Albert Van de Vorst

ON THE PRODUCTION OF NITROXIDE RADICALS BY SINGLET OXYGEN REACTION: AN EPR STUDY

Abstract— The production of free radicals by reaction of 2,2,6,6‐tetramethyl‐4‐piperidinol with singlet oxygen was studied by EPR spectroscopy. The rate constant of the amine was found to be equal to 8 ×105M‐1s‐1 in ethanol and to 4 × 107M‐1s‐1 in phosphate buffer (pH 8). Competition experiments were performed with singlet oxygen quenchers such as NaN3, DABCO and the quenching rate constants were found to be consistent with the literature values. The EPR method proved to be a valuable technique to study the reaction of singlet O2 with the sterically hindered amine without any interfering effect.

TYPE I AND TYPE II MECHANISMS IN THE PHOTOOXIDATION OF L-TRYPTOPHAN AND TRYPTAMINE SENSITIZED BY HEMATOPORPHYRIN IN THE PRESENCE AND ABSENCE OF SODIUM DODECYL SULPHATE MICELLES

Abstract— Oxygenated aqueous solutions (pH 10) of L‐tryptophan or tryptamine containing hematopor‐phyrin and/or specific quenchers of the possible reactive intermediates were irradiated with visible light in the presence and in the absence of dispersions of sodium dodecyl sulphate micelles. The rate of loss of the indole derivatives was followed over several min. In the absence of dispersed micelles, indole derivatives appeared to be photooxidized largely by a type I mechanism involving electron transfer from triplet hematoporphyrin to the indole moiety. A type II (lO2‐involving) mechanism was of minor importance. The presence of sodium dodecyl sulphate micelles, where the porphyrin was solubilized in a monomeric state, exerted only a slight influence on the competition between the two reaction pathways described above in the case of tryptophan, which was essentially all present in the aqueous phase. On the other hand, tryptamine, when dissolved within micelles, underwent photooxidative attack exclusively by 1O2 which was generated within a micelle, diffused through the aqueous phase and penetrated into another (tryptamine‐containing) micelle; the reduced efficiency of the latter photoprocess was probably a consequence of the low probability of 1O2 penetration into charged micelles.

COMPETITION BETWEEN THE SINGLET OXYGEN AND ELECTRON TRANSFER MECHANISMS IN THE PORPHYRIN‐SENSITIZED PHOTOOXIDATION OF l‐TRYPTOPHAN AND TRYPTAMINE IN AQUEOUS MICELLAR DISPERSIONS

Abstract— Kinetic studies of the hematoporphyrin–sensitized photooxidation of l‐tryptophan and tryptamine at pH 10 in either homogeneous aqueous solutions or in aqueous dispersions of Triton X–100 and cetyltrimethylammonium bromide micelles indicate that the indole substrates are attacked via a mixed type I (electron transfer from triplet dye)/type II (1O2‐involving) mechanism. Both reactive intermediates, generated by micelle‐solubilized hematoporphyrin, can diffuse to attack substrate molecules located in either the bulk aqueous phase or a different micelle. In particular, incorporation of the substrate into a micelle has only minor effects on its reactivity toward1O2, although the 1O2—indole interaction appears to be more efficient in cationic micelles owing to a favourable orientation of the target with respect to the attacking species. On the other hand, the electron transfer from triplet porphyrin to a micellized substrate is virtually non‐operative when the latter is located in an anionic micelle, whereas in neutral or cationic micelles, the efficiency of the process is again controlled by the substrate orientation. Studies of tryptamine photooxidation sensitized by meso‐tetra‐(4‐sulfonato‐phenyl) porphine in the presence of sodium dodecylsulphate micelles lend further support to the abovementioned conclusions.

Photosensitization by hematoporphyrin: ESR evidence for free radical induction in unsaturated fatty acids and for singlet oxygen production

Abstract Hematoporphyrin ability to photoreact by type I and type II mechanisms was investigated in some model systems. At room temperature, visible irradiation of hematoporphyrin-unsaturated fatty acids and hematoporphyrin-cholesterol systems resulted in the Electron Spin Resonance (ESR) spectrum of the hematoporphyrin free radical. Triplet state hematoporphyrin is shown to be involved in the electron transfer from the lipid moiety. Moreover an ESR method to monitor the singlet oxygen production by hematoporphyrin was used. β-carotene effect on both mechanisms (type I and type II) was tested.

