【JACS】光照秒变超分辨荧光染料！通过C–H活化定制光活化氧杂嗪，定位精度达2.6纳米
文章标题：Phenoxazines with a Phototransferable N-Acetyl Group and Acrylate Linker: Assembly by C–H Activation, Photoconversion to Fluorescent Dyes, Biolabeling, and Super-Resolution Imaging
通讯作者：Mariano L. Bossi, Vladimir N. Belov, Lutz Ackermann, Stefan W. Hell
文章概要
引言
光活化荧光染料作为生物成像与超分辨纳米显微技术的核心工具，在极高时空分辨率下追踪细胞动态过程和观察生物标本纳米级组织结构方面发挥着至关重要的作用。传统的常规吩噻嗪或氧杂嗪类染料凭借其极高的亮度、远红外发射以及优异的单分子光稳定性，在荧光显微镜中得到了广泛的应用，但长期以来科学界一直缺乏一种能够兼顾多种发射颜色与活性反应基团的光活化氧杂嗪通用合成路径。由于天然的氧杂嗪核心缺乏类似于罗丹明核心的羧酸等易于偶联的官能团，导致传统的不对称修饰合成路线极其冗长繁琐。为了打破这一瓶颈，研究团队创新性地引入了过渡金属催化的后期C–H键活化策略，利用极其简单易得的对称前体或商业化试剂，开发出了一条极其高效、简短且具备高度区域选择性的光活化氧杂嗪染料合成新路线，成功为超分辨成像领域带来了高性能的新型分子荧光标签。
Scheme 1. Irradiation with Light Transforms Phenoxazines Introduced in This Work into Fluorescent Products
主要实验及结论
研究人员首先探索了光活化氧杂嗪染料分子结构的精妙设计与高效合成路线。通过巧妙利用弱配位的N10-乙酰基的邻位定向效应，成功在催化剂体系下实现了吩噻嗪核心C1位的选择性氧化烯烃化反应。实验测试表明，在C1位直接引入丙烯酸酯连接基团能够有效扩展分子的共轭体系，使吸收光谱产生大约50纳米的显著红移，从而将吸收带成功扩展至400纳米以上的可见光波段，为温和的光活化提供了先决条件。在活化机制方面，光解监测证实该系列分子在紫外或紫光照射下，N10位的乙酰基发生同位断裂并迅速通过特殊的1,5-自由基迁移被邻位的丙烯酸酯双键高效捕捉，随后经过进一步的 photooxidation（光氧化）过程，最终原位转化为具有强荧光发射的非笼变化合物。这一创新的分子设计不仅省去了传统化学合成中极其繁琐的异构体高效液相色谱分离步骤，更通过将羧酸基团转化为酰胺或各种功能性配体，成功制备出了涵盖橙色到远红外光谱区间的系列光笼型分子标签。
Scheme 2. Photolysis of N10-Acyl Phenoxazines 1-R in Acetonitrile Solution; R = Me, CBr3, C6H5, 4-BrC6H4, 3,5-Br2C6H3
Scheme 3. 3,7-Bis(N,N-diethylamino)phenoxazines 2-R and 3 with an N10-Acetyl “Photocaging” Group and an Ethenyl or Alkyl Linker to COOH Reactive Site
为了验证这些光笼型染料在生物标本中的靶向标记性能与光活化动态表现，研究团队将其进一步衍生为了能够特异性识别融合蛋白的配体。研究人员通过体外重组蛋白结合实验，深入测试了包括CA1到CA4在内的四种具有不同氨基取代基的ω-氯代烷烃酰胺衍生物在自由状态下以及与HaloTag受体蛋白特异性共价结合后的光谱变化规律。令人振奋的是，这些化合物在结合目标蛋白后表现出了极其优异的环境自适应性，不仅光活化速率得到了显著的提升，而且光解脱笼后的氧杂嗪荧光产物也展现出更长的荧光寿命和更高的发光对比度，完全消除了酰胺键断裂导致染料脱落的潜在风险。紧接着在活细胞和固定细胞的成像实验中，研究人员将这些探针精准靶向递送至内质网膜蛋白、线粒体、波形蛋白及核纤层等多种亚细胞结构中，利用405纳米激光进行局部扫描激活，观察到了从近乎无荧光的极暗“关闭”状态向极亮远红外荧光“开启”状态的爆发式转变，展现出了高标靶特异性与极佳的光子产率对比。
Scheme 7. N-(ω-Chloroalkyl) Amides 8a-Halo, 10-H-Halo, 2-Halo, 10-Me-Halo (CA1-CA)─ Ligands for HaloTag Protein Labeling─and Amide 10-Me-NH-PEG-BG for SNAP-Tag Labeling
Figure 1. Absorption changes upon photoactivation of compound 10-Me-Halo = CA4 in buffered aqueous solutions (100 mM phosphate buffer, pH = 7) in a free state (A) and after binding with HaloTag HT7 protein (B). The LCMS plots of the solution before (top) and after (bottom) photoactivation of compound 10-Me-Halo; the structure and the molecular masses (as M+H, or M+) of the starting compound CA4 and the photolysis product are indicated in (A, C). (D) Transients obtained at 650 nm from photoactivation in cases (A) and (B). (E) Half-lives for the activation reactions of compounds 8a-Halo, 10-H-Halo, 2-Halo, and 10-Me-Halo (CA1, CA2, CA3, and CA4, respectively) in a free state (light gray bars) and bound to HT7 protein (dark gray bars), showing acceleration of the photolysis upon covalent binding with HT7 protein for all compounds. (F) Emission spectra of photoactivated oxazines bound to the protein (8a-Halo in orange, 10-H-Halo in red, 2-Halo in blue, and 10-Me-Halo in magenta). (G) SDS-page gels of the solutions obtained after the photoactivation detected by fluorescence (top) and after Coomassie staining (bottom).
