Synthesis and In Vitro Photodynamic Activities of an Integrin‐Targeting cRGD‐Conjugated Zinc(II) Phthalocyanine

A 1,4‐disubstituted zinc(II) phthalocyanine conjugated with a cyclic Arg‐Gly‐Asp‐D ‐Phe‐Lys (cRGDfK) moiety through a triazole linker was prepared and characterized by UV/Vis spectroscopy and high‐resolution ESI‐MS. The conjugate showed a relatively weak fluorescence emission in N,N‐dimethylformamide (Φ_F=0.08), but it was a very efficient singlet oxygen generator (Φ_Δ=0.80) as a result of the di‐α‐substituted structure. Owing to the presence of the cyclic peptide sequence cRGDfK, which is a well‐known α_vβ₃‐integrin antagonist, this conjugate exhibited significantly higher cellular uptake toward the α_vβ₃⁺ U87‐MG cells compared with the α_vβ₃^? MCF‐7 cells, as determined by flow cytometry and fluorescence microscopy. The photocytotoxicity of this compound against these two cell lines, however, was comparable owing to the similar efficiency of intracellular reactive oxygen species generation. Confocal microscopic studies also revealed that this conjugate localized preferentially in the lysosomes, but not in the nucleus, endoplasmic reticulum, and mitochondria of the U87‐MG cells.     

A phthalocyanine-peptide conjugate with high in vitro photodynamic activity and enhanced in vivo tumor-retention property

A novel zinc(II) phthalocyanine conjugated with a short peptide with a nuclear localization sequence, Gly-Gly-Pro-Lys-Lys-Lys-Arg-Lys-Val, was synthesized by click chemistry and a standard Fmoc solid-phase peptide synthesis protocol. The conjugate was purified by HPLC and characterized with UV/Vis and high-resolution mass spectroscopic methods. Both this compound and its non-peptide-conjugated analogue are essentially non-aggregated in N,N-dimethylformamide and can generate singlet oxygen effectively with quantum yields (Φ(Δ)) of 0.84 and 0.81, respectively, relative to unsubstituted zinc(II) phthalocyanine (Φ(Δ) =0.56). Conjugation of the peptide sequence, however, can enhance the cellular uptake, efficiency in generating intracellular reactive oxygen species, and photocytotoxicity of the phthalocyanine-based photosensitizer against HT29 human colorectal carcinoma cells. The IC(50) value of the conjugate is as low as 0.21 μM. In addition, the conjugate shows an enhanced tumor-retention property in tumor-bearing nude mice. After 72 h post-injection, the dye concentration in the tumor was significantly higher than that in other organs. The results suggest that this phthalocyanine-peptide conjugate is a highly promising photosensitizer for photodynamic therapy.     

Synthesis,Supramolecular Behavior,and In Vitro Photodynamic Activities of Novel Zinc(II) Phthalocyanines “Side‐strapped” with Crown Ether Bridges

Two new tetra‐ or di‐α‐substituted zinc(II) phthalocyanines 5 and 6 have been prepared through a “side‐strapped” method. In the molecules, the adjacent benzene rings of the phthalocyanine core are linked at α‐position through a triethylene glycol bridge to form a hybrid aza‐/oxa‐crown ether. The tetra‐α‐substituted phthalocyanine 5 shows an eclipsed self‐assembly property in CH₂Cl₂ and the effect on the di‐α‐substituted analogue 6 is significantly weakened. Furthermore, the crown ethers of these compounds can selectively complex with Fe³⁺ or Cu²⁺ ion in DMF, leading to formation of J‐aggregated nano‐assemblies, which can be disaggregated in the presence of some organic or inorganic ligands, such as triethylamine, tetramethylethylenediamine, CH₃COO^?, or OH^?. In addition, both compounds are efficient singlet oxygen generators with the singlet oxygen quantum yields (Φ_Δ) of 0.54‐0.74 in DMF relative to unsubstituted zinc(II) phthalocyanine (Φ_Δ=0.56). They exhibit photodynamic activities toward HepG2 human hepatocarcinoma cells, but the compound 6 , which has more than 40‐fold lower IC₅₀ value (0.08 μM ) compared to the analogue 5 (IC₅₀=3.31 μM ), shows remarkablely higher in vitro photocytotoxicity due to its significantly higher cellular uptake and singlet oxygen generation efficiency. The results suggest that these compounds can serve as promising multifunctional materials both in (opto)electronic field and photodynamic therapy.     

