Department of Chemistry & Biochemistry

Title: beta-Cyclodextrin Complexation of 6PPD and its Hydrolysis

Authors: Taylor Adams, Kaitlyn Scheldorf, Emily Wymer, Edward Collett, Jaylee Wolford, and Patrick M. Hare

Department: Chemistry & Biochemistry

Abstract: N–(1,3-dimethylbutyl)-N’-phenyl-p-phenylenediamine (6PPD), a common tire anti-degradant, has an ozonolysis product that is known to be toxic to certain fish species. However, 6PPD also hydrolyzes in water to ultimately form 4-hydroyxdiphenylamine (4HDPA). This project examines whether host-guest binding with beta-cyclodextrin (bCD) alters the hydrolysis pathway of 6PPD. Binding was analyzed using isothermal titration calorimetry, UV-Vis spectroscopy and liquid chromatography-mass spectrometry of 6PPD and 4HDPA in pH 7.4 buffer and bCD solutions. Hydrolysis of 6PPD in 15 mM bCD solutions was slower relative to buffer, while bCD had little effect on 4HDPA kinetics. Calorimetry results suggest that 4HDPA’s binding with bCD is weaker than 6PPD’s binding to bCD.

Title: Comparison of Cycloheptanone and Tropinone Derivatives from Claisen-Schmidt Condensations

Authors: Joseph Land, Guinn Harler, Lili Ma Ph.D

Department: Chemistry & Biochemistry

Abstract: Benzaldehyde and methoxy-substituted benzaldehydes were reacted with cycloheptanone and tropinone using NaOH-catalyzed Claisen–Schmidt condensations. Reactions were monitored by TLC, and products were isolated, purified by recrystallization, and characterized by IR and NMR. The effects of methoxy substitution on reaction outcomes and drug-like properties were compared.

Title: Determination of the effect on translation of 2’-O-methylation at positions 32 and 34 in eukaryotic tRNA

Authors: Danika Maki, Dipshika Rai, Madisyn Hayes, Holly M. Funk, Anh Bui, Linh T Le, and Michael P. Guy

Department: Chemistry & Biochemistry

Abstract: Post-transcriptional modification of tRNA is vital for protein translation. Mutations in the widely conserved eukaryotic methyltransferase TRM7 are associated with intellectual disability in humans and slow cell growth in yeast. Trm7 interacts with Trm732 and Trm734 to catalyze 2′-O-methylation at positions C32 and G34 of tRNAPhe. While loss of both modifications causes growth defects, loss of either modification alone results in no defects. Because the codons UUU and UUC both encode phenylalanine, it remains unclear whether Cm32 and Gm34 differentially influence their translation. We are using an RNA-ID reporter system to compare translation of UUU, UUC, UGG, and UUA codons to determine whether Trm7-dependent translational effects are specific to phenylalanine or extend to other Trm7-modified tRNAs, including tRNATrp and tRNALeu in trm7Δ,  trm732Δ, trm734Δ, and additional tRNA modification mutant strains. Understanding how conserved tRNA modifications regulate codon-specific translation may reveal mechanisms underlying TRM7-associated intellectual disability in humans.

Title: Determining the Presence of Toxic Substances in UV and Non-UV Tattoo Inks

Authors: Tessa Sangermano and Charlisa Daniels

Department: Chemistry & Biochemistry

Abstract: UV tattoo ink has increased in popularity over the past decade, but safety concerns continue to plague some brands on the market. This study investigated the possible presence of polyaromatic hydrocarbons (PAHs) in yellow, red, and white non-UV and UV inks. PAHs are fused aromatic rings typically formed from incomplete combustion of organic material. These have devastating effects on health due to their carcinogenic, mutagenic, and teratogenic nature. High Performance Liquid Chromatography (HPLC) was used to identify PAHs and compare UV and non-UV inks. Various HPLC methods were tested throughout the research to improve data analysis and allow for precise and accurate identifications.

Title: Determining the Role of Yeast tRNA Body Modifications using LEU2 Stop-Codon Readthrough

Authors: Alisha Detmer, Mubassil Chaudhry, Madisyn Hayes, and Michael P. Guy, Ph.D.

Department: Chemistry & Biochemistry

Abstract: Post-transcriptional modifications are critical for tRNA function and are generally conserved between yeast and humans. Modification defects cause human diseases such as intellectual disability through unclear mechanisms. Cellular defects caused by loss of modifications on individual tRNAs can be investigated in yeast using tRNAs that read through stop codons in the LEU2 gene. Elimination of the tRNA modification enzyme Dus2 prevented suppressor tRNA function, resulting in loss of growth on media lacking leucine. To determine whether restoration of growth was due to reversion of the engineered stop codons, several suppressor colonies were isolated and the LEU2 gene was sequenced. Sequencing results confirmed both stop codons remained intact in all colonies tested. Future work will determine whether the suppressor growth is caused by compensatory mutations in the suppressor tRNA or mutations in the degradation pathways that compensate for Dus2 modification.

