Theoretical Study of Phosphodiester Hydrolysis and

Jan 26, 2016 - Synopsis. The reaction mechanisms of phosphodiester hydrolysis and transesterification catalyzed by a dinuclear zinc complex mimicking ...
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Theoretical Study of Phosphodiester Hydrolysis and Transesterification Catalyzed by an Unsymmetric Biomimetic Dizinc Complex Henrik Daver,† Biswanath Das,‡ Ebbe Nordlander,‡ and Fahmi Himo*,† †

Department of Organic Chemistry, Arrhenius Laboratory, Stockholm University, SE-106 91 Stockholm, Sweden Inorganic Chemistry Research Group, Chemical Physics, Center for Chemistry and Chemical Engineering, Lund University, Box 124, SE-221 00 Lund, Sweden

‡

S Supporting Information *

ABSTRACT: Density functional theory calculations have been used to investigate the reaction mechanisms of phosphodiester hydrolysis and transesterification catalyzed by a dinuclear zinc complex of the 2-(N-isopropyl-N-((2-pyridyl)methyl)aminomethyl)-6-(N-(carboxylmethyl)-N-((2-pyridyl)methyl)aminomethyl)-4-methylphenol (IPCPMP) ligand, mimicking the active site of zinc phosphotriesterase. The substrates bis(2,4)-dinitrophenyl phosphate (BDNPP) and 2-hydroxypropyl-p-nitrophenyl phosphate (HPNP) were employed as analogues of DNA and RNA, respectively. A number of different mechanistic proposals were considered, with the active catalyst harboring either one or two hydroxide ions. It is concluded that for both reactions the catalyst has only one hydroxide bound, as this option yields lower overall energy barriers. For BDNPP hydrolysis, it is suggested that the hydroxide acts as the nucleophile in the reaction, attacking the phosphorus center of the substrate. For HPNP transesterification, on the other hand, the hydroxide is proposed to act as a Brønsted base, deprotonating the alcohol moiety of the substrate, which in turn performs the nucleophilic attack. The calculated overall barriers are in good agreement with measured rates. Both reactions are found to proceed by essentially concerted associative mechanisms, and it is demonstrated that two consecutive catalytic cycles need to be considered in order to determine the rate-determining free energy barrier.

I. INTRODUCTION Phosphodiester bonds are present in both nucleosidic linkages and the energy currency of the cell, adenosine triphosphate (ATP). During the last century, phosphotriester bonds have also been introduced by man in nature in the form of pesticides (e.g., malathion, parathion, diazinon) and warfare nerve agents (e.g., Sarin, Tabun, and Soman). Thus, catalyst-mediated effective cleavage of phosphoester bonds in order to break down such hazardous residues are of great interest. Such catalysts can also be relevant for purposes where selective DNA and RNA cleavage is desired. Phosphatases are enzymes that have evolved to catalyze the hydrolysis of phosphoester bonds.1,2 Many phosphatases have been found to encompass a dizinc active site.3 Examples include phosphotriesterase (PTE), which hydrolyzes organophosphorus triesters but for which no natural substrate has yet been identified,4 and ecto-5′-nucleotidases, e.g., E. coli 5′-nucleotidase,1,5 an enzyme that hydrolyzes all 5′-ribonucleotides and 5′deoxyribonucleotides, including di- and triphosphates. In recent years, great efforts have been put into synthesizing biomimetic complexes that mimic the catalytic effect of such dinuclear zinc enzymes.6−15 The study of these compounds gives insights into the enzymatic reaction mechanism(s) and may also have © 2016 American Chemical Society

practical applications, e.g., for the hydrolysis of organophosphorus-based pesticides and nerve gases (vide supra). For dizinc phosphatases as well as for biomimetic complexes of such enzymes either a bridging13,16−18 or a terminal hydroxide15,19−22 (or in some cases methoxide23) has been proposed to be the active nucleophile in the mechanism. Alternatively, the hydroxide24 (or methoxide23,25) has also been proposed to act as a base in the reaction, deprotonating either an external solvent molecule (water or methanol) or the substrate itself to activate a nucleophile. These possible scenarios are summarized in Scheme 1. Another aspect of the hydrolysis mechanism(s) that has not been fully elucidated is the coordination mode(s) of the organophosphate substrate throughout the reactions. Recently, a number of theoretical mechanistic studies have been conducted on dizinc complexes that have been designed to function as phosphatase biomimetics.7,13,20−26 These studies have often focused on the hydrolysis of various organophosphoesters that function as models for nucleic acids (vide infra). Special interest has been invested in resolving the Received: November 30, 2015 Published: January 26, 2016 1872

