【作者】 王丽秋；

【导师】 彭孝军；

【作者基本信息】 大连理工大学 ， 应用化学， 2003， 博士

【摘要】 随着生命科学的发展，迫切需要高灵敏度的分析检测方法。生物分子的荧光标记是继同位素标记后出现的快速方便的生物分子分析手段。二十世纪八十年代中期发展起来的基于荧光标记的DNA自动测序技术，已成为生命科学研究的重要里程碑。但目前仍需在荧光分析灵敏度和可靠性上进一步提高。3H-吲哚菁染料具有摩尔消光系数大(10~5L/mol·cm)、与生物基质结合后荧光增强(如与DNA结合后荧光增强25倍)、最大吸收波长可在400-1700 nm、调谐范围大、易于合成得到近红外荧光染料等优点，已成为新一代高灵敏荧光标示剂。但光稳定性差，影响分析的可靠性，是其不足。 本文以吲哚菁染料为母体，希望在分子拥有良好的荧光性能的同时，从两个方面改变染料的结构以提高耐光稳定性。首先将已有的N-羧戊基改变为N-对羧苄基，设计了具有下列通式中的1-3类系列线性多甲川3H-吲哚菁染料；在引入对羧苄基后，将方酸环或六元环引入分子的多甲川链中，设计了新型方酸菁染料和含氯六元桥环吲哚菁染料4-5，其结构通式如下： 这些新型3H-吲哚菁染料化合物的结构特点是分子中吲哚环N原子上的取代基至少有一个为对羧苄基，羧基与5位上的磺酸基团使分子具有良好的水溶性，其中的羧基为染料提供能与含游离氨基化合物共价结合的基团。 本文从对氨基苯磺酸出发，经重氮化、还原、与3-甲基-2-丁酮环合、进一步转化为钾盐后，以对氯甲基苯甲酸为吲哚环中氮原子上的季铵盐化试剂，首先合成了新染料中间体N-对羧苄基-2，3，3-三甲基-3H-吲哚啉-5-磺酸钾。通过该中间体与不同的缩合剂及其自身或另一种中间体进行缩合反应，合成得到了相应的上述系列目标染料化合物，并研究出适宜的分离与制备方法，经分离提 大连理工大学博士论文纯后，通过’H NMR、ESI－MS等对染料进行了结构表征。 光谱性能测试结果表明：染料的摩尔消光系数在 10’L／mol（m以上，新型三甲川蓄染料的最大吸收及荧光发射波长在540．600 urn之间，五甲）］l和六甲川染料的波长在近红外区。在溶液状态下，分子中氮原子上的取代基、甲 j！！链的长短、链中桥环及所用的溶剂影响着染料的吸收及荧光发射的波长和强度。研究表明，氮原子上的取代基改为对装等基后，线性多甲）h3H一吃呢蓄染料的最大吸收及荧光发射波长红移：引入方酸环后蓝移：引入含氯六元桥环时红移。在DMF和 DMSO溶剂中染料的吸收及荧光发射波长较大，在水中最小。在4。pH、10范围内N一对簸等基方酿着染料水溶液的最大吸收及荧光发射波长保持不变，吸收及发射强度却随pH值的不同而发生改变；与在水溶液中相比，在碳酸盐缓冲溶液中染料的荧光发射强度有所增大，在牛血清白蛋白缓冲溶液中吸收及荧光发射波长发生红移，吸收强度最小，荧光则最强。 无机氧化物是研究染料在固态下光谱性能的良好基质。与在水溶液中相比，染料在 SIOZ和 TIOZ固态基质中的最大吸收及荧光发射波长红移；在 SIOZ基质中荧光增强；在纳米 n。中，荧光强度减弱或粹灭，N一对装书基方酸美染料的最大吸收出现了双重峰，双峰的吸收强度随光照次数增加同时发生相对反方向的变化。经实验证明，在光的作用下，N一对枝节基方酸脊染料分于中的对接节基、方酸环和TIOZ导电玻璃的纳米孔穴彼此之间的相互作用产生了分于的结构或电荷分离态形式的不同，是产生这种光致效应的主要原因。 在距 40 W灯 125 cm处照射 75 h后的稳定性测试结果表明，与己有 N．按成基呗保蓄染料相比，系列新型N一对核等基一3H一吼跺蓄染料具有较好的光稳定性，这可能是因为对核书基的空间位阻较大，减少了单线态氧进攻甲川链的几率，从而使染料的光稳定性增加。 将近红外N一对核茉基一3H一哨跺方酸著染料转化为摇滚酸亚胺酯，在常温下用于赖氨酸、牛磺酸、个服及牛血清白蛋白的荧光标记。质谱、蛋白电泳及HPLC荧光检测分析结果证实，该染料可用于这类样品的荧光标记，对赖氨酸。牛磺酸和千胺的标记反应转化率为 100％。牛血清白蛋白的荧光标记结果证实产物具有很高的荧光检测响应值，最低平均检测限可达 1．44X 10”‘’mol几。 设计、合成的新荧光染料具有较好的水溶性、光稳定性和荧光性能，可望在生物分子标记、分析和分离等领域中得到重要应用。更多还原

