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A method of direct borylation of vinyl-substituted porphyrinoids (porphyrins and chlorins) has been developed based on the copper catalyzed vinylic C–H activation.

Five new bifunctional conjugates of pyropheophorbide a with 17-substituted testosterone, dihydrotestosterone and epitestosterone differing in the length of linker (1–5) and two new complex conjugates 6 and 7 (containing three functional units: pyropheophorbide a, 17α-substituted testosterone, and lipophylic hexadecyl chain) were synthesized. Mutual influence of steroidal and macrocyclic fragments in conjugates 1–7 was established by analysis of 1H NMR spectra and molecular models of conjugates. The uptake and internalization of conjugates 1–5 by prostate carcinoma cells were dependent on the stereochemical configuration of 17-hydroxyl group in steroidal moiety, and the length of linker. Conjugates 1–5 significantly decreased the LNCaP and PC-3 cells growth and proliferation at 96 h incubation; the anti-proliferative activity of epitestosterone derivative comprising short linker 3 was superior. Irradiation of cells labeled with conjugates with light (λ = 660 nm) significantly increased cytotoxicity. Trifunctioal conjugates 6 and 7 easily formed mixed micells with phosphatidyl choline and pluronic F68; these mixed micelles efficiently internalized by human hepatocarcinoma Hep G2 cells, herewith the internalization was dependent on the conjugate structure, rather than on the method of solubilization.

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 β-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-β-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.

Cell analysis by optochemical sensing represents large and important niche in life and biomedical sciences. We present advanced multi-modal, multi-analyte sensing platform and dedicated materials for cell analysis based on the substituted phosphorescent Pt(II)- or Pd(II)-porphyrin indicator dyes bearing dual O2 and pH sensing functionality (MePor-SB). The study includes screening of the different host matrices for the sensor, synthesis and evaluation of new MePor-SB derivatives with altered protonation behaviour, development of the ratiometric version of the pH sensor, assessment of photoluminescent signal enhancement options, deposition of sensor coatings on common cell analysis substrates and their demonstration of sensor performance in Oxygen Consumption Rate (OCR) and Extracellular Acidification (ECA) measurements with relevant cell models. The main outcomes include the elaboration of the structure-function relationships for this biosensor system, development of the self-referenced tandem O2/pH and OCR/ECA sensing system which enables calibration-free operation, demonstration of the sensor operation on common plastic substrates in cell analysis, benchmarking against the existing platforms and real-life experiments with cells. This new sensing platform shows potential for wide practical use.

Conjugates of 17α-substituted testosterone (1 and 2) and 17β-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 17β-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.

The reaction between coproporphyrin I tetraethyl ester and ruthenium(II) dodecacarbonyl in toluene is investigated. The formation of two different products, complexes 2 and 3 of ruthenium(II) with coproporphyrin I tetraethyl ester, studied by means of mass spectrometry, electronic absorption spectroscopy, NMR, X-ray diffraction, and thermogravimetric analysis, is revealed. Structures are proposed for the products, of which (2) is a monocarbonyl complex of ruthenium(II) porphyrin that exists as a coordination polymer formed owing to intermolecular axial bonding between the oxygen atoms of carboethoxyl groups and ruthenium(II). The structure proposed for second product (3) is in the form of the corresponding monomer of a monocarbonyl complex of ruthenium(II) porphyrin. It is established that polymeric complex 2 transforms into monomeric complex 3 when it is heating in pyridine.

A new approach to functionalize porphyrinoids was developed based on the Vilsmeier formylation followed by the azine formation and a new class of the compounds namely azines of porphyrinoids was obtained. Variously substituted azine porphyrins, chlorins and their dyads were synthesized, and their structural and electronic parameters were studied with X-ray diffraction analysis, electronic absorption and emission spectra, electrochemistry and DFT calculations. Azine represents a very peculiar bridge with an unclear conjugation ability affecting optical properties of chromophores. This work was aimed to reveal azine properties and the influence of the azine bridge on the chromophores linked. The results of investigation allowed to conclude that azine bridge can partly be regarded as a conjugation switch with a high threshold level of switching, where limited conjugation in the ground state can be greatly increased at higher energy states of the azine molecule.

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 themesopositions of β-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.

The previously unknown transformation of coproporphyrins to coprochlorins was achieved via reduction of coprohemins I and II with sodium in isoamyl alcohol to give the corresponding novel coprochlorins I and II. Preliminary mechanistic studies using DFT calculations suggested a reduction pathway leading first to the porphodimethene followed by rearrangement to the chlorin.

