Fluolab【JACS】破局1000纳米！实现深层组织高特异性生物共轭标记的无催化红外化学反应
文章标题：TagC-RED: An Infrared-Triggered Retro-Ene Reaction for Deep-Tissue Bioconjugation
通讯作者：Robert S. Paton*, Monika Raj*, Tianning Diao*
文章概要
引言
生物共轭反应是原位研究生物大分子功能不可或缺的工具。尽管光活化生物共轭技术具备优异的时空控制能力，但现有的方法面临着巨大的实际局限。传统技术普遍依赖高能紫外光或可见光，这不仅带来了严重的细胞光毒性风险，而且由于光散射和吸收限制了在活体深层组织中的穿透深度。此外，许多反应还需要依赖具有细胞毒性的重金属催化剂，或者必须引入复杂的非天然官能团。理论上，能够穿透深层组织的长波长红外光（大于1000纳米） 是理想的激发源，然而红外光子的能量通常低于典型共价键的断裂能，直接引发化学键剪切面临巨大的化学挑战。为了攻克这一瓶颈，研究团队成功开发出一种名为TagC-RED的全新光反应平台。该反应利用重氮化合物在红外光激发下发生的逆烯（Retro-Ene）型周环重排，实现了无需催化剂、高度特异性、定量且快速的半胱氨酸共轭标记，为活体内的化学生物学研究和精准诊断开辟了全新路径。
Figure 1. Challenges and solutions to photobioconjugation. (A) Existing challenges in photobioconjugation associated with the necessity of high-energy irradiation. (B) Chemical challenges of using infrared light for triggering reactions involving covalent bond cleavage. (C) Mechanistic hypothesis of the retro-ene reaction for catalyst-free photobioconjugation triggered by infrared light.
主要实验及结论
研究人员首先合成了一种带有邻位硫醚和炔基锚定基团的水溶性重氮化合物作为反应探针，并在水相环境中以含有多种亲核性侧链的八肽进行条件筛选。实验结果表明，在无需任何光催化剂的情况下，该体系在绿色可见光、红色可见光以及1040纳米脉冲红外激光照射下均能表现出极其高效的转化率，在数分钟内定量形成半胱氨酸特异性的二硫键共轭产物。得益于半胱氨酸在天然蛋白质中较低的丰度以及该反应极高的化学选择性，即便在面对赖氨酸、丝氨酸、酪氨酸等常规亲核性氨基酸时，探针也没有产生任何副反应。
Figure 2. Bioconjugation of peptides with infrared irradiation. (A) Development of conditions for the bioconjugation of Cys-containing peptides. (B) HPLC trace of the crude reaction mixture. (C) Application of TagC-RED to label peptides with d-biotin.
随后，研究团队将TagC-RED反应成功应用到了生物大分子和复杂细胞环境中。在蛋白质水平上，无论是泛素突变体、牛血清白蛋白还是临床相关的单克隆抗体（如曲妥珠单抗），该反应均能实现快速且配比精准的化学修饰。令人惊叹的是，当反应容器被4毫米厚的培根肉包裹以模拟真实生物组织屏障时，1040纳米的红外激光依然能够轻松穿透屏障，驱动定量转化，这有力证明了其卓越的深层组织穿透能力。在细胞实验中，探针展现出良好的细胞膜渗透性，在红外光照射下成功实现了HeLa细胞内特定内体蛋白质的荧光和生物素标记，并能够通过细胞内半胱氨酸表达水平的差异来区分不同的细胞系。更进一步地，活体小鼠实验和小鼠全脑外源标记实验证实，在1064纳米红外光引导下，该反应可以在活体小鼠肝脏及小鼠大脑深层结构中实施精准的时空选择性生物共轭标记，展现出巨大的转化医学潜力。
Figure 3. Application of TagC-RED for protein labeling. (A) Conjugation of Ub-K63C with a “Click” handle under 660 nm irradiation. (B) Conjugation of BSA with Biotin under 660 or 1040 nm irradiation. Western blot: Lane 1: BSA in the presence of 6 under ambient light; Lane 2: BSA in the presence of 6 exposed to pulsed 1040 nm IR irradiation; Lane 3: BSA in the presence of diazonium 6, wrapped with 4 mm-thick bacon, exposed to pulsed 1040 nm IR laser irradiation; Lane 4: BSA alone in the absence of 6. (C) Conjugation of BSA with fluorescein under 660 nm irradiation. SDS-PAGE: Lane 1: BSA alone in the absence of 9; Lane 2: BSA in the presence of 9 with 660 nm red light irradiation; Lane 3: BSA in the presence of 9 in the dark. (D) Conjugation of trastuzumab S239C with d-biotin under 660 nm red light irradiation.
