CN107417714A - 一种基于bodipy的高灵敏荧光探针及其合成方法和应用 - Google Patents
一种基于bodipy的高灵敏荧光探针及其合成方法和应用 Download PDFInfo
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- CN107417714A CN107417714A CN201710545494.5A CN201710545494A CN107417714A CN 107417714 A CN107417714 A CN 107417714A CN 201710545494 A CN201710545494 A CN 201710545494A CN 107417714 A CN107417714 A CN 107417714A
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- bodipy
- probe
- highly sensitive
- fluorescence probe
- detection
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F5/00—Compounds containing elements of Groups 3 or 13 of the Periodic Table
- C07F5/02—Boron compounds
- C07F5/022—Boron compounds without C-boron linkages
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F5/00—Compounds containing elements of Groups 3 or 13 of the Periodic Table
- C07F5/02—Boron compounds
- C07F5/025—Boronic and borinic acid compounds
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
- C07F9/02—Phosphorus compounds
- C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
- C07F9/6596—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having atoms other than oxygen, sulfur, selenium, tellurium, nitrogen or phosphorus as ring hetero atoms
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Abstract
本发明涉及一种基于BODIPY的高灵敏荧光探针及其合成方法和应用。其结构通式如(I)所示;式中,Trigger为刺激物触发基团:或;R1、R2为调控探针荧光发射波长和引入细胞器靶向的基团:R1、R2分别为:、或。本发明能够在不改变探针母核结构的情况下,只改换不同的Trigger基团,就能实现对不同底物的检测。此外,根据不同的波长和靶向的需要,能够对探针母核结构进行快速的修饰。通过改变R1、R2,不但能改变探针的最大荧光发射波长,并能使探针靶向到线粒体,实现线粒体内活性物质的检测。本发明的探针都具有良好的生物相容性,因此能够用于生物体系的检测。这些荧光探针在炎症或肿瘤组织过表达的生物活性小分子和蛋白酶检测方面的应用价值,具有潜在的社会效益和经济效益。
Description
技术领域
本发明涉及一种基于BODIPY的高灵敏荧光探针及其合成方法和应用,具体为基于BODIPY的高灵敏荧光探针的合成及其在生物分析和成像方面的应用。这些荧光探针在肿瘤或炎症组织过表达的生物活性小分子和蛋白酶检测方面具有潜在的应用价值。
背景技术
