Handling a tricycle: Orthogonal versus random oxidation of the tricyclic inhibitor cystine knotted peptide gurmarin

Publication: Research - peer-reviewJournal article – Annual report year: 2012

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Handling a tricycle: Orthogonal versus random oxidation of the tricyclic inhibitor cystine knotted peptide gurmarin. / Eliasen, Rasmus; Andresen, Thomas L.; Conde-Frieboes, Kilian W.

In: Peptides, Vol. 37, No. 1, 2012, p. 144-149.

Publication: Research - peer-reviewJournal article – Annual report year: 2012

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Eliasen, Rasmus; Andresen, Thomas L.; Conde-Frieboes, Kilian W. / Handling a tricycle: Orthogonal versus random oxidation of the tricyclic inhibitor cystine knotted peptide gurmarin.

In: Peptides, Vol. 37, No. 1, 2012, p. 144-149.

Publication: Research - peer-reviewJournal article – Annual report year: 2012

Bibtex

@article{70920f3322614c4790510b999e271ad6,
title = "Handling a tricycle: Orthogonal versus random oxidation of the tricyclic inhibitor cystine knotted peptide gurmarin",
keywords = "Gurmarin, Peptide synthesis, Inhibitor cystine knot, Disulfide formation, Orthogonal oxidation",
publisher = "Elsevier Inc.",
author = "Rasmus Eliasen and Andresen, {Thomas L.} and Conde-Frieboes, {Kilian W.}",
year = "2012",
doi = "10.1016/j.peptides.2012.06.016",
volume = "37",
number = "1",
pages = "144--149",
journal = "Peptides",
issn = "0196-9781",

}

RIS

TY - JOUR

T1 - Handling a tricycle: Orthogonal versus random oxidation of the tricyclic inhibitor cystine knotted peptide gurmarin

A1 - Eliasen,Rasmus

A1 - Andresen,Thomas L.

A1 - Conde-Frieboes,Kilian W.

AU - Eliasen,Rasmus

AU - Andresen,Thomas L.

AU - Conde-Frieboes,Kilian W.

PB - Elsevier Inc.

PY - 2012

Y1 - 2012

N2 - Gurmarin is a 35 amino acid peptide with three disulfide bridges in an inhibitor cystine knot. It is found in the plant Gymnema sylvestre, and has been identified as a sweet taste inhibitor in rodents. In this article we provide an efficient route for the synthesis of gurmarin by a controlled random oxidation strategy. We compared two oxidation procedures to form the three disulfide bridges. In the first, based on random oxidation, reduced gurmarin was synthesized using trityl for cysteine protection, and oxidized for 48h in a Tris–HCl buffer containing cystamine and reduced glutathione to facilitate disulfide scrambling. The second was based on step-wise deprotection followed by oxidation in which the cysteine pairs are orthogonally protected with tert-Butylthio, trityl and acetamidomethyl. To verify that the native gurmarin oxidation product was obtained, thermolysin cleavage was used. Cleavage of random oxidized gurmarin showed two possible disulfide combinations; the native and a non-native gurmarin disulfide isomer. The non-native isomer was therefore synthesized using the orthogonal deprotection-oxidation strategy and the native and the non-native gurmarin isomers were analyzed using UPLC. It was found that the random oxidation procedure leads to native gurmarin in high yield. Thus, the synthetic route was simple and significantly more efficient than previously reported syntheses of gurmarin and other cysteine rich peptides. Importantly, native gurmarin was obtained by random oxidation, which was confirmed by a synthetic approach for the first time.

AB - Gurmarin is a 35 amino acid peptide with three disulfide bridges in an inhibitor cystine knot. It is found in the plant Gymnema sylvestre, and has been identified as a sweet taste inhibitor in rodents. In this article we provide an efficient route for the synthesis of gurmarin by a controlled random oxidation strategy. We compared two oxidation procedures to form the three disulfide bridges. In the first, based on random oxidation, reduced gurmarin was synthesized using trityl for cysteine protection, and oxidized for 48h in a Tris–HCl buffer containing cystamine and reduced glutathione to facilitate disulfide scrambling. The second was based on step-wise deprotection followed by oxidation in which the cysteine pairs are orthogonally protected with tert-Butylthio, trityl and acetamidomethyl. To verify that the native gurmarin oxidation product was obtained, thermolysin cleavage was used. Cleavage of random oxidized gurmarin showed two possible disulfide combinations; the native and a non-native gurmarin disulfide isomer. The non-native isomer was therefore synthesized using the orthogonal deprotection-oxidation strategy and the native and the non-native gurmarin isomers were analyzed using UPLC. It was found that the random oxidation procedure leads to native gurmarin in high yield. Thus, the synthetic route was simple and significantly more efficient than previously reported syntheses of gurmarin and other cysteine rich peptides. Importantly, native gurmarin was obtained by random oxidation, which was confirmed by a synthetic approach for the first time.

KW - Gurmarin

KW - Peptide synthesis

KW - Inhibitor cystine knot

KW - Disulfide formation

KW - Orthogonal oxidation

U2 - 10.1016/j.peptides.2012.06.016

DO - 10.1016/j.peptides.2012.06.016

JO - Peptides

JF - Peptides

SN - 0196-9781

IS - 1

VL - 37

SP - 144

EP - 149

ER -