Induction of Enzyme-like Peroxidase Activity in an Iron Porphyrin

Article pubs.acs.org/IC

Cite This: Inorg. Chem. XXXX, XXX, XXX−XXX

Induction of Enzyme-like Peroxidase Activity in an Iron Porphyrin Complex Using Second Sphere Interactions Snehadri Bhakta, Abhijit Nayek, Bijan Roy, and Abhishek Dey* Department of Inorganic Chemistry, Indian Association for the Cultivation of Science, Kolkata, India 700032

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S Supporting Information *

ABSTRACT: Emulating enzymatic reactivity using small molecules has been a long-time challenging pursuit of the scientific community. Peroxidases, ubiquitous heme enzymes that are involved in hormone synthesis and the immune system, have been a prime target of such efforts due to their tremendous potential in the chemical industry as well as in wastewater treatment. Here it is demonstrated that inclusion of a second sphere guanidine moiety in an iron porphyrin not only makes this small molecule a veritable peroxidase catalyst but also offers an auxiliary binding site for organic substrates, facilitating their rapid oxidation with a green oxidant like H2O2. This small molecule analogue exhibits a “ping−pong” mechanism and Michaelis−Menten type kinetics, which is generally typical of metallo-enzymes and follows a mechanism of the natural enzyme in its entirety, including the formation of compound I as the primary oxidant.

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INTRODUCTION

of horseradish peroxidases to oxidize organic molecules with a green oxidant like hydrogen peroxide makes it a key target of the chemical industry. It has also been hailed as a potential way to decontaminate wastewater of toxic phenol and aniline wastes, which are converted to their nontoxic derivatives by HRP.5 The prolific reactivity of peroxidase is due to the generation of a versatile high valent oxidant, namely, compound I, upon the reaction of peroxides with a histidine bound ferric heme active site (Figure 1B).6 The facile generation of compound I requires participation of distal residues such as Arg38 and His42 as acid−base catalysis required for the heterolytic O−O bond cleavage, leading to the formation of compound I, the “pull effect”.7 Compound I is formally two-electron oxidized relative to the resting ferric state and is best described as a FeIVO (ferryl) species bound to a one-electron oxidized porphyrin π-cation radical (P+), [P+FeIVO].8,9 The Arg38 residue is also proposed to stabilize both the compound I and II intermediates by hydrogen bonding to the ferryl oxygen (Figure 1B).10 Substantial research has gone into understanding the mechanistic details of peroxidases and making its functional mimics (Figure 2).11−14 Various metallo-porphyrin synthetic models of ferric peroxo,15 ferric hydroperoxide16 and, most importantly, compound I17 have been synthesized. The electronic structure of compound I could be tuned by modifying the porphyrin ring and as well as by varying axial ligands/counterions. Unfortunately, the synthetic metalloporphyrins require organic per-acids for the generation of compound I.18 Water-soluble iron and manganese porphyrins

Heme peroxidases are ubiquitous in nature. They catalyze oxidation of organic substrates using hydrogen peroxide and participate in the synthesis of prostanoids (cyclooxygenase),1 defend plant and animals against pathogens (horseradish peroxidase and myeloperoxidase),2 as well as reduce the concentration of free peroxide protecting organisms from oxidative damage (glutathione peroxidase).3 Ascorbate peroxidase, cytochrome c peroxidase, and horseradish peroxidase (HRP) are the most efficient H2O2 activator among this vast variety, and all have strikingly similar active site structures (Figure 1A). After Planche’s discovery in 1810,4 HRP got the attention of the scientific community in terms of structure, kinetics, mechanism, and practical applications. The capability

Figure 1. (A) The active site structure of HRP (PDB ID 1ATJ). The His-42 and Arg-38 constitute a second sphere of HRP, while His-176 provides an axial ligand.6 (B) The crystal structure of compound I (PDB ID 1HCH) showing hydrogen-bonding between ferryl oxygen and Arg-38.10 © XXXX American Chemical Society

Received: September 24, 2018

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DOI: 10.1021/acs.inorgchem.8b02707 Inorg. Chem. XXXX, XXX, XXX−XXX

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Inorganic Chemistry

Figure 2. Current status of the development of artificial peroxidase in terms of catalytic efficiency (η) toward H2O2 activation. Multiple undefined active sites per nanoparticles.

