Tuning Electron-Accepting Properties of Phthalocyanines for Charge Transfer Processes.

Phthalocyanines play fundamental roles as electron-acceptors in many different fields; thus, the study of structural features affecting electron-accepting properties of these macrocycles is highly desirable. A series of low-symmetry zinc(II) phthalocyanines, in which one, three, or four benzene rings were replaced for pyrazines, was prepared and decorated with electron-neutral (alkylsulfanyl) or strongly electron-withdrawing (alkylsulfonyl) groups to study the role of the macrocyclic core as well as the effect of peripheral substituents. Electrochemical studies revealed that the first reduction potential (Ered1) is directly proportional to the number of pyrazine units in the macrocycle. Introduction of alkylsulfonyl groups had a very strong effect and resulted in a strongly electron-deficient macrocycle with Ered1 = -0.48 V vs SCE (in THF). The efficiency of intramolecular-charge transfer (ICT) from the peripheral bis(2-methoxyethyl)amine group to the macrocycle was monitored as a decrease in the sum of ΦΔ + ΦF and correlated well with the determined Ered1 values. The strongest quenching by ICT was observed for the most electron-deficient macrocycle. Importantly, an obvious threshold at -1.0 V vs SCE was observed over which no ICT occurs. Disclosed results may substantially help to improve the design of electron-donor systems based on phthalocyanines.

Thermal stabilisation of the short DNA duplexes by acridine-4-carboxamide derivatives.

The short oligodeoxynucleotide (ODN) probes are suitable for good discrimination of point mutations. However, the probes suffer from low melting temperatures. In this work, the strategy of using acridine-4-carboxamide intercalators to improve thermal stabilisation is investigated. The study of large series of acridines revealed that optimal stabilisation is achieved upon decoration of acridine by secondary carboxamide carrying sterically not demanding basic function bound through a two-carbon linker. Two highly active intercalators were attached to short probes (13 or 18 bases; designed as a part of HFE gene) by click chemistry into positions 7 and/or 13 and proved to increase the melting temperate (Tm) of the duplex by almost 8°C for the best combination. The acridines interact with both single- and double-stranded DNAs with substantially preferred interaction for the latter. The study of interaction suggested higher affinity of the acridines toward the GC- than AT-rich sequences. Good discrimination of two point mutations was shown in practical application with HFE gene (wild type, H63D C > G and S65C A > C mutations). Acridine itself can also serve as a fluorophore and also allows discrimination of the fully matched sequences from those with point mutations in probes labelled only with acridine.

Monomerization of Phthalocyanines in Water via Their Supramolecular Interactions with Cucurbiturils.

Aggregation of phthalocyanines (Pcs) represents a problematic feature that decreases the potential of these macrocycles in a number of applications. In this work, we present a supramolecular approach based on the interaction of aminoadamantyl-substituted Pcs with bulky and hydrophilic cucurbit[7]uril (CB[7]) to increase the levels of Pc monomers in water. A series of zinc(II) Pcs substituted at positions α or ß by an aminoadamantyl substituent (with a different level of alkylation of nitrogen) were prepared from the corresponding phthalonitriles. A 1H nuclear magnetic resonance study of the interaction of phthalonitriles with CB[7] in water confirmed the formation of an inclusion complex with an aminoadamantyl moiety with Ka values of ∼1012 M-1. The interaction of CB[7] with Pcs in water substantially weakened H-type aggregation and improved both fluorescence and singlet oxygen production, confirming that this approach is efficient for the monomerization of Pcs. In vitro evaluation of the photodynamic activity of prepared Pcs led to EC50 values in the submicromolar range on HeLa and SK-MEL-28 cells. However, the activity decreased for at least an order of magnitude after host-guest interaction with CB[7] despite better photophysical properties. This was attributed to a much lower uptake by cells due to the very bulky and hydrophilic character of the Pc-CB[7] assembly.

Comparison of Quenching Efficiencies in Long Triple-Labeled and Double-Labeled TaqMan Oligodeoxynucleotide Probes.

