CN112159396A - 一种检测γ-谷氨酰转肽酶近红外荧光分子探针及其制备方法与应用 - Google Patents
一种检测γ-谷氨酰转肽酶近红外荧光分子探针及其制备方法与应用 Download PDFInfo
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Abstract
Description
技术领域
本发明属于荧光分子探针技术领域,具体涉及一种用于检测γ-谷氨酰转肽酶的近红外荧光分子探针及其制备方法和应用。
背景技术
癌症是严重危害人类健康的三大疾病之一。目前,晚期癌症仍然难以攻克。早期发现和治疗仍然是治疗癌症的最有效手段。因此,早期发现癌症对提高癌症患者的生存率具有重要意义。科学家们也做了大量的研究,发现了一些肿瘤标志物,包括γ-谷氨酰转肽酶。γ-谷氨酰转肽酶作为谷胱甘肽代谢的关键酶之一,广泛分布于人体各组织器官。γ-谷氨酰转肽酶在原发性和转移性肿瘤中的表达异常高。γ-谷氨酰转肽酶的高表达与患者恶性程度高、预后差有显著关系。近年来,人们认为血清和外分泌液中γ-谷氨酰转肽酶的高活性可增加多发性恶性肿瘤的发病风险,提示γ-谷氨酰转肽酶可能是肿瘤治疗的潜在靶点和肿瘤诊断及预后的标志物,对其进行精准检测非常重要。
传统检测γ-谷氨酰转肽酶的荧光探针多数都是短波长的,这种探针在与γ-谷氨酰转肽酶响应后产生的荧光很难透过活体呈现出来,并且存在很多自体荧光的干扰。但是如何得到性能更加优异的探针,对活体安全无害,且荧光强度较高,易于辨别,还需要进一步研究。
发明内容
发明目的:针对现有技术存在的问题,本发明提供了一种具有良好的选择性检测γ-谷氨酰转肽酶的近红外荧光分子探针,其能对γ-谷氨酰转肽酶进行快速荧光响应检测,并且还可以本着安全性原则将该探针运用在细胞和活体中γ-谷氨酰转肽酶检测。
本发明还提供了该荧光分子探针的制备方法和应用。
技术方案:为实现上述发明目的,本发明所述一种检测γ-谷氨酰转肽酶近红外荧光分子探针,其分子式为C36H38N3O4 +,结构式如下:
本发明所述的检测γ-谷氨酰转肽酶近红外荧光分子探针的制备方法,其特征在于,包括如下步骤:
(1)在惰性气体保护条件下,将化合物1加入到二氯甲烷中,搅拌,继续加入Boc-L-谷氨酸-1-叔丁酯,搅拌;
(2)在惰性气体保护条件下,继续加入N,N-二异丙基乙胺,室温条件下搅拌;
(3)步骤(2)得到的反应液加入三氟乙酸,室温下搅拌;
(4)对步骤(3)得到的反应液进行萃取,将所得水相冷冻干燥,所得固体即为近红外荧光分子探针;
其反应路线如下所示:
所述化合物1的合成参考文献:X.Yao,W.Liu,W.Zhu,A.Tiemuer,F.Zhou,S.Yang,et al.,Near-infrared fluorescent chemodosimeter for real-time in vivoevaluation of H2S-release efficiency of prodrug,Chem Commun(Camb),56(2020)8111-4.
作为优选,所述惰性气体选自氮气。
其中,步骤(1)所述化合物1与二氯甲烷的比例为1mmol:10-15mL。
其中,所述化合物1、Boc-L-谷氨酸-1-叔丁酯、N,N-二异丙基乙胺和三氟乙酸的比例为1mmol:8-10mmol:1.2-1.5mmol:15-20μL。
其中,步骤(3)所述搅拌反应直到溶液变深蓝色,通常搅拌24小时。
作为优选,步骤(4)所述萃取的方法为:反应液中加入蒸馏水,混匀,静置分液,取上层水层。
本发明所述的检测γ-谷氨酰转肽酶近红外荧光分子探针在选择性检测γ-谷氨酰转肽酶中的应用。
本发明所述的检测γ-谷氨酰转肽酶近红外荧光分子探针在检测溶液中、细胞中或者活体中γ-谷氨酰转肽酶中的应用。
本发明的荧光分子探针可以制备成用于检测溶液中、细胞中或者活体中γ-谷氨酰转肽酶中的工具或者试剂。
