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Metallovesicular catalytic hydrolysis of p-nitrophenyl picolinate catalyzed by zinc(II) complexes of pyridyl ligands in vesicular solution
Colloids and Surfaces A: Physicochem. Eng. Aspects 392 (2011) 110–115
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Metallovesicular catalytic hydrolysis of p-nitrophenyl picolinate catalyzed by zinc(II) complexes of pyridyl ligands in vesicular solution Yongwei Song a,b , Xinting Han a , Xiaoli Guo a , Qing Zeng c , Fubin Jiang a,∗ a b c
Department of Chemistry, Beijing Normal University, Beijing 100875, People’s Republic of China Ordos School attached to Beijing Normal University, Ordos, The Inner Mongolia Autonomous 017000, People’s Republic of China School of Chinese Pharmacy, Beijing University of Chinese Medicine, Beijing 100102, People’s Republic of China
a r t i c l e
i n f o
Article history: Received 20 June 2011 Received in revised form 25 September 2011 Accepted 30 September 2011 Available online 8 October 2011 Keywords: Enzyme Metallovesicular Ternary complex Kinetics Catalytic hydrolysis
a b s t r a c t The hydrolysis of p-nitrophenyl picolinate (PNPP) catalyzed by the Zn(II) complexes of 6-(nbutyloxymethyl)-2-(hydroxymethyl)pyridine and 6-(n-dodecyloxymethyl)-2-(hydroxymethyl)pyridine was kinetically investigated by observation of the rates of release of p-nitrophenol in a vesicular solution at different pH values and temperatures. A 1:1 ligand:Zn2+ stoichiometry was found to produce the most active species based on a kinetic version of the Job plot analysis. Experimental results also showed that the complex formed from ligand 2 and Zn2+ exhibited a more remarkable rate acceleration effect on the hydrolysis of PNPP in vesicular solution than that formed from ligand 1 and Zn2+ . © 2011 Elsevier B.V. All rights reserved.
1. Introduction Metalloenzymes play an important role in the catalytic hydrolysis of carboxylic acid esters. A number of hydrolytically active metalloenzymes have been found in nature. Metallomicelles made up of ligand surfactants and transition metallic ions have been extensively reported as artificial hydrolytic metalloenzymes [1–9]. However, increasing the catalytic efficiency and achieving the activity of natural enzymes remain major challenges to researchers. If the catalytic efficiency in such systems is increased, we can not only mimic the enzymatic active centers of natural enzymes, but also simulate their hydrophobic microenvironments [9]. Vesicles are bilayer shells, with each shell enclosing a pool of solvent in the interior [10]. They have been considered as models of biological membranes and are similar to micelles in that they can concentrate the reactants and accelerate chemical reactions. Chemical reactions can be catalyzed effectively if the reactants can be concentrated on the interface between the vesicles’ bilayer and water layer [11]. Vesicles can adsorb more reactants compared with micelles because they have a somewhat larger specific surface area and a faster aggregation velocity than micelles. Moreover, vesicles can reach a higher catalytic efficiency than micelles [12]. Thus, extensive investigations on vesicles have been carried out in recent
years. However, few studies have focused on catalytic reactions in vesicular solutions. In our laboratory, an efficient and novel model in enzymatic catalysis, namely, metallic vesicles, has been developed. Metallovesicular systems are aggregates made of or containing lipophilic metallic complexes. In the current paper, two types of lipophilic pyridine-containing alkanol ligands are synthesized, namely, 6-(n-butyloxymethyl)-2-(hydroxymethyl)pyridine (1) and 6-(n-dodecyloxymethyl)-2-(hydroxymethyl)pyridine (2). The catalytic hydrolytic ability of the Zn(II) complexes of these ligands on p-nitrophenyl picolinate (PNPP) in vesicular solutions is investigated, and the effects of the alkyl chain length, different temperatures, and vesicles formed from different ratios and concentrations of surfactants on the catalytic hydrolysis of PNPP are discussed. Vesicles can be spontaneously formed in mixtures of the anionic surfactant sodium dodecylbenzenesulfonate (SDBS) with the cationic surfactant cetyltrimethylammonium bromide (CTAB) [13,14]. The current study aims to investigate the catalytic mechanism in terms of the ternary complex kinetic model [9]. Some kinetic and thermodynamic parameters of catalytic ternary complexes are discussed here in detail. 2. Experimental 2.1. Materials
∗ Corresponding author. Tel.: +86 010 5880 2850. E-mail address: [email protected] (F. Jiang). 0927-7757/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfa.2011.09.041
Zn(NO3 )2 ·6H2 O, KNO3 , acetonitrile, HCl, CTAB, SDBS, and tri(hydroxymethyl) aminomethane (Tris) were analytical-grade
Y. Song et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 392 (2011) 110–115
commercial products and used without further purification. PNPP was synthesized according to the literature [15]. The PNPP stock solution for the kinetics study was prepared in acetonitrile to prevent it from decomposing too rapidly. Tris–TrisH+ buffer was used in all cases to avoid the influence of chemical components from different buffers; its pH was adjusted by adding analytically pure HCl in all runs. The ionic strength was maintained at 0.1 with KNO3 . 2.2. Preparation of the ligands Ligands 1 and 2 were prepared according to published procedures [16].
H
O
N
O
Ligand 1: 6-(n-butyloxymethyl)-2-(hydroxymethyl)pyridine
H
O
N
111
88.45- and 186.40-fold in a 1:4 vesicular solution (CTAB:SDBS) and 49.25- and 95.75-fold in a 1:2 vesicular solution, respectively. This may be ascribed to the different combinations when vesicles were formed from different surfactant ratios [14]. Vesicles are differentially formed by the electrostatic and hydrophobic interactions between the anionic surfactant and the cationic surfactant [17]. From the data in Table 1, it can be seen that the reaction rate of the vesicle is one orders of magnitude larger than the micelle. This indicates vesicles can reach a higher catalytic efficiency than micelles. The kobsd of ligand 2 is larger than that of ligand 1. This indicates that the catalytic efficiency of ligand 2 is more powerful than that of ligand 1, which may be attributed to the different structures of the two ligands. The longer the alkyl chain of the ligand is, the higher the catalytic efficiency [4], which is to be rationalized by the kinetic and thermodynamic parameters in Table 4. It is shown that the value of kN of ligand 2 is larger than that of kN of ligand 1 in vesicular solution.
