Evaluation of transnitrosating ability of N-nitrosoguanidines to alkyl thiols and thiol amino acids

Article abstract of DOI:10.1016/j.tet.2016.01.008

The transfer of the nitroso group from 1-nitroso-1-methyl-3-tolylsulfonylguanidine (NOTSG) and 1-nitroso-1-methyl-3-benzoylguanidine (NOBMG) to some thiols, including the amino acid cysteine, was studied in a pH range between 7 and 12. The measured apparent bimolecular rate constant of transnitrosation (k_tr^app) revealed a bell-shaped pH dependence that clearly indicates that both nitrosoguanidines react through the corresponding neutral form, and the nucleophiles in the thiolate anion form to give the corresponding S-nitrosothiol. Regarding cysteine, the existence of three macroscopic acidity constants influenced the kinetic behavior of the transnitrosation reaction. Transnitrosation rates (k_tr) of the two possible nucleophilic species were obtained and it was found that NOBMG has lower thiol transnitrosation capacity due to the lower electron-withdrawing effect of benzoyl group and to the possible stabilization of the anionic structure as a consequence of the establishment of the intramolecular hydrogen bond. The k_tr values of the studied nucleophiles were calculated and a Br?nsted-type plot was established giving unexpected negatives β_nuc(β_nuc(NOBMG)=-0,17 and β_nuc(NOTSG)=-0,11). The atypical β_nuc values were attributed to the need for previous desolvation of the nucleophile.

Full text of DOI:10.1016/j.tet.2016.01.008

Tetrahedron 72 (2016) 1177e1184
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Evaluation of transnitrosating ability of N-nitrosoguanidines to alkyl
thiols and thiol amino acids
,
b
a
Lara Ribeiro a , Luis García-Río , M. Eduarda Araujo
*
ꢀ
a
^
Centro de Química e Bioquímica and Departamento de Química e Bioquímica, Faculdade de Ciencias, Universidade de Lisboa, 1749-016 Lisboa,
Portugal
b
ꢀ
ꢀ
Departamento de Química Física, Centro de Investigacion en Química Biologica y Materiales Moleculares (CIQUS), Universidad de Santiago de
~
Compostela, 15782 Santiago de Compostela, Espana
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 23 July 2015
Received in revised form 15 December 2015
Accepted 5 January 2016
Available online 9 January 2016
The transfer of the nitroso group from 1-nitroso-1-methyl-3-tolylsulfonylguanidine (NOTSG) and 1-
nitroso-1-methyl-3-benzoylguanidine (NOBMG) to some thiols, including the amino acid cysteine, was
studied in a pH range between 7 and 12.
The measured apparent bimolecular rate constant of transnitrosation (katrpp) revealed a bell-shaped pH
dependence that clearly indicates that both nitrosoguanidines react through the corresponding neutral
form, and the nucleophiles in the thiolate anion form to give the corresponding S-nitrosothiol. Regarding
cysteine, the existence of three macroscopic acidity constants influenced the kinetic behavior of the
transnitrosation reaction. Transnitrosation rates (ktr) of the two possible nucleophilic species were ob-
tained and it was found that NOBMG has lower thiol transnitrosation capacity due to the lower electron-
withdrawing effect of benzoyl group and to the possible stabilization of the anionic structure as a con-
sequence of the establishment of the intramolecular hydrogen bond. The ktr values of the studied nu-
cleophiles were calculated and a Brønsted-type plot was established giving unexpected negatives
Keywords:
Transnitrosation
S-nitrosothiols
N-nitrosocompounds
NO donors
bnuc
(
bnuc(NOBMG)¼ꢀ0,17 and bnuc(NOTSG)¼ꢀ0,11). The atypical bnuc values were attributed to the need
for previous desolvation of the nucleophile.
Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction
However, there is little information about S-nitrosation using
transnitrosating ability of nitrosocompounds in the presence of
sulfur nucleophiles such as cysteine (Cys). Only two mechanistic
studies about MNTS reactivity in the presence of cysteine, at several
pH values, were found in the literature that showed the S-nitro-
socysteine obtention by a transnitrosation process.9,14
The discovery of physiological properties of nitric oxide (NO),
particularly those of vasodilation and inhibition of platelet
aggregation,1e3 is actually responsible for the major interest in the
chemistry of nitrosothiols (RSNOs).
