This research project is focused on the use of chemical ecology to locate novel sources of bioactive natural products from microbial sources. The first stage of this research involves studying the ecology of a system, developing a bioassay, fermentation of the microbe and purification of the bioactive chemical(s). Once a new bioactive substance has been purified and determined to be responsible for the activity, characterization of the compound must be undertaken. In the final stages of structure elucidation, a high field FT-NMR is required for both advanced 1-D and 2-D NMR techniques. Interpretation of NMR spectra resulting from these experiments is an essential component of the characterization of unknown natural products.
The quest for new antifungal drugs is critical for two reasons. First, fungal infections are often topical but can become systemic and life threatening when the host is immuno-compromised. An aging population, AIDS and medical advances such as organ transplants and tumor treatments have all contributed to the rise in immune deficiencies in recent years. The current increase in the number of immune-suppressed individuals in society has led to a large population in need of potent and non-toxic antifungal drugs.
The rationale for exploiting fungi for antifungal drugs stems from the capability of fungi to produce a wide array of secondary metabolites. Presently, fungal natural products present a large and relatively untapped source of antifungal drugs. There are over one million species of fungi worldwide, but only a few species have their secondary metabolites analyzed. Many of these metabolites are believed to have developed in order to inhibit the proliferation of competing species. Thus, biorationale would predict fungi to be a good source of new and interesting fungistatic or fungicidal chemicals.
A number of possible sources of novel fungi have been located. These fungi include insect-associated fungi and endophytic fungi. The fungi were evaluated based upon their ability to inhibit the growth of a variety of fungi and pathogenic yeasts using plug assays and standard diffusion assays. The most successful fungi were selected for characterization of their secondary metabolites. The project now entails the purification and structure elucidation of possible antifungal drugs.
Candida albicans is a dimorphic, pathogenic fungus. In fact, pathogenicity in Calbicans appears to be linked to its ability to switch rapidly between morphological forms. Therefore, an understanding of the biochemical regulation may prove useful in understanding pathogenicity and in the design of new antifungal drugs. A bioassay was developed to follow the growth modulating activity. An extracellular, organic compound was found to exhibit biological activity in the morphological control of dimorphism in Candida albicans and has been partially purified through bioassay-guided fractionation. This project will entail further purification of the active compound as well as structure elucidation. Further studie will then be done on the active component. In addition, the active component of other species of dimorphic fungi will also need to be analyzed.
Plants have to deal with attack by various herbivores during their life cycles. A plants' response to the presence of insect herbivores is determined by the defense system.A typical factor that causes plants to be resistant to insects include chemical repellents or feeding deterrents. There is significant genetic variation among the 35 genotypes of goldenrod, Solidago altissima, in their resistance to insect colonization and defoliation. This project will entail the determination of the genetic variability in the chemicals of goldenrod and relate this information to data on insect colonization and defoliation of goldenrods.
Transition-metal systems catalyze several important transformations of relevance to the petroleum industry, such as hydrocarbon cracking and the synthesis of artificial fuels (Fischer Tropsch Synthesis). While the catalysts responsible for these transformations are frequently hard to characterize, the reactions proceed through elementary steps which can be studied in detail using simpler, model systems.
For instance, formation of a C-H bond from an alkyl and a hydride bonded to the same metal center, referred to as reductive elimination, is one of the fundamental reactions of organometallic chemistry. However, attempts to systematically study the steric and electronic influence of different alkyl groups on the course of the reaction have been limited, largely due to synthetic constraints. 1
We have developed a promising synthetic route which should make a number of simple ruthenium alkyl hydride compounds easily accessible.2 Ruthenium is an interesting metal system because of its relevance to Fischer Tropsch catalysts. Ongoing work in this area will entail the synthesis and characterization of a number of alkyl derivatives and kinetic investigations of the reductive elimination reaction.
