CN111333576A - 一类高稳定性的免洗Halo-tag探针及其合成方法和生物应用 - Google Patents
一类高稳定性的免洗Halo-tag探针及其合成方法和生物应用 Download PDFInfo
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- CN111333576A CN111333576A CN201811550971.8A CN201811550971A CN111333576A CN 111333576 A CN111333576 A CN 111333576A CN 201811550971 A CN201811550971 A CN 201811550971A CN 111333576 A CN111333576 A CN 111333576A
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Abstract
本发明提供了一类高稳定性的免洗Halo‑tag探针及其合成方法和生物应用，该探针为基于萘酰亚胺染料，设计合成的一类高稳定性的免洗Halo‑tag探针，其结构式如(1)所示，通过刚性的环胺对分子内扭转的抑制，该类Halo‑tag探针能够保持高的量子产率与稳定性，在水中量子产率达到0.80以上。此外，该类探针能够实现对活细胞内融合有Halo‑tag的目标蛋白特异性标记，实现免洗荧光成像。该类探针在单分子检测、超分辨荧光成像等领域有较好的应用前景。
Description
技术领域
本发明属于荧光标记领域，具体涉及一类高稳定性的免洗Halo-tag探针及其合成方法和生物应用。
背景技术
对目标蛋白融合携带有功能的标签蛋白的方式已经成为目前对蛋白检测、分析的重要技术。荧光蛋白的引入，能够实现目标蛋白的荧光标记，从而可以通过荧光信号的采集在原位、实时监测蛋白的分布、数量、功能等。但是，荧光蛋白的改造较为复杂、颜色种类并不丰富，这为许多研究工作者带来了巨大困难。SNAP-tag、Halo-tag等标签蛋白与有机小分子荧光探针的协同发展很大程度上克服了这种问题。通过颜色丰富、结构简小的有机小分子荧光探针合理设计，可以实现荧光分子与标签蛋白的特异性、共价键连接，从而达到对目标蛋白的稳定标记。
Halo-tag是紫红红球菌中脱卤素酶的变异体，能够以共价键形式与卤素代烷烃结合。原生脱卤素酶中，卤代脂肪烃在天冬氨酸(Asn41)与色氨酸(Trp107)的催化下(N-H-X形式的氢键)，受到天冬氨酸(Asp106)的羧基亲核进攻后生成烷基酯。烷基酯在天冬氨酸相邻的组氨酸(His272)催化下，进一步水解成醇。基于水解这一过程，将组氨酸(His272)突变为苯丙氨酸(Phe272)。当卤素脱去后生成的酯无法在苯丙氨酸存在下水解，即将反应停止在中间体酯，形成脱卤素酶与底物的稳定共价键。此外，由于Halo-tag并不是人源的酶经突变而来，故其在细胞内的特异性更高，与在细胞内非特异性标记几率很低。
基于有机小分子荧光染料的Halo-tag探针已经被广泛应用于蛋白标记中，但超分辨荧光成像技术及单分子检测技术的迅速发展对探针的稳定性、亮度提出了更高的要求。尤其在STED、STORM等超分辨技术中荧光染料的稳定性及信噪比直接影响成像的准确性与分辨率。目前，现有Halo-tag探针稳定性与亮度仍然不足，仍需要开发多波段的高稳定Halo-tag探针。
发明内容
本发明的目的之一是提供一类高稳定性的免洗Halo-tag探针，该类探针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类高稳定性的免洗Halo-tag探针的制备方法，该方法步骤简单、容易分离、原料价廉等优点。
本发明一类高稳定性的免洗Halo-tag探针，通过对萘酰亚胺分子内扭转的强力限制使分子达到荧光稳定性、亮度的大幅度提升，探针分子在水中量子产率最高可达0.80。
一类高稳定性的免洗Halo-tag探针，该类Halo-tag荧光探针具有如下结：
其中，R1与R2分别为H、
但若R1为H，则R2不为H，R3为C1-4烷基。
一类高稳定性的免洗Halo-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-(2-(2-羟基)-乙氧基)乙基-4-溴-5-硝基-1,8萘酰亚胺(OAN-NBr)的合成：
