Catecholamine-induced release of nitric oxide from N-nitrosotryptophan derivatives: A non-enzymatic method for catecholamine oxidation

Article abstract of DOI:10.1039/b513857d

In recent years, interest in the physiological functions of S-nitrosothiols has strongly increased owing to the potential of these compounds to release nitric oxide. In contrast, little is known about similar functions of N-nitrosated (N-terminal-blocked) tryptophan derivatives, which can be also formed at physiological pH. Utilizing N-acetyl-N-nitrosotryptophan (NANT) and N-nitrosomelatonin (NOMela) as model compounds, we have studied their reaction with catechol and catecholamines such as epinephrine and dopamine. In these reactions, NANT was quantitatively converted to N-acetyltryptophan (NAT), and nitric oxide was identified as a volatile product. During this process, ortho-semiquinone-type radical anions deriving from catechol and dopamine, were detected by ESR spectrometry. The catechol radical concentration was about eight times higher under normoxia than under hypoxia and a similar relationship was found for the decay rates of NANT under these conditions. An epinephrine-derived oxidation product, namely adrenochrome, but not a catechol-derived one, was identified. These observations strongly indicate that N-nitrosotryptophan derivatives transfer their nitroso-function to an oxygen atom of the catecholamines, and that the so-formed intermediary aryl nitrite may decompose homolytically with release of nitric oxide, in addition to a competing hydrolysis reaction to yield nitrite and the corresponding catechol. These conclusions were supported by quantum chemical calculations performed at the CBS-QB3 level of theory. Since nitric oxide is non-enzymatically released from N-nitrosotryptophan derivatives on reaction with catecholamines, there might be a possibility for the development of epinephrine-antagonizing drugs in illnesses like hypertension and pheochromocytoma. The Royal Society of Chemistry 2006.

Full text of DOI:10.1039/b513857d

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Catecholamine-induced release of nitric oxide from N-nitrosotryptophan
derivatives: A non-enzymatic method for catecholamine oxidation
a
b
a
a
Anna Kytzia, Hans-Gert Korth, Herbert de Groot and Michael Kirsch*
Received 29th September 2005, Accepted 8th November 2005
First published as an Advance Article on the web 30th November 2005
DOI: 10.1039/b513857d
In recent years, interest in the physiological functions of S-nitrosothiols has strongly increased owing to
the potential of these compounds to release nitric oxide. In contrast, little is known about similar
functions of N-nitrosated (N-terminal-blocked) tryptophan derivatives, which can be also formed at
physiological pH. Utilizing N-acetyl-N-nitrosotryptophan (NANT) and N-nitrosomelatonin
(
NOMela) as model compounds, we have studied their reaction with catechol and catecholamines such
as epinephrine and dopamine. In these reactions, NANT was quantitatively converted to
N-acetyltryptophan (NAT), and nitric oxide was identified as a volatile product. During this process,
ortho-semiquinone-type radical anions deriving from catechol and dopamine, were detected by ESR
spectrometry. The catechol radical concentration was about eight times higher under normoxia than
under hypoxia and a similar relationship was found for the decay rates of NANT under these
conditions. An epinephrine-derived oxidation product, namely adrenochrome, but not a
catechol-derived one, was identified. These observations strongly indicate that N-nitrosotryptophan
derivatives transfer their nitroso-function to an oxygen atom of the catecholamines, and that the
so-formed intermediary aryl nitrite may decompose homolytically with release of nitric oxide, in
addition to a competing hydrolysis reaction to yield nitrite and the corresponding catechol. These
conclusions were supported by quantum chemical calculations performed at the CBS-QB3 level of
theory. Since nitric oxide is non-enzymatically released from N-nitrosotryptophan derivatives on
reaction with catecholamines, there might be a possibility for the development of
epinephrine-antagonizing drugs in illnesses like hypertension and pheochromocytoma.
Introduction
(NOMela) (Scheme 1). These surprising reactions can be explained
satisfactorily by proposing the intermediacy of short-lived aryl
nitrites.
Catecholamines are known to be oxidized in vivo mainly enzymat-
ically by monoaminoxidase and catechol-O-methyltransferase.
1
Although catecholamines are well known for their vasoconstrictor
activity, there are only a few reports about their involvement in
endogenous nitric oxide production. Girgin et al. hypothesized
Results
2
an indirect way of enhancement of nitric oxide production in
the presence of elevated catecholamine levels, since an increased
monoaminoxidase activity would increase endogenous L-arginine
Decomposition of NANT
At pH 7.4, NANT is not stable in aqueous solution and decays hy-
3
drolytically with an apparent first-order rate constant of khydrolysis
=
concentrations. Rigobello et al. reported on the release of nitric
−
5
−1
(
6.6 ± 0.1) × 10
s
to produce stochiometric amounts of N-
oxide during reaction of S-nitrosoglutathione with epinephrine in
the presence of copper ions, pointing out that the latter would be
absolutely necessary for nitric oxide formation.
−
7
acetyltryptophan (NAT) and NO
the N NMR spectrum (Fig. 2A), showing partial hydrolysis of
00 mM N-NANT in phosphate buffer within 24 hours at 20 C
2
(Fig. 1). This is confirmed by
1
5
1
5
◦
1
Since N-nitrosotryptophan derivatives are known for both
4
(nitrite/ammonium sulfate ratio 40 : 60). The presence of either
catechol or epinephrine (100 lM each) accelerated the decay of
NANT (100 lM) with regard to the hydrolysis reaction (Fig. 1).
The decomposition of NANT in the presence of catechol follows
their vasodilatative activity and their nitric oxide releasing
5,6
potency, the question arises whether catecholamines can re-
lease nitric oxide from N-nitrosotryptophan derivatives in the
absence of copper ions. In this paper we report on the highly
complex nitric oxide yielding mechanism of the reaction between
catecholamines and N-nitrosotryptophan derivatives, such as
N-acetyl-N-nitrosotryptophan (NANT) and N-nitrosomelatonin
−
4
−1
a clean first-order rate law with kcatechol = (1.9 ± 0.2) × 10 s .
