CN116396304B - 一种靶向荧光探针分子及其合成方法和应用 - Google Patents
一种靶向荧光探针分子及其合成方法和应用 Download PDFInfo
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Abstract
本发明公开了一种靶向荧光探针分子及其合成方法和应用。该分子可以选择性的识别半胱氨酸上的巯基,该分子本身带有荧光基团,可以借助荧光偏振实验方法灵敏的检测出探针分子与蛋白之间的结合。其用于药物筛选的优势在于:1)结合巯基的基团空间较小,可以结合到蛋白凹槽深处的半胱氨酸;2)探针分子和靶标蛋白的巯基是通过形成S‑C共键价而结合在一起,不怕还原剂的干扰,更加稳定;3)本发明探针筛选药物的方法荧光偏振实验,其检测的灵敏度是DTNB实验紫外分光光度法的上千倍;4)速度快、通量高,带有荧光偏振模块的酶标仪能在毫秒内完成对1个样品荧光偏振值的读取,这种快速的检测方法结合多孔板的实验设计,即可完成药物的高通量筛选。
Description
技术领域
本发明涉及靶向荧光探针分子技术领域,具体涉及一种靶向荧光探针分子及其合成方法和应用。
背景技术
靶向荧光探针,能够在术中实时点亮癌细胞,帮助医生判断肿瘤边界和发现转移灶,这一概念也被称为荧光引导手术。靶向荧光探针通常由三部分组成:识别基团(recognition element)、报告基团(fluorophore)和连接体(linker)。
靶向药物由于其精准性高、活性强、副作用低等原因已经成为新药研究的主要方向。随着人类基因组学的完成和各种现代生物技术的发展,越来越多的蛋白质功能和结构被挖掘。基于疾病靶标的高通量药物筛选是靶向药物发现的第一步。因此针对某种疾病设计一些通用型高、靶向性强、通量高的筛选方法,来针对靶向药物的发现具有主要意义。然而,疾病靶标因其种类繁多并没有一个统一的药物筛选模式。针对新型的疾病靶标,如何建立一个快速的药物筛选方法仍然面临挑战。
氨基酸不仅是靶标蛋白的组成部分,它们不同的化学性质还决定了酶的催化活性、蛋白质的半衰期和控制蛋白质功能的多种翻译后修饰。半胱氨酸是其中与蛋白结构和功能相关性较大的一种氨基酸,它除了合成蛋白时形成蛋白质必要的二硫键外,多种蛋白活性位点的半胱氨酸还通过其巯基的氧化态和还原态之间的转换或与附近的赖氨酸形成LYS-CYS氧化还原开关来参与细胞内和细胞间信号传导。
研究发现多种疾病中经常会发生关键位点半胱氨酸的突变(半胱氨酸的获得或丢失)或周围氨基酸的突变来改变半胱氨酸巯基的pKa,以改变巯基的反应活性,这让众多含有半胱氨酸(CYS)的位点成为药物靶向作用的活性位点。此外,CYS上的巯基处于还原态(-SH)时易与缺电子的亲电基团(如α,β-不饱合酯基)发生麦克尔加成反应而形成共价结合,这成为药物学家设计共价结合药物的一种思路。如KRASG12C是癌症中最常发生突变的基因,并编码肿瘤发生中的关键信号蛋白,在肺腺癌患者中的突变率达到了13%。KRAS蛋白通过在结合GDP的非活化态和结合GTP的活化态之间的转化来发挥分子开关的作用,活化的KRAS蛋白通过结合下游效应蛋白来调控下游信号通路,如MAPK通路。当12位甘氨酸(G)突变成半胱氨酸(C)后会抑制对GTP的水解,从而增加KRAS-GTP的水平,并导致KRAS调控的下游通路的异常激活,最终导致肿瘤细胞的增殖和迁移。KRAS因为和GDP/GTP之间的强亲和力和光滑的蛋白表面而被一直认为是一个不可成药(undruggable)的蛋白。