Synthesis of poly(ethylene glycol)‐bound NADP by selective modification at the 6‐amino group of NADP

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Abstract
The N-1 position of the adenine ring of NADP was selectively alkylated by the reaction of 2'',3''-cyclic NADP with 3-propiolactone to yield 2'',3''-cyclic 1-(2-carboxyethyl)-NADP (I). Derivative I was converted to a mixture of the isomers of N6-(2-carboxyethyl)-NADP with their phosphate groups at the 2'' or 3''-position (IIa and IIb) by chemical reduction, alkaline rearrangement and chemical reoxidation. Carbodiimide coupling of the mixture of IIa and IIb to .alpha.,.omega.-diaminopoly(ethylene glycol) gave the 2'',3''-cyclic derivative of poly(ethylene glycol)-bound NADP(III), which was enzymically hydrolyzed to yield poly(ethylene glycol)-bound NADP (PEG-NADP). PEG-NADP has good cofactor activity (16-100% of that of NADP) for NADP-specific and NAD(P)-specific dehydrogenases except isocitrate and glucose dehydrogenases. For NAD-specific enzymes, PEG-NADP has higher cofactor activity than NADP: for horse liver alcohol dehydrogenase, the cofactor activity of PEG-NADP is 40 times that of NADP and 14% of that of NAD. Kinetic studies show that for most of enzymes tested, Km values for PEG-NADP are higher than those for NADP and V values for PEG-NADP are similar to those for NADP. PEG-NADP proved to be applicable in a continuous enzyme reactor, in which reactions of glutamate dehydrogenase and glucose-6-phosphate dehydrogenase were coupled by the recycling of PEG-NADP.