CN113200975B - 基于靛红衍生物专一响应onoo-的水溶性荧光探针、制备方法及应用 - Google Patents
基于靛红衍生物专一响应onoo-的水溶性荧光探针、制备方法及应用 Download PDFInfo
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- C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
- C07D413/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings linked by a chain containing hetero atoms as chain links
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Abstract
Description
技术领域
本发明涉及荧光探针领域,具体涉及一种基于靛红衍生物专一响应ONOO‒的水溶性荧光探针、制备及应用。
背景技术
过氧亚硝酸盐(ONOO‒)作为活性氮物种家族中的一员,具有稳定含量低(纳摩级)和寿命短(∼10ms)的特点。生物体内的ONOO‒是由一氧化氮(NO)和超氧阴离子(O2▪-)通过自由扩散反应在线粒体中产生。ONOO‒及其次生代谢产物(▪NO2,▪CO3 ‒,▪OH)可以与蛋白质、脂类和核酸等多种生物分子发生反应,导致线粒体功能障碍和细胞凋亡从而引起多种疾病。到目前为止,越来越多的研究表明,ONOO‒参与了大量的细胞信号传递和病理过程,包括炎症,神经退行性疾病,阿尔茨海默氏病,帕金森氏病,心血管,缺血性再灌注损伤和癌症等。因此,开发一种有效检测ONOO‒的方法并探讨其对于疾病诊断和各种病理生理功能的研究至关重要。
目前已经探索出了多种方法用于ONOO‒的检测,其中包括电化学分析、免疫组织化学、光致发光和化学发光等方法。荧光探针法由于具有响应速度快,灵敏度高,可视化能力强和生物创伤性低等优点,在化学分析、生物分析以及医学研究中得到了广泛的应用。近年来,基于化学反应型的荧光探针主要基于以下几种机理:硼酸或硼酸酯的氧化,α-酮酰胺的氧化,膦酰/次膦酰酯的氧化,酰肼的氧化,C=C双键的裂解,硒的氧化还原反应,氧化N-脱芳基化和其他反应策略等。其中,α-酮酰胺类似物因能在温和条件下与ONOO‒平稳反应而引起了研究人员的兴趣。构建基于靛红衍生物专一响应ONOO‒的水溶性荧光探针,实现快速、高选择性地以及高灵敏度地检测ONOO‒且具有良好的水溶性,并实现在细胞以及活体层面的应用具有重要意义。
发明内容
本发明提出了一种基于靛红衍生物专一响应ONOO‒的水溶性荧光探针、制备方法及应用,该探针制备方法操作方便,原料易得,能够实现细胞内ONOO‒的专一性检测。检测机制是ONOO‒亲核进攻1-三乙二醇单甲醚靛红基团的羰基,先发生亲核加成反应,再发生分子内重排和水解反应生成邻甲酸苯胺衍生物,最后进行一步自消除反应,释放荧光团引起荧光信号变化。
实现本发明的技术方案是:
一种基于靛红衍生物专一响应ONOO‒的水溶性荧光探针,化学分子式为:C28H26N2O8,命名为RF-PN2,探针的结构式为:
所述的荧光探针的制备方法,步骤如下:
(1)冰水浴下,将三乙二醇单甲醚溶于四氢呋喃中,加入氢氧化钠溶液,再加入对甲基苯磺酰氯的四氢呋喃溶液,二氯甲烷萃取,柱层析分离得到化合物1;
(2)在N2保护下,将5-甲基靛红与碳酸钾溶于无水N,N-二甲基甲酰胺中,再加入步骤(1)制得的化合物1的无水N,N-二甲基甲酰胺溶液,50~100 ℃加热反应,二氯甲烷萃取,柱层析分离得到化合物2;
(3)步骤(2)制得的化合物2在N2保护下,溶于四氯化碳中,加入N-溴代丁二酰亚胺和过氧化苯甲酰,50~100 ℃加热搅拌,冷凝回流,然后减压去除溶剂,柱层析分离得到化合物3;
(4)N2保护下,先将试卤灵和碳酸钾溶于无水N,N-二甲基甲酰胺中,再加入步骤(3)所得化合物3,加热搅拌,二氯甲烷萃取,柱层析分离得到荧光探针。
所述步骤(1)中三乙二醇单甲醚和对甲基苯磺酰氯按照当量为1:(0.5~2)的比例添加,冰浴下搅拌2~8h。
所述步骤(2)中化合物1、5-甲基靛红和碳酸钾按照当量为1:(1~4):(2~3)比例添加,加热搅拌5~10h。
所述步骤(3)中化合物2、N-溴代丁二酰亚胺和过氧化苯甲酰按照当量为1:(1~4):(0.1~0.3)比例添加,加热搅拌4~8h。
所述步骤(4)中试卤灵、化合物3和碳酸钾按照当量为1:(1~4):(1~3)比例添加,加热搅拌8~14h。
制备的荧光探针在制备细胞/活体中专一检测ONOO‒试剂中的应用。
