Characterization and analysis of biphalin: an opioid peptide with a palindromic sequence

Among the many opioid peptides developed to date as nonaddictive analgesics , biphalin has exhibited extraordinary high potency and many other desirabl e characteristics. Biphalin is an octapeptide consisting of two monomers of a modified enkephalin, attached via a hydrazine bridge, and with the amino acids assembled in a palindromic sequence. Its structure is (Tyr-D-Ala-Gly -Phe-NH-)-(2). However, this unique peptide, like any other synthetic pepti de, needs strict quality control because of certain drawbacks associated wi th peptide synthesis. This paper discusses our approaches to characterizing and analyzing biphalin. Many techniques were used, including elemental ana lysis, amino acid analysis, amino acid sequence analysis (AASA), mass spect rometry (MS), H-1-NMR, H-1-correlated spectroscopy (COSY)-NMR, high-perform ance liquid chromatography (HPLC) and capillary electrophoresis (CE). Elect rospray ionization (ESI) mass spectrometry, which included both ESI-MS and ESI-MS/MS, was performed to confirm the full sequence because AASA results alone verified only the monomer sequence, and not the full sequence. Althou gh the H-1-NMR results led to a preliminary assignment of many protons, the H-1 COSY-NMR results allowed for unequivocal assignment of almost all prot ons. Peptide purity was determined using two techniques, reversed-phase HPL C and CE. The counter-ion of the peptide, trifluoroacetic acid, was determi ned by CE, using an indirect detection method developed previously in our l aboratory. This paper illustrates successful application of nonconventional techniques to characterize and analyze a structurally modified peptide, bi phalin, when standard techniques for peptide analysis are inadequate.