A related area of interest is the study of reductive elimination in metal oxide systems. These systems are relevant to the heterogeneous catalysts used in catalytic cracking of hydrocarbons. Our initial attempts in this area have been hampered by the low solubility of the target compounds, but future work will employ ligands which have been demonstrated to bestow greater solubility on metal oxide compounds.3
Bimetallic catalysts (those which contain two different metals) are widely used in industry, particularly in the synthesis of starting materials in polymer chemistry and petroleum refining. Bimetallics are often used because it has been found that, in many cases, the addition of a second metal to a catalyst makes the reaction faster, changes the product distribution, or allows the reaction to occur under milder conditions. An intriguing aspect of these catalysts is that there is often little understanding of reaction mechanisms or the manner in which one metal affects the chemistry of the other.
The intent of this project is to synthesize new bimetallic compounds and to study their chemistry. The complexes will be synthesized using the ligand 2[bis(diphenylphosphino)methyl]pyridine, or "PNP" which is shown below. PNP may be bidentate (bound to transition metals through both phosphorus atoms or one phosphorus atom and the nitrogen atom) or tridentate. When bidentate, a dangling arm of the ligand is available to bind to a second metal. Furthermore, as a heterofunctional ligand (two different types of donor atoms), coordination to two different metals may be especially favorable in some cases. Because of the variety of binding modes and the relatively small number of bimetallic compounds containing PNP that have been reported to date, this ligand represents a unique opportunity to synthesize new compounds and to gain insight into the behavior of two metals held in close proximity.
The experimental work can be divided into three stages. These include the synthesis of PNP-containing monometallic complexes, synthesis of bimetallic complexes, and examination of the reaction chemistry of the bimetallic complexes. Characterization methods include phosphorus and proton NMR, infrared spectroscopy, solution conductivity, and mass spectrometry.
Many important enzymes contain transition metals in their active sites. One particular family of these enzymes is the multicopper oxidases. As the name suggests, these enzymes contain at least four copper atoms and couple the four electron reduction of dioxygen to oxidation of substrate. As with most enzymes, it is difficult to isolate and purify useful amounts of the native enzyme to study. Our approach will be to use a small-molecule model for the actual enzyme. The initial stages of the project will involve organic synthesis, as we prepare a ligand that will bind copper ions in a manner that mimics the multicopper oxidases. Second, we will prepare copper complexes of the ligand and characterize them using a variety of techniques. Finally, with our copper-containing model complex in hand, we will begin a series of spectroscopic and kinetic studies that will provide insight into the properties and mode of action of the native enzyme. This research will be done in collaboration with Dr. William Tolman at the University of Minnesota.
I have two main areas of research both broadly dealing with how small molecules affect biological systems and organisms. The focus of the first area is to examine the biological activation mechanisms of small molecules that result in the formation of DNA adducts, to determine the significance of these adducts in carcinogenesis, to understand structure-carcinogenicity relationships, and to develop methods that can be used detect relevant metabolites and/or DNA adducts in humans. With the success of the human genome project, current opinion is that cancer is based on modifications at the DNA level. A recent report in The New England Journal of Medicine studying over 44,000 sets of heterozygous and homozygous twins has concluded that roughly 80 to 90 percent of human cancers are due to environmental factors such as environmental and dietary exposure to carcinogens.1 Covalent DNA modification with carcinogens or their metabolites can be the initial step of chemical carcinogenesis.1 If not repaired before DNA replication, DNA adducts can cause mispairing resulting in genetic damage. Numerous studies have demonstrated that the carcinogenic potency of many chemicals depends on their ability to modify DNA nucleobases.1
The second area of study focuses on developing novel antibacterial agents to combat drug resistant bacteria. The increasing occurrence of drug resistant bacteria in both livestock and humans has received much attention in the media. Traditionally effective antibiotics are, unfortunately, losing their effectiveness leading to the development of an increasing number of resistant bacterial strains. For patients infected with the drug-resistant organisms, there are few, if any, therapeutic options. In the late 1980's, efforts were made to expand the repertoire of antibacterial agents used to treat infections. Zidovudine (AZT, 3'-azido-3'-deoxythymidine), an effective antiviral agent used in the treatment of AIDS patients, was shown to be very effective against gram-negative bacteria such as Escherichia coli (E-coli.) and Salmonella dublin (MIC50 = 0.125 and 0.5 ?g/mL, respectively).2 Unfortunately, these bacterial strains also develop stable high-level AZT resistance after short incubations with the drug in vitro at concentrations up to 100 times the minimum inhibitory concentration (MIC).3 This resistance to AZT is caused by a deficiency or down regulation of an essential enzyme used to metabolize AZT to its active form.3 A drug delivery system was developed at the University of Minnesota to circumvent this type of resistance in cancerous and virally infected cells by using amino acid phosphoramidate conjugates of various chemotherapeutic nucleosides to deliver the corresponding nucleotide.4 We are currently applying this approach to bacterial resistance.
