Oxidation of N-4-chlorophenyl-N′-hydroxyguanidine to N-(4-chlorophenyl)urea and nitric oxide by photoexcited iron porphyrins

Article abstract of DOI:10.1002/ejic.200400056

Photochemical excitation (λ > 350 nm) of chloro[meso-tetrakis(2, 6-dichlorophenyl)porphyrin]iron(III) [Fe^III(TDCPP)Cl] and chloro[meso-tetra(α,α,α,α-pivalamidophenyl)porphyrin] -iron(III) [Fe^III(TpivPP)Cl] induces the oxidation of coordinated N-(4-chlorophenyl)-N′-hydroxyguanidine (1) by molecular oxygen, to give iminoxyl radicals and the Fe^II(O₂) [or Fe ^III(O₂^.)] adduct. This complex can be accumulated in significant amounts using [Fe^III(TpivPP)Cl]. The primary photoproducts give rise to secondary reactions that lead to the formation of N-4-chlorophenyl)urea (2) as the main end-product of 1. The conversion of 1 into 2 is accompanied by the formation of NO, as revealed both by an ESR spin-trapping technique and in the form of its stable end-products NO₂^- and NO₃^-. The presence of 1-methyl imidazole (1-MeIm) coordinated in axial position has a significant positive effect on the photoinduced production of 2 and NO from the hydroxyguanidine 1. The observation that radical scavengers inhibit the photooxidation process strongly supports the possibility that the reaction pathway resembles a radical-type autoxidation mechanism, where the very fast reaction of O₂ with the ferrous porphyrin in the presence of the photo-generated iminoxyl radical should yield an iron-peroxo intermediate as precursor of the urea 2 and NO. Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004.

Full text of DOI:10.1002/ejic.200400056

FULL PAPER
Oxidation of N-(4-Chlorophenyl)-NЈ-hydroxyguanidine to
N-(4-Chlorophenyl)urea and Nitric Oxide by Photoexcited Iron Porphyrins
[
a]
[a]
[a]
[a]
Andrea Maldotti,* Alessandra Molinari, Irene Vitali, Elena Ganzaroli,
[b]
[b]
[b]
Pierrette Battioni, Delphine Mathieu, and Daniel Mansuy
Keywords: OϪO activation / Nitric oxide / Photooxidation / Porphyrins / Protein models
−
and NO
3
−. The presence of 1-methyl imidazole (1-
Photochemical excitation (λ Ͼ 350 nm) of chloro[meso-tetra-
NO
2
III
kis(2,6-dichlorophenyl)porphyrin]iron(III
)
[Fe (TDCPP)Cl]
MeIm) coordinated in axial position has a significant positive
effect on the photoinduced production of 2 and NO from the
hydroxyguanidine 1. The observation that radical scavengers
inhibit the photooxidation process strongly supports the
possibility that the reaction pathway resembles a radical-
type autoxidation mechanism, where the very fast reaction
and chloro[meso-tetra(α,α,α,α-pivalamidophenyl)porphyrin]-
III
iron(III) [Fe (TpivPP)Cl] induces the oxidation of coordinated
N-(4-chlorophenyl)-NЈ-hydroxyguanidine (1) by molecular
II
oxygen, to give iminoxyl radicals and the Fe (O
2
) [or
III
·
Fe (O
2
)] adduct. This complex can be accumulated in signi-
ficant amounts using [Fe (TpivPP)Cl]. The primary of O
III
2
with the ferrous porphyrin in the presence of the photo-
photoproducts give rise to secondary reactions that lead to
the formation of N-(4-chlorophenyl)urea (2) as the main end-
product of 1. The conversion of 1 into 2 is accompanied by
the formation of NO, as revealed both by an ESR spin-trap-
ping technique and in the form of its stable end-products
generated iminoxyl radical should yield an iron-peroxo inter-
mediate as precursor of the urea 2 and NO.
( Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2004)
II
Introduction
tremely fast subsequent reaction of O with Fe (P) in the
2
·
presence of R may lead to the formation of alkyl-peroxo
The photochemical excitation of iron porphyrin com- complexes according to Equation (3). In all cases, the
[
2]
III
plexes [Fe (P)] is an important tool for obtaining biomi- photoassisted activation of O
metic model systems of interest in oxygenase-like catalytic since the starting iron porphyrin is restored after its reoxi-
occurs in a catalytic manner,
2
[
1]
oxidation. All these systems are characterized by common dation and after coordination of a new equivalent of axial
primary photoprocesses. Irradiation in axial ligand (L) to ligand.
