Synthesis and characterization of alkanethiolate-coordinated iron porphyrins and their dioxygen adducts as models for the active center of cytochrome p450: Direct evidence for hydrogen bonding to bound dioxygen

Article abstract of DOI:10.1021/ja003430y

Two kind of novel cytochrome P450 models, which have alkanethiolate axial ligands and hydroxyl groups inside molecular cavities, were designed and synthesized as functional O₂ binding systems. A superstructured porphyrin, designated as "twin-coronet" porphyrin, was used as the common framework of the model complexes. This porphyrin bears four binaphthalene bridges on the both sides and forms two pockets surrounded by the bulky aromatic rings. Thiobenzyloxy and thioglycolate moieties, which contain an alkanethiolate group exhibiting various electron-donating abilities and degrees of bulkiness, were covalently linked to twin-coronet porphyrin to yield thiolate-coordinated hemes, TCP-TB and TCP-TG (twin-coronet porphyrin with thiobenzyloxy and thioglycolate groups), respectively. Both ferric complexes exhibited high stability during usual experimental manipulation under air and were characterized by MS, UV/vis, ESR spectroscopies, and CV. The ESR spectra exhibited low-spin signals (TCP-TB: g = 2.334, 2.210, 1.959; TCP-TG: g = 2.313, 2.209, 1.966). The cyclic voltammogram of TCP-TB in CH₃CN gave a quasi-reversible wave which corresponds to the Fe^III/Fe^II redox couple: E_p/2 = -1.35 V (vs Fc/Fc⁺). On the other hand, TCP-TG showed a fine reversible wave: E_1/2 (Fe^III/Fe^II) = -1.12 V. The stable dioxygen adducts were formed in the reaction of the ferric complexes with KO₂ under an oxygen atmosphere and characterized by UV/vis and resonance Raman (RR) spectroscopies. In the RR spectra, the v(O-O) bands of the dioxygen adducts were observed at 1138 cm^-1 (TCP-TB) and 1137 cm^-1 (TCP-TG). The hypothesis that hydrogen bonding between the bound oxygen and the hydroxyl groups of the binaphthyl moieties could increase their stability was verified by RR spectroscopy. When all hydroxyl groups were deuterated, only the frequencies of the v(O-O) bands were upshifted by 2 cm^-1 without any perturbation in the porphyrin skeleton. This work shows the first direct evidence for a hydrogen bond to dioxygen in an oxy form of a thiolate-coordinated heme model system. These results are discussed in context of the process of dioxygen binding and activation in cytochrome P450.

Full text of DOI:10.1021/ja003430y

J. Am. Chem. Soc. 2001, 123, 1133-1142
1133
Synthesis and Characterization of Alkanethiolate-Coordinated Iron
Porphyrins and Their Dioxygen Adducts as Models for the Active
Center of Cytochrome P450: Direct Evidence for Hydrogen Bonding
to Bound Dioxygen
Fumito Tani,1a Mikiya Matsu-ura,1a Shinya Nakayama,1a Mari Ichimura,1b
Nobuhumi Nakamura,1a and Yoshinori Naruta*,1a
Contribution from the Institute for Fundamental Research of Organic Chemistry, Kyushu UniVersity,
Higashi-ku, Fukuoka 812-8581, Japan, and Department of Chemistry, Graduate School of Science,
Kyoto UniVersity, Sakyo-ku, Kyoto 606-8502, Japan
ReceiVed September 19, 2000
Abstract: Two kinds of novel cytochrome P450 models, which have alkanethiolate axial ligands and hydroxyl
groups inside molecular cavities, were designed and synthesized as functional O2 binding systems. A
superstructured porphyrin, designated as “twin-coronet” porphyrin, was used as the common framework of
the model complexes. This porphyrin bears four binaphthalene bridges on the both sides and forms two pockets
surrounded by the bulky aromatic rings. Thiobenzyloxy and thioglycolate moieties, which contain an
alkanethiolate group exhibiting various electron-donating abilities and degrees of bulkiness, were covalently
linked to twin-coronet porphyrin to yield thiolate-coordinated hemes, TCP-TB and TCP-TG (twin-coronet
porphyrin with thiobenzyloxy and thioglycolate groups), respectively. Both ferric complexes exhibited high
stability during usual experimental manipulation under air and were characterized by MS, UV/vis, ESR
spectroscopies, and CV. The ESR spectra exhibited low-spin signals (TCP-TB: g ) 2.334, 2.210, 1.959;
TCP-TG: g ) 2.313, 2.209, 1.966). The cyclic voltammogram of TCP-TB in CH3CN gave a quasi-reversible
wave which corresponds to the FeIII/FeII redox couple: Ep/2 ) -1.35 V (vs Fc/Fc+). On the other hand, TCP-
TG showed a fine reversible wave: E1/2 (FeIII/FeII) ) -1.12 V. The stable dioxygen adducts were formed in
the reaction of the ferric complexes with KO2 under an oxygen atmosphere and characterized by UV/vis and
resonance Raman (RR) spectroscopies. In the RR spectra, the ν(O-O) bands of the dioxygen adducts were
observed at 1138 cm-1 (TCP-TB) and 1137 cm-1 (TCP-TG). The hypothesis that hydrogen bonding between
the bound oxygen and the hydroxyl groups of the binaphthyl moieties could increase their stability was verified
by RR spectroscopy. When all hydroxyl groups were deuterated, only the frequencies of the ν(O-O) bands
were upshifted by 2 cm-1 without any perturbation in the porphyrin skeleton. This work shows the first direct
evidence for a hydrogen bond to dioxygen in an oxy form of a thiolate-coordinated heme model system.
These results are discussed in context of the process of dioxygen binding and activation in cytochrome P450.
Introduction
heme in P450 causes distinct spectroscopic signatures as well
as being implicated as a critical structural factor influencing
P450’s unique reactivity. Both of these distinctive functional
and structural features of cytochrome P450 have prompted
chemists to prepare synthetic model systems5 in addition to
studying the enzyme itself.
A limited number of chemical methodologies2 have been
reported for well-controlled oxygenation of organic substrates
by dioxygen with concomitant consumption of a stoichiometric
amount of a sacrificial substrate. On the other hand, cytochrome
P450 isozymes readily utilize dioxygen to catalyze oxygenation
in various biosyntheses of endogenous organic compounds and
in detoxification of exogenous ones, generally with high
efficiency and selectivity.3 Gaining an understanding of the
oxygenation reaction mechanism of cytochrome P450 could
contribute to the design of new oxidation catalysts. Cytochrome
P450 as a hemoprotein is structurally distinguished from the
large majority of hemoproteins by the presence of a unique
thiolate group as an axial ligand to the heme.4 The strong
electron-donor character of the cysteinyl thiolate group to the
In most reactions mediated by cytochrome P450’s, one
oxygen atom of a heme-bound dioxygen molecule is inserted
into a substrate via heterolysis of the O-O bond, with the other
(4) Poulos, T. L.; Finzel, B. C.; Gunsalus, I. C.; Wagner, G. C.; Kraut,
J. J. Biol. Chem. 1985, 260, 16122.
(5) For typical examples, (a) Ogoshi, H.; Sugimoto, H.; Yoshida, Z.
Tetrahedron Lett. 1975, 27, 2289. (b) Collman, J. P.; E. Groh, S. J. Am.
Chem. Soc. 1982, 104, 1391. (c) Patzelt, H.; Woggon, W. D. HelV. Chim.
Acta 1992, 75, 523. (d) Higuchi, T.; Uzu, S.; Hirobe, M. J. Am. Chem.
Soc. 1990, 112, 7051. (e) Ueyama, N.; Nishikawa, N.; Yamada, Y.;
Okamura, T.; Nakamura, A. J. Am. Chem. Soc. 1996, 118, 12826. (f) Volz,
H.; Holtzbecher, M. Angew. Chem., Int. Ed. Engl. 1997, 36, 1442. (g)
Wagenknecht, H. A.; Woggon, W. D. Angew. Chem., Int. Ed. Engl. 1997,
36, 390. (h) Aissaoui, H.; Bachmann, R.; Schweiger, A.; Woggon, W. D.
Angew. Chem., Int. Ed. 1998, 37, 7, 2998. (i) Tang, S. C.; Koch, S.;
Papaefthymiou, G. C.; Foner, S.; Frankel, R. B.; Ibers, J. A.; Holm, R. H.
J. Am. Chem. Soc. 1976, 98, 2414.
(1) (a) Kyushu University. (b) Kyoto University.
(2) (a) Mukaiyama, T.; Yamada, T. Bull. Chem. Soc. Jpn. 1995, 68, 17.
(b) Naota, T.; Takaya, H.; Murahashi, S.-I. Chem. ReV. 1998, 98, 2599.
(3) (a) Ortiz de Montellano, P., Ed. Cytochrome P-450. Structure,
Mechanism, and Biochemistry, 2nd ed.; Plenum Press: New York, 1995.
(b) Watanabe, Y.; Groves, J. T. In The Enzymes; Sigman, D. S., Ed.;
Academic Press: San Diego, 1992; Vol. 20, p 405.