本发明荧光分子的结构为R-Glu。其中R基(化合物1)为近红外荧光团,能吸收近红外的光并发出波长更长的荧光。原理为具有吸电子能力的谷氨酰基团(Boc-L-谷氨酸-1-叔丁酯),链接在近红外荧光团R基上之后,通过光致电子转移会使荧光团R基失去荧光效应,而当谷氨酰基团被γ-谷氨酰转肽酶特异性切除后生成的给电子基氨基会恢复荧光基团的荧光能力。与γ-谷氨酰转肽酶反应前后的近红外荧光分子探针荧光能力的变化使得其可以精准的检测γ-谷氨酰转肽酶。该荧光分子探针通过细胞以及活体成像实验显著性效果,可以看到该分子探针应用于临床之中的潜力。
本发明检测γ-谷氨酰转肽酶的近红外荧光分子探针具有以下优势:
第一、本发明的近红外荧光分子探针易于进入细胞,具有较小的生物毒性。
第二、本发明以谷氨酰基团作为响应基团检测γ-谷氨酰转肽酶具有非常优异的选择性。
第三、本发明的近红外荧光分子探针吸收和发射均在近红外区具有较强的生物体穿透性,还能减少生物体自发荧光的干扰。
第四、本发明的近红外荧光分子探针具有较好的细胞和活体成像效果。
有益效果:与现有技术相比,本发明具有如下优点:
本发明提供了一种全新结构的近红外荧光分子探针,安全性良好对于γ-谷氨酰转肽酶选择性强,灵敏度高,响应时间短,重复性好,并且其细胞毒性低,生物相容性好,细胞渗透能力强,近红外吸收与荧光的发射的特性使其极大的避免了生物体自发荧光的干扰,可以检测溶液中、细胞中或者活体中γ-谷氨酰转肽酶,能很好的运用于细胞或者活体成像等。此外,本发明合成工艺简单,原料易得,成本低,收率高。
附图说明
图1是本发明近红外荧光分子探针的核磁氢谱表征(氘代甲醇);
图2是本发明近红外荧光分子探针与γ-谷氨酰转肽酶响应的荧光变化图;
图3是本发明近红外荧光分子探针的选择性实验数据图;
图4是本发明近红外荧光分子探针的MTT实验数据图;
图5是本发明近红外荧光分子探针的共聚焦显微镜细胞成像图。
具体实施方式
下面结合附图和实施例对本发明作进一步说明。
本发明中使用的实验方法如无特殊说明,均为常规方法。实验所用的材料、试剂等,如无特殊说明,均可从商业途径得到。实施例中所选用的以下所有试剂皆为市售分析纯或化学纯。
Boc-L-谷氨酸-1-叔丁酯选购自bidepharmatech。
γ-谷氨酰转肽酶选购自WAKO。
实施例1
检测γ-谷氨酰转肽酶近红外荧光分子探针的制备:
将二氯甲烷(12mL)加入到圆底烧瓶中,在氮气保护条件下缓慢加入化合物1(1mmol),开始搅拌,搅拌状态下在氮气保护的条件下缓慢加入Boc-L-谷氨酸-1-叔丁酯(8mmol),待搅拌15分钟之后,在氮气保护的条件下缓慢加入N,N-二异丙基乙胺(1.2mmol),室温下搅拌反应30min左右,向反应液中滴加三氟乙酸(18μL),室温下搅拌24h左右,待溶液变成深蓝,将瓶中反应液倒入分液漏斗中加入蒸馏水,充分混匀之后,静置,待分液漏斗中溶液分层之后,取上层水相溶液冷冻干燥得蓝色固体,即为目标探针。
实施例1制备的探针质谱以及核磁氢谱如图1所示1H NMR(400MHz,MeOD)δ8.85(t,J=12.0Hz,1H),8.36(t,J=9.5Hz,1H),8.16(d,J=8.9Hz,1H),8.09(d,J=1.8Hz,2H),7.82(d,J=8.9Hz,1H),7.78–7.72(m,1H),7.66–7.60(m,1H),7.44(t,J=9.1Hz,1H),7.37–7.31(m,2H),6.60(dd,J=16.0,8.2Hz,1H),4.60–4.51(m,2H),3.76–3.70(m,1H),2.82–2.79(m,2H),2.74(dd,J=13.3,6.7Hz,4H),2.30–2.25(m,2H),2.10(s,6H),2.00–1.94(m,2H),1.57(d,J=7.3Hz,3H).MS(ESI+):calcd for C36H38N3O4 +,576.29[M]+;found,576.4[M]+.
