AN AFFINITY CHROMATOGRAPHIC METHOD FOR THE PURIFICATION OF WATER-INSOLUBLE PEPTIDES

A simple and efficient affinity chromatographic method is described for the purification of hydrophobic and/or protected synthetic peptides in organic solvent. This affinity method entails the use of a new protecting group that contains a UV-active disulfide moiety ([2-[(2-nitrophenyl)dithio]-1-phenylethoxy]carbonyl, NpSSPeoc). NpSSPeoc is electrophilic and can be immobilized by a thiolate-disulfide interchange reaction on a thiol-containing solid support. Alternatively, NpSSPeoc can be converted into a nucleophilic thiol ((2-mercapto-1-phenylethoxy)carbonyl, HSPeoc) and immobilized by an electrophilic solid support. NpSSPeoc can be quantified via an Ellman-type assay to measure the amount of full-length peptide at the completion of a solid-phase synthesis before and/or after cleavage from the synthesis resin. NpSSPeoc can be removed with 25% trifluoroacetic acid in methylene chloride. Four peptides incorporating NpSSPeoc at the N-terminus were synthesized. Affinity chromatography was performed on these peptides using 1 % cross-linked polystyrene supports which incorporate a mercury(II) trifluoroacetate, disulfide, alkyl thiol, or iodoacetamide functionality. The results indicate that the highest and most consistent yields of purified peptide are obtained using the combination of HSPeoc and a polystyrene support incorporating the iodoacetamide functionality.