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.     

Transient tropoelastin nanoparticles are early-stage intermediates in the coacervation of human tropoelastin whose aggregation is facilitated by heparan sulfate and heparin decasaccharides

Tropoelastin assembly is a key step in the formation of elastin. We consider how nanoscale intracellular assemblies of tropoelastin can congregate in an extracellular environment to give microscale aggregates. We describe novel 200–300 nm spherical particles that serve as intermediates in the formation of the coacervate. Their aggregation gives 800 nm to 1 µm species. This process is facilitated by heparan sulfate and dermatan sulfate interactions which effectively lower the critical concentration to facilitate this transition. This coacervation process was examined using a panel of heparin chains of various lengths and showed greatest efficacy for the decasaccharide, followed by the octasaccharide, while the hexasaccharide displayed the shortest efficacious length. We propose that these oligosaccharide interactions enable the charge-mediated aggregation of positively charged tropoelastin. This biochemistry models glycosaminoglycan interactions on the cell surface during elastogenesis which is characterized by the clustering of nascent tropoelastin aggregates to form micron-sized spherules.     

The impact of binding of macrocyclic metal complexes on amyloid fibrillization of insulin and lysozyme

Amyloid fibrils are insoluble protein aggregates whose accumulation in cells and tissues is connected with a range of pathological diseases. We studied the impact of 2 metal complexes (axially coordinated Hf phthalocyanine and iron (II) clathrochelate) on aggregation of insulin and lysozyme. For both proteins, the host‐guest interaction with these compounds changes the kinetics of fibrillization and affects the morphology of final aggregates. The Hf phthalocyanine is a very efficient inhibitor of insulin fibrillization; in its presence, only very low amounts of fibrils with the diameters of 0.8 to 5 nm and spherical aggregates were found. Effective concentration of fibrillization inhibition (IC₅₀) was estimated to be 0.11 ± 0.04 μM. The clathrochelate induced the formation of thin fibrils with the diameters of 0.8 to 2.5 nm; IC₅₀ was estimated as 20 ± 9 μM. The lysozyme fibrillization remained quite intensive in the presence of the studied compounds; they induced the formation of long filaments (the length up to 2.5 μm, the diameters of 1.5‐3.5 nm). These fibrils noticeably differed from those of free lysozyme short linear species (the diameters of 3‐5 nm, the length up to 0.6 μm). Thinning and elongation of fibrils suggest that the metal complexes bind mainly to the grooves of protofilaments; this hinders the stacking of early aggregates or protofilaments together but does not hinder their growth. The image of the fibril separated into 2 protofilaments allows suggesting that the fibril formation occurs via the growth of the parallel protofilaments with their subsequent twisting in the fibril. The changes of the lysozyme intrinsic fluorescence indicate that both metal complexes interact with the protein during the stage of the fibrillar seeds formation.     

Studies of anti-fibrillogenic activity of phthalocyanines of zirconium containing out-of-plane ligands

Series of phthalocyanines of zirconium containing lysine, citric, nonanoic acid residues and dibenzolylmethane groups as out-of-plane ligands are firstly studied as inhibitors of fibrillogenesis using cyanine-based fluorescent inhibitory assay. It was shown that studied phthalocyanines at concentration of 20μM inhibited aggregation reaction on 38.5-57.6% and inhibitory activity of phthalocyanines depended on the chemical nature of out-of-plane ligand. For the most active compound PcZrLys(2) (zirconium phthalocyanine containing lysine fragment) the efficient inhibitor concentration was estimated to be 37μM. AFM studies have shown that in the presence of PcZrLys(2) the inhibition of fibrils formation and formation of spherical oligomeric aggregates took place. Due to the ability of phthalocyanines to decrease efficiently protein aggregation into the amyloid fibrils, modification of phthalocyanine molecules via out-of-plane substitutions was proposed as approach for design of anti-fibrillogenic agents with required properties.     

