CN111333618A - 一种488nm激发的免洗SNAP-tag探针及其制备方法 - Google Patents
一种488nm激发的免洗SNAP-tag探针及其制备方法 Download PDFInfo
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Abstract
本发明提供了一种488nm激发的免洗SNAP‑tag探针及其制备方法,该免洗SNAP‑tag探针是在4,5‑位双取代的萘酰亚胺染料中引入螺环刚性结构并设计合成的一系列可用于488nm激发的免洗SNAP‑tag探针,其结构式如(1)所示。刚性结构的引入不仅限制了分子内扭转,提高了荧光分子的稳定性及亮度,还增加了探针分子的平面性。这导致SNAP‑tag探针分子在水中形成强烈π‑π作用,导致荧光的淬灭。而与SNAP‑tag结合后,探针分子荧光得到释放,最高可增强28倍。该系列探针能够实现活细胞内对SNAP‑tag的特异性标记,达到免洗荧光成像。此外,由于稳定性及亮度的提升,该系列探针还可用于SIM(结构光照明显微镜),STED(受激辐射损耗)等超分辨荧光成像领域。
Description
技术领域
本发明属于蛋白标记领域,具体涉及一种488nm激发的免洗SNAP-tag探针及其制备方法。
背景技术
荧光成像技术已经逐渐成为在细胞层次到个体水平研究蛋白质功能的强有力工具。由于具有尺寸小、荧光发射光谱宽泛、可选择荧光颜色多样等优点,有机小分子荧光染料在蛋白标记领域逐渐成为荧光蛋白的替代者。但是有机小分子染料是外源物种,存在的问题是不能够像荧光蛋白一样由细胞源生,所以无法控制在细胞中的数量以及细胞中的位置。为了解决这个问题,化学家发展了多种生物正交的方法将小分子染料共价连接到目标蛋白上,从而可以进一步跟踪研究目标蛋白的位置和功能。其中,目前应用最广泛的是蛋白标签技术,该技术通过遗传编码的方法首先将标签蛋白融合到目标蛋白上,然后这种蛋白标签与荧光底物发生专一的酶促共价连接反应,从而实现将小分子荧光染料特异性连接到目标蛋白的目的。
利用蛋白标签技术实现活细胞蛋白成像时,荧光底物需要满足多种性能要求,至少包括荧光底物具有良好的细胞透膜性、与蛋白标签之外的生物大分子无结合,与蛋白标签反应快、蛋白标记前后荧光有明显变化以提高信噪比等。其中,自由的荧光染料无荧光或很弱,与蛋白标签标记后荧光显著增强,这样的荧光信号能够有效避开背景荧光以及没有反应的荧光底物的干扰。这一性能使染料在进行活细胞成像时可以省掉多次洗细胞这样的有损细胞的操作。
SNAP-tag标签法已经成为目前应用最为广泛的蛋白质标记技术,通过其与目标蛋白的融合可以对目标蛋白进行示踪、功能的监测等。目前,已有多种商业的SNAP-tag荧光染料被开发出来,其主要是基于环境不敏感的罗丹明与花菁染料。这类染料能够与SNAP-tag能够达到很高的反应速率,但是反应前后荧光变化较小(通常增加1-2倍)。因此,在对细胞着色后需要多次洗涤才能达到比较好的染色效果。而对于环境敏感型染料,由SNAP-tag结合前水环境到结合后的疏水的蛋白表面会使其荧光增强倍数增加,有可能会达到免洗的效果;此外SNAP-tag探针还可以基于聚集诱导淬灭、PET等机制实现荧光背底的减弱。由此可见,对于SNAP-tag探针的设计有多种方式可以遵循,如何通过合适的方式达到最佳识别效果一直是科研工作者研究的重要方向。尤其在单个蛋白功能研究的今天,单分子和超分辨成像等技术对荧光染料稳定性和亮度提出高要求的同时,对荧光信号真实性也提出了更高的需求,而背景增高会造成错误信号的增加,无法得到可信的成像结果。因此,设计出光稳定更好、反应速率更高的免洗SNAP-tag荧光探针显得尤为重要。
发明内容
本发明的目的之一是提供一种488nm激发的免洗SNAP-tag探针,该系列探针与SNAP-tag蛋白结合后荧光增强倍数可达28倍,可实现活细胞内的免洗荧光成像。
本发明的另一目的是提供一种488nm激发的免洗SNAP-tag探针的制备方法,该方法具有通用性,相比于目前嘌呤引入方法拥有步骤简单、易于提纯等优点。
本发明提供一种488nm激发的免洗SNAP-tag探针,以萘酰亚胺为荧光团,通过4,5-位刚性结构的调节使萘酰亚胺的荧光稳定性、亮度得到大幅度提升,实现了SNAP-tag蛋白的免洗超分辨荧光成像。此外,萘酰亚胺由环境敏感型染料蜕变为环境不敏感型荧光染料,在与SNAP-tag结合前后荧光峰型及波长不随极性变化而发生变化,保持了荧光信号的准确性。
一种488nm激发的免洗SNAP-tag探针,该系列荧光探针具有如下结构:
R2为H、C1-4烷基、(CH2CH2O)nH等;
R3为H、C1-4烷基、(CH2CH2O)nH。n位0,1,2,3.
