Cysteine Thiosulfinate Ester and Cysteine
charge effect for such nucleophilic/electrophilic reactions.19,38,39
Perhaps there is a coincidental cancellation of charge effects
whereby CyS2- is a better nucleophile, but CyS- experiences
less charge repulsion in its reaction with CyS(dO)SCy2-. In
contrast to the insensitivity of the thiolate with respect to charge,
the reactivity of CyS(dO)SCy shows a marked sensitivity to
the protonation states of its amine groups. Given the magnitude
of the reactivity differences of CyS(dO)SCy0 and CyS(dO)SCy-
versus CyS(dO)SCy2-, it appears likely to us that the increase
in reactivity that is observed upon protonation of the amine
groups is a consequence of specific acid catalysis, perhaps vis-
a`-vis an intramolecular hydrogen bond:
investigated the oxidation of CySH with HOX (X ) Cl, Br),
which is a more facile means of generating CySOH.34 This
reaction has allowed us to extend the pH range downward to
further explore the chemistry of CySOH.34
Experimental Section
Reagents. All chemicals were A.C.S. certified grade or better.
Water was doubly distilled in glass. Solutions of NaOH, mostly
free of CO2 contamination, were quantified by titration with
potassium hydrogen phthalate or standardized HCl, HClO4 solutions
using phenolphthalein as an indicator. HClO4 and HCl were
standardized against bicarbonate. The buffer solutions were prepared
from the solids K3PO4, NaH2PO4‚H2O, Na2HPO4, and Na3PO4‚12
H2O; the ionic strength was adjusted with NaClO4; and the pH/pD
was adjusted with NaOH, NaOD, HClO4, or DCl. L-Cysteine,
L-cystine, L-cysteinesulfinic acid monohydrate, L-cysteic acid
monohydrate, peracetic acid, deuterium chloride (35 wt % solution
in D2O), NaOD (40 wt % solution in D2O), NaClO4, K3PO4, NaH2-
PO4‚H2O, Na2HPO4, and Na3PO4‚12H2O were used as received.
The synthesis of CyS(dO)SCy was accomplished using a published
procedure.43
pH/pD Measurements. The [OH-] for the unbuffered solutions
was determined by acid-base titration against standardized HCl
or standardized HClO4 solutions. The [H+] of the buffered solutions
was determined with an ion analyzer using a Ag/AgCl combination
pH electrode. The ionic strength was kept constant at 1.0 M for all
H2O solutions (NaClO4 + NaOH/HClO4 + iP/Tris). To obtain the
[H+] or [OH-] of the buffered solutions from the measured pH
values, all pH measurements were corrected for the “Irving factor”44
and the ionic product of water (pKw) that were measured by titration
of a 1.0 M NaClO4 solution by a standardized solution of 0.1 M
NaOH (in 1.0 M NaClO4); pD measurements in D2O were made
using the same pH electrode by adding 0.4 units to the measure-
ment.45
We note that Kice et al. have previously observed acid
catalysis of nucleophilic reactions of aromatic thiosulfinate
esters.40
A Comparison of the Different Methods of Generating
CySOH. Scheme 4 summarizes possible reactions of CySOH
with the five redox derivatives that we have considered in this
contribution. The opposite reactions can potentially offer
methods of generating CySOH. We have shown in a previous
study that hydrolysis of CyS(dO)SCy is too slow to permit the
study of CySOH (Scheme 4, 1a),19 and we report herein that
hydrolysis of CySSCy (Scheme 4, -2) is even slower. The
present study exploits the facile reaction of CyS(dO)SCy with
CySH (Scheme 4, 1b) at high pH to generate CySOH. The
reaction of CyS(dO)2SCy with CySH (Scheme 4, -1d) remains
unexplored. However, an alternative approach to generating
CySOH in situ is the oxidation of CySH by reactions that are
capable of delivering an oxygen atom. Sluggish oxidants like
hydrogen peroxide are ineffective for producing CySOH at a
sufficient rate for studying the subsequent reactions.41,42 How-
ever, we have recently discovered that CySOH can be generated
by the facile reaction of CySH with hypohalous acids (HOX,
X ) Cl or Br).34 As forecasted in the data presented in Figure
3, the reaction mechanisms that are associated with the latter
reaction are consistent with the present study.
NMR Studies. 1H NMR spectra were recorded at 20((0.5) °C.
Deuterated buffers were prepared from D2O solutions of anhy-
drous K3PO4 by adding DCl, by dilution of a 40 wt % NaOD
solution with D2O, or by dilution of a 35 wt % DCl solution with
D2O. The chemical shifts (parts per million) were referenced to
sodium 2,2-dimethyl-2-silapentane-5-sulfonate (DSS, δ ) 0.015
ppm). Turbulent mixing of reagents was necessary to ensure
homogeneity of the reaction mixtures in the time frame of the
chemical reactions, and this was achieved for the NMR studies by
employing a hand mixer comprising two gastight syringes and a
T-mixer. Failure to quickly mix solutions produced different,
irreproducible results.
UV/Vis Spectroscopy. Electronic spectra were measured using
a diode array spectrophotometer using quartz cells with calibrated
1 mm, 2 mm, and 1 cm path lengths at 20 °C or the monochromator
of the stopped-flow instrument with a 1 cm path length using a Xe
arc lamp and a PMT detector at 18 °C.
General Description of the Stopped-Flow Studies. Kinetic
measurements were made with a stopped-flow spectrophotometer
using a Xe arc lamp and a PMT detector. A double-mixing mode
was used for some of the experiments. All the stopped-flow
measurements were made at a temperature of 18 °C maintained in
the observation cell with a circulator. The adiabatic temperature
increases that were associated with the pH-jump experiments were
determined experimentally to be less than 1 °C.
Conclusions
The reaction of CyS(dO)SCy with CySH produces CySSCy
and the reactive species CySOH. Above pH 12, this reaction is
sufficiently facile that the subsequent comproportionation of
CySOH with CySH can be observed. We report herein an
investigation of the latter reaction, which is apparently the first
detailed mechanistic study of a reaction of CySOH. Unfortu-
nately, the resulting kinetics can only be described in terms of
the variable KCa ySOHof the sulfenic acid moiety of CySOH
(because the value of the acid dissociation constant for CySOH
apparently lies below pH 12). In a parallel study, we have
Reaction of CyS(dO)SCy with CySH. The pH and [CySH]
dependencies of the reaction rates were investigated under pseudo-
first-order conditions using excess [CySH] over [CyS(dO)SCy]
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(43) Walti, M.; Hope, D. B. J. Chem. Soc., Perkin Trans. 1 1971, 12,
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J. Org. Chem, Vol. 72, No. 23, 2007 8845