Derivatives Of Protoporphyrin Research Articles

An engineered thermally tolerant apo-cytochrome scaffold for metal-less incorporation of heme derivative.

Cytochrome c552 from Thermus thermophilus is one of the hot topics for creating smart biomaterials as it possesses remarkable stability, is tolerant to multiple mutations and has therefore been recently reported for a number of functionalizations upon substitution of the original prosthetic group with an artificial prosthetic group. However, all of the substitutions were driven by the coordination through the axial ligands followed by complete reconstitution with a metal-porphyrin complex. This limits the scope of the cytochrome c for incorporating a metal-less non-natural heme species that could improve the versatility of cytochrome c for a new generation of engineered cytochrome proteins for further enhancement in their functionalities such as biocatalysts. In this connection, a new variant of Cytochrome c (rC552 C14A) from Thermus thermophilus was reported, where an easy approach to remove the original prosthetic group was achieved, followed by the incorporation of a number of metal-PPIX derivatives that ultimately led to the formation of artificial c-type cytochromes through covalent bonding. The apo-cytochrome was found to be thermally tolerant and to possess a distinctive overall structure as that of the wild type, as was evident from the corresponding CD spectra, which ultimately encouraged reconstitution with a metal-less protoporphyrin derivative for better understanding the role of axial ligands in the reconstitution process. Successful reconstitution was achieved, resulting in a new type of Cytochrome b-type artificial protein without the metal in its active site, indicating the non-involvement of the axial ligand. In order to prove the non-involvement of the axial ligand, a subsequent double mutant (C14A/M69A) was constructed, replacing the methionine at 69 position with non-coordinating alanine residue. Accordingly, the apo-C14A/M69A was prepared and found to be extremely stable as the earlier mutants and the WT showed no signs of denaturation, even at the elevated temperature of 98°C. Subsequently, heme b was successfully incorporated into the apo-C14A/M69A, which demonstrated itself as a highly thermally tolerant protein scaffold for incorporating a metal-less artificial prosthetic group in the absence of the axial ligand. Further improvement in the reconstitution process is achieved by replacing the methionine at 69 position with phenyl alanine (C14A/M69F mutant), resulting in further stabilization of heme species, possibly through non-covalent π-interactions, as corroborated by molecular docking.

Effect of amide protoporphyrin derivatives on immune response in Apis mellifera

The intracellular microsporidian parasite Nosema ceranae is known to compromise bee health by induction of energetic stress and downregulation of the immune system. Porphyrins are candidate therapeutic agents for controlling Nosema infection without adverse effects on honeybees. In the present work, the impact of two protoporphyrin IX derivatives, i.e. PP[Asp]2 and PP[Lys]2, on Apis mellifera humoral immune response has been investigated in laboratory conditions in non-infected and N. ceranae-infected honeybees. Fluorescence spectroscopy analysis of hemolymph showed for the first time that porphyrin molecules penetrate into the hemocoel of honeybees. Phenoloxidase (PO) activity and the expression of genes encoding antimicrobial peptides (AMPs: abaecin, defensin, and hymenoptaecin) were assessed. Porphyrins significantly increased the phenoloxidase activity in healthy honeybees but did not increase the expression of AMP genes. Compared with the control bees, the hemolymph of non-infected bees treated with porphyrins had an 11.3- and 6.1-fold higher level of PO activity after the 24- and 48-h porphyrin administration, respectively. Notably, there was a significant inverse correlation between the PO activity and the AMP gene expression level (r = − 0.61696, p = 0.0143). The PO activity profile in the infected bees was completely opposite to that in the healthy bees (r = − 0.5118, p = 0.000), which was related to the changing load of N. ceranae spores in the porphyrin treated-bees. On day 12 post-infection, the spore loads in the infected porphyrin-fed individuals significantly decreased by 74%, compared with the control bees. Our findings show involvement of the honeybee immune system in the porphyrin-based control of Nosema infection. This allows the infected bees to improve their lifespan considerably by choosing an optimal PO activity/AMP expression variant to cope with the varying level of N. ceranae infection.

