Hydrolysis of p-nitrophenyl picolinate catalyzed by divalent metal ion complexes containing imidazole groups in micellar solution

Colloids and Surfaces A: Physicochem. Eng. Aspects 235 (2004) 145–151

Hydrolysis of p-nitrophenyl picolinate catalyzed by divalent metal ion complexes containing imidazole groups in micellar solution Bing-ying Jiang a , Yan Xiang b , Juan Du a , Jia-qing Xie a , Chang-wei Hu a , Xian-cheng Zeng a,∗ a

Sichuan Key Laboratory of Green Chemistry and Technology, Faculty of Chemistry, Sichuan University, P.O. Box 74, Chengdu 610064, PR China b Department of Environmental Engineering, School of Material Science and Engineering, Beihang University, Beijing 100083, PR China Received 13 March 2003; accepted 17 October 2003

Abstract The hydrolysis of p-nitrophenyl picolinate (PNPP) catalyzed by Zn(II), Cu(II) and Co(II) complexes of imidazole groups have been investigated kinetically in the pH range 6.0–8.0 at the presence of three kinds of surfactants and 25.0 ± 0.01 ◦ C, respectively. The results indicated that Zn(II) complex exhibit a great catalytic function in micellar solution with higher pH value, but when reactions were performed in weak acid solutions, Cu(II) complex catalyzed the hydrolysis of PNPP more efficiently than Zn(II) and Co(II) complexes did, which may be attributable to the different ionization states of the corresponding complexes in micellar media with different pH values and the different nucleophilic ability of active species in different complexes. In addition, these complexes showed more reactivity in zwitterionic (LSS) and nonionic (Brij35) micelles than that in cationic (CTAB) micelle, which may be explained by the electrostatic interaction between metal ions of these complexes and the head groups of surfactants or the substrate. The relative kinetic and thermodynamic parameters were obtained by kinetic analysis employing ternary complex model for metallomicellar catalysis. © 2003 Published by Elsevier B.V. Keywords: Hydrolysis of PNPP; Metallomicellar catalysis; Imidazole groups; Ionization states of complexes; Electrostatic interaction

1. Introduction Metal ions catalyzed reactions of carboxylic acid derivatives have been extensively investigated in recent years as model reactions of metalloenzymes such as carboxypeptidase A, carbonic anhydrase and related enzymes [1–6]. It is well known that a zinc ion is coordinated to two imidazoles and one carboxyl group at the active site of carboxypeptidase A [6–11] and to three imidazole groups at the active site of carbonic anhydrase [6,12–14]. In these enzymes, metal ion (zinc ion), is considered to act as catalytic active center for bringing substrate towards the complexes, which formed by ligands and metal ions, or to activate the metal-bound water molecules to nucleophiles. About two decades ago, bis(imidazolyl)cyclodextrin [15] and some triimidazolyl derivatives [16] were reported to be effective ∗

Corresponding author. E-mail address: [email protected] (X.-c. Zeng).

0927-7757/$ – see front matter © 2003 Published by Elsevier B.V. doi:10.1016/j.colsurfa.2003.10.019

ligands for the modeling of carbonic anhydrase. In the following years, Tagaki et al. [17] investigated the effect of cocatalysis on hydrolysis of PNPP by imidazolyl and hydroxyl groups, and the results suggested that metal-bound imidazolate and hydroxide ions are very reactive nucleophiles [18]. The presence of the imidazole groups makes the ligand suitable for use in the synthesis and investigation of mimics for histidine-containing active sites of metalloproteins. Moving along these lines, to better elucidate the features of the active site of carbxypeptidase A containing two imidazole groups, we made our efforts to study the hydrolysis of PNPP catalyzed by complexes of Zn(II), Cu(II) and Co(II) with imidazole derivatives (N,N-bis(2-ethyl-5-methyl-imidazole-4-ylmethyl) aminopropane (biap)) kinetically. It is also well known that micelles can provide hydrophobic microenvironment for artificial enzymatic mimicry and the rates and pathways of all kinds of chemical reactions can be altered in many cases by performing the reactions in micellar

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media instead of pure bulk solutions [19]. Thus, in order for better understanding the effects of different metallomicelles formed by different surfactants and complexes on hydrolysis of PNPP, kinetic study was examined, respectively, in the presence of three surfactants: cetyltrimethylammonium bromide (CTAB), polyoxyethylene(23) lauryl ether (Brij35) and n-lauroylsarcosine sodium (LSS). Thermodynamic and kinetic parameters of reactions were obtained by employing the ternary complex kinetic model for metallomicellar catalysis [20–23] and the catalytic mechanism was discussed in this paper.

