ACS Puget Sound Section: Undergraduate Research Symposium

A FLUORESCENT METHOD FOR DETECTION OF TELOMERASE ACTIVITY. Jennifer Tolzmann, Department of Chemistry, Pacific Lutheran University, Tacoma, WA, and Wade K. Aldous, Nathaniel R. Grabil, James R. Wright, Corey Keene, and Rodger K. Martin, Madigan Army Hospital, Tacoma, WA

Recent research in the area of telomerase has indicated a possible link between telomerase activity and cancer cell proliferation. If telomerase activity can be definitely associated with cancerous tumors, telomerase activity could be used as a diagnostic tool in cancers. In addition, inhibition of telomerase could be a safe and effective cancer therapy. A method for detecting telomerase activity, the telomere repeat amplification protocol (TRAP) was recently developed. The TRAP method identifies telomeric repeats by detection of incorporated radionucleotides using autoradiography. The TRAP assay is currently the method of choice to detect telomerase activity in protein extracts. Several drawbacks to this method have been recognized including the time required to complete the assay, the resolution of the results, and the hazards of using radioactive material. A new flourescent method of detecting telomerase was developed to alleviate these problems. This modification to the TRAP method identifies telomeric repeats by the incorporation of flourescein- labeled primers during amplification and detection with an automated DNA sequencer. This talk will discuss the possible role of telomerase in cancer tumor growth and the optimization of the TRAP assay. Figures will include telomerase assay results from various cancer tumors as well as results of those assays used in the TRAP optimization.

MATHEMATICAL MODELS OF THE BELOUSOV-ZHABOTINSKII REACTION UNDER REACTION DIFFUSION CONDITIONS. Alan Burningham and Steven Neshyba, University of Puget Sound Chemistry Department, Tacoma, WA

Various mathematical models of reaction-diffusion systems are applied to the Belousov-Zhabotinskii reaction. The objectives are to simulate oscillating chemical reactions in a diffusive medium and to compare these simulations to linear models of the system and experimental observations. This comparison will help us to predict flow reactor parameters necessary for the experimental generation of stationary Turing patterns.

CONSTRUCTION AND USE OF A LASER CLOUD POINT INSTRUMENT. J. Chris Bock and Dean A. Waldow, Department of Chemistry, Pacific Lutheran University, Tacoma, WA 98447

A lot of published information exists about the thermodynamics involved in binary polymer blends, however little information can be found on tertiary blends. To determine phase diagrams for binary and tertiary polymer blends, a laser cloud point instrument was constructed. The instrument makes use of the temperature dependance for polymer mixtures on phase diagrams. By detecting the amount of scattered laser light from a He-Ne laser versus temperature over time a phase diagram for a polystyrene/polybutadiene blend has been completed. This talk will include information on how the instrument was constructed and show current data that has been taken to date.

THIOPHENE HYDRODESULFURIZATION OVER ALUMINA-SUPPORTED MOLYBDENUM CARBIDE AND NITRIDE CATALYSTS: PREPARATION, CATALYTIC EVALUATION, AND NATURE OF THE ACTIVE SURFACE Samantha Glazier and Mark E. Bussell, Department of Chemistry, MS-9150, Western Washington University, Bellingham, WA 98225

Early transition metal carbides and nitrides have shown promise as low cost alternatives to noble metal catalysts for a number of important catalytic processes. This research is directed toward determining the potential of alumina-supported Mo2C and Mo2N catalysts for use in the hydrodesulfurization (HDS) process. Catalysts have been prepared by carburization (20% CH4/H2) and nitridation (NH3) of MoO3/Al2O3 at 950 K. Thiophene (C4H4S) HDS activities have been measured in an atmospheric pressure flow reactor equipped with a gas chromatograph for on-line analysis of products. The following trend of HDS activities was observed: sulfided Mo < Mo2N < Mo2C. Catalyst characterization is accomplished in ultrahigh vacuum using infrared (IR) spectroscopy. After treatments in a mixture of 5.0 Torr C4H4S and 750 Torr H2 at temperatures in the range 350 - 750 K, IR studies of carbon monoxide adsorbed on the catalysts were carried out. The results suggest "sulfide-like" sites form on the surface of the catalysts following C4H4S/H2 treatments.

