CN110483398A - 一种含有生物可降解基团的光亲和链接体及制备方法和应用 - Google Patents
一种含有生物可降解基团的光亲和链接体及制备方法和应用 Download PDFInfo
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Abstract
一种含有生物可降解基团的光亲和链接体及制备方法和应用,带Boc保护基氨基的光亲和链接体的中间产物在三氟乙酸条件下脱除保护基,得到含有氨基末端的光亲和链接体;将含有氨基末端的光亲和链接体与亚二硫基二乙酸在EDC·HCl的缩合作用下,得到含有生物可降解基团的光亲和链接体。本发明中的生物可降解光亲和链接体分子可以对生物大分子进行化学共价修饰构建探针分子,进而通过传统的光亲和标记技术对目标分子进行标记,后通过触发可释放基团释放体积较大的诱饵分子,从而达到对目标分子的准确标记示踪。
Description
技术领域
本发明涉及一种含有生物可降解基团的光亲和链接体及制备方法和应用。
背景技术
光亲和标记技术(PAL)是结合现代分子生物学、细胞生物学、药物化学、分析化学等多学科的优势,运用合成的光亲和探针分子,在特定波长光的照射下产生高活性的中间体,其可以直接与药物分子特异性结合的蛋白质进行不可逆共价交联从而实现对药物靶标蛋白分子的捕获。它是分子水平上研究配体与受体相互作用的核心工具之一,对阐明配体与受体相互作用机理以及药物新靶点的发现都有着巨大的推动作用。
近些年来这项技术主要应用于活性小分子靶标蛋白的示踪及确证,其主要基于光交联技术、生物正交技术以及相关的生物分析技术等,实现药物分子靶标的捕获及其示踪。其中光亲和探针分子的设计合成是在不影响分子活性的基础上直接进行化学修饰,在其非活性部位上引入光亲和链接体设计合成光亲合探针分子,其在特定波长光的照射下特异性不可逆的共价捕获与其相互作用的靶标蛋白。再接下来通过生物正交反应来实现所捕获的靶标蛋白的示踪及鉴定。通过对光亲和标记技术工作原理的研究发现,发现该技术也可用于研究生物大分子间的相互作用。众所周知,生物大分子的相互作用在细胞生物信号传导等方面占有重要地位。基于此,选择在生物大分子上引入光亲和链接体设计合成探针分子,利用光交联技术研究与生物大分子发生相互作用的生物分子。但是,当该技术直接用于研究生物大分子相互作用时,存在着一些问题,例如,诱饵分子体积较大,对目标分子地示踪产生干扰;对于体内丰度较低的分子分析能力差,灵敏度低。这些不足均对所捕获的猎物分子的鉴定和分离造成影响。因此,需要设计合成一种可释放性的光亲和链接体。当诱饵蛋白捕获与其相互作用的生物分子后,在一定的条件触发可释放的链接体,释放诱饵大分子,以减少其对猎物分子研究的干扰。
发明内容
本发明的目的在于提供一种含有生物可降解基团的光亲和链接体及制备方法和应用,生物可降解光亲和链接体分子用于化学修饰生物大分子,并且可以用于验证含有生物可降解基团二硫键的光亲和链接体所构建的探针进行生物标记示踪的可行性。
为达到上述目的,本发明采用以下技术方案:
一种含有生物可降解基团的光亲和链接体,其结构式如下:
一种含有生物可降解基团的光亲和链接体的制备方法,包括以下步骤:
a)4-[3-(三氟甲基)-3H-双吖丙啶-3-基]苄胺盐酸盐与2-(叔丁基氧基)-4-戊炔酸在EDC·HCl的缩合下,得到带Boc保护基氨基的光亲和链接体的中间产物;