荧光探针的合成路线如下:
本发明基于1-三乙二醇单甲醚靛红基团专一检测ONOO‒的水溶性荧光探针,该探针在溶液测试中,利用紫外和荧光光谱可判断出探针与ONOO‒的反应时间,浓度依赖性关系;在通过选择性和抗干扰能力测试中,结果表明探针可专一性检测ONOO‒,不与其他活性氧化物反应且抗干扰能力强;并且探针的pH稳定性强,细胞毒性小。同时通过荧光成像技术达到对HepG-2细胞中的ONOO‒检测的目的。
本发明的有益效果是:
(1)本发明基于一种靛红衍生物专一响应ONOO‒的水溶性荧光探针,合成方法简单,操作方便;
(2)本发明的检测方法可以实现ONOO‒专一性检测,而且不受其他活性氮和活性氧化物类的干扰;该检测过程简便、快速、灵敏,检测限为87nM;
(3)本发明探针在靛红基团上引入三乙二醇单甲醚链修饰提高其水溶性,使其具有良好的水溶性和细胞相容性,可以检测细胞中的ONOO‒,检测信号明显,为长波长增强型荧光探针。
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为荧光探针RF-PN2的核磁氢谱图。
图2为荧光探针RF-PN2的核磁碳谱图。
图3为荧光探针RF-PN2与ONOO‒作用的紫外吸收变化。
图4为荧光探针RF-PN2与ONOO‒作用的时间响应变化。
图5为荧光探针RF-PN2测定ONOO‒浓度滴定实验荧光变化。
图6为荧光最强发射波长586 nm与ONOO‒的浓度线性拟合图。
图7为常见活性氧对探针RF-PN2检测ONOO‒的荧光选择性。
图8为常见活性氧对探针RF-PN2检测ONOO‒的荧光干扰性。
图9为荧光探针RF-PN2和探针加ONOO‒在不同pH缓冲溶液中最大荧光强度变化图。
图10为探针RF-PN2检测ONOO‒的细胞毒性。
图11为荧光探针RF-PN2检测外源性ONOO‒的HepG-2细胞成像图。
图12为荧光探针RF-PN2检测内源性ONOO‒的HepG-2细胞成像图。
图13为荧光探针RF-PN2检测ONOO‒小鼠活体成像。
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
探针的制备步骤如下:
(1)化合物1的制备
冰水浴下,将三乙二醇单甲醚和对甲基苯磺酰氯按照当量为1:1.1比例溶于四氢呋喃中,加入氢氧化钠溶液,冰浴下搅拌5h,二氯甲烷萃取,柱层析分离得到化合物1;
(2)化合物2的制备
在N2保护下,将5-甲基靛红与碳酸钾按照当量1:2.5的比例溶于无水N,N-二甲基甲酰胺中,再加入步骤(1)制得的化合物1的无水N,N-二甲基甲酰胺溶液,80 ℃加热反应7h,二氯甲烷萃取,柱层析分离得到化合物2;
(3)化合物3的制备
N2保护下,步骤(2)制得的化合物2、N-溴代丁二酰亚胺和过氧化苯甲酰按照当量为1:2:0.2比例溶于四氯化碳中,80 ℃加热搅拌,冷凝回流6h,然后减压去除溶剂,二氯甲烷萃取,柱层析分离得到化合物3;
(4)探针RF-PN2的制备
N2保护下,先将试卤灵和碳酸钾按照当量为1:2:2比例溶于无水N,N-二甲基甲酰胺中,再加入步骤(3)所得化合物3,80 ℃加热搅拌11h,二氯甲烷萃取,柱层析分离得到荧光探针RF-PN2。
1H NMR (400 MHz, CDCl3) δ 7.74 (d, J = 8.9 Hz, 1H), 7.67 (d, J = 6.2Hz, 1H), 7.65 (s, 1H), 7.43 (d, J = 9.8 Hz, 1H), 7.18 (d, J = 8.1 Hz, 1H),6.99 (dd, J = 8.9, 2.7 Hz, 1H), 6.91 – 6.79 (m, 2H), 6.33 (d, J = 2.0 Hz,1H), 5.12 (s, 2H), 3.95 (t, J = 5.3 Hz, 2H), 3.77 (t, J = 5.4 Hz, 2H), 3.63(t, J = 2.8 Hz, 2H), 3.58 (t, J = 2.6 Hz, 2H), 3.56 (t, J = 2.4 Hz, 2H), 3.50(t, J = 4.4 Hz, 2H), 3.36 (s, 3H).13C NMR (101 MHz, DMSO) δ 185.78, 183.63,162.53, 158.75, 151.43, 150.13, 145.79, 145.65, 138.40, 135.37, 134.23,131.82, 131.44, 128.50, 124.56, 117.94, 114.77, 112.00, 106.15, 101.71,71.70, 70.27, 70.17, 70.06, 69.86, 67.75, 58.48.
