Abstracts from the Department of Chemistry and Biochemistry
Undergraduate Summer Research Symposium August 9, 2006

Ordered alphabetically by student's last name

Bourreza Hullman Karpowicz Kotowski Meyers Passarelli Schnitker Silverio Thornley
Dignan Jegede Knerr Koyoshi Omondi Patel, A Shuman
Smith, A Zony, C


Chemical Complementation of Androgen Receptor Mutations Associated with Androgen Independent Prostate Cancer
Aly Bourreza
, John T. Koh, Paula McGinley
Department of Chemistry and Biochemistry

Prostate cancer remains the second leading cause of cancer death in men.  As prostate tissue is dependent on androgens for growth and homeostasis, anti-androgens, such as bicalutamide (Casodex®), used alone or in conjunction with chemical castration have been used in the treatment of prostate cancer for decades.  However, as many as 30% -40% of patients treated with anti-androgens become resistant to anti-androgens within five years of treatment.  Androgen receptor mutations have been found in androgen independent prostate cancer.  They cause anti-androgens to act as agonists with the mutant.  It is believed that anti-androgens can be synthesized in such a way that they remain functional antagonists in mutant forms of the androgen receptor. The novel compounds were synthesized based on molecular modeling designs and crystallography structures of hormones and the androgen receptor.  The androgenic and anti-androgenic effects of novel compounds in competition with synthetic androgen methyltrienolone (R1881) will be measured with luciferase reporter gene assays.  This research may lead to new, effective treatments for patients with androgen-independent prostate cancer. Supported 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 presumably not involved at the catalytic site. Six of these residues are close enough to form unwanted disulfide bonds upon oxidation: Cys266, Cys304, Cys305, Cys180, Cys172, and Cys173. It is possible to stabilize human ASL by keeping it in solution with reducing agents like dithiothreitol (DTT), but this is cumbersome and often impractical. In order to stabilize the enzyme, serine residues were systematically substituted for cysteine through site-directed mutagenesis and overexpression in E. coli. A Vmax and Km were obtained for the WT, C266S, C304S, C305S, and C180S. 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. The thermostability of the mutants was tested at 60ºC and compared to the wild type (WT). C266S lost activity within 5 minutes, while the rest only lost activity over a period of hours.  (Funding provided by HHMI.)

Synthesis of New Alkaline Earth and Pnicogen Compounds

Jonathan E. Hullmann and Svilen Bobev
Department of Chemistry and Biochemistry

While pursuing the synthesis of compounds in the Zintl phase structure type Ca11GaSb9 two new alkaline earth and pnicogen compounds, Sr4Sb3 and Ba4Sb3, were serendipitously discovered.  Previous attempts to synthesize these two compounds using stoichiometric ratios had failed.  The use of In and Ga fluxes as a solvent to lower the melting point of other phases allowed for the evasion of the thermodynamically stable and exceedingly prevalent AE11Pn10 phase (AE= Ca, Sr, Ba; Pn= Sb, Bi).  These new compounds belong to the anti-Th3P4 structure type and crystallize in the space group I-43d, number 220.  The crystallographic unit cell parameters were found using single crystal X-ray diffraction to be a=9.91(1) Å, and a=10.28(2) Å respectively.  These results were then confirmed by powder diffraction.  Attempts to synthesize the remaining compounds in this series are underway. Supported by NSF Summer Research Program in Solid State and Materials Chemistry

Identification of a Small Nucleolar RNA in Microsporidia

Yewande Jegede1
, Lisa Frerichs2, Debra Barninger3, Peter DiMaria2
Department of Biology, Columbia University, 2Department of Chemistry, Delaware State University, and  3Department of Biology, Wesley College