Regioselective bromination of methyl pyropheophorbide a at the C32-position of the terminal double bond has been carried out as a one-pot two-step addition/elimination process. The elimination occurs with 100% stereoselectivity and bromovinyl 4 has E-configuration of the C3-double bond. The reactivity of unsaturated bromide 4 has been evaluated in the series of the Pd -catalyzed coupling reactions.

The effective synthesis of Pd(II) monofunctional azomethine derivatives of coproporphyrin isomers I and II is reported. The proposed method is based on the interaction of a “phosphorus complex”, generated in situ by Vilsmeier-Haack reaction, with various amines. The reaction with palladium complex of coproporphyrin II gave Schiff bases as a mixture of two structural isomers in 4:1 ratio. The structure of the obtained compounds was determined using 1H, 13C, 1H-1H NOESY and UV-vis spectroscopy and mass spectrometry data.

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 β-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(C

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.

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.

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.

New types of chlorin e 6 derivatives containing organo-phosphorus units were synthesized under microwave irradiation.

Synthesis of 13′[(cholest-5-en)-3β-yloxyethoxycarbamoyl]-chlorin e6 starting from methylpheophorbide and 3β(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.

Several phosphorescent platinum(II) and palladium(II) complexes of tetrapyrrole dyes are prepared in an attempt to improve the compatibility with red laser diodes. The nature of meso-substituents in benzoporphyrin macrocycle is found to affect mainly the position of the Soret band and has little influence on the Q-bands. In rigid polymeric matrices these dyes possess more favourable photochemical properties than corresponding meso-tetraphenyltetrabenzoporphyrins. Platinum(II) chlorins show good compatibility with 635 nm laser diode and 632.8 line of He–Ne laser, and have moderate brightness with emission yields ∼2%. Photophysical properties and photostability of these dyes is assessed and compared with the known NIR-emitting oxygen indicators.

In this work the synthesis, optical and luminescence spectroscopic characterization of the new ytterbium complexes with asymmetric porphyrins of hematoporphyrin group, modified by two β-diketone fragments are presented. All ligands and complexes are investigated by the methods of molecular mechanics, elemental analysis, UV-vis, IR, luminescence, mass and 1H NMR spectroscopy. Influence of some factors (such as location of β-diketones substituents, value of charges on ytterbium ion, etc.) on the value of 4f-luminescence is discussed.

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 Ca2+ 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.

Quenching of phosphorescent platinum(II) and palladium(II) coproporphyrin (MeCP) labelled oligonucleotides was investigated. Strong hybridization-specific quenching was observed in duplex DNA structures with a variety of quenchers and with two identical porphyrin labels when in close proximity. Classical resonance energy transfer mechanism was ruled out, since quenching did not correlate with spectral overlaps and lifetime changes were insignificant. Quenching of MeCP by the free quenchers in solution revealed that porphyrin-porphyrin quenching is predominantly static while other dyes quench dynamically. The results suggest that the quenching in DNA duplex proceeds via direct contact.

Base-assisted ring opening in 1,2-oxazinochlorin derivatives led selectively to the corresponding meso-cyanohydroxychlorin derivatives. The latter could then undergo acid-mediated carbocation formation followed by nucleophilic treatment to give different products, depending on the nature of the nucleophile reagent. Treatment of (E)- and/or (Z)-2-ethylidene-3-hydroxy-5-cyano-3,7,8,12,13,17,18-heptaethylchlorin nickel complex with a mixture of 5% trifluoroacetic acid and dichloromethane yielded a novel type porphyrin-porphyrin dimer with a condensed cyclohexane ring in an almost quantitative yield. The structure of this dimer was determined by single crystal X-ray analysis.

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.

The synthesis has been carried out of nickel complexes of oximes of two isomers of meso-formyletioporphyrin-II and their conversion in the heterophase system methylene chloride-water into nickel complexes of tripyrrolylisoxazoles has been studied. The structure of one of them was established by X-ray structural analysis.

