CN116606334A - 一种荧光染料及其制备方法和制得的荧光探针 - Google Patents
一种荧光染料及其制备方法和制得的荧光探针 Download PDFInfo
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Abstract
本发明提出了一种荧光染料及其制备方法和制得的荧光探针,属于荧光探针技术领域。所述荧光染料的结构:,其中,R=N(CH3)2、CH3、C2H5、C3H7、中的至少一种。本发明荧光探针具有响应时间短,成本低、实时监测、原位检测、可视化检测、非破坏性检测,灵敏度高、选择性好、操作简单快速、重现性好、取样量少的优点,在生物分子检测领域具有广阔的应用前景。
Description
技术领域
本发明涉及荧光探针技术领域,具体涉及一种荧光染料及其制备方法和制得的荧光探针。
背景技术
荧光探针作为一种有效的、便捷的、快速的工具已经广泛的应用于小分子检测、蛋白质质分析、以及药物分析。目前主要的荧光染料有罗丹明、香豆素、荧光素等化合物。但这些化合物都存在其显著的缺点:细胞相容性差、合成困难、荧光强度低、难于修饰。因此对于新荧光染料的开发已经引起越来越多研究者的关注。
β-半乳糖苷酶属于糖苷水解酶,由4个亚基四聚体组成,在食品工业、基因工程、酶工程、蛋白质工程、生物医学等领域发挥着重要作用。近年来,β-半乳糖苷酶作为一种关键酶开始广泛应用于医药领域,如用作细胞衰老和原发性卵巢癌生物标志物,人转移性卵巢癌细胞SKOV3和OVCAR3相对于正常人细胞表现出更高水平的β-半乳糖苷酶,检测β-半乳糖苷酶有助于卵巢癌的早期诊断。此外,β-半乳糖苷酶常被用作研究生物系统中基因表达和调控的报告酶,特别是对于大肠杆菌β-半乳糖苷酶,它具有很好的特征并由lacZ基因编码。常用的β-半乳糖苷酶检测方法有比色法、电化学法、磁共振分子显像、单光子发射计算机断层扫描成像和正电子发射断层扫描成像等。这些方法各有优缺点,甚至有些缺点非常明显,如MR、SPECT、PET等方法成本高、操作繁琐、灵敏度低,不能实现生物系统中β-半乳糖苷酶的实时原位无损检测。为了监测体外和体内的β-半乳糖苷酶,研究人员致力于开发高选择性、高灵敏度的化学和生物检测技术。荧光探针因高灵敏度、实时成像、高分辨率及无损实时监测等优势,为活细胞和生物体中的动态生物过程可视化提供了有效途径。
发明内容
本发明的目的在于提出一种荧光染料及其制备方法和制得的荧光探针,具有响应时间短,成本低、实时监测、原位检测、可视化检测、非破坏性检测,灵敏度高、选择性好、操作简单快速、重现性好、取样量少的优点,在生物分子检测领域具有广阔的应用前景。
本发明的技术方案是这样实现的:
本发明提供一种荧光染料,所述荧光染料的结构式如式I所示:
式I;
其中,R=N(CH3)2、CH3、C2H5、C3H7、中的至少一种。
作为本发明的进一步改进,所述荧光染料为下列至一:
式II、/>式III、式IV、/>式V、式VI。
本发明进一步保护一种上述荧光染料的制备方法,包括以下步骤:
S1.将4-溴-1,8-萘二甲酸酐、取代乙胺溶于乙醇中,加热回流反应,冷却至室温,减压除去溶剂,柱层析色谱分离,制得中间体,结构如下:;
S2.将中间体、β-D-吡喃半乳糖苷、碱溶于二氯甲烷中,加热回流反应,冷却至室温,减压除去溶剂,柱层析色谱分离,制得产物。
作为本发明的进一步改进,步骤S1中所述取代乙胺选自正丁胺、正戊胺、正己胺、N-(2-氨基乙基)吗啉、N,N'-二甲基乙二胺中的至少一种。
作为本发明的进一步改进,步骤S1中所述4-溴-1,8-萘二甲酸酐、取代乙胺的摩尔比为1:1.1-1.2,所述反应的时间为5-7h。
作为本发明的进一步改进,步骤S2中所述碱选自三乙胺、二乙胺、碳酸钠、碳酸钾、碳酸氢钠、碳酸氢钾、NaOH、KOH中的至少一种,所述中间体、β-D-吡喃半乳糖苷、碱的摩尔比为1:1.1-1.2:3-5,所述反应的时间为2-3h。
作为本发明的进一步改进,具体包括以下步骤:
S1.将1摩尔当量4-溴-1,8-萘二甲酸酐、1.1-1.2摩尔当量取代乙胺溶于乙醇中,加热回流反应5-7h,冷却至室温,减压除去溶剂,柱层析色谱分离,洗脱液为二氯甲烷:乙醇的体积比为20-30:1,制得中间体;
S2.将1摩尔当量中间体、1.1-1.2摩尔当量β-D-吡喃半乳糖苷、3-5摩尔当量碱溶于二氯甲烷中,加热回流反应2-3h,冷却至室温,减压除去溶剂,柱层析色谱分离,洗脱液为石油醚:乙醇的体积比为10-12:1,制得产物。
本发明进一步保护一种上述荧光染料在制备β-半乳糖苷酶荧光探针中的应用。
作为本发明的进一步改进,所述β-半乳糖苷酶荧光探针为:
式II。
作为本发明的进一步改进,所述β-半乳糖苷酶荧光探针采用乙醇-PBS缓冲溶液配制成用于β-半乳糖苷酶检测用的组合物。
本发明具有如下有益效果:
如图2,本发明荧光探针以萘酰亚胺为荧光母体。荧光母体在外界光作用后由基态到激发态再回到基态产生荧光效应,可以通过改变分子共轭结构调整发光性能;当该荧光探针在与β-半乳糖苷酶发生反应时,特异性降解荧光探针,半乳糖水解游离,通过连接基团引起荧光基团发生变化,出现强的荧光改变。
本发明的萘酰亚胺为荧光母体,具有大π共轭刚性平面结构的化合物,具有荧光量子产率高、Stokes位移大、光化学稳定性好等优点。并通过引入取代乙胺结构,该取代乙胺结构可以延长探针在细胞中的保留时间、提高保留能力,也可以有效地用于生理pH值下与β-半乳糖苷酶反应调控荧光成像,本发明制得的荧光探针具有较强的细胞穿透能力。
本发明荧光探针具有响应时间短,成本低、实时监测、原位检测、可视化检测、非破坏性检测,灵敏度高、选择性好、操作简单快速、重现性好、取样量少的优点,在生物分子检测领域具有广阔的应用前景。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动性的前提下,还可以根据这些附图获得其他的附图。
图1为本发明荧光染料的合成示意图;
图2为本发明荧光探针的检测原理图;
图3为本发明测试例5中细胞成像结果对比图。
实施方式
下面将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在没有做出创造性劳动前提下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
如图1,本实施例提供一种荧光染料,其制备方法具体包括以下步骤:
S1.将0.1mol 4-溴-1,8-萘二甲酸酐、0.11mol N-(2-氨基乙基)吗啉溶于100mL乙醇中,加热回流反应5h,冷却至室温,减压除去溶剂,柱层析色谱分离,洗脱液为二氯甲烷:乙醇的体积比为20:1,制得中间体;ESI-MS计算值:C18H18BrN2O3(M+H)+ 389.04,实测值:389.0,收率为80.7%。
核磁结果:1H NMR(300MHz,CDCl3)δ8.45(d,J=6.2Hz,1H),8.12(d,J=5.8Hz,1H),7.9-7.92(m,2H),7.75(dd,J=6.1Hz,5.8Hz,1H),3.67(t,4H),3.30(t,2H),2.65(t,2H),2.37(t,4H).
S2.将0.1mol中间体、0.11mol β-D-吡喃半乳糖苷、0.3mol溶于100mL二氯甲烷中,加热回流反应2h,冷却至室温,减压除去溶剂,柱层析色谱分离,洗脱液为石油醚:乙醇的体积比为10:1,制得产物。ESI-MS计算值:C24H29N2O9(M+H)+ 489.18,实测值:489.2,收率为92.5%。
核磁结果:1H NMR(300MHz,CDCl3)δ8.44(d,J=6.0Hz,1H),8.02(d,J=5.6Hz,1H),7.9(d,J=6.4Hz,1H),7.59(dd,J=6.0Hz,5.7Hz,1H),6.88(d,J=6.4Hz,1H),5.40(d,1H),4.18(m,1H),3.97-4.10(m,2H),3.66(t,4H),3.49(m,2H),3.40(m,1H),3.30(t,2H),2.66(t,2H),2.37(t,4H),2.0(br,4H).
