新型有机双光子探针的制备及其在生物成像中的应用
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摘要
自从1990年W. Denk等人第一次将双光子激发引入到荧光显微镜后,双光子激发荧光显微成像逐渐引起了人们极大的关注。相对于单光子荧光显微成像,双光子荧光显微成像具有很多优点,双光子激发只发生在焦点附近的极小区域,不需要共焦小孔便可实现三维高分辨成像。同时,双光子激发采用近红外激光作为激发光源,不仅可以提高穿透深度,并且能减少生物样品的光毒性和背景荧光的影响。
双光子荧光显微成像要求双光子荧光探针有较大的双光子激发截面和较好特异性能等。近二十年来,虽然很多具有优良的双光子特性的分子被报道,但是这些被报道的双光子吸收分子很少能应用到双光子荧光显微成像中。在本论文中,设计和合成了一些在双光子荧光显微成像中有较好应用前景的双光子探针。主要的内容和结果如下:
(1)设计和合成了一系列的基于2-羟基嘧啶的单枝(D-π-A,D:电子给体,π:π-共轭键桥, A:电子受体)和双枝类型(D-π-A-π-D,或Y型)的有机双光子探针分子(1a-4a和1b-4b)。系统研究了这些分子的光物理特性及Hep G2细胞的单光子和双光子荧光显微与成像的应用。结果表明,双枝分子(2a-4a和2b-4b)的双光子吸收截面是相应单枝分子(1a和1b)的12倍以上。另外,从疏水性分子(酯类, 1a-4a)到相应的两亲性分子(羧酸钠类型, 1b-4b),分子的单光子和双光子光谱特性没有很大的变化。但是,在相同的实验条件下,两亲性分子能对Hep G2细胞的细胞质部分进行有效的染色,而疏水性分子不易进入到Hep G2细胞内。
(2)设计和绿色合成了三个基于2-氨基嘧啶的有机双光子探针(5-7)。探针分子是在没有任何有机溶剂参与的条件下通过Aldol缩合反应制备,化合物(5-7)的制备具有反应时间短(化合物5-7分别为1 h、1.5 h和0.5 h)、产率高(化合物5-7分别为84%、75%和94%)及分离和纯化简单的特点。探针5-7具有较大的双光子吸收截面,例如探针7在甲苯溶液中的双光子吸收截面值达到819 GM,是相应标准物荧光素的22倍。另外,这三个分子在质子化或去质子化的作用下,分子的单光子和双光子荧光光谱的发射波长和强度都发生了很大的变化,探针的单光子和双光子荧光光谱随pH值的敏感性质使得这些探针将有可能被应用为pH传感器。
(3)合成了一个基于2-异硫氰嘧啶的和生物分子上的氨基能进行共价结合的有机双光子荧光探针8,探针8和D-氨基葡萄糖连接生成模型化合物9。探针8和模型化合物9的单光子特性和荧光量子产率没有很大的改变,但是其双光子吸收截面值却急剧的增大(在极性溶剂氯仿中),是探针8的3倍以上。探针8和转铁蛋白偶联后的产物(Tf-8)和转铁蛋白的竞争成像实验表明,连接探针8没有改变转铁蛋白的特性,依然能够在细胞表面的转铁蛋白受体的介导作用下进入到细胞内,并主要分布在细胞质部分。
(4)为了研究炔烃的衍生物对紫外光的光稳定性能,设计和合成了两个不对称(D-π-A)含炔烃的有机双光子探针(10-11)。系统研究了分子10和11的光物理特性、光稳定性和抗光漂白性能及Hep G2细胞的单光子和双光子荧光显微与成像的应用。结果表明,分子10和11对紫外光(λirr = 365 nm; I0 = 4 mW/cm2)有较好的光稳定性。这两个炔烃的衍生物具有较大的双光子吸收截面值,例如分子10在甲苯溶液中达到690 GM。
(5)设计和合成了四个对称结构(D–π–D)含炔烃的有机双光子吸收分子。通过调节分子π-共轭键的长度来调控分子的双光子吸收波长。发现在π-共轭键桥中间插入蒽环后分子的双光子吸收峰在820 nm附近,处在飞秒激光的最佳工作波长(800 nm)附近。为新型双光子探针的设计及性能调控提供了新的策略。
Since W. Denk et al. introduced the two-photon excitation method into fluorescence microscopy in 1990, there is an increasing interest in two-photon excitation fluorescence microscopic imaging (2PM). 2PM has some significant advantages over conventional single-photon fluorescence imaging techniques, including a capacity for a highly confined excitation, intrinsic three-dimensional resolution and the possibility of imaging at an increased penetration depth in tissue with reduced photodamage and background fluorescence.