O
Ligand 2: 6-(n-dodecyloxymethyl)-2-(hydroxymethyl)pyridine 2.3. Preparation of the vesicles Samples were prepared by mixing stock solutions of CTAB–Tris and SDBS–Tris at desired concentrations and molar ratios to yield blue translucent solutions. The solutions were not subjected to any type of mechanical agitation except slight shaking. 2.4. Kinetic measurements Kinetic studies were carried out using UV–vis methods with a TU-1901 UV–vis spectrophotometer equipped with a thermostatic cell holder at a series of pH and different temperatures. The reactions were initiated by adding 30 L of a 0.005 mol dm−3 stock solution of PNPP into 3 mL of buffer solution containing the desired reagents using a microsyringe. The rates were followed by monitoring the release of p-nitrophenol at 400 nm. The pseudo-first-order rate constants were obtained from the spectrophotometer with a computer data processing system. Each pseudo-first-order rate constant is the average of three determinations; its average relative standard deviation is smaller than 3.0%. 3. Results and discussion 3.1. Pseudo-first-order rate constants for the hydrolysis of PNPP at 25 ◦ C Ligands 1 and 2 were hardly soluble in water, but they could be solubilized in vesicular solutions when heated. Moreover, they could not be precipitated after cooling at room temperature. Thus, the kinetics study was carried out in a vesicular solution. The pseudo-first-order rate constants (kobsd ) were obtained under excess ligand and metallic ion conditions. 3.1.1. Pseudo-first-order rate constants for the hydrolysis of PNPP in vesicular and micellar solution Table 1 shows that the pseudo-first-order rate constant (kobsd ) for the hydrolysis of PNPP in buffer solution is 2.0 × 10−4 s−1 at pH 7.50 and 25 ◦ C. The vesicular system itself showed almost no rate-enhancing effect. In the absence of metallic ions, the rate enhancement by the ligand alone was relatively small under vesicular conditions. In the presence of Zn2+ , the rate increased ca. 9.80and 11.40-fold in micellar solution. While the rate increased ca.
From the values given in Table 1, it can be seen that the catalytic hydrolysis rate in a 1:4 vesicular solution (CTAB:SDBS) with the 1.4 × 10−2 mol L−1 concentration has a higher catalytic efficiency than that with the 2.8 × 10−2 mol L−1 concentration. The phenomena may have resulted from the structural differences of the vesicles formed from the different surfactant concentrations. Thus, the changes in the microenvironment may, to some extent, have an effect on the catalytic efficiency. 3.1.2. Pseudo-first-order rate constants for the hydrolysis of PNPP by metallic complexes with different concentrations at 25 ◦ C and different pH values Table 2 indicates that the kobsd of ligand 2 is larger than that of ligand 1 at the same concentration and pH value. This may have resulted from the structural differences of the ligands. The longer the alkyl chains of the ligands are, the higher the solubility of the complexes in vesicular solution. The rate constants are also found to increase with increasing concentrations of complexes at a constant pH. The phenomena may be attributed to the increase in the collision frequency of the reactants. When the concentration of the complexes was held constant, the rate constants initially increased with increasing pH values. This may be caused by the increase in the nucleophilicity of the hydroxyl groups of the complexes, which is more favorable for substrate attack. Further increases in the pH values caused a decrease in the rate constants, which may be attributed to the hydrolysis of Zn2+ , which causes a decrease in the concentration of the metallic complexes. At pH 7.80, the highest rate constants for the hydrolysis of PNPP were obtained when the concentration of the complexes was 2.00 × 10−3 mol L−1 . 3.1.3. Pseudo-first-order rate constants for the hydrolysis of PNPP by metallic complexes with different temperatures at pH 7.50 Table 3 shows the pseudo-first-order rate constants for the hydrolysis of PNPP catalyzed by the mixed system of Zn2+ and ligand 1 or 2 in a vesicular solution at pH 7.50 and at different temperatures. As can be seen from Table 3, the altered activity observed with changes to reaction temperature due to changes in the pH of the vesicular solution. Because the pseudo-first-order rate constants for the hydrolysis of PNPP are not observed with great changes at different temperatures in buffer. It can be seen that the kobsd of ligand 2 is larger than that of ligand 1 at the same temperature and concentration, which may be caused by the structural differences of the ligands. The rate constants were also seen
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Table 1 Pseudo-first-order rate constants (103 × kobsd /s−1 ) for the hydrolysis of PNPP in vesicular and micellar systems at 25 ◦ C and pH 7.50. Systems
Buffer (1a)
Zn2+
(1a)
Buffer Micelle 1 + micelle 2 + micelle Vesicle 1 + vesicle 2 + vesicle
0.20 0.57 0.46 0.80 0.26 0.40 0.53
2.42 2.81 1.96 2.28 5.68 18.25 31.63
Buffer (1b)
Zn2+
(1b)
0.20 0.57 0.46 0.80 0.24 0.36 0.80
2.42 2.81 1.96 2.28 5.35 9.85 19.15
Buffer (2c)
Zn2+
(2c)
0.20 0.57 0.46 0.80 0.29 0.67 0.80
2.42 2.81 1.96 2.28 6.72 16.68 31.19
Note. In 0.01 mol L−1 Tris–HCl buffer; = 0.1 mol L−1 (KCl). (1) [CTAB] = [SDBS] = 0.014 mol L−1 : (a) CTAB:SDBS = 1:4; (b) CTAB:SDBS = 1:2. (2) [CTAB] = [SDBS] = 0.028 mol L−1 : (c) CTAB:SDBS = 1:4. Micelle = [CTAB] = 0.01 mol L−1 ; [PNPP] = 5 × 10−5 mol L−1 ; [ligand] = [Zn2+ ] = 1 × 10−3 mol L−1 .
Table 2 Pseudo-first-order rate constants (103 × kobsd /s−1 ) for the hydrolysis of PNPP by metallic complexes with different concentrations at 25 ◦ C and different pH values. C (mol L−1 )
0.50 × 10−3 0.75 × 10−3 1.00 × 10−3 1.50 × 10−3 2.00 × 10−3
pH 7.20
7.50
7.80
8.00
8.20
4.42(11.15) 8.04(18.46) 12.02(29.37) 22.64(40.37) 36.96(52.58)
7.41(14.23) 9.80(23.34) 18.25(31.63) 26.95(63.52) 39.49(68.92)
8.52(17.87) 10.27(24.32) 19.74(34.30) 23.28(66.09) 40.48(113.77)
8.29(17.66) 9.09(19.84) 12.37(30.47) 22.55(54.87) 38.62(87.79)
7.43(14.53) 7.98(19.07) 11.01(22.04) 20.27(40.41) 27.76(77.28)
Note. In 0.01 mol L−1 Tris–HCl buffer; = 0.1 mol L−1 (KCl). [CTAB] = [SDBS] = 0.014 mol L−1 ; CTAB:SDBS = 1:4. [PNPP] = 5 × 10−5 mol L−1 ; the rate constants by ligand 2 are listed in brackets.
