CN110272437A - 可见光光控的snap蛋白标签类耐酸荧光分子开关及其合成 - Google Patents

可见光光控的snap蛋白标签类耐酸荧光分子开关及其合成 Download PDF

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CN110272437A
CN110272437A CN201810219060.0A CN201810219060A CN110272437A CN 110272437 A CN110272437 A CN 110272437A CN 201810219060 A CN201810219060 A CN 201810219060A CN 110272437 A CN110272437 A CN 110272437A
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徐兆超
祁清凯
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Abstract

本发明提供一种可见光光控的SNAP蛋白标签类耐酸荧光分子开关及其合成。该分子开关具体结构以3‑氨基或乙酰氨基取代的罗丹明螺酰胺为基本结构，将SNAP蛋白标签识别基团苄基鸟嘌呤(BG)共价连接到罗丹明螺酰胺荧光开关分子上，其结构式如(1)所示，将上述连接有SNAP标签的荧光开关探针特异性标记到细胞内的微管蛋白上，通过STORM技术重叠重构出微管蛋白的超分辨成像。本发明里可见光光控的SNAP蛋白标签类耐酸荧光分子开关不仅具有耐酸的性能，而且保留了可见光激活性能。因此这类可见光激活的耐酸性荧光开关染料可以应用于超分辨成像技术中不受酸性环境的干扰。

Description

可见光光控的SNAP蛋白标签类耐酸荧光分子开关及其合成
技术领域
本发明属于分子开关领域，具体涉及一种可见光光控的SNAP蛋白标签类耐酸荧光分子开关及其合成。
背景技术
近年来发展的一系列超高分辨率成像技术，其中基于单分子定位的光激活定位显微技术(PLAM)和随机光学重构显微技术(STORM或dSTORM)使光学显微镜的空间分辨达到了前所未有的高度。目前超分辨显微成像技术已经被广泛应用到生命科学研究中，然而尽管超分辨显微成像技术取得了巨大的进步，将荧光显微镜的空间分辨率推进到了20纳米，但是超分辨显微成像技术仍然面临诸多技术问题，其中之一的技术问题就是荧光染料的性能不够完美。基于单分子定位的超分辨显微成像技术需要染料不仅满足光稳定性好和荧光亮度高的优点，还需要其具有光致荧光“开-关”功能，这样才能够实现单分子的检测及定位。因此开发高荧光强度和光稳定性，具有光致荧光开关功能的新型荧光染料是超分辨荧光成像的迫切需求和当前热点。
开发生物成像用单分子定位超分辨荧光染料，目前最好的方法是在高荧光强度和光稳定性的染料中引入光开关功能。罗丹明类染料由于其突出的光性能，是目前超分辨中用的最多的一类染料，特别是硅罗丹明的性能更加突出，在超分辨中的应用受到广泛关注。罗丹明染料的荧光“明-暗”状态是基于酰胺螺环开关，传统罗丹明螺酰胺在紫外光辐照下，会由不发光的闭环结构变为强荧光发射的开环结构。S.W.Hell等人最早利用这一独特的光化学反应将罗丹明螺酰胺标记在固定的PtK2细胞的微丝骨架上，利用单分子定位技术实现了超分辨成像。但是，包括罗丹明螺酰胺在内，光开关分子在细胞内应用所面临的共同难题是需要紫外光作为“开-关”激发光，例如罗丹明螺酰胺需要用波长小于375nm的光来将闭环结构打开变为有荧光的开环结构，而紫外光会对细胞产生严重的光毒性，难以在活细胞中应用。为了改善激活光波长，W.E.Moerner等人将酰胺取代基修饰为较大的共轭体系，将吸收波长向长波长移动，首次将开关激发光延长到可见光区(>400nm)，实现了对细菌表面的三维超分辨荧光成像。由此，罗丹明螺酰胺在超分辨成像中表现出巨大潜力。
