CN112940020A - 一类405nm激发的高亮度免洗脂滴荧光探针及其合成方法与应用 - Google Patents
一类405nm激发的高亮度免洗脂滴荧光探针及其合成方法与应用 Download PDFInfo
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Abstract
本发明开发出了一类405nm激发的高亮度免洗脂滴荧光探针及其合成方法与应用,结构特点是在BODIPY母体的meso‑位引入含氮取代基。该类探针合成方法简单,合成原料低廉易得。探针在质子性溶剂如水中荧光量子产率小于0.01,而在非质子性溶剂如二氯甲烷中荧光量子产率最高可达到0.9,探针对环境的荧光敏感度有效地降低了成像时的背景。实验表明该类探针能够快速透过细胞膜并富集在脂滴内,成像时具有很高的亮度及很低的成像背景,在细胞生物学研究中具有广阔的应用前景。
Description
技术领域
本发明属于荧光探针和成像领域,具体涉及一类高亮度、可用405nm激发的高亮度免洗脂滴荧光探针的结构、合成方法及其在荧光成像领域中的应用。
背景技术
脂滴是一种广泛存在于大多数细胞和生物体系中的动态细胞器,参与了许多重要的生理功能,包括脂质代谢、膜合成和转移、信号转导、蛋白质降解等。近年来研究发现,脂滴与肥胖、糖尿病、炎症和癌症等紧密相关。因此,开发出有效的方法对活细胞中脂滴的直接和选择性可视化和监测显得格外重要。荧光成像具有高灵敏度、高时空分辨率等优势,在生命科学领域有着广阔的应用前景。开发可用于脂滴原位荧光成像的工具对于深入理解脂滴结构与功能有着重要的意义。
目前商品化的激光器主要为405nm,488nm,及561nm,广泛使用的脂滴探针BODIPY493和尼罗红分别匹配488nm和561nm激光器,而少有适用于405nm激发的脂滴探针问世,且这些探针大多存在荧光强度低的缺陷。BODIPY类探针具有较高的亮度,常用于活细胞成像中,然而BODIPY为环境不敏感探针,在成像时容易产生背景荧光的干扰,成像时需要多次更换新鲜的培养基,费时费力。因此,亟需开发一种能够匹配405nm激发且具有高亮度的荧光探针以满足对脂滴进行免洗荧光成像的需求。
发明内容
本发明开发出了一类不对称的BODIPY类荧光探针,该探针在不对称的BODIPY探针母体meso-位引入不同含氮取代基,使探针在弱极性溶剂中有较高的荧光量子产率,而在大极性溶剂及质子性溶剂中荧光量子产率较低。研究发现这类探针有合适的亮度能够快速透过细胞并富集于脂滴中,成像信噪比,借助共聚焦荧光显微镜实现了对脂滴的荧光成像。
本发明采用的技术方案为:
本发明提供的高亮度、能用于405nm激发的免洗脂滴探针,是以不对称BODIPY为结构单元,其结构式如下所示:
同时,本发明提供了一种405nm激发的高亮度免洗荧光脂滴探针的合成方法,meso-N取代BODIPY类探针的一般合成方法,合成步骤如下:
具体合成步骤如下:
(1)中间体2'-吡咯基-2'-(3,5-二烷基)吡咯基硫酮(S-1)的合成
将不同的吡咯衍生物P-1,P-2溶于干燥的四氢呋喃中,冰浴下加入硫光气反应10-60分钟。减压除去溶剂,硅胶柱色谱分离提纯得到中间体S-1。
(2)中间体不对称-meso-甲硫基-BODIPY(S-2)合成
将步骤(1)得到的中间体S-1溶于干燥的二氯甲烷中,加入碘甲烷在室温下反应5-24小时,而后加入三乙胺、三氟化硼乙醚,室温下反应1-5小时后减压除去溶剂,硅胶色谱柱分离提纯得到中间体S-2。
(3)脂滴探针不对称-meso-N取代BODIPY(B-1)的合成
将步骤(2)得到的中间体S-2溶于二氯甲烷中,加入对应伯胺或仲胺,室温下搅拌1-5小时。减压除去溶剂,硅胶柱色谱分离提纯得到脂滴探针不对称-meso-N取代BODIPY(B-1)。
步骤(1)中,吡咯衍生物P-1,吡咯衍生物P-2与硫光气的质量比为1:1-5:1-3,吡咯衍生物P-1的质量与四氢呋喃的体积比为1:100-200g/mL。