2. Experimental section 2.1. Materials All reagents, unless otherwise indicated, were analytical or better grade commercial products and used without further purification. CTAB was recrystallized from ethanol before use, and p-nitrophenyl picolinate(PNPP) was synthesized according to literature [24]. PNPP stock solution for kinetic study was prepared in acetonitrile. To avoid the influence of chemical components of different buffers, Tris–TrisH+ buffer was used in all runs and its pH value was adjusted by adding analytically concentrated hydrochloric acid. Doubly distilled deionized water was used through out the experiments. 2.2. Synthesis of ligand and complexes Ligand (Scheme 1), N,N-bis(2-ethyl-5-methyl-imidazole4-ylmethyl)aminopropane (biap), was synthesized in doubly distilled deionized water according to literature [25,26] at room temperature. The white solid was formed, washed with acetonitrile and diethyl ether and dried in vacuo. Anal. Cald. for ligand (C17 H29 N5 ·H2 O): C, 63.5; H, 9.7; N, 21.8%. Found: C, 63.39; H, 9.6; N, 21.7%. MS: 304 (M + + 1). IR (KBr pellet): 3161 (br,vs), 2975 (vs), 2934 (vs), 2791 (vs), 1614 (s), 153 (s), 1452 (vs,br), 1374 (s), 1324 (s), 1284 (m), 1242 (w), 1176 (w), 1120 (m), 1071 (s,br), 974 (m), 960 (w), 850 and 792 (m,vbr) cm−1 . The divalent metal complexes, Cu(biap)Cl2 , Zn(biap)Cl2 (H2 O) and Co(biap)Cl2 were prepared also following the above literatures. Characterization of these complexes was performed by C, H, N elemental analysis (Perkin–Elmer

Scheme 1.

240 elementary analyzer), IR (Perkin–Elmer 1000 FTIR spectrophotometer) and ICP (IRIS Advantage ER/S) techniques. Anal. Calc. for C17 H29 CuCl2 N5 : C, 46.6; H, 6.67; N, 16.0; Cu, 14.5%. Found: C, 46.3; H, 6.51; N, 16.2; Cu, 14.8%. IR (KBr pellet): 3200–2700 (vs, multiple band), 1644 (m), 1542 (m), 1464 (vs), 1389 (w), 1369 (w), 1352 (w), 1326 (w), 1286 (m), 1237 (w), 1137 (w), 1100 (m), 1086 (s), 1048 (s), 996 (m), 958 (m), 918 (m), 854 (w), 826 (w), 802 (s), 786 (s), 732 (w), 564 (m). Anal. Calc. for C17 H29 ZnCl2 N5 (H2 O): C, 44.6; H, 6.78; N, 15.3; Zn, 13.7%. Found: C, 44.3; H, 6.62; N, 15.5; Zn, 13.5%. IR (KBr pellet): 3214-2700 (vs, multiple band), 1627 (m), 1541 (m), 1460 (vs), 1389 (w), 1369 (w), 1352 (w), 1322 (w), 1281 (m), 1237 (w), 1137 (w), 1100 (m), 1085 (s), 1051 (s), 979 (m), 958 (m), 918 (m), 854 (w), 802 (s), 753 (s), 584 (m). Anal. Calc. for C17 H29 CoCl2 N5 : C, 47.1; H, 6.70; N, 16.2; Co, 13.6%. Found: C, 47.4; H, 6.73; N, 16.5; Co, 13.7%. IR (KBr pellet): 3225–2727 (vs, multiple band), 1639 (m), 1533 (m), 1453 (vs), 1389 (w), 1369 (w), 1323 (w), 1286 (m), 1237 (w), 1137 (w), 1115 (m), 1083 (s), 1053 (s), 994 (m), 905 (m), 844 (w), 755 (w), 571 (m). 2.3. Method The isoelectronic point of LSS was determined to be 5.20 through solubility method using a ZD-2 acid–base orientation titration apparatus [22]. Kinetic measurements of hydrolysis of PNPP were carried out spectrophotometrically at 25.0 ± 0.01 ◦ C, employing a GBC 916 UV-Vis spectrophotometer with a thermostatic cell holder. Reactions were initiated by injecting 30 ␮l of PNPP stock solution into a 1-cm cuvette containing 3 ml of desired reagents. The reaction rate was followed by monitoring the liberation of p-nitrophenyl at 400 nm under the conditions of excess complexes over substrate (at least ca.10 folds). Pseudo-first-order rate constants were obtained by linear least-square fits of the plots of ln(A − At ) versus time. Each value is the average of three determinations; its average relative standard deviation is smaller than 2.5%.

