水溶性吲哚方酸菁染料的合成及性能研究
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摘要
在近红外区内,荧光分析技术在组织细胞体内应用更充分,获得检测结果更理想。菁染料光谱性能优良,可通过增加甲川链长度,获得近红外菁染料。但随着甲川链的增长,染料光稳定性下降。另外,由于被标示的基质溶在水中,有机溶剂对敏感的基质会产生有害影响,所以生物分子荧光探针应为水溶性的。为此本论文合成了一系列水溶性对称和不对称吲哚方酸菁荧光染料,研究了分子结构与染料光谱性能,尤其是稳定性之间的关系。为了性能对比,合成了非水溶性吲哚方酸菁和链状五甲川菁染料。在合成过程中发现,固相法合成水溶性不对称吲哚方酸菁染料,产率接近传统两步法的2倍,为一种更适合的不对称染料的合成方法。
不同溶剂中,方酸菁染料最大吸收和发射波长在628～672 nm之间,Stokes位移在10～17 nm之间；链状五甲川菁染料的最大吸收和发射波长在646～687 nm之间,Stokes位移在18～27 nm之间；非水溶性对称方酸菁染料最大吸收和发射波长在621～658 nm之间,N-苄基取代染料Stokes位移在7-19 nm之间。
本论文所合成菁染料摩尔消光系数均大于105 mol-1·cm-1·L,水中荧光量子产率低于在其它极性有机溶剂中荧光量子产率。质子性溶剂中,染料表现为负向溶剂化效应。光稳定性实验证明,染料吲哚环N位脂烷基长度增加,染料稳定性下降,N位取代基上引入羧基,稳定性提高。向甲川链上引入方酸环、向吲哚环N位引入空间体积较大的苄基,都能够提高染料的光稳定性,苄基上连有吸电子基时,染料的稳定性得到进一步改善。N-羧苄基水溶性吲哚方酸菁染料的光漂白常数k约为商品化的荧光探针N-羧戊基染料的1／3,采用重结晶方法即可获得高纯度染料,为后续生物分析的实际应用奠定了基础。
水溶性对称吲哚方酸菁染料在阳离子和非离子表面活性剂溶液中,随着胶束的形成,染料的最大吸收和发射光谱发生红移、荧光强度和荧光量子产率大幅度增加、光稳定性提高,阳离子表面活性剂对染料的作用更明显,连有羧基的染料受影响程度最大。提出染料在表面活性剂胶束中的嵌入和吸附模型,对染料在胶束中的行为进行了解释。
水溶性不对称吲哚方酸菁染料在强酸强碱性水溶液中吸光度明显减小,pH=4时发生较明显的聚集。非水溶性菁染料在生物标记常用的有机溶剂DMF和DMSO的水溶液中,聚集随水量的增加而增强。含羧基染料在酸性溶液中形成较强烈的聚集。非水溶性菁染料在DMF中的光稳定性高于在甲醇中的光稳定性。通过对比,非水溶性菁染料在DMF中的光稳定性比相应的水溶性方酸菁染料高,而甲醇中水溶性方酸菁染料更稳定。
Fluorescence analysis technique can be fully applied in tissue cells with acceptable experiment results in the region of near-infrared. Cyanine dyes possess excellent spectral properties. Near-infrared cyanine dyes can be obtained by increasing length of conjugated methine chain in dye molecules. However, the photo-stability of cyanine dyes could be decreased when conjugated methine chain increases. The fluorescence probes in labeling the bio-substrates should be water-soluble because bio-substrates are dissolved in water and the organic solvents could be harmful to sensitive bio-substrates. In this paper, symmetrical and asymmetrical water-soluble squarylium indocyanine dyes were synthesized, the relationship between molecular structures and their spectral properties, especially in the aspect of the stability, was studied. Water-insoluble and chainlike pentamethine indocyanine dyes were synthesized for reference.
In the process of synthesizing of asymmetrical water-soluble squarylium indocyanine dyes, it is found that the yields of solid synthesis method are 2 times of that of traditional two-step method. Solid synthesis method is more suitable to synthesize water-soluble asymmetric squarylium indocyanine dyes.
