Direct C-H borylation of vinylporphyrins via copper catalysis.

A method of direct borylation of vinyl-substituted porphyrinoids (porphyrins and chlorins) has been developed based on the copper catalyzed vinylic C-H activation. Ni(II) complexes of meso- and ß-vinylporphyrinoids have been transformed to the corresponding pinacolboronated derivatives with good yields and high (E)-stereoselectivity. The method provides an easy and direct access to the valuable synthons which were shown to act as nucleophylic partners in the Suzuki cross-coupling building tetrapyrrole derivatives with π-conjugation through the carbon-carbon double bond.

Carbene functionalization of porphyrinoids through tosylhydrazones.

Here, we investigated methods for carbene functionalization of porphyrinoids through metal catalyst-free thermal decomposition of their tosylhydrazones. For the first time, tetrapyrrolyl substituted carbenes were obtained via thermolysis of tosylhydrazones of the corresponding tetrapyrrolyl aldehydes and ketones in the presence of a base. The carbenes formed reacted thermally with substrates without a metal catalyst or light irradiation. Carbenes at the ß-pyrrolic position of porphyrinoids reacted with styrene leading to cyclopropane derivatives of tetrapyrroles. Carbenes also reacted with 1,4-dioxane with their insertion into the C-H bond yielding a tetrapyrrole 1,4-dioxane conjugate. Thermolysis of tosylhydrazones of meso-formyl-ß-octaalkylporphyrinoids led exclusively to the corresponding cyclopentane fused porphyrinoids via intramolecular carbene C-H insertion. A plausible reaction mechanism was discussed based on DFT calculations of the intermediates. The tetrapyrrolyl carbenes were found to be considerably more stable than other carbenes. The products of the functionalization of porphyrinoids via hydrazone formation and subsequent carbene reactions exhibited modified optical spectra. The method for carbene functionalization of porphyrinoids through thermal decomposition of their tosylhydrazones created a new synthetic pathway for tailoring the perimeter of tetrapyrrolic macrocycles. Moreover, this method allows the obtainment of dyes with controllable spectral optical properties. In particular, new tetrapyrrole derivatives possessing phytoporphyrin carbon skeletons which have not been accessible were obtained using a convenient straightforward procedure.

Conjugates of 17-substituted testosterone and epitestosterone with pyropheophorbide a differing in the length of linkers.

Conjugates of 17α-substituted testosterone (1 and 2) and 17ß-substituted epitestosterone (3 and 4) with pyropheophorbide a were synthesized. The scheme consisted of synthesis of 17α-hydroxy-3-oxopregn-4-en-21-oic and 17ß-hydroxy-3-oxopregn-4-en-21-oic acids, and their coupling with pyropheophorbide a by means of either ethylene diamine, or 1,5-diamino pentane linkers. Mutual influence of steroidal and macrocyclic fragments in conjugates molecules was dependent on configuration of C17 and length of linker, that was established by analysis of 1H NMR spectra and molecular models of conjugates. Studies of interaction of conjugates with prostate carcinoma cells revealed that their uptake and internalization were independent on the androgen receptor activity, but dependent on the structure of conjugates, decreasing in the following row: 3 > 4 ≥ 1 > 2. Conjugates significantly decreased the LNCaP and PC-3 cells growth at 96 h incubation. Epitestosterone derivatives 3 and 4 also showed superior anti-proliferative activity versus testosterone ones. Conformationally more rigid conjugates 1 and 3, comprising short linkers, were more active than those with long linkers; conjugate 3 was the most potent.

Structural explanation of the spectral features of the nonsymmetrical complex {2,3,7,8,12,13,17,18-octaethyl-5-[(methylimino)methyl]porphyrinato-κ4N21,N22,N23,N24}palladium(II).