尽管罗丹明螺酰胺作为光激活染料已经可以用于超分辨荧光成像，但是这类染料分子还有一些不足还需要改进，首先是酸激活荧光干扰，通常情况下酸激活和光激活是两种都能够打开罗丹明酰胺螺环的方式。细胞内存在许多偏酸性的环境，如溶酶体，酸性蛋白等，当罗丹明螺酰胺染料使用于这些酸性环境中，其酸激活产生的荧光会严重干扰甚至导致光激活性能完全失效，因此在酸性环境中基于这类染料的荧光探针目前无法应用于超分辨荧光成像。另外，目前报道的绝大多数罗丹明螺酰胺都只能用紫外光(＜375nm)辐照来实现光激活荧光，而紫外光对生物体具有光毒性不利于活细胞超分辨成像。尽管S.W.Hell等人使用了长波长的双光子激光激活罗丹明螺酰胺的荧光并应用于超分辨成像，但是双光子激光功率要比单光子激光大几个数量级，这也会对成像的生物体造成不可修复的光损伤。W.E.Moerner等人开发的可见光激活染料最大吸收波长约为380nm，仅在405nm左右有一点吸收带边，因此也不能高效地利用405nm激光实现光激活。综上所述，开发一类具有耐酸性、可见光激活的罗丹明螺酰胺类荧光开关染料对于活细胞超分辨荧光成像显得尤为迫切和重要。
发明内容
本发明提供了一种可见光光控的SNAP蛋白标签类耐酸荧光分子开关及其合成，具体结构以3-氨基或乙酰氨基取代的罗丹明螺酰胺为基本结构单元，将SNAP蛋白标签识别基团苄基鸟嘌呤(BG)共价连接到罗丹明螺酰胺荧光开关分子上，将上述连接有SNAP标签的荧光开关探针特异性标记到细胞内的微管蛋白上，通过STORM技术重构出微管蛋白的超分辨图像。
本发明所述的一种SNAP蛋白标签类耐酸光控荧光分子开关，其结构式如下所示：
其中：R为H或COCH3。
本发明还提供了所述一种SNAP蛋白标签类耐酸光控荧光分子开关的合成方法，合成路线如下：
具体的合成方法步骤如下：
(1)将3-硝基罗丹明和三氯氧磷按物质的量比1:3-20溶解于1,2-二氯乙烷，升温至84℃回流，搅拌1-3小时后蒸除溶剂，将粗酰氯中间体溶于无水二氯甲烷，随后逐滴加入三乙胺和6-(4-氨基苯乙炔基)萘酐混合溶液，其中粗酰氯中间体、三乙胺和6-(4-氨基苯乙炔基)萘酐三者的物质的量比为1:0.5-2:1-2，室温搅拌8-24小时后减压蒸除溶剂，产物通过硅胶柱色谱分离提纯得到中间体P1；
(2)取步骤(1)中的产物P1与二水合氯化亚锡和浓盐酸分别按物质的量比1:1.5-5:0.1-2溶解于混合于无水乙醇中，回流搅拌5-8小时后减压蒸除溶剂，产物通过硅胶柱色谱分离提纯得到中间体P2；
(3)取步骤(2)中的产物P2与乙酰氯按物质的量比1:1-30混合于无水二氯甲烷中，室温搅拌反应0.5-3小时后，产物通过硅胶柱色谱分离提纯得到中间体P3；
(4)取步骤(2)中的产物P2与SNAP蛋白标签识别受体分子6-(4-氨甲基苄氧基)-9H-嘌呤-2-胺按物质的量比例1:1-10混合并置于无水乙醇中，升温至78℃回流2-10小时，最后通过硅胶柱色谱提纯即可得到产物P4；
(5)取步骤(3)中的产物P3与SNAP蛋白标签识别受体分子6-(4-氨甲基苄氧基)-9H-嘌呤-2-胺按物质的量比例1:1-10混合并置于无水乙醇中，升温至78℃回流2-10小时，最后通过硅胶柱色谱提纯即可得到产物P5。
一种SNAP蛋白标签类耐酸光控荧光分子开关的应用，基于其耐酸性或可见光激活的优点及蛋白标签标记应用在超分辨荧光成像或被作为荧光探针分子用于生物及化学物质的传感及检测等诸多领域。
