Article Cite This: Inorg. Chem. 2017, 56, 13166-13173
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Oxygen-Centered Radicals Formed in the Reaction Mixtures Containing Chloroiron Tetraphenylporphyrin, Iodosylbenzene, and Ethanol Daisuke Nishizaki and Hideo Iwahashi* Department of Chemistry, Wakayama Medical University, 580 Mikazura, Wakayama 6410011, Japan
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ABSTRACT: Heme and nonheme high-valent FeIVO can mediate reactions of olefin epoxidation, alkane hydroxylation, aromatic hydroxylation, S-oxidation, P-oxidation, N-dealkylation, alkylaromatic oxidation, and alcohol oxidation. Bromocycloheptane forms as a product in the reaction of high-valent FeIVO with cycloheptane, suggesting that a cycloheptyl radical reacts with CCl3Br. However, the cycloheptyl radical has not yet been directly detected. To directly detect the radical intermediate in the reaction of the high-valent FeIVO, we analyzed reaction mixtures containing chloroiron tetraphenylporphyrin, iodosylbenzene, ethanol, and α-(4-pyridyl1-oxide)-N-tert-butylnitrone (4-POBN) in 1,2-dichloroethane by an electron spin resonance (ESR) spin-trapping method. As a spin-trapping reagent, we used 4-POBN. Prominent ESR signals were observed in the reaction mixtures. To determine the structure of the radical, the reaction was performed using ethanol-1-13C (or ethanol-2-13C) instead of ethanol. ESR spectra with no additional hyperfine splitting were observed, indicating that the radical formed in complete reaction mixtures of the porphyrin π-cation-radical species (TPP)•+FeIVO (TPP = 5,10,15,20-tetraphenyl-21H,23H-porphine) with ethanol has an unpaired electron at neither the α-carbon nor the β-carbon. When the reaction mixture containing ethanol-d6 instead of ethanol was analyzed using high-performance liquid chromatography−ESR−mass spectrometry, the ions m/z 240 (4-POBN/•OCH2CH3) shifted to m/z 245 (4-POBN/•OCD2CD3). Thus, the radical formed in the complete reaction mixture of (TPP)•+FeIVO with ethanol has an unpaired electron at the oxygen atom in ethanol. We detected and identified the ethanol-derived oxygen-centered radicals in the reaction of (TPP)•+FeIVO with ethanol for the first time in this study.
1. INTRODUCTION Heme and nonheme high-valent FeIVO can mediate reactions of olefin epoxidation, alkane hydroxylation, aromatic hydroxylation, S-oxidation, P-oxidation, N-dealkylation, alkylaromatic oxidation, and alcohol oxidation.1−9 Of the heme and nonheme high-valent FeIVO, high-valent iron(IV) oxo porphyrin π-cation-radical species [(Porp)•+FeIV(O)] are generally accepted to be the key reactive intermediates in a variety of oxidation reactions by heme-containing enzymes such as cytochromes P450, peroxidases, and catalases.9,10 In 1979, Groves et al. first published their study on the catalytic olefin epoxidation and alkane hydroxylation by Fe(TPP)Cl (TPP = 5,10,15,20-tetraphenyl-21H,23H-porphine) and iodosylbenzene (PhIO).11 The reaction of Fe(TPP)Cl with PhIO produces (TPP)•+FeIVO, which is involved in olefin epoxidation and alkane hydroxylation.12,13 During the oxidation of cycloheptane using PhIO and Fe(TPP)Cl in the presence of bromotrichloromethane (CCl3Br) under an Ar atmosphere, bromocycloheptane was observed as the product, indicating that a cycloheptyl radical appears to react with CCl3Br.1,14 The substrate radical has not yet been directly detected. Furthermore, in the reaction mixture of high-valent FeIVO with alcohols, an iron(II) alcohol intermediate and an iron(III) alkoxide species were observed.3,15 © 2017 American Chemical Society
Yamazaki and Piette detected 5,5-dimethyl-1-pyrroline Noxide (DMPO)/OH and DMPO/α-hydroxyethyl radical adducts in the Fenton reactions with and without ethanol. On the basis of quantitative analysis of the sum of DMPO/OH and DMPO/α-hydroxyethyl radical adducts formed in the reaction mixtures, the α-hydroxyethyl radical was reported to form through the reaction not only with •OH but also with other species, presumably ferryl ion.16 It has been reported that the α-hydroxyethyl radicals form in the reactions of ethanol with deermouse microsomes or a perferryl complex of nitric oxide synthase.17,18 Formation of the α-hydroxyethyl radicals was confirmed using α-13C-ethanol as a substrate by electron spin resonance.17,19 Thus, many papers have proposed that radical intermediates form in the reactions of [(Porp)•+FeIV(O)] and Fenton’s reagent. In this study, we aimed to detect radicals formed in the reaction of [(Porp)•+FeIV(O)]. Furthermore, we determined the chemical structures of radical intermediates in order to clarify the difference in the reaction pathways of the two reactions. We employed high-performance liquid chromatography−electron spin resonance−mass spectrometry (HPLC− ESR−MS).20 Using ESR, HPLC−ESR, and HPLC−ESR−MS Received: July 31, 2017 Published: October 10, 2017 13166
