Substrate ionization energy influences the epoxidation of m-substituted styrenes catalyzed by chloroperoxidase from Caldariomyces fumago

Catalysis Communications 77 (2016) 52–54

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Substrate ionization energy influences the epoxidation of m-substituted styrenes catalyzed by chloroperoxidase from Caldariomyces fumago Fabio A. Muñoz-Guerrero a, Sergio Águila b, Rafael Vazquez-Duhalt b, Cristian H. Campos a, Cecilia C. Torres a, Joel B. Alderete a,⁎ a b

Departamento de Química Orgánica, Universidad de Concepción, Edmundo Larenas 129, Concepción, Chile Centro de Nanociencia y Nanotecnología, Universidad Nacional Autónoma de México, Ensenada, Baja California, México

a r t i c l e

i n f o

Article history: Received 7 December 2015 Received in revised form 13 January 2016 Accepted 20 January 2016 Available online 22 January 2016 Keywords: Chloroperoxidase Epoxidation m-Substituted styrene Ionization energies

a b s t r a c t Chloroperoxidase from the fungus Caldariomyces fumago is a versatile heme-peroxidase, which is able to catalyze olefin epoxidation. In this report, the epoxidation of m-substituted styrene catalyzed by chloroperoxidase was studied. The catalytic data fit the Hill's model, and the activity rate (kcat) constants were strongly dependent of nature of substituents. The epoxidation kcat values varied as follows: m-aminostyrene N styrene N mmethylstyrene N m-chlorostyrene N m-nitrostyrene. The catalytic rate constant for m-aminostyrene, an electron-releasing substituent, was 298 times higher than these for m-nitrostyrene, an electron-withdrawing compound. These results were quantitatively analyzed and a good linear correlation between the experimental catalytic constants of m-substituted styrenes and their ionization energies, calculated at B3LYP level of theory, was demonstrated. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Chloroperoxidase from the fungus Caldariomyces fumago (CPO; E.C. 1.11.1.10) is a versatile heme-peroxidase that uses hydrogen peroxide and halide ions for halogenation reactions of a variety of organic substrates. In addition, of halogenation reactions, CPO is able to catalyze different reactions including hydrogen peroxide dismutation, sulfide and alcohol oxidations, benzylic and allylic hydroxylation, and olefin epoxidations [1,2]. This enzyme catalyzes the epoxidation of a variety of olefin substrates with high yields, and several strategies have been employed in order to enhance its catalytic performance [3–6]. CPOmediated catalytic epoxidation shows several advantages because this enzyme is the fastest peroxidase known so far, it does not need cofactors, and the final electron acceptor is the environment friendly compound hydrogen peroxide. Moreover, the enzyme can be produced in large quantities and it is stable to storage conditions. CPO-mediated epoxidation of olefins has been objet of intensive research due to the importance of chiral epoxides in pharmaceutical and fine chemical industries [7,8]. The catalytic cycle of CPO peroxidase-like oxidations, using hydrogen peroxide or suitable alkyl peroxides, start with the formation of the highly reactive intermediate iron(IV)-oxo radical, named Compound I (Cpd I) [9]. Subsequently, the reaction proceeds in a stepwise

⁎ Corresponding author. E-mail address: [email protected] (J.B. Alderete).

http://dx.doi.org/10.1016/j.catcom.2016.01.017 1566-7367/© 2016 Elsevier B.V. All rights reserved.

manner with the C–O bond formation on the organic substrate, followed by C–O–C ring formation. The ring formation step is barrier-less for the low spin pathway and the C–O bond formation is the rate determining step [10,11]. The correlation between the ionization potential of substrate and the enzymatic transformation rate has been reported for peroxidases [12–14] and as well for CPO in the oxidation of organosulfur compounds [15]. Recently, the rate constants (kcat) of CPO-catalyzed oxidation on a variety of substrates have been reported [16]. In particular, for p-substituted styrene derivatives a linear Hammett relationship between log kcatX/kcatH and σ-Hammett constants have been found. Linear free energy relationship has also been established in the oxidation of styrene derivatives catalyzed by divers biomimetic porphyrin compounds [17]. Moreover, epoxidations of olefin substrates by these biomimetic iron(IV)-oxo porphyrin compounds has been extensively studied during the last decades through experimental and theoretical approaches [18,19]. Sainna et al. [20] have performed a comprehensive study on the epoxidation of alkenes using biomimetic iron(IV)-oxo porphyrin models. These authors have shown that the experimental rate constants are correlated with the ionization energies of olefin substrates. This correlation has been previously established on theoretical grounds using Density Functional Theory (DFT) calculations for the epoxidation of a variety of olefin substrates mediated by Cpd I model [21]. In order to evaluate if this correlation is also observed in the CPO-catalyzed epoxidations, the enzymatic reaction kinetics with divers m-substituted styrenes were determined and correlated with the substrate ionization energies calculated at DFT level.

