【作者】 侯瑞珍；

【导师】 张学忠；

【作者基本信息】 吉林大学 ， 生物化学与分子生物学， 2007， 博士

【摘要】 RGD三肽序列是细胞外基质及体内多种粘附蛋白分子所共有的细胞粘附和分子识别位点，是一类含有Arg，Gly和Asp三种氨基酸的小肽。RGD三肽及其衍生物在抗肿瘤、抗血栓、治疗急性肾衰、抗炎、治疗骨质疏松、皮肤再生及生物材料工程等方面的研究已有越来越多的报道。由于RGD具有广泛的应用前景，所以已经成为极具研究价值的生物活性肽之一。本论文主要围绕RGD肽的合成、制剂及生物活性方面进行了研究。一、Alcalase酶催化下的RGD肽合成本文以工业用碱性蛋白酶alcalase作为催化剂，在动力学控制下合成了RGD的前体三肽Bz-RGD-（-NH₂）-OH，RGDS的前体二肽Bz-Arg-Gly-NH₂和Z-Asp-Ser-NH₂。通过考察溶剂体系种类、水含量、pH值、温度、反应时间、底物摩尔比等因素对酶促肽合成产率的影响，得出最佳的反应条件。Bz-RGD-（-NH₂）-OH合成是通过酶法和化学法相结合来完成的。这里所采用的化学法可以低成本、大规模地合成GD-（NH₂）₂。Bz-Arg-OEt和GD-（NH₂）₂之间的键合是通过酶法完成的。以alcalase为催化剂，Bz-Arg-OEt和GD-（NH₂）₂为底物，在水-有机溶剂双相体系中合成目标产物。合成该三肽的最佳反应条件是：Bz-Arg-OEt·HCl（0.05M），GD-（NH₂）₂（0.25M），三乙胺（35μl／ml体系），乙醇／0.1M Tirs-HCl pH 8.0缓冲液体系（85：15，V／V），35℃，反应时间8小时，产率达73.6％。Bz-Arg-Gly-NH₂、Z-Asp-Ser-NH₂是合成RGDS的两个前体二肽，本文以alcalase为催化剂，分别以Bz-Arg-OEt·HCl、Z-Asp-OMe为酰基供体，以Gly-NH₂、Ser-NH₂为亲核试剂，在有机溶剂／水双相体系中合成目标产物。Bz-Arg-Gly-NH₂合成的最佳反应条件：Bz-Arg-OEt·HCl（0.05M），Gly-NH₂（0.35M），三乙胺（14μl／ml体系），乙腈／0.1M Na₂CO₃-NaH CO₃ pH10.0缓冲液（90：10，V／V），45℃，反应时间1小时，产率达82.9％。Z-Asp-Ser-NH₂合成的最佳反应条件：Z-Asp-OMe（0.05M）and Ser-NH₂（0.35M），三乙胺（28μl／ml体系），乙腈／0.1 M Na₂CO₃-NaHCO₃ pH 10.0缓冲液（90：10，V／V），35℃，反应时间6小时，产率达75.5％。由上述结果我们可以得出如下结论：Alcalase酶在含有少量水的亲水性溶剂中（如乙醇、乙腈等）有很好的稳定性和催化活性，使亲水性小肽酶促合成可以在亲水性的溶剂中进行，解决了在亲水性小肽合成中需要兼顾底物溶解性和酶的活性及稳定性的问题。此外，从经济成本的角度出发，Alcalase是工业化酶，成本低，离心很容易沉淀下来，易于与产物分离并可以考虑重复使用的问题，适合规模化生产。尽管Alcalase酶目前还没有广泛用于肽合成中，但是它有望成为亲水性小肽合成中很有前景的催化剂。二、木瓜蛋白酶催化下的RGD肽合成本文研究了在纯水相体系中，碱性pH条件下，木瓜蛋白酶催化的Bz-RGD-OMe的合成，尽管在反应过程中合成的亲水性肽溶于反应体系中，但肽产物的二次水解不显著。最佳反应条件：Bz-RG（-OEt）和D（-OMe）₂·HCl分别为0.05M和0.15M，0.1M KCl／NaOH溶液作为反应介质（含有8mM EDTA和2mM DTT），pH8.5，40℃，反应时间为60min，最大转化率为62％。上述结果进一步证实，对于木瓜蛋白酶来说，采用动力学控制，在pH＞8时其水解酶的活性受到抑制，酯酶活性却很明显。在这个条件下能够合成出可溶性肽，并且不会发生二次水解。三、RGD肽的化学法合成本文根据RGD结构上有一个Gly这个特点，巧妙地设计了RGD合成的新路线。GD二肽合成采用了新颖的化学法，包括天冬氨酸的氯乙酰化和酰化产物的氨解。三肽的合成采用N-羧基内酸酐法，首先将苄氧羰酰精氨酸用催化剂催化使生成N-羧基内酸酐精氨酸，然后与游离的GD二肽缩合生成RGD三肽。这一路线中采用了较少保护的氨基酸，路线低成本、简单、快速，产率为62％。四、脂质体制备本文采用逆相蒸发法制备了RGD脂质体，在制备过程中对各种影响因素进行了考察，采用正交实验进行优化，确定最佳的工艺条件：即卵磷脂／胆固醇比例为5：1，RGD 16mg，PBS 8ml，超声时间5min。制得的脂质体平均粒径为473nm。包封率62.99％。稳定性实验证明RGD脂质体在4℃低温存放时较稳定，温度升高稳定性下降。随时间的延长包封率有下降的趋势。尤其是超过两个月包封率急剧下降。体外释放度实验证明RGD脂质体有一定的缓释效果。五、RGD抗肿瘤作用本文对上述化学法合成RGD肽进行了活性检测，细胞增殖实验、细胞粘附实验、细胞迁移实验证明：RGD肽对人纤维肉瘤细胞HT1080、人小细胞肺癌细胞NCI-H446、人肝癌细胞SMMC-7721增殖、在FN上粘附及迁移有一定的抑制作用。更多还原

【Abstract】 Many adhesive proteins such as fibronectin, vitronectin, collagens, and osteopontin are present in extracellular matrices and in the blood cells, which contain the Arg-Gly-Asp （RGD） sequence as their cell recognition sites. The RGD motif plays a key role in mediating integrin-matrix interaction and RGD-sequence containing peptides are known to be potent anti-adhesive molecules, as they compete for the integrin-matrix recognition process, their anti-metastatic、anti-thrombotic and anti-inflammatory effects、acute renal failure、osteoporosis and skin regeneration therapy have been largely investigated and showed relevant potential clinical applications. Therefore, the characteristic tripeptide sequence Arg-Gly-Asp has been attracting much attention of investigators.It is difficult to isolate RGD from whole tissue directly, so the adhesiveness of exogenetic RGD has been target in drug design. Many researchers tried to synthesize RGD and RGD-containing peptides by chemical or enzymatic methods. Chemical and enzymatic method has different characteristic and suit different synthesis scale. Chemical method is suitable for synthesis the moderate length peptide and with large-scale. An enzymatic method is suitable for synthesis the short peptide and with small-scale. As compared with the chemical method, the important benefits of enzymatic peptide synthesis are: a) the mild conditions of the reaction; b) the high regiospecifity of enzyme