Fluolab【Angew.Chem.】四川大学余志鹏、胡常伟|协同光控新高度：双波长加速 光点击反应效率提升达 27 倍
通讯作者： Changwei Hu, Zhipeng Yu
文章概要
引言
在现代化学领域，光响应分子开关因其卓越的时空分辨率而成为操控生物行为、设计智能材料及光学工程的核心工具。点击化学作为一种高效的共价键合手段，在引入光活化前体后演变为光点击化学，极大地提升了反应的精确度。然而，如何在复杂的生物环境中平衡中间体的稳定性和反应活性，一直是该领域的重大挑战。尽管目前已有利用单一光开关作为偶极子或亲偶极子的尝试，但利用两个正交光开关前体实现双波长协同控制的研究仍处于前沿阶段。本研究提出了一种创新的双波长协同光点击平台，通过结合P型光开关（二芳基茚酮环氧化物）与T型光开关（二苯并硫氮杂卓二氧化物），实现了反应速率的显著提升和更高维度的光控精度。
Comparison of [5+2] cycloaddition reactions between the DIO-PY photoswitch and ring-strain-preloaded dipolarophiles versus the photoswitchable 7-membered cyclic dibenzothiadiazepines: (a) State-of-the-art photoclick strategy utilizing single photoswitch, DIO⇌PY and BCN-OH; (b) novel photoclick strategies comprise a pair of two orthogonal photoswitches, DBTD versus DBTDD as dipolarophile in this work.
主要实验及结论
研究团队首先构建了一个功能强大的噻吩融合二芳基茚酮环氧化物（DIOs）库。通过在茚酮核心上策略性地引入噻吩单元，成功调节了分子的前线轨道能量，使其激发波长显著红移至可见光范围（405-490 nm）。实验观察到，这些 DIO 衍生物在可见光照射下会发生光致开环，生成具有特定色彩的介离子氧化吡喃鎓叶立德（PY）。这一过程表现出优异的多色光致变色性能，在聚合物基质中通过顺序照射可实现图案的精确打印与擦除。更重要的是，PY 作为一种高活性的偶极子，能与特定设计的亲偶极子发生 [5+2] 环加成反应。
The DIO derivative library and their photophysical/chemical properties in CH3CN/H2O = 1/1 mixed solvent (unless otherwise specified). (a) Chemical structure and overall yield of each DIO; (b) UV/Vis absorption spectra of DIOs at 50 µM; (c) Photoisomerization between 2d and 2dPY, UV/Vis spectra with molar extinction coefficients (ε) for 2d or 2dPY (extrapolated dotted-curve) at 100 µM, PSS405 for 2d⇌2dPY equilibrium (dash-curve); (d) Photoisomerization between 3a and 3aPY, UV/Vis spectra and ε for 3a or 3aPY (extrapolated dotted-curve) at 100 µM, PSS445 for 3a⇌3aPY (dash-curve); (e) Photoisomerization between 5d and 5dPY, UV/Vis spectra and ε for 5d or 5dPY (extrapolated dotted-curve) at 50 µM in THF, PSS445 for 5d⇌5dPY (dash-curve). Laser power density at 405 nm PD405 = 150 mW cm−2, PD445 = 200 mW cm−2.
