CN109180694B - 一种用于细胞成像的罗丹明衍生物荧光探针及其制备方法 - Google Patents

一种用于细胞成像的罗丹明衍生物荧光探针及其制备方法 Download PDF

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CN109180694B
CN109180694B CN201811064932.7A CN201811064932A CN109180694B CN 109180694 B CN109180694 B CN 109180694B CN 201811064932 A CN201811064932 A CN 201811064932A CN 109180694 B CN109180694 B CN 109180694B
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何文英
吴禄勇
李建玲
刘艳萍
冯华杰
丁国华
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Abstract

本发明涉及一种用于细胞成像的罗丹明衍生物荧光探针及其制备方法，所述制备方法包括如下步骤：
Figure DDA0001797071290000011
将化合物1与化合物2溶于CH2Cl2中，在Fe(acac)3和Et3N的作用下得到式I化合物；其中化合物1与化合物2的摩尔比为1:0.8‑1.0，Fe(acac)3的摩尔用量为化合物1的0.10‑0.12倍，每毫摩尔化合物1使用CH2Cl2的体积为12‑15mL，使用Et3N的体积为0.5‑0.6mL。反应温度优选在室温下进行，反应时间优选20‑24小时。

Description

一种用于细胞成像的罗丹明衍生物荧光探针及其制备方法
技术领域
本发明属于有机合成及细胞成像领域，具体涉及一种用于细胞成像的罗丹明衍生物荧光探针及其制备方法。
背景技术
随着工业的迅速发展，带来经济效应的同时，所带来的污染对环境也构成了极大的威胁。而重金属离子Hg2+的污染更是不容忽视。汞作为最具毒害的重金属离子之一，被排放到土壤或水体中，通过食物链富集生命体造成威胁，因此而引发的各类疾病如阿尔茨海默症、水俣病、肢端疼痛症、尿毒症，已严重影响到人们的生活。基于1,2,3-三氮唑-罗丹明的荧光探针已有报道，但是尚未有该系列探针应用于细胞成像的报道。本发明提供一种由罗丹明酰肼与三氮唑羧酸酯一步法制备高灵敏度的Hg2+离子识别探针，并将该探针应用于细胞成像。
发明内容
本发明提供一种式I结构的罗丹明衍生物荧光探针，其特征在于式I结构如下：
Figure GDA0002723855050000011
本发明的另一实施方案提供上述式I化合物的制备方法，其特征在于包括如下步骤：
Figure GDA0002723855050000012
将化合物1与化合物2溶于CH2Cl2中，在Fe(acac)3和Et3N的作用下得到式I化合物；
其中化合物1与化合物2的摩尔比为1:0.8-1.0，Fe(acac)3的摩尔用量为化合物1的0.10-0.12倍，每毫摩尔化合物1使用CH2Cl2的体积为12-15mL，使用Et3N的体积为0.5-0.6mL。反应温度优选在室温下进行，反应时间优选20-24小时。
本发明的另一实施方案提供式I化合物在检测Hg2+中的应用。
本发明的另一实施方案提供式I化合物在HeLa细胞成像中的应用。
与现有技术相比，本发明的优点在于：本发明利用乙酰丙酮铁(Fe(acac)3)作为催化剂成功地实现了一步法合成罗丹明衍生物探针，且本发明合成的上述衍生物对Hg2+的检测灵敏度高，并能成功用于HeLa细胞成像。
附图说明
图1是式I化合物的1H NMR图；
图2是式I化合物的13C NMR图；
图3是式I化合物的HRMS图；
图4是式I化合物的荧光选择性图，激发波长为563nm；荧光滴定图，插图为584nm处体系的荧光强度变化；
图5是本发明实施例4的HeLa细胞活细胞荧光成像图。
具体实施方式
为了便于对本发明的进一步理解，下面提供的实施例对其做了更详细的说明。但是这些实施例仅供更好的理解发明而并非用来限定本发明的范围或实施原则，本发明的实施方式不限于以下内容。
