CN113788830B - 荧光探针化合物及其制备方法和应用 - Google Patents
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Abstract
Description
技术领域
本发明涉及生物分析领域,具体地,涉及一种荧光探针化合物及其制备方法和应用。
背景技术
雌激素相关受体α(Estrogen Related Receptorα,ERRα),属于核受体家族成员,其内源性的配体仍未得到解析,被列为孤儿核受体,由于其DNA序列与经典的雌激素核受体ER具有很高的相似性,因而被命名为雌激素相关受体。虽然天然雌激素并不能与ERRα直接结合,但是ERRα在信号传导过程中,可以与经典的雌激素受体ER竞争性结合雌激素响应原件以及共激活因子,从而影响雌激素相关的生理功能。研究表明,ERRα在乳腺癌细胞的雌激素信号通路中发挥着重要作用,其表达与乳腺癌患者的不良预后具有相关性,因此,ERRα被视为乳腺癌潜在的生物标志物以及药物治疗靶标。ERRα在卵巢癌中的表达也比正常组织要高,并且ERRα的表达与卵巢癌的存活时间具有相关性,表明ERRα可能也是治疗卵巢癌的新靶点。此外,ERRα在一些高能量需求的组织具有高表达,在线粒体能量代谢过程发挥重要调控作用。
与经典的核受体ER不同,ERRα在无配体结合的情况下就能维持自发活性,但已有研究表明一些外源人工合成的小分子均能与ERRα结合,并且激活或者抑制该受体的转录活性,但是这些合成配体与ERRα的结合大多采用基于共激活因子或共抑制因子招募的荧光偏振/荧光共振能量转移的检测方法,并且已有商业化的检测试剂盒。但是该试剂盒比较昂贵,操作比较繁琐,只能筛选出具有共激活因子或共抑制因子招募增强或者抑制效应的配体,对于有结合能力但是对共激活因子招募无影响的配体,该方法不能检出,而这些配体也很有可能与其它激活或者抑制剂竞争结合受体从而影响受体的功能。
因此,建立简便易得且经济可行的方法,用于高通量筛选能与ERRα结合的药物分子或者未知环境污染物具有重要的意义。
发明内容
本发明的目的是为了克服现有技术存在的筛选与ERRα结合的配体操作繁琐、准确性低的问题,提供一种荧光探针化合物及其制备方法和应用,该荧光探针化合物可有效检测待测样品与ERRα的结合能力,且准确性高、操作简单,可用于高通量快速筛查。
为了实现上述目的,本发明第一方面提供一类荧光探针化合物,具有式(I)所示的结构:
其中,R为具有与氨基反应活性的有机荧光分子基团。
优选地,所述荧光探针化合物中R选自如下含有异硫氰酸酯或琥珀酰亚胺酯的有机荧光分子所形成的有机荧光分子基团中的一种:
优选地,所述荧光探针化合物为:
本发明第二方面提供一种如式(I)所示的荧光探针化合物的制备方法,包括以下步骤:
在反应溶剂I和催化剂I存在的条件下,将式(II)所示的化合物和具有与氨基反应活性的有机荧光分子进行反应I,
优选地,式(II)所示的化合物的制备方法,包括以下步骤:
(1)在反应溶剂II和催化剂II存在的条件下,将式(III)所示的化合物与2,4-噻唑烷二酮进行反应II,得到式(IV)所示的化合物;
(2)在反应溶剂III和催化剂III存在的条件下,将式(IV)所示的化合物与(5-羟基戊基)氨基甲酸叔丁酯进行反应III,得到式(V)所示的化合物;
(3)将式(V)所示的化合物与三氟乙酸和/或二氯甲烷进行反应IV,得到式(II)所示的化合物;
优选地,所述反应溶剂II为乙腈,所述催化剂II为乙酸钠和/或乙酸钾,所述反应溶剂III为四氢呋喃,所述催化剂III为三苯基磷和偶氮二甲酸二异丙酯;
步骤(1)中,相对于100mL所述反应溶剂II,式(III)所示的化合物的用量为5-8g、2,4-噻唑烷二酮的用量为2-3g、所述催化剂II的用量为12-18g;
步骤(2)中,相对于100mL所述反应溶剂III,式(IV)所示的化合物的用量为4-6g、(5-羟基戊基)氨基甲酸叔丁酯的用量为2-3g、所述催化剂III的用量为7-10g;
步骤(3)中,相对于10mL所述三氟乙酸和/或二氯甲烷,式(V)所示的化合物的用量为0.5-2.2g。