b)带Boc保护基氨基的光亲和链接体的中间产物在三氟乙酸条件下脱除保护基,得到含有氨基末端的光亲和链接体;
c)将含有氨基末端的光亲和链接体与亚二硫基二乙酸在EDC·HCl的缩合作用下,得到含有生物可降解基团的光亲和链接体,结构式如下:
本发明进一步的改进在于,步骤a)的具体过程如下:将2-(叔丁基氧基)-4-戊炔酸、EDC·HCl、HOBT溶于无水二氯甲烷中,0℃下搅拌均匀后,滴加DIPEA,反应2h后,加入4-[3-(三氟甲基)-3H-双吖丙啶-3-基]苄胺盐酸盐进行反应,得到带有Boc保护基的光亲和链接体中间产物。
本发明进一步的改进在于,所述步骤a)的具体过程为:将2-(叔丁基氧基)-4-戊炔酸0.4768mmol、EDC·HCl 0.7151mmol、HOBT 0.5721mmol溶于无水二氯甲烷中,0℃下搅拌均匀后,滴加DIPEA 3.9730mmol,反应2h后,加入4-[3-(三氟甲基)-3H-双吖丙啶-3-基]苄胺盐酸盐0.3973mmol进行反应24h反应,得到带有Boc保护基的光亲和链接体中间产物。
本发明进一步的改进在于,步骤b)的具体过程为:将步骤a)得到的带有Boc保护基的光亲和链接体中间产物溶于无水二氯甲烷溶液中,在0℃搅拌1h后滴加三氟乙酸,0℃下反应1h,室温下搅拌24h后,进行处理,得到含有氨基末端的光亲和链接体。
本发明进一步的改进在于,所述步骤b)的具体过程为:将步骤a)得到的带有Boc保护基的光亲和链接体中间产物0.3168mmol溶于无水二氯甲烷溶液中,在0℃搅拌1h后滴加三氟乙酸2mL,0℃下反应1h,室温下搅拌24h后,进行处理,得到含有氨基末端的光亲和链接体。
本发明进一步的改进在于,步骤c)的具体过程为:将亚二硫基二乙酸、EDC·HCl、NHS溶于无水二氯甲烷中,0℃搅拌1h后滴加三乙胺,反应2h后加入含有氨基末端的光亲和链接体,室温反应24h,经处理后,得到含有生物可降解基团二硫键的光亲和链接体。
本发明进一步的改进在于,所述步骤c)的具体过程为,将亚二硫基二乙酸0.3483mmol、EDC·HCl 0.4180mmol、NHS 0.4180mmol溶于3mL无水二氯甲烷中,0℃搅拌1h后滴加三乙胺0.4180mmol,反应2h后加入含有氨基末端的光亲和链接体,室温反应24h,经处理后,得到含有生物可降解基团二硫键的光亲和链接体。
一种基于含有生物可降解基团的光亲和链接体在光亲和标记技术方面的应用。
一种含有生物可降解基团的光亲和链接体在细胞原位荧光示踪方面的应用。
与现有技术相比,本发明具有以下有益效果:
本发明通过将光亲和基团三氟甲基取代的双吖丙啶、炔基生物正交手柄以及生物可降解基团二硫键连接,获得生物可降解光亲和链接体分子。该链接体分子能够化学修饰生物大分子进而构建生物大分子光亲和探针,接下来探针分子中的生物正交手柄与含有荧光素或者生物素的另一生物正交手柄进行点击反应以示踪及确证相互作用分子。本发明的生物可降解光亲和链接体分子制备方法简单,易于实现,并且收率较高。