were used to investigate the push−pull effects.14,19 Despite such great efforts, to date the reactivities of most of these artificial molecular catalysts are found to be very sluggish with the exception of nonheme Fe-TAML (Figure 2) and its derivatives which were meticulously designed to withstand oxidative damage.20,21 On the other hand, several functional mimics of peroxidase such as Fe2O3 MNPs,22 Ru NPs,23 Pd@ pt nanoplates,24 Cu(OH)2SCs,25 GQDs-BrPE,26 popularly known as nanozymes, have shown promising outcomes in terms of the reaction rate.27 Unfortunately, nanozymes face several critical issues primarily from biocompatibility, low binding affinity, and having an unknown mechanism which restricts their rational designing.28 Heme-peptide complexes12 can be employed to achieve peroxidase activity. Notable successes came through several approaches such as utilizing μperoxidase,29 mimochrome,30 re-engineered Mb,31 and, very recently, from de novo c-type cytochrome maquettes (CTMs).32 Design of de novo protein is very challenging33 to ensure an appropriate orientation of the second sphere residues to result in a desirable orientation. On the other hand, molecular complexes are much more amenable to modification of the design of the ligand allowing emulation of the second sphere interactions in protein active site.34 One of the limitations of the synthetic models resulting from erstwhile efforts is that none of them include the guanidine residue present in the distal site of peroxidases which relay the proton affecting rapid O−O heterolysis of the initial ferric hydroperoxide (Compound 0), resulting in the formation of the oxidant Compound I. In particular, the arginine residue which has a guanidine (more specifically guanidinium) side chain plays a catalytically important role in peroxidases, chlorite dismutases,35 nitric oxide synthase,36 and cytochrome C-nitrite reductase37 and is generally assumed to translocate proton and stabilize the intermediate by hydrogen bonding and electrostatic interaction [Figure 1].10,38 Recently mononuclear iron porphyrin appended with the distal amine groups were found to reduce O2 rapidly and selectively to H2O. A facile decay of the O−O bond in a ferric peroxide intermediate, orchestrated by a distal amine group, has been proposed to be key for this desirable activity.34 The role of Arg38 in HRP, on the other hand, has been established since the early 1980s but have not been modeled in synthetic systems so far.8 In this manuscript, a structural, as well as a functional mimic of horseradish peroxidase, is reported. The biomimetic model

described here (FeIIICl-MARG) includes a guanidinium moiety in a second sphere which is covalently attached to porphyrin to mimic the Arg-38 residue present in the active site of HRP. This complex shows the highest enzyme-like activity reported for any synthetic molecular catalysts against HRP substrates, using H2O2, to date. The guanidinium residue capable of forming a strong hydrogen bond acts as a binding site for the substrates, and as a result, this complex exhibits an enzyme-like ping−pong mechanism.39 Rapid kinetics experiments show a generation of compound I within a millisecond time scale, which like native HRP is demonstrated to be primary oxidant.

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RESULTS 1. Synthesis. o-Monoguanidinotetraphenyliron(III)porphyrin(FeIIICl-MARG): o-monoaminotetraphenyl porphyrin (1, Figure 3), and boc-protected thiourea were taken as

Figure 3. Rational designing of FeIIICl-MARG from HRP’s active site.

starting materials to execute a CN disconnection strategy. Stoichiometric Hg2+ was used to desulfurize the boc-protected thiourea which yielded a Boc-protected carbodiimide intermediate.40 The electrophilic carbodiimide reacted with the NH2 group of o-monoaminotetraphenyl porphyrin, slowly forming a protected guanidine moiety which upon deprotection gave the desired ligand (3). The ligand was metalated with FeBr2 using a standard protocol.41 Purple-colored rectangular crystals of MARG, suitable for XRD analysis were grown by slow vapor diffusion of diethyl ether into a methanolic solution of MARG. MARG crystallized in the monoclinic space group C2/c. The guanidinium moiety in MARG is protonated, which is counterbalanced by a chloride counterion. The solid-state structure also revealed several hydrogen bonding interactions between the pendant B


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Inorganic Chemistry

fence) (Figure 7A, green) and meso-tetraphenylporphyrin iron(III) chloride (Fe-TPP) (Figure 7A, pink), which clearly demonstrates the role of the pendant guanidinium group in accelerating catalysis. It is important to note that the proton has marked effect on catalysis. When the same experiments are performed without acetic acid (Figure 7A, blue), no TMB oxidation is observed. In order to evaluate the generality of FeIIICl-MARG’s peroxidase activity, different oxidants such as m-CPBA and H2O2 along with different substrates tetramethylbenzidine (TMB), o-phenylenediamine (OPD), and 2,4,6-tri-tert-butylphenol (TBPH) were evaluated [Figure 8A,B].45,46 The results show that FeIIICl-MARG is an indiscriminate peroxidase. We also revised the peroxidase activity with a different sterically demanding organic hydroperoxide47 [Figure S-6]. Enzyme-like Kinetics. To investigate the kinetics of catalysis by FeIIICl-MARG in detail, TMB was chosen as a model substrate. The rate of TMB oxidation increases linearly with the concentration of catalyst, suggesting that the reaction is first order with respect to the catalyst (Figure 9A). The dependence of rate on the concentration of H2O2 is linear at low concentrations of H2 O 2 (i.e., first order at low concentration) but shows saturation at higher concentrations, suggesting a substrate binding pre-equilibrium before the irreversible rate determining chemical step (Figure 9B). The oxidation of TMB, catalyzed by FeIIICl-MARG, follows substrate saturation kinetics with respect to TMB as well (Figure 10). The Michaelis−Menten type saturation is observed for FeIIICl-MARG over a reasonable range of TMB and H2O2 H2O2 concentration ([TMB] < KTMB M , [H2O2] < KM ), yielding a hyperbolic curve between initial rate and substrate concentration.43 A MM type saturation is an extremely rare observation in the case of molecular systems, as artificial catalysts are not known to form a catalyst−substrate (CS) complex analogous enzyme−substrate (ES) complex.48 The rate of TMB oxidation (VTMB) which relates VTMB with the concentration of catalyst [E0], TMB (CTMB) and with varying hydrogen peroxide concentration (CH2O2) can be expressed in a Lineweaver−Burk plot (Figure 11A). A secondary plot varying CTMB and CH2O2 yields the M−M kinetic parameters. A double reciprocal plot (Figure 11A) of initial velocity (VTMB) versus TMB concentration (CTMB) which is acquired for different H2O2 concentrations shows near parallel lines, which is the diagnostic of the ping−pong mechanism for substrate oxidation similar to an enzyme like HRP.49

Figure 4. Synthetic scheme for FeIIICl-MARG.

guanidinium group, the chloride anion, and a methanol solvent molecule in the distal cavity (Figure 5).

