Anti-fibrillogenic properties of phthalocyanines: Effect of the out-of-plane ligands

The axially-coordinated phthalocyanines were previously reported as agents possessing strong anti-fibrillogenic properties. In the presented study we used the atomic force microscopy to investigate the intermediates and the products of insulin aggregation reaction formed in the presence of Zr and Hf phthalocyanine complexes that contain out-of-plane ligands of different size and nature. It is shown that while phthalocyanine-free insulin generated mostly amyloid fibrils with a diameter of 2–8 nm and a length of up to 5 μm, the presence of phthalocyanines with spatial bulky ligands (PcZrDbm₂) leads to the redirection of the fibrillization reaction to the formation of the spherical oligomer aggregates with a diameter of 4–12 nm. At the same time the phthalocyanine complex PcHfCl₂ having the small-volume ligands induces the formation of large size insulin aggregates with a height of about 100 nm that are supposed to be amorphous species. The study of the aggregation intermediates showed the certain similarity of the reaction passing for phthalocyanine-free insulin and insulin in the presence of PcZrDbm₂. The large-size amorphous species were observed at the beginning of reaction, later they dissociated, leading to the formation and growth of the smaller size particles. The amyloid-sensitive cyanine dye 7519 demonstrates the strong fluorescent response both in the presence of fibrils and spherical oligomers, while it is non-sensitive to amorphous aggregates.     

Study of anti-fibrillogenic activity of iron(II) clathrochelates

The macrocyclic compounds mono- and bis-iron(II) clathrochelates were firstly studied as potential anti-fibrillogenic agents using fluorescent inhibitory assay, atomic force microscopy and flow cytometry. It is shown that presence of the clathrochelates leads to the change in kinetics of insulin fibrillization reaction and reduces the amount of formed fibrils (up to 70%). The nature of ribbed substituent could determine the activity of clathrochelates—the higher inhibitory effect is observed for compounds containing carboxybenzenesulfide groups, while the inhibitory properties only slightly depend on the size of complex species. The mono- and bis-clathrochelate derivatives of meta-mercaptobenzoic acid have close values of IC₅₀ namely 16 ± 2 and 24 ± 5 μM, respectively. The presence of clathrochelates decreases the fibril diameter from 5-12 nm for free insulin fibrils to 3–8 nm for these formed in the clathrochelate presence, it also prevents the lateral aggregation of mature fibrils and formation of superfibrillar clusters. However the addition of clathrochelate results in more heterogeneous (both by size and structure) insulin aggregates population as compared to the free insulin. This way, cage complexes—iron(II) clathrochelates are proposed as efficient agents able to suppress the protein aggregation processes.     

Pitfalls associated with the use of Thioflavin-T to monitor anti-fibrillogenic activity

Thioflavin-T (ThT) is a cationic benzothiazole dye that displays enhanced fluorescence upon binding to amyloid fibrils. This property makes ThT the current reagent of choice for the quantification of amyloid fibrils. Herein, we investigate the main pitfalls associated with the use of ThT-based assays to monitor the fibrillation of α-synuclein (α-syn), a protein linked to Parkinson’s disease and other α-synucleinopathies. We demonstrated for the first time that ThT interacts with α-syn disordered monomer and accelerates the protein fibrillation in vitro. As a consequence, misleading conclusions may arise from the use of ThT-based real-time assays in the evaluation of anti-fibrillogenic compounds. Interestingly, NMR experiments indicated that C-terminal domain of α-syn is the main region perturbed by ThT interaction, similarly to that found for the pesticide paraquat, a well-documented accelerator of α-syn fibrillation. Moreover, we demonstrated that certain potent inhibitors of α-syn fibrillation, such as oxidized catecholamines and polyphenols, undergo spontaneous oxidation in aqueous solution, generating compounds that strongly quench ThT fluorescence. In light of these findings, we alert for possible artifacts associated to the measure of the anti-fibrillogenic activity based only on ThT fluorescence approach.     

In vitro antimicrobial activity of light-activated phthalocyanines

Background

Photodynamic antimicrobial therapy (PACT) is proposed as a topical, non-invasive approach suitable for treatment of locally occurring infection. Research of photosensitizers, (PS) as well as their development, is aimed at finding effective antimicrobial substances which would have a broad-spectrum potency. The aim of this paper is to evaluate the antimicrobial effect of phthalocyanine (Pc) derivatives.

