CN110724523B - 一种具有肿瘤靶向的水溶性荧光探针、合成方法及其应用 - Google Patents
一种具有肿瘤靶向的水溶性荧光探针、合成方法及其应用 Download PDFInfo
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Abstract
本发明涉及生物技术领域，公开了一种具有肿瘤靶向的水溶性荧光探针、合成方法及其应用。本发明以吡咯并吡咯二酮衍生物为原料，通过铜(I)催化的“click”反应将带正电的炔丙基[12]aneN3基团引入到骨架中合成荧光探针1。该探针在多种磷酸腺苷存在下，可以特异性的识别ATP，随着ATP浓度的增大，探针的荧光强度逐渐增强，并且该探针可以实时监测细胞内ATP的变化和小鼠体内ATP的分布。因此该探针在评估体内与ATP相关的能量代谢方面具有较大的应用潜力。
Description
技术领域
本发明涉及生物技术领域，具体涉及一种肿瘤靶向的水溶性荧光探针的合成方法及其对细胞和活体中ATP的检测。
背景技术
能量代谢与机体功能密切相关。肿瘤需要大量的ATP和NADH，不仅是肿瘤细胞转移和增殖所必需的，也是肿瘤细胞生存所必需的。ATP过表达与肿瘤恶性程度、侵袭性、不良预后密切相关。ATP由核糖，腺嘌呤和磷酸基团的三部分组成，支撑着地球上几乎所有的生物体。作为一种能源，它在能量传导，细胞呼吸，酶催化和信号传导等生命过程中起着至关重要的作用。此外，ATP还作为神经传递的符号，调节分子运动和离子通道，降低ATP水平会导致低血糖、缺血、帕金森氏症。因此，实时监测细胞内ATP水平具有重要意义。
肿瘤在假缺氧状态，依赖糖酵解获得ATP的作用进一步增强，导致肿瘤呈侵袭性表型。ATP减少可使癌症的能量产生脱轨。利用葡萄糖转运体抑制剂，可以降低肿瘤细胞对葡萄糖的摄入量和肿瘤细胞内ATP水平，从而抑制糖酵解和肿瘤细胞的生长。因此，能量状态的检测和跟踪对于了解机体功能和疾病的诊断和治疗，特别是对疾病的早期诊断和治疗具有实际的指导意义。
随着空间和时间分辨率成像技术的不断发展和进步，科学家们开辟了研究能量代谢的新领域。荧光传感器作为一种将检测物质转化为荧光信号的有效方法，为分析物提供了精确的定量检测。结合激光共聚焦显微镜和活体成像技术，其应用范围扩展到了活细胞，组织甚至整个动物的活体成像。最近，已经开发了几种荧光ATP探针并将其应用于活细胞成像。大多数用于ATP的荧光探针都是基于与带负电荷的tris-磷酸基团的静电或氢键相互作用，与核糖环上的羟基形成硼酸酯或与腺嘌呤碱基的π-π相互作用。
发明内容
本发明的内容是提供一种肿瘤靶向的水溶性荧光探针、合成方法及其应用。
本发明的主要技术方案是：一方面提供一种肿瘤靶向的水溶性ATP探针1，具有如下结构：
本发明的第二方面，提供荧光探针1的合成路径，合成方法如下：
第一步：对溴苯腈与琥珀酸二异丙酯在叔戊醇钠和三氯化铁的作用下生成溴代吡咯并吡咯二酮；
第二步：溴代吡咯并吡咯二酮与1,6-二溴己烷在叔丁醇钾的作用下，发生亲核取代反应生成化合物2；
第三步：化合物2与4-硼酸三苯胺在四三苯基膦钯的作用下发生Suzuki偶联反应，生成化合物3；
第四步：化合物3与叠氮化钠反应生成化合物4；
第五步：化合物4与Boc酸酐保护的炔丙基[12]aneN3在溴化亚铜作用下发生click反应，利用柱层析法进行分离后，生成化合物5。