在反应伴侣的选择上，研究人员对比了传统的 DBTD 与氧化后的 DBTDD（二苯并硫氮杂卓二氧化物）。实验结果证明，得益于砜基的强吸电子效应，DBTDD 在光激发下的反应活性远高于 DBTD。通过 405 nm 与 445 nm 双波长的协同刺激，该体系展现了惊人的动力学加速效应，表观反应速率常数高达 1.6 × 10⁵ M⁻¹ s⁻¹，相比单波长条件实现了 27 倍的速率提升。此外，该体系在蛋白质标记实验中也表现出色，利用双波长正交控制实现了对溶菌酶的高效荧光标记，标记效率高达 38% 至 92%，且在黑暗条件下完全无背景反应。
Photochromism of the DIO derivatives. (a) Exemplary photochromism of DIO⇌PY at 2.0 mM in THF solution; (b) Various photochromic patterns generated by doping DIO into PS film after irradiation employing photomasks for 10 s. Left to right: the emblem of Sichuan University, “LOVE”, the text “chemistry”, a shape of a panda; (c) The structural pattern of 3aPY can be printed in a 3a-containing PS film using Digital Light Processing (DLP) for spatial control in 405 nm channel; (d) The structural pattern of 5dPY developed in a 5d-containing PS film using DLP in 452 nm channel; e) Multicolor photochromism cycles via writing and erasing of QR code patterns by sequential narrowband irradiations in a single PS matrix doping both 3a and 5d; (f) Dual-color spatial patterning via sequential irradiation in a single PS matrix containing both 3a and 5d. Light yellow background color: the color of 3a and 5d, dark green: the color of 5aPY, violet-black: the mixed color of 3aPY and 5dPY, pink: the color of 3aPY, scale bar = 1.0 cm.
Screening on DIO/DBTD(D) dual-photoswitch combinations for the photo-initiated cycloaddition under biocompatible conditions. (a) The reaction schemes. (b) The chemical structures of the DBTDD family versus DBTD family. (c) SXRD structures of 3a and 3a6a; (d) The photoclick reaction conditions for screening, and the gradient color code for characteristics of each reaction and color-coded HPLC-MS screening results. The red color represents the desired cycloadducts with conversion in positive values, and the indigo color for by-products from photolysis of DIO with negative values. Experiments were carried out at 298 K, irradiated with the indicated light sources. (e) Heatmap for results under 405 nm laser. (f) Heatmap for results under 445 nm laser.
研究团队还通过密度泛函理论（DFT）计算和 CASSCF 分析深入揭示了反应机制。计算发现 DIO 到 PY 的光转换遵循无势垒的单重态途径，而 PY 表现出的单重态双自由基特征增强了其热稳定性。对于点击反应，理论分析证实了 E-DBTDD 具有更低的活化能垒，这从分子层面解释了其在环加成反应中的动力学优势。这种双光开关配对模式不仅避免了激发光谱的重叠，还通过功率密度依赖的协同效应，为生物偶联提供了极高的灵活性和精准度。
Synergistic dual-λ photoclick system utilizing DIO⇌PY and Z_⇌_E-DBTDD photoswitch pairs. (a) Scheme for the dual-λ photo-induced competitive reactions between DIO 2d and DBTDD 6b versus BCN-PNP 8a. (b) Photo-decay of the in situ formed [5+2] cycloadduct from 2d with various dipolarophiles under varying 405 nm laser intensities for 120 s. (c) Correlation between PSS of both the photoswitches and incident laser power density (PD). Top: Relationship of PSDPY (cyan curve obtained via characteristic Δabsorbance of 2dPY) versus PD at 405 nm (PD405). Back-switching of 2dPY to 2d via 532 nm laser was essential in each cycle. Bottom: relationship of PSD_E_ of 6b (orange curve obtained via Δabsorbance of E-6b minus Z-6b versus PD445). (d) Qualitative λ-dependent absorbance changes of the photoswitchable active species, 2dPY (cyan line) and E-6b (orange line). 2dPY is generated upon 405 nm irradiation but not under 445 nm, and its reversion to 2d requires 532 nm excitation due to the long half-life of 2dPY. E-6b can be populated under either 405 or 445 nm, and rapidly relaxed to Z-6b in the dark. Simultaneous 405 and 445 nm excitation offered PSD of both 2dPY and E-6b remain at higher levels. (e) 3D Bar graph depicting λ-orthogonal PD-dependent photo-conversion of 6b in photoclick reactions at both 405 (100–1000 mW cm−2 scale) and 445 (1000–4000 mW cm−2 scale) nm dimensions, with SF values showing on each bar, (f) a zoom-in scale at 90–180 mW cm−2 for 405 nm and 300–700 mW cm−2 for 445 nm, t = 60 s. (g) The algorithm to obtain SF, and value-range annotations. (h) Bar graph showing dipolarophile conversions under single 405 nm laser activation (Figure S65). (i) 3D Bar graph of λ-orthogonal PD-dependent yield ratio of 2d6b/2d8a in photoclick reactions at 405 and 445 nm dimensions, (j) and a zoom-in scale, t = 60 s. (k) 3D Bar graph of λ-orthogonal PD-dependent rate-acceleration (apparent _k_2) between 2dPY and 6b at 405 and 445 nm dimensions. Conditions: MeCN:H2O (v/v) = 1:1, [2d] = 50 µM, [6b] = [8a] = 100 µM, irradiation duration: PD405 = 0, t = 600 s; PD405 = 0.10 W cm−2, t = 300 s; PD405 = 0.40 W cm−2, t = 120 s; PD405 = 0.70 W cm−2, t = 60 s; PD405 = 1.0 W cm−2, t = 30 s.