10.1021/ja003430y CCC: $20.00 © 2001 American Chemical Society
Published on Web 01/18/2001

1134 J. Am. Chem. Soc., Vol. 123, No. 6, 2001
Tani et al.
oxygen atom being reduced to water with two electrons and
two protons.6 Reductive activation of dioxygen by cytochrome
P450cam, the most thoroughly studied P450, has been postulated
to be assisted not only by the heme itself but also by the
peripheral amino acids such as Cys-357 and Thr-252, etc., which
are conserved in practically all P450 isozymes and are consid-
ered to be essential for enzymatic oxygen activation. The thiolate
group of Cys-357 axially coordinates to the central iron atom
to provide a strong electron donor that increases the electron
density of the terminal oxygen atom of the bound dioxygen.
The hydroxyl group of Thr-252 and a number of water
molecules form a hydrogen-bonding network to relay protons
to the terminal oxygen atom.7,8 The cooperative functions
(push-pull effect)9 of these amino acids could enhance the
heterolysis of the dioxygen, and discourage homolysis, so that
an active oxidizing species is generated efficiently. However,
the entire molecular mechanism of the catalytic cycle, especially
the activation of dioxygen, has not yet been fully verified
because all of the crucial intermediates have not yet been
identified. The dioxygen adduct of P450 is the final intermediate
to be observed directly in the catalytic oxygen activation process
so far. Preparation of an analogous adduct has been pursued in
model studies. However, only a limited number of examples of
dioxygen adducts of thiolate-coordinated hemes have been
reported10 due to their characteristic instability. In contrast, there
are numerous oxy heme model systems with a nitrogen axial
ligand which have been prepared as globin models.11 The most
well-known adducts10a utilized extremely electron-deficient
arenethiolates as the fifth axial ligand in order to prevent ligand
oxidation, instead of an electron-rich alkanethiolate as in the
enzyme. To realize the formation of a stable oxygen adduct
and to simulate the push-pull effect on the O-O heterolysis,
the requisites for the design of a more advanced model are the
following: (1) A hydrophobic pocket for oxygen binding to
iron, (2) axial coordination of a well-sheltered alkanethiolate
anion for the strong electron demand at the stage of O-O bond
cleavage, and (3) hydrogen bonding to the bound dioxygen for
the stabilization of the adduct and for proton donation. A
hydrogen bond to the bound dioxygen has never been observed
for previous models for P450. Thus, we designed and prepared
two kinds of alkanethiolate-coordinated model hemes,12 that
fulfill the above requirements. The stable dioxygen adducts were
prepared from the ferric complexes and characterized by UV/
vis and resonance Raman (RR) spectroscopies. In addition, direct
evidence for hydrogen bonding to the bound dioxygen was
obtained. These complexes increase our understanding of the
mechanism of dioxygen activation by cytochrome P450.
Results and Discussion
Design and Synthesis of P450 Models. The following are
the most important features of our design of TCP-TB and TCP-
TG (twin coronet porphyrins13 with thiobenzyloxy and thio-
glycolate groups): (1) On both sides of the porphyrin plane,
bulky binaphthyl moieties can form hydrophobic molecular
cavities, which are suitable for the creation of an oxygen binding
site to prevent irreversible autoxidation of the iron center. (2)
In one of the cavities, an alkanethiolate group is covalently fixed
to coordinate axially to the central iron and is sterically protected
from undesirable disulfide formation and other oxidative
decomposition. (3) In the opposite cavity, two naphtholic
hydroxyl groups are oriented toward the center above the heme
with the objective of forming a hydrogen bond to bound
dioxygen. (4) To prepare hemes having an alkanethiolate ligand
of different steric and electronic character, two kinds of
alkanethiolates were chosen. The thiolate moiety of TCP-TG
is more compact and less electron-donating due to its ester-
carbonyl group than that of TCP-TB.
First, the major framework, twin-coronet porphyrin (TCP),
is synthesized by making bridges between the ortho positions
of the adjacent meso aryl rings with binaphthyl derivatives
through ethereal linkages (Scheme 1). Next, a protected thiolate
moiety is covalently fixed into one of the cavities. Finally, metal
insertion and the deprotection of the thiolate group are ac-
complished. Hence, meso-tetrakis(2,6-dihydroxylphenyl)por-
phine 1, optically active binaphthyl derivative 5, and protected-
thiolate derivatives 7 or 8 are necessary.
The porphyrin 1 was obtained from meso-tetrakis(2,6-
dimethoxyphenyl)porphine, which was synthesized by clay-
mediated condensation14 between 2,6-dimethoxybenzaldehyde
and pyrrole. Montmorillonite K10 clay functions as an acid
catalyst and an efficient template for macrocycle formation from
linear pyrrole oligomers. In terms of simplicity and yield, the
procedures using K10 are superior to previous ones13a,15 which
utilized Adler16 or Lindsey17 methods. The demethylation
reaction was carried out with pyridinium hydrochloride to give
1 in a satisfactory yield.
To avoid formation of many isomers in the reaction of 1 and
5, optically pure (S)-1,1′-bi-2-naphthol was used as a starting
compound for the synthesis of 5 (Scheme 2). On the basis of
the following requirements, the methoxymethyl (MOM) group
was chosen for the protection of the hydroxyl groups. (1) It
should be stable during the condensation reaction under basic
and high-temperature conditions. (2) It should be compact
enough to avoid possible steric repulsion between the porphyrin
and the binaphthyl derivatives. (3) It should be selectively
removable under mild conditions, because similar ether groups,
phenyl benzyl ether, exist in TCP 9a.
(6) (a) Sono, M.; Roach, M. P.; Coulter, E. D.; Dawson, J. H. Chem.
ReV. 1996, 96, 2841. (b) Shimada, H.; Sligar, S. G.; Xeom, H.; Ishimura,
Y. In Oxygenase and Model Systems; Funabiki, F., Ed.; Kluwer Aca-
demic: Dordrechet, 1997; p 195.
(7) (a) Imai, M.; Shimada, H.; Watanabe, Y.; Matsushima-Hibiya, Y.;
Makino, R.; Koga, H.; Horiguchi, T.; Ishimura, Y. Proc. Natl. Acad. Sci.
U.S.A. 1989, 86, 7823. (b) Gerber, N. C.; Sligar, S. G. J. Am. Chem. Soc.
1992, 114, 8742. (c) Vidakovic, M.; Sligar, S. G.; Li, H.; Poulos, T. L.
Biochemistry 1998, 37, 9211.
Even very careful ortho lithiation and formylation of 2 gave
unavoidably a mixture of dialdehyde 3 and monoaldehyde as a
(8) Schlichting, I.; Berendzen, J.; Chu, K.; Stock, A. M.; Maves, S. A.;
Benson, D. E.; Seet, R. M.; Ringe, D.; Petsko, G. A.; Sligar, S. G. Science
2000, 287, 1615.
(9) (a) Dawson, J. H. Science 1988, 240, 433. (b) Poulos, T. L. AdV.
(13) We already reported that similar TCP complexes catalyzed asym-
metric oxidation of olefins and sulfides with high enantio selectivity: (a)
Naruta, Y.; Tani, F.; Ishihara, N.; Maruyama, K. J. Am. Chem. Soc. 1991,
113, 6865. (b) Naruta, Y.; Tani, F.; Maruyama, K. Tetrahedron: Asymmetry
1991, 2, 533.
(14) Onaka, M.; Shinoda, T.; Izumi, Y.; Nolen, E. Tetrahedron Lett. 1993,
34, 2625.
(15) (a) Tsuchida, E.; Hasegawa, E.; Komatsu, T.; Nakata, T.; Nagano,
K.; Nishida, H. Bull. Chem. Soc. Jpn. 1991, 64, 888. (b) Collman, J. P.;
Lee, V. J.; Zhang, X. Inorg. Synth. 1996, 31, 117.
Inorg. Biochem. 1988, 7, 1.
(10) (a) Schappacher, M.; Ricard, L.; Fisher, J.; Weiss, R.; Bill, E.;
Montiel-Montoya, R.; Winkler, H.; Trautwein, A. X. Eur. J. Biochem. 1987,
168, 419. (b) Kasmi, D. EI.; Tetreau, C.; Lavatte, D.; Momenteau, M. J.
Am. Chem. Soc. 1995, 117, 6041.
(11) Momenteau, M.; Reed, C. A. Chem. ReV. 1994, 94, 659 and
references therein.
(12) For our preliminary reports, see: (a) Tani, F.; Nakayama, S.;
Ichimura, M.; Nakamura, N.; Naruta, Y. Chem. Lett. 1999, 729. (b) Matsu-
ura, M.; Tani, F.; Nakayama, S.; Nakamura, N.; Naruta, Y. Angew. Chem.,
Int. Ed. 2000, 39, 1989.
(16) Adler, A. D. J. Org. Chem. 1967, 32, 476.
(17) Lindsey, J. S.; Wagner, R. W. J. Org. Chem. 1989, 54, 828.

EVidence for H-Bonding to Bound Dioxygen
J. Am. Chem. Soc., Vol. 123, No. 6, 2001 1135
Scheme 1a
a Reagents and conditions: (a) K2CO3, THF, NMP, 110 °C, 19%. (b) trimethylsilyl bromide, CH2Cl2, -40 °C, 96%. (c) pivaloyl chloride,
pyridine, CH2Cl2, 97%. (d) 7, K2CO3, NMP, 100 °C, 62%. (e) Fe(CO)5, I2, toluene, 50 °C, 78%. (f) BuNH2, CH3CN, 48%. (g) 8, 1-(3-dimethylamino-
propyl)-3-ethylcarbodiimide hydrochloride, 4-(dimethylamino)pyridine, CH2Cl2, 36%. (h) Fe(CO)5, I2, toluene, 50 °C, 41% (TCP-TG), 16% (15).