Photodynamic Action Mechanism Mediated by Zinc(II) 2,9,16,23‐Tetrakis[4‐(N‐methylpyridyloxy)]phthalocyanine in Candida albicans Cells

The photoreaction type I/type II pathways mediated by zinc(II) 2,9,16,23‐tetrakis[4‐(N‐methylpyridyloxy)]phthalocyanine (ZnPPc⁴⁺) was studied in Candida albicans cells. This photosensitizer was strongly bound to C. albicans cells at short times. After 30 min irradiation, 5 μM ZnPPc⁴⁺ produced ~5 log decrease in cell viability. Different probes were used to detect reactive oxygen species (ROS) in cell suspensions (~10⁶ CFU mL^?1). Singlet molecular oxygen, O₂(¹Δ_g), was observed by the reaction with 9,10‐dimethylanthracene (DMA) and tetrasodium 2,2‐(anthracene‐9,10‐diyl)bis(methylmalonate) (ABMM), whereas the nitro blue tetrazolium (NBT) method was used to sense superoxide anion radical (). Moreover, the effects produced by an anoxic atmosphere and cell suspensions in D₂O, as well as the addition of sodium azide and mannitol as ROS trapping were evaluated in the PDI of C. albicans. These investigation indicates that O₂(¹Δ_g) is generated in the cells, although a minor extension other radical species can also be involved in the PDI of C. albicans mediated by ZnPPc⁴⁺.     

A novel lutetium(III) acetate phthalocyanine directly substituted with N,N’-dimethylaminophenyl groups via CC bonds and its water-soluble derivative for photodynamic therapy

In this study, the novel 4-(N,N′-dimethylamino)phenyl substituted lutetium(III) acetate phthalocyanine (2) and its quaternized derivative (3) were synthesized via a Suzuki-Miyaura coupling reaction between tetrakis(iodo) lutetium(III)acetate phthalocyanine (1) and 4-(N,N-dimethylamino)phenylboronic acid, and subsequent quaternization using dimethyl sulfate, respectively. The obtained phthalocyanine 3 exhibited excellent solubility in water which is important for photodynamic therapy applications. Photophysical properties such as fluorescence quantum yield and fluorescence lifetime, and photochemical properties such as singlet oxygen generation and photostability were investigated to determine their suitability for photodynamic therapy. The lutetium(III) phthalocyanines, especially quaternized derivative 3, showed promising properties as potential photosensitizers for the treatment of cancer, producing higher singlet oxygen (Φ_Δ = 0.59) than motexafin lutetium (Φ_Δ = 0.31) which is a clinically used lutetium texaphyrin photosensitizer.     

Synthesis and photochemical characteristics of novel tribenzoporphyrazines possessing peripherally annulated tetrahydrodiazepine and diazepine rings

Novel tribenzoporphyrazines possessing peripherally annulated tetrahydrodiazepine and diazepine rings were synthesized and characterized, and the substituent effects on their absorption spectra in various solvents and on singlet oxygen generation were studied. Solvatochromic effects of tribenzoporphyrazines dissolved in a range of protic and aprotic solvents were evaluated by monitoring the changes in the UV-Vis spectra. The correlation between the Q-band shift towards longer wavelengths and the refractive index of the solvent indicated that the solvatochromic effects are mainly a result of solvation rather than coordination processes. The potential photosensitizing activity of novel tribenzoporphyrazines was evaluated by measuring the ability of singlet oxygen production, which is the result of the interaction between an activated photosensitizer and oxygen. This experiment proves promising photosensitizing activity of novel styryldiazepinotribenzoporphyrazine, which is an efficient singlet oxygen generator with a Φ_Δ value of 0.44, although this value is a little lower than that of zinc phthalocyanine.     

