Abstracts from the Department of Chemistry and Biochemistry
Undergraduate Summer Research Symposium August 8, 2007

Ordered alphabetically by student's last name

Bourreza Dignan Knerr Mlynarski
Rhoades Styer White
Bazzoli Carter Dreher
Koyoshi Nuzzio Pirnot
Schnitker Tereniak Zony
Currier Hullmann
Reed Spencer
 Wagner Zucker

Neurite Growth Preference between Laminin and Fibronectin Patterning
Peter Adelman
, Soonmoon Yoo, Bill Theilacker, Jeffery Twiss, and Thomas P. Beebe
Department of Chemistry and Biochemistry and the A.I. duPont Hospital for Children

Following an injury, neurons in the peripheral nervous system (PNS) are capable of re-extending neurites or axons and restoring function. The central nervous system (CNS) only shows regeneration under very special circumstances.  If the mechanism for this regenerative growth could be understood, then it might be possible to facilitate PNS regeneration and greatly improve outcomes for CNS regeneration (e.g., spinal cord injury).  In large part, axon growth is guided by extracellular matrix (ECM) macromolecules, which can encourage or inhibit neurite growth.  Here, I am setting the groundwork to ask how axons distinguish between common ECM components found in the PNS and CNS. We have devised a means to determine how neurons show preference between the two supportive matrices, laminin and fibronectin. To accomplish this, we micro-contact printed two dimensional growth substrates with the laminin and fibronectin proteins in adjacent stripes.  These substrates were analyzed for surface chemistry and then for neurite growth promotion.  Earlier studies from our lab had shown that axons of rat sensory neurons prefer laminin to fibronectin, though recent literature suggests neurite outgrowth may favor the protein-protein interaction between laminin and fibronectin, rather than having a preference for one of the two (Hodgkinson, et al., 2007).  To more closely address this issue of axonal substrate preference, dissociated adult rat dorsal root ganglia were plated onto engineered coverslips with 40 µm wide stripes of fibronectin and laminin.  These cultures consist of a mixture of sensory neurons and the non-neuronal 'Schwann' cells. The number of axon/neurite crossing from one substrate to the other was quantitated and correlated with Schwann cell contact. Supported by HHMI.

Identifying Biomarkers of the MHC Gene to Investigate Chemosensory Discrimination

Tyler C. Bazzoli and Steven D. Brown
Department of Chemistry and Biochemistry 

Variability in the highly polymorphic genes of the major histocompatibility complex (MHC) plays a fundamental role in influencing the odor of urinary volatiles in the common house mouse (Mus musculus). To better understand the mechanisms underlying MHC-dependent chemosensory signals, it is critical to characterize the chemical nature of MHC-dependent odorants. Urine samples from MHC-genotyped mice were analyzed using capillary gas chromatography. By employing multivariate analysis and pattern recognition techniques on the chromatographic data, it is possible to classify mice by their MHC genotype and to explore which chemical compounds are biomarkers capable of discrimination. Soft Independent Modeling of Class Analogy (SIMCA) of the data was performed and indicated that the observed genotypes were easily distinguishable and well separated, a strong indication that specific compounds differentiated the genotypes. Based on the SIMCA model, a graph of the discriminating power of each compound within the model was generated. The compounds that exhibited a high level of genotype-discriminating power were dimethyldisulfide, methyl methylsulfenylmethyl disulfide, Z-5,5-Dimethyl-2-ethylidenetetrahydrofuran, and E-5,5-Dimethyl-2-ethylidenetetrahydrofuran.The structural similarity between the two disulfide compounds along with the similarity of the two tetrahydrofuran compounds suggest that there may be a biochemical explanation for the role that these four compounds play in differentiating mice with dissimilar MHC genes. The ability to qualitatively determine specific biomarkers associated with genetic differences has an invaluable application in the field of genomic medicine. Further exploration of the relationship between chemistry and genetics promises new treatments for gene-linked ailments. Supported by the Howard Hughes Medical Institute.

Development of a Fluorescent Sensor of ErbB2 Kinase Activity for Application in Breast Cancer Research

Ann Benavidez
and  Neal Zondlo
Department of Chemistry and Biochemistry

When breast cancer develops in an individual, ErbB2 kinase signaling becomes deregulated. The resultant uncontrolled phosphorylation leads to functional changes in multiple proteins, altering cellular behavior. To explore ErbB2 kinase signaling, we sought to develop fluorescent sensors of ErbB2 activity. Using solid-phase peptide synthesis, chemical phosphorylation, and reverse purification techniques, two tyrosine kinase-inducible domains were created which contain ErbB2 recognition sites and have varying electrostatics at the N-terminus. The sequences are based off of a EF hand motif, modified so as to respond to phosphorylation. Since peptides bind terbium ions and fluoresce when phosphorylated and are unstructured when nonphosphorylated, I hypothesized that the phosphorylated peptides would bind Tb3+ well and display great terbium luminescence, while the nonphosphorylated peptides would poorly bind Tb3+ and display correspondingly weaker terbium luminescence. Accordingly, I have sought to purify phosphorylated and nonphosphorylated versions of the peptides to investigate this phenomenon. In the future, phosphorylation behavior upon exposure to ErbB2 will be examined and the necessary modifications will be performed in order to build a tyrosine kinase-inducible domain with optimal specificity for ErbB2. (Funded by Chemistry Alumni Scholars Program).

