CN115232152B - 检测次氯酸的荧光探针及其制备方法和应用 - Google Patents
检测次氯酸的荧光探针及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种检测次氯酸的荧光探针及其制备方法和应用,该探针如结构式(I)所示,对次氯酸的检测具有高特异性和高灵敏度,而且具有长波长、选择性高、检测限低等特点,与次氯酸反应后伴随有明显的颜色变化及开启型的荧光响应。该探针可用于细胞和斑马鱼中内源性和外源性次氯酸的检测和成像。
Description
技术领域
本发明涉及一种检测次氯酸的荧光探针及其制备方法和应用,属于荧光探针技术领域。
背景技术
次氯酸作为生物体内存在的一种重要的活性氧物种,它可以参与到生物体重要的生理、病理等过程中,在维持生物体正常生命活动起着重要的作用。生物体内源性的次氯酸通常在髓过氧化物酶的催化下,由过氧化氢和氯离子反应产生。它能够有效地杀灭入侵生物体的各种细菌和病原体,在生物体防御免疫系统中占据着重要地位。但是,生物体内过量的次氯酸的产生和累积会破坏氨基酸、DNA和蛋白质,从而引起一系列的疾病,例如心血管疾病,炎症,甚至癌症等。次氯酸的强氧化性也使得它在家庭以及工业领域也常被用作杀菌剂和漂白剂,如泳池消毒和衣物漂白等,这些应用也增加了人与次氯酸大量接触的风险。大量证据表明,过度暴露于次氯酸环境中会导致相关的疾病,如哮喘,膀胱癌。因此,寻找一种快速、准确、实时的检测次氯酸的方法具有重要的意义。
发明内容
本发明的目的在于提供一种检测次氯酸的荧光探针,该探针具有选择性好、检测限低、特异性识别次氯酸的特点。
本发明的另一个目的是提供该荧光探针的制备方法及其应用,特别是在细胞和斑马鱼中的检测和生物成像。
本发明实现过程如下:
结构式(I)所示的化合物,
结构式(I)所示的化合物的制备方法,包括以下步骤,
(1)制备2-溴-1-环己烯甲醛(1)
(2)制备10-乙基-2-甲氧基-10H-吩噻嗪(2)
(3)制备10-乙基-2-甲氧基-10H-吩噻嗪-3-甲醛(3)
(4)制备10-乙基-2-羟基-10H-吩噻嗪-3-甲醛(4)
(5)制备7-乙基-1,2,3,7-四氢色烯并[2,3-b]吩噻嗪-4-甲醛(5)
(6)制备2-((7-乙基-1,2,3,7-四氢色烯[2,3-b]吩噻嗪-4-基)亚甲基)丙二腈(DHX-4)
上述化合物作为次氯酸荧光探针的应用,荧光检测中该探针的激发波长为490nm,发射波长为600 nm,该探针为比色检测和开启型荧光响应探针,上述化合物适合细胞和斑马鱼中内源性和外源性次氯酸的检测和成像。
本发明的荧光探针响应机理如下:富电子性吩噻嗪部分在探针DHX-4激发态时容易诱导产生分子内光诱导电子转移(Photo-induced Electron Transfer, PET )效应,导致荧光团二氢氧杂蒽电子受体部分荧光受到了抑制,从而实现了对整个探针荧光的调控。相反,探针DHX-4与次氯酸反应后吩噻嗪结构中的硫原子被氧化为亚砜,吩噻嗪部分的电子效应降低,PET效应同时受到了抑制,使得荧光团的荧光得以恢复。因此,探针荧光信号受到了PET过程的调控和基于HClO氧化介导的探针开启荧光响应。
本发明的优点:(1)与传统的检测方法相比,荧光探针检测法具有高效、便捷、选择性高、检测限低等显著优点。(2)本发明吩噻嗪衍生物的N和S原子具有高的电子云密度,促使整个结构呈现蝶形构象的非平面结构,这不仅有利于阻止分子的π-π聚集,还能够抑制分子间振动的形成和聚集态的荧光自猝灭现象。(3)与其他活性氧类物种相比,吩噻嗪中具有富电子特性的硫原子能够高选择性参与对次氯酸的识别,具有优异的特异性,基于吩噻嗪设计的响应次氯酸的荧光探针在环境分析和生物成像中具有独特的优势。(4)本发明荧光探针对次氯酸的检测特异性高、灵敏度好,而且具有结构新颖、选择性高、检测限低、长波长等优点,该探针可以用于细胞和斑马鱼中内源/外源性次氯酸的检测和成像,且该检测不受其他分析物的干扰。
附图说明
图1为DHX-4的1H NMR谱图;
图2为DHX-4的13C NMR谱图;
图3为DHX-4对次氯酸响应前后的紫外吸收光谱图;
图4为DHX-4对次氯酸的浓度滴定荧光谱图;
图5为DHX-4对次氯酸的浓度滴定线性曲线;
图6为DHX-4在不同PH下对次氯酸响应前后的曲线图;
