Abstracts from the Department of Chemistry and Biochemistry
Undergraduate Summer Research Symposium August 10, 2005

Ordered alphabetically by student's last name

Conformational Effects of Protein Phosphorylation   

Agata Bielska and Neal Zondlo
Department of Chemistry and Biochemistry

Phosphorylation-induced conformational change is an important tool used by cells to regulate metabolic pathways, signal transduction pathways, and transcription.  The objective of this research is to characterize the specific conformational effects of protein phosphorylation using the proteins tau and cJun as a model. Hyperphosphorylation of tau has been shown to cause its dissociation from microtubules and subsequent precipitation into neurofibrillary tangles (NFTs) in Alzheimer’s disease.  In order to study mechanisms by which phosphorylation could lead to NFT formation, various tau-derived peptides were synthesized.  Phosphorylated and non-phosphorylated versions of the peptides were then analyzed using circular dichroism (CD) and NMR spectroscopy.  Significant conformational changes were found in regions which, when hyperphosphorylated, are associated with NFT formation.   These conformational changes were consistent with an increase in type two polyproline helix (PPII) character.  The oncoprotein, cJun, is a transcription factor regulated by phosphorylation.   Peptides derived from cJun were synthesized and analyzed in non-phosphorylated and phosphorylated forms. Upon phosphorylation, significant conformational changes were observed favoring PPII formation.  The magnitude of these changes increased as the number of phosphorylation sites increased. Supported by an HHMI .

Winner of the Sigma Xi Award for the best oral presentation at the 2005 Undergraduate Research Symposium.

Glutathione S-Transferase Pi: A Monomer or Dimer?

Katrice Brooks1, Stephanie Misquitta, and Roberta F. Colman
1Spelman College and Department of Chemistry and Biochemistry, University of Delaware

Glutathione S-Transferases (GSTs) constitutes a family of detoxifying enzymes that play a role in metabolism of endogenous and xenobiotic compounds.  Mammalian GST has been subdivided into eight classes (alpha, kappa, mu, omega, pi, sigma, theta, and epsilon) based on chemical and physical properties, and can exist as a homo or heterodimer.  Recombinant human GST of the Pi class is purified using affinity chromatography.  Purification by a S-Hexyl column has resulted in low yields of the enzyme.  Earlier studies have shown that salts like potassium chloride affect the monomer-dimer equilibrium of the enzyme.  In an effort to optimize purification, the salt concentration was changed for each trial.  It was found that there is a direct proportion between the sodium chloride concentration and the percentage of enzyme purified.  To investigate if sodium chloride had any affect on the monomer-dimer equilibrium of wild-type human GST Pi, light scattering was used to determine the molecular weight at various sodium chloride concentrations at pH 6.5, and pH 8.  Our studies found that a high salt concentration resulted in a lower molecular weight at pH 8, indicating that the monomer-dimer equilibrium was shifted completely toward the monomer.  These results suggest that the electrostatic interactions may exist between the two subunits, which are disrupted in the presence of NaCl leading to the formation of a monomer. (Supported by HHMI)

The Role of Arg-181 of the E. coli Pseudouridine Synthase TruB

Kirsten H. Butterfoss, Christopher S. Hamilton, and Eugene G. Mueller
Department of Chemistry and Biochemistry

The isomerization of uridine to pseudouridine on tRNA is catalyzed by pseudouridine synthases.  These enzymes contain an aspartic acid residue (Asp-48 in E. coli TruB) that is essential for pseudouridine synthase function.  The crystal structure of E.coli TruB reveals a salt bridge between Arg-181 and Asp-48.  To determine the role of Arg-181, site specific mutagenesis was used create R181K TruB.  A tritium release assay will be used to compare the activity of wild type to R181K TruB by determining values kcat and Km for R181K TruB. The preparation of substrate tRNA by in vitro transcription and the initial characterization of R181K TruB will be described. This project was funded by HHMI.

