CN113717544B - 基于胺基芴骨架的快速及长时间溶酶体或细胞核靶向的近红外染色试剂及其制备方法和应用 - Google Patents
基于胺基芴骨架的快速及长时间溶酶体或细胞核靶向的近红外染色试剂及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种基于胺基芴骨架的快速及长时间溶酶体或细胞核靶向的近红外染色试剂及其制备方法和应用,本发明以胺基芴骨架作为溶酶体或细胞核染料的基础,通过不同形式的烷基胺的调控设计,得到基于胺基芴骨架的溶酶体或细胞核靶向的近红外染色试剂,该类试剂在快速靶向染色的同时,具有超高的稳定性,有利于长时间监测,同时具有较好的生物相容性,不会对活细胞的生理活动产生明显的干扰。由于该染色试剂具有超大斯托克斯位移的特性,在进行生物成像时,基本不会受到激发光所造成的成像干扰,使得该类染料具有高信噪比成像的优势。本发明的制备方法收率高、反应条件温和,制得的染色试剂稳定性好、发射波长较长、斯托克斯位移大、靶向性高。
Description
技术领域
本发明涉及溶酶体及细胞核靶向染色技术领域,具体涉及到一种基于胺基芴骨架的快速及长时间溶酶体或细胞核靶向的近红外染色试剂及其制备方法和应用。
背景技术
溶酶体是分解蛋白质、核酸、多糖等生物大分子的细胞器,内含多种水解酶,是0.025~0.8微米的泡状结构。溶酶体在细胞中的功能,是分解从外界进入到细胞内的物质,也可消化细胞自身的局部细胞质或细胞器。溶酶体内酸性的微环境(pH 4.5-5.5)能够保证水解酶的活性,使得细胞内消化和降解的过程可以顺利进行。溶酶体也是细胞内吞过程的“终点站”,与细胞死亡的三个通路:细胞凋亡(Apoptosis)、II型细胞程序化死亡(Type IIProgrammed cell death)和细胞坏死(Necrosis)都有一定程度的关联。因此,开发高选择性、高灵敏度以及高稳定性的溶酶体靶向荧光染料以准确对活细胞内溶酶体进行实时成像,对于探索与解决溶酶体相关生物医学基本问题具有非常深远的意义。
细胞核是真核细胞中最大、最重要的亚细胞器,是细胞遗传和新陈代谢的活动中心,在细胞的代谢、生长、分化等过程中起着不可替代的作用。细胞核的异常也与很多生物过程密切相关,比如细胞凋亡,有丝分裂等过程。凋亡过程中的细胞核的形态明显出现畸变以及皱缩,核仁变大等形态学特征。而有丝分裂过程中的核膜会出现破裂、DNA进行自我复制等的现象。因此,开发高选择性、高灵敏度以及高稳定性的细胞核靶向荧光染料以准确对活细胞内的细胞核进行实时成像,对于探索与解决与细胞核相关的生物医学等基本问题具有重要的意义。
现有的市售溶酶体染色染料通常为基于氟硼二吡咯结构的荧光染料,该类荧光染料光稳定性较差,同时活细胞内的谷胱甘肽等还原性生物硫醇容易造成干扰,染料的斯托克斯位移太小,成像信噪比较低,成像所需时间较长等,这些不足限制了其进一步应用。因此,开发一种具有大斯托克斯位移、光稳定性优异和染色速度快的溶酶体靶向荧光染料具有重要意义。而现有的市售细胞核染色染料通常具有价格高,荧光染料的光稳定性差,荧光发射的波长短,灵敏性较低等缺陷。因此,开发一种具有合成方法简单、光稳定性优异,靶向性好,信噪比高的细胞核靶向荧光染料具有重要意义。
发明内容
针对上述的不足,本发明的目的是提供一种基于胺基芴骨架的快速及长时间溶酶体或细胞核靶向的近红外染色试剂及其制备方法和应用,可有效解决现有溶酶体以及细胞核靶向染料因其合成繁琐、发射波长短、斯托克斯位移小,成像时间长等导致的操作过程复杂、成本高、成像结果准确性低、耗时长等的问题。
为达上述目的,本发明采取如下的技术方案:
本发明提供一种基于胺基芴骨架的快速及长时间溶酶体或细胞核靶向的近红外染色试剂,其结构如式Ⅰ所示:
式Ⅰ中:
R1为C1-C10烷基链或芳香基团;
进一步地,在本发明较佳的实施例中:
R1为C3烷基链;
