Reactivity of peroxoiron(III) porphyrin complexes: Models fordeformylation reactions catalyzed by cytochrome P-450

Yoshio Goto a,b, Senji Wada a, Isao Morishima b, Yoshihito Watanabe a,*

a Institute for Molecular Science, Okazaki 444-0867, Japanb Department of Molecular Engineering, Graduate School of Engineering, Kyoto University, Kyoto 606-8217, Japan

Received 24 June 1997; received in revised form 30 September 1997; accepted 21 October 1997

Abstract

Oxoiron (IV) porphyrin p-cation radical is considered as a common reactive intermediate in cytochrome P-450 catalyzed mono-

oxygenation. However, a ferric peroxo complex has been also proposed as a reactive intermediate in deformylation reactions cat-

alyzed by cytochrome P-450s. In spite of the importance of the peroxo complex in biological functions, reactivities of the peroxo

complex have not been well elucidated. Thus, we employed several peroxo±FeIII TPP derivatives to examine the reactions with alkyl

aldehydes. A reaction of [TPPFeIIIO2]ÿK� (TPP� tetrakis-5,10,15,20-phenylporphyrin dianion) with cyclohexanecarboxyaldehyde

in acetonitrile under He atmosphere gave cyclohexanone as a deformylated compound, along with 3-cyclohexylacrylonitrile, which

was formed by the condensation of the aldehyde with acetonitrile. Similar reactions were also observed when other aldehydes were

employed as the substrate. We propose that the ferric peroxo porphyrin complex attacks the aldehyde carbon as a nucleophile in

these reactions. Those aldehydes were simply oxidized to the corresponding carbonic acids when O@FeIVTMP (TMP, tetrakis

5,10,15,20-mesitylporphyrin dianion) p-cation radical was used as the oxidant. The results by these model studies indicate possible

involvement of the peroxo complex in deformylation reactions observed in P-450 systems. Ó 1998 Elsevier Science Inc. All rights

reserved.

Keywords: Peroxoiron(III); Cytochrome P-450; Aromatase; Deformylation

1. Introduction

Cytochrome P-450 is a family of heme containingmonooxygenases which reductively activate molecularoxygen to metabolize xenobiotics and steroids by hy-droxylation, epoxidation, or N- and O-dealkylation [1].In the catalytic cycle of cytochrome P-450, an oxoferryl(FeIV@O) porphyrin p-cation radical (or its equivalent)through the heterolytic O±O cleavage of a ferric peroxocomplex (FeIII±O±Oÿ) has been postulated as the reac-tive intermediate. Most of the oxidations catalyzed bycytochrome P-450 can be explained by assuming theoxo-ferryl species [2]. In the case of aldehyde oxidation

by cytochrome P-450, two di�erent types of oxidationhave been observed. One is the straightforward oxida-tion of aldehyde to the carboxylic acid (RCHO !RCO2H) [2±4]. Examples of this type of reactions are cy-tochrome P-450 catalyzed oxidation of acetaldehyde [5],saturated aliphatic aldehydes [6,7], a,b-unsaturated ali-phatic aldehydes [6,8±10].

The second type of the oxidation is rather unusual[2,11±15]. The ferric peroxo intermediate is proposedto attack the aldehyde carbon directly as a nucleophile.A peroxo±substrate adduct ®nally yields formic acid anddeformylated products. Key pieces of evidence support-ing this mechanism include the ®nding that the reactionis supported both by NADPH/O2 and by H2O2 but notby alkylhydroperoxides, peracids, and iodosobenzene[11]. For example, the cytochrome P-450 catalyzed de-carboxylation of aldehydes was observed in the demeth-ylation reactions by lanosterol 14-demethylase [12] andaromatase [13].

Placental aromatase is responsible for the transfor-mation of androgens 1 to estrogens 3 at the expense of

Journal of Inorganic Biochemistry 69 (1998) 241±247

Abbreviations: TPP, tetrakis-5,10,15,20-phenylporphyrin dianion;

TDCPP, tetrakis-5,10,15,20-(2,6-dichrolophenyl)porphyrin dianion;

TMP, tetrakis-5,10,15,20-mesitylporphyrin dianion; m-CPBA, 3-chlor-

operbenzoic acid* Corresponding author. Tel.: +81 564 55 7430; fax: +81 564 54 2254;

e-mail: yoshi@ims.ac.jp.