When catechol was used in large excess (100 mM catechol and
100 lM NANT, data not shown), the decay of NANT followed
further a clean pseudo-first-order rate law with an apparent rate
−
4
−1
constant of 7.6 × 10 s , i.e., the rate constant was increased only
about four-fold by increasing the catechol concentration 1000-
fold. Since initially a complete conversion of NANT should have
occured at this high excess of catechol, and because a first-order
rate law was additionally found at equimolar concentrations, a
a
Institut f u¨ r Physiologische Chemie, Universit a¨ tsklinikum Essen, Hufeland-
strasse 55, 45122, Essen, Germany. E-mail: michael.kirsch@uni-essen.de;
Fax: (+49) 201-723 5943; Tel: (+49) 201-723 4108
b
Institut f u¨ r Organische Chemie, Universit a¨ t Duisburg-Essen, Univer-
sit a¨ tsstrasse 5, 45117, Essen, Germany
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Scheme 1
1
5
15
Fig. 2
N NMR spectra. (A) The 24 h hydrolysis of 100 mM N-NANT
15
−
15
in 70 mM phosphate buffer yielded mainly NO
B) In contrast, the reaction of 100 mM catechol and 100 mM N-NANT
in 70 mM phosphate buffer yielded only small amounts of NO
after complete decomposition (3 h) when continuously purged with N
2
3
and some NO .
15
(
15
−
2
2
.
N
15
15
(
NH
4
)
2
SO
2
was added as an internal standard after the above times.
15
15
NMR data (phosphate buffer): N-NANT: d = 165.2/179.6 ppm; NO
d = 229.6 ppm.
2
:
to 1800 s) did the decay trace seem to follow a first-order process,
−
4
−1
with a rate constant of k = (4.3 ± 0.9) × 10 s , i.e., ca. 10 times
higher than the first-order constant of the hydrolysis reaction of
NANT. In addition, the decay of the optical density at 335 nm
varied significantly with pH (Fig. 3). The apparent first-order
behavior at the final stage of the reaction increased with decreasing
Fig. 1 The decay of 100 lM NANT in phosphate buffer (50 mM, pH 7.4,
◦
3
7 C), monitored spectrophotometrically at 335 nm in the absence and in
the presence of either 100 lM catechol or 100 lM epinephrine. Each curve
represents the mean of at least three experiments.
complex mechanism with a pre-equilibrium should operate, where
the rate constant k−1 of the backward reaction is much faster
than the rate constants k
2
and k of the formation of products
3
(
Scheme 2). An even more complicated situation was evident in the
presence of epinephrine, because the decay of NANT follows an
unusual “wavy” time profile. Such behavior can be explained by the
occurrence of an intermediate having a rather strong absorptivity
compared to NANT. Only at the final stage of the reaction (1100
Fig. 3 pH Dependence of the reaction of 100 lM epinephrine with
◦
1
00 lM NANT in phosphate buffer (50 mM, 37 C, pH 6.5, pH 7.5,
and pH 8). The decay of NANT was monitored by recording the optical
density at 335 nm. Representative measurements for each pH are shown.
Scheme 2
2
58 | Org. Biomol. Chem., 2006, 4, 257–267
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+
[
H ] because it was most prominent at pH 8. At pH values lower
than 6.5, the NANT hydrolysis reaction competed effectively with
the bimolecular reaction and this limited the application of the
acid pH value to 6.5.
1
5
The N NMR spectrum shows products other than those
produced from the hydrolysis reaction. Only small amounts of
1
5
−
15
−
NO
Fig. 2B) were formed. As the reaction mixture was continously
purged with oxygen-free nitrogen during the course of the reaction,
2
(nitrite/ammonium sulfate ratio 5 : 95) and NO
3
(
1
5
the formation of a volatile N product, most probably nitric oxide,
is indicated (see below). The low amounts of nitrite then represent
the fraction of NANT hydrolysis within the 3 h reaction period.
In line with these findings, under normoxic conditions the NANT-
dependent production of nitrite, as determined by the Griess assay,
corresponded to 100% and 70% in the absence and in the presence
of 100 lM epinephrine, respectively. Thus, the catechol-dependent
change in the product pattern clearly demonstrates the occurence
of a direct reaction between NANT and catechol.
Notably, the reaction with catechol and catecholamines was spe-
cific for N-nitrosotryptophan derivatives, because under copper-
free conditions GSNO did reacted neither with epinephrine nor
catechol (data not shown) in agreement with the results of
3
Rigobello et al.
Formation of NAT
As the product pattern of the reaction between catechol and
NANT differed significantly from that of the hydrolysis of NANT,
we investigated whether NAT was formed quantitatively in the
former reaction. Formation of NAT was monitored by following
its fluorescence at 358 nm because NANT did not fluoresce at this
wavelength (Fig. 4A). In fact, the increase in the fluorescence of
NAT (kexc = 270 ± 5 nm, kem = 358 ± 5 nm) corresponded well with
the decrease of the NANT concentration (Fig. 4B), demonstrating
that NANT was quantitatively converted to NAT.
Fig. 4 (A) Fluorescence spectra of NAT and NANT (kexc = 270 ± 5 nm,
k
em = 358 ± 5 nm) in 50 mM phosphate buffer. (B) The formation of
NAT from reaction of 200 lM NANT with 200 lM epinephrine was
monitored spectrofluorophotometrically in phosphate buffer (50 mM,
room temperature). The fluorescence intensity was determined from a
1
0-fold diluted aliquot of the reaction mixture. The decay of NANT was
determined simultaneously by recording the absorption at 335 nm from a
2-fold diluted aliquot of the reaction mixture. Each value represents the
mean of four experiments.
