CN114605432B - 基于花菁染料的靶向性半胱氨酸荧光探针的制备和应用 - Google Patents

基于花菁染料的靶向性半胱氨酸荧光探针的制备和应用 Download PDF

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CN114605432B
CN114605432B CN202210320936.7A CN202210320936A CN114605432B CN 114605432 B CN114605432 B CN 114605432B CN 202210320936 A CN202210320936 A CN 202210320936A CN 114605432 B CN114605432 B CN 114605432B
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李春艳
顾青松
费俊杰
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Abstract

本发明涉及基于花菁染料的靶向性半胱氨酸荧光探针的制备和应用，该荧光探针的结构式为：
Figure DDA0003559150390000011
本发明提供了以花菁荧光染料、D‑生物素、3,4‑二甲氧基苯硫酚等为原料合成该荧光探针的制备方法；该荧光探针是一种具有近红外发射、高选择性和能够靶向细胞的半胱氨酸荧光探针。首先，该荧光探针对Cys表现出很高的灵敏度，探针与Cys反应之后荧光显著增强；其次，该荧光探针对Cys表现出很好的选择性，不受其他常见无机离子、活性氧、活性氮、活性硫、氨基酸以及生物硫醇的干扰；此外，该荧光探针已成功用于细胞中的Cys成像，可以检测细胞中的半胱氨酸水平。
Figure DDA0003559150390000012

