含苯并咪唑基配體的鋅配合物的晶體結構和催化對硝基苯磷酸酯水解的活性研究

張 勇 舒威虎 王繼猛 馬志剛 陳雪梅

(湖北理工學院化學與材料工程學院，黃石 435003)

Alkaline phosphatase (AP, EC 3.1.3.1) is a nonspecific phosphomonoesterase found in organisms from all kingdoms of life. Its a zinc metalloenzyme that catalyzes the hydrolysis and, in the presence of a phosphate acceptor, transphosphorylation of a wide variety of phosphate monoesters. The enzymatic reaction proceeds through a covalent phosphoseryl intermediate to produce inorganic phosphate or to transfer the phosphoryl group to alcohols[1]. Recently,in order to mimic the Zn2+coordination structure as well as the function of the Zn2+ion at the active site,most model complexes have been designed and investigated to elucidate the detailed reaction mechanism of AP[2-5]. Benzimidazole-containing ligands are often used in the preparation of model metalbased catalyst complexes because these ligands are structurally related to the biologically important imidazole group found in histidine, and the basicity of benzimidazole approximates that of histidine (pKb：histidine=7.96 and benzimidazole=8.47)[6-7]. So many synthetic mononuclear and dinuclear zinc(Ⅱ)complexes of ligands incorporating benzimidazoles have drawn much attention[8-11]. We show great interest in preparing phosphoester model complexes that are expected to study catalytic hydrolysis of p-nitrophenyl phosphate(PNPP). Herein, to continue this work, we report the crystal structure and catalytic activity of a phosphoester model complex[Zn(L1)(L2)]·H2O in this paper(L1=bis(benzimidazol-2-yl-methyl)amine, L2=5-dimethylaminonaphthalene-1-sulphonylglycine).

1 Experimental

All reagents were purchased from commercial companies and directly used unless stated otherwise.Solvents were purified by the most used methods. The melting point was determined with an XT4A micromelting point apparatus and was uncorrected.The IR spectra were measured on a Perkin-Elmer Spectrum BX FT-IR instrument in tablets with potassium bromide. Elemental analyses were carried out on a Vario EL Ⅲinstrument. UV-Vis spectra were recorded on a Analytik jena Specord 210 spectrophotometer.

1.1 Preparation of [Zn(L1)(L2)]H2O

The ligands L1 and L2 were prepared as the literature method[12-13]. A mixture of ligands L1 (0.28 g,1 mmol), L2 (0.31 g, 1 mmol) and zinc chloride (0.14 g, 1 mmol) were stirred in ethanol (50 mL) for 6 h at room temperature. The resulting solution was filtered and allowed to stand for a week to give yellow crystals.Yield： 65%. m.p. 284～285 ℃. UV-Vis spectra (λmax,nm (ε, L·mol-1·cm-1))： (MeOH solution) 278(9 750),350(12 980).IR(KBr)：2 945,1 585,1 465,1 380,1 136,1 088, 954, 843, 736 cm-1. Anal. Calcd. for C30H31N7O5SZn(%)： C, 53.97, H, 4.65; N, 10.49; Found(%)： C,53.55; H, 4.45; N, 15.53.

1.2 X-ray crystallography

Yellow crystals of the complex for X-ray diffraction were got by slow evaporation at room temperature.The complex having approximate dimensions of 0.2 mm×0.10 mm×0.10 mm was mounted on a glass fibre in a random orientation at 298(2) K. The determination of unit cell and the data collection were performed with Mo Kα radiation (λ=0.071 073 nm) on a Bruker Smart APEX-CCD diffactometer with a φ-ω scan mode. A total of 46 626 reflections were collected in the range of 1.84°＜θ＜26.00° at room temperature. The structures were solved by direct methods and semiempirical absorption corrections were applied. The non-hydrogen atoms were located by direct phase determination and full-matrix least-squares refinement on F2, while the hydrogen atoms for non-water protons were treated using the riding mode. All calculations were carried out on a PC using SHELXS-97 and SHELXL-97 programs[14-15].The detailed crystallographic data are listed in Table 1, and the selected bond parameters are given in Table 2.

CCDC： 867155.

Table 1 Crystal data and structure refinements of the complex

Table 2 Selected bond lengths (nm) and bond angles (°) of the complex

1.3 Hydrolysis of p-nitrophenyl phosphate(PNPP)

All kinetic studies were carried in 75% ethanol solution according to the reported procedure[16-18]. The ionic strength was adjusted to 0.1 with NMe4NO3. The pH was adjusted and kept constant by addition of dilute KOH to the thermostatic cell manually. The hydrolysis of PNPP was followed by taking 3.0 mL aliquots from the cell at the appropriate times, and experiments were monitored by increasing UV-Vis absorbance of 4-nitrophenol at 400 nm. Freshly prepared stock solutions of the complexe (1 ～10 mmol·L-1) and PNPP (1～10 mmol·L-1) were used in the measurements. All reactions were found to obey Michaelis-Mentent model. In each kinetic trial 5 ～8 points were recorded to obtain the kinetic data.Reactions rates were corrected by blank experiments which were made up similarly but without the addition of the zinc complex.