The wavelengths of absorption and fluorescence emission maximum of the squarylium indocyanine dyes are from 628 to 672 nm. The Stokes shifts of the squarylium indocyanine dyes in the range of 10～17 nm are smaller than those of the chainlike pentamethine indocyanine dyes, which are in the range of 18～27 nm. Chainlike pentamethine indocyanine dyes display the absorption and fluorescence emission maximum from 646 to 687 nm. Water-insoluble symmetrical squarylium indocyanine dyes exhibit absorption and emission maximum from 621 to 658 nm. The Stokes shifts of these water-insoluble dyes with N-benzyl are in the range of 7～19 nm.
All dyes synthesized in this paper show a high molar absorption coefficientsεat least 105 mol-1·cm-1·L and lower values of fluorescence quantum yields in the aqueous medium compared with that in organic solvents. The absorption spectra of these dyes exhibit negative solvatochromism in protonic solvents.
The results of photo-stability tests demonstrate that photo-stability increases when shortening the length of alkyl group on N-position and introducing carboxyl groups on the alkyl group, squaric acid ring on conjugated methine chain or large benyzl ring in the N-substituents. Furthermore, the electron-withdrawing groups on the benzyl group improve the photo-stability compared with electron-donating groups. Photo-fading constant k of the squarylium indocyanine dye with N-carboxyl benzyl which is obtained with high purity only by recrystallizing is less than one-third of that of the squarylium indocyanine dyes with N-carboxyl pentyl that is commercially available. This lays the groundwork for subsequent bio-analytical applications of the dye with N-carboxyl benzyl.
In cationic and non-ionic surface active agents, water-soluble squarylium indocyanine dyes exhibit a red shift on their absorption and emission maximum, a significant increase in their fluorescence yields, and an enhancement on photo-stabilities along with the formation of micelles especially to the dye with carboxyl. The interaction between non-ionic surfactant and the dyes is weaker than that between cationic surfactant and the dyes. Embeding model and adsorption model were presented in this paper to explain the behaviors of dyes in surface active agents.
In aqueous solution with pH=2 and pH=12, absorption peak values of asymmetrical water-soluble sqarylium indocyanine dyes are reduced significantly. In aqueous solution with pH=4, aggregation of dyes molecules is apparent.
The aggregation of water-insoluble symmetrical squarylium indocyanine dyes can be enhanced with the increasing of water content in DMF and DMSO-aqueous medium. Only the dye with carboxyl could be influenced by pH value and exhibit obvious aggregation in acidic medium. The photo-stabilities of water-insoluble dyes in DMF are better compared with that in methanol. By contrast, the water-insoluble squaraylium indocyanine dyes express better photo-stabilities than that of water-soluble squaraylium indocyanine dyes in DMF, and opposite in methanol.
不同溶剂中,方酸菁染料最大吸收和发射波长在628～672 nm之间,Stokes位移在10～17 nm之间；链状五甲川菁染料的最大吸收和发射波长在646～687 nm之间,Stokes位移在18～27 nm之间；非水溶性对称方酸菁染料最大吸收和发射波长在621～658 nm之间,N-苄基取代染料Stokes位移在7-19 nm之间。
本论文所合成菁染料摩尔消光系数均大于105 mol-1·cm-1·L,水中荧光量子产率低于在其它极性有机溶剂中荧光量子产率。质子性溶剂中,染料表现为负向溶剂化效应。光稳定性实验证明,染料吲哚环N位脂烷基长度增加,染料稳定性下降,N位取代基上引入羧基,稳定性提高。向甲川链上引入方酸环、向吲哚环N位引入空间体积较大的苄基,都能够提高染料的光稳定性,苄基上连有吸电子基时,染料的稳定性得到进一步改善。N-羧苄基水溶性吲哚方酸菁染料的光漂白常数k约为商品化的荧光探针N-羧戊基染料的1／3,采用重结晶方法即可获得高纯度染料,为后续生物分析的实际应用奠定了基础。
水溶性对称吲哚方酸菁染料在阳离子和非离子表面活性剂溶液中,随着胶束的形成,染料的最大吸收和发射光谱发生红移、荧光强度和荧光量子产率大幅度增加、光稳定性提高,阳离子表面活性剂对染料的作用更明显,连有羧基的染料受影响程度最大。提出染料在表面活性剂胶束中的嵌入和吸附模型,对染料在胶束中的行为进行了解释。
水溶性不对称吲哚方酸菁染料在强酸强碱性水溶液中吸光度明显减小,pH=4时发生较明显的聚集。非水溶性菁染料在生物标记常用的有机溶剂DMF和DMSO的水溶液中,聚集随水量的增加而增强。含羧基染料在酸性溶液中形成较强烈的聚集。非水溶性菁染料在DMF中的光稳定性高于在甲醇中的光稳定性。通过对比,非水溶性菁染料在DMF中的光稳定性比相应的水溶性方酸菁染料高,而甲醇中水溶性方酸菁染料更稳定。
Fluorescence analysis technique can be fully applied in tissue cells with acceptable experiment results in the region of near-infrared. Cyanine dyes possess excellent spectral properties. Near-infrared cyanine dyes can be obtained by increasing length of conjugated methine chain in dye molecules. However, the photo-stability of cyanine dyes could be decreased when conjugated methine chain increases. The fluorescence probes in labeling the bio-substrates should be water-soluble because bio-substrates are dissolved in water and the organic solvents could be harmful to sensitive bio-substrates. In this paper, symmetrical and asymmetrical water-soluble squarylium indocyanine dyes were synthesized, the relationship between molecular structures and their spectral properties, especially in the aspect of the stability, was studied. Water-insoluble and chainlike pentamethine indocyanine dyes were synthesized for reference.