Dual-λ photo-acceleration for protein labeling by the photoclick reaction via fluorescent DIO probes. [Protein] = 10 µM and [Probe] = 20 µM in H2O with MeCN as co-solvent, illuminated for 10 s. (a) Cartoon structure for 6a-tagged lysozyme, structures of the fluorophore-linked DIO probes, and also photo-labeled lysozymes. (b) SDS-PAGE imaging for the photoclick labeling of Lyso-6a with 2d-Cy5 utilizing co-irradiation with 445 nm at a constat PD = 0.20 W cm−2 + an increasing PD of 405 nm laser, in both Cy5 and Coomassie brilliant blue (CBB) channels. (c) Histogram of normalized Cy5 intensity of the protein bands versus PD405. (d) SDS-PAGE imaging for the photoclick labeling of Lyso-6a with 2d-Cy5 using co-irradiation of 405 nm at a constat PD = 50 mW cm−2 + an increasing 445 nm laser PD, in both Cy5 and CBB channels. (e) Histogram of the normalized Cy5 intensity of the protein bands versus PD445. (f) Deconvoluted mass spectra of Lyso-6a (40 µM) before and after photo-labeling with probe 3a-Cy5 (200 µM) using single 445 nm laser (PD445 = 500 mW cm−2). (g) Deconvoluted mass spectra of Lyso-6a before and after photo-labeling with probe 2d-Cy5 under various illumination conditions. (h) Table summarizing the protein photo-labeling efficiency, conjugating toward Lyso-6a under various illumination conditions. Notably, thermal labeling by the DIO probes was detected during the denaturation procedure at 95°C for 8.0 min, which was mostly blocked by adding excess BCN-OH (10 eq.) as a DIO quencher prior to denaturation. For stringent negative controls to confirm no labeling in the dark at 298 K (Figures S75 and S76). (i) SDS-PAGE imaging for the dual-λ orthogonal-controlled photoclick labeling of Lyso-6a with 2d-Cy5 using co-irradiation of 405 + 445 nm laser with stringent controls.
(a) Scheme presenting computational results on the photochemical and thermal transformation in the DIO⇌PY photoswitch system. Relative electronic energies (in kcal/mol) were given. (b) PES plot for the photoisomerization cycles between path I and III, with key species characterized by C1-C2 bond lengths (BC1-C2, Å) and C1-O3-C2 bond angles (AC1-O3-C2, degree). (c) Gibbs energy profiles for thermal relaxation of E-DBTD versus E-DBTDD, and their cycloaddition with PY, comparing E/Z-DBTD and E/Z-DBTDD isomers.
总结及展望
本研究成功开发了一种基于双波长协同控制的高效光点击反应平台。通过将可调谐的 DIO 偶极子光开关与高性能的 DBTDD 亲偶极子光开关相结合，不仅突破了传统光点击反应在反应速率和控制维度上的局限，还为多模式光致变色材料和精准生物正交标记开辟了新途径。这种协同光控策略在三维空间精准修饰生物分子方面展现了巨大潜力。未来，开发具有更长激发波长、更高热稳定性的 DIO 类似物，将是推动该技术在复杂生物系统中广泛应用的关键所在。