Although triple labeling of molecular beacons has been documented to improve quenching efficiencies and studies generally assume similar benefits at long TaqMan probes, a limited number of works have studied this issue in TaqMan probes. We therefore prepared a series of long triple-labeled oligodeoxynucleotide probes with 6-carboxyfluorescein as a fluorophore at the 5'-end and BlackBerry (BBQ-650) or azaphthalocyanine quenchers at the 3'-end and in the intrastrand position and systematically compared their quenching efficiencies with those of the corresponding double-labeled probes including important control probes. A model polymerase chain reaction (PCR) assay enabled the determination of the quenching efficiencies of static and Förster resonance energy transfer (FRET) quenching in the target probes. The type of probe had no effect on the static quenching ability. Importantly, FRET quenching of double-labeled probes with a quencher at the 3'-end showed a statistically insignificant difference from the control probe without any quencher, indicating the need to shift the quencher closer to the fluorophore in long probes. Shortening the distance between the fluorophore and the quencher played a key role in FRET quenching, whereas the introduction of an additional quencher only slightly improved the quenching efficiency. BBQ-labeled probes had lower quenching efficiencies than azaphthalocyanine probes. The methodologies and relationships described above seem, however, to be universal and applicable to any quencher.

Tuning Photodynamic Properties of BODIPY Dyes, Porphyrins' Little Sisters.

The photodynamic properties of a series of non-halogenated, dibrominated and diiodinated BODIPYs with a phthalimido or amino end modification on the phenoxypentyl and phenoxyoctyl linker in the meso position were investigated. Halogen substitution substantially increased the singlet oxygen production based on the heavy atom effect. This increase was accompanied by a higher photodynamic activity against skin melanoma cancer cells SK-MEL-28, with the best compound reaching an EC50 = 0.052 ± 0.01 µM upon light activation. The dark toxicity (toxicity without light activation) of all studied dyes was not detected up to the solubility limit in cell culture medium (10 µM). All studied BODIPY derivatives were predominantly found in adiposomes (lipid droplets) with further lower signals colocalized in either endolysosomal vesicles or the endoplasmic reticulum. A detailed investigation of cell death indicated that the compounds act primarily through the induction of apoptosis. In conclusion, halogenation in the 2,6 position of BODIPY dyes is crucial for the efficient photodynamic activity of these photosensitizers.

[Comparing traditional and commercial molecular biology detection of gastrointestinal pathogens with AusDiagnostics panels in Motol University Hospital].

BACKGROUND: The aim was to do an internal audit of gastrointestinal pathogen detection at the Department of Medical Microbiology, Motol University Hospital between the years 2014 and 2018 and to test two commercial multiplex molecular biology assays potentially improving the diagnostic process and reducing costs. MATERIAL AND METHODS: Based on data from a laboratory information system (LIS), a total of 45,888 samples were identified which had been tested for the presence of gastrointestinal pathogens using culture, immunochromatographic, microscopic and molecular biology techniques between 2014-2018. Novel multiplex molecular biology detection was used to test 182 nucleic acid isolates obtained from stool samples with the Enteric Viruses (8-well) assay (Viral Panel, EVP) or Faecal Pathogens M (16-well) assay (Microbial Panel, FPM) manufactured by AusDiagnostics. RESULTS: The LIS data showed 6.2 % of positive pathogens causing diarrhea from all tested samples (detection rates: 4.5 % for bacterial agents, 21.6 % for viral agents and 0.4 % for parasitic agents). Valid samples (98.9 % of all tested samples) tested by the molecular biology technique yielded, in descending order: C. difficile toxin B (19 %), norovirus (9 %), astrovirus (8 %), Campylobacter (7 %), sapovirus (6 %), Yersinia enterocolitica (6 %), rotavirus (4 %), enterovirus (3 %), Aeromonas (3 %), adenovirus (2 %) and Salmonella (1 %). There was found at least 1 additional new positive detection in 27 % of stools tested by the Viral Panel and in 40 % of stools tested by the Microbial Panel in comparison with the traditional approach. Introducing the panels into routine diagnostic practice will not reduce the costs. CONCLUSIONS: The introduction of novel multiplex molecular biology assays for detecting gastrointestinal pathogens will considerably increase pathogen detection rates even though the costs will be higher for the Department of Medical Microbiology.

Efficient Synthesis of a Wide-Range Absorbing Azaphthalocyanine Dark Quencher and Its Application to Dual-Labeled Oligonucleotide Probes for Quantitative Real-Time Polymerase Chain Reactions.

