Reactivity of peroxynitrite with melatonin as a function of pH and CO2 content

Article abstract of DOI:10.1002/1099-0690(200301)2003:1<172::AID-EJOC172>3.0.CO;2-W

Peroxynitrite is known to be a strong oxidant and a nitrating agent of aromatic phenolic or heterocyclic rings, depending on its form in aqueous solutions: the anion ONOO-, its conjugate acid ONOOH, or its CO₂ adduct ONOOCO₂-. Various reactions have been observed with melatonin (1), a tryptophan derivative, in phosphate-buffered solutions. Melatonin (1) is recognized as a scavenger of several strong oxidants (HO', H₂O₂,...) accounting for its biological and pharmacological effects. Here we describe two oxidation routes that give rise to indol-2-ones 2 (probably via a 2,3-epoxyindole) and kynuramines 6 (by cleavage of the pyrrole ring), attributable to reactions of ONOOH and ONOO-, respectively, according to the effects of pH and CO₂ content. At pH = 7.6 and in the presence of CO₂, an important conversion is the cyclization of the lateral amide function, giving 3-substituted pyrroloindoles 4. At neutral pH, therefore, all routes coexist, with a balance between indol-2-ones 2 and pyrroloindoles 4 on the one side and kynuramines 6 on the other, depending on the CO₂ content. Furthermore, under specific conditions substitutions of the hydrogen atom on the pyrrole nitrogen atom, affording the 1-nitro-(5) and the unstable 1-nitrosomelatonin (7), are among the major transformations: formation of the nitrosation product, together with that of kynuramines 6, rises sharply when the pH of the medium increases, confirming the implication of ONOO-in their synthesis; conversely, both reaction yields decrease with increasing CO₂ content, to favor 1-nitromelatonin (5). Finally, nitration by aromatic substitution occurring essentially on C-4 becomes important at acidic pH, and also at pH = 7.6 over a narrow range of CO₂ concentrations. Most of the reactions are typical of the indole moiety, suggesting that melatonin (1) is a model of potential use for investigation of tryptophan chemistry with peroxynitrite. Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003.

Full text of DOI:10.1002/1099-0690(200301)2003:1<172::AID-EJOC172>3.0.CO;2-W

FULL PAPER
Reactivity of Peroxynitrite with Melatonin as a Function of pH and
CO2 Content
[a]
[a]
[a]
[a]
´ `
Fabienne Peyrot, Marie-Therese Martin, Julie Migault, and Claire Ducrocq*
Keywords: Indole / Nitration / Nitrosation / Oxidation / Oxidoperoxidonitrate(1Ϫ)
Peroxynitrite is known to be a strong oxidant and a nitrating
agent of aromatic phenolic or heterocyclic rings, depending
CO2 content. Furthermore, under specific conditions substi-
tutions of the hydrogen atom on the pyrrole nitrogen atom,
on its form in aqueous solutions: the anion ONOO−, its con- affording the 1-nitro- (5) and the unstable 1-nitrosomelatonin
jugate acid ONOOH, or its CO2 adduct ONOOCO2−. Various
reactions have been observed with melatonin (1), a trypto-
phan derivative, in phosphate-buffered solutions. Melatonin
(1) is recognized as a scavenger of several strong oxidants
(7), are among the major transformations: formation of the
nitrosation product, together with that of kynuramines 6,
rises sharply when the pH of the medium increases, con-
firming the implication of ONOO− in their synthesis; con-
(HO·, H2O2, ...) accounting for its biological and pharmacolo- versely, both reaction yields decrease with increasing CO2
gical effects. Here we describe two oxidation routes that give
content, to favor 1-nitromelatonin (5). Finally, nitration by
rise to indol-2-ones 2 (probably via a 2,3-epoxyindole) and
aromatic substitution occurring essentially on C-4 becomes
kynuramines 6 (by cleavage of the pyrrole ring), attributable important at acidic pH, and also at pH = 7.6 over a narrow
to reactions of ONOOH and ONOO−, respectively, according
to the effects of pH and CO2 content. At pH = 7.6 and in the
presence of CO2, an important conversion is the cyclization
range of CO2 concentrations. Most of the reactions are typ-
ical of the indole moiety, suggesting that melatonin (1) is a
model of potential use for investigation of tryptophan chem-
of the lateral amide function, giving 3-substituted pyrroloin- istry with peroxynitrite.
doles 4. At neutral pH, therefore, all routes coexist, with a
balance between indol-2-ones 2 and pyrroloindoles 4 on the
one side and kynuramines 6 on the other, depending on the
( Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2003)
Introduction
such as urate and vitamins C and E. In specific cellular
compartments, it is believed to intervene as a biological me-
Oxidoperoxidonitrate(1Ϫ) (ONOOϪ) is formed in vivo diator during differentiation and development, or in re-
and in vitro from the diffusion-controlled reaction (k ϭ sponse to stress. However, it also appears to act as a toxic
·
·Ϫ
·
4Ϫ19 ϫ 109 Ϫ1sϪ1) of NO and O2 radicals, both pro- agent in numerous diseases in which synthesis of both NO
duced by enzymatic reactions.[1] In acid solution, isomeriz- and O2·Ϫ is considerably increased. This occurs in infection,
ation affords NO3Ϫ, via the conjugate acid ONOOH, with inflammation, and chronic degenerative processes, with
a rate constant of 1 sϪ1. The ONOOϪ anion is much more markers being detectable in tissues, such as 3-nitrotyrosine
stable, but reacts with CO2 (k ϭ 3 ϫ 104 Ϫ1sϪ1) to produce residues in proteins, 8-nitroguanine in DNA, and thiyl rad-
Ϫ [2]
ONOOCO2
.
The name ‘‘peroxynitrite’’ is used for the icals and disulfide bridges and/or linkages between peptides
mixture of ONOOH, ONOOϪ, and ONOOCO2Ϫ, the pro-
portions of which depend on the pH value (pKa ϭ 6.8) and
the presence of CO2. It has been proposed that peroxynitr-
ite chemistry is governed by radicals generated from
or proteins. These effects are related to the peroxynitrite
formation and can be counteracted by administration of ox-
idant scavengers and/or inhibitors of NO-synthases. How-
ever, the reactivity of peroxynitrite is far from well under-
stood. It was therefore of interest to study the reactions
of peroxynitrite with a natural indole compound known to
decrease the effects of oxidative stress.[7]
Ϫ
ONOOH and ONOOCO2 homolysis and their prod-
ucts.[3,4]
Peroxynitrite has been shown in vitro to be an oxidant of
lipids, proteins, and nucleic acids, and also a nitrating agent
of phenolic and heterocyclic compounds.[5,6] Under physio-
logical conditions, it reacts with reduced transition metal
containing compounds, thiols, and other natural scavengers
We investigated the interaction of peroxynitrite with the
tryptophan derivative melatonin {N-[2-(5-methoxy-1H-in-
dol-3-yl)ethyl]acetamide, 1}. Melatonin is secreted in mam-
mals by the pineal gland, following a circadian cycle, and
by some other tissues such as the retina and the gastrointes-
tinal tract.[7] This lipophilic hormone spreads throughout
the body, whereas peroxynitrite is a locally formed effector.