Methods

Fifteen different Pc compounds were investigated. Their photokilling activity was tested on Staphylococcus aureus, Escherichia coli and Candida albicans. After treating of microbial cells with Pc at the concentrations: 1 mg/l, 2 mg/l, 4 mg/l, 8 mg/l for 30 minutes, the cultures were irradiated with low-power laser light at a wavelength of 670 nm (20 J/cm², 40 J/cm²). The effectiveness of photoinactivation was evaluated based on the decrease in number (log₁₀) of viable bacteria.

Results

Eight Pc compounds tested showed antibacterial effects against S. aureus, but only four were effective against E. coli and two against C. albicans. The most effective photosensitizers were amphiphilic sulphonated zinc Pc compounds [(3-diethylammonium)-propylsulphonamide citrate (Pc3) and cationic tetramethylenepyridinium chloride of hydroxyaluminum Pc (Pc7)].

Conclusions

The most efficient phthalocyanines (Pc3, Pc7) cause a significant decrease in viable counts of all tested microbes.     

Photosensitizing activity of water- and lipid-soluble phthalocyanines on Escherichia coli

Escherichia coli, as most Gram-negative bacteria, is insensitive to the photosensitizing action of both lipid-soluble Zinc-phthalocyanine (Zn-Pc) and water-soluble Zinc-mono/disulfonated phthalocyanine (Zn-PcS). Photosensitivity can be induced by alteration of the outer membrane, as obtained by either induction of competence or treatment with Tris-EDTA. Both phthalocyanines largely bind at the level of the cytoplasmic membrane; however, Zn-PcS shows a superior photosensitizing activity as compared with Zn-Pc. Biochemical analyses performed on irradiated cells suggest that the cytoplasmic membrane is an important target of the photoprocess, while DNA is not involved.     

Titanium, zirconium and hafnium halide complexes of trithioether and triselenoether ligands

The reaction of TiX₄(X=Cl or Br) with the tripodal ligands MeC(CH₂SMe)₃ or MeC(CH₂SeMe)₃, (L³) in anhydrous n-hexane or CH₂Cl₂ produced the extremely moisture sensitive complexes [TiX₄(L³)]. These were characterised by microanalysis, IR, UV-Vis and variable temperature ¹H,¹³C{¹H} and ⁷⁷Se NMR spectroscopy. The NMR studies showed that in solution in CH₂Cl₂ the complexes contain L³ bound as bidentates, and that pyramidal inversion and exchange between the free and coordinated chalcogen donors is rapid at room temperature. Ligand dissociation/exchange increases TiCl₄<TiBr₄ and MeC(CH₂SMe)₃<MeC(CH₂SeMe)₃ and attempts to isolate TiI₄ analogues were unsuccessful. The reactions of [MCl₄(Me₂S)₂] (M=Zr or Hf) with (L³) in anhydrous CH₂Cl₂ produces white or cream 7-coordinate [MCl₄(L³)], which are insoluble in chlorocarbon solvents. The reactions of TiX₄ (X=Cl, Br or I) with the trithia-macrocycles [9]aneS₃ and [10]aneS₃ produced [TiX₃([n]aneS₃)]X, whilst reaction of TiCl_4, SbCl₅ and [9]aneS₃ in anhydrous CH₂Cl₂ gave [TiCl₃([9]aneS₃)]SbCl₆. Spectroscopic studies suggest these macrocyclic compounds contain 6-coordinate cations, [TiX₃([n]aneS₃)]⁺ (n=9 or 10) but with Zr and Hf the complexes [MCl₄([n]aneS₃)] are 7-coordinate and neutral.     

Synthesis and photodynamic activity of novel asymmetrically substituted fluorinated phthalocyanines

A series of asymmetrically substituted dodecafluorinated phthalocyanines has been synthesized via the Kobayashi ring expansion reaction of the corresponding dodecafluorinated boron subphthalocyanine with differently substituted 1,3-diiminoisoindolines. The mild reaction conditions employed during this ring expansion reaction gave rise exclusively to 3:1 asymmetrically substituted dodecafluorinated phthalocyanines. Metal insertion into the metal-free phthalocyanines was accomplished by heating at 40 degrees C in N,N-dimethylformamide in the presence of zinc bromide. The resulting zinc dodecafluorophthalocyanines were formulated as Cremophor EL oil-water emulsions and evaluated as photosensitizers in vitro against EMT-6 mouse mammary tumor cells. As compared to the previously studied zinc hexadecafluorophthalocyanine, these new asymmetrical zinc dodecafluorophthalocyanines exhibited improved photodynamic activity.     