In the process of synthesizing of asymmetrical water-soluble squarylium indocyanine dyes, it is found that the yields of solid synthesis method are 2 times of that of traditional two-step method. Solid synthesis method is more suitable to synthesize water-soluble asymmetric squarylium indocyanine dyes.
The wavelengths of absorption and fluorescence emission maximum of the squarylium indocyanine dyes are from 628 to 672 nm. The Stokes shifts of the squarylium indocyanine dyes in the range of 10～17 nm are smaller than those of the chainlike pentamethine indocyanine dyes, which are in the range of 18～27 nm. Chainlike pentamethine indocyanine dyes display the absorption and fluorescence emission maximum from 646 to 687 nm. Water-insoluble symmetrical squarylium indocyanine dyes exhibit absorption and emission maximum from 621 to 658 nm. The Stokes shifts of these water-insoluble dyes with N-benzyl are in the range of 7～19 nm.
All dyes synthesized in this paper show a high molar absorption coefficientsεat least 105 mol-1·cm-1·L and lower values of fluorescence quantum yields in the aqueous medium compared with that in organic solvents. The absorption spectra of these dyes exhibit negative solvatochromism in protonic solvents.
The results of photo-stability tests demonstrate that photo-stability increases when shortening the length of alkyl group on N-position and introducing carboxyl groups on the alkyl group, squaric acid ring on conjugated methine chain or large benyzl ring in the N-substituents. Furthermore, the electron-withdrawing groups on the benzyl group improve the photo-stability compared with electron-donating groups. Photo-fading constant k of the squarylium indocyanine dye with N-carboxyl benzyl which is obtained with high purity only by recrystallizing is less than one-third of that of the squarylium indocyanine dyes with N-carboxyl pentyl that is commercially available. This lays the groundwork for subsequent bio-analytical applications of the dye with N-carboxyl benzyl.
In cationic and non-ionic surface active agents, water-soluble squarylium indocyanine dyes exhibit a red shift on their absorption and emission maximum, a significant increase in their fluorescence yields, and an enhancement on photo-stabilities along with the formation of micelles especially to the dye with carboxyl. The interaction between non-ionic surfactant and the dyes is weaker than that between cationic surfactant and the dyes. Embeding model and adsorption model were presented in this paper to explain the behaviors of dyes in surface active agents.
In aqueous solution with pH=2 and pH=12, absorption peak values of asymmetrical water-soluble sqarylium indocyanine dyes are reduced significantly. In aqueous solution with pH=4, aggregation of dyes molecules is apparent.
The aggregation of water-insoluble symmetrical squarylium indocyanine dyes can be enhanced with the increasing of water content in DMF and DMSO-aqueous medium. Only the dye with carboxyl could be influenced by pH value and exhibit obvious aggregation in acidic medium. The photo-stabilities of water-insoluble dyes in DMF are better compared with that in methanol. By contrast, the water-insoluble squaraylium indocyanine dyes express better photo-stabilities than that of water-soluble squaraylium indocyanine dyes in DMF, and opposite in methanol.
引文
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