Description

基于花菁染料的靶向性半胱氨酸荧光探针的制备和应用
技术领域
本发明属于荧光探针技术领域，具体涉及基于花菁染料的靶向性半胱氨酸荧光探针的制备和应用。
背景技术
半胱氨酸(Cys)是一种重要的生物硫醇，在多种生命活动中扮演者重要角色(S.Y.Zhang,C.N.Ong and H.M.Shen,Cancer Lett.,2004,208,143-153；A.Sharma,M.G.Lee,M.Won,S.Koo,J.F.Arambula,J.L.Sessler,J.Am.Chem.Soc.,2019,141,15611-15618)。科学研究表明，Cys的不正常代谢与许多疾病有关，包括癌症、造血功能减退、牛皮癣、水肿、肝脏损伤等(L.Cui,Y.Baek,S.Lee,N.Kwon,J.Yoon,J.Mater.Chem.C,2016,4,2909-2914；G.Liu,D.Liu,X.Han,X.Sheng,Z.Xu,S.H.Liu,L.Zeng,J.Yin,Talanta,2017,170,406-412)。然而，Cys在活细胞中含量极低，在生物环境中检测Cys仍然是一个巨大的挑战(N.Karton-Lifshin,E.Segal,L.Omer,M.Portnoy,R.Satchi-Fainaro,D.Shabat,J.Am.Chem.Soc.,2011,133,10960-10965；X.Liu,L.Wang,T.Bing,N.Zhang,S.Dihua,ACSAppl.Bio Mater.,2019,2,1368-1375)。因此，发展一种高灵敏度检测Cys的策略具有重要意义。
到目前为止，已经开发了许多检测Cys的方法，比如：电化学法(Y.N.Zhong,M.H.Lin,J.D.Zhou,Y.J.Liu,Chin.J.Anal.Chem.,2010,38,229-232)，流动注射分析法(A.Waseem,M.Yaqoob,A.Nabi,Chem.Res.Chinese Universities.,2010,26,893-898)，质谱法(N.Burford,M.D.Eelman,D.E.Mahony,M.Morash,Chem.Commun.,2003,1,146-147)等。相比于这些传统的方法，荧光分析方法具有高灵敏度，实时监测和高分辨率成像生物样本等优势。到目前为止，有许多检测Cys的荧光探针被报道(P.Yue,X.Yang,P.Ning,X.Xi,H.Yu,Y.Feng,R.Shao,X.Meng,Talanta.,2018,178,24-30；N.Karton-Lifshin,E.Segal,L.Omer,M.Portnoy,R.Satchi-Fainaro,D.Shabat,J.Am.Chem.Soc.,2011,133,10960-10965；Y.B.Gan,G.X.Yu,T.Yin,Y.Y.Zhang and P.Yin,Talanta.,2020,210,120612-120619；J.Q.Hou,P.F.Cai,C.Y.Wang,Y.J.Shen,Tetrahedron Lett.,2018,59,2581-2585)。但是，这些Cys探针存在一些不足：(1)分析波长较短，因此容易被活体内生物分子产生的自发荧光信号干扰，且组织穿透能力较弱，从而限制了在生物体内的应用；(2)不能主动靶向细胞和组织，难以在特定组织聚集，分散在各处的探针会降低检测效率并使背景荧光进一步增强。因此，设计和合成具有长波长和靶向能力的Cys荧光探针是非常有意义的。
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，它具有摩尔吸光系数大、光稳定性高等优势，最重要的是，具有近红外发射性能。近红外发射能够穿透更深的组织，不易受到生物自体荧光的干扰，对生物成像更有利。生物素(维生素B7)是一种很好的靶向肿瘤细胞的结构，据报道，各种癌细胞，如宫颈癌、乳腺癌、肺癌和卵巢癌，都过度表达生物素受体(Y.Singh,K.K.Durga-Rao-Viswanadham,A.Kumar-Jajoriya,J.G.Meher,K.Raval,S.Jaiswal,J.Dewangan,H.K.Bora,S.K.Rath,J.Lal,Mol.Pharmaceutics.,2017,14,2749–2765；K.Li,L.Qiu,Q.Liu,G.Lv,X.Zhao,S.Wang,J.Lin,J.Photochem.Photobiol.,2017,174,243–250；N.U.Deshpande,M.Jayakannan,Biomacromolecules.,2018,19,3572–3585)，在探针中引入生物素结构是实现肿瘤特异性分布的一种有效策略，探针在细胞和组织的特异性分布有望提升细胞中Cys检测的灵敏度和信噪比。但是，现在还没有能同时靶向细胞和检测Cys的荧光探针。因此，设计和合成一种基于花菁染料的靶向性半胱氨酸荧光探针，作为检测细胞中Cys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，提供了一种基于花菁染料的靶向性半胱氨酸荧光探针
本发明的技术方案是，一种基于花菁染料的靶向性半胱氨酸荧光探针，其结构式如下：
一种基于花菁染料的靶向性半胱氨酸荧光探针的制备方法。步骤如下：
将1当量的Cy-Cl溶解到10～20mL无水DMF中，加入50mL的圆底烧瓶中，在-10～0℃条件下搅拌均匀；将4～6当量的3,4-二甲氧基苯硫酚溶解在4～6mL无水DMF中，缓慢滴加到上述体系；接着，将反应体系用氮气保护并升温到60～80℃，继续搅拌3～4h；反应完成后，冷却至室温，粗产物在减压条件下除去溶剂，用体积比为30:1～10:1的CH2Cl2/CH3OH洗脱剂纯化，得到墨绿色固体化合物Cy-S，即为所述的荧光探针。
