CN110498786A - 一种检测半胱氨酸/同型半胱氨酸和谷胱甘肽的新型比值型荧光探针 - Google Patents
一种检测半胱氨酸/同型半胱氨酸和谷胱甘肽的新型比值型荧光探针 Download PDFInfo
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Abstract
本发明公开一种检测细胞和斑马鱼体内半胱氨酸/同型半胱氨酸和谷胱甘肽的新型比值型荧光探针，其结构式为：该探针具有灵敏度高、选择性好，检测限低，对生物体毒性小等优点，是一种够对活细胞和斑马鱼体内的三种主要细胞生物硫醇进行快速监测及细胞成像的荧光探针，具有良好的生物应用前景。
Description
技术领域
本发明属于化学分析检测技术领域，具体涉及一种检测细胞和斑马鱼体内半胱氨酸/同型半胱氨酸和谷胱甘肽的新型比值型荧光探针及其应用。
背景技术
氨基酸是构成蛋白质的基本物质，并且与生物的生命活动有着密切的连写。半胱氨酸(Cys)、同型半胱氨酸(Hcy)、谷胱甘肽(GSH)作为细胞内三种主要的硫醇，在许多生理病理过程中发挥着重要作用。例如，半胱氨酸与生物催化、蛋白质合成、翻译后修饰密切相关等。谷胱甘肽与氧化应激、炎症、信号转导和凋亡相关，在维持细胞内氧化还原稳态方面发挥着至关重要的作用。因此，半胱氨酸、同型半胱氨酸、谷胱甘肽异常水平可导致肝损伤、生长缓慢、水肿、嗜睡、神经毒性和心血管疾病。重要的是，人们发现这三种硫醇在其生产和代谢过程中有着密切的共生关系。为了阐明半胱氨酸/同型半胱氨酸与谷胱甘肽的复杂功能及其相互之间的关系，迫切需要开发一种有效的方法，在生物系统中对半胱氨酸/同型半胱氨酸与谷胱甘肽进行鉴别检测。目前已经应用的技术包括高效液相色谱法、毛细管电泳法、电化学检测、光学分析和质谱鉴定，这些方法可以再体外检测半胱氨酸/同型半胱氨酸和谷胱甘肽。与上述方法相比，荧光探针却是一种比较理想的体内检测手段，因为它具有检测方便，响应时间快，灵敏度高，检测限低，对细胞损害小等优点。由于这三种氨基酸都含有巯基(-SH)并且在结构和反应活性上相差较小，所以这类荧光探针很难将半胱氨酸/同型半胱氨酸和谷胱甘肽区分开，因此研发能够输出不同响应信号的该类荧光探针是有必要的。
发明内容
本发明目的之一是提供一种合成简单、反应条件温和、成本较低的荧光探针合成方法；目的之二是提供一种灵敏度高、选择性好，抗干扰能力强，比值型，能够对细胞内或斑马鱼的半胱氨酸/同型半胱氨酸和谷胱甘肽进行快速监测或者细胞成像的荧光探针，其结构如下：
其合成路线为：
具体合成方法：化合物1和化合物4是根据文献合成。(a)将化合物1(65mg,0.2mmol)和化合物2(31mg,0.2mmol)以及1.2倍当量的三乙胺加入到3mL无水乙腈中，在氩气保护下室温搅拌2小时，将反应液旋干后通过柱层析纯化得化合物3，黄色固体(67.0mg,产率72％)。(b)将化合物3(49mg,0.1mmol)和化合物4(34mg,0.1mmol)以及EDCI(19mg,0.1mmol)加入到2mL无水二氯甲烷中，再加入DMAP(1mg,0.008mmol)作为催化剂，在氩气保护下室温反应12小时，将反应液旋干柱层析得到红色固体(53mg,产率65％)，即为探针CPR。
本发明的探针的机理如下：
探针CPR在半胱氨酸或同型半胱氨酸作用下，分子中的香豆素4位的硫键以及碳碳双键会被进攻导致分子分为两部分，香豆素部分重排产物为Cou-N-Cys/Cou-N-Hcy显示蓝色荧光(最大发射波长472nm)，另一部分双键被进攻，分子共轭体系消失导致红色荧光消失,产物为PR-S-Cys/PR-S-Hcy，而与谷胱甘肽作用下香豆素部分不会发生重排，显示香豆素部分的黄色荧光(最大发射波长542nm),探针CPR本身的红色荧光(最大发射波长584nm)在与半胱氨酸/同型半胱氨酸和谷胱甘肽的反应中显示不同的荧光，从而达到特异性比率型区分检测半胱氨酸/同型半胱氨酸和谷胱甘肽。
