CN105968170B - 一种二肽基肽酶iv的荧光探针底物及制备方法及应用 - Google Patents
一种二肽基肽酶iv的荧光探针底物及制备方法及应用 Download PDFInfo
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Abstract
本发明公开了一种二肽基肽酶IV的荧光探针底物及制备方法及应用，该荧光探针底物具有如下式的结构式：合成方法以下6‑氨基‑2‑萘甲酸为原料合成。采用所述的二肽基肽酶IV的荧光探针底物，与含二肽基肽酶IV的生物样品混合后进行酶促反应，通过定量检测单位时间内的底物消除率或其去二肽基产物的生成率来定量测定不同生物体系中DPP‑IV的活性。本发明的探针底物作为特异性的DPP‑IV单酶的荧光探针底物，该化合物可以用来检测DPP‑IV的活性。
Description
技术领域
本发明涉及一种荧光探针底物及制备方法及应用，特别涉及一种二肽基肽酶IV的荧光探针底物及制备方法及应用，属于化学领域。
背景技术
二肽基肽酶-IV（DPP-IV, CD26, EC 3.4.14.5）是以二聚体形式存在的Ⅱ型跨膜丝氨酸蛋白酶，广泛分布于哺乳动物脏器的上皮细胞及血管内皮细胞，其中在肾脏中表达最高，也能以溶解形式存在于体液中。作为重要的I相代谢酶，DPP-IV能特异性地催化多肽链N末端第2 位的氨基酸残基脯氨酸（Pro）或丙氨酸（Ala）肽键水解断裂，从而参与体内多种生物活性多肽的激活、部分或完全失活，如肠促胰岛素、神经肽、胃泌素释放肽、生长激素释放激素等。DPP-IV也在糖代谢过程中扮演重要角色，已经成为了治疗2型糖尿病新靶点，同时也被FDA批准为重要的药物作用靶点。另外它还是细胞膜受体和共刺激分子，从而参与机体的免疫调节、细胞移行、细胞黏附和细胞调亡过程，故与多种疾病的发生发展密切相关。研究表明，DPP-IV在一些肿瘤细胞中高表达，如结肠癌、原发性肺癌、前列腺癌、卵巢癌等，利用DPP-IV代谢活化机制，以DPP-IV为潜在靶点，可以设计靶向性的抗癌前药。临床研究表明许多疾病如口腔癌、结肠癌、肺癌、肾病、肝病等会引起患者血液或尿液DPP-IV含量显著改变，使血液或尿液中的DPP-IV可作为这些疾病诊断的标志物。因此，开发DPP-IV检测方法具有广阔的临床应用前景，特别是血液、尿液中DPP-IV活性检测对疾病的早期诊断，活细胞和活组织层面开展DPP-IV实时定量检测和功能成像，高效筛选DPP-IV抑制剂作为糖尿病治疗的候选药物，及DPP-IV功能异常与疾病发生发展间的关系等研究等。无论是疾病的诊断和治疗，还是对疾病发病机理的研究，都必须要借助于精准高效的检测手段。传统的代谢酶的定量评估技术主要有以下三种：1）基于抗体的Elisa或Western检测技术；2）借助LC-MS/MS技术对目标酶特异性肽段进行分析及定量的蛋白组学技术；3）借助特异性探针分子的目标酶活性定量评估技术。与传统代谢酶的定量评估技术相比，荧光探针检测具有以下突出优势：1）操作简单、易高通量；2）高灵敏度、样品需求量小，检测灵敏度可达皮摩尔级；3）荧光成像技术可以实现对活细胞或组织水平的精确定位及实时动态检测；4）借助荧光探针分子可实现亚毫秒时间和亚纳米空间的高分辨率检测。因此，开发实用的DPP-IV荧光探针检测具有重要的应用价值。
发明内容
本发明的目的在于提供一种二肽基肽酶IV的荧光探针底物及制备方法及应用。
本发明所提供的一种二肽基肽酶IV的荧光探针底物，所述的荧光探针底物具有如下式的结构式：
R为甲基或乙基或丙基或丁基。
本发明所提供的一种二肽基肽酶IV的荧光探针底物的制备方法，包括以下步骤：
（1）以下式所示的6-氨基-2-萘甲酸为起始原料，经过酸催化酯化反应合成下式所示的6-氨基-2-萘甲酸酯类化合物，反应式如下：