Several RSNOs like S-nitrosocysteine, S-nitrosoglutathione, S-
nitrosoalbumins and S-nitrosohemoglobin, have been detected
in vivo4 and are believed to be responsible for the NO transport and
release around the body.5e7
RSNOs are very readily generated in solution by nitrosation of
the corresponding thiols using conventional nitrosation methods8
or transferring the nitroso group from a nitrosocompound to thi-
olate ions (transnitrosation).9e11
From all RSNOs with biological relevance, S-nitrosocysteine is
probably one of the most important and therefore this S-nitro-
sothiol has been the aim of several mechanistic studies concerning
its decomposition with simultaneous NO generation.4,8,11e13
It is known that N-nitrosocompounds, like MNTS (Fig. 1), have
a high cytotoxic activity due the possible formation of electrophilic
species by a deamination process15 and the possible transfer of the
nitroso group to amines increases the risk of carcinogenicity of
these compounds,16 however, in the presence of sulfur nucleophiles
O
N
O
NH2
NNO2
S
N
O
N
CH3
O
N
H3C
CH3
MNNG
MNTS
Fig. 1. N-methyl-N0-nitro-N-nitrosoguanidine (MNNG) and N-methyl-N-nitroso-p-tol-
* Corresponding author. E-mail address: laramarques@sapo.pt (L. Ribeiro).
uenosulfonamide (MNTS) structures.
http://dx.doi.org/10.1016/j.tet.2016.01.008
0040-4020/Ó 2016 Elsevier Ltd. All rights reserved.

1178
L. Ribeiro et al. / Tetrahedron 72 (2016) 1177e1184
usually more reactive than amines, these compounds can assume
a new and interesting role as NO donors.
In the reactions performed at pHꢁ11 it was also detected the
presence of benzoic acid, or p-toluenesulfonic acid if the nitrosating
agent was NOBMG or NOTSG.
The presence of free guanidines BMG and TSG is consistent with
the possible direct transfer of nitroso group to the thioles, and the
acid benzoic (or p-toluenesulfonic acid) anions formation at pHꢁ11
is in agreement with the competition from basic hydrolysis of
nitrosoguanidines.24
Due to this duality, our group has been interested in the
chemistry and reactivity of some N-nitrosocompounds, particularly
nitrososulfonamides, nitrosothioureas and nitrosoguanidines. After
confirming the ability of these compounds to transnitrosate to
amines15,17e19 and based on some studies realized with MNTS9,14
and MNNG20 (Fig. 1) involving nitric oxide and S-nitrosocysteine
formation, it was our goal to study the nitroso group transfer from
two N-nitrosoguanidines containing electro-withdraw groups
(NOTSG and NOBMG) to thiolate ions including cysteine.
2.2. Transnitrosation to simple thioles (RSH type)
To accomplish our purpose, the transnitrosation ability of
NOBMG and NOTSG to sulfur nucleophiles was kinetically evalu-
ated, first with thioles containing only the thiol function with
ionization capacity, and then with cysteine an amino acid that in
The influence of ME concentration on the observed rate con-
stants, kobs, of its reaction with the N-nitrosoguanidines was stud-
ied at different pH values. Figs. 3 and 4 illustrate the good linear
addition to the thiol function has two other ionizable functions:
carboxilate and -amino.
The thioles used (Table 1) were chosen based on their basicity.
The complete kinetic study was accomplished using the nucleo-
phile 2-mercaptoethanol (ME).
a-
6
a
pH=8.66
5
pH=9.40
4
3
2
1
0
pH=9.73
pH=10.13
Table 1
pKa values of studied sulfur nucleophiles (RSH)
RSH
Trivial name
pKRaSH
CH3O2CCH2SH
CH3O2CCH2CH2SH
OHCH2CH2SH
CH3CH2SH
Methylhtioglycolate (MTG)
Methyl 3-mercaptopropionate (MMP)
2-mercaptoethanol (ME)
Ethanothiol (EtSH)
7,8121
9,3322
9,6022,23
10,621
0
0,1
0,2
0,3
T
[ME] / M
2. Results and discussion
Fig. 3. Influence of 2-mercaptoethanol total concentration on kobs in transnitrosation
reaction with NOBMG at 25 ꢂC.
2.1. Preliminary analysis of the transnitrosation products
The transnitrosating ability of N-nitrosoguanidines (Fig. 2) to
some thioles at different pH values was previously studied under
similar conditions of those described for the kinetics studies de-
veloped in this work.
14
pH = 8.78
12
pH = 9.41
10
pH = 9.67
O
S
NH
O
NH
8
CH3
pH = 10.07
CH3
N
H
N
H
N
H
N
H
O
6
4
2
0
H3C
BMG
TSG
O
NH
O
NH
CH3
CH3
S
N
H
N
N
H
N
0
0,1
0,2
0,3
0,4
0,5
0,6
O
N=O
N=O
T
[ME] / M
H3C
NOBMG
NOTSG
Fig. 4. Influence of 2-mercaptoethanol total concentration on kobs in transnitrosation
reaction with NOTSG at 25 ꢂC.