一种488nm激发的免洗SNAP-tag探针的制备方法,此系列荧光探针合成路线,如下:
具体合成步骤如下:
(1)中间体N-(4-羟甲基)苄基-4-溴-5-硝基-1,8-萘酰亚胺(BA-NBr)的合成:
将4-溴-5-硝基-1,8-萘酐和4-氨甲基苄醇溶于无水乙醇中。将反应液加热至40-90℃,搅拌1-10h。将反应液泠却至室温后,减压除去溶剂后,硅胶柱分离,以体积比为800-100:1的二氯甲烷和甲醇为洗脱剂,减压除去溶剂得米白色固体N-(4-羟甲基)苄基-4-溴-5-硝基-1,8-萘酰亚胺(BA-NBr);
(2)中间体N-(4-羟甲基)苄基-4,5-二脂肪胺基-1,8-萘酰亚胺的合成:
将N-(4-羟甲基)苄基-4-溴-5-硝基-1,8-萘酰亚胺溶于乙二醇甲醚中,并向其中加入脂肪环胺;将反应液缓慢升温至100-140℃,并在氮气保护下反应10-24h;减压除去溶剂,硅胶柱分离,以体积比为400-30:1的二氯甲烷和甲醇为洗脱剂,除去溶剂,得棕黄色固体N-(4-羟甲基)苄基-4,5-二脂肪胺基-1,8-萘酰亚胺;
(3)SNAP-tag探针的合成
将N-(4-羟甲基)苄基-4,5-脂肪胺基-1,8-萘酰亚胺、叔丁醇钾和2-氨基-6-(N-甲基)四氢吡咯基鸟嘌呤置于史莱克瓶中,氮气置换2-5次后加入干燥的N,N-二甲基甲酰胺;室温反应3-10h后,加压出去溶剂,硅胶柱分离,以体积比为100-10:1的二氯甲烷和甲醇为洗脱剂,除去溶剂得靶向SNAP-tag蛋白的荧光探针;一类如权利1所述的一系列488nm激发的免洗SNAP-tag探针在细胞、组织及活体内对目标蛋白的荧光成像的应用。
步骤(1)中:4-溴-5-硝基-1,8-萘酐:4-氨甲基苄醇的质量比为1:0.5-2;4-溴-5-硝基-1,8-萘酐的质量与无水乙醇的体积比为1:20-80g/mL;
步骤(2)中:N-(4-羟甲基)苄基-4-溴-5-硝基-1,8-萘酰亚胺与脂肪环胺的质量比为1:1-3;N-(4-羟甲基)苄基-4-溴-5-硝基-1,8-萘酰亚胺的质量与乙二醇甲醚的体积比为1:50-200g/mL。
脂肪环胺为氮丙啶、氮杂环丁烷、四氢吡咯、乙二胺衍生物等。
步骤(3)中:N-(4-羟甲基)苄基-4,5-脂肪胺基-1,8-萘酰亚胺、叔丁醇钾、2-氨基-6-(N-甲基)四氢吡咯基鸟嘌呤的质量比为1:1-5:1-5;N-(4-羟甲基)苄基-4,5-脂肪胺基-1,8-萘酰亚胺的质量与N,N-二甲基甲酰胺的体积比为1:80-200g/mL。
上述一系列免洗SNAP-tag探针对SNAP-tag蛋白具有高度选择性,能够在复杂环境中对SNAP-tag进行特异性识别。
一类488nm激发的免洗SNAP-tag探针在识别与检测SNAP-tag蛋白中的应用。
一类488nm激发的免洗SNAP-tag探针在超分辨成像中的应用。
一类488nm激发的免洗SNAP-tag探针在细胞、组织及活体内对目标蛋白的荧光成像的应用。
本发明具有以下特点:
本发明涉及的SNAP-tag探针拥有合成原料低价、方法简单通用等优点。
本发明涉及的SNAP-tag探针在水中分子间发生聚集而导致荧光淬灭,而在结合SNAP-tag蛋白后,探针逐渐被解聚,荧光恢复。因此,该系列探针在与SNAP-tag结合前后达到off-on的效果,荧光增强倍数可达28倍。