[a]
Institut de Chimie des Substances Naturelles, C. N. R. S,
Avenue de la Terrasse, 91190 Gif-sur-Yvette, France
Fax: (internat.) ϩ 33-1/69077247
·
E-mail: Claire.Ducrocq@cnrs.icsn-gif.fr
The scavenging properties of melatonin with OH radicals
172
 2003 WILEY-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim 1434Ϫ193X/03/0101Ϫ0172 $ 20.00ϩ.50/0 Eur. J. Org. Chem. 2003, 172Ϫ181

Reactivity of Peroxynitrite with Melatonin
FULL PAPER
have been demonstrated,[8] but only a few reactions with
peroxynitrite have been described. The first, well-docu-
mented, step in the reaction between melatonin and peroxy-
nitrite is the formation of the melatoninyl radical cation
through one-electron transfer to peroxynitrite.[9Ϫ11] Only a
few stable products were identified by GC-MS and 1H
NMR.[12] In our laboratory, we have previously demon-
strated the formation of unstable derivatives such as 1-hy-
droxy- and 1-nitrosomelatonin, with yields dependent on
physicochemical factors.[13] Here we have completed the
identification of a series of products and have measured
their respective yields under various sets of physicochemical
conditions. Taking into account the complexity and the
duration of the chemical transformations of melatonin by
peroxynitrite in aqueous solutions, we have distinguished
different types of reactions and assigned the relevant react-
ive form of peroxynitrite for each of them. The results de-
scribed and the proposed mechanisms afford new insights
into reactions between peroxynitrite and the indole moiety.
Results and Discussion
Figure 1. A: Typical preparative RP-HPLC profile of products of
the reaction between peroxynitrite and melatonin at pH ϭ 7.6; the
reaction was carried out in a final volume of 100 mL of phosphate
(0.4 )/NaHCO3 (0.1 ) buffered solution containing melatonin
(2 m) and initiated by addition of peroxynitrite (2 mmol) while
stirring; the mixture was chromatographed 30 min later; B: the time
course of the RP-HPLC analysis of the reaction mixture at pH ϭ
7.6; peroxynitrite (10 m) was allowed to react with melatonin
(1 m) in 1 mL of phosphate buffer and NaHCO3 (60 m); 25-
µL aliquots were injected 1, 5, and 20 min afterwards; RP-HPLC
methods are described in the Exp. Sect.
Results
When synthetic peroxynitrite was injected into a phos-
phate-buffered solution of melatonin at pH ϭ 7.6 and 25
°C, more than 13 products were detected and resolved by
RP-HPLC after decay of the peroxynitrite (half-life of 1 s)
(Figure 1, part A).
A tenfold excess of peroxynitrite was necessary to obtain
80% melatonin transformation at pH ϭ 7.6. This peroxyn-
itrite/melatonin ratio gave only 50% melatonin conversion
at pH ϭ 6.5, but complete conversion either at pH ϭ 8Ϫ9
(even without addition of NaHCO3) or at neutral pH in the
presence of HCO3Ϫ. It is noteworthy that the melatonin was
C-Nitromelatonin
Ϫ
not modified by similar amounts of H2O2, NO2 or end-
products of spontaneous peroxynitrite decomposition at
pH ϭ 7.6.
Two stable nitration products 3a and 3b were identified
by their characteristic absorption bands at 380 and 370 nm,
1
by mass spectrometry, and by H NMR. The major prod-
At pH ϭ 7.6, the transformation products of melatonin
were distributed among several fractions resolved by RP-
HPLC. Analysis of the reaction mixture indicated that some
peaks appeared in the following minutes and decomposed
afterwards. For instance, the peak with tR ϭ 32 min ap-
peared in the first few minutes and then decreased within
20 min, while the peak with tR ϭ 20 min appeared (see B
in Figure 1). The latter reached its maximum after 1 h and
then decayed over the following days. To preserve most of
the products formed, neutral conditions were used in pre-
parative HPLC and each collected fraction was immediately
analyzed by spectrophotometry, analytical HPLC, and mass
uct, 4-nitromelatonin (3a), was more concentrated than 3b,
which had a low yield (below 1%) throughout the pH scale
(see A in Figure 2). Reduction of the pH from 7.6 to 6 pro-
duced a sixfold increase in the yield of 3a, from 1 to 6%.
At pH ϭ 7.6, nitration was favored between 10 and 20 m
NaHCO3 but dramatically decreased above 40 m (see B
in Figure 2).
Kynuramines
Of the oxidized derivatives of melatonin, the most stable
spectrometry. Analysis classified the products into five typ- was formylkynuramine (6a), with close to 7% yield under
ical groups: three oxidized derivatives Ϫ pyrroloindoles, ky- preparative conditions, together with a minor deformylated
nuramines, and indol-2-ones Ϫ and C-nitration and N-sub- product 6b. The UV absorption spectrum shows character-
stitution melatonin derivatives. Their yields, determined by istic bands at 234, 270, and 340 nm (see A in Figure 5). The
the net weights obtained at pH ϭ 7.6 with addition of yields of these kynuramines increased with rising pH and
NaHCO3 (100 m), were 20, 7, 10, 5, and 16%, respectively. decreased upon addition of NaHCO3 (Figure 3).
Eur. J. Org. Chem. 2003, 172Ϫ181
173

F. Peyrot, M.-T. Martin, J. Migault, C. Ducrocq
FULL PAPER
Figure 3. Effects of pH and addition of NaHCO3 on the RP-HPLC
peak areas of 1-nitrosomelatonin (7, closed circles) and formylkyn-
uramine (6a, open triangles) obtained from the reaction between
peroxynitrite and melatonin; effective yields of 3% for 7 and 7%
for 6a were obtained under preparative scale conditions [ONOOϪ/
melatonin (10:1), pH ϭ 7.6, 100 m NaHCO3]; A: the reaction was
carried out at pH ϭ 6Ϫ9 in a final volume of 1 mL of phosphate
(0.4 ) buffer, melatonin (1 m), and peroxynitrite (10 m) without
added NaHCO3; B: the reaction was carried out at pH ϭ 7.6 in a
final volume of 1 mL of phosphate buffer that also contained
NaHCO3 (0Ϫ200 m), melatonin (1 m), and peroxynitrite
(10 m) (see Exp. Sect. for details)
Figure 2. Effects of pH and of addition of NaHCO3 on the RP-
HPLC peak area of 4-nitromelatonin (3a, closed squares) and 6-
nitromelatonin (3b, open squares) obtained from the reaction be-
tween peroxynitrite and melatonin; the reaction was initiated in all
cases by addition of peroxynitrite; the RP-HPLC analysis condi-
tions were as described in the Exp. Sect.; effective yields of 5 and
1% were obtained under preparative scale conditions [ONOOϪ/me-
latonin (10:1), pH ϭ 7.6, 100 m NaHCO3]; A: the reaction was
carried out at pH ϭ 6Ϫ9 in a final volume of 1 mL of phosphate
(0.4 ) buffer that also contained NaHCO3 (60 m), melatonin
(1 m), and peroxynitrite (10 m); B: the reaction was carried out
at pH ϭ 7.6 in a final volume of 1 mL of phosphate buffer that
also contained NaHCO3 (0Ϫ200 m), melatonin (1 m), and per-
oxynitrite (10 m)
and decreasing much more slowly afterwards at pH ϭ 7.6
(t1/2 ϭ 40 h) to yield the final stable products 2a, 2b, and 2c.