Fig. 2. Free guanidines structures: 1-methyl-3-benzoylguanidine (BMG) and 1-
methyl-3-tolylsulfonylguanidine (TSG) and their correspondent N-nitrosated de-
rivatives: 1-nitroso-1-methyl-3-benzoylguanidine (NOBMG) and 1-nitroso-1-methyl-
3-tolylsulfonylguanidine NOTSG).
relationship with a zero intercept between kobs and the total thiol
concentration, [ME]T, indicating the absence of NOBMG (or NOTSG)
basic hydrolysis competition.9
It is also evident, for both N-nitrosoguanidines, the slope in-
creases with the increasing pH, which is consistent with the Eq. 1,
app
The obtained reaction mixtures were treated as described in the
experimental section and analyzed by TLC that showed the deni-
trosated guanidines BMG and TSG (Fig. 2), respectively, as the
principal final products of these reactions.
where k
is an apparent bimolecular rate constant since it in-
tr
cludes the different acid-base equilibrium constants of the nucle-
ophile and, [RSH]T represents in this case the total concentration of
ME.

L. Ribeiro et al. / Tetrahedron 72 (2016) 1177e1184
1179
the corresponding pKa values:24 pKa(NOTSG)¼10.6ꢄ0.1 and
pKa(NOBMG)¼11.5ꢄ0.1.
app
kobs ¼ k ½RSHꢃT
(1)
tr
For NOTSG, when pH is about 10, the anionic form is pre-
dominant in solution, however for NOBMG, at the same pH values,
concentration of the neutral and the anionic forms are similar.
When pH increases until z12.5 the equilibrium shifts towards the
anionic form formation, which becomes predominant after that
Only when pH was higher than 10, interceptions between kobs
and [ME]T were different than zero, indicating that basic hydrolysis
of N-nitrosoguanidines takes place competing with the trans-
nitrosation process, kobs should be expressed as:
h
i
point. Thus, it seems that the appearance in solution of the anionic
app
tr
kobs ¼ kOHꢀ OHꢀ þ k ½RSHꢃT
(2)
app
tr
forms of the N-nitrosoguanidines explains the decrease of k
.
Experimental results can be quantitatively explained by a re-
action mechanism involving nitroso group transfer from the neutral
forms of N-nitrosoguanidines to the thiolate ion of ME (Scheme 2).
where kꢀOH is the bimolecular rate constant for the N-nitro-
soguanidines hydrolysis catalyzed by the hydroxide ion existent in
solution. However, basic hydrolysis studies of these N-nitro-
soguanidines24 showed, for similar pH ranges, kOHꢀ values between
From Scheme 2, the following expressions (Eqs. 3 and 4) for the
app
tr
apparent bimolecular rate constant k
were obtained:
10ꢀ5and10ꢀ4
compared with the obtained k
order to express kobs we considered only Eq. 1.
The influence of pH on k
shape curve with a progressive increase of k
M
ꢀ1sꢀ1, which are not considered significant when
app
rate constants. For this reason, in
tr
kobs
app
tr
k
¼
¼
½MEꢃT
app
rate constants (Fig. 5) reveal a bell-
0
1
A
tr
ꢀ
ꢁ
app
tr
kꢅ Hþ ꢅKaRSH
values until pH is
@
ꢂ
ꢃ
ꢂ
ꢃ
(3)
(4)
ꢀ
ꢁ
ꢀ
ꢁ
2
KaXNO ꢅKaRSH þ KaXNO þKaRSH ꢅ Hþ þ Hþ
22
20
18
16
14
12
10
8
ꢀ
ꢁ
h
i
h
i
Hþ
KaXNO KaXNO þ KaRSH
1
2
¼
þ
ꢅ Hþ
þ
ꢅ Hþ
app
tr
k ꢅ KaRSH
k ꢅ KaRSH
k
k
where the equilibrium constants (KaRSH and KaXNO) refer to the thiol
and N-nitrosoguanidines acid-base dissociations previously de-
scribed (Table 1 and Scheme 1) and k is the rate constant corre-
sponding to the transnitrosation reaction shown in Scheme 2.
The experimental results (Fig. 5) fitted very well to Eq. 3 as
shown by the curve plotted and the second-degree polynomial
equation expressed by Eq. 4 is also in good agreement with the
observed kinetic behavior as we can see in Fig. 6.
6
4
2
0
10-12 10-11 10-10
10-9
10-8
10-7
10-6
Transnitrosation velocity constants (k) and macroscopic equi-
librium acidity constants (pKa(NOTSG); pka(NOBMG) and pKa(RSH))
were obtained from the fit of the kinetic results to Eqs. 3 and 4
(Table 2).
