S-nitrosothiol and disulfide formation through peroxynitrite-promoted oxidation of thiols

Article abstract of DOI:10.1002/1099-0690(200101)2001:1<131::AID-EJOC131>3.0.CO;2-N

Peroxynitrite reacts with thiols 1 at acidic pH to give the corresponding S-nitrosothiols 2 and disulfides 3. The formation of nitrosothiols 2, the yield of which is strongly pH-dependent, can be rationalized in terms of acid-catalyzed decomposition of the undissociated HPN, probably through the intermediacy of the protonated form H₂PN⁺, leading to a species, X-NO, capable of nitrosating the thiol function. In contrast, the formation of disulfides 3 occurs in a manner independent of the pH, without the intermediacy of sulfanyl radicals. Under basic conditions, the peroxynitrite anion (PN) oxidizes the thiolate ion to sulfanyl radicals, eventually leading to disulfide 3, or undergoes a thiol-catalyzed decomposition. The former is the exclusive reaction exhibited by peroxynitrite at pH > 13.

Full text of DOI:10.1002/1099-0690(200101)2001:1<131::AID-EJOC131>3.0.CO;2-N

FULL PAPER
S-Nitrosothiol and Disulfide Formation through Peroxynitrite-Promoted
Oxidation of Thiols
Loris Grossi,*[a] Pier Carlo Montevecchi,*[a] and Samantha Strazzari[a][‡]
Keywords: Peroxynitrite / Thiols / Oxidations / Nitrosation / S-Nitrosothiol
Peroxynitrite reacts with thiols 1 at acidic pH to give the cor-
trast, the formation of disulfides 3 occurs in a manner
responding S-nitrosothiols 2 and disulfides 3. The formation independent of the pH, without the intermediacy of sulfanyl
of nitrosothiols 2, the yield of which is strongly pH-depend- radicals. Under basic conditions, the peroxynitrite anion (PN)
ent, can be rationalized in terms of acid-catalyzed decom- oxidizes the thiolate ion to sulfanyl radicals, eventually lead-
position of the undissociated HPN, probably through the in-
termediacy of the protonated form H2PN+, leading to a spe-
cies, X−NO, capable of nitrosating the thiol function. In con-
ing to disulfide 3, or undergoes a thiol-catalyzed decomposi-
tion. The former is the exclusive reaction exhibited by per-
oxynitrite at pH Ͼ 13.
Introduction
It would seem that the mechanism of peroxynitrite-pro-
moted thiol oxidation is far from being fully understood.
Except for the case of the aforementioned work by Radi,[7]
who studied the oxidation of cysteine and bovine serum al-
bumin in the pH range 6.5Ϫ10, all the available data in the
literature concern thiol oxidations carried out at physiolo-
gical pH (i.e. 7.4). At this value, both PN and HPN forms
are present in equilibrium, hence it is not easy to ascertain
the actual species responsible for the thiol oxidation. In our
opinion, in order to achieve a better understanding of the
reaction mechanism, we first need to study the behavior
exhibited by peroxynitrite under conditions of acidic and
basic pH, at which the undissociated HPN and the dissoci-
ated PN forms, respectively, largely predominate. To this
end, we have explored the reaction of simple thiols 1 with
peroxynitrite carried out under conditions of acidic (0Ϫ6)
and basic (10.5Ϫ13.5) pH.