Table 3 Pseudo-first-order rate constants (103 × kobsd /s−1 ) for the hydrolysis of PNPP by metallic complexes at different temperatures and pH 7.50. pH
7.50
C (mol L−1 )
Buffer 0.50 × 10−3 0.75 × 10−3 1.00 × 10−3 1.50 × 10−3 2.00 × 10−3
T (◦ C) 25
37
50
0.20 7.41(14.23) 9.80(23.34) 18.25(31.63) 26.95(63.52) 39.49(68.92)
0.21 8.84(18.52) 18.97(38.91) 29.97(72.01) 54.21(110.04) 81.07(144.45)
0.23 12.45(20.26) 27.43(42.05) 36.08(81.03) 85.31(163.12) 102.99(192.61)
Note. In 0.01 mol L−1 Tris–HCl buffer; = 0.1 mol L−1 (KCl). [CTAB] = [SDBS] = 0.014 mol L−1 ; CTAB:SDBS = 1:4. [PNPP] = 5 × 10−5 mol L−1 ; the rate constants by ligand 2 are listed in brackets.
to increase with increasing temperature at a constant concentration. The activation energy (Ea ) and pre-exponential factor (A) of the metallovesicular catalytic reaction can be estimated. The activation energy (Ea ) and pre-exponential factor (A) for the mixed system with Zn2+ and ligands 1 and 2 are 27.79 kJ/mol and 1668 s−1 , and 24.67 kJ/mol and 1670 s−1 , respectively. The higher the activation energy (Ea ) is, the slower is the reaction rate. The activation energy (Ea ) of ligand 1 is higher than that of ligand 2. Thus, the kobsd followed the order, ligand 2 > ligand 1.
the hydrolytic reaction in vesicular systems and obtain a better understanding of the catalytic mechanism [22]. Equilibrium exists between the ligands (L), the metallic ions (M), and the substrates (S). Based on the phase-separation model [9], metallovesicular catalytic reactions can be assumed to take place simultaneously in the
0.018 0.016
The kinetic version of the Job plots was utilized to determine the chelating stoichiometry of the kinetically active species. The pseudo-first-order rate constants (kobsd ) are plotted as a function of the molar fraction (r) of ligands or metallic ions (Fig. 1), keeping the total concentration constant [18–20]. As can be seen, the r-values for ligands 1 and 2 corresponding to the maximum kobsd are approximately 0.50, suggesting that the 1:1 (metal:ligand) complexes could be the active species.
0.014
kobsd /s-1
3.2. Determination of the chelating ratio of metallic ions to ligands for the active species of the reactions
0.012 0.01 0.008 0.006 0.004 0.002 0 0
3.3. The determination of kinetic and thermodynamic parameters The kinetic effects of the vesicles on the pseudo-first-order rate constant can be analyzed based on the pseudophase model [21]. The kinetic model of the ternary complex was applied to study
0.2
0.4
0.6
0.8
1
r=[Zn]/([Zn]+[L]) Fig. 1. Job plots for ligands 1 (䊉), 2 (), and Zn2+ ion complexes as measured by the rates of the hydrolysis of PNPP at pH 7.50 and 25 ◦ C in a vesicular system (CTAB:SDBS = 1:4); [CTAB] = [SDBS] = 0.014 mol L−1 , [L] + [Zn2+ ] = 1 × 10−3 mol L−1 , [PNPP] = 5 × 10−5 mol L−1 .
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113
N
N
Ka
O(H)
RO
Zn2+
Zn2+
O
N
KN
O
RO
P O
N
NO2
NO2 O
O
[T-]
[TH] Scheme 1.
bulk phase and the metallovesicular phase to form the product (P). KM
nL + mM Mm Ln KM =
KM
mM + S−→P
kL
nL + S−→P
[Mm Ln ]
(1)
[M]m [L]n
that the complex formed from Zn2+ and the ligands are considerably stable. The association constant (KS ) of the ternary complex formed by the substrate with the metallic complex is small, indicating that the substrates with the metallic complexes are connected by weak chemical bonds and react on intramolecular nucleophilic reactions. 3.4. The mechanism of the reaction
KS
kN
Mm Ln + SMm Ln S−→P KS =
k0
S−→P
[Mm Ln S] [Mm Ln ][S]
(2)
where KM is the association constant among m metallic ions and n ligands, KS is the association constant between a binary complex (Mm Ln ) and a substrate, kN and k0 are the pseudo-first-order rate constants for the product formation in the vesicular phase and in the bulk phase, respectively, k0 is the rate constant of the buffer, and kL and kM are the second-order rate constants of the ligand and the metallic ion alone. According to the rate law, the rate equation can be written as:
v = kobsd [S]t = kN [Mm Ln S] + k0 [S]
(3)
[S]t = [S] + [Mm Ln S]
(4)
where [S]t and [Mm Ln S] are the concentrations of the substrate and ternary complex, respectively, when the reaction proceeds to the moment t. k0 = k0 + kM [M]T + kL [L]T , where [L]T and [M]T are the total concentrations of the ligand and metallic ions, respectively. [Mm Ln ] is the concentration of m metallic ions or n ligands in the vesicular phase. Combining Eqs. (2)–(4) and rearranging give Eq. (5): kobsd =
k0 + kN KS [Mm Ln ] 1 + KS [Mm Ln ]
(5)
Table 4 shows the kN , KS , and KM values for the hydrolysis of PNPP catalyzed by the mixed system of Zn2+ and ligand 1 or 2 in vesicular solution at 25 ◦ C. As can be seen, at low pH values (<7.80), kN increased with increasing pH until a maximum point was reached. Further increases in the pH value caused a decrease in the rate constants. This may be ascribed to the hydrolysis of Zn2+ with the increasing pH value and the reduction in the concentration of complexes, resulting in a decrease in the reaction rate. In addition, the association constant (KM ) of Zn2+ with the ligand is high, indicating
3.4.1. pH-rate profile The pH value plays an important role in the hydrolytic reaction. Thus, the effect of the changes of pH values on the hydrolytic reaction was investigated. As reported in metallomicelle solution previously, many hydrolytic processes in enzymes involve metal ions that are assumed to activate a water molecule acting as a nucleophilic group in a ternary complex, but the hydroxyl group of the ligand acting as a nucleophilic group in our system, as shown in Scheme 1. According to rate law, the rate equation can be expressed as: r = kN [Mm Ln S] = kN [T− ]
(6)
From Table 4, it can be seen that it was obviously shown that kN is pH-dependent and kN is related to the acid dissociation constant (Ka ) of the hydroxyl group of the ternary complex in the reaction system. [Mm Ln S] = [TH] + [T],
Ka =
[T− ][H+ ] [TH]
(7)
where TH is the undissociated complex, T− is the dissociated complex anion assumed to be the active species in vesicular phase and kN is the first-order rate constant which is pH-independent. We have 1 1 1 = + [H+ ] kN kN kN ka
(8)
Fig. 2 shows the pH-rate profile of the hydrolysis of PNPP catalyzed by the metallic complexes in vesicular solution. The apparent rate constants for the hydrolysis of PNPP were at first seen to increase with increasing pH value, and then decrease with further increases in the pH. The inflection points of the curve are at about 7.38 and 7.52, respectively, which correspond to the pKa of Zn2+ in vesicular solution. The pKa values are all lower than those of corresponding metal hydrates [23], indicating that the Zn2+ ion induces the dissociation of the ligands’ hydroxyl groups to form the active species in
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Table 4 kN , KS and KM values for the hydrolysis of PNPP in vesicular (CTAB:SDBS = 1:4) systems at 25 ◦ C. L1
pH
kN (s−1 )
10−3 KS (L mol−1 )
10−5 KM (L mol−1 )
7.20 7.50 7.80 8.00 8.20
0.06 0.10 0.19 0.03 0.02
2.93 0.75 0.32 1.90 2.57
2.42 5.27 3.73 52.80 80.40
L2
pH
kN (s−1 )
10−3 KS (L mol−1 )
10−5 KM (L mol−1 )
7.20 7.50 7.80 8.00 8.20
0.10 0.17 0.29 0.07 0.03
3.05 2.98 0.38 2.84 4.79
5.15 4.04 4.00 46.25 60.51
Note. In 0.01 mol L−1 Tris–HCl buffer; = 0.1 (KCl). [CTAB] = [SDBS] = 0.014 mol L−1 ; [PNPP] = 5 × 10−5 mol L−1 .