但是药物学家基于突变后CYS的性质设计了多个能够共价结合在CYS巯基上的候选药物,如AMG510,此类药物共价结合在突变的CYS后可诱导KRAS结合GDP/GTP的位点的构象发生变化,使其对GDP的结合力大于GTP,从而使异常激活的KRAS回归到失活的状态。此外还有武田(Takeda)开发的口服酪氨酸激酶抑制剂(TKI)mobocertinib是一种专门针对EGFR外显子20插入突变设计的药物,它能够共价结合在EGFR胞内区酪氨酸激酶催化位点的CYS797上而持续抑制EGFR的酪氨酸激酶活性。目前已被FDA批准用于治疗携带EGFR外显子20插入突变的非小细胞肺癌患者。可见靶标蛋白活性位点的CYS不仅对蛋白功能发挥主要作用,而且其巯基还是设计共价结合药物的关键点。
在已报道的蛋白结构中,活性位点含有关键CYS的蛋白数量较多。通过提取PDB数据库所有人源蛋白活性口袋中的信息,发现含有CYS的蛋白多达600多个,这意味着设计一些针对CYS的药物筛选方法具有重要意义。
目前针对活性位点含有半胱氨酸的靶标蛋白的共价结合药物筛选方法主要有质谱法、DTNB实验法(即Ellman实验)等,其中质谱法通过检测药物结合前后靶标蛋白的分子量变化来检测药物是否共价结合在靶标蛋白上,此种方法精确度高,但是通量低,需要价格昂贵的仪器设备,不适合高通量药物的筛选。DTNB实验法是通过DTNB和靶标蛋白半胱氨酸上的巯基结合之后释放出在412nm处具有紫外吸收的TNB,来判断药物是否结合在半胱氨酸上的一种方法,此方法由于DTNB分子体积的大小及刚性,对于那些包埋在蛋白凹槽深处的半胱氨酸无法靠近,使得检测范围较小;还有反应有还原性试剂后会破坏巯基和DTNB之间形成的二硫键;此外检测方法用的紫外分光光度法,需要样品量较大,灵敏度较低。
综上所述,活性位点含有半胱氨酸的疾病靶标蛋白是一类可用于设计药物或筛选药物的重要药物靶标,对其设计一些特异的药物筛选方法可加快药物的研发速度。
发明内容
基于上述技术问题,本发明的目的是提供一种靶向荧光探针分子及其合成方法和应用。
本发明保护一种靶向荧光探针分子,所述靶向荧光探针分子为P1或P2,其结构式分别如下所示:
其中,P1中n1表示碳链中碳的数量,1≤n1≤20;P2中n2表示乙二醇PEG的数量,1≤n2≤15。
进一步的,所述靶向荧光探针分子P1或P2在激发波长492±30nm,吸收波长518±30nm处具有荧光吸收;所述靶向荧光探针分子P1或P2以共价结合方式结合在蛋白裸露的半胱氨酸上来标记蛋白。
优化的,所述共价结合方式为靶向荧光探针分子P1或P2中的末端双键通过迈克尔加成反应结合在半胱氨酸的巯基上。
本发明还保护上述靶向荧光探针分子P1或P2的合成方法,具体包括如下步骤:
所述靶向荧光探针分子P1的合成方法:
取0.2g,1.12mmol的化合物1与0.2g,1.12mmol的化合物2加入THF和H2O中,加入0.13g,1.12mmol的锌粉,室温下搅拌过夜,TLC检测反应完全;加入20mL水,EA萃取,旋干得化合物3;将化合物3粗产品溶于20mL DCM中,加入0.2g,1.12mmol的对甲苯磺酸(PTSA),室温下搅拌1h,TLC检测反应完成,加入20mL饱和碳酸氢钠溶液淬灭反应,DCM萃取,EA萃取,旋干柱层析得到0.23g,0.97mmol的化合物4;化合物4溶于20mL