CN111333649A - 一种基于SNAP-tag技术的细胞膜荧光探针及其制备和应用 - Google Patents
一种基于SNAP-tag技术的细胞膜荧光探针及其制备和应用 Download PDFInfo
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Abstract
本发明提供了一种基于SNAP‑tag技术的细胞膜荧光探针及其制备和应用,该荧光探针基于罗丹明6G小分子荧光染料、连接SNAP蛋白标签BG基团,其结构式如(1)所示。本发明涉及的荧光探针具有小分子荧光染料优异的光稳定性、荧光亮度的优势,同时与现有的利用小分子染料双亲性(亲水亲油性)识别细胞膜不同,该探针可以专一的共价键结合细胞膜SNAP蛋白,结合更为稳固,可长时间成像观测;且其合成步骤简单、原料廉价易得,在细胞膜成像领域中有着巨大的应用前景。
Description
技术领域
本发明属荧光成像领域,具体涉及一种基于SNAP-tag技术的细胞膜荧光探针及其制备和应用。
背景技术
基于有机小分子荧光染料的SNAP-tag蛋白标签技术,具有独特的优点:蛋白标签与底物的反应速度快且特异性高;通过共价键的结合蛋白标记的稳定性好,即使在体外分析SDS-PAGE的变性条件下仍可稳定标记;通过模块化的设计将标签特异性底物BG基团与功能染料结合,灵活性高;便于衍生多样化的荧光染料,通用性强,可满足各种荧光成像研究的需求。目前,SNAP-tag在细胞内蛋白质标记、体外分析及靶向荧光的检测的研究中获得了非常广泛的应用。尽管如此,这种方法也存在些许不足:为了达到更高的信号强度、提高信噪比,需要充分的洗涤去除非特异性染色,这也限制了其在活细胞实时成像中的应用。
细胞膜又称质膜,其主要是由磷脂构成的富有弹性的半透性膜,在生命活动过程中占据着十分重要的位置,如选择性地交换物质,吸收营养物质,排出代谢废物,分泌与运输蛋白质。但是目前关于细胞膜成像的荧光探针主要以分子双亲性(亲水亲油性)插入到细胞膜上,无法人为控制其细胞膜的渗透性,进行长时间的成像观察。小分子荧光染料的SNAP-tag蛋白标签技术则可以完美的解决了这一缺陷,有望实现对细胞复杂的生命过程中细胞膜变化的实时追踪。因此,开发设计出基于有机小分子荧光染料的SNAP-tag蛋白标签技术的细胞膜荧光探针显得十分迫切。
发明内容
本发明的目的在于提供一种基于SNAP-tag技术的细胞膜荧光探针及其制备和应用,该细胞膜荧光探针光稳定性高、亮度高,以及较高的细胞非渗透性,可以长时间进行细胞膜的成像研究。
本发明一种基于SANP-tag技术的细胞膜荧光探针,其以罗丹明6G为荧光母体,对氨基鸟嘌呤为识别基团,具体的化学结构如下:
其中:n=0,1,2,3或4。
本发明的荧光探针由于其较高的非渗透性,较难以进入细胞中,同时其能够特异性的与细胞膜表面转染的SNAP蛋白相结合,从而在细胞膜上聚集,实现快速、清晰的细胞膜蛋白成像的目的。
一种基于SANP-tag技术的细胞膜荧光探针的制备方法,其合成路线如下:
具体合成步骤如下:
(1)中间体罗丹明6G-羧酸的合成
罗丹明6G溶于乙醇中,加入强碱试剂的水溶液,回流反应5-10h后,减压除去有机溶剂,硅胶柱分离,洗脱剂为体积比20-5:1的二氯甲烷和甲醇,减压除去溶剂后得中间体。
(2)中间体罗丹明6G-琥珀酰亚胺羧酸酯的合成
将罗丹明6G-羧酸,N-羟基琥珀酰亚胺,缩合试剂1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐依次加入到二氯甲烷中,室温搅拌10-16h,减压除去溶剂得粗产品。
(3)中间体罗丹明6G-甲胺烷基酸得合成