实施例2
近红外荧光分子探针与γ-谷氨酰转肽酶体外响应的荧光发射测量实验:
称取5.6mg实施例1制备的探针溶于1mL DMSO,配置成10mM探针母液。取0.14mg的γ-谷氨酰转肽酶(30U/4.2mg)溶于10mL PBS缓冲液,配置成100U/L的γ-谷氨酰转肽酶母液。准备6个5mL的离心管,依次在每个离心管加入2mL PBS缓冲液(pH=7.4)后,再加入2μL的探针母液,此时每个离心管探针浓度为10μM。待充分混匀后在每个离心管中分别加入0、4、8、12、16、20μL的γ-谷氨酰转肽酶母液,反应20分钟后,依次加入比色皿中进行荧光发射的检测(激发光波长为658nm),所得数据经origin软件处理得图2,如图2所示,随着酶浓度的增加,在740nm处的荧光信号不断增加,表明探针在溶液中与γ-谷氨酰转肽酶的响应效果好。
此外,探针的近红外荧光的发射波长在740nm,组织穿透能力强,在近红外通道的成像时可以有效地避免生物体自发荧光的干扰。
实施例3
近红外荧光分子探针的选择性实验:
通过与浓度均为(100μM)的不同底物:GGT(γ-GT)、KCl、NaCl、MgCl2、CaCl2、半胱氨酸、GSH、谷氨酸、MnO2、Vc、KMnO4孵育来检测实施例1制备的探针(10μM)对GGT的选择性。如图3所示,只有GGT具有诱导荧光信号显著增强的能力,而其他物质不能诱导荧光信号显著变化。结果明显表明,本发明探针是一个高度特异的探针,对GGT的响应效果远超过其他不同的物质。
实施例4
近红外荧光分子探针与U87细胞进行MTT生物相容性实验:
在96孔板的外围一圈加入PBS溶液,在剩余内部孔中铺满U87细胞,37℃培养12小时。称取5.6mg实施例1制备的探针溶于1mL生物用DMSO溶液,配置成10mM生物用探针母液。配置5mg/mL MTT溶液。准备5个5mL的离心管,均加入2mL DMEM培养基,依次在离心管中加入0、4、8、12、16μL生物用探针母液。将5个离心管中溶液吸取190μL依次每一横排等浓度加入5排孔中孵育24h后,在每个孔中加入10μL MTT溶液。37℃继续孵育4小时,倒掉培养基,在每个孔中均加入150μL DMSO溶液。待充分溶解后,进行紫外吸收检测(检测波长为490nm)。用origin进行数据处理得图4,由图可以看出探针浓度达到80μM(16μL生物用探针母液)时,细胞存活率依旧有95%以上,表明探针的生物适用性较好,细胞毒性小。
实施例5
近红外荧光分子探针与γ-谷氨酰转肽酶响应的共聚焦显微镜细胞实验:
在两个共聚焦皿中铺适当浓度的U87细胞,培养至细胞状态优良,将共聚焦皿中培养基吸取干净。吸取实施例4中生物用探针母液1μL加入1mLDMEM培养基中充分混匀,全部加入一个共聚焦皿中,在另一个皿中只加入等体积的DMEM培养基作为对照。溶液继续孵育20分钟后,倒掉培养基。用多聚甲醛溶液固定10分钟后,加入DAPI染色液分别进行染色5分钟,PBS缓冲液(pH=7.4)冲洗3次加入多聚甲醛溶液继续固定保存。共聚焦显微镜拍摄图片如图5,不加探针的一组近红外通道没有观察到荧光信号,添加了探针的一组在近红外通道可以观察到强烈的荧光信号,表明探针在细胞中能有很好的成像效果。
实施例6
实施例6与实施例1的制备方法相同,化合物1与二氯甲烷的比例为1mmol:10mL。化合物1、Boc-L-谷氨酸-1-叔丁酯、N,N-二异丙基乙胺和三氟乙酸的比例为1mmol:10mmol:1.5mmol:20μL。
实施例7
实施例7与实施例1的制备方法相同,化合物1与二氯甲烷的比例为1mmol:15mL。化合物1、Boc-L-谷氨酸-1-叔丁酯、N,N-二异丙基乙胺和三氟乙酸的比例为1mmol:8mmol:1.2mmol:15μL。
Claims (9)
3.根据权利要求2所述的检测γ-谷氨酰转肽酶近红外荧光分子探针的制备方法,其特征在于,所述惰性气体优选自氮气。
4.根据权利要求2所述的检测γ-谷氨酰转肽酶近红外荧光分子探针的制备方法,其特征在于,步骤(1)所述化合物1与二氯甲烷的比例为1mmol:10-15mL。
5.根据权利要求2所述的选择性检测γ-谷氨酰转肽酶近红外荧光分子探针的制备方法,其特征在于,所述化合物1、Boc-L-谷氨酸-1-叔丁酯和N,N-二异丙基乙胺的比例为1mmol:8-10mmol:1.2-1.5mmol:15-20μL。
6.根据权利要求2所述的检测γ-谷氨酰转肽酶近红外荧光分子探针的制备方法,其特征在于,步骤(3)所述搅拌反应直到溶液变深蓝色。
7.根据权利要求2所述的检测Γ-谷氨酰转肽酶近红外荧光分子探针的制备方法,其特征在于,步骤(4)所述萃取的方法为:反应液中加入蒸馏水,混匀,静置分液,取上层水层。
8.一种权利要求1所述的检测γ-谷氨酰转肽酶近红外荧光分子探针在选择性检测γ-谷氨酰转肽酶中的应用。
9.一种权利要求1所述的检测γ-谷氨酰转肽酶近红外荧光分子探针在检测溶液中、细胞中或者活体中γ-谷氨酰转肽酶中的应用。
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