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Publication Synthesis of Nanoparticles to Modulate Astrocyte Lysosomal Activity(2023-08-04) Tianchen WangThe accumulation of partially degraded waste material in astrocytes is increasingly recognized as contributing to age-dependent neurodegenerations, and increased pH of the lysosomal lumen is a likely cause. Acidic nanoparticles are predicted to lower the lysosomal pH and reduce this accumulation; their ability to be internalized to lysosomes and non-toxic to astrocytes makes this a potentially long-term solution. In order to track nanoparticle delivery to lysosomes and optimize their incubation conditions, fluorescent-labeled nanoparticles are necessary. Here, we describe an improved method for fluorescent labeling of acid nanoparticles to determine their delivery to lysosomes. We synthesized nanoparticles from polymers in which 502 H PLGA was covalently bonded to the fluorescent dye Cyanine3. These stable fluorescent nanoparticles enabled detection of colocalization between lysosomes and nanoparticles in astrocytes, with retention up to two weeks. The time course and concentration of nanoparticles had a significant effect on their delivery efficiency. We also examine the ability of acid nanoparticles to recover lysosomal pH or functions. In summary we developed an improved method to label acid nanoparticles which gave enhance detection of delivery to lysosomes.Publication Highly conserved residues of coronavirus nsp1 skew translational equilibrium toward viral protein production(2023-08-04) Nicholas A. ParentiCoronaviruses cause diseases in many species of animal including humans and have been a major cause of lethal human disease outbreaks for the past twenty years. There have been three major outbreaks of human coronaviruses in 2002, 2012, and 2019 by SARS-CoV, MERS-CoV, and SARS-CoV-2 respectively that cause severe disease and have a high rate of mortality. Beyond the severe pathogenesis of these diseases, their rapid spread and unpredictable spillover into the human population is equally worrisome. This underscores the need for therapeutics to treat and prevent current and future coronavirus outbreaks. To this end, it is paramount to understand the basic biochemistry of coronavirus replication and innate immune evasion. The coronavirus non- structural protein 1 (nsp1) is of particular interest to this work due to its ability to inhibit host protein synthesis. There are two important functional domains of nsp1: the ribosome-binding domain (KH/KY motif) and the genome-recognition domain (LLRK motif). The KH/KY motif confers binding to the 40S subunit of the ribosome and inhibits host translation. It has been hypothesized that a conserved, structured region of the 5’UTR interacts with the LLRK motif, releasing nsp1 from the 40S subunit, and thereby allows preferential translation of viral genome and mRNAs. Conversely, various cellular stressors active the integrated stress response (ISR), which phosphorylates the alpha subunit of eukaryotic initiation factor 2 (eIF2a) to reduce translation during times of stress. We hypothesized that the viruses evade the ISR by competitively inhibiting host protein synthesis, impeding expression of key stress and innate immune genes. A reverse genetic approach was employed to generate viruses with point mutations in either of these motifs to investigate their influence on the ISR.Publication Asymmetric Oxidative Coupling of 2-Hydroxycarbazoles(2016-12-21) Sung, PaulThe biscarbazole skeleton is present in compounds produced by the plants of the Rutaceae family with over 20 naturally occurring molecules. Bishydroxycarbazoles, an important class of these alkaloids, are garnering increased interest for their potential antimalarial, cytotoxic, anti-HIV, and antimicrobial activity. Previous methods to form biscarbazoles are limited to racemic couplings to form the biaryl linkage. The goal of the project is to use a chiral vanadium catalyst and oxygen to achieve high enantioselectivity in a more efficient oxidative coupling. For a range of five substrates, enantioselectivies of 72-79% ee and yields up to 87% were obtained. This technique provides a potential route to various natural products, such as bismurrayfoline E. With the ability to synthesize these natural products, avenues are opened for further study of their biological activity.Publication Peptide Synthesis and Modification as a Versatile Strategy for Probes Construction(2017-08-11) Wang, JieliangPeptide synthesis and modification is a versatile chemical biology strategy to construct probes and sensors of a variety of types of biological activity, including protein/protein interactions, protein localization, and proteolysis. In my thesis work, I have made probes for three distinct biological applications. To do so, I have used a combination of solid phase peptide synthesis (SPPS), native chemical ligation (NCL), protein expression, and S-alkylation to construct probes with desired functional groups, while minimizing the perturbation to the native structure. In the first