CN113501818A - 一种荧光探针分子及其制备方法与应用 - Google Patents
一种荧光探针分子及其制备方法与应用 Download PDFInfo
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Abstract
本发明提供一种荧光探针分子,涉及有机功能材料技术领域,用于同时监测次氯酸和pH;一种荧光探针分子为亚甲基蓝‑4‑氨基萘酰亚胺衍生物,具有与次氯酸发生反应的酰胺基团,以及与pH值响应的邻苯二胺基团;HClO具有较强的氧化性,可以使荧光探针分子中的酰胺键断裂,生成不稳定的中间体,最后释放出带正电荷的亚甲基蓝荧光团,在近红外光区出现荧光(690nm);当质子与4‑位邻苯二胺结合,PET效应得到抑制,从而出现黄绿色荧光(525nm),从而实现双识别位点选择性识别和同时检测次氯酸和pH值;本发明还提供一种荧光探针分子的制备方法和应用;所述荧光探针分子用于检测多细胞内的次氯酸与pH。
Description
技术领域
本发明涉及有机功能材料技术领域,更具体地,涉及一种荧光探针分子及其制备方法与应用。
背景技术
光探针分子成像技术在外科手术和生物医学领域有着良好的应用前景。近年来,荧光成像技术取得了快速发展,由于其对生物样本具有便捷、无损伤、实时原位动态可视监测以及高灵敏度、高时空分辨率的特点,逐渐成为生物荧光探针研究领域的重要工具。
针对生物活性小分子(活性氧(ROS)、活性氮(RNS)、活性硫(RSS)等)检测的荧光探针及生物活体荧光成像研究是目前的研究热点。相比于单一检测荧光探针,设计和合成同时检测两种关联性生物活性小分子的双位点协同响应荧光探针及荧光成像研究更是目前的难点和发展趋势。基于双识别位点检测不同或相同活性物种的荧光探针已经有一些报道,然而已报道的双识别位点检测荧光探针所检测的活性物种还比较有限,主要集中在生物硫醇(Cys、Hcy、GSH)的选择性识别以及不多的活性氧和活性硫物种检测上。
如中国专利“一种同时或分别检测细胞溶酶体内硫化氢和次氯酸的荧光探针及其制备方法与应用”(申请号:201610263094.0,公开日:2016.07.20)公开了一种探针,实现了单一探针对多种靶标分子(H2S、HClO和H2S/HClO)的同时或分别检测。
又如“抗肿瘤诊疗前药以及双识别位点荧光探针的设计、合成以及生物应用”(陈宇,华南师范大学,2020.6.3)公开的荧光探针分子可用于同时检测次氯酸和硫化氢。
但上述仅公开了荧光探针可以同时检测次氯酸和硫化氢,但针对另外一些重要关联性物质的检测还比较缺乏,如次氯酸和pH。
次氯酸对人体免疫***的重要防线,由髓过氧化物酶(MPO)催化的氯离子和过氧化氢反应产生的。次氯酸水平异常与心血管、神经性弱化、肺损伤、甚至癌症等密切相关。线粒体、溶酶体、内质网等细胞器会保持特定的pH值,以便其在生物过程中保持各自的功能。细胞内pH的异常变化可能会诱发心肺和神经退行性等疾病,因此设计合成一种同时监测HClO和pH的荧光探究,研究HClO和pH在生理和病理过程上的关系,对疾病的预防和诊断具有重要的意义。
发明内容
本发明旨在克服上述现有技术的至少一种缺陷(不足),提供一种荧光探针分子,可同时监测次氯酸和pH。
本发明的另一目的在于提供一种荧光探针分子的制备方法。
本发明的又一目的在于提供荧光探针分子的应用。
本发明采取的技术方案是,一种荧光探针分子,具有如下结构:
