Metalloporphyrins as cytochrome P450 models for chlorhexidine metabolite prediction

Article abstract of DOI:10.1016/j.apcata.2012.08.026

The catalytic oxidation of chlorhexidine (CHX, a strong microbicidal agent) mediated by ironporphyrins has been investigated by using hydrogen peroxide, mCPBA, tBuOOH, or NaOCl as oxidant. All of these oxygen donors yielded p-chloroaniline (pCA) as the main product. The higher pCA yields amounted to 71% in the following conditions: catalyst/oxidant/substrate molar ratio of 1:150:50, aqueous medium, FeTMPyP as catalyst. The medium pH also had a strong effect on the pCA yields; in physiological pH, formation of this product was specially favored in the presence of the catalysts, with yields 58% higher than those achieved in control reactions. This provided strong evidence that CHX is metabolized to pCA upon ingestion.

Full text of DOI:10.1016/j.apcata.2012.08.026

Applied Catalysis A: General 447–448 (2012) 7–13
Contents lists available at SciVerse ScienceDirect
Applied Catalysis A: General
journal homepage: www.elsevier.com/locate/apcata
Metalloporphyrins as cytochrome P450 models for chlorhexidine
metabolite prediction
Vinicius Palaretti, Joicy Santamalvina dos Santos, Débora Fernandes Costa Guedes,
Luiz Alberto Beraldo de Moraes, Marilda das Dores Assis∗
Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Av. Bandeirantes 3900, 14040-901 Ribeirão Preto, SP, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 26 June 2012
Received in revised form 16 August 2012
Accepted 17 August 2012
Available online 3 September 2012
The catalytic oxidation of chlorhexidine (CHX, a strong microbicidal agent) mediated by ironporphyrins
has been investigated by using hydrogen peroxide, mCPBA, tBuOOH, or NaOCl as oxidant. All of these
oxygen donors yielded p-chloroaniline (pCA) as the main product. The higher pCA yields amounted to
71% in the following conditions: catalyst/oxidant/substrate molar ratio of 1:150:50, aqueous medium,
FeTMPyP as catalyst. The medium pH also had a strong effect on the pCA yields; in physiological pH,
formation of this product was specially favored in the presence of the catalysts, with yields 58% higher
than those achieved in control reactions. This provided strong evidence that CHX is metabolized to pCA
upon ingestion.
Keywords:
Biomimetic models
Metalloporphyrins
Cytochrome P450
Chlorhexidine
© 2012 Elsevier B.V. All rights reserved.
1. Introduction
transformations, including countless reactions like alkene epox-
idation, n-dealkylation of secondary and tertiary amines, o-
Chlorhexidine (CHX, Fig. 1) is a bis-guanidine with bactericidal
and fungicidal properties. It is commonly used in surgical [1–3],
neonatal treatments, periodontal treatments [4,5], and oral rinses
[6], and it is also employed as additive in chicken and pig food,
among other applications. Although the skin absorption of CHX is
not significant, as documented in the literature [7], the use of this
compound as preservative in chicken meat or food as well as in rinse
solutions might lead to the generation of toxic metabolites [8] such
as p-chloroaniline (pCA) and p-chloronitrobenzene (pCNB) [8–12]
when such food is consumed.
The oxidative metabolism of exogenous compounds in plants,
animals, bacteria, and fungi is mediated by a super family of
cytochrome P450 enzymes [13], which have an iron protoporphyrin
IX as the prosthetic group (Fig. 2).
The iron(IV)-oxo porphyrin ␲-cation, a highly eletrophilic
species, is assumed to be the most important catalytic intermedi-
ate in reactions catalyzed by cytochrome P450 enzymes. However,
the general consensus nowadays is that this species may not be
the only catalytic intermediate responsible for the large number of
reactions mediated by the cytochromes P450, as reported in recent
works [14,15]. The existence of different catalytic species enables
the cytochromes P450 to carry out a wide variety of chemical
dealkylation, and hydroxylation of aromatic compounds, among
others [13,16].
A number of biomimetic systems that are able to mimic the
function of P450 enzymes have been developed, in order to con-
tribute to a better understanding of the action mechanisms of these
enzymes [13,17]. Synthetic metalloporphyrins have been success-
fully used as P450 models for the oxidation of many endogenous
and exogenous compounds, mainly for comparison purposes and
identification of the metabolites formed in in vivo systems and/or
as an alternative method for the production of these metabolites.
The toxicity of the organochloride metabolites of chlorexidine
[18–20] justifies studies involving CHX degradation or CHX metab-
olization by cytochrome P450 in living organisms. However, the
great complexity inherent to the study of in vivo systems, the
way metalloporphyrins successfully mimic the cytochrome P450
enzymes, and our ongoing interest in this field have prompted
the present investigation on the use of metalloporphyrins as
cytochrome P450 models for the prediction and identification of
the possible metabolites generated from the antimicrobial agent
CHX.
2. Experimental
2.1. Physical measurements
∗
UV–vis spectra were obtained on a Hewlett–Packard 8452A
diode array spectrometer. Analytical HPLC analyses were
Corresponding author. Tel.: +55 16 3602 3799.
E-mail address: mddassis@usp.br (M. das Dores Assis).
0926-860X/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.apcata.2012.08.026