第六步：化合物5在盐酸乙酸乙酯溶液中脱去Boc酸酐，得到荧光探针1。
进一步的合成方法包括如下步骤：
第一步：将金属钠溶于叔戊醇中，加入催化量的FeCl3，90℃反应2h，冷却至50℃，加入对溴苯甲腈，继续加热至90℃后，逐滴加入丁二酸二异戊酯，加热搅拌24h，冷却至50℃，加入乙酸，在120℃回流30min，冷却至室温，抽滤，滤饼用热水热甲醇洗涤数次，得到溴代吡咯并吡咯二酮；
第二步：将第一步得到的溴代吡咯并吡咯二酮，叔丁醇钾，溶解在N,N二甲基甲酰胺中，加热到60℃，慢慢加入1,6-二溴己烷，反应24h，浓缩有机相，柱层析分离纯化得到化合物2；
第三步：将化合物2,4-硼酸三苯胺，四三苯基膦钯，碳酸钾水溶液加入四氢呋喃中,氩气保护下，回流24h，二氯甲烷萃取三次，柱层析分离纯化得到化合物3；
第四步：将化合物3，NaN3加入到无水N，N二甲基甲酰胺中，氩气保护下80℃搅拌24h，反应结束后，不断加入乙醇直至大量固体析出，得到化合物4；
第五步：将化合物4，炔丙基[12]aneN3，溴化亚铜加入到二氯甲烷中，50℃回流搅拌12h，柱层析分离纯化得到化合物5；
第六步：将化合物5加入盐酸乙酸乙酯溶液中，室温搅拌2h，抽滤，乙醚洗涤得到荧光探针1。荧光探针1的结构经1H NMR、13C NMR和高分辨质谱鉴定。
本发明的第三方面，提供一种荧光探针1对ATP检测的机制。
从分子结构可知，探针1含有正电单元和平面单元，可以与ATP中的三磷酸根发生静电作用，和腺嘌呤的碱基发生π-π相互作用，从而导致探针1的荧光增强。
本发明的第四方面，提供一种ATP荧光探1针对体外ATP的检测。
阳离子探针1通过静电作用和疏水作用与ATP结合后，导致探针1的聚集，随着ATP浓度的增大，探针1的荧光逐渐增强，对ATP的最低检测限量为24nM。为了评估探针1对ATP的选择性，研究了探针1对其他分析物的荧光响应。结果表明，只有在ATP存在下，探针1出现明显的荧光增强，而其他的生物阴离子如ADP,AMP,CMP,GTP,GMP,UTP,PPI，无机阴离子PO4 3-,HPO4 2-,H2PO4 -,Cl-,NO3 -,HSO3 -均没有产生任何荧光的变化。
本发明的第五方面提供一种ATP荧光探针对细胞和活体中ATP的检测应用。
实时追踪Hela细胞中ATP的变化，利用糖酵解抑制剂2-脱氧葡萄糖来降低细胞中ATP水平。将Hela细胞在共聚焦小皿中培养60h，待细胞富集程度达到50％后，一组加入20μM探针1，一组加入20μM探针1和10mM 2-脱氧葡萄糖，放置于细胞培养箱中37℃恒温孵化1h，然后用PBS缓冲溶液清洗细胞3次，去除未进入细胞的探针，并添加新的PBS缓冲溶液。利用激光共聚焦显微镜观察细胞中荧光成像。结果表明，随着2-脱氧葡萄糖刺激时间的延长，细胞中的红色荧光逐渐降低，随着额外补加ATP后，荧光恢复，说明探针1可实时监控细胞中ATP的变化。
本发明进一步探索了探针1的肿瘤靶向性。准备一只正常的裸鼠和皮下移植Hela瘤裸鼠，通过腹腔注射探针1的PBS溶液，通过活体成像仪观察裸鼠的荧光强度分布。结果表明与正常裸鼠相比，荷瘤裸鼠的肿瘤区域有明显的荧光增强现象，而正常裸鼠在注射部位表现出较强的荧光。研究结果表明探针1具有肿瘤靶向性，可用于有效的体内能量代谢监测。
本发明设计合成了一个具有聚集诱导荧光增强性质的水溶性ATP探针1，具有一下特点：
(1)该探针对ATP具有较高的灵敏性和特异性。