N
N
-PNPO(H)
O(H)
RO Zn2+
RO Zn2+
OH(H) O
N
2+
Zn
O
N
O
N
NO2
O(H)
RO
O
O
N O-
PNPP Scheme 2. The proposed mechanism of the catalysis of PNPP hydrolysis by a ternary complex in a vesicular system.
the reaction system, increasing the rates of hydrolysis of PNPP by an effective nucleophilic attack on the substrate.
3.4.2. The mechanism of the catalytic reaction The mechanism of the catalytic reaction can be deduced based on the experimental data and relevant literature [24], as represented by the structure in Scheme 2. The mechanism involves: (i) the formation of a ternary complex (ligand/metallic ion/substrate); (ii) the pseudo-intramolecular attack of the activated hydroxyl on the carboxyl group of the ester, resulting in its acylation; and (iii) the metallic ion-mediated hydrolysis of the acylated intermediate. The product is then formed and the catalytic reaction ends.
45 40
103*kobsd
35 30 25 20 15 10
7.2
7.4
7.6
7.8
8.0
8.2
pH Fig. 2. Profiles of kobsd vs. pH for the hydrolysis of PNPP catalyzed by ligands 1 (䊉), 2 (), and Zn2+ ion complexes in a vesicular system (CTAB:SDBS = 1:4); [CTAB] = [SDBS] = 0.014 mol L−1 , [L] = [Zn2+ ] = 1 × 10−3 mol L−1 , [PNPP] = 5 × 10−5 mol L−1 , T = 25 ◦ C.
4. Conclusions The effects of the alkyl chain length, different temperatures, and vesicles formed from different ratios and concentrations of surfactants on the hydrolysis of PNPP in a vesicular solution were investigated. Results showed that the metallic vesicular system not only mimics the active center of the enzymatic catalysis, but also effectively simulates the hydrophobic microenvironment. Some experimental results should also be noted:
(1) On the basis of a kinetic version of a Job plot analysis, a 1:1 ligand:Zn2+ stoichiometry was found to produce the most active species. (2) The reaction rate of the vesicle is one order of magnitude larger than the micelle. The catalytic hydrolysis rate of the vesicles formed from 1:4 CTAB:SDBS is larger than that of the system formed from 1:2 CTAB:SDBS. When 1:4 CTAB:SDBS was used, the catalytic hydrolysis rate of the system with 1.4 × 10−2 mol L−1 concentration showed a higher catalytic efficiency than with 2.8 × 10−2 mol L−1 concentration. This indicates that the changes in microenvironments can affect the catalytic reaction. (3) The pseudo-first-order rate constants for the hydrolysis of PNPP increased with increasing temperature at a constant concentration. The activation energy (Ea ) of ligand 1 is higher than that of ligand 2. Thus, the kobsd followed the order, ligand 2 > ligand 1. (4) The pseudo-first-order rate constants for the hydrolysis of PNPP increased with increasing concentrations at a constant pH. When the concentration of the metallic complexes is the same, the pseudo-first-order rate constants increased with increasing pH value until a maximum point is reached. Further increases in the pH value caused a decrease in the rate constants. (5) The association constant (KM ) of Zn2+ with the ligand is high, whereas the association constant (KS ) of the ternary complex
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formed by the substrate with the metallic complex is low, indicating that the substrates formed by the metallic complex are connected by weak chemical bonds and react on intramolecular nucleophilic reactions. Acknowledgements The authors would like to thank the financial supports from the Chinese National Science Foundation (No. 21175014, 20975016). References [1] S.Q. Cheng, X.C. Zeng, X.G. Meng, X.Q. Yu, Metallomicellar catalysis hydrolysis of p-nitrophenyl picolinate catalyzed by copper(II), nickel(II), and zinc(II) complexes of long alkyl pyridine ligands in micellar solution, Journal of Colloid and Interface Science 224 (2000) 333–337. [2] R. Jairam, M.L. Lau, J. Adorante, P.G. Potvin, Ester hydrolysis with 2,6-di(pyrazol3-yl) pyridines and their CoII complexes in homogeneous and micellar media, Journal of Inorganic Biochemistry 84 (2001) 113–118. [3] Y. Xiang, B.Y. Jiang, X.C. Zeng, J.Q. Xie, Metallomicellar catalysis: catalytic cleavage of p-nitrophenyl picolinate by Cu2+ complex of 4-chloride-2,6bis(N-hydroxyethylaminomethyl)-benzophenol in micellar solution, Journal of Colloid and Interface Science 247 (2002) 366–371. [4] F.B. Jiang, L.Y. Huang, X.G. Meng, J. Du, X.Q. Yu, Y.F. Zhao, X.C. Zeng, Metallomicellar catalysis: hydrolysis of phosphate monoester and phosphodiester by Cu(II), Zn(II) complexes of pyridyl ligands in CTAB micellar solution, Journal of Colloid and Interface Science 303 (2006) 236–242. [5] L.G. Qiu, A.J. Xie, Y.H. Shen, Metallomicellar catalysis: hydrolysis of pnitrophenyl picolinate catalyzed by Cu(II) complexes of triazole-based ligands in cationic Gemini surfactant micelles, Journal of Molecular Catalysis A: Chemical 244 (2006) 58–63. [6] A. Polyzos, A.B. Hughes, J.R. Christie, Catalysis of aryl ester hydrolysis in the presence of metallomicelles containing a copper(II) diethylenetriamine derivative, Langmuir 23 (2007) 1872–1879. [7] J. Zhang, X.G. Meng, X.C. Zeng, X.Q. Yu, Metallomicellar supramolecular systems and their applications in catalytic reactions, Coordination Chemistry Reviews 253 (2009) 2166–2177. [8] F. March, P. Scrimin, P. Tecilla, U. Tonellato, Amphiphilic metalloaggregates: catalysis, transport, and sensing, Coordination Chemistry Reviews 253 (2009) 2150–2165. [9] S. Bhattacharya, N. Kumari, Metallomicelles as potent catalysts for the ester hydrolysis reactions in water, Coordination Chemistry Reviews 253 (2009) 2133–2149.
