Porphyrin Binding Research Articles

Porphyrin Induced Multimerization of Solution-State Proteins

Interactions between charged porphyrins and complimentary or similarly charged proteins provide important models systems for studies of electron transfer processes, artificial photosynthesis, and control of protein-protein interactions. Typically, the experimental results are analyzed and discussed assuming that the proteins exist in a monodisperse state. Here, we explored interaction of four solution-state proteins (horse heart cytochrome c, hen egg-white lysozyme, 3-heme c-type cytochrome PpcA from Geobacter sulfurreducens, 2-heme cyt c4 from Pseudomonas stutzeri) with several cationic and anionic water-soluble derivatives of tetraphenylporphyrin. Combined small- and wide-angle X-ray scattering experiments revealed formation of multimers with a wide range of complex sizes. Thermodynamic interaction parameters and complex binding stoichiometries were established with isothermal calorimetry. Locations of porphyrin binding sites were determined with heteronuclear single quantum coherence (HSQC) and total correlation spectroscopy (TOCSY) NMRs for PpcA and cytochrome c, while covalent labeling shielding experiments followed by LC-MS analysis of tryptic digests were used to map ligand binding sites on cyt c4 and lysozyme surfaces. The obtained results demonstrate that multimerization of solution-state proteins by large water-soluble ligands appears to be a wide-spread phenomenon controlled by a delicate interplay of electrostatic and hydrophobic forces. Molecular level mapping of the binding sites allows us to build a theory explaining the size of the formed complexes and provides opportunities for targeted design and assembly of multi-subunit protein complexes.

Tryptophan-Rich Sensory Protein/Translocator Protein (TSPO) from Cyanobacterium Fremyella diplosiphon Binds a Broad Range of Functionally Relevant Tetrapyrroles

Tryptophan-rich sensory protein/translocator protein (TSPO) is a membrane protein involved in stress adaptation in the cyanobacterium Fremyella diplosiphon. Characterized mammalian and proteobacterial TSPO homologues bind tetrapyrroles and cholesterol ligands. We investigated the ligand binding properties of TSPO from F. diplosiphon (FdTSPO1), which was functionally characterized in prior genetic studies. Two additional TSPO proteins (FdTSPO2 and FdTSPO3) are present in F. diplosiphon; they are similar in size to reported bacterial TSPOs and smaller than FdTSPO1. The longer cyanobacterial TSPO1 is found almost exclusively in filamentous cyanobacteria and has a relatively low degree of homology to bacterial and mammalian TSPO homologues with confirmed tetrapyrrole binding. To probe distinctions of long-form TSPOs, we tested the binding of porphyrin and bilin to FdTSPO1 and measured binding affinities in the low micromolar range, with the highest binding affinity detected for heme. Although tetrapyrrole ligands bound FdTSPO1 with affinities similar to those previously reported for proteobacterial TSPO, binding of cholesterol to FdTSPO1 was particularly poor and was not improved by introducing an amino acid motif known to enhance cholesterol binding in other bacterial TSPO homologues. Additionally, we detected limited binding of bacterial hopanoids to FdTSPO1. Cyanobacterial TSPO1 from the oxygenic photosynthetic F. diplosiphon, thus, binds a range of tetrapyrroles of functional relevance with efficiencies similar to those of mammalian and proteobacterial homologues, but the level of cholesterol binding is greatly reduced compared to that of mammalian TSPO. Furthermore, the ΔFdTSPO1 mutant exhibits altered growth in the presence of biliverdin compared to that of wild-type cells under green light. Together, these results suggest that TSPO molecules may play roles in bilin homeostasis or trafficking in cyanobacteria.

Interaction of a tricationic meso-substituted porphyrin with guanine-containing polyribonucleotides of various structures