0162-0134/98/$19.00 Ó 1998 Elsevier Science Inc. All rights reserved.

PII: S 0 1 6 2 - 0 1 3 4 ( 9 7 ) 1 0 0 2 9 - 0

3 mol each of NADPH and O2 according to threestepwise reactions shown in Scheme 1 [13]. The reactionis initiated by C-19 hydroxylation of 1 and subsequentoxidation gives a C-19 oxo intermediate 2. The ®nal stepin the aromatase reaction is the oxidative deformylationof 2 yielding 3 and formic acid. If a high valent oxo spe-cies is the reactive intermediate in the ®nal step, theproducts are expected to be derivatives of the 19-carbox-ylic acid intermediate [14,15b]. Thus, direct participa-tion of the peroxoiron (III) porphyrin complex for thetransformation of 2 to 3 has been proposed [15±17].Not only the case of lanosterol 14-demethylase and aro-matase, the decarboxylation of xenobiotic aldehydes hasnow been observed in the oxidation of isobutyralde-hyde [18], trimethylacetaldeyde [18], citronellal [18],cyclohexanecarboxaldehyde [11], and 3-oxodecalin-4-ene-10-carboxaldehyde [19].

In order to understand more detail about the deform-ylation by the peroxo-iron(III) intermediate, studies byemploying synthetic model systems seem to be very im-portant. Unfortunately, a few works [20±23] have beenreported since the ®rst successful preparation of per-oxo±FeIIITPP and peroxo±FeIIIOEP by Valentine et al.[24]. In this paper, we report the reactions of peroxo±FeIIITPP derivatives and a series of aldehydes.

2. Experimental

2.1. General procedure

Due to instability of peroxoiron(III) porphyrin com-plexes upon exposure to moisture, all reactions includ-ing preparation of the ferric peroxo complexes werecarried out in a glove box ®lled with dry helium(99.9999%) unless otherwise noted. Acetonitrile was rig-orously dried before use: HPLC grade acetonitrile wasstirred over KO2 (Aldrich) in a glove box for 1 h andsubsequently passed through Super I acidic alumina(ICN).

2.2. Instruments

UV±VIS spectra were measured on a SHIMADZUUV1200 spectrometer in a glove box ®lled with dry he-lium, or on a SHIMADZU UVPC2400 spectrometerwith screw-capped cells. Electron paramagnetic reso-nance (EPR) measurements were carried out at X-band(9.15 GHz) microwave frequency on a Bruker ESP300EElectron Spin Resonance spectrometer with X-band

cavity at 4 K, or on a JEOL JES-FE2XG Electron SpinResonance spectrometer with X-band cavity at 77 K, byoperating with 100-kHz magnetic ®eld modulation.Product analyses were carried out by a SHIMADZUQP-5000 Gas Chromatography Mass Spectrometer(Shimadzu capillary column: HiCap-CBP1, 25m) withelectron ionization voltage at 1.5 eV.

2.3. Materials

Commercially available reagents from Aldrich, WakoChemical and Nacalai Tesque were used without furtherpuri®cation unless otherwise noted. Phenyl acetaldehydeand 2-phenylpropionaldehyde were distilled under re-duced pressure and stored in a glove box. Tetraphenyl-porphyrin [TPPH2], tetramesitylporphyrin [TMPH2],and tetrakis(2,6-dichlorophenyl)porphyrin [TDCPPH2]were prepared by modi®cation of methods reported[25]. Iron was inserted into the porphyrins to form ferricporphyrin chloride complexes by a standard method [26].

