Endothelin (ET-1), a 21-residue peptide, characterized from aortic endothelial cells by Yanagisawa, et al., has a potent vasoconstrictor effect. Studies on cloning and sequencing of prepro ET cDNA have revealed the existence of the putative pecuraor of ET-21, named big endothelin (big ET), consisting of 38 amino acid residues. In addition, other types of ET were found and named ET-II, and ET-III. Big ET could be further processed to ET-21 by a unique endopeptidase named "enodothelin converting enzyme" (ECE). In order to evaluate the physiological significance of ET and clarify the chemical nature of ECE, we have synthesized ETs (ETI,II,III,) human big ET and their related peptides by two alternative solid phase method. In the course of this synthetic studies, a new Sdeprotecting reagent, silver trifluoromethanesulphonate, and a new two step hard acid deprotection/cleavage procedure [1._3SiBr deprotection and 2. HF(or Me_3SiOSO_2CF_3) cleavage] were developed.
Boc-based solid phase synth
esis of ET-1 was carried out by using automatic synthesizer. As amino acid derivatives, Cys(Acm), Asp(OBzl), Glu(OBzl), Ser(Bzl), Thr(Bzl), Trp(CHO), His(Tos), Tyr(BrZ), Lys(CIZ), and Arg(Mts) were employed. In the final step, the above two new methods were successfully employed to give a biologically active ET-I. In essentially the same manner, human big endothelin (ET-38) and C-terminally elongated homologs of ET-I, such as ET-22, ET-23, ET-31, and ET-36 were prepared. ET-II and ET-III were synthesized manually by Fmoc-based solid phase synthesis according to the principle of Sheppard, et al., using S-p-methoxybenzyl (MBzl) protection for 4 Cys residues. ETs, big ET, and their related peptides thus synthesized were assessed to the several bioassay systems.
The in vitro vasoconstrictor activity of human big ET was two orders of magnitude less than that of ET-I. However, in vivo assay in rats, human big ET caused a dose dependent rise of arterial pressure after the cumulative administration in essentially the same potency as that of ET-I. The activities of C-terminally elongated peptides, ET-22, ET-23, ET-31, and ET-36, were comparable to that human big ET. Thus, it was suggested that big-ET and all of C-terminally elongated peptides might be processed directly to ET-21 by ECE in animal body.
3.ビッグエンドセリン1及びエンドセリン1のC末端をいろいろな長さ延長した誘導体の合成 システインの側鎖保護基にメトキシベンジル基を用い、Boc固相合成法で合成した。これらの合成品のラットへ反復投与したところ、どのペプチドもエンドセリン1とほぼ同程度の容量依存性の血圧上昇効果を示した。この結果により、ビッグエンドセリン1及びエンドセリン1のC末端を延長した誘導体は動物体内で直接エンドセリン1にプロセッシングされる可能性が示唆された。 Less