New methods of regulating protein functions

一种具有高空间和时间分辨率的调节蛋白质功能的方法将为研究细胞化学提供一种新的工具。笼化化合物已被认为是一种强有力的工具，用于阐明各种生物过程具有高空间和时间分辨率。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降解保护基团，并发现Bhc笼的神经递质和第二信使具有提高的光解效率。为了扩大六六六笼化化合物的应用，我们在这里报道了在其侧链官能团上具有六六六保护基团的笼化氨基酸、肽和蛋白质的合成和光化学。选择了具有不同侧链功能的四种氨基酸（Asp、Tyr、Lys和Ser）。所有合成的Bhc及其7-甲氧基型bmc -笼氨基酸的光化学效率都比传统的2-硝基苯笼化合物的光化学效率高一个数量级。结果表明，Bhc和Bmc基团将使我们能够开发出新的笼化化合物，这些化合物可以在弱光照射下产生活性蛋白质和肽，从而最大限度地减少细胞损伤。利用体外转录系统，选择性地将链霉亲和素中的赖氨酸-83替换为bhc笼型赖氨酸。该方法可作为构建光照射激活生物功能的笼化蛋白的通用方法。此外，利用Fmoc固相化学方法，将εPKC易位和功能的选择性拮抗剂-八肽（Glu-Ala-Val-Ser-Leu-Lys-Pro-Thr）的Lys残基替换为bmc笼型Lys。合成的笼状肽在350 nm照射下释放亲本八肽，光解量子产率（Φ）为0.082，总光解效率（Φε）为435，这对于活细胞应用来说必须足够高。

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.