Physical chemistry of porphyrins and their interactions with membranes: the importance of pH

The physicochemical properties of dicarboxylic porphyrins are examined with particular emphasis on acid-base equilibria. Interactions with membranes appear to be dominated by the charge of the propionic acid chains and the hydrophobic character of other peripheral substituents. A physicochemical basis for the effect of pH on the incorporation of porphyrins within membranes is given. The effect of lowered tumour pH on the incorporation of porphyrins within membranes and on porphyrin retention is discussed.     

Inactivation of erythrocytic, lymphocytic and myelocytic leukemic cells by photoexcitation of endogenous porphyrins

The photodynamic sensitization of leukemic cells (erythrocytic, myelocytic and lymphocytic) via light activation of endogenous porphyrins is described. Human myelocytic-erythrocytic K562 cells and murine Friend erythroleukemia (FELC) and T-cell lymphoma Eb-Esb cells were stimulated to synthesize and accumulate porphyrins. K562 cells accumulated high amounts of protoporphyrin by stimulation with 5-aminolevulinic acid (ALA) plus sodium butyrate or hemin. For Friend and Eb-Ebs cells ALA was an adequate stimulator. The high-metastatic Esb lymphoma cells accumulated comparatively more porphyrin than the low-metastatic Eb cell line. Maximal porphyrin accumulation produced mortality rates of more than 99% after 10 min of photoactivation of the three leukemic lines. Thymidine incorporation was inhibited by the photodynamic effect depending on porphyrin concentration. These results confirm the photodynamic ability of endogenous porphyrins to inactivate cancer cells of different origins.     

DNA as supramolecular scaffold for porphyrin arrays on the nanometer scale

Tetraphenyl porphyrin substituted deoxyuridine was used as a building block to create discrete multiporphyrin arrays via site specific incorporation into DNA. The successful covalent attachment of up to 11 tetraphenyl porphyrins in a row onto DNA shows that there is virtually no limitation in the amount of substituents, and the porphyrin arrays thus obtained reach the nanometer scale (approximately 10 nm). The porphyrin substituents are located in the major groove of the dsDNA and destabilize the duplex by deltaT(m) 5-7 degrees C per porphyrin modification. Force-field structure minimization shows that the porphyrins are either in-line with the groove in isolated modifications or aligned parallel to the nucleobases in adjacent modifications. The CD signals of the porphyrins are dominated by a negative peak arising from the intrinsic properties of the building block. In the single strands, the porphyrins induce stabilization of a secondary helical structure which is confined to the porphyrin modified part. This arrangement can be reproduced by force-field minimization and reveals an elongated helical arrangement compared to the double helix of the porphyrin-DNA. This secondary structure is disrupted above approximately 55 degrees C (T(p)) which is shown by various melting experiments. Both absorption and emission spectroscopy disclose electronic interactions between the porphyrin units upon stacking along the outer rim of the DNA leading to a broadening of the absorbance and a quenching of the emission. The single-stranded and double-stranded form show different spectroscopic properties due to the different arrangement of the porphyrins. Above T(p) the electronic properties (absorption and emission) of the porphyrins change compared to room temperature measurements due to the disruption of the porphyrin stacking at high temperature. The covalent attachment of porphyrins to DNA is therefore a suitable way of creating helical stacks of porphyrins on the nanometer scale.     

Porphyrin effects on zwitterionic HPS micelles as investigated by small-angle X-ray scattering (SAXS) and electron paramagnetic resonance (EPR)