Title: Developing Safe and Effective Antidotes for Low-Molecular-Weight Heparin Anticoagulants

Authors: Methmi Kasturiratna, and Kebede Gemene, Ph.D.

Department: Chemistry & Biochemistry

Abstract: Low molecular weight heparins (LMWHs) are widely used as safe anticoagulants, but the antidote protamine only partially neutralizes their activity. This project investigates an arginine rich low molecular weight peptide (LMWPs), FRRRFRRFVRRF-NH2, as a potential antidote for LMWH. Chronopotentiometric polyion selective sensors were developed to measure LMWHs and peptides and quantify their binding ratio. Three LMWHs, Dalteparin, Enoxaparin, and Tinzaparin were evaluated. The transition times of the peptide were monitored first, followed by LMWH addition, which produced measurable decreases in transition time consistent with peptide - LMWH complex formation. The binding ratio was calculated by dividing the slope of the calibration curves of LMWHs by that of LMWPs and were found to be 1.95, 1.90, and 1.80 for Dalteparin, Enoxaparin, and Tinzaparin respectively. These results show that arginine rich LMWPs are strong antidote candidates and demonstrate the utility of chronopotentiometric polyion selective sensors for evaluating anticoagulant - antidote binding affinity and ratio.

Title: Estrogen emission and photochemistry

Authors: Edward Collett, Jaylee Wolford, Kaitlyn Scheldorf, and Patrick M. Hare

Department: Chemistry & Biochemistry

Abstract: Steroidal estrogens are naturally occurring hormones that undergo photochemical reactions upon exposure to ultraviolet light, making it important to understand their excited-state behavior and environmental fate. Estrone and 17β-estradiol share a phenolic chromophore that absorbs at ~280 nm and fluoresces at ~304 nm; however, estrone exhibits a lower fluorescence quantum yield, a biexponential fluorescence lifetime, and an additional carbonyl emission at ~410 nm. This project investigated the photophysical properties of estrone, estradiol, and methoxyestrone in methanol, acetonitrile, tetrahydrofuran, and water using UV/Vis absorption, steady-state fluorescence, and fluorescence lifetime spectroscopy. Complementary photodegradation studies monitored changes in these compounds following controlled UV exposure over varying time intervals. Together, these measurements demonstrate solvent-dependent excited-state dynamics, energy transfer, and photochemical behavior of environmentally relevant steroidal estrogens.

Title: Green Chemistry Synthesis and Computer Modeling of Tropinone Derivatives

Authors: Austin Clifton, Daniela Coronado Pablo, Jessica Forbeck, Guinn Harler, Joseph Land, and Lili Ma

Department: Chemistry & Biochemistry

Abstract: This study focuses on the development of environmentally sustainable synthetic pathways for novel tropinone-based drug candidates and the evaluation of their binding affinity with aromatase through computational modeling. Green chemistry techniques, including solvent-free conditions and mechanochemistry, were employed to optimize reaction conditions and to improve the sustainability of the synthesis. All compounds were fully characterized using NMR spectroscopy and HRMS. By integrating green synthetic strategies with analytical characterization and computational prediction tools, this research provides new insights into tropinone-based drug candidates, and supports more sustainable practices in medicinal chemistry.

Title: Identifying Important Residues for Trm732 Function via Random Mutagenesis and Next Generation Sequencing

Authors: Johannes Smal, Rylee Schurger, Maira Faisal, Natalie N. Creech, Madisyn Hayes, Holly M. Funk, and Michael P. Guy

Department: Chemistry & Biochemistry

Abstract: Mutations in the tRNA methyltransferase gene FTSJ1 cause non-syndromic, X-linked intellectual disability.  FTSJ1 needs the auxiliary protein THADA to modify tRNA, and this gene has been linked to polycystic ovary syndrome, type II diabetes, and certain cancers. Our focus is on the homologous genes in yeast. Specifically, Trm7, and its auxiliary protein, Trm732. This protein complex performs 2’-O-methylation of tRNAPhe, which is critical for proper tRNA function and protein translation. Three key motifs have been identified in Trm732, but the protein’s full mechanism is not yet known. This project seeks to discover essential residues within Trm732. A library of mutated TRM732 plasmids was transformed into a knockout yeast strain, and this strain was then subjected to selective conditions to force utilization of the mutated plasmids. Mutations in critical regions correspond to slower growth and next generation sequencing of the plasmids before and after selection will identify critical Trm732 residues.