The features of porphyrins defining their functionality are related to their conformational flexibility. The degree of nonplanarity of metalloporphyrins depends directly on the number of substituents, their size and their location. The introduction of substituents in the meso positions of ß-substituted porphyrins increases the steric interaction and leads to distortions of the porphyrin core. Increasing the distortion of the porphyrin core would augment the bathochromic (red) shift of the electronic absorption spectra. A new nonsymmetrical 2,3,7,8,12,13,17,18-octaethyl-5-[(methylimino)methyl]porphyrin complex of palladium(II), [Pd(C38H47N5)], was synthesized and characterized by NMR, mass spectrometry and X-ray analysis. The features of the electronic absorption spectrum of the synthesized complex are explained by the planarity of the porphyrin core and the π-system of the imino group orthogonal to it.

Polymeric structure of a coproporphyrin I ruthenium(II) complex: a powder diffraction study.

Porphyrin complexes of ruthenium are widely used as models for the heme protein system, for modelling naturally occurring iron-porphyrin systems and as catalysts in epoxidation reactions. The structural diversity of ruthenium complexes offers an opportunity to use them in the design of multifunctional supramolecular assemblies. Coproporphyrins and metallocoproporphyrins are used as sensors in bioassay and the potential use of derivatives as multiparametric sensors for oxygen and H+ is one of the main factors driving a growing interest in the synthesis of new porphyrin derivatives. In the coproporphyrin I RuII complex catena-poly[[carbonylruthenium(II)]-µ-2,7,12,17-tetrakis[2-(ethoxycarbonyl)ethyl]-3,8,13,18-tetramethylporphyrinato-κ5N,N',N'',N''':O], [Ru(C44H52N4O8)(CO)]n, the RuII centre is coordinated by four N atoms in the basal plane, and by axial C (carbonyl ligand) and O (ethoxycarbonylethyl arm from a neighbouring complex) atoms. The complex adopts a distorted octahedral geometry. Self-assembly of the molecules during crystallization from a methylene chloride-ethanol (1:10 v/v) solution at room temperature gives one-dimensional polymeric chains.

Lipophilic derivatives of natural chlorins: synthesis, mixed micelles with phospholipids, and uptake by cultured cells.

The chemical synthesis of six lipophilic conjugates of chlorins was carried out, in which lipophilic fragment (either hexadecyl- or cholest-5-en-3ß-yloxyethyl-) bound to 13(1)-, 15(2)-, 17(3)-positions of macrocycle by formation of related carboxamides. Structure of synthesized conjugates was studied by spectral methods and molecular modeling. Lipophilic conjugates of chlorins, being mixed with egg yolk phosphatidyl choline, formed mixed micelles stable in aqueous media under physiological conditions. Mixed micelles of conjugates with phosphatidyl choline differing in stoichiometric compositions were prepared and characterized by absorption spectra, electron microscopy and laser scattering. These micelles were found to bind and internalized by human breast carcinoma MCF-7 cells. The presented data reveal that modification of macrocycle with lipophilic substituents, solubilization of obtained conjugates in aqueous medium as mixed micelles with phospholipids, and transfer of mixed micelles to cells is simple approach for targeting of chlorin derivatives, which apparently may be used in photodynamic therapy.

Aluminum-substituted heme domain of P450BM-3 (BMP): introducing a heme-derived fluorescent probe for studies of substrate binding and protein-protein interactions in cytochromes P450.

We describe a novel technique for heme removal and replacement in the heme domain of P450BM-3 (BMP). The method was applied to obtain the aluminum-protoporphyrin IX (Al-PP) substituted derivative of BMP (Al-BMP). The overall yield of the purified Al-BMP was about 15% as related to the initial amount of the hemeprotein. Al-BMP possesses extensive fluorescence in the 550-650 nm region with excitation in the porphyrin absorbance bands. The protein was shown to bind substrates of P450BM-3 (palmitic, arachidonic, and cis-parinaric acids) with affinities similar to those of the native enzyme (3-6 µM). However, the substrate-induced changes in fluorescence of Al-PP reveal the existence of a second, low-affinity substrate-binding site, which cannot be detected by the spin shift in the native, heme-containing BMP. Using fluorescence resonance energy transfer, we have demonstrated that Al-BMP forms a complex with the flavoprotein domain of P450BM-3 labeled with 7-ethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin maleimide, revealing the affinity similar to that of native BMP (Kd = 5 µM at 0.06 M ionic strength). Therefore, aluminum-substituted BMP may serve as a valuable tool in studies on the mechanisms of interactions of P450s with their substrates and protein partners.

