CN112110946A - 基于氟硼骨架的大Stokes位移的荧光探针TQBF-NBD及其制备方法和应用 - Google Patents
基于氟硼骨架的大Stokes位移的荧光探针TQBF-NBD及其制备方法和应用 Download PDFInfo
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Abstract
Description
技术领域
本发明属于荧光探针领域,涉及一种基于氟硼骨架的荧光探针,特别是指基于氟硼骨架的大Stokes位移的荧光探针TQBF-NBD及其制备方法和应用。
背景技术
生物硫醇是生物体内含巯基化合物的总称,广泛地存在于生物体内,最具代表性的生物硫醇为三种巯基化合物:半胱氨酸 (Cys)、同型半胱氨酸 (Hcy) 和谷胱甘肽(GSH)。巯基化合物是生物体酶发挥作用后和生理活动中的重要信号分子,同时巯基化合物也可以调节细胞的正常氧化还原状态,在生物体的生理活动中具有重要的功能。生物体内巯基化合物的浓度异常与许多种疾病有关,Cys的缺乏与生长缓慢、肝损伤、毛发褪色、皮肤病、昏睡以及水肿等疾病有关;血浆中Hcy浓度过高与心血管疾病、阿兹海默症和骨质疏松症有关;GSH能够保护机体免受氧化及自由基介导的氧应激损伤,其浓度与白血病、癌症及艾滋病等有关。因此,检测生物体内巯基化合物浓度对研究细胞功能和进行疾病早期诊断具有重要的意义。
关于生物硫醇的研究中,本课题组的前期研究专利CN201911011150.1,发现了一类红光发射且具有大Stokes位移的不对称氟硼染料,其发射波长大于600nm,且具有120nm的Stokes位移,但是如何能应用于检测生物硫醇,仍是需要研究探索的问题。
而专利CN201811182560.8公开了一种新型生物硫醇荧光探针及其制备方法和应用,该生物硫醇荧光探针是以4-[7-(二乙氨基)-2,2-二氟苯并[e][1,3,2]恶唑硼烷-3-基]苯酚为荧光团、以2,4-二硝基苯磺酰基为识别单元,该探针的最低检测限为:半胱氨酸为44.7nM,谷胱甘肽为65.4nM,同型半胱氨酸为106.0nM。该探针在检测生物硫醇时,所述混合溶液发出蓝色荧光且均在474nm处,不能很好的将Cys/Hcy与GSH区分开。本课题组前期也对将Cys/Hcy与GSH区分进行研究,制备的探针发出的是弱的蓝绿光,荧光强度荧光波长较短,对生物组织损失较大,不利于生物应用。
相对于其它传统的检测技术,荧光分析法具有操作简单、选择性好、灵敏度高等优点,尤其可以用于细胞及生物活体成像,因此近年来利用荧光探针来检测离子及生物分子的研究受到了广泛的关注。
发明内容
本发明提出一种基于氟硼骨架的大Stokes位移的荧光探针TQBF-NBD及其制备方法和应用,解决了现有生物硫醇检测灵敏度低、不能将Cys/Hcy与GSH快速、明显的区分开检测的技术问题 。
本发明的技术方案是这样实现的:
基于氟硼骨架的大Stokes位移的荧光探针TQBF-NBD,所述荧光探针TQBF-NBD具有如下结构式:
上述的荧光探针TQBF-NBD的制备方法,步骤如下:
(a)将2,4-二乙基-1,2,3,4-四氢喹喔啉水杨醛、对氨基苯酚和TsOH溶于乙醇中,在氩气保护下加热回流2小时,停止反应后,将反应液冷却至室温,旋干得到暗棕色油状液体粗产物,技术路线为:
(b)将步骤(a)制得的暗棕色油状液体粗产物溶于1,2-二氯乙烷中,再加入DIEA和BF3•OEt2,在氩气保护下80 ℃反应4 h,停止反应后,向反应液中加入1,2-二氯乙烷,再将反应液倒入NaHCO3溶液中,分出有机相,水相用二氯甲烷萃取3次,合并有机相,用无水硫酸钠干燥,减压旋干得到油状液体,再经柱层析分离得到化合物TQBF-OH,技术路线为:;