（2）采用式所示的1当量的6-氨基-2-萘甲酸酯类化合物在0.5-2当量的N-羟基苯并三氮唑、0.5-2当量的1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐、1-3当量的2-(7-偶氮苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯存在下，与1-3当量的叔丁氧羰基甘氨酸基脯氨酸，在20-30℃搅拌反应24-48小时，生产式所示的产物，反应式如下：
（3）将步骤（2）生成的产物经柱层析纯化，再经三氟乙酸酸催化，20-30℃搅拌反应4-10小时脱除叔丁氧羰基生成式所示的二肽基肽酶IV的荧光探针底物；反应式如下：
上述各反应式中，R为甲基或乙基或丙基或丁基。
本发明的一种二肽基肽酶IV的荧光探针底物的应用，所述的探针底物具有式所述的结构式，采用式所示的化合物作为二肽基肽酶IV的荧光探针底物，与含二肽基肽酶IV的生物样品混合后进行酶促反应，通过定量检测单位时间内的底物消除率或其去二肽基产物的生成率来定量测定不同生物体系中DPP-IV的活性。
本发明的一种二肽基肽酶IV的荧光探针底物的应用，所述的探针底物具有式所述的结构式，所述的探针底物可用于DPP-IV抑制剂或诱导剂的快速筛选；或DPP-IV抑制及诱导能力的定量评估，
进一步的，上述应用中具体的测定方法如下：
A探针底物浓度为1/10~10 Km，Km为酶促反应速度达到最大反应速度一半时所对应的底物浓度；
B 以PBS缓冲液作为DPP-IV孵育体系，孵育体系pH值介于5.5~10.5之间，孵育温度在20~60℃之间，在该体系中加入步骤A浓度的探针底物进行反应；
C.反应时间为5~120分钟，确保以上底物相应的N-去二肽基化产物达到定量限且底物转化率不超过20%时终止反应；
D.测定单位时间内底物减少量或N-去二肽基化产物生成量作为DPP-IV活性的评价指标；
进一步的，步骤B中孵育温度为37℃，孵育体系pH值为7.4。
本发明的积极有益技术效果在于：本发明的探针底物作为特异性的DPP-IV单酶的荧光探针底物，该化合物可以用来检测DPP-IV的活性，尤其适合用于对细菌、昆虫细胞、哺乳动物细胞以及酵母菌克隆表达体系生产的DPP-IV的酶活测定，以及多种哺乳动物组织器官来源的微粒体、S-9等制备物中DPP-IV的活性标定。选用本发明的探针底物及测定方法检测DPP-IV单酶体外活性具有以下突出优势：
(1)特异性：该探针底物可被DPP-IV高特异性地代谢成一个代谢产物，即N-去二肽化产物；
(2)廉价易得：GP-ANC可经化学合成获得，合成工艺简单易行；
(3)易高通量检测：可在实验室常见各类荧光酶标仪及临床大生化仪上测定，可利用96或386微孔板进行批量检测。
附图说明
图1是 6-(甘氨酸-脯氨酸)-氨基-2-萘甲酸甲酯(GP-ANC)的合成路线。
图2是 6-(甘氨酸-脯氨酸)-氨基-2-萘甲酸甲酯(GP-ANC)的1H-NMR谱图。
图3是6-(甘氨酸-脯氨酸)-氨基-2-萘甲酸甲酯(GP-ANC)的13C-NMR谱图。
图4是6-(甘氨酸-脯氨酸)-氨基-2-萘甲酸甲酯(GP-ANC)的选择性图。
图5是 DPP-IV的定量标准曲线。
具体实施方式
为了更充分的解释本发明的实施，提供本发明的实施实例，这些实施实例仅仅是对本发明的阐述，不限制本发明的范围。
本发明实施例中所采用的设备及其型号为：荧光发射/激发光谱是由SynergyH1全功能微孔板检测仪检测完成；NMR谱图是由核磁共振波谱仪（ Avance II 400MHz）检测完成。本发明中Boc-Gly-Pro-OH指N-[叔丁氧羰基]甘氨酰-L-脯氨酸，其中文同义词还有：BOC-甘氨酸-L-脯氨酸；Boc指叔丁氧羰基 .