优选地,步骤(1)中所述反应II的条件至少满足:温度为80-120℃、时间为20-30h;
步骤(2)中所述反应III的条件至少满足:温度为0-40℃、时间为2-4h;
步骤(3)中所述反应IV的条件至少满足:温度为0-40℃、时间为2-4h。
优选地,所述反应溶剂I为N,N-二甲基甲酰胺,所述催化剂I为N,N-二异丙基乙胺,所述具有与氨基反应活性的有机荧光分子选自:
相对于100mL所述反应溶剂I,式(II)所示的化合物的用量为2-3g,具有与氨基反应活性的有机荧光分子为2-3g,所述催化剂I的用量为5-8g;
所述反应I的条件至少满足:温度为40-60℃、时间为8-15h。
本发明第三方面提供上述的荧光探针化合物、上述的的制备方法制得的荧光探针化合物在检测待测样品与ERRα蛋白结合能力中的应用;
优选地,所述ERRα蛋白为如下(a1)或(a2)或(a3)所述的蛋白质:
(a1)具有SEQ ID NO.1所示氨基酸序列的蛋白质;
(a2)在SEQ ID NO.1所示氨基酸序列的氨基末端和/或羧基末端连接有标签的氨基酸序列所示的蛋白质;
(a3)SEQ ID NO.1所示的氨基酸序列经过取代、缺失或添加一个或几个氨基酸残基且仍具有相同功能的氨基酸序列所示的蛋白质。
本发明第四方面一种检测待测样品与ERRα蛋白结合能力的方法,包括以下步骤:将所述待测样品、荧光探针化合物和所述ERRα蛋白混合孵育后,进行荧光偏振检测,所述荧光探针化合物为上述的荧光探针化合物、上述的制备方法制得的荧光探针化合物;
优选地,所述荧光探针化合物与所述ERRα蛋白的用量比为1:1.5-10。
通过上述技术方案,本发明的有益效果为:本发明提供的荧光探针化合物与ERRα蛋白的结合位点明确,且与ERRα蛋白的结合能力强,通过表征待测样品相对于荧光探针化合物,与ERRα蛋白的竞争结合情况,能够准确、有效地检测出待测样品与ERRα蛋白的结合能力;本发明提供的检测待测样品与ERRα蛋白结合能力的方法中,荧光探针化合物的表征采用荧光偏振检测,属于均相检测,无需繁琐的物理分离过程等,检测方便快速,稳定性好、对人体基本无危害性,并且可用于高通量快速筛查。本发明提供的荧光探针化合物及检测待测样品与ERRα蛋白结合能力的方法,对于高通量筛选能够与ERRα蛋白结合的药物分子或者未知环境污染物具有重要的意义。
有关本发明的其它技术特征和技术效果,将在下文的具体实施方式中进一步说明。
附图说明
图1是本发明中提供的5FB-FITC的制备流程图;
图2是本发明的实施例1中制得的5FB-FITC的HPLC检测谱图;
图3是本发明的实施例1中制得的5FB-FITC的MS检测谱图;
图4是本发明的实施例1中制得的5FB-FITC的1H NMR谱图;
图5是本发明中提供的5FB-FITC荧光偏振值与ERRα蛋白浓度的拟合曲线图;
图6是本发明中提供的5FB-FITC相对荧光偏振值与待测样品浓度的拟合曲线图。
具体实施方式
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。
第一方面,本发明提供了一类荧光探针化合物,具有式(I)所示的结构:
其中,R为具有与氨基反应活性的有机荧光分子基团。
根据本发明,式(I)所示的通式部分形成为能够与ERRα蛋白具有特异性相互作用的配体;式(I)中的R基团用于提供荧光检测信号,R基团为具有与氨基反应活性的有机荧光分子形成的对应基团,其不仅具有与氨基反应活性,还能够产生荧光检测信号。
本发明中,利用该类荧光探针化合物的配体部分与ERRα蛋白进行特异性结合,在待测样品加入后,如待测样品含有能够与ERRα蛋白结合的化合物,则可将荧光探针化合物从ERRα蛋白的结合位点竞争下来,成为游离态,使荧光探针化合物的自由旋转加快,进而旋转弛豫时间减少,从而导致荧光探针化合物的荧光偏振值逐渐降低,反映出待测样品与ERRα蛋白结合能力;如待测样品不含有能够与ERRα蛋白结合的化合物,则荧光探针化合物的荧光偏振值不变。
优选情况下,所述荧光探针化合物中R选自如下含有异硫氰酸酯或琥珀酰亚胺酯的有机荧光分子所形成的有机荧光分子基团中的一种:
在本发明的一种优选的实施方式中,R基团由5(6)-羟基荧光素(FITC)形成,所述荧光探针化合物为:
本发明第二方面提供一种如式(I)所示的荧光探针化合物的制备方法,包括以下步骤:
在反应溶剂I和催化剂I存在的条件下,将式(II)所示的化合物和具有与氨基反应活性的有机荧光分子进行反应I,
根据本发明,式(II)所示的化合物能够提供与ERRα蛋白具有特异性相互作用的配体,可以根据应用需求选择不同的具有与氨基反应活性的有机荧光分子,以与式(II)所示的化合物形成相应的荧光探针化合物。
优选情况下,所述具有与氨基反应活性的有机荧光分子选自:
在本发明中,所述反应溶剂I为N,N-二甲基甲酰胺,所述催化剂I为N,N-二异丙基乙胺。
在本发明中,相对于100mL所述反应溶剂I,式(II)所示的化合物的用量为2-3g,具有与氨基反应活性的有机荧光分子为2-3g,所述催化剂I的用量为5-8g;所述反应I的条件至少满足:温度为40-60℃,例如可以为40℃、45℃、50℃、55℃、60℃以及这些点值中的任意两个所构成的范围中的任意值;时间为8-15h,例如可以为8h、10h、12h、14h、15h以及这些点值中的任意两个所构成的范围中的任意值。发明人发现,在该优选的具体实施方式下,本发明的方案具有进一步提高反应I的反应速率、提高式(I)所示的化合物的收率的优势。
本发明中,式(II)所示的化合物可以是商购得到,也可以自行合成获得。
示例性地,式(II)所示的化合物的制备方法,包括以下步骤:
(1)在反应溶剂II和催化剂II存在的条件下,将式(III)所示的化合物与2,4-噻唑烷二酮进行反应II,得到式(IV)所示的化合物;
(2)在反应溶剂III和催化剂III存在的条件下,将式(IV)所示的化合物与(5-羟基戊基)氨基甲酸叔丁酯进行反应III,得到式(V)所示的化合物;
(3)将式(V)所示的化合物与三氟乙酸和/或二氯甲烷进行反应IV,得到式(II)所示的化合物;
根据本发明,式(III)所示的化合物可以采用现有技术中公知的方法合成,也可以是商购得到。示例性地,式(III)所示的化合物的制备过程可以包括:以K2CO3和/或Na2CO3作为催化剂,以二甲基甲酰胺作为反应溶剂,将4-氟-3-(三氟甲基)苯腈与4-羟基-3-甲氧基苯甲醛进行加热反应后,用冰水稀释,再用乙酸乙酯萃取获得萃取液,干燥浓缩后通过硅胶柱柱层析制得。其中,相对于100mL二甲基甲酰胺,4-氟-3-(三氟甲基)苯腈的用量为8-12g、4-羟基-3-甲氧基苯甲醛的用量为7-9g、所述催化剂II的用量为18-25g;加热反应的温度为60-70℃、时间为8-15h。
根据本发明,所述反应II、反应III在反应结束后,也需要分别进行反应溶剂去除、硅胶柱柱层析等纯化过程,以获得纯度较高的相应化合物。具体地,反应溶剂去除可以采用减压蒸馏、旋转蒸发等常规的方式,硅胶柱柱层析的洗脱液根据需要选用不同比例的乙酸/正己烷或者乙酸乙酯/正己烷。
根据本发明,步骤(1)中,优选地,所述反应溶剂II为乙腈,所述催化剂II为乙酸钠和/或乙酸钾;相对于100mL所述反应溶剂II,式(III)所示的化合物的用量为5-8g、2,4-噻唑烷二酮的用量为2-3g、所述催化剂II的用量为12-18g;所述反应II的条件至少满足:温度为80-120℃,例如可以为80℃、85℃、90℃、95℃、100℃、105℃、110℃、115℃、120℃以及这些点值中的任意两个所构成的范围中的任意值,优选为95-105℃;时间为20-30h,例如可以为20h、22h、24h、26h、28h、30h以及这些点值中的任意两个所构成的范围中的任意值,优选为22-26h。发明人发现,在该优选的具体实施方式下,本发明的方案具有进一步提高式(IV)所示的化合物的收率的优势。
根据本发明,步骤(2)中,优选地,所述反应溶剂III为四氢呋喃,所述催化剂III为三苯基磷和偶氮二甲酸二异丙酯,其中,三苯基磷与偶氮二甲酸二异丙酯的质量比为1-1.5:1;相对于100mL所述反应溶剂III,式(IV)所示的化合物的用量为4-6g、(5-羟基戊基)氨基甲酸叔丁酯的用量为2-3g、所述催化剂III的用量为7-10g;所述反应III的条件至少满足:温度为0-40℃,例如可以为0℃、5℃、10℃、15℃、20℃、25℃、30℃、35℃、40℃以及这些点值中的任意两个所构成的范围中的任意值;时间为2-4h,例如可以为2h、2.5h、3h、3.5h、4h以及这些点值中的任意两个所构成的范围中的任意值。发明人发现,在该优选的具体实施方式下,本发明的方案具有进一步提高式(V)所示的化合物的收率的优势。
