Peroxide decoloration of CI acid orange 7 catalyzed by manganese chlorophyll derivatives at the surfaces of micelles and lipid bilayers

Article abstract of DOI:10.1021/la904574m

Manganese-substituted chlorophyll a derivatives (MnChls) were synthesized. We first report peroxidative oxidation of an azo dye, CI Acid Orange 7, catalyzed by MnChls at the surfaces of micelles and lipid bilayers with hydrogen peroxide (H₂O₂) under mild conditions (pH 8.0, 25 °C). Peroxide decoloration depended upon the structures of MnChls, surfactants, lipids, and the presence of imidazole. Surprisingly, a largest decoloration rate was observed for MnChls dimer, MnPChlide a-K(MnPChlide a)-His 5 in cetyltrimethylammonium bromide (CTAB) micellar solution, especially when imidazole was present: this observation is analogous to the decoloration using horseradish peroxidase (HRP). Interestingly, the dimer complexes showed enhanced decoloration in comparison to the corresponding MnChls monomer in the micellar solution. In contrast, the MnChls monomer showed enhanced decoloration in comparison with the MnChls dimer in liposomal suspensions. Further, the imidazole residue covalently linked to the MnChls plays an important role in increasing the decoloration in both micellar and liposomal suspensions as well as in addition of imidazole into the solutions. It is interesting that the electron paramagnetic resonance (EPR) spectra of MnPChlide a ME 2, MnPChlide a-His 3, and MnMPMME-His 7 have 16 peaks around g = 2 in Egg PC or DMPC liposomal suspension with H₂O₂, which is typical of a mixed-valence Mn(III)-Mn(IV) complex with coupling between two ions. The higher decoloration performance obtained by the monomer porphyrin compounds at the surface of the lipid bilayers appears to be related to the stability of this mixed-valence Mn(III)-Mn(IV) species formed in the lipid bilayers. This finding should provide useful information to note that MnChls, which are easily found in a number of biological systems, are involved in functions such as hydrogen peroxide decomposition in bacteria and the oxidation of water during photosynthesis as well as the peroxidases function such as the peroxidative decoloration as bleaching agents.

Full text of DOI:10.1021/la904574m

pubs.acs.org/Langmuir
©
2010 American Chemical Society
Peroxide Decoloration of CI Acid Orange 7 Catalyzed by Manganese
Chlorophyll Derivatives at the Surfaces of Micelles and Lipid Bilayers
†
†
†
†
†
Shuichi Ishigure, Tatsuro Mitsui, Shingo Ito, Yuji Kondo, Shigeki Kawabe,
†
†
‡
‡
,†
Masaharu Kondo, Takehisa Dewa, Hiroyuki Mino, Shigeru Itoh, and Mamoru Nango*
†
Department of Life and Materials Engineering, Nagoya Institute of Technology, Gokiso-cho, Showa-ku,
Nagoya, Aichi 466-8555, Japan, and Division of Material Science, Graduate School of Science,
Nagoya University, Furo-cho, Chikusa-ku Nagoya, Aichi 464-8602, Japan
‡
Received December 4, 2009. Revised Manuscript Received January 18, 2010
Manganese-substituted chlorophyll a derivatives (MnChls) were synthesized. We first report peroxidative oxidation
of an azo dye, CI Acid Orange 7, catalyzed by MnChls at the surfaces of micelles and lipid bilayers with hydrogen
peroxide (H O ) under mild conditions (pH 8.0, 25 °C). Peroxide decoloration depended upon the structures of
2 2
MnChls, surfactants, lipids, and the presence of imidazole. Surprisingly, a largest decoloration rate was observed for
MnChls dimer, MnPChlide a-K(MnPChlide a)-His 5 in cetyltrimethylammonium bromide (CTAB) micellar solution,
especially when imidazole was present: this observation is analogous to the decoloration using horseradish peroxidase
(HRP). Interestingly, the dimer complexes showed enhanced decoloration in comparison to the corresponding MnChls
monomer in the micellar solution. In contrast, the MnChls monomer showed enhanced decoloration in comparison
with the MnChls dimer in liposomal suspensions. Further, the imidazole residue covalently linked to the MnChls plays
an important role in increasing the decoloration in both micellar and liposomal suspensions as well as in addition of
imidazole into the solutions. It is interesting that the electron paramagnetic resonance (EPR) spectra of MnPChlide a
ME 2, MnPChlide a-His 3, and MnMPMME-His 7 have 16 peaks around g = 2 in Egg PC or DMPC liposomal
2 2
suspension with H O , which is typical of a mixed-valence Mn(III)-Mn(IV) complex with coupling between two ions.
The higher decoloration performance obtained by the monomer porphyrin compounds at the surface of the lipid
bilayers appears to be related to the stability of this mixed-valence Mn(III)-Mn(IV) species formed in the lipid
bilayers. This finding should provide useful information to note that MnChls, which are easily found in a number of
biological systems, are involved in functions such as hydrogen peroxide decomposition in bacteria and the oxidation of
water during photosynthesis as well as the peroxidases function such as the peroxidative decoloration as bleaching
agents.
8
Introduction
hydrogen peroxide (H O ) decomposition in bacteria and the
2 2
9
oxidation of water during photosynthesis. Further, some peroxi-
dases such as lignin peroxidase, manganese peroxidase, and
horseradish peroxidase have been used to catalyze the decolora-
tion of azo dyes because not only are they more active than those
bleaching agents currently available but also environmentally
The porphyrin model is useful for providing insights into
possible reactions of porphyrin complexes. In these models,
porphyrin pigments play a key role in oxidative electron-transfer
1-6
systems such as peroxidases and cytochromes.
It has been
reported that many oxidation systems, metalloporphyrins, can be
used to mimic cytochrome P-450-dependent monooxygenases
and peroxidases, and the quantitative epoxidations of alkenes
and alkanehydroxylation can becatalyzed bysynthetic porphyrin
1
0-12
safe.
