CN116478188A - 荧光探针及其制备方法和应用 - Google Patents
荧光探针及其制备方法和应用 Download PDFInfo
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- CN116478188A CN116478188A CN202211608612.XA CN202211608612A CN116478188A CN 116478188 A CN116478188 A CN 116478188A CN 202211608612 A CN202211608612 A CN 202211608612A CN 116478188 A CN116478188 A CN 116478188A
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- fluorescent probe
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Abstract
本发明公开了一种荧光探针及其制备方法和应用。该荧光探针的化学结构式为:式中,R1选自取代或未取代的烷基、取代或未取代的苯基、取代或未取代的吡啶、取代或者未取代的苯甲酸、取代或未取代的苯甲醛、取代或未取代的苯酚中的任意一种;R2选自取代的苯基,苯基的取代基团选自氰基、硝基、三氟甲基、氟、甲酸甲酯中的至少一种。该探针可特异性识别GSH,即可有效地区分GSH,半胱氨酸,同型半胱氨酸和其他生物硫醇类。同时在酸性条件下,表现出与GSH更高的反应活性。在高选择性、高灵敏度的检测和可视化追踪生物系统中,该探针对于检测复杂生物体系中的GSH意义重大,对于酸性肿瘤微环境中升高的GSH检测具有很大的应用前景。
Description
技术领域
本发明涉及生物成像技术领域,具体而言,涉及一种荧光探针及其制备方法和应用。
背景技术
谷胱甘肽(GSH),是哺乳动物细胞中最常见的非蛋白质硫醇,也是真核细胞中最丰富的低分子量肽。GSH是重要的还原剂和抗氧化剂,组织中的氧化还原平衡在一定程度上受还原性GSH及其氧化二硫化物对应物的相对浓度控制,可影响基因表达、细胞分化、增殖和凋亡。GSH同时也是自由基清除剂,在细胞周期调控和微管相关机制中发挥作用。GSH是半胱氨酸的生理储存库,调节钙离子稳态,通过硫醇-二硫化物交换反应调节蛋白质功能和基因表达,调节淋巴细胞功能和免疫反应,并参与连接细胞死亡激活的线粒体机制。GSH水平和代谢的改变与很多疾病密切相关,包括癌症、神经退行性疾病、衰老、心脏病和糖尿病等。
肿瘤细胞中的GSH与很多调控相关,比如致癌机制,对细胞毒性药物、电离辐射和某些细胞因子的敏感性,DNA合成和细胞增殖。肿瘤细胞中GSH水平明显高于正常细胞,尤其是非小细胞肺癌中GSH水平可达10mM(Estrela,J.M.,Ortega,A.,Obrador,E.,Glutathionein Cancer Biology and Therapy,Crit.Rev.Cl.Lab.Sci.,2006,43,143-181;Gamcsik,M.P.,Kasibhatla,M.S.,Teeter,S.D.,Colvin,O.M.,Glutathione levels in humantumors,Biomarkers,2012,17,671-691)。荧光探针作为一种实时成像工具,在生物化学分析中得到了广泛的应用,在GSH检测中具有一定的优越性。但是目前大多数荧光探针很难有效地区分GSH,Cys和Hcy,很多探针由于波长较短,很难应用于活体检测。因此,开发选择特异性识别GSH的近红外荧光探针,可以有效地检测复杂生物体内的GSH,同时对于疾病的早期检测,尤其是肿瘤早期检测具有重要的意义。
