寡糖合成的新策略—固相酶法合成和ManNAc立体选择性合成的研究
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摘要
寡糖是生物体内重要的生物分子,与核酸,蛋白质等生物大分子一样在信号传导,细胞间的识别与结合,细胞的生长与增殖,免疫应答和病原体的感染等过程中有着重要的作用。与蛋白质和核酸这两类生物大分子不同,寡糖的合成是非模板依赖型的合成。在这个过程中,不同糖基转移酶和多种糖基供体构建了一个结构复杂的糖世界。近些年,随着分子克隆技术的日渐成熟和对模式生物基因组的深入了解,酶促合成寡糖技术日渐成熟。因其高度的区域选择性和立体选择性的特点,这种技术愈来愈被人们重视。同时,在寡糖的酶法合成领域中,很多问题需要解决与改进。我们将这些问题归纳为“5S”。即：1.酶的特异性、2.底物的水溶性、3.制备稀有的底物与辅助因子、4.分离纯化、5.规模化。本文就是围绕这5个问题开展课题的。本文的研究内容主要有以下几方面：
本文介绍了两种利用固相技术简化了寡糖酶法合成的分离过程。第一种方法是以pH敏感的水溶性线性高分子作为固相载体的化学酶法合成技术。这种载体有效的克服了固相载体影响酶与载体相互接触的问题。利用这种载体,本文使用一锅三酶体系原位生成稀有糖基供体CMP-Sia,成功地合成了Sia-LN三糖衍生物。这种三糖衍生物在流感病毒侵染宿主过程中起着决定性的作用,是下游分子生物学及药物开发研究中的重要研究对象。
基于第一种方法的优点与不足,我们发展了第二代基于固相萃取技术的酶法合成策略。本策略的特点在于利用简单的“标签”技术无须任何前处理即可实现对酶法合成产物的纯化。对比前人的研究,本方法有以下几个优点：1.使用的“标签”分子简单易得,便于扩大规模；2.相较于传统的固相载体,本方法反应速率更快,反应进程更容易监控；3.本方法后处理过程中不需要传统方法中的脱盐,浓缩等操作,也不需要传统固相萃取方法中的后期纯化,缩短了分离纯化过程所需时间。为了证实方法的可行性,本文共利用了6种糖基转移酶合成了5种不同的具有重要生物学意义的寡糖分子。更重要的是,通过引入另外2种水解酶,本方法首次实现了在固定相上对目标化合物的纯化,这一思路可以帮助未来人们设计出更加高效的固相萃取策略。
为了更加高效地合成N-乙酰氨基甘露糖ManNAc及含有唾液酸的寡糖分子,本论文通过对底物扭张力的控制,首次报道了利用叠氮自由基对葡萄糖烯糖加成制备ManNAc衍生物的方法。通过高精度的计算,证实了本课题的设计思想及对反应机理的假设。首先扭张力在动力学控制的自由基立体选择性加成过程中控制并决定了过渡态的选择性,从而决定了产物的立体选择性。其次,在热力学控制的立体选择性加成过程中,反应中心处扭转角的构型决定了中间体的选择性,最终ManNAc的产物为最有利产物。最后通过对异头碳自由基平均电离能的研究,不同取代基的电子效应决定了动力学控制和热力学控制在产物选择性中所占的比例。在有机方法学角度,本文展示了通过控制底物的扭转角和取代基,可以实现对自由基活性的调节,从而实现对自由基反应路径及立体选择性的控制。基于我们的发现与技术,本文简单高效地合成了ManNAc衍生物,并将其用于酶法合成了含有非天然唾液酸的三糖分子。此分子是重要的CD22的配体,可以用于B细胞相关疾病的机理研究和药物开发。这项工作在有机理论研究和实际合成唾液酸衍生物方面都有一定的价值。
Oligosaccharide along with DNA and protein plays the crucial roles in the signal pathway, communication between cells, proliferation, recognition of pathogen and infection. Differing from the biosynthesis of DNA and protein, the biosynthesis of oligosaccharide is a template-independent process. In this process, a series of glycotransferases and various glycosylation donors build a diversity world of oligosaccharides. Recently, incited by the achievement in molecular clone technology and research in the genome of model organisms, enzymatic synthesis of oligosaccharides is becoming more and more attractive due to its high regioselectivity and stereoselectivity. At the meantime, some defects in this technology are required prompt solution. We classify these problems into "5S":1. Specificity of enzymes;2.Solubility of substrates;3.Preparation of special cofactors and substrates;4.Separation;5. Scale up.