DOI: 10.1021/acs.inorgchem.7b01949 Inorg. Chem. 2017, 56, 13166−13173
Article
Inorganic Chemistry
without further processing for the complete reaction mixture of Fenton’s reagent with ethanol. For HPLC−ESR and HPLC−ESR−MS analyses of the complete reaction mixture of Fenton’s reagent with ethanol, residual ethanol was removed from the reaction mixture using the centrifugal concentrator cc-105 after the reaction was finished. Determination of Hyperfine Coupling Constants of the ESR Spectra Observed in the Complete Reaction Mixtures of (TPP)•+FeIVO with Ethanol (or Ethanol-1-13C or Ethanol-2-13C) and Fenton’s Reagent with Ethanol (or Ethanol-1-13C or Ethanol-2-13C). ESR spectra of the respective peaks were measured by stopping the HPLC flow when the peaks appeared on the HPLC− ESR elution profiles of the complete reaction mixtures of (TPP)•+FeIVO with ethanol (or ethanol-1-13C or ethanol-2-13C) and Fenton’s reagent with ethanol (or ethanol-1-13C or ethanol-2-13C). Kinetic Isotope Effect Experiments. Kinetic isotope effect experiments to probe the hydrogen-bonding interaction were performed. The magnetic field of the ESR spectrometer was fixed at the third ESR peak in the characteristic six-line spectrum of the 4POBN radical adduct observed in the complete reaction mixture of (TPP)•+FeIVO with ethanol. The ESR peak height of the complete reaction mixture was monitored at 25 °C from 30 s after the addition of PhIO.
combined with spin trapping, we analyzed the radicals formed in the reaction of (TPP)•+FeIVO with alcohols such as ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, and tertbutyl alcohol, in addition to the reaction of Fenton’s reagent with ethanol.
2. EXPERIMENTAL SECTION Materials. 5,10,15,20-Tetraphenyl-21H,23H-porphine iron(III) chloride ([Fe(TPP)Cl], commonly known as chloro iron tetraphenylporphyrin) was purchased from Sigma-Aldrich Co. LLC. Ethanol and 1,2-dichloroethane were purchased from Kishida Chemical Co., Ltd. (Osaka, Japan). Iodosylbenzene (PhIO), ethanol-d 6 (CD3CD2OD), ethanol-d3 (CD3CH2OH), ethanol-d1 (CH3CH2OD), 1-propanol, 2-propanol, 1-butanol, 2-butanol, tert-butyl alcohol, and α(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) were obtained from Tokyo Kasei Kogyo, Ltd. (Tokyo, Japan). Ethanol-1-13C and ethanol2-13C were from Cambridge Isotope Laboratories, Inc. (Andover, MA). Water was purified by distillation and passed through an Autopure WT101UV (Yamato Scientific Co. Ltd., Tokyo, Japan). All other chemicals were of analytical grade. ESR, HPLC−ESR, and HPLC−ESR−MS Analyses. We performed ESR, HPLC−ESR, and HPLC−ESR−MS analyses as described previously.20 By aspirating the reaction mixtures into a Teflon tube centered in a microwave cavity, we performed ESR analyses. HPLC− ESR analyses were performed with two solvent systems, an acid solvent system [solvent A, 50 mM acetic acid; solvent B, 50 mM acetic acid/acetonitrile (20:80, v/v)], or a neutral solvent system [solvent A, 50 mM ammonium acetate; solvent B, 50 mM ammonium acetate/ acetonitrile (36:64, v/v)]. Unless otherwise noted, we used the acid solvent system. HPLC−ESR−MS analyses of all of the respective peaks except for 1-butanol were performed with the acid solvent system after the respective peaks were partially purified using HPLC− ESR with the neutral solvent system. HPLC−ESR−MS analysis of the complete reaction mixture of 1-butanol was performed directly with the acid solvent system. Complete Reaction Mixtures of (TPP)•+FeIVO with Some Alcohols. The complete reaction mixture of (TPP)•+FeIVO contained 600 μL of ethanol (1-propanol, 2-propanol, 1-butanol, 2butanol, or tert-butyl alcohol), 17.5 mM 4-POBN, 