CN115232064A - 一种两亲性双位点受体的合成及其荧光指示剂置换法识别atp与生物硫醇 - Google Patents
一种两亲性双位点受体的合成及其荧光指示剂置换法识别atp与生物硫醇 Download PDFInfo
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Abstract
本发明公开了一种两亲性双位点受体的合成及其荧光指示剂置换法识别ATP与生物硫醇。该双位点受体含有ATP识别基团联吡啶盐正离子及生物硫醇作用位点二硫键。受体分子与荧光指示剂在水溶液中能自组装成纳米组装体,且发生指示剂荧光猝灭。加入ATP和生物硫醇后纳米组装体发生荧光恢复,对ATP检测限达到7.14nM,生物硫醇以谷胱甘肽为例,检测限也能达到9.83nM。该纳米聚集体有较低细胞毒性和高细胞通透性,适用于活细胞中ATP和GSH水平的成像。
Description
技术领域
本发明属于化学合成与生物应用领域,具体涉及一种双位点自组装材料的合成,及利用指示剂置换法在检测ATP和生物硫醇中的应用。
背景技术
三磷酸腺苷作为“细胞的能量货币”,在细胞分裂、细胞内信号转导、细胞膜转运、蛋白质合成等功能中发挥着重要作用。人体细胞中ATP/AMP和ATP/ADP的比值与ATP稳态密切相关。并且细胞内ATP水平的异常已被证明与多种疾病密切相关,包括癌症和帕金森病。
生物硫醇主要包括半胱氨酸(Cys)、同型半胱氨酸(Hcy)和谷胱甘肽(GSH),这些硫醇与生物系统中的代谢和运输重要的酶和蛋白质有关。这些硫醇的内源性浓度表明了相应的酶和蛋白质的功能状态,并且它们的异常水平与疾病相关。例如,半胱氨酸水平的异常与肝损伤、皮肤损伤和生长迟缓等有关。GSH是细胞内最丰富的非蛋白硫醇。游离GSH及其氧化态谷胱甘肽二硫醚(通常为>100:1)的比例是反映相应酶活性和细胞氧化还原状态的指标。GSH的异常水平也是许多疾病的信号,如艾滋病、癌症、肝和肺损伤和帕金森病。因此,对ATP和生物硫醇等多分析物的同时检测具有重要意义。
近年来,自组装纳米结构材料以其高灵敏度和优异的生物相容性等优点而受到广泛关注。多位点材料的设计因其能够同时检测和可视化活细胞中的两种或多种分析物从而提高诊断的全面性和准确性已逐渐成为研究热点。已经报道的有关ATP和生物硫醇的自组装纳米结构材料多是单一检测的,那么将双位点引入自组装纳米材料以实现同时区分检测ATP和生物硫醇的目的是很有必要的。
发明内容
针对现有技术中的不足与难题,本发明的目的在于提供一种两亲性双位点受体的合成及其荧光指示剂置换法识别ATP与生物硫醇。
为实现上述目的,本发明采用的技术方案是:
本发明提供了一种用于荧光指示剂置换法识别ATP和生物硫醇双位点两亲性双位点受体,缩写为Bp-SS-C13,其结构如下:
本发明还提供了上述的识别ATP和生物硫醇双位点两亲性双位点受体的制备方法,包括以下步骤:
(1)中间化合物1的合成:胱胺盐酸盐和三乙胺溶于甲醇中,冰浴条件下缓慢滴加二碳酸二叔丁酯,反应结束后,真空除去溶剂得到固体,并用乙醚洗涤,真空干燥得到白色固体化合物1;
(2)中间化合物2的合成:将化合物1和十四酸溶解在二氯甲烷中,加入二环己基碳二亚胺和4-二甲氨基吡啶,在常温条件下进行反应,反应结束后分离提纯得到白色固体化合物2;
(3)中间化合物3的合成:将化合物2溶在二氯甲烷中,加入三氟乙酸,常温反应三个小时,加入甲苯真空除去溶剂得到淡黄色液体,冷冻得到淡黄色固体化合物3;
(4)中间化合物4的合成:将化合物3溶在二氯甲烷中,冰浴条件下缓慢滴加溴乙酰溴,反应结束后,经分离提纯得到白色固体化合物4;
