The Synthesis of Adenine‐Substituted Derivatives of NADP⁺ and Their Potential as Active Coenzymes and Affinity Adsorbents

Abstract

Alkylation of NADP' with iodoacetic acid at pH 6.5 yielded N'-carboxymethyl-NADP' which, following enzymic reduction, alkaline rearrangement and enzymic reoxidation, gave N6-carboxymethyl-NADP' . Condensation of these .analogues with 1,6-diaminohexane in the presence of a water-soluble carbodiimide generated the N' and N6 N-(6-aminohexyl)-acetamide derivatives of NADP' respectively. The coenzymic activities of the N' and N 6 adenine-substituted carboxymethyl and N-(6-aminohexyl)-acetamido derivatives of NADP' were tested with several NADP'-dependent dehydrogenases. The carboxymethyl derivatives were more active (up to 145 %) relative to unsubstituted NADP' than the corresponding N-(6-aminohexyl)-acetamido compounds (up to 10 %). NADP+-N6-[N-(6-aminohexyl)-acetamide] was coupled to a soluble dextran and to Sepharose 4Bby the cyanogen bromide technique. The dextran-bound derivative contained 85 pmol NADP' analogue/g dry dextran and was reduced at approximately 35% the rate of native NADP' with glucose-6-phosphate dehydrogenase. The dextran-bound NADP' cycled at a rate of about 30 % relative to free NADP+ in a system comprising glucose-6-phosphate dehydrogenase and L-glutamate dehydrogenase. Sepharose-bound NADP+ proved to be an effective biospecific adsorbent for affinity chromatography and was competent for the resolution of a mixture of bovine albumin, lactate dehydrogenase and glucose-6-phosphate dehydrogenase. The latter was eluted almost quantitatively with a pulse of 0.5 mM NADP'.

Immobilised enzymes

“…Two synthetic approaches have been followed for nucleotide coenzymes:(a) the nucleotide analogue consisting of nucleotide and spacer has been synthesized first and then coupled to the matrix, (b) the nucleotide has been coupled to a preformed matrix which already has the spacer attached• The first mentioned pre-assembly approach is in my opinion advantageous since well defined preparations can thus be obtained, whereas with the other approach unsubstituted spacer arms are likely to be left on the matrix. Of the various adenine nucleotide analogues described in particular NAD(H) and NADP(I-I), those substituted at the exocyclic amino group [79,81 ] ( fig.9) and those substituted at position C-8 in the adenine moiety [80] retain most of their 'coenzymic activity'. In this essay I will have to restrict myself and I will only give one example to illustrate the usefulness of immobilised coenzymes.…”

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

“…The inactivity with isocitrate dehydrogenase of all our derivatives (Table 1) and of all but one of the N-1 and N6 derivatives prepared by Lowe and Mosbach [7] indicates high specificity requirements of this enzyme for the cofactor structure. Also the activity of the N-1 carboxymethyl derivative of Lowe and Mosbach [7], when compared to the inactivity of our N-I carboxylic analogue Ia, shows how stringent these structure requirements are.…”

“…Also the activity of the N-1 carboxymethyl derivative of Lowe and Mosbach [7], when compared to the inactivity of our N-I carboxylic analogue Ia, shows how stringent these structure requirements are.…”

“…The synthesis and coenzymic activity of macromolecular soluble NAD' [l-61 and NADP' [7] 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.…”

New Coenzymically‐Active Soluble and Insoluble Macromolecular NAD + Derivatives

“…Recently the synthesis and coenzymic activity of macromolecular soluble NAD' [l -51 and NADP' [6] derivatives from these and other laboratories has been reported. In all these derivatives the site of attachment, through a suitable linkage, of the cofactor molecule to the polymer was the exocyclic adenine amino group (P).…”