在本发明中,该荧光探针分子一种结构清晰、光性质稳定的对次氯酸和pH同时响应的亚甲基蓝-4-氨基萘酰亚胺衍生物;具有与次氯酸发生反应的酰胺基团,以及与pH值响应的邻苯二胺基团。HClO具有较强的氧化性,可以使荧光探针分子中的酰胺键断裂,生成不稳定的中间体,最后释放出带正电荷的亚甲基蓝荧光团,在近红外光区出现荧光(690nm);当质子与4-位邻苯二胺结合,PET效应得到抑制,从而出现黄绿色荧光(525nm),从而实现双识别位点选择性识别和同时检测次氯酸和pH值。该探针分子最初设计思路是首先具有与次氯酸发生反应的酰胺基团,其次萘酰亚胺4-位具有与一氧化氮发生反应的邻苯二胺基团,但光谱研究发现该探针分子的邻苯二胺基团对一氧化氮不响应,而意外发现该基团对pH值响应。
一种如上述的荧光探针分子的制备方法,包括如下步骤:
S1:将亚甲基蓝和碳酸钠溶于蒸馏水和二氯甲烷中,搅拌,再缓慢加入硫代硫酸钠水溶液,继续搅拌至溶液变黄,将上述溶液冷却,向其中缓慢滴加二(三氯甲基)碳酸酯的二氯甲烷溶液,继续搅拌,然后倒入冰水中,再用二氯甲烷萃取,干燥有机相,过滤,减压蒸馏,收集,经硅胶层析柱,洗脱、分离、提纯,得到化合物4;
S2:将S1得到的化合物4溶于二氯甲烷中,向其中缓慢滴入乙二胺的二氯甲烷溶液继续搅拌,倒入水中,经萃取、干燥、减压蒸馏,收集,经硅胶层析柱,洗脱、分离、提纯,得到化合物3;
S3:将S2得到的化合物3和4-溴-1,8-萘二甲酸酐溶于无水乙醇中,在氮气保护下,回流搅拌;蒸发、析柱,洗脱、分离、提纯,得到化合物2。
S4:将S3得到的化合物2和邻苯二胺溶于无水甲苯中,再依次加入三(二亚苄基丙酮)二钯的氯仿加合物,4,5-双二苯基膦9,9-二甲基氧杂蒽和碳酸铯。在氮气保护下,加热回流,蒸发、析柱,洗脱、分离、提纯,得到如权利要求1所述的荧光探针分子。
进一步地,在S1中,具体步骤为:将亚甲基蓝和碳酸钠溶于蒸馏水和二氯甲烷中,常温搅拌35分钟,然后向其中慢慢加入硫代硫酸钠水溶液,继续搅拌至溶液变黄,将上述溶液用冰水浴冷却后,向其中缓慢滴加二(三氯甲基)碳酸酯的二氯甲烷溶液,继续搅拌1小时,然后倒入冰水中,再用二氯甲烷萃取,干燥有机相,过滤,减压蒸馏,收集,经硅胶层析柱,洗脱、分离、提纯,得到化合物4;
或在S2中,具体步骤为:将S1得到的化合物4溶于二氯甲烷中,向其中缓慢滴入乙二胺的二氯甲烷溶液,在室温下继续搅拌2小时,倒入水中,经萃取、干燥、减压蒸馏,收集固体混合物,经硅胶层析柱,洗脱、分离、提纯,得到化合物3;
或在S3中,具体步骤为:将S2得到的化合物3和4-溴-1,8-萘二甲酸酐溶于无水乙醇中,在氮气保护下,回流搅拌3小时,减压蒸发溶剂,粗产物经硅胶层析柱,洗脱、分离、提纯,得到化合物2;
或在S4中,具体步骤为:将S3得到的化合物2和邻苯二胺溶于无水甲苯中,再依次加入三(二亚苄基丙酮)二钯的氯仿加合物,4,5-双二苯基膦9,9-二甲基氧杂蒽和碳酸铯,在氮气保护下,加热回流10小时,减压蒸发溶剂,粗产物经硅胶层析柱,洗脱、分离、提纯,得到如权利要求1所述的荧光探针分子。
进一步地,在步骤S1中,所述硫代硫酸钠、亚甲基蓝、碳酸钠、二(三氯甲基)碳酸酯的物质的量比为6.66:1.72:6.66:1;所述洗脱剂为正己烷与二氯甲烷的混合物,其体积比为3:1。
进一步地,在步骤S2中,所述化合物4与乙二胺的物质的量比为1:2.5,所述洗脱剂为二氯甲烷与甲醇的混合物,其体积比为10:1。
进一步地,在步骤S3中,所述4-溴-1,8-萘二甲酸酐与化合物3的质量比为228.0mg:296.0mg,所述无水乙醇体积为8.00mL,所述洗脱剂为二氯甲烷与甲醇的混合物,其体积比为100:1。