Indol-2-ones
Two other types of oxidation products were character-
ized: indol-2-ones 2 and pyrroloindoles 4. In the case of
indol-2-ones, a total 10% yield from melatonin was re-
Pyrroloindoles
Several peaks representing roughly 20% of transforma-
corded. Stable 3-hydroxyindol-2-one 2a, and two classes of tion under preparative conditions (pH ϭ 7.6, 100 m
corresponding dimers, a C-3ϪOϪC-3 ether-bridged dimer NaHCO3) contained mixtures of 3a-hydroxypyrroloindole
2b and two C-3ϪC-3 dimers 2c, were identified by MS, isomers 4a and of 3a-nitro- or more probably 3a-nitritopyr-
NMR, and their characteristic absorption bands were at roloindole 4b (nitrito: ϪOϪNϭO), beside others partially
265 and 300 nm (see A in Figure 5). They derived from a identified by NMR. All showed characteristic absorption
labile common precursor observed as a peak with tR ϭ 27 bands at 235 and 300 nm (see A in Figure 5). Structural
and 20 min in preparative and analytical HPLC, respect- identification of 4a and 4b was based on MS and NMR
ively, which showed a similar absorption spectrum. MS results, especially on the characteristic δ value for the pro-
analyses indicated addition of one oxygen atom to mela- ton on C-8a (δ ϭ 5Ϫ6 ppm) and on the shift in the δ value
tonin. According to HPLC analyses of the reaction mixture for C-3a. The compounds 4a and 4b are certainly formed
(see B in Figure 1), this precursor increased quickly after a as their respective pairs of enantiomers, but the enantiom-
few minutes of reaction, reaching a maximum within 1 h eric ratios could not be established under our conditions.
174
Eur. J. Org. Chem. 2003, 172Ϫ181

Reactivity of Peroxynitrite with Melatonin
FULL PAPER
Pyrroloindoles could not be identified in HPLC analysis of
the reaction mixture. However, a transient peak (tR
ϭ
32 min) with a similar absorption spectrum (A315/A234 ϭ
0.21), but with an additional band at 285 nm (A285/A234 ϭ
0.28), reached a maximum within 5 min (see B in Figure 1).
LC-ESI-MS showed the acetonitrile adduct m/z ϭ 341 ([M
ϩ CH3CN ϩ Na]ϩ) of a nitro or a nitrito derivative of
melatonin and a signal m/z ϭ 294 ([M Ϫ HNO2 ϩ CH3CN
ϩ Na]ϩ) showing that NO2 is a leaving group. Once isolated
from the reaction mixture, it yielded the well-characterized
pyrroloindole 4b.
N-Substitution Products
Of the three detected N-substitution products, 1-hydroxy-
melatonin, which was described previously,[13] was obtained
in very low yield (Ͻ 3%) whatever the conditions. While
we can consider 1-hydroxylation almost negligible, nitration
and nitrosation on the same position were highly dependent
upon experimental conditions and became major trans-
formations of melatonin under specific conditions de-
scribed below.
1-Nitrosomelatonin (7) was described previously.[13,14] Its
structure was confirmed by comparison with authentic 1-
nitrosomelatonin.[13] Its absorption spectrum (see B in Fig-
ure 5) and its HPLC behavior were very similar to those
1
of 1-nitromelatonin (5). H NMR spectroscopy, however,
showed two conformers in solution. Additional support for
the structural assignments was obtained from the FT-IR
spectrum, which exhibited an absorption band at 1435
cmϪ1 [ν(NϪNO)], characteristic of a compound bearing a
nitroso group on the nitrogen atom of an indole nucleus, in
agreement with the literature.[15] The 3% yield of 7 obtained
when the reaction between melatonin and peroxynitrite was
carried out at pH ϭ 7.6 in the presence of NaHCO3
(100 m) increased in the absence of additional CO2 and
with increasing pH (Figure 3). Independently of NaHCO3
addition, yields reached a significant 45% at pH ϭ 9. In the
reaction mixture, 7 decomposes with a half-life of 2 h but
is stable as a solid.[16]
Figure 4. Effects of pH and addition of NaHCO3 on the RP-HPLC
peak areas of indol-2-one precursor 2 (open circles), pyrroloindole
precursor 4 (closed triangles), and 1-nitromelatonin (5, open dia-
monds) obtained from the reaction between peroxynitrite and mel-
atonin (peak areas for 2 and 4 recorded when the peaks reached
their maxima); effective yields of 10% for 2, 20% for 4, and 13%
for 5 were obtained under preparative scale conditions [ONOOϪ/
melatonin (10:1), pH ϭ 7.6, 100 m NaHCO3]; A: the reaction was
carried out at pH ϭ 6Ϫ9 in a final volume of 1 mL of phosphate
(0.4 ) buffer that also contained NaHCO3 (60 m), melatonin
(1 m), and peroxynitrite (10 m); B: the reaction was carried out
at pH ϭ 7.6 in a final volume of 1 mL of phosphate buffer that
also contained NaHCO3 (0Ϫ200 m), melatonin (1 m), and per-
oxynitrite (10 m) (see Exp. Sect. for details)
1-Nitromelatonin (5) was eluted just after 7 in preparat-
ive and analytical HPLC (tR ϭ 68 and 48 min, respectively).
MS indicated that 5 was a nitro derivative of melatonin.
Discussion
1
The H NMR spectrum showed the same δ values as 7 but
only one conformer. The IR spectrum confirmed the posi-
Our results show that peroxynitrite acts upon the indole
tion of the nitro group on the pyrrole nitrogen atom by the moiety simultaneously as an oxidizing, a nitrating, and a
characteristic absorption band at 1287 cmϪ1 [ν(NϪNO2)] nitrosating agent. The melatonin lateral chain is only im-
as reported in the literature.[15] The absorption spectrum of plicated in the cyclization into pyrroloindoles 4. The differ-
7 (230, 275, and 340 nm) was almost identical to that of 5 ent types of reactions varied with respect to the pH and
(see B in Figure 5). In our previous paper[13] there was con- CO2 content of the medium. Three typical oxidation path-
fusion between these two derivatives, which have almost ways of indole compounds (pyrroloindoles 4, indol-2-ones
identical HPLC behavior and absorption spectra. Both de- 2, and kynuramines 6) appeared to be preponderant within
composed in the reaction medium, albeit at different rates the pH range of 7.5Ϫ8 in the presence of CO2. Substitu-
(t1/2 ϭ 7 d for 5, compared to 2 h for 7). The 10% yield tions by nitro or nitroso groups are also important modi-
obtained under preparative conditions was optimum, as ad- fications; both types of nitration either on the benzene car-
dition of NaHCO3 at pH ϭ 7.6 promoted the nitration and bon atoms or on the pyrrole nitrogen atom seem to compete
inhibited nitrosation on the indole nitrogen atom (Fig- with the N-nitrosation reaction. The latter occurs at neutral
ure 4). With a sufficient amount of CO2, 5 was formed in pH in the absence of added bicarbonate, or at alkaline pH
place of 7.
independently of the CO2 content. 1-Nitrosomelatonin (7),
Eur. J. Org. Chem. 2003, 172Ϫ181
175

F. Peyrot, M.-T. Martin, J. Migault, C. Ducrocq
FULL PAPER
Scheme 1. Products obtained from the reaction between peroxynitrite and melatonin in phosphate (0.4 ) buffered solutions; structural
assignments are presented in the Exp. Sect.
