CN111073634A - 基于硝基还原、硫氮转位的硝基还原酶荧光探针及其制备方法 - Google Patents
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Abstract
本发明属于化学分析技术领域,涉及小分子荧光探针,具体涉及基于硝基还原、硫氮转位的硝基还原酶荧光探针,本发明的荧光探针是一种氟硼二吡咯(BODIPY)结构类的硝基还原酶荧光探针,本发明公开了该类探针的制备应用及对硝基还原酶识别的新型反应机制。本发明的荧光探针光物理活性稳定,对硝基还原酶响应的灵敏度高,并可应用于缺氧肿瘤细胞的荧光成像检测。
Description
技术领域
本发明属于属于化学分析技术领域,涉及小分子荧光探针,具体涉及基于硝基还原、硫氮转位的硝基还原酶荧光探针及其制备方法及应用,尤其是一种氟硼二吡咯(BODIPY)结构类的硝基还原酶荧光探针其制备方法及应用。
背景技术
现有技术公开了缺氧与多种疾病如心脏缺血、中风、炎症疾病、肿瘤等有关,尤其在实体瘤中氧的含量不足4.4%,肿瘤的缺氧状态直接关系到肿瘤的发展、转移、耐药性和治疗的耐受性。
有研究表明组织或细胞在缺氧状态下硝基还原酶会过表达,硝基还原酶可作为衡量细胞缺氧状态的重要指标,因此对硝基还原酶的检测可以一定程度上反映生物体系的缺氧状态。
研究显示,荧光分析法具有高灵敏度、高选择性、高时空分辨率、操作简单并可实现对生物活性分子原位无损伤、实时快速的可视化成像等优点,已成为近年来的研究热点。目前,已有多种硝基还原酶荧光探针被陆续报道,但是现有技术的硝基还原酶探针仍然是基于多米诺分解反应或者硝基还原为氨基后的电子推拉效应所设计,尚不能满足有关研究及实际操作的需要。因此,研发新的监测肿瘤细胞和生物体缺氧状态的方法对于医学诊断和肿瘤研究具有重要意义。
基于现有技术的现状,本申请的发明人拟提供一种硝基还原酶探针,具体涉及硝基还原、硫氮转位的硝基还原酶荧光探针及其制备方法,该探针能对硝基还原酶实现高选择性和高灵敏度的检测,一定程度的满足有关研究及实际操作的需要。
发明内容
本发明的目的在于基于现有技术的现状,针对现有技术存在的问题,提供一种硝基还原酶探针,尤其涉及基于硝基还原、硫氮转位的硝基还原酶荧光探针,该探针能对硝基还原酶实现高选择性和高灵敏度的检测,一定程度的满足有关研究及实际操作的需要。
具体的,本发明的第一个目的是提供一种基于新的反应机制的硝基还原酶荧光探针。
本发明第二个目的提供所述硝基还原酶荧光探针的制备方法。
本发明进一步目的是提供所述荧光探针对硝基还原酶实现高选择性和高灵敏度的检测以及应用于缺氧肿瘤细胞的荧光成像研究。
本发明的目的通过下述技术方案施现:
一种基于硝基还原、硫氮转位的硝基还原酶荧光探针,其特征在于,硝基还原酶检测的识别基团为硝基处于邻、间、对位的苯硫醚,其结构为:
所述荧光基团为氟硼二吡咯类染料,其结构为:
本发明所述基于硝基还原、硫氮转位反应机制的硝基还原酶探针其结构为:
本发明提供所述硝基还原酶探针的合成路线为:
通过下述制备方法:将化合物0.5mmol A、1.5mmol二硫苏糖醇及1.5mmol三乙胺溶于5mL无水四氢呋喃中,室温下搅拌3小时,加入水和二氯甲烷萃取,有机相经无水硫酸钠干燥,浓缩的粗产品经快速柱层析(石油醚∶乙酸乙酯=10∶1)分离得化合物B;将化合物0.338mmol C和化合物0.667mmol B溶于3mL干燥二氯甲烷中,加入1.016mmol DMAP,氮气保护下室温搅拌5小时,反应液浓缩后经硅胶柱层析(石油醚∶乙酸乙酯∶二氯甲烷=20∶1∶1)分离得化合物D。
本发明所述基于新型反应机制的硝基还原酶荧光探针可通过下述方式实现对硝基还原酶的特异性检测,以及实现缺氧肿瘤细胞的特异性荧光成像检测;
所述基于新型反应机制的硝基还原酶荧光探针其结构为:
本发明所述基于新型反应机制的硝基还原酶荧光探针其本身没有荧光,作为硝基还原酶特异性底物被还原后的产物荧光强度显著增强,可实现对硝基还原酶灵敏和选择性检测。