Aly took third place in the Sigma Xi competition.

Evaluating Anti-Androgen Resistance by In-Vitro Selection
Aly Bourreza and John Koh
Department of Chemistry and Biochemistry

Anti-androgens, such as flutamide and bicalutamide (Casodex®), used alone or in conjunction with chemical castration, have been used in the treatment of prostate cancer (PCa) for decades.  However, as many as 30%- 40% of patients treated with anti-androgens acquire a therapy-resistant phenotype after one to three years of treatment.  Furthermore, some patients experience clinical improvement upon cessation of anti-androgens, a condition known as anti-androgen withdrawal syndrome.  Anti-androgen withdrawal syndrome has been associated with mutations to the AR that cause an agonist response to anti-androgens and is one of the most difficult forms of PCa to treat.  The goal of this project is to redesign anti-androgens to evade molecular mechanisms that lead to anti-androgen withdrawal syndrome in androgen independent PCa.  To accomplish this, novel analogs, PLM1 and PLM6, were developed that remain antagonists towards three AR mutants associated with anti-androgen withdrawal syndrome.  In this study, long-term growth analyses of the human LNCaP cell line in the presence of PLM1 and PLM6 versus bicalutamide have been performed to identify potential AR mutants that would represent an anti-androgen withdrawal phenotype.  After approximately 4- 6 weeks, resistant colonies developed and were selected.  DNA sequence data of the resistant colonies show mutations to the AR.  Current studies are underway to examine the effects of drug withdrawal and changing the drug treatment.  This work is supported by the Howard Hughes Medical Institute Undergraduate Science Education program and the National Institutes of Health, NIDDK; 3-R01-DK054257-09.

A Study of the Effect of a t-Butyl Group on the α-Carbon of Chloroformate Esters
Carter1, Malcolm J. D’Souza1, Dennis N. Kevill2
Department of Chemistry, Wesley College, Dover, Delaware,  2Department of Chemistry & Biochemistry, Northern Illinois University, DeKalb, Illinois

Alkyl chloroformates are broadly used in peptide synthesis for protecting amino groups as their alkoxycarbonyl derivatives.  Additionally bulky alkyl chloroformate esters are efficient precursors in the syntheisis of a wide variety of commercial products.   For example, neopentyl chloroformate is used as a protecting group to develop an extremely inert photoresist resisn poly(4-neopentyloxycarbonyloxystyrene) NPOCST. Here, we study what we believe is the missing link in published results for the influence of solvent variation in the study of alkyl chloroformate esters neopentyl chloroformate, a compound containing a t-butyl group on the α-carbon.  We have determined the rate constants and analyzed them using the Grunwald-Winstein equations to determine its utility in studies of mechanism of solvolyses of chloroformate esters. Supprted by INBRE.

Evaluation of Complementation Among Mutants of Human Adenylosuccinate Lyase

Jenna M. Currier, Peychii Lee, and Roberta F. Colman
Department of Chemistry and Biochemistry

Adenylosuccinate lyase (ASL) is an enzyme that catalyzes two distinct reactions in the de novo synthesis of purine nucleotides.  The metabolic importance of this enzyme is shown by the observation of autism, mental retardation, developmental delay, epilepsy and muscle wasting in patients with ASL deficiency.  ASL deficiency is caused by single point mutations of the gene encoding ASL, with many patients being heterozygous for two different point mutations.  We now seek to determine whether human ASL subunits with different point mutations can form hybrids to restore enzymatic activity.  The disease-associated mutations R194C and R396C were constructed by site-directed mutagenesis, expressed in E. coli and purified.  R396C has a specific activity only 27% that of WT human enzyme, but is comparable to WT enzyme in stability; while R194C has similar specific activity to WT, but is more thermally unstable than is the normal enzyme.  Each enzyme has a 6-histidine tag at the N-terminus, allowing it to bind reversibly to a nickel-NTA column.  To separate hybrids as a result of complementation, the His-tag from R396C was cleaved with thrombin, rendering it unable to bind to the Ni-NTA column.  Complementation experiments were conducted by brief exposure of an enzyme mixture (R194C + R396C) to 1.5M guanidine HCl followed by a  five-fold dilution, causing dissociation then reassociation of the 4 subunits.  Using an imidazole gradient on a Ni-NTA column, the hybrid enzymes were isolated, as confirmed through N-terminal sequencing.  Kinetic and stability measurements are now being performed.  (Supp. by HHMI.)