本发明的荧光探针本身荧光发射峰在584nm,与半胱氨酸和同型半胱氨酸作用后荧光发射峰在472nm处，与谷胱甘肽作用后荧光发射峰出现在542nm处。
附图说明
图1为本发明的荧光探针(5.0×10-6mol/L)在pH为7.4的PBS缓冲溶液(10.0mM,VDMF/VPBS＝3/7)中，与0.3mM的半胱氨酸响应后在40分钟内的紫外吸收变化图，横坐标为波长，纵坐标分别为吸收强度。
图2为本发明的荧光探针(5.0×10-6mol/L)在pH为7.4的PBS缓冲溶液(10.0mM,VDMF/VPBS＝3/7)中，与0.3mM的同型半胱氨酸响应后在102分钟内的紫外吸收变化图，横坐标为波长，纵坐标分别为吸收强度。
图3为本发明的荧光探针(5.0×10-6mol/L)在pH为7.4的PBS缓冲溶液(10.0mM,VDMF/VPBS＝3/7)中，与0.1mM的谷胱甘肽响应后在38分钟内的紫外吸收变化图，横坐标为波长，纵坐标分别为吸收强度。
图4为本发明的荧光探针(5.0×10-6mol/L)在pH为7.4的PBS缓冲溶液(10.0mM,VDMF/VPBS＝3/7)中，与不同浓度的半胱氨酸作用后的荧光光谱变化，横坐标为波长，纵坐标分别为荧光强度。小图是与半胱氨酸浓度的线性关系，横坐标为浓度，纵坐标为荧光强度的比值。
图5为本发明的荧光探针(5.0×10-6mol/L)在pH为7.4的PBS缓冲溶液(10.0mM,VDMF/VPBS＝3/7)中，与不同浓度的同型半胱氨酸作用后的荧光光谱变化，横坐标为波长，纵坐标分别为荧光强度。小图是与半胱氨酸浓度的线性关系，横坐标为浓度，纵坐标为荧光强度的比值。
图6为本发明的荧光探针(5.0×10-6mol/L)在pH为7.4的PBS缓冲溶液(10.0mM,VDMF/VPBS＝3/7)中，与不同浓度的谷胱甘肽作用后的荧光光谱变化，横坐标为波长，纵坐标分别为荧光强度。小图是与半胱氨酸浓度的线性关系，横坐标为浓度，纵坐标为荧光强度的比值。
图7为本发明的荧光探针(5.0×10-6mol/L)在pH为7.4的PBS缓冲溶液(10.0mM,VDMF/VPBS＝3/7)中，与半胱氨酸作用过程中472nm,584nm处荧光强度与探针本身在472nm,584nm处荧光强度的比值(I/I0)随时间的变化，横坐标为时间，纵坐标为荧光强度的比值。
图8为本发明的荧光探针(5.0×10-6mol/L)在pH为7.4的PBS缓冲溶液(10.0mM,VDMF/VPBS＝3/7)中，与同型半胱氨酸作用过程中472nm,584nm处荧光强度与探针本身在472nm,584nm处荧光强度的比值(I/I0)随时间的变化，横坐标为时间，纵坐标为荧光强度的比值。
图9为本发明的荧光探针(5.0×10-6mol/L)在pH为7.4的PBS缓冲溶液(10.0mM,VDMF/VPBS＝3/7)中，与谷胱甘肽作用过程中542nm,584nm处荧光强度与探针本身在542nm,584nm处荧光强度的比值(I/I0)随时间的变化，横坐标为时间，纵坐标为荧光强度的比值。
图10为本发明的荧光探针(5.0×10-6mol/L)在不同pH值缓冲溶液中，与半胱氨酸作用前后的荧光强度，横坐标为pH值，纵坐标为荧光强度的比值。
图11为本发明的荧光探针(5.0×10-6mol/L)在不同pH值缓冲溶液中，与同型半胱氨酸作用前后的荧光强度，横坐标为pH值，纵坐标为荧光强度的比值。
图12为本发明的荧光探针(5.0×10-6mol/L)在不同pH值缓冲溶液中，与谷胱甘肽作用前后的荧光强度，横坐标为pH值，纵坐标为荧光强度的比值。
