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Chemistry

Characterization of the Mechanism of NPSR From Shewanella Loichia PV-4 Using Primary Kinetic Isotope Effects

Raj Bhanvadia ('10); E.J. Crane III

NADH-dependent polysulfide reductase (Npsr) is an enzyme from the microorganism Shewanella. Although members of this genus are aerobes, under anaerobic conditions this organism can also respire through dissimaltory sulfur reduction. Npsr has a flavoprotein domain similar to the NAD(P)H-dependent coenzyme A disulfide reductase/sulfur reductase (CoADR) from Pyrococcus and a smaller domain similar to the Sud polysulfide carrier protein of Wolinella succinogenes. This project aims to further characterize the mechanism of this enzyme and to examine the hydride transfer step of the reductive half reaction through primary kinetic isotope effects as well as pre-steady state and steady state kinetics. I have synthesized (4S)-[4-2H]NADH by the reduction of NAD+ with [ l-2H]glucose via glucose dehydrogenase from Escherichia coli . Initial steady state characterization resulted in a P.K.I.E. of 1.2 on both Vmax and Vmax/Km (for 1H/2H NADH) for the NADH-dependent reduction of bis-dithionitrobenzoate (DTNB). I have recently begun characterizing the P.K.I.E.
Funding provided by: Pomona College Chemistry Dept.

Radical Chemistry and C6H6 Isomer Formation on Titan

Adam Chaimowitz ('10); Fred Grieman

Titan, one of Saturn's moons, shows similar characteristics to early Earth. Titan is the only planetary body besides Earth in which clear evidence of surface and subsurface liquid has been found. It has comparable surface topography, seasonal climate patterns, and a dense nitrogen rich atmosphere. Understanding Titan’s atmosphere could reveal key insights into the evolution of Earth, as well as other planetary bodies. The Infrared Space Observatory (ISO) and Cassini Composite Infrared Spectrometer have detected benzene and polycyclic aromatic hydrocarbons (PAHs) in Titan’s stratosphere. We studied the formation mechanisms of benzene using 1,5-hexadiyne (HC≡C-CH2-CH2-C≡CH) pyrolysis and the propargyl (C3H3) radical at conditions similar to Titan’s atmosphere. We are currently working on determining energy pathways for the propargyl recombination reaction. In the future we hope to understand the mechanisms behind PAH formation in Titan’s atmosphere.
Funding provided by: Jet Propulsion Laboratory; NASA; California Institute of Technology; Pomona College Chemistry Dept.

Determination of Equilibrium Constants for the Reactions of Carbonyl Compounds With HO2 Using Infrared Kinetic Spectroscopy (IRKS)

Casey Davis-Van Atta ('10); Kira Watson ('10); Fred J. Grieman; Aaron C. Noell*; Mitchio Okumura*; Stanley P. Sander**
*California Institute of Technology, Pasadena, CA; **NASA Jet Propulsion Laboratory, Pasadena, CA

Previous computational studies have suggested that the primary sink for carbonyl compounds in the upper troposphere may be oxidation by the hydroperoxy radical (HO2). We have, for the first time, determined the equilibrium constant for the reaction of HO2 with acetone experimentally. An  excimer laser 14 (λ=308nm) was fired in a 2m flow cell containing an N2/O2/Cl2/CH3OH mixture in order to generate HO2 in the presence of acetone at several concentrations. Highly sensitive infrared and ultraviolet absorption techniques were used to monitor the decay of HO2 over time. An analysis  method was developed that permitted calculation of the equilibrium constant from these data. The experiment was conducted over 215-273K, allowing a van’t Hoff plot that yielded thermodynamic parameters for the reaction. We found that ΔrxnH° = -30.1 kJ mol-1 and ΔrxnS° = -65.6 J K-1 mol-1. Preliminary work concerning the reaction of acetaldehyde with HO2 has also been accomplished.
Funding provided by: Pomona College SURP (CDVA); The Paul K. Richter and Evalyn E. Cook Richter Award (KW); National Aeronautics and Space Administration; California Institute of Technology;

Mechanistic Aspects of Polyelectrolyte Adsorption and Effects of Salt on Multilayer Assembly

