CN105623647A - 一种检测细胞内co的荧光探针及其制备方法和应用 - Google Patents
一种检测细胞内co的荧光探针及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种检测细胞内CO的荧光探针及其制备方法和应用,一种检测细胞内CO的荧光探针,结构式如下:其中,R为H、-CH3、-CH2CH3或-COOEt。本发明合成新型环钯基团能够对CO的响应性更快更灵敏,其与BODIPY结合后不仅提高了对CO的选择性,而且实现更快更灵敏的检测CO。其制备方法是对硝基苯甲酰氯、二甲基吡咯和三氟化硼***,或对硝基苯甲酰氯、二甲基吡咯衍生物和三氟化硼***反应生成硝基苯基氟硼二吡咯(硝基苯基BODIPY),然后将硝基苯基BODIPY中的硝基还原成氨基,再加入亚硝酸盐和3,5-二甲基苯酚偶氮化反应,最后加入钯盐进行环钯化反应得到目标产物,即检测细胞内CO的荧光探针(ACP-CO)。本发明合成方法简单,适于工业化生产。
Description
技术领域
本发明涉及一种荧光探针,特别涉及一种BODIPY类荧光探针、其合成方法及其在检测细胞内CO的应用,属于检测技术领域。
背景技术
多年来,一氧化碳(carbonmonoxide,CO)因其极易与血红蛋白结合,形成碳氧血红蛋白,使血红蛋白丧失携氧的能力和作用,造成组织窒息,严重时死亡。一直以来,CO被人们认为是一种有毒气体。20世纪70年代,人们发现机体可以内源性产生CO,它在体内主要是由血红素氧化酶酶解血红素产生。CO与其它气体信号小分子NO、H2S一样,在机体调节的生理和病理方面都起着重要作用。CO的主要生理作用是为心肌局部缺血和再灌注时组织损伤提供保护。相比传统直接给机体吸入CO气体,过渡金属羰基复合物的CO释放分子(CORMs)被认为是临床上防止心肌局部缺血和再灌注时组织损伤的潜在药物。内源性气体信号分子因其具有持续产生、传播迅速等特别。目前的CO检测技术,如硫酸钯-钼酸铵比色式法、电化学法、气相色谱法、红外线一氧化碳检测法等,往往需要延后处理或破坏的组织或产生细胞裂解产物。
近年来,用于检测体内CO的荧光探针,比较少见。如以BODIPY为荧光母体(BrianW.Michel,AlexanderR.Lippert,andChristopherJ.Chang,J.Am.Chem.Soc.2012,134,15668-15671)探针,以及以双光子染料香豆素衍生物为荧光母体(K.Zheng,W.Lin,L.Tan,H.ChenaandH.Cuia,Chem.Sci.,2014,5,3439-3448.)的荧光探针。以上两种探针的响应基团都是以苄胺形成的环钯化物,虽然选择性好,但反应时间长(1h或30min)。
发明内容
为克服现有技术的缺陷,本发明提供了一种检测细胞内CO的荧光探针及其制备方法和应用,该类荧光探针结构新颖、灵敏度高、光稳定性好。
为实现上述目的,本发明的技术方案为:
一种检测细胞内CO的荧光探针,结构式如下:
其中,R为H、-CH3、-CH2CH3或-COOEt。
本发明合成新型环钯基团能够对CO的响应性更快更灵敏,其与BODIPY结合后不仅提高了对CO的选择性,而且实现更快更灵敏的检测CO。
一种检测细胞内CO的荧光探针的制备方法,将对硝基苯甲酰氯、二甲基吡咯和三氟化硼***,或对硝基苯甲酰氯、二甲基吡咯衍生物和三氟化硼***反应生成硝基苯基氟硼二吡咯(硝基苯基BODIPY),然后将硝基苯基BODIPY中的硝基还原成氨基,再加入亚硝酸盐和3,5-二甲基苯酚偶氮化反应,最后加入钯盐进行环钯化反应得到目标产物,即检测细胞内CO的荧光探针(ACP-CO)。