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in Scheme 2).6,27 A pH−potentiometric speciation study indicated that the catalyst has two hydroxides coordinated in solution. In the same study, it was suggested that when a substrate molecule binds to the catalyst, it might substitute one of the hydroxide ions.27 Hence, it is not fully ascertained whether one or two hydroxides are present in the catalyst− substrate complexes during the reaction. In the present study, the mechanisms of reactions 1 and 2 with the Zn2 complex of the IPCPMP ligand have been investigated using density functional theory (DFT) calculations. For both substrates studied two scenarios are considered in which either one or two hydroxides are bound to the catalyst in the catalyst−substrate complex. Very recently, we used a similar approach to study the hydrolysis of BDNPP with a dizinc complex of the ligand 2-(N(3-((bis((pyridin-2-yl)methyl)amino)methyl)-2-hydroxy-5methylbenzyl)-N-((pyridin-2-yl)methyl)amino)acetic acid (DPCPMP), which is related to IPCPMP in that the isopropyl moiety is substituted for a pyridyl.7 The current results will be compared with those obtained for the Zn2(DPCPMP) catalyst. Some of the recent computational studies are also of special relevance to the current work.13,20−25 These will be discussed in the appropriate sections below, and similarities and differences between those mechanisms and the current results will be highlighted.

Scheme 1. Summary Description of Proposed Mechanisms of Phosphoester Hydrolysis Catalyzed by Dinuclear Zinc Enzymes and/or Model Complexesa

a

The nucleophile may be (1) a bridging hydroxide, (2) a terminal hydroxide, or (3) a hydroxide ion generated by the deprotonation of water by a terminal hydroxide. The phosphate substrate may be coordinated to the active site in a bridging mode (left) or terminally coordinated to one zinc ion (right).

identity of the nucleophile and whether the reaction occurs in a stepwise or a concerted manner. Also, it has been discussed how the intramolecular zinc−zinc distance affects the reaction mechanism.9,23 The current study focuses on a particular biomimetic catalyst that has been prepared by coordinating two zinc ions to the IPCPMP ligand (Figure 1).6 The ligand is unsymmetric as it

II. COMPUTATIONAL DETAILS The calculations were carried out with the B3LYP functional28 as implemented in the Gaussian 09 program.29 For the geometry optimizations, the LANL2DZ30 effective core potential was used for Zn, the 6-311+G(d) basis set for the phosphorus and the oxygens coordinated to it (including the nucleophilic oxygen), and the 631G(d,p) basis set for all other atoms. To obtain more accurate energies, single-point calculations were performed on these optimized geometries with the 6-311+G(2d,2p) basis set on all atoms except Zn, for which LANL2DZ was used. Solvation effects were evaluated by single-point calculations on the optimized geometries at the same level of theory as the geometry optimizations using the conductor-like polarized continuum model (C-PCM)31 with UAKS atomic radii and parameters taken from Gaussian 03.32 Experimentally, the reactions were performed in a 1:1 water:acetonitrile mixture.27 In the calculations, water (ε = 78.36) was used as the surrounding solvent. Frequency calculations were performed at the same level of theory as the geometry optimizations to obtain free energy corrections at 298.15 K and 1 atm pressure as well as to confirm the nature of the stationary points. The rigid-rotor harmonic-oscillator model was

Figure 1. IPCPMP ligand (2-(N-isopropyl-N-((2-pyridyl)methyl)aminomethyl)-6-(N-(carboxylmethyl)-N-((2-pyridyl)methyl)aminomethyl)-4-methylphenol). Atom numbering for relevant donor atoms is indicated.

imposes different coordination environments for each metal ion: one tetradentate pocket comprised of the N1, N2, O1, and O2 donors and one tridentate pocket formed by the O2, N3, and N4 donor atoms (cf. Figure 1). The binuclear complex has been found to catalyze hydrolysis of the DNA analogue bis(2,4)-dinitrophenyl phosphate (BDNPP, 1, reaction 1 in Scheme 2) and transesterification of the RNA analogue 2hydroxypropyl-p-nitrophenyl phosphate (HPNP, 4, reaction 2 Scheme 2. Reactions Investigated in the Present Study

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Inorganic Chemistry employed to calculate the entropic contributions of rotational and vibrational degrees of freedom. A shortcoming of this method is that as the vibrational frequency approaches zero, the associated contribution to the entropy approaches infinity.33 Therefore, the influence of the low vibrational modes on the total free energies is overestimated. To correct for this, the rotational entropies for these modes were calculated instead of the vibrational entropies, following the quasiRRHO approach suggested by Grimme.34 Dispersion effects are also taken into account by applying the DFT-D3BJ correction.35 In the gas phase, the standard state refers to 1 atm pressure, which corresponds to a volume of 24.5 L/mol for an ideal gas at room temperature. Since the reactions take place in solution, where the standard state is 1 M concentration for all solutes and 55.4 M for water at room temperature, a correction of RT ln (24.5 L/mol × 1 mol/L) = 1.9 kcal/mol is added for all species, except for water for which RT ln (24.5 L/mol × 55.4 mol/L) = 4.3 kcal/mol is added. The final energies reported here are thus Gibbs free energies corrected for solvation effects, dispersion effects, and standard state. For comparison, we also recalculated the energies using different computational schemes, such as different density functionals (with and without dispersion corrections) and different solvation models and dielectric constants. The results are reported in the Supporting Information.

