Coordination Studies of Iron(II) Protoporphyrin IX and Iron(II) Hematoporphyrin IX with 4,4′-Dipyridyl and other Nitrogenous Bases

Abstract

The results of spectral studies of iron(II) protopor-phyrin IX and iron(II) hematoporphyrin IX with several substituted pyridines are reported. The existence in solution of an iron(II) porphyrin complex coordinated to a water molecule and to a substituted pyridine was shown by isolation of the complex from solution. The complex isolated was dimeric inono-4,4′-dipyridyl diaquo iron(II) hematoporphyrin. Addition of ethanol to the aqueous solvent inhibits coordination of iron(II) porphyrins with substituted pyridines. The protoporphyrin ring enhances coordination relative to the hematoporphyrin ring.     

Complexes of Manganese Hematoporphyrin IX With Picoline and other Nitrogenous Bases

Abstract

The results of polarography and cyclic voltammetry studies of hematoporphyrin IX with eight different added ligands (other than H₂O and OH^?) in aqueous solvent are reported. Two nitrogenous ligand molecules were found to coordinate with both manganese (III) and manganese(II) hematoporphyrin IX. Oxygenated ligands such as acetate and pyrophosphate showed no tendency to coordinate with either manganese (III) or manganese (II) porphyrin. Porphyrin adsorption on the mercury electrode is quite evident. The adsorption mechanism appears to be complicated.     

Antioxidative effect of several porphyrins on lipid peroxidation in rat liver homogenates

The effect of several porphyrins on Fe2(+)-ascorbic acid-stimulated lipid peroxidation was examined in rat liver homogenates. Not only protoporphyrin IX (PP) but also mesoporphyrin IX and hematoporphyrin inhibited the lipid peroxidation. Some porphyrins, in which 6- and 7-carboxyethyl groups were esterified with a methyl group, such as protoporphyrin IX dimethyl ester and mesoporphyrin IX dimethyl ester, had no antioxidative effect. Hemin and zinc protoporphyrin IX, which are metal-chelated porphyrins, inhibited the lipid peroxidation while cobalt protoporphyrin IX and tin protoporphyrin IX showed no antioxidative effect. Thus, some of the porphyrins used in the present study showed an antioxidative effect as did PP, but the others did not show such an effect.     

Study of reaction of poly(butyl acrylate) radical with cobalt-porphyrin

Conclusions The reaction of poly(butyl acrylate) radical with a cobalt complex of dimethyl esterdimethyl ether of hematoporphyrin IX leads to the formation of a Co-C bond.Translated from Izvestiya Akademii Nauk SSSR, Seriya Khimicheskaya, No. 12, pp. 2830–2833, December, 1987.     

THE EFFECT OF OXYGEN ON THE INCORPORATION OF MANGANESE,IRON AND COBALT INTO HEMATOPORPHYRIN IX IN GLACIAL ACETIC ACID

Abstract

The results of polarography studies for the reaction of hematoporphyrin IX with Mn(II), Fe(II) and Co(II) ion in acetic acid in the absence and the presence of oxygen are reported. The metal incorporation reaction in the presence of oxygen is quantitative for Mn and Co and incomplete for Fe. In the absence of oxygen, the Mn reaction does not proceed at all, whereas, both the Fe and Co reactions are quantitative.     

Photosensitizing Effect of Hematoporphyrin IX on Immature Stages of Ceratitis capitata (Diptera: Tephritidae)

Immature stages of Ceratitis capitata were tested as a model for hematoporphyrin IX (HP IX) phototoxicity. The lethal concentration 50 (LC₅₀) of HP IX in the food was determined during postembryonic development until adult emergence as 0.173 mm (95% CI: 0.138–0.209). The corresponding HP IX LC₅₀ during the dispersal period alone was 0.536 mm (95% CI: 0.450–0.633). HP IX toxicity was compared against Phloxine B (PhB) (0.5 mm ). HP IX elicited a mortality of 90.87%, which was mainly concentrated during prepupal and early pupal stages. PhB mortality was much lower (56.88%) and occurred mainly during the adult pharate stage. A direct correlation between light-dependent HP IX mortality, evidence of reactive oxygen species (ROS) and lipid peroxidation (conjugated dienes and thiobarbituric acid reactive substances) was established in C. capitata larvae. ROS were found to be very significant in both the brain and in the gut.     

