Ben Bixenmann

Thesis Title: Structural analysis and crystallization of deoxyuridine 5’-triphosphate nucleotidohydrolase (dUTPase).

Thesis Abstract: The structural analysis and crystallization of deoxyuridine 5’-triphosphate nucleotidohydrolase (dUTPase) has been conducted in hopes of identifying structural factors that accompany optimal temperature differences between functioning enzymes. The salting out method along with the hanging drop vapor diffusion technique was used to crystallize the dUTPase enzyme isolated from the Paramecium bursaria Chlorella virus (PBCV-1). Successful crystallization conditions were found within a mother liquor solution of 8% polyethyleneglycol 4000 (PEG4k) and 10% iso-propanol and a dUTPase protein solution of 20 mg/mL. These crystals were then successfully diffracted to a 2.7 angstrom resolution, from which the final electron map has not yet been calculated. Moreover, a heavy atom derivative of the PBCV-1 crystal was created while being saturated in a 4 mM solution of HgI2 for 5 hours 30 minutes. In addition to the PBCV-1 crystallization, successive crystallizations of a similar Chlorella virus strand of the dUTPase enzyme, NY-2A, were conducted. This was done with a dUTPase protein solution of 5.4552 mg/mL and the same mother liquor solution of 8% PEG4k and 10% iso-propanol. The initial structural analysis experiments for the enzyme were conducted through primary protein sequence comparisons of numerous species. Results have identified a two amino acid insertion within the highly conserved motif 3 among all five bacterial species that is not present among any of the other species studied. The importance of this insertion has been shown to shift one of the binding residues within this substrate-binding motif. Therefore, signaling an evolutionarily conserved deviation among the dUTPase protein sequences of bacteria.

Future Plans: Ben is currently enrolled in medical school at the University of Nebraska Medical Center.

James Eicher

Thesis Title: A New Model for the Active Site of Hemocyanin.

Thesis Abstract: The binucleating ligand HB66 was synthesized for the purpose of creating an organic mimic for the oxygen transport protein hemocyanin that will reversibly bind with dioxygen. The ligand was then analyzed electrochemically via cyclic voltametry (CV) and Oster-Young square wave voltammetry (OSWV) to determine electrochemical reversibility of the ligand. The difference between the anodic and cathodic peaks for the first Cu2+ ion was approximately 76 mV, indicating that it is electrochemically reversible. For the second Cu2+ ion, the difference was 115 mV, indicating that it is quasi-reversible. These electrochemical results indicate that the complex shows good potential to also be chemically reversible.

Future Plans: James is currently enrolled in medical school at Ross University.

Cheryl Heitzman

Thesis Title: Neighboring Group Effects on C(sp3)SiMe2Ph Protiodesilylation.

Thesis Abstract: Protiodesilylation, the process of removing a silicon functionality and replacing it with a proton, occurs readily on many different types of silicon groups. However, the substrate scope and mechanism of the reaction has been unclear and even contradictory in some literature. Siloxanes and silanols have all been proposed as intermediates, with no definite reaction pathways determined, and only very harsh conditions proven to protiodesilylate with any regularity. The protiodesilylation of C(sp3)SiMe2Ph bonds has been shown to proceed via mild conditions (tetrabutylammonium fluoride in dimethylformamide) without the availability of an adjacent hydroxyl in an 83% yield. An intermediate silanol was isolated with a 56% yield, demonstrating that protiodesilylation need not proceed through a siloxane intermediate. Further investigation using deuterium labelled dimethylsulfoxide and deuterated water demonstrated that the stereochemistry of the carbon in question is preserved. Nuclear Overhauser effect experiments with nuclear magnetic resonance spectroscopy were also conducted, providing further evidence that the stereochemistry is preserved with protiodesilylation. As for the reaction mechanism, a process involving the intramolecular delivery of a proton seems to be the most likely direction for protiodesilyation, though a determination of which groups could actually be transferred to the tetrahydrofuran ring has not been conducted yet.

Future Plans: Cheryl is currrently enrolled in the chemical education Ph.D. program at Illinois Institute of Technology.

Violet Lee

Thesis Title: Separation and Quantification of Vasopressin and Oxytocin and Their Degradation Products by Use of a Nitrogen-Specific HPLC Detector.

