本文關(guān)鍵詞： 氨肽酶N 可激活型熒光探針 熒光抑制劑 化學(xué)合成 活性測(cè)定 熒光成像　出處：《山東大學(xué)》2014年博士論文　論文類(lèi)型：學(xué)位論文

【摘要】：氨肽酶N (APN, CD13)廣泛表達(dá)于哺乳動(dòng)物的多個(gè)組織和器官,能夠從蛋白質(zhì)的N末端降解肽鏈并釋放氨基酸。與正常細(xì)胞相比,該酶在某些腫瘤細(xì)胞表面過(guò)度表達(dá),對(duì)腫瘤的侵襲、血管生成具有重要作用。另外,該蛋白酶可作為許多病毒(如引起新生豬急性胃腸炎的播散性胃腸炎病毒以及引起人上呼吸道感染的冠狀病毒HCV229E)的受體參與相應(yīng)的生理反應(yīng)。它還能夠表達(dá)于抗原遞呈細(xì)胞表面,降解多種免疫活性物質(zhì),使機(jī)體免疫力下降,削弱巨噬細(xì)胞和NK細(xì)胞對(duì)腫瘤細(xì)胞的識(shí)別和殺傷能力等。由于APN在腫瘤生長(zhǎng)、侵襲和轉(zhuǎn)移過(guò)程中發(fā)揮著重要的作用,以APN為靶點(diǎn)的抗腫瘤藥物研究一直是藥物化學(xué)領(lǐng)域的熱點(diǎn)。自首個(gè)APN抑制劑Bestatin上市以來(lái),數(shù)以千計(jì)的APN抑制劑先后被報(bào)道。此外,因?yàn)锳PN在腫瘤組織過(guò)度表達(dá),被稱(chēng)作腫瘤標(biāo)記物,以APN為靶點(diǎn)的腫瘤診斷劑也一直是受到化學(xué)生物學(xué)家的關(guān)注,比如以APN天然配體NGR(Asp-Gly-Arg)為載體的熒光探針或者放射性探針已經(jīng)能夠?qū)崿F(xiàn)活體動(dòng)物的腫瘤組織成像。此外,以APN抑制劑為先導(dǎo)化合物的小分子熒光探針的研究也越來(lái)越受到人們的關(guān)注,并取得了一定進(jìn)展。 雖然人們對(duì)APN抑制劑的開(kāi)發(fā)投入了大量的精力,然而目前上市的APN抑制劑只有Bestatin一個(gè)。目前以NGR為載體的腫瘤組織檢測(cè)探針已經(jīng)能夠?qū)崿F(xiàn)活體動(dòng)物的腫瘤組織成像,但APN酶活檢測(cè)方法研究較少。大多數(shù)藥物化學(xué)工作者仍然在使用亮氨酸氨肽酶(LAP)的底物對(duì)APN進(jìn)行酶活測(cè)試和抑制劑的篩選,這也是限制APN抑制劑上市的一個(gè)重要因素。本文的研究?jī)?nèi)容之一就是設(shè)計(jì)開(kāi)發(fā)可用于APN酶活檢測(cè)的熒光探針：通過(guò)對(duì)APN晶體結(jié)構(gòu)及催化機(jī)理的研究,選擇容易被APN識(shí)別的氨基酸作為識(shí)別基團(tuán),直接或者間接(通過(guò)可自發(fā)降解的連接基團(tuán))與特定的熒光團(tuán)連接,可設(shè)計(jì)合成一系列具有參比波長(zhǎng)性質(zhì)的熒光探針。在特定的激發(fā)波長(zhǎng)下,探針自身和探針被APN降解后釋放的熒光團(tuán)均能夠發(fā)射波長(zhǎng)不同的熒光,兩個(gè)波長(zhǎng)處的熒光值在作用過(guò)程中一增一減,通過(guò)二者的比值可以反映APN的水解活性,而且可以扣除外界環(huán)境對(duì)測(cè)量結(jié)果的影響。我們可針對(duì)不同的測(cè)試要求,通過(guò)對(duì)熒光團(tuán)和連接基團(tuán)的調(diào)整對(duì)探針進(jìn)行結(jié)構(gòu)優(yōu)化,以期發(fā)現(xiàn)準(zhǔn)確度高、操作方便、應(yīng)用范圍廣的APN酶活測(cè)試方法。