2 Results and discussion

2.1 Crystal structure of the complex

Fig.1 Crystal structure of the complex [Zn(L1)(L2)]·H2O with 50% probability for thermal ellipsoids

AP is a hydrolytic enzyme with a two-metal-ion Zn2+catalytic core, and each Zn2+is coordinated by N(histidine) and O (aspartate) atoms[19-20]. To keep the structural similarity of the active site of AP and invistigate the function of mononuclear Zn2+complexe，benzimidazole-containing ligand L1, carboxylatecontaining ligand L2 and Zn2+were used to model the biomimetic compound. The complex was prepared by the reaction of two ligands with an equimolar amount of ZnCl2in ethanol. In the molecular structure of[Zn(L1)(L2)]·H2O, the Zn2+ion displays a distorted trigonal-bipyramidal coordination geometry provided by the ligands L1 and L2： two benzimidazolyl N atoms(N2 and N4) of L1 and one sulfonamide N atom (N7)of L2 make up the trigonal plane, one amine N atom(N1) of L1 and one carboxylate O atom ( O3) of L2 occupy the axial position (Fig.1). The Zn-N bond distances range from 0.200 1(3) to 0.231 7(3) nm, and the amino N atom (N1) is slightly further away from the Zn2+ion than the benzimidazolyl N atoms (N2 and N4) and sulfonamide N atom(N7) (Table 1). The O(3)atom of the carboxylate group of L2 is 0.206 2 (3) nm from the Zn2+ion. The dihedral angle between the two benzimidazolyl ring systems is 80.20° , while that between the naphthalene ring and each benzimidazolyl ring is 85.91°, 47.63°, respectively. Atoms N6 and S1 are located approximately in the naphthalene ring plane with their deviations being 0.006 9 and 0.010 3 nm. The sulfonamide N atom (N7) of L2 is found to be deprotonized, analogous to a related structure[Cu(L2)(bipy)(CH3OH)] (bipy=2,2′-bipyridine)[13].

2.2 Kinetic studies of PNPP hydrolysis

The kinetic studies of PNPP hydrolysis were conducted by monitoring the formation of the pnitrophenolate at 400 nm using UV-Vis spectrophotometry. The initial velocity (Vi) method was carried out in the experiment. When different concentrations of substrate (PNPP, S) was varied and other factors unchanged, Viwas found to be linear independent on low PNPP concentrations. However, at higher PNPP concentrations, the catalyst (model complex, M) was saturated and Vifollowed saturation kinetics, namely,Viwas zero-order with respect to substrate concentrations cS, and the maximum velocity (Vmax) is also obtained at the same time. So a treatment on the basis of Michaelis-Menten model, originally developed for enzyme kinetics, can be applied. Several kinetic parameters Vmax, Kmand kcatwere obtained under different conditions (Table 2). Here, Kmwas Michaelis constant, kcatwas the catalytic constant, namely, the amounts (mole) of substrate converted to product per minute per mole of mimetic enzyme. Fig.2 showed 1/V is linear with 1/cSfor the complex, implying a firstorder dependence. The slope of the straight line was Km/Vmax, and the intercept on the vertical axis was 1/Vmax.

The results in Table 2 were evaluated from Lineweaver-Burk plots. Moreover, it′s found that the catalytic activity of the complex was strongly influenced by the temperature and pH of the reaction mixture. Fig.3a showed a remarkable pH-catalytic constant profile via bell shaped curve between pH=6.5 and 10, the optimal reactivity was reached at about pH=8.5. A temperature versus catalytic constant profile was shown in Fig.3b, and the maximum rate was found at 35 ℃. So the maximal catalytic activity was observed at a temperature of 35°C and pH 8.5 when other factors were unchanged (Table 2). The results were similar to the reported phosphoester model complexes[7,21]. For the mononuclear zinc(Ⅱ)complex, a probable explanation for phosphateester reactivity showed by the complex can come from the coordination environment of this species. Maybe it had one five-coordinated zinc (Ⅱ) ion with an unsaturated vacancy, and it could interact with the substrate coordinated to the Zn(Ⅱ)ion.

Fig.2 Lineweaver-Burk plot for PNPP hydrolysis catalyzed by the complex at pH=8.50 and 35 ℃

Fig.3 Catalytic constant profiles dependent on the variation of pH value (a, T=35 ℃) and temperature (b, pH 8.5)

Table 3 Kinetic data of the complexe

3 Conclusions

In summary, we have synthesized a phosphoester model complex [Zn(L1)(L2)]·H2O, its structure was confirmed by single crystal X-ray diffraction. The hydrolysis of PNPP catalyzed by the zinc complex obeyed Michaelis-Mentent model.The maximal catalytic rate constant kobswas observed at a temperature of 35 ℃and pH=8.5.

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