Hage et al. reported that non-heme manganese ion
complex catalyzed the low-temperature decoloration of tea-co-
lored cotton when hydrogen peroxide was used as a bleaching
1
3
5-7
agent, and Namboodri et al. reported the decoloration of dyes
with hot peroxide catalyzed by a copper phthalocyanine-based
complexes.
It is interesting to note that binuclear and multi-
nuclear manganese containing enzymes, which are found in a
number of biological systems, are involved in functions such as
1
4
reactive blue 21 dye. Recently, Sheriff et al. reported that
selective dye oxidation using in situ generated hydrogen peroxide
1
5
catalyzed by manganese(II) ions. Hodges et al. reported the
results of kinetic and mechanistic investigations focusing on the
oxidation of 1-arylazohydroxynaphthalene-6-sulfonate dyes by
*
Corresponding author: Tel þ81-52-735-5226; Fax þ81-52-735-5208;
e-mail nango@nitech.ac.jp.
1) Nango, M.; Hikita, T.; Nakano, T.; Yamada, T.; Nagata, M.; Kurono, Y.;
Ohtsuka, T. Langmuir 1998, 14, 407–416.
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R.; Kondo, M.; Ohtsuka, T.; Nango, M. Thin Solid Films 2005, 474, 310–321.
3) Nango, M.; Iwasaki, T.; Takeuchi, Y.; Kurono, Y.; Tokuda, J.; Oura, R.
Langmuir 1998, 14, 3272–3278.
4) Kondo, M.; Kawashima, N.; Mitsui, T.; Ito, S.; Ishigure, S.; Hashimoto, T.;
(
3-chloroperoxybenzoic acid in the presence of the sterically
(
(
(9) Naruta, Y.; Sasayama, M.; Sasaki, T. Angew. Chem., Int. Ed. Engl. 1994, 33,
1839–1841.
(10) Chivukula, M.; Spadarao, J. T.; Renganathan, V. Biochemistry 1995, 34,
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(
Umemura, R.; Dewa, T.; Yamashita, K.; Mino, H.; Itoh, S.; Nango, M. Chem.
Lett. 2005, 34, 1592–1593.
(11) Heinfling, A.; Martınez, M. J.; Martınez, A. T.; Bergbauer, M.; Szewzyk,
´ ´
U. Appl. Environ. Microbiol. 1998, 64, 2788–2793.
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(13) Hage, R.; Iburg, J. E.; Kerschner, J.; Koek, J. H.; Lempers, E. L. M.;
Martens, R. J.; Racherla, U. S.; Russell, S. W.; Swarthoff, T.; van Vliet, M. R.;
Warnaar, J. B.; van der Wolf, L.; Krijnen, B. Nature 1994, 369, 637–639.
(14) Namboodri, C. G.; Walsh, W. K. Am. Dyest. Rep. 1995, 84, 86–95.
(15) Sheriff, T. S.; Cope, S.; Ekwegh, M. Dalton Trans. 2007, 5119–5122.
(
5) Collman, J. P.; Kodadek, T.; Raybuck, S. A.; Meunier, B. Proc. Natl. Acad.
Sci. U.S.A. 1983, 80, 7039–7041.
6) Nolte, R. J. M.; Razenberg, J. A. S. J.; Schurman, R. J. Am. Chem. Soc. 1986,
08, 2751–2752.
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Mansuy, D. J. Am. Chem. Soc. 1988, 110, 8462–8470.
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(
1
(
(
7
774 DOI: 10.1021/la904574m
Published on Web 02/25/2010
Langmuir 2010, 26(11), 7774–7782

Ishigure et al.
Article
Chart 1. Structure of Manganese Chlorophyll a Derivatives and Manganese Porphyrin Derivatives
Chart 2. Structure of Azo Dye
(Scheme 1) with decomposition of hydrogen peroxide under mild
conditions. Manganese chlorophyll a derivatives (MnChls), man-
ganese-substituted chlorophyll a (MnChl a) 1, MnPChlide a ME
2, MnPChlide a-His 3, MnPChlide a-K(MnPChlide a)-OMe 4,
and MnPChlide a-K(MnPChlide a)-His 5 (Chart 1), were synthe-
sized. MnChls with hydorgen peroxide (H O ) efficiently cata-
2
2
lyzed the peroxide oxidation of CI Acid Orange 7 in micellar
solutions and liposomal suspensions under mild conditions; in
these reactions, the imidazole residue covalently linked to the
MnChls played an important role in increasing decoloration.
Electron paramagnetic resonance (EPR) spectra of MnChls
were also measured to elucidate the catalytic mechanism of the
MnChls for decoloration.
A key aspect of the peroxidative decoloration is its usefulness in
providing in insights into the catalytic effect of porphyrin struc-
tures on decoloration at the surface of the micelllar and lipid
bilayers. MnChls were selected because of their environmental
safety, usefulness, well-defined biological properties, and indus-
trial applications.
hindered anionic oxoiron(IV) tetra(2,6-dichloro-3-sulfonatophe-
1
6
nyl)porphyrin in aqueous solution. In previous papers, we
reported the peroxide decoloration of azo dyes catalyzed by
3
,4,17
manganese porphyrins.
However, fewer studies, if any, have
investigated the catalytic effect of chlorophyll complexes upon the
peroxidative decoloration of azo dyes with decomposition of
H O under mild conditions such as neutral pH at 25 °C. In this
2
2
study, we first report the peroxidative oxidation of the azo dye
(
(
Chart 2) catalyzed by manganese chlorophyll a derivatives
Chart 1) in various micellar solutions and liposomal suspensions
Future detergents will be routinely required to contain bleach-
ing agents that are not only more active than those currently
available but are also environmentally safe and cost-effective.