The interaction of a tricationic water-soluble meso-(N-methylpyridinium)-substituted porphyrin, TMPyP3+, derived from classic TMPyP4, with double-stranded poly(G) ⋅ poly(C) and four-stranded poly(G) polyribonucleotides has been studied in aqueous buffered solutions, pH 6.9, of low and near-physiological ionic strengths in a wide range of molar phosphate-to-dye ratios (P/D). To clarify the binding modes of TMPyP3+ to biopolymers various spectroscopic techniques, including absorption and polarized fluorescence spectroscopy, Raman spectroscopy, and resonance light scattering, were used.As a result, two competitive binding modes were revealed. In solution of low ionic strength outside binding of the porphyrin to the polynucleotide backbone with self-stacking prevailed at low P/D ratios (P/D < 3.5). It manifested itself by the substantial quenching of porphyrin fluorescence. Also the formation of large-scale porphyrin aggregates was observed near the stoichiometric binding ratio. The spectral changes observed at P/D > 30 including emission enhancement were supposed to be caused by the embedding of partially stacked porphyrin J-dimers into the polymer groove. TMPyP3+ binding to poly(G) induced a fluorescence increase 2.5 times as large as that observed for poly(G) ⋅ poly(C). In solution of near-physiological ionic strength the efficiency of external porphyrin binding was reduced substantially due to the competitive binding of Na+ ions with the polymer backbone. The spectroscopic characteristics of porphyrin bound to polynucleotides at different conditions were compared with those for free porphyrin.

Interaction of Mesotetrakis (2,6,dimethoxyphenol) Porphyrin with AuTiO2 Nanoparticles: A Spectroscopic Approach.

The combination of nanoparticles with the photosensitizing molecules will assist in developing new approach for their biological applications. In this paper work, we have studied the interaction of photosensitising mesotetrakis (2,6,dimethoxyphenol) porphyrin molecule (P1) with AuTiO2 nanoparticles using absorption, fluorescence and time resolved measurements. An isosbestic point is appeared in the absorption spectrum of P1 on increasing the concentration of AuTiO2 nanoparticles indicates the interaction of P1 with AuTiO2 nanoparticles. Static type of quenching is observed in the fluorescence quenching measurement which is confirmed through lifetime measurements. Energy level calculations and Rehm Weller methods confirms the electron transfer mechanism from the excited P1 to the AuTiO2 nanoparticles. The observed effective binding and electron transfer property of porphyrin with AuTiO2 nanoparticles has great potential to be applied in the field of photodynamic therapy.

Multimerization of Solution-State Proteins by Water Soluble Porphyrins

Abstract: Interactions between charged porphyrins and complimentary or similarly charged proteins provide important models systems for studies of electron transfer processes, artificial photosynthesis, and control of protein-protein interactions. Typically, the experimental results are analyzed and discussed assuming that the proteins exist in a monodisperse state. Here, we explored interaction of four solution-state proteins (horse heart cytochrome c, hen egg-white lysozyme, 3-heme c-type cytochrome PpcA from Geobacter sulfurreducens, 2-heme cyt c4 from Pseudomonas stutzeri) with several cationic and anionic water-soluble derivatives of tetraphenylporphyrin. Combined small- and wide-angle X-ray scattering experiments revealed quick formation of mutlimers with a wide range of complex sizes. Thermodynamic interaction parameters and complex binding stoichiometries were established with isothermal calorimetry. Locations of porphyrin binding sites were determined with heteronuclear NMR and chemical modification shielding experiments. The obtained results demonstrate that multimerization of solution-state proteins by large water-soluble ligands appears to be a wide-spread phenomenon controlled by a delicate interplay of electrostatic and hydrophobic forces. Molecular level mapping of the binding sites allows us to build a theory explaining the size of the formed complexes and provides opportunities for targeted design and assembly of multi-subunit protein complexes.

Porphyrin Binding to Gun4 Protein, Facilitated by a Flexible Loop, Controls Metabolite Flow through the Chlorophyll Biosynthetic Pathway

In oxygenic phototrophs, chlorophylls, hemes, and bilins are synthesized by a common branched pathway. Given the phototoxic nature of tetrapyrroles, this pathway must be tightly regulated, and an important regulatory role is attributed to magnesium chelatase enzyme at the branching between the heme and chlorophyll pathway. Gun4 is a porphyrin-binding protein known to stimulate in vitro the magnesium chelatase activity, but how the Gun4-porphyrin complex acts in the cell was unknown. To address this issue, we first performed simulations to determine the porphyrin-docking mechanism to the cyanobacterial Gun4 structure. After correcting crystallographic loop contacts, we determined the binding site for magnesium protoporphyrin IX. Molecular modeling revealed that the orientation of α6/α7 loop is critical for the binding, and the magnesium ion held within the porphyrin is coordinated by Asn-211 residue. We also identified the basis for stronger binding in the Gun4-1 variant and for weaker binding in the W192A mutant. The W192A-Gun4 was further characterized in magnesium chelatase assay showing that tight porphyrin binding in Gun4 facilitates its interaction with the magnesium chelatase ChlH subunit. Finally, we introduced the W192A mutation into cells and show that the Gun4-porphyrin complex is important for the accumulation of ChlH and for channeling metabolites into the chlorophyll biosynthetic pathway.