本发明的有益效果是，一种基于花菁染料的靶向性半胱氨酸荧光探针的良好的光谱响应性能。首先，研究该探针的荧光光谱性质。荧光探针本身没有明显的近红外发射峰；加入Cys后，在760nm处出现了明显的近红外发射峰。并且随着Cys浓度的增大，探针的近红外荧光强度不断增强。该探针的检测范围从3μM到100μM，检测限为0.07μM，这说明该探针对Cys有高灵敏度。接着，研究探针的紫外吸收光谱。探针本身在795nm附近有吸收带，加入Cys后，795nm附近的吸收峰明显减小，在645nm附近出现新的吸收峰，溶液颜色从浅绿色变为蓝色。然后，研究探针的选择性。考察了探针与无机离子(Mg2+,K+,Fe2+,Na+)，活性氧(H2O2,ClO-,O2 -)，活性氮(NO2 -,NO3 -)，活性硫(H2S,SO3 2-,SO4 2-)，常见氨基酸(Phe,Lys,Leu,Val,Trp,Ile,Met,Thr,His)以及生物硫醇(Hcy,GSH,Cys)的荧光响应情况。结果发现，只有Cys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Cys的影响，随着pH值增加，760nm处的荧光逐渐增强，在pH＝7.4的生理环境下，荧光强度增强了200倍以上，表明此荧光探针可以灵敏地检测溶液中的Cys。
基于花菁染料的靶向性半胱氨酸荧光探针的应用。未经探针染色的肝癌细胞不发出荧光；在细胞中加入荧光探针Cy-S，荧光明显增强；随后，将细胞用NEM(细胞内Cys抑制剂)处理，再用探针Cy-S染色进行成像，发现几乎没有荧光产生；细胞经NEM处理后再用Cys处理，最后使用探针Cy-S染色成像，产生了很强的荧光。这些结果说明探针Cy-S能够灵敏地检测细胞内的Cys，这为监控人体内Cys相关病变提供了一种可靠的手段。
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Cys作用后的荧光光谱图。
横坐标为波长，纵坐标为荧光强度。荧光探针的浓度为10μM，Cys浓度分别为：0,3,5,7,10,20,30,40,50,60,70,80,90,100μM。荧光激发波长为645nm。
图3为荧光探针对不同Cys浓度的荧光线性响应图。
图4为荧光探针与Cys作用前后的紫外可见吸收光谱图。
荧光探针的浓度为10μM，Cys浓度为100μM。
图5为荧光探针的选择性图。
荧光探针的浓度为10μM，Cys浓度为100μM，其它分析物浓度均为200μM。
图6为pH对荧光探针的影响图。
荧光探针的浓度为10μM，Cys浓度为100μM。
图7为荧光探针与Cys作用的响应时间的测定。
荧光探针的浓度为10μM，Cys浓度为0,40,70,100μM。
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Cys作用的细胞成像图。
(a)细胞未经探针染色；(b)细胞用探针Cy-S染色30min；(c)细胞先用NEM处理20min，再用探针Cy-S染色30min；(d)细胞先经NEM处理20min，接着用Cys处理20min，再用探针Cy-S染色30min；(e)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。Cys荧光探针(Cy-S)的合成：在50mL的圆底烧瓶中，将Cy-Cl(85mg,0.1mmol)溶解到15mL无水DMF中，在-5℃条件下搅拌均匀；将3,4-二甲氧基苯硫酚(73μL,0.5mmol)溶解在5mL无水DMF中，缓慢滴加到上述体系；接着，将反应体系用氮气保护并升温到70℃，继续搅拌3.5h；反应完成后冷却至室温，粗产物在减压条件下除去溶剂，用体积比为20:1的CH2Cl2/CH3OH洗脱剂纯化，得到墨绿色固体产物(产率55％)，即为荧光探针Cy-S。1H NMR(400MHz,DMSO-d6)δ8.68(d,J＝14.2Hz,1H),8.61(d,J＝13.9Hz,1H),7.95(t,J＝5.7Hz,1H),7.54(dd,J＝15.0,7.4Hz,2H),7.46–7.31(m,4H),7.29–7.16(m,2H),6.96(d,J＝2.2Hz,1H),6.88(d,J＝8.5Hz,1H),6.69(dd,J＝8.5,2.2Hz,1H),6.42(s,1H),6.36(t,J＝7.2Hz,2H),6.22(d,J＝14.1Hz,1H),4.31–4.17(m,3H),4.15–4.06(m,3H),3.73(s,3H),3.63(s,3H),3.45(t,J＝5.3Hz,2H),3.15(t,J＝3.6Hz,2H),3.06(d,J＝10.2Hz,1H),2.82–2.67(m,5H),2.54(d,J＝12.5Hz,2H),2.07(t,J＝6.9Hz,2H),1.93–1.73(m,4H),1.47(d,J＝5.5Hz,12H),1.27(t,J＝7.2Hz,6H).13CNMR(100MHz,DMSO-d6)δ172.2,171.2,162.7,150.6,149.3,147.4,145.9,144.5,142.1,141.6,141.3,140.9,133.5,132.8,128.6,128.5,127.8,125.3,124.7,122.5,122.4,118.5,113.2,111.4,111.0,110.5,102.0,101.0,72.0,70.0,61.1,60.3,59.2,55.7,55.6,55.5,49.0,48.6,41.5,36.0,35.3,31.4,28.4,28.1,27.3,27.1,25.9,25.3,20.6,18.8,13.8,12.3.HRMS(ESI-MS):m/z found,900.4563(calcd for C53H66N5O4S2 +,900.4551)。
