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 separ ation and characterization of synthetic peptide libraries was investig ated using a model system based on a mixture of all 27 tripeptide comb inations of alanine (A), methionine (M) and tyrosine (Y) as the C-term inal amides. The H-1 NMR spectra were obtained in real time using on-f low HPLC-NMR spectroscopy at 600 MHz and 21 of the 27 peptides were id entified on the basis of chemical shifts and coupling constants, inclu ding 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. Th e 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 meth od for investigating the relative efficiency of resin-based peptide sy nthesis for different amino acid combinations and should rapidly provi de data suitable for deriving structure-retention time relationships f or peptides.