CN111592472A - 一种荧光染料及其制备方法和用途 - Google Patents

一种荧光染料及其制备方法和用途 Download PDF

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CN111592472A
CN111592472A CN201910352348.XA CN201910352348A CN111592472A CN 111592472 A CN111592472 A CN 111592472A CN 201910352348 A CN201910352348 A CN 201910352348A CN 111592472 A CN111592472 A CN 111592472A
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iii
compound
alkyl
fluorescence
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CN111592472B (zh
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朱麟勇
杨弋
张大生
陈显军
林秋宁
苏倪
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Naying Shanghai Biotechnology Co ltd
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Shanghai Gaochi Asset Management Co ltd
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Priority to CN201910352348.XA priority Critical patent/CN111592472B/zh
Priority to US17/607,005 priority patent/US20220214351A1/en
Priority to JP2021564230A priority patent/JP7321584B2/ja
Priority to PCT/CN2020/087311 priority patent/WO2020221217A1/zh
Priority to EP20798794.2A priority patent/EP3964496A4/en
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Abstract

一种荧光染料及其制备方法和用途，所述荧光染料对粘度响应敏感并且特异，本底荧光低等优点，还可以作为荧光激活点亮型探针，用于蛋白、酶或核酸的荧光标记、定量或检测。

Description

一种荧光染料及其制备方法和用途
技术领域
本发明涉及荧光染料技术领域，具体涉及一种粘度响应性、本底荧光低的荧光染料及其制备方法和用途。
背景技术
分子转子是一类荧光强度随微环境粘度变化的染料，它们在激发后分子发生构象扭曲形成TICT分子内电荷转移态，激发态能量主要以非辐射形式释放，而当它们处于粘度较大或者较刚性的微环境时，此类分子的分子构象扭曲被限制，此时染料激发态能量主要以辐射发光的形式释放，即该类分子的荧光性质被激活，重要的是，该类分子的荧光强度随微环境的粘度的变化而变化，能够实时、原位、灵敏、可视化地显示微环境的粘度变化。
目前，基于限制分子转子的构象扭曲除了用于粘度检测领域之外，还被广泛应用于荧光激活探针的构建，如，分子转子与BSA结合后，分子构象被蛋白限制，荧光被点亮，而未与蛋白结合的染料的激发态能量仍然以非辐射形式耗散，从而达到蛋白实时检测、定量的效果。再如，噻唑橙与DNA、RNA结合前处于荧光淬灭的状态，当与DNA或RNA结合后分子构象被限制，从而荧光被激活，被广泛用于DNA、RNA的检测与示踪；孔雀绿等分子转子被抗体包裹，限制分子的构象变化，用于蛋白激活型荧光成像；DHBI与适配体结合，构建用于RNA示踪的荧光蛋白模拟物；再如，与淀粉样蛋白结合，限制分子的构象变化，用于艾滋海默病的检测与研究等。
然而，目前的分子转子普遍存在荧光本底高的缺点，即分子转子在自由状态下的荧光强度较高，较难适用于样本量少、成分复杂、待测物丰度低的样品检测与标记，如生物样品中的内源性蛋白、核酸、代谢物等，因此，发展一类本底荧光低的分子转子能够进一步拓展目前荧光转子的用途。
发明内容
本发明的目的在于提供一种具有粘度响应性、本底荧光低的荧光染料。
本发明一方面，提供一种荧光染料，所述荧光染料如式(I)所示，
Figure BDA0002043227710000021
其中：
D-为HO-或N(X1)(X2)-，X1、X2各自独立地选自氢、烷基和改性烷基；X1，X2任选相互连接，与N原子一起形成脂杂环；
R选自氰基、羧基、酰胺基、酯基、亚砜基、砜基、磺酸酯基或磺酰胺基；Ar1和Ar2各自独立地选自亚芳基，亚杂芳基；其中，Ar1、Ar2中的氢原子任选各自独立地被卤原子、羟基、醛基、羧基、酯基、酰胺基、氰基、磺酸基、磷酸基、氨基、伯氨基、仲氨基、烷基或改性烷基取代；
X1、X2任选独立地与Ar1形成脂杂环；
其中：所述“烷基”各自独立地为C1-C10直链或支链烷基；可选地，为C1-C7直链或支链烷基；可选地，为C1-C5直链或支链烷基；可选地，选自甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、仲丁基、正戊基，1-甲基丁基、2-甲基丁基、3-甲基丁基、异戊基、1-乙基丙基、新戊基、正己基、1-甲基戊基、2-甲基戊基、3-甲基戊基、异己基、1，1-二甲基丁基、2，2-二甲基丁基、3，3-二甲基丁基、1，2-二甲基丁基、1，3-二甲基丁基、2，3-二甲基丁基、2-乙基丁基、正庚基、2-甲基己基、3-甲基己基、2，2-二甲基戊基、3，3-二甲基戊基、2，3-二甲基戊基、2，4-二甲基戊基、3-乙基戊基或2，2，3-三甲基丁基；
所述“改性烷基”各自独立地为烷基的任意碳原子被选自卤原子、-OH、-CO-、-O-、-CN、-S-、-SO2-、-(S＝O)-、叠氮基、伯氨基、仲氨基、叔氨基、季铵盐基的一种或多种基团置换所得的基团，所述改性烷基具有1-10个碳原子，其中碳碳单键任选独立地被碳碳双键或碳碳三键置换；
所述的碳原子被置换，是指碳原子或碳原子与其上的氢原子一起被相应的基团置换；
所述“卤原子”各自独立地为F、Cl、Br或I；
所述“脂杂环”为环上含有N、O、S或Si中的一种或多种杂原子的饱和或不饱和的4～15元单环或多环脂杂环，所述脂杂环上含有S原子时，其为-S-、-SO-或-SO2-；所述脂杂环任选被卤原子、烷基、芳基或改性烷基取代；
所述“亚芳基”为5～13元单环或双环或稠合双环或稠合多环的亚芳香基团；
所述“亚杂芳基”为环上含有选自N、O、S或Si中的一种或多种杂原子的5～13元单环或双环或稠合双环或稠合多环的亚杂芳香基团；
所述“酯基”为R′(C＝O)OR″基团；
所述“酰胺基”为R′CONR″R″′基团；
所述“磺酸基”为R′SO3H基团；
所述“磺酸酯基”为R′SO2OR″基团；
所述“磺酰胺基”为R′SO2NR″R″′基团；
所述“磷酸基”为R′OP(＝O)(OH)2基团；
所述“砜基”为R′SO2R″基团；
所述“亚砜基”为R′SOR″基团；
所述“伯氨基”为R′NH2基团；
所述“仲氨基”为R′NHR″基团；
所述“叔氨基”为RNR″R″′基团；
所述“季铵盐基”为R′R″R″′R″″N+基团；
各个R′、R″、R″′、R″″各自独立地为单键、氢、烷基、亚烷基、改性烷基或改性亚烷基；
所述“亚烷基”为C1-C1010的直链或支链的亚烷基；可选地，为C1-C7直链或支链亚烷基；可选地，为C1-C5直链或支链亚烷基；
所述“改性亚烷基”为C1-C10(优选为C1-C6)亚烷基的任意碳原子被选自-O-、-OH、-CO-、-CS-、-(S＝O)-中的基团置换所得的基团；
可选地，所述“改性烷基”为含有选自-OH、-O-、乙二醇单元(-(CH2CH2O)n-)、单糖单元、-O-CO-、-NH-CO-、-SO2-O-、-SO-、Me2N-、Et2N-、-S-S-、-CH＝CH-、F、Cl、Br、I、氰基中的一种或多种基团；
可选地，Ar1和Ar2各自独立地为选自下式(II-1)～(II-22)中的结构：
Figure BDA0002043227710000031
Figure BDA0002043227710000041