metal charge-transfer bands (λ ϭ 300Ϫ400 nm) induces a
III
homolytic cleavage of the Fe (L) bond, which causes oxi-
·
dation and detachment of L as a radical species and re-
duction of Fe to Fe [Equation (1)]. It has been demon-
(
(
1)
2)
III
II
strated that O is able to effectively react with the ferrous
2
intermediate, thus inhibiting solvent-cage effects [back reac-
tion in Equation (1)]. A peculiar aspect of this process is
that a monoelectronic reductive activation of O2 occurs
II
through the formation of a dioxyiron complex Fe (P)(O )
2
III
·
or Fe (P)(O ) according to Equation (2). A two-electron
2
reduction of O may also be achieved when the described
2
photochemical process is carried out in the presence of suit-
able hydrocarbons (RH). Under these conditions, alkyl rad-
·
(
3)
icals (R ) may be formed as a consequence of the reaction
·
between the photogenerated L radicals and RH. The ex-
[
[
a]
b]
Dipartimento di Chimica Universit a` di Ferrara, Sezione
ISOFϪCNR,
Chloro[meso-tetrakis(2,6-dichlorophenyl)porphyrin]-
iron() [Fe (TDCPP)Cl] has been often employed in the
Via Luigi Borsari 46, 44100 Ferrara, Italy
III
Laboratoire de Chimie et Biochimie Pharmacologiques
Toxicologiques, UMR 8601 CNRS, Universit e´ Paris V,
photocatalytic activation of O as the bulky chlorine atoms
2
4
5 rue des Saints P e` res, 75270 Paris Cedex 06, France
in its meso-aryl groups prevent the well-known bimolecular
Eur. J. Inorg. Chem. 2004, 3127Ϫ3135
DOI: 10.1002/ejic.200400056
 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
3127

A. Maldotti et al.
FULL PAPER
II
reaction of the Fe dioxy complex to give µ-oxo dimers as redox function in the second step of NO synthesis. Stuehr
[
3]
[9]
the final product. Moreover, they provide a steric protec- et al. have proposed that the cofactor BH may be able to
4
III
·
tion of the porphyrin ring against its radical-induced oxi- provide a second electron to Fe (P)(O ) to yield an iron-
dative degradation.
2
[
4]
peroxo intermediate as ultimate oxidant able to react with
One intriguing and up-to-date aspect of the photocata- bound N-hydroxy--arginine according to Scheme 1 (steps a
ϩ·
lytic processes described above is their possibility to gener- and b). The subsequent back electron transfer to the BH4
III
·
III
ate reactive intermediates such as Fe (P)(O ), Fe (P)- radical should yield the final products NO and -citrulline
OOR) and radical species that might be relevant also to together with BH and Fe (P) in their starting conditions
2
III
(
4
the mechanism of nitric oxide synthases (NOSs), as ex- (step c).
plained below.
The formation of radical intermediates has been pro-
All the three NOS isoforms in mammals catalyse the posed by Groves et al.[5d,10] for model systems that mimic
stepwise oxidation of -arginine to nitric oxide (NO) by O
many important properties of the NOS-catalysed oxidation
2
[
5]
and NADPH. This process occurs in the NOS oxygenase of N-hydroxy--arginine. This mechanism, summarized in
domain, which contains heme and tetrahydrobiopterin Scheme 2, involves the oxidation of N-hydroxy--arginine
[6]
(
usually indicated as BH ) as cofactors. The NO synthesis to the corresponding iminoxyl radical with the simul-
4
III
II
mechanism is complex and not completely understood. A taneous reduction of Fe (P) to Fe (P) (step a). The inser-
first step is likely a classical monooxygenation of -arginine tion of O between the ferrous porphyrin and the radical in
2
to give N-hydroxy--arginine, which occurs with the con- its α-nitroso form would generate an iron-peroxo intermedi-
sumption of one mol of O and one mol of NADPH [first ate (steps b and c). The subsequent homolytic cleavage of
2
step in Equation (4)]. The generally accepted P450 reaction its OϪO bond may lead to the formation of NO, -citrul-
IV
mechanism can account for the stoichiometry and the prod- line and Fe (P)(O) (step d), which can immediately un-
[
7]
uct formation. Reduction of ferric heme is followed by dergo a single-electron reduction with, possibly, the involve-
II
the formation of a Fe dioxy complex that accepts a second ment of BH (step e).
4
electron from NADPH to form an iron-peroxo intermedi-
ate. The subsequent OϪO bond scission yields a presumed
iron-oxo species that is able to hydroxylate a guanidinic
nitrogen of -arginine.
The very limited number of substrates for NOSs known
so far suggests that highly specific structural features are
required for NO generation by these enzymes. However, it
has been recently shown that NOSs are also able to catalyse
the oxidation of some analogues of N-hydroxy--argi-
[
11]
nine. In particular, it has been found that mixtures of N-
arylurea, N-arylcyanamide and NO are formed when sev-
eral N-aryl-NЈ-hydroxyguanidines are incubated in the pres-
ence of NOS. These substrates are of particular interest in
mechanistic studies with model systems since, contrary to
N-hydroxy--arginine, their oxidation products are rela-
tively stable and easy to analyse.
(
4)
In this work, we have used as oxidizable substrate a sim-
ple N-aryl-NЈ-hydroxyguanidine: N-(4-chlorophenyl)-NЈ-
The second step involves an oxidative cleavage of the Cϭ hydroxyguanidine (1, see Scheme 3). We have investigated
NOH bond of N-hydroxy--arginine to give the final prod- its redox reactivity in the presence of photoexcited iron por-
ucts NO and -citrulline through the consumption of one phyrins with the aim of inducing the reductive activation
mol of O and only one electron coming from 0.5 mol of of O in order to obtain intermediates analogous to those
2
2
NADPH [second step in Equation (4)].[8] A monoelectronic involved in the NOS-dependent oxidation of N-hydroxyar-
reduction of ferric heme enables O binding and forms an ginine. The possibility that the photoinduced redox pro-
2
II
III
·
iron-dioxy complex [Fe (P)(O ) or Fe (P)(O )]. A number cesses may be affected by the presence of N-methyl imidaz-
2
2
of experimental results are consistent with BH having a ole (1-MeIm) has also been evaluated.
