氨肽酶N小分子荧光探针的设计、合成与活性评价

发布时间：2018-01-20 08:25

本文关键词： 氨肽酶N 可激活型荧光探针荧光抑制剂化学合成活性测定荧光成像　出处：《山东大学》2014年博士论文　论文类型：学位论文

【摘要】：氨肽酶N (APN, CD13)广泛表达于哺乳动物的多个组织和器官,能够从蛋白质的N末端降解肽链并释放氨基酸。与正常细胞相比,该酶在某些肿瘤细胞表面过度表达,对肿瘤的侵袭、血管生成具有重要作用。另外,该蛋白酶可作为许多病毒(如引起新生猪急性胃肠炎的播散性胃肠炎病毒以及引起人上呼吸道感染的冠状病毒HCV229E)的受体参与相应的生理反应。它还能够表达于抗原递呈细胞表面,降解多种免疫活性物质,使机体免疫力下降,削弱巨噬细胞和NK细胞对肿瘤细胞的识别和杀伤能力等。由于APN在肿瘤生长、侵袭和转移过程中发挥着重要的作用,以APN为靶点的抗肿瘤药物研究一直是药物化学领域的热点。自首个APN抑制剂Bestatin上市以来,数以千计的APN抑制剂先后被报道。此外,因为APN在肿瘤组织过度表达,被称作肿瘤标记物,以APN为靶点的肿瘤诊断剂也一直是受到化学生物学家的关注,比如以APN天然配体NGR(Asp-Gly-Arg)为载体的荧光探针或者放射性探针已经能够实现活体动物的肿瘤组织成像。此外,以APN抑制剂为先导化合物的小分子荧光探针的研究也越来越受到人们的关注,并取得了一定进展。虽然人们对APN抑制剂的开发投入了大量的精力,然而目前上市的APN抑制剂只有Bestatin一个。目前以NGR为载体的肿瘤组织检测探针已经能够实现活体动物的肿瘤组织成像,但APN酶活检测方法研究较少。大多数药物化学工作者仍然在使用亮氨酸氨肽酶(LAP)的底物对APN进行酶活测试和抑制剂的筛选,这也是限制APN抑制剂上市的一个重要因素。本文的研究内容之一就是设计开发可用于APN酶活检测的荧光探针：通过对APN晶体结构及催化机理的研究,选择容易被APN识别的氨基酸作为识别基团,直接或者间接(通过可自发降解的连接基团)与特定的荧光团连接,可设计合成一系列具有参比波长性质的荧光探针。在特定的激发波长下,探针自身和探针被APN降解后释放的荧光团均能够发射波长不同的荧光,两个波长处的荧光值在作用过程中一增一减,通过二者的比值可以反映APN的水解活性,而且可以扣除外界环境对测量结果的影响。我们可针对不同的测试要求,通过对荧光团和连接基团的调整对探针进行结构优化,以期发现准确度高、操作方便、应用范围广的APN酶活测试方法。此外,虽然已有两篇文献报道了具有荧光性质的APN抑制剂,但是它们对APN的抑酶活性均不是很强。本文的另一个研究方向是设计开发具有高抑制活性的APN荧光抑制剂。在本课题组数十年APN抑制剂的研究基础上,选择IC50数值小,对APN亲和力高的脲基异羟肟酸类抑制剂作为先导化合物,设计合成一系列自身具有荧光的APN抑制剂,该抑制剂中的荧光团也是药效团,避免了常规荧光探针中荧光团对抑制剂与酶结合的影响,并通过活性测试结果进行结构优化。在探针设计的基础上,根据所设计的目标化合物的结构以及本课题组多年的类肽合成经验,通过逆合成分析设计合成路线。合成中根据氨基的活性强弱,采用了多种不同的缩合方法,如对于亲核性高的氨基,我们选择反应条件温和、后处理简单的缩合剂EDCI,或者将酸酐直接与氨基反应；对于电负性相对较弱的吡啶芳胺,我们选择酰氯法；对于电负性较弱并且空间位阻大的萘胺衍生物,我们先将羧基转变为酰氯,然后再与萘胺衍生物在高温回流以及酸碱催化的条件下进行缩合。对于氨基的保护,可根据不同要求,选择对碱稳定的Boc保护或者是对酸稳定的Fmoc保护；对于羧基的保护,根据不同要求,选择甲酯保护或者苄酯保护。对于脲基的合成,根据参与反应的两个氨基的特点选择先将哪一个氨基变为异氰酸酯,并且根据异氰酸酯中间体的稳定情况,选择先分离后缩合或者是一锅法反应。在得到目标化合物以后,本文根据所设计探针的不同用途,采取了不同的评价方法。对于可激活型APN荧光探针主要进行了探针的酶识别试验、酶促动力学测试、光学性质研究、细胞成像研究以及探针在IC5o测试中的应用研究；对于具有荧光性质的APN抑制剂,主要进行了体外ICso测定、MTT试验、体内抗肿瘤试验和肿瘤细胞成像研究。氨基酸-自发降解连接基团-香豆素类探针中以对氨基苄醇为连接基团的探针能够被APN降解,而以邻氨基苄醇或者是3-氨基吡啶-2-甲醇为连接基团的探针不能够被APN降解。探针1.1-7a具有参比波长性质,能够降低环境因素对测量结果的影响,提高了测试准确度。而且值得注意的是,该探针具有较高的灵敏度,降低了APN抑制剂IC50测试中酶的用量,从而降低了测试成本。该探针还简化了细胞水平APN酶活的测试方法,为APN抑制的筛选提供了一种准确、方便、经济的测试方法。但是由于探针1.1-7a在降解前后的波长均为蓝色荧光,不适合在细胞成像中的使用,因此我们在此探针的基础上进一步优化得到了氨基酸-萘二酰亚胺类探针；氨基酸-萘二酰亚胺类探针被APN降解前发蓝色荧光,降解后发绿色荧光。在细胞成像时,根据细胞中绿色和蓝色荧光的强度比值可以定量地反映APN的活性。该类探针的缺点是与APN反应的速度太慢,这限制了其在抑制剂筛选中的使用：接下来,我们在氨基酸和萘二酰亚胺中间加上自发降解连接基团后得到的氨基酸-自发降解连接基团-萘二酰亚胺类探针,与APN的反应速度提高了8倍,使其能够成功地应用于APN抑制剂的IC50测定。这三个系列的探针不仅能够对非荧光抑制剂进行IC50测定,并且通过联合使用,能够对常规方法无法测量的荧光抑制剂进行ICs0测定。此外,本文还设计合成了三个系列共21个具有荧光性质的APN抑制剂,并对所合成的抑制剂进行了体外抑酶活性测试以及计算机辅助的分子对接研究。系列1和系列2大部分化合物的活性均强于上市药物Bestatin,化合物4.1-3a,4.1-3b,4.1-3k的活性高于先导化合物4q,其中化合物4.1-3k的活性比先导化合物高出了一个数量级,比Bestatin高出了三个数量级。分子对接的结果显示,活性相差较大的化合物与APN的结合模式不同。其次,4.1-3k能够对APN高表达的肿瘤细胞A549进行染色,而对非肿瘤细胞HEK-293的染色强度明显较弱,并且染色强度能够被APN抑制剂4q显著减弱。综上所述,本文共设计合成了3个系列可激活型APN荧光探针,它们能够准确、快捷地测试不同条件下的APN酶活性,并且能够准确地进行非荧光抑制剂和荧光抑制剂的IC50测定,为今后APN抑制剂的初步活性筛选提供了一套完整的测试方法。此外,本文所设计合成的荧光抑制剂4.1-3k,其抑酶活性比上市药物强100-1000倍,并具有显著的抗肿瘤作用。而且能够选择性地标记APN高表达的肿瘤细胞,将有望被开发为既有治疗作用又能够用作疾病诊断的APN荧光抑制剂。
