2-{[4-氨基-5-(3,4,5-三甲氧基-苄基)-嘧啶-2-亚胺基]-甲基}-6-甲氧基-苯酚与酵母细胞和牛血清白蛋白的相互作用

doi:10.7503/cjcu20130797

高等学校化学学报 ›› 2014, Vol. 35 ›› Issue (4): 831.doi: 10.7503/cjcu20130797

2-{[4-氨基-5-(3,4,5-三甲氧基-苄基)-嘧啶-2-亚胺基]-甲基}-6-甲氧基-苯酚与酵母细胞和牛血清白蛋白的相互作用

蒋建宏¹, 李旭¹, 肖圣雄¹, 谷惠文², 李传华¹, 杨平¹, 魏得良¹, 何笃贵¹, 李爱桃¹, 李霞¹, 姚飞虹¹, 李强国¹()

1. 湘南学院湘南稀贵金属化合物及其应用湖南省重点实验室, 化学与生命科学系, 郴州 423043
2. 湖南大学化学生物传感与计量学国家重点实验室, 化学化工学院, 长沙 410082

收稿日期:2013-08-16 出版日期:2014-04-10 发布日期:2014-02-27
作者简介:联系人简介: 李强国, 男, 教授, 主要从事热化学和生物热化学研究. E-mail: liqiangguo@163.com
基金资助:
国家自然科学基金(批准号: 20973145, 21273190)、湖南省教育厅科研计划基金(批准号: 11A112, 10C1233)和湖南省科技厅计划基金(批准号: 2011TP4016-1, 2012TP4021-5, 2013FJ3033)资助

Interaction of 2-{[4-Amino-5-(3,4,5-trimethoxy-benzyl)-pyrimidin-2-ylimino]-methyl}-6-methoxy-phenol with S. pombe Cells and BSA^†

JIANG Jianhong¹, LI Xu¹, XIAO Shengxiong¹, GU Huiwen², LI Chuanhua¹, YANG Ping¹, WEI Deliang¹, HE Dugui¹, LI Aitao¹, LI Xia¹, YAO Feihong¹, LI Qiangguo^1,^*()

1. Hunan Provincial Key Laboratory of Xiangnan Rare-Precious Metals Compounds and Applications, Department of Chemistry and Life Science, Xiangnan University, Chenzhou 423043, China
2. State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, China

Received:2013-08-16 Online:2014-04-10 Published:2014-02-27
Contact: LI Qiangguo E-mail:liqiangguo@163.com
Supported by:
† Supported by the National Natural Science Foundation of China(No.20973145, 21273190), the Educational Ministry Foundation of Hunan Province, China(No.11A112, 10C1233) and the Science and Technology Ministry Foundation of Hunan Province, China(No.2011TP4016-1, 2012TP4021-5, 2013FJ3033)

摘要/Abstract

摘要：

用等摩尔的邻香草醛(C₈H₈O₃)与甲氧苄胺嘧啶(C₁₄H₁₈N₄O₃)反应, 合成了一种新的化合物2-{[4-氨基-5-(3,4,5-三甲氧基-苄基)-嘧啶-2-亚胺基]-甲基}-6-甲氧基-苯酚(APYP). 通过元素分析、核磁共振、摩尔电导、红外光谱和紫外光谱等手段对其进行了表征, 其化学式为C₂₂H₂₄N₄O₅. 采用微量热法测定了APYP在301.15 K时对粟酒裂殖酵母细胞的生长代谢的产热曲线, 计算得到了粟酒裂殖酵母细胞生长速率常数k, 抑制率I 以及半抑制浓度IC₅₀等热动力学参数. 实验结果表明, 随着APYP浓度的增加, 粟酒裂殖酵母细胞的生长代谢速率常数k减小, 抑制率I增加, 半抑制浓度IC₅₀为123.2 mg/L(或2.96×10^-4 mol/L), 即APYP对粟酒裂殖酵母细胞有抑制作用. 同时利用荧光光谱和紫外光谱等方法研究了APYP与牛血清白蛋白(BSA)在接近生理条件下的相互作用, 探讨了荧光猝灭机理, 计算了反应的结合常数、结合位点、结合距离及热力学参数, 研究了APYP与BSA之间的能量转移情况; 并通过同步荧光光谱考察了APYP对BSA二级结构的影响.