实施例2
如图1,本实施例提供一种荧光染料,其制备方法具体包括以下步骤:
S1.将0.1mol 4-溴-1,8-萘二甲酸酐、0.12mol N,N'-二甲基乙二胺溶于100mL乙醇中,加热回流反应7h,冷却至室温,减压除去溶剂,柱层析色谱分离,洗脱液为二氯甲烷:乙醇的体积比为30:1,制得中间体;ESI-MS计算值:C16H16BrN2O2(M+H)+ 347.03,实测值:347.0,收率为75.3%。
核磁结果:1H NMR(300MHz,CDCl3)δ8.44(d,J=6.1Hz,1H),8.10(d,J=5.5Hz,1H),7.94-7.90(m,2H),7.75(dd,J=6.1Hz,5.5Hz,1H),3.31(t,2H),2.65(t,2H),2.27(s,6H).
S2.将0.1mol中间体、0.12mol β-D-吡喃半乳糖苷、0.5mol溶于100mL二氯甲烷中,加热回流反应3h,冷却至室温,减压除去溶剂,柱层析色谱分离,洗脱液为石油醚:乙醇的体积比为12:1,制得产物。ESI-MS计算值:C22H27N2O8(M+H)+ 447.17,实测值:447.2,收率为93.1%。
核磁结果:1H NMR(300MHz,CDCl3)δ8.45(d,J=6.0Hz,1H),8.03(d,J=5.5Hz,1H),7.91(d,J=6.2Hz,1H),7.59(dd,J=6.0Hz,5.5Hz,1H),6.87(d,J=6.2Hz,1H),5.42(d,1H),4.17(m,1H),3.96-4.09(m,2H),3.50(m,2H),3.41(m,1H),3.30(t,2H),2.65(t,2H),2.27(t,6H),2.0(br,4H).
实施例3
如图1,本实施例提供一种荧光染料,其制备方法具体包括以下步骤:
S1.将0.1mol 4-溴-1,8-萘二甲酸酐、0.115mol正丁胺溶于100mL乙醇中,加热回流反应6h,冷却至室温,减压除去溶剂,柱层析色谱分离,洗脱液为二氯甲烷:乙醇的体积比为25:1,制得中间体;ESI-MS计算值:C16H15BrNO2(M+H)+ 332.02,实测值:332.0,收率为65.2%。
核磁结果:1H NMR(300MHz,CDCl3)δ8.45(d,J=6.2Hz,1H),8.10(d,J=5.7Hz,1H),7.93-7.90(m,2H),7.75(dd,J=6.2Hz,5.7Hz,1H),3.20(t,2H),1.59(m,2H),1.33(m,2H),0.96(t,3H).
S2.将0.1mol中间体、0.115mol β-D-吡喃半乳糖苷、0.4mol溶于100mL二氯甲烷中,加热回流反应2.5h,冷却至室温,减压除去溶剂,柱层析色谱分离,洗脱液为石油醚:乙醇的体积比为11:1,制得产物。ESI-MS计算值:C22H26NO8(M+H)+ 432.16,实测值:432.2,收率为91.7%。
核磁结果:1H NMR(300MHz,CDCl3)δ8.44(d,J=6.1Hz,1H),8.02(d,J=5.4Hz,1H),7.90(d,J=6.4Hz,1H),7.59(dd,J=6.1Hz,5.5Hz,1H),6.88(d,J=6.4Hz,1H),5.41(d,1H),4.20(m,1H),3.96-4.10(m,2H),3.49(m,2H),3.40(m,1H),3.20(t,2H),2.0(br,4H),1.59(m,2H),1.33(m,2H),0.97(t,3H).
实施例4
如图1,本实施例提供一种荧光染料,其制备方法具体包括以下步骤:
S1.将0.1mol 4-溴-1,8-萘二甲酸酐、0.115mol正己胺溶于100mL乙醇中,加热回流反应6h,冷却至室温,减压除去溶剂,柱层析色谱分离,洗脱液为二氯甲烷:乙醇的体积比为25:1,制得中间体;ESI-MS计算值:C18H19BrNO2(M+H)+ 360.05,实测值:360.0,收率为64.3%。
核磁结果:1H NMR(300MHz,CDCl3)δ8.44(d,J=6.1Hz,1H),8.10(d,J=5.8Hz,1H),7.93-7.90(m,2H),7.75(dd,J=6.2Hz,5.8Hz,1H),3.20(t,2H),1.58(m,2H),1.28-1.34(m,6H),0.95(t,3H).