two hydroxide ions, in accordance with evidence from speciation studies and mass spectrometry (vide supra). The resulting complex with the lowest energy is found to have one hydroxide bridging the two zinc ions and one hydroxide bound terminally to Zn2 (see Figure 2 for atom numbering). The substrate (1) can then either replace one of the hydroxides, forming a neutral catalyst−substrate complex, or bind to the complex with two hydroxides to yield an overall anionic complex. We will first present the results with the neutral species. As the substrate can coordinate to the catalyst in different ways, a number of substrate binding modes have been evaluated as well as a large number of rotamers for each binding mode in order to ensure that the most stable structures are located. In the catalyst−substrate complex with lowest energy, the substrate coordinates via a phosphate oxygen in a monodentate fashion to Zn2 (C·1, Scheme 3 and Figure 3). This binding Scheme 3. Schematic Representations of Three Binding Modes of BDNPP to the Catalysta

III. RESULTS AND DISCUSSION In this section we first discuss the hydrolysis reaction of BDNPP (reaction 1, Scheme 2) using the dinuclear zinc complex of the IPCPMP ligand. The results will be compared with those obtained previously with the corresponding dinuclear zinc complex of the DPCPMP ligand7 and with other computational studies on related systems. In the second part, we focus on the transesterification of HPNP (reaction 2, Scheme 2) using [Zn2(IPCPMP)]2+, and comparisons are also made to recent computational studies on the same reaction with different dizinc catalysts. III.A. Hydrolysis of BDNPP. The starting point for the calculations is the X-ray structure determined by Jarenmark et al. (Figure 2).6 This [{(Zn2(IPCPMP) (OAc)}2]2+ complex is a

a

Atom numbering is indicated. DNP = 2,4-dinitrophenyl.

mode is 4.8 kcal/mol more stable than when the substrate coordinates in a bidentate bridging fashion and as much as 14.4 kcal/mol more stable compared to monodentate coordination of the substrate to Zn1 (Scheme 3). This can be explained by the more electrophilic nature of Zn2 as there is no terminal carboxylate ligand bonded to it (in the monomer). In the calculations, it was found that prior to the nucleophilic attack the substrate binds in a bridging bidentate coordination mode to the catalyst while the hydroxide coordinates in a terminal fashion to Zn2 in a transient intermediate called C·1B (Figure 3). The energy of this complex is 7.2 kcal/mol higher than the catalyst−substrate complex C·1. The Zn−Zn distance is longer than in C·1 (3.40 vs 3.08 Å) because now it is the substrate rather than the hydroxide that bridges the two metals. Next, the nucleophilic hydroxide attacks the phosphorus center. The calculated energy of the transition state (1-TS1, Figure 3) is 10.8 kcal/mol relative to C·1. The resulting pentacoordinate intermediate C·1Int (Figure 3) is geometrically quite similar to 1-TS1 and has a calculated energy of 9.4 kcal/mol relative to C·1, i.e., only 1.4 kcal/mol lower than the transition state. From C·1Int, dissociation of the leaving group occurs at 1TS2 (Figure 3). The distance between the phosphorus center and the oxygen of the leaving group is 2.43 Å, 0.3 Å longer than the distance between the phosphorus center and the nucleophile in 1-TS1. As discussed in the Computational Details, the transition state was optimized in the gas phase. When all effects are included (i.e., corrections for large basis set, solvation, free energy, and dispersion), 1-TS2 was found to be 0.8 kcal/mol lower than C·1Int. Interestingly, most of the effect stems from solvation. In the gas phase, the leaving group

Figure 2. Schematic representation and X-ray structure of the [{(Zn2(IPCPMP)(OAc)}2]2+dimer. In the schematic representation, the studied monomer is colored and bonds that are broken upon solvation are indicated. PF6− counterion in the crystal structure is not shown.

tetranuclear dimer that dissociates into two dinuclear monomers in solution. The dissociation is supported by, inter alia, IR spectroscopic and mass spectrometric evidence.6,27 In the calculations, the [{(Zn2(IPCPMP) (OAc)}2]2+ dimer structure is used to generate the monomer that is assumed to be the active catalyst. The bridging acetate is substituted for 1874

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Figure 3. Optimized geometries of stationary points along the reaction pathway for the hydrolysis of BDNPP (1) with the IPCPMP ligand. Interatomic distances (Angstroms) are given according to the atom numbering indicated in Scheme 3.