The peroxynitrite ion ONOOϪ (PN) is a relatively stable
species capable of oxidizing biological targets according to
either a one- or two-electron mechanism.[1]
At physiological to acidic pH,[1,2] PN is in equilibrium
with its conjugate acid ONOOH (HPN) (pka ϭ 6.8).[1b] The
latter rapidly undergoes OϪO bond scission (τ1/2 Ͻ 1 s)[3]
with formation of an ‘‘in cage’’ radical pair, which can act
as a source of hydroxyl radicals and NO2 (30%) or re-
arrange to nitric acid (70%).[2,4] However, it has been re-
ported that HPN can also oxidize suitable substrates, either
according to a direct one- or two-electron transfer mechan-
ism or by an indirect one-electron mechanism.[1b,5]
Most notably, peroxynitrite[6] has been shown to be in-
volved in the in vitro biological oxidation of the thiol func-
tion. In 1991, Radi et al.[7] reported that cysteine was oxi-
dized to cystine: the authors proposed the involvement of a
two-electron transfer process between peroxynitrite and the
undissociated form of the thiol. Accordingly, the second-
order rate constant was found to be pH-dependent with a
maximum at 6.8, notwithstanding that the yield of disulfide
increased with pH up to 10.
Many intermediates have been proposed for the forma-
tion of disulfides in the peroxynitrite-promoted thiol oxida-
tion, e.g. sulfenic acids,[1a] sulfanyl radicals,[8] thionitrates,
and sulfenyl nitrites.[1a] It has also been reported that at
physiological pH peroxynitrite can give rise to the forma-
tion of small amounts of S-nitrosothiols, which can behave
as slow NO carriers.[9] However, the mechanism of thiol
nitrosation remains somewhat unclear, even though a direct
electrophilic nitrosation has been suggested.[10]
Results and Discussion
Reactions under acidic conditions were carried out at
5Ϫ10 °C by adding 2 mL of a 0.5  aqueous solution of
peroxynitrite (pH ϭ 13.5) to a 0.1  acetonitrile solution
(10 mL) of the appropriate thiol 1a؊d containing 0.15 mL
of 12  hydrochloric acid. Under these conditions, all the
solutions immediately became colored due to the formation
of the corresponding S-nitrosothiol 2a؊d.[11]
When benzylthiol 1a was used, the solution became
bright red. The yield of S-nitrosothiol 2a increased over a
period of 20 min, as monitored by spectrophotometric anal-
ysis at λ ϭ 550 nm.[12] The reaction mixture was sub-
sequently extracted with diethyl ether and the organic layer
[a]
`
Dipartimento di Chimica Organica ‘‘A. Mangini’’, Universita
1
was separated and analyzed by H NMR. The spectrum
di Bologna,
obtained showed the formation of the S-nitrosothiol 2a and
the disulfide 3a in a 70:30 ratio in Ͼ 90% overall yield.
Subsequent column chromatography allowed the isolation
of 2a in 55% yield, which rapidly decomposed upon
Viale Risorgimento 4, 40136 Bologna, Italy
E-mail: grossi@ms.fci.unibo.it
montevec@ms.fci.unibo.it
[‡]
In partial fulfillment of the requirements for the Ph.D. degree
in Chemical Sciences, University of Bologna.
Eur. J. Org. Chem. 2001, 131Ϫ135
WILEY-VCH Verlag GmbH, 69451 Weinheim, 2001
1434Ϫ193X/01/0101Ϫ0131 $ 17.50ϩ.50/0
131

L. Grossi, P. C. Montevecchi, S. Strazzari
FULL PAPER
standing at room temperature in air to give a ca. 1:1 mix- the crude residue showed the formation of the disulfide 3c
ture of the disulfide 3a and the benzyl thiosulfinate 5. The and the S-nitrosothiol 2c in a 40:60 ratio as the only detect-
formation of the thiosulfinate 5 would suggest the interme- able products. S-Nitrosothiol 2c was found to be stable in
diacy of the sulfenic acid, PhCH2SOH,[13] possibly originat- acetonitrile solution for several days; as a consequence, we
ing from 2a through air oxidation followed by hydrolysis can infer that the disulfide 3c did not derive from decom-
due to the presence of moisture (see Scheme 1).
position of 2c, but was rather directly formed from thiol 1c
in competition with the formation of the S-nitrosothiol 2c.