图13为本发明的荧光探针(5.0×10-6mol/L)在pH为7.4的PBS缓冲溶液(10.0mM,VDMF/VPBS＝3/7)中存在(1)PBS,(2)Cys,(3)Hcy,(4)GSH,(5)Lys,(6)Phe,(7)Thr,(8)Met,(9)Val,(10)His,(11)Gly,(12)Ser,(13)Ala,(14)Tyr,(15)Arg,(16)Glu,(17)Ile,(18)KI,(19)KCl,(20)Na2SO4,(21)NaNO3,(22)KO2,(23)NaNO2,(24)H2O2等不同物质时与半胱氨酸响应选择性的条形图，纵坐标为荧光强度。黑色条形是472nm荧光强度，红色条形是584nm荧光强度。
图14为本发明的荧光探针(5.0×10-6mol/L)在pH为7.4的PBS缓冲溶液(10.0mM,VDMF/VPBS＝3/7)中存在(1)PBS,(2)Cys,(3)Hcy,(4)GSH,(5)Lys,(6)Phe,(7)Thr,(8)Met,(9)Val,(10)His,(11)Gly,(12)Ser,(13)Ala,(14)Tyr,(15)Arg,(16)Glu,(17)Ile,(18)KI,(19)KCl,(20)Na2SO4,(21)NaNO3,(22)KO2,(23)NaNO2,(24)H2O2等不同物质时与同型半胱氨酸响应选择性的条形图，纵坐标为荧光强度。黑色条形是472nm荧光强度，红色条形是584nm荧光强度。
图15为本发明的荧光探针(5.0×10-6mol/L)在pH为7.4的PBS缓冲溶液(10.0mM,VDMF/VPBS＝3/7)中存在(1)PBS,(2)Cys,(3)Hcy,(4)GSH,(5)Lys,(6)Phe,(7)Thr,(8)Met,(9)Val,(10)His,(11)Gly,(12)Ser,(13)Ala,(14)Tyr,(15)Arg,(16)Glu,(17)Ile,(18)KI,(19)KCl,(20)Na2SO4,(21)NaNO3,(22)KO2,(23)NaNO2,(24)H2O2等不同物质时与谷胱甘肽响应选择性的条形图，纵坐标为荧光强度。黑色条形是542nm荧光强度，红色条形是584nm荧光强度。
图16为本发明的荧光探针细胞适用性的毒性研究实验，横坐标为探针浓度，纵坐标为细胞存活率。
图17为本发明的荧光探针细胞内区分检测半胱氨酸/同型半胱氨酸和谷胱甘肽。
图18为本发明的荧光探针斑马鱼内区分检测半胱氨酸/同型半胱氨酸和谷胱甘肽。
具体实施实例
实施例1：探针分子的合成
化合物3的合成
将化合物1(65mg,0.2mmol)和化合物2(31mg,0.2mmol)以及1.2倍当量的三乙胺加入到3mL无水乙腈中，在氩气保护下室温搅拌2小时，将反应液旋干后通过柱层析纯化得化合物3，黄色固体(67.0mg,产率72％)。探针分子的结构表征如下：1H NMR(300MHz,CDCl3)δ8.01(d,J＝7.9Hz,2H),7.58(d,J＝9.3Hz,1H),7.46(d,J＝8.0Hz,2H),7.38(t,J＝7.3Hz,2H),7.24(s,1H),7.16(d,J＝7.6Hz,2H),6.54(s,2H),3.48(q,J＝7.1Hz,4H),1.23(t,J＝6.4Hz,6H).13C NMR(75MHz,CDCl3)δ170.7,162.8,157.8,156.0,151.9,150.6,147.4,141.1,131.1,129.4,128.8,126.2,121.5,119.7,109.7,107.4,97.4,45.0,12.4.HRMS(ESI)m/z:calcd.for C27H23NO6S[M+Na]+:512.1138；found 512.1143.