Detection of Antigens in Pesticide-treated Rat and Mice Livers

Meghan Gelburd
1, Stephen Ramick1, Sue Snajdr2, Brian Shertz2, John O Connor2, and Christine Glatt2
1University of Delaware and  2DuPont-Haskell Lab

Twenty five female rats and twenty five male rats, along with twenty five female mice and twenty five male mice were treated with a certain compound to test its toxicity.  After treatment, the animals were sacrificed and their livers removed for testing.  Following a standard procedure, the livers were prepared.  Using the samples prepared from the mouse and rat livers, several different tests were run to determine the toxicity of the compound.  A Bio-Rad Protein Assay was run in order to determine the concentration of the protein in the liver, followed by a Western Blot to identify and measure any antigens present.  Using these methods it was determined that the compound was not toxic and the western blots showed that it had no effect on the Cytochrome P450 isozyme.

Europium-Substituted Polyoxometalates in Thin Films

Kate Frysinger, Wenlin Huang, Tom Dugan, and Tatyana Polenova
Department of Chemistry & Biochemistry

Polyoxometalates (POMs) are attractive for design of new materials, due to diverse chemistry, favorable structural and electronic properties.  Lanthanide-substituted polyoxometalates are becoming of special interest due to their excellent luminescent characteristics and the possibility to fine tune their electronic properties via synthesis of mixed-addenda anions or use of different counter cations.  Fluorescent and luminescent polyoxometalate materials are typically fabricated as thin solid films of hybrid organic/inorganic materials. It has been recently demonstrated that luminescence properties of thin polyoxometalate films differ from those of the bulk solids. An interaction between the polyoxoanion and the organic matrix leading to the altered site symmetry of the rare earth atom in the polyoxometalate, has been proposed based on the bulk changes detected in the excitation and emission spectra.  However, no further details are available from these measurements, due to the intrinsic lack of site resolution.  The goal of this research is development of 31P solid-state NMR spectroscopy as a sensitive site-specific probe of the paramagnetic rare earths substituted polyoxometalates prepared as thin films. Europium-substituted polyoxometalates of the Keggin and Wells-Dawson families have been synthesized and their structure verified by 31P solution NMR.  The polyoxometalates were processed into organic/inorganic thin polymer films, creating a polyoxometalate/polymer bilayer on a quartz substrate with a precursor film.  The process was monitored by UV-visible spectroscopy to ensure that the polyoxometalates were deposited.  Solid-state NMR spectroscopy investigations are under way.  This research was funded by the University of Delaware, the HHMI Undergraduate Research Program, the National Institute of Health, and the National Science Foundation.

Bimetallic Complexes as Bifunctional Lewis Acid-Base Reaction Sites

Margaret A. Goodell1, Andrea E. Martin1,2, and John E. Bulkowski1
1Department of Chemistry and Biochemistry, University of Delaware and 2Widner University

The objective of the project is the synthesis and reactivity study of a neopentyl derivative of a 22-membered hexaamine 1,4,7,12,15,18-hexaazacyclodocosane, L4.  The first phase involved thee synthesis of the L4 macrocycle by a six step procedure.  The macrocycle is then converted to the neopentyl derivative where the macrocyclic hexaamine is fully substituted with six neopentyl groups.  The procedure involves reacting L4 with the acid chloride and subsequent reduction with diborane.  In the final phase of the project, dihydroxy zinc and copper complexes of these macrocyclic ligands will be synthesized.   The rings hold the metals at fixed distances from each other and create a hydrophobic region where the metals can react with oxygen, hydroxide, nitrite, and other small molecules.  These interactions will be characterized to determine the effectiveness of the metal complexes as bifunctional acid-base reaction centers.   Funding by Howard Hughes Medical Institute.
      Neopentyl Derivative of L*

Spectroscopic Elucidation of Selected Beta-hairpin Turn Sequences

Eric D. Hardter, Karthikan Rajagopal, and Joel Schneider
Department of Chemistry and Biochemistry

