CN107814796B - 一种基于苯并呋咱的环境敏感染料及其制备方法和应用 - Google Patents
一种基于苯并呋咱的环境敏感染料及其制备方法和应用 Download PDFInfo
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- CN107814796B CN107814796B CN201710958413.4A CN201710958413A CN107814796B CN 107814796 B CN107814796 B CN 107814796B CN 201710958413 A CN201710958413 A CN 201710958413A CN 107814796 B CN107814796 B CN 107814796B
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- C07D413/06—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
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- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
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Abstract
Description
技术领域
本发明属于有机生物技术领域,尤其是涉及一种基于苯并呋咱的环境敏感染料及其制备方法和应用。
背景技术
环境敏感探针是一类新型的探针,其荧光强度随着外界微环境的变化(例:极性,粘度,分子排序等)而改变,而且环境敏感的染料荧光背景低,此类探针常被用于蛋白质的检测,为了开发新型的环境敏感的染料。我们设法改造传统的硝基取代的苯并呋咱染料,此染料作为一种常用的环境敏感染料,但是它只发出波长较短的绿色荧光,常与细胞或组织本身的自发荧光产生干扰,而且这些短波的光穿透力差,无法有效地透过深层组织,限制其广泛的应用。我们假设通过用其它强的拉电子基团的替换硝基从而延伸了荧光团的共轭系统产生出一种新的基于分子内电荷转移(ICT)的荧光染料,基于这一猜想,我们用三种具有强拉电子的阳离子基团,这三种基团分别为1,4-二甲基吡啶,1,2,3,3-四甲基吲哚,1,1,2,3-四甲基苯并吲哚,与苯并呋咱相交联。这些新的荧光团具有推拉电子体系,具有强的环境敏感性质,在水中荧光非常弱,然而在小极性和高粘度环境中表现出强烈的远红外荧光。更有趣的是,这些含阳离子的染料能够靶向线粒体。通过体外数据分析,1,2,3,3-四甲基吲哚衍生出的染料相比其它两种染料对环境更加敏感,响应倍比更高。
线粒体在许多基本的细胞活动中发挥重要作用,包括脂肪酸的合成,钙稳态和细胞凋亡信号转导。细胞内活性氧物种(ROS)主要来源于线粒体,线粒体易于受到氧化损伤,正常调控氧化过程是细胞器正常工作的关键之处。氧化还原代谢的异常会诱发许多疾病如神经退行型疾病,糖尿病和癌症。邻二巯基蛋白(VDPs)是调节线粒体的氧化还原平衡的关键物种,通过与活性氧,活性氮和亲电试剂物种相互作用维持这一过程。蛋白质中邻二巯基会有一个氧化和还原态的互变,这一机制调节着VDPS的功能同时也是维持线粒体氧化还原信号的重要机制。VDPs维持蛋白质的稳定结构,也和蛋白质的功能有着密切关系。因此,检测和跟踪细胞线粒体中VDPs对氧化还原相关疾病的诊断和病理生理学研究具有重大的作用。
荧光探针是一种有用的检测细胞内邻二巯基蛋白的方法。这些探针的一般策略用三价砷作为识别基团,对邻二巯基具有很高的特异性。一种常用的方法就是将一个荧光团与含砷的五元环相结合,用于检测细胞内的VDPs。这种设计策略最大的问题就是荧光背景高,在细胞成像前,需要进行复杂的清洗过程。
发明内容
针对现有技术的不足,本发明旨在提出一种基于苯并呋咱的环境敏感染料及其制备方法和应用,筛选出了一种对极性和粘度响应倍比更高的染料,构建了高灵敏度和快速响应VDPs的小分子荧光探针,并以此探针作为工具用于VDPs的生理学和生物学特征的体内和体外研究,为VDPs的研究提供了更方便、更有效的研究手段。
为达到上述目的,本发明的技术方案是这样实现的:
一种基于苯并呋咱的环境敏感染料,其结构式如下:
一种基于苯并呋咱的环境敏感染料的制备方法包括以下步骤:
(1)称取4-氯苯并呋咱3~15g于真空封管当中,依次加入50mL乙醇、10~25g二甲胺盐酸盐、25~45mL三乙胺,在110~170℃加热条件下搅拌48h,冷却至室温,加入70~130mL 2M氢氧化钠溶液,用乙酸乙酯萃取后干燥、过滤,得到化合物1;
