Diphenyl-Furanones and Diphenyl-Oxopyrrole Derivatives: From Analytical Reagents for Amino Groups to New Fluorochromes for Cytochemical Staining of Chromatin DNA and Chromosomes: Proposal for Intercalative Binding and Fluorescence Mechanism

Abstract

Diaryl-furanones are specific analytical reagents for the biochemical detection of primary amines by fluorescence techniques. Well-known reagents are fluorescamine (Fluram) and 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF), yielding fluorescent products with λem at 480–490 nm. Although the reaction products claim to be pyrrolinones, recent studies show that they are really 3-oxopyrrole (pyrrolone) derivatives. Both reagents have been used for the cytochemical demonstration of primary amines. In this work, we have applied the fluorescent products of MDPF with amines (n-butylamine, BA; glucosamine, GA; and spermine, Sp), which showed interesting fluorescence reactions with chromatin DNA. 2,4-diphenyl-3-oxopyrrole products (diPOPy) can be easily synthesized according to well-known procedures, by mixing solutions of MDPF in acetone with water at pH 9 containing the amino compounds. DiPOPy derivatives of BA, GA, and Sp were used for spectroscopic, microscopic, and molecular modeling studies, showing a bright and selective blue–green fluorescence on DNA substrates, mainly chromatin, kinetoplast DNA, and stretched chromatin fibers. The cationic diPOPy fluorophore is planar, with a high partial positive charge in the N atom, and suitable for intercalative binding to DNA. A mechanism of fluorescamine fluorescence due to an inner-salt isomeric form is proposed, and an astonishing correlation between adenine–thymine-rich centromeric heterochromatin in mouse metaphase chromosomes after reaction of the fluorescamine reagent with protein amino groups is also discussed.