CN106866727A - 一种2‑磷酸苄基甲酸酯类化合物及其制备方法和作为荧光探针在检测碱性磷酸酶的应用 - Google Patents
一种2‑磷酸苄基甲酸酯类化合物及其制备方法和作为荧光探针在检测碱性磷酸酶的应用 Download PDFInfo
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Abstract
本发明公开了一种2‑磷酸酯苄基甲酸酯类化合物及其制备方法。所述2‑磷酸酯苄基甲酸酯类化合物的结构式如下式所示:其中,X为O、NH或NCH3,Dye为荧光基团。由于2‑磷酸酯苄基甲酸酯类化合物具有邻位苄基结构,对于碱性磷酸酶的特异性识别能力,不与其他磷酸酶发生反应。本发明2‑磷酸酯苄基甲酸酯类化合物可作为荧光探针用于检测碱性磷酸酶,是一种简单、快速、灵敏且专一性的碱性磷酸酶荧光探针,可以应用于人体血液及细胞等内的碱性磷酸酶活性检测,在生物分子检测领域具有广阔的应用前景。
Description
技术领域
本发明属于分析化学技术领域,更具体地,涉及一种2-磷酸酯苄基甲酸酯类化合物及其制备方法和作为荧光探针在检测碱性磷酸酶的应用。
背景技术
在临床诊断上碱性磷酸酶可作为一个重要的生物指标,在很多疾病中如骨骼疾病,肝脏功能障碍,卵巢癌,乳腺癌,前列腺癌,白血病和糖尿病等中均可通过检测碱性磷酸酶的异常水平来进行初步诊断。碱性磷酸酶可来源于不同哺乳动物的骨、肝、胎盘和肠道,作为常见的分析酶,能催化水解多样的磷酸化合物并且具有底物特异性。目前有很多方法用于该碱性磷酸酶检测,但对碱性磷酸酶的检测缺少选择性,不能对人体血液或细胞内的碱性磷酸酶进行选择性检测,特别是细胞内含有的多种磷酸酶,而其中酪氨酸磷酸酶,色氨酸磷酸酶等,通常都可以与一般磷酸酶探针发生反应,进而影响检测的准确性。因此,设计对于碱性磷酸酶具有特异性识别的荧光探针具有重要的意义。
发明内容
本发明的目的在于克服现有技术的缺点与不足,提供一种2-磷酸酯苄基甲酸酯类化合物。该化合物具有邻位磷酸苄基基团,对于碱性磷酸酶的特异性识别能力,是一种简单,快速,灵敏,专一的碱性磷酸酶检测试剂。
本发明另一个目的是提供上述2-磷酸酯苄基甲酸酯类化合物的制备方法。
本发明再一个目的是提供上述2-磷酸酯苄基甲酸酯类化合物的应用。
本发明上述目的是通过以下技术方案予以实现:
一种2-磷酸酯苄基甲酸酯类化合物,所述化合物的结构式如式(I)所示:
其中,X为O、NH或NCH3,Dye为荧光基团。
优选地,所述荧光基团的结构式如式2-12所示:
上述2-磷酸酯苄基甲酸酯类化合物的制备方法,包括如下具体步骤:
S1.将2-羟甲基-苯基磷酸二乙酯和吡啶加入到二氯甲烷中,降温到0℃后,加入三氯甲基碳酸酯,氮气保护下反应4~8小时;
S2.用TLC板监测反应,反应完全后,用氮气吹干多余的三氯甲基碳酸酯;然后加入荧光基团,并反应12~24小时;
S3.用TLC板监测反应,反应完全后,加入二氯甲烷和水萃取,无水硫酸钠干燥,旋干溶剂,并用洗脱剂进行硅胶柱分离,得到化合物A;
S4.将化合物A溶解在二氯甲烷中,室温下加入三甲基溴硅烷,氮气保护,反应8~12小时;
S5.用TLC板监测反应,反应完全后,旋干溶剂,并用洗脱剂进行硅胶柱分离,得到2-磷酸酯苄基甲酸酯类化合物。
优选地,步骤S1中所述2-羟甲基-苯基磷酸二乙酯、吡啶、二氯甲烷、三氯甲基碳酸酯的摩尔比为1:(2~5):(15~30):(0.5~1.5)。
优选地,步骤S2中所述荧光基团与步骤S1中所述2-羟甲基-苯基磷酸二乙酯的摩尔比为1:(0.8~1.5)。
优选地,步骤S3中所述洗脱剂为乙酸乙酯和正己烷,所述乙酸乙酯和正己烷的体积比为1:(1~3)。
优选地,步骤S4中所述化合物A、二氯甲烷、三甲基溴硅烷的比例关系1:(20~30):(3~5)。