Figure 5. Capped stick representation of the solid-state structure of MARG ligand.

2. Reactivity. Peroxidase Activity. 3,3′,5,5′-Tetramethylbenzidine (TMB) was used as the substrate for the peroxidase activity measurements (other substrates vide infra).42−44

1 VTMB

=

TMB KM K H2O2 1 1 1 + M + Vmax C TMB Vmax C H2O2 Vmax

(1)

2O2 Secondary plot provides Vmax = 5 × 10−5 M s−1, KH = 4.76 M TMB −1 ± 0.5 mM, KM = 3.4 ± 0.4 mM, kcat = 25 ± 5 s , catalytic 3 −1 −1 2O2 efficiency (ηH2O2 = kcat/KH s , ηTMB = kcat/ M ) = 5.2 × 10 M TMB 3 −1 −1 KM ) = 7.35 × 10 M s . The catalytic rate of substrate oxidation by HRP and related peroxidases vary dramatically (even for the same protein) depending on the substrate, and hence the rates of catalysis by different catalysts (enzyme, molecules, etc.) are difficult to compare.50 The most relevant kinetic parameters to compare peroxidase activities of different 2O2 enzymatic and molecular entities are KH and the catalytic M H2O2 TMB efficiency (ηH2O2). The KM and KM of FeIIICl-MARG are

Figure 6. Different oxidation states and colors of TMB. Two electron oxidized TMB is dubbed as diimine.

Kinetic traces were recorded by monitoring the increase of the 466 nm absorption band (ε ≈ 30 250 M−1 cm−1 in a 5% v/ v H2O−acetonitrile mixture) (Figure 7B), which is characteristic of the oxidized di-imine formation.42 TMB is rapidly and catalytically oxidized by FeIIICl-MARG (Figure 7A, purple), and the rate is much faster than that of meso-tetra(α, α,α,α-opivalamidophenyl) porphyrin iron(III) chloride (Fe-picket C


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Inorganic Chemistry

Figure 7. (A) Catalytic oxidation of TMB by FeIIICl-MARG at different catalyst concentrations. The blue line represents catalysis by FeIIICl-MARG in the absence of AcOH. (B) Evolution of the 466 nm band, characteristics of the di-imine formation with time. Experimental conditions: TMB/ H2O2/catalyst = 0.5 mM: 3 mM: 1 μg in 1 mL of a 5% v/v H2O−acetonitrile mixture, whereas the HRP-catalyzed assay was performed in pH 5 buffer.

Figure 8. (A) Catalytic oxidation of OPD with FeIIICl-MARG. Evolution of the 470 nm band, characteristics of 2,3-diaminophenazine formation. Experimental conditions; OPD: H2O2: FeIIICl-MARG = 0.5 mM: 3 mM: 2.5 μM. Time traces shows kinetics with 3 mM, 6 mM, and 9 mM H2O2. (B) Catalytic oxidation of TBPH with FeIIICl-MARG. Evolution of the 398 and 630 nm band, characteristics of 2,4,6-tri-tert-butylphenoxyl (TBP) free radical formation. Experimental conditions: TBPH: m-CPBA: FeIIICl-MARG = 2 mM: 1.25 mM: 1 μM in MeOH at 298 K. Kinetic trace of 630 nm (ε = 400) with violet: 1 mM TBPH, red: 1.5 mM TBPH green: 2 mM TBPH keeping FeIIICl-MARG − 1 μM and mCPBA-1.5 mM (inset).

Figure 9. (A) First-order rate constant of TMB oxidation catalyzed by FeIIICl-MARG plotted against catalyst concentration varying from 1.25 μM to 7.5 μM in a 5% v/v H2O−acetonitrile mixture. Kinetic traces of the 466 nm band with 1.25 μM to 7.5 μM catalyst respectively (inset), (B) The pseudo-first-order rate constant with respect to the concentration of H2O2 concentration (CH2O2) varying from 0.75 mM to 12 mM in 5% v/v H2O−acetonitrile mixture, time traces of the 466 nm band with 0.75 mM, 1.5 mM, 2.25 mM, 3 mM, and 5 mM (inset) catalysis runs.

