Rational design and evaluation of improved o-phthalaldehyde-like fluorogenic reagents

• Published in: Analytical biochemistry Vol. 144; no. 1; pp. 233 - 246
• Main Authors: Sternson, Larry A., Stobaugh, John F., Repta, A.J.
• Format: Journal Article
• Language: English
• Published: San Diego, CA Elsevier Inc 1985; Elsevier
• Subjects: Aldehydes; Amines - analysis; amino acid analysis; Aminoacids, peptides. Hormones. Neuropeptides; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Chemical Phenomena; Chemistry; Chromatography, High Pressure Liquid; fluorescence; Fundamental and applied biological sciences. Psychology; Indicators and Reagents - chemical synthesis; Indoles - analysis; Indoles - chemical synthesis; Isomerism; Kinetics; o-Phthalaldehyde; Proteins; Spectrometry, Fluorescence; Temperature; amino acid analysis; fluorescence; o-phthalaldehyde; Primary amine; Aminoacid; Analysis; Fluorogenic reagent
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/0003-2697(85)90111-3

Evidence was presented suggesting that the fluorescent isoindole produced by reaction of o-phthalaldehyde (OPA), ethanethiol, and primary amine was formed by initial imine formation follwed by conversion to an α-alkylaminobenzylsulfide and subsequent ring closure to form the isoindole nucleus. This mechanism suggested that the minimum structural requirement for condensation to an isoindole was an o-diacyl benzene in which one of the carbonyl groups was aldehydic. A major drawback of OPA as an analytical reagent is the limited stability of the fluorescent 1,2-disubstituted isoindole. Since isoindole instability is related to autoxidation at C-3, the use of o-(formyl) arylketones as alternatives to OPA is attractive in increasing the lifetime of the fluorescent species in that such reagents would form 1,2,3-trisubstituted isoindoles. Two compounds, o-acetylbenzaldehyde (OAB) and o-benzoylbenzaldehyde (OBB), were synthesized and evaluated as potential fluorogenic reagents. Both formed fluorescent products. The rate of formation of isoindole from the latter was too slow to make it of practical analytical value; however, OAB formed isoindoles with t 1 2 < 10 s and offered markedly improved stability over that observed with OPA.