2-Mercaptoethanol → m-Maleimidobenzoyl-N-hydroxysuccinimide

HRID 1358175Notes

Reaction equation

HRID 1358175 reaction equation
[OH-].[Na+].C1(=O)OC(=O)C=C1C.P([O-])([O-])([O-])=O.[Na+].[Na+].[Na+].[CH2:19]1[C:24](=[O:25])[N:23]([O:26][C:27]([C:29]2[CH:34]=[CH:33][CH:32]=[C:31]([N:35]3[C:40](=[O:41])[CH:39]=[CH:38][C:36]3=[O:37])[CH:30]=2)=[O:28])[C:21](=[O:22])[CH:20]1S(O)(=O)=O.SCCO>O>[CH2:19]1[C:24](=[O:25])[N:23]([O:26][C:27]([C:29]2[CH:34]=[CH:33][CH:32]=[C:31]([N:35]3[C:36](=[O:37])[CH:38]=[CH:39][C:40]3=[O:41])[CH:30]=2)=[O:28])[C:21](=[O:22])[CH2:20]1

Participants

7

Reactant 5

HCID 15672-Mercaptoethanol2×HCID 12012Citraconic AnhydridesHCID 14798Sodium HydroxideHCID 24243Trisodium Phosphate2×HCID 124851m-Maleimidobenzoyl-N-hydroxysulfosuccinimide ester

Product 1

HCID 93861m-Maleimidobenzoyl-N-hydroxysuccinimide

Reagents, catalysts & solvents 1

HCID 962WaterSolvent3×

Procedure

To 500 μl of H2O were added 5 mg of the peptide and the pH was adjusted to 8.5 with NaOH, to obtain a final concentration of 10 mg/ml. Citraconic anhydride was diluted in H2O to a concentration of 10 mg/ml. 500 μl of the anhydride solution were added to the peptide solution 100 μl at a time with adjustment of the pH to 8.5 between each addition. The solution was then stirred constantly at room temperature for 1 hour. This was followed by the addition of 100 μl of 1M sodium phosphate buffer (pH 7.2) and then 900 μl of 100 mM sodium phosphate buffer (pH 7.2). Sulfo-MBS was diluted in H2O to a concentration of 25 mg/ml and 400 μl of this solution were added to the peptide solution to obtain an MBS concentration of about 5 mg/ml. This solution was stirred constantly at room temperature for 30 minutes. 6 μl of β-mercaptoethanol were added for a final β-mercaptoethanol concentration of 35 mM. The solution was stirred constantly at room temperature for 1 hour. KLH was dissolved in PBS at 3 mg/ml and 2.5 ml were added to the peptide solution. The solution was stirred constantly at room temperature for 3 hours and then dialysed against 1 litre of PBS, with three changes of the PBS. The final peptide concentration was about 1 mg/ml and the final KLH concentration was about 1.5 mg/ml.

Workup

  1. customto obtain a final concentration of 10 mg/ml
  2. customto obtain an MBS concentration of about 5 mg/ml
  3. stirringThis solution was stirred constantly at room temperature for 30 minutes
  4. stirringThe solution was stirred constantly at room temperature for 1 hour
  5. dissolutionKLH was dissolved in PBS at 3 mg/ml and 2.5 ml
  6. stirringThe solution was stirred constantly at room temperature for 3 hours
  7. customdialysed against 1 litre of PBS, with three changes of the PBS

Sources & evidence

ORD record
ord-203374a78ff74139860cc4060ff23cbf
Source dataset
uspto-grants-2003_07
Patent number
US06596498B1

Reaction notes

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