图7为DHX-4对次氯酸的选择性荧光谱图;
图8为DHX-4对次氯酸的响应时间图;
图9为DHX-4对细胞内源性和外源性次氯酸特异性识别的成像图;
图10为DHX-4对斑马鱼内源性和外源性次氯酸特异性识别的成像图。
具体实施方式
下述实施例将有助于理解本发明,但不限于本发明。本发明的荧光探针测试方法如下:将探针分子溶解在PBS(pH=7 .4,10 mM)与乙醇混合溶液中,室温下进行测试。
实施例1:荧光探针的合成
(1) 反应瓶中加入5.6 mL DMF和25 mL氯仿,将混合溶液置于0 oC,搅拌下加入三溴化磷 (5.25 mL),搅拌反应45 min后,向反应液中加入环己酮 (2.5 mL), 然后将化合物置于室温搅拌16 h。反应完成后,将反应液倒入冰水中,并用氢氧化钠溶液调节至pH=7。随后,用DCM萃取混合液,收集有机相,旋蒸除去溶剂,得化合物1为棕黄色油状物。
(2) 向反应瓶中加入2-甲氧基吩噻嗪 (13.08 mmol,1.0 equiv),并用15 mL DMF溶解。随后向反应体系中加入氢氧化钠 (4.0 equiv) ,抽真空,补氩气。最后,向上述体系中加入10.0 mL碘乙烷 (10.0 equiv) ,并置于100 oC下搅拌,反应4 h。反应完成后,将体系冷却至室温,并加入大量的水,用DCM萃取反应体系,收集有机相,无水Na2SO4干燥,减压旋除溶剂得到白色固体的粗产物,柱快速纯化得化合物2白色固体3.2 g,产率:96%。1H NMR(400 MHz, DMSO-d 6 ) δ 7.23 – 7.09 (m, 2H), 7.06 – 6.96 (m, 2H), 6.92 (t, J =7.3 Hz, 1H), 6.55 (dd, J = 7.5, 2.5 Hz, 2H), 3.90 (q, J = 7.0 Hz, 2H), 3.74(s, 3H), 1.29 (t, J = 6.9 Hz, 3H)。
(3) 将2.0 mL DMF加入到反应瓶中,抽真空,补氩气,后置于冰浴下搅拌。随后,将三氯氧磷 (46.6 mmol,1.0 equiv) 缓慢加入到反应体系中,加毕,移至室温继续反应15min。之后,将化合物2 (11.65 mmol,0.4 equiv) 用DMF溶解,缓慢加入到反应体系中,加毕,升温至60℃,继续搅拌6 h。反应完毕,将体系冷却至室温,随后缓慢入水,淬灭反应。用DCM 萃取反应体系,分离收集有机相,无水硫酸钠干燥,减压旋除溶剂得粗产物,柱层析,得化合物3黄色固体2.8 g,产率84%。1H NMR (400 MHz, DMSO-d6) δ 10.09 (s, 1H), 7.33(s, 1H), 7.23 (t, J = 7.9 Hz, 1H), 7.13 (dd, J = 21.4, 8.0 Hz, 2H), 7.00 (t,J = 7.5 Hz, 1H), 6.67 (s, 1H), 4.06 (q, J = 7.0 Hz, 2H), 3.95 (s, 3H), 1.34(t, J = 7.0 Hz, 3H)。
(4) 将铝粉 (43.8 mmol,1.0 equiv) 加入到装有20 mL乙腈的反应瓶中,形成混悬溶液体系。向体系持续通入氩气的情况下,将称量好的碘单质 (18.4 mmol,0.42 equiv)分不同批次加入到反应体系中,加毕,安装冷凝管。后在室温的条件下,将用乙腈溶解的化合物3 (8.76 mmol,0.2 equiv) 加入到反应体系中,随后氩气保护下,加热回流6 h。反应完毕,将体系冷却至室温,随后加入大量的水。用DCM 萃取反应体系,分离收集有机相,无水硫酸钠干燥,减压旋除溶剂得粗产物,柱层析,得化合物4黄色固体2.2 g,产率93%。1H NMR(400 MHz, CDCl3) δ 11.39 (s, 1H), 9.59 (s, 1H), 7.19 – 7.07 (m, 3H), 6.94(dd, J = 23.5, 7.9 Hz, 2H), 6.38 (s, 1H), 3.98 – 3.86 (m, 2H), 1.45 (t, J =7.0 Hz, 3H)。