本发明涉及的SNAP-tag探针分子由于4,5-位刚性结构对分子内扭转的限制使探针在结合SNAP-tag蛋白后荧光量子产率均大于0.80,亮度高、光稳定性好。该系列SNAP-tag探针在结合SNAP-tag蛋白后荧光波长及峰型不随极性变化而改变,能够保持荧光信号的准确性。
本发明涉及的SNAP-tag探针能够对活细胞内SNAP-tag蛋白进行特异性识别,并实现免洗荧光成像。此外,探针可用于SIM,STED等超分辨荧光成像。
附图说明
图1实施例1制备的BA-DAze的核磁谱图氢谱。
图2实施例1制备的SNAP-DAze的核磁谱图氢谱。
图3实施例1制备的探针SNAP-DAze的核磁谱图碳谱。
图4实施例1制备的探针SNAP-DAze在不同溶剂中归一化的荧光发射谱图,横坐标为波长,纵坐标为归一化荧光强度,荧光染料的浓度为10μM。
图5实施例4制备的探针SNAP-DAC在PBS中与1μM SNAP-tag蛋白结合前后荧光光谱,横坐标为波长,纵坐标为荧光强度,荧光探针的浓度为1μM。
图6实施例1制备的探针SNAP-DAze在PBS中与1μM SNAP-tag蛋白结合的动力学曲线图,横坐标为时间,纵坐标为荧光强度,荧光探针的浓度为1μM。
图7实施例1制备的探针SNAP-DAze在在转染的HEK293细胞荧光共聚焦成像图,荧光探针的浓度为1μM。
图8实施例1制备的探针SNAP-DAze在在转染的HEK293细胞结构光照明显微镜成像图,荧光探针的浓度为1μM。
图9实施例4制备的探针SNAP-DAC在在转染的Hela细胞结构光照明显微镜成像图,荧光探针的浓度为1μM。
具体实施方式
实施例1
SNAP-DAze的合成:
中间体N-(4-羟甲基)苄基-4-溴-5-硝基-1,8萘酰亚胺(BA-NBr)的合成:
4-溴-5-硝基-1,8-萘酰亚胺(1.00g,3.11mmol)溶于50mL乙醇中,并向其中加入4-氨甲基苄醇(500mg,3.60mmol)。80℃下10h后,减压蒸馏除去溶剂,残余物经硅胶柱(石油醚:二氯甲烷=3:1-二氯甲烷:甲醇=200:1,V/V)分离得米白色固体480mg,产率35%。其核磁谱图氢谱数据如下:
1H NMR(400MHz,DMSO-d6)δ8.69(d,J=8.1Hz,2H),8.50–8.39(m,2H),7.35(d,J=8.1Hz,2H),7.25(d,J=7.9Hz,2H),5.23(s,2H),5.13(t,J=5.8Hz,1H),4.45(d,J=5.5Hz,2H).
BA-DAze的合成
将BA-NBr(300mg,0.68mmol)溶于30mL乙二醇甲醚中,并向其中加入氮杂环丁烷300mg。将反应液缓慢加热至120℃,并反应10h。减压除去乙二醇甲醚,残余物经硅胶柱分离残余物(二氯甲烷:甲醇=60:1,V/V),得黄色固体52mg,产率18%。其核磁谱图氢谱如图1所示,具体数据如下:
1H NMR(400MHz,CDCl3)δ8.38(d,J=8.5Hz,2H),7.50(d,J=7.8Hz,2H),7.25(d,J=5.9Hz,2H),6.38(d,J=8.5Hz,2H),5.36(s,2H),4.61(s,2H),4.11(s,8H),2.44(s,4H).
