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. Widlanski4

1Mount Union College, Department of Chemistry, 1972 Clark Ave., Alliance, OH 44601; 2Indiana University, Bloomington, Department of Geology, Bloomington, IN, 47405; 3University of Washington, Department of Chemistry, Seattle, WA 98195-

1700; 4Indiana University, Bloomington, Department of Chemistry, Bloomington, IN, 47405.

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

SO

O OO

SO

O OR

HO

SO

O OR H2O

OS

O

O OR

H

R O O HH H

SO

O O

H2OO

SO

O OR

H+

H+

ROH + HOS

OH

O O

HOS

OH

O O

ROH +

O NO2SO

OO

1 N DClHSO4

-+ HO NO2

D2O

O NO2SO

OO

1 N HClHSO4

-+ HO NO2

H2O

BaSO4

Elemental Analyzer

Combustion Reactor

GC ColumnSO2

Stable IsotopeMass Spectroscopy

"" values: isotopic ratio compared to a standard

Materials and Methods

OCH3

OSO

OO

O NO2SO

OO 1 N HCl

HSO4-

+ HO NO2

1 N HCl+ HO

CH3

OHSO4

-

BaCl2BaSO4

BaCl2BaSO4

N

O

O

OS

O

OOH

HO

H

S-34 KIE ?

• 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 - Rstd)/Rstd]

a = RP/RSM or

a= (1000+ P)/( 1000+ SM)

KIE= log(1-f)/log(1-f*a)

Kinetic Isotope Effect determined from delta values:

AbstractA 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.

Substrate KIE SIE

1.54±0.02 % 0.51 +/- 0.02

1.72±0.03 %

SO

O OR O O H

HOH

H HO

SOH

O OR

HOHROH + H2SO4

2. Concerted Dissociative Mechanism: Proton Transfer in TS

SO

O OO

SO

O OR

HO

SO

O OR H2O

OS

O

O OR

H

R O O HH H

SO

O O

H2OO

SO

O OR

H+

H+

ROH + HOS

OH

O O

HOS

OH

O OROH +

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

OS

O

O OR

H

H2OR O O H

H HSO

OOO

SO

O OR

H+

OS

O

O OH

HO

SO

O OH

H+- ROH

1. Associative Mechanism: Pentavalent Intermediate

O RSO

OOHO R

SO3. pyridinium

pyridine

NH

Silica gel chromatography2% TEA/acetonitrile

O RSO

OO(Et)3NH

O RSO

OOK

AG50W-X8 Dowex.potassium form

R = -NO2, or -COCH3,

Data Acquisition2

Introduction

The two mechanistic possibilities for sulfate ester hydrolysis most consistent with data:

N

O

O

OS

O

OO

15k = 0.02%18k nonbridge =

0.83% (2.3%)

18k bridging = 0.97%

HH

OH

• 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 proposed1

AcknowledgmentsThis work has been funded by NIH/NCI grant RO1CA71736 (TSW) and NSF grant EAR-978267 (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:

1Hoff, R. H.; Larsen, P.; Hengge, A. C. J. Am. Chem. Soc. 2001, 123, 9338-9344.

2Giesemann, A.; Jager, H.-J.; Norman, A. L.; Krouse, H. R.; Brand, W. A. Anal. Chem. 1994, 66,

2816-2819.

O NO2SO

OO

OSO

OO

O

CH3