Failure sequences in the solid phase synthesis of polypeptides

Bayer, Ernst; Eckstein, Heiner; Haegele, K. D.; Koenig, W. A.; Bruening, W.; Hagenmaier, Hanspaul; Parr, W.

doi:10.1021/ja00709a053

Cited by 112 publications

(18 citation statements)

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“…(1) The work clearly illustrates some of the drawbacks of solid state synthesis discussed by Bayer et al 6 and Rudihbger. 7 Li and Yamashiro had no reason to be surprised that their synthetic product had only 10%°b of the biological activity of the purest natural growth hormone, because it was impossible to know to what extent it might be contaminated with a host of error peptides.…”

supporting

confidence: 52%

Hiccup

Jonas¹

1971

BMJ

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supporting

confidence: 52%

Hiccup

Jonas¹

1971

BMJ

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“…A cycle for the incorporation of each amino acid into the growing peptide chain involved the following washing (W, for 10 min) and reaction (R) steps (8 ml of solvent/g of starting resin was used throughout): (a) 3 X W, dioxane; (b) R, 2 N HCl in dioxane, 20 min; (c) 3 X W, dioxane; (d) 3 X W, dimethylformamide; (e) R, 10% triethylamine in dimethylformamide, 10 min; (f) 3 X W, dimethylformamide; (g) 3 X W, methylene chloride; (h) addition of 3.3-5 equivalents of tert-butyloxycarbonylamino acid in methylene chloride and mixing for 10 min; (i) R, 3-5 equivalents of dicyclohexylcarbodiimide, methylene chloride, 3-15 hr; (j) 3 X W, methylene chloride. 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).…”

Section: Methodsmentioning

confidence: 99%

“…for C7IH91N13016S3 (1526.8): C, 59.0; H, 6.01; N, 11.9. Found: C, 58.6; H, 5.94; N, 11.6. 18-Lysine]-vasopressin, L-hemicystyl-Ltyrosyl-L-phenylalanyl-L-glutaminyl-L-asparaginyl-L-hemicystyl-Lprolyl-L-lysyl-glycinamide 1,6-disulfide (9) Recrystallized protected nonapeptide 7 (80 mg, 48 Amol) was dissolved in liquid ammonia (150 ml) that had been distilled from sodium. Sodium was added in small quantities to the boiling-ammonia solution over a period of 20 min until a blue color remained for 30 sec.…”

Section: Methodsmentioning

confidence: 99%

“…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.…”

mentioning

confidence: 99%

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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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“…47 Some workers thought that reactions will be less efficient close to the polymer backbone 48 while others thought that there would be significant declines in yields as the peptide is elongated, due to temporary steric occlusion of peptide chains within the polymer network. 49 In 1971, I proposed an approach that would allow testing of coupling or synthetic efficiency in SPPS.…”

Section: Solid-phase Peptide Synthesis 223mentioning

confidence: 99%

Studies in solid‐phase peptide synthesis: A personal perspective

Mitchell

2008

Biopolymers

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By the early 1970s it had became apparent that the solid‐phase synthesis of ribonuclease A could not be generalized. Consequently, virtually every aspect of solid‐phase peptide synthesis (SPPS) was reexamined and improved during the decade of the 1970s. The sensitive detection and elimination of possible side reactions (amino acid insertion, Nα‐trifluoroacetylation, Nαϵ‐alkylation) were examined. The quantitation of coupling efficiency in SPPS as a function of chain length was studied. A new and improved support for SPPS, the “PAM‐resin,” was prepared and evaluated. These and many other studies from the Merrifield laboratory and elsewhere increased the general acceptance of SPPS leading to the 1984 Nobel Prize in Chemistry for Bruce Merrifield. © 2007 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 90:215–233, 2008.This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprintversion. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

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Failure sequences in the solid phase synthesis of polypeptides

Cited by 112 publications

References 1 publication

Hiccup

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

Studies in solid‐phase peptide synthesis: A personal perspective

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