4.76 ± 0.5 mM and 3.4 ± 0.4 mM, while those for HRP are 3.7 mM and 0.43 mM, respectively (Table 1).39,22 Thus, not only do H2O2 and TMB bind to FeIIICl-MARG but also the binding KM of these two substrates is similar to that of native HRP (Table 1). The catalytic rate toward TMB oxidation of FeIIIClMARG is 25 ± 5 s−1, and the ηH2O2 is 5.2 × 103 M−1 s−1. These values are substantially greater than any reported synthetic complexes and compare well with most peptide mimics or artificial proteins. Thus, the presence of a pendant guanidinium

group in the iron porphyrin not only mimics the HRP structurally but also mimics it functionally. Note that while the MM parameters like KM are like that of native HRP, the catalytic rate for TMB oxidation is slower in the artificial catalyst than in HRP.22 It is difficult to compare the molecular enzyme and peptide-based catalyst with NP-based catalysts as the NPs have multiple undefined numbers of active sites and grossly overestimate the catalytic efficiency per active site of HO the catalyst. As a result, despite having KM2 2 values much D


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Inorganic Chemistry

Table 1. Comparison of Kinetic Parameters of Oxidation of Organic Substrates Using H2O2 catalyst Fe(III)-MC6 E2L(TD)-MC6 protein-86 protein-K5 CTMs-C45 μ-peroxidase reconstitutedMb antibody−heme complex peroxidase (HRP, MPO) FeIIICl-MARG

Figure 10. Saturation kinetics of TMB oxidation by FeIIICl-MARG with respect to substrate (TMB) concentration varying from 25 μM to 400 μM with 3 mM H2O2 in 5% v/v H2O−acetonitrile mixture, at 298 K; initial rates were collected at the first second of the reaction.

2O2 KH M (mM)

44 ± 2 31 ± 2 n.d.

H O2

−1

kcat (s )

ηH2O2 = kcat/KM2 (M−1 s−1)

ref

7.4 ± 0.5

370 ± 10 780 ± 60 ∼ 280 ∼217 1200 21 1.1 ± 0.1

8.4 ± 0.6 × 103 25 ± 3 × 103 500 125 1.3 × 104 680 1.4× 102

24

6.56

274

51

3.7

0.01−2100a

4.6 × 106

22, 50

4.76

25

5.2 × 103

this work

94

30a 51 32 52 31

a

Rates depend on the substrate tested. TMB oxidation by MPO has a rate of 180 s−1.

lower than nanozymes (Table S-1) and a 1000 fold higher Vmax, a direct comparison is avoided. Inhibition is a well-established phenomenon of enzyme catalysis. Azide inhibits several heme enzymes like cytochrome oxidase, catalase, and HRP itself by binding reversibly or irreversibly to the heme cofactor.53,54 HRP is inhibited by sodium azide competitively as azide has a high binding affinity for the active ferric porphyrin where the oxidant peroxide binds.55 The catalytic oxidation of TMB by FeIIICl-MARG is strongly inhibited by azide (Figure 11B). A double reciprocal plot at different azide concentrations yields the inhibition constant, KI, of azide (Figure 11B). The plot reflects that the KM for H2O2 decreases with increasing azide concentration retaining the original Vmax. Such behavior indicates competitive inhibition of peroxidase activity of FeIIICl-MARG by azide. A KI of azide inhibition of 0.045 ± 0.04 mM can be estimated from the data (eq 256). Horseradish peroxidase shows KI = 1.47 mM for azide inhibition, and higher KI for HRP indicates lower binding affinity of N3− toward HRP.55

associated with the rate-limiting heterolytic O−O bond cleavage, generating compound I, the reactive species in HRP. The reactivity and KSIE implies a similar oxidant in FeIIICl-MARG [Figure S-5]. Reactive Intermediate. A reasonable peroxidase activity inevitably hints at compound I (for FeIIICl-MARG) as the reactive intermediate. Since m-CPBA is a two-electron oxidant and has a higher solubility in the organic medium than H2O2 and undergoes the same O−O heterolysis, it is used as a surrogate of H2O2. Rapid kinetics measurement at room temperature of the reaction of m-CPBA with FeIIICl-MARG shows a new absorption feature at 650 nm within 24 ms, resulting in a green solution that is characteristics of compound I (Figure 12A).60−63 The rate of formation of compound I (for FeIIICl-MARG) at room temperature64 is 100 s−1 (Figure 12B). The decay of the reactive Compound I (for FeIIIClMARG) is completed by 4 s with first-order rate constants of k1 = 2.3 s−1 (Figure S-7). The X-band EPR data of the 0.5 mM FeIIICl-MARG in THF shows an axial HS signal at g = 6.0 (Figure 13A), which suggests an axial HS ground state of FeIIICl-MARG resembling with the HS Fe ground state of HRP. The FeIIICl-MARG complex is reacted with m-CPBA in DCM−methanol (5:1 v/ v) solvent at −80 °C, and EPR analysis of the solution, frozen in liquid N2 immediately after the addition of m-CPBA (within ∼3 s), shows a broad signal at g ≈ 2, suggesting the formation

ij [I] yzz K mapp = K mfreejjj1 + z j KI zz{ (2) k H/D Isotope Effect. To understand the mechanism in detail, the kinetic solvent isotope effect was evaluated in various deuterated solvents.57 KSIE 3.7 ± 0.07 is slightly higher than the solvent isotope effect observed in HRP (KSIE = 1.6 ± 0.1),58 but close to that of chloroperoxidase (3.66 ± 0.62) from the demethylation reaction.59 In HRP, the KSIE is