Computational chemistry/Quantum chemistry
Computational chemistry has become a recognized sub-discipline of chemistry in the last fifteen years because of the explosive improvements in both computer hardware and software. The chemistry department currently has ten Silicon Graphics workstations and site licenses for Mathcad and Spartan.
Mathcad provides an excellent programming environment for routine problem solving, small-scale quantum mechanical calculations, and testing numerical algorithms. Quantum mechanical principles are most easily learned with the aid of computer exercises that illustrate the relationship between the conceptual framework of quantum theory and its computational methodologies. Exercises have been prepared and published in the following areas: numerical integration of Schrodinger's equation in one, two, and three dimensions; applications of the variational theorem; the self-consistent field method for atoms and molecules; numerical Huckel calculations; LCAO-MO methods; group theory; and matrix mechanics. A project of current interest involving Mathcad is to develop an algorithm for the numerical solution of the time-dependent Schrodinger equation and then to use it to model the visible spectra of conjgated organic molecules.
Spartan is a comprehensive program for doing molecular mechanics, semi-empirical and ab initio quantum mechanical calculations on small to intermediate sized organic, inorganic and bio-molecules. The primary goal of the research projects using Spartan is the calculation of the electronic structures of such molecules in order to understand their geometry, chemical reactivity, and interaction with electromagnetic radiation.
I have worked on several research projects over the last several years.
1. Oxidized LDL: quantitation and effect on endothelial cells: (In collaboration with Dr. Amy Olson, Nutrition Department, and Dr. Mani Campos, Biology Department) Oxidized low density lipoprotein (LDL) has been implicated as a risk factor in the development of cardiovascular disease. Most clinical test assessing cardiovascular risk measure normal lipoproteins (LDL, HDL) but not oxidatively damaged LDL. We are developing methods (including an enzyme-linked immunoassay - ELISA) to identify and quantitative levels of oxidative damage of lipids and proteins in human blood lipoproteins. The project involves purification of lipoprotein using differential ultracentrifugation and analysis using chemical, spectroscopic, immunologic and chromatographic techniques. Once a reliable, sensitive, and reproducable assay is developed, we will measure levels of oxidized LDL in human blood and correlate it with known levels of cardiovascular disease risk.
In contrast to LDL which is taken up by cells through the LDL receptor (which exhibits regulated binding), oxdized LDL is taken up in a non-regulated fashion by scavenger receptors found on cells like macrophages and endothelial cells. We are trying to quantitate the extent and affinity of binding of oxidized LDL and LDL to appropriate cellular receptors using fluorescent probes and a spectrofluorometer for which we just received NSF funding. In collaboration with Dr. Campos, we will also try to look at effects of internalized or extracellular oxidized LDL on gene expression in endothelial cells.
2. Seine Protease Inhibitors: I am developing a multi-course set of laboratory experiences to study the interaction of the protease trypsin, with inhibitors. This research involves the synthesis of trypsin inhibitors, and developing methods used to study the interaction of the inhibitor with the enzyme. Laboratory results will be correlated with molecular modeling using Insight II/Charmm, molecular modeling and computational program for the Silicon Graphics network. When developed, these laboratory experiences will be used in General Chemistry, Organic Chemistry, Analytical Chemistry, and Biochemistry. Inhibitors will be tested with other homologous serine proteases such as thrombin, the enzyme which directly initiates blood clotting by activating platelets and cleaving fibrinogen (which then forms a fibrin clot). I have extensive experience in the purification and enzymology of blood coagulation proteins. Trypsin inhibitors should prove to all inhibit thrombin, albeit with different efficiency. Molecular modeling will be used to explain any differences in the interaction of the inhibitors with the enzymes. Students involved in this part of the project will perform many of the techniques used in modern drug design. I would also like to extend this study to another serine protease, cocoonase, in collaboration with Dr. David Mitchell, Biology Dept.