本发明中的生物可降解光亲和链接体分子可以对生物大分子进行化学共价修饰构建探针分子,进而通过传统的光亲和标记技术对目标分子进行标记,后通过触发可释放基团释放体积较大的诱饵分子,从而达到对目标分子的准确标记示踪。利用该链接体所构建的探针分子可以改善传统光亲和标记技术研究生物大分子间相互作用的缺点。原有的标记技术不能有效的释放诱饵分子,存在的诱饵大分子会由于其较大的分子体积对所标记的分子造成干扰,其次,由于体积较大,其对生物体中丰度较低的大分子难以标记,即其灵敏度低。而通过可降解光亲和链接体所构建的探针分子在进行特异性共价捕获后,可以通过触发可释放基团释放体积较大的诱饵分子实现生物正交基团的转移,以改善传统光亲和标记技术应用于研究大分子相互作用的不足。本发明的生物可降解光亲和链接体能够用于构建生物大分子探针以及验证该探针进行靶标示踪的可行性。
附图说明
图1为本发明提供的含有生物可降解基团二硫键的光亲和链接体的合成路线图;
其中,化合物1为4-[3-(三氟甲基)-3H-双吖丙啶-3-基]苄胺盐酸盐,化合物2为2-(叔丁基氧基)-4-戊炔酸,化合物3为(1-氧代-1-(4-(3-(三氟甲基)-3H-二氮杂萘-3-基)苄基)氨基)戊-4-炔-2-基)氨基甲酸叔丁酯,化合物4为(1-氧代-1-(4-(3-(三氟甲基)-3H-双吖丙啶-3-基)苄基)氨基)戊-4-炔-2-基)氨基,化合物5为亚二硫基二乙酸,化合物6为2-(2-氧代-2-((1-氧代-1-((4-(3-(三氟甲基)-3H-双吖丙啶-3-基)苄基)氨基)戊-4-炔-2-基)氨基)乙基)二硫烷基)乙酸。
试剂与条件:(a)EDC·HCl,HOBT,DIPEA,DCM,0℃ to rt,24h;(b)TFA,DCM,0℃ tort,24h;(c)EDC·HCl,NHS,TEA,DCM,0℃ to rt,24h。
图2为细胞成像结果,其中,(a)为未加入还原剂,(b)为加入还原剂释放VEGF分子。
具体实施方式
下面结合附图和具体的实施例对本发明做进一步的详细说明,所述是对本发明的解释而不是限定。
本发明通过使用4-[3-(三氟甲基)-3H-双吖丙啶-3-基]苄胺盐酸盐、2-(叔丁基氧基)-4-戊炔酸、亚二硫基二乙酸等设计合成生物可降解光亲和链接体分子,该链接体可以用于化学修饰生物大分子以构建生物大分子光亲合探针。
本发明提供的具有生物可降解二硫键的光亲和链接体,其结构如下:
本发明所述的具有生物可降解基团二硫键的光亲和链接体名称如下:
2-(2-氧代-2-((1-氧代-1-((4-(3-(三氟甲基)-3H-双吖丙啶-3-基)苄基)氨基)戊-4-炔-2-基)氨基)乙基)二硫烷基)乙酸。
下面结合图1中所示的合成路线和具体的合成实施例来详细说明本发明提供的具有构建生物大分子光亲合探针的光亲和链接体的制备。
参见图1,一种含有生物可降解基团二硫键的光亲和链接体分子的合成路线,包括以下步骤:
a)2-(叔丁基氧基)-4-戊炔酸、[3-(三氟甲基)-3H-双吖丙啶-3-基]苄胺盐酸盐在EDC·HCl缩合作用下生成含有Boc保护基的中间产物。具体操作为:
将2-(叔丁基氧基)-4-戊炔酸EDC·HCl、HOBT(羟基苯并三唑)溶于无水二氯甲烷中,0℃下搅拌一段时间待体系冷却后,缓慢滴加DIPEA,反应2h后,加入4-[3-(三氟甲基)-3H-双吖丙啶-3-基]苄胺盐酸盐进行反应,得到带有Boc保护基的光亲和链接体中间产物,经饱和碳酸氢钠洗涤三次、饱和氯化钠洗涤、无水硫酸钠干燥有机相,柱色谱分离即得;
b)将带有Boc保护基的中间产物在三氟乙酸的作用下脱除保护基,得到含有氨基末端的光亲和链接体中间体;具体操作为:
将带有Boc保护基的光亲和链接体中间产物溶于无水二氯甲烷中,加在0℃搅拌1h后缓慢滴加三氟乙酸,0℃下反应1h,室温下搅拌24h后,经饱和碳酸氢钠中和体系至中性,萃取后,经饱和氯化钠、无水硫酸钠干燥有机相,旋干即得带有氨基末端的光亲和链接体;
c)将含有氨基末端的光亲和链接体与亚二硫基二乙酸在EDC·HCl缩合剂的作用下生成含有生物可降解基团二硫键的光亲和链接体。具体操作为:
将亚二硫基二乙酸、EDC·HCl、NHS(N-羟基琥珀酰亚胺)溶于无水二氯甲烷中,0℃搅拌1h待体系冷却后缓慢滴加三乙胺,反应2h后加入含有氨基末端的光亲和链接体,室温反应24h,经饱和碳酸氢钠洗涤三次、饱和氯化钠洗涤、无水硫酸钠干燥有机相,柱色谱分离。
上述含有生物可降解基团二硫键的光亲和链接体在构建生物大分子光亲合探针方面的应用。
实施例1
参见图1,该含有生物可降解集团二硫键的光亲和链接体中的制备过程如下:
将2-(叔丁基氧基)-4-戊炔酸、[3-(三氟甲基)-3H-双吖丙啶-3-基]苄胺盐酸盐在EDC·HCl缩合作用下生成含有Boc保护基的中间产物,具体过程如下:
将2-(叔丁基氧基)-4-戊炔酸(0.102g,0.4768mmol),EDC·HCl(0.137g,0.7151mmol、HOBT(0.077g,0.5721mmol)于3mL无水二氯甲烷中,0℃下搅拌一段时间待体系冷却后,缓慢滴加DIPEA(0.5135g.3.9730mmol),反应2h后,加入4-[3-(三氟甲基)-3H-双吖丙啶-3-基]苄胺盐酸盐(0.1g,0.3973mmol)进行反应24h,用二氯甲烷萃取,萃取的有机相经饱和食盐水洗涤、无水硫酸钠干燥后减压蒸去溶剂,得粗品,用层析柱分离纯化粗品,使用石油醚/乙酸乙酯(V/V=10/1)洗脱得到目标化合物,重0.13g,收率79.7%,到带有Boc保护基的光亲和链接体中间体;
将带有Boc保护基的光亲和链接体中间产物溶于无水二氯甲烷溶液中,加在0℃搅拌1h后缓慢滴加三氟乙酸,0℃下反应1h,室温下搅拌24h后,进行处理,得到含有氨基末端的光亲和链接体分子,其具体实施过程如下:
将带有Boc保护基的光亲和链接体中间产物(0.13g,0.3168mmol)溶于无水二氯甲烷中,加在0℃搅拌1h后缓慢滴加三氟乙酸2mL,0℃下反应1h,室温下搅拌24h后,采用饱和碳酸氢钠调节至中性,二氯甲烷萃取,分别经饱和氯化钠、无水硫酸钠干燥有机相,减压旋干,重0.09g,收率91.6%,得到带有即得含有氨基末端的光亲和链接体中间体。
LC-MS(ESI,m/z):311.10[M+H]+,309.15[M-H]--。
将亚二硫基二乙酸、EDC·HCl、NHS溶于无水二氯甲烷中,0℃搅拌1h待体系冷却后缓慢滴加三乙胺,反应2h后加入含有氨基末端的光亲和链接体,室温反应24h,经饱和碳酸氢钠洗涤三次、饱和氯化钠洗涤、无水硫酸钠干燥有机相,柱色谱分离,其具体实施过程如下:
将亚二硫基二乙酸(0.08g,0.3484mmol),EDC·HCl(0.0760g,0.4180mmol)、NHS(0.0481g,0.4180mmol)溶于3mL无水二氯甲烷中,0℃搅拌1h待体系冷却后缓慢滴加三乙胺(0.0565g,0.4180mmol),反应2h后加入含有氨基末端的光亲和链接体(0.09g,0.2903mmol),室温反应24h,经饱和碳酸氢钠洗涤三次、饱和氯化钠洗涤、无水硫酸钠干燥有机相,减压蒸去溶剂,得粗品,用层析柱分离纯化粗品,使用石油醚/乙酸乙酯(V/V=1/1)洗脱得到目标化合物,重0.07g,收率50.9%,得到含有生物可降解基团二硫键的光亲和链接体分子。
