Solid-Phase Synthesis with Attachment of Peptide to Resin through an Amino Acid Side Chain: [8-Lysine]-Vasopressin

Abstract

It is proposed that the scope of solid-phase peptide synthesis could be considerably broadened by attaching peptides to the solid-phase through functional side-chain groups rather than through the commonly used a-carboxyl groups. Side-chain attachment offers the use of a large variety of chemical linkages to solid supports. Attachment through the e-amino group of the lysine residue to a polystyrene resin has been applied to a solid-phase synthesis of lysine-vasopressin. Na-tert-butyloxycarbonyl-L-lysyl-glycinamide was condensed with chloroformoxymethyl polystyrene-2% divinylbenzene resin. After removal of the Na-protecting tert-butyloxycarbonyl group, the peptide chain was elongated by standard Merrifield procedures to give Tos-Cys(Bzl)-Tyr-Phe-Glu-(NH2) -Asp(NH2) -Cys(Bzl) -Pro -Lys(Z -resin) -Gly-NH2. Cleavage from the resin with HBr in dioxane or trifluoroacetic acid gave a partially protected nonapeptide hydrobromide. For purification, it was converted into a fully protected peptide by treatment with benzyl p-nitrophenyl carbonate and crystallized. Deprotection by sodium in liquid ammonia, oxidative cyclization, IRC-50 desalting, and ion-exchange chromatography gave lysinevasopressin with high potency in a rat-pressor assay.The solid-phase method of peptide synthesis, developed by Merrifield (1, 2), makes possible the chemical syntheses of proteins. Synthetic achievements to date include syntheses of bovine insulin (3) in 1966, an analog of horse heart cytochrome c (4) and bovine pancreatic ribonuclease A (5) in 1969, and human growth hormone (6) in 1970. The advantages of the solid-phase method lie in (a) unprecedented speed, (b) simplicity of operation, (c) elimination of solubility problems, (d) minimal danger of racemization, and (e) possibility for automation. However, at its present state of development, the procedure suffers from several serious shortcomings. Incomplete peptide-bond formation (a), potentially occurring in each peptide-coupling step, results in so-called failure sequences and truncated sequences (7). The reagents (b) that must be used to remove the final product from the solid support often give incomplete cleavage and (or) partial degradation of the peptide. Analytical methods (c) sensitive enough to accurately monitor the process and to assess the purity of the products are not yet available.Some of the many efforts to improve the solid-phase method have focused on variations of the solid-phase (1, 8-16), or on facilitating formation of the commonly used benzyl-ester bond (17)(18)(19) between the starting C-terminal amino acid and the standard resin (chloromethyl polystyrene-2% divinylbenzene). In all of these investigations, the attachment to the solid-phase has been made through the a functional groups of amino acids or peptides (2). We wish to suggest that attachment to a solid-phase through functional groups in sidechains of amino acids could, (a) considerably broaden the chemical scope of such investigations, and (b) eliminate any danger of partial racemization of the am...

Design, Synthesis, Characterization and Preliminary Anticancer Study for Methotrexate Silibinin Conjugates

“…After 5 min, a cooled mixture of paranitrophenylchloroformate (2.17 mmol,0.443 g) in toluene (5 ml)was added drop wise . The mixture was stirred for 2 hr at -10°C and for 15 hr at room temperature, filtered,and evaporated to dryness under reduced pressure.The residual oil was crystallized twice from diethyl ether (150ml) to give a yellow powder (26) .…”