2-(Hydroxymethyl)-6-(4-nitrophenoxy)oxane-3,4,5-triol → p-Nitrophenyl beta-D-glucuronide
Reaction equation
C([O-])(=[O:3])C.[Na+].[CH:6]1[C:11]([N+:12]([O-:14])=[O:13])=[CH:10][CH:9]=[C:8]([O:15][CH:16]2[O:21][CH:20]([CH2:22][OH:23])[CH:19]([OH:24])[CH:18]([OH:25])[CH:17]2[OH:26])[CH:7]=1.NCC(O)=O.[OH-].[Na+]>>[CH:10]1[C:11]([N+:12]([O-:14])=[O:13])=[CH:6][CH:7]=[C:8]([O:15][C@@H:16]2[O:21][C@H:20]([C:22]([OH:3])=[O:23])[C@@H:19]([OH:24])[C@H:18]([OH:25])[C@H:17]2[OH:26])[CH:9]=1Participants
4
Procedure
The reagent solutions were: NaAc buffer (200 mM sodium acetate buffer pH 4.5); Substrate (50 mM p-nitrophenyl-α-D-glucopyranoside (Sigma N-1377) in NaAc buffer (0.3 g/20 ml)) and stop solution (800 mM glycine-NaOH buffer pH 10). 30 μl filtered supernatant was placed in a fresh 96-well flat bottom MTP. To each well 50 μl NaAc buffer and 120 μl substrate was added and incubated for 30 minutes at 50° C. (Thermolab systems iEMS Incubator/shaker HT). The reaction was terminated by adding 100 μl stop solution. The absorbance was measured at 405 nm in a MTP-reader (Molecular Devices Spectramax 384 plus) and the activity was calculated using a molar extinction coefficient of 0.011 μM/cm.
Workup
- filtration30 μl filtered supernatant
- additionTo each well 50 μl NaAc buffer and 120 μl substrate was added
- customThe reaction was terminated
- additionby adding 100 μl
Sources & evidence
- ORD record
- ord-e953530c27044291912712b1cdb69482
- Source dataset
- uspto-grants-2013_11
- Patent number
- US08592194B2
Reaction notes
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