allowing the use of minimally protected substrates; c) the reaction being stereospecificity without racemization. At present, many hydrophobic small peptides were synthesized in high yield using proteases in organic media as largely reported. However, the formation of peptide bonds between hydrophilic amino acids faced many problems. Hydrophilic substrate has low solubility in hydrophobic organic solvents, hindering us to use hydrophobic organic solvents where the enzyme usually shows better activity and selectivity than in hydrophilic organic solvents. Hydrophilic organic solvents are suitable as the reaction media to enhance the solubility of hydrophilic substrates. However, hydrophilic solvents are often harmful to enzyme because it has a greater tendency to strip the tightly bound essential water from the enzyme molecules. The appropriate reaction system should be selected considering the balance of the solubility of substrates and enzymatic activity.In this study, RGD（S） is a good hydrophilic peptide model containing three charged or polar residues （Arg, Asp and Ser） and a neutral one （Gly）. Considering the solubility of hydrophilic substrates, We selected hydrophilic organic solvents such as ethanol、acetonitrile as the reaction media. A new and practical enzymatic procedure for peptide synthesis using alcalase as a biocatalyst was studied. Chen et al. successfully synthesized a number of peptides in anhydrous alcohol with yields up to 94% in 1992. Recently, Klein et al also reported the synthesis of peptide bonds catalyzed by subtilisin Carlsberg in different hydrophilic organic solvents with variable water concentration. So we can deduce alcalase has better stability and activity in polar organic solvents such as alcohols, acetonitrile, DMF, etc. Alcalase is a suitable biocatalyst to catalyze peptide bond formation in hydrophilic organic solvents. Alcalase was used to catalyze the synthesis of Bz-RGD-（-NH₂）-OH （precursor tripeptide of RGD）、Bz-Arg-Gly-NH₂和Z-Asp-Ser-NH₂ （precursor dipeptides of RGDS） under kinetic control condition in water-organic cosolvents systems. The synthesis reaction conditions were optimized by examining the effects of several factors including water content, temperature, pH, and reaction time on the yields. The results showed①The optimum conditions for Bz-RGD-（-NH₂）-OH synthesis were Bz-Arg-OEt·HCl （0.05M）, GD-（NH₂）₂ （0.25M）, triethylamine （35μl/ml system）, in ethanol/0.1M This-HCl pH8.0 buffer system （85:15, V/V）, 35℃, 8h with the maximum yield of 73.6%.②The optimum conditions for Bz-Arg-Gly-NH₂ synthesis were Bz-Arg-OEt·HCl （0.05M）, Gly-NH₂ （0.35M）, triethylamine （14μl/ml system）, in acetonitrile/0.1M Na₂CO₃-NaHCO₃ pH 10.0 buffer system （90:10, V/V）, 45℃, 1h with the dipeptide yield of 82.9%.③The optimum conditions for Z-Asp-Ser-NH₂ synthesis were Z-Asp-OMe （0.05M）, Ser-NH₂ （0.35M）, triethylamine（28μl/ml system）, in acetonitrile/0.1M Na₂CO₃-NaHCO₃ pH 10.0 buffer system （90:10, V/V）, 35℃, 6h with the dipeptide yield of 75.5%.In this paper, synthesis of Bz-RGD-OMe catalyzed by papain at alkaline pH under the kinetic control in full aqueous medium is described. The substrates are N-benzoyl-argininylglycine ethyl ester and asparagine dimethyl ester. 