1
13
15
H, C and N NMR product studies
In order to identify the formation of other tryptophan- and/or
catechol-derived reaction products, e.g., nitrated or nitrosated
intermediates and products, NMR spectra were performed with
adrenochrome, could easily be detected during reaction of
epinephrine with NANT or N-nitrosomelatonin (NOMela). Re-
action of 100 lM epinephrine with 100 lM NANT yielded
1
5
100 mM catechol and 100 mM N-NANT or NANT, respectively,
in phosphate buffer. These spectra showed that catechol reacted
with NANT with stoichiometric formation of NAT (Fig. 5), in line
with the earlier experiments (Fig. 4B). Nitrosation and nitration
◦
adrenochrome concentrations of 12.1 ± 1.01 lM at 25 C and
◦
of 26.9 ± 1.76 lM at 37 C, respectively. Under the applied
products of NAT such as 1-NO
2
-NAT, 6-NO -NAT, or other C-
2
experimental conditions, the reaction periods were too short to
allow autoxidation of epinephrine. A maximum concentration
of 2.9 ± 0.99 lM adrenochrome was formed by autoxidation
nitroso-NAT derivatives, as reported in ref. 8, were not detected
in phosphate buffer, nor were any other products found at levels
1
3
exceeding ca. 8%. Additionally, reaction of 10 mM C-catechol
◦
of 100 lM epinephrine after 30 minutes of incubation at 37 C
1
5
13
with 10 mM N-NANT in phosphate buffer was monitored by C
NMR in order to detect possible nitration of catechol in aqueous
solution (data not shown). Again, these spectra did not show any
significant formation of products deriving from catechol.
(
Fig. 6).
Characteristic UV-vis spectra from the reaction between NANT
◦
and epinephrine at 37 C are collected in Fig. 7. Since isosbestic
points were identified (236 nm, 276 nm, 309 nm, 410 nm)
during the reaction, the formation of an intermediate with a
long lifetime at substantial concentrations can be safely excluded.
Nevertheless, the formation of an intermediate, i.e., a short-
lived aryl nitrite, which either regenerates the reactants, or yields
products according to Scheme 2 is still possible.
Adrenochrome formation
In contrast to the experiments performed with catechol, sub-
stantial formation of an epinephrine-derived product, namely
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1
3
Fig. 5
C NMR spectrum of the products from the reaction of 100 mM
N-NANT with 100 mM catechol in phosphate buffer pH 7.4, after
four days. Signals of catechol and NAT are labelled “C” and “N”, respec-
15
13
tively. C NMR data (phosphate buffer): NANT: d = 22.06/22.1 (CH
7.6/27.4 (b-CH ), 54.6/54.8 (a-CH), 113.5/111.3 (C-3), 115.7/119.1
C-7), 120.2/121.8 (C-6), 125.6/125.56 (C-5), 126.3/122.6 (C-4),
26.8/127.8 (C-2), 129.7/129.5 (C-3a), 135.4/128.4 (C-7a), 173.5/173.4
), 27.6 (b-CH ),
3
),
2
(
1
(
5
1
2
Fig. 7 UV-vis spectra from the reaction of 100 lM NANT with 100 lM
◦
epinephrine in phosphate buffer (50 mM, pH 7.4, 37 C), taken every
C=O), 177.6/177.8 (COOH) ppm. NAT: d = 22.1 (CH
5 min for an overall reaction period of 30 min. Data for a representative
measurement are shown.
3
2
6.1 (a-CH), 110.5 (C-3), 112.0 (C-7), 118.7 (C-5), 119.3 (C-6), 121.9 (C-4),
24.4 (C-2), 127.4 (C-3a), 136.3 (C-7a), 173.4 (C=O), 178.7 (COOH) ppm.
Catechol: d = 116.5 (C-3, C-6), 121.2 (C-4, C-5), 144.3 (C-1, C-2) ppm.
should be flawed by the adrenochrome formation at least for the
last 12.5 min. This conclusion, however, is in striking contrast
to the fact that during the reaction of NANT with epinephrine
(
Fig. 1) the decrease of the optical density at 335 nm was strongly
enhanced during the last 12.5 min of reaction. This discrepancy
can be only explained by the occurrence of an additional reaction
(see below).
•NO-release
As NANT was almost quantitatively converted to NAT by
1
5
15
catechol but no N-labelled product could be detected in the
N
NMR experiment (see above), nitric oxide was expected as the
second product, similar to what has been observed from reaction
5
of NANT with ascorbate. Nitric oxide formation was monitored
•
◦
◦
with an NO-sensitive electrode at 25 C and 37 C. Epinephrine
Fig. 6 Adrenochrome formation from 100 lM epinephrine in the pres-
ence and absence of 100 lM NANT, monitored spectrophotometrically at
(
2
500 lM) yielded a steady-state nitric oxide concentration of
.4 ± 0.3 lM at 25 C and 3.0 ± 0.8 lM at 37 C. At the
◦
◦
k
max = 487 nm. Autoxidation of 100 lM epinephrine in 50 mM phosphate
same concentrations, dopamine and catechol produced lower
◦
buffer (50 mM, pH 7.4, 37 C) yielded ca. 3% adrenochrome. In the
presence of 100 lM NANT, adrenochrome formation increased to ca.
steady-state concentrations of nitric oxide, viz. 1.2 ± 0.1 lM
◦
from catechol at 25 C and 2.7 ± 0.4 lM from dopamine at
2
7 lM. Each value represents the mean of at least three experiments.
◦
3
7 C. The steady-state nitric oxide concentration obtained from
◦
reaction of epinephrine with 100 lM NANT at 37 C followed
the epinephrine concentration in an exponential manner and
leveled off at ca. 2.8 lM NO at epinephrine concentrations
>250 lM. The concentration-dependence of nitric oxide release
from dopamine and catechol, respectively, was similar to the one
found for epinephrine (data not shown).
Identification of hidden peaks
•
In order to evaluate whether the region at around 335 nm is
influenced by adrenochrome, the UV-vis spectra were analyzed
in detail. The deconvolution of the adrenochrome UV-vis spectra
predicted in fact the existence of a UV-vis resonance at around
3
35 nm (Table 1), and this finding was confirmed by TD-DFT
Similar results were obtained when 100 lM NANT was
replaced by 100 lM NOMela. Now nearly the same steady-state
calculations. Since UV-vis spectra of both adrenochrome and
NANT were accurately analyzed, peak-fitting software was used
to re-analyze kinetic runs, an example of which is outlined in
Fig. 7. The deconvolution of the UV-vis spectra predicted that the
amount of NANT should fall within the reaction period of 30 min
•
◦
concentration of NO was detected, viz. 2.4 ± 0.2 lM at 25 C
•
◦
and 3.0 ± 0.5 lM NO at 37 C with 500 lM epinephrine. Thus,
•
the epinephrine-stimulated release of NO is not restricted to the
model compound NANT.
