CN114933555A - 一种检测小分子硫醇的近红外荧光探针及其制备方法 - Google Patents
一种检测小分子硫醇的近红外荧光探针及其制备方法 Download PDFInfo
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Abstract
本发明公开了一种检测小分子硫醇的近红外荧光探针及其制备方法,具体为:以异佛尔酮和丙二腈为原料,通过Knoevenagel缩合反应合成中间体TEM;将TEM和6‑羟基‑2‑萘甲醛进行反应,生成荧光团TZ‑OH;将荧光团TZ‑OH和2,4‑二硝基苯磺酰氯进行反应,生成荧光探针TZ4。探针TZ4能够对硫醇进行特异性识别响应,它的发射波长是631nm,斯托克斯位移为185nm。
Description
技术领域
本发明属于分析检测技术领域,具体涉及一种检测小分子硫醇的近红外荧光探针,还涉及该近红外荧光探针的制备方法。
背景技术
小分子生物硫醇是一类含有巯基的分子,主要包括Cys、Hcy、GSH三种,在许多生理和病理过程中扮演着不可忽视的角色,包括防止细胞氧化应激和维持细胞稳态方面。它们的浓度水平异常会引发相关疾病,如心血管病、癌症、阿尔兹海默症、肝损伤和骨质疏松等。近年来许多科学研究者以全花菁、香豆素、罗丹明、四苯乙烯、萘酰亚胺、氟硼吡咯荧光团结构为母核设计了对硫醇响应的荧光探针,均达到了很好的效果。因此,生物体内Cys/Hcy/GSH的检测,对于理解疾病的产生和发展将产生积极的、有意义的影响。
发明内容
本发明的目的在于提供一种检测小分子硫醇的近红外荧光探针,该荧光探针分子合成简单,对小分子硫醇能进行特异性识别响应。
本发明所采用的技术方案是,一种检测小分子硫醇的近红外荧光探针,该荧光探针具有如下式(Ⅰ)所示的结构式:
本发明所采用的另一技术方案是,一种检测小分子硫醇的近红外荧光探针的制备方法,具体按照以下步骤实施:
步骤1,以异佛尔酮和丙二腈为原料,通过Knoevenagel缩合反应合成中间体TEM;
步骤2,将TEM和6-羟基-2-萘甲醛进行反应,生成荧光团TZ-OH;
步骤3,将荧光团TZ-OH和2,4-二硝基苯磺酰氯进行反应,生成荧光探针TZ4。
本发明的特点还在于,
步骤1中,具体为:
将无水乙醇、异氟尔酮、丙二腈以及催化剂哌啶混合,在60℃的条件下,持续加热12h;之后冷却至室温,用旋转蒸发仪旋干溶剂,进行柱层析分离,洗脱剂为体积比为3:1的二氯甲烷和正己烷,得到中间体TEM;无水乙醇、异氟尔酮、丙二腈以及哌啶的质量比为7105:100:209:1。
步骤2中,具体为:
将无水乙醇、TEM、6-羟基-2-萘甲醛混合均匀,加入哌啶,在85℃条件下冷凝回流反应11-12h,再通过柱层析分离,得到橙黄色产物,即为荧光团TZ-OH;无水乙醇、TEM、6-羟基-2-萘甲醛和哌啶的质量比为779446:10909:9090:1。
步骤3中,具体为:
将无水二氯甲烷、TZ-OH混合,滴加三乙胺,室温搅拌15min后,将2,4-二硝基苯磺酰氯溶于二氯甲烷溶液中,逐滴滴入反应液中,并在室温条件下反应15-16h,待反应完成后,通过柱层析分离得到橙黄色产物,洗脱剂为体积比为3:1的二氯甲烷和石油醚,即可得到近红外荧光探针TZ4;二氯甲烷、TZ-OH、三乙胺、2,4-二硝基苯磺酰氯、二氯甲烷的质量比为1065:1:2:2:106。
本发明的有益效果是:本发明的荧光探针,以异氟尔酮为荧光团母体设计合成了探针TZ4,探针TZ4能够对硫醇进行特异性识别响应,它的发射波长是631nm,斯托克斯位移为185nm。在DMSO比H2O=1:1的溶剂条件下,当三个硫醇浓度在0~100μmol/L时,随着硫醇浓度的增加,探针TZ4的荧光强度呈线性增强,在30min内荧光强度达到饱和不再增强,且选择性较好,不受其它氨基酸分子的干扰。
