CN110498786B - 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
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***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。小图是与同型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。小图是与谷胱甘肽浓度的线性关系,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。
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