0.5 mM Fe(TPP)Cl, and 60 mg of PhIO in 1,2-dichloroethane. The total volume of the complete reaction mixture was 3.0 mL. 4-POBN was used as a spin-trapping reagent. The complete reaction mixture was initiated by the addition of PhIO and allowed to proceed for 10 min at 25 °C. ESR measurements were performed for the complete reaction mixture without further processing. For HPLC−ESR and HPLC−ESR−MS analyses of all of the complete reaction mixtures except for 1-butanol, the following procedures were carried out. After the reaction was finished, we added 3.0 mL of 0.2 M phosphate buffer (pH 7.4) to the complete reaction mixture and stirred the resulting reaction mixture vigorously. We collected 2 mL from the upper phase of the phase-separated reaction mixture. We further added 3.0 mL of 0.2 M phosphate buffer (pH 7.4) to the residue and stirred. Again, we collected 3 mL of the upper layer. Residual 1,2-dichloroethane was completely removed from the combined two upper layers using a centrifugal concentrator cc-105 (Tomy Seiko Co., Ltd., Tokyo, Japan). The following procedures were carried out for the complete reaction mixture of 1butanol. After the reaction was finished, we added 5 mL of 0.2 M phosphate buffer (pH 7.4) to the complete reaction mixture. Using a rotary evaporator model RE-46 (Yamato Scientific Co., Ltd., Tokyo, Japan), 1,2-dichloroethane was removed from the reaction mixture. Using the centrifugal concentrator cc-105, residual 1,2-dichloroethane was completely removed from the reaction mixture. Complete Reaction Mixture of Fenton’s Reagent with Ethanol. The complete reaction mixture of Fenton’s reagent with ethanol contained 600 μL of ethanol, 1.0 mM Fe(SO4)2(NH4)2, 1.0 mM H2O2, and 20 mM 4-POBN in a total volume of 3.0 mL. The reaction was initiated by the addition of Fe(SO4)2(NH4)2 and allowed to proceed for 1.0 min at 25 °C. We performed ESR measurements
3. RESULTS ESR Measurements of the Complete Reaction Mixture of (TPP)•+FeIVO with Ethanol. ESR measurements of the complete reaction mixture of (TPP)•+FeIVO with ethanol showed a distinguished triplet of doublets (αN = 1.37 mT and αH = 0.20 mT; Figure 1a), suggesting that a radical
Figure 1. ESR spectra of the complete reaction mixture of (TPP)•+FeIVO with ethanol. Reaction and ESR conditions were as described in the Experimental Section. (a) Complete reaction mixture of (TPP)•+FeIVO with ethanol. The complete reaction mixture of (TPP)•+FeIVO contained 600 μL of ethanol, 17.5 mM 4POBN, 0.5 mM Fe(TPP)Cl, and 60 mg of PhIO in 1,2-dichloroethane. The total volume of the complete reaction mixture was 3.0 mL. (b) As in part a but with ethanol omitted. (c) As in part a but with Fe(TPP)Cl omitted. (d) As in part a except with PhIO omitted.
intermediate forms in the complete reaction mixture of (TPP)•+FeIVO with ethanol. ESR peak heights of the complete reaction mixtures without ethanol, Fe(TPP)Cl, or PhIO decreased to 11 ± 1%, 32 ± 1%, or 0 ± 0% (n = 3) of the complete reaction mixture of (TPP)•+FeIVO with ethanol, respectively (Figure 1b−d). These results indicate that ethanol, Fe(TPP)Cl, and PhIO are essential for radical formation. HPLC−ESR Analyses of the Complete Reaction Mixture of (TPP)+•FeIVO with Ethanol. HPLC−ESR analyses were performed with the acid solvent system. A prominent peak with a retention time of 31.9 min (peak 1) was 13167
DOI: 10.1021/acs.inorgchem.7b01949 Inorg. Chem. 2017, 56, 13166−13173
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Inorganic Chemistry observed on the HPLC−ESR elution profile of ethanol (Figure 2a). Weak peaks were observed at retention times of 32.1 and
Figure 3. HPLC−ESR analyses of the complete reaction mixture of ethanol with (TPP)•+FeIVO or Fenton’s reagent. The reaction and HPLC−ESR conditions were as described in the Experimental Section. (a) Complete reaction mixture of (TPP)•+FeIVO with ethanol. (b) Complete reaction mixture of Fenton’s reagent with ethanol. (c) Mixture of fractions of peaks 1 and 3. The fraction of peak 1 in part a and the fraction of peak 3 in part b were collected, and the two fractions were mixed at a ratio of 5 (peak 1) to 1 (peak 3). After acetonitrile was completely removed from the mixture using the centrifugal concentrator cc-105, HPLC−ESR analysis of the mixture was performed.