In this work, small-angle X-ray scattering (SAXS) and electron paramagnetic resonance (EPR) studies on the interaction of three anionic mesotetrakis (4-sulfonatophenyl) porphyrins, TPPS4, FeTPPS4, and ZnTPPS4, at concentrations in the 2-10 mM range, with micelles of the zwitterionic surfactant 3-(N-hexadecyl-N,N-dimethylammonium) propane sulfonate (HPS, 30 mM) at pH 4.0 and 9.0 are reported. The SAXS results demonstrate that, upon addition of all species of porphyrins, the HPS micelle of prolate shape reduces its axial ratio from 1.8 +/- 0.2 (in the absence of porphyrin) to 1.5 +/- 0.1. Such an effect is accompanied by a shrinking of the paraffinic shortest semiaxis from 22.5 +/- 0.5 A to 18.0 +/- 0.2 A. This shows that the micellar hydrophobic core is affected by porphyrin incorporation, independent of the type of porphyrin and pH. Concurrently, EPR results demonstrate an increase in the micellar packing as noticed from the increase in motional restriction for both nitroxides. Furthermore, increase of the porphyrin concentration induces the appearance of a repulsive interference function over the SAXS curve of zwitterionic micelles, which is typical of an interaction between surface-charged micelles. Such a finding gives strong evidence that the negatively charged porphyrin molecule must accommodate in the HPS micelle dipole layer close to the inner positive charges (near the hydrophobic core), inducing a surface charge (probably a negative one associated with the HPS sulfonate external groups) in the original zwitterionic (overall neutral) micelle. Such a porphyrin location is favored by both electrostatic and hydrophobic contributions, giving rise to binding constant values that are quite large compared to the binding of cationic drugs to HPS micelles (Caetano, W.; Barbosa, L. R. S.; Itri, R.; Tabak, M. J. Coll. Int. Sci. 2003, 260, 414).     

CLEARANCE TIMES OF PORPHYRIN DERIVATIVES FROM MICE AS MEASURED BY in vivo FLUORESCENCE SPECTROSCOPY

The clearance times of 17 different porphyrin derivatives from SKH:HR-1 mice have been measured using the technique of in vivo fluorescence spectroscopy. This technique monitors the in vivo porphyrin fluorescence observed from the external skin surface. Most hydrophilic porphyrin derivatives show relatively short clearance times, in the order of 2.5-6 h. The dicarboxylic acid porphyrins, proto-, hydroxyethylvinyldeutero- and hematoporphyrin IX have clearance times of 7.8, 12.2 and 14.7 h respectively. The mixture hematoporphyrin derivative has an intermediate clearance time of 12.6 h. N-methylated porphyrins show clearance times in the vicinity of 15-22 h. Monoaspartyl chlorin e6 shows the longest clearance time of all porphyrin derivatives measured (30.3 h).     

CLEARANCE TIMES OF PORPHYRIN DERIVATIVES FROM MICE AS MEASURED BY in vivo FLUORESCENCE SPECTROSCOPY

Abstract
The clearance times of 17 different porphyrin derivatives from SKH:HR-1 mice have been measured using the technique of in vivo fluorescence spectroscopy. This technique monitors the in vivo porphyrin fluorescence observed from the external skin surface. Most hydrophilic porphyrin derivatives show relatively short clearance times, in the order of 2.5–6 h. The dicarboxylic acid porphyrins, proto-, hydroxyethylvinyldeutero-and hematoporphyrin IX have clearance times of 7.8, 12.2 and 14.7 h respectively. The mixture hematoporphyrin derivative has an intermediate clearance time of 12.6 h. N -methylated porphyrins show clearance times in the vicinity of 15–22 h. Monoaspartyl chlorin e₆ shows the longest clearance time of all porphyrin derivatives measured (30.3 h).     

Sequential energy and electron transfer in aggregates of tetrakis[oligo(p-phenylene vinylene)] porphyrins and C60 in water

Chiral aggregation of oligo(p-phenylene vinylene)-functionalized Zn and free-base porphyrins is observed in water. The formation of mixed assemblies containing both porphyrins results in sequential energy transfer from OPV via zinc porphyrin to free-base porphyrin. Furthermore, the incorporation of C60 as electron acceptor yields a charge separated state by ultimate electron transfer.     

UV-visible and fluorescence spectral study on a pH controlled transfer process of an amphiphilic porphyrin in nonionic micelle

5,15-di(4-hydroxyphenyl)-10,20-di(hexadecyloxyphenyl) porphyrin P was solubilized in nonionic polyoxyethylene(9.5) octylphenol (Triton X-100 or TX-100) micelle solutions. By means of analyzing the UV-visible and fluorescence spectra of the synthesized amphiphilic porphyrin P in different solvent environments, and the relationship between the solubilizing location of the porphyrins in TX-100 micelle and the microenvironment polarity, P is shown to involve in a transfer process for the porphyrin moiety from inner to the outer surface of TX-100 micelle as the pH is increased. The kinetic study of porphyrin incorporate with Cu(II) shows that metalation rate of porphyrin increases with the pH increasing, indicating that metalation rate could be controlled by changing pH.     

