CN114957271A - 具有强特异性的半胱氨酸荧光分子探针及其合成与应用、以及含有其的试剂盒 - Google Patents
具有强特异性的半胱氨酸荧光分子探针及其合成与应用、以及含有其的试剂盒 Download PDFInfo
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Abstract
一种具有强特异性的半胱氨酸荧光分子探针及其合成与应用、以及含有其的试剂盒，所述分子探针具有式Ⅰ所示的结构式：
所示荧光分子探针合成路线简单，成本低廉，操作简便，对半胱氨酸的检测具有优异的特异性，且具有快速的响应速度，在对半胱氨酸的快速检测方面具有较高的应用价值。
Description
技术领域
本发明涉及一种且具有强特异性的半胱氨酸荧光分子探针及其合成与应用、以及含有其的试剂盒，属于有机小分子荧光探针技术领域。
背景技术
半胱氨酸(cysteine，Cys)、谷胱甘肽(glutathione，GSH)、同型半胱氨酸(homocysteine，Hcy)等生物硫醇在生物系统中发挥着重要作用，其水平与癌症、代谢疾病、精神退行性疾病等疾病密切相关。其中，半胱氨酸被认为是蛋白质形成、抗氧化剂生成、金属离子结合的必需氨基酸。因此，其检测具有重要的声明科学和医学价值。目前，已经开发了几种用于检测生物条件下半胱氨酸的分析方法，其中，基于荧光的分子传感系统因其操作简单和生物相容性高而备受关注。基于其巯基的亲核性和还原性，已有了多种半胱氨酸反应型荧光探针被报道，然而，它们大多容易受到其他生物硫醇的干扰。因此，开发强特异性、高灵敏度的半胱氨酸荧光分子探针具有重要的意义。
发明内容
本发明针对当前存在的问题，提出了一种具有强特异性的半胱氨酸荧光分子探针及其合成与应用、以及含有其的试剂盒，能够快速准确地检测出半胱氨酸。
本发明为解决上述问题所采用的技术手段为：一种具有强特异性的半胱氨酸荧光分子探针，所述分子探针具有式Ⅰ所示的结构式：
一种具有强特异性的半胱氨酸荧光分子探针的合成，包括以下步骤：
(1)将化合物TPC溶解于二氯甲烷，加入乙二酰氯和DMF，反应结束后浓缩干燥；然后再溶解于二氯甲烷，加入苯酚、4-二甲氨基吡啶和三乙胺至反应结束，用饱和碳酸氢钠溶液淬灭反应，萃取后干燥浓缩，最后层析纯化，得式Ⅱ所示中间物：
其中乙二酰氯和DMF的加入方式为0-5℃搅拌加入，在室温下继续搅拌至反应结束后减压浓缩、然后真空干燥，且1mmol TPC优选加入5-10mL二氯甲烷、两滴即0.1mL DMF，加入的乙二酰氯与TPC量的比优选为1.2:1；反应结束且干燥后再次溶解时1mmol TPC对应的产物中加入的二氯甲烷优选为2-3mL，苯酚优选为1-1.2mmol，4-二甲氨基吡啶优选为0.1-0.5mmol，三乙胺优选为3-4mmol，萃取时先用DCM或乙酸乙酯萃取，再用饱和氯化钠溶液萃取有机层；层析纯化采用硅胶柱层析纯化；
(2)式Ⅱ中间物与Lawesson试剂、对二甲苯反应至结束，浓缩后层析纯化得到式Ⅰ所示目标分子探针：
其中反应时在140℃惰性气体保护下搅拌反应至结束，惰性气体优选为氩气；浓缩方式采用减压浓缩，层析纯化采用柱层析纯化；1mmol式Ⅱ化合物中优选加入1.4-1.5mmolLawesson试剂，1.5-2mL对二甲苯。
一种具有强特异性的半胱氨酸荧光分子探针在半胱氨酸检测试剂盒中的应用。
一种半胱氨酸检测试剂盒，所述试剂盒包括结构式为式Ⅰ的半胱氨酸荧光分子探针，以及半胱氨酸检测组合物。
进一步地，半胱氨酸检测组合物包括半胱氨酸标准品和溶剂。