Figure 4. Application of TagC-RED to intracellular bioconjugation and tissue imaging. (A) Schematic of the intracellular protein labeling protocol with TagC-RED. (B) Western blot analysis of HeLa cells treated with TagC-RED biotin-labeling with or without 1040 nm IR irradiation. (C) Confocal images of HeLa cells treated with TagC-RED fluorescent labeling probe 9 with and without 660 nm red light irradiation. Depicted scale bar is 10 μm. (D) Tissue imaging of mice organs via TagC-RED. Fluorescence and brightfield imaging of mice brain tissue after the whole brain was treated with TagC-RED with and without 740 nm IR irradiation. No fluorescence observed without light treatment. Scale bar represents 200 μm. Experiments were repeated in triplicate with similar imaging results. (E) in vivo labeling of mice with probe 1 followed by ex vivo labeling with Cy5 azide via Click chemistry. IVIS Imaging and quantification of livers exposed in vivo to probe 1 with or without light. Top row = 1064 nm light exposure, bottom = no light exposure. Experiments were repeated in triplicate.
深入的机理研究和密度泛函理论（DFT）计算揭示了这一奇迹背后的化学本质。实验数据和光谱分析表明，半胱氨酸与重氮探针会在溶液中自发组装形成电子供体-受体（EDA）配合物。这种电荷转移复合物的吸收光谱大幅红移，直接延伸到了红外光区，从而赋予了低能量红外光子诱导电子转移的能力。当受到红外光激发后，体系会发生一步法自由基过程，引发高驱动力的氢原子转移，进而诱导逆烯重排并释放硫苯自由基，最终与半胱氨酸衍生自由基高效结合。理论计算进一步表明，质子溶剂对电荷分离状态的稳定作用以及激发态自由基对极慢的热回迁电子转移速度，共同保障了该反应高达0.55的反应量子产率，这使其在光子通量高度受限的活体深层组织中依然能够高效运转。
Figure 5. Studies of the stability of the disulfide linkage and the mechanism. (A) Stability test of 14 (6 mM) in H2O, 1× PBS, or 1× MEM, all three with added glutathione (30 mM). (B) UV–vis studies of the interaction between 1 and Cys: Cys = 1, 2, 3, 4, and 5 equiv relative to [1]. (C) Quantum yield and control experiments.
Figure 6. Computational modeling of donor–acceptor complex formation and photochemistry. (A) Two classes of donor–acceptor complexes are observed: ion-collapsed 19 and charge-separated 20. Complex 20 is favored upon inclusion of explicit water solvent molecules, highlighting the role of hydrogen bonding in stabilizing charge separation. (B) Lowest-energy absorption wavelengths and oscillator strengths for various conformers of complexes 19 and 20. Complex 19 absorbs predominantly in the visible region below 550 nm, while complex 20 exhibits near-IR absorption across its conformers. (C) Thermodynamic driving force (ΔG°) and reorganization energy (λ) associated with back electron transfer (BET) from the excited singlet state (S1) to the ground state (S0). The large λ leads to high activation barriers and a long half-life for the BET process.
总结及展望
这项研究成功建立了基于红外光驱动的TagC-RED生物共轭反应平台。该反应彻底摆脱了传统光反应对高能有害光源和外源催化剂的依赖，完美兼顾了生物相容性、高特异性以及无与伦比的深层组织穿透性。作为一种可在活体系统内部署的通用工具，TagC-RED技术在蛋白质实时精准标记、复杂生物大分子相互作用示踪、活体动态过程监测以及新一代靶向药物输送和疾病诊疗策略的设计中，都将发挥极其核心的推动作用。