34S Isotope Effect on Sulfate Ester Hydrolysis: Mechanistic Implications

Article abstract of DOI:10.1021/ja0279747

In this communication, we report the first determination of ³⁴S kinetic isotope effects (KIEs) for the hydrolysis of sulfate monoesters. The method involves the conversion of the inorganic sulfate, acquired at partial extent of reaction, to SO₂, followed by isotope ratio determination by mass spectrometry. The KIEs determined for p-nitrophenyl sulfate and p-acetylphenyl sulfate are 1.0154 (±0.0002) and 1.0172 (±0.0003), respectively. These results, together with previous peripheral ¹⁸O KIE values, are inconsistent with an associative mechanism. The isotope effect method we report should also prove useful for studying the mechanism of other sulfuryl group transfers, including sulfatase and sulfotransferase reactions, as well as sulfate hydrolyses under other conditions. Copyright

Full text of DOI:10.1021/ja0279747

Published on Web 10/07/2003
34S Isotope Effect on Sulfate Ester Hydrolysis: Mechanistic Implications
Benjamin T. Burlingham,† Lisa M. Pratt,‡ Ernest R. Davidson,† Vernon J. Shiner, Jr.,† Jon Fong,‡ and
Theodore S. Widlanski*,†
Department of Chemistry and Department of Geology, Indiana UniVersity, Bloomington, Indiana 47405
Received August 1, 2002; E-mail: twidlans@indiana.edu
Sulfate and phosphate monoesters are among the most biologi-
cally ubiquitous esters. Because of this, a variety of techniques have
been developed to study the hydrolysis of these compounds,
including elegant methods for determining bridging and nonbridging
18O isotope effects by remote labeling.1,2 Notably absent from this
panoply of techniques are methods for determining isotopic
discrimination associated with the central heavy atom, be it
phosphorus or sulfur. In this communication we describe the first
Figure 1. Acid hydrolysis of aryl sulfate monoesters.
determination of a 34S isotope effect on sulfate ester hydrolysis
The 34S isotope discrimination during acid-catalyzed sulfate ester
and examine the mechanistic implications of this result in light of
available data and theoretical predictions.
hydrolysis10,11 (Figure 1) was determined as follows. A 10 mM
solution of the sulfate ester was incubated in 1.0 N HCl at 22 °C,
and the extent of reaction was determined spectrophotometrically.
At various times, 5 µL aliquots were dissolved in 995 µL of 2 N
NaOH, and the amount of free phenoxide was determined by
measuring the absorbance change at 405 and 325 nm for 3 and 4,
respectively. The sample was then treated with excess BaCl2,
leading to the rapid precipitation of the inorganic sulfate product
as BaSO4. The barium sulfate was then converted to SO2, and the
isotope ratio was determined by mass spectrometry. Three inde-
pendent runs were conducted for each compound, and inorganic
sulfate samples were collected at ∼50% extent of reaction.
Experimental isotopic fractionation value determinations for each
run were done in triplicate. The KIE determined for p-NPS (1) is
1.0154 ((0.0002), and the KIE determined for p-APS is 1.0172
((0.0003). The p-acetyl compound was further analyzed at ∼30
and ∼70% reaction, and the data are in agreement with the values
determined at 50% reaction (See Supporting Information.)
On the basis of the available data, a number of different reaction
mechanisms have been proposed for acid-catalyzed sulfate ester
hydrolysis. Any mechanism must account for the relatively small
18O leaving group isotope effect measured by Hengge’s group.5 A
priori, such an observation suggests an associative type of mech-
anism (Figure 2C), in which formation of a pentavalent intermediate
of some kind (either what is shown in 2C, or some kind of protomer
or tautomer) is partially rate determining, and cleavage of the S-O
bond takes place subsequently. However, this mechanism is
inconsistent with the bulk of previously available data. A concerted
process has also been proposed (Figure 2A).5 In this mechanism,
proton transfer from the sulfate ester to the leaving-group oxygen
is mediated by an intervening water molecule, and attack on sulfate
is concerted (though not necessarily synchronous) with the breaking
of the S-O bond. Protonation of the leaving-group oxygen
suppresses the intrinsic oxygen isotope effect associated with S-O
cleavage. This mechanism may also account for the small, but
experimentally significant, difference in 18O nonbridging isotope
effects associated with sulfate hydrolysis under differing HCl
concentrations, and for the difference in secondary 18O isotope
effects for the acidic and basic hydrolysis reactions. On the basis
of symmetry arguments, mechanism 2A may be found to be
incomplete. To avoid generating a zwitterionic protonated sulfate
Highly accurate isotopic ratios may be determined by use of
stable isotope ratio mass spectrometry (IRMS). This technique may
be used to determine the isotopic composition of a given species,
provided that it can be converted to a gas, such as N2, CO2, etc.3
It is also possible to use this technique to determine isotopic ratios
associated with the sulfur of SO2, provided that a dedicated isotope
