Sordaricin is a biologically derived natural product that has been shown to have anti-fungal and limited anti-protozoan activity in laboratory testing. The Taber group has set out to find a synthetic route to this natural product ((+)-sordaricin) that allows for enantiomeric selectivity. Setting the seterochemistry at one of the critical carbons requires the enantioface-selective insertion of a menthyl diazoester to form the bicyclic ketone. Our work has included investigating what proportion of diasteriomers is produced by both rhodium-mediated and copper mediated reactions and the characterization of these intermediates by NMR, optical rotation, IR and GC/MS. Funding provided by the Chemistry & Biochemistry Alumni Scholar’s Program.

Previous studies have shown that the effects of solvent variation of the available specific rates of solvolysis of frequently used pharmaceutical precursors can be analyzed using the extended Grunwald-Winstein equation. There are also many instances for solvents rich in fluoroalcohols where simple G-W plots lead to points lying above the correlation line. Here it is shown that this behavior can be treated by the addition of a new term called the aromatic ring parameter, to the extended G-W equation. By measuring the rate of solvolysis of cinnamyl chloride and applying the extended G-W equation with the aromatic ring parameter, this research will show that our studies of solvolytic acylation mechanisms could lead to better understanding of the pathways involved in the acylation of the hydroxyl groups of potential pharmaceuticals to increase their water solubility. It also helps us to understand mechanisms of reaction of cinnamyl halides, which are common precursors in the synthesis of useful haloenol lactones that are often used in treatment of drug resistant cancer and prevention of herbicide resistance in weeds. This project was supported by Grant Number 2 P20 RR016472-08 under the INBRE Program of the National Center for Research Resources (NCRR), National Institutes of Health (NIH).

The gp41 protein of the HIV virus plays an integral role in CD4+ cellular membrane fusion and subsequent infection. Before membrane fusion occurs, gp41 assumes a fusogenic state, exposing C- and N- terminal alpha-helical heptad repeats (HR) in the protein’s sub-structure. At this stage in gp41’s entry mechanism, these trimeric coiled coil HRs provide an attractive site for HIV inhibition. A highly conserved hydrophobic pocket near the N-terminus of gp41 has been of particular interest in HIV inhibitory molecule modeling. Previous studies targeting the fusogenic state of HIV have employed short complimentary alpha-helical peptides. In our studies we seek to take advantage of the correspondent distances between the functional groups on a face of the alpha-helix and a designed tetrahydronaphthalene molecular scaffold. By optimizing the functional groups attached to this scaffold, we hope to obtain a high affinity molecule capable of inhibiting HIV-1 viral membrane fusion. To test this, we will utilize a bipyridally connected trimer of IZN17 peptides to mimic the N region of gp41. Along with a fluoroscein labeled C14 peptide of known binding constant, our small molecules will be placed in a competitive binding assay to measure our mimics’ respective potencies. While we are still in the process of synthesizing small molecules and the IZN17 trimeric coiled coil mimic, we have synthesized the C14 peptide a.

Self-assembling peptide hydrogel scaffolds have the potential for use in tissue regenerative therapies. Hydrogels provide an ideal, hydrated, porous environment for tissue growth. However, for the implantation of a biomaterial, many design considerations and precautions must be met, in particular preventing the introduction of infection. We have designed a lysine-rich peptide, MAX1, which self-assembles to form a hydrogel whose surface is active against Gram-positive (Staphylococcus epidermidis, Staphylococcus aureus and Streptococcus pyogenes) and Gram-negative (Klebsiella pneumoniae and Escherichia coli) bacteria, all prevalent in hospital settings. Although detrimental towards bacteria, the surface is cytocompatible towards a variety of mammalian cells, making these hydrogels attractive candidates as tissue engineering scaffolds. This study will focus on determining the effect of the length of the lysine side chains of MAX1 on the material properties as well as the antibacterial activity of the hydrogel surface. A new peptide sequence, HPL1, was designed, in which the lysine residues were homogenously replaced by ornithine, a methylene deficient derivative of lysine. The biophysical properties of HPL1 will be analyzed using circular dichroism spectroscopy and oscillatory shear rheology and compared to that of the parent peptide, MAX1. Next, the antibacterial activity of HPL1 surfaces will be assessed to determine the efficacy of antibacterial activity. Supported by the Howard Hughes Medical Institute