实施例1
将化合物1(1.0mmol)与化合物2(1.0mmol)溶于CH2Cl2(15mL)中，室温下加入Fe(acac)3(0.12mmol)和Et3N(0.6mL)，搅拌反应20小时，TLC检测原料完全消失，反应液浓缩后，进行硅胶柱层析(200-300目硅胶)，用石油醚/乙酸乙酯(V:V＝3:1～4:1)为洗脱剂，得式I化合物592mg，产率为92.2％。如图1所示：1H NMR(400MHz,CDCl3)δ8.40(s,1H),7.98(d,J＝6.4Hz,1H),7.56–7.43(m,5H),7.38–7.35(m,2H),7.12(d,J＝6.6Hz,1H),6.78(d,J＝8.5Hz,2H),6.36(s,3H),6.33(s,1H),3.34(q,J＝7.0Hz,8H),2.49(s,3H),1.16(t,J＝7.0Hz,12H)；13C NMR(100MHz,CDCl3)δ165.14,158.87,153.66,152.36,149.01,137.82,136.95,135.51,133.15,129.98,129.65,129.26,128.69,128.16,125.27,124.03,123.50,108.17,104.39,97.89,77.40,77.08,76.76,66.04,44.36,12.68,9.77.HRMS(ESI)(m/z):[M+H]+calcd for C38H40N7O3,642.3193；found,642.3199。
实施例2
将化合物1(1.0mmol)与化合物2(1.0mmol)溶于CH2Cl2(15mL)中，室温下加入Et3N(0.6mL)，搅拌反应20小时后，TLC显示除化合物1和2外，未产生新的点(即没有发生反应)。
实施例3
如图4所示为检测在DMF-水(v/v＝1:1,Tris–HCl,pH＝7.4)溶液中，各离子(4×10-5mol/L)存在下式I探针(1×10-5mol/L)的荧光选择性图和对Hg2+荧光滴定光谱(inset)584nm处体系的荧光强度变化。荧光选择性图中在563nm激发光照射下，除了Hg2+，其他离子的加入均没有明显特征发射峰及颜色变化。加入Hg2+后，在584nm处荧光强度明显增强并伴有粉红色变化。荧光强度变化图式I探针(1×10-5mol/L)逐渐滴加Hg2+时的荧光光谱图。随着Hg2+的浓度增大，体系的荧光强度逐渐增大，当Hg2+浓度达到4.33×10-5mol/L时，体系荧光强度达到最大，此时Hg2+浓度为式I探针浓度的4.33倍。而当Hg2+浓度超过该浓度时，随着Hg2+浓度增大，体系荧光强度降低，在2.67×10-5～4.33×10-5mol/L浓度范围内荧光强度与Hg2+浓度呈线性相关，R2＝0.95108。
实施例4细胞成像应用
在37℃的CO2细胞培养箱中培养HeLa细胞24小时进行实验。如图5所示，A.在DMEM培养液下HeLa细胞的亮场成像；B.在DMEM培养液下HeLa细胞的荧光成像；C.式I化合物在HeLa细胞的亮场成像；D.式I化合物在HeLa细胞的荧光成像；E.式I化合物在HeLa细胞对Hg2+的亮场成像；F.式I化合物在HeLa细胞对Hg2+的荧光成像的效果。式I化合物的浓度为1.0×10-5mol/L，加入Hg2+的浓度为2.67×10-5mol/L。上述实验结果表明本发明的式I化合物可用于细胞成像。

Claims (1)

1.一种式I结构的罗丹明衍生物荧光探针的制备方法，其特征在于包括如下步骤：
Figure FDA0002485350890000011
将化合物1与化合物2溶于CH2Cl2中，在Fe(acac)3和Et3N的作用下得到式I化合物；化合物1与化合物2的摩尔比为1:0.8-1.0，Fe(acac)3的摩尔用量为化合物1的0.10-0.12倍，每毫摩尔化合物1使用CH2Cl2的体积为12-15mL，使用Et3N的体积为0.5-0.6mL；反应温度选自在室温下进行，反应时间为20-24小时。
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