步骤(2)中,中间体S-1与碘甲烷的质量与体积比为1:15-50g/mL;中间体S-1与三乙胺的质量与体积比为1:10-50g/mL;中间体S-1与三氟化硼乙醚的质量与体积比为1:10-50g/mL;中间体S-1与二氯甲烷的质量与体积比为1:50-200g/mL。
步骤(3)中,中间体S-2与对应伯胺或仲胺的质量与体积比为1:2-20g/mL;中间体S-2与二氯甲烷的质量与体积比为1:50-200g/mL。
上述荧光探针能够在活细胞内对脂滴进行特异性标记并用实现荧光成像。
本发明有益效果:
这类探针拥有合成原料低价、方法简单且易于衍生等优点。
本发明开发出了一类不对称的BODIPY类荧光探针,该探针在不对称的BODIPY探针母体meso-位引入不同N-取代基,探针在弱极性溶剂中有较高的荧光量子产率,而在大极性溶剂及质子性溶剂中荧光量子产率较低。这一性质使探针在进行脂滴成像时有较高的亮度及很低的成像背景,能够实现对活细胞脂滴的免洗荧光成像。
该探针可以实现对细胞中脂滴的精准定位,可用于细胞或组织中脂滴及各种脂质的成像、传感、检测等领域的研究。
附图说明
图1:实施例1制备得到的中间体S-2的核磁氢谱;
图2:实施例1制备得到的脂滴探针BODIPY-NH的核磁氢谱;
图3:实施例1制备得到的脂滴探针BODIPY-NH的核磁碳谱;
图4:实施例2制备得到的脂滴探针BODIPY-N5在二氯甲烷中归一化荧光激发与发射谱图,横坐标为波长,纵坐标为归一化的荧光强度与吸收强度,荧光探针的浓度为10μM;;
图5:实施例3制备的脂滴探针BODIPY-NN的HeLa细胞共聚焦显微成像图;
具体实施方式
下面的实施例将对本发明予以进一步的说明,但并不因此而限制本发明。
实施例1.
脂滴探针BODIPY-NH的合成。
中间体S-1合成路线和产物结构如下:
将吡咯(100mg,1.49mmol)和2,4-二甲基吡咯(240mg,2.98mmol)溶于10mL干燥的四氢呋喃中,冰浴下缓慢加入硫光气(260mg,2.25mmol),0℃下反应15min后,加入10mL甲醇后并升温至室温反应30min淬灭反应。减压蒸馏除去溶剂,残余物经硅胶柱(石油醚/乙酸乙酯=10/1,V/V)分离得到产物136mg,为紫色固体,产率45%。
其高分辨质谱数据如下:
HRMS(ESI):m/z:[M+H]+:计算值:205.0799,实验值:205.0661。
经检测,其结构如上式S-1所示。
中间体S-2的合成路线和产物结构如下:
在氮气氛围中,将S-1(100mg,0.49mmol)溶于干燥的10mL二氯甲烷中,加入碘甲烷(610μL,9.8mmol)。在室温下反应12小时后,加入5mL三乙胺及5mL三氟化硼乙醚,室温下反应30min。减压蒸馏除去溶剂,残余物经硅胶柱(石油醚/乙酸乙酯=10/1,V/V)分离得到产物66.5mg,为橙红色固体,产率为51%。
其核磁谱图氢谱如下图1所示,具体数据如下:
1H NMR(400MHz,CDCl3)δ7.51(s,1H),7.17(s,1H),6.38(s,1H),6.12(s,1H),2.68(s,3H),2.50(s,6H).
经检测,其结构如上式S-2所示。
脂滴探针BODIPY-NH的合成路线和产物结构如下:
称取S-2(50mg,0.19mmol)溶于5mL干燥的二氯甲烷中,用移液枪加入乙胺(120μL,1.9mmol),室温下反应2h后,减压蒸馏除去溶剂,残余物经硅胶柱(石油醚/二氯甲烷=1/1,V/V)分离得到产物40mg,为黄色固体,产率82%。
其核磁谱图氢谱如下图2所示,具体数据如下:
1H NMR(400MHz,CDCl3)δ7.57(s,1H),6.98(s,1H),6.45(s,1H),6.14(s,1H),5.94(s,1H),3.68–3.55(m,2H),2.49(s,3H),2.33(s,3H),1.43(t,J=7.0Hz,3H).