可选地，式(I)所示的化合物选自下式化合物：
Figure BDA0002043227710000042
Figure BDA0002043227710000051
本发明的第二个方面是提供制备上述的荧光染料的方法，其特征在于，包括式(a)化合物与式(b)化合物发生羟醛缩合反应的步骤。
Figure BDA0002043227710000052
本发明的第三个方面是提供上述的荧光染料在粘度测试、蛋白荧光标记、核酸荧光标记、蛋白定量或检测、或者核酸定量或检测中的用途，所述用途为非用于疾病的诊断方法的用途。
本发明的第四个方面是提供上的荧光染料在制备用于粘度测试、蛋白荧光标记、核酸荧光标记、蛋白定量或检测、或者核酸定量或检测的试剂中的用途。
本发明的第五个方面是提供一种荧光激活点亮型探针，其包括上述荧光染料。
本发明的第六个方面是提供上述的荧光激活点亮型探针在蛋白荧光标记、核酸荧光标记、蛋白定量或检测、或者核酸定量或检测中的用途，所述用途为非用于疾病的诊断方法的用途。
本发明的第七个方面是提供上述的荧光激活点亮型探针在制备用于蛋白荧光标记、核酸荧光标记、蛋白定量或检测、或者核酸定量或检测的试剂中的用途。
本发明所得荧光染料可用于测定样品的粘度，例如适用于微观粘度的测试。根据另一方面的具体实施方式，所得荧光染料可与对应的抗体、适配体或淀粉样蛋白等特异性结合，或者通过配体或抑制剂与蛋白标签或酶键合，获得系列荧光激活点亮型探针，用于蛋白、酶或核酸的荧光标记、定量或监测。
附图说明
图1为分子转子III-3(1×10-5M)在不同粘度条件下荧光发射强度图；
图2为分子转子III-3(1×10-5M)粘度条件与荧光强度的线性关系图；
图3为分子转子III-4(1×10-5M)在不同粘度条件下荧光发射强度图；
图4为分子转子III-4(1×10-5M)粘度条件与荧光强度的线性关系图；
图5为分子转子III-28(1×10-5M)在不同粘度条件下荧光发射强度图；
图6为分子转子III-28(1×10-5M)粘度条件与荧光强度的线性关系图；
图7为分子转子III-34(1×10-5M)在不同粘度条件下荧光发射强度图；
图8为分子转子III-34(1×10-5M)粘度条件与荧光强度的线性关系图；
图9为分子转子III-11与III-36(1×10-6M)在PBS中的荧光本底对比图；
图10为分子转子III-34与III-37(1×10-6M)在PBS中的荧光本底对比图；
图11为分子转子III-31、III-32、III-33与III-38(1×10-6M)在PBS中的荧光本底对比图；
图12为分子转子III-3与III-39(1×10-6M)在PBS中的荧光本底对比图；
图13为分子转子III-21与III-40(1×10-6M)在PBS中的荧光本底对比图；
图14为分子转子III-28、III-29、III-30与III-41(1×10-6M)在PBS中的荧光本底对比图；
图15为分子转子III-3与III-42(1×10-6M)在PBS中的荧光本底对比图；
图16为分子转子III-3与III-43(1×10-6M)在PBS中的荧光本底对比图；
图17为分子转子III-3、III-4、III-6、III-7、III-8、III-18、III-21用于细胞内RNA适配体的标记，其中A是表达了目标RNA适配体的细胞，B是未表达目标RNA适配体的细胞；
图18为分子转子III-3、III-43用于标记细胞内mRNA。
具体实施方式
实施例1：
化合物III-1：
Figure BDA0002043227710000071
4-N，N-二甲基-苯甲醛(0.35g，2.3mmol)，4-氰基-苯乙腈(0.4g，2.8mmol)于100ml圆底烧瓶中，加入40ml无水乙醇溶解，加入二滴哌啶，Ar保护条件下油浴加热回流2h，反应完毕，冷却至室温，大量固体析出，过滤，冷乙醇冲洗滤饼三次，真空烘干的橙色固体(0.60g，95％)。1H NMR(400MHz，DMSO-d6)：δ＝3.05(s，6H)，6.83(d，J＝9.2Hz，2H，)，7.84-7.94(m，6H)，8.02ppm(s，1H).HRMS(ESI-TOF)：Calcd.For C18H16O3[M+H]+：274.1344.Found：274.1345.
实施例2：
化合物III-2：
Figure BDA0002043227710000072
参照化合物III-1的合成方法，(0.34g，89％)。1H NMR(400MHz，DMSO-d6)：δ＝1.23(t，J＝7.60Hz，6H)，3.05(t，J＝7.60Hz，4H)，6.84(d，J＝9.2Hz，2H，)，7.84-7.95(m，6H)，8.09ppm(s，1H).HRMS(ESI-TOF)：Calcd.For C20H20O3[M+H]+：302.1657.Found：302.1658.
实施例3：
化合物III-3：
Figure BDA0002043227710000073
参照化合物III-1的合成方法，(0.33g，95％)。1H NMR(400MHz，DMSO-d6)：δ＝7.96(s，1H)，7.85(d，J＝16.0Hz，6H)，6.81(d，J＝8.0Hz，2H)，4.77(s，1H)，3.55(d，J＝28.0Hz，4H)，3.04(s，1H).HRMS(ESI-TOF)：Calcd.For C19H18N3O[M+H]+：304.1450.Found：304.1451.
实施例4：
化合物III-4：
Figure BDA0002043227710000081
化合物III-3(0.61g，2.0mmol)于40mL干燥DCM中，加入TEA(0.25g，2.2mmol)，0℃条件下缓慢加入对甲苯磺酰氯的(0.38g，2.0mmol)的10ml DCM溶液，Ar保护条件下缓慢升至室温，反应完毕，加入2mL水淬灭反应，分出有机相，Na2SO4干燥，减压条件下除去有机溶剂，残余物不经进一步处理，直接用于下一步。
残余物溶于20ml乙腈中，加入1ml甲胺的甲醇溶液，体系在Ar保护条件下油浴加热回流过夜，反应完毕，加压除去溶剂，体系溶于50ml DCM中，分别用水、饱和食盐水洗涤(2×100ml)，有机相用Na2SO4干燥，加压除去溶剂，残余物经柱色谱分离得橙红色固体(0.54g，82％)。1H NMR(400MHz，CDCl3)：δ＝7.88(d，J＝9.0Hz，2H)，7.74-7.65(m，4H)，7.48(s，1H)，6.73(d，J＝9.1Hz，2H)，3.60-3.55(m，2H)，3.08(s，3H)，2.57-2.52(m，2H)，2.34(s，6H).HRMS(ESI-TOF)：Calcd.For C21H23N4[M+H]+：331.1923.Found：331.1925.
实施例5：
化合物III-5：
Figure BDA0002043227710000082
4-羟基-3，5-二氟-苯甲醛(0.32g，2.0mmol)，4-氰基-苯乙腈(0.35g，2.4mmol)于100ml圆底烧瓶中，加入40ml无水乙醇溶解，加入二滴哌啶，Ar保护条件下油浴加热回流2h，反应完毕，冷却至室温，大量固体析出，过滤，冷乙醇冲洗滤饼三次，真空烘干的橙色固体。1H NMR(400MHz，CDCl3)：δ＝7.80(d，J＝9.0Hz，2H)，7.74-7.66(m，4H)，7.48(s，1H).HRMS(ESI-TOF)：Calcd.For C16H9F2N2O[M+H]+：283.0683.Found：283.0684.
实施例6：
化合物5-(N-甲基-N-羟乙基)氨基-吡嗪-2-甲醛：
Figure BDA0002043227710000083
4-N-甲基-N-羟乙基胺(2.6g，35mmol)，5-氯-吡嗪-2-甲醛(0.50g，3.5mmol)于100ml圆底烧瓶中，加入20ml无水乙腈溶解，加入K2CO3(0.71g，5.3mmol)，Ar保护条件下油浴加热回流24h，反应完毕，冷却至室温，过滤，真空除去溶剂，残余物溶于100ml DCM中，分别用水、饱和食盐水洗涤(2×100m1)，有机相用Na2SO4干燥，除去有机溶剂，残余物经柱色谱分离得5-(N-甲基-N-羟乙基)-吡嗪-2-醛(0.48g，76％)。1H NMR(400MHz，CDCl3)：δ9.88(s，1H)，8.62(d，J＝1.2Hz，1H)，8.14(d，J＝1.1Hz，1H)，3.92(m，2H)，3.88-3.83(m，2H)，3.28(s，3H).HRMS(ESI-TOF)：Calcd.For C8H12N3O2[M+H]+：182.1.Found：182.1.
化合物III-6：
Figure BDA0002043227710000091
参照化合物III-1的合成方法，(0.36g，96％)。1H NMR(400MHz，CDCl3)：δ8.39(s，1H)，8.30(s，1H)，7.80(d，J＝8.5Hz，2H)，7.72(d，J＝8.4Hz，2H)，7.51(s，1H)，3.93(t，J＝4.9Hz，2H)，3.88-3.83(m，2H)，3.29(s，3H).HRMS(ESI-TOF)：Calcd.For C17H16N5O[M+H]+：306.1355.Found：306.1357.
实施例7：
化合物III-7：
Figure BDA0002043227710000092
参照化合物III-4的合成方法，(0.21g，67％)。1H NMR(400MHz，DMSO-d6)：δ8.37(d，J＝5.2Hz，2H)，8.06(s，1H)，8.00-7.85(m，4H)，3.77(t，J＝6.5Hz，2H)，3.20(s，3H)，2.56(m，2H)，2.23(s，6H).HRMS(ESI-TOF)：Calcd.For C19H21N6[M+H]+：333.1828.Found：333.1829.
实施例8：
化合物6-(N-甲基-N-羟乙基)氨基-吡嗪-3-醛：
Figure BDA0002043227710000093
按照化合物5-(N-甲基-N-羟乙基)氨基-吡嗪-2-醛的合成方法，(0.45g，68％)。1HNMR(400MHz，CDCl3)：δ＝9.69(s，1H)，8.43(d，J＝2.1Hz，1H)，7.86(dd，J＝9.0，2.3Hz，1H)，6.56(d，J＝9.1Hz，1H)，3.86-3.79(m，4H)，3.15(s，3H).HRMS(ESI-TOF)：Calcd.ForC9H13O2N2[M+H]+：181.1.Found：181.1.
化合物III-8：
Figure BDA0002043227710000101
参照化合物III-1的合成方法，(0.39g，89％)。1H NMR(400MHz，DMSO-d6)：δ＝8.54(d，J＝4.0Hz，1H)，8.30(dd，J＝9.3，2.5Hz，1H)，8.03(s，1H)，7.92(d，J＝8.0Hz，2H)，7.85(d，J＝8.0Hz，2H)，6.84(d，J＝8.0Hz，1H)，4.77(t，J＝5.4Hz，1H)，3.67(t，J＝5.3Hz，2H)，3.60(q，J＝5.4Hz，2H)，3.15(s，3H).HRMS(ESI-TOF)：Calcd.For C18H27N4O[M+H]+：305.1402.Found：305.1401.
实施例9：
化合物III-9：
Figure BDA0002043227710000102
参照化合物III-4的合成方法，(0.31g，92％)。1H NMR(400MHz，DMSO-d6)：δ＝8.55(d，J＝4.0Hz，1H)，8.31(dd，J＝9.3，2.5Hz，1H)，8.05(s，1H)，7.93(d，J＝8.0Hz，2H)，7.84(d，J＝8.0Hz，2H)，6.85(d，J＝8.0Hz，1H)，4.78(t，J＝5.4Hz，1H)，3.67(t，J＝5.3Hz，2H)，3.60(q，J＝5.4Hz，2H)，3.17(t，J＝8.0Hz，4H)，1.17(t，J＝8.0Hz，6H).HRMS(ESI-TOF)：Calcd.For C22H26N5[M+H]+：360.2188.Found：360.2187.
实施例10：
4-N，N-二甲基-6醛基-吡啶：
Figure BDA0002043227710000103
参照化合物4-N-甲基-N-(2-N’，N’-二甲基-乙基)-苯甲醛的合成方法，(0.31g，49％)。1H NMR(400MHz，DMSO-d6)：δ＝9.86(d，J＝0.6Hz，1H)，8.17(d，J＝2.9Hz，1H)，7.83(d，J＝8.9Hz，1H)，6.94(dd，J＝8.8，2.9Hz，1H)，3.10(s，6H).HRMS(ESI-TOF)：Calcd.ForC8H11N2O[M+H]+：151.1.Found：151.1.
化合物III-10：
Figure BDA0002043227710000104
参照化合物III-1的合成方法，(0.36g，96％)。1H NMR(400MHz，DMSO-d6)：δ＝9.86(d，J＝0.6Hz，1H)，8.26(s，1H)，8.17(d，J＝2.9Hz，1H)，7.83(d，J＝8.9Hz，1H)，7.46(m，4H)，6.94(dd，J＝8.8，2.9Hz，1H)，3.10(s，6H).HRMS(ESI-TOF)：Calcd.For C17H15N4[M+H]+：275.1297.Found：275.1298.
实施例11：
2-(N-甲基-N-羟乙基)氨基-5-醛基-嘧啶：
Figure BDA0002043227710000111
参照化合物4-N-甲基-N-(2-N’，N’-二甲基-乙基)-苯甲醛的合成方法，(0.42g，72％)。1H NMR(400MHz，DMSO-d6)：δ＝9.89(s，1H)，8.73(s，2H)，3.64(t，J＝8.9Hz，2H)，3.45(t，J＝8.8Hz，2H)，3.10(s，3H).HRMS(ESI-TOF)：Calcd.For C8H12N3O[M+H]+：182.1.Found：182.1.
化合物III-11：
Figure BDA0002043227710000112
参照化合物III-1的合成方法，(0.36g，96％)。1H NMR(400MHz，DMSO-d6)：δ＝8.26(s，1H)，8.73(s，2H)，7.64(m，4H)，3.64(t，J＝8.9Hz，2H)，3.44(t，J＝8.8Hz，2H)，3.11(s，3H).HRMS(ESI-TOF)：Calcd.For C17H16N5O[M+H]+：306.1355.Found：306.1356.
实施例12：
5-(N-甲基-N-羟乙基)氨基-2-醛基-嘧啶：
Figure BDA0002043227710000113
参照化合物4-N-甲基-N-(2-N’，N’-二甲基-乙基)-苯甲醛的合成方法，(0.42g，72％)。1H NMR(400MHz，DMSO-d6)：δ＝9.98(s，1H)，8.21(s，2H)，3.64(t，J＝8.9Hz，2H)，3.44(t，J＝8.8Hz，2H)，3.12(s，3H).HRMS(ESI-TOF)：Calcd.For C8H12N3O2[M+H]+：182.1.Found：182.1.
1-氰基-1-(4-苯乙腈)-2-2-(5-(N-甲基-N-羟乙基)氨基-)嘧啶-乙烯：
Figure BDA0002043227710000114
参照化合物III-1的合成方法，(0.56g，89％)。1H NMR(400MHz，DMSO-d6)：δ＝8.21(s，2H)，7.99(s，1H)，7.64(s，4H)，3.64(t，J＝8.9Hz，2H)，3.44(t，J＝8.8Hz，2H)，3.12(s，3H).HRMS(ESI-TOF)：Calcd.For C17H16N5O[M+H]+：306.1.Found：306.1.
化合物III-12：
Figure BDA0002043227710000121
参照化合物III-4的合成方法，(0.36g，96％)。1H NMR(400MHz，DMSO-d6)：δ＝8.21(s，2H)，7.99(s，1H)，7.64(s，4H)，3.77(t，J＝6.5Hz，2H)，3.20(s，3H)，2.56(m，2H)，2.23(s，6H).HRMS(ESI-TOF)：Calcd.For C19H21N6[M+H]+：333.1828.Found：333.1829.
实施例13：
2-乙腈-5-氰基-吡啶：
Figure BDA0002043227710000122
2-溴甲基-5-氰基吡啶(0.50g，2.5mmol)于100ml圆底烧瓶中，加入50ml THF溶解，Ar保护条件下加入10ml NaCN的2M的水溶液，油浴加热回流12h，反应完毕，体系冷却至室温，DCM萃取(3×100ml)，合并有机相，分别用水、饱和食盐水洗涤(2×100ml)，有机相用NaSO4干燥，减压除去溶剂，残余物经柱色谱纯化分离得2-乙腈-5-氰基吡啶(0.19g，56％)。1HNMR(400MHz，DMSO-d6)：δ＝8.78(s，1H)，7.95(m，1H)，7.56(m，1H)，4.01(s，2H).HRMS(ESI-TOF)：Calcd.For C8H6N3[M+H]+：144.1.Found：144.1.
化合物III-13：
Figure BDA0002043227710000123
参照化合物III-1的合成方法，(0.45g，95％)。1H NMR(400MHz，DMSO-d6)：δ＝8.78(s，1H)，8.21(s，1H)，7.94(m，1H)，7.86(d，J＝8.0Hz，2H)，7.57(m，1H)，6.80(d，J＝8.0Hz，2H)，3.64(t，J＝8.9Hz，2H)，3.44(t，J＝8.8Hz，2H)，3.12(s，3H).HRMS(ESI-TOF)：Calcd.For C18H17N4O[M+H]+：305.1402.Found：305.1403.
实施例14：
2-氰基-5-乙腈-吡嗪：
Figure BDA0002043227710000131
2-氯-吡嗪-5-乙腈(0.32g，2.0mmol)、CuCN(0.93g，10.0mmol)于100ml圆底烧瓶中，加入30ml干燥DMSO溶解，Ar保护条件下80℃油浴加热12h，反应完毕，体系倒入100ml水中，DCM萃取(4×50ml)，合并有机相，分别用水、饱和食盐水洗涤(2×100ml)，有机相用Na2SO4干燥，减压除去有机溶剂，残余物经柱色谱分离得2-氰基-吡嗪-5-乙腈(0.20g，69％)。1H NMR(400MHz，DMSO-d6)：δ＝8.60(s，1H)，8.48(s，1H)，3.92(s，2H).HRMS(ESI-TOF)：Calcd.For C7H5N4[M+H]+：145.1.Found：145.1.
化合物III-14：
Figure BDA0002043227710000132
参照化合物III-1的合成方法，(0.25g，91％)。1H NMR(400MHz，DMSO-d6)：δ＝8.60(s，1H)，8.48(s，1H)，8.11(s，1H)，7.81(d，J＝8.2Hz，2H)，6.84(d，J＝8.2Hz，2H)，3.60(t，J＝9.2Hz，2H)，3.46(t，J＝9.2Hz，2H)，3.12(s，3H).HRMS(ESI-TOF)：Calcd.For C17H16N5O[M+H]+：306.1355.Found：306.1354.
实施例15：
化合物III-15：
Figure BDA0002043227710000133
参照化合物III-1的合成方法，(0.25g，91％)。1H NMR(400MHz，DMSO-d6)：δ＝8.22(s，1H)，8.00(d，J＝9.1Hz，1H)，7.77-7.69(m，1H)，7.43-7.34(m，1H)，6.88(d，J＝9.1Hz，1H)，4.81(t，J＝5.2Hz，1H)，3.31-3.25(m，4H)，2.66-2.63(m，4H)，1.89-1.81(m，4H).HRMS(ESI-TOF)：Calcd.For C22H20N3[M+H]+：326.1657.Found：326.1658.
实施例16：
化合物III-16：
Figure BDA0002043227710000141
参照化合物III-1的合成方法，(0.29g，94％)。1H NMR(400MHz，DMSO-d6)：δ＝8.11(2H，d，J＝10.4Hz)，7.99(3H，dd，J＝8.6，3.0Hz)，7.54(1H，dd，J＝8.0，8.0Hz)，7.44(1H，dd，J＝8.0，8.0Hz)，6.88(2H，d，J＝9.2Hz)，4.82(1H，bt，t，J＝5.2Hz)，3.01-3.08(m，2H)，3，53-3.60(m，2H)，2.89(s，3H).HRMS(ESI-TOF)：Calcd.For C19H16N3[M+H]+：286.1344.Found：286.1345.
实施例17：
6-甲胺-苯并[b]噻吩-2-甲醛：
Figure BDA0002043227710000142
6-溴苯并[b]噻吩-2-甲醛(0.42g，1.7mmol)、二甲基乙胺(40％水溶液，1g，8.9mmol)、CuI(13.9mg，0.073mmol)、K3PO4·H2O(155.4mg，0.73mmol)、甲胺(33％水溶液，1g)于100ml耐压瓶中，密封条件下60℃油浴加热12h，体系冷却至室温，加入50ml水，DCM萃取(3×100ml)，合并有机相，Na2SO4干燥，减压除去有机溶剂，残余物经柱色谱分离纯化(0.23g，68％)。1HNMR(400MHz，DMSO-d6)：δ＝9.92(1H，s)，8.14(1H，s)，7.82(1H，d，J＝9.1Hz)，7.18(1H，d，J＝2.1Hz)，7.01(1H，dd，J＝9.1，2.3Hz)，3.05(3H，s).HRMS(ESI-TOF)：Calcd.For C10H10NOS[M+H]+：192.0.Found：192.0.
化合物III-17：
Figure BDA0002043227710000143
参照化合物III-1的合成方法，(0.29g，94％)。1H NMR(400MHz，DMSO-d6)：δ＝8.45(s，1H)，7.92(d，J＝8.6Hz，2H)，7.85(d，J＝8.3Hz，3H)，7.73(dd，J＝8.6，3.9Hz，1H)，7.21(d，J＝1.9Hz，1H)，7.21(d，J＝1.9Hz，1H)，6.96(dd，J＝9.1，2.3Hz，1H)，3.05(s，3H).HRMS(ESI-TOF)：Calcd.For C19H14N3S[M+H]+：360.1171.Found：360.1173.
实施例18：
6-N-甲基-N-羟乙基-苯并[b]噻吩-2-甲醛：
Figure BDA0002043227710000151
参照化合物6-甲胺-苯并[b]噻吩-2-甲醛的合成方法，(0.54g，79％)。1H NMR(400MHz，DMSO-d6)：δ＝9.91(s，1H)，8.14(s，1H)，7.81(d，J＝5.2Hz，1H)，7.17(d，J＝2.0Hz，1H)，7.01(dd，J＝2.0，8.8Hz，1H)，4.76(t，J＝5.6Hz，1H)，3.58(t，J＝4.2Hz，2H)，3.52(t，J＝4.2Hz，2H)，3.04(s，3H).HRMS(ESI-TOF)：m/z Calcd.For C12H14NO2S，[M+H]+：235.1.Found 236.1.
化合物III-18：
Figure BDA0002043227710000152
参照化合物III-1的合成方法，(0.21g，95％)。1H NMR(400MHz，DMSO-d6)：δ＝8.45(s，1H)，7.92(d，J＝8.6Hz，2H)，7.85(d，J＝8.3Hz，3H)，7.73(dd，J＝8.6，3.9Hz，1H)，7.21(d，J＝1.9Hz，1H)，7.21(d，J＝1.9Hz，1H)，6.96(dd，J＝9.1，2.3Hz，1H)，3.63-3.57(m，2H)，3.52(t，J＝5.7Hz，2H)，3.05(s，3H).HRMS(ESI-TOF)：Calcd.For C21H19N3OS[M+H]+：360.1171.Found：360.1173.
实施例19：
5-N，N-二甲胺-2-醛基并二噻吩：
Figure BDA0002043227710000153
参照化合物6-N-甲基-N-羟乙基-苯并[b]噻吩-2-甲醛的合成方法，(0.54g，79％)。1H NMR(400MHz，DMSO-d6)：δ＝9.66(s，1H)，8.05(s，1H)，6.30(s，1H)，4.88(bt，1H)，3.07(s，6H).HRMS(ESI-TOF)：m/z Calcd.For C9H12NOS2[M+H]+：214.0；found 214.0.
化合物III-19：
Figure BDA0002043227710000154
参照化合物III-1的合成方法，(0.31g，90％)。1H NMR(400MHz，DMSO-d6)：δ＝8.34(s，1H)，7.86(d，J＝8.0Hz，2H)，7.81(s，1H)，7.77(d，J＝8.0Hz，2H)，6.32(s，1H)，4.88(t，J＝4.0Hz，1H)，3.08(s，6H).HRMS(ESI-TOF)：Calcd.For C18H14N3S2[M+H]+：336.0629.Found：336.0630.
实施例20：
5-N，N-二乙胺-2-醛基并二噻吩：
Figure BDA0002043227710000161
参照化合物6-N-甲基-N-羟乙基-苯并[b]噻吩-2-甲醛的合成方法，(0.44g，75％)。1H NMR(400MHz，DMSO-d6)：δ＝9.78(s，1H)，8.09(s，1H)，6.30(s，1H)，4.87(bt，1 H)，3.27(t，J＝8.4Hz，4H)，1.26(t，J＝8.4Hz，4H).HRMS(ESI-TOF)：m/z Calcd.For C9H12NOS2[M+H]+：214.0；found 214.0.
化合物III-20：
Figure BDA0002043227710000162
参照化合物III-1的合成方法，(0.31g，90％)。1H NMR(400MHz，DMSO-d6)：δ＝8.34(s，1H)，7.86(d，J＝8.0Hz，2H)，7.81(s，1H)，7.77(d，J＝8.0Hz，2H)，6.32(s，1H)，4.88(t，J＝4.0Hz，1H)，3.27(t，J＝8.4Hz，4H)，1.26(t，J＝8.4Hz，4H).HRMS(ESI-TOF)：Calcd.ForC20H18N3S2[M+H]+：364.0942.Found：364.0943.
实施例21：
5-(N-甲基-N-羟乙基)氨基-2-醛基并二噻吩：
Figure BDA0002043227710000163
参照化合物6-N-甲基-N-羟乙基-苯并[b]噻吩-2-甲醛的合成方法，(0.44g，75％)。1H NMR(400MHz，DMSO-d6)：δ＝9.66(s，1H)，8.05(s，1H)，6.30(s，1H)，4.88(bt，1 H)，3.64(t，J＝5.6Hz，2H)，3.44(t，J＝5.6Hz，2H)，3.07(s，3H).HRMS(ESI-TOF)：m/zCalcd.For C10H12NO2S2[M+H]+：241.0；found 242.0.
化合物III-21：
Figure BDA0002043227710000171
参照化合物III-1的合成方法，(0.31g，90％)。1H NMR(400MHz，DMSO-d6)：δ8.34(s，1H)，7.86(d，J＝8.0Hz，2H)，7.81(s，1H)，7.77(d，J＝8.0Hz，2H)，6.32(s，1H)，4.88(t，J＝4.0Hz，1H)，3.65(q，J＝5.5Hz，2H)，3.44(t，J＝5.5Hz，2H)，3.34(s，1H)，3.08(s，3H).HRMS(ESI-TOF)：Calcd.For C19H16N3OS2[M+H]+：366.0735.Found：366.0736.
实施例22：
化合物III-22：
Figure BDA0002043227710000172
参照化合物III-1的合成方法，(0.31g，90％)。1H NMR(400MHz，DMSO-d6)：δ＝3.04(s，6H)，6.82(d，J＝9.2Hz，2H，)，7.59(d，J＝9.1Hz，2H)，7.84-7.94(m，6H)，8.02ppm(s，1H).HRMS(ESI-TOF)：Calcd.For C24H19O3[M+H]+：350.1657.Found：350.1656.
实施例23：
化合物III-23：
Figure BDA0002043227710000173
参照化合物III-1：1H NMR(400MHz，DMSO-d6)：δ＝3.02(s，6H)，6.72(d，J＝8.0Hz，2H，)，7.24(d，J＝4.0Hz，1H)，7.49(d，J＝8.8Hz，2H)，7.55(d，J＝8.0Hz，1H)，7.69(d，J＝8.8Hz，2H)，8.02ppm(s，1H).HRMS(ESI-TOF)：Calcd.For C22H18N3S[M+H]+：356.1221.Found：356.1220.
实施例24：
化合物III-24：
Figure BDA0002043227710000181
参照化合物化合物III-1的合成方法，其中化合物1(参照Chem.Commun.2011，47，985-987.的合成方法)：1H NMR(400MHz，DMSO-d6)：δ＝3.63(m，16H)，3.77(m，4H)，6.76(d，J＝8.8Hz，2H，)，7.38(d，J＝4.0Hz，2H)，7.49(d，J＝8.8Hz，2H)，7.59(d，J＝8.8Hz，2H)，7.72(m，4H)，8.28(s，1H).HRMS(ESI-TOF)：Calcd.For C30H32O3N4S[M+H]+：530.2114.Found：530.2115.
实施例25：
化合物III-25：
Figure BDA0002043227710000182
参照化合物化合物III-1的合成方法，其中化合物2(参照J.OrR.Chem.2008，73，6587-6594.的合成方法)：1H NMR(400MHz，DMSO-d6)：δ＝1.23(t，J＝7.2Hz，6H)，3.35(m，J＝7.2Hz，4H)，5.78(d，J＝4.0Hz，1H)，6.92(d，J＝4.0Hz，1H)，7.12(d，J＝4.0Hz，1H)，7.49(d，J＝8.8Hz，2H)，7.56(d，J＝4.0Hz，1H)，7.69(d，J＝8.8Hz，2H)，8.28(s，1H).HRMS(ESI-TOF)：Calcd.For C30H32O3N4S[M+H]+：390.1099.Found：390.1097.
实施例26：
化合物III-26：
Figure BDA0002043227710000183
参照化合物化合物III-1的合成方法，1H NMR(400MHz，DMSO-d6)：δ＝3.30(s，6H)，5.71(d，J＝4.0Hz，1H)，6.93(d，J＝4.0Hz，1H)，7.15(d，J＝4.0Hz，1H)，7.47(d，J＝8.8Hz，2H)，7.56(d，J＝4.0Hz，1H)，7.64(d，J＝8.8Hz，2H)，8.28(s，1H).HRMS(ESI-TOF)：Calcd.For C20H17O2N2S2[M+H]+：381.0731.Found：381.0730.
实施例27：
化合物III-27：
Figure BDA0002043227710000191
参照化合物III-1的合成方法，其中化合物4(参照Heterocycles，1997，46，489-501.)1H NMR(400MHz，CDCl3)：δ2.07(m，4H)，3.33(t，J＝6.6Hz，4H)，4.2(s，3H)，5.70(d，J＝4.4Hz，1H)，6.92(d，J＝4.0Hz，1H)，7.15(d，J＝4.0Hz，1H)，7.43(d，J＝8.2Hz，2H)，7.51(d，J＝8.2Hz，2H)，7.57(d，J＝4.0Hz，1H)，8.10(s，1H).HRMS(ESI-TOF)：Calcd.ForC23H21O2N2S2[M+H]+：421.1044.Found：521.1042.
实施例28：
化合物III-28：
Figure BDA0002043227710000192
参照化合物III-1的合成方法，其中化合物5(参照WO2018014821).1H-NMR(400MHz，DMSO-d6)：δ＝7.84(s，1H)，7.59(d，J＝8.8Hz，2H)，7.49(d，J＝8.8Hz，2H)，7.24(s，1H)，3.78(t，2H，J＝4.80Hz)，3.44(t，2H，J＝4.80Hz)，3.02(s，3H)。HRMS(ESI-TOF)：Calcd.For C21H16ON3S3.[M+H]+：422.0455.Found：422.0456.
实施例29：
化合物III-29：
Figure BDA0002043227710000193
参照化合物III-1的合成方法，其中化合物6(参照WO2018014821)。1H-NMR(400MHz，DMSO-d6)：δ＝7.84(s，1H)7.59(d，J＝8.8Hz，2H)，7.49(d，J＝8.8Hz，2H)，7.24(s，1H)，3.56(q，J＝4.0Hz，2H)，3.01(s，6H)，1.21(t，J＝4.0Hz，3H).HRMS(ESI-TOF)：Calcd.For C22H19O2N2S3.[M+H]+：439.0609.Found：439.0610.
实施例30：
化合物III-30：
Figure BDA0002043227710000201
参照化合物III-1的合成方法，其中化合物7(WO 2014048547)。1H-NMR(400MHz，DMSO-d6)：δ＝7.84(s，1H)，7.59(d，J＝8.8Hz，2H)，7.49(d，J＝8.8Hz，2H)，7.24(s，1H)，3.10(s，3H)，3.01(s，6H).HRMS(ESI-TOF)：Calcd.For C21H17O1N2S4.[M+H]+：429.0024.Found：429.0026.
实施例31：
化合物III-31：
Figure BDA0002043227710000202
参照化合物III-1的合成方法，其中化合物9(J.Chem.Phann.Res.，2012，4，1661-1669.)。1H-NMR(400MHz，DMSO-d6)：δ＝7.84(s，1H)，7.59(d，J＝8.8Hz，2H)，7.49(d，J＝8.8Hz，2H)，7.24(s，1H)，3.14(s，3H)，3.01(s，6H).HRMS(ESI-TOF)：Calcd.ForC22H23O2N2S3Si.[M+H]+：471.0691.Found：471.0690.
实施例32：
化合物III-32：
Figure BDA0002043227710000203
参照化合物III-1的合成方法。1H-NMR(400MHz，DMSO-d6)：δ＝7.84(s，1H)，7.59(d，J＝8.8Hz，2H)，7.49(d，J＝8.8Hz，2H)，7.24(s，1H)，3.77(t，2H，J＝4.80Hz)，3.41(t，2H，J＝4.80Hz)，3.00(s，3H).HRMS(ESI-TOF)：Calcd.For C22H24O3N3S3Si.[M+H]+：502.0749.Found：502.0752.
实施例33：
化合物III-33：
Figure BDA0002043227710000211
参照化合物III-1的合成方法。1H-NMR(400MHz，CDCl3)：δ＝7.89(s，1H)，7.59(d，J＝8.8Hz，2H)，7.49(d，J＝8.8Hz，2H)，7.18(s，1H)，6.96(d，2H，J＝5.6Hz)，3.85(t，2H，J＝4.80Hz)，3.46(t，2H，J＝4.80Hz)，3.06(s，3H)，0.46(s，6H).Calcd.For C23H22ON3S2Si.[M+H]+：448.0974.Found：448.0972.
实施例34：
化合物III-34：
Figure BDA0002043227710000212
参照化合物III-1的合成方法。1H-NMR(400MHz，CDCl3)：δ＝7.83(s，1H)，7.59(d，J＝8.8Hz，2H)，7.49(d，J＝8.8Hz，2H)，7.11(s，1H)，3.85(t，2H，J＝4.80Hz)，3.46(t，2H，J＝4.80Hz)，3.06(s，3H)，1.46(s，6H).HRMS(ESI-TOF)：Calcd.For C24H24O2N3S2[M+H]+：450.1310.Found：450.1311.
实施例35：
Figure BDA0002043227710000213
化合物12：
参照文献公开方法(K.T.Arun et.al.J.Phys.Chem.A.2005，109，5571-5578.)1H-NMR(400MHz，CDCl3)：δ＝10.01(s，1H)，7.89(s，1H)，7.18(s，1H)，6.96(d，2H，J＝5.6Hz)，3.52-3.65(m，20H)，3.37(s，3H)，2.97(s，3H).HRMS(ESI-TOF)：Calcd.For C24H22ON3S2Si.[M+H]+：432.1204.Found：432.1203.Calcd.For C24H36O6N1S2.[M+H]+：497.3.Found：497.3.
化合物III-35：
参照化合物III-1的合成方法。1H-NMR(400MHz，CDCl3)：δ＝7.89(s，1H)，7.59(d，J＝8.8Hz，2H)，7.49(d，J＝8.8Hz，2H)，7.18(s，1H)，6.96(d，2H，J＝5.6Hz)，3.52-3.65(m，20H)，3.37(s，3H)，2.97(s，3H).HRMS(ESI-TOF)：Calcd.For C33H39O5N3S2.[M+H]+：622.2409.Found：622.2409.
对比例1：
化合物III-36：
Figure BDA0002043227710000221
参照化合物III-1的合成方法，(0.25g，91％)。1H NMR(400MHz，DMSO-d6)：δ＝8.21(s，2H)，7.99(s，1H)，7.64(s，4H)，3.64(t，J＝8.9Hz，2H)，3.44(t，J＝8.8Hz，2H)，3.12(s，3H).HRMS(ESI-TOF)：Calcd.For C16H17N4O4S[M+H]+：361.0971.Found：361.0970
对比例2：
化合物III-37：
Figure BDA0002043227710000222
参照化合物III-1的合成方法，(0.39g，91％)。1H NMR(400MHz，DMSO-d6)：δ＝7.83(s，1H)，7.59(d，J＝8.8Hz，2H)，7.49(d，J＝8.8Hz，2H)，7.11(s，1H)，3.85(t，2H，J＝4.80Hz)，3.46(t，2H，J＝4.80Hz)，3.05(s，3H)，1.46(s，6H).HRMS(ESI-TOF)：Calcd.ForC23H23N2O4S3[M+H]+：487.0820.Found：487.0821.
对比例3：
化合物III-38：
Figure BDA0002043227710000223
参照化合物III-1的合成方法，其中化合物11(CN 106349105.)。1H-NMR(400MHz，DMSO-d6)：δ＝7.84(s，1H)，7.59(d，J＝8.8Hz，2H)，7.49(d，J＝8.8Hz，2H)，7.24(s，1H)，3.78(t，2H，J＝4.80Hz)，3.44(t，2H，J＝4.80Hz)，3.01(s，3H).HRMS(ESI-TOF)：Calcd.ForC22H23O4N2S3Si.[M+H]+：503.0589.Found：203.0588.
对比例4：
化合物III-39：
Figure BDA0002043227710000231
参照化合物III-1的合成方法。1H-NMR(400MHz，DMSO-d6)：δ＝7.84(s，1H)，7.59(d，J＝8.8Hz，2H)，7.49(d，J＝8.8Hz，2H)，3.78(t，2H，J＝4.80Hz)，3.44(t，2H，J＝4.80Hz)，3.01(s，3H).HRMS(ESI-TOF)：Calcd.For C18H19O4N2S.[M+H]+：359.1066.Found：359.1065.
对比例5：
化合物III-40：
Figure BDA0002043227710000232
参照化合物III-1的合成方法。1H-NMR(400MHz，DMSO-d6)：δ＝8.34(s，1H)，7.59(d，J＝8.0Hz，2H)，7.81(s，1H)，7.49(d，J＝8.0Hz，2H)，6.32(s，1H)，4.88(t，J＝4.0Hz，1H)，3.65(q，J＝5.5Hz，2H)，3.44(t，J＝5.5Hz，2H)，3.34(s，1H)，3.08(s，3H).HRMS(ESI-TOF)：Calcd.For C18H17O4N2S3.[M+H]+：421.0350.Found：421.0351.
对比例6：
化合物III-41：
Figure BDA0002043227710000233
参照化合物III-1的合成方法.1H-NMR(400MHz，DMSO-d6)：δ＝7.85(s，1H)，7.59(d，J＝8.8Hz，2H)，7.47(d，J＝8.8Hz，2H)，7.24(s，1H)，3.79(t，2H，J＝4.80Hz)，3.43(t，2H，J＝4.80Hz)，3.01(s，3H)。HRMS(ESI-TOF)：Calcd.For C20H17O4N2S4.[M+H]+：477.0071.Found：477.0070.
对比例7：
化合物III-42：
Figure BDA0002043227710000241
参照化合物III-1的合成方法，(0.25g，91％)。1H NMR(400MHz，DMSO-d6)：δ＝8.22(s，1H)，8.00(d，J＝9.1Hz，1H)，7.77-7.69(m，1H)，7.43-7.34(m，1H)，6.88(d，J＝9.1Hz，1H)，4.81(t，J＝5.2Hz，1H)，3.64-3.52(m，3H)，3.09(s，1H).LR-HRMS(ESI-TOF)：Calcd.ForC19H18N3O2[M+H]+：320.1399.Found：320.1397.
对比例8：
化合物III-43：
Figure BDA0002043227710000242
参照化合物III-1的合成方法，(0.29g，94％)。1H NMR(400MHz，DMSO-d6)：δ＝8.11(2H，d，J＝10.4Hz)，7.99(3H，dd，J＝8.6，3.0Hz)，7.54(1H，dd，J＝8.0，8.0Hz)，7.44(1H，dd，J＝8.0，8.0Hz)，6.88(2H，d，J＝9.2Hz)，4.82(1H，bt，t，J＝5.2Hz)，3.60(2H，t，J＝5.2Hz)，3.56(2H，t，J＝5.2Hz)，3.09(3H，s).LR-HRMS(ESI-TOF)：Calcd.For C19H18N3OS[M+H]+：336.1171.Found：336.1170.
试验例1：
将实施例1-35制备的荧光染料(分子转子)分别溶于二甲基亚砜中，各自制得浓度为1×10-2M的母液，将各母液分别加入甘油和甲醇中，混合均匀，各配制终浓度为1×10-5M的溶液，根据荧光染料不同，依次用各荧光染料最大激发波长在相同条件下检测其荧光发射图谱，结果如表1所示，表明本发明荧光染料粘度变化响应灵敏。
表1
Figure BDA0002043227710000243
Figure BDA0002043227710000251
试验例2：
分子转子III-3、III-4、III-28、III-34加入二乙醇-甘油混合溶液中，配制成终浓度为1×10-5M的溶液，480nm激发，不同粘度条件下荧光发射光谱如图1、3、5、7所示，相同浓度的分子转子在不同粘度条件下荧光强度逐渐增大，说明分子转子荧光强度随环境粘度的增大荧光增强，荧光强度的对数和溶剂粘度的对数关系符合哈夫曼方程，具有很好的线性关系，如图2、4、6、8所示，证明分子转子对粘度反应灵敏，并且可以用于未知样品的粘度测试。
试验例3：
分子转子III-11与III-36；III-34与III-37；III-31、III-32、III-33与III-38；III-3与III-39；III-21与III-40；III-28、III-29、III-30与III-41；III-3与III-42；III-3与III-43加入PBS溶液中，配置终浓度为1×10-6M的溶液，分别用各化合物最大激发波长激发，检测他们在PBS中的荧光强度，以各组最强荧光为100对各样品进行归一化，分别如图9、图10、图11、图12、图13、图14、图15和图16所示。结果表明：与拉电子基团上的芳香环上有磺酸基取代的分子转子以及没有取代的分子转子相比较，本申请拉电子基团上的芳香环上具有氰基、酯基、亚砜、砜、磺酰胺取代的分子转子具有更低的本底荧光。
试验例4：
化合物III-3、III-4、III-6、III-7、III-8、III-18、III-21与RNA适配体(序列10：F30-8Pepper-5RNA适配体序列
UUGCCAUGUGUAUGUGGGUUCGCCCACAUACUCUGAUGAUCCCCAAUCGUGGCGUGUCGGCCUCUCCCAAUCGUGGCGUGUCGGCCUCUCCCAAUCGUGGCGUGUCGGCCUCUCCCAAUCGUGGCGUGUCGGCCUCUCCCAAUCGUGGCGUGUCGGCCUCUCCCAAUCGUGGCGUGUCGGCCUCUCCCAAUCGUGGCGUGUCGGCCUCUCCCAAUCGUGGCGUGUCGGCCUCUCUUCGGAGAGGCACUGGCGCCGGAGAGGCACUGGCGCCGGAGAGGCACUGGCGCCGGAGAGGCACUGGCGCCGGAGAGGCACUGGCGCCGGAGAGGCACUGGCGCCGGAGAGGCACUGGCGCCGGAGAGGCACUGGCGCCGGGAUCAUUCAUGGCAA)特异性结合，结合后的化合物荧光被显著激活，在合适波长激发光的激发下发射明亮的荧光，结合后的光学性质见表2，化合物还可以与细胞内与该适配体结合，转录了该RNA适配体的细胞具有明亮的荧光，如图17A，未表达该RNA适配体的细胞无荧光，如图17B，说明该系列染料可以用于核酸的标记。
表2
Figure BDA0002043227710000261
Figure BDA0002043227710000271
注：荧光量子产率以罗丹明6G为标准(QY＝0.94)，采用相对法测得。
试验例5：
构建了以适配体(ACTB-4Pepper RNA适配体序列
AUGGAUGAUGAUAUCGCCGCGCUCGUCGUCGACAACGGCUCCGGCAUGUGCAAGGCCGGCUUCGCGGGCGACGAUGCCCCCCGGGCCGUCUUCCCCUCCAUCGUGGGGCGCCCCAGGCACCAGGGCGUGAUGGUGGGCAUGGGUCAGAAGGAUUCCUAUGUGGGCGACGAGGCCCAGAGCAAGAGAGGCAUCCUCACCCUGAAGUACCCCAUCGAGCACGGCAUCGUCACCAACUGGGACGACAUGGAGAAAAUCUGGCACCACACCUUCUACAAUGAGCUGCGUGUGGCUCCCGAGGAGCACCCCGUGCUGCUGACCGAGGCCCCCCUGAACCCCAAGGCCAACCGCGAGAAGAUGACCCAGAUCAUGUUUGAGACCUUCAACACCCCAGCCAUGUACGUUGCUAUCCAGGCUGUGCUAUCCCUGUACGCCUCUGGCCGUACCACUGGCAUCGUGAUGGACUCCGGUGACGGGGUCACCCACACUGUGCCCAUCUACGAGGGGUAUGCCCUCCCCCAUGCCAUCCUGCGUCUGGACCUGGCUGGCCGGGACCUGACUGACUACCUCAUGAAGAUCCUCACCGAGCGCGGCUACAGCUUCACCACCACGGCCGAGCGGGAAAUCGUGCGUGACAUUAAGGAGAAGCUGUGCUACGUCGCCCUGGACUUCGAGCAAGAGAUGGCCACGGCUGCUUCCAGCUCCUCCCUGGAGAAGAGCUACGAGCUGCCUGACGGCCAGGUCAUCACCAUUGGCAAUGAGCGGUUCCGCUGCCCUGAGGCACUCUUCCAGCCUUCCUUCCUGGGCAUGGAGUCCUGUGGCAUCCACGAAACUACCUUCAACUCCAUCAUGAAGUGUGACGUGGACAUCCGCAAAGACCUGUACGCCAACACAGUGCUGUCUGGCGGCACCACCAUGUACCCUGGCAUUGCCGACAGGAUGCAGAAGGAGAUCACUGCCCUGGCACCCAGCACAAUGAAGAUCAAGAUCAUUGCUCCUCCUGAGCGCAAGUACUCCGUGUGGAUCGGCGGCUCCAUCCUGGCCUCGCUGUCCACCUUCCAGCAGAUGUGGAUCAGCAAGCAGGAGUAUGACGAGUCCGGCCCCUCCAUCGUCCACCGCAAAUGCUUCUAGCACUCGCUAGAGCAUGGUUAAGCUUCCCACGGAGGAUCCCCAAUCGUGGCGUGUCGGCCUCUCCCAAUCGUGGCGUGUCGGCCUCUCCCAAUCGUGGCGUGUCGGCCUCUCCCAAUCGUGGCGUGUCGGCCUCUCCCAAUCGUGGCGUGUCGGCCUCUCUUCGGAGAGGCACUGGCGCCGGAGAGGCACUGGCGCCGGAGAGGCACUGGCGCCGGAGAGGCACUGGCGCCGGGAUCCUCCGUGGG)融合骨架蛋白mRNA的稳定细胞株(293T/17细胞株)，在常规哺乳动物细胞培养条件下(37℃，5％二氧化碳，100％相对湿度)，待该细胞株与对照细胞(293T/17)生长至细胞汇合度达90％后，将细胞消化下来，800rpm离心，并使用含有0.2μM III-3和0.2μMIII-43分子的PBS重悬细胞并孵育5分钟后进行流式检测，检测结果可见图18，III-3分子转子在表达了目标RNA的细胞株中可特异性标记骨架蛋白的mRNA，且无明显本底荧光(如图18a)，而III-43分子本底荧光高于III-3，无法清晰区分ACTB是否表达(如图18b)。
SEQUENCE LISTING
<110> 华东理工大学
<120> 一种荧光染料及其制备方法和用途
<130> 2019
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 390
<212> RNA
<213> 人工序列
<400> 1
uugccaugug uauguggguu cgcccacaua cucugaugau ccccaaucgu ggcgugucgg 60
ccucucccaa ucguggcgug ucggccucuc ccaaucgugg cgugucggcc ucucccaauc 120
guggcguguc ggccucuccc aaucguggcg ugucggccuc ucccaaucgu ggcgugucgg 180
ccucucccaa ucguggcgug ucggccucuc ccaaucgugg cgugucggcc ucucuucgga 240
gaggcacugg cgccggagag gcacuggcgc cggagaggca cuggcgccgg agaggcacug 300
gcgccggaga ggcacuggcg ccggagaggc acuggcgccg gagaggcacu ggcgccggag 360
aggcacuggc gccgggauca uucauggcaa 390
<210> 2
<211> 1373
<212> RNA
<213> 人工序列
<400> 2
auggaugaug auaucgccgc gcucgucguc gacaacggcu ccggcaugug caaggccggc 60
uucgcgggcg acgaugcccc ccgggccguc uuccccucca ucguggggcg ccccaggcac 120
cagggcguga uggugggcau gggucagaag gauuccuaug ugggcgacga ggcccagagc 180
aagagaggca uccucacccu gaaguacccc aucgagcacg gcaucgucac caacugggac 240
gacauggaga aaaucuggca ccacaccuuc uacaaugagc ugcguguggc ucccgaggag 300
caccccgugc ugcugaccga ggccccccug aaccccaagg ccaaccgcga gaagaugacc 360
cagaucaugu uugagaccuu caacacccca gccauguacg uugcuaucca ggcugugcua 420
ucccuguacg ccucuggccg uaccacuggc aucgugaugg acuccgguga cggggucacc 480
cacacugugc ccaucuacga gggguaugcc cucccccaug ccauccugcg ucuggaccug 540
gcuggccggg accugacuga cuaccucaug aagauccuca ccgagcgcgg cuacagcuuc 600
accaccacgg ccgagcggga aaucgugcgu gacauuaagg agaagcugug cuacgucgcc 660
cuggacuucg agcaagagau ggccacggcu gcuuccagcu ccucccugga gaagagcuac 720
gagcugccug acggccaggu caucaccauu ggcaaugagc gguuccgcug cccugaggca 780
cucuuccagc cuuccuuccu gggcauggag uccuguggca uccacgaaac uaccuucaac 840
uccaucauga agugugacgu ggacauccgc aaagaccugu acgccaacac agugcugucu 900
ggcggcacca ccauguaccc uggcauugcc gacaggaugc agaaggagau cacugcccug 960
gcacccagca caaugaagau caagaucauu gcuccuccug agcgcaagua cuccgugugg 1020
aucggcggcu ccauccuggc cucgcugucc accuuccagc agauguggau cagcaagcag 1080
gaguaugacg aguccggccc cuccaucguc caccgcaaau gcuucuagca cucgcuagag 1140
caugguuaag cuucccacgg aggaucccca aucguggcgu gucggccucu cccaaucgug 1200
gcgugucggc cucucccaau cguggcgugu cggccucucc caaucguggc gugucggccu 1260
cucccaaucg uggcgugucg gccucucuuc ggagaggcac uggcgccgga gaggcacugg 1320
cgccggagag gcacuggcgc cggagaggca cuggcgccgg gauccuccgu ggg 1373

Claims (10)

1.一种荧光染料，其结构式如式(I)所示，
Figure FDA0002043227700000011
其中：
D-为HO-或N(X1)(X2)-，X1、X2各自独立地选自氢、烷基和改性烷基；X1，X2任选相互连接，与N原子一起形成脂杂环；
R选自氰基、羧基、酰胺基、酯基、亚砜基、砜基、磺酸酯基或磺酰胺基；
Ar1和Ar2各自独立地选自亚芳基，亚杂芳基；其中，Ar1、Ar2中的氢原子任选各自独立地被卤原子、羟基、醛基、羧基、酯基、酰胺基、氰基、磺酸基、磷酸基、氨基、伯氨基、仲氨基、烷基或改性烷基取代；
X1、X2任选独立地与Ar1形成脂杂环；
其中：
所述“烷基”各自独立地为C1-C10直链或支链烷基；可选地，为C1-C7直链或支链烷基；可选地，为C1-C5直链或支链烷基；可选地，选自甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、仲丁基、正戊基，1-甲基丁基、2-甲基丁基、3-甲基丁基、异戊基、1-乙基丙基、新戊基、正己基、1-甲基戊基、2-甲基戊基、3-甲基戊基、异己基、1，1-二甲基丁基、2，2-二甲基丁基、3，3-二甲基丁基、1，2-二甲基丁基、1，3-二甲基丁基、2，3-二甲基丁基、2-乙基丁基、正庚基、2-甲基己基、3-甲基己基、2，2-二甲基戊基、3，3-二甲基戊基、2，3-二甲基戊基、2，4-二甲基戊基、3-乙基戊基或2，2，3-三甲基丁基；
所述“改性烷基”各自独立地为烷基的任意碳原子被选自卤原子、-OH、-CO-、-O-、-CN、-S-、-SO2-、-(S＝O)-、叠氮基、伯氨基、仲氨基、叔氨基、季铵盐基的一种或多种基团置换所得的基团，所述改性烷基具有1-10个碳原子，其中碳碳单键任选独立地被碳碳双键或碳碳三键置换；
所述的碳原子被置换，是指碳原子或碳原子与其上的氢原子一起被相应的基团置换；
所述“卤原子”各自独立地为F、Cl、Br或I；
所述“脂杂环”为环上含有N、O、S或Si中的一种或多种杂原子的饱和或不饱和的4～15元单环或多环脂杂环，所述脂杂环上含有S原子时，其为-S-、-SO-或-SO2-；所述脂杂环任选被卤原子、烷基、芳基或改性烷基取代；
所述“亚芳基”为5～13元单环或双环或稠合双环或稠合多环的亚芳香基团；
所述“亚杂芳基”为环上含有选自N、O、S或Si中的一种或多种杂原子的5～13元单环或双环或稠合双环或稠合多环的亚杂芳香基团；
所述“酯基”为R′(C＝O)OR″基团；
所述“酰胺基”为R′CONR″R″′基团；
所述“磺酸基”为R′SO3H基团；
所述“磺酸酯基”为R′SO2OR″基团；
所述“磺酰胺基”为R′SO2NR″R″′基团；
所述“磷酸基”为R′OP(＝O)(OH)2基团；
所述“砜基”为R′SO2R″基团；
所述“亚砜基”为R′SOR″基团；
所述“伯氨基”为R′NH2基团；
所述“仲氨基”为R′NHR″基团；
所述“叔氨基”为R′NR″R″′基团；
所述“季铵盐基”为R′R″R″′R″″N+基团；
各个R′、R″、R″′、R″″各自独立地为单键、氢、烷基、亚烷基、改性烷基或改性亚烷基；
所述“亚烷基”为C1-C10的直链或支链的亚烷基；可选地，为C1-C7直链或支链亚烷基；可选地，为C1-C5直链或支链亚烷基；
所述“改性亚烷基”为C1-C10(优选为C1-C6)亚烷基的任意碳原子被选自-O-、-OH、-CO-、-CS-、-(S＝O)-中的基团置换所得的基团。
2.根据权利要求1所述的荧光染料，其特征在于，
所述“改性烷基”为含有选自-OH、-O-、乙二醇单元、单糖单元、-O-CO-、-NH-CO-、-SO2-O-、-SO-、Me2N-、Et2N-、-S-S-、-CH＝CH-、F、Cl、Br、I、氰基中的一种或多种基团。
3.根据权利要求1或2所述的荧光染料，其特征在于，
Ar1和Ar2各自独立地为选自下式(II-1)～(II-22)中的结构：
Figure FDA0002043227700000031
4.根据权利要求1-3任一项所述的荧光染料，其特征在于，式(I)所示的化合物选自下式化合物：
Figure FDA0002043227700000032
Figure FDA0002043227700000041
5.制备权利要求1-4任一项所述的荧光染料的方法，其特征在于，包括式(a)化合物与式(b)化合物发生羟醛缩合反应的步骤，
Figure FDA0002043227700000042
6.权利要求1-4任一项所述的荧光染料在粘度测试、蛋白荧光标记、核酸荧光标记、蛋白定量或检测、或者核酸定量或检测中的用途，所述用途为非用于疾病的诊断方法的用途。
7.权利要求1-4任一项所述的荧光染料在制备用于粘度测试、蛋白荧光标记、核酸荧光标记、蛋白定量或检测、或者核酸定量或检测的试剂中的用途。
8.一种荧光激活点亮型探针，其包括权利要求1-4任一项所述的荧光染料。
9.权利要求8所述的荧光激活点亮型探针在蛋白荧光标记、核酸荧光标记、蛋白定量或检测、或者核酸定量或检测中的用途，所述用途为非用于疾病的诊断方法的用途。
10.权利要求8所述的荧光激活点亮型探针在制备用于蛋白荧光标记、核酸荧光标记、蛋白定量或检测、或者核酸定量或检测的试剂中的用途。
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