将4-溴-5-硝基-1,8-萘酐，二甘醇胺溶于无水乙醇中。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100～800:1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-2-(2-羟基)-乙氧基)乙基-4-溴-5-硝基-1,8-萘酰亚胺(OAN-NBr)；
(2)中间体N-(2-(2-羟基)-乙氧基)乙基-4-溴-1,8萘酰亚胺(OAN-Br)的合成
将4-溴-1,8-萘酐，二甘醇胺溶于无水乙醇中。将反应液加热至40-90℃，搅拌1-6h。将反应液泠却至室温后，过滤后干燥得灰白色固体N-(2-(2-羟基)-乙氧基)乙基-4-溴-1,8-萘酰亚胺(OAN-Br)。
(3)中间体N-(2-(2-羟基)-乙氧基)乙基-4,5-取代-1,8-萘酰亚胺的合成
将(1)或(2)中所得中间体，溶于乙二醇甲醚中，并向其中加入脂肪胺。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，以二氯甲烷和甲醇为洗脱剂，除去溶剂，得棕黄色固体N-(2-(2-羟基)-乙氧基)乙基-4,5-二取代-1,8-萘酰亚胺。
(4)Halo-tag探针的合成
将N-(2-(2-羟基)-乙氧基)乙基-4,5-取代-1,8-萘酰亚胺与NaH置于史莱克瓶中，并氮气置换2-5次。将1-碘-6-氯己烷溶于干燥的N,N-二甲基甲酰胺后，加入反应液中。室温下搅拌1-5h后，减压除去溶剂，硅胶柱分离，以体积比为400:1-100:1的二氯甲烷和甲醇为洗脱剂，除去溶剂得到靶向Halo-tag蛋白的荧光探针。
步骤(1)中，4-溴-5-硝基-1,8-萘酐与二甘醇胺的质量比为1:0.5-1；4-溴-5-硝基-1,8-萘酐的质量与乙醇的体积比为1:20-80；
步骤(2)中，4-溴-1,8-萘酐与二甘醇胺的质量比为1:0.5-1；4-溴-1,8-萘酐的质量与乙醇的体积比为1:20-80g/mL；
步骤(3)中，(1)或(2)中所得中间体与脂肪胺的质量比为1:1-3；(1)或(2)中所得中间体与脂肪胺的质量与乙二醇甲醚的体积比为10-20:1mg/mL；
脂肪胺为氮丙啶、氮杂环丁烷、四氢吡咯、乙二胺及环己二胺衍生物。
步骤(4)中，N-(2-(2-羟基)-乙氧基)乙基-4,5-取代-1,8-萘酰亚胺与NaH的质量比为5-10:1；N-(2-(2-羟基)-乙氧基)乙基-4,5-取代-1,8-萘酰亚胺的质量与1-碘-6-氯己烷体积比为0.5-1mg/μL；N-(2-(2-羟基)-乙氧基)乙基-4,5-取代-1,8-萘酰亚胺的质量与N,N-二甲基甲酰胺体积比为5-20:1mg/mL。
上述一类高稳定性的免洗Halo-tag探针对Halo-tag蛋白具有高度选择性，能够在活细胞等复杂环境中对Halo-tag进行特异性识别。
一类高稳定性的免洗Halo-tag探针在活细胞及组织内对Halo-tag及其融合蛋白成像领域的应用。
一类高稳定性的免洗Halo-tag探针在Halo-tag蛋白的识别与检测领域的应用。
一类高稳定性的免洗Halo-tag探针在单分子检测中的应用。
一类高稳定性的免洗Halo-tag探针在STED及SIM超分辨成像中的应用。本发明具有以下特点：
本发明涉及的Halo-tag探针拥有合成原料低价、方法简单、易分离等优点。
本发明涉及的Halo-tag探针分子在结合Halo-tag蛋白后荧光量子产率均大于0.80，亮度高、光稳定性好。
本发明涉及的Halo-tag探针能够对活细胞内Halo-tag蛋白进行特异性识别，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Halo-DAze的核磁谱图氢谱。
图2实施例2制备的Halo-DAC的核磁谱图氢谱。
图3实施例4制备的Halo-Aze的高分辨质谱。
图4实施例1制备的探针Halo-DAze在500W钨灯照射下495nm处荧光强度随时间变化图，选取商业罗丹明123、荧光素作为参比染料，横坐标为时间，纵坐标为归一化荧光强度。
图5实施例2制备的探针Halo-DAC在水中归一化的荧光激发光谱与荧光发射谱图，横坐标为波长，纵坐标为归一化强度，荧光染料的浓度为10μM。
图6实施例1制备的探针Halo-DAze在转染的pHALOf-H2B的HeLa细胞荧光共聚焦成像图，荧光探针的浓度为1μM。