本发明以3-氨基或乙酰氨基取代的罗丹明螺酰胺为基本结构，将SNAP蛋白标签识别基团苄基鸟嘌呤(BG)共价连接到罗丹明螺酰胺荧光开关分子上，将上述连接有SNAP标签的荧光开关探针特异性标记到细胞内的微管蛋白上，通过STORM技术重叠重构出微管蛋白的超分辨成像。本发明里可见光光控的SNAP蛋白标签类耐酸荧光分子开关不仅具有耐酸的性能，而且保留了可见光激活性能(如图6所示)。因此这类可见光激活的耐酸性荧光开关染料可以应用于超分辨成像技术中不受酸性环境的干扰。
附图说明
图1：实施例4制备得到产品P4的核磁氢谱；
图2：实施例5制备得到产品P5的核磁氢谱；
图3：P4和P5溶解在DMSO溶液中的紫外可见吸收光谱，浓度为10-5M；
图4：P4标记的U2OS细胞中微管的宽场荧光图像及对应的超分辨图像；
图5：P5标记的U2OS细胞中微管的宽场荧光图像及对应的超分辨图像；
图6：光诱导的耐酸性3-伯胺或仲胺取代的罗丹明螺酰胺分子的螺环及荧光开关的示意图。
具体实施方式
本发明给出了一种耐酸及可见光激活的能够靶向目标蛋白的染料的合成方法及其作为光激活荧光染料应用于微管蛋白的超分辨荧光成像技术领域。
实施例1
中间体分子(P1)合成路线和产物结构如下：
合成步骤及表征：将3-硝基罗丹明(2.92g，6mmol)和三氯氧磷(5.6mL，60mmol)置于1,2-二氯乙烷(150mL)，升温至84℃回流，搅拌2小时后蒸除溶剂得到暗紫红色油状液体。将粗酰氯产物溶于二氯甲烷(100mL)，随后逐滴加入三乙胺(3mL)和6-(4-氨基苯乙炔基)萘酐(1.88g，6mmol)混合溶液中，室温搅拌24小时后减压蒸除溶剂，残余物通过柱色谱(硅胶，二氯甲烷/乙酸乙酯，30:1v/v)分离得到黄色粉末产物P1(2.44g，52％)。对黄色粉末产物进行了核磁和质谱的表征：
1H NMR(400MHz,CDCl3)δ8.75(d,J＝8.4Hz,1H),8.65(d,J＝7.1Hz,1H),8.55(d,J＝7.7Hz,1H),7.92(d,J＝7.6Hz,1H),7.90–7.82(m,1H),7.75(d,J＝7.7Hz,1H),7.61(t,J＝7.8Hz,1H),7.46(d,J＝8.6Hz,2H),7.36(d,J＝7.6Hz,1H),7.11(d,J＝8.6Hz,2H),6.68(d,J＝8.5Hz,2H),6.40–6.24(m,4H),3.34(dd,J＝14.0,6.8Hz,8H),1.17(t,J＝7.0Hz,12H).13C NMR(101MHz,CDCl3)δ162.61,160.40,160.12,155.81,152.86,149.11,146.15,137.83,133.85,133.78,133.65,132.56,132.34,131.68,130.88,130.18,129.25,128.33,128.03,127.85,125.99,122.74,121.48,119.52,119.04,117.77,108.34,104.38,100.50,97.83,86.03,66.95,44.33,12.52.LC-MS(ESI):m/z:calcd:782.2740；found:783.2810[M+H]+。
经上述检测，鉴定其结构为P1所示。
实施例2
中间体分子(P2)合成路线和产物结构如下：
合成步骤及表征：将P1(1.56g，2mmol)，二水合氯化亚锡(1.80g，8mmol)和浓盐酸(9mL)置于无水乙醇(50mL)中，升温至78℃回流，搅拌8小时后减压蒸除溶剂，粗产物通过柱色谱(硅胶，乙酸乙酯/石油醚，1:3v/v)分离得到黄色固体P2(1.27g，85％)。对黄色固体产物进行了核磁和质谱的表征：