<Microsporidia are eukaryotic obligate intracellular parasites that are interesting for a number of reasons.  Medically, they are opportunistic pathogens in AIDS patients and other immunocompromised individuals.  These organisms also are highly novel in terms of their molecular biology.  This includes the presence of ribosomal RNAs (rRNAs), which are prokaryotic-like in terms of their size and other characteristics.  In other eukaryotes, it is well established that small nucleolar RNAs (snoRNAs) act as guides in directing the variety of nucleotide modifications associated with rRNAs.  These include the Box C/D and Box H/ACA snoRNAs which are responsible for 2’-O-nucleoside methylation and pseudouridylation, respectively.  However, little is known about snoRNAs and rRNA modification in microsporidia.  In the present study, we report the identification of the first example of a snoRNA in a microsporidian (Vairimorpha necatrix).  The identified RNA, designated RNA1, was characterized at both the gene and RNA level.  RNA1 is estimated to be 63 nt in size and contains sequence elements associated with the Box C/D family of snoRNAs. These include C and D boxes and a guide sequence.  RNA1 appears to be an ortholog to snoRNAs, snR38 (yeast and mouse) and snR38A, B, and C (human).   These snoRNAs are responsible for the guide-directed 2’-O-methylation of a specific guanosine residue within a region of the large subunit (LSU) rRNA, whose sequence is highly conserved among eukaryotic species including microsporidia.  Further studies will ascertain whether this site is similarly modified in microsporidian LSU rRNAs.  This project was supported by NIH grant to Delaware 2P20 RR016472-06 from the NCRR.

Progress on the Synthesis of Asymmetric Metallofoldamer Catalysts
Richard Karpowicz,
Dorthey Dong, and Joseph M. Fox
Department of Chemistry and Biochemistry

Described is the concept of using metallofoldamers in the design of asymmetric catalysts. It is widely known that salen 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.  Recent progress has been on the synthesis of a second generation 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 a chiral primary coordination sphere. Supported by a Pfizer Undergraduate Research Fellowship.

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 intramolecular folding from random coil to β-hairpin conformation, triggered by specific environmental conditions.  The folded, amphiphilic β-hairpins then self-assemble to form an interlaced fibrillar network, converting the material from a liquid to a self-supporting, rigid hydrogel.  Such responsive materials show great promise in tissue engineering, serving as scaffolds which mimic the extracellular matrix by allowing cellular adhesion and proliferation.  Past work has demonstrated that pH, temperature, ionic strength and light can be utilized to stimulate the folding event.  We are currently interested in developing a new trigger: metal-induced hydrogelation.  Unnatural, multidentate α-amino acids have been synthesized to chelate strongly to divalent metal ions, most notably calcium or zinc.  These ligands are subsequently incorporated into the primary sequence of a 20 residue peptide composed of two strand regions connected through a four residue type II’ β-turn.  This affords peptides in which folding and self-assembly is prevented at physiological conditions in the absence of the metal ion, but triggered when the ion is present.  Funding for this work has been supplied by the Arnold and Mabel Beckman Foundation.

Determination of Microbes in Ambient Air using MALDI-TOF MS and Proteomics

Don Kotowski
, Ann M. O’Brien, and Murray V. Johnston
Department of Chemistry and Biochemistry 

In this study proteomics and mass spectrometry was used to identify proteins from unknown microbial communities to classify naturally occurring microbes in ambient air. Previous studies show that unknown microbes can be identified through identification of the microbe’s proteins. Air samples from the University of Delaware campus were collected on SKC Biocassettes® containing Luria-Bertani (LB) agar. The samples were incubated at room temperature for 3 days to promote microbial growth and streaked to isolate the colonies. Individual colonies were picked, added to LB broth, and further cultured in an orbital shaker at 37ºC overnight. The cultures were centrifuged for 20 minutes at 5K rev/min to pellet out the cells, which were then washed twice with Millipore water. The cells were then lysed via French Press.  The soluble protein fraction collected and concentrated for one dimensional (1D) sodium dodecyl sulphate (SDS) polyacrylamine gel electrophoresis (PAGE). The 1D SDS-PAGE served to separate the proteins according to their molecular weight therefore simplifying the sample matrix prior to mass spectral analysis.  Individual protein spots were excised and an in-gel tryptic digestion was performed. Peptide mass fingerprinting was used to identify the unknown proteins.  MALDI spectra of the protein digests were obtained on a BRUKER Omniflex (Bellerica, MA). Peak lists were generated for each peptide digest and searched against known protein databases with the MASCOT search engine (www.matrixscience.com). Identification of a family of proteins resulted in species level identification of the collected airborne microbes. This project was funded by NSF EPSCoR under grant number EPS-0447610.  

