The direct biotransformation of (4a) into (Sa), however, proceeded in only low yields owing to the instability of the aminoketone (5a), the amino group being free. Protection of the amino function by a benzyloxycarbonyl group proved especially favorable, since both (46) as well as (5b) crystallize particularly well and deblocking of (5b) can be achieved in one step with ring-closure to give the l-deoxynojirimycin ( I ) .Reaction of (4a) with benzyloxycarbonyl chloride in water at pH=8-10 afforded (4b), which could be quantitatively oxidized with Gluconobacter oxydans to (Sb). The yield was more than 90%, for a conversion of 60-80 g per liter of culture medium. The hydrogenation of (Sb) in methanollwater led stereoselectively[61, with removal of the protecting group and ring-closure, to 1-deoxynojirimycin Using the same synthetic sequence, we were also able to prepare some N-alkyl-1 -deoxynojirimycin derivatives[31 and-starting from D-mannose-the manno-isomer of (I), 1,5-dideoxy-1,5-imino-~-mannitol~'~ (yield 14%). (1j.
Procedure(56): 7 L of nutrient medium (H20, 5% sorbitol, 2% Ohly yeast extract, 0.4% K2HP04, pH=6.5 with KOH) is autoclaved in a 1OL fermenter for 45 min at 121"C, inocculated with Gluconobacter oxydans''] (250 mL pre-culture, same medium), and incubated for 24 h at 30°C, 10 L air/ min, 500 rpm. The culture is treated 5 times at intervals of 24 h with a hot (80-90°C) solution of 100 g (46) (m.p. 142-144 "C, from water) in 750 mL of water. After 2d (56) begins to crystallize out, after 6d the fermentation is finished. The precipitate is filtered off by suction, dissolved in 6 L of methanol and the cell residues separated; the solvent is removed in a rotary evaporator and the residue recrystallized from 2-propanol. Yield 455 g (92v0), m.p.
