Barbara Czochralska

Oxidation of excited-state NADH and NAD dimer in aqueous medium involvement of O2− as a mediator in the presence of oxygen

Abstract It has been shown that direct excitation of NADH (or NADPH) in aqueous medium at 254 nm, or at wavelengths longer than 320 nm (where only the reduced nicotinamide moiety absorbs), leads to generation of NAD+ (or NADP+). The reaction proceeds both in the presence and absence of oxygen. Under aerobic conditions the reaction is accompanied by formation of H2O2 at a level equimolar with that of the NADH present in solution. On irradiation at wavelengths longer than 320 nm, conversion of NADH to enzymatically active NAD+ is about 75%. Under analogous irradiation conditions, the dimers (NAD)2 and (NADP)2 undergo disproportionation to NAD+ and NADP+, respectively, to the extent of 90%. Both physicochemical and enzymatic criteria were employed to formulate mechanisms for the photooxidation of NADH and the photodisproportionation of the dimer (NAD)2.

Electrochemical properties of 4-thiouracil derivatives

A study has been made of the polarographic behaviour of 4-thiouracil and its N-methylated derivatives, and of 4-thiouridine. Similar electrochemical properties are exhibited by all the compounds with a 2-keto-4-thione structure. In acid medium they give a typical catalytic hydrogen discharge wave, which disappears completely at pH values above 6. At pH values above 4.2 a second catalytic wave appears. Maximal catalytic activity is exhibited at pH 7.1, and decreases with increasing pH. Both catalytic waves possess pronounced surface characteristics, most likely due to adsorption of the molecules with differing orientations on the electrode surface. The second catalytic wave overlaps the reduction wave, which is placed in evidence under conditions where the catalytic effect is absent. The reduction wave is a 4e−/4H+ process involving reduction of the 4-thiouracil ring to 5,6-dihydropyrimidone-2. The same product is formed by a 2e−/2H+ reduction of 5,6-dihydro-4-thiouracil. The potential applications of the electrochemical properties of 4-thiouracil to studies on tRNA structure are discussed.

Electrochemical reduction of 4-thiomethyluracil derivatives

Summary The polarographic reduction of 4-thiomethyluracil, 1-methyl-4-thiomethyluracil and 4-thiomethyluridine was investigated by d.c. and a.c. polarography, cyclic voltammetry at HMDE, preparative electrolysis, and coulometry. The reduction mechanisms of the compouds studied were found to be similar, i.e. a two-step reduction in the pH range 1.0–6.0, and merging of the two steps into one at higher pH. In the 1st two-electron reduction step HSCH3 was eliminated. Subsequently, in the 2nd one-electron reduction step, a free radical was formed, which underwent either dimerization to 6,6′-bis-(3,6-dihydropyrimidone-2) or a further one-electron reduction to 3,6-dihydropyrimidone-2. The products obtained were identical to those formed by polarographic reduction of pyrimidone-2 and cytosine. The compounds investigated and their reduction products were strongly absorbed on mercury.

Photochemically reversible pyrimidine dimer product of electrochemical reduction of pyrimidone-2.

Abstract Pyrimidone-2 gives a single one-electron polarographic wave in aqueous buffered medium, and two one-electron waves of equal height in aqueous unbuffered (CH3)4NBr. The reduction product in neutral aqueous medium, identical with that corresponding to wave I in (CH3)4NBr, was isolated by macroelectrolysis, and shown to consist of a dimer of 3,6-dihydropyrimidone-2, the structure of which was identified as 6,6′-bis-(3,6-dihydropyrimidone-2). This dimer reduction product could be readily oxidized polarographically to regenerate the original pyrimidone-2. On irradiation in neutral aqueous medium at 254 nm, the dimer reduction product was quantitatively photo-dissociated to the parent pyrimidone-2 with a quantum yield of about 0.1. The reduction product corresponding to wave II in (CH3)4NBr was also isolated and shown to be 3,6-dihydropyrimidone-2. The mechanism for electroreduction of pyrimidone-2 to give these two products has been formulated. Both of these products are also formed during electroreduction of cytosine in aqueous medium at pH approx. 4.5. The dimer reduction product has been identified as a component of some photoproducts of nucleic acid constituents and, possibly, of nucleic acids and its photobiological significance is discussed.

