Chloroisothiocyanatoquinolines as fluorogenic derivatizing agents for primary and secondary amines

Two new prechromatographic LC fluorogenic derivatizing agents, 2- and 4-chloro-3-isothiocyanatoquinoline (4 and 5) have been synthesized and shown to react smoothly with primary and secondary amines to produce fluorescent thiazoloquinolines. Compounds 4 and 5 hydrolyse in aqueous base and the rates of this reaction compared with the rates of derivatizations with amino acids indicate that the hydrolysis reaction interferes with derivatization when UV detection is used. The kinetics of derivatization with ordinary amines indicate that this reaction is quite facile, although less nucleophilic amines, e.g. aniline, react slowly. The pKas for first protonations indicate that derivatives of 4 would be unprotonated and those of 5 would be protonated with typical RP-LC mobile phases. The Stokes shift for protonated derivatives of 5 is nearly 200 nm. The excess of unreacted derivatizing reagent interferes with UV detection of some analytes; but when fluorescence detection is used this excess produces only a small negative peak. With fluorescence detection the sensitivity of this method is about 0.8 microM at S/N of 2, and the response of peak height to concentration is linear over at least two decades of concentration.