CN114685463B - 一种异鼠李素光亲和探针及其合成方法和应用 - Google Patents
一种异鼠李素光亲和探针及其合成方法和应用 Download PDFInfo
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- C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
- C07D405/12—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings linked by a chain containing hetero atoms as chain links
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
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- G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
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Abstract
本发明涉及一种异鼠李素光亲和探针及其合成方法和应用,该探针以异鼠李素为活性基团,双吖丙啶为光交联基团,炔烃为生物正交反应基团,具有如下所示结构:与现有技术相比,本发明保留了异鼠李素的抑制肿瘤细胞增殖活性,且具有良好的细胞膜透过性;利用双吖丙啶作为光交联基团,以及分子结构带有的炔烃作为生物正交反应基团,可以实现对靶蛋白共价结合及细胞内原位靶点钓取,可用于异鼠李素抗癌作用靶点的发现。
Description
技术领域
本发明属于化学生物学技术领域,涉及一种异鼠李素光亲和探针及其合成方法和应用。
背景技术
异鼠李素是一种具有多种生物活性的天然产物,近年来,因其在多种癌症中的抗增殖特性而受到关注。异鼠李素通过诱导G2/M阶段阻滞抑制人肝癌,乳腺癌,结肠癌和膀胱癌细胞的增殖(Asian Pacific journal of cancer prevention:APJCP,2015,16(7):3035-3042),并在多种癌细胞中显示出依赖死亡受体(DR)外在途径和线粒体内在途径的凋亡诱导作用(General Physiology and Biophysics,2019,38(6):473-484;MolecularMedicine Reports,2015,12(4):5796-5806)。异鼠李素的抗癌作用伴随着对多种细胞信号通路的干扰,其中PI3K/Akt/mTOR/p70S6K通路和MAPK通路在其介导的细胞周期阻滞以及诱导的细胞凋亡和自噬中起着关键作用(Scientific Reports,2018,8;Nutrition andCancer-an International Journal,2015,67(7):1191-1200)。异鼠李素等黄酮类化合物与靶蛋白之间多为非共价的弱相互作用,且生物活性往往是多靶点作用的结果,因而目前对于异鼠李素的作用靶点知之甚少。
基于活性的蛋白质谱分析(ABPP)已被用于大规模研究蛋白质-小分子相互作用,但是ABPP无法捕获弱的非共价结合的蛋白质(Natural Product Reports,2016,33(5):731-733)。光亲和标记探针是识别天然产物非共价结合靶蛋白的一种有效策略。此前设计的一种异鼠李素生物素探针(2020,CN112300191A),以异鼠李素骨架结构为光反应基团,生物素为亲和标签,可以用于靶蛋白的发现,但对于靶蛋白的标记模式比较复杂,而且探针末端直接连接生物素取代基体积较大,影响化合物的细胞透过性,在原位标记中的应用存在限制。
