FACILE RESOLUTION OF N-TERT-BUTOXY-CARBONYL AMINO-ACIDS - THE IMPORTANCE OF ENANTIOMERIC PURITY IN PEPTIDE-SYNTHESIS

Twenty five pairs of N-tert-butoxycarbonyl (N-t-Boc) protected racemic amino acids were separated using a hydroxypropyl derivatized beta-cyclodextrin bonded phase column in the reversed-phase mode. Most of the N-t-Boc-amino acids are base-line separated in 30 minutes or less. The retention behavior, the effect of structure on chiral recognition, and the effect of mobile phase composition are discussed. It was found there is a maximum resolution value when the mobile phase contains from 5% to 10% acetonitrile (or approximately 20% methanol) for many of the N-t-Boc-amino acids. Others were found to resolve better with acetonitrile concentrations between 90 - 97%. It was found that the higher the pH, the shorter the retention and the better the resolution. The optimum concentration of buffer solution was 1% triethylammonium acetate. N-t-Boc-amino acids are used extensively in peptide synthesis. Relatively small enantiomeric impurities in these starting materials can translate into peptide products containing significant, often unacceptable impurities. The relationship between the enantiomeric purity of the starting material (N-t-Boc-amino acids), the number of synthetic steps and the stereochemical purity of the final product is discussed.