1H NMR(400MHz,CDCl3)δ8.75(d,J＝8.3Hz,1H),8.64(d,J＝7.2Hz,1H),8.54(d,J＝7.7Hz,1H),7.90(d,J＝7.7Hz,1H),7.85(t,J＝7.8Hz,1H),7.44(d,J＝8.5Hz,2H),7.22(t,J＝7.7Hz,1H),7.13(d,J＝8.6Hz,2H),6.76(d,J＝8.5Hz,2H),6.60(d,J＝8.0Hz,1H),6.37(d,J＝7.4Hz,1H),6.35–6.24(m,4H),5.44(s,2H),3.32(q,J＝7.0Hz,8H),1.16(t,J＝7.0Hz,12H)。13C NMR(101MHz,CDCl3)δ169.69,160.45,160.16,154.88,152.57,148.73,145.74,138.85,134.52,133.91,133.73,132.58,132.28,131.66,130.76,130.19,129.48,128.53,127.77,125.45,119.00,118.41,117.57,113.36,112.35,111.95,108.16,106.83,101.06,97.69,85.74,67.17,44.26,12.57。LC-MS(ESI)：m/z：计算值：752.2999；实验值：753.3073[M+H]+。
经上述检测，鉴定其结构为P2所示。
实施例3
中间体P3合成路线和产物结构如下：
合成步骤及表征：将P2(0.75g，1mmol)和乙酰氯(0.12g，1.5mmol)混合于二氯甲烷(10mL)，搅拌2小时后减压蒸除溶剂，粗产物通过柱色谱(硅胶，乙酸乙酯/石油醚，1:3v/v)分离得到黄色粉末产物P3(0.76g，96％)。对黄色粉末产物进行了核磁和质谱的表征：
1H NMR(400MHz,CDCl3)δ10.58(s,1H),8.75(d,J＝8.2Hz,1H),8.65(d,J＝7.2Hz,1H),8.55(d,J＝7.7Hz,1H),8.51(d,J＝8.2Hz,1H),7.92(d,J＝7.7Hz,1H),7.90–7.82(m,1H),7.56–7.43(m,3H),7.00(d,J＝8.5Hz,2H),6.81(d,J＝7.6Hz,1H),6.67(d,J＝8.8Hz,2H),6.37–6.26(m,4H),3.33(q,J＝7.0Hz,8H),2.31(s,3H),1.17(t,J＝7.0Hz,12H)。13CNMR(100MHz,CDCl3)δ169.31,168.94,160.38,160.11,153.44,152.94,148.99,137.79,137.43,134.99,133.82,133.77,132.55,132.43,131.70,130.91,130.20,129.24,128.48,127.84,126.34,119.68,119.10,118.08,117.81,115.18,108.26,105.41,100.48,97.77,86.08,67.99,44.32,24.98,12.55。LC-MS(ESI)：m/z：计算值：794.3104；实验值：795.3177[M+H]+。
经上述检测，鉴定其结构为P3所示。
实施例4
分子(P4)合成路线和产物结构如下：
合成步骤及表征：P2(0.37g，0.5mmol)和BG-NH2(0.19g，1.5mmol)混合于无水乙醇(10mL)，升温至78℃回流，搅拌10小时后减压蒸除溶剂。粗产物通过柱色谱(硅胶，乙酸乙酯/石油醚，1:2v/v)分离提纯得到黄色粉末产物P4(0.42g，84％)。
