Chemical Biology Research Group, Institute of Organic Chemistry, ELKH Research Centre for Natural Sciences, Magyar Tudósok Krt 2, H-1117 Budapest, Hungary.
Molecular Cell Biology Research Group, Institute of Enzymology, ELKH Research Centre for Natural Sciences, Magyar Tudósok Krt 2, H-1117 Budapest, Hungary.
Molecules. 2021 Aug 18;26(16):4988. doi: 10.3390/molecules26164988.
Bioorthogonal click-reactions represent ideal means for labeling biomolecules selectively and specifically with suitable small synthetic dyes. Genetic code expansion (GCE) technology enables efficient site-selective installation of bioorthogonal handles onto proteins of interest (POIs). Incorporation of bioorthogonalized non-canonical amino acids is a minimally perturbing means of enabling the study of proteins in their native environment. The growing demand for the multiple modification of POIs has triggered the quest for developing orthogonal bioorthogonal reactions that allow simultaneous modification of biomolecules. The recently reported bioorthogonal [4 + 1] cycloaddition reaction of bulky tetrazines and sterically demanding isonitriles has prompted us to develop a non-canonical amino acid (ncAA) bearing a suitable isonitrile function. Herein we disclose the synthesis and genetic incorporation of this ncAA together with studies aiming at assessing the mutual orthogonality between its reaction with bulky tetrazines and the inverse electron demand Diels-Alder (IEDDA) reaction of bicyclononyne () and tetrazine. Results showed that the new ncAA, bulky-isonitrile-carbamate-lysine () is efficiently and specifically incorporated into proteins by genetic code expansion, and despite the slow [4 + 1] cycloaddition, enables the labeling of outer membrane receptors such as insulin receptor (IR) with a membrane-impermeable dye. Furthermore, double labeling of protein structures in live and fixed mammalian cells was achieved using the mutually orthogonal bioorthogonal IEDDA and [4 + 1] cycloaddition reaction pair, by introducing through GCE and through a HaloTag technique.
生物正交点击反应是一种理想的方法，可以选择性和特异性地用合适的小合成染料标记生物分子。遗传密码扩展 (GCE) 技术能够有效地在感兴趣的蛋白质 (POI) 上选择性地安装生物正交接头。非天然氨基酸的生物正交化是一种最小干扰的方法，可以在其天然环境中研究蛋白质。对 POI 进行多次修饰的需求不断增长，这促使人们寻求开发正交生物正交反应，以允许同时修饰生物分子。最近报道的大体积四嗪和空间要求苛刻的异腈的生物正交 [4+1] 环加成反应促使我们开发了一种带有合适异腈官能团的非天然氨基酸 (ncAA)。本文介绍了该 ncAA 的合成和遗传掺入，并进行了旨在评估其与大体积四嗪反应的相互正交性以及双环壬炔 () 和四嗪的逆电子需求 Diels-Alder (IEDDA) 反应的研究。结果表明，新的 ncAA，大体积异腈-氨基甲酸酯-赖氨酸 () 通过遗传密码扩展被有效地特异性掺入蛋白质中，尽管 [4+1] 环加成反应缓慢，但能够用不透膜的染料标记胰岛素受体 (IR) 等外膜受体。此外，通过引入 GCE 中的 并通过 HaloTag 技术引入 ，使用相互正交的生物正交 IEDDA 和 [4+1] 环加成反应对活细胞和固定哺乳动物细胞中的蛋白质结构进行了双重标记。