图7实施例1制备的探针Halo-DAze在在转染的Hela细胞SIM超分辨荧光成像图，荧光探针的浓度为1μM。
图8实施例2制备的探针Halo-DAC在转染的pSNAPf-H2B的Hela细胞荧光共聚焦成像图，荧光探针的浓度为1μM。
图9实施例4制备的探针Halo-Aze在转染的pSNAPf-H2B的Hela细胞荧光共聚焦成像图，荧光探针的浓度为1μM。
具体实施方式
实施例1
Halo-DAze的合成
中间体N-(2-(2-羟基)-乙氧基)乙基-4-溴-5-硝基-1,8萘酰亚胺(OAN-NBr)的合成
4-溴-5-硝基-1,8-萘酰亚胺(2.00g,6.24mmol)溶于40mL乙醇中，并向其中滴加二甘醇胺(2.0g，19.0mmol)。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＝3:1-0:1，V/V)分离得米白色固体996mg，产率39％。其核磁谱图氢谱与碳谱数据如下：
1H NMR(400MHz,CDCl3/DMSO-d6)δ8.70(d,J＝7.8Hz,1H),8.50(d,J＝7.9Hz,1H),8.28(d,J＝7.9Hz,1H),8.13(d,J＝7.8Hz,1H),4.37(t,J＝5.9Hz,2H),4.21(s,1H),3.78(t,J＝5.9Hz,2H),3.58(s,4H).13C NMR(101MHz,CDCl3/DMSO)δ167.48,166.76,155.77,140.86,137.11,136.25,135.21,130.44,128.84,128.34,127.29,125.60,77.28,72.13,65.73.
其高分辨质谱数据如下：高分辨质谱理论值C16H14BrN2O6[M+H]+409.0035,实际值409.0031.
中间体N-(2-(2-羟基)-乙氧基)乙基-4,5-二氮杂环丁基-1,8萘酰亚胺(OAN-DAze)的合成
将OAN-Br(50mg，0.12mmol)溶于5mL乙二醇甲醚中，并向其中加入氮杂环丁烷150mg。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＝80:1，V/V)，得黄色固体25mg，产率52％。其核磁谱图氢谱与碳谱数据如下：
1H NMR(400MHz,CDCl3)δ8.37(d,J＝8.5Hz,2H),6.38(d,J＝8.5Hz,2H),4.42(d,J＝5.3Hz,2H),4.09(s,8H),3.83(t,J＝5.4Hz,2H),3.68(s,4H),2.42(s,4H).13C NMR(101MHz,CDCl3)δ164.74,155.77,133.31,133.16,109.85,107.84,106.39,72.18,68.91,61.94,55.25,38.99,16.89.
其高分辨质谱数据如下：高分辨质谱理论值C22H26N3O4[M+H]+396.1923,实测值396.1919.
Halo-DAze的合成
将Halo-OH(30mg，0.08mmol)与NaH(6mg，0.25mmol)置于10mL史莱克瓶中，用氮气置换三次。将15μL 1-碘-6-氯己烷溶于6mL干燥的DMF后，并加入反应液。室温下搅拌5h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＝200:1，V/V)，得棕色固体20mg，产率50％。实施例1制得的Halo-DAze核磁谱图氢谱如图1所示，氢谱与碳谱具体数据如下：
1H NMR(400MHz,CDCl3)δ8.37(d,J＝8.5Hz,2H),6.38(d,J＝8.5Hz,2H),4.41(t,J＝6.5Hz,2H),4.07(s,8H),3.78(t,J＝6.5Hz,2H),3.71–3.65(m,2H),3.60–3.54(m,2H),3.43(t,J＝6.6Hz,2H),2.43(s,4H),2.02(dd,J＝14.1,7.1Hz,2H),1.80–1.70(m,2H),1.54(dd,J＝13.8,6.9Hz,2H),1.41(dd,J＝15.2,7.8Hz,2H).13C NMR(101MHz,CDCl3)δ164.41,155.61,133.22,132.94,110.11,108.02,107.86,106.32,77.22,71.21,70.13,68.21,54.55,38.61,33.56,29.70,26.74,25.42,25.38.
