【作者】 张金亮；

【导师】 王继东；

【作者基本信息】 吉林大学 ， 生物化学与分子生物学， 2006， 硕士

【摘要】 细胞凋亡是维持正常的免疫系统和正常的组织更新所必要的。Caspase在细胞凋亡这种重要的生理过程中位于中心地位,现在,已经有很多人工合成的肽类抑制剂,它们能够特异性地抑制蛋白酶使得减缓或抑制疾病的发展。所以,研制caspases酶的抑制剂将有望成为非常有效的治疗性药物。本论文中基于已报道的广泛性的Caspase抑制剂Z-VAD-CHO为模型,设计出了一种新型的Caspase抑制剂。在设计中我们用D型氨基酸去代替L型氨基酸,这样侧链空间结构得到了保留,只是肽键了方向有了改变,这将增强其对蛋白酶水解的抗性。另外,设计中保留了醛基,因此,这个抑制剂属于可逆的肽醛类抑制剂。我们已经合成出了的二肽醛和三肽醛。并进行了酶抑制活性的检测。更多还原

【Abstract】 The caspase (cysteinyl-aspartate protease) family represents a class ofintracellular proteases playing a critical role in apoptotic cell death pathways andactivation of pro-inflammatory cytokines. Their enzymatic properties are governedby a nearly absolute specificity for substrates containing aspartic acid at the P1 siteand by the use of a cysteine side-chain for peptide-bond hydrolysis. To date, 11human caspases have been identified. Since activation of caspase-dependentapoptotic cell death has been implicated in the etiology of many harmful humandisorders, such as immunodeficiency, Alzheimer′s, Parkinson′s, and Huntington′sdiseases, as well as ischemia, brain trauma, and amyotrophic lateral sclerosis,inhibitor of caspase is believed to be a valuable therapeutic approach.There are now many designed potent and selective protease inhibitors. Althoughcaspase inhibitors display a diverse range of biological properties, As effective andpractical drugs, they still have some deficiencies to be overcome, such asinstability, low bioavailability and poor pharmacological profiles. Therefore theseprotease inhibitors need to have minimal peptide characters, high stability tononselective proteolytic degradation, good membrane permeability, long lifetimesin the bloodstream and in cells, low susceptibility to elimination, high selectivityfor a protease, and good bioavailability (preferably by oral delivery).For many years intense work has been focused on the synthesis of peptideanalogues in the search for mimics with enhanced activity and biological half-lives.Examples of modifications introduced in peptides are the placement of L-aminoacid residues by D-amino acids (retro-inverso transformation) or by unnaturalresidues (e.g., sarcosine and β-alanine) and the modification of peptide bonds.These changes provide pseudopeptides or peptidomimetics with a higher metabolicstability, since most natural proteases cannot cleave D-amino acid residues andnonpeptide bonds. At present, the retro-inverso transformation remains animportant pseudopeptidic modification undertaken by numberous bioorganicchemists. Many partially modification retro-inverso (PMRI) analogs of peptideswere synthesized and tested. It was reported that reversed peptide not onlydisplayed stability toward enzymatic degradation but also improved bioavailabilityand potency. The remarkable resistance of PMRI analogs to proteolyticdegradation combined with retention of high biological activity following oral orintravenous administration provides a strong impetus for the continuing efforts onthis modification.In this paper, we designed the novel PMRI caspase-3 inhibitors based on theinhibitor Z-VAD-CHO. We replaced L-amino acids with D-amino acids, whichresulted in all the amide bonds reversed. However, C-terminal P1 site remained.Our synthesis progress consisted of three major steps, C-terminal synthesis,coupling and deprotection of the inhibitor and completion of the inhibitor. Amongthem, C-terminal compound synthesis is the key step. Part 1: succinic anhydridereacted with benzyl alcohol to give monobenzyl succinate. the product was treatedwith Lithium bis(trimethyl-silyl) amide in dry tetrahydrofurane. The ethyl formatewas added to afford the α-formyl α-benzyl succinate . Then, the aldehyde andcarboxyl groups were protected by reacting with the trimethyl orthoformate in thepresence of p-toluene sulfonic acid as the catalyst. Part 2: the benzyl group wasremoved by catalytic hydrogenation using 5% Pd/c. Peptide aldehyde derivativeswere formed by using the BOP method. Part 3 : the protective group of aldehydewas removed using 50% aqueous trifluoroacetic acid. In order to stabilizealdehyde group, we transformed them into its bisulfite adduct by adding saturatedsolution of sodium bisulfite. Now, we have already successfully synthesized theanalogues of Z-VAD-CHO and every product have been purified and characterizedby 1~H NMR or MS.The analogues have been tested for their ability to inhibit caspase-3 catalyzedproteolytic breakdown of its fluorogenic substrate, DEVD-AFC. In our experiment,we made use of the product of Biovision company, Caspase-3 Inhibitor DrugScreening Kit. We have demonstrated inhibitory activity ( IC50 ) of analogues are71μM and 43μM,according to the method provided by kit. Parallel experimentsdemonstrated that the IC50 value for Z-VAD-FMK, a potent tripeptide inhibitor ofcaspase-3 was equal to 1.7μM under the same experimental conditions.In summary, we have developed a general procedure for preparation ofretro-inverso peptidyl aldehydes, and synthesized PMRI caspase-3 inhibitor whichhas never been reported before. Bioassays shows the inhibitors have moderateactivity compared with that of the inhibitors reported previously. This is only ourprimary work, and the result is encouraging, suggesting that caspase-3 inhibitorsbuilt on this novel scaffold have the potential for future drug development.更多还原