【Nat.Chem.】精准控制在17微米内！仅需16 μW/mm²低功率辐照，利用基因靶向有机染料实现细胞特异性光催化合成#

[!summary] 本研究开发了一种基因靶向的光催化有机染料策略，旨在解决活体细胞内非天然产物合成缺乏时空与细胞特异性的难题。研究团队利用可见光与 HaloTag 标签，在特定活细胞表面及内部实现了低功率、单细胞级分辨率（17 µm 半径内）的 Anabolic（同化/合成）C-H 键活化偶联。该系统还成功原位构建了可逆转蛋白质转运方向的重定位工具，为未来在活体生物中进行精密定向的非天然化学合成奠定了基础。

哺乳动物细胞自身具备高度复杂的代谢与合成网络，但其天然的生物催化能力普遍局限于内源性的生物化学反应。如何扩展活体生物系统中生理兼容的成键催化反应反应类型，实现在特定活细胞甚至特定亚细胞位点原位（in situ）同化合成非天然的非生物有机化合物，一直是化学生物学领域的重大挑战。如果能够精准操纵这些异源催化反应，科学家们将能够把生物体内的特定细胞视为独立的“反应釜”，从而实现局部给药、调节细胞行为或赋予细胞非天然的功能。

近期，来自斯坦福大学的Spencer Zhao、Kang Yong Loh以及Karl Deisseroth和鲍哲南等研究团队，在《Nature Chemistry》上发表了题为“Genetically targeted photocatalytic organic dyes for spatiotemporally controlled organic synthesis in specific living cells”的研究论文。研究人员开发了一种将异源有机光催化染料基因靶向锚定至活细胞表面的通用策略，在极低功率的可见光刺激下，成功在活细胞上实现了单细胞分辨率的非天然有机同化合成。

1. 研究背景与核心问题#

传统的活细胞非生物催化主要依赖于过渡金属催化剂（如Hoveyda-Grubbs催化剂、钯催化剂、金(III)配合物等）或人工金属酶。此外，通过定向进化技术，研究人员也成功将酶催化扩展到了非天然的 C-H 键功能化反应中。然而，这些方法在异质性极强的活体生物系统中应用时，通常表现出“泛滥性”催化，极度缺乏微米级的时空精准度与细胞类型特异性。

近年来发展的“基因靶向化学组装”（GTCA）技术虽能通过启动子门控的病毒递送将催化酶引入特定细胞，但在以往的研究中，该方法大多局限于氧化同聚反应。更重要的是，这类基因编码的蛋白质光敏剂（如miniSOG、KillerRed）往往需要极高的光照强度（如miniSOG需要20 mW/mm²），极易引发细胞毒性与氧化应激。

光催化有机染料（如曙红Y、Janelia Fluor类染料、亚甲蓝等）具有无金属构筑、绿光至红光吸收波长可调、光触发高时空控制能力强等优点。然而，如何将这些外源有机染料选择性地、稳固地结合到特定种类的细胞上，并使其在复杂的生理环境中保持高效的同化合成催化活性，此前尚未得到解决。

Fig. 1: Genetic targeting of photocatalytic dyes.#

Genetic targeting of photocatalytic eosin Y, JF-567 or methylene blue organic dyes to the mammalian expression of human codon-optimized IgK-HT9-PDGFR, driven by the elongation factor 1α promoter for membrane localization. These dyes drive wavelength-defined photocatalysis (cat) localized to specific cells.#

2. 基于基因靶向与光催化的化学分子设计#

为了克服这一瓶颈，研究团队利用了蛋白质-配体共价结合标签技术。他们设计并转染了人胚胎肾细胞（HEK293FT），使其表达融合蛋白 IgK-HT9-PDGFR 。该融合蛋白通过血小板衍生生长因子受体（PDGFR）跨膜结构域将HaloTag-9（HT9）蛋白质标签定向引导并展示在细胞外膜表面，同时通过内含子核糖体进入位点（IRES）基序同步表达胞质荧光蛋白（如BFP、GFP或oScarlet），用于可视化追踪转 transfected 阳性细胞（图1）。