catalyst−substrate complex C·1. The free energy is calculated to increase by 1.8 kcal/mol when the leaving group anion is dissociated completely from the catalyst (C·2+), indicating that C·2·3 is the structure with lowest energy in the reaction, constituting the resting state of the catalytic cycle. In proceeding from C·1B to C·2·3, the hydroxide forms a hydrogen bond to the carboxylate moiety of IPCPMP. This is reminiscent of the hydrolysis of dimethyl 4-nitrophenyl phosphate catalyzed by phosphotriesterase (PTE), where a Zn-coordinated aspartate has been suggested to assist in the mechanism.16 The aspartate accepts a hydrogen bond from the

transition state is calculated to be 6.1 kcal/mol higher in energy than the pentacoordinate intermediate, but the solvation effect stabilizes 1-TS2 by 6.9 kcal/mol relative to C·1Int. Thus, the calculations indicate that this stepwise associative mechanism can be considered as effectively concerted in solution, as the leaving group is predicted to dissociate without or with a very low barrier after the nucleophilic attack has taken place. After its dissociation, the leaving group is found to coordinate to Zn2 in a bidentate manner via both the phenolic oxygen and an oxygen of one of the nitro groups (C·2·3, Figure 3). This structure is 20.2 kcal/mol lower in energy than the 1875

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Figure 4. Calculated free energy profile for the hydrolysis of BDNPP (1).

Figure 5. Optimized transition states for alternative mechanisms considered for the hydrolysis of BDNPP. (A) The hydroxide acts as a base in the reaction. (B) Hydrolysis with two hydroxides bound to the catalyst.

cycle is estimated to be endergonic by 20.2−10.0 = 10.2 kcal/ mol. As the combined product release and catalyst regeneration process is calculated to be endergonic, the free energy profile for two consecutive catalytic cycles needs to be considered in order to determine the overall free energy barrier that is to be compared to the experimentally determined rate constant. The rate-determining barrier of the reaction becomes the one from C·2·3 in one catalytic cycle to 1-TS1 in the next (see Figure 4), which is calculated to be 21.0 kcal/mol. Experimentally, the rate constant for this reaction has been measured to 6.4 × 10−4 s−1 at room temperature, which can be converted to a ratedetermining barrier of ca. 22 kcal/mol.27 The calculated barrier is thus in good agreement with experiments. Here, it is important to point out that if only one catalytic cycle had been considered, the barrier would have been predicted to be from C·1 to 1-TS1, i.e., only 10.8 kcal/mol, which is greatly underestimated as compared to the experiments. It is interesting to compare to the findings of a very recent experimental study on a dinuclear FeIIFeIII complex that

nucleophile in the transition state for nucleophilic attack and is then proposed to deprotonate the product in a separate, consecutive, step. For IPCPMP, calculations were carried out where the carboxyl group of the ligand deprotonates the product 2, but no stable structure could be located in which the proton is transferred to the carboxyl moiety; the proton was always found to return to the oxygen of the hydroxide moiety during the geometry optimization. Closing the catalytic cycle requires the release of the hydrolysis products to the solution and the coordination of a hydroxide and a new substrate to the catalyst. These steps are difficult to study accurately in detail with the current methodology. However, as discussed in previous studies,7,36,37 the overall energetics of this process can be estimated by realizing that the reaction free energy of reaction 1, calculated to be −10.0 kcal/mol,38 is not changed by the catalyst. One full catalytic cycle should be exergonic by the same amount, such that C·1 in one catalytic cycle is 10.0 kcal/mol lower in energy than the same complex in the preceding cycle. Hence, the regeneration process from C·2·3 in one cycle to C·1 in the next 1876

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Inorganic Chemistry catalyzes hydrolysis of BDNPP.39 A hydroxide that binds terminally to FeIII was concluded to be the nucleophile in the reaction, and furthermore, the catalyst−product complex turned out to be stable enough to be isolated and characterized by X-ray crystallography.39 This supports the notion that the resting state of the catalytic cycle is the catalyst−product complex, as argued in the present work. Two alternative mechanistic scenarios have also been considered in the current study. In the first, the hydroxide is calculated to function as a Brønsted base rather than a nucleophile, deprotonating a water molecule from the solution, which performs the nucleophilic attack on the phosphorus in a concerted fashion. The optimized transition state for this mechanism is shown in Figure 5A. The energy of this transition state is calculated to be 17.2 kcal/mol relative to (C·1 + H2O), i.e., 6.4 kcal/mol higher than 1-TS1, which renders this possibility less likely. In the other mechanistic scenario considered here, the substrate binds to the catalyst without displacing a hydroxide, such that two hydroxides are bound to the catalyst rather than one throughout the reaction. In the lowest energy transition state, one hydroxide binds in a bridging position as before, while the second hydroxide is found to bind terminally to Zn2, trans to the pyridyl nitrogen (N4). The terminal hydroxide attacks the phosphorus center, and dissociation of the leaving group occurs concomitantly, as shown in Figure 5B. The overall barrier for hydrolysis is calculated to be 28.8 kcal/mol (also here two consecutive cycles have to be considered), which should be compared to the barrier of 21.0 kcal/mol obtained for the mechanism with only one hydroxide. These results suggest that the mechanism with two hydroxides is also less likely, see details in the Supporting Information. To summarize the obtained mechanism, the calculations show that the bridging hydroxide becomes terminal in the transition state and attacks the phosphorus center of the substrate. The calculated energy of the transition state for the dissociation of the leaving group is slightly lower than the pentacoordinate intermediate when all corrections are included (vide supra), which indicates that the nucleophilic attack and the dissociation can be considered to take place in an effectively concerted manner. The calculations further show that the resting state of the reaction is the catalyst−product complex and that two catalytic cycles need to be considered in order to obtain the rate-limiting barrier for the overall process. Here, it is interesting to compare the results to those obtained previously for the DPCPMP ligand.7 The reaction mechanism was found to be the same as that described above for IPCPMP. The overall barrier was calculated to be 30.4 kcal/ mol in that case. However, the computational scheme is somewhat different in the present paper, including corrections for standard state and for the influence of the low-lying frequencies on the entropy, which should improve the obtained energies (see Computational Details). Redoing the DPCPMP calculations with the same computational scheme as employed here, the rate-determining free energy barrier becomes 25.0 kcal/mol, which is in good agreement with the experimentally measured barrier for that ligand.7 The revised free energies and further information for the reaction with the DPCPMP ligand are given in the Supporting Information. Other relevant comparisons are to recent DFT studies on similar reactions employing dinuclear zinc complexes. Maxwell et al. studied the methanolysis of methyl p-nitrophenyl phosphate and the transesterification of HPNP catalyzed by a

