Asparagine coupling in Fmoc solid phase peptide synthesis

Abstract

To investigate side reactions during the activation of side chain unprotected asparagine in Fmoc‐solid phase peptide synthesis the peptide Met‐Lys‐Asn‐Val‐Pro‐Glu‐Pro‐Ser was synthesized using different coupling conditions for introduction of the asparagine residue. Asparagine was activated by DCC/HOBt, BOP (Castro's reagent) or introduced as the pentafluorophenyl ester. The resulting peptide products were analyzed by HPLC, mass spectrometry and Edman degradation. In the crude products varying amounts of β‐cyano alanine were found, which had been formed by dehydration of the side chain amide during carboxyl activation of Fmoc‐asparagine. A homogeneous peptide was obtained by using either side chain protected asparagine derivatives with BOP mediated activation or by coupling of Fmoc‐Asn‐OPfp. Fmoc‐Asn(Mbh)‐OH and Fmoc‐Asn(Tmob)‐OH were coupled rapidly and without side reactions with BOP. For the side chain protected derivatives the coupling was as fast as that of other Fmoc‐amino acid derivatives, whereas couplings of Fmoc‐Asn‐OH proceed more slowly. However, during acidolytic cleavage both protection groups, Mbh and Tmob, generate carbonium ions which readily alkylate tryptophan residues in a peptide. Tryptophan modification was examined using the model peptide Asn‐Trp‐Asn‐Val‐Pro‐Glu‐Pro‐Ser. Alkylation could be reduced by addition of scavengers to the TFA during cleavage and side chain deprotection. A homogeneous peptide containing both, asparagine and tryptophan, was obtained only by coupling of Fmoc‐Asn‐OPfp.