Figure 2. HPLC−ESR analyses of the complete reaction mixture of (TPP)•+FeIVO with ethanol. The reaction and HPLC−ESR conditions were as described in the Experimental Section. (a) Complete reaction mixture of (TPP)•+FeIVO with ethanol. The complete reaction mixture of (TPP)+•FeIVO contained 600 μL of ethanol, 17.5 mM 4-POBN, 0.5 mM Fe(TPP)Cl, and 60 mg of PhIO in 1,2-dichloroethane. The total volume of the complete reaction mixture was 3.0 mL. (b) As in part a but with ethanol omitted. (c) As in part a but with Fe(TPP)Cl omitted. (d) As in part a but with PhIO omitted. (e) As in part a except HPLC−ESR used the neutral solvent system [solvent A, 50 mM ammonium acetate; solvent B, 50 mM ammonium acetate/acetonitrile (36:64, v/v)]. (f) Same as that in part b except HPLC−ESR used the neutral solvent system.
39.1 min in the absence of ethanol (Figure 2b) and 31.8 min in the absence of Fe(TPP)Cl (Figure 2c). Peaks were hardly observed in the absence of PhIO (Figure 2d). Furthermore, HPLC−ESR analyses were performed with the neutral solvent system. A prominent peak with a retention time of 31.8 min and two feeble peaks with retention times of 18.2 and 28.6 min were observed on the HPLC−ESR elution profile of ethanol (Figure 2e). The two feeble peaks were observed at retention times of 18.1 and 39.5 min in the absence of ethanol (Figure 2f). HPLC−ESR Analysis of the Complete Reaction Mixture of Fenton’s Reagent with Ethanol. To compare (TPP)•+FeIVO with Fenton’s reagent with regard to the ethanol reaction, ESR and HPLC−ESR analyses were performed for the complete reaction mixture of Fenton’s reagent with ethanol (Figures 3 and S1). Prominent peaks with retention times of 25.2 min (peak 2) and 28.2 min (peak 3) were observed on the HPLC−ESR elution profile of ethanol with Fenton’s reagent (Figure 3b). The retention times are different from that of peak 1, which was observed for the complete reaction mixture of ethanol with (TPP)•+FeIVO (Figure 3a). To confirm that peaks 1 and 3 are different, fractions of them were mixed and analyzed by HPLC−ESR. The two fractions were eluted at different retention times (Figure 3c). HPLC−ESR−MS Analyses of the Complete Reaction Mixture of (TPP)•+FeIVO with Ethanol. To identify peak 1, HPLC−ESR−MS analyses were performed for the complete reaction mixture of (TPP)•+FeIVO with ethanol. An ion at m/z 240 was detected for peak 1, which corresponded to [M + H]+ of 4-POBN/ethanol-derived radical adducts (Figure 4a). A
Figure 4. HPLC−ESR−MS analyses of the complete reaction mixture of (TPP)•+FeIVO with ethanol (or ethanol-d6). The reaction and HPLC−ESR−MS conditions were as described in the Experimental Section. (a) Complete reaction mixture of (TPP)•+FeIVO with ethanol. (b) Complete reaction mixture of (TPP)•+FeIVO with ethanol-d6.
fragment ion at m/z 153 corresponds to the loss of [(CH3)3C(O)N] from the protonated molecular ion [M + H]+. An ion at m/z 460 corresponds to the 4-POBN/ethanolderived radical adduct complexed with PhIO, [M + PhIO + H]+. To determine the structure of the radical, the reaction was performed using ethanol-d6. If α-carbon-centered radicals [•C(OD)DCD3] form, deuterium in the hydroxyl group is displaced by hydrogen in H2O (eq 2).21 Meanwhile, no deuterium is displaced in the oxygen-centered radicals 13168
DOI: 10.1021/acs.inorgchem.7b01949 Inorg. Chem. 2017, 56, 13166−13173
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Inorganic Chemistry
Figure 5. HPLC−ESR−MS analyses of the complete reaction mixture of Fenton’s reagent with ethanol (or ethanol-d6 or ethanol-d3). The reaction and HPLC−ESR−MS conditions were as described in the Experimental Section. (a) Peak 2 obtained from the complete reaction mixture of Fenton’s reagent with ethanol. (b) Peak 3 obtained from the complete reaction mixture of Fenton’s reagent with ethanol. (c) Peak 2 obtained from the complete reaction mixture of Fenton’s reagent with ethanol-d6. (d) Peak 3 obtained from the complete reaction mixture of Fenton’s reagent with ethanol-d6. (e) Peak 2 obtained from the complete reaction mixture of Fenton’s reagent with ethanol-d3. (f) Peak 3 obtained from the complete reaction mixture of Fenton’s reagent with ethanol-d3.