进一步地，溶剂为PBS缓冲液和乙腈的混合溶液。
进一步地，溶剂中PBS缓冲溶液(0.01mol/L，pH＝7.4)和乙腈的体积比为7：3。
本发明所述的半胱氨酸检测试剂盒的检测原理为：
由于强吸电子碳硫双键降低了派嗪的给电子能力，减弱了式Ⅰ探针中类香豆素母核的推拉电子效应，导致探针荧光减弱。而当半胱氨酸存在时，探针中的碳硫双键通过选择性亲核加成反应与半胱氨酸的N端形成具有天然酰胺键的稳定五元环，恢复探针的推拉电子效应并使其荧光显著增强，同时这种反应具有高选择性，Hcy、GSH等巯基化合物并不会发生反应，故探针具有优异的特异性。探针的检测原理如下所示：
本发明的有益效果是：
1.本发明荧光分子探针合成路线简单，成本低廉，操作简便。
2.本发明荧光分子探针对半胱氨酸的检测具有优异的特异性，生物硫醇(Hcy、GSH)、氨基酸(L-谷氨酰胺、L-赖氨酸)、金属离子(Na+、K+、Mg2+)等均不会对其有影响。
3.本发明荧光分子探针对半胱氨酸的检测具有快速的响应速度，仅需6.5分钟即可完成响应。
附图说明
图1为实施例二中荧光分子探针的荧光强度随半胱氨酸浓度变化的发射光谱图；
图2为实施例二中荧光分子探针的荧光强度和半胱氨酸浓度的线性关系；
图3为实施例三中荧光分子探针对半胱氨酸的选择性图；
图4为实施例四中荧光分子探针对半胱氨酸的时间响应图；
图5为实施例五中荧光分子探针对活细胞中内源性半胱氨酸的成像图。
具体实施方式
下面结合附图对本发明进一步说明。
一种具有强特异性的半胱氨酸荧光分子探针的合成，合成路线如下：
(1)式Ⅱ所示中间物的合成
取化合物TPC(604.66mg,2.0mmol)置于25mL圆底烧瓶中，0℃下向其加入二氯甲烷10mL，并向反应体系滴加乙二酰氯(206μL,2.4mmol)和2滴约0.1mL DMF。0℃下继续搅拌30分钟后，置于室温下继续反应，TLC监测至反应结束。减压除去所有挥发性成分后真空下干燥3h，随后将粗其溶解在4mL二氯甲烷中。0℃下向反应体系中加入苯酚(188.22mg，2.0mmol)、4-二甲氨基吡啶(DMAP，24.43mg，0.2mmol)和三乙胺(838μL、6.0mmol)在室温下搅拌过夜，TLC监测反应至结束，用饱和碳酸氢钠溶液淬灭反应体系，并用DCM萃取，再用饱和氯化钠溶液萃取有机层，干燥，减压浓缩。最后用硅胶柱层析进行纯化，得产物381.46mg，产率为50.4％。1H NMR(400MHz,DMSO-d6):δ8.55(s,1H),7.44(t,J＝8.0Hz,2H),7.33-7.31(m,3H),6.78(s,1H),6.31(s,1H),3.57(t,J＝4.0Hz,4H),3.40(q,J＝7.2Hz,4H),1.12(t,J＝7.1Hz,6H)。
(2)式Ⅰ荧光分子探针的合成
在5mL圆底烧瓶中加入式Ⅱ所示化合物(378.42mg，1mmol)和Lawesson试剂(163.81mg，1.4mmol)，并加入对二甲苯(1.5mL)使其完全溶解。在140℃氩气保护下搅拌反应24h。反应结束后，减压旋蒸除去溶剂，用柱层析纯化粗混合物，得到所需化合物PTPC-Cys(78.11mg，19.8％)。1H NMR(400MHz,CDCl3):δ7.56(s,1H),7.30(t,J＝8.2Hz,2H)7.06(t,J＝8.0Hz,1H),6.99(d,J＝8.2Hz,2H),6.94(s,1H),6.35(s,1H),3.59(t,J＝4.0Hz,4H),3.46(q,J＝7.1Hz,4H),1.20(t,J＝7.0Hz,6H).HRMS-ESI(C22H22N2O3S)m/z:calcd.for,[M+H]+395.4890；found,395.5131。