(2)将盛有10~35mL无水DMF的烧瓶置于冰浴中,加入5~20mL POCl3,搅拌均匀,加入3~10g化合物1,室温下搅拌,将50mL冰水倾入到烧瓶中,用10%的NaOH溶液调pH约等于9,用乙酸乙酯萃取,干燥并过滤,得到化合物2;
(3)将50~120mg化合物2与120~220mg强拉电子供体化合物溶解在10mL的无水乙醇中,加入1~15mg哌啶,加热回流16h,溶液逐渐由黄色变成蓝色,反应结束后冷却至室温,得到产物。
进一步的,所述强拉电子供体化合物为1,2,3,3-四甲基-3H-吲哚-1-碘鎓或1,2,3,3-四甲基-3H-苯并[g]吲哚-1-碘鎓或1,4-二甲基吡啶-1-碘鎓中的一种。
进一步的,所述1,2,3,3-四甲基-3H-吲哚-1-碘鎓的制备方法如下:将2~5g 2,3,3-三甲基-3H-吲哚和2~8g碘甲烷溶于20mL乙腈中回流反应72h,反应结束后冷却、过滤、干燥,得到产物。
进一步的,所述1,2,3,3-四甲基-3H-苯并[g]吲哚-1-碘鎓的制备方法如下:将1~5g 2,3,3-三甲基苯并吲哚和1~5g碘甲烷溶于30mL乙腈中回流反应48h,反应结束后冷却、过滤、干燥,得到产物。
进一步的,所述1,4-二甲基吡啶-1-碘鎓的制备方法如下:将0.1~1.5g4-甲基哌啶和0.5~4g碘甲烷混合于20mL甲苯中,室温搅拌8~12h,反应结束后冷却、过滤、干燥,得到产物。
一种基于苯并呋咱的环境敏感染料在检测蛋白质及生物成像中的应用,其中蛋白质分子荧光探针结构通式如下:
R2为蛋白质识别位点。
进一步的,蛋白质分子为邻二巯基蛋白,邻二巯基蛋白荧光探针结构通式如下:
相对于现有技术,本发明所述的一种基于苯并呋咱的环境敏感染料及其制备方法和应用具有以下优势:
(1)本发明所述的基于苯并呋咱的环境敏感染料制备的探针分子的荧光性质随着周围环境的改变而发生变化,尤其是其荧光会因周围环境的极性减少或者粘度增大而显著增强;
(2)本发明所述的基于苯并呋咱的环境敏感染料制备的探针分子与蛋白质分子结合时荧光会大大增强,能够很容易地实现了受体蛋白的可视化,提高了信噪比,避免了繁琐的洗涤步骤,大大节省了时间;
(3)本发明所述的基于苯并呋咱的环境敏感染料制备的探针分子的荧光的波长可调范围大,可进入红光区,能够有效地避开生物体系的自发荧光干扰,从而极大地提高了探针的灵敏度,避免了干扰;
(4)本发明所述基于苯并呋咱的环境敏感染料制备的探针分子在兼顾灵敏度的同时,显示出快速的响应速率,能特异性地响应蛋白质分子,以该探针作为工具实现了细胞成像,亚细胞定位和细胞表达水平检测等生理学特征的研究。
附图说明
构成本发明的一部分的附图用来提供对本发明的进一步理解,本发明的示意性实施例及其说明用于解释本发明,并不构成对本发明的不当限定。在附图中:
图1.其中图1a-1c染料BDI,BDB,BDP在不同比例的水和1,4-二氧六环混合溶剂中的荧光光谱曲线;图1d-1f染料BDI,BDB,BDP在不同比例的水和甘油混合溶剂中的荧光光谱曲线。
图2.其中图2a 1μm BDI probe本身,及分别加入2.5μm oBSA和2.5μm rBSA的荧光光谱曲线;
图2b SDS-PAGE验证探针对VDPs的特异性;
图2c 1μm BDI probe当加入0.9μm rBSA实时动力学曲线;
图2d 1μm BDI probe在0-2.5μm rBSA的响应荧光光谱曲线。
图3共聚焦荧光成像MCF-7细胞中的VDPs,
其中图3a 1μm BDI probe孵育细胞,共聚焦细胞成像图,
图3b 1μm Control probe孵育细胞,共聚焦细胞成像图;
图3c先用10mM DTT孵育细胞,再用1μm BDI probe孵育细胞,共聚焦细胞成像图;
图3d先用30μM PAO孵育细胞,再用1μm BDI probe孵育细胞,进行共聚焦细胞成像图。
图4 MCF-7细胞共定位实验,共聚焦细胞成像图。
其中图a1-c1 Mito-Tracker Green(1.0μM)孵育细胞,共聚焦细胞成像图;
图a2-c2 Lyso-Tracker Green(0.05μM)孵育细胞,共聚焦细胞成像图;
图a3-c3 Hoechst 33342(0.5μg mL-1)孵育细胞,共聚焦细胞成像图;
图d1-d3是c1、c2、c3中画白线的荧光叠加图,c1中的x是线粒体定位试剂的,c2中x是溶酶体定位试剂,c3中的x是细胞核染料的,y是探针荧光曲线。
具体实施方式
下面结合实施例及附图来详细说明本发明。
实例1
BDI,BDB,BDP的合成
反应试剂与条件:(a)二甲胺盐酸盐,乙醇,三乙胺,150℃(b)三氯氧磷,N,N-二甲基甲酰胺,0℃,(c)乙腈,碘甲烷,回流,(d)2,哌啶,乙醇,回流(e)乙腈,碘甲烷,回流(f)甲苯,碘甲烷,回流,