本发明还提供上述基于胺基芴骨架的快速及长时间溶酶体或细胞核靶向的近红外染色试剂的制备方法,包括以下步骤:
步骤(1):将3,6-二溴芴酮和烷基胺溶于第一有机溶剂中,除氧后于惰性气体保护下加入催化剂、配体和碱,加热搅拌,得到第一中间体;
步骤(2):将步骤(1)所得的第一中间体加入到含第二有机溶剂的反应容器中搅拌,随后加入草酰氯,浓缩后加入到含有机碱和R1取代的有机胺的第二有机溶剂中搅拌,制得基于胺基芴骨架的快速及长时间溶酶体或细胞核靶向的近红外染色试剂;
或将步骤(1)所得的第一中间体加入到含有机碱的第二有机溶剂中搅拌,加入三氟甲磺酸酐,随后加入R1取代的有机胺搅拌,制得基于胺基芴骨架的快速及长时间溶酶体或细胞核靶向的近红外染色试剂;
其中,R1为C1-C10烷基链或芳香基团;
烷基胺的结构式如下所示:
进一步地,步骤(1)中3,6-二溴芴酮、烷基胺、催化剂、配体和碱的摩尔比为1:(2.2~3.0):(0.05~0.1):(0.08~0.2):(3.0~7.0);优选为1:2.2:0.05:0.08:6.6。
进一步地,步骤(2)中第一中间体、草酰氯、有机碱和R1取代的有机胺的摩尔比为1:(10.0~100.0):(20.0~50.0):(5.0~20.0);优选为1:30.0:20.0:10.0。
进一步地,步骤(2)中第一中间体、有机碱、三氟甲磺酸酐和和R1取代的有机胺的摩尔比为1:(8.0~30.0):(5.0~10.0):(5.0~20.0);优选为1:8.0:5.0:10.0。
进一步地,步骤(1)中第一有机溶剂为乙腈、四氢呋喃、二氯甲烷、三氯甲烷、二甲基亚矾、邻二氯苯、氯苯、甲苯、二甲苯、均三甲苯、1,4-二氧六环、1,2-二氯乙烷、N,N-二甲基甲酰胺和N,N-二甲基乙酰胺中的至少一种,优选为甲苯;步骤(1)中惰性气体包括氩气和氮气中的一种或两种组合;步骤(1)中催化剂包括双二亚苄基丙酮钯和三(二亚苄基丙酮)二钯中的一种或两种组合;步骤(1)中配体包括三叔丁基膦和三正丁基膦中的一种或两种组合;步骤(1)中碱包括叔丁醇钾和叔丁醇钠中的一种或两种组合。
进一步地,步骤(2)中第二有机溶剂包括二氯甲烷、乙腈和四氢呋喃中的至少一种;步骤(2)中有机碱包括三乙胺和吡啶中的一种或两种组合。
进一步地,步骤(1)中的加热温度为100-120℃,步骤(2)中的搅拌温度为0-25℃。
本发明还提供上述基于胺基芴骨架的快速及长时间溶酶体或细胞核靶向的近红外染色试剂在生物体溶酶体或细胞核内荧光成像中的应用。
一种近红外荧光造影剂,包括上述的基于胺基芴骨架的快速及长时间溶酶体或细胞核靶向的近红外染色试剂。
综上所述,本发明具有以下优点:
1、本发明提供了一种基于胺基芴骨架的快速及长时间溶酶体或细胞核靶向的近红外染色试剂,本发明以胺基芴为基础骨架,通过进行不同形式的烷基胺的修饰,设计并合成了近红外、光稳定性好、染色时间快速以及斯托克斯大的溶酶体或细胞核靶向的近红外染色试,可用于体外培养细胞、组织细胞的溶酶体或细胞核染色。
2、本发明所制得的近红外染色试剂具有较长的荧光发射(>700nm)以及较大的斯托克斯位移(>140nm),可以有效地避免背景光的干扰;本发明的染色试剂对于溶酶体的染色具有超快速染色、高稳定性成像的特性,能够对细胞进行长时间的监测;对于细胞核的染色具有较高的清晰度以及较高的稳定性,可以对细胞核的形态进行追踪研究;解决了现有溶酶体以及细胞核靶向染料因其合成繁琐、发射波长短、斯托克斯位移小,成像时间长等导致的操作过程复杂、成本高、成像结果准确性低、耗时长等的问题。
附图说明
图1为本发明制备方法的合成路线图。
图2为实施例1的染色试剂的氢谱。
图3为实施例1的染色试剂的碳谱。
图4为实施例2的染色试剂的氢谱。
图5为实施例2的染色试剂的碳谱。
图6为实施例3的染色试剂的氢谱。
图7为实施例3的染色试剂的碳谱。