2.4. Preparation and reactions of peroxo complexes underHe atmosphere

K�[PorFeIIIO2]ÿ was prepared in situ by stirring Por-FeIIICl (3.75 mM) with two equivalent of 18-crown-6ether and a large excess of KO2 powder in dry acetonit-rile for 15 min followed by ®ltration to remove unreactedKO2 [24]. After con®rmation of the peroxo complex for-mation by UV±VIS measurement, a dry acetonitrile solu-tion of a substrate was added to the K�[PorFeIIIO2]ÿ

solution (0.5 ml, 3.75 mM) under He atmosphere atroom temperature (®nal concentration, 2.5 mM forK�[PorFeIIIO2]ÿ and 5 mM for the substrate). The reac-tion mixture was stirred for 1 h under He atmosphereand the products were analyzed by GC±MS.

2.5. Reaction of Fe(IV)@O porphyrin p-cation radicalwith aldehyde

To a methylene chloride solution of TMPFeIIICl (1.0mM) was added four equivalents of mCPBA at )80°Cand the reaction mixture was stirred. After con®rmationof TMPFe(IV)�O p-cation radical formation [27] bythe solution color change to green, two equivalents ofcyclohexanecarboxyaldehyde was added and stirred for30 min. The reaction mixture was then treated withdiazomethane and submitted to GC±MS to identifycyclohexanecarboxylic acid as a methyl ester.

Scheme 1.

242 Y. Goto et al. / J. Inorg. Biochem. 69 (1998) 241±247

2.6. Preparation of substrates and authentic samples ofoxidation products

2.6.1. 2-Cyclohexylacrylonitrile (6)To a THF (35 ml) suspension of sodium hydride (585

mg. 14.6 mmol; commercial 60% oil dispersion) was add-ed dropwise a solution of diethyl cyanomethylphosphon-ate (2.14 ml, 14.1 mmol) in THF (17.5 ml) at 0°C andstirred for 15 min. Then the solution was warmed toRT and a THF (17.5 ml) solution of cyclohexanecar-boxyaldehyde (1.26 ml, 10.5 mmol) was added dropwiseand stirred at RT for 3 h. The reaction mixture waswashed with water. The organic layer was extracted withdichloromethane and the solvent was evaporated. Theremaining liquid was puri®ed by distillation to give col-orless oil (50% yield). 1HNMR (CDCl3): d 1.05±1.80(m, 10H), 2.5±2.75 (m, 1H), 5.20 (d, J� 11 Hz, 1H),6.31 (t, J� 10 Hz, 1H).

2.6.2. 2-Methyl-2-phenylpropionaldehyde (10)To a THF (50 ml) suspension of sodium hydride (880

mg, 22 mmol; commercial 60% oil dispersion) was addeddropwise 2-phenylpropionaldehyde (2.65 ml, 20 mmol)in THF (10 ml) at 0°C. After stirring for 5 min, iodo-methane (1.37 ml, 22 mmol) in THF (10 ml) was addeddropwise and stirred at room temperature for 10 h. Thereaction mixture was washed with water, then organiclayer was extracted with diethylether and the solventwas evaporated. The remaining liquid was puri®ed bydistillation under reduced pressure (20 mm Hg, 82±85°C) to give colorless oil (50% yield). 1HNMR(CDCl3): d 1.47 (s, 6H), 7.20±7.40 (m, 5H), 9.51 (s, 1H).

2.6.3. 2-(Benzyldimethyl)methylacrylonitrileTo a THF (17 ml) suspension of sodium hydride (240

mg, 6.0 mmol; commercial 60% oil dispersion) was add-ed dropwise diethyl cyanomethylphosphonate (0.88 ml,5.8 mmol) in THF (8.5 ml) at 0°C and stirred for 10min. Then 2,2-dimethyl-3-phenylpropionaldehyde (754mg, 4.65 mmol) in THF (8.5 ml) was added dropwiseand stirred at room temperature for 20 h. The reactionmixture was washed with water and the organic layerwas extracted with dichloromethane, followed by evap-oration of the solvent. The residue was submitted to acolumn chromatograph (silica gel, hexane:ethyl ace-tate� 5:1) to give a cis- and trans-isomer as colorless liq-uid (50% yield). 1HNMR (CDCl3): d for trans-isomer,1.06 (s, 6H), 2.64 (s, 2H), 5.10 (d, J� 16 Hz, 1H), 6.74(d, J� 16 Hz, 1H), 7.05±7.30 (m, 5H), for cis-isomer,1.28 (s, 6H), 2.75 (s, 2H), 5.28 (d, j� 12 Hz, 1H), 6.32(d, J� 12 Hz, 1H), 7.05±7.30 (m, 5H).

