Faculty Research Descriptions
Faculty Research Projects
Mike Ross - Environmental Chemistry
The focus of my research is on the photodecomposition of drugs and personal care products in surface water. This project includes: a) the study of the photodecomposition rate of these materials by direct as well as indirect mechanisms; b) a study to look at the levels of these materials within East Gemini Lake, which is the place the SJU Waste Water Plant dumps its outflow.
The first part of the project systematically looks at the photodecomposition rate and mechanisms for a select number differing types of pharmaceutical and personal care products (PPCP) by looking at the decomposition rates under artificial illumination and under direct exposure to the sun. This summer I hope to finish a couple of projects that have been started on a series of antidepressants. I have had former students do much of the photodecomposition under artificial illumination. I would like to repeat some of these experiments, but more importantly, I want to run the experiments under natural lighting as well in order to compare our results to the results of others doing this kind of research.
The second part of this project fits well within our summer research program where there is a need for longer stretches of time focused on an experiment. SJU also provides the perfect setting for conducting these experiments since East Gemini Lake receives much of its water from the outflow of the waste water treatment plant. The experiments will require separation and pre-concentration of the lake water samples before analysis, but because we can prescreen the effluent as it enters the lake, we will know the compounds that are entering the lake and therefore have a better chance to quantitate their amounts in the lake water. Since we can also sample the outflow of the lake, we will be able to look at the efficiency with which these compounds are removed by lake processes. We will be sorking with Dr. Michael Heroux and his students (Computer Science) in an attempt to model the processes taking place in the lake and generate an efficiency for the removal.
T. Nicholas Jones - Organic Chemistry
Synthesis of small molecules; synthetic methodolgy development; design and preparation of copper enzyme model systems; use of computational chemistry to assist synthetic goals; analysis by 1H- 13C-NMR, IR, and GC-MS.
Henry Jakubowski - Biochemistry
Low Molecular Weight Protein Tyrosyl Phosphatase (PTP)
Enzymes that cleave phosphate groups from proteins are abundant in all cells. These enzymes, called phosphatases, regulate the activity of phosphorylated proteins, rendering the dephosphorylated proteins either active or inactive. Phosphatases are key proteins in the regulation of cells growth and metabolic state. I am studying human low molecular protein tyroysl phosphatase (PTP). Dr. McIntee (Chemistry Dept) and I are collaborating to study the natural protein substrates of this enzyme and to develop drugs to inhibit its activity.
The protein is made from a cDNA inserted into a plasmid, PGEX-6P1 which is used to transform E. Coli. Gene expression is induced by addition of IPTG, which activated the lac promoter, leading to the formation of a GST-PTP fusion protein. The active site of the enzyme, where the chemical cleavage of the phosphate group occurs, must bind phosphate groups which are covalently attached to target proteins. The activity of the enzyme can be monitored easily in solution using p-nitrophenol phosphate which is cleaved by the enzyme to produce a yellow solution. An active site cysteine acts as a nucleophile in the cleavage reaction. The enzyme binds phosphate in the active site, which competitively inhibits the enzyme.
My research group has made a series of mutations in the DNA for PTP and we are presently studying the effect of these mutants on its activity. The mutants can be divided into two groups:
Active site mutant: The active site Cys 12 (C12) is replaced for a Ser (C12S). This makes the enzyme catalytically inactive, unable to cleave phosphate groups from proteins. However, this mutant will still be able to bind phospho-proteins. We will use this mutant in future research to bind to natural phospho-proteins in epithelial and fat cells, and to identify the binding sites on those phospho-proteins for PTP
Nonactive site mutant: We have made two different mutants, changing a single W to phenyalanine (F) in each one, thereby producing two mutants that contain only one W residue. We have changed an amino acid that is near the active site and which fluoresces, to F which does not fluoresce. We have also made a second mutant to change W39, located on the opposite side from the active site, to a phenylalanine. Fluorescence from the mutant with only a single W at position 49 will be sensitive to the environment of the active site, while the other mutant, containing a single W at position 39, will be used to detect changes in protein structure away from the active site and serve as a control.
The activity of the two mutant proteins, as measured by cleavage of p-nitrophenyl phosphate and fluorescence, in the presence and absence of different inhibitors of the enzyme, will be used to better understand how the structure of the enzyme influences its activity. In addition, the stability and unfolding of the protein will be studied used fluorescence from the single tryptophan-containing mutants.