In this dissertation, two improved methods based on solid phase technique were introduced. Firstly we utilized a pH-responsive water-soluble polymer as support in chemo-enzyme synthesis. This method facilitates the interaction between substrates and enzyme. Based on this strategy, we utilized a "one-pot three enzymes system" to generate precious CMP-Sia in situ and synthesized sia-LN successfully. This trisaccharide which is very usefulresearch tool in the molecular biology and pharmaetical research plays a significant role in the infection of flu.
To improve the above method, second generation strategy was developed. The advantage of this strategy is that using easily available tag and solid phase exactraction, pure target compounds can be obtained without any pre-treatment. The advantage pf this method including:1."tag" molecule is cheap and easily available, which is suitable for scale up;2. the reaction rate is fast compared to traditional method, the conversion is easy to monitor;3. there is no need to "de-salt" inpretreatment or addional purification step used in traditional solid phase extraction method. In this project,6enzymes were introduced to afford5different oligosaccharides with great biological importance. Furthermore, this is the first time thatthe target products can be purified with tags on the solid phase by introducingtwo special hydrolases, this stratedgy can help people develop more efficient solid phase extraction stratedgy.
By control the torsional strain of substrates, to the best of our knowledge, this is the first report that2-N-acetamido-2-deoxy-mannose derivatives were obtained from glucals with satisfactory selectivity.Experimental results and high-accuracy computational methods have proven that our proposal is reliable. Firstly, torsional strain plays a decisive role in the kinetic control process. It determines which transition state is preferred by the process. Secondly, in the thermodynamic control process, the ManNac conformation adopted staggered conformation in reaction center is the more stable intermediate, and is also the favored product. Finally, average local ionization energy of intermediate suggests that the substrates follow the thermodynamic process is deficient in electron and hard to undergo further oxidation. Based on this result a trisaccharide which is an important ligand to CD22was synthesized. It shows the potential usage of our research both in academic area and synthetic application.
本文介绍了两种利用固相技术简化了寡糖酶法合成的分离过程。第一种方法是以pH敏感的水溶性线性高分子作为固相载体的化学酶法合成技术。这种载体有效的克服了固相载体影响酶与载体相互接触的问题。利用这种载体,本文使用一锅三酶体系原位生成稀有糖基供体CMP-Sia,成功地合成了Sia-LN三糖衍生物。这种三糖衍生物在流感病毒侵染宿主过程中起着决定性的作用,是下游分子生物学及药物开发研究中的重要研究对象。
基于第一种方法的优点与不足,我们发展了第二代基于固相萃取技术的酶法合成策略。本策略的特点在于利用简单的“标签”技术无须任何前处理即可实现对酶法合成产物的纯化。对比前人的研究,本方法有以下几个优点：1.使用的“标签”分子简单易得,便于扩大规模；2.相较于传统的固相载体,本方法反应速率更快,反应进程更容易监控；3.本方法后处理过程中不需要传统方法中的脱盐,浓缩等操作,也不需要传统固相萃取方法中的后期纯化,缩短了分离纯化过程所需时间。为了证实方法的可行性,本文共利用了6种糖基转移酶合成了5种不同的具有重要生物学意义的寡糖分子。更重要的是,通过引入另外2种水解酶,本方法首次实现了在固定相上对目标化合物的纯化,这一思路可以帮助未来人们设计出更加高效的固相萃取策略。
为了更加高效地合成N-乙酰氨基甘露糖ManNAc及含有唾液酸的寡糖分子,本论文通过对底物扭张力的控制,首次报道了利用叠氮自由基对葡萄糖烯糖加成制备ManNAc衍生物的方法。通过高精度的计算,证实了本课题的设计思想及对反应机理的假设。首先扭张力在动力学控制的自由基立体选择性加成过程中控制并决定了过渡态的选择性,从而决定了产物的立体选择性。其次,在热力学控制的立体选择性加成过程中,反应中心处扭转角的构型决定了中间体的选择性,最终ManNAc的产物为最有利产物。最后通过对异头碳自由基平均电离能的研究,不同取代基的电子效应决定了动力学控制和热力学控制在产物选择性中所占的比例。在有机方法学角度,本文展示了通过控制底物的扭转角和取代基,可以实现对自由基活性的调节,从而实现对自由基反应路径及立体选择性的控制。基于我们的发现与技术,本文简单高效地合成了ManNAc衍生物,并将其用于酶法合成了含有非天然唾液酸的三糖分子。此分子是重要的CD22的配体,可以用于B细胞相关疾病的机理研究和药物开发。这项工作在有机理论研究和实际合成唾液酸衍生物方面都有一定的价值。