Will Fletcher ('12); Thomas J. Lane ('10); Malkiat Johal

Aqueous polyelectrolytes spontaneously self-assemble to form films when exposed to a complementary columbically charged surface. Details of the physical and chemical mechanisms underlying the solution and solid-phase behavior of the formation of these films has remained mysterious, likely due to the large number of variables associated with film formation. Recently, we have capitalized on sensitive surface techniques, specifically the quartz crystal microbalance (QCM-D) and dual polarization interferometer (DPI) in order to elucidate the mechanistic aspects of polyelectrolyte adsoption that leads to stable film formation. These studies have revealed that polymers adsorb in a distinct three-stage pattern, osmotic forces are present in the initial stages of adsorption, and solution ionic strength affects the magnitude of adsoption in a non-monotonic fashion. We have developed a series of mathematical models to attribute subtle features to well-documented and predictable physical forces and conventional solution theory, shining light on the unexplained features of polyelectrolyte behavior.
Funding provided by: Howard Hughes Medical Institute (WF); Arnold and Mabel Beckman Foundation

Ab Intio Structure Prediction of SRBC-64 and SRBC-66 GPCRS in C. Elegans

Ulysses Gomez ('11)

G-protein-coupled receptors (GPCRs) play a crucial role in cellular activity due to their vital involvement in many physiological processes. Since GPCRs can be associated with a numerous amount of diseases, the elucidation of their structures can provide insight for the development of pharmaceuticals. Recently, it has been found ascarosides affect mating and developmental behavior in Caenorhabditis elegans by activating SRBC-64 and SRBC-66 GPCRs. High population density or high temperature increases ascaroside concentrations, which halts larvae development as it enters a non-feeding and strengthening stage known as the dauer response. The dauer response attracts special interest since signaling pathways that regulate metabolism, aging and development in higher eukaryotes control it. By utilizing Dr. William A. Goddard’s computational software, GEnSeMBLE, we elucidated the SRBC-64 and SRBC-66 structures. Upon optimization of these predicted structures, we used the GenDock method to dock various ascarosides to see their relative affinities toward these GPCRs.
Funding provided by: Minority Undergraduate Research Fellowship at Caltech

Structure and Function Studies of Coenzyme a Disulfide Reductase of Pyrococcus Horikoshii

Sanna Herwald ('10); Aaron Kaufman ('10); Grace Huang ('09); E.J. Crane III; Karlo Lopez; Matthew Sazinsky

Abstract removed upon request.

Characterization of N-Terminus FEOB

Calvin Kagan ('10); John Gelber ('10); Grant Gucinski ('10); Justin Moser ('11); Jessica Lee ('09); Matthew Sazinsky

Abstract removed upon request.

The Effect of Ligand Chemistry on Vanadium Oxide Thin Film Morphology: A Study of an Inorganic (Vanadium (V) Oxynitrate) Versus an Organometallic (Vanadium (V) Oxy-tri-propoxide) Precursor

Heidi Leonard ('12); Charles Taylor; Tyler Moersch

Thin films of vanadium oxide were created on oxidized single-crystal silicon substrates via chemical vapor deposition using two different chemical precursors, vanadium oxynitrate, (VO(NO3)3), and vanadium oxy-tri-propoxide, (VO(OC3H7)3). The vanadium oxynitrate was synthesized in-house from vanadium (V) oxide and dinitrogen pentoxide, while the corresponding oxy-tri-propoxide was commercially obtained. Films were deposited at substrate temperatures ranging from 300°C to 500°C. Comparisons of film microstructures were made using X-ray diffraction and scanning electron microscopy to evaluate crystallinity and topography of the deposited films.
Funding provided by: Pomona College Chemistry Dept.

Inhibiting Polymer Degradation Through Dendrimer Complexation

Jenny Lin ('11); Jeremy Treger ('09); Malkiat Johal

This project investigates the complexation between a water soluble anionic polymer poly{2,5-methoxy- propyloxy sulfonate phenylene vinylene} (MPS-PPV) and a cationic dendrimer (generation 4) and its effect on inhibiting photo-oxidation. We observe an increase in the rates of decay for both initial exponential and slow linear components with increasing dendrimer concentrations. Specifically, the exponential decay rate starts to decrease drastically at 1:1 polymer/dendrimer ratio, and the trend continues with increasing dendrimer concentrations. Further experiments are needed to see whether the jump in the decay rate reaches a maximum at higher dendrimer concentrations. This work contributes to the construction of longer-lasting PPV-based photovoltaic and electroluminescent devices.
Funding provided by: The Craddock-McVicar Award

Reactiton Efficiency of Diffusion in Dendrimeric Systems

Albert Liu ('12); Jae Won Saw (‘12); Steven Chau (‘12); Joyce Lee (‘12); Joseph Tseng (‘12); Roberto A. Garza-López; John J. Kozak*
*Chemistry Dept, DePaul University, Chicago, IL

