CN113308131A - 一种羧基修饰的近红外方酸染料及其制备方法与应用 - Google Patents
一种羧基修饰的近红外方酸染料及其制备方法与应用 Download PDFInfo
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Abstract
本发明公开了一种羧基修饰的近红外方酸染料及其制备方法与应用,其是以羧基修饰的2‑甲基苯并噻唑衍生物和二氰乙烯基方酸衍生物为原料制得。该近红外方酸染料具有较好的稳定性及优异的光学性能,尤其是羧酸基团可增强染料的水溶性。将该近红外方酸染料用于平行、混合型G‑四联体的检测时,G‑四联体可与染料分子相互作用,在不引起G‑四联体拓扑结构改变的同时,染料吸收光谱和荧光发射光谱发生改变,从而可作为平行、混合型G‑四联体检测的荧光探针。
Description
技术领域
本发明属于分析化学领域,具体涉及一种羧基修饰的近红外方酸染料制备方法与其在平行、混合型G-四联体的荧光检测上的应用。
背景技术
G-四联体是由富含鸟嘌呤的核酸序列形成的一种非典型结构。G-四联体在体内发挥着重要作用,如端粒维持、转录和DNA复制。G-四分体是在阳离子存在时形成的四个鸟嘌呤碱基平面,其进一步堆积成四链体螺旋结构G-四联体。一般来说,金属阳离子,如钾和钠,通过在G-四分体中的协调来稳定G-四联体。G-四联体可以根据不同的拓扑结构分为三类:平行、反平行、混合型G-四联体。平行G-四联体即四条链都相互平行;混合型G-四联体指三条链平行,一条链反平行;反平行G-四联体指两条链平行而另外两条链与之反平行的结构。
到目前为止,已经有多种分析技术被报道用于检测G-四联体,例如x射线晶体学,核磁共振波谱学和圆二色谱学(CD)等。荧光探针也是检测G-四联体常用的方法之一,因其选择性好、灵敏度高、使用方便、成本低以及最重要的是对于G-四联体的折叠不会有影响等优点逐渐成为探究G-四联体的重要工具之一。
方酸染料是由方酸或其酯与含富电子基团物质,如芳胺、酚类或含氮杂环化合物,发生缩合反应生成的于近红外区有强烈吸收的1,3-二取代衍生物。该类化合物的显著特征是在可见光至近红外区有狭窄而强的吸收带和较好的光稳定性。相对于其它有机染料而言,方酸染料由于其优异的荧光发射性能、良好的光学稳定性及易修饰等特点,拥有更广的应用前景。
本发明通过对方酸染料的结构进行优化,合成了羧基修饰的方酸染料,增强了其水溶性,并实现了其在平行、混合型G-四联体荧光检测中的应用,具有良好的应用前景。
发明内容
本发明的目的在于提供一种羧基修饰的近红外方酸染料制备方法与其在平行、混合型G-四联体荧光检测中的应用。
为了实现上述目的,本发明采用如下的技术方案:
一种羧基修饰的近红外方酸染料,其结构式如下:
所述羧基修饰的近红外方酸染料的制备方法包括以下步骤:
(2)冷却至室温,减压除去溶剂,得粗产品;
(3)经硅胶柱层析纯化,得到所述的羧基修饰的近红外方酸染料;
其中,步骤(1)中所用溶剂为正丁醇:甲苯:喹啉按体积比5: 5: 1组成的混合溶液,所述回流的温度为110℃,时间为15小时;
步骤(3)所述硅胶柱层析采用体积比为10: 1的二氯甲烷和甲醇的混合溶液作为洗脱剂。
进一步地,所述羧基修饰的2-甲基苯并噻唑衍生物的合成方法包括如下步骤:
(1)将6-溴己酸与2-甲基苯并噻唑混合,加热反应;
(2)冷却至50℃,加入5 mL乙酸乙酯,再加入25 mL丙酮溶液,搅拌至粘稠物质全部溶解;
(3)冷却至室温,析出白色固体,抽滤,用甲醇多次洗涤,得到白色粉末,为所述羧基修饰的2-甲基苯并噻唑衍生物;
其中所述反应的温度为110℃,反应时间为8小时。
(2)反应完成后,将反应瓶置于冰箱冷却,然后用二氯甲烷萃取,减压除去溶剂,得所述二氰乙烯基方酸衍生物;
其中步骤(1)所用溶剂为苯,搅拌反应时间为6小时;
步骤(2)中冷却的时间为24小时。
进一步地,所述方酸二乙酯的合成方法包括如下步骤:
(1)将方酸溶于溶剂中,加热回流反应3小时;
(2)旋蒸除去溶剂,加入溶剂,加热回流反应30分钟;
(3)重复步骤(2)三次,反应结束冷却至室温;
(4)所得产物经硅胶柱层析纯化,得到所述方酸二乙酯;
其中所用溶剂为乙醇,回流温度为80℃;
所述硅胶柱层析采用体积比为2: 1的石油醚和乙酸乙酯的混合溶液作为洗脱剂。