(•OCD2CD3; eq 1) in H2O. When the reaction was performed using ethanol-d6 instead of ethanol, the ions at m/z 240, 153, and 460 shifted to m/z 245, 158, and 465, respectively. Thus, we demonstrated that ethanol-derived oxygen-centered radicals (•OCD2CD3) form in the reaction mixture using ethanol-d6 instead of ethanol (Figure 4b).
2-13C) and Fenton’s reagent with ethanol (or ethanol-1-13C or ethanol-2-13C). ESR measurements of peaks 1−3 showed distinct triplet of doublets (αN = 1.37 mT and αH = 0.20 mT for peak 1; αN = 1.57 mT and αH = 0.25 mT for peak 2; αN = 1.55 mT and αH = 0.26 mT for peak 3; Figure 6a−c). To further determine the structure of the radical, the complete reaction mixtures of (TPP)•+FeIVO with ethanol-1-13C (or ethanol-2-13C) and Fenton’s reagent with ethanol-1-13C (or ethanol-2-13C) were analyzed (Figure 6). When ethanol-1-13C was used instead of ethanol, ESR spectra of peaks 2 and 3 showed additional splitting due to hyperfine coupling of an unpaired electron with the 13C nucleus in ethanol-1-13C (Figure 6e,f). We observed no additional hyperfine splitting for Fenton’s reagent with ethanol-2-13C (Figure 6h,i). These results indicate that the radical formed in Fenton’s reagent with ethanol has an unpaired electron at the α-carbon atom in ethanol. On the other hand, no additional hyperfine splitting was observed in the case of the complete reaction mixtures of (TPP)•+FeIVO with ethanol-1-13C (or ethanol-2-13C), suggesting that the radical formed in the complete reaction mixture of (TPP)•+FeIVO with ethanol has an unpaired electron at neither α-carbon nor β-carbon (Figure 6d,g). Thus, the radical formed in the complete reaction mixture of (TPP)•+FeIVO with ethanol has an unpaired electron at the oxygen atom in ethanol. ESR Measurements of the Complete Reaction Mixtures of (TPP)•+FeIVO with 1-Propanol, 2-Propanol, 1-Butanol, 2-Butanol, or tert-Butyl Alcohol. We performed ESR measurements of the control reaction mixtures of (TPP)•+FeIVO with 1-propanol, 2-propanol, 1-butanol, 2butanol, or tert-butyl alcohol to shed light on the relationship between the reactivity and structure (Figures 7 and S2−S6). ESR measurements of the complete reaction mixtures of (TPP)•+FeIVO with 1-propanol, 2-propanol, 1-butanol, 2butanol, or tert-butyl alcohol showed distinguished triplet of doublets (αN = 1.37 mT and αH = 0.22 mT for 1-propanol; αN = 1.35 mT and αH = 0.20 mT for 2-propanol; αN = 1.35
4‐POBN + •OCD2 CD3 H 2O
⎯⎯⎯→ 4‐POBN/•OCD2 CD3 (m /z 245)
(1)
4‐POBN + •C(OD)DCD3 H 2O
⎯⎯⎯→ 4‐POBN/•C(OH)DCD3 (m /z 244)
(2)
HPLC−ESR−MS Analyses of the Complete Reaction Mixture of Fenton’s Reagent with Ethanol. To identify peaks 2 and 3 (Figure 3b), HPLC−ESR−MS analyses were performed for the complete reaction mixture of Fenton’s reagent with ethanol. Ions at m/z 240 were detected for peaks 2 and 3, which correspond to [M + H]+ of 4-POBN/ethanolderived radical adducts (Figures 5a and 5b). When the reaction was performed using ethanol-d6 instead of ethanol, the ions at m/z 240 of peaks 2 and 3 shifted to m/z 244 (Figure 5c,d). Furthermore, the ions at m/z 240 of peaks 2 and 3 shifted to m/z 243 for the reaction using ethanol-d3 [C(OH)H2CD3] instead of ethanol (Figure 5e,f). Thus, we demonstrated that diastereomers of the 4-POBN/α-hydroxyethyl radical [•C(OH)HCH3] adducts form in the complete reaction mixture of Fenton’s reagent with ethanol. ESR Measurements of the Complete Reaction Mixtures of (TPP)•+FeIVO with Ethanol (or Ethanol1-13C or Ethanol-2-13C) and Fenton’s Reagent with Ethanol (or Ethanol-1-13C or Ethanol-2-13C). ESR spectra of the respective peaks were measured by stopping the HPLC flow at the same time that the peaks appeared on the HPLC− ESR elution profiles of the complete reaction mixtures of (TPP)•+FeIVO with ethanol (or ethanol-1-13C or ethanol13169
DOI: 10.1021/acs.inorgchem.7b01949 Inorg. Chem. 2017, 56, 13166−13173
Article
Inorganic Chemistry
Figure 7. ESR spectra of the complete reaction mixtures of (TPP)•+FeIVO with some alcohols: (a) ethanol; (b) 1-propanol; (c) 2-propanol; (d) 1-butanol; (e) 2-butanol; (f) tert-butyl alcohol. The reaction and ESR conditions were as described in the Experimental Section.
