Extreme rate acceleration by axial thiolate coordination on the isomerization of endoperoxide catalyzed by iron porphyrin

Article abstract of DOI:10.1002/anie.200800137

(Chemical Equation Presented) A coordinated effort: The isomerization mechanism of prostaglandin H₂ (PGH₂), which is catalytically isomerized to prostacyclin or thromboxane A₂ by cytochrome P450s, was investigated using a hemethiolate complex and an endoperoxide. Isomerization of endoperoxides proceeded very rapidly with this complex, whereas imidazole- or chloride-ligated heme had slight or no catalytic activity (see scheme).

Full text of DOI:10.1002/anie.200800137

Communications
DOI: 10.1002/anie.200800137
Enzyme Mechanisms
Extreme Rate Acceleration by Axial Thiolate Coordination on the
Isomerization of Endoperoxide Catalyzed by Iron Porphyrin**
Takehiro Yamane, Kohei Makino, Naoki Umezawa, Nobuki Kato, and Tsunehiko Higuchi*
Cytochrome P450s catalyze various types of oxidative reac-
tions involved in steroid biosynthesis, the metabolism of fatty
acids, and the detoxification of xenobiotic compounds.[1] Most
of the reactions are monooxygenase-type NADPH/NADH-
and O2-dependent ones, but when the endoperoxide (EP)
isomerizing P450 works as a prostacyclin synthase or throm-
boxane synthase it is neither NADPH/NADH- nor O2-
dependent, and is known generally as prostaglandin H2
(PGH2) isomerase.[2] Malondialdehyde (MDA) is one of the
major products from the isomerization of PGH2 by throm-
boxane synthase, and the reaction is accompanied by the
formation of 12-hydroxyheptadeca-5,8,10-trienoic acid
(HHT, Scheme 1).[3] Prostacyclin (PGI2) and thromboxane A2
atoms. It would be of interest to know why nature has selected
the heme-thiolate structure, from the wide range of metal
complexes, for the isomerization of EPs. Several proposed
reaction mechanisms for the catalysis are based largely on the
structure of the product and by analogy with the general
mechanism of P450;[3a,4] more direct evidence is needed to test
these ideas. PGH2 isomerases contain the heme-thiolate
structure in their active sites, whereas most peroxidases
have imidazole-ligated heme structures. Therefore, we
focused on the role of the thiolate ligand in the isomerase
catalysis. Metalloporphyrins have been extensively investi-
gated as chemical models of P450 and/or peroxidases to
understand the catalysis by P450 and peroxidase in the
presence of hydroperoxides as oxidants.[5] There have
been two reports describing the isomerization of EPs
in the presence of synthetic metalloporphyrins,[4,6] but
the effect of the axial ligand on the isomerization was
not considered.
We have already succeeded in the synthesis of the
first synthetic heme thiolate (SR complex, Scheme 2),
in which thiolate coordination is retained during the
catalytic oxidation, and have observed several
remarkable effects of the thiolate axial ligand.[7] We
report here the first isomerization of an EP catalyzed
by a stable synthetic heme-thiolate complex and the
large enhancing effect of the thiolate ligand on the
reaction.
Scheme 1. Isomerization of PGH2 with cytochrome P450 enzymes.
A simple EP 1, which is a partial structure of
PGH2, was prepared by modification of the reported
method.[9] Reactions of 1 with iron porphyrins were moni-
tored by 1H NMR spectroscopic measurement of samples
(TXA2) are regulatory factors of platelet aggregation as well
as of vaso- and bronchodilation and constriction. Both
compounds are derived from the common precursor PGH2
endoperoxide.
Hydroperoxides are widely used as oxidants by heme-
containing peroxidases and P450. However, no metalloen-
zyme other than the P450-type one is known to catalyze
reactions involving EPs that do not have active hydrogen
[*] T. Yamane, K. Makino, Dr. N. Umezawa, Dr. N. Kato,
Prof. Dr. T. Higuchi
Graduate School of Pharmaceutical Sciences
Nagoya City University
3-1 Tanabe-dori, Mizuho-ku, Nagoya 467-8603 (Japan)
Fax: (+81)52-836-3435
E-mail: higuchi@phar.nagoya-cu.ac.jp
[**] This work was supported in part by Grants-in-Aid for Scientific
Research (Nos. 18390039 and 19028054) from the Ministry of
Education, Science, Sports, and Culture (Japan). We are grateful to
Dr. Hidehiko Nakagawa for his help with ESR measurements.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.200800137.