发明内容
本发明的目的就是为了克服上述现有技术存在的缺陷而提供一种异鼠李素光亲和探针及其合成方法和应用。该探针具有与异鼠李素相似的抗癌作用,并具有在细胞原位标记异鼠李素靶蛋白的能力,包括弱的非共价结合靶蛋白。
本发明的目的可以通过以下技术方案来实现:
本发明第一方面提供一种异鼠李素光亲和探针,该探针以异鼠李素为活性基团,双吖丙啶为光交联基团,炔烃为生物正交基团,具有如下所示结构:
本发明第二方面提供一种所述的异鼠李素光亲和探针的合成方法,包括以下步骤:
S1:将槲皮素加入无水二氯甲烷和DMF混合溶液中搅拌溶解,在冰浴下加入N,N-二异丙基乙胺和氯甲基甲基醚,搅拌0.5~1.5h,然后恢复至室温反应16~18h,反应完成后,旋蒸除去溶剂、萃取、干燥、柱层析纯化得到中间体1,中间体1的结构式如下所示:
S2:将步骤S1得到的中间体1和K2CO3加入无水DMF溶液中搅拌溶解,向溶液中加入光交联基团(3-(3-炔-1-丁基)-3-(2-碘乙基)-3H-双吖丙啶),并在62~68℃加热回流反应48~96h,旋蒸除去溶剂、萃取、干燥、柱层析纯化得到中间体2,中间体2的结构式如下所示:
S3:将步骤(2)所得中间体2加入丙酮溶液中搅拌溶解,在冰浴下向溶液中加入2.5~3.5N HCl,搅拌0.5~1.5h,然后恢复至室温反应5~7d;反应结束后加入水淬灭反应,萃取、洗涤、干燥,旋干溶剂,柱层析纯化得到化合物3,化合物3的结构式如下所示:
化合物3即为所述的异鼠李素光亲和探针。
优选地,步骤S1中,包括以下条件中的任一项或多项:
(i)槲皮素、N,N-二异丙基乙胺和氯甲基甲基醚的摩尔之比为1:(3~4):8;
(ii)槲皮素与(无水二氯甲烷和DMF混合溶液)的用量之比为1g:(200~300)ml;
(iii)无水二氯甲烷和DMF混合溶液中,无水二氯甲烷和DMF的体积之比为(20~15):1。
优选地,步骤S2中,包括以下条件中的任一项或多项:
(i)中间体1、K2CO3和光交联基团(3-(3-炔-1-丁基)-3-(2-碘乙基)-3H-双吖丙啶)的摩尔之比为1:(1.2~1.3):2;
(ii)中间体1与无水DMF溶液的用量之比为10mg:(0.5~1)mL。
优选地,步骤S3中,包括以下条件中的任一项或多项:
(i)丙酮与HCl的体积之比为(5~7):1;
(ii)中间体2与丙酮的用量之比为10mg:(0.5~1)mL。
本发明第三方面提供所述的异鼠李素光亲和探针在癌细胞成像中的应用。
优选地,所述的癌细胞包括乳腺癌细胞MDA-MB-231、结肠癌细胞HCT-116或前列腺癌细胞PC-3。
本发明第四方面提供所述的异鼠李素光亲和探针在癌细胞原位鉴定异鼠李素直接作用靶蛋白中的应用。
优选地,所述的靶蛋白与探针共价结合,通过紫外照射,实现探针中的双吖丙啶基团与包括癌细胞增殖、侵袭在内的生物过程相关蛋白的共价交联。
优选地,所述的癌细胞包括乳腺癌细胞MDA-MB-231、结肠癌细胞HCT-116或前列腺癌细胞PC-3。
本发明保留了异鼠李素的抑制肿瘤细胞增殖活性,且具有良好的细胞膜透过性;利用双吖丙啶作为光交联基团,以及分子结构带有的炔烃作为生物正交反应基团,可以实现对靶蛋白共价结合及细胞内原位靶点钓取,可用于异鼠李素抗癌作用靶点的发现。
与现有技术相比,本发明采用“极简的光交联基团”修饰3’位的-OH,通过常用的探针设计方法,以延长碳链的方式得到异鼠李素的同系物,保留异鼠李素的结构骨架,同时降低了对异鼠李素原有生物活性的影响,且具有良好的细胞膜透过性和原位标记效力。以异鼠李素为反应基团,双吖丙啶为光交联基团,可以实现对靶蛋白的共价结合,推动异鼠李素靶蛋白和抗癌作用机制的发现。
附图说明
图1(A)为异鼠李素的结构,(B)为异鼠李素光亲和探针的结构。