Thesis Abstract: High-performance liquid chromatography (HPLC) coupled with chemiluminescent nitrogen detection (CLND) has been shown to be a qualitative and quantitative method in the separation and detection of underivatized free amino acids and degradation products of Arginine Vasopressin (AVP) and Oxytocin (OT). Optimized separation of polar amino acids used 1mM 18-Crown-6 Ether and 10mM NFPA diluted in 3% methanol (MeOH) run isocratically for 14 minutes, resulting in clear separation of all early eluting peaks. Thermally degraded OT samples were separated and detected by previously established separation schemes. The mass balance of these degraded samples was evaluated. The results indicated poor mass balance. There was a loss of 18.4% in degraded AVP over 77 hours and a loss of 19% in degraded OT over 96 hours. Dithiothritol (DTT) treatment of degraded and fresh OT samples did not have an affect on samples and retention times.

Future Plans: Violet is currently enrolled in the Ph.D. program at the University of Illinois and is specializing in Analytical chemistry.

Zachary Osborne

Thesis Title: Comparison of Ab Initio Software Packages: Gaussian 03 and Gaussian 98 and Characterization of Pentachlorocylopentadiene.

Thesis Abstract: This investigation consisted of two parts: an analysis of pentachlorocyclopentadiene, and a comparison of Gaussian 03 and Gaussian 98. The analysis of pentachlorocyclopentadiene included the calculation of energies and bond lengths, as well as the calculation of vibrational frequencies and their assignment. The second phase analyzed various molecules using both Gaussian 98 and Gaussian 03. The molecules analyzed included: H2O, CCl4, CHCl3, CH2Cl2, CH3Cl, C6H6, C6H6+, PF5, SF6, CH4, NH3, NF, and H2. In the second phase, three methods/basis set combinations B3LYP/6-31G(d), B3P86/6-311G(3d,p), and HF/6-31G(d) were used, while in the first phase B3P86/6-311G(3d,p), B3P86/6-311+G(3d,p), B3P86/6-311G++(3d,p) were used as well as the other methods RHF, and B3LYP were used for each basis set. The calculations of pentachlorocyclopentadiene at the B3P86/6-311G(3d,p), B3P86/6-311+G(3d,p), and B3P86/6-311++G(3d,p) level yielded theoretical energies of –67862.508, –67862.508, and –67862.416 eV respectively. These values supported the hypothesis about the characteristics of a chlorinated cyclopentadiene ring, by increasing the energy of the cyclopentadiene anion. The comparison phase using Gaussian 03 and Gaussian 98 calculated molecules with no differences, small differences, and major differences in all characteristics examined. The point group of the H2O optimization for G98 and G03 was found to be much different (C2v, and Cs, respectively). The bond lengths of CH4, CClH3, and CCl2H2 yielded 1.0837 Å, 1.7846 Å and 1.7680 Å; respectively at the RHF/6-31G(d) level all demonstrated differences on the order of 10-3 Å. The molecules NF, NH3, PF5 and SF6 demonstrated no difference between Gaussian 03 and Gaussian 98.

Future Plans: Zach is currently enrolled in medical school at Ross University.

Elizabeth Seutter

Thesis Title: Effects of Ethanol on Gallus gallus Embryonic Brains Using Nuclear Magnetic Resonance Spectroscopy and Gas Chromatography-Mass Spectrometry.

Thesis Abstract: The prevalent use of ethanol in alcoholic beverages has led to the study of ethanol metabolism, especially on fetal development due to the rise in Fetal Alcohol Syndrome. Through the use of Gallus gallus chick embryos and 13C labeled ethanol, the link between the fetal damage and oxidative and nonoxidative metabolism of ethanol was investigated. Using the Folch extraction technique followed by analysis with 13C nuclear magnetic resonance spectroscopy (NMR) and gas chromatography-mass spectroscopy (GC-MS), it was hoped that fatty acid ethyl esters could be isolated from the brain tissue of ethanol exposed embryos. Unfortunately, this did not give the desired results because no fatty acid ethyl esters were detected as a result of ethanol metabolism. Using the perchloric acid extraction, the lipid fraction was also analyzed through 13C NMR and GC-MS. This analysis indicated that the 13C labeled ethanol was indeed incorporated into the fatty acids. In order to determine the fatty acid composition of the extracts, the lipid fractions from both extraction techniques were methylated with good yields and, then, compared to standards.

Future Plans: Elizabeth is currently enrolled in the accelerated nursing program at St. Mary's College.