4
Scheme 1. Proposed mechanism for NOS-assisted oxidation of N-hydroxy--arginine
3128
 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Eur. J. Inorg. Chem. 2004, 3127Ϫ3135

Oxidation of N-(4-Chlorophenyl)-N-hydroxyguanidine by Photoexcited Iron Porphyrins
FULL PAPER
Scheme 2. Proposed radical-type mechanism
Results and Discussion
Biomimetic Photooxidation of N-(4-Chlorophenyl)-NЈ-
hydroxyguanidine (1)
The photochemical experiments described below were
III
carried out by irradiating CH CN solutions of [Fe -
3
Scheme 3. N-(4-Chlorophenyl)-NЈ-hydroxyguanidine (1), N-(4-
chlorophenyl)urea (2) and N-(4-chlorophenyl)cyanamide (3)
Ϫ5
Ϫ3
(
TDCPP)Cl] (1 ϫ 10 ) and 1 (1 ϫ 10 ) in the pres-
ence of 1-MeIm, (5 ϫ 10 ) [Fe (TDCPP)Cl/1/1-MeIm
system] or without 1-MeIm [Fe (TDCPP)Cl/1 system]
Ϫ5
III
III
with light of wavelength higher than 350 nm. First of all,
we report some UV/Vis results shedding light on the nature
of the main light-absorbing species present in these systems.
More specifically, we were interested in defining the nature
of the iron porphyrin axial ligand that may be involved in
the primary photochemical process in Equation (1).
The iron porphyrins employed are shown in Figure 1.
Fe (TDCPP)Cl] has been chosen because of its stability
III
[
under photocatalytic conditions. The chloro[meso-
III
tetra(α,α,α,α-pivalamidophenyl)porphyrin]iron()
[Fe -
(
TpivPP)Cl] is expected to give important information
II
about the possible formation and reactivity of Fe (P)(O )
2
It has been reported previously that numerous N-
hydroxyguanidines such as 1 are able to bind through their
oxygen atom to the ferric heme group of both microperoxi-
adducts as it has a protective enclosure for binding oxygen
on one side of the porphyrin ring and it is able to form a
stable adduct with O2.[
12]
[
13]
dase-8 and NOS itself.
The consequent formation of
stable, low-spin complexes is accompanied by significant
changes in the UV/Vis spectra. In particular, the coordi-
nation of N-hydroxyguanidines causes red shifts of the So-
ret peak. Table 1 (runs a and b) provides evidence that the
III
addition of 1 to a [Fe (TDCPP)Cl] solution induces an
analogous shift of the Soret band from 412 nm to 422 nm,
thus indicating that 1 is coordinated in the axial position to
III
the metal centre to give the complex [Fe (TDCPP)(1)Cl].
Table 1 (run c) and Figure 2 show that different spectral
variations were obtained when both 1 and 1-MeIm were
III
added to [Fe (TDCPP)Cl]. This result, together with the
observation that addition of 1-MeIm alone causes other
spectral changes (run d) allows us to infer that solution
III
equilibria in the [Fe (TDCPP)Cl]/1/1-MeIm system lead
mainly to the formation of a porphyrin complex that con-
tains hydroxyguanidine and 1-MeIm bound in the two axial
positions (step a in Scheme 4).
Figure 1. Structure of the investigated iron porphyrins
www.eurjic.org
Eur. J. Inorg. Chem. 2004, 3127Ϫ3135
 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
3129

A. Maldotti et al.
FULL PAPER
Table 1. UV/Vis spectroscopic data of [FeIII(TDCPP)Cl]
III
The [Fe (TDCPP)Cl]/1/1-MeIm system was irradiated
for 30 minutes in the presence of 760 torr of O . HPLC
Absorption maxima (nm) analyses revealed the formation of both N-(4-chloro-
2
Run System[a]
phenyl)urea (2) and N-(4-chlorophenyl)cyanamide (3;
III
a
b
c
d
e
Fe (TDCPP)Cl
325, 412, 570
Scheme 3), by comparison of their retention times with
422, 541
III
Fe (TDCPP)Cl/1
III
those of authentic samples, and by co-injection with the
426, 535
Fe (TDCPP)Cl/1/1-MeIm
III
Fe (TDCPP)Cl/1-MeIm
327, 413, 569
authentic compounds.
III
Fe (TDCPP)Cl/1/1-MeIm/hν 416, 505, 546
Table 2 reports the yields of the described photoprocess
in terms of amount of detected products. The first row of
this table provides evidence that the urea 2 is the main end-
product of the hydroxyguanidine 1. In agreement with an
NOS-like oxidation mechanism, the conversion of 1 to 2
approximately matches the formation of NO, which was re-
[
a]
FeIII(TDCPP)Cl] (CH CN): 1 ϫ 10Ϫ5 ; [1]: 1 ϫ 10Ϫ3 ; [1-
3
MeIm]: 5 ϫ 10 . Irradiation in run e was carried out at 22 Ϯ
°C in the presence of 760 Torr of oxygen; excitation wavelength:
λ Ͼ 350 nm; irradiation time: 30 min.