中间体1:N,N-二甲基苯并[c][1,2,5]噁二唑-4-胺
称取4-氯苯并呋咱(6.0g,0.039mol)于100mL真空封管当中,加入50mL乙醇将其完全溶解,加入二甲胺盐酸盐(18g,0.22mol),再滴加三乙胺(36mL),迅速盖上自封管,在150℃加热条件下搅拌48h。反应完毕,冷却至室温,减压蒸馏除溶剂。加入氢氧化钠溶液(2M,100mL),用乙酸乙酯萃取(60mL×3),有机相用浓盐水洗,无水硫酸镁干燥,过滤,减压蒸馏除溶剂,得到纯产物1(5.5g),产率86.2%。Mp:31-32℃.1H NMR(400MHz,CDCl3)δ(ppm):3.30(s,6H,N(Me)2),6.06(d,J=7.5Hz,1H,Ar-H),7.04(d,J=8.9Hz,1H,Ar-H),7.22(dd,J=7.5Hz,Ar-H).13C NMR(100MHz,CDCl3)δ(ppm):41.78,101.60,104.63,133.41,139.62,145.58,150.78.ESI-MS(positive mode):m/z,Calcd.164.18,found 164.07for[M+H]+。
中间体2:7-(二甲基胺)苯并[c][1,2,5]噁二唑-4-醛基
将盛有20mL无水DMF的圆底烧瓶置于冰浴中,POCl3(12mL,0.13mol)缓慢加入,搅拌均匀,再将溶有化合物1(7.1g,0.044mol)的20mL DMF加入。撤去冰浴,室温下搅拌,用TLC检测反应。化合物1反应完全后,用50mL冰水倾入到混合物中猝灭反应,用10%的NaOH溶液调pH约等于9,用乙酸乙酯萃取,无水硫酸镁干燥有机相,过滤,减压蒸馏除溶剂。粗产品经柱层析分离[V(石油醚):V(乙酸乙酯)=2:1],得红色固体产物DBDC(6.2g),产率75.0%。Mp:113-115℃.1H NMR(400MHz,CDCl3)δ(ppm):3.51(s,6H,N(Me)2),6.15(d,J=8.1Hz,1H,Ar-H),7.89(d,J=8.4Hz,1H,Ar-H),10.02(s,1H,CHO).13C NMR(100MHz,CDCl3)δ(ppm):42.88,102.32,111.28,142.38,144.84,145.02,147.60,185.89.ESI-MS(positive mode):m/z,Calcd.192.19,found 192.08for[M+H]+。
中间体3:1,2,3,3-四甲基-3H-吲哚-1-碘鎓
将2,3,3-三甲基-3H-吲哚(3.3g,21.0mmol)和碘甲烷(4g,12.3mmol)溶于20mL乙腈中回流反应72h,反应结束后冷却,有沉淀析出,过滤,用少量冷的乙酸乙酯淋洗几遍沉淀固体,置于真空干燥箱干燥,得白色固体粉末1(3.1g),收率78.6%。产物纯度较高,可不经进一步纯化而直接用于下一步反应。
中间体4:1,2,3,3-四甲基-3H-苯并[g]吲哚-1-碘鎓
将2,3,3-三甲基苯并吲哚(2.0g,12.6mmol)和碘甲烷(2.4g,7.4mmol)溶于30mL乙腈中回流反应48h,反应结束后冷却,有沉淀析出,过滤,用少量冷的乙酸乙酯淋洗几遍沉淀固体,置于真空干燥箱干燥,得白色固体粉末2(18.6g),收率78.6%。产物纯度较高,可不经进一步纯化而直接用于下一步反应。
中间体5:1,4-二甲基吡啶-1-碘鎓
将4-甲基哌啶(0.93g,10mmol)和碘甲烷(1.75g,12.5mmol)混合在20mL甲苯中,室温搅拌一整晚,反应结束后,冷却至室温,有沉淀析出,过滤,固体沉淀用乙醚清洗几遍,置于真空干燥箱干燥,得黄色固体粉末5(2.1g,20mmol),收率89%。产物纯度较高,可不经进一步纯化而直接用于下一步反应。1H NMR(400MHz,DMSO-d6)δ(ppm):8.82(d,J=6.0Hz,2H),7.95(d,J=6.0Hz,2H),4.27(s,3H),2.58(s,3H).ESI-MS:m/z 108.12[M-I-]+(calcd.108.08)。
环境敏感染料BDI:(E)-2-(2-(7-(二甲基胺)苯并[c][1,2,5]噁二唑-4-基)乙烯基)-1,3,3-三甲基-3H-吲哚-1-碘鎓