MAX1 is a 20 amino acid Beta-hairpin comprised of valine-lysine repeats and a type II' turn sequence of -VDPPT-. This peptide undergoes triggered folding from random coil to Beta-hairpin conformation. Subsequent self-assembly of the hairpins affords hydrogel material which is currently being investigated for use in tissue engineering. Circular dichroism is used extensively to assess the secondary structure of these peptides during folding and self-assembly. However, it is not known definitively how the turn sequence contributes to the spectroscopic data obtained. To elucidate this matter, a series of 6 residue turns (C-X-X-X-T-C) has been synthesized, and subjected to circular dichroism, nuclear magnetic resonance, and infrared spectroscopies. The contribution of the turn region to the overall spectroscopic signal of the hairpin will be discussed. Furthermore, all experiments were performed under both oxidizing and reducing conditions, so as to further determine the impact of a disulfide bond on these turns. Funding by Howard Hughes Medical Institute.

Progress on the Synthesis of an Asymmetric Metallofoldamer Catalyst

Richard J. Karpowicz Jr., Dorthey Dong, and Joseph M. Fox
Department of Chemistry and Biochemistry

We are currently developing the concept of using metallofoldamers in the design of enantioselective catalysts. It is widely known that salen and salophen catalysts with inherently chiral primary coordination spheres have yielded enantioselectivity in Diels-Alder reactions, non-terminal alkene epoxidations and various other common reactions. These catalysts derive their selectivity from an inherently chiral diamine backbone. Their limitation is the relatively small pool of chiral molecules which can be incorporated into these multidentate ligands as compared to the large number of achiral analogs. Described is the nearly complete synthesis of a helical metallofoldamer with a chiral secondary structure that will mimic the stereocontrol found in enzymes. Here, the chiral binding pocket is strongly influenced by unbound residues which define the local secondary structure instead of an inherently chiral primary coordination sphere. Steric, electronic and bite angle changes can be
made to the primary sphere and thus alter the reactivity of the catalyst. This project is currently being funded by the Howard Hughes Medical Institute and startup funds provided by the University of Delaware.

Construction of a Two-Helix Fragment of the Adenosine A2A Receptor

Kellie Machlus
1, Jennifer Kreider
1, and Clifford Robinson1,2,3
1Department of Chemistry and Biochemistry, 2Delaware Biotechnology Institute, 3Department of Chemical Engineering

Although much is known about the structure and corresponding function of soluble proteins, the folding mechanisms and pathways of G protein-coupled receptors (GPCRs) are highly unknown.  In my research, the Adenosine A2A Receptor is studied as a model GPCR.  Like all GPCRs, A2A contains seven membrane-spanning alpha-helical domains.   The goal of my research project is to use recombinant DNA to create a fragment of the A2A receptor that contains trans-membrane helices two and three, and five and six.  Ultimately, we will use these and similar constructs to learn about the assembly pathway and structure of A2A.  Once this peptide is sufficiently expressed in E. coli, it will be purified by Ni-Chelate chromatography.  We will then characterize the structure of the two-helix fragment, and measure its interactions with other trans-membrane helices from A2A.  This project was funded by the Howard Hughes Medical Institute.


A New Route to Enantiomerically Pure Alpha Amino Acids

Timothy J. Martin and  Douglass F. Taber
Department of Chemistry and Biochemistry

We are exploring a new method for the synthesis of enantiomerically pure natural and unnatural alpha-amino acids.  We have prepared the diazo ester 1 from commercially available L-menthol and diketene.  The key step focused on the N-H insertion of o-anisidine to the diazo ester 1, using Rhodium octanoate dimer as the catalyst.  The diastereomeric products 2a and 2b were separable by silica gel chromatography.  Oxidation of the aryl group employing iodobenzene diacetate gave the free amine, which was then protected with Fmoc.  The ester was cleaved using trifluoroacetic acid without racemization, resulting in the enantiomerically-pure Fmoc alpha-amino acid.