2.6.4. 2,2-Dimethyl-3-phenylpropionaldehyde (11)To a suspension of pyridinium chlorochromate (6.47

g, 30 mmol) in dry dichloromethane (40 ml) was added2,2-dimethyl-3-phenylbutanol (3.29 g, 20 mmol) in drydichloromethane (4 ml) in one portion with vigorousstirring. After stirring at room temperature for 90 min,

100 ml of diethyl ether was added and the resulting so-lution was passed through a silica gel column for remov-al of solid residues. The solvent was evaporated andthen the remaining oil was puri®ed by distillation underreduced pressure (20 mm Hg, 103±106°C) to give color-less liquid (56% yield). 1HNMR (CDCl3): d 1.05 (s, 6H),2.78 (s, H), 7.07±7.30 (m, 5H).

2.6.5. 2-(Dimethylphenyl)methylacrylonitril (2-cum-ylacrylonitril)

To a THF (10 ml) suspension of sodium hydride (240mg, 6.0 mmol; commercial 60% oil dispersion) was add-ed dropwise diethyl cyanomethylphosphonate (0.88 ml,5.8 mmol) in THF (8.5 ml) at 0°C and stirred for 10min. Then 2,2-dimethyl-3-phenylpropionaldehyde (740mg, 5.0 mmol) in THF (8.5 ml) was added dropwiseand stirred at room temperature for 20 h. The reactionmixture was washed with water and the organic layerwas extracted with dichloromethane, followed by evap-oration of the solvent. The residue was submitted to acolumn chromatograph (silica gel, hexane:ethylace-tate� 5:1) to give colorless liquid as a mixture of cis-and trans-isomer (48% yield). 1HNMR (CDCl3): d forcis-isomer, 1.64 (s, 6H), 5.36 (d, J� 12 Hz, 2H), 6.53(d, J� 12 Hz, 1H), 7.24±7.38 (m, 5H), for trans-isomer,1.45 (s, 6H), 5.28 (d, J� 17 Hz, 1H), 6.32 (d, J� 17 Hz,1H), 7.24±7.38 (m, 5H).

3. Results and discussion

Peroxoiron(III) species in the catalytic cycle of cyto-chrome P-450 has been postulated as a key intermediateto a�ord a high valent species equivalent to compound Iof peroxidases. In 1980, Valentine et al. reported thepreparation of peroxo±FeIII(TPP) and peroxo±FeIII

(OEP) by the reactions of KO2 with FeIII(TPP)Cl andFeIII (OEP)Cl in aprotic solvents [24]. Examination ofthe complexes by NMR, ESR, UV, IR, and EXAFSsupports the structure of the complexes to be ferric±per-oxo species with side-on structure as shown below [24].The same peroxo complexes could be obtained eitherby the reaction Fe(I) porphyrin with O2 or by the reduc-tion of oxy complexes [28]. The latter reaction is the ex-act model reaction of the cytochrome P-450 catalyzedoxygen activation. Similar reactions were also appliedfor the preparation of peroxo±Mn porphyrin complexes[29]. The X-ray structure of peroxo-MnIII(TPP) revealedan unusually domed structure of the complex [30]. Reac-tions of this type of peroxo complexes might be goodmodels for the peroxo intermediate in cytochrome P-450.