0.1M KCl/NaOH aqueous solution containing 8mM EDTA and 2mM DTT was selected as the reaction medium. The synthesized hydrophilic tripeptide was soluble in the reaction medium during the reaction process, however, the secondary hydrolysis of the tripeptide product was not considerable. The effects of different factors, including water content, temperature, reaction time and molar ratio of the substrates, on the yield of Bz-Arg-Gly-Asp-OMe were examined. The optimal reaction conditions are 0.05M Bz-Arg-Gly-OEt and 0.15M Asp（-OMe）₂·HCl in 0.1M KCl/NaOH solution（pH 8.5）, 40℃and reaction time of 60 min with the maximum conversion yield of 62%. As far as papain is concerned, its peptidase activity at pH＞8 is negligible, while the esterase activity is still significant, it is possible to synthesize soluble peptides in aqueous medium under kinetic control and alkaline pH value without obvious secondary hydrolysis of peptide product.We attempted to design a simple chemical synthesis route. RGD can be synthesized from N-terminal to C-terminal or from C-terminal to N-terminal. In previous literature, mixed anhydrides method、DCC method and activated ester method were generally applied, it is necessary that certain functional groups of amino acid such as the amino groups, the carboxyl groups and side chain groups must be blocked, otherwise this can be the source of serious complications and side reactions will occur. In addition, the protecting groups need to be removed after the peptide bond formation. In our study, we designed a novel and simple synthesis route, Firstly, Gly-Asp was synthesized by a novel chemical method in two steps including chloroacetylation of L-aspartic acid and ammonolysis of chloroacetyl L-aspartic acid. Secondly, N₀-Z-L-Arginine was reacted with Gly-Asp to yield RGD tripeptide by the N-carboxyanhydride method. Less protected amino acids were used in this synthesis. This method possessed low cost、simple and rapidity with a reasonable yield 62% calculated from arginine. In additional, compared with above method, a conventional solid phase method was also used to synthesize RGD in this paper, the yield was 75% calculated from the first amino acid anchored to resin.RGD have small molecular weight and can be easily decomposed by enzyme, their half life in vivo are very short. Some efforts had been made to increase circulating time and enhance their stability by modificating RGD’s structure. The synthetic substance such as RGD repetitive sequence, RGD-conjugated with polyethylene glycol or CM-chitin; RGD-containing pseudopeptide and peptidomimetics. However, RGD amounts these linear peptides contained are limited. In this paper, RGD liposome was designed. Liposome is a new drug delivery. It has been widely used in medical area. Liposome has many characters: longest effect, target effect, slow release, reducing dose frequency. RGD liposome was prepared by reverse phase evaporation method. The impact of every factor on the encapsulation efficiency was investigated, and the factors were optimized by orthogonal design The optimum conditions for RGD liposome preparation: the mass ratio of egg phosphatidylcholine to. cholesterol 5:1, RGD 16mg, the volume of phosphate buffer 8ml, sonicating 5min, its mean size was 473nm. the highest encapsulation efficiency was 62.99%. The liposomes should be stored under lower temprerature, because the stability is not good enough under higher temperature. There is no any phenomenon of layering and emulison damage after storing under 4℃, 25℃respectively for 2 months. There is a big decrease of encapsulation efficiency when time is more than 2 months. In vitro, the release test showed RGD liposome had slow release effect.The effects of the synthetic tripeptide RGD on the proliferation、adherence and motility of HT1080、NCI-H446 and SMMC-7721 cells have been studied. The results indicated that RGD peptide had inhibitory effects on the proliferation and adherence of HT1080、NCI-H446 and SMMC-7721 cells to fibronectin. It also could restrain motility of HT1080、NCI-H446 and SMMC-7721 cells on fibronectin.更多还原