CN108276442B - 一种线粒体靶向甲醛荧光探针及其制备方法和应用 - Google Patents
一种线粒体靶向甲醛荧光探针及其制备方法和应用 Download PDFInfo
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Abstract
本发明提供了一种反应灵敏、检测限低、特异性好的可检测甲醛靶向线粒体的荧光探针Na‑FA‑Mito,化学名称为溴化N‑(2‑甲酰胺基乙基‑N‑(3‑三苯基磷基丙基))‑4‑肼基‑1,8‑萘二甲酰亚胺盐。所述荧光探针可用于检测溶液或细胞中的甲醛。
Description
技术领域
本发明涉及一种检测甲醛的靶向线粒体的荧光探针及其制备方法和应用,属于有机小分子荧光探针领域。
背景技术
甲醛是一种常见的工业原料,其在化学化工、木材加工、纺织工业等领域都有广泛的应用,而环境中的甲醛污染也大都来源于人类的生产排放。相关统计数据表明,接触甲醛较多的从业人员的患癌风险明显高于普通人,2017年,在WHO整理的致癌物质清单中,甲醛被列为一类致癌物。除了其致突变风险,过多的直接接触甲醛还会对眼、皮肤、呼吸道有强烈的刺激作用,进而引起眼睛过敏、皮肤炎症、肺水肿等病症。甲醛的这些危害使人们常常谈“醛”色变。在这种背景下,人们迫切需求一种方法来方便快捷的检测环境中的甲醛,而甲醛荧光探针无疑满足这种需求。
有趣的是,就在人们积极预防甲醛对人体造成危害的同时,很多研究已经证明甲醛可以伴随很多代谢过程而内源产生,例如:一些含有N-甲基的氨基酸去甲基化过程、DNA和RNA碱基的N-甲基去甲基化过程、体内甲胺物质通过SSAO的代谢过程等等。另一方面,甲醛在体内是一种重要信号因子,参与体内的碳循环过程,其中一个典型的例子就是,甲醛参与到了叶酸调节的线粒体单碳循环过程。在这个过程中,二甲基甘氨酸在线粒体酶DMGDH以及SARDH作用下产生甘氨酸并释放出两分子的甲醛,产生的甲醛会与线粒体内部的叶酸生成5,10-亚甲基叶酸,用于进一步参与到细胞质以及细胞核中的单碳循环过程,最终生产出用于维持细胞正常生理功能的嘌呤核苷酸、胸苷酸、蛋氨酸、丝氨酸等物质,而细胞单碳循环的异常往往伴随某些疾病发生如发育异常、癌症等等。有研究表明,过多的甲醛会触发线粒体半胱天冬酶凋亡过程使细胞死亡。而甲醛在人体内,特别是线粒体内部的含量波动对于人体生理和病理的影响还是不够明确。因此,研究一个性能优异的靶向线粒体的甲醛荧光探针对于揭露甲醛在人体内部的这种信号-损伤的双重作用的机理是必要的。
发明内容
针对缺乏线粒体靶向的荧光探针的问题,本发明提供一种反应灵敏、检测限低、特异性好的线粒体靶向甲醛荧光探针。
本发明的另一目的是提供一种简便地合成上述线粒体靶向甲醛荧光探针的方法。
为实现上述目的,本发明采用如下技术方案。
一种线粒体靶向检测甲醛的荧光探针,化学名称为溴化N-(2-甲酰胺基乙基-N-(3-三苯基磷基丙基))-4-肼基-1,8-萘二甲酰亚胺盐,简称为Na-FA-Mito,具有如式(I)所示的结构:
式(I)。
一种上述荧光探针的合成方法,包括以下步骤:
(1)溴丙胺盐与三苯基膦在乙腈中加热搅拌回流,得到化合物1 (3-氨基丙基三苯基磷鎓);
(2)4-溴-1,8-萘二甲酸酐与β-氨基丙酸在乙醇中加热回流,得化合物2 (N- (2-甲酸基乙基) -4-溴-1,8-萘二甲酰亚胺);
(3)化合物2与化合物1在二氯甲烷中,在失水剂与催化剂存在下生成化合物3 (溴化N- (2-甲酰胺基乙基-N- (3-三苯基磷基丙基)) -4-溴-1,8-萘二甲酰亚胺盐);
(4)水合肼与化合物3在乙醇中加热搅拌回流,生成化合物溴化N- (2-甲酰胺基乙基-N- (3-三苯基磷基丙基)) -4-肼基-1,8-萘二甲酰亚胺盐,即荧光探针。
作为优选,各步骤还包括分离纯化的步骤;所述分离纯化步骤可以采用化学领域分离及纯化操作,如色谱柱分离、重结晶。
所述步骤(1)的回流时间为16-24h;步骤(2)的回流时间为4-8h;步骤(3)的反应时间为16-24h;步骤(4)的回流时间为6-10h。