研究人员筛选了三种具备高光氧化活性的外源有机染料，并对其进行了HaloTag配体功能化改性（图1）：

EY-HT：基于黄绿光吸收的曙红Y（Eosin Y）；
JF-567-HT：基于绿光吸收的碘代Janelia Fluor类罗丹明染料；
MB-HT：基于红光吸收的亚甲蓝（Methylene Blue）。

实验表明，当使用EY-HT处理转染细胞时，染料能够特异性地共价锚定在表达HT9的细胞膜上（图1）。定量分析显示，平均每株成功转染的细胞表面可锚定约 $(3.20 \pm 0.28) \times 10^6$ 个EY分子，且光催化剂在细胞表面的分布密度与胞内荧光蛋白的表达量呈现出良好的线性正相关（ $R^2 = 0.62$ ）。

3. 单细胞分辨率下的空间与波长正交光催化#

研究团队首先选择了一个经典的生物共轭模型反应来评估原位光催化活性：利用红光或呫吨类染料催化，将二氢四嗪（DHTz）光氧化前体转化为具备点击化学活性的双吡啶取代四嗪（Tz）配体（图2a）。

在水相、有氧的模拟生理条件下，使用低功率（90 μW/mm²）的可见光（对应530 nm、565 nm或660 nm LED）照射DHTz，EY-HT、JF-567-HT和MB-HT均展现出显著的Tz催化生成速率，溶液在415 nm处的特征吸收峰显著持续上升（图2b）。通过高效液相色谱-电喷雾电离飞行时间质谱（HPLC-ESI-TOF-MS）分析，检测到四嗪点击加合物的高分辨质谱信号（加氢峰 $m/z = 613.7373$ ），确认了光催化产物的化学结构与功能性（图2c）。

为了评估细胞水平的空间精准度，研究人员建立了细胞表面原位Tz追踪测定法：先通过糖代谢标记在全体细胞表面引入叠氮基团，随后借助加成反应在全球细胞膜上装饰DHTz前体；然而，光催化剂染料仅装配在HT9阳性细胞上（图2d）。

Fig. 2: Genetic targeting of photocatalytic organic dye to the cell surface.#

a, Photo-oxidative synthesis of Tz from DHTz as a measure of photocatalytic activity. r.t., room temperature. b, Tz production catalysed by EY-HT, JF-567-HT and MB-HT under aqueous, aerobic conditions and illuminated by 530, 565 and 660 nm LEDs (90 μW mm−2), respectively. c, Extracted ion chromatograms from HPLC-ESI-TOF-MS analysis of unligated Cy5-TCO ([M + H]+, m/z = 959.3605) (i) and ligated Cy5-TCO-Tz ([M + 2H]2+, m/z = 613.7373) (ii) after treatment with DHTz and EY-HT, JF-567-HT, MB-HT or no photocatalyst (no PC) (Supplementary Fig. 2). The arrows indicate the decrease in unligated Cy5-TCO and increase in ligated Cy5-TCO-Tz in the presence of photocatalyst. d, Assay to measure the local synthesis of Tz on the cell surface. DHTz is immobilized on the membrane by tetraacetylated 2-[(azidoacetyl)amino]-2-deoxy-D-mannose glycoprotein metabolic labelling. Tz is produced only in the presence of photocatalyst and is detected by Cy5-TCO. e, Under illumination at 565 nm (column 2), transfected and fixed HEK293FT cells (row 1) labelled with JF-567-HT (row 2) produced Tz, as visualized by Cy5-TCO labelling (row 3). Row 4 shows an overlay of the images. In the absence of stimulation (column 1), no Cy5-TCO labelling occurs. Scale bars, 50 μm. f,g, Cell-by-cell β values for illuminated (+hν; n = 36 cells) and non-illuminated transfected cells (−h__ν; n = 49 cells) (f) and transfected (+HT; n = 39 cells) and non-transfected cells (−HT; n = 40 cells) under constant illumination (g). The data are presented as the mean ± 95% confidence interval (CI) for a representative experiment; similar results were achieved in triplicate. Significance was assessed by the two-way unpaired t-test. **P < 0.0001; exact P value: <0.0001 for both f and g. h, Co-culture of transfected HEK293FT cells (ii) and non-transfected glia, labelled with glial fibrillary acidic protein (GFAP) antibody (i), with Tz visualized by Cy5-TCO (iii). An overlay of these images is also shown (iv). Scale bars, 50 μm. i, Individual glia and HEK293FT cells and their respective Cy5-TCO channels. Scale bars, 10 μm. j, Average Tz production on transfected HEK293FT cells and glia, compared with the background substrate. The data are presented as the mean ± standard deviation for n = 3 biological replicates. Significance was assessed by one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test. NS (not significant) P > 0.05, *P < 0.001; exact P values (left-right): 0.0005 and 0.8578.#