An Evaluation of Solvolytic Mechanisms at a Trigonal Carbon
Fumie Koyoshi1, Malcolm J. D’Souza1, Dennis N. Kevill2
1Department of Chemistry, Wesley College, Dover, Delaware
2Department of Chemistry & Biochemistry, Northern Illinois University, DeKalb, Illinois

Background:  Acyl halides are important pharmaceutical intermediates.  As a result there has been there has been significant interest in their hydrolysis, alcoholysis, and aminolysis processes, as such reactions are useful models for enzymatic mechanisms.  For example a recent study (Klis, W. A.; Sarver, J. G.; Erhardt, P. W.; Tetrahedron Letters, 42, 2001, 7747-7750), proposed a concerted (SN2) process for the methanolysis of paclitaxel-related derivatives having a chloroacetyl substituent on the C2’ oxygen.  Methods:  The effects of solvent variation of the available specific rates of solvolysis of phenyl chloroformate, acetyl, chloroacetyl, and α-methoxy-α-(trifluoromethyl)phenyl acetyl chlorides, are analyzed in terms of the extended Grunwald-Winstein equation using the NT scale of solvent nucleophilicity (S-methyldibenzothiophenium ion) combined with a YCl scale based on 1-adamantyl chloride solvolysis. Results:  Analysis of the data for phenyl chloroformate, chloroacetyl, and α-methoxy-α-(trifluoromethyl)phenyl acetyl chlorides, leads to similar sensitivities for l (the sensitivity to changes in solvent nucleophilicity) and m (the sensitivity to changes in solvent ionizing power).  This indicates that all three substrates follow an addition-elimination pathway, whereas, magnitude of l and m values obtained for acetyl chloride can be rationalized in terms of an ionization pathway (SN1) with appreciable nucleophilic solvation of the incipient carbocation. Conclusions:  The results obtained are consistent with our earlier suggestion that acid chlorides of monoesters of carbonic acid and of carboxylic acids tend to solvolyze with competing addition-elimination (with rate-determining addition) and ionization SN1 (assisted by nucleophilic solvation) pathways.  [Grant support: NIH grant 2 P20 RR016472-04 from the NCRR]

Synthesis and Characterization of Novel REGeSn Compounds (RE = Y, Gd - Tm)

John J. Meyers, Jr.
and Svilen Bobev
Department of Chemistry and Biochemistry

Recent work has afforded a new series of intermetallic, solid state compounds with unique and interesting chemical and structural properties.  Our recent work in the rare-earth series REGe1+xSn1-x (0 < x < 0.3) has provided much insight.  Crystallizing in the orthorhombic space group Cmcm (No. 63), the structure can be regarded as alternating planes of zigzag chains of Ge atoms and a square-shaped network of Sn atoms extending down the crystallographic b-axis.  These planes are separated by the rare-earth cations.  Single-crystal and powder X-ray diffraction data has allowed for structural refinements showing no vacancies and a slight mixing of Sn-Ge sites within the crystal structure.  At present time, we are working for a more systematic synthetic route for optimization of procedures as well as obtaining low-temperature magnetic and calorimetric measurements.

Use of LFERS to Evaluate the Solvolytic Mechanism of Isopropenyl Chloroformate

Arnold O. Omondi1, Kevin E. Shuman1, Malcolm J. D’Souza1, Dennis N. Kevill2
Department of Chemistry, Wesley College, Dover, DE 19901
Department of Chemistry & Biochemistry, Northern Illinois University,   DeKalb, IL 60115 

Since World War I there has been significant interest in Isopropenyl Chloroformate due to its ability to cause sharp pain in the eyes upon exposure of the evaporating gas.  In 1915 Phosgene was first employed as a war gas because it readily dissolved in Acetone to form Isopropenyl Chloroformate which undergoes rapid hydrolysis to produce corrosive HCl.