所述步骤(3)中,反应温度为室温。失水剂优选为二环己基碳二亚胺(DCC)或1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDCI);催化剂优选为4-二甲氨基吡啶(DMAP)或1-羟基苯并三唑(HOBT)。
合成路线如下:
一种上述荧光探针在检测溶液或细胞中甲醛的应用。
本发明中荧光探针的识别机理如下:
本发明所述检测甲醛的荧光探针Na-FA-Mito本身由于肼基的供电子能力导致a-PET效应而淬灭分子的荧光,当探针与甲醛分子作用后,化合物Na-FA-Mito上的肼基与甲醛发生亲和加成反应,转变成腙的结构,a-PET作用被抑制,导致荧光强度得到大幅度提升:
本发明具有以下优点:
本发明的荧光探针以萘酰亚胺为荧光团,以肼基为识别位点,该位点极易与甲醛发生亲核加成反应生成腙,从而使得探针识别前后的分子前线轨道能量降低,进而导致PET(光致电子转移)终止,荧光增强,探针在识别前后荧光增强45倍,性能优异。通过与商品化线粒体定位染料相比,本发明的荧光探针可以成功定位于线粒体,可将其应用于细胞线粒体检测内外源甲醛的荧光成像;而且本发明的荧光探针具有双光子效应,可降低对生物细胞的光损伤。
附图说明
图1为Na-FA-Mito的1H NMR谱图;
图2为Na-FA-Mito的13C NMR谱图;
图3为Na-FA-Mito的质谱图;
图4为不同浓度甲醛下化合物Na-FA-Mito的荧光强度;
图5为不同反应时间下Na-FA-Mito的荧光强度变化;
图6为Na-FA-Mito对不同干扰物质的选择性;
图7为Na-FA-Mito外源甲醛细胞成像;
图8为Na-FA-Mito内源甲醛细胞成像;
图9为Na-FA-Mito的线粒体共定位细胞成像。
具体实施方式
下面结合实施例和附图对本发明做进一步说明,但本发明不受下述实施例的限制。
实施例1 荧光探针的合成
(1)化合物3-氨基丙基三苯基磷鎓的合成:在100 mL圆底烧瓶中,加入3-溴丙胺5mmol,三苯基膦6 mmol,再加入乙腈30 mL,加热回流24 h后,冷却至室温,减压蒸干溶剂,硅胶色谱柱分离 (DCM: CH3OH=10:1),真空干燥,得白色固体3-氨基丙基三苯基磷鎓 (1)。产率:60 %。1H NMR (400 MHz, DMSO-d 6 ) δ 7.97 – 7.76 (m, 15H), 3.83 – 3.68 (m, 2H),2.99 (t, J = 6 Hz, 2H), 1.84 (m, 2H);13C NMR (101 MHz, DMSO-d 6 ) δ 135.59,135.56, 134.15, 134.04, 130.91, 130.79, 118.96, 118.11, 20.58, 19.11, 18.58。
(2)化合物N-(2-甲酸基乙基)-4-溴-1,8-萘二甲酰亚胺的合成:在100 mL的圆底烧瓶中,将化合物4-溴-1,8-萘二甲酸酐5 mmol加入到含有β-氨基丙酸的乙醇溶液中回流搅拌6 h,冷却至室温,抽滤,滤饼用无水乙醇重结晶,得到灰色粉末状固体N- (2-甲酸基乙基) -4-溴-1,8-萘二甲酰亚胺 (2)。产率80%。1H NMR (400 MHz, DMSO-d 6 ): δ 8.45-8.51(m, 2H), 8.25-8.27 (d, J=7.6 Hz, 1H), 8.15-8.17 (d, J= 7.6 Hz, 1H), 7.94 (t,1H), 4.20-4.24 (t, 2H), 2.57-2.61 (t, 2H). 13C NMR (101 MHz, DMSO): δ 173.0,163.1, 163.0, 132.9, 131.9, 131.7, 131.2,130.0, 129.6, 129.1, 128.4, 123.0,122.1, 36.4, 32.6。