8
V. Palaretti et al. / Applied Catalysis A: General 447–448 (2012) 7–13
Fig. 1. Chlorhexidine, CHX.
performed on
a
SHIMADZU liquid chromatograph equipped
Oganics. Hydrogen peroxide (H2O2, 30% in water) was supplied by
Fluka and stored at 5 ◦C, and it was periodically titrated for confir-
mation of its purity. Acetonitrile (ACN) HPLC grade was obtained
from Mallinckrodt. Water used in the experiments was purified by
a Milli-Q, Millipore System.
with an LC-10AS solvent pump, an SPD-M 10A VP spectro-
photometric detector (ꢀ = 216 nm for pCA and pCNB, and 234 nm
for CHX) coupled to a CTO-10A VP column oven, and an SCL-10A VP
system controller. Separation of CHX and the oxidation products
pCA and pCNB was carried out in a C18 Shim-pack CLC-ODS (M)
column with a particle size of 5 ␮m (250 mm × 4 mm) supplied
by Merck, using a trifluoroacetic acid 0.08% acetonitrile/aqueous
solution (v/v) as eluent. The analytical column was protected
2.3. Oxidation reactions
Reactions were carried out in a 3-mL vial containing a screw cap.
Briefly, CHX (56 ␮L, 1.25 × 10−5 mol), 2.5 × 10−7 mol of the metal-
loporphyrin solubilized in 50 ␮L water, and the oxidant (mCPBA,
tBuOOH, or H2O2, 5.0 × 10−5 mol) in 2 mL water were added to a
reaction vial. Reactions were carried out for 24 h, under magnetic
stirring at room temperature, at a catalyst/oxidant/CHX molar ratio
of 1:200:50, which was the initial condition in our studies. Other
catalyst/oxidant/CHX molar ratios were also employed. At the end
of the reaction, magnetic stirring was interrupted, and an aliquot
of the reaction mixture (50 ␮L) was withdrawn and analyzed by
high-performance liquid chromatography (HPLC) or GC–mass. The
pH effect was investigated in buffered aqueous solution. Reactions
at pH 3 and 10 were performed in acetic acid 0.1 mol L−1, and in
carbonate buffer, respectively. The pH of the reaction solution was
by
a
Lichrospher guard column (4 mm × 4 mm). GC–MS was
conducted on a QP2010 mass spectrometer (Shimadzu) fitted
with a GC17A gas chromatograph (Shimadzu). The ionization
voltage was 70 eV. Gas chromatography was accomplished in
the temperature-programming mode, using a DB-5MS column
(30 m × 0.25 mm × 0.25 ␮m). Reaction products were identified
by comparison of their retention times with known reference
compounds, and by comparing their mass spectra to fragmenta-
tion patterns obtained from the NIST spectral library stored in
the computer software (version 1.10 beta, Shimadzu) of the mass
spectrometer.
2.2. Materials
adjusted by adding either HCl (0.5 mol L−1) or NaOH (0.5 mol L−1
solutions whenever necessary.
)
Unless otherwise stated, all the compounds used herein were
purchased from Aldrich or Merck and were of analytical grade.
The porphyrins 5,10,15,20-tetrakis(4-carboxyphenyl)porphyrin,
H2(TCPP), and 5, 10, 15, 20 tetrakis(4-N-methylpyridil)porphyrin,
H2(TMPyP), were acquired from Mid-Century. Iron insertion into
these free base-porphyrins was carried out by using the method
of Adler et al. [21]. tert-Butyl hydroperoxide (70 wt.% solution in
water) and 3-chloroperoxybenzoic acid were provided by Acros
The oxidation products were identified by comparison of their
retention times with those of authentic standards. Yields (or con-
version) are based on the added drug and were determined by
means of a calibration curve. Other minor products were identi-
fied by mass spectrometry, although their structural elucidation is
still not conclusive.
Control reactions were carried out in the absence of the catalyst,
under the same conditions as the catalytic runs.
3. Results and discussion
The metalloporphyrins FeTMPyP and FeTCPP (Fig. 3a and b,
respectively) were chosen for these studies because they are com-
mercially available and display good catalytic activity, as well
described in the literature [23,24]. Moreover, FeTMPyP and FeTCPP
exhibit appreciable water solubility, which enables their use in
studies carried out in aqueous medium. Hydrogen peroxide was the
oxidant of choice since it is clean, yields only water as byproduct,
and provides useful information for mechanism proposition.
The initial reactions for the investigation of CHX oxidation by
H2O2 were carried out using 2.5 × 10−7 mol catalyst at a cata-
lyst/oxidant/CHX ratio of 1:200:50, which had been previously
determined as standard conditions for other catalytic systems [22],
in aqueous medium. Reaction products were analyzed by HPLC. In
these conditions, one compound was detected as the main degra-
dation product, and it displayed the same elution time and UV/Vis
spectrum as an authentic p-chloroaniline (pCA) sample. Some other
minor products were also verified.
To confirm that pCA was the main product and in an attempt
to identify the minor products, the reaction products were also
analyzed by GC–MS. An authentic sample of pCA was also injected
Fig. 2. Iron-protoporphyrin IX.