本发明合成方法简单,适于工业化生产。
优选的,所述二甲基吡咯为2,4-二甲基吡咯。
其反应过程如下:
其中,R为H、-CH3、-CH2CH3或-COOEt。
优选的,步骤如下:
(1)将对硝基苯甲酰氯和二甲基吡咯,或对硝基苯甲酰氯和二甲基吡咯衍生物溶解于溶剂1中,加热回流后冷却,加入溶剂2,加入三氟化硼***升温反后得到5,5-二氟-1,3,7,9-四甲基-10-(4-硝基苯基)-5H-4λ4,5λ4-二吡咯[1,2-c:2',1'-f][1,3,2]二氮杂硼,即硝基苯基BODIPY;
(2)将硝基苯基BODIPY和甲酸铵溶于溶剂中,然后加入Pd/C搅拌反应后即得氨基苯基BODIPY;
(3)将氨基苯基BODIPY和浓盐酸溶于溶剂中,加入亚硝酸盐溶液,搅拌后,加入3,5-二甲基苯酚的碱溶液搅拌后得到偶氮BODIPY;
(4)将偶氮BODIPY染料溶解于溶剂中,然后加入PdCl2,室温搅拌过夜后,即得检测细胞内CO的荧光探针。
进一步优选的,所述步骤(1)的具体步骤为:将对硝基苯甲酰氯和二甲基吡咯,或对硝基苯甲酰氯和二甲基吡咯衍生物溶解于CH2Cl2中,加热回流1-2h,冷却后加入三乙胺、甲苯,待反应10-20分钟后加入三氟化硼***,升温至50-60℃反应2-4小时,旋干,用色谱柱分离,所得固体即硝基苯基BODIPY;
所述的硝基苯甲酰氯、二甲基吡咯或二甲基吡咯衍生物、三乙胺、甲苯、三氟化硼***及CH2Cl2的用量比为7-10:7-20:3.5-5.5:30-50:3-6:20-50(g:g:mL:mL:mL:mL);色谱柱分离洗脱剂比例为V石油醚:V二氯甲烷=4-6:1。提高原料的转化率,同时提高硝基苯基BODIPY的产率和纯度。
进一步优选的,所述步骤(2)的具体步骤为:将硝基苯基BODIPY、甲酸铵溶于CH2Cl2,然后加入Pd/C,常温搅拌1-3小时后过滤,将滤液旋干得氨基苯基BODIPY;所述的硝基苯BODIPY、甲酸铵、Pd/C、CH2Cl2的用量比例为:5~10:50~70:1~5:10-30(g:g:g:mL)。提高原料的转化率,同时提高氨基苯基BODIPY的产率和纯度。
进一步优选的,所述步骤(3)的具体步骤为:将氨基苯基BODIPY、浓盐酸溶于DMF中,加入亚硝酸钠水溶液,在0-5℃下搅拌3-4h,然后将其缓慢滴加到3,5-二甲基苯酚的NaOH溶液中,在0-5℃下搅拌4-5h,过滤后的滤渣色谱柱分离,得黄色固体偶氮BODIPY;
所述的氨基苯基BODIPY、浓盐酸、DMF、亚硝酸钠、3,5-二甲基苯酚、NaOH、水的用量比为1-3:3-5:10-15:3-5:3-5:4-8:20-30(g:mL:mL:g:g:g:mL)。色谱柱分离洗脱剂比例为V二氯甲烷:V石油醚=1-3:2。提高原料的转化率,同时提高偶氮BODIPY的产率和纯度。
进一步优选的,所述步骤(4)中的溶剂为CH3OH;所述的偶氮BODIPY、CH3OH、PdCl2的用量比例为5~10:10~30:10~20(g:mL:g)。提高原料的转化率,同时提高检测细胞内CO的荧光探针的产率和纯度。
一种上述检测细胞内CO的荧光探针在检测细胞内CO中的应用。
一种检测细胞内CO的方法,步骤为:
(1)将上述检测细胞内CO的荧光探针溶解于生理盐水、缓冲溶液或者培养液中,配制成储备液储存待用;
(2)将步骤(1)中的储备液加入含有细胞组织的适当缓冲液进行测试。
一种检测活细胞内CO的方法,步骤为:
(1)将含有上述检测细胞内CO的荧光探针的缓冲溶液5-10μM加入培养好的细胞放于培养箱是孵育8-12分钟;