实施例2
探针的制备步骤如下:
(1)化合物1的制备
冰水浴下,将对甲基苯磺酰氯溶于四氢呋喃溶剂中,再加入6mL三乙二醇单甲醚的四氢呋喃溶液,将三乙二醇单甲醚和对甲基苯磺酰氯按照当量为1:0.5比例反应,然后加入氢氧化钠溶液,冰浴下搅拌2h,二氯甲烷萃取,柱层析分离得到化合物1;
(2)化合物2的制备
在N2保护下,将5-甲基靛红与碳酸钾按照当量1:2的比例溶于无水N,N-二甲基甲酰胺中,再加入步骤(1)制得的化合物1的无水N,N-二甲基甲酰胺溶液,50 ℃加热反应5h,二氯甲烷萃取,柱层析分离得到化合物2;
(3)化合物3的制备
N2保护下,步骤(2)制得的化合物2、N-溴代丁二酰亚胺和偶氮二异丁腈按照当量为1:1:0.1比例溶于四氯化碳中,80 ℃加热搅拌,冷凝回流4h,然后减压去除溶剂,柱层析分离得到化合物3;
(4)探针RF-PN2的制备
N2保护下,先将试卤灵和碳酸钾按照当量为1:1:1比例溶于无水N,N-二甲基甲酰胺中,再加入步骤(3)所得化合物3,50 ℃加热搅拌8h,二氯甲烷萃取,柱层析分离得到荧光探针RF-PN2。
实施例3
探针的制备步骤如下:
(1)化合物1的制备
冰水浴下,将三乙二醇单甲醚溶于四氢呋喃溶剂中,再加入对甲基苯磺酰氯的四氢呋喃溶液,将三乙二醇单甲醚和对甲基苯磺酰氯按照当量为1:2比例反应,然后加入氢氧化钠溶液,冰浴下搅拌8h,二氯甲烷萃取,柱层析分离得到化合物1;
(2)化合物2的制备
在N2保护下,将5-甲基靛红与碳酸钾按照当量4:3的比例溶于无水N,N-二甲基甲酰胺中,再加入步骤(1)制得的化合物1的无水N,N-二甲基甲酰胺溶液,100 ℃加热反应10h,二氯甲烷萃取,柱层析分离得到化合物2;
(3)化合物3的制备
N2保护下,步骤(2)制得的化合物2、N-溴代丁二酰亚胺和偶氮二异丁腈按照当量为1:4:0.3比例溶于四氯化碳中,100 ℃加热搅拌,冷凝回流8h,然后减压去除溶剂,二氯甲烷萃取,柱层析分离得到化合物3;
(4)探针RF-PN2的制备
N2保护下,先将试卤灵和碳酸钾按照当量为1:4:3比例溶于无水N,N-二甲基甲酰胺中,再加入步骤(3)所得化合物3,100 ℃加热搅拌14h,二氯甲烷萃取,柱层析分离得到荧光探针RF-PN2。
利用实施例1中的探针进行性能测试
(1)溶液中检测ONOO‒的荧光探针RF-PN2的反应时间测试
用二甲基亚砜(DMSO)配制1 mM RF-PN2的荧光探针储备液;探针RF-PN2(5 μM)与ONOO‒(100 μM)在溶液体系为PBS缓冲液(10 mM,pH 7.4,1% DMSO)中进行反应,如图4所示,从时间动力学曲线观察RF-PN2在2min内与ONOO‒快速响应,并在10 min内达到最大荧光值,反应曲线达到平台;同时,在紫外吸收图谱上反应前后的吸收峰发生了96 nm的红移。(见图3所示)。
(2)溶液中检测ONOO‒的荧光探针RF-PN2的浓度滴定测试以及浓度线性关系
在浓度滴定实验中,发现随着ONOO‒的浓度逐渐增加,586 nm处的荧光峰也逐渐增强,其荧光强度在ONOO‒浓度至100 μM时达到最大。(见图5所示)。
以ONOO‒浓度为横坐标,探针RF-PN2在586 nm处的荧光强度为纵坐标,绘制图并进行线性拟合,得到该探针的线性回归方程为:Y = 3.908 X + 3.301,线性相关系数R2 =0.994并计算出检测限为87nM。(见图6所示)。
(3)干扰性和抗干扰性离子实验