V. Palaretti et al. / Applied Catalysis A: General 447–448 (2012) 7–13
9
Fig. 3. (a) 5,10,15,20-tetrakis(4-N-methylpyridil)porphyrin iron (III), FeTMPyP; (b) 5,10,15,20 tetrakis(4-carboxyphenyl)porphyrin iron (III), FeTCPP.
under the same conditions, for comparison of the elution times and
fragmentation patterns in relation to the main reaction product.
There was a perfect correlation between the retention times and
the fragmentation patterns obtained for the main reaction product
and the authentic pCA sample (retention times of 8.00 and 7.98 min
respectively and mass fragments of 65, 92 and 127/129 u for both
samples), thus confirming that the main CHX degradation product
in these conditions was pCA.
Once pCA formation was ratified, this product was quantified by
HPLC under different reaction conditions, by means of a calibration
curve constructed from authentic pCA samples.
Table 1 summarizes the pCA yields under different conditions.
pCA formation was also detected in control reactions conducted in
the absence of the catalyst. Hence, the last column in Table 1 as
well as in the other tables included in this work refers to the pCA
formed exclusively through catalyst action and was calculated via
the following Eq. (1):
reaction by UV/Vis spectroscopy at regular time intervals. After
4 h of reaction, the Soret band at 426 nm, characteristic of the
catalyst, disappeared, thus corroborating destruction of the met-
alloporphyrin.
These results attested to the fact that, although pCA formation
occurred even in the absence of the catalyst, this formation was
considerably higher when the reaction was catalyzed by metal-
loporphyrins. In conditions where there was a balance between
oxidant concentration and catalyst lifetime (i.e., 1:2500:50), the dif-
ference between the catalyzed and the non-catalyzed reaction was
more significant, as well as the total pCA yield. Considering these
results, the 1:2500:50 molar ratio was used for evaluation of the
effects of other variables on the CHX oxidation, unless otherwise
stated.
Catalytic pCA formation may take place by different mecha-
nisms, depending on the generated active intermediate species.
As reported in the literature, two catalytic species can generally
be formed as a result of the interaction of the metalloporphyrin
with hydrogen peroxide, as shown in Fig. 4 [14,23,24]. The main
one is the iron(IV)-oxo-porphyrin ␲-cation radical, Fe(IV)OP•+ (II,
Fig. 4), which acts as a strong electrophile and performs oxidations
of various functional groups such as alcohols and alkenes, among
ꢀ
ꢁ
[pCA]cat − [pCA]ncat
pCA yield (%) =
× 100%,
(1)
[pCA]ncat
where [pCA]cat = pCA concentration in the catalyzed reaction and
[pCA]ncat = pCA concentration in the non-catalyzed reaction.
Increasing oxidant concentration implied in higher pCA yields
others [13,25,26]. The other possible catalytic species is the iron(III)
-
hydroperoxo porphyrin, Fe(III)–OOH(P) (I, Fig. 4), a nucleophilic
species that can be formed when the proton donation that leads
to Fe(IV)OP•+ is inhibited [14,23,25]. Considering these species, one
possible mechanism accounting for pCA formation in the studied
systems could involve the oxidation of the CHX iminic bond by
the highly electrophilic intermediate Fe(IV)OP•+, yielding a oxaziri-
dine ring (1a, Fig. 4), which hydrolyzes and furnishes pCA and a
N-(hydroxy)amide (1b, Fig. 4).
Another possibility involves the nucleophilic intermediate iron-
hydroperoxo porphyrin (I, Fig. 4), which preferably attacks the CHX
iminic carbon, thereby affording intermediate 2a, Fig. 4. An intra-
molecular rearrangement of intermediate 2a furnishes pCA and an
amide (2b, Fig. 4).
up to
a catalyst/oxidant/substrate molar ratio of 1:2500:50
(Table 1). The pCA yields decreased thereafter, when there was
fast discoloration of the reaction medium, which indicated cata-
lyst degradation. This was confirmed through monitoring of the
Table 1
pCA yields obtained from the CHX oxidation by H2O2 catalyzed by FeTCPP in aqueous
medium.
Catalyst/oxidant/substrate
molar ratioa
Total pCA yield
pCA yieldsc (%)
b
([pCA]cat
)
(%)
1:200:50
1:300:50
1:2500:50
1:5000:50
1:10,000:50
15
18
60
65
92
49
49
58
41
10
As neither amide 2b (Fig. 4) nor the N-(hydroxy)amide 1b could
be isolated, there was no conclusive evidence of which mecha-
nism is responsible for pCA formation in these systems. However,
some indication about which mechanism predominates in CHX
a
Catalyst = 2.5 × 10−7 mol, yields after 24 h of reaction.
Total pCA yields obtained in reactions.
pCA yields as determined by Eq. (1).
b
c