[
Ϫ5
1
Ϫ
vealed in the form of its stable end-products NO2 and
Ϫ
NO3 . Table 2 shows that the cyanamide 3 is a second
minor photoproduct. Literature data show that the oxi-
dation of the hydroxyguanidine 1 to the corresponding cy-
Ϫ
anamide is accompanied by the formation of NO instead
[
14]
of NO.
This intermediate, after dimerization, pro-
tonation and dehydration would release gaseous N O with-
2
Ϫ
Ϫ
out the formation of NO2 and NO3
.
Control experiments indicated that the formation of 2, 3,
Ϫ
Ϫ
NO2 and NO3 from 1 did not occur in appreciable
III
amounts in the dark or in the absence of either [Fe -
(
TDCPP)Cl] or O . We also verified that the photoexciting
2
wavelengths employed here did not cause any appreciable
degradation of 2 and 3. Finally, we found UV/Vis evidence
that neither 2 nor 3 competes with 1 in binding the metal
centre at the concentrations that they typically reach during
the photochemical experiment.
[Fe (TDCPP)Cl] underwent significant spectral changes
when irradiated in the presence of 1 and 1-MeIm. As shown
Figure 2. A) UV/Vis spectrum of a CH
3
CN solution of [FeIII
CN solu-
-
Ϫ5
III
(
TDCPP)Cl] (1 ϫ 10 ); B) UV/Vis spectrum of a CH
3
III
Ϫ5
tion of [Fe (TDCPP)Cl] (1 ϫ 10 ) in the presence of 1 (1 ϫ
Ϫ3
Ϫ5
10
), and 1-MeIm (5 ϫ 10 )
in Table 1 (compare runs c and e), we observed a blue shift
Scheme 4. Proposed photooxidation mechanism
130
3
 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Eur. J. Inorg. Chem. 2004, 3127Ϫ3135

Oxidation of N-(4-Chlorophenyl)-N-hydroxyguanidine by Photoexcited Iron Porphyrins
Table 2. Oxidation of 1 by photoexcited [FeIII(TDCPP)Cl] and by the [FeIII(TpivPP)(O ·
FULL PAPER
2
)(1-MeIm)] adduct
System
Products[a] (µ)
Urea 2
Ϫ
Ϫ
Cyanamide 3
NO
2
NO
3
III
[b]
Fe (TDCPP)Cl/1/1-MeIm
6.6 Ϯ 0.6
1.6 Ϯ 0.1
3.5 Ϯ 0.3
1 Ϯ 0.1
1.5 Ϯ 0.1
1.5 Ϯ 0.1
1.5 Ϯ 0.1
Ͻ0.2
2.2 Ϯ 0.2
Ͻ0.6
1.2 Ϯ 0.2
Ͻ0.6
2.7 Ϯ 0.2
Ͻ0.6
1.3 Ϯ 0.2
Ͻ0.6
III
[b]
Fe (TDCPP)Cl/1
III
[b]
Fe (TDCPP)Cl/1/1-MeIm/PBN
III
[c]
2
Fe (TpivPP)(O ·)/1/1-MeIm
[
[
a]
b]
Results are expressed in µmol dmϪ3 of products formed and are averages (Ϯ standard deviation) of between three and five experiments.
Irradiations were carried out at 22 Ϯ 1 °C in the presence of 760 Torr of oxygen. Excitation wavelength: λ Ͼ 350 nm. Irradiation time:
III
Ϫ5
Ϫ3
Ϫ5
Ϫ4
[c]
III
·
3
2
0 minutes. [Fe (P)]: 1 ϫ 10 ; [1]: 1 ϫ 10 ; [1-MeIm]: 5 ϫ 10 ; [PBN]: 1 ϫ 10 . The [Fe (TpivPP)(O )]/1-MeIm adduct
III Ϫ5
was obtained by dissolving [Fe (TpivPP)Cl] (1 ϫ 10 ) in a mixture of acetone and ethanol (95:5, v/v) in the presence of 1-MeIm (5
ϫ 10 ). The solution was degassed (less than 1 ϫ 10 Torr), irradiated (90 s) and put in contact with O at low temperature (Ϫ5 to
2
Ϫ8 °C). Then, 1 (1 ϫ 10 ) was added to the obtained sample.
Ϫ5
Ϫ5
Ϫ3
of the porphyrin Soret band from 426 nm to 416 nm, as this statement, blank experiments carried out without 1 did
well as the formation of two new bands at 505 and 546 nm. not give any ESR signal.
These spectral variations may be ascribed to axial coordi-
Ϫ
nation of the end-product NO2 anion on the basis of lit-
erature data concerning the effect of binding of this anion
on the spectroscopic properties of iron porphyrin com-
[
15]
plexes
and by the observation that progressive addition
III
of NaNO to the [Fe (TDCPP)]/1/1-MeIm system induced
2
analogous spectral variations.
The data reported in the second line of Table 2 show that
the photoinduced oxidation of 1 occurred to some extent
also in the absence of 1-MeIm. On the other hand, a signifi-
cant reduction in the production of 2 and NO was observed
in this case, indicating the main role of coordinated 1-MeIm
in the photoinduced formation of these products.
