CN112939934A - 一种高稳定性、高亮度的Halo-tag探针及其合成方法与应用 - Google Patents
一种高稳定性、高亮度的Halo-tag探针及其合成方法与应用 Download PDFInfo
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Abstract
本发明提供了一类高稳定性、高亮度Halo‑tag荧光探针及其合成方法与在双光子荧光成像中的生物应用。该类探针以萘酰亚胺为荧光团,以氯代脂肪烃为识别基团,结构如式(1)所示。4位引入的氮杂环酮类取代基,使该Halo‑tag探针能够保持高的量子产率与稳定性。其中R为氮杂环酮类取代基,氮杂环包括四元氮杂环、五元氮杂环、六元氮杂环等。该类探针能够对活细胞的Halo‑tag蛋白标签特异性标记,在双光子荧光成像等领域有很好的应用前景。
Description
技术领域
本发明属于荧光探针标记领域,具体涉及一种高稳定性、高亮度的Halo-tag探针及其合成方法与应用。
背景技术
Halo-tag是由紫红红球菌的脱卤素酶改造而成的蛋白标签,能够高亲和力、高特异性地与连有不同功能基团的卤代脂肪烃类探针共价结合。目的蛋白与Halo-tag蛋白标签构成表达载体,可以保证遗传标记的特异性;同时Halo-tag探针的多样性能够实现不同的研究目的(比如:活细胞荧光成像、蛋白质动力学分析、探测细胞氧化还原微扰及自噬的诱导等)。
Halo-tag探针主要由两部分组成:一个是识别基团,一般为氯代己烷类衍生物;另一个是满足不同研究需求的功能基团,如荧光素、香豆素、罗丹明等荧光染料。目前这类halo-tag探针通常存在膜透性差、背景荧光高、功能单一等问题。而双光子荧光成像技术具有组织穿透性深、空间分辨率高、背景信号低等优势,能够弥补传统halo-tag探针的不足。因此,Halo-tag探针设计与双光子荧光成像技术的结合,必将成为生命过程研究中的强有力工具,开发高性能的Halo-tag双光子荧光探针显得尤为迫切。
发明内容
本发明提供了一类高稳定性、高亮度的Halo-tag探针,该探针能够实现活细胞内的双光子荧光成像。
本发明的另一目的是提供一种高稳定性、高亮度的Halo-tag探针的合成方法,该方法步骤简单、原料低廉、产物易于分离等优点。
本发明的Halo-tag探针,通过在萘酰亚胺分子4位引入氮杂环酮类取代基,使得其适合应用于双光子荧光成像。
本发明一类高亮度、高稳定性Halo-tag荧光探针,其结构式如下所示:
其中R为氮杂环酮类取代基,氮杂环包括四元氮杂环、五元氮杂环、六元氮杂环等。
该类探针结构式(1)具体为下列中的一种,
一类用于Halo-tag标记的荧光探针的合成方法,其合成路线如下,
具体合成步骤如下:
(1)中间体Halo-Br的合成
将4-溴-1,8-萘酐溶于乙醇中,而后向该反应液中加O-(6-氯己基)二甘醇胺。将反应液加热至50-100℃,搅拌2-7h。冷却降温后,减压除去溶剂,残余物经硅胶柱分离(展开剂为石油醚:二氯甲烷=10:1-2:1)得无色油状产物Halo-Br。
步骤(1)中:4-溴-1,8-萘酐与O-(6-氯己基)二甘醇胺的质量比为1:0.7-1.5;4-溴-1,8-萘酐与无水乙醇的质量与体积比为1:20-60g/mL。
(2)荧光探针的合成
Halo-Br、氮杂环酮RH、Cs2CO3、G3-Xantphos(XantPhos Pd G3 95%)置于双口瓶中并用氮气置换3-4次。向反应体系中加入干燥的二氧六环,并加热至70-140℃。2-7h后减压除去溶剂,残余物经硅胶柱分离得固体(荧光探针)。
步骤(2)中:Halo-Br与氮杂环酮(RH)的质量比为1:0.25-0.75;Cs2CO3与氮杂环酮(RH)的摩尔比为1:1;G3-Xantphos(XantPhos Pd G3 95%)为Halo-Br的1mol%;Halo-Br与二氧六环质量与体积比为1:80-240g/mL。
上述一类高稳定性、高亮度Halo-tag探针对Halo-tag蛋白特异性识别。
一类高稳定性、高亮度Halo-tag探针在细胞、组织及活体内的荧光成像应用。
一类高稳定性、高亮度Halo-tag探针用于Halo-tag蛋白的识别与检测。
一类高稳定性、高亮度Halo-tag探针在单分子检测中的应用。
一类高稳定性、高亮度Halo-tag探针在双光子荧光成像中的应用。
本发明优点和有益效果:
该类Halo-tag探针具有制备简单、原料低廉、产物易分离等优点。
该类Halo-tag探针分子亮度高、光稳定性。
该类Halo-tag探针能够对活细胞内Halo-tag蛋白进行特异性识别,并进行双光子荧光成像应用。
附图说明
图1为实施例1制备的Halo-Br的核磁氢谱。
图2为实施例1制备的Halo-Br的核磁碳谱。
图3为实施例1制备的Halo-405的核磁氢谱。
图4为实施例1制备的Halo-405的核磁碳谱。
图5为实施例1制备的荧光探针Halo-405在水中归一化的荧光激发光谱与发射谱图,横坐标为波长,纵坐标为归一化强度。
图6为实施例1制备的探针Halo-405在Hela细胞中的单光子荧光成像图,荧光探针的浓度为1μM。
图7为实施例1制备的探针Halo-405在Hela细胞中的双光子荧光成像图,荧光探针的浓度为1μM。
具体实施方式
本发明提供了一类Halo-tag的荧光探针及其制备方法和在荧光方面的应用。
下面将对本发明实施例中的方案进行清楚、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。
下面三个实施例(实施例1-3)为Halo-405的制备,其反应式如下所示:
实施例1
(1)中间体Halo-Br的合成
将4-溴-1,8-萘酐(277.07mg,1.00mmol)溶于5.54mL乙醇中,而后向该反应液中加O-(6-氯己基)二甘醇胺(193.95mg,0.87mmol)。将反应液加热至50℃,搅拌2h。冷却降温后,减压除去溶剂,残余物经硅胶柱分离(展开剂为石油醚:二氯甲烷=10:1-2:1)得无色油状产物Halo-Br 310.82mg,产率74%。
其核磁谱图氢谱、碳谱如图1、2所示,具体数据如下:
1H NMR(400MHz,CDCl3)δ8.61(dd,J=7.3,1.0Hz,1H),8.52(dd,J=8.5,1.0Hz,1H),8.37(d,J=7.9Hz,1H),8.00(d,J=7.9Hz,1H),7.82(dd,J=8.4,7.4Hz,1H),4.42(t,J=6.1Hz,2H),3.83(t,J=6.1Hz,2H),3.69(dd,J=5.7,3.9Hz,2H),3.54(dd,J=5.6,3.9Hz,2H),3.50(t,J=6.7Hz,2H),3.38(t,J=6.6Hz,2H),1.77–1.67(m,2H),1.53–1.44(m,2H),1.43–1.33(m,2H),1.32–1.21(m,2H).
经检测,其结构如上式Halo-Br所示。
(2)探针Halo-405的合成
Halo-Br(28.97mg,0.06mmol),2-氮杂环丁酮(7.24mg,0.10mmol),Cs2CO3(32.58mg,0.10mmol),G3-Xantphos(XantPhos Pd G3 95%)(1mol%)置于双口瓶中并用氮气置换3-4次。向反应体系中加入2.32mL干燥的二氧六环,并加热至70℃。2h后减压除去溶剂,残余物经硅胶柱分离(展开剂为二氯甲烷:甲醇=200:1-30:1)得白色固体21.00mg,产率74%。
其核磁谱图氢谱、碳谱如图3、4所示,具体数据如下:
1H NMR(400MHz,CDCl3)δ8.82(dd,J=8.6,1.0Hz,1H),8.62(dd,J=7.3,1.0Hz,1H),8.54(d,J=8.1Hz,1H),7.75(dd,J=8.6,7.3Hz,1H),7.59(d,J=8.1Hz,1H),4.42(t,J=6.2Hz,2H),4.06(t,J=4.8Hz,2H),3.83(t,J=6.2Hz,2H),3.69(dd,J=5.7,3.9Hz,2H),3.55(dd,J=5.7,3.9Hz,2H),3.50(t,J=6.7Hz,2H),3.39(t,J=6.6Hz,2H),3.32(t,J=4.8Hz,2H),1.79–1.66(m,2H),1.53–1.43(m,2H),1.42–1.35(m,2H),1.32–1.20(m,2H).13CNMR(101MHz,CDCl3)δ165.32,164.18,163.58,140.86,131.90,131.79,131.32,129.47,126.28,123.74,122.64,119.12,116.02,71.20,70.18,70.13,67.89,45.10,41.09,39.11,36.21,32.53,29.46,26.69,25.37.
