocks, dolomite, CaMg(CO$3$)$2$, is abundant in the geologic record while elusive in modern environments due to kinetic barriers in formation. Geochemical signals measured in dolomite, however, are frequently used as evidence of changes to the global carbon cycle. To refine interpretations of ancient dolomite, this thesis investigates dolomite precipitation in the Coorong region of southern Australia, examining how dolomite geochemistry encodes environmental information. To that end, I create mineralogical and geochemical fingerprints of 49 samples collected from the carbonate-precipitating lakes. I develop a method for quantifying the relative abundances of carbonates from XRD spectra. I also measure carbon and oxygen isotope ratios and elemental composition (Mg, Ca, Sr, U), and, on a subset of samples, clumped carbonate isotopes to constrain formation temperatures. I use these fingerprints to evaluate whether dolomite precipitates record signals from a global carbon reservoir or are instead influenced by non-global controls like carbonate mixing processes and local environmental conditions on geochemistry. I identify two groups of isotopically and elementally distinct dolomite precipitates—one in equilibrium with atmospheric CO$2$, and one that is not. Finally, I use the fingerprints to test hypotheses about dolomite formation and show that varying levels of seawater-groundwater mixing can explain the observed chemical differences in dolomite precipitates.

Poly(vinyl chloride), or PVC, is the world’s third-most produced thermoplastic but one of the least recycled materials. A deleterious tendency to release HCl (g) upon heating and high plasticizer content is among the most significant hurdles toward a more circular economy. Leveraging photothermal heating initiated by carbon black, this work productively utilizes this HCl (g) to functionalize styrene into (1-chloroethyl)benzene in up to 89 % yield with minimal side-products. This versatile product was further upcycled into 1-phenylethanol, a perfume additive and precursor to the commodity chemical acetophenone, as well as fendiline, a common heart medication. Crucially, the system proved highly tolerant of plasticizers and amenable to commercial samples in up to one-gram total loading without solvent processing. Additionally, the PVC system was able to hydro-chlorinate various olefins into the corresponding chloroalkanes in good yields. Finally, the dechlorinated carbon content of PVC, DHPVC, also found utility as a photothermal agent to depolymerize polystyrene, while further study of its photothermal capabilities revealed a new approach towards TiO2-catalyzed small molecule transformations. Overall, this work presents a novel strategy for tapping an overlooked portion of the plastic waste stream as a chemical feedstock in the hopes of better managing global PVC waste.

Improperly treated wastewater can be harmful to human health and aquatic ecosystems due to residual pollutants with aromatic compounds, such as dyes and pharmaceuticals, being particularly difficult to degrade. Dyes are attractive probes for other more toxic compounds, as they are widely available and have many functional groups that are present in other pollutants. Many treatment methods are used for this application, such as the homogeneous Fenton process involving the reaction between iron salt and an oxidant, but such catalysts are often non-recoverable. Iron-based metal-organic frameworks (MOFs) are the focus of this work due to their stabilization of iron that could allow them to react in a Fenton-like process with less leaching. MIL-100(Fe) is part of a reportedly water-stable series of MOFs, and in this work is synthesized as a catalyst for the degradation of several dyes, with a focus on methylene blue (MB) dye. In its as-synthesized state, MIL-100(Fe) does not greatly improve MB degradation rates relative to a non-MOF control. Thermal activation under vacuum at conditions of 493 K and 393 K, as well as “fresh” non-activated MIL-100(Fe), are compared, with activation conditions improving both MB adsorption and degradation rates. The leaching of active species into solution is also explored, with differences in rates batch-to-batch after MOF removal.

