Elimination and exchange of trifluoroacetate counter‐ion from cationic peptides: a critical evaluation of different approaches

Most synthesized peptides are nowadays produced using solid‐phase procedures. Due to cleavage and purification conditions, they are mainly obtained in the presence of trifluoroacetic acid (TFA) and, for cationic peptides, as trifluoroacetate (TF‐acetate) salts. However, TF‐acetate interferes with physicochemical characterizations using infrared spectroscopy and might significantly affect the in vivo studies. Thus, TF‐acetate exchange by another counter‐ion is often required. Up to now, the classical procedure has consisted of freeze‐drying the peptide several times in the presence of an excess of a stronger acid than TFA (pKa ∼0): generally HCl (pKa = − 7). This approach means that working at pH < 1 can induce peptide degradation. We therefore tested three different approaches to exchange the tightly bound TF‐acetate counter‐ion from the dicationic octapeptide lanreotide: (i) reverse‐phase HPLC, (ii) ion‐exchange resin, and (iii) deprotonation/reprotonation cycle of the amino groups. The first two approaches allow the partial to almost complete exchange of the TF‐acetate counter‐ion by another ion from an acid weaker than TFA, such as acetic acid (pKa = 4.5), and the third requires a basic solution that permits the complete removal of TF‐acetate counter‐ion. The efficiency of these three procedures was tested and compared by using different analytical techniques such as 19 F‐NMR, 1 H‐NMR and attenuated total reflectance Fourier transformed infrared spectroscopy (ATR FT‐IR). We also show that ATR‐IR can be used to monitor the TFA removal. The counter‐ion exchange procedures described in this study are easy to carry out, fast, harmless and reproducible. Moreover, two of them offer the very interesting possibility of exchanging the initial TF‐acetate by any other counter‐ion. Copyright © 2007 European Peptide Society and John Wiley & Sons, Ltd.