研究酮酸之去羧基醯胺化反應以建立醣疫苗製備之新途徑

Abstract

傳統的疫苗大多利用來自病原體的蛋白質或胜肽片段作為免疫原。由於許多病原在其表面表現具有特異性的醣類分子，利用這類醣分子作為抗原以誘發抗體達到治療效果，也提供疫苗研究一個新策略。然而這些醣類分子需要與蛋白質連結以增加其免疫原性；因此，在醣疫苗的開發過程中，醣聯抗原的合成方式便成為一個關鍵的因素。在細菌表面的多醣中，多聚唾液酸(PSA)和脂多醣(LPS)是兩種帶有還原端酮酸醣的獨特多醣，由末端酮酸醣衍生的醣聯抗原也能夠保留多醣分子的抗原性。儘管已經有許多方法被報導可以用來建構醣聯抗原，但針對酮酸醣進行衍生之醣聯抗原則較為少見。 在先前的研究中我們利用酮酸醣與鄰苯二胺的縮合反應及硫醇對馬來醯亞胺(maleimide)的Michael加成反應來連接酮酸醣與載體蛋白，並成功將這套方法應用到醣聯蛋白抗原的合成，利用大腸桿菌及沙門氏鼠傷寒桿菌表面之脂多醣建構相對應之醣聯蛋白抗原。然而，此類醣聯抗原中的苯環結構可能會誘發非專一性免疫反應，進而降低醣聯抗原作為疫苗的保護效果。因此，我們試圖利用不同的連結方法來修正醣聯抗原的結構。在本研究中我們測試了幾種酮酸與胺類分子的連結方法，包含酮酸-烴胺(KAHA)醯胺鍵形成反應、過氧化氫促進之去羧基醯胺化反應以及碘促進之去羧基醯胺化反應。測試結果顯示，碘促進之去羧基醯胺化反應可能適合進一步應用到醣聯抗原的製備。

In traditional vaccines, protein or peptide fragments from pathogens are chosen as the immunogen. Because massive and specific carbohydrates are present on the surface of various pathogens, utilizing the highly conservative glycan on the surface of pathogens as the immunogen has illuminated another direction in vaccine research. However, glycans need to be conjugated to proteins to enhance the immunogenicity; therefore, the synthesis of glycan conjugates becomes a critical issue in the glycan vaccinology. Among bacterial surface polysaccharides, polysialic acid (PSA) and lipopolysaccharide (LPS) are unique since they possess α-ketoacid saccharides at the terminal position, and the derivatization from these terminal α-ketoacid saccharides retains the immunogenic property for immune cell recognition. Although there are many methods for the construction of glycan conjugates, the corresponding conjugation methods for α-ketoacid -derivatized glycan conjugates are less common. In a previous study, our lab has developed a method for the synthesis of Kdo-derivatized glycan conjugates by using a condensation reaction of α-ketoacid with o-phenylenediamine and Michael addition of thiol to maleimide. We have also successfully utilized the LPS of Escherichia coli and Salmonella typhimurium to construct the corresponding glycan conjugates. However, the aromatic subunits in the above-prepared conjugates may induce non-specific immune response. Therefore, we try to modify the structure of the glycan conjugates by using different ligation methods. In this study, we examine distinct methods for conjugation of α-ketoacid saccharides with amines, including ketoacid-hydroxylamine (KAHA) amide-forming reaction, hydrogen peroxide-promoted decarboxylative amidation and iodine-promoted decarboxylative amidation. The results show that the iodine-promoted decarboxylative amidation would be the superior choice for further application in the derivatization of α-ketoacid-terminated polysaccharides.