All estimated pKa values for both N-Nitrosoguanidines com-
pared well with those reported in the literature24 with the excep-
tion of the NOBMG pKa value obtained by fitting the results to Eq. 3.
In fact, considering the value near 11.5, the lack of experimental
results close the ionization pH range may be the reason for this
discrepant value. The fitted values show less acidity behavior for
NOBMG, which is consistent with fact that amides are indeed more
basic than sulfonamides due to the smaller electron-withdrawing
effect of the benzoyl group.
[H+]/M
app
Fig. 5. Influence of pH on k
rate constants for the transfer of the nitroso group from
tr
N-Nitrosoguanidines to 2-mercaptoethanol at 25 ꢂC.
near 10. After this point, apparent bimolecular rate constants de-
crease with the continuous increase of alkalinity.
Considering the acid-base equilibrium of ME (see pKaRSH on Table
1) the concentration of thiolate in solution is increasing with the
increasing pH, reaching a maximum value when pHz10.6. This fact
app
tr
justifies the k
increase for both reactions until pH is near 10
however, if the thiolate concentration was the unique determining
rate factor, katrpp values should stabilize when pH>10 but instead we
observed decreasing values leading us to consider the following
acid-base equilibrium (Scheme 1) for both N-nitrosoguanidines and
For ME pKa values are in agreement with the literature value
(Table 1) except the estimated value of 8.3 obtained from the ki-
netic results fitted to Eq. 4 that describes a second-degree poly-
nomial behavior (y¼aþbxþcx2). The fitting process with this
NH
NH
CH3
X
XNO
CH3
pKa
X
H+
N
N
_
N
N
+
N
O
N
O
Y
Y
_
XNO
HXNO
NOBMG: X=CO; Y=H
NOTSG: X=SO2;Y=CH3
Scheme 1. Acid-base equilibrium of N-nitrosoguanidines.

1180
L. Ribeiro et al. / Tetrahedron 72 (2016) 1177e1184
NH
O
S
O
N
CH3
N
NH
O
O
N
H
H
H
CH3
S
H3C
N
TSG
N
O
k
H3C
S
N O
HO
NOTSG
+
S
HO
+
NH
O
NH
O
k
CH3
CH3
N
N
H
N
H
N
H
N
O
BMG
NOBMG
Scheme 2. Reaction mechanism involving nitroso group transfer from the neutral forms of N-nitrosoguanidines to the thiolate ion of cysteine.
Fig. 6. Influence of pH on the quotient ½Hþꢃ=ktarpp
.
Table 2
The lower reactivity of NOBMG can also be explained by the
possible stabilization of the anion resonant structure 2 (Scheme 3),
due to the establishment of the intramolecular hydrogen bond24
(Fig. 7, structure I).
Estimated transnitrosation velocity constants (k) and equilibrium constants for the
studied thiol and N-nitrosoguanidines
XNO
Fit to Eq. 3
Fit to Eq. 4
pKa
K/Mꢀ1sꢀ1
pKaRSH
pKa
K/Mꢀ1sꢀ1
pKaRSH
NOTSG
NOBMG
10.73
10.81
2.88ꢅ10ꢀ1
9.14
9.39
10.73
11.29
2.33ꢅ10ꢀ1
8.30
9.16
H
2.48ꢅ10ꢀ1
1.84ꢅ10ꢀ1
-O
N
O
S
R2
R3
CH3
R1
N
+
N
N
equation was only possible considering constant the parameter
a previously obtained by a linear fit of the results with a linear
behavior. This assumption and the mathematic calculations needed
to get the final values may be the cause of the observed deviation.
Analysing the transnitrosation rate constants we can see that
both fitting results gave values in good agreement and comparing
transnitrosating velocities of NOTSG with NOBMG we can see that
O
N
O
I
II
Fig. 7. NOBMG anion stabilization by the establishment of the intramolecular hydro-
gen bond (I); possible resonant structure for sulfonamides (II).
For NOTSG, the same stabilization is highly improbable because
various studies carried out with sulfonamides indicate that struc-
ture II (Fig. 7) has no significant contribution in its stabilization.
there is a very little difference between obtained values: kNOTSG
NOBMGz1.2.
Although there is a slight difference of reactivity between
/
k
NOTSG and NOBMG, the first one shows more reactivity in the
transnitrosation process. This result is compatible with the greater
electron-withdrawing capacity given by the sulfonyl group that
generates a positive charge towards the guanidine group leading to
a fast nucleophilic attack of the thiolate ion.