附图说明
图1本发明方法制备的荧光探针检测小分子硫醇的原理图;
图2是DMSO:H2O(5:5,V:V)溶液中探针分子TZ1(10μmol/L)在加入三种硫醇前后的荧光强度变化图;
图3是DMSO:H2O(5:5,V:V)溶液中探针分子TZ2(10μmol/L)在加入三种硫醇前后的荧光强度变化图;
图4是DMSO:H2O(5:5,V:V)溶液中探针分子TZ3(10μmol/L)在加入三种硫醇前后的荧光强度变化图;
图5是DMSO:H2O(5:5,V:V)溶液中探针分子TZ4(10μmol/L)在加入三种硫醇前后的荧光强度变化图;
图6是不同有机溶剂与水(5:5,V:V)混合溶液中探针分子TZ4(10μmol/L)荧光发射光谱图;
图7是不同有机溶剂与水(5:5,V:V)混合溶液中探针分子TZ4(10μmol/L)与Cys响应的荧光发射光谱图;
图8是DMSO与H2O不同体积比溶液中探针分子TZ4(10μmol/L)与Cys响应的荧光发射光谱图;
图9是DMSO:H2O(5:5,V:V)溶液中探针分子TZ4(10μmol/L)在不同pH下与Cys响应荧光变化图;
图10是DMSO:H2O(5:5,V:V)溶液中探针分子TZ4(10μmol/L)对生物硫醇选择性响应的荧光发射光谱图;
图11是DMSO:H2O(5:5,V:V)溶液中探针分子TZ4(10μmol/L)与Cys及其他不同种氨基酸的竞争响应的荧光强度柱状图;
图12是DMSO:H2O(5:5,V:V)溶液中探针分子TZ4(10μmol/L)与Hcy及其他不同种氨基酸的竞争响应的荧光强度柱状图;
图13是DMSO:H2O(5:5,V:V)溶液中探针分子TZ4(10μmol/L)与GSH及其他不同种氨基酸的竞争响应的荧光强度柱状图;
图14是DMSO:H2O(5:5,V:V)溶液中探针TZ4与不同浓度Cys响应荧光发射光谱图;
图15是探针响应前后荧光强度与Cys浓度的线性拟合图;
图16是DMSO:H2O(5:5,V:V)溶液中探针TZ4与不同浓度Cys响应紫外吸收光谱图;
图17是DMSO:H2O(5:5,V:V)溶液中探针TZ4与不同浓度Hcy响应荧光发射光谱图;
图18是探针TZ4响应前后荧光强度与Hcy浓度的线性拟合图;
图19是DMSO:H2O(5:5,V:V)溶液中探针TZ4与不同浓度Hcy响应紫外吸收光谱图;
图20是DMSO:H2O(5:5,V:V)溶液中探针TZ4与不同浓度GSH响应荧光发射光谱图;
图21是探针TZ4响应前后荧光强度与GSH浓度的线性拟合图;
图22是DMSO:H2O(5:5,V:V)溶液中探针TZ4与不同浓度GSH响应紫外吸收光谱图。
具体实施方式
下面结合具体实施方式和附图对本发明进行详细说明。
本发明一种检测小分子硫醇的近红外荧光探针,荧光探针具有如下式(Ⅰ)所示的结构式:
本发明一种检测小分子硫醇的近红外荧光探针的制备方法,具体按照以下步骤实施:
步骤1,以异佛尔酮和丙二腈为原料,通过Knoevenagel缩合反应合成如下式(Ⅱ)所示的中间体TEM;
具体为:将无水乙醇、异氟尔酮、丙二腈以及催化剂哌啶混合,在60℃的条件下,持续加热12h;之后冷却至室温,用旋转蒸发仪旋干溶剂,进行柱层析分离,洗脱剂为体积比为3:1的二氯甲烷和正己烷,得到中间体TEM;
无水乙醇、异氟尔酮、丙二腈以及哌啶的质量比为7105:100:209:1;
步骤2,将TEM和6-羟基-2-萘甲醛进行反应,生成荧光团TZ-OH,如式(Ⅲ)所示;
具体为:将无水乙醇、TEM、6-羟基-2-萘甲醛混合均匀,加入哌啶,在85℃条件下冷凝回流反应11-12h,再通过柱层析分离,得到橙黄色产物,即为荧光团TZ-OH;
无水乙醇、TEM、6-羟基-2-萘甲醛和哌啶的质量比为779446:10909:9090:1;