(
16) Hodges, G. R.; Lindsay, Smith J. R.; Oakes, J. J. Chem. Soc., Perkin Trans.
998, 2, 617–627.
17) Tokuda, J.; Oura, R.; Nango, M. Textile Res. J. 1999, 69, 956–960.
1
(
Langmuir 2010, 26(11), 7774–7782
DOI: 10.1021/la904574m 7775

Article
Ishigure et al.
Scheme 1. Decoloration of the Azo Dye Catalyzed by Manganese Chlorophyll Derivatives in Micllar or Liposomal Solutions
Hydrogen peroxide, a traditional bleaching agent, loses its
activity as the washing temperature decreases. It is considered
that manganese or ferric complexes are relatively more favorable
azo dye. To obtain each decoloration curve, each of the solutions
was placed as quickly as possible in a covered optical quartz cell,
and the concentration of the coloring matter in the bleaching
solution was determined by a spectrophotometric method using a
Hitachi U-3500 spectrophotometer equipped with a thermostat;
the concentration was measured at appropriate time intervals and
the desired temperature. At the absorption maximum of the
coloring matter, there is no interference from products formed
during decomposition. Under these conditions, the calibration
line follows Beer’s law. Absorptions of CI Acid Orange 7 was
measured at 484 nm.
3
,10,17
for catalytic bleaching than hydrogen peroxide alone.
Thus,
studying the catalytic effect of MnChls on the rate of decoloration
of azo dyes by hydrogen peroxide is useful for providing insights
into the favorable catalytic bleaching properties of horseradish
peroxidase (HRP) as well as binuclear and multinuclear manga-
nese-containing enzymes, which are easily found in a number of
biological systems, are involved in functions such as hydrogen
peroxide decomposition in bacteria and the oxidation of water
during photosynthesis.
Preparation of Liposomal Membrane Containing Man-
ganese Chlorophyll a Derivatives or Manganese Porphyrin
Derivatives. Manganese chlorophyll a derivativrs (MnChls) or
manganese porphyrin derivatives (MnPorphyrins) (2.4 μmol) and
Egg PC (143 μmol) (MnPorphyrin/lipid = 1/60, mol/mol) in
choloroform was transferred to a flask. The organic solvent was
removed using rotary evaporator, resulting in a thin lipid film
containing MnChls or MnPorphyrins. The last traces of solvent
were then removed under reduced pressure using vacuum pump
for 12 h. To the dried film was added 2.5 mL of 10 mM Tris-HCl
buffer (pH 8.0), and the mixture was vortexed for 2 min. The
liposomal suspension was subjected to five freeze/thaw cycles
(77 K/325 K) and was then sonicated for 10 min in an ice bath
conditions to make small unilamellar vesicles. After sonication,
the liposomal suspension was purified by gel filtration (Sephadex
G-50). After gel filtration, liposomal membrane containing
MnChls and MnPorphyrins was obtained. The concentrations
of MnChls and MnPorphyrins are calculatedbya UV/visspectro-
meter to ajust sample concentration for catalytic and EPR
experiments.
Materials and Methods
General Materials. All reactions and chromatographic se-
parations were carried out at minimum room light. Tetrahydro-
furan (THF) and triethylamine were distilled and stored over
molecular sieves. The other solventsusedinthisstudy were atleast
of spectral grade. The silica gel used for column chromatography
1
˚
was silica gel, 70-230 mesh, 60 A, obtained from Aldrich. H
NMR spectra were obtained using the Varian Gemini-300 NMR
spectrometer, with tetramethylsilane used as an internal standard
3
for CDCl . The UV absorption spectra were recorded on Hitachi
U-3500 spectrophotometers. Mass spectra (MS) were obtained in
R-cyano-4-hydroxycinnamic acid (CHCA), using a matrix-as-
sisted laser desorption/ionization mode-time of flight (MALDI-
TOF) mass spectrometer (Perseptive Biosystems Voyager RN).
Hydrogen peroxide was provided by Mitubishigasu Chemicals as
a 31% solution.
The pH was adjusted with tris(hydroxymethyl)aminomethane
and 1.0 N HCl(aq). The azo dye, CI Acid Orange 7, was provided
by Tokyo Kasei and purified by recrystallization from aqueous
ethanol. Triton X-100 and sodium dodecyl sulfate (SDS) were
obtained from Nacalai Tesque, Inc. Cetyltrimethylammonium
bromide (CTAB) was obtained from Wako Pure Chemical
Industries, Ltd. Egg yolk phosphatidylcholine (Egg PC) and
dimyristoylphosphatidylcholine (DMPC) were gifts from Nippon
Fine Chemical Co.
Cyclic Voltammogram Measurement. Cyclic voltammo-
gram (CV) measurements were conducted using a Hokuto Denko
HZ-3000. A standard Ag/AgCl electrode served as reference
electrode, glassy carbon served as the working electrode, and
platinum functioned as the counter electrode. Solutions contain-
ing MnChls or MnPorphyrins were prepared in DMSO contain-
ing 0.1 M tetra-n-butylammomium perchlorate (TBAP). The
solutions were degassed by argon bubbling. The data are sum-
marized in Table 2.
3
Peroxidative Decoloration of Azo Dye. The peroxide
oxidation of azo dyes by hydrogen peroxide in the presence of
MnChls and manganese porphyrin derivatives (MnPorphyrins)
was evaluated by determining the decoloration curve of the dye in
Electric Paramagnetic Resonance Measurement. The
EPR was measured on a Bruker ESP 300E spectrometer at
9.487 GHz. Solutions containing MnChls and MnPorphyrins
were prepared in Egg PC liposomal suspension. The solutions
were degassed by argon bubbling and cooled by liquid helium.
