Synthesis, characterization, and transfer hydrogenation catalysis of mid-row transition metals and the investigations of regulatory mechanisms of the OAS-RNase Land PKR pathways for RNA sensing

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Authors
Keuk, Channita
Issue Date
2024-05
Type
Thesis
Keywords
University of the South , Chemistry Department , Sewanee Senior Honors Theses 2024 , Chemistry , catalysis , biochemistry , molecular biology
Abstract
A. The synthesis of a series of chlorine-substituted acetylacetonate cobalt(III) complexes was attempted by varying the stoichiometric ratios of the starting materials in solution. Following the synthesis, the complexes were purified using column chromatography and subsequently characterized using ultraviolet-visible, infrared, and nuclear magnetic spectroscopy and x-ray crystallography to analyze the effects of these chlorine substitutions on the Co(III) metal center. Based on the spectroscopic analysis of the complexes, these substitutions yielded discernible differences in the electronic properties of the Co(III) center. Characterization of these complexes can lead to its potential applications in catalysis and photochemical studies. B. Alcohols play an extremely important role in the synthesis of larger organic compounds such as perfumes, sweeteners, and pharmaceuticals. The reduction of ketones to alcohols via traditional metal-catalyzed hydrogenation has multiple drawbacks such as the use of pressurized hydrogen gas and requiring high temperatures. Organometallic catalysts can eliminate some of these by utilizing alternative reaction conditions that are more environmentally friendly and can occur at much lower temperatures. Ruthenium(II) metal complexes with 4-aminopyridine (ampy) and diphenylphosphinobutane (dppb) were synthesized with the replacement of the para-hydrogen on the pyridine ring with a chlorine (Cl) group and a methoxy (OMe) group to study the effects of electron withdrawing groups (EWG) and electron donating groups (EDG) on the electronic profiles of the ruthenium center. NMR spectroscopy, UV-Vis spectroscopy, cyclic voltammetry, and X-ray crystallography were used to characterize each compound. Studies have shown that the ampy based ruthenium(II) metal complexes are capable of isomerization; thus, the rates of trans-to-cis isomerization were. The transfer hydrogenation catalysis data shows that these ampy-ruthenium(II) systems are highly capable of transfer hydrogenation, with selectivity for ketone functional groups. C. N-Heterocyclic carbenes (NHC) are excellent ligands for the coordination and reactivity of cobalt complexes in the transfer hydrogenation catalysis of ketone substrates. The traditional synthesis of such ligands is a time-consuming process that hinders efficient synthesis of metal complexes. Thus, an alternative synthetic method via a microwave reactor for several N-substituted imidazole-based NHC pincer ligands was developed to reduce reaction time from three days to under two hours. Seven different compounds were synthesized and characterized using 1H and 13C nuclear magnetic resonance spectroscopy. Several compounds resulted in mixtures of unreacted starting material and the desired products, which were then cleanly isolated using column chromatography purification. D. Transfer hydrogenation offers an alternative to selectively target specific functional groups on a molecule while keeping the others intact. An abundance of previously published research focuses on expensive transition metal catalysts of ruthenium, platinum, and more. Given the reactivity of ruthenium(II) systems in transfer hydrogenation, the shift to utilizing cobalt(II)-based catalysts systems allows for a more cost-efficient method due to the natural abundance of these first-row transition metals. The synthesis of a series of imidazole-based N-heterocyclic carbene (NHC) cobalt(II) complexes are being explored in order to generate a diverse range of metal complexes with varying electron-donating and withdrawing substitutions, as well as sterically bulky R-groups, on the imidazole ligand. Catalytic studies are also being done to determine if these changes in electron density around the metal center can have an effect on the rates of catalysis. E. In the field of transition metal catalysis, specific analytical techniques must be employed in order to quantitatively assess the rates and percent yield of catalytic conversion of the substrates. For studies involving paramagnetic metal complex species, the common method of NMR spectroscopy proves to be challenging as it requires various additional steps for sample preparation. Thus, an alternatively strategy using the gas chromatography mass spectrometer (GC-MS) allows for quantitative assessment of the amount of product converted by the catalyst through the use of a calibration curve. A series of calibration curves using benzophenone and benzhydrol were generated in an attempt to accurately assess the percent yields of the products in Co(II) imidazole-NHC catalyzed transfer hydrogenation. Studies are currently still ongoing to find the optimal sample preparation techniques to generate an accurate calibration curve. F. The vaccinia virus (VACV) utilizes the hypophosphorylation of serine/arginine-rich (SR) proteins, which are essential to the host alternative RNA splicing machinery, to induce host shut-off. In response to viral infections, the Protein Kinase R (PKR) pathway is activated in order to prevent viral RNA translation. To explore whether defects in RNA splicing generates ligands for PKR activation, western blots were used to probe for PKR activation in A549 cells during SR inhibition. Upon treatment with SR inhibitors and infection with the vaccinia virus for 24 hours, A549 cells were collected and probed for PKR activity via western blot. Contrary to published literature, it was found that VACV infection did not suppress SR phosphorylation based on the western blot analysis, and chemical inhibition of SR proteins do not induce PKR activation. G. As dsRNAs are the hallmark of viral infections, RNA sensing becomes an important component of host innate immunity. The oligoadenylate synthetase (OAS)-RNase L system, a dsRNA-sensing pathway of interest, provides a mechanism of action against dsRNAs within the body. Given the potency of its activation, this pathway is not only highly regulated through cellular mechanisms but also heavily antagonized by viruses. To test whether regulation of the OAS/RNase L pathway occurs at the post translational level, RNase L wildtype and K684R mutant plasmids were generated to test the effect of acetylation of the enzyme and its overexpression on its activation using bioanalyzer assays. Based on the assays conducted, there appears to be no activation of RNase L due to overexpression of the mutant plasmids. However, due to experimental errors, the assays comparing the transfection of PCDEF3 empty vector, wildtype RNase L, and RNase L K684R mutant followed by polyIC and 2-5A treatments failed to show any discernable differences in RNA degradation. Further studies are needed in order to accurately assess the RNA integrity in the bioanalyzer assays studying the effects of hyperacetylation of RNase L on its activation.
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