The reaction mixture obtained with thiophenol 1b imme-
diately became deep brown-red;[11] TLC analysis showed
the formation of the disulfide 3b and S-nitrosothiol 2b as
the exclusive products. On standing at 10 °C for 15 min,
the color faded; TLC and GC/MS analysis of the resulting
mixture showed the formation of the disulfide 3b and the
thiosulfonate 4b as the only detectable products. Sub-
sequent column chromatography allowed the separation of
these products in yields of 45% and 15%, respectively.
Similarly to 1b, the reaction of allylthiol 1d gave the S-
nitrosothiol 2d,[11] which was found to be unstable under
the reaction conditions. The bright-red color faded within
1
30Ϫ45 min. Subsequent H NMR analysis of the reaction
mixture showed the formation of the disulfide 3d as the
major product (60%), together with minor amounts of
other unidentified compounds.
The overall findings indicate that the peroxynitrite-pro-
moted reaction of thiols 1 under acidic conditions generally
gives S-nitrosothiols 2 and disulfides 3 as the exclusive reac-
Scheme 1
From repeated experiments, we found that the reaction tion products. However, the finding that the yields of prod-
mixture slowly decomposed upon standing at room temper- ucts 2a and 2c increased over a period of 20 min (vide su-
ature over a period of 72 h to give mainly the disulfide 3a pra) clearly indicates that HPN itself (τ1/2 Ͻ 1 s)[3] is not
together with trace amounts of the benzyl thiosulfonate 4a the actual species responsible for the nitrosation of the
and benzaldehyde 7. When the reaction mixture was re- thiol function.
fluxed, decomposition of 2a was found to be complete
within 20 min. Thereafter, GC/MS and H NMR analyses ent pH values in the range 0Ϫ6. In each case, S-nitrosothiol
The reaction of thiol 1a with HPN was repeated at differ-
1
showed the presence of the disulfide 3a, benzaldehyde 7, 2a and disulfide 3a were found to be the only reaction prod-
1
and dibenzyl sulfone (PhCH2SO2CH2Ph) in a 6:2:1 ratio. ucts, as detected by H NMR analysis. We observed that
The formation of 4a and 7 can be rationalized in terms the yield of the disulfide 3a (30%, based on starting thiol)
of further oxidation of the S-nitrosothiol 2a. Specifically, was essentially independent of the pH in the aforemen-
oxidation of 2a might lead to thioaldehyde 6, and then to tioned range.
aldehyde 7, through an oxidation/deprotonation process fol-
In contrast, the yield of the S-nitrosothiol 2a rapidly de-
lowed by nitric oxide loss, whereas the formation of 4a creased at pH Ͼ 1 (Figure 1). We suggest that at acidic pH
might be accounted for in terms of the intermediacy of the the undissociated HPN might undergo, in competition with
sulfinic acid, PhCH2SO2H,[13] as depicted in Scheme 1. At the fast decomposition pathway to nitrate ion,[2Ϫ4] an acid-
present, the formation of dibenzyl sulfone remains unclear. catalyzed decomposition, leading to some species, XϪNO,
As for the disulfide 3a, at first sight this product might be capable of behaving as the actual nitrosating agent. This
viewed as being derived from decomposition of 2a which, in acid-catalyzed decomposition, which probably occurs
all cases examined, afforded 3a as the main product to- through the intermediacy of the protonated form, H2PNϩ,
gether with minor amounts of other compounds, the nature would then prevail over the decomposition to nitrate ion
of which was found to depend on the mode of decomposi- under very acidic conditions (pH Ͻ 1). On the other hand,
tion, as outlined above. However, we found S-nitrosothiol we cannot exclude that the protonated form itself could be
2a to be rather stable under the reaction conditions; on the in part responsible for the formation of the S-nitrosothiol
other hand, we found that the disulfide 3a was formed in 2a, through direct nucleophilic attack at the nitrogen atom
30% yield after just 20 min. On this basis, it seems likely by thiol
1 with displacement of hydrogen peroxide
that disulfide 3a was formed, at least in part, from peroxy- (Scheme 1).
nitrite-promoted reaction of 1a in competition with the
formation of S-nitrosothiol 2a.