图2.异鼠李素光亲和探针对癌细胞的生长抑制作用。异鼠李素和光亲和探针对乳腺癌细胞系MDA-MB-231(A),结肠癌细胞系HCT-116(B)前列腺癌细胞系PC-3(C)的生长抑制作用。用化合物(0-300μM)处理MDA-MB-231细胞48h,处理HCT-116和PC-3细胞72h,使用MTT比色法测定评估化合物对细胞的存活率的影响的IC50曲线,数据表示为平均值±SD,n=3;
图3异鼠李素光亲和探针在乳腺癌MDA-MB-231细胞内的荧光定位。向MDA-MB-231细胞添加20μM(A)或30μM(B)探针分子后,使用共聚焦显微镜在488nm激发波长下,500~560nm的发射波长下在细胞内显示的荧光。
图4凝胶内荧光分析实验显示异鼠李素光亲和探针标记异鼠李素的靶蛋白。30μM异鼠李素光亲和探针与MDA-MB-231细胞作用4h后,紫外光照射,裂解细胞,通过CUAAC反应连接叠氮罗丹明基团,SDS-PAGE凝胶电泳分离后,采用化学成像仪观察光亲和探针结合的靶蛋白荧光。右图为相应蛋白条带考马斯亮蓝染色结果。
图5异鼠李素光亲和探针对蛋白质的富集实验。30μM异鼠李素光亲和探针与MDA-MB-231细胞作用4h后,紫外光照射,裂解细胞,通过CUAAC反应连接叠氮生物素基团,与链霉亲和素琼脂糖珠孵育富集生物素连接的蛋白,SDS-PAGE凝胶电泳分离后,银染观察光亲和探针结合的蛋白。
具体实施方式
一种异鼠李素光亲和探针,该探针以异鼠李素为活性基团,双吖丙啶为光交联基团,炔烃为生物正交基团,具有如下所示结构:
上述异鼠李素光亲和探针的合成方法,包括以下步骤:
S1:将槲皮素加入无水二氯甲烷和DMF混合溶液中搅拌溶解,在冰浴下加入N,N-二异丙基乙胺和氯甲基甲基醚,搅拌0.5~1.5h,然后恢复至室温反应16~18h,反应完成后,旋蒸除去溶剂、萃取、干燥、柱层析纯化得到中间体1,中间体1的结构式如下所示:
S2:将步骤S1得到的中间体1和K2CO3加入无水DMF溶液中搅拌溶解,向溶液中加入光交联基团(3-(3-炔-1-丁基)-3-(2-碘乙基)-3H-双吖丙啶),并在62~68℃加热回流反应48~96h,旋蒸除去溶剂、萃取、干燥、柱层析纯化得到中间体2,中间体2的结构式如下所示:
S3:将步骤(2)所得中间体2加入丙酮溶液中搅拌溶解,在冰浴下向溶液中加入2.5~3.5N HCl,搅拌0.5~1.5h,然后恢复至室温反应5~7d;反应结束后加入水淬灭反应,萃取、洗涤、干燥,旋干溶剂,柱层析纯化得到化合物3,化合物3的结构式如下所示:
化合物3即为所述的异鼠李素光亲和探针。
步骤S1中,优选槲皮素、N,N-二异丙基乙胺和氯甲基甲基醚的摩尔之比为1:(3~4):8;优选槲皮素与(无水二氯甲烷和DMF混合溶液)的质量:体积之比为1g:(200~300)mL;优选无水二氯甲烷和DMF混合溶液中,无水二氯甲烷和DMF的体积之比为(20~15):1。
步骤S2中,优选中间体1、K2CO3和光交联基团(3-(3-炔-1-丁基)-3-(2-碘乙基)-3H-双吖丙啶)的摩尔之比为1:(1.2~1.3):2;优选中间体1与无水DMF溶液的用量之比为10mg:(0.5~1)mL。
步骤S3中,优选丙酮与HCl的体积之比为(5~7):1;中间体2与丙酮的用量之比为10mg:(0.5~1)mL。
上述异鼠李素光亲和探针可用在癌细胞成像中。癌细胞优选包括乳腺癌细胞MDA-MB-231、结肠癌细胞HCT-116或前列腺癌细胞PC-3。
上述异鼠李素光亲和探针还可用在癌细胞原位鉴定异鼠李素直接作用靶蛋白中。优选靶蛋白与探针共价结合,通过紫外照射,实现探针中的双吖丙啶基团与包括癌细胞增殖、侵袭在内的生物过程相关蛋白的共价交联。优选癌细胞包括乳腺癌细胞MDA-MB-231、结肠癌细胞HCT-116或前列腺癌细胞PC-3。
