CN113234014B - 一种检测氨肽酶n的聚集诱导发射荧光探针及其制备 - Google Patents
一种检测氨肽酶n的聚集诱导发射荧光探针及其制备 Download PDFInfo
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- C07D215/02—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom
- C07D215/12—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom with substituted hydrocarbon radicals attached to ring carbon atoms
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Abstract
本发明公开了一种检测氨肽酶N的聚集诱导发射荧光探针及其制备方法,所述的探针化合物结构如式Ⅰ所示。该探针分子由三个主要部分组成:基于喹啉‑丙二腈的聚集诱导发射荧光团,可自我裂解的连接基团和氨肽酶N(APN)特异性识别基团。当探针化合物N‑末端丙氨酰位点被APN准确水解为氨基后,暴露出的氨基作为供电子基团,促进了共轭体系的电子推拉效应,从而增强了荧光。优势在于,该探针响应速度快,灵敏度高,斯托克位移大,发射波长较长,当溶液浓度越高时,荧光发射越强,有效避免了聚集诱导荧光猝灭的问题。
Description
技术领域
本发明涉及小分子荧光探针原位检测活性酶在细胞中的位置和表达水平,更具体地说是一种基于聚集诱导发射的荧光探针对内源性氨肽酶N含量的精确检测,属于荧光探针技术领域。
背景技术
氨基肽酶N(APN/CD13),称为膜结合锌离子依赖性II型金属蛋白酶,可以降解蛋白多肽链N端的中性或碱性氨基酸。APN相应地参与了体内许多重要的生理和病理过程,例如信号转导,免疫调节,生物活性肽的水解以及冠状病毒的入侵过程。许多研究表明,与正常细胞相比,APN在大多数肿瘤细胞的表面高度表达,同时APN在恶性肿瘤的生长,侵袭,转移和血管生成中起着至关重要的作用。因此,APN具有成为肿瘤细胞生物标志物的潜力,开发一种可追踪体内APN活性的有效方法具有重大医学意义。
近年来,小分子荧光成像技术引起了人们极大的兴趣,荧光成像在快速检测、高选择性、高灵敏度、出色的时间、空间分辨率和良好的生物相容性等方面具有明显的优势。到目前为止,已经报道了一些荧光探针来监测和可视化细胞中的APN。然而,目前常规的荧光探针大多都具有平面结构的疏水特征。此类探针在浓溶液或聚集状态下会出现聚集引起的荧光猝灭问题(ACQ),这导致它们的发射减弱甚至猝灭。与普通的ACQ染料相反,聚集诱导发射(AIE)荧光染料具有可旋转的结构或扭曲的螺旋桨形构象,由于分子内运动的限制,这些染料可以产生高荧光发射。特别是,当溶液浓度越高时,它们的荧光发射越强。因此,AIE荧光团是检测生物体中APN的理想候选物。此外,发射波长较长(>600纳米)的探针具有很小的光损伤和更深组织穿透能力,可以最大限度地减少背景荧光所产生的不必要影响。然而,据我们所知,一种可激活的长发射波长检测APN的基于聚集诱导发射荧光探针几乎没有报道。
本发明公开了一种检测氨肽酶N的聚集诱导发射荧光探针及其制备方法,所述的探针化合物结构如式Ⅰ所示。该探针分子由三个主要部分组成:基于喹啉-丙二腈的聚集诱导发射荧光团,可自我裂解的连接基团和氨肽酶N(APN)特异性识别基团。当探针化合物N-末端丙氨酰位点被APN准确水解为氨基后,暴露出的氨基作为供电子基团,促进了共轭体系的电子推拉效应,从而增强了荧光。优势在于,该探针响应速度快,灵敏度高,斯托克位移大,发射波长较长,当溶液浓度越高时,荧光发射越强,有效避免了聚集诱导荧光猝灭的问题。
发明内容
