绿光光控释放和荧光增强响应的CO给体设计、合成及应用

doi:10.6023/cjoc202407023

摘要/Abstract

一氧化碳(CO)不仅是一种有毒气体, 同时也是重要的气体信号传递分子, 在多种生理和病理过程中发挥重要的调节作用, 包括神经传递、血管扩张、减缓炎症反应、抗癌等. 发展可控释放并能产生荧光信号定量示踪的CO给体分子对于研究其信号传导和量效关系尤为重要. 以3-羟基黄酮醇(HF)为光控释放CO基团, 通过在B环4'位以双键共轭连接方式引入氟硼二吡咯(BODIPY)荧光团, 设计合成了一种新型绿光光解和荧光示踪的光控CO给体分子BODIPY-Fla-1. 它能在绿色发光二极管(LED)光源照射的有氧条件下光解生成CO, 同时在562 nm处产生绿色荧光增强响应, 可通过荧光成像对其在细胞内的光控释放CO进行示踪, 也在细胞氧化应激模型中, 验证了BODIPY-Fla-1光控释放CO可有效降低细胞内超氧自由基水平, 缓解氧化应激. 这种以双键共轭的方式连接荧光团的策略为进一步开发光解波长更长可荧光示踪的3-黄酮醇光控CO给体分子提供了新的分子设计思路.

关键词: 一氧化碳, CO给体, 光控释放, 氧化应激, 绿色荧光

Carbon monoxide (CO) is not only a toxic gas, but also an important gas signaling molecule that plays crucial regulatory roles in various physiological and pathological processes, which include neurotransmission, vasodilation, mitigation of inflammatory response, and anti-cancer activities. Therefore, development of CO donors for controlled release of CO with simutaneous fluorescence signal generation for quantification and tracking purpose is particularly useful for CO signal transduction and quantity-activity relationship studies. Herein, a novel photo-controlled CO donor BODIPY-Fla-1 was designed and synthesized using 3-hydroxyflavone (HF) as the photo-triggered CO-releasing moiety, which is conjugated to a boron-dipyrromethene (BODIPY) fluorophore at its 4' position of B ring via a double-bond linkage. It can achieve photo- triggered CO release under green light-emitting diode (LED) light irradiation and aerobic condition with simutaneous fluorescence turn-on responses at 562 nm for convenient tracking CO release via fluorescence imaging at green channel in live cell studies. It was also verified that the CO released by photolysis of BODIPY-Fla-1 can effectively reduce the level of intracellular superoxide radical and alleviate oxidative stress. This incorporation of a conjugated fluorophore moiety via a double bond linkage provides a new design strategy for development of HF-based CO donors with longer photolysis light and fluorescence response wavelength.

Key words: carbon monoxide, CO donor, photo-triggered release, oxiditive stress, green fluorescence

引用此文

邢皖晋, 张逸蓉, 陈军伟, 励扬, 徐缓, 王卫, 楼开炎. 绿光光控释放和荧光增强响应的CO给体设计、合成及应用[J]. 有机化学, 2025, 45(5): 1698-1706.

Wanjin Xing, Yirong Zhang, Junwei Chen, Yang Li, Huan Xu, Wei Wang, Kaiyan Lou. Design, Synthesis and Application of CO Donor with Green Light Controlled Release and Fluorescence Enhancement Response[J]. Chinese Journal of Organic Chemistry, 2025, 45(5): 1698-1706.

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图/表 9

图1. (a)已报道的基于3-羟基黄酮醇结构衍生的光控CO给体; (b)光解波长在可见区或近红外区的3-羟基黄酮醇衍生物的代表性结构; (c)本工作: 3-羟基黄酮醇(HF)通过在B环4'位以双键共轭的连接方式与氟硼二吡咯(BODIPY)荧光团连接

Figure 1. (a) Reported 3-HF derivatives for photo-controlled CO donors; (b) Representative structures of 3-HF derivatives with photolysis wavelength at visible or near-infrared region; (c) This work: incorporation of a boron-dipyrromethene (BODIPY) fluorophore conjugated to 4' position of 3-hydroxyflavone (HF) via a double-bond linkage

图式1. 探针BODIPY-Fla-1的合成路线

Scheme 1. Synthesis route of probe BODIPY-Fla-1

图2. (a) BODIPY-Fla-1 (10 μmol/L)随光解时间变化的紫外吸收光谱; (b) BODIPY-Fla-1 (10 μmol/L)随光解时间变化的荧光发射光谱图(λex=520 nm)

Figure 2. (a) Time-dependent UV absorption spectra of BODIPY-Fla-1 (10 μmol/L) upon photolysis; (b) Time-dependent fluorescence emission spectrum of BODIPY-Fla-1 (10 μmol/L, λex=520 nm) upon photolysis All measurements were performed in 10 mmol/L pH=7.4 PBS buffer. Photolysis reactions were performed under a 15 W green LED light.