将化合物2(96mg,0.5mmol)和化合物3(181mg,0.6mmol)混合溶解在10mL的无水乙醇中,加入8mg哌啶,加热回流16h,溶液逐渐由黄色变成蓝色,反应结束后冷却至室温,减压蒸馏除溶剂,粗产品经柱层析分离[V(二氯甲烷):V(甲醇)=100:1],得蓝色固体产物BDI(150mg),产率54.2%。1H NMR(400MHz,d6-DMSO)δ(ppm):1.80(s,6H),3.67(s,6H),3.93(s,3H),6.64-6.66(d,1H,J=8.0Hz),7.47-7.58(m,3H),7.75-7.81(m,2H),8.33-8.36(d,1H,J=12.0Hz),8.51-8.55(d,1H,J=16.0Hz).13C NMR(100MHz,d6-DMSO)δ(ppm):26.67,33.32,44.09,51.16,106.58,107.86,108.44,114.04,141.79,123.17,128.02,129.23,142.56,142.97,145.66,146.29,147.22,148.97,149.33,179.81.HRMS(EI):calcd.For C21H23N4O+347.1866;found 347.1806。
环境敏感染料BDB:(E)-2-(2-(7-(二甲基胺)苯并[c][1,2,5]噁二唑-4-基)乙烯基)-1,3,3-三甲基-3H-苯并[g]吲哚-1-碘鎓
将化合物2(96mg,0.5mmol)和化合物4(211mg,0.6mmol)混合溶解在10mL的无水乙醇中,加入8mg的哌啶,加热回流16h,溶液逐渐由黄色变成蓝色,反应结束后冷却至室温,减压蒸馏除溶剂,粗产品经柱层析分离[V(二氯甲烷):V(甲醇)=100:1],得蓝色固体产物BDB(168mg),产率54.7%。1H NMR(400MHz,CD3OD)δ(ppm):2.10(s,6H),3.71(s,6H),4.11(s,3H),6.56-6.58(d,1H,J=8.0Hz),7.62-7.65(t,1H,J=8.0Hz),7.74-7.77(d,1H,J=8.0Hz),7.81-7.87(m,2H),8.10-8.13(t,2H,J=4.0Hz),8.16-8.18(d,1H,J=8.0Hz,8.35-8.37(d,1H,J=8.0Hz),8.53-8.57(d,1H,J=16.0Hz).13C NMR(100MHz,CD3OD)δ(ppm):25.61,32.27,42.63,52.95,105.53,107.68,108.98,111.65,122.76,126.26,127.48,127.91,129.86,130.85,133.29,136.56,139.49,145.44,145.95,146.14,148.49,148.74,181.49.HRMS(EI):calcd.For C25H25N4O+397.2023;found 397.2006。
环境敏感染料BDP:(E)-4-(2-(7-(二甲基胺)苯并[c][1,2,5]噁二唑-4-基)乙烯基)-1-甲基吡啶-1-碘鎓
将化合物2(96mg,0.5mmol)和化合物5(142mg,0.6mmol)混合溶解在10mL的无水乙醇中,加入8mg哌啶,加热回流16h,溶液逐渐由黄色变成红色,反应结束后冷却至室温,减压蒸馏除溶剂,粗产品经柱层析分离[V(二氯甲烷):V(甲醇)=100:1],得红褐固体产物BDP(86mg),产率36.1%。1H NMR(400MHz,d6-DMSO)δ(ppm):3.45(s,6H),4.22(s,3H),6.35-6.37(d,1H,J=8.0Hz,),7.47-7.51(d,1H,J=16.0Hz),7.68-7.70(d,1H,J=8.0Hz),8.09-8.12(d,1H,J=12.0Hz),8.16-8.17(d,2H,J=4.0Hz),8.75-8.76(d,2H,J=4.0Hz.13C NMR(100MHz,d6-DMSO)δ(ppm):42.81,46.96,105.09,109.24,121.43,122.87,137.98,140.84,141.79,144.95,145.62,148.70,153.59.HRMS(EI):calcd.For C9H10N4O2 281.1397;found281.1307。邻二巯基蛋白探针(BDI probe)及控制探针(Control probe)的合成
反应试剂和条件:(a)苯肼,甲醇,回流,60min;(b)乙二硫醇,乙醇,回流,10min,61%;(c)PAO-EDT,DMF,2-(7-氧化苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯,N,N-二异丙基乙胺,24h,室温,81%;(d)三氟乙酸,二氯甲烷,室温,2h,87%;(e)乙醇,哌啶,回流,16h,65%;(f)2,DMF,2-(7-氧化苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯,N,N-二异丙基乙胺,24h,室温,49%;(g)苯胺,DMF,2-(7-氧化苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯,N,N-二异丙基乙胺,24h,室温,90%;(h)三氟乙酸,二氯甲烷,室温,2h,84%;(i)6,DMF,2-(7-氧化苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯,N,N-二异丙基乙胺,24h,室温,54%。