本发明要解决的技术问题:一是提供一种检测氨肽酶N的聚集诱导发射荧光探针及其制备方法,所述探针化合物具有对癌细胞和组织内源性APN进行聚集诱导荧光原位成像的优点。二是提供一种荧光诊断试剂对过表达APN的肝癌细胞和正常细胞进行有效,准确的区分。三是提供一种灵敏度高、选择性好的荧光探针,可以改善探针组织穿透深度浅、背景荧光高的缺陷。
为了解决上述技术问题,采取的技术方案如下:
本发明提供一种检测氨肽酶N的聚集诱导发射荧光探针,具有如下分子结构式:
化合物MQ-N
本发明还提供一种检测氨肽酶N的聚集诱导发射荧光探针的制备方法,步骤包括:
(1)(a)在N2气氛下将2-甲基喹啉(1 eq)和碘甲烷(2.5-3 eq)溶解在乙腈中,并将反应液回流10-15小时,减压浓缩得到化合物1;(b)将化合物1(1 eq)和丙二腈(2.5-3 eq)溶于无水乙醇中,在0°C下不断搅拌,加入乙醇钠后反应5-8小时,将混合物倒入冰水中,调节pH(7.5-8.5)后,将产生的沉淀物过滤,得到化合物2;(c)将化合物2(1 eq)和对羟基苯甲醛(1.2-1.8 eq)溶解在的乙腈中,然后将哌嗪加入到该溶液中,将混合物在N2气氛下回流8-12小时,柱纯化(二氯甲烷/石油醚)得到化合物3;
(2)(d)在0°C下,将Boc-L-丙氨酸(1 eq),HATU(1-1.2 eq)和DIPEA(2-3 eq)溶于无水二氯甲烷溶液中,在室温下搅拌0.5-1.5小时,在反应混合物中加入对氨基苯甲醇(1-1.2 eq),并在室温下进一步搅拌24-30小时,柱纯化(二氯甲烷/甲醇)得到化合物4;(e)将化合物4(1 eq)溶解于无水四氢呋喃中,并在0°C下加入PBr3(1.2-1.6 eq),将混合物搅拌2-5小时,柱纯化(石油醚/乙酸乙酯)得到化合物5;
(3)(f)将化合物3(1 eq)和化合物5(1.1-1.2 eq)溶解无水DMF溶液中,然后加入K2CO3,并在120°C下搅拌6-8小时,柱纯化(石油醚/乙酸乙酯)得到化合物MQ-N。
本发明的优点:
本发明的荧光探针分子,具有聚集诱导荧光发射的性质,可以有效降低背景荧光对检测信号的干扰,提高活体成像中的组织穿透深度。
本发明的荧光探针分子,对氨肽酶N的响应速度非常快,在20分钟内即可完全响应,可以应用于快速检测复杂样品中的酶含量。
本发明的荧光探针分子,具有良好的灵敏度和选择性,荧光信号仅在氨肽酶N存在的条件下发生,其它常见的无机盐、氨基酸、生物酶等均无法使探针溶液产生荧光光谱的变化。
因此,本发明为非侵入性监控活体内亮氨酸氨肽酶活性的变化提供了一种可靠的手段。在生物分析检测领域具有广阔的应用前景。
具体实施方式
下面结合具体实施例对本发明做进一步的详细说明。
实施例1
一种检测氨肽酶N的聚集诱导发射荧光探针的制备方法,步骤包括:
1)化合物1的合成:
在N2气氛下将2-甲基喹啉(1.79 g,12.5 mmol)和碘甲烷(5.50 g,35 mmol)溶解在乙腈(25 mL)中,并将反应液回流10小时,减压蒸发除溶剂,得到化合物1,产率75%。
化合物2的合成:
将化合物1(1.74 g,6 mmol)和丙二腈(1.20 g,18 mmol)溶于无水乙醇(15 mL)中,在0°C下不断搅拌,加入乙醇钠后反应6小时,将混合物倒入冰水中,调节pH为8后,将产生的沉淀物过滤,用水洗涤并真空干燥,得到化合物2,产率85%。
化合物3的合成:
将化合物2(1.40 g,7.5 mmol)和对羟基苯甲醛(1.65 g,13.5 mmol)溶解在的乙腈(30 mL)中,然后将哌嗪加入到该溶液中,将混合物在N2气氛下回流12小时,减压蒸发除溶剂,柱纯化(二氯甲烷/石油醚=2/1)得到化合物3,产率65%。
2)化合物4的合成:
在0°C下,将Boc-L-丙氨酸(0.17 g,0.9 mmol),HATU(0.42 g,1.1 mmol)和DIPEA(0.37 mL,2.3 mmol)溶于无水二氯甲烷溶液(30 mL)中,在室温下搅拌1.2小时,在反应混合物中加入对氨基苯甲醇(0.14 g,1.1 mmol),并在室温下进一步搅拌25小时,减压蒸发除溶剂,柱纯化(二氯甲烷/甲醇=20/1)得到化合物4,产率70%。
