CN113880821A - 一类双光子荧光探针对癫痫脑内次氯酸特征成像的荧光探针设计及其合成方法 - Google Patents
一类双光子荧光探针对癫痫脑内次氯酸特征成像的荧光探针设计及其合成方法 Download PDFInfo
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Abstract
本发明属于分子生物学、医学等领域,尤其涉及本发明涉及一类以喹啉为母核的针对癫痫脑内次氯酸动态变化特征成像的荧光探针的设计及合成方法。荧光探针化合物结构如下式所示:
Description
技术领域
本发明属于分子生物学、医学等领域,尤其涉及本发明涉及一类以喹啉为母核的针对癫痫脑内次氯酸动态变化特征成像的荧光探针的设计及合成方法及应用说明书。
背景技术
荧光成像已被广泛应用于生物医学和临床诊断领域,小分子探针是通过荧光检测和/或成像监测特定细胞过程的强大工具。这些分子探针在响应特定分析物或环境时会改变其荧光发射。它们已广泛应用于细胞成像、药物筛选和医学治疗等多个领域。基于小分子探针的检测方法具有特定的优势,包括:具有良好灵敏度和选择性的可测量荧光发射、细胞和动物成像中的空间和时间分辨率以及易于获取的荧光团。与无机荧光探针相比,有机荧光探针具有安全性高、生物相容性好、光学稳定性强等优点。因此有机荧光探针辅助的荧光成像可为研究者提供生物样品的结构和动态信息,是当前光学、化学、生物医学等多学科交叉研究领域的热点。结合单光子(OP)探针和单光子显微镜(OPM)是针对各种生物分子和细胞器的最常用工具。常见的荧光团包括香豆素、BODIPY、荧光素和罗丹明,已用于使用适当的设计策略构建OP探针。这些荧光团通常由OPM使用紫外-可见光激发波长(350-650nm)激发。因此, OP探针在实际应用中受到限制。主要原因包括:(1)短波长激发光限制了生物样品中的穿透深度;(2)光漂白,使探头不适合长时间成像;(3)对生物样品的光损伤;(4)来自干扰荧光信号的生物物种的自发荧光。双光子激发荧光(TPEF)可以在不同程度上克服OPM的这些问题。值得注意的是,表现出TPEF的荧光团通常表现出单光子激发荧光(OPEF)。最常用的OP荧光团的激发光谱在400到500nm范围内,而使用双光子显微镜(TPM)激发相同荧光团的波长在800到1000nm之间。因此,TPM减少了光漂白并增加了渗透深度,并提供了比OPM更高的空间分辨率。
癫痫(epilepsy)即俗称的“羊角风”或“羊癫风”,是大脑神经元突发性异常放电,导致短暂的大脑功能障碍的一种慢性疾病。据中国最新流行病学资料显示,国内癫痫的总体患病率为 7.0‰,年发病率为28.8/10万,1年内有发作的活动性癫痫患病率为4.6‰。据此估计中国约有900万左右的癫痫患者,其中500~600万是活动性癫痫患者,同时每年新增加癫痫患者约 40万,在中国癫痫已经成为神经科仅次于头痛的第二大常见病。癫痫在我国农村地区的死亡率也居高不下。发明内容
发明目的:
1.提供一个对次氯酸响应的新化合物,以开发一种新的小分子荧光探针。
2.提供上述小分子荧光探针的制备方法。
3.提供该探针检测癫痫内环境中次氯酸(HClO)浓度的初步应用。
技术方案:HClO小分子荧光探针HCOP的合成与表征:
探针HCOP的结构式:
探针HCOP的合成过程:
化合物2:二氧化硒(5g,36mmol)在1,4-二氧六环/水(200mL/20mL)中的混合物在80℃加热30分钟,然后加入化合物1(5g,23mmol)并80℃回流,4h。反应结束后,冷却至室温后,混合物通过硅藻土过滤,然后用少量二氯甲烷多次冲洗滤渣。滤液通过减压浓缩得到粗产物,经硅胶柱纯化(PE∶EA=6∶1v/v)得到相应的化合物2,为黄色固体(1.5克,产率:30%)。
化合物3:将碳酸铯(2.7g,8.28mmol)、2,2′双(二苯基膦)-1,1′-联萘(0.119g,0.019mmol)和乙酸钯(II)(0.057g,0.0255mmol)溶解在溶液中甲苯(5mL),然后加入化合物2(1.5g, 5.67mmol)和哌啶(12.3mmol),并将反应进一步在100℃回流6h。冷却至室温后,溶液用硅藻土过滤,乙酸乙酯萃取,减压浓缩。所得残余物通过硅胶柱色谱法(PE∶EA=6∶1v/v)纯化以获得所需化合物3。(338.3mg,产率:22.1%)。1HNMR(600MHz,DMSO-d6)δ10.01(s,1H),8.23(d,J=8.5Hz,1H),7.98(d,J=9.4Hz,1H),7.83(d,J=8.5Hz,1H),7.73(dd,J=9.4,2.8 Hz,1H),7.24(d,J=2.8Hz,1H),3.44(t,J=5.0Hz,4H),1.67-1.60(m,J=4.8Hz,6H).