(3)化合物溴化N- (2-甲酰胺基乙基-N- (3-三苯基磷基丙基)) -4-溴-1,8-萘二甲酰亚胺盐的合成:在50 mL的圆底烧瓶中,将化合物 (1) 2 mmol,化合物 (2) 2 mmol,DCC 3mmol,DMAP 0.1 mmol加入到10 mL无水二氯甲烷溶液中室温搅24 h,减压除掉溶剂,硅胶色谱柱分离 (二氯甲烷/甲醇 (V/V=30:1)),真空干燥,得到灰白色晶状固体化合物(3)。产率:65%。1H NMR (400 MHz, CDCl3) δ 8.87 (s, 1H), 8.52 (d, J = 2.8 Hz, 1H),8.50 (d, J = 1.3 Hz, 1H), 8.25 (d, J = 8.0 Hz, 1H), 7.95 (d, J = 7.6 Hz, 1H),7.87 – 7.75 (m, 9H), 7.78 – 7.68 (m, 6H), 4.52 (t, J = 7.2 Hz, 2H), 3.85 (m,2H), 3.47 (s, 2H), 2.89 (t, J = 7.2 Hz, 2H), 1.90 (s, 2H).13C NMR (101 MHz,CDCl3) δ 171.97, 163.44, 163.42, 135.13, 135.10, 133.65, 133.55, 133.06,131.87, 131.04, 130.92, 130.64, 130.52, 130.01, 129.00, 127.93, 123.18,122.34, 118.77, 117.91, 38.70, 38.52, 37.08, 33.85, 22.52。
(4)化合物溴化N-(2-甲酰胺基乙基-N-(3-三苯基磷基丙基))-4-肼基-1,8-萘二甲酰亚胺盐的合成:在25 mL的圆底烧瓶中,将化合物 (3) 1 mmol加入到含有水合肼10mmol的乙醇溶液中回流搅拌8 h,减压除掉溶剂,硅胶色谱柱分离 (二氯甲烷/甲醇(V/V=20:1)),真空干燥,得到橙色固体化合物 (4)。产率:63%。1H NMR (400 MHz, DMSO-d 6 ) δ8.62 (d, J = 8.4 Hz, 1H), 8.26 (d, J = 6.8 Hz, 1H), 8.14 (d, J = 8.7 Hz, 1H),8.10 (d, J = 5.6 Hz, 1H), 7.97 – 7.87 (m, 3H), 7.87 – 7.70 (m, 12H), 7.59 (t,J = 7.9 Hz, 1H), 7.19 (d, J = 8.8 Hz, 1H), 4.70 (s, 2H), 4.21 (t, J = 7.3 Hz,2H), 3.59 (m, 2H), 3.20 (m, 2H), 2.42 (t, J = 7.3 Hz, 2H), 1.67 (m, 2H). 13CNMR (101 MHz, DMSO-d 6 ) δ 170.83, 164.19, 163.27, 153.65, 135.45, 135.42,134.61, 134.12, 134.02, 130.92, 130.81, 130.69, 129.80, 128.95, 124.46,122.09, 119.28, 118.88, 118.43, 107.56, 104.43, 55.41, 36.64, 34.47, 22.69.质谱: 601.2326 m/z。
实施例2 不同浓度的甲醛下Na-FA-Mito的荧光强度变化
配制10 mL浓度为100 mM、10 mM、1 mM、0.1 mM甲醛的水溶液及浓度为1 mM的实施例1制备的荧光探针母液作为备用。
探针浓度为5 μM的PBS缓冲溶液 (pH=7.4,5% DMSO) 中分别加入不同浓度的甲醛(0 – 250 μM) 中相互作用,作用一个小时后进行荧光光谱测试,(λex = 440 nm, λem =450-700 nm),检测各体系中荧光强度,以荧光强度-甲醛浓度作曲线,如图4中大图所示,以539nm荧光强度-甲醛浓度作曲线,如图4中小图所示:随着甲醛浓度的增加,反应体系荧光强度逐渐增加,当甲醛浓度达到150 μM时,反应体系荧光强度达到饱和状态。
实施例3 不同反应时间下Na-FA-Mito的荧光强度变化
配制10 mL浓度为100 mM甲醛的水溶液及浓度为1 mM的实施例1制备的荧光探针母液作为备用。
