開發新穎的生物正交反應並評量唾液酸蛋白在細胞表面的視覺化

Abstract

近年來逐漸發展出一種結合化學方法的生物標定成像工具，稱為Bioorthogonal chemical reporter，可以用於觀察一些生物分子在生物系統上的功能、代謝，例如蛋白質、核酸、醣類等。但這項工具應用於生理環境下存在著許多限制，由其對於標定醣類生物分子，至今只有疊氮化合物發展成很好的Bioorthogonal chemical reporter。 於是我們利用click chemistry、Diels–Alder reaction and addition-elimination這三個反應設計為唾液酸生合成細胞標定的Bioorthogonal反應，於是分別設計合成帶有疊氮、1,2,4,5-tetrazine以及1,2 diketone的甘露醣胺衍生物為Bioorthogonal chemical reporter (化合物5、45和55)，以及另一部份帶有螢光的反應化合物(16，39，48，52和62)。然而從這三個反應的合成過程中，成功突破了化合物35的反應性得到化合物45，也成功改善了化合物39的水溶性得到化合物48，但其中化合物48螢光基團對生物的緩衝液1X PBS溶解性不好而且對細胞染色並沒有專一性。不過最後成功合成出含有生物素的化合物52，可以穩定的存在於生物的環境中。接著和化合物45利用流式細胞儀去測試在MDA-MB-231乳癌細胞表面上的唾液酸標定量，但很可惜的並無成功的偵測到被標定的唾液酸，不過，藉由以上的經驗我們已經找到一個適合在生物環境中做Bioorthogonal reaction的方向，接下來只要在Bioorthogonal chemical reporter的設計上有所改良且能為唾液酸生合成之酵素所接受並催化，讓它能成功的表現在細胞表面，就可以順利表達出我們要標定的目標生物分子唾液酸。A bioorthogonal chemical reporter is a tool for visualizing and tagging biological molecules, observing their functional behaviour in living systems, however it is physiologically demanding and challenging for limited use in vivo. To our knowledge, few bioorthogonal chemical reporters (except azide) with an inert property towardcell’s metabolic enzymes have been reported so far. Here we describe the exploration and synthesis of azide-, 1,2,4,5-tetrazine-, and 1,2 diketone-containing N-alpha-acetylmannosamines (compounds 5, 45 and 55), which employ methods of click chemistry、Diels–Alder reaction and addition-elimination to facilitate their chemical ligations with fluorescent conjugate-partners (16, 39, 48, 52 and 62), and demonstrate their potential abilities of molecular imaging in vitro. Interestingly, the poor ligation with fluorescent conjugate-partners was initially found for 35; an improvement in this was successfully accomplished by the discovery of 45. On the other hand, an increase of solubility in aqueous buffer is solved by modifying fluorescent conjugate 39 into compound 48, which does display multiple/random recognitions toward bimolecular donors on the surface of cells. In order to provide an unique binding affinity toward 1,2,4,5-tetrazine-containing N-alpha-acetylmannosamines (45) rather than other bimolecular donors, we have identified and synthesized a biotin-norbornene conjugate 52 with suitable properties in cell culture. Building on these effective ligation and unique recognition studies in vitro (45 and 52), we want to investigate the ability of imaging sialic acid on cell surfaces by treating MDA-MB-231 cells with 1,2,4,5-tetrazine-containing N-alpha-acetylmannosamines (45), followed by sequential visualization (by reaction with 52) and analysis with flow cytometry. Unfortunately, this technique has not enabled visualization of sialic acid in living cells, not successful recognitions of 45 by these enzymes during the process of biosynthesis. However, the results demonstrated here should pave the way for the redesign of new chemical reporters in the near future.