中间体PAO:(4-氨基苯基)亚砷酸
将对氨基苯砷酸(10.85g,50mmol)溶解在60mL甲醇中,加热回流,向反应液中逐滴滴加苯肼(10.3mL,100mmol)。当溶液不再产生氮气时,继续回流1小时,反应结束后,将反应液在80℃条件下浓缩。先加85mL的水,然后加入60mL 0.1M NaOH溶液,然后用150mL的乙醚萃取,在往水溶液中加入40mL 5M NH4Cl,将溶液置于0℃1h,有白色沉淀析出,过滤,用冰水洗涤产物得白色晶体(4.31g),产率39%。
中间体PAO-EDT:4-(1,3,2-二硫环砷-2-基)苯胺
将PAO(4.2g,22.5mmol)溶解在33mL无水乙醇中,加热回流。然后往溶液中逐滴加入(2.4mL,30mmol)乙二硫醇,继续加热搅拌10min。随后,将混合物置于冰水中冷却,过滤得到粗产品,在乙醇中进行重结晶得到白色固体(3.5g),产率61%。1H NMR(400MHz,DMSO-d6)δ(ppm):3.19-3.24(m,2H),3.26-3.30(m,2H),5.39(s,2H),6.55-6.57(d,J=4.0Hz,2H),7.25-7.27(d,J=4.0Hz,2H).13C NMR(DMSO-d6,100MHz)δ(ppm):41.61,114.07,127.75,132.45,150.65.HRMS(EI):m/z calcd for C9H10AsNS2[M]258.9471;found:258.9471。
中间体6:叔丁基(4-((4-(1,3,2-二硫环砷-2-基)苯基)氨基)-4-氧丁基)氨基甲酸盐(6)
将PAO-EDT(0.65g,2.5mmol),N-BOC-γ-氨基丁酸(0.51g,2.5mmol),2-(7-氧化苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯分别溶解在无水的DMF中,向溶液中滴加0.45mL N,N-二异丙基乙胺,在N2保护条件下室温搅拌24h,蒸除溶剂得粗产品,硅胶柱层析纯化得白色固体(0.9g),产率81%。1H NMR(CDCl3,400MHz)δ(ppm):1.46(s,9H),2.04(s,1H),2.36-2.39(t,J=8.0Hz,2H),3.13-3.19(m,2H),3.23-3.24(m,2H),3.31-3.37(m,2H),4.84(s,1H),7.57-7.63(m,4H),9.02(s,1H);13C NMR(CDCl3,100MHz)δ(ppm):171.42,171.17,157.39,139.45,138.20,131.44,119.50,79.95,39.19,34.63,28.40,27.44,21.04.HRMS(EI):calcd.For C17H25AsN2O3S2 444.0523;found 444.0515。
中间体7:N-(4-(1,3,2-二硫环砷-2-基)苯基)-4-氨基丁酰胺(7)
将中间体6(444mg,1.0mmol)溶解在1mL三氟乙酸和5mL二氯甲烷的混合溶剂中,室温搅拌2小时,反应完全后,旋干除去溶剂,得粗产品,硅胶柱层析纯化得白色固体(300mg),产率87%。1H NMR(DMSO-d6,400MHz)δ(ppm):1.82-1.90(m,2H),2.41-2.45(t,J=8.0Hz,1H),2.83-2.87(t,J=8.0Hz,2H),3.12-3.20(m,2H),3.32-3.39(m,4H),7.55-7.63(m,2H),10.17(s,1H);13C NMR(DMSO-d6,100MHz)δ(ppm):175.88,145.29,142.51,136.54,124.01,46.60,43.70,38.16,28.15.HRMS(EI):calcd.For C12H17AsN2OS2 343.9998;found343.9987。
中间体:81-(2-羧乙基)-2,3,3-三甲基-3H-吲哚-1-碘鎓(8)