其高分辨质谱数据如下：高分辨质谱理论值C28H37ClN3O4[M+H]+514.2473,实测值514.2477.
经检测，其结构如上式Halo-DAze所示，在水中的紫外吸收波长为484nm，荧光发射波长为493nm，能够用于Halo-tag的荧光标记。
实施例2
Halo-DAC的合成
中间体N-(2-(2-羟基)-乙氧基)乙基-4-溴-5-硝基-1,8萘酰亚胺(OAN-NBr)的合成
4-溴-5-硝基-1,8-萘酰亚胺(1.00g,3.12mmol)溶于80mL乙醇中，并向其中滴加二甘醇胺(0.5g，9.5mmol)。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＝3:1-0:1，V/V)分离得米白色固体677mg，产率53％。
N-十二烷基-4,5-(1,2-环己二胺)基-1,8萘酰亚胺(DDAN-DAC)的合成
将OAN-NBr(100mg，0.24mmol)溶于5mL乙二醇甲醚中，并向其中加入1,2-环己二胺100mg。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＝70:1，V/V)，得黄色固体34mg，产率35％。其核磁谱图氢谱与碳谱数据如下：
1H NMR(400MHz,DMSO-d6)δ8.04(d,J＝8.6Hz,21H),7.54(s,2H),6.83(d,J＝8.7Hz,2H),4.59(t,J＝4.7Hz,1H),4.15(t,J＝6.8Hz,2H),3.56(t,J＝6.8Hz,2H),3.46(s,4H),3.15(d,J＝9.4Hz,2H),2.20(d,J＝12.0Hz,2H),1.73(d,J＝7.2Hz,2H),1.43–1.22(m,4H).13C NMR(101MHz,DMSO d6)δ163.43,154.65,134.84,133.40,110.63,107.62,106.40,72.53,67.69,60.66,59.48,46.17,32.07,23.63.
其高分辨质谱数据如下：高分辨质谱理论值C22H26N3O4[M+H]+396.1923,实测值396.1919.
Halo-DAC的合成
将OAN-DAC(50mg，0.13mmol)与NaH(10mg，0.42mmol)置于10mL史莱克瓶中，用氮气置换三次。将50μL 1-碘-6-氯己烷溶于5mL干燥的DMF后，并加入反应液。室温下搅拌1h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＝100:1，V/V)，得棕色固体36mg，产率56％。实施例2制备的Halo-DAC的核磁批图氢谱如图2所示，氢谱与碳谱具体数据如下：
1H NMR(400MHz,CDCl3)δ8.25(d,J＝8.3Hz,2H),6.52(d,J＝8.3Hz,2H),5.00(s,2H),4.39(t,J＝6.2Hz,2H),3.81(t,J＝6.2Hz,2H),3.70(s,2H),3.58(d,J＝4.4Hz,2H),3.41(dd,J＝11.1,6.3Hz,2H),3.21(d,J＝7.7Hz,2H),2.13(d,J＝11.4Hz,2H),1.86(d,J＝7.5Hz,2H),1.79–1.64(m,2H),1.56–1.27(m,10H).13C NMR(101MHz,CDCl3)δ164.33,152.36,133.81,114.40,110.73,110.57,107.77,71.20,70.18,70.12,68.15,59.46,45.18,38.69,33.58,32.67,32.56,29.51,26.75,25.41,23.61.
其高分辨质谱数据如下：高分辨质谱理论值C28H37ClN3O4[M+H]+514.2473,实测值514.2477.
