CN111072694B - 一种硫化氢识别检测荧光探针及其制备方法与应用 - Google Patents
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Abstract
本发明公开了属于化学分析技术领域,涉及小分子荧光探针,具体涉及式(1)结构的硫化氢荧光探针及其制备方法与应用,本发明的探针属于氟硼二吡咯(BODIPY)结构类荧光探针,具有良好的稳定性及水溶性,并能对硫化氢进行快速灵敏的检测。
Description
技术领域:
本发明属于化学分析技术领域,涉及小分子荧光探针,具体涉及一种硫化氢荧光探针及其制备方法与应用,本发明的探针属于氟硼二吡咯(BODIPY)结构类荧光探针,具有良好的稳定性及水溶性,并能对硫化氢进行快速灵敏的检测。
技术背景:
现有技术公开了硫化氢是继一氧化氮之后的又一气体信号分子,它是一种最简单的生物硫醇,在人体中可以通过酶催化或非酶催化的途径产生。研究显示,硫化氢与其他活性硫物质在维持细胞健康状态的一系列生理反应中扮演着重要的角色。有研究表明,硫化氢水平的异常可能会导致多种疾病,如糖尿病、帕金森症和阿尔茨海默氏症。尽管越来越多的研究发现硫化氢对细胞和动物的生理过程有诸多重要影响,但是具体的分子作用机制目前仍不清晰。
据报道,硫化氢分子本身具有非常特殊的化学性质,它既是一种有力的还原剂,还是一种良好的亲核试剂,由于硫化氢的活泼的理化性质,使若干关于硫化氢生物浓度的报道受到质疑,也对检测生命系统中硫化氢的水平带来新的挑战性。本领域技术人员关注到所存在的问题,所以有必要开发可以在细胞、组织乃至动物体内检测硫化氢的新技术,以帮助人们更好的理解这一短暂的生物分子是如何对病理生理过程发挥作用。传统的硫化氢检测技术如比色法和气相色谱法可以检测到硫化氢的产生,但却无法对活细胞和组织中硫化氢进行选择性、灵敏性的检测。为了应对这一挑战,基于反应型的H2S荧光探针应运而生,荧光探针具有诸多优点,如很高灵敏度和选择性、可实现实时可视化检测以及具有良好的生物相容等。因此,研究和开发更加符合实际应用要求的硫化氢荧光探针仍具有重要的科研价值。
基于现有技术的现状,本申请的发明人拟提供一种硫化氢荧光探针及其制备方法与应用。
发明内容:
本发明的目的在于,基于现有技术的现状,提供一种高灵敏度硫化氢探针的制备方法,本发明的探针可应用于硫化氢的检测识别。本发明的探针属氟硼二吡咯(BODIPY)结构类荧光探针,具有良好的稳定性及水溶性,并能对硫化氢进行快速灵敏的检测。
本发明所述的硫化氢识别探针,其结构通式为:
其中,R1为甲基或烯丙基。
本发明中,通式(1)探针具体的制备路线如下:
其中,n1=1或2,R1甲基或烯丙基。
本发明中,通式(1)探针的制备方法包括:
1)将N,N-二甲基甲酰胺溶于二氯甲烷中,0℃下缓慢滴加三氯氧磷的二氯甲烷溶液,将化合物异吲哚啉-1-酮的二氯甲烷溶液滴加入上述溶液中,45℃下回流反应6h冰水浴下调节pH至8,萃取,有机层用饱和氯化钠溶液洗3次,无水硫酸镁干燥,中性氧化铝过柱得亮黄色固体L2-1;
2)将化合物L2-1溶于4M的NaOH乙醇溶液中,80℃下回流3h。反应液浓缩后于冰水浴下调节PH为5-6,将析出的固体过滤,经甲醇和水重结晶得化合物L2-2;
3)将化合物L2-2溶于二氯甲烷中,0℃氮气保护下加入吡咯的二氯甲烷溶液和三氯氧磷二氯甲烷溶液,0℃下搅拌30min,然后加入三乙胺搅拌10min,最后加入三氟化硼乙醚溶液并于室温下反应20h。反应液加饱和碳酸氢钠溶液淬灭,二氯甲烷萃取,合并有机相浓缩后经柱层析得化合物L2-3;
4)将化合物A溶于的三氟乙酸中,加入三苯甲醇,室温下搅拌2h。冰水浴下加入乙醚并调节pH为4-5,后用10%的碳酸钠溶液调节pH5-6,将析出的白色固体过滤干燥后得到化合物L2-4;
5)在圆底烧瓶中加入叠氮化钠、水、二氯甲烷,冰水浴剧烈搅拌下缓慢滴加三氟甲磺酸酐,反应液于0℃下反应2h,加入饱和碳酸氢钠溶液淬灭反应,用二氯甲烷萃取,有机层加入饱和碳酸氢钠溶液洗3次,合并有机相直接用于下一步;在圆底烧瓶中依次加入L2-4、硫酸锌、甲醇和水,冰水浴下滴加三乙胺,滴加上述TfN3溶液,TLC检测至反应结束调节溶液pH为2,水相用二氯甲烷萃取,有机相浓缩后经柱层析分离得化合物L2-5;