Unsymmetrical dialkylamino-substituted zinc azaphthalocyanine (AzaPc) exhibits unique spectral and photophysical properties for dark quenchers of fluorescence in DNA hybridization probes. The panchromatic light absorption of AzaPc from 300 nm up to at least 700 nm and its lack of fluorescence make it an ideal candidate for a universal dark quencher. To prove this experimentally, oligodeoxyribonucleotide probes were labeled at the 3'-end by this AzaPc and at the 5'-end by a fluorophore used in the polymerase chain reaction (PCR)-that is, fluorescein, CAL Fluor Red 610, and Cy5. AzaPc showed a significantly higher quenching efficiency compared to the commercially available dark quenchers (BHQ-1, BHQ-2, BBQ-650) in a developed model of TaqMan PCR assay. The AzaPc-labeled probe proved to also be useful in a practical PCR assay for the quantification of the SLCO2B1 transporter gene expression. The constructed calibration curves indicated linearity in the range from 102 to 107 of target copies.

Photodynamic properties of aza-analogues of phthalocyanines.

The replacement of benzene rings in phthalocyanines with various N-heterocycles produces a number of aza-analogues, azaphthalocyanines. This review summarizes their properties important for photodynamic therapy with a focus on (but not limited to) the most studied derivatives, i.e. tetrapyrazinoporphyrazines, tetra(2,3-pyrido)porphyrazines and tetra(3,4-pyrido)porphyrazines. Specifically, the spectral properties in both organic and aqueous solutions are discussed, with an emphasis on the prevention of undesirable aggregation, which typically leads to a loss of the photodynamic effects. Photophysical properties, such as the quantum yield of singlet oxygen production, may provide insights into the potential of azaphthalocyanines to cause cell death, whereas fluorescence quantum yields may refer to their role in cancer visualization. The main part of this review summarizes published results on the in vitro evaluation of these aza-analogues for anticancer, antifungal, and antimicrobial treatments as well as their interactions with biological materials.

An experimental and computational study on isomerically pure, soluble azaphthalocyanines and their complexes and boron azasubphthalocyanines of a varying number of aza units.

Herein, we present a series of isomerically pure, peripherally alkyl substituted, soluble and low aggregating azaphthalocyanines as well as their new, smaller hybrid homologues, azasubphthalocyanines. The focus lies on the effect of the systematically increasing number of aza building blocks [-N[double bond, length as m-dash]] replacing the non-peripheral [-CH[double bond, length as m-dash]] units and their influence on the physical and photophysical properties of these chromophores. The absolute and relative HOMO-LUMO energies of azaphthalocyanines were analyzed using UV-Vis and CV and compared to the density functional theory calculations (B3LYP, TD-DFT). The lowering of the HOMO level is revealed as the determining factor for the trend in the adsorption energies by electronic structure analysis. Crystals of substituted subphthalocyanines, N2-Pc*H2 and N4-[Pc*Zn·H2O], were obtained out of DCM. For the synthesis of the valuable tetramethyltetralin phthalocyanine building block a new highly efficient synthesis involving a nearly quantitative CoII catalyzed aerobic autoxidation step is introduced replacing inefficient KMnO4/pyridine as the oxidant.

OFF-ON-OFF Red-Emitting Fluorescent Indicators for a Narrow pH Window.

A unique combination of two independent mechanisms of fluorescence quenching, namely intramolecular charge transfer (ICT) from a peripheral donor and protonation of azomethine nitrogen atoms in zinc tetrapyrazinoporphyrazines (TPyzPz), provides a new possibility for sensing pH in a specific range. The pH selectivity was controlled by the different basicities of the donor for ICT (dimethylaminoaryl), which was connected to the macrocycle by π-extended linkers of different lengths. ICT and protonation have been studied in detail by photophysical, spectral (UV/Vis and MCD spectra), and electrochemical measurements, and further supported by theoretical calculations (DFT, TDDFT). The pH-sensing properties of the TPyzPzs have been investigated in THF and in water after anchoring the TPyzPzs to liposomes. The salient pKa values were around 1.3 (azomethine nitrogen) and 2.29-4.76 (donor for ICT). The lead indicators (sensing over a pH range of 1.0-2.5) with fairly steep sensing profiles exhibited increases in fluorescence between the OFF/ON states of more than 20-fold and strong absorption in the red region (Q-band maximum >650 nm, ϵ≈2×106 m-1 cm-1 ).