The finding that the formation of the disulfide 3a shows
no pH dependence was somewhat surprising. At first sight,
This hypothesis is strongly supported by results obtained we might suppose that HPN can either afford the nitrosat-
with tert-butylthiol 1c. In this case, the reaction mixture ing species and then S-nitrosothiol 2a, through the interme-
immediately became reddish-green.[11] 1H NMR analysis of diacy of the protonated form H2PNϩ, or decompose lead-
132
Eur. J. Org. Chem. 2001, 131Ϫ135

S-Nitrosothiol and Disulfide Formation through Peroxynitrite-Promoted Oxidation of Thiols
FULL PAPER
At pH ϭ 13.5, thiols 1a,b were found to be totally con-
sumed within 5 min, leading to the corresponding disulfides
3a,b as the only detectable products in Ͼ 90% yield. When
the reaction of 1b was carried out in the presence of a two-
fold excess of phenylacetylene, it gave instead a mixture of
the disulfide 3b and the thiol/alkyne adduct 8 in an 80:20
ratio as the only detectable products, as determined by GC/
1
MS and H NMR analyses. The formation of the thiol/al-
kyne adduct 8 indicated the presence of sulfanyl radicals
and suggests that these species are likely to be involved as
intermediates in the formation of disulfides 3 under basic
conditions (Scheme 2).
Figure 1. Influence of pH on the yield of S-nitrosothiol 2a in the
reaction of peroxynitrite with thiol 1a under acidic conditions
Scheme 2
ing to some species[2,4] capable of oxidizing 1a to disulfide
3a. If this were to be the case, the decrease in the yield of
2a on increasing the pH would be expected to be compens-
ated by an increase in the yield of disulfide 3a, which in-
stead was found to be independent of the yield of 2a. Thus,
two different species are seemingly responsible for the
formation of 2a and 3a.
The reaction with thiol 1a was repeated at various pH
values in the range 10.5Ϫ13. The desired pH was attained
by adding appropriate amounts of 12  hydrochloric acid
to the peroxynitrite solution (pH ϭ 13.5). Under these con-
ditions, peroxynitrite proved to be fairly stable, but immedi-
ately decomposed (within 30 s) when thiol 1a was added,
as could be monitored by spectrophotometric analysis.
However, the conversion of 1a dramatically decreased on
decreasing the pH, as shown in Figure 2. In all cases, the
disulfide 3a was found to be the exclusive reaction product
(Ͼ 90% yield, based on reacted thiol).
In the mid 1990s, Pryor et al.[1b] suggested that, at acidic
pH, the protonation of PN leads to two isomeric species,
HPN and HPN*. On this basis, we might hypothesize that
the HPN form could give the nitrosating species, XϪNO,
under very acidic conditions, or decompose,[2Ϫ4] whereas
the HPN* isomer could be responsible for the oxidation
of the thiol function, affording the disulfide 3 (Scheme 1).
However, at the present time, many doubts exist concerning
the identity and even the actual existence of this HPN* spe-
cies and, therefore, the question of the formation of the
disulfide 3 remains open.
In order to obtain evidence for the possible intermediacy
of sulfanyl radicals in the formation of disulfides 3, we car-
ried out the reactions of thiols 1b and 1c at pH ϭ 0 in the
presence of a twofold excess of phenylacetylene. Phenylacet-
ylene is known to be a good scavenger for benzenesulfanyl
radicals, which add to the CϪC triple bond to eventually
give a thiol/alkyne adduct through vinyl radical interme-
diates.[14] We found that in neither case was a thiol/alkyne
adduct formed, suggesting that no sulfanyl radicals are in-
volved as intermediates. At present, the formation of disulf-
ides 3 in the peroxynitrite-promoted reaction of thiols 1 un-
der acidic conditions remains unclear.