Synthesis, characterization and comparative studies on the photophysical and photochemical properties of metal-free and zinc(II) phthalocyanines with phenyloxyacetic acid functionalities

The synthesis and characterization of new peripherally and non-peripherally tetra-substituted metal-free and zinc(II) phthalocyanines with 2-, 3- and 4-phenyloxyacetic acid functionalities are described for the first time in this study. The new compounds have been characterized by elemental analysis, FT-IR, UV-Vis, MALDI-TOF and ¹H-NMR spectra. Photodegradation, singlet oxygen and fluorescence quantum yields, and fluorescence lifetimes of these compounds are studied in dimethylformamide (DMF). The influence of the substituent position on the phthalocyanine framework (non-peripherally or peripherally), central metal ion (metal-free or zinc) and the position of the COOH group (2-, 3- or 4-position on the phenyloxyacetic acid) on the spectroscopic, photophysical and photochemical properties have been investigated. Non-peripherally zinc(II) phthalocyanines (1b and 2b) and peripherally zinc(II) phthalocyanine (4b) gave good singlet oxygen quantum yields (Φ_Δ) (0.37, 0.39 and 0.38, respectively) which indicate the potential of the complexes as photosensitizers in applications of PDT.     

A Panchromatic Supramolecular Fullerene‐Based Donor–Acceptor Assembly Derived from a Peripherally Substituted Bodipy–Zinc Phthalocyanine Dyad

A panchromatic 4,4‐difluoro‐4‐bora‐3a,4a‐diaza‐s‐indacene –zinc phthalocyanine conjugate (Bodipy–ZnPc) 1 was synthesized starting from phthalocyanine aldehyde 4 , via dipyrromethane 3 and dipyrromethene 2 . Conjugate 1 represents the first example in which a Bodipy unit is tethered to the peripheral position of a phthalocyanine core. Electrochemical and optical measurements provided evidence for strong electronic interactions between the Bodipy and ZnPc constituents in the ground state of 1 . When conjugate 1 is subjected to photoexcitation in the spectral region corresponding to the Bodipy absorption, the strong fluorescence characteristic of the latter subunit is effectively quenched (i.e., ≥97 %). Excitation spectral analysis confirmed that the photoexcited Bodipy and the tethered ZnPc subunits interact and that intraconjugate singlet energy transfer occurs with an efficiency of ca. 25 %. Treatment of conjugate 1 with N‐pyridylfulleropyrrolidine ( 8 ), an electron‐acceptor system containing a nitrogen ligand, gives rise to the novel electron donor–acceptor hybrid 1 ? 8 through ligation to the ZnPc center. Irradiation of the resulting supramolecular ensemble within the visible range leads to a charge‐separated Bodipy–ZnPc^.+–C₆₀^.? radical‐ion‐pair state, through a sequence of excited‐state and charge transfers, characterized by a remarkably long lifetime of 39.9 ns in toluene.     

Effect of the Media on the Quantum Yield of Singlet Oxygen (O2(1Δg)) Production by 9H‐Fluoren‐9‐one: Microheterogeneous Systems

The quantum yield (Φ_Δ) of singlet oxygen (O₂(¹Δ_g) production by 9H‐fluoren‐9‐one (FLU) is very sensitive to the nature of the solvent (0.02 in a highly polar and protic solvent, such as MeOH, to 1.0 in apolar solvents). This high sensitivity has been used for probing the interaction of FLU with micellar media and microemulsions based on anionic (sodium dodecyl sulfate, SDS; bis‐(2‐ethylhexyl)sodium sulfosuccinate, AOT), cationic (cetyltrimethylammonium chloride, CTAC) and nonionic (Triton X‐100, TX) surfactants. Values of Φ_Δ of FLU vary in a wide range (0.05–1.0) in both microheterogeneous media and neat solvent, and provide information on the microenvironment of FLU, i.e., on its localization within organized media. In ionic and nonionic micellar media, as well as in four‐component microemulsions, FLU is, to various extents, exposed to solvation by the polar and protic components of the microheterogeneous systems (water and/or butan‐1‐ol) in the micellar interfacial region (Φ_Δ=0.05–0.30). In contrast, in AOT reverse micelles (consisting of AOT as surfactant, cyclohexane as hydrophobic component, and water), FLU is located in the hydrophobic continuous pseudophase, and is totally separated from the micellar water pools (Φ_Δ≈1.0).     