My research this summer in solid-state synthesis aims to discover the structure-property relationships for intermetallic rare earth compounds. My project was extrapolated from a family of RESn_1+xGe_1-x compounds recently discovered by my research group, prepared by utilizing the flux growth method. My work was focused on extending the chemistry of the nearly equiatomic rare-earth tin germanide compounds, replacing the tin flux with bismuth in order to prepare isostructural analogues. The attempts were successful in that a new family of compounds was discovered by employing bismuth flux. The new structures crystallized with the orthorhombic space group Cmcm (No. 63) and can be described with the formulae RE[Bi_xGe_1-x]₂ (RE = Y, Nd, Pr, Gd-Ho, 0 < x < 0.15). Their structures have been established from single crystal X-ray crystallography and can be built from the same fragments just in different stacking arrangements: one-dimensional germanium zig-zag chains and rare-earth atoms separated by square sheets of germanium containing a small admixture of bismuth. Further studies currently underway include temperature dependent magnetic susceptibility measurements as well as attempting to extend the chemistry to rare-earth antimony germanides. I would like to thank David A. Plastino for funding my research for the summer through a Chemistry Alumni Scholarship.

Isofurans are compounds formed from arachidonic acid via a nonenzymatic free radical mechanism in the presence of oxygen. Isoprostanes, another class of arachidonic acid derived compounds also forms under oxygen tension; however, the formation of isofurans is favored as oxygen tension increases. These molecules can be used a metric for the degree of oxidative stress in an organism. A strategy towards the synthesis of these compounds was devised with a key intermediate that allows access to many isofuran structures. As these arachidonic acid derived compounds have high biological activity, this synthesis aims to aid those performing physiological experimentation with this class of compounds. Funded by a Chemistry Alumni Scholarship..

Hydrogels are heavily hydrated, elastic-like, porous materials that show considerable promise as artificial extracellular matrices for use in tissue regenerative therapies. Unfortunately, not only do hydrogels provide ideal environments for cell proliferation, but for opportunistic bacteria as well. To combat the threat of infection, hydrogels are often modified to display antibacterial activity, usually by impregnating the gel with antibiotic agents or covalently attaching them to the gel surface. The development of hydrogels that are inherently antibacterial has been of great interest to the hydrogel research community. We have developed MAX1, a self-assembling, twenty amino acid peptide hydrogel whose surface exhibits inherent antibacterial activity against several gram-negative and gram-positive bacteria prevalent in hospital settings. Under physiological conditions, MAX1 folds into an amphiphilic beta-hairpin and subsequently self-assembles into a highly-crosslinked hydrogel network composed of fibrils rich in beta-sheet. The resultant hydrogel is mechanically rigid and cytocompatible. This study aims to investigate the possible contributions of a cation-pi interaction to the antibacterial activity of a MAX1-like peptide hydrogel. Cation-pi interactions between arginine and tryptophan pairs are a common feature of many conventional antibacterial peptides, where they appear to assist in the binding and disruption of bacterial membranes. A new peptide sequence (RWMAX1) was designed and synthesized, incorporating a cross-strand R/W pair into MAX1. Preliminary studies using circular dichroism assessed RWMAX1’s folding and self-assembly kinetics. Future studies will explore RWMAX1’s material rigidity and antibacterial properties against E. coli and S. aureus. Funded by a Beckman Scholarship.

