SEPARATION AND CHARACTERIZATION OF COMPONENTS OF PEPTIDE LIBRARIES USING ON-FLOW COUPLED HPLC-NMR SPECTROSCOPY

The efficiency of directly coupled HPLC-NMR spectroscopy for the separation and characterization of synthetic peptide libraries was investigated using a model system based on a mixture of all 27 tripeptide combinations of alanine (A), methionine (M) and tyrosine (Y) as the C-terminal amides. The H-1 NMR spectra were obtained in real time using on-flow HPLC-NMR spectroscopy at 600 MHz and 21 of the 27 peptides were identified on the basis of chemical shifts and coupling constants, including the use of diagnostic values derived from H-1 NMR spectra and H-1-C-13 and H-1-N-15 HMQC studies on authentic A-A-A-OH and Y-Y-Y-OH. The effects on H-1 NMR chemical shifts of pH and of varying proportions of acetonitrile and water as occurred during the gradient elution HPLC run were evaluated. The HPLC-NMR approach could provide a facile method for investigating the relative efficiency of resin-based peptide synthesis for different amino acid combinations and should rapidly provide data suitable for deriving structure-retention time relationships for peptides.