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).     

High-performance liquid chromatographic separation of hematoporphyrin and its nickel,copper and zinc complexes on octadecyl-bonded stationary phase

Summary The feasibility of reversed-phase high-performance liquid chromatography for the separation of metal complexes of hematoporphyrin IX (Hp) is described. The retention order, Zn-complex<Hp (free acid)< Ni-complex<Cu-complex, is regular on an octadecylbonded stationary phase with different compositions of an aqueous methanol mobile phase. These four compounds can be successfully separated within about 8 min on a LiChrosorb RP-18 column (250×4-mm i.d.) with a 85:15 (vol/vol) mixture of methanol and phosphate buffer (pH 3) at a flow rate of 1 ml/min.     

PROPERTIES OF A NEW STATE OF HEMATOPORPHYRIN IN DILUTE AQUEOUS SOLUTION

Abstract —Dilute aqueous hematoporphyrin undergoes a transformation after standing at room temperature leading to a shift of the Soret peak from 395 nm to 405 nm, disappearance of visible bands I (610 nm) and IV (503 nm), a shift of the first emission band from 615 nm to 580 nm, and fluorescence polarization at room temperature. The transformation was accelerated by heating and was faster for more dilute solutions. It did not take place in ethanol, 99:1 glycerol-water, or in small egg lecithin liposomes. It was rapid in SDS micelles and did not occur in CTAB and Triton X-100 micelles. Low concentrations of divalent zinc and copper increased the rate of transformation, but the product was not the corresponding metal ion ligand. The transformation was fast with uroporphyrin I and fast but incomplete with hematoporphyrin derivative. The methyl esters of hematoporphyrin IX and uroporphyrin I did not transform. The tentative identification of the transformed product is a tightly-bound linear micelle, oriented such that the central hydrogens of the free base monomer units are anti-parallel.     

Spectroscopic and Biological Testing of Photobleaching of Porphyrins in Solutions*

The photobleaching of protoporphyrin IX (PP IX) and hematoporphyrin derivative (HpD) solutions was followed using three different methods: spectrophotometry, fluorometry and photodynamically induced cytotoxicity. The latter entails photoirradiation of HT29 human colon adenocarcinoma cells in the presence of preirradiated solutions of HpD and PP IX (λ 415 nm). The highest cytotoxicity was observed in the presence of unirradiated dye and decreased with the time of preirradiation. This decay in photocytotoxicity was further used to determine the porphyrin photobleaching kinetics in solution. For both sensitizers, quantum yields of photobleaching obtained by matching fluoresence were higher than that obtained from absorbance measurements (10 and 11 times for HpD and PP IX, respectively). This difference reflects preferential photobleaching of photolabile monomeric forms compared to aggregates. The highest quantum yield was obtained in the biological test (decay in cytotoxicity) which was 14 times higher for HpD and 30 times higher for PP IX than the quantum yield obtained from absorbance measurements. The absence of correlation between biological and fluorescence measurements has to be taken into account in the in vivo situation. Dark storage of preirradiated sensitizers (37°C, 24 h) completely restored photocytotoxity for PP IX but only partially for HpD, whereas fluorescence patterns were partially restored for both sensitizers.     

Reversed-phase high-performance thin-layer chromatography of hematoporphyrin and its nickel,copper and zinc complexes

Summary The thin-layer chromatographic mobility of hematoporphyrin IX (Hp) and its metal complexes on octyl (C₈)- or octadecyl (C₁₈)-bonded silica gel plate is described. The mobility order for those compounds is regular on both kinds of plate with various compositions of methanol-phosphate buffer mixtures as the developing solvents; thus, Cu-complex < Ni-complex < Hp (free acid) < Zn-complex. The combination of a C₁₈-plate with an 85∶15 (vol/vol) mixture of methanol and phosphate buffer (pH 3) is recommended for the successful separation of these four compounds.     

