Preparation and Coenzymic Activity of Soluble Polyethyleneimine‐Bound NADP⁺ Derivatives

Abstract

Alkylation at N-1 of the NADP' adenine ring with 3,4-epoxybutanoic acid gave 1-(2-hydroxy-3-carboxypropy1)-NADP+ . Enzymic reduction of the latter, followed by alkaline Dimroth rearrangement and enzymic reoxidation, gave N6-(2-hydr~xy-3-carboxypropyl)-NADP+. On the other hand, bromination at C-8 of the NADP+ adenine ring, followed by reaction with' the disodium salt of 3-mercaptopropionic acid, gave 8-(2-carboxyethylthio)-NADP+. Carbodiimide coupling of the three carboxylic NADP' derivatives to polyethyleneimine afforded the corresponding macromolecular NADP+ analogues.The carboxylic and the polyethyleneimine derivatives synthesized have been shown to be coenzymically active with yeast glucose-6-phosphate dehydrogenase, liver glutamate dehydrogenase and yeast aldehyde dehydrogenase. The degree of efficiency relative to NADP' with the three enzymes ranged from 17 "/, to 100 % for the carboxylic derivatives and from 1 "/, to 36 % for the polyethyleneimine analogues. On comparing the efficiencies with the three enzymes of the N-1 derivatives to the one of the corresponding N6 and C-8 analogues, the order of activity was N-1 > N6 > C-8, except in the case of the carboxylic compounds with glutamate dehydrogenase, where this order was inverted. None of these modified cofactors were active with pig heart isocitrate dehydrogenase.The synthesis and coenzymic activity of macromolecular soluble NAD' derivatives, with potential applications in the industrial, analytical or biomedical field as active cofactors for dehydrogenase systems entrapped in porous structures, have been reported in recent times from these and other laboratories.In our two approaches to coenzymically active macromolecular NAD+ derivatives [4,6] the bonds involved in the linking of the coenzyme to the polymer have been shown to be hydrolytically stable. The same did not hold for the polymer-bound cofactors synthesized in the other laboratories ([l-3,7]; for the instability of the linking obtained by the cyanogen bromide coupling technique see also [S]), thus limiting the applicability of these modified coenzymes. In view of this we were interested in applying our macromolecularization methods also to NADP+ with the aim of obtaining coenzymically active, operationally stable cofactor analogues.The present paper describes the synthesis of three new NADP + analogues functionalized with a carboxyl group, i.e., 1-(2-hydroxy-3-carboxypropyl)-NADP+

Synthesis, Characterization and Preliminary Crystallographic Data of N6‐(6‐carbamoylhexyl)‐FAD‐d‐amino‐acid Oxidase from Pig Kidney, a Semi‐Synthetic Oxidase

“…An improved version of this approach using diphenyl phosphochloridate [27] has been shown to be superior with respect to the reaction yield, even in the case of modified AMP derivatives [38,391. The alternative route of specifically modifying FAD at the N1 position of the adenine ring by nucleophilic displacement followed by Dimroth rearrangement to prepare W-functionalized FAD has been described by several authors [40,41,181. Although the latter method seems to be more straightforward than the coupling approach, this method is disadvantageous, if the modification should lead to controlling the spacer length by -CH,-increments.…”

Self-complex formation of nicotinamide-modified dextran with carboxymethyl dextran using their degradation products

“…Firstly, the reduction of 4 by sodium ditionate was studied. Sodium dithionate is known to be a strong reductant toward NAD(P) C and other nicotinamide derivatives [19,20]. During the reduction of 4 with 16 or 26 NA C per 100 glucose units with sodium dithionate, the solution became turbid.…”

Synthesis and application of water-soluble macromolecular derivatives of the redox coenzymes NAD(H), NADP(H) and FAD

“…Bromination with Br 2 of the C(8) position of the adenine in aqueous solution followed by specific chemical reduction with NazS204 to C(8)-brominated NADH or NADPH and nucleophilic displacement of Br with 1,6-diaminohexane, introducing a spacered primary amino group, and enzymatic oxidation is similar in length to the traditional preparation of N6-alkylated -NAD(P). Similarly, Zappelli et al 72,73) introduced a carboxyl function at the C(8) position of the adenine of NAD(P) by reacting 8-bromo-adenine NAD(P) with the disodium salt of 3-mercaptopropionic acid in dimethyl sulfoxide without previous reduction, to give 8-(2-carboxyethylthio)-adenine-NAD(P). C(8)-functionalized coenzymes can generally be obtained in good overall yield (60-70 ~o) except for 8-(2-carboxyethylthio)-adenine-NADP (25 ~) 73) Compared to native NAD and NADP, N6-functionalized -and C(8)-functionalized NAD and NADP generally maintain acceptable coenzyme activities with respect to the usual dehydrogenases from different sources such as alcohol dehydrogenases, lactate dehydrogenases, malate dehydrogenases, glucose-6-phosphat dehydrogenases, glutamate dehydrogenases, etc.…”