Yang Byungseop, Kwon Kiyoon, Jana Subhashis, Kim Seoungkyun, Avila-Crump Savanna, Tae Giyoong, Mehl Ryan A, Kwon Inchan
School of Materials Science and Engineering, Gwangju Institute of Science and Technology (GIST), Gwangju 61005, Republic of Korea.
Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon 97331, United States.
Bioconjug Chem. 2020 Oct 21;31(10):2456-2464. doi: 10.1021/acs.bioconjchem.0c00497. Epub 2020 Oct 9.
An inverse-electron-demand Diels-Alder (IEDDA) reaction using genetically encoded tetrazine variants enables rapid bioconjugation for diverse applications and . However, bioconjugation using genetically encoded tetrazine variants is challenging, because the IEDDA coupling reaction competes with rapid elimination of reaction partners . Here, we tested the hypothesis that a genetically encoded phenylalanine analogue containing a hydrogen-substituted tetrazine (frTet) would increase the IEDDA reaction rate, thereby allowing for successful bioconjugation . We found that the IEDDA reaction rate of superfolder green fluorescent protein (sfGFP) containing frTet (sfGFP-frTet) was 12-fold greater than that of sfGFP containing methyl-substituted tetrazine (sfGFP-Tet_v2.0). Additionally, sfGFP variants encapsulated with chitosan-modified, pluronic-based nanocarriers were delivered into nude mice or tumor-bearing mice for imaging. The -delivered sfGFP-frTet exhibited almost complete fluorescence recovery upon addition of trans-cyclooctene via the IEDDA reaction within 2 h, whereas sfGFP-Tet_v2.0 did not show substantial fluorescence recovery. These results demonstrated that the genetically encoded frTet allows an almost complete IEDDA reaction upon addition of trans-cyclooctene, enabling temporal control of bioconjugation in a very high yield.
使用基因编码的四嗪变体进行的逆电子需求狄尔斯-阿尔德（IEDDA）反应能够实现快速生物共轭，以用于多种应用。然而，使用基因编码的四嗪变体进行生物共轭具有挑战性，因为IEDDA偶联反应会与反应伙伴的快速消除相竞争。在这里，我们测试了这样一个假设，即含有氢取代四嗪的基因编码苯丙氨酸类似物（frTet）会提高IEDDA反应速率，从而实现成功的生物共轭。我们发现，含有frTet的超折叠绿色荧光蛋白（sfGFP-frTet）的IEDDA反应速率比含有甲基取代四嗪的sfGFP（sfGFP-Tet_v2.0）快12倍。此外，用壳聚糖修饰的基于普朗尼克的纳米载体包裹的sfGFP变体被递送至裸鼠或荷瘤小鼠体内进行成像。通过IEDDA反应在2小时内加入反式环辛烯后，递送的sfGFP-frTet几乎完全恢复荧光，而sfGFP-Tet_v2.0没有显示出明显的荧光恢复。这些结果表明，基因编码的frTet在加入反式环辛烯后能够实现几乎完全的IEDDA反应，从而能够以非常高的产率对生物共轭进行时间控制。