Regioselective reactivity of an asymmetric tetravalent di[dihydroxotin(IV)] bis-porphyrin host driven by hydrogen-bond templation

Local molecular environment effects on the rates of ligand exchange at an asymmetric di[dihydroxotin(IV)] bis-porphyrin 5 are examined. The host 5 possesses four non-equivalent tin(IV)-ligand binding sites that are distinguished by their position relative to a shallow cavity, by the steric environment at each binding site and by electronic-structure differences between the constituent porphyrin and quinoxalinoporphyrin macrocycles. These design features of the asymmetric host are confirmed by X-ray crystal structure analysis. Binding experiments with monodentate carboxylic acids and bidentate dicarboxylic acids show significant differences in the rate of ligand exchange at each of the four tin(IV) binding sites. For monodentate carboxylic acids, binding preferentially occurs at the exterior porphyrin site. Further addition of carboxylic acid results in sequential binding at the quinoxalinoporphyrin sites and lastly at the interior site on the porphyrin, with high regioselectivity. These selective binding outcomes are immediately apparent by NMR spectroscopy. A series of 2D NMR spectroscopy experiments allowed identification of the preferred binding sites at the host. This positively identifies that steric hindrance and electron-withdrawing functionality on the porphyrin macrocycle impede ligand exchange. However, these effects are overcome by dicarboxylic acid guests, which form ditopic hydrogen-bond interactions between the intracavity hydroxo ligands in the initial stage of ligand exchange, leading to regioselective binding between the tin(IV) sites within the cavity. It is envisaged that the factors identified herein that define regioselective ligand exchange at host 5 will find wider application in supramolecular systems incorporating tin(IV) porphyrins.     

A novel pH-controlled transfer process of 5,10,15-tri(4-hydroxyphenyl)-20-(4-hexadecyloxyphenyl) porphyrin in CTAB micelles

By analysis of the UV-visible and fluorescence spectra of 5,10,15-tri(4-hydroxyphenyl)-20-(4-hexadecyloxyphenyl)porphyrin (P) in different microenvironments of micelle and solvent solutions, a novel pH-controlled transfer process of P in CTAB micelle was reported. In neutral CTAB micelles, porphyrins may locate at the inner layers of micelles. With pH increases to 11.19, the porphyrin can be completely deprotonated and transfers to the outer surface of CTAB micelle. The investigation of kinetics of porphyrin complexing with Cu(II) indicates that the metallation rate of porphyrins in CTAB micelles could also be controlled by changing pH.     

PORPHYRIN-LIPID INTERACTIONS AT THE AIR-WATER INTERFACE IN SPREAD MONOLAYERS. A FLUORESCENCE STUDY.

In membrane systems, carboxylic porphyrins may interact with both the lipid pseudophase and the adjacent aqueous environment through their hydrophobic core and their polar acid chains, respectively. These interactions are monitored in model membrane systems, i.e. spread monolayers of dioleoylphosphatidylcholine as functions of lipid organization and pH of the aqueous subphase using steady state and time resolved fluorescence techniques. In all cases contact between porphyrin and aqueous subphase, as indicated through quenching by I^-, is observed at low surface pressure. This contact decreases and becomes almost insignificant as the monolayer approaches maximum organization through compression. On deprotonation of the monocarboxylic porphyrin, methylpyrroporphyrin, increased contact with water is observed in liquid compressed monolayers. In liquid expanded layers, however, it appears that organization of lipid molecules surrounding this dissymmetric charged form affords some isolation from water. The effect of esterification of carboxylic chains is also examined.     