生物新陈代谢过程有许多部分组成,既包括生物活性小分子(如过氧化氢、硫化氢等),也包括生物活性大分子(如蛋白酶等)。几乎所有疾病的发生都伴随着某些活性物质表达量的异常,因此,检测这些活性物质可以用于疾病的诊断。荧光成像技术作为一种强大的工具,具有时空分辨率高,无创检测等优势。当前,已有很多荧光探针用于体外或体内的检测,但它们的最大荧光发射波长几乎都是固定的。为了更好的提高时空分辨率,同时检测多种物质而互不干扰,开发最大荧光发射波长从可见光到近红外光可随意调节的技术,是当前研究的热点。
过氧化氢是生物体内活性氧之一,具有很高氧化活性,参与到需要正常的生物学过程和病理学过程。硫化氢做一种内源性气体信号分子,不但能起调节细胞生长、保护心血管等作用,而且与一些常见的疾病相关。当前,分别检测这两种小分子的荧光探针已有很多,但如何快速的在两种探针之间转换,依旧比较有挑战。此外,这两种活性分子只是体内众多物质的两个代表,如何开发一种策略,能够通过简单的化学基团的改变,实现对其它物质的检测,是当前的热门和必要研究的方向之一。
生物体内的代谢,无论正常与否,都与细胞内的酶息息相关。在新陈代谢过程中,有很多酰胺键的形成和断裂,各种酶都起着至关重要的作用。炎症、癌变等病变的发生,往往伴随着某些酶过量的表达。利用酶的专一性,通过开发与这些过量表达的酶相关的荧光探针,能使我们实现对炎症和肿瘤的可视化诊断。但也正是这种专一性,使得一种酶探针,很难便捷的转换为另一种酶的探针,因为新的探针几乎需要从头设计。这就使得亟须开发一种通用策略,只需修饰探针的一部分,就能使其在多种酶响应探针之间转化。
细胞器是生物细胞的重要组成部分,对其研究是认识细胞结构和功能的必要手段。线粒体是细胞的能源站,是细胞内尤为重要的细胞器,参与几乎所有的新陈代新过程中。许多疾病都与线粒体功能异常有关,因此开发线粒体相关的细胞器靶向荧光探针技术具有中的生物基础研究和临床研究意义。细胞器膜具有很高的负的膜电位,能够与带正电荷的三苯基膦基团相作用,使其在膜上面定位。基于这种特性,开发了很多这种细胞器靶向的荧光探针,又由于三苯基膦具有较高的脂溶性,导致其水溶性差、细胞膜通透性不足等缺点。因此开发生物相容性、水溶性和细胞通透性都良好的荧光探针很有必要。除了线粒体,细胞内的溶酶体、内质网、高尔基体等细胞器也是研究细胞代谢的重要组成,研究其靶向探针,也尤为重要。通过改变荧光探针上不同的靶向基团,来实现不同细胞器的定位,这是亟须解决多细胞器定位的策略之一。
综上,无论是研究生物活性小分子,还是生物活性大分子,或是研究不同细胞器,都需要一种便捷、简单、通用的策略,为基础生物研究和临床研究提供一种好的探针平台。
发明内容
本发明的目的在于提供基于BODIPY的高灵敏荧光探针及其合成方法和应用。本发明合成方法简单高效,能够在不改变探针母核结构的情况下,只改换不同的Trigger基团,就能实现对不同底物的检测,并对这种高效的制备策略进行了代表性验证,这表明其在生物医学基础研究和疾病临床诊断方面具有潜在应用价值。此外,根据不同的波长和靶向的需要,能够对探针母核结构进行快速的修饰。本发明的探针都具有良好的生物相容性,因此能够用于生物体系的检测。即这些荧光探针在炎症或肿瘤组织过表达的生物活性小分子和蛋白酶检测方面具有潜在的应用价值。
本发明提供的基于BODIPY的高灵敏荧光探针是以BODIPY为母核的生物荧光探针,其结构通式如下:
式中,Trigger为刺激物触发基团:
R1、R2为调控探针发射波长和引入细胞器靶向的基团:R1、R2分别为:
上述三种不同的Trigger基团能分别高选择性的与过氧化氢、硫化氢、青霉素G酰化酶等三种有代表性的生物活性小分子和蛋白酶作用,生成羟基或氨基,然后通过自降解化学,转化成具有高量子产率的荧光团,从而实现荧光的打开。
通过改变R1、R2,不但能改变探针的最大荧光发射波长,为探针引入水溶性,并能使探针靶向到线粒体,实现线粒体内活性物质的检测。
本发明提供的基于BODIPY的高灵敏荧光探针的总的合成步骤见附图1。
本发明提供的一种基于BODIPY的高灵敏荧光探针是以BODIPY为母核的生物荧光探针的合成,它是以模块化的方式进行的,其具体步骤如下:
1)BODIPY母核的合成
在室温下,以二氯甲烷为溶剂,草酰氯和苯甲醇反应制备得到单草酰氯甲酯。旋蒸除去溶剂后,直接用于下一步反应。然后分别于2,4-二甲基吡咯和三氟化硼乙醚反应,制备得到 BODIPY母核。
2)三种Trigger基团的合成
通过NBS/AIBN、CBr4/PPh3、PBr3三种溴代方式,能高效的分别制备出过氧化氢、硫化氢、青霉素G酰化酶等的三种Trigger。
3)不同的生物荧光探针的合成
通过Knoevenagel反应,BODIPY母核的3、4位甲基与不同苯甲醛类缩合,然后在碘化锂作用下脱去甲基,制备得到不同发射波长的羧基BODIPY。对于线粒体靶向的羧基BODIPY,只需一步高效的点击化学就能得到。最后通过酯化反应,分别合成出不同的生物荧光探针。
值得强调的是,三种不同发射波长的羧基BODIPY都能与不同的Trigger基团进行酯化反应,我们仅选择性地合成了六种生物荧光探针为代表。
本发明提供了一种基于BODIPY的高灵敏荧光探针,能应用于肿瘤或炎症组织过表达的生物活性小分子和蛋白酶的检测,既代表性的检测过氧化氢、硫化氢、青霉素G酰化酶,其应用价值具有潜在的社会效益和经济效益。
附图说明
图1是本发明提供的基于BODIPY的高灵敏荧光探针的总的合成步骤。
图2是在PBS缓冲溶液中(10mM,pH 7.4,37℃)中,0.5μM的过氧化氢探针B-H2O2(λex=480nm)与不同浓度的过氧化氢(0-50μM)反应30min后的荧光发射光谱。
图3是在PBS缓冲溶液中(10mM,pH 7.4,37℃)中,0.5μM的过氧化氢探针B-TEG2-H2O2(λex=550nm)与不同浓度的过氧化氢(0-50μM)反应30min后的荧光发射光谱。
图4是在PBS缓冲溶液中(10mM,pH 7.4,37℃)中,0.5μM的过氧化氢探针B-TEG4-H2O2(λex=620nm)与不同浓度的过氧化氢(0-50μM)反应30min后的荧光发射光谱。
图5是在PBS缓冲溶液中(10mM,pH 7.4,37℃)中,线粒体靶向的过氧化氢探针 B-mito-H2O2(0.5μM)与100μM的过氧化氢作用下的时间依赖荧光发射光谱(λex=620 nm)。
图6是在PBS缓冲溶液中(10mM,pH 7.4,37℃)中,硫化氢探针B-TEG4-H2S(0.5μM)与100μM的硫化氢作用下的时间依赖荧光发射光谱(λex=620nm)。
图7是在PBS缓冲溶液中(10mM,pH 7.4,37℃)中,青霉素G酰化酶探针B-TEG4-PGA(0.5μM)与2U/mL的青霉素G酰化酶作用下的时间依赖荧光发射光谱(λex=620nm)。
具体实施方式
下面结合具体实施例,进一步详细阐述本发明。实施例中未注明具体条件的实验方法,通常按照常规条件以及手册中所述的条件,或按照制造厂商所建议的条件;所用设备、材料、试剂等,如无特殊说明,均可从商业途径得到。
实施例1:
BODIPY母核的合成路线如下:
合成方法如下:
冰水浴下,将甲醇(1.60g,50.0mmol)缓慢滴加到草酰氯(6.35g,50.0mmol)的二氯甲烷 (CH2Cl2,100mL)溶液中,然后室温搅拌30min,旋蒸除去溶剂,得到的草酰氯甲酯直接用于下一步反应。先将2,4-二甲基吡咯(7.14g,75.0mmol)溶于二氯甲烷(140mL),冷却至-78℃,缓慢滴加草酰氯甲酯(3.67g,30.0mmol)的二氯甲烷(80mL)溶液,然后-78℃下继续搅拌2h,依次缓慢加入三乙胺(Et3N,12.14g,120.0mmol)、三氟化硼乙醚(BF3·Et2O,22mL),升至室温,继续搅拌2h。旋蒸除去溶剂,用石油醚和二氯甲烷(V:V=2:1)作为淋洗剂过柱子,分离得到B-Me(3.67g,收率为40.0%)。
实施例2:
Alk-Ba和Azide-tpp的合成路线如下:
合成方法如下:
将3,4-二羟基苯甲醛(2.76g,20.0mmol)、碳酸钾(2.76g,20.0mmol)于丙酮(30mL)中搅拌10min,室温下,缓慢滴加炔丙溴(2.38g,20.0mmol)的丙酮(50mL)溶液两个小时,搅拌 14小时。过滤,旋蒸滤液除去丙酮,用乙酸乙酯(100mL)溶解,依次用蒸馏水(100mL)、饱和氯化钠溶液(100mL)洗涤一次,有机层用无水硫酸钠干燥,旋蒸除溶剂,用体积比1:1的二氯甲烷:石油醚混合溶剂作为淋洗剂过柱子,分离得到3-羟基-4-炔丙氧基苯甲醛(1.73g,收率为49.1%)。
将3-羟基-4-炔丙氧基苯甲醛(1.70g,10.0mmol)、碳酸钾(2.76g,20.0mmol)于丙酮(100mL) 中搅拌10min,加入2-溴乙烷(4.36g,40.0mmol),回流搅拌15小时。过滤,旋蒸滤液除去丙酮,用乙酸乙酯(100mL)溶解,依次用蒸馏水(100mL)、饱和氯化钠溶液(100mL)洗涤一次,有机层用无水硫酸钠干燥,旋蒸除溶剂,得到Alk-Ba(1.95g,收率为95.6%)。
将1,4-二溴丁烷(4.32g,20.0mmol)、三苯基膦(5.24g,20.0mmol)溶于甲苯(50mL)中,加热搅拌回流,慢慢有白色固体析出,反应16小时,冷却后过滤得到白色固体,少量乙醚洗涤后干燥,产物Br-tpp(8.95g,收率为93.6%)。
将Br-tpp(4.78g,10.0mmol)、叠氮化钠(1.30g,20.0mmol)溶于30mL的乙醇/水(V/V=1:1)混合液中,加热搅拌回流反应14小时,旋蒸除去乙醇,用二氯甲烷(20mL×3) 萃取,无水硫酸钠干燥,旋蒸得到无色蜡状固体Azide-tpp(4.01g,收率为95.2%)。
实施例3:
B-TEG4-COOH的合成路线如下:
合成方法如下:
0℃下,将对甲苯磺酰氯(20.97g,110.0mmol)的四氢呋喃(100mL)溶液缓慢滴加到三乙二醇单甲醚(16.42g,100.0mmol)和三乙胺(22.26g,220.0mmol)的二氯甲烷(300mL)溶液中,然后室温搅拌14小时,过滤,将滤液分别用蒸馏水(150mL;1次)、饱和氯化氨溶液(150mL×3次)洗涤,有机层用无水硫酸钠干燥,旋蒸除去溶剂,用石油醚和乙酸乙酯(V:V=10:1) 作为淋洗剂过柱子,分离得到Ts-TEG(30.88g,收率为97.0%)。
将3,4-二羟基苯甲醛(1.38g,10.0mmol)、碳酸钾(K2CO3,6.91g,50.0mmol)、碘化钾(KI, 0.30g)于丙酮(200mL)中搅拌10min,加入Ts-TEG(8.00g,25.0mmol),然后回流搅拌16小时。过滤,旋蒸滤液除去丙酮,用二氯甲烷(150mL)溶解,依次用蒸馏水(100mL)、饱和氯化钠溶液(100mL)洗涤一次,有机层用无水硫酸钠干燥,旋蒸除溶剂,用乙酸乙酯作为淋洗剂过柱子,分离得到Ph-TEG2(4.05g,收率为94.2%)。
将Ph-TEG2(1.29g,3.0mmol)溶于无水乙腈(CH3CN,15mL)中,加入活化的分子筛、哌啶(1mL)、乙酸(1mL),室温搅拌10min,加入B-Me(0.31g,1.0mmol),然后回流搅拌14小时,旋蒸除去溶剂,用二氯甲烷和甲醇(V:V=20:1)作为淋洗剂过柱子,分离得到 B-TEG4-Me(0.57g,收率为50.4%)。
B-TEG4-Me(0.34g,0.3mmol)溶于干燥的乙酸乙酯(EA,5mL),加入碘化锂(LiI)(0.04g, 0.3mmol),回流搅拌14-16小时,冷却至室温,滴加0.1M的稀盐酸(0.1mL),搅拌1h,然后直接过柱子,用二氯甲烷和甲醇(V:V=10:1)作为淋洗剂,分离得到B-TEG4-COOH(0.23g,收率为68.7%)。
用制备B-TEG4-COOH类似的方法,制备得到B-COOH、B-TEG2-COOH和B-Alk-COOH。
实施例4:
BODIPY-TEG4-H2O2的合成路线如下:
合成方法如下:
将无水硫酸镁(40.02g,300.0mmol)、对甲苯硼酸(13.60g,100.0mmol)、频哪醇(11.81g, 100.0mmol)加入到无水乙醚(200mL)中,常温搅拌14-16小时,过滤,旋蒸除去溶剂,用石油醚作为淋洗剂过柱子,分离得到对甲苯硼酸频哪醇酯(20.90g,收率为95.8%)。