Oligosaccharide along with DNA and protein plays the crucial roles in the signal pathway, communication between cells, proliferation, recognition of pathogen and infection. Differing from the biosynthesis of DNA and protein, the biosynthesis of oligosaccharide is a template-independent process. In this process, a series of glycotransferases and various glycosylation donors build a diversity world of oligosaccharides. Recently, incited by the achievement in molecular clone technology and research in the genome of model organisms, enzymatic synthesis of oligosaccharides is becoming more and more attractive due to its high regioselectivity and stereoselectivity. At the meantime, some defects in this technology are required prompt solution. We classify these problems into "5S":1. Specificity of enzymes;2.Solubility of substrates;3.Preparation of special cofactors and substrates;4.Separation;5. Scale up.
In this dissertation, two improved methods based on solid phase technique were introduced. Firstly we utilized a pH-responsive water-soluble polymer as support in chemo-enzyme synthesis. This method facilitates the interaction between substrates and enzyme. Based on this strategy, we utilized a "one-pot three enzymes system" to generate precious CMP-Sia in situ and synthesized sia-LN successfully. This trisaccharide which is very usefulresearch tool in the molecular biology and pharmaetical research plays a significant role in the infection of flu.
To improve the above method, second generation strategy was developed. The advantage of this strategy is that using easily available tag and solid phase exactraction, pure target compounds can be obtained without any pre-treatment. The advantage pf this method including:1."tag" molecule is cheap and easily available, which is suitable for scale up;2. the reaction rate is fast compared to traditional method, the conversion is easy to monitor;3. there is no need to "de-salt" inpretreatment or addional purification step used in traditional solid phase extraction method. In this project,6enzymes were introduced to afford5different oligosaccharides with great biological importance. Furthermore, this is the first time thatthe target products can be purified with tags on the solid phase by introducingtwo special hydrolases, this stratedgy can help people develop more efficient solid phase extraction stratedgy.
By control the torsional strain of substrates, to the best of our knowledge, this is the first report that2-N-acetamido-2-deoxy-mannose derivatives were obtained from glucals with satisfactory selectivity.Experimental results and high-accuracy computational methods have proven that our proposal is reliable. Firstly, torsional strain plays a decisive role in the kinetic control process. It determines which transition state is preferred by the process. Secondly, in the thermodynamic control process, the ManNac conformation adopted staggered conformation in reaction center is the more stable intermediate, and is also the favored product. Finally, average local ionization energy of intermediate suggests that the substrates follow the thermodynamic process is deficient in electron and hard to undergo further oxidation. Based on this result a trisaccharide which is an important ligand to CD22was synthesized. It shows the potential usage of our research both in academic area and synthetic application.
引文
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