鉴于此,特提出本发明。
发明内容
本发明的目的在于提供一种荧光探针及其制备方法和应用,以改善上述技术问题。
本发明是这样实现的:
第一方面,本发明提供了一种荧光探针,该荧光探针的化学结构式为:
式中,R1选自取代或未取代的烷基、取代或未取代的苯基、取代或未取代的吡啶、取代或者未取代的苯甲酸、取代或未取代的苯甲醛、取代或未取代的苯酚中的任意一种;R2选自取代的苯基,所述苯基的取代基团选自氰基、硝基、三氟甲基、氟、甲酸甲酯中的至少一种。
第二方面,本发明还提供了一种上述荧光探针的制备方法,包括以下合成步骤:
将三溴氧磷、N,N-二甲基甲酰胺和异吲哚啉-1-酮进行反应,制备并分离得到第一中间体,第一中间体的化学结构式为:
将第一中间体和2-羟基苯硼酸在碱性条件和催化剂的作用下进行反应,制备并分离得到第二中间体,第二中间体的化学结构式为:
将第二中间体、3-乙基-2,4-二甲基吡咯以及三氯氧磷进行反应,再与甲基硼酸反应,制备并分离得到第三中间体,第三中间体的化学结构式为:
将第三中间体、4-羟基苯甲醛、哌啶以及对甲基苯磺酸进行反应,制备并分离得到第四中间体,所述第四中间体的化学结构式为:
将第四中间体、2-氰基-4-硝基-苯磺酰氯以及碳酸铯进行反应,制备并分离得到所述荧光探针,荧光探针的化学结构式为:
第三方面,本发明还提供了上述荧光探针在制备对谷胱甘肽特异性识别的荧光成像染料中的应用。
本发明具有以下有益效果:本发明的荧光探针与GSH反应前没有荧光,反应后生成近红外荧光产物,近红外荧光穿透力强,对于活体荧光成像具有很大的应用前景,且该荧光探针对GSH有高度的选择性,能够在GSH,Cys,Hcy,胱氨酸和其他生物硫醇类等物质中特异性结合GSH,为复杂生物体内GSH的检测提供了有效的工具。此外,在弱酸性条件下,可以更好地和GSH反应,对肿瘤弱酸性环境中GSH的检测具有重要意义和实用性。
附图说明
为了更清楚地说明本发明实施例的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,应当理解,以下附图仅示出了本发明的某些实施例,因此不应被看作是对范围的限定,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他相关的附图。
图1~图10为实施例1~实施例5中合成的荧光探针的核磁氢谱和碳谱;
图11~图15为实施例1~实施例5中合成的荧光探针的质谱;
图16为试验例1中荧光探针与GSH反应的机理图;
图17为试验例2中荧光探针对多种生物相关干扰物的选择性结果图;
图18~图22为试验例3中荧光探针在中性(A和C)和微酸性(B和D)条件下与GSH反应的吸收光谱(A和B)和荧光光谱(C和D);
图23为试验例4中荧光探针对肿瘤细胞毒性;
图24为试验例5中荧光探针应用于多种肿瘤细胞中GSH检测,比例尺25μm;
图25为试验例6中荧光探针应用于A549荷瘤小鼠肿瘤微环境中GSH的荧光可视化。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚,下面将对本发明实施例中的技术方案进行清楚、完整地描述。实施例中未注明具体条件者,按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者,均为可以通过市售购买获得的常规产品。
下面对本发明提供的一种荧光探针及其制备方法和应用进行具体说明。
本发明的一些实施方式提供了一种荧光探针,该荧光探针的化学结构式为:
式中,R1选自取代或未取代的烷基、取代或未取代的苯基、取代或未取代的吡啶、取代或者未取代的苯甲酸、取代或未取代的苯甲醛、取代或未取代的苯酚中的任意一种;R2选自取代的苯基,所述苯基的取代基团选自氰基、硝基、三氟甲基、氟、甲酸甲酯中的至少一种。
一些实施方式中,R2为二取代苯基,苯基上的两个取代位置不限,较佳的实施方式中,R2中,两个取代基团分别为氰基和硝基。
为了获得更佳的GSH结合性能和结合后更佳的荧光效果,一些实施方式中,R2为
一些较佳的实施方式中,R1选自C1~C10的烷基,示例性地,R1选自C1~C5的烷基,优选地,R1为甲基。
一些实施方式中,荧光探针的化学结构式选自以下结构式中的任意一种:
一些实施方式中,荧光探针的化学结构式为:(NG2)。该化学结构式的荧光探针能够对谷胱甘肽具有较佳的特异性响应,能够用于检测肿瘤细胞内的GSH以及区分不同种类肿瘤细胞中的GSH水平。此外,其还可以用于活体肿瘤部位的荧光成像。
本发明的一些实施方式还提供了一种上述荧光探针的制备方法,包括以下合成步骤:
将三溴氧磷、N,N-二甲基甲酰胺和异吲哚啉-1-酮进行反应,制备并分离得到第一中间体,第一中间体的化学结构式为:
将第一中间体和2-羟基苯硼酸在碱性条件和催化剂的作用下进行反应,制备并分离得到第二中间体,第二中间体的化学结构式为:
将第二中间体、3-乙基-2,4-二甲基吡咯以及三氯氧磷进行反应,再与甲基硼酸反应,制备并分离得到第三中间体,第三中间体的化学结构式为:
将第三中间体、4-羟基苯甲醛、哌啶以及对甲基苯磺酸进行反应,制备并分离得到第四中间体,所述第四中间体的化学结构式为:
将第四中间体、2-氰基-4-硝基-苯磺酰氯以及碳酸铯进行反应,制备并分离得到荧光探针,荧光探针的化学结构式为:
进一步地,本发明的一些实施方式的荧光探针的制备方法具体包括以下合成步骤:
(1)三溴氧磷(20mmol,5.73g)溶解于5mL无水二氯甲烷中,在0℃温度条件下,逐滴加入溶解于15mL无水二氯甲烷的N,N-二甲基甲酰胺(20mmol,1.46g)混合溶液。滴加结束后,混合反应液在室温条件下继续搅拌30分钟。随后,在0℃温度条件下,滴加溶于50mL无水二氯甲烷的异吲哚啉-1-酮(10mmol,1.33g)溶液。最后,反应混合液回流反应6小时。反应结束后,冷却,减压浓缩去除溶剂。加入冰水搅拌,用5M的氢氧化钠水溶液调pH到8左右,产生黑色沉淀。继续搅拌过夜,收集黑色沉淀,即为第一中间体。化学结构式如下图所示:
(2)第一中间体和2-羟基苯硼酸溶于50mL甲苯,并加入1M碳酸钠水溶液,以四(三苯基膦)钯为催化剂,惰性气体保护下,75℃温度下反应24小时,反应结束后,经萃取得到粗品。粗品用4M氢氧化钠溶液处理,经萃取和柱层析法得到第二中间体。其化学结构式如下图所示:
(3)3-乙基-2,4-二甲基吡咯(10.4mmol,1.281g)溶于15mL二氯甲烷中,在0℃温度条件下,滴加三氯氧磷(5.2mmol,0.797g)。随后,在0℃温度条件下,滴加溶解于25mL二氯甲烷的第二中间体(5.2mmol,1.233g)溶液。混合溶液室温反应4小时后,溶液经柱层析,浓缩得到粗品。无需纯化,粗品溶于50mL乙酸乙酯,加入甲基硼酸(52mmol,3.112g)回流反应3小时。反应结束后,减压浓缩挥去溶剂,经柱层析得到第三中间体。其化学结构式如下图所示:
(4)第三中间体(1mmol,366.19mg)和4-羟基苯甲醛(1.2mmol,146.544mg)溶于100mL甲苯,加入4mL哌啶和100mg对甲基苯磺酸,反应混合液在140℃温度下反应48小时。反应结束后,冷却,减压浓缩挥去溶剂,经柱层析得到第四中间体。其化学结构式如下图所示:
(5)第四中间体(1mmol,479.379mg)和2-氰基-4-硝基-苯磺酰氯(4mmol,986.51mg)溶解于20mL二氯甲烷中,加入碳酸铯(0.5mmol,162.91mg),室温搅拌24小时。反应结束后,减压浓缩挥去溶剂,经柱层析得到所需的荧光探针。
本发明的一些实施方式还公开了上述荧光探针在制备对谷胱甘肽特异性识别的荧光成像染料中的应用。
具体地,荧光成像染料为实现以下任意功能的荧光成像染料:
a.检测肿瘤细胞内的谷胱甘肽;
b.区分不同种类肿瘤细胞中的谷胱甘肽水平;
c.活体肿瘤部位谷胱甘肽的荧光成像。
需要说明的是,荧光成像染料还可以为在酸性溶液中对谷胱甘肽特异性识别的荧光成像染料,其在酸性环境下与谷胱甘肽的反应活性相对于中性环境更佳,具体地,酸性条件为pH=5.5~6.5,例如,pH可为5.5、6或6.5,优选pH=6.5。
以下结合实施例对本发明的特征和性能作进一步的详细描述。
实施例1
本实施例提供了一种荧光探针,其化学结构为:
其制备包括以下合成步骤:
(1)三溴氧磷(20mmol,5.73g)溶解于5mL无水二氯甲烷中,在0℃温度条件下,逐滴加入溶解于15mL无水二氯甲烷的N,N-二甲基甲酰胺(20mmol,1.46g)混合溶液。滴加结束后,混合反应液在室温条件下继续搅拌30分钟。随后,在0℃温度条件下,滴加溶于50mL无水二氯甲烷的异吲哚啉-1-酮(10mmol,1.33g)溶液。最后,反应混合液回流反应6小时。反应结束后,冷却,减压浓缩去除溶剂。加入冰水搅拌,用5M的氢氧化钠水溶液调pH到8左右,产生黑色沉淀。继续搅拌过夜,收集黑色沉淀,即为第一中间体。化学结构式如下图所示:
(2)第一中间体和2-羟基苯硼酸溶于50mL甲苯,并加入1M碳酸钠水溶液,以四(三苯基膦)钯为催化剂,惰性气体保护下,75℃温度下反应24小时,反应结束后,经萃取得到粗品。粗品用4M氢氧化钠溶液处理,经萃取和柱层析法得到第二中间体。其化学结构式如下图所示:
(3)3-乙基-2,4-二甲基吡咯(10.4mmol,1.281g)溶于15mL二氯甲烷中,在0℃温度条件下,滴加三氯氧磷(5.2mmol,0.797g)。随后,在0℃温度条件下,滴加溶解于25mL二氯甲烷的第二中间体(5.2mmol,1.233g)溶液。混合溶液室温反应4小时后,溶液经柱层析,浓缩得到粗品。无需纯化,粗品溶于50mL乙酸乙酯,加入甲基硼酸(52mmol,3.112g)回流反应3小时。反应结束后,减压浓缩挥去溶剂,经柱层析得到第三中间体。其化学结构式如下图所示:
(4)第三中间体(1mmol,366.19mg)和4-羟基苯甲醛(1.2mmol,146.544mg)溶于100mL甲苯,加入4mL哌啶和100mg对甲基苯磺酸,反应混合液在140℃温度下反应48小时。反应结束后,冷却,减压浓缩挥去溶剂,经柱层析得到第四中间体。其化学结构式如下图所示:
(5)第四中间体(1mmol,479.379mg)和2-氰基-4-硝基-苯磺酰氯(4mmol,986.51mg)溶解于20mL二氯甲烷中,加入碳酸铯(0.5mmol,162.91mg),室温搅拌24小时。反应结束后,减压浓缩挥去溶剂,经柱层析得到所需的荧光探针。1H NMR(400MHz,DMSO)δ9.18(t,J=3.7Hz,1H),8.69(dd,J=8.7,2.4Hz,1H),8.42(d,J=8.3Hz,1H),8.35–8.27(m,2H),8.23(d,J=8.0Hz,1H),8.00(s,1H),7.90(d,J=16.9Hz,1H),7.67(dd,J=17.1,8.4Hz,3H),7.51(ddd,J=4.9,3.6,2.0Hz,2H),7.28(dd,J=12.9,7.6Hz,3H),7.17–7.06(m,2H),2.81–2.64(m,2H),2.28(s,3H),1.20(t,J=11.0Hz,3H),-0.16(s,3H).13C