◦
when 100 lM NANT is treated with 100 lM epinephrine at 37 C
The initial rates of nitric oxide release were evaluated from the
•
•
(
data not shown). After a reaction period of 17.5 min, the UV-vis
initial slopes of the NO-time profiles. The rates of NO release
from 100 lM NANT increased in the presence of epinephrine in
a non-linear fashion with the epinephrine concentration (Fig. 8),
resonances of adrenochrome, but not of any other intermediate,
were clearly distinguishable. Thus, the optical density at 335 nm
2
60 | Org. Biomol. Chem., 2006, 4, 257–267
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Table 1 Evaluation of peaks in the optical density UV-vis spectra of 100 lM adrenochrome and of 100 lM NANT in 50 mM phosphate buffer were
ꢀ
R
evaluated with the PeakFit program as well as with the TD-DFT: MPW1B95/6–311 + + (G,d,p)//O3LYP/6-311 + G(2d,p) density functional theory
calculations
Adrenochrome
NANT
TD-DFT
Exp.
Deconvol.
TD-DFT
Exp.
Deconvol.
Z-Conformer
E-Conformer
a
k/nm
k/nm
Area
69.9
k/nm
f
k/nm
k/nm
Area
65.9
k/nm
f
k/nm
f
2
3
18.9
03.8
219.4
223.9
0.004
0.231
0.021
0.305
0.000
—
—
—
0.074
0.000
—
201.0
199.7
212.8
225.6
236.7
241.0
299.9
—
0.015
0.079
0.013
0.399
0.218
—
210.8
228.9
235.7
240.7
—
320.5
365.8
432.3
0.013
0.088
0.010
0.368
—
0.203
0.061
0.001
2
28.3
2
71.9
8.5
35.5
7.1
4.9
9.6
10.3
10.4
8.2
278.3
296.7
323.1
—
—
—
492.8
513.9
—
236.0
264.8
308.3
335.5
365.5
17.0
26.0
11.0
15.7
12.0
303.5
265.7
334.8
3
3
4
4
32.4
68.7
35.9
83.8
364.5
429.0
0.041
0.001
4
86.6
487.5
5
5
23.2
77.6
2.6
a
f = Oscillator frequency.
◦
◦
3
1
7 C than from NANT but the rate was lower at 25 C (about
−
1
6 nM s ). Due to the leveling off in the rates of nitric oxide release
at higher epinephrine concentrations, the experimental rates were
evaluated only for epinephrine concentrations ≤100 lM. In addi-
tion, kinetic measurements were performed in order to evaluate
the corresponding NANT decay rates.
Kinetic measurements
The initial decomposition of NANT or NOMela, respectively,
was observed spectrophotometrically by monitoring the decay at
3
35 nm or 346 nm for the first 210 seconds of the reaction. The
NANT decay rate was almost identical to the NOMela decay
rate at equimolar conditions. When 100 lM epinephrine were
•
◦
Fig. 8 The initial rates of NO production from the reaction of 100 lM
added to 100 lM NANT at 25 C, NANT decayed at 14.8 ±
NANT with epinephrine, as a function of epinephrine concentration
25–1000 lM), determined in phosphate buffer (50 mM, pH 7.4, 25 C).
Each value represents the mean of at least four experiments.
−1
−1
4
.1 nM s and NOMela at 16.7 ± 4.2 nM s . These decay rates of
◦
(
the N-nitrosotryptophan derivatives corresponded very well with
the observed NO-releasing rates shown in Fig. 8. In order to verify
•
whether the reaction of NANT and catechol follows a second-
−
1
◦
−1
◦
to level off at about 21 nM s (25 C) and 27 nM s (37 C),
order reaction, 700 lM epinephrine was added to 100 lM NANT
◦
−1
respectively, at an epinephrine concentration of about 100 lM
data not shown). Epinephrine (100 lM) was somewhat faster
35 nM s ) in releasing nitric oxide from N-nitrosomelatonin at
at 25 C (Table 2). The observed decay rate of 30.2 ± 3.1 nM s was
only about two times faster than the corresponding decay rate at
equimolar concentrations, whereas a factor of 7 was expected for
(
(
−
1
Table 2 Decay rates of reactions between epinephrine and NANT. Varying amounts of NANT were added to varying amounts of epinephrine in
◦
5
3
0 mM phosphate buffer, pH 7.4. The decay of NANT was determined spectrophotometrically at 335 nm by taking measurements every 30 s at 25 C or
◦
0 C. The decay rate was evaluated from the first linear 210 s of data. Experiments were also performed in the presence of superoxide dismutase as well
as under hypoxic conditions within an argon-flushed glove bag. Each data represents the mean of at least four experiments
◦
−1
Reactants
T/ C
Decay rate/nM s
1
1
1
1
7
7
7
1
1
00 lM NANT + 100 lM epinephrine
00 lM NANT + 100 lM epinephrine + 300 U ml SOD
00 lM NANT + 700 lM epinephrine
00 lM NANT + 700 lM epinephrine + 300 U ml SOD
00 lM NANT + 100 lM epinephrine
00 lM NANT + 100 lM epinephrine + 300 U ml SOD
00 lM NANT + 700 lM epinephrine
25
25
25
25
25
25
25
30
30
14.8 ± 4.1
20.5 ± 2.9
30.2 ± 3.1
32.1 ± 5.0
53.5 ± 10.1
41.9 ± 5.1
95.8 ± 4.7
24.0 ± 2.5
10.0 ± 1.6
−
1
−
1
−
1
00 lM NANT + 100 lM epinephrine
00 lM NANT + 100 lM epinephrine under hypoxia
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a true second-order process. When 700 lM NANT was reacted
this reaction (data not shown) by monitoring the decay of the
absorption at 487 nm. The reaction of NANT with adrenochrome
yielded NAT, in line with the reaction between NANT and
epinephrine. In contrast to the latter reaction, nitric oxide was
not detected with the NO-sensitive electrode (data not shown), so
that the reaction mechanism between NANT and adrenochrome
is different from the reaction of NANT with epinephrine.