下面结合附图和具体实施例对本发明进行详细说明。以下实施例在以本发明技术方案为前提下进行实施,给出了详细的实施方式和具体的操作过程,但本发明的保护范围不限于下述的实施例。除有定义外,以下实施例中所用的技术术语具有与本发明所属领域技术人员普遍理解的相同含义,以下实施例中所用的实验试剂,如无特殊说明,均为常规生化试剂;所述实验方法,如无特殊说明,均为常规方法。
实施例1一种异鼠李素光亲和探针,其合成方法包括以下步骤:
具体来说,包括以下步骤:
(1)合成中间体1[5-羟基-2-(3-羟基-4-(甲氧基甲氧基)苯基)-3,7-双(甲氧基甲氧基)-4H-苯并吡喃-4-酮]
反应式1
将1g槲皮素(3.3mmol,1eq)加入250mL无水DCM和15mL无水DMF混合溶液中溶解,并用冰浴降温至0℃左右,向溶液中加入4.5mL DIPEA(26.4mmol,8eq),将0.75mL MOMCl(9.9mmol,3eq)稀释溶解于40mL DCM溶液中,使用恒压滴液漏斗滴加,并在0℃下搅拌1h,然后恢复至室温反应过夜。反应结束后,旋蒸除去溶剂,加入50mL饱和食盐水,再加入50mL乙酸乙酯萃取三遍,得有机层用无水硫酸钠干燥,经柱层析纯化(石油醚/乙酸乙酯=6:1至3:1),得到黄色固体中间体1(290mg),产率为20.23%。1H NMR(CDCl3,400MHz):δ 12.52(s,1H),7.68(d,J=2.12Hz,1H),7.61(dd,J=8.6,2.16Hz,1H),7.19(d,J=8.6Hz,1H),6.61(d,J=2.12Hz,1H),6.45(d,J=2.12Hz,1H),5.98(s,1H),5.29(s,2H),5.23(s,2H),5.18(s,2H),3.54(s,3H),3.49(s,3H),3.25(s,3H)ppm;
(2)合成中间体2[2-(3-(2-(3-(丁-3-炔-1-基)-3H-双吖丙啶-3-基)乙氧基)-4-(甲氧基甲氧基)苯基)-5-羟基-3,7-双(甲氧基甲氧基)-4H-苯并吡喃-4-酮]
反应式2
将200mg中间体1(0.46mmol,1eq),127.14mg无水K2CO3(0.92mmol,2eq)加入10mL无水DMF溶液中,搅拌2min使其溶解,向溶液中加入82mL(0.552mmol,1.2eq)3-(3-炔-1-丁基)-3-(2-碘乙基)-3H-双吖丙啶,65℃加热回流反应3d,旋蒸除去DMF,加入50mL水,再加入50mL乙酸乙酯萃取三次,得有机层用无水硫酸钠干燥,经柱层析纯化(石油醚/乙酸乙酯=10:1至3:1),得到黄色固体中间体2(50mg),产率为19.6%。1H NMR(CDCl3,400MHz): δ12.51(s,1H),7.66(dd,J=8.56,2Hz,1H),7.62(d,J=2Hz,1H),7.23(d,J=8.56Hz,1H),6.63(d,J=2.16Hz,1H),6.46(d,J=2.16Hz,1H),5.31(s,2H),5.24(s,2H),5.18(s,2H),3.98(t,J=6.2Hz,2H),3.55(s,3H),3.50(s,3H),3.24(s,3H),2.12(dt,J=7.72,2.64Hz,2H),1.98(t,2.62Hz,1H),1.95(t,J=6.16Hz,2H),1.80(t,J=7.6Hz,2H)ppm;13C NMR(100MHz,CDCl3)δ178.73,163.13,162.04,156.83,156.73,149.33,148.46,135.77,124.51,123.20,116.06,114.34,106.77,99.95,98.05,95.30,94.35,94.21,83.02,77.16,69.29,64.02,57.94,56.60,56.55,33.17,32.90,26.82,13.45ppm.