图8为实施例1的染色试剂在PBS溶液中的紫外吸收光谱。
图9为实施例2的染色试剂在PBS溶液中的紫外吸收光谱。
图10为实施例3的染色试剂在PBS溶液中的紫外吸收光谱。
图11为实施例1的染色试剂在PBS溶液中的荧光发射光谱。
图12为实施例2的染色试剂在PBS溶液中的荧光发射光谱。
图13为实施例3的染色试剂在PBS溶液中的荧光发射光谱。
图14为实施例1、2、3的染色试剂的CCK-8细胞毒性实验。
图15为实施例1、2、3的染色试剂在HepG2细胞中的溶酶体染色激光共聚焦实验。
图16为实施例2、3的染色试剂在HepG2细胞中的细胞核染色激光共聚焦实验。
图17为实施例1、2、3的染色试剂在HepG2细胞中溶酶体染色的光稳定性激光共聚焦实验。
图18为实施例2、3的染色试剂在HepG2细胞中细胞核染色的光稳定性激光共聚焦实验。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白,以下结合实施例,对本发明进行进一步详细说明。应当理解,此处所描述的具体实施例仅用以解释本发明,并不用于限定本发明,即所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。
因此,以下对提供的本发明的实施例的详细描述并非旨在限制要求保护的本发明的范围,而是仅仅表示本发明的选定实施例。基于本发明的实施例,本领域技术人员在没有做出创造性劳动的前提下所获得的所有其他实施例,都属于本发明保护的范围。
本发明实施例中,3,6-二溴芴酮、各类溶剂、催化剂、配体、碱购于阿拉丁科技有限公司,细胞株购于ATCC(American Type Culture Collection),10%胎牛血清(FBS)购于Hyclone,DMEM培养基购于美国Gibco。
本发明实施例的合成路线如图1所示,过程包括:
(1)将3,6-二溴芴酮和烷基胺溶于第一有机溶剂中,除氧后用惰性气体保护,加入催化剂、配体和强碱加热搅拌,得到第一中间体;
(2)将所述第一中间体加入到含第二有机溶剂中搅拌,随后加入草酰氯,浓缩后加入到含有有机碱和R1取代的有机胺的第二有机溶剂中搅拌制得基于胺基芴骨架的快速及长时间溶酶体或细胞核靶向的近红外染色试剂;或者将所述第一中间体加入到含有有机碱的第二有机溶剂中搅拌,加入三氟甲磺酸酐,随后加入R1取代的有机胺搅拌制得基于胺基芴骨架的快速及长时间溶酶体或细胞核靶向的近红外染色试剂。
下面结合实施例对本发明进一步说明。
实施例1:
本实施例的以胺基芴为骨架的快速及长时间溶酶体靶向的近红外染色试剂1的制备方法,包括以下步骤:
(1)合成第一中间体:3,6-双(二乙胺基)-9氢-芴-9-酮。
合成路线如下:
向250mL双颈瓶中加入80mL甲苯,除氧后在氩气保护下加入二亚苄基丙酮钯(86.25mg,0.15mmol)和三叔丁基膦(486μL,0.24mmol,10%w/v in toluene),室温搅拌10分钟后加入3,6-二溴芴酮(1.024g,3.0mmol)、二乙胺(483mg,6.6mmol)和叔丁醇钠(1.9g,19.8mmol),加热到110℃回流18个小时。随后冷却到室温,用硅藻土过滤,乙酸乙酯洗涤三次后再用二氯甲烷洗涤,直至洗涤完全。合并有机相,浓缩后用200-300目硅胶柱层析纯化。用石油醚/乙酸乙酯(20:1)梯度洗脱,得到第一中间体为橙色固体,产率为67%。
1H NMR(400MHz,Chloroform-d)δ7.47(d,J=8.4Hz,2H),6.72(d,J=2.3Hz,2H),6.42(dd,J=8.5,2.3Hz,2H),3.47(q,J=7.1Hz,8H),1.24(t,J=7.1Hz,12H);13C NMR(101MHz,Chloroform-d)δ191.2,152.0,146.2,125.4,123.6,109.6,102.3,44.7,12.8.