Human Platelet-Activating Factor Acetylhydrolase Type II (PAF-AH II) is a 44 kD monomeric enzyme that belongs to group VII of the PLA2 superfamily. A medically important enzyme, PAF-AH II plays a role in inflammation events such as sepsis, atherosclerosis, and asthma. Often thought of as a scavenger, PAF-AH II seeks and subsequently hydrolyses oxidized phospholipids, Platelet-Activating Factor (PAF), and other structurally similar molecules that contribute to inflammation events in vivo. Using a recently constructed homology model of the enzyme, it was predicted that PAF-AH II contains five hydrophobic amino acid residues, namely Leu-76, Leu-79, Leu-327, Ile-328, and Phe-331 that embed themselves in the inner membrane. Presumably, the nature of these hydrophobic residues is the cause of aggregation observed when the wild-type protein is overexpressed, making PAF-AH II exceedingly difficult to crystallize. The five aforementioned hydrophobic residues of interest were targeted for site-directed mutagenesis. The main aim of this project was to systematically change each of the five hydrophobic residues to serine, a compatible hydrophilic amino acid residue in an effort to keep the protein from aggregating. Polymerase Chain Reaction (PCR) was implemented in creating the mutants. The work done thusfar represents the first step in the eventual crystallization of mutant PAF-AH II. Funding provided by the Science and Engineering Scholars Program and the following NIH grants: COBRE 2P20RR015588 and 1R01HL084366.

Aliphatic chlorothioformate esters are used to prepare low molecular weight thiocarbamate inhibitors of the enzyme elastase in mammals. Such small molecules are therapeutically effective in treating chronic inflammatory diseases such arthritis, asthma, colitis, and emphysema. Because of convenient rates of reaction at temperatures close to ambient, the specific rates of solvolysis of Isopropyl chlorothioformate are analyzed using the extended Grunwald-Winstein equation. Previously, in a comparison of chloroformate ester and chlorothioformate ester solvolyses by the ionization mechanism, it was assumed that sulfur would have a smaller tendency to use its electrons in a stabilizing interaction with the adjacent electron deficient carbon of an incipient carbocation. Kevill and D’Souza have shown that in ethyl chloroformate solvolysis, an addition-elimination channel dominates and only in the more ionizing and least nucleophilic solvents does the principal reaction channel involve ionization. For ethyl chlorothioformate, the relative importance of the two reaction channels is reversed, and for the majority of solvents, the ionization pathway is dominant. Thus it will be of great interest to observe the characteristics introduced by a bulkier isopropyl group from the isopropyl thioester.

Classical statistical mechanical modeling, such as molecular dynamics (MD) simulations, uses the atomic-level interactions between atoms and molecules (Angstrom scale) to compute continuum, or macroscopic system properties accessible via experiment. Biologically relevant lipid bilayer membranes and the proteins that function as an integral part of such environments are an important target of study using MD due to their prevalence in the human genome and their association with disease and system dysfunction. Towards understanding the relation between the microscopic structure and interactions in such systems to their functional roles using theoretical approaches, it is important to develop physically relevant models. Current models represent electrostatic interactions using fixed charges, though quantum mechanics and experiments suggest biological systems encompass vastly differing dielectric (vis-à-vis, electrostatic) environments (cell membrane interior versus cytoplasm). In this experiment an electrostatically polarizable interaction model for hexane (as a model of the lipid bilayer environment), refined in our lab to better fit ab initio calculations of torsional energy, heat of vaporization, density, and electrostatic interactions, was used to perform MD simulations to calculate the free energy of transfer for water, hexane, and methanol molecules across the water-hexane interface. For transfer from water to hexane, the present calculations predict transfer free energies of 4.5, 5.4, 4.0, 3.8 kcal/mole for water, hexane, methanol, ethanol, respectively. These values are similar to experimental results and are increasing in similarity as more data is collected and the values converge. Moreover, our results for water transfer free energetics are in good agreement with water distributions obtained in independent MD simulations of a fully hydrated DMPC bilayer Furthermore, we observe that the amphiphilic alcohols exhibit a shallow free energy minimum in the interfacial region, a surfactant-like effect also observed with micelles at the air-water interface. Future extensions of the methodologies developed will investigate partitioning and distribution of solutes in hydrated lipidic systems. Supported by the Howard Hughes Medical Institute.