New antitumor titanocene derivatives containing hydrophilic ligands

Four titanocene derivatives containing hydrophilic ligands were tested for antiproliferative activity against Ehrlich ascites tumor in mice. The new compounds (C₅H₅)₂TiCl(p-SC₆H₄NH₃⁺Cl^?) (I) and (C₅H₅)₂Ti(p-SC₆H₄NH₃⁺Cl^?)₂ (II), containing hydrochlorinated p-aminothiophenolate ligands, and the known compounds (C₅H₅)₂Ti(cis-OOCCHCHCOOH)₂ (III) and (C₅H₅)₂Ti(OOCCCl₃)₂ (IV) containing the carboxylic acid anions hydrogen- maleinate and trichloroacetate as acido ligands, induced maximum cure rates of 100%. The T.I. values amounted to 4.4–4.6 (I), 3.5–4.1 (II), 3.7– 3.8 (III) and 5.5 (IV), and were slightly increased in comparison to (C₅H₅)₂TiCl₂ (T.I. = 3.3). The complexes I–III were rather soluble in water and equally active in a DMSO/saline (1/9, v/v) mixture, in pure saline and in buffered solutions. In the case of IV, the toxicity was considerably low (LD₅₀,440 mg/kg; LD₁₀₀, 500 mg/kg) in relation to (C₅H₅)₂TiCl₂ (LD₅₀, 100 mg/kg; LD₁₀₀, 140 mg/kg).     

Iridium and rhodium complexes containing dichalcogenoimidodiphosphinato ligands

Treatment of [MCl(CO)(PPh₃)₂] with K[N(R₂PQ)₂] afforded [M{N(Ph₂PQ)₂}(CO)(PPh₃)] (M = Ir, Rh; Q = S, Se). The IR C=O stretching frequencies for [M(CO)(PPh₃){N(Ph₂PQ)₂}] were found to decrease in the order S > Se. Treatment of [M(COD)Cl]₂ with K[N(Ph₂PQ)₂] afforded [M(COD){N(Ph₂PQ)₂}] (COD = 1,5-cyclooctadiene; M = Ir, Rh; Q = S, Se). Treatment of [Ir(ol)₂Cl] with afforded (ol = cyclooctene COE, C₂H₄; Q = S, Se). Oxidative addition of [Ir(CO)(PPh₃){N(Ph₂PS)₂}] and [Ir(COD){N(Ph₂PS)₂}] with HCl afforded [Ir(H)(Cl)(CO)(PPh₃){N(Ph₂PS)₂}] and trans-[Ir(H)(Cl)(COD){N(Ph₂PS)₂}], respectively. Oxidative addition of [Ir(CO)(PPh₃){N(Ph₂PS)₂}] with MeI afforded [Ir(Me)(I)(CO)(PPh₃){N(Ph₂PS)₂}]. Treatment of [Ir(COE)₂Cl]₂ with K[N(R₂PO)₂] afforded [Ir(COE)₂{N(Ph₂PO)₂}] that reacted with MeOTf (OTf = triflate) to give [Ir{N(Ph₂PO)₂}(COE)₂(Me)(OTf)]. The crystal structures of [Ir(CO)(PPh₃){N(Ph₂PS)₂}], [M(COD){N(Ph₂PS)₂}] (M = Ir, Rh), (ol = COE, C₂H₄), trans-[Ir(H)(Cl)(COD){N(Ph₂PS)₂}], and [Ir(COE)₂{N(Ph₂PO)₂}] have been determined.     