Effect of the Media on the Quantum Yield of Singlet Oxygen (O2(1Δg)) Production by 9H‐Fluoren‐9‐one: Solvents and Solvent Mixtures

We have investigated the effect of a series of 18 solvents and mixtures of solvents on the production of singlet molecular oxygen (O₂(¹Δ_g), denoted as ¹O₂) by 9H‐fluoren‐9‐one (FLU). The normalized empirical parameter E derived from E_T(30) has been chosen as a measure of solvent polarity using Reichardt's betaine dyes. Quantum yields of ¹O₂ production (Φ_Δ) decrease with increasing solvent polarity and protic character as a consequence of the decrease of the quantum yield of intersystem crossing (Φ_ISC). Values of Φ_Δ of unity have been found in alkanes. In nonprotic solvents of increasing polarity, Φ_ISC and, therefore, Φ_Δ decrease due to solvent‐induced changes in the energy levels of singlet and triplet excited states of FLU. This compound is a poor ¹O₂ sensitizer in protic solvents, because hydrogen bonding considerably increases the rate of internal conversion from the singlet excited state, thus diminishing Φ_Δ to values much lower than those in nonprotic solvents of similar polarity. In mixtures of cyclohexane and alcohols, preferential solvation of FLU by the protic solvent leads to a fast decrease of Φ_Δ upon addition of increasing amounts of the latter.     

Peptide‐Coated Platinum Nanoparticles with Selective Toxicity against Liver Cancer Cells

Peptide‐stabilized platinum nanoparticles (PtNPs) were developed that have significantly greater toxicity against hepatic cancer cells (HepG2) than against other cancer cells and non‐cancerous liver cells. The peptide H‐Lys‐Pro‐Gly‐d Lys‐NH₂ was identified by a combinatorial screening and further optimized to enable the formation of water‐soluble, monodisperse PtNPs with average diameters of 2.5 nm that are stable for years. In comparison to cisplatin, the peptide‐coated PtNPs are not only more toxic against hepatic cancer cells but have a significantly higher tumor cell selectivity. Cell viability and uptake studies revealed that high cellular uptake and an oxidative environment are key for the selective cytotoxicity of the peptide‐coated PtNPs.     

Oligolysine‐Conjugated Zinc(II) Phthalocyanines as Efficient Photosensitizers for Antimicrobial Photodynamic Therapy

A series of zinc(II) phthalocyanines conjugated with an oligolysine chain (n=2, 4, and 8) were synthesized and characterized by using various spectroscopic methods. As shown by using UV/Vis and fluorescence spectroscopic methods, these compounds were nonaggregated in N,N‐dimethylformamide, and gave a weak fluorescence emission and high singlet oxygen quantum yield (Φ_Δ=0.86–0.89) as a result of their di‐α‐substitution. They became slightly aggregated in water with 0.05 % Cremophor EL, but they could still generate singlet oxygen effectively. The antimicrobial photodynamic activities of these compounds were then examined against various bacterial strains, including the Gram‐positive methicillin‐sensitive Staphylococcus aureus ATCC 25923 and methicillin‐resistant Staphylococcus aureus ATCC BAA‐43, and the Gram‐negative Escherichia coli ATCC 35218 and Pseudomonas aeruginosa ATCC 27853. Generally, the dyes were much more potent toward the Gram‐positive bacteria. Only 15 to 90 nM of these photosensitizers was required to induce a 4 log reduction in the cell viability of the strains. For Escherichia coli, the photocytotoxicity increased with the length of the oligolysine chain. The octalysine derivative showed the highest potency with a 4 log reduction concentration of 0.8 μM . Pseudomonas aeruginosa was most resistant to the photodynamic treatment. The potency of the tetralysine derivative toward a series of clinical strains of Staphylococcus aureus was also examined and found to be comparable with that toward the nonclinical counterparts. Moreover, the efficacy of these compounds in photodynamic inactivation of viruses was also examined. They were highly photocytotoxic against the enveloped viruses influenza A virus (H1N1) and herpes simplex virus type 1 (HSV1), but exhibited no significant cytotoxicity against the nonenveloped viruses adenovirus type 3 (Ad3) or coxsackievirus (Cox B1). The octalysine derivative also showed the highest potency with an IC₅₀ value of 0.05 nM for the two enveloped viruses.     

