1,2-Dichloroethane → n-hydroxysulfosuccinimide sodium salt

HRID 469924

Reaction equation

HRID 469924 reaction equation
O.[OH:2][N:3]1[C:7](=[O:8])[CH2:6][CH:5]([S:9]([OH:12])(=[O:11])=[O:10])[C:4]1=[O:13].C(Cl)CCl.[Na+:18].[Cl-]>Cl.C(O)C(O)C.C1N(CCS(O)(=O)=O)CCOC1>[CH2:6]1[C:7](=[O:8])[N:3]([OH:2])[C:4](=[O:13])[CH:5]1[S:9]([O-:12])(=[O:11])=[O:10].[Na+:18]

Participants

8

Reactant 4

HCID 111,2-DichloroethaneHCID 962WaterHCID 5234Sodium ChlorideHCID 133909N-Hydroxysulfosuccinimide

Product 1

HCID 3520574n-hydroxysulfosuccinimide sodium salt

Reagents, catalysts & solvents 3

HCID 313Hydrochloric AcidSolventHCID 1030Propylene GlycolSolventHCID 781654-Morpholineethanesulfonic acidSolvent

Procedure

Recombinant rat PDGF-AA was purchased from R&D Systems (Minneapolis, Minn., USA) and dissolved in 4 mM HCl with 10% 1,2-propanediol at a concentration of 400 μg/ml (40 μg/100 μl). The water-soluble carboimide, 1-ethyl-3-(3-dimethylaminopropyl) carboimide hydrochloride (EDC, 50 μl; Sigma-Aldrich) and N-Hydroxysulfosuccinimide (sulfo-NHS, 50 μl; Pierce) in 0.1 M 2-(N-morpholino)ethanesulfonic acid (MES) buffer, 1 M NaCl, pH 6 (reaction buffer) were added to the PDGF-AA solution to obtain a final concentration of 11 mM EDC and 12 mM sulfo-NHS. After mixing and reacting the above reagents at room temperature for 15 min, Alexa Fluor 350 hydrazide sodium salt (5 mM) in dimethyl sulfoxide (DMSO) and 4-(4-N-maleimidophenyl) butyric acid hydrazide (MPBH, 7 mM) in 0.1 M sodium acetate pH 5.5 (200 μl) were added to the PDGF-AA for 75 min at room temperature. After adding 20 mM HCl (80 μl), the modified PDGF-AA (MI-PDGF-AA) was purified with AKTA-FPLC (Amersham Pharmacia) using Sephadex G-25 (Sigma-Aldrich) column (10×200 mm) (Amersham Pharmacia) equilibrated in 4 mM HCl pH 2.3, 150 mM NaCl, 5% propanediol. Elution of MI-PDGF-AA was monitored at 280 nm using UV-Vis monitor (UPC-900 Pharmacia Biotech) equipped on the FPLC. Fluorescently tagged MI-PDGF-AA was confirmed by measuring fluorescent emission at 445 nm on a fluorescence plate reader (345 nm excitation, Molecular Device). The concentration of MI-PDGF-AA was determined using the PDGF-AA enzyme-linked immunosorbent assay (ELISA; R&D Systems). Assays were performed according to the manufacturer's instructions. The yield of MI-PDGF-AA was determined to be 30.4 μg (7.6 μg/ml), yielding 76% recovery. To confirm the conjugation of MPBH to PDGF-AA, the reaction described above, excluding the addition of Alexa Fluor 350 was performed. The molecular weight of conjugated PDGF-AA-MPBH was determined using MALDI-TOF.

Workup

  1. additionwere added to the PDGF-AA solution

Sources & evidence

ORD record
ord-78ea46df97e54139b8efe0d0a3e39a4a
Source dataset
uspto-grants-2014_01
Patent number
US08629197B2

Linked notes

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