Dinuclear ruthenium complexes containing tripodal dithiophosphonate ligands

Treatment of [(η⁶-p-cymene)RuCl(μ-Cl)]₂ with Lawesson’s reagent [ArP(S)(μ-S)]₂ (Ar = p-C₆H₄OMe) in the presence of ammonium hydroxide afforded the dinuclear complex [(η⁶-p-cymene)Ru{μ-η¹(S),η²(S,S′)-ArP(O)S₂}]₂ (1) in which the tripodal [ArP(O)S₂]²⁻ ligands bind to the ruthenium atom in both bridging and chelating modes with two non-coordinating PO groups. Interaction of [RuHCl(CO)(PPh₃)₃] with [ArP(S)(μ-S)]₂ and bis(diphenylphosphino)methane (dppm) in the presence of ammonium hydroxide gave the dinuclear complex [Ru(CO){μ₃-η¹(O),η²(S,S′)-ArP(O)S₂}(dppm)]₂ (2) in which the tripodal [ArPOS₂]²⁻ ligands bind the two Ru atoms via both sulfur and oxygen atoms. Treatment of [Ru(PPh₃)₃Cl₂] with [ArP(S)(μ-S)]₂ at reflux in the presence of ammonium hydroxide led to the formation of the dinuclear mixed valence complex [Ru₂Cl₂(μ-S){μ₃-η¹(O),η¹(S),-η²(S,S′)-ArP(O)S₂}(PPh₃)₃] (3), which contains a [Ru^II(PPh₃)₂Cl]⁺ and [Ru^IV(PPh₃)Cl]³⁺ moieties by the tripodal [ArPOS₂]²⁻ ligand in a μ₃-η¹(O),η¹(S),η²(S,S′) coordination mode and the μ-S²⁻ anion. The crystal structures of 1, 2, and 3·CH₂Cl₂ along with their spectroscopic and electrochemical properties are reported.     

Studies of antibacterial activity of binuclear rhodium (II) complexes with heterocyclic nitrogen ligands

Binuclear rhodium (II) complexes, [Rh2(OOCPh)2(phen)2(H2O)2] (OOCPh)2 (1), [Rh2(OOCPh)2(bpy)2(H2O)2] (OOCPh)2 (2), [Rh2(OOCBu(n))2 (bpy)2(H2O)2] (OOCBu(n)2 (3), and [Rh2(OOCPr(n)2 (phen)2(H2O)2] (OOCPr(n)2 (4) (Phen = 1,10-phenanthroline and bpy = 2,2'-bipyridine), have been synthesized and characterized using NMR, IR and electronic spectra. Activity of these compounds against Gram-positive bacteria decreases in the order: 1?2?3 > 4. Complex 1 is active against many Staphylococcus strains resistant to commonly used antibiotics. The complexes 1-4 are much less active agents against Gram-negative bacteria.     

Hydrophobicity, topography in membranes and photosensitization of silicon phthalocyanines with axial ligands of varying lengths.

Six amphiphilic silicon phthalocyanines (SiPc's) axially linked to a dimethylated amino alkyl group of varying length have been examined for their potential suitability as photosensitizers for photodynamic therapy (PDT). This group of molecules was chosen because the length of the axial ligand might place the chromophoric part of the molecule at different vertical depths in the membrane and possibly affect the extent of membrane localized damage caused by singlet oxygen. We tested the relative penetration depth of the SiPc groups in the membrane by the extent to which their fluorescence was quenched by external iodide ions. We also measured singlet oxygen quantum yields of the SiPc's in a liposome membrane, using the fluorescent target for singlet oxygen, 9,10-dimethylanthracene. The hydrophobicity parameters, LogP, were calculated and were also measured. Some correlation was found between them and Kb's, the binding constants for liposomes. The effect of the axial ligand's length is less striking than in similar cases with hematoporphyrins and protoporphyrins. We link this smaller effect with a bending of the linker chain that enables, sterically, a better positioning of the sensitizer molecules within the ordered lipid layer structure.     

Hybrid scorpionate/cyclopentadienyl titanium and zirconium complexes with alkoxide and imido ligands

The reactivity of hybrid scorpionate/cyclopentadienyl ligand-containing trichloride zirconium complexes [ZrCl₃(bpzcp)] (1) [bpzcp = 2,2-bis(3,5-dimethylpyrazol-1-yl)-1,1-diphenylethylcyclopentadienyl] and [ZrCl₃(bpztcp)] (2) [bpztcp = 2,2-bis(3,5-dimethylpyrazol-1-yl)-1-tert-butylethylcyclopentadienyl] toward several lithium alkoxides has been carried out. Thus, alkoxide-containing complexes [ZrCl₂(OR)(bpzcp)] (R = Me, 3; Et, 4; ⁱPr, 5; (R)-2-Bu, 6), [ZrCl₂(OR)(bpztcp)] (R = Me, 7; Et, 8; ⁱPr, 9; (R)-2-Bu, 10) and [Zr(OR)₃(bpztcp)] (R = Et, 11; ⁱPr, 12) were prepared by deprotonation of the appropriate alcohol group with BuⁿLi followed by reaction with 1 or 2. In addition, the imido-complex [Ti(N^tBu)Cl(bpztcp)(py)] (13) were also prepared. The structures of these complexes have been proposed on basis of spectroscopic and DFT methods.     