(c)将步骤(b)制得的产物TQBF-OH、NBD-Cl和无水三乙胺溶于无水乙腈中,室温下反应过夜,有棕色固体析出,将其过滤,用无水乙醇洗涤2次后,经柱层析分离得到产物TQBF-NBD,技术路线为:
所述步骤(a)中2,4-二乙基-1,2,3,4-四氢喹喔啉水杨醛、对氨基苯酚和TsOH的物质的量比为1:(1-1.2):0.05。
所述步骤(b)中粗产物、DIEA和BF3•OEt2的体积比为(2-2.5):1:1。
所述步骤(c)中每10μL无水三乙胺中添加0.1mmol 的化合物TQBF-OH和0.11 mmol的NBD-Cl。
所述步骤(b)、(c)中柱层析采用的洗脱剂为V二氯甲烷/V乙醇 =100:(0-1)
上述的荧光探针TQBF-NBD在制备定量检测生物硫醇的试剂中的应用,室温下,将荧光探针TQBF-NBD溶解在pH=7.4的PBS缓冲溶液中配制成10 μM的检测试剂,加入待测样品,观察荧光变化,根据荧光强度随硫醇加入量的线性变化规律及检测线计算公式:D= 3σ/k (D为检测线,σ为标准偏差,k为斜率),定量检测生物硫醇。
所述PBS缓冲溶液的浓度为0.01M,PBS缓冲溶液中含有体积分数20%的乙腈。
所述应用以在620nm处监测到红光确定待测样品含有GSH、以分别在620nm处和550nm处监测到强烈的红色荧光和绿色荧光确定待测样品中含有Cys/Hcy。
本发明的荧光探针的响应机理如下:探针TQBF-NBD以NBD为识别基团,由于荧光团与NBD之间产生光诱导电子转移 (PET) 过程,荧光被猝灭,所以探针TQBF-NBD无荧光。如下图所示,巯基化合物与探针发生亲核取代反应,NBD部分从探针分子上脱离并释放出具有红色荧光的TQBF-OH和无荧光的NBD-S-Hcy/Cys/GSH。随后,中间产物NBD-S-Hcy/Cys能够发生Smiles重排反应,最终生成有绿色荧光的化合物NBD-N-Hcy/Cys。探针与GSH反应时,生成的产物NBD-S-GSH,分子内的重排需要经过动力学不稳定的十元环过渡态,重排反应不能发生,因此无绿色荧光信号。因此,探针TQBF-NBD与Cys/Hcy反应时,产生红色荧光和绿色荧光两种混合信号,与GSH反应时,探针溶液仅发射出单一红色荧光荧光信号,所以探针可以成功地将Cys/Hcy与GSH区分开。探针分子的响应过程见说明书附图6。
本发明具有以下有益效果:
1、本发明的荧光探针本身没有荧光,与GSH响应后发射强烈的红色荧光,最大发射峰在620nm处;与Cys/Hcy响应后发射强烈的红色荧光和绿色荧光,最大发射峰分别在620nm处和550nm处。
2、如附图4所示,从620 nm处的荧光强度与Cys、Hcy和GSH浓度变化可以得出,荧光强度与浓度在0-100 μM(10eqv)之间有很好的线性关系,线性相关系数分别为Cys的线性方程为:y=118.1195+12.2614x,R = 0.9919;Hcy的线性方程为:y=133.5+6.922x,R =0.9964;GSH的线性方程为:y=98.695+10.868x,R = 0.993。根据信噪比S/N = 3,可以算出探针QCBF-NBD对Cys、Hcy和GSH的检测限分别为0.202μmol/L、0.359μmol/L和 0.228 μmol/L。而且可以实现快速、定量检测生物硫醇。
3、本申请的荧光探针TQBF-NBD与Cys/Hcy/GSH响应后得到红色荧光产物TQBF-OH,测得其最大吸收波长为445nm, 最大发射波长为620nm,Stokes位移为175nm,具有大Stokes位移。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1为本发明的荧光探针在氘代氯仿中核磁共振氢谱,横坐标化学位移,纵坐标为强度。