3. Results and discussion 3.1. A survey of hydrolytic rates of PNPP at pH 7.00 and 25 ◦ C Because of the low solubility of these complexes in water, the kinetic study was performed in buffered micellar solutions. According to the previous report [22], the CMC values of CTAB, Brij35 and LSS at 25 ◦ C are 9.2 × 10−4 , 5.22 × 10−5 and 4.61 × 10−4 mol dm−3 , respectively. Thus, the prepared surfactant concentrations in kinetic runs were all higher than their CMC values to ensure the formation of micelles in buffer.

B.-y. Jiang et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 235 (2004) 145–151 Table 1 Apparent rate constants (103 kobsd (s−1 )) for the hydrolysis of PNPP in different solutions at pH 7.00 ± 0.01, 25 ◦ Ca Entry

Systems

103 kobsd (s−1 )

1 2 3 4 5 6 7 8 9 10 11 12 13

None CTAB Brij35 Lss CuL + CTAB ZnL + CTAB CoL + CTAB CuL + Brij35 ZnL + Brij35 CoL + Brij35 CuL + Lss ZnL + Lss CoL + Lss

0.0178 0.0213 0.0258 0.0197 1.530 1.020 1.070 3.840 3.100 1.010 4.270 2.170 1.710

In 0.01 mol dm−3 Tris–TrisH+ buffer (µ = 0.1 KNO3 ). [CTAB] = 0.01 mol dm−3 , [Brij35] = 6 × 10−4 mol dm−3 , [Lss] = 6 × 10−4 mol dm−3 , [ML] = 1 × 10−3 mol dm−3 , [PNPP] = 2 × 10−5 mol dm−3 . a

Pseudo-first-order rate constants of the hydrolytic reaction were determined by observing the release of p-nitrophenyl from the substrate spectrophotometrically as shown in Table 1. From the results, the rates of the hydrolysis of PNPP in metallomicelles depended on both the concentrations of the complexes and the pH value of the reaction systems, as exemplified in Fig. 1 for Zn(II) complex catalyzing the hydrolysis of PNPP in LSS micellar solution. Table 1 suggested that the rate constant of spontaneous hydrolysis of PNPP in buffered solution is k0 = 1.780 × 10−5 s−1 at pH 7.00 and 25 ◦ C. In micellar solutions, three kinds of surfactants all showed very little catalytic efficacy on the hydrolysis of PNPP. In the presence of any complexes, the pronounced rate acceleration was observed in micellar solution, especially the metallomicelles formed by Cu(II)