在不同的荧光比色皿中,分别加入4 mL PBS缓冲液(10 mM,pH 7.4,1% DMSO)和40μL荧光探针储备液,如图7所示,当探针RF-PN2分别加入上述选择的活性氮以及活性氧化物分析物种(100 M)后(0: free, 1: ONOO‒, 2: OCl‒, 3: H2O2, 4: ˙OH, 5: ˙OtBu, 6:TBHP, 7: O2▪-, 8: H2S, 9: 1O2, 10: NO2 ‒, 11: Hcy, 12: GSH, 13: Cys, 14: HS‒, 15:SO4 2‒, 16: SO3 2‒, 17: Ca2+, 18:Mg2+,19: Fe3+),探针RF-PN2可以对ONOO‒进行专一性识别,与ONOO‒反应后在586 nm处出现明亮的红色荧光,而跟其它种类分析物反应后没有荧光出现,因此,探针可以实现专一性响应ONOO‒。当RF-PN2(5 μM)在分别加入上述分析物(0:free, 1: OCl‒, 2: H2O2, 3: ˙OH, 4: ˙OtBu, 5: TBHP, 6: O2˙‒, 7: H2S, 8: 1O2, 9:NO2 ‒, 10: Hcy, 11: GSH, 12: Cys, 13: HS‒, 14: SO4 2‒, 15: SO3 2‒, 16: Ca2+, 17:Mg2 +,18: Fe3+)后再加入100 μM ONOO‒反应10分钟后,可以看出ONOO‒在与各种分析物共存情况下,RF-PN2在复杂的溶液体系内依然能够专一性检测到ONOO‒,且荧光程度较强。实验证明,RF-PN2能够在响应ONOO‒时不受其它物质的干扰(见图8所示)。
(4)pH值的响应性实验
将探针RF-PN2溶于二甲基亚砜中得到1 mM探针母液,配置pH为4.0、5.0、6.0、7.0、8.0、9.0、10.0的溶液,对探针以及探针和ONOO‒反应后荧光强度的变化进行了研究。
结果如图9所示,探针荧光强度在pH值为4.0-10.0的溶液中基本保持不变;在加入ONOO‒后,在不同pH值条件下,其位于586 nm处荧光强度保持稳定。实验证明探针RF-PN2能够适应生物体内的pH环境,且具有良好的稳定性。
(5)MTT细胞毒性实验
利用探针RF-PN2对HepG-2细胞的MTT细胞毒性实验,结果如图10所示。HepG-2细胞用含有不同浓度的探针(1.25, 2.5, 5.0, 10, 20 μM)培养液孵育后,计算细胞的存活百分比。结果如图10所示,低浓度下细胞存活率可高达90%,探针几乎没有细胞毒性。
(6)检测ONOO‒的荧光探针对HepG-2细胞外源性ONOO‒的检测性能测试
检测ONOO‒的荧光探针RF-PN2在小鼠肝癌细胞(HepG-2细胞)中ONOO‒的荧光共聚焦成像。结果如图11所示,(a)和(b)为探针RF-PN2(10 μM)和HepG-2细胞一起孵育2小时后,用PBS缓冲液洗涤三次,再加入Hoechst 33342(10 μL)共孵育10分钟后,用PBS缓冲液洗涤三次后显微镜下荧光成像;(d)和(e)为探针RF-PN2(10 μM)和HepG-2细胞孵育2小时后,用PBS缓冲液洗涤三次,再加入SIN-1(100 μM)共孵育30分钟,然后加入Hoechst 33342(10 μL)共孵育10分钟后,用PBS缓冲液洗涤三次后显微镜下荧光成像;(c)和(f)为蓝色通道和红色通道荧光成像图的重叠。通过共聚焦红色通道观察,检测外源性ONOO‒的荧光探针在HepG-2细胞中产生明亮的红色荧光;对照组实验中,HepG-2细胞只与RF-PN2共同孵育,由成像实验结果可以观察红色通道荧光响应微弱。由此说明探针可以检测细胞外源性ONOO‒。
(7)检测ONOO‒的荧光探针对HepG-2细胞内源性ONOO‒的检测性能测试
