合成血球凝集素辨認之醣分子與其衍生物作為抗流感病毒之材料

Synthesis of Hemagglutinin Binding Glycans and Their Conjugation for Anti-Influenza

10.6342/NTU.2012.2012.03415

葉先偉

紅血球凝集素 ； 抗流感 ； 醣類 ； Hemagglutinin ； anti-influenza ； carbohydrate

國立臺灣大學藥學研究所學位論文

2012年

碩士

梁碧惠

英文

流感（Influenza）是一種高感染性的病毒，每年在全球造成約五百萬人感染以及約五十萬人死亡，其主要以H1N1及H3N2亞型流行於人類之間，流感病毒藉由表面的兩種醣蛋白 − 血球凝集素（HA）以及神經胺酸酶（NA），與人類宿主細胞上具特異性α2-6的末端唾液酸 （sialic acid）作用而達到流感病毒感染及散播的目的。然而，家禽流感則是辨認α2-3的末端唾液酸。前人的研究中，Neu5Ac-α(2-6)Gal-β(1-4)Glc認為是被人類流感病毒HA所辨認的醣分子，但近期的醣晶片以及蛋白質結晶研究顯示Neu5Ac-α(2-6)Gal-β(1-4)GlcNAc和Neu5Ac-α(2-6)Gal-β(1-4)-6-SO3-GlcNAc這兩個醣分子對流感病毒HA具有更強的結合力。由於NA會迅速的切斷唾液酸與半乳糖之間的氧原子鍵結，我們希望利用硫原子取代氧原子以提升分子的穩定性。因此S-linked Neu5Ac-α(2-6)Gal-β(1-4)GlcNAc將可用來與流感病毒上的HA辨識，我們合成了S-linked Neu5Ac-α(2-6)Gal-β(1-4)GlcNAc（38, 20步, 總產率8%）和S-linked Neu5Ac-α(2-6)Gal-β(1-4)-6-SO3-GlcNAc（39, 21步, 總產率7%），並將這些合成的醣分子及單一個唾液酸分別接上DLPE形成DLPE鍵結分子45-47，並進一步使用此DLPE鍵結分子作成微脂粒（liposome）做為流感抑制劑。流感病毒抑制實驗結果顯示這些DLPE鍵結分子45-47對抑制流感的EC50約在70-180 μM之間。有趣的是，這些DLPE鍵結分子可以自行形成微胞（micelle），並以穿透式電子顯微鏡（TEM）檢驗證明，而這些微胞對流感病毒的抑制活性比形成liposome的組別來得更高。

Influenza, a highly pathogenic virus, accounts for about five million cases of severe illness, and about half-million deaths annually. Currently, the subtypes of H1N1 and H3N2 prevail over humans. The binding between the virus and the human host cell is dependent on the interaction of viral glycoprotein (HA and NA) with specific terminal Sia-α(2-6)Gal linkage glycans. In contrast, avian HA prefers to bind to host glycans with sia-α(2-3)Gal linkage. Previous research indicated that Neu5Ac-α(2-6)Gal-β(1-4)Glc was found to be the human HA binding glycan. Through the recent glycan arrays and protein co-crystal studies, two glycans − Neu5Ac-α(2-6)Gal-β(1-4)GlcNAc and Neu5Ac-α(2-6)Gal-β(1-4)-6-SO3-GlcNAc have been identified to be the most important HA-binding glycans. As NA cleaves O-sialosides rapidly, we substituted the glycosidic oxygen atom by sulfur to enhance the stability of the glycosidic linkage. In fact, this strategy has been proved to inhibit the activity of glycosidases because S-glycosides show similar conformational space to O-glycosides. Owing to the metabolic stability, S-linked Neu5Ac-α(2-6)Gal-β(1-4)GlcNAc should serve as a novel synthetic recognition molecule of HA. An efficient synthesis of S-Neu5Ac-α(2-6)Gal-β(1-4)GlcNAc (38, 20 steps, 8% overall yield) and S-Neu5Ac-α(2-6)Gal-β(1-4)-6-SO3-GlcNAc (39, 21 steps, 7% overall yield) were achieved. These molecules and mono sialic acid were further conjugated with DLPE to form DLPE conjugations 45-47 and also using these DLPE conjugations to form liposome, as competitors to block HA of influenza virus. The virus inhibitory assay showed that DLPE conjugations 45-47 have EC50 around 70-180 μM. Interestingly, these DLPE conjugations can form self-assembly micelles, which was confirmed by TEM and have higher anti-influenza activity comparing to their liposome forms.

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