根据本发明,步骤(3)中,优选地,相对于10mL所述三氟乙酸和/或二氯甲烷,式(V)所示的化合物的用量为0.5-2.2g;所述反应IV的条件至少满足:温度为0-40℃,例如可以为0℃、5℃、10℃、15℃、20℃、25℃、30℃、35℃、40℃以及这些点值中的任意两个所构成的范围中的任意值;时间为2-4h,例如可以为2h、2.5h、3h、3.5h、4h以及这些点值中的任意两个所构成的范围中的任意值。发明人发现,在该优选的具体实施方式下,本发明的方案具有进一步提高式(II)所示的化合物的收率的优势。
本发明第三方面提供上述的荧光探针化合物、上述的的制备方法制得的荧光探针化合物在检测待测样品与ERRα蛋白结合能力中的应用。
根据本发明,待测样品可以为化合物,也可以是含有某种化合物的混合物或者溶液等。利用该类荧光探针化合物的配体部分与ERRα蛋白进行特异性结合后,将待测样品加入后,利用荧光偏振检测荧光偏振值是否减少,以判定待测样品是否具有与ERRα蛋白结合的能力。
本发明中,ERRα蛋白为雌激素相关受体α,可以为由Invitrogen公司提供的ERRα蛋白配体结合域,也可以为由专业的蛋白合成公司订制合成的ERRα蛋白,比如,上海吉凯基因有限公司订制合成的ERRα配体结合域蛋白。优选地,所述ERRα蛋白为如下(a1)或(a2)或(a3)所述的蛋白质:
(a1)具有SEQ ID NO.1所示氨基酸序列的蛋白质;
(a2)在SEQ ID NO.1所示氨基酸序列的氨基末端和/或羧基末端连接有标签的氨基酸序列所示的蛋白质;
(a3)SEQ ID NO.1所示的氨基酸序列经过取代、缺失或添加一个或几个氨基酸残基且仍具有相同功能的氨基酸序列所示的蛋白质。
本发明第四方面一种检测待测样品与ERRα蛋白结合能力的方法,包括以下步骤:将所述待测样品、荧光探针化合物和所述ERRα蛋白混合孵育后,进行荧光偏振检测,所述荧光探针化合物为上述的荧光探针化合物、上述的制备方法制得的荧光探针化合物。
根据本发明,所述孵育的条件至少满足:温度为0-40℃,例如可以为0℃、5℃、10℃、15℃、20℃、25℃、30℃、35℃、40℃以及这些点值中的任意两个所构成的范围中的任意值;时间为8-12min,例如可以为8min、9min、10min、11min、12min以及这些点值中的任意两个所构成的范围中的任意值。
优选地,所述荧光探针化合物与所述ERRα蛋白的用量比为1:1.5-10。示例性地,所述混合孵育的初始时刻,所述荧光探针化合物的浓度可以为50-120nmol/L(如50nmol/L);所述ERRα蛋白的浓度可以为200-600nmol/L(如200nmol/L);所述待测样品的浓度可为0-10000nmol/L(如0nmol/L、0.1nmol/L、1nmol/L、10nmol/L、100nmol/L、1000nmol/L或10000nmol/L)。
所述荧光偏振检测的具体方法如下:将孵育完成的混合液转移至四面透光的石英比色皿或表面经non-binding处理的微孔板,采用配备有荧光偏振检测模块的任意荧光分光光度计(如Horiba Fluoromax-4spectrofluorimeter)或酶标仪(如TECAN Spark 20Mmicroplate reader)进行荧光检测。
根据本发明,检测待测样品与ERRα蛋白结合能力的方法还包括:将荧光偏振检测得到的荧光偏振变化值对所述待测样品的浓度作图,得到竞争曲线;对所述竞争曲线进行Sigmoidal拟合,即得到待测样品的半抑制浓度,然后按照下述公式计算得到待测样品与ERRα蛋白的解离常数:
解离常数=半抑制浓度×A/B;
其中,A为所述荧光探针化合物与ERRα蛋白的解离常数,B为所述荧光探针化合物的浓度。
示例性地,当荧光探针化合物为下式所示的化合物时,
