階段型合成醣核苷與其應用於合成聚 N-乙醯乳糖胺

Sequencial Enzymatic Synthesis of Sugar Nucleotides and Its Application on Poly-LacNAc Synthesis

簡薇庭(Wei-Ting Chien)

指導教授 ： 林俊成

國立清華大學/理學院/化學系/博士(2012年)

摘要

中文摘要
醣類與醣基化衍生物在生物體中調節許多生理反應，扮演著許多重要的角色，如細胞間辨識、訊息傳遞與病毒入侵等。儘管文獻中已有許多醣體合成的研究陸續被開發發表，但由於醣體本身結構之多樣性，使得其在複雜寡醣體多醣體的合成上，仍具有相當挑戰。由於酵素反應具有高度選擇性與位向專一性，因此提供另一個有效的合成策略。其中，醣基轉移酶已廣泛的被應用在多醣體的合成，可精確地控制醣體合成的鍵結與位向；而研究中顯示，取自於細菌體的醣基轉移酶，其對受質的容忍度較高，在合成醣體衍生物上，更具有合成應用價值。
在本論文中，建構表達重組蛋白磷酸葡萄糖胸苷轉移酶、β-1,4-半乳糖轉移酶、N-乙醯基己胺糖激酶與 β-1,3-N-乙醯葡萄糖胺轉移酶等酵素，並對這些酵素進行定性分析與反應條件優化篩選。利用表達之磷酸葡萄糖胸苷轉移酶，在55 oC中，以二價金屬鎂離子作為輔助因子，可在二小時內製備多種高單價之醣核苷：尿苷二磷酸半乳糖、尿苷二磷酸 N-乙醯基葡萄糖、尿苷二磷酸葡萄糖與胸苷二磷酸葡萄糖。將表達之磷酸葡萄糖胸苷轉移酶以天然化學黏合法，位向專一的固化於磁性奈米粒子上，重複回收利用，經十次循環反應後，固化之磷酸葡萄糖胸苷轉移酶仍保有 95% 活性。結合半乳糖機酶與N-乙醯基己胺糖激酶進行一鍋化反應，可從相對較低價之起始物合成尿苷二磷酸半乳糖與尿苷二磷酸N-乙醯基葡萄糖，經過離子交換樹脂與凝膠層析的方式得到高純度的醣核苷酸予體。
論文中，磷酸葡萄糖胸苷轉移酶催化生成之尿苷二磷酸半乳糖、尿苷二磷酸N-乙醯基葡萄糖醣予體經結合β-1,4-半乳糖轉移酶與 β-1,3-N-乙醯葡萄糖胺轉移酶，可進行階段性一鍋化合成聚N-乙醯乳糖胺寡糖體，省去醣予體純化步驟。一般自然界中取得之聚N-乙醯乳糖胺寡糖體為混合物，經由本系統可合成固定已知鏈長之聚N-乙醯乳糖胺寡糖體單體，將其分別經由α-2,3-唾液酸轉移酶 與 α-2,6-唾液酸轉移酶修飾後，可得一系列結構多樣性之唾液酸基化聚N-乙醯乳糖胺寡糖體衍生物。值得一提的是，不同於文獻中所描述，唾液酸基化一般位於末端半乳糖，我們成功的合成出修飾有兩個唾液酸的聚N-乙醯乳糖胺的六醣體 (4-26-2) 與三個唾液酸的聚N-乙醯乳糖胺的九醣體 (6-26-3)，此二化合物利用化學合成亦是非常困難的。藉由所建構之酵素系統，快速有效合成多種聚N-乙醯乳糖胺多醣體衍生物，將有助於醣體生物學之研究。
最後，利用磷酸葡萄糖胸苷轉移酶催化生成之醣核苷酸予體與磷酸葡萄糖胸苷轉移酶進行共結晶，成功解出九種不同的結晶晶體，對此進行結構分析。未來可藉此進行胺基酸點突變，增進磷酸葡萄糖胸苷轉移酶之受質容忍度與催化效率。