We have also made a double mutant: C12S/W39F, producing a protein that can not cleave phosphates from target proteins (C12S) and which has a single W at position 49 (W39F) which will be used to monitor phospho-protein binding to the double mutant by monitoring fluorescence changes in W49. P>
Additionally, we are studying the protein and its interaction with inhibitors through in silico computer modeling of the protein uisng VMD/NAMD and Autodock
Applications of Fluorescence in Biochemistry
My second project involves using the spectrofluorometer to study a range of biological questions. When molecules absorb UV or visible light, electrons are excited to higher energy levels. The excited electrons can “relax” back to lower energy levels by losing energy through collisions or by emitting photons of lower energy than the original excitation photons. This emission is called fluorescence. In contrast to simple absorbance properties, fluorescence emission is extremely sensitive to the environment of the fluorophore, the molecule that emits. The properties of biological molecules can be studied using fluorescence. Two types of fluorophores are used. Intrinsic fluorophores are part of the actual molecule (for example a tryptophan side chain in a protein). Extrinsic fluorophores (like fluorescein) can be attached (covalently or noncovalently) to proteins, lipid aggregates and DNA. We develop research projects using fluorescence to study the structural transition and binding interactions of proteins, lipids, and DNA.
Kate Graham - Natural Products Chemistry
Isolation of antifungal compounds; fungal growth regulatory compounds fungal culture techniques, advanced extraction techniques, column chromatography, high performance liquid chromatography, pulsed NMR experiments.
Ed McIntee - Medicinal Chemistry
LMW-PTP inhibitor project: Protein kinases and phosphatases work in an intricate manner to act as cellular signaling mechanisms. The focus of this research is on modulating the activity of human low molecular weight protein tyrosine phosphatase (LMW-PTP). It has been shown that complexation of LMW-PTP to the Eph receptor is important for promotion (up regulation) of endothelial capillary-like assembly and cell adhesion. In addition, LMW-PTP has been shown to be overexpressed in many oncogene-transformed or tumor derived mammary epithelial cells. Overexpression of LMW-PTP is sufficient to convert normal epithelial cells into cancerous cells. Other studies have shown that when nude mice received NIH-3T3 cells expressing an active form of LMW-PTP they developed sarcomas more readily and had lower levels of phosphorylated- EphA2 receptors than mice expressing a dominant-negative form of LMW-PTP. The EphA2 receptor is also overexpressed in a large number of cancers, including breast, prostate, and lung carcinomas together with melanomas and sarcomas. In addition to its overexpression, EphA2 is predominantly tyrosine phosphorylated in nontransformed (normal) cells, while it is mainly dephosphorylated in transformed (cancerous) cells. Inhibitors of LMW-PTP hence may possess potential anti-tumor properties by inhibiting dephosphorylation of the EphA2 receptor. One natural product that is a very good inhibitor of LMW-PTP is pyridoxal 5’-phosphate (PLP). PLP is actually just a poor substrate for LMW-PTP because in time it is also dephosphorylated. What we are proposing is to synthesize non-hydrolysable PLP analogs, specifically phosphonate analogs of PLP.
Antibacterial pro-drug project: One area that has recently received much attention in the media is the increasing occurrence of drug resistant bacteria in both livestock and humans. Traditionally effective antibiotics such as trimethoprim, sulfamethoxazole, second and third generation cephalosporins, and carbapenem and monobactam antibiotics are, unfortunately, losing their effectiveness leading to the development of an increasing number of resistant bacterial strains. The problem has health experts quite concerned. For patients infected with the drug-resistant organisms, there are few, if any, therapeutic options. Creative approaches must be utilized to circumvent resistance or develop new antibacterial drugs. One of the areas that bacteria develop resistance to drugs (specifically to nucleoside based drugs) is the mutation or down regulation of thymidine kinase. Thymidine kinase is often the first enzyme needed to activate nucleoside- based drugs before they have any biological effect. One way to overcome this type of resistance is to circumvent the enzyme needed to activate the drug. With this in mind, a unique kinase bypass system has been developed by researchers at the University of Minnesota. The general idea behind this approach was to use amino acid phosphoramidate conjugates of various chemotherapeutic nucleosides to deliver the corresponding nucleotide. We want to apply this approach with the nucleoside 5-fluoro-2?-deoxyuridine (FUdR). The proposed phosphoramidates are shown on the right.
