Solid phase peptide synthesis: a simple esterification procedure

Marglin, A.

doi:10.1016/s0040-4039(01)97113-8

Cited by 24 publications

(6 citation statements)

References 14 publications

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“…Methylene chloride (CH2Clz) was further purified by passage through a column of alumina (Alumina Adsorption, Fisher Scientific Co.). N~'-dimethylformamide was filtered over a column of 4A molecular sieves (Matheson, Coleman & Bell) and stored with flesh sieves for 72 h before use (8). C5a was prepared by tryptic digestion (9,10) of purified C5 prepared from human plasma (11).…”

Section: Methodsmentioning

confidence: 99%

Structural determinants of the eosinophil: chemotactic activity of the acidic tetrapeptides of eosinophil chemotactic factor of anaphylaxis.

Goetzl¹,

Austen²

1976

The Journal of Experimental Medicine

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The acidic tetrapeptides of ECF-A, Ala/Val-Gly-Ser-Glu, exhibit peak in vitro chemotactic activity for human eosinophils at concentrations of 3 X 10(-8) M to 10(-6) M, and rapidly deactivate eosinophils to homologous and other stimuli at concentrations as low as 10(-10) M. The analogue Leu-Gly-Ser-Glu reaches peak activity at 10(-8)M-10(-7)M, while Phe-Gly-Ser-Glu requires 10(-4)M to elicit a peak response. Although inversion of the order of glycine and serine does not alter the eosinophil chemotactic activity of the tetrapeptides, deletion of glycine increases by 10-fold the concentration required for peak chemotactic activity, indicating the critical nature of the spacing between NH2- and COOH-terminal residues. The substituent COOH-terminal tripeptide, which is only marginally chemotactic, irreversibly suppresses eosinophil chemotactic responsiveness at a concentration 10,000-fold higher than concentrations necessary for deactivation by the intact tetrapeptide. The high concentration of tripeptide required for this cell directed effect, which is assumed to be analogous to deactivation, is attributed to the absence of the NH2-terminal residue which would facilitate effective interaction with the eosinophil. A substituent NH2-terminal tripeptide and amides of the NH2-terminal amino acids, which are devoid of chemotactic and deactivating activities, reversibly inhibit the tetrapeptide stimulus in a dose-response fashion. The additional finding that the NH2-terminal tripeptide protects the eosinophil from deactivation by the intact tetrapeptide confirms that the competitive interaction is stimulus specific.

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Section: Methodsmentioning

confidence: 99%

Structural determinants of the eosinophil: chemotactic activity of the acidic tetrapeptides of eosinophil chemotactic factor of anaphylaxis.

Goetzl¹,

Austen²

1976

The Journal of Experimental Medicine

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“…Boc-Ala-resin ester was prepared essentially as described in the literature. 41 The hydrolysis of 20 mg of the sample with 6 M HC1 in dioxane yielded 0.28 mmol of Ala per gram of resin.…”

Section: Methodsmentioning

confidence: 99%

Studies on the solid-phase synthesis of bovine pancreatic trypsin inhibitor (Kunitz) and the characterization of the synthetic material

Tan¹,

Kaiser²

1976

J. Org. Chem.

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A resin-bound protected linear polypeptide of 58 residues with the sequence of the bovine pancreatic trypsin inhibitor (Kunitz) was synthesized by the solid-phase method. The polypeptide was removed from the solid support by cleavage with HF and purified, and the three disulfide bonds were formed by air oxidation of the reduced form. The, synthetic inhibitor was purified by gel filtration, trypsin-Sepharose affinity chromatography, and finally by ion-exchange chromatography. A highly purified inhibitor which inhibited trypsin stoichiometrically was isolated. The synthetic trypsin inhibitor was indistinguishable from natural trypsin inhibitor by chromatography on CMSephadex, polyacrylamide gel electrophoresis, amino acid analysis, peptide maps of tryptic digests, and circular dichroism spectra. The dissociation constant for the trypsin-synthetic trypsin inhibitor complex also agreed well with that for the trypsin-native trypsin inhibitor complex.The methodology of Merrifield solid-phase ~ynthesisl-~ has gained considerable acceptance, particularly in the preparation of peptides of moderate molecular weight. This has encouraged a number of investigators to attempt the synthesis of large biologically active polypeptides by the solid-phase approach. However, the difficulties associated with a stepwise solid-phase strategy are expected to increase in magnitude with increases in the length of the polypeptide chain.We have undertaken studies on solid-phase peptide synthesis with a view toward eventually using this method in the design of polypeptide model enzymatic catalysts. In this paper, we present the results of our solid-phase synthesis of a polypeptide with the amino acid sequence of bovine pan-'creatic trypsin inhibitor (Kunitz) and of our characterization of the synthetic species. We selected this inhibitor (BPTI) for synthesis because its amino acid chain length (58) is in the same range as the model enzymes we plan to prepare and because it is a very stable, well-characterized protein which has been studied in great detail in many laboratorie~.~-6 Both its amino acid sequence7-l0 and crystallographic structurel1-l3 have been determined. Also, it has been established with the native inhibitor that the denatured, reduced peptide chain can be reoxidized and refolded into a structure possessing full inhibitory activity.] 4-16 Furthermore, the extraordinarily low dissociation constant (6 X M t pH 8,25 "C) which has been measured for the trypsin inhibitor-trypsin complex17 provides a stringent criterion by which the purity of synthetic material can be assessed. Finally, if success in the solid-phase synthesis of a peptide possessing the amino acid sequence of native BPTI and meeting high standards of purity can be achieved, this could open the way to the preparation of analogues with variations in the amino acid composition in the vicinity of the "reactive site" which would be useful in mechanistic studies of the inhibition process.

