Siegler R, Sternson L A, Stobaugh J F
Department of Pharmaceutical Chemistry, University of Kansas, Lawrence 66045.
J Pharm Biomed Anal. 1989;7(1):45-55. doi: 10.1016/0731-7085(89)80066-4.
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.
DTAF已成功用于制备用于荧光偏振免疫分析的荧光标记试剂。评估了其作为伯胺和仲胺直接荧光分析衍生试剂的适用性。结果表明，DTAF的光谱特性与荧光素非常相似，并且显然对三嗪核的存在不敏感。光谱测定的pKa值也与荧光素和其他氨基芳基-s-三嗪的pKa值非常相似。DTAF易于水解降解，反应速率随pH值升高而增加，直到达到一个pH值，此时连接两个芳核的胺发生电离；在此pH值下，反应速率达到平稳值。伯胺和仲胺都能与DTAF有效反应，反应活性随胺反应物碱性的增加而增加。该反应依赖于pH值，在DTAF的“桥连胺”和胺底物均未电离的pH值下反应最有效。甲基取代的仲胺始终比相应的伯胺更具反应性，但在胺氮周围进一步增加空间位阻会显著降低胺对DTAF的反应性。在空间位阻最小的情况下，DTAF似乎是一种适用于直接分析应用的荧光标记试剂。
