Phenol → 4-Nitrophenol

HRID 1406877Notes

Reaction equation

HRID 1406877 reaction equation
Cl[C:2]1[CH:13]=[C:12]([N+:14]([O-:16])=[O:15])[CH:11]=[CH:10][C:3]=1[O:4]C(C)C([O-])=O.ClC1C=C([N+]([O-])=O)C=CC=1O.C1(O)C=CC=CC=1.C1N(CCCS(O)(=O)=O)CCOC1.O=C1O[C@H]([C@H](CO)O)C([O-])=C1O.[Na+].O=C(CCC([O-])=O)C([O-])=O>CS(C)=O>[N+:14]([C:12]1[CH:13]=[CH:2][C:3]([OH:4])=[CH:10][CH:11]=1)([O-:16])=[O:15]

Participants

8

Reactant 6

HCID 996PhenolHCID 12074NitrofunginHCID 70807MopsHCID 164533Alpha-Ketoglutarate2×HCID 23667548Sodium AscorbateHCID 57371537HCID 57371537, compound4×

Product 1

HCID 9804-Nitrophenol

Reagents, catalysts & solvents 1

HCID 679Dimethyl SulfoxideSolvent

Procedure

A convenient assay of AAD-12 was devised using 2-(2-chloro,4-nitrophenoxy)propionate (CNPP) as substrate. Cleavage of CNPP by AAD-12 releases 2-chloro,4-nitrophenol. This phenol has a bright yellow absorbance at 410 nm at pH 7 enabling the reaction to be followed continuously or by endpoint analysis. The presence of AAD-12 activity can be monitored visually without the need for addition of further reagents. Microplate-based spectrophotometric assays were performed in a total volume of 0.2 ml 20 mM MOPS pH 6.75 containing 200 μM NH4FeSO4, 200 μM sodium ascorbate, 1 mM α-ketoglutarate, the appropriate amount of CNPP (added from a 10 mM stock made up in DMSO), and enzyme. Assays were initiated by addition of CNPP, enzyme, or α-ketoglutarate at time zero and the increase in absorbance followed for 10 min at 410 nm in a microplate reader. The first 2 min of the reaction was used to determine initial rates. A calibration curve performed under standard assay conditions (200 μl final assay volume) indicated that an absorbance at 410 nm of 0.1 was obtained from 25.1 μM 2-chloro, 4-nitrophenol. Using this assay, the kinetic constants for CNPP as a substrate were determined to be Km=31±5.5 μM and kcat=16.2±0.79 min−1

Workup

  1. customthe reaction
  2. additionThe presence of AAD-12 activity can be monitored visually without the need for addition of further reagents
  3. temperaturethe increase in absorbance
  4. customThe first 2 min of the reaction

Sources & evidence

ORD record
ord-c85f39d1f471498aa1873bccb4c7ab9c
Source dataset
uspto-grants-2014_12
Patent number
US08916752B2

Reaction notes

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