进一步地,在步骤S4中,所述化合物2与邻苯二胺、三(二亚苄基丙酮)二钯的氯仿加合物、4,5-双二苯基膦9,9-二甲基氧杂蒽及碳酸铯的质量比为308mg:158.8mg:21.0mg:12mg:483.0mg,所述无水甲苯体积为5.00mL;所述柱层析洗脱剂为二氯甲烷与甲醇混合物,其体积比为50:1。
一种如上述的荧光探针分子用于同时检测次氯酸与pH。
进一步地,所述荧光探针分子用于检测多细胞内的次氯酸与pH。
进一步地,所述多细胞为RAW264.7,HIBEC,CT26,QBC939,HuCCT1中的一种。
与现有技术相比,本发明的有益效果为:
(1)本发明提供一种新的荧光探针分子,可同时监测次氯酸和pH,灵敏性高,选择性良好;
(2)本发明的荧光探针分子合成方法步骤简单,收率高;
(3)本发明荧光探针分子能够非常好地选择识别次氯酸和pH,当探针中加入HClO和其它活性氧时,通过各个活性物种在690nm荧光强度值的柱形图对比(如图5所示),在690nm处该荧光探针分子仅明显地增强HClO荧光,表现出高度的荧光选择性,具有良好的抗干扰性。当缓冲溶液的pH=7.4时,探针分子在475nm处有一个较小的荧光发射峰;当缓冲溶液的pH=7.0时,在475nm处的荧光发射峰消失;当缓冲溶液的pH继续降低,在525nm出现一个新的荧光发射峰,且荧光强度随pH的降低而增强(如图8所示);探针分子在525nm处的荧光强度随pH循环变化(如图9所示),探针分子在pH为5.5或7.0时荧光强度几乎无变化,说明荧光探针分子有良好的pH适应性,能够用于循环检测内环境中pH的变化。
(4)本发明荧光探针分子物理化学性质稳定性好。
(5)本发明荧光探针分子穿透细胞的渗透性好,可在多细胞荧光成像。
附图说明
图1为本发明荧光探针分子的合成路线。
图2为本发明荧光探针分子的1H-NMR谱图。
图3为本发明荧光探针分子的13C-NMR谱图。
图4为本发明荧光探针分子的HRMS(ESI)谱图。
图5为本发明荧光探针分子在乙醇水溶液体系中对次氯酸响应的荧光光谱变化图和不同干扰离子在690nm处荧光强度柱状图。
图6为本发明荧光探针分子对不同浓度次氯酸的荧光光谱变化图。
图7为本发明荧光探针分子在690nm处荧光强度与相对应的次氯酸浓度拟合曲线图。
图8为本发明荧光探针分子对pH识别位点荧光检测变化图。
图9为本发明荧光探针分子在525nm处的荧光强度随pH循环变化图。
图10为本发明荧光探针分子在525nm处荧光强度与相对应的不同pH值的拟合曲线图。
图11为本发明荧光探针分子在多细胞体内荧光成像图。其中,a1-e1为绿色荧光通道图;a2-e2为红色荧光通道图;a3-e3为明场图;a1-a3为RAW264.7细胞;b1-b3为HIBEC;图c1-c3为CT26;图d1-d3为QBC939;图e1-e3为HuCCT1。
具体实施方式
本发明实施例仅用于示例性说明,不能理解为对本发明的限制。
实施例1
基于亚甲基蓝-4-氨基萘酰亚胺衍生物的荧光探针分子的合成(合成路线见图1)
(1)化合物4的制备:将亚甲基蓝(5.0g,15.66mmol)和碳酸钠(6.64g,62.52mmol)溶于蒸馏水(30mL)和二氯甲烷(20mL)中,常温搅拌35分钟,然后向其中慢慢加入硫代硫酸钠(10.98g,62.52mmol)水(40mL)溶液,继续搅拌至溶液变黄,将上述溶液用冰水浴冷却后,向其中缓慢滴加二(三氯甲基)碳酸酯(2.78g,9.38mmol)的二氯甲烷(13mL)溶液,继续搅拌1小时,然后倒入冰水中,再用二氯甲烷萃取,干燥有机相,过滤,减压蒸馏,收集,所得粗产物经硅胶层析柱,用正己烷:二氯甲烷=3:1洗脱,得到2.7g白色固体化合物4,产率50%。
