CN112457303B - 一种荧光化合物及其制备方法、用途 - Google Patents
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Abstract
本发明公开了一种荧光化合物及其制备方法、用途,涉及荧光有机化合物技术领域。该荧光化合物的制备,具体包括:取4‑羟基‑苯并[b]噻吩‑7‑羧醛、乙基苯并吲哚加入的正丁醇/甲苯的混合溶液搅拌溶解,加热回流反应;反应结束后除去溶剂,在水和二氯甲烷的混合体系中静置分层,有机相进一步纯化,得到的荧光化合物。本发明制得的荧光化合物在不同的酸碱环境中,均具有敏锐的响应;基于此制得的荧光探针,可用于检测环境中的ALP,排除其它物质的影响,具有高选择性、专一性和灵敏度。
Description
技术领域
本发明属于荧光有机化合物技术领域,具体涉及一种荧光化合物及其制备方法、用途。
背景技术
荧光,是指一种光致发光的冷发光现象。当常温物质经某种波长的入射光(通常是紫外线或X射线)照射后,吸收光能,进入激发态,并发出比入射光的波长长的出射光(通常波长在可见光波段)。物质的荧光非常容易受外界条件刺激(如热、气体、水等)而发生变化(如红移,蓝移或者强度变化等)。
在包括化学和生物学的许多领域,荧光技术受到的关注日益增多。在某些情况中,荧光分子可用于检测食品和环境样品中是否存在分析物或化学品。在离体生化分析(如DNA测序和血糖定量)中,需要一些灵敏的荧光定量检测设备。在一些情况中,具有合适的光化学性质的荧光探针可用于示踪活细胞中的分子和生理学事件,以及用于高通量筛选。一般地,荧光技术比其他类型的光学测量更简单,并可提供更高的灵敏度和更多的分子信息。荧光测量一般是高度灵敏的,因为大多数化学和生物样品中发现的荧光背景一般处于低水平。随着荧光仪器(如共焦和多光子荧光显微镜)领域最近取得的进展,细胞事件的实时三维成像和生物物质的实时动力学研究已经变得可能。尽管有以上提及的优点,但将荧光技术用于特定应用的可行性往往可受到适当荧光分子的可利用性的限制。
磷酸盐被认为是藻类和细菌最重要的磷的来源,海水中的磷的缺乏会限制藻类的生长,限制海域的初级生产力,进而影响整个生态循环系统。早有研究表明,当海水缺乏无机磷时,水体中溶解有机磷(DOP)化合物可以作为磷营养源被藻类和细菌所利用,这便是海水中ALP的贡献。最近,各种检测ALP检测手段已经被开发,包括电化学、色谱、比色技术及肽微阵列分析等等。虽然这些为ALP检测提供了有效的方法,但是对于底物,并没有良好的专一性,其中,有一个不可忽视的威胁来自于磷酸单酯酶中的酸性磷酸酶,这是由于碱性磷酸酶和酸性磷酸酶是一类对底物没有专一性水解功能的酶类,他们有着不同的最适pH值。
与传统花菁染料相比,半花菁染料耐光性好,荧光量子产率高,更有利于成像。半花菁染料已被设计合成,并用于检测硒化氢,肼,半胱氨酸,pH,一氧化氮,β-内酰胺酶,硒醇,超氧根离子,硝基还原酶等,而基于pH值响应的荧光探针检测环境中的ALP活性几乎未见有报道,因此设计一种基于pH值的具有高选择性的荧光探针CyP用于检测ALP活性是非常重要的。
发明内容
本发明的目的在于提供一种荧光化合物及其制备方法、用途,该荧光化合物在较宽的pH范围内具有敏锐的信号响应,制得的荧光探针对ALP的检测具有较高的选择性、特异性和灵敏度。
本发明为实现上述目的所采取的技术方案为:
一种荧光化合物,其结构式如下所示:
本发明获得的荧光化合物,使得半花菁的荧光发射信号红移,在pH变化范围2.5~9.5时,有着敏锐的比率信号的改变,在pH为8的环境下,化合物在550nm的激发波长下,具有明显的信号强度,随着pH值的变化,其吸光度明显衰退,且信号强度具有明显的区别,可适用不同酸碱性环境;相比于现有技术,信号强度更高,增强其灵敏度。因而可用于设计基于pH值响应的荧光强度信号改变的荧光探针,高选择性检测环境中的碱性磷酸酶(ALP)活性,在保证ALP的最大活力的同时,降低了其它物质的干扰,具有优异的选择性。本发明荧光化合物采用一锅法即可制得,方法简单,且产率较高。
上述荧光化合物的制备方法,包括:
S1:取4-羟基-苯并[b]噻吩-7-羧醛、乙基苯并吲哚加入的正丁醇/甲苯的混合溶液搅拌溶解,加热回流反应;
S2:反应结束后除去溶剂,在水和二氯甲烷的混合体系中静置分层,有机相进一步纯化,得到的荧光化合物Cy5-M。
优选地,步骤S1中4-羟基-苯并[b]噻吩-7-羧醛与乙基苯并吲哚的质量比为1:1.8~2.2。
优选地,步骤S1中反应过程用到正丁醇/甲苯的混合溶液,两者体积比为7:2~4;上述混合溶液与4-羟基-苯并[b]噻吩-7-羧醛的液固比为3~4mL:1mg。
优选地,加热回流反应时间为3~4h。
本发明的又一目的在于提供一种荧光化合物在制备荧光探针中的用途。
本发明的又一目的在于提供一种荧光化合物在检测碱性磷酸酶活性中的用途。
一种基于上述荧光化合物的荧光探针,其结构式如下所示:
本发明基于上述制得的荧光化合物设计了新的探针CyP,具有高选择性,能够通过pH值来专一响应ALP而不受其它物质的干扰,具有优异的特异选择性。在对探针CyP光物理学特性的研究中,探针在响应ALP后,拥有在550nm处有强烈的紫外吸收峰,和570nm处强烈的荧光强度。本发明荧光探针光稳定性好,且能够适应不同环境下单一检测ALP,相比于现有技术,特异性、灵敏度和选择性更高。
上述荧光探针的制备方法,包括:
将上述制得的Cy5-M溶解在吡啶中,滴加三氯氧磷,室温下搅拌0.5~1h后,在反应体系中加入水继续搅拌0.5~1h。反应结束后除去溶剂,得到的粗产物在二氯甲烷和水的混合液中进行萃取,保留水相,并在减压下收集浓缩。产物通过反相硅胶C18色谱柱纯化(75~80%甲醇/水),得到的探针CyP。
优选地,荧光化合物与三氯氧磷的固液比为1.6~1.8mg:1μL。
相比于现有技术,本发明具有如下有益效果:
本发明获得的荧光化合物,使得半花菁的荧光发射信号红移,在pH变化范围2.5到9.5时,有着更加敏锐的比率信号的改变。其中在pH为8的环境下,化合物在550nm的激发波长下,具有明显的信号强度,随着酸碱性的改变,信号强度有着明显的变化,可适用不同酸碱性环境。因而可用于设计基于pH值响应的荧光强度信号改变的荧光探针,相比于现有技术,具有高选择性,且具有更高的灵敏度,能够通过pH值来专一响应ALP而不受其它物质的干扰。本发明荧光探针光稳定性好,且能够适应不同环境下单一检测ALP,特异性、灵敏度和选择性高。本发明荧光化合物采用一锅法即可制得,方法简单,且产率较高。
因此,本发明提供了一种荧光化合物及其制备方法、用途,该荧光化合物在较宽的pH范围内具有敏锐的信号响应,制得的荧光探针对ALP的检测具有较高的选择性、特异性和灵敏度。
附图说明
图1为本发明实施例1中荧光化合物合成路线;
图2为本发明实施例1中紫外吸收测试结果示意图;
图3为本发明实施例1中550nm处激发时荧光光谱图;
图4为本发明实施例2中探针合成路线;