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complex with zwitterionic micelle (LSS) and nonionic micelle (Brij35) exhibit marked catalytic function on the hydrolytic reaction. From Table 1, a notable rate enhancement of more than 200-fold was found in both zwitterionic metallomicelles and nonionic metallomicelles. However, rate acceleration of only less than 100-fold was obtained in cationic metallomicelle (CTAB) for the hydrolysis of PNPP at pH 7.00 and 25 ◦ C with [complex] = 1.00 × 10−3 mol dm−3 , which may be explained by the partition of PNPP between the bulk solvent and the micellar pseudo-phase, while the complexes partially remains in the bulk solution due to the electrostatic repulsion between metal ions of these complexes and the cationic head groups of the CTAB micelle [27]. On the opposite, zwitterionic micelle presents some characteristics similar to that of anionic micelle when pH value of the micellar solution is higher than its isoelectronic point (pI = 5.20), thus the Stern layer of LSS micelle is concentrated with negative charge, and then the complexes mainly residues into this reaction region through electrostatic attraction, which involved in the rate enhancement of the hydrolysis of PNPP. 3.2. Kinetic investigation for complex catalysis in micellar solution One of the most important processes leading to micellar effects on reactions is the solubilization of substrate in micellar interiors. Therefore, micelle-dependent reaction is generally assumed to take place simultaneously in bulk solvent and micellar phase to afford the products (P) on the basis of the phase-separation model of micelle [28]. The kinetic results reported in this paper can be explained assuming the operation of the following processes [22], Ks

kN

ML + S ↔MLS − →P

(1)

Fig. 1. Pseudo-first-order rate constants for the hydrolysis of PNPP as the function of [ZnL] in LSS Micellar System at 25 ◦ C. [Lss] = 6 × 10−4 mol dm−3 , [PNPP] = 2 × 10−5 mol dm−3 , (䊏) pH 6.00, (䊊) pH 6.50, (䉱) pH 7.00, ( ) pH 7.50, (䉬) pH 8.00.

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k0

S− →P Ks =

[MLS] [ML][S]

(2)

Table 3 pH dependencies of kN and Ks for the hydrolysis of PNPP by different compounds in Brij35 micellar solution at 25 ◦ Ca

(3)

Systems

pH

103 kN (s−1 )

Ks (mol dm−3 )

ZnL + Brij35

6.0 6.5 7.0 7.5 8.0 6.0 6.5 7.0 7.5 8.0 6.0 6.5 7.0 7.5 8.0

0.299 0.897 2.128 10.17 49.17 3.532 5.350 6.426 7.837 9.680 0.252 0.552 1.360 3.590 5.870

275.8 302.1 322.3 521.8 601.0 534.2 1128 1982 3243 3500 1121 2105 2740 3822 2619

k0 = k0 + km [m]

(4)

[S]t = [S] + [MLS]

(5)

where [ML] and [MLS] the concentrations of the complex and the ternary complex formed by metal ion, ligand and substrate in the micellar phase, respectively, [m] the concentration of micelle and [S] the concentration of substrate in bulk phase, [S]t the total concentration of substrate, k0 the first-order-rate constant due to the buffer, k0 the apparent first-order-rate constant of reaction in bulk phase without the complex, km the second-order-rate constants of reaction in micellar solution, and Ks the binding constant between the complex and the substrate, kN the apparent first-order-rate constant for intracomplex nucleophilic reaction in the ternary complex. Therefore, based on the rate equation, the pseudo-firstorder rate constant (kobsd ) can be expressed as: kobsd =

kN [MLS] + k0 [S] [S]t

(6)

Inserting Eqs. (3) and (5) into Eq. (6) rearranging, then give rise to 1 1 1 1 = (7) + · kobsd − k0 kN − k 0 (kN − k0 )Ks [ML]t Based on the above equation, the value of kN and Ks can be evaluated from the slopes and the intercepts of the plots of 1/(kobsd − k0 ) versus 1/[ML]t by using the linear regression method, and all linear coefficients are no less than 0.985. This indicated that the kinetic model for the metallomicellar Table 2 pH dependencies of kN and Ks for the hydrolysis of PNPP by different compounds in CTAB micellar solution at 25 ◦ Ca Systems

pH

103 kN (s−1 )

Ks (mol dm−3 )

ZnL + CTAB

6.0 6.5 7.0 7.5 8.0 6.0 6.5 7.0 7.5 8.0 6.0 6.5 7.0 7.5 8.0

−a 0.628 1.979 3.481 3.790 1.390 2.046 2.642 3.011 3.263 0.417 0.920 2.290 3.300 3.650

−a 499.3 895.6 2111 5957 1057 1274 2288 3326 4427 157.5 634.4 979.4 1507 3788

CuL + CTAB

CoL + CTAB

a [CTAB] = 0.01 mol dm−3 , [PNPP] = 2 × 10−5 mol dm−3 ; −a, cannot be determined.