6)将化合物L2-5溶于二氯甲烷中,加入三氟乙酸和三乙基硅烷,氮气保护下室温反应4h。反应结束后加水洗,二氯甲烷萃取,有机相干燥浓缩后直接用于下一步;
7)将化合物L 2-3和上述浓缩液溶于二氯甲烷中,氮气保护下室温1.5h,反应液浓缩经柱层析得通式(1)探针。
本发明的探针的水溶性好,在PBS(10mM,pH7.4)缓冲溶液中,室温下可实现对硫化氢的检测识别。
本发明的探针对硫化氢的响应速度快,反应时间5min。
本发明的探针对硫化氢的响应为荧光淬灭型或比率型,紫外下亦可检测到明显的荧光颜色变化。
说明书附图
图1,探针(1)的1H NMR谱图;
图2,探针(1)的13CNMR谱图;
图3,探针(1)与硫化氢响应的紫外吸收光谱变化;
图4,探针(1)与硫化氢响应的荧光光谱变化。
具体实施方式:
下面结合实施例对本发明做进一步说明,本发明的实施例仅用于说明本发明的技术方案,本发明不受具体实施例的限制。
实施例1
制备通式(1)的化合物:
按下述步骤:
1)制备化合物L2-1:
将DMF(2.1mL,27.3mmol)溶于5mL二氯甲烷中,0℃下缓慢滴加三氯氧磷(2.3mL,25mmol)的二氯甲烷溶液,将化合物A(1.65g,12.4mmol)的二氯甲烷溶液滴加入上述溶液中,45℃下回流反应6h。反应冷却至室温,冰水浴下加入5M的NaOH溶液调节PH至8,分液,有机层用饱和氯化钠溶液洗3次,无水硫酸镁干燥,中性氧化铝过柱(PE∶EA=8∶1),得亮黄色固体L2-1 1.35g,产率53%。LC-MS(ESI)m/z:207.1[M]+;
2)制备化合物L2-2:
将化合物L2-1(898mg,5mmol)溶于NaOH的乙醇溶液(4M,65mL)中,80℃下回流3h。反应液浓缩后于冰水浴下加2M HCl溶液调节PH为5-6,有大量固体析出,过滤,滤饼水洗,干燥,得淡黄色固体粉末,经甲醇∶水(1∶3,v/v)重结晶,得化合物L2-2,720mg,产率80%。LC-MS(ESI)m/z:181[M+H]+;1H NMR(400MHz,DMSO-d6)δ9.83(s,1H),8.12(d,J=8.5Hz,1H),7.65(d,J=8.5Hz,1H),7.39(t,J=7.6Hz,1H),7.25(d,J=8.1Hz,1H);
3)制备化合物L2-3:
将化合物L2-2(298mg,1.65mmol)溶于8mL二氯甲烷中,0℃,氮气保护下加入吡咯(111mg,1.65mmol)的二氯甲烷0.5mL和三氯氧磷(253mg,1.65mmol)的二氯甲烷溶液0.5mL,0℃下搅拌30min,然后加入2.3mL的三乙胺搅拌10min,最后加入三氟化硼乙醚溶液2.3mL,于室温反应20h。反应液加饱和碳酸氢钠溶液淬灭,二氯甲烷萃取,合并有机相用饱和氯化钠溶液洗,无水硫酸钠钠干燥,旋蒸后过柱(PE∶EA=10∶1-5∶1)得化合物L2-3,210mg,产率46%。LC-MS(ESI)m/z:277.0[M+H]+;1H NMR(400MHz,CDCl3)δ7.85-7.80(m,2H),7.67-7.60(m,2H),7.50-7.44(m,2H),6.92(d,J=3.7Hz,1H),6.44(d,J=3.4Hz,1H);
4)制备化合物L2-4B:
将L-半胱氨酸甲酯盐酸盐(5.2g,20mmol)溶于49mL TFA中,加入三苯甲醇(5.2g,20mmol),室温下搅拌2h。冰水浴下加入乙醚2mL和4N的氢氧化钠调节PH4-5,然后用10%的碳酸钠溶液调节PH5-6,水相用乙醚萃取2次,有机相用无水硫酸钠干燥,过滤,旋蒸除去溶剂后得粗品化合物L2-4B为3.14g,直接用于下一步;
5)制备化合物L2-5B:
于10mL圆底烧瓶中依次加入叠氮化钠(1.17g,18mmol),3mL水,3mL二氯甲烷,冷却至0℃,剧烈搅拌下缓慢滴加三氟甲磺酸酐(3mL,18mmol),滴加结束后反应液于0℃下搅拌反应2h,反应结束后加入饱和碳酸氢钠溶液3mL,水层加二氯甲烷萃取(2*3mL),有机层加入饱和碳酸氢钠洗(2*3mL),所得有机相的TfN3直接用于下一步;