Stabilization of Human Adenylosuccinate Lyase Through Site-Directed Mutagenesis of Cysteine Residues
Michael Dignan
, Lushanti De Zoyza Ariyananda, and Roberta F. Colman
Department of Chemistry and Biochemistry

Adenylosuccinate lyase (ASL) is a metabolically important enzyme that catalyzes two distinct reactions in the purine nucleotide biosynthesis pathway: conversion of adenylosuccinic acid to adenosine monophosphate (AMP) and fumarate, and conversion of succinylaminoimidazole carboxamide ribotide (SAICAR) to aminoimidazole carboxamide ribotide (AICAR) and fumarate. Human ASL exhibits marked instability compared to the ASL of other species because it is easily oxidized and is cold-inactivated. It contains 13 cysteine residues that are not conserved among other species and that are not involved at the catalytic site. Seven of these residues are close enough to form unwanted disulfide bonds upon oxidation: Cys266, Cys304, Cys305, Cys98, Cys99, Cys172, and Cys173. It is possible to stabilize human ASL by keeping it in the presence of reducing agents like dithiothreitol (DTT), but this is cumbersome and often impractical. In order to stabilize the enzyme, alanine residues were systematically substituted for cysteine through site-directed mutagenesis and overexpressed in E. coli. The Vmax and Km for Adenylosuccinate were obtained for the WT, C266/304/305A, C98/99A, C266/304/305/172A, and C266/304/305/173A. All of these enzymes had comparable values for both the Km and Vmax. Circular dichroism was used to compare the secondary structure of the enzymes, and the activity of the WT and mutants were tested under oxidative stress at 37ºC with hydrogen peroxide and oxidized glutathione. (Funding provided by HHMI.)

Bulk Material Properties of β–Bulge Peptide Hydrogels For Tissue Engineering

Stephanie Dreher1, Daphne Salick2, Lisa Haines-Butterick2, and Joel P. Schneider2
Department of Biological Sciences1 and Department of Chemistry & Biochemistry2

Hydrogels are porous, physically rigid, three dimensional matrices primarily composed of water which show promise in the field of biomedical technology as scaffolds to aid in tissue regeneration. Our laboratory has developed a class of β-hairpin peptides that self-assemble into a hydrogel in response to specific stimuli, such as light, pH, temperature, ionic strength, and cell culture media. The parent peptide, MAX1, consists of alternating valine and lysine residues on each β-strand flanking a type II´ β-turn (-VDPPT-). However, the kinetics of self-assembly for MAX1 under cellular encapsulation conditions are too slow, causing cellular sedimentation. Therefore, MAX8 was designed to hasten hydrogelation, resulting in homogeneous gel-cell constructs. These constructs can be prepared in a syringe and subsequently shear thin delivered to target sites while maintaining cellular homogeneity. Although it has been demonstrated that MAX8 is a suitable scaffold for tissue engineering applications, this sequence must be prepared via Fmoc-based peptide synthesis due to the unnatural amino acid DProline. In an attempt to make this material more cost efficient for large-scale application, I have helped design a sequence, BBH8, containing all natural amino acids which can be overexpressed in Escherichia coli. I have synthesized, purified, and characterized the bulk material properties of BBH8 under physiological and cell culture conditions. Initial data show that BBH8 behaves analogously to MAX8, demonstrating that it is an excellent candidate for tissue engineering. Future research entails studying cellular viability and the homogeneity of cellular distribution throughout the material before and after shear-thinning delivery. Funding for my research was provided by NSF CHEM0348323 and the Howard Hughes Medical Institute.

Synthesis and Characterization of New Alkaline-Earth Metal Indium-Antimonides
Jonathan Hullmann
and Svilen Bobev
Department of Chemistry and Biochemistry