(5) 称取化合物4 (7.37 mmol,1.0 equiv)于反应瓶中,加入10 mL DMF作为反应溶剂,逐滴加入用DMF稀释的上述油状物3-1 (9.66 mmol,1.3 equiv),再加入碳酸铯(58.96 mmol,8.0 equiv),迅速将反应瓶内抽真空,补氩气,室温下搅拌反应24 h。反应完毕,向体系中加入100 mL水,用DCM萃取,分离收集有机相,无水硫酸钠干燥,减压旋除溶剂得粗产物,柱层析,得化合物5红棕色固体1.2 g,产率64%。1H NMR (400 MHz, CDCl3) δ10.28 (s, 1H), 7.21 – 7.07 (m, 2H), 6.98 – 6.82 (m, 3H), 6.56 (d, J = 16.4Hz, 2H), 3.93 (q, J = 7.0 Hz, 2H), 2.55 (s, 2H), 2.44 (t, J = 6.0 Hz, 2H),1.70 (p, J = 6.0 Hz, 2H), 1.48 – 1.37 (m, 3H). 13C NMR (101 MHz, CDCl3) δ187.3 , 160.8 , 152.5 , 146.8 , 143.5 , 127.5 , 127.4 , 127.0 , 126.2 , 124.0, 123.6 , 123.1 , 119.1 , 116.1 , 115.5 , 112.7 , 102.2 , 42.4 , 30.0 , 21.6, 20.4 , 12.9. HRMS (APCI-MS) m/z: C22H20NO2S+ [M+H]+ 理论计算值362.1209; 实际值 362.1207。
(6) 依次称取化合物5(2.77 mmol,1.0 equiv)、丙二腈 (5.54 mmol,2.0 equiv)于反应瓶中,加入乙腈作为反应溶剂,并加入2.0 mL的哌啶,抽真空,补氩气,加热搅拌反应12 h。反应完毕,减压旋除溶剂得粗产物,柱层析,得化合物DXH-4紫色固体0.3 g,产率27%。1HNMR (400 MHz, CDCl3)δ 8.03 (s, 1H), 7.18 (td, J = 7.8, 1.7 Hz, 1H), 7.09 (dd,J = 7.7, 1.6 Hz, 1H), 7.01 – 6.89 (m, 3H), 6.78 (s, 1H), 6.69 (s, 1H), 3.98(q, J = 7.0 Hz, 2H), 2.86 (t, J = 6.1 Hz, 2H), 2.58 (t, J = 5.7 Hz, 2H), 1.82(p, J = 6.2 Hz, 2H), 1.49 (t, J = 7.0 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ158.6 , 153.3 , 150.0(2) , 147.6 , 142.8 , 129.9 , 127.8 , 127.4 , 126.3 ,123.9 , 123.4 , 122.9 , 120.7 , 116.2 , 117.9 , 115.8 , 115.6 , 110.0 , 101.8, 42.7 , 29.1 , 24.7 , 20.5 , 12.8. HRMS (APCI-MS) m/z: C25H20N3OS+ [M+H]+ 理论计算值410.1322; 实际值410.1321。
实施例2:荧光探针DHX-4与次氯酸响应前后的紫外吸收光谱的测定
准确称量探针DHX-4 4.2 mg,用DMSO溶解后再定容至10 mL容量瓶中,配制成为浓度为1.0 mM的探针母液,保存于4~8 ℃备用;用蒸馏水配制10 mM的次氯酸标准溶液。
在10 μM探针的PBS和乙醇的混合溶液中加入100 μM的次氯酸溶液,反应30 min,检测该混合体系在600 nm处的荧光强度。如图3所示,纯探针体系在483 nm处显示出最大吸收峰,且溶液体系呈现紫色。当加入次氯酸钠溶液反应30 min后,探针的吸收峰在515 nm显示出最大吸收峰,且溶液体系呈现粉色。通过对探针与次氯酸钠反应后体系的最大吸收波长范围,确定了探针最佳的激发波长为490 nm。
实施例3:荧光探针DHX-4对次氯酸的浓度滴定荧光谱图