(2)可实时监测细胞中ATP的变化。
(3)具有肿瘤靶向性可实现活体肿瘤部位ATP的检测。
附图说明
图1为在10μM荧光探针1的水溶液中加入不同浓度的ATP，荧光发射光谱的变化；
图2为10μM荧光探针1对10μM的ATP选择性实验；
图3为将HeLa细胞与10μM探针1孵育30分钟，然后用10mM 2-脱氧葡萄糖处理(A):0，(B):5，(C):10，(D):20分钟；(E):(D)组的细胞加入10mM ATP；(F):明场的共聚焦显微镜荧光图像。(G):从细胞图像A–E计算出荧光强度；
图4为经腹膜内注射探针1后裸鼠和荷瘤裸鼠的体内成像图。
具体实施方式
以下结合附图和具体实施例，对本发明作进一步说明。应理解，以下实施例仅用于说明本发明而非用于限定本发明的范围。
下述具体实施例中的方法，如无特殊说明，均为常规方法。
实施例1：荧光探针1的合成：
(1)溴代吡咯并吡咯二酮的合成：将金属钠(1.26g，54.5mmol)溶于26mL的叔戊醇中，加入1.00g催化量的FeCl3，90℃反应2h，冷却至50℃，加入对溴苯甲腈(5.00g,27.6mmol)，继续加热至90℃，后逐滴加入丁二酸二异戊酯(2.20g，10.5mmol)(溶解到12mL叔戊醇中，滴加时间不少于2h)，加热搅拌24h，冷却至50℃，加入15mL乙酸，在120oC回流30min，冷却至室温，抽滤，滤饼用热水，热甲醇洗涤数次，固体在110℃烘干8h，得到溴代吡咯并吡咯二酮，产率77％。
(2)化合物2的合成：将0.45g(1mmol)的溴代吡咯并吡咯二酮，0.25g叔丁醇钾，溶解在5mL的NMP中，加热到60℃，慢慢加入1mL的1,6-二溴己烷(4mmol)，反应24h，冷却至室温，向反应液中加入50mL甲苯，大量水洗除去NMP，浓缩有机相，柱层色谱分离，得到化合物2，产率14％。1H NMR(500MHz,CDCl3)δ7.70(s,8H),3.76(s,4H),3.37(s,4H),1.80(s,4H),1.60(s,5H),1.40(s,4H),1.28(s,4H)。
(3)化合物3的合成：将化合物2(0.0386g,0.05mmol),4-硼酸三苯胺(0.0578g,0.2mmol),Pd(PPh3)4(0.007g,0.007mmol)加入到100mL圆底烧瓶中，再加入5mL THF,1mL 2MK2CO3水溶液，Ar保护下，60℃回流24h，反应结束后，DCM萃取三次，柱层色谱分离(DCM/PE＝3/1v/v)得到化合物3，产率80％。1H NMR(400MHz,CDCl3)δ7.89(d,4H),7.72(s,4H),7.53(d,4H),7.28(t,9H),3.83(s,4H),3.33(s,4H),1.79(s,4H),1.66(s,4H),1.39(s,4H),1.30(s,4H).13C NMR(125MHz,CDCl3)δ162.87,148.09,147.99,147.45,143.37,133.19,129.40,129.23,127.81,126.86,126.34,124.83,123.36,123.31,109.81,41.90,33.73,32.54,29.30,27.65,25.94.MS:m/z calcd.[M+H]+for C66H61Br2N4O2,1101.306；found,1101.201.