关键词: 邻香草醛, 甲氧苄氨嘧啶, 热动力学参数, 牛血清白蛋白, 粟酒裂殖酵母细胞

Abstract:

A new compound 2-{[4-amino-5-(3,4,5-trimethoxy-benzyl)-pyrimidin-2-ylimino]-methyl}-6-methoxy-phenol(APYP) was synthesized using equivalent moles of o-vanillin(C₈H₈O₃) and trimethoprim(C₁₄H₁₈N₄O₃). Elemental analysis, molar conductivity, UV-Visual, IR and NMR spectroscopy were employed to characterize the composition and structure of APYP. The analytical results showed that the molecular formula of the compound is C₂₂H₂₄N₄O₅. The thermokinetic properties of the action of APYP on growth meta-bolism of S. pombe were studied by a TAM air isothermal calorimeter at 301.15 K. The microbial growth rate constant(k), inhibition ratio(I) and half inhibition concentration(IC₅₀) were obtained. Experimental results show that the k values of S. pombe decreased and I values of S. pombe increased with the increase in the concentrations of APYP and the IC₅₀ value of APYP was found to be 123.20 mg/L. The APYP has good inhibition activity on the growth of S. pombe. Besides, fluorescence spectroscopy and UV-Vis absorption spectroscopy were employed to investigate the interactions of APYP with bovine serum albumin(BSA) under the approximatively physiological conditions. The binding constants, binding sites, binding distance, and the thermodynamic parameters were calculated. The energy transfer between the donor(BSA) and acceptor(APYP) was investigated. Furthermore, synchronous fluorescence spectroscopy was used to investigate the structural change of BSA molecules with additions of APYP.

Key words: o-Vanillin, Trimethoprim, Thermokinetic property, Bovine serum albumin, S. pombe cell

中图分类号:

O641
O633.12

TrendMD:

蒋建宏, 李旭, 肖圣雄, 谷惠文, 李传华, 杨平, 魏得良, 何笃贵, 李爱桃, 李霞, 姚飞虹, 李强国. 2-{[4-氨基-5-(3,4,5-三甲氧基-苄基)-嘧啶-2-亚胺基]-甲基}-6-甲氧基-苯酚与酵母细胞和牛血清白蛋白的相互作用. 高等学校化学学报, 2014, 35(4): 831.

JIANG Jianhong, LI Xu, XIAO Shengxiong, GU Huiwen, LI Chuanhua, YANG Ping, WEI Deliang, HE Dugui, LI Aitao, LI Xia, YAO Feihong, LI Qiangguo. Interaction of 2-{[4-Amino-5-(3,4,5-trimethoxy-benzyl)-pyrimidin-2-ylimino]-methyl}-6-methoxy-phenol with S. pombe Cells and BSA^†. Chem. J. Chinese Universities, 2014, 35(4): 831.

图/表 12

Fig.1 Power-time curves for the growth of S. pombe affected by APYPConcentrations of APYP/(mg·L-1): a. 0; b. 40; c. 60; d. 80; e. 100; f. 120; g. 140.

Table 1 Thermokinetic parameters of the growth of S.pombe affected by different concentrations of APYP at 301.15 K

c(APYP)/(mg·L^-1)	k^*/s^-1	R	I	IC₅₀/(mg·L^-1)
0	7.20×10^-5±5×10^-8	0.9994	0
40	6.26×10^-5±4×10^-8	0.9992	13.1
60	5.37×10^-5±4×10^-8	0.9993	25.4
80	4.76×10^-5±2×10^-8	0.9998	33.9	123.2
100	4.10×10^-5±7×10^-9	1.0000	43.1
120	3.63×10^-5±1×10^-8	0.9995	49.6
140	3.40×10^-5±2×10^-8	0.9991	52.8

Fig.2 Rate constants of growth(k) of S. pombe with concentration of APYP(c)

Fig.3 Inhibitive ratio(I) of S. pombe with concentration of APYP(c)

Fig.4 Quenching effects of APYP on BSA fluore-scence intensity at 298.15 Kλex=280 nm, λem=340 nm, c(BSA)=2.0×10-6 mol/L, c(APYP)=2.0×10-6 mol/L. c(APYP)/c(BSA), a—k: 0, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0.