EPR STUDIES ON SINGLET OXYGEN PRODUCTION BY PORPHYRINS

Abstract— EPR studies of the porphyrin‐sensitized photooxidation of 2, 2, 6,6‐tetramethyl‐piperidine to the nitroxide demonstrate that all the porphyrins examined are able to generate 1O2, although the efficiency of the photoprocess is dependent on the nature of the side chains. Incorporation of metal ions into the porphyrin molecule depresses or even inhibits the formation of 1O2. Comparison of these results with previously obtained kinetic data points out that the efficiency of porphyrins as photosensitizes is controlled by the lifetime of their lowest triplet state.

Induction of breaks in deoxyribonucleic acid by photoexcited promazine derivatives.

Near-u.v. photoexcited promazine and three of its derivatives are shown to induce single-strand breaks in phi X174-DNA replicative form. The mechanisms of this DNA breakage depend upon the various photochemical properties of the promazine derivatives. Chlorpromazine is shown to act predominantly via the photodechlorination reaction both in aerobic and anaerobic conditions. The three other promazine derivatives (promazine, trifluopromazine and methoxypromazine) display two mechanisms for DNA breakage. One of them occurs through the cation radical, which is formed during near-u.v. irradiation of promazine derivatives. The second mechanism is demonstrated to act via an hydroxyl radical-dependent pathway. Acepromazine is without photoactivated action. EPR-spin-trapping studies of irradiated mixtures, containing the drugs and 5,5-dimethyl-1-pyrroline-N-oxide (as spin trap), suggest the production of superoxide radical by photoexcited promazines. When DNA is present in the irradiation mixture, this superoxide radical is converted into hydroxyl radical probably via a Haber Weiss-type reaction, catalysed by DNA-iron complexes.

ESR and optical absorption evidence for free radical involvement in the photosensitizing action of furocoumarin derivatives and for their singlet oxygen production

Frozen aqueous solutions of thymine and its derivatives were irradiated with visible light (lambda greater than 320 nm) in the presence of various furocoumarins. ESR analysis revealed the induction of hydrogen adduct free radicals at C-6 position of thymine, only with those furocoumarin derivatives which show a skin-photosensitizing ability. It has been shown, moreover, that the photocycloaddition of psoralen to thymine, which is responsible for the biological effects of this dye, is inhibited when the induction of free radicals in thymine moiety has been prevented by electron scavengers. It is suggested that the free radicals observed could be involved in the biological photosensitization. The mechanism of free radical generation and singlet oxygen production by furoccoumarins were also investigated.

PRODUCTION OF BREAKS IN SINGLE- AND DOUBLE-STRANDED FORMS OF BACTERIOPHAGE øx174 DNA BY PROFLAVINE AND LIGHT TREATMENT

Abstract— Irradiation at 440 + 360 nm and a fluence rate of 3.8 kJm‐2 min‐1, of both complexes previously formed between proflavine and either øX circular single‐stranded (ss) DNA or øX supercoiled duplex (RFI)DNA, induces single‐strand scissions in the two DNAs under consideration. Linear øXSS DNA molecules are detected by sedimentation through alkaline sucrose gradients. After treatment of the øXRFI DNA, however, the degree of degradation is the same whether it is measured under neutral or alkaline conditions, indicating that alkaline‐labile bonds are not created; moreover, double‐strand breaks can only be detected after accumulation of single‐strand breaks.

Biomolecular photoalterations mediated by phenothiazine derivatives.

This survey focuses on recent developments in the field of the ultraviolet photochemistry and photobiology of phenothiazine derivatives. One of the major alterations introduced by this kind of photosensitized reaction is a covalent addition of the photosensitizer or one of its photoproducts onto the macromolecular target. This reaction has been observed with soluble and membrane proteins, lipids and DNA. In the latter case, the addition occurs at the level of guanine residues and leads to inhibition of DNA replication.

PHOTOSENSITIZATION OF AMINO ACIDS BY DI-CYAN-HEMIN: KINETIC AND EPR STUDIES

Abstract— The low spin hemin derivative di‐cyan‐hemin can sensitize the photooxidation of amino acids. The efficiency of this dye as a photosensitizer is generally lower than that of other porphyrins such as hematoporphyrin in aqueous solution, but it is significantly enhanced by addition of ethanol. On the basis of the identified photooxidation end‐products of some amino acids, photooxidation kinetic data and EPR measurements, it appears that methionine is quantitatively converted to methionine sulfoxide by a pure Type II (singlet oxygen) mechanism, whereas in the case of cysteine and tryptophan a mixed Type ILType II mechanism is probably operative.