对黄色粉末产物进行了核磁氢谱(如图1)、核磁碳谱和高分辨质谱表征。1H NMR(400MHz,DMSO)δ12.53(s,1H),8.75(d,J＝8.4Hz,1H),8.56(d,J＝7.3Hz,1H),8.46(d,J＝7.7Hz,1H),8.01(d,J＝7.7Hz,1H),7.99–7.90(m,1H),7.76(s,1H),7.58(d,J＝8.7Hz,2H),7.40(dd,J＝21.1,8.1Hz,3H),7.32–7.09(m,3H),6.63(dd,J＝10.1,6.4Hz,2H),6.46(s,2H),6.37(dd,J＝8.9,2.4Hz,1H),6.32(d,J＝2.4Hz,1H),6.26(s,2H),6.13–5.96(m,2H),5.68(dd,J＝8.9,2.9Hz,1H),5.41(s,2H),5.25(s,2H),3.29(dd,J＝14.2,7.0Hz,8H),2.54(s,2H),1.06(t,J＝6.9Hz,12H)。LC-MS(ESI)：m/z：计算值：1004.4122，实验值：503.2012[M+2H]2+/2。经上述检测，鉴定其结构为P4所示。
产物P4溶于DMSO中配成溶液(浓度为10-5M)，测试溶液的紫外可见吸收光谱，最大吸收波长为400nm左右(图3)。
向P4的DMSO溶液(浓度为10-5M)中加入2.3μL三氟乙酸(1000eq)，测定加酸前后时间分辨的紫外可见吸收光谱，结果显示P4的最大吸收波长处的吸光度没有随着酸化时间的增长而增加，表明P4分子具有耐酸的特性。
将SNAP-微管蛋白融合质粒通过标准的基因工程方法转染U2OS细胞，培养24小时后取10uL P4的DMSO溶液(浓度为10-3M)加入1mL培养基中染色培养U2OS细胞1小时，随后弃去培养基溶液并加入1mLPBS缓冲溶液清洗掉未染色的多余染料，反复清洗5-8次后，加入1mL4％的多聚甲醛PBS溶液固定细胞10分钟后，用PBS溶液洗涤3-4次后即可置于STORM显微镜下，选择常用的405nm激光作为激活光，561nm激光作为激发光，通过随机光激活以及重叠重构技术即可得到超分辨图像，如图4所示，超分辨成像得到的微管图像比宽场荧光图像分辨率大大提高。
实施例5
分子(P5)合成路线和产物结构如下：
合成步骤及表征：P3(0.39g，0.5mmol)和BG-NH2(0.19g，1.5mmol)混合于无水乙醇(10mL)，升温至78℃回流，搅拌10小时后减压蒸除溶剂。粗产物通过柱色谱(硅胶，乙酸乙酯/石油醚，1:2v/v)分离提纯得到黄色粉末产物P5(0.44g，84％)。
对黄色粉末产物进行了核磁氢谱(如图2)、核磁碳谱和高分辨质谱表征。1H NMR(400MHz,DMSO)δ12.40(s,1H),10.42(s,1H),8.71(d,J＝8.5Hz,1H),8.55(d,J＝6.9Hz,1H),8.45(d,J＝7.7Hz,1H),8.33(d,J＝8.2Hz,1H),7.99(d,J＝7.7Hz,1H),7.92(t,J＝7.8Hz,1H),7.77(s,1H),7.60(d,J＝8.6Hz,2H),7.52(t,J＝7.9Hz,1H),7.43(d,J＝8.2Hz,2H),7.38(d,J＝8.2Hz,2H),7.09(d,J＝8.6Hz,2H),6.71–6.64(m,3H),6.39(dd,J＝9.0,2.4Hz,2H),6.32(d,J＝2.3Hz,2H),6.25(s,1H),5.75(s,1H),5.42(s,2H),5.24(s,2H),3.29(dd,J＝13.9,6.8Hz,8H),2.24(s,3H),1.06(t,J＝6.9Hz,12H)。