(4)化合物4的合成：将化合物3(0.02g 0.018mmol),NaN3(0.0068g 0.1mmol)加入到2mL无水DMF中，Ar保护下80℃搅拌24h，反应结束，不断加入乙醇直至大量固体析出，抽滤,得到化合物4，直接进行下一步反应。
(5)化合物5的合成：将0.0195mmol 0.02g化合物4和0.0391mmol 0.016g炔丙基[12]aneN3加入到CH2Cl2/H2O/tert-butanol(21mL,10:10:1,v/v/v)中，同时加入0.004mmol，0.004g CuBr(PPh3)3,50℃回流搅拌12h，停止反应，冷却至室温，CH2Cl2萃取三次。柱层色谱分离(DCM/MeOH＝20/1)，得到化合物5，产率：83％。1H NMR(400MHz,CDCl3)δ7.88(s,4H),7.76(s,4H),7.53(s,4H),7.28(d,J＝12.9Hz,12H),7.15(d,J＝7.1Hz,10H),7.07(s,4H),4.27(s,6H),3.79(d,J＝23.8Hz,6H),3.31(s,14H),2.41(s,6H),2.04(s,3H),1.85(s,10H),1.65(s,8H),1.26(s,36H),0.87(d,J＝7.6Hz,12H).13C NMR(125MHz,CDCl3)162.04,156.39,148.12,147.97,147.42,143.38,129.40,129.50,127.78,124.84,123.39,123.24,109.76,50.14,49.46,45.43,44.00,41.83,29.70,29.67,29.32,28.50,26.03,14.14.MS:m/z calcd.[M+2H]2+for C110H140N16O10,923.041；found,923.152.
(6)探针1的合成：取50mg,0.027mmol化合物5，加入5mL HCl/EA，室温搅拌2h，得到探针1，产率82％。1H NMR(400MHz,DMSO)δ9.64(d,J＝36.9Hz,6H),8.36(s,1H),8.07(s,1H),7.89(d,J＝16.9Hz,6H),7.72(s,4H),7.35(s,6H),7.18–6.82(m,14H),3.75(s,20H),3.54–2.94(m,18H),2.02(dd,J＝63.3,35.6Hz,10H),1.72(s,4H),1.46(s,4H),1.18(s,8H).13C NMR(125MHz，DMSO)162.16，148.12，147.78，147.32，142.57，132.55，130.24，129.81，128.34，126.75，126.50，125.13，124.21，,123.04,109.31,60.21,50.99,49.99,49.04,46.95,41.40,29.97,29.08,26.31,25.89,25.70.21.06,19.32,17.28.HR-MS:m/zcalcd.[M+2H]2+for C90H108N16O2,722.4415；found,722.4408.
实施例2:ATP探针性能的测试
(1)荧光探针1对ATP的荧光滴定测试
取20μL荧光探针1的DMSO溶液于2mL水中，依次加入2,4,6,8,10,12μL的ATP水溶液(1mM),测试其荧光发射光谱，激发波长为520nm，如图1所示。随着ATP浓度的增大，探针1的荧光逐渐增强，对ATP的最低检测限为24nM。
(2)荧光探针1对ATP的选择性测试
取20μL荧光探针1的DMSO溶液(1mM)于2mL水中，然后加入20μL的ADP,AMP,CMP,GTP,GMP,UTP,PPI，无机阴离子PO4 3-,HPO4 2-,H2PO4 -,Cl-,NO3 -,HSO3 -水溶液(1mM)，然后测定溶液的荧光发射光谱，激发波长为520nm。结果表明，只有在ATP存在下，探针1出现明显的荧光增强，而其他的生物阴离子如ADP,AMP,CMP,GTP,GMP,UTP,PPI，无机阴离子PO4 3-,HPO4 2-,H2PO4 -,Cl-,NO3 -,HSO3 -均没有产生任何荧光的变化。