结果表明，在565 nm LED（90 μW/mm²）辐射30分钟后，经Cy5-TCO探针染色，强烈的Cy5荧光仅选择性地共交于表达GFP与JF-567的转染细胞中（图2e）。图像分割算法分析显示，辐照组细胞的反应比例常数（ $\beta$ 值为 $1.43 \pm 0.17$ ）与皮尔逊相关系数显著高于未辐照组（图2f）。在HEK293FT细胞与小鼠海马胶质细胞（Glia）的异质共培养体系中，光催化反应展现出高度的细胞特异性，胶质细胞区域未检测到明显的非特异性信号（图2h、2i、2j）。

更进一步地，借助共聚焦激光显微镜重复扫描，研究人员成功实现了单细胞分辨率的光图案化（Photopatterning）催化（图3a）。在EY标签细胞上，低至16 μW/mm²的530 nm激光功率密度便足以产生超过背景的显着Tz产物信号（图3c）。当激光照射被局限于 $24 \text{ }\mu\text{m} \times 24 \text{ }\mu\text{m}$ 的单细胞微区时（图3e），光化学反应被严格限制在距离刺激中心17微米的空间半径内（图3f）。在该激发范围内，细胞死亡率与对照组无异，证实了极高的生物相容性。

Fig. 3: Single-cell resolution photopatterning of photocatalysis on live HEK293FT cells.#

a, Photopatterned photocatalysis on live cells by confocal laser microscopy. b, Tz conversion as detected by post-stimulation (post-stim) Cy5-labelling within an illuminated (567 nm) millimetre-scale square (outlined in orange) zone of JF-567-labelled HEK293FT cells (i, JF-567; ii, Cy5). Magnified image of (ii) at the boundary (dashed white line) of the illuminated and non-illuminated zones (iii). Scale bars, 200 μm (i, ii) and 100 μm (iii). c,d, Quantification of Tz on EY-labelled HEK293FT cells with increasing 530-nm illuminant intensity at 20 laser scans (c) or with increasing scan number at 34 μW mm−2 (d). Images were segmented to include only photocatalyst-expressing cells before image analysis. The data are presented as the mean ± standard deviation for n = 3 biological replicates. Significance was assessed by one-way ANOVA with Dunnett’s multiple comparisons test. NS P > 0.05, P < 0.05; P < 0.01, P < 0.001, **P < 0.0001; exact P values (left-right): 0.5325, 0.0030, 0.0013, 0.0013, 0.0004 and <0.0001 (c), and 0.0247, 0.0030 and 0.0003 (d). e, Individual cell resolution patterning of Tz synthesis with four 24 μm × 24 μm illumination zones (red squares) (i, EY overlaid onto brightfield; ii, Cy5). Examples of cell Tz production targeted to individual cells (iii, EY; iv, Cy5). Scale bars, 200 μm (i,ii) and 50 μm (iii,iv). f, Dependence of β on distance from illumination. The green and grey data points represent cells that are within or beyond the edge of the illumination zone (red line), respectively. Inset: β values of cells within the stimulation zone (0–20 μm from the centre of stimulation; n = 18 cells), cells outside but close to the stimulation zone (20–30 μm; n = 17 cells) and cells outside and far from the stimulation zone (120–130 μm; n = 10 cells). The data are presented as the mean ± 95% CI for a representative experiment; similar results were achieved in triplicate. Significance was assessed by Welch’s ANOVA with Dunnett’s T3 multiple comparisons test (NS P > 0.05, *P < 0.001; exact P values (top-bottom): 0.3615 and 0.0001).#

此外，由于不同有机染料的激发光谱相互独立，研究人员在溶液和细胞体系中均实现了“波长正交”（颜色多路复用）的光催化控制（图4a、4b）。EY-HT和JF-567-HT在660 nm红光照射下完全不具备催化活性，而MB-HT在530 nm绿光下同样保持惰性，这为在同一生物环境中实现多通道逻辑控制提供了可能（图4c、4d）。