经检测，其结构如上式Halo-DAC所示，其荧光性能如下：
将Halo-DAC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，以检测其荧光光谱变化。
Halo-DAC在水中荧光激发与发射光谱测试。每次取20μL Halo-DAC母液加入4mL水中，配制成10μM的荧光染料测试液，进行荧光激发与发射光谱的测试。
Halo-DAC在水中荧光激发与发射光谱如图5所示：Halo-DAC在水光发射波长在490nm左右，激发波长在480nm，荧光半峰宽只有40nm。
实施例3
Halo-Aze的合成
中间体N-(2-(2-羟基)-乙氧基)乙基-4-溴-1,8-萘酰亚胺(OAN-Br)的合成
4-溴-1,8-萘酐(1.0g，3.6mmol)与二甘醇胺1000mg置于80mL乙醇中，并缓慢加热至40℃。10h后停止加热，将反应液冷却至室温。过滤得灰白色固体1.1g，产率85％。
OAN-Aze的合成
将OAN-Br(200mg，0.55mmol)溶于15mL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140℃，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＝100:1，V/V)，得黄色固体122mg，产率61％。其核磁谱图氢谱与碳谱数据如下：
Halo-Aze的合成
将OAN-Aze(50mg，0.15mmol)与NaH(10mg，0.40mmol)置于10mL史莱克瓶中，用氮气置换三次。将40μL 1-碘-6-氯己烷溶于5mL干燥的DMF后，并加入反应液。室温下搅拌2h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＝150:1，V/V)，得棕色固体22mg，产率32％。
经检测，其结构如上式Halo-Aze所示，在水中的紫外吸收波长在475nm，荧光发射波长为556nm。
实施例4
Halo-Aze的合成
中间体N-(2-(2-羟基)-乙氧基)乙基-4-溴-1,8-萘酰亚胺(OAN-Br)的合成
4-溴-1,8-萘酐(1.40g，5.0mmol)与二甘醇胺700mg置于28mL乙醇中，并缓慢加热至90℃。1h后停止加热，将反应液冷却至室温。过滤得灰白色固体1.5g，产率83％。其核磁谱图氢谱与碳谱数据如下：
1H NMR(400MHz,DMSO-d6)δ8.46(d,J＝7.2Hz,1H),8.40(d,J＝8.4Hz,1H),8.21(d,J＝7.9Hz,1H),8.11(d,J＝7.9Hz,1H),7.90(t,J＝7.9Hz,1H),4.59(s,1H),4.20(t,J＝6.5Hz,2H),3.66(t,J＝6.5Hz,2H),3.48(s,4H).13C NMR(101MHz,DMSO-d6)δ163.25,163.20,133.01,132.00,131.73,131.36,130.07,129.62,129.16,128.53,122.95,122.17,72.57,67.26,60.65.
OAN-Aze的合成
将OAN-Br(200mg，0.55mmol)溶于10mL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8h。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＝100:1，V/V)，得黄色固体135mg，产率72％。其核磁谱图氢谱与碳谱数据如下：
1H NMR(400MHz,CDCl3)δ8.55(dd,J＝7.3,0.8Hz,1H),8.39(d,J＝8.5Hz,1H),8.25(dd,J＝8.5,0.8Hz,1H),7.51(dd,J＝8.3,7.5Hz,1H),6.39(d,J＝8.5Hz,1H),4.51(t,J＝7.5Hz,4H),4.42(t,J＝5.6Hz,2H),3.85(t,J＝5.6Hz,2H),3.74–3.61(m,4H),2.76(s,1H),2.64–2.46(m,2H).13C NMR(101MHz,CDCl3)δ165.12,164.38,152.66,133.63,131.42,130.73,130.30,123.74,122.50,120.89,109.88,106.30,72.23,68.69,61.91,55.40,39.26,17.07.
其高分辨质谱数据如下：高分辨质谱理论值C19H21N2O4[M+H]+341.1501,实测值341.1492.