Table 1. 1H NMR Chemical Shifts of Selected Protons of
Scheme 2
Twin-Coronet Porphyrinsa
compound
pyrrole â-H
piv-CH3
NH
9a
9b
10
11
12
8.69, 8.50 (each s)
8.67, 8.49 (each d)
8.74, 8.54 (each s)
8.77, 8.41 (each s)
8.73, 8.70, 8.58, 8.57
8.54, 8.50, 8.16, 8.11
(each d)
-
-
-
-2.42
-2.99
-2.81
-3.01
-3.62
0.51
0.77, 0.74
0.28, 0.17
14
8.82, 8.79, 8.77, 8.76
8.62, 8.55, 8.30, 8.27
(each d)
0.73, 0.68
0.33, 0.23
-2.94
a The detailed H NMR data of 9a-11 are included in Supporting
1
minor product. Fortunately, 3 was purified by recrystallization
from ether to give fine, pale-yellow crystals.18 Reduction of 3
and subsequent chlorination of 4 with NCS under neutral
conditions19 afforded the corresponding dichloride 5 in a good
yield.
Information.
be eclipsed 9a and staggered 9b,20 respectively. Only the
eclipsed isomer 9a was further used in the following reactions.
The MOM-protecting groups were removed with trimethylsilyl
bromide21 in an excellent yield (96%), which more than doubled
the previous yield of ca. 45% observed when pyridinium
p-toluenesulfonate (PPTS) was used as a deprotecting reagent.
Deprotection by PPTS22 resulted in the formation of various
byproducts and a low yield with poor reproducibility. The
selective reprotection of the outer four hydroxyl groups of 10
was accomplished with bulky pivaloyl chloride, while the inner
four hydroxyl groups remained free. Since these porphyrin
molecules 9-11 have high symmetry (D2); the four binaphtha-
According to the procedure of the previous report13a with
some improvements, the major framework of the binaphthalene-
bridged TCP was prepared by the condensation of the octahy-
droxylated TPP 1 and the binaphthyl dichloride 5 under basic
conditions (Scheme 1). A THF-NMP solution of 1 and 5 was
heated at 115-120 °C with K2CO3 under a high-purity nitrogen
atmosphere. The resultant two isomers were separated carefully
by column chromatography and distinguished by characteristic
1H NMR signals of the pyrrole â-protons (Table 1). The less
polar isomer showed signals of the â-protons as a pair of
singlets, while two doublet peaks appeared in the case of the
more polar isomer. On the basis of their spectroscopic features,
the less polar isomer and the more polar one were assigned to
1
lene moieties of each compound are equivalent. The H NMR
spectra of 9-11 showed simpler patterns than those of porphy-
rins 12 and 14, which have no symmetry due to their protected-
thiolate group substituents.
(20) The staggered isomer 9b is omitted in Scheme 1. In 9b, the positions
of the binaphthalene bridges are different between the upper and lower sides
of the porphyrin plane. See ref 13a.
(21) Hanessian, S.; Delorme, D.; Dufresne, Y. Tetrahedron Lett. 1984,
25, 2515.
(22) Monti, H.; Leandri, G.; Klos-Ringquet, M.; Corriol, C. Synth.
Commun. 1983, 13, 1021.
(18) The three-dimensional structure of 3 was revealed by X-ray
crystallographic analysis. Tachi, Y.; Nakayama, S.; Tani, F.; Ueno, G.;
Naruta, Y. Acta Crystallogr. 1999, C55, 1351.
(19) Since MOM-protecting group is labile under acidic conditions,
conventional chlorinating reagents, such as SOCl2 and PCl3, cannot be used.
Corey, E. J.; Kim, C. U.; Takeda, M. Tetrahedron Lett. 1972, 13, 4339.

1136 J. Am. Chem. Soc., Vol. 123, No. 6, 2001
Tani et al.
Scheme 3
The thiolate moiety was designed with the following con-
siderations: (1) It should contain another functional group,
which can be linked to one of the inner hydroxyl groups. (2) It
should not be too large to fit inside the sterically hindered cavity.
(3) The sulfur atom should favorably coordinate to the iron.
(4) The protecting group of the thiolate should be intact at the
stage of the condensation reaction and should be selectively
removable at the final stage under mild conditions, keeping the
other potentially labile groups undamaged, such as the resulting
thiolate group, the O-pivaloyl ester, and the linkage between
the thiolate moiety and the porphyrin. The syntheses of two
different protected thiolate derivatives, 7 and 8, which satisfy
the above requirements, were carried out as follows (Scheme
3). The reaction of thiobenzoic acid with 1,2-bis(chloromethyl)-
benzene was accomplished under basic conditions23 to give
2-(chloromethyl)benzyl thiobenzoate 6. Two electrophilic carbon
atoms in 6, the chloro-substituted carbon and the thioester
carbonyl carbon, are potentially reactive to the nucleophilic
naphtholate anion.24 To enhance the reactivity of the halogen-
substituted carbon, 6 was converted to the corresponding iodide
7 via a halogen-exchange reaction. Ethyldisulfanylacetic acid,
8, was obtained by the reaction of thioglycolic acid and ethyl
disulfide at 50 °C.25 Disulfide protection can be cleaved by
reductive halogenation with I2 as a mild nucleophile.26 The use
of Fe(CO)5 and I2 in the final step of the synthesis of TCP-TG
allows iron insertion and the deprotection to be accomplished
simply and efficiently in one pot.
Figure 1. ESR spectra of (a) TCP-TB and (b) TCP-TG in THF at 77
K. Microwave frequency (a) 9.24924 GHz, (b) 9.24946 GHz, power 1
mW, modulation amplitude 1 mT, modulation frequency 100 kHz.
For the preparation of TCP-TG, ethyldisulfanylacetic acid,
8, was connected to one of the inner hydroxyl groups of 11
through an ester linkage. The resultant coupling product 14 also
shows a 1H NMR spectrum as complicated as that of 12.
Chemical shifts of the thiolate moiety, which was strongly
shielded by the porphyrin ring current, are significantly shifted
to upper field. Chemical shifts for the ethyl group are δ )
-0.44, -1.20 and for the methylene group are δ ) -2.15,
-2.72. It is noteworthy that, at the final step in this synthesis,
the iron insertion and the deprotection of the thiolate group were
simultaneously achieved to yield the thiolate-coordinated ferric
heme TCP-TG with concomitant production of the thiolate-
protected iron complex 15. When the same reaction was carried
out at ambient temperature, 15 was obtained as a major product.
Both of these complexes exhibited sufficient stability for usual
manipulation under aerobic conditions. Compared to the thiolate-
protected ferric complexes, 13 and 15, TCP-TB and TCP-TG
were less polar on silica gel TLC and were easily purified by
column chromatography. Generally, a ferric heme having no
anionic group bears a counteranion and consequently shows high
polarity. On the other hand, a thiolate-coordinated ferric heme
is a neutral molecule due to the negative charge of the
intramolecularly coordinated thiolate anion. This characteristic
TLC behavior provides a convenient probe for the screening
thiolate-coordinated ferric heme complexes.
The coupling reaction between porphyrin 11 and 2-(iodo-
methyl)benzyl thiobenzoate,7, in the presence of K2CO3 gave
the desired 12, where the protected thiolate moiety is connected
1
through an ether linkage. The H NMR spectrum of 12 shows
fairly complicated patterns; the eight pyrrole â-protons appear
separately as eight doublets, and the four pivaloyl groups show
four distinct singlets. This indicates complete loss of the
symmetry in 12. The free base porphyrin 12 was converted to
the corresponding ferric complex 13 with Fe(CO)5 and I2.
Nucleophilic attack of a primary amine to the carboxyl carbon
of a S-ester is favored over that of an O-ester, resulting in the
corresponding thiol and amide.27 On the basis of this knowledge,
the selective deprotection of the thioester group in 13 was
achieved with BuNH2, without any cleavage of the pivaloyl ester
groups, to afford the thiolate-coordinated ferric complex TCP-
TB. This compound was stable enough to allow purification
by column chromatography on silica gel under air.
Spectroscopic Characterization of the Thiolate-Ligated
Model Complexes. ESI-MS spectra of the thiolate-ligated model
complexes showed molecular ion peaks at m/z ) 2509.8 (TCP-
TB) and 2447.0 (TCP-TG), respectively. The isotopic patterns
were in good accordance with their simulation. Moreover, the
observed mass numbers in high-resolution FAB-MS were also
in good agreement with the calculated mass numbers. These
experimental data indicate the successful preparation of the
desired complexes. Electronic spectra of ferric TCP-TB and
TCP-TG in CH2Cl2 were essentially identical, exhibiting Soret
bands at λmax ) 416-418 nm and visible bands at λmax ) 513-
515 nm. The thiolate-protected ferric complexes, 13 and 15,
showed Soret bands at λmax ) 421-423 nm, which are slightly
red-shifted compared to those of the thiolate-ligated complexes.