Oxygen‐Dependent Photochemistry and Photophysics of “MiniSOG,” a Protein‐Encased Flavin

Selected photochemical and photophysical parameters of flavin mononucleotide (FMN) have been examined under conditions in which FMN is (1) solvated in a buffered aqueous solution, and (2) encased in a protein likewise solvated in a buffered aqueous solution. The latter was achieved using the so‐called “mini Singlet Oxygen Generator” (miniSOG), an FMN‐containing flavoprotein engineered from Arabidopsis thaliana phototropin 2. Although FMN is a reasonably good singlet oxygen photosensitizer in bulk water (?_Δ = 0.65 ± 0.04), enclosing FMN in this protein facilitates photoinitiated electron‐transfer reactions (Type‐I chemistry) at the expense of photosensitized singlet oxygen production (Type‐II chemistry) and results in a comparatively poor yield of singlet oxygen (?_Δ = 0.030 ± 0.002). This observation on the effect of the local environment surrounding FMN is supported by a host of spectroscopic and chemical trapping experiments. The results of this study not only elucidate the behavior of miniSOG but also provide useful information for the further development of well‐characterized chromophores suitable for use as intracellular sensitizers in mechanistic studies of reactive oxygen species.     

Spontaneous Single‐Crystal‐to‐Single‐Crystal Evolution of Two Cross‐Laminated Polymers

Two cases of spontaneous evolution of monomers to linear polymers having novel cross‐laminated topology are reported. We synthesized two peptide monomers N₃‐Gly‐Gly‐NH‐CH₂‐CCH and N₃‐Gly‐Gly‐Gly‐CH₂‐CCH and solved their crystal structures by single‐crystal X‐ray diffraction. They adopt H‐bonded crisscrossed layered packing in their crystals such that: (a) the monomers are aligned head‐to‐tail in 1D‐chain‐like arrays and parallel arrangement of such arrays forms a layer; (b) the proximally placed azide and alkyne motifs are in an orientation apt for their regiospecific cycloaddition; (c) each monomer having x peptide bonds is H‐bonded with 2x monomers disposed in intersecting arrangement, which pre‐organize 1D‐chain‐like arrays in adjacent layers in perpendicular orientation. These crystals underwent spontaneous single‐crystal‐to‐single‐crystal (SCSC) polymerization via azide–alkyne cycloaddition reaction to form triazolyl‐polyglycines, at room temperature. The crisscrossed arrangement of monomers in adjacent layers ensured the formation of cross‐laminated polymers.     

Photophysics of Zinc (II) Phthalocyanine Polymer and Gel Formulation

The photophysical properties of lipophilic phthalocyanines encapsulated into a polymer and two different gels were studied in order to predict their photosensitizing efficacy in vivo. Photophysical techniques for solid phase were adapted for light dispersing samples. Gel formulation of two tetrasubstituted phthalocyanines, tetra-t-butylphthalocyaninato zinc(II) ( 1 ), tetrakis(1,1-dimethyl-2-phthalimido)ethylphtalocyaninatozinc(II) ( 2 ) and two octasubstituted phthalocyanines, 2,3,9,10,16,17,23,24-octakis(decyloxy)phthalocyaninatozinc(II) ( 3 ) and 2,3,9,10,16,17,23,24-octakis[(N,N-dimethylamino)ethylsulfanyl]phthalocyaninatozinc(II) ( 4 ) were investigated for their possible use in photodynamic therapy for topical purposes. Supporting the fact that gel formulation improves the photophysical properties of phthalocyanines, singlet molecular oxygen quantum yield (Φ_Δ) values for 1 – 4 zinc(II) phthalocyaninates in Lutrol^® F 127-Cremophor^® RH 40 were 0.60, 0.60, 0.20 and 0.26, respectively. Permeation studies showed that no release of phthalocyanines occurs, thus indicating there should be no risk of generalized skin photosensitivity in areas other than the dye-deposition site.     