3. Secondary structure in oligonucleotides: We have purchased and studied an oligonucleotide, s(5'CGCGTTGTTCGCG3') which can form an intramolecular hairpin mediated by H-bond formation among the terminal four nucleotides, and compared it to a control nucleotide s(5'CCCCTTGTTCCCC3'), which can't form secondary structure. We are attempting to demonstrate secondary structure formation through urea gradient gel electrophoresis, UV spectrophotometry, fluorescence spectroscopy (using fluorescence resonance energy transfer) and eventually 2-D NMR techniques. We are trying to correlate laboratory result with molecular modeling studies. In addition, we are studying the thermodyanmics of urea-induced denaturation of the hairpin oligonucleotide.
4. Human adipocyte acid phosphatase: We have obtained a plasmid containing this gene, expressed as a fusion protein with glutathione-S-transferase, expressed it in E. Coli, and are characterizing its enzymatic activity. We are presently attempting to develop a more effective purification procedure for the protein. This protein is homologous to a family of intracellular phosphatases. We can address questions about how the structure of the protein mediates its function by changing specific amino acids in the protein. We can do this by performing site-specific mutagenesis on the plasmid DNA which encodes the protein, expressing the mutant protein, and comparing the enzymology of the mutant and wild-type protein. In addition, we are interested in developing fluorescent inhibitors of this phosphatase to help in studying the inteaction of the phosphatse with othe protein modulators.
Sauk River Project
My principle research interest has been in the area of environmental analysis. Of special interest at this time is the analysis of surface water for pesticides. These pesticides may be either herbicides or insecticides, but my major focus is on herbicides. Since we live in an agricultural area, the herbicides used by the local farmers on their fields have been a study for the past three years. We have worked closely with the Sauk River Watershed District, sampling at ten sites within the district, both directly from the Sauk River, and from several of its tributaries.
Several different methods are used for this analysis. Most of our work has been in preconcentration and analysis by Gas Chromatography/ Mass Spectrometery. This gives us quantitative information on the amount of pesticide in the water, and also a high level of certainty as to the identity of the pesticide found. Current and future work expands the techniques used to High Performance Liquid Chromatography and Capillary Electrochromatography. With both of these techniques, we are trying to minimize the amount of sample preparation necessary to obtain reproducible results for the analysis.
Watab Creek Project
In an effort to be better stewards of our environment, Saint John's Abbey, Saint John's University and the MN Department of Natural Resources have embarked on a study of the Watab Creek Watershed. Watab Creek runs directly into the Mississippi River. Tributaries of Watab Creek run through the Saint John's campus and adjacent to the Saint Benedict's campus.
Beginning the summer of 1996, groups within the chemistry department and biology departments will start looking at the Watab Creek watershed in terms of the overall water quality. My initial work will be in looking at the nitrate/nitrite and phospate levels in the soil and water, particularly in the areas of Stumpf Lake and the Gemini Lakes. As the project continues, other water quality measures will be made (similar to the Sauk River Project) as well.
Capillary Electrophoresis with Laser Enhanced Fluorescence Detection
During the Spring Semester of 1996, I spent my sabbatical time developing a capillary electrophoresis system with laser enhanced fluorescence detection. This system was created for two reasons: to have capillary electrophoresis capabilities within our department; and, to explore the use of capillary electrochromatography as a means of analysis for herbicides in surface water. The use of laser enhancement in the detection of these herbicides hopefully will lead to a simplification of sample preparation, greater recoveries of the herbicide from the water and a better analysis technique for these compounds
Computer Controlled Instrumentation
Most instrumentation used today has built into it some kind of computer control. This does not mean that there is no longer a need to be able to interface computers with scientific instrumentation. That need is still very important and so another part of my research effort is in the integration of computers with stand-alone instruments which either have no computer control, or which have computer control and we now want to have communication between this computer and another instrument. Being part of the instructional staff for the Computer Science Department continues to make this interest an important part of my study.