Scheme 2. Structures of various heme complexes.
6438
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2008, 47, 6438 –6440

Angewandte
Chemie
obtained after quenching the reaction by adding coordinative
[D5]pyridine to aliquots of the reaction mixture at regular
intervals. In the presence of [Fe(tpp)Cl]or hemin dimethyl
ester, 1 was completely unchanged in CDCl3 at 258C for 24 h
(Table 1). In contrast, SR showed extremely high catalytic
first report of the redox potential (FeIII/FeIV) of synthetic
heme-thiolate. The ready accessibility of the high-valent iron
form of heme thiolate is considered critical for cleavage of the
À
O O bond of the EP, which has no active hydrogen atom.
Our previous study showed that the initial rate of 2,4,6-tri-
tert-butylphenol (TBPH) oxidation with mCPBA cata-
lyzed by SR was only 70-fold higher than that with SR-
Im, while [Fe(tpp)Cl]could catalyze the oxidation with
RCOOOH. Thus, the difference in the axial ligand effect
on the isomerization of EPs is much greater than that on
Table 1: Comparison of reaction rates, product profiles, and redox potentials
with various heme complexes.[a]
Catalyst
Ligand
Endoperoxide Yields [%][b] TOF [sÀ1 [c]
] Redox
potentials
FeIII/FeIV
E1/2 [V][g]
3[d]
4
the oxidation with a peroxycarboxylic acid. The remark-
able difference in the axial ligand effect provides
2
unambiguous mechanistic insight into the cleavage of
SR[e]
SR-Im
[Fe(tpp)Cl]
hemin
dimethyl
ester
R-SÀ
1
1
1
1
40 17 56 10.5
0.69
III
imidazole
34
2
44 1.1”10À2 0.86
À
the O O bond by heme (Fe ). SR showed much lower
ClÀ
no reaction
no reaction
0.84
0.77
activity for the isomerization of EP 5. This result suggests
ClÀ
À
that steric hindrance around the O O bond has a major
influence on the reaction, and that coordination of the
oxygen atom of the EP is necessary for subsequent
6
33
3[d]
2
7
À
cleavage of the O O bond, since the electronic state of
SR
R-SÀ
5
58 3.4”10À4 0.69
(5.0 mol%)[f]
the oxygen atoms of 5 would be similar to 1. No clear
solvent effect on the isomerization of 1 with SR was
observed (see the Supporting Information). This result
suggests that the reaction does not involve the formation
of a polar intermediate in the rate-determining step.
Furthermore, the presence of tetrahydrofuran (at four
times the concentration of 1), which would be more
strongly coordinating than 1, had little effect on the rate
of isomerization of 1. Therefore, it is likely that the rate-
[a] Reactions were carried out in CDCl3 at 258C for 24 h under Ar. Substrate:
3.5m m, catalyst: 1.0 mol%. [b] Determined by 1H NMR spectroscopy.
[c] Turnover frequency (TOF) was calculated from the initial rate of the
reaction. [d] Yields of 3 would have been underestimated because ethylene was
gradually released from the solution into the gas phase. [e] Reaction was
carried out for 15min. [f] 1 mol% SR isomerized 5 too slowly. [g] Iron
porphyrin: 0.1 mm, tetra-n-butylammonium perchlorate: 100 mm, solvent:
CH2Cl2, working electrode: Pt, reference electrode: Ag/AgCl.
À
determining step is cleavage of the O O bond, and the
large difference in the reaction rate among the iron
porphyrins should directly reflect the effect of the axial ligand
activity for the isomerization/breakdown of 1 under the same
conditions. Most of 1 was converted into malondialdehyde
(2), ethylene (3), and epoxyaldehyde (4) within 10 s
(Scheme 3). It was confirmed by checking the ESR spectrum
À
on the cleavage of the O O bond. The product profile in the
case of SR-Im resembled that obtained with SR. Interestingly,
the same type of conversion of 1 into 2 and 3 occurs in the
reactions of PGH2 with thromboxane synthase, prostacyclin
[3]
synthase, and cytochrome P450CAM
.