Green synthesis of silver nanoparticles using Andean blackberry fruit extract

Green synthesis of nanoparticles using various plant materials opens a new scope for the phytochemist and discourages the use of toxic chemicals. In this article, we report an eco-friendly and low-cost method for the synthesis of silver nanoparticles (AgNPs) using Andean blackberry fruit extracts as both a reducing and capping agent. The green synthesized AgNPs were characterized by various analytical instruments like UV–visible, transmission electron microscopy (TEM), dynamic light scattering (DLS), X-ray diffraction (XRD) and Fourier transform infrared (FTIR) spectroscopy. The formation of AgNPs was analyzed by UV–vis spectroscopy at λ_max = 435 nm. TEM analysis of AgNPs showed the formation of a crystalline, spherical shape and 12–50 nm size, whereas XRD peaks at 38.04°, 44.06°, 64.34° and 77.17° confirmed the crystalline nature of AgNPs. FTIR analysis was done to identify the functional groups responsible for the synthesis of the AgNPs. Furthermore, it was found that the AgNPs showed good antioxidant efficacy (>78%, 0.1 mM) against 1,1-diphenyl-2-picrylhydrazyl. The process of synthesis is environmentally compatible and the synthesized AgNPs could be a promising candidate for many biomedical applications.     

Solution conditions define morphological homogeneity of α-synuclein fibrils

The intrinsically disordered human α-synuclein (αSyn) protein exhibits considerable heterogeneity in in vitro fibrillization reactions. Using atomic force microscopy (AFM) we show that depending on the solvent conditions, A140C mutant and wild-type αSyn can be directed to reproducibly form homogeneous populations of fibrils exhibiting regular periodicity. Results from Thioflavin-T fluorescence assays, determination of residual monomer concentrations and native polyacrylamide gel electrophoresis reveal that solvent conditions including EDTA facilitate incorporation of a larger fraction of monomers into fibrils. The fibrils formed in 10 mM Tris–HCl, 10 mM NaCl and 0.1 mM EDTA at pH 7.4 display a narrow distribution of periodicities with an average value of 102 ± 6 nm for the A140C mutant and 107 ± 9 nm for wt αSyn. The ability to produce a homogeneous fibril population can be instrumental in understanding the detailed structural features of fibrils and the fibril assembly process. Moreover, the availability of morphologically well-defined fibrils will enhance the potential for use of amyloids as biological nanomaterials.     

Activity of Zn and Mg phthalocyanines and porphyrazines in amyloid aggregation of insulin

Formation of the deposits of protein aggregates—amyloid fibrils in an intracellular and intercellular space—is common to a large group of amyloid‐associated disorders. Among the approaches to develop of therapy of such disorders is the use of agents preventing protein fibrillization. Polyaromatic complexes—porphyrins and phthalocyanines—are known as compounds possessing anti‐fibrillogenic activity. Here, we explore the impact of related macrocyclic complexes—phthalocyanines (Pc) and octaphenyl porphyrazines (Pz) of Mg and Zn—on aggregation of amyloidogenic protein insulin. Pz complexes are firstly reported as compounds able to affect protein fibrillization. The effect of Pc and Pz complexes on the kinetics and intensity of insulin aggregation was studied by the fluorescent assay using amyloid sensitive cyanine dye. This has shown the impact of metal ion on the anti‐fibrillogenic properties of macrocyclic complexes—the effect on the fibrillization kinetics of Mg‐containing compounds is much more pronounced comparing to that of Zn analogues. Scanning electron microscopy experiments have demonstrated that filamentous fibrils are the main product of aggregation both for free insulin and in the presence of macrocyclic complexes. However, those fibrils are distinct by their length and proneness to lateral aggregation. The Pc complexes cause the increase in variation of fibrils length 0.9 to 2.7 nm in opposite to 1.4 to 2.0 nm for free insulin, whereas Pz complexes cause certain shortening of the fibrils to 0.8 to 1.6 nm. The averaged size of the fibrils population was estimated by dynamic light scattering; it correlates with the size of single fibrils detected by scanning electron microscopy.     

