Intramolecular oxidative o-demethylation in a per-o-methylated β-cyclodextriniron porphyrin inclusion complex in aqueous solution

Article abstract of DOI:10.1246/cl.130678

The reaction of a supramolecular heme enzyme model (methemoCD2), a 1:1 inclusion complex of 5,10,15,20-tetrakis(4-sulfonatophenyl)porphinatoiron(III) (Fe^IIITPPS) and a per-Omethylated β-cyclodextrin dimer having a OCH₂PyCH₂O (Py: pyridin-3,5-diyl) linker (Py2CD), with peroxy acid (m-chloroperbenzoic acid or peracetic acid) caused intramolecular mono- and di-O-demethylations of the OCH₃ group(s) of Py2CD. The O-demethylation seemed to proceed through the hydroxylation of a CH bond of OCH₃ by an oxoiron(IV) porphyrin π-cation radical (O=Fe ^IVP^·+).

Full text of DOI:10.1246/cl.130678

doi:10.1246/cl.130678
1
366
Published on the web August 22, 2013
Intramolecular Oxidative O-Demethylation in a Per-O-methylated β-Cyclodextrin­Iron
Porphyrin Inclusion Complex in Aqueous Solution
Takunori Ueda, Saki Kumeda, Hiroaki Kitagishi, and Koji Kano*
Department of Molecular Chemistry and Biochemistry, Faculty of Science and Engineering,
Doshisha University, Kyotanabe, Kyoto 610-0321
(
Received July 22, 2013; CL-130678; E-mail: kkano@mail.doshisha.ac.jp)
The reaction of a supramolecular heme enzyme model (met-
hemoCD2), a 1:1 inclusion complex of 5,10,15,20-tetrakis(4-
III
sulfonatophenyl)porphinatoiron(III) (Fe TPPS) and a per-O-
methylated β-cyclodextrin dimer having a OCH2PyCH2O (Py:
pyridin-3,5-diyl) linker (Py2CD), with peroxy acid (m-chlo-
roperbenzoic acid or peracetic acid) caused intramolecular
mono- and di-O-demethylations of the OCH3 group(s) of
Py2CD. The O-demethylation seemed to proceed through the
hydroxylation of a C­H bond of OCH3 by an oxoiron(IV)
IV •+
porphyrin π-cation radical (O=Fe P ).
The hydroxylation of C­H bonds catalyzed by cytochrome
P450 (Cyt P450) is an important chemical reaction among
Figure 1. Structures of Py3CD, Py2CD, met-hemoCD1, and
met-hemoCD2.
1
metabolic processes. An oxoiron(IV) porphyrin π-cation radical
IV •+
(
O=Fe P , Cpd I), known to be the active species in Cyt P450-
1
,2
catalyzed oxidation, is formed via the heterolytic cleavage of
the O­O bond of a hydroperoxoiron(III) porphyrin (HOO­Fe P,
(•OH) generated by the homolysis of the O­O bond of HOO­
III
III 15,17
Fe P.
Protection of the porphyrin ring by the CD dimer
Cpd 0) produced by reductive activation of an oxyiron(II)
enabled us to observe the formation of the high-valent oxoiron
porphyrin complex in aqueous solution. In the present study, we
investigated the reaction of met-hemoCD2 with m-CPBA and
peracetic acid (PAA) to explore whether O=Fe P is formed in
the CD cage.
II
2­4
complex (O ­Fe P). Strong electron donation from an axial
2
thiolate ligand to the iron is considered to be essential for the O­
O bond heterolysis in Cpd 0. A number of model reactions have
been studied. In the model systems, m-chloroperbenzoic acid (m-
CPBA) has been widely used as an oxidant to produce the Cpd I-
like active species, because the electron-attracting nature of the
RCO-group of an acylperoxoiron(III) porphyrin (RC(O)OO­
IV •+
The addition of m-CPBA (20 equiv) to met-hemoCD1
III
¹5
([Fe TPPS] = [Py3CD] = 1 © 10 M) in 0.05 M phosphate
buffer at pH 7 and 25 °C caused significant bleaching due to
decomposition of the porphyrin ring (Figure 2A). In addition,
because met-hemoCD1 has two thioether bonds at the linker
position of the CD dimer, Py3CD may be directly oxidized to the
III
Fe P) causes heterolytic cleavage of the O­O bond even in the
absence of a thiolate ligand.3 Previous model studies revealed
­8
that the oxoiron(IV) porphyrin π-cation radicals are the active
8
18
reaction intermediates in the hydroxylation of alkanes and
sulfoxide and/or sulfone by m-CPBA. Previously, we observed
IV
oxidative N- and O-demethylations of amines and ethers,
intramolecular oxygen transfer from O=Fe P to a sulfide bond
6
,9
16
respectively.