Figure 2. Influence of pH on the yield of disulfide 3a in the reaction
of peroxynitrite with thiol 1a under basic conditions
These results indicate that two competing reactions are
Peroxynitrite-promoted oxidation of the thiol function operative: a thiol-catalysed decomposition of PN and a PN-
under basic conditions was studied in the pH range promoted oxidation of thiol to disulfide. The latter prevails
10.5Ϫ13.5, in which the dissociated PN form largely pre- under strongly basic conditions (pH Ͼ 13), where the thiol-
dominates.
ate ion is clearly prevalent over the undissociated thiol (Fig-
Eur. J. Org. Chem. 2001, 131Ϫ135
133

L. Grossi, P. C. Montevecchi, S. Strazzari
FULL PAPER
mixture immediately became colored due to the formation of the
respective S-nitrosothiol 2a؊d.
ure 2). This indicates that the oxidation occurs on the thiol-
ate ion, rather than the thiol, leading first to the formation
of sulfanyl radicals and then to the disulfide. In the pH
range 10.5Ϫ13, in which both thiolate and thiol exist,[15]
thiolate oxidation competes with the thiol-catalysed decom-
Reaction with 1a: A bright-red coloration immediately developed[11]
and slowly intensified over a period of 20 min, as determined by
spectrophotometric measurements at 550 nm. After this time, the
position of PN, which possibly occurs through the forma- reaction mixture was extracted with diethyl ether, and the com-
bined organic phases were washed with water and dried with
Na2SO4. The solvent was removed under reduced pressure. 1H
NMR analysis of the residue showed the formation of S-nitroso-
thiol 2a[12] and disulfide 3a as the exclusive products in a 70:30
ratio. Subsequent column chromatography on silica gel gave, on
elution with petroleum ether (b.p. 40Ϫ70 °C), the S-nitrosothiol
2a (85 mg, 55%), identified by its UV/Vis spectrum[11] [1H NMR
(200 MHz): δ ϭ 4.65 (br. s, 2 H), 7.11Ϫ7.42 (m, 5 H)] and dibenzyl
disulfide 3a (35 mg, 30%). S-Nitrosothiol 2a decomposed within
2 h on standing in air to give a ca. 1:1 mixture of disulfide 3a
tion of a hydrogen-bonded PN/thiol complex (Scheme 2).
Conclusions
Our results indicate that at very acidic pH the undissoci-
ated HPN undergoes acid-catalyzed decomposition, prob-
ably through the intermediacy of the protonated form
H2PNϩ, leading to some species, XϪNO, capable of nitro-
sating the thiol function. The latter reaction affords S-nitro-
sothiols 2 in yields that are strongly dependent on the pH
(up to 70% at pH ϭ 0) and represents a convenient syn-
thetic route to these compounds, alternative to that re-
ported in the literature.[13] In competition with the forma-
tion of nitrosothiols 2, the formation of disulfides 3 occurs
in a manner independent of the pH, without the interme-
diacy of sulfanyl radicals.
1
and benzyl phenylmethanethiosulfinate 5,[18] as determined by H
NMR analysis. Ϫ In a repeated reaction, the reaction mixture was
split into two portions. One of these was allowed to stand at room
temperature. The bright-red coloration disappeared within 72 h.
The resulting mixture was extracted with diethyl ether, the com-
bined organic phases were washed with water, and the solvent was
evaporated. The residue was found to consist mainly of the disulf-
ide 3a, contaminated with trace amounts of benzyl phenylmeth-
anethiosulfonate 4a[19] and benzaldehyde 7, as determined by 1H
NMR and GC/MS analyses. The second portion of the reaction
mixture was refluxed for 20 min and then worked up as described
above. The residue was found to consist of the disulfide 3a, dibenzyl
sulfone (PhCH2SO2CH2Ph), and benzaldehyde 7 in a 6:1:2 ratio, as
Under basic conditions, the PN form either oxidizes the
thiolate ion to sulfanyl radicals or undergoes thiol-catalyzed
decomposition. The former is the exclusive reaction exhib-
ited by PN at pH Ͼ 13.