将2,3,3-三甲基-3H-吲哚(5g,31.4mmol)和3-碘丙酸(6.6g,31.4mmol)溶解在5mL甲苯中,在N2保护条件下,加热到100℃回流24h后。旋干蒸除溶剂,在超声条件下缓慢加入乙酸乙酯,有大量沉淀析出。过滤,乙酸乙酯洗涤沉淀干燥后得白色固体(8.14g),产率72%。1H NMR(DMSO-d6,400MHz)δ(ppm):1.53(s,6H),2.86(s,3H),2.97-3.0(t,J=4.0Hz,2H),4.64-4.67(t,J=4.0Hz,2H),7.61-7.63(m,2H),7.83-7.85(m,1H),7.98-8.0(m,1H).13C NMR(DMSO-d6,100MHz)δ(ppm):198.39,171.99,142.23,141.31,129.83,129.40,123.95,116.04,54.74,44.01,31.57,22.35,14.84.MS(ESI):m/z calcd for C14H18O2N[M]+232.1;found:232.2。
中间体9:(E)-1-(2-羧基乙基)-2-(2-(7-(二甲基胺)苯并[c][1,2,5]噁二唑-4-基)乙烯基)-3,3-二甲基-3H-吲哚-1-碘鎓(9)
将中间体8(0.72g,2mmol)和7-(二甲基胺)-苯并[c][1,2,5]噁二唑-4-醛基(2mmol,0.38g)溶解在30mL无水乙醇中,再向反应液中滴加哌啶(30μL)作为催化剂,在N2保护条件下加热回流16h,将反应液置于冰浴中冷却,过滤得蓝色粗产品,乙醇重结晶得深蓝色固体(0.69g),产率65%。1H NMR(CD3OD,400MHz)δ(ppm):1.84(s,6H),2.88(s,2H),3.71(s,6H),4.69(s,2H),6.55-6.57(d,J=8.0Hz,1H),7.48-7.55(m,2H),7.65-7.71(m,2H),7.82-7.85(d,J=6.0Hz,1H),8.10-8.12(d,J=4.0Hz,2H),8.45-8.49(d,J=8.0Hz,1H).13CNMR(CD3OD,100MHz)δ(ppm):25.93,42.72,46.96,47.18,47.39,47.60,47.81,48.03,48.24,51.00,99.99,105.69,107.63,109.00,113.39,122.30,127.58,128.78,141.25,141.71,142.60,145.34,146.11,146.57,148.81,149.62,179.87.MS(ESI):m/z calcd forC23H25N4O3[M]+405.2;found:405.2。
邻二巯基蛋白BDI probe:(E)-1-(3-((4-((4-(1,3,2-二硫环砷-2-基)苯基)氨基)-4-氧丁基)氨基)-3-氧丙基)-2-(2-(7-(二甲基胺)苯并[c][1,2,5]噁二唑-4-基)乙烯基)-3,3-二甲基-3H-吲哚-1-碘鎓(BDI probe)
将中间体5(53.2mg,0.1mmol),中间体2(34.4mg,0.1mmol)和2-(7-氧化苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(45.5mg,0.12mmol)溶解在无水DMF中,向溶液中滴加N,N-二异丙基乙胺(34μL,0.2mmol),在N2保护室温条件下搅拌24h。反应完全后,旋干蒸除溶剂,硅胶柱层析纯化得深蓝固体(35mg),产率49%。1H NMR(D-CD3OD,400MHz)δ(ppm):1.74(s,6H),1.92-1.94(m,2H),2.07-2.11(t,J=8.0Hz,2H),2.78-2.81(t,J=8.0Hz,2H),2.98-3.02(t,J=8.0Hz,2H),3.04-3.09(m,2H),3.10-3.16(m,2H),3.56(s,5H),4.62-4.65(t,J=4.0Hz,2H),6.37-6.40(d,J=6.0Hz,2H),7.33-7.47(m,6H),7.54-7.57(t,J=8.0Hz,2H),7.69-7.72(d,J=6.0Hz,1H),7.96-7.98(d,J=4.0Hz,1H),8.34-8.38(d,J=8.0Hz,1H);13C NMR(CDCl3,100MHz)δ(ppm):27.26,27.50,31.46,31.96,34.56,35.96,48.76,41.71,51.27,55.05,106.19,113.59,119.49,122.48,128.26,129.53,129.89,131.19,137.62,139.66,141.04,141.96,144.99,146.46,169.55,171.63,179.76.HRMS(ESI):m/z calcd for C35H40AsN6O3S2[M]+731.1814;found:731.1811。
中间体10:叔丁基(4-氧-4-(苯氨基)丁基)氨基甲酸盐(10)