(23) (a) Ono, N.; Yamada, T.; Saito, T.; Tanaka, K.; Kaji, A. Bull. Chem.
Soc. Jpn. 1978, 51, 2401. (b) Rao, C. G. Org. Prep. Proced. Int. 1980, 12,
225.
(24) In fact, the use of the chloride 6 led only to the formation of the
corresponding benzoyl ester of the naphthol group through ester exchange
reaction instead of O-alkylation.
(25) Armitage, D. A.; Clark, M. J.; Tso, C. C. J. Chem. Soc., Perkin
Trans. I 1972, 681.
(26) Pryor, W. A. Mechanisms of Sulfur Reactions; McGraw-Hill: New
York, 1962; p 60.
ESR spectra of TCP-TB and TCP-TG were measured in
THF at 77 K (Figure 1 and Table 2). The ESR spectra of these
complexes clearly showed near-axial rhombic low-spin signals.
The two crystal field parameters, tetragonality |µ/λ| and rhom-
(27) Fuchs, G. Acta Chem. Scand. 1965, 19, 1490.

EVidence for H-Bonding to Bound Dioxygen
J. Am. Chem. Soc., Vol. 123, No. 6, 2001 1137
Scheme 4
Table 2. ESR Spectroscopic Data of Thiolate-Ligated Hemes
Table 3. Reduction Potentials of TCP-TB, TCP-TG, and
FeTMPCla
ESR g value
crystal field parameter
reduction potentials/V, Fe(III)/Fe(II)
g1
g2
g3
|µ/λ|
8.230
8.512
5.998
|R/µ|
0.414
0.370
0.469
complex
vs Fc/Fc+c
vs NHE
TCP-TB
TCP-TG
P450cam
2.334
2.313
2.45
2.210
2.209
2.26
1.959
1.966
1.91
TCP-TB; Ep/2
TCP-TG; E1/2
FeTMPCl;b E1/2
P450cam;d E1/2
-1.35
-1.12
-0.73
-
(-0.95)
(-0.72)
(-0.33)
-0.33
a
a Reference 5i.
bicity |R/µ|, both of which are calculated from g values, have
been used to evaluate rhombic types of low-spin ferric hemo-
proteins and their model complexes.28 By Bohan’s method,29
two crystal field parameters, |µ/λ| and |R/µ|, were determined
from the three observed g values to be 8.230, 0.414 (TCP-TB)
and 8.512, 0.370 (TCP-TG), respectively. According to the
classification of axial donor ligands based on the crystal field
parameters,30 these values are included in the range of six-
coordinate low-spin ferric hemes with a thiolate axial ligand
and an oxygen-donor ligand.31 Compared with cytochrome
P450cam, TCP-TB and TCP-TG showed higher tetragonality
and lower rhombicity values, indicating relatively high axial
symmetry and strong electron donation from the thiolate ligands.
A previous alkanethiolate-coordinated model complex exhibited
very similar ESR features (g ) 2.32, 2.21, 1.96).5d
(28) (a) Griffith, J. S. Nature 1957, 180, 30. (b) Kotani, M. Suppl. Prog.
Theor. Phys. 1961, 17, 4. (c) Peisach, J.; Blumberg, W. E.; Alder, A. Ann.
N.Y. Acad. Sci. 1973, 206, 310. (d) Blumberg, W. E.; Peisach, J. In Probes
of Structure and Function of Macromolecules and Membranes; Chance,
B., Yonetani, T., Mildoon, A. S., Eds.; Academic: New York, 1971; Vol.
II, p 215. (e) Blumberg, W. E.; Peisach, J. In Bioinorganic Chemistry; Gould,
R. F., Ed.; American Chemical Society: Washington, DC, 1971; p 271.
(29) Bohan, T. L. J. Magn. Reson. 1977, 26, 109.
a 0.1 M Bu4NBF4/CH3CN with Pt working/counter electrodes, [Fe-
por] ) 0.5 mM, at scanning rate 50 mV/sec. b FeTMPCl) iron meso-
tetramesityl-porphyrin chloride. c Fc ) ferrocene. d Reference 6a.
The reduction potentials of TCP-TB and TCP-TG were
determined by cyclic voltammetry (Table 3). A quasi-reversible
FeIII/FeII couple was observed in the voltammogram of TCP-
TB, while TCP-TG showed a reversible couple. The slightly
positive-shifted potential of TCP-TG is ascribed to the coor-
dination of the less electron-donating thiolate ligand relative to
that of TCP-TB. The potentials versus NHE of TCP-TB and
TCP-TG were estimated to be -0.95 and -0.72 V, respectively,
from the observed potentials. This indicates that TCP-TG has
a potential closer to that of low-spin cytochrome P450cam6a than
TCP-TB. The more negative potentials of both TCP-TB and
TCP-TG compared to those of iron meso-tetramesitylporphyrin
chloride and cytochrome P450cam, indicate that strong electron
donation occurs mainly from the thiolate ligands to the iron
atoms.
Formation and Characterization of Dioxygen Adducts.
Valentine and co-workers reported reactions of superoxide
anion, O2-, with ferric porphyrins and found that two types of
reactions occurred which depend on conditions: one-electron
reduction of an iron center or formation of a dioxygen adduct.32
Using their methods, we have obtained dioxygen adducts
directly from the ferric forms of TCP-TB and TCP-TG by
addition of an equimolar amount of KO2 under an oxygen
(30) Sakurai, H.; Yoshimura, T. J. Inorg. Biochem 1985, 24, 75.
(31) Under these ESR conditions in THF, the solvent molecule itself
was likely to the sixth ligand of TCP-TB and TCP-TG, based on the
experimental results. Their UV/vis spectra in THF were quite different from
those in a noncoordinating solvent such as toluene. Nonetheless, addition
of even a small amount (2-3% volume) of THF to their toluene solutions
turned their spectra identical to those in THF, indicating the axial
coordination of THF. The assumption that THF is the sixth ligand is also
consistent with the observed ESR parameters. UV/vis; TCP-TB, λmax
(toluene) 325, 350, 421, 512, 624 nm; TCP-TG, λmax (toluene) 325, 340,
418, 511, 575, 649 nm.
(32) McCandlish, E.; Miksztal, A. R.; Nappa, M.; Sprenger, A. Q.;
Valentine, J. S.; Stong, J. D.; Spiro, T. G. J. Am. Chem. Soc. 1980, 102,
4268.

1138 J. Am. Chem. Soc., Vol. 123, No. 6, 2001
Tani et al.
Figure 3. High-frequency region of resonance Raman spectra of the
dioxygen adduct of TCP-TG. THF/CH3CN, -60 °C, 413.1 nm
excitation, 20 mW: trace A, K16O2 + 16O2; trace B, K18O2 + 18O2.
The RR spectra of the dioxygen adduct of TCP-TP are included in
Supporting Information.
Figure 2. UV/vis spectra of O2, CO, ferric complexes of TCP-TG in
THF at -80 °C: full line, O2 complex; dash-dot line, CO complex;
broken line, ferric complex.
Table 4. Electronic Absorption Data for Ferric, CO, and O2
Complexesa
Table 5. Summary of ν(O-O) Frequencies for Dioxygen Adducts
of Thiolate-Coordinated Hemes
complex
λmax/nm
(TCP-TB)FeIII
(TCP-TB)Fe-CO
(TCP-TB)Fe-O2
(TCP-TG)FeIII
(TCP-TG)Fe-CO
(TCP-TG)Fe-O2
340, 423, 532
complex
ν(O-O)a/cm-1
reference
342, 360, 445, 529
339, 424, 532, 642
337, 424, 530
366, 444, 537
339, 421, 544, 629
(P450cam)Fe-O2/camphor
(P450cam)Fe-O2/adamantanone
(C6HF4S-)(TPpivP)Fe-O2
(C6F5S-)(TPpivP)Fe-O2
(TCP-TB)Fe-O2
1140 (1074)
1147 (1080)
1140 (1080)
1147 (1084)
1138 (1074)
1137 (1073)
36, 37
36
36, 38
36
this work
this work
a In THF, -80 °C.
