Reaction #348330

Reaction equation

(6R,7R)-3-hydroxymethyl-7-[2-methoxyimino-2-(fur-2-yl) acetamido]ceph-3-em-4-carboxylic acid

carbamoyl phosphate

ATP

magnesium chloride

hydrochloric acid

potassium hydroxide

→

(6R,7R)-3-Carbamoyloxymethyl-7-[2-methoxyimino-2-(fur-2-yl) acetamido] ceph-3-em-4-carboxylic acid

Solvents

acetic acid

Conditions

Workup

1. 1
   ConcentrationConcentrated enzyme (0.5 ml)
2. 2
   Otherwas incubated at 28°
3. 3
   ExtractionThe supernatant was extracted with ethylacetate (2.5 ml)
4. 4
   OtherThe organic phase was collected
5. 5
   Otherevaporated to dryness
6. 6
   OtherThe 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. 7
   Otherwere detected by their absorption at 270 nm

Procedure

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.

Source