THF中,冰浴下滴加2.0M,1.5mL的硼烷-二甲硫醚溶液,搅拌2h后,缓慢加入0.5mL水,再加入0.45g,2.95mmol的过硼酸钠,搅拌2h后,硅藻土抽滤,柱层析得到0.1g,0.39mmol的化合物5;化合物6和咪唑按1:1摩尔比例溶解于2mL二氯甲烷中,然后在冰浴中加入等比例TBSCL,搅拌反应10min,反应物用水洗,粗提物用硅胶纯化得到化合物7(得率85%);化合物5、化合物7、EDCl和DMAP按1:1.2:2:0.5反应比溶解于二氯甲烷中,室温搅拌反应20h,反应液加盐酸水溶液洗涤,用氯仿萃取,氯仿部位浓缩后用硅胶柱分离得化合物8(得率95%);化合物8溶解于THF,加入1.2:2.0比例的TBAF和乙酸,室温搅拌过夜,然后用硅胶柱进行纯化等目标化合物P1,收率为70%;其中,所述化合物1-8的化学式结构式分别为:
所述靶向荧光探针分子P2的合成方法:
取0.9g的化合物9与0.9g的三乙胺溶于10mL DCM中,内温0度滴加0.36g的丙烯酰氯,用100ml二氯甲烷稀释,5%柠檬酸洗,饱和食盐水洗,干燥旋干过柱得0.6g淡黄色油状化合物10(得率67%);0.6g的化合物10溶于DCM中,通氯化氢气体,反应完后旋干得0.6g的化合物11(得率100%),0.6g的化合物11溶于DMF中,加入三乙胺,20min后加入0.76g的化合物12(FAM SE),反应完后旋干DMF,拌样过柱得0.5g的目标化合物P2(得率83%);
其中,所述化合物9-12的化学结构式分别为:
本发明还保护上述靶向荧光探针分子的应用,所述靶向荧光探针分子P1或P2用于靶向药物筛选的试剂盒,所述靶向药物筛选的试剂盒由靶标蛋白、靶向荧光探针分子P1或P2、测试缓冲液及阳性药物分子组成。
进一步的,所述测试缓冲液由蛋白缓冲液加1mM的还原剂配置而得,所述还原剂包含但不限于DTT或β巯基乙醇;所述靶向荧光探针分子P1或P2和阳性药物分子以1mM/L的浓度溶解于DMSO中。
进一步的,所述靶标蛋白为活性位点包含半胱氨酸的疾病靶标。
优化的,所述疾病靶标为癌症靶标KRAS(G12C)、结核杆菌药物靶标Ldtmt2、新冠病毒药物靶标Mpro、抗癌药物靶标EGFR外显子20插入突变体、炎症药物靶标Ubc13、抗癌药物靶标Pin1、白血病药物靶标Menin-MLL、抗癌药物靶标FGFR4、抗癌药物靶标SMYD3、自身免疫药物靶标BTK或抗癌药物靶标DCN1-UBC12。
本发明还保护上述试剂盒的药物筛选方法,具体包括如下步骤:
阳性对照组:测试缓冲液+等体积靶标蛋白+等比例靶向荧光探针分子P1或P2;阴性对照组:测试缓冲液+等比例靶向荧光探针分子P1或P2;阳性药物组:测试缓冲液+等体积靶标蛋白+等比例阳性药物分子+等比例靶向荧光探针分子P1或P2;药物筛选组:测试缓冲液+等体积靶标蛋白+等比例待筛选药物+等比例靶向荧光探针分子P1或P2;加入待筛选药物后先孵育30min,然后再加入靶向荧光探针分子P1或P2孵育30min,最后在激发波长492nm,吸收波长518nm处检测荧光偏振值;药物的抑制率=[mP(药物筛选组)-mP(阴性对照组)]÷[mP(阳性对照组)-mP(阴性对照组)]×100%。
相比于现有的技术,本发明具有如下有益效果:
本发明的靶向荧光探针分子可以选择性的识别半胱氨酸上的巯基,此外由于该探针分子本身带有荧光基团,可以借助荧光偏振实验方法灵敏的检测出探针分子与蛋白之间的结合。本发明的靶向荧光探针分子用于药物筛选的优势在于:1)结合巯基的基团空间较小,可以结合到蛋白凹槽深处的半胱氨酸,其中,DTNB中分子含有苯环,其基团的大小为而P1或P2中用于结合半胱氨酸的基团大小只有/>这对于空间较小的活性位点,P1或P2更利于结合;2)探针分子和靶标蛋白的巯基是通过形成S-C共键价而结合在一起,不怕还原剂的干扰,更加稳定;3)DTNB实验筛选药物的方法是紫外分光光度法,而本发明探针筛选药物的方法是荧光偏振实验,其检测的灵敏度是紫外分光光度法的上千倍;4)速度快、通量高,带有荧光偏振模块的酶标仪能在毫秒内完成对1个样品荧光偏振值的读取,这种快速的检测方法结合多孔板的实验设计,即可完成药物的高通量筛选。