Chris Schaller - Organometallic Chemistry
New Catalysts or Initiators for Polymerization of Lactide
Polylactide (PLA, 1) is a biodegradable polymer produced by the ring-opening polymerization of lactide (LA, 2). Because LA can be obtained through fermentation of corn and soybeans, production of PLA does not depend on the consumption of petrochemical feedstocks but is instead based on renewable resources.[i] PLA is currently produced commercially for food packaging; a number of biomedical applications have also been developed.
The most successful process for PLA production employs toxic tin octoate as an initiator; hence, the development of more benign initiators or catalysts is warranted. Ideally, an LA polymerization catalyst should not only induce rapid polymerization of LA, but should also demonstrate control of PLA molecular weight and stereochemistry. A number of recent efforts have focused on the development of single site metal alkoxides for this purpose.[ii] A Lewis acidic metal ion promotes a controlled reaction by binding and activating carbonyl oxygen, whereas an alkoxide serves as the nucleophile for ester cleavage.
In our continuing studies of N-heterocyclic carbene (NHC) zinc complexes and LA polymerization [iii] we have pursued a number of different avenues designed to address issues of catalyst stability and selectivity. Because the relative lability of zinc complexes raised the possibility that the NHC ligand could dissociate from these initiators, we are investigating the use of bidentate phenoxy-NHC ligands (3). It is hoped that these complexes will remain stable even in solvent-free melt polymerizations at elevated temperatures.
[i] (a) R.W. Drumwright, P.R. Gruber, D.E. Henton, Adv. Mat. 12 (2000) 1841. (b) M. Okada, Prog. Polym. Sci. 27 (2002) 87.
[ii] (a) O. Dechy-Cabaret, B. Martin-Vaca, D. Bourissou, Chem. Rev. 104 (2004) 6147. (b) B.J. O'Keefe, M.A. Hillmyer, W.B. Tolman, J. Chem. Soc. Dalton Trans. (2001) 2215.
[iii] (a) T.R. Jensen, L.E. Breyfogle, M.A. Hillmyer, W.B. Tolman, J. Chem. Soc. Chem. Commun. 21 (2004) 2504. (b) T.R. Jensen, C.P.Schaller, M.A. Hillmyer, W.B. Tolman,, J. Organomet. Chem. 690 (2005) 5881-5891.
Brian Johnson - Inorganic Chemistry
Synthesis of Model Compounds for the Trinuclear site in Multicopper Oxidases
Multicopper oxidases, including laccase, ascorbate oxidase and ceruloplasmin, couple the oxidation of substrate molecules to the four electron reduction of dioxygen to water. These enzymes contain a triangular array of three copper atoms as well as one or more coppers at a distance of about 13 Å from the trinuclear site. Dioxygen binding and its subsequent reduction to water occur at the trinuclear center, while the function of the other copper atoms is to transfer electrons. The reduced form of a trinuclear cluster is shown in Figure 1. Two of the copper atoms are bound to three histidines in a trigonal planar environment, while the third vertex of the copper triangle is occupied by a copper which has two histidine ligands and water molecule bound to it.
The chemistry of these enzymes has been studied by a variety of techniques, yet significant uncertainty remains in the mechanism by which they operate. Three mechanisms have been proposed, differing in several ways including the identity of the initial species formed when dioxygen reacts with the tricopper array. All three mechanisms also differ in the identity of the subsequent oxygen intermediates as water is formed. Despite the interesting and unusual nature of the trinuclear arrangement of coppers in multicopper oxidases, uncertainty about the mechanism by which these enzymes operate and the potential relevance of these enzymes to copper oxidation chemistry in industrial catalysis, there have been relatively few reported syntheses of trimetallic copper clusters in the literature.
In order to gain insight into the mechanism of these enzymatic reactions and the nature of the copper-oxygen intermediates involved, we are synthesizing a series of complexes which will model the trinuclear site in the multicopper oxidases. One approach will involve the synthesis of models for the trinuclear site based on the 1,3,5- 2,4,6-hexasubstituted benzene ligands shown below. In these molecules, the even- numbered ethyl substituents have been shown to occupy one side of the benzene ring, while the odd-numbered substituents which will bind the coppers are on the other. This will provide a framework by which the copper atoms can be held in close proximity. It will also allow us to prepare a number of variants in which the steric bulk, electron donating ability of the nitrogenous bases and the distance between coppers can be varied.
Richard White - Physical Chemistry
Microwave spectroscopy; analysis of polymer blends by NMR spectroscopy and calorimetry
Frank Rioux - Computational Chemistry
Foundations of quantum theory with special emphasis on computational studies of atomic and molecular structure and stability. Prerequisite: a course in quantum mechanics