(5)化合物Bp-SS-C13的合成:将化合物4,碘化钾和4,4-联吡啶溶解在乙腈中,在氮气氛围下加热回流,反应完全后冷却至室温,经分离提纯得到黄色固体化合物Bp-SS-C13。
优选地,步骤(4)所述的化合物3和溴乙酰溴的摩尔比为1:2-2.3,反应时间三个小时。步骤(5)所述的4,4-联吡啶和化合物4的摩尔比为1:2-2.5,反应时间72小时。
本发明还提供了一种识别ATP和生物硫醇双位点自组装纳米材料,是上述的Bp-SS-C13与荧光指示剂在水溶液中自组装成纳米聚集体,纳米聚集体不发光。
优选地,荧光指示剂包括荧光素钠(UD)、署红(EY)、溶剂绿7(HPTS)、玫瑰红和4,4',4”,4”'-(卟啉-5,10,15,20-四基)四苯磺酸中的一种或几种。
本发明的双位点自组装纳米材料检测ATP和生物硫醇的测试条件:无特殊说明,选取荧光素钠(UD)为指示剂,生物硫醇以谷胱甘肽为例,在HEPES缓冲溶液(10mMpH=7.4)中进行测试。
本发明的双位点自组装纳米材料的作用机理,包含两部分:一是识别ATP的特征在于联吡啶盐和ATP的磷酸部分的静电引力、氢键、Л-Л作用更强,可以将指示剂置换出来;二是识别生物硫醇的特征在于其巯基可以有效的剪断探针的硫硫键,打破组装。当ATP存在时,可以有效置换出荧光素钠(UD),荧光明显增强。当生物硫醇存在时,可以有效的剪断探针的硫硫键,指示剂释放出来荧光增强。
本发明的双位点自组装纳米材料的选择性好。含有联吡啶盐猝灭基团可以有效猝灭阴离子指示剂荧光素钠(UD)的荧光,加入ATP、生物硫醇后荧光恢复。
本发明的双位点自组装纳米材料与UD络合时,加入10eq的ATP和生物硫醇,在515nm处的荧光明显增强,而加入其他阴离子(ADP,PPi,COO-,NO3-,Cl-,Pi,Br-,SO4 2-,CO3 2-,AMP,F-,I-)和氨基酸(Glu,Arg,His,Met,Thr,Ser,Ala,Asp,Leu,Gly,PHe,Tyr,Val,Trp,Lys)荧光没有明显增强。
本发明的双位点自组装纳米材料抗干扰能力较强,在其他分析物(ADP,PPi,COO-,NO3-,Cl-,Pi,Br-,SO4 2-,CO3 2-,AMP,F-,I-,Glu,Arg,His,Met,Thr,Ser,Ala,Asp,Leu,Gly,PHe,Tyr,Val,Trp,Lys)的存在下几乎不影响检测ATP和生物硫醇的效果。
本发明的双位点自组装纳米材料对ATP和生物硫醇具有较低的检测限,低达10-11。
本发明的双位点自组装纳米材料对生物硫醇的响应时间较短,在四十分钟左右就可以达到平衡。
本发明的双位点自组装纳米材料pH应用范围较宽,7-10均可检测。
本发明的双位点自组装纳米材料通过荧光共聚焦显微镜成像技术,证明了可用于检测细胞内的ATP和生物硫醇。
与现有技术相比,本发明有益效果是:
本发明提供了一种选择性好、灵敏度高的双位点材料的合成方法,并且可以自主装成纳米粒子,具有较低的细胞毒性和良好的生物相容性。
本发明制备的双位点自组装材料具有合成原料便宜,合成工艺简单,分离程序容易,产率高,稳定易保存等优点。
本发明的双位点自组装纳米材料,利用指示剂置换法结合不同的指示剂达到对ATP和生物硫醇的特异性识别,避免了单一识别的缺陷。
附图说明
图1为化合物Bp-SS-C13的核磁氢谱(DMSO);
图2为化合物Bp-SS-C13对UD的荧光滴定;
图3为化合物Bp-SS-C13/UD对ATP和生物硫醇的竞争性;
图4为化合物Bp-SS-C13/UD对ATP和生物硫醇的检测限;
图5为化合物Bp-SS-C13/UD生物硫醇的响应时间;
图6为化合物Bp-SS-C13/UD/ATP、GSH的细胞成像图。
具体实施方式