·
obtained by use of nitrous acid or NO in the presence of
oxygen at pH ϭ 7.4,[13,14] was also described by us from
the reaction with peroxynitrite.[13] In contrast, the 1-nitro
derivative 5 has never been reported.
Indol-2-ones and Pyrroloindoles
HPLC analyses of the reaction mixture over prolonged
periods (minutes or days) showed that some pyrroloindoles
4 and indol-2-ones 2 are derived from corresponding pre-
cursors that are the main products observed at pH ϭ 7.6 in
the presence of CO2 (see B in Figure 1). Both give substi-
tuted derivatives on C-3 of the indole ring, indicating that
their precursors should have reactive functions on C-2 and/
or on C-3. Hydroperoxide (ϪOOH), epoxide, organic ni-
trite (ϪOϪNϭO), or nitrosoperoxide (ϪOOϪNϭO) could
be postulated. However, the formation of hydroperoxide
should depend on the oxygen reaction with radicals. Such
formation of hydroperoxide appears unlikely, since analysis
of the whole reaction mixture performed either under air
or under argon revealed identical amounts of detected
products (results not shown).
The precursor of 2 corresponds, according to ESI and
APCI mass spectrometry, to the addition of an oxygen
atom to melatonin. Its decomposition is accelerated in the
presence of ferric chloride, giving rise to the three indol-2-
one derivatives 2a, 2b, and 2c, identified by LC-APCI-MS.
Their absorption spectra (260 and 300 nm) are similar to
one another and to their precursor. We postulate that the
precursor of the indol-2-ones 2 is melatonin 2,3-epoxide.
Epoxides have been proposed as short-lived transients in
chemical and biological oxidations of indoles, and it has
been demonstrated that they rearranged quantitatively to
indol-2-ones above 0 °C.[21,22] Furthermore, oxidation of
tryptophan[23] and of other molecules by peroxynitrite
The effects of pH and CO2 allow the reactions to be di-
vided into three classes according to the ONOOϪ-derived
reagent (ONOOH, ONOOϪ, ONOOCO2Ϫ; Scheme 1). As
ONOOCO2 decay generates NO2 and CO3 radicals, it
is likely to induce oxidation in indol-2-ones 2 and pyrrolo-
indoles 4 as well as nitration in various nitromelatonins.
Ϫ
·
·Ϫ
Effect of CO2
Oxidations to indol-2-ones 2 and pyrroloindoles 4 and
C- and N-nitrations are both favored by the presence of
CO2. With regard to nitration, an equimolar amount of
NaHCO3 with respect to initial peroxynitrite is optimal for
aromatic substitutions. Since 25 m NaHCO3 releases
around 1 m CO2 at pH ϭ 7.4, addition of 10 m
NaHCO3 at pH ϭ 7.6 does not allow total conversion into
Ϫ
ONOOCO2 from 10 m peroxynitrite. A small amount is
sufficient to induce the nitroaromatic substitutions. On the
other hand, an excess of NaHCO3 is necessary to obtain
the maximum degree of conversion into 1-nitromelatonin
Ϫ
(5), suggesting that ONOOCO2 is the reagent of N-nitra-
tion.
With regard to the numerous oxidation reactions
achieved by peroxynitrite, CO2 has been reported to acceler-
ate some[2,17Ϫ19] and to inhibit others.[12,20] CO2 concentra-
tion is probably determinant. In the case of melatonin, CO2
promotes its transformation and, more specifically, the ox-
idation routes towards pyrroloindoles and indol-2-ones as has been reported, with increasing yields upon CO2
well as nitrations.
addition.[24]
176
Eur. J. Org. Chem. 2003, 172Ϫ181

Reactivity of Peroxynitrite with Melatonin
FULL PAPER
cursor 4, derived from melatonin. The formation of
Ϫ
ONOOCO2 is optimum at pH ഠ 7.5 in the presence of
NaHCO3 (60 m) [Equation (1)].
Ϫ
ONOOϪ ϩ CO2 Ǟ ONOOCO2
(1)
(2)
(3)
(4)
·Ϫ
ONOOCO2Ϫ Ǟ ·NO2 ϩ CO3
Mel ϩ CO3·Ϫ Ǟ Mel·ϩ ϩ CO3
2Ϫ
Mel·ϩ ϩ ·NO2 Ǟ NO2Mel ϩ Hϩ
1-Nitromelatonin (5) is fairly stable in solution, but its
slow decomposition quantitatively releases nitrite anions
(results obtained by Griess test). However, the stable 4-
nitro- (3a) and 6-nitromelatonin (3b) are formed mostly at
acidic pH (Figure 2, part A) and are favored by a small
amount of CO2 (10 m) at neutral pH (Figure 2, part B).
A similar peroxynitrous acid dependent reaction has been
described for tryptophan,[23] with a 12% yield at pH ϭ 5.
The pyrroloindole precursor is more labile than the pre-
cursor of 2. It decomposes in solution within a few minutes.
Identification of the precursor of pyrroloindoles thus re-
mains difficult, and its structure can only be investigated by
MS and diode array detection coupled to HPLC. Isolated
from the reaction mixture, it gives the 3-nitro- or 3-nitrito-
pyrroloindole 4b. The precursor absorption spectrum shows
a 285-nm band in addition to the 235- and 295-nm bands
characteristic of pyrroloindoles 4, which are lost during the
conversion, suggesting that cyclization occurs in this step.
Thus, 4b should derive from 3-nitro- or, more probably, 3-
nitrito-3H-indole, which is a tautomer:
Kynuramines and 1-Nitrosomelatonin
The oxidation routes towards indol-2-ones 2 and pyrrolo-
indoles 4 decrease in favor of kynuramines 6 with decreas-
ing CO2 content. In a parallel manner, the formation of 1-
nitromelatonin (5) switches to that of 1-nitrosomelatonin
(7). The yields of kynuramines 6 and 1-nitrosomelatonin (7)
are 10 and 20%, respectively, at pH ϭ 7.6. Both increase
strongly with increasing pH [1-nitrosomelatonin (7) reaches
45% at pH ϭ 9]. In the literature, formation of radical inter-
mediates has been demonstrated in the course of the reac-
tion of the indole moiety with peroxynitrite, as well as in
peroxynitrite decay itself.[4,11Ϫ13] The reaction of radicals
with ONOOϪ should be considered.[26] As the natural
amount of CO2 provided by air is sufficient to promote
nitrosation and cleavage to kynuramines by ONOOϪ, initial
oxidation of ONOOϪ to the nitrosating radical ONOO· has
been postulated [Equation (5)]. An excess of CO2 converts
Ϫ
all ONOOϪ into ONOOCO2 [Equation (1)], giving rise to
In the same manner, 3-(hydroperoxy)indole derived from
tryptophan decays to 3-(hydroperoxy)pyrroloindole.[25]
Finally, this accounts for the fact that this NO2 addition
on C-3 occurred under the same conditions as the forma-
tion of 1-nitromelatonin (5) (see below).
the peroxynitrite nitration and oxidation.