将罗丹明6G-琥珀酰亚胺羧酸酯粗产品、甲胺丁酸盐酸盐、碳酸钾依次加入到乙腈中,室温搅拌过夜;减压除去溶剂,硅胶柱分离,洗脱剂为体积比50-10:1的二氯甲烷和甲醇,减压除去溶剂后得紫色固体。
(4)探针的合成
将罗丹明6G-甲胺烷基酸、氨基鸟嘌呤、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐、1-羟基苯并三唑、碳酸钾依次加入到N,N-二甲基甲酰胺中,室温搅拌两天;减压除去溶剂,硅胶柱分离,洗脱剂为体积比40-3:1的二氯甲烷和甲醇,减压除去溶剂后得紫色固体。
步骤(1)中:罗丹明6G与强碱的质量比为1:0.2-0.4,水与乙醇的体积比为1:5-8,罗丹明6G与乙醇的质量与体积比为1:20-40g/mL。
步骤(2)中:罗丹明6G-羧酸与N-羟基琥珀酰亚胺、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐的质量比为1:0.2-0.4:0.6-0.8,罗丹明6G-羧酸与二氯甲烷的质量与体积比为1:40-60g/mL。
步骤(3)中:罗丹明6G-琥珀酰亚胺羧酸酯与甲胺烷基酸盐酸盐、碳酸钾的质量比为1:0.3-0.6:0.2-0.4,罗丹明6G-琥珀酰亚胺羧酸酯与乙腈的质量与体积比为10-25:1mg/mL。
步骤(4)中:罗丹明6G-甲胺丁酸、氨基鸟嘌呤、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐、1-羟基苯并三唑、碳酸钾的质量比为1:0.4-1:0.4-0.6:0.3-0.5:1-2,罗丹明6G-甲胺丁酸与N,N-二甲基甲酰胺的质量与体积比为5-10:1mg/mL。
步骤(1)中:强碱试剂为KOH、NaOH、LiOH,反应时间以点板检测为基准,直到原料基本反应完全就停止反应。
步骤(2)中:缩合试剂为DCC,EDC,HOBt中的一种或多种。
本发明提供了一种基于SANP-tag技术的细胞膜荧光探针,该荧光探针具有光稳定性高、荧光亮度高、非渗透性高等特点,可以进行快速、免洗的细胞膜成像。
本发明一种基于SANP-tag技术的小分子细胞膜荧光探针的制备方法,该方法具有原料低廉、操作简便等特点。
本发明涉及的荧光探针具有小分子荧光染料优异的光稳定性、荧光亮度的优势,同时与现有的利用小分子染料双亲性(亲水亲油性)识别细胞膜不同,该探针可以专一的共价键结合细胞膜SNAP蛋白,结合更为稳固,可长时间成像观测;且其合成步骤简单、原料廉价易得,在细胞膜成像领域中有着巨大的应用前景。
附图说明
图1实施例得到的探针Rho6G-SNAP高分辨质谱;
图2实施例得到的探针Rho6G-SNAP在DMSO中的紫外吸收图,横坐标为波长,纵坐标为荧光强度,荧光探针的浓度为10μM;
图3实施例得到的探针Rho6G-SNAP在DMSO中的荧光发射图,横坐标为波长,纵坐标为荧光强度,荧光探针的浓度为10μM;
图4实施例得到的探针Rho6G-SNAP在转染SNAP蛋白的细胞膜成像图,成像浓度为0.5μM。
具体实施方法
实施例1
用于细胞膜成像的小分子荧光探针Rho6G-SNAP的合成方法。
中间体罗丹明6G-羧酸的合成:
将1g的罗丹明6G溶于30mL乙醇中,300mg的氢氧化钠溶于5mL水中后缓慢加入到反应混合物中,加热搅拌回流。6h后,点板监测,待反应原料反应完全,停止反应。旋蒸除去溶剂,硅胶柱分离,用二氯甲烷:甲醇=5:1(体积比)作为洗脱剂,减压除去溶剂得紫色固体700mg,产率80%。其核磁谱图氢谱数据如下:
1H NMR(400MHz,CD3OD),δ8.22(d,J=6.4Hz,1H),7.80(m,2H),7.36(d,J=6.8Hz,1H),6.89(d,J=10.4Hz,4H),3.50(q,J=7.2Hz,4H),2.15(s,6H),1.35(t,J=6.8Hz,6H).
