CN113121599B - 一种靶向线粒体g-四链体dna的荧光探针及其制备方法和应用 - Google Patents
一种靶向线粒体g-四链体dna的荧光探针及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种靶向线粒体G‑四链体DNA的荧光探针及其制备方法和应用。所述荧光探针的结构如式(Ⅰ)所示；其中，R1为H、F、Cl、Br或I；R2为‑O(CH2)n三苯基磷，其中n为2～10中任意一个整数；R3为N‑甲基哌嗪、‑NR4R5或‑NH(CH2)mR6，其中m为1～8中任意一个整数，R4和R5各自独立地为氢、C1～8烷基或C1～8卤代烷基，R6为胺基、C1～8烷胺基或C1～8烷氧基取代胺基；A‑为N甲基化阴离子、卤离子、对甲苯磺酸离子或三氟甲磺酸离子。所述荧光探针可特异性地检测识别线粒体G‑四链体DNA结构，不受其他组分的干扰；同时其具有较好的化学稳定性、光稳定性、溶解性和生物兼容性，在线粒体G‑四链体DNA生物学功能研究上具有广阔的应用空间。
Description
技术领域
本发明涉及G-四链体DNA检测技术领域，更具体地，涉及一种靶向线粒体G-四链体DNA的荧光探针及其制备方法和应用。
背景技术
G-四链体(G-quadruplex)DNA是由富含鸟嘌呤的核酸序列通过hoogsteen氢键首先形成G-四分体，G-四分体进一步堆积折叠形成G-四链体DNA。生物信息学分析发现，人类基因组中约有37万个具有形成G-四链体可能性的序列，包括端粒末端鸟嘌呤重复序列启动子区、核糖体DNA(rDNA)、转录起始位点(TSS)、非翻译区(UTR)等区域，预示着G-四链体在人类基因组和转录组中扮演着重要的角色，参与了许多重要生命过程的调控。近十年来，对G-四链体的研究主要集中于核DNA G-四链体；近年的研究发现线粒体DNA同样能形成G-四链体结构，然而对线粒体G-四链体DNA的生物学功能知之甚少。因此，在细胞内能够特异性地检测出线粒体G-四链体DNA的存在，对于研究线粒体G-四链体DNA相关生物学功能以及对阐明与线粒体受损有关的细胞功能障碍及疾病的发病机制有着重要的意义。
目前，在细胞内能检测DNA G-四链体结构的研究取得了一些进展。已有一些荧光探针能够实现G-四链体DNA细胞内的检测。然而能够特异性识别线粒体G-四链体DNA的荧光探针未见报道。而且由于体内大大过量的其他核酸二级结构的存在，以及复杂的细胞内环境，使得细胞内特异性检测线粒体G-四链体DAN相对于核G-四链体DNA检测需要解决更多的难题。
因此，亟待于提供一种细胞内特异性识别线粒体G-四链体DNA的荧光探针。
发明内容
本发明的目的在于针对现有技术中缺少可特异性识别线粒体G-四链体DNA的荧光探针的不足，提供一种靶向线粒体G-四链体DNA的荧光探针。本发明所述荧光探针可在细胞内特异性的识别线粒体G-四链体DNA，而不受细胞内其他组分的干扰，特别是其他位置G-四链体结构的干扰，例如核G-四链体DNA、G-四链体RNA，进而能够准确的检测和实时示踪活细胞中线粒体G-四链体DNA。
本发明的另一目的在于提供所述靶向线粒体G-四链体DNA的荧光探针的制备方法。
本发明的再一目的在于提供所述靶向线粒体G-四链体DNA的荧光探针的应用。
本发明的上述目的是通过以下方案予以实现的：
一种靶向线粒体G-四链体DNA的荧光探针，其结构如式(Ⅰ)所示：
其中，R1为H、F、Cl、Br或I；R2为-O(CH2)n三苯基磷，其中n为2～10中任意一个整数；R3为N-甲基哌嗪、-NR4R5或-NH(CH2)mR6，其中m为1～8中任意一个整数，R4和R5各自独立地为氢、C1～8烷基或C1～8卤代烷基，R6为胺基、C1～8烷胺基或C1～8烷氧基取代胺基；A-为N甲基化阴离子、卤离子、对甲苯磺酸离子或三氟甲磺酸离子。
优选地，所述R2为-O(CH2)n三苯基磷，其中n为2～6中任意一个整数；R3为N-甲基哌嗪、-NR4R5或-NH(CH2)mR6，其中m为1～5中任意一个整数，R4和R5各自独立地为氢、C1～4烷基或C1～4卤代烷基，R6为胺基、C1～4烷胺基或C1～4烷氧基取代胺基。
优选地，所述R2为-O(CH2)n三苯基磷，其中n为2～6中任意一个整数；R3为-NR4R5，R4和R5各自独立地为氢、C1～4烷基或C1～4卤代烷基。
优选地，所述R2为-O(CH2)n三苯基磷，其中n为2～6中任意一个整数；所述R3为-NR4R5，R4和R5各自独立地为氢、C1～4烷基或C1～4卤代烷基。
优选地，所述R2为-O(CH2)4三苯基磷。
优选地，所述R3为-NR4R5，R4和R5各自独立地为氢、甲基、乙基、丙基、三氟甲基或三氟乙基。
优选地，所述A-为卤离子、对甲苯磺酸离子或三氟甲磺酸离子。
优选地，所述荧光探针的结构如下结构之一所示：
本发明同时还保护所述靶向线粒体G-四链体DNA的荧光探针的制备方法，包括如下步骤：
S1.2-吡咯烷酮与式2化合物在三氯氧磷存在条件下反应，得式3化合物；
S2.式3化合物在甲醇钠在甲醇作用下反应，得式4化合物；再脱去甲基，得式5化合物；
S3.式5化合物与Br(CH2)n Br发生取代反应，得式6化合物，然后再与三苯基磷发生取代反应，得式7化合物；
S4.式7化合物与甲基化试剂反应，得式8化合物，然后与式9化合物反应，制备得到式(Ⅰ)化合物；
其中式2至式9化合物的结构如下所示，其中n为2～10中任意一个整数；
所述靶向线粒体G-四链体DNA的荧光探针在检测线粒体G-四链体DNA中的应用也在本发明的保护范围之内。
与现有技术相比，本发明具有以下有益效果：
本发明提供的荧光探针在活细胞内可以特异性地检测识别线粒体G-四链体DNA结构，检测过程不受其他组分的干扰；同时所述荧光探针具有较好的化学稳定性、光稳定性，较好的溶解性和生物兼容性，制备过程简单、成本低廉在线粒体G-四链体DNA生物学功能研究上具有广阔的应用空间。
附图说明
图1为荧光探针MitoISCH-1在Tris-盐酸缓冲液的紫外可见吸收光谱。
图2为向荧光探针MitoISCH-1在Tris-HCl缓冲溶液中滴加不同量的线粒体G-四链体DNA Mito27的紫外可见吸收光谱。其中，荧光探针的浓度为5μM。
图3为荧光探针MitoISCH-1中滴加不同线粒体DNA样品的荧光光谱图。其中，荧光探针的浓度为1μM，线粒体DNA样品的浓度为3μM。
图4为荧光探针MitoISCH-1在Tris-HCl缓冲溶液中滴加不同线粒体DNA样品的荧光滴定曲线。其中，荧光探针的浓度为1μM。
图5为荧光探针MitoISCH-1检测活细胞内线粒体G-四链体DNA的激光共聚焦显微成像。
具体实施方式
下面结合具体实施例对本发明做出进一步地详细阐述，所述实施例只用于解释本发明，并非用于限定本发明的范围。下述实施例中所使用的试验方法如无特殊说明，均为常规方法；所使用的材料、试剂等，如无特殊说明，为可从商业途径得到的试剂和材料。
实施例1化合物MitoISCH-1，MitoISCH-2，MitoISCH-3的合成
化合物MitoISCH-1，MitoISCH-2，MitoISCH-3的具体制备过程如下：
(1)取10g 2-氨基-4，5-二氟苯甲酸和18g 2-吡咯烷酮溶于150mL干燥甲苯中，搅拌至充分溶解后；室温下，缓慢滴加35mL POCl3。室温下搅拌5h。旋蒸出甲苯和三氯氧磷，将浓缩液倒入冰水中，调节pH至弱碱性，析出大量白色固体。抽滤干燥得10.47g白色固体，即化合物(a)。
(2)取5g化合物(a)和5g甲醇钠溶于50mL甲醇中，60℃回流反应24h，冷却至室温，减压抽滤，蒸干滤液得4.6g白色固体，即化合物(b)。
(3)取4g化合物(b)溶于20mL冰乙酸和20mL氢溴酸，加热140℃回流反应48h，抽滤，蒸干滤液得3.4g白色固体，即化合物(c)。
(4)将2g化合物(c)，2.5g碳酸钾和2.4mL 1,4-二溴丁烷溶于40mL丙酮，60℃回流搅拌24h,减压抽滤，蒸干滤液。以甲醇︰二氯甲烷(体积比1︰250)为洗脱剂通过硅胶层析纯化，分离得到2.8g白色固体，即化合物(d)。
(5)将1g化合物(d)和1.3g三苯基膦溶于20mL乙腈中,90℃回流搅拌24h。冷却至室温，蒸干乙腈，以甲醇︰二氯甲烷(体积比1︰50)为洗脱剂通过硅胶层析纯化，分离得到1.4g白色固体,即化合物(e)。
(6)将0.5合物(e)溶于2mL乙腈中，加入1mL碘甲烷，70℃下反应
24h，冷至室温，抽滤，用无水乙醚洗后真空干燥得0.5g浅黄色固体，即化合物(f)。
(7)将0.4g即化合物(f)和0.18g 7-(二乙基氨基)香豆素醛溶于20mL乙醇中,加入催化量的哌啶，90℃回流搅拌过夜。冷却至室温，蒸干乙醇，以甲醇︰二氯甲烷(体积比1︰25)为洗脱剂通过硅胶层析纯化，分离得到0.37g棕褐色固体，即化合物(MitoISCH-1)。
(8)将0.4g即化合物(f)和0.18g 7-氨基-香豆素醛溶于20mL乙醇中,加入催化量的哌啶，90℃回流搅拌过夜。冷却至室温，蒸干乙醇，以甲醇︰二氯甲烷(体积比1︰20)为洗脱剂通过硅胶层析纯化，分离得到0.32g棕褐色固体，即化合物(MitoISCH-2)。
(9)将0.4g即化合物(f)和0.18g 7-(甲基哌嗪)-香豆素醛溶于20mL乙醇中,加入催化量的哌啶，90℃回流搅拌过夜。冷却至室温，蒸干乙醇，以甲醇︰二氯甲烷(体积比1︰15)为洗脱剂通过硅胶层析纯化，分离得到0.28g棕褐色固体，即化合物(MitoISCH-3)。
化合物MitoISCH-1的结构和核磁共振氢谱数据如下所示：
1H NMR(400MHz,MeOD)δ8.21(s,1H),8.05–8.01(m,2H),7.92–7.71(m,15H),7.60(d,J＝9.1Hz,1H),7.52(d,J＝6.7Hz,1H),6.85(dd,J＝9.1,2.3Hz,1H),6.61(d,J＝2.1Hz,1H),4.48(t,J＝5.8Hz,2H),4.39–4.32(m,5H),3.65-3.52(m,6H),3.48–3.41(m,2H),2.24-2.15(m,2H),2.03-1.91(m,2H),1.25(t,J＝7.1Hz,6H).13C NMR(126MHz,DMSO)δ160.00,159.03,156.64,155.84(d,JC,F＝2.3Hz),153.02(d,JC,F＝12.1Hz),152.51,150.94(d,JC,F＝251.6Hz),144.77,139.03,138.46,134.62(d,4JC,P＝2.9Hz,3C),133.21(d,2JC,P＝10.1Hz,6C),131.37,129.93(d,3JC,P＝12.5Hz,6C),126.68,118.12(d,1JC,P＝85.7Hz,3C),112.73,112.53(d,JC,F＝21.0Hz),112.02(d,JC,F＝7.5Hz),110.13,108.21,104.31,96.31,68.81,46.38,44.18(2C),40.93,28.54(d,2JC,P＝16.8Hz),27.32,20.12(d,1JC,P＝50.7Hz),18.22(d,3JC,P＝3.7Hz),12.06(2C).19F NMR(376MHz,DMSO)δ-131.36.31P NMR(162MHz,DMSO)δ24.06.Purity:99.4％by HPLC.HRMS(ESI):calcd for[(M-2I)/2]2+389.6639,found389.6626.
1H NMR(400MHz,MeOD)δ8.23(s,1H),8.04–8.02(m,2H),7.94–7.70(m,15H),7.63(d,J＝9.0Hz,1H),7.51(d,J＝6.8Hz,1H),6.85(dd,J＝9.0,2.4Hz,1H),6.63(d,J＝2.2Hz,1H),4.45(t,J＝5.6Hz,2H),4.39–4.32(m,5H),3.90(s,2H),3.65-3.52(m,2H),3.47–3.41(m,2H),2.25-2.13(m,2H),2.03-1.91(m,2H).13C NMR(126MHz,DMSO)δ160.05,159.01,156.62,155.83(d,JC,F＝2.4Hz),153.03(d,JC,F＝12.3Hz),152.52,150.96(d,JC,F＝251.8Hz),144.72,139.02,138.44,134.61(d,4JC,P＝2.9Hz,3C),133.22(d,2JC,P＝10.2Hz,6C),131.36,129.94(d,3JC,P＝12.6Hz,6C),126.67,118.14(d,1JC,P＝85.8Hz,3C),112.74,112.51(d,JC,F＝21.1Hz),112.03(d,JC,F＝7.6Hz),110.12,108.23,104.34,96.33,68.80,46.36,40.91,28.52(d,2JC,P＝16.8Hz),27.34,20.15(d,1JC,P＝50.7Hz),18.23(d,3JC,P＝3.8Hz).19F NMR(376MHz,DMSO)δ-131.20.31P NMR(162MHz,DMSO)δ24.00.Purity:98.2％byHPLC.HRMS(ESI):calcd for[(M-2I)/2]2+361.6325,found 361.6332.
1H NMR(400MHz,MeOD)δ8.23(s,1H),8.03–8.02(m,2H),7.91–7.70(m,15H),7.61(d,J＝9.0Hz,1H),7.50(d,J＝6.6Hz,1H),6.86(dd,J＝9.0,2.4Hz,1H),6.63(d,J＝2.2Hz,1H),4.47(t,J＝5.8Hz,2H),4.40–4.33(m,5H),3.67-3.51(m,6H),3.49–3.40(m,2H),2.39-2.30(m,4H),2.26-2.14(m,2H),2.22(s,3H),2.03-1.91(m,2H).13C NMR(126MHz,DMSO)δ160.07,159.05,156.56,155.79(d,JC,F＝2.5Hz),153.13(d,JC,F＝12.3Hz),152.59,150.83(d,JC,F＝251.7Hz),144.68,139.13,138.53,134.67(d,4JC,P＝3.0Hz,3C),133.25(d,2JC,P＝10.3Hz,6C),131.43,129.86(d,3JC,P＝12.8Hz,6C),126.69,118.23(d,1JC,P＝85.9Hz,3C),112.79,112.59(d,JC,F＝21.3Hz),112.13(d,JC,F＝7.8Hz),110.17,108.21,104.38,96.30,68.89,46.43,46.08,44.21(2C),40.87,28.59(d,2JC,P＝16.9Hz),27.39,20.19(d,1JC,P＝50.7Hz),18.27(d,3JC,P＝3.8Hz),12.06(2C).19F NMR(376MHz,DMSO)δ-131.42.31P NMR(162MHz,DMSO)δ24.09.Purity:95.8％by HPLC.HRMS(ESI):calcd for[(M-2I)/2]2+403.1693,found 403.1686.
实施例2性能测试实验
以荧光探针MitoISCH-1为代表，检测荧光探针MitoISCH-1识别线粒体G-四链体DNA的性能。
一、测试荧光探针MitoISCH-1对线粒体G-四链体DNA的体外识别作用
测试的线粒体DNA样品序列包括：
Mito27：5’-(AGGTCGGGGCGGTGATGTAGAGGGTGATGGT)-3’
Mito160：
5’-GGGCTTGATGTGGGGAGGGGTGTTTAAGGGGTTGGCTAGGGTATAATTGTCTGGG-3’
Mito27mut：5’-AGGTCGAAGCGGTGATGTAGAGAGTGATAGT-3’
Mito160mut：
5’-GAGCTTGATGTGAAGAGAAGTGTTTAAGAAGTTGGCTAGAGTATAATTGTCTGAG-3’
MitoHP19：
5’-CAGTATCTGTCTTTGATTCTTTTTTGAATCAAAGACAGATACTG-3’
其中，Mito27、Mito160为线粒体G-四链体DNA结构,MitoHP19是线粒体双链DNA结构，Mito27mut和Mito160为单链DNA结构。DNA样品购自上海生工。将DNA样品适量溶于Tris-HCl的缓冲液中(PH7.4，10mM Tris，100mM KCl)，超微量紫外定浓，在95℃下加热5min后缓慢冷却退火到室温作为储存液，4℃储存待用。
以化合物MitoISCH-1为测试荧光探针，将其用二甲基亚砜溶解，配成10mM的储存液，再用Tris-HCl的缓冲液中(pH 7.4，10mM Tris，100mM KCl)分别稀释成5uM或1uM浓度的荧光探针溶液用于测试。
(1)将配置好的荧光5uM探针溶液进行吸光度测试，结果如图1所示，荧光探针MitoISCH-1在525nm左右有最大紫外吸收强度。
(2)以Mito27为测试线粒体G-四链体DNA，向配置好的5uM的荧光探针MitoISCH-1的缓冲溶液中滴加Mito27线粒体G-四链体DNA，随着滴加量的增加，混合液的吸光度结果如图2所示。
测试结果为：荧光探针MitoISCH-1的最大紫外吸收峰由530nm处红移至600nm左右，并且在570nm处出现等色点。
(3)分别向1uM的荧光探针MitoISCH-1的缓冲溶液中滴加上述不同的线粒体DNA样品，DNA的终浓度为3uM，检测荧光探针MitoISCH-1中加入不同线粒体DNA后的荧光强度，测试结果如图3所示。
测试结果为荧光探针MitoISCH-1对线粒体G-四链体DNA(Mito27,Mito160)的荧光响应明显高于其他核酸二级结构。由此可见，荧光探针MitoISCH-1能够特异性识别线粒体G-四链体DNA结构。
(4)分别向1uM的荧光探针MitoISCH-1的缓冲溶液中滴加上述不同线粒体DNA测试液，测试随着DNA浓度的升高，混合液的荧光强度变化情况，测试结果如图4所示。
结果显示在相同浓度下，探针对线粒体G-四链体Mito27和Mito160的荧光响应明显高于其他二级结构，且随着浓度的增加，荧光强度越来越强，与其他二级结构的区别也越来越明显。证明荧光探针MitoISCH-1特异性识别线粒体G-四链体DNA结构。
二、测试荧光探针MitoISCH-1对活细胞内线粒体G-四链体DNA的特异性识别作用
(1)对线粒体中G-四链体DNA的荧光显微成像
将Hela(人宫颈癌细胞)细胞放在培养基(DMEM培养液和10％胚牛血清)中，放置于条件为37℃、5％CO2和20％O2的培养箱中培养24～48h。取荧光探针MitoISCH-1(用Hela细胞培养基配制1.0μM MitoISCH-1)加入到培养皿中继续培养3h后，使用培养基清洗样本3次，进行荧光成像，结果见图5。
实验结果发现，染有MitoISCH-1的细胞线粒体中呈现出较强的荧光，实验结果表明MitoISCH-1具有较好的细胞膜透过性，能够定位于细胞的线粒体中。预先用G-四链体配体PDS(10μM)培养24h后的Hela细胞，线粒体中的荧光强度明显增强，表明MitoISCH-1能够用于线粒体中G-四链体DNA的检测，并且在测试过程中细胞保持较高的活性。
上述实验结果表明本发明所述荧光探针具有特异性识别活细胞线粒体中G-四链体DNA的作用，能够实时检测线粒体中G-四链体DNA的变化。
本发明提供的荧光探针，其结构如式(Ⅰ)所示，结构中Isaindigotone结构和香豆素形成的共轭体系是探针能够特异性识别G-四链体DNA的母体结构。其中R1，R3取代基发生变化时，对于活性的影响不大。R2为线粒体定位基团，其中连接三苯基膦的linker的长度不影响探针在线粒体中的定位和对线粒体中G-四链体DNA特异性识别，因此化合物MitoISCH-2和MitoISCH-3同样具有靶向线粒体G-四链体DNA的作用。
最后所应当说明的是，以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制，对于本领域的普通技术人员来说，在上述说明及思路的基础上还可以做出其它不同形式的变化或变动，这里无需也无法对所有的实施方式予以穷举。凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明权利要求的保护范围之内。
Claims (3)
1.一种靶向线粒体G-四链体DNA的荧光探针，其特征在于，其结构如式(Ⅰ)所示：
其中，R1为F；R2为-O(CH2)4三苯基膦；R3为N-甲基哌嗪、-NH2或-N(CH2CH3)2；A-为I离子。
2.权利要求1所述靶向线粒体G-四链体DNA的荧光探针的制备方法，其特征在于，包括如下步骤：
S1.2-吡咯烷酮与式2化合物在三氯氧磷存在条件下反应，得式3化合物；
S2.式3化合物在甲醇钠和甲醇作用下反应，得式4化合物；再脱去甲基，得式5化合物；
S3.式5化合物与Br(CH2)n Br发生取代反应，得式6化合物，然后再与三苯基膦发生取代反应，得式7化合物；
S4.式7化合物与甲基化试剂反应，得式8化合物，然后与式9化合物反应，制备得到式(Ⅰ)化合物；
其中式2至式9化合物的结构如下所示，其中n为4；
3.权利要求1所述靶向线粒体G-四链体DNA的荧光探针在制备检测线粒体G-四链体DNA探针中的应用。
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