ONOOϪ ϩ CO3·Ϫ Ǟ ONOO· ϩ CO3
(5)
2Ϫ
Since ONOO· (A ϭ 410Ϫ420 nm) and melatoninyl rad-
Nitromelatonins
icals (A ϭ 335 and 515 nm) are transiently formed in the
·Ϫ
Nitration of aromatic rings by peroxynitrite is well docu-
mented for phenolic compounds and tyrosine residues, in
which hydroxyl is oxidized in a first step, and the radical
course of the first steps of the reactions between CO3
,
ONOOϪ, and melatonin,[13] the most likely common path-
way is a radical mechanism[27Ϫ29] affording 6 and 1-nitroso-
melatonin (7, Scheme 2). Addition of ONOO· to the mela-
toninyl radical in the same manner as in the formation of
1- and 3-nitro derivatives is proposed, resulting either in
nitrosomelatonin or in kynuramines. Likewise, kynurenines
is coupled with NO2.[2Ϫ5] A similar mechanism could be
·
proposed in the case of the indole ring, giving a melatoninyl
radical cation delocalized on C-3 and on the pyrrole nitro-
gen atom [Equation (3)]. Such a mechanism would be in
agreement with the formation of 1-nitromelatonin (5) and are formed by the reaction of tryptophan with superox-
3-nitro- or 3-nitrito-3H-indole, the postulated unstable pre- ide,[30] which is as mild an oxidant as ONOO·. It is note-
Eur. J. Org. Chem. 2003, 172Ϫ181
177

F. Peyrot, M.-T. Martin, J. Migault, C. Ducrocq
FULL PAPER
dissolved in 5  HCl solution and diluted in the buffer solution at
pH ϭ 7.4 according to the method of Zhang et al.,[12] the trans-
formations of melatonin after peroxynitrite addition were similar,
showing that interactions of peroxynitrite with the solvent are un-
likely.
worthy that these two melatonin derivatives have been
shown to act as oxidant scavengers.[18,31,32]
Peroxynitrite Synthesis: Peroxynitrite synthesis was performed in a
two-phase system with isoamyl nitrite and hydrogen peroxide by
the method of Uppu and Pryor.[39] Peroxynitrite was stored at Ϫ20
°C and its concentration was determined by measurement of the
absorbance at 302 nm (ε ϭ 1670 Ϫ1·cmϪ1) of samples diluted in
NaOH (0.01 ). The contamination of the crude oil containing
peroxynitrite (2.3 ) by H2O2 was estimated as 55 m. Further
treatment with MnO2 was carried out to eliminate residual H2O2,
but no difference in the HPLC profiles of melatonin transforma-
tions was noted for use of untreated or MnO2-treated peroxynitrite
(results not shown).
Reaction between Peroxynitrite and Melatonin: Peroxynitrite solu-
tion in NaOH (0.01 ) was added to buffered solutions of mela-
tonin as a bolus, while stirring at room temperature. A constant
0.2 unit pH rise was recorded. Henceforth only final pH values are
given. For preparative purposes, peroxynitrite (20 m) was added
to a melatonin (2 m, 46.4 mg) solution in phosphate-buffered so-
lutions (0.4 , 100 mL) in the presence of NaHCO3 (100 m). The
final pH was 7.6. After 30 min, the reaction products were separ-
ated by HPLC. Analytical experiments were performed with mela-
tonin (1 m) in phosphate-buffered solution treated with peroxyn-
itrite at an ONOOϪ/melatonin ratio of 10:1 in 1-mL open tubes.
In a first set of experiments, the final pH was varied between 6 and
9, with or without addition of NaHCO3 (60 m). In a second series
Scheme 2. Proposed mechanism for the formation of formylkynur-
amine (6a) and 1-nitrosomelatonin (7)
The oxidized derivatives of melatonin have been already
described as catabolites, or markers of oxidative stress in
vivo. For instance, pyrroloindole 4a has been detected in
the urine of rats and humans and at increased levels after
subjection to ionizing radiation. It has been proposed as a
biomarker of oxidative stress in various diseases. Formylky-
with the pH set at 7.6, NaHCO3 was added up to 200 m. When
Ϫ
nuramine 6a has been described as being formed by dioxy- NaHCO3 was used, the equilibrium between HCO3 and CO2 was
genase in the brain[33] or by reaction with superoxide anion.
allowed to establish itself for 5 min before peroxynitrite addition.
HPLC analyses were performed just after peroxynitrite had reacted
and repeated after a few hours in order to evaluate the products’
stabilities at room temperature. Each experiment was repeated at
least three times independently.
This catabolism is recognized, together with the production
of 6-sulfatomelatonin, as a part of melatonin elimination.
Transformations of melatonin are typical of indoles and
could help understanding of what occurs with free trypto-
phan or tryptophan residues when exposed to peroxynitrite.
Kynurenines and indol-2-ones (indol-2-one, 5-hydroxyin- recorded with a double-beam device (Uvikon 942, Kontron Instru-
UV/Vis Absorption Spectrophotometry: All absorption data were
ments) in 1-cm quartz cells.
dol-2-one, indole-2,3-dione) are metabolites of indole found
in mammalian body fluids and tissues.[34] Some of them
could be produced by peroxynitrite reactions in addition to
6-nitrotryptophan.[25,35] Treatment of proteins with peroxy-
nitrite resulted in an extinction of the fluorescence, indicat-
ing that tryptophan residues are affected. Nitration and/or
Reversed-Phase HPLC (RP-HPLC) Devices: The preparative chro-
matography system consisted of a Waters Delta Prep 300 injector
and a gradient pump (Waters 600E System controller) provided
with a reversed-phase C18 Si, 10 µm, Waters 1000 Prep PAK col-
umn and a Waters Lambda Max Model 480 LC spectrophoto-
oxidation of tryptophan residues by peroxynitrite have been meter. Fractions were collected with an ISCO-FOXY fraction sep-
reported for albumin,[36] collagen,[37] and human plasma
proteins. Recently, a Cu/Zn superoxide dismutase was
arating system. Elution was carried out with a 10Ϫ30% gradient
of acetonitrile for 30 min, followed by a 30Ϫ37% gradient for
15 min and a 37% isocratic mode for 25 min with a flow rate of
shown to be inactivated by peroxynitrite, which modifies a
50 mL/min. The mixture was filtered through a 0.2 µm Acrodisc
specific tryptophan residue.[38] Analysis of the various reac-
tions of peroxynitrite with 3-substituted indole might help
to provide insight into peroxynitrite-dependent reactions
with tryptophan residues in proteins.
filter and injected on the column. Each fraction was analyzed (ana-
lytical RP-HPLC, MS, chemical tests, ...) and concentrated under
reduced pressure and lyophilized (Ϫ50 °C, Ͻ 0.1 mbar). Analytical
HPLC was used to identify products by matching their retention
times with those of authentic compounds when available, with a
Waters 717plus auto sampler fitted with a Waters 600E Multisolv-
Experimental Section
ent gradient pump, and linked to a Hypersil reversed-phase column
(C18 Si, 5 µm, 250 ϫ 4.6 mm) and a Waters 2487 Dual λ ab-
Chemicals and Reagents: Melatonin and isoamyl nitrite were from
Sigma and Acros Organics, respectively. Melatonin was first dis-
solved either in DMSO or in methanol (100 m) before dilution in
a phosphate-buffered (0.4 ) solution. When melatonin was first
sorbance detector. The column was eluted with a 10Ϫ50% gradient
of acetonitrile for 60 min at a flow rate of 1 mL/min. Waters Mil-
lenium32 chromatography manager software was used for data
treatment. 25-µL samples of the reaction mixtures from melatonin
178
Eur. J. Org. Chem. 2003, 172Ϫ181

Reactivity of Peroxynitrite with Melatonin
FULL PAPER
(1 m) solutions were injected. Other analytical systems coupled
the chromatography system described above either with a Waters
996 photodiode array detector or with a mass spectrometer.