Table 1. ESR Peak Heights of the Complete Reaction Mixtures of (TPP)+ FeIVO with Some Alcoholsa compound ethanol 1-propanol 2-propanol 1-butanol 2-butanol tert-butyl alcohol
peak height (cm) 6.6 10.5 1.2 8.8 3.4 0.8
± ± ± ± ± ±
0.4 0.5 0.2 0.2 0.3 0.1
The complete reaction mixture of (TPP)+ FeIVO contained 600 μL of ethanol (1-propanol, 2-propanol, 1-butanol, 2-butanol, or tert-butyl alcohol), 17.5 mM 4-POBN, 0.5 mM Fe(TPP)Cl, and 60 mg of PhIO in 1,2-dichloroethane. The total volume of the complete reaction mixture was 3.0 mL. The results are the mean ± standard deviation of three experiments.
a
Butanol. HPLC−ESR analyses were performed to examine the radical adducts formed in the complete reaction mixtures of (TPP)+•FeIVO with 1-propanol or 1-butanol. Two peaks were detected at retention times of 36.3 min (peak 4; distinguished) and 32.1 min (weak) for the complete reaction mixture of (TPP)•+FeIVO with 1-propanol (Figure 8b). A prominent peak with a retention time of 40.8 min (peak 5) was observed on the HPLC−ESR elution profile of the complete
Figure 6. ESR spectra of the complete reaction mixtures of (TPP)•+FeIVO with ethanol (or ethanol-1-13C or ethanol-2-13C) and Fenton’s reagent with ethanol (or ethanol-1-13C or ethanol-2-13C). The reaction and ESR conditions were as described in the Experimental Section. (a) Peak 1 obtained from the complete reaction mixture of (TPP)•+FeIVO with ethanol. (b) Peak 2 obtained from the complete reaction mixture of Fenton’s reagent with ethanol. (c) Peak 3 obtained from the complete reaction mixture of Fenton’s reagent with ethanol. (d) Peak 1 obtained from the complete reaction mixture of (TPP)•+FeIVO with ethanol-1-13C. (e) Peak 2 obtained from the complete reaction mixture of Fenton’s reagent with ethanol1-13C. (f) Peak 3 obtained from the complete reaction mixture of Fenton’s reagent with ethanol-1-13C. (g) Peak 1 obtained from the complete reaction mixture of (TPP)•+FeIVO with ethanol-2-13C. (h) Peak 2 obtained from the complete reaction mixture of Fenton’s reagent with ethanol-2-13C. (i) Peak 3 obtained from the complete reaction mixture of Fenton’s reagent with ethanol-2-13C.