附图说明
图1为本发明靶向荧光探针分子P1的结构式;
图2为本发明靶向荧光探针分子P2的结构式;
图3为本发明靶向荧光探针分子P1的合成方法;
图4为本发明靶向荧光探针分子P1的合成方法(n1=8);
图5为本发明靶向荧光探针分子P1的H谱图;
图6为本发明靶向荧光探针分子P2的合成方法;
图7为本发明靶向荧光探针分子P2的合成方法(n2=2);
图8为本发明靶向荧光探针分子P2的H谱图;
图9为本发明靶向荧光探针分子P1和P2与LdtMt2蛋白的共价结合质谱结果;
图10为LdtMt2蛋白与本发明靶向荧光探针分子P1和P2结合力测试结果;
图11为检验阳性药物美罗培南与LdtMt2蛋白的结合情况。
具体实施方式
下面将结合实施例对本发明的技术方案进行清楚、完整地描述,显然,所描述的实施例仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例1
一种靶向荧光探针分子,所述靶向荧光探针分子为P1或P2,其结构式分别如附图1和2所示,其中,P1中n1表示碳链中碳的数量,1≤n1≤20;P2中n2表示乙二醇PEG的数量,1≤n1≤15;所述靶向荧光探针分子P1或P2在激发波长492±30nm,吸收波长518±30nm处具有荧光吸收;所述靶向荧光探针分子P1或P2以共价结合方式结合在蛋白裸露的半胱氨酸上来标记蛋白,其中,所述共价结合方式为靶向荧光探针分子P1或P2中的末端双键通过迈克尔加成反应结合在半胱氨酸的巯基上。该探针分子共价结合在药物靶标活性位点中的半胱氨酸后引起荧光偏振值的变化,然后根据荧光偏振值变化的大小来测试被筛选药物的结合率。
实施例2
靶向荧光探针分子P1或P2的合成方法。
(1)靶向荧光探针分子P1的合成方法,具体包括如下步骤:
取0.2g,1.12mmol的化合物1与0.2g,1.12mmol的化合物2加入THF和H2O中,加入0.13g,1.12mmol的锌粉,室温下搅拌过夜,TLC检测反应完全;加入20mL水,EA萃取,旋干得化合物3;将化合物3粗产品溶于20mL DCM中,加入0.2g,1.12mmol的对甲苯磺酸(PTSA),室温下搅拌1h,TLC检测反应完成,加入20mL饱和碳酸氢钠溶液淬灭反应,DCM萃取,EA萃取,旋干柱层析得到0.23g,0.97mmol的化合物4;化合物4溶于20mL THF中,冰浴下滴加2.0M,1.5mL的硼烷-二甲硫醚溶液,搅拌2h后,缓慢加入0.5mL水,再加入0.45g,2.95mmol的过硼酸钠,搅拌2h后,硅藻土抽滤,柱层析得到0.1g,0.39mmol的化合物5;化合物6和咪唑按1:1摩尔比例溶解于2mL二氯甲烷中,然后在冰浴中加入等比例TBSCL,搅拌反应10min,反应物用水洗,粗提物用硅胶纯化得到化合物7(得率85%);化合物5、化合物7、EDCl和DMAP按1:1.2:2:0.5反应比溶解于二氯甲烷中,室温搅拌反应20h,反应液加盐酸水溶液洗涤,用氯仿萃取,氯仿部位浓缩后用硅胶柱分离得化合物8(得率95%);化合物8溶解于THF,加入1.2:2.0比例的TBAF和乙酸,室温搅拌过夜,然后用硅胶柱进行纯化等目标化合物P1,收率为70%(详见附图3)。