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[10] F.B. Bruno, E.F. Silva, U.O. Marques, Unusual vesicle-micelle transitions in a saltfree catanionic surfactant: temperature and concentration effects, Langmuir 24 (2008) 10746–10754. [11] Z.G. Zhao, Miceller Catalysis and Miroemulsion Catalysis, 2006, Beijing. [12] J.H. Fendler, W.L. Hinze, Reactivity control in micelles and surfactant vesicles. Kinetics and mechanism of base-catalyzed hydrolysis of 5,5 -dithiobis(2nitrobenzoic acid) in water, hexadecyltrimethylammonium bromide micelles, and dioctadecyldimethylammonium chloride surfactant vesicles, Journal of the American Chemical Society 103 (1981) 5439–5441. [13] E.W. Kaler, A.K. Murthy, B.E. Rodrigueze, J.A. Zasadzinski, Spontaneous vesicle formation in aqueous mixtures of single-tailed surfactants, Science 245 (1989) 1371. [14] N. Yu, D. Sun, Z.R. Han, C.L. Liu, Y. Li, L.L. Cui, Spontaneous formation and polymerization of CTAB/SDBS vesicles, Acta Chimica Sinica 66 (2008) 315–320. [15] D.S. Sigman, C.T. Jorgensen, Models for metalloenzymes. The zinc(II)-catalyzed transesterification of N-(p-hydroxyethyl) ethylenediamine by p-nitrophenyl picolinate, Journal of the American Chemical Society 94 (1972) 1724. [16] F.B. Jiang, B.Y. Jiang, Y.S. Cao, X.G. Meng, X.Q. Yu, X.C. Zeng, Hydrolysis of phosphate monoester by Cu(II), Zn(II), Ni(II) and Co(II) complexes of pyridyl ligands in CTAB micellar solution, Colloids and Surfaces A: Physicochemical and Engineering Aspects 254 (2005) 91–97. [17] J. Yuan, X.T. Bai, M.W. Zhao, L.Q. Zheng, C12 mimBr ionic liquid/SDS vesicle formation and use as template for the synthesis of hollow silica spheres, Langmuir 10 (2010) 1021–1221. [18] T. Fejita, Y. Inaba, K. Dgino, W. Tagaki, Hydrolytic metalloenzyme models effects of AOT reverse micelles on the hydrolysis of p-nitrophenyl picolinate catalyzed by Zn2+ and Cu2+ complexes of N-alkyl-2-hydroxymethylimidazoles, Bulletin of the Chemical Society of Japan 61 (1988) 1661. [19] F. Mancin, P. Tecilla, U. Tonellato, Metallomicelles made of Ni(II) and Zn(II) complexes of 2-pyridinealdoxime-based ligands as catalyst of the cleavage of carboxylic acid esters, Langmuir 16 (2000) 227–233. [20] S. Bhattacharya, K. Snehalatha, V.P. Kumar, Synthesis of new Cu(II)-chelating ligand amphiphiles and their esterolytic properties in cationic micelles, Journal of Organic Chemistry 68 (2003) 2741–2747. [21] F.M. Menger, C.E. Portnoy, On the chemistry of reactions proceeding inside molecular aggregates, Journal of the American Chemical Society 89 (1967) 4698. [22] F. Liu, Z.C. Liu, Q.X. Xiang, Y.Q. Zhang, D.H. Wan, Kinetic research on the hydrolysis of p-nitrophenyl picolinate catalysed by Cu2+ complexes of Schiff bases macrocyclic polyamine, Journal of Sichuan Normal University (Natural Science) 31 (2008) 748–753. [23] K. Wang, Microelement of Life Science, 1991, Beijing. [24] P. Scrimin, P. Tecilla, U. Tonellato, Metallomicelles as catalysts of the hydrolysis of carboxylic and phosphoric acid esters, Journal of Organic Chemistry 56 (1991) 161–166.
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Colloids and Surfaces A: Physicochemical and Engineering Aspects journal homepage: www.elsevier.com/locate/colsurfa
Metallovesicular catalytic hydrolysis of p-nitrophenyl picolinate catalyzed by zinc(II) complexes of pyridyl ligands in vesicular solution Yongwei Song a,b , Xinting Han a , Xiaoli Guo a , Qing Zeng c , Fubin Jiang a,∗ a b c
Department of Chemistry, Beijing Normal University, Beijing 100875, People’s Republic of China Ordos School attached to Beijing Normal University, Ordos, The Inner Mongolia Autonomous 017000, People’s Republic of China School of Chinese Pharmacy, Beijing University of Chinese Medicine, Beijing 100102, People’s Republic of China
a r t i c l e
i n f o
Article history: Received 20 June 2011 Received in revised form 25 September 2011 Accepted 30 September 2011 Available online 8 October 2011 Keywords: Enzyme Metallovesicular Ternary complex Kinetics Catalytic hydrolysis
a b s t r a c t The hydrolysis of p-nitrophenyl picolinate (PNPP) catalyzed by the Zn(II) complexes of 6-(nbutyloxymethyl)-2-(hydroxymethyl)pyridine and 6-(n-dodecyloxymethyl)-2-(hydroxymethyl)pyridine was kinetically investigated by observation of the rates of release of p-nitrophenol in a vesicular solution at different pH values and temperatures. A 1:1 ligand:Zn2+ stoichiometry was found to produce the most active species based on a kinetic version of the Job plot analysis. Experimental results also showed that the complex formed from ligand 2 and Zn2+ exhibited a more remarkable rate acceleration effect on the hydrolysis of PNPP in vesicular solution than that formed from ligand 1 and Zn2+ . © 2011 Elsevier B.V. All rights reserved.
1. Introduction Metalloenzymes play an important role in the catalytic hydrolysis of carboxylic acid esters. A number of hydrolytically active metalloenzymes have been found in nature. Metallomicelles made up of ligand surfactants and transition metallic ions have been extensively reported as artificial hydrolytic metalloenzymes [1–9]. However, increasing the catalytic efficiency and achieving the activity of natural enzymes remain major challenges to researchers. If the catalytic efficiency in such systems is increased, we can not only mimic the enzymatic active centers of natural enzymes, but also simulate their hydrophobic microenvironments [9]. Vesicles are bilayer shells, with each shell enclosing a pool of solvent in the interior [10]. They have been considered as models of biological membranes and are similar to micelles in that they can concentrate the reactants and accelerate chemical reactions. Chemical reactions can be catalyzed effectively if the reactants can be concentrated on the interface between the vesicles’ bilayer and water layer [11]. Vesicles can adsorb more reactants compared with micelles because they have a somewhat larger specific surface area and a faster aggregation velocity than micelles. Moreover, vesicles can reach a higher catalytic efficiency than micelles [12]. Thus, extensive investigations on vesicles have been carried out in recent
years. However, few studies have focused on catalytic reactions in vesicular solutions. In our laboratory, an efficient and novel model in enzymatic catalysis, namely, metallic vesicles, has been developed. Metallovesicular systems are aggregates made of or containing lipophilic metallic complexes. In the current paper, two types of lipophilic pyridine-containing alkanol ligands are synthesized, namely, 6-(n-butyloxymethyl)-2-(hydroxymethyl)pyridine (1) and 6-(n-dodecyloxymethyl)-2-(hydroxymethyl)pyridine (2). The catalytic hydrolytic ability of the Zn(II) complexes of these ligands on p-nitrophenyl picolinate (PNPP) in vesicular solutions is investigated, and the effects of the alkyl chain length, different temperatures, and vesicles formed from different ratios and concentrations of surfactants on the catalytic hydrolysis of PNPP are discussed. Vesicles can be spontaneously formed in mixtures of the anionic surfactant sodium dodecylbenzenesulfonate (SDBS) with the cationic surfactant cetyltrimethylammonium bromide (CTAB) [13,14]. The current study aims to investigate the catalytic mechanism in terms of the ternary complex kinetic model [9]. Some kinetic and thermodynamic parameters of catalytic ternary complexes are discussed here in detail. 2. Experimental 2.1. Materials
∗ Corresponding author. Tel.: +86 010 5880 2850. E-mail address: [email protected] (F. Jiang). 0927-7757/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfa.2011.09.041
Zn(NO3 )2 ·6H2 O, KNO3 , acetonitrile, HCl, CTAB, SDBS, and tri(hydroxymethyl) aminomethane (Tris) were analytical-grade
Y. Song et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 392 (2011) 110–115
commercial products and used without further purification. PNPP was synthesized according to the literature [15]. The PNPP stock solution for the kinetics study was prepared in acetonitrile to prevent it from decomposing too rapidly. Tris–TrisH+ buffer was used in all cases to avoid the influence of chemical components from different buffers; its pH was adjusted by adding analytically pure HCl in all runs. The ionic strength was maintained at 0.1 with KNO3 . 2.2. Preparation of the ligands Ligands 1 and 2 were prepared according to published procedures [16].