Metal-Cation Recognition in Water by a Tetrapyrazinoporphyrazine-Based Tweezer Receptor.

A series of zinc azaphthalocyanines with two azacrowns in a rigid tweezer arrangement were prepared and the fluorescence sensing properties were investigated. The size-driven recognition of alkali and alkaline earth metal cations was significantly enhanced by the close cooperation of the two azacrown units, in which both donor nitrogen atoms need to be involved in analyte binding to switch the sensor on. The mono- or biphasic character of the binding isotherms, together with the binding stoichiometry and magnitude of association constants (KA ), indicated specific complexation of particular analytes. Water solvation was shown to play an important role and resulted in a strong quenching of sensor fluorescence in the ON state. The lead compound was embedded into silica nanoparticles and advantageous sensing properties towards K(+) were demonstrated in water (λF =671 nm, apparent KA =82 m(-1) , increase of 17×), even in the presence of (supra)physiological concentrations of Na(+) and Ca(2+) .

Phenol-substituted tetrapyrazinoporphyrazines: pH-dependent fluorescence in basic media.

Tetrapyrazinoporphyrazines (TPyzPzs) bearing one, two, four or eight 3,5-di(tert-butyl)-4-hydroxyphenol moieties were synthesized as zinc(II) complexes and metal-free derivatives. The deprotonation of the phenol using tetrabutylammonium hydroxide induced the formation of a strong donor for intramolecular charge transfer that switched OFF the red fluorescence (λF ∼660 nm) of the parent zinc TPyzPzs. The changes were fully reversible for TPyzPzs with one to four phenolic moieties, and an irreversible modification was observed for TPyzPzs substituted with eight phenols. The sensors were anchored to lipophilic particles in water, and a pKa approximately 12.5-12.7 was determined for the phenolic hydroxyl based on fluorescence changes in different buffers. In addition, a novel concept for fluorescence OFF-ON-OFF switching in metal-free TPyzPzs bearing phenolic moieties upon addition of specific amounts of base was demonstrated.

Heteroatom-substituted tetra(3,4-pyrido)porphyrazines: a stride toward near-infrared-absorbing macrocycles.

A synthesis procedure for heteroatom-substituted tetra(3,4-pyrido)porphyrazines that absorb light near 800 nm was developed. Based on the observed relationships between the structure and photophysical parameters, a novel highly photodynamically active (IC50 = 0.26 µM) compound was synthesized and biologically characterized.

Role of steric hindrance in the Newman-Kwart rearrangement and in the synthesis and photophysical properties of arylsulfanyl tetrapyrazinoporphyrazines.

Conditions for the Newman-Kwart rearrangement of phenols into thiophenols were investigated in relation to the bulkiness of substituents at the 2 and 6 positions of the starting phenol derivative with an emphasis on eliminating side reactions. Thiophenols with different 2,6-disubstitution patterns (including hydrogen, methyl, isopropyl or tert-butyl groups) were used for the synthesis of 5,6-bis(arylsulfanyl)pyrazine-2,3-dicarbonitriles that underwent cyclotetramerization leading to the corresponding zinc tetrapyrazinoporphyrazines (TPyzPz), aza-analogues of phthalocyanines. Several methods for the cyclotetramerization were attempted to eliminate problematic side reactions. Magnesium butoxide was found as the most suitable cyclotetramerization agent and afforded TPyzPzs in reasonable yields of approximately 30% under mild conditions. The varying arrangements of the peripheral substitutions resulting from the different bulkiness of the substituents were demonstrated by the X-ray structures of the pyrazine-2,3-dicarbonitriles. The prepared zinc arylsulfanyl TPyzPzs showed an absorption maximum at a Q-band over 650 nm, fluorescence quantum yields between 0.078 and 0.20, and singlet oxygen quantum yields ranging 0.58-0.69. TPyzPzs with isopropyl groups were found to be the best derivatives in this series as they combined facile cyclotetramerization, no aggregation, and good photophysical properties, which makes them potentially suitable for photodynamic therapy.