所述荧光探针化合物与ERRα蛋白的解离常数具体为140nmol/L,待测样品与ERRα蛋白的解离常数越小,则待测样品与ERRα蛋白的结合能力越强。
本发明还提供的该类荧光探针化合物还可以用于制备诊断肿瘤药物和治疗肿瘤药物。具体地,该类荧光探针化合物可用于药物小分子化合物的筛选检测,例如,用于制备诊断ERRα蛋白作用靶点的药物。
作为本发明进一步改进的技术方案,所述诊断肿瘤药物和治疗肿瘤药物还包括一种或多种药学上可接受的载体或赋形剂。
以下将通过实施例对本发明进行详细描述。
以下实施例中,荧光偏振值采用酶标仪TECAN Spark 20M microplate reader进行检测;在没有特别说明的情况下,所用原料均采用市售产品,所述室温为25±5℃,所述过夜的时间为10h。
制备例1
(1)将1g的4-氟-3-(三氟甲基)苯腈和0.8g的4-羟基-3-甲氧基苯甲醛溶解于10mL二甲基甲酰胺(DMF),然后加入2.2g的K2CO3,加热至65℃过夜得到反应液I,将反应液I用1mL冰水稀释后,再用10mL乙酸乙酯萃取三次,合并三次萃取液后,经干燥浓缩,通过硅胶柱柱层析(洗脱液为乙酸/正己烷,体积比为1:10)得到1.4g淡黄色的式(III)所示的化合物;
(2)将1.3g式(III)所示的化合物、515mg的2,4-噻唑烷二酮和3.3g的乙酸钠(NaOAc)加入到20mL乙腈(ACN)中,在温度为100℃的条件下反应24h得到反应液II,将反应液II的有机相旋干,再通过硅胶柱柱层析(洗脱液为乙酸/正己烷,体积比为1:2)纯化得到857mg黄色固体,为式(IV)所示的化合物;
(3)将800mg式(IV)所示的化合物、406mg的(5-羟基戊基)氨基甲酸叔丁酯和735mg三苯基磷(PPh3)溶于15mL四氢呋喃(THF)中,并用氮气保护,冰水浴,将566mg偶氮二甲酸二异丙酯(DIAD)滴加到上述溶液中,然后升到室温反应3h后,加入1mL水淬灭反应得到反应液III,将反应液III减压蒸馏除去溶剂,再通过硅胶柱柱层析(洗脱液乙酸乙酯/正己烷,体积比为1:1)纯化得到667mg白色固体,为式(V)所示的化合物;
(4)将605mg式(V)所示的化合物溶于3mL的CF3COOH(TFA),室温搅拌3h,减压蒸馏除去溶剂后,得到620mg式(II)所示的化合物。
制备例2
(1)将0.8g的4-氟-3-(三氟甲基)苯腈和0.7g的4-羟基-3-甲氧基苯甲醛溶解于10mL二甲基甲酰胺,然后加入1.8g的Na2CO3,加热至60℃过夜得到反应液I,将反应液I用1mL冰水稀释后,再用10mL乙酸乙酯萃取三次,合并三次萃取液后,经干燥浓缩,通过硅胶柱柱层析(洗脱液为乙酸/正己烷,体积比为1:10)得到淡黄色的式(III)所示的化合物;
(2)将1g式(III)所示的化合物、0.4g的2,4-噻唑烷二酮和2.4g的乙酸钾加入到20mL乙腈中,在温度为80℃的条件下反应30h得到反应液II,将反应液II的有机相旋干,再通过硅胶柱柱层析(洗脱液为乙酸/正己烷,体积比为1:2)纯化得到黄色固体,为式(IV)所示的化合物;
(3)将0.6g式(IV)所示的化合物、0.3g的(5-羟基戊基)氨基甲酸叔丁酯和525mg三苯基磷溶于15mL四氢呋喃(THF)中,并用氮气保护,冰水浴,将525mg偶氮二甲酸二异丙酯滴加到上述溶液中,然后升到室温反应2h后,加入1mL水淬灭反应得到反应液III,将反应液III减压蒸馏除去溶剂,再通过硅胶柱柱层析(洗脱液乙酸乙酯/正己烷,体积比为1:1)纯化得到白色固体,为式(V)所示的化合物;
(4)将2.2g式(V)所示的化合物溶于3mL的CF3COOH,室温搅拌4h,减压蒸馏除去溶剂后,得到式(II)所示的化合物。
制备例3
(1)将1.2g的4-氟-3-(三氟甲基)苯腈和0.9g的4-羟基-3-甲氧基苯甲醛溶解于10mL二甲基甲酰胺,然后加入2.5g的Na2CO3,加热至70℃过夜得到反应液I,将反应液I用1mL冰水稀释后,再用10mL乙酸乙酯萃取三次,合并三次萃取液后,经干燥浓缩,通过硅胶柱柱层析(洗脱液为乙酸/正己烷,体积比为1:10)得到淡黄色的式(III)所示的化合物;