Androgen-dependent prostate cancer can be treated with anti-androgens, however, in time many cancers become hormone refractory and no longer respond to the drugs. Mutations on the androgen receptor (AR) have been identified to play a major role in anti-androgen resistance. In some cases anti-androgens activate the androgen receptor stimulating the growth of the cancer, which has proved difficult to treat.The Koh group, is involved in designing compounds that target mutant hormone receptors. A new anti-androgen PLM6 was found not to form resistant clones in culture. PLM6 may evade resistance mechanisms or may simply be more toxic. By growing the LNCaP cell line, we studied their proliferation in the presence of different concentrations of bicalutamide and PLM6 over a short-term growth period. Two growth assays (Cyquant and titer blue) are used to evaluate their growth. We also did site-directed mutagenesis where we created some AR mutations that were found in resistant cells. The findings of this study indicate that PLM6 is toxic rather that evading the AR resistance mechanisms. Funded by DoD grant.

The cytoskeleton is an essential structural feature of all eukaryotic cells. Cytoskeleton-associated proteins (CAPs) mediate interactions between filaments present in the cytoskeleton. These protein complexes have a variety of roles including sustaining cell polarity, participating in mitosis, and partaking in intracellular transport and signaling. The cytoskeleton-associated protein-glycine rich (CAP-Gly) domain is found in several proteins including the p150glued subunit of dynactin, CLIP-170, the tumor suppressor CYLD, and KIF13B. The primary goal of our work is to determine the structure of the CAP-Gly domain in order to further understand the mechanism of this protein in its microtubule context. Isotope labeling of the target protein is necessary to effectively characterize the protein using solution NMR. Once the structure and function of these microtubule associated motor proteins are understood, eventual treatments for motor protein disorders can be discovered. We have prepared 15N uniformly labeled CAP-Gly samples using an Escherichia coli overexpression system. We have successfully purified several CAP-Gly samples and quantified the concentration of the purified protein. 2-D solution NMR spectroscopy has been performed on the samples to obtain Heteronuclear Single Quantum Coherence (HSQC) spectra. Solid state NMR spectroscopy will also be used to determine the structures of the CAP-Gly domain in both its free and bound states. Funding for this research was provided through the Howard Hughes Medical Institute Scholarship.

Lycopladine A, a compound that is extracted from the club moss Lycopodium complanatum, displays cytoxic activity towards murine Lyphoma L1210 cells (IC50, 7ug/mL) in vitro, making it a promising lead compound for cancer treatment. Lycopladine A also has a unique skeleton, which differs from all other C16N-type alkaloids. Thus, this compound is an interesting synthetic target. A previous synthesis of lycopladine A included a gold(I)-catalyzed 5-endo-dig cyclization to construct the hydrindanone core of lycopladine A. Subsequently, the pyridine in the compound was assembled. Our synthetic scheme presents a more efficient and environmentally safe means of synthesizing lycopladine A. Rather than building the pyridine, 2-methyl pyridine is inserted into the cyclohexenone intermediate. Recently, an article in Heterocycles indicated that attempted 1,4 conjugate addition of 2-methylpyridine with cyclohexenone failed. Only the 1,2 addition product was observed. The only other report, in the Journal of Heterocyclic Chemistry in 1972, noted that 1,4 conjugate addition of 2-cyclopentenone was possible, but only in an abysmal yield of 9%. Through a series of reactions, the 1,4 conjugate addition of 2-methylpyridines has been optimized. Funding for this project was provided by Pfizer’s Summer Undergraduate Research Fellowship Program.