Figure 11. (A) Double reciprocal plot of peroxidase activity of FeIIICl-MARG with respect to CTMB (varied from 1.5 mM to 3 mM H2O2). (B) The corresponding reciprocal plot varying CH2O2 with respect to intercept from plot “A” (orange). Study shows azide binding orchestrates competitive inhibition at 0.0125 mM (violet) and 0.025 mM (green). E


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Inorganic Chemistry

Figure 12. (A) Kinetics of formation of compound I (for FeIIICl-MARG) showing the growth in the 650 nm band. (B) Simulation of the trace of absorbance at 650 nm with time with a first-order process. The rate constant obtained (k1) is 100 s−1 for compound I (for FeIIICl-MARG) generation. The timing of intermediate trapping for EPR spectroscopy at 213 K by a rapid freezing technique after 1 s (inset).

Figure 13. (A) X-band EPR spectra of 0.5 mM FeIIICl-MARG S = 5/2, g = 6 in THF at 77 K. (B) The stable phenoxyl radical generated on scavenging of intermediate with 0.5 mM FeIIICl-MARG, 0.5 mM TBPH, and 0.5 mM m-CPBA (red line) and with 0.5 mM FeIIICl-MARG, 1 mM TBPH, and 0.5 mM m-CPBA (blue line) and 0.5 mM FeIIICl-MARG with 0.5 mM m-CPBA (green line).

Figure 14. Plausible mechanism of FeIIICl-MARG catalyzed substrate oxidation. Note that the Cl− ion is likely to be exchanged by OH− under the reaction conditions (5% v/v H2O/CH3CN). F


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Inorganic Chemistry of compound I (for FeIIICl-MARG). Note that the signal from compound I (for FeIIICl-MARG) at 77 K is very weak as expected. High valent ferryl species, such as compound I, are known for orchestrating substrate oxidation via several pathways such as electron transfer (ET), concerted hydrogen atom transfer (HAT), or stepwise electron−proton transfer (ET/PT), etc.65 The product of HAT from TBPH is the stable 2,4,6-tri-(tbutyl)phenoxyl radical, which can be quantified in EPR (Figure 13B). The 0.5 mM FeIIICl-MARG complex is reacted with 0.5 mM m-CPBA and 0.5 mM TBPH in MeOH at −80 °C, and EPR analysis of the solution, frozen in liquid N2 immediately after the addition of m-CPBA (within 1 s) showed typical organic radical signal (g = 2.00). Spin quantification of the radical signal against 1 mM Cu2+ standard yields 0.35 mM spin when the ratio of catalyst (0.5 mM): TBPH: m-CPBA is 1:1:1. The same experiment is repeated with 0.5 mM FeIIICl-MARG complex, 0.5 mM m-CPBA, and 1 mM TBPH in MeOH at −80 °C and spin quantification yields 0.8 mM spin (catalyst: TBPH: m-CPBA = 1:2:1). Thus, one equivalent of compound I (for FeIIICl-MARG) can oxidize two equivalents of TBPH to the corresponding radical, suggesting likely by two consecutive oxidations by compound I (for FeIIICl-MARG) and the ensuing compound II (for FeIIICl-MARG) (Figure 14).

for enzyme-like kinetics exhibited by this complex. In HRP the first substrate, i.e., H2O2, binds to the heme and forms the active catalyst E* (compound I) via O−O bond heterolysis. The second substrate binds the protein to form the active [ES] complex via H-bonding to the distal Arg38 residue.68,69 In that, 2O2 the KH increases 2−3 order in magnitude when the arginine M is mutated with lysine, suggesting that Arg-38 plays a crucial role in facilitating the rapid binding of H2O2 to HRP.38 Similarly, the Arg38 is also responsible for the tight binding of organic substrates via hydrogen bonding to the active site of HRP68 lowering KTMB M . With a combination of these effects of 2O2 Arg38, the enzyme exhibits very low KH and KMsubstrate M characteristic of HRP. An analogous ping−pong mechanism (Figure 14) is also operative in this case where the substrate binds the distal guanidine to form a supramolecular substrate− catalyst complex which then reacts with H2O2 to generate the oxidant compound I. The compound I generated then oxidizes two equivalents of a substrate to regenerate the resting ferric state. Note that the Cl− counterion of the reactant is likely to be replaced by more abundant OH− during catalysis. The HO ping−pong mechanism, the similar KM2 2 and KTMB value, and M even competitive inhibition kinetics observed in FeIIICl-MARG clearly suggest that the distal guanidinium group metamorphoses the synthetic iron porphyrin allowing it to behave like a miniature enzyme both qualitatively as well as quantitatively. Geometry optimized density functional theory (DFT) calculations indicate that the guanidine group is strongly hydrogen bonded to an axial hydroperoxide group (Figure S8A), and the hydrogen bonding is enhanced when the guanidine is protonated, leading to substantial elongation of the O−O bond (Figure S8B). The calculations are consistent with the rapid O−O bond cleavage observed experimentally. Because the reaction requires water to proceed, it is possible that the hydrogen bonding is mediated/enhanced by water molecules. Generally, small analogues of metalloenzymes cannot fashion an elegant aqueduct of water molecules which deliver the protons necessary for the O−O bond heterolysis in HRP.70 Such organized proton transfer channels result in low KSIE on the O−O bond heterolysis step, and the lack of it results in slightly elevated KSIE as is observed here. Additionally, the second key residue His42 is not involved in this synthetic analogue and can be anticipated to enhance the reactivity further to a synthetic system, particularly, in lowering 2O2 the KH and enhancing the catalytic rate and is currently M under investigation.