symmetric dizinc complex.23 For the former substrate, a methoxide monodentately bound to one of the zinc ions was concluded to act either as a nucleophile, attacking the phosphorus center, or as a base, activating a methanol molecule from the bulk which in turn attacks the phosphorus. The corresponding barriers were calculated to be of similar magnitude, in contrast to the findings with the IPCPMP ligand in the current study, for which it was found that the base mechanism is associated with a substantially higher barrier (vide supra). However, it should be pointed out that apart from the differences in the catalyst structure, the computational scheme adopted by Maxwell et al. is somewhat different from the one used in the present study, which may be a source of some of the differences in energies. Sanyal et al. studied the hydrolysis of p-nitrophenyl phosphate with a dizinc complex of a Mannich-based ligand system.13 In the most favorable mechanism, the nucleophile was calculated to be a bridging hydroxide that attacks a bidentately bridging substrate. In the current calculations with the IPCPMP ligand, no transition state could be located with a bridging hydroxide attacking the phosphorus. In the geometry optimization, the bridging hydroxide always becomes terminally bound to Zn2. In a series of studies, Zhao and co-workers investigated the hydrolysis of p-nitrophenyl phosphate and bis-p-nitrophenyl phosphate catalyzed by dizinc complexes of both a symmetric ligand (L1) and an unsymmetric ligand (L6).20−22 In all cases, a hydroxide ion terminally bound to one of the zinc ions is proposed to be the nucleophile in the reactions. An interesting finding in this context is that for the phosphomonoester hydrolysis with the L1-complex, a low barrier was obtained with a “sandwich” complex in which two binuclear catalysts are bridged by the substrate.21 On this basis, the “sandwich” complex was proposed to be operational at high zinc concentrations, which could explain the deviations from linearity observed when the rate of hydrolysis was measured as a function of catalyst concentration. Quite interestingly, in cases where stepwise mechanisms were considered, it was found that the solvation effects stabilized the transition state for dissociation of the leaving group to be lower in energy than the preceding intermediate.20−22 A similar effect was observed in our calculations with the IPCPMP complex discussed above. Finally, comparing the energies obtained in the current study on Zn2IPCPMP with the other calculations cited above,13,20−23 it is interesting to note that we find a significantly lower energy for the transition state associated with nucleophilic attack relative to the catalyst−substrate complex. On the other hand, the energy of the catalyst−product complex is here predicted to be much lower, which results in an overall barrier of similar magnitude. III.B. Transesterification of HPNP. In this section we will discuss the transesterification mechanism of HPNP (reaction 2, Scheme 2).40 In this intramolecular reaction, the alcohol moiety of the HPNP substrate acts as the nucleophile in the reaction, attacking the phosphorus center. In order to activate the nucleophile, it can be deprotonated by either the catalyst or the solvent. Although the pKa value of the OH group is quite high (for isopropanol in water it is 16.5)41 when coordinated to Zn, the substrate will be acidified such that solvent-assisted deprotonation might be plausible in the pH range of the experiments (up to 11). On the basis of the experiments, a preequilibrium involving deprotonation of HPNP before the ratedetermining step has been suggested for the catalyst.27 1877

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Inorganic Chemistry Scheme 4. Schematic Representation of Various Binding Modes of HPNP to the Catalysta

a

Atom numbering is indicated. NP = p-nitrophenyl.

Figure 6. Optimized geometries of the stationary points in the reaction pathway for transesterification of HPNP (4). Atom numbering is indicated in Scheme 4.