此外,雖然已有兩篇文獻(xiàn)報(bào)道了具有熒光性質(zhì)的APN抑制劑,但是它們對(duì)APN的抑酶活性均不是很強(qiáng)。本文的另一個(gè)研究方向是設(shè)計(jì)開(kāi)發(fā)具有高抑制活性的APN熒光抑制劑。在本課題組數(shù)十年APN抑制劑的研究基礎(chǔ)上,選擇IC50數(shù)值小,對(duì)APN親和力高的脲基異羥肟酸類(lèi)抑制劑作為先導(dǎo)化合物,設(shè)計(jì)合成一系列自身具有熒光的APN抑制劑,該抑制劑中的熒光團(tuán)也是藥效團(tuán),避免了常規(guī)熒光探針中熒光團(tuán)對(duì)抑制劑與酶結(jié)合的影響,并通過(guò)活性測(cè)試結(jié)果進(jìn)行結(jié)構(gòu)優(yōu)化。 在探針設(shè)計(jì)的基礎(chǔ)上,根據(jù)所設(shè)計(jì)的目標(biāo)化合物的結(jié)構(gòu)以及本課題組多年的類(lèi)肽合成經(jīng)驗(yàn),通過(guò)逆合成分析設(shè)計(jì)合成路線。合成中根據(jù)氨基的活性強(qiáng)弱,采用了多種不同的縮合方法,如對(duì)于親核性高的氨基,我們選擇反應(yīng)條件溫和、后處理簡(jiǎn)單的縮合劑EDCI,或者將酸酐直接與氨基反應(yīng)；對(duì)于電負(fù)性相對(duì)較弱的吡啶芳胺,我們選擇酰氯法；對(duì)于電負(fù)性較弱并且空間位阻大的萘胺衍生物,我們先將羧基轉(zhuǎn)變?yōu)轷Ｂ?然后再與萘胺衍生物在高溫回流以及酸堿催化的條件下進(jìn)行縮合。對(duì)于氨基的保護(hù),可根據(jù)不同要求,選擇對(duì)堿穩(wěn)定的Boc保護(hù)或者是對(duì)酸穩(wěn)定的Fmoc保護(hù)；對(duì)于羧基的保護(hù),根據(jù)不同要求,選擇甲酯保護(hù)或者芐酯保護(hù)。對(duì)于脲基的合成,根據(jù)參與反應(yīng)的兩個(gè)氨基的特點(diǎn)選擇先將哪一個(gè)氨基變?yōu)楫惽杷狨?并且根據(jù)異氰酸酯中間體的穩(wěn)定情況,選擇先分離后縮合或者是一鍋法反應(yīng)。 在得到目標(biāo)化合物以后,本文根據(jù)所設(shè)計(jì)探針的不同用途,采取了不同的評(píng)價(jià)方法。對(duì)于可激活型APN熒光探針主要進(jìn)行了探針的酶識(shí)別試驗(yàn)、酶促動(dòng)力學(xué)測(cè)試、光學(xué)性質(zhì)研究、細(xì)胞成像研究以及探針在IC5o測(cè)試中的應(yīng)用研究；對(duì)于具有熒光性質(zhì)的APN抑制劑,主要進(jìn)行了體外ICso測(cè)定、MTT試驗(yàn)、體內(nèi)抗腫瘤試驗(yàn)和腫瘤細(xì)胞成像研究。氨基酸-自發(fā)降解連接基團(tuán)-香豆素類(lèi)探針中以對(duì)氨基芐醇為連接基團(tuán)的探針能夠被APN降解,而以鄰氨基芐醇或者是3-氨基吡啶-2-甲醇為連接基團(tuán)的探針不能夠被APN降解。探針1.1-7a具有參比波長(zhǎng)性質(zhì),能夠降低環(huán)境因素對(duì)測(cè)量結(jié)果的影響,提高了測(cè)試準(zhǔn)確度。而且值得注意的是,該探針具有較高的靈敏度,降低了APN抑制劑IC50測(cè)試中酶的用量,從而降低了測(cè)試成本。該探針還簡(jiǎn)化了細(xì)胞水平APN酶活的測(cè)試方法,為APN抑制的篩選提供了一種準(zhǔn)確、方便、經(jīng)濟(jì)的測(cè)試方法。