Acetate → (8R)-3-(4-Hydroxy-5-(Hydroxymethyl)Oxolan-2-Yl)-7,8-Dihydro-4H-Imidazo(4,5-D)(1,3)Diazepin-8-Ol

HRID 442619Notes

Reaction equation

HRID 442619 reaction equation
[C:1]([O-])(=[O:3])C.[Cl-].[Mg+2].[Cl-].P([O:20][CH2:21][C@H:22]1[O:26][C@@H:25]([N:27]2[C:36]3[N:35]=[CH:34][N:33]=[C:31](N)[C:30]=3[N:29]=[CH:28]2)[C@H:24](O)[C@@H:23]1[OH:38])(OP(OP(O)(O)=O)(O)=O)(=O)O.C([O-])(=O)C.[Na+].NO>C(O)C(N)(CO)CO.O>[CH2:31]1[N:33]=[CH:34][NH:35][C:36]2[N:27]([CH:25]3[O:26][CH:22]([CH2:21][OH:20])[CH:23]([OH:38])[CH2:24]3)[CH:28]=[N:29][C:30]=2[C@@H:1]1[OH:3]

Participants

8

Reactant 5

HCID 175AcetateHCID 787HydroxylamineHCID 59575'-AtpHCID 517045Sodium AcetateHCID 5360315Magnesium Chloride2×

Product 1

HCID 40926(8R)-3-(4-Hydroxy-5-(Hydroxymethyl)Oxolan-2-Yl)-7,8-Dihydro-4H-Imidazo(4,5-D)(1,3)Diazepin-8-Ol

Reagents, catalysts & solvents 2

HCID 962WaterSolventHCID 6503Tris(hydroxymethyl)aminomethaneSolvent

Procedure

Acetate kinase levels were measured essentially according to the method described by Brown et al., Journal of General Microbiology 102:327-336; 1977. Briefly, reaction constituents (all from Sigma chemicals) were prepared, and added to a microcentrifuge tube as follows: 12.5 μl of 200 mM magnesium chloride, 50 μl of 100 mM ATP, 30 μl of 200 mM sodium acetate, 50 μl of hydroxylamine solution (see below), and 30 μl of water. All of these solutions except for magnesium chloride are made in 50 mM Tris buffer, pH 8.0. The tube was placed into a 37° C. dry block and equilibrated for 3 minutes. Next, 100 μl of diluted cell-free extract (dilutions range between 1:2 and 1:50) was added, the contents mixed, and the reaction allowed to proceed for 10 minutes. The reaction was stopped by adding 450 μl of ferric chloride reagent (see below), and the tube was placed on ice for 10 min. The tube was then centrifuged in a microcentrifuge for 2 min, and absorbance at 540 nanometers determined versus a blank containing all of the above constituents except for 100 μl of deionized water in place of the enzyme preparation.

Workup

  1. customBriefly, reaction constituents (all from Sigma chemicals)
  2. customwere prepared
  3. additionadded to a microcentrifuge tube
  4. customwas placed into a 37° C.
  5. customdry block
  6. extractionNext, 100 μl of diluted cell-free extract (dilutions range between 1:2 and 1:50)
  7. additionwas added
  8. additionthe contents mixed
  9. waitto proceed for 10 minutes
  10. additionby adding 450 μl of ferric chloride reagent (see below)
  11. waitthe tube was placed on ice for 10 min
  12. waitThe tube was then centrifuged in a microcentrifuge for 2 min
  13. additiona blank containing all of the above constituents except for 100 μl of deionized water
  14. customin place of the enzyme preparation

Sources & evidence

ORD record
ord-95c734f0097b4bf79f50b4b3023ee2a5
Source dataset
uspto-grants-1999_04
Patent number
US05891686

Reaction notes

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