[Abstract]:Aminopeptidase N (APN, CD13) more widely expressed in mammalian tissues and organs, from the end of the N degradation and release of amino acid peptide protein. Compared with normal cells, overexpression of the enzyme on the surface of some tumor cells, tumor invasion, angiogenesis plays an important role. In addition, the protease can be as many viruses (such as acute gastroenteritis caused by neonatal pig disseminated gastroenteritis virus and cause upper respiratory tract infection of coronavirus HCV229E) receptors participate in physiological responses. It can also be expressed on antigen-presenting cell surface degradation, immune activity of various substances, make the body immunity, reduce macrophage and NK cells to identify tumor cells and killing ability. Because the APN in tumor growth, plays an important role in the process of invasion and metastasis, APN on antitumor drug targets is a drug The hot field of chemistry. Since the first APN inhibitor Bestatin APN inhibitor listed, thousands were reported. In addition, because of the excessive expression of APN in tumor tissue, called tumor markers, with APN as the diagnostic agent for tumor targeting has been subjected to chemical biologists concern, such as the natural ligand of APN NGR (Asp-Gly-Arg) the tumor tissue imaging carrier fluorescence probe or radioactive probe has been able to achieve a live animal. In addition, a APN inhibitor of small molecule fluorescent probe compounds have attracted more and more attention, and has made some progress.
Although it is development of APN inhibitors have invested a lot of energy, however, the APN inhibitor listed only a Bestatin. At present NGR tumor imaging detection probe vector for tumor tissue has been able to achieve a living animal, but APN activity detection method is less. Most drug chemists still use leucine aminopeptidase (LAP) substrate screening test and enzyme inhibitors of APN, which is an important factor limiting APN inhibitors listed. One of the research content of this paper is to design and development can be used for fluorescence probe APN activity detection: through the research on the crystal structure of APN and the catalytic mechanism, selection of easy recognition of amino acid APN as the recognition group, directly or indirectly (through the connection group spontaneous degradation) connected with the specific fluorophore, to design and synthesize a series of ginseng has long waves The nature of the fluorescent probe. The excitation wavelength under the