Development of A Recycling Approach for Carbohydrate Derivatization with Aminooxy-based Labeling Reagents and Its Application to Purification and Identification of Oligosaccharides from Natural Source

擁有特定結構的寡醣在許多生物性功能上扮演關鍵的角色，如細胞與細胞間的辨識、細胞激素的表現和生物標記分子等。在近數十年研究中，藉由生物正交反應引入發色團或螢光團已經被發展成為具有化學選擇性的醣類衍生方法。然而，絕大部分的化學選擇性連結方法在中性環境之反應速率較慢，又由於其為不可逆的化學修飾，進而限制其應用性。 在本篇論文中，我們利用胺氧基為標識試劑，以2-萘甲氧基胺及其衍生物N-甲基-2-萘甲氧基胺, N-甲基-9-蔥甲氧基胺等，來發展一套可逆的生物正交型醣類結合反應。此衍生化的醣類可藉由偵測萘分子的螢光來提升分離效率。另外，這類O-萘甲基肟的衍生物可以透過鈀催化的氫解反應以及N-溴琥珀醯亞胺的脫去肟反應來再生原本的醣分子。另一方面，醣的肟醚類衍生物在碳5位置有氫氧基，氫解反應則產生N-氫氧基-呱喃醣胺的中間體，其在酸性環境下易被水解而可直接得到原本的醣分子。本文結果證明了此方法可以適用在單醣、雙醣以及寡醣上的分離以及回收。同時此衍生方法擁有化學選擇性反應、好的溶劑容忍度與發光團的高度可塑性等優勢。 霍山石斛(Dendrobium huoshanense)為一種名貴的傳統中草藥，被用於抗發炎、防止視力惡化以及提升自體免疫能力等用途。含有天然乙醯基(acetyl group)的霍山石斛黏液多醣可誘使小鼠巨噬細胞表現白血球生長因子(granulocyte colony-stimulating factor, G-CSF)是一種醣蛋白可透過NF-$kappa$b訊息傳導路徑來刺激單核白血球細胞成熟。我們利用液態層析儀(liquid chromatography, LC)、介質輔助雷射脫附游離飛行時間質譜儀(matrix-assisted laser desorption/ionization-time of flight mass spectroscopy, MALDI-TOF MS)、逆轉錄聚合酶連鎖反應(reverse transcription-polymerase chain reaction, RT-PCR)和酵素連結免疫吸附法(enzyme-linked immunosorbent assay, ELISA)等工具分析多醣體中具有生物活性的結構。在初步的結果中，我們發現含有七個甘露糖組成之$ etaup$-1,4-寡醣，無論有沒有乙醯基均可刺激G-CSF產生，可能是具生物活性的最小寡醣單元；而更大的甘露聚醣，如：多至12個單元，則有更高的G-CSF表現。進一步地使用我們所開發的N-甲基-2-萘甲氧基胺可循環式醣類衍生法來分離石斛寡醣混合物，成功地得到具有較高分子量的寡醣部分。如此，透過這樣的衍生方法分離具生物活性之寡醣分子將成為開發中草藥的一項有力工具。Oligosaccharides with specific structures play a critical role in many biological functions such as cell-cell recognition, cytokine expressions and biomarkers. Many chemoselective ligation methods for carbohydrate have been developed to incorporate chromophores/fluorophores; however, most of them are often limited by slow reaction rate at neutral pH and non-reversibility of the modified compounds to their parental sugars for further studies. In this study, we utilized the aminooxy-based labeling reagents, O-(2-naphthylmethoxy)amine, N-methyl-(2-naphthylmethoxy)amine and N-methyl-(9-anthrylmethoxy) amine, to develop a reversible bioorthogonal glycoconjugation. Separation of these glycoconjugates was facilitated by easy detection of the fluorophore of naphthyl or anthryl groups. The naphthylmethyl oxime derivatives underwent a palladium-catalyzed hydrogenolysis, followed by deoximation with NBS, to regenerate their parental sugars. The oxime ether derivative of saccharide bearing a free OH group at the C-5 position, underwnet the hydrogenolysis reaction to an N-hydroxy glycopyranosylamine intermediate, which was labile to hydrolysis under acidic conditions to give directly the parental sugar. These results demonstrated that our approach allowed the separation and regeneration of many types of mono-, di-, and oligosaccharides. This oxime-based derivatization method has several advantageous features including high chemoselectivity, high tolerance to di erent solvents, and high plasticity of chromogenic moiety. Dendrobium huoshanense, a famous traditionally Chinese herb, has been utilized to prevent inflammation and deterioration of eyesight and to promote self-immunity. The mucilage polysaccharide with acetyl groups from the stem of D. huoshanense showed to enhance the expression of granulocyte colony-stimulating factor (G-CSF), a glycoprotein which can stimulate the mature of monogranulocytes via a NF- b-mediated signal pathway in Raw 264.7 macrophages. A serious of experiment to determine the bioactive structure from polysaccharide mixture, including liquid chromatography, matrix-assisted laser desorption/ionization-time of flight mass spectroscopy (MALDITOF MS), reverse-transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), have been carried out. Our preliminary analyses from MS, RT-PCR and ELISA assays implied that a -1,4-oligomannoside containing seven units of mannosides, with or without acetylation, was a possible smallest bioactive oligosaccharide for the production of G-CSF. The higher oligomannosides, e.g. up to 12 DP exhibited even higher activity of G-CSF induction. Furthermore, separation of D. huoshanense oligosaccharide mixture by recycling derivatization with N-methyl- (2-naphthylmethoxyl)amine was also successfully performed to obtain the portions of higher-molecular-weight oligosaccharide. This strategy will be a useful tool for the isolation of the bioactive oligosaccharide from medicinal herbs