Modification Of Coenzyme I(NAD～+) By Poly(Ethyleneglycol) (PEG)

Macromolecular NAD⁺ derivative-PEG-N⁶-(2-amnioethyl)-NAD⁺ was prepared from carboxylation, alkylation, Dimroth rearrangement and covalent coupling of NAD⁺ and poly(ethylene glycol) monomethyl ether (MPEG) in this paper.The primary optimization results of each reaction were obtained and the kinetic properties of PEG-N⁶-(2-amnioethyl)-NAD⁺ and NAD⁺ were also studied. The carboxylation was carried out in room temperature by using Jone's reagent (CrO₃ and H₂SO₄) as the oxidizing agent for 16h, the mole ratio of -OH in MPEG to Cr in the Jone's reagent was 1:1, the oxidation by-products were removed by adsorbing onto activated charcoal for 3h, poly(ethylene glycol) monomethyl ether carboxylic acid (MPEG-COOH) was obtained by recrystallization in acetone at 4℃, yield of MPEG-COOH was 89.9%, yield of recrystallization was 97.4%, the overall yield was 87.6%, the acid value was 0.185mmol -COOH/g MPEG-COOH, and the melting point was 66～67℃. The alkylation was carried out in 35℃, pH 3.5 for 50h in the dark with a yield 81.7%. The crude product was purificated by JK110 cation exchange resin, and the isolated yield of N(1)-alkylated NAD⁺ derivative was 67.3%. The Dimroth rearrangement was carried out in 50℃, pH 6.8 for 5h in the dark with a yield 88.2% (78.6%). The crude product was purificated by JK110 cation exchange resin, and the isolated yield of N⁶-alkylated NAD⁺ derivative was 69.3%. The covalent coupling was carried out in room temperature, the ratio of N⁶-alkylated NAD⁺ derivative to MPEG-COOH to DCCI was 1: 1.1: 2. and the reaction completed within 5h with a yield 93.9%. The crude product was purificated by Sephadex G-50 column, and the isolated yield of PEG-N⁶-alkylated NAD⁺ derivative was 84.8%. The stability of this macromolecular NAD⁺ derivative was studied at alkline conditions with a pH 10.0, after three weeks the activity was remained above 90%; in the same condition, the activity of NAD⁺ was remained lower than 40%.The kinetic constants measured in pH 8.0 Tris-HCl at room temperature of alchol dehydrogenase (ADH) for this macromolecular NAD⁺ derivative were: V_max' = 14.8μmol/L·min, K_m' = 738μmol/L; in the same condition, the kinetic constants of alchol dehydrogenase (ADH) for NAD⁺ were: V_max = 19.6μmol/L·min, K_m = 127μmol/L。...