步骤3,将荧光团TZ-OH和2,4-二硝基苯磺酰氯进行反应,生成荧光探针TZ4,如式(Ⅳ)所示;
具体为:将无水二氯甲烷、TZ-OH混合,滴加三乙胺,室温搅拌15min后,将2,4-二硝基苯磺酰氯溶于少量的二氯甲烷溶液中,逐滴滴入反应液中,并在室温条件下反应15-16h,待反应完成后,通过柱层析分离得到橙黄色产物,洗脱剂为体积比为3:1的二氯甲烷和石油醚,即可得到近红外荧光探针TZ4;
二氯甲烷、TZ-OH、三乙胺、2,4-二硝基苯磺酰氯、二氯甲烷的质量比为1065:1:2:2:106;
本发明方法制备的荧光探针检测小分子硫醇的原理如图1所示,在DMSO:H2O(5:5,V:V)混合溶液中,探针溶液本身几乎不发射荧光信号。当向溶液中加入小分子硫醇分子后,在446nm激发波长,630nm处荧光强度表现出明显的提升,呈现出荧光信号的“开-关”响应机制。并且,“裸眼”观察下溶液颜色由黄色转变为深蓝色。
实施例
本发明一种检测小分子硫醇的近红外荧光探针的制备方法,具体按照以下步骤实施:
步骤1,以异佛尔酮和丙二腈为原料,通过Knoevenagel缩合反应合成中间体TEM;
具体为:用量筒量取150mL无水乙醇于200mL的圆底烧瓶中,加入异氟尔酮(5g,36.18mmol)和丙二腈(5g,75.70mmol)以及催化剂哌啶(112mg,0.362mmol),在60℃的条件下,持续加热约12h;之后冷却至室温,用旋转蒸发仪旋干溶剂,进行柱层析分离;
步骤2,将TEM和6-羟基-2-萘甲醛进行反应生成荧光团TZ-OH;
具体为:用量筒量取10.0mL无水乙醇于50mL圆底烧瓶中,加入TEM(0.447g,2.4mmmol),6-羟基-2-萘甲醛(0.344g,2.0mmol),充分搅拌,加入哌啶21.9μL,将反应加热到85℃,冷凝回流反应过夜。通过柱层析分离,得到橙黄色产物,;
步骤3,将荧光团TZ-OH和2,4-二硝基苯磺酰氯反应,生成荧光探针TZ4;
具体为:用量筒量取20mL的无水二氯甲烷于100mL圆底烧瓶中,加入TZ-OH(0.1g,0.293mmmol),滴加0.1mL三乙胺,室温搅拌15min后,将2,4-二硝基苯磺酰氯(0.586mmol)溶于少量的二氯甲烷溶液中,逐滴滴入反应液中,室温反应过夜。待反应完成后,通过柱层析分离得到橙黄色产物,洗脱剂为二氯甲烷比石油醚=3:1,得到探针TZ4;
采用上述相同的方法,将2,4-二硝基苯磺酰氯分别改为间硝基苯磺酰氯、邻硝基苯磺酰氯、对硝基苯磺酰氯制备探针TZ1、TZ2和TZ3,如式(V)、(VI)、(VII)所示;
其中,产物表征如下:
TZ1:1H NMR(400MHz,DMSO-d6)δ8.65(ddt,J=4.7,2.4,1.2Hz,1H),8.54(dd,J=3.8,1.8Hz,1H),8.29(ddt,J=4.3,2.1,1.1Hz,1H),8.18(d,J=6.7Hz,1H),7.97(dd,J=4.7,3.2Hz,1H),7.93(dd,J=7.8,3.5Hz,1H),7.72(t,J=3.7Hz,1H),7.63–7.53(m,1H),7.43(d,J=16.2Hz,1H),7.31–7.22(m,1H),6.94(d,J=6.2Hz,1H),2.64(s,1H),2.59(s,1H),1.03(s,1H).13C NMR(100MHz,DMSO-d6)δ170.8,156.0,148.7,147.4,137.4,136.1,135.2,134.7,133.9,132.5,132.3,131.4,131.3,130.2,129.1,128.9,125.8,123.9,123.5,122.1,120.5,114.3,113.5,77.4,42.8,38.7,32.2,27.9.HRMS:Calcd.forC29H23N3O5S[M-H]-:524.1275;Found:524.1290.