其高分辨质谱数据如下:高分辨质谱理论值C26H26N3O3[M+H]+428.1974,实际值428.1997.
SNAP-DAze的合成
将BA-DAze(40mg,0.09mmol)、BG+(40mg,0.16mmol)、叔丁醇钾(40mg,0.36mmol)置于10mL史莱克瓶中,用氮气置换三次并加入5mL干燥DMF。室温下搅拌6h后减压除去溶剂,残余物经硅胶柱分离残余物(二氯甲烷:甲醇=20:1,V/V),得棕色固体24mg,产率45%。实施例1制备的核磁谱图氢谱与碳谱分别如图2、3所示,具体数据如下:
1H NMR(400MHz,DMSO-d6)δ12.40(s,1H),8.17(d,J=8.4Hz,2H),7.79(s,1H),7.41(d,J=7.4Hz,2H),7.28(d,J=7.6Hz,2H),6.48(d,J=8.5Hz,2H),6.26(s,2H),5.42(s,2H),5.22(s,2H),4.14(s,8H),2.38(s,4H).13C NMR(101MHz,DMSO-d6)δ163.47,160.31,160.09,156.01,155.65,138.90,138.22,135.60,133.37,132.87,128.87,127.74,113.97,108.21,106.94,106.73,56.50,54.63,42.37,19.02.
其高分辨质谱数据如下:高分辨质谱理论值C31H29N8O3[M+H]+561.2363,实际值561.2380.
经检测,其结构如上式SNAP-DAze所示,其荧光性能如下:
将SNAP-DAze溶解于DMSO溶液中,配制成2mM母液,根据需要配制成不同浓度测试溶液,以检测其荧光光谱。
SNAP-DAze在乙腈、氯仿、二甲基亚砜、乙醇、水中荧光发射光谱测试。每次取20μLSNAP-DAze母液加入4mL乙腈、氯仿、二甲基亚砜、乙醇、水中,配制成10μM的荧光染料测试液,进行荧光发射光谱的测试。
SNAP-DAze在乙腈、氯仿、二甲基亚砜、乙醇、水中归一化荧光发射光谱如图4所示:SNAP-DAze在乙腈、氯仿、二甲基亚砜、乙醇、水光发射波长在490nm左右,且随着极性的变化荧光发射波长及荧光峰型均没有明显变化。
实施例2
SNAP-DAzo的合成
中间体N-(4-羟甲基)苄基-4-溴-5-硝基-1,8萘酰亚胺(BA-NBr)的合成:
4-溴-5-硝基-1,8-萘酰亚胺(1.00g,3.11mmol)溶于50mL乙醇中,并向其中加入4-氨甲基苄醇(2.00g,14.4mmol)。90℃下1h后,减压蒸馏除去溶剂,残余物经硅胶柱(石油醚:二氯甲烷=3:1-二氯甲烷:甲醇=200:1,V/V)分离得米白色固体880mg,产率66%。
中间体BA-DAzo的合成
将BA-NBr(100mg,0.23mmol)溶于20mL乙二醇甲醚中,并向其中加入四氢吡咯300mg。将反应液缓慢加热至100℃,并反应24h。减压除去乙二醇甲醚,残余物经硅胶柱分离残余物(二氯甲烷:甲醇=80:1,V/V),得黄色固体46mg,产率45%。其核磁谱图氢谱数据如下:
1H NMR(400MHz,CDCl3)δ8.34(d,J=8.7Hz,2H),7.51(d,J=7.9Hz,2H),7.25(d,J=8.4Hz,2H),6.66(d,J=8.7Hz,2H),5.37(s,2H),4.61(d,J=5.8Hz,2H),3.61(s,2H),3.40(s,2H),3.29(s,2H),2.71(s,2H),2.21(s,2H),1.98(dt,J=15.9,7.8Hz,4H),1.62(s,2H),1.50(t,J=6.0Hz,1H).
