SYNTHESIS OF ALPHA-D-GLCP-(1 -] -ALPHA-D-GLCP-(1-]3)-ALPHA-D-GLCP-O-(CH2)(8)COOCH3 FOR USE IN THE ASSAY OF ALPHA-GLUCOSIDASE-I ACTIVITY

The chemical synthesis of alpha-D-Glcp-(1 --> 2)-alpha-D-Glc p-(1 --> 3)-alpha-D-Glcp-O-(CH2)(8)COOCH3 (9), a substrate specific for cr-gluc osidase I, is reported. This enzyme removes the terminal alpha-D-Glcp unit to produce alpha-D-Glcp-(1 --> 3)-alpha-D-Glcp-O-(CH2)(8)COOCH3 ( 10). This is the first synthetic substrate described for glucosidase I that allows kinetic evaluation of substrates and inhibitors of this e nzyme. Tetramethylrhodamine was coupled to 9 through an ethylenediamin e linker to produce a brilliant red derivative. Addition of this fluor escent dye did not affect enzyme binding to the substrate, as determin ed by a comparison of the K-m value (1.3 mM). The fluorescent label al lows visual detection of 2-3 pmol of product by TLC. (C) 1996 Elsevier Science Ltd.