化合物4:将化合物3(360mg,1.5mmol溶解于10mL无水乙醇中,并加入对甲基苯乙酮(201mg, 1.5mmol),催化量的哌啶加入其中并于80℃回流6h,TLC监测反应进行,结束后,冷却至室温,减压浓缩。所得残余物通过硅胶柱色谱法纯化。得到化合物4.(438.2mg,81%)。1H NMR(500MHz,Chloroform-d)δ8.05(dd,J=7.5,1.5Hz,1H),7.99-7.93(m,2H),7.74(dd,J= 7.5,0.7Hz,1H),7.75-7.65(m,1H),7.59-7.52(m,1H),7.44(d,J=7.4Hz,1H),7.31-7.25(m, 2H),7.10(t,J=1.7Hz,1H),6.93(dd,J=7.5,1.5Hz,1H),3.45-3.38(m,4H),2.42(d,J=0.9Hz, 3H),1.73-1.57(m,6H).
化合物4:二氧化硒(250mg,1.8mmol)在1,4-二氧六环/水(20mL/2mL)中的混合物在80℃加热30分钟,然后加入化合物4(427.4mg,1.2mmol)并80℃回流,4h。反应结束后,冷却至室温后,混合物通过硅藻土过滤,然后用少量二氯甲烷多次冲洗滤渣。滤液通过减压浓缩得到粗产物,通过硅胶柱色谱法纯化。得到化合物5.(323.3mg,72.8%).1H NMR(500MHz,Chloroform-d)δ9.94(s,1H),8.10(dd,J=7.5,1.5Hz,1H),8.07-8.01(m,2H),8.01-7.95(m, 2H),7.74(d,J=7.4Hz,1H),7.68(d,J=15.1Hz,1H),7.58-7.52(m,1H),7.44(d,J=7.5Hz, 1H),7.08(t,J=1.4Hz,1H),6.93(dd,J=7.5,1.5Hz,1H),3.46-3.39(m,4H),1.72-1.58(m, 7H).
化合物HCOP:将醛(1.0mmol)、巯基乙醇(1.2mmol)和PPA/SiO2(0,25g)在二氯乙烷(5 mL)中的混合物在室温下搅拌30分钟。反应完成后,过滤除去催化剂,滤液通过减压干燥,所得的残余物通过柱色谱纯化得到探针HCOP。(116.1mg,27%)1H NMR(500MHz,Chloroform-d)δ8.06(dd,J=7.5,1.5Hz,1H),7.92-7.86(m,2H),7.74(d,J=7.5Hz,1H),7.67 (d,J=15.2Hz,1H),7.63-7.57(m,1H),7.51-7.43(m,3H),7.08(t,J=1.4Hz,1H),6.94(dd,J =7.5,1.5Hz,1H),5.69(d,J=0.9Hz,1H),3.97(td,J=7.1,1.3Hz,2H),3.41(dt,J=8.6,7.0Hz, 4H),3.08-3.00(m,2H),1.72-1.64(m,4H),1.68-1.56(m,3H).
实施例一:
探针HCOP的体外应用说明:
体外活性的测试:使用DMSO配制HCOP浓储母液(10hM),体外活性测试一律于200uL终体积的PBS buffer(包括2%DMSO,10uM HCOP)中进行,荧光测试采用HITACHI F7000 荧光光谱仪。激发:450nm,发射:565nm.紫外吸收测试采用HITACHI UV2550.