Title: Identifying Mutations That Restore Growth in Dus2-KO1 Yeast

Authors: Alisha Detmer, Mubassil Chaudhry, Madisyn Hayes, and Michael P. Guy, Ph.D

Department: Chemistry & Biochemistry

Abstract: Post-transcriptional modifications are critical for tRNA function and are generally conserved between yeast and humans. Modification defects cause human diseases such as intellectual disability through unclear mechanisms. Cellular defects caused by loss of modifications on individual tRNAs can be investigated in yeast using tRNAs that read through stop codons in the LEU2 gene. Elimination of the tRNA modification enzyme Dus2 prevented suppressor tRNA function, resulting in loss of growth on media lacking leucine. To determine whether restoration of growth was due to reversion of the engineered stop codons, several suppressor colonies were isolated and the LEU2 gene was sequenced. Sequencing results confirmed both stop codons remained intact in all colonies tested. Future work will determine whether the suppressor growth is caused by compensatory mutations in the suppressor tRNA, mutations in tRNA degradation pathways, or in other cellular pathways.

Title: Influence of Methoxy Groups on the Potential Bioactivity of Tropinone Derivatives from Claisen-Schmidt Condensations

Authors: Guinn Harler, Lili Ma

Department: Chemistry & Biochemistry

Abstract: This study investigates the Claisen-Schmidt condensation of tropinone and various methoxy-substituted benzaldehydes. Mono-, di-, and tri-substituted methoxy benzaldehydes were introduced to obtain insights on their influence upon the synthetic outcomes and bioactivity potentials. Improving synthetic and purification methods of these molecules by understanding the influence of different methoxy groups can lead to the development of new anticancer drugs and contribute to the field of cancer research.

Title: Insights into the Synthesis and Purification of Claisen-Schmidt Condensation Products Derived from Cycloheptanone and Tropinone Scaffolds

Authors: Jessica Forbeck and Lili Ma

Department: Chemistry & Biochemistry

Abstract: Tropinone is a molecule known for its application in pharmaceutical industry, and cycloheptanone serves as its structural model due to their close resemblance. The Claisen-Schmidt products derived from these two scaffolds offer a valuable starting point for comparing synthetic accessibility, purification efficiency, and crystallization behavior. Detailed procedures for obtaining pure crystals of these compounds are presented, providing practical insights to guide future synthetic and medicinal chemistry studies.

Title: Optimizing Crystallization Conditions in Chorismate Synthase from Neurospora Crassa

Authors: Herrison Sizemore, Isabella Ingram, Julia Hageman, Rachel Combs, and Catherine Shelton

Department: Chemistry & Biochemistry

Abstract: Chorismate synthase is the final enzyme in the shikimate pathway, catalyzing the conversion of 5-enolpyruvylshikimate 3-phosphate (EPSP) into chorismate. Although the enzyme has been characterized in other organisms, the crystal structure of chorismate synthase from N. crassa has not yet been experimentally determined. In this study, crystals were grown using hanging drop vapor diffusion plates under multiple conditions. After examining the crystallization plates, there were multiple occurrences of crystal formations. Moving forward, any determined crystals will be harvested and flash-frozen to prepare for further imaging.

Title: Photodegradation kinetics of 6PPD and 4HDPA

Authors: Jonah Steveson, Emily Wymer, Taylor Adams, and Patrick M. Hare

Department: Chemistry & Biochemistry

Abstract: 6PPD is a tire antidegradant that can be found in aquatic environments, where it undergoes transformation through oxidation, hydrolysis, or through exposure to UV light. Comparing the influence of different solvent properties on its UV photodegradation is important for predicting the behavior of these molecules in real-world environments. This research investigated the effect of solvent on the photodegradation of 6PPD and its hydrolysis product, 4HDPA. Solutions of each compound were prepared in multiple solvents at different initial concentrations as determined by UV-Vis spectroscopy. Samples were exposed to UV light for 30 s to over 20 min, and changes were monitored by UV-Vis spectroscopy and liquid chromatography–mass spectrometry. Photodegradation rates differed among solvents, with photodegradation in water being much slower for 4HDPA than in alcohols, demonstrating that solvent strongly influences photodegradation kinetics.

Title: Potentiometric and Chronopotentiometric Ion-selective Electrodes for Simple Cations and Anions.

Authors: Ava Reed, Dr. Kebede Gemene Ph.D

Department: Chemistry & Biochemistry

Abstract: We demonstrate potentiometric and chronopotentiometric measurements of simple cations and anions using ionophore-free ion-selective electrodes (ISEs). Unlike ionophore-based ISEs, these membranes rely primarily on intrinsic ion-exchange and partitioning properties, resulting in selectivity that generally follows the Hofmeister series, which reflects differences in ion lipophilicity and membrane affinity:

• For anions, ClO₄⁻ > SCN⁻ > I⁻ > NO₃⁻ > Br⁻ > Cl⁻ > F⁻,

• For cations, NH₄⁺ > Cs⁺ > K⁺ > Na⁺ > Li⁺ > Ca²⁺ > Mg²⁺.

