A mechanism for the fluorogenic reaction of amino groups with fluorescamine and MDPF.
Fluorescamine and 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF) react with primary amines at alkaline pH to yield highly fluorescent products, claimed to be diaryl-2-hydroxy-pyrrolinones. However, it seems to have been overlooked that these products appear as the pseudobase of coplanar and cationic diarylpyrrolones. Horse blood smears subjected to fluorogenic MDPF and fluorescamine reactions showed eosinophil granules with bright blue-white fluorescence, which required washing at neutral pH and was dependent on the presence of amino groups. The fluorescence of MDPF-butylamine product was abolished at alkaline pH and by bisulphite, suggesting that nucleophilic attacks to the pyrrolone ring (with formation of carbinol (pseudobase) and sulfonate derivatives, respectively), destroy the planarity and conjugation of the whole molecule, thus abolishing the emission at long wavelength. Analysis of the correlation between the largest conjugated fragment (LCF) values and the emission peaks of several fluorophores (fluorescamine- and MDPF-butylamino products, non-rigid fluorochromes) showed the best-fit when the cationic pyrrolones were considered. Although the pseudobases of fluorescamine- and MDPF-amino derivatives are formed at alkaline pH, a full conjugated, coplanar and cationic molecule is suggested to be the true fluorescent product.
Stockert
Juan C
JC
Department of Biology, Faculty of Sciences, Autonomous University of Madrid, Cantoblanco, 28049 Madrid, Spain. juancarlos.stockert@uam.es
Blázquez
Alfonso
A
Galaz
Sergio
S
Juarranz
Angeles
A
D000588:Amines / Q000737:chemistry*; D005450:Fluorescamine / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry*; D005663:Furans / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D015394:Molecular Structure
D000588:Amines; D005456:Fluorescent Dyes; D005663:Furans; D005450:Fluorescamine; C008394:2-methoxy-2,4-diphenyl-3(2H)-furanone