【作者】 杨龙飞；

【导师】 董守良；

【作者基本信息】 兰州大学 ， 生物化学与分子生物学， 2010， 硕士

【摘要】 目的：脯氨酸是蛋白质序列中常见的天然氨基酸之一,对蛋白质高级结构的形成具有重要作用。在生物大分子的研究中,可以用脯氨酸的衍生物替代它,通过其特定的侧链基团引入各种标记分子用于生物学行为的跟踪。氨基脯氨酸是常用的脯氨酸衍生物,其侧链基团为NH2,氨基与亚氨基的高效、快速的保护策略对其后期的应用价值具有重要意义。胶原蛋白中含有大量的脯氨酸与羟基脯氨酸,在结构上与氨基脯氨酸极其相似,我们以合成的双保护4-氨基脯氨酸作为中间连接体,将标记性小分子引入到胶原模型肽中,以期通过小分子的特征性变化来揭示胶原的体外动力学特征。方法：本文以非天然氨基酸(2S,4R)-氨基脯氨酸为原料,氨基与亚氨基经苄氧羰基(CBZ)临时性保护、羧基经苄基(Bn)临时性保护,再利用氨基与亚氨基结构上特有的差异,在氨基上引入第二个保护基叔丁氧羰基(Boc),然后经催化氢化脱除氨基与亚氨基上的CBZ与羧基上的Bn,最终使用9-芴甲氧羰基(Fmoc)保护亚氨基,采用此全新的方法合成了氨基与亚氨基选择性双保护的(2S,4R)-氨基-脯氨酸。产物经ESI-MS、1H-NMR、13C-NMR、比旋光度鉴定。通过固相合成的方法将其引入人工合成的胶原模型23肽Ac-(Gly-Pro-Hyp)3-Gly-Pro-Yaa-(Gly-Pro-Hyp)3-Gly-Gly-NH2的Yaa上,利用酰胺化反应其将标记性小分子自由基与荧光素分别引入到胶原肽单链中的侧链自由NH2上得到自由基或荧光素标记的胶原模型肽,以期通过自由基与荧光素的光谱学特征来研究胶原三螺旋的折叠与解折叠动力学过程。结果与结论：经五步反应最终合成的Fmoc-(2S,4R)-Amp(Boc)-OH总产率可达到47.23%,这比以往以羟基脯氨酸为原料经叠氮途径合成相比具有高产率、高效率、反应步骤精简等优点,可以在短时间内得到选择性双保护的氨基脯氨酸。我们成功地将其引入到胶原模型23肽序列中,并以其4位裸露的NH2为目标位点将标记性小分子3-羧基氮氧自由基与5-羧基荧光素连接到此胶原肽序列上。更多还原

【Abstract】 Objective:Proline is one of the common canonical amino acids which plays an important role in protein stucture. For convenient connection of labeling molecules to large biomoleculars for tracking various biological activities, derivatives with specific side chains of proline were introduced to large moleculars as replacement of proline. Amino proline is a commonly used proline derivative whose side chain is amino group, and amino and imino groups should be efficiently and conveniently protected for application. Collagen contains large amounts of hydroxy proline which is very similar to amino proline in structure, therefore double-protected 4-amino-proline was synthesized as an intermediate for introducing small labeling molecules into collagen model peptides, and changes of small labling molecules after replacing hydroxy proline with amino proline were measured in order to reveal the dynamic kinetics of the structure change of collagen in vitro.Methods:A non-canonical amino acid (2S,4R)-amino-proline whose amino and imino group were protected by benzyloxycarbonyl (CBZ), and carboxyl group by benzyl (Bn) temporarily was used. The second protecting group tert-Butoxycarbonyl (Boc) was added to amino group because of the specific structure difference of amino and imino group. Then CBZ and Bn were deprotected by hydrogenation. Finally, imino group was protected by 9-Fluorenylmethyloxycarbonyl (Fmoc). Then a selective dual-protection (2S, 4R)-amino-proline were obtained by the totally new strategy above and then it was characterized by ESI-MS,1H-NMR,13C-NMR and specific rotation. The dual-protected (2S,4R)-amino-proline was introduced into Yaa position of collagen model 23 peptide [Ac-(Gly-Pro-Hyp)3-Gly-Pro-Yaa-(Gly-Pro-Hyp)3-Gly-Gly-NH2] which was synthesized by solid-phase peptide synthesis. Then the amidation reaction was used to conjunct the free radical and fluorescein molecule with the free amino group of collagen model peptide to create free radical or fluorescein labeled collagen model peptide, as to study the kinetic process of folding and unfolding of collagen triple helix through spectroscopy. Results andConclusion:Synthesis of Fmoc-(2S,4R)-Amp(Boc)-OH reaches a total yield of 47.23% via five-steps. Compared to other methods utilizing hydroxyproline as substrate, this new route has several advantages as higher yield, efficiency and much more convenience. In our study, (2S,4R)-amino-proline were introduced into the 23 peptide collagen model successfully, and 3-carboxy nitroxyl radical and 5-Carboxyfluorescein were conjuncted to the peptide via the exposed 4-NH2.更多还原