19 min in analytical HPLC; m/z ϭ 533 ([M ϩ Na]ϩ) by APCI-MS.
1H NMR (CD3OD, 27 °C): δ ϭ 1.81 [s, 3 H, NHC(O)Me], 2.08
(m, 2 H, 2-H), 3.12 (m, 2 H, 1-H), 3.79 (s, 3 H, ArOMe), 6.79 (m,
1 H, 7Ј-H), 6.81 (m, 1 H, 6Ј-H), 7.00 (m, 1 H, 4Ј-H) ppm; HMBC
correlations give δ values for C-2Ј and C-3Ј 182.5 and 83.3 ppm,
respectively. Two isomer dimers 2c: tR ϭ 23 and 24 min in analyt-
ical HPLC; m/z ϭ 495 ([M ϩ H]ϩ) and 517 ([M ϩ Na]ϩ) by LC-
APCI-MS. 1H NMR (CD3OD, 27 °C): δ ϭ 1.88 [s, 3 H,
NHC(O)Me], 2.05Ϫ2.12 (m, 2 H, 2-H), 3.22Ϫ3.34 (m, 2 H, 1-H),
3.76 (s, 3 H, ArOMe), 6.78 (m, 1 H, 7Ј-H), 6.81 (m, 1 H, 6Ј-H),
6.98 (m, 1 H, 4Ј-H) ppm. HMBC correlations reveal 13C signals at
δ ϭ 22.7 [NHC(O)Me], 31.1 (C-2), 37.6 (C-1), 45.7 (C-3Ј), 56.4
(ArOMe), 111.4 (C-7Ј), 112.5 (C-4Ј), 114.1 (C-6Ј), 132.7 (C-3Јa),
137.0 (C-7Јa), 157.9 (C-5Ј), 173.8 [NHC(O)Me], 182.1 (C-2Ј) ppm.
Mass Spectrometry: All ESI-MS, LC-ESI-MS, and LC-APCI-MS
(Atmospheric Pressure Chemical Ionization) measurements were
performed with a quadrupole mass spectrometer (Navigator Ther-
mofinnigan) operating in positive ionization mode. The analysis
conditions for ESI-MS were: infusion flow (10 µL/min); capillary
and cone voltages (3.5 kV and 20 V respectively); probe T 150 °C;
nebulizing gas nitrogen. LC separations were performed by use of
a P1000XR pump equipped with a UV 2000 absorbance detector
and an AS100XR autosampler (all devices were from Thermo Sep-
aration Products). 100 µL was injected on a Hypersil reversed-
phase column (C18 Si, 5 µm, 250 ϫ 4.6 mm) with absorbance de-
tection at 215 nm; the flow rate (1 mL/min) was split prior to the
mass spectrometer so as to obtain a flow rate of 300 µL/min in the
ion source. The eluents and gradient sequence are described in the
HPLC section. Capillary and cone voltages were set to 3.5 kV and
20 V, respectively. The probe T was 180 °C for ESI and was in-
creased to 300 °C for APCI. Nitrogen was used as nebulizing gas
in both experiments. IC-MS measurements were performed at the
´
Ecole Normale Superieure, Paris. The instrument used was a mag-
netic mass spectrometer (JEOL MS 700) operating in positive
chemical ionization mode with methane as ionizing gas. HR-
MALDI-TOF-MS (High Resolution Ϫ Matrix Assisted Laser De-
sorption Ionization Ϫ Time Of Flight Ϫ Mass Spectrometry) was
used in positive ionization mode with α-cyano-4-hydroxycinnamic
acid (α-CHCA) as matrix and internal reference. The spectrometer
was a Voyager-DE STR device from PerSeptive Biosystems.
NMR Measurements: All experiments (1H, 13C, H-H COSY,
HMQC, HMBC, NOESY) were carried out at 300 K with a
400 MHz AMX Bruker spectrometer. Compounds were dissolved
1
in CD3OD. The H and 13C chemical shifts are expressed in ppm
relative to SiMe3. Compounds have been numbered as indicated
in Scheme 1.
Nitrite Detection by Griess Reaction: In a microplate well, a frac-
tion (50 µL) was mixed with sulfanilamide (5% in 20% HCl solu-
tion, 25 µL) and N-(1-naphthyl)ethylenediamine dihydrochloride
(0.5% in 20% HCl solution, 25 µL). Absorption at 540 nm was
monitored after 20 min incubation. A linear calibration curve was
obtained from NaNO2 solutions (1Ϫ100 µ).
Indol-2-ones 2: The fraction (tR ϭ 27 min) obtained by preparative
HPLC (5 mg, 10% yield) was characterized by a peak eluted at
20 min in analytical HPLC, by m/z ϭ 271 ([M ϩ Na]ϩ) in ESI-MS,
and by an absorption spectrum exhibiting 260- and 300-nm bands
with a ratio of absorbance A300/A260 ϭ 0.17 (Figure 5, part A).
HR-MALDI-TOF-MS: calcd. for C13H17N2O3 249.1239, found
249.1242 [M ϩ H]ϩ. Analysis of the reaction mixture by HPLC
showed that this product is unstable over a period of hours. When
purified or isolated in aqueous solution and incubated in the pres-
ence of ferric chloride, its decomposition gives a mixture of four
stable indol-2-one derivatives, all presenting the same absorptions
at 265 and 315 nm (A315/A265 ϭ 0.2). Monomer N-[2-(3-hydroxy-
Figure 5. A: Absorption spectra of formylkynuramine 6a (solid
line), pyrroloindole mixture 4 (dashed line), and indol-2-one pre-
cursor 2a (dotted line); B: absorption spectra of 1-nitromelatonin
(5, solid line) and 1-nitrosomelatonin (7, dashed line); all spectra
were recorded in methanol
5-methoxy-2-oxo-2,3-dihydro-1H-indol-3-yl)ethyl]acetamide (2a): C-Nitromelatonins 3: 4-Nitromelatonin {N-[2-(5-methoxy-4-nitro-
tR ϭ 11 min in analytical HPLC; m/z ϭ 265 ([M ϩ H]ϩ) and 287 1H-indol-3-yl)ethyl]acetamide (3a), C13H15N3O4, MW ϭ 277} and
([M ϩ Na]ϩ) by LC-APCI-MS. 1H NMR (CD3OD, 27 °C): δ ϭ 6-nitromelatonin {N-[2-(5-methoxy-6-nitro-1H-indol-3-yl)ethyl]-
1.81 [s, 3 H, NHC(O)Me], 2.08 (m, 2 H, 2-H), 3.12 (m, 2 H, 1-H), acetamide (3b)} eluted with tR ϭ 47 min and 43 min in preparative
3
3.79 (s, 3 H, ArOMe), 6.79 (d, JH,H ϭ 8.4 Hz, 1 H, 7Ј-H), 6.85 and 37 min and 32.5 min in analytic HPLC, respectively. Yields ob-
4
(dd, JH,H ϭ 2.5, 8.4 Hz, 1 H, 6Ј-H), 7.00 (d, JH,H ϭ 2.5 Hz, 1 H,
4Ј-H); δ values obtained by HMBC correlations for C-2Ј and C-3Ј
are 182.5 and 77.5 ppm, respectively. Ether-bridged dimer 2b: tR ϭ
tained by preparative HPLC were 4.5% (2.5 mg) and 0.5% (0.3 mg),
respectively. ESI-MS: m/z ϭ 278 [M ϩ H]ϩ, 300 [M ϩ Na]ϩ, 316
[M ϩ K]ϩ and 577 [2M ϩ Na]ϩ. HR-MALDI-TOF-MS: 3a: calcd.
