CN114736213B - 一种羧基-四氯-荧光素的制备方法 - Google Patents
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Abstract
本发明提供一种羧基‑四氯‑荧光素的制备方法,以对氯间苯二酚和二氯偏三酸酐为原料,通过直接环合反应直接制得5,6‑羧基‑四氯‑荧光素,将该异构体混合物先通过重结晶纯化,再利用两种异构体在酸性条件下,在不同有机溶剂中的溶解度不同,实现两者的分离,分别得到高纯度的5‑羧基‑四氯‑荧光素和6‑羧基‑四氯‑荧光素。整个制备工艺具有操作简便,对设备要求低,成本低,设备使用效率高等特点,达到工业化生产应用的要求。
Description
技术领域
本发明涉及含氯荧光素制备技术领域,尤其是一种羧基-四氯-荧光素的制备方法。
背景技术
自1871年Bayer首次合成荧光素以来,荧光素及其衍生物被广泛用于各种荧光探针,如药理学、生理学、分子生物学、细胞生物学、分子遗传学、环境化学、单个分子检测、信息科学、荧光标记、激光染料等方面。荧光素及其衍生物属于呫吨类结构,即由一个桥氧键将两个苯环固定在同一个平面上,这样的刚性平面结构使得荧光素及其衍生物分子具有长波长(吸收和发射都在可见光区),其高摩尔吸光系数和高量子产率与其它常用荧光染料相比,具有对生物体友好无毒或低毒的优点。
然而,荧光素的这种特殊结构也给其带来的特殊性质,例如,对pH敏感,在生物体内的生理环境下荧光量子效率急剧下降,对光敏感,抗光漂白能力弱,在强光下荧光衰减明显,检测特异性不强等。这些特殊性质也极大地限制了其进一步的应用。
为了改善荧光素类染料的光学性能,国内外许多生物及化学工作者在荧光素的底环或顶环上引入不同功能的化学基团,对其结构进行修饰以达到不同的功能。例如,1963年,Guilbault成功地应用荧光素二丁酸酯检测了脂酶的活性,Miller合成出四种乙基取代的荧光素衍生物用于测定白鼠肝细胞中细胞色素P-450的活性;1997年,Riondet利用羧基荧光素检测大肠埃希氏菌细胞内部的pH值,得到满意结果,Kharma设计合成了氯代羧基荧光素衍生物,发现氯代羧基荧光素增加羧基荧光素的荧光强度、降低其pKa值,荧光对pH值敏感度明显酸移;后来科学家们又合成四氯-5(6)-羧基荧光素,发现随着荧光素结构中氯原子的增加,最大吸收波长和最大发射波长都明显向长波长方向移动,氯原子个数增加,移动愈明显,且随着氯原子增加,抗光漂白的能力进一步增强,荧光对pH值敏感度酸移越明显。
这种相似分子结构的微小改变可以调整分子吸收/发射波长,有助于平行合成不同标记的DNA或蛋白质,这一特性使得含氯荧光素衍生物在DNA合成和测序中得到广泛应用。但是,现有的羧基-四氯-荧光素的制备方法收率低(通常只有百分之十几)和纯化过程繁琐(制备柱纯化),因此开发出更适合工业化生产的羧基-四氯-荧光素合成方法,降低生产成本有着十分重要的意义。
目前国内外合成羧基-四氯-荧光素的方法主要有以下两种:
1、直接缩合合成法
此路线是荧光素类荧光染料的最经典合成方法,最早由瑞士化学家MauriceCeresole开发,整个反应过程需要两个连续的Friedel-Crafts型亲电芳香取代反应形成呫吨骨架;以对氯间苯二酚苯酚和二氯偏三酸为原料,在路易斯酸(ZnBr2)中高温缩合制得。优点是路线简单,主要缺点有:⑴反应条件比较苛刻,通常需要高温(180℃)甚至熔融;⑵收率低,通常只有百分之十几;⑶生成5,6位的异构体,这类异构体混合物物理化学性质相似,常规方法很难分离纯化,需采用硅胶柱层析分离方法,不仅成本高外,而且只能得到克级甚至毫克级的产品,难以工业化批量制备。