NMR(101MHz,DMSO)δ157.13,151.27,148.14,144.88,144.70,140.95,138.16,135.36,134.54,133.57,133.32,133.25,132.27,131.27,130.00,129.55,128.89,128.56,127.73,127.59,127.26,124.41,123.24,122.13,121.44,120.78,120.66,118.43,117.28,114.21,112.43,18.64,14.62,9.33.Maldi Tof:Calcd for C38H29BN4O6S[M+H]+,681.5500;found 681.833。
实施例2~实施例5
实施例2~实施例5与实施例1不同之处,仅在于更换不同的苯磺酰氯取代物,按照步骤(5)合成其他不同取代的荧光探针。
实施例1~实施例5制备得到的荧光探针(NG1~NG5)的核磁氢谱,碳谱和质谱依次如图1~图15所示。
试验例1
荧光探针对谷胱甘肽响应机理
具体步骤如下:将实施例1所制备的荧光探针溶解在DMSO中,制成10mM的储备液。GSH溶解于水中,制备浓度为500mM的储备液。除非特别指出,以下实验中所使用的样品均通过储备液稀释得到。用乙醇和水的混合溶液(v/v=2/1,含2% PEG 400)将荧光探针储备液稀释成2mL终浓度为10μM的溶液,加入少量GSH储备液,使得GSH的浓度为10mM。混合溶液37℃水浴振荡孵育1小时。取少量样品,用质谱进行分析。结果如图16所示,荧光探针中的2-氰基-4-硝基苯-1-磺酰基被GSH移除,可在质谱中找到相应产物的分子量,进一步确认反应机理。
试验例2
荧光探针对生物干扰物的响应
具体步骤如下:干扰物,如氯化钠、氯化钾、氯化镁、氯化铁、硫酸亚铁、氯化铜、氯化锌、碘化亚铜、硫化氢、硫酸钠、亚硫酸钠、亚硫酸氢钠、硫代硫酸钠、胱氨酸、谷氨酸、精氨酸、苯丙氨酸、N-乙酰-L-半胱氨酸、N-乙酰谷氨酸、谷胱甘肽、半胱氨酸、同型半胱氨酸、过氧亚硝酸盐、次氯酸钠、双氧水和抗坏血酸钠,溶解于水中制备100mM的储备液。用乙醇和水的混合溶液(v/v=2/1,含2% PEG 400)将实施例1制备的近红外荧光探针储备液稀释成2mL终浓度为10μM的溶液,加入少量干扰物储备液,使得终浓度为50μM,其中GSH终浓度为1mM,Cys终浓度为200μM,双氧水终浓度为100μM。所有混合溶液37℃水浴振荡孵育1小时。测定荧光光谱,激发波长为550nm。结果如图17所示(1.荧光探针,2.钠离子,3.钾离子,4.钙离子,5.镁离子,6.锌离子,7.亚铜离子,8.亚铁离子,9.氯离子,10.硫化氢,11.硫酸根,12.亚硫酸根,13.亚硫酸氢根,14.硫代硫酸根,15.胱氨酸,16.谷氨酸,17.精氨酸,18.苯丙氨酸,19.N-乙酰-L-半胱氨酸,20.N-乙酰谷氨酸,21.谷胱甘肽,22.半胱氨酸,23.同型半胱氨酸,24.过氧亚硝酸根,25.次氯酸根,26.双氧水,27.抗坏血酸),仅在加入谷胱甘肽的溶液中,703nm处的荧光峰明显增强。结果表明,荧光探针中的2-氰基-4-硝基苯-1-磺酰基只能被谷胱甘肽移除,生成近红外荧光的产物,即本发明实施方式制备的探针对GSH具有高度的选择特异性。
试验例3
荧光探针在中性和酸性条件下对GSH的响应