(2)将1.6g式(III)所示的化合物、0.6g的2,4-噻唑烷二酮和3.6g的乙酸钾加入到20mL乙腈中,在温度为105℃的条件下反应20h得到反应液II,将反应液II的有机相旋干,再通过硅胶柱柱层析(洗脱液为乙酸/正己烷,体积比为1:2)纯化得到黄色固体,为式(IV)所示的化合物;
(3)将0.9g式(IV)所示的化合物、0.45g的(5-羟基戊基)氨基甲酸叔丁酯和0.9g三苯基磷溶于15mL四氢呋喃(THF)中,并用氮气保护,冰水浴,将0.6g偶氮二甲酸二异丙酯滴加到上述溶液中,然后升到室温反应4h后,加入1mL水淬灭反应得到反应液III,将反应液III减压蒸馏除去溶剂,再通过硅胶柱柱层析(洗脱液乙酸乙酯/正己烷,体积比为1:1)纯化得到白色固体,为式(V)所示的化合物;
(4)将0.5g式(V)所示的化合物溶于10mL的二氯甲烷,室温搅拌2h,减压蒸馏除去溶剂后,得到式(II)所示的化合物。
实施例1
将250mg制备例1制得的式(II)所示的化合物、231mg的5(6)-羟基荧光素(FITC-NCS)以及645mg的N,N-二异丙基乙胺(DIPEA)溶解于10mL的N,N-二甲基甲酰胺(DMF),在温度为50℃的条件下搅拌过夜得到反应液IV,将反应液IV减压旋蒸除去溶剂,再采用反相制备色谱纯化得到21mg淡黄色的最终产物。
将得到的最终产物进行纯度分析,采用配有Waters XBridgeTMC18的反相柱(2.1mm×50mm,Waters公司产品)的高效液相色谱仪(Agilent Technologies产品,型号为HP-1100)进行纯度检测,具体操作过程如下:流动相由甲醇(A)和水(B)组成,流速为0.8mL/min,使用以下洗脱条件:在0~10min内,流动相中甲醇的体积百分含量由10%匀速增至100%进行线性梯度洗脱,检测波长为254nm,HPLC的检测结果见图2,采用低分辨液相-质谱(Agilent Technologies)进行分析。
将得到的最终产物进行分子量的鉴定,采用正电荷模式电喷射离子化对样品进行分析,分子量为894,MS的检测结果见图3。
将得到的最终产物的结构采用1H NMR进行表征,1H NMR谱图见图4,表征结果分析如下:
1H NMR(400MHz,DMSO)δ10.09(s,2H),9.85(s,1H),8.31-8.30(d,1H),8.19(s,1H),8.08(s,1H),8.00-7.97(dd,1H),7.96(s,1H),7.70-7.68(d,1H),7.52-7.51(d,1H),7.36-7.34(m,1H),7.28-7.25(dd,1H),7.15-7.12(d,1H),6.90-6.88(d,1H),6.64-6.63(d,1H),6.59-6.51(m,4H),3.76(s,3H),3.67-3.64(t,2H),3.47-3.45(m,2H),1.63-1.56(m,4H),1.34-1.29(m,2H)。
经上述质谱(见图3)和1H NMR谱(见图4)验证,实施例1得到的最终产物的结构式如下式所示(简称5FB-FITC),实施例1得到的最终产物中5FB-FITC的纯度>99%(见图2所示的HPLC分析结果),
测试例1荧光偏振检测5FB-FITC与ERRα蛋白的解离常数
将溶于Tris-HCl buffer(50mM Tris-HCl,100mM NaCl,pH 7.4)的50nmol/L荧光探针化合物5FB-FITC与不同浓度的ERRα蛋白(0nmol/L、50nmol/L、100nmol/L、50nmol/L、200nmol/L、250nmol/L)混合并加入384微孔板(总体积20μL),室温孵育10min后,采用酶标仪测定荧光偏振值,每个浓度点重复三次实验。
随着ERRα蛋白浓度的增加,5FB-FITC与ERRα蛋白的结合越多,由于ERRα蛋白结合口袋对5FB-FITC的束缚作用,使探针5FB-FITC的自由旋转减慢,因而旋转弛豫时间增加,荧光偏振值增加,ERRα蛋白浓度达200nmol/L时,荧光偏振值增加达到饱和,将荧光偏振值对ERRα蛋白浓度作图,得到结合曲线。