Cell-penetrating conjugates of coproporphyrins with oligoarginine peptides: rational design and application for sensing intracellular O2.

A panel of phosphorescent oligoarginine conjugates of tetracarboxylic Pt(II)-coproporphyrin I dye (PtCP), monosubstituted with long peptides or tetra-substituted with short peptides and having different linkers and peripheral groups, is described. Their photophysical properties, cell loading efficiency, and mechanisms of transport into the cell were investigated and compared. The conjugates were seen to rely on endocytotic mechanisms of cell entry, which are different from that of the unconjugated oligoarginine peptide, and show diverse patterns of intracellular distribution. On the basis of this study, the tetra-substituted PtCP conjugate displaying whole cell distribution was selected for the sensing of intracellular O(2). This probe has been tested in biological experiments on a fluorescence plate reader, including the monitoring of in situ oxygenation of respiring cells and their responses to metabolic stimulation. Similar conjugates of the phosphorescent Pd(II)-coprorphyrin and fluorescent coproporphyrin-ketone were also synthesized and assessed for the sensing of low levels intracellular O(2) and ratiometric pH-sensing, respectively. The results produced and the structure-activity relationships determined can facilitate the rational design of new bioconjugates of porphyrin dyes tailored to specific applications.

O(2)/pH multisensor based on one phosphorescent dye.

A new sensor is described based on a phosphorescent metalloporphyrin dye incorporated in a polymeric membrane, which allows measurement of the two important analytes, oxygen and pH. In such a sensor, the bifunctional dye is quenched by O(2) altering its phosphorescence intensity and lifetime (0-21 kPa O(2)), whereas protonation of the dye causes a major change in the absorption spectrum and also reduces the phosphorescence intensity. As a result, quantification and continuous monitoring of the two analytes can be achieved by (i) simultaneous phosphorescence intensity (O(2), pH) and lifetime (O(2)) measurements and (ii) parallel phosphorescence lifetime (O(2)) and ratiometric absorbance (pH) measurements. Although the first method generally requires sensor calibration in intensity mode, the second provides internal referencing and calibration-free capabilities for both analytes. This approach can be extended to sensing of some other analytes.

Bactenecin 7 peptide fragment as a tool for intracellular delivery of a phosphorescent oxygen sensor.

Research on cell-penetrating peptides for the intracellular delivery of porphyrin compounds has mainly focused on the use of trans-activator of transcription (TAT)-derived peptides and, to a lesser extent, on proline-rich peptides and phosphorescent metalloporphyrins. In this article, we describe a novel phosphorescent oxygen-sensitive probe for intracellular use which comprises a bactenecin 7 peptide fragment (15-24) conjugated with the uncharged monofunctional derivative of Pt(II) coproporphyrin I (PEPP0). This probe provides efficient loading of various mammalian cells, including PC12, HCT116, SH-SY5Y and HeLa, via cell-type-dependent uptake mechanisms. The conjugate displays a similar distribution in cytoplasm and mitochondria which allows local oxygen levels to be monitored. Respiratory responses of PC12 cells loaded with the conjugate, measured on a time-resolved fluorescent reader, showed significant cell deoxygenation in response to uncoupling by carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone and external hypoxia. Treatment with mitochondrial inhibitors led to a decrease in cell deoxygenation. Although the biophysical properties of this conjugate are similar to those of the phosphorescent intracellular oxygen-sensitive probes described previously, it possesses a number of advantages, including ease of synthesis, high loading efficiency and reliability in physiological experiments with cells. This intracellular probe can be employed for the measurement of intracellular O(2) levels in samples containing mammalian cells using the phosphorescence quenching technique. In addition, the responses to metabolic stimuli can be assessed in a wide range of cells, as can the levels of relative cell oxygenation under external hypoxia.

Chlorin e6-cholesterol conjugate and its copper complex. Simple synthesis and entrapping in phospholipid vesicles.

