42 Years of Research. Fun with Free Radicals!

Forty-two Years of Research at Hampden-Sydney:
Fun with Free Radicals!

Herb Sipe working with students in the labMy graduate education and research at the University of Wisconsin was in physical chemistry with my thesis work being the study of electron spin resonance (ESR) spectra of organometal-substituted aromatic molecules. ESR data may be interpreted in terms of models for the structure and bonding in molecules that have unpaired electrons (free radicals), and my specific interest was in how organosilicon- and organogermanium-substituents influence the molecular orbitals of the molecules on which they are substituted. Soon after I joined the HSC faculty in 1968, a senior physics major, Richard Wobus '69, approached me about doing a senior research project to reconfigure a donated microwave relay system into an ESR spectrometer. Never daunted by a challenge, we undertook this task with almost no money and -in hindsight- a foolhardy disregard for the difficulties involved. By the end of the year, Richard succeeded in producing an instrument that could detect a sample of 100% radicals by noting the complete absorption of a microwave signal when the magnet of the "ESR" was tuned by hand through the resonance condition. Fortunately, the Research Corporation accepted my proposal for $5,500 to buy real microwave components and an ESR cavity for use with the magnet that was part of the newly-constructed Gilmer Science Center. With those funds, my students -Bruce Berger '72, Bennie Good '73, and A. C. Buchanan '73- and I assembled the first of three ESR spectrometers that have been used here over the years. Our efforts were aided significantly by Harrison DeLancy who was at that time the science division instrument technician and machinist. In 1976, the component-assembled ESR instrument was replaced by a Varian instrument that was cobbled together with an instrument console from Peter Smith's research group at Duke University, a magnet from a retired Varian HA-60 NMR instrument, and a power supply from Cornell University. In 1992, a National Science Foundation grant enabled us to replace the obsolescent vacuum tube instrument with a modern solid state model (with a 30-fold increase in sensitivity!).

My free radical research interests currently fall into two broad categories: ENDOR spectroscopy and free radical metabolites of xenobiotic compounds.

Electron Nuclear Double Resonance (ENDOR) Spectroscopy is a variant of ESR spectroscopy that I have been interested in since the mid 1970s. This technique provides higher resolution spectra than does ESR. Many students have worked in the area of developing ENDOR instrument components at HSC. Of note is the attempt by Preston Lovelace '00 to construct an ENDOR microwave cavity as his senior honors project. Although he became adept at the required machining of ceramics, we could never figure out how to couple microwave energy into the cavity reliably. Ultimately we purchased a commercial ENDOR cavity from JEOL, the supplier of our ESR spectrometer. Nate Stasko '02 did a summer research project developing software to operate the ENDOR components added to our ESR spectrometer. Cory Jaques '04 devoted a summer research period and his senior honors research to developing free radical reference samples and getting all the ENDOR apparatus to work. Thus, after about 25 years of effort by many students, we are now able to measure ENDOR spectra. Related to the ENDOR work, we have developed our own software to control computer data acquisition of ESR and ENDOR spectra. Our first efforts were made by Pat Anonick '86 who used a DOS-based programming language called ASYST. Because of improvements in computer technology and operating systems and the acquisition of a new ESR spectrometer, Ryan Henry '96 wrote new software to control the spectrometer using the ORIGIN programming environment. The current software package that we are using was written in the LabVIEW programming environment by Dan Armata '99, Chris Lea '00, and Robert George '05. These students reported on their research efforts by presenting posters at regional meetings held by the Virginia Section of the American Chemical Society. Our recent efforts at developing instrumentation have benefitted immensely from the assistance of Mr. Irvin Robertson, who is officially the laboratory technician for the Department of Physics and Astronomy.

Since taking the first of three sabbaticals at the Laboratory of Molecular Biophysics (now Laboratory of Pharmacology and Chemistry) at the National Institute of Environmental Health Sciences at Research Triangle Park, NC in 1987-88, my research interests have shifted from organometallic chemistry to physical biochemistry. At NIEHS, in collaboration with a Wisconsin grad school colleague, Dr. Ronald P. Mason, I learned techniques to generate and study (by ESR, of course) radicals produced by biochemical systems. The broad research area of the Mason research group has been the study (by ESR and some ancillary techniques) of free radical metabolites of xenobiotic compounds. Xenobiotic compounds are those that are not part of an organism's ordinary environment. For humans, xenobiotic compounds include pharmaceuticals, food additives, pesticide residues, and environmental pollutants. As a result of these sabbaticals, I have developed interests in medicinal chemistry and toxicology and have been able to offer courses for HSC undergraduate science majors in these areas. I have also developed a course for nonscience majors, Chemistry 105 - Toxic Chemicals and Society that addresses similar issues but for the non-specialist. This course has been very popular and is almost always fully subscribed when offered.