Proteins play crucial roles as molecular machines in various biological processes. While they are abundant, naturally occurring proteins are only a small portion of the vast range of potential sequences. De novo proteins, which are synthesized in the lab and haven't been shaped by natural selection, provide a unique opportunity to explore new protein structures and functions. These proteins could have wide-ranging applications, from medical treatments to material engineering. One such protein, Syn-F4, was the first de novo enzyme shown to be active both in vitro and in vivo, and it successfully rescues auxotrophic bacteria. Rescuer 6 is another de novo protein, created by duplicating, fusing, and diversifying Syn-F4 according to Dayhoff’s hypothesis. Earlier studies indicated that Rescuer 6 exhibited greater biological activity than Syn-F4. However, this thesis examines the in vitro enzymatic activity of Rescuer 6 and finds it to be less effective than Syn-F4. To enhance its activity, directed evolution was used, and through random mutagenesis and selection, several truncated proteins with better rescuing abilities were identified. Three of these proteins were chosen for further analysis of their catalytic functions. Two of these proteins were found to be more efficient enzymes than both Rescuer 6 and Syn-F4. These experiments demonstrate that just a few rounds of directed evolution can lead to de novo enzymes with improved biological and catalytic functions. The fact that the best-performing proteins were truncated suggests that gene or protein duplication can serve as an intermediate step toward developing smaller, more efficient proteins.

C−H bond functionalization is an important transformation of broad utility for chemical synthesis. Using transition metal catalysts, like cobalt, allows for the synthesis of new molecules from electron-rich arenes. This research reports the synthesis of 6-coordinated arene-chromium tricarbonyl complexes that are able to undergo cobalt-catalyzed C−H borylation. Furthermore, this work finds that these 6-coordinated complexes can catalytically compete with known reactive substrates, like fluoroarenes. These reactions can be used to synthesize new molecules from aromatic molecules with electron-withdrawing and electron-donating substituents, which has previously been proven very difficult. These new molecules have applications in the pharmaceutical, agriculture, and materials industries.

Intrinsically disordered soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins are the conserved machinery that facilitate membrane fusion in eukaryotic cells. The assembly of SNAREs into folded four-SNARE bundles bridging two membranes to be fused provides the driving force for fusion. Two families of protein complexes, multisubunit tethering complexes (MTC) and Sec1/Munc18 (SM) proteins, both regulate the complementarity of the SNARE combinations that are formed to ensure that fusion on occurs on-pathway, and to accelerate the rate by which SNARE assembly and fusion occur. After fusion occurs, SNARE complexes are pried apart by the conserved NSF (N-ethylmaleimide-sensitive factor) and αSNAP (soluble NSF attachment protein) proteins and individual SNAREs are recycled. Rather unexpectedly, biochemical studies implicate NSF and αSNAP in catalyzing pre-fusion SNARE assembly and accelerating fusion through synergistic action with MTCs and SM proteins. Through a series of size-exclusion chromatography (SEC)-based binding assays, we establish that the Saccharomyces cerevisiae NSF and αSNAP homologs Sec18 and Sec17 cooperatively and stably associate with Dsl1 and Sly1—the MTC and SM of the retrograde cis-Golgi/endoplasmic reticulum (ER), respectively— in a SNARE-dependent manner. Coupled with preliminary cryoelectron microscopy (cryoEM) analysis of the Dsl1 • SNARE complex • Sec17/Sec18 complex, the data suggests that the MTC and Sec17/Sec18 may not interact directly at all, with the MTC acting at N-terminus of the SNARE complex and Sec17/Sec18 at the opposite, C-terminal end. This is the first-ever study aimed at determining the structure of a pre-fusion NSF/αSNAP complex.

Ethers are a ubiquitous functional group in natural products and drug compounds due to their high stability and mild polarity. However, robust synthetic methods to access ether functionalities with wide functional group tolerance remain scarce. Herein, a method to synthesize dialkyl ethers from aliphatic diols under mild conditions is disclosed, utilizing N-heterocyclic carbene reagents to generate alkyl radicals from alcohols and copper catalysis for C O bond formation. Studies were conducted on the mechanism of this reaction that indicate a triplet sensitization cycle is operative, and a preliminary isolated scope is presented.