经检测,其结构如上式Halo-405所示。
将该类探针分别溶于DMSO中,配制成2mM母液。Halo-405探针在水中荧光激发与发射光谱测试。将20μL Halo-405母液加入到4mL水中,配制成10μM的测试液,进行荧光激发与发射光谱的测试。
探针Halo-405的荧光激发、发射光谱如图5所示,其中激发波长在373nm,发射波长在478nm左右。
实施例2
(1)中间体Halo-Br的合成
将4-溴-1,8-萘酐(277.07mg,1.00mmol)溶于11.08mL乙醇中,而后向该反应液中加O-(6-氯己基)二甘醇胺(304.78mg,1.36mmol)。将反应液加热至75℃,搅拌4.5h。冷却降温后,减压除去溶剂,残余物经硅胶柱分离(展开剂为石油醚:二氯甲烷=10:1-2:1)得无色油状产物Halo-Br 376.58mg,产率78%。
(2)探针Halo-405的合成
Halo-Br(28.97mg,0.06mmol),2-氮杂环丁酮(14.49mg,0.20mmol),Cs2CO3(65.16mg,0.20mmol),G3-Xantphos(XantPhos Pd G3 95%)(1mol%)置于双口瓶中并用氮气置换3-4次。向反应体系中加入4.64mL干燥的二氧六环,并加热至105℃。4.5h后减压除去溶剂,残余物经硅胶柱分离(展开剂为二氯甲烷:甲醇=200:1-30:1)得白色固体19.30mg,产率68%。
经检测,其结构为Halo-405,其荧光性能如下:
Halo-405在水中荧光激发波长在373nm,发射波长在478nm。
实施例3
(1)中间体Halo-Br的合成
将4-溴-1,8-萘酐(277.07mg,1.00mmol)溶于16.62mL乙醇中,而后向该反应液中加O-(6-氯己基)二甘醇胺(415.61mg,1.86mmol)。将反应液加热至100℃,搅拌7h。冷却降温后,减压除去溶剂,残余物经硅胶柱分离(展开剂为石油醚:二氯甲烷=10:1-2:1)得无色油状产物Halo-Br 495.90mg,产率82%。
(2)探针Halo-405的合成
Halo-Br(28.97mg,0.06mmol),2-氮杂环丁酮(21.73mg,0.31mmol),Cs2CO3(101.00mg,0.31mmol),G3-Xantphos(XantPhos Pd G3 95%)(1mol%)置于双口瓶中并用氮气置换3-4次。向反应体系中加入6.95mL干燥的二氧六环,并加热至140℃。7h后减压除去溶剂,残余物经硅胶柱分离(展开剂为二氯甲烷:甲醇=200:1-30:1)得白色固体21.85mg,产率77%。
经检测,其结构如Halo-405所示,其荧光性能如下:
Halo-405在水中荧光激发波长在373nm,发射波长在478nm。
实施例4
本实施例进行荧光探针Halo-52的制备,其反应式如下所示:
(1)中间体Halo-Br的合成
将4-溴-1,8-萘酐(277.07mg,1.00mmol)溶于11.08mL乙醇中,而后向该反应液中加O-(6-氯己基)二甘醇胺(304.78mg,1.36mmol)。将反应液加热至75℃,搅拌4.5h。冷却降温后,减压除去溶剂,残余物经硅胶柱分离(展开剂为石油醚:二氯甲烷=10:1-2:1)得无色油状产物Halo-Br 376.58mg,产率78%。
1H NMR(400MHz,CDCl3)δ8.61(dd,J=7.3,1.0Hz,1H),8.52(dd,J=8.5,1.0Hz,1H),8.37(d,J=7.9Hz,1H),8.00(d,J=7.9Hz,1H),7.82(dd,J=8.4,7.4Hz,1H),4.42(t,J=6.1Hz,2H),3.83(t,J=6.1Hz,2H),3.69(dd,J=5.7,3.9Hz,2H),3.54(dd,J=5.6,3.9Hz,2H),3.50(t,J=6.7Hz,2H),3.38(t,J=6.6Hz,2H),1.77–1.67(m,2H),1.53–1.44(m,2H),1.43–1.33(m,2H),1.32–1.21(m,2H).
(2)探针Halo-52的合成
Halo-Br(28.97mg,0.06mmol),2-吡咯烷酮(14.49mg,0.17mmol),Cs2CO3(55.39mg,0.17mmol),G3-Xantphos(XantPhos Pd G3 95%)(1mol%)置于双口瓶中并用氮气置换3-4次。向反应体系中加入4.64mL干燥的二氧六环,并加热至105℃。4.5h后减压除去溶剂,残余物经硅胶柱分离得固体21.91mg,产率75%。
1H NMR(400MHz,CDCl3)δ8.81(dd,J=8.6,1.0Hz,1H),8.61(dd,J=7.3,1.0Hz,1H),8.52(d,J=8.1Hz,1H),7.76(dd,J=8.6,7.3Hz,1H),7.57(d,J=8.1Hz,1H),4.41(t,J=6.2Hz,2H),4.11(t,J=4.8Hz,2H),4.07(t,J=5.1Hz,2H),3.68(dd,J=5.7,3.9Hz,2H),3.56(dd,J=5.7,3.9Hz,2H),3.51(t,J=6.7Hz,2H),3.38(t,J=6.6Hz,2H),2.42(t,J=5.4Hz,2H),2.14-2.06(m,2H),1.78–1.67(m,2H),1.54–1.44(m,2H),1.43–1.36(m,2H),1.31–1.21(m,2H).