(3)合成化合物3[2-(3-(2-(3-(丁-3-炔-1-基)-3H-双吖丙啶-3-基)乙氧基)-4-羟基苯基)-3,5,7-三羟基--4H-苯并吡喃-4-酮]
反应式3
将40mg中间体2(0.072mmol,1eq)加入2.4mL丙酮溶液中,搅拌2min使其溶解,并用冰浴降温至0℃左右,向溶液中加入0.4mL 3N HCl,并在0℃下搅拌1h,然后恢复至室温反应6d。向反应液中加入水终止反应,饱和碳酸氢钠溶液调节PH至中性,再加入10mL乙酸乙酯萃取三次,合并有机层,无水硫酸钠干燥,经柱层析纯化(石油醚/乙酸乙酯=5:1至3:1),得到黄色固体化合物3(12.56mg),产率为41.3%。
1H NMR(DMSO-d6,400MHz):δ 12.46(s,1H),10.78(s,1H),9.78(s,1H),9.43(s,1H),7.72(d,J=1.92Hz,1H),7.70(dd,J=8.44,2.04Hz,1H),6.96(d,J=8.36Hz,1H),6.47(d,J=2Hz,1H),6.19(d,J=2.04Hz,1H),3.89(t,J=6.22Hz,2H),2.83(t,2.62Hz,1H),2.06(dt,J=7.4,2.6Hz,2H),1.89(t,J=6.2Hz,2H),1.71(t,J=7.4Hz,2H)ppm;13C NMR(176MHz,DMSO-d6)δ175.91,163.94,160.71,156.18,149.29,146.52,146.24,135.86,122.26,121.98,115.79,113.79,103.05,98.22,93.61,83.34,71.80,63.68,39.52,32.14,31.81,27.08,12.71ppm.
异鼠李素的结构如图1所示。本发明得到的异鼠李素光亲和探针的结构如图1(B)所示。
实施例2异鼠李素光亲和探针对肿瘤细胞系增殖抑制实验
1.细胞培养
所有肿瘤细胞系(MDA-MB-231,HCT-116,PC-3)均购于ATCC(美国菌种保藏中心)。细胞在DMEM完全培养基(高糖DMEM培养基,加入10%胎牛血清,100units/mL青霉素,100mg/mL链霉素)中培养。细胞在37℃,5%CO2中培养。
2.MTT测定
将细胞种于96孔板中,加入不同浓度的化合物,与MDA-MB-231细胞作用48h,与HCT-116,PC-3细胞作用72h后,每孔加入20μL MTT(5mg/mL)并孵育4h。吸去上清,每孔加入150μL DMSO。酶标仪(Thermo Varioskan Flash)读取490nm下吸光度(OD)。每个化合物在每个浓度下设三个复孔。
计算化合物的增殖抑制率:细胞增殖抑制率=(OD阴性对照-OD实验)/(OD阴性对照-OD空白)×100%。使用GraphPad Prism 5软件,以不同浓度化合物对细胞增殖抑制率作图可得到剂量反应曲线和半数抑制浓度IC50值。
由图2可知,本发明异鼠李素光亲和探针对乳腺癌MDA-MB-231细胞系、结肠癌HCT-116细胞系和前列腺癌PC-3细胞系的增殖均具有抑制的作用。由抑制率曲线和IC50值可以看出探针分子与异鼠李素具有相似的癌细胞生长抑制作用,说明探针在3’位羟基的修饰基本不影响异鼠李素的原有生长抑制活性,可以充分模拟异鼠李素在细胞内的作用,能够作为有效的异鼠李素靶点发现工具。
实施例3异鼠李素光亲和探针细胞内荧光定位实验
将MDA-MB-231细胞接种于铺有细胞爬片的24孔板,贴壁过夜后,加入20或30μM异鼠李素光亲和探针,4小时后,弃去培养基,PBS洗涤,每孔加入100μL的2μM环孢霉素A(CsA),4%多聚甲醛固定细胞5min,使用封片液封片。使用荧光共聚焦显微镜(Leica SP8),在63×油镜下观察细胞内荧光,激发波长为488nm,发射波长为500-560nm,分辨率为1024×1024,行扫描频率为600Hz。
