Identification and reactivity of the triplet excited state of 5-hydroxytryptophan.

Both the neurotransmitter serotonin and the unnatural amino acid 5-hydroxytryptophan (5HT), contain the 5-hydroxyindole chromophore. The photochemistry of 5HT is being investigated in relation to the multiphoton excitation of this chromophore to produce a characteristic photoproduct with green fluorescence ('hyperluminescence'). Laser flash photolysis (308 nm) of 5HT in aqueous solution at neutral pH produces both the neutral 5-indoloxyl radical (lambda(max) 400-420 nm) and another transient absorption with lambda(max) 480 nm and lifetime of 2 micros in deaerated solutions. Based on quenching by oxygen and beta-carotene, the species at 480 nm is identified as the triplet excited state of 5HT. In acidic solution a new oxygen-insensitive intermediate with lambda(max) 460 is assigned to the radical cation of 5HT. Time-resolved measurements of luminescence at 1270 nm have shown that the triplet state of 5HT is able to react with oxygen to form singlet excited oxygen (1O2*) with a quantum yield of approximately 0.1. However, 5HT has also been found to be an effective quencher of singlet oxygen with a second order rate constant of 1.3 x 10(8) dm3 mol(-1) s(-1). The results are discussed in the light of recent observations on the multiphoton-excited photochemistry of serotonin.

Structure of the radical from one-electron oxidation of 4-hydroxycinnamate.

Radicals from one-electron oxidation of 4-hydroxycinnamate, ferulate and 3,4-dihydroxycinnamate have been formed by reaction with the oxidising triplet state of duroquinone. All three compounds react with triplet duroquinone with second order rate constants close to the diffusion-controlled limit. The identity of the resulting radicals is confirmed by observation of their characteristic visible absorption spectra. Time-resolved resonance Raman (TR3) spectra of the radical from 4-hydroxycinnamate were measured using a probe laser wavelength of 600 nm, to be in resonance with the long wavelength absorption band of the radical. The TR3 spectra contain prominent bands ascribed to the C-O and ring C-C stretching vibrations. The spectra are interpreted as indicating strong delocalisation of the radical site to the double bond in conjugation with the aromatic ring in 4-hydroxycinnamate. This contributes to the low reduction potential of the radical and the antioxidant properties of hydroxycinnamates.

Wavelength-programmed solute release from photosensitive liposomes.

Liposomes of dipalmitoylphosphatidylcholine containing a photochromic lipid "Bis-Azo PC" release entrapped solutes on exposure to UV light. We have now demonstrated that on addition of cholesterol (up to 25 mol%) to the liposomal membrane the liposomes also release their contents in response to visible light in the region of 470 nm, to which liposomes lacking steroid are insensitive. In a mixed population of liposomes prepared with and without cholesterol, this enables wavelength-dependent release of entrapped solutes on sequential exposure to visible and UV light. Furthermore, the cholesterol-containing liposomes allow stepped partial release of entrapped solute following multiple periods of short visible illumination. It is suggested that the cholesterol-containing liposomes may be potentially useful for drug delivery and for "caging" of reagents.

Active uptake of drugs into photosensitive liposomes and rapid release on UV photolysis.

Liposomes containing high concentrations of the anticancer drug doxorubicin, prepared by active-loading techniques, have been intensively investigated as potential agents for chemotherapy. The present study investigates the possibility of active uptake and photoinduced release of such solutes from liposomes incorporating a photoisomerizable lipid. The active loading of acridine orange and doxorubicin was investigated using liposomes containing entrapped ammonium sulfate. The liposomes were prepared with dipalmitoyl-L-alpha-phosphatidyl choline (DPPC) and a photochromic lipid, (1,2-(4'-n-butylphenyl)azo-4'-(gamma-phenylbutyroyl))-glycero-3- phosphocholine (Bis-Azo PC), which isomerizes on exposure to near-UV light with resulting changes in membrane permeability to solutes. The rate of loading of the vesicles below the phase transition temperature of DPPC was investigated as a function of Bis-Azo PC and cholesterol concentrations in the liposome. The rate of doxorubicin uptake was found to be greatly decreased in the presence of cholesterol, while below 30 degrees C the rate of acridine orange uptake was increased in the presence of cholesterol. On exposure to a single UV laser pulse, actively loaded acridine orange was rapidly released from liposomes containing Bis-Azo PC at a rate similar to that found for the indicator dye calcein. However while cholesterol had previously been shown to greatly enhance the rate of photo-induced calcein leakage, it had no significant effect on the rate of acridine orange release. After active loading into DPPC vesicles containing Bis-Azo PC, doxorubicin was also released after exposure to a single laser pulse, but at a rate slower than for acridine orange and calcein. The difference in behavior between these systems is ascribed to the interactions of acridine orange and doxorubicin with the liposome bilayer. Photoinduced release of pharmacologically active materials from sensitized liposomes might provide a useful adjunct or alternative to conventional photodynamic therapy.

Photosensitive liposomes as 'cages' for laser-triggered solute delivery: the effect of bilayer cholesterol on kinetics of solute release.