识别机理如下:
荧光探针分子中邻位硝基被还原为氨基,氨基进攻硫醚形成五元过渡中间态,进一步生成硫氮转位产物。
本发明中硝基还原酶荧光探针的具体特点为:
本发明所述荧光探针可根据所述反应机制,即硝基还原、硫氮转位机理实现对硝基还原酶的检测。
本发明所述荧光探针的紫外最大吸收为505nm,激发波长为480nm,荧光最大发射为550nm。
本发明所述硝基还原酶荧光探针本身具没有或有非常弱的荧光,在37℃下,25%乙腈的Tris缓冲液中,加入NADH后荧光不发生变化,而再加入硝基还原酶后可产生了7倍的荧光信号增强。
本发明所述硝基还原酶荧光探针在各种分析物,如无机盐、氨基酸、糖类、维生素、硫醇、还原物质存在下不受干扰,可实现对硝基还原酶的特异性检测。
本发明所述硝基还原酶探针的生物毒性较低,可实现缺氧肿瘤细胞的特异性荧光成像检测。
附图说明
图1为探针(1)的核磁1H NMR图谱;
图2为探针(1)的核磁13C NMR图谱;
图3为探针(1)与硝基还原酶反应前后的紫外吸收光谱图;
图4为探针(1)与硝基还原酶前后的荧光发射光谱图;
图5为探针(1)与硝基还原酶反应的动力学实验图;
图6为探针(1)与不同浓度硝基还原酶反应的浓度滴定实验结果图;
图7为探针(1)与硝基还原酶及各种干扰物质反应的选择性结果图;
图8为探针(1)与硝基还原酶反应的pH稳定性结果图;
图9为探针(1)检测缺氧细胞成像图。
具体实施方式
下面通过实施例和附图对本发明进行说明,但本发明的内容不受具体实施例的限制。
实施例1.
合成探针(1),其结构为:
探针(1)的合成工艺为:
将化合物C(122mg,0.338mmol)和化合物e(105mg,0.667mmol)溶于干燥二氯甲烷中,加入DMAP(124mg,1.016mmol),氮气保护下室温搅拌5小时,反应液浓缩后经硅胶柱层析(石油醚∶乙酸乙酯∶二氯甲烷=20∶1∶1)得化合物(1),62mg,产率38.5%。LC-MS(ESI)m/z:477.1[M+H]+;1H NMR(400MHz,DMSO-d6)δ8.21-8.15(m,1H),7.60-7.48(m,4H),7.46-7.42(m,2H),7.35(t,J=7.8Hz,1H),6.90(d,J=8.2Hz,1H),6.57(d,J=3.9Hz,1H),6.26(d,J=3.8Hz,1H),2.47(s,3H),2.30(q,J=7.6Hz,2H),1.38(s,3H),0.90(t,J=7.5Hz,3H);13CNMR(151MHz,Chloroform-d)δ165.83,144.24,142.40,139.71,138.80,137.04,136.59,135.92,133.02,132.67,128.89,128.78,128.18,127.97,124.85,124.57,124.23,123.33,16.51,13.44,13.02,11.79。
实施例2.探针(1)与硝基还原酶反应前后的紫外吸收光谱图变化
将NADH配制成0.15M的Tris溶液,现用现配;将探针配制成3mM的乙腈溶液,备用;将硝基还原酶(1mg,90%)用去离子水配制成3g/L的储备液,分装-20℃储存,备用。在3mL的石英比色皿中加入3mL含25%乙腈的Tris(pH 7.4,50mM)缓冲溶液,再依次加入10μL的探针、10μL的NADH和5μL的硝基还原酶。37℃下反应180min,测量其吸收光谱。由图3可以看出,探针(1)在反应前后,505nm处的紫外最大吸收强度变弱。
实施例3.探针(1)与硝基还原酶反应前后的荧光发射光谱变化
在3mL的石英比色皿中加入3mL含25%乙腈的Tris(50mM,pH 7.4)缓冲溶液,再依次加入10μL的探针、10μL的NADH和5μL的硝基还原酶(NTR)。37℃下反应180min,测量其荧光发射光谱。激发波长470nm,激发狭缝宽10nm,发射狭缝宽10nm,增益700V。由图4可以看出,探针(1)本身荧光微弱,与硝基还原酶反应后荧光最大发射为560nm,荧光增强约7倍。