Photochemical redox transformations of dimers of NAD+ and N′-methylnicotinamide

Abstract Electrochemically generated dimers of N′ -methylnicotinamide and NAD + both undergo photooxidation on irradiation at 254 nm in aqueous medium to yield the respective parent monomers. For the NAD dimer the quantum yield for photodissociation was about 0.01, whether irradiated at 254 nm or at wavelengths to the red of 320 nm. Irradiation at the latter wavelengths, where the NAD + monomer itself does not absorb and is not photosensitive, led to quantitative regeneration of coenzyme activity in the alcohol dehydrogenase system. The photodissociation reaction exhibited no oxygen effect. The photochemically generated dimer of N′ -methylnicotinamide was also photooxidized to the parent monomer by irradiation at 254 nm at pH 9.5. The foregoing process of electrochemical (or photochemical) reduction and photochemical oxidation, comprising a closed cycle of electron and proton transport, is similar to that previously observed for a number of pyrimidine analogs. Furthermore, the NAD dimer is a substrate of snake venom nucleotide pyrophosphatase and is hydrolyzed to release NMN dimer which, on irradiation at 254 nm, also undergoes photooxidation to the parent NMN + monomer. A mechanism for the photooxidation reaction is formulated and relevant biological implications of the foregoing are presented.

Photochemical and enzymatic redox transformations of reduced forms of coenzyme NADP

Abstract— The two reduced forms of NADP+, NADPH and its dimer (NADP), on irradiation in aqueous medium at 365 nm, are converted to enzymatically active NADP+, with accompanying formation of H2O2. The rate of photooxidation of NADPH is strongly dependent on the presence of oxygen, but that of (NADP)2 is similar under aerobic and anaerobic conditions. In the presence of oxygen, but not in its absence, O2 is an intermediate in the reaction. Generation of H2O2 under anaerobic conditions, confirmed by the fact that presence of peroxidase in irradiated solutions of (NADP)2 enhances photooxidation of the latter, is ascribed to attack on water of the excited dimer. Under anaerobic conditions at pH 9.5, Fe(EDTA)2+ and Fe(CN)4 increase the rate of photooxidation of NADP dimer two‐fold. γ ‐Irradiation of (NADP)2 at pH 9.5 in the presence of N2O results in 80% conversion to enzymatically active NADP + . A mechanism for photooxidation of (NADP)2 under anaerobic conditions is suggested, and some relevant biological implications are presented.

Red-ox transformations of NAD+ model compounds

Abstract The electrochemical reduction of four N1-substituted nicotinamides, and the properties of their electrochemically generated dimers, have been examined. Bearing in mind previously reported data for dimers of NAD + , NMN + , N1-methylnicotinamide and the parent nicotinamide, it is shown that the nature of the N1-substituent exerts a marked effect on the stability of the dimers and their conversion products. All dimers undergo photo-oxidation to the parent monomers with quantum yields of the order of 0.1. The rate of oxidation by molecular oxygen, and by an enzyme extract from mung beans, is much more rapid for dimers of N 1 -substituted nicotinamide then for dimers of NAD + and NMN + . The lability of the dimer of the phosphate ester of N 1 -hydroxyethylnicotinamide was particularly marked, due in this case to phosphate-catalysed, so-called acid hydration which, for this derivative, is so rapid that it occurs during electrolysis at pH 9.5. The phosphate group also influences the electrode process for reduction of the nicotinamide moiety on wave I, leading to a pH-dependence of U 1 2 . Esterification of the secondary phosphate hydroxyl liquidates both effects. The 1 H NMR spectrum of the NMN dimer indicates that it consists of several stereoisomers of the 4-4′ linked dimer. The foregoing data are discussed in relation to the choice of suitable simpler model analogues for NAD + .

Stereochemistry of the electroreduction of the CC1 bond

Abstract The electroreduction mechanism of α-halogen derivatives of organic compounds in proton donating solvents was investigated on the example of optically active 2-phenyl-2-chloropropionic acid. It was shown that the electroreduction occurs with inversion of configuration according to the SN 2 mechanism.

Electrooxidation of the antileukemic 2-chloro-2′-deoxyadenosine and related compounds

Abstract The electrochemical oxidation process of 2-halogenoderivatives: 2-chloro-2′-deoxy- adenosine (2CldAdo), 2-bromo-2′-deoxyadenosine (2BrdAdo) and 2′-deoxyadenosine (2′dAdo) has been studied in aqueous buffered solutions by DPV and CV using a glassy-carbon electrode. All compounds investigated exhibit one-step electrochemical oxidation in the pH range 2–9. Macroelectrolyses at the potential corresponding to DPV oxidation peaks have been performed in acetate buffer (pH 4.5) using a pyrolytic graphite electrode. While oxidation of 2BrdAdo occurred with elimination of Br 2 , no liberation of Cl atom was found for oxidation of 2CldAdo. The oxidation products of the compounds investigated have been examined by UV spectroscopy, electrochemical and HPLC analysis. The mechanism of electrochemical oxidation of halogenated deoxyadenosines is proposed.

Electrochemical Analysis of 3′-Azidothymidine (AZT)

Abstract 3′-Azido-3′-deoxythymidine (AZT) exhibits a two-electron diffusion—controlled polarographic reduction wave, with conversion to 3′−amino-3′-deoxythymidine. The mechanism of reduction, analytical and clinical applications, and its use for one-step synthesis of amino from azido nucleosides, are described.