Mechanism of action of uridine diphoglucose dehydrogenase. Evidence for an essential lysine residue at the active site.

UDP-Glucose Analogues as Inhibitors and Mechanistic Probes of UDP-Glucose Dehydrogenase

“…The resin was removed by filtration, washed with MeOH, and solvent was removed in vacuo. Preparative TLC (44% CHCl 3/44% MeOH/ 12% H2O) followed by passage through cation-exchange resin (sodium form) and lyophilization gave 8a as a white powder (21 mg, 29%): 1 H NMR (400 MHz, D2O) δ 5.45 (dd, 1H, J ) 6.9, 3.4), 4.10 (dq, 1H, J ) 6.7, 1.5), 3.71 (dd, 1H, J ) 9.5, 9.4), 3.60 (dd, 1H, J ) 10.1, 1.4), 3.49-3.45 (m, 2H,), 1.22 (d, 1H, J ) 6.6); 13 Uridine 5′-(7-deoxy-L-glycero-r-D-gluco-heptopyranosyl Diphosphate), Sodium Salt (4a). Compound 8a (22 mg, 0.074 mmol) was converted to its pyridinium counterion form and maintained in vacuo overnight.…”

“…Flash chromatography (50% Et2O/50% hexanes) yielded an analytically pure mixture of anomers as a white solid (4β:1R by 1 H NMR, 478 mg, 83%). Successive recrystallizations from Et2O gave the pure β-anomer 7a (274 mg, 48%): [R] 20 D ) -15.2 (c ) 0.798, CHCl3); 1 H NMR (400 MHz, CDCl3) δ 5.63 (d, 1H, J ) 8.3), 5.20 (dd, 1H, J ) 9.3, 9.3), 5.13 (dd, 1H, J ) 9.2, 8.3), 5.11 (dd, 1H, J ) 9.6, 9.4), 5.03 (dq, 1H, J ) 6.6, 2.0), 3.56 (dd, 1H, J ) 9.8, 2.0), 2.10 (s, 3H, Ac), 2.04 (s, 3H, Ac), 2.01 (s, 3H, Ac), 1.98 (s, 3H, Ac), 1.98 (s, 3H, Ac), 1.27 (d, 3H, J ) 6.6); 13 7-Deoxy-L-glycero-r-D-gluco-heptopyranosyl Phosphate, Sodium Salt (8a). Compound 7a (100 mg, 0.25 mmol) and 0.5 mL of Et2O were quickly added to crystalline phosphoric acid (210 mg) that had been dried overnight in vacuo over P2O5.…”

“…Compound 8b (16 mg, 0.053 mmol) was subject to coupling with UMP-morpholidate and purification as described for the preparation of 4a. This procedure yielded 4b as a white powder (8.6 mg, 26%): 1 H NMR (400 MHz, D2O) δ 7.93 (d, 1H, J ) 8.1), 5.99-5.98 (m, 2H), 5.60 (dd, 1H, J ) 7.3, 3.4), 4.37-4.17 (m, 5H), 4.13 (dq, 1H, J ) 6.7, 2.6), 3.93 (dd, 1H, J ) 10.2, 2.4), 3.75 (dd, 1H, J ) 9.6, 9.3), 3.52 (ddd, 1H, J ) 9.8, 3.3, 3.1), 3.39 (dd, 1H, J ) 10.1, 9.2), 1.21 (d, 3H, J ) 6.6); 13 C NMR (75 MHz, D2O) δ 166.6, 152.1, 141.8, 102.9, 95.8 (d, J ) 6.5), 88.6, 83.5 (d, J ) 8.9), 74.9, 74.0, 73.3, 71.8 (d, J ) 8.2), 70.8, 69.9, 66.8, 65.2 (d, J ) 5.0), 15.6; 31 P NMR (121.5 MHz, D2O) δ -10.8 (d, 1P, J ) 20.3), -12.5 (d, 1P, J ) 20.8); HR-LSI(-) MS (thioglycerol matrix) m/z calcd for C16H24N2O17P2Na 601.0448, found 601.0461. 6,7-Dideoxy-1,2:3,5-di-O-isopropylidene-6-C-methyl-r-D-gluco-hept-6-enofuranose (10).…”

“…Preparative TLC gave the acetylated free acid of 8b (73 mg), which was immediately subjected to O-deacetylation and purification as described. This procedure gave 8b as a white powder (23 mg, 31%): 1 H NMR (400 MHz, D2O) δ 5.43 (dd, 1H, J ) 7.1, 3.7), 4.09 (dq, 1H, J ) 6.7, 2.4), 3.90 (dd, 1H, J ) 10.1, 2.3), 3.71 (dd, 1H, J ) 9.4, 9.4), 3.48-3.45 (m, 1H), 3.32 (dd, 1H, J ) 10.0, 9.3), 1.16 (d, 3H, J ) 6.8); 13 C NMR (75 MHz, D2O) δ 94.4 (d, J ) 5.3), 74.2, 73.6, 72.1 (d, J ) 6.9), 71.0, 66.7, 15.5; 31 P NMR (121.5 MHz, D2O) δ 1.18 (s); HR-LSI(-) MS (thioglycerol matrix) m/z calcd for C7H14O9P 273.0375, found 273.0373.…”

“…Flash chromatography (50% Et2O/50% hexanes) gave 7b and its R-anomer (3β:1R by 1 H NMR, 688 mg, 80%). Successive recrystallizations (Et2O/hexanes) gave 7b (429 mg, 50%): [R] 20 D ) +23.4 (c ) 0.798, CHCl3); 1 H NMR (400 MHz, CDCl3) δ 5.68 (d, 1H, J ) 8.2), 5.20 (dd, 1H, J ) 9.3, 9.3), 5.05 (dd, 1H, J ) 9.4, 8.2), 5.00 (dd, 1H, J ) 10.0, 9.2), 4.90 (dq, 1H, J ) 6.7, 2.3), 3.76 (dd, 1H, J ) 10.1, 2.4), 2.09 (s, 3H, Ac), 2.03 (s, 3H, Ac), 2.03 (s, 3H, Ac), 2.00 (s, 3H, Ac), 1.98 (s, 3H, Ac), 1.23 (d, 3H, J ) 6.7); 13 7-Deoxy-D-glycero-r-D-gluco-heptopyranosyl phosphate, Sodium Salt (8b). Compound 7b (100 mg, 0.25 mmol) was treated according to the modified MacDonald procedure described for the preparation of 8a.…”