New methods of regulating protein functions

一种高时空分辨率的蛋白质功能调控方法将为细胞化学研究提供新的工具。笼状化合物已被认为是解释各种生物过程的强大工具，具有很高的空间和时间分辨率。我们报道了6-溴-7-羟基香豆素-4-甲基(BHC)基团作为一种新型的光可移除保护基团，发现BHC笼状神经递质和第二信使可以提高光解效率。为了扩大六六六笼状化合物的用途，我们报道了侧链上带有六六六保护基团的笼状氨基酸、多肽和蛋白质的合成和光化学。选择了四种具有不同侧链功能的氨基酸(天冬氨酸、酪氨酸、赖氨酸和丝氨酸)。所有合成的六六六及其7-甲氧基变体BMC笼状氨基酸的光化学效率都比传统的2-硝基苄基笼状化合物高一个数量级。结果表明，BHC和BMC基团将允许我们开发新的笼式化合物，这些化合物可以在低光强的紫外线照射下产生活性蛋白质和多肽，从而将细胞损伤降至最低。利用体外转录系统，链霉亲和素中的赖氨酸-83被六六六笼养的赖氨酸选择性地取代。这种方法可以作为构建笼状蛋白的通用程序，其生物学功能可以在光照射下被激活。此外，八肽的赖氨酸残基(Glu-Ala-Val-Ser-Leu-Lys-Pro-Thr)也通过Fmoc固相化学取代了ε蛋白激酶C转位和功能的选择性拮抗剂。合成的笼状肽在350nm光照射下释放了亲本八肽，光解量子产率(Φ)为0.082，总光解效率(Φε)为435%，足以满足活细胞应用的需要。

A method of regulating protein function with high spatial and temporal resolution would offer a new tool of investigating cellular chemistry. Caged compounds have been recognized as a powerful tool for elucidating various biological processes with high spatial and temporal resolution. We have reported 6-bromo-7-hydoroxycoumarin-4-ylmethyl (Bhc) group as a novel photoremovable protecting group and found that Bhc-caged neurotransmitters and second messengers have improved photolysis efficiencies. To extend the utility of the Bhc-caged compounds, we report here the synthesis and photochemistry of caged ammo acids, peptides and proteins having the Bhc protecting group on their side chain functional groups. Four types of amino acids (Asp, Tyr, Lys, and Ser) that have different side chain functionality were selected. All of the synthesized Bhc and its 7-methoxy variant Bmc-caged amino acids showed about an order of magnitude larger photochemical efficiencies than those of the conventional 2-nitrobenzyl-caged compounds. The results suggest that the Bhc and Bmc groups would allow us to develop new caged compounds that can generate active proteins and peptides upon UV irradiation with low light intensity to minimize cell damages. Using an in vitro transcription system, lysine-83 in streptavidin was selectively replaced by Bhc-caged Lys. The method could be a general procedure to construct caged proteins whose biological functions can be activated upon photo-irradiation. In addition, Lys residue of octa-peptide (Glu-Ala-Val-Ser-Leu-Lys-Pro-Thr), a selective antagonist of εPKC translocation and function, was also replaced by Bmc-caged Lys by utilizing Fmoc solid-phase chemistry. The synthetic caged peptide released the parent octa-peptide on 350-nm irradiation with photolysis quantum yield (Φ) of 0.082 and overall photolysis efficiency (Φε) of 435, which must be high enough for live cell applications.