吩噻嗪-荧光蛋白生色团类似物的合成、S原子促进的光动力治疗及双光子荧光成像

doi:10.6023/cjoc202104040

摘要/Abstract

绿色荧光蛋白(GFP)生色团由于良好的生物相容性、低暗毒性和光稳定性而备受关注, 然而, 将GFP生色团用于光动力治疗鲜有报道. 以N-丁基吩噻嗪-3-甲醛、甘氨酸叔丁酯盐酸盐和亚胺酯为原料, 通过[2+3]环加成反应合成了一种吩噻嗪荧光蛋白生色团类新型光敏剂(Ptz-FP), Ptz-FP是一种不含重原子、由S原子促进的具有优异单线态氧产生效果的光敏剂, 可用于光动力治疗. 在二甲基亚砜(DMSO)溶液中, Ptz-FP光敏剂的吸收波长位于436 nm, 此处的摩尔消光系数为1.4×10⁴ L•mol^–1•cm^–1, 发射波长在626 nm, Stokes位移高达190 nm, 荧光量子产率为1.5%. 以Ru(bpy)₃Cl₂为参比, 测得光敏剂Ptz-FP在甲醇溶液中单线态氧产率为33.1%. 通过噻唑蓝(MTT)法测试了光敏剂Ptz-FP的暗毒性和光毒性, 结果表明其具有较低的细胞毒性(≥89.9%)和较高的光毒性, 能在30 min内杀死50%以上的A-549细胞. 此外, 通过活性氧检测试剂盒DCFH-DA在15 min内成功检测到A-549细胞中活性氧的产生, 并由AO/EB活死细胞染色剂监测到A-549细胞凋亡过程. 最后, 光敏剂Ptz-FP成功应用于双光子荧光成像. 光敏剂Ptz-FP有望为今后荧光成像指导的荧光蛋白生色团类光敏剂的开发奠定基础.

关键词: 荧光蛋白生色团, 含S原子的光敏剂, 光动力治疗, 双光子荧光成像

The green fluorescent protein (GFP) chromophore has attracted much attention due to its good biocompatibility, low dark cytotoxicity and high photostability. However, there are rare reports about GFP chromophore analogues in photodynamic therapy (PDT). In this paper, a new photosensiter phenothiazine fluorescent protein (Ptz-FP) chromophore was synthesized by [2+3] cycloaddition reaction using N-butylphenothiazine-3-carbaldehyde, tert-butyl glycine hydrochloride and imine ester as raw materials. Ptz-FP was a heavy-atom free photosensitizer that promoted by S atom and showed excellent singlet oxygen generation which can be used in PDT. In dimethyl sulfoxide (DMSO) solution, the absorption wavelength of Ptz-FP photosensitizer was at 436 nm, the molar extinction coefficient was 1.4×10⁴ L•mol^–1•cm^–1, the emission wavelength was at 626 nm, stokes shift was 190 nm, and the fluorescence quantum yield was 1.5%. The singlet oxygen yield of Ptz-FP was 33.1% using Ru(bpy)₃Cl₂ as a reference in methanol solution. The dark cytotoxicity and phototoxicity of the photosensitizer Ptz-FP were tested by methyl thiazolyl tetrazolium (MTT) assay. The results showed that it had negligible dark toxicity (≥89.9%) and excellent phototoxicity. Besides that, DCFH-DA (ROS indicator) successfully detected the production of reactive oxygen in A-549 cells within 30 min, and the apoptosis process of A-549 cells was monitored by the AO/EB stain assay. Finally, the photosensitizer Ptz-FP was successfully used in two-photon fluorescence imaging. Therefore, the photosensitizer Ptz-FP is expected to lay a foundation for FP chromophore based photosensitizers on fluorescence imaging guided PDT in the future.

Key words: fluorescent protein chromophore, containing S atom photosensitizer, photodynamic therapy, two-photon fluorescence imaging

引用此文

项雯晖, 张磊, 支旭, 钱鹰. 吩噻嗪-荧光蛋白生色团类似物的合成、S原子促进的光动力治疗及双光子荧光成像[J]. 有机化学, 2021, 41(9): 3578-3584.

Wenhui Xiang, Lei Zhang, Xu Zhi, Ying Qian. Synthesis, S Atom Promoted Photodynamic Therapy and Two- Photon Fluorescence Imaging of Phenothiazine Fluorescent Protein Chromophore Analogue[J]. Chinese Journal of Organic Chemistry, 2021, 41(9): 3578-3584.