在成功解决特异性细胞标记与高效光活化问题的基础上，研究团队将该系列高性能光笼染料推向了极具挑战性的超分辨纳米显微成像应用。研究人员首先利用常规的单分子定位超分辨技术（PALM）对固定细胞内的内质网动态网络进行了精确制图，活化后的单分子发光体平均每个闪烁周期能够释放多达数百至近两千个光子，从而在无需任何复杂特殊闪烁缓冲液的纯水相环境中，直接绘制出了极其锐利、清晰的高空间分辨率纳米结构图像。不仅如此，利用该系列染料独特的颜色可调性，研究人员通过组合不同发光波长的酰胺探针与活化抗体标记物，在双通道荧光检测下完美实现了线粒体外膜与线粒体DNA的高分辨特异性双色共定位成像，信号互不干扰且色彩分离度极高。最终，该研究更是将该成果应用到了代表当前光学显微镜时空分辨极限的MINFLUX（最小光通量）纳米显微镜中，通过确定性的多轮定位迭代，仅需探测极少量的光子便成功实现了对内质网膜蛋白簇前所未有的超高细节刻画，最终将成像的空间定位精度（标准差）硬生生推进到了2.6纳米的单数字纳米级别。
Figure 2. Confocal images of live U-2 OS cells expressing Halo7-Sec61β, labeled with compounds 2-Halo (A), 10-Me-Halo (B), 10-H-Halo (C), and 8a-Halo (D), and then irradiated with 405 nm light. Staining was performed with 1 μM solutions, except for 2-Halo (250 nM). Cells were washed and imaged in supplemented FluoroBrite DMEM medium. Images before photoactivation are displayed in the top-right corners. Samples were additionally stained with a blue nuclear stain marker. Scale bars: 5 μm.
Figure 3. Super-resolution PALM images of fixed U-2 OS cells expressing Halo7-Sec61β, labeled with amides 8a-Halo (A), 10-H-Halo (B), 2-Halo (C), and 10-Me-Halo (D). Staining was performed on live cells with 1 μM solutions, except for 2-Halo (250 nM). Imaging was performed on samples mounted in aqueous PBS buffer. Scale bars: 2 μm.
Figure 4. Two-color imaging on fixed cells. Tomm20-Halo cells costained with 8a-Halo (live-cell labeling) and a combination of a primary antibody against dsDNA and a secondary antibody labeled with 10-Me-NHS, and imaged in a confocal microscope (A-D) or a camera-based (PALM) superresolution microscope (E-G). (A) Confocal green channel (8a-Halo on Tom20), (B) Confocal red channel (10-Me-NHS on dsDNA). Upper left corners show the images before activation, and the ROIs indicated are enlarged in (C) and (D), respectively. PALM green channel (8a-Halo on Tom20), (F) PALM red channel (10-Me-NHS on dsDNA). The inset shows the enlarged ROI indicated in (G). Scale bars: 5 μm (A-B and E-G), 1 μm (C-D), 500 nm (insets in E-G).
Figure 5. MINFLUX images in fixed U-2 OS cells expressing Halo7-Sec61β. (A-B) 560 nm MINFLUX of a sample labeled with 8a-Halo; (C-D) 640 nm MINFLUX of a sample labeled with 10-Me-Halo. Labeling was performed on live cells with 1 μM solutions, and imaging was performed after fixation in aqueous PBS buffer without additives. (B, D) Enlarged ROIs indicated in A and C, respectively. Scale bars: 1 μm (A, C) and 200 nm (B, D).
总结及展望
该项研究成功利用金属催化的后期C–H活化与分子 assembly 策略，开辟了一条简短、经济且高度原子利用率的光活化氧杂嗪类纳米成像染料合成新范式。合成的系列新型光笼染料在光解前处于完美的荧光猝灭状态，激活后发光表现优异，且能与目前主流的Halo-Tag和SNAP-Tag等自标记酶体系以及标准免疫荧光技术完美兼容，在从 diffraction 限制的常规共聚焦到超越衍射极限的MINFLUX纳米显微成像中均展现出了无与伦比的应用潜力。展望未来，这种通过在芳香酰胺邻位精准引入丙烯酸酯捕获基团的区域选择性催化策略，有望被进一步推广到香豆素、罗丹明以及碳罗丹明等更多经典、对称的通用染料核心骨架中。这不仅能够大幅降低高性能不对称功能化荧光探针的工业化与实验室合成门槛，更为开发具备更高溶解性、更强吸光能力以及更多分子功能维度的下一代超分辨多色纳米成像标签提供了极其强有力的化学技术支撑。