图2为本发明的荧光探针在氘代氯仿中核磁共振碳谱,横坐标化学位移,纵坐标为强度。
图3为本发明的荧光探针(10 μM)在PBS缓冲液 (0.01 M,pH = 7.4,20% CH3CN)中相关氨基酸的响应(Ala、Glu、Asp、Gly、His、Iso、Arg、Leu、Thr、Trp、Met、Lys、Phe、Ser、Val、Tyr 、Hcy、Cys和GSH,浓度为100 µM),横坐标为波长,纵坐标为荧光强度。
图4为探针QCBF-NBD对Cys、Hcy和GSH的灵敏度检测图。
图5为探针QCBF-NBD检测Cys/Hcy和GSH的最佳反应时间测试图。
图6本申请荧光探针的的响应过程机理图。
具体实施方式
下面将结合本发明实施例,对本发明的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有付出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例1
基于氟硼骨架的大Stokes位移的荧光探针TQBF-NBD的制备方法,技术路线如下:
制备步骤如下:
(a)将2,4-二乙基-1,2,3,4-四氢喹喔啉水杨醛(234mg, 1.0 mmol) ,对氨基苯酚(121mg, 1.2 mmol) 和催化量的TsOH溶于5 mL乙醇中,在氩气保护下加热回流2小时,停止反应,将反应液冷却至室温,旋干得到暗棕色油状液体粗产物;
(b)将200μL粗产物溶于10mL1,2-二氯乙烷中,再加入100μL DIEA和100μLBF3•OEt2,在氩气保护下80 ℃反应4 h,停止反应,向反应液加入30mL1,2-二氯乙烷,将其倒入100mLNaHCO3溶液中,分出有机相,水相用二氯甲烷萃取3次 (3× 30 mL),合并有机相,用无水硫酸钠干燥,减压旋干得到油状液体,再经柱层析 (洗脱剂:V二氯甲烷/V乙醇 = 100/0至100/1) 分离得到产物TQBF-OH,90mg;产率25.6%;
(c)将化合物TQBF-OH (37mg,0.1mmol),NBD-Cl(22mg,0.11 mmol)和10μL无水三乙胺溶于2 mL无水乙腈中,在室温下反应过夜,有大量棕色固体析出,将其过滤,用无水乙醇洗涤2次后,经柱层析 (洗脱剂:V二氯甲烷/V乙醇= 100/0至100/1) 分离得到产物TQBF-NBD,30mg,产率56%。
表征数据如下:1H NMR (400 MHz, DMSO-d 6 ) δ 8.69 (d, J = 8.3 Hz, 1H),8.59 (s, 1H), 7.72 (d, J = 8.3 Hz, 2H), 7.53 (d, J = 8.3 Hz, 2H), 6.79 (d, J= 8.3 Hz, 1H), 6.66 (s, 1H), 6.14 (s, 1H), 3.56 (d, J = 17.7 Hz, 2H), 3.50(d, J = 6.9 Hz, 2H), 3.25 (d, J = 6.8 Hz, 2H), 3.14 (s, 2H), 1.23-1.03 (m,6H). 13C NMR (100 MHz, DMSO-d 6 ) δ 158.1 (s), 157.4 (s), 153.59 (s), 151.6-151.3 (m), 148.1 (s), 145.8 (s), 144.9 (s), 142.0 (s), 136.0 (s), 130.9 (s),129.9 (s), 125.1 (s), 122.1 (s), 110.4 (d, J = 17.0 Hz), 106.8-106.3 (m),96.0 (s), 48.1 (s), 46.1 (s), 45.1 (s), 44.3 (s), 11.1 (s), 9.8 (s).