实施例2：
荧光探针和Cys溶液配制
探针溶液的制备：称取一定量探针溶解在二甲基亚砜(DMSO)中，配成1×10-4M的探针溶液。Cys储备溶液的制备：将一定量的Cys超声溶解在二次蒸馏水中，配成1×10-3M的Cys溶液。将1mL的探针溶液、不同体积的Cys储备溶液加入10mL容量瓶，用pH＝7.4的PBS缓冲溶液定容。得到含有1.0×10-5M荧光探针、3.0×10-6～1.0×10-4M的Cys的待测溶液。
实施例3：
荧光探针与Cys作用的荧光光谱的测定
图2为荧光探针与Cys作用的荧光光谱，荧光探针的浓度为10μM，Cys浓度分别为：0,3,5,7,10,20,30,40,50,60,70,80,90,100μM。荧光激发波长为645nm，发射波长范围为700～820nm。激发和发射狭缝宽度均为5nm，所用的荧光测定仪器为日立F4600荧光分光光度计。从图中可以看出，由于3,4-二甲氧基苯硫基团的淬灭作用，荧光探针本身没有明显的近红外发射峰，加入Cys后，在760nm处出现了明显的近红外发射峰。这是由于Cys取代了荧光探针的3,4-二甲氧基苯硫基，释放了花菁荧光团，从而产生近红外荧光。并且，随着Cys浓度的增大，探针分子的近红外荧光强度不断增强。图3为探针对不同Cys浓度的线性响应图。荧光强度跟Cys的浓度呈现线性关系，线性范围是3～100μM，检测限是0.07μM。这说明该探针可以高灵敏地检测Cys。
实施例4：
荧光探针与Cys作用的紫外可见吸收光谱的测定
图4为荧光探针与Cys作用后的紫外可见吸收光谱图，荧光探针的浓度为10μM，Cys的加入量为100μM。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。从图中可以看出，探针本身在795nm处有吸收带，加入Cys之后，795nm处的吸收峰降低，在645nm附近出现新的吸收峰。
实施例5：
荧光探针对Cys测定的选择性
图5为荧光探针对Cys测定的选择性图。考察在浓度为10μM的荧光探针溶液中加入Cys(100μM)以及生物体内常见的无机离子(Mg2+,K+,Fe2+,Na+)，活性氧(H2O2,ClO-,O2 -)，活性氮(NO2 -,NO3 -)，活性硫(H2S,SO3 2-,SO4 2-)，常见氨基酸(Phe,Lys,Leu,Val,Trp,Ile,Met,Thr,His)以及生物硫醇(Hcy,GSH)(200μM)的荧光响应情况。从图中可以看出，只有Cys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Cys有较好的选择性。
实施例6：
溶液pH值对荧光探针测定Cys的荧光性质的影响
考察pH值对荧光探针测定Cys的荧光光谱的影响，其结果如图6。我们研究的pH范围为4.0～10.0，荧光探针的浓度为10μM，Cys的浓度为100μM。从图中可以看出，荧光探针随着pH的增加，荧光始终处于关闭状态，说明pH对探针本身没有很大的影响。然而，加入Cys之后，随着pH的增加，荧光强度显著增强，在pH值为7.0～8.0的生理pH范围内，荧光强度基本稳定。综上所述，该探针具有生物样品测试的潜力。
实施例7：
荧光探针与Cys作用的响应时间的测定
我们研究了荧光探针对Cys的响应时间，其结果如图7。从图中可以看出，该探针对Cys的响应时间为30min，这能够满足在实际样品中进行实时监测的要求。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0～30μM荧光探针，细胞的成活率均在90％以上。这可以说明，该荧光探针毒性较小，可应用于检测活细胞内的Cys。然后，我们研究荧光探针在活细胞中的应用，选择肝癌细胞进行共聚焦显微成像，结果如图9所示。未经染色的肝癌细胞不发出荧光(图9中的a)；细胞用探针Cy-S染色30min后，荧光明显增强(图9中的b)；随后，我们先将细胞用NEM处理20min，然后用探针Cy-S染色30min进行成像，发现几乎没有荧光产生(图9中的c)；细胞经NEM处理20min后再用Cys处理20min，最后使用探针Cy-S染色30min成像，产生了很强的荧光(图9中的d)。图9中的e为相对荧光强度图，这些结果说明探针Cy-S能够灵敏地检测细胞内的Cys，这为监控人体内Cys相关病变提供了一种可靠的手段。

Claims (3)

1.一种基于花菁染料的靶向性半胱氨酸荧光化合物，即Cy-S，其结构如下：
Figure FDA0004036114320000011
2.根据权利要求1所述的一种基于花菁染料的靶向性半胱氨酸荧光化合物的制备方法，其特征在于，反应步骤如下：
将1当量的Cy-Cl溶解到10～20mL无水DMF中，加入50mL的圆底烧瓶中，在-10～0℃条件下搅拌均匀；将4～6当量的3,4-二甲氧基苯硫酚溶解在4～6mL无水DMF中，缓慢滴加到上述体系；接着，将反应体系用氮气保护并升温到60～80℃，继续搅拌3～4h；反应完成后，冷却至室温，粗产物在减压条件下除去溶剂，用体积比为30:1～10:1的CH2Cl2/CH3OH洗脱剂纯化，得到墨绿色固体化合物Cy-S，即为所述的荧光化合物，其中，Cy-Cl的结构如下：
Figure FDA0004036114320000012
3.根据权利要求1所述的一种基于花菁染料的靶向性半胱氨酸荧光化合物在制备探针中的应用，其特征在于，所述荧光化合物应用于细胞内半胱氨酸含量的检测。
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