合成具有多價性醣體之新穎光親和性探針並用於探討碳水化合物與凝集素間之交互作用

Synthesis and Evaluation of a Novel Photoactive Probe with Multivalent Carbohydrates for Capturing Carbohydrate-Lectin Interaction

張宗哲(Tsung-Che Chang)

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

凝集素與醣類分子的作用關係在生物體內所影響的生物機制相當廣泛,包含了細胞與細胞間的辨識、細胞的附著、細胞間的訊號傳 遞、醣蛋白的清除、免疫系統與組織發炎,病毒、細菌對細胞的感染, 這些過程均是透過醣類與凝集素的交互作用才得以進行。研究凝集素 的最佳工具就是醣類分子,但是它們之間的作用力為非共價鍵性結 合、可逆的鍵結方式、微弱結合能力均是造成以醣分子為探針研究凝 集素的困難。
雖然醣體光親和性探針可以解決上述的問題,然而現今大部分的 醣體光親和性探針目標導向能力不足,導致無法專一地光標記凝集 素;缺乏多功能化的光親和性探針骨架,無法快速的建構多價醣體與 標籤於光親和性探針上;光標記凝集素後鮮少利用質譜分析鑑定.等 缺點。因此本論著重於開發新穎醣體光親和性探針,以提升光標記凝 集素的能力與後續分析的準確性。
本論研究分為兩個部份,第一部份為合成雙功能化光親和性探針 骨架,利用兩個 orthogonal 反應,Copper(I)-Click 反應與 Amide bond formation,快速的建構三價半乳醣光親和性探針化合物 43。化合物 43 能在蛋白質池與大腸桿菌溶解產物當中專一的光標記 RCA120(Ricinus Communis Agglutinin),並結合質譜分析近一步確認標
記的 RCA120;化合物 43 也可直接於完整、活的 HepG2 細胞膜上光標 記 ASGP-R(Asialoglycoprotein receptor)。
第二部份為設計與合成第二 代醣類光親和性探針,此探針由化合物 66 與 45 組成,利用多價醣體 光親和性探針、bio-orthogonal chemistry、光斷裂型官能基與功能化磁 性奈米粒子,達到專一地光標記並濃縮凝集素,藉以提升後續質譜分 析的偵測極限與準確性。

關鍵字

光親和性探針 ； 凝集素 ； 碳水化合物

並列摘要

Carbohydrate-lectin interactions involve in various biological recognitions and mediate many processes such as cellular recognition, adhesion, signal transduction, glycoprotein clearance, immunomodulation, inflammation, and host-pathogen recognition. To fully understand the biological implications of carbohydrate-lectin interactions in living organisms, it is imperative to investigate and profile their functions under pathological conditions.
However, it is quite difficult to probe the carbohydrate interacting protein(s) due to the intrinsic characters of carbohydrate-lectin interaction: non-covalent, reversible, and weak binding affinity.
Thus, for low affinity interaction between carbohydrate ligand and lectin, the monovalent carbohydrate ligand conjugated with photoaffinity probe may not be suitable for target lectin labeling. The multivalent carbohydrate structures which greatly enhance the weak affinity of individual mono-ligands to their binding lectins is a solution to overcome the weak affinity problem.
We designed a novel and multifuntional photoaffinity scaffold that derived from a glutamic acid core, carried a powerful photo-cross-linker, 3-trifluoromethyl-3-phenyl-diazirine, and two well-defined orthogonal synthetic handles that empower subsequent conjugations. Multivalent carbohydrate and tag of the probe were conveniently conjugated via orthogonal reactions, Cu(I) catalyzed click reaction and amide bond formation. The tri-valent (compound 43) galactose photoaffinity probe demonstrate that the designed probe is able to selectively label the target
lectin, RCA120 (Ricinus communis Agglutinin) in E. Coli. lysates and ASGP-R (Asialoglycoprotein receptor) on intact HepG2 cell membrane.
Moreover, we had synthesized a second generation carbohydrate photoaffinity probe for enrichment of lectin by combination of multivalent carbohydrate, bio-orthogonal chemistry, photochemical cleavage group, and functional magnetic nanoparticle.

並列關鍵字

photoaffinity group ； lectin ； carbohydrate ； photoaffinity probe

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