衍生Alpha-鹵代酮之可觸發侷限基團並應用於光調控螢光與反應性

Abstract

標靶型共價抑制劑可藉由與目標生物分子形成共價鍵而使其失活，而Alpha-鹵代酮是其中一類常用的反應基團。標靶型共價抑制劑在臨床和生醫研究上皆扮演著重要的角色，但由於其較高的化學反應性，脫靶效應和其導致的生物毒性是一大為人詬病之處。 可控釋放可能是這個問題的解答。藉由可控的侷限基團屏蔽其原先的反應性，使活性基團必須在特定條件下才能被釋出。同時，由於增強型螢光(Turn on fluorescence)在分子探針上的優勢，我們希望進一步衍生原本的侷限基團為一雙官能化的侷限基團，使其能同時屏蔽分子探針的螢光與反應性。 我們先利用三種簡單的模型分子驗證可控釋放和屏蔽反應性的可行性，並將光觸發的侷限基團應用於實際的模型標靶型共價抑制劑上。我們以全合成得到FMK及其類似物CMK，並將其接上侷限基團。我們以HPLC色譜圖證實了CMK可以藉由UV照射成功被釋放出來；同時，西方墨點法也證實侷限基團能屏蔽抑制劑對RSK2蛋白自磷酸化的抑制效果。雙官能化侷限基團的合成仍在進行且即將完成，我們希望能藉此探針即時顯影細胞中RSK2的分布情形。

Targeted covalent inhibitors (TCIs) are small molecules designed to inactivate their biological targets by covalent binding, and alpha-haloketone is one of the electrophilic warheads commonly used. For years, TCIs have played important roles in both clinical trials and biological researches. However, due to intrinsic reactivity of the electrophilic warheads, off-target effect is one of the main concerns for TCIs, which results in cytotoxicity. Controlled releasing could be a strategy to overcome this problem. By masking reactivity with a triggerable protecting group, the warhead can be regenerated only under specific condition. Furthermore, for turn-on fluorescence is another powerful tool in molecular probes, we also aim to derive the original capping group into a dual-functional version, masking both fluorescence and reactivity. We first confirmed the viability of controlled releasing and reactivity attenuation by model alpha-haloketones. The light-responsive capping group was further applied to the real TCI model. FMK and its analog CMK was total synthesized and capped. HPLC chromatograph showed successful releasing of the original inhibitor CMK under UV irradiation, and we also confirmed the attenuation of inhibition reactivity toward autophosphorylation of RSK2 on Ser386 by Western blot analysis. Synthesis of the dual-functional trigger is on-going, and we hope to utilize this probe for real-time imaging of endogenous RSK2.