优选地,步骤S5中所述洗脱剂为二氯甲烷和甲醇,所述二氯甲烷和甲醇的体积比为1:(0.2~1)。
上述2-磷酸酯苄基甲酸酯类化合物作为荧光探针在检测碱性磷酸酶的应用。
优选地,所述碱性磷酸酶存在于人体血液或细胞中。
本发明以2-磷酸酯苄基甲酸酯类化合物作为荧光探针,用以碱性磷酸酶的特异性识别。由于邻位磷酸苄基基团对于碱性磷酸酶的特异性识别能力,甲酸酯的吸电子效应,荧光基团的荧光被猝灭。碱性磷酸酶选择性识别并切除磷酸基团,产生的酚羟基负离子经过电子重排,最终释放出自由的荧光基团,产生荧光。
与现有技术相比,本发明具有以下有益效果:
1.本发明利用特殊的空间位阻效应,提供一种具有邻位磷酸苄基基团,即2-磷酸酯苄基甲酸酯,对于碱性磷酸酶具有特异性识别的官能团,此结构可以实现对于碱性磷酸酶的特异性识别。而且2-磷酸酯苄基甲酸酯不与其他磷酸酶发生反应,例如肾小球上皮细胞蛋白(GELPP),蛋白酪氨酸磷酸酶1B(PTP1B)等,只与碱性磷酸酶反应,产生荧光,进而提高检测结果的准确性。
2.本发明2-磷酸酯苄基甲酸酯类化合物是一种简单,快速,灵敏,专一的碱性磷酸酶检测试剂,作为荧光探针可对模拟溶液中或细胞内碱性磷酸酶进行实时定性及定量的无损检测,在生物分子检测领域具有广阔的应用前景。
附图说明
图1为实施例1合成的TP-Phos的化学结构式。
图2为实施例1合成的TP-Phos的1H NMR图谱。
图3为实施例1合成的TP-Phos的13C NMR图谱。
图4为实施例1合成的TP-Phos的31P NMR图谱。
图5为实施例1合成的TP-Phos与碱性磷酸酶反应前后的紫外吸收(a)及荧光发射(b)光谱。
图6为实施例1合成的TP-Phos对不同的磷酸酶的选择性柱状图数据。
图7为实施例1合成的TP-Phos检测细胞内源性碱性磷酸酶荧光成像图。
具体实施方式
下面结合具体实施例进一步说明本发明的内容,但不应理解为对本发明的限制。若未特别指明,实施例中所用的技术手段为本领域技术人员所熟知的常规手段。除非特别说明,本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
实施例1化合物TP-Phos的合成
1.合成步骤:
(1)将2-羟甲基-苯基磷酸二乙酯(0.5mmol,130mg,1eq)和吡啶(1.5mmol,121μL,3eq)加入到20mL二氯甲烷中,降温到0℃后,加入1eq的三氯甲基碳酸酯(0.5mmol,148mg,1eq),氮气保护下反应4小时。
(2)用TLC板监测反应,反应完全后,用氮气吹干多余三氯甲基碳酸酯,然后加入化合物10(0.55mmol,85mg,1.1eq),并反应24小时。
(3)用TLC板监测反应,反应完全后,加入二氯甲烷和水萃取,无水硫酸钠干燥,旋干溶剂,并用配比为乙酸乙酯:正己烷=1:1洗脱剂进行硅胶柱分离,硅胶颗粒大小为200目,得到化合物13,反应过程如式(1)所示,产率为34%。
(4)将化合物13(0.2mmol,97mg,1eq)溶解在5mL二氯甲烷中,室温下加入三甲基溴硅烷(1.0mmol,130μL,5eq),氮气保护,反应12小时。
(5)用TLC板监测反应,反应完全后,加入甲醇(3mL)反应2小时后停止反应。旋干溶剂,并用配比为二氯甲烷:甲醇=5:1的洗脱剂进行硅胶柱分离,硅胶颗粒大小为200目,得到化合物TP-Phos,反应过程如式(2)所示,产率为44%。
2.测试分析:图1为实施例1合成的TP-Phos的化学结构式。图2为实施例1的1HNMR图谱。从图2中可知化学位移为8.65,8.10,7.96,7.29,7.09的共十个氢为芳香环区域的氢位移,化学位移为5.21的两个氢为苄基基团上的氢位移,化学位移为3.41和2.69的六个氢为甲基上的氢位移,以上说明化合物TP-Phos化学结构上的氢位移位置正确。图3为实施例1的13C NMR图谱。其中碳的数目与化学位移与化合物TP-Phos相对应,进一步证明了化合物TP-Phos的结构正确。图4为实施例1的31P NMR图谱,只有一个位于-5.91位置的磷峰,说明化合物TP-Phos只含有一个五价的磷酸酯结构。综上从核磁1H NMR、13C NMR和31P NMR证明了化合物TP-Phos的化学结构如图1所示。