(2)化合物3的制备:将284mg(0.76mmol)化合物4溶于8mL二氯甲烷中,向其中缓慢滴入乙二胺(228mg,1.9mmol)的二氯甲烷(5mL)溶液,在室温下继续搅拌2小时,倒入水中,经萃取、干燥、减压蒸馏,收集,所得粗产物经硅胶层析柱,用二氯甲烷:甲醇=10:1洗脱,得到196mg固体化合物3,产率70%。
(3)化合物2的制备:4-溴-1,8-萘二甲酸酐(228.0mg,0.82mmol),化合物3(296.0mg,0.8mmol)溶解于无水乙醇(8mL)中,在氮气保护下,回流搅拌3小时,减压蒸发溶剂,洗脱纯化(二氯甲烷/甲醇=100:1,v/v),得到红色固体化合物2(189.3mg,75%产率)。1H NMR(500MHz,CDCl3)δ8.66(dd,J=7.3,0.9Hz,1H),8.60(dd,J=8.5,0.9Hz,1H),8.41(d,J=7.8Hz,1H),8.05(t,J=7.0Hz,1H),7.87(dd,J=8.4,7.4Hz,1H),7.20(d,J=8.8Hz,2H),6.63(d,J=2.4Hz,2H),6.54(dd,J=8.8,2.6Hz,2H),5.39(t,J=5.4Hz,1H),4.42–4.33(m,2H),3.66(dd,J=11.3,5.6Hz,2H),2.92(s,12H).13C NMR(125MHz,CDCl3)δ163.87,163.83,156.10,148.86,134.07,133.34,132.21,131.40,131.07,130.61,130.33,129.12,128.35,128.06,127.25,122.98,122.12,111.29,110.86,40.77,39.81,39.64.
(4)基于亚甲基蓝-4-氨基萘酰亚胺衍生物双识别位点荧光探针分子1的合成:将化合物2(308mg,0.49mmol),邻苯二胺(158.8mg,1.47mmol)溶解在无水甲苯(5mL)中,再依次加入三(二亚苄基丙酮)二钯的氯仿加合物(21.0mg,0.02mmol),4,5-双二苯基膦9,9-二甲基氧杂蒽(12mg,0.02mmol),碳酸铯(483.0mg,1.35mmol),在氮气保护下,加热回流10小时。减压蒸发溶剂,洗脱纯化(二氯甲烷/甲醇=50:1,v/v),得到210mg黄绿色固体化合物1,产率50%。1H NMR(500MHz,DMSO-d6)δ9.01(s,1H),8.91(d,J=8.5Hz,1H),8.52(d,J=7.1Hz,1H),8.25(d,J=8.5Hz,1H),7.79(t,J=7.8Hz,1H),7.76–7.71(m,2H),7.69(dd,J=5.7,3.1Hz,2H),7.13(d,J=8.9Hz,1H),7.10–7.07(m,1H),6.88(d,J=7.8Hz,1H),6.67(t,J=7.5Hz,1H),6.63(d,J=2.5Hz,1H),6.45(dd,J=8.0,4.6Hz,2H),6.18(t,J=5.5Hz,1H),5.05(s,2H),4.23(t,J=6.5Hz,1H),4.18(d,J=5.2Hz,1H),4.02(d,J=6.5Hz,2H),2.84(s,12H).13C