Spin-trap experiments were then carried out to examine
the formation of radical species. Compound 1 was treated
with SR (2.5 mol%) for 0.5 s at 258C in benzene and then
3,3,5,5-tetramethylpyrroline-N-oxide (TMPO) was added to
the mixture. The ESR spectrum of the reaction mixture
clearly showed a sextet signal (Figure 1) that could be
assigned to a product derived from an alkoxyl radical, since
its hyperfine coupling constants (aN: 1.32 mT, abH: 0.58 mT)
were in good agreement with those of the TMPO-tert-butoxyl
Scheme 3. Isomerization/beakdown of EPs with SR.
of the reaction mixture that most of the SR remained intact
after the reaction when 10 mol% of SR was used, whereas
considerable decomposition of the SR complex was observed
at a lower concentration (1.0 mol%). The SR-Im complex,[7b]
which is a monoimidazole-ligated iron porphyrin (UV/Vis
spectroscopic analysis has already confirmed that SR-Im is
almost entirely in the monoimidazole form), showed much
lower activity than the SR complex. The initial rate of
isomerization/breakdown by SR was almost 1000-fold higher
than that by SR-Im. The coordination of a chloride anion had
no positive effect on the isomerization. This is the first
example in which the axial ligand effect of a thiolate on the
isomerization of an EP has been unambiguously evaluated.
The redox potential (FeIII/FeIV or the electronic isomer) of SR
was lower than those of the other complexes. This is also the
Figure 1. ESR spectrum of the radical adduct with TMPO. The reaction
was carried out in benzene at 258C. Substrate 1: 6.0 mm, SR:
2.5mol%, TMPO: 60 m m.
Angew. Chem. Int. Ed. 2008, 47, 6438 –6440
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org 6439

Communications
radical adduct (aN: 1.33 mT, abH: 0.58 mT).[10] This is the first
In summary, we have found an extremely strong enhanc-
ing effect of an axial thiolate ligand on the isomerization of
EPs catalyzed by heme in hydrophobic media. This axial
ligand effect is the largest found in heme–peroxide reactions.
The thiolate ligand is suggested to play a critical role in
direct evidence for the formation of an oxy radical inter-
mediate in the isomerization of an EP catalyzed by heme.
The presence of 10 equivalents of TBPH did not alter the
product profile in the isomerization of 1 with SR (2.5 mol%).
However, the reaction in the presence of 100 equivalents of
TBPH afforded 1,3-cyclopentanediol 8 (33%) as well as 2, 3,
and 4 (Scheme 4)
À
cleavage of the O O bond in the absence of an active
hydrogen atom, which hydroperoxides have. The present
results provide strong evidence for the critical role played by
the heme-thiolate structure for the effective function of PGH2
isomerase in a hydrophobic environment. Further investiga-
tion using PGH2 is in progress.
Received: January 11, 2008
Revised: May 5, 2008
Published online: July 11, 2008
Keywords: cytochrome P450 · endoperoxides · enzyme models ·
.
heme · ligand effects
[1]a) Cytochrome P450: Structure, Mechanism, and Biochemistry,
3rd ed. (Ed.: P. R. Ortiz de Montellano), Kluwer Academic/
Plenum Publishers, New York, 2004; b) M. Sono, M. P. Roach,
E. D. Coulter, J. H. Dawson, Chem. Rev. 1996, 96, 2841 – 2888;
c) I. G. Denisov, T. M. Makris, S. G. Sligar, I. Schlichting, Chem.
Rev. 2005, 105, 2253 – 2278.
Scheme 4. Radical trapping with TBPH during the isomerization of 1.
Differential UV/Vis spectroscopy indicated the formation
of TBPC (lmax = 630 nm). The TBPH would thus reduce the O-
radical intermediate and the high-valent iron species to give 8
[2]a) V. Ullrich, L. Castle, P. Weber, Biochem. Pharmacol. 1981, 30,
2033 – 2036; b) D. L. DeWitt, W. L. Smith, J. Biol. Chem. 1983,
258, 3285 – 3293.
