Asparagine coupling in Fmoc solid phase peptide synthesis.
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 beta-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.
Gausepohl
H
H
European Molecular Biology Laboratory, Heidelberg, West Germany.
Kraft
M
M
Frank
R W
RW
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis*; D001216:Asparagine / Q000032:analysis*; D055598:Chemical Phenomena; D002621:Chemistry; D002851:Chromatography, High Pressure Liquid; D005449:Fluorenes; D007202:Indicators and Reagents; D008969:Molecular Sequence Data; D009943:Organophosphorus Compounds; D010455:Peptides / Q000138:chemical synthesis*; D014364:Tryptophan / Q000032:analysis
D000596:Amino Acids; D005449:Fluorenes; D007202:Indicators and Reagents; C016456:N(alpha)-fluorenylmethyloxycarbonylamino acids; D009943:Organophosphorus Compounds; D010455:Peptides; C031032:benzotriazol-1-yloxy-tris(dimethylamino)phosphonium; D001216:Asparagine; D014364:Tryptophan