The Radical {S}-adenosylmethionine (RaS) enzyme superfamily catalyzes highly diverse and versatile chemical modifications in all kingdoms of life. One notable subset of this powerful enzyme superfamily installs chemically intriguing and diverse modifications onto Ribosomally synthesized and Post-translationally modified Peptide (RiPP) natural products. These RaS enzyme-modified RiPPs (RaS-RiPPs) contain complex structures and exhibit potent biological activities, thus holding clinical relevance as medicinal chemistry scaffolds and drug progenitors. One bioinformatically predicted RaS-RiPP gene cluster encoded in Nitrosomonas oligotropha, termed RGI after its consensus motif, was explored in this work with the intent of expanding the chemical library of RaS enzyme modifications and identifying a new natural product. This investigation indeed yielded evidence for an unprecedented transformation, the attachment of 5'-thioadenosine through a thioether bond onto a RiPP precursor. This modification expands the chemical space of RiPP natural products and highlights RaS-RiPPs as a source of new biosynthetic and small molecule chemistry.

The field of \textit{de novo} protein design carries immense potential for creating novel proteins guided by the principles of nature. Despite the challenges in designing proteins, recent research has leveraged the driving forces of protein folding, particularly the burial of hydrophobic residues, to computationally design new complex topologies using binary patterning.$\mathrm{<mrow\; data-mjx-texclass="ORD">1</mrow>}$ This work studies the structural tolerance of \textit{de novo} proteins to mutations within their hydrophobic cores by investigating variants from a combinatorial library. Differential scanning calorimetry (DSC), a technique that measures the melting temperature and enthalpy of a protein unfolding event, demonstrated high thermal stability of two mutant proteins. Hydrogen-deuterium exchange measured by mass spectrometry (HDX-MS) analyzed the conformational behavior of these mutants. While the mutants had varying degrees of isotopic exchange, only a fraction of the available hydrogens underwent exchange in the three most promising mutants. A consistent trend can be drawn from the results of both techniques: the most thermodynamically stable mutant of DSC incorporated the fewest deuterium atoms in HDX-MS experiments, suggesting a correlation between thermal and conformational stability. These findings demonstrate that \textit{de novo} proteins of a combinatorial library can be successfully designed with novel backbone structures, tolerating variations within their hydrophobic cores. This presents a promising avenue for true \textit{de novo} design, achieving complexity and stability approaching that of natural proteins.

Immunomodulatory drugs (IMiDs), including thalidomide, pomalidomide, and lenalidomide, are essential therapeutics for multiple myeloma (MM) throughout the course of treatment. However, most patients with MM eventually develop IMiD resistance and relapsed/refractory MM. Although IMiDs are known to target Cereblon (CRBN), an E3 ubiquitin ligase substrate receptor, and induce neo-substrate degradation of critical MM proteins, the mechanisms of resistance development and IMiD action are not fully understood. Here, novel endogenous interactors of CRBN identified via the µMap proximity labeling platform reveal a novel mechanism of action of IMiDs in inhibiting native CRBN co-chaperone function with heat shock proteins (HSPs). Independently of induced neo-substrate degradation, IMiDs destabilize CRBN interactors in lenalidomide-sensitive MM lines and increase apoptotic markers. In contrast, CRBN’s stabilization of proteins and nascent protein synthesis is maintained in lenalidomide-resistant MM lines, which attenuate the IMiD-mediated destabilization effect by upregulating critical HSPs. Combination treatment with HSP inhibitors and IMiDs can leverage this mechanism to sensitize IMiD-resistant MM cells to IMiD treatment. These data establish a novel mechanism of action and nominate native CRBN interactors and chaperone function as therapeutic targets for next-generation MM therapeutics.