Ultra-fine Particle Emissions from Trains

Stephen Miller and Murray Johnston
Department of Chemistry and Biochemistry

It has been well established that particulate matter (PM10, PM2.5) has many detrimental health effects on humans.  Over the last several years there has been increasing evidence that many of these health effects are associated with the ultra-fine mode, referring to particles with diameters less than 100 nm.  Because of this fact, it is important to determine from what sources ultra-fine particulate matter is emitted into the atmosphere so that an effort can be made to reduce or eliminate these emissions.  It is suspected that trains are a major source of ultra-fine particles.  The goal of this study was to determine whether or not this is true.  From an air quality monitoring station in Wilmington Delaware, situated about 35 meters from a railway, particle size distribution spectra were measured continuously in order to record any changes at or around the time when a train passed by.  As suspected, for some types of trains, specifically diesel trains, major peaks in the size distribution spectra were observed very near the time that the train passed the site.  These peaks all occurred in the 10-30 nm particle size range and corresponded to an increase in the total particle number concentration by a factor of about 1.5-10.  Often a second peak was observed a few minutes after the train had passed that is likely due to the formation of new particles through the nucleation of the exhaust gases from the passing trains.  These peaks all occurred in the 0-15 nm particle size range. Source of Funding: Science and Engineering  Scholars Program.

Correlation of the Rates of Solvolysis of IsopropylSulfonyl Chloride and 2,2,2-Trifloroethanesulfonyl Chloride

Stacey L. Mlynarski; †Lamia Yaakoubd; †*Malcolm J. D’Souza; §Dennis N. Kevill
†Department of Chemistry, Wesley College, Dover, DE
§Department of Chemistry & Biochemistry, Northern Illinois University, DeKalb, IL

Alkyl Sufonyl Chlorides (RSO2Cl) are versatile acid chlorides that are important synthetic intermediates in the preparation of pharmaceuticals, agrochemicals, dyes, plastics, etc.  Substitution reactions involving these compounds basically bridge organic and inorganic chemistry.  Recently there has been much dispute over their kinetics of solvolysis.  Here, we study the rates of solvolysis of methyl, isopropyl and 2,2,2-trifloroethane sulfonyl chlorides and show differences in solvolytic behavior when compared to previously detailed solvolysis of N,N-dimethylcarbamoyl chloride, n-propyl & isopropyl chloroformate. Supported by the INBRE Program.

Synthesis of Protected γ-dicarboxyglutamic Acid for Use in Solid Phase Peptide Synthesis

James Nelson, Lisa Capriotti, and Joel Schneider
Dept. of Chemistry and Biochemistry

Osteocalcin, a protein component of bone, is believed to bind tightly to the surface of hydroxyapatite due to the presence of three dicarboxyglutamic acid residues in its amine terminal α-helix.  Despite its hypothesized affinity for hydroxyapatite, dicarboxyglutamic acid has not been widely used in de novo peptide design.  In order to prepare an α-helical peptide which binds to bone, we designed a short peptide containing five dicarboxyglutamic acid residues.  The synthesis of this peptide is contingent on the successful preparation of the precursor Fmoc-Gla amino acid.  This synthesis was undertaken through the substitution of the hydroxyl group of L-serine with malonate and the protection of the amine group with an Fmoc group to afford the Fmoc-protected dicarboxyglutamic acid. Funded by a grant from the Howard Hughes Medical Institute.