实施例二
荧光分子探针的荧光强度与Cys浓度的变化
配制浓度为1mmol/L的式I荧光分子探针的乙腈测试母液溶液待用。量取20μL的此母液分别滴加到不同浓度半胱氨酸的PBS缓冲溶液(0.01mol/L，pH＝7.4)中，并用相应的PBS缓冲溶液(0.01mol/L，pH＝7.4)定容到2mL，使得测试液中，探针的浓度为10μmol/L，半胱氨酸浓度为2μmol/L、5μmol/L、10μmol/L、20μmol/L、30μmol/L、50μmol/L、100μmol/L、200μmol/L、300μmol/L激发光波长355nm处进行荧光检测。得各体系中荧光强度，建立如图2所示的荧光强度与半胱氨酸浓度标准曲线。如图1所示，随着半胱氨酸浓度的增加，527nm处荧光强度逐渐增加，此外，在低浓度下，荧光强度和半胱氨酸的浓度(0-50μmol/L)之间呈现良好的线性关系(R2＝0.99086)，探针PTPC-Cys对半胱氨酸的检测限可达61nmol/L。
实施例三
荧光分子探针对半胱氨酸的选择性实验
配制浓度为1mmol/L的式Ⅰ荧光分子探针的乙腈测试母液溶液待用。配制浓度为10mmol/L的生物硫醇(Hcy、GSH)、氨基酸(L-谷氨酰胺、L-赖氨酸)、金属离子(Na+、K+、Mg2+)的溶液作为备用。量取10μL的上述母液分别滴加到生物硫醇(Hcy、GSH)、氨基酸(L-谷氨酰胺、L-赖氨酸)、金属离子(Na+、K+、Mg2+)PBS缓冲溶液(0.01mol/L，pH＝7.4)中，并用相应的PBS缓冲溶液(0.01mol/L，pH＝7.4)定容到10mL，使得测试液中，探针的浓度为10μmol/L，半胱氨酸的浓度为10μmol/L，而生物硫醇(Hcy、GSH)、氨基酸(L-谷氨酰胺、L-赖氨酸)、金属离子(Na+、K+、Mg2+)的浓度为30μmol/L。进行荧光检测得各体系中荧光强度，建立527nm处荧光强度与各离子的柱状图。如图3所示，其他生物硫醇、氨基酸及金属离子对式Ⅰ探针的荧光几乎没有影响。
实施例四
荧光分子探针对半胱氨酸的时间响应实验
配制浓度为1mmol/L的式Ⅰ荧光分子探针的乙腈测试母液溶液待用。量取20μL的此母液滴加到20μL浓度为1mmol/L的半胱氨酸的母液中，并用1960μLPBS缓冲溶液(0.01mol/L，pH＝7.4)定容到2mL，使得其中探针的浓度为10μmol/L，半胱氨酸浓度为10μmol/L；然后快速放入荧光分光光度计中，分别记录下0min、2min、4min、6min、8min、10min、12min、14min、16min、18min、20min、25min、30min、40min、50min、60min后检测体系的荧光光谱数据，并对数据进行处理，用527nm处的荧光强度作为纵坐标，时间作为横坐标，得出式Ⅰ探针对半胱氨酸的时间响应情况如图4所示。从图4中可以看到，一加入Cys，探针的荧光强度就出现增长，而到了15min的时候式Ⅰ探针与半胱氨酸响应完全。因此，该探针在对半胱氨酸的快速检测方面具有较高的应用价值。
实施例五
荧光分子探针对活细胞中内源性半胱氨酸的成像实验
