【作者】 刘栋；

【导师】 陈兴娟；

【作者基本信息】 哈尔滨工程大学 ， 应用化学， 2005， 硕士

【摘要】 细胞内游离钙离子在生物生理过程中起着非常重要的作用,它广泛参与细胞的控制和新陈代谢,并且是一种重要的第二信使。鉴于钙离子的重要作用,需要有完善的分析技术来测量完整组织细胞内的钙离子浓度,其中¹⁹F-NMR是一种能无损伤检测生物组织细胞内钙离子浓度的理想方法。 应用¹⁹F-NMR法检测钙离子需要氟代指示剂,5F-BAPTA-AM是应用最广的氟代指示剂,它对钙离子有很高的选择亲和力,并且受细胞内pH值影响很小。另外,用5F-BAPTA检测时,细胞内较高的荧光背景不会对检测结果造成很大的影响。 在合成这种指示剂的过程中,首先利用2,4-二氟硝基苯与氢氧化钾在45℃反应合成了原料5-氟-2-硝基酚,并用乙酰溴与多聚甲醛（摩尔比1:1）反应合成了乙酸溴甲酯;等摩尔的5-氟-2-硝基酚与氢氧化钠在无水甲醇内反应得到对应的钠盐;该钠盐与过量的1,2—二溴乙烷在162℃反应得到1,2-二（5-氟-2-硝基苯氧基）乙烷;该产物可通过铁粉还原或氢化还原两种途径还原,氢化还原高效快捷,但对设备要求较高;还原产物与过量的溴乙酸甲酯（摩尔比1:6）在氮气环境下反应,反应温度91℃,产物用柱色谱分离后用95%乙醇重结晶;重结晶产物在无水甲醇内与等量的氢氧化钾,回流反应完成后提纯;在氮气环境下,5F-BAPTA钾盐与过量乙酸溴甲酯（摩尔比1:5）在无水乙腈中反应,反应温度91℃,产物用柱色谱分离后在无水甲醇中加热完全溶解,在-4℃冷冻室内析出无色粒状结晶,即5F-BAPTA-AM。 利用高效液相色谱HPLC检测了产物纯度,并利用质谱、紫外光谱、核磁氢谱等检测手段鉴定原料5-氟-2-硝基酚和最终产物5F-BAPTA-AM结构,并与文献中资料进行对照,确定了两种化合物的结构,为了确定实验过程的正确性,还对部分过程产物进行了质谱检测,并进行了分析,证明了反应过程的正确性和可行性。更多还原

【Abstract】 Intracellular free calcium ion has important effects on physiological functions. It is recognized that free calcium ion is involved in cell regulation, metabolism, and may serve as a second messenger. Because of its important functions, there is increasing demand for flawless analytical techniques for measuring intracellular calcium in intact viable tissues, in which ¹9F-NMR is a non-invasive technique which allows us to examine the intracellular free calcium ion inside living tissue.Fluorinated indicators are needed in ¹9F-NMR , in which 5F-BAPTA-AM is the most widely used probe for studying cytosolic free calcium ion ,which has high selectively for free calcium ion ,is not particularly pH sensitive. In addition, determinations with 5F-BAPTA are not sensitive to cells which have a high background fluorescence.During the process of synthesis of 5F-BAPTA-AM. we synthesized 5-fluoro-2-nitrophenol from 2,4-difluoro nitrobenzene and potassium hydroxide at 45 ℃ and bromomethyl acetate was synthesized from acetyl bromide and paraformaldehyde（1:1）. 5-fluoro-2-nitrophenol was converted to the corresponding sodium salt in aqueous with equal moles of sodium hydroxide. The salt was reacted with superfluous 1,2-dibromoethane in refluxing DMF. The nitro group of the product can be reduced with H₂ or by iron powder reduction. The progress of the reduction with H₂ was highly active, but complicated equipment was needed. The amine product was with superfluous methyl bromoacetate （1:6） in aqueous acetonitrile The product was puried by column chromatography and recrystallized from aqueous ethanol. The tetraester with aqueous methanol containing equivalents of KOH gave the tetrapotassium carboxylate which wassufficiently pure for the last step. The final conversion was effected by the esterification of the tetrapotassium carboxylate with superfluous bromomethy acetate （1:5） to give the acetoxymethy ester. The final compound was purified by column chromatography and the resulting syrup was then resoluted at high temperature. At -4°C, colorless needle-shaped crystal separated out, which was 5F-BAPTA-AM.The purity of the compounds was detected by HPLC spectrum , and the constitution of 5-fluoro-2-nitrophenol and 5F-BAPTA-AM was identified by El spectrum, ultraviolet spectrum and ’H-NMR spectrum ,and compared with the spectrum in the literature. In corroboration of the correctness of the process, the intermediate products were detected by El spectrum.The successful synthesis of 5F-BAPTA-AM prepared the ground for 19F-NMR in measuring intracellular calcium, and we piled up experience for synthesis of the derivatives of BAPTA.更多还原