Structure-activity relationships for DNA photocleavage by cationic porphyrins.

The influence of molecular structure and DNA binding mode on the ability of cationic porphyrins to photosensitize DNA strand break formation has been studied for a series of meso-substituted pyridinium porphyrins using electrophoretic and DNA sequencing techniques. Porphyrins substituted with pyridyl groups in which the heterocyclic nitrogen is in the para or meta position vis-à-vis the substitution point are capable of intercalative binding and are considerably more efficient DNA photosensitizers than the corresponding non-intercalating ortho compounds. Within each group of porphyrins the photosensitizer efficiency increases with the number of positive charges. Using DNA sequencing experiments, we have demonstrated that photomodification occurs primarily at the guanine and thymine bases, and that alkali-labile sites produced by photo-oxidation are as important as direct cleavage events. The kinetics of strand degradation in aerated and degassed solution suggest that type II reactions (probably mediated by singlet oxygen) occur with significantly higher yield than type I reactions and are responsible for the formation of alkali-labile sites in aerated systems. These observations seem to confirm the hypothesis that those structural features which influence the strength and mode of binding also serve to establish favourable porphyrin-DNA interactions for photosensitization.

Escherichia coli and herpes-simplex-virus ribonucleotide reductase R2 subunit. Compared reactivities of the redox centres.

Protein R2, the small subunit of ribonucleotide reductase, contains a diferric centre and a tyrosyl radical absolutely required for enzyme activity. The reduction of the tyrosyl radical and the mobilization of the iron centre result in the inhibition of the enzyme and thus of DNA synthesis. The chemical reactivity of the iron-radical centre of Escherichia coli and herpes simplex virus has been studied by u.v.-visible and e.p.r. spectroscopies. The tyrosyl radical is efficiently scavenged by hydroxamic acids and phenols during reactions controlled by steric hindrance and hydrophobic interactions. The reaction with o-disubstituted phenols yields the corresponding diphenoquinones. The reactivity of the bacterial radical greatly contrasts with that of the viral radical, and the iron centre in herpes-simplex-virus R2 is much more labile than that in E. coli R2, as shown from the facile mobilization of iron by chelators such as catechol. These results suggest that the active sites of the two enzymes are significantly different and might be useful for designing new antiviral agents.

Mechanisms of inactivation of lipoxygenases by phenidone and BW755C.

Inhibition of soybean lipoxygenase (L-1) and potato 5-lipoxygenase (5-PLO) by the pyrazoline derivatives phenidone and BW755C only occurs after oxidation of these compounds by the peroxidase-like activity of the lipoxygenases. There is a clear relationship between this oxidation and the irreversible inactivation of L-1. The final product of phenidone oxidation by L-1, 4,5-didehydrophenidone, is not responsible of this inactivation, but the species derived from a one-electron oxidation of phenidone plays a key role in L-1 inactivation. In the absence of O2, inactivation of 1 mol of L-1 occurs after the oxidation of 34 mol of phenidone and the covalent binding of 0.8 mol of phenidone-derived metabolite(s) to L-1. In the presence of O2, inactivation of 1 mol of L-1 occurs already after oxidation of 11 mol of phenidone and only involves the covalent binding of 0.4 mol of phenidone-derived metabolite(s) to L-1. A mechanism is proposed for L-1 inactivation by phenidone, which involves the irreversible binding of a phenidone metabolite to the protein and the oxidation of an L-1 amino acid residue (in the presence of O2).

Vitamin E derivatives as new potent inhibitors of microsomal lipid peroxidation.

Several synthetic Vitamin E derivatives are strong inhibitors of lipid peroxidation induced in rat liver microsomes either chemically by ferrous ions and ascorbate or enzymatically by NADPH and carbon tetrachloride. The relative activities of these inhibitors are consistent with their intrinsic antioxidant properties, as peroxyl radicals scavengers. Among them, a 3,4-dihydro-6-hydroxy-2H-1-naphtopyran with IC50 around 0.08 microM is one of the most potent yet known inhibitor of lipid peroxidation.

Peroxidase-like activity of lipoxygenases: different substrate specificity of potato 5-lipoxygenase and soybean 15-lipoxygenase and particular affinity of vitamin E derivatives for the 5-lipoxygenase.

Potato 5-lipoxygenase (5-PLO) catalyzes the reduction of 13(S)-hydroperoxy-9Z,11E-octadecadienoic acid (13-HPOD) in the presence of vitamin E. I mol of vitamin E is required to consume 2 mol of 13-HPOD. The mechanism of the 5-PLO-catalyzed oxidation of vitamin E by 13-HPOD is similar to that previously established for the soybean 15-lipoxygenase (L-1)-catalyzed oxidation of phenidone by 13-HPOD, and seems to involve a one-electron reduction of the O-O bond of 13-HPOD. 5-PLO and L-1 exhibit very different substrate specificities and pH profiles for their peroxidase-like activity. Actually, among the 20 compounds containing various reducible functions and the 10 derivatives of vitamin E which have been studied, only four products containing hydrophobic long chains, ascorbic acid 6-palmitate, the trolox esters of octanol and undecanol, and vitamin E exhibit high peroxidase-like activities for 5-PLO. On the contrary, much more compounds, even not very hydrophobic, are good substrates for the peroxidase-like activity of L-1.