实施例1：6-(甘氨酸-脯氨酸)-氨基-2-萘甲酸甲酯(GP-ANC)的合成，即式中的R为甲基，附图1给出了其合成路线。
附图1中化合物2的合成
室温下，将6-氨基-2-萘甲酸（561.2 mg, 3 mmol）加入 甲醇（50 mL）溶液中，搅拌下滴加浓硫酸（0.5 mL），加完后回流反应，反应完毕后，除溶剂，饱和碳酸氢钠溶液调pH值至7-8，每次采用50 mL乙酸乙酯萃取，共萃取3次，采用30 mL水洗、30 mL饱和氯化钠溶液洗，采用无水硫酸钠干燥，干燥充分后，蒸除溶剂，得附图1中的化合物2，为淡褐色固体，产率85-95%，ESI-MS m/z 188.1 [M+H]+；
附图1中化合物3的合成：
室温，将化合物2（201.2 mg, 1 mmol）、N-[叔丁氧羰基]甘氨酰-L-脯氨酸即Boc-Gly-Pro-OH（544.6 mg, 2.0 mmol），、N-羟基苯并三氮唑（135.2 mg, 1 mmol）、1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐（191.8mg, 1 mmol）、2-(7-偶氮苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯（760.4 mg, 2.0 mmol）依次加入到N,N-二甲基甲酰胺（10 mL）溶液中反应，薄板层析（TLC）监测反应，反应36 h后，反应体系冷却到0-5 ℃，加水（25 mL），每次采用30 mL乙酸乙酯萃取，共萃取三次，合并有机相每次采用15 mL水洗，水洗三次，采用20ml的5%碳酸氢钠溶液洗乙醇，15ml水洗一次、20ml饱和氯化钠溶液洗一次，无水硫酸钠干燥，蒸除溶剂，粗产物柱层析（二氯甲烷/甲醇 = 50/1）得化合物3，为淡黄色固体，产率80-90%,，ESI-MS m/z 202.1 [M+H]+；
附图1化合物4（GP-ANC）的合成：
室温，将化合物3（227.8 mg, 0.5 mmol）加入到二氯甲烷（16 mL）与三氟乙酸（4mL）混合溶液中反应，薄板层析（TLC）监测反应。反应6 h后，蒸除反应溶剂，粗产物加入15mL水中，饱和碳酸氢钠溶液调pH 值= 7-8，每次采用25 mL二氯甲烷萃取，共萃取三次，合并有机相采用15ml水进行水洗，20ml的饱和氯化钠溶液洗，无水硫酸钠干燥，蒸除溶剂，粗产物柱层析（二氯甲烷/甲醇 = 4/1）得化合物4，即GP-ANC，产率80-90%，其核磁谱图如附图2、附图3所示，该产物为白色固体，化合物4的核磁共振波谱具体如下：
1H NMR (400 MHz, CDCl3) δ 10.03 (s, 1H), 8.44 (s, 1H), 8.18 (s, 1H),7.97 (d, J = 7.8 Hz, 1H), 7.91 (brs, 2H), 7.73 (d, J = 6.9 Hz, 1H), 7.68 (d,J = 7.6 Hz, 1H), 7.50 (d, J = 7.9 Hz, 1H), 4.38 (brs, 1H), 3.97 (s, 3H), 3.65(brs, 1H), 3.31 (brs, 1H), 2.86 (brs, 1H), 2.54 (brs, 1H), 1.88 (brs, 2H),1.66 (brs, 1H), 1.43 (brs, 1H). 13C NMR (101 MHz, CDCl3) δ 171.4, 165.4,161.6, 137.5, 135.9, 130.5, 129.5, 127.7, 126.0, 120.8, 117.9, 116.4, 115.0,61.2, 52.3, 46.2, 40.7, 29.4, 24.3. ESI-MS m/z 356.2 [M+H]+ 。
实施例2：附图1中的化合物4（6-(甘氨酸-脯氨酸)-氨基-2-萘甲酸甲酯，GP-ANC）体外测定人重组DPP-IV单酶的选择性