(2)将细胞用生理PBS缓冲液洗去多余的探针,然后进行激光共聚焦显微成像,如果是检测内源性物质,刚是直接进行共聚焦成像,而如果是外加检测的,刚是加了待检测物质后,再次孵育8-12分钟,然后进行成像。
与现有技术相比,本发明的有益效果是:
1.合成简单,检测速度快,检测过程中不需要长时间的孵育;
2.选择性高,其它生物活性小分子不干扰;
3.检测波长512nm处于可见光区,对细胞损伤小。有利细胞内进行CO的检测。
附图说明
图1是本发明的探针ACP-CO荧光强度和CORM-2浓度变化的关系;横坐标是波长(nm),纵坐标是荧光强度;该图为512nm处的荧光光谱;
图2是本发明的探针ACP-CO的相对于生物体内常见的活性小分子的选择性实验;
图3是本发明的探针ACP-CO的相对荧光强度和CORM-2浓度的工作曲线;横坐标是CORM-2的浓度,纵坐标为相对荧光强度;
图4是本发明的探针ACP-CO对人肝癌细胞(HepG2)的激光共聚显微成像;图a与图b为细胞成像图,图c为前两者的叠加图,图d为细胞明场图。
具体实施方式
下面结合附图和实施例对本发明作进一步说明。
实施例1
(1)将对硝基苯甲酰氯(7g)、2,4-二甲基吡咯(7g)溶解CH2Cl2(20mL)中,加热回流1h。冷却后加入三乙胺(3.5g)、甲苯(30ml)。待反应15分钟后加入三氟化硼***(3g),升温至50度反应3小时,旋干,用色谱柱分离(洗脱剂:V石油醚:V二氯甲烷=5:1),旋干得橙色固体硝基苯基BODIPY。
核磁及质谱表征:
1HNMR(400MHz,CDCl3):δ=1.36(s,6H),2.57(s,6H),6.02(s,2H),7.54(d,J=8Hz,2H),8.39(d,J=8Hz,2H)
ESI-MS:calculatedfor[M-H]-=368.1,found368.1。
(2)将硝基苯基BODIPY(5g)、甲酸铵(50g)溶于CH2Cl2(10mL),然后加入Pd/C12(1g),常温搅拌2小时,过滤,滤液旋干得氨基苯基BODIPY。
核磁及质谱表征:
1HNMR(400MHz,CDCl3):δ=1.49(s,6H),2.54(s,6H),3.89(s,2H),5.97(s,2H),6.79(d,J=4Hz,2H),7.01(d,J=8Hz,2H)
ESI-MS:calculatedfor[M+H]+=340.2,found340.2。
(3)将氨基苯基BODIPY(1g)、浓HCl(3mL)溶于DMF(10mL)中,加入亚硝酸钠(3g)水溶液(10mL),在0-5℃下搅拌3h,然后将其缓慢滴加到3,5-二甲基苯酚(3g)的NaOH(4g)溶液中,在0-5℃下搅拌4h,过滤,滤渣溶解旋干,色谱柱分离(洗脱剂:V二氯甲烷:V石油醚=1:1),得黄色固体偶氮BODIPY。
核磁及质谱表征:
1HNMR(400MHz,DMSO-d6):δ=1.44(s,6H),2.47(s,12H),6.21(s,2H),6.63(s,2H),7.56(d,J=8Hz,2H),7.95(d,J=8Hz,2H),10.07(s,1H)
ESI-MS:calculatedfor[M-H]-=471.2,found471.2。
(4)将偶氮BODIPY(5g)溶解于CH3OH(10mL)中,然后加入PdCl2(10g),室温搅拌过夜,过滤得黄色固体。
核磁表征:
1HNMR(400MHz,DMSO-d6):δ=1.50(s,6H),2.20(s,6H),2.47(s,6H),6.23(s,2H),6.60(s,2H),7.35(d,J=8Hz,1H),7.56(s,1H),8.12(d,J=8Hz,1H),9.75(s,1H)。