Y. Goto et al. / J. Inorg. Biochem. 69 (1998) 241±247 243

3.1. Reaction of alkyl aldehydes with peroxoiron (III)porphyrin complexes

A reaction of [TPPFeIII(O2)]ÿ and cyclohexanecar-boxyaldehyde 4 took place in acetonitrile at room tem-perature under dry helium atmosphere. According tothe reaction, the UV±VIS spectral change of[TPPFeIII(O2)]. was observed (Fig. 1). The oxidationproducts of 4 were determined by GC±MS. Cyclohexa-none 5 was identi®ed (yield, 15%) as the deformylatedproduct along with cis- and trans-isomers of 3-cyclohex-yl acrylonitrile 6 (total yield, 20%) (run 1 in Table 1,Eq. (1)), which are formed by condensation of 4 andacetonitrile. Similar reactions of 4 with a series of per-oxoiron(III) porphyrin complexes were also carriedout and the results are summarized in Table 1. Whilethe deformylated product 5 was obtained in the reactionof 4 with [FeIIITDCPP(O2)]ÿ under He atmosphere,[FeIIITMP(O2)]ÿ gave a trace amount of 5. As shownin Table 1, the reaction of KO2 and 4 also gave 6, thus,possible participation of free KO2 in the peroxo±Fecomplex reactions was examined. However, ESR exam-ination of the solution clearly showed no contaminationof superoxide anion in the solution (Fig. 2).

�1�Deformylation in the same manner was also observed

in the reactions of peroxo±iron(III) complexes withphenylacetaldehyde (7) and 2-phenylpropionaldehyde(8). The reactions of 7 and peroxo±iron (III) complexesgave a trace amount of benzaldehyde, while 15±30%yields of acetophenone (9) were observed for theoxidation of 8. Incidentally. the reactions of 8 withKO2 gave 9 in yield of 25%. In reactions of[TPPFeIII(O2)]ÿ and aldehydes bound to the tertiary car-bon such as 2-methyl-3-phenylpropionaldehyde (10) or2,2-dimethyl-3-phenylpropionaldehyde (11), the corre-sponding condensation products with acetonitrile wereobtained without deformylated compound (Table 2).This indicates a-hydrogen is crucial for the deformylat-ion reaction. Apparently, the yields of deformation reac-tions are very much dependent on the structure of bothsubstrate and porphyrin.

The results summarized in Tables 1 and 2 indicatethat the peroxo complexes serve as a base as well asthe deformylating reagent. Though the peroxo±Fe com-plexes readily react with proton derived from a traceamount of such as methanol and H2O to a�ord hydro-xo±FeIII porphyrins, the peroxo complexes are stablein dry acetonitrile, indicating the peroxo-oxygen is notable to abstract a proton from acetonitrile. Thus, the re-action of the peroxo complex with aldehyde must a�ordvery basic intermediate. Scheme 2 shows a plausiblemechanism for the formation of condensation products.The initial nucleophilic attack of the peroxo complex toaldehyde is consistent with its reaction with acyl halides[21], sulfur dioxide [22], and carbon dioxide [23].

The peroxo complexes were also prepared in THF toavoid the involvement of the solvent into the products.Unfortunately, the peroxo complexes did not react withaldehydes in THF at room temperature.

Fig. 2. ESR spectra (at 77 K) before (solid) and after (dotted) the ad-

dition of 4 to a CH3CN solution of [FeIIITPP(O2)]ÿ at room tempera-

ture.

Fig. 1. UV±VIS spectral change in the reaction of [FeIIITPP(O2)]ÿ up-

on the addition of 4 at room temperature. ±±±±±±: before the reaction,

- - - : 30 min after the addition of 4.

244 Y. Goto et al. / J. Inorg. Biochem. 69 (1998) 241±247

In order to gain mechanistic details of deformylationreactions, ESR and UV±VIS spectral changes upon ad-dition of aldehydes to the peroxo complexes were mea-sured (Figs. 1 and 2). To an acetonitrile solution of[FeIIITPP(O2)]ÿ (2.5 mM) two equivalents of 4 was add-ed and stirred for 30 min. Then, the ESR spectrum ofthe reaction mixture was examined. A typical rhombicESR signal for the peroxo complexes (g� 9.5, 4.2, 1.3)[24] disappeared and the solution became EPR silent.This spectral change corresponds to the Soret band shiftfrom 432 to 429 nm shown in Fig. 1. These results sug-gest that [FeIIITPP(O2]ÿ was changed to Fe(II) TPP ac-cording to the reaction with aldehydes.

