生物荧光标识用菁染料的研究
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摘要
随着蛋白质组时代的来临,荧光检测技术结合高效液相(HPLC)或凝胶电泳技术的应用大大推动了蛋白质组学的进步。但生物检测的灵敏度仍有待于进一步提高。菁染料是目前最主要的蛋白质荧光标示剂之一,该类染料的主要问题在于容易发生光漂白和自聚集导致荧光淬灭。本论文主要针对吲哚类菁染料进行合成、性能以及蛋白质荧光标记的应用研究。
为了开发出蛋白质标记效果更好的菁染料,合成了系列水溶性三甲川菁染料,其中N-对羧苄基-N'-磺丁基-5’-磺酸基-三甲川-3H-吲哚菁染料(Ⅱe)标记牛血清白蛋白(BSA)的检测限为4.8×10-10 mol L-1,达到文献报道的较灵敏的荧光试剂的水平。同时应用高效液相色谱和聚丙烯酰胺凝胶电泳(SDS-PAGE)两种分析技术研究染料结构对蛋白质标记的影响。结果表明,在染料的琥珀酰亚胺活性酯(NHS)一侧应为疏水的,而在染料另一侧引入磺酸基来提供必要的水溶性,这样的染料结构更有利于蛋白质标记反应。
由于近红外染料可以有效避开生物自荧光所造成的干扰,合成了四个水溶性不对称五甲川菁染料(647-680 nm),结构未见报道。光稳定性实验结果表明,在吲哚环N位引入刚性苯环结构或空间体积较大的磺丁基等基团,能够提高染料的光稳定性能。应用其中具有较好光稳定性的N-对羧苄基-N'-磺丁基-5,5'-磺酸基-五甲川-3H-吲哚菁染料(Ⅲj)标记BSA,最低检测限达到1.2×10-8mol L-1,可比商品化染料荧光素异硫氰酸酯(FITC)检测更低丰度的蛋白质,与紫外检测相比检测灵敏度可以提高近二个数量级。
针对近红外五甲川菁染料光稳定性差的问题,设计合成系列水溶性方酸菁染料,其中两个不对称染料结构未见报道。光稳定性实验结果表明,方酸菁染料的稳定性优于五甲川菁染料的稳定性,不同N位取代基染料光稳定性大小顺序依次为对羧苄基>苄基>乙基>羧戊基,可见,在吲哚环N位引入体积大、具有刚性苯环结构的基团,能够阻止单重态氧的进攻,从而提高菁染料的光稳定性。而对于同是苯环结构的对羧苄基和苄基来说,苯环对位具有吸电性的羧基更有利于提高菁染料的光稳定性。
此外,针对菁染料易于聚集的问题,合成了新型多氟取代三甲川菁染料,同时优化了反应条件,提高中间体和目标产物的收率。通过与传统无氟菁染料对比,多个氟原子的引入提高了染料在水中的光稳定性,减弱了染料的自聚作用。
In the post-genome sequencing era, protein analysis has been paid more and more attention. Fluorescence detection was readily applied in high performance liquid chromatography (HPLC) or electrophoresis techniques, especially the differential in-gel electrophoresis (DIGE), enabling qualitative and quantitative analysis of proteins. For proteomics, the improvement on the detection sensitivity of proteins is an imperative task. Cyanine dyes have found widespread use as fluorescent labels for biomolecules, particularly for fluorescent labeling proteins. However, two common problems encountered by cyanine dyes are their susceptibility to form nonfluorescent aggregates and their tendency to undergo photobleaching. In this thesis, cyanine dyes based on 2,3,3-trimethyl-3H-indolium are synthesized and their corresponding properties and biological applications are studied.