Structural factors influencing the intramolecular charge transfer and photoinduced electron transfer in tetrapyrazinoporphyrazines.

A series of unsymmetrical tetrapyrazinoporphyrazines (TPyzPzs) from the group of azaphthalocyanines with one peripherally attached amino substituent (donor) were synthesized, and their photophysical properties (fluorescence quantum yield and singlet oxygen quantum yield) were determined. The synthesized TPyzPzs were expected to undergo intramolecular charge transfer (ICT) as the main pathway for deactivating their excited states. Several structural factors were found to play a critical role in ICT efficiency. The substituent in the ortho position to the donor center significantly influences the ICT, with tert-butylsulfanyl and butoxy substituents inducing the strongest ICTs, whereas chloro, methyl, phenyl, and hydrogen substituents in this position reduce the efficiency. The strength of the donor positively influences the ICT efficiency and correlates well with the oxidation potential of the amines used as the substituents on the TPyzPz as follows: n-butylamine < N,N-diethylamine < aniline < phenothiazine. The ICT (with conjugated donors and acceptors) in the TPyzPz also proved to be much stronger than a photoinduced electron transfer in which the donor and the acceptor are connected through an aliphatic linker.

Subphthalocyanines as fluorescence sensors for metal cations.

Subphthalocyanines (SubPcs) and their aza-analogues (SubTPyzPzs) are fluorophores with strong orange fluorescence emission; however, their sensing ability towards metal cations remains uncharted. To fill this gap, we have developed an efficient method for introducing aza-crown moieties at the axial position of SubPcs and SubTPyzPzs to investigate the structure-activity relationship for sensing alkali (Li+, Na+, K+) and alkaline earth metal (Ca2+, Mg2+, Ba2+) cations. SubPcs showed better photostability than SubTPyzPzs and even a commonly utilized dye, 6-carboxyfluorescein. Selectivity toward metal cations was driven by the size of the aza-crown, irrespective of the counter anion. The stoichiometry of binding was found to be 1 : 1 in all cases, and the interaction between SubPcs and cations was characterized by the corresponding apparent binding constants (Ka). Notably, an unusually strong interaction of all sensoric SubPcs with Ba2+ compared to other studied cations was demonstrated. The role of the surrounding environment, i.e. the addition of water or methanol, in sensing cations is shown in detail as well. Selectivity towards K+ over Na+ was demonstrated in aqueous media with SubPcs bearing the 1-aza-6-crown-18 moiety in Tween 80 micelles. In this case, a 5-fold increase of the fluorescence quantum yield was observed upon binding K+ ions. The high brightness, photostability, and sensing activity in aqueous media make SubPc macrocycles promising fluorophores for metal cation sensing.

Photophysical properties of CdSe quantum dot self-assemblies with zinc phthalocyanines and azaphthalocyanines.

The formation of self-assemblies between CdSe quantum dots (QDs) and Zn phthalocyanines (Pc) and azaphthalocyanines (AzaPc) bearing alkylsulfanyl substituents and the photophysical properties of these assemblies were studied using both steady-state and time-resolved luminescence/absorption spectroscopy. The formation of the self-assemblies was accompanied by a blue shift of the Q band of the dyes and by a quenching of the CdSe QDs luminescence. The largest spectral shift of the Q-band was approximately 7 nm and was observed for pentan-3-ylsulfanyl-functionalised phthalocyanine (). Assuming a 1 : 1 stoichiometry, the calculated binding constant was 4 × 10(4) M(-1). Pc substituted with the bulky tert-butylsulfanyl groups (1) exhibited a smaller shift of the Q band. The quenching of the CdSe QDs luminescence by 1 was more effective than that observed for 3. The results indicated that the luminescence quenching may be due to a photoinduced charge transfer between 1 or 3 and the CdSe QDs. In contrast, the AzaPc (2) with the same substituents as 1 had little effect on the QDs luminescence. For all cases, we found an inefficient resonance energy transfer between the attached dyes and the CdSe QD. The formation of the self-assemblies had negligible effects on the photogeneration of the singlet oxygen, O2((1)Δg), that was fully controlled only by the absorption of the light by the macrocycles.

Near-Infrared Probes for Biothiols (Cysteine, Homocysteine, and Glutathione): A Comprehensive Review.