S2.将0.1mol中间体、0.115mol β-D-吡喃半乳糖苷、0.4mol溶于100mL二氯甲烷中,加热回流反应2-3h,冷却至室温,减压除去溶剂,柱层析色谱分离,洗脱液为石油醚:乙醇的体积比为11:1,制得产物。ESI-MS计算值:C24H30NO8(M+H)+ 460.19,实测值:460.2,收率为90.8%。
核磁结果:1H NMR(300MHz,CDCl3)δ8.44(d,J=6.1Hz,1H),8.02(d,J=5.4Hz,1H),7.90(d,J=6.4Hz,1H),7.59(dd,J=6.1Hz,5.4Hz,1H),6.88(d,J=6.4Hz,1H),5.42(d,1H),4.22(m,1H),3.96-4.11(m,2H),3.49(m,2H),3.40(m,1H),3.20(t,2H),2.0(br,4H),1.58(m,2H),1.29-1.34(m,6H),0.95(t,3H).
实施例5
如图1,本实施例提供一种荧光染料,其制备方法具体包括以下步骤:
S1.将0.1mol 4-溴-1,8-萘二甲酸酐、0.115mol正戊胺溶于100mL乙醇中,加热回流反应6h,冷却至室温,减压除去溶剂,柱层析色谱分离,洗脱液为二氯甲烷:乙醇的体积比为25:1,制得中间体;ESI-MS计算值:C17H17BrNO2(M+H)+ 346.04,实测值:346.0,收率为64.8%。
核磁结果:1H NMR(300MHz,CDCl3)δ8.45(d,J=6.2Hz,1H),8.10(d,J=5.7Hz,1H),7.93-7.90(m,2H),7.75(dd,J=6.2Hz,5.7Hz,1H),3.20(t,2H),1.59(m,2H),1.28-1.33(m,4H),0.97(t,3H).
S2.将0.1mol中间体、0.115mol β-D-吡喃半乳糖苷、0.4mol溶于100mL二氯甲烷中,加热回流反应2.5h,冷却至室温,减压除去溶剂,柱层析色谱分离,洗脱液为石油醚:乙醇的体积比为11:1,制得产物。ESI-MS计算值:C23H28NO8(M+H)+ 446.17,实测值:446.2,收率为91.2%。
核磁结果:1H NMR(300MHz,CDCl3)δ8.45(d,J=6.2Hz,1H),8.02(d,J=5.4Hz,1H),7.90(d,J=6.4Hz,1H),7.59(dd,J=6.2Hz,5.5Hz,1H),6.88(d,J=6.4Hz,1H),5.42(d,1H),4.21(m,1H),3.96-4.10(m,2H),3.49(m,2H),3.40(m,1H),3.20(t,2H),2.0(br,4H),1.59(m,2H),1.29-1.33(m,4H),0.97(t,3H).
测试例1 选择性
将本发明实施例1-5制得的荧光探针(10 µM)用生物分子处理,包括空白组(未添加生物分子),β-半乳糖苷酶,谷胱甘肽,丙氨酸,天冬氨酸,半胱氨酸,谷氨酸,丝氨酸,H2O2,Zn2+,Fe3+,Cu2+,检测510nm处的荧光强度,结果见表1。
表1
由上表可知,本发明实施例1-5制得的荧光探针对β-半乳糖苷酶具有较好的选择性,其中,实施例1制得的荧光探针对β-半乳糖苷酶的选择性最佳。
测试例2 细胞毒性
将本发明实施例1-5制得的荧光探针采用MTT法处理宫颈癌HeLa细胞和人卵巢癌OVCAR-3细胞,孵育48h,检测体系中细胞的存活率,结果见表2。
将宫颈A549细胞和和人卵巢癌OVCAR-3细胞接种到96孔板中,分别添加50μmol/L(最终浓度)实施例1-5制得的荧光探针,将细胞在37℃,空气的环境中培养48h。然后用PBS缓冲液洗涤细胞,加入RPMI1640培养基(100μL),将MTT(10μL,5mg/mL)注入到每个孔中孵育4h。用H2O-DMF混合物中的十二烷基硫酸钠溶液(100μL)处理产生紫色甲臜,测量溶液的吸光度。以无荧光探针细胞的细胞活力为100%来确定细胞活力。
表2
由上表可知,本发明实施例1-5制得的荧光探针对细胞毒性较小。
测试例3 响应时间