−
1
with 100 lM epinephrine, a decay rate of 53.5 ± 10.1 nM s
was measured. The observed rates depended more strongly on the
concentration of NANT than on the concentration of epinephrine.
Employing 700 lM NANT and 700 lM epinephrine, the decay rate
•
−
1
increased only 6.5-fold (95.8 ± 4.7 nM s (Table 2)) whereas a 49-
fold increase would be expected from a true second-order reaction.
Further decay rates are collected in Table 2. These results clearly
indicate that the reaction between epinephrine and NANT does
not follow a simple second-order rate law.
Catechol radical anion and dopamine radical anion
Since under our experimental conditions epinephrine might
yield small amounts of superoxide, which is well-known to
The release of the radical nitric oxide via homolysis of an
epinephrine-derived aryl nitrite necessarily implicates the forma-
tion of a second radical. Since the solubility of epinephrine was
too low for the ESR experiments, this compound was replaced
by dopamine. In fact, after mixing 7.5 mM NANT and 7.5 mM
dopamine in phosphate buffer at pH 7.4, the characteristic ESR
signal of the dopamine radical anion was immediately detected
(Fig. 10A). The ESR signal decayed, in correspondence with the
foregoing kinetic experiments. The catechol radical anion was
detected by ESR on mixing NANT with catechol under similar
conditions. In order to exclude the possibility that the catechol-
type radicals were exclusively formed from reaction of catechol
with nitrogen dioxide, which may be rapidly formed by autoxida-
9–11
react with S-nitrosothiols,
one may suspect that our kinetic
experiments might be somewhat flawed by the intermediacy of
−
1
this radical. However, additional experiments with 300 units ml
superoxide dismutase in the presence of varying concentrations
◦
of NANT and epinephrine at 25 C revealed that the decay rates
did not depend significantly on the presence of SOD (Table 2).
Thus, superoxide is obviously not involved in the reaction between
epinephrine and NANT. On the contrary, the decay rates are
influenced by molecular oxygen. Under normoxia, a decay rate of
−
1
2
4.0 ± 2.5 nM s was found from reaction of 100 lM epinephrine
with 100 lM NANT, which was about 2.4 times faster than under
•
12
hypoxic conditions, as outlined in Table 2.
tion of NO, additional experiments were performed under strict
oxygen-free conditions. Although the signal intensity was about
eight times lower than under normoxic conditions, the generation
Adrenochrome–NANT reaction
The “wavy” time profile (Fig. 1) of the NANT–epinephrine
reaction not only derived from the interference with the absorption
of adrenochrome at 335 nm, but also was affected by a reaction
of adrenochrome with NANT. In order to simulate the conditions
achieved during reaction of epinephrine and NANT, in which
1
00 lM NANT and 100 lM epinephrine yielded about 27 lM
◦
adrenochrome at 37 C, the reaction of 25 lM adrenochrome
with 100 lM NANT was observed in phosphate buffer (Fig. 9).
At 37 C, a decay at 335 nm as well as at 487 nm was observed
◦
spectrophotometrically. Since the optical density at 335 nm is
flawed by adrenochrome, the NANT concentration cannot be
deduced spectrophotometrically, but it can be safely assumed
that ca. 4.93 ± 0.32 lM adrenochrome was consumed during
Fig. 10 (A) ESR spectrum of the dopamine radical anion recorded after
rapid mixing of 7.5 mM dopamine and 7.5 mM NANT in phosphate
buffer (50 mM, pH 7.4, room temperature). (B) Under similar conditions,
the ESR spectrum of the catechol radical anion was recorded from 7.5 mM
catechol and 7.5 mM NANT under normoxic and hypoxic conditions . The
mixtures were supplemented by 100 lM DTPA to avoid any influence of
metal ions.
Fig. 9 The decay of 100 lM NANT in the presence and absence of
2
5 lM adrenochrome, followed by monitoring the optical density at 335 nm
in phosphate buffer (50 mM, pH 7.4, room temperature). Each value
represents the mean of at least three experiments.
2
62 | Org. Biomol. Chem., 2006, 4, 257–267
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of the radicals in the absence of oxygen was unequivocally demon-
strated (Fig. 10B). This observation points clearly to the intermedi-
acy of the short-lived aryl nitrite. A possible influence of metal ions
was excluded by supplemention of 100 lM DTPA to the reaction
mixture, after which no significant effect on the steady-state con-
centration of catechol radical ion was observed (data not shown).
for 4-methyl-O-nitrosocatechol (Table 3, entries 4 and 5), in
22–24
line with the experimental observations.
The hydrolysis of O-
nitrosocatechol is expected to compete with the homolysis reac-
tion, as it is calculated to be exergonic for both O-nitrosocatechol
−
1
(−4.3 kcal mol , Table 3, entry 6) and for 4-methyl-O-
−
1
nitrosocatechol (−4.0 kcal mol , Table 3, entry 7). The alternative
mechanism, direct homolysis of NANT to give nitric oxide and the
NAT aminyl radical (Table 3, entry 8), can be excluded because of
Discussion
14
its endergonic character, in line with the data of Zhu et al. Notice-
ably, reaction of N-nitrosoindole compounds with catechol anions
to yield the corresponding O-nitrosocatechols seems to be ther-
mochemically favored (Scheme 3). Although the formation of the
O-nitrosocatechol is predicted to be slightly endergonic (3–4 kcal
Our findings demonstrate for the first time the release of nitric
oxide on reaction of catechols (ortho-hydroxyphenols) and cate-
cholamines with N-nitrosoindoles like NANT or NOMela. Prod-
uct formation proceeds either by initial electron transfer or via an
intermediate aryl nitrite. Since catechol has an oxidation potential
−
1
−1
mol ), the homolysis (−11 to −14 kcal mol ) as well as the hy-
13
−1
of Eox = 0.3 V it is unlikely to reduce N-nitrosotryptophan
drolysis (ca. −12 kcal mol ) of these aryl nitrites are calculated to
derivatives to the corresponding N-nitrosoindole radical anions
be clearly exergonic, so that the reaction between N-nitrosoindole
compounds and catechol anions appears feasible. The CBS-
QB3 calculations further predict that the homolysis reaction
1
4
(
E
red = −0.6 V). Therefore, initial electron transfer can be
safely excluded. N-Nitrosotryptophan derivatives are known to
1
5
6
16
17
−1
nitrosate butanol, thiols, H
2
O
2
,
primary aromatic amines,
(−14.3 kcal mol ) is thermodynamically slightly favored over
18
−1
and tryptophan-derivatives; hence, a similar transnitrosation
reaction between NANT and the catechol/catecholamines ap-
the hydrolysis reaction (−11.8 kcal mol ) for 4-methyl-O-nitroso-
catechol anion but not for O-nitrosocatechol anion (Scheme 3).