Binding of Metallated Porphyrin-Imidazophenazine Conjugate to Tetramolecular Quadruplex Formed by Poly(G): a Spectroscopic Investigation.

The binding of telomerase inhibitor ZnTMPyP(3+)-ImPzn, Zn(II) derivative of tricationic porphyrin-imidazophenazine conjugate, to tetramolecular quadruplex structure formed by poly(G) was studied in aqueous solutions at neutral pH and near physiological ionic strength using absorption and polarized fluorescent spectroscopy techniques. Three binding modes were determined from the dependences of the fluorescence intensity and polarization degree for the porphyrin and phenazine moieties of the conjugate on molar polymer-to-dye ratio (P/D). The first one is outside electrostatic binding of positively charged porphyrin fragments to anionic phosphate groups of the polymer which prevails only at very low P/D values and manifests itself by substantial fluorescence quenching. It is suggested that the formation of externally bound porphyrin dimers occurs. The other two binding modes observed at high P/D are embedding of the ZnTMPyP(3+) moiety into the groove of poly(G) quadruplex accompanied by more than 3-fold enhancement of the conjugate emission, and simultaneous intercalation of the phenazine fragment between the guanine bases accompanied by the increase of its fluorescence polarization degree up to 0.25. Thus Zn(II) conjugate seems to be promising ligand for the stabilization of G-quadruplex structures since porphyrin binding to poly(G) is strengthened by additional intercalation of phenazine moiety.

Molecular docking and dynamics simulations on the interaction of cationic porphyrin–anthraquinone hybrids with DNA G-quadruplexes

A series of cationic porphyrin–anthraquinone hybrids bearing either pyridine, imidazole, or pyrazole rings at the meso-positions have been investigated for their interaction with DNA G-quadruplexes by employing molecular docking and molecular dynamics simulations. Three types of DNA G-quadruplexes were utilized, which comprise parallel, antiparallel, and mixed hybrid topologies. The porphyrin hybrids have a preference to bind with parallel and mixed hybrid structures compared to the antiparallel structure. This preference arises from the end stacking of porphyrin moiety following G-stem and loop binding of anthraquinone tail, which is not found in the antiparallel due to the presence of diagonal and lateral loops that crowd the G-quartet. The binding to the antiparallel, instead, occurred with poorer affinity through both the loop and wide groove. All sites of porphyrin binding were confirmed by 6 ns molecular dynamics simulation, as well as by the negative value of the total binding free energies that were calculated using the MMPBSA method. Free energy analysis shows that the favorable contribution came from the electrostatic term, which supposedly originated from the interaction of either cationic pyridinium, pyrazole, or imidazole groups and the anionic phosphate backbone, and also from the van der Waals energy, which primarily contributed through end stacking interaction.

Amino acid-linked porphyrin-nitroimidazole antibiotics targeting Porphyromonas gingivalis.

The periodontal pathogen Porphyromonas gingivalis requires porphyrin supplementation for growth. Previously, in order to inhibit P. gingivalis growth, we synthesised very effective 'Trojan horse' ester and amide-linked deuterporphyrin-nitroimidazole (DPIX-Nim) adducts that exploited this requirement to transport metronidazole-derived antibiotics with excellent antimicrobial selectivity and recognition by the HA2 porphyrin binding site. Herein, in the context of developing topical agents to target P. gingivalis, l-amino acids are incorporated into adducts as linkers to improve uptake. Ten 13- and 17-propionic amide regioisomers of l-amino acid-linked deuterporphyrin-nitroimidazole adducts were synthesised using a peptide coupling approach. DPIX-Lys regioisomers without attached nitroimidazole were also synthesised as comparison compounds. All the porphyrin adducts bound (Kd50 7 to 20 nM) to a recombinant HA2 receptor with similar binding affinity to haem, except the lysine-proline linked DPIX-Lys(Boc)Pro-Nim adducts (Kd50 300 nM) and the DPIX-Lys(Nim)-Nim adducts (Kd50 200 nM), both of which have large appended groups. DPIX-Lys(Boc)-Nim, DPIX-Lys(OH)-Nim, and DPIX-Pro-Nim adducts were shown to be very effective against P. gingivalis. DPIX-Lys(Boc)Pro-Nim adducts and DPIX-Lys(Nim)-Nim adducts showed weak activity. Importantly, DPIX-Lys(Boc)-Nim adducts were selective for P. gingivalis and, unlike metronidazole, did not kill a range of other anaerobic bacteria isolated from the human gastrointestinal tract.