NMR(125MHz,DMSO-d6)δ166.97(s),164.16(s),163.36(s),155.02(s),149.70(s),148.31(s),144.99(s),133.85(s),133.00(s),130.75(s),129.77(s),129.22(s),128.06(s),127.76(s),127.28(s),124.46(s),123.38(s),122.24(s),120.65(s),116.52(s),115.69(s),111.04(s),110.19(s),109.53(s),65.06(s),40.23(s),38.64(s).HRMS:Calcd for C37H35N7O3S[M+H]+=658.2595,m/z found,658.2597.其中,CDCl3为氘代三氯甲烷;DMSO-d6为氘代二甲亚砜。相关谱图见图2~4。
实施例2
本实施例考察基于亚甲基蓝-4-氨基萘酰亚胺衍生物对次氯酸识别位点荧光检测的选择性。
采用DMF:PBS(0.01mol/L,pH=7.4)=9:1(v:v)溶液控制实验条件。
将基于亚甲基蓝-4-氨基萘酰亚胺衍生物加入DMF溶剂中,配制成荧光探针分子浓度为20μmol/L的溶液。
将样品瓶分为14组,每组各样品瓶分别加入20μmol/L的基于亚甲基蓝-4-氨基萘酰亚胺衍生物荧光探针分子溶液,第一瓶溶液作为空白组,其它13组分别加入1200μmol/L的HClO、NO、HSO3 -、SO3 2-、H2S、H2O2、NO3 -、ONOO-、·OH、1O2、Cys、GSH、Hcy溶液。在室温下配制完待测液后,将各测试工作液转移至1cm×1cm的标准石英比色皿中,测定其荧光光谱。激发波长为620nm,发射波长为690nm。基于亚甲基蓝-4-氨基萘酰亚胺荧光探针分子对次氯酸荧光选择性检测如图5a所示。可见基于亚甲基蓝-4-氨基萘酰亚胺衍生物荧光探针分子在690nm处仅对HClO有明显的荧光增强现象,我们选用了在690nm处各个离子的荧光强度值做了一个柱形图(如图5b所示),图5b可以直观地看出探针对次氯酸的荧光选择性非常好。
实施例3
本实施例考察基于亚甲基蓝-4-氨基萘酰亚胺衍生物荧光探针分子对次氯酸的定量荧光检测。
采用乙醇:PBS(0.01mol/L,pH=7.4)=9:1(v:v)溶液控制实验条件。
将基于亚甲基蓝-4-氨基萘酰亚胺衍生物荧光探针分子配制成浓度为20μmol/L的溶液。将不同溶度的HClO溶液加入其中。在室温下配制完待测液后,将各测试工作液转移至1cm×1cm的标准石英比色皿中,测定其荧光光谱。荧光测试光栅缝隙大小为5nm×5nm。图6为本发明所涉及的基于亚甲基蓝-4-氨基萘酰亚胺衍生物荧光探针分子在含水溶液体系中随次氯酸浓度变化的荧光光谱图。将荧光光谱690nm处的荧光强度与相应次氯酸浓度进行拟合,在次氯酸浓度为0μmol/L~1200μmol/L范围内得到一拟合曲线(如图7所示),表明本发明所涉及的基于亚甲基蓝-萘酰亚胺荧光探针分子可以定量检测次氯酸浓度。
实施例4
本实施例考察基于亚甲基蓝-4-氨基萘酰亚胺衍生物对pH识别位点荧光检测。
采用DMF:PBS(0.01M,pH=X)=9:1(v:v)溶液控制实验条件。
