Peptide purification using the chemoselective reaction between N-(methoxy)glycine and isothiocyanato-functionalized resin.

An efficient peptide purification strategy is established, comprising the selective reaction of an N-terminal N-(methoxy)glycine residue of the peptide and isothiocyanato-functionalized resins, and subsequent Edman degradation. These reactions take place in acidic media; in particular, the Edman degradation proceeds smoothly in media containing more than 50% trifluoroacetic acid (v/v). These acidic conditions offer increased solubility, making them advantageous for the purification of hydrophobic and aggregation-prone peptides. The effectiveness of this method, together with scope and limitations, is demonstrated using model peptides and the practical purification of the loop region of the human dopamine D2 receptor long isoform (residues 240-272). Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Site-specific labeling of synthetic peptide using the chemoselective reaction between N-methoxyamino acid and isothiocyanate.

Site-specific labeling of synthetic peptides carrying N-methoxyglycine (MeOGly) by isothiocyanate is demonstrated. A nonapeptide having MeOGly at its N-terminus was synthesized by the solid-phase method and reacted with phenylisothiocyanate under various conditions. In acidic solution, the reaction specifically gave a peptide having phenylthiourea structure at its N-terminus, leaving side chain amino group intact. The synthetic human ß-defensin-2 carrying MeOGly at its N-terminus or the side chain amino group of Lys(10) reacted with phenylisothiocyanate or fluorescein isothiocyanate also at the N-methoxyamino group under the same conditions, demonstrating that this method is generally useful for the site-specific labeling of linear synthetic peptides as well as disulfide-containing peptides.