Reaction #1178400

ord-c67d553bfeb947c3b1f988699f125973

Reaction equation

CC(=O)N[C@H]1[C@@H](OP(=O)(O)OP(=O)(O)OC[C@H]2O[C@@H](n3ccc(=O)[nH]c3=O)[C@H](O)[C@@H]2O)O[C@H](CO)[C@H](O)[C@@H]1O
UDP-GalNAc
CC(=O)N[C@H]1[C@@H](OP(=O)(O)OP(=O)(O)OC[C@H]2O[C@@H](n3ccc(=O)[nH]c3=O)[C@H](O)[C@@H]2O)O[C@H](CO)[C@H](O)[C@@H]1O
UDP-GalNAc
O=C([O-])[C@H]1O[C@H](OP(=O)([O-])OP(=O)([O-])OC[C@H]2O[C@@H](n3ccc(=O)[nH]c3=O)[C@H](O)[C@@H]2O)[C@H](O)[C@@H](O)[C@@H]1O.[Na+].[Na+].[Na+]
UDP-GlcA
→
CC(=O)N[C@H]1C(O)O[C@H](CO)[C@H](O)[C@@H]1O
GalNAc

Reactants

CC(=O)N[C@H]1[C@@H](OP(=O)(O)OP(=O)(O)OC[C@H]2O[C@@H](n3ccc(=O)[nH]c3=O)[C@H](O)[C@@H]2O)O[C@H](CO)[C@H](O)[C@@H]1O
UDP-GalNAc
—
( 1 )
CC(=O)N[C@H]1[C@@H](OP(=O)(O)OP(=O)(O)OC[C@H]2O[C@@H](n3ccc(=O)[nH]c3=O)[C@H](O)[C@@H]2O)O[C@H](CO)[C@H](O)[C@@H]1O
UDP-GalNAc
O=C([O-])[C@H]1O[C@H](OP(=O)([O-])OP(=O)([O-])OC[C@H]2O[C@@H](n3ccc(=O)[nH]c3=O)[C@H](O)[C@@H]2O)[C@H](O)[C@@H](O)[C@@H]1O.[Na+].[Na+].[Na+]
UDP-GlcA

Reagents

None

Solvents

O
water

Conditions

Detailed conditions
See reaction.notes.procedure_details.

Workup

  1. 1
    workup.ADDITIONadding the K4CP enzyme
  2. 2
    workup.WAITthe reaction was performed again at 30° C. for 5 hours
  3. 3
    workup.WAITThermal inactivation was performed for 1 minute
  4. 4
    Otherthe reaction mixture was subjected to ethanol precipitation
  5. 5
    workup.DISSOLUTIONthe residue was dissolved again in 200 μl of distilled water
  6. 6
    FiltrationThe solution was subjected to gel filtration chromatography in a Superdex Peptide HR10/30 column
  7. 7
    Otherwere collected every 1 minute (1 ml)
  8. 8
    workup.DISSOLUTIONeach of the lyophilized fractions was dissolved again in 10 μl of distilled water
  9. 9
    Otherto obtain a sample for MALDI-TOF-MS

Procedure

A reaction was performed in the same manner as that of (1) mentioned above under conditions of 30° C. and pH 7.2 for 8 hours by using CH6, UDP-GalNAc and UDP-GlcA as substrates and adding the K4CP enzyme. Then, 300 nmole of UDP-GalNAc was added again, and the reaction was performed again at 30° C. for 5 hours. Thermal inactivation was performed for 1 minute in boiling water, the reaction mixture was subjected to ethanol precipitation, and the residue was dissolved again in 200 μl of distilled water. The solution was subjected to gel filtration chromatography in a Superdex Peptide HR10/30 column using 0.2 M ammonium acetate as a developing buffer. The mixture was loaded at a flow rate of 1 ml/minute, and fractions were collected every 1 minute (1 ml). The obtained fractions were lyophilized, and each of the lyophilized fractions was dissolved again in 10 μl of distilled water. The solution was loaded on a small amount of Dowex 50 XW8 (H+ form) gel to obtain a sample for MALDI-TOF-MS.

Source

DOI: 10.6084/m9.figshare.5104873.v1Patent: US08129148B2uspto-grants-2012_03