将本发明实施例1-5制得的荧光探针(10 µM)在生物体体温(37℃)的PBS缓冲溶液中加入了β-半乳糖苷酶(12 U),测试510nm处荧光强度达到最强时的响应时间,结果见表3。
表3
由上表可知,本发明实施例1-5制得的荧光探针对β-半乳糖苷酶的响应时间短。
测试例4 pH稳定性
在2mL的不同pH溶液中加入本发明实施例1-5制得的荧光探针(10μM,终浓度)、β-半乳糖苷酶(12 U,终浓度),测定不同pH溶液在510nm处的荧光强度。结果见表4。
表4
由上表可知,本发明实施例1-5制得的荧光探针在pH=3-10范围内可以稳定存在,在pH=5-10范围内可以很好的识别β-半乳糖苷酶,应用范围广,适合在生理环境下进行检测。
测试例5 细胞成像
以OVCAR-3细胞和H2O2诱导的A549衰老细胞作为β-半乳糖苷酶高表达的细胞模型,以正常A549细胞为阴性对照。如图2,本发明荧光探针以萘酰亚胺为荧光母体。荧光母体在外界光作用后由基态到激发态再回到基态产生荧光效应,可以通过改变分子共轭结构调整发光性能;当该荧光探针在与β-半乳糖苷酶发生反应时,特异性降解荧光探针,半乳糖水解游离,通过连接基团引起荧光基团发生变化,出现强的荧光改变。
如图3所示,当细胞与本发明实施例1制得的荧光探针(10μΜ)孵育1h后,在OVCAR-3细胞中观察到荧光,但在正常A549细胞中没有荧光。而经H2O2诱导A549细胞衰老后,在衰老的A549细胞中也能观察到荧光。这些结果表明本发明实施例1制得的荧光探针可用于检测卵巢癌细胞以及衰老细胞中的β-半乳糖苷酶。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (10)
1.一种荧光染料,其特征在于,所述荧光染料的结构式如式I所示:
式I;
其中,R=N(CH3)2、CH3、C2H5、C3H7、中的至少一种。
2.根据权利要求1所述荧光染料,其特征在于,所述荧光染料为下列至一:
式II、/>式III、式IV、/>式V、式VI。
3.一种如权利要求1或2所述荧光染料的制备方法,其特征在于,包括以下步骤:
S1.将4-溴-1,8-萘二甲酸酐、取代乙胺溶于乙醇中,加热回流反应,冷却至室温,减压除去溶剂,柱层析色谱分离,制得中间体,结构如下:;
S2.将中间体、β-D-吡喃半乳糖苷、碱溶于二氯甲烷中,加热回流反应,冷却至室温,减压除去溶剂,柱层析色谱分离,制得产物。
4.根据权利要求3所述的制备方法,其特征在于,步骤S1中所述取代乙胺选自正丁胺、正戊胺、正己胺、N-(2-氨基乙基)吗啉、N,N'-二甲基乙二胺中的至少一种。
5.根据权利要求3所述的制备方法,其特征在于,步骤S1中所述4-溴-1,8-萘二甲酸酐、取代乙胺的摩尔比为1:1.1-1.2,所述反应的时间为5-7h。
6.根据权利要求3所述的制备方法,其特征在于,步骤S2中所述碱选自三乙胺、二乙胺、碳酸钠、碳酸钾、碳酸氢钠、碳酸氢钾、NaOH、KOH中的至少一种,所述中间体、β-D-吡喃半乳糖苷、碱的摩尔比为1:1.1-1.2:3-5,所述反应的时间为2-3h。
7.根据权利要求3所述的制备方法,其特征在于,具体包括以下步骤:
S1.将1摩尔当量4-溴-1,8-萘二甲酸酐、1.1-1.2摩尔当量取代乙胺溶于乙醇中,加热回流反应5-7h,冷却至室温,减压除去溶剂,柱层析色谱分离,洗脱液为二氯甲烷:乙醇的体积比为20-30:1,制得中间体;
S2.将1摩尔当量中间体、1.1-1.2摩尔当量β-D-吡喃半乳糖苷、3-5摩尔当量碱溶于二氯甲烷中,加热回流反应2-3h,冷却至室温,减压除去溶剂,柱层析色谱分离,洗脱液为石油醚:乙醇的体积比为10-12:1,制得产物。
8.一种如权利要求1或2所述荧光染料在制备β-半乳糖苷酶荧光探针中的应用。
9.根据权利要求8所述的应用,其特征在于,所述β-半乳糖苷酶荧光探针为:
式II。
10.根据权利要求8所述的应用,其特征在于,所述β-半乳糖苷酶荧光探针采用乙醇-PBS缓冲溶液配制成用于β-半乳糖苷酶检测用的组合物。
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