Synthesis of deaminooxytocin by the solid phase method

Takashima, Herbert; Vigneaud, Vincent du; Merrifield, R. B.

doi:10.1021/ja01007a038

Cited by 77 publications

(30 citation statements)

References 3 publications

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“…Deprotection of the Boc group was achieved with 4 M HCl in dioxane, except in the case of glutamine where tri- fluoroacetic acid was used to minimize the possibility of pyroglutamyl chain termination (17). At the tryptophan and all following amino acid addition steps, 1% mercaptoethanol was added to the 4 M HCl-dioxane reagent, and to subsequent dioxane washes, to minimize oxidation of the labile tryptophan residue (18).…”

Section: Materials and Methods Chemicalmentioning

confidence: 99%

Synthesis of a Biologically Active N-Terminal Tetratriacontapeptide of Parathyroid Hormone

Potts

Tregear

Keutmann

et al. 1971

Proc. Natl. Acad. Sci. U.S.A.

201

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Determination of the amino acid sequence of bovine parathyroid hormone has led to the synthesis of a tetratriacontapeptide corresponding to the aminoterminal 1-34 residues of the native molecule. The specific biological effects of this synthetic peptide on bone and kidney are qualitatively identical to those of the native hormone in classical bioassays in vivo and in several systems in vitro. Potency of the synthetic peptide equals or exceeds that of a biologically active fragment of comparable size isolated from the native hormone; the synthetic and natural peptides show complete immunological crossreactivity. Thus, essential requirements for the physiological actions of the peptide on both skeletal and renal tissue are contained within the 34 amino-terminal amino acids. The potency of the synthetic peptide, relative to that of the native (84-amino acid) polypeptide, is greater in vitro than in vivo; this suggests that the carboxyl terminal two-thirds of the native hormone may protect the circulating polypeptide from rapid metabolic degradation.

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Section: Materials and Methods Chemicalmentioning

confidence: 99%

Synthesis of a Biologically Active N-Terminal Tetratriacontapeptide of Parathyroid Hormone

Potts

Tregear

Keutmann

et al. 1971

Proc. Natl. Acad. Sci. U.S.A.

201

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“…In the activeester cycles (Boc-Asn-ONp, Boc-Gln-ONp) step g was: R, 3.3-5 equivalents of active ester, dimethylformamide, [4][5][6][7][8][9][10][11][12][13][14][15][16] hr. The tert-butyloxycarbonyl protecting group was removed from the asparagine and glutamine residues with anhydrous trifluoroacetic acid (step b, 15 min) (24). After completion of the protected nonapeptide, the peptide-resin was removed from the reaction vessel, thoroughly washed with methylene chloride, ethanol, and ether, and dried under reduced pressure (P205 and KOH).…”

Section: Methodsmentioning

confidence: 99%

“…The C-terminal dipeptide derivative, Na-tert-butyloxycarbonyl-NE-benzyloxycarbonyl-ilysyl-glycinamide (3) was catalytically hydrogenated to yield the free e-amino group and condensed with 1. For chain elongation, the general procedure (22) for solid-phase synthesis was used execpt that (23) the reaction times were generally longer and the N-protecting groups were removed from the asparagine and glutamine components by treatment with anhydrous trifluoroacetic acid (24). Dicyclohexylcarbodiimide (25) was used as the coupling reagent for tert-butyloxycarbonyl-iproline (26,27), tertbutyloxycarbonyl-i4phenylalanine (26,27), tert-butyloxycarbonyl-ityrosine (26,27), and S-benzyl-N-tosyl-icysteine (28,29).…”

mentioning

confidence: 99%

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

Meienhofer

Trzeciak

1971

Proc. Natl. Acad. Sci. U.S.A.

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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...

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“…Ammonolysis Peptide amides have been synthesized by ammonolysis of polymerbound benzyl esters prepared from nitrated Merrifield linker 2.12 [92] or 4-hydroxymethyl-benzoylaminomethyl resin (HMBA) linker 2.8 [87]. The sensitivity towards nucleophile-mediated cleavage was increased by introducing an electron-withdrawing group on the aromatic ring of the benzyl ester.…”

Section: Cleavage From the Resinmentioning

confidence: 99%

“…The sensitivity towards nucleophile-mediated cleavage was increased by introducing an electron-withdrawing group on the aromatic ring of the benzyl ester. Nitration of the Merrifield resin facilitates the release of resin-bound deamino-oxytocin using a saturated solution of ammonia in MeOH at 0 C [92]. Cleavage from the HMBA linker 2.8 by ammonolysis was achieved using a solution of ammonia in 2-propanol.…”

Section: Cleavage From the Resinmentioning

confidence: 99%