specific probe itself and the probe is released from the degradation of APN fluorophores can different fluorescence emission wavelengths, two wavelengths of fluorescence in the process a minus, by the ratio of the two may reflect the APN hydrolysis activity, but also can be deducted the influence of external environment on the measurement results. We can according to different test requirements, the fluorophore and adjust the connection groups to optimize the structure of the probe, in order to find the high accuracy, convenient operation, wide application of the APN enzyme live test method. In addition, there are two reported APN inhibitors with fluorescence in nature, but they are on the APN inhibitory activity were not very strong. Another research direction of this paper is to design and development of APN fluorescent inhibitor with high inhibitory activity. In this study group for decades APN inhibitors Based on the selection of IC50 value is small, the APN high affinity urea hydroxamic acid inhibitors as a lead compound, APN inhibitor has its own design and synthesize a series of fluorescence, fluorescence of the inhibitor in the pharmacophore, avoid the influence of conventional fluorescence fluorescence probe group of inhibitor and enzyme combination, and through the activity the test results of structure optimization.
On the basis of probe design, according to the structure of the target compound design and the class of this group for many years experience in peptide synthesis, through inverse synthetic analysis and design. According to the synthetic route of synthesis activity of amino, the condensation of several different methods, such as the nuclear high pro -, we choose mild reaction conditions postprocessing, condensation agent EDCI, or directly reacts with amino acid anhydride; pyridine for aromatic amine electronegativity is relatively weak, we choose the electronegativity of acyl chloride; weak and large steric hindrance of naphthylamine derivatives, we divide into carboxyl acyl chloride, and then naphthylamine derivatives in high temperature and reflux under the condition of acid catalytic condensation. For the protection of amino group, according to different requirements, selection of Boc protective alkali stable or Fmoc protection of acid stable; for carboxyl protection, according to the Different requirements, choose methyl benzyl protection or protection. For the synthesis of urea groups, according to the characteristics of two amino groups involved in the reaction the first one into amino isocyanate, and according to the stability of isocyanate intermediates, the choice of the first after the separation or condensation is a pot reaction.