2、分步合成法
Matthew H.Lyttle改进了直接缩合合成法,新方法以对氯间苯二酚苯酚和二氯偏三酸为原料,在甲磺酸中150℃缩合制得。此方法显著提高了5,6羧基-四氯-荧光素(5,6-TET)的收率(79%左右),但HPLC纯度仅为74%,还需通过中性氧化铝柱层析继续纯化,得到HPLC纯度为90%左右的混合物。
此方法借鉴了之前羧基荧光素的分离方法,采用特戊酰氯保护羟基,然后用二异丙胺成盐,利用两种衍生物异构体的盐在溶剂中溶解度的不同进行拆分,拆分出的盐通过浓氨水脱保护,并用盐酸脱盐得到两种异构体,但异构体的纯度只能在90%左右。
发明内容
针对现有羧基-四氯-荧光制备方法存在的技术缺陷,本发明提供一种纯度高、成本低、操作简便、原子经济性高、易于大规模生产的羧基-四氯-荧光素制备方法。
一种羧基-四氯-荧光素的制备方法,以对氯间苯二酚和二氯偏三酸酐为原料,通过环合反应制得5,6-羧基-四氯-荧光素粗品,利用重结晶方法对5,6-羧基-四氯-荧光素粗品进行提纯,得到5,6-羧基-四氯-荧光素纯品;
将5,6-羧基-四氯-荧光素纯品溶解于有机溶剂Ⅰ中,室温下逐滴滴入酸溶液,充分反应后过滤,洗涤滤饼并烘干,得到5-羧基-四氯-荧光素,向滤液中缓慢加入异丙醚,充分反应后过滤,洗涤滤饼并烘干得到6-羧基-四氯-荧光素;
所述有机溶剂Ⅰ为乙腈、四氢呋喃、二氧六环、N,N-二甲基甲酰胺、乙二醇二甲醚中的一种,所述酸溶液为盐酸、硫酸、甲磺酸、三氟甲磺酸、三氟乙酸、醋酸中的一种。
进一步的,5,6-羧基-四氯-荧光素纯品与有机溶剂Ⅰ的投料比为1:2-10。进一步的,线性结构的聚氨酯树脂/丙烯酸树脂与网状结构的聚氨酯树脂/丙烯酸树脂的混合比例为1:2-2:1。
进一步的,从5,6-羧基-四氯-荧光素纯品分离出5-羧基-四氯-荧光素、6-羧基-四氯-荧光素包括以下步骤:
步骤1,5,6-羧基-四氯-荧光素、有机溶剂Ⅰ加入玻璃反应釜,70℃温度下搅拌2小时,待固体充分溶解后停止加热,反应体系逐渐冷却至室温;
步骤2,逐滴滴入酸溶液,体系逐渐变为黄红色溶液,滴加完毕继续搅拌1小时,体系变为悬浮液,停止搅拌;
步骤3,体系冷却至0℃后静置12小时,有大量黄色固体析出,过滤;
步骤4,滤饼加入冰水搅拌1小时,过滤,重复操作2次;
步骤5,滤饼在室温下用丙酮打浆1小时,过滤;
步骤6,烘干滤饼,烘干温度40℃,烘干时长12小时,得到5-羧基-四氯-荧光素;
步骤7,将步骤3所得滤液冷却至0℃搅拌,缓慢加入异丙醚,停止搅拌,体系在0℃温度下静置12小时,有大量黄色固体析出,过滤;
步骤8,滤饼加入冰水搅拌1小时,过滤,重复操作2次;
步骤9,滤饼在室温下用丙酮打浆1小时,过滤;
步骤10,烘干滤饼,烘干温度40℃,烘干时长12小时,得到6-羧基-四氯-荧光素。
进一步的,以对氯间苯二酚和二氯偏三酸酐为原料,以质子酸为催化剂,进行环合反应,反应温度120-220℃,反应时间2-8小时;对氯间苯二酚和二氯偏三酸酐投料比为2.05-2.5:1。
进一步的,所述质子酸为甲磺酸、三氟甲磺酸、硫酸、磷酸中的一种,与二氯偏三酸酐投料比为1-10:1。
进一步的,将5,6-羧基-四氯-荧光素粗品加入至有机溶剂Ⅱ中,加热温度40-80℃,加热时间45-90分钟,固体溶解完全后停止加热,继续搅拌冷却至室温,再缓慢滴入正己烷,滴加过程中有固体析出,0℃冷却,静置24小时,过滤、洗涤、烘干得5,6-羧基-四氯-荧光素纯品。