Understanding embryonic development has broad implications, ranging from treating congenital defects to growing organs in vitro. During embryogenesis, temporal and spatial variation in the proteome is critical for regulating developmental processes. Typically, it is assumed that differences in protein abundance result primarily from differential protein expression in distinct cell types. However, it is also possible that asymmetric protein distribution throughout the embryo contributes to the formation of unique cellular proteomes. While this mechanism is widely accepted in fly embryos, its existence in vertebrates remains unclear. Previous proteomic analyses in the Wühr lab have been performed on whole embryos at various stages, combining measurements from all cells. Here, I investigated intracellular and intercellular proteome heterogeneity in developing Xenopus laevis embryos from the one-cell (NF 1) to eight-cell stage (NF 4). My colleague, Argit Marishta, measured intracellular protein distribution along the animal-vegetal (top-bottom) axis in unfertilized eggs. I developed a single-blastomere proteomics method to quantify relative protein abundance distributions up to the eight-cell stage. Interestingly, significant animal-vegetal asymmetry was observed at eight-cell stage, correlating with the intracellular protein distribution present in the unfertilized egg. This finding suggests that cellular differentiation during vertebrate development may partially result from asymmetric maternal protein distribution, rather than exclusively from cell-specific transcription, translation, or protein degradation.

One of the goals of synthetic biology is to isolate novel protein sequences that support essential biological functions. By using both rational and combinatorial design, the Hecht group has successfully discovered multiple synthetic sequences capable of sustaining life. Most of these functional de novo sequences form stable, well-folded protein structures, but the Rescuer 4 protein enables the survival of auxotrophic ∆metC E. coli cells in minimal media despite being disordered and insoluble. Preliminary experimental results suggested that the Rescuer 4 protein may rescue ∆metC through gene regulation, by co precipitating with MetJ, a transcriptional repressor protein. In order to investigate the rescue mechanism of Rescuer 4, multiple variants of the sequence were created through site-directed mutagenesis. These sequences were then transformed into ∆metC cells for life-or-death screening and growth analysis. Most variants exhibited reduced or abolished rescue function, verifying that the protein—rather than the RNA—is responsible for the rescue of ∆metC and confirming the interaction between MetJ and Rescuer 4 plays a key part in the mechanism. In addition, a variant sequence with enhanced rescue efficiency was discovered. Proteomics analysis of this strain points to an upregulation of chaperone proteins. These findings provide further insight into the mechanism of Rescuer 4 and demonstrate how de novo proteins can interact with natural biomolecules to support life-sustaining functions.

Modified nucleotides in tRNA are diverse and abundant, and aberrations are implicated in several human diseases. However, the physiological role of these modified nucleotides remains poorly understood. Previous studies have shown that S. cerevisiae strains containing multiple knockouts of tRNA modifying enzymes display temperature sensitive growth due to degradation of hypomodified tRNAs through the rapid tRNA decay (RTD) pathway. Based on high throughput screens indicating negative genetic interactions, five single knockout and five double knockout strains were generated. Single knockout strains pus1-Δ, trm1-Δ, tan1-Δ, trm8-Δ, and maf1-Δ and double knockout strains pus1-Δ dus3-Δ and trm1-Δ dus2-Δ did not exhibit temperature sensitive growth. In contrast, double knockout strains, trm8-Δ dus3-Δ, tan1-Δ dus2-Δ, and maf1-Δ dus2-Δ exhibited temperature sensitive growth. tRNA sequencing was used to determine tRNA abundances to assess whether tRNA stability was affected in the tan1-Δ dus2-Δ strain. While sequencing results were largely inconclusive, this work provides a characterization of several knockout strains and a basis for further investigation into the tan1-Δ dus2-Δ strain.