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DISCUSSION A functional model of heme peroxidase active site bearing a guanidinium moiety covalently attached to the porphyrin ring has been synthesized. The synthetic design achieves the goal of incorporating a distal guanidinium residue in an iron porphyrin mimicking the Arg38 in the active site of HRP. The crystal structure of the free ligand indicates a suitable orientation of the guanidinium group with respect to the metal center in the porphyrin. The iron complex is a veritable mimic of peroxidase and can catalytically oxidize most of the HRP substrates tested using H2O2. Most synthetic porphyrin catalysts need m-CPBA to generate compound I with reasonable yields; otherwise there is very slow compound I formation from H2O2 at lower pH.66,67 The FeIIICl-MARG, on the other hand, forms compound I using a green oxidant like H2O2 rapidly. The substrate oxidation shows nonsequential multiple substrate binding in nature, typical of enzymes, and the KM for both H2O2 and TMB could be obtained, suggesting a “ping−pong” mechanism in a molecular complex. This unique attribute sets it apart from all reported synthetic analogues of peroxidase to date. The catalytic efficiency of H2O2 activation by FeIIIClMARG which is easy to synthesize is higher than any known peptide scaffolds, engineered myoglobin, and even μperoxidase systems (Table-1) and only slightly lower than the natural enzyme. The complex FeIIICl-MARG also possesses the highest initial rate Vmax/1 μg of the catalyst among the nanozymes, which shows its advantage over other known systems (Table S-1). The rapid kinetics data and EPR data indicate that the reactive intermediate is compound I (for FeIIICl-MARG), which is formed due to the heterolysis of the O−O bond of a ferric peroxide species. Unsubstituted iron TPP does not show either rapid peroxidase activity or formation of compound I at such fast time scales, illustrating the advantage of the pendant guanidinium (protonated under neutral pH) group. The guanidinium group is a strong hydrogen bond donor and can hydrogen bond to the substrates used here, e.g., TMB, OPD, and TBPH. This strong H-bonding interaction is responsible

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CONCLUSION In summary, a prototype of structural as well as functional mimic of horseradish peroxidase is developed by including a guanidinium group in the second sphere. The FeIIICl-MARG catalyst was able to demonstrate a “pull effect” of the distal guanidinium moiety. To the best of our knowledge, FeIIIClMARG is the only known molecular catalyst to utilize a “ping− pong” mechanism for substrate oxidation. Overall, these results bespeak the advantage of including a second sphere guanidinium residue in catalyzing the oxidation of organic substrate with H2O2.

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EXPERIMENTAL DETAILS

Materials and Methods. All reagents were of the highest grade commercially available product. Thiourea, sodium hydride (60% in mineral oil), Boc anhydride, HgCl2, triethylamine, hydrochloric acid, collidine Na2SO4, NaN3 , and pyrrole were purchased from G


