Oxazole-based tagging reagents for analysis of secondary amines and thiols by liquid chromatography with fluorescence detection

Abstract

The reactions of three fluorescent tagging reagents, 2-chloro-4,5-diphenyloxazole (DICLOX), 2-fluoro-4,5-diphenyloxazole (DIFOX) and 2-chloro-4,5-bis(p-N,N-dimethylaminosulfonylphenyl)oxazole (SAOX-CI), with thiols and amines are reported. Emission maxima for the diphenyloxazole (DIOX) and SAOX derivatives of amines were 420 nm (λ_ex 320 nm) and 485 nm (λ_ex 360 nm), respectively. The emission wavelengths for the DIOX- and SAOX-thiols are 390 nm (λ_ex 310 nm) and 425 nm (λ_ex 330 nm), respectively. In all cases, the derivatives exhibited strong fluorescence whereas the reagents themselves exhibited only weak fluorescence. The labelled derivatives are very stable, less than 5% decomposition occurs after heating at 60 °C for 2 h. Fluorescence intensities of the amine derivatives were higher in neutral and alkaline than in acidic solutions and were virtually independent of solvent polarity. The thiol derivatives exhibited fluorescence intensities that were relatively constant under all conditions studied. The relative reaction rate toward both thiols and amines was DIFOX > SAOX-CI > DICLOX. The reaction of proline with DIFOX was complete after 60 min at room temperature at pH 9.3. However, the yield with SAOX-CI was only 70% at 60 °C after 3 h, and only a small amount of proline could be derivatized with DICLOX (less than 3%). Thiols, on the other hand, reacted relatively rapidly with SAOX-CI. Therefore, SAOX-CI was used for the determination of thiols and DIFOX was employed for amines in all subsequent studies. Detection limits (signal-to-noise ratio = 2) for authentic DIOX-amines ranged from 3.7 to 28.4 fmol, and SAOX-thiols ranged from 1.2 to 1.9 fmol.