of Py3CD. It was concluded, therefore, that met-hemoCD1 was
IV •+
We have been studying new types of supramolecular heme
protein models composed of 5,10,15,20-tetrakis(4-sulfonatophe-
not suitable for studying the formation of O=Fe P in the
reaction with m-CPBA. In the present study, we used met-
III
nyl)porphinatoiron (FeTPPS) and per-O-methylated β-cyclo-
hemoCD2 (a 1:1 complex of Fe TPPS and Py2CD, Figure 1), in
dextrin dimers having nitrogenous axial ligands.1
0­14
We
which the two cyclodextrin units were bound together by a
OCH2PyCH2O (Py: pyridin-3,5-diyl) linker. Unlike in the case
of met-hemoCD1, no bleaching occurred and biphasic spectral
changes were observed by UV­vis spectroscopy for the reaction
of met-hemoCD2 with m-CPBA (Figure 2B). An intermediate
species with ­max values of 400, 531, and 700 nm (Cpd X)
appeared immediately after the addition of m-CPBA to met-
hemoCD2 (­max = 409 and 569 nm) in 0.05 M phosphate buffer
at pH 7 and 25 °C. The spectrum of Cpd X gradually changed to
show ­max at 422 and 558 nm, indicating the formation of the
designed two types of supramolecular complexes, namely,
II
III
Fe TPPS and Fe TPPS called hemoCDn (n = 1 and 2) and met-
hemoCDn, respectively (Figure 1). The FeTPPS in these models
is surrounded by hydrophobic cyclodextrin cavities, similar to
the manner in which heme or hemin is surrounded by proteins.
1
1­13
Such supramolecules behave similar to myoglobin
and met-
1
0,14
myoglobin
the reaction of an inclusion complex of Fe TPPS and Py3CD
met-hemoCD1) with hydrogen peroxide yielded a very stable
in aqueous solution. We recently found that
III
(
IV
IV
15
IV
oxoiron(IV) porphyrin (O=Fe P) through the homolytic cleav-
age of the O­O bond of HOO­Fe P in the cyclodextrin (CD)
In the absence of Py3CD, the porphyrin ring of
Fe TPPS was rapidly decomposed upon the addition of
hydrogen peroxide due to the attack of the hydroxyl radical
oxoiron(IV) complex (O=Fe P). The resulting O=Fe P
III
19
gradually returned to Cpd X upon standing for 30 h (Figure S1).
The results suggested that Cpd X was not O=Fe P .
1
5,16
IV •+
cage.
III
The UV­vis spectrum of Cpd X at pH 7.0 was almost
identical to that of a monoaqua form of met-hemoCD2 at pH 5
Chem. Lett. 2013, 42, 1366­1368
© 2013 The Chemical Society of Japan
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1
367
Figure 3. (A) HPLC traces for the CD component extracted
from the aqueous solution containing Cpd X. (B) MALDI-TOF
mass spectra for the fractionated solutions of (A). The calculated
mass number of Py2CD (C131H225NO70) is 2932.
analyzed by reverse-phase HPLC (Figure 3A). The MALDI-
TOF mass spectra of peaks A and B in the HPLC trace
(
Figure 3B) corresponded to demethylated Py2CD and Py2CD
itself, respectively. The mass peaks of fraction A were observed
at m/z 2957 and 2941, which corresponded to [Py2CD +
+
+
K ¹ 14] and [Py2CD + Na ¹ 14] , respectively. The results
strongly suggested that the compound corresponding to peak A
OH
was a monohydroxylated Py2CD (Py2CD ), wherein an OCH3
¹
5
Figure 2. UV­vis spectra of met-hemoCD1 (1 © 10 M) (A)
¹
5
group of Py2CD was converted to an OH group. The reactions
of met-hemoCD2 with excess PAA (2­5 equiv) yielded dihy-
and met-hemoCD2 (1 © 10 M) (B) before (green) and after
¹
4
addition of m-CPBA (2 © 10 M, black) in 0.05 M phosphate
buffer at pH 7 and 25 °C. The spectra after the addition of m-
CPBA were recorded at time intervals of 20 s. Inset of (B) shows
time course of the absorbances at 400 and 422 nm.
