Reaction of Cysteine with Nitrous Acid
been taken into account. As reported above, NO is a good
nitrosating agent in the presence of air, but almost
unreactive in anaerobic conditions: actually, no evidence
of CySNO formation was evidenced. It has been also
reported that NO slowly reacts with thiols through a
reaction catalyzed by traces of iron ions, invoked to be
present in the reaction medium;13 however, results of our
preliminary tests on the role of iron ions seem not to
confirm this behavior.
A direct involvement of NO2 until now had not been
explored. Toward this aim, we ran experiments, directly
preparing NO2 by addition of NO to a sealed tube
containing dioxygen. The brown-colored nitrogen dioxide,
instantaneously formed, was added with a 0.04 M CySH
aqueous solution, buffered at pH ) 2.5. Two experiments
with 1 and 2 molar equiv of NO2 showed that the
conversion of CySH was 45% and 90%, respectively,
showing a stoichiometry of 1:2 CySH/NO2.
In both cases CySNO and CySSCy were formed in a
78:22 and 80:20 ratio, respectively. These values are
those forecasted for the reaction of 0.04 M CySH with
0.04 M NaNO2 at pH 2.5, suggesting that HNO2, and
then NO+ (or H2O-NO+), is once again the reactive
species. In fact, HNO2 was expected to be derived from
the disproportionation reaction of NO2 with water (2 NO2
+ H2O f HNO2 + HNO3).
However, no evidence of formation of the cysteine radi-
cal, CyS•, could be achieved. At the present time we can-
not exclude that the oxidation of CySH could give some
intermediates (for instance CySOH and/or CySNO2),
which by decomposition can lead to CySSCy.
Exp er im en ta l Section
The reaction were conducted in a thermostated bath at 37
°C. The formation of CySNO was constantly monitored by
measuring the absorbance at λ ) 543 nm (ꢀ ) 16.8 M-1 cm-1),
flowing the reacting solution, by a peristaltic pump, in a UV/
vis spectrophotometer equipped with a flow-cell. The CySNO
percent yield was calculated on the basis of reacted CySH.
The formation of CySSCy cannot be spectrophotometrically
monitored. However, CySNO and CySSCy being the only
reaction products (see below), the CySSCy yield was calculated
as CySSCy % ) (100 - CySNO%).
Capillary electrophoresis analysis was performed with an
instrument equipped with a UV-detector at 200 nm. A fused-
silica capillary column (75 µm i.d., 67 cm length), a 100 mM
boric acid/25 mM Tris background electrolyte (pH 8.2), and a
30 kV voltage were used. Under these conditions the migration
time of CySSCy was 3.43 ( 0.01 min.
The following buffer solutions were used: H2C2O4/NaHC2O4
(pH ) 1.5), H3PO4/KH2PO4 (pH ) 2.0 and 2.5), phthalic acid/
monosodium phthalate (pH ) 3.3 and 3.5), NaHC2O4/Na2C2O4
(pH ) 4.0), acetic acid/sodium acetate (pH ) 4.65), monoso-
dium phthalate/disodium phthalate (pH ) 5.3), KH2PO4/
K2HPO4 (pH ) 7.0).
Rea ction of 0.04 MCySH w ith 0.04 M Na NO2 a t p H )
0.5. An aqueous solution (24.0 mL) containing CySH (1.0
mmol, 121 mg) and NaNO2 (1.0 mmol, 69 mg) was added with
12 M HCl (1.0 mL); a deep red color, due to the formation of
CySNO, immediately developed. From Amax ) 0.670, reached
in less than 30 s, the CySNO yield was calculated to be 100%
(100% conversion yield). The absorbance quickly decreased,
and in less than 1 h the solution was completely faded.
Capillary electrophoresis analysis showed the presence of
CySSCy alone.
Rea ction of 0.04 MCySH w ith 0.04 M Na NO2 in Bu ff-
er ed Solu tion . An aqueous solution buffered at the appro-
priate pH (25.0 mL) containing CySH (1.0 mmol, 121 mg)
was added with NaNO2 (1.0 mmol, 69 mg), and the forma-
tion of CySNO monitored spectrophotometrically. For reac-
tions carried out at pH 1.5-4.65 the Amax values were reached
within 1-30 min and were found not to decrease over 2 h.
The reaction mixtures were assayed to assess the conversion
of CySH (see below), which in all cases was found to be
quantitative. Reactions carried out at pH ) 5.2 and 6.3 were
stopped after 2 h; after this time conversion of CySH was
found to be 50% and 30%, respectively. The reaction car-
ried out at pH ) 7.0 showed no conversion at all of CySH after
60 min.
Assessm en t of CySH Con ver sion . After the Amax value
was reached, an extra aliquot of NaNO2 (0.5 molar equiv)
was added and then 12 M HCl to set the pH at 0.5. Under
these conditions the CySH is quantitatively converted into
CySNO, as reported before. The amount of unreacted CySH
can be calculated from the enhancement of absorbance. After
complete decomposition of CySNO occurred, the reaction
mixture was analyzed by capillary electrophoresis. In all
experiments CySSCy was the only detected product in 100%
yield.
Rea ction s of CySH 0.04M w ith NO2. A 35 mL glass tube
was filled with dioxygen, and then 23 mL of gaseous NO was
added at atmospheric pressure: the brown color of NO2
immediately appeared. The reaction tube was placed in a
thermostated bath, and a CySH (1 mmol, 121 mg) aqueous
solution (25 mL), buffered at pH ) 2.5, was then added. The
Amax value was reached after 15 min. The reaction mixture
was tested to determine the conversion of CySH (45%). From
Con clu sion s
The reaction of CySH with NaNO2 in aqueous solution
leads to CySNO and CySSCy with a rate of formation,
and relative yield, strongly dependent on the pH. The
rate of formation of CySNO (VCySNO) as well as CySSCy
(VCySSCy) decreases by increasing the pH, while the ratio
CySSCy,%/CySNO,% largely increases with the pH.
Different species seem to be involved as nitrosating and
oxidizing species. Under strong acidic conditions the main
reactive species is NO+ (or H2O-NO+), deriving from
nitrous acid through an acid-promoted reaction. This
species mainly leads to the nitrosation product, CySNO,
and, to a lesser extent, to the oxidation product, CySSCy.
It is important to underline that the formation of both
CySNO and CySSCy takes place on the unprotonated
CySH instead of the protonated form, CySH2+, which
prevails at pH < 1.7.
By increasing the pH the acid-catalyzed reaction
leading to the nitrosating/oxidizing species NO+ (and/or
H2O-NO+) is progressively inhibited, and HNO2 and
N2O3 are mainly involved as oxidizing and nitrosating
agent, respectively. The kinetic equation obtained from
experiments carried out at pH 4.65 (VCySNO ) k2[HNO2]2,
k2 ) (3.3 ( 0.1) × 103 L mol-1 min-1) suggests that the
formation of N2O3 from HNO2 is the rate-determing step
in the nitrosation of CySH.
The reason HNO2 behaves as oxidant, while N2O3 as
nitrosating agent, is not clear. We can hypothesise that
both species are involved in an initial SET process
leading to the formation of a radical ion pair, which in
cage behaves differently depending on the nature of the
radical anion. In the former case proton transfer from
CySH•+ might lead to the free CyS• radical and then to
CySSCy by dimerization. In the latter case radical ion
coupling, followed by loss of HNO2, would lead to the
nitrosated product, CySNO (Scheme 2).
J . Org. Chem, Vol. 67, No. 24, 2002 8629