化合物5的合成:
(e)将化合物4(0.59 g,2 mmol)溶解于无水四氢呋喃(10 mL)中,并在0°C下加入PBr3(0.23 mL,2.4 mmol),将混合物搅拌3小时,减压蒸发除溶剂,柱纯化(石油醚/乙酸乙酯=9/1)得到化合物5,产率40%。
3)化合物MQ-N的合成:
将化合物3(0.37 g,1.1 mmol)和化合物5(0.43 g,1.2 mmol)溶解无水DMF溶液(5mL)中,然后加入K2CO3,并在120°C下搅拌7小时,减压蒸发除溶剂,柱纯化(石油醚/乙酸乙酯=10/1)得到化合物MQ-N,产率65%。
实施例2
探针MQ-N吸收光谱和荧光光谱的测量
取实施例1合成的MQ-N于试管中,制备探针MQ-N在DMSO中的储备液(1.0 mM)。在蒸馏水中制备NaCl,KCl,CaCl2,ZnCl2,MgCl2,CuCl2,H2O2,NaClO,葡萄糖,GSH,Cys,Hcy,碱性磷酸酶,γ-谷氨酰转肽酶,硝基还原酶,羧酸酯酶等原液。以上储备液均用磷酸盐缓冲液将最终体积调节至5 mL。对于光谱实验,将MQ-N储备溶液与一定量的分析物在pH=7.4下在石英比色皿中孵育30分钟后,取3 mL反应溶液转移至1 cm石英皿中,将激发设置为480 nm,发射波长为500-700 nm。同时,制备没有氨肽酶N的对照组,并在相同条件下进行比较。MQ-N在350 nm和425 nm附近显示出双吸收峰,向胶束探针中添加APN后,以350 nm为中心的吸收峰减弱,随后在425 nm处的峰显著增加。然后评估探针MQ-N在不同浓度的APN处理下的特性,随着APN的增加,荧光强度在630 nm处逐渐增加。实验数据表明,MQ-N对APN具有相当高的灵敏度,并且可以定量检测APN浓度。
实施例3
评估不同pH值、温度对探针MQ-N荧光的影响
取实施例2中MQ-N储备溶液(10 μM),探针在pH范围为4.0-8.0和温度范围为23-40°C时几乎是无荧光的,这表明探针具有很高的稳定性。与氨肽酶N反应后,尽管最大荧光强度出现在6.0-7.4的pH范围内,但探针在整个测试的pH范围中都产生了显着的荧光响应。另一方面,反应溶液的最强荧光出现在约37°C时,这与酶通常在37°C下具有最大活性的事实相符。结果表明探针MQ-N在正常生理条件下具有良好的功能。
实施例4
探针MQ-N对APN的特异性检测
取实施例2中MQ-N储备溶液(10 μM),测试了探针对APN的荧光响应以及一些潜在的干扰物,例如一些可能与探针或APN形成复合物的金属离子(Na+,K+,Ca2+,Zn2+,Mg2+和Cu2 +),可能会氧化探针的活性氧(NO2 -,NO,HNO,1O2,H2O2,ClO-,TBHP,·OH和O2 -),生物分子(GSH,Hcy和Cys)和APN相关的酶(碱性磷酸酶,γ-谷氨酰转肽酶,硝化还原酶,羧酸酯酶,β-半乳糖苷酶等)。当APN加入MQ-N溶液中后,在630 nm处的荧光强度明显增强,但是其他物质未能引起明显的荧光变化。随后对APN和其他干扰物质进行进一步的竞争性实验,结果表明APN诱导的荧光响应不受其他干扰的影响。因此,探针MQ-N在各种竞争性分析物中对APN具有优越的选择性。
实施例5
MQ-N对癌细胞的毒性及内源性APN的测定
通过使用线粒体脱氢酶将MTT还原的方法来测试细胞活力。将HepG-2细胞以1×105细胞/ mL的密度接种在96孔微孔板中,其中含有LBS的培养基。细胞附着24小时后,然后将板用PBS洗涤,将细胞在含有0、1、2、5和10 μM探针MQ-N的培养基中培养24小时。同时,设置未与探针MQ-N培养的细胞用作对照。将MTT(10 μL,5 mg/mL)注入每个孔中,并将板在5%CO2湿润的培养箱中孵育4小时。然后除去培养基,利用酶标仪于490 nm下测定溶液的光密度,评估细胞活力。当用不同浓度MQ-N处理细胞24小时后,仍有90%以上的细胞存活,证明了探针对细胞具有低毒性。随后,通过共聚焦荧光显微镜评估了MQ-N在追踪癌细胞和正常细胞中内源性APN能力。在MQ-N(5μM)和37°C的5%CO2中对细胞进行0.5小时预处理,正常细胞中几乎没有荧光。相比之下,在探针与癌细胞孵育后,红色荧光信号急剧增加。因此,这些数据表明,探针MQ-N表现出良好的膜通透性,可以有效区分癌细胞和正常细胞。