对甲苯硼酸频哪醇酯(10.91g,50.0mmol)、溴代丁二酰亚胺(NBS,9.91g,55.0mmol)、偶氮二异丁腈(AIBN,0.44g,2.0mmol)溶于干燥的四氯化碳(CCl4,200mL),回流搅拌14 小时,过滤,旋蒸除去溶剂,乙酸乙酯(200mL)溶解,依次用蒸馏水(100mL)、饱和氯化钠水溶液(100mL)洗涤一次,无水硫酸钠干燥,旋蒸,最后以石油醚作为淋洗剂过柱子,分离得到4-溴甲基苯硼酸频哪醇酯(11.30g,收率为76.1%)。
将B-TEG4-COOH(0.11g,0.1mmol)、1,8-二氮杂二环十一碳-7-烯(DBU,30.0mg,0.2mmol) 溶干燥的四氢呋喃(THF,10mL),然后加入4-溴甲基苯硼酸频哪醇酯(25.1g,0.12mmol),室温搅拌14小时。旋蒸除去溶剂,二氯甲烷(100mL)溶解,饱和氯化铵水溶液(50mL)洗涤两次,有机层用无水硫酸钠干燥,过滤,旋蒸,最后用二氯甲烷和甲醇(V:V=25:1)作为淋洗剂过柱子,分离得到B-TEG4-H2O2(0.11g,收率为82.5%)。1H NMR(400MHz)δ(ppm)7.83(d,J =7.6Hz,2H),7.59–7.38(m,4H),7.26–7.05(m,6H),6.93(d,J=8.4Hz,2H),6.66(s,2H),5.42 (s,2H),4.23(dt,J=10.1,4.8Hz,8H),3.94–3.86(m,8H),3.81–3.74(m,8H),3.72–3.61(m, 16H),3.54(dt,J=9.3,4.6Hz,8H),3.38(s,6H),3.36(s,6H),2.09(s,6H),1.35(s,12H)。13C NMR(100MHz,CDCl3)δ(ppm)165.53,154.23,150.51,148.98,139.66,137.27,136.70,135.31, 131.04,130.21,128.19,125.17,121.83,117.79,117.48,114.60,114.35,84.12,72.05,72.04,70.99, 70.81,70.68,70.65,69.91,69.77,69.34,68.82,68.36,59.17,59.12,24.99,13.08。
用制备B-TEG4-H2O2的类似方法,制备得到B-H2O2,1H NMR(400MHz,CDCl3)δ(ppm)7.82(d,J=7.8Hz,2H),7.42(d,J=7.8Hz,2H),6.03(s,2H),5.40(s,2H),2.52(s,6H),2.02(s, 6H),1.35(s,12H)。13C NMR(100MHz,CDCl3)δ(ppm)165.13,157.76,141.30,136.54,135.32, 128.92,128.23,121.34,84.14,68.45,29.85,25.00,14.93,12.89.ElementalAnalysis calcd for C27H32B2F2N2O4:C,63.82;H,6.35;N,5.51;Found:63.64;H,6.22;N,5.35。
用制备B-TEG4-H2O2的类似方法,制备得到B-TEG2-H2O2,1H NMR(400MHz,CDCl3) δ(ppm)7.83(d,J=7.9Hz,2H),7.48–7.40(m,3H),7.22–7.12(m,3H),6.89(d,J=8.6Hz,1H),6.64(s,1H),6.05(s,1H),5.41(s,2H),4.22(dt,J=11.9,5.0Hz,4H),3.93–3.85(m,4H),3.80–3.74(m,4H),3.72–3.64(m,8H),3.58–3.52(m,4H),3.38(s,3),3.37(s,3),2.56(s,3H),2.08 (s,3H),2.04(s,3H),1.35(s,12H)。