[NH2:1][CH:2]1[C:14](=[O:15])[N:4]2[C:5]([C:11]([OH:13])=[O:12])=[C:6]([CH2:9][OH:10])[CH2:7][S:8][C@H:3]12.P([O-])([O-])([O:18][C:19](=O)[NH2:20])=O.S([O-])([O-])(=O)=O.[Mg+2]>[Cl-].[Mn+2].[Cl-].C(O)(=O)C>[NH2:1][CH:2]1[C:14](=[O:15])[N:4]2[C:5]([C:11]([OH:13])=[O:12])=[C:6]([CH2:9][O:10][C:19](=[O:18])[NH2:20])[CH2:7][S:8][C@H:3]12
7-amino-3-hydroxymethylceph-3-em-4-carboxylic acid (40 μg), [14C] carbamoyl phosphate (150 μg, 7.5 μCi), magnesium sulphate (20 μg), manganese chloride (20 μg) and pipes buffer (0.5 μmole, pH 6.75) were incubated with 0-transcarbamoylase as described in Example 9(i). After incubation the mixture was acidified with glacial acetic acid (10 μl), clarified by centrifugation and the supernatant dried by rotary evaporation. This material was taken up in acetate/formate buffer [glacial acetic acid (5 ml), formic acid (0.2 ml) in methanol/water (40:60 v/v, 1 liter), pH adjusted to 3.9 with 4 N sodium hydroxide] (30 μl) and a portion (10 μl) was applied to a Partisil 10 SAX column (250 mm×4.6 mm) and eluted with the same buffer at 100 psi (1 ml/minute). The U.V. absorption at 260 nm and radioactivity of the eluate were monitored. A new U.V. absorption peak was detected after 12 minutes which also contained radioactivity. This peak corresponded to that obtained with authentic title compound. The conversion of cephalosporin starting material into title compound was 13%.
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