图5为本发明实施例2中紫外吸收测试结果示意图;
图6为本发明实施例2中550nm处激发时荧光光谱图;
图7为本发明实施例2中ALP紫外测试结果示意图;
图8为本发明实施例2中ALP荧光测试结果示意图。
具体实施方式
以下结合具体实施方式和附图对本发明的技术方案作进一步详细描述:
本发明实施例所用正丁醇(n-bμtyl alcohol),甲苯(tolμene),二氯甲烷(CH2Cl2),乙酸乙酯(EtOAc),甲醇(CH3OH),吡啶(pyridine),三氯氧磷(phosphorμsoxychloride)均购自国药集团化学试剂有限公司。
实施例1:
一种荧光化合物的制备,其合成路线如图1,具体制备方法为:
S1:将4-羟基-苯并[b]噻吩-7-羧醛(40mg,0.33mmol),乙基苯并吲哚(80.5mg,0.33mmol)加入到250mL的三口烧瓶中,再加入150mL的正丁醇/甲苯(7:3,V/V)的混合溶液搅拌溶解。反应体系加热回流3.5h,期间用薄层色谱监测反应进度;
S2:反应结束后在真空中除去溶剂,粗产品在水和二氯甲烷的混合体系中静置分层,保留有机相,重复操作三次。粗产物用硅胶柱层析于进一步纯化,洗脱剂为乙酸乙酯/甲醇(3:1,V/V)。最终得到的产物Cy5-M是橘黄色固体,产率86%。1H NMR(400MHz,DMSO):δ8.49(d,1H,Ar-H),8.23-8.35(d,2H,Ar-H),8.05-8.07(d,2H,S-CH=CH-),7.83(d,1H,Ar-H),7.73-7.76(m,2H,Ar-H),7.63(d,1H,Ar-H),7.34(d,1H,Ar-H),6.97(d,1H,-CH=CH-),5.01(d,1H,-CH=CH-),4.87(s,1H,-OH),4.36(m,2H,-CH2),2.01(s,6H,-CH3),1.54(t,3H,-CH3);13C NMR(400MHz,DMSO):δ183.1,162.8,155.3,146.1,139.1,133.4,132.7,131.8,130.4,129.2,128.1,127.9,126.7,125.3,122.6,120.1,116.7,113.9,109.1,61.1,54.3,35.4,26.4,19.3,14.5。MS(TOF)m/z 398.21。结果表明所得产物正确。
荧光化合物的pH的独特响应
为了调查荧光化合物Cy5-M的pH的独特响应,其在不同pH缓冲溶液中的紫外和荧光光谱(λex=550nm、λem=570~750nm和λex=405nm、λem=450~700nm)得到调查。紫外吸收测试结果如图2所示,pH值比率响应的Cy5-M,在405nm和550nm处有两个最高的吸收峰。并且,伴随着pH值的上升,在405nm处的吸收强度随之减弱,而550nm处的吸收强度随之升高。当Cy5-M在400nm的激发下,其不同pH值下荧光峰的分离并不明显。当选用550nm处激发时,其荧光光谱如图3所示,荧光强度分离明显,当pH处于小于4的酸性条件下,其荧光强度可以忽略不计。
实施例2:
一种荧光探针的制备,其合成路线如图4所示,具体制备方法为:
将实施例1得到的Cy5-M(17.2mg,5mmol)溶解在15mL的吡啶中。然后将三氯氧磷(10μL,5mmol)滴加到混合溶液中。室温下搅拌0.5h后,在反应体系中加入5mL水继续搅拌0.5h。最后用旋转蒸发器除去溶剂。得到的粗产物在二氯甲烷和水的混合液中进行萃取。保留水相,并在减压下收集浓缩。产物通过反相硅胶C18色谱柱纯化(流动相:75%甲醇/水,进样量15μL;色谱柱为Eclipse XDB-C18(9.4mm×250mm,5μm);洗脱速度2mL/min;)。得到的探针CyP是一个黄色的油状体,产率72%。1H NMR(400MHz,DMSO):δ11.42(s,2H,-OH),8.99(d,1H,Ar-H),8.53-8.65(d,2H,Ar-H),8.35~8.37(d,2H,S-CH=CH-),8.13(d,1H,Ar-H),8.03-8.06(m,2H,Ar-H),7.93(d,1H,Ar-H),7.64(d,1H,Ar-H),7.26(d,1H,-CH=CH-),5.20(d,1H,-CH=CH-),4.56(m,2H,-CH2),2.11(s,6H,-CH3),1.64(t,3H,-CH3);MS(TOF)m/z478.21。
1、不同pH值对探针CyP的影响
为了探究不同pH值对探针CyP的影响,CyP(10μM)在不同pH缓冲溶液中保持1h后,测定其紫外吸收和荧光光谱(λex=550nm、λem=570~750nm和λex=405nm、λem=450~700nm)。如图5~6所示,可见无论处于何种pH条件下,其紫外吸收和荧光光谱均没有响应。
2、探针CyP属性测试
探针CyP的光谱特性是在温度37℃,模拟优化的生理条件下进行研究。大肠杆菌碱性磷酸酶(Escherichia coliALP,ECAP)被选作为ALP模型。工作液选用了50mM Tris-HCl bμffer(pH=8.0)并添加1mM MgCl2以保证酶处于最大的活性。结果如图7所示,探针CyP展现出几乎没有任何明显的吸收光;而当ALP加入(100Μ/L)10min后,有一个肉眼可见的黄色褪去的变化,此时在550nm处出现了强烈的吸收峰,这是典型的荧光化合物Cy5-M的吸收谱图。其荧光光谱(λex=550nm、λem=570~750nm)也得到了相同的结果,如图8所示。因此,可以肯定CyP是一个OFF-ON属性的探针。
实施例3:
一种荧光化合物的制备:
S1:将4-羟基-苯并[b]噻吩-7-羧醛(40mg,0.33mmol),乙基苯并吲哚(85mg,0.33mmol)加入到250mL的三口烧瓶中,再加入155mL的正丁醇/甲苯(7:2.5,V/V)的混合溶液搅拌溶解。反应体系加热回流3.2h,期间用薄层色谱监测反应进度;
S2:反应结束后在真空中除去溶剂,粗产品在水和二氯甲烷的混合体系中静置分层,保留有机相,重复操作三次。粗产物用硅胶柱层析于进一步纯化,洗脱剂为乙酸乙酯/甲醇(3:1,V/V)。最终得到的产物Cy5-M是橘黄色固体,产率82.3%。
一种荧光探针的制备:
将得到的Cy5-M(18mg,5mmol)溶解在15mL的吡啶中。然后将三氯氧磷(10μL,5mmol)滴加到混合溶液中。室温下搅拌0.6h后,在反应体系中加入6mL水继续搅拌0.6h。最后用旋转蒸发器除去溶剂。得到的粗产物在二氯甲烷和水的混合液中进行萃取。保留水相,并在减压下收集浓缩。产物通过反相硅胶C18色谱柱纯化(75%甲醇/水)。得到的探针CyP是一个黄色的油状体,产率68.3%。
实施例4:
一种荧光化合物的制备:
S1:将4-羟基-苯并[b]噻吩-7-羧醛(38.3mg,0.33mmol),乙基苯并吲哚(81.4mg,0.33mmol)加入到250mL的三口烧瓶中,再加入152mL的正丁醇/甲苯(7:2,V/V)的混合溶液搅拌溶解。反应体系加热回流3.5h,期间用薄层色谱监测反应进度;