Bioactivity studies of porphyrinoids against microsporidia isolated from honeybees

Microsporidian infections are dangerous to honeybees due to the absence of an efficient treatment for nosemosis. In the present work, the abilities of several porphyrins to directly inactivate microsporidia derived from Nosema-infected honeybees were studied in vitro. Amide derivatives of protoporphyrin IX (PPIX) conjugated with one and two amino acid moieties were synthesized, and their activities were compared with those of two cationic porphyrins, TMePyP and TTMePP. The most active porphyrins, PP[Lys-Asp]2, PP[Lys-TFA]2, PP[Asp(ONa)2]2 and PP[Lys-Lys]2 at concentrations as low as 10–50 µM exerted significant effects on microsporidia, reducing the number of spores by 67–80% compared to the control. Live-cell imaging of the spores treated with porphyrins showed that only 1.6% and 3.0% of spores remained alive after 24 h-incubation with 50 µM PP[Asp(ONa)2]2 and PP[Lys-Asp]2, respectively. The length of the amino acid side chains and their identity in the PPIX molecules affected the bioactivity of the porphyrin. Importantly, the irradiation of the porphyrins did not enhance their potency in destroying Nosema spores. We showed that the porphyrins accumulated inside the living spores but not inside dead spores, thus the destruction of the microsporidia by non-metallated porphyrins is not dependent on photosensitization, but is associated with their active transport into the spore cell. When administered to honeybees in vivo, PPIX[Lys-TFA]2 and PPIX[Lys-Lys]2 reduced spore loads by 69–76% in infected individuals. They both had no toxic effect on honeybees, in contrast to zinc-coordinated porphyrin.

Mitochondria-Targeting Polyamine-Protoporphyrin Conjugates for Photodynamic Therapy.

Two polyamine derivatives of protoporphyrin IX (PPIX) were tested as photodynamic therapy (PDT) agents in HT29 colorectal cancer and HEP3B liver cancer cell lines. These compounds exhibit excellent singlet oxygen quantum yields and show strong in vitro PDT efficacy after 660 nm laser irradiation, whereas exogenous PPIX itself exhibits much weaker PDT effects. Confocal microscopy imaging studies reveal that a protoporphyrin derivative with eight amine moieties has excellent water solubility, and localizes mainly in the mitochondria of both HT29 and HEP3B cells, whereas the cellular distribution of a protoporphyrin derivative with four amine moieties is not as specific. This work demonstrates that polyamine moieties on macrocycles can enhance PDT efficacy by targeting mitochondria.

Antimicrobial photodynamic therapy combined to periodontal treatment: Experimental model

BackgroundAntimicrobial photodynamic therapy (aPDT) has been used as an adjuvant treatment for periodontitis. It combines a photosensitizer with a light source to induce reactive oxygen species and kill microbial cells. PpNetNI is a protoporphyrin derivative, and it has a chemical binding site at biofilm and great affinity to microbial cells. The aim of this study was to investigate the effects of aPDT as an adjuvant treatment for periodontitis. MethodsThirty healthy male rats Wistar (Rattus norvegicus) were used in this study (Approved by UNINOVE Ethical committee AN0029/2015). Periodontitis was induced by placing a cotton ligature around the first mandibular molar in a subgengival position. The contralateral mandibular first molar received neither a ligature nor any treatment, and was used as a control. After 7 days, the ligature was removed and all animals received scaling and root planing (SRP) and were divided according to the following treatments: SRP group (received SRP and irrigation with PpNetNI, 10μM) and aPDT group (PpNetNI 10μM followed by LED irradiation). aPDT was performed with a LED (630nm) with an output power of 400mW (fluence-rate 200mW/cm2; fluence 18J/cm2). Rats were euthanized at 24h, 48h and 7days postoperatively. The area of bone loss in vestibular region of the first molar was evaluated by Optical Coherence Tomography (OCT, THORLABS LTD., Ely, UK). Data were analyzed statistically (ANOVA and Tukey tests, p<0.05). ResultsThe animals treated by aPDT showed bone gain of approximately 30% compared to the SRP group following 7days from the treatment. ConclusionaPDT promoted bone recovery 7days after periodontal intervention.