Spin-Trapping Investigation and Proposed Reaction
Pathways
Figure 3. ESR spin-trapping spectrum obtained during irradiation
III
Ϫ5
3
of a CH CN solution of [Fe (TDCPP)Cl] (1 ϫ 10 ) in the
Ϫ3 Ϫ5
Evidence for the oxidation of bound N-hydroxyguanidine
presence of 1 (1 ϫ 10 ), 1-MeIm (5 ϫ 10 ) and PBN (5 ϫ
(1) to radical species was provided by an ESR spin-trapping
Ϫ3
10
)
investigation. This technique is a powerful tool for detecting
the formation of short-lived radicals[ and has been fruit-
fully employed in photochemical studies on iron porphy-
16]
Ϫ
Row 3 in Table 2 reports the concentration of 2, 3, NO
2
[
2a,4]
Ϫ
III
rins.
It is based on the ability of some molecules, such and NO
as α-phenyl-N-tert-butylnitrone (PBN), to trap radicals (R ) (TDCPP)Cl]/1/1-MeIm system carried out in the presence
3
after 30 minutes irradiation of the [Fe -
·
Ϫ4
and give paramagnetic nitroxides stable enough to be stud- of PBN (1 ϫ 10 ). The radical scavenger did not affect
ied by ESR spectroscopy [Equation (5)].
at all the formation of the cyanamide 3, while it partially
Ϫ
Ϫ
inhibited the production of the urea 2, NO2 and NO3
.
The above results are consistent with a reaction pathway
for the light-induced oxidation of the hydroxyguanidine 1
III
by [Fe (TDCPP)Cl] that resembles the radical-type autox-
(
5)
[5d,10]
idation mechanism mentioned in the introduction.
In
line with the general mechanism shown in Equations
1)Ϫ(3), peroxidic intermediates should be formed as a
(
III
The [Fe (TDCPP)Cl]/1/1-MeIm system was irradiated consequence of the very fast reaction of O with the photo-
2
Ϫ3
in the presence of PBN (5 ϫ 10 ) directly in the cavity generated ferrous porphyrin in the presence of iminoxyl
of an ESR spectrometer. Figure 3 shows that the spectrum radicals (steps c and d in Scheme 4). The formation of the
obtained consists of a triplet of doublets, with hyperfine urea 2 and NO likely occurs as a consequence of a homo-
splitting constants aN ϭ 14.75 G and aH ϭ 2.77 G. This lytic cleavage of the OϪO bond according to steps e and f.
signal may be ascribed to the trapping of a radical species The possibility that photogenerated alkyl hydroperoxides
originating from the photooxidation of the bound hydroxy- may decompose by the above reactions has been reported
[
1c,2]
guanidine 1 according to step b in Scheme 4. In line with previously.
It seems likely that the well-known ability of
Eur. J. Inorg. Chem. 2004, 3127Ϫ3135
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 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
3131

A. Maldotti et al.
FULL PAPER
bound 1-MeIm to stabilize this photogenerated ferrous-di-
The ESR spin-trapping technique was also used to obtain
III
· [12b]
oxy intermediate Fe (O )
may be the reason for the direct evidence for the formation of NO. We employed 2-
2
observed positive effect of this nitrogen base on the final phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO)
[
18]
product yields.
as an NO-specific reactant. The ESR spectrum of PTIO
An additional possibility to take into account is that oxi- consists of a 1:2:3:2:1 quintet due to the interaction of one
dation of 1 may occur as a consequence of its reaction with unpaired electron with two equivalent nitrogen atoms (a ϭ
N
O2· , which could originate from Fe (O ) dissociation 8.2 G). Nitric oxide reacts with this compound according
Ϫ
III
·
2
(
steps g and h). Indeed, it has been already demonstrated to Equation (6) to give an imino-nitroxide (PTI), in which
·
Ϫ
that O2 can react with 1 to give the cyanamide 3 as the two different nitrogen atoms generate a different and more
[
17]
The low yield of this product indicates complex spectrum with nine lines (aN1 ϭ 9.8 G and aN2
ϭ
major product.
that this possibility is only a minor process in the photoind- 4.4 G).
uced oxidation of 1 described here. Curve A in Figure 4 shows the five-line ESR spectrum of
Ϫ6
Ϫ3
PTIO (2 ϫ 10 ) in the presence of 1 (1 ϫ 10 ),
III
Ϫ5
Ϫ5
[
Fe (TDCPP)Cl] (1 ϫ 10 ) and 1-MeIm (5 ϫ 10 ).
III
The same signals were obtained when 1 or [Fe -
6) (TDCPP)Cl] or 1-MeIm were omitted from the reaction
mixture. Ten-minute photoexcitation led to a decrease of
the intensity of this spectrum together with the growth of
new signals, as shown by Curve B in Figure 4. The position
of these signals corresponds very well to that expected for
(
[
17]
the nitroxide PTI.
Negligible spectral changes were ob-
served in the dark when the photochemical experiment was
III
carried out in the absence of [Fe (TDCPP)Cl] or 1.
