Thiamine (vitamin B1) is essential for energy metabolism, and interruption of its utilization pathways is linked to various disease states. Thiamine pyrophosphate (TPP, the bioactive form of ) functions as a coenzyme of a variety of enzymes. To understand the role of vitamin B1 in these diseases, a chemical approach is to use coenzyme analogues to compete with TPP for the enzyme active site, which abolishes the coenzyme function. Exemplified by oxythiamine and triazole hydroxamate , chemical probes require the coenzyme analogues to be membrane-permeable and of broad inhibitory activity to the enzyme family (rather than being too selective to particular TPP-dependent enzymes). In this study, using biochemical assays, we show that changing the hydroxamate metal-binding group of to a 1,3-dicarboxylate moiety leads to the potent inhibition of multiple TPP-dependent enzymes. We further demonstrate that this dianionic thiamine analogue when masked in its diester form becomes membrane-permeable and can be unmasked by esterase treatment. Taken together, our inhibitors are potentially useful chemical tools to study the roles of vitamin B1, using a prodrug mechanism, to induce the effects of thiamine deficiency in cell-based assays.