Liquid chromatography-mass spectrometry study towards the pH and temperature-induced N-acyl migration in polymyxin B

In the course of the synthesis and purification of new polymyxins and analogues, formation of a by-product with identical mass was observed and it was believed that this might be the result of acyl migration from the Nalpha- to the Ngamma-position of residue alpha,-gamma-diaminobutyric acid 1 (Dab1) under acidic conditions. Therefore, a LC-MS/MS study was initiated to establish the stability of polymyxin B3 in aqueous solution at room temperature and 60 degrees C, as well as different pH values (i.e. 1.4, 4.4 and 7.4). It was shown that the by-product, which is actually formed in the course of the purification of polymyxin B3 after evaporation in acidic medium, has a retention time similar to Ngamma-polymyxin B3. Acyl-migration occurred most rapidly at 60 degrees C and pH 7.4. Furthermore, it was established that migration of the acyl from the Nalpha- to the Ngamma-position of residue Dab1 is reversible and that the equilibrium seems to be in favor of the Nalpha-acylated compound.