The biological activities of synthetic calciseptine and FS2, a homologous peptide from snake venom, were determined using in vitro and in vivo preparations. Calciseptine and FS2 produced dose-dependent relaxation in pre-constricted rat aorta, pulmonary artery and trachea. The onset and duration pattern of these relaxing effects were similar to those caused by nifedipine, an L-type Ca2+ channel blocker. Calciseptine relaxed the contraction of rat aorta provoked by an L-type channel agonist, Bay K 8644. This relaxation was not affected by NG-nitro-L-arginine, indomethacin or propranolol. Calciseptine and FS2 inhibited the contraction caused by acetylcholine in guinea pig ileal longitudinal muscle. In case of in vivo study using anesthetized rats, calciseptine, FS2 and nifedipine showed depressor effects. The hypotensive effects of the two peptides were more potent and sustained than that of nifedipine. These findings show that both synthetic calciseptine and FS2 have similar biological activities like nifedipine, an L-type Ca2+ channel blocker. In addition, these two peptides with large molecular weights may be unique and useful tools for studying the Ca2+ channel.

Calciseptine, an L-type specific calcium channel blocker consisting of 60 amino acid residues with four intramolecular disulfide bonds, was synthesized by the solution procedure applying our maximum protection strategy. The recently developed solvent systems used were a mixture of chloroform or dichloromethane and trifluoroethanol; they were useful for the synthesis of slightly soluble protected peptides. After removal of all the protecting groups by our two-step HF procedure followed by treatment with mercuric acetate, the peptide was folded into its native form by air oxidation, in which redox reagents were necessary to accelerate the correct disulfide bond formation. The product was purified to homogeneity and found to block contractions of the rat thoracic aorta induced by 40 mM K+ with the same potency as that of the natural product. In addition, this peptide was found to block contractions of various rat smooth muscle preparations as well as increase in cytosolic free calcium concentration induced by a high concentration of extracellular K+.

Our maximum protection strategy for the synthesis of human parathyroid hormone(1-84) indicates that fully protected peptide segments in the form of Boc-peptide phenacyl (Pac) ester are relatively soluble in ordinary organic solvents such as DMF, NMP or DMSO, which are suitable for coupling segments. However, about 1% of such segments synthesized were found to be insoluble even in the most polar solvent, DMSO. Thus, a more powerful solvent which can be used for their peptide synthesis was pursued. Among the solvent systems tested, a mixture of trifluoroethanol (TFE) or hexafluoroisopropanol (HFIP) and trichloromethane (TCM) or dichloromethane (DCM) was found to be most powerful for dissolving such sparingly-soluble protected peptides. These solvent systems were confirmed to be useful for the removal reaction of the carboxy-terminal Pac esters from the sparingly-soluble segments. They were then tested for the coupling reactions of fully protected Boc-peptides with other sparingly-soluble peptide esters. The TFE/TCM or TFE/DCM system was extremely useful for coupling segments without danger of racemization and of trifluoroester formation, if WSCI was used as the coupling reagent in the presence of 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (HOOBt).

When L-proline or O-benzyl-trans-4-hydroxy-L-proline phenacyl ester was coupled with Boc-amino acids in dimethylformamide using water-soluble carbodiimide (WSCI) in the presence of anhydrous 1-hydroxybenzotriazole (HOBt) as coupling reagents, extensive racemization was observed at the C alpha of the proline or hydroxy-proline residue. The extent of racemization was measured by HPLC after the coupling with Boc-L-Leu-OH in the presence or absence of HOBt. The extent of racemization increased when HOBt was added to the reaction mixture, but greatly decreased when it was not, indicating that HOBt was needed for inducing racemization. Almost no racemization was observed when the coupling reaction was carried out by the mixed anhydride procedure in tetrahydrofuran or by the carbodiimide method in dichloromethane without using HOBt. In the case of coupling reactions with ordinary L-amino acid phenacyl esters, no racemization was observed. Examination of some model systems yielded sufficient evidence to prove that HOBt is an efficient catalyst for racemizing proline or hydroxy-proline phenacyl ester not only in the stage of cyclic intermediate formation but also in the opening of the ring structure. Thus, the racemization reaction was found to be closely related to the formation of the cyclic carbinol-amine derivative.

Charybdotoxin, a 37 amino acid peptide which is a minor component of Leiurus quinquestriatus venom, was synthesized by the solution procedure applying our maximum protection strategy. After formation of the three disulfide bonds, for which a redox buffer was necessary, the final product was purified to homogeneity and found to have similar biological potency to that reported by others for the natural product. The disulfide bond configuration was found to be: Cys7-Cys28; Cys13-Cys33; Cys17-Cys35. Conformational analysis by 1H-NMR showed that the molecule exists as a very tightly folded structure, in which residues 1–7 and 24–37 form a triple-srranded β-sheet, with a turn at positions 30–31. The region from 11–20 appears to adopt an α-helical conformation.