Eur. J. Org. Chem. 2003, 172Ϫ181
179

F. Peyrot, M.-T. Martin, J. Migault, C. Ducrocq
FULL PAPER
for C13H16N3O4 278.1141, found 278.1148 [M ϩ H]ϩ; 3b: calcd. for
δ ϭ 23.2 [NHC(O)Me], 26.5 (C-2), 40.5 (C-1), 57.0 (ArOMe), 104.8
(C-4Ј), 115.9 (C-6Ј), 117.3 (C-7Ј), 121.7 (C-3Ј), 122.6 (C-2Ј), 128.8
C13H16N3O4 278.1141, found 278.1148 [M ϩ H]ϩ. Characteristic
absorption bands were at 280 and 380 nm (A380/A280 ϭ 0.4) for (C-7Јa), 131.9 (C-3Јa), 160.1 (C-5Ј), 174.4 [NHC(O)Me] ppm. IR
3a and at 270 and 370 nm (A370/A270 ϭ 0.12) for 3b. 3a: 1H NMR
absorption at 1287 cmϪ1 agrees with ν(NϪNO2). The absorption
bands are at 230, 275 and 340 nm. Obtained with 10% yield
3
(CD3OD, 27 °C): δ ϭ 1.91 [s, 3 H, NHC(O)Me], 2.72 (t, JH,H
ϭ
3
7.1 Hz, 2 H, 2-H), 3.32 (t, JH,H ϭ 7.1 Hz, 2 H, 1-H), 3.90 (s, 3 H, (6.6 mg) under the preparative conditions from melatonin (2 m),
3
ArOMe), 7.04 (d, JH,H ϭ 8.9 Hz, 1 H, 5Ј-H), 7.24 (s, 1 H, 2Ј-H),
peroxynitrite (20 m), NaHCO3 (100 m) at pH ϭ 7.6, it decom-
poses in the reaction mixture very slowly (t1/2 ϭ 7 d) to give mela-
tonin.
3
7.49 (d, JH,H ϭ 8.9 Hz, 1 H, 6Ј-H). 3b: 1H NMR (CD3OD, 27
3
°C): δ ϭ 1.90 (s, 3 H, NHC(O)Me), 2.93 (t, JH,H ϭ 7.3 Hz, 2 H,
3
2-H), 3.45 (t, JH,H ϭ 7.3 Hz, 2 H, 1-H), 3.96 (s, 3 H, ArOMe),
Kynuramines
oxopropyl}acetamide (6a, C13H16N2O4, MW ϭ 264) is eluted with
R ϭ 32 and 23 min in preparative and analytic HPLC, respectively.
6:
N-{3-[2-(Formylamino)-5-methoxyphenyl]-3-
7.30 (s, 1 H, 4Ј-H), 7.36 (s, 1 H, 2Ј-H), 7.93 (s, 1 H, 7Ј-H) ppm.
HMBC correlations reveal 13C signals at δ ϭ 26.1 (C-2), 41.7 (C-
1), 110.7 (C-7Ј), 114.4 (C-3Ј), 130.2 (C-2Ј), 131.1 (C-7Јa), 133.2 (C-
3Јa), 137.7 (C-6Ј), 148.8 (C-5Ј), 173.7 [NHC(O)Me] ppm.
t
The yield obtained by preparative HPLC was 7% (3.7 mg). It shows
absorption bands at 234, 270 (A270/A234 ϭ 0.4) and 338 nm
(A338/A234 ϭ 0.16) (Figure 5, part A). ESI-MS: m/z ϭ 265 [M ϩ
H]ϩ, 287 [M ϩ Na]ϩ, 551 [2M ϩ Na]ϩ. HR-MALDI-TOF-MS:
calcd. for C13H17N2O4 265.1188, found 265.1181 [M ϩ H]ϩ. 1H
NMR (CD3OD, 27 °C): δ ϭ 1.92 [s, 3 H, NHC(O)Me], 3.32 (t,
3JH,H ϭ 6.3 Hz, 2 H, 2-H), 3.55 (t, 3JH,H ϭ 6.3 Hz, 2 H, 1-H), 3.86
(s, 3 H, ArOMe), 7.19 (dd, JH,H ϭ 3.0, 9.1 Hz, 1 H, 4Ј-H), 7.49 (d,
4JH,H ϭ 3.0 Hz, 1 H, 6Ј-H), 8.35 [s, 1 H, ArNHC(O)H], 8.41 (d,
3JH,H ϭ 9.1 Hz, 1 H, 3Ј-H) ppm. HMBC correlations reveal 13C
signals at δ ϭ 22.6 [NHC(O)Me], 35.6 (C-1), 39.6 (C-2), 56.2 (Ar-
OMe), 116.4 (C-6Ј), 120.6 (C-4Ј), 124.3 (C-3Ј), 125.8 (C-1Ј), 132.9
(C-2Ј), 156.9 (C-5Ј), 162.5 (ArNHCHO), 173.8 [NHC(O)Me], 204.0
(C-3) ppm. N-[4-(2-Amino-5-methoxyphenyl)-3-oxopropyl]acet-
amide (6b): 6b is a minor product coeluting with its derivative 6a.
