Photoinactivation of the thiamin transport system in Saccharomyces cerevisiae with azidobenzoyl derivatives of thiamin

In an attempt to obtain a potent inhibitor for thiamin transport of Saccharomyces cerivisiae three novel thiamin derivatives having an arylazido substituent in the thiazole moiety have been synthesized. The derivatives prepared were 4-azidobenzoylthiamin (ABT), 4-azidobenzoylthiamin disulfide (ABTD), and 4-azido-2-nitrobenzoylthiamin disulfide (ANBTD). Among the newly prepared photoreactive azidobenzoyl derivatives of thiamin, ANBTD showed the strongest competitive inhibition with an apparent Ki of 7.9 nM against thiamin uptake by S. cerevisiae IFO-2375. The Ki values for ABT, 4-azido-2-nitrobenzoylthiamin (ANBT), and ABTD were 187 nM, 83 nM, and 15 nM, respectively. When exposed to visible light, ANBTD inactivated in a time- and concentration-dependent manner the uptake of 14C]thiamin by yeast protoplasts as well as intact cells. Remaining activities of the thiamin uptake by the intact cells were 71.9%, 27.3%, 40.1%, and 15.0% after visible light irradiation for 15 min in the presence of 1 microM ABT, ANBT, ABTD, and ANBTD, respectively. The inactivation by ANBTD (0.05 microM) was partially prevented by previous addition of an excessive amount of thiamin (5 microM). Furthermore, it was found that ANBTD (0.5 microM) irreversibly inactivated 70.6% of the thiamin-binding activity of the membrane fraction from S. cerevisiae IFO-2375. These results suggest that ANBTD can inhibit yeast thiamin transport by photoinactivation of membrane-bound thiamin-binding protein in the plasma membrane which may be a functional component involved in the thiamin transport system of S. cerevisiae.