A facile synthesis and application of protoporphyrin derivatives on reducing the tobacco specific N-nitroamines levels of smoke

Two protoporphyrin derivatives were prepared by a facile method using inexpensive hemin as starting material. They were added to cigarette filters to reduce the carcinogenic tobacco specific N-nitroamines (TSNAs), especially toward NNK (4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone) and NNN (N-nitrosonornicotine) for environment protection and public health. The reduction level of TSNAs was reached to 37.6% from MSS, with greater reductions when more porphyrin was included in the filter. The decrease level for NNK by protoporphyrin derivatives is more effective than NNN. The interaction between protoporphyrin derivatives and TSNAs (NNK and NNN) were investigated by fluorescence spectra and UV-visible titration. The correlation coefficients were 0.978~0.997 and the binding constants was the scope from 1.26 × 103 to 4.04 × 104. The interaction mechanisms between protoporphyrin derivatives and, NNK and NNN are possibly the co-interaction of hydrogen bond binding and strong π–π stacking.

Supramolecular self-assembled nanowires by the aggregation of a protoporphyrin derivative in low-polarity solvents

The self-assembly of a simple tetraalkyl-substituted protoporphyrin derivative into fibril wire-like nanostructures from CHCl3/cyclohexane solvent mixes is explored. The protoporphyrin IX derivative is synthesised in two efficient steps by amidation of the two propylenic acid groups followed by a cross metathesis of the vinyl groups using Grubbs' second-generation catalyst. The self-assembly of the functionalised protoporphyrin is sensitive to solvent and under favourable conditions forms ‘nanowires’ of micrometre length. The nanowires were characterised by absorption and fluorescence spectroscopy, transmission electron microscopy and atomic force microscopy.

Natural dyes based on chlorophyll and protoporphyrin derivatives

The utility of natural dyes (chlorophyll, protoporphyrin, and their derivatives) as ecologically pure new-generation dyes that are safer than common synthetic arene-based dyes is discussed. New dyes were obtained from chlorophyll and protoporphyrin derivatives such as pheophorbide (a+b), chlorin e6, 6-N-(2-aminoethylamido)chlorin e6 dimethyl ester, and 6-N-(2-aminoethylamido)deuteroporphyrin methyl ester and tested in coloration of wool, cotton, and acetate fibers.

Relationship between structure and photoactivity of porphyrins derived from protoporphyrin IX

Protoporphyrin (Pp IX) derivatives were prepared to study the relationship between photosensitizer structure and photoactivity, with an emphasis on understanding the role of membrane interactions in the efficiency of photosensitizers used in photodynamic therapy (PDT). The synthetic strategies described here aimed at changing protoporphyrin periferic groups, varying overall charge and oil/water partition, while maintaining their photochemical properties. Three synthetic routes were used: (1) modification of Pp IX at positions 31and 81by addition of alkyl amine groups of different lengths (compounds 2–5), (2) change of Pp IX at positions 133and 173, generating alkyl amines (compounds 6 and 7, a phosphate amine (compound 8, and quarternary ammonium compounds (compounds 9 and 10), and (3) amine-alkylation of Hematoporphyrin IX (Hp IX) at positions 31, 81, 133and 173(compound 12). Strategy 1 leads to hydrophobic compounds with low photocytotoxicity. Strategy 2 leads to compounds 6–10 that have high levels of binding/incorporation in vesicles, mitochondria and cells, which are indicative of high bioavailability. Addition of the phosphate group (compound 8), generates an anionic compound that has low liposome and cell incorporation, plus low photocytotoxicity. Compound 12 has intermediate incorporation and photocytotoxic properties. Compound modification is also associated with changes in their sub-cellular localization: 30% of 8 (anionic) is found in mitochondria as compared to 95% of compound 10 (cationic). Photocytotoxicity was shown to be highly correlated with membrane affinity, which depends on the asymmetrical and amphiphilic characters of sens, as well as with sub-cellular localization.