实施例3:ATP探针细胞成像
将Hela细胞在共聚焦小皿中培养60h，待细胞密度达到50％后，一组加入20μM探针1，一组加入20μM探针1和10mM 2-脱氧葡萄糖，放置于细胞培养箱中37oC恒温孵化1h，然后用PBS缓冲溶液清洗细胞3次，去除未进入细胞的探针，并添加新的PBS缓冲溶液。利用激光共聚焦显微镜观察细胞中荧光成像。结果表明，随着2-脱氧葡萄糖刺激时间的延长，细胞中的红色荧光逐渐降低，随着额外补加ATP后，荧光恢复，说明探针1可实时监控细胞中ATP的变化。
实施例4:ATP探针的肿瘤靶向性
将1×106个Hela细胞注射到6周左右的雌性裸鼠的侧翼腋下区，待肿瘤长体积到200mm3时，通过腹腔注射探针1的PBS溶液，通过活体成像仪观察裸鼠的荧光强度分布，未接种肿瘤的正常裸鼠为对照组。通过活体成像仪观察裸鼠的荧光强度分布。结果表明与正常裸鼠相比，荷瘤裸鼠的肿瘤区域有明显的荧光增强现象，而正常裸鼠在注射部位表现出较强的荧光。研究结果表明探针1具有肿瘤靶向性，可用于有效的体内能量代谢监测。
以上已对本发明创造的较佳实施例进行了具体说明，但本发明创造并不限于所述实施例，熟悉本领域的技术人员在不违背本发明创造精神的前提下还可作出种种的等同的变型或替换，这些等同的变型或替换均包含在本申请权利要求所限定的范围内。
Claims (8)
1.一种水溶性ATP探针1，其特征在于，所述探针结构如下图所示：
2.一种权利要求1所述的水溶性ATP探针1的合成方法，其特征在于：所述合成方法以吡咯并吡咯二酮衍生物为原料，通过铜(I)催化的“click”反应将带正电的炔丙基[12]aneN3基团引入到骨架中合成荧光探针1。
3.根据权利要求2所述的水溶性ATP探针1的合成方法，所述合成方法包括如下步骤：
第一步：对溴苯腈与琥珀酸二异丙酯在叔戊醇钠和三氯化铁的作用下生成溴代吡咯并吡咯二酮；
第二步：溴代吡咯并吡咯二酮与1,6-二溴己烷在叔丁醇钾的作用下，发生亲核取代反应生成化合物2；
第三步：化合物2与4-硼酸三苯胺在四三苯基膦钯的作用下发生Suzuki偶联反应，生成化合物3；
第四步：化合物3与叠氮化钠反应生成化合物4；
第五步：化合物4与Boc酸酐保护的炔丙基[12]aneN3在溴化亚铜作用下发生click反应，利用柱层析法进行分离后，生成化合物5；
第六步：化合物5在盐酸乙酸乙酯溶液中脱去Boc酸酐，得到荧光探针1。
4.根据权利要求3所述的水溶性ATP探针1的合成方法，其特征在于：包括以下步骤：
第一步：将金属钠溶于叔戊醇中，加入催化量的FeCl3，90℃反应2h，冷却至50℃，加入对溴苯甲腈，继续加热至90℃后，逐滴加入丁二酸二异戊酯，加热搅拌24h，冷却至50℃，加入乙酸，在120℃回流30min，冷却至室温，抽滤，滤饼用热水热甲醇洗涤数次，得到溴代吡咯并吡咯二酮；
第二步：将第一步得到的溴代吡咯并吡咯二酮，叔丁醇钾，溶解在N,N二甲基甲酰胺中，加热到60℃，缓慢加入1，6-二溴己烷，反应24h，浓缩有机相，柱层析分离纯化得到化合物2；
第三步：将化合物2,4-硼酸三苯胺，四三苯基膦钯，碳酸钾水溶液加入四氢呋喃中,氩气保护下，回流24h，二氯甲烷萃取三次，柱层析分离纯化得到化合物3；
第四步：将化合物3，NaN3加入到无水N，N二甲基甲酰胺中，氩气保护下80℃搅拌24h，反应结束后，不断加入乙醇直至大量固体析出，得到化合物4；
第五步：将化合物4，炔丙基[12]aneN3，溴化亚铜加入到二氯甲烷中，50℃回流搅拌12h。柱层析分离纯化得到化合物5；
第六步：将化合物5加入到盐酸乙酸乙酯溶液中，室温搅拌2h，抽滤，乙醚洗涤得到荧光探针1。
5.一种如权利要求1所述的荧光探针1在ATP荧光检测中的应用。
6.根据权利要求5所述荧光探针1在ATP荧光检测中的应用，其特征在于：所述ATP荧光探针1应用于体外ATP的检测。
7.根据权利要求5所述荧光探针1在ATP荧光检测中的应用，其特征在于：所述ATP荧光探针1应用于细胞和活体中ATP的检测。
8.根据权利要求5所述荧光探针1在ATP荧光检测中的应用，其特征在于：所述ATP荧光探针1应用于活体肿瘤部位ATP的监测。
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