The object of this work is to prepare artificial substrates of sphingolipid hydrolases that can detect enzyme activity within intact cells. Chromogenic substrates synthesized earlier in this section have proven highly useful for enzyme measurements in vitro. However, the requirements of in vivo measurement of enzyme activity necessitate the preparation of fluorogenic glycolipids. The substrates required for such an approach should be nonfluorescent prior to enzymatic cleavage of the glycoside bond. The released product should then be fluorescent at lysosomal pH. A group of strongly fluorescent phenols, obtained by 0-4 monoalkylation of 2,3-dicyanohydroquinone (DCH) were synthesized for this project. These were converted into glucosides and galactosides which were then tested as enzyme substrates. Most interesting was the beta-galactoside of a lysosomotropic derivative of DCH. The membrane-permeant tetraacetate of this substrate was hydrolysed within intact human fibroblasts. Moreover, beta-galactosidase deficient fibroblasts derived from patients with G(m1) gangliosidosis hydrolysed this compound at 6-17% of the rate seen with normal cells. Attempts to apply these techniques to the beta-glucocerebrosidase which is deficient in Gaucher's disease are in progress. Ultimately, these probes will be used to measure the efficiency of gene-transfer experiments with lysosomal hydrolase genes. In this setting, in vivo fluorogenic substrates have the potential to greatly increase the sensitivity for detection of gene expression in transformed cells. In conjunction with fluorescence activated cell sorting, fluorogenic probes should also expand the possibilities for study of the developmental biology of these recombinant cells.