2.3. Transnitrosation to cysteine
Since an identical transnitrosation kinetic profile was found for
both N-nitrosoguanidines with only a slight difference in terms of

L. Ribeiro et al. / Tetrahedron 72 (2016) 1177e1184
1181
-
O
N
H
O
N
H
O
N H
CH3
CH3
CH3
N
N
N
N
N
N
-
N
O
N O
N
O
2
1
3
Scheme 3. Stabilization of the anion resonant structure of NOBMG.
reactivity, the kinetic study of transnitrosation reactions to the bi-
ological aminoacid cysteine (Cys) was performed using only NOTSG
as NO donor.
As previously mentioned for NOTSG, due to the existence of an
acidic proton when pH is between z9.6 and 11.6, two chemical
species coexist in solution (see Scheme 1) but when pH>11.6 the
anionic one becomes predominant.
As observed with ME, the influence of total Cys concentration,
[Cys]T, on the observed rate constants, kobs, in its reaction with the
NOTSG at different pH values (8.48<pH<11.11) show good linear
relationships and visible slope increase with the increasing pH
(Fig. 8). Only when pH is higher than 10.5, interceptions between
kobs and [Cys]T are different than zero indicating the basic hydro-
lysis of N-nitrosoguanidines competition but, as in the previous
With respect to cysteine, according to Scheme 4 and in the pH
range studied, the presence of three possible chemical species in
solution can be considered: (H2Cys), (HCys)ꢀ and (Cys2ꢀ).
app
tr
Analyzing experimental results, k
increases with the in-
creasing pH reaching a maximum value when pH is near 10.4. In
what concerns cysteine, this basicity increase in solution means the
gradual appearance of the anionic form (HCys)ꢀ that becomes
predominant when pHz9.3. If (HCys)ꢀ was the unique nucleophilic
form of cysteine, and both neutral and anionic forms of NOTSG
were equally reactive, from this point rate constants would be ex-
pected to become pH independent but instead we observe de-
creasing values with the increasing appearance of (Cys)2ꢀ and
NOTSG anionic form.
study, kꢀOH constants for similar pH values can be disregarded when
app
tr
compared with the obtained k
rate constants for the trans-
nitrosation process leading us to consider Eq. 1 (in this case [RSH]T
refers to [Cys]T) in all studied pH range.
Fig. 9 a) illustrates the obtained pH-rate profile and as it is ev-
ident the pH influence on the apparent bimolecular rate constants
(katrpp) also follows a bell-shape behavior but now with a less pro-
app
tr
nounced increase of k
with the increasing basicity. The wider
The proposed mechanism (Scheme 5) involving nitroso transfer
from neutral form of N-nitrosoguanidine to the anionic species of
bell-shape form probably may indicate the presence of more
chemical species in the transnitrosation reaction.
cysteine (HCys)ꢀ and (Cys)2ꢀ lead to the following expression for
app
tr
k
(Eq. 5):
h
þ
i
k1 ꢅ Ka2 ꢅ Hþ þ ꢅk2 ꢅ Ka3 ꢅ Ka2
app
tr
k
¼
h
i
h
i
h
i
ꢂ
ꢃ
Ka3 ꢅ Ka2 ꢅ KaXNO
3
2
a ꢅ Hþ þ b ꢅ Hþ þ c ꢅ Hþ
Ka3 ꢅ Ka2 þ Ka2 ꢅ KaXNO
þ
ꢀ
ꢁ
Hþ
(5)
a ¼ Ka1
b ¼ Ka1 ꢅ KaXNO þ 1
c ¼ Ka2 þ KaXNO
pH = 8.48
pH = 8.79
pH = 9.18
pH = 9.71
pH = 10.36
Experimental results fit very well to the deduced Eq. 5 as is
3
2
1
0
made clear by the plotted curve in Fig. 9b but in this case, due to the
complexity of the equation, macroscopic acidity constants of cys-
teine were kept constant for the fitting process: pKa2¼8.3 and
pKa3¼10.2.
The kinetic data analysis yields a value for the acidity constant of
NOTSG: pKa¼10.51, which compares well with the described value
in the literature.24 Transnitrosation velocity constants obtained,
k1¼9.07ꢅ10ꢀ2 Mꢀ1 sꢀ1 and k2¼2.36ꢅ10ꢀ1 Mꢀ1 sꢀ1, showed higher
reactivity for the anion (Cys)2ꢀ. Compatible results were obtained
(k1¼9.87ꢅ10ꢀ2 Mꢀ1
s
ꢀ1; k2¼1.71ꢅ10ꢀ1 Mꢀ1
s
ꢀ1) when the same
data was fitted to a rearranged form of Eq. 5 (Eq. 6) showing clear
app
tr
linear dependence between the product (k ꢅEq. 5 denominator)
and [Hþ] (Fig. 10).
h
i
0
0,05
0,1
0,15
0,2
0,25
0,3
app
tr
k
ꢅðequation5denominatorÞ ¼ k1 ꢅKa2 ꢅ Hþ þk2 ꢅKa3 ꢅKa2
T
[Cys] / M
|fflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflffl{zfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflffl} |fflfflfflffl{zfflfflfflffl} |fflffl{zfflffl} |fflfflfflfflfflfflfflfflfflffl{zfflfflfflfflfflfflfflfflfflffl}
m
x
Y
b
Fig. 8. Influence of cysteine total concentration on kobs in transnitrosation reaction
(6)
with NOTSG at 25 ꢂC.