（1）预先准备198 µL DPP-IV代谢反应体系，包括pH 值7.4的PBS缓冲液（50 mM）、碳酸酐酶（CA，10 μg/mL），胰蛋白酶（trypsin，10 μg/mL），胃蛋白酶（pepsin，10 μg/mL），丁酰胆碱酯酶（BChE，10 μg/mL），乙酰胆碱酯酶（AChE，10 μg/mL），羧酸酯酶（hCE1，hCE2，10 μg/mL），人血清白蛋白（HAS，10 μg/mL），重组人DPP-IX单酶 (1 μg/mL)，重组人DPP-VIII单酶 (1 μg/mL)，重组人成纤维细胞活化蛋白单酶 (FAP，1 μg/mL)，重组人DPP-IV单酶 (1 μg/mL)于37℃条件下震荡预孵3分钟；
（2）向反应体系中加入2 µL浓度为10 mM的GP-ANC起始反应；
（3）30分钟后，加入200 µL冰乙腈，剧烈震荡后，终止反应；
（4）用高速冷冻离心机在4℃，20,000×g的条件下，高速离心20分钟后，取上清，进行荧光检测（Ex = 310/330 nm，Em = 385/445 nm）；结果如附图4所示，实验结果表明GP-ANC为探针地物的重组人DPP-IV酶的选择性较好。
实施例3：附图1中的化合物4（6-(甘氨酸-脯氨酸)-氨基-2-萘甲酸甲酯，GP-ANC）体外测定DPP-IV的检测下限测定
实验在酶标仪上使用96孔板进行测定，GP-ANC 100 μM， DPP-IV单酶0.5 μg/mL~1.2 μg/mL，pH 值7.4的PBS缓冲液50 mM，总体积为200 μL，37℃下孵育1 h后通过荧光酶标仪分析，每组的平均值与不加DPP-IV的对照组比较，通过产物的荧光强度与DPP-IV含量做标准曲线，如附图5所示，计算结果表明DPP-IV的检测下限为1 ng/mL。
实施例4：作为DPP-IV抑制剂筛选
以GP-ANC的水解代谢为探针反应，借助人肠微粒体体外孵育体系，测定五环三萜化合物甘草次酸、熊果酸、齐墩果酸、白桦脂酸、对DPP-IV抑制的IC50：
a. 200微升体外代谢反应体系中，含有pH为7.4的磷酸缓冲液，人肠微粒体蛋白浓度为10 μg/ml，抑制剂终浓度范围为0.01μM-100μM，于37℃条件下震荡预孵10分钟；
b. 向反应体系中加入底物（终浓度100μM），起始反应；于37℃条件下反应30分钟后，加入200 µl乙腈，剧烈震荡后，终止反应；
c. 采用高速冷冻离心机，在20,000 ×g的条件下，高速离心上述体系5分钟后，取上清，进行酶标仪检测分析；对代谢水解产物进行定量检测，检测数据如下表一所示，从所得实验数据可以看出，白桦脂酸对DPP-IV具有一定的抑制活性。
在详细说明本发明的实施方式之后，熟悉该项技术的人士可清楚地了解，在不脱离上述申请专利范围与精神下可进行各种变化与修改，凡依据本发明的技术实质对以上实施例所作的任何简单修改、等同变化与修饰，均属于本发明技术方案的范围，且本发明亦不受限于说明书中所举实例的实施方式。
Claims (1)
1.一种二肽基肽酶IV的荧光探针底物用于快速筛选DPP-IV抑制剂的应用，所述的探针底物具有式所述的结构式，
R为甲基或乙基或丙基或丁基； 其特征在于：快速筛选方法如下：
以GP-ANC的水解代谢为探针反应，借助人肠微粒体体外孵育体系，测定所要筛选的抑制剂对DPP-IV抑制的IC50：步骤如下：
a. 200微升体外代谢反应体系中，含有pH为7.4的磷酸缓冲液，人肠微粒体蛋白浓度为10 μg/ml，抑制剂终浓度范围为0.01μM-100μM，于37℃条件下震荡预孵10分钟；
b. 向反应体系中加入底物，终浓度100μM，起始反应；于37℃条件下反应30分钟后，加入200 µl乙腈，剧烈震荡后，终止反应；
c. 采用高速冷冻离心机，在20,000 ×g的条件下，高速离心上述体系5分钟后，取上清，进行酶标仪检测分析；对代谢水解产物进行定量检测。
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