TZ2:1H NMR(400MHz,DMSO-d6)δ8.29–8.17(m,1H),8.08(t,J=7.8Hz,1H),8.04–7.94(m,2H),7.86(t,J=7.7Hz,1H),7.78(s,1H),7.60(d,J=16.1Hz,1H),7.44(d,J=16.1Hz,1H),7.31(dd,J=9.0,1.7Hz,1H),6.95(s,1H),2.61(d,J=18.8Hz,2H),1.03(s,3H).13C NMR(100MHz,DMSO-d6)δ170.9,156.1,148.5,147.4,137.7,137.4,135.2,134.6,133.9,133.7,132.4,131.5,131.3,129.2,128.9,126.6,126.1,125.9,123.9,121.8,120.3,114.4,112.1,55.5,38.7,32.3,27.9.HRMS:Calcd.for C29H23N3O5S[M-H]-:524.1275;Found:524.1222.
TZ3:1H NMR(400MHz,DMSO-d6)δ8.45(d,J=8.5Hz,1H),8.26–8.13(m,1H),7.97(dd,J=15.9,8.9Hz,1H),7.69(s,1H),7.59(d,J=16.1Hz,1H),7.43(d,J=16.2Hz,1H),7.22(d,J=9.1Hz,1H),6.95(s,1H),2.59(t,J=18.8Hz,1H),1.03(s,1H).13C NMR(100MHz,DMSO-d6)δ170.8,156.0,152.7,151.6,139.9,135.2,132.3,131.4,131.3,130.6,129.1,128.9,125.8,125.6,123.9,121.9,120.3,114.3,113.5,77.3,42.8,38.7,32.2,27.9.HRMS:Calcd.for C29H23N3O5S[M-H]-:524.1275;Found:524.1298.
TZ4:1H NMR(400MHz,DMSO-d6)δ9.15(s,1H),8.60(t,J=11.6Hz,1H),8.26(t,J=8.5Hz,1H),8.22(s,1H),7.99(t,J=7.3Hz,4H),7.79(s,1H),7.60(d,J=16.1Hz,1H),7.44(d,J=16.2Hz,1H),7.33(t,J=10.2Hz,1H),6.95(s,1H),2.61(d,J=18.8Hz,5H),1.03(s,7H).13C NMR(100MHz,DMSO-d6)δ170.8,155.9,152.1,148.7,147.2,137.3,135.4,134.2,133.9,132.5,131.7,131.4,131.3,129.2,128.9,128.0,125.9,123.9,121.7,120.3,114.3,113.5,77.4,42.8,38.7,32.2,27.98.HRMS:Calcd.for C29H22N4O7S[M-H]-:569.1125;Found:569.1114.
探针测试溶剂筛选
为了研究探针TZ1-TZ4是否对硫醇具有识别响应,如图2-5所示,选择了在有机溶剂DMSO:水=1:1的条件下,分别测试了四个探针TZ1-TZ4在加入三种硫醇前后的荧光强度变化值,发现只有探针TZ4有较好的识别响应且荧光发射波长为631nm接近近红外区;探针TZ1-TZ3对硫醇几乎没有识别响应并且荧光发射波长较短为570nm,这也与报道的2,4-二硝基是较好的离去基团,容易发生亲核反应相应证。
在实施例的基础之上,测试了探针(10μmol/L)在不同有机溶剂与水混合溶液中的荧光发射性能。如图6所示,通过对不同有机溶剂中荧光发射强度进行比较。发现在选择有机溶剂为DMSO时,探针自身具有较小的背景荧光发射。进一步的,如图7所示,在测试了探针TZ4(10μmol/L)在不同有机溶剂与水混合溶液中与Cys(5.0equiv.)响应之后的荧光发射性能。当有机溶剂为DMSO时表现出明显的荧光信号的增长。
对于探针TZ4(10μmol/L)在混合溶剂DMSO和水在不同体积比存在下对Cys(5.0equiv.)响应情况进行了测试,如图8所示,发现当探针TZ4在DMSO:H2O比例为1:1混合溶剂体系下具有较好的荧光发射。