mT and αH = 0.20 mT for 1-butanol; αN = 1.37 mT and αH = 0.20 mT for 2-butanol; αN = 1.31 mT and αH = 0.16 mT for tert-butyl alcohol). The relative peak heights of the ESR signals of the alcohols increased as follows, 1-propanol > 1-butanol > ethanol > 2-butanol > 2-propanol > tert-butyl alcohol (Table 1). HPLC−ESR Analyses of the Complete Reaction Mixtures of (TPP)•+FeIVO with 1-Propanol or 1-
Figure 8. HPLC−ESR analyses of the complete reaction mixture of (TPP)•+FeIVO with some alcohols: (a) ethanol; (b) 1-propanol; (c) 1-butanol. The reaction and HPLC−ESR conditions were as described in the Experimental Section except for the volume of 1-butanol added to the complete reaction mixture. The volume of 1-butanol was 300 μL. 13170
DOI: 10.1021/acs.inorgchem.7b01949 Inorg. Chem. 2017, 56, 13166−13173
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Inorganic Chemistry reaction mixture of (TPP)•+FeIVO with 1-butanol (Figure 8c). HPLC−ESR−MS Analyses of the Complete Reaction Mixtures of (TPP)•+FeIVO with 1-Propanol or 1Butanol. To identify peaks 4 and 5, HPLC−ESR−MS analyses were performed and ions at m/z 254 and 268 were detected (Figure 9a,b). The ions correspond to protonated molecular
Figure 10. Kinetic isotope effect experiments of the complete reaction mixture of (TPP)•+FeIVO with ethanol or ethanol-d1. The reaction and ESR conditions were as described in the Experimental Section. Kinetic isotope effect experiments to probe the hydrogen-bonding interaction were performed. The ESR peak height of the complete reaction mixture was monitored at 25 °C from 30 sec after the addition of PhIO. (a) Relative peak height of ethanol. (b) Relative peak height of ethanol-d1. Inset: Changes in the ESR peak height in the initial stage of the reaction. The results are the means of three experiments.
4. DISCUSSION Peak 1, corresponding to the protonated molecular ion [M + H]+ of 4-POBN/ethoxyl radical adducts, was observed at the retention time of 31.9 min in the HPLC−ESR elution profile of the complete reaction mixture of (TPP)•+FeIVO with ethanol (Figure 2a). However, a peak with a retention time similar to that of peak 1 was observed at a retention time of 32.1 min for the complete reaction mixture of (TPP)•+FeIVO without ethanol (Figure 2b). To examine whether or not the peak is the same as that of peak 1, HPLC−ESR analyses were performed using the neutral solvent system. While peak 1 was observed at a retention time of 31.8 min (Figure 2e) on the HPLC−ESR elution profile using the neutral solvent system, no peaks were observed around 31.8 min on the HPLC−ESR elution profile of the complete reaction mixture of (TPP)•+FeIVO without ethanol (Figure 2f), showing that the peak is not the 4-POBN/ ethoxyl radical adduct. Peaks with retention times similar to those of peak 1 were also observed at a retention time of 32.1 min for the complete reaction mixtures of (TPP)•+FeIVO with 1-propanol (Figure 8b) or 1-butanol (Figure 8c). Because ethanol is not contained in the complete reaction mixture, the peak with a retention time similar to that of peak 1 is not the 4POBN/ethoxyl radical adduct, supporting the claim described above. The reaction of Fe(TPP)Cl with PhIO has been known to produce (TPP)•+FeIVO 1 (Scheme 1).10−13 In spite of the higher bond-dissociation energy of OH (104.7 kcal/mol) compared to that of α-CH (96.1 kcal/mol) in an alcohol
Figure 9. HPLC−ESR−MS analyses of the complete reaction mixture of (TPP)•+FeIVO with 1-propanol or 1-butanol. The reaction and HPLC−ESR−MS conditions were as described in the Experimental Section except for the volume of 1-butanol added to the complete reaction mixture. The volume of 1-butanol was 300 μL. (a) Peak 4 of 1-propanol. (b) Peak 5 of 1-butanol.