中间体罗丹明6G-琥珀酰亚胺羧酸酯的合成:
称取500mg的罗丹明6G-羧酸和345mg的1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐于25mL的二氯甲烷中,再加入N-羟基琥珀酰亚胺125mg,在室温下搅拌12h。停止反应,旋干溶剂得粗产品。其核磁谱图氢谱数据如下:
1H NMR(400MHz,DMSO-d6)δ8.27(d,J=7.7Hz,1H),7.91(t,J=6.9Hz,1H),7.84(t,1H),7.79(s,1H),7.46(d,J=7.5Hz,1H),6.93(s,1H),6.78(s,2H),3.53–3.46(m,J=13.5,6.8Hz,2H),2.50(s,3H),2.10(s,2H),1.27(t,J=7.2Hz,3H).
中间体罗丹明6G-甲胺丁酸的合成:
称取288mg的罗丹明6G-琥珀酰亚胺羧酸酯和80mg碳酸钾溶解到15mL的乙腈中,再向混合液中加入4-甲氨基丁酸盐酸盐130mg,室温搅拌过夜。减压除去有机溶剂,硅胶柱分离(200-300目),用二氯甲烷:甲醇=40-10:1(体积比)为洗脱剂,减压除去有机溶剂得紫色固体199mg,产率69%。其核磁谱图氢谱数据如下:
1H NMR(400MHz,DMSO-d6)δ7.88(s,2H),7.77–7.70(m,2H),7.69–7.64(m,1H),7.55–7.49(m,1H),6.94(s,2H),6.86(s,2H),3.48(d,J=6.9Hz,4H),3.14–3.06(m,2H),2.90(s,3H),2.15(s,6H),1.68(t,J=7.3Hz,2H),1.25(t,J=7.1Hz,8H).
荧光探针Rho6G-SNAP的合成:
依次称取羧基罗丹明6G-甲胺丁酸103mg,氨基鸟嘌呤56mg,1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐46mg,1-羟基苯并三唑32mg,碳酸钾110mg,溶于DMF中室温搅拌两天。减压除去DMF后,硅胶柱分离,用二氯甲烷:甲醇=30-5:1(体积比)为洗脱剂,旋干溶剂得紫色固体66mg,产率42%。其核磁谱图氢谱数据如下:
1H NMR(400MHz,CD3OD)δ8.08(s,0H),8.08(s,0H),8.08(s,1H),8.08(s,1H),7.98(d,J=8.1Hz,2H),7.66(d,J=8.1Hz,1H),7.53(d,J=7.6Hz,1H),7.29(d,J=8.0Hz,1H),7.02(d,J=9.1Hz,2H),6.78(d,J=5.5Hz,1H),5.59(d,J=7.7Hz,1H),4.24(s,1H),3.49–3.40(m,3H),3.22(s,1H),2.18(s,3H),2.09–2.00(m,1H),1.74–1.68(m,1H),0.91(d,J=6.8Hz,3H).
实施例1中得到的目标探针Rho6G-SNAP的高分辨质谱如图1所示,具体数据为:高分辨质谱理论值C44H48N9O4[M+H]+766.3829,实测值766.38424.