(TCP-TG)Fe-O2
a Values in parentheses are for 18O2 adducts.
atmosphere (Scheme 4).33 Addition of KO2 to a THF solution
of the ferric complex (TCP-TB, TG)FeIII at -80 °C induced
an immediate and drastic change in the UV/vis spectrum (Figure
2 and Table 4). The absorbance of the Soret band slightly
decreased, and the intensity of Q-band around 530 nm signifi-
cantly decreased. When the atmosphere was replaced with CO,
the dioxygen adducts were converted to the corresponding CO
adducts, which exhibit typical hyperporphyrin spectra with split
Soret bands. The reversibility of the O2/CO exchange indicates
that the ferrous oxidation state and the axial coordination of
the thiolate remain under these conditions without any autoxi-
dation or other oxidative decomposition. The thermal stability
of these dioxygen adducts are fairly high, especially in the case
of TCP-TG. The UV/vis spectra of the adducts were stable
and unchanged at elevated temperatures up to -20 °C for TCP-
TB (t1/2 at -20 °C ≈ 1 h) and 0 °C for TCP-TG (t1/2 at 0 °C
≈ 5 h). The differences in thermal stability of the two complexes
could be due to differences in electron density at the iron,
resulting from the strength of the electron donation from the
axial thiolate ligand, as already shown in the electrochemical
data.
hemoproteins and their model systems.34 RR spectra have also
been obtained for cytochrome P450 enzymes from various
origins.35 We have applied RR spectroscopy to the dioxygen
adducts of TCP-TB and TCP-TG in order to verify the
existence of the Fe-O2 moiety. The RR spectra of the dioxygen
adducts obtained with Soret excitation exhibited a strong line
at 1138 cm-1 for TCP-TB and 1137 cm-1 for TCP-TG (Figure
3). By replacing K16O2 and 16O2 with K18O2 and 18O2, these
lines shifted to 1074 and 1073 cm-1, respectively. The observed
isotopic shifts upon 18O2 substitution are in good agreement with
the value calculated from the harmonic oscillator approximation
of the O-O stretching vibration (∆obs(16O2/18O2) ) 64 cm-1
;
∆
) 65 cm-1). These bands are therefore assigned to the
calc
ν(O-O) mode. The ν(O-O) frequencies of oxy thiolate-
coordinated hemes determined by RR spectroscopy, are sum-
marized in Table 5. The observed frequencies of the ν(O-O)
36,37
modes are very close to those of oxy-cytochrome P450cam
(34) Spiro, T. G. Ed. Biological Applications of Raman Spectroscopy;
John Wiley: New York, 1988; Vol. 3.
(35) (a) Champion, P. M.; Gunsalus, I. C.; Wagner, G. C. J. Am. Chem.
Soc. 1978, 100, 3743. (b) Ozaki, Y.; Kitagawa, T.; Kyogoku, Y.; Imai, Y.;
Hashimoto-Yutsudo, C.; Sato, R. Biochemistry 1978, 17, 5827. (c) Shimizu,
T.; Kitagawa, T.; Mitani, F.; Iizuka, T.; Ishimura, Y. Biochim. Biophys.
Acta 1981, 670, 236.
(36) Hu, S.; Schneider, A. J.; Kincaid, J. R. J. Am. Chem. Soc. 1991,
113, 4815.
(37) (a) Bangchaoenpaurpong, O.; Rizos, A. K.; Champion, P. M.; Jollie,
D.; Sligar, S. J. Biol. Chem. 1986, 261, 8089. (b) Macdonald, I. D. G.;
Sligar, S. G.; Christian, J. F.; Unno, M.; Champion, P. M. J. Am. Chem.
Soc. 1999, 121, 376. (c) Egawa, T.; Ogura, T.; Makino, R.; Ishimura, Y.;
Kitagawa, T. J. Biol. Chem. 1991, 266, 10246.
Resonance Raman (RR) spectroscopy is a very powerful
technique for elucidating the detailed active-site structures of
(33) A dioxygen adduct of a heme is generally prepared stepwise through
reduction of a ferric complex to the ferrous one under aqueous conditions
and subsequent addition of dioxygen gas. However, during the reduction
of ferric TCP-TB and TCP-TG under protic conditions, such as with
aqueous dithionite solution, the thiolate-ferrous complexes are easily
protonated to turn into the thiol-coordinated ones with the neutralization of
the unstable negative charge. Therefore, we selected the direct aprotic
method by the use of KO2. See ref 12a.

EVidence for H-Bonding to Bound Dioxygen
J. Am. Chem. Soc., Vol. 123, No. 6, 2001 1139
and oxy-model complexes.36,38 A Raman active ν(O-O) mode
has been found to be characteristic of the dioxygen adduct of a
thiolate-coordinated heme. In contrast, the ν(O-O) mode of
an oxy heme with an axial ligand of a nitrogen-base is Raman
inactive.39 Simultaneous enhancement of M-O2 and O-O
stretching vibrations was first observed by Yu and co-workers
in oxycobalt myoglobin and hemoglobin.40 They suggested that
the enhancement was attributed to the charge-transfer transition
2
from the occupied π*(pg*/dxz) to empty antibonding σ*(dz /pg*)
orbital. For an oxy iron porphyrin, it is impossible to have charge
transfer from π* to σ*, since the π* orbital is no longer
populated. However, RR enhancement of ν(Fe-O2) and
ν(O-O) was found in a thiolate-coordinated oxy heme and
explained as follows: the thiolate ligand increases the electron
density of porphyrin π* through pπ and dπ to promote the
charge-transfer transition from the π* to the empty Fe-O π*
orbital of the oxy form which then leads to the enhancement of
ν(O-O) band. Therefore, the present RR results reveal suc-
cessful formation of the desired dioxygen adducts of the thiolate-
coordinated hemes. However, no oxygen-sensitive bands,
corresponding to ν(Fe-O2) and δ(Fe-O-O), were detectable
in the low-frequency region in the RR spectra of both the
adducts with Soret excitation (413.1, 441.6 nm)
Figure 4. Selected region of resonance Raman spectra of the dioxygen
adduct of TCP-TG on H/D exchange experiment. THF/CH3CN, -80
°C, 413.1 nm excitation, 20 mW: trace A, CH3OH; trace B, CD3OD;
top, difference spectrum A - B. The RR spectra of the dioxygen adduct
of TCP-TB on H/D exchange experiment are included in Supporting
Information.
Table 6. Comparison of ν(O-O) Frequencies on H/D Exchange
Experiment
It is notable that the oxidation marker band (ν4) of oxy TCP-
TB and TCP-TG appeared at 1368 cm-1. This value is lower
∆(H/D)/
complex
CoMbO2
CoMbO2
CoHbO2
CoHbO2
ν(O-O)/cm-1
cm-1
ref
41a
41b
41a
41b
this work
this work
36
by 6 cm-1 than that of the oxy-cytochrome P450cam
.
The
1134 (H2O)
1136 (D2O)
1139 (D2O)
1138 (D2O)
1140 (D2O)
2
3
5
4
2
2
vibrational modes of porphyrin skeleton are empirically cor-
related with heme core size and are very useful for determining
the spin and oxidation state of the heme iron. The ν4 band, which
can be clearly identified without interference from other modes,
is sensitive to the electron population in the π* antibonding
orbitals of the porphyrin ring. When the π* orbital increases in
population, the frequency of ν4 decreases. Hence, the electron
densities of the iron centers of our dioxygen adducts are
1136 (H2O)
1133 (H2O)
1136 (H2O)
(TCP-TB)Fe-O2 1138 (CH3OH) 1140 (CD3OD)
(TCP-TG)Fe-O2 1137 (CH3OH) 1139 (CD3OD)
independently reported the upshifts of ν(O-O) mode of
oxycobalt myoglobin and hemoglobin on replacement of H2O
with D2O.41 However, their interpretations of the ν(O-O) mode
upshifts were different. Kitagawa et al. considered the upshifts
as evidence for hydrogen bonding of bound dioxygen to the
distal histidine of oxycobalt myoglobin and hemoglobin.41a On
the other hand, Kincaid et al. observed the vibrational coupling
of ν(O-O) and the internal modes of the proximal histidinyl
imidazole trans to the dioxygen and concluded that the observed
upshifts were derived from the changes of the coupling because
of an alteration in the internal modes of the imidazole group
(which has also an exchangeable proton) due to H/D exchange.41b,c
In the present case, the possibility of the coupling effect can be
ruled out by the following experimental results: (1) The -64
cm-1 shift upon 18O substitution matches well the shift of -65
cm-1 predicted by Hooke’s law for a diatomic O-O stretch.
(2) In the structures of both TCP-TB and TCP-TG complexes,
exchangeable protons exist only in the inner hydroxyl groups
of the binaphthalene moieties near the iron center, not in the
trans axial ligand. We therefore conclude that the bound oxygen
in TCP-TB and TCP-TG interacts with the adjacent exchange-
able protons and that the dioxygen adducts of these complexes
are stabilized by the hydrogen bonds between the dioxygen and
the inner hydroxyl groups, as predicted in the molecular design.
considered to be higher than that of oxy-cytochrome P450cam
as predicted from the electrochemical data.
,
Evidence for Hydrogen Bonding to Bound Dioxygen. The
thermal stability of these dioxygen adducts was also confirmed
by RR spectroscopy. The ν(O-O) bands were successfully
observed at -20 °C (TCP-TB) and 0 °C (TCP-TG), respec-
tively. Despite the coordination of the strongly electron-donating
alkanethiolate anions, these dioxygen adducts exhibited high
thermal stability. We postulated that an intramolecular hydrogen
bond between the bound oxygen and the hydroxyl groups of
the binaphthalene moieties was mainly responsible for this
higher stability. To verify this prediction, we investigated the
frequencies of the O-O stretching modes of the dioxygen adduct
upon deuterium substitution of the exchangeable protons. After
ferric TCP-TB and TCP-TG were kept in CH3OH/CH3CN or
CD3OD/CH3CN for several hours and dried in vacuo overnight,
the reaction with KO2 was carried out (Scheme 4). In the IR
spectra of ferric TCP-TB and TCP-TG, ν(O-H) signals (TCP-
TB: 3489, 3443 cm-1, TCP-TG: 3453 cm-1) were replaced
with lower ν(O-D) bands (TCP-TB: 2588, 2549 cm-1, TCP-
TG: 2558 cm-1) upon H/D exchange treatment, and no other
bands were shifted. In the RR spectra of both oxy-TCP-TB
and TCP-TG, only the ν(O-O) modes were upshifted by 2
cm-1 upon exchange of CH3OH by CD3OD (Figure 4 and Table
6). No other vibrations exhibited frequency shifts. These results
indicate that there is no perturbation in the porphyrin skeleton
upon H/D exchange. Using RR spectroscopy, two groups have
Conclusions
Synthesis and characterization of novel iron porphyrins,
designated TCP-TB and TCP-TG, with an alkanethiolate axial
(38) Chottard, G.; Schapracher, M.; Ricard, L.; Weiss, R. Inorg. Chem.