EPR measurements were performed in quartz tubes (d = 5 mm,
l = 150 mm) placed into a dewar with liquid helium. While
studying the temperature dependence of the micropower satura-
tion, the likely influence of the modulation field amplitude on P1/2
was examined. The data are summarized in Table 5.
-
2
aqueous solution (1 ꢀ 10 [M] Tris-HCl buffer) at pH 8.0 at
25 °C. The required amount of an aqueous solution of the azo dye
adjusted to the desired pH was added to 25 mL of the bleaching
solution at 25 °C so as to obtain the initial concentrations of the
-4
azo dye of1 ꢀ 10 M. MnChls solubilized bysurfactants orlipids
were mixed with the azo dye in aqueous solution, and hydrogen
peroxide was finally added to mixture solution of MnChls and the
7
776 DOI: 10.1021/la904574m
Langmuir 2010, 26(11), 7774–7782

Ishigure et al.
Article
Scheme 2. Synthesis of Chlorophyll a Derivatives
1
Results and Discussion
absorption, H NMR spectroscopies, and MALDI-TOF mass
spectroscopies. The manganese complexes were prepared as
Synthesis and Characterization of Manganese Chloro-
phyll a Derivatives. Synthetic sequences leading to the com-
pounds, manganese chlorophyll a derivatives (MnChls), MnChl a
3,4
described in previous papers. MALDI-TOF data showed the
presence of a characteristic MnChls, as shown in the Supporting
Information. Tables 1 and 2 show the absorption maximum of
Soret and Qy bands and CV potential values of MnChls in the
absence and presence ofimidazole. When imidazolecoordinates to
Mn, theλmax ofSoret and Qy bands andCV potential are changed.
Table 1 presents that the Soret and Qy bands are red-shifted by
around 1 -3 nm (except for the Soret band of MnPChlide a-
K(MnPChlide a)-OMe 4, which is shiftedby12 nm), depending on
the structures of MnChls and the concentration of imidazole. For
1
, MnPChlide a ME 2, MnPChlide a-His 3, MnPChlide a-
K(MnPChlide a)-OMe 4, and MnPChlide a-K(MnPChlide a)-
His 5 (Chart 1), were preparedbythe followingsynthetic sequence
of steps as shown in Scheme 2. Pheophytin a, PPheide a ME,
PPheide a-His, PPheide a-K(PPheide a)-OMe, and PPheide a-
K(PPheide a)-His were prepared from Chl a as described in the
Supporting Information. Characterization and verification of the
compounds synthesized were performed by mean of UV/vis
Langmuir 2010, 26(11), 7774–7782
DOI: 10.1021/la904574m 7777

Article
Ishigure et al.
Table 1. λmax of Manganese(III) Chlorophyll a Derivatives in the
Presence of Imidazole in CTAB Micelle Solution at 25 °C and pH 8.0
Table 3. Rate Constants for Decoloration Reaction of CI Acid Orange
7 Catalyzed by Manganese Chlorophyll a Derivatives and Manganese
a
a
2 2
Porphyrin Derivatives with H O at 25 °C and pH 8.0
λ
max/nm
2
-1
k
obs (ꢀ 10 min
)
catalyst
imidazole/mM
none
Soret band
Qy band
surfactant
lipid
Egg PC
2
367
369
370
364
367
367
358
363
370
364
683
683
685
685
685
686
688
691
690
688
catalyst
CTAB
Triton X-100
SDS
DMPC
1
1
0
00
1
2
3
4
6
7
252
188
438
608
1.75
53.5
191
5.76
1.06
5.11
0.42
3
4
none
124
88.1
67.9
264
1
1
0
00
none
b
b
b
27.9
2.79
0.247
1
1
0
00
b
215
c
d
d
none
a
0.84
0.07
1.33
-2
5
none
-4
-4
a
-5
[CI Acid Orange 7] = 1.0 ꢀ 10 M, [H O ] = 3.0 ꢀ 10 M,
2
2
[
CTAB] = 8.0 ꢀ 10 M, [2] = [3] = 1.0 ꢀ 10 M, and [4] = [5] =
-
- -4
4
6
[CTAB] = 8.0 ꢀ 10 M, [Triton X-100] = 1.5 ꢀ 10 M, [SDS] = 1.0 ꢀ
- -4 -4
10
5
.0 ꢀ 10 M.
3
b
M, [Egg PC] = 9.0 ꢀ 10 M, [DMPC] = 9.0 ꢀ 10 M. Cited
c
d
2 2
from ref 4. Control data no catalyst with H O . Cited from ref 25.
Table 2. Redox Potential of Manganese Chlorophyll a Derivatives
and Manganese Porphyrin Derivatives in the DMSO Solution at
Room Temperature
a
Table 4. Rate Constants for Decoloration Reaction of CI Acid Orange
Catalyzed by Manganese Chlorophyll a Derivatives and Manganese
7
a
Porphyrin Derivatives with H
2
O
2
at 25 °C and pH 8.0
redox potential/V vs
Ag/AgCl (0.1 M KClaq) Mn(II)/Mn(III)
catalyst
imidazole/M
2
-1
k
obs (ꢀ 10 min
imidazole/mM
)
1
2
0
0
2
5
0
0
2
5
0
2
5
0
0
-0.296
-0.264
-0.302
-0.350
-0.259
-0.265
-0.314
-0.336
-0.456
-0.496
-0.520
-0.463
-0.475
catalyst
none
10
3
4
1
2
3
4
5
6
7
8
9
252
188
438
608
777
827
941
1019
1113
797
646
705
957
6
7
b
27.9
b
b
b
c
215
165
389
8
a
-3
-3
HRP
a
[
1100
[
1]=[2]=[3]=[7] =[6] =5.0ꢀ 10 Mand[4] =[8] =2.5ꢀ 10 M.