THIOPHENE HYDRODESULFURIZATION OVER ALUMINA-SUPPORTED MOLYBDENUM CARBIDE AND NITRIDE CATALYSTS: QUANTITATION OF ACTIVE SITES AND UNDERSTANDING TRENDS IN ACTIVITY John W. Logan and Mark E. Bussell, Department of Chemistry, MS-9150, Western Washington University, Bellingham, WA 98225

The purpose of this study has been to correlate the thiophene hydrodesulfurization (HDS) activities of alumina-supported Mo2C, Mo2N, and sulfided Mo catalysts with their CO and O2 chemisorption capacities. Using a pulse chemisorption instrument, CO and O2 chemisorption capacities were measured for freshly prepared and sulfided catalysts. HDS catalytic activities correlate most closely with the chemisorption capacities of the sulfided catalysts, providing additional evidence that the surface of the catalysts become sulfided under HDS reaction conditions. X-ray diffraction patterns of carbide and nitride catalysts before and after use in the flow reactor indicate the bulk structure of the catalysts does not change during HDS catalysis. A model is proposed to describe the structure of the carbide and nitride catalysts under HDS reaction conditions in which the carbide or nitride particles act as secondary supports for a thin layer of highly dispersed sulfided Mo.

13C SPIN-LATTICE RELAXATION MEASUREMENTS OF POLYMER BLENDS IN SOLUTION. Shayne Williams, Department of Chemistry, Pacific Lutheran University, Tacoma, WA 98447

Using variable temperature 13C spin-lattice relaxation (T1) measurements, the temperature dependent correlation time (tc) for the C-H reorientation in the polybutadiene polymer chain has been determined for polybutadiene and butadiene-styrene blended polymer in solution. With tc being a measurement of the local segmental motion of the polymers, the temperature dependence of molecular motion was analyzed for both the blend and homopolymer Data was acquired in the extreme narrowing regime of the relaxation pathway, and non-Kramers behavior for polymer-solvent coupling effects were taken into account using a power law viscosity dependence. Particular focus was on the change in tc as a function of the proximity to the critical point in the polymer blend in comparison to that of the homopolymer solution.

REACTION OF ORGANOCOBALOXIMES WITH TRIBROMOACETALDEHYDE: PREPARATION OF UNCONJUGATED 2,2-DIBROMOALDEHYDES. B. Colclazier and B. Smits, Department of Chemistry, Western Washington University, Bellingham, Washington 98225

2-Methyl-2-propenylbis(dimethylglyoximato)pyridinecobalt(III) was pre- pared by an Sn2 reaction of a cobalt(I) species with 3-bromo-2-methyl -1-propene. Cobaloximes resemble the natural Vitamin B12 in that they undergo free radical reactions with radical precursors. The proton NMR spectrum at 260 K gives sharp lines for the vinyl protons. At 298 K, the vinyl protons are not clearly seen. It is assumed that cobaloximes dissociate into radicals. This process is reversible. 2-Methyl-2-prop- enyl cobaloxime reacts with tribromoacetaldehyde at room temperature yielding 2,2-dibromo-4-methyl-4-pentenal in 91% yield. Infrared analysis indicates an unconjugated aldehyde group. The compound shows two halogen atoms in the mass spectrum. Proton and carbon NMR spectra are consistent with the assigned structure. Reduction with titanium(III) chloride in acetonitrile, buffered with ammonium acetate yields 4-methyl-4-pentenal. There is evidence that some 2-bromo-4-methyl-4-pentenal was formed as an intermediate. We have developed an unusual method of preparing unconj- ugated aldehydes. The research was sponsored by the Murdock Foundation.

SOLID PHASE PROTEIN SYNTHESIS OF SURFACTIN. Duc Vo, Department of Chemistry, University of Puget Sound, Tacoma, WA 98416

The purpose of this research is to determined the steps for the synthesis of surfactin using solid phase protein synthesis methods. Previously surfactin has been isolated from bacterial cultures of a marine Bacillus. Surfactin has known surface active properties and is a cyclic depsipeptide consisting of L-aspartic acid, L-glutamic acid, L-valine, L-leucine, D-leucine(1:1:1:2:2) and a ß-hydroxy fatty acid. One aspect of the project involved determining conditions to improve D-leucine couplings using Fmoc peptide synthesis methods. The second aspect of the project involves bonding a b-hydroxybutyric acid with Fmoc-L-leu to form an ester bonded intermediate. In the future the entire b-hydroxybutarate analogue of surfactin can be synthesized using the improved coupling conditions and the Fmoc protected ester linked L-leu intermediate.

SOLID PHASE SYNTHESIS OF BETA-ALANINE SURFACTIN ANALOGUE. Jill Hanck, Department of Chemistry, University of Puget Sound, Tacoma, WA 98416.