S2:反应结束后在真空中除去溶剂,粗产品在水和二氯甲烷的混合体系中静置分层,保留有机相,重复操作三次。粗产物用硅胶柱层析于进一步纯化,洗脱剂为乙酸乙酯/甲醇(3:1,V/V)。最终得到的产物Cy5-M是橘黄色固体,产率80.6%。
一种荧光探针的制备:
将得到的Cy5-M(16.5mg,5mmol)溶解在15mL的吡啶中。然后将三氯氧磷(10.5μL,5mmol)滴加到混合溶液中。室温下搅拌1h后,在反应体系中加入5mL水继续搅拌1h。最后用旋转蒸发器除去溶剂。得到的粗产物在二氯甲烷和水的混合液中进行萃取。保留水相,并在减压下收集浓缩。产物通过反相硅胶C18色谱柱纯化(75%甲醇/水)。得到的探针CyP是一个黄色的油状体,产率69.4%。
实施例5:
一种荧光化合物的制备:
S1:将4-羟基-苯并[b]噻吩-7-羧醛(41.5mg,0.33mmol),乙基苯并吲哚(80.5mg,0.33mmol)加入到250mL的三口烧瓶中,再加入150mL的正丁醇/甲苯(7:3,V/V)的混合溶液搅拌溶解。反应体系加热回流4h,期间用薄层色谱监测反应进度;
S2:反应结束后在真空中除去溶剂,粗产品在水和二氯甲烷的混合体系中静置分层,保留有机相,重复操作三次。粗产物用硅胶柱层析于进一步纯化,洗脱剂为乙酸乙酯/甲醇(3:1,V/V)。最终得到的产物Cy5-M是橘黄色固体,产率84.5%。
一种荧光探针的制备:
将得到的Cy5-M(17.8mg,5mmol)溶解在15mL的吡啶中。然后将三氯氧磷(10.8μL,5mmol)滴加到混合溶液中。室温下搅拌0.5h后,在反应体系中加入6mL水继续搅拌0.6h。最后用旋转蒸发器除去溶剂。得到的粗产物在二氯甲烷和水的混合液中进行萃取。保留水相,并在减压下收集浓缩。产物通过反相硅胶C18色谱柱纯化(75%甲醇/水)。得到的探针CyP是一个黄色的油状体,产率63.3%。
上述实施例中的常规技术为本领域技术人员所知晓的现有技术,故在此不再详细赘述。
以上所述,仅为本发明的具体实施方式,但本发明的保护范围并不局限于此,任何熟悉本技术领域的技术人员在本发明揭露的技术范围内,可轻易想到变化或替换,都应涵盖在本发明的保护范围之内。因此,本发明的保护范围应以所述权利要求的保护范围为准。
Claims (8)
2.一种如权利要求1所述的荧光化合物的制备方法,包括:
S1:取4-羟基-苯并[b]噻吩-7-羧醛、乙基苯并吲哚加入到正丁醇/甲苯的混合溶液搅拌溶解,加热回流反应;
S2:反应结束后除去溶剂,在水和二氯甲烷的混合体系中静置分层,有机相进一步纯化,得到的荧光化合物。
3.根据权利要求2所述的荧光化合物的制备方法,其特征在于:所述步骤S1中4-羟基-苯并[b]噻吩-7-羧醛与乙基苯并吲哚的质量比为1:1.8~2.2。
4.根据权利要求2所述的荧光化合物的制备方法,其特征在于:所述步骤S1中反应过程用到正丁醇/甲苯的混合溶液,两者体积比为7:2~4;所述混合溶液与4-羟基-苯并[b]噻吩-7-羧醛的液固比为3~4mL:1mg。
5.根据权利要求2所述的荧光化合物的制备方法,其特征在于:所述加热回流反应时间为3~4h。
6.权利要求1所述的荧光化合物在制备荧光探针中的用途。
8.权利要求7所述的荧光探针在检测碱性磷酸酶活性中的用途。
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