In order to improve the protein labeling effect, a series of water-soluble trimethine 3H-indocyanine (Cy3) dyes were synthesized. The limit of detection of the asymmetric dye II e for BSA decreased to 4.8×10-10 mol L-1. Two methods, high performance liquid chromatography and gel electrophoresis (SDS-PAGE), were used to evaluate the influence of different dyes'structures on protein labeling. The results indicate the asymmetric dyes can benefit protein labeling, which possess the carboxyl group on the hydrophobic side of the dyes to synthesize the NHS ester and sulfo-groups on the other side to provide the dyes with necessary water-solubility.
Four novel water-soluble asymmetric Cy5 dyes were synthesized because their spectra reach the near-infrared (NIR) region, where a biological matrix exhibits the least absorption and autofluorescence background. It is demonstrated that the benzyl group and sulfo-group on the N-position of 3H-indolium pentamethine cyanine dyes improved the photostability in aqueous solutions. In addition, the limit of detection of dye IIIj for BSA was 1.2×10-8 mol L-1 about 100-fold lower than that by UV detection. Compared to commercial fluorescein isothiocyanate (FITC), more proteins with low concentrations could be labeled, resulting in improved detection sensitivity for protein analysis. All results demonstrated that asymmetric dye IIIj possesses better photostability, which can be a good fluorescent labeling reagent for protein labeling.
Focus on the poor photostability of pentamethine cyanine dyes, a series of water-soluble squarylium cyanine dyes were synthesized, including two novel asymmetric dyes. The result is shown that their photostabilities of squarylium cyanine dyes reported here are better than that of pentamethine cyanine dyes. The effect of different N-substituents on the photostability of the dyes can be placed in the order:p-carboxylbenzyl group> benzyl group> ethyl group > carboxylpentanyl group. Therefore, the benzyl group on the N-position of 3H-indolium pentamethine cyanine dyes improved the photostability in aqueous solutions, and reduced reactivity toward singlet oxygen, and the electron-withdrawing group on the benzyl group on nitrogen atom of cyanine dyes is good for improving photostability.
Finally, focus on the problem of aggregation, a novel polyfluorinated cyanine dye was synthesized and its reaction conditions were optimized to imcrease the yields of intermediates and product. Compared with the nonfluorinated analogue, the novel dye exhibits significantly reduced aggregation in aqueous media, enhanced fluorescence quantum yield, greater resistance to photobleaching.
为了开发出蛋白质标记效果更好的菁染料,合成了系列水溶性三甲川菁染料,其中N-对羧苄基-N'-磺丁基-5’-磺酸基-三甲川-3H-吲哚菁染料(Ⅱe)标记牛血清白蛋白(BSA)的检测限为4.8×10-10 mol L-1,达到文献报道的较灵敏的荧光试剂的水平。同时应用高效液相色谱和聚丙烯酰胺凝胶电泳(SDS-PAGE)两种分析技术研究染料结构对蛋白质标记的影响。结果表明,在染料的琥珀酰亚胺活性酯(NHS)一侧应为疏水的,而在染料另一侧引入磺酸基来提供必要的水溶性,这样的染料结构更有利于蛋白质标记反应。
由于近红外染料可以有效避开生物自荧光所造成的干扰,合成了四个水溶性不对称五甲川菁染料(647-680 nm),结构未见报道。光稳定性实验结果表明,在吲哚环N位引入刚性苯环结构或空间体积较大的磺丁基等基团,能够提高染料的光稳定性能。应用其中具有较好光稳定性的N-对羧苄基-N'-磺丁基-5,5'-磺酸基-五甲川-3H-吲哚菁染料(Ⅲj)标记BSA,最低检测限达到1.2×10-8mol L-1,可比商品化染料荧光素异硫氰酸酯(FITC)检测更低丰度的蛋白质,与紫外检测相比检测灵敏度可以提高近二个数量级。
针对近红外五甲川菁染料光稳定性差的问题,设计合成系列水溶性方酸菁染料,其中两个不对称染料结构未见报道。光稳定性实验结果表明,方酸菁染料的稳定性优于五甲川菁染料的稳定性,不同N位取代基染料光稳定性大小顺序依次为对羧苄基>苄基>乙基>羧戊基,可见,在吲哚环N位引入体积大、具有刚性苯环结构的基团,能够阻止单重态氧的进攻,从而提高菁染料的光稳定性。而对于同是苯环结构的对羧苄基和苄基来说,苯环对位具有吸电性的羧基更有利于提高菁染料的光稳定性。
此外,针对菁染料易于聚集的问题,合成了新型多氟取代三甲川菁染料,同时优化了反应条件,提高中间体和目标产物的收率。通过与传统无氟菁染料对比,多个氟原子的引入提高了染料在水中的光稳定性,减弱了染料的自聚作用。