13C NMR(100MHz,CDCl3)δ(ppm)165.35,156.62,155.20, 150.57,149.06,140.72,140.17,138.74,137.72,136.63,135.32,129.98,129.13,128.21,127.01, 122.43,121.11,117.77,117.18,114.10,113.48,84.13,72.06,71.01,70.98,70.85,70.82,70.69, 69.88,69.74,69.14,68.78,68.41,59.20,59.18,25.00,15.02,13.09,12.90.Elemental Analysis calcd for C48H64B2F2N2O12:C,62.62;H,7.01;N,3.04;Found:C,62.38;H,6.92;N,3.06. Elemental Analysis calcd forC69H96B2F2N2O20:C,62.17;H,7.26;N,2.10;Found:C,62.05;H, 7.30;N,2.07。
实施例5:
B-TEG4-H2S的合成路线如下:
合成方法如下:
对硝基苯甲醇(15.30g,100.0mmol)和0.31g钯碳(10%Pd/C)加入到无水甲醇(150mL)中,缓慢滴加水合肼(N2H4·H2O)20mL,然后室温搅拌16小时,旋蒸除去甲醇,用乙酸乙酯萃取三次,无水硫酸钠干燥,旋蒸除去乙酸乙酯,得到的对氨基苯甲醇(11.70g,收率为95.0%) 直接进行下一步反应。
将对氨基苯甲醇(1.20g,9.7mmol)溶于蒸馏水(3mL)中,加入浓盐酸(36%HCl,3mL),在0℃下缓慢滴加亚硝酸钠(NaNO2,0.69g,10.0mmol)的水溶液(10mL),30min后,缓慢滴加叠氮化钠(NaN3,0.78g,12.0mmol)的水溶液(10mL),然后室温搅拌14-16小时,用乙酸乙酯萃取三次,干燥,旋蒸除去乙酸乙酯,得到的对叠氮苯甲醇(1.32g,收率为88.5%)直接用于下一步反应。
对叠氮苯甲醇(0.30g,2.0mmol)溶于二氯甲烷(20mL),冷却至0℃,依次加入四溴化碳 (CBr4,1.33g,4.0mmol)、三苯基膦(PPh3,1.05g,4.0mmol),TLC点板跟踪反应。完全反应后,旋蒸除去二氯甲烷,用石油醚和乙酸乙酯(V:V=10:1)作为淋洗剂过柱子,分离得到对叠氮苄溴(0.38g,收率为90.3%)。
用制备B-TEG4-H2O2类似的方法,制备得到B-TEG4-H2S。1H NMR(400MHz,CDCl3) δ(ppm)7.54–7.42(m,4H),7.26–7.15(m,4H),7.12(s,2H),7.05(d,J=8.2Hz,2H),6.93(d,J= 8.4Hz,2H),6.66(s,2H),5.37(s,2H),4.23(dt,J=9.9,4.8Hz,8H),3.93–3.86(m,8H),3.80– 3.74(m,8H),3.66(dt,J=15.0,5.0Hz,16H),3.54(dt,J=9.2,4.6Hz,8H),3.38(s,6H),3.36(s, 6H),2.07(s,6H)。13C NMR(100MHz,CDCl3)δ(ppm)165.52,154.29,150.59,149.02,141.06, 139.53,137.35,131.05,130.54,130.21,128.91,125.08,121.86,119.45,117.81,117.47,114.68, 114.41,72.05,70.99,70.82,70.68,70.65,69.92,69.78,69.38,68.85,67.91,59.15,59.11,13.06. ElementalAnalysis calcd forC63H84BF2N5O18:C,60.62;H,6.78;N,5.61;Found:C,60.38;H,6.67; N,5.65。
实施例6:
BODIPY-TEG4-PGA的合成路线如下:
合成方法如下:
对氨基苯甲醇(0.43g,3.5mmol)、乙酸钾(CH3COOK,6.9g,7.0mmol)加入到 DCM/DMF(30mL/10mL)混合液中,冷却至-60℃,然后缓慢滴加苯乙酰氯(0.54g,3.5mmol) 的二氯甲烷溶液(30mL),然后升至室温,搅拌2h,旋蒸除去溶剂,用石油醚和乙酸乙酯 (V:V=2:1)作为淋洗剂过柱子,分离得到PGA-OH(0.69g,收率为81.7%)。
PGA-OH(0.48g,2.0mmol)溶于干燥的二氯甲烷中(15mL),0℃下缓慢滴加三溴化磷(PBr3,0.65g,2.4mmol)的二氯甲烷溶液(5mL),点板跟踪反应。待PGA-OH完全反应后,加入饱和的碳酸氢钠水溶液(5mL)后,搅拌1h,然后分液,除去水层,有机层用蒸馏水(10mL) 洗涤一次,无水硫酸钠干燥,旋蒸除溶剂,用石油醚和乙酸乙酯(V:V=2:1)作为淋洗剂过柱子,分离得到PGA-Br(0.55g,收率为90.4%)。
用制备B-TEG4-H2O2类似的方法,制备得到B-TEG4-PGA。1H NMR(400MHz,CDCl3)δ(ppm) 7.53–7.44(m,4H),7.44–7.28(m,8H),7.25–7.15(m,4H),7.11(s,2H),6.92(d,J=8.4Hz,2H), 6.64(s,2H),5.33(s,2H),4.22(dt,J=9.6,5.0Hz,8H),3.92–3.85(m,8H),3.80–3.72(m,10H), 3.71–3.61(m,16H),3.57–3.51(m,8H),3.38(s,6H),3.35(s,6H),2.06(s,6H)。13C NMR(100 MHz,CDCl3,Figure S40)δ(ppm)169.27,165.53,154.23,150.51,148.97,139.64,138.52,137.29, 134.34,131.01,130.21,129.71,129.66,129.47,129.41,128.75,127.90,125.23,121.83,119.88, 117.78,117.47,114.59,114.33,72.06,72.04,70.99,70.82,70.68,70.66,69.91,69.77,69.32,68.81, 68.15,59.18,59.13,44.97,13.10.Elemental Analysis calcd for C71H92BF2N3O19:C,63.62;H,6.92; N,3.14;Found:C,63.02;H,6.94;N,3.18。
实施例7:
B-mito-H2O2的合成路线如下:
合成方法如下:
B-Alk-COOH(27.0mg,0.03mmol)、Azide-tpp(22.0mg,0.05mmol)、抗坏血酸钠(2.0mg,0.01mmol)、无水硫酸铜(1.0mg,0.005mmol)溶于THF/H2O(5mL/5mL),脱气除氧,在N2下反应24h,旋蒸除去THF,用二氯甲烷萃取两次(20mL×3),无水硫酸钠干燥,旋蒸除溶剂后得到的产物直接用于下一步反应。
将上一步产物和DBU(9.0mg,0.06mmol)溶干燥THF(10mL),然后加入4-溴甲基苯硼酸频哪醇酯(15.0mg,0.05mmol),25℃搅拌14-16小时。旋蒸除去溶剂,用体积比25:1 的二氯甲烷:甲醇作为淋洗剂过柱子,分离得到B-mito-H2O2(24.0mg,收率为52.6%)。1H NMR(400MHz,CDCl3)δ(ppm)7.98(s,1H),7.88–7.72(m,11H),7.71–7.59(m,6H),7.53–7.38 (m,4H),7.25–7.13(m,3H),7.12–6.96(m,4H),6.89(d,J=8.2Hz,1H),6.67(s,2H),5.42(s,2H), 5.28(s,2H),4.67(s,2H),4.28–4.09(m,6H),4.01–3.85(m,6H),3.81–3.72(m,4H),3.72–3.60(m, 8H),3.58–3.49(m,4H),3.38(s,3H),3.35(s,3H),2.39(s,2H),2.09(s,6H),1.55(s,2H),1.47(t,J =7.0Hz,3H),1.35(s,12H)。