Abstract

1. Abstract The Water Quality Act of 1972 was amended in 1990 to include testing waterways utilizing “biocriteria” by fiscal year 1991-1993 triennium. Means to measure the ecological health of waterways were developed to help standardize field methods, but measuring ecological health needed region-based quantitative definitions to account for natural geographic variation. In this study, the Index of Biotic Integrity (IBI) developed for Maryland will be used to look at the population of macroinvertebrates that reside in the Trout and Salisbury River. Three sites will be compared and analyzed using the IBI criteria. The study is being used to determine if the Brockton Waste Water Treatment Plant (BWWTP) is assisting the health of the waterways after the treatment location. The waterway after the treatment plant is being compared to two human impact locations above the location of the BWWTP. It is anticipated that the population after the BWWTP will be better than the population before the plant.

2. Introduction • The Glyoxalase system (Figure 1) is a metabolic pathway that consists of two enzymes: Glyoxalase I (GxI) and Glyoxalase II (GxII), which are responsible for the detoxification of methylglyoxal and regulation of cell growth (1,2) by converting the cytotoxic methylgloxal to D-lactate, with glutathione (GSH) as a “co-substrate.” • The presence of the GxII enzyme activity has been found in all cells of eukaryotic and prokaryotic organisms (3). • GxII activity has also been detected in tumors (3) with a high concentration of lactic acid and a deficiency of methylglyoxal, which suggests that the cell no longer has the ability to maintain the necessary balance of methylglyoxal causing the cell to grow at an uncontrollable rate (7).

3. Figure 1. Known Inhibitors of GxII S-N-hydroxy-N-bromophenylcarbamoyl)glutathione N,S-bis-fluorenylmethoxycarbonylglutathione S-(Nitrocarbobenzyoxy)glutathione 5-p-tolylazotropolone 4,5-benzotropolone

4. Research Question & Objectives The long term goal of my research project is to synthesize S-glutathionyl-3-bromooxindole acetic acid (SGB) from 3-bromooxindole acetic acid ethyl ester (BOAA-EE) and evaluate the potential inhibition capabilities of SGB on the enzyme Glyoxalase II. The prioritized objectives are: • Synthesize BOAA-EE by acid catalyzed esterification of BOAA; • Evaluate purity by thin layer chromatography and HPLC; purify if necessary by silica gel flash chromatography; • Identify structure by proton NMR; • Synthesize SGB by ester exchange of BOAA-EE & glutathione; • Carry out assay of glyoxalase II and inhibition studies; • Evaluation of assay results and overall inhibition; • Submission of the SGB ester for anti-cancer screening.

5. Significance • BOAA-EE is a precursor to the synthesis of the potential GxII inhibitor, SGB. In this research, BOAA-EE has been successfully synthesized by the acid-catalyzed esterification of BOAA. • BOAA-EE was characterized and purity analyzed by TLC, HPLC, and H-1 NMR (Figure 6) at various stages of the procedure. • The Rf of the ester was calculated at 0.90, however several other spots were also seen on the TLC plate. Additional purification was accomplished on a RediSep, 4g flash chromatography column, using methylene chloride:ethylacetate (80:20). • The final weight of the BOAA-EE product after being purified was 0.2708g, which is a percent yield of 35.7%.

6. Methodology • The synthesis of 3-bromooxindole acetic acid ethyl ester was performed by acid catalyzed esterification of 3-bromooxindole acetic acid with ethanolic HCl (Figure 5). • 0.6861g (2.54mmol) of pure BOAA and 30mL of 1.86M ethanolic HCl was stirred at room temperature for two days in a 50mL round bottom flask completely covered with aluminum foil to protect it from light damage. • Molecular sieves were added to the reaction in order to absorb the water formed from the esterification reaction. • On the third day, the mixture was centrifuged for ten minutes to remove the molecular sieves, then placed on the HighVac for 90 minutes to evaporate to dryness. • Once dry, 150mL of fresh ethyl acetate was added to the product along with 75mL of a 10% solution of NaHCO3.

7. Acknowledgements I would like to acknowledge receipt of a course semester grant from the BSC Center for Sustainability.

8. Works Cited 1. “N,S-Bis-Fluorenylmethoxycarbonylglutathione: A New, Very Potent Inhibitor of Mammalian Glyoxalase II”, A.C. Ella, M.K. Chyan, G.B. Principato, E. Giovannini, G. Rosi and S.J. Norton, Biochem. Mol. Biol. Int. (1995) 35,763-771. 2. “Small molecules probes of glyoxalase I and glyoxalase II”, John F. Barnard, David L. Vander Jagt, John F. Honek, Biochimica et Biophysica Acta (1994) 1208, 127-135. 3. “The glyoxalase system in health and disease”, P.J. Thornalley, Mol. Asp. Med. (1993) 14, 287-371. 4. “The active-site residue Tyr-175 in humans glyoxalase II contributes to binding of glutathione derivatives”, Marianne Ridderström, Per Jemth, Alexander D. Cameron, Bengt Mannervik, Biochimica et Biophysica Acta (2000) 1481, 344-348. 5. “The glyoxalase system in higher plants: Regulation in growth and differentiation”, R. Deswal, T.N. Chakarvarty and S.K. Sopory, Biochem. Soc. Trans. (1993) 21, 527-530. 6. “Explaining the inhibition of glyoxalase II by 9-fluorenylmethoxycarbonyl-protected glutathione derivatives”, Ke-Wu Yang, D.N. Sobieski, A. L. Carenbauer, P.A. Crawford, C.A. Makaroff and M. W. Crowder, Arch. Biochem. Biophys. (2003) 414, 271-278.