II
Reactivity of Photogenerated [Fe (TpivPP)(O )(1-MeIm)]
2
III
Irradiation of [Fe (TpivPP)Cl] in the presence of 1, 1-
MeIm and O gave results very similar to those obtained
2
III
with the [Fe (TDCPP)Cl]/1/1-MeIm system from a quali-
tative point of view. However, experiments carried out with
this porphyrin allowed us to accumulate only traces of the
end-products, because it undergoes a very fast degradation
under aerobic photochemical conditions to give small
colourless fragments. This instability may be ascribed to the
lack of hindered groups able to protect the meso positions
from oxidative attacks.
In spite of this limitation, which forced us to limit the
photocatalytic characterization previously described to
III
III
[
Fe (TDCPP)Cl], with [Fe (TpivPP)Cl] we could obtain
direct experimental evidence of the photoinduced formation
II
of a [Fe (TpivPP)(O )(1-MeIm)] adduct, which, in the pre-
2
III
vious discussion, has only been supposed for [Fe -
(
TDCPP)Cl].
II
The first step for obtaining the [Fe (TpivPP)(O )(1-
2
MeIm)] adduct was the photoinduced reduction of the fer-
ric porphyrin under anaerobic conditions. This result was
obtained by irradiating (for 90 s, λ Ͼ 350 nm) deoxygenated
III
Ϫ5
solutions of [Fe (TpivPP)Cl] (1 ϫ 10 ) containing 1-
Ϫ5
MeIm (5 ϫ 10 ). We decided to use an acetone/ethanol
(95:5) mixture as solvent knowing that, in these conditions,
ethanol works as coordinating solvent and that the primary
photochemical process is expected to be the reduction of
III
II
Fe to Fe by the bound ethanolate, in line with the gen-
[
2,4]
eral mechanism shown in Equation (1).
Accordingly, the
UV/Vis spectrum observed after irradiation (Curve A in
Figure 5) presents the typical absorption at 567 nm of the
ferrous porphyrin. The spectral changes (Curve B) that ac-
company the subsequent oxygenation of the sample at low
Ϫ6
Figure 4. A) ESR spectrum of PTIO (2 ϫ 10 ) in a CH CN
Ϫ5
3
Ϫ3
III
solution containing 1 (1 ϫ 10 ), [Fe (TDCPP)Cl] (1 ϫ 10 )
Ϫ5
and 1-MeIm (5 ϫ 10 ); B) curve A after ten minutes irradiation temperature (Ϫ5 to Ϫ8 °C) are consistent with the forma-
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Eur. J. Inorg. Chem. 2004, 3127Ϫ3135

Oxidation of N-(4-Chlorophenyl)-N-hydroxyguanidine by Photoexcited Iron Porphyrins
FULL PAPER
II
III
tion of [Fe (TpivPP)(O )(1-MeIm)] or [Fe (TpivPP)-
2
[12]
(
O ·)(1-MeIm)] adduct.
2
Figure 5. A) UV/Vis spectrum of an acetone/ethanol (95:5) solution
Scheme 5. Oxidation of 1 by the [FeIII(TpivPP)(O ·
duct
2
)(1-MeIm)] ad-
III
Ϫ5
Ϫ5
of [Fe (TpivPP)Cl] (1 ϫ 10 ) and 1-MeIm (5 ϫ 10 ) after
9
0 s irradiation under anaerobic conditions; B) curve A after sub-
sequent oxygenation of the sample at Ϫ8 °C; C) curve B after a
second deoxygenation
The formation of urea as a major product of the reaction
II
between [Fe (TpivPP)(O )(1-MeIm)] and 1 was confirmed
2
II
by an additional experiment where the [Fe (TpivPP)(O )(1-
2
MeIm)] adduct was prepared with a chemical reducing ag-
A second deoxygenation of this solution regenerated the
[12b]
ent according to previous work (see Exp. Sect.).
Ad-
spectrum of the ferrous porphyrin deprived of O . Figure 5
2
dition of the hydroxyguanidine 1 to the chemically obtained
shows that this oxygenation/deoxygenation cycle is not
II
[
Fe (TpivPP)(O )(1-MeIm)] led to the formation of a de-
2
completely reversible, probably due to irreversible oxidation
tectable amount of the urea 2 with a yield of about the 65%
with respect to the sum of 2 and 3. This chemoselectivity
II
of the porphyrin ring. The O binding reversibility to Fe -
2
(
TpivPP) has been demonstrated and discussed in depth by
in the production of urea is in line with that obtained using
[12]
Collman in previous fundamental papers. The novelty of
this work is that we observe the same binding reversibility
using light rather than chemical reducing agents.
II
light to form [Fe (TpivPP)(O )(1-MeIm)]. The observed
2
small differences of the 2:3 concentration ratios may be as-
cribed to the experimental error inherent in the HPLC
analysis and to the different experimental conditions that
we were forced to employ in the two approaches.
The possibility of accumulating significant amounts of
II
the [Fe (TpivPP)(O )(1-MeIm)] adduct enabled us to inves-
2
tigate its possible reactivity towards the hydroxyguanidine
II
1
. In fact, it should be taken into account that the [Fe (T-
pivPP)(O )(1-MeIm)] species might undergo reduction by 1
2
Conclusion
to give an iron-peroxo intermediate,[ although a number
19]
of results seem to argue against a reaction between the
The aforementioned results show that photochemical ex-
citation of iron porphyrins is a suitable means of inducing
III
·
[9a,9b,20]
Fe (P)(O ) of NOS and N-hydroxy--arginine.