SNAP-DAzo的合成
将BA-DAzo(30mg,0.07mmol)、BG+(150mg,0.63mmol)、叔丁醇钾(150mg,0.91mmol)置于10mL史莱克瓶中,用氮气置换四次并加入6mL干燥DMF。室温下搅拌6h后减压除去溶剂,残余物经硅胶柱分离残余物(二氯甲烷:甲醇=25:1,V/V),得棕色固体23mg,产率60%。其核磁谱图氢谱数据如下:
1H NMR(400MHz,DMSO-d6)δ12.60(s,1H),8.27(d,J=8.4Hz,2H),7.78(s,1H),7.41(d,J=7.4Hz,2H),7.28(d,J=7.6Hz,2H),6.48(d,J=8.5Hz,2H),6.26(s,2H),5.42(s,2H),5.22(s,2H),4.62(d,J=5.8Hz,2H),3.71(s,2H),3.45(s,2H),3.39(s,2H),2.71(s,2H),2.21(s,2H),1.98(dt,J=15.9,7.8Hz,4H),1.62(s,2H).
经检测,其结构如上式SNAP-DAzo所示,其在水中的荧光发射波长为495nm,吸收波长为485nm左右,能有用于488nm激光激发。
实施例3
SNAP-DMEDA的合成
中间体N-(4-羟甲基)苄基-4-溴-5-硝基-1,8萘酰亚胺(BA-NBr)的合成:
4-溴-5-硝基-1,8-萘酰亚胺(1.00g,3.11mmol)溶于80mL乙醇中,并向其中加入4-氨甲基苄醇(1.00g,7.2mmol)。40℃下10h后,减压蒸馏除去溶剂,残余物经硅胶柱(石油醚:二氯甲烷=3:1-二氯甲烷:甲醇=200:1,V/V)分离得米白色固体573mg,产率43%。
BA-DMEDA的合成
将BA-NBr(150mg,0.34mmol)溶于10mL乙二醇甲醚中,并向其中加入N,N’-二甲基乙二胺200mg。将反应液缓慢加热至100℃,并反应24h。减压除去乙二醇甲醚,残余物经硅胶柱分离残余物(二氯甲烷:甲醇=90:1,V/V),得黄色固体88mg,产率65%。其核磁谱图氢谱与碳谱数据数据如下:
1H NMR(400MHz,DMSO-d6)δ8.25(d,J=8.6Hz,2H),7.26(d,J=8.3Hz,2H),7.22(d,J=8.3Hz,2H),6.86(d,J=8.7Hz,2H),5.18(s,2H),5.11(t,J=5.7Hz,2H),4.43(d,J=5.7Hz,2H),3.62(s,4H),3.12(s,6H).13C NMR(101MHz,DMSO-d6)δ163.65,155.89,141.53,137.07,132.95,127.72,126.86,116.10,110.62,110.37,63.14,57.85,42.52,41.66.
SNAP-DMEDA的合成
将BA-DMDEA(50mg,0.12mmol)、BG+(95mg,0.37mmol)、叔丁醇钾(100mg,0.89mmol)置于10mL史莱克瓶中,用氮气置换四次并加入4mL干燥DMF。室温下搅拌3h后减压除去溶剂,残余物经硅胶柱分离残余物(二氯甲烷:甲醇=25:1,V/V),得棕色固体27mg,产率40%。其核磁谱图氢谱数据如下:
1H NMR(400MHz,DMSO-d6)δ12.40(s,1H),8.25(d,J=8.6Hz,2H),7.79(s,1H),7.42(d,J=7.8Hz,2H),7.32(d,J=8.1Hz,2H),6.86(d,J=8.7Hz,2H),6.27(s,2H),5.42(s,2H),5.22(s,2H),3.63(s,4H),3.12(s,6H).