Recently Isopropenyl Chloroformate was used in the esterification of pMOBA (peptidyl p-methoxybenzoic acid).  This use was advantageous compared to the more commonly used Isobutyl- and Ethyl Chloroformates, because the former can produce acetone. This knowledge has been directed in the development of selective inactivators for serine proteases that have been known to play major roles in human health e.g. protein turnover, coagulation, fibriolysis and complement activation, which in most cases lead to the offset of diseases such as emphysema, cystic fibrosis, cancer, systemic lupus erythematosus, and myocardial infarction. Here we expand previous kinetic studies of Isopropenyl Chloroformate in predominantly aqueous alcohol solvents with varying solvent nucleophilicity and solvent ionizing power. This analysis includes 1,1,1,3,3,3- Hexafluoro-2- Propanol (HFIP) and 2,2,2-Trifluoroethanol (TFE) that exhibit strong hydrogen bonding and can dissolve a large number of biological molecules with receptive sites such as oxygen, double bonds or amine groups.  [This project was supported by NIH NCRR INBRE grant number; 2 P20 RR016472-06]


Bromomandelate Formation and Addition of Ethyl Acrylate to a Cyclic Bromomandelate

Andrea J. Passarelli
and Douglass F. Taber
Department of Chemistry and Biochemistry

We have been studying the formation of bromomandelates and the addition of ethyl acrylate to a cyclic bromomandelate.  The bromomandelation technique has been optimized in an easy, one-pot synthesis using (S)-mandelic acid, a nitrogen-containing base, N-bromosuccinamide, and an alkene in dry CH2Cl2.  Our most significant discovery was the high selectivity of the base 2,6-lutidine towards adding the mandelate to the secondary position of the bromonium ion. We were also able to successfully form a carbon-carbon bond by adding ethyl acrylate to a cyclic bromomandelate.  This was done using a free radical chain reaction to abstract the bromine atom and add the ethyl acrylate by reduction of the double bond.  After oxidation with Dess-Martin Periodinane and lactonization with K2CO3, we were able to determine that this carbon-carbon bond formation proceeded with about 2:1 selectivity towards the trans diastereomer.  We would like to thank the Howard Hughes Medical Institute for funding this project.

DLC8 Determination using Solid-state NMR

Anant Patel
, Shangin Sun, and Tatyana Polenova
Department of Chemistry

Dynein light chain, the 8 kDa protein, is a crucial subunit in the motor protein complex of cytoplasmic dynein.  The cytoplasmic dynein complex is used in transporting various cellular cargoes amongst cells via microtubules.  The light chain subunits are directly responsible, but not limited solely, for binding the various cargoes onto the larger motor protein complex.  Recent studies have shown the light chains abilities to regulate different biological processes by means of binding various proteins and enzymes.  The studies conducted recently to discover such functions of the light chain can be attributed to the solution-state NMR technique used for the structural determination of the protein.  The focus of this research is to perform solid-state NMR studies on the DLC8 in order to obtain results highly similar if not the same to that of the solution-state NMR study.  The protocols for expression and purification of the unlabeled DLC8 protein have been successfully formulated.  Construction of isotopically labeled samples of DLC8 are under way and necessary for solid-state NMR analysis.  Funding for this project was from the Howard Hughes Medical Institute.

Synthesis of a Novel Redox-Active Scorpionate Ligand

Jennifer B. Schnitker
and Klaus H. Theopold
Department of Chemistry and Biochemistry

The scorpionates are an ever-growing class of versatile chemical compounds that have common applications in catalysis, enzyme modeling, metal extraction, and biomedicine.  These tridentate ligands are frequently complexed with metals, binding in such a fashion that allows for the control of the remaining coordination sites on the metal.  Additionally, the pyrazol ring can be modified with substituents, whose steric effects often have significant impacts on the chemistry of the ligand.  The goal of current research is to synthesize a scorpionate ligand with ferrocene and methyl groups attached at the 3 and 5 sites of the pyrazol, respectively.  With this ligand, an examination of the effects of the redox-active ferrocene groups can occur.  By successively oxidizing the ferrocene groups, we can evaluate the chemical impact of an increasingly positively charged environment on the scorpionate ligand.  Funding from the Howard Hughes Medical Institute and from the Department of Energy is gratefully acknowledged. 