H
O
N
O
Ligand 1: 6-(n-butyloxymethyl)-2-(hydroxymethyl)pyridine
H
O
N
111
88.45- and 186.40-fold in a 1:4 vesicular solution (CTAB:SDBS) and 49.25- and 95.75-fold in a 1:2 vesicular solution, respectively. This may be ascribed to the different combinations when vesicles were formed from different surfactant ratios [14]. Vesicles are differentially formed by the electrostatic and hydrophobic interactions between the anionic surfactant and the cationic surfactant [17]. From the data in Table 1, it can be seen that the reaction rate of the vesicle is one orders of magnitude larger than the micelle. This indicates vesicles can reach a higher catalytic efficiency than micelles. The kobsd of ligand 2 is larger than that of ligand 1. This indicates that the catalytic efficiency of ligand 2 is more powerful than that of ligand 1, which may be attributed to the different structures of the two ligands. The longer the alkyl chain of the ligand is, the higher the catalytic efficiency [4], which is to be rationalized by the kinetic and thermodynamic parameters in Table 4. It is shown that the value of kN of ligand 2 is larger than that of kN of ligand 1 in vesicular solution.
O
Ligand 2: 6-(n-dodecyloxymethyl)-2-(hydroxymethyl)pyridine 2.3. Preparation of the vesicles Samples were prepared by mixing stock solutions of CTAB–Tris and SDBS–Tris at desired concentrations and molar ratios to yield blue translucent solutions. The solutions were not subjected to any type of mechanical agitation except slight shaking. 2.4. Kinetic measurements Kinetic studies were carried out using UV–vis methods with a TU-1901 UV–vis spectrophotometer equipped with a thermostatic cell holder at a series of pH and different temperatures. The reactions were initiated by adding 30 L of a 0.005 mol dm−3 stock solution of PNPP into 3 mL of buffer solution containing the desired reagents using a microsyringe. The rates were followed by monitoring the release of p-nitrophenol at 400 nm. The pseudo-first-order rate constants were obtained from the spectrophotometer with a computer data processing system. Each pseudo-first-order rate constant is the average of three determinations; its average relative standard deviation is smaller than 3.0%. 3. Results and discussion 3.1. Pseudo-first-order rate constants for the hydrolysis of PNPP at 25 ◦ C Ligands 1 and 2 were hardly soluble in water, but they could be solubilized in vesicular solutions when heated. Moreover, they could not be precipitated after cooling at room temperature. Thus, the kinetics study was carried out in a vesicular solution. The pseudo-first-order rate constants (kobsd ) were obtained under excess ligand and metallic ion conditions. 3.1.1. Pseudo-first-order rate constants for the hydrolysis of PNPP in vesicular and micellar solution Table 1 shows that the pseudo-first-order rate constant (kobsd ) for the hydrolysis of PNPP in buffer solution is 2.0 × 10−4 s−1 at pH 7.50 and 25 ◦ C. The vesicular system itself showed almost no rate-enhancing effect. In the absence of metallic ions, the rate enhancement by the ligand alone was relatively small under vesicular conditions. In the presence of Zn2+ , the rate increased ca. 9.80and 11.40-fold in micellar solution. While the rate increased ca.
From the values given in Table 1, it can be seen that the catalytic hydrolysis rate in a 1:4 vesicular solution (CTAB:SDBS) with the 1.4 × 10−2 mol L−1 concentration has a higher catalytic efficiency than that with the 2.8 × 10−2 mol L−1 concentration. The phenomena may have resulted from the structural differences of the vesicles formed from the different surfactant concentrations. Thus, the changes in the microenvironment may, to some extent, have an effect on the catalytic efficiency. 3.1.2. Pseudo-first-order rate constants for the hydrolysis of PNPP by metallic complexes with different concentrations at 25 ◦ C and different pH values Table 2 indicates that the kobsd of ligand 2 is larger than that of ligand 1 at the same concentration and pH value. This may have resulted from the structural differences of the ligands. The longer the alkyl chains of the ligands are, the higher the solubility of the complexes in vesicular solution. The rate constants are also found to increase with increasing concentrations of complexes at a constant pH. The phenomena may be attributed to the increase in the collision frequency of the reactants. When the concentration of the complexes was held constant, the rate constants initially increased with increasing pH values. This may be caused by the increase in the nucleophilicity of the hydroxyl groups of the complexes, which is more favorable for substrate attack. Further increases in the pH values caused a decrease in the rate constants, which may be attributed to the hydrolysis of Zn2+ , which causes a decrease in the concentration of the metallic complexes. At pH 7.80, the highest rate constants for the hydrolysis of PNPP were obtained when the concentration of the complexes was 2.00 × 10−3 mol L−1 . 3.1.3. Pseudo-first-order rate constants for the hydrolysis of PNPP by metallic complexes with different temperatures at pH 7.50 Table 3 shows the pseudo-first-order rate constants for the hydrolysis of PNPP catalyzed by the mixed system of Zn2+ and ligand 1 or 2 in a vesicular solution at pH 7.50 and at different temperatures. As can be seen from Table 3, the altered activity observed with changes to reaction temperature due to changes in the pH of the vesicular solution. Because the pseudo-first-order rate constants for the hydrolysis of PNPP are not observed with great changes at different temperatures in buffer. It can be seen that the kobsd of ligand 2 is larger than that of ligand 1 at the same temperature and concentration, which may be caused by the structural differences of the ligands. The rate constants were also seen
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Table 1 Pseudo-first-order rate constants (103 × kobsd /s−1 ) for the hydrolysis of PNPP in vesicular and micellar systems at 25 ◦ C and pH 7.50. Systems
Buffer (1a)
Zn2+
(1a)
Buffer Micelle 1 + micelle 2 + micelle Vesicle 1 + vesicle 2 + vesicle
0.20 0.57 0.46 0.80 0.26 0.40 0.53
2.42 2.81 1.96 2.28 5.68 18.25 31.63
Buffer (1b)
Zn2+
(1b)
0.20 0.57 0.46 0.80 0.24 0.36 0.80
2.42 2.81 1.96 2.28 5.35 9.85 19.15
Buffer (2c)
Zn2+
(2c)
0.20 0.57 0.46 0.80 0.29 0.67 0.80
2.42 2.81 1.96 2.28 6.72 16.68 31.19
Note. In 0.01 mol L−1 Tris–HCl buffer; = 0.1 mol L−1 (KCl). (1) [CTAB] = [SDBS] = 0.014 mol L−1 : (a) CTAB:SDBS = 1:4; (b) CTAB:SDBS = 1:2. (2) [CTAB] = [SDBS] = 0.028 mol L−1 : (c) CTAB:SDBS = 1:4. Micelle = [CTAB] = 0.01 mol L−1 ; [PNPP] = 5 × 10−5 mol L−1 ; [ligand] = [Zn2+ ] = 1 × 10−3 mol L−1 .