实施例2
基于氟硼骨架的大Stokes位移的荧光探针TQBF-NBD的制备方法,技术路线如下:
制备步骤如下:
(a)将2,4-二乙基-1,2,3,4-四氢喹喔啉水杨醛(234mg, 1.0 mmol) ,1mmol的对氨基苯酚和0.05mmol的TsOH溶于5 mL乙醇中,在氩气保护下加热回流2小时,停止反应,将反应液冷却至室温,旋干得到暗棕色油状液体粗产物;
(b)将250μL粗产物溶于10mL1,2-二氯乙烷中,再加入100μL DIEA和100μLBF3•OEt2,在氩气保护下80 ℃反应4 h,停止反应,向反应液加入30mL1,2-二氯乙烷,将其倒入100mLNaHCO3溶液中,分出有机相,水相用二氯甲烷萃取3次 (3× 30 mL),合并有机相,用无水硫酸钠干燥,减压旋干得到油状液体,再经柱层析 (洗脱剂:V二氯甲烷/V乙醇 = 100/0至100/1) 分离得到产物TQBF-OH,90mg;产率25.6%;
(c)将化合物TQBF-OH (37mg,0.1mmol),NBD-Cl(22mg,0.11 mmol)和10μL无水三乙胺溶于2 mL无水乙腈中,在室温下反应过夜,有大量棕色固体析出,将其过滤,用无水乙醇洗涤2次后,经柱层析 (洗脱剂:V二氯甲烷/V乙醇= 100/0至100/1) 分离得到产物TQBF-NBD,30mg,产率56%。
实施例3
基于氟硼骨架的大Stokes位移的荧光探针TQBF-NBD的制备方法,技术路线如下:
制备步骤如下:
制备步骤如下:
(a)将2,4-二乙基-1,2,3,4-四氢喹喔啉水杨醛(234mg, 1.0 mmol) ,1.1 mmol的对氨基苯酚和0.05 mmol的TsOH溶于5 mL乙醇中,在氩气保护下加热回流2小时,停止反应,将反应液冷却至室温,旋干得到暗棕色油状液体粗产物;
(b)将230μL粗产物溶于10mL1,2-二氯乙烷中,再加入100μL DIEA和100μLBF3•OEt2,在氩气保护下80 ℃反应4 h,停止反应,向反应液加入30mL1,2-二氯乙烷,将其倒入100mLNaHCO3溶液中,分出有机相,水相用二氯甲烷萃取3次 (3× 30 mL),合并有机相,用无水硫酸钠干燥,减压旋干得到油状液体,再经柱层析 (洗脱剂:V二氯甲烷/V乙醇 = 100/0至100/1) 分离得到产物TQBF-OH,90mg;产率25.6%;
(c)将化合物TQBF-OH (37mg,0.1mmol),NBD-Cl(22mg,0.11 mmol)和10μL无水三乙胺溶于2 mL无水乙腈中,在室温下反应过夜,有大量棕色固体析出,将其过滤,用无水乙醇洗涤2次后,经柱层析 (洗脱剂:V二氯甲烷/V乙醇= 100/0至100/1) 分离得到产物TQBF-NBD,30mg,产率56%。
实施效果例
荧光探针检测生物硫醇研究:将实施例1制备的探针溶于PBS (0.01 M, pH 7.4, 20%CH3CN)缓冲溶液中,配制成10 μM的溶液,加入三种生物硫醇可以迅速观测到620nm和550nm处的荧光变化。其检测时间较短,可以实现定性、定量检测生物硫醇:
1、探针的选择性实验:
选择性试验中用到的氨基酸包括Ala、Arg、Asp、Glu、Gly、His、Iso、Leu、Lys、Met、Phe、Ser、Thr、Trp、Tyr、Val、Cys、Hcy和GSH。在乙腈:10 mM PBS = 2:8,v:v,pH = 7.4的体系中,10 μM的探针与10倍当量的各种氨基酸反应20分钟后。如附图3所示,该探针分子在溶液中几乎没有荧光,当加入Cys、Hcy后,探针溶液在550 nm和610 nm出出现两个荧光峰,分别属于NBD-Cys、NBD-Hcy和荧光母体QCBF的;当加入GSH后,只在610 nm处出现荧光峰,属于QCBF荧光母体;而加入其它种类的氨基酸后,溶液荧光并没有荧光产生。因此,根据荧光信号的不同,可以成功地将Cys、Hcy、GSH与其它种类的氨基酸区分开来,并且可以根据信号的不同把Cys/Hcy和GSH区分开来。
2、探针的灵敏度实验