其核磁谱图碳谱如下图3所示,具体数据如下:
13C NMR(101MHz,CDCl3)δ148.09,145.42,133.21,130.64,122.68,121.99,120.71,117.65,113.86,42.33,15.40,14.70,13.82.
经检测,其结构如上式BODIPY-NH所示,能够准确定位活细胞脂滴中进行荧光成像。
实施例2.
脂滴探针BODIPY-N5的合成。
中间体S’-1合成路线和产物结构如下:
将吡咯(100mg,1.49mmol)和2,4-二乙基吡咯(280mg,2.98mmol)溶于10mL干燥的四氢呋喃中,冰浴下缓慢加入硫光气(260mg,2.25mmol),0℃下反应15min后,加入10mL甲醇后并升温至室温反应30min淬灭反应。减压蒸馏除去溶剂,残余物经硅胶柱(石油醚/乙酸乙酯=10/1,V/V)分离得到产物143mg,为紫色固体,产率45%。
其高分辨质谱数据如下:
HRMS(ESI):m/z:[M+H]+:计算值:233.1112,实验值:233.1112。
经检测,其结构如上式S’-1所示。
中间体S’-2的合成路线和产物结构如下:
在氮气氛围中,将S’-1(100mg,0.43mmol)溶于干燥的10mL二氯甲烷中,加入碘甲烷(610μL,9.8mmol)。在室温下反应12小时后,加入5mL三乙胺及5mL三氟化硼乙醚,室温下反应30min。减压蒸馏除去溶剂,残余物经硅胶柱(石油醚/乙酸乙酯=10/1,V/V)分离得到产物66.5mg,为橙红色固体,产率为51%。
其高分辨质谱数据如下:
HRMS(ESI):m/z:[M+H]+:计算值:295.1252,实验值:295.1233。
经检测,其结构如上式S’-2所示。
脂滴探针BODIPY-N5的合成路线和产物结构如下:
称取S’-2(50mg,0.19mmol)溶于5mL干燥的二氯甲烷中,用移液枪加入四氢吡咯(85μL,0.95mmol),室温下反应2h后,减压蒸馏除去溶剂,残余物经硅胶柱(石油醚/二氯甲烷=1/1,V/V)分离得到产物36mg,为黄色固体,产率66%。
其高分辨质谱数据如下:
HRMS(ESI):m/z:[M+H]+:计算值:318.1953,实验值:318.1967。
经检测,其结构如上式BODIPY-N5所示,能够准确定位活细胞脂滴中进行荧光成像。
将待测探针溶解于二甲基亚砜溶液中,配制成2mM母液,根据需要制配成不同浓度测试溶液,对其光谱及细胞成像进行检测。
BODIPY-N5在二氯甲烷中的光谱测试。取20μL BODIPY-N5母液,加入4mL二氯甲烷中,配制成10μM的荧光探针测试液,并进行紫外吸收光谱和荧光发射光谱的测试。
BODIPY-N5在二氯甲烷中的归一化紫外吸收光谱和荧光发射光谱如图4所示,其中荧光探针浓度为10μM,BODIPY-N5在二氯甲烷中量子产率达到0.9,具有较高的亮度。
实施例3.
脂滴探针BODIPY-NN的合成。
中间体S-1合成路线和产物结构如下:
将吡咯(100mg,1.49mmol)和2,4-二甲基吡咯(1.2g,14.9mmol)溶于20mL干燥的四氢呋喃中,冰浴下缓慢加入硫光气(780mg,6.75mmol),0℃下反应15min后,加入15mL甲醇后并升温至室温反应30min淬灭反应。减压蒸馏除去溶剂,残余物经硅胶柱(石油醚/乙酸乙酯=10/1,V/V)分离得到产物180mg,为紫色固体,产率60%。
其高分辨质谱数据如下:
HRMS(ESI):m/z:[M+H]+:计算值:205.0799,实验值:205.0661。
经检测,其结构如上式S-1所示。
中间体S-2的合成路线和产物结构如下:
在氮气氛围中,将S-1(100mg,0.49mmol)溶于干燥的10mL二氯甲烷中,加入碘甲烷(300μL,4.8mmol)。在室温下反应20小时后,加入2mL三乙胺及2mL三氟化硼乙醚,室温下反应60min。减压蒸馏除去溶剂,残余物经硅胶柱(石油醚/乙酸乙酯=10/1,V/V)分离得到产物46mg,为橙红色固体,产率为35%。
其核磁谱图氢谱如下图1所示,具体数据如下:
1H NMR(400MHz,CDCl3)δ7.51(s,1H),7.17(s,1H),6.38(s,1H),6.12(s,1H),2.68(s,3H),2.50(s,6H).