经检测,其结构如上式SNAP-DMEDA所示,其在水中的荧光发射波长为510nm左右,吸收波长为458nm左右,能快速特异性识别SNAP-tag。
实施例4
SNAP-DAC的合成
BA-DAC的合成
将BA-NBr(200mg,0.45mmol)溶于30mL乙二醇甲醚中,并向其中加入1,2-环己二胺400mg。将反应液缓慢加热至140℃,并反应12h。减压除去乙二醇甲醚,残余物经硅胶柱分离残余物(二氯甲烷:甲醇=80:1,V/V),得黄色固体93mg,产率48%。其核磁谱图氢谱与碳谱数据如下:
1H NMR(400MHz,DMSO-d6)δ8.05(d,J=8.6Hz,21H),7.56(s,2H),7.24(d,J=8.2Hz,2H),7.20(d,J=8.3Hz,2H),6.83(d,J=8.7Hz,2H),5.14(s,2H),5.10(t,J=5.7Hz,2H),4.42(d,J=5.7Hz,2H),3.16(d,J=9.2Hz,2H),2.19(d,J=12.0Hz,2H),1.72(d,J=7.3Hz,2H),1.49–1.18(m,4H).13C NMR(101MHz,DMSO-d6)δ163.39,154.73,141.39,137.45,134.91,133.49,127.79,126.77,110.69,107.60,106.41,63.16,59.47,42.36,32.06,23.62
SNAP-DAC的合成
将BA-DAC(40mg,0.09mmol)、BG+(95mg,0.37mmol)、叔丁醇钾(84mg,0.75mmol)置于10mL史莱克瓶中,用氮气置换四次并加入3mL干燥DMF。室温下搅拌10h后减压除去溶剂,残余物经硅胶柱分离残余物(二氯甲烷:甲醇=25:1,V/V),得棕色固体28mg,产率53%。其核磁谱图氢谱与碳谱数据如下:
1H NMR(400MHz,DMSO-d6)δ12.39(s,1H),8.05(d,J=8.6Hz,2H),7.79(s,1H),7.56(s,2H),7.40(d,J=8.0Hz,2H),7.30(d,J=8.0Hz,2H),6.84(d,J=8.7Hz,2H),6.27(s,2H),5.41(s,2H),5.17(s,2H),3.16(d,J=8.5Hz,2H),2.19(d,J=11.3Hz,2H),1.73(d,J=6.6Hz,2H),1.40–1.25(m,4H).13C NMR(101MHz,DMSO-d6)δ163.39,160.30,160.09,155.65,154.76,138.94,138.22,135.61,134.96,133.53,128.86,127.99,113.94,110.71,107.56,106.40,99.99,66.98,59.47,42.38,32.06,23.62.
其高分辨质谱数据如下:高分辨质谱理论值C31H29N8O3[M+H]+561.2363,实际值561.2380.
经检测,其结构如上式SNAP-DAC所示,其在水中的荧光发射波长为485nm,吸收波长为479nm左右,能够对SNAP-tag进行免洗标记。
将该类探针分别溶解于DMSO溶液中,配制成不同染料的2mM母液,根据需要配制成不同浓度测试溶液,以检测其荧光光谱变化及细胞内荧光成像。
实施例5
SNAP-DAC在PBS中与1μM SNAP-tag蛋白结合前后荧光光谱测试。取0.5μL SNAP-DAC母液溶于1mL PBS中进行荧光光谱测试,而后加入等浓度SNAP-tag蛋白半小时后进行荧光光谱测试。测试温度为37℃。
SNAP-DAC在PBS中与1μM SNAP-tag蛋白结合前后荧光光谱图如图5所示:SNAP-DAC在与SNAP-tag蛋白结合后逐渐被分散,荧光强度增加28倍。荧光发射波长及峰型没有明显变化。
实施例6
SNAP-DAze在PBS中与1μM SNAP-tag蛋白结合的动力学曲线测试。取0.5μL SNAP-DAze母液溶于1mL PBS中,而后加入等浓度蛋白后检测485nm处荧光强度,激发波长为440nm。
SNAP-DAze在PBS中与1μM SNAP-tag蛋白结合的动力学曲线图如图6所示:SNAP-DAze在加入SNAP-tag后逐渐与蛋白发生特异性结合,荧光迅速增强,荧光强度在2分钟内达到稳定。SNAP-DAze与SNAP-tag反应常数大于8000M-1S-1,t1/2=9s。