由图3可知,本发明异鼠李素光亲和探针具有在肿瘤细胞中成像,显示探针在细胞中的定位与分布的能力,化合物浓度越高,荧光越强。说明探针分子剂量依赖性地进入细胞,具有良好的膜透过性,因此可以进行细胞原位靶点钓取。在30μM探针作用4小时后,荧光成像显示细胞内的化合物浓度较高,且细胞保持了基本形态,说明30μM是合适的细胞原位靶点钓取的化合物浓度。
实施例4异鼠李素光亲和探针凝胶内荧光分析实验
将MDA-MB-231细胞铺在6孔板中,贴壁过夜后,实验组,紫外对照组,竞争组均加入30μM异鼠李素光亲和探针,竞争组另加入100μM异鼠李素竞争,空白对照组加入等量DMSO。4小时后,6孔板置于冰上在365nm的紫外灯光下照射25min(紫外灯功率:40W,6孔板距紫外灯10cm),紫外对照组不进行紫外照射。胰酶裂解收集细胞,PBS洗涤2次,每组加入120μL细胞裂解液(含0.1%Triton X-100的PBS溶液),混匀置于冰上裂解45min。4℃下12000r/min离心10min,收集上层细胞裂解液。
BCA法测定蛋白质溶液浓度,调节蛋白浓度至1mg/mL。将细胞裂解液与60μM罗丹明叠氮化物,100μM三(苄基三唑基甲基)胺(TBTA),1mM三(2-羧乙基)膦(TCEP),1mM CuSO4在室温下混合作用2h,然后加入500μL预冷的丙酮,-20℃放置20min沉淀蛋白质。4℃下12000r/min离心10min,收集沉淀用甲醇洗涤两次,100μL裂解液重悬蛋白,加入5×上样缓冲液,95℃加热10min以变性蛋白。
将样品通过SDS-PAGE分离,在90-150V电压下电泳约1.5个小时。使用ChemiDoc MP(Bio-Rad)扫描显示凝胶荧光条带,然后用考马斯亮蓝对凝胶进行染色,以证明每条泳道的蛋白量相等。
由图4可知,异鼠李素光亲和探针可用于点击化学反应连接荧光基团,通过SDS-PAGE凝胶电泳之后进行荧光扫描成像,探针结合的蛋白条带显现荧光。说明异鼠李素光亲和探针表现出了在细胞原位标记靶蛋白的能力(泳道2),与UV对照组(泳道3)相比,紫外光照实现了探针分子与靶蛋白的共价连接,标记了相互作用的靶点;与竞争组(泳道4)相比,异鼠李素作为竞争化合物几乎去掉了探针标记的所有条带,说明二者具有相似的靶蛋白,探针具有良好的标记异鼠李素作用靶点的能力,通过与对照组和竞争组对比,可以去除非特异性的背景结合以及假阳性靶点蛋白。
实施例5异鼠李素光亲和探针的蛋白质组标记和富集实验
MDA-MB-231细胞在10cm培养皿中生长至80-90%密度,实验组,紫外对照组,竞争组均加入30μM异鼠李素光亲和探针,竞争组另加入100μM异鼠李素竞争,空白对照组加入等量DMSO。4小时后,培养皿置于冰上在365nm的紫外灯光下照射25min,紫外对照组不进行紫外照射。胰酶裂解收集细胞,PBS洗涤2次,每组加入1.2mL细胞裂解液,冰上裂解45min。4℃下12000r/min离心10min,收集上层清液。
BCA法测定蛋白质溶液浓度,调节蛋白浓度至1mg/mL。将细胞裂解液与60μM生物素叠氮化物,100μM TBTA,1mM TCEP,1mM CuSO4在室温下混合2h,然后加入500μL预冷的丙酮,-20℃放置20min沉淀蛋白质。4℃下12000r/min离心10min,收集沉淀用甲醇洗涤两次,将沉淀的蛋白再溶解于1mL 1%SDS中,将重悬的样品与NeutrAvidinTM琼脂糖珠(Pierce)在室温下孵育3小时。然后将珠子用将1%SDS,PBS各洗涤3次。将琼脂糖珠用7.5M尿素重悬,加入5×上样缓冲液,95℃加热10min以释放生物素结合的蛋白。
将样品通过SDS-PAGE分离,在90-150V电压下电泳约1.5个小时。使用银染试剂盒(碧云天)对凝胶进行银染。