1
determined by H NMR and GC/MS analyses. Ϫ In independent
experiments, aliquots of a 0.50  aqueous solution of peroxynitrite
(2.0 mL) were added at 5Ϫ10 °C under stirring to portions of a
0.10  acetonitrile solution of thiol 1a (10.0 mL) containing vari-
able amounts of 12  hydrochloric acid. The apparent final pH
values were 0.0, 0.7, 0.8, 1.8, 4.3, and 6.0. The reaction mixtures
were stirred for 20 min, and then the absorption at λ ϭ 550 nm
was measured. S-Nitrosothiol 2a yields were determined on the
basis of a 70% yield at pH ϭ 0 (see Figure 1). Disulfide 3a yields
Experimental Section
General: NMR spectra were recorded with a Varian Gemini 200
instrument using Me4Si as an internal standard. Ϫ GC-MS ana-
lyses were performed with a Carlo Erba QMD 1000 instrument.
Ϫ UV/Vis spectra were recorded with a PerkinϪElmer Lambda
12 instrument.
1
were determined by H NMR analysis using an internal standard.
Materials: Thiols 1a؊d and phenylacetylene were commercial
products and were used as received. Peroxynitrite was synthesized
following a procedure described in the literature.[16] According to
this method, a solution of 30% H2O2 (10.0 mL) was diluted to a
volume of 50 mL with water and chilled to 4 °C. To this solution,
5.0  NaOH solution (18 mL) and a 0.04  solution of diethyl-
enetriaminepentaacetic acid (DTPA) in 0.05  NaOH (5.0 mL)
were added and the resulting mixture was diluted to a total volume
of 100 mL with water (the final pH was ca. 13.5). This solution
was then allowed to react with an equimolar amount of isoamyl
nitrite (12.3 mL) under vigorous stirring for 3Ϫ4 h at ca. 15 °C.
Thereafter, the aqueous phase was washed with chloroform (5 ϫ
75 mL) to remove the contaminating isoamyl alcohol and the un-
changed nitrite. The resulting yellow solution was treated with an
excess of MnO2 in order to destroy the unchanged H2O2, and then
filtered. The concentration of peroxynitrite (usually in the range
Reaction with 1b: A deep brown-red coloration immediately de-
veloped.[11] TLC analysis performed after 2 min showed the forma-
tion of the S-nitrosothiol 2b[13] and the disulfide 3b as the exclusive
products. After stirring for a further 15 min, the color faded. The
reaction mixture was then extracted with diethyl ether, the com-
bined organic phases were washed with water, and the solvent was
evaporated in vacuo. TLC analysis of the residue showed the pres-
ence of the disulfide 3b and the thiosulfonate 4b as the main reac-
tion products. Subsequent silica gel column chromatography gave,
on gradual elution with petroleum ether/diethyl ether, diphenyl dis-
ulfide 3b (50 mg, 45%) and phenyl benzenethiosulfonate 4b (20 mg,
15%). The reaction was then repeated, but with the addition of
phenylacetylene (200 mg, 2.0 mmol) to the acetonitrile solution of
1b. TLC and GC/MS analyses of the residue showed the presence
of 3b and 4b as the only detectable products.
0.45Ϫ0.50 ) was determined spectrophotometrically (ε302
ϭ
Reaction with 1c: A reddish-green coloration immediately de-
veloped.[11] After 20 min, the reaction mixture was extracted with
diethyl ether, and the combined organic phases were washed with
1670 Ϫ1cmϪ1).[17] Solutions kept at Ϫ18 °C showed little sign of
decomposition over several weeks.