Isopropyl Chloroformate decomposes on heating or on contact with acid producing toxic and corrosive fumes including chlorine and phosgene. Yet this highly flammable explosive compound has found pharmaceutical use as a precursor in the synthesis of molecules useful in treating prophylaxis, diabetes, atherosclerosis, cardiovascular diseases, hyperuricemia, inflammatory bowel diseases, or skin disorders related to an anomaly of differentiation of epidermic cells. Hence, understanding its mechanism of reaction in a variety of solvents will give real insight for the production of synthetically useful pharmaceutical products. Here the specific rates of solvolysis Isopropyl Chloroformate are analyzed using Grunwald-Winstein equations. Previous studies found that Alkyl or Alkenyl Chloroformates solvolyze in most of the commonly studied solvents by an addition-elimination mechanism with the addition step being rate-determining. In fluoroalcohols for Ethyl, and n-Propyl chloroformate, an ionization mechanism was indicated with up to 50% water content in HFIP-H2O and with up to 10% water content in TFE-H2O mixtures. Therefore in this project we provide kinetic studies of Isopropyl Chloroformate including 1,1,1,3,3,3- Hexafluoro-2- Propanol (HFIP) and 2,2,2-Trifluoroethanol (TFE). This project was supported by Grant Number 2 P20 RR016472-08 under the INBRE Program of the National Center for Research Resources (NCRR), National Institutes of Health (NIH).

The Rad5 protein is required for postreplicative DNA repair. Rad5 possesses both DNA helicase and ubiquitin ligase activities. This dual activity has been suggested as essential for the regression of the stalled DNA replication fork. However, little is known with respect to the specific domain(s) of Rad5 utilized in this process. The goal of this project is to express and characterize the conserved HIRAN domain of Rad5 as an independent entity, and to test the DNA structures to which it binds. When expressed as a standalone form in E. coli, the HIRAN domain was shown to be insoluble, presumably forming insoluble protein aggregates. To generate soluble protein the denatured form of HIRAN domain was purified, and refolded following a rapid dilution protocol. The affinity to single-stranded DNA (ssDNA) of the refolded HIRAN was tested. It was found that the refolded protein did not bind to ssDNA. We now try to obtain soluble HIRAN domain by fusing it to a maltose-binding protein. The HIRAN sequence was inserted into the pMAL vector that bears the maltose-binding protein sequence to generate a fusion protein. Two clones with varying amino acid linker size have been created. Once expressed, fluorescence anisotropy studies with Oregon Green 488 labeled DNA oligoes will be performed to determine the HIRAN DNA binding properties. For this purpose, DNA structures that resemble the stalled DNA replication fork will be generated and tested. This research was funded by the Howard Hughes Medical Institute (HHMI).

Kinetoplastid parasites are responsible for three important diseases: Human African Trypanosomiasis (African sleeping sickness), Chagas’ disease, and Leishmaniasis. Worldwide, these diseases infect millions, and kill over 110,000 people yearly. All three are classified by the World Health Organization as “Neglected Tropical Diseases” because they disproportionately effect the poorest of the poor. Of the neglected tropical diseases, these three classes of single-celled flagellates are some of the hardest to treat because current drugs are expensive and toxic. These parasites have a unique structure called a kinetoplast in their single mitochondrion. It contains thousands of DNA minicircles (0.5-2 kb) and dozen(s) of maxicircles (3-5 kb) which are linked together like medieval chain mail. Each time the kDNA replicates, all of these must be unlinked, replicated, and linked together again into mother and daughter networks. This requires remarkable replication machinery, where a kinetoplast-localized topoisomerase II (topo II) is very important. Topo II enzymes link and unlink DNA (catenate and decatenate) in an ATP-dependant manner by inducing transient double-stranded breaks in one DNA strand, pulling the other DNA strand through the break, and then ligating the break. Many topo II drugs (including some antitrypanosomals, as well as many cancer and antimicrobial drugs) stabilize the cleavage complex, leading to fragmented DNA and initiating apoptosis. My research focuses on using in vitro methods to study the unique structure and function of kinetoplast topo II from the model organism, Crithidia fasciculata. This will enhance understanding of these parasite’s beautiful biology and drug mechanism and design. Supported by A Beckman Scholarship.

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. 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 effect of the ligand on the reactivity of the nickel complex modeling the distal nickel active site of ACS was sought. The target of the first part of this project was to synthesize and isolate a new ligand containing phosphine and thioether donors, ‘PS^iPr₂’ (PS^iPr₂ = PhP(CH₂CH₂SPrⁱ)₂). Herein is reported the synthesis and characterization of the PS^iPr₂ ligand. The next part of the project involved synthesizing nickel(II) complexes supported by the PS^iPr₂ ligand. The characterization of [(PS^iPr₂)NiCl]BPh₄ will be presented. The Howard Hughes Medical Institute Undergraduate Research Scholarship Program is thanked for funding this project. References (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.