We performed three types of calculations on our molecular lattices. Calculation of the average walklength was performed twice, one assuming that there were no biases such as dipole-dipole interactions or other extraneous forces, and the other assuming that dipole-dipole forces were present. This required solving certain equations that depicted the walker’s path, constructed using the probabilities and valency of each site. We used Maple 11 to solve these simultaneous equations and obtain the values of these random walks. Once we built the matrix containing the coefficients, we found the random walks using linear algebra by creating an identity matrix and inverting our matrix with the equation coefficients. It is important to note that once the walker reached the trap, the probability became zero since the diffusion had become irreversible. Finally, we calculated the eigenvalues of the matrices we created in order to establish a relationship between these values and the random walklengths.
Funding provided by: Pomona College SURP (AL, JWS); Howard Hughes Medical Institute (JT); Pomona College Chemistry Dept.

Investigating Integral Membrane Protein FEOB: Optomizing GTPASE Conditions and Purifying the G-Protein Domain

Justin Moser ('11); Matthew Sazinsky

Abstract removed upon request.

Towards a Series of Potent and Selective Inhibitors of Dihydrofolate Reductase

Amanda Peel ('10); Cynthia Selassie; Rajeshwar Verma

Because of its essential role in cell growth and replication in all organisms, the enzyme dihydrofolate reductase (DHFR) is a frequently exploited chemotherapeutic target. This work is part of an ongoing project aimed at developing novel inhibitors of the DHFR of the protozoal organisms P. carinii and T. gondii, which are often responsible for opportunistic infections in immunocomprimised individuals. Prior to this work, a series of compounds containing a 2,4-diaminopyrimidine moiety identical to that of the antibiotic Trimethoprim have been synthesized with various structural modifications designed to improve both their potency and selectivity. I have nearly completed the synthesis of several 2,4-diamino-5-(4-X- anilinomethyl) pyrimidines by coupling para-substituted anilines with previously synthesized 2,4- diaminopyrimidine-5-carboxaldehyde in an acid-catalyzed Schiff Base reaction. Once these potential inhibitors exhibit sufficient variation in both the hydrophobicity and size of their X-groups, we will use QSAR to analyze the effects of these para ring substituents on binding to DHFR.
Funding provided by: Pomona College SURP

Synthesis of Carbocylic Skeletons Via Radical-cation Intermediates in a Tandem Pinacol Rearrangement Radical Cyclization

Vanessa Ramirez ('10); Zulimar Nevarez

This project is intended to expand the chemistry of radical cations in a tandem Pinacol-rearrangement radical cyclization reaction to provide synthetically relevant structures. Such methodology requires homolytic cleavage of a radical precursor followed by heterolytic fragmentation of an α-leaving group to generate a radical cation/anion pair. The in situ generated intermediate may undergo [1,2]-migration of an R substituent followed by a radical cyclization to yield a carbocyclic skeleton. The design and progress of the syntheses of the phosphorylated nitro alcohol substrates needed to test the tandem strategy will be presented as well as future plans to overcome synthetic challenges.
Funding provided by: GlaxoSmithKline Fellowship

Soil Analysis of Lead Concentrations at Bracket Airifield

Jamie Shannon ('11); Dejo Kotevski ('10); Charles Taylor

Although the use of leaded motor gasoline (MoGas) has since been banned in automobiles, lead continues to be a major component in many aviation fuels. Aviation Gasoline (AvGas) fuels can contain up to two to four grams of lead per gallon, which are released into the atmosphere upon take-off, flight, and landing. Soil samples in the area adjacent to and surrounding Brackett Airport in La Verne, California were collected and then digested using Harvey Mudd College’s microwave digestion system. Flame and Graphite Furnace Atomic Absorption Spectroscopy (FAAS and GFAAS, respectively) were employed to determine the concentrations of lead in the soil samples. The majority of the samples did not exceed the EPA’s allotted limits; however, one sample was well over the limit while four others approached the limit. Although slightly elevated lead levels were observed in the area surrounding Brackett Airport, the area can be deemed safe for its residents.
Funding provided by: Schultz Environmental Studies Award