Extracellular biosynthesis of silver nanoparticles using Rhizopus stolonifer

Synthesis of silver nanoparticles (AgNPs) has become a necessary field of applied science. Biological method for synthesis of AgNPs by Rhizopus stolonifer aqueous mycelial extract was used. The AgNPs were identified by UV–visible spectrometry, X-ray diffraction (XRD), transmission electron microscopy (TEM) and Fourier transform infrared spectrometry (FT-IR). The presence of surface plasmon band around 420 nm indicates AgNPs formation. The characteristic of the AgNPs within the face-centered cubic (fcc) structure are indicated by the peaks of the X-ray diffraction (XRD) pattern corresponding to (1 1 1), (2 0 0) and (2 2 0) planes. Spherical, mono-dispersed and stable AgNPs with diameter around 9.47 nm were prepared and affirmed by high-resolution transmission electron microscopy (HR-TEM). Fourier Transform Infrared (FTIR) shows peaks at 1426 and 1684 cm⁻¹ that affirm the presence of coat covering protein the AgNPs which is known as capping proteins. Parameter optimization showed the smallest size of AgNPs (2.86 ± 0.3 nm) was obtained with 10⁻² M AgNO₃ at 40 °C. The present study provides the proof that the molecules within aqueous mycelial extract of R. stolonifer facilitate synthesis of AgNPs and highlight on value-added from R. stolonifer for cost effectiveness. Also, eco-friendly medical and nanotechnology-based industries could also be provided. Size of prepared AgNPs could be controlled by temperature and AgNO₃ concentration. Further studies are required to study effect of more parameters on size and morphology of AgNPs as this will help in the control of large scale production of biogenic AgNPs.     

Silver glyconanoparticles functionalized with sugars of sweet sorghum syrup as an antimicrobial agent

Bio-directed synthesis of metal nanoparticles is gaining importance due to their biocompatibility, low toxicity and eco-friendly nature. We used sweet sorghum syrup for a facile and cost-effective green synthesis of silver glyconanoparticles. Silver nanoparticles were formed due to reduction of silver ions when silver nitrate solution was treated with sorghum syrup solutions of different pH values. The nanoparticles were characterized by UV–vis, TEM (transmission electron microscopy), DLS (dynamic light scattering), EDAX (energy dispersive X-ray spectroscopy), FT-IR (fourier transform infrared spectroscopy) and XRD (X-ray diffraction spectroscopy). The silver glyconanoparticles exhibited a characteristic surface plasmon resonance around 385 nm. At pH 8.5, the nanoparticles were mono-dispersed and spherical in shape with average particle size of 11.2 nm. The XRD and SAED studies suggested that the nanoparticles were crystalline in nature. EDAX analysis showed the presence of elemental silver signal in the synthesized glyconanoparticles. FT-IR analysis revealed that glucose, fructose and sucrose present in sorghum syrup acted as capping ligands. Silver glyconanoparticles prepared at pH 8.5 had a zeta potential of ?28.9 mV and were anionic charged. They exhibited strong antimicrobial activity against Gram-positive, Gram-negative and different Candida species at MIC values ranging between 2 and 32 μg ml^?1. This is first report on sweet sorghum syrup sugars-derived silver glyconanoparticles with antimicrobial property.     

