反应 #348330
ord-deb97bd49c524110a64ea3ae885506b7
反应方程式
反应物
反应条件
后处理
- 1浓缩Concentrated enzyme (0.5 ml)
- 2其他was incubated at 28°
- 3萃取The supernatant was extracted with ethylacetate (2.5 ml)
- 4其他The organic phase was collected
- 5其他evaporated to dryness
- 6其他The cephalosporins it contained were then separated by hplc [100 mm×5 diam. column Partisil 5 eluted at 1500 psi with chloroform:methanol:90% formic acid (25:2:1 v/v) saturated with water]
- 7其他were detected by their absorption at 270 nm
实验过程
Concentrated enzyme (0.5 ml) was incubated at 28° in a final volume of 1.0 ml water containing (6R,7R)-3-hydroxymethyl-7-[2-methoxyimino-2-(fur-2-yl) acetamido]ceph-3-em-4-carboxylic acid (syn-isomer) (0,1 μmole), carbamoyl phosphate (3 μmoles), ATP (1 μmole), magnesium chloride (1 μmole), manganese chloride (1 μmole) & pipes buffer (50 μmoles) adjusted to pH 6.0 with potassium hydroxide. After 2 hr the reaction was stopped by the addition of acetic acid (20 μl). The pH was then adjusted to 2.0 with M hydrochloric acid and the precipitated protein was centrifuged off. The supernatant was extracted with ethylacetate (2.5 ml). The organic phase was collected and evaporated to dryness. The cephalosporins it contained were then separated by hplc [100 mm×5 diam. column Partisil 5 eluted at 1500 psi with chloroform:methanol:90% formic acid (25:2:1 v/v) saturated with water] and were detected by their absorption at 270 nm. Their retention time was compared with standard samples. The size of the U.V. absorbing peaks showed that 20% of the cephalosporin starting material had been converted to title compound.