project, I constructed photo-crosslinking probes to study the difference in protein-protein interactions of N-terminal acetylated (N-ac) histone H4 peptide versus non-acetylated histone H4 peptide. One protein was identified by Western blot with binding preference to N-Ace histone H4 peptide. In the second separate project, I constructed probes to study the toxicity mechanism of proline/arginine dipeptide PRx from amyotrophic lateral sclerosis (ALS) associated gene C9ORF72. The preliminary result suggested the PRx peptide toxicity on proteasome depends on the length of the (PR)x peptide. In the third project, I synthesized fluorescence sensors to study the positional effects of thioamide on the proteolysis process of chymotrypsin. From hydrolysis studies, my coworkers and I determined that thioamide incorporation at the P1 or P2 positions can greatly inhibit chymotrypsin proteolysis.Publication Molecular Mechanism of Copper Regulation of Mitogen-Activated Protein Kinase (MEK1)(2017-05-19) Schibrowsky, Natalie AMutated constitutively activated forms of the BRAF kinase have been associated with various cancers, such as melanoma and leukemia. One such common BRAF mutant, BRAFV600E is found in about 50% of melanomas. BRAF is part of the MAPK signaling cascade that contains the downstream target MEK1/2, which phosphorylates ERK1/2 and subsequently increases cell proliferation. While the majority of the therapeutic inhibitors of the MAPK pathway have been designed to target BRAF, some have also targeted MEK1/2 as well as other kinases within the pathway such as ERK1/2. Recently, MEK1 has been observed to interact with copper (Cu). This element and copper transporter 1 (CTR1) were found to be important for kinase activity and when absent, decreased the ability of BRAFV600E to signal and mediate tumorigenesis. In this study, work was done to further characterize the MEK1-Cu interaction through crystallography, and carried out associated biochemical and enzymatic studies in order to characterize the functional consequence of the interaction and its effects on downstream ERK phosphorylation. A crystal structure of a MEK1-Cu complex was obtained revealing a Cu binding site to a regulatory site of the kinase that would be predicted to inhibit kinase activity. This structural observation is supported by functional studies with bacterially produced recombinant homogeneously purified MEK1 demonstrating that Cu inhibits MEK1 kinase activity in vitro. Interestingly, crudely purified MEK1 did show Cu-mediated MEK1 activation suggesting another mode of Cu-mediated MEK1 activation that is yet to be characterized. These results suggest that MEK1 is a metal-sensitive enzyme and that targeting these metals may be exploited to inhibit MAPK signaling for therapy.Publication The Study of Sulfenate and Sterically Hindered Chalcogenide Anions as Organocatalysts for the Construction of C-C Bonds(2017-08-01) Annunziato, Christopher MChalcogenides are highly redox active and biologically potent nucleophiles with significant unexplored catalytic potential. In particular, sulfenate anions potential as organocatalyst has largely been overlooked in the literature due to their esoteric status. A unique sulfenate anion catalyzed Baylis Hillman (BH) reaction forming sp2-sp3 C-C bonds is in development. In this two-part study, part one focuses on screening conditions suitable for both cleavage of a precatalyst and subsequent BH reaction. Whereas part two focuses on the proclivity of chalcogenide oxyanions to disproportionate. Sterically hindered analogs were investigated, to prevent catalyst decomposition. The sulfenate anion catalyzed (20 mol %) BH reaction was conducted in the presence of α,β-unsaturated nitriles, ketones and esters to generate allylic alcohols in yields up to 37%. Precatalyst cleavage studies were an integral part to the realization of smooth sulfenate anion generation. Efforts to optimize the BH reaction conditions are ongoing.Publication Synthesis of Novel Tröger’s Base-Derived Helical Scaffolds(2017-05-01) Goel, RahulTröger’s base (TB) is a chiral V-shaped molecule in which the aromatic rings are nearly perpendicular. The overarching goal of this project is to utilize the unique chirality and inherent shape of the Tröger’s base monomer to design, synthesize and study dimeric, tetrameric and octameric TB oligomers, which will form helical structures. We describe here the methodology for the synthesis of novel Tröger’s base diester monomer 13, which is highly soluble in most organic solvents compared to TB systems with methylene bridges. Chiral HPLC resolution of TB monomer 18, using a semiprep chiral AD-H column, gave access to pure enantiomers of the TB monomer. The (-)-enantiomer of 18 was used to synthesize the novel syn diester TB dimer 20, via double Buchwald-Hartwig coupling based phenazine formation. Energy minimization modeling of the syn dimer 20 using Web MO shows a potential binding cleft, which can ultimately be applied for the synthesis of desired tetrameric and octameric scaffolds. The chiral HPLC resolution of TB