实施例3化合物TP-Phos的合成
1.合成步骤:
(1)将2-羟甲基-苯基磷酸二乙酯(0.5mmol,130mg,1eq)和吡啶(1.5mmol,121μL,3eq)加入到20mL二氯甲烷中,降温到0℃后,加入1eq的三氯甲基碳酸酯(0.5mmol,148mg,1eq),氮气保护下反应8小时。
(2)用TLC板监测反应,反应完全后,用氮气吹干多余三氯甲基碳酸酯,然后加入化合物10(0.55mmol,85mg,1.1eq),并反应12小时。
(3)用TLC板监测反应,反应完全后,加入二氯甲烷和水萃取,无水硫酸钠干燥,旋干溶剂,并用配比为乙酸乙酯:正己烷=1:3洗脱剂进行硅胶柱分离,硅胶颗粒大小为300目,得到化合物13。
(4)将化合物13(0.2mmol,97mg,1eq)溶解在5mL二氯甲烷中,室温下加入三甲基溴硅烷(1.0mmol,130μL,5eq)。氮气保护,反应8小时。
(5)用TLC板监测反应,反应完全后,加入甲醇(3mL)反应2小时后停止反应。旋干溶剂,并用配比为二氯甲烷:甲醇=1:1的洗脱剂进行硅胶柱分离,硅胶颗粒大小为300目,得到化合物TP-Phos。
实施例4反应能力测试
将实施例1中制备的TP-Phos溶于二甲亚砜(DMSO)中,制成5mmol/L的储备液。从储备液中取出1μL加入到含有1mL Tris-HCl缓冲溶液(50mM,pH 7.4)的离心管中,加入1μL的10U/mL碱性磷酸酶并使最终浓度为0.01U/mL。10min后检测紫外吸收光谱及荧光发射光谱变化。图5为TP-Phos与碱性磷酸酶反应前后的紫外吸收(a)及荧光发射(b)光谱。其中,TP-Phos表示实施例1的荧光探针(5μM),TP-Phos+ALP表示实施例1的荧光探针(5μM)与碱性磷酸酶(0.01U/mL)的混合液;反应条件为Tris-HCl缓冲溶液(50mM,pH 7.4)。由图5(a)可知,反应前后溶液的最大紫外吸收光谱由300nm转移至365nm。同时,通过5(b)可以发现,荧光光谱强度由于碱性磷酸酶的加入显著增强。说明碱性磷酸酶可以与探针TP-Phos发生反应,使探针释放荧光。
实施例5选择性测试
将实施例1中制备的TP-Phos溶于二甲亚砜(DMSO)中,制成5mmol/L的储备液。从储备液中取出1μL加入到含有1mL Tris-HCl缓冲溶液(50mM,pH7.4)的离心管中,分别加入不同种类的磷酸酶作为竞争分子,其中一个加入碱性磷酸酶。10min后以365nm为激发光检测溶液的荧光发射光谱变化。图6为TP-Phos对不同的磷酸酶的选择性柱状图数据。荧光强度值为发射波长在500nm处的荧光值。其中,TP-Phos(5μM),碱性磷酸酶(10μM),GLEEP(50μM),PTP1B(50μM),MEG-2(50μM),LYP(50μM),VHR(50μM),SSH2(50μM),PPM1A(50μM),PPM1B(50μM),PPM1G(50μM),反应条件为Tris-HCl缓冲溶液(50mM,pH 7.4)。由图6可知其它代表性的磷酸酶对化合物TP-Phos的荧光几乎没有影响,而碱性磷酸酶的加入使荧光显著增强。以上实验说明TP-Phos只与碱性磷酸酶发生反应,不与其他的磷酸酶发生反应,显示了探针对于碱性磷酸酶极强的选择性能力。
实施例6细胞内源性碱性磷酸酶的检测