2
OH
droxylated Py2CD (Py2CD ), along with the monohydroxy-
OH
19
lated product (Py2CD ) (Figure S6). Interestingly, more
hydroxylated Py2CD products were not observed despite the
presence of 14 primary (6-OCH3) and 26 secondary OCH3
Figure S2).19 Because the pK value of the axial H O of met-
groups (2- and 3-OCH ) in Py2CD. The H NMR spectrum of
1
(
a
2
3
1
2
¹
OH
hemoCD2 is 6.9, both H2O and OH can act as the sixth axial
ligand of met-hemoCD2 before the reaction at pH 7. Therefore,
it was predicted that Cpd X would have a structure similar to
met-hemoCD2, but the pKa of the axial H2O of Cpd X would be
higher than that of met-hemoCD2. The EPR spectrum of met-
hemoCD2 at 77 K (Figure S3) showed a pattern typical for a
high-spin iron(III) porphyrin,15 with a pH-dependent g¦ signal.
At pH 7, split peaks at g¦ = 6.09 and 6.34 were observed,
whereas a single peak at g¦ = 6.09 was measured at pH 5.
Py2CD isolated by HPLC was quite complex because of its
dissymmetric structure, but showed marked changes in the
signals of the secondary OCH3 groups while slight changes
were observed in the signals of the primary OCH3 groups
1
9
(Figure S7). The H­H COSY spectrum indicated that the
signal at 5.07 ppm could be ascribed to an OH group at the
3-position, formed by the demethylation reaction (Figure S7). It
was found that the reaction of met-hemoCD2 with peroxy acids
such as m-CPBA and peracetic acid caused the conversion of
19
These results suggested that the signals at g = 6.09 and 6.34
could be assigned to the H2O- and OH -coordinated met-
hemoCD2s, respectively. The EPR spectrum of Cpd X, showing
one and/or two OCH groups at the 3-position(s) of Py2CD to
¦
¹
3
OH group(s) through oxidative O-demethylations.
2
0,21
Oxidative N- and O-demethylations catalyzed by Cpd I
6
,9
a single peak at g = 6.09 at pH 7, was quite similar to that of
and its mimics are known. The plausible mechanism for the
oxidative O-demethylation reaction of met-hemoCD2 is shown
¦
met-hemoCD2 at pH 5, indicating that the iron center of Cpd X
was in the iron(III) state with an axial H2O ligand. The pH
titration for Cpd X (Figure S4)19 showed spectral changes
in Scheme 1. The O-demethylation reaction proceeds through
IV •+
the hydroxylation of the OCH group by O=Fe P , followed
3
1
5
ascribable to an acid­base equilibrium of the axial H2O at
around pH 7­10 (pKa = ca. 8.2, vide infra). The increase in the
pK value of the axial H O of Cpd X suggested a dramatic
by the elimination of formaldehyde from the resultant acetal. In
the present system, formaldehyde was detected in the reaction
1
9
mixture of met-hemoCD2 with PAA (Figure S8). Furthermore,
a
2
II
change in the coordination environment at the distal site of the
iron porphyrin.
O-demethylation did not occur when Py2CD or a Zn TPPS/
Py2CD complex was reacted with m-CPBA (data not shown).
These results indicate that the iron center of met-hemoCD2 plays
an essential role in the O-demethylation reactions. The fact that
To identify the structure of Cpd X, the CD dimer component
was extracted from the aqueous solution of Cpd X with CHCl3.
In order to exclude the oxidant and its derivative from the
CHCl3 extract, water-soluble peracetic acid (PAA), which
showed the same behavior as m-CPBA (Figure S5),19 was used.
The reaction mixture obtained by mixing met-hemoCD2
IV
an oxoiron(IV) derivative of hemoCD1 (O=Fe P) did not
1
5,16
induce demethylation of Py3CD
strongly suggests that
IV •+
O=Fe P is the reactive intermediate. A stopped-flow analysis
did not reveal the formation of the O=Fe P intermediate,
IV •+
III
¹4
(
1
[Fe TPPS] = [Py2CD] = 1.0 © 10 M) with PAA (1.5 ©
0
but provided a pseudo-first-order rate constant (kobs) for the
¹
4
19
M) at room temperature for 5 min was extracted with
formation of Cpd X (Figure S9). A linear relationship between
CHCl3. The organic layer was concentrated, and the residue was
kobs and [m-CPBA] indicated that the formation of an acylperoxo
Chem. Lett. 2013, 42, 1366­1368
© 2013 The Chemical Society of Japan
www.csj.jp/journals/chem-lett/

1
368
Table 1. O2 binding affinities (P1/2) and half-lifetimes for
II
OH
autoxidation (t1/2) of hemoCD2 and Fe TPPS/Py2CD
.05 M phosphate buffer at pH 7 and 25 °C
in
0
P1/2/Torr
t1/2/h
Ref
II
Fe TPPS/Py2CD
hemoCD2)
176
¨
12
(
II
OH
Fe TPPS/Py2CD
17
4
this work
cyclodextrin cavity and effect the water-catalyzed autoxidation
II
OH
23
of the O2­Fe TPPS/Py2CD complex.