上述仅为本发明的优选具体实施方式,并非是对本发明作其它形式的限制,任何熟悉本专业的技术人员可能利用上述揭示的技术内容加以变更或改型为等同变化的等效实施例。但本发明的设计构思并不局限于此,凡利用此构思对本发明进行非实质性的改动,均应属于侵犯本发明保护范围的行为。
Claims (4)
1.一种检测氨肽酶N的聚集诱导发射荧光探针的制备方法,其特征在于,所述化合物结构如式Ⅰ所示:
式Ⅰ;
该方法的制备步骤如下:
(1)(a)在N2气氛下将2-甲基喹啉和碘甲烷溶解在乙腈中,并将反应液回流10-15小时,减压浓缩得到化合物1;(b)将化合物1和丙二腈溶于无水乙醇中,在0°C下不断搅拌,加入乙醇钠后反应5-8小时,将混合物倒入冰水中,调节pH后,将产生的沉淀物过滤,得到化合物2;(c)将化合物2和对羟基苯甲醛溶解在的乙腈中,然后将哌嗪加入到该溶液中,将混合物在N2气氛下回流8-12小时,柱纯化得到化合物3;
(2)(d)在0°C下,将Boc-L-丙氨酸,HATU和DIPEA溶于无水二氯甲烷溶液中,在室温下搅拌0.5-1.5小时,在反应混合物中加入对氨基苯甲醇,并在室温下进一步搅拌24-30小时,柱纯化得到化合物4;(e)将化合物4溶解于无水四氢呋喃中,并在0°C下加入PBr3,将混合物搅拌2-5小时,柱纯化得到化合物5;
(3)(f)将化合物3和化合物5溶解无水DMF溶液中,然后加入K2CO3,并在120°C下搅拌6-8小时,柱纯化得到化合物MQ-N
2.根据权利要求1所述的一种检测氨肽酶N的聚集诱导发射荧光探针的制备方法,其特征在于:所述的步骤(1)(a)中2-甲基喹啉和碘甲烷摩尔比范围为1:(2.5~3);2-甲基喹啉溶于乙腈溶液的摩尔浓度范围为0.35~0.55 mol·L-1;(b)中化合物1和丙二腈摩尔比范围为1:(2.5~3);化合物1溶于乙醇溶液的摩尔浓度范围为0.3~0.45 mol·L-1;调节pH范围为7.5~8.5;(c)中化合物2和对羟基苯甲醛摩尔比范围为1:(1.2~1.8);化合物2溶于乙腈溶液的摩尔浓度范围为0.2~0.35 mol·L-1;柱纯化中二氯甲烷和石油醚体积比为(2~1):1。
3. 根据权利要求1所述的一种检测氨肽酶N的聚集诱导发射荧光探针的制备方法,其特征在于:所述的步骤(2)(d)中Boc-L-丙氨酸,对氨基苯甲醇,HATU和DIPEA摩尔比范围为1:(1~1.2):(1~1.2):(2~3);Boc-L-丙氨酸溶于无水二氯甲烷溶液的摩尔浓度范围为0.025~0.035 mol·L-1;柱纯化中二氯甲烷和甲醇体积比为(20~15):1;(e)中化合物4和PBr3摩尔比范围为1:(1.2~1.6);化合物4溶于无水四氢呋喃溶液摩尔浓度范围为0.2~0.26 mol·L-1;柱纯化中石油醚和乙酸乙酯体积比为(10~5):1。
4. 根据权利要求1所述的一种检测氨肽酶N的聚集诱导发射荧光探针的制备方法,其特征在于:所述的步骤(3)(f)中化合物3和化合物5摩尔比范围为1:(1.1~1.2);化合物3溶于无水DMF溶液摩尔浓度范围为0.2~0.24 mol·L-1;柱纯化中石油醚和乙酸乙酯体积比为(10~6):1。
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CN110330505B (zh) * | 2019-07-30 | 2021-07-09 | 济南大学 | 一种用于氨肽酶n检测的双光子比率型荧光探针化合物及其制备方法 |
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