Iodine Promoted Condensation of Aldoses and α-Ketoacid- Terminated Saccharides with o-Phenylenediamines for Synthesis of Glycoconjugates

細胞以外層表面上的醣蛋白作為標記分子，在生物辨識的過程中扮演重要的角色。除了與蛋白質連接的核心醣序列很類似外，不同種的細菌會有其特定的多醣序列。在革蘭氏陽性菌中，GalNAc是作為連接蛋白質與多醣序列的核心；而在革蘭氏陰性菌中，其核心變為Kdo (3-deoxy-D-manno-octulosonic acid)。目前有很多方法可以去修飾未保護的尾端GalNA，但是對於Kdo而言，仍然沒有一個有效的方法可以針對性修飾並將多醣序列與其他攜帶蛋白連接。 我們在這裡描述了一個新的方法，用碘在醋酸的環境下，可以將未保護的醣用鄰苯二胺修飾得到咪唑化合物。這個方法可以應用在單醣，雙糖甚至是三醣上。此外，我們也發現這個反應可以應用在酮酸醣上，在類似的條件下可以得到喹喔啉酮類衍生物。由多醣衍生出來的喹喔啉酮化合物可以經由螢光光譜去鑑定其結構。運用適當的二胺連結化合物，我們可以將去掉lipid A的多醣與胺基包覆的聚苯乙烯膠體連結。用簡單的苯酚–硫酸染色法就可以鑑定聚苯乙烯上的多醣。我們發展的方法提供了一個途徑去合成含有革蘭氏陰性菌外層多醣體的醣聯化合物。這個方法將可應用於多醣與攜帶蛋白質的連結，做為一個很好的抗原，去引發抗體來預防或治療有抗藥性的細菌感染。Carbohydrate molecules act as markers on the outer membrane of cell surface to play a central role in biological recognition events. Different species of bacteria have different types of polysaccharides except for the core structure of oligosaccharide that links to protein. GalNAc is the linking saccharide for most Gram-positive bacteria, whereas Kdo (3-deoxy-D-manno-octulosonic acid) is for Gram-negative bacteria. Various methods have been applied for modification of the unprotected terminal GalNAc, but there is no effective method has been explored for Kdo conjugation. We report here a novel method for condensation of unprotected saccharides with o-phenylenediamines using iodine in acetic acid solution to form aldo-benzimidazoles. This method is applied to mono-, di-, and trisaccharides. We also found that α-keto acids reacted with o-phenylenediamines using iodine in acetic acid solution to form the corresponding quinoxalinones. The formation of quinoxalinones from polysaccharides was verified by fluorescence spectra. By using an appropriate diamine linker, we demonstrated the conjugation of lipid A-free polysaccharides with aminated polystyrene gel. The content of glycoconjugates was determined by a simple phenol–sulfuric acid method. This method provides a convenient way to synthesize glycoconjugates containing the polysaccharide from the outer surface of Gram-negtive bacteria. This method is potentially useful in conjugation of carrier proteins with polysaccharides, which can be a good antigen for inducing antibodies against antibiotic-resistance bacteria.Acknowledgement ........................................................................................................Ibstract in Chinese ...................................................................................................IIIbstract in English .....................................................................................................Vable of Contents .....................................................................................................VIIndex of Schemes ........................................................................................................Xndex of Figures .......................................................................................................XIIndex of Tables ........................................................................................................XIIIbbreviations ..........................................................................................................XIVhapter 1. Introduction ..............................................................................................1.1 Importance of Benzimidazole ......................................................................1.2 Glycosyl Benzimidazoles Act as Nucleoside Mimics .................................2.3 Previous Synthetic Methods for Glycosyl Benzimidazoles .......................2.4 Methods for Formation of Imidazoles Directly from Aldehydes and iamines .........................................................................................................5.5 Importance of Saccharide Benzimidazoles in Carbohydrate Chemistry 6.6 The Family of α-Ketoacid Carbohydrates .................................................7.7 Condensation of o-Phenylenediamines with α-Ketoacids .........................8.8 Importance of Polysaccharides on the Bacterial Surface .........................9.9 Carbohydrate-Based Vaccines ..................................................................10.10 Methods for Glycoconjugates Synthesis .................................................11 1.10.1 Conjugation via Reductive Amination ......................................12 1.10.2 Conjugation via Oxime Formation ............................................16 1.10.3 Conjugation via NHS Activation ................................................16 1.10.4 Conjugation via Bifunctional Linker .........................................18.11 Outline of my research .............................................................................18hapter 2. Results and Discussion ...........................................................................21.1 Previous Research in Our Laboratory .....................................................21.2 Optimization of the Reaction of Aldoses with -Phenylenediamines ..................................................................................23.3 Expansion of Reaction Scope ....................................................................27.4 Proposed Mechanism .................................................................................29.5 pH Value Consideration .............................................................................31.6 Iodine Promoted Condensation Reaction of α-Ketoacid Saccharides ...32.7 Design and Synthesis of Diamine Linkers ................................................38.8 Fluorescence Spectral Study of the Quinoxalinones Formed by α-Ketoacid Saccharides with o-Phenylenediamines ..............................41.9 Conjugation of Polysaccharides with Phenylenediamine-linked Carriers .......................................................................................................45.10 Conjugation of Polysialic Acid with Aminated Solid Support .............54.11 Conclusion .................................................................................................55.12 Future Application ...................................................................................56hapter 3. Experimental Section .............................................................................59.1 General Part ................................................................................................59.2 General Procedure for Oxidative Condensation of Aldoses with-Phenylenediamines ..................................................................................60.3 General Procedure for Condensation Reaction of α-Ketoacid Saccharides with o-Phenylenediamines ....................................................61.4 Fluorescence Spectral Study ......................................................................62.5 LPS–Induced Inflammatory Assay ...........................................................62.6 Phenol–Sulfuric Acid Assay .......................................................................62.7 Physical and Spectroscopic Properties of Compounds ...........................63eferences ..................................................................................................................91ppendices .................................................................................................................9