酵素有機合成 GDP-L-Fucose 衍生物以作為岩藻糖轉移酵素抑制劑之開發

Chemoenzymatic Synthesis of GDP-L-Fucose Derivatives for the Development of α-1,3-Fucosyltransferase Inhibitors

林宥穠(Yu-Nong Lin)

指導教授 ： 林俊宏

國立臺灣大學/生命科學院/生化科學研究所/博士(2012年)

https://doi.org/10.6342/NTU.2012.10782

摘要

無資料

關鍵字

酵素 ； 酵素有機合成 ； 抑制劑 ； 岩藻糖轉移酵素

並列摘要

Fucosylated glycans are critical to a variety of cell events such as cell-cell adhesion, immune response, bacterial infection and tumor progression. Fucosyltransferases (FucTs) usually catalyze the final steps of glycosylation and are critical to many biological processes. High levels of specific FucT activities are often associated with various cancers. It is thus a promising approach to develop FucT inhibitors for potential anti-inflammatory and anti-tumor therapies is therefore a promising strategy.

Sugar nucleotide analogues are often potent glycosyltransferase inhibitors. Several GDP-L-Fucose (GDP-Fuc) analogues have been synthesized for the development of FucT inhibitors. However, it is notoriously difficult to synthesize and purify these molecules by conventional chemical synthesis. In this thesis, we developed one-step enzymatic synthesis of GDP-Fuc and analogues by utilizing a bifunctional enzyme fucokinase/GDP-fucose pyrophosphorylase (FKP) from Bacteroides fragilis. FKP contains a kinase domain and a pyrophosphorylase domain in a single peptide chain that catalyzes the phosphorylation of L-fucose to form L-fucose-1-phosphate and the subsequent conversion to GDP-Fuc. The process is simple as compared to the chemical method and is suitable for the synthesis at a preparative scale. FKP is a promiscuous enzyme for its broad substrate specificity toward the modifications at C-5 position of L-fucose. Successful synthesis of GDP-Fuc and its analogues including GDP-D-arabinose, GDP-L-galactose, GDP-N3-L-fucose and GDP-alkynyl-L-fucose was achieved with high yields (60-95%) and purities (>98% by HPLC). A multienyme synthesis involving CMP-sialic acid synthetase (NeuA), α-2,3-sialyltransferase (SiaT), α-1,3-FucT and FKP was carried out in one-pot and two-steps with in-situ cofactor regeneration for preparing of sLex. After simple steps of purification, sLex was obtained in a 90% yield.

To discover promising FucT inhibitors, we developed a three-step chemoenzymatic method for rapid assembly of various GDP-Fuc derivatives from 6-azido-L-fucose. 6-Azido-L-fucose was converted to GDP-6-azido-L-fucose by FKP, followed by reduction to produce GDP-6-amino-L-fucose. The product was subjected to diversity-oriented synthesis, i.e., the amide-forming reactions with 94 carboxylic acids. The obtained GDP-Fuc derivatives were screened for inhibitory activity against H. pylori and human α-1,3-FucTs. Compounds incorporating appropriate hydrophobic moieties were identified from the initial screening, individually synthesized, purified and characterized for their inhibition kinetics. Compound 29 had a Ki of 29 nM for human FucT-VI, and is 269 and 11 times more selective than for H. pylori FucT (Ki = 7.8 μM) and for human FucT-V (Ki = 0.31 μM).

並列關鍵字

enzyme ； chemoenzymatic synthesis ； inhibitor ； fucosyltransferase

參考文獻

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