CN115160391B - 一种靶向性过氧化亚硝基荧光探针的制备和应用 - Google Patents
一种靶向性过氧化亚硝基荧光探针的制备和应用 Download PDFInfo
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Abstract
本发明涉及了一种靶向性过氧化亚硝基荧光探针的制备和应用。该荧光探针的结构式为:本发明提供了以Cy‑OH、溴代四乙酰基半乳糖、碳酸钾等为原料合成该荧光探针的制备方法。该荧光探针是一种近红外、靶向性的比率过氧化亚硝基荧光探针。首先,该荧光探针对ONOO‑表现出很高的灵敏度,荧光强度比值(F477/F717)增强872倍。其次,该荧光探针对ONOO‑表现出很高的选择性,不受其他活性氧、活性氮、活性硫、生物硫醇以及常见氨基酸的干扰。并且,该荧光探针与ONOO‑作用快速,响应时间在15分钟左右。此外,该荧光探针已应用于细胞成像研究,可以检测细胞内ONOO‑含量的变化。
Description
技术领域
本发明属于荧光探针技术领域,具体涉及一种靶向性过氧化亚硝基荧光探针的制备和应用。
背景技术
过氧化亚硝基(ONOO-)是一种重要的活性氧(ROS),是由一氧化氮(NO)和超氧阴离子(O2 .-)反应生成的产物(R.Radi,J.Biol.Chem.,2013,288,26464-26472)。作为一种强氧化剂和强亲核试剂,ONOO-能够与多种生物分子发生反应,比如蛋白质、脂质、核酸等,最终导致细胞死亡(L.Liaudet,G.Vassalli,P.Pacher,Front.Biosci.,2009,14,4809-4814)。并且,ONOO-还可作为信号分子参与信号传导过程(B.C.Dickinson,C.J.Chang,Nat.Chem.Biol.,2011,7,504-511)。此外,过氧化亚硝基的水平异常和一些常见的疾病相关,如心血管疾病、神经退行性疾病、糖尿病和炎症型肠病(J.S.Beckman,M.Carson,C.D.Smith and W.H.Koppenol,Nature,1993,364,584–585;H.Wiseman and B.Halliwell,Biochem.J.,1996,313,17–29;P.Pacher,J.S.Beckman and L.Liaudet,Physiol.Rev.,2007,87,315–424;S.M.Schieke,K.Briviba,L.O.Klotz and H.Sies,FEBS Lett.,1999,448,301–303)。由于过氧化亚硝基有着重要的生理及临床意义,因此设计有效的方法去精确检测它的含量就显得非常必要。
近年来,由于荧光探针具有操作简单,灵敏度高,对生物样品无损害、可实现的时空分辨等优点,引起了人们的广泛关注(H.Zhu,J.L.Fan,J.J.Du,X.Peng,J.Acc.Chem.Res.,2016,49,2115-2126)。到目前为止,利用ONOO-的强氧化性和亲核性,开发了许多荧光探针来检测ONOO-(J.Zhou,Y.Li,J.N.Shen,Q.Li,R.Wang,Y.F.Xu,X.H.Qian,RSC Adv.,2014,4,51589-51592;X.F.Yang,X.Q.Guo,Y.B.Zhao,Talanta.,2002,57,883-890;F.B.Yu,P.Li,B.S.Wang,K.L.Han,J.Am.Chem.Soc.,2013,135,7674-7680;T.Peng,N.K.Wong,X.Chen,Y.K.Chan,D.H.Ho,Z.Sun,J.J.Hu,J.Shen,H.EI-Nezami,D.Yang,J.Am.Chem.Soc.,2014,136,11728-11734;Z.N.Sun,H.L.Wang,F.Q.Liu,Y.Chen,P.K.H.Tam,D.Yang,Org.Lett.,2009,11,1887-1890;Y.Li,X.Xie,X.Yang,M.Li,X.Jiao,Y.Sun,X.Wang,B.Tang,Chem.Sci.,2017,8,4006-4011)。但是,这些探针存在一些问题:(1)探针的荧光团(如荧光素、萘酰亚胺、氟硼吡咯等)的发射波长较短,这限制了这些探针在生物体内的应用;(2)以前所报道的这些荧光探针大多是以单个荧光发射峰强度变化作为响应信号的,容易受到一些环境因素(如设备效率,温度,探针浓度等)的影响;(3)这些探针缺乏靶向性,这些探针没有靶向团,在细胞成像时,缺乏对炎症细胞的准确靶向识别能力,导致探针非特异性地进入正常细胞产生假阳性信号。因此,设计和合成具有长波长发射,靶向性的比率型荧光探针是非常有意义的。
半花菁染料是目前荧光探针领域中应用比较广泛的一类染料,它具有化学性质稳定、荧光量子产率高等优点。特别是,基于半花菁类的探针具有近红外发射,因此组织穿透力强,不易受到生物自体荧光的干扰,对生物成像更有利。据文献报道,半花菁类荧光探针已被用来检测pH、活性氧、生物硫醇和各种酶(L.L.Wu,Y.Wang,T.D.James,N.Q.Jia,C.S.Huang,Chem.Commun.2018,54,5518-5521;X.Xie,X.Yang,T.Wu,Y.Li,M.Li,Q.Tan,X.Wang,B.Tang,Anal.Chem.2016,88,8019-8025;C.S.Park,T.H.Ha,S.A.Choi,D.N.Nguyen,S.Noh,O.S.Kwon,C.S.Lee,H.Yoon,Biosens.Bioelectron.2017,89,919-926;S.J.Li,C.Y.Li,Y.F.Li,J.J.Fei,P.Wu,B.Yang,J.Ou-Yang,S.X.Nie,Anal.Chem.2017,89,6854-6860)。在探针中引入半乳糖结构是实现靶向炎症性肠病(IBD)的一种有效策略,探针在细胞和组织的特异性分布有望提升细胞中ONOO-检测的灵敏度和信噪比。但是,现在还没有能同时靶向IBD和检测ONOO-的荧光探针。因此,设计和合成一种基于半花菁染料的靶向性过氧亚硝基荧光探针,作为检测细胞中ONOO-的有效工具,是非常必要的。
发明内容
根据所提出的要求,本发明人对此进行了深入研究,在付出了大量创造性劳动后,提供了一种靶向性过氧化亚硝基荧光探针。
本发明的技术方案是,一种靶向性过氧化亚硝基荧光探针,其结构式如下:
一种靶向性过氧化亚硝基荧光探针的制备方法,步骤如下:
在100mL圆底烧瓶中,将1当量的Cy-OH,3~5当量的碳酸铯溶于二氯甲烷中,室温下,在氮气中搅拌10~20分钟;然后,通过注射器加入3~5当量的溴代四乙酰基半乳糖,继续在室温下搅拌10~12小时;通过减压蒸馏除去溶剂,粗产品用体积比为30:1~12:1的CH2Cl2/CH3OH洗脱剂进行柱层析,得到蓝色固体物质;随后,将其溶解在无水甲醇中,加入3~5当量的碳酸钾脱保护,在室温下搅拌反应混合物2~3小时;然后,减压蒸馏除去溶剂,所得粗产品用体积比为15:1~10:1的CH2Cl2/CH3OH洗脱剂柱层析分离得到蓝色固体Cy-Gal,即为荧光探针。
本发明的有益效果是,一种靶向性过氧化亚硝基荧光探针的良好性能。首先,研究了该探针的荧光光谱性质,加入ONOO-之前,荧光探针有近红外(717nm)的荧光发射峰;加入ONOO-之后,在可见区(477nm)出现了蓝色发射峰。并且随着ONOO-浓度的增大,探针分子的近红外荧光强度不断降低,蓝色荧光强度不断增强。当加入40μM的ONOO-时,荧光强度比值(F477/F717)增强872倍,因此可以比率检测ONOO-。该探针的检测范围从1.0μM到40.0μM,检测限为0.06μM,这说明该探针可以高灵敏的检测ONOO-。其次,研究了探针的紫外吸收光谱,在没有加入ONOO-时,探针在680nm处有吸收带;加入ONOO-后,680nm处的吸收峰减小,在345nm附近出现新的吸收峰,溶液颜色从蓝色变为无色。接着,研究了探针的选择性,考察了探针与活性氧(ClO-,H2O2),活性氮(NO2 -,NO3 -),活性硫(SO3 2-,HSO3 -),生物硫醇(Cys,Hcy,GSH)以及常见氨基酸(Met,Lys,Trp,Phe,Thr,Ile,Leu,Val)的荧光响应情况。结果发现,只有ONOO-能引起荧光光谱的改变,其他检测物对探针的荧光光谱没有明显的影响。最后,研究了pH值对荧光探针测定ONOO-的影响,当pH值在6.0到8.6之间时,不影响荧光探针对ONOO-的测定。此外,该荧光探针响应较快,响应时间在15min以内。
一种靶向性过氧化亚硝基荧光探针的应用。在细胞中加入荧光探针,红色通道可以观察到的较强的荧光,而在蓝色通道几乎没有荧光,这说明细胞中的ONOO-较低。细胞用脂多糖(LPS)和干扰素-γ(IFN-γ)处理,然后用探针染色,发现蓝色通道荧光明显增强,而红色通道荧光减弱;用氨基胍盐酸盐(AG)处理抑制细胞内ONOO-的产生,发现蓝色通道的荧光减弱,红色通道的荧光增强。这些结果说明荧光探针Cy-Gal能监控细胞内ONOO-含量的变化,这为监控人体内和过氧化亚硝基相关病变提供一种可靠的手段。
附图说明
图1为荧光探针的合成路线。
图2为荧光探针与不同浓度的ONOO-作用后的荧光光谱图。
横坐标为波长,纵坐标为荧光强度。荧光探针的浓度均为10μM,ONOO-浓度分别为:0,1.0,2.0,3.0,4.0,5.0,7.5,10.0,12.5,15.0,20.0,22.5,25.0,27.5,30.0,32.5,35.0,37.5,40.0μM。发射波长为717nm对应的激发波长为680nm,发射波长为477nm对应的激发波长为345nm。
图3为荧光探针对不同ONOO-浓度的荧光线性响应图。
图4为荧光探针与ONOO-作用后的紫外可见吸收光谱图。
横坐标为波长,纵坐标为吸光度。荧光探针的浓度为10μM,ONOO-浓度为40μM。
图5为荧光探针的选择性图。
荧光探针的浓度均为10μM,ONOO-浓度为40μM,其它分析物浓度均为200μM。
图6为pH对荧光探针的影响图。
图7为荧光探针与ONOO-作用后荧光强度随时间变化的关系曲线图。
图8为细胞毒性试验。横坐标为荧光探针的浓度,纵坐标为细胞的存活率。
图9荧光探针与ONOO-作用的细胞成像图。(a)细胞用探针染色0.5h。(b)细胞用LPS和IFN-γ处理10h,然后用探针染色0.5h。(c)细胞用LPS、IFN-γ和AG处理10h,然后用探针染色0.5h。(d)相对荧光强度图。
具体实施方式
下面结合附图和具体实施例对本发明进行详细说明,但不限于此。
实施例1:
荧光探针的合成
合成路线如图1。在100mL圆底烧瓶中,将1当量的Cy-OH,4当量的碳酸铯溶于二氯甲烷中,室温下,在氮气中搅拌15分钟;然后,通过注射器加入4当量的溴代四乙酰基半乳糖,继续在室温下搅拌12小时;通过减压蒸馏除去溶剂,粗产品用体积比为12:1的CH2Cl2/CH3OH洗脱剂进行柱层析,得到蓝色固体物质;随后,将其溶解在无水甲醇中,加入4当量的碳酸钾脱保护,在室温下搅拌反应混合物2小时;然后,减压蒸馏除去溶剂,所得粗产品用体积比为10:1的CH2Cl2/CH3OH洗脱剂柱层析分离得到蓝色固体Cy-Gal(产率60%),即为荧光探针。1H NMR(400MHz,MeOD)δ8.77(d,J=15.4Hz,1H),7.70(d,J=7.3Hz,1H),7.62–7.50(m,2H),7.50–7.42(m,2H),7.35(s,1H),7.20(s,1H),7.10(d,J=8.3Hz,1H),6.54(d,J=15.0Hz,1H),5.03(d,J=7.7Hz,1H),4.43(d,J=7.3Hz,2H),3.98(d,J=3.2Hz,1H),3.85(d,J=9.2Hz,3H),3.66(d,J=4.8Hz,1H),2.86–2.63(m,4H),1.92(d,J=6.7Hz,2H),1.83(d,J=8.5Hz,5H),1.49(t,J=7.2Hz,3H),1.28(d,J=3.7Hz,5H).13C NMR(100MHz,MeOD)δ178.5,161.7,154.7,146.8,143.1,141.8,133.7,130.2,129.4,127.9,125.9,118.3,117.6,115.3,113.2,104.2,102.3,76.8,74.1,71.4,69.6,62.0,51.5,41.0,32.4,29.5,27.6,41.6,24.3,23.0,20.9,13.8,12.3.
实施例2:
荧光探针和ONOO-溶液配制
探针溶液的制备:称取一定量探针溶解在DMSO中,配成4×10-4M的探针溶液。ONOO-溶液的配制:将0.70M的H2O2溶液、0.60M的HCl溶液、0.60M的NaNO2溶液混合,并迅速加入1.5M的NaOH溶液,过量的H2O2用二氧化锰除去,保存在-20℃的冷冻环境中。使用前化冻使用,ONOO-浓度的确定,需要测定该溶液在302nm处的吸光度A,计算公式为:CONOO -=A/1.67(mM)。
实施例3:
荧光探针与ONOO-作用的荧光光谱的测定
图2为荧光探针与ONOO-作用的荧光光谱,荧光探针的浓度为10μM,ONOO-的浓度依次为0,1.0,2.0,3.0,4.0,5.0,7.5,10,12.5,15,20,22.5,25,27.5,30,32.5,35,37.5,40μM。第一个激发波长为680nm,发射波长范围为693~850nm;第二个激发波长为345nm,发射波长范围为400~520nm。狭缝宽度为5.0nm/5.0nm,所用的荧光测定仪器为日立F4600荧光分光光度计。从图2可以看出,加入ONOO-之前,荧光探针有近红外(717nm)的荧光发射峰;加入ONOO-之后,在可见区(477nm)出现了蓝色发射峰。这是因为探针分子被ONOO-氧化,导致断裂,共轭结构变小,从而产生短波长的蓝色荧光。并且,随着ONOO-浓度的增大,探针分子的近红外荧光强度不断降低,蓝色荧光强度不断增强。当加入30μM的ONOO-时,荧光强度比值(F477/F717)增强872倍,因此可以比率检测ONOO-。图3为探针对不同ONOO-浓度的线性响应图。荧光强度跟ONOO-的浓度呈现线性关系,线性范围是1.0μM~40.0μM,检测限是0.06μM。这说明该探针可以高灵敏的检测ONOO-。
实施例4:
荧光探针与ONOO-作用的紫外可见吸收光谱的测定
图4为荧光探针与ONOO-作用后的紫外可见吸收光谱图,荧光探针的浓度为10μM,ONOO-的浓度为40μM。紫外可见吸收光谱测定用的仪器为安捷伦Cary60紫外可见分光光度计。从图2中可以看出,在没有加入ONOO-时,探针在680nm处有吸收带;加入ONOO-后,680nm处的吸收峰减小,在345nm附近出现新的吸收峰,溶液颜色从蓝色变为无色。
实施例5:
荧光探针对ONOO-测定的选择性
图5为荧光探针对ONOO-测定的选择性图。考察在浓度为10μM的荧光探针溶液中加入ONOO-(40μM)及其与活性氧(ClO-,H2O2),活性氮(NO2 -,NO3 -),活性硫(SO3 2-,HSO3 -),生物硫醇(Cys,Hcy,GSH)以及常见氨基酸(Met,Lys,Trp,Phe,Thr,Ile,Leu,Val)(200μM)的荧光响应情况。从图5中可以看出,只有ONOO-能引起荧光光谱的改变,其他检测物对探针的荧光光谱没有明显的影响。这些结果表明,荧光探针对ONOO-有较好的选择性。
实施例6:
溶液pH值对荧光探针测定ONOO-的荧光性质的影响
考察pH值对荧光探针测定ONOO-的荧光光谱的影响,其结果如图6。我们研究的pH范围为4.5~10.0,荧光探针的浓度为10μM,ONOO-的浓度为40μM。从图中可以看出,荧光探针随着pH的变化,荧光强度比值(F477/F717)基本不变,说明pH对探针本身没有很大的影响。然而,加入ONOO-之后,当pH<6时,荧光强度随pH的下降而降低;当pH为6.0~8.6范围时,荧光强度比值基本不变;当pH>8.6时。荧光强度比值逐渐下降。综上所述,当pH值在6.0到8.6之间时,不影响荧光探针对ONOO-的测定,是比较合适的pH值范围,这非常有利于该探针用于实际样品中ONOO-的测定。
实施例7:
荧光探针与ONOO-作用的响应时间的测定
我们研究了荧光探针对ONOO-的响应时间,其结果如图7。从图中可以看出,该探针对ONOO-的响应时间为15min,这能够满足在实际样品中进行实时监测时对响应时间的要求。从图7我们还可以看出,荧光强度达到最大值后,在之后的时间里,荧光强度不再发生变化,这表明此荧光探针光稳定性较好。
实施例8:
荧光探针在活细胞中的应用
首先,我们做了细胞毒性试验,如图8所示。当加入0~40μM ONOO-探针,细胞的成活率均在90%以上,因此可以说明,该荧光探针毒性较小,可应用于检测活细胞内的ONOO-。然后,我们研究荧光探针在活细胞中的应用,选择HepG2细胞进行共聚焦显微成像,结果如图9所示。在细胞中加入荧光探针,红色通道可以观察到的较强的荧光,而在蓝色通道几乎没有荧光,这说明细胞中的ONOO-较低(图9中的a)。文献报道脂多糖(LPS)和干扰素-γ(IFN-γ)可以共同刺激肝癌细胞释放OONO-。细胞用LPS和IFN-γ预先处理10h,然后用探针染色0.5h,蓝色通道荧光明显增强,而红色通道荧光减弱(图9中的b);细胞用LPS、IFN-γ和AG处理10h,然后用探针染色0.5h,蓝色通道的荧光减弱,红色通道的荧光增强(图9中的c)。图9中的d为相对荧光强度图,这些结果说明荧光探针能监控细胞内ONOO-含量的变化,为监控人体内和过氧化亚硝基相关病变提供一种可靠的手段。
Claims (3)
1.一种靶向性过氧化亚硝基荧光探针,其特征在于,结构如下:
2.根据权利要求1所述的一种靶向性过氧化亚硝基荧光探针的制备方法,其特征在于,反应步骤如下:
在100mL圆底烧瓶中,将1当量的Cy-OH,3~5当量的碳酸铯溶于二氯甲烷中,室温下,在氮气中搅拌10~20分钟;然后,通过注射器加入3~5当量的溴代四乙酰基半乳糖,继续在室温下搅拌10~12小时;通过减压蒸馏除去溶剂,粗产品用体积比为30:1~12:1的CH2Cl2/CH3OH洗脱剂进行柱层析,得到蓝色固体物质;随后,将其溶解在无水甲醇中,加入3~5当量的碳酸钾脱保护,在室温下搅拌反应混合物2~3小时;然后,减压蒸馏除去溶剂,所得粗产品用体积比为15:1~10:1的CH2Cl2/CH3OH洗脱剂柱层析分离得到蓝色固体Cy-Gal,即为荧光探针。
3.根据权利要求1所述的一种靶向性过氧化亚硝基荧光探针的应用,其特征在于,所述荧光探针应用于非疾病诊断与治疗为目的的细胞内过氧化亚硝基检测。
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