经检测,其结构如上式S-2所示。
脂滴探针BODIPY-NN的合成路线和产物结构如下:
称取S-2(50mg,0.19mmol)溶于10mL干燥的二氯甲烷中,用移液枪加入氨的二氧六环溶液(120μL,1.9mmol),室温下反应2h后,减压蒸馏除去溶剂,残余物经硅胶柱(石油醚/二氯甲烷=1/1,V/V)分离得到产物40mg,为黄色固体,产率65%。
其高分辨质谱数据如下:
HRMS(ESI):m/z:[M+H]+:计算值:236.1171,实验值:236.1183。
经检测,其结构如上式BODIPY-NN所示,能够准确定位活细胞脂滴中进行荧光成像。
实施例4.
BODIPY-NN对活细胞染色后的荧光成像显微成像实验。将待测探针溶解于二甲基亚砜溶液中,配制成2mM母液。取1μL BODIPY-NN母液溶于1mL细胞培养液中,37℃,5%CO2下孵育15分钟后进行共聚焦荧光显微成像。
BODIPY-NN终浓度为2μM的细胞培养液孵育HeLa细胞15分钟后荧光显微成像图,如图5所示,HeLa细胞内圆型的脂滴清晰可见。
Claims (7)
2.如权利要求1所述的荧光脂滴探针,其特征在于,该探针在不对称的BODIPY探针母体meso-位引入不同含氮取代基。
3.一种如权利要求1所述的405nm激发的高亮度免洗荧光脂滴探针的合成方法,其特征包含步骤如下:
(1)中间体2'-吡咯基-2'-(3,5-二烷基)吡咯基硫酮(S-1)的合成
将不同的吡咯衍生物P-1,P-2溶于干燥的四氢呋喃中,冰浴下加入硫光气反应10-60分钟;减压除去溶剂,硅胶柱色谱分离提纯得到中间体S-1;
(2)中间体不对称-meso-甲硫基-BODIPY(S-2)合成
将步骤(1)得到的中间体S-1溶于干燥的二氯甲烷中,加入碘甲烷在室温下反应5-24小时,而后加入三乙胺、三氟化硼乙醚,室温下反应1-5小时后减压除去溶剂,硅胶色谱柱分离提纯得到中间体S-2;
(3)脂滴探针不对称-meso-N取代BODIPY(B-1)的合成
将步骤(2)得到的中间体S-2溶于二氯甲烷中,加入对应伯胺或仲胺,室温下搅拌1-5小时,减压除去溶剂,硅胶柱色谱分离提纯得到脂滴探针。
4.如权利要求3中所述的合成方法,其特征在于:
步骤(1)中:吡咯衍生物P-1,吡咯衍生物P-2与硫光气的质量比为1:1-5:1-3,吡咯衍生物P-1的质量与四氢呋喃的体积比为1:100-200g/mL。
5.如权利要求3中所述的合成方法,其特征在于:
步骤(2)中:中间体S-1与碘甲烷的质量与体积比为1:15-50g/mL;中间体S-1与三乙胺的质量与体积比为1:10-50g/mL;中间体S-1与三氟化硼乙醚的质量与体积比为1:10-50g/mL;中间体S-1与二氯甲烷的质量与体积比为1:50-200g/mL。
6.如权利要求3中所述的合成方法,其特征在于:
步骤(3)中:中间体S-2与对应伯胺或仲胺的质量与体积比为1:2-20g/mL;中间体S-2与二氯甲烷的质量与体积比为1:50-200g/mL。
7.一种如权利要求1所述的脂滴探针在荧光成像、荧光传感、生化检测等领域的应用。
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US20120253050A1 (en) * | 2009-12-10 | 2012-10-04 | Universidad De Guanajuato | Synthesis of 8-amino boron dipyrromethenes having blue fluorescence |
WO2019132479A1 (ko) * | 2017-12-27 | 2019-07-04 | 주식회사 엘지화학 | 함질소 화합물, 이를 포함하는 색변환 필름, 및 이를 포함하는 백라이트 유닛 및 디스플레이장치 |
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