由图5可知,异鼠李素光亲和探针可通过点击化学反应连接生物素基团,利用NeutrAvidinTM琼脂糖珠即可富集探针结合的靶蛋白,通过SDS-PAGE凝胶电泳分离和银染能实现富集靶蛋白的可视化。与对照组和竞争组相比,探针组富集了更多的蛋白。可以结合LC-MS分析,鉴定出探针显著富集的靶蛋白。结合图4凝胶内荧光分析的结果可知,异鼠李素光亲和探针是发现异鼠李素靶蛋白的有力工具。
上述对实施例的描述是为便于该技术领域的普通技术人员能理解和使用发明。熟悉本领域技术的人员显然可以容易地对这些实施例做出各种修改,并把在此说明的一般原理应用到其他实施例中而不必经过创造性的劳动。因此,本发明不限于上述实施例,本领域技术人员根据本发明的揭示,不脱离本发明范畴所做出的改进和修改都应该在本发明的保护范围之内。
Claims (8)
1.一种用于细胞内原位靶点钓取的异鼠李素光亲和探针,其特征在于,该探针以异鼠李素为活性基团,双吖丙啶为光交联基团,炔烃为生物正交基团,具有如下所示结构:
。
2.一种如权利要求1所述的异鼠李素光亲和探针的合成方法,其特征在于,包括以下步骤:
S1:将槲皮素加入无水二氯甲烷和DMF混合溶液中搅拌溶解,在冰浴下加入N,N-二异丙基乙胺和氯甲基甲基醚,搅拌0.5~1.5 h,然后恢复至室温反应16~18 h,反应完成后,旋蒸除去溶剂、萃取、干燥、柱层析纯化得到中间体1,中间体1的结构式如下所示:
;
S2:将步骤S1得到的中间体1和K2CO3加入无水DMF溶液中搅拌溶解,向溶液中加入光交联基团 (3-(3-炔-1-丁基)-3-(2-碘乙基)-3H-双吖丙啶),并在62~68 ℃加热回流反应48~96 h,旋蒸除去溶剂、萃取、干燥、柱层析纯化得到中间体2,中间体2的结构式如下所示:
;
S3:将步骤S2所得中间体2加入丙酮溶液中搅拌溶解,在冰浴下向溶液中加入 2.5~3.5N HCl,搅拌0.5~1.5 h,然后恢复至室温反应5~7 d;反应结束后加入水淬灭反应,萃取、洗涤、干燥,旋干溶剂,柱层析纯化得到化合物3,化合物3的结构式如下所示:
;
化合物3即为所述的异鼠李素光亲和探针。
3.根据权利要求2所述的一种异鼠李素光亲和探针的合成方法,其特征在于,步骤S1中,包括以下条件中的任一项或多项:
(i)槲皮素、N,N-二异丙基乙胺和氯甲基甲基醚的摩尔之比为1 : (3~4) : 8 ;
(ii)槲皮素与无水二氯甲烷和DMF混合溶液二者总量的质量:体积之比为1 g : (200~300) mL;
(iii)无水二氯甲烷和DMF混合溶液中,无水二氯甲烷和DMF的体积之比为(20~15):1。
4.根据权利要求2所述的一种异鼠李素光亲和探针的合成方法,其特征在于,步骤S2中,包括以下条件中的任一项或多项:
(i)中间体1、K2CO3和光交联基团(3-(3-炔-1-丁基)-3-(2-碘乙基)-3H-双吖丙啶)的摩尔之比为1: (1.2~1.3) : 2 ;
(ii)中间体1与无水DMF溶液的用量之比为10 mg: (0.5~1) mL。
5.根据权利要求2所述的一种异鼠李素光亲和探针的合成方法,其特征在于,步骤S3中,包括以下条件中的任一项或多项:
(i)丙酮与HCl的体积之比为(5~7) :1;
(ii)中间体2与丙酮的用量之比为10 mg: (0.5~1) mL。
6.一种如权利要求1所述的异鼠李素光亲和探针在非疾病诊断和治疗目的的癌细胞原位鉴定异鼠李素直接作用靶蛋白中的应用。
7.根据权利要求6所述的异鼠李素光亲和探针的应用,其特征在于,所述的靶蛋白与探针共价结合,通过紫外照射,实现探针中的双吖丙啶基团与包括癌细胞增殖、侵袭在内的生物过程相关蛋白的共价交联。
8.根据权利要求6或7所述的异鼠李素光亲和探针的应用,其特征在于,所述的癌细胞包括乳腺癌细胞MDA-MB-231、结肠癌细胞HCT-116或前列腺癌细胞PC-3。
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