THE EFFECTS OF PERIPHERAL SUBSTITUENTS ON THE KINETICS OF ZINC ION INCORPORATION AND ACID CATALYZED REMOVAL FROM WATER SOLUBLE SULFONATED PORPHYRINS

Abstract

The kinetics of Zn²⁺ and Zn(OH)⁺ incorporation into and the kinetics of the acid catalyzed removal of Zn(II) from twelve water-soluble, sulfonated derivatives of tetraphenylporphyrin with alkyl or halogen groups in the para, ortho or di-ortho positions were investigated. While the incorporation reactions showed little dependence on porphyrin basicity, the Zn-P (P = porphyrin derivative) acid solvolysis reactions were faster the higher the basicity of the free base (H₂-P) compound. Equilibrium constants for the formation of cadmium porphyrins decreased with an increase in porphyrin basicity. The predeformed tetrakis(4-sulfonatophenyl)-β-octabromo-porphyrin reacted with Zn²⁺ about 10³ times faster than porphyrins of similar basicity. These results indicate how substituents on the phenyl and beta-pyrrole rings influence the solution chemistry of water soluble porphyrins.     

光源的选择对水溶性磺酸卟啉光催化对萘二酚类物质催化性能的影响

制备了水溶性磺酸卟啉(TPPS, TMPPS)及其铁配合物(FeTPPS). 以过氧化氢为氧源、 碘钨灯为光源, 水溶性磺酸卟啉可以高效光催化氧化1,5-萘二酚, 产物为5-羟基-1,4-萘二醌. 测定了卟啉的荧光量子产率及寿命, 分析了催化机理. 选择波长在350~650 nm区间内6个波段和功率在0~20 W区间内4种功率的光源, 探索了不同波段及功率的光源对卟啉光催化活性的影响. 研究结果表明, 不同波段的光源对卟啉光催化1,5-萘二酚的催化活性顺序为λ_380—385>λ_360—370>λ_580—585>λ_620—630>λ_492—577>λ_450—470, 该活性顺序与卟啉的紫外-可见吸收密切相关; 当使用相同波段光源时, 卟啉配体催化氧化1,5-萘二酚的反应速率常数与功率呈良好的线性关系; 卟啉铁配合物随着光源功率的增大, 其催化活性与光解程度有直接关系.     

Synthetic porphyrins at interfaces; photosensitization and related reactions of atropisomers of o-substituted tetraphenylporphine derivatives in films, reversed micelles and membranes

Abstract— Previous studies from our laboratories and elsewhere have shown that amides between fatty acids and the synthetic α, α, α, α -tetra (o-aminophenyl)-porphyrins exhibit good surfactant properties which facilitate their incorporation into structured assemblies characterized by hydrophobic-hydrophilic compartmentalization. This paper will focus on a number of aspects of our studies of these porphyrins at different interfaces. The α, α, α, α (4,0) isomers are readily incorporated into Langmuir-Blodgett films as either free base or metal complexes. Studies of assemblies containing free base and palladium (II) complexes have been carried out in which the porphyrin is irradiated in the presence of oxygen and nonexcited but oxidizable substrates. Much of the reactivity observed can be attributed to ¹O₂* generation. These studies reveal the migration range and reactivity of activated oxygen in a structure related to biomem-branes. Several of these synthetic porphyrins have also been examined in cell suspensions and in synthetic reversed micelle solutions. Studies in the former have shown that the porphyrins can mediate the photoinactivation of several enzymes located inside and within the mitochondrial membrane in tumor cells extracted from rats. They are found to compare favorably to hematoporphyrin derivative in effectiveness. Studies of the same porphyrins in their reactivity towards copper ion incorporation in anionic reversed micelles indicate striking rate differences which can be interpreted in part to structural variations between the porphyrins as well as to their orientation at the hydrophobic-hydrophilic interface.     