In the post-genome sequencing era, protein analysis has been paid more and more attention. Fluorescence detection was readily applied in high performance liquid chromatography (HPLC) or electrophoresis techniques, especially the differential in-gel electrophoresis (DIGE), enabling qualitative and quantitative analysis of proteins. For proteomics, the improvement on the detection sensitivity of proteins is an imperative task. Cyanine dyes have found widespread use as fluorescent labels for biomolecules, particularly for fluorescent labeling proteins. However, two common problems encountered by cyanine dyes are their susceptibility to form nonfluorescent aggregates and their tendency to undergo photobleaching. In this thesis, cyanine dyes based on 2,3,3-trimethyl-3H-indolium are synthesized and their corresponding properties and biological applications are studied.
In order to improve the protein labeling effect, a series of water-soluble trimethine 3H-indocyanine (Cy3) dyes were synthesized. The limit of detection of the asymmetric dye II e for BSA decreased to 4.8×10-10 mol L-1. Two methods, high performance liquid chromatography and gel electrophoresis (SDS-PAGE), were used to evaluate the influence of different dyes'structures on protein labeling. The results indicate the asymmetric dyes can benefit protein labeling, which possess the carboxyl group on the hydrophobic side of the dyes to synthesize the NHS ester and sulfo-groups on the other side to provide the dyes with necessary water-solubility.
Four novel water-soluble asymmetric Cy5 dyes were synthesized because their spectra reach the near-infrared (NIR) region, where a biological matrix exhibits the least absorption and autofluorescence background. It is demonstrated that the benzyl group and sulfo-group on the N-position of 3H-indolium pentamethine cyanine dyes improved the photostability in aqueous solutions. In addition, the limit of detection of dye IIIj for BSA was 1.2×10-8 mol L-1 about 100-fold lower than that by UV detection. Compared to commercial fluorescein isothiocyanate (FITC), more proteins with low concentrations could be labeled, resulting in improved detection sensitivity for protein analysis. All results demonstrated that asymmetric dye IIIj possesses better photostability, which can be a good fluorescent labeling reagent for protein labeling.
Focus on the poor photostability of pentamethine cyanine dyes, a series of water-soluble squarylium cyanine dyes were synthesized, including two novel asymmetric dyes. The result is shown that their photostabilities of squarylium cyanine dyes reported here are better than that of pentamethine cyanine dyes. The effect of different N-substituents on the photostability of the dyes can be placed in the order:p-carboxylbenzyl group> benzyl group> ethyl group > carboxylpentanyl group. Therefore, the benzyl group on the N-position of 3H-indolium pentamethine cyanine dyes improved the photostability in aqueous solutions, and reduced reactivity toward singlet oxygen, and the electron-withdrawing group on the benzyl group on nitrogen atom of cyanine dyes is good for improving photostability.
Finally, focus on the problem of aggregation, a novel polyfluorinated cyanine dye was synthesized and its reaction conditions were optimized to imcrease the yields of intermediates and product. Compared with the nonfluorinated analogue, the novel dye exhibits significantly reduced aggregation in aqueous media, enhanced fluorescence quantum yield, greater resistance to photobleaching.
引文
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