1182
L. Ribeiro et al. / Tetrahedron 72 (2016) 1177e1184
12
10
8
b)
a)
6
4
10-9
2 x 10-9 3 x 10-9 4 x 10-9
[H+] / M
8,5
9
9,5
10 10,5 11
pH
app
tr
Fig. 9. a) Influence of pH on k
rate constants for the transfer of the nitroso group from NOTSG to cysteine at 25 ꢂC. b) Fitted results to Eq. 5.
-
-
-
COO
COO
COO
COOH
pKa3=10,2
pKa2=8,3
-
pKa1=1,8
-
+
+
S
+
SH
S
SH
H2N
H3N
H3N
H3N
-
2 -
+
(H2Cys)
(H3Cys)
(Cys)
(HCys)
Scheme 4. Acidebase equilibrium of cysteine: possible cysteine ions in solution.
-
COO
-
+
S
k1
-
H3N
COO
+
-
S
N=O
NH
(HCys)
H3N
O O
NH
CH3
S
O
O
N
H
N
CH3
S
+
N
N
H
+
H3C
N
O
H
-
H3C
TSG
COO
k2
NOTSG
-
S
N=O
COO
H2N
-
S
H2N
2 -
(Cys)
Scheme 5. Proposed mechanism for nitroso transfer from neutral form of N-nitrosoguanidine to the anionic species of cysteine (HCysꢀ and (Cys)2ꢀ
.
The higher reactivity of (Cys)2ꢀ wasnot a surprising result since
Therefore, since NOBMG is structurally identical to NOTSG we
it is expected a more basic character of (Cys)2ꢀ then (HCys)ꢀ.
Similar studies of cysteine transnitrosation using MNTS as NO do-
nor accomplished by C. Adam et al.9 also showed the same pattern
of reactivity between (Cys)2ꢀ and (HCys)ꢀ however, in this last case,
obtained transnitrosation velocity constants are 100 times higher.
This huge difference compared to our study can only be explained
by the highest electron-withdrawing effect towards the nitroso
group in the MNTS (see Fig. 1) since it is directly bounded to the
sulfonyl group. In the case of our NO donor, NOTSG we have the
guanidine function between the sulfonyl and the nitroso group that
attenuates the electron-withdrawing effect giving lower trans-
nitrosation velocity constants.
predict a similar mechanistic behavior but less reactive since the
electron-withdrawing effect of benzoyl group is lower
when compared with the same effect of sulfonyl group.24
2.4. Structure-reactivity correlations
The influence of the nucleophilic reactivity of studied thiolate
ions with their basicity was evaluated (Fig. 11) and as we can see,
for both N-nitrosoguanidines obtained Brønsted exponents are
negative (bnuc (NOBMG)¼ꢀ0.17 and bnuc (NOTSG)¼ꢀ0.11) sug-
gesting a reactivity decrease with the increasing basicity strength,

L. Ribeiro et al. / Tetrahedron 72 (2016) 1177e1184
1183
Fig. 10. Fitted results to Eq. 6.
Kd
which is an apparent contradiction when we are considering
a typical nucleophilic attack mechanism.
-
-
.
RS
solvent
RS solvent
+
In fact, for SN reactions, expected Brønsted exponents are pos-
itive, although, some described cases in the literature also show
negative or near zero bnuc values as a consequence of the des-
olvation effect on the reaction rate or on the reactivity-structure
correlations.
The same behavior has been observed for some phosphoryl
transfer reactions to amines25 and for reactions of highly reactive
carbocations with amines26 and also for reactions of thiolate ions
with Fisher carbene complexes.27
Likewise, Brønsted exponents near zero were found for reactions
of diphenylketene with amines28 and for transnitrosation reactions
from MNTS9 and N-nitrososulfonamides18 to thiolate ions.
