MECHANISTIC STUDIES OF SULFATE HYDROLYSIS Benjamin T. Burlingham, 1 Lisa M. Pratt, 2 Ernest R. Davidson, 3 Vernon J. Shiner, Jr., 4 Jon Fong, 2 Theodore S. Widlanski 4 1 Mount Union College, Department of Chemistry, 1972 Clark Ave., Alliance, OH 44601; 2 Indiana University, Bloomington, Department of Geology, Bloomington, IN, 47405; 3 University of Washington, Department of Chemistry, Seattle, WA ; 4 Indiana University, Bloomington, Department of Chemistry, Bloomington, IN, Conclusions A new procedure for sulfur-34 isotope effect determination is presented S-34 KIE suggest a dissociative mechanism for acidic sulfate hydrolysis Solvent isotope effects inconsistent with a concerted dissociative mechanism S-34 KIE determinations may be useful for the investigation of sulfatases and sulfotransferases Materials and Methods Clear up mechanistic ambiguities by giving full picture of heavy atom isotope effects No synthetic isotopic labeling of substrate necessary Wider range of substrates—no requirement for nitrogen in substrate Methodology for determination of central heavy atom isotope effects would be valuable:  = 1000[(R - R std )/R std ]  = R P /R SM or  = (1000+  P )/(  SM ) KIE= log(1-f)/log(1-f*  ) Kinetic Isotope Effect determined from delta values: Abstract A stable isotope mass spectrometry method for the determination of S-34 kinetic isotope effects in sulfate monoester hydrolysis is described. Hydrolysis of aryl sulfates under acidic conditions give large, normal S-34 kinetic isotope effect (KIE) data. These data, along with inverse solvent isotope effects, are inconsistent with the currently proposed concerted mechanism involving simultaneous cleavage and proton transfer in the transition state. This method for the acquisition of S-34 kinetic isotope effects may also prove useful for studying the mechanism of other sulfuryl group transfers, including sulfatase and sulfotransferase catalyzed reactions. SubstrateKIESIE 1.54±0.02 %0.51 +/ ±0.03 % 2. Concerted Dissociative Mechanism: Proton Transfer in TS 3. Stepwise Dissociative Mechanisms: Pre- equilibrium protonation Results Discussion Distinguishing between mechanisms: – Central atom KIE consistent with dissociative mechanism – Inverse SIE suggests no proton transfer in transition state – Of listed mechanisms, stepwise dissociative mechanism most consistent with data Synthesis of pure sulfate monoesters Partial acid hydrolysis of sulfate monoesters Solvent Isotope Effects Rate determined via visible spectroscopy 1. Associative Mechanism: Pentavalent Intermediate Data Acquisition 2 Introduction The two mechanistic possibilities for sulfate ester hydrolysis most consistent with data: Sulfate ester hydrolysis important in physiological processes such as desulfation of estrone sulfate Mechanism of sulfate hydrolysis still not completely understood O-18 and N-15 KIE previously reported and a mechanism proposed 1 Acknowledgments This work has been funded by NIH/NCI grant RO1CA71736 (TSW) and NSF grant EAR (LMP). We would like to thank Alvan Hengge for helpful discussions. References % completion determined by visible spectroscopy Inorganic sulfate product precipitated with barium Product collected at multiple points of hydrolysis No satisfactory theoretical transition state structure yet attained Greater than 1% KIE considered qualitatively large for sulfur Possible mechanism of acid hydrolysis of sulfate monoesters: 1 Hoff, R. H.; Larsen, P.; Hengge, A. C. J. Am. Chem. Soc. 2001, 123, Giesemann, A.; Jager, H.-J.; Norman, A. L.; Krouse, H. R.; Brand, W. A. Anal. Chem. 1994, 66,