图3. (a) BODIPY-Fla-1 (10 μmol/L)绿光照射15 min后加入探针NF-APC (10 μmol/L)与Na2PdCl4 (60 μmol/L)后荧光发射谱图随时间的变化(λex=620 nm); (b)探针BODIPY-Fla-1 (50 μmol/L)与血红蛋白(4.2 μmol/L)共孵育条件下的紫外吸收光谱随绿光照射时间的变化(每10 min测一次); (c)相应生成的碳氧血红蛋白浓度计算值随光照时间的变化

Figure 3. (a) Time-dependent fluorescence emission spectra of 10 μmol/L BODIPY-Fla-1 preirradiated with green LED light for 15 min incubated with NF-APC (10 μmol/L) and Na2PdCl4 (60 μmol/L) (λex=620 nm); (b) Time-dependent absorption spectra of the probe BODIPY-Fla-1 (50 μmol/L) coincubated with Hb (4.2 μmol/L) upon irradiated with green LED light; (c) Corresponding time-dependent calculated COHb concentration upon photoirradiation All measurements were performed in 10 mmol/L pH=7.4 PBS buffer, the light source is green LED, 15 W

图4. BODIPY-Fla-1光解释放CO的反应式

Figure 4. Reaction scheme of CO release from photolysis of BODIPY-Fla-1

图5. 探针BODIPY-Fla-1 (10 μmol/L)在PBS缓冲液(10 mmol/L, pH=7.4)中每隔15 min周期性照射(15 W, 绿色LED灯)后在562 nm处(λex=520 nm)的荧光强度随时间变化

Figure 5. Time-dependent of fluorescent intensity changes of probe BODIPY-Fla-1 (10 μmol/L) at 562 nm (λex=520 nm) in PBS buffer (10 mmol/L, pH=7.4) after the periodic irradiation with green LED (15 W) in every 15 min

图6. (a)不同浓度的BODIPY-Fla-1作用于HeLa细胞8 h后的细胞存活率; (b)不同浓度的BODIPY-Fla-1作用于RAW264.7细胞8 h后的细胞存活率

Figure 6. (a) Percentage of viable HeLa cells after treated with different concentrations of BODIPY-Fla-1 for 8 h; (b) Percentage of viable RAW264.7 cells after treated with different concentrations of BODIPY-Fla-1 for 8 h

图7. 探针BODIPY-Fla-1 (10 μmol/L)在RAW 264.7细胞中的CO释放荧光成像图: (a)细胞在正常培养基中孵育; (b)探针BODIPY-Fla-1 (10 µmol/L)孵育10 min后的细胞荧光图像; (c)探针BODIPY-Fla-1 (10 µmol/L)孵育10 min后再光照30 min后的细胞荧光图像; (d~f)相应的明场图像; (g)不同细胞组中荧光图像的相对荧光强度(于λex=340~380 nm, λem>425 nm处采集荧光图像, 光解所用光源为15 W绿色LED灯, 误差棒表示三个重复的标准偏差, ****P≤0.0001, ***P≤0.001)

Figure 7. Fluorescence imaging of CO-release with probe BODIPY-Fla-1 in living RAW 264.7 cells. (a) Cells were incubated in normal media; (b) Fluorescence image of cells incubated in 10 μmol/L probe BODIPY-Fla-1 for 10 min; (c) Fluorescence image of cells incubated in 10 μmol/L probe BODIPY-Fla-1 for 10 min followed by 30 min of irradiation; (d~f) Corresponding bright-field images; (g) Relative fluorescence intensities of fluorescence images in different cell groups (Fluorescence images were captured with λex=340~380 nm and λem>425 nm; irradiation light source: green LED,15 W. The error bar represents the standard deviation of three repetitions, ****P≤0.0001, ***P≤0.001)

图8. 不同浓度的BODIPY-Fla-1在绿光照射或黑暗条件下对A549细胞的抗氧化应激作用

Figure 8. Anti-oxidative stress effect of different concentrations of BODIPY-Fla-1 on A549 cells in dark or under green light irradiation Error bars represent standard deviations of three replicate images in the same group, ***P≤0.001, *P≤0.1, ns: no significant difference.

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