Synthesis of 13'[(cholest-5-en)-3beta-yloxyethoxycarbamoyl]-chlorin e6 starting from methylpheophorbide and 3beta(2-hydroxy)-ethoxycholest-5-ene is presented, as well as the preparation of related copper complex. Both conjugates obtained may be simply incorporated in phosphatidyl choline vesicles.

Evaluation of the derivates of phosphorescent Pt-coproporphyrin as intracellular oxygen-sensitive probes.

Several new derivatives of the phosphorescent Pt(II)-coproporphyrin (PtCP) were evaluated with respect to the sensing of intracellular oxygen by phosphorescence quenching. Despite the more favorable molecular charge compared to PtCP, self-loading into mammalian cells was rather inefficient for all the dyes, while cell loading by facilitated transport using transfection reagents produced promising results. The PtCP-NH(2) derivative, which gave best loading efficiency and S/N ratio, was investigated in detail including the optimisation of loading conditions, studies of sub-cellular localization, cytotoxicity, oxygen sensitivity and long-term signal stability. Being spectrally similar to the macromolecular MitoXpress™ probe currently used in this application, the PtCP-NH(2) demonstrated higher loading efficiency and phosphorescent signals, suitability for several problematic cell lines and a slightly increased lifetime scale for the physiological range (0-200 µM O(2)). In physiological experiments with different cell types, mitochondrial uncouplers and inhibitors performed on a time-resolved fluorescence plate reader, this probe produced the anticipated profiles of intracellular oxygen concentration and responses to cell stimulation. Therefore, PtCP-NH(2) represents a convenient probe for the experiments and applications in which monitoring of cellular oxygen levels is required.

Intracellular oxygen-sensitive phosphorescent probes based on cell-penetrating peptides.

Probing of molecular oxygen in mammalian cells is important for the analysis of mitochondrial function, metabolic responses, and energetic status of the cells. We describe a new panel of intracellular O(2)-sensitive probes based on phosphorescent porphyrin dyes conjugated to cell-penetrating peptides. The probes comprising the uncharged derivatives of Pt(II)-coproporphyrin I covalently linked to positively charged TAT-derived peptides are shown to effectively load live mammalian cells without any transfection reagents. The probes work well with all cell types tested, show similar subcellular localization, and produce characteristic responses to cell stimulation with mitochondrial uncouplers and inhibitors. They provide a simple and versatile tool for O(2) monitoring in live cells and in tissue, and an alternative to the existing O(2) probes which require facilitated transport into the cell.

Analysis of intracellular oxygen and metabolic responses of mammalian cells by time-resolved fluorometry.

A simple, minimally invasive methodology for the analysis of intracellular oxygen in populations of live mammalian cells is described. Loading of the cells with the phosphorescent O(2)-sensing probe, MitoXpress, is achieved by passive liposomal transfer or facilitated endocytosis, followed by monitoring in standard microwell plates on a time-resolved fluorescent reader. Phosphorescence lifetime measurements provide accurate, real-time, quantitative assessment of average oxygen levels in resting cells and their alterations in response to stimulation. Analytical performance of the method is examined, optimized, and then demonstrated with different suspension and adherent cell lines including Jurkat, PC12, A549, HeLa, SH-SY5Y, and C2C12, by monitoring responses to mitochondrial uncouplers, inhibitors, plasma membrane depolarization, and Ca(2+) effectors. The assay provides relevant, information-rich data on cellular function and metabolism. It allows monitoring of small, rapid, and transient changes in cell respiration and screening of new chemical entities.

Sensing intracellular oxygen using near-infrared phosphorescent probes and live-cell fluorescence imaging.

The development and application of a methodology for measurement of oxygen within single mammalian cells are presented, which employ novel macromolecular near infrared (NIR) oxygen probes based on new metalloporphyrin dyes. The probes, which display optimal spectral characteristics and sensitivity to oxygen, excellent photostability, and low cytotoxicity and phototoxicity, are loaded into cells by simple transfection procedures and subsequently analyzed by high-resolution fluorescence microscopy. The methodology is demonstrated by sensing intracellular oxygen in different mammalian cell lines, including A549, Jurkat, and HeLa, and monitoring rapid and transient changes in response to mitochondrial uncoupling by valinomycin and inhibition by antimycin A. Furthermore, the effect of ryanodine receptor-mediated Ca(2+) influx on cellular oxygen uptake is shown by substantial changes in the level of intracellular oxygen. The results demonstrate the ability of this technique to measure small, rapid, and transient changes in intracellular oxygen in response to different biological effectors. Moreover, this technique has wide ranging applicability in cell biology and is particularly useful in the study of low oxygen environments (cellular hypoxia), mitochondrial and cellular (dys)function, and for therapeutic areas, such as cardiovascular and neurological research, metabolic diseases, and cancer.