13C NMR(100MHz,CDCl3)δ(ppm)165.50,154.14,150.56,149.50, 149.29,148.98,143.90,139.72,137.31,137.27,136.65,135.32,135.14,135.11,133.84,133.74, 131.03,130.65,130.52,130.41,130.13,128.21,125.18,124.19,121.84,121.33,118.54,117.80, 117.69,117.40,114.84,114.51,114.30,112.79,84.12,72.04,70.98,70.80,70.67,70.64,69.88, 69.74,69.31,68.81,68.40,64.94,63.12,59.17,59.13,48.88,24.99,22.82,19.28,19.12,15.08,13.09.Elemental Analysis calcd for C82H97B2BrF2N5O14P:C,63.66;H,6.32;N,4.53;Found:C, 63.39;H,6.40;N,4.46。
实施例8:
分别配制0.5μM的B-H2O2、B-TEG2-H2O2和B-TEG4-H2O2的PBS缓冲溶液(10mM, pH7.4),分别与不同浓度的过氧化氢(0-50μM)在37℃反应30min,然后用日立F-4600荧光分光光度计,在激发波长分别为480nm、550nm和620nm条件下,测试其荧光发射光谱,结果分别如附图2、3、4所示。
实施例9:
分别配制0.5μM的B-mito-H2O2、B-TEG4-H2S和B-TEG4-PGA的PBS缓冲溶液(10 mM,pH7.4),分别与50μM的过氧化氢、50μM的硫氢化钠和2U/mL的青霉素G酰化酶在 37℃反应,在不同的反应不同的时间点(0min、30min、60min、90min、120min),用日立 F-4600荧光分光光度计,在激发波长为620nm条件下,测试其荧光发射光谱,结果分别如附图5、6、7所示。
值得强调的是,三种不同发射波长的羧基BODIPY都能与不同的Trigger基团进行酯化反应,我们仅选择性地合成了六种近红外生物荧光探针为代表。
所有上述实施例仅用于阐明本发明制备的几种具体代表性探针,而非限制本发明策略。
Claims (4)
1.一种基于BODIPY的高灵敏荧光探针,其特征在于它是以BODIPY为母核的生物荧光探针,其结构通式如下:
。
2.根据权利要求1所述的基于BODIPY的高灵敏荧光探针,其特征在于:
R1、R2都为:时,最大荧光发射波长为509 nm;
R1、R2分别为:时,最大荧光发射波长为585 nm;
R1、R2都为:时,最大荧光发射波长为660nm;
R1、R2分别为:时,最大荧光发射波长为660 nm。
3.权利要求1所述的基于BODIPY的高灵敏荧光探针的合成方法,其特征在于包括以下步骤:
1)BODIPY母核的合成
在室温下,以二氯甲烷为溶剂,草酰氯和苯甲醇反应制备得到单草酰氯甲酯;旋蒸除去溶剂后,直接用于下一步反应,然后分别于2,4-二甲基吡咯和三氟化硼乙醚反应,制备得到BODIPY母核;
2)三种Trigger基团的合成
通过NBS/AIBN、CBr4/PPh3、PBr3三种溴代方式,分别高效的制备出过氧化氢、硫化氢、青霉素G酰化酶等的三种Trigger前体;
3)不同的生物荧光探针的合成
通过Knoevenagel反应,BODIPY母核的3、4位甲基与不同苯甲醛类缩合,然后在碘化锂作用下脱去甲基,制备得到不同发射波长的羧基BODIPY;对于线粒体靶向的羧基BODIPY,只需一步高效的点击化学就能得到,最后通过酯化反应,分别合成出不同的生物荧光探针。
4.权利要求1所述的基于BODIPY的高灵敏荧光探针在检测过氧化氢、硫化氢或青霉素G酰化酶中的应用。
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