将基于亚甲基蓝-4-氨基萘酰亚胺衍生物加入DMF溶剂中,配制成浓度为20μmol/L的溶液。将样品瓶分为13组,第一瓶溶液pH为7.4,其它12组溶液pH分别7.0,6.5,6.0,5.5,5.0,4.5,4.0,3.5,3.0,2.5,2.0,1.5。在室温下配制完待测液后,将各测试工作液转移至1cm×1cm的标准石英比色皿中,测定其荧光光谱。基于亚甲基蓝-4-氨基萘酰亚胺荧光探针分子对pH识别位点荧光检测变化如图8所示。可见当缓冲溶液的pH=7.4时,在475nm处有一个较小的荧光发射峰;当缓冲溶液的pH=7.0时,在475nm处的荧光发射峰消失;当缓冲溶液的pH继续降低,在525nm出现一个新的荧光发射峰,且荧光强度随pH的降低而增强。图9为探针分子在525nm处的荧光强度随pH循环变化,探针分子在pH为5.5或7.0时荧光强度几乎无变化,表明荧光探针分子有良好pH适应性,能够用于循环检测内环境中pH的变化。将荧光光谱525nm处的荧光强度与不同pH值进行拟合,在pH=4.0,4.5,5.0,5.5,6.0,6.5范围内得到一拟合曲线(如图10所示),表明本发明所涉及的基于亚甲基蓝-4-氨基萘酰亚胺荧光探针分子在pH值在4.0-6.5的范围内对pH具有良好的线性关系。
上述表明本发明所涉及的基于亚甲基蓝-4-氨基萘酰亚胺衍生物荧光探针分子能很好的识别内环境的酸性程度。
实施例5
本实施例考察基于亚甲基蓝-4-氨基萘酰亚胺衍生物荧光探针分子多细胞荧光成像。
本实验采用来自西南医科大学重点实验室的RAW264.7(小鼠巨噬细胞),HIBEC(正常胆管细胞),CT26(结肠癌细胞),QBC939(胆管癌细胞),HuCCT1(胆管癌细胞)进行荧光成像研究。拍摄条件:Green Channel:λex=405nm(410nm),λem=485-565nm(525nm),激光输出功率5%;Red Channel:λex=638nm(620nm),λem=650-730nm(690nm),激光输出功率5%。目镜放大倍数10X,物镜放大倍数20X。操作如下:首先用移液枪移去培养液,再用PBS缓冲溶液洗涤,重复上述操作三次。然后用浓度为20μmol/L的探针分子孵育24h。最后用移液枪移去探针溶液,用PBS缓冲溶液洗涤,重复上述操作三次。将孵育后的细胞在共聚焦激光扫描显微镜下进行荧光成像。结果见图11。a1-e1为绿色荧光通道图;a2-e2为红色荧光通道图;a3-e3为明场图;a1-a3为RAW264.7细胞;b1-b3为HIBEC;图c1-c3为CT26;图d1-d3为QBC939;图e1-e3为HuCCT1。结果发现在QBC939和HuCCT1细胞中,其绿色荧光通道和红色荧光通道,均出现明显的荧光增强,说明了QBC939和HuCCT1细胞内环境中酸性较强且HClO的含量较高。该实验表明本发明所涉及的基于亚甲基蓝-4-氨基萘酰亚胺衍生物荧光探针分子可以在QBC939和HuCCT1细胞内对HClO2和pH进行识别成像。
显然,本发明的上述实施例仅仅是为清楚地说明本发明技术方案所作的举例,而并非是对本发明的具体实施方式的限定。凡在本发明权利要求书的精神和原则之内所作的任何修改、等同替换和改进等,均应包含在本发明权利要求的保护范围之内。
Claims (10)
2.一种如权利要求1所述的荧光探针分子的制备方法,其特征在于,包括如下步骤:
S1:将亚甲基蓝和碳酸钠溶于蒸馏水和二氯甲烷中,搅拌,再缓慢加入硫代硫酸钠水溶液,继续搅拌至溶液变黄,将上述溶液冷却,向其中缓慢滴加二(三氯甲基)碳酸酯的二氯甲烷溶液,继续搅拌,然后倒入冰水中,再用二氯甲烷萃取,干燥有机相,过滤,减压蒸馏,收集,经硅胶层析柱,洗脱、分离、提纯,得到化合物4;
S2:将S1得到的化合物4溶于二氯甲烷中,向其中缓慢滴入乙二胺的二氯甲烷溶液继续搅拌,倒入水中,经萃取、干燥、减压蒸馏,收集,经硅胶层析柱,洗脱、分离、提纯,得到化合物3;