2
Table 2 shows that the addition of 1 to the photo- the oxidation of the hydroxyguanidine 1. This reaction pre-
III
·
generated [Fe (TpivPP)(O )(1-MeIm)] led to a detectable sents important similarities with the oxidation of the en-
2
amount of urea 2 with a yield of 80% with respect to the dogenous N-hydroxy--arginine by heme-containing metal-
sum of 2 and 3. A possible reaction pathway explaining this loenzyme nitric oxide synthases (NOSs) and their model
III
·
result is given in Scheme 5. The [Fe (TpivPP)(O )- systems.
2
(1-MeIm)] intermediate might abstract the hydrogen atom
Coordinated hydroxyguanidine 1 presents a photoreactiv-
of the hydroxyguanidine hydroxy group to furnish a hydro- ity that typically characterizes many axial ligands, under-
peroxo complex (step a), whose well-known nucleophilicity going oxidation to radical species. The simultaneous forma-
III
·
should allow it to react with the iminoxy radical (step tion of Fe (O ) is a key point for the oxidation of 1.
2
b).[
19,21]
Finally, a subsequent pericyclic reaction of the per-
The two iron porphyrin complexes employed here have
oxy intermediate is expected to form NO and 2 according provided complementary results. Photochemical excitation
III
to step c. The possibility that the conversion of 1 to 2 and of [Fe (TDCPP)Cl] in the presence of O and 1-MeIm re-
2
III
NO upon irradiation of the [Fe (TDCPP)Cl]/1/1-MeIm vealed that the urea 2 is the main end-product of 1. In
system may also occur, at least in part, through the agreement with an NOS-like oxidation mechanism, the con-
described mechanism cannot be ruled out.
version of 1 to 2 approximately matches the formation of
Eur. J. Inorg. Chem. 2004, 3127Ϫ3135
www.eurjic.org
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3133

A. Maldotti et al.
FULL PAPER
NO, as revealed by both ESR spin-trapping experiments, 760 Torr of O
2
. UV/Vis spectra were recorded during the prep-
Ϫ
Ϫ
aration of the solutions after the addition of each component and
at the end of the irradiation. HPLC analysis was performed on the
irradiated sample. Separation and detection of 1, 2 and 3 was
and in the form of its stable end-products NO2 and NO3
A significant reduction in the production of 2 and NO was
.
observed in the absence of 1-MeIm, due to the main role
achieved by eluting with a mixture of H
the presence of CH COO NH buffer (10 m), while HPLC analy-
was realised using a water solution of acetic
2 3
O and CH CN (85:15) in
III
·
of coordinated 1-MeIm in stabilizing the Fe (O ) inter-
2
3
4
mediate.
Ϫ
Ϫ
sis of NO
2
and NO
3
The ability of radical scavengers to inhibit the production
of both the urea 2 and NO strongly supports the possibility
acid (pH 3) for elution. In both cases the flow rate was 1 mL/min.
Compounds 1, 2 and 3 were identified by injection of authentic
that the reaction pathway resembles a radical-type autoxid- samples. Quantitative analysis was performed using calibration
ation mechanism, where the very fast reaction of O with curves obtained from the authentic compounds. Control HPLC
2
the ferrous porphyrin in the presence of the photogenerated experiments were carried out in order to verify that no reaction
occurred when irradiation was carried out in the absence of
iminoxyl radical should yield an iron-peroxo intermediate
III
2
[Fe (TDCPP)Cl] or O . Moreover, we verified that no thermal re-
as precursor of 2 and NO.
III
action occurred when the reaction mixture was kept in the dark for
several hours.
The use of [Fe (TpivPP)Cl] provides the first experimen-
tal evidence for the photoinduced formation of an iron-di-
III
oxy adduct. Moreover, the observed reactivity of the ESR Spin-Trapping: CH CN solutions of [Fe (TDCPP)Cl] (1 ϫ
3
II
Ϫ5
Ϫ3
Ϫ5
[
Fe (TpivPP)(O )(1-MeIm)] complex indicates that iron- 10 ) containing 1 (1 ϫ 10 ), 1-MeIm (5 ϫ 10 ) and PBN
2
Ϫ3
peroxo species able to generate the urea 2 may be also (5 ϫ 10 ) were irradiated (λ Ͼ 350 nm, room temperature) in-
III
Ϫ·
side the ESR cavity using a flat quartz cell. No ESR signal was
obtained when performing the described experiment in the absence
of 1.
formed as a consequence of the reduction of the Fe (O2
)
intermediate by the hydroxyguanidine 1 itself.
The described results give important indications about
the reactivity of iron-dioxy complexes that are in good
agreement with current opinion on the second step of NOS
catalysis. In particular, our results are in line with the hy-
pothesis that the formation of iron-peroxo intermediates is
essential for the oxidation of N-hydroxy--arginine to -ci-
trulline and NO.
For NO detection experiments, CH
3
CN solutions of [FeIII-
Ϫ5
Ϫ5
(
TDCPP)Cl] (1 ϫ 10 ), 1-MeIm (5 ϫ 10 ) and PTIO (2 ϫ
Ϫ6
) with and without 1 (1 ϫ 10Ϫ3 ) were irradiated (λ Ͼ
10
360 nm) inside the ESR cavity for some minutes.