进一步的,所述有机溶剂Ⅱ为乙腈、四氢呋喃、二氧六环、乙醇、乙酸乙酯中的一种。
本发明以对氯间苯二酚和二氯偏三酸酐为原料,在质子酸的催化下,直接环合反应得到5位取代基和6位取代基的异构体混合物,将该异构体混合物先通过重结晶纯化,再利用两种异构体在酸性条件下,在不同有机溶剂中的溶解度不同,实现两者的分离,分别得到高纯度的5-羧基-四氯-荧光素和6-羧基-四氯-荧光素。
在该异构体混合物的分离操作上,彻底变革了现有技术通过保护羟基,然后成盐,再脱除保护基的操作,极大降低了分离成本,简化了操作步骤,从而使羧基-四氯-荧光素工业化生产的原子经济性和生产成本显著降低。
利用重结晶方法对5,6-羧基-四氯-荧光素粗品进行提纯,避免了传统的柱层析分离纯化,显著地提高反应转化率和提纯操作的简便性,使得规模化生产成为可能。
整个制备工艺具有操作简便,对设备要求低,成本低,设备使用效率高等特点,达到工业化生产应用的要求。
具体实施方式
下面结合具体实施例对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员所获得的所有其他实施例,都属于本发明保护的范围。
实施例1制备5,6-羧基-四氯-荧光素粗品
4320g对氯间苯二酚、4000g二氯偏三酸酐、12L甲磺酸加入50L玻璃反应釜,抽去反应釜中的空气,通入氮气,反应体系逐渐升温至160℃,反应6小时。对反应物取样检测,液相相色谱结果表明,二氯偏三酸酐的转化率为99%以上。
反应停止后,待体系冷却到室温,加入24L冰水至反应釜中,充分搅拌2小时,搅拌过程中有大量黄色固体析出,停止搅拌,过滤,收集滤饼;将滤饼置入24L冰水中搅拌,过滤,重复操作两次,收集滤饼并烘干,得到6135g 5,6-羧基-四氯-荧光素粗品。
实施例2制备5,6-羧基-四氯-荧光素纯品
6135g 5,6-羧基-四氯-荧光素粗品、12L乙酸乙酯(有机溶剂Ⅱ)加入50L玻璃反应釜,抽去反应釜中的空气,通入氮气,反应体系逐渐升温至60℃,热熔约2小时,体系中的固体溶解完全后停止加热,继续搅拌冷却至室温。
缓慢滴入18L正己烷,滴加过程中有固体析出,0℃冷却,静置24小时,过滤;滤液浓缩后再重复这一过程,合并两次收集的滤饼,用2L冷却的正己烷洗涤两次,过滤、烘干得5218g黄色固体,即5,6-羧基-四氯-荧光素。经检测,收率66.2%,纯度92.84%,其中5-TET占比52.08%,6-TET占比40.77%。
实施例3分离5-羧基-四氯-荧光素、6-羧基-四氯-荧光素
5140g 5,6-羧基-四氯-荧光素、15L乙腈加入50L玻璃反应釜,70℃温度下搅拌2小时,待固体充分溶解后停止加热,反应体系逐渐冷却至室温;逐滴滴入3000g三氟甲磺酸,体系逐渐变为黄红色溶液,滴加完毕继续搅拌1小时,体系变为悬浮液,停止搅拌;体系冷却至0℃后静置12小时,有大量黄色固体析出,过滤,得滤液和滤饼。
其中,滤饼加入15L冰水搅拌1小时,过滤,重复操作2次;滤饼在室温下用10L丙酮打浆1小时,过滤;烘干滤饼,烘干温度40℃,烘干时长12小时,得到5-羧基-四氯-荧光素1793g。经检测,收率67.1%,HPLC 98.97%。
1H-NMR:(400MHz,d6-DMSO,ppm)δ:14.068(b,1H),11.097(s,2H),8.102(s,1H),7.213(s,2H),6.873(s,2H).,核磁共振氢谱如图1所示。
ESI-MS(M+H):514.95.,ESI-MS分析数据如图2所示。