A Glutathione‐Activated Phthalocyanine‐Based Photosensitizer for Photodynamic Therapy

A zinc(II) phthalocyanine substituted with a 2,4‐dinitrobenzenesulfonate group has been prepared. Its fluorescence emission and reactive oxygen species generation can be greatly enhanced by glutathione in phosphate‐buffered saline and inside MCF‐7 cells. This compound thus functions as a highly efficient molecular‐based activatable photosensitizer.     

Effects of pluronic silica nanoparticles on the photophysical and photodynamic therapy behavior of triphenyl-p-phenoxy benzoic acid metalloporphyrins

5, 10, 15, Triphenyl-20-p-phenoxy benzoic acid porphyrins (P) containing Zn (ZnP), Ga (GaP), and Si (SiP) were synthesized and conjugated to pluronic-silica (PluS) nanoparticles (NPs) where the fluorescence and singlet oxygen generating behavior of the porphyrins were investigated. The highest singlet oxygen quantum yield (Φ_Δ) was obtained for ZnP. When the porphyrins were conjugated to the PluS NPs, the Φ_Δ was quenched and fluorescence was enhanced. The pore size of the NPs upon conjugation decreased from 18.9 nm for PluS NPs to 2.4 nm (for ZnP as an example) as determined by applying the Brunauer–Emmett–Teller method. The porphyrin complexes and their conjugates were tested for their photodynamic therapy (PDT) activity on MCF-7 breast cancer cells. It was found that ZnP and its conjugate showed the highest PDT activity. The p > 0.05 indicated that ZnP is significantly different than GaP and SiP.     

The achiral tetrapeptide Z‐Aib‐Aib‐Aib‐Gly‐OtBu

The title achiral peptide N‐benzyloxycarbonyl‐α‐aminoisobutyryl‐α‐aminoisobutyryl‐α‐aminoisobutyrylglycine tert‐butyl ester or Z‐Aib‐Aib‐Aib‐Gly‐OtBu (Aib is α‐aminoisobutyric acid, Z is benzyloxycarbonyl, Gly is glycine and OtBu indicates the tert‐butyl ester), C₂₆H₄₀N₄O₇, is partly hydrated (0.075H₂O) and has two different conformations which together constitute the asymmetric unit. Both molecules form incipient 3₁₀‐helices. They differ in the relative orientation of the N‐terminal protection group and at the C‐terminus. There are two 4→1 intramolecular hydrogen bonds.     

A Novel Stable High‐Nitrogen Energetic Compound: Copper(II) 1,2–Diaminopropane Azide

The multi‐ligand coordination compound copper(II) 1,2‐diaminopropane (pn) azide, [Cu(pn)(N₃)₂]_n ( 1 ), was synthesized using pn and azido groups. It was characterized by X‐ray single crystal diffraction, elemental analysis, and FT‐IR spectroscopy. The crystal structure of 1 belongs to the monoclinic system, space group C2/c. The copper(II) cation is six‐coordinated by one pn molecule and four azido ligands with μ‐1 and μ‐1,1,3 coordination modes. Thermogravimetric investigations with a heating rate of 10 K · min^–1 under nitrogen showed one main exothermic stage with a peak temperature of 215.7 °C in the DSC curve. The non‐isothermal kinetics parameters were calculated by Kissinger and Ozawa methods, respectively. The heat of combustion was measured by oxygen bomb calorimetry, and the enthalpy of formation, the critical temperature of thermal explosion, the entropy of activation (ΔS^≠), the enthalpy of activation (ΔH^≠), and the free energy of activation (ΔG^≠) were calculated. The measurements showed that 1 has very high impact, friction, and flame sensitivities.     

Crystallographic report: A coordination polymer containing [Zn(NO3)(H2O)2(btp)2]+ units bridged by btp ligands (btp = 2,6‐bis(N′‐1,2,4‐triazolyl)pyridine)

A zinc(II) coordination polymer has been formed from Zn(NO₃)₂ and 2,6‐bis(N′‐1,2,4‐triazolyl)pyridine (btp) ligands in which each zinc(II) atom is coordinated by three nitrogen donor atoms from btp and three oxygen donor atoms from a nitrate and two water molecules. Copyright © 2003 John Wiley & Sons, Ltd.