Suitability of DTAF as a fluorescent labelling reagent for direct analysis of primary and secondary amines--spectral and chemical reactivity considerations.

DTAF has been used successfully to prepare fluorescent labelled reagents for fluorescence polarization immunoassays. Its applicability as a derivation reagent for direct fluorescence analysis of primary and secondary amines was evaluated. DTAF was shown to have spectral properties that closely resemble those of fluorescein and that are apparently insensitive to the presence of the triazine nucleus. Spectrally determined pKa values also closely resemble those of fluorescein and other amino aryl-s-triazines. DTAF is prone to hydrolytic degradation with the rate of reaction increasing with increasing pH, until a pH value is reached at which ionization of the amine bridging the two aromatic nuclei occurs; at this pH the rate reaches a plateau value. Both primary and secondary amines react efficiently with DTAF, and the reactivity increases with the increasing basicity of the amine reactants. The reaction is pH-dependent, proceeding most efficiently at pH values at which both the "bridging amine" of DTAF and the amine substrate are unionized. Methyl substituted secondary amines were consistently more reactive than the corresponding primary amine, but further imposition of steric bulk about the amine nitrogen significantly reduced the reactivity of amines toward DTAF. In cases where such steric bulk is minimal. DTAF appears to be a suitable fluorescent labelling reagent for direct analytical applications.