SYNTHESIS AND CHARACTERIZATION OF 1-C-13-D.LEU-12,LEU-14 GRAMICIDIN-A

Abstract

The 13C-D-Leu12,14 gramicidin A was synthesized by the solid phase method incorporating 13C-D-leucine in positions 12 and 14 with about 25 and 50% enrichment, respectively. The pentadecapeptide was removed from the resin by ethanolamine treatment, with the N-protecting group (Boc) still on. After removal of the protecting group, the peptide was formylated and purified by preparative TLC to obtain 13C-D-Leu12,14 gramicidin A in a very pure state in an overall yield of about 12.5%. The peptide was then thorougly characterized by HPLC [high performance liquid chromatography] which gave one single peak with the same retention time as that of Val1-gramicidin A of the natural gramicidin mixture. The CD [circular dichroism] spectra of the synthetic and the HPLC purified natural Val1-GA were obtained and found to be identical, indicating the optical purity of the sample. The synthetic GA was characterized by 13C MMR spectrum and compared with that of natural GA. Single channel conductance parameters of the synthetic GA were determined and were indistinguishable from those of natural Val1-GA in lipid bilayer membranes and the mean channel lifetime was found to be as reported earlier by others.