Halo-Aze的合成
将OAN-Aze(50mg，0.15mmol)与NaH(12mg，0.51mmol)置于10mL史莱克瓶中，用氮气置换三次。将30μL 1-碘-6-氯己烷溶于4mL干燥的DMF后，并加入反应液。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＝150:1，V/V)，得棕色固体22mg，产率32％。其核磁谱图氢谱与碳谱数据如下：
1H NMR(400MHz,CDCl3)δ8.55(d,J＝7.3Hz,1H),8.39(d,J＝8.5Hz,1H),8.25(d,J＝8.5Hz,1H),7.61–7.45(m,1H),6.40(d,J＝8.5Hz,1H),4.55–4.49(m,4H),4.42(t,J＝6.3Hz,2H),3.81(t,J＝6.4Hz,2H),3.73–3.64(m,2H),3.56(dd,J＝5.7,4.0Hz,2H),3.50(t,J＝6.8Hz,2H),3.40(td,J＝6.6,2.5Hz,2H),2.66–2.48(m,2H),2.01(dd,J＝14.3,7.2Hz,1H),1.78–1.68(m,1H),1.51(dq,J＝13.4,6.8Hz,2H),1.42–1.19(m,4H).
实施例4制备的Halo-Aze的高分辨质谱如图3所示，具体数据如下：高分辨质谱理论值C25H32ClN2O4[M+H]+459.2051,实测值459.2027.
经检测，其结构如上式Halo-Aze所示，在水中的紫外吸收波长在475nm，荧光发射波长为556nm。
将该类染料分别溶解于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
Halo-DAze在500W钨灯照射下荧光强度随时间变化测试。取20μL BuAN-DAze及商业染料母液加入4mL PBS(磷酸缓冲液，pH 7.4)中，而后加入螺纹比色皿中，正面放置于钨灯50cm处，分别采取0,,1,,2,3,4,6,8,10h为时间节点进行荧光光谱测试，并选取各自染料荧光发射峰值对时间进行曲线图。
Halo-DAze在500W钨灯照射下荧光强度随时间变化图如图4所示：Halo-DAze荧光强度在光照10h后仍然能够保持较高强度(96％)，而商业罗丹明123、荧光素等荧光强度均大幅降低，这说明Halo-DAze光稳定性极高，有望用于超分辨荧光成像。
实施例6
该类探针在转染细胞中荧光共聚焦成像及超分辨成像。取0.5μL Halo-tag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。激发波长为488nm，采集为500-550nm。
Halo-DAze对表达融合有Halo-tag的H2B的HeLa细胞的共聚焦成像图如图6所示：探针Halo-DAze能够对融合有Halo-tag的H2B进行特异性标记，达到对细胞核免洗成像，细胞核轮廓清晰，信噪比较高。
Halo-DAze对表达融合有Halo-tag的H2B的HeLa细胞的结构光照明荧光成像图如图7所示：1μM探针Halo-DAze通道染色效果图(采集500-550nm)探针能够对融合有Halo-tag的H2B进行特异性标记，染料稳定性的提升使其能够应用于SIM成像，达到更高的分辨率。
Halo-DAC对表达融合有Halo-tag的H2B的HeLa细胞的共聚焦荧光成像图如图8所示：探针Halo-DAC能够对融合有Halo-tag的H2B进行特异性标记，达到对细胞核免洗成像，细胞核轮廓清晰，信噪比较高。100倍物镜下细胞核内荧光强度达到细胞质内的6倍。
Halo-Aze对表达融合有Halo-tag的H2B的HeLa细胞的共聚焦成像图如图9所示：1μM探针Halo-Aze通道染色效果图(采集500-550nm)探针能够对融合有Halo-tag的H2B进行特异性标记，细胞核轮廓清晰。
Claims (12)
1.一类高稳定性的免洗Halo-tag探针，其特征在于其结构如下：
其中，R1与R2分别为H、、
若R1为H，则R2不为H，R3为C1-4烷基。