In conclusion, we found that the reaction of a 1:1 inclusion
III
complex of Fe TPPS and Py2CD (met-hemoCD2) with m-
CPBA or PAA generated a very active oxoiron(IV) porphyrin
π-cation radical (O=Fe P ), which oxidized a neighboring
Scheme 1. Schematic representation for the reaction of met-
hemoCD2 with peroxy acids.
IV •+
OCH group in Py2CD, leading to demethylation and affording
3
III
OH
the Fe TPPS/Py2CD complex. The only structural difference
between Py2CD and Py2CD was the OH group introduced
III
OH
intermediate (RC(O)OO­Fe P) was the rate-determining step.
III
Very rapid heterolysis of the O­O bond of RC(O)OO­Fe P
into the 3-position of the per-O-methylated cyclodextrin dimer.
The single hydroxylation of Py2CD drastically altered the
properties of its FeTPPS complexes, such as the pKa of the axial
H O, the dioxygen affinity, and the autoxidation rate of the O
IV •+
afforded O=Fe P , which then oxidized the OCH3 group to
III
yield the acetal (OCH2OH) and Fe P. The acetal decomposed to
the alcohol (Cpd X) and formaldehyde. In the presence of excess
oxidant, two OCH3 groups at the 3-positions of Py2CD were
converted to the OH groups. Because further demethylation
was impossible due to the inadequate positional relationships
2
2
adduct.
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1
2
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3
IV •+
IV •+
O=Fe P , subsequently formed O=Fe P seemed to be
2
IV
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3
4
W. Nam, Acc. Chem. Res. 2007, 40, 522.
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IV •+
IV
reduction of O=Fe P in Cpd X to O=Fe P seemed to
IV •+
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IV
5
6
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III
HPLC formed an inclusion complex with Fe TPPS. The pKa
1
997, 119, 12008.
III
OH
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8
9
1
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OH
Fe TPPS/Py2CD
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2
004, 126, 15202.
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OH
II
OH
the Fe TPPS/Py2CD
(
complex (O ­Fe TPPS/Py2CD
)
2
Ed. 2005, 44, 435.
1
9
II
OH
Figure S11). This O2­Fe TPPS/Py2CD
complex could
1
2 K. Kano, Y. Itoh, H. Kitagishi, T. Hayashi, S. Hirota, J. Am. Chem.
Soc. 2008, 130, 8006.
II
OH
II
be converted to the Fe TPPS/Py2CD
and CO­Fe TPPS/
OH
Py2CD complexes under N and CO atmospheres, respec-
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2
II
tively. The oxygen binding affinity (P1/2) of the Fe TPPS/
OH
Py2CD
complex and the autoxidation rate of the O2­
5
2, 6894.
II
OH
Fe TPPS/Py2CD complex were quite different from those
of hemoCD2 (Table 1). P1/2 is the partial O2 pressure, at which
half of the O2-receptor molecules are oxygenated. The P1/2 of
the Fe TPPS/Py2CD complex was determined to be 17 Torr
in phosphate buffer at pH 7.0 and 25 °C, whereas that of
hemoCD2 was 176 Torr. It was noteworthy that the O2 affinity
dramatically increased upon the modification of only one OCH3
group in Py2CD to an OH group. Although we need to carry out
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1
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II
OH
1
1
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1
9 Supporting Information is available electronically on the CSJ-
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II
OH
higher O2 affinity of the Fe TPPS/Py2CD
complex is
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2
1 Y. Goto, T. Matsui, S.-i. Ozaki, Y. Watanabe, S. Fukuzumi, J. Am.
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hydrogen bonding between the OH group at the 3-position of
OH
II
Py2CD and the O2 bound to Fe TPPS, leading to a slower
2
2 F. Tani, M. Matsu-ura, K. Ariyama, T. Setoyama, T. Shimada, S.
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II
OH
22
dissociation rate of the O2­Fe TPPS/Py2CD complex. The
replacement of a single OCH group by an OH group in Py2CD
3
may allow the surrounding water molecules to penetrate into the
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Chem. Lett. 2013, 42, 1366­1368
© 2013 The Chemical Society of Japan
www.csj.jp/journals/chem-lett/