采用Origin软件对结合曲线进行Sigmoidal曲线拟合,拟合曲线见图5,得到荧光探针化合物5FB-FITC与ERRα蛋白的解离常数为140nmol/L。
测试例2检测已知配体XCT790与ERRα的结合能力
将溶于Tris-HCl buffer(50mM Tris-HCl,100mM NaCl,pH 7.4)的50nmol/L荧光探针化合物5FB-FITC,200nmol/L的ERRα蛋白和不同浓度的配体XCT790(0nmol/L、1nmol/L、10nmol/L、100nmol/L、1000nmol/L、10000nmol/L、50000nmol/L)加入384微孔板(总体积20μL),室温孵育10min后,采用酶标仪测定荧光偏振值,每个浓度点重复三次实验。
随着配体XCT790浓度的增加,5FB-FITC被逐渐从ERRα蛋白结合位点竞争下来,成为游离态,使荧光探针化合物5FB-FITC的自由旋转加快,因而旋转弛豫时间较少,从而导致探针荧光偏振值逐渐降低。将荧光偏振检测得到的荧光偏振变化值(相对荧光偏振值)对配体XCT790的浓度作图,得到竞争曲线,结果见图6。
采用sigmaplot软件对竞争曲线进行Sigmoidal拟合,得到配体XCT790的半抑制浓度,然后按照下述公式计算得到配体XCT790与ERRα蛋白的解离常数:
解离常数=半抑制浓度×A/B;
其中,A为荧光探针化合物5FB-FITC与ERRα蛋白的解离常数,B为荧光探针化合物5FB-FITC的浓度。
结果表明,荧光探针化合物5FB-FITC浓度为50nmol/L时,配体XCT790的半抑制浓度为720nmol/L,计算得到,配体XCT790与ERRα蛋白的解离常数为2000nmol/L。
测试例3检测非活性化合物雌二醇(E2)与ERRα的结合能力
将溶于Tris-HCl buffer(50mM Tris-HCl,100mM NaCl,pH 7.4)的50nmol/L荧光探针化合物5FB-FITC,200nmol/L的ERRα蛋白和不同浓度的雌二醇(E2)(0nmol/L、10nmol/L、100nmol/L、1000nmol/L、10000nmol/L)加入384微孔板(总体积20μL),室温孵育10min后,采用酶标仪测定荧光偏振值,每个浓度点重复三次实验。
随着雌二醇(E2)浓度的增加,5FB-FITC的荧光偏振值没有显著降低趋势,说明雌二醇(E2)并不能与探针竞争结合ERRα蛋白,结果见图6。由于雌二醇并非ERRα蛋白的配体,并不能与ERRα蛋白直接结合,因此,进一步从侧面证实,荧光探针化合物5FB-FITC检测待测样品与ERRα蛋白蛋白结合能力的特异性。
以上详细描述了本发明的优选实施方式,但是,本发明并不限于此。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,包括各个技术特征以任何其它的合适方式进行组合,这些简单变型和组合同样应当视为本发明所公开的内容,均属于本发明的保护范围。
SEQUENCE LISTING
<110> 湖南农业大学
<120> 荧光探针化合物及其制备方法和应用
<130> 2021.7.19
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 244
<212> PRT
<213> 人工合成
<400> 1
Pro Leu Ala Val Ala Gly Gly Pro Arg Lys Thr Ala Ala Pro Val Asn
1 5 10 15
Ala Leu Val Ser His Leu Leu Val Val Glu Pro Glu Lys Leu Tyr Ala
20 25 30
Met Pro Asp Pro Ala Gly Pro Asp Gly His Leu Pro Ala Val Ala Thr
35 40 45
Leu Cys Asp Leu Phe Asp Arg Glu Ile Val Val Thr Ile Ser Trp Ala
50 55 60
Lys Ser Ile Pro Gly Phe Ser Ser Leu Ser Leu Ser Asp Gln Met Ser