将本发明探针用于HeLa细胞中内源性碱性磷酸酶的荧光成像应用。具体操作步骤如下:将5μM TP-Phos的DMSO溶液加入到育有HeLa细胞的培养液中,在二氧化碳培养箱中培养30min后用共聚焦显微镜进行成像。首先进行明场成像,可以观察到细胞的轮廓。然后用蓝光进行激发观察荧光成像情况,可以发现细胞有绿色荧光发出。作为对照实验,首先在HeLa细胞中加入矾酸钠抑制细胞中的磷酸酶活性,孵育20min。然后加入5μM TP-Phos的DMSO溶液加入到育有HeLa细胞的培养液中,在二氧化碳培养箱中培养30min。用共聚焦显微镜的蓝光进行激发观察荧光成像情况,图7为TP-Phos检测细胞内源性碱性磷酸酶荧光成像图。其中,图7(a)为先加入磷酸酶抑制剂矾酸钠(1mM)到HeLa细胞中培养20min,再加入5μM探针到HeLa细胞中培养30min后荧光成像图;图7(b)为探针浓度为5μM加入到HeLa细胞中培养30min后荧光成像图。从图7中可知当HeLa细胞内的碱性磷酸酶被磷酸酶抑制剂矾酸钠所抑制后,探针TP-Phos不显示荧光。当不含有抑制剂时,探针TP-Phos与HeLa细胞内的碱性磷酸酶特异性的反应产生绿色荧光。结果表明TP-Phos可以用作荧光探针用于检测人体血液和细胞内源性碱性磷酸酶。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合和简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
Claims (10)
1.一种2-磷酸酯苄基甲酸酯类化合物,其特征在于,所述化合物的结构式如式(I)所示:
其中,X为O、NH或NCH3,Dye为荧光基团。
2.根据权利要求1所述2-磷酸酯苄基甲酸酯类化合物,其特征在于,所述荧光基团的结构式如式2-12所示:
3.根据权利要求1或2所述2-磷酸酯苄基甲酸酯类化合物的制备方法,其特征在于,包括如下具体步骤:
S1.将2-羟甲基-苯基磷酸二乙酯和吡啶加入到二氯甲烷中,降温到0℃后,加入三氯甲基碳酸酯,氮气保护下反应4~8小时;
S2.用TLC板监测反应,反应完全后,用氮气吹干多余的三氯甲基碳酸酯;然后加入荧光基团,并反应12~24小时;
S3.用TLC板监测反应,反应完全后,加入二氯甲烷和水萃取,无水硫酸钠干燥,旋干溶剂,并用洗脱剂进行硅胶柱分离,得到化合物A;
S4.将化合物A溶解在二氯甲烷中,室温下加入三甲基溴硅烷,氮气保护,反应8~12小时;
S5.用TLC板监测反应,反应完全后,旋干溶剂,并用洗脱剂进行硅胶柱分离,得到2-磷酸酯苄基甲酸酯类化合物。
4.根据权利要求3所述2-磷酸酯苄基甲酸酯类化合物的制备方法,其特征在于,步骤S1中所述2-羟甲基-苯基磷酸二乙酯、吡啶、二氯甲烷、三氯甲基碳酸酯的摩尔比为1:(2~5):(15~30):(0.5~1.5)。
5.根据权利要求3所述2-磷酸酯苄基甲酸酯类化合物的制备方法,其特征在于,步骤S2中所述荧光基团与步骤S1中所述2-羟甲基-苯基磷酸二乙酯的摩尔比为1:(0.8~1.5)。
6.根据权利要求3所述2-磷酸酯苄基甲酸酯类化合物的制备方法,其特征在于,步骤S3中所述洗脱剂为乙酸乙酯和正己烷,所述乙酸乙酯和正己烷的体积比为1:(1~3)。
7.根据权利要求3所述2-磷酸酯苄基甲酸酯类化合物的制备方法,其特征在于,步骤S4中所述化合物A、二氯甲烷、三甲基溴硅烷的比例关系1:(20~30):(3~5)。
8.根据权利要求3所述2-磷酸酯苄基甲酸酯类化合物的制备方法,其特征在于,步骤S5中所述洗脱剂为二氯甲烷和甲醇,所述二氯甲烷和甲醇的体积比为1:(0.2~1)。
9.权利要求1或2所述2-磷酸酯苄基甲酸酯类化合物作为荧光探针在检测碱性磷酸酶的应用。
10.权利要求9所述2-磷酸酯苄基甲酸酯类化合物作为荧光探针在检测碱性磷酸酶中的应用,其特征在于,所述碱性磷酸酶存在于人体血液或细胞中。
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