II
)(1-MeIm)] Adduct: [FeIII(T-
Preparation of the [Fe (TpivPP)(O
2
Ϫ5
pivPP)Cl] (1 ϫ 10 ) dissolved in a mixture of acetone and etha-
nol (95:5, v/v) in the presence of 1-MeIm (5 ϫ 10 ) was de-
gassed to less than 1 ϫ 10 Torr by means of five vacuum-line
Ϫ5
Ϫ5
freeze-pump-thaw cycles. Subsequently, it was irradiated (for 90 s)
at low temperature (Ϫ5 to Ϫ8 °C) at wavelengths higher than
Experimental Section
350 nm. After irradiation, the solution was put in contact with O
2
Materials: The complexes [FeIII(TDCPP)Cl] and [FeIII(TpivPP)Cl]
at the same low temperature. Finally, a new deoxygenation cycle
was carried out. UV/Vis spectra were recorded after each step of
the procedure. HPLC analysis was carried out in order to investi-
were prepared and purified as described elsewhere.[
22,12b]
The
hydroxyguanidine 1, the urea 2 and the cianamide 3 were synthe-
sized following previous procedures.[
MeIm), α-phenyl-N-tert-butylnitrone (PBN) and NaNO
commercial products (Aldrich), 2-phenyl-4,4,5,5-tetramethylimida-
zoline-1-oxyl 3-oxide (PTIO) was purchased from Alexis Biochemi- from [Fe (TpivPP)(Br)] and following the procedure already re-
11a]
N-Methyl imidazole (1-
Ϫ3
gate the reactivity of the hydroxyguanidine 1 (1 ϫ 10 ) with the
II
were Fe (TpivPP)(O
2
) adduct.
) adduct was also chemically prepared starting
2
II
The Fe (TpivPP)(O
2
III
ported by Collman et al.[
12b]
Here, we employed zinc amalgam as
cals. They were all used as received. Acetonitrile, acetone and etha-
nol were spectroscopic-grade solvents.
III
a reducing agent instead of [Cr(acac)
2
]
2
. [Fe (TpivPP)(Br)] (1 ϫ
Ϫ3
Ϫ3
10
) and 1-MeIm (3 ϫ 10 ) dissolved in acetone were de-
Apparatus: UV/Vis spectra were recorded with a Kontron Model
Uvikon 943 spectrophotometer and X-Band electron spin reson-
ance (ESR) spectra with a Bruker 220 SE spectrometer that was
calibrated with α,αЈ-diphenylpicrylhydrazyl. HPLC analysis was
carried out with a Thermo Quest instrument, equipped with a UV
detector, using a Discovery HS PEG column (Supelco, 25 cm ϫ
gassed by means of three vacuum-line freeze-pump-thaw cycles. A
drop of zinc amalgam was then introduced into the solution kept
under argon atmosphere. After a while, the solution became red in
colour and the appearance of an intense band at 535 nm was in-
II
dicative of the formation of the [Fe (TpivPP)(1-MeIm)
2
] complex.
The solution containing the ferrous complex, separated from the
4.6 mm, 5 µm).
zinc amalgam, in a bath of ethanol/liquid nitrogen at Ϫ60 °C was
Irradiation was carried out with a medium-pressure Hanau Q 400
II
put in contact with O
pivPP)(O
2
. Evidence for the formation of the [Fe (T-
2
mercury lamp (15 mW/cm ). The required wavelength interval was
2
)(1-MeIm)] complex was obtained at room temperature
selected by using cut-off filters. When necessary, solutions were de-
by UV/Vis spectroscopy (observation of a new band at 548 nm).
Ϫ5
gassed to less than 1 ϫ 10 Torr by means of five vacuum-line
Ϫ2
Addition of the hydroxyguanidine 1 (2 ϫ 10 ) to an acetone
freeze-pump-thaw cycles. Low temperature experiments were per-
formed in a Julabo F12 (MP) cryostat.
II
Ϫ3
2
solution of [Fe (TpivPP)(O )(1-MeIm)] (1 ϫ 10 ) was carried
in an ice bath at 0 °C. The reaction mixture was stirred and kept
at the same temperature for 30 min before the HPLC analysis.
Photooxidation of N-(4-Chlorophenyl)-NЈ-hydroxyguanidine (1):
III
III
Ϫ5
[
Fe (TDCPP)Cl] or [Fe (TpivPP)Cl] (1 ϫ 10 ) was dissolved
Ϫ3
3
in a CH CN solution (3 mL) containing 1 (1 ϫ 10 ), with or
Acknowledgments
Financial support from MIUR and CNR is gratefully acknowl-
edged.
Ϫ5
Ϫ4
without 1-MeIm (5 ϫ 10 ). When necessary PBN (1 ϫ 10 )
was also added. The solutions thus obtained were irradiated for 30
minutes with wavelengths higher than 350 nm in the presence of
3134
 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Eur. J. Inorg. Chem. 2004, 3127Ϫ3135

Oxidation of N-(4-Chlorophenyl)-N-hydroxyguanidine by Photoexcited Iron Porphyrins
FULL PAPER
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