Reaction of o-phthalaldehyde and thiols with primary amines: Fluorescence properties of 1-alkyl(and aryl)thio-2-alkylisoindoles

The fluorescence properties of 1-alkyl(and aryl)thio-2-alkylisoindoles, formed by the reaction of o-phthalaldehyde (OPTA) and thiols with primary amines, are reported. Variations in thiol and amine substituents and solvent polarity have large effects on the isoindole fluorescence spectra. These parameters, in addition to 3-thiol substitution of the isoindoles, pH, and the use of phosphate vs borate aqueous buffers, were found to have dramatic effects on the corrected relative fluorescence intensity. Low concentrations and nonaqueous solvents apparently stabilized most adducts while aqueous solutions, especially at low pH, caused pseudo-first-order decomposition, probably via hydrolysis to the corresponding 2,3-dihydro-1H-isoindole-1-one. However, 3.3 × 10⁻⁸ solutions of the more intensely fluorescent adducts (total adduct 5 pmol) were readily detected if the fluorescence was determined shortly after adding the isoindole to pH 9.2 borate buffer. The adduct formed using ethanethiol and n-propylamine possessed spectral properties which were the most responsive to changes in solvent polarity and was the most stable under the various conditions employed. Finally, arguments are presented that these isoindoles are the products in several other fluorogenic assays using OPTA.