(2)合成基于胺基芴骨架的快速及长时间溶酶体靶向染色试剂:N-(6-(二乙胺基)-9-(丙胺基)-3氢-芴-3-亚基)-N-乙基乙铵。
合成路线如下:
在二氯甲烷(10mL)中加入化合物2(64.5mg,0.2mmol),冰浴下缓慢滴加过量的草酰氯(0.5mL),反应30分钟后减压浓缩,浓缩物加适量二氯甲烷溶解后缓慢加入到丙胺(118mg,2.0mmol)、三乙胺(404mg,4.0mmol)和二氯甲烷(8mL)的混合体系中,室温搅拌2小时后减压蒸馏除去溶剂,用200-300目硅胶柱层析纯化。二氯甲烷/甲醇(30:1)作洗脱剂,得到紫色固体溶酶体染色试剂1,产率为88%。
1H NMR(400MHz,Chloroform-d)δ10.44–10.14(m,1H),7.94(d,J=8.8Hz,1H),7.40(d,J=9.0Hz,1H),6.78(d,J=2.4Hz,1H),6.67(d,J=2.3Hz,1H),6.42(dd,J=8.9,2.4Hz,1H),6.37(dd,J=9.0,2.5Hz,1H),3.80(q,J=6.7Hz,2H),3.53(q,J=7.4Hz,4H),3.48(q,J=7.3Hz,4H),1.92(h,J=7.3Hz,2H),1.28(t,J=7.1Hz,6H),1.24(t,J=7.1Hz,6H),1.05(t,J=7.4Hz,3H);13C NMR(101MHz,Chloroform-d)δ166.79,153.34,153.27,148.33,144.27,130.86,128.24,118.58,115.74,110.90,109.78,104.65,103.38,48.37,45.30,45.22,22.31,12.94,11.45.
本实施例制得的以胺基芴为骨架的溶酶体靶向染色试剂1的氢谱和碳谱分别如图2和图3所示。
实施例2:
本实施例的以胺基芴为骨架的快速及长时间溶酶体或细胞核靶向染色试剂2的制备方法,包括以下步骤:
(1)合成第一中间体:3,6-二吡咯烷基-9氢-芴-9-酮。
合成路线如下:
向250mL双颈瓶中加入80mL甲苯,除氧后在氩气保护下加入二亚苄基丙酮钯(86.25mg,0.15mmol)和三叔丁基膦(486μL,0.24mmol,10%w/v in toluene),室温搅拌10分钟后加入3,6-二溴芴酮(1.024g,3.0mmol)、四氢吡咯(470mg,6.6mmol)和叔丁醇钠(1.9g,19.8mmol),加热到110℃回流18个小时。随后冷却到室温,用硅藻土过滤,乙酸乙酯洗涤三次后再用二氯甲烷洗涤,直至洗涤完全。合并有机相,浓缩后用200-300目硅胶柱层析纯化。用石油醚/乙酸乙酯(20:1)梯度洗脱,得到第一中间体为橙色固体,产率为65%。
1H NMR(400MHz,Chloroform-d)δ7.46(d,J=8.2Hz,2H),6.58(d,J=2.1Hz,2H),6.26(dd,J=8.3,2.1Hz,2H),3.38–3.41(m,4H),2.09–1.94(m,4H);13C NMR(101MHz,Chloroform-d)δ191.6,151.7,145.9,125.1,123.7,109.9,103.1,47.9,25.4.