monomer 18 is expensive, time-consuming and has low scalability. This problem was solved by the synthesis of a menthone-based chiral auxiliary 27, which allows easy access to the enantiopure monomers of TB. The chirality of 27 was utilized to form the diastereomers of menthone TB 33, which were readily separable by column chromatography. These diastereomers were then hydrolyzed to give pure enantiomers of diol TB monomer 34.Publication Janus Dendritic Ligands for Nanoparticle Assemblies(2016-12-22) Elbert, Katherine; Murray, Christopher BThis project was conducted in the laboratory of Professor Christopher B. Murray at the University of Pennsylvania. The project described herein includes the synthesis of a Janus dendrimer as well as complimentary dendrimers of hydrophobic and hydrophilic nature to study the self-assembly and organizational properties of these molecules on gold surfaces. A complete synthesis and characterization of these molecules is described, as well as grafting the molecules onto both gold nanoparticle and thin film surfaces. How the different dendritic molecules guide self-assembly of the nanoparticles and how the Janus molecule assembles itself on a gold surface was studied. To characterize these systems, TEM and solid state UV-vis were employed, and general trends are described herein.Publication Employing Confidence Intervals in Electron Microscopy Digital Image Analysis to Promote Better Analytical Practices(2021-05-01) Arndt, Diana sAs the range of applications for nanoparticle systems continues to expand, the importance of providing reliable, informative, quantitative characterization for nanomaterial categorization and quality control continues to increase. Transmission Electron Microscopy (TEM) is a standard characterization technique that provides nanoscale image representations of a thin nanomaterial sample and has the potential to provide quantitative information with substantial digital image analysis. Modern semi-automatic TEM image analysis processes, such as the popular software ImageJ, aim to improve on outdated manual processes by incorporating user input with automated algorithms, decreasing the potential for human error and time expense. These processes segregate particles from the background in grayscale TEM images based on pixel intensity or particle shape, then calculate attributes such as size parameters from the derived shapes. However, in attempting to create processes that require little effort and time for the user, current image analysis procedures often undervalue the user’s knowledge of the system. Because the automated algorithms are expected to incorporate information about the system more suited to human determination, particles are often misidentified, resulting in inaccurate calculations. Furthermore, the automated algorithms are hidden from the user, making discussion of data manipulation biases arduous. To address these limitations, a novel TEM image analysis process was developed by integrating a traditional data analytics perspective. To provide feedback on the reliability of quantified size parameters, the presented process introduces confidence intervals based on image contrast and noise. These percentages represent the clarity of the image and variability between particles, allowing the user to choose what areas of the image they want to include in analysis. Encompassed in an easy-to-use MATLAB® App dashboard, the process allows the user to customize a more effective TEM image analysis process based on confidence interval feedback. In this project, a GdF3:Yb/Er rhombus nanoplatelet system and two Fe3O4 spherical systems were analyzed with the MATLAB® App to quantify size parameters comparable to standard ImageJ analysis. Additionally, the application’s ability to reveal image properties using confidence intervals is showcased by probing the effects of pre-processing regimes, magnification variation, and shape matching on size parameter reliability.Publication Effect of the Anchoring Groups on the Photostability of Ruthenium(II) Polypyridyl Sensitizers(2021-01-01) Gao, YunhanRuthenium(II) polypyridyl derivatives are widely used as sensitizer molecules in water-splitting dye-sensitized photoelectrochemical cells (WS-DSPECs) to harvest solar energy and generate hydrogen and oxygen gas from water. The desorption of sensitizer molecules from the TiO2 surface during long-term illumination is a significant problem for the phoatostability of these cells. Another factor that limits the efficiency of the cells is the low injection yield from the dye excited state to the electrode at neutral pH. To address these two problems, the effects of different anchoring groups (carboxylate or phosphonate) at the 4,4’-positions of bipyridine were systematically studied. In this report, three ruthenium(II) polypyridyl sensitizers were synthesized and characterized. Results from stability measurements showed that the phosphonate anchoring groups can enhance the photostability of ruthenium(II) sensitizers over the pH range from 4.9 to 6.9 compared with carboxylate substitutions.