These data suggest that hydrolysis and homolytic fragmentation
represent competitive pathways with a slight preference for hydrol-
19
pears feasible. In line with this, Darbeau et al. reported on a
transfer of the nitroso function from N-nitrosoamides to alcohols.
Although our NMR and UV-vis spectrometric experiments failed
to identify intermediary formation of O-nitrosocatechols, they
should be the key intermediates. The formation of n-butyl nitrite
from the reaction of NANT with butanol, and regeneration
•
ysis of O-nitrosocatechol, but enhanced formation of NO and the
epinephrine-derived phenoxyl radical from O-nitrosoepinephrine.
The additional release of catechol-type radicals is further predicted
to enforce the decay of NANT (Table 3, entries 9 and 10). Since
under aerobic conditions a higher NANT concentration leads to
1
5,20
of NAT was described in 1977.
Likewise, NANT effectively
1
6
12
O-nitrosates hydrogen peroxide. Aryl nitrites are unstable to-
an enhanced release of nitric oxide, and thus of nitrogen dioxide,
21
wards rearrangement and transnitrosation and O-nitrosophenols
the catechol radicals are additionally produced by reaction of
25
are known for their short lifetimes, as they are in equilibrium
catechol with nitrogen dioxide, in line with an increased ESR-
signal intensity of the catechol radical (Fig. 10B) and a faster
decay of NANT (Table 2) under normoxic conditions. Hence,
the reaction of NANT with catechol compounds cannot follow a
simple second-order process, as was shown above. In the case of
epinephrine the system is further complicated by the fact that
the formed adrenochrome (Fig. 7) also enhances significantly
the decay of NANT, albeit slowly. Since during this reaction
•
22–24
with NO and the corresponding phenoxyl-type radical.
High-
level quantum mechanical calculations performed with the multi-
level method CBS-QB3, using N-nitrosoindole as placeholder
for NANT and 4-methylcatechol as placeholder for epinephrine,
indeed predict that the formation of O-nitrosocatechol and 4-
methyl-O-nitrosocatechol are moderately endergonic processes
(
Table 3, entries 1–3). In aqueous solution, the aryl nitrite of
•
catechol is predicted not to be a stable compound because the
dissociation into nitric oxide and the corresponding catechol
nitric oxide release could not be detected by the NO-sensitive
electrode, we assume that adrenochrome supports the hydrolysis
−
1
26
radical is calculated to be slightly endergonic by 0.8 kcal mol
of NANT. As adrenochrome is mainly formed at alkaline pH, this
for O-nitrosocatechol and slightly exergonic by −0.4 kcal mol−
1
would explain the apparent first-order behaviour of NANT decay
Table 3 Quantum chemical calculations. Complete basis set (CBS-QB3) computations were carried out with Gaussian 03 (revision AM64L-G03RevC0.2)
suite of programs. Gibbs free energies of solvation for water were estimated for the optimized gas-phase geometries with the CPCM-UAHF procedure
incorporated in Gaussian 03
G/kcal mol−
1
D
E
solv/kcal mol
−1
−1
Entry
Reactant
D
R
R
R
D Gsolv/kcal mol
1
2
3
4
5
6
7
8
9
E-NO-indole + cat → indole + NO-cat
Z-NO-indole + cat → indole + NO-cat
E-NO-indole + mecat → indole + NO-mecat
6.2
6.2
5.8
0.4
0.9
1.0
1.0
0.4
7.1
7.2
6.8
0.8
−0.4
−4.3
−4.0
13.7
•
NO-cat → NO + cat-radical
•
NO-mecat → NO + mecat-radical
−0.8
−0.4
NO-cat + H
NO-mecat + H
2
O → trans-HNO
2
+ cat
+ mecat
1.2
−5.5
2
O → trans-HNO
2
1.6
−5.6
•
E-NO-indole → indole-radical + NO
15.5
−1.7
−1.8
−1.8
•
E-NO-indole + cat-radical → indole + NO + cat ox
−11.9
−13.6
•
1
0
E-NO-indole + mecat-radical → indole + NO + mecat ox
−11.7
−13.5
cat = catechol, mecat = methylcatechol, NO-cat = O-nitrosocatechol, NO-mecat = 4-methyl-O-nitrosocatechol, cat-radical = catechol radical, mecat-
radical = methylcatechol radical, cat ox = oxidized catechol (catechol dichinon), mecat ox = oxidized methylcatechol (quinone of 4-methylcatechol).
This journal is © The Royal Society of Chemistry 2006
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Scheme 3
at pH 8 at later stages of the reaction (Fig. 3). Adrenochrome
formation can be explained by the intermediate formation of
the corresponding quinone of epinephrine. The full reaction
mechanism is shown in Scheme 4.
In order to avoid any misunderstandings, we do not suggest
the application of NANT to patients with hypertensive diseases.
Nevertheless, one might speculate that in the future an optimized,
yet unavailable N-nitrosotryptophan derivative may be found
which will be both an effective vasodilatative and epinephrine-
decreasing drug.