Rapid routes of synthesis of chemically reactive and highly radioactively labeled alpha- and beta-oligonucleotide derivatives for in vivo studies.

Development of the antisense oligonucleotide strategy for the regulation of gene expression in vivo poses several problems: the stability of oligonucleotides toward intracellular nucleases, labeling of oligonucleotides with high specific radioactivity, improvements of penetration of oligonucleotides into living cells, and enhancement of antisense action by coupling of chemically active groups. In the present paper synthesis of highly radioactively labeled [32P]- and [35S]oligonucleotide derivatives is described starting from both natural (beta) and nuclease-resistant (alpha) anomers of oligonucleotides. Conditions for preparative phosphorylation and thiophosphorylation suitable for oligonucleotides of various lengths, base composition, and anomeric forms were established. The stability of the phosphoramide bond under in vivo experimental conditions was checked. The methods of terminal phosphate chemical activation and terminal thiophosphate alkylation were applied to synthesize oligonucleotides equipped with hydrophobic, intercalating, alkylating, and photoactivatable groups. In the case of porphyrin-oligonucleotide conjugates, a series of new monofunctional porphyrin derivatives bearing a free aliphatic amino group was developed.

Interaction of cationic porphyrins with DNA: importance of the number and position of the charges and minimum structural requirements for intercalation.

Thirty-three porphyrins or metalloporphyrins corresponding to the general formula [meso-[N-methyl-4(or 3 or 2)-pyridiniumyl]n(aryl)4-nporphyrin]M (M = H2, CuII, or ClFeIII), with n = 2-4, have been synthesized and characterized by UV-visible and 1H NMR spectroscopy and mass spectrometry. These porphyrins differ not only in the number (2-4) and position of their cationic charges but also in the steric requirements to reach even temporarily a completely planar geometry. In particular, they contain 0, 1, 2, 3, or 4 meso-aryl substituents not able to rotate. Interaction of these porphyrins or metalloporphyrins with calf thymus DNA has been studied and their apparent affinity binding constants have been determined by use of a competition method with ethidium bromide which was applicable not only for all the free base porphyrins but also for their copper(II) or iron(III) complexes. Whatever their mode of binding may be, their apparent affinity binding constants were relatively high (Kapp between 1.2 x 10(7) and 5 x 10(4) M-1 under our conditions), and a linear decrease of log Kapp with the number of porphyrin charges was observed. Studies of porphyrin-DNA interactions by UV and fluorescence spectroscopy, viscosimetry, and fluorescence energy transfer experiments showed that not only the tetracationic meso-tetrakis[N-methyl-4(or 3)-pyridiniumyl]porphyrins, which both involved four freely rotating meso-aryl groups, but also the corresponding tri- and dicationic porphyrins were able to intercalate into calf thymus DNA. Moreover, the cis dicationic meso-bis(N-methyl-2-pyridiniumyl)diphenylporphyrin, which involved only two freely rotating meso-aryl groups in a cis position, was also able to intercalate. The other meso-(N-methyl-2-pyridiniumyl)n(phenyl)4-nporphyrins, which involved either zero, one, or two trans freely rotating meso-aryl groups, could not intercalate into DNA. These results show that only half of the porphyrin ring is necessary for intercalation to occur.

Soybean lipoxygenase-catalyzed oxidations by linoleic acid hydroperoxide: different reducing substrates and dehydrogenation of phenidone and BW 755C.

Phenidone is not a substrate for dioxygenation by soybean lipoxygenase-1 (L1) but reduces L1Fe(III) into L1Fe(II), as shown by EPR spectroscopy. L1 catalyzes the oxidation of phenidone by 13-HPOD, the hydroperoxide formed by dioxygenation of linoleic acid by L1, with formation of 4,5-dehydrophenidone. Two moles of 13-HPOD are used per mole of phenidone dehydrogenated. Other pyrazoline derivatives such as BW 755C, but also, in a more general manner, different compounds containing phenol, aniline, hydrazine, hydroxylamine or hydrazide functions act as reducing substrates for decomposition of 13-HPOD by L1.

Mode of interaction and apparent binding constants of meso-tetraaryl porphyrins bearing between one and four positive charges with DNA.

Meso-substituted porphyrins, ((4-N-methyl-pyridyl)n(Ph)4-n)PH2, n = 1 to 4, bearing between 1 and 4 positive charges have been synthetized and studied for their interaction with Calf Thymus DNA. Competition binding experiments using ethidium bromide or one of its dimers show that these porphyrins and some of their Cu(II) or Fe(III)Cl complexes have apparent binding constants between 3 10(5) and 5 10(7) M-1. Fluorescence energy transfer experiments show that not only the tetracationic previously described porphyrin but also the tri- and dicationic porphyrins are able to intercalate into DNA. These data indicate a greater importance of the polyaromatic porphyrin ring than of the number or position of the positive charges for meso-tetra-arylporphyrin interaction with DNA.