实施例2
(1)将对硝基苯甲酰氯(0.14g)、2,4-二甲基吡咯(0.14g)溶解CH2Cl2(2mL)中,加热回流1h。冷却后加入三乙胺(0.35g)、甲苯(3ml)。待反应15分钟后加入三氟化硼***(0.3g),升温至50度反应3小时,旋干,用色谱柱分离(洗脱剂:V石油醚:V二氯甲烷=5:1),旋干得橙色固体硝基苯基BODIPY。
核磁及质谱表征:
1HNMR(400MHz,CDCl3):δ=1.36(s,6H),2.57(s,6H),6.02(s,2H),7.54(d,J=8Hz,2H),8.39(d,J=8Hz,2H)
ESI-MS:calculatedfor[M-H]-=368.1,found368.1。
(2)将硝基苯基BODIPY(10g)、甲酸铵(100g)溶于CH2Cl2(20mL),然后加入Pd/C12(2g),常温搅拌2小时,过滤,滤液旋干得氨基苯基BODIPY。
核磁及质谱表征:
1HNMR(400MHz,CDCl3):δ=1.49(s,6H),2.54(s,6H),3.89(s,2H),5.97(s,2H),6.79(d,J=4Hz,2H),7.01(d,J=8Hz,2H)
ESI-MS:calculatedfor[M+H]+=340.2,found340.2。
(3)将氨基苯基BODIPY(2g)、浓HCl(6mL)溶于DMF(20mL)中,加入亚硝酸钠(6g)水溶液(10mL),在0-5℃下搅拌3h,然后将其缓慢滴加到3,5-二甲基苯酚(6g)的NaOH(8g)溶液中,在0-5℃下搅拌4h,过滤,滤渣溶解旋干,色谱柱分离(洗脱剂:V二氯甲烷:V石油醚=1:1),得黄色固体偶氮BODIPY。
核磁及质谱表征:
1HNMR(400MHz,DMSO-d6):δ=1.44(s,6H),2.47(s,12H),6.21(s,2H),6.63(s,2H),7.56(d,J=8Hz,2H),7.95(d,J=8Hz,2H),10.07(s,1H)
ESI-MS:calculatedfor[M-H]-=471.2,found471.2。
(4)将偶氮BODIPY(15g)溶解于CH3OH(30mL)中,然后加入PdCl2(30g),室温搅拌过夜,过滤得黄色固体。
核磁表征:
1HNMR(400MHz,DMSO-d6):δ=1.50(s,6H),2.20(s,6H),2.47(s,6H),6.23(s,2H),6.60(s,2H),7.35(d,J=8Hz,1H),7.56(s,1H),8.12(d,J=8Hz,1H),9.75(s,1H)。
实施例3
本发明与实施例1相同,不同之处在于:将2,4-二甲基吡咯改为2,3,4-三甲基吡咯。
实施例4
本发明与实施例1相同,不同之处在于:将2,4-二甲基吡咯改为2,4-二甲基-3-乙基吡咯。
实施例5
本发明与实施例1相同,不同之处在于:将2,4-二甲基吡咯改为2,4-二甲基-3-吡咯甲酸乙酯。
效果试验:
1.分别用5.0μM实施例1的探针ACP-CO对0,10,20,30,40,50,60,70,80和90μMCROM-2进行荧光响应测试,得到探针ACP-CO的荧光强度和CORM-2浓度变化的关系;实验条件:激发/发射波长=495/512nm,50mMPBS/DMSO(pH7.4,7:3,v/v),37℃下孵育20min。横坐标为波长(nm),纵坐标为荧光强度。探针的激发波长为495nm,发射波长为512nm,测试效果显示如图1,可以看出,在512nm处荧光随着CORM-2浓度增大,荧光逐渐增强。可以说明本探针可以用于CO的检测,其孵育时间仅为20min,时间短,检测速度快。