This behavior can produce membrane-history effects. That is, exposure of the membrane to a more lipophilic ion can hinder subsequent exchange with a less lipophilic ion, leading to hysteresis effect and apparent interference. We show that chronopotentiometry can overcome this limitation by electrochemically regenerating the membrane during a recovery step. As a result, reproducible responses to subsequently measured ions can be obtained even after exposure to more strongly lipophilic ions.

Title: Quantifying the Role of tRNA Body Modifications in Yeast Utilizing an RNA-ID Reporter System

Authors: Drew Scott, Mubassil Chaudhry, Ryan Peterson, Martin Ding, Alisha Detmer, Madisyn Hayes, and Michael P. Guy

Department: Chemistry & Biochemistry

Abstract: Post-transcriptional tRNA modifications are critical for protein translation. In humans, the absence of specific modifications can result in a variety of diseases. Many of the modifications are conserved between yeast and humans; the effect of these modifications on translation can be measured utilizing a reporter system in yeast cells by quantifying GFP expression. A stop codon was introduced into the gene encoding GFP in yeast cells, which normally terminates translation. Since many modification enzymes act on multiple tRNAs, several suppressor tRNAs were developed to identify specific tRNAs correlated with translation efficiency. These tRNAs have engineered anticodon loops that read through the stop codon, allowing translation of GFP in yeast. If a modification is important for the function of a given tRNA, GFP expression will be reduced in cells without the modification. Investigation of important tRNAs and modifications in yeast can provide insight into which defective tRNAs drive disease in humans.

Title: Site-Directed Mutagenesis to Probe Cofactor Binding of Chorismate Synthase

Authors: Julia Hageman, Rachel Combs, Herrison Sizemore, Isabella Ingram, and Catherine Shelton

Department: Chemistry & Biochemistry

Abstract: Chorismate synthase is a bifunctional enzyme from the organism Neurospora crassa that catalyzes the final step of the shikimate pathway. This reaction converts 5-enoylpyruvylshikimate-3-phosphate (EPSP) to chorismate. Chorismate is a key starting material for aromatic amino acid biosynthesis in plants, fungi, and bacteria. In this homologue Arg129 is hypothesized to contribute to the binding of the cofactors FMN and NADPH. To test this, site-directed mutagenesis was used to create three different mutations, R129K, R129A, and R129E. Sequencing confirmed the presence of the R129K and R129A mutations. R129K was then further purified.

Title: Synthesis and Purification of Lumiestrone

Authors: Brendan McPheron

Department: Chemistry & Biochemistry

Abstract: The photophysical and photochemical properties of estrogens are of interest due to their relatively unknown nature and the biological activity of 13α-steroids. 13α-estrone, or lumiestrone, is not readily available or possible to purify with UV photolysis and must be synthesized and purified chemically. Syntheses in the past have resulted in an impurity at m/z 361 which may interfere with the photochemistry of lumiestrone. Lumiestrone and lumiestrone 3-methyl ether were synthesized from estrone and estrone 3-methyl ether, respectively. Purification techniques including flash column chromatography and acid-base extraction were employed to isolate this impurity and to obtain clean lumiestrone fractions. Acid-base extractions failed to isolate the impurity at m/z 361 but flash column chromatography resulted in fractions free of the m/z 361 impurity but still containing the starting material. No chemically pure lumiestrone was obtained but progress was made in the purification of lumiestrone through the removal of the impurity at m/z 361.

Title: The Effect of Body Modifications on S. pombe Protein Translation

Authors: Julia Verhoff, Lexi Giordano, Martin Ding, Alisha Detmer, Madisyn Hayes, and Michael P. Guy

Department: Chemistry & Biochemistry

Abstract: Post-transcriptional modifications must occur for tRNA to translate proteins properly. Previous research has shown that Saccharomyces cerevisiae lacking tRNATyr(UUA) body modifications (D20, m2,2G26, or m5C48) experience a decrease in protein translation. The effect of the missing tRNA body modifications in Schizosaccharomyces pombe will be quantified using a translation reporter system that uses flow cytometry. Since S. cerevisiae and S. pombe genetically diverged 600 million years ago, similarities in tRNA modifications could indicate a conserved role in other eukaryotes like humans. This is important because it will allow the identity of defective tRNAs responsible for causing human diseases to be determined. Currently, we are setting up the reporter system by expressing green fluorescent protein (GFP) and red fluorescent protein (RFP) into S. pombe cells. Once expression is verified, we will integrate these reporter genes into the genome and test the effect of tRNA modifications on protein expression.