Phosphorescent platinum(II)-coproporphyrin label (PtCP) was evaluated in post-PCR detection of nucleic acids by time-resolved fluorescence (TR-F) using three common formats. PtCP-labelled oligonucleotide primers and PtCP-dUTP were incorporated in a PCR to produce labelled amplified target −173 or 305 bp DNA. Alternatively, aminoallyl-dUTP was incorporated in a PCR and the product was subsequently labelled with PtCP. The resulting PCR mixtures containing labelled dsDNA were separated on 1.5% agarose gels and then analysed by ethidium bromide staining and by direct detection of PtCP label on a commercial TR-F plate reader Victor 2 (Perkin Elmer Life Sciences) used in scanning mode. In all cases label incorporation and high yields of amplified DNA were observed. Direct TR-F detection of PtCP-labelled DNA from a gel provided high sensitivity and signal to noise ratio, with limits of detection in the range of 9–22 pg for all three formats. The sensitivity achieved with PtCP label was considerably better than that achieved with ethidium bromide staining (∼1 ng of dsDNA) or with conventional fluorescent label FITC. Neither the FITC label nor ethidium bromide staining interfered with PtCP detection, thus allowing multiplexed detection.

A preparative procedure is proposed for the synthesis of α-aminophosphonates of natural porphyrins by the microwave-assisted Kabachnik—Fields reaction.

Starting from the nickel meso-formyloctaethylporphyrin oxime complex, the meso-cyanooctaethylporphyrin N-oxide complex was synthesized for the first time. The new complex enters into 1,3-cycloaddition to olefins. The double addition of the nitrile oxide to 2,5-norbornadiene affords a porphyrin dimer, whose structure was established by X-ray diffraction analysis.

The extensive published data on methods for the modification of peripheral substituents in chlorophylls a and b and their various semisynthetic derivatives are classified and analyzed. Special attention is paid to specific transformations arising from the presence of the unique macrocyclic tetrapyrrole fragment. Data on certain main pathways to modification of the exocyclic fragment of chlorophylls are also presented.

The first synthesis of α-amino phosphonates comprising porphyrin core was accomplished. Three methods of obtaining α-amino phosphonates 5-8 were compared. Conventional heating of formylporphyrins 1-4 with t-BuNH 2 and (EtO) 2 P(O)H in various solvents was ultimately unsuccessful for preparing 5-8 whereas the use of microwave irradiation made it possible to obtain 5-8 in good yields. Regioselective preparation of 5-8 in excellent yields was achieved by combining microwave-assisting conditions and catalysis with CdI 2 . Efficient synthetic procedures of obtaining formylporphyrins 3,4 in large scale were also proposed.

Synthesis of monofunctional derivatives of platinum(II) and palladium(II) coproporphyrin-II with the isothiocyanato reactive group is presented. The compounds exhibit strong phosphorescence at room temperature in deoxygenated aqueous solutions and they enable facile covalent labeling of proteins and other biomolecules under slightly alkaline conditions. The performance of the compounds as phosphorescent labeling reagents is studied and the results are considered in relation to the results for the respective labeling reagent of coproporphyrin-I isomer. The results show that the derivatives of coproporphyrin-II exhibit similar reactivity and emission efficiency to that of the derivatives of coproporphyrin-I isomers. Thus, the coproporphyrin-II derivatives serve as alternative labels to coproporphyrin-I in the design of sensitive bioassays based on phosphorescence and time-resolved fluorescence detection.

A method is described for preparation of monosubstituted derivatives of platinum and palladium coproporphyrin-I. The method is based on chemical differentiation of one of the four alkoxycarbonyl groups of the starting coproporphyrin-I tetraalkyl ester by selective acid hydrolysis. The hydrolysis results after chromatographic purification to pure mono hydroxycarbonyl coproporphyrin, which can be subsequently coupled with a chemical species carrying a functional group of interest. The described method is used for preparation of phosphorescent metalloporphyrin labeling reagents having an isothiocyanato reactive group.

The photodynamic effects of 6 new deuteroporphyrin IX derivatives with different amphiphilicity and lipophilicity, as well as effects of known hematoporphyrin derivatives Photofrin II and Photoheme on isolated crayfish mechanoreceptor neurons were studied. After 30 min photosensitization, neurons were irradiated with He-Ne laser (632.8 nm, 0.3 W/cm(2)), and changes in their firing frequency were recorded. Neuron firing was shown to be very sensitive to photodynamic effect of the studied deuteroporphyrin IX derivatives causing irreversible firing abolition at pikomolar concentrations while Photoheme and Photofrin II were effective in the nanomolar range. The most effective sensitizers were 4-(1-methyl-3-hydroxybutyl)- and 4-(1-methyl-2-acetyl-3-oxobutyl)-deuteroporphyrins. Extinction and amphiphilicity were shown to be the most important properties determining photodynamic efficiency of the studied photosensitizers.

