使用鄰苯二胺連接劑連結酮酸醣與蛋白質之方法以研究製備對抗B型腦膜炎雙球菌之疫苗

Abstract

細菌表面之多醣與細菌感染中之致病機轉有關而常被選作醣疫苗之目標，但這些多醣需與蛋白質連結才能夠引發T 細胞媒介之免疫反應。在細菌表面之多醣中，多聚唾液酸和脂多醣是兩種具有還原端之酮酸醣的獨特多醣。儘管已經有許多方法可以應用在醣蛋白的合成，但針對酮酸醣之醣蛋白的合成方法則較為少見。 我們在這裡描述一個方法，藉由丙酮酸和鄰苯二胺的縮合反應及硫醇對馬來醯亞胺(maleimide)的Michael 加成反應來連結酮酸醣與蛋白質。我們的結果指出酮酸醣單醣、雙醣和多醣都可與帶有末端胺基的鄰苯二胺連接劑有效率地進行縮合反應，而產生之喹喔啉酮(quinoxalinone)衍生物可再修飾上末端的硫醇，最後的Michael 加成反應即可將帶有末端硫醇之唾液酸喹喔啉酮衍生物與修飾有馬來醯亞胺的牛血清蛋白進行連結。在多聚唾液酸的例子中，我們可成功得到其帶有末端硫醇之喹喔啉酮衍生物，但多聚唾液酸與牛血清蛋白的連結卻失敗。未來多聚唾液酸與牛血清蛋白之連結物的製備仍會繼續嘗試，而這樣的多聚唾液酸醣蛋白有希望可以應用於對抗B 型腦膜炎雙球菌之疫苗。

Bacterial surface polysaccharides are virulent agents for bacterial infection and often chosen as the target of carbohydrate-based vaccines; however, these polysaccharides need to be conjugated to proteins to elicit T-cell mediated immune response. Among bacterial surface polysaccharides, polysialic acid (PSA) and lipopolysaccharide (LPS) are unique in that their terminal saccharides are α-ketoacids. Although there are many methods for conjugation of glycoproteins, the corresponding conjugation methods for α-ketoacid-based glycoproteins are less common. We report here a method for conjugation of α-ketoacid saccharides with proteins by using condensation reactions of α-ketoacid with o-phenylenediamine (OPD) and Michael addition of thiol to maleimide (MA). Our results showed that α-ketoacid mono-, di-, and polysaccharides were efficiently condensed with the OPD linker bearing a terminal amino group. These saccharide quinoxalinone derivatives were then modified with a terminal thiol, and the subsequent Michael addition furnished the conjugation of thiol-terminated sialic acid–quinoxalinone derivative to MA-modified bovine serum albumin (MA–BSA). In the case of NPrPSA, a α-ketoacid polysaccharide, its quinoxalinone derivative bearing terminal thiol was prepared, but the subsequent conjugation to MA–BSA failed with unclear reason. The preparation of NPrPSA–BSA conjugate as a vaccine against group B Neisseria meningitidis may be attempted in a different approach.