65 70 75 80
Val Leu Gln Ser Val Trp Met Glu Val Leu Val Leu Gly Val Ala Gln
85 90 95
Arg Ser Leu Pro Leu Gln Asp Glu Leu Ala Phe Ala Glu Asp Leu Val
100 105 110
Leu Asp Glu Glu Gly Ala Arg Ala Ala Gly Leu Gly Glu Leu Gly Ala
115 120 125
Ala Leu Leu Gln Leu Val Arg Arg Leu Gln Ala Leu Arg Leu Glu Arg
130 135 140
Glu Glu Tyr Val Leu Leu Lys Ala Leu Ala Leu Ala Asn Ser Asp Ser
145 150 155 160
Val His Ile Glu Asp Ala Glu Ala Val Glu Gln Leu Arg Glu Ala Leu
165 170 175
His Glu Ala Leu Leu Glu Tyr Glu Ala Gly Arg Ala Gly Pro Gly Gly
180 185 190
Gly Ala Glu Arg Arg Arg Ala Gly Arg Leu Leu Leu Thr Leu Pro Leu
195 200 205
Leu Arg Gln Thr Ala Gly Lys Val Leu Ala His Phe Tyr Gly Val Lys
210 215 220
Leu Glu Gly Lys Val Pro Met His Lys Leu Phe Leu Glu Met Leu Glu
225 230 235 240
Ala Met Met Asp
Claims (8)
4.根据权利要求3所述的制备方法,其特征在于,所述反应溶剂II为乙腈,所述催化剂II为乙酸钠和/或乙酸钾,所述反应溶剂III为四氢呋喃,所述催化剂III为三苯基磷和偶氮二甲酸二异丙酯;
步骤(1)中,相对于100mL所述反应溶剂II,式(III)所示的化合物的用量为5-8g、2,4-噻唑烷二酮的用量为2-3g、所述催化剂II的用量为12-18g;
步骤(2)中,相对于100mL所述反应溶剂III,式(IV)所示的化合物的用量为4-6g、(5-羟基戊基)氨基甲酸叔丁酯的用量为2-3g、所述催化剂III的用量为7-10g;
步骤(3)中,相对于10mL所述三氟乙酸和/或二氯甲烷,式(V)所示的化合物的用量为0.5-2.2g。
5.根据权利要求3所述的制备方法,其特征在于,步骤(1)中所述反应II的条件至少满足:温度为80-120℃、时间为20-30h;
步骤(2)中所述反应III的条件至少满足:温度为0-40℃、时间为2-4h;
步骤(3)中所述反应IV的条件至少满足:温度为0-40℃、时间为2-4h。
6.根据权利要求2至5中任意一项所述的制备方法,其特征在于,所述反应溶剂I为N,N-二甲基甲酰胺,所述催化剂I为N,N-二异丙基乙胺;
相对于100mL所述反应溶剂I,式(II)所示的化合物的用量为2-3g,具有与氨基反应活性的有机荧光分子为2-3g,所述催化剂I的用量为5-8g;
所述反应I的条件至少满足:温度为40-60℃、时间为8-15h。
7.权利要求1所述的荧光探针化合物、权利要求2至6中任意一项所述的制备方法制得的荧光探针化合物在制备诊断ERRα蛋白作用靶点的药物中的应用。
8.根据权利要求7所述的应用,其特征在于,所述ERRα蛋白为如下(a1)或(a2)或(a3)所述的蛋白质:
(a1)具有SEQ ID NO.1所示氨基酸序列的蛋白质;
(a2)在SEQ ID NO.1所示氨基酸序列的氨基末端和/或羧基末端连接有标签的氨基酸序列所示的蛋白质;
(a3)SEQ ID NO.1所示的氨基酸序列经过取代、缺失或添加一个或几个氨基酸残基且仍具有相同功能的氨基酸序列所示的蛋白质。
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