用乙醇和HEPES缓冲溶液的混合溶液(v/v=2/1,含2% PEG 400,pH=7.4)和乙醇和BP缓冲溶液的混合溶液(v/v=2/1,含2% PEG 400,pH=6.5)分别将试验例1的荧光探针储备液稀释成2mL终浓度为10μM的溶液,加入少量GSH储备液,使得GSH的浓度为10mM。混合溶液37℃水浴振荡孵育1小时。反应结束后,测定吸收光谱和荧光光谱,激发波长为550nm。结果如图18~图22所示,与探针本身的吸收峰或者荧光峰相比,加入GSH的吸收峰出现明显的红移,而703nm处的荧光峰明显增强,且酸性BP缓冲溶液中的荧光峰强于中性HEPES缓冲溶液中的荧光峰。结果表明,荧光探针在弱酸性条件下,对GSH表现出更好的反应性。
试验例4
荧光探针对细胞毒性检测
具体操作步骤如下:取对数生长期的HeLa细胞,A549细胞,MCF-7细胞和U87细胞,将其消化后调整细胞密度为1×105个/mL,分别地均匀接种于96孔板上,置于37℃,5% CO2的培养箱中培养24小时,吸去培养基,进行荧光探针干预实验:分别加入终浓度为0.5μM,5μM,10μM,25μM,50μM实施例1的荧光探针的完全DMEM培养基100μL,每个浓度均设6个复孔,该板置于37℃,5% CO2的培养箱中培养24小时后,吸弃培养基,用PBS洗三次。每孔加入CCK-8溶液10μL于37℃,5% CO2的培养箱中培养4小时后,于37℃恒温振荡10分钟,全自动酶标仪测定各孔吸光度A。根据实验结果,计算细胞的存活率。结果如图23所示。
试验例5
荧光探针检测不同肿瘤细胞内谷胱甘肽水平
具体步骤如下:取对数生长期的HeLa细胞,A549细胞,MCF-7细胞和U87细胞,接种于共聚焦皿(2mL,1.5×105个细胞/孔),在37℃,含5%CO2的培养箱中过夜培养。随后不同的肿瘤细胞给与荧光探针干预,采用共聚焦显微镜观察不同的时间点近红外通道(λEm=700nm~750nm)荧光变化。结果如图24所示,不同种类肿瘤细胞的近红外荧光随着培养时间的延长而增强,但是A549细胞中荧光强度明显强于其他细胞,说明A549细胞内GSH含量高于其他肿瘤细胞。
试验例6
活体A549荷瘤小鼠肿瘤部位GSH成像
清洁级雌性Nu/J裸鼠3只,3~4周,体重18~20g,由澳门大学动物实验中心提供。所有动物实验由澳门大学实验动物管理和使用委员会批准。所有小鼠均可自由获得食物和水,并置于无菌条件下,室温明暗周期为12h/12h,适应性饲养1周。A549荷瘤小鼠模型通过皮下注射A549细胞(5×106个细胞,50μL,PBS:Matrigel=1:1)建立。注射结束20天后,肿瘤部位注射溶于无菌PBS缓冲溶液的荧光探针,注射量为5ng每个瘤子。不同的时间点记录活体小鼠肿瘤部位的近红外荧光(λEm=710nm)。结果如图25所示,随着时间的延长,小鼠肿瘤部位的近红外荧光不断增强,说明所制备的荧光探针可被肿瘤部位的GSH脱保护生成近红外荧光产物。
综上所述,本发明实施方式制备的可选择特异性检测GSH的荧光探针,可以有效地区分GSH,半胱氨酸(Cys),同型半胱氨酸(Hcy)和其他生物硫醇类。同时在酸性条件下,表现出与GSH更高的反应活性。成功的应用于区分A549肺癌细胞和其他肿瘤细胞中GSH水平。在高选择性、高灵敏度的检测和可视化追踪生物系统中,本发明实施方式制备的探针对于检测复杂生物体系中的GSH意义重大,对于酸性肿瘤微环境中升高的GSH检测具有很大的应用前景。
以上所述仅为本发明的优选实施例而已,并不用于限制本发明,对于本领域的技术人员来说,本发明可以有各种更改和变化。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (10)
1.一种荧光探针,其特征在于,所述荧光探针的化学结构式为:
式中,R1选自取代或未取代的烷基、取代或未取代的苯基、取代或未取代的吡啶、取代或者未取代的苯甲酸、取代或未取代的苯甲醛、取代或未取代的苯酚中的任意一种;R2选自取代的苯基,所述苯基的取代基团选自氰基、硝基、三氟甲基、氟、甲酸甲酯中的至少一种。