4
Zhang et al. observed a vasorelaxation of aorta rings from
male New Zealand white rabbits when treated with the nitrosated
dipeptide N-nitroso-Gly-Trp. In that study the vessel rings were
prepared in the presence of 1 lM phenylepinephrine. Our above
results now provide an explanation for the observed vasorelaxation
because, similarly to NANT, N-nitroso-Gly-Trp is expected to
react with phenylepinephrine in the aortic tissues with the release
of nitric oxide.
Experimental
Chemicals and solutions
N-Acetyl-DL-tryptophan, melatonin, catechol, epinephrine,
The reaction between N-nitrosated tryptophan derivatives and
catechols might offer a starting point for the development of a
new class of drugs for the treatment of diseases in which high
catecholamine levels were found, as for instance in pheochro-
mocytoma. This neuroendocrine tumor exhibits catecholamine
adrenochrome, dopamine, and chelex 100 were obtained from
1
3
Sigma (Taufkirchen, Germany).
6
C -Catechol (99%) was pur-
chased from Cambridge Isotope Laboratories Inc. (Andover,
USA). Superoxide dismutase from bovine erythrocytes was
from Roche (Mannheim, Germany). N-Acetyl-N-nitroso-DL-
−
1
concentrations up to 200 nmol l in the corresponding adrenal
1
5
tryptophan (NANT), N-NANT, N-nitrosomelatonin, and S-
nitrosoglutathione were prepared as described in ref. 18. Stock
solutions were prepared daily and their concentrations were spec-
trophotometrically determined at 335 nm and 346 nm, respectively,
and checked at least every 6 h. All other chemicals were of the
highest purity commercially available.
27
vein, and the general plasma catecholamine levels are 10 times
28
higher than in the control group. Due to these high catecholamine
concentrations, pheochromocytoma patients can develop life-
29
threatening hypertensive crisis. It is conceivable that the ap-
plication of a drug based on the reaction mechanism presented
might be a useful therapeutical intervention in these illnesses
because the catecholamines can be non-enzymatically destroyed
with simultaneous release of nitric oxide (see Scheme 4).
Experimental conditions
30
On the other hand, Venitt et al. reported on the mutagenicity of
N-acetyl-N-nitrosotryptophan at high concentrations (>120 lM)
in bacteria which would counteract pharmacological use. How-
ever, one should keep in mind that a variety of drugs are harmful
Since nitrosation reactions are highly sensitive to the presence
of metal ions, phosphate buffer solutions (50 mM) were treated
with the heavy metal scavenger resin Chelex 100 (0.5 g in 15 ml).
After gently shaking, the solution was stored overnight and was
then carefully decanted from the resin. Afterwards, the pH of all
31,32
at high concentrations, e.g. the drug sodium nitroprusside,
but
that lower doses can sucessfully be applied.
2
64 | Org. Biomol. Chem., 2006, 4, 257–267
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Scheme 4
−
1
−1
36
solutions was readjusted to pH 7.4 ± 0.1 by the addition of 50 mM
PO or 50 mM K PO
catechol, e346 = 7070 M cm for N-nitrosomelatonin, and
1
−1
37
H
3
4
3
4
.
e
336 = 770 M− cm for GSNO. The reaction between cate-
chol/catecholamines and the nitrosated tryptophan derivatives
•
(
both 100 lM at pH 7.4) was monitored for 30 min by taking
Determination of nitric oxide with an NO sensitive electrode
◦
◦
◦
recordings every 30 s at 25 C, 30 C, and 37 C. Experiments
with epinephrine and NANT were performed also at pH 6.5,
pH 7, pH 7.5, and pH 8. For the determination of the reaction
order, the reaction between equimolar concentrations of catechol
and NANT (100 lM and 700 lM) was monitored. Further, both
compounds were reacted under pseudo-first-order conditions, i.e.,
at 100 mM catechol and 100 lM NANT. In addition, 700 lM
epinephrine was reacted with 100 lM NANT and vice versa.
The possible influence of superoxide anions on this reaction was
checked by adding 300 units ml of superoxide dismutase (SOD)
to the reaction mixture. Similar experiments (100 lM NANT and
00 lM epinephrine) were performed under hypoxic conditions in
an argon-flushed glove box at 30 C after the pO
•
Nitric oxide formation was determined using an NO sensitive elec-
trode (ISO-NO; World Precision Instruments, Sarasota, Florida)
as described in ref. 33. The reaction mixtures were continously
stirred throughout the measurements, and the temperature was
◦
◦
kept at 25 ± 1 C or 37 ± 1 C. The electrode was calibrated
•
daily, and NO production was quantified according to the man-
ufacturer’s instructions, employing potassium iodide (100 mM) in
H
2
SO
of NaNO
00 lM of the nitrosated tryptophan derivative was applied to
4
(0.1 M) as a calibration solution to which varying amounts
2
(0.5 mM) were added. A standard concentration of
−1
1
varying concentrations of catechols.
1
◦
2
of the buffer
Decay kinetic experiments
had dropped to <4.7 kPa (from 202–205 kPa at normoxia) as
ꢀ
R
determined with a LICOX MCB Oxygen Monitor (GMS, Kiel-
The catechol- and catecholamine-induced decay of N-acetyl-
N-nitrosotryptophan and N-nitrosomelatonin, respectively, was
monitored on a SPECORD S 100 spectrophotometer from
38
Mielkendorf, Germany).
Detection of NAT and NANT
Analytic Jena (Jena, Germany) using the molar absorptivity e487
=
−
1
−1
34
−1
−1
2
800 M cm for adrenochrome,
e
335 = 6100 M cm for
The formation of NAT and the decrease of NANT were simulta-
neously monitored from a solution of 200 lM NANT and 200 lM
35
−1
−1
−1
−1
NANT,
e
335 < 70 M cm for NAT, e335 < 155 M cm for
This journal is © The Royal Society of Chemistry 2006
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1
3
15
epinephrine in 50 mM phosphate buffer at room temperature
for 100 min. The formation of NAT was determined by 10-
fold dilution of the sample and recording of the fluorescence
from 10 mM C
6
-catechol and 10 mM N-NANT were measured
in phosphate buffer pH 7.45. In order to quantify the amount
of nitrite formed during the decay of 100 mM N-NANT in
phosphate buffer as well as during the reaction of 100 mM
1
5
(
kexc = 270 ± 5 nm, kem = 358 ± 5 nm) on a Shimadzu
1
5
15
15
spectrofluorophotometer. The decomposition of NANT was spec-
trophotometrically determined by 1 : 1 dilution of the samples and
reading the optical density at 335 nm.