Porphyrins and chlorins containing acetylacetone residues in the peripheral substituents [-CH(Me)CHAc2] are converted under alkaline conditions into the corresponding deacetylated compounds with [-CH(Me)CH2Ac] residues. Reduction of the latter as acids using sodium borohydride gives the corresponding alcohols and, after esterification, their acetates with branched peripheral [-CH(Me)CH2CH(OAc)Me] substituents. Porphyrins and chlorins with such substituents in water-soluble form may hold interest as new photosensitizers in photodynamic cancer therapy.

We have obtained various adducts, including homodimers and heterodimers of ethanebisporphyrins and ethanebischlorins containing pyrrole and dipyrrylmethane insertions, by reaction of mesodimethylaminomethylporphyrins and chlorins with α-unsubstituted pyrrole derivatives in the presence of methyl iodide.

Porphyrins which contain 1-hydroxy(alkoxy)ethyl or alkoxymethyl substituents on the periphery of the macrocycle (i.e. formally able to yield “benzyl type” carbocations) react with nucleophiles (4-tert-butylphenol or the β-diketones acetylacetone, benzoylacetone, dibenzoylmethane, or 4,4,4-trifluoro-1-(2-thienyl)-1,3-butanedione) in the presence of excess zinc acetate. They give high yields of the corresponding addition products, which are the zinc complexes of porphyrins with a 1-(4-tert-butylphenoxy)ethyl substituent or with substituents containing β-diketone residues.

Spectral-luminescent properties and quenching behavior of the covalent conjugate of the platinum(II) complex of coproporphyrin-I and bovine serum albumin (PtCP–BSA) was investigated. Quenching of phosphorescence by sulfite was studied in detail in an acidic range as a function of pH. Strong dynamic quenching of the phosphorescence by sulfite occurred at pH below 4.0 which was attributed to the formation of sulfur dioxide. The quenching reached its maximal level at pH below 1.5, Stern–Volmer quenching constants being as high as 2000 M−1. A new flow-injection system for the determination of sulfite was developed based on the PtCP–BSA conjugate immobilized on a preactivated Biodyne ABC membrane and placed in a flow-cell, with an acidic carrier buffer and fiber-optic phosphorescent detector. Such a solid-state phosphorescent probe is quenchable by sulfite and allowed rapid on-line detection of sulfite with a detection limit of about 10 μM. Performance of the new system and possible interferences were investigated.

New sensor technology and materials for luminescence-based oxygen sensors are described, which utilise microporous light-scattering support in sensor fabrication. Such materials display a number of new features and advantages over conventional oxygen sensor membranes, such as, significantly increased levels of luminescent signals obtainable from the sensor, better mechanical, physical and optical properties and a high capacity for loading biomaterial. The new oxygen sensor technology enables simple and robust procedures of sensor fabrication, as well as saving of the sensing materials, bringing down sensor photobleaching, and improvement in reliability of measurements and signal resolution. Biosensors developed on the basis of the above luminescent oxygen-sensitive materials used as chemical transducers, are described. Direct immobilisation of oxidase enzymes of the oxygen sensor membranes allows simple assays of metabolites. In particular, new batch-type enzyme biosensor for glucose has been investigated, using a new heterogeneous assay format. The latter allows simple, fast and highly reproducible analysis of important metabolites.

5,5′-Ethylenebis(octaethylporphyrin) 1 and its metallocomplexes with various combination of metals (Zn, Cu, and Ni) 2—10 were prepared. On the basis of 1H and 13C NMR as well as MCD spectra, it was concluded that interactions between the two intramolecular porphyrins become attractive when at least one of the two rings is converted to zinc porphyrin. The phenomenon was explained by dipole–dipole interaction arising from the large polarization in Zn complexes.

Protomeric forms of the porphyrin-ketones (or oxo-chlorins) are investigated by optical spectral methods. The main spectral forms of octaethylporphine-ketone and coproporphyrin-ketone tetramethyl ester appearing in organic solvents in the presence of trifluoroacetic acid, in SDS micellar solution and in sulfuric acid, namely, the free base, monocation, dication and trication, are identified and described in detail. Their mutual transformations are shown to proceed differently for the two dyes. The possibility of protonation of the macrocycle is also proven for the porphyrin-ketone complexes. Two new spectral forms of platinum(II) and palladium(II) complexes of the porphyrinketones appearing in organic solvents on addition of TFA are described. Possible mechanisms of protonation, including formation of oxonium ions and carbocations, are discussed for these dyes.