Internal versus external binding of cationic porphyrins to single-stranded DNA.

Absorbance, induced circular dichroism, and emission studies establish that the tetrasubstituted cationic porphyrin Cu(T4) preferentially binds externally to single-stranded (ss) DNA sequences, except in a purine-rich system like 5'-(dA)10-3' where a degree of internalization occurs. On the other hand, the sterically friendly, disubstituted Cu(tD4) system exclusively binds to ss DNA by internalization, that is, pseudointercalation. By and large the results show that double-stranded DNA hosts decisively outcompete more flexible ss hosts for the uptake of a porphyrin, regardless of the binding motif. The findings are relevant because ss domains of DNA appear during replication, in different types of DNA-secondary structure, and as products of the disassembly of multistranded forms.

Functionalized porphyrin derivatives for solar energy conversion

In this review, we discuss a collection of three sets of porphyrin-based systems recently synthesized in our group. In the first set we improve the light harvesting ability of porphyrins by preparing arrays in which strongly absorbing boron dipyrrin (BDP) chromophores are combined with porphyrins, either by covalent attachment to the periphery of the porphyrin ring or by axial coordination to a tin(IV) porphyrin. We observe that BDP increases significantly the light absorption capability of porphyrins by efficient BDP to porphyrin excitation energy transfer. Further functionalization of the arrays with redox active groups such as phenolate ligands or fullerenes gives rise to interesting electron transfer dynamics in the excited state. In the second set, we use porphyrins as sensitizers in photocatalytic hydrogen production schemes with cobaloxime catalysts either in supramolecular or diffusion controlled systems. Photocatalytic experiments, detailed spectroscopic studies on our systems and recent literature results show that attachment of a photosensitizer to a catalyst is in fact detrimental to H2 production. Diffusion controlled systems show considerably increased photocatalytic activity mainly because deleterious effects such as back electron transfer are avoided. In the final set, porphyrins are used as light harvesting dyes in dye-sensitized solar cells (DSSCs). Aiming to extend the light absorption range of the sensitizing dyes, to achieve enhanced solar cell performances, appropriately functionalized porphyrins with carboxylic acid anchoring groups are synthesized. These include porphyrins with π-conjugated groups at meso positions, “push–pull” porphyrins, porphyrins with bridges between the macrocycle and the anchoring group, and covalently linked arrays of porphyrins and other chromophores. Porphyrins with pyridyl anchoring groups are also utilized, in order to get insight into the effect of the nature and strength of the porphyrin binding on the solar cell efficiency. Devices with photovoltaic efficiencies higher than the average values of solar cells sensitized by simple porphyrin and other hybrid-porphyrin derivatives are obtained, particularly after reduction of dye-aggregation by means of chemical co-adsorbents, and by using co-sensitization, modified photoanodes, molten electrolytes and electrolyte additives.

Anticancer activity of cationic porphyrins in melanoma tumour-bearing mice and mechanistic in vitro studies