10
V. Palaretti et al. / Applied Catalysis A: General 447–448 (2012) 7–13
Fig. 4. Possible mechanisms of pCA formation from CHX degradation by H2O2 mediated by metalloporphyrins, in aqueous medium: (1) electrophilic pathway involving
Fe(IV)OP•+ and (2) nucleophilic pathway through the Fe(IV)OOH(P) species.
degradation can be obtained from the studies of reaction variables
such as pH and oxidant nature.
metalloporphyrins. This corroborated the fact that these complexes
actively participated in pCA formation, and that this participation
was also pH-dependent. Results listed in Table 2 revealed a reduc-
tion in or absence of pCA at both extreme pH values for reactions
accomplished in the presence of the catalyst.
Table 2 presents the pCA yields attained from CHX oxidation
reactions mediated by FeTCPP under different pH values.
The pH had little effect on pCA yields for the non-catalyzed reac-
tions (36% pCA in acid pH vs 38% in neutral pH), but it played
a significant role in reactions occurring in the presence of the
An alkaline pH probably reduces the availability of CHX in the
reaction medium, because of the minor solubility of this substrate in

V. Palaretti et al. / Applied Catalysis A: General 447–448 (2012) 7–13
11
iron(IV)-oxo-porphyrin ␲-cation radical is considerably slower. In
conclusion, the pH effects suggest that the nucleophilic attack pre-
vails, since the catalytic yields rose substantially under neutral
medium, where the iron-hydroperoxo species has longer half-life.
The utilization of different oxidants is also interesting because
each one favors a different catalytic intermediate, thus provid-
ing some useful information about the reaction mechanisms.
In this context, H2O2 was substituted by m-chloroperbenzoic
acid (mCPBA), tert-butyl-hydroperoxyde (tBuOOH), or sodium
hypochlorite (NaOCl) in equimolar amounts, and FeTMPyP (Fig. 3a)
was employed as the catalyst, for evaluation of their effects on CHX
oxidation.
Table 2
pCA yields from CHX oxidation by H2O2 mediated by FeTCPP under different pH
values, in aqueous medium.
pH
Total pCA yield (%)
Non-catalyzed
pCA yieldb (%)
Catalyzeda
3
7
10
36
38
2
41
60
2
13
58
0
Catalyst/oxidant/substrate 1:2500:50. Catalyst = 2.5 × 10−7 mol, yields after 24 h of
reaction.
a
pCA yields obtained in catalyzed reactions.
pCA yields as determined by Eq. (1).
b
Table 3 shows that although the H2O2 was not the best oxi-
dant in terms of total pCA yields, it was the most efficient in the
case of the catalyzed reactions. It also accounts for the nucleophilic
mechanism, because this oxidant is the only reagent that is able to
generate iron-hydroperoxo, the active species in the nucleophilic
mechanism (Fig. 4).
The high pCA yields observed for mCPBA in the non-catalyzed
reactions can be explained by the direct epoxidation of the iminic
double bond, to yield an oxaziridine [27]. Epoxide ring-opening
induced by a nucleophilic attack of a water molecule would yield a
N-(hydroxy)amide and pCA similarly as shown in Fig. 4, I where the
electrophilic mechanism in the presence of the metalloporphyrins
these conditions. As a consequence, the pCA yields are considerably
diminished in this case (Table 2).
Reduced pCA formation under acid medium is coherent with
the nucleophilic mechanism (2, Fig. 4), since higher proton con-
centration favors protonation of the nucleophilic iron-hydroperoxo
species (I, Fig. 4), leading to fast consumption of this intermedi-
ate and consequent Fe(IV)OP•+ production, thereby reducing the
probability of a nucleophilic attack on the CHX molecules.
In neutral medium, the nucleophilic mechanism is also favored
as a result of the higher lifetime of the hydroperoxo species,
since the cleavage of the O O bond that culminates in the
Fig. 5. Main catalytic species probably involved in the CHX oxidation reactions by different oxidants catalyzed by ironporphyrins.