取n1=8,采用上述方法,合成靶向荧光探针分子P1的过程(详见附图4)。所得产品的鉴定数据结果为:1H-NMR(600MHz,CDCl3):δ(ppm)8.17(m,1H),8.04(d,1H),7.05(d,1H),7.07(d,2H),6.49(d,2H),6.43(m,2H),6.28(s,1H),5.56(s,1H),4.60(m,1H),4.33(m,2H),2.31-2.06(m,2H),1.78(m,2H),1.43(m,2H),1.30-1.25(m,8H);13C-NMR(125MHz,CDCl3)δ(ppm):170.0,169.5,165.9,156.0,155.7,155.7,152.4,152.4,135.1,134.7,130.3,129.3,129.3,128.6,128.0,127.1,109.5,109.5,107.7,107.7,105.7,105.7,84.8,77.7,64.8,34.4,33.3,31.7,29.3,29.3,29.4,29.4,29.6,29.7,25.8.(详见附图5)
(2)靶向荧光探针分子P2的合成方法,具体包括如下步骤:
取0.9g的化合物9与0.9g的三乙胺溶于10mL DCM中,内温0度滴加0.36g的丙烯酰氯,用100ml二氯甲烷稀释,5%柠檬酸洗,饱和食盐水洗,干燥旋干过柱得0.6g淡黄色油状化合物10(得率67%);0.6g的化合物10溶于DCM中,通氯化氢气体,反应完后旋干得0.6g的化合物11(得率100%),0.6g的化合物11溶于DMF中,加入三乙胺,20min后加入0.76g的化合物12(FAM SE),反应完后旋干DMF,拌样过柱得0.5g的目标化合物P2(得率83%)(详见附图6)。
取n2=2,采用上述方法,合成靶向荧光探针分子P2的过程(详见附图7)。所得产品的鉴定数据结果为:MS[M+1]+:605.31;1H-NMR(600MHz,CDCl3):δ(ppm)8.4(m,1H),8.2(d,1H),7.3(d,1H),6.7-6.6(m,7H),6.2(m,2H),5.6(m,1H),5.5(m,1H),3.7-3.2(m,16H).13C-NMR(125MHz,CDCl3)δ(ppm):169.5,167.5,166.8,155.7,155.1,152.4,132.3,131.6,131.1,129.3,128.2,127.9,127.3,126.8,109.5,107.7,105.7,84.8,70.4,70.2,70.1,39.9.(详见附图8)
实施例3
靶向荧光探针分子P1或P2与LdtMt2蛋白的共价结合
LdtMt2为D,L-转肽酶,是细菌细胞壁中肽聚糖层合成的控制酶,抑制其活性可以抑制细菌的生长,因此经常被用于抗菌药物筛选的作用靶标,LdtMt2在活性位点含有一个半胱氨酸,为催化必须的半胱氨酸,根据结合原理,设计的探针分子P1或P2可以通过麦克加成共价结合在这个活性位点的半胱氨酸上。
(1)实验方法:纯度大于95%的LdtMt2(34-408)蛋白与探针分子1和2分别孵育30min,蛋白与小分子探针的浓度比为:20uM:1mM,然后用ESI-Q-TOF进行测试其分子量。
(2)实验结果:分子量结果如表1和附图7所示。孵育后增加的分子量正好等于探针分子P1或P2的分子量,证明探针分子P1或P2通过共价作用结合在LdtMt2蛋白的结合位点。同时通过荧光偏振测试了探针分子P1和P2与LdtMt2蛋白的结合力,Kd值分别为12.84nM和9.89nM质谱结果和结合力实验结果如附图8所示。