检测ONOO‒的荧光探针RF-PN2在小鼠肝癌细胞(HepG-2细胞)中ONOO‒的荧光共聚焦成像。结果如图12所示,(a)和(b)为HepG-2细胞先用脂多糖(LPS)和γ-干扰素(IFN-γ)(LPS和IFN-γ联用可刺激小鼠单核巨噬细胞产生内源性的ONOO‒)孵育12小时,用PBS缓冲液洗涤三次,RF-PN2(10 μM)再与细胞共孵育2小时,再加入Hoechst 33342(10 μL)共孵育10分钟后,用PBS缓冲液洗涤三次后显微镜下荧光成像;(d)和(e)为HepG-2细胞先用脂多糖(LPS)和γ-干扰素(IFN-γ)(LPS和IFN-γ联用可刺激小鼠单核巨噬细胞产生内源性的ONOO‒)孵育12小时,再加入尿酸(UA)(ONOO‒清除剂)孵育30分钟,用PBS缓冲液洗涤三次,RF-PN2(10 μM)再与细胞共孵育2小时,然后加入Hoechst 33342(10 μL)共孵育10分钟后,用PBS缓冲液洗涤三次后显微镜下荧光成像;(c)和(f)为蓝色通道和红色通道荧光成像图的重叠。通过共聚焦红色通道观察,检测ONOO‒的荧光探针在HepG-2细胞中产生明亮的红色荧光;对照组实验中,加入尿酸使细胞与RF-PN2共同孵育,由成像实验结果可以观察红色通道荧光响应微弱。由此说明探针可以检测细胞内源性ONOO‒。该结果也表明,本发明的荧光探针具有检测HepG-2细胞内ONOO‒的良好应用前景。
(8)检测ONOO‒的荧光探针对活体内源性ONOO‒的成像能力测试
通过小鼠胫跗关节皮下注射方式将LPS(200 μL,1 mg/mL,生理盐水)注射到裸鼠左侧胫跗关节(左脚踝)孵育12小时作为实验组;将等量生理盐水注射到裸鼠右侧胫跗关节(右脚踝)孵育12小时作为对照组,将探针RF-PN2(50 μL,500 μM,DMSO/生理盐水=1/9)原位注射到裸鼠左、右侧胫跗关节(左脚踝和右脚踝)。如图13所示,注射前/注射后进行成像系统内荧光监测,通过共聚焦红色通道观察,检测ONOO‒的荧光探针在LPS刺激的小鼠发炎模型左腿中产生明亮的红色荧光,并在5分钟时即可达到最大荧光值;对照组实验中,仅用生理盐水和探针孵育的右腿成像实验结果中可以观察红色通道荧光响应微弱。由此说明探针可以快速地原位监测小鼠活体内源性ONOO‒的产生。
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 (7)
2.权利要求1所述的荧光探针的制备方法,其特征在于,步骤如下:
(2)在N2保护下,将5-甲基靛红与碳酸钾溶于无水N,N-二甲基甲酰胺中,再加入步骤(1)制得的化合物1的无水N,N-二甲基甲酰胺溶液,50~100℃加热反应,二氯甲烷萃取,柱层析分离得到化合物2;步骤(2)中化合物2的结构式如下:;
(3)步骤(2)制得的化合物2在N2保护下,溶于四氯化碳中,加入N-溴代丁二酰亚胺和过氧化苯甲酰,50~100℃加热搅拌,冷凝回流,然后减压去除溶剂,柱层析分离得到化合物3;步骤(3)中化合物3的结构式如下:;
(4)N2保护下,先将试卤灵和碳酸钾溶于无水N,N-二甲基甲酰胺中,再加入步骤(3)所得化合物3,加热搅拌,二氯甲烷萃取,柱层析分离得到荧光探针。
3.根据权利要求2所述的荧光探针的制备方法,其特征在于:所述步骤(1)中三乙二醇单甲醚和对甲基苯磺酰氯按照当量为1:(0.5~2)的比例添加,冰浴下搅拌2~8h。
4.根据权利要求2所述的荧光探针的制备方法,其特征在于:所述步骤(2)中化合物1、5-甲基靛红和碳酸钾按照当量为1:(1~4):(2~3)比例添加,加热搅拌5~10h。
5.根据权利要求2所述的荧光探针的制备方法,其特征在于:所述步骤(3)中化合物2、N-溴代丁二酰亚胺和过氧化苯甲酰按照当量为1:(1~4):(0.1~0.3)比例添加,加热搅拌4~8h。
6.根据权利要求2所述的荧光探针的制备方法,其特征在于:所述步骤(4)中试卤灵、化合物3和碳酸钾按照当量为1:(1~4):(1~3)比例添加,加热搅拌8~14h。
7.权利要求2-6任一项制备方法制备的荧光探针在制备细胞/活体中专一检测ONOO‒试剂中的应用。
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