Fig. 4: Orthogonal in vitro photocatalysis in the green-to-red range of the visible spectrum.#

a, The concept of colour multiplexed photocatalysis, where cells labelled with one dye are photocatalytically active only when illuminated with the correct wavelength. b, Colour-orthogonal absorption spectra of EY-HT, JF-567-HT and MB-HT. The dashed lines indicate the peak emissions of 530, 565 and 660 nm LEDs used for stimulation. c, Tz production, measured by UV–visible absorbance at 415 nm, catalysed by EY-HT (top), JF-567-HT (middle) or MB-HT (bottom) in solution subjected to repeated cycles of illumination at 530, 565 and 660 nm for 1 min each. The coloured bars superimposed on the plots indicate the wavelength of the LED used in that time interval: green, 530 nm; yellow, 565 nm; red, 660 nm. d, Cy5-TCO labelling of HEK293FT cells previously tagged with different photocatalysts and illuminated with different laser wavelengths at the same illuminance. Cy5-TCO was replaced with Alexa Fluor 594-TCO for all MB-HT-labelled cells due to spectral overlap. Scale bars, 200 μm.#

4. 活细胞表面原位同化C-H键功能化反应#

有机光催化染料最引人瞩目的应用之一在于直接激活相对惰性的 C-H 键。研究团队将该体系成功拓宽到了具有挑战性的活细胞表面同化 C-H 键交叉脱氢偶联反应上（图5a）。

该反应基于自由基-极性交叉（radical-polar crossover）机制：激发的曙红Y（EY*）与 $N$ -苯基-四氢异quinoline（PTHIQ, 1）底物发生单电子转移（SET），随后发生氢原子转移（HAT），生成稳定的亚胺离子中间体；该亲电性亚胺中间体继而与生理介质中的亲核试剂发生偶联；体系中的分子氧则充当最后的电子汇（sink），再生光催化剂（图5b）。

研究人员选择了热力学烯醇式极其稳定的二甲酮（Dimedone, 2）作为亲核试剂，在完全基于活HEK293FT细胞表面的有氧、中性缓冲液环境中（530 nm LED, 90 μW/mm², 37°C 反应6小时），通过逆相HPLC-ESI-TOF-MS在细胞裂解液及培养基中成功检测到了预期的 C-C 偶联产物——胺类加合物 3a 和进一步被氧化的亚胺加合物 3b（图5c、5d、5e）。

定量分析表明，该反应在活细胞体系中的催化产出效率极高（图5f）：

产物 3a：生成量达 $370 \pm 60 \text{ nmol} / 10^6 \text{ cells}$ ；
产物 3b：生成量达 $1577 \pm 885 \text{ nmol} / 10^6 \text{ cells}$ 。

这一产出水平显著超越了无光照、无催化剂等对照组（图5f）。此外，该活细胞表面脱氢偶联反应展现出较好的底物普适性，当采用乙酰丙酮、吲哚或2,4-二甲基吡咯作为亲核试剂时，也分别获得了 $156 \pm 56 \text{ nmol}$ （产物 4）、 $468 \pm 66 \text{ nmol}$ （产物 6）和 $134 \pm 48 \text{ nmol}$ （产物 7）的单细胞基准产率（每 $10^6$ 细胞）（图5f）。底物电性控制实验表明，当使用带有强吸电子基团的 $p$ -cyano-PTHIQ 时，产物 9 的产率骤降至 $15 \pm 5 \text{ nmol}$ ，而富电子的 $p$ -methoxy-PTHIQ 则由于其自由基阳离子陷入热力学深谷而完全无法检测到产物（图5f）。

