SELECTIVE FLUOROGENIC DERIVATIZATION OF PRO2-LYS PEPTIDES WITH NAPHTHALENE-2,3-DICARBOXALDEHYDE CYANIDE

The potential for fluorescence quenching due to bis-derivatization of Lys peptides with naphthalene-2,3-dicarboxaldehyde/cyanide was confirmed. Kinetic investigation revealed that derivatization at the epsilon-amino sites proceeded significantly faster than at alpha-amino sites. After characterization of pH and co-solvent (methanol) effects on the absolute and relative reaction rate at epsilon- and alpha-amine sites, it was concluded that by using a combination of pH control and timing that the formation of the analytically useful mono-derivative at the epsilon-amino site was feasible. Using this kinetic information, derivatization at pH 10 with 80% methanol as co-solvent for 2 min resulted in the formation of a highly fluorescent mono-derivative of the Pro2-Lys peptide, Substance P. When this derivatization protocol was used in conjunction with liquid chromatography and fluorescence detection for Substance P, a linear calibration plot resulted (50-500 nM range; 1.94-19.4 mol injected; R.S.D. less-than-or-equal-to 2% at each point).