探针适用pH范围测试
在DMSO:H2O(5:5,V:V)溶液中对不同pH(1~11)范围内探针对Cys响应的影响进行测试,如图9所示。发现:探针本身在强碱性溶剂中其荧光会出现明显的增长,但在响应之后荧光强度值在中性和碱性溶液中会趋向稳定的状态。所以,该探针测试适用于生理条件。
探针的选择性测试
在DMSO:H2O(5:5,V:V)溶液中,比较了在探针TZ4(10μmol/L)中加入各种氨基酸的荧光发射光谱,其中包括:100μmol/L,Arg,Val,Thr,Lys,Ile,Met,His,Phe,Ser,Tyr,Leu,Ala,Hcy,GSH,Cys。如图10所示,除三种硫醇分析物外,在激发波长为446nm下,其它氨基酸并未对荧光强度产生明显的增长。结果表明,探针TZ对该三种生物硫醇表现出了较好的选择性检测效果。
探针的抗干扰能力测试
在10μmol/L探针TZ4中加入上述实例中各种氨基酸300μmol/L形成干扰氛围。之后,加入分析物100μmol/L Cys、Hcy和GSH后测试其荧光光谱的变化情况。如图11、12和13所示,分别对其它各种氨基酸的荧光响应和加入Cys、Hcy和GSH(100μmol/L,)后荧光强度进行了比较。激发波长为446nm下其荧光发射强度在630nm处均表现出明显的改变,结果表明,探针TZ4在其它各种氨基酸的存在下对三种生物硫醇的识别几乎不受影响。
探针的滴定实验
如图14所示,在446nm波长激发下,探针TZ4溶液本身不没有明显的荧光发射信号。然而,当存在Cys时,溶液颜色发生了转变。并且,其在630nm处荧光强度数值表现出了极大的增加。此外,当分析物达到100μmol/L时,其荧光强度提升了约5倍。同时,从紫外可见吸收光谱图16可以得到,在446nm处有相似于荧光增长的趋势。如图17、19、20和22所示,探针对Hcy和GSH表现出类似于Cys的现象。
进一步的,如图15所示,探针TZ4对Cys响应与Cys的浓度在0~50μmol/L表现出较好的线性相关。探针TZ4的回归方程为y=11.40+28.97x(R2=0.9925)。如图18和21所示,探针TZ对Hcy(GSH)响应与GSH(Hcy)的浓度在0~70μmol/L表现出较好的线性相关,其回归方程为y=535.72+19.15x(R2=0.9922),y=187.58+26.30x(R2=0.9910)。
Claims (5)
2.一种如权利要求1所述的检测小分子硫醇的近红外荧光探针的制备方法,其特征在于,具体按照以下步骤实施:
步骤1,以异佛尔酮和丙二腈为原料,通过Knoevenagel缩合反应合成中间体TEM;
步骤2,将TEM和6-羟基-2-萘甲醛进行反应,生成荧光团TZ-OH;
步骤3,将荧光团TZ-OH和2,4-二硝基苯磺酰氯进行反应,生成荧光探针TZ4。
3.如权利要求2所述的一种检测小分子硫醇的近红外荧光探针的制备方法,其特征在于,所述步骤1中,具体为:
将无水乙醇、异氟尔酮、丙二腈以及催化剂哌啶混合,在60℃的条件下,持续加热12h;之后冷却至室温,用旋转蒸发仪旋干溶剂,进行柱层析分离,洗脱剂为体积比为3:1的二氯甲烷和正己烷,得到中间体TEM;无水乙醇、异氟尔酮、丙二腈以及哌啶的质量比为7105:100:209:1。
4.如权利要求2所述的一种检测小分子硫醇的近红外荧光探针的制备方法,其特征在于,所述步骤2中,具体为:
将无水乙醇、TEM、6-羟基-2-萘甲醛混合均匀,加入哌啶,在85℃条件下冷凝回流反应11-12h,再通过柱层析分离,得到橙黄色产物,即为荧光团TZ-OH;无水乙醇、TEM、6-羟基-2-萘甲醛和哌啶的质量比为779446:10909:9090:1。
5.如权利要求2所述的一种检测小分子硫醇的近红外荧光探针的制备方法,其特征在于,所述步骤3中,具体为:
将无水二氯甲烷、TZ-OH混合,滴加三乙胺,室温搅拌15min后,将2,4-二硝基苯磺酰氯溶于二氯甲烷溶液中,逐滴滴入反应液中,并在室温条件下反应15-16h,待反应完成后,通过柱层析分离得到橙黄色产物,洗脱剂为体积比为3:1的二氯甲烷和石油醚,即可得到近红外荧光探针TZ4;二氯甲烷、TZ-OH、三乙胺、2,4-二硝基苯磺酰氯、二氯甲烷的质量比为1065:1:2:2:106。
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