Thermoelectric materials may provide a unique solution to the continual problem of increased energy demand due to their ability to convert thermal energy into electricity, and vise versa.   These materials can be utilized in refrigeration units without the need for environmentally harmful CFC’s.  Another promising application of these materials is the conversion of low-level waste heat from automobile engines to electricity; decreasing the use of fossil fuels.   However, thermoelectric materials do not have widespread application today because they are inefficient.  In 2006, the intermetallic compound Yb14MnSb11 was synthesized and found to have four times greater efficiency than the thermoelectric material currently used by NASA, SiGe.   This discovery prompted renewed interest in structurally related antimonides.  Intrigued, we began investigating similar compounds.  Until now we have synthesized and structurally characterized three new materials: Eu11InSb9, Yb11InSb9 and Sr11InSb9, the properties of which are currently being studied. During these investigations, several other new compounds were also discovered: Ba4Sb3, Sr4Sb3 and Eu4Sb3.  These three compounds cannot be made using the traditional solid-state approaches, suggesting In inclusions or substitutions in the crystal structure.  In addition, another “first-of-a-kind” compound, Ba4In3Sb4, was recently discovered.  Exploration of the synthetic conditions for Ba4In3Sb4 versus the still unknown Ba14InSb11 is ongoing.  This research was funded by the Howard Hughes Medical Institute.

Pat was the winner of the Sigma Xi oral presentation competition.

Metal-triggered Hydrogelation of Designed β-hairpin Peptides
Patrick J. Knerr, Christopher Micklitsch, Colin Thorpe, and Joel P. Schneider

Department of Chemistry and Biochemistry

Peptides have been designed which undergo an environmentally-triggered folding transition from random coil to a β-hairpin conformation, responsive to changes in temperature, pH and ionic strength.  These folded, amphiphilic β-hairpins subsequently self-assemble to form a crosslinked, fibrillar network, yield a self-supporting, rigid hydrogel.  This work reports a new folding trigger: zinc-induced hydrogelation.  An unnatural, negatively-charged α-amino acid with a strong propensity to chelate zinc was synthesized and incorporated into the hydrophobic face of a β-hairpin sequence composed of two strand regions connected by a four residue type II’ β-turn.  The negative charge of this residue interferes with hydrophobic interactions necessary for folding and self-assembly; hydrogelation can only be accomplished when a zinc ion chelates to the residue and neutralizes charge to allow adoption of the amphiphilic β-hairpin.  With further sequence modulation, a peptide was designed which can fold and self-assemble at physiological pH only when a stoichiometric amount of the zinc trigger is added.  Such a responsive design is of particular interest in sensor technologies to detect toxic levels of zinc in the environment, in bioremediation and in the design of microfluidic devices and novel nanomaterials.  Funding has been supplied by the National Science Foundation and the Arnold and Mabel Beckman Foundation.

Synthesis of 1,1’- Diaminoferrocene

James Lansing
, John Young, Wes Monillas, David Grieco, and Klaus Theopold
Department of Chemistry and Biochemistry

1,1’-Diaminoferrocene (DAFc) is a redox active ligand whose derivatives have been employed in several uses, such as an olefin polymerization catalyst.[1]  Following an improved literature procedure, its synthesis has become significantly easier and made it accessible as a ligand.[2]  As of now, its uses when complexed to a cobalt cation have yet to be explored.  We hope to complex it to a cobalt cation and oxidize the resulting coordination compound, with the hopes of preparing cobalt in a high oxidation state and with the potential for Proton Coupled Electron Transfer.

The first step in the synthesis of DAFc involved a TMEDA/ n-butyl lithium complex to form 1,1’-dilithioferrocene(1).  It was seen that the addition of even small amounts of air cause immediate decomposition of the lithiated product, thus employing Schlenk line techniques and inert gas protection were of utmost importance.  After workup was performed in a nitrogen atmosphere, 1 was reacted with 1,1,2,2-tetrabromoethane to brominates the species, forming 1,1’-dibromoferrocene(2).  Both temperature and rate of addition of tetrabromoethane played a role in the reaction, which optimized to a 70% yield.  Once 2 was formed, it was reacted with cuprous chloride in ethanol and aqueous sodium azide.  Following the formation of 1,1’-diazidoferrocene(3), a hydrogenation with a palladium on carbon catalyst should yield the diamino product.  As of yet, our efforts to produce and isolate the azide and subsequent amino derivative have failed, but further exploration is being undertaken. Supported by Chemistry Alumni Undergraduate Research Fellowship Program.
[1]Arnold, J., Shafir, A.  Ferrocene-Based Olefin Polymerization Catalysts: Activation, Structure, and Intermediates.  Organometallics 2003, 22, 567-575
[2]Arnold, John, et al.  Synthesis, Structure, and Process of 1,1’-Diamino- and 1,1’-Diazidoferrocene, Organometallics 2000, 19, 3978-3982

Synthesis of a Redox-Active Scorpionate Ligand

Stacey L. Mlynarski
, Jennifer B. Schnitker, and Klaus Theopold
Department of Chemistry and Biochemistry

Scorpionates are one of the most used classes of ligands in modern chemistry; they find applications in materials science, catalysis, and bioinorganic chemistry.  Tris(pyrazolyl)borate ligands (i. e. Tp) are the most common tridentate scorpionates.  These ligands are useful due to their versatility which is made possible by the modification of the substituents on the pyrazolyl ring, thereby altering the steric effects exerted upon the metal center.  Here, we study the synthesis of a new scorpionate ligand with ferrocenyl and methyl groups attached to the pyrazole ring, i. e. tris(3-ferrocenyl-5-methyl pyrazolyl)borate, TpFc,Me).  This ligand will be used to elucidate the effects of the redox-active ferrocene groups.  Sequential oxidation of the three ferrocenyl moieties is expected to dramatically effect the reactivity of the metal atom coordinated by the new ligand. Funded by INBRE.