探针浓度为5 μM的PBS缓冲溶液 (pH=7.4,5% DMSO) 中加入150 μM甲醛或等量PBS缓冲液,每隔2 min测试一次,测试1 h,激发波长为440nm,取其最大发射峰位置λem=539nm处的荧光强度绘制其随时间变化的动力学曲线,如图5所示,探针在PBS缓冲溶液 (pH=7.4,5% DMSO) 中照射1 h,其荧光强度并没有明显变化;而探针与甲醛 (150 μM) 的动力学实验表明,荧光强度大约在40 min时趋于饱和。
实施例4 Na-FA-Mito荧光探针对不同分子或离子的选择性
配制10 mL浓度为100 mM的各种常规的离子及氨基酸的水溶液及浓度为1 mM实施例制得的荧光探针母液作为备用。
探针浓度为5 μM的PBS缓冲溶液 (pH=7.4,5% DMSO) 中分别加入常规离子浓度为1.0 mM;氨基酸的浓度为1.0 mM;活性氧和活性氮浓度为100 μM;醛酮浓度为150 μM。摇匀并作用40 min后进行荧光光谱测试,取其最大发射峰位置λem=539 nm处的荧光强度做条形图如图6所示,其中1 - 25加入的离子分别为:空白,乙二醛,甲基乙二醛,丙酮酸钠,对二甲氨基苯甲醛,三氯乙醛,乙醛,对硝基苯甲醛,丙酮,次氯酸钠,过氧化氢,过氧化二叔丁基,过氧化叔丁醇,一氧化氮,氯化钙,氯化镁,硝酸钾,硫酸钠,亚硝酸钠,亚硫酸氢钠,硫氢化钠,半胱氨酸,谷胱甘肽,葡萄糖,甲醛。通过对比发现,其他物质对探针Na-FA-Mito的荧光几乎没有影响,而甲醛的加入使化合物Na-FA-Mito的荧光显著增强。
实施例5 Na-FA-Mito荧光探针检测外源甲醛细胞成像
将本发明实施例1中的荧光探针Na-FA-Mito应用于HeLa细胞中进行荧光成像,得图7,具体操作步骤如下:
(1)将4份密度为3 × 105 个/mL的HeLa细胞接种到灭菌的铺有盖玻片 (22 mm ×22 mm) 的35 mm培养皿中,在CO2培养箱 (37 oC,5% CO2) 中培养至细胞贴壁;
(2)4份细胞分别进行如下操作:a.空白细胞组;b.向细胞培养液中加入10 μM探针孵育20 min;c.向细胞培养液中加入300 μM甲醛孵育20 min后,加入10 μM探针作用40min;d.向细胞培养液中加入300 μM甲醛孵育20 min后,加入600 μM的亚硫酸氢钠孵育30min,再加入10 μM探针孵育40 min;
(3)各样品用PBS缓冲液冲洗细胞3次,制样后在荧光显微镜下分别明场、单光子FITC通道成像、明场与单光子FITC 通道叠加(Merge)、双光子通道;单光子激发波长为488nm,双光子激发波长为800 nm,发射波段为500-550 nm。
图7中:a1) – a4) 分别为HeLa细胞的明场图,FITC通道,merge图,以及双光子通道;b1) – b4) 分别为HeLa细胞与探针共孵育的明场图,FITC通道,merge图,以及双光子通道;c1) – c2) HeLa细胞与甲醛、探针共孵育的明场图,FITC通道,merge图,以及双光子通道;d1) –d4) HeLa细胞的甲醛负控制实验:HeLa细胞与甲醛20 min,后加SO2孵育30 min,后加探针孵育40 min的明场图,FITC通道,merge图,以及双光子通道。由图7可知,第(3)组的HeLa细胞在绿色通道发出荧光。而在负控制实验中,加入的SO2与甲醛发生亲和加成反应,外加的甲醛被消耗掉,绿色通道无荧光。
实施例6 Na-FA-Mito荧光探针检测内源甲醛细胞成像
将本发明实施例1中的荧光探针Na-FA-Mito应用于HeLa细胞中进行荧光成像,得图8,具体操作步骤如下:
(1)将4份密度为3 × 105 个/mL的HeLa细胞接种到灭菌的铺有盖玻片 (22 mm ×22 mm) 的35 mm培养皿中,在CO2培养箱 (37 oC,5% CO2) 中培养至细胞贴壁;
(2)4份细胞分别进行如下操作:a.空白细胞组;b.向细胞培养液中加入10 μM探针孵育40 min;c.向细胞培养基中加入20 μM毒胡萝卜素 (TG) 培养1 h,再加入10 μM的探针孵育40 min;d.向细胞培养基中加入20 μM毒胡萝卜素 (TG) 培养1 h,细胞外加600 μM亚硫酸氢钠孵育30 min 后再外加5 μM探针孵育40 min;