下面结合实施例和附图对本发明作进一步详细的描述,但本发明的实施方式不限于此。
实施例1
本发明Bp-SS-C13的合成方法,包括以下步骤:
(1)中间化合物4的合成:将化合物3(0.543g,1.5mmol)溶在二氯甲烷中,冰浴条件下缓慢滴加溴乙酰溴(0.561g,3mmol),反应结束后,真空除去溶剂得到白色固体,以石油醚:二氯甲烷(1:10)为洗脱剂,通过柱层析法获得0.644g白色固体化合物4,收率89%。
(2)化合物Bp-SS-C13的合成:将化合物4(0.576g,1.2mmol),碘化钾(0.0016g,0.01mmol)和4,4-联吡啶(0.078g,0.5mmol)溶解在乙腈中,在氮气氛围下加热回流反应72小时,反应完全后冷却至室温,经过滤,乙腈洗涤得到黄色固体化合物Bp-SS-C13(0.482g),收率86%。
图1为化合物Bp-SS-C13的核磁氢谱(DMSO)。上述所得的黄色固体化合物Bp-SS-C13通过核磁共振仪器(Varianinstrument400MHz)测定,数据如下所示:
1HNMR(400MHz,DMSO-d6)δ(ppm):9.26(d,J=6.3Hz,2H),8.81(d,J=6.5Hz,4H),7.96(dd,J=11.5,5.9Hz,2H),5.53(s,2H),3.45(q,J=6.5Hz,3H),2.82(t,J=6.6Hz,7H),2.75(p,J=6.5Hz,7H),2.02(t,J=7.4Hz,5H),1.44(t,J=7.2Hz,5H),1.20(s,43H),0.83(t,J=6.5Hz,6H).
实施例2
荧光光谱测试实验:将Bp-SS-C13溶解在甲醇溶液中,配成浓度为4mM的溶液,UD也用甲醇溶液配置成同样的浓度,ATP和生物硫醇配置成10mM的水溶液,测试都在HEPES缓冲溶液(10mMpH=7.4)中进行。
图2为化合物Bp-SS-C13对UD的荧光滴定。对UD的荧光滴定实验:取30μl的指示剂UD母液加入到2ml的缓冲液中,加入不同当量(0-6eq)的Bp-SS-C13,在515nm处的荧光强度都逐渐降低。
实施例3
化合物Bp-SS-C13/UD对ATP和生物硫醇的选择性:取30μl的指示剂UD母液/180μl的Bp-SS-C13母液加入到2ml的缓冲液中,加入10eq的ATP和GSH及其他分析物(ADP,PPi,COO-,NO3-,Cl-,Pi,Br-,SO4 2-,CO3 2-,AMP,F-,I-,Glu,Arg,His,Met,Thr,Ser,Ala,Asp,Leu,Gly,PHe,Tyr,Val,Trp,Lys),通过荧光光谱变化明显观察到ATP和生物硫醇在515nm处的荧光强度最强,其他没有明显变化,表现出良好的选择性。
图3为化合物Bp-SS-C13/UD对ATP和生物硫醇的竞争性。化合物Bp-SS-C13/UD对ATP和生物硫醇的竞争性:取30μl的指示剂UD母液/180μl的探针母液加入到2ml的缓冲液中,加入10eq的其他分析物后再加入10eq的ATP和GSH,通过荧光光谱变化明显观察到加入ATP和GSH后,在515nm处的荧光明显增强,表现出良好的竞争性。
实施例4
图4为化合物Bp-SS-C13/UD对ATP和生物硫醇的检测限。取30μl的指示剂UD母液/180μl的Bp-SS-C13母液加入到2ml的缓冲液中,加入不同当量的ATP(0-3.5eq),通过荧光光谱变化明显观察到在515nm处的荧光强度逐渐增强,检测限低达0.0175nM。同样的,取30μl的指示剂UD母液/180μl的Bp-SS-C13母液加入到2ml的缓冲液中,加入不同当量的GSH(0-6eq),37℃恒温水浴孵育四十分钟,通过荧光光谱变化明显观察到在515nm处的荧光强度逐渐增强,检测限低达0.0248nM。