12
V. Palaretti et al. / Applied Catalysis A: General 447–448 (2012) 7–13
Table 3
strong evidence that this product may be a CHX metabolite when
the antiseptic is ingested.
pCA yields from CHX oxidation by different oxidants mediated by FeTMPyP in aque-
ous medium.
Based on the pCA yields under different reaction conditions, it
is difficult to unambiguously determine which of the mechanisms
is responsible for pCA formation, one involving the iron(IV)-oxo-
porphyrin ␲-cation radical intermediate or another mediated by
a nucleophilic attack of the iron(III)-hydroperoxo-porphyrin. How-
ever, an alert remains: under all the studied conditions pCA was
detected as the main product. Considering the success of the metal-
loporphyrins as a synthetic analog of the cytochromes P450, the
higher catalytic activity of these complexes in physiological pH
demonstrates the strong possibility of pCA formation when CHX is
ingested. Since pCA is highly toxic, application of CHX as preser-
vative in chicken meat or as food additive may require revised
regulation and reinforces the need for more detailed studies on
CHX toxicity.
Oxidant
Total pCA yielda (%)
pCA yieldsb (%)
H2O2
12
24
21
4
71
9
mCPBA
NaOCl
tBuOOH
−23
33
Catalyst/oxidant/substrate 1:150:50, 24 h.
a
pCA yields for catalyzed reactions.
Calculated using Eq. (1).
b
is described. In this case, however, the electrophilic species is the
mCPBA instead of the Fe(IV)OP•+
.
Although NaOCl led to high pCA yields, these results were lower
compared to those achieved in the case of the non-catalyzed reac-
tions in the same conditions (Table 3). pCA formation in this case
may have taken place through CHX alkaline hydrolysis, as sug-
gested in the literature [8,28,29]. Hydroxyl ions originated from
OCl− hydrolysis in aqueous medium may have attacked the CHX
iminic carbon, thereby generating pCA. Part of the OCl− ions was
consumed in the presence of the metalloporphyrins, to form the
iron(IV)-oxo-porphyrin ␲-cation radical, as depicted in Fig. 5. This
reaction probably competed with the fast CHX hydrolysis, thus
resulting in lower pCA yield.
Acknowledgements
We thank FAPESP, CAPES, and CNPq for financial support. We are
grateful to Dr. Cynthia Maria de Campos Prado Manso for linguistic
advice.
Appendix A. Supplementary data
Lower pCA yields were also achieved in the CHX oxidation by
tBuOOH, as compared to H2O2 (Table 3). These results can also be
explained by analysis of the main intermediate yielded by this oxi-
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/j.
apcata.2012.08.026.
dant. Alkylhydroperoxides often tend to undergo homolytic O
O
bond cleavage upon coordination to the metalloporphyrin [30,31],
thereby giving rise to a radical (tBuO.) and the Fe(IV)POH species
(III, Fig. 5), which is not able to epoxidize or perform a nucleophilic
attack on CHX [25].
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aqueous medium, FeTMPyP as catalyst.
It is also remarkable that under physiological pH, pCA formation
is specially favored in the presence of the metalloporphyrins (with
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