Comparison and optimization of chromatographic conditions for separation of cyclic analogues of Dynorphin A from linear by-products

Abstract

In this study, chromatographic conditions for the separation of the lactam analogue of dynorphin A (Dyn A)(1-13)NH2 cyclo[D-Asp(2),Orn(5)]Dyn A(1-13)NH2 from the tetramethylguanidinium by-product [D-Asp(2),Orn(Tmg)(5)]Dyn A(1-13)NH2 and linear peptide [D-Asp(2),Orn(5)]Dyn A(1-13)NH2 were compared. The by-product was obtained following the cyclization of the peptides with 2-(1-H-benzotriazo-1-yl)-1,1,3,3tetramethyluronium hexafluorophosphate. Two methods of high performance liquid chromatography, reverse phase and ion-exchange chromatography, were examined. A number of factors affected the resolutionand the elution profile of peptides, including the ion-pairing agent, the mobile phase pH and the organic solvent. These factors were varied to obtain optimum separation.