Selective Preparation of 3-Aryl Indoles

Daniel L. Silverio, Weiwei Tian, and Douglass F. Taber
Department of Chemistry and Biochemistry

A new method for creating indoles using an azirine has been developed in the Taber group.  The first goal of my project is to synthesize 1,1-diphenyl-2-propanone(1) in order to create a 3-aryl indole (3). Subsequently, many asymmetric diaryl-2-propanone species will be synthesized.  The goal here is to attach functional groups to one of the aryl groups that may direct selectivity of the indole formation towards one ring over the other.  By determining the selectivity, we are hoping to find clues to how the mechanism proceeds.  With the mechanism in hand, we will be better able to optimize the reaction.  I would like to thank the Howard Hughes Medical Institute for providing my funding.

Synthesis of Polyguanidiniums via Alpha Guanidino Acids

Alfred Smith, Shalini Balakrishnan, and Neal Zondlo
Department of Chemistry and Biochemistry
The guanidinium functionality is biologically active, because of its ability to bind to anionic regions of proteins and nucleic acids.  Due to its role in protein-protein and protein-RNA interactions, guanidiniums have become attractive targets in pharmaceuticals.  In order to examine these biological properties, chemistry was developed for the synthesis of alpha guanidino acids, which are amino acid derivatives in which the amine of an amino acid is converted into a guanidinium. The methyl esters valine, leucine, glycine, and phenylalanine were guanylated.  The chemistry used bis-boc-thiourea and Mukaiyama’s reagent to attach the guanidine functionality to the amine of an amino acid methyl-ester.  The methyl ester was then saponified using LiOH.  These guanidino acid derivatives will be coupled to diamines and various peptides.  Funding for this project was from the Howard Hughes Medical Institute.

Biochemical Investigations of the Novel Histidine-Rich Protein MAHRP-1 from Plasmodium falciparum

Tarjani Thaker, William Perea, and Tatyana Polenova
Department of Chemistry and Biochemistry

The Plasmodium falciparum parasite is responsible for spreading the infectious disease, malaria.  Associated with this parasite are a variety of proteins that assist in the organization and sorting of proteins necessary for the parasite’s survival.  The recently discovered MAHRP-1 protein from P. falciparum is the first membrane associated histidine rich protein of its kind.  It has been found to bind the toxic hemoglobin degradation product, ferriprotoporphyrin (FP), at a stoichiometric ratio determined by the number of histidine-containing DHGH repeats located at the C-terminus.  These binding properties, as well as other chemical properties associated with the high density of histidines found within the P. falciparum parasite derived protein, are of great interest.  Moreover, MAHRP-1 is of interest as a potential drug target.  However, little is known about the structural characteristics of MAHRP-1.  The focus of this research is structural characterization of MAHRP-1 by solution and solid-state NMR spectroscopy.  As the first step, we have successfully established protocols for expression and purification of isotopically enriched MAHRP-1.  Subsequently, the MAHRP1-membrane interactions as well as metal binding will be addressed.  These and structural NMR investigations are under way. Funding for this research provided by HHMI

Phosphorylation-dependent Protein Design: Protein Kinase-inducible Domains

Blair S. Thornley and Neal J. Zondlo
Department of Chemistry and Biochemistry

Protein kinases modify other proteins by phosphorylating hydroxyl groups on serine, threonine and tyrosine residues, resulting in functional and conformational changes of the target protein.  Kinases are highly regulated, and misregulation of protein kinases is closely associated with human diseases, including cancer, Alzheimer’s disease and heart disease.  Protein design is a technique that implements the fundamental principles of protein folding, structure, function, and activity and applies them to the development of new combinations of structure and/or function.  We chose to focus mainly on the observation that many human proteins are functionally dependent on phosphorylation.  We designed a protein kinase-inducible domain (pKID) such that it will be structured only in the presence of specific kinase activity, but that in the absence of a specific kinase, the protein will be unstructured. Thus, the structure serves as an indicator of successful phosphorylation of the protein.  The design of these pKIDs included a recognition site for protein kinase A.  The peptides were synthesized using solid phase peptide synthesis and characterized by HPLC and ESI mass spectrometry.  The peptides were phosphorylated chemically, as well as enzymatically using protein kinase A.  We demonstrated that the structure and fluorescence of these pKIDs were dependent upon phosphorylation, showing minimal fluorescence when non-phosphorylated and strong fluorescence when phosphorylated.  Funding for this project was provided by the Science and Engineering Scholars program and the American Heart Association

