Glycosynthases catalyze the formation of a glycosidic bond between a glycosyl fluoride donor substrate and a glycosyl acceptor substrate with high yield, thus providing a valuable approach for the synthesis of carbohydrates and glycoconjugates. Chemical complementation can be used to link glycosynthase activity to the transcription of a reporter gene in vivo, providing a selection for the directed evolution of glycosynthase enzymes with improved properties. In this approach, glycosynthase activity is detected as covalent coupling between a small molecule disaccharide acceptor substrate and a small molecule disaccharide alpha-fluoro donor substrate. Here we report the optimized design and synthesis of these small molecule substrates. These optimized substrates are shown to give a robust, glycosynthase-dependent transcriptional read-out in the chemical complementation assay. The full synthesis and characterization of these substrates are reported for the first time. These optimized chemical dimerizer substrates should allow the potential of chemical complementation for the directed evolution of glycosynthases with diverse substrate specificities and improved properties to be fully realized.