According to Jencks,25 these anomalous bnuc values are a result
of the requirement of nucleophile desolvation prior to the reaction,
a pre-equilibrium occurring in a separated step as it is shown in
Scheme 6:
Assuming this partial desolvation, the experimental value of the
rate constant for the nucleophilic attack (transnitrosation) corre-
sponds to the product Kdk0, where Kd is the equilibrium constant for
the partial desolvation of the thiolate ion and observed bnuc will be
given by Eq. 7:
k '
-
RS
N-nitrosoguanidine
+
Products
k -1
Scheme 6. Partial desolvation pre-equilibrium of the thiolate ion and N-nitro-
soguanidine reaction with free thiolate ion.
dðlog kÞ
dðlog Kdk0Þ
dðlog KdÞ
dðlog k0Þ
ꢂ
ꢃ
ꢂ
ꢃ
ꢂ
ꢃ
ꢂ
ꢃ
bnuc
¼
¼
¼
¼
þ
d pKaRSH
d pKaRSH
d pKaRSH
d pKaRSH
bd
þ
b0nuc
(7)
Considering a more difficult desolvation with the higher basicity
of thiolate ions (RSꢀ),
b
d<0 it is expected. Thus if b0nuc is low, in-
dicating a transition state with little bond formation, observed bnuc
values may be dominated by bd and be close to zero or even neg-
ative like those obtained in study.
3. Conclusion
Although N-nitrosocompounds may be potential carcinogens
when they decompose by a deamination reaction due to their
N-alkylating ability, it was found that N-nitrosoguanidines
studied, N-nitroso-1-methyl-3-benzoylguanidine (NOBMG) and
N-nitroso-1-methyl-3-tolylsulfonylguanidine (NOTSG), react
faster with thiol type sulfur nucleophiles in neutral and basic
media, transferring directly the nitroso group to the corre-
sponding thiolate ion forming S-nitrosotiol. Thus, it can be
considered that these N-nitrosoguanidines are potential nitric
oxide donors.
0
NOBMG
NOTSG
-0,2
-0,4
-0,6
-0,8
4. Experimental
4.1. General
7
8
9
10
11
All reactants used in the synthesis were of the highest purity
commercially available and used without further purification. All
reagents used for the kinetics studies were analytical grade and
deionized water was used throughout this study.
RSH
a
pK
Fig. 11. Influence of the nucleophilic reactivity of studied thiolate ions with their ba-
sicity for transnitrosation reactions with NOBMG and NOTSG.

1184
L. Ribeiro et al. / Tetrahedron 72 (2016) 1177e1184
2. Ignarro, L. J. J. Physiol. Pharmacol. 2002, 53 (4), 503e514.
1-methyl-3-benzoylguanidine (BMG) was prepared as de-
3. Williams, D. L. H. Org. Biomol. Chem. 2003, 1, 441e449.
4. Nitrosation Reactions and the Chemistry of Nitric Oxide; Williams, D. L. H., Ed.;
Elsevier: B. V.: Amsterdam, 2004; pp 137e160.
5. Hogg, N. Free Radic. Biol. Med. 2000, 28 (10), 1478e1486.
6. Hogg, N. Annu. Rev. Pharmacol. Toxicol. 2002, 42, 585e600.
7. Richardson, G.; Benjamin, N. Clin. Sci. 2002, 102, 99e105.
8. Adam, C.; García-Río, L.; Leis, J. R.; Ribeiro, L. J. Org. Chem. 2005, 70 (16),
6353e6361.
9. Adam, C.; García-Río, L.; Leis, J. R. Org. Biomol. Chem. 2004, 2, 1181e1185.
10. Noble, D. R.; Williams, D. L. H. J. Chem. Soc., Perkin Trans. 2 2001, 13e17.
11. Peterson, L. A.; Wagener, T.; Sies, H.; Stahl, W. Chem. Res. Toxicol. 2007, 20,
721e723.
12. Dicks, A. P.; Herves Beloso, P.; Williams, D. L. H. J. Chem. Soc., Perkin Trans. 2
1997, 1429e1434.
scribed in the literature for 1-methyl-3-tolylsulfonylguanidine
(TSG)29 and nitrosation was accomplished mixing the free guani-
dines with acidic aqueous solution of sodium nitrite as described
for the nitrosation of clonidine.30 The nitrosoguanidines obtained
showed similar spectroscopic data by 1H NMR analysis as described
previously in the literature.17
Transnitrosation reactions at 25 ꢂC were monitored following
the formation of the correspondent S-nitrosothiol at 330 nm using
a UVevis spectrophotometer and a stopped-flow for faster re-
actions. All kinetic experiments were triggered using a small ali-
quot of acetonitrile solution of N-nitrosoguanidines. The final
percentage of acetonitrile in the reaction mixtures was always 3,
33% (v/v) and the final N-nitrosoguanidines concentration was
(4e5)ꢅ10ꢀ5 M.