应用:所得羧基修饰的近红外方酸染料可制成荧光响应的平行、混合型G-四联体探针,用于溶液中平行、混合型G-四联体的荧光检测。
本发明的显著优点在于:
本发明将羧基修饰的2-甲基苯并噻唑衍生物连接到二氰乙烯基方酸骨架上,得到了一种羧基修饰的对称型近红外方酸染料。其中,羧基的引入大大提高了染料的水溶性,且可能与G-四联体上的碱基存在氢键作用;二氰乙烯基的引入在提升染料光稳定性的同时,还能使染料具有顺式构象,这种V型构象能提升染料对平行G-四联体的选择性。当染料单独存在于缓冲溶液中时,在极性水分子的作用下,处于H-聚集状态,此时荧光猝灭,一旦加入平行或混合型G-四联体,则会与之相互作用,染料解聚集,荧光恢复,从而实现对平行、混合型G-四联体的检测。
本发明所得的方酸染料合成简单,对平行、混合型G-四联体有较好的选择性,且在检测G-四联体的同时不会破坏G-四联体的拓扑结构,即便在存在大量双链DNA的缓冲溶液中,也能选择性地与平行G-四联体结合,能很好地区分平行、混合型G-四联体与反平行G-四联体、单、双链DNA。利用荧光滴定实验得出,染料对平行、混合型G-四联体的检测限为14 -60 nM。
附图说明
图1为在羧基修饰的方酸染料(6 μM)的缓冲溶液中滴加不同浓度DNA(0-20 μM)时的吸收光谱图;
图2为在羧基修饰的方酸染料(6 μM)的缓冲溶液中滴加不同浓度DNA(0-20 μM)时的荧光光谱图(λex= 650 nm, slit= 5 nm/ 5 nm, PMT= 650 V);
图3-1为羧基修饰的方酸染料(6 μM)在712 nm处的荧光强度与c-myc浓度的线性关系图(λex= 650 nm, slit= 5 nm/ 5 nm, PMT= 650 V);
图3-2为羧基修饰的方酸染料(6 μM)在712 nm处的荧光强度与PW.17浓度的线性关系图(λex= 650 nm, slit= 5 nm/ 5 nm, PMT= 650 V);
图3-3为羧基修饰的方酸染料(6 μM)在712 nm处的荧光强度与EAD浓度的线性关系图(λex= 650 nm, slit= 5 nm/ 5 nm, PMT= 650 V);
图3-4为羧基修饰的方酸染料(6 μM)在712 nm处的荧光强度与G3T3浓度的线性关系图(λex= 650 nm, slit= 5 nm/ 5 nm, PMT= 650 V);
图3-5为羧基修饰的方酸染料(6 μM)在712 nm处的荧光强度与C-Kit1浓度的线性关系图(λex= 650 nm, slit= 5 nm/ 5 nm, PMT= 650 V);
图4为1 小时内羧基修饰的方酸染料(6 μM)溶液中加入平行G-四联体c-myc(20 μM)后712 nm处的荧光强度随时间变化图(λex= 650 nm, slit= 5 nm/ 5 nm, PMT= 650 V);
图5为不同比例羧基修饰的方酸染料与平行G-四联体c-myc的圆二色谱图;
图6为羧基修饰的方酸染料与不同G-四联体的Job’s plot图(λex= 650 nm, slit=5 nm/ 5 nm, PMT= 650 V);
图7为羧基修饰的方酸染料(2 μM)中加入平行G-四联体c-myc(2 μM)的712 nm处的荧光强度随体系中双链DNA ds12浓度变化图(λex= 650 nm, slit= 5 nm/ 5 nm, PMT=650 V)。
具体实施方式
为了使本发明所述的内容更加便于理解,下面结合具体实施方式对本发明所述的技术方案做进一步的说明,但是本发明不仅限于此。
实施例1
在50 mL圆底烧瓶中先加入20 mL乙醇,再加入方酸(2.00 g, 17.5 mmol),80℃下回流反应3小时,待方酸全部溶解后减压旋蒸除去乙醇。再次加入20 mL乙醇于烧瓶中,回流0.5小时,旋蒸除去溶剂,如此反复操作三次。反应结束后,用硅胶柱层析分离。洗脱剂为石油醚:乙酸乙酯(2: 1, v/v),得到浅黄色油状液体产物2.09 g,产率为70%。1H NMR (400MHz, CDCl3) δ 4.74 (q, J=7.1 Hz, 4H), 1.48 (t, J=7.1 Hz, 6H)。
实施例2