After getting the target compound, according to different uses design of the probe, adopt different evaluation methods for activation of type APN fluorescent probe of enzyme identification test probe, enzyme kinetics test, optical properties, application of cell imaging research and probe in the IC5o test for APN inhibitors with; the fluorescence properties, mainly in vitro ICso assay, MTT test, test and in vivo tumor cell imaging. Anti tumor amino acid linkers - the spontaneous degradation of coumarin probe to amino benzyl alcohol as a probe connected groups can be APN degradation, and 2-aminobenzyl alcohol or 3- amino pyridine -2- methanol the probe is connected groups can not be degraded. APN probe 1.1-7a with reference wavelength properties, can reduce the influence of environmental factors on the measurement results, and improve testing accuracy. It is worth noting that the probe has high sensitivity, reduced APN inhibitor IC50 test the dosage of enzyme, which reduces the test cost. The probe also simplifies the testing method of cell level APN activity, for the screening of APN inhibition provides an accurate, convenient, test method of economy. But due to probe in the 1.1-7a before and after degradation were wavelength blue fluorescence, not suitable for use in cell imaging, so we probe further on the basis of optimized amino acid - naphthalene imide two - two amino acid probe; naphthalene imide probe by APN degradation before the blue fluorescence, after the degradation of green fluorescence in the cell. When imaging according to the intensity ratio of the green and blue fluorescence in the cells can quantitatively reflect the activity of APN and APN. The reaction speed is too slow is the probe faults, which limits its in the screening of inhibitors Use: next, we in the middle of two amino acid - amino acid and naphthalene imide with spontaneous degradation linkers obtained after spontaneous degradation of linkers - naphthalene imide two probe and reaction rate of APN increased by 8 times, so that it can be successfully applied to APN inhibitors of IC50 determination of the three probe. The series can not only determine the IC50 inhibitor of non fluorescence, and through the combined use of fluorescence measurement of the conventional method of the inhibitor is unable to determine the ICs0. In addition, this paper also designs three series a total of 21 has the fluorescence properties of APN inhibitors and inhibitors of the synthesis, the synthesis of molecular docking studies on enzyme activity the computer aided test and inhibition in vitro. Series 1 and 2 most active compounds are stronger than the listed drug Bestatin, compound 4.1-3a, 4.1-3b, 4.1-3k was higher than that of the lead compound 4q, which Compound 4.1-3k activity than the lead compound is higher than an order of magnitude higher than Bestatin by three orders of magnitude. Molecular docking results show that the combination of model compounds with APN activity differ larger difference. Secondly, the tumor cell A549 4.1-3k high expression of APN staining, while the non tumor cells HEK-293 the staining intensity was weaker, and the staining intensity can be APN inhibitor 4q significantly weakened.
In summary, the paper has designed and synthesized 3 series of activated type APN fluorescent probe, which can accurately and quickly test the activity of APN under different conditions, and can accurately carry out non fluorescent inhibitors and inhibitors of IC50 fluorescence determination, a complete set of test methods for screening with initial activity of APN inhibitors in the future. In addition, the fluorescence of 4.1-3k synthesis inhibitors designed in this paper, the inhibition of enzyme activity is 100-1000 times stronger than the listed drugs, and has significant anti-tumor effects. And selectively labeled APN highly expressed in tumor cells, is expected to be developed as APN fluorescent inhibitors have therapeutic effect and can be used for disease diagnosis.

【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R914;R96

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