实施例4.探针(1)(10μM)与硝基还原酶反应的动力学实验
在37℃下,探针(1)(10μM)与NADH(0.5mM)、NTR(9μg/mL)在含25%乙腈的Tris(50mM,pH 7.4)溶液中反应。激发波长470nm,激发狭缝宽10nm,发射狭缝宽10nm,增益700V。结果如图5所示,再加入硝基还原酶后1min即检测到540nm处的荧光增强了9倍,并且在前5min内变化明显,之后随着时间的延长,荧光缓慢增强,反应180min后荧光强度趋于稳定,至此荧光信号增强了13倍,同时荧光最大发射波长也由540nm位移至560nm处。
实施例5.探针(1)与硝基还原酶的浓度滴定实验
在3mL的石英比色皿中加入3mL含25%乙腈的Tris(50mM,pH 7.4)缓冲溶液,再依次加入10μL的探针和各种浓度(0,1,3,5,7,9,11μg/mL)的硝基还原酶,NDAH浓度均为0.5mM。37℃下反应,180min,测量其荧光发射光谱。激发波长450nm,激发狭缝宽10nm,发射狭缝宽10nm,增益700V。由图6可以看出,随着硝基还原酶浓度的增大,560nm处的荧光强度也不断增强,在0-11μg/mL的浓度范围内,荧光强度与硝基还原酶浓度呈良好的线性关系。线性方程为Y=105.96X+149.29,R2=0.9833。
实施例6.探针(1)与各种干扰分析物反应的选择性实验
各分析物KCl,NaCl,CaCl2,NaNO2,NaSH,H2O2,NaClO,Na2S2O3,Na2S2O5,Vitamin C,Vitamin B6,胎牛血清(BSA),酪氨酸(Tyr),谷氨酸(Glu),苏氨酸(Thr),丙氨酸(Ala),色氨酸(Trp),精氨酸(Arg),亮氨酸(Leu),半胱氨酸(Cys),缬氨酸(Val),脯氨酸(Pro),赖氨酸(Lys),高半胱氨酸(GSH),同型半胱氨酸(Hcy),D-葡萄糖(D-glucose),二硫苏糖醇(DTT)均配制成3M的浓溶液,备用。在3mL的石英比色皿中加入3mL含25%乙腈的Tris(pH 7.4,50mM)缓冲溶液,再依次加入10μL的探针、5μL的硝基还原酶或100equiv的各种分析物,NADH浓度均为0.5mM。37℃下反应180min,测量其荧光发射光谱。激发波长450nm,激发狭缝宽10nm,发射狭缝宽10nm,增益700V。由图6可知,在其分析物存在下探针(1)的荧光基本不发生改变,而加入硝基还原酶后荧光强度大幅度提升,这说明L3-e可对硝基还原酶的检测不受其他物质干扰,可对硝基还原酶进行选择性检测。
实施例7.探针(1)与硝基还原酶反应的pH稳定性实验
在37℃下,将10μM的探针(1)与0.5mM的NADH,5μg/mL的NTR在不同pH(7.1,7.2,7.3,7.4,7.5,7.7,7.9,8.1,8.3)的Tris缓冲溶液中反应180min后,测定在530nm处的荧光强度值。结果如下图8所示,探针(1)在pH 7.1-8.3的范围内对NTR响应的荧光信号强度较为稳定,说明(1)在该pH范围内可实现对NTR稳定的检测识别。
实施例8.探针(1)检测缺氧细胞成像图
将A549细胞以3×105个/mL的浓度接种到35mm的共聚焦培养皿中,在37℃,5%CO2的培养箱中孵育12小时后,将细胞分成两组,分别加入10μM的探针(1),对照组在正常氧含量条件下继续培养6小时,实验组在缺氧条件(1%O2)下培养6小时,然后吸掉培养液,并用PBS洗涤3次,加入无血清培养基,在荧光显微镜下观察。激发波长488nm,发射波段500-580nm。结果如图9所示,正常氧含量下培养的细胞没有荧光,而缺氧条件下培养的可观测到明显的荧光。
Claims (4)
3.权利要求1的基于硝基还原、硫氮转位的硝基还原酶荧光探针在制备对硝基还原酶特异性检测,及缺氧肿瘤细胞荧光成像检测的制剂中的用途。
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