Design and efficient synthesis of a new scaffold based on unsymmetrical protoporphyrin IX derivatives for use in SPPS

Fmoc-protected amine derivatives of Protoporphyrin IX were synthesized by two original methods using solid-phase chemistry. The new compounds represent unsymmetrical scaffolds suitable for the generation of a large range of peptidic porphyrin derivatives through SPPS strategy.

Photodynamic therapy with 5‐aminolevulinic acid and diamino acid derivatives of protoporphyrin IX reduces papillomas in mice without eliminating transformation into squamous cell carcinoma of the skin

Photodynamic therapy (PDT) is used to treat malignant and nonmalignant diseases. It is also used for cosmetological skin treatment. PDT is generally considered to have a low risk of carcinogenicity. However, instances of nonmalignant human tumors turning malignant have been linked to PDT. In this study, we used 5-aminolevulinic (ALA) acid and 3 water soluble photosensitizers-PP(Arg)(2), PP(Ser)(2)Arg(2), PP(Ala)(2)Arg(2), all diamino acid derivatives of protoporphyrin IX-to treat benign papillomas in FVB/N mice induced by 7,12-dimethylbenz(a)anthracene (DMBA)-12-O-tetradecanoyl-phorbol-13-acetate (TPA). Of these drugs, ALA and PP(Arg)(2) were found the most efficient. PDT reduced the number of papillomas, but with increasing effectiveness of the drugs, the risk of malignant transformation of the papillomas into squamous cell carcinomas increased. The underlying mechanisms are not clear and further investigations are needed.

Diamino acid derivatives of PpIX as potential photosensitizers for photodynamic therapy of squamous cell carcinoma and prostate cancer: In vitro studies

Photodynamic therapy (PDT) is an alternative treatment modality involving light activated drugs, called photosensitizers (PSs), to treat cancer and non-cancerous conditions. The search for new compounds which might become effective PSs is the major direction for PDT development. In the present work we have studied the dark toxicity, intracellular localization and photodynamic properties of four potential, water soluble, second generation PSs – PP(Arg) 2, PP(Ser) 2Arg 2, PP(Ala) 2Arg 2, PP(Phe) 2Arg 2, all diamino acid derivatives of protoporphyrin IX. Human prostate cancer (DU-145) and squamous carcinoma (A431) cells were used as experimental model. Among investigated compounds PP(Ser) 2Arg 2 exhibited the lowest dark toxicity and the highest PDT effectiveness towards both cell lines. Fluorescence microscopy revealed the time-dependent changes in intracellular localization of the PS which were related to the phototoxicity. The results show that PP(Ser) 2Arg 2 may be a potential PS for PDT.

Interactions of peripheral-type benzodiazepine receptor with diamino acid derivatives of protoporphyrin IX

The aim of this study was to examine and determine binding affinities of the peripheral benzodiazepine receptor (PBR) with diamino acid derivatives of protoporphyrin IX with general structure PP(AA) 2 in a function of environmental pH changes. Fluorescence intensity was used as a measure of protein association constant. Decrease in protein fluorescence, after titration with increasing concentration of porphyrins, gave evidence of interactions between PBR and examined ligands. Experimental data were fitted to theoretical curves, assuming different models of interactions. All examined ligands were best fitted to weak binding model. Increase in environmental acidity resulted in changes in association constants. For all examined derivatives association constants were at least twice higher in pH 5.5 as compared to pH 7.4 and 6.5.