Biothiols (cysteine, homocysteine, and glutathione) are an important class of compounds with a free thiol group. These biothiols plays an important role in several metabolic processes in living bodies when present in optimum concentration. Researchers have developed several probes for the detection and quantification of biothiols that can absorb in UV, visible, and near-infrared (NIR) regions of the electromagnetic spectrum. Among them, NIR organic probes have attracted significant attention due to their application in in vivo and in vitro imaging. In this review, we have summarized probes for these biothiols, which could work in the NIR region, and discussed their sensing mechanism and potential applications. Along with focusing on the pros and cons of the reported probes we have classified them according to the fluorophore used and summarized their photophysical and sensing properties (emission, response time, limit of detection).

Silicon(IV) complexes of octaaryl-substituted porphyrazines and corrolazines: influence of macrocycle contraction on spectral-luminescence, acid-base, and redox properties.

The template cyclomerization of the iminoimide derivatives (obtained by the treatment of diarylfumarodinitriles with NH3 in methanol containing catalytic amounts of dissolved Na) in the presence of SiCl4 in pyridine leads to silicon(IV) octaarylporphyrazine complexes ((HO)2SiPzAr8, Ar = Ph, tBuPh) as a main reaction product. In the case of phenyl-substituted derivative, the formation of a distinctive Si(IV) complex as a byproduct was observed, which according to mass-spectroscopy measurement contains the macrocycle with five diphenylpyrrolic units. The treatment of bishydroxy complexes with tripropylchlorosilane in the presence of magnesium in pyridine leads to the formation of axially siloxylated porphyrazines (Pr3SiO)2SiPzAr8, followed by the reductive contraction of the macrocycle and formation of the corresponding corrolazine complexes (Pr3SiO)SiCzAr8. It is shown that the addition of acid (trifluoroacetic acid, TFA) facilitates the detachment of one siloxy group in (Pr3SiO)2SiPzAr8 and is essential for its Pz → Cz transformation. In the presence of TFA, only one meso-nitrogen is protonated in the porphyrazine complexes (Pr3SiO)2SiPzAr8 (stability constant of the protonated form pKs1 = -0.45 for Ar = Ph; pKs1 = 0.68 for Ar = tBuPh), while two consequent protonation stages are observed for the more basic corrolazine complex (Pr3SiO)SiCzPh8 (pKs1 = 0.93, pKs2 = 0.45). Both types of Si(IV) complexes are poorly fluorescent (ΦF < 0.07). The porphyrazine complexes have low ability to generate singlet oxygen (ΦΔ < 0.15), whereas the corrolazine derivative (Pr3SiO)SiCzPh8 is a very efficient photosensitizer (ΦΔ = 0.76).

Domino-like effect of C112R mutation on ApoE4 aggregation and its reduction by Alzheimer's Disease drug candidate.

BACKGROUND: Apolipoprotein E (ApoE) ε4 genotype is the most prevalent risk factor for late-onset Alzheimer's Disease (AD). Although ApoE4 differs from its non-pathological ApoE3 isoform only by the C112R mutation, the molecular mechanism of its proteinopathy is unknown. METHODS: Here, we reveal the molecular mechanism of ApoE4 aggregation using a combination of experimental and computational techniques, including X-ray crystallography, site-directed mutagenesis, hydrogen-deuterium mass spectrometry (HDX-MS), static light scattering and molecular dynamics simulations. Treatment of ApoE ε3/ε3 and ε4/ε4 cerebral organoids with tramiprosate was used to compare the effect of tramiprosate on ApoE4 aggregation at the cellular level. RESULTS: We found that C112R substitution in ApoE4 induces long-distance (> 15 Å) conformational changes leading to the formation of a V-shaped dimeric unit that is geometrically different and more aggregation-prone than the ApoE3 structure. AD drug candidate tramiprosate and its metabolite 3-sulfopropanoic acid induce ApoE3-like conformational behavior in ApoE4 and reduce its aggregation propensity. Analysis of ApoE ε4/ε4 cerebral organoids treated with tramiprosate revealed its effect on cholesteryl esters, the storage products of excess cholesterol. CONCLUSIONS: Our results connect the ApoE4 structure with its aggregation propensity, providing a new druggable target for neurodegeneration and ageing.