2.如权利要求1所述的一类高稳定性的免洗Halo-tag探针，特征在于：通过对萘酰亚胺分子内扭转的强力限制使分子达到荧光稳定性、亮度的大幅度提升，探针分子在水中量子产率最高可达0.80。
3.如权利要求1所述的一类高稳定性的免洗Halo-tag探针的合成方法，其特征包含步骤如下：
(1)中间体N-(2-(2-羟基)-乙氧基)乙基-4-溴-5-硝基-1,8萘酰亚胺(OAN-NBr)的合成：
将4-溴-5-硝基-1,8-萘酐，二甘醇胺溶于无水乙醇中。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100～800:1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-2-(2-羟基)-乙氧基)乙基-4-溴-5-硝基-1,8-萘酰亚胺(OAN-NBr)；
(2)中间体N-(2-(2-羟基)-乙氧基)乙基-4-溴-1,8萘酰亚胺(OAN-Br)的合成
将4-溴-1,8-萘酐，二甘醇胺溶于无水乙醇中；将反应液加热至40-90℃，搅拌1-6h；将反应液泠却至室温后，过滤后干燥得灰白色固体N-(2-(2-羟基)-乙氧基)乙基-4-溴-1,8-萘酰亚胺(OAN-Br)；
(3)中间体N-(2-(2-羟基)-乙氧基)乙基-4,5-取代-1,8-萘酰亚胺的合成
将(1)或(2)中所得中间体，溶于乙二醇甲醚中，并向其中加入脂肪胺；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，以二氯甲烷和甲醇为洗脱剂，除去溶剂，得棕黄色固体N-(2-(2-羟基)-乙氧基)乙基-4,5-二取代-1,8-萘酰亚胺；
(4)Halo-tag探针的合成
将N-(2-(2-羟基)-乙氧基)乙基-4,5-取代-1,8-萘酰亚胺与NaH置于史莱克瓶中，并氮气置换2-5次；将1-碘-6-氯己烷溶于干燥的N,N-二甲基甲酰胺后，加入反应液中；室温下搅拌1-5h后，减压除去溶剂，硅胶柱分离，以体积比为100～400:1的二氯甲烷和甲醇为洗脱剂，除去溶剂得到靶向Halo-tag蛋白的荧光探针。
4.根据权利要求3所述的一类高稳定性的免洗Halo-tag探针的合成方法，其特征在于步骤(1)中，4-溴-5-硝基-1,8-萘酐与二甘醇胺的质量比为1:0.5-1；4-溴-5-硝基-1,8-萘酐的质量与乙醇的体积比为1:20-80。
5.根据权利要求1所述的一类高稳定性的免洗Halo-tag探针的合成方法，其特征在于步骤(2)中，4-溴-1,8-萘酐与二甘醇胺的质量比为1:0.5-1；4-溴-1,8-萘酐的质量与乙醇的体积比为1:20-80g/mL。
6.根据权利要求1所述的一类高稳定性的免洗Halo-tag探针的合成方法，其特征在于步骤(3)中，(1)或(2)中所得中间体与脂肪胺的质量比为1:1-3；(1)或(2)中所得中间体与脂肪胺的质量与乙二醇甲醚的体积比为10-20:1mg/mL。
7.根据权利要求1所述的一类高稳定性的免洗Halo-tag探针的合成方法，其特征在于步骤(4)中，N-(2-(2-羟基)-乙氧基)乙基-4,5-取代-1,8-萘酰亚胺与NaH的质量比为5-10:1；
N-(2-(2-羟基)-乙氧基)乙基-4,5-取代-1,8-萘酰亚胺的质量与1-碘-6-氯己烷体积比为0.5-1mg/μL；
N-(2-(2-羟基)-乙氧基)乙基-4,5-取代-1,8-萘酰亚胺的质量与N,N-二甲基甲酰胺体积比为5-20:1mg/mL。
8.根据权利要求1所述的一类高稳定性的免洗Halo-tag探针的合成方法，其特征在于所述的脂肪胺为：氮丙啶、氮杂环丁烷、四氢吡咯、乙二胺或环己二胺衍生物。
9.如权利1所述的一类高稳定性的免洗Halo-tag探针在活细胞及组织内对Halo-tag及其融合蛋白成像领域的应用。
10.如权利1所述的一类高稳定性的免洗Halo-tag探针在Halo-tag蛋白的识别与检测领域的应用。
11.如权利1所述的一类高稳定性的免洗Halo-tag探针在单分子检测中的应用。
12.如权利1所述的一类高稳定性的免洗Halo-tag探针在STED及SIM超分辨成像中的应用。
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