Enantiomeric perylene-glycerolipids as fluorogenic substrates for a dual wavelength assay of lipase activity and stereoselectivity

A new type of fluorogenic alkyldiacyl glycerols was synthesized and used as fluorogenic substrates for the analysis of lipase activities and stereoselectivities. These compounds contain perylene as a fluorophore and the trinitrophenylamino (TNP) residue as a quencher. Both substituents are covalently bound to the omega-ends of the sn-2 and sn-1(3) acyl chains, respectively. Upon glycerolipid hydrolysis, the residues are separated from each other thus allowing determination of lipase activity by the continuous increase in fluorescence intensity which is caused by dequenching. Using enantiomeric pairs of these compounds, we were able to analyze lipase stereoselectivity depending on the reaction medium. Mixtures of enantiomeric fluorogenic alkyldiacyl glycerols, selectively labelled with pyrene or perylene as fluorophores, can be used for a dual-wavelength ''stereoassay'' of lipases. Since absorption and emission maxima of both labels are clearly separated, hydrolysis of the respective enantiomeric substrates can be determined simultaneously, and the difference in the rates of hydrolysis can be taken as a parameter for the stereopreference of a lipase. Hydrolysis rates measured with perylene-substituted lipids are generally lower than those obtained with the pyrene analogs. Thus, with a mixture of perylene and pyrene-substituted lipids, we observe a higher apparent stereoselectivity of lipases since we measure a combination of stereo- and substrate selectivity. In the presence of albumin, all microbial lipases tested so far exhibit stereopreference for the sn-1 glycerol position. In our assay, the apparent stereoselectivities are highest if in the presence of albumin, the sn-1 position carries pyrene and the sn-3 position is substituted with perylene. The lipase stereoselectivity assay described here requires the simultaneous measurement of the fluorescence intensities at two different wavelengths in a single cuvette and can thus be carried out using existing and cheap instrumentation that was developed for the fluorimetric analysis of Ca++ concentrations. (C) 1996 Wiley-Liss, Inc.