(2)合成基于胺基芴骨架的快速及长时间溶酶体或细胞核靶向染色试剂2。
合成路线如下:
在二氯甲烷(10mL)中加入化合物3(63.7mg,0.2mmol)和吡啶(126.6mg,1.6mmol),冰浴下缓慢滴加三氟甲磺酸酐(282mg,1.0mmol),继续搅拌30分钟。然后将丙胺(118mg,2.0mmol)加入到体系之中,在室温下搅拌6小时,以薄层色谱监控反应完全后,减压蒸馏除去溶剂,粗品经200-300目硅胶柱层析纯化,用二氯甲烷/甲醇(30:1)洗脱,得到紫色固体溶酶体或细胞核染色试剂2,产率为54%。
1H NMR(400MHz,Chloroform-d)δ10.08(s,1H),7.82(d,J=8.7Hz,1H),7.30(d,J=8.8Hz,1H),6.76(d,J=2.3Hz,1H),6.63(d,J=2.2Hz,1H),6.23–6.17(m,2H),3.68(d,J=9.5Hz,2H),3.47–3.55(m,4H),3.44–3.35(m,4H),2.12–2.07(m,4H),2.05–2.00(m,4H),1.89(h,J=7.4Hz,2H),1.03(t,J=7.4Hz,3H);13C NMR(101MHz,Chloroform-d)δ166.9,152.7,152.6,147.8,143.8,130.4,127.5,118.6,115.7,111.0,110.2,105.9,104.6,48.3,48.1,25.3,25.3,22.1,11.3.
本实施例制得的以胺基芴为骨架的快速及长时间溶酶体或细胞核靶向染色试剂2的氢谱和碳谱分别如图4和图5所示。
实施例3:
本实施例与实施例2基本相同,区别在将起始原料四氢吡咯替换为氮杂环丁烷制得溶酶体或细胞核染色试剂3,其合成路线如下:
得到紫色固体溶酶体或细胞核染色试剂3,两步反应的产率分别为94%和33%。
化合物4:1H NMR(400MHz,Chloroform-d)δ7.46(d,J=8.1Hz,2H),6.42(d,J=2.0Hz,2H),6.12(dd,J=8.2,2.1Hz,2H),4.03(t,J=7.3Hz,8H),2.43(p,J=7.3Hz,4H);13CNMR(101MHz,Chloroform-d)δ191.5,155.3,145.6,125.1,124.7,108.9,101.9,51.7,16.5.
溶酶体或细胞核染色试剂3:1H NMR(400MHz,Chloroform-d)δ10.16(t,J=4.9Hz,1H),7.81(d,J=8.5Hz,1H),7.28(d,J=8.7Hz,1H),6.49(d,J=2.2Hz,1H),6.36(d,J=2.1Hz,1H),5.98(dd,J=6.4,2.2Hz,1H),5.96(dd,J=6.4,2.2Hz,1H),4.18(t,J=7.5Hz,4H),4.05(t,J=7.4Hz,4H),3.75–3.64(m,2H),2.51(p,J=7.5Hz,2H),2.43(p,J=7.3Hz,2H),1.89(h,J=7.4Hz,2H),1.04(t,J=7.4Hz,3H);13C NMR(101MHz,Chloroform-d)δ167.0,155.2,155.0,147.5,143.4,130.4,127.4,119.1,115.9,109.0,108.0,103.8,102.7,51.3,48.4,22.0,16.2,11.3.
本实施例制得的以胺基芴为骨架的快速及长时间溶酶体或细胞核靶向染色试剂3的氢谱和碳谱分别如图6和图7所示。
试验例1紫外吸收光谱
将上述实施例1-3制得的基于胺基芴骨架的快速及长时间溶酶体或细胞核靶向的近红外染色试剂分别配制成浓度为10mM的DMSO母液。把溶酶体靶向的染色试剂的母液稀释成浓度为1.25、2.5、5.0、10.0、20.0μM的PBS溶液,把溶酶体或细胞核靶向的染色试剂2和3分别稀释成浓度为1、2、4、6、8、10、12μM的PBS溶液,分别扫描其紫外吸收值并绘制吸收曲线。实施例1的染色试剂的紫外吸收光谱如图8所示,实施例2的染色试剂的紫外吸收光谱如图9所示,实施例3的染色试剂的紫外吸收光谱如图10所示。如图所示,实施例1、2和3均有三个吸收峰,峰值λ分别在340nm、410nm以及560nm附近。