The NIEHS-based research has involved studying free radicals produced from biochemical action of enzymes on such molecules as ∃-estradiol and phenolphthalein (formerly used widely in over-the-counter laxatives), and acetaminophen. HSC chemistry majors have extended some of these studies. Keith Williams '05 developed a fast-flow system to allow us to study short-lived free radicals by producing them in small but steady-state concentrations by mixing two precursor solutions in the ESR spectrometer's cavity. This apparatus has been used by Stephen Wiese '05, Shea Duerring '06, and Brad Benedetti '06 to study the phenoxyl radical produced by oxidation of acetaminophen, a principal ingredient in many over-the-counter analgesic products. Taken in overdose, acetaminophen causes liver toxicity and is often fatal unless treated with an antidote, N-acetylcysteine. We believe that acetaminophen toxicity arises from acetaminophen phenoxyl free radicals produced when acetaminophen in high concentration is activated metabolically by liver enzymes such as the cytochromes. My students have been studying by ESR spectroscopy the interaction of N-acetylcysteine with the acetaminophen phenoxyl free radical. Although we can study more stable radicals by producing them with enzymes such as horse radish peroxidase (HRP) activated by hydrogen peroxide as models for liver enzyme action, the fast flow experiments would be prohibitively expensive with HRP. Instead we are using solutions of hemoglobin activated by hydrogen peroxide and obtain reasonable concentrations of radicals under fast-flow conditions at acceptable costs. Recently C. W. Clemmons '05 has developed a non-enzymatic system for reducing nitro-substituted molecules to radical anions using ascorbate (Vitamin C) ion in anaerobic basic solution. This technique shows promise of allowing us to study a wide variety of chemical substances. Several student poster presentations have been made at regional meetings such as MARCUS [Middle Atlantic Conference on Undergraduate Scholarship] held annually at Sweet Briar College and at meetings of the Virginia Section of the American Chemical Society.

My current research interests continue to focus on electron spin resonance [ESR] spectroscopic studies of free radical metabolites of xenobiotic compounds. These investigations continue to involve undergraduate coworkers from among the College's chemistry students. We have been using an innovative technique to generate and observe unstable free radicals: by mixing two solutions at high flow rates at the entrance of the ESR sample holder, we can observe spectra for steady-state concentrations of short-lived free radicals, and by recording and superimposing multiple, duplicate spectra using computer software, we can enhance even weak signals. This fast-flow ESR technique has allowed us to observe radicals with millisecond lifetimes at sub-micromolar concentrations. Student coworkers have optimized the experimental apparatus and developed the computer software as part of summer research projects and the junior/senior advanced laboratory projects in chemistry. Kevin Thompson '07 and Mike Antolini '08 have used this approach to observe high quality ESR spectra for the unstable phenoxyl radicals produced by oxidizing phenolphthalein (previously widely used as an over-the-counter laxative ingredient) with a biochemical oxidizing system of hemoglobin and hydrogen peroxide and a chemical oxidizing system of cerium(IV) in sulfuric acid. Chris Arnatt '09, with support from the Honors Council during summer 2006 and 2007, has used the fast-flow ESR technique and the biochemical and chemical oxidizing systems to investigate the widely used anti-convulsant drug, Dilantin. Teratogenic side effects of Dilantin have been attributed to one of its metabolites, and Chris has studied ESR spectra of phenoxyl radicals from that metabolite. He synthesized a chemical analog of Dilantin to help understand the ESR results he obtained in summer 2006, and he used the Gaussian state-of-the-art quantum chemical software to perform calculations to rationalize the ESR results. In a summer 2006 research project supported by the Hampden-Sydney Honors Council, Nick Bandy '09 also used the cerium(IV) radical-generating system and ESR spectroscopy to observe unstable phenoxyl radicals from the widely-used plasticizer, Bisphenol-A and chemically-related compounds. Sean Platt '09, who was also supported in summer 2006 by the Honors Council, took a different approach to studying unstable phenoxyl radicals from some pharmaceuticals by observing their ability to consume oxygen to produce superoxide radicals. His studies used a computer-interfaced polarographic Clarke electrode to monitor the changes in oxygen levels as a function of time in a small volume reaction vessel. The phenoxyl radicals reacted with natural protectant reductant molecules such as glutathione to produce intermediate radicals which subsequently reacted with ambient oxygen in the reaction solution.

Many other students have contributed to our research in free radical chemistry over the past 39 years, and work in progress and planned shows promise of being as exciting as the past studies described above.

- Herb Sipe
Spalding Professor of Chemistry

Department of Chemistry
Hampden-Sydney College

August 2010