CuL + Brij35

CoL + Brij35

a

[Brij35] = 6 × 10−4 mol dm−3 , [PNPP] = 2 × 10−5 mol dm−3 .

catalysis employed in the reaction system mentioned in this paper is conformed to be reasonable. The results obtained are summarized in Tables 2–4. From Tables 2–4, as for any complex, it is obvious that the rate constants are all pH-dependent in three kinds of micelles, and moreover, they increased apparently with the increase in pH value of micellar solution, which indicated that the cleavage of PNPP promoted by the complexes is an acid–base catalytic process and the dissociated complex is more active than the undissociated complex. According to the results listed in Tables 2–4, it can be seen that in any micellar solution with low pH value investigated in this paper, among the three complexes, Cu(II) complex showed relatively high catalytic efficacy on the hydrolytic reaction. However, in any micellar solution with high pH value, it is worth noting that the maximum first-order-rate constants (kN ) in different micellar solutions obtained are Table 4 pH dependencies of kN and Ks for the hydrolysis of PNPP by different compounds in LSS micellar solution at 25 ◦ Ca Systems

pH

103 kN (s−1 )

Ks (mol dm−3 )

ZnL + Lss

6.0 6.5 7.0 7.5 8.0 6.0 6.5 7.0 7.5 8.0 6.0 6.5 7.0 7.5 8.0

0.754 1.754 5.333 12.28 22.70 3.157 4.919 6.880 7.373 8.270 0.472 1.173 3.184 3.957 8.369

446.6 649.5 731.8 883.6 1325 1278 1592 1827 2138 2783 655.5 771.9 961.0 2097 2904

CuL + Lss

CoL + Lss

a

[LSS] = 6 × 10−4 mol dm−3 , [PNPP] = 2 × 10−5 mol dm−3 .

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Scheme 2.

3.790 × 10−3 s−1 in CTAB, 4.917 × 10−2 s−1 in Brij35 and 2.270 × 10−2 s−1 in LSS micelles, respectively, which are all for cleavage of PNPP catalyzed by Zn(II) complex at pH 8.00 and 25 ◦ C. The rate increases by ca. 213-, 2762and 1275-fold, respectively, when compared with that of the spontaneous hydrolysis of PNPP in buffered solution at pH 7.00 and 25 ◦ C. As for binding constants (Ks ) in all systems studied, they enhanced clearly with the increasing in the pH values of micellar solutions, which is favorable to the increase of the first-order-rate constants (kN ) for the hydrolysis of PNPP. That is to say, as to the same complex in the same micellar solution, the bigger the binding constants, the tighter the association between the substrate and the complex, and then leads to higher reactivity and rate constants because the hydrolytic reaction of PNPP in the metallomicelle is a intramolecular nucleophilic attack.

Fig. 2. pH-rate profile for the liberation of p-nitrophenyl from PNPP in CTAB micelle at 25 ◦ C: (䉬) Zn(II) complex; (䉱) Co(II) complex; (䊏) Cu(II) complex.

3.3. pH-rate profile From the results, the magnitude of kN , Ks are also dependent on pH values of reaction media. To further acquire the insight into the catalytic hydrolytic mechanism of PNPP, it is necessary to investigate the pH-independent first-order-rate ) and constant of the reaction in the metallomicellar phase (kN the acidic ionization equilibrium constant (Ka ) for the hydroxyl group of the ternary complex served as nucleophilic groups in hydrolytic reaction. From Scheme 2, we have [20–23], 1 1 1 = + [H+ ] kN kN k N Ka

Fig. 3. pH-rate profile for the liberation of p-nitrophenyl from PNPP in Brij35 micelle at 25 ◦ C: (䉬) Zn(II) complex (䉱) Co(II) complex; (䊏) Cu(II) complex.

