基于二硫代氨基甲酸盐自组装的糖芯片制备与表征

摘要

Carbohydrates chips are powerful tools for quantitatively studying protein-carbohydrate interactions.Typically,carbohydrate chips are prepared using the self-assembly of carbohydrate thiol/disulfide,which always requires multiple hydroxyl group protection/deprotection steps resulting low conversion in the preparation.In this paper,a kind of carbohydrate derivatives containing dithiocarbamate (DTC) group was synthesized through a two-step reaction to prepare self-assembled monolayers presenting carbohydrate (glycol-DTC SAMs).The glycol-DTC SAMs was characterized using X-ray photoelectron spectroscopy (XPS) and the protein binding activity was quantitatively analysized using surface plasma resonance (SPR) and enzyme linked lectin assay (ELLA).By mixed self-assembly of carbohydrate dithiocarbamate and sarcosine dithiocarbamate,Glycol-DTC SAMs with different glucose density were prepared.The protein binding kinetics was monitored in real time and the thermodynamics was calculated.Interestingly,a 1:1 binding of concanavalin A (Con A) was obtained on the SAMs prepared in solution containing 1％ glucose-DTC,as the dissociation constant (Kd) was calculated to be (39.10 ± 0.12) μmol· L-1.A 1:2 binding of Con A was obtained on the SAMs prepared in solutions containing ＞ 2％ glucose-DTC,as the Kd was calculated to be (1.17 ± 0.18) μmol·L-1.By simply mixed self assembly,multivalent binding of Con A can be realized and separate kinetic parameters can be obtained.Our work would promote the study of protein-carbohydrate interactions and be helpful for revealing the relevant biological progress.%糖基传感芯片是定量研究糖-蛋白相互作用的有力工具.传统糖基传感芯片的制备过程通常涉及糖基硫醇衍生物的合成,过程复杂且产率较低.本文采用脱氧氨基糖与二硫化碳温和条件下一步反应合成了一类新型糖基自组装功能分子-糖基二硫代氨基甲酸盐(DTC)化合物,进而在金衬底芯片上构筑了糖基传感功能膜.采用X射线光电子能谱(XPS)分析了该糖基传感功能膜的元素组成和元素化学环境;采用表面等离子体共振(SPR)和酶联凝集素分析(ELLA)技术定量分析了其在蛋白质水平的糖生物学活性.通过混合自组装的方法,制备了一系列表面葡萄糖密度不同的糖基传感功能膜并测定了伴刀豆球蛋白(Con A)吸附的热力学和动力学数据.通过调控表面密度,我们观察到了蛋白在葡萄糖表面吸附的多价态现象.当自组装溶液中葡萄糖-DTC摩尔分数低于1％时,ConA呈现单价态吸附,其解离平衡常数(K4)为(39.10±0.12) μmol·L-1;当自组装溶液中葡萄糖-DTC摩尔分数高于2％时,ConA呈现多价态吸附,解离平衡常数降至(1.17±0.18)μmol· L-1.本文所发展的糖基自组装功能分子合成方法快速便捷、适用范围广,通过混合自组装可以实现蛋白结合价态的调控,是一种深入研究基于糖-蛋白相互作用的诸多生物过程的有效工具.