A pseudo-enantiomeric reaction was developed which was monitored on-line via 2D-fluorescence spectroscopy. During enzymatic reactions the enantiomeric excess can be followed on-line. Fluorescence spectroscopic detectable substrates, L-/D-phenylaianine-7-amido-4-methylcoumarine (L-/D-PheAMC) and L-/D-phenylalanine-7-amido-4-trifluoro-methylcoumarine (L-/D-PheAFC) were synthesized and deployed for applications in aqueous systems. Several proteases and esterases were tested to detect suitable biocatalysts for the enzymatic hydrolysis of the coumarine substrates. With a-chymotrypsin and the esterase from porcine liver, every coumarine substrate was hydrolyzed in an aqueous system and the determined enzymatic parameters were compared. The enantioselectivity and the enantiomeric excess of the enzymatic reactions were investigated. In simultaneous applications of the L- and D-substrates, the reactions were monitored on-line and the enantioselectivities, enantiomeric excesses and kinetic parameters were examined. To display the versatility of the on-line method, the enzymatic reactions were transferred to the organic solvent toluene. After the optimisation of the biocatalysis in toluene, the kinetic parameters were measured and the ee-parameters for the substrates and products were examined in off-line and on-line investigations. (c) 2005 Elsevier Inc. All rights reserved.