Table 2 Pseudo-first-order rate constants (103 × kobsd /s−1 ) for the hydrolysis of PNPP by metallic complexes with different concentrations at 25 ◦ C and different pH values. C (mol L−1 )
0.50 × 10−3 0.75 × 10−3 1.00 × 10−3 1.50 × 10−3 2.00 × 10−3
pH 7.20
7.50
7.80
8.00
8.20
4.42(11.15) 8.04(18.46) 12.02(29.37) 22.64(40.37) 36.96(52.58)
7.41(14.23) 9.80(23.34) 18.25(31.63) 26.95(63.52) 39.49(68.92)
8.52(17.87) 10.27(24.32) 19.74(34.30) 23.28(66.09) 40.48(113.77)
8.29(17.66) 9.09(19.84) 12.37(30.47) 22.55(54.87) 38.62(87.79)
7.43(14.53) 7.98(19.07) 11.01(22.04) 20.27(40.41) 27.76(77.28)
Note. In 0.01 mol L−1 Tris–HCl buffer; = 0.1 mol L−1 (KCl). [CTAB] = [SDBS] = 0.014 mol L−1 ; CTAB:SDBS = 1:4. [PNPP] = 5 × 10−5 mol L−1 ; the rate constants by ligand 2 are listed in brackets.
Table 3 Pseudo-first-order rate constants (103 × kobsd /s−1 ) for the hydrolysis of PNPP by metallic complexes at different temperatures and pH 7.50. pH
7.50
C (mol L−1 )
Buffer 0.50 × 10−3 0.75 × 10−3 1.00 × 10−3 1.50 × 10−3 2.00 × 10−3
T (◦ C) 25
37
50
0.20 7.41(14.23) 9.80(23.34) 18.25(31.63) 26.95(63.52) 39.49(68.92)
0.21 8.84(18.52) 18.97(38.91) 29.97(72.01) 54.21(110.04) 81.07(144.45)
0.23 12.45(20.26) 27.43(42.05) 36.08(81.03) 85.31(163.12) 102.99(192.61)
Note. In 0.01 mol L−1 Tris–HCl buffer; = 0.1 mol L−1 (KCl). [CTAB] = [SDBS] = 0.014 mol L−1 ; CTAB:SDBS = 1:4. [PNPP] = 5 × 10−5 mol L−1 ; the rate constants by ligand 2 are listed in brackets.
to increase with increasing temperature at a constant concentration. The activation energy (Ea ) and pre-exponential factor (A) of the metallovesicular catalytic reaction can be estimated. The activation energy (Ea ) and pre-exponential factor (A) for the mixed system with Zn2+ and ligands 1 and 2 are 27.79 kJ/mol and 1668 s−1 , and 24.67 kJ/mol and 1670 s−1 , respectively. The higher the activation energy (Ea ) is, the slower is the reaction rate. The activation energy (Ea ) of ligand 1 is higher than that of ligand 2. Thus, the kobsd followed the order, ligand 2 > ligand 1.
the hydrolytic reaction in vesicular systems and obtain a better understanding of the catalytic mechanism [22]. Equilibrium exists between the ligands (L), the metallic ions (M), and the substrates (S). Based on the phase-separation model [9], metallovesicular catalytic reactions can be assumed to take place simultaneously in the
0.018 0.016
The kinetic version of the Job plots was utilized to determine the chelating stoichiometry of the kinetically active species. The pseudo-first-order rate constants (kobsd ) are plotted as a function of the molar fraction (r) of ligands or metallic ions (Fig. 1), keeping the total concentration constant [18–20]. As can be seen, the r-values for ligands 1 and 2 corresponding to the maximum kobsd are approximately 0.50, suggesting that the 1:1 (metal:ligand) complexes could be the active species.
0.014
kobsd /s-1
3.2. Determination of the chelating ratio of metallic ions to ligands for the active species of the reactions
0.012 0.01 0.008 0.006 0.004 0.002 0 0
3.3. The determination of kinetic and thermodynamic parameters The kinetic effects of the vesicles on the pseudo-first-order rate constant can be analyzed based on the pseudophase model [21]. The kinetic model of the ternary complex was applied to study
0.2
0.4
0.6
0.8
1
r=[Zn]/([Zn]+[L]) Fig. 1. Job plots for ligands 1 (䊉), 2 (), and Zn2+ ion complexes as measured by the rates of the hydrolysis of PNPP at pH 7.50 and 25 ◦ C in a vesicular system (CTAB:SDBS = 1:4); [CTAB] = [SDBS] = 0.014 mol L−1 , [L] + [Zn2+ ] = 1 × 10−3 mol L−1 , [PNPP] = 5 × 10−5 mol L−1 .
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113
N
N
Ka
O(H)
RO
Zn2+
Zn2+
O
N
KN
O
RO
P O
N
NO2
NO2 O
O
[T-]
[TH] Scheme 1.
