CN109608495B - 一种检测hno的化合物及其制备方法和应用 - Google Patents
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Abstract
Description
技术领域
本发明属于生物化工领域,涉及构建一种用于检测细胞及斑马鱼内次硝酸(HNO)的近红外荧光分子探针,具体涉及一种检测次硝酸(HNO)的近红外荧光分子探针及其制备方法和生物应用。
背景技术
一氧化氮(NO)是哺乳动物系统中重要的生物信号分子,它在许多生理和病理过程中起着至关重要的作用,包括神经传递调节,血管舒张和免疫反应。次硝酸(HNO)是一种单电子还原和质子化的一氧化氮(NO)衍生物,具有与NO不同的生物效应。 在生命系统中,作为亲电子体的HNO可以氧化蛋白质硫醇并直接抑制醛脱氢酶,组织蛋白酶P和一些其他含巯基的酶;通过介导心脏组织中的钙,HNO也可以作为心力衰竭的新疗法;此外,一些生化研究表明HNO和NO可能在适当的条件下相互转化。因此,开发能够监测生物硝酰基的有效分析方法是非常重要的。
迄今为止,已经开发了几种分析方法,包括电化学分析,质谱,NMR,HPLC和比色法,用于检测各种样品中的HNO。尽管它具有高灵敏度分析,但这些方法不适用于检测生命系统中的HNO。最近,荧光探针由于其超灵敏度,非侵入性检测以及对体外和体内靶标的优异时空分析而引起广泛关注。迄今为止,已开发出各种荧光探针用于HNO的识别,主要是基于Cu(Ⅱ)还原为Cu(Ⅰ),或氮氧化物还原为羟胺。尽管这些荧光探针在检测HNO方面具有一些优势,但它们往往受到生物系统中丰富的生物还原剂如谷胱甘肽(GSH)和抗坏血酸的干扰。为了解决这个问题,已经开发了几种不含金属的荧光探针,它们基于HNO和三芳基膦之间的反应,它们不受生物还原剂的干扰。然而,这些无金属荧光探针中的大多数受到短发射波长的限制,这可能诱导光漂白,细胞自发荧光和对组织或细胞器的光损伤,使得它们不适合体内成像。此外,相当多的报道的荧光探针具有反应时间长和检测限高的缺点。为了解决这些缺陷,迫切需要一种能够克服上述缺点的近红外(> 650nm)无金属荧光探针。因此,开发一种能够克服上述缺点的新型荧光探针显得尤为必要。
发明内容
本发明第一个目的是提供一种检测HNO的荧光探针。
本发明第二个目的是提供一种检测HNO的荧光探针的制备方法。
本发明第三个目的是提供一种检测HNO的荧光探针在检测细胞内HNO的应用。
本发明第四个目的是提供一种检测HNO的荧光探针在检测斑马鱼体内HNO的应用。
本发明第五个目的是提供一种检测HNO的化合物。
本发明第六个目的是提供一种检测HNO的化合物的制备方法。
本发明第七个目的是提供一种检测HNO的化合物在检测细胞内HNO的应用。
本发明第八个目的是提供一种检测HNO的化合物在检测斑马鱼体内HNO的应用。
本发明的技术方案如下:
本发明由近红外荧光染料NF-OH和HNO识别基团(三芳基膦)通过酯键连在一起,合成近红外荧光分子探针NF-P,用于体外和体内特异性监测HNO,并成功用于对活细胞及斑马鱼中痕量的HNO进行跟踪检测。
所述近红外荧光分子探针和化合物NF-P,其结构通式如下:
其中R1、R2、R3、R4、R5、R6、R7、R8、R9、R10、R11均为引入的取代基,可用于调节荧光探针电子效应使其荧光发生变化。
本发明优选R5为氧原子,R1、R2、R3、R4、R6、R7、R8、R9、R10、R11为氢。
所述化合物NF-OH,具有如下结构:
其中R1、R2、R3、R4、R5、R6、R7、R8、R9、R10、R11均为引入的取代基,可用于调节荧光探针电子效应使其荧光发生变化。本发明优选R5为氧原子,R1、R2、R3、R4、R6、R7、R8、R9、R10、R11为氢。
根据本发明所述检测HNO的荧光探针具有发射波长较长(>600 nm)和水溶性较好的特点。
根据本发明所述检测HNO的荧光探针,所述化合物荧光团为NF-OH,识别基团为三芳基膦,并由酯键连在一起。
根据本发明所述检测HNO的荧光探针,所述R1、R2、R3、R4、R5、R6、R7、R8、R9、R10、R11均为引入的取代基,调节R基团可使荧光探针在检测HNO时荧光发射波长发生变化。
本发明提供一种近红外荧光分子探针的制备方法,该方法包括下述步骤:
(1)三芳基膦与NF-OH通过酯化反应,得到化合物NF-P;
上述化合物具体结构式如下:
所述检测HNO的近红外荧光分子探针的制备方法与合成路线如下:
本发明独创性是基于近红外荧光染料NF-OH,并通过酯键将其与识别基团(三芳基膦)桥连在一起,如所预期的,NF-P被酯键部分的吸电子羰基猝灭。在NF-P于HNO相互作用时,产生氮杂萘并随后发生分子内亲核攻击,释放出近红外荧光团NF-OH,从而实现荧光增强,达到检测HNO的目的。所得荧光探针NF-P具有生物兼容性好、检测灵明度高、更长的发射波长、抗干扰能力强等特点。
本发明还提供一种检测HNO的化合物,其特征在于,所述化合物NF-P,其结构通式如下:
其中R1、R2、R3、R4、R5、R6、R7、R8、R9、R10、R11均为引入的取代基,可用于调节荧光探针电子效应使其荧光发生变化。
本发明还提供一种检测HNO的化合物的制备方法,该方法包括下述步骤:
(1)三芳基膦与NF-OH通过酯化反应,得到化合物NF-P;
本发明进一步提供一种检测HNO的近红外荧光分子探针在检测细胞及斑马鱼内HNO的应用。
本发明进一步提供一种检测HNO的化合物在检测细胞及斑马鱼内HNO的应用。
本发明提供的检测HNO的近红外荧光分子探针和化合物的详细制备方法:
根据所述用于检测HNO的小分子化合物NF-P,优选R5为氧原子,R1、R2、R3、R4、R6、R7、R8、R9、R10、R11为氢,为例进行说明。
合成步骤如下:
以NF-OH为原料经过亲核取代反应制得NF-P。
将化合物NF-OH(50mg,0.16mmol)和2-(二苯基膦基)苯甲酸(161.7mg,0.528mmol)溶解在二氯甲烷中,然后加入4-(二甲基氨基) - 吡啶-4-甲苯磺酸酯(141.3mg,0.48mmol)和N,N'-二异丙基碳二亚胺(60.6mg,0.48mmol)。 在氩气保护下,将反应体系在室温下搅拌5小时。然后用乙酸乙酯萃取,分离有机相,无水硫酸钠干燥,柱层析,得到产物:NF-P。
术语:
Absorbance为吸收值。
FL intensity为荧光强度。
有益技术效果:
本发明提供一种能够在近红外区对硝酰氧基(HNO)进行跟踪监测的荧光探针,该探针克服了相关技术中存在的一些缺陷,如该类荧光探针不受生物还原剂的干扰,短发射波长的限制,此外,相当多的报道的荧光探针具有反应时间长和检测限高的缺点。在本发明中,通过利用近红外荧光染料NF-OH和HNO识别基团(三芳基膦)通过酯键连在一起,合成了近红外荧光分子探针NF-P, 从而解决了该类荧光探针分子存在的上述缺陷;该荧光探针对硝酰氧基(HNO)具有很好的选择性,表现出抗干扰能力强,灵敏度高等特点。同时,本发明所述的近红外荧光探针可以应用于对细胞及斑马鱼内HNO的实时跟踪监测。
本发明提供一种检测硝酰氧基(HNO)的化合物NF-P,可用于检测细胞及斑马鱼内硝酰氧基,化合物NF-P选择性较好,抗干扰能力更强,具有更高的灵敏性。
附图说明
图1 (A)是荧光探针NF-P在PBS/DMSO 缓冲溶液(7: 3, v / v, pH=7.4)中与HNO反应前后的紫外吸收光谱图,图(B)为NF-P在PBS/DMSO 缓冲溶液(7: 3, v / v, pH=7.4)中与HNO反应前后的荧光发射光谱图;
图2 为荧光探针NF-P表现出的高选择性测试图,其中a.空白组;b. NaClO;c.GSH;d. Na2S;e. HA;f. FeCl3;g. H2O2;h. AA;i. NO2 -;j. NO3 -;k. GSNO;l. NOC7;m.NOR1;n. ONOO-;o. SNAP;p. AS (Angeli盐(AS)被用作HNO供体)。
图3 为荧光探针NF-P对A549细胞内HNO的检测效果图。
图4 为荧光探针NF-P对斑马鱼内HNO的检测效果图。
图5为化合物NF-P的表征图谱(氢谱),氘代试剂为d 6-DMSO;
图6 为化合物NF-P的表征图谱(碳谱),氘代试剂为d 6-DMSO;
图7为化合物NF-P的高分辨质谱图。
具体实施方式
为了更好地理解本发明,下面结合实施例进一步阐明本发明的内容,但本发明的内容不仅仅局限于下面的实施例。
实施例:近红外荧光分子探针NF-P的制备方法, (本发明优选R5为氧原子,R1、R2、R3、R4、R6、R7、R8、R9、R10、R11为氢)。
将化合物NF-OH(50mg,0.16mmol)和2-(二苯基膦基)苯甲酸(161.7mg,0.528mmol)溶解在二氯甲烷中,然后加入4-(二甲基氨基) - 吡啶-4-甲苯磺酸酯(141.3mg,0.48mmol)和N,N'-二异丙基碳二亚胺(60.6mg,0.48mmol)。 在氩气保护下,将反应体系在室温下搅拌5小时。然后用乙酸乙酯萃取,分离有机相,无水硫酸钠干燥,柱层析,得到产物:NF-P。
化合物NF-P的氢谱(如图5),氘代试剂为d 6-DMSO。