【Abstract】 With the development of life science, there is a growing requirement for high sensitive detection technologies in analysis. Fluorescence label is a convenient method. Indocyanines with larger extinction coefficients (105 L/mol-cm), good fluorescent properties and fluorescence enhancement combining with biomass have been used as a new generation of fluorescent label compouds in biological analysis. However, photostability of these dyes has been a problem. In this paper the substituents on the nitrogen atoms in the heterocyclic rings of these indocyanines were designed as p-carboxybenzyl groups, and series novel straight chain polymethine indocyanines 1-3 were synthesized. Introduction of rigid rings into the methine chain resulted in novel 3H-indocyanines 4 and 5.They contained at least.one H-carboxybenzyl group on the nitrogen atoms in the heterocyclic rings. The sulfonate and carboxyl groups made them become water soluble, and the carboxyl group was employed as a linker for covalently linking to biomolecules in their biological applications.They have been synthesized by condensation of their intermediates(quaternary salt of indolenine) with corresponding condensating agents, respectively. The intermediates were prepared by the quaternization of 2,3,3-trimethylindoleninium-5-sulfonate with p-(chloromethyl)benzoic acid, 6-bromohexanoic acid or ethyl iodide. The 2,3,3-trimethyl-3H-indoleninium-5-sulfonate was from p-aminobenzoic acid. The dyes were separated, purified, and confirmed by ’HNMR and ESI-MS.The absorption and emission maxima of the novel dyes are between 540 nm and 850 nm,molar extinction is above 10~5 L/mol-cm. Compared to those of the corresponding straight pentamethine and heptamethine indocyanines, the absorption and emission maxima of these dyes become blue shift while the squaric ring was introduced into the methine chain, and red shift while the introduction of six-membered ring. The absorption and emission wavelength of these dyes were shifted with the changing of solvents. Within 4<pH<10, the difference in the pH values of the N-/7-carboxybenzyl-squaraine in water could make the intensity of absorption and emission of the squarine changed, but not the wavelength and shape of the peak.In this paper, the dyes were firstly embedded into a matrix of silica gel. Compared to those in water, the dyes in SiO2 gel slices exhibited longer absorption and fluorescence emission wavelength, stronger absorption and fluorescence intensity, and narrower absorption bands. The absorption spectrum of the N-b-carboxybenzyl-squaraine dye adsorbed on TiO2 film exhibited double peaks, one was blue shift (588 nm), another was red shift (637 nm). The absorption intensity of the blue shift one was stronger than that of the red shift one at the beginning of the testing, then with the increasing of times of irradiation on it the absorption intensity of the red shift peak increased, simultaneously that of the blue one decreased at the same quantity.The photostability of these dyes was tested under irradiation (40 W light) about 125 cm away. Compared to the series indocyanines containing N-carboxypentynyl group, the novel N-p-carboxybenzyl-3H-indocyanine dyes were stable.The NHS-carboxyl squaraine was prepared from the esterification of N-hydroxysuccinimide with carboxyl squaraine, and the NHS-carboxyl squaraine was used to conjugate with taurine, benzylamine, lysine and BSA in a pH 9.5 bicarbonate buffer, respectively. The conjugates were separated and detected by ESI-MS, SDS-PAGE, CE and HPLC, and the result indicated that the dye could couple covalently to biomass containing free NH2 group, and the detectability of BSA-sqauraine conjugate was 1.44 X 10-17 mol/L.These novel indocyanines hav ing good water solubility, fluorescent properties and photostability, can be used in biological analysis as fluorescent labeling reagents.更多还原