向DMEM培养基中加入10％胎牛血清，将HeLa细胞置于该培养基中于37℃，5％CO2下进行培养，12h后，从孔中取出细胞培养基，用D-Hanks洗涤细胞三次。将一部分细胞实验组a)用20μM式Ⅰ探针处理30分钟。对于对照组b)，则将N-乙基马来酰亚胺(NEM，一种广泛使用的硫醇封闭剂)加入到培养基中，然后PBS洗涤细胞除去剩余的NEM，并在1.0mL新鲜培养基中与式Ⅰ探针(20μmol/L)一起孵育30分钟后进行荧光成像。结果如图5所示，实验组a)可见明显荧光信号，而对照组b)的荧光信号微弱，表明式Ⅰ探针可用于活细胞中内源性半胱氨酸的检测。
需要说明的是，化合物TPC(见Dyes and Pigments 198(2022)109971)英文名称1,4-diethyl-7-oxo-2,3,4,7-tetrahydro-1H-pyrano[2,3-g]quinoxaline-8-carboxylicacid，采用1,4-diethyl-7-hydroxy-1,2,3,4-tetrahydroquinoxaline-6-carbaldehyde(CAS.1194709-71-1，结构式如下)合成：
以上实施例仅供说明本发明之用，而非对本发明的限制，有关技术领域的技术人员在不脱离本发明的精神和范围的情况下，还可以做出各种变化或变换，因此所有等同的技术方案也应该属于本发明的保护范围，本发明的保护范围应该由各权利要求限定。
Claims (10)
1.一种具有强特异性的半胱氨酸荧光分子探针，所述分子探针具有式Ⅰ所示的结构式：
2.一种具有强特异性的半胱氨酸荧光分子探针的合成，其特征在于：包括以下步骤：
(1)将化合物TPC溶解于二氯甲烷，加入乙二酰氯和DMF，反应结束后浓缩干燥，然后再溶解于二氯甲烷，加入苯酚、4-二甲氨基吡啶和三乙胺至反应结束，用饱和碳酸氢钠溶液淬灭反应，萃取后干燥浓缩，最后层析纯化，得式Ⅱ所示中间物：
(2)式Ⅱ中间物与Lawesson试剂、对二甲苯反应至结束，浓缩后层析纯化得到式Ⅰ所示目标分子探针：
3.如权利要求2所述的具有强特异性的半胱氨酸荧光分子探针的合成，其特征在于：所述步骤(1)中乙二酰氯和DMF的加入方式为0-5℃搅拌加入，然后在室温下继续搅拌至反应结束。
4.如权利要求2所述的具有强特异性的半胱氨酸荧光分子探针的合成，其特征在于：所述步骤(1)中1mmol TPC加入5-10mL二氯甲烷、0.1mL DMF，加入的乙二酰氯与TPC量的比为1.2:1；反应结束且干燥后再次溶解时1mmol TPC对应的产物中加入的二氯甲烷为2-3mL，苯酚为1-1.2mmol，4-二甲氨基吡啶为0.1-0.5mmol，三乙胺为3-4mmol。
5.如权利要求2所述的具有强特异性的半胱氨酸荧光分子探针的合成，其特征在于：所述步骤(1)中萃取时先用DCM或乙酸乙酯萃取，再用饱和氯化钠溶液萃取有机层。
6.如权利要求2所述的具有强特异性的半胱氨酸荧光分子探针的合成，其特征在于：所述步骤(2)中反应时在140℃惰性气体保护下搅拌反应至结束。
7.如权利要求2所述的具有强特异性的半胱氨酸荧光分子探针的合成，其特征在于：所述步骤(2)中1mmol式Ⅱ化合物中加入1.4-1.5mmol Lawesson试剂，1.5-2mL对二甲苯。
8.一种权利要求1所述的具有强特异性的半胱氨酸荧光分子探针的应用：其特征在于，所述荧光分子探针在半胱氨酸检测试剂盒中的应用。
9.一种半胱氨酸检测试剂盒，其特征在于：所述试剂盒包括权利要求1所述的荧光分子探针，以及半胱氨酸检测组合物。
10.如权利要求9所述的半胱氨酸检测试剂盒，其特征在于：半胱氨酸检测组合物包括半胱氨酸标准品和溶剂，其中溶剂为PBS缓冲液和乙腈的混合溶液。
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