F.A. Muñoz-Guerrero et al. / Catalysis Communications 77 (2016) 52–54

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2. Materials and methods 2.1. Chemicals Hydrogen peroxide (30% w/w) and 2-methyl-2-propanol (2M2P) were obtained from Merck KGaA (Darmstadt, Germany). Styrene and m-substituted derivatives were purchased from Sigma-Aldrich (St. Louis, MO). Chloroperoxidase from C. fumago was obtained and purified as previously reported [22]. The enzyme preparation contained 6530 U mL−1, with a RZ = 1.0 and an enzyme concentration of 289 μmol L−1. All other chemicals, unless otherwise stated, were supplied by Sigma-Aldrich. 2.2. Enzyme activity and determination of catalytic parameters of CPO Peroxidase activity of CPO was determined spectrophotometrically using guaiacol as substrate at pH 6.0 and monitoring the absorbance increase at 470 nm. The transformation rate was estimated by using an extinction coefficient of 26,600 M−1 cm−1, as guaiacol is converted to tetraguaiacol [23]. Epoxidation reactions were performed in a 60 mM phosphate buffer, pH 6.0, containing substrate (0.10 to 10.0 mM), 15% (v/v) co-solvent (2M2P) and 800 nM CPO final concentration. The reaction was started by adding hydrogen peroxide solution (40 mM) at a rate of 0.2 μmol/min flow until reach 1 mM of final concentration (saturating concentration). The reactions were stopped after 5 min by adding 1 mL acetonitrile and rapid cooling in ice/water bath. Then, the reaction mixtures were analyzed in a Knauer high-resolution liquid chromatography (HPLC) equipped with Smartline 2850 Photodiode Array Detector (HPLC-PDA). The elution was performed with a mobile phase of 65:35 (v/v) acetonitrile-water, with a flow of 1.0 mL min−1, through a reverse phase C18 column 5 μm Eurospher 100–5 (250 × 4.6 mm). The detection wavelengths were set at 207 nm and 216 nm to measure the generated diols and epoxides, respectively. All extracts obtained from different experiments were filtered using 0.22 μm nylon syringe filters (Membrane Solutions), prior to their analysis by HPLC. Enzyme assays and other UV–VIS experiments were performed with an UV–VIS spectrophotometer UV2310II (Techcomp). Prior to catalytic tests, calibration curves for each substrate and their corresponding epoxides were obtained. Hill's equation (Eq. (1)) was used as adjustment model to determine the catalytic parameters of CPO-catalyzed epoxidation of styrene derivatives [24]   ν ½S n ¼ kcat n 0 Et K þ ½S 

ð1Þ

where v is the reaction rate calculated as total concentration produced of epoxides per minute, Et is the total concentration of enzyme and kcat represents the catalytic rate constant. The initial concentration of substrate is defined by [S], n corresponds to the substrate cooperativity to the enzyme and K′ is a constant including different interaction factors and the intrinsic dissociation constant KS of enzyme-substrate complex. CPO specific activity is defined as v/Et ratio, expressed in min−1 units. 2.3. Computational methods DFT calculations were performed at UB3LYP/6–311 + g(2d,p) level of theory [25] for all styrene substrates. Geometry optimizations were carried out without symmetry restrictions for both neutral and ionic species. Ionization energies (IE) were evaluated as the energy difference between the radical-cation and neutral species, according the following equation: S → S+ + e− since has been demonstrated that B3LYP functional give precise estimations for both electronic and energetic properties of organic compounds [26].

Fig. 1. Specific activity of CPO for the epoxidation of styrene (♦), m-aminostyrene (▲) and m-chlorostyrene (●) derivatives. The pointed lines correspond to the fit of experimental data to Hill's equation.