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图/表 9

图1. (a)近年来报道的荧光蛋白生色团类似物; (b)本工作合成的光敏剂Ptz-FP及其在A-549细胞内的光动力治疗

Figure 1. (a) Recent reported GFP chromophore analogues; (b) the photosensitizer Ptz-FP synthesized in this work and its photodynamic therapy in A-549 cells

图2. (a)含S原子光敏剂Ptz-FP单线态氧产生能级图和(b) Ptz-FP分子的合成路线

Figure 2. (a) Energy level diagram of singlet oxygen generation in Ptz-FP photosensitizer containing S atom; (b) Synthetic route of Ptz-FP

图3. Ptz-FP (20 μmol•L–1)在DMSO溶液中的紫外吸收和荧光发射光谱

Figure 3. Absorbance spectrum and fluorescence spectrum of Ptz-FP (20 μmol•L–1) in DMSO

Compd.	λmax abs/nm^a	ε^max/(10⁴ L•mol^–1•cm^–1)^b	λmax em/nm^c	Stokes shift/nm	φ_F^d	IC₅₀^e/(μmol•L^–1)
Ptz-FP in DMSO	436	1.4	626	190	1.5%	3
Ptz-FP in CH₂Cl₂	436	1.8	626	190	28%	—

表1. 光敏剂Ptz-FP的光物理参数

图4. Ptz-FP在A-549细胞内的单光子荧光成像(Group A, 2 μmol•L–1, Ex: 488 nm, Em: 577~677 nm)和Ptz-FP在A-549细胞内的双光子荧光成像(Group B, 2 μmol•L–1, Ex: 800 nm).

Figure 4. One-photon fluorescence imaging of Ptz-FP in A-549 cells (Group A, 2 μmol•L–1, Ex: 488 nm, Em: 577~677 nm), and two-photon fluorescence imaing of Ptz-FP in A-549 cells (Group B, 2 μmol•L–1, Ex: 800 nm) Scale bar: 100 μm

图5. (a)在460 nm (4 mW/cm2)光源照射下Ptz-FP与DPBF溶液(CH3OH)中DPBF在414 nm处的吸光度降低图; (b) Ptz-FP与DPBF溶液中DPBF在414 nm处吸光度和相应的时间拟合曲线图; (c)在460 nm (4 mW/cm2)光源照射下Ru(bpy)3Cl2与DPBF溶液(CH3OH)中DPBF在414 nm处的吸光度降低图; (d) Ru(bpy)3Cl2与DPBF的溶液中DPBF在414 nm处吸光度和相应的时间拟合曲线图

Figure 5. (a) Absorbance decrease at 414 nm of DPBF in the presence of presence of Ptz-FP in CH3OH and blue light (460 nm, 4 mW/cm2) irradiation; (b) Linear fitting of the absorption at 414 nm of DPBF in the presence of Ptz-FP; (c) Absorbance decrease at 414 nm of DPBF in the presence of Ru(bpy)3Cl2 in CH3OH and blue light (4 mW/cm2) irradiation; (d) Linear fitting of the absorption at 414 nm of DPBF in the presence of Ru(bpy)3Cl2

图6. 在黑暗和光照(460 nm) 10 min和30 min下不同浓度Ptz-FP处理过的A-549细胞存活率

Figure 6. Cell viability of A-549 cells treated with various concentrations of Ptz-FP in the dark and exposed to light (460 nm) for 10 and 30 min

图7. Ptz-FP (3 μmol•L–1, Ex: 488 nm, Em: 577~677 nm)在A- 549细胞内的活性氧产生实验

Figure 7. ROS generation in Ptz-FP (3 μmol•L–1, Ex: 488 nm, Em: 577~677 nm) treated A-549 cells

图8. Ptz-FP的活死细胞染色实验

Figure 8. AO/EB staining assay of Ptz-FP to A-549 cells Group a: No light+Ptz-FP (3 μmol•L–1)+AO/EB; Group b: Blue light (irradiation time:15 min, 460 nm, 4 mW/cm2)+AO/EB; Group c: Ptz-FP (3 μmol•L–1)+blue light (irradiation time: 5 min, 460 nm, 4 mW/cm2)+AO/EB; Group d: Ptz-FP (3 μmol•L–1)+blue light (irradiation time:10 min, 460 nm, 4 mW/cm2)+AO/EB; Group e: Ptz-FP (3 μmol•L–1)+blue light (irradiation time: 15 min, 460 nm, 4 mW/cm2)+AO/EB. Scale bar: 100 μm

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