Role of hydrogen bonding in the photophysical properties of isomeric tetrapyridylporphyrins in aprotic solvent

Extensive photophysical properties of isomeric tetra-2-pyridylporphyrin (TpyP(2)), tetra-3-pyridylporphyrin (TpyP(3)), and tetra-4-pyridylporphyrin (TpyP(4)) have been studied in the presence of a series of phenols of increasing hydrogen bonding power in dichloromethane solution by employing UV/vis spectroscopy; steady-state, time-resolved fluorescence spectroscopy; and transient absorption spectroscopic techniques. The change of absorption spectra of all three porphyrins as a function of different phenol concentrations established the preference of hydrogen bonded complex formation to the peripheral pyridyl nitrogen rather than the pyrrole nitrogen of the porphyrin macrocycle. The fluorescence behaviors of the porphyrins which were observed upon addition of different phenols point to a marked dependence on the nature of the added phenols. Phenols with an electron withdrawing group do not quench the fluorescence of porphyrins, whereas phenols with an electron donating group quench the singlet porphyrin both in static and dynamic pathways. A remarkable difference in quenching behaviors of singlet excited porphyrin by 4-methylphenol (4-MePhOH) and 4-MeOPhOH/4-EtOPhOH (4-EtOPhOH = 4-ethoxyphenol) are observed. The quenching of singlet excited porphyrins by 4-MePhOH is attributed to be purely static in nature, and the H-bond provides a strong nonradiative channel to singlet excited porphyrins. However, the quenching of singlet excited porphyrins by 4-MeOPhOH/4-EtOPhOH is mostly dynamic, and it is ascribed to be the reductive quenching of single excited porphyrins. Picosecond transient absorption study with TpyP(2) and 4-MeOPhOH provides the evidence of porphyrin radical anion and phenol radical cation of equal lifetime, which indicates the fact that electron transfer occurs from phenol to singlet excited porphyrin. The temperature effect on dynamic quenching by 4-MeOPhOH/4-EtOPhOH and kinetic deuterium isotope effect established the reaction to be a photoinduced concerted proton coupled electron transfer.     

Porphyrin profiles in blood, urine and faeces by HPLC/electrospray ionization tandem mass spectrometry

A high-resolution high-performance liquid chromatography/electrospray ionization quadrupole time-of-flight tandem mass spectrometry method is described for the analysis of porphyrins in blood, urine and faeces. The gradient elution reversed-phase HPLC system using acetonitrile-methanol-1 m ammonium acetate/acetic acid buffer (pH 5.16) as gradient solvent mixtures was able to separate all porphyrin metabolites, including the type I and type III isomers of uroporphyrin, hepta-, hexa- and penta-carboxylic acid porphyrins and coproporphyrin. The porphyrins were positively identified by the protonated molecules [M+H](+) and further characterized by tandem mass spectrometric analysis with each porphyrin giving a characteristic collisioninduced dissociation product ion spectrum. The mass chromatograms obtained by HPLC/ESI MS are useful for the differential diagnosis of the porphyrias, since each type of porphyria has a typical porphyrin excretion pattern.     

Light-harvesting energy transfer and subsequent electron transfer of cationic porphyrin complexes on clay surfaces

A novel energy-transfer system involving nonaggregated cationic porphyrins adsorbed on an anionic-type clay surface and the electron-transfer reaction that occurs after light harvesting are described. In the clay-porphyrin complexes, photochemical energy transfer from excited singlet zinc porphyrins to free-base porphyrins proceeds. The photochemical electron-transfer reaction from an electron donor in solution (hydroquinone) to the adsorbed porphyrin in the excited singlet state was also examined. Because the electron-transfer rate from the hydroquinone to the excited singlet free-base porphyrin is larger than that to the excited singlet zinc porphyrin, we conclude that the energy transfer accelerates the overall electron-transfer reaction.     