试验例2荧光光谱光谱
将实施例1、2、3制得的染色试剂配成浓度为10mM的DMSO母液。随后分别稀释成浓度为10μM的PBS溶液,并测定其荧光光谱,得到荧光发射曲线。在PBS的溶液中,待测物实施例1、2、3的最大发射波长发生了明显的红移,到了近红外发射区域。其中,实施例1的染色试剂的荧光强度如图11,实施例2的染色试剂的荧光强度如图12,实施例3的染色试剂的荧光强度如图13。
试验例3 CCK-8细胞毒性实验
处于对数生长期的HepG2细胞接种于96孔板中,每孔接种约10000个细胞,用含10%胎牛血清(FBS)、1%双抗的(青霉素-链霉素,1000KU/L)的DMEM(H)培养基在37℃和5%CO2条件下培养24小时。待细胞完全贴壁,加入不同浓度梯度的实施例1、2、3制得的染色试剂,每个浓度设3个复孔,同时设空白对照组。加染色试剂后继续培养24小时,用CCK-8毒性试剂盒检测细胞的存活率,结果如图14所示。在8μM的高浓度下,实施例1、2、3对HepG2细胞都有较高细胞毒性。然而,当浓度降低到到4μM时,细胞存活率在80%左右。
试验例4对HepG2细胞溶酶体染色的激光共聚焦成像
将HepG2细胞在35毫米培养皿中培养一夜。用一定浓度的实施例1、2、3染色一定时间后(在1mL PBS中加入1μL浓度为1mM的化合物母液),在激光共聚焦显微镜下用适当的激发和发射滤光片对染料进行成像:实施例1、2和3的激发波长和收光范围均是λex=543nm,λem=600-740nm。结果如图15所示。由于探针具有水溶性,预计探针可以均匀地分散在水溶液中,探针的正电荷特性可以使其快速进入到溶酶体中。基于此,我们可以实现溶酶体成像。
试验例5对HepG2细胞细胞核染色的激光共聚焦成像
将HepG2细胞在35毫米培养皿中培养一夜。用一定浓度的实施例2、3染色一定时间后(在1mL PBS中加入4μL浓度为1mM的化合物母液),在激光共聚焦显微镜下用适当的激发和发射滤光片对染料进行成像:实施例2和3的激发波长和收光范围均是λex=543nm,λem=600-740nm。结果如图16所示。由于探针具有较高的平面性和刚性,预计探针可以与DNA进行结合,而高浓度的探针会从溶酶体中逃逸出来并进入到细胞核中。基于此,我们可以实现细胞核成像。
试验例6对HepG2细胞溶酶体染色的光稳定性激光共聚焦成像
将HepG2细胞在35毫米培养皿中培养一夜。用一定浓度的实施例1、2、3染色一定时间后(在1mL PBS中加入1μL浓度为1mM的化合物母液),在激光共聚焦显微镜下用100%强度的激发光和发射滤光片对染料进行成像:实施例1、2和3的激发波长和收光范围均是λex=543nm,λem=600-740nm,每隔5秒拍摄一张,共拍摄50张。结果如图17所示。探针具有较高的光稳定性,我们可以基于此实现溶酶体的长时间成像。
试验例7对HepG2细胞细胞核染色的光稳定性激光共聚焦成像
将HepG2细胞在35毫米培养皿中培养一夜。用一定浓度的实施例2、3染色一定时间后(在1mL PBS中加入4μL浓度为1mM的化合物母液),在激光共聚焦显微镜下用100%强度的激发光和发射滤光片对染料进行成像:实施例2和3的激发波长和收光范围均是λex=543nm,λem=600-740nm,每隔5秒拍摄一张,共拍摄50张。结果如图18所示。由于探针具有较高的平面性和刚性,预计探针可以与DNA进行结合,而高浓度的探针会从溶酶体中逃逸出来并进入到细胞核中。同时探针较高的光稳定性,基于此,我们可以实现细胞核的长时间成像。