[3]a) M. Hecker, V. Ullrich, J. Biol. Chem. 1989, 264, 141 – 150; b) S.
Hara, A. Miyata, C. Yokoyama, H. Inoue, R. Brugger, F.
Lottspeich, V. Ullrich, T. Tanabe, J. Biol. Chem. 1994, 269,
19897 – 19903.
À
and TBPC. The results suggest that 1) cleavage of the O O
bond occurs prior to cleavage of the C C bond, and 2) the
À
observed ESR signal is not derived from the radicals formed
by direct oxidation of TMPO (only 10 equiv to 1), because
TBPH is a much better substrate for the oxidant than TMPO.
We propose that the mechanism for the isomerization of
an EP by heme-thiolate is as follows (Scheme 5). Rapid
[4]V. Ullrich, Arch. Biochem. Biophys. 2003, 409, 45 – 51.
[5]a) B. Meunier, A. Robert, G. Pratviel, J. Bernadou in The
Porphyrin Handbook, Vol. 4 (Eds.: K. M. Kadish, K. M. Smith,
R. Guilard), Academic Press, San Diego, 2000, pp. 119 – 187;
b) J. T. Groves, K. Shalyaev, J. Lee in The Porphyrin Handbook,
Vol. 4 (Eds.: Kadish, K. M. Kadish, K. M. Smith, R. Guilard),
Academic Press, San Diego, 2000, pp. 17 – 40.
[6]K. Takahashi, M. Kishi, Tetrahedron 1988, 44, 4737 – 4746.
[7]a) T. Higuchi, S. Uzu, M. Hirobe, J. Am. Chem. Soc. 1990, 112,
7051 – 7053; b) T. Higuchi, K. Shimada, N. Maruyama, M.
Hirobe, J. Am. Chem. Soc. 1993, 115, 7551 – 7552; c) Y. Urano,
T. Higuchi, M. Hirobe, T. Nagano, J. Am. Chem. Soc. 1997, 119,
12008 – 12009; d) N. Suzuki, T. Higuchi, Y. Urano, K. Kikuchi, H.
Uekusa, Y. Ohashi, T. Uchida, T. Kitagawa, T. Nagano, J. Am.
Chem. Soc. 1999, 121, 11571 – 11572; e) T. Ohno, N. Suzuki, T.
Dokoh, Y. Urano, K. Kikuchi, M. Hirobe, T. Higuchi, T. Nagano,
J. Inorg. Biochem. 2000, 82, 123 – 125; f) N. Suzuki, T. Higuchi, T.
Nagano, J. Am. Chem. Soc. 2002, 124, 9622 – 9628; other
synthetic heme-thiolate complexes are reviewed in refer-
ence [8].
À
homolytic cleavage of the O O bond of the EP coordinated
to the iron atom occurs with the assistance of the potent
electronic “push effect” of the thiolate ligand.[7a,11] The
biradical then cleaves to give products 2 and 3. An alternative
pathway involves rearrangement of the oxy radical coordi-
nated to the heme to afford unsymmetrical product 4.
À
Heterolysis of the O O bond would also be a possible
reaction pathway for the formation of 4. The remarkable
effect of an axial thiolate ligand on the isomerization of EPs
À
probably arises because cleavage of the O O bond of the EPs
without any active hydrogen atoms is purely dependent on the
donation of p electrons from the axial ligand.
[8]W.-D. Woggon, Met. Ions Life Sci. 2007, 3, 27 – 55.
[9]a) W. Adam, H. J. Eggelte, J. Org. Chem. 1977, 42, 3987 – 3988;
b) D. J. Coughlin, R. G. Salomon, J. Am. Chem. Soc. 1977, 99,
655 – 657.
[10]G. J. Edward, V. S. Raghav, M. K. Susan, Can. J. Chem. 1981, 59,
756 – 758.
[11]J. H. Dawson, Science 1988, 240, 433 – 439.
Scheme 5. Proposed mechanism for the isomerization of an EP.
6440 www.angewandte.org
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2008, 47, 6438 –6440