选择性,干扰性实验分别选用CO:PBS;1:GSH 2:SO32-;3:PO43-;4:NO3-;5:Cl-;6:Br-;7:H2S;8:Cys;9:NO2-;10NO;11:·OH;12:H2O2;13:O2·-;14:ONOO-; 15:1O2;16:BrO-;17:ClO-)进行测试。PH影响情况,采用上述体系,并将PBS置换为不同PH值的缓冲溶液。
附图说明
图1为本发明的核磁共振氢谱示意图
图2为本发明的核磁共振碳谱示意图
图3为本发明的浓度梯度响应测试及线性关系示意图
图4为本发明的紫外吸收测试示意图
图5为本发明选择性测试示意图
图6为本发明的干扰性测试示意图
图7为本发明的pH干扰测试示意图
图8为本发明的细胞成像测试示意图
图9为本发明的癫痫脑组织成像测试示意图
探针HCOP的氢谱:
见附图1
探针HCOP的碳谱:
见附图2
生物活性初步评价
探针HCOP浓度梯度响应测试及线性关系
见附图3
附图说明:该探针(10uM)与不同HClO产生响应,0-500uM的HClO的浓度梯度响应良好,且R2为0.9935,线性关系良好。
探针HCOP反应前后的紫外吸收测试
见附图4:
附图说明:探针HCOP在460nm处左右具有最大的紫外吸收。且反应前后的对比具有显著差异。
探针HCOP的选择性测试:
见附图5
附图说明:该探针(10uM)仅对HClO响应,其他底物几乎无响应。
探针HCOP干扰性测试:
见附图6
附图说明:该探针(10uM)不受其他底物的干扰。
探针HCOP的pH干扰性测试
见附图7
附图说明:探针HCOP在pH范围在2-12的范围内性质保持稳定,不受pH的变化干扰。
探针对细胞内源性的HClO的成像测试
见附图8
附图说明:KA诱导细胞产生癫痫,随后对癫痫模型的细胞进行成像,可以看出探针HCOP 对诱导产生癫痫的细胞具有明显的荧光增强。
探针对癫痫脑组织的成像测试
见附图9
附图说明:对癫痫模型的小鼠和正常鼠的脑组织切片分别孵育HCOP成像,发现癫痫脑组织切片的荧光强度明显强于正常鼠,表明探针可以对癫痫鼠的脑组织进行成像。
该小分子荧光探针生物活性的初步评价均表明该探针具有检测HClO的作用,可以应用于HClO过表达的癫痫模型细胞的原位检测。
以上详细描述了本发明的优选实施方式及生物活性评价,但是,本发明并不限于上述实施方式中的具体细节,在本发明的技术构思范围内,可以对本发明的技术方案进行多种等同变换,这些等同变换均属于本发明的保护范围。另外需要说明的是,在上述具体实施方式中所描述的各个具体技术特征,在不矛盾的情况下,可以通过任何合适的方式进行组合。为了避免不必要的重复,本发明对各种可能的组合方式不再另行说明。此外,本发明的各种不同的实施方式之间也可以进行任意组合,只要其不违背本发明的思想,其同样应当视为本发明所公开的内容。
Claims (2)
2.权利要求1所述的用于检测次氯酸的荧光探针的制备方法,其特征是它由以下步骤组成:
步骤1:二氧化硒(5g,36mmol)在1,4-二氧六环/水(200mL/20mL)中的混合物在80℃加热30分钟,然后加入化合物1(5g,23mmol)并80℃回流,4h。反应结束后,冷却至室温后,混合物通过硅藻土过滤,然后用少量二氯甲烷多次冲洗滤渣。滤液通过减压浓缩得到粗产物,经硅胶柱纯化(PE∶EA=6∶1v/v)得到相应的化合物2,为黄色固体(1.5克,产率:30%);
步骤2:将碳酸铯(2.7g,8.28mmol)、2,2′双(二苯基膦)-1,1′-联萘(0.119g,0.019mmol)和乙酸钯(II)(0.057g,0.0255mmol)溶解在溶液中甲苯(5mL),然后加入化合物2(1.5g,5.67mmol)和哌啶(12.3mmol),并将反应进一步在100℃回流6h。冷却至室温后,溶液用硅藻土过滤,乙酸乙酯萃取,减压浓缩。所得残余物通过硅胶柱色谱法(PE∶EA=6∶1v/v)纯化以获得所需化合物3。(338.3mg,产率:22.1%)。;
步骤3:将化合物3(360mg,1.5mmol溶解于10mL无水乙醇中,并加入对甲基苯乙酮(201mg,1.5mmol),催化量的哌啶加入其中并于80℃回流6h,TLC监测反应进行,结束后,冷却至室温,减压浓缩。所得残余物通过硅胶柱色谱法纯化。得到化合物4;
步骤4:二氧化硒(250mg,1.8mmol)在1,4-二氧六环/水(20mL/2mL)中的混合物在80℃加热30分钟,然后加入化合物4(427.4mg,1.2mmol)并80℃回流,4h。反应结束后,冷却至室温后,混合物通过硅藻土过滤,然后用少量二氯甲烷多次冲洗滤渣。滤液通过减压浓缩得到粗产物,通过硅胶柱色谱法纯化。得到化合物5.(323.3mg,72.8%);
步骤5:将醛(1.0mmol)、巯基乙醇(1.2mmol)和PPA/SiO2(0,25g)在二氯乙烷(5mL)中的混合物在室温下搅拌30分钟。反应完成后,过滤除去催化剂,滤液通过减压干燥,所得的残余物通过柱色谱纯化得到探针。
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