-4
-2
CI Acid Orange 7] = 1.0 ꢀ 10 M, [H
2
O
2
] = 3.0 ꢀ 10 M,
-5
-4
[
CTAB] =8.0ꢀ 10 M, [1] =[2] =[3] =[6] =[7] =1.0ꢀ 10 M, [4] =
- -5
6
b
[
ref 4. Without CTAB.
5] = [8] = [9] = 5.0 ꢀ 10 M, and [HRP] = 1.0 ꢀ 10 M. Cited from
c
Peroxidative Decoloration of Azo Dye in Micellar Solu-
tions. Effect of Surfactants. To examine the effects of sur-
factants and the porphyrin structures on the chemoselectivity
variations in the peroxide oxidation, we investigated the decolora-
tion of CI Acid Orange 7 catalyzed by MnChls in micellar
solutions, as shown in Scheme 1. For example, Figure 1 shows
that for various micellar solutions semilogarithmic plots of C /Ct
0
against decoloration time gave a straight line passing through the
origin in case of CI Acid Orange 7 in the presence and absence of
MnPChlide a ME 2. C and C are the dye concentrations in the
initial solution and at time t, respectively. The decoloration of azo
dye was evidently observed in the presence of 2 with surfactants.
The decoloration rates were first order with respect to the dye
concentration for the times as shown in Figure 1. Equation 1
provides numerical values that can be used later to compare the
compounds and the influence of various conditions, such as pH
and temperature:
Figure 1. Plot of ln(C
Orange 7 catalyzed by MnPChlide a ME 2 in micellar solution at
0 t
/C ) against decoloration time for CI Acid
0
t
-4
2
3
5 °C and pH 8.0 ([CI Acid Orange 7] = 1.0 ꢀ 10 M, [H
2 2
O ] =
-2
-5
-4
.0 ꢀ 10 M, [2] = 1.0 ꢀ 10 M, [Triton X-100] = 1.5 ꢀ 10 M,
-
3
-4
[
SDS] = 1.0 ꢀ 10 M, [CTAB] = 8.0 ꢀ 10 M): CTAB (circles),
SDS (triangles), Triton X-100 (squares), only H without 2
cross).
2 2
O
(
example, the Qy band of MnPChlide a ME 2 is red-shifted by 2 nm
due to the presence of 100 mM imizazole, consistent with
MnPChlide a-His 3, implying that the Mn atom of the MnChls
lnðC
0
=C
t
Þ ¼ kobs
t
ð1Þ
3
coordinated with the N atom of imizazole. The CV potential
The rate constant k based on first-order kinetics is given in
obs
-1
values of MnChls become negative with increasing the presence of
imidazole as shown in Table 2, this is an observation that is
consistent with the red shift of the Soret or Qy band as shown
units of min and summarized in Tables 3 and 4. The effect of
surfactants on the decoloration rate of the dye has been examined
at pH 8.0 to provide an insight into the charge effect of the
dye-surfactant interaction as shown in Figure 1 and Table 3.
These results presented that the decoloration rate increased in the
3
Table 1. A higher oxidation potential was observed for
MnPChlide a ME 2 than for MnMPDME 6.
7
778 DOI: 10.1021/la904574m
Langmuir 2010, 26(11), 7774–7782

Ishigure et al.
Article
Figure 2. EPR spectra of manganese chlorophyll derivatives, 1 (a), 2 (b), 3 (c), and 4 (d) and manganese porphyrin derivatives, 6 (e) and 7 (f)
-
3
-4
in Egg PC liposomal solution at pH 8.0, 5 K. [1] = [2] = [3] = [6] = [7] = 1 ꢀ 10 M, [4] = 5 ꢀ 10 M, and [H
2 2
O ] = 1.5 M. The spectra
O
2 2
2 2
prior to H addition are given in black, with the spectra after H O presented in red.
Table 5. EPR Spectra of Manganese Chlorophyll a Derivatives and
Manganese Porphyrin Derivatives in Egg PC or DMPC Liposome
Solution at 5 K and pH 8.0
comparison to those in Triton X-100 and SDS, indicating that the
role of CTAB could be important for favoring complex formation
between MnChls and azo dyes, with hydrogen peroxide getting
oxidized. This result indicates that the decoloration rates depend on
the electrostatic interaction between the dye (anion) and the
a
-1
catalyst surfactant split width/G
A
1
/G
A
2
/G
A
1
/A
2
J/cm
1
2
Egg PC
Egg PC
DMPC
Egg PC
DMPC
Egg PC
Egg PC
Egg PC
6
16
16
16
16
6
510
1083
1088
1085
1087
518
hydrophilic part of the surfactant, consistent with the result as
146
148
147
147
70
70
70
70
2.1
2.1
2.1
2.1
-15.3
-45.0
3,4,17
described previously.
Mitsuishi et al. reported that the interac-
tions between methyl orange anion and dodecyltrimethylammo-
3
18
nium cation were hindered due to the presence of sodium chloride.
Their observation is also consistent with our present results.
4
6
7
6
16
510
1071
Effect of Strucutures of Manganese Chlorophyll a Deriva-
tives. Table 4 shows that in CTAB micellar solutions the
decoloration rates increase in the order MnMPDME 6 <
MnMPMME-K(MnMPMME)-OMe 8 < MnPChlide a ME 2 <
MnMPMME-His 7 < MnChl a 1 < MnMPMME-K-
145
69
2.1
-4
-25.5
O ] =
2 2
a
-3
[
.5 M,and [Egg PC] = [DMPC] = 0.1 M.
1] = [2] = [3] = [6] = [7] = 1 ꢀ 10 M, [4] = 5 ꢀ 10 M, [H
1
order CTAB (cation) > Triton X-100 (neutral) > SDS (anion).
