摘要
【目的】本课题旨在通过引入可诱导cis/trans异构变化的伪脯氨酸(ΨPro),构建高cis构象含量的昆虫激肽类似物,并用蟑螂后肠收缩活性检测来阐明C端五肽H-Phe-Xaa-Yaa-Trp-Gly-NH_2的优势活性构象。Pseudoprolines(ΨPro)是由Ser/Thr和Cys与醛或酮环化缩合而成的Pro类似物,其脯氨酰环中的C_γH_2基被氧或硫原子取代。将ΨPro引入多肽中能够提高Xaa-Pro肽键中的cis构象的比率。【方法】本文描述了由Ser和Cys经过甲醛和2,2-二甲氧基丙烷经缩合环化反应形成伪脯氨酸Ser(~(Me,Me)Pro)-OH、Cys(~(H,H)Pro)-OH和Cys(~(Me,Me)Pro)-OH的过程,同时还描述了昆虫激肽H-Phe~1-Phe~2-Pro~3-Trp~4-Gly~5-NH_2及其ΨPro~3代类似物通过Fmoc固相合成方法的合成,用ESI-MS和HPLC对昆虫激肽类似物的结构和纯度进行分析。【结果】通过用不同ΨPro取代的昆虫激肽类似物进行促蟑螂后肠收缩活性研究,结果表明Pro-kinin、Ser(Me,Me)-kinin、Cys(H,H)-kinin和Cys(Me,Me)-kinin分别保留了昆虫激肽Achetakinin 1在1.1×10~7M时62%、93%、68%和88%的最大活性;而与之对应的EC_(50)值分别为2.81×10~(-8)M,3.74×10~(-8)M,2.12×10~(-8)M和1.79×10~(-8)M。【结论】以上实验结果表明,含具有诱导高顺式构象的双甲基取代的ΨPro昆虫激肽类似物有更高的活性,有利于形成Phe~1-Trp~4 cis-ProⅥ型β-转角的cis构象是昆虫激肽类似物产生生理活性的优势活性构象。
[Objective]In this work,pseudoprolines(ΨPro) was introduced to substitute the Pro residue in Incsect Kinin Core pentapetide H-Phe~1-Phe~2-Pro~3-Trp~4-Gly~5-NH_2 for forming a Xaa_(i-1)-Pro_i cis peptide bond,which can be strongly enhanced by the introduction of aΨPro residue depending on its C(2) substituents,to elucidate the bioactive conformation of the H-Phe~1-Phe~2-Pro~3-Trp~4-Gly~5-NH_2 using the myotropic activities on isolated cockroach hindgut.[Methods]Ser(~(Me,Me) Pro)-OH,Cys(~(H,H) Pro)-OH and Cys(~(Me,Me) Pro)-OH were obtained by cyclocondensation of the amino acids cysteine,threonine,or serine with aldehydes or ketones.Incsect Kinin and its analogs were synthesized by Fmoc solid peptide synthesis methods,the structures were analyzed with ESI-MS and HPLC.[Results]After measured with the cockroach hindgut myotropic activity of theΨPro analogs,the results indicated that Pro-kinin, Ser(Me,Me)-kinin,Cys(H,H)-kinin and Cys(Me,Me)-kinin have the activity of AchetakininⅠ62%,93%,68%and 88%,respectively,shown that the 2,2-dimethylated neuropeptide analogues appeared higher effect than others. [Conclusion]The cisΨPro~3-kinin conformer is the bioactive conformation when interact with kinin receptor on cockroach hindgut.Theβ-turn preference of residues Phe~1-Phe~2-Pro~3-Trp~4 implicates this as the bioactive conformation.
引文
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