Systemic suppression of the contact hypersensitivity by the products of protoporphyrin IX photooxidation.

Photodynamic therapy (PDT) is frequently accompanied by induction of systemic immunosuppression. Photochemical mechanisms underlying this effect are not completely understood. Here, we demonstrate the immunosuppressive activity of photooxidation products of protoporphyrin IX dimethyl ester (PPIX) in a murine model of contact hypersensitivity (CHS) to 2,4-dinitrofluorobenzene (DNFB). Intravenous injection of the preirradiated solution of PPIX to mice resulted in fluence-dependent suppression of the CHS. The samples of photodecomposed PPIX with suppressive effect on the CHS contained chlorin-type products, namely, two isomers of photoprotoporphyrin (pPP1 and pPP2) as main photoproducts. Concentration-dependent suppression of the CHS was also induced when purified pPP1 or pPP2 were injected to mice intravenously. These purified photoproducts exerted equal immunosuppressive activity. The highest suppression of the CHS was induced when pPP1 was injected 20 h before sensitization with DNFB. The lowest suppression was at its injection time 24 h before challenge. The pPP1-induced suppression of the CHS was adoptively transferable and was associated with generation of cells with suppressive functions. These suppressor cells inhibited the efferent phase of the CHS. Our results strongly indicate that induction of systemic immunosuppression by PDT with PPIX may proceed through photobleaching of photosensitizer and generation of photoprotoporphyrins, which can affect T cell immunity.

Homogeneous assays for cellular proteases employing the platinum(II)-coproporphyrin label and time-resolved phosphorescence.

Phosphorescent platinum(II) coproporphyrin label (PtCP) is evaluated for the detection of cellular proteases by time-resolved fluorescence in homogeneous format. An octameric peptide containing the recognition motif for the caspase-3 enzyme was dual labeled with a new maleimide derivative of PtCP and with the dark quencher dabcyl. Following photophysical characterization, the quenched substrate was employed in cleavage assays for caspase-3 using Jurkat and HL60 cell lines treated with proapoptotic stimuli performed on a commercial plate reader. Dose-response and time course assays for the drug camptothecin were obtained for comparison with conventional fluorometric detection.

Chiral Bis-chlorin: enantiomer resolution and absolute configuration determination.

[structure: see text] A racemic mixture of the ethane-bridged bis(zinc octaethylchlorin) was successfully resolved for the first time to yield two enantiomers that exhibit substantial CD signals in the regions of chlorin B and Q transitions. The absolute configuration of the corresponding enantiomers was assigned and the origin of its high optical activity was rationalized through a combined spectral, crystallographic, and theoretical analysis.

Influence of linker unit on performance of palladium(II) coproporphyrin labelling reagent and its bioconjugates.

In this paper we describe the preparation of a series of new phosphorescent labelling reagents, based on monosubstituted palladium(II) coproporphyrin-I and the isothiocyanato reactive group. The labelling reagents differ with respect to the chemical composition of the linker unit that combines the reactive group and the porphyrin chromophore. Altogether, seven different labelling reagents are prepared. The new labelling reagents are conjugated with monoclonal mouse IgG to yield label conjugates with variable degrees of conjugation. The effect is studied of linker unit on: (a) the conjugation reaction kinetics; (b) the biological activity of the resulting IgG conjugates; and (c) the efficiency of phosphorescence emission. The results show that an increase in the length of the linker unit has a positive effect on both the reactivity of the label and the biological activity of the resulting conjugates. In addition, the results indicate that the labels with the most hydrophilic linker units exhibit the highest phosphorescence emission efficiencies.