1984, 23, 4557.
(39) Brunner, H. Naturwissenschaften 1974, 61, 129.
(40) Tsubaki, M.; Yu, N.-T. Proc. Natl. Acad. Sci. U.S.A. 1981, 78, 3581.
(41) (a) Kitagawa, T.; Ondrias, M. R.; Rousseau, D. L.; Ikeda-Saito, M.;
Yonetani, T. Nature 1982, 298, 869. (b) Bruha, A.; Kincaid, J. R. J. Am.
Chem. Soc. 1988, 110, 6006. (c) Proniewicz, L. M.; Kincaid, J. R. Coord.
Chem. ReV. 1997, 161, 881.

1140 J. Am. Chem. Soc., Vol. 123, No. 6, 2001
Tani et al.
circulator. Infrared spectra were recorded on a BIO RAD FTS-6000
spectrometer. ESR spectra were obtained at 77 K in 5.0 mm diameter
ESR quartz tubes on a JEOL JES-TE 300 spectrometer. The magnetic
field strength was calibrated by the hyperfine coupling constants (hfcc)
of Mn(II) ion doped in MgO powder (86.9 G). Redox potentials of
TCP-TB, TCP-TG, and FeTMPCl (0.5 mM) in dried CH3CN contain-
ing 0.1 M tetra-n-butylammonium tetrafluoroborate (Bu4NBF4) as a
supporting electrolyte were determined at room temperature by cyclic
voltammetry using a three-electrode system under deaerated conditions
and a BAS 100B electrochemical analyzer. Platinum wires were used
as working and counter electrodes. The reversibility of the electro-
chemical processes was evaluated by standard procedures, and all
potentials were recorded against an Ag/Ag+ reference electrode, which
was calibrated using the ferrocene/ferrocenium redox couple. Resonance
Raman spectra were obtained on a SpectraPro-300i (Acton Research
Co.) spectrograph (operating at a 2400-groove grating) using a Spectra-
Physics Beamlok 2060 Kr ion laser (413.1 nm) or a KIMMON Electric
IK4152R-G He-Cd laser (441.6 nm), a Kaiser Optical holographic
supernotch filter, and a Princeton Instruments (LN-1100PB) liquid-
N2-cooled CCD detector. Spectra were collected on solution samples
in spinning cells (2-cm diameter, 1500 rpm) with a laser power of 20
mW, 90°-scattering geometry, and 5-min data accumulation. The
temperature was monitored with a thermocouple at the sample point
and controlled by the flow rate of cold nitrogen gas, which flowed
through liquid N2 to a cell in a quartz Dewar. Peak frequencies were
calibrated relative to indene and CCl4 standards and were accurate to
(1 cm-1. During each Raman experiment, UV/vis spectra were
simultaneously collected on a HAMAMATSU PMA-11 CCD spectro-
photometer with Photal MC-2530 as a light source (D2/W2).
ligand were carried out to mimic the active center of cytochrome
P450. Ferric TCP-TB and TCP-TG showed sufficient stability
for usual manipulation under air and were characterized by
means of HR-FAB-MS, UV/vis, ESR, and CV. The correspond-
ing stable dioxygen adducts were obtained by reaction of the
ferric complexes with an equimolar amount of KO2 under an
oxygen atmosphere. The frequencies of ν(O-O) bands in the
RR spectra were determined to be 1138 cm-1 for TCP-TB and
1137 cm-1 for TCP-TG. Finally, direct evidence for hydrogen
bonding of the bound dioxygen to the distal hydroxyl groups
was obtained also by RR spectroscopy.
It has been long accepted that the hydrogen bonds to bound
dioxygen stabilize the oxy-complexes of globins and their
nitrogen-base coordinated models.11,42 In contrast, oxy com-
plexes of thiolate-ligated iron porphyrins are much less stable.
However, an analogous hydrogen bond in oxy cytochrome P450
has been very recently revealed by cryocrystallographic tech-
niques.8 To the best of our knowledge, the present work also
shows the first clear evidence for a hydrogen bond to dioxygen
in a thiolate-coordinated model. These results have implications
in studies of the process of binding and activation of dioxygen
by cytochrome P450. These cytochrome P450 model dioxygen
adducts could be promising candidates which may lead up to
isolation of peroxy-, hydroperoxy-, and high-valent oxo species,
which correspond to long-sought, but not-yet-identified, inter-
mediates in the catalytic cycle of cytochrome P450.
Synthesis of TCP-TB and TCP-TG complexes. 9a and 9b. The
octahydroxylated porphyrin 1 (281 mg, 0.38 mmol) and the chloride 5
(688 mg, 1.46 mmol) were separately dried in vacuo (<10-6 Torr)
overnight. They were introduced into a glovebox and mixed in an
autoclave (300 mL) together with K2CO3 (2.31 g), dry degassed THF
(195 mL) and dry degassed NMP (39 mL). The autoclave was taken
out, and heated for 4 days at 115 °C. The chloride 5 (each ∼160 mg)
was further added to the mixture subsequently 2 and 4 days later,
depending on the progress of the coupling reaction. The aliquot was
taken and checked by ESI-MS. After 4 days, THF was evaporated,
and the residue was poured into water to afford purple solids, followed
by suction filtration. The precipitates were purified by alumina column
chromatography (CH2Cl2/AcOEt ) 9/1, v/v). The crude product was
further purified by flash column chromatography (silica gel, CH2Cl2/
MeOH ) 50/1, v/v). The less polar fraction contained the eclipsed
isomer 9a (171 mg, 7.3 × 10-2 mmol, 19%), and the more polar one
included the staggered one 9b (88 mg, 3.8 × 10-2 mmol, 10%). Each
of the title compounds, eclipsed and staggered isomers, was obtained
as purple solids (total yield 29%).
Experimental Section
General. All reagents and solvents were of the commercial reagent
grade and were used without further purification except where noted.
Purification was made by the standard methods.43 Preparation and
handling of air-sensitive materials were carried out under N2 in a
glovebox (M. Braun 150B-G-II or VAC Dri-Lab-08/85) equipped with
a circulating purifier (O2, H2O < 1 ppm). Dry ether and THF were
obtained by predrying over CaCl2 or KOH, and distillation from
benzophenone ketyl under N2 atmosphere. Absolute THF was obtained
by redistillation over potassium in a glovebox. Acetonitrile was distilled
from P2O5 twice and redistilled from K2CO3. Then, dry CH3CN was
obtained by redistillation from CaH2 in a glovebox. Dry THF and dry
CH3CN were treated in the same manner as that described by Selke et
al.44 These solvents were stirred over KO2 (Aldrich) for ∼1 h and
subsequently passed through activated alumina. Activated alumina
(Wako) was dried at 220 °C in vacuo for 8 h before use. Deoxygenation
of solvents was achieved by a freeze-and-thaw technique under high
vacuum conditions (<10-5 Torr). K18O2 was prepared according to the
literature.45
Instruments. 1H NMR spectra were recorded on a JEOL JMX-
GX400 (400 MHz) or a Bruker DRX600 (600 MHz) spectrometer.
Chemical shifts were reported on d-scale relative to tetramethylsilane
(TMS). High-resolution mass (HRMS) spectra were recorded on a JEOL
LMS-HX-110 spectrometer. FAB-MS spectra were measured with
3-nitrobenzyl alcohol (NBA) or magic bullet as matrices. Electrospray
ionization mass (ESI-MS) spectra were obtained on a Perkin-Elmer
Sciex API 300 mass spectrometer. Scanning was in 0.1-Da steps and
a 10-ms dwell time per step. The orifice voltage was controlled at +60
V. UV/vis electronic spectra were recorded on a Shimadzu UV-3100PC
spectrophotometer or a HAMAMATSU PMA-11 CCD spectropho-
tometer with a Photal MC-2530 as a light source (D2/W2). The
temperature was controlled by a NESLAB ULT-95 low-temperature
The eclipsed isomer 9a: UV/vis (CH2Cl2) λmax (10-3 ꢀ, M-1‚cm-1
)
316 (24.3), 329 (25.7), 407 (57.7), 424 (304), 517 (13.3), 549 (4.59),
591 (5.38), 648 nm (1.97); IR (neat) 3318, 3059, 2933, 2876, 1586,
1453, 1356, 1233, 1204, 1155, 1069, 976, 913, 794, 750, 718 cm-1
;
HRMS (FAB, NBA, C148H119N4O24) calcd 2335.8214, found 2335.8215.