Fig. 5: Genetically targeted in situ cross-coupled C–H functionalization.#

a, Mannich reaction between PTHIQ and a nucleophile on the surface of cells genetically targeted with photocatalyst. b, Photocatalysed radical–polar crossover mechanism via a stabilized iminium intermediate and using O2 as an electron sink. c, Formation of Mannich adducts amine 3a and iminium 3b from PTHIQ (1) and dimedone (2), photocatalysed by EY genetically targeted to HEK293FT cells. Standard conditions: 1.25 mM 1, 2.5 mM 2, D10 medium, 37 °C, 6 h, 530 nm LED, 90 μW mm−2. d, ESI-TOF mass spectra of 3a and 3b. Found masses (3a: m/z = 348.1966; 3b: m/z = 346.1796) conform to the calculated (calcd) [M + H]+ of 3a (C23H26NO2: m/z = 348.1963) and [M]+ of 3b (C23H24NO2: m/z = 346.1807). The counts are separately normalized for the 3a and 3b spectra. e, Extracted ion chromatograms at m/z = 346.18 and 348.20 from reversed-phase HPLC-ESI-TOF-MS analysis of 3a and 3b from cell lysate and purified standards of 3a and 3b. The counts are separately normalized for the 3a and 3b spectra. f, Output of products 3a, 3b, 4, 6, 7 and 9 in the presence or absence of EY and light (h__ν), indicating that the reaction is specific to the photocatalytic mechanism. Products 5, 8 and 10 were not detected (n.d.). The data are presented as the mean ± standard deviation of n = 4 biological replicates. Significance was assessed by one-way ANOVA with Dunnett’s multiple comparisons test. P < 0.01, *P < 0.001, ****P < 0.0001; the exact P values are reported in Supplementary Table 3.#

5. 特异性细胞器内化学工具原位构建与蛋白反向易位#

为了证明活细胞内原位合成非天然化学handle能够直接转录或组装为具有功能性的化学生物学工具，研究人员进一步将目光投向了胞内细胞器定向的“蛋白原位易位”（Protein relocalization）控制上。

传统的小分子诱导三元复合物形成技术，通常由外源加入线性连接的双功能配体来启动。而在本设计中，研究团队将易位剂拆分为两个化学碎片：一个是偶联有DHTz的FKBP12(F36V)结构域配体（AP1867-DHTz），另一个是偶联有TCO的大肠杆菌二氢叶酸还原酶（ecDHFR）结构域配体（TMP-TCO）（图6a）。

研究人员构建了双质粒共表达体系：

核内靶向结构域：HT9-FKBP12(F36V)-GFP-NMNAT1，该蛋白天然常驻于细胞核内；
胞质常驻结构域：BFP-ecDHFR-NES，该蛋白由于携带强核出口序列（NES），天然被排斥在细胞核外（图6b、6e）。

当利用膜渗透性的有机染料 JF-567-HT 标记核内的 HaloTag 标签后，加入两种前体加合碎片，并施加 565 nm LED（65 μW/mm²）微弱刺激 10 分钟（图6f、6g）。核内原位发生的光催化氧化反应快速将 AP1867-DHTz 转化为活性的四嗪加合物（AP1867-Tz），进而在核内直接与 TMP-TCO 发生点击偶联，原位组装出完整的双功能化学易位剂 AP1867-TMP（图6a、6f）。

令人惊奇的是，在刺激结束3小时后，研究人员观察到了一种与传统外源递送完全相反的生物表型：原本常驻于胞质的荧光蛋白 BFP-ecDHFR-NES 表现出了显着的净核输入（Net import），而核内的荧光分布依然保持（图6g）。定量分析显示，只有在光照、催化剂和前体碎片同时存在的实验组中，细胞核与细胞质的荧光密度比值（ $L_N / L_C$ ）才显著增加至 $1.26 \pm 0.15$ （图6i）。

这种现象揭示了原位催化合成的独特空间解耦优势（图6f）：

“由于HaloTag被融合锚定在核内，新生成的易位剂配体在核内最先饱和结合核内的FKBP12融合蛋白，继而捕获从胞质被动扩散进入核内的BFP-ecDHFR-NES，从而在核内固化并强制执行核本地化网络。相反，在常规的外源小分子给药（Exogenous delivery）方式下，AP1867-TMP从外膜进入细胞时首先在胞质中遇到大量的BFP-ecDHFR-NES，从而导致了截然相反的核外排（Net export）表型。”

