Quinoline-Based Photolabile Protection Strategy Facilitates Efficient Protein Assembly
Quinoline-Based Photolabile Protection Strategy Facilitates Efficient Protein Assembly
复制标题
基于喹啉的光不稳定保护策略促进高效的蛋白质组装
DOI：
10.1021/jacs.1c10324
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发表时间：
2022
期刊：
影响因子：
--
通讯作者：
Ping Wang
中科院分区：
文献类型：
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作者：
Siyao Wang;Qingqing Zhou;Yunxue Li;Bingcheng Wei;Xinliang Liu;Jie Zhao;Farong Ye;Zhongneng Zhou;Bei Ding;Ping Wang
天然化学连接(NCL)为组装具有精确化学特征的蛋白质提供了一个强大的解决方案，这使得详细研究蛋白质结构与功能的关系成为可能。作为NCL的延伸，脱硫法和表达蛋白连接(EPL)技术的发现极大地扩展了通过化学连接获得大的或具有挑战性的蛋白质序列的有效途径。尽管NCL-脱硫协议具有优异的可靠性，但它需要正交保护策略，以允许在本地半胱氨酸存在的情况下选择性脱硫，这对其合成应用至关重要。与传统的硫醇保护基团不同，耐光性保护基团(PPG)在照射后被移除，简化了蛋白质组装，因此对多肽支架的扰动最小。然而，目前的PPG策略主要局限于硝基-苄基衍生物，这与NCL-脱硫是不兼容的。在这里，我们首次提出了基于喹啉的半胱氨酸PPG可以促进各种连接策略，包括迭代的NCL和EPL-脱硫法。7-(哌嗪-1-基)-2-(甲基)喹啉(PPZQ)笼化蛋白质序列中的多个半胱氨酸残基可以通过后期修饰容易地引入，而PPZQ的无痕迹去除通过在水中的缓冲液中的光解是高效的。此外，PPZQ基团与自由基脱硫剂相容。γ-突触核蛋白和磷酸化胱抑素-S通过一锅迭代连接和EPL-脱硫法的合成凸显了该策略的有效性。此外，成功地对表达的IL-34片段进行了六重保护和去保护，显示了该策略在蛋白质笼化/去中心化研究中的巨大潜力。
Native chemical ligation (NCL) provides a powerful solution to assemble proteins with precise chemical features, which enables a detailed investigation of the protein structure–function relationship. As an extension to NCL, the discovery of desulfurization and expressed protein ligation (EPL) techniques has greatly expanded the efficient access to large or challenging protein sequences via chemical ligations. Despite its superior reliability, the NCL-desulfurization protocol requires orthogonal protection strategies to allow selective desulfurization in the presence of native Cys, which is crucial to its synthetic application. In contrast to traditional thiol protecting groups, photolabile protecting groups (PPGs), which are removed upon irradiation, simplify protein assembly and therefore provide minimal perturbation to the peptide scaffold. However, current PPG strategies are mainly limited to nitro-benzyl derivatives, which are incompatible with NCL-desulfurization. Herein, we present for the first time that quinoline-based PPG for cysteine can facilitate various ligation strategies, including iterative NCL and EPL-desulfurization methods. 7-(Piperazin-1-yl)-2-(methyl)quinolinyl (PPZQ) caging of multiple cysteine residues within the protein sequence can be readily introduced via late-stage modification, while the traceless removal of PPZQ is highly efficient via photolysis in an aqueous buffer. In addition, the PPZQ group is compatible with radical desulfurization. The efficiency of this strategy has been highlighted by the synthesis of γ-synuclein and phosphorylated cystatin-S via one-pot iterative ligation and EPL-desulfurization methods. Besides, successful sextuple protection and deprotection of the expressed Interleukin-34 fragment demonstrate the great potential of this strategy in protein caging/uncaging investigations.