The staggered isomer 9b: UV/vis (CH2Cl2) λmax (10-3 ꢀ, M-1‚cm-1
)
329 (34.6), 407 (80.9), 423 (391), 516 (20), 546 (5.5), 591 (7.2), 646
nm (2.1); HRMS (FAB, NBA, C148H119N4O24) calcd 2335.8214, found
2335.8184.
10. The eclipsed isomer 9a (50 mg, 2.1 × 10-2 mmol) was dried in
vacuo for 1-2 h, and then placed under Ar. Dry dichloromethane was
added to the flask, and then bromotrimethylsilane (88 mL, 0.68 mmol)
was added dropwise at -40 °C. The formation of title compound was
monitored by TLC (silica gel, dichloromethane), and the reaction
temperature was gradually raised to -20 °C. After 4-5 h later, the
reaction mixture was poured into cold saturated aqueous NaHCO3 and
extracted with CH2Cl2. The combined organic layer was washed with
saturated aqueous NaHCO3 and brine and then dried over Na2SO4. After
evaporation of the solvent, the crude product was dried in vacuo for 1
h and then washed with methanol. Insoluble purple solids were filtered
to give the title compound (40 mg, 0.02 mmol, yield 96%). UV/vis
(CH2Cl2) λmax (10-3 ꢀ, M-1‚cm-1) 325 (30), 337 (33.7), 407 (52), 424
(285), 518 (13.5), 550 (3.7), 591 (4.3), 646 nm (1.3); IR (neat) 3458,
(42) Jameson, G. B.; Ibers, J. A. In Bioinorganic Chemistry; Bertini, I.,
Gray, H. B., Lippard, S., Valentine, J., Eds.; University Science Books:
California, 1994; pp 167-252.
(43) Perrin, D. D.; Armarego, W. L. F. Purification of Laboratory
Chemicals, 3rd ed.; Pergamon Press: Oxford, 1988.
(44) Selke, M.; Sisemore, M. F.; Valentine, J. S. J. Am. Chem. Soc. 1996,
118, 2008.
(45) Brauer, G. Ed. In Handbook of PreparatiVe Inorganic Chemistry;
Academic Press: New York, 1963; Vol. 1, p 981.

EVidence for H-Bonding to Bound Dioxygen
J. Am. Chem. Soc., Vol. 123, No. 6, 2001 1141
3068, 2959, 2929, 1629, 1595, 1454, 1384, 1369, 1259, 1232, 1111,
1083, 982, 966, 792, 749, 724 cm-1; HRMS (FAB, NBA, C132H87N4O16)
calcd 1983.6117, found 1983.6115.
1 mmol) were added, and the mixture was stirred for 2 days at room
temperature in a glovebox. The reaction mixture was quenched with
an aqueous solution (5 mL) of p-toluenesulfonic acid (500 mg, 2.90
mmol). The flask was taken out from the glovebox and immediately 1
N HCl (2 mL) was added. The mixture was extracted with CH2Cl2.
The combined organic layer was washed with water until the aqueous
layer became neutral and then with brine, and was dried over Na2SO4.
The crude product was purified by flash column chromatography (silica
gel, CH2Cl2/MeOH ) 300/1, v/v). TCP-TB was obtained as brown
solids (2.1 mg, 8.4 × 10-4 mmol, yield 48%). UV/vis (CH2Cl2) λmax
(10-3 ꢀ, M-1‚cm-1) 326 (32.8), 338 (31.3), 416 (Soret, 61.8), 515 (9.83)
and 657 (2.69) nm; IR (neat) 3501, 3444, 3059, 2963, 2926, 2855,
11. The deprotected porphyrin 10 (65 mg, 3.3 × 10-2 mmol) was
dried in vacuo for 1 h, and then placed under Ar. Dry CH2Cl2 (40 mL)
was added to the reaction flask and cooled to 0 °C under N2, and
pyridine (4.0 mL, 50 mmol) was added. Then, pivaloyl chloride (1.3
mL, 10 mmol) was added to the mixture dropwise. After stirring
overnight at room temperature, the reaction mixture was cooled again
to 0 °C, quenched with 1 N HCl, and then extracted with CH2Cl2. The
combined organic layer was washed with 1 N HCl, saturated aqueous
NaHCO3 (three times), water, and brine and then dried over Na2SO4.
The crude product was purified by flash column chromatography (silica
gel, CH2Cl2/EtOH ) 200/1, v/v). The title compound 11 was obtained
as purple solids (74 mg, 3.2 × 10-2 mmol, yield 97%). UV/vis (CH2-
Cl2) λmax (10-3 ꢀ, M-1‚cm-1) 325 (29.7), 338 (28.9), 375 (17.4), 405
(55.1), 423 (308), 517 (15), 547 (5), 588 (6.3), 642 nm (2.4); IR (neat)
3457, 3316, 3061, 2973, 2934, 2874, 1745, 1631, 1585, 1455, 1368,
1261, 1231, 1111, 1083, 1007, 893, 789, 749, 719 cm-1; HRMS (FAB,
NBA, C152H119N4O20) calcd 2319.8418, found 2319.8376.
1743, 1585, 1456, 1366, 1261, 1084, 1026, 871, 766, 749, 709 cm-1
;
ESR (THF, 77 K) g ) 2.334, 2.210, 1.959; HRMS (FAB, NBA,
C160H124N4O20SFe) calcd 2508.7879, found 2508.7866; elemental
analysis (C160H123N4O20SFe‚6H2O) calcd C 73.41, H 5.20, N 2.14; found
C 73.57, H 5.61, N 2.34.
14. To a CH2Cl2 solution (5 mL) of 11 (29.7 mg, 12.8 × 10-3 mmol),
4-(dimethylamino)pyridine (15.6 mg, 0.128 mmol), 1-(3-dimethylami-
nopropyl)-3-ethylcarbodiimide hydrochloride (22.4 mg, 0.128 mmol),
and ethyldisulfanylacetic acid 8 (15.3 mL, 0.128 mmol) were added
and stirred at room temperature in the dark. After 1 h, the portion of
the solution was taken, washed with 1 N HCl and saturated aqueous
NaHCO3, and then extracted with CH2Cl2. The formation of the title
compound was checked by ESI-MS. Before the formation of the
undesired compound, in which the two hydroxyl groups were esterified,
the reaction mixture was quenched with 1 N HCl, and then extracted
with CH2Cl2. The combined organic layer was washed successively
with saturated aqueous NaHCO3 and brine and then dried over Na2-
SO4. After evaporation of the solvent, the crude product was purified
by flash column chromatography (silica gel, CH2Cl2/EtOH). The title
compound 14 was obtained as purple solids (11.2 mg, 4.56 × 10-3
12. The porphyrin 11 (15.8 mg, 6.8 × 10-3 mmol) and 2-(iodo-
methyl)benzyl thiobenzoate 7 (27.4 mg, 74.3 × 10-3 mmol) was dried
in vacuo for 1 h, and then dry K2CO3 (20 mg, 145 × 10-3 mmol) and
NMP (2 mL) were added into the flask. The reaction mixture was heated
at 90 °C for 15 min. Then the solvent was removed by heating under
reduced pressure. A portion was checked by TLC. If the reaction was
not complete, NMP was added, and the mixture was heated again. The
mixture was finally dried in vacuo, and purified by flash column
chromatography (silica gel). The title compound 12 was obtained as
1
purple solids (10.8 mg, 4.2 × 10-3 mmol, yield 62%). H NMR (400
MHz, CDCl3) 8.73 (d, J ) 4.9 Hz, 1H, pyrrole â-H), 8.70 (d, J ) 4.4
Hz, 1H, pyrrole â-H), 8.58 (d, J ) 4.4 Hz, 1H, pyrrole â-H), 8.57 (d,
J ) 4.4 Hz, 1H, pyrrole â-H), 8.54 (d, J ) 4.4 Hz, 1H, pyrrole â-H),
8.50 (d, J ) 4.9 Hz, 1H, pyrrole â-H), 8.16 (d, J ) 4.4 Hz, 1H, pyrrole
â-H), 8.11 (d, J ) 4.9 Hz, 1H, pyrrole â-H), 8.00 (s, 1H), 7.90 (d, J
) 7.8 Hz, 1H), 7.83 (s, 1H), 7.81 (d, J ) 9.8 Hz, 1H), 7.71 (m, 4H),
7.53 (m, 10H), 7.44 (s, 1H), 7.38-7.13 (m, 12H), 7.08-6.98 (m, 6H),
6.83 (m, 4H), 6.68 (m, 5H), 6.52 (m, 3H), 6.41 (d, J ) 8.3 Hz, 1H),
