New Strategy For Synthesis Of Oligosaccharides:Solid Phase Enzymatic Synthesis And Stereoselective Synthesis Of ManNAc

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.