CPR的合成
将化合物3(49mg,0.1mmol)和化合物4(34mg,0.1mmol)以及EDCI(19mg,0.1mmol)加入到2mL无水二氯甲烷中，再加入DMAP(1mg,0.008mmol)作为催化剂，在氩气保护下室温反应12小时，将反应液旋干柱层析得到红色固体CPR(53mg,产率65％)。探针分子结构表征如下：1H NMR(300MHz,DMSO-d6)δ8.80(s,1H),7.91(d,J＝9.3,3.0Hz,2H),7.63(d,J＝9.2Hz,1H),7.55(d,J＝8.4Hz,2H),7.50-7.43(m,4H),7.40-7.22(m,3H),7.08(d,J＝6.3Hz,3H),6.92(s,1H),6.75(d,J＝9.3Hz,1H),6.66(d,J＝2.2Hz,1H),3.83-3.62(m,8H),3.46(d,J＝13.4,6.5Hz,8H),1.23(t,J＝6.9Hz,12H).13C NMR(75MHz,DMSO-d6)δ168.9,163.1,158.4,157.9,157.8,157.0,156.1,156.1,152.4,150.6,147.9,146.6,135.4,134.9,134.3,133.8,130.3,129.7,129.1,126.4,121.8,118.2,115.5,115.1,114.9,114.3,110.5,107.0,97.69,97.2,96.3,46.0,44.7,12.8.HRMS(ESI)m/z:calcd.forC48H47N4O6S[M]+:807.3211；found 807.3218.
实施例2：本发明的荧光探针的应用
荧光探针(5.0×10-6mol/L)在pH为7.4的PBS缓冲溶液(10.0mM,VDMF/VPBS＝3/7)中加入氨基酸(Lys,Phe,Thr,Met,Val,His,Gly,Ser,Ala,Tyr,Arg,Glu,Ile,)后没有引起探针本身发射峰的明显变化，而加入氨基酸(Cys,Hcy,GSH)后，则引起了荧光变化，说明该探针具有优良的选择性，并且能够区分半胱氨酸/同型半胱氨酸和谷胱甘肽。而细胞毒性实验，细胞和斑马鱼中成像实验说明探针具有好的生物适用前景。
Claims (1)
1.一种检测半胱氨酸/同型半胱氨酸和谷胱甘肽的新型比值型荧光探针，其结构式如下:
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| CN112110903A (zh) * | 2020-09-30 | 2020-12-22 | 河南凯普瑞生物技术有限公司 | 一种定量区分检测Cys、Hcy、GSH和H2S的荧光探针及其制备方法和应用 |
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| CN112209942B (zh) * | 2020-10-14 | 2023-10-31 | 中南大学 | 一种区分检测半胱氨酸、同型半胱氨酸和谷胱甘肽的荧光探针 |
| CN112939918A (zh) * | 2021-02-05 | 2021-06-11 | 山西大学 | 一种香豆素衍生物ctt及其合成方法和应用 |
| CN112939918B (zh) * | 2021-02-05 | 2022-07-19 | 山西大学 | 一种香豆素衍生物ctt及其合成方法和应用 |
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