BackgroundPorphyrin TMPyP4 (P4) and its C14H28-alkyl derivative (C14) are G-quadruplex binders and singlet oxygen (1O2) generators. In contrast, TMPyP2 (P2) produces 1O2 but it is not a G-quadruplex binder. As their photosensitizing activity is currently undefined, we report in this study their efficacy against a melanoma skin tumour and describe an in vitro mechanistic study which gives insights into their anticancer activity.MethodsUptake and antiproliferative activity of photoactivated P2, P4 and C14 have been investigated in murine melanoma B78-H1 cells by FACS, clonogenic and migration assays. Apoptosis was investigated by PARP-1 cleavage and annexin-propidium iodide assays. Biodistribution and in vivo anticancer activity were tested in melanoma tumour-bearing mice. Porphyrin binding and photocleavage of G-rich mRNA regions were investigated by electrophoresis and RT-PCR. Porphyrin effect on ERK pathway was explored by Western blots.ResultsThanks to its higher lipophylicity C14 was taken up by murine melanoma B78-H1 cells up to 30-fold more efficiently than P4. When photoactivated (7.2 J/cm2) in B78-H1 melanoma cells, P4 and C14, but not control P2, caused a strong inhibition of metabolic activity, clonogenic growth and cell migration. Biodistribution studies on melanoma tumour-bearing mice showed that P4 and C14 localize in the tumour. Upon irradiation (660 nm, 193 J/cm2), P4 and C14 retarded tumour growth and increased the median survival time of the treated mice by ~50% (P <0.01 by ANOVA), whereas porphyrin P2 did not. The light-dependent mechanism mediated by P4 and C14 is likely due to the binding to and photocleavage of G-rich quadruplex-forming sequences within the 5′-untranslated regions of the mitogenic ras genes. This causes a decrease of RAS protein and inhibition of downstream ERK pathway, which stimulates proliferation. Annexin V/propidium iodide and PARP-1 cleavage assays showed that the porphyrins arrested tumour growth by apoptosis and necrosis. C14 also showed an intrinsic light-independent anticancer activity, as recently reported for G4-RNA binders.ConclusionsPorphyrins P4 and C14 impair the clonogenic growth and migration of B78-H1 melanoma cells and inhibit melanoma tumour growth in vivo. Evidence is provided that C14 acts through light-dependent (mRNA photocleavage) and light-independent (translation inhibition) mechanisms.

Formation and thermodynamic stability of (polymer + porphyrin) supramolecular structures in aqueous solutions

Optical properties of porphyrins can be tuned through (polymer+porphyrin) (host+guest) binding in solution. This gives rise to the formation of supramolecular structures. In this paper, the formation, thermodynamic stability and spectroscopic properties of (polymer+porphyrin) supramolecular structures and their competition with porphyrin self-association were investigated by both isothermal titration calorimetry (ITC) and absorption spectroscopy. Specifically, reaction enthalpies and equilibrium constants were measured for meso-tetrakis(4-sulfonatophenyl) porphyrin (TPPS) self-association and TPPS binding to the polymer poly(vinylpyrrolidone) (PVP, 40kg/mol) in aqueous solutions at pH 7 and three different temperatures (12, 25 and 37°C). ITC, compared to spectroscopic techniques, provides two independent means to determine reaction enthalpies: direct measurements and Van’t Hoff plot. This was used as a criterion to assess that (1) self-association of TPPS is limited to the formation of dimers and (2) TPPS binds to PVP in its monomeric state only. The formation of TPPS dimers and (PVP+TPPS) supramolecular structures are both enthalpically driven. However, (polymer+porphyrin) binding was found to be entropically favored compared to dimerization. Furthermore, the reaction enthalpies of these two processes significantly depend on temperature. This behavior was attributed to hydrophobic interactions. Finally, the limiting absorption spectra of monomeric, dimeric and polymer-bound states of TPPS were extracted from our spectroscopic measurements combined with the thermodynamic parameters obtained by ITC. The observed spectral shifts indicate that the two hydrogens in the central porphyrin are involved in (PVP+TPPS) binding. This work provides valuable information on thermodynamic stability of (polymer+porphyrin) supramolecular nanostructures and the general understanding of complex competing associative processes in solution.

Conformations of DNA in the presence of nanomole concentrations of Co+2 ions and mezo-tetra(4-N-oxyethylpiridil) porphyrin

Conformation changes of calf thymus DNA induced by nanomole concentrations of Co+2 and TOEPyP4 and influence of this ion on porphyrin binding mode with DNA duplex were studied at two NaCl concentrations (10 and 100 mM) using circular dichroism spectroscopy and absorption data. It was shown that addition of 85 nM Co+2 into TOEPyP4/DNAb.p. = .015 solution caused an immediate change of the intercalative binding mode of porphyrin to external binding modes at low ionic strengths (10 mM NaCl). Increase of Co+2 concentration from 165 to 650 nM caused a cooperative transition of DNA from B form to C-like conformation. Similar changes in binding mode and DNA conformation were observed at 100 mM NaCl, but they took place at higher concentrations of Co+2 ions. Incubation of TOEPyP4/DNAb.p. = .015 in the presence of nanomole concentrations of Co+2 ions at 36.6 °C showed a transition of DNA-B form into Z-like conformation. Maximal percent of Z-like DNA was observed when the incubation time was from 6 to 8 h. Longer incubation times of [DNA-TOEPyP4]+Co+2 complexes caused a considerable recovery of CD spectra in UV region and a strong decrease in absorption at 260 nm. Here, we discuss some mechanisms of transition from B-DNA to C- and Z-like conformations.