2.本发明实施例1的探针ACP-CO的选择性实验。将5.0μM的探针ACP-CO分别与不同的生物活性物质如活性氧.OH、HS-、O1、NO、H2O2、ClO-和巯基小分子Hcy、Cys、GSH以及还原性物质Vc、Fe2+孵育,将其荧光强度与探针孵育CORM-2对比。测试结果如图2,可以说明本发明的探针ACP-CO的选择性高,其它生物活性小分子不干扰。
3.本发明实施例1的探针ACP-CO的相对荧光强度和CORM-2的浓度的工作曲线,如图3;横坐标为CORM-2的浓度,纵坐标为相对荧光强度。
4.本发明实施例1的探针ACP-CO对人肝癌细胞(HepG2)的激光共聚显微成像;HepG2细胞由高糖的DMEM培养液培养,成像前,细胞贴壁于盖玻片上,然后加入5μM探针的PBS缓冲液,37℃孵育10分钟,加入200μMCORM-2,然后进行激光共聚焦显微成像。其结果如图4,图a为405nm激光器激发下采集430-480nm的细胞核染料DAPI的细胞成像图,可以观察到细胞核被染色,细胞保持很好的活性。图b为488nm激光器激发下采集500-550nm的细胞成像图,探针ACP-CO其在细胞质有明显的染色。图c为图a与图b的叠加图,图d为细胞明场图,以表明细胞在整个实验过程中仍保持良好的细胞活力。
上述虽然结合附图对本发明的具体实施方式进行了描述,但并非对发明保护范围的限制,所属领域技术人员应该明白,在本发明的技术方案的基础上,本领域技术人员不需要付出创造性劳动即可做出的各种修改或变形仍在本发明的保护范围内。
Claims (10)
1.一种检测细胞内CO的荧光探针,其特征是,结构式如下:
其中,R为H、-CH3、-CH2CH3或-COOEt。
2.一种检测细胞内CO的荧光探针的制备方法,其特征是,将对硝基苯甲酰氯、二甲基吡咯和三氟化硼***,或对硝基苯甲酰氯、二甲基吡咯衍生物和三氟化硼***反应生成硝基苯基氟硼二吡咯,然后将硝基苯基BODIPY中的硝基还原成氨基,再加入亚硝酸盐和3,5-二甲基苯酚偶氮化反应,最后加入钯盐进行环钯化反应得到目标产物,即检测细胞内CO的荧光探针。
3.如权利要求2所述的一种检测细胞内CO的荧光探针的制备方法,其特征是,步骤如下:
(1)将对硝基苯甲酰氯和二甲基吡咯,或对硝基苯甲酰氯和二甲基吡咯衍生物溶解于溶剂1中,加热回流后冷却,加入溶剂2,加入三氟化硼***升温反后得到5,5-二氟-1,3,7,9-四甲基-10-(4-硝基苯基)-5H-4λ4,5λ4-二吡咯[1,2-c:2',1'-f][1,3,2]二氮杂硼,即硝基苯基BODIPY;
(2)将硝基苯基BODIPY和甲酸铵溶于溶剂中,然后加入Pd/C搅拌反应后即得氨基苯基BODIPY;
(3)将氨基苯基BODIPY和浓盐酸溶于溶剂中,加入亚硝酸盐溶液,搅拌后,加入3,5-二甲基苯酚的碱溶液搅拌后得到偶氮BODIPY;
(4)将偶氮BODIPY染料溶解于溶剂中,然后加入PdCl2,室温搅拌过夜后,即得检测细胞内CO的荧光探针。
4.如权利要求3所述的一种检测细胞内CO的荧光探针的制备方法,其特征是,所述步骤(1)的具体步骤为:将对硝基苯甲酰氯和二甲基吡咯,或对硝基苯甲酰氯和二甲基吡咯衍生物解于CH2Cl2中,加热回流1-2h,冷却后加入三乙胺、甲苯,待反应10-20分钟后加入三氟化硼***,升温至50-60℃反应2-4小时,旋干,用色谱柱分离,所得固体即硝基苯基BODIPY;
所述的硝基苯甲酰氯、二甲基吡咯或二甲基吡咯衍生物、三乙胺、甲苯、三氟化硼***及CH2Cl2的用量比为7-10:7-20:3.5-5.5:30-50:3-6:20-50,g:g:mL:mL:mL:mL;色谱柱分离洗脱剂比例为V石油醚:V二氯甲烷=4-6:1。