Therefore, the reaction was modeled with two different starting structures: the substrate bound as either an alcohol or an alkoxide. The results with the alcohol are discussed first. The possible binding modes of HPNP to the catalyst differ from BDNPP as the former has one more potentially coordinating group, namely, its alcohol moiety. The most

stable catalyst−substrate binding conformers are given in Scheme 4. In the binding mode with lowest energy (C·4, Scheme 4 and Figure 5), the substrate is bridging the two zinc ions in a bidentate coordination mode and the hydroxide is bound terminally to Zn2 and accepts a hydrogen bond from the hydroxyl moiety of 4 that is coordinated to Zn1. It is interesting 1878

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Figure 7. Free energy profile for transesterification of HPNP (4).

Figure 8. Transition states for alternative mechanisms evaluated for HPNP. (A) Specific-base mechanism. (B) Transesterification with the deprotonated substrate.

to note that HPNP is calculated to bind to the catalyst 8.1 kcal/ mol more strongly than BDNPP.42 This is consistent with the larger binding affinity observed experimentally for HPNP as compared to BDNPP.27 The C·4 structure is reminiscent of the catalyst−substrate complex structure calculated by Gao and co-workers for an analogous catalyst.24 In the present study, despite many attempts to optimize transition states for deprotonation of the substrate by the hydroxide or transition states for nucleophilic attack by the alcohol on the phosphorus center, no such transition states could be located. It was rather found that for the reaction to proceed, the substrate must bind as in the transient C·4B intermediate shown in Figure 6. In this geometry, which is calculated to be 7.9 kcal/mol higher than C· 4, the hydroxide binds to Zn2 in the same position as in C·1B (cf. Figure 3) and the substrate bridges the two zinc ions. The isopropanol moiety of the substrate forms a hydrogen bond to the hydroxide. Next, deprotonation of the alcohol group occurs in a concerted fashion with the nucleophilic attack on the phosphorus center in the 4-TS1 transition state (Figure 6).

This transition state is 16.4 kcal/mol higher in energy than C·4, and the distances between the phosphorus center and the nucleophilic and leaving group oxygens are very similar to those in 1-TS1 (cf. Figure 3). In the resulting pentacoordinate intermediate C·4Int (Figure 6), a water molecule is formed on Zn2 upon deprotonation of the substrate, and it forms a hydrogen bond to the alkoxide moiety of the substrate. This structure is calculated to be 13.2 kcal/mol higher in energy than C·4, i.e., 3.2 kcal/mol lower than the transition state. Numerous attempts were made to find a stepwise mechanism in which the substrate is deprotonated in a separate step before the nucleophilic attack, but no such transition states could be located. We could further optimize a transition state in which the leaving group dissociates (4-TS2, Figure 6). The distance between the phosphorus and the oxygen of the leaving group is similar to the distance from the nucleophile to the phosphorus in 4-TS1 (2.29 vs 2.17 Å). In the gas phase and at the level of theory of the geometry optimizations, 4-TS2 is calculated to be 2.5 kcal/mol higher in energy than C·4Int. However, when all corrections are added (i.e., large basis set, solvation, free energy, 1879

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dinuclear zinc complexes.23,24 As mentioned above, the binding mode of the substrate in the C·4 complex of the present study is similar to that calculated by Gao et al.24 With that catalyst, deprotonation of the substrate by a terminally bound hydroxide was found to occur in a pre-equilibrium step before the ratedetermining nucleophilic attack. In the current study we found that the two steps occur concertedly. Incidentally, the barrier calculated by Gao et al. (16.5 kcal/mol) is very similar to the barrier found here (from C·4 to 4-TS1, 16.4 kcal/mol). However, the formation of the catalyst−product complex C·5+ in the present study is calculated to be considerably more exergonic than the corresponding reaction with the symmetric catalyst studied by Gao et al. (−9.2 kcal/mol compared to −1.7 kcal/mol). Thus, the product is more stabilized with the IPCPMP ligand, which results in a slower overall reaction. Transesterification of HPNP was also investigated by Maxwell et al. using the same catalyst as employed for the methyl p-nitrophenyl phosphate hydrolysis discussed above.23 In that computational model the deprotonation of the alcohol moiety of the substrate was calculated to take place in a separate step prior to the nucleophilic attack and dissociation of the leaving group. The base was suggested to be the methoxide bridging the zinc ions.23

