This program covers the synthesis of enzyme substrates and enzyme inhibitors which are used to study sphingolipid metabolism. The major ongoing project is the preparation of fluorescent substrates of lysosomal hydrolases that can detect enzyme activity within intact cells. The ultimate goal of these studies is to measure the activity of beta-glucocerebrosidase within living cells in order to study the outcome of gene-transfer experiments involving this enzyme or other lysosomal hydrolases. A number of beta-glucosides and beta-galactosides of 2,3-dicyanohydroquinone (DCH) have been prepared as fluorescent enzyme substrates. These compounds have alkyl groups attached at the 4-position to increase their hydrophobicity (n-octyl or n-octadecyl substituents) or to concentrate the substrate in lysosomes (4-imidazoyl-butyl substituent, """"""""Im-DCH""""""""). The latter approach, which employs a lysosomatropic amine, has proven to be the most useful with intact cells. The tetra-acetate of Im-DCH-beta-Gal has been studied most thoroughly using normal human fibroblasts and beta-galactosidase deficient fibroblasts that are derived from patients with GM1-gangliosidosis. Studies with these cells and with fibroblasts expressing other lysosomal storage disorders demonstrate that this fluorescent substrate is taken up by intact cells and hydrolysed intracellularly by beta-galactosidase. The development of techniques for slowing the efflux of the fluorescent product, Im-DCH, from cells is in progress. High specific activity [3H]-dihydrosphingosine was prepared for use in the study of sphingosine metabolism of Type C Niemann Pick disease. A synthesis of dihydrosphingosine containing 4 deuterium atoms per molecule was also carried out. This will be used as an internal standard for quantitating sphingosine and dihydrosphingosine in biologic samples by thermospray HPLC/Mass Spectroscopy. Experiments have begun on a new project in which inhibitors of the enzyme sphingosine-1 phosphate lyase are designed and synthesized. This enzyme catalyzes the final step of sphingosine catabolism. An irreversible inhibitor would be especially useful for studying the effects on sphingolipid metabolism and cell activation (via protein kinase C) caused by blocking sphingosine degradation.