5.如权利要求3所述的一种检测细胞内CO的荧光探针的制备方法,其特征是,所述步骤(2)的具体步骤为:将硝基苯基BODIPY、甲酸铵溶于CH2Cl2,然后加入Pd/C,常温搅拌1-3小时后过滤,将滤液旋干得氨基苯基BODIPY;所述的硝基苯BODIPY、甲酸铵、Pd/C、CH2Cl2的用量比例为:5~10:50~70:1~5:10-30,g:g:g:mL。
6.如权利要求3所述的一种检测细胞内CO的荧光探针的制备方法,其特征是,所述步骤(3)的具体步骤为:将氨基苯基BODIPY、浓盐酸溶于DMF中,加入亚硝酸钠水溶液,在0-5℃下搅拌3-4h,然后将其缓慢滴加到3,5-二甲基苯酚的NaOH溶液中,在0-5℃下搅拌4-5h,过滤后的滤渣色谱柱分离,得黄色固体偶氮BODIPY;
所述的氨基苯基BODIPY、浓盐酸、DMF、亚硝酸钠、3,5-二甲基苯酚、NaOH、水的用量比为1-3:3-5:10-15:3-5:3-5:4-8:20-30,g:mL:mL:g:g:g:mL;色谱柱分离洗脱剂比例为V二氯甲烷:V石油醚=1-3:2。
7.如权利要求3所述的一种检测细胞内CO的荧光探针的制备方法,其特征是,所述步骤(4)中的溶剂为CH3OH;所述的偶氮BODIPY、CH3OH、PdCl2的用量比例为5~10:10~30:10~20,g:mL:g。
8.一种如权利要求1所述的检测细胞内CO的荧光探针在检测细胞内CO中的应用。
9.一种检测细胞内CO的方法,其特征是,步骤为:
(1)将权利要求1所述的检测细胞内CO的荧光探针溶解于生理盐水、缓冲或者培养液中,配制成储备液储存待用;
(2)将步骤(1)中的储备液加入含有细胞组织的适当缓冲液进行测试。
10.一种检测活细胞内CO的方法,其特征是,步骤为:
(1)将含有权利要求1所述的检测细胞内CO的荧光探针的缓冲溶液5-10μM加入培养好的细胞放于培养箱是孵育8-12分钟;
(2)将细胞用生理PBS缓冲液洗去多余的探针,然后进行激光共聚焦显微成像,如果是检测内源性物质,刚是直接进行共聚焦成像,而如果是外加检测的,刚是加了待检测物质后,再次孵育8-12分钟,然后进行成像。
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CN107141319A (zh) * | 2017-06-08 | 2017-09-08 | 济南大学 | 一种检测一氧化碳的荧光探针及其制备方法和应用 |
CN107141319B (zh) * | 2017-06-08 | 2019-07-09 | 济南大学 | 一种检测一氧化碳的荧光探针及其制备方法和应用 |
CN111748003A (zh) * | 2020-06-24 | 2020-10-09 | 河北大学 | N-乙酰胺基半乳糖修饰的3-硝基邻苯二甲酰亚胺衍生物、制备方法与应用及肝靶向探针 |
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CN115651005A (zh) * | 2022-10-26 | 2023-01-31 | 天津大学 | 一类具有光响应性质的偶氮基-氮杂氟硼二吡咯染料分子及制备方法 |
CN115651005B (zh) * | 2022-10-26 | 2023-10-31 | 天津大学 | 一类具有光响应性质的偶氮基-氮杂氟硼二吡咯染料分子及制备方法 |
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