Synthesis of Mutant-Targeting Thyroid Hormone Receptor PROTACS

Katie Vodra, Jianfei Zheng, Quamrul Hassan, and John Koh.
Department of Chemistry & Biochemistry

Resistance to thyroid hormone (RTH) is a genetic disease caused by mutants of the thyroid hormone receptor (TR).  TR is a nuclear hormone that controls the expression of genes that regulate growth, development and metabolism.  Currently, the Koh group is developing a new application of PROTACS (PROteolysis TArgeting Chimeric moleculeS), that can selectively destroy RTH-associated mutants of TR that act in a dominant negative fashion when expressed with wild-type TR.   Our PROTAC design is composed of a thyroid hormone analogs that can selectively bind mutant vs. wild-type TR and recruit ubiquitin ligase activity.  The goal of my project is to develop a synthesis of  a methoxymethyl (MOM) and triisopropylsilyl (TiPS) protected bisphenol core of the thryomimetic GC-1.  This key intermediate will ultimately be used to further the understanding of the new application of PROTACS. This project was funded in part by the Howard Hughes Medical Institute Undergraduate Science Education program.

Investigation of Vanadium Haloperoxidases using Magic Angle Spinning Solid-State NMR

Chris Willis, Neela Pooransingh and Tatyana Polenova
Department of Chemistry

Vanadium chloroperoxidase was expressed in Saccharomyces cerevisiae cells producing the apo form of the enzyme. The latter was activated by the addition of the vanadate cofactor. In order to study the vanadium active site of this protein, 51V solid-state magic angle spinning solid-state NMR spectroscopy has been employed. Vanadium (51V) possesses a half-integer spin 7/2, has high gyromagnetic ratio and small quadrupolar moment. 51V solid-state NMR spectroscopy is therefore an excellent probe for studying the electrostatic and geometric environment of the “spectroscopically silent” vanadium (V) states. Developing 51V solid-state NMR spectroscopy will make it possible to investigate the metal sites in vanadium chloroperoxidase and other vanadium proteins. Funding from the Life Science Scholar Fund.

Correlation of the Rates of Solvolysis of Phenylmethanesulfonyl Chloride

Lamia Yaakoubd; †Stacey L. Mlynarski; †*Malcolm J. D’Souza; §Dennis N. Kevill
†Department of Chemistry, Wesley College, Dover, DE
§Department of Chemistry & Biochemistry, Northern Illinois University, DeKalb, IL

Benzenesulfonyl and Phenylmethanesulfonyl Chlorides are thought to solvolyze by similar mechanisms.  Here the effect of solvent variation of the specific rates of solvolysis of substituted Benzenesulfonyl Chlorides (XPhSO2Cl) and Phenylmethanesulfonyl Chloride (PhCH2SO2Cl) are compared and analyzed in terms of the extended Grunwald-Winstein Equation using the NT scale (based on S-methyldibenzothiophenium solvolysis) of solvent nucleophilicity, combined with a YCl scale (based on 1-adamantyl chloride solvolysis).  The specific rates and sensitivities to changes in solvent nucleophilicity and ionizing power suggests that XPhSO2Cl solvolyzes via a tight SN2 mechanism, whereas a duality of mechanisms may exist in PhCH2SO2Cl. Supported by the INBRE Program.

Links: Summer 2005 Undergraduate Research Symposium, Symposium Abstracts from other Colleges and Departments,
Undergraduate Research Summer Enrichment ProgramUnversity of Delaware Undergraduate Research Program, Howard Hughes Undergraduate Program.
Created  5 August 2005. Last up dated 25 August 2005 by Hal White
Copyright 2005, University of Delaware