One of possible mechanisms for the formation ofFeIIITPP is a homolytic cleavage of Fe±O bond of theferric peroxy hemiacetal anionic intermediate [A] as

shown in Scheme 3. The resulting alkyl peroxy hemiac-etal anion radical species could also be an intermediateformed by the direct reaction of 4 and superoxide anion.If this is the case, the deformylation and condensationwith solvent share the same intermediate [A] and the fol-lowing competitive Fe±O cleavage (Scheme 3) and de-protonation (Scheme 2) a�ord a mixture of products.In order to detect radical species present in the solution,radical trap experiments were carried out. However, thereactions of 4 and [FeIIITPP(O2)]ÿ in the presence ofphenyl-tert-butylnitrone (PBN) as a radical trap reagentunder He atmosphere (Table 1, run 2) a�orded 5 with-out appreciable e�ect. This result implies no stable rad-ical species in the solution. In addition. UV±VIS spectralchanges according the reaction of [TPPFeIII(O2)]ÿ andaldehyde are very di�erent from that obtained by the re-action of [TPPFeIII(O2)]ÿ and proton derived from suchas methanol, benzoic acid, or H2O. Therefore, directproton abstraction from the substrate by the peroxocomplex is a quite unlikely process.

While we could not observe formic acid to be formedalong with deformylated ketones, we have obtained acomparable amount of a formic acid equivalent, benzoicacid, to propiophenone when we used 2-phenyl-butyrophenone as the substrate (Eq. (2)).

3.2. Reaction of Fe(IV)@O porphyrin p-cation radicalwith alkyl aldehydes

It has been reported that aldehydes were readily oxi-dized to carboxylic acids by an oxoferryl porphyrin p-cation radical species [15], a model complex for the ac-tive species responsible for oxidations by peroxidaseand cytochrome P-450. Indeed, the reaction of 4 andO@FeIVTMP p-cation radical at )80°C generated cyclo-hexane carboxylic acid in a quantitative yield. Thus,high valent oxo-species are not responsible for the de-formylation. Our results are rather suggestive of the per-oxo±FeIII complexes being the active species for thedeformylation process. However, there is the crucial dif-ference in the deformylation reactions catalyzed by P450and our system; i.e., the model system failed to yield

Scheme 2

Y. Goto et al. / J. Inorg. Biochem. 69 (1998) 241±247 245

ole®ns. Not only the deformylation, oxidation of hydro-carbons catalyzed by iron porphyrin complexes is usedto give alcohols and ketones but not ole®ns even thoughdehydration (ole®n formation) can be observed in hemeenzymes catalyzed oxidations.

Very recently, Valentine and coworkers reported ep-oxidation of a,b-unsaturated ketone by peroxo±FeIIITMP [20]. The reaction could be explained by theinitial nucleophilic Michael addition of the peroxo oxy-gen as observed in the epoxidation of a,b-unsaturatedketone by H2O2 under basic conditions [31]. These re-sults suggest that the peroxo±FeIII intermediate in thecatalytic cycle of P-450 could be either trapped by thoseelectrophilic substrates or further activated to high va-lent oxo-species by its reaction with proton.

In summary, we have examined the reactions of per-oxo±FeIII porphyrin complexes with a series of alde-hydes. While condensation of aldehydes with solventacetonitrile masked the oxidation process, we haveshown that the deformylation can be conducted onlyby the peroxo species but not by an oxo±ferryl porphyr-in p-cation radical.

Note added in proof. After the submission of thismanuscript, D. Wertz and co-workers reported thedetection of formic acid by NMR under the conditionvery similar to this report (10th International Conferenceon Cytochrome P450, San Francisco, August, 1997).

Acknowledgements

This work was supported by Grant-in-Aid for Scien-ti®c Research on Priority Areas, Molecular Biometallics(I.M. and Y.W.).

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