实施例7
探针分子在转染细胞中荧光共聚焦成像及超分辨成像。取0.5μL探针母液溶于1mL培养液中,而后置于37℃下孵育30分钟后进行荧光成像。
SNAP-DAze对转染的HEK293细胞荧光共聚焦成像图如图7所示:通过pSNAPf-H2B诱导HEK293细胞表达融合有SNAP-tag的H2B。1μM探针SNAP-DAze能够对融合有SNAP-tag的H2B进行特异性标记,从而达到对细胞核免洗成像,细胞核轮廓清晰。
SNAP-DAze对转染的HEK293细胞结构光照明成像图如图8所示:图8中通过pSNAPf-H2B诱导HEK293细胞表达融合有SNAP-tag的H2B。1μM探针SNAP-DAze能够对融合有SNAP-tag的H2B进行特异性标记,实现了SIM超分辨荧光成像。
SNAP-DAC对转染的HeLa细胞结构光照明成像图如图9所示::图9中通过pSNAPf-H2B诱导HeLa细胞表达融合有SNAP-tag的H2B。1μM探针SNAP-DAC能够对融合有SNAP-tag的H2B进行特异性标记,由于染料稳定性及亮度的提升,该探针实现了SIM超分辨成像。
Claims (8)
2.如权利要求1所述的一种488nm激发的免洗SNAP-tag探针的合成方法,其特征包含步骤如下:
(1)中间体N-(4-羟甲基)苄基-4-溴-5-硝基-1,8-萘酰亚胺的合成:
将4-溴-5-硝基-1,8-萘酐和4-氨甲基苄醇溶于无水乙醇中;将反应液加热至40-90℃,搅拌1-10h;将反应液泠却至室温后,减压除去溶剂后,硅胶柱分离,以体积比为800-100:1的二氯甲烷和甲醇为洗脱剂,减压除去溶剂得米白色固体N-(4-羟甲基)苄基-4-溴-5-硝基-1,8-萘酰亚胺;
(2)中间体N-(4-羟甲基)苄基-4,5-二脂肪胺基-1,8-萘酰亚胺的合成:
将N-(4-羟甲基)苄基-4-溴-5-硝基-1,8-萘酰亚胺溶于乙二醇甲醚中,并向其中加入脂肪环胺;将反应液缓慢升温至100-140℃,并在氮气保护下反应10-24h;减压除去溶剂,硅胶柱分离,以体积比为400-30:1的二氯甲烷和甲醇为洗脱剂,除去溶剂,得棕黄色固体N-(4-羟甲基)苄基-4,5-二脂肪胺基-1,8-萘酰亚胺;
(3)SNAP-tag探针的合成
将N-(4-羟甲基)苄基-4,5-脂肪胺基-1,8-萘酰亚胺、叔丁醇钾和2-氨基-6-(N-甲基)四氢吡咯基鸟嘌呤置于史莱克瓶中,氮气置换2-5次后加入干燥的N,N-二甲基甲酰胺;室温反应3-10h后,加压出去溶剂,硅胶柱分离,以体积比为100-10:1的二氯甲烷和甲醇为洗脱剂,除去溶剂得靶向SNAP-tag蛋白的荧光探针。
3.根据权利要求1所述的一系列488nm激发的免洗SNAP-tag探针的制备方法,其特征在于步骤(1)中:4-溴-5-硝基-1,8-萘酐:4-氨甲基苄醇的质量比为1:0.5-2;4-溴-5-硝基-1,8-萘酐的质量与无水乙醇的体积比为1:20-80g/mL。
4.根据权利要求1所述的一系列488nm激发的免洗SNAP-tag探针的制备方法,其特征在于步骤(2)中:N-(4-羟甲基)苄基-4-溴-5-硝基-1,8-萘酰亚胺与脂肪环胺的质量比为1:1-3;N-(4-羟甲基)苄基-4-溴-5-硝基-1,8-萘酰亚胺的质量与乙二醇甲醚的体积比为1:50-200g/mL;
脂肪环胺为氮丙啶、氮杂环丁烷、四氢吡咯或乙二胺衍生物。
5.根据权利要求1所述的一系列488nm激发的免洗SNAP-tag探针的制备方法,其特征在于步骤(3)中:N-(4-羟甲基)苄基-4,5-脂肪胺基-1,8-萘酰亚胺、叔丁醇钾、2-氨基-6-(N-甲基)四氢吡咯基鸟嘌呤的质量比为1:1-5:1-5;N-(4-羟甲基)苄基-4,5-脂肪胺基-1,8-萘酰亚胺的质量与N,N-二甲基甲酰胺的体积比为1:80-200g/mL。
6.一类如权利1所述的一系列488nm激发的免洗SNAP-tag探针在识别与检测SNAP-tag蛋白中的应用。
7.一种如权利1所述的488nm激发的免洗SNAP-tag探针在超分辨成像中的应用。
8.一种如权利1所述的488nm激发的免洗SNAP-tag探针在细胞、组织及活体内对目标蛋白的荧光成像的应用。
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