Organo-Group 15 derivatives of triosmium clusters containing naphthyl ligands

Reactions of the clusters Os₃(μ-H)₂(μ₃-1-OC₁₀H₆)(CO)₉, 1, and Os₃(μ-H)(μ-2-OC₁₀H₇)(CO)₁₀, 2, with the group 15 ligands EPh₃ (E=P, As, Sb) generally afforded the mono- and, or disubstituted derivatives. These derivatives tend to decompose during chromatographic separations on silica gel; thus one of the decomposition products from the reaction of 1 with PPh₃ has been identified as Os(H)₂(CO)₂(PPh₃)₂, 3. The molecular structures of 3, as well as the derivatives Os₃(μ-H)₂(μ₃-1-OC₁₀H₆)(CO)₈(AsPh₃), 4, Os₃(μ-H)₂(μ₃-1-OC₁₀H₆)(CO)₇(SbPh₃)₂, 5c, Os₃(μ-H)(μ-2-OC₁₀H₇)(CO)₈(AsPh₃)₂, 7b, and Os₃(μ-H)(μ-2-OC₁₀H₇)(CO)₈(SbPh₃)₂, 7c, have been determined by single crystal X-ray diffraction studies.     

Some coordination compounds of titanium and zirconium halides with tertiary phosphines and related ligands

Various new adducts of TiCl₄, (PhO)₂TiCl₂, TiCl₃ and ZrCl₄ with phosphine and amine ligands are reported including, (PhO)₂TiCl₂·2L (or L′) (L = PMe₃, PPh₃; L′ = dmpe, dbpe, tmed); MCl₄·L (M = Ti, Zr; L = dbpe): TiCl₃·L (L = dmpe, dbpe, tmed): TiCl₃·tmed·THF and ZrCl₄·1.5tmed. The solution properties of some of the TiCl₄ adducts are discussed, as deduced from ³¹P NMR spectra.     

Studies on synthesis, characterization, and G-quadruplex binding of Ru(II) complexes containing two dppz ligands

In this work, the interaction between the guanine-rich single-strand oligomer AG₃(T₂AG₃)₃ quadruplex and two Ru(II) complexes, [Ru(L¹)(dppz)₂](PF₆)₄ (1) and [Ru(L²)(dppz)₂](PF₆)₄ (2) (L¹ = 5,5′-di(1-(trimethylammonio)methyl)-2,2′-dipyridyl cation, L² = 5,5′-di(1-(triethylammonio)methyl)-2,2′-dipyridyl cation, dppz = dipyrido[3,2-a:2′,3′-c] phenazine), has been studied by UV-Visible, fluorescence, DNA melting, and circular dichroism in K⁺ buffer. The two complexes after binding to G-quadruplex have shown different DNA stability and fluorescence enhancement. The results show that both complexes can induce the stabilization of quadruplex DNA. ΔT_m values of complexes 1 and 2 at [Ru]/[DNA] ratio of 1:1 were 9.4 and 7.0, respectively. Binding stoichiometry along with the quadruplex was investigated through a luminescence-based Job plot. The major inflection points for complexes 1 and 2 were 0.49 and 0.46, respectively. The data were consistent with the binding mode at a [quadruplex]/[complex] ratio of 1:1. In addition, the conformation of G-quadruplex was not changed by the complexes at the high ionic strength of K⁺ buffer.     

Synthesis of multifunctional polyvinylsaccharide containing controllable amounts of biospecific ligands

In the present study, the attempt to synthesize a multibiofunctional polymeric vector to be used for construction of composite scaffolds for bone tissue engineering has been undertaken. The polymers based on 2-deoxy-2-methacrylamido- d-glucose were functionalized by a growth factor (BMP-2), GRGDSP peptide, and poly( l-lysine) using aldehyde chemistry. The covalent modification process was quantitatively studied, and a polymer conjugate containing all these ligands was formed. In addition, the impacts of coupled ligands toward the adsorption of polymers on the commercial mineral macroporous matrix Sponceram used in cell culture applications were studied.