Isolation and Purification of Vanadium Haloperoxidase Mutants for Characterization by 51V Solid-State NMR
Kristin M. Nuzzio, Stephanie E. Bolte and Tatyana Polenova
Department of Chemistry & Biochemistry

Vanadium haloperoxidases are a specific class of vanadium containing enzymes commonly found in marine algae, lichens and terrestrial fungi. These enzymes are the most efficient halide oxidants known to date. Vanadium containing compounds have shown excellent potential in the treatment of diabetes, particularly as insulin enhancing compounds, as well as in the treatment of some forms of cancer. However, in order for these compounds to be useful in biomedical applications, the structure of the vanadium active sites and the mechanism of their biochemical activity need to be determined. The focus of our work is to understand the catalytic mechanism of vanadium haloperoxidases and their active site mutants by utilizing 51V solid-state NMR. This knowledge is expected to be important in the design of artificial vanadium enzymes with tuned halogenating activities. We have established protocols for the expression, isolation and purification of recombinant vanadium chloroperoxidase (VCPO) using a Saccharomyces cerevisiae overexpression system (Strain BJ1991 with the vector PTNT14). We have successfully isolated and purified two VCPO active site mutants: R360A and R490A. We have quantified the concentration of the purified protein and begun to crystallize the wild type protein using the hanging drop method to form well-ordered crystals. These protocols are necessary for preparation of the protein samples for subsequent 51V solid-state NMR spectroscopy studies. 51V solid-state NMR will be used to directly probe the diamagnetic “spectroscopically” silent vanadium sites in biological systems, which will encompass both insulin mimetics and vanadium haloperoxidases. Funding for this research was provided through the Chemistry & Biochemistry Alumni Scholars Program.

Alkyne Metathesis in Natural Product Synthesis
Andrea J. Passarelli and Douglass F. Taber
Department of Chemistry and Biochemistry

Developing new methodologies for organic synthesis is on the forefront of chemistry research today.  Organic molecules can be used in many arenas, from pharmaceuticals to agriculture to cosmetics. We are seeking a new route to the synthesis of medium-sized rings, specifically muscone and exaltone, two naturally occurring 15-membered ring compounds.  Currently, precursor molecules have been synthesized in preparation for the ring-closing step.  We have successfully created a 15-carbon di-alkyne alcohol in a two-step synthesis.  Current work is focused on creating an isoxazolone ring with a bromine-containing side chain which will be used to synthesize muscone.  We would like to thank the Howard Hughes Medical Institute for funding of this project.

Preparation and Pasteur Resolution of Racemic Hydrobenzoin

Michael T. Pirnot and Douglass F. Taber
Department of Chemistry and Biochemistry

In 1849, Louis Pasteur separated racemic tartrate through crystallization and physical separation of the enantiomerically-pure crystals. This has come to be called “Pasteur resolution.”  It was also reported many years ago that racemic hydrobenzoin also resolved spontaneously in crystallization. Thinking that this would make a good organic teaching lab experiment, we were faced with two questions: 1. How to prepare racemic hydrobenzoin; and 2. How to carry out the crystallization so the enantiomeric crystals could be separated. Funding for this research was provided through the Chemistry & Biochemistry Alumni Scholars Program.

Understanding the Mechanisms of Reaction of Aryl Chloroformates 
Darneisha Reed1, Fumie Koyoshi, Malcolm J. D’Souza1, Dennis N. Kevill2
1Department of Chemistry, Wesley College, Dover, Delaware
Department of Chemistry & Biochemistry, Northern Illinois University, DeKalb, Illinois

Aryl chloroformates are extensively used in the synthesis of polyurethanes, optical polycarbonates, diaryl carbonates, and carbamates.  Thus, gaining mechanistic insight of such bioactive molecules by examining the effects of solvent variation on their rates of reaction is important to expand our understanding of many biological processes. Here we present correlation analysis of the specific rates of solvolysis of p-methoxyphenyl chloroformate (1) in 44 solvents using the Kevill-D’Souza three-term extension of the Grunwald-Winstein equation (previously proposed for compounds containing an aromatic ring adjacent to the reaction center).  The experimental results for p-methoxyphenyl chloroformate (1) when compared to those obtained for phenyl chloroformate (2), suggests very similar mechanistic characteristics for the solvolyses of these two chloroformate esters. Supported by INBRE.