and dispersion), the transition state is calculated to be +10.8 kcal/mol relative to C·4, i.e., 2.4 kcal/mol lower than C·4Int. Thus, similarly to the case of BDNPP hydrolysis discussed above, the reaction can be considered as effectively concerted. After the dissociation of the leaving group, a complex is formed in which the leaving group coordinates to Zn2. This structure is called C·5·6 and is 9.0 kcal/mol lower in energy than C·4. Upon complete dissociation of the anionic leaving group, complex C·5+ (Figure 6) can be formed, only 0.2 kcal/ mol lower in energy than C·5·6. Complex C·5+ does thus constitute the lowest point in the free energy profile (Figure 7). Similarly to the case of BDNPP hydrolysis, a hydrogen bond from the nucleophile to the carboxyl moiety of IPCPMP is formed on proceeding from C·4B to C·5+. Reaction 2 is calculated to be exergonic by 6.1 kcal/mol.38 Hence, the catalyst−substrate complex C·4 in one catalytic cycle should be 6.1 kcal/mol lower in energy than C·4 in the preceding cycle. Using the same procedure as above to determine the overall free energy barrier, from Figure 6 it is concluded to be associated with proceeding from the catalyst− product complex C·5+ in one catalytic cycle to 4-TS1 in the next cycle. The overall free energy barrier becomes 19.5 kcal/ mol, in good agreement with the experimental value of ca. 23 kcal/mol.27 We optimized the structure for an alternative transition state in which the proton transfer from the substrate to the hydroxide proceeds via a shuttling water molecule (Figure 8A). However, the energy turned out to be 19.9 kcal/mol higher in energy than (C·4 + H2O), i.e., 3.5 kcal/mol higher than 4-TS1. Furthermore, it should be mentioned that also for this reaction the mechanism with two hydroxides bound to the complex throughout the reaction has been considered. In this case, the additional hydroxide can act as either a base, deprotonating the alcohol group of HPNP, or as a nucleophile, attacking the phosphorus center. In both cases, it is calculated that the hydroxide is bound terminally to the Zn1 center, in contrast to the case of BDNPP hydrolysis discussed above, where the second hydroxide was found to bind to Zn2 instead. Interestingly, the two mechanisms yield rather similar overall barriers, both of which, however, are considerably higher (more than 12 kcal/mol) than the case with one hydroxide ion discussed above. See the Supporting Information for further details. Turning to the starting structure with the substrate being in the alkoxide form, the calculations show that the deprotonated substrate binds very similarly as in the neutral form (C·4). The alkoxide moiety coordinates to Zn2, and an intramolecular nucleophilic attack of the alkoxide on the phosphorus center takes place (Figure 8B). However, the overall free energy barrier of this scenario is calculated to be 29.3 kcal/mol, which is significantly higher than the barrier of 19.5 kcal/mol calculated with the alcohol substrate. A detailed free energy graph is provided in the Supporting Information. To summarize the results for the transesterification of HPNP, a general-base mechanism is proposed in which the hydroxide bound to the catalyst deprotonates the alcohol group of the substrate, which concomitantly attacks the phosphorus center. The calculated free energy barrier is in good agreement with the experimentally measured rate constant and is also quite similar to the calculated barrier for hydrolysis of BDNPP. It is interesting to compare these results with recent DFT studies on the transesterification of HPNP with different

IV. CONCLUSIONS In the present work, we used DFT methodology to study the reaction mechanism of hydrolysis of BDNPP and transesterification of HPNP using an unsymmetric dizinc complex with the IPCPMP ligand. The starting point of the calculations is the crystal structure, which is a tetranuclear dimer that, according to spectroscopic studies, is found to dissociate into an active dinuclear complex. Different substrate−catalyst binding modes were considered, and various mechanistic pathways were investigated, with the active catalyst binding one or two hydroxide ions. For both reactions, the calculations indicate that the catalyst with one hydroxide yields lower overall energy barriers, i.e., that [Zn2(IPCPMP) (OH)]+ is the active catalyst. For the hydrolysis of BDNPP, the calculations demonstrate that the bridging hydroxide first becomes terminal prior to performing the nucleophilic attack on the phosphorus center of the substrate to form a pentacoordinate intermediate. The calculations furthermore suggest that this intermediate dissociates without or with a very low energy barrier, making this mechanism essentially concerted. The resulting catalyst− product complex is calculated to be rather low in energy, constituting thus the resting state of the catalytic cycle. For the transesterification of HPNP, it is concluded that a terminal hydroxide acts as a Brønsted base, activating the alcohol group of the substrate, which performs a nucleophilic attack on the phosphorus center. Also here, the calculations indicate that the nucleophilic attack and the dissociation of the leaving group occur in an effectively concerted manner. For both reactions considered here the calculated overall barriers are in good agreement with available experimental rate constants. It is argued that two consecutive catalytic cycles have to be considered in order to obtain the rate-determining barriers of the processes. 1880