ratio mass spectrometer and inlet system is available. Since the
conversion of sulfate to sulfur dioxide is fairly routine,4 and has
been used for some time to study isotopic constitutions in minerals,
a simple extension of this technique would be to determine 32S/34S
discrimination as a function of extent conversion during the
hydrolysis of sulfate esters. The measured isotope effect would then
provide an important piece of data for evaluating the mechanism
of sulfate ester hydrolysis.
In testing the feasibility of this technique, two sulfate esters were
of initial interest to us. 18O isotope effects for p-nitrophenyl sulfate
(p-NPS) (1) have been measured,5 and we thought that 34S isotope
data would be of additional benefit in testing the proposed
mechanism. Because of our interest in studying the enzyme-
catalyzed mechanism of sulfate hydrolysis, we also chose to study
p-acetylphenyl sulfate (p-APS), which has been used routinely in
sulfatase assays.6,7
The accurate determination of heavy atom kinetic isotope effects
(KIEs) requires the availability of highly pure starting sulfate ester,
free of both free phenol and inorganic sulfate. A number of
syntheses of these compounds have been reported,7-9 but we
thought that a modification in the purification procedure would lead
to a more facile isolation of pure compound. Mixing the phenol of
interest with sulfur trioxide-pyridinium complex in pyridine yielded
the pyridinium salts of 1 and 2. These salts could be purified by
silica gel chromatography (2% triethylamine/acetonitrile) to give
the triethylammonium salts, free of inorganic sulfate. Ion exchange
gave the potassium salts of 1 and 2 as white solids. No phenol was
1
detectable by H NMR, and analysis by UV/vis spectroscopy at
405 and 325 nm for 1 and 2, respectively, suggested that there was
less than 0.5% phenol. Elemental analysis confirms this level of
purity (see Supporting Information.)
† Chemistry Department.
‡ Geology Department.
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C O MMU N I C A T I O N S
maximum C-S KIE at 1.8%.12 This suggests that our measured
isotope effects are near the upper limit of what would be predicted
for S-O bond cleavage. Mechanistically, our observation of a large
34S kinetic isotope effect associated with acid-catalyzed hydrolysis
suggests that cleavage of the S-O bond does take place during
the rate-limiting step. This probably rules out an associative
mechanism (Figure 2C), since such a mechanism requires two
isotopically sensitive steps that would be of opposing magnitudes.
However, it is consistent with the other mechanisms shown, all of
which contain an isotope sensitive S-O bond cleavage event.
In summary, measurement of the 34S isotope effect on sulfate
ester hydrolysis suggests that an associative mechanism is unlikely.
The isotope effect method we report should also prove useful for
studying the mechanisms of sulfate hydrolyses under various
conditions, other sulfuryl group transfers, and a variety of enzyme-
catalyzed reactions involving sulfur-containing compounds.
Acknowledgment. This work has been funded by NIH/NCI
Grant RO1CA71736 (T.S.W.) and NSF Grant EAR-978267 (L.M.P.).
We thank Alvan Hengge for helpful discussions.
Supporting Information Available: Synthesis and characterization
of 1 and 2; general procedure for isotopic data acquisition; general
procedures for KIE calculations (PDF). This material is available free
of charge via the Internet at http://pubs.acs.org.
Figure 2. Possible mechanisms for the hydrolysis of sulfate esters. (A)
This transition state has been previously proposed to account for 18O KIE’s.
(B) Modification of the transition state in A; this figure takes into account
the demands of microscopic reversibility. (C) Associative mechanism utilizes
a pentavalent intermediate. (D) Concerted mechanism. (E) Stepwise
mechanism. (F) Two analogous zwitterionic species.
References
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7422.
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(6) analogous to the zwitterionic protonated sulfate ester (5) (see
Figure 2F) another water molecule must be added to the reaction
mechanism to give a new version (Figure 2B). Such an argument
hinges on whether the zwitterionic form of sulfuric acid (6) and
the zwitterionic intermediate 5 are of similar energy, something
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Although no specific S-O bond breaking data have been reported
to date, data for the C-S KIEs are available. Such measurements
are in the range of 1.0-1.8%. Simple calculations also put the
(6) Anderson, C. J.; Lucas, L. J. H.; Widlanski, T. S. J. Am. Chem. Soc.
1995, 117, 3889-3890.
(7) Anderson, C.; Freeman, J.; Lucas, L. H.; Farley, M.; Dalhoumi, H.;
Widlanski, T. S. Biochemistry 1997, 36, 2586-2594.
(8) Benkovic, S. J.; Dunikoski Jr., L. K. Biochemistry 1970, 9, 1390-1397.
(9) Burkhardt, N. G.; Lapworth, A. J. Chem. Soc. 1926, 684-690.
(10) Fendler, E. J.; Fendler, J. H. J. Org. Chem. 1968, 33, 3852-3859.
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