Reaction of Thiols 1a؊d with Peroxynitrite under Acidic Conditions
؊ General Procedure: To a 0.10  acetonitrile solution of thiol duced pressure. H NMR analysis of the residue using an internal
1a؊d (10.0 mL), 12.0  hydrochloric acid (0.15 mL) was added at
5Ϫ10 °C. A 0.50  aqueous solution of peroxynitrite (2.0 mL) was
then added with stirring. The apparent final pH was 0. The reaction
water and dried with Na2SO4. The solvent was removed under re-
1
standard showed the formation of S-nitrosothiol 2c[13] and disulf-
ide 3c in yields of 40% and 30%, respectively. The reaction was
then repeated, but with the addition of phenylacetylene (200 mg,
134
Eur. J. Org. Chem. 2001, 131Ϫ135

S-Nitrosothiol and Disulfide Formation through Peroxynitrite-Promoted Oxidation of Thiols
FULL PAPER
[1c]
1
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S. Pfeiffer, B. Mayer, B. Hemmens, Angew.
2.0 mmol) to the acetonitrile solution of 1c. H NMR analysis of
the residue showed the presence of 2c and 3c as the only detectable
products in a 60:40 ratio.
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Reaction with 1d: A bright-red coloration immediately developed[11]
due to the formation of S-nitrosothiol 2d [UV/Vis: λmax ϭ 550,
518, and 416 nm]. The color faded within 30Ϫ45 min. The reaction
mixture was then extracted with diethyl ether. The combined or-
ganic phases were washed with water, dried with Na2SO4, and the
solvent was removed under reduced pressure. 1H NMR analysis
using an internal standard showed the formation of the disulfide
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[6]
The term ‘‘peroxynitrite’’ has been used throughout this paper
to indicate both the unprotonated and the protonated forms.
PN and HPN have been used to specifically indicate the
ONOOϪ and ONOOH forms, respectively.
Reaction of Thiols 1a,b with Peroxynitrite under Basic Conditions:
To portions of a 0.10  acetonitrile solution of thiol 1a,b (10.0 mL),
aliquots of a 0.50  aqueous solution of peroxynitrite (2.0 mL)
(pH ϭ 13.5) were added with stirring at 5Ϫ10 °C. Spectrophoto-
metric analysis showed complete consumption of the peroxynitrite
within 5 min. After this time, the reaction mixtures were extracted
with diethyl ether and the combined organic phases were washed
with water. GC/MS analysis using an internal standard showed that
the starting thiols 1a,b had been consumed and that the corres-
ponding disulfides 3a,b had been formed in 90% yield. Ϫ The reac-
tion with 1b was then repeated in the presence of phenylacetylene
(200 mg, 2.0 mmol). 1H NMR and GC/MS analyses of the reaction
mixture showed the formation of the disulfide 3b and β-(phenyl-
thio)styrene (8)[14] in an 80:20 ratio. Ϫ The reaction with thiol 1a
was repeated at various pH values by using peroxynitrite solutions
at pH ϭ 13.0, 12.6, 12.0, 11.0, and 10.5; these were obtained from
the 0.5  solution at pH ϭ 13.5 by the addition of appropriate
amounts of 12.0  hydrochloric acid. In all cases, spectrophoto-
metric analysis showed the consumption of peroxynitrite within
30 s. The reaction mixtures were worked up as described above and
analyzed by GC/MS using an internal standard to determine the
conversion of thiol 1a (see Figure 2) and the yield of disulfide 3a
(90% in all cases).
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R. Radi, J. S. Beckman, K. M. Bush, B. A. Freeman, J.
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A more convenient and reliable identification of S-nitrosothiols
is provided by their characteristic visible spectra. The bright-
red S-nitrosothiol 2a shows λmax at 550 nm (ref.[13]); the red-
dish-green S-nitrosothiol 2c shows λmax at 552, 562, 598, and
605 nm (ref.[14]); the brown-red S-nitrosothiol 2b shows λmax at
530 and 570 nm (ref.[14]); the bright-red S-nitrosothiol 2d shows
λmax at 550 and 518 nm.
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Acknowledgments
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[18]
`
This work was supported by the Ministero dell’Universita e della
Ricerca Scientifica e Tecnologica (MURST) (funds 60% and 40%)
and by the University of Bologna (funds for selected research topics
A. A. 1997Ϫ99).
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Received July 18, 2000
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135