2PM of biological systems will benefit from probes with large two-photon excitation cross-sections and good specificity. Over the past 20 years, numerous organic two-photon absorption (2PA) molecules with good 2PA properties have been reported. However, few efforts have been made on using efficient 2PA molecules as organic probes in practical 2PM. In this thesis, some 2PA probes with good prospects in 2PM application have been designed and synthesized. The main contents and results are summarized as follows:
(1) A series of 2-hydroxy-pyrimidine-based mono-substituted D-π-A (D: donor,π:π-conjugation, A: acceptor) and disubstituted D-π-A-π-D type (Y shape) organic 2PA probes (1a-4a and 1b-4b) have been designed and synthesized. Photophysical properties and sing- and two-photon fluorescence Hep G2 Cell imaging of the molecules are investigated systematically. The results indicate that the two-photon absorption cross-sections were found to have significant (up to 12 times) increase when changing from monosubstituted molecules(1a and 1b) to their disubstituted counterparts (2a-4a and 2b-4b). In addition, no significant difference in the single- and two-photon spectroscopic properties of these molecules can be observed when comparing the hydrophobic molecules (ethyl ester, 1a-4a) to the corresponding amphiphilic molecules (carboxylic acid salt, 1b-4b). However, single- and two-photon fluorescence microscopic imaging experiments revealed that the disubstituted amphiphilic molecules can efficiently stain the cytoplasmic region of HepG2 cells, while little or no dye uptake happened when incubating those cells with the hydrophobic molecules under the same experimental conditions.
(2) Three new organic 2PA probes (5-7) based on 2-amino-pyrimidine core were designed and greenly synthesized by Aldol condensation in the absence of any organic solvents. Compounds 5-7 could be prepared after short reaction times (1 h, 1.5 h and 0.5 h for compounds 5-7, respectively), high yields (84%, 75% and 94% for compounds 5-7, respectively) and simple separation and purification. Probes 5-7 were found to have large two-photon absorption cross-sections. Probe 7 has the largest two-photon absorption cross-section value (819 GM) in toluene, which is 22 times larger than that of fluorescein standard. In addition, strong modulation of single-photon and two-photon fluorescent spectra of these molecules by (de)protonation makes them promising candidates for two-photon pH sensor.
(3) An amine-reactive organic 2PA probe (8) based on 2-pyrimidinyl- isothiocyanate for covalent attachment onto amine-containing biomolecules was prepared. A model probe-adduct (9) was prepared by reacted (8) with D-glucosamine. Although no significant changes in the single-photon spectroscopic properties and the quantum yield could be observed between 8 and the modle adduct, the 2PA cross-sections of molecule 9 sharp increases in polar CHCl3 solvent, which is 3 times larger than that of probe 8. Tf-8 conjugates were produced by coupling molecule 8 to transferrin (Tf). Competirion experiment between Tf-8 and Tf indicate that Tf-8 was recognized by and bound to the transferrin receptor on the membrane, and delivered into cytoplasm.
(4) In order to study the photostability of alkynes derivatives under UV light, two asymmetrical (D-π-A) organic 2PA probes with substituted acetylene as central rigid elongated conjugation have been designed and synthesized. Photophysical properties, photostability, antiphotobleaching and sing- and two-photon fluorescence Hep G2 Cell imaging of the molecules 10 and 11 are investigated systematically. The results indicate that the acetylene-substituted molecules (10 and 11) were found to have good photostability under UV light (λirr = 365 nm; I0 = 4 mW/cm2). In addition, these acetylene-substituted molecules were found to have high two-photon absorption cross-sections (for example, 690 GM for molecule 10 in toluene).
