Mixed anhydrides in peptide synthesis. A study of urethane formation with a contribution on minimization of racemization

Abstract

A study of the factors contributing to urethane formation during the coupling of N-alkoxycarbonylamino acids with an amino acid ester by the mixed carboxylic–carbonic acid anhydride method has been carried out, using nuclear magnetic resonance spectroscopy and high-performance liquid chromatography for the quantitation of products. Urethane formation is associated primarily with reactions of activated hindered residues such as isoleucine and N-methylamino acids. Of prime importance in dictating the amount of urethane generated is the tertiary amine/solvent combination. N-Methylpiperidine/dichloromethane is the best combination for minimizing urethane formation, N-methylmorpholine/tetrahydrofuran is a good combination, while triethylamine/dichloromethane is a particularly bad one. In dimethylformamide, the differences between these amines are marginal. Aqueous dimethylformamide is a good solvent for mixed anhydride generation and coupling. A small excess of substrate reduces the amount of urethane. Less racemization accompanies the coupling of peptide acids in tetrahydrofuran than in halogen-containing solvents, N-methylpiperidine being the superior base in these solvents, but not in dimethylformamide. Racemization is reduced by one half when menthyl chloroformate instead of isobutyl chloroformate is used in the couplings.