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Inorganic Chemistry spectrochem Ltd. Diethyl ether, THF, acetonitrile, dichloromethane, DMF, chlorobenzene, acetic acid, and methanol were purchased from Merck. Anhydrous ferrous bromide (FeBr2), 2,4,6-collidine, 3,3′,5,5′tetramethylbenzidine (TMB), 2,4,6-tritertbutylphenol (TBPH), mCPBA, HRP (type-IV), o-phenylenediamine (OPD), and H2O2 from Sigma-Aldrich chemical company. The column chromatography was carried out over neutral alumina. Synthesis of FeIIICl-MARG. o-Monoaminotetraphenyl porphyrin (629 mg, 1 mmol), Diboc thiourea (330 mg, 1.2 mmol), 3.5 equiv of triethylamine (726 μL, 5.25 mmol), and 1.2 equiv of mercury(II) chloride (489 mg, 1.8 mmol) were added in dimethylformamide (5 mL) at 273 K in stirring condition. The temperature was maintained at 273 K for 30 min and was stirred continuously for next 24 h at room temperature. The reaction mixture was filtered through a Celite column to isolate the ligand from HgS. The Celite column was rinsed with Et2O, and the organic layer was washed first with saturated ammonium chloride and then with brine (50 mL). The organic layer was dried over anhydrous MgSO4. Dark purple-colored boc-protected MARG (2) was collected by evaporating Et2O. We avoided any chromatographic separation at this stage. Ligand 2 was dissolved with 5 mL of MeOH followed by addition of 1 mL of 5 N HCl and was stirred continuously for 12 h. The reaction mixture was neutralized by 33% v/v ammonia solution followed by evaporation of MeOH. The reaction mixture was separated with DCM from a DCM−water bilayer. The organic layer was dried over Na2SO4 and concentrated under a vacuum. To a solution of the MARG (65 mg, 0.08 mmol) in 20 mL of dry degassed THF, 2,4,6-collidine (25 μL, 0.195 mmol) was added and was stirred for 10 min in a glovebox. FeBr2 (70 mg, 0.328 mmol) dissolved in THF was added dropwise and was stirred for 20 h. The reaction mixture was worked up with 0.5 N HCl followed by the addition of DCM. The organic layer was washed with brine solution and collected. It was dried over anhydrous Na2SO4 and purified by column chromatography with neutral alumina using 5% v/ v MeOH−DCM mixture as eluent. Yield: (62 mg, 90 ± 2%). MARG: Elemental analysis calculated (%) (C45H34N7Cl): C 76.31, H 4.84, N 13.84. Found: C 76.10, H 5.01, N 13.79. 1H NMR (500 MHz, CDCl3, 25 °C): δ, ppm = 8.98−9.12 (m, 8H), 8.540−8.55 (d, 1H), 8.41−8.46 (dd, 6H), 7.91−7.95 (m, 10H), 7.54−7.57 (m, 2H), 5.28 (s, 1H), 7.86 (s, 2H) − 2.37 (s, 2H) ESI-MS (positive ion mode in ACN): m/z (%) = 672.09(100). FeIIICl-MARG: Elemental analysis calculated. (%) (C45H32N7Cl2Fe): C 67.77, H 4.04, N 12.29 Found: C 67.50, H 4.10, N 12.20. 1H NMR (500 MHz, CDCl3,25 °C): δ, ppm = 78.80 (β-pyrrolic protons). UV−vis (ACN) λmax = 415, 510, 588, 645 nm. UV−vis (5%v/v H2O-ACN) λmax = 415, 510, 567, 620 nm. ESI-MS (positive ion mode in ACN): m/z (%) = 725.156(100). Crystallization of MARG. A single crystal of compound X suitable for X-ray diffraction analysis was obtained by slow vapor diffusion of diethyl ether into a methanolic solution of MARG. The purple-colored crystal of “MARG” crystallized in monoclinic C2/c space group, which shows eight molecules of X partially contributing to the unit cell resulting in a Z value of 4. A suitable single crystal of X was mounted on a crystal mounting loop with the help of Paratone oil, and the intensity data were collected on a Bruker SMART APEX CCD diffractometer using graphite-monochromatic Mo−Kα radiation (0.7107 Å) at 293 K. The structures were solved by direct methods using SHELX-2013 incorporated in WinGX. The non-hydrogen atoms in the main fragments were refined with anisotropic displacement coefficient, and hydrogen atoms were fixed at the geometrical positions suggested by the software (Table S2). Absorption Spectra of Complex. Absorption spectra were recorded with ∼2.5 μL of 1 mM FeIIICl-MARG complex diluted with 1 mL of acetonitrile where path length was 1 cm. Absorption spectra were obtained by a UV−vis diode array spectrophotometer (Agilent 8453). Preparation of Stock Solution. 4.8 mg of 3,5,3′,5′-tetramethylbenzidine (TMB) was dissolved in 1 mL of acetonitrile to make 20 mM primary stock solution. Most importantly, the entire stock was frozen every time in liquid N2 to prevent aerial oxidation of TMB. Freshly recrystallized 2.7 mg of o-phenylenediamine (OPD) was dissolved in 1 mL of acetonitrile to make 50 mM of initial stock

solution. OPD was recrystallized from an acetonitrile−pentane mixture. 2,4,6-Tri-tert-butylphenol (TBPH) was used without further purification. For the preparation of the primary stock of 200 mM 2,4,6-tri-tert-butylphenol (TBPH), 5.24 mg of TBPH was dissolved in 100 μL of methanol. Analytical grade H2O2 (30% v/v) was obtained from Merck. H2O2 concentration was determined spectrophotometrically as 8.8 M by calculating absorbance at 240 nm (ε = 43.6 M−1 cm−1). After that, it was diluted with acetonitrile to make 300 mM H2O2. 12.5 mM 1 mL m-CPBA was prepared by dissolving 2.8 mg of m-CPBA in methanol. Kinetic Data. All kinetic spectrophotometric measurements were carried out on a UV−vis diode array spectrophotometer (Agilent 8453). Peroxidase activity was recorded by following the absorbance change at the Soret maximum (461 nm), which is typical of oxidized TMB. Unless mentioned, all steady state kinetics were carried out at 298 K, and the kinetic trace was recorded at the time interval of 0.5 s. Baseline of the UV spectrum was corrected at 335, 590, and 1100 nm. Peroxidase Activity. For the relative study of different catalysts except HRP, steady-state kinetics were carried out with 1 μg of catalyst, 2.5 μL of 200 mM of TMB, 10 μL of glacial acetic acid, and 10 μL of 300 mM of H2O2 in a 5% v/v H2O−acetonitrile mixture (Figure 6A), and the final volume was 1 mL. Blank kinetics were also recorded in the same condition. For HRP-catalyzed reaction, we performed kinetics taking 1 μg of HRP in 1 mL of 0.1 M sodium acetate−acetic acid buffer (pH = 5). Concentration Dependence. To investigate concentration dependence of [FeIIICl-MARG], we fixed [TMB] = 200 μM and [H2O2] = 3 mM while varying the catalyst concentration from 1.25 to 7.5 μM. Concentration dependence of H2O2 was recorded as [FeIIIClMARG] = 2.5 μM and [TMB] = 200 μM, while the concentration of H2O2 was varied from 0.75 mM to 12 mM. Rate constants (s−1) for H2O2 dependence and catalyst dependence were calculated using the equation y = y0(1 − exp−kt). Concentration dependence of TMB was recorded as [FeIIICl-MARG] = 2.5 μM and [H2O2] = 300 μM, while the concentration of TMB was varied from 25 μM to 400 μM. Enzyme-like Kinetics. To investigate “ping−pong” kinetics except azide inhibition, we kept the volume at 1 mL as 920 μL of ACN, 10 μL of glacial acetic acid, 10 μL of 20 mM TMB aliquot in acetonitrile, and 40 μL of nanopure water followed by 10 μL of 0.2 mM of the FeIIICl-MARG complex, and finally a gastight syringe, charged with 10 μL of 300 mM H2O2 in acetonitrile, was injected in the solution. In order to investigate the mechanism by altering [TMB] and [H2O2], we adjusted the respective volumes to make 1 mL every time. Initial velocity was calculated using vTMB =