13C NMR(101MHz,DMSO)δ169.90,169.09,164.28,163.98,160.59,155.01,153.07,149.54,138.74,138.07,137.97,136.04,133.30,132.57,131.89,131.31,129.61,129.30,128.63,128.47,127.30,126.19,123.45,122.80,119.92,118.62,118.20,115.43,109.27,105.82,99.34,98.16,87.62,67.76,55.90,44.62,25.61,13.38。LC-MS(ESI)：m/z：计算值：1046.4228；实验值：524.2137[M+2H]2+/2。经上述检测，鉴定其结构为P5所示。
将产物P5溶于DMSO中配成溶液(浓度为10-5M)，测试溶液的紫外可见吸收光谱，其最大吸收波长为400nm左右(图3)。
向P5的DMSO溶液(浓度为10-5M)中加入2.3μL三氟乙酸(1000eq)，测定加酸前后时间分辨的紫外可见吸收光谱，结果显示P5的最大吸收波长处的吸光度没有随着酸化时间的增长而增加，表明P5分子仍然具有耐酸的特性。
将SNAP-微管蛋白融合质粒通过标准的基因工程方法转染U2OS细胞，培养24小时后取10uL P5的DMSO溶液(浓度为10-3M)加入1mL培养基中染色培养U2OS细胞1小时，随后弃去培养基溶液并加入1mLPBS缓冲溶液清洗掉未染色的多余染料，反复清洗5-8次后，加入1mL4％的多聚甲醛PBS溶液固定细胞10分钟后，用PBS溶液洗涤3-4次后即可置于STORM显微镜下，选择常用的405nm激光作为激活光，561nm激光作为激发光，通过随机光激活以及重叠重构技术即可得到超分辨图像，如图5所示，超分辨成像得到的微管图像比宽场荧光图像分辨率大大提高。

Claims (3)

1.一种可见光光控的SNAP蛋白标签类耐酸荧光分子开关，其特征在于：其结构式如下所示，
其中：R为H或COCH3。
2.根据权利要求1所述可见光光控的SNAP蛋白标签类耐酸荧光分子开关的合成方法，其特征在于：其合成的具体方法如下，
(1)将3-硝基罗丹明和三氯氧磷按物质的量比1:3-20溶解于1,2-二氯乙烷，升温至84℃回流，搅拌1-3小时后蒸除溶剂，将粗酰氯中间体溶于无水二氯甲烷，随后逐滴加入三乙胺和6-(4-氨基苯乙炔基)萘酐混合溶液，其中粗酰氯中间体、三乙胺和6-(4-氨基苯乙炔基)萘酐三者的物质的量比为1:0.5-2:1-2，室温搅拌8-24小时后减压蒸除溶剂，产物通过硅胶柱色谱分离提纯得到中间体P1；
(2)取步骤(1)中的产物P1与二水合氯化亚锡和浓盐酸分别按物质的量比1:1.5-5:0.1-2溶解于混合于无水乙醇中，回流搅拌5-8小时后减压蒸除溶剂，产物通过硅胶柱色谱分离提纯得到中间体P2；
(3)取步骤(2)中的产物P2与乙酰氯按物质的量比1:1-30混合于无水二氯甲烷中，室温搅拌反应0.5-3小时后，产物通过硅胶柱色谱分离提纯得到中间体P3；
(4)取步骤(2)中的产物P2与SNAP蛋白标签识别受体分子6-(4-氨甲基苄氧基)-9H-嘌呤-2-胺按物质的量比例1:1-10混合并置于无水乙醇中，升温至78℃回流2-10小时，最后通过硅胶柱色谱提纯即可得到产物P4；
(5)取步骤(3)中的产物P3与SNAP蛋白标签识别受体分子6-(4-氨甲基苄氧基)-9H-嘌呤-2-胺按物质的量比例1:1-10混合并置于无水乙醇中，升温至78℃回流2-10小时，最后通过硅胶柱色谱提纯即可得到产物P5。
3.根据权利要求1所述的可见光光控的SNAP蛋白标签类耐酸荧光分子开关在超分辨荧光成像、分子探针及荧光传感及其领域的应用。
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