(5) Four symmetrical organic 2PA molecules (D–π–D) with substituted acetylene as central rigid elongated conjugation have been designed and synthesized. One derivative with great enhancement on the two-photon absorption cross-section in 820 nm, which is among the best output wavelength range of a typical Ti:Sapphire femtosecond laser, could be obtained by inserting an anthracene ring into the rigid elongatedπ-conjugation. Such kind of structure modification is expected to be helpful in designing better organic 2PA probes for biological imaging.
双光子荧光显微成像要求双光子荧光探针有较大的双光子激发截面和较好特异性能等。近二十年来,虽然很多具有优良的双光子特性的分子被报道,但是这些被报道的双光子吸收分子很少能应用到双光子荧光显微成像中。在本论文中,设计和合成了一些在双光子荧光显微成像中有较好应用前景的双光子探针。主要的内容和结果如下:
(1)设计和合成了一系列的基于2-羟基嘧啶的单枝(D-π-A,D:电子给体,π:π-共轭键桥, A:电子受体)和双枝类型(D-π-A-π-D,或Y型)的有机双光子探针分子(1a-4a和1b-4b)。系统研究了这些分子的光物理特性及Hep G2细胞的单光子和双光子荧光显微与成像的应用。结果表明,双枝分子(2a-4a和2b-4b)的双光子吸收截面是相应单枝分子(1a和1b)的12倍以上。另外,从疏水性分子(酯类, 1a-4a)到相应的两亲性分子(羧酸钠类型, 1b-4b),分子的单光子和双光子光谱特性没有很大的变化。但是,在相同的实验条件下,两亲性分子能对Hep G2细胞的细胞质部分进行有效的染色,而疏水性分子不易进入到Hep G2细胞内。
(2)设计和绿色合成了三个基于2-氨基嘧啶的有机双光子探针(5-7)。探针分子是在没有任何有机溶剂参与的条件下通过Aldol缩合反应制备,化合物(5-7)的制备具有反应时间短(化合物5-7分别为1 h、1.5 h和0.5 h)、产率高(化合物5-7分别为84%、75%和94%)及分离和纯化简单的特点。探针5-7具有较大的双光子吸收截面,例如探针7在甲苯溶液中的双光子吸收截面值达到819 GM,是相应标准物荧光素的22倍。另外,这三个分子在质子化或去质子化的作用下,分子的单光子和双光子荧光光谱的发射波长和强度都发生了很大的变化,探针的单光子和双光子荧光光谱随pH值的敏感性质使得这些探针将有可能被应用为pH传感器。
(3)合成了一个基于2-异硫氰嘧啶的和生物分子上的氨基能进行共价结合的有机双光子荧光探针8,探针8和D-氨基葡萄糖连接生成模型化合物9。探针8和模型化合物9的单光子特性和荧光量子产率没有很大的改变,但是其双光子吸收截面值却急剧的增大(在极性溶剂氯仿中),是探针8的3倍以上。探针8和转铁蛋白偶联后的产物(Tf-8)和转铁蛋白的竞争成像实验表明,连接探针8没有改变转铁蛋白的特性,依然能够在细胞表面的转铁蛋白受体的介导作用下进入到细胞内,并主要分布在细胞质部分。
(4)为了研究炔烃的衍生物对紫外光的光稳定性能,设计和合成了两个不对称(D-π-A)含炔烃的有机双光子探针(10-11)。系统研究了分子10和11的光物理特性、光稳定性和抗光漂白性能及Hep G2细胞的单光子和双光子荧光显微与成像的应用。结果表明,分子10和11对紫外光(λirr = 365 nm; I0 = 4 mW/cm2)有较好的光稳定性。这两个炔烃的衍生物具有较大的双光子吸收截面值,例如分子10在甲苯溶液中达到690 GM。
(5)设计和合成了四个对称结构(D–π–D)含炔烃的有机双光子吸收分子。通过调节分子π-共轭键的长度来调控分子的双光子吸收波长。发现在π-共轭键桥中间插入蒽环后分子的双光子吸收峰在820 nm附近,处在飞秒激光的最佳工作波长(800 nm)附近。为新型双光子探针的设计及性能调控提供了新的策略。
Since W. Denk et al. introduced the two-photon excitation method into fluorescence microscopy in 1990, there is an increasing interest in two-photon excitation fluorescence microscopic imaging (2PM). 2PM has some significant advantages over conventional single-photon fluorescence imaging techniques, including a capacity for a highly confined excitation, intrinsic three-dimensional resolution and the possibility of imaging at an increased penetration depth in tissue with reduced photodamage and background fluorescence.
