Sandberg B E, Lee C M, Hanley M R, Iversen L L
Eur J Biochem. 1981 Feb;114(2):329-37. doi: 10.1111/j.1432-1033.1981.tb05152.x.
Six analogues of substance P were synthesized with the aim of developing a metabolically stable peptide that would retain the biological activity of substance P. A recently isolated and characterized substance-P-degrading enzyme from human brain with a high specificity for substance P described in the preceding paper in this journal was used as a model for the enzymatic inactivation of substance P. The synthetic analogues were designed to protect the peptide bonds on the carboxyl side of residues 6, 7 and 8 of substance P, which represent the sites of cleavage by substance-P-degrading enzyme. To test for increased enzymatic resistance, the analogues were incubated with the enzyme, the digests were separated on a high-performance liquid chromatography reverse-phase column and the peptide fragments were collected and identified by amino acid analysis. Of the analogues described, an heptapeptide analogue of residues 5-11, less than Glu-Gln-Phe-MePhe-MeGly-Leu-MetNH2, showed almost complete resistance both towards substance-P-degrading enzyme and to degradation on exposure to rat hypothalamic slices. This analogue was about a third as potent as substance P in competing for binding to receptor sites for this peptide in rat brain membranes and a tenth as potent in eliciting contractions of the guinea pig ileum. The peptides were synthesized using the solid-phase technique with polydimethylacrylamide as a solid support and the coupling was achieved with pre-formed symmetrical anhydrides in dimethylacetamide. Fluorenylmethyloxycarbonyl was used as an alpha-amino protecting group in conjunction with t-butyloxycarbonyl as an epsilon-amino protecting group. Ammoniolytic cleavage from the resin was followed by stepwise elution from an SP-Sephadex column, deprotection with trifluoroacetic acid and chromatography on a Bio-Rex 70 ion-exchanger. The peptides were finally purified on a semi-preparative reverse-phase column.
合成了P物质的六种类似物，目的是开发一种代谢稳定的肽，该肽将保留P物质的生物活性。在本期刊上一篇论文中描述的最近从人脑中分离并表征的对P物质具有高特异性的P物质降解酶，被用作P物质酶促失活的模型。设计合成类似物以保护P物质残基6、7和8羧基侧的肽键，这些肽键代表P物质降解酶的切割位点。为了测试酶抗性的增加，将类似物与酶一起孵育，消化物在高效液相色谱反相柱上分离，肽片段被收集并通过氨基酸分析进行鉴定。在所描述的类似物中，残基5-11的七肽类似物，少于Glu-Gln-Phe-MePhe-MeGly-Leu-MetNH2，对P物质降解酶和暴露于大鼠下丘脑切片时的降解几乎都表现出完全抗性。在与大鼠脑膜中该肽的受体位点竞争结合方面，该类似物的效力约为P物质的三分之一，在引发豚鼠回肠收缩方面的效力为十分之一。使用以聚二甲基丙烯酰胺为固相载体的固相技术合成肽，并用在二甲基乙酰胺中的预制对称酸酐实现偶联。芴甲氧羰基用作α-氨基保护基，叔丁氧羰基用作ε-氨基保护基。从树脂上进行氨解裂解，然后从SP-葡聚糖凝胶柱上逐步洗脱，用三氟乙酸脱保护，并在Bio-Rex 70离子交换剂上进行色谱分离。肽最终在半制备反相柱上纯化。