Liposomes containing acyl chains incorporating azobenzene chromophores have been investigated as potential 'caging' agents for fast solute release. On photolysis, trapped marker dye can be released from gel-phase liposomes within milliseconds. Solute release is markedly sensitive to the presence of cholesterol in the bilayer. Phospholipids bearing one saturated acyl chain and an azobenzene-substituted chain are ineffective as sensitisers unless cholesterol is present, while doubly substituted phospholipids sensitise release in its absence. Cholesterol markedly affects the temperature profile of solute release depending on the host phospholipid chain length. Solute release is not seen for lipid hosts with unsaturated acyl chains.

Fast laser-induced solute release from liposomes sensitized with photochromic lipid: effects of temperature, lipid host, and sensitizer concentration.

Liposomes of gel-phase phospholipid have been prepared containing a photochromic lipid sensitizer. A fast UV laser pulse isomerizes the sensitizer destabilizing the lipid bilayer structure and causing release of trapped solute. The kinetics of solute release have been investigated as a function of host lipid chain length, sensitizer concentration, and temperature, and the limits of liposome stability have been established. At low concentrations of sensitizer, pulsed laser irradiation induces some solute release when continuous UV illumination is ineffective. Although rates of solute release usually increase with temperature, at low sensitizer concentration in a rigid host, leakage at first increases but then decreases rapidly above a threshold temperature. The results presented are relevant to the design of photostimulated drug delivery systems and to potential applications of photosensitive liposomes as caging agents for biological effectors.

Antioxidant reactions of dihydrolipoic acid and lipoamide with triplet duroquinone.

The oxidation of the antioxidants dihydrolipoate and lipoamide by triplet duroquinone (3DQ) has been studied by laser flash photolysis and time-resolved resonance Raman (TR3) spectroscopy. Reaction of 3DQ with lipoamide by electron transfer [k(H2O)/k(D2O approximately 1] was more rapid than with dihydrolipoate, in which a proton is also involved [k(H2O)/k(D2O approximately 2]. For dihydrolipoate at neutral pH the undeprotonated form was the major reactive species with k approximately 10(9) dm3 mol-1 s-1. At higher pH values the reaction of ionised (thiolate) forms was observed with k > or = 4 x 10(9) dm3 mol-1 s-1. The electron transfer mechanism of reaction between 3DQ and lipoamide was confirmed by TR3 spectra in which formation of the durosemiquinone radical anion and lipoamide disulfide radical cation (RSS+.) was observed.

The free radical site in pea seedling copper amine oxidase probed by resonance Raman spectroscopy and generated by photolysis of caged substrate.

Resonance Raman spectra were obtained of the free radical site in substrate reduced anaerobic samples of pea seedling copper amine oxidase (PSAO). The spectra differ significantly from those reported previously for E. coli copper amine oxidase [Moenne-Loccoz et al. (1995) Biochemistry 34, 7020]. The spectra were found to be independent of substrate (benzylamine, spermidine or methylamine) used to reduce the TOPA quinone cofactor, however, several of the peaks in the Raman spectrum displayed small shifts on using [15N]benzylamine, proving incorporation of the substrate nitrogen atom onto the cofactor radical. Changes in the spectrum were also observed when measured in D2O solution indicating a strongly bound proton in the radical. The spectra were independent of pH values between 5 and 9 and are interpreted as showing that the radical exists as a semiiminoquinone radical monoanion. Benzylamine and phenethylamine have been caged with 2-nitrobenzaldehyde and shown by laser flash photolysis to uncage on a sub-millisecond timescale. Preliminary experiments have shown the formation of the enzyme radical intermediate on laser flash photolysis of 2-nitrobenzyl-caged benzylamine in the presence of enzyme. This should permit time-resolved resonance Raman spectral investigations of the catalytic cycle of copper amine oxidases.

Quenching of reactive oxidative species by probucol and comparison with other antioxidants.

One-electron oxidation of the antiatherogenic and antiatherosclerotic drug probucol has been studied in relation to its activity as an antioxidant. Oxidation by triplet excited states of duroquinone and benzophenone, and by the inorganic radicals Br2.- and N3., lead to the formation of a transient absorption at 500 nm. This was identified by time-resolved resonance Raman spectroscopy as the phenoxyl radical from probucol, formed by hydrogen atom or electron plus proton loss from one of the phenolic groups of probucol. The reactivity of probucol with triplet duroquinone and triplet benzophenone, and as a quencher of singlet oxygen, was compared with the reactivities of other antioxidants (alpha-tocopherol, palmitoyl ascorbic acid, dihydrolipoic acid and N-acetyl cysteine). In quenching of the triplet states the reactivity of probucol was comparable with that of alpha-tocopherol, whereas as a quencher of singlet oxygen probucol (k < 10(6) M-1 s-1) was less effective than alpha-tocopherol (k = 2.0 x 10(8) M-1 s-1) by more than two orders of magnitude. This difference in reactivity may allow the contribution of singlet oxygen towards oxidative stress to be quantified separately.

Fast solute release from photosensitive liposomes: an alternative to 'caged' reagents for use in biological systems.

The kinetics of release of soluble marker trapped in liposomes of gel phase phospholipid containing a photoisomerisable phospholipid analogue have been investigated. Marker release is triggered by UV laser flash photolysis at 355 nm. A markedly temperature-dependent release rate is seen, and above 25 degrees C millisecond release kinetics can be achieved. These results suggest that such liposomes might find application as an alternative to conventional 'caged' reagents for photo-triggered reagent release in biological research.