将苯胺(0.3g,3.2mmol),N-BOC-γ-氨基丁酸(0.51g,2.5mmol),2-(7-氧化苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯分别溶解在无水的DMF中,向溶液中滴加0.45mL N,N-二异丙基乙胺,在N2保护条件下室温搅拌24h,蒸除溶剂得粗产品,硅胶柱层析(洗脱液:石油醚/乙酸乙酯=5/1,v/v)纯化得白色固体(0.8g),产率90%。1H NMR(CD3Cl,400MHz)δ(ppm):1.46(s,9H),1.84-1.91(m,2H),2.36-2.40(t,J=8.0Hz,2H),3.24-3.25(m,2H),4.83(s,1H),7.06-7.10(t,J=8.0Hz,1H),7.26-7.33(m,2H),7.59-7.61(d,J=4.0Hz,2H),8.72(s,1H);13C NMR(CD3Cl,100MHz)δ(ppm):23.32,33.03,39.12,120.26,124.01,128.66,138.08,171.58.HRMS(EI):calcd.For C15H22N2O3 278.1630;found 278.1616。
中间体11:4-氨基-N-苯基丁酰胺(11)
将中间体1(444mg,1.0mmol)溶解在2mL三氟乙酸和4mL二氯甲烷的混合溶剂中,室温搅拌2小时,反应完全后,旋干除去溶剂,得粗产品,硅胶柱层析纯化得白色固体(150mg),产率84%。1H NMR(CD3OD,400MHz)δ(ppm):1.99-2.06(m,2H),2.52-2.55(t,J=4.0Hz,2H),3.00-3.03(t,J=8.0Hz,2H),7.07-7.10(m,2H),7.28-7.32(t,J=8.0Hz,2H),7.56-7.58(d,J=8.0Hz,2H),10.17(s,1H);13C NMR(DMSO-d6,100MHz)δ(ppm):23.32,33.03,39.12,120.26,124.01,128.66,138.08,171.58.HRMS(EI):calcd.For C10H14N2O 178.1106;found178.1099。
控制探针:(E)-2-(2-(7-(二甲基胺)苯并[c][1,2,5]噁二唑-4-基)乙烯基)-3,3-二甲基-1-(3-氧-3-((4-氧-4-(苯氨基)丁基)氨基)丙基)-3H-吲哚-1-碘鎓(Controlprobe)
将中间体5(53.2mg,0.1mmol),中间体7(17.8mg,0.1mmol)和2-(7-氧化苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(45.5mg,0.12mmol)溶解在无水DMF中,向溶液中滴加N,N-二异丙基乙胺(34μL,0.2mmol),在Ar保护室温条件下搅拌24h。反应完全后,旋干蒸除溶剂,硅胶柱层析纯化得深蓝固体(31mg),产率54%。Yield:31mg(54%).1H NMR(D-CD3OD,400MHz)δ(ppm):1.46(s,9H),2.04(s,1H),3.12-3.18(m,2H),3.31-3.38(m,2H),3.93-3.94(d,2H),5.58(s,1H),4.84(s,1H),7.48-7.57(m,4H),8.66(s,1H);
13C NMR(CDCl3,100MHz)δ(ppm):168.18,156.67,138.98,138.45,131.54,119.75,45.46,41.85,29.71,28.34.HRMS(ESI):m/z calcd for C33H37N6O3 +[M]+565.2922;found:565.2960。
测定周围环境对荧光分子性质的影响
测量溶剂的极性和粘度对三种荧光团荧光性质的影响,从图1可以看出,探针在PBS缓冲溶液中荧光强度非常弱,三种荧光团的荧光强度随着溶剂的极性的减少而逐渐增强(图1a-c),同样地,随着溶液粘度的增加而增加(图1d-f)。说明该类分子属于环境敏感染料。但是,对比三种荧光团的环境敏感性质,我们发现染料BDI在小极性和高粘度的介质中荧光增强的倍比更高,所以,我们选择染料BDI作为荧光报告基团,用于邻二巯基蛋白的检测。邻二巯基蛋白探针(BDI probe)对rBSA的响应情况