The decoloration rate on CTAB and Triton X-100 depended
upon it is concentration, where the maximum decoloration rate
was observed at the critical micelle concentration. Enhanced
decoloration of the dye was observed in the CTAB micelle in
(
MnMPMME)-His 9 < MnPChlide a-His 3 < MnPChlide
(
18) Mitsuishi, M.; Yoshida, D.; Urata, S.; Hamada, K.; Ishiwatari, T. J. Soc.
Fiber Sci. Tech. Jpn 1993, 49, 176–181.
Langmuir 2010, 26(11), 7774–7782
DOI: 10.1021/la904574m 7779

Article
Ishigure et al.
a-K(MnPChlide a)-OMe 4 < MnPChlide a- K(MnPChlide
a)-His 5. The decoloration rate depended on the structures of
the porphyrin derivatives. Comparing the decoloration rate
and the structures of these MnChl a 1 or manganese chlor-
ophyll derivatives, an enhanced decoloration rate was ob-
served for MnChl a 1 in comparison with MnPChlide a ME 2.
This result indicates that the phytol group contributes to
increasing the rate, suggesting that the phytol group plays an
important role in hydrophobic domain formation with
CTAB through the hydrophobic interaction in the micellar
reaction. Further, in these chlorophyll derivatives, the imi-
dazole residue covalently linked to the MnChls for
MnPChlide a-His 3 and MnPChlide a-K(MnPChlide a)-His
decoloration rate depended on the surfactants, structures of the
MnChls, lipids, and the presence of imidazole, as shown in
Tables 3 and 4.
Furthermore, as is apparent from Table 3, the decoloration
rates increase in the order Egg PC < DMPC. Egg PC extracted
from egg yolk is a mixture and bearing verious acyl chains (Tm
∼
- 10 ( 5 °C), while the synthetic phospholipid, DMPC, is single
component with saturated acyl chain (T ∼ 23.6 ( 1.5 °C).
m
Yoshimoto et al. reported that the packing density of phospha-
1
9
tidylcholine (PC) molecules influences the reactivity in which
hydrogen peroxide was more easily decomposed at the surface of
DMPC lipid bilayers than at the surface of unsaturated phos-
pholipid bilayers. Therefore, the interface of liposomal membrane
plays a key role in the catalytic reaction.
9
plays an important role in increasing decoloration due to
coordination between the Mn atom in the porphyrin ring and
the imidazole group of MnPChlide a-His 3. This coordina-
tion causes the red shift of the Soret band of MnChls and the
decrease in the CV potential values as shown in Tables 1 and
Effect of Ligand. The rate of decoloration of CI Acid Orange
7 with hydrogen peroxide catalyzed by MnChls was examined in
the presence of imidazole (10 mM) at pH 8.0, as shown in Table 4.
The decoloration rate increased with the addition of imidazole for
MnChls in micellar solutions. A similar addition effect of imida-
zole on decoloration was observed in liposomal suspensions. It is
interesting to note that the effect of imidazole effect on the
decoloration rate depended on the structures of MnChls in
liposomal suspension as well as in micellar solutions. Surprisingly,
the largest decoloration rate was observed for MnPChlide a-
K(MnPChlide a)-His 5 in CTAB micellar solution, especially
when imidazole was present; this result is analogous to of the
results obtained horseradish peroxidase (HRP) as shown in
Table 4. Thus, interactions between these MnChls and imidazole
play a crucial role in influencing the decoloration rate at the
surface of lipid as well as micellar bilayers.
2
. This imidazole moiety can effectively enhance the deco-
3
,4
loration as described previously. Surprisingly, the MnChls
dimer 4 and 5 enhance decoloration much more effectively
than the corresponding MnChls monomer; this result is
consistent with the result obtained for MnPorphyrins dimer
4
8
and 9. Alternatively, a large increased rate was observed
for MnPChlide a ME 2 compared with MnMPDME 6 in
CTAB micellar solution, as shown in Table 4, in which a high
oxidation potential was observed for MnPChlide a ME 2
(
-0.264 V) than for MnMPDME 6 (-0.456 V) as described in
Table 2. This high oxidation potential of CV is likely to
contributes to the enhanced rate for MnPChlide a ME 2 in
3
,4
comparison to the rate for MnMPMDME 6.
The probable role of imidazole is that it coordinates with Mn as
7
Peroxidative Decoloration of Azo Dye in Liposomal
Suspensions. The effects of lipids and the structures of MnChls
and MnPorphyrins on the peroxidative decoloration of CI Acid
Orange 7 catalyzed by MnChls and MnPorphyrins in liposomal
suspensions were investigated, as shown in Scheme 1.
a ligand and also works as the base catalyst, in which manganese
porphyrins decompose H O and transfer more or less than one
2
2
oxygen atom to the azo dye presumably via a high valent MndO
intermediate such as Mn(IV)dO in the bleaching solution when
imidazole is present. As an axial Mn ligand, imidazole plays an
important role by favoring the heterolytic cleavage of H O ,
The decoloration rates in Egg PC solution increased in the
order MnMPDME 6 < MnChl a 1 < MnMPMME-His 7 <
MnPChlide a-K(MnPChlide a)-OMe 4 , MnPChlide a ME 2 <
MnPChlide a-His 3, as shown in Table 3. Interestingly, less
enhanced decoloration, if at all, was observed in liposomal
suspension for MnChl a 1 and MnPChlide a-K(MnPChlide a)-
OMe 4 incomparison with MnPChlide a ME 2. This resultis quite
different from the order in CTAB micellar solutions and can
probably be attributed to the decrease of the mobility due to the
phytol group of MnChl a 1 or the large size of the dimer molecules
in MnPChlide a-K(MnPChlide a)-OMe 4 in comparison with the
corresponding monomer derivatives, which contributes to the
decreased rate in the lipid bilayers. A similar enhanced rate was
observed for MnPChlide a-His 3 in comparison with the case for
MnPChlide a ME 2 in a liposomal suspension; this result is
consistent with the data for micellar solution and indicates again
that coordination between the Mn atom in the porphyrin ring and
the imidazole group in MnChls plays an important role in
2
2
which yields the MndO intermediate and H O, over a possible
2
homolytic cleavage, which may yield a hydroxyl radical; in
addition, imidazole favors monooxygenation of azo dye over
H O dismutation at the level of an imidazole-MndO inter-
2
2
mediate. Its role as a base catalyst could be important for the
formation of a MndO intermediate from Mn(III) and H O .