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“…The resin (BioBeads SX I , BioRad) was chloromethylated according to Merrifield (14). and Boc-Ala was esterified to the chloromethyl resin by a new method (20). The peptides were elongated by published procedures (14), except that the Boc groups were removed with 12.5 % trifluoroacetic acid in methylene chloride (v/v).…”

Section: Reduction With Sodium In Liquid Ammonia and Analysis Of The mentioning

confidence: 99%

“…The peptides were elongated by published procedures (14), except that the Boc groups were removed with 12.5 % trifluoroacetic acid in methylene chloride (v/v). The desired peptides were removed from the resin with HBr/TFA-CH,CI, (13c), anhydrous H F (12, 21), or NH,/ CH,OH (20). The peptides were then diluted in water.…”

Section: Reduction With Sodium In Liquid Ammonia and Analysis Of The mentioning

confidence: 99%

“…Another aliquot was applied directly t o the analyzer column as described, and from the known concentration the color values were calculated. Although the peptides were reacted with sodium in liquid ammonia without preliminary purification, they were actually homogeneous by paper electrophoresis and tlc (20), and those that were amenable to chromatography on the analyzer appeared as single symmetrical peaks (see Fig. 1); for example, the chromatograms of II, X and XI showed less than 0.3% VIII.…”

Section: Reduction With Sodium In Liquid Ammonia and Analysis Of The mentioning

confidence: 99%

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Factors That Influence Acyl Proline Cleavage by Sodium in Liquid Ammonia

Marglin

1972

International Journal of Peptide and Protein Research

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The reductive cleavage of aminoacyl proline bonds by sodium in liquid ammonia was compared by a simple chromatographic technic for various protected and unprotected analogs of the C‐terminal tetrapeptide of the insulin B‐chain, ‐Thr‐Pro‐Lys‐Ala‐OH. The literature suggests that the Thr‐Pro bond in this peptide is especially sensitive to Na‐NN 3, compared to various other acyl proline bonds. The present study shows that this is true regardless of whether the sidechains, the α‐amino, and the α‐carboxyl groups are protected or free when they are introduced into liquid ammonia; there is no difference in the amount of reductive Thr‐Pro cleavage despite the different numbers of protons released into liquid ammonia by the functional groups of the residues surrounding the Thr‐Pro bond. The C‐terminal tetrapeptide and its blocked derivatives, prepared by solid phase synthesis, were reduced by Na‐NH 3 to the same extent as similarly synthesized B‐chains and natural B‐chain. Thr‐Pro cleavage could be minimized by sharply limiting the amount of sodium and the time of exposure of the peptide to it. The results are therefore useful in planning synthetic approaches to the B‐chain. The Ser‐Pro bond shares the lability of the Thr‐Pro bond to Na‐NH 3 reduction, whereas the Val‐Pro bond is only half as labile.

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Solid phase peptide synthesis: a simple esterification procedure

Cited by 24 publications

References 14 publications

Structural determinants of the eosinophil: chemotactic activity of the acidic tetrapeptides of eosinophil chemotactic factor of anaphylaxis.

Structural determinants of the eosinophil: chemotactic activity of the acidic tetrapeptides of eosinophil chemotactic factor of anaphylaxis.

Studies on the solid-phase synthesis of bovine pancreatic trypsin inhibitor (Kunitz) and the characterization of the synthetic material

Factors That Influence Acyl Proline Cleavage by Sodium in Liquid Ammonia

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