3. Results and discussion The enzymatic activity of CPO in the epoxidation of m-subtituted styrenes was evaluated at pH 6.0. It is well known, that CPO is more active as halogenase at low pH, while the peroxidase activity is favored at pH 6.0 [27]. In most of cases, the kinetic data fitted a sigmoidal behavior as can be observed in the Fig. 1. Styrene derivatives with electronwithdrawing substituent showed lower catalytic activities than those derivatives with electron-donor substituents (Fig.1), as in the cases of m-nitro and m-amino styrene derivatives, respectively. The experimental data fit the Hill equation from which the catalytic constants were determined (Table 1). The substrate cooperativity to the enzyme (n) and the “substrate affinity” (K′) are also shown in Table 1. In the cases where n was equal to 1.0, the Hill equation becomes the rectangular hyperbolic equation and therefore K′ became the Michaelis–Menten constant (Km) corresponding to the substrate concentration that yields half-maximal velocity. The nitro derivative showed the lower catalytic constant (13 min−1), whereas the amino derivative showed the highest k cat value (3869 min− 1). A deeper quantitative analysis was performed. Initially, the kinetic data was correlated with σ-Hamett constant, since previous report has showed a linear correlation between σ constant Table 1 Catalytic parameters of CPO epoxidation of m-substituted styrenes obtained from Hill's equation and substrate ionization energies (IE) calculated at UB3LYP/6–311(2d,p) level of theory. m-Substituent

kcat (min−1)

K′ (Moln L−n)

n

Hill model adjustment (R2)

IE (eV)

H NO2 Cl CH3 NH2

90 ± 6 13 ± 2 25 ± 1 58 ± 1 3869 ± 309

0.7 ± 0.2 6.7 ± 1.2 0.39 ± 0.04 0.26 ± 0.03 0.42 ± 0.08

1.1 ± 0.1 1.2 ± 0.2 1.2 ± 0.2 2.0 ± 0.2 0.9 ± 0.1

0.999 0.994 0.981 0.985 0.992

8.159 8.762 8.302 7.974 7.109

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4. Conclusion The catalytic activity of CPO for the epoxidation of m-substituted styrene is determinate by the electronic nature of substituent group. Thus, substituted styrene with electron-withdrawing groups showed very low activities, while styrene derivatives with electron-releasing groups present higher activities. A logarithmic correlation model is able to predict the epoxidation activity of CPO based in the IE of the substrates. Acknowledgments F. A. Muñoz-Guerrero thanks to CONICYT for a graduate fellowship (number 63105502). Authors are grateful for the support of CONACYTCONICYT International Cooperation Program (Number PCCI130043) México — Chile governments and Red Doctoral REDOC MINEDUC Project UCO 1202.

Fig. 2. Experimental log(km/kH) v.s. B3LYP calculated IE of substrates.

of p-substituted styrenes and the catalytic constants for epoxidation reaction catalyzed by CPO [4]. However, σ-Hamett constant showed a poor linear correlation (R2 = 0.576) in the case of m-substituted styrenes. Then, a correlation analysis between the calculated IE of substrates and the experimental catalytic constants was performed. The rational of this analysis was based on the de Visser's group results on biomimetic iron(IV)-oxo porphyrin compounds and DFT theoretical calculations [26], which have found a linear correlation between the IE of olefins and the catalytic epoxidation constant, and on the clear correlation between ionization potential and peroxidase transformation rate of different substrates [12–15]. Table 1 shows that the catalytic activities are related with the calculated ionization energy of the m-substituted styrene derivatives. Styrene derivatives with lower ionization energy, showed higher catalytic rate constant. A good linear correlation (R2 = 0.935) between the experimental log (kcatX/kcatH) and the B3LYP calculated ionization energy (IE) was found (Fig. 2), where X indicate a meta-substituent and H is referred to styrene molecule. The following logarithmic equation describes the relationship between (kcatX/kcatH) and the IE of the substrates, showing to be a statistically accurate model with 99% confidence (p = 0.0072):

References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17]

kX log ¼ 12:2−1:516  IE: kH

ð2Þ

These results clearly indicate that the catalytic activities of chloroperoxidase-mediated epoxidation of m-substituted styrenes are determined by the IE values of substrates. Free radical intermediates are involved in the catalytic mechanism of peroxidases, as well in the CPO catalytic cycle. Thus, substrates that easily lose one electron (low ionization energy) are fast transformed by the oxidizing radical intermediates of the enzyme. Here, we have extended these concepts to the epoxidation of m-substituted styrene derivatives catalyzed by CPO from C. fumago.

[18] [19] [20] [21] [22] [23] [24] [25] [26] [27]

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