(8)

the pKa values of undissociated complexes (HA) and the kN − values of dissociated complex anions (A ) could be evaluated according to Eq. (8) as shown in Figs. 2–4. The results calculated are shown in Table 5. From Table 5, the pKa values for these divalent metal complexes studied mean that the free water molecules bound to metal ions can easily deprotonated to hydroxyl groups acting as nucleophiles in micellar solutions with neutral pH values. In enzymatic catalysis, the crucial factor determining the catalytic efficacy of acid–base catalyst is that, under certain conditions, whether catalyst presents effective (or proper) ionization state (protonated or deprotonated state) or not

Fig. 4. pH-rate profile for the liberation of p-nitrophenyl from PNPP in LSS micelle at 25 ◦ C: (䉬) Zn(II) complex; (䉱) Co(II) complex; (䊏) Cu(II) complex.

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Table 5 kN and pKa of the hydrolysis of PNPP in the metallomicellar solutionsa

CTAB

Brij35

Lss

a

Systems

(s−1 ) 103 kN

pKa

ZnL CuL CoL ZnL CuL CoL ZnL CuL CoL

6.666 3.053 3.733 23.59 8.389 3.448 15.13 7.802 6.569

7.47 6.09 6.91 7.89 6.24 7.12 7.29 6.18 7.12

All correlation coefficients ≥0.98.

[29], i.e, acid acts as effective acid catalyst only when it is in acid-form, and base catalyst exhibits high catalytic efficacy when it is in base-form. In micellar solution with relatively low pH value, the complexes with low pKa ’s are more active on reaction than those with high pKa ’s because the catalysts (with high pKa values) itself with high reactivity were mainly protonated when pH values of reaction systems is lower than pKa of the catalysts. Therefore, among the three complexes in this paper, the lower pKa value for Cu(II) complex compared with that for Zn(II) and Co(II) complex suggests the higher ability of Cu(II) complex to make the water molecules coordinated to the complexes deprotonate completely to hydroxyl groups serving as nucleophile even in the lower pH value than that of Zn(II) and Co(II) complexes in the experimental conditions. Consequently, without doubt, Cu(II) complex, other than Zn(II) and Co(II) complex, presents the best catalytic function on the hydrolytic reaction under the same experimental conditions of low pH value. On the contrary, the increase of pKa from Cu(II) to Zn(II) and Co(II) complexes would provide stronger conjugate base [30], the hydroxyl, which acts as nucleophilic species in the reaction. In conclusion, two competitive effects coexisted in the catalytic hydrolysis of PNPP, one was the ability of the complexes to provide a larger fraction but less nucleophilic species and the other was the opposite case. From the results, it can be readily concluded that, in the present reaction conditions, the effect of providing more nucleophilic species was prevailing, which made the Zn(II) complex become a more reactive species than the Cu(II) and Co(II) complex in enhancing the hydrolytic rate of PNPP [31]. In addition, the ligand investigated in this paper mimics the active sites of carboxypeptidase A, the activity of the complex for the hydrolysis of PNPP maybe affected after Zn(II) was replaced by other divalent metal ions. This may be the reason that, even with nearly pKa value with Zn(II) complex, Co(II) complex showed much lower activity than that Zn(II) complex to catalyze hydrolytic reaction. ), it can be noted that As to first-order-rate constant (kN the maximum values for Zn(II) complex in Brij35 and LSS micelles are up to 23.59 × 10−3 and 15.13 × 10−3 s−1 , their magnitude is in accordance with the values of kN , and the rate enhancement is about 1000-fold comparing with the