關鍵字

醣核苷 ； 聚N-乙醯乳糖胺 ； 唾液酸

並列摘要

Abstract
Carbohydrates and their glycoconjugates are important in mediating structural and functional roles in numerous physiological processes, including various disease states. Despite significant advancement in the field like programmable one-pot assembly of carbohydrates, at the recent time, synthesis of complex carbohydrates and glycoconjugates remains elusive than that of other biomolecules. To simplify the synthesis of carbohydrates, enzymes provide an alternative means that are likely to be synthetically viable to chemists. In this regard, enzymes like glycosyltransferases and glycosidases have proven useful biocatalysts in constructing stereo- and regiospecific glycosidic linkages in complex carbohydrate structures. However, in the preparative-scale synthesis point, glycosyltransferases from microbial sources may exhibit greater flexibility because of their ability to synthesize a large range of oligosaccharide analogues at relatively high yields.
My dissertation describes the expression of various recombinant bacterial enzymes; thymidylyltransferase (RmlA) of Aneurinibacillus thermoaerophilus, N-acetylhexosamine-1-kinase (NahK) of Bifidobacterium longum, β-1,3-N-acetyl-glucosaminyltransferase of Helicobacter pylori (HpGnT) and β-1,4-galactosyltransferase of Neisseria meningitides (NmGalT), from Escherichia coli. We determined that use of magnesium (Mg2+) as a cofactor and at 55 oC, numerous sugar nucleotides were effectively synthesized in milligram-scale by RmlA in two hours, and these include uridine 5′-diphosphate galactose (UDP-Gal), uridine 5′-diphosphate N-acetylglucosamine (UDP-GlcNAc), uridine 5′-diphosphate glucose (UDP-Glc) and thymidine 5′-diphosphate glucose (TDP-glucose). Additionally, RmlA was site-specifically and covalently immobilized on an MNP using a combination of intein-mediated protein expression and NCL, and found that Rm1A-MNP retains almost 95% of its activity following ten consecutive enzyme assays. We also demonstrated synthesis of UDP-GlcNAc and UDP-Gal by using corresponding kinases from relatively cheap starting materials such as GlcNAc and Gal. All sugar nucleotides were purified by ion-exchange column for analytical purposes.
The dissertation also demonstrates oligo-LacNAc synthesis in a cost-effective way. Normally, oligo-LacNAcs exist as an inseparable mixtures isomer in nature. By using our newly developed enzymatic system, defined lengths of oligo-LacNAcs were synthesized in a one-pot fashion by employing expressed NmGalT and HpGnT in the presence of UDP-Gal and UDP-GlcNAc. Also, we have demonstrated the versatility of the method by incorporating structurally more complex sialic acid residues with different linkages at the hitherto unknown internal Gal unit of oligo-LacNAc backbone in combination with α-2,3-sialyltransferase and α-2,6-sialyltransferase. Thus, we have achieved the synthesis of sialyl-oligo-LacNAcs; a hexa-saccharide with two repeating sialyl-LacNAc unit (4-26-2) and a nona-saccharide with three repeating sialyl-LacNAc unit (6-26-3), the attachment of which at the internal galactose unit was otherwise difficult by chemical means. With the enzymatic system, we can efficiently and quickly produce oligo-LacNAc derivatives.
Finally to gain insights into the structure-activity studies, we have determined nine crystal structures of RmlA complexed with NDP-sugars, which we have synthesized enzymatically. Therefore, with the analysis of these structures, we can create amino acid mutation to improve the substrate tolerance and the catalytic efficiency of RmlA for accelerating progress in glycobiology.

並列關鍵字

Sugar Nucleotides ； Poly-LacNAc ； Sialic acid

參考文獻

63. Logan, S. M.; Altman, E.; Mykytczuk, O.; Brisson, J. R.; Chandan, V.; Schur, M. J.; St Michael, F.; Masson, A.; Leclerc, S.; Hiratsuka, K.; Smirnova, N.; Li, J.; Wu, Y.; Wakarchuk, W. W., Novel biosynthetic functions of lipopolysaccharide rfaJ homologs from Helicobacter pylori. Glycobiology 2005, 15, 721-733.

49. Hindsgaul, O.; Kaur, K. J.; Srivastava, G.; Blaszczyk-Thurin, M.; Crawley, S. C.; Heerze, L. D.; Palcic, M. M., Evaluation of deoxygenated oligosaccharide acceptor analogs as specific inhibitors of glycosyltransferases. J. Biol. Chem. 1991, 266, 17858-17862.

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