2
2
These results imply that the hydroxyl radicals react with the
3
,10
organic azo coloring agent, destroying the chromophore.
However, further investigations will be necessary to determine
the various roles indicated by imidazole in oxidations by H O
2
2
catalyzed by MnChls.
Electron Paramagnetic Spectroscopy Measurement.
Characterization of High Valence Manganese Complex.
All the manganese chlorophyll a derivatives proved to be excellent
catalysts under mild conditions, suggesting the formation of
similar active species of MnChls with decomposition of H O .
2
2
The mechanism of catalyzed decoloration was further investi-
gated by EPR in micellar solution and liposomal suspensions.
Figures 2 and 3 show X-band EPR spectra at 5 K for MnChls,
MnChl a 1, MnPChlide a ME 2, MnPChlide a-His 3, and
MnPChlide a-K(MnPChlide a)-OMe 4, and for MnPorphyrins,
MnMPDME 6 and MnMPMME-His 7, in Egg PC and
DMPC liposomal suspensions. Table 5 presents detailed EPR
spectral data, including the hyperfine valence constant, hyperfine
3
,4
enhancement of the decoloration rate.
The precise mechanism governing the peroxide decoloration
of dyes with hydrogen peroxide in such micellar solution and
liposomal suspension is not fully understood, but it is thought
to be due to hydroxyl radical, reacting with the organic color-
ing agent and destroying the chromophore at the surface of
3
,4
micellar and lipid bilayers. For example, after the decom-
position of CI Acid Orange 7, the dye decoloration products
were 4-sulfophenyldiazene, 4-nitrosobenzenesulfonic acid, and
(
19) Yoshimoto, M.; Miyazaki, Y.; Umemoto, A.; Walde, P.; Kuboi, R.;
3
,10
quinone intermediates.
It is interesting to note that the
Nakao, K. Langmuir 2007, 23(18), 9416–9422.
7
780 DOI: 10.1021/la904574m
Langmuir 2010, 26(11), 7774–7782

Ishigure et al.
Article
Figure 3. EPR spectra ofmanganese chlorophyll derivatives, 2 (a) and 3 (b) inEgg PC(black line) and DMPC (red line) liposomalsolution at
-
3
pH 8.0, 5 K. [Catalyst] = 1 ꢀ 10 M, [H
2 2
O ] = 1.5 M.
Table 5 shows that the ratio of the hyperfine valence constant
data (A /A ) equals 2.1 for manganese complexes. Talsi et al.
1
2
indicate that the ratio of Mn(III)-Mn(IV) complexes corre-
sponds to A /A = 2/1, while the ratio of Mn(II)-Mn(III)
1
2
2
0
complexes corresponds to A /A = 7/4. Thus, these hyperfine
1
2
valence constants imply that the mixed-valence Mn complexes
formed from for MnMPMME-His 7, MnPChlide a ME 2, and
MnPChlide a-His 3 are likely to be Mn(III)-Mn(IV) complexes
rather than Mn(II)-Mn(III) complexes. Dismukes et al. reported
that the Mn(III)-Mn(IV) complex has a total hyperfine band-
width of 1070 G, while the Mn(II)-Mn(III) complex has a
2
1
bandwidth of 1410 G. Table 5 shows that the bandwidth for
MnPChlide a ME 2, MnPChlide a-His 3, and MnMPMME-His 7
is between 1071 and 1088 G, which corresponds the bandwidth of
an Mn(III)-Mn(IV) complex.
Figure 4. Semilogarithmic plot of P1/2 vs 1/T. [Catalyst] = 1 ꢀ
-3
10
M, [H
2 2
O ] = 1.5 M; 2 (triangles), 3 (squares), and 7 (circles).
Stability of the Mixed-Valence Manganese(III)-Manga-
nese(IV) Complex. Figure 4 shows the semilogarithmic plots of
P1/2 vs 1/T prepared from the representative microwave satura-
tion data in the temperature range of 4-30 K for manganese
complexes.
bandwidth, and representative microwave saturation, for MnChls
-2
and MnPorphyrins in Egg PC and DMPC liposome in 1 ꢀ 10
M
Tris-HCl buffer with pH 8.0. Figure 2 and Table 5 show that when
H O was added, EPR spectra of MnPChlide a ME 2, MnPChlide
2
2
a-His 3, and MnMPMME-His 7 contained 16peaks around g = 2
22,23
According to eq 2
in Egg PC or DMPC liposomal suspension, which are typical of a
2
0,21
mixed-valence complex with coupling between two ions.
No
P1=2 µ 1=T
1
¼ expð-Δ=kTÞ
ð2Þ
detectable change in 16 peaks was observed for MnChl a 1,
MnPChlide a-K(MnPChlide a)-OMe 4, and MnMPDME 6 in the
liposomal suspensions. These results suggest that MnPChlide a
ME 2, MnPChlide a-His 3, and MnMPMME-His 7 react with
P1/2 is the microwave power for half-saturation, T1 is the
spin-lattice relaxation time, Δ is the zero-field splitting constant
(
Δ = -3J), k is the Boltzmann constant, and T is the temparature.
-1
J (cm ) values are summarized in Table 5.
