A. Casini

Dimers of nicotinamide adenine dinucleotide: New evidence for the structure and the involvement in an enzymatic redox process

Abstract The composition and the structure of the product from the known electrochemical dimerization of the NAD+ have been conclusively demonstrated. A detailed analysis of the 1H and 13C nmr spectra has in fact led to the conclusion that the product contains three diastereoisomeric dimers of the 4,4′-tetrahydrobipyridyl type. Furthermore, the cytoplasmic fraction obtained from a standard mitochondrial preparation of rat liver has been shown to catalyze the oxygen uptake by the dimers. A 1 : 1 molar ratio of the reagents in the redox process is indicated by manometric data on oxygen uptake complemented by spectrophotometric analysis of the oxidized substrates, suggesting that H2O2 is the reduction product. NAD+ was identified as the oxidation product by an enzymatic method.

Photocatalyzed oxidation of NAD dimers

Abstract A mixture of dimers of nicotinamide adenine dinucleotide, largely 4,4−-linked, obtained by electrochemical reduction of NAD + , can be photooxidized back to NAD + in the presence of oxygen. Oxygen is consumed during the photooxidation process with the production of hydrogen peroxide. The oxidation is almost pH independent and is stimulated by light whose wavelength exceeds 300 nm. Lactate dehydrogenase and alcohol dehydrogenase added to the solutions under irradiation increased the oxygen uptake by the NAD dimers in a concentration-dependent way. These observations suggest that light induces the homolytic cleavage of NAD dimers to NAD radicals which in turn are oxidized to NAD + by oxygen.

Evidence for binding of NAD dimers to NAD-dependent dehydrogenases.

The binding of dimers of nicotinamide adenine dinucleotide, (NAD)2, to lactate, malate and alcohol dehydrogenase has been studied by the fluorescence quenching technique. While the alcohol dehydrogenase shows a low binding ability, malate and lactate dehydrogenases have been found to bind (NAD)2 in a specific way with high affinity. Malate dehydrogenase binds (NAD)2 more than NADH. All three dehydrogenases are inhibited by (NAD)2, which behaves as a competitive inhibitor with respect to both NAD+ and NADH. The results show that (NAD)2 is bound to the nucleotide-specific binding site of the dehydrogenases. (NAD)2 was found to stoichiometrically react with ferricyanide at variance with NADH. The specific interactions with the NAD-dependent dehydrogenases and the ability to enter in monoelectronic redox cycles suggest possible physiological roles for (NAD)2.

On the regio- and stereoselectivity of pyridinyl radical dimerization

The present work was carried out in order to elucidate the combined effects of the electron spin density on the ring carbons and the steric hindrance of the ring substituents upon the regio- and stereoselectivity of the dimerization of 3-carbamoyl- and 3-cyanopyridinyl radicals. To this purpose the composition of mixtures of diastereoisomeric dimers arising from the one-electron electrochemical reduction of several 3-carbamoyl and 3-cyano substituted pyridinium salts has been studied by 1H NMR spectroscopy. In some cases, single diastereoisomers have been isolated from mixtures by preparative HPLC.The results show that: (a) hindering steric effect of substituents at coupling sites prevails over electron spin density on coupling carbons in governing regioselectivity of 3-carbamoylpyridinyl radical dimerization; (b) large bulky N-ring substituents produce a significant shielding effect on the adjacent dimerization site; (c) the relative amounts of diastereoisomers in the mixtures of 4,4′- and 6,6′-linked dimers indicate that the dimerization process is largely stereoselective; (d) otherwise, nearly equal amounts of 4,4′- and 4,6′-linked dimers, and relative diastereoisomers as well, arise from the reduction of 3-cyano substituted pyridinium salts. This finding indicates that the presence of the carbamoyl substituent at the 3 position is a primary factor in inducing the regio- and stereoselectivity of pyridinyl radical dimerization.

1H-NMR study and structure determination of 4,4- and 4,6-dimers from electrochemical reduction of NADP+

The products arising from one-electron electrochemical reduction of the coenzyme nicotinamide adenine dinucleotide phosphate (NADP+) have been studied by HPLC chromatography and 1H-NMR spectroscopy. HPLC and NMR analyses have shown seven dimeric species, the most abundant of which (40%) has been isolated and has resulted to be an NADP 4,4-linked dimer. The other two diastereoisomeric 4,4-dimers present for the 25% and 10%, respectively, have been detected in the crude reaction mixture, but have not been isolated. The 4,4-tetrahydrobipyridine structure and the stereochemistry at the ring-ring junction for these three isomers have been determined on the basis of their NMR parameters. Preparative HPLC chromatography also led to two fractions enriched in another four dimers, present in the crude mixture, which turned out to have a 4,6-tetrahydrobipyridine structure. All the chemical shifts and the H,H coupling constants of the 4,4- and 4,6-tetrahydrobipyridine systems have been obtained for the seven compounds. For the most abundant among the 4,4-dimers the NMR analysis also gave the coupling constant values of the ribose-diphosphate chain.

Electrochemical reduction of 2-cyano-1-methylpyridinium ion

Abstract 2-Cyano-1-methylpyridinium ion, I, exhibits three well-defined polarographic waves in Britton-Robinson buffer at pH 5 to 8. Electrolysis at the plateau potential of the most positive wave, involving a one-electron uptake, leads to the formation of a pyridinyl radical which probably dimerizes to a 2,4′-linked structure, VIII. Electrolysis at the plateau potential of the second wave, involving a number of electrons variable between nearly 4 (at pH≈5) and ≈3 (at pH≈8), leads to the formation of a mixture of 2-aminomethyl-1-methylpyridinium ion and 1-methylpyridinium ion. At the third wave potentials the second wave products are further reduced to give basic species with catalytic activity.

Adriamycin-catalyzed aerobic photooxidation of NAD dimers to NAD+

The photooxidation of the dimers of nicotinamide adenine dinucleotide, (NAD)2, is catalyzed by adriamycin under aerobic conditions. (NAD)2 and O2 react in 1:1 molar ratio to yield 2 mol of NAD+. Experiments carried out by irradiating at 340 and 485 nm, corresponding to the absorption maxima of (NAD)2 and adriamycin, respectively, clearly indicate that the process is primed by photoexcitation of adriamycin. The key step of the process is the redox reaction between (NAD)2 and adriamycin with formation of the semiquinone radical anion, identified by the EPR spectrum. The semiquinone is then oxidized back to adriamycin by oxygen with formation of the superoxide radical.

Photocatalyzed Anaerobic Oxidation of Nicotinamide Coenzyme Dimers to NAD+ by Adriamycin

The nicotinamide adenine dinucleotide dimers (NAD)2 obtained by electrochemical reduction of NAD+ are oxidized by adriamycin in anaerobic photocatalyzed reaction yielding NAD+ and 7-deoxyadriamycinone. Under the same conditions NADH is not oxidized.

An electron spin resonance and voltammetric investigation of some 2-mono-and 2,4-di-substituted pyridinyl radicals

The chemical reduction of some 2-monosubstituted and 2,4-disubstituted pyridinium ions has been investigated by means of e.s.r. spectroscopy and the results compared with those of an electrochemical study on the same species. By combining the electrochemical and e.s.r. data, a scale of stability has been determined for the investigated pyridinyl radicals. N-Methyl-2-cyanopyridinyl, which was found to be too unstable to be observed, eventually evolved to the 2,4-dicyano-substituted radical. E.s.r. studies have also been carried out on the structurally similar, although more persistent, N-germylpyridinyls.