Synthesis and applications of phosphonoacetic derivatives.

In this paper, the synthesis of new phosphonoacetic acid derivatives and their applications in fields of biotechnological interest are discussed. Phosphonoacetic acids are competitive inhibitors of alkaline phosphatase, an enzyme widely used in diagnostics, as colorimetric detection tool. The phosphonoacetic acid's inhibition activity has been exploited by us for the obtainment of an innovative technique for non-radioactive DNA probes detection, the last being based on DNA labeling with the enzyme inhibitor, followed by detection by means of the chromogenic enzyme and substrate. Moreover, we have found a further application of phosphonoacetic acids, by the preparation of an affinity chromatography support that has been revealed to be very effective in the purification of alkaline phosphatase. Finally, phosphonoacetic acid derivatives have been tested also for their antiviral activity. Some of them, examined in preliminary in vitro experiments, have been found very active against Herpes simplex virus.

Alkaline phosphatase inhibitors as labels of DNA probes.

A new approach to nucleic acid labeling was developed by preparing bifunctional reagents containing, in addition to the DNA-linking group, a competitive inhibitor of the chromogenic enzyme alkaline phosphatase. The nucleic acids labeled in such a way were able to bind themselves to the enzyme, whose activity was restored in the presence of a chromogenic substrate. Five phosphonic-acid-containing reagents were synthesized and coupled to linearized pBR322 plasmid DNA by different condensation methods. Eight probes thus obtained were assayed in a modified dot-blot detection procedure obtaining the best nucleic acid detection sensitivity of 25 pg. Finally, five of the above probes were tested in hybridization experiments, reaching sensitivity of 50 pg.

Preparation of coenzymic activity of soluble polyethyleneimine-bound NADP+ derivatives.

Alkylation at N-1 of the NADP+ adenine ring with 3,4-epoxybutanoic acid gave 1-(2-hydroxy-3-carboxypropyl)-NADP+. Enzymic reduction of the latter, followed by alkaline Dimroth rearrangement and enzymic reoxidation, gave N6-(2-hydroxy-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-mercaptroproionic acid, gave 8-(2-carboxyethylthio)-NADP+. Carbodimide 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 co-enzymically 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 efficiences with the three enzymes of the N-1 derivatives to the one of the corresponding N6 anc C-8 analogues, the order of activity was N-1 greater than N6 greater 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.

New coenzymically-active soluble and insoluble macromolecular NAD+ derivatives.

Reaction in dimethyl sulfoxide of nicotinamide 8-bromoadenine dinucleotide with the disodium salt of 3-mercaptopropionic acid afforded nicotinamide-8-(2-carboxyethylthio)adenine dinucleotide, a new NAD+ analogue functionalized at the adenine C-8 position by an omega-carboxylic side chain. Carbodimide coupling of the latter derivative to high-molecular-weight water-soluble (polyethyleneimine, polylysine) and insoluble (aminohexy)-Sepharose) polymers gave the corresponding macromolecular NAD+ analogues. These derivatives have been shown to be enzymically reducible. The polyethyleneimine analogue showed a substantial degree of efficiency relative to free NAD+ with yeast alcohol dehydrogenase (47%) but a considerably lower one with rabbit muscle lactate dehydrogenase (3%); the polylysine analogue showed a low degree of efficiency with both enzymes (5-6%).

Synthesis of coenzymically active soluble and insoluble macromolecularized NAD+ derivatives.

Alkylation at N-1 of the NAD+ adenine ring with 3,4-epoxybutanoic acid, followed by chemical reduction to the alkali-stable NADH form and alkaline Dimroth rearrangement, gave the NADH derivative alkylated at the exocyclic adenine amino group. Enzymic reoxidation of the latter derivative gave nicotinamide-6-(2-hydroxy-3-carboxypropylamino)purine dinucleotide, a functionalized NAD+ analogue carrying an omega-carboxyalkyl side-chain at the exocyclic adenine amino group. Carbodiimide coupling of the latter derivative to high-molecular-weight water-soluble (polyethyleneimine, polylysine) and insoluble (aminohexyl-Sepharose) polymers gave the corresponding macromolecularized NAD+ analogues. These derivatives have been shown to be enzymically reducible. The polyethyleneimine and polylysine analogues showed a substantial degree of efficiency relative to free NAD+ with rabbit muscle lactate dehydrogenase (60 and 25% respectively) but a lower one with yeast alcohol dehydrogenase and Bacillus subtilis alanine dehydrogenase (2-7%). The polyethyleneimine derivative entrapped in cellulose triacetate fibres together with the lactate dehydrogenase was operationally stable during repetitive use.