这一发现清晰地表明，非天然化合物分子“在何处合成”本身就编码了极其关键的生物学功能与表型。

Fig. 6: Organelle-specific in situ synthesis directs targeted protein relocalization.#

a, AP1867 ligand of FKBP12F36V and TMP ligand of ecDHFR are functionalized with DHTz and TCO, respectively. The in situ photocatalytic synthesis of Tz enables click assembly of the bifunctional linker for protein translocation. b, Cytoplasmic and nuclear constructs to investigate localization control of ecDHFR and NMNAT1 (top), and the sender–receiver assay to study in situ synthesized AP1867-TMP (bottom). EY-functionalized HEK293FT cells photocatalysed Tz production from AP1867-DHTz to facilitate the formation of bifunctional linker AP1867-TMP. The linker was purified from media and subsequently re-spiked into a receiver culture expressing both the cytosolic and nuclear constructs in HEK293FT cells. c, Net nuclear export of FKBP12F36V-GFP-NMNAT1 on treatment with pre-synthesized AP1867-TMP. d, Extracted ion chromatograms of AP1867-Tz ([M + H]+, m/z = 1,101.5046) extracted from EY-functionalized HEK293FT cells in the presence or absence of EY and light, indicating that the reaction is specific to photocatalysis. e, Representative receiver culture images for FKBP12F36V-GFP-NMNAT1 (row 2) relocalization by BFP-ecDHFR-NES (row 1), driven by pre-in situ synthesized AP1867-TMP 3 h after treatment. The white arrows indicate induced locations of induced NMNAT1 export. Scale bars, 25 μm. f, Net nuclear import of BFP-ecDHFR-NES upon in situ nuclear synthesis of AP1867-TMP. g, Representative culture images for BFP-ecDHFR-NES (column 1) import by HT9-GFP-FKBP12F36V-NMNAT1 (column 2), driven by the in situ synthesis of AP1867-TMP by nuclear JF-567-labelled HEK293FT cell culture treated with AP-1867-DHTz and TMP-TCO. Overlaid images are also shown (column 3). The imaging was performed 3 h after stimulation for 10 min (567 nm, 65 μW mm−2). The white arrows indicate the locations of induced import. Scale bars, 10 μm. h, Net import of BFP-ecDHFR-NES occurs only when light, photocatalyst, and the AP1867-DHTz and TMP-TCO ligand fragments are all present. The white arrows indicate locations of induced import. Scale bars, 50 μm. i, The ratio of nuclear (_L_N) to cytoplasmic (_L_C) density of BFP-ecDHFR-NES increases only when light, photocatalyst and the ligand fragments are all present. The data are presented as the mean ± standard deviation for n = 3 biological replicates. Significance was assessed by one-way ANOVA with Tukey’s multiple comparisons test. P < 0.01, *P < 0.001; exact P values (left-right): 0.0011, 0.0008 and 0.0006.#

6. 局限性与未来展望#

尽管该工作在细胞特异性及低功率单细胞光催化合成领域取得了突破性的进展，但从严谨的科研视角审视，该体系目前仍存在数项亟待解决的方法学局限：

持续催化带来的光毒性与氧化应激窗口限制：研究指出，短时间（10分钟）的可见光辐照能够安全驱动蛋白易位，但在长时间（>30分钟）的连续辐照下，由于有机染料介导的胞内持续氧化应激，会不可避免地对核内富集的核出口蛋白（如CRM1）造成不对称损伤。这种内在的自由基副反应会导致配体非依赖性的蛋白分布异常，限制了该体系在极长时程生物反应中的连续应用。
底物电子效应与普适性的制约：活细胞表面的脱氢 C-H 键功能化偶联高度依赖于中间体自由基阳离子的热力学稳定性。由于生理环境的极度稀释与有氧限制，底物的 Hammett 常数对成键效率有着极强的控制权，吸电子或过强的供电子底物均会导致产率严重下降或完全不反应，这极大地缩窄了可供选择的非天然同化合成底物范围。
非催化背景反应的干扰：尽管光催化反应占据主导，但在活细胞表面依然能检测到少量的未催化自发背景反应（图5f）。在未来向更复杂的活体生物系统（In vivo）或药理学载体转化时，如何通过对底物分子进行膜局部限域设计以完全消除这些背景杂信号，将是实现更高精度的精密非生物化学同化合成的关键改进方向。

参考文献#

Zhao, S., Loh, K.Y., Tyson, J. et al. Genetically targeted photocatalytic organic dyes for spatiotemporally controlled organic synthesis in specific living cells. Nat. Chem. (2026). https://doi.org/10.1038/s41557-026-02195-6