1H NMR (d 6-DMSO, 405 MHz): δ 8.74 (d, J = 8.1 Hz, 1H), δ 8.25 (dd, J =8.1 Hz, 4.0 Hz, 1H), δ 7.96-7.92 (m, 1H), δ 7.79 (d, J = 12.1 Hz, 3H), δ 7.74(s, 1H), δ 7.65-7.59 (m, 3H), δ 7.54 (d, J = 16.2 Hz, 2H), δ 7.42-7.40 (m,5H), δ 7.25-7.21 (m, 4H), δ 7.09 (d, J = 8.1 Hz, 2H), δ 7.06 (d, J = 8.1 Hz,1H), δ 6.94-6.90 (m, 1H);
化合物NF-P的碳谱(如图6),氘代试剂为d 6-DMSO。
13C NMR (d 6-DMSO, 150 MHz): δ 170.79, 165.10, 158.40, 153.50, 152.52,152.04, 140.78, 140.50, 137.91, 137.65, 137.53, 135.98, 134.13, 133.92,133.41, 131.90, 131.59, 129.81, 129.51, 129.30, 129.23, 126.70, 125.16,122.84, 122.80, 120.47, 119.57, 117.58, 116.23, 61.05, 60.21;
化合物NF-P的高分辨质谱(如图7)
HRMS (ESI) calcd for C39H26N2O3P+: 601.1675, Found: 601.1675 [M + H]+。
效果例:
参考图1:在生理条件下(PBS/DMSO缓冲溶液(7: 3, v / v),10 mM,pH = 7.4,37℃),测试NF-P对HNO(Angeli盐(AS)被用作HNO供体)的光学响应。由图1(A):NF-P(10 μM)在425 nm处产生主要吸收带。在与AS(100 μM)孵育后,425 nm处的原始吸收带逐渐减少,并且在 556 nm处观察到伴随的红移新吸收峰出现。同时,反应体系颜色由黄色变为红色。此外,由图1(B):NF-P在 530 nm光激发下在 688 nm处显示出相对弱的荧光(Φ= 0.001)。在于AS(100 μM)孵育后,在 688 nm处观察到显着的荧光增强信号,并且荧光强度在约10分钟达到顶点。
参考图2:是近红外荧光探针NF-P在PBS/DMSO 缓冲溶液(7: 3, v / v, pH=7.4)中的干扰测试图。由图可以看出NF-P对HNO具有很好的选择性。
参考图3:是近红外荧光探针NF-P在A549细胞中与HNO的成像效果图。参考图4:是近红外荧光探针NF-P在斑马鱼中与HNO的成像效果图。
本发明提供一种近红外区检测硝酰氧基(HNO)的荧光探针NF-P可用于检测细胞及斑马鱼内HNO,该探针克服了相关技术中存在的一些缺陷,如该类荧光探针不受生物还原剂的干扰,短发射波长的限制,此外,相当多的报道的荧光探针具有反应时间长和检测限高的缺点。在本发明中,通过利用近红外荧光染料NF-OH和HNO识别基团(三芳基膦)通过酯键连在一起,合成了近红外荧光分子探针NF-P, 从而解决了该类荧光探针分子存在的上述缺陷;该荧光探针对硝酰氧基(HNO)具有很好的选择性,表现出抗干扰能力强,灵敏度高等特点。同时,本发明所述的近红外荧光探针可以应用于对细胞及斑马鱼内HNO的实时跟踪监测。
本发明提供一种在近红外区检测硝酰氧基(HNO)的的化合物NF-P,可用于在近红外区检测细胞及斑马鱼内HNO,化合物NF-P对HNO选择性好,抗干扰能力强,具有高的灵敏性。
以上显示描述了本发明的基本原理、主要特征和本发明的主要用途。本行业的技术人员应该了解,本发明不受上述实施例的限制,上述实施例和说明书中描述的只是说明本发明的原理,在不脱离本发明精神和范围的前提下本发明还会有各种变化和改进,这些变化和改进都落入要求保护的本发明范围内。本发明要求保护范围由所附的权利要求书及其等同物界定。
Claims (5)
3.根据权利要求2所述化合物的制备方法,其特征在于:将化合物NF-OH和2-(二苯基膦基)苯甲酸溶解在二氯甲烷中,然后加入4-二甲基氨基吡啶对甲苯磺酸盐和N,N'-二异丙基碳二亚胺;在氩气保护下,将反应体系在室温下搅拌5小时;然后用乙酸乙酯萃取,分离有机相,无水硫酸钠干燥,柱层析,得到产物:化合物NF-P。
4.权利要求1所述化合物作为荧光探针用于非疾病的诊断与治疗目的的检测次硝酸HNO的应用。
5.权利要求1所述化合物作为荧光探针用于非疾病的诊断与治疗目的的检测细胞及斑马鱼内次硝酸HNO的应用。
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