Pyrroloindoles 4: Monomeric isomers of 1-acetyl-5-methoxy-
2,3,8,8a-tetrahydropyrrolo[2,3-b]indol-3a(1H)-ol (4a, C13H16N2O3,
MW ϭ 248) were collected by preparative HPLC at tR ϭ 20, 25 min
and 3a-nitro- or 3a-nitritopyrroloindole 4b (C13H15N3O4, MW ϭ
277) at 48 min. A total yield of 20% (10 mg) was obtained under
preparative conditions. They each have two characteristic absorp-
tion bands: at 236 and 306 nm (A306/A236 ϭ 0.33) for 4a and at
235 and 315 nm (A315/A235 ϭ 0.3) for 4b (Figure 1). ESI-MS:
m/z ϭ 249 [M ϩ H]ϩ, 271 [M ϩ Na]ϩ, 519 [2 M ϩ Na]ϩ corres-
ponding to 4a. HR-MALDI-TOF-MS: calcd. for C13H16N2O3
1
248.1165, found 248.1164 [M]ϩ. H NMR of 4a (CD3OD, 27 °C):
δ ϭ 2.06/2.18 [s, 3 H, NHC(O)Me], 2.38/2.30 (m, 2 H, 3-H),
3.37Ϫ3.81/3.11Ϫ3.91 (m, 2 H, 2-H), 3.75/3.74 (s, 3 H, ArOMe),
3
1
ESI-MS: m/z ϭ 237 [M ϩ H]ϩ. H NMR (CD3OD, 27 °C): δ ϭ
5.29/5.31 (s, 1 H, 8a-H), 6.60/6.64 (d, JH,H ϭ 8.5 Hz, 1 H, 7-H),
4
1.92 (s, 3 H, [NHC(O)Me], 3.22 (t, 3JH,H ϭ 6.5 Hz, 2 H, 2-H), 3.54
6.75/6.77 (m, 1 H, 6-H), 6.91/6.90 (d, JH,H ϭ 2.5 Hz, 1 H, 4-H)
ppm. HMBC correlations reveal 13C signals at
δ ϭ 22.4
3
(t, JH,H ϭ 6.5 Hz, 2 H, 1-H), 3.83 (s, 3 H, ArOMe), 7.04 (d,
3JH,H ϭ 7.0 Hz, 1 H, 3Ј-H), 7.15 (dd, JH,H ϭ 2.7, 7.0 Hz, 1 H, 4Ј-
[NHC(O)Me], 38.8/38.1 (C-3), 48.5/46.5 (C-2), 56.6 (ArOMe), 84.4
(C-8a), 88.7/90.9 (C-3a), 110.6 (C-4), 113.1 (C-7), 117.5 (C-6), 133.3
(C-3b), 144.7 (C-7b), 156.1 (C-5), 173.3 [NHC(O)Me] ppm. Con-
formational equilibrium NOESY spots for 4a show 2 conformers
due to rotation of the acetyl group on N-1. 4b: Eluted in analytical
HPLC (tR ϭ 39 min), it gives a signal m/z ϭ 300 [M ϩ Na]ϩ and
a fragment m/z ϭ 253 [M Ϫ HNO2 ϩ Na]ϩ by ESI-MS. HR-
MALDI-TOF-MS: calcd. for C13H16N3O4 278.1141, found
3
H), 7.42 (d, JH,H ϭ 2.7 Hz, 1 H, 6Ј-H) ppm. HMBC correlations
reveal 13C signals at δ ϭ 22.8 [NHC(O)Me], 36.7 (C-1), 40.3 (C-2),
56.8 (ArOMe), 116.4 (C-6Ј), 123.3 (C-4Ј), 123.8 (C-3Ј), 124.2 (C-
1Ј), 138.2 (C-2Ј), 156.1 (C-5Ј), 173.5 [NHC(O)Me], 202.3 (C-3)
ppm.
1-Nitrosomelatonin (7): Under our conditions, N-[2-(5-methoxy-1-
nitroso-1H-indol-3-yl)ethyl]acetamide (7, C13H15N3O3, MW ϭ
261) eluted just before 1-nitromelatonin (5), with tR ϭ 62 min and
46 min in preparative and analytic HPLC, respectively. The yield
obtained by preparative HPLC was 2.5% (1.3 mg). Structural as-
signments are by comparison with the product synthesized by the
Bravo method.[40] LC-ESI-MS gives the molecular mass m/z ϭ 284
[M ϩ Na]ϩ and shows the radical m/z ϭ 254 [M Ϫ NO ϩ Na]ϩ
due to homolytic rupture of the NϪNO bond. An equilibrium be-
tween two conformers due to rotation of the nitroso group is evid-
enced by conformational equilibrium NOESY spots. HMBC cor-
relations reveal 13C signals at δ ϭ 23.2 [NHC(O)Me], 26.2 (C-2),
40.0/40.2 (C-1), 56.6 (ArOMe), 105.1/104.4 (C-4Ј), 115.9/115.1 (C-
6Ј), 113.4/118.3 (C-7Ј), 126.5/124.7 (C-3Ј), 114.2/127.1 (C-2Ј),
131.8/125.5 (C-7Јa), 132.9/133.2 (C-3Јa), 160.5/160.9 (C-5Ј), 174.3
[NHC(O)Me] ppm. The UV absorption spectrum of 7 is very sim-
ilar to that of 5, showing a characteristic band at 243 nm besides
279 and 343 nm (Figure 5, part B). IR absorption at 1435 cmϪ1
corresponds to ν(NϪNO).
1
278.1136 [M ϩ H]ϩ. H NMR (CD3OD, 27 °C): δ ϭ 2.07/2.21 [s,
3 H, NHC(O)Me], 2.82Ϫ3.02/2.78Ϫ2.89 (m, 2 H, 3-H), 3.35Ϫ3.96/
3.00Ϫ4.12 (m, 2 H, 2-H), 3.75/3.76 (s, 3 H, ArOMe), 6.17/6.22 (s,
3
1 H, 8a-H), 6.68/6.75 (d, JH,H ϭ 8.7 Hz, 1 H, 7-H), 6.89/6.93 (m,
4
1 H, 6-H), 7.04/7.03 (d, JH,H ϭ 2.7 Hz, 1 H, 4-H) ppm. HMBC
correlations reveal 13C signals at δ ϭ 23.0 [NHC(O)Me], 38.0/36.6
(C-3), 49.0/46.5 (C-2), 57.4 (ArOMe), 81.2/82.2 (C-8a), 102.9/104.7
(C-3a), 111.8/111.0 (C-4), 114.0/115.2 (C-7), 120.9 (C-6), 126.4/
128.1 (C-3b), 147.7 (C-7b), 156.6/157.3 (C-5), 173.7 [NHC(O)Me]
ppm. Conformational equilibrium NOESY spots show two con-
formers due to rotation of the acetyl group on N-1.
1-Nitromelatonin (5): N-[2-(5-Methoxy-1-nitro-1H-indol-3-yl)ethyl]-
acetamide (5, C13H15N3O4, MW ϭ 277) is eluted in preparative
and analytical HPLC (tR ϭ 64 and 47 min, respectively). IC-MS:
m/z ϭ 278 [M ϩ H]ϩ, ESI-MS confirms the molecular mass and
shows the radicals m/z ϭ 232 [M Ϫ NO2 ϩ H]ϩ and 254 [M Ϫ
NO2 ϩ Na]ϩ due to homolytic rupture of the NϪNO2 bond. HR-
MALDI-TOF-MS: calcd. for C13H16N3O4 278.1141, found
1
1
278.1138 [M ϩ H]ϩ. H NMR shows the same δ values as 7: H
1-Hydroxymelatonin (8): This is a minor product, described previ-
NMR (CD3OD, 27 °C): δ ϭ 1.92 [s, 3 H, NHC(O)Me], 2.89 (t, ously.[13] Eluted with tR ϭ 52 and 43 min in preparative and analyt-
3JH,H ϭ 7.0 Hz, 2 H, 2-H), 3.50 (t, 3JH,H ϭ 7.0 Hz, 2 H, 1-H), 3.91
ical HPLC, respectively, it has characteristic absorption bands at
230, 280 and 310 nm. As its formation diminishes with decreasing
CO2 content and does not vary with pH, the yield (Ͻ 3%) remains
very low whatever the conditions used.
(s, 3 H, ArOMe), 7.03 (dd, JH,H ϭ 2.4, 9.0 Hz, 1 H, 6Ј-H), 7.18 (d,
3
4JH,H ϭ 2.4 Hz, 1 H, 4Ј-H), 7.85 (s, 1 H, 2Ј-H), 8.05 (d, JH,H
ϭ
9.0 Hz, 1 H, 7-H) ppm. HMBC correlations reveal 13C signals at
180
Eur. J. Org. Chem. 2003, 172Ϫ181

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