A Novel Approach to Altering Estrogen Receptor Function

Tara Rhoades and John Koh
Department of Chemistry and Biochemistry

Many compounds have been developed for the purpose of altering the function of the estrogen receptor (ER) in order to treat ovarian and breast cancers as well as other ailments.  While most are drugs that either target ER by mimicking the function of the natural ligand estrogen (agonists) or by blocking the binding of the hormone (antagonists), this project explores the question of whether small molecules can be designed to alter ER function by acting as synthetic acetyl transferases.  Recently, it has been shown that ER acetylation is an important mechanism for regulating estrogen-dependent activities that have been linked to the pathology of ER-dependent cancers.  In order to test this new paradigm of ER-regulation, an artificial acetyl transferase was designed, one end of which consists of the artificial ER ligand tamoxifen.  At the opposite end of the molecule is a thioester, the same functional group which executes ER acetylation in nature.  The two ends are tethered by a molecular arm which extends from the ER ligand binding domain (to which tamoxifen should bind as usual) toward the lysines of the receptor that are known to naturally undergo acetylation (which should also occur artificially by means of the terminal thioester of this molecule).  While the effects on gene transcription and expression due to this modification of the estrogen receptor are unknown, if successful, this approach would represent an entirely new way to alter ER function by a drug-like molecule.  This project is supported by HHMI.

An Investigation of Cr(II) Pyrrolide Diimine Ligand Complexes
Jennifer B. Schnitker
, John F. Young, and Klaus H. Theopold
Department of Chemistry and Biochemistry

Pyrrolide diimine ligand complexes have shown potential as transition-metal catalysts for olefin polymerization.  These ligands can bind in mono-, bi-, or tridentate fashion to the metal atom to stabilize higher oxidation states through nitrogen donor atoms, and also, the addition of substituted aryl groups to imine nitrogens can provide steric protection to the metal site. Current research has focused on exploring the 2,5-bis{[(2,6-diisopropylphenyl)imino]methyl}pyrrol-1-ide ligand (L) and was started with the previously synthesized Cr(II)L2 complex.  Work with this complex has been done to create a high valent chromium atom through oxidation, to further determine chemical characteristics which may make it suitable for group transfer catalysis.  Also, a synthesis of the Cr(II)LX complex has been in development this summer.  Following confirmation of the synthesis of the mono-ligand complex, further investigation of its capabilities as a low-valent compound will be undertaken.  This should give insight in its potential for ethylene trimerization and small-molecule activation. Funding from the Howard Hughes Medical Institute and from the Department of Energy is gratefully acknowledged. 

Dual-Domain Dynamic Multivariate Spectrometer

Jachin Spencer and Sharon Neal

Department of Chemistry and Biochemistry

The goal of this project is to develop a Dual-Domain Dynamic Multivariate Spectrometer, by expanding a time domain spectrometer system to include frequency domain measurements.  The advantage of taking data in either the time domain or frequency domain is determined by what you are trying to measure.  Because the frequency domain and time domain are related by a Fourier transform it is a common practice to simply convert to the desired domain if necessary.  The objective of the Dual-Domain Dynamic Multivariate Spectrometer is to develop a system that takes measurements in either domain natively.  This poster is summary of my work on this project so far adapting the spectrometer.  The project is divided into two phases.
Phase 1:  Developing an independent remote control system for the ISS K2 Optical Bench.
<>Phase 2:  Adapting the Picostar HRI to interact with a third party signal generator, collection of Dual Domain Data.
This poster summarizes my progress thus far through phase one of the project.  A custom code solution was chosen to best satisfy the phase 1 objective, because of the time and technical requirements.  Funded through the NIH Bridges Program between University of Delaware and Delaware Technical and Community College.