其中,滤液冷却至0℃搅拌,缓慢加入12L异丙醚,停止搅拌,体系在0℃温度下静置12小时,有大量黄色固体析出,过滤;滤饼加入10L冰水搅拌1小时,过滤,重复操作2次;滤饼在室温下用8L丙酮打浆1小时,过滤;烘干滤饼,烘干温度40℃,烘干时长12小时,得到6-羧基-四氯-荧光素1319g。经检测,收率81%,HPLC 98.41%。
1H-NMR:(400MHz,d6-DMSO,ppm)δ:14.181(b,1H),11.083(s,2H),8.051(s,1H),7.212(s,2H),6.870(s,2H).,核磁共振氢谱如图3所示。
ESI-MS(M+H):514.95.,ESI-MS分析数据如图4所示。
最后,还需要注意的是,以上列举仅是本发明一个具体实施例。显然,本发明不限于以上实施例,还可以有许多变形。本领域的普通技术人员能从本发明公开的内容直接导出或联想到的所有变形,均应认为是本发明的保护范围。
Claims (1)
1.一种羧基-四氯-荧光素的制备方法,以对氯间苯二酚和二氯偏三酸酐为原料,通过环合反应制得5,6-羧基-四氯-荧光素粗品,其特征在于,环合反应以质子酸为催化剂,反应温度120-220℃,反应时间2-8小时,对氯间苯二酚和二氯偏三酸酐投料比为2.05-2.5:1,与二氯偏三酸酐投料比为1-10:1;
利用重结晶方法对5,6-羧基-四氯-荧光素粗品进行提纯,得到5,6-羧基-四氯-荧光素纯品,具体操作为将5,6-羧基-四氯-荧光素粗品加入至有机溶剂Ⅱ中,加热温度40-80℃,加热时间45-90分钟,固体溶解完全后停止加热,继续搅拌冷却至室温,再缓慢滴入正己烷,滴加过程中有固体析出,0℃冷却,静置24小时,过滤、洗涤、烘干得5,6-羧基-四氯-荧光素纯品;
从5,6-羧基-四氯-荧光素纯品分离出5-羧基-四氯-荧光素、6-羧基-四氯-荧光素,具体包括以下步骤:
步骤1,5,6-羧基-四氯-荧光素、有机溶剂Ⅰ加入玻璃反应釜,5,6-羧基-四氯-荧光素纯品与有机溶剂Ⅰ的投料比为1:2-10,70℃温度下搅拌2小时,待固体充分溶解后停止加热,反应体系逐渐冷却至室温;
步骤2,逐滴滴入酸溶液,体系逐渐变为黄红色溶液,滴加完毕继续搅拌1小时,体系变为悬浮液,停止搅拌;
步骤3,体系冷却至0℃后静置12小时,有大量黄色固体析出,过滤;
步骤4,滤饼加入冰水搅拌1小时,过滤,重复操作2次;
步骤5,滤饼在室温下用丙酮打浆1小时,过滤;
步骤6,烘干滤饼,烘干温度40℃,烘干时长12小时,得到5-羧基-四氯-荧光素;
步骤7,将步骤3所得滤液冷却至0℃搅拌,缓慢加入异丙醚,停止搅拌,体系在0℃温度下静置12小时,有大量黄色固体析出,过滤;
步骤8,滤饼加入冰水搅拌1小时,过滤,重复操作2次;
步骤9,滤饼在室温下用丙酮打浆1小时,过滤;
步骤10,烘干滤饼,烘干温度40℃,烘干时长12小时,得到6-羧基-四氯-荧光素;
所述有机溶剂Ⅰ为乙腈,所述酸溶液为三氟甲磺酸,所述有机溶剂Ⅱ为乙酸乙酯,所述质子酸为甲磺酸。
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CN111286320A (zh) * | 2020-01-22 | 2020-06-16 | 公安部物证鉴定中心 | 可用于九色荧光str分型的荧光染料、特异性扩增引物对和分型方法 |
CN113461651A (zh) * | 2021-08-20 | 2021-10-01 | 山东绅联药业有限公司 | 一种高纯度荧光素的纯化方法 |
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