There is an increasing need for sp3 carbons in industrial and pharmaceutical sectors. A higher sp3 content can be achieved via hydrogenation reactions that require catalysts with high chemo- and enantioselectivities, typically based on precious metals. Previous work in the Chirik group has developed pre-catalysts of oxazoline imino(pyridine) (OIP) and pyridine (diimine) (PDI) ligand frameworks based on the earth-abundant metal molybdenum. This work aims to integrate features of both PDI and OIP catalysts into the PDI ligand framework for the asymmetric hydrogenation of naphthalene. A pathway for the ligand synthesis of C1-symmetric PDI ligand was developed. C1- and C2-symmetric PDI(Mo)COD pre-catalysts were synthesized, and their catalytic activity was evaluated for 2,6- substituted naphthalene. Results revealed that the C1- and C2-symmetric 4-tBu-((S)-Cy,MePDI)Mo(COD) were active hydrogenation catalysts for 2,6-dimethylnaphthalene, and selective for the partially reduced product. While the C2-symmetric catalyst induced no enantioselectivity, the C1-symmetric 4-tBu-((S)-Cy,MePDI)Mo(COD) achieved an enantiomeric excess of 13%. Preliminary work has been done on developing C1-symmetric PDI ligands substituted with chiral anilines to increase enantioselectivity.

Suzuki-Miyaura cross-coupling is one of the most important named chemical reactions used in industrial synthesis. Although palladium catalysts are most often used for these reactions, catalysts that instead use first-row metals are of interest in part because they may offer complementary reactivity to the shortcomings of palladium catalysts, in addition to being more sustainable, lower cost, and usually less toxic. This work highlights nickel catalysts bearing phenoxyimine (FI) and phenoxythiazoline (FTz) ligands for C(sp$2$)–C(sp$3$) cross-coupling reactions, specifically expanding the scope of the Chirik group’s previously reported nickel-catalyzed Suzuki-Miyaura cross-coupling method from boronic acids and boronic neopentyl glycol esters to boronic pinacol esters. These substrates are of particular interest given their ease of installment on highly functionalized scaffolds via Miyaura borylation. Reaction conditions are optimized for this specific type of reaction, the reactivity of FI and FTz ligands are compared, and the expanded nucleophile scope is briefly investigated. The results herein demonstrate the applications these nickel complexes have toward C(sp$2$)–C(sp$3$) Suzuki-Miyaura cross-coupling and establish a method that can be used to cross-couple boronic pinacol esters.

Traditional approaches for improving the reactivity and selectivity of homogenous molecular catalysts center on fine tuning the steric and electronic properties of the ancillary ligands. Beyond shaping the catalytic environment, various research groups have pursued the incorporation of non-covalent interactions within the secondary coordination sphere of transition metal catalysts to facilitate challenging chemical transformations. Conventional strategies for modifying the secondary coordination sphere have explored the use of hydrogen bond donors, Lewis acidic and basic functional groups, and cation sequestration sites within the ligand architecture. However, the design of latent ligand-centered radicals offers a distinct approach for the activation of persistent organometallic complexes. In particular, this research presents a strategy for activating kinetically inert nickel—trifluoromethyl (Ni—CF3) bonds, as CF3 functional groups are valuable structural motifs in medicinal chemistry and drug discovery processes. Notably, homolytic cleavage of both Ni—CF3 bonds within a fluorenone-appended bis(pyridine-2-ylmethyl)amine bis(trifluoromethyl)nickel(II) complex was observed under visible light irradiation, leading to controlled CF3 radical generation and Csp²—CF3 bond formation. Through a combination of electrochemical, experimental, and spectroscopic studies, the reactivity of the fluorenone-appended bis(trifluoromethyl)nickel(II) complex under visible light irradiation is demonstrated to be fundamentally distinct from that of the intermolecular system employing exogenous diaryl ketone under light irradiation and from reactions with stoichiometric oxidant under thermal heating. The ability to activate Ni—CF3 bonds formally at Ni(II) was found to be dictated by the photophysical properties of the diaryl ketone, the choice of solvent, and whether the organic chromophore is appended or not. Moreover, insights into the effect of coordination number on the reactivity of the fluorenone-appended system in the presence of coordinating solvent were further obtained through chemical oxidation of [(MeNNN)NiII(CF₃)₂] to a paramagnetic [(MeNNN)NiIII(CF3)2(MeCN)]BF4 complex.