DNA Binding of Porphyrin Conjugates: Characteristics and Consequences

Recently cationic porphyrin-peptide conjugates were synthesized to enhance cellular uptake of porphyrins and to deliver peptide moieties to the close vicinity of nucleic acids.The aim of our work was to characterize the binding of porphyrin-peptide conjugates to nucleic acids and nucleoprotein complexes (NP) and describe structural changes induced by the interaction.Porphyrin-peptide conjugates were synthesized, linking tetra-peptides to tris(N-methylpyridyl)carboxyphenyl-porphyrin (TMPCP) and bis(methylpyridyl)carboxyphenyl-porphyrin (BMPCP). These structures can be considered as a typical monomer unit corresponding to the branches of porphyrin-branched chain polypeptide conjugates. DNA was isolated from T7 bacteriophage; as nucleoprotein complex the complete phage particle was used (NP).Binding modes were studied by comprehensive spectroscopic methods such as absorption spectroscopy, CD spectroscopy and fluorescence lifetime measurements. To look for possible structural changes of NP and DNA caused by the porphyrin binding CD spectroscopy was used and the thermal stability of DNA/NP was detected by optical melting method.Our results show that the interaction of porphyrin conjugates with DNA can be either intercalative or external binding. The dominant factor in the interaction with isolated DNA proved to be the charge distribution, with binding affinities higher for tri-cationic molecules than their di-cationic counterparts. Presence of protein capsid opposes but does not inhibit DNA-porphyrin interaction. In the case of NP, binding affinity of conjugates decreases and the mode of interaction is mostly external binding.CD spectra recorded in 200-300 nm range revealed hypochromicity in the DNA bands and alteration of protein bands in the presence of conjugates that can be attributed to the distortion of helical structure and loosening of capsid structure, respectively. Absorption melting showed that the porphyrin binding stabilizes the helical structure, i.e. increases the strand separation temperature of DNA, but does not influence DNA - protein interaction in NP.

Synthesis and photophysical characterization of porphyrin and porphyrin–Ru(ii) polypyridyl chromophore–catalyst assemblies on mesoporous metal oxides

A layer-by-layer procedure has been used to prepare chromophore–catalyst assemblies consisting of phosphonate-derivatized porphyrin chromophores and a phosphonate-derivatized Ru(II) water oxidation catalyst on the surfaces of SnO2 and TiO2 mesoporous, nanoparticle films. In the procedure, initial surface binding of the phosphonate-derivatized porphyrin is followed in sequence by reaction with ZrOCl2 and then with the phosphonate-derivatized water oxidation catalyst [RuII(2,6-bis-(1-methylbenzimidazole-2-yl)pyridine)(2,2′-bipyridine-4,4′-hydroxymethylphosphonate)(H2O)]2+, [RuII(Mebimpy)(4,4′-(PO(OH)2–CH2)2-bpy)(OH2)]2+. Fluorescence from both the free base and Zn(II) porphyrin derivatives on SnO2 is quenched; substantial emission quenching of the Zn(II) porphyrin occurs on TiO2. Transient absorption difference spectra provide direct evidence for appearance of the porphyrin radical cation on SnO2via excited-state electron injection. For the chromophore–catalyst assembly on SnO2, transient absorption difference spectra demonstrate rapid intra-assembly electron transfer oxidation of the catalyst following excitation and injection by the porphyrin chromophore.