将1.77 g 的丙二腈(26.8 mmol)溶于35 mL干燥苯中,置于100 mL圆底烧瓶中,并逐滴滴入化合物方酸二乙酯(5.00 g, 29.4 mmol),再将三乙胺(2.98 g, 29.8 mmol)慢慢加入上述反应液中,室温搅拌6小时。反应完成后,将反应瓶置于冰箱中冷却过夜,用适量CH2Cl2萃取,取有机层真空浓缩,得到黄色固体产物5.31 g,产率为62%。1H NMR (400 MHz,CDCl3) δ 9.50 (s, 1H), 4.78 (q, J = 6.9 Hz, 2H), 3.65-2.88 (q, J = 6.9 Hz,6H), 1.50 (t, J = 7.0 Hz, 3H), 1.39 (t, J = 6.9 Hz, 9H)。
实施例3
于50 mL二颈烧瓶中加入2-甲基苯并噻唑(601 mg, 4.03 mmol),6-溴己酸(782mg, 4.03 mmol),通10分钟氮气,于110℃下反应8小时后,有白色粘稠物质产生,将反应液冷却至50℃,往瓶中加入5 mL乙酸乙酯溶液,再加入25 mL丙酮,于50℃环境下搅拌至粘稠物质全部溶解,冷却至室温,抽滤,得白色粉末,用甲醇多次洗涤白色粉末。白色粉末为目标化合物899 mg,产率为65%。1H NMR (400 MHz, CD3OD) δ 8.32 (dd, J = 19.2, 8.4 Hz,2H), 7.95 (m, 1H), 7.85 (m, 1H), 4.80 (t, J = 7.8 Hz, 2H), 3.27 (s, 3H), 2.37(t, J = 7.0 Hz, 2H), 2.08-1.95 (m, 2H), 1.81-1.68 (m, 2H), 1.65-1.52 (m, 2H); 13C NMR (100 MHz, CD3OD) δ 176.52, 175.77, 141.22, 129.66, 129.37, 128.36,124.02, 116.60, 49.53, 33.14, 27.73, 25.68, 24.10。
实施例4
于50 mL接有Dean-Stark蒸馏器的二颈烧瓶中加入羧基修饰的2-甲基苯并噻唑衍生物(549 mg, 1.60 mmol),二氰乙烯基修饰的方酸衍生物(233 mg, 0.80 mmol),8 mL甲苯溶液,8 mL正丁醇溶液,1.60 mL喹啉溶液,通氮气保护,于110℃环境下回流反应15小时。反应结束,冷却至室温,减压旋蒸除去溶剂,硅胶柱提纯,洗脱剂为二氯甲烷:甲醇=10: 1(v/v),得到墨绿色固体277 mg,产率为53%。1H NMR (400 MHz, d6-DMSO) δ 12.02 (s,2H), 7.81 (d, J = 7.8 Hz, 2H), 7.59 (d, J = 8.3 Hz, 2H), 7.42 (t, J = 7.7 Hz,2H), 7.24 (t, J = 7.6 Hz, 2H), 6.02 (s, 2H), 4.19-4.03 (m, 4H), 2.20 (t, J =7.2 Hz, 4H), 1.8-1.63 (m, 4H), 1.54 (dt, J = 16.7, 8.2 Hz, 4H), 1.41 (dd, J =18.2, 11.5 Hz, 4H); 13C NMR (100 MHz, d6-DMSO) δ 174.79, 173.29, 162.95,161.01, 160.37, 140.93, 128.03, 124.91, 123.03, 118.92, 113.34, 86.41, 46.70,33.99, 27.17, 26.16, 24.60; HRMS(ESI): Calcd for C35H33N4O5S2 +([M+H]+):653.1887, Found: 653.1882。
应用实施例
选取9种DNA,其中包括三种平行G-四联体(c-myc:TGAGGGTGGGTAGGGTGGGTAA、PW.17:GGGTAGGGCGGGTTGGG、EAD:CTGGGTGGGTGGGTGGGA);两种混合G-四联体(G3T3:GGGTTTGGGTTTGGGTTTGGG、C-Kit1:GGGAGGGCGCTGGGAGGAGGG);两种反平行G-四联体(TBA:GGTTGGTGTGGTTGG、Q1:GGTTAGGTTAGGTTAGG);一种单链DNA(dt21:TTTTTTTTTTTTTTTTTTTTT);一种双链DNA(ds12:GCGCAATTGCGC)进行测试。
1. 在含有6 μM实施例4所得近红外方酸染料的缓冲溶液中,进行DNA的吸收滴定实验,结果见图1。测定DNA浓度为:0, 1, 3, 5, 7, 10, 12, 15, 20 μM。
如图1可见,当近红外方酸染料单独存在于缓冲溶液(10 mM Tris-HCl, 100 mMKCl, pH=7.4)中时,处于聚集状态,随着体系中加入平行、混合型G-四联体,其680 nm附近的吸收增强,但是即便加入20 μM的反平行G-四联体或者单、双链DNA,680 nm附近吸收也无明显增强。
2. 在含有6 μM实施例4所得近红外方酸染料的缓冲溶液中,进行DNA的荧光滴定实验,结果见图2、图3-1至图3-5。测定DNA浓度为:0, 1, 3, 5, 7, 10, 12, 15, 20 μM。
由图2可以看出,当近红外方酸染料单独存在于缓冲溶液(10 mM Tris-HCl, 100mM KCl, pH=7.4)中时,荧光猝灭,随着体系中平行、混合型G-四联体浓度的逐渐增加,其荧光强度也逐渐恢复,当体系中c-myc、PW.17、EAD这三种G-四联体的浓度达到20 μM时,其712nm处荧光强度分别提高了约40、54、36倍。
如图3-1至图3-5所示,近红外方酸染料对不同平行、混合型G-四联体的检测限及线性范围为:c-myc(23 nM,1-15 μM);PW.17(14 nM,1-10 μM);EAD(36 nM,1-20 μM);G3T3(60 nM,1-20 μM);C-Kit1(50 nM,1-12 μM)。
3. 如图4所示,为了确定测试前的孵育时长,我们测定了1小时内6 μM 方酸染料与20 μM c-myc在缓冲溶液中相互作用的荧光强度变化,在加入G-四联体的30分钟后,体系712 nm处荧光强度基本不变 ,相互作用达到稳定,因而将孵育时长定为30分钟。
4.如图5所示,我们测定了不同比例下的G-四联体c-myc(2.5 μM)与方酸染料的圆二色谱图,证明了方酸染料与G-四联体的结合并不会影响到G-四联体的拓扑结构类型。
5. 为了探究方酸染料与G-四联体结合的化学计量比,我们对其进行了再一次的滴定实验。保持方酸染料与G-四联体的总摩尔浓度为10-5 M,以方酸染料的摩尔分数χSQ-1为横坐标,对应测定的712 nm处的荧光强度变化为纵坐标,绘制Job’s plot图,从而计算方酸染料与不同G-四联体的结合化学计量比如图6。
由图6可知,c-myc、C-Kit1与方酸染料的结合化学计量比为1: 1,PW.17、EAD、G3T3与方酸染料的结合化学计量比为2: 1。
6. 我们测定了缓冲溶液中存在不同浓度双链DNA ds12存在条件下,2 μM 方酸染料与2 μM c-myc的荧光图,以确定存在双链DNA环境下,方酸染料仍然能与平行G-四联体有较好的相互作用(图7)。
由图7可知,即便存在高浓度的双链DNA的干扰,方酸染料对G-四联体仍有较好的特异性。
本领域的技术人员容易理解,以上所述仅为本发明的较佳实例而已,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、等同替换和改进,均应包含在本发明的保护范围之内。
Claims (6)
3.根据权利要求2所述的制备方法,其特征在于:所述羧基修饰的2-甲基苯并噻唑衍生物的合成方法包括如下步骤:
(1)将6-溴己酸与2-甲基苯并噻唑混合,加热反应;
(2)冷却至50℃,加入5 mL乙酸乙酯,再加入25 mL丙酮,搅拌至粘稠物质全部溶解;
(3)冷却至室温,析出白色固体,抽滤,用甲醇多次洗涤,得到白色粉末,为所述羧基修饰的2-甲基苯并噻唑衍生物;
其中步骤(1)所述反应的温度为110℃,反应时间为8小时。
5.根据权利要求4所述的制备方法,其特征在于:所述方酸二乙酯的合成方法包括如下步骤:
(1)将方酸溶于溶剂中,加热回流反应3小时;
(2)旋蒸除去溶剂,加入溶剂,加热回流反应30分钟;
(3)重复步骤(2)三次,反应结束冷却至室温;
(4)所得产物经硅胶柱层析纯化,得到所述方酸二乙酯;
其中所用溶剂为乙醇,回流温度为80℃;
所述硅胶柱层析采用体积比为2: 1的石油醚和乙酸乙酯的混合溶液作为洗脱剂。
6.一种如权利要求1所述的羧基修饰的近红外方酸染料在平行、混合型G-四联体荧光检测中的应用,其特征在于:将所述羧基修饰的近红外方酸染料制成响应平行、混合型G-四联体的荧光探针。
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