经检测,其结构如上式Rho6G-SNAP所示,可以进行细胞膜成像,其光性能如下:
染料分子Rho6G-SNAP在二甲亚砜中的吸收与发射光谱测试。取实施例1中得到的罗丹明类染料分子Rho6G-SNAP,溶解于DMSO中配置成2mM的母液。取20μL的母液溶于4mLDMSO溶液中,进行吸收和发射光谱的测试。
Rho6G-SNAP在二甲亚砜中的吸收与发射谱图分别如图2、图3所示:图2和图3分别为实施例1中得到的荧光探针Rho6G-SNAP在DMSO中的吸收和荧光发射图,其中吸收为539nm,发射波长为568nm。
实施例2
用于细胞膜成像的小分子荧光探针Rho6G-SNAP的合成方法。
中间体罗丹明6G-羧酸的合成:
将1g的罗丹明6G溶于20mL乙醇中,200mg的氢氧化钠溶于5mL水中后缓慢加入到反应混合物中,加热搅拌回流。5h后,点板监测,待反应原料反应完全,停止反应。旋蒸除去溶剂,硅胶柱分离,用二氯甲烷:甲醇=5:1(体积比)作为洗脱剂,减压除去溶剂得紫色固体600mg,产率69%。
中间体罗丹明6G-琥珀酰亚胺羧酸酯的合成:
称取500mg的罗丹明6G-羧酸和300mg的1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐于20mL的二氯甲烷中,再加入N-羟基丁二酰亚胺100mg,在室温下搅拌10h。停止反应,旋干溶剂得粗产品。
中间体罗丹明6G-甲胺丁酸的合成:
称取200mg的罗丹明6G-丁二酰亚胺羧酸酯和40mg碳酸钾溶解到10mL的乙腈中,再向混合液中加入4-甲氨基丁酸盐酸盐60mg,室温搅拌过夜。减压除去有机溶剂,硅胶柱分离,用二氯甲烷:甲醇=40-10:1(体积比)为洗脱剂,减压除去有机溶剂得紫色固体120mg,产率60%。
荧光探针Rho6G-SNAP的合成:
依次称取羧基罗丹明6G-甲胺丁酸100mg,氨基鸟嘌呤40mg,1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐40mg,1-羟基苯并三唑30mg,碳酸钾100mg,溶于10mL的DMF中室温搅拌两天。减压除去DMF后,硅胶柱分离,用二氯甲烷:甲醇=30-5:1(体积比)为洗脱剂,旋干溶剂得紫色固体65mg,产率42%。
经检测,其结构如上式Rho6G-SNAP所示,可以进行细胞膜成像,其光性能如下:
荧光探针Rho6G-SNAP在DMSO中的吸收和荧光发射波长分别为:吸收波长为539nm,发射波长为568nm。
实施例3
用于细胞膜成像的小分子荧光探针Rho6G-SNAP的合成方法。
中间体罗丹明6G-羧酸的合成:
将1g的罗丹明6G溶于40mL乙醇中,400mg的氢氧化钠溶于5mL水中后缓慢加入到反应混合物中,加热搅拌回流。6h后,点板监测,待反应原料反应完全,停止反应。旋蒸除去溶剂,硅胶柱分离,用二氯甲烷:甲醇=5:1(体积比)作为洗脱剂,减压除去溶剂得紫色固体672mg,产率77%。
中间体罗丹明6G-琥珀酰亚胺羧酸酯的合成:
称取500mg的罗丹明6G-羧酸和400mg的1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐于30mL的二氯甲烷中,再加入N-羟基丁二酰亚胺200mg,在室温下搅拌16h。停止反应,旋干溶剂得粗产品。
中间体罗丹明6G-甲胺丁酸的合成:
称取200mg的罗丹明6G-丁二酰亚胺羧酸酯和80mg碳酸钾溶解到20mL的乙腈中,再向混合液中加入4-甲氨基丁酸盐酸盐120mg,室温搅拌过夜。减压除去有机溶剂,硅胶柱分离,用二氯甲烷:甲醇=40-10:1(体积比)为洗脱剂,减压除去有机溶剂得紫色固体142mg,产率71%。
荧光探针Rho6G-SNAP的合成:
依次称取羧基罗丹明6G-甲胺丁酸100mg,氨基鸟嘌呤100mg,1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐60mg,1-羟基苯并三唑50mg,碳酸钾200mg,溶于20mL的DMF中室温搅拌两天。减压除去DMF后,硅胶柱分离,用二氯甲烷:甲醇=30-5:1(体积比)为洗脱剂,旋干溶剂得紫色固体70mg,产率44%。
经检测,其结构如上式Rho6G-SNAP所示,可以进行细胞膜成像,其光性能如下:
荧光探针Rho6G-SNAP在DMSO中的吸收和荧光发射波长分别为:吸收波长为539nm,发射波长为568nm。
实施例4
Rho6G-SNAP对活细胞染色后荧光共聚焦成像测试。取实施例1得到的荧光探针Rho6G-SNAP母液加入到转染有细胞膜SNAP蛋白的HeLa细胞培养皿中,探针终浓度为0.5μM。孵育5min后进行共聚焦成像。
Rho6G-SNAP对活细胞染色5min后荧光共聚焦成像如图4所示:Rho6G-SNAP荧光探针在转染细胞膜SANP蛋白的清晰细胞膜成像,说明该探针Rho6G-SNAP可以在细胞中进行转染细胞膜SANP蛋白染色。
Claims (9)
2.根据权利要求1所述一种基于SNAP-tag技术的细胞膜荧光探针的制备方法,其特征在于按照如下步骤进行:
(1)中间体罗丹明6G-羧酸的合成
罗丹明6G溶于乙醇中,加入强碱试剂的水溶液,回流反应5-10h后,减压除去有机溶剂,硅胶柱分离,洗脱剂为体积比20-5:1的二氯甲烷和甲醇,减压除去溶剂后得中间体;
(2)中间体罗丹明6G-琥珀酰亚胺羧酸酯的合成
将罗丹明6G-羧酸,N-羟基琥珀酰亚胺,缩合试剂1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐依次加入到二氯甲烷中,室温搅拌10-16h,减压除去溶剂得粗产品;
(3)中间体罗丹明6G-甲胺烷基酸得合成
将罗丹明6G-琥珀酰亚胺羧酸酯粗产品、甲胺丁酸盐酸盐、碳酸钾依次加入到乙腈中,室温搅拌过夜;减压除去溶剂,硅胶柱分离,洗脱剂为体积比50-10:1的二氯甲烷和甲醇,减压除去溶剂后得紫色固体;
(4)探针的合成
将罗丹明6G-甲胺烷基酸、氨基鸟嘌呤、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐、1-羟基苯并三唑、碳酸钾依次加入到N,N-二甲基甲酰胺中,室温搅拌两天;减压除去溶剂,硅胶柱分离,洗脱剂为体积比40-3:1的二氯甲烷和甲醇,减压除去溶剂后得紫色固体。
3.根据权利要求2所述一种基于SNAP-tag技术的细胞膜荧光探针的制备方法,其特征在于步骤(1)中:罗丹明6G与强碱的质量比为1:0.2-0.4,
水与乙醇的体积比为1:5-8,
罗丹明6G与乙醇的质量与体积比为1:20-40g/mL。
4.根据权利要求2所述一种基于SNAP-tag技术的细胞膜荧光探针的制备方法,其特征在于步骤(2)中:罗丹明6G-羧酸与N-羟基琥珀酰亚胺、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐的质量比为1:0.2-0.4:0.6-0.8,
罗丹明6G-羧酸与二氯甲烷的质量与体积比为1:40-60g/mL。
5.根据权利要求2所述一种基于SNAP-tag技术的细胞膜荧光探针的制备方法,其特征在于步骤(3)中:罗丹明6G-琥珀酰亚胺羧酸酯与甲胺烷基酸盐酸盐、碳酸钾的质量比为1:0.3-0.6:0.2-0.4,
罗丹明6G-琥珀酰亚胺羧酸酯与乙腈的质量与体积比为10-25:1mg/mL。
6.根据权利要求2所述一种基于SNAP-tag技术的细胞膜荧光探针的制备方法,其特征在于步骤(4)中:罗丹明6G-甲胺丁酸、氨基鸟嘌呤、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐、1-羟基苯并三唑、碳酸钾的质量比为1:0.4-1:0.4-0.6:0.3-0.5:1-2,
罗丹明6G-甲胺丁酸与N,N-二甲基甲酰胺的质量与体积比为5-10:1mg/mL。
7.根据权利要求3所述中一种基于SNAP-tag技术的细胞膜荧光探针的制备方法,其特征在于:步骤(1)中所述强碱试剂为KOH、NaOH、LiOH,反应时间以点板检测为基准,直到原料基本反应完全就停止反应。
8.根据权利权利要求3所述中一种基于SNAP-tag技术的细胞膜荧光探针的制备方法,其特征在于:步骤(2)中所使用的缩合试剂为DCC,EDC,HOBt中的一种或多种。
9.一种如权利要求1所述基于SNAP-tag技术的细胞膜荧光探针的应用,其特征在于:该探针应用于细胞膜的成像领域中,该探针荧光亮度高,且具有高度的不透膜性,特异性的与SNAP蛋白结合,结合后在细胞膜表面聚集,荧光亮度增强,从而实现快速、免洗的细胞膜成像。
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