1.0μm BDI probe溶液荧光很弱,往探针溶液中加入2.5μm rBSA时,荧光显著增强,加入2.5μm oBSA时,荧光强度增加的并不明显(图2a)。为了证明探针响应邻二巯基蛋白的选择性,我进行了十二烷基硫酸钠聚丙烯酰胺凝胶电泳实验,发现探针与rBSA结合后,有强的红色荧光条带,然而探针与BSA和oBSA混合后,并没有强的荧光信号,而且控制探针(Control probe)与rBSA混合后也没有荧光信号,考马斯亮蓝染色实验再次证明BDI probe与rBSA发生共价交联形成混合体,从而发出荧光(图2b)。接下来做一个BDI probe实时响应rBSA的工作曲线。图2c表明当探针加入0.9μm rBSA后荧光强度迅速增强,在30s时荧光强度达到一个平衡,对比之前的邻二巯基蛋白探针,说明探针的效应速度非常快。BDI probe能够高灵敏度地检测邻二巯基蛋白,随着rBSA浓度的增加,荧光信号逐渐增强,在2.5μm时达到荧光强度最大,有35倍的荧光信倍比(图2d)。
BDI probe用于检测MCF-7细胞内的邻二巯基蛋白
当用1.0μm BDI probe培育MCF-7细胞,然后进行共聚焦荧光成像,细胞有强的荧光信号(图3a1-3a2),而当用1.0μm Control probe培育MCF-7细胞时,细胞并没有产生强的荧光信号。以上结果说明,BDI probe能够特异性检测细胞内的邻二巯基蛋白。当用一定量的DTT和PAO提前培育细胞一段时间后,然后用BDI probe培育细胞,在同一条件下进行共聚焦荧光成像发现用DTT培育过的细胞,荧光强度明显增强,而用PAO处理过的细胞,荧光强度明显减弱。这是因为DTT是二硫键的还原剂,能提高细胞内邻二巯基蛋白的含量,PAO能够特异性地和邻二巯基蛋白发生反应,阻止了BDI probe与邻二巯基蛋白的反应。上述结果证明,BDI probe能够特异性地检测活细胞内邻二巯基蛋白的含量。
BDI probe亚细胞器共定位实验
为了测试我们的探针能靶向检测线粒体内的邻二巯基蛋白,进一步进行BDIprobe在MCF-7细胞中亚细胞共定位的荧光成像实验。线粒体绿,溶酶体绿和Hoechst 33342蓝分别用于线粒体,溶酶体和细胞核的染色成像实验。首先用BDI probe培育细胞,然后分别用线粒体绿,溶酶体绿和Hoechst 33342蓝培育细胞一段时间,然后进行共聚焦荧光成像实验,我们发现探针的荧光信号与线粒体的荧光信号基本上是完全重合的,然而,BDIprobe荧光信号与溶酶体绿和细胞核染的重叠程度非常低,以上结果证明BDI probe能够特异性地检测到MCF-7细胞线粒体中的邻二巯基蛋白。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (7)
2.一种制备如权利要求1所述的一种基于苯并呋咱的环境敏感染料的方法,其特征在于:所述制备方法包括以下步骤:
(1)称取4-氯苯并呋咱3~15g于真空封管当中,依次加入50mL乙醇、10~25g二甲胺盐酸盐、25~45mL三乙胺,在110~170℃加热条件下搅拌48h,冷却至室温,加入70~130mL 2M氢氧化钠溶液,用乙酸乙酯萃取后干燥、过滤,得到化合物1,化合物1为N,N-二甲基苯并[c][1,2,5]噁二唑-4-胺;
(2)将盛有10~35mL无水DMF的烧瓶置于冰浴中,加入5~20mL POCl3,搅拌均匀,加入3~10g化合物1,室温下搅拌,将50mL冰水倾入到烧瓶中,用10%的NaOH溶液调pH约等于9,用乙酸乙酯萃取,干燥并过滤,得到化合物2,化合物2为7-(二甲基胺)苯并[c][1,2,5]噁二唑-4-醛基;
(3)将50~120mg化合物2与120~220mg强拉电子供体化合物溶解在10mL的无水乙醇中,所述所述强拉电子供体化合物为1,2,3,3-四甲基-3H-吲哚-1-碘鎓或1,2,3,3-四甲基-3H-苯并[g]吲哚-1-碘鎓或1,4-二甲基吡啶-1-碘鎓中的一种,加入1~15mg哌啶,加热回流16h,溶液逐渐由黄色变成蓝色,反应结束后冷却至室温,得到产物。
3.根据权利要求2所述的一种基于苯并呋咱的环境敏感染料的制备方法,其特征在于:所述1,2,3,3-四甲基-3H-吲哚-1-碘鎓的制备方法如下:将2~5g 2,3,3-三甲基-3H-吲哚和2~8g碘甲烷溶于20mL乙腈中回流反应72h,反应结束后冷却、过滤、干燥,得到产物。
4.根据权利要求2所述的一种基于苯并呋咱的环境敏感染料的制备方法,其特征在于:所述1,2,3,3-四甲基-3H-苯并[g]吲哚-1-碘鎓的制备方法如下:将1~5g 2,3,3-三甲基苯并吲哚和1~5g碘甲烷溶于30mL乙腈中回流反应48h,反应结束后冷却、过滤、干燥,得到产物。
5.根据权利要求2所述的一种基于苯并呋咱的环境敏感染料的制备方法,其特征在于:所述1,4-二甲基吡啶-1-碘鎓的制备方法如下:将0.1~1.5g 4-甲基哌啶和0.5~4g碘甲烷混合于20mL甲苯中,室温搅拌8~12h,反应结束后冷却、过滤、干燥,得到产物。
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