Surfactin, the strongest surfactant known, is a compound that has been isolated from cultures of B. substilis . Surfactin is a cyclic compound that contains seven amino acids(asp:glu:L-leu:D-Leu:val in 1:1:2:2:1) and a long chain alpha-hydroxy fatty acid. This talk will deal with the synthesis of a surfactin analogue using F-moc solid phase protein synthesis(SPPS) on a Wang resin. The surfactin analogue contains the seven amino acids and a alpha-amino acid in place of the alpha-hydroxy fatty acid. This alteration simplifies the SPPS because it does not require an ester bond incorporation during the synthesis. Aspartic acid, with an allyl blocking group at the a-carboxylic acid, was bonded to the resin via its side chain carboxylic acid. A straight chain peptide was synthesized by coupling successive amino acids to the free amino group of the resin bound intermediates. Finally, the allyl and F-moc protecting groups were removed from the octapeptide, the peptide was cyclized and then hydrolyzed from the resin. Analysis of the product(s) is being done at this time.

PROBE DEVELOPMENT FOR THE DETECTION OF PACIFIC OYSTER NOCARDIA. M.C. Strehlow, Department of Chemistry, Pacific Lutheran University, Tacoma, WA 98444

Pacific Oyster Nocardiosis is a bacterial oyster disease. The epidimiological parameters of this disease are unknown. Often when infective lesions are detected on animals, it is too late to save the harvest. We have designed PCR primers/oligonucleotide probe for detecting Nocardia, the bacterial pathogen. The design is based on the 16S (small subunit RNA) sequence information. Sets of primer/probe oligonucleotides were constructed in order to detect Nocardia bacteria by PCR amplification.

AUTOXIDATIVE REARRANGEMENTS OF VALENCE-TAUTOMERIC EPOXYCYCLOPENTENONES AND SYNTHESIS OF A MODEL COMPOUND. B. C. Bales, Department of Chemistry, Western Washington University, Bellingham, Washington 98225

The goal of this project is to discover the mechanism and the electronic and steric factors that influence the autoxidative rearrangements in a series of valence- tautomeric epoxycyclopentenones. Key in this series is 4,5-epoxy-2,4,5- triphenyl-2-cyclopentenone, the synthesis of which will be discussed.

AUTOXIDATIVE REARRANGEMENTS OF VALENCE-TAUTOMERIC EPOXYCYCLOPENTENONES. SYNTHESIS, STRUCTURE, 1H AND 13C NMR OF SOME MODEL COMPOUNDS. A. Powers, Department of Chemistry, Western Washington University, Belligham, Washington 98225

The focus of this work is to clarify the structures and 1H and 13C NMR spectra of a series of valence-tautomeric epoxycyclopentenones. The goal is the discovery of the mechanism and the electronic and steric factors that influence the valence tautomerization and subsequent autoxidative rearrangements in this series of compounds. The discussion will center on 4,5-epoxy-3,4,5-triphenyl-2-cyclopentenone as an example.

THE DETERMINATION OF THE THERMODYNAMIC PARAMETERS ASSOCIATED WITH THE REVERSIBLE ENOLIZATION OF PYRUVATE. Aaron Lukas, (Dr. John E. Meany), Department of Chemistry, Seattle University, Seattle, WA 98122

Pyruvate plays a significant role in many biological reactions. It is the substrate for numerous enzymatic processes, and plays a major role in the glycolytic pathway. In particular, the ketonization of enol pyruvate contributes strongly to the exergonicity of glycolysis. It is the goal of the present investigation to quantitatively determine the position of equilibrium between keto and enol pyruvate, Kenol, in aqueous solution. Uptake of triiodide ion by enol pyruvate was monitored at 353nm using a Shimadzu UV 2101 spectrophotometer. These studies allow the determination of Kenol=3.06*10-5 at 25°C and ionic strength, m=0.35 M. This result differs greatly from Kenol values determined in other laboratories. The present work suggests that the larger values of Kenol determined previously in earlier studies was due to the existence of a triiodide scavenging impurity formed via the condensation of pyruvate. In the present investigation pyruvate is initially recrystallized and then further purified by a titrimetric method to remove any triiodide scavenging impurity which may have reformed in solution. The thermodynamic measurements carried out as a function of absolute temperature allow the evaluation of the parameters DG°, DH°, and DS°.

