Factors affecting the stability of fluorescent isoindoles derived from reaction of o-phthalaldehyde and hydroxyalkylthiols with primary amines.

The stability of a series of fluorescent isoindole derivatives formed in situ under analytical conditions following the reaction of o-phthalaldehyde (OPA) and 2-mercaptoethanol (2-ME) with a series of primary amines are reported. Increasing the bulk and degree of substitution at C-10 of the resulting isoindole resulted in substantial increases in product stability. The effects of excess OPA and 2-ME on isoindole stability were examined and OPA was observed to catalyze isoindole degradation while 2-ME had no effect. Previously proposed degradation mechanisms were reexamined in light of the present data and an alternate degradation pathway is proposed. 3-Mercapto-1-propanol (3-MP) was found to be a superior thiol for use in the fluorogenic OPA reaction. The OPA/3-MP reagent combination was utilized to derive several amino acids and offered detection limits (S/N = 2) of less than 200 fmol.