Preparation and in vitro photodynamic activity of amphiphilic zinc(II) phthalocyanines substituted with 2-(dimethylamino)ethylthio moieties and their N-alkylated derivatives

Treatment of 4,5-dichlorophthalonitrile with 2-(dimethylamino)ethanethiol hydrochloride and K₂CO₃ afforded 4,5-bis[2-(dimethylamino)ethylthio]phthalonitrile or a heterocycle-fused phthalonitrile depending on the reaction temperature. The latter has been spectroscopically and structurally characterized. Both compounds underwent mixed cyclization with 3 equiv of unsubstituted phthalonitrile in the presence of Zn(OAc)₂·2H₂O and 1,8-diazabicyclo[5.4.0]undec-7-ene to give the corresponding 2,3-disubstituted zinc(II) phthalocyanines. N-methylation or pentylation of the bis[2-(dimethylamino)ethylthio] substituted analogue resulted in the formation of the respective dicationic phthalocyanines. For comparison, the octa-substituted analogues were also prepared by base and zinc-promoted self-cyclization of 4,5-bis[2-(dimethylamino)ethylthio]phthalonitrile followed by N-methylation. The spectroscopic and basic photophysical properties of these di- and octa-substituted phthalocyanines were examined in N,N-dimethylformamide. All of them remained essentially non-aggregated, showed moderate fluorescence emission, and could generate singlet oxygen, except the heterocycle-fused analogue, of which the singlet excited state was reductively quenched by the amino substituent. The photocytotoxicity of these compounds was also evaluated against HepG2 human hepatocarcinoma cells and HT29 and T84 human colon adenocarcinoma cells. The disubstituted amphiphilic phthalocyanines are particularly potent with IC₅₀ values down to 0.08 μM. Fluorescence microscopic studies revealed that the non-ionic derivative has selective affinity to the mitochondria of HT29 cells, while its di-N-methylated analogue shows preferential localization in the cell membrane.     

Monomerization and aggregation of β-lactoglobulin under adverse condition: A fluorescence correlation spectroscopic investigation

β-Lactoglobulin is one of the major components of bovine milk and it remains in a dimeric form under physiological conditions. The present contribution elucidates the structural change of β-lactoglobulin at pH 7.4 under the action of guanidine hydrochloride (GnHCl) and heat at the single molecular level. The only free cysteine (Cys-121) of β-lactoglobulin has been tagged with 7-diethylamino-3-(4-maleimidophenyl)-4-methylcoumarin (CPM) for this purpose. The dimeric structure of β-lactoglobulin found to undergoes a monomerization prior to the unfolding process upon being subjected to GnHCl. The hydrodynamic diameter of the native dimer, native monomer and the unfolded monomer has been estimated as ~ 55 Å, ~ 29 Å and ~ 37 Å, respectively. The free energy change for the monomerization and denaturation are respectively 1.57 kcal mol^− 1 and 8.93 kcal mol^− 1. With change in temperature, development of two types of aggregates (small aggregates and large aggregates) was observed, which is triggered by the formation of the monomeric structure of β-lactoglobulin. The hydrodynamic diameters of the smaller and larger aggregates have been estimated to be ~ 77 Å and ~ 117 Å, respectively. The formation of small aggregates turns out to be reversible whereas that of larger aggregates is irreversible. The free energy associated with these two steps are 0.69 kcal mol^− 1 and 9.09 kcal mol^− 1. Based on the size parameters, the smaller and larger aggregates have been proposed to contain ~twenty and ~sixty monomeric units. It has also been concluded that the monomeric subunits retain their native like secondary structure in these aggregates.     

Trispyrazolylmethane piano stool complexes of iron(II) and cobalt(II)

A series of cationic trispyrazolylmethane complexes of the general form [Tm^RM(CH₃CN)₃]²⁺ (Tm = tris(pyrazolyl)methane, 1, R = 3,5-Me₂, M = Fe(II); 2, R = 3-Ph, M = Fe(II); 3, R = 3,5-Me₂, M = Co(II); 4, R = 3-Ph, M = Co(II)) with ‘piano-stool’ structures was prepared by the reaction of the N₃tripodal ligands (Tm^R)with [(CH₃CN)₆M](BF₄)₂ in a 1:1 stoichiometric ratio. Magnetic susceptibility measurements indicate that all four complexