CN114958025A - 一种有机小分子荧光染料及其制备方法和用途 - Google Patents
一种有机小分子荧光染料及其制备方法和用途 Download PDFInfo
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Abstract
本发明提供了一种有机小分子荧光染料及其制备方法和用途,所示荧光染料具有如下式I表示的结构,或其药学上可接受的盐,或其对映异构体、非对映异构体、互变异构体或溶剂化物。本发明的荧光染料应用于寡核苷酸修饰时,由于本发明的有机小分子染料具有90至200nm斯托克斯位移,从而解决了测量中因串扰以及后向散射效应带来的自淬灭和测量误差的问题,同时使生物偶联方法更有效。
Description
相关申请的交叉引用
本申请要求于2021年2月26日提交的专利申请号为202110220384.8、发明名称为“一种有机小分子荧光染料及其制备方法和用途”的专利申请的优先权,其全部内容通过引用并入本文。
技术领域
本发明属于荧光染料领域,更具体地,本发明通常涉及一种有机小分子荧光染料及其制备方法和用途。
背景技术
有机小分子荧光染料是一种广泛用于药物研发及临床研究的物质。荧光修饰物具有若干优点,例如,结构与光学性质的灵活可调控性、可非侵入性测量、高细胞兼容性、实时响应及高灵敏度和多种转导方法(荧光猝灭或增强,荧光各向异性,荧光寿命,荧光共振能量转移和准分子-单体光转换)1-5。
市场中有诸多商业化染料,如罗丹明(Rhodamine)系列、BODIPY系列、荧光素(FITC)系列、香豆素(Coumarin)系列、花青素(cyanine)系列、恶嗪(oxazine)、ATTO系列、AleaxFluor和LightCycler系列等。然而,这些染料的斯托克斯位移(Stokes shift)大都小于35纳米(nm),由于激发光与发射光之间的串扰以及后向散射效应(backscatteringeffects)的影响,在用于测量过程中会产生严重的自淬灭和测量误差6-8。
另外,医学生物成像要求染料最好在650-900纳米的光学范围内,因为这个范围内光散射效应、自发荧光以及器官组织的吸收现象可以大大减少9,10。因此,为了满足市场应用于生物检测的要求11,12,需要开发在650-900纳米范围内斯托克斯位移大于35纳米的荧光染料分子13–15。
目前,最常用的染料标记寡核苷酸是通过手动的两步程序进行的。第一步为通过固相合成手段合成带活性基团的寡核苷酸,第二步为将活化的染料与寡核苷酸的活性基团连接。活化的染料通常是引入反应性基团,该反应性基团有助于染料与寡核苷酸的共价连接。尽管这种偶联方法在大多数情况下是有效的,但这种两步法耗时(需要4-5天),操作繁琐,试剂昂贵且通常会产生不需要的有机副产物。另一种替代的、更方便的一步方法是,将荧光染料转化为亚磷酰胺结构的染料分子,并在寡核苷酸的固相合成过程中用于直接标记寡核苷酸,这种方法不需要合成后的修饰,从而提高偶联的效率降低成本16。因此,开发在650-900纳米范围内斯托克斯位移大于35纳米的荧光染料分子、更特别地是其亚磷酰胺结构分子的需求巨大。
发明内容
本发明的目的在于克服目前市面上的染料因斯托克斯位移大都小于35nm,造成在测量过程中会产生严重的自淬灭和测量误差的缺陷,从而提供一系列用于寡核苷酸固相合成的新型结构并拥有90-200nm斯托克斯位移的有机小分子染料,这可解决测量中上述因斯托克斯位移小于35nm导致的串扰以及后向散射效应带来的自淬灭和测量误差的问题,同时使生物偶联方法更有效。
为了实现上述目的,在一方面,本发明提供了一种有机小分子荧光染料,其具有如下式I表示的结构,或其药学上可接受的盐,或其对映异构体、非对映异构体、互变异构体或溶剂化物:
[式I]
其中,
R1选自氢、或者取代或未取代的C1-C6烷基、C2-C6烯基、C2-C6炔基或C2-C6烷酰基;R2-R4各自独立选自氢、羟基、氰基、卤素、或者取代或未取代的氨基、C1-C6烷基、C1-C6烷氧基、C3-C6环烷基、C1-C6羧基或C2-C6烷酰基;R5-R8各自独立选自氢、羟基、氰基、卤素、或者取代或未取代的氨基、C1-C6烷基、C1-C6烷氧基、C3-C6环烷基、C1-C6羧基或C2-C6烷酰基,或者R5-R6和R7-R8中的一者与其所连接的碳原子一起形成取代或未取代的苯环;并且R9选自氢、或者取代或未取代的C1-C6烷基、C2-C6烯基、C2-C6炔基、C2-C6烷酰基、C1-C6烷基硅基或亚磷酰胺基。
如本文所用,术语“药学上可接受的盐”包括药学上可接受的酸加成盐和药学上可接受的碱加成盐。其中,所述药学上可接受的酸加成盐是指能够保留游离碱的生物有效性而无其它副作用的与无机酸或有机酸所形成的盐,例如无机酸盐包括但不限于盐酸盐、氢溴酸盐、硫酸盐、硝酸盐、磷酸盐等;有机酸盐包括但不限于甲酸盐、乙酸盐、三氟乙酸盐、丙酸盐、己酸盐、水杨酸盐等。药学上可接受的碱加成盐是指能够保持游离酸的生物有效性而无其它副作用的与无机碱或有机碱所形成的盐,例如无机碱盐包括但不限于钠盐、钾盐、锂盐、铵盐、钙盐、镁盐、铁盐、锌盐、铜盐、锰盐、铝盐等;有机碱盐包括但不限于伯胺类、仲胺类及叔胺类盐。这些盐可通过本专业已知的方法制备。
在本发明的优选实施方式中,R1可以选自氢、或者取代或未取代的C1-C6烷基或C2-C6烷酰基;R2-R4可以各自独立选自氢、羟基、氰基、卤素、或者取代或未取代的氨基、C1-C6烷基、C1-C6烷氧基或C3-C6环烷基;R5-R8可以各自独立选自氢、羟基、氰基、卤素、或者取代或未取代的氨基、C1-C6烷基、C1-C6烷氧基或C3-C6环烷基,或者R5-R6和R7-R8中的一者可以与其所连接的碳原子一起形成取代或未取代的苯环;并且R9可以选自氢、或者取代或未取代的C1-C6烷基、C2-C6烷酰基、C1-C6烷基硅基或亚磷酰胺基。对于上述R1-R9的优选选择方式可以单独选择或组合选择。
在本发明的优选实施方式中,R1可以选自氢、或者取代或未取代的甲基、乙基、丙基、异丙基、乙酰基、丙酰基或叔丁基酰基;R2-R4可以各自独立选自氢、羟基、氰基、卤素、或者取代或未取代的氨基、甲基、乙基、丙基、异丙基、甲烷氧基、乙烷氧基、环丙基或环丁基;R5-R8可以各自独立选自氢、羟基、氰基、卤素、或者取代或未取代的氨基、甲基、乙基、丙基、异丙基、甲烷氧基、乙烷氧基、环丙基或环丁基,或者R5-R6和R7-R8中的一者可以与其所连接的碳原子一起形成取代或未取代的苯环;并且R9可以选自氢、或者取代或未取代的甲基、乙基、丙基、异丙基、乙酰基、丙酰基、叔丁基酰基、叔丁基硅基或亚磷酰胺基,最优选地为亚磷酰胺基。对于上述R1-R9的优选选择方式可以单独选择或组合选择。
在本发明的优选实施方式中,所述取代可以为被卤素(例如氟、氯或溴)、羟基、氨基、氰基、C1-C3烷基(例如甲基、乙基、丙基或异丙基)、C1-C3烷氧基(例如甲烷氧基、乙烷氧基或丙烷氧基)或C1-C3卤代烷基(例如卤代甲基、卤代乙基或卤代丙基)取代,但不限于此。另外,所述取代可以包括一个或多个取代,取代数量的上限可以取决于氢的数量,即全部取代。
在本发明的一个优选实施方式中,所述亚磷酰胺基可以具有如下结构:
在本发明的一个优选实施方式中,所述有机小分子荧光染料可以进一步地表示为具体的化合物,例如,其可以具有如下所示结构中的一种:
在本发明的优选实施方式中,该有机小分子荧光染料可具有90至200nm(例如105nm、106nm、122nm、123nm、145nm、147nm、149nm、150nm或170nm等)、优选地,具有105nm至170nm、更优选地,具有105至123nm(更优选为亚磷酰胺类荧光染料)斯托克斯位移。
在另一方面,本发明还提供了一种制备上述有机小分子荧光染料的方法,其包括:使式II化合物与式III化合物接触,
[式II]
[式III]
其中,R1-R9如上述所定义。
在本发明的优选实施方式中,所述式II化合物可以由式IV化合物和式V化合物制备得到,
[式IV]
[式V]
其中,R1-R9如上述所定义。
在本发明的优选实施方式中,所述式III化合物可以由式VI化合物和式VII化合物制备得到,
[式VI]
[式VII]
其中,R1-R9如上述所定义。
对于本发明提供的上述制备方法中所使用的各反应原料,其可以通过商业购买或各种本领域已知的合成工艺得到,而没有特别地限制。另外,本发明的有机小分子荧光染料还可以通过已由本发明制备方法得到的另一种有机小分子荧光染料经过侧基的取代而转变得到。也就是说,在本发明范围内的各种有机小分子荧光染料在理想的情况下也可以通过互相转变的方式得到,而不用重新合成制备。
具体地,在优选的实施方式中,本发明的制备方法可以通过如下所示的两种示例性合成路线中的任一种来进行:
其中,具体地,路线(I)示出了从小分子原料开始合成本发明的荧光染料的示例性方法,而路线(II)示出了通过产品之间的相互转变而合成本发明的荧光染料的示例性方法。
对于上述两种示例性合成路线中的反应条件,可以根据本领域技术人员的经验来实现,优选地,所示条件可以如下所示设置:a)PBr3,CH2Cl2,DMF,RT,16h;b)Cs2CO3,DMF,RT,16h;c)90℃,18h;d)吡啶/THF,TBDMSCl,RT,5h;e)哌啶,EtOH,回流,16h;f)2-氰乙基N,N,N',N'-四异丙基磷酰胺,BTT,CH2Cl2,RT,2h;g)PivCl,Et3N,CH2Cl2,0℃,2h;h)Et3N/3HF,DMF,16h。
在又另一方面,本发明还提供了上述有机小分子荧光染料在寡核苷酸修饰中的用途。
在又另一方面,本发明还提供了上述有机小分子荧光染料在多肽修饰中的用途
在又另一方面,本发明还提供了上述有机小分子荧光染料在核酸药物合成中的用途。
在将本发明的有机小分子荧光染料应用于寡核苷酸修饰和核酸药物合成中时,由于本发明的有机小分子染料具有90至200nm,优选地,具有105至170nm斯托克斯位移,更优选地,具有105至123nm斯托克斯位移,从而解决了测量中因串扰以及后向散射效应带来的自淬灭和测量误差的问题,同时使生物偶联方法更有效。
附图说明
附图是用来提供对本发明的进一步理解,并且构成说明书的一部分,与下面的具体实施方式一起用于解释本发明,但并不构成对本发明的限制。在附图中:
图1示出了DT-4在pH=7.0水溶液中的吸收光及发射光谱;
图2示出了DT-4-Ph在pH=7.0水溶液中的吸收光及发射光谱;
图3示出了DT-6在pH=7.0水溶液中的吸收光及发射光谱;
图4示出了DT-6-Ph在pH=7.0水溶液中的吸收光及发射光谱;
图5示出了DT-8在pH=7.0水溶液中的吸收光及发射光谱;
图6示出了DT-8-Ph在pH=7.0水溶液中的吸收光及发射光谱;
图7示出了DT-5与寡核苷酸偶联后纯化后的产物HPLC图谱;
图8示出了DT-5与寡核苷酸偶联后的产物质谱;
图9示出了DT-5与寡核苷酸偶联后的产物在pH=7.0水溶液中的吸收光及发射光谱;
图10示出了DT-5-Ph与寡核苷酸偶联后纯化后的产物HPLC图谱;
图11示出了DT-5-Ph与寡核苷酸偶联后的产物质谱;
图12示出了DT-5-Ph与寡核苷酸偶联后的产物在pH=7.0水溶液中的吸收光及发射光谱;
图13示出了DT-9与寡核苷酸偶联后纯化后的产物HPLC图谱;
图14示出了DT-9与寡核苷酸偶联后的产物质谱;
图15示出了DT-9与寡核苷酸偶联后的产物在pH=7.0水溶液中的吸收光及发射光谱;
图16示出了DT-9-Ph与寡核苷酸偶联后纯化后的产物HPLC图谱;
图17示出了DT-9-Ph与寡核苷酸偶联后的产物质谱;并且
图18示出了DT-9-Ph与寡核苷酸偶联后的产物在pH=7.0水溶液中的吸收光及发射光谱。
具体实施方式
以下对本发明的具体实施方式进行详细说明。应当理解的是,此处所描述的具体实施方式仅用于说明和解释本发明,并不用于限制本发明。
在本文中所披露的范围的端点和任何值都不限于该精确的范围或值,这些范围或值应当理解为包含接近这些范围或值的值。对于数值范围来说,各个范围的端点值之间、各个范围的端点值和单独的点值之间,以及单独的点值之间可以彼此组合而得到一个或多个新的数值范围,这些数值范围应被视为在本文中具体公开。
在以下实施例中,部分所使用的化学品的来源如下表1所示:
表1
名称 | 厂家 | 货号 |
环己酮 | 上海阿拉丁生化科技股份有限公司 | C116449 |
三溴化磷 | 上海阿拉丁生化科技股份有限公司 | P108403 |
三溴化硼 | 上海阿拉丁生化科技股份有限公司 | 104688 |
N,N-二甲基甲酰胺 | 百灵威科技有限公司 | 983353 |
氯仿 | 国药集团化学试剂有限公司 | 10006818 |
碳酸铯 | 上海阿拉丁生化科技股份有限公司 | C105061 |
2-羟基-4-甲氧基苯甲醛 | 上海皓鸿生物医药科技有限公司 | 1039550 |
4-甲基吡啶 | 上海阿拉丁生化科技股份有限公司 | P105226 |
5-溴戊酸 | 上海阿拉丁生化科技股份有限公司 | B106966 |
无水乙醇 | 国药集团化学试剂有限公司 | 100092683 |
二氯甲烷 | 国药集团化学试剂有限公司 | 800473603 |
双(二异丙基氨基)(2-氰基乙氧基)膦 | 上海毕得医药科技有限公司 | BD40171 |
5-苄硫基四氮唑 | 百灵威科技有限公司 | 295494 |
勒皮啶 | 百灵威科技有限公司 | 131729 |
实施例1化合物的制备
化合物DT-1的合成
根据上述示例性合成路线,向两口圆底烧瓶中加入43.8g N,N-二甲基甲酰胺(DMF,600mmol)和100mL二氯甲烷(CH2Cl2),将体系至于冰浴中,在磁子搅拌条件下滴加135.2g PBr3(500mmol),约45min滴加完毕,反应1h后加入19.64g环己酮(200mmol),撤去冰浴,自然升温至室温,搅拌反应24h后,将反应液倒入冰水中,并加入固体碳酸氢钠(NaHCO3)直至pH至为7左右,分离有机相,水相用CH2Cl2萃取,合并有机层并用无水硫酸钠(NaSO4)干燥,过滤,浓缩除去溶剂,得到红色油状物无需纯化直接投入下一步反应。
化合物DT-2的合成
根据上述示例性合成路线,将上步反应产物(理论上为9.68mmol)溶解在15mL DMF中,磁子搅拌,加入1.23g(8.1mmol)2-羟基-4-甲氧基苯甲醛和7.88g(24.2mmol)碳酸铯(Cs2CO3),室温反应16h。反应完成后过滤,滤液浓缩,浓缩后的产物溶于DCM中,并用水洗两次,有机层用无水硫酸钠干燥,过滤,减压浓缩除去溶剂,硅胶柱层析(石油醚:乙酸乙酯(PE:EtOAc)=5:1,Rf=0.6),得到亮黄色固体产物0.71g(2.9mmol),第一步和第二步反应总收率36.2%。质谱(ESI):[M+H]+:243.27。1H NMR(400MHz,CDCl3)δ10.32(s,1H),7.08(d,J=9.1Hz,1H),6.76–6.57(m,3H),3.84(s,3H),2.66–2.51(m,2H),2.45(t,J=6.0Hz,2H),1.78–1.68(m,2H)。
化合物DT-2-OH的合成
向反应管中加入11.2g(80mmol)2,4-二羟基苯甲醛、100mmol DT-1和300mL DMF,然后加入98g(300mmol)碳酸铯,在室温下搅拌反应过夜。反应后过滤,滤液浓缩,浓缩后的产物溶于DCM中,并用水洗涤两次,有机层用无水硫酸钠干燥,过滤,减压浓缩除去溶剂,硅胶柱层析纯化。得到亮黄色固体产物5.1g(22.4mmol),收率28%。质谱(ESI):[M+H]+:228.24。1H NMR(400MHz,DMSO-d6)δ9.35(d,J=6.6Hz,2H),7.85(d,J=6.3Hz,2H),4.92(t,J=7.5Hz,2H),3.60(t,J=5.9Hz,2H),2.67(s,3H),2.08–2.04(m,2H),1.61–1.40(m,6H)。
化合物DT-3的合成
向圆底烧瓶中加入2.37g(25mmol)4-甲基吡啶和4.50g(25mmol)5-溴戊酸,搅拌条件下,油浴加热至90℃,反应过夜。冷却至室温,加入乙酸乙酯,油状物逐渐变为固体,将固体粉碎,用大量乙酸乙酯洗。过滤即得的目标产物DT-3为土黄色产物4.5g(16.9mmol),收率67.6%。质谱(ESI):[M-Br]+:194.24。1H NMR(400MHz,CDCl3)δ8.95(d,J=6.6Hz,2H),8.00(d,J=6.3Hz,2H),4.55(t,J=7.2Hz,2H),2.61(s,3H),2.27(t,J=7.4Hz,2H),1.97–1.82(m,2H),1.50-1.44(m,2H)。
化合物DT-3-Ph的合成
根据上述示例性合成路线,将3.58g(25mmol)勒皮定和4.50g(25mmol)5-溴-1-戊酸加入圆底烧瓶中,由于加热至110℃,反应过夜。停止反应,加入50mL乙酸乙酯,将生成的固体破碎,用乙酸乙酯和丙酮洗至无原料,过滤,干燥。所得固体用乙酸乙酯与甲醇重结晶的方法进行重结晶。得到DT-3-Ph为浅绿色固体6.6g(20.6mmol),产率82.3%;质谱(ESI):[M-Br]+:244.35。1H NMR(400MHz,DMSO)δ9.46(d,J=7.0,1H),8.61(d,J=8.9Hz,1H),8.56(d,J=8.5,1H),8.27(d,J=8.7,1H),8.12–8.03(m,2H),5.03(t,J=7.5Hz,2H),3.37(t,J=6.1Hz,2H),3.02(s,3H),2.05–1.88(m,2H),1.53–1.29(m,6H)。
化合物DT-3-TBDMS的合成
向圆底烧瓶中加入5.0g(15.4mmol)DT-3,并加入20mL吡啶和20mL四氢呋喃,搅拌条件下,加入4.6g(30.8mmol)叔丁基二甲基甲硅烷基氯(TBDMSCl)和5.2g(30.8mmol)硝酸银(AgNO3),室温反应7h。反应完成后将反应液过滤,减压浓缩后无需纯化直接投入下一步反应。质谱(ESI):[M-Br]+:308.20。1H NMR(400MHz,DMSO)δ8.99(d,J=6.3Hz,2H),8.07–8.02(d,J=6.3Hz,2H),4.55(t,J=7.4Hz,2H),3.36(t,J=6.3Hz,2H),2.61(s,3H),1.89(m,J=7.3Hz,2H),1.35–1.18(m,6H),0.83(s,9H),-0.05(s,6H)。
化合物DT-3-Ph-TBDMS的合成
向圆底烧瓶中加入3.24g(10mmol)DT-3-Ph-OH,并加入12mL吡啶和12mL四氢呋喃,在搅拌条件下,加入3.0g(20.0mmol)TBDMSCl和3.4g(20.0mmol)AgNO3,室温反应7h。反应完成后将反应液过滤,减压浓缩后无需纯化直接投入下一步反应。质谱(ESI):[M-Br]+:359.13。1H NMR(400MHz,DMSO)δ9.40(d,J=6.0Hz,1H),8.59(d,J=8.9Hz,1H),8.45(tt,J=7.8,1.7Hz,1H),8.27(ddd,J=8.7,6.9,1.5Hz,1H),8.10–8.05(m,2H),5.00(t,J=7.5Hz,2H),3.37(t,J=6.2Hz,2H),3.01(s,3H),1.95(m,2H),1.50–1.30(m,6H),0.84(s,9H),-0.04(s,3H)。
化合物DT-4的合成
向圆底烧瓶中加入2.2g(8mmol)DT-3和2.4g(10mmol)DT-2,然后加入30mL无水乙醇,搅拌条件下加入两滴哌啶,回流反应过夜。反应完成后将反应液减压浓缩,硅胶柱层析分离纯化,得到紫色产物2.9g(5.8mmol),收率73.1%。质谱(ESI):[M-Br]+:418.27。1H NMR(400m,CDCl3)δ8.91(d,J=6.9Hz,2H),8.12(d,J=15.6Hz,1H),7.78(d,J=6.9Hz,2H),6.97(d,J=8.5Hz,1H),6.86(d,J=2.3Hz,1H),6.58(dd,J=8.5,2.4Hz,1H),6.49(s,1H),6.27(d,J=15.6Hz,1H),4.60(t,J=7.4Hz,2H),3.87(s,3H),3.60(t,J=6.0Hz,2H),2.47(dt,J=26.3,5.7Hz,4H),1.98(dd,J=12.9,6.2Hz,2H),1.82–1.72(m,2H),1.54–1.33(m,6H)。取2.1mg DT-4溶于2.5mL乙腈中,然后加入纯水(pH=7.0)至50mL,配成100μM溶液,再取10mL加入40mL纯水(pH=7.0)稀释至20μM。溶液在pH=7.0条件下,经过紫外吸收及荧光发射检测,如图1所示,其最大吸收波长为523nm,最大发射波长为670nm,斯托克斯位移为147nm。
化合物DT-4-Ph的合成
向圆底烧瓶中加入1.6g(4.9mmol)DT-3-Ph和1.45g(6mmol)DT-2,然后加入20mL无水乙醇,搅拌条件下加入两滴哌啶,回流反应过夜。反应完成后将反应液减压浓缩,硅胶柱层析分离纯化,得到紫色产物2.0g(5.8mmol),收率73.7%。质谱(ESI):[M-Br]+:468.27。1HNMR(400MHz,CDCl3)δ9.91(d,J=6.8Hz,1H),8.54(d,J=15.0Hz,1H),8.46(d,J=8.6Hz,1H),8.27(d,J=6.8Hz,1H),8.05–7.99(m,2H),7.78(m,1H),7.13–7.02(m,3H),6.74–6.66(m,2H),5.03(t,J=7.5Hz,2H),3.97(s,3H),3.68(t,J=5.7Hz,2H),2.65(dt,J=19.6,6.0Hz,4H),2.14–2.02(m,2H),1.90(p,J=6.2Hz,2H)。取2.3mg DT-4-Ph溶于2.5mL乙腈中,然后加入纯水(pH=7.0)至50mL,配成100μM溶液,再取10mL加入40mL纯水(pH=7.0)稀释至20μM。溶液在pH=7.0条件下,经过紫外吸收及荧光发射检测,如图2所示,其最大吸收波长为559nm,最大发射波长为708nm,斯托克斯位移为149nm。
化合物DT-5的合成
将50mg(0.1mmol)DT-4-Ph用无水乙腈和无水乙醚旋蒸除水3次,然后溶于2mL无水二氯甲烷中,搅拌条件下向该混合物中加入48μL(0.15mmol)2-氰基乙基-N,N,N',N'-四异丙基亚磷酰胺试剂和31mg(0.17mmol)BTT,室温反应1h。反应完成后减压浓缩除去大部分溶剂,然后加入正己烷洗涤3次,固体减压浓缩至干,直接用于寡核苷酸修饰。质谱(ESI):[M-Br]+:618.13。1H NMR(400MHz,CDCl3)δ8.66(t,J=5.7Hz,2H),8.11(d,J=15.5Hz,1H),7.64(d,J=6.1Hz,2H),6.98(d,J=8.5Hz,1H),6.80(d,J=2.4Hz,1H),6.58(dd,J=8.4,2.4Hz,1H),6.50(s,1H),6.21(d,J=7.0Hz,1H),4.45(dt,J=15.2,7.5Hz,2H),4.19–4.01(m,4H),3.83(s,3H),3.74(q,J=6.6Hz,2H),3.47(dp,J=18.3,6.8Hz,4H),2.72(t,J=6.2Hz,3H),2.49(t,J=6.2Hz,2H),2.43(t,J=6.1Hz,2H),1.95–1.78(m,4H),1.76(t,J=6.1Hz,2H),1.23(d,J=6.3Hz,14H)。31P NMR(162MHz,CDCl3)δ147.11,146.88。
化合物DT-5-Ph的合成
将54mg(0.1mmol)DT-4-Ph用无水乙腈和无水乙醚旋蒸除水3次,然后溶于2mL无水二氯甲烷中,在搅拌条件下向该混合物中加入48μL(0.15mmol)2-氰基乙基-N,N,N',N'-四异丙基亚磷酰胺试剂和31mg(0.17mmol)BTT,室温反应1h。反应完成后减压浓缩除去大部分溶剂,然后加入正己烷洗涤3次,固体减压浓缩至干,直接用于寡核苷酸修饰。质谱(ESI):[M-Br]+:668.20。1H NMR(400MHz,CDCl3)δ9.21(dd,J=7.1,3.4Hz,1H),8.50(d,J=15.0Hz,1H),8.44(d,J=8.4Hz,1H),8.00(t,J=6.0Hz,1H),7.95(d,J=7.5Hz,2H),7.77(s,1H),7.12(m,J=8.0Hz,2H),7.07(d,J=8.4Hz,1H),6.74–6.67(m,2H),4.73(t,J=7.6Hz,2H),4.28(m,2H),4.11(m,3H),3.90(s,2H),3.63–3.46(m,2H),2.85–2.73(m,4H),2.62(t,J=6.4Hz,4H),1.91(m,6H),1.69(q,J=6.9Hz,2H),1.28(d,J=6.2Hz,12H)。31P NMR(162MHz,CDCl3)δ147.038,147.029。
化合物DT-6的合成
向圆底烧瓶加入2.2g DT-2-OH(9.6mmol)和4.4g DT-3-TBDMS(10mmol),加入30mL无水乙醇,在搅拌条件下加入两滴哌啶,回流反应过夜。反应完成后将反应液减压浓缩,硅胶柱层析分离纯化,得到紫色产物2.4g(4.0mmol),收率41.8%。质谱(ESI):[M-Br]+:519.67。1H NMR(400MHz,DMSO)δ10.12(s,1H),8.70(d,J=6.7Hz,2H),8.23(d,J=15.7Hz,1H),8.06(d,J=6.7Hz,2H),7.13(d,J=8.3Hz,1H),6.77(d,J=2.0Hz,2H),6.67–6.54(m,2H),4.40(t,J=7.3Hz,2H),3.56(t,J=6.3Hz,2H),2.59–2.52(m,2H),1.88(m,2H),1.75(t,J=6.2Hz,2H),1.45(m,2H),1.38–1.17(m,6H),0.85(s,9H)。取2.6mg DT-6溶于2.5mL乙腈中,然后加入纯水(pH=7.0)至50mL,配成100μM溶液,再取10mL加入40mL纯水(pH=7.0)稀释至20μM。溶液在pH=7.0条件下,经过紫外吸收及荧光发射检测,如图3所示,其最大吸收波长为528nm,最大发射波长为673nm,斯托克斯位移为145nm。
化合物DT-6-Ph的合成
向圆底烧瓶中加入1.4g DT-2-OH(6.1mmol)和2.9g DT-3-Ph-TBDMS(6.7mmol),加入20mL无水乙醇,在搅拌条件下加入两滴哌啶,回流反应过夜。将反应液减压浓缩,硅胶柱层析分离纯化,得到紫色产物1.22g(1.9mmol),收率30.9%。Mass(ESI):[M-Br]+:569.93。1H NMR(400MHz,DMSO)δ10.25(s,1H),9.00(d,J=6.8Hz,1H),8.84(dd,J=8.8,1.4Hz,1H),8.51(d,J=15.0Hz,1H),8.35(dd,J=15.5,7.8Hz,2H),8.14(ddd,J=8.6,6.9,1.3Hz,1H),7.89(dd,J=8.4,7.0Hz,1H),7.34(d,J=15.1Hz,1H),7.19(d,J=8.4Hz,1H),6.96–6.80(m,2H),6.64(dd,J=8.3,2.3Hz,1H),4.84(t,J=7.4Hz,2H),3.55(t,J=6.2Hz,2H),2.71(t,J=6.1Hz,2H),2.60(t,J=6.1Hz,2H),2.00–1.86(m,2H),1.80(t,J=6.3Hz,2H),1.53–1.26(m,6H),0.83(s,9H)。取2.9mg DT-6-Ph溶于2.5mL乙腈中,然后加入纯水(pH=7.0)至50mL,配成100μM溶液,再取10mL加入40mL纯水(pH=7.0)稀释至20μM。溶液在pH=7.0条件下,经过紫外吸收及荧光发射检测,如图4所示,其最大吸收波长为604nm,最大发射波长为726nm,斯托克斯位移为122nm。
化合物DT-7的合成
向圆底烧瓶中加入1.17g(1.96mmol)DT-6和25mL无水二氯甲烷,将体系置于冰浴,在搅拌条件下加入0.26g Piv-Cl(2.2mmol)和0.22g(2.2mmol)三乙胺,保持冰浴温度,反应2h。反应完成后减压浓缩除去溶剂,无需纯化直接投入下一步反应。
化合物DT-7-Ph的合成
向圆底烧瓶中加入0.66g(1.0mmol)DT-6-Ph和15mL无水二氯甲烷,将体系置于冰浴,在搅拌条件下加入0.13g Piv-Cl(1.1mmol)和0.11g(1.1mmol)三乙胺,保持冰浴温度,反应2h。反应完成后减压浓缩除去溶剂,无需纯化直接投入下一步反应。
化合物DT-8的合成
向DT-7(约1.96mmol)中加入20mL DMF和1.0mL Et3N/3HF,搅拌反应室温过夜。反应完成后减压浓缩除去溶剂,硅胶柱层析分离纯化,得到紫色产物0.59g(1.05mmol),收率53.4%。质谱(ESI):[M-Br]+:489.40。1H NMR(400MHz,DMSO)δ8.75(d,J=6.7Hz,2H),8.25(d,J=15.8Hz,1H),8.13(d,J=6.8Hz,2H),7.33(d,J=8.3Hz,1H),7.16(d,J=2.2Hz,1H),6.87(dd,J=8.3,2.3Hz,1H),6.82(s,1H),6.70(d,J=15.8Hz,1H),4.43(t,J=7.3Hz,2H),3.44–3.35(m,2H),2.59(t,J=6.0Hz,2H),1.88(p,J=7.5Hz,2H),1.77(t,J=6.1Hz,2H),1.42(m,2H),1.33(s,9H),1.32–1.21(m,6H)。取2.5mg DT-8溶于2.5mL乙腈中,然后加入纯水(pH=7.0)至50mL,配成100μM溶液,再取10mL加入40mL纯水(pH=7.0)稀释至20μM。溶液在pH=7.0条件下,经过紫外吸收及荧光发射检测。溶液在pH=7.0条件下,经过紫外吸收及荧光发射检测,如图5所示,吸收波长红移为500nm,最大发射波长红移为650nm,斯托克斯位移为150nm。
化合物DT-8-Ph的合成
向DT-7-Ph(约1.0mmol)中加入20mL DMF和1.0mL Et3N/3HF,搅拌反应室温过夜。反应完成后减压浓缩除去溶剂,硅胶柱层析分离纯化,得到紫色产物0.54g(0.88mmol),收率88.8%。质谱(ESI):[M-Br]+:539.07。1H NMR(400MHz,DMSO)δ9.14(d,J=6.7Hz,1H),8.85(d,J=8.7Hz,1H),8.47(d,J=15.2Hz,1H),8.43–8.34(m,2H),8.15(ddd,J=8.7,6.9,1.2Hz,1H),7.95–7.87(m,1H),7.46–7.31(m,2H),7.23(d,J=2.2Hz,1H),6.92–6.84(m,2H),4.89(t,J=7.5Hz,2H),3.38(t,J=6.1Hz,2H),2.71(t,J=6.1Hz,2H),2.62(t,J=6.0Hz,2H),1.92(d,J=7.8Hz,2H),1.81(t,J=6.2Hz,2H),1.47–1.35(m,6H),1.34(s,9H)。取2.7mg DT-8-Ph溶于2.5mL乙腈中,然后加入纯水(pH=7.0)至50mL,配成100μM溶液,再取10mL加入40mL纯水(pH=7.0)稀释至20μM。溶液在pH=7.0条件下,经过紫外吸收及荧光发射检测。溶液在pH=7.0条件下,经过紫外吸收及荧光发射检测,如图6所示,吸收波长红移为538nm,最大发射波长红移为708nm,斯托克斯位移为170nm。
化合物DT-9的合成
将57mg(0.1mmol)DT-4-Ph用无水乙腈和无水乙醚旋蒸除水3次,然后溶于2mL无水二氯甲烷中,在搅拌条件下向该混合物中加入48μL(0.15mmol)2-氰基乙基-N,N,N',N'-四异丙基亚磷酰胺试剂和31mg(0.17mmol)BTT,室温反应1h。反应完成后减压浓缩除去大部分溶剂,然后加入正己烷洗涤3次,固体减压浓缩至干,直接用于寡核苷酸修饰。质谱(ESI):[M-Br]+:689.40。1H NMR(400MHz,CDCl3)δ8.55(d,J=6.7Hz,1H),8.04(dd,J=15.7,2.2Hz,1H),7.59(d,J=6.7Hz,2H),7.09(d,J=8.3Hz,1H),6.97(d,J=2.3Hz,1H),6.75(d,J=5.7Hz,2H),6.52(s,1H),6.32(d,J=15.7Hz,1H),4.40(d,J=8.0Hz,2H),4.35(s,9H),4.25(dt,J=8.6,6.0Hz,2H),4.05(dd,J=8.7,6.3Hz,4H),2.63(q,J=6.8Hz,2H),2.54(t,J=6.1Hz,2H),2.47(t,J=6.2Hz,2H),1.93–1.72(m,6H),1.69–1.59(m,4H),1.35(d,J=3.1Hz,12H).31P NMR(162MHz,CDCl3)δ147.16,147.02。
化合物DT-9-Ph的合成
将62mg(0.1mmol)DT-4-Ph用无水乙腈和无水乙醚旋蒸除水3次,然后溶于2mL无水二氯甲烷中,搅拌条件下向该混合物中加入48μL(0.15mmol)2-氰基乙基-N,N,N',N'-四异丙基亚磷酰胺试剂和31mg(0.17mmol)BTT,室温反应1h。减压浓缩除去大部分溶剂,然后加入正己烷洗涤3次,固体减压浓缩至干,直接用于寡核苷酸修饰。质谱(ESI):[M-Br]+:738.97。1H NMR(400MHz,DMSO)δ8.99(d,J=6.6Hz,1H),8.79(d,J=8.9Hz,1H),8.54(d,J=15.1Hz,2H),8.41(s,2H),8.10(t,J=8.5Hz,1H),7.86(t,J=7.8Hz,1H),7.79(s,1H),7.04(d,J=2.5Hz,1H),6.90(s,1H),6.73(dd,J=8.5,2.4Hz,1H),4.78(d,J=7.2Hz,2H),4.25(s,9H),4.19-4.15(m,3H),4.06-4.01(m,4H),3.79–3.71(m,2H),2.72(d,J=6.2Hz,4H),1.80(q,J=5.9Hz,6H),1.54(d,J=6.8Hz,2H),1.41(s,12H)。31PNMR(162MHz,DMSO)δ146.44,146.39。
实施例2荧光染料用于寡核苷酸修饰
荧光标记的寡核苷酸探针通过类似转换器作用将生物识别(杂交,配体结合等)转化为荧光信号。荧光标记具有若干优点,例如,高灵敏度和多种转导方法(荧光猝灭或增强,荧光各向异性,荧光寿命,荧光共振能量转移和准分子-单体光转换)。这些多种信号选择与识别元件(DNA,RNA,PNA,LNA)的设计灵活性和各种标记策略相结合,有助于开发多种选择性和敏感的生物检测。
为了验证本发明中有机小分子荧光染料可以修饰于寡核苷酸中,将本发明得到的亚磷酰胺类染料被用于连接在一个包含一端是亚磷酰胺一端是羟基的C18连接子(在一些其他的具体实施例中,C18连接子也可以不添加)的长度为21个碱基的寡核苷酸的5’端(5’-染料-C18-CTCTATGGGCAGTCGGTGAAT-OH-3’)。将包含连接子、染料亚磷酰胺和寡核苷酸序列(CTCTATGGGCAGTCGGTGAAT)的位置信息编辑完毕后,上载于自动化Dr.Oligo48 DNA合成仪。单体A、C、G、T亚磷酰胺单体溶解于乙腈,浓度为0.06M;连接子溶解于乙腈,浓度为0.10M;染料亚磷酰胺溶解于乙腈,浓度为0.15M;分别置于合成仪单独的对应合成通道后,固相合成及切割脱保护的方法同常规的寡核苷酸固相合成参见(Beaucage等人,J.TetrahedronLetters.22.20:1859-1862(1981),其中,染料亚磷酰胺偶联时间为180s×2次,在得到含有染料分子的寡核苷酸链后,经过HPLC分离纯化回收。HPLC条件流动相为缓冲液A:0.1MTEAA,pH=7.0;缓冲液B:95%H2O,5%缓冲液A,pH=7.0。HPLC梯度洗脱条件为0-15min,乙腈浓度为10%-60%;15.01-17min,乙腈浓度为88%;17.01-19min,乙腈浓度为88%-10%。结果如下所示:
1)DT-5修饰寡核苷酸:纯化后的产物总产率:17.1%,纯度较高(保留时间:5.291min,纯度为97.80%,如图7所示),及质谱分析与理论值吻合(图8所示),经过修饰的寡核酸的6.6μM水溶液(pH=7.0)最大吸收峰在558nm,最大发射在680nm,斯托克斯位移达到122nm,如图9所示。
2)DT-5-Ph修饰寡核苷酸:纯化后的产物总产率:14.8%,纯度较高(保留时间:5.734min,纯度为99.15%,如图10所示),及质谱分析与理论值吻合(图11所示),经过修饰的寡核酸的8.2μM水溶液(pH=7.0)最大吸收峰在635nm,最大发射在740nm,斯托克斯位移达到105nm,如图12所示。
3)DT-9修饰寡核苷酸:纯化后的产物总产率:6.1%,纯度较高(保留时间:6.080min纯度为98.80%,如图13所示),及质谱分析与理论值吻合(图14所示),经过修饰的寡核酸的2.5μM水溶液(pH=7.0)最大吸收峰在561nm,最大发射在684nm,斯托克斯位移达到123nm,如图15所示。
4)DT-9-Ph修饰寡核苷酸:纯化后的产物总产率:2.5%,纯度较高(纯度为96.1%,如图16所示),及质谱分析与理论值吻合(图17所示),经过修饰的寡核酸的2.5μM水溶液(pH=7.0)最大吸收峰在640nm,最大发射在746nm,斯托克斯位移达到106nm,如图18所示。
以上详细描述了本发明的优选实施方式,但是,本发明并不限于上述实施方式中的具体细节,在本发明的技术构思范围内,可以对本发明的技术方案进行多种简单变型,这些简单变型均属于本发明的保护范围。
另外需要说明的是,在上述具体实施方式中所描述的各个具体技术特征,在不矛盾的情况下,可以通过任何合适的方式进行组合,为了避免不必要的重复,本发明对各种可能的组合方式不再另行说明。
此外,本发明的各种不同的实施方式之间也可以进行任意组合,只要其不违背本发明的思想,其同样应当视为本发明所公开的内容。
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Claims (12)
1.一种有机小分子荧光染料,其具有如下式I表示的结构,或其药学上可接受的盐,或其对映异构体、非对映异构体、互变异构体或溶剂化物:
[式I]
其中,
R1选自氢、或者取代或未取代的C1-C6烷基、C2-C6烯基、C2-C6炔基或C2-C6烷酰基;
R2-R4各自独立选自氢、羟基、氰基、卤素、或者取代或未取代的氨基、C1-C6烷基、C1-C6烷氧基、C3-C6环烷基、C1-C6羧基或C2-C6烷酰基;
R5-R8各自独立选自氢、羟基、氰基、卤素、或者取代或未取代的氨基、C1-C6烷基、C1-C6烷氧基、C3-C6环烷基、C1-C6羧基或C2-C6烷酰基,或者R5-R6和R7-R8中的一者与其所连接的碳原子一起形成取代或未取代的苯环;并且
R9选自氢、或者取代或未取代的C1-C6烷基、C2-C6烯基、C2-C6炔基、C2-C6烷酰基、C1-C6烷基硅基或亚磷酰胺基。
2.根据权利要求1所述的有机小分子荧光染料,其中,
R1选自氢、或者取代或未取代的C1-C6烷基或C2-C6烷酰基;
R2-R4各自独立选自氢、羟基、氰基、卤素、或者取代或未取代的氨基、C1-C6烷基、C1-C6烷氧基或C3-C6环烷基;
R5-R8各自独立选自氢、羟基、氰基、卤素、或者取代或未取代的氨基、C1-C6烷基、C1-C6烷氧基或C3-C6环烷基,或者R5-R6和R7-R8中的一者与其所连接的碳原子一起形成取代或未取代的苯环;并且
R9选自氢、或者取代或未取代的C1-C6烷基、C2-C6烷酰基、C1-C6烷基硅基或亚磷酰胺基。
3.根据权利要求2所述的有机小分子荧光染料,其中,
R1选自氢、或者取代或未取代的甲基、乙基、丙基、异丙基、乙酰基、丙酰基或叔丁基酰基;
R2-R4各自独立选自氢、羟基、氰基、卤素、或者取代或未取代的氨基、甲基、乙基、丙基、异丙基、甲烷氧基、乙烷氧基、环丙基或环丁基;
R5-R8各自独立选自氢、羟基、氰基、卤素、或者取代或未取代的氨基、甲基、乙基、丙基、异丙基、甲烷氧基、乙烷氧基、环丙基或环丁基,或者R5-R6和R7-R8中的一者与其所连接的碳原子一起形成取代或未取代的苯环;并且
R9选自氢、或者取代或未取代的甲基、乙基、丙基、异丙基、乙酰基、丙酰基、叔丁基酰基、叔丁基硅基或亚磷酰胺基。
4.根据权利要求1-3中任一项所述的有机小分子荧光染料,其中,所述取代为被卤素、羟基、氨基、氰基、C1-C3烷基、C1-C3烷氧基或C1-C3卤代烷基取代。
6.根据权利要求1所述的有机小分子荧光染料,其具有90至200nm,优选地,具有105至170nm斯托克斯位移,更优选地,具有105至123nm斯托克斯位移。
11.根据权利要求1-7中任一项所述的有机小分子荧光染料在寡核苷酸修饰中的用途。
12.根据权利要求1-7中任一项所述的有机小分子荧光染料在核酸药物合成中的用途。
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