DOI: 10.1021/acs.inorgchem.5b02733 Inorg. Chem. 2016, 55, 1872−1882

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(19) Jackson, C. J.; Foo, J.-L.; Kim, H.-K.; Carr, P. D.; Liu, J.-W.; Salem, G.; Ollis, D. L. J. Mol. Biol. 2008, 375, 1189−1196. (20) Zhang, X.; Zhu, Y.; Zheng, X.; Phillips, D. L.; Zhao, C. Inorg. Chem. 2014, 53, 3354−3361. (21) Zhang, X.; Xu, X.; Xu, H.; Zhang, X.; Phillips, D. L.; Zhao, C. ChemPhysChem 2014, 15, 1887−1898. (22) Zhang, X.; Zheng, X.; Phillips, D. L.; Zhao, C. Dalton Trans. 2014, 43, 16289−16299. (23) Maxwell, C. I.; Mosey, N. J.; Brown, R. S. J. Am. Chem. Soc. 2013, 135, 17209−17222. (24) Gao, H.; Ke, Z.; DeYonker, N. J.; Wang, J.; Xu, H.; Mao, Z.-W.; Phillips, D. L.; Zhao, C. J. Am. Chem. Soc. 2011, 133, 2904−2915. (25) Guo, N.; Zhong, J.-Y.; Chen, S.-L.; Liu, J.-Q.; Min, Q.; Shi, R.-X. Chem. Phys. 2015, 457, 70−77. (26) Fan, Y.-B.; Gao, Y.-Q. Acta Phys.-Chim. Sin. 2010, 26, 1034− 1042. (27) Jarenmark, M.; Csapó, E.; Singh, J.; Wöckel, S.; Farkas, E.; Meyer, F.; Haukka, M.; Nordlander, E. Dalton Trans. 2010, 39, 8183− 8194. (28) (a) Becke, A. D. J. Chem. Phys. 1993, 98, 5648−52. (b) Lee, C.; Yang, W.; Parr, R. G. Phys. Rev. B: Condens. Matter Mater. Phys. 1988, 37, 785−789. (29) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H. P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Montgomery, J. A. Jr.; Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin, K. N.; Staroverov, V. N.; Keith, T.; Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega, N.; Millam, J. M.; Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Zakrzewski, V. G.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Dapprich, S.; Daniels, A. D.; Farkas, O.; Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox, D. J. Gaussian 09, Revision A.02; Gaussian: Wallingford, CT, 2009. (30) Hay, P. J.; Wadt, W. R. J. Chem. Phys. 1985, 82, 270−283. (31) Cossi, M.; Rega, N.; Scalmani, G.; Barone, V. J. Comput. Chem. 2003, 24, 669−681. (32) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Montgomery, J. A. Jr.; Vreven, T.; Kudin, K. N.; Burant, J. C.; Millam, J. M.; Iyengar, S. S.; Tomasi, J.; Barone, V.; Mennucci, B.; Cossi, M.; Scalmani, G.; Rega, N.; Petersson, G. A.; Nakatsuji, H.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Klene, M.; Li, X.; Knox, J. E.; Hratchian, H. P.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Ayala, P. Y.; Morokuma, K.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Zakrzewski, V. G.; Dapprich, S.; Daniels, A. D.; Strain, M. C.; Farkas, O.; Malick, D. K.; Rabuck, A. D.; Raghavachari, K.; Foresman, J. B.; Ortiz, J. V.; Cui, Q.; Baboul, A. G.; Clifford, S.; Cioslowski, J.; Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.; Komaromi, I.; Martin, R. L.; Fox, D. J.; Keith, T.; Al-Laham, M. A.; Peng, C. Y.; Nanayakkara, A.; Challacombe, M.; Gill, P. M. W.; Johnson, B.; Chen, W.; Wong, M. W.; Gonzalez, C.; Pople, J. A. Gaussian 03, Revision D.01; Gaussian: Wallingford, CT, 2004. (33) See, for example: Jensen, F. Introduction to Computational Chemistry; John Wiley & Sons Ltd.: New York, 2007. (34) Grimme, S. Chem. - Eur. J. 2012, 18, 9955−9964. (35) (a) Grimme, S.; Antony, J.; Ehrlich, S.; Krieg, H. J. Chem. Phys. 2010, 132, 154104−22. (b) Grimme, S.; Ehrlich, S.; Goerigk, L. J. Comput. Chem. 2011, 32, 1456−1465. (36) Das, B.; Daver, H.; Singh, A.; Singh, R.; Haukka, M.; Demeshko, S.; Meyer, F.; Lisensky, G.; Jarenmark, M.; Himo, F.; Nordlander, E. Eur. J. Inorg. Chem. 2014, 2014, 2204−2212.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.5b02733. Methodological tests, results concerning hydrolysis of BDNPP with two hydroxides, results for the DPCPMP ligand, alternative mechanisms for transesterification of HPNP, calculated absolute energies and corrections, and Cartesian coordinates of all structures (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Phone: +46 8 161094. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support from the Swedish Research Council, the Göran Gustafsson Foundation, and the Knut and Alice Wallenberg Foundation is acknowledged. Computer time was provided by the Swedish National Infrastructure for Computing. B.D. thanks the European Commission for an Erasmus Mundus predoctoral fellowship.

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DOI: 10.1021/acs.inorgchem.5b02733 Inorg. Chem. 2016, 55, 1872−1882