ions [M + H]+ of 4-POBN/1-propanol-derived and 4-POBN/ 1-butanol-derived radical adducts. Fragment ions at m/z 167 and 181 correspond to the loss of [(CH3)3C(O)N] from the respective protonated molecular ions [M + H]+. Ions at m/z 474 and 488 correspond to 4-POBN/1-propanol-derived radical adducts complexed with PhIO [M + PhIO + H]+ and 4-POBN/1-butanol-derived radical adducts complexed with PhIO [M + PhIO + H]+. The 4-POBN/1-propanol (or 1butanol)-derived radical adducts complexed with PhIO [M + PhIO + H]+ were also detected as the reaction of ethanol. Kinetic Isotope Effect Experiments. Kinetic isotope effect experiments to probe the hydrogen-bonding interaction were performed for ethanol and ethanol-d1 (Figures 10 and S7). The ESR peak height of ethanol increased more rapidly than that of ethanol-d1. Using the changes in the ESR peak height in the initial stage of the reaction, the kinetic isotope effect (kH/kD) was estimated to be 1.34 ± 0.09 (n = 3). 13171
DOI: 10.1021/acs.inorgchem.7b01949 Inorg. Chem. 2017, 56, 13166−13173
Article
Inorganic Chemistry Scheme 1. Possible Reaction Path for the Formation of the Ethanol-Derived Oxygen-Centered Radical Formed in the Complete Reaction Mixture of (TPP)•+FeIVO with Ethanol
Scheme 2. Possible Reaction Path for Formation of the Ethanol-Derived Carbon-Centered Radical Formed in the Complete Reaction Mixture of Fenton’s Reagent with Ethanol
molecule,22,23 hydrogen-atom abstraction from the OH group by (TPP) • + Fe I V O seems to occur because the (TPP)•+FeIVO···H−O−CH2CH3 intermediate 2 is stable. Indeed, the kinetic isotope effect (kH/kD) was 1.34 ± 0.09 (n = 3) for the complete reaction mixtures of (TPP)•+FeIVO with ethanol or ethanol-d1 (Figures 10 and S7). (TPP)FeIVOH 3 and ethoxyl radicals 4 have been hypothesized to form through the (TPP)•+FeIVO···H−O−CH2CH3 intermediate 2.15,24 FeIVOH was reported as a ferryl intermediate in compound II of a heme peroxidase.25 The ethoxyl radicals were first trapped by 4-POBN in this paper. Compared with 1-butanol and 2-butanol, weak ESR signals were observed for the complete reaction mixture of (TPP)•+FeIVO with tert-butyl alcohol. The relative intensity of the ESR signal could be explained based on the O−H bonddissociation energies of CH3CH2OH (104.7 kcal/mol), (CH3)2CHOH (105.7 kcal/mol), and (CH3)3COH (106.3 kcal/mol).22,23 We have detected ethanol-derived oxygen-centered radicals in the reaction mixture of (TPP)•+FeIVO with ethanol for the first time. A mechanism suggests that an alkoxyl radical forms in the P450scc (CYP11A)-catalyzed conversion of cholesterol to pregnenolone and 4-methylpentanal. A hydrogen atom may be abstracted from one of the alcohols by the ferryl species to form the alkoxyl radical.9 It was reported that a diiron(IV) complex generated by electrochemical oxidation attacked not only the C−H bond but also the O−H bond.26 Density functional theory (DFT) calculation of diiron(IV) oxo complexes revealed the mechanism of C−H and O−H bond activation in detail.27 A DFT study also suggested that oxidation of the hydroxyl group of proclavaminic acid by clavaminic acid synthase forms an oxygen radical.28 Furthermore, in the reaction of 2-hydroxyethylphosphonate dioxygenase with 2-hydroxyethylphosphonate, the O−H activation was proposed based on the accumulation of an iron(IV) oxo complex in the presence of deuterium oxide.24 On the other hand, an aminyl radical cation and amine radical were also proposed as intermediates in the reaction of 1-aminocyclopropane 1-carboxylic acid oxidase with alternating cyclic and acyclic substrates.29,30 Meanwhile, we detected and identified diastereomers of the 4-POBN/α-hydroxyethyl radical [•C(OH)HCH3] adducts using ESR and HPLC−ESR−MS, showing that an unpaired electron locates on the α-carbon atom of the ethanol-derived radical formed in the complete reaction mixture of Fenton’s reagent with ethanol (Figures 5 and 6). α-Hydrogen-atom abstraction seems to occur by the hydroxyl radicals formed in the reaction of Fenton’s reaction (Scheme 2a). Abstraction occurs at the α-hydrogen atom because of a relatively weak
bond-dissociation energy (96.1 kcal/mol) compared with that of the O−H bond (104.7 kcal/mol) in an ethanol molecule22,23 (Scheme 2b). Because a complex like the (TPP)•+FeIVO··· H−O−CH2CH3 intermediate 2 is unstable in Fenton’s reaction, hydrogen-atom abstraction from the OH group could not occur.
5. CONCLUSION Using the ESR and HPLC−ESR−MS combined use of the spin-trapping technique, we have detected and identified the ethanol-derived oxygen-centered radicals in the reaction mixture of Fe(TPP)Cl, PhIO, and ethanol for the first time. The reaction of Fe(TPP)Cl with PhIO produces (TPP)•+FeIVO, which seems to be involved in the formation of the ethanol-derived oxygen-centered radicals. The results provide important mechanistic insight into a variety of oxidation reactions by heme-containing enzymes such as cytochromes P450, peroxidases, and catalases.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.7b01949. ESR spectra and kinetic isotope effect experiments (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Hideo Iwahashi: 0000-0002-9097-8795 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank the Department of Clinical Research, Wakayama Medical University, for proofreading and editing the manuscript.
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REFERENCES
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