Fluolab【JACS】新型红移芳香阳离子探针，绿色光照也能给蛋白质“贴标签”
通讯作者： Michael T. Taylor (University of Arizona)
文章概要
引言
光介导的蛋白质标记技术是化学生物学研究中的核心工具，其通过空间和时间上的精准控制，为揭示生命活动中的蛋白质功能提供了可能。然而，传统的标记策略往往依赖高能紫外光或紫光，这不仅容易产生细胞毒性，也限制了光线在组织中的穿透深度。为了克服这一瓶颈，研发能够被长波长可见光激活的生物相容性化学反应变得至关重要。虽然近年来在红移成像染料和光开关方面取得了显著进展，但针对蛋白质共价连接的红移光化学反应开发却相对滞后。Michael T. Taylor团队此前曾报道过利用N-取代吡啶盐通过光诱导电子转移（PET）机制实现色氨酸（Trp）的选择性标记，但在如何保持探针分子量微小、膜渗透性良好且激发波长进一步红移之间寻找平衡，依然是极具挑战的课题。
Figure 1. (A) Photoinduced electron transfer-driven protein ligation using N-substituted pyridinium salts. (B) Design considerations for N-substituted pyridinium salts for protein ligation triggered at longer wavelengths of light. (C) This work: design principles and applications of N-substituted pyridinium salts for photoinduced protein ligation at longer wavelengths of light.
主要实验及结论
研究人员首先通过系统的结构-活性关系研究，探索了多种供体-受体型吡啶盐的电子特性。实验发现，简单的共轭延伸并不一定能转化为有效的标记能力，例如带有强供电子胺基的衍生物虽然吸收波长显著红移，却因扭曲分子内电荷转移（TICT） 或光异构化等非辐射衰减途径而失去了光化学活性。为了解决这一矛盾，团队提出了构型限制的设计理念，开发出一系列具有色烯（Chromene）供体核心的新型探针，如探针8、9和11。这些探针不仅成功将吸收光谱推向了420-530 nm的可见光区，同时维持了极小的分子体积，甚至比前代产品更小。在针对溶菌酶和糜蛋白酶原的实验中，这些新型芳香阳离子展现出了优异的色氨酸位点选择性，并在生理条件下表现出稳健的标记效率。
Figure 2. (A) Select photophysical and calculated properties of probes 1-11. Values were measured in 20 mM Na2HPO4 (pH 7.4) except where stated otherwise. (B) Labeling of lysozyme with probes 1-11. (C) Comparisons of the absorption maxima and calculated molecular volume of select probes. _a_Lowest-energy transition. b Measured in pH 3.5 20 mM Na2HPO4. c Measured in water. d from ref (6)c. _e_Calculated at the CAMB3LYP 6–31g+(d,p) SMD = H2O level of theory. f Estimated using experimental _E_0,0 values and calculated reduction potentials. (16) g Volume calculated from DFT-optimized structures.
Figure 3. (A) Retrosynthetic strategies for pyridinium salts. (B) General synthetic strategy for pyridinium probes 7-11. Analogs 8a–11a, as well as 8b and 11b were accessed using this route (Figures 6 and 7)._a_DMF was used for 10.
Figure 4. (A) 11 displays reversible, pH-dependent changes to electronic structure. (B) Observation by LC-MS of hydrate formation of 11 after incubation in pH 7.4 Na2HPO4 buffer. (C) pH-dependent changes to absorption at 467 nm. (D) Proposed mechanism of pH-sensing by 11 via reversible, pH-dependent, hydrate formation. (E) pH-dependent labeling of lysozyme using 11. (F) Turn-on fluorescence effect of 10 in sodium dodecyl sulfate (SDS).
深入的机理研究揭示了探针11独特的pH敏感性。该探针在生理pH环境下会形成一种光惰性的水合物，而这种平衡会随着酸性的增加向具有活性的阳离子状态偏移。利用这一特性，研究人员实现了免洗、活细胞溶酶体成像，能够长时间观察酸性细胞器的形态变化而不产生传统染料的碱化干扰。在化学生物学应用层面，团队合成了带有生物素或叠氮基团的功能化探针，成功在530 nm绿光下完成了活细胞内的蛋白质组学标记。质谱分析结果显示，探针8b和11b富集的蛋白亚群具有高度的互补性，前者倾向于定位在线粒体和高尔基体，而后者则表现出明显的内质网选择性。这种通过分子结构微调实现的细胞器特异性标记，为研究特定微环境下的蛋白功能提供了精准的“雷达”。
Figure 5. Absorption/emission spectra of pyridinium probes in 20 mM pH 7.4 NaH2PO4: (A) 8, (B) 10, (C) 11. (D) Confocal imaging of live HeLa cells using compounds 8-11. (E) Live cell confocal imaging using 8 (λex = 405 nm). (F) Live HeLa cell imaging under wash and wash-free conditions using 10 (λex = 561 nm). (G) Wash-free live cell imaging using 11. (λex = 488 nm) (H). The photobleaching profile of 11 was obtained using 488 nm excitation at 5% intensity (25 mW, Zeiss LSM 880 Argon laser). _a_Cells were washed 3× with PBS buffer. _b_Wash-free imaging.
Figure 6. (A) Photophysical properties of chemical proteomic pyridinium probes 8a and 8b in 20 mM Na2HPO4 (pH 7.4). (B) Proteome profiling of HEK293T lysates using biotinylated probes. (C) Western analysis of proteome HEK293T lysate profiling with probes 1a, 8a, 9a, and 11a. _a_Lowest-energy transition. _b_Measured in water.
- Figure 7. (A) Live HeLa cell labeling using 8b and 11b with 530 nm irradiation. Adapted from NIAID Visual & Medical Arts, 10/7/2024. Low Friction Flask. Source: bioart.niaid.nih.gov/bioart/303. (B) Volcano plot showing the enrichment of 659 protein entities using 8b. (C) Top 10 biological processes from EnRichr GO analysis of enriched proteins from 8b (Mt = mitochondria). (D) Top 10 orphan disease associations of enriched proteins from 8b. (E) Volcano plot showing the enrichment of 344 protein entities using 50 μM 11b. (F) Top 10 GO Cellular components for proteins enriched with 11b. (G) Venn diagram of proteomic subsections enriched by 8b and 11b. _a_Performed with 8b only.
总结及展望
该研究成功构建了一套高效的红移芳香阳离子探针库，实现了低能量可见光驱动的蛋白质高效偶联。通过引入构型限制的策略，研究人员不仅解决了光物理衰减与反应活性之间的冲突，还赋予了探针独特的微环境感知能力。这一成果将蛋白质标记、活细胞成像与质谱蛋白组学分析有机统一在同一套分子体系中，极大地扩展了芳香阳离子在化学生物学中的应用维度。未来，这种基于长波长激发的标记策略有望在更高层级的复杂生物系统乃至多细胞生物中大显身手，为精准医疗和特定疾病状态下的微环境蛋白质分析奠定坚实的技术基础。