6.31 (d, J ) 8.8 Hz, 1H), 6.13 (d, J ) 8.3 Hz, 1H), 6.09 (d, J ) 8.8
Hz, 1H), 5.80 (t, J ) 7.6 Hz, 1H), 5.41 (d, J ) 9.8 Hz, 1H), 5.35 (d,
J ) 9.3 Hz, 1H), 5.25 (m, 3H), 5.12 (m, 2H), 5.03 (s, 1H), 4.85 (m,
3H), 4.71 (m, 3H), 4.59 (s, 1H), 4.21 (d, J ) 13.7 Hz, 1H), 4.16 (d, J
) 7.8 Hz, 1H), 4.14 (d, J ) 13.7 Hz, 1H), 4.02 (t, J ) 7.1 Hz, 1H),
3.66 (t, J ) 7.6 Hz, 1H), 3.13 (d, J ) 7.3 Hz, 1H), 2.74 (d, J ) 13.2
Hz, 1H), 2.53 (d, J ) 13.7 Hz, 1H), 1.90 (d, J ) 13.7 Hz, 1H), 1.39
(d, J ) 11.2 Hz, 1H), 1.21 (d, J ) 10.7 Hz, 1H), 0.77 (s, 9H, Piv-
CH3), 0.76 (d, J ) 13.7 Hz, 1H), 0.74 (s, 9H, Piv-CH3), 0.28 (s, 9H,
Piv-CH3), 0.17 (s, 9H, Piv-CH3), -3.62 (s, 2H, NH); UV/vis (CH2-
Cl2): λmax (10-3 ꢀ, M-1‚cm-1) 325 (22.7), 406 (25.8), 427 (272) and
553 nm (14.0); IR (neat) 3483, 3310, 3060, 2961, 2929, 2874, 1746,
1
mmol, 36%). H NMR (400 MHz, CDCl3) 8.82 (d, J ) 4.4 Hz, 1H,
pyrrole â-H), 8.79 (d, J ) 4.9 Hz, 1H, pyrrole â-H), 8.77 (d, J ) 4.4
Hz, 1H, pyrrole â-H), 8.76 (d, J ) 4.9 Hz, 1H, pyrrole â-H), 8.62 (d,
J ) 4.9 Hz, 1H, pyrrole â-H), 8.55 (d, J ) 4.4 Hz, 1H, pyrrole â-H),
8.30 (d, J ) 4.4 Hz, 1H, pyrrole â-H), 8.27 (d, J ) 4.9 Hz, 1H, pyrrole
â-H), 7.88-6.50 (m, 46H, aromatic-H), 6.43 (d, J ) 8.8 Hz, 1H,
aromatic-H), 6.34 (d, J ) 8.3 Hz, 1H, aromatic-H), 6.10 (d, J ) 8.3
Hz, 1H, aromatic-H), 6.08 (d, J ) 5.9 Hz, 1H, aromatic-H), 5.87 (m,
2H, aromatic-H), 5.34-4.70 (m, 11H, benzyl-CH2), 4.34 (d, J ) 14.2
Hz, 1H, benzyl-CH2), 4.22 (d, J ) 13.7 Hz, 1H, benzyl-CH2), 4.21 (d,
J ) 13.2 Hz, 1H, benzyl-CH2), 2.66 (d, J ) 13.2 Hz, 1H, benzyl-
CH2), 2.52 (d, J ) 14.2 Hz, 1H, benzyl-CH2), 0.73 (s, 9H, Piv-CH3),
0.68 (s, 9H, Piv-CH3), 0.33 (s, 9H, Piv-CH3), 0.23 (s, 9H, Piv-CH3),
-0.44 (m, 1H, SCH2CH3), -0.50 (t, J ) 6.8 Hz, 3H, SCH2CH3), -1.20
(m, 1H, SCH2CH3), -2.15 (d, J ) 18.6 Hz, 1H, COCH2S), -2.72 (d,
J ) 18.6 Hz, 1H, COCH2S), -2.94 (s, 2H, NH); UV/vis (CH2Cl2)
λ
max (10-3 ꢀ, M-1‚cm-1) 325 (19.1), 338 (18.3), 402 (39), 422 (211.6),
518 (11.8), 587 (4), 640 nm (1.4); IR (neat) 3458, 3333, 3305, 3056,
2958, 2926, 2875, 2853, 1747, 1585, 1457, 1367, 1260, 1231, 1110,
1082, 1025, 796, 748, 719 cm-1; HRMS (C156H125O21N4S2) calcd
2453.8278, found 2453.8284.
1585, 1457, 1365, 1260, 1230, 1111, 1082, 892, 791, 748, 718 cm-1
;
HRMS (FAB, NBA, C167H131N4O21S) calcd 2559.9027, found 2559.9016.
13. The porphyrin 12 (5.3 mg, 2.1 × 10-3 mmol) was dried in vacuo
for 2 h. Dry toluene (10 mL) was added to the reaction flask. The
mixture was heated in an oil bath at 50 °C, and Fe(CO)5 (100 mL,
7.45 × 10-1 mmol) and a toluene solution (100 mL) of I2 (6.5 mg, 2.6
× 10-2 mmol) was added. The reaction mixture was stirred for 1 day
at 50 °C under N2 in the dark. An aliquot was taken and checked by
TLC (CH2Cl2/MeOH ) 100/1, v/v) and ESI-MS. The reaction mixture
was quenched with water and extracted with CH2Cl2. The combined
organic layer was washed with 0.5 N HCl and brine and then dried
over Na2SO4. After evaporation of the solvent, the residue was purified
by flash column chromatography (silica gel, CH2Cl2/MeOH ) 100/1,
v/v). The title compound 13 was obtained as brown solids (4.2 mg,
TCP-TG. 14 (5.3 mg, 2.16 × 10-3 mmol) was dissolved in dry
toluene (10 mL) and heated at 50 °C under N2. Fe(CO)5 (105 mL,
0.78 mmol) and a toluene solution of I2 (6.8 mg, 26.8 × 10-3 mmol)
were added. The mixture was stirred overnight in the dark, quenched
with water, and then extracted with CH2Cl2. After removal of the solvent
and drying, the residue was purified by flash column chromatography
(silica gel, CH2Cl2). TCP-TG was obtained as brown solids (3.6 mg,
1.47 × 10-3 mmol, 41%) and the thiolate-protected complex 15 was
also obtained as a minor product (1.5 mg, 0.59 × 10-3 mmol, 16%).
TCP-TG: UV/vis (CH2Cl2) λmax (10-3 ꢀ, M-1‚cm-1) 325 (18.2), 339
(18.7), 363 (17.1), 418 (31.6), 513 (5.8), 575 (2.7), 658 nm (1.7); IR
(neat) 3453, 3062, 2957, 2927, 2873, 1746, 1585, 1456, 1367, 1260,
1229, 1111, 1085, 997, 788, 747, 719 cm-1; ESR (THF, 77 K) g )
2.313, 2.209, 1.966; HRMS (C154H118O21N4SFe) calcd 2446.7359, found
2446.7417; elemental analysis (C154H117O21N4SFe‚8H2O) calcd C 71.37,
H 5.17, N 2.16; found C 71.66, H 5.49, N 1.94. 15: UV/vis (CH2Cl2)
1.6 × 10-3 mmol, yield 78%). UV/vis (CH2Cl2) λmax (10-3 ꢀ, M-1‚cm-1
)
327 (37.1), 423 (77.3), 518 (9.4), 583 (2.7) and 653 (2.0) nm; IR (neat)
3446, 3058, 2965, 2928, 2856, 1745, 1586, 1456, 1367, 1261, 1231,
1110, 1079, 1026, 1003, 895, 797, 750, 720 cm-1; HRMS (FAB, Magic
Bullet, C167H128N4O21SFe) calcd 2612.8141, found 2612.8159.
TCP-TB. The iron complex 13 (4.6 mg, 1.8 × 10-3 mmol) was
dried in vacuo for 2 h. Dry CH3CN (2 mL) and dry BuNH2 (0.1 mL,
λ
max (10-3 ꢀ, M-1 ‚cm-1) 325 (19.1), 338 (18.3), 402 (39), 422 (211.6),
518 (11.8), 587 (4) and 640 (1.4) nm; IR (neat) 3474, 3057, 2960,

1142 J. Am. Chem. Soc., Vol. 123, No. 6, 2001
Tani et al.
2928, 2874, 1747, 1586, 1456, 1365, 1261, 1232, 1110, 1085, 999,
890, 794, 748, 717 cm-1; HRMS (FAB, NBA, C156H123N4O21S2Fe) calcd
2507.7471, found 2507.7478.
Reaction of TCP-TB and TCP-TG with KO2. TCP-TB, or TCP-
TG (0.2 mg, 8.2 × 10-5 mmol) was dissolved in THF (0.6 mL, 1.37
× 10-4 M) in a glovebox. The solution was cooled to -80 °C, and
then a solution of KO2 (50 µL, CH3CN, 1.65 × 10-3 M), which was
dissolved with cryptand [2.2.2], was added into the porphyrin solution.
After the replacement of the atmosphere to O2, the resulting solution
was subjected to UV/vis absorption and resonance Raman spectroscopy.
11228207 to Y.N.), and for Encouragement of Young Scientists
(No. 08740500 to F.T.) from the Ministry of Education, Science
and Culture, Japan and by Research Grants to Y.N. from
Sumitomo Foundation and to F.T. from Takeda Science
Foundation and Otsuka Chemical Co. Ltd.
Supporting Information Available: Experimental details
relative to the syntheses and characterization of the binaphthyl
derivatives (2-5), the porphyrin compounds and the protected
1
thiolate compounds (6-8), H NMR data of 9a-11, and RR
spectra of the dioxygen adduct of TCP-TB (PDF). This material
Acknowledgment. This research was financially supported
by Grant-in-Aids for COE Research (No. 08CE2005 to Y.N.),
for Scientific Research on Priority Areas (No. 09235225 and
is available free of charge via the Internet at http://pubs.acs.org.
JA003430Y