spontaneous rate of the hydrolysis of PNPP in buffered solution. In all systems studied in this paper, the distinguished first-order rate constant was found in metallomicelle made of Zn(II) complex and nonionic surfactant, which may be caused by the electrostatic attraction between the metal ions of the complex and the substrate in Brij35 micelle. As a result, this electrostatic interaction could increase the local concentration of reactants and then enhance the collision frequencies of reactive molecules, which involved in notable rate enhancement of the hydrolytic reaction in nonionic micelle. In LSS micellar system, in spite of the good solubilization of complex into LSS micelle, the substrate molecule exhibits electronegativity, while the LSS micelle shows anionic characteristics in the pH range under experimental conditions because of its isoelectronic point of 5.20, so the solubilizing amount of PNPP into LSS micelle is not so enough to meet the need of reaction because of the electrostatic repulsion. Thus, the solubilization and the electrostatic interaction between reactants may also be involved in the effect of micelle on hydrolysis of PNPP. The schematic representation of the mechanism of the hydrolysis of PNPP catalyzed by complexes is shown in Scheme 2. In Scheme 2, the M2+ ions play a very important role in catalyzing the hydrolysis of PNPP: (1) M2+ ion serves as a template upon which PNPP is able to coordinate and the metal center masks the anionic character of the bound substrate, which otherwise electrostatically inhibits the approach of the hydroxide nucleophile. The geometry of the resulting ternary complex facilitates the pseudo-intramolecular nucleophilic attack of the hydroxyl groups on the carbonyl of substrate; (2) Another catalytic function of the M2+ ions in the metallomicelle is to lower the pKa values of the water molecules bound to metal center, providing a high concentration of the effective hydroxyl nucleophile at neutral pH for reaction; (3) The metal ion can stabilize the negative charges developed on the carbonyl oxygen atom in the transition state. In the ternary complex shown in Scheme 2, an intramolecular nucleophilic attack of the hydroxyl on the carbonyl group of the activated ester [32,33], yields the penta-coordinate intermediate, and then releases the good leaving group (p-nitrophenyl). Finally, the catalyst is regenerated from the acylated intermediate for another catalytic process. In summary, the metallomicelles formed by divalent metal complexes with different micelles operates via metal-hydroxide mechanism to complete the catalytic reaction of hydrolysis of PNPP. As for the rate constants are not so high as expected, it may be due to the fact that the steric hindrance of the two ethyl groups in the complexes could hold back the approach of the substrate to metal ion, which is unfavorable for the hydrolytic cleavage of PNPP.

4. Conclusion In this paper, the effect of metallomicelles formed by different surfactants and complexes on the hydrolysis of PNPP

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was investigated kinetically. There are some interesting results should be noticed: (1) in any micellar solution, the apparent rate acceleration was observed in the presence of any complex, especially the metallomicelles formed by complexes with nonionic surfactant and zwitterionic surfactant exhibit highly catalytic function on the hydrolytic reaction. However, remarkable rate acceleration as reported previously was not obtained in cationic metallomicelle for hydrolysis of PNPP even with [complex] = 1 × 10−3 mol dm−3 at pH 7.00 and 25 ◦ C. (2) In spite of the lowest pKa value of Cu(II) complex, the hydrolysis of PNPP catalyzed by it efficiently only in micellar solutions with low pH value, but in reaction systems with slightly high pH values, Zn(II) complex catalyzed the reaction more effectively than Cu(II) complex did due to their different ionization states of corresponding complexes in micellar solutions with different pH values and the different nucleophilic ability of active species in different complexes. The results of this paper indicated that metallomicelles influence the chemical reaction both by micellar microenvironment and by active site similar in enzyme, which provide the information on design of better enzyme model and interpretation of cleavage mechanism of hydrolytic reaction. Acknowledgements This work was supported by the National Natural Science Foundation of China (Grant: 29873031 and 20173038). References [1] T.C. Bruice, S.J. Benkovic, Bioorganic Mechanism, vol. 1, Benjamin, New York, 1966. [2] W.P. Jencks, Catalysis in Chemistry and Enzymology, Mcgraw-Hill, New York, 1966. [3] M.L. Bender, Mechanism of Homogeneous Catalysis from Protons to Proteins, Wiley-Interscience, New York, 1971. [4] A. Midvan, Enzyme 2 (1970) 446. [5] J.E. Coleman, in: E.T. Kaiser, F.J. Kezdy (Eds.), Progress in Bioorganic chemistry, vol. 1, Wiley-Interscience, New York, 1971, 159.

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