但是由于探針1.1-7a在降解前后的波長(zhǎng)均為藍(lán)色熒光,不適合在細(xì)胞成像中的使用,因此我們?cè)诖颂结樀幕A(chǔ)上進(jìn)一步優(yōu)化得到了氨基酸-萘二酰亞胺類(lèi)探針；氨基酸-萘二酰亞胺類(lèi)探針被APN降解前發(fā)藍(lán)色熒光,降解后發(fā)綠色熒光。在細(xì)胞成像時(shí),根據(jù)細(xì)胞中綠色和藍(lán)色熒光的強(qiáng)度比值可以定量地反映APN的活性。該類(lèi)探針的缺點(diǎn)是與APN反應(yīng)的速度太慢,這限制了其在抑制劑篩選中的使用：接下來(lái),我們?cè)诎被岷洼炼啺分虚g加上自發(fā)降解連接基團(tuán)后得到的氨基酸-自發(fā)降解連接基團(tuán)-萘二酰亞胺類(lèi)探針,與APN的反應(yīng)速度提高了8倍,使其能夠成功地應(yīng)用于APN抑制劑的IC50測(cè)定。這三個(gè)系列的探針不僅能夠?qū)Ψ菬晒庖种苿┻M(jìn)行IC50測(cè)定,并且通過(guò)聯(lián)合使用,能夠?qū)ΤＲ?guī)方法無(wú)法測(cè)量的熒光抑制劑進(jìn)行ICs0測(cè)定。此外,本文還設(shè)計(jì)合成了三個(gè)系列共21個(gè)具有熒光性質(zhì)的APN抑制劑,并對(duì)所合成的抑制劑進(jìn)行了體外抑酶活性測(cè)試以及計(jì)算機(jī)輔助的分子對(duì)接研究。系列1和系列2大部分化合物的活性均強(qiáng)于上市藥物Bestatin,化合物4.1-3a,4.1-3b,4.1-3k的活性高于先導(dǎo)化合物4q,其中化合物4.1-3k的活性比先導(dǎo)化合物高出了一個(gè)數(shù)量級(jí),比Bestatin高出了三個(gè)數(shù)量級(jí)。分子對(duì)接的結(jié)果顯示,活性相差較大的化合物與APN的結(jié)合模式不同。其次,4.1-3k能夠?qū)PN高表達(dá)的腫瘤細(xì)胞A549進(jìn)行染色,而對(duì)非腫瘤細(xì)胞HEK-293的染色強(qiáng)度明顯較弱,并且染色強(qiáng)度能夠被APN抑制劑4q顯著減弱。 綜上所述,本文共設(shè)計(jì)合成了3個(gè)系列可激活型APN熒光探針,它們能夠準(zhǔn)確、快捷地測(cè)試不同條件下的APN酶活性,并且能夠準(zhǔn)確地進(jìn)行非熒光抑制劑和熒光抑制劑的IC50測(cè)定,為今后APN抑制劑的初步活性篩選提供了一套完整的測(cè)試方法。此外,本文所設(shè)計(jì)合成的熒光抑制劑4.1-3k,其抑酶活性比上市藥物強(qiáng)100-1000倍,并具有顯著的抗腫瘤作用。而且能夠選擇性地標(biāo)記APN高表達(dá)的腫瘤細(xì)胞,將有望被開(kāi)發(fā)為既有治療作用又能夠用作疾病診斷的APN熒光抑制劑。
[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.

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R914;R96

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 張_",王毅,褚泰偉,劉新起,胡少文,王祥云;潛在的新型腫瘤顯像劑~(99)mTc-MAG_3-GGCNGRC的合成及生物分布[J];高等學(xué)校化學(xué)學(xué)報(bào);2005年05期

2 于欣;喬守怡;;腫瘤干細(xì)胞研究進(jìn)展[J];中國(guó)生物工程雜志;2010年01期

相關(guān)博士學(xué)位論文 前1條

1 王學(xué)健;小分子類(lèi)肽APN抑制劑的活性評(píng)價(jià)及其抗腫瘤作用機(jī)制研究[D];山東大學(xué);2010年

，

本文編號(hào)：1447538