bulk phase and the metallovesicular phase to form the product (P). KM
nL + mM Mm Ln KM =
KM
mM + S−→P
kL
nL + S−→P
[Mm Ln ]
(1)
[M]m [L]n
that the complex formed from Zn2+ and the ligands are considerably stable. The association constant (KS ) of the ternary complex formed by the substrate with the metallic complex is small, indicating that the substrates with the metallic complexes are connected by weak chemical bonds and react on intramolecular nucleophilic reactions. 3.4. The mechanism of the reaction
KS
kN
Mm Ln + SMm Ln S−→P KS =
k0
S−→P
[Mm Ln S] [Mm Ln ][S]
(2)
where KM is the association constant among m metallic ions and n ligands, KS is the association constant between a binary complex (Mm Ln ) and a substrate, kN and k0 are the pseudo-first-order rate constants for the product formation in the vesicular phase and in the bulk phase, respectively, k0 is the rate constant of the buffer, and kL and kM are the second-order rate constants of the ligand and the metallic ion alone. According to the rate law, the rate equation can be written as:
v = kobsd [S]t = kN [Mm Ln S] + k0 [S]
(3)
[S]t = [S] + [Mm Ln S]
(4)
where [S]t and [Mm Ln S] are the concentrations of the substrate and ternary complex, respectively, when the reaction proceeds to the moment t. k0 = k0 + kM [M]T + kL [L]T , where [L]T and [M]T are the total concentrations of the ligand and metallic ions, respectively. [Mm Ln ] is the concentration of m metallic ions or n ligands in the vesicular phase. Combining Eqs. (2)–(4) and rearranging give Eq. (5): kobsd =
k0 + kN KS [Mm Ln ] 1 + KS [Mm Ln ]
(5)
Table 4 shows the kN , KS , and KM values for the hydrolysis of PNPP catalyzed by the mixed system of Zn2+ and ligand 1 or 2 in vesicular solution at 25 ◦ C. As can be seen, at low pH values (<7.80), kN increased with increasing pH until a maximum point was reached. Further increases in the pH value caused a decrease in the rate constants. This may be ascribed to the hydrolysis of Zn2+ with the increasing pH value and the reduction in the concentration of complexes, resulting in a decrease in the reaction rate. In addition, the association constant (KM ) of Zn2+ with the ligand is high, indicating
3.4.1. pH-rate profile The pH value plays an important role in the hydrolytic reaction. Thus, the effect of the changes of pH values on the hydrolytic reaction was investigated. As reported in metallomicelle solution previously, many hydrolytic processes in enzymes involve metal ions that are assumed to activate a water molecule acting as a nucleophilic group in a ternary complex, but the hydroxyl group of the ligand acting as a nucleophilic group in our system, as shown in Scheme 1. According to rate law, the rate equation can be expressed as: r = kN [Mm Ln S] = kN [T− ]
(6)
From Table 4, it can be seen that it was obviously shown that kN is pH-dependent and kN is related to the acid dissociation constant (Ka ) of the hydroxyl group of the ternary complex in the reaction system. [Mm Ln S] = [TH] + [T],
Ka =
[T− ][H+ ] [TH]
(7)
where TH is the undissociated complex, T− is the dissociated complex anion assumed to be the active species in vesicular phase and kN is the first-order rate constant which is pH-independent. We have 1 1 1 = + [H+ ] kN kN kN ka
(8)
Fig. 2 shows the pH-rate profile of the hydrolysis of PNPP catalyzed by the metallic complexes in vesicular solution. The apparent rate constants for the hydrolysis of PNPP were at first seen to increase with increasing pH value, and then decrease with further increases in the pH. The inflection points of the curve are at about 7.38 and 7.52, respectively, which correspond to the pKa of Zn2+ in vesicular solution. The pKa values are all lower than those of corresponding metal hydrates [23], indicating that the Zn2+ ion induces the dissociation of the ligands’ hydroxyl groups to form the active species in
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Table 4 kN , KS and KM values for the hydrolysis of PNPP in vesicular (CTAB:SDBS = 1:4) systems at 25 ◦ C. L1
pH
kN (s−1 )
10−3 KS (L mol−1 )
10−5 KM (L mol−1 )
7.20 7.50 7.80 8.00 8.20
0.06 0.10 0.19 0.03 0.02
2.93 0.75 0.32 1.90 2.57
2.42 5.27 3.73 52.80 80.40
L2
pH
kN (s−1 )
10−3 KS (L mol−1 )
10−5 KM (L mol−1 )
7.20 7.50 7.80 8.00 8.20
0.10 0.17 0.29 0.07 0.03
3.05 2.98 0.38 2.84 4.79
5.15 4.04 4.00 46.25 60.51
Note. In 0.01 mol L−1 Tris–HCl buffer; = 0.1 (KCl). [CTAB] = [SDBS] = 0.014 mol L−1 ; [PNPP] = 5 × 10−5 mol L−1 .
N
N
-PNPO(H)
O(H)
RO Zn2+
RO Zn2+
OH(H) O
N
2+
Zn
O
N
O
N
NO2
O(H)
RO
O
O
N O-
PNPP Scheme 2. The proposed mechanism of the catalysis of PNPP hydrolysis by a ternary complex in a vesicular system.
the reaction system, increasing the rates of hydrolysis of PNPP by an effective nucleophilic attack on the substrate.
3.4.2. The mechanism of the catalytic reaction The mechanism of the catalytic reaction can be deduced based on the experimental data and relevant literature [24], as represented by the structure in Scheme 2. The mechanism involves: (i) the formation of a ternary complex (ligand/metallic ion/substrate); (ii) the pseudo-intramolecular attack of the activated hydroxyl on the carboxyl group of the ester, resulting in its acylation; and (iii) the metallic ion-mediated hydrolysis of the acylated intermediate. The product is then formed and the catalytic reaction ends.
45 40
103*kobsd
35 30 25 20 15 10
7.2
7.4
7.6
7.8
8.0
8.2
pH Fig. 2. Profiles of kobsd vs. pH for the hydrolysis of PNPP catalyzed by ligands 1 (䊉), 2 (), and Zn2+ ion complexes in a vesicular system (CTAB:SDBS = 1:4); [CTAB] = [SDBS] = 0.014 mol L−1 , [L] = [Zn2+ ] = 1 × 10−3 mol L−1 , [PNPP] = 5 × 10−5 mol L−1 , T = 25 ◦ C.
4. Conclusions The effects of the alkyl chain length, different temperatures, and vesicles formed from different ratios and concentrations of surfactants on the hydrolysis of PNPP in a vesicular solution were investigated. Results showed that the metallic vesicular system not only mimics the active center of the enzymatic catalysis, but also effectively simulates the hydrophobic microenvironment. Some experimental results should also be noted:
(1) On the basis of a kinetic version of a Job plot analysis, a 1:1 ligand:Zn2+ stoichiometry was found to produce the most active species. (2) The reaction rate of the vesicle is one order of magnitude larger than the micelle. The catalytic hydrolysis rate of the vesicles formed from 1:4 CTAB:SDBS is larger than that of the system formed from 1:2 CTAB:SDBS. When 1:4 CTAB:SDBS was used, the catalytic hydrolysis rate of the system with 1.4 × 10−2 mol L−1 concentration showed a higher catalytic efficiency than with 2.8 × 10−2 mol L−1 concentration. This indicates that the changes in microenvironments can affect the catalytic reaction. (3) The pseudo-first-order rate constants for the hydrolysis of PNPP increased with increasing temperature at a constant concentration. The activation energy (Ea ) of ligand 1 is higher than that of ligand 2. Thus, the kobsd followed the order, ligand 2 > ligand 1. (4) The pseudo-first-order rate constants for the hydrolysis of PNPP increased with increasing concentrations at a constant pH. When the concentration of the metallic complexes is the same, the pseudo-first-order rate constants increased with increasing pH value until a maximum point is reached. Further increases in the pH value caused a decrease in the rate constants. (5) The association constant (KM ) of Zn2+ with the ligand is high, whereas the association constant (KS ) of the ternary complex
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