S3:将S2得到的化合物3和4-溴-1,8-萘二甲酸酐溶于无水乙醇中,在氮气保护下,回流搅拌;蒸发、析柱,洗脱、分离、提纯,得到化合物2。
S4:将S3得到的化合物2和邻苯二胺溶于无水甲苯中,再依次加入三(二亚苄基丙酮)二钯的氯仿加合物,4,5-双二苯基膦9,9-二甲基氧杂蒽和碳酸铯。在氮气保护下,加热回流,蒸发、析柱,洗脱、分离、提纯,得到如权利要求1所述的荧光探针分子。
3.根据权利要求2所述的一种荧光探针分子的制备方法,其特征在于,在S1中,具体步骤为:将亚甲基蓝和碳酸钠溶于蒸馏水和二氯甲烷中,常温搅拌35分钟,然后向其中慢慢加入硫代硫酸钠水溶液,继续搅拌至溶液变黄,将上述溶液用冰水浴冷却后,向其中缓慢滴加二(三氯甲基)碳酸酯的二氯甲烷溶液,继续搅拌1小时,然后倒入冰水中,再用二氯甲烷萃取,干燥有机相,过滤,减压蒸馏,收集,经硅胶层析柱,洗脱、分离、提纯,得到化合物4;
或在S2中,具体步骤为:将S1得到的化合物4溶于二氯甲烷中,向其中缓慢滴入乙二胺的二氯甲烷溶液,在室温下继续搅拌2小时,倒入水中,经萃取、干燥、减压蒸馏,收集固体混合物,经硅胶层析柱,洗脱、分离、提纯,得到化合物3;
或在S3中,具体步骤为:将S2得到的化合物3和4-溴-1,8-萘二甲酸酐溶于无水乙醇中,在氮气保护下,回流搅拌3小时,减压蒸发溶剂,粗产物经硅胶层析柱,洗脱、分离、提纯,得到化合物2;
或在S4中,具体步骤为:将S3得到的化合物2和邻苯二胺溶于无水甲苯中,再依次加入三(二亚苄基丙酮)二钯的氯仿加合物,4,5-双二苯基膦9,9-二甲基氧杂蒽和碳酸铯,在氮气保护下,加热回流10小时,减压蒸发溶剂,粗产物经硅胶层析柱,洗脱、分离、提纯,得到如权利要求1所述的荧光探针分子。
4.根据权利要求2或3所述的一种荧光探针分子的制备方法,其特征在于,在步骤S1中,所述硫代硫酸钠、亚甲基蓝、碳酸钠、二(三氯甲基)碳酸酯的物质的量比为6.66:1.72:6.66:1;所述洗脱剂为正己烷与二氯甲烷的混合物,其体积比为3:1。
5.根据权利要求2或3所述的一种荧光探针分子的制备方法,其特征在于,在步骤S2中,所述化合物4与乙二胺的物质的量比为1:2.5,所述洗脱剂为二氯甲烷与甲醇的混合物,其体积比为10:1。
6.根据权利要求2或3所述的一种荧光探针分子的制备方法,其特征在于,在步骤S3中,所述4-溴-1,8-萘二甲酸酐与化合物3的质量比为228.0mg:296.0mg,所述无水乙醇体积为8.00mL,所述洗脱剂为二氯甲烷与甲醇的混合物,其体积比为100:1。
7.根据权利要求2或3所述的一种荧光探针分子的制备方法,其特征在于,在步骤S4中,所述化合物2与邻苯二胺、三(二亚苄基丙酮)二钯的氯仿加合物、4,5-双二苯基膦9,9-二甲基氧杂蒽及碳酸铯的质量比为308mg:158.8mg:21.0mg:12mg:483.0mg,所述无水甲苯体积为5.00mL;所述柱层析洗脱剂为二氯甲烷与甲醇混合物,其体积比为50:1。
8.一种如权利要求1-7任一所述的荧光探针分子用于同时检测次氯酸与pH。
9.根据权利要求8所述的用于同时检测次氯酸与pH的荧光探针分子,其特征在于,所述荧光探针分子用于检测多细胞内的次氯酸与pH。
10.根据权利要求9所述的用于同时检测次氯酸与pH的荧光探针分子,其特征在于,所述多细胞为RAW264.7,HIBEC,CT26,QBC939,HuCCT1中的一种。
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