Fig.5 Stem-Volmer plots for the fluorescence quenching of BSA by APYP at different temperaturesa. 298.15 K, slope=1.0622, R=0.9996; b. 302.15 K, slope=1.0194, R=0.9985; c. 306.15 K, slope=0.9414, R=0.9987; d. 310.15 K, slope=0.9133, R=0.9977.

Fig.6 Lineweaver-Burk plots for the fluorescence quenching of BSA by APYP at different temperaturesa. 298.15 K, slope=0.00277, R=0.9976; b. 302.15 K, slope=0.00301, R=0.9875; c. 306.15 K, slope=0.00379, R=0.9962; d. 310.15 K, slope=0.00425, R=0.09960.

Table 2 Relative thermodynamic parameters of APYP-BSA system

Temperature/K	K_LB	$Δ H 0 —$ /(kJ·mol^-1)	$Δ G 0 —$ /(kJ·mol^-1)	$Δ S 0 —$ /(J·mol^-1·K^-1)
298.15	1.317×10⁶	-22.997	-34.929	40.020
302.15	1.241×10⁶	-22.997	-35.248	40.546
306.15	1.013×10⁶	-22.997	-35.198	39.853
310.15	9.198×10⁵	-22.997	-35.409	40.019

Table 2 Relative thermodynamic parameters of APYP-BSA system

Temperature/K	K_LB	$Δ H 0 —$ /(kJ·mol^-1)	$Δ G 0 —$ /(kJ·mol^-1)	$Δ S 0 —$ /(J·mol^-1·K^-1)
298.15	1.317×10⁶	-22.997	-34.929	40.020
302.15	1.241×10⁶	-22.997	-35.248	40.546
306.15	1.013×10⁶	-22.997	-35.198	39.853
310.15	9.198×10⁵	-22.997	-35.409	40.019

Fig.7 Double-log plots of APYP quenching effect on BSA fluorescence at different temperatures

Table 3 Binding constants and number of binding sites of APYP-BSA system

Temperature/K	K_A/(L·mol^-1)	n	R	Temperature/K	K_A/(L·mol^-1)	n	R
298.15	3.3´10⁵	0.9026(≈1)	0.9968	306.15	7.5´10⁵	0.9820(≈1)	0.9980
302.15	5.6´10⁵	0.9521(≈1)	0.9951	310.15	1.0´10⁶	1.0139(≈1)	0.9971

Fig.8 Spectral overlap between the absorption spectrum of APYP(a) and fluorescence emission spectrum of BSA(b) at 298.15 Kc(BSA)=2.0×10-6 mol/L; c(APYP)=2.0×10-6, 5 mmol/L. Tris-HCl buffer, pH=7.4. Overlapped region is highlighted.

Fig.9 Synchronous fluorescence spectra of BSA at 298.15 K(A) Δλ=15 nm; (B) Δλ=60 nm. c(BSA)=c(APYP)=2.0×10-6 mol/L. c(APYP)/c(BSA), a—e: 0, 1.0, 2.0, 4.0, 5.0.

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2-{[4-氨基-5-(3,4,5-三甲氧基-苄基)-嘧啶-2-亚胺基]-甲基}-6-甲氧基-苯酚与酵母细胞和牛血清白蛋白的相互作用

Interaction of 2-{[4-Amino-5-(3,4,5-trimethoxy-benzyl)-pyrimidin-2-ylimino]-methyl}-6-methoxy-phenol with S. pombe Cells and BSA^†

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