1,2-Dichloroethane → L-Aspartic Acid

HRID 1943540

Reaction equation

HRID 1943540 reaction equation
[CH3:1][NH:2][CH2:3][C@H:4]([NH2:8])[C:5]([OH:7])=[O:6].C(Cl)CCl.S(C1C[C:21](=[O:23])N(O)C1=O)(O)(=O)=O.[OH:25]P([O-])(O)=O.[K+]>OP([O-])([O-])=O.[K+].[K+]>[CH3:1][NH:2][CH2:3][C@H:4]([NH2:8])[C:5]([OH:7])=[O:6].[NH2:8][C@H:4]([C:5]([OH:7])=[O:6])[CH2:3][C:21]([OH:23])=[O:25]

Participants

7

Reactant 4

HCID 111,2-Dichloroethane4×HCID 105089beta-(N-Methylamino)-L-alanine4×HCID 133909N-HydroxysulfosuccinimideHCID 516951Monobasic potassium phosphate

Product 2

HCID 5960L-Aspartic AcidHCID 105089beta-(N-Methylamino)-L-alanine

Reagents, catalysts & solvents 1

HCID 24450Potassium Phosphate, DibasicSolvent

Procedure

Conjugates of BMAA with BSA or with HRP were prepared by activating, in the absence of BMAA, the carboxylic groups of BSA or HRP with EDC thereby forming BSA-EDC or HRP-EDC. This was followed by the coupling of the activated carboxylate groups on the carrier protein to the primary amino group of BMAA. The procedure was as follows: 50 mg of EDC and 5 mg of sulfo-NHS (Sulfo-N-hydroxysuccinimide) were added to 15 mg of BSA or HRP pre-dissolved in 5 ml of 10 mM KH2PO4, pH 5.0. The reaction mixture was stirred for 2-3 minutes, thereby forming BSA-EDC or HRP-EDC, followed by the addition of 5 mL of a 1% solution of BMAA in 0.2 M K2HPO4, pH 8.5. After the solution was stirred overnight, the conjugate (BSA-EDC-BMAA or HRP-EDC-BMAA) was dialyzed exhaustively against PBS-7.4 (0.14 M NaCl, 10 mM K2HPO4, pH 7.4) or TBS-8 (tris-buffered saline, pH 8). Conjugates were stored at ≦20° C. (20° C. or less) By this procedure the conjugate was formed by linkage of BMAA to the carrier protein predominantly via the glutamic and aspartic acid residues of the carrier protein.

Workup

  1. stirringAfter the solution was stirred overnight
  2. customthe conjugate (BSA-EDC-BMAA or HRP-EDC-BMAA) was dialyzed exhaustively against PBS-7.4 (0.14 M NaCl, 10 mM K2HPO4, pH 7.4) or TBS-8 (tris-buffered saline, pH 8)
  3. customwere stored at ≦20° C.
  4. custom(20° C. or less)

Sources & evidence

ORD record
ord-081eacc0d5f947e5a4ca457b505093bc
Source dataset
uspto-grants-2013_03
Patent number
US08394596B1

Linked notes

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