Syntheses and testing of substrates and mechanism-based inactivators for xylanases

The syntheses of the 2,5- and 3,4-dinitrophenyl β-xylobiosides by two separate routes are described, as well as the syntheses of the 2,4-dinitrophenyl β-glycosides of 2-chloro-2-deoxyxylobiose and 2-deoxy-2-fluoro-xylobiose. Both the 3,4- and 2,5-dinitrophenyl β-xylobiosides proved to be good substrates for the Bacillus subtilis xylanase, with kcat/Km values of 1.0 and 34.4 mM−1 s−1, respectively. Excellent time-dependent inactivation of the exoxylanase/glucanase from Cellulomonas fimi was provided by 2,4-dinitrophenyl 2-deoxy-2-fluoro-β-xylobioside, according to inactivation parameters of ki = 0.057 min−1 and Ki = 0.0035 mM.