2PM of biological systems will benefit from probes with large two-photon excitation cross-sections and good specificity. Over the past 20 years, numerous organic two-photon absorption (2PA) molecules with good 2PA properties have been reported. However, few efforts have been made on using efficient 2PA molecules as organic probes in practical 2PM. In this thesis, some 2PA probes with good prospects in 2PM application have been designed and synthesized. The main contents and results are summarized as follows:
(1) A series of 2-hydroxy-pyrimidine-based mono-substituted D-π-A (D: donor,π:π-conjugation, A: acceptor) and disubstituted D-π-A-π-D type (Y shape) organic 2PA probes (1a-4a and 1b-4b) have been designed and synthesized. Photophysical properties and sing- and two-photon fluorescence Hep G2 Cell imaging of the molecules are investigated systematically. The results indicate that the two-photon absorption cross-sections were found to have significant (up to 12 times) increase when changing from monosubstituted molecules(1a and 1b) to their disubstituted counterparts (2a-4a and 2b-4b). In addition, no significant difference in the single- and two-photon spectroscopic properties of these molecules can be observed when comparing the hydrophobic molecules (ethyl ester, 1a-4a) to the corresponding amphiphilic molecules (carboxylic acid salt, 1b-4b). However, single- and two-photon fluorescence microscopic imaging experiments revealed that the disubstituted amphiphilic molecules can efficiently stain the cytoplasmic region of HepG2 cells, while little or no dye uptake happened when incubating those cells with the hydrophobic molecules under the same experimental conditions.
(2) Three new organic 2PA probes (5-7) based on 2-amino-pyrimidine core were designed and greenly synthesized by Aldol condensation in the absence of any organic solvents. Compounds 5-7 could be prepared after short reaction times (1 h, 1.5 h and 0.5 h for compounds 5-7, respectively), high yields (84%, 75% and 94% for compounds 5-7, respectively) and simple separation and purification. Probes 5-7 were found to have large two-photon absorption cross-sections. Probe 7 has the largest two-photon absorption cross-section value (819 GM) in toluene, which is 22 times larger than that of fluorescein standard. In addition, strong modulation of single-photon and two-photon fluorescent spectra of these molecules by (de)protonation makes them promising candidates for two-photon pH sensor.
(3) An amine-reactive organic 2PA probe (8) based on 2-pyrimidinyl- isothiocyanate for covalent attachment onto amine-containing biomolecules was prepared. A model probe-adduct (9) was prepared by reacted (8) with D-glucosamine. Although no significant changes in the single-photon spectroscopic properties and the quantum yield could be observed between 8 and the modle adduct, the 2PA cross-sections of molecule 9 sharp increases in polar CHCl3 solvent, which is 3 times larger than that of probe 8. Tf-8 conjugates were produced by coupling molecule 8 to transferrin (Tf). Competirion experiment between Tf-8 and Tf indicate that Tf-8 was recognized by and bound to the transferrin receptor on the membrane, and delivered into cytoplasm.
(4) In order to study the photostability of alkynes derivatives under UV light, two asymmetrical (D-π-A) organic 2PA probes with substituted acetylene as central rigid elongated conjugation have been designed and synthesized. Photophysical properties, photostability, antiphotobleaching and sing- and two-photon fluorescence Hep G2 Cell imaging of the molecules 10 and 11 are investigated systematically. The results indicate that the acetylene-substituted molecules (10 and 11) were found to have good photostability under UV light (λirr = 365 nm; I0 = 4 mW/cm2). In addition, these acetylene-substituted molecules were found to have high two-photon absorption cross-sections (for example, 690 GM for molecule 10 in toluene).
(5) Four symmetrical organic 2PA molecules (D–π–D) with substituted acetylene as central rigid elongated conjugation have been designed and synthesized. One derivative with great enhancement on the two-photon absorption cross-section in 820 nm, which is among the best output wavelength range of a typical Ti:Sapphire femtosecond laser, could be obtained by inserting an anthracene ring into the rigid elongatedπ-conjugation. Such kind of structure modification is expected to be helpful in designing better organic 2PA probes for biological imaging.
引文
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