综上所述,本发明以胺基芴为骨架作为溶酶体或细胞核染料的基础,通过合理的烷基胺的调控设计,得到了基于胺基芴骨架的快速及长时间溶酶体或细胞核靶向的近红外染色试剂。在低浓度下,该类试剂可以快速靶向到溶酶体中,能够对溶酶体进行较长时间的成像监测;而在高浓度下,该类试剂可以从溶酶体中逃逸出来进入到细胞核中,可以对细胞核进行较长时间的成像监测。此外,该染色试剂具有近红外发射以及较大斯托克斯位移的特性,使其具有背景荧光低,成像信噪比高的表现。本发明的制备方法简单、收率高,制得的染色试剂斯托克斯位移大、稳定性好以及靶向性高。
以上内容仅是对本发明中较佳实施例所作的举例和说明,所属本领域的技术人员不经创造性劳动即对所描述的具体实施例做的修改或补充或采用类似的方式替代仍属本专利的保护范围。
Claims (10)
2.如权利要求1所述的基于胺基芴骨架的快速及长时间溶酶体或细胞核靶向的近红外染色试剂,其特征在于,所述R1为C3烷基链。
3.如权利要求1所述的基于胺基芴骨架的快速及长时间溶酶体或细胞核靶向的近红外染色试剂,其特征在于,所述R为C2烷基链;所述n为1或2。
4.权利要求1-3任一项所述的基于胺基芴骨架的快速及长时间溶酶体或细胞核靶向的近红外染色试剂的制备方法,其特征在于,包括以下步骤:
步骤(1):将3,6-二溴芴酮和烷基胺溶于第一有机溶剂中,除氧后于惰性气体保护下加入催化剂、配体和碱,加热搅拌,得到第一中间体;
步骤(2):将步骤(1)所得的第一中间体加入到含第二有机溶剂的反应容器中搅拌,随后加入草酰氯,浓缩后加入到含有机碱和R1取代的有机胺的第二有机溶剂中搅拌,制得基于胺基芴骨架的快速及长时间溶酶体或细胞核靶向的近红外染色试剂;
或将步骤(1)所得的第一中间体加入到含有机碱的第二有机溶剂中搅拌,加入三氟甲磺酸酐,随后加入R1取代的有机胺搅拌,制得基于胺基芴骨架的快速及长时间溶酶体或细胞核靶向的近红外染色试剂;
其中,R1为C1-C10烷基链或芳香基团;
烷基胺的结构式如下所示:
5.如权利要求4所述的基于胺基芴骨架的快速及长时间溶酶体或细胞核靶向的近红外染色试剂的制备方法,其特征在于,所述步骤(1)中3,6-二溴芴酮、烷基胺、催化剂、配体和碱的摩尔比为1:(2.2~3.0):(0.05~0.1):(0.08~0.2):(3.0~7.0);所述步骤(2)中第一中间体、草酰氯、有机碱和R1取代的有机胺的摩尔比为1:(10.0~100.0):(20.0~50.0):(5.0~20.0);所述步骤(2)中第一中间体、有机碱、三氟甲磺酸酐和R1取代的有机胺的摩尔比为1:(8.0~30.0):(5.0~10.0):(5.0~20.0)。
6.如权利要求4或5所述的基于胺基芴骨架的快速及长时间溶酶体或细胞核靶向的近红外染色试剂的制备方法,其特征在于,所述步骤(1)中第一有机溶剂为乙腈、四氢呋喃、二氯甲烷、三氯甲烷、二甲基亚矾、邻二氯苯、氯苯、甲苯、二甲苯、均三甲苯、1,4-二氧六环、1,2-二氯乙烷、N,N-二甲基甲酰胺和N,N-二甲基乙酰胺中的至少一种;所述步骤(1)中催化剂包括双二亚苄基丙酮钯和三(二亚苄基丙酮)二钯中的一种或两种组合;所述步骤(1)中配体包括三叔丁基膦和三正丁基膦中的一种或两种组合;所述步骤(1)中碱包括叔丁醇钾和叔丁醇钠中的一种或两种组合。
7.如权利要求4或5所述的基于胺基芴骨架的快速及长时间溶酶体或细胞核靶向的近红外染色试剂的制备方法,其特征在于,所述步骤(2)中第二有机溶剂包括二氯甲烷、乙腈和四氢呋喃中的至少一种;所述步骤(2)中有机碱包括三乙胺和吡啶中的一种或两种组合。
8.如权利要求4所述的基于胺基芴骨架的快速及长时间溶酶体或细胞核靶向的近红外染色试剂的制备方法,其特征在于,所述步骤(1)中的加热温度为100-120℃;所述步骤(2)中的搅拌温度为0-25℃。
9.权利要求1-3任一项所述的基于胺基芴骨架的快速及长时间溶酶体或细胞核靶向的近红外染色试剂在生物体溶酶体或细胞核内荧光成像中的应用。
10.一种近红外荧光造影剂,包括权利要求1-3任一项所述的基于胺基芴骨架的快速及长时间溶酶体或细胞核靶向的近红外染色试剂。
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