在50mL圆底烧瓶中加入L2-4B(754mg,2mmol),硫酸锌(6.4mg,0.04mmol),甲醇13.4mL,水2.6mL,混合物冷却至0℃后缓慢加入三乙胺1.1mL,然后滴加上述TfN3溶液,后于0℃反应,TLC检测至反应结束。加入PH为3的磷酸缓冲液,后用稀盐酸调节溶液PH为2,分液,水相用二氯甲烷溶液萃取,有机相经饱和食盐水洗,无水硫酸钠干燥,过滤,旋蒸除去溶剂后过硅胶柱(PE∶EA=50∶1),得L2-5B,600mg,产率74%。1H NMR(400MHz,CDCl3)δ7.45(dd,J=7.6,1.8Hz,6H),7.31(dd,J=8.3,6.7Hz,6H),7.23(t,J=7.4Hz,3H),3.70(s,3H),3.20(dd,J=8.1,6.0Hz,1H),2.70(dd,J=13.4,5.9Hz,1H),2.56(dd,J=13.4,8.1Hz,1H);
6)制备化合物L2-6B:
L2-5B(500mg,2mmol)溶液5ml的二氯甲烷溶液中,后加入TFA(327uL,1.5mmol)和三乙基硅烷(194uL,0.6mmol),氮气保护下室温搅拌4h。反应结束后加水洗,二氯甲烷萃取,有机相用无水硫酸钠干燥,过滤,浓缩后直接用于下一步;
7)制备通式(1)的化合物
将化合物L2-5B(55mg,0.1mmol)和上述浓缩液溶于二氯甲烷中,氮气保护下室温搅拌2h,反应液减压旋蒸除去溶剂,经硅胶柱层析(PE∶EA=100∶1),得化合物L2-B 11.5mg,产率12%。LC-MS(ESI)m/z:424.1[M+Na]+;1H NMR(400MHz,CDCl3)δ7.90(d,J=8.2Hz,1H),7.82(d,J=8.0Hz,1H),7.64-7.57(m,2H),7.47-7.41(m,2H),6.89(d,J=3.8Hz,1H),6.43(dd,J=3.9,2.2Hz,1H),4.32(dd,J=7.9,5.1Hz,1H),3.85(dd,J=14.0,5.1Hz,1H),3.77(s,3H),3.68(dd,J=13.9,7.9Hz,1H);13C NMR(151MHz,Chloroform-d)δ162.22,151.97,142.63,142.37,135.74,133.96,133.14,130.34,130.28,129.07,129.01,95.15,90.20,57.36,12.84。
实施例2 探针(1)与硫化氢响应的紫外吸收光谱变化
在石英比色皿中加入3mL PBS(10mM,pH7.4),进行基线调零,然后加入10μL的L2-B乙腈储备液使其终浓度为10μM,再加入5倍当量的NaSH溶液,测试不同时间探针L2-B与硫化氢反应的紫外吸收变化。由图3可知,随着时间延长,探针在555nm处的吸收峰逐渐降低,而470nm处的紫外吸收逐渐增强,15min达到峰值。
实施例3 探针(1)与硫化氢响应的荧光光谱变化
在石英比色皿中加入3mL PBS(10mM,pH7.4),然后加入10μL的L2-B乙腈储备液使其终浓度为10μM,再加入2.5倍当量的NaSH溶液,检测探针L2-B与硫化氢响应的荧光-时间变化。由图4可以看出,随着反应时间延长,探针在576nm处的荧光逐渐淬灭,并在542nm处出现新的峰,15min时反应达到平衡。
Claims (3)
2.权利要求1所述的检测硫化氢的荧光探针的制备方法,其特征在于,其制备路线如下:
其中R1为甲基或烯丙基;
所述制备方法其包括步骤:
(1)L2-4与硫酸锌溶于水和甲醇和二氯甲烷的混合溶剂其体积比为1:5:2,混合物冷却至0℃后缓慢加入三乙胺,滴加TfN3溶液,0℃反应,TLC检测至反应结束;加入pH=3的磷酸缓冲液,用稀盐酸调节溶液pH为2,水相用二氯甲烷萃取,有机相经硅胶柱层析,制得化合物L2-5;
(2)L2-5溶于二氯甲烷中,加入TFA和三乙基硅烷,氮气保护下室温搅拌反应;反应结束后加水洗,二氯甲烷萃取,有机相用无水硫酸钠干燥,过滤,滤液浓缩制得L2-6,直接用于下一步骤;
(3)将L2-3和L2-6浓缩液溶于二氯甲烷中,氮气保护下室温搅拌1.5h,反应液浓缩经柱层析得通式(1)探针。
3.权利要求1所述的通式(1)探针在制备用于硫化氢识别检测荧光探针中的用途。
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