经检测,其结构如Halo-52所示。
实施例5
本实施例进行荧光探针Halo-62的制备,其反应式如下所示:
(1)中间体Halo-Br的合成
将4-溴-1,8-萘酐(277.07mg,1.00mmol)溶于11.08mL乙醇中,而后向该反应液中加O-(6-氯己基)二甘醇胺(304.78mg,1.36mmol)。将反应液加热至75℃,搅拌4.5h。冷却降温后,减压除去溶剂,残余物经硅胶柱分离(展开剂为石油醚:二氯甲烷=10:1-2:1)得无色油状产物Halo-Br 376.58mg,产率78%。
13C NMR(101MHz,CDCl3)δ163.57,163.54,133.22,132.02,131.20,131.05,130.53,130.23,128.96,128.04,123.01,122.15,71.20,70.16,70.14,67.82,45.06,39.22,32.52,29.44,26.69,25.37.
(2)探针Halo-62的合成
Halo-Br(28.97mg,0.06mmol),2-氮己环酮(14.49mg,0.15mmol),Cs2CO3(48.87mg,0.15mmol),G3-Xantphos(XantPhos Pd G3 95%)(1mol%)置于双口瓶中并用氮气置换3-4次。向反应体系中加入4.64mL干燥的二氧六环,并加热至105℃。4.5h后减压除去溶剂,残余物经硅胶柱分离得固体22.85mg,产率76%。
Halo-62的质谱具体数据如下:
C27H33ClN2O5[M+H]+理论值:501.2134,实际值:501.2103。
经检测,其结构如Halo-62所示。
实施例6
荧光探针Halo在转染细胞中单光子和双光子荧光成像。取0.5μL Halo-405母液溶于1mL培养液中,而后置于37℃下孵育30分钟后进行荧光成像。探针Halo-405终浓度为1μM的细胞培养液孵育表达融合有Halo-tag的H2B的Hela细胞10分钟后单光子荧光成像图如图6所示,双光子荧光成像图如图7所示。可以观察到,探针能够对融合有Halo-tag的H2B进行特异性标记,且适合做双光子荧光成像,有高度局部激发、延长了观察时间、深层组织穿透、高空间分辨率、低背景信号之类的优势。
Claims (9)
3.一种如权利要求1-2任一所述Halo-tag探针的制备方法,其特征在于,该方法包含以下合成步骤:
(1)中间体Halo-Br的合成
将4-溴-1,8-萘酐溶于乙醇中,而后向该反应液中加O-(6-氯己基)二甘醇胺;将反应液加热至50-100℃,搅拌2-7h;冷却降温后,减压除去溶剂,残余物经硅胶柱分离(展开剂为石油醚:二氯甲烷=10:1-2:1)得无色油状产物Halo-Br;
(2)Halo-tag探针的合成
Halo-Br、氮杂环酮RH、Cs2CO3、G3-Xantphos(XantPhos Pd G3 95%)置于双口瓶中并用氮气置换3-4次;向反应体系中加入干燥的二氧六环,并加热至70-140℃;2-7h后减压除去溶剂,残余物经硅胶柱分离得固体(荧光探针)。
5.如权利要求3所述的Halo-tag探针的制备方法,其特征在于:步骤(1)中:4-溴-1,8-萘酐与O-(6-氯己基)二甘醇胺的质量比为1:0.7-1.5;4-溴-1,8-萘酐与无水乙醇的质量与体积比为1:20-60g/mL。
6.如权利要求3所述的Halo-tag探针的制备方法,其特征在于:步骤(2)中,Halo-Br与氮杂环酮(RH)的质量比为1:0.25-0.75;Cs2CO3与氮杂环酮(RH)的摩尔比为1:1;G3-Xantphos(XantPhos Pd G3 95%)为Halo-Br的1mol%;Halo-Br与二氧六环质量与体积比为1:80-240g/mL。
7.一种如权利要求1所述的Halo-tag探针在细胞、组织及活体内的荧光成像的应用。
8.一种如权利要求1所述的Halo-tag探针在Halo-tag蛋白的识别与检测中的应用。
9.一种如权利要求1所述的Halo-tag探针在双光子荧光成像中的应用。
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