Phosphate → HCID 9836845, compound

HRID 2374657Notes

Reaction equation

HRID 2374657 reaction equation
P([O-])([O-])([O-])=O.[Na+].[Cl-].C(N(CC(O)=O)CC(O)=O)CN(CC(O)=O)CC(O)=O.[CH3:28][C:29](=[N:31][NH2:32])[CH3:30].[NH:33]([C:35]1[CH:50]=[CH:49][C:38]([C:39]([O:41][N:42]2[C:46](=[O:47])[CH2:45][CH2:44][C:43]2=[O:48])=[O:40])=[CH:37][N:36]=1)[NH2:34].CN(C=O)C>CN(C=O)C>[CH3:28][C:29](=[N:31][NH2:32])[CH3:30].[NH:33]([C:35]1[CH:50]=[CH:49][C:38]([C:39]([O:41][N:42]2[C:43](=[O:48])[CH2:44][CH2:45][C:46]2=[O:47])=[O:40])=[CH:37][N:36]=1)[NH2:34]

Participants

6

Reactant 4

HCID 1061PhosphateHCID 5234Sodium ChlorideHCID 6049Ethylenediaminetetraacetic AcidHCID 131724604HCID 131724604, compound

Product 1

HCID 9836845HCID 9836845, compound

Reagents, catalysts & solvents 1

HCID 6228DimethylformamideSolvent

Procedure

A 5 mg/mL solution of ovalbumin in PBS (100 mM phosphate, 150 mM NaCl, pH 7.4) and 2 mM EDTA (200 μL; 1 mg protein) was prepared. A solution of succinimidyl 6-hydrazinonicotinate acetone hydrazone (SANH)(EXAMPLE 2)(2 mg) in DMF (50 μL) is prepared. To the protein solution was added the SANH/DMF solution (15 eq.). The reaction mixture was incubated at room temperature for 4 hrs. The modified protein was isolated and buffer exchanged by placing the reaction mixture in a 30K ultra-free centrifugation device and washing three times with 0.1 M MES, 0.9% NaCl, pH 4.7 (3×400 μL). The purified protein was quantified for protein concentration (BCA assay; Pierce Chemical Co., Rockford, Ill.) and for hydrazine modification level by incubation of an aliquot of protein in a 0.5 mM 4-nitrobenzaldehyde in 0.1 M MES, 0.90% NaCl, pH 4.7 and measuring the absorbance at 360 nm (molar extinction coefficient 22,000).

Workup

  1. customThe modified protein was isolated
  2. washwashing three times with 0.1 M MES, 0.9% NaCl, pH 4.7 (3×400 μL)
  3. concentrationThe purified protein was quantified for protein concentration (BCA assay; Pierce Chemical Co., Rockford, Ill.) and for hydrazine modification level by incubation of an aliquot of protein in a 0.5 mM 4-nitrobenzaldehyde in 0.1 M MES, 0.90% NaCl, pH 4.7

Sources & evidence

ORD record
ord-6f6dea105b0846b1a4cae76e3b25a0b1
Source dataset
uspto-grants-2008_12
Patent number
US07462689B2

Reaction notes

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