Hiefinger Caroline, Marcon Michela, Pape Verena, Casadevall Guillem, Lahmy Ranit, Haag Christoph, Nazet Julian, Bartl Michael, Bruckmann Astrid, Osuna Sílvia, König Burkhard, Hupfeld Andrea
Institute of Biophysics and Physical Biochemistry and Regensburg Center for Biochemistry, University of Regensburg, Universitätsstraße 31, D-93053, Regensburg, Germany.
Institute of Organic Chemistry, University of Regensburg, Universitätsstraße 31, D-93053, Regensburg, Germany.
Angew Chem Int Ed Engl. 2025 Sep 15;64(38):e202508562. doi: 10.1002/anie.202508562. Epub 2025 Aug 4.
Photoswitchable unnatural amino acids (psUAAs) play a crucial role in the engineering of light-sensitivity in enzymes, which holds significant promise for diverse applications such as biotherapy and biocatalysis. Besides near-quantitative photoconversion, the success and expediency of a psUAA for a certain application is defined by its interaction potential with the enzyme, its thermal stability and its effective wavelength of irradiation. To establish high versatility in the current repertoire, we have designed and synthesized six psUAAs based on azobenzene, arylazopyrazole, arylazothiazole, hemithioindigo and spiropyran photoswitches. The resulting psUAAs exhibit an enhanced interaction potential within an enzyme owing to their capacity for hydrogen bonding, ionic interactions and metal ion coordination. Moreover, we observed diverse photochemical behaviors among the psUAAs, with four of them reversibly switching between the isomers with purely visible light. Notably, we identified orthogonal aminoacyl-tRNA synthetases that facilitate the incorporation of five of the six psUAAs co-translationally and computationally analyzed the synthetase-psUAA interactions. Finally, we evaluated the photochemical behavior of the five psUAAs within an enzymatic model and tested the photocontrol of catalysis confirming their diversity. Ultimately, our findings significantly expanded the repertoire of psUAAs and demonstrated their feasibility for enzyme engineering studies.
光开关非天然氨基酸（psUAA）在酶的光敏感性工程中起着关键作用，这对于生物治疗和生物催化等多种应用具有重要前景。除了近乎定量的光转化外，一种psUAA在特定应用中的成功与便捷性取决于其与酶的相互作用潜力、热稳定性以及有效的照射波长。为了在当前的库中建立高通用性，我们基于偶氮苯、芳基偶氮吡唑、芳基偶氮噻唑、半硫代靛蓝和螺吡喃光开关设计并合成了六种psUAA。由于它们具有氢键、离子相互作用和金属离子配位的能力，所得的psUAA在酶内表现出增强的相互作用潜力。此外，我们观察到psUAA之间存在多样的光化学行为，其中四种在纯可见光下在异构体之间可逆切换。值得注意的是，我们鉴定出了正交氨酰 - tRNA合成酶，其有助于六种psUAA中的五种共翻译掺入，并通过计算分析了合成酶 - psUAA相互作用。最后，我们在酶模型中评估了五种psUAA的光化学行为，并测试了催化的光控，证实了它们的多样性。最终，我们的研究结果显著扩展了psUAA的库，并证明了它们在酶工程研究中的可行性。