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).     

A NEW ELECTRONIC ABSORBANCE BAND IN CONCENTRATED AQUEOUS SOLUTIONS OF HEMATOPORPHYRIN IX DETECTED BY PHOTOACOUSTIC SPECTROSCOPY

Abstract— Photoacoustic (PA) spectroscopy reveals a new electronic transition in concentrated aqueous solutions of hematoporphyrin IX (Hp). This new band has a maximum near 440 nm. Its intensity is sensitive to pH, concentration, PA chopping frequency, and the presence of sodium dodecyl sulfate (SDS). Data presented support the assignment of this new band to a dimeric form of Hp. The pH effects on this new band are discussed in terms of their implications for the selective biodistribution of certain porphyrin-based photochemotherapeutic agents.     

Synthesis and study of the spectral properties of diquinone derivatives of hematoporphyrin IX

Diquinone derivatives of hematoporphyrin IX with different structures of the quinone fragments were synthesized by the method of mixed anhydrides. The compounds obtained were investigated by UV, IR, PMR, and fluorescence spectroscopy. Pronounced quenching of the fluorescence of porphyrin in the porphyrin quinones, which depends on the acceptor properties of the quinones and the spatial orientation of the donor and acceptor, was observed.Translated from Khimiya Geterotsiklicheskikh Soedinenii, No. 10, pp. 1324–1330, October, 1991,     

Porphyrins. 33. Proton magnetic resonance spectra of hematoporphyrin derivatives. properties of a dimethoxy derivative (dimegin) in aqueous solution

The PMR spectra of derivatives of hematoporphyrin IX have been investigated in organic and aqueous solution. It has been shown that the presence of the two chiral centers at positions 2 and 4 of the macrocycle is displayed in the specific splitting of the signals of the meso protons. The structures of dimeric associates of 2,4-di(-methoxyethyl)-deuteroporphyrin IX (dirnegin) have been studied in aqueous solution over a wide pH range and have the structure of a skewed sandwich according to PMR data.For Communication 32, see [1].Translated from Khimiya Geterotsiklicheskikh Soedinenii, No. 2, pp. 198–204, February, 1996.     

Laser photolysis study of the hematoporphyrin IX—ℓ-tryptophan system in solvent mixtures at different polarity

The oxygen and ?-tryptophan (TRP) effect on the triplet state of hematoporphyrin IX (HP) has been investigated by means of the laser photolysis technique at room temperature, in various pH 7.4 buffered aqueous/formamide or methanol mixtures with different polarity. The quenching of the HP triplet state by oxysgen is not affected by the medium polarity (k_q O₂ = 1.5 × 10⁹ M^?1 s^?1), whereas the quenching by TRP appears dependent on the composition. The mechanism of the HP-sensitized photooxidation of TRP is discussed.     

pH-dependent spectral properties of HpIX, TPPS2a, mTHPP and mTHPC

Lower extracellular pH in tumors as compared to normal tissues has been proposed to be a factor contributing to the tumor selective uptake of several photosensitizers. Therefore, the pH dependence of absorption and fluorescence spectral properties of four different drugs relevant for photodynamic therapy (hematoporphyrin IX [HpIX], disulfonated meso-tetraphenylporphine [TPPS2a], meso-tetra(3-hydroxyphenyl)porphine [mTHPP] and meso-tetra(3-hydroxyphenyl)chlorin [mTHPC]) has been examined. Spectral analysis of the dyes dissolved in phosphate buffered saline (PBS) indicates pH-dependent modification in the physiologically important region (6.0-8.0) only in the case of HpIX. This modification is probably related to the protonation of carboxylic groups. Spectral changes of HpIX in PBS observed at acidic pH values < 5, as well as those of the rest of the drugs (inflection points of titration curves occurred at about 5.1, 3.8 and 2.4 for TPPS2a, mTHPP and mTHPC, respectively), are likely to be due to the protonation of imino nitrogens. The tumor localizing properties of mTHPP and mTHPC reported in the literature appear to be due to factors other than pH-dependent changes in the lipophilicity of the drugs.     