(3)各样品用PBS缓冲液冲洗细胞3次,制样后在荧光显微镜下分别明场、单光子FITC通道成像、明场与单光子FITC 通道叠加(Merge)、双光子通道;单光子激发波长为488nm,双光子激发波长为800 nm,发射波段为500-550 nm。
图8中:e1) – e4) 分别为HeLa细胞的明场图,FITC通道,merge图,以及双光子通道;f1) – f4) 分别为HeLa细胞与探针共孵育的明场图,FITC通道,merge图,以及双光子通道;g1) – g2) HeLa细胞与毒胡萝卜素 (TG) 共孵育1 h后加探针孵育40 min的明场图,FITC通道,merge图,以及双光子通道;h1) – h4) HeLa细胞的甲醛负控制实验:HeLa细胞与毒胡萝卜素共孵育1 h,后加SO2共孵育30 min,最后后加探针孵育40 min的明场图,FITC通道,merge图,以及双光子通道。由图8可知,第(3) 组的HeLa细胞在绿色通道有荧光。而在负控制实验中,加入的SO2与甲醛发生亲和加成反应,外加的甲醛被消耗掉,绿色通道无荧光。现象与外源性甲醛负控制实验一致,证明内源性甲醛的产生以及探针对内源性甲醛的可检测性。
实施例7 Na-FA-Mito荧光探针与Mito Tracker Deep Red的线粒体共定位
将本发明实施例1中的荧光探针Na-FA-Mito与商业线粒体定位染料Mito TrackerDeep Red共同应用于癌细胞中进行荧光成像,得图9,具体操作步骤如下:
(1)将密度为3 × 105 个/mL的HeLa细胞接种到灭菌的铺有盖玻片 (22 mm × 22mm) 的35 mm培养皿中,在CO2培养箱 (37 oC,5% CO2) 中培养至细胞贴壁;
(2)向细胞培养液中加入150 μM甲醛,孵育20 min后,将探针Na-FA-Mito 5 μM加入到细胞培养皿中,细胞培养箱内孵育40 min,将商业线粒体定位染料Mito Tracker DeepRed 5 μM 加入到细胞培养皿中孵育2 min;
(3)样品用PBS缓冲液冲洗细胞3次,制样后在荧光显微镜下成像;Na-FA-Mito的激发波长为488 nm,发射波段为500-550 nm;Mito Tracker Deep Red的激发波长为647 nm,发射波段为663-738 nm。
图9中:a) HeLa细胞与Mito Tracker Deep Red、甲醛探针Na-FA-Mito以及甲醛共孵育的绿色通道;b) HeLa细胞与Mito Tracker Deep Red、甲醛探针Na-FA-Mito以及甲醛共孵育的红色通道;c) HeLa细胞与Mito Tracker Deep Red、甲醛探针Na-FA-Mito以及甲醛共孵育的红色和绿色重叠通道;d) 绿色通道和红色通道的强度散点图;e) 绿色通道和红色通道的线性相关系数陪面图。经过测试,Pearson's Coefficient为90.87%。说明Na-FA-Mito能够有效的检测线粒体内的甲醛。
Claims (7)
3.根据权利要求2所述的合成方法,其特征在于,各步骤还包括分离纯化的步骤。
4.根据权利要求2所述的合成方法,其特征在于,所述步骤(1)的回流时间为16-24h;步骤(2)的回流时间为4-8h;步骤(3)的反应时间为16-24h;步骤(4)的回流时间为6-10h。
5.根据权利要求2所述的合成方法,其特征在于,所述步骤(3)中,反应温度为室温。
6.根据权利要求2所述的合成方法,其特征在于,所述步骤(3)中,失水剂选自二环己基碳二亚胺或1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐;催化剂选自4-二甲氨基吡啶或1-羟基苯并三唑。
7.一种如权利要求1所述的荧光探针在制备检测溶液或细胞中甲醛试剂中的应用。
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