3-AMINO-5-HYDROXYBENZOIC ACID (AHBA) SYNTHASE: CHEMICAL SYNTHESIS OF PURPORTED INTERMEDIATES. J. R. Bohle, Department of Chemistry, Pacific Lutheran University, Tacoma, Washington 98447

3-Amino-5-hydroxybenzoic acid (AHBA) synthase is an enzyme involved in the shikmic acid pathway. The role of AHBA synthase is the conversion of 5-amino-3-dehydroshikimic acid (amino-DHS) into AHBA. AHBA is involved in the biosynthesis of secondary metabolites, including the ansmycins (e.g. rifamycin). In a recent study (Fryhle, C. B., Kim, C. G., and Floss, H. G.) using electrospray ionization mass spectrometry the presence of pyridoxal phosphate as coenzyme was proven in the AHBA synthase reaction. From that data a five step mechanism has been proposed. Currently the synthesiS of the purported intermediates of this mechanism has begun. Once the intermediates are synthesized, the purported mechanism will be tested by tritium labeling of intermediates during the enzymatic reaction.

THE EFFECTS OF TRANS C18:1 FATTY ACIDS ON THE EXCITABLE MEMBRANE OF PARAMECIUM TETRAURELIA, Shane O'Mahony, Department of Chemistry, Seattle University, Broadway & Madison, Seattle, Washington 98122

Research has established a correlation between trans fatty acids resulting from partial hydrogenation of oils and coronary heart disease. We have used Paramecium Tetraurelia as a model for excitable cells to determine some effects of trans fatty acids at the cellular level. Paramecium grow well and exhibit normal swimming behavior when grown in axenic media supplemented with phospholipids and free oleic acid. We grew paramecium in media containing varying levels of oleic acid (C18:1 cis) and elaidic acid (C18:1 trans) to establish the dose/response relationships between the trans fatty acid levels and paramecium growth and swimming behavior. The swimming behavior of paramecium is determined by their membrane ion channel activity. Ion channel activity has been shown to be effected by the free fatty acids in vitro. Our results indicated that increasing the levels of free trans fatty acid in the media cause a decrease in growth and an increase in altered backward swimming behavior.

UPTAKE OF TRANS-FATTY ACIDS INTO PARAMECIUM TETRAURELIA, Paul W. Riley, Department of Chemistry, Seattle University, Broadway & Madison. Seattle, Washington 98122

Past research has shown that Paramecium tetraurelia, when cultured in axenic media (media without bacteria, but rich in vitamins, saturated fatty acids, and other nutrients) with small quantities of trans-fatty acids instead of the cis-fatty acid equivalents produced backward swimming responses for shorter periods of time. The intention set forth in this paper is to find out if the uptake of the trans-fatty acids occurs in the cells displaying the response. The lipid fractions from the cells, once isolated, are then derivitized to methyl esters using sodium methoxide, which has been shown not to derivitize any free fatty acids from the media. Derivitized samples are run on a HPLC with a silver nitrate impregnated silica column, along with a GC-MS equipped with a (1:1) phenyl silicine/methyl silicone capillary column to determine the presence of trans-fatty acids. The presence of trans-fatty acids in the cells grown in media containing trans-fatty acids, when compared to the absence of trans-fatty acids in cells grown in media containing cis-fatty acids would show that the uptake of trans-fatty acids into the cells may be related to the behavioral response described above. The findings of the current research will be discussed, along with the relationship to past findings, all of which may be applied to current nutritional science research concerning the effect of trans-fatty acids on eukaryotic organisms of all kinds.

QUANTUM MECHANICAL SCATTERING OF METHANE BY ARGON. Mark Mitchell, University of Puget Sound

The objective of my thesis is to calculate the scattering of methane (CH4) after a collision with argon. The scattering matrix describes how methane's angular momentum changes during a collision. This matrix was calculated using a quantum rigid rotor model for methane's wavefunctions with the perturbing atom, Ar, treated as a classical particle. The collision between CH4 and Ar is interesting in part because the intermolecular forces are best described as if each atom of CH4 interacted independently with Ar, the so-called atom-atom model. Little is known about such forces. However, the larger significance of this research is its importance to atmospheric spectroscopy. When an excited molecule collides with another molecule before it can deexcite, the result is the broadening of sharp spectroscopic lines. Information provided about line broadening phenomena is used in remote sensing experiments to determine the temperature and chemical concentration gradients of the atmosphere. Methane is an important greeenhouse gas, therefore understadning relevant spectroscopic affects is necessary for understanding atmospheric changes.