Metalloporphyrin phototoxicity

The phototoxicities of six metalloporphyrin dimethylesters (i.e. cobalt (Co), copper (Cu), manganese (Mn), nickel (Ni), tin (Sn) and zinc (Zn) were investigated. Hemolysis of human erythrocytes and inactivation of two enzymes (acetylcholinesterase and beta-galactosidase) were used to assess the phototoxic efficacy of these metal chelates. Tin protoporphyrin (SnPP), the only porphyrin found to hemolyze erythrocytes at a concentration of 40 microM (radiation dose, 230 kJ m-2), was much less efficient than either free protoporphyrin IX or hematoporphyrin. SnPP completely inactivated beta-galactosidase at concentrations above 15 microM (radiation dose, 75 kJ m-2) and drastically interfered with acetylcholinesterase activity at a concentration of 150 microM (radiation dose, 75 kJ m-2). CoPP, CuPP, MnPP, NiPP and ZnPP were ineffective photohemolytic agents at 40 microM (radiation dose, 230 kJ m-2), but inactivated acetylcholinesterase and beta-galactosidase activity to varying degrees. These results suggest that (i) metal ions reduce the phototoxicity of protoporphyrin IX, (ii) different metal ions reduce the phototoxic activity of protoporphyrin IX to different degrees and (iii) the biological activities of the various metal complexes vary in different assay systems.     

Time-resolved fluorescence spectroscopy of hematoporphyrin, mesoporphyrin, pheophorbide a and chlorin e6 in ethanol and aqueous solution

The fluorescence decay I(t) and time-resolved spectra I(lambda, t) of some porphyrins and chlorins in ethanol and phosphate-buffered aqueous solution were investigated with a time-correlated single-photon-counting apparatus with a mode-locked Ar+ laser (514.5 nm) as the excitation source. The fluorescence of hematoporphyrin, mesoporphyrin and pheophorbide aa is considerably influenced by the conditions of aggregation (these compounds undergo aggregation in phosphate-buffered solution but not in ethanolic solution). The fluorescence decay of chlorin e6 which remains monomeric in both solvents is single exponential in all cases. The fluorescence spectra of hematoporphyrin, mesoporphyrin and pheophorbide a in phosphate-buffered solution are shifted with respect to the spectra obtained in ethanol; moreover, a new emission band (X band) appears, whose intensity increases on increasing the amount of equilibrium aggregates and shows a fast fluorescence decay. For hematoporphyrin and mesoporphyrin the appearance of the X band emission appears to be correlated with irreversible photoprocesses leading to fluorescent photoproducts. Analysis of the reported fluorescence spectra of cancer cells after incubation with hematoporphyrin derivative suggests that the fluorescent photoproducts might be formed also in vivo.     

A ROLE FOR STEROLS IN THE PORPHYRIN MEDIATED PHOTOSENSITIZATION OF YEAST

The yeast Saccharomyces cerevisiae was used as a model system to determine the role of sterols in the porphyrin mediated photosensitization of yeast. A sterol auxotroph, RD5-R, was grown on sterols with different levels of unsaturation and assayed for photosensitivity in the presence of either protoporphyrin IX or hematoporphyrin (both at 100 micrograms/ml). Cells grown on the completely saturated sterol (stanol), cholestanol, were substantially more resistant to the photosensizing effects of the porphyrin. We hypothesize that this resistance arises from the inability of the porphyrin to mediate the oxidation of the membrane sterol. Our results indicate that photodegradation of the native yeast sterol, ergosterol, can account for substantial losses of cell viability.