Fluorescence properties of o-phthaldialdehyde derivatives of amino acids

Abstract

The fluorescence properties of the products formed by reaction of o-phthaldialdehyde with amino acids and their derivatives, in the presence of thiol compounds, have been studied. The emission spectra, quantum yields, and lifetimes depend on the primary amine and thiol compound used; the observations confirm the report (Simsons, S.S., Jr. and Johnson, D.F. (1978) J. Org. Chem. 43, 2886–2891) that the product incorporates molecules of all three types of compounds. The fluorescence quantum yields of o-phthaldialdehyde derivatives of the naturally occuring amino acids ranged from 0.33 to 0.47, using 2-mercaptoethanol as the thiol compound. The fluorescence lifetimes were about 18–20 ns. Lower quantum yields were obtained when mercaptoethanol was replaced by dithiothreitol or ethnethiol. Derivatives of amino acid amides and peptides had quantum yields as low as 0.03, due to quenching by the carboxamide group. The intramolecular quenching was relieved by the detergent, sodium dodecyl sulfate, and by dimethylsulfoxide. Monosubstituted lysine exhibited a normal fluorescence, but the di-substituted product was largely quenched, presumably due to interaction between the two isoindole fluorophors. Fluorescence stopped-flow experiments showed that the α- and ϵ-amino groups reacted at different rates, with the ϵ-amino group reacting 10 times faster, with a t 1 2 of about 6 s under pseudo first order conditions at pH 9.0 with 10 −3 M o-phthaldialdehyde. The amount of instability shown by the o-phthaldialdehyde derivatives depended on the thiol compound used, the primary amine involved, and the solvent. Cysteine and o-phthaldialdehyde reacted to give an unstable, weakly fluorescent product; but cysteine could be assayed normally if its sulfhydryl was blocked. The o-phthaldialdehyde reagent was discussed in relation to fluorescamine, another reagent for primary amines.