13. Gu, J.; Lewis, R. S. Ann. Biomed. Eng. 2007, 9 (35), 1554e1560.
14. Oh, S.; Williams, D. L. H. J. Chem. Soc., Perkin Trans. 2 1989, 755e758.
~
ꢀ
15. Faustino, C. M. C. Benzoílnitrosoureias-Reacc¸ oes de Hidrolise e de Trans-Nitro-
~
ꢀ
sac¸ ao Tese de Mestrado; Faculdade de Farmacia da Universidade de Lisboa,
2002; p 71.
In most reactions pH was controlled by using the nucleophile
itself as buffering agent. When pH was between 6 and 8, reactions
were carried out using H2PO4Na/HPO4Na2 buffer and for pH values
higher than 10, 5 aqueous NaOH solution was used to fix pH.
At the end of all the studied reactions the presence of nitrite ion
was qualitatively determined using a modification of Shinn’s
method.31
16. Loeppky, R. N.; Michejda, C. J. In Nitrosoamines and Related N-Nitroso Com-
pounds, Chemistry and Biochemistry ACS Symposium Series; 1994; 553, pp 1e15
Washington, DC.
ꢀ
ꢀ
17. Moreira, J. A. S. Estudio Cinetico de Reacciones de Transferencia Protonica, del
ꢀ
Grupo Nitroso y de Solvolisis de Cloruros de Benzoílo en Diversos Medios Tese de
Doutoramento; Faculdade de Química da Universidade de Santiago de Com-
postela, 2000; p 104.
18. Adam, C.; García-Río, L.; Leis, J. R.; Moreira, J. A. Tetrahedron 2006, 62,
8822e8829.
19. L. García Río, L. Ribeiro unpublished results.
20. Santala, T.; Fishbein, J. C. J. Am. Chem. Soc. 1992, 114, 8852e8857.
21. pKa Data Compiled by R. Williams: research.chem.psu.edu/brpgroup/pKa_
compilation.pdf.
22. Llinas, A.; Donoso, J.; Vilanova, B.; Frau, J.; Munoz, F.; Page, M. I. J. Chem. Soc.,
Perkin Trans. 2 2000, 1521e1525.
In order to carry out the reactions under pseudo-first order
conditions, all the experiments were performed with a large excess
of nucleophile relative to the N-nitrosoguanidines following every
run to at least 90% of the reaction. In the experiments followed by
stopped-flow each run was repeated at least five times and the
average value of the pseudo-first order rate constant, kobs, was
obtained. In all cases, obtained kobs values were reproducible with
a standard error less than 3%.
ꢀ
~
23. Brocklehurst, K.; Kowlessur, D.; O’ Driscoll, M.; Patel, G.; Qenby, S.; Salih, E.;
Templeton, W.; Thomas, M. W.; Willenbrock, F. Biochem. J. 1987, 244, 173e181.
ꢀ
24. García-Río, L.; Leis, J. R.; Moreira, J. A.; Araujo, E.; Norberto, F.; Ribeiro, L. J. Org.
Chem. 2003, 68 (11), 4330e4337.
25. Jencks, W. P.; Haber, M. T.; Herschlag, D.; Nazaretian, K. L. J. Am. Chem. Soc. 1986,
108, 479e483.
26. (a) Richard, J. P. J. Chem. Soc. Commun. 1987, 1968; (b) McClelland, R. A.; Ka-
nagasabapathy, V. M.; Banait, N. S.; Steenken, S. J. Am. Chem. Soc. 1992, 114,
1816e1823.
27. Bernasconi, C. F.; Kittredge, K. W.; Flores, F. X. J. Am. Chem. Soc. 1999, 121,
6630e6639.
28. Andraos, J.; Kresge, A. J. Am. Chem. Soc. 1992, 114, 5643e5646.
29. Leis, J. R.; Norberto, F.; Moreira, J. A.; Iley, J. J. Chem. Res. Synop. 1997, 88e89.
30. Iley, J.; Norberto, F.; Rosa, E. in part, V. Cardoso and C. Rocha J. Chem. Soc, Perkin
Trans. 2 1993, 591e594.
Acknowledgements
~
^
This work was supported by Fundac¸ ao para a Ciencia e a Tec-
nologia (FCT), Portugal (PEst-OE/QUI UI0612/2013). Lara Ribeiro
thanks FCT for postdoctoral grant (SFRH/BD/64404/2009)
References and notes
31. Berry, C. N.; Challis, B. C. J. Chem. Soc, Perkin Trans. 2 1974, 1638e1644.
1. Butler, A. R.; Williams, D. L. H. Chem. Soc. Rev. 1993, 22, 233e241.