YC-1-like potentiation of NO-dependent activation of soluble guanylate cyclase by derivatives of protoporphyrin IX

The influence of protoporphyrin IX derivatives--2,4-di(1-methoxyethyl)-deuteroporphyrin IX disodium salt (dimegin) and hematoporphyrin IX (HP)--on the activation of human platelet soluble guanylate cyclase by sodium nitroprusside was investigated. Dimegin and HP, like 1-benzyl-3-(hydroxymethyl-2-furyl)indazole (YC-1), produce synergistic effects on the activation of soluble guanylate cyclase by sodium nitroprusside. The synergistic activation of the enzyme by the combination of 10 microM sodium nitroprusside and 5 microM dimegin (or 5 microM HP) was 190 +/- 19 and 134 +/- 10%, respectively. The synergistic activation of guanylate cyclase by 3 microM YC-1 and 10 microM sodium nitroprusside was 255 +/- 19%. Dimegin and HP had no effect on the activation of guanylate cyclase by YC-1; they did not change the synergistic effect of YC-1 (3 microM) and sodium nitroprusside (10 microM) on guanylate cyclase activity. The synergistic activation of NO-stimulated guanylate cyclase activity by dimegin and HP represents a new biochemical effect of these compounds that may have important pharmacotherapeutic and physiological significance.

Verteporfin

Photodynamic therapy (PDT) with verteporfin (Visudyne), a photosensitising protoporphyrin derivative, is used in the management of subfoveal choroidal neovascularisation (CNV) secondary to age-related macular degeneration (AMD) or pathological myopia (PM). PDT with verteporfin over 1 and 2 years reduces the decline in visual acuity in patients with classic-containing subfoveal CNV secondary to AMD. Verteporfin is generally well tolerated by most patients. Verteporfin is also effective in patients with CNV secondary to PM, although data in this indication are limited and further controlled studies are required. Although verteporfin has shown efficacy in patients with occult AMD-related subfoveal CNV lesions in early trials, data are currently limited on its first-line use in this indication; fully published data from the Verteporfin In Occult (VIO) trial are therefore awaited with interest. Verteporfin should be considered as a first-line treatment in patients with predominantly classic subfoveal CNV secondary to AMD, and in patients with smaller minimally classic subfoveal CNV lesions. It may also be considered an option for the treatment of patients with occult AMD-related subfoveal CNV in whom visual acuity decreases or predominantly classic features develop over time.

Synthesis of New Derivatives of Protoporphyrin IX and Chlorophyll a

The vinyl groups in protoporphyrin IX and chlorophyll a derivatives were selectively transformed into hydroxymethyl and acetoxymethyl substituents. The reactivities of β-hydroxymethyl and β-acetoxymethyl groups in porphyrins and chlorins toward nucleophilic reagents were compared for the first time using the reaction with acetylacetone as an example. Peripheral acetylacetone moieties in porphyrins and chlorins were shown to be promising as building blocks for generation of exo heterocyclic structures.

Influence of diamino acid derivatives of protoporphyrin IX on mouse immunological system: Preliminary results

Immunological response related to photodynamic therapy (PDT) is one of the basic elements that influence on the efficiency of this cancer treatment method. Diamino acid derivatives of protoporphyrin IX are promising photosensitizing agents that are intended to be the components of new anti-tumor drug. The influence of three derivatives – PP(Ser) 2Arg 2, PP(Ala) 2Arg 2, PP(Phe) 2Arg 2 and a mixture of these compounds called Sensyphyrine ® on mouse immunological system was investigated where animals were exposed and unexposed to the laser irradiation. Porphyrins solutions were injected intravenously, mice were irradiated with the red diode laser at λ = 632 nm. Cells and blood samples were taken at time intervals after irradiation. The levels of interleukin-6, interleukin-1β and the production of reactive forms of nitrogen by macrophages were determined. The results show that all of the diamino acid derivatives of protoporphyrin IX indicate an immunological response when the light is applied. Each of the porphyrin revealed different impact on mice immunological system. The most potent stimulant properties disclosed PP(Phe) 2Arg 2 derivative for which the highest values of IL-1β, IL-6 and NO 2 - were noticed. The weakest immunological activation revealed PP(Ser) 2Arg 2 derivative.

Pyrazolinyl and cyclopropyl derivatives of protoporphyrin IX and chlorins related to chlorophyll a

Diazomethane reacts regioselectively with peripheral vinyl substituents of pyropheophorbide a and purpurin-18 N-methylimide to produce corresponding 1′-pyrazolinyl-substituted derivatives as a main product. Similarly, treatment of protoporphyrin IX gave a mixture of mono- and di-substituted pyrazolinyl analogs, which were isolated as individual products. Thermolytic decomposition of the pyrazolinyl derivatives produced cyclopropyl-substituted chlorins and porphyrins. 1,3-Dipolar cycloaddition mechanisms of the formation of 1′-pyrazolinyl derivatives are discussed.