2.根据权利要求1所述的荧光探针,其特征在于,所述R2为二取代苯基;优选地,所述R2中,两个取代基团分别为氰基和硝基。
3.根据权利要求2所述的荧光探针,其特征在于,所述R2为
4.根据权利要求1所述的荧光探针,其特征在于,R1选自C1~C10的烷基,优选地,R1选自C1~C5的烷基,更优选地,R1为甲基。
5.根据权利要求1所述的荧光探针,其特征在于,所述荧光探针的化学结构式选自以下结构式中的任意一种:
优选地,所述荧光探针的化学结构式为:
6.如权利要求1~5任一项所述的荧光探针在制备对谷胱甘肽特异性识别的荧光成像染料中的应用。
7.根据权利要求6所述的应用,其特征在于,所述荧光成像染料为实现以下任意功能的荧光成像染料:
a.检测肿瘤细胞内的谷胱甘肽;
b.区分不同种类肿瘤细胞中的谷胱甘肽水平;
c.活体肿瘤部位谷胱甘肽的荧光成像。
8.根据权利要求6所述的应用,其特征在于,所述荧光成像染料为在酸性溶液中对谷胱甘肽特异性识别的荧光成像染料;优选地,所述酸性溶液的pH=5.5~6.5,更优选pH=6.5。
9.一种荧光探针的制备方法,其特征在于,包括以下合成步骤:
将三溴氧磷、N,N-二甲基甲酰胺和异吲哚啉-1-酮进行反应,制备并分离得到第一中间体,所述第一中间体的化学结构式为:
将所述第一中间体和2-羟基苯硼酸在碱性条件和催化剂的作用下进行反应,制备并分离得到第二中间体,所述第二中间体的化学结构式为:
将所述第二中间体、3-乙基-2,4-二甲基吡咯以及三氯氧磷进行反应,再与甲基硼酸反应,制备并分离得到第三中间体,所述第三中间体的化学结构式为:
将所述第三中间体、4-羟基苯甲醛、哌啶以及对甲基苯磺酸进行反应,制备并分离得到第四中间体,所述第四中间体的化学结构式为:
将所述第四中间体、2-氰基-4-硝基-苯磺酰氯以及碳酸铯进行反应,制备并分离得到所述荧光探针,所述荧光探针的化学结构式为:
10.根据权利要求9所述的制备方法,其特征在于,包括以下合成步骤:
将三溴氧磷溶解于无水二氯甲烷中,在-5℃~5℃温度条件下,逐滴加入溶解于无水二氯甲烷的N,N-二甲基甲酰胺混合溶液,滴加结束后,混合反应液在室温条件下继续搅拌25~35分钟,随后,在-5℃~5℃温度条件下,滴加溶于无水二氯甲烷的异吲哚啉-1-酮溶液,最后,反应混合液回流反应5~7小时,反应结束后,去除溶剂,加入冰水搅拌,用调pH到7.8~8.5,产生黑色沉淀,收集黑色沉淀,获得第一中间体;
将所述第一中间体和2-羟基苯硼酸溶于甲苯,并加入碳酸钠水溶液,以四(三苯基膦)钯为催化剂,惰性气体保护下,70℃~80℃温度下反应20~28小时,反应结束后,经萃取得到粗品,粗品用碱性溶液处理,经萃取和柱层析法得到第二中间体;
将3-乙基-2,4-二甲基吡咯溶于二氯甲烷中,在-5℃~5℃温度条件下,滴加三氯氧磷,随后,在-5℃~5℃温度条件下,滴加溶解于二氯甲烷的第二中间体溶液,混合溶液室温反应3.5~4.5小时后,溶液经柱层析,浓缩得到粗品,溶于乙酸乙酯,加入甲基硼酸回流反应2.5~3.5小时,反应结束后,减压浓缩去除溶剂,经柱层析得到第三中间体;
将所述第三中间体和4-羟基苯甲醛溶于甲苯,加入哌啶和对甲基苯磺酸,反应混合液在130℃~150℃温度下反应45~50小时,反应结束后,冷却,减压浓缩去除溶剂,经柱层析得到第四中间体;
将所述第四中间体和2-氰基-4-硝基-苯磺酰氯溶解于二氯甲烷中,加入碳酸铯,室温搅拌20~28小时,应结束后,减压浓缩去除溶剂,经柱层析得到所述荧光探针。
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