Factors influencing the distribution pattern of porphyrins in cell membranes

The mechanism of the sensitizer-membrane interactions has been studied by following the distribution properties of selected porphyrins, including haematoporphyrin (HP) and protoporphyrin (PP), into unilamellar liposomes of dipalmitoyl phosphatidylcholine (DPPC). The endomembrane distribution of HP and PP has been checked as a function of the membrane fluidity and composition by fluorescence polarization and quenching techniques. At porphyrin concentrations below 0.5 microM, HP and PP exclusively localize in the inner phospholipid monolayer; at higher concentrations, the outer monolayer also becomes populated. The porphyrin binding sites in liposomes, however, are different for HP and PP: HP preferentially distributes into water-accessible lipid regions, while PP localizes in the most hydrophobic loci of the lipid matrix. A porphyrin redistribution occurs when the fluidity properties of the liposomes are changed by addition of cholesterol or cardiolipin. In DPPC-cholesterol vesicles, all HP molecules dissolve in DPPC-rich regions while all PP molecules partition in cholesterol-rich environments. In DPPC-cardiolipin vesicles both porphyrins preferentially localize in regions accessible to the external medium. The effect of the nature of the carrier on porphyrin distribution in membranes has been studied by following the uptake and photosensitization properties of free and DPPC-incorporated PP and HP with rat liver mitochondria. The porphyrin photosensitizing efficiency has been checked by following the impairment of the respiratory function of mitochondria upon irradiation. Liposome-bound HP is less active than aqueous HP in determining membrane photodamage in mitochondria. On the contrary, aqueous PP is a very poor sensitizer as compared to a DPPC liposome-entrapped drug.(ABSTRACT TRUNCATED AT 250 WORDS)     

Doubly charged porphyrin ion tandem mass spectrometry: Implications for structure elucidation

Under electron ionization (EI) conditions, porphyrins yield unusually high intensities of doubly charged molecular and fragment ions. These doubly charged ions offer unique opportunities for the structure elucidation of porphyrins by tandem mass spectrometry (MS/MS). First, they fragment to a greater extent than the corresponding singly charged ions under both EI/MS and EI/MS/MS conditions. Second, doubly and singly charged porphyrin ions often fragment via different pathways, and can therefore yield different structural information. This paper describes several ways in which analyses of doubly charged porphyrin ions with a triple quadrupole tandem mass spectrometer can be useful in structure elucidation of porphyrins. The effect of the metal atom on the fragmentation of metalloporphyrins in an EI source is demonstrated by correlating the extent of doubly charged fragment ion formation to a stability index. Doubly charged porphyrin ions are shown to yield predominantly doubly charged daughter ions upon collisionally activated dissociation (CAD), and are also shown to fragment to a greater extent than corresponding singly charged porphyrin ions. Advantages and disadvantages of doubly charged porphyrin ion MS/MS for structure elucidation are discussed.     

Three-dimensionally arranged windmill and grid porphyrin arrays by AgI-promoted meso-meso block oligomerization

The syntheses of soluble windmill and grid porphyrin arrays through the AgI-promoted coupling reaction of 1,4-phenylene-bridged linear porphyrin arrays, which are comprised of a central ZnII beta-free porphyrin and flanking peripheral NiII beta-octaalkylporphyrins, are described. The coupling reaction is advantageous in light of its high regioselectivity occurring only at the meso-position of the ZnII beta-free porphyrin as well as its easy extension to large porphyrin arrays. The windmill porphyrin arrays in turn serve as an effective substrate for further coupling reactions, to give three-dimensionally arranged grid porphyrin arrays. Further the grid porphyrin 12-mer (a tetramer of the linear porphyrin trimer) was also coupled to afford grid porphyrins (24-mer, 36-mer, and 48-mer). These porphyrin arrays were isolated in a discrete form by repetitive GPC/HPLC (GPC= gel-permiation chromatography). Competitive experiments with three linear porphyrin trimers bearing different peripheral metalloporphyrins (ZnII, NiII, and Cull), and the trapping experiment of the radical cation at the peripheral porphyrin with AgNO2, suggested that an initial one-electron oxidation of the easily oxidizable peripheral ZnII beta-octaalkylporphyrin with an AgI ion and a subsequent endothermic hole transfer assist the generation of the radical cation at the central ZnII beta-free porphyrin. In all ZnII-metallated windmill porphyrin arrays, the energy level of the S1 state of the meso-meso-linked diporphyrin core is lower than that of the peripheral porphyrins, thereby allowing an energy flow from the peripheral porphyrins to the central diporphyrin core; this has been confirmed by measurements of fluorescence lifetimes and picosecond time-resolved fluorescence spectra. The excitation energy transfer in the arrays encourages their potential use as an light-harvesting antenna.