Surface-mediated differentiation of osteoblastic stem cells
using a novel hydroxyapatite-binding peptide conjugated with osteoinductive ligands

Amy Styer
, Lisa Capriotti, Bill Theilacker, Joel P. Schneider, Thomas P. Beebe
Department of Chemistry and Biochemistry

Bone tissue engineering employs osteoinductive (inducing bone stem cell differentiation or maturation) or osteoconductive (providing favorable surfaces for bone growth) materials to facilitate the repair of large breaks, or bone ravaged by cancer or osteoporosis.  This research investigates the bone tissue engineering potential of a novel peptide designed by Lisa Capriotti in the laboratories of Thomas Beebe and Joel Schneider. The peptide, JAK1, resembles osteocalcin, the most abundant non-collagenous protein in bone, in its surface-induced conformational folding at the surface of hydroxyapatite (HA), a model for bone apatite.  Both JAK1 and osteocalcin contain acidic gamma-carboxyglutamic acid residues (Gla) whose two carboxyl groups bind to surface calcium inducing peptide folding.  The Schneider lab specializes in solid-phase peptide synthesis (SPPS), used for JAK1 synthesis.  SPPS requires amino acid residues with special protecting groups. I synthesized each product of the first four steps of the eleven-step synthesis of Fmoc and t-butyl protected L-Gla several times at the twenty-gram scale.   The peptide BFP4 is an osteoinductive peptide based on a receptor-binding region of bone morphogenetic protein-2.  In the Beebe lab, 3-mercaptopropyltrimethoxyilane (MTS) and N-gamma-malimidobutyryloxy succinimide ester (GMBS) are used to covalently attach proteins and peptides to glass substrates.  This attachment can be characterized by contact angle goniometry, X-ray Photoelectron Spectrometry (XPS), and Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS).  After working out the techniques, MTS/GMBS chemistry will be used to attach BFP4 to surfaces for controlled tests of osteoblastic stem cell differentiation.  Finally, BFP4 will be conjugated to JAK1 for surface induced bioactivity. Funded by the Undergraduate Research Program’s Science and Engineering Scholars

Synthesis and Characterization of a New Tridentate Phosphine-Thioether Ligand
for Coordination Chemistry and Bioinorganic Modeling
Stephen J. Tereniak, Matthew T. Kieber-Emmons, Nathan A. Eckert, and Charles G. Riordan
Department of Chemistry and Biochemistry

Certain acetogenic, methanogenic, and sulfate-reducing organisms use a bifunctional enzyme, acetyl coenzyme A synthase/carbon monoxide dehydrogenase (ACS/COdH), to synthesize and break down acetate (1).  The mechanism of the final step of the synthesis of acetyl coenzyme A is a point of contention among scholars in the field of bioinorganic chemistry.  Recently, the Riordan group used the ligand triphos (triphos = PhP(CH2CH2PPh2)2) in metal complex syntheses to model the final step of the synthesis of acetyl coenzyme A (2).  The target of the first half of this project, begun in the spring of 2007, was to synthesize and isolate a new ligand containing phosphine and thioether donors, ‘PS2iPr’ (PS2iPr = PhP(CH2CH2SPri)2).  Herein is reported the synthesis and characterization of the PS2iPr ligand by proton and phosphorus nuclear magnetic resonance spectroscopies and chemical ionization mass spectrometry (CI/MS).  The goal of the second half of the project involved synthesizing cobalt and nickel complexes supported by the PS2iPr ligand.  Progress towards this latter goal will be presented. Supported by Chemistry Alumni Undergraduate Research Fellowship Program.
(1) Ragsdale, S. W. Crit. Rev. Biochem. Mol. Bio. 2004, 39, 165.
(2) Eckert, N. A.; Dougherty, W. G.; Yap, G. P. A.; Riordan, C. G. J. Am. Chem. Soc. 2007, 129, 9286.

Synthesis and Applications of Complex Molecules with
a-Substituted Guanidiniums
Sasha J. Wagner and Neal J. Zondlo
Department of Chemistry and Biochemistry

The guanidinium functional group enables and mediates interactions between proteins, DNA, and RNA.  Therefore, guanidiniums have a variety of applications in molecular recognition and pharmaceutical chemistry.  To encourage specific recognition, an amino acid is coupled to a chiral base molecule and the amino acid is guanylated.  This process converts the free amine to a guanidinium.  Specific activity of the previously-described molecule can be modified by manipulating the identity and stereochemistry of the base molecule, such as a 1,2-diaminocyclohexane, and the side chain and stereochemistry of the amino acid.  The characteristics of the diamine and the properties of the amino acid side chain allow different parts of the overall molecule to work in cooperation with one another to increase its affinity for specific target areas.  We have isolated both R,R and S,S-(1,2)-diaminocyclohexane and coupled them to Boc-protected L-phenylalanine.  After Boc deprotection, the free amines were guanylated using Moroder's reagent.  It was imperative that the Boc-protected guanidinium groups were deprotected under mildly acidic conditions, in order to minimize epimerization.  These complex molecules, with -substituted guanidiniums, can be used for specific recognition of target areas, such as RNA or telomeric G-quadruplexes, whose elongation is involved in the majority of cancers. (Funded by Chemistry Alumni Scholars Program).

Design of a Protein Kinase-Inducible Domain for CDK5.