ΔA Δt

×

106 εTMB ACN

μ M s−1.

−1 εTMB AcCN = 30 250 M cm . For determination of solvent kinetic isotope effect (KSIE) AcOH and H2O are replaced by AcOD and D2O, respectively. Inhibition. We adjust the reaction sequence as 920 μL of ACN, 10 μL of glacial acetic acid, 10 μL of 20 mM TMB aliquot in acetonitrile, and 35 μL of nanopure water, 5 μL of 2.5 mM sodium azide, followed by 10 μL of 0.2 mM of the FeIIICl-MARG complex, and finally, 10 μL of 300 mM H2O2 in acetonitrile was injected in the solution. The same experiment was repeated with 30 μL of nanopure water and 10 μL of 2.5 mM sodium azide. Catalytic Oxidation of o-Phenylenediamine (OPD). The oxidation of OPD by the FeIIICl-MARG using H2O2 as oxidant was conducted by observing 2,3-diaminophenazine formation at 470 nm in time scan mode. We set up the reaction sequence as 920 μL of ACN, 10 μL of glacial acetic acid, 10 μL of 50 mM OPD aliquot in acetonitrile, and 40 μL of nanopure water followed by 10 μL of 0.25 mM of the FeIIICl-MARG complex, and finally 10 μL of 300 mM H2O2 in acetonitrile was injected in the solution. Catalytic Oxidation of 2,4,6-tri-tert-butylphenol (TBPH). Catalytic oxidation of TBPH was performed at methanol using mCPBA as oxidant instead of H2O2. For steady-state kinetics, 10 μL of 200 mM TBPH aliquot and 4 μL of 0.25 mM of the FeIIICl-MARG complex were mixed in 980 μL of MeOH followed by addition of 10 μL of 12.5 mM m-CPBA. The course of the reaction was monitored

H


Article

Inorganic Chemistry with the rising 398 and 630 nm (ε = 400 M cm−1) absorption band of tri-tert-butyl phenoxyl radical. Reactive Intermediate. Compound I formation kinetics of FeIIICl-MARG using m-CPBA was performed on an SFM 4000 stopped-flow absorption spectrophotometer (light source Xe lamp). The reactions were performed by rapid mixing of 75 μL of 0.25 mM of FeIIICl-MARG with 75 μL of 1 mM m-CPBA in 5%v/v H2O− acetonitrile mixture at room temperature. 10 μL of 5 mM FeIIICl-MARG and 10 μL of 5 mM TBPH were taken in a 4 cm EPR tube, and 70 μL of methanol was further added to make an overall volume of 90 μL. An EPR tube was cooled down to −80 °C. To the solution of 0.5 mM FeIIICl-MARG and 0.5 mM TBPH, a gastight syringe filled with 10 μL of 5 mM m-CPBA (catalyst/TBPH/m-CPBA = 1:1:1) was injected instantly. An EPR tube was immediately (within 1 s) plunged into liquid N2. The same reaction set was prepared with 20 μL of 5 mM TBPH (catalyst/ TBPH/m-CPBA = 1:2:1). EPR spectra were obtained with a JEOL FA200 spectrometer. Freq ≈ 9.13 GHz, power ≈ 1 mW, mod. width = 3 gauss, amplitude = 2, time constant = 0.03 s, sweep time = 60 s.

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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.8b02707. Absorbance spectra of FeIIICl-MARG, ΔTMB ACN determination, reciprocal plots of azide inhibition, H2O2 concentration dependence of Fe-TPP, reactivity with respect to nanozymes, determination of isotope effects, catalysis with other peroxides, Decay of compound I (for FeIIICl-MARG), crystallographic data, and characterization spectra (PDF) Accession Codes

CCDC 1869092 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by emailing [email protected], or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Abhishek Dey: 0000-0002-9166-3349 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was funded by the Department of Science and Technology, India (DST), and Council for Scientific and Industrial Research (CSIR). S.B. and A.N. thank CSIR SPMF and CSIR JRF, respectively.

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