利用半乳糖及葡萄糖衍生物探討尿苷二磷酸糖焦磷酸化酶的專一性

Abstract

醣類跟醣軛合物在生物體中是很重要的分子。舉例來講，醣類分子在癌症細胞生長 轉移擴散中扮演著很重要的角色。雖然近十幾年來很多化學合成的方法被研究發展出來，但是醣類軛合物的合成，尤其是有立體選擇性的醣基化反應還是有很多問題需要被解決。相對而言，利用醣基轉移酶來作酵素合成反應在立體選擇性上還有綠色化學上有很大的優勢。Arabidopsis thaliana UDP-sugar pyrophosphorylase (AtUSP) 是一種可以催化磷酸化單醣(sugar-1-phosphate)變成尿苷二磷酸醣(UDP-sugar)的酵素。 近來的研究指出AtUSP對不同的磷酸化單醣有很廣泛的專一性。我們對使用酵素合成非天然醣軛合物有很大的興趣。我們的目標是利用激酶將非天然的單醣轉化成1-磷酸化醣，然後用AtUSP催化尿苷二磷酸化醣的生成。而這些尿苷二磷酸化醣將來或許可以再用糖轉移酶合成我們想要的寡醣或多醣。為了測試AtUSP的專一性，我們合成了一些葡萄糖跟半乳糖的衍生物。這些衍生物在六號碳的位置做官能基的修飾，將原本的ㄧ級醇轉化成氟、疊氮、碳碳單鍵、雙鍵、還有三鍵。我們也合成了乙醯葡萄糖胺、乙醯半乳糖胺的衍生物，我們將乙醯基取代變成不同長度的末端疊氮烷醯基和5-戊炔醯基。我們利用胜肽鍵偶合反應來得到這些想要的產物。往後我們所利用的這些方法也可以拿來合成其它類似的分子。最後我們利用激酶想要拿到磷酸化糖以便於作往下的AtUSP專一性測試。不過我們發現我們所合成的衍生物反應速率太低了，沒有辦法拿到我們想要的1-磷酸化醣。或許接下來我們可以多試其它不同的激酶或者利用化學合成方法合成想要的1-磷酸化醣以便於可以很快速的了解AtUSP的對不同單醣衍生物的專一性。

Glycans and glycoconjugates are important biomolecules, which participate in various cellular processes, e.g. cancer growth, infiltration, and metastasis. Although many chemical synthetic methods have been developed during last decade, the synthesis of glycoconjugates, especially the stereoselective glycosylation, remains to be problematic. In contrast, enzymatic synthesis using glycosyltransferases is advantageous in terms of not only chemical selectivities but also green chemistry. In this study, we looked into arabidopsis thaliana UDP-sugar pyrophosphorylase (AtUSP), an enzyme that catalyses the conversion of monosaccharide-1-phosphate to the respective UDP-sugars with broad specificity, and its application for the synthesis of glycoconjugate containing non-natural sugar. We attempted to use kinase to transfer non-nature monosacharides to monosacharide-1-phosphate followed by AtUSP-catalyzed transfermation of these monosacharides-1-phosphates to the respective UDP-sugars. To examine the substrate tolerance of AtUSP, we synthesized glucose and galactose analogues containing azido, fluorous, methyl, vinyl, and alkynyl groups at the C-6 position. The azido and fluorous ananlogues were prepared form C-6 OH free derivatives by SN2 reaction and fluorination, respectively, using DAST. Alkenyl and alkynyl mimics were synthesized from the corresponding aldehydes via Wittig olefination and Bestman-ohira reaction. Finally, methyl analogues were obtained by hydrogenation of corresponding alkenyl derivatives. We also synthesized GlcNAc and GalNAc analogues with different azidoalkylcarbonyl groups and pentynoyl groups. We applied amide bond formation to synthesize these compounds. This methodology is straightforward and may be applicable for the preparation of other sugar analogues. The evaluation of the synthesized analogues as substrates is under way. However, when we used kinase to catalyze the formation of monosaccharide-1-phosphates, the reaction rate is too slow to obtain any kinetic data. The problem may be solved by using more kinases or chemical method to synthesize these monosaccharide-1-phosphates to test AtUSP more quickly.