在10 μM探针的PBS溶液(EtOH: PBS = 1:1,10 mM, pH=7.4)中加入不同浓度(20-200 μM)的次氯酸溶液,反应30min,检测该混合体系在600 nm处的荧光强度。如图4所示,检测到600 nm处的荧光强度随次氯酸溶液浓度的增加而逐渐增强。且次氯酸浓度在20-200 μM范围内具有较好的荧光强度-浓度线性关系,如图5,线性曲线Y=175.03X+5687.27, R2=0.9942。
实施例4:荧光探针DHX-4在不同PH下对次氯酸响应前后的曲线图
配制不同PH(2-12)的PBS缓冲溶液,在10 μM探针的不同PH溶液(EtOH:PBS=3:7,20 mM, pH=2-12)中加入100 μM的次氯酸溶液,得到600 nm发射波长处的荧光强度与不同PH的荧光强度变化曲线图。如图6,探针在强酸与强碱环境下不会发生显著的荧光变化,但在2<pH<7范围内,探针在600 nm处荧光信号逐渐增强,且在pH=7的环境下,表现出了对次氯酸强的荧光性能以及响应能力。以上实验结果表明,探针DHX-4可以用于生理条件下对次氯酸的检测。
实施例5:荧光探针DHX-4对次氯酸的选择性荧光谱图
用蒸馏水配制10 mM的各种分析物溶液(GSH,Cys,Met,F-,S2O5 2-,S2O4 2-,S2-,S2O3 2-,CO3 2-,HCO3 -,SCN−,·OH,ONOO-,NO2 -,1O2,TBHP,H2O2,ClO4 -,Hcy,ClO-)。
在10 μM探针的PBS溶液(EtOH: PBS = 1:1,10 mM, pH=7.4)中分别加入不同的分析物溶液,如图7所示,在探针溶液中加入其他分析物后,没有观察到明显的荧光强度变化。然而将次氯酸加入到探针溶液中后,可在600 nm处观察到明显增强的荧光信号。
实施例6:荧光探针DHX-4对次氯酸的响应时间图
在10 μM探针的PBS溶液(EtOH:PBS=1:1, 10 mM, pH=7.4)中加入100 μM的次氯酸溶液,得到600 nm发射波长处的荧光发射强度与检测时间的曲线图。如图8所示,在探针溶液中加入次氯酸溶液后,即可产生明显的荧光响应。并且随着检测时间的延长,观察到600nm处的荧光发射强度也逐渐增强,并在反应30 min后荧光强度增至最大并达到平衡。
实施例7:荧光探针DHX-4对细胞内源性和外源性次氯酸的成像
利用激光共聚焦显微镜研究了探针DHX-4对HeLa细胞内源性和外源性次氯酸的响应情况。细胞成像实验分为5组,其中A为直接用10 μM探针溶液孵育30 min后的细胞成像图,B为先用10 μM探针溶液孵育30 min后,加入50 μM次氯酸溶液的细胞成像图,C为先用10μM探针溶液孵育30 min后,加入100 μM次氯酸溶液的细胞成像图,D为先用2 μg/mL脂多糖(LPS) 培养液孵育2 h,之后用10 μM探针溶液孵育30 min后的细胞成像图,E为先用2 μg/mL脂多糖 (LPS) 培养液孵育2 h,之后加入活性氧清除剂NAC (20 mM) 再孵育1h后,再用10 μM探针溶液孵育30 min后的细胞成像图。如图9所示,B组、C组和D组可以观察到红色通道显著的红色荧光信号且浓度增加荧光信号增强,而A组和E组在红色通道内仅能观察到微弱的红色荧光信号,说明探针可以对细胞内源性和外源性次氯酸进行检测和成像。
实施例8:荧光探针DHX-4对斑马鱼内源性和外源性次氯酸的成像
利用激光共聚焦显微镜研究了探针DHX-4对斑马鱼内源性和外源性次氯酸的响应情况。成像实验分为3组,其中第一组斑马鱼用含有10 μM探针DHX-4的培养液培养斑马鱼30min后进行成像,第二组斑马鱼先用10 μM探针的培养液培养斑马鱼30 min,之后加入100 μM次氯酸培养液孵育30min后进行成像,第三组斑马鱼先用2 μg/mL脂多糖 (LPS) 培养液孵育2 h,之后用10 μM探针溶液孵育30 min后进行共聚焦成像。如图10所示,第二组和第三组可以观察到明显的红色荧光信号,而第一组在红色通道内仅能观察到微弱的红色荧光信号,说明探针可以对斑马鱼内源性和外源性次氯酸进行检测和成像。
Claims (5)
3.权利要求1所示化合物在制备次氯酸荧光探针中的应用。
4.根据权利要求3所述应用,其特征在于荧光检测中该探针的激发波长为490nm,发射波长为600nm。
5.根据权利要求3所述应用,其特征在于该探针为比色检测和开启型荧光响应探针。
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