为了考察探针QCBF-NBD对Cys、Hcy和GSH的灵敏度,我们研究了QCBF-NBD随各浓度的Cys、Hcy和GSH的荧光变化情况。如附图4所示,随着Cys浓度的逐渐增加,550 nm和620 nm处的荧光强度也逐步上升,当Cys浓度到达200 μM(20 equiv.)时,荧光强度达到最大值,反应平衡;当加入Hcy溶液时,荧光强度的变化情况与Cys类似;随着GSH溶液浓度的增加,620 nm处荧光强度不断加强,在250 μM(25 equiv.)到达平衡,但是550 nm处的荧光相对于620 nm处只有小幅增加。从620 nm处的荧光强度与Cys、Hcy和GSH浓度变化可以得出,荧光强度与浓度在0-100 μM(10eqv)之间有很好的线性关系,线性相关系数分别为Cys的线性方程为:y=118.1195+12.2614x,R = 0.9919;Hcy的线性方程为:y=133.5+6.922x,R = 0.9964;GSH的线性方程为:y=98.695+10.868x,R = 0.993。根据信噪比S/N = 3,可以算出探针QCBF-NBD对Cys、Hcy和GSH的检测限分别为0.202μmol/L、0.359μmol/L和 0.228 μmol/L。实验结果表明,探针QCBF-NBD可以在一定浓度内定性和定量地检测Cys、Hcy和GSH,由检出限可知其灵敏度较高。
3、探针的响应时间实验
为了考察探针QCBF-NBD检测Cys/Hcy和GSH的最佳反应时间,提高检测的灵敏度,缩短检测时间,我们进行了探针的最佳时间的测试。实验以10 μM的探针溶液和10倍当量的三种生物硫醇(Cys、Hcy和GSH)反应时550 nm和620 nm处的荧光强度随时间的变化情况。如附图5所示,探针QCBF-NBD与生物硫醇的反应基本上在10分钟内已经反应完全,反应从最开始便可以检测出荧光强度的变化。Cys和GSH的反应速率明显快于Hcy,这可能是由于NBD重排引起的。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (9)
2.权利要求1所述的荧光探针TQBF-NBD的制备方法,其特征在于,步骤如下:
(a)将2,4-二乙基-1,2,3,4-四氢喹喔啉水杨醛、对氨基苯酚和TsOH溶于乙醇中,在氩气保护下加热回流2小时,停止反应后,将反应液冷却至室温,旋干得到暗棕色油状液体粗产物;
(b)将步骤(a)制得的暗棕色油状液体粗产物溶于1,2-二氯乙烷中,再加入DIEA和BF3•OEt2,在氩气保护下80 ℃反应4 h,停止反应后,向反应液中加入1,2-二氯乙烷,再将反应液倒入NaHCO3溶液中,分出有机相,水相用二氯甲烷萃取3次,合并有机相,用无水硫酸钠干燥,减压旋干得到油状液体,再经柱层析分离得到化合物TQBF-OH;
(c)将步骤(b)制得的产物TQBF-OH、NBD-Cl和无水三乙胺溶于无水乙腈中,室温下反应过夜,有棕色固体析出,将其过滤,用无水乙醇洗涤2次后,经柱层析分离得到产物TQBF-NBD。
3.根据权利要求2所述的荧光探针TQBF-NBD的制备方法,其特征在于:所述步骤(a)中2,4-二乙基-1,2,3,4-四氢喹喔啉水杨醛、对氨基苯酚和TsOH的物质的量比为1:(1-1.2):0.05。
4.根据权利要求2所述的荧光探针TQBF-NBD的制备方法,其特征在于:所述步骤(b)中粗产物、DIEA和BF3•OEt2的体积比为(2-2.5):1:1。
5.根据权利要求2所述的荧光探针TQBF-NBD的制备方法,其特征在于:所述步骤(c)中每10μL无水三乙胺中添加0.1mmol 的化合物TQBF-OH和0.11 mmol的NBD-Cl。
6.根据权利要求2所述的荧光探针TQBF-NBD的制备方法,其特征在于:所述步骤(b)、(c)中柱层析采用的洗脱剂为V二氯甲烷/V乙醇 =100:(0-1)。
7.权利要求1所述的荧光探针TQBF-NBD在制备定量检测生物硫醇的试剂中的应用,其特征在于:室温下,将荧光探针TQBF-NBD溶解在pH=7.4的PBS缓冲溶液中配制成10 μM的检测试剂,加入待测样品,观察荧光变化,根据荧光强度随硫醇加入量的线性变化规律及检测线计算公式:D= 3σ/k ,定量检测生物硫醇,其中D为检测线,σ为标准偏差,k为斜率。
8.根据权利要求7所述的应用,其特征在于:所述PBS缓冲溶液的浓度为0.01M,PBS缓冲溶液中含有体积分数20%的乙腈。
9.根据权利要求7所述的应用,其特征在于:所述应用以在620nm处监测到红光确定待测样品含有GSH、以分别在620nm处和550nm处监测到强烈的红色荧光和绿色荧光确定待测样品中含有Cys/Hcy。
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