Characterization of an Isolate from the Mud Volcanoes of Salton Sea

Johnson Trang ('11); EJ Crane III

Further tests were performed to characterize an isolate from the mud volcanoes of Salton Sea. This unique bacterium is able to grow at high temperatures, high pHs and high salt concentrations. Its 16S rRNA gene sequence show that this isolate is a member of the Thermoanaerobacter family. Previous studies have shown this anaerobic organism can reduce soluble iron. Iron-reduction is an important process in the iron cycle, the geochemistry of mineral formation and breakdown, and in the bioremediation of polluted sites. It was found that the organism is able to reduce Fe(III) in the form of insoluble ferrihydrite quickly and prolonged growth in the presence of ferrihydrite seemed to result in physiological changes in the bacteria. Tests were conducted to determine the pathway for the reduction of iron. A large scale growth of the thermoanaerobe was prepared for studies of the ability of its membranes to reduce Fe(III).
Funding provided by: The Paul K. Richter and Evalyn E. Cook Richter Award

Characterization of the Novel Sulfur-Reducing Enzyme NADH-Dependent Polysulfide Reductase (NPSR) From Shewanella Loichia PV-4

Megan Warner ('10); Vinita Lukose ('08); Edward J. Crane III; Karlo Lopez

Bacteria in the genus Shewanella have the ability to utilize a variety of different electron acceptors such as chromium, uranium, and sulfur. We have identified a unique sulfur-reducing enzyme in some Shewanella species which we refer to as Npsr (NADH-dependent polysulfide reductase). The protein consists of two domains: a flavoprotein domain homologous to other sulfide reductases and a smaller C- terminal tail domain homologous to small polysulfide carrier proteins. We have discovered two candidates for the in vivo substrates of Npsr: coenzyme A bound persulfide (CoA—S-S-) and coenzyme A bound polysulfide (CoA–Sn – S-). This discovery explains the previously observed need for coenzyme A during sulfur reduction. The cysteine located in the tail is critical for the CoA sulfide substrates while it is not required for the enzyme’s DTNB reductase activity. This suggests that the tail region plays a key role in the catalysis performed by this enzyme in vivo.
Funding provided by: Pomona College Chemistry Dept.

Modeling the Kinetics of the HO2-Acetone Reaction at Tropospheric Conditions: Implications for the Production of Smog

Kira Watson ('10); Casey Davis-Van Atta ('10); Fred Grieman; Stan Sanders*; Mitichio Okamura**; Aaron Noell**; Aileen Hui**
*Jet Propulsion Lab, Pasadena, CA; **CalTech, Pasadena, CA

Acetone present in the troposphere has been implicated as a significant sink of the HO2 radical. Computational studies predict that at tropospheric temperatures the acetone-HO2 reaction rapidly converts into a molecular complex, which possibly isomerizes to the hydroxyisoproplyperoxy adduct. Grieman et al., using the Infrared Kinetic Spectroscopy method, determined the equilibrium constant (Keq) for this rapid reaction using only the drop in [HO2] at time = ~0. I have modeled the subsequent decay of [HO2] with the expected reactions and fit the data using the FACSIMILE program using Keq and rate coefficients of known reactions, and varying theoretical rate coefficients for the acetone-HO2 product reactions in order to determine the likely product and the forward rate constant (kf) of the acetone-HO2 reaction. To directly measure kf, we are currently attempting to install astigmatic Herriott cell mirrors that will give higher signal to noise by increasing path length of the system.
Funding provided by: Pomona College SURP (KDVA); The Paul K. Richter and Evalyn E. Cook Richter Award (KW)

Detecting Changes in SDS Adsorption on a Hydrophobic Surface Using QCM-D

Ellen Yang ('11); Malkiat S. Johal

An important characteristic of surfactant molecules is their ability to self-assemble into nano-scale agglomerates known as micelles. The onset of micellization occurs at a characteristic concentration known as the critical micelle concentration (CMC). My research goal was to investigate surfactant adsorption on a hydrophobic surface in the vicinity of the CMC using the quartz crystal microbalance with dissipation monitoring (QCM-D). This involved exposing various aqueous concentrations of sodium dodecylsulfate (SDS) to a hydrophobic octadecane thiol (ODT)-coated crystal, and determining the mass of the resulting monolayer by correcting for viscosity effects using a general mathematical model. The ODT substrates were initially cleaned with 10mM SDS in the QCM-D liquid cell, followed by a water rinse, the addition of a SDS solution, and another water rinse. The QCM-D data indicate that as the concentration of SDS increases, frequency decreases. Dissipation, however, increases and begins to level off above the known CMC.
Funding provided by: Rose Hills Foundation

Research at Pomona