MECHANISTIC STUDIES OF SULFATE HYDROLYSIS
1
Burlingham,
2
Pratt,
3
Davidson,
4
Jr.,
2
Fong,
4
Widlanski
Benjamin T.
Lisa M.
Ernest R.
Vernon J. Shiner,
Jon
Theodore S.
1Mount Union College, Department of Chemistry, 1972 Clark Ave., Alliance, OH 44601; 2Indiana University, Bloomington,
3
Department of Geology, Bloomington, IN, 47405; University of Washington, Department of Chemistry, Seattle, WA 981954
1700; Indiana University, Bloomington, Department of Chemistry, Bloomington, IN, 47405.
Abstract
Materials and Methods
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.
Introduction
• 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
Substrate
Synthesis of pure sulfate monoesters
SO3. pyridinium
HO
NH
R
pyridine
O
O S O
O
R
Silica gel chromatography
2% TEA/acetonitrile
K
O
O S O
O
AG50W-X8 Dowex.
potassium form
R
O
O S O
O
(Et)3NH
R
R = -NO2, or -COCH3,
Partial acid hydrolysis of sulfate monoesters
O
O S O
O
NO2
O
O S O
O
O
1 N HCl
-
HSO4
1 N HCl
-
HSO4
CH3
HO
+
NO2
O
+
Conclusions
Results
O
O S O
O
NO2
O
O S O
O
O
KIE
SIE
1.54±0.02 % 0.51 +/- 0.02
O O
R
S
O
O
1.72±0.03 %
CH3
• No satisfactory theoretical transition state
structure yet attained
• Greater than 1% KIE considered qualitatively
large for sulfur
BaSO4
BaCl2
BaSO4
HO
Possible mechanism of acid hydrolysis of
sulfate monoesters:
CH3
1. Associative Mechanism: Pentavalent Intermediate
O O
R
S
O
O
H+
Data Acquisition2
O O
R
S
O
O
H
H2O
O
S
H
R O
O
H
O H
- ROH
O
O O
H
S
O
O
H
H+
O O
H
S
O
O
Elemental Analyzer
2. Concerted Dissociative Mechanism: Proton
Transfer in TS
18
O
S
O
Combustion Reactor
BaSO4
O
O
O
H O
18
k nonbridge =
SO2
O
k = 0.02%
"" values: isotopic
ratio compared to a
standard
0.83% (2.3%)
Methodology for determination of central
heavy atom isotope effects would be valuable:
O
O
O
O
H O
H
H2O
O O
R
S
O
O
H
R O
H O
O
S
O
ROH +
ROH +
O H
O H
S
O
O
H2O
O O
S
HO
OH
O O
S
HO
OH
• 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
R O
H
O
S
O
O
O H
H
ROH + H2SO4
O H
References
Stable Isotope
Mass Spectroscopy
3. Stepwise Dissociative Mechanisms: Preequilibrium protonation
1Hoff,
R. H.; Larsen, P.; Hengge, A. C. J. Am. Chem.
Soc. 2001, 123, 9338-9344.
Kinetic Isotope Effect determined from delta values:
O O
R
S
O
O
a = RP/RSM or
O
S
H+
H
 = 1000[(R - Rstd)/Rstd]
S-34 KIE ?
HOH
O O
R
S
O
OH
15
H
H
O O
R
S
O
O
H
GC Column
N
H
O O
R
S
O
O
+
Discussion
BaCl2
• % completion determined by visible spectroscopy
• Inorganic sulfate product precipitated with barium
• Product collected at multiple points of hydrolysis
k bridging = 0.97%
The two mechanistic possibilities for sulfate
ester hydrolysis most consistent with data:
H+
O O
R
S
O
O
H
H2O
R O
H O
O
S
O H
O H
ROH +
O O
S
HO
OH
a= (1000+ P)/( 1000+ SM)
N
H
•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
O O
R
S
O
O
KIE= log(1-f)/log(1-f*a)
O
Solvent Isotope Effects
O
O S O
O
O
O S O
O
NO2
1 N DCl
HSO4-
+
HO
NO2
D2O
NO2
1 N HCl
HSO4-
H+
O O
R
S
O
O
H
O
ROH +
S
O
O
H2O
HO
Distinguishing between mechanisms:
NO2
H2O
Rate determined via visible spectroscopy
Jager, H.-J.; Norman, A. L.; Krouse,
H. R.; Brand, W. A. Anal. Chem. 1994, 66,
2816-2819.
O O
S
HO
OH
– Central atom KIE consistent with dissociative mechanism
+
2Giesemann, A.;
– Inverse SIE suggests no proton transfer in transition state
– Of listed mechanisms, stepwise dissociative mechanism
most consistent with data
Acknowledgments
This 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.