N-NANT with 100 mM catechol in N NMR, 50 mM N-
ammonium sulfate (Sigma Aldrich) was added as an internal
standard. To avoid the autoxidation of nitric oxide, the latter
reaction mixture was continously flushed with nitrogen for 3 h.
Measurements of nitrite
The amounts of nitrite formed from both the reaction of
Peak deconvolution software
1
00 lM catechol or catecholamines with 100 lM NANT and
the decomposition of NANT were quantified as follows: A
0 ll aliquot of the reaction mixture (NANT and catechol)
In order to detect hidden peaks in the performed spectrophoto-
metrical measurements, the PeakFit program (SeaSolve Program
Inc., Framingham, MA) was applied. Spectra from NANT,
ꢀ
R
5
and 250 ll phosphate buffer (pH 7.4) were added to 750 ll of
the Griess reagent. The Griess reagent was prepared daily from
adrenochrome, and from the reaction of NANT with epinephrine
◦
(
both 100 lM at 37 C) were deconvoluted with the peak-fitting
0
.1% naphthylethylenediamine and 1% sulfanilamide (both from
Sigma). After a reaction period of 10 min, the optical density at
42 nm was recorded and the nitrite concentration was evaluated
software.
5
by means of a calibration curve. All experiments were performed
in quadruplicate 24 h after mixing the reactants.
Quantum-chemical calculations
Density functional theory (DFT) and complete basis set (CBS-
QB3) computations were carried out with the Gaussian 03 (Rev
ESR measurements
40
C0.2) suite of programs. Geometries were fully optimized to sta-
tionary points, using the O3LYP/6-311 + G(2d,p) level in the DFT
calculations. Vertical excitation energies were computed on these
optimized structures using the protocol of the time-dependent
The catechol radical anion as well as the dopamine radical
anion were identified by ESR spectrometry. ESR spectra were
◦
recorded at 18 C on a Bruker ESP-300E X-band spectrometer
(
Bruker, Rheinstetten, Germany) equipped with a TM110 wide-
41
density functional theory (TD-DFT) with MPW1B95/6-311 +
bore cavity. Solutions were prepared from 1 ml of the buffer
solution (pH 7.4) containing catechol and dopamine, respectively,
and NANT (7.5 mM each). The reaction solutions were quickly
transferred to a 0.4 mm aqueous solution quartz cell (Willmad,
Buena, NY). Experiments with catechol were performed under
normoxic and under hypoxic conditions. For the latter, a glove
bag (Roth, Karlsruhe, Germany) flushed with argon was used.
To check on the influence of metal ions, ESR spectra were also
recorded in the presence of 100 lM DTPA (diethylenetriamine
pentaacetic acid; Sigma). Recording conditions were as follows:
42
+
G(d,p) as model for the first 10 excited states, in order
to reproduce the UV-visible spectra of adrenochrome and N-
nitrosoindole, respectively. To take into account solute–solvent-
dependent spectral shifts in the UV-visible spectra, solvent effects
were evaluated for all calculations of electronic spectra with the
CPCM method. In addition, Gibbs free energies of solvation
for water were estimated for the CBS-QB3 optimized gas-phase
geometries with the CPCM-UAHF procedure incorporated in
Gaussian 03. Both the CBS-QB3 and CPCM/(U)HF/6-31 + G(d)
methodologies are known to provide thermodynamic estimates
microwave frequency, 9.79 GHz; modulation, 0.04 mT; signal gain,
−1 43,44
within “chemical accuracy” (± 1 kcal mol ).
5
5
2
× 10 ; sweep range, 10 mT; microwave power, 2 mW; sweep time,
.8 min. Spectrum simulation was performed using the WinSim
program.
39
Statistics
All experiments were repeated at least three times, except the ESR
and NMR measurements which were performed twice. The results
are expressed as means ± S.D.
1
13
15
H, C and N NMR measurements
NMR experiments were performed on a Bruker AVANCE DRX
5
5
00 instrument (Bruker Biospin, Rheinstetten, Germany) at
1
13
00 MHz for H NMR, 125.71 MHz for C NMR, and 50.67 MHz
1
5
1
13
Abbreviations
The abbreviations used are: •NO, nitric oxide; NOS, ni-
tric oxide synthase; GSNO, S-nitrosoglutathione; NAT, N-
acetyltryptophan; SOD, superoxide dismutase; NANT, N-acetyl-
N-nitrosotryptophan; NOMela, N-nitrosomelatonin; DTPA, di-
ethylenetriamine pentaacetic acid;
for N NMR spectra. H and C NMR chemical shifts (d) are
given in ppm relative to TMS (d = 0) as external standard. For
1
5
N NMR spectrometry, neat nitromethane (d = 0) was used as
1
5
external standard. N NMR measurements were performed on
a mixture of 0.9 M catechol and 0.9 M N NANT in a 9 :
1
obtained from 1 M NANT and 1 M catechol in DMSO/D
DMSO 9 : 1 (v/v).
1
5
1
3
1
DMSO/D -DMSO mixture. C and H NMR spectra were
6
6
-
To elucidate the effect of water, similar measurements were
performed with 100 mM catechol and 100 mM N-NANT in
Acknowledgements
1
5
7
1
0 mM phosphate buffer solution at pH 7.2 in the presence of
Anna Kytzia was supported by an IFORES grant from the
Universit a¨ t Duisburg-Essen.
1
3
0% D
2
O as an internal lock. In addition, C NMR spectra
2
66 | Org. Biomol. Chem., 2006, 4, 257–267
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