H O via electron transfer, yielding a mixed-valence Mn complex
2
The J values in Table 5 indicate that the stability of the mixed-
valence Mn species(as shown in Supporting Information Chart
in the lipid bilayers. Furthermore, the 16 peaks obtained for
MnPChlide a-His 3 or MnMPMME-His 7 were clearer than those
for MnPChlide a ME 2 or MnMPDME 6, respectively, indicating
that the imidazole moiety of the compound contributes to the
formation of themixed-valence Mn complex. In contrast, MnChls
and MnPorphyrins did not provide such EPR spectra in micellar
solutions with H O under this condition.
2
1,24
S1) formed
in the lipid bilayers can be evaluated. The absolute
values increase in the order MnPChlide a ME 2 < MnMPMME-
His 7 < MnPChlide a-His 3. The larger value observed for
MnPChlide a-His 3 than for MnMPMME-His 7 indicates that
Mn(III)-Mn(IV) complexes of the MnChls are more stable than
the MnPorphyrins. In addition, a larger value is also observed for
MnPChlide a-His 3 than for MnPChlide ME 2, thereby indicating
that Mn(III)-Mn(IV) complexes of MnPChlide a-His 3 are more
stable than those of MnPChlide a ME 2 and implying that the
histidine residue contributes to the stability of the mixed-valence
Mn species. These results are consistent with the results obtained
for the X-band EPR spectra described above (Figure 2).
2
2
Interestingly, Figure 3 shows that the EPR spectra of
MnPChlide a ME 2 and MnPChlide a-His 3 contained 16 peaks
in DMPC liposomal suspension as well as in Egg PC when H O
2
2
was added; the peaks were more clearly observed for DMPC
liposomal suspension than for the Egg PC liposomal suspension.
This result indicates that molecular packing by DMPC lipid
bilayers influences on the formation of a mixed-valence Mn
complex; this is consistent with the effective decomposition of
1
9
(22) Yim, M. B.; Kuo, L. C.; Makinen, M. W. J. Magn. Reson. 1982, 46, 247–
56.
H O at the surface of the DMPC lipid bilayers.
2
2
2
(
23) Sarrou, J.; Ioannidis, N.; Deligiannakis, Y.; Petrouleas, V. Biochemistry
(
20) Bryliakov, K. P.; Kholdeeva, O. A.; Vanina, M. P.; Talsi, E. P. J. Mol.
Catal. A 2002, 178, 47–53.
21) Dismukes, G. C.; Sheats, J. E.; Smegali, J. A. J. Am. Chem. Soc. 1987, 109,
202–7203.
1998, 37, 3581–3587.
(24) Mukhopadhyay, S.; Mandal, S. K.; Bhaduri, S.; Armstrong, W. H. Chem.
Rev. 2004, 104, 3981–4026.
(25) Tokuda, J.; Ohura, R.; Nango, M. Textile Res. J. 1999, 69, 456–462.
(
7
Langmuir 2010, 26(11), 7774–7782
DOI: 10.1021/la904574m 7781

Article
Altogether, the EPR results suggest that the higher decolora-
tion performance obtained by these monomer porphyrin com-
pounds at the surface of the lipid bilayers appears to be related to
the stability of this mixed-valence Mn(III)-Mn(IV) species
formed in the lipid bilayers.
Ishigure et al.
MnChls monomer showed enhanced decoloration in comparison
with the MnChls dimer in liposomal suspensions.
Furthermore, when H O was added, the EPR spectra of
2
2
MnMPMME-His 7, MnPChlide a ME 2, and MnPChlide a-His
3 presented the typical of mixed-valence Mn(III)-Mn(IV) com-
plexes with coupling between two ions. The higher decoloration
performance obtained by these monomer porphyrin compounds in
the liposomal suspension appears to be related to the stability of this
mixed-valence Mn(III)-Mn(IV) species formed in lipid bilayers.
This finding should provide useful information to note that
MnChls, whichare easilyfound ina number of biologicalsystems,
are involved in functions such as hydrogen peroxide decomposi-
tion in bacteria and the oxidation of water during photosynthesis
as well as the peroxidases function such as the peroxidative
decoloration as bleaching agents.
Conclusion
MnChls, MnPChlide a ME 2, MnPChlide a-His 3, MnPChlide
a-K-(MnPChlide a)-OMe 4, and MnPChlide a-K(MnPChlide a)-
His 5, and Mn-substituted chlorophyll a (MnChl a) 1 (Chart 1)
were synthesized. MnChls with H O in the presence of imidazole
2
2
efficiently catalyzed the peroxide oxidation of azo dyes (Chart 2)
in micellar solutions and liposomal suspensions under mild
conditions such as pH 8.0 and 25 °C. The peroxidation depended
upon the structures of surfactant, lipids, and MnChls and the
presence of imidazole. The effects of surfactant, lipids, and the
chlorophyll structures on the peroxidation are discussed.
The kinetics of the decoloration of the azo dye by hydrogen
peroxide catalyzed by MnChls in the presence and absence of
imidazole were evaluated. Interestingly, the imidazole residue
covalently linked with the Mn complexes and the added imidazole
play an important role in increasing the decoloration in both
micellar solutions and liposomal suspensions. The dimer com-
plexes enhanced decoloration in comparison with the correspond-
ing monomer derivatives in the micellar solution. In contrast, the
Acknowledgment. M.N. thanks Dr Alastair T. Gardiner,
University of Glasgow, for helpful discussions. The present work
was partially supported by a Grand-in-Aid from the Ministry of
Education, Science and Culture, Japan.
Supporting Information Available: The structure of [Mn-
(III)-Mn(IV)] μ-oxo chlorophyll dimer on EPR conditions
(Chart S1) and experimental details. This material is avail-
able free of charge via the Internet at http://pubs.acs.org.
7
782 DOI: 10.1021/la904574m
Langmuir 2010, 26(11), 7774–7782