LC-MS(ESI,m/z):475.35[M+H]+,473.30[M-H]--。
含有生物可降解基团二硫键的光亲和链接体在光亲和标记技术方面的应用,具体如下:
利用该可释放光亲和链接体化学修饰VEGF细胞因子,构建了VEGF光亲和探针分子,并且利用所构建的VEGF光亲合探针分子对一些高表达VEGFR受体的细胞进行示踪定位成像分析。
VEGF细胞因子光亲合探针的构建:(1)配制I液:VEGF细胞因子溶于1mL三蒸水中,4℃保存2h;(2)配制II液:将光亲和链接体、EDC、NHS溶于1mL三蒸水中。4℃活化2h;将II液缓慢加入I液中,4℃反应24h后,4℃下透化24h,冷冻干燥,即得VEGF光亲和探针分子。
含有生物可降解基团二硫键的光亲和链接体在细胞原位荧光示踪方面的应用。
利用上述设计合成的VEGF光亲合探针分子对EA.hy926细胞进行成像分子。
具体细胞成像步骤:
1.细胞种板:每孔细胞密度为4.3*105细胞/ml,37℃孵育过夜
2.细胞给药:每孔探针浓度为1ug/ml,37℃孵育2h;
3.洗涤:利用PBS洗涤三次细胞,除去过量探针分子;
4.光交联及细胞固定:洗涤完成后,将细胞置于距离紫外等3cm下的冰盘中紫外照射30min,光交联完成后,在室温下,在细胞中加入1ml 3.7%多聚甲醛(925ul 4%多聚甲醛+75ul PBS),固定30分钟,用PBS洗涤两次(轻微搅拌1-2分钟),并在室温下用含有0.1%Triton X-100的PBS溶液透化10分钟。然后用PBS将细胞洗涤两次,缓慢搅拌1分钟,在室温下,用含2%BSA的PBS(含0.05%Tween-20)封闭30分钟,并用PBS(含0.05%Tween-20)洗涤两次。每次5分钟,轻轻晃动。
5.点击成像:
5.1各孔中,分别按顺序添加以下试剂:82.5ul PBS,5ul Cy3-N3(0.8mmol DMSO溶液),5.8ul TCEP(170mmol水溶液,新鲜制备)和6.7ul TBTA(15mmol),震荡混合。
5.2加入10ul CuSO4·5H2O(0.1mol水溶液)开始反应。在室温下,避光孵育60分钟。
5.3向一组细胞中加入还原剂;另一组细胞中不加还原剂,室温下反应1h;
5.4 PBS洗涤细胞3次,每次5分钟。
6.PI复染
各孔中均加入10ul 1ug/ml PI,染色10min,PBS洗涤3次,20min/次
7.封片:在各组加入5ul抗荧光淬灭封片剂,周围加指甲油封片。
8.荧光显微镜进行检测
细胞成像结果如图2中的(a)和(b)所示:探针浓度为1μg/ml,探针与香豆素物质的量的比例为1:5。
从图2中可以看出,未加入还原剂时,细胞的状态比较差,加入还原剂后,释放外来引入的蛋白分子后,细胞的状态较好,说明本发明的引用可释放基团后对细胞进行成像分析时,对细胞正常生命活动影响较小。
Claims (10)
1.一种含有生物可降解基团的光亲和链接体,其特征在于,其结构式如下:
2.一种含有生物可降解基团的光亲和链接体的制备方法,其特征在于,包括以下步骤:
a)4-[3-(三氟甲基)-3H-双吖丙啶-3-基]苄胺盐酸盐与2-(叔丁基氧基)-4-戊炔酸在EDC·HCl的缩合下,得到带Boc保护基氨基的光亲和链接体的中间产物;
b)带Boc保护基氨基的光亲和链接体的中间产物在三氟乙酸条件下脱除保护基,得到含有氨基末端的光亲和链接体;
c)将含有氨基末端的光亲和链接体与亚二硫基二乙酸在EDC·HCl的缩合作用下,得到含有生物可降解基团的光亲和链接体,结构式如下:
3.根据权利要求2所述的一种含有生物可降解基团的光亲和链接体的制备方法,其特征在于:步骤a)的具体过程如下:将2-(叔丁基氧基)-4-戊炔酸、EDC·HCl、HOBT溶于无水二氯甲烷中,0℃下搅拌均匀后,滴加DIPEA,反应2h后,加入4-[3-(三氟甲基)-3H-双吖丙啶-3-基]苄胺盐酸盐进行反应,得到带有Boc保护基的光亲和链接体中间产物。
4.根据权利要求3所述的一种含有生物可降解基团的光亲和链接体的制备方法,其特征在于,所述步骤a)的具体过程为:将2-(叔丁基氧基)-4-戊炔酸0.4768mmol、EDC·HCl0.7151mmol、HOBT 0.5721mmol溶于无水二氯甲烷中,0℃下搅拌均匀后,滴加DIPEA3.9730mmol,反应2h后,加入4-[3-(三氟甲基)-3H-双吖丙啶-3-基]苄胺盐酸盐0.3973mmol进行反应24h反应,得到带有Boc保护基的光亲和链接体中间产物。
5.根据权利要求2所述的一种含有生物可降解基团的光亲和链接体的制备方法,其特征在于:步骤b)的具体过程为:将步骤a)得到的带有Boc保护基的光亲和链接体中间产物溶于无水二氯甲烷溶液中,在0℃搅拌1h后滴加三氟乙酸,0℃下反应1h,室温下搅拌24h后,进行处理,得到含有氨基末端的光亲和链接体。
6.根据权利要求5所述的一种含有生物可降解基团的光亲和链接体的制备方法,其特征在于,所述步骤b)的具体过程为:将步骤a)得到的带有Boc保护基的光亲和链接体中间产物0.3168mmol溶于无水二氯甲烷溶液中,在0℃搅拌1h后滴加三氟乙酸2mL,0℃下反应1h,室温下搅拌24h后,进行处理,得到含有氨基末端的光亲和链接体。
7.根据权利要求2所述的一种含有生物可降解基团的光亲和链接体的制备方法,其特征在于,步骤c)的具体过程为:将亚二硫基二乙酸、EDC·HCl、NHS溶于无水二氯甲烷中,0℃搅拌1h后滴加三乙胺,反应2h后加入含有氨基末端的光亲和链接体,室温反应24h,经处理后,得到含有生物可降解基团二硫键的光亲和链接体。
8.根据权利要求7所述的一种含有生物可降解基团的光亲和链接体的制备方法,其特征在于,所述步骤c)的具体过程为,将亚二硫基二乙酸0.3483mmol、EDC·HCl 0.4180mmol、NHS 0.4180mmol溶于3mL无水二氯甲烷中,0℃搅拌1h后滴加三乙胺0.4180mmol,反应2h后加入含有氨基末端的光亲和链接体,室温反应24h,经处理后,得到含有生物可降解基团二硫键的光亲和链接体。
9.一种如权利要求2-8中任意一项方法制备的基于含有生物可降解基团的光亲和链接体在光亲和标记技术方面的应用。
10.一种如权利要求2-8中任意一项方法制备的含有生物可降解基团的光亲和链接体在细胞原位荧光示踪方面的应用。
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