表1探针分子与LdtMt2孵育前后的分子量差异
实施例4
靶向荧光探针分子的应用
以结核杆菌药物靶标LdtMt2为例,研究靶向荧光探针分子P1和P2在靶向药物筛选试剂盒的应用。
结核杆菌药物靶标LdtMt2为D,L-转肽酶,是细菌细胞壁中肽聚糖层合成的控制酶,抑制其活性可以抑制细菌的生长,因此经常被用于抗菌药物筛选的作用靶标,LdtMt2在活性位点含有一个半胱氨酸(CYS408),为LdtMt2催化必须的半胱氨酸,发现抗生素药物美罗培南通过β内酰胺环开环并共价结合在CYS408的巯基上,是一种共价结合模式的药物。根据实施例1中的靶向荧光探针分子P1或P2可以通过麦克加成共价结合在这个活性位点的半胱氨酸上。因此设计以下实验来验证靶向荧光探针分子P1或P2在LdtMt2蛋白抑制剂筛选中的效果,检验美罗培南与LdtMt2蛋白的结合情况。
(1)实验方法:按以下表格2所示的量比进行反应准备。筛选分为阳性对照组(测试缓冲液+等体积靶标蛋白+等比例靶向荧光探针分子P1或P2)、阴性对照组(测试缓冲液+等比例靶向荧光探针分子P1或P2),阳性药物组(测试缓冲液+等体积靶标蛋白+等比例阳性药物美罗培南+等比例靶向荧光探针分子P1或P2)三个组。其中蛋白缓冲液为:0.15M NaCl,25mM Tris-HCl pH 8.0,加入待筛选药物后先孵育30min,测试缓冲液的组成为:0.15MNaCl,25mM Tris-HCl pH 8.0,1mM DTT;反应顺序为:在96孔板中先加入50微升1uM/L的蛋白,再加入50微升浓度为10uM的靶向荧光探针分子P1或P2,孵育30min,然后加入不同浓度的阳性药物美罗培南,药物先配置成10mM的DMSO溶液,然后用测试缓冲液稀释成不同浓度。最后在激发波长492nm,吸收波长518nm处检测荧光偏振值。药物的抑制率=[mP(药物筛选组)-mP(阴性对照组)]÷[mP(阳性对照组)-mP(阴性对照组)]×100%。
表2药物筛选反应量比
2)实验结果:药物美罗培南已经被证实是作用于LdtMt2的阳性药物,本实验中通过上述实验方法重新测试了美罗培南针对LdtMt2蛋白的抑制率,结果如附图9所示。证实美罗培南针对LdtMt2蛋白的抑制率IC50为896.7nM,结果和文献报道相符。由此可见本发明中的探针分子结合荧光偏振实验可以有效的用于LdtMt2蛋白抑制剂的筛选实验中。
尽管已经示出和描述了本发明的实施例,对于本领域的普通技术人员而言,可以理解在不脱离本发明的原理和精神的情况下可以对这些实施例进行多种变化、修改、替换和变型,本发明的范围由所附权利要求及其等同物限定。
Claims (9)
1.一种靶向荧光探针分子,其特征在于,所述靶向荧光探针分子为P1或P2,其结构式分别如下所示:
其中,P1中n1表示碳链中碳的数量,1≤n1≤20;P2中n2表示乙二醇PEG的数量,1≤n2≤15。
2.根据权利要求1所述的一种靶向荧光探针分子,其特征在于,所述靶向荧光探针分子P1或P2在激发波长492±30nm,吸收波长518±30nm处具有荧光吸收;所述靶向荧光探针分子P1或P2以共价结合方式结合在蛋白裸露的半胱氨酸上来标记蛋白。
3.根据权利要求2所述的一种靶向荧光探针分子,其特征在于,所述共价结合方式为靶向荧光探针分子P1或P2中的末端双键通过迈克尔加成反应结合在半胱氨酸的巯基上。
4.根据权利要求1或2所述的一种靶向荧光探针分子的合成方法,其特征在于,所述靶向荧光探针分子P1或P2的合成方法,具体包括如下步骤:
所述靶向荧光探针分子P1的合成方法:
取0.2g,1.12mmol的化合物1与0.2g,1.12mmol的化合物2加入四氢呋喃和H2O中,加入0.13g,1.12mmol的锌粉,室温下搅拌过夜,TLC检测反应完全;加入20mL水,乙酸乙酯萃取,旋干得化合物3;将化合物3粗产品溶于20mL二氯甲烷中,加入0.2g,1.12mmol的对甲苯磺酸,室温下搅拌1h,TLC检测反应完成,加入20mL饱和碳酸氢钠溶液淬灭反应,二氯甲烷萃取,乙酸乙酯萃取,旋干柱层析得到0.23g,0.97mmol的化合物4;化合物4溶于20mL四氢呋喃中,冰浴下滴加2.0M,1.5mL的硼烷-二甲硫醚溶液,搅拌2h后,缓慢加入0.5mL水,再加入0.45g,2.95mmol的过硼酸钠,搅拌2h后,硅藻土抽滤,柱层析得到0.1g,0.39mmol的化合物5;化合物6和咪唑按1:1摩尔比例溶解于2mL二氯甲烷中,然后在冰浴中加入等比例叔丁基二甲基氯硅烷,搅拌反应10min,反应物用水洗,粗提物用硅胶纯化得到化合物7;化合物5、化合物7、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐和4-二甲氨基吡啶按1:1.2:2:0.5反应比溶解于二氯甲烷中,室温搅拌反应20h,反应液加盐酸水溶液洗涤,用氯仿萃取,氯仿部位浓缩后用硅胶柱分离得化合物8;化合物8溶解于四氢呋喃,加入1.2:2.0比例的四丁基氟化铵和乙酸,室温搅拌过夜,然后用硅胶柱进行纯化等目标化合物P1,收率为70%;其中,所述化合物1-8的化学式结构式分别为:
所述靶向荧光探针分子P2的合成方法:
取0.9g的化合物9与0.9g的三乙胺溶于10mL二氯甲烷中,内温0度滴加0.36g的丙烯酰氯,用100ml二氯甲烷稀释,5%柠檬酸洗,饱和食盐水洗,干燥旋干过柱得0.6g淡黄色油状化合物10;0.6g的化合物10溶于二氯甲烷中,通氯化氢气体,反应完后旋干得0.6g的化合物11,0.6g的化合物11溶于二甲基甲酰胺中,加入三乙胺,20min后加入0.76g的化合物12,反应完后旋干二甲基甲酰胺,拌样过柱得0.5g的目标化合物P2;
其中,所述化合物9-12的化学结构式分别为:
5.根据权利要求1或2所述的一种靶向荧光探针分子的应用,其特征在于,所述靶向荧光探针分子P1或P2用于靶向药物筛选的试剂盒,所述靶向药物筛选的试剂盒由靶标蛋白、靶向荧光探针分子P1或P2、测试缓冲液及阳性药物分子组成。
6.根据权利要求5所述的一种靶向荧光探针分子的应用,其特征在于,所述测试缓冲液由蛋白缓冲液加1mM的还原剂配置而得,所述还原剂包含但不限于二硫苏糖醇或β巯基乙醇;所述靶向荧光探针分子P1或P2和阳性药物分子以1mM/L的浓度溶解于二甲基亚砜中。
7.根据权利要求5所述的一种靶向荧光探针分子的应用,其特征在于,所述靶标蛋白为活性位点包含半胱氨酸的疾病靶标。
8.根据权利要求7所述的一种靶向荧光探针分子的应用,其特征在于,所述疾病靶标为癌症靶标KRAS G12C、结核杆菌药物靶标Ldtmt2、新冠病毒药物靶标Mpro、抗癌药物靶标EGFR外显子20插入突变体、炎症药物靶标Ubc13、抗癌药物靶标Pin1、白血病药物靶标Menin-MLL、抗癌药物靶标FGFR4、抗癌药物靶标SMYD3、自身免疫药物靶标BTK或抗癌药物靶标DCN1-UBC12。
9.根据权利要求5所述的一种靶向荧光探针分子的应用,其特征在于,所述试剂盒的药物筛选方法,具体包括如下步骤:
阳性对照组:测试缓冲液+等体积靶标蛋白+等比例靶向荧光探针分子P1或P2;阴性对照组:测试缓冲液+等比例靶向荧光探针分子P1或P2;阳性药物组:测试缓冲液+等体积靶标蛋白+等比例阳性药物分子+等比例靶向荧光探针分子P1或P2;药物筛选组:测试缓冲液+等体积靶标蛋白+等比例待筛选药物+等比例靶向荧光探针分子P1或P2;加入待筛选药物后先孵育30min,然后再加入靶向荧光探针分子P1或P2孵育30min,最后在激发波长492nm,吸收波长518nm处检测荧光偏振值;药物的抑制率=[mP(药物筛选组)-mP(阴性对照组)]÷[mP(阳性对照组)-mP(阴性对照组)]×100%。
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Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
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JP2013025149A (ja) * | 2011-07-22 | 2013-02-04 | Shin Etsu Chem Co Ltd | ポジ型レジスト材料及びパターン形成方法 |
KR20150090673A (ko) * | 2014-01-29 | 2015-08-06 | 한국과학기술원 | 플루오레세인을 기반으로 한 시스테인/호모시스테인에 선택적으로 반응하는 화합물 및 형광 프로브 |
CN106995451A (zh) * | 2017-04-26 | 2017-08-01 | 许昌学院 | 一种反应型半胱氨酸探针及其制备方法 |
CN107253961A (zh) * | 2017-06-29 | 2017-10-17 | 湖南科技大学 | 一种可比率检测半胱氨酸的水溶性荧光传感器的制备及应用 |
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Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2013025149A (ja) * | 2011-07-22 | 2013-02-04 | Shin Etsu Chem Co Ltd | ポジ型レジスト材料及びパターン形成方法 |
KR20150090673A (ko) * | 2014-01-29 | 2015-08-06 | 한국과학기술원 | 플루오레세인을 기반으로 한 시스테인/호모시스테인에 선택적으로 반응하는 화합물 및 형광 프로브 |
CN106995451A (zh) * | 2017-04-26 | 2017-08-01 | 许昌学院 | 一种反应型半胱氨酸探针及其制备方法 |
CN107253961A (zh) * | 2017-06-29 | 2017-10-17 | 湖南科技大学 | 一种可比率检测半胱氨酸的水溶性荧光传感器的制备及应用 |
Non-Patent Citations (1)
Title |
---|
Chen, Zhixing等.Second-Generation Covalent TMP-Tag for Live Cell Imaging.Journal of the American Chemical Society.2012,第134卷(第33期),13692-13699. * |
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