Sara E. White
and Neal J. Zondlo
Department of Chemistry and Biochemistry

<>Many human diseases are linked to phosphorylation leading to changes in protein kinase activity. To measure these changes, protein kinase inducible domains employ the use of a canonical motif that is sequence-specific, and will undergo a structural change when phosphorylated. These peptide sequences are non-florescent when not phosphorylated and highly florescent when phosphorylated as detected through terbium binding.  A protein kinase inducible domain has been developed for CDK5, a protein that is involved in phosphorylation of postmitotic neurons and is a strong candidate for therapeutics for Alzheimer’s disease. Two peptide sequences phosphorylated by CDK5 have been identified from Src and the His H1. Kinase-Inducible domain peptides were designed to include a metal-binding loop and a CDK5 recognition sequence. Peptides were synthesized via Fmoc solid-phase peptide synthesis and characterized by HPLC and ESI-MS. Trityl-protected serine and threonine were incorporated into the peptide sequences to allow for selective phosphorylation. In the presence of Tb3+ increased florescence was detected for both peptides when phosphorylated as compared to the non-phosphorylated species, thus indicating that peptide-metal complex formation is dependent on phosphorylation.  This suggests that a pKID for CDK5 may act as a sensor of protein kinase activity and enable the investigation of mechanisms involved in phosphorylation-induced structural changes of proteins. Supported by the Science and Engineering Scholars Program.  

Isolation and Characterization of a Complex Between Glutathione S-Transferase Pi and c-Jun N-Terminal Kinase
Chati L. Zony, Anastasia.G. Fuzaylova and Roberta F. Colman
Department of Chemistry and Biochemistry

Glutathione S-Transferase pi (GST pi) plays a vital role in cell detoxification and has recently been shown to interact with c-Jun N-terminal kinases (JNKs), a kinase family involved in the Mitogen Activated Protein Kinase cascade. Normally, JNKs are essential components involved in the initiation of apoptosis triggered during periods of cellular stress. In cancerous cells, GST pi is overexpressed and has been postulated to inactivate JNKs through the formation of a complex, which in turn inhibits apoptosis, leading to tumor survival and proliferation. The complex has been observed in vivo, but isolation and characterization of this complex in vitro has not been accomplished.  We now seek to isolate the complex of GST pi and two isoforms of JNK : JNK1a2 and JNK2a2. Current work with JNK2a2 involves its incubation with a histidine-tagged GST pi in HEPES buffer (pH 7.8), containing 12.5% 1,6-hexanediol to dissociate the dimers, followed by dialysis in the same buffer but lacking 1,6-hexanediol, thus allowing complex formation. The complex is purified using a nickel- nitriloacetic acid agarose column and an imidazole gradient. Preliminary results show that under current conditions, the complex is formed but dissociates during the imidazole gradient. Studies are currently underway to determine more favorable conditions to stabilize and purify the complex. We have also inserted the cDNA for JNK1a2 into pET15b, a bacterial vector which yields the target protein with an N-terminal histidine-tag. This new construct will be used to express and purify JNK1a2 for similar experiments in complex isolation. By understanding the basis for the JNK/GST pi interaction, inhibitors of the interaction may be designed which should enhance the beneficial effects of apoptosis to tumor tissues. Funded by the Ronald E. McNair Scholars Program and the Howard Hughes Medical Institute.

Aerosol Analysis Using A Scanning Mobility Particle Sizer

Steven M. Zucker, Julie A. Lloyd, Murray V. Johnston
Department of Chemistry and Biochemistry

The goal of atmospheric aerosol research is to understand the source, transformation and fate of airborne particles.  Laboratory generated particles play an important role in this work, serving both as well-characterized reactants in laboratory studies of gas-particle reactions and as test particles for evaluating and checking the performance of instrumentation.  The goal of this project is to generate and characterize test particles for a new instrument that analyzes organic and biological components in particles.  A Scanning Mobility Particle Sizer (SMPS) was used to measure the number and mass concentrations of erythromycin particles.  Solutions were prepared at various concentrations and all particles were generated with a constant output atomizer.  It was observed that as the concentration of solute increases, the mass concentration of particles increases linearly, but the number of particles increases logarithmically.  The effect of solvent on particle generation was also studied.  The number of erythromycin particles was found to be the same whether using 100% methanol or 100% ethanol.  When the erythromycin solute was 50% water by volume, the number of particles generated decreased significantly.  The above results were obtained with a constant output atomizer.  Current and future work will focus on the use of other aerosol generation methods such as the electrospray ionization generator and the Collison Nebulizer.  Funding for this research was provided by the Delaware Space Grant Consortium.

Links: Summer 2007 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  19 July 2007. Last up dated 21 August 2007 by Hal White
Copyright 2007, University of Delaware