The Depth of Porphyrin in a Membrane and the Membrane’s Physical Properties Affect the Photosensitizing Efficiency

Photosensitized biological processes, as applied in photodynamic therapy, are based on light-triggered generation of molecular singlet oxygen by a membrane-residing sensitizer. Most of the sensitizers currently used are hydrophobic or amphiphilic porphyrins and their analogs. The possible activity of the short-lived singlet oxygen is limited to the time it is diffusing in the membrane, before it emerges into the aqueous environment. In this paper we demonstrate the enhancement of the photosensitization process that is obtained by newly synthesized protoporphyrin derivatives, which insert their tetrapyrrole chromophore deeper into the lipid bilayer of liposomes. The insertion was measured by fluorescence quenching by iodide and the photosensitization efficiency was measured with 9,10-dimethylanthracene, a fluorescent chemical target for singlet oxygen. We also show that when the bilayer undergoes a melting phase transition, or when it is fluidized by benzyl alcohol, the sensitization efficiency decreases because of the enhanced diffusion of singlet oxygen. The addition of cholesterol or of dimyristoyl phosphatydilcholine to the bilayer moves the porphyrin deeper into the bilayer; however, the ensuing effect on the sensitization efficiency is different in these two cases. These results could possibly define an additional criterion for the choice and design of hydrophobic, membrane-bound photosensitizers.

Binding specificity of the Porphyromonas gingivalis heme and hemoglobin receptor HmuR, gingipain K, and gingipain R1 for heme, porphyrins, and metalloporphyrins.

Previous genetic and biochemical studies have confirmed that hemoglobin and hemin utilization in Porphyromonas gingivalis is mediated by the outer membrane hemoglobin and heme receptor HmuR, as well as gingipain K (Kgp), a lysine-specific cysteine protease, and gingipain R1 (HRgpA), one of two arginine-specific cysteine proteases. In this study we report on the binding specificity of the recombinant P. gingivalis HmuR protein and native gingipains for hemoglobin, hemin, various porphyrins, and metalloporphyrins as assessed by spectrophotometric assays, by affinity chromatography, and by enzyme-linked immunosorbent assay. Protoporphyrin, mesoporphyrin, deuteroporphyrin, hematoporphyrin, and some of their iron, copper, and zinc derivatives were examined to evaluate the role of both the central metal ion and the peripheral substituents on binding to recombinant HmuR and soluble gingipains. Scatchard analysis of hemin binding to Escherichia coli cells expressing recombinant membrane-associated six-His-tagged HmuR yielded a linear plot with a binding affinity of 2.4 x 10(-5) M. Recombinant E. coli cells bound the iron, copper, and zinc derivatives of protoporphyrin IX (PPIX) with similar affinities, and approximately four times more tightly than PPIX itself, which suggests that the active site of HmuR contains a histidine that binds the metal ion in the porphyrin ring. Furthermore, we found that recombinant HmuR prefers the ethyl and vinyl side chains of the PPIX molecule to either the larger hydroxyethyl or smaller hydrogen side chains. Kgp and HRgpA were demonstrated to bind various porphyrins and metalloporphyrins with affinities similar to those for hemin, indicating that the binding of Kgp and HRgpA to these porphyrins does not require a metal within the porphyrin ring. We did not detect the binding of RgpB, the arginine-specific cysteine protease that lacks a C-terminal hemagglutinin domain, to hemoglobin, porphyrins, or metalloporphyrins. Kgp and HRgpA, but not RgpB, were demonstrated to bind directly to soluble recombinant six-His-tagged HmuR. Several possible mechanisms for the cooperation between outer membrane receptor HmuR and proteases Kgp and HRgpA in hemin and hemoglobin binding and utilization are discussed.