The effect of pyridyl substituents on the thermodynamics of porphyrin binding to G-quadruplex DNA

Most of the G-quadruplex interactive molecules reported to date contain extended aromatic flat ring systems and are believed to bind principally by π–π stacking on the end G-tetrads of the quadruplex structure. One such molecule, TMPyP4, (5,10,15,20-tetra(N-methyl-4-pyridyl)porphyrin), exhibits high affinity and some selectivity for G-quadruplex DNA over duplex DNA. Although not a realistic drug candidate, TMPyP4 is used in many nucleic acid research laboratories as a model ligand for the study of small molecule G-quadruplex interactions. Here we report on the synthesis and G-quadruplex interactions of four new cationic porphyrin ligands having only 1, 2, or 3 (N-methyl-4-pyridyl) substituents. The four new ligands are: P(5) (5-(N-methyl-4-pyridyl)porphyrin), P(5,10) (5,10-di(N-methyl-4-pyridyl)porphyrin), P(5,15) (5,15-di(N-methyl-4-pyridyl)porphyrin), and P(5,10,15) (5,10,15-tri(N-methyl-4-pyridyl)porphyrin). Even though these compounds have been previously synthesized, we report alternative synthetic routes that are more efficient and that result in higher yields. We have used ITC, CD, and ESI-MS to explore the effects of the number of N-methyl-4-pyridyl substituents and the substituent position on the porphyrin on the G-quadruplex binding energetics. The relative affinities for binding these ligands to the WT Bcl-2 promoter sequence G-quadruplex are: KTMPyP4≈KP(5,15)>KP(5,10,15)>>>KP(5,10), KP(5). The saturation stoichiometry is 2:1 for both P(5,15) and P(5,10,15), while neither P(5) nor P(5,10) exhibit significant complex formation with the WT Bcl-2 promoter sequence G-quadruplex. Additionally, binding of P(5,15) appears to interact by an ‘intercalation mode’ while P(5,10,15) appears to interact by an ‘end-stacking mode’.

Regulation of dynamic structure of cyclophanes by their complexation with the porphyrin

Dithia[3.3]metacyclophanes which consist of the pyridine unit connecting to the different positions of the parent cyclophane skeleton have been prepared. Conformational change has been observed for the cyclophane having a 4-substituted pyridine unit by binding to the porphyrin. In contrast the porphyrin binding has no influence on conformational behavior of the cyclophane having a 3-substituted pyridine unit.

Unusual Stepwise Protonation and J‐Aggregation of meso‐Tetrakis(N‐methylpyridinium‐4‐yl)porphine on Binding Poly(sodium vinylsulfonate)

The binding of a tetracationic porphyrin to a highly charged polymer like poly(sodium vinylsulfonate) has been investigated over a wide pH range and under various experimental conditions. We present evidence that, depending on the pH, the high electrostatic field exerted by the polymer stabilizes the diprotonated form of the free base porphyrin at unusual pH values or otherwise causes the formation of H-type aggregates. In particular, at a low polymer concentration, lowering the pH at first allows the formation of the diacid species then it determines its reorganization in close-packed J-type aggregates. The employment of various metallo-derivatives of the title porphyrin enables a better insight into the nature of all the detected species.

Calculation of cooperativity and equilibrium constants of ligands binding to G-quadruplex DNA in solution

Equilibrium model of a ligand binding with DNA oligomer has been considered as a process of small molecule adsorption onto a lattice of multiple binding sites. An experimental example has been used to verify the assertion that during saturation of the macromolecule by a ligand should expect effect of cooperativity due to changes in DNA conformation or the mutual influence between bound ligands. Such phenomenon cannot be entirely described by the classical stepwise complex formation model. To evaluate a ligand binding affinity and cooperativity of ligand–oligomer complex formation the statistical approach has been proposed. This new computational approach used to re-examine previously studded ligand binding towards DNA quadruplexes targets with multiple binding sites. The intrinsic equilibrium constants K1−3 of the mesotetrakis-(N-methyl-4-pyridyl)-porphyrin (TMPyP4) binding with the [d(T4G4)]4 and with the [AG3(T2AG3)3] quadruplexes and the correction for the mutual influence between bound ligands (cooperativity parameters ω) was determined from the Job plots based upon the nonlinear least-squares fitting procedure. The re-examination of experimental curves reveals that the equilibrium is affected by the positive cooperative (ω>1) binding of the TMPyP4 ligand with tetramolecular [d(T4G4)]4. However for an intramolecular antiparallel–parallel hybrid structure [AG3(T2AG3)3] the weak anti-cooperativity of TMPyP4 accommodation (ω<1) onto two from three nonidentical sites was detected.