Cyclin Dependent Kinase 5 is an essential regulator for neuronal migration and has been implicated in the pathological neurodegeneration of Alzheimer ’s disease. Unregulated and mislocalized CDK5 activity promotes hyperphosphorylation of tau protein that will accumulate as neurofibrillary tangles (NFTs) the hallmarks of AD. The mechanisms of NFT formation are poorly understood and difficult to discern amidst the multitude of neuronal cell signaling pathways. To aid in the study of these mechanisms we have designed a series of genetically encodable protein motifs that measuring specific CDK5 activity. These motifs, dubbed protein Kinase Inducible Domains (pKIDs), consist of a metal-binding loop, modeled from an EF-hand, and a CDK5 recognition sequence. pKIDs undergo a structural change when phosphorylated that is detectable through a florescent reporter element. Five pKIDs have been developed for CDK5 from the Src and His H1. Each pKID was synthesized via Fmoc solid phase synthesis and characterized by HPLC and ESI-MS. In the presence of Terbium increased florescence was detected for all peptides when phosphorylated as compared to the non-phosphorylated species, thus implying that peptide-metal complex formation is dependent on phosphorylation. pKID-SW5 exhibits the largest differentiation in florescence between phosphorylated and non-phosphorylated species, demonstrating complete structural switching upon phosphorylation. CDK5/p25 readily phosphorylates pKID-SW5, as determined by increased terbium luminescence for pKIDs incubated with the kinase. These results suggest that CDK5-pKIDs may act as sensors of protein kinase activity and could be applied to the study of enzymatic misregulation in Alzheimer ’s disease. Supported by the Howard Hughes Memorial Institute.

Peroxiredoxin VI (Prdx6) is an antioxidant enzyme highly expressed in the lungs. Its antioxidant properties is due to its ability to reduce hydroperoxides found in lung surfactants and thus prevent the toxicity associated with hyperoxia. Prdx6 is a bifunctional protein that contains two distinct active sites. One active site catalyzes a phospholipase A2 (PLA2) type hydrolysis of phospholipids, and the second active site catalyzes the reduction of lipid hydroperoxides typical of 1-cys peroxiredoxins. The structure of Prdx6 has been solved; however, there is no structural evidence to elucidate its catalytic mechanism. This study is designed to solve the structure of Prdx6 in complex with MJ33, a PLA2 transition-state inhibitor, in an effort to understand the mechanism and conformational change Prdx6 undergoes during its catalytic cycle. Here we report that Prdx6 has been successfully purified to homogeneity. Our crystallization screens have produced protein crystals that have diffracted to a resolution of 2.8 Å. This research has been funded by the Chemistry Alumni Fellowship.

The goal of this project was to determine the sources of carbonaceous airborne particulate matter in Wilmington, Delaware. A Sunset Laboratory Model-4 Semi Continuous OC/EC Field Analyzer was used to measure the mass concentration of airborne organic and elemental carbon (OC and EC, respectively) at the State of Delaware Air Quality Monitoring Site in Wilmington. Measurements were made at one hour intervals over a two week period. Previous work has shown that the ratio of OC to EC in air can be used to distinguish primary from secondary organic aerosol (POA and SOA, respectively) and combustion from non-combustion POA. This approach, called the EC tracer method, was applied to the Wilmington measurements to distinguish the various types of carbonaceous particulate matter. The results give a detailed understanding of Wilmington aerosol that may assist the development of pollution control strategies. New Castle County does not meet the federal PM2.5 (mass concentration of airborne particles 2.5 µm and smaller) standard for air quality. As a result, the State must develop a plan to overcome this problem. Funding for this research was provided by the Chemistry and Biochemistry Alumni Scholars Program and a grant from the Environmental Protection Agency.