A New Method for the Deprotection of Aryl Mesylates.
Daniel L. Silverio and Douglass F. Taber
Department of Chemistry and Biochemistry

The mesyl group has been shown to be useful for protecting the hydroxyl group in phenols.  A key issue with any protecting group is the development of a method to remove it that is facile, efficient, and preserves functionality elsewhere in the molecule.  A new method developed in the Taber group uses heated diethylamine in a high pressure reaction vial to accomplish this goal.  A major advantage of this is that the diethylamine acts as both the solvent and the reagent in the reaction. Funding provided by the Howard Hughes Medical Institute. 

Synthesis and Applications of Arginine Mimetics

Alfred Smith, Shalini Balakrishnan, and Neal Zondlo
Department of Chemistry and Biochemistry

Protein-protein, protein-RNA, and protein-DNA interactions are broadly mediated through the guanidinium functionality of arginine residues.  However, specific recognition by arginine is limited, because the guanidinium functionality is attached to a linear alkyl group.  To achieve specific molecular recognition, arginine mimetics are used, which place functional groups adjacent to a guanidinium.  In order to specifically target arginine-mediated recognition, we developed convenient syntheses of alpha- guanidino acids, in which the amine of an amino acid is converted into a guanidinium.  The alpha-substituted guanidiniums of guanidino acids and the side chain of the amino acid work synergistically toward molecular recognition with greater affinity for the target site.  We have designed arginine mimetics for specific and high affinity molecular recognition by coupling protected guanidino acids to alcohol and amine nucleophiles. Protected guanidino acids of Gly, Phe, Val, and Leu were readily synthesized from methyl esters of alpha-amino acids by guanylation of the amine with bis-boc-thiourea and Mukaiyama’s reagent.  Protected guanidino acids, with a free carboxylic acid for coupling to nucleophiles, were generated by saponification of the methyl ester using LiOH.  Arginine mimetics were synthesized by coupling protected guanidino acids to hydroxyl and amino groups to generate complex alpha-substituted guanidiniums.  Molecules containing alpha-guanidino acids will be applied to specific protein and RNA recognition.  Funding from the National Institute of Health and Howard Hughes Medical Institute Undergraduate Science education Program.

Phosphorylation-Dependent Protein Design: Minimal 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 Ca.  The peptides were synthesized using solid phase peptide synthesis and characterized by HPLC and ESI mass spectrometry.  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.

Elucidation of the Subunit Interface in the Heterodimer of Glutathione S-Transferase pi and 1-Cysteine Peroxiredoxin

Chati Lum Zony, Luis Ralat, and Roberta F. Colman
Department of Chemistry and Biochemistry

Glutathione S-transferase pi (GST pi) has been shown to reactivate 1-cysteine peroxiredoxin (1-Cys Prx) by the formation of a heterodimer complex [Ralat, Manevich, Fischer and Colman Bioc (2006), 45, 360-372]. A model of the heterodimer has been postulated with the glutathione binding site of the subunit interface. We now seek to evaluate this model of GST pi – 1-Cys Prx interaction by testing the ability of peptides, derived from various regions of GST pi, to inhibit the formation of the heterodimer complex. GST pi is digested with V8 protease in ammonium bicarbonate buffer at pH 7.8 and the peptides purified by HPLC. The heterodimer complex is formed by incubation of the two proteins at pH 8.0 in buffer containing 20% 1,6-hexanediol to dissociate the homodimers, followed by dialysis against a buffer containing 2.5mM glutathione (GSH) but lacking l,6-hexanediol. The heterodimer canbe purified by chromatography on a nickel-nitroloacetic acid agarose column in the presence of GSH.  We are now comparing the amount of heterodimer complex formed when the incubation of the two proteins and dialysis to remove hexanediol are conducted in the absence and presence of various purified, sequenced peptides of GST pi. The studies are expected to reveal the regions of GST pi which interact with 1-Cys Prx. Supported by HHMI and McNair programs.

Links: Summer 2006 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  1 August 2006. Last up dated 16 August 2006 by Hal White
Copyright 2006, University of Delaware