实施例5
图5为化合物Bp-SS-C13/UD生物硫醇的响应时间。Bp-SS-C13/UD与GSH的动力学实验,Bp-SS-C13/UD的荧光强度与加入饱和当量的GSH孵育时间的关系:取30μl的指示剂UD母液/180μl的Bp-SS-C13母液加入到2ml的缓冲液中,加入饱和当量的GSH(6eq),随着时间的增加,在515nm处的荧光强度逐渐增强,四十分钟达到饱和。
实施例6
对实施例1中所得Bp-SS-C13用MTT法进行了其对细胞的毒性测试实验,在探针浓度高达40μM的情况下细胞存活率仍然较高,具有较低的细胞毒性。如图6所示,探针/UD成功的对细胞内ATP和GSH成像,在生物化学,分析检测等领域极具价值。
以上所述仅表达了本发明的优选实施方式,其描述较为具体和详细,但并不能因此而理解为对本发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形、改进及替代,这些都属于本发明的保护范围。因此,本发明专利的保护范围应以所附权利要求为准。
Claims (9)
3.根据权利要求2所述的识别ATP和生物硫醇双位点两亲性双位点受体的制备方法,其特征在于,所述溶剂为超干乙腈,催化剂为碘化钾,4,4-联吡啶和化合物4的摩尔比为1:2~2.5。
4.根据权利要求2所述的识别ATP和生物硫醇双位点两亲性双位点受体的制备方法,其特征在于,包括以下步骤:
(1)中间化合物1的合成:胱胺盐酸盐和三乙胺溶于甲醇中,冰浴条件下缓慢滴加二碳酸二叔丁酯,反应结束后,真空除去溶剂得到固体,并用乙醚洗涤,真空干燥得到白色固体化合物1;
(2)中间化合物2的合成:将化合物1和十四酸溶解在二氯甲烷中,加入二环己基碳二亚胺和4-二甲氨基吡啶,在常温条件下进行反应,反应结束后分离提纯得到白色固体化合物2;
(3)中间化合物3的合成:将化合物2溶在二氯甲烷中,加入三氟乙酸,常温反应三个小时,加入甲苯真空除去溶剂得到淡黄色液体,冷冻得到淡黄色固体化合物3;
(4)中间化合物4的合成:将化合物3溶在二氯甲烷中,冰浴条件下缓慢滴加溴乙酰溴,反应结束后,经分离提纯得到白色固体化合物4;
(5)化合物Bp-SS-C13的合成:将化合物4,碘化钾和4,4-联吡啶溶解在乙腈中,在氮气氛围下加热回流,反应完全后冷却至室温,经分离提纯得到黄色固体化合物Bp-SS-C13。
5.根据权利要求4所述的识别ATP和生物硫醇双位点两亲性双位点受体的制备方法,其特征在于,步骤(4)所述的化合物3和溴乙酰溴的摩尔比为1:2-2.3,反应时间三个小时;步骤(5)所述的4,4-联吡啶和化合物4的摩尔比为1:2-2.5,反应时间72小时。
6.一种识别ATP和生物硫醇双位点自组装纳米材料,其特征在于,所述双位点自组装纳米材料是权利要求1所述的Bp-SS-C13或通过权利要求2-5任一项所述的制备方法得到的Bp-SS-C13与荧光指示剂在水溶液中自组装成纳米聚集体。
7.根据权利要求6所述的识别ATP和生物硫醇双位点自组装纳米材料,其特征在于,所述荧光指示剂包括荧光素钠、署红、溶剂绿7、玫瑰红和4,4',4”,4”'-(卟啉-5,10,15,20-四基)四苯磺酸中的一种或几种。
8.一种如权利要求6或7所述的识别ATP和生物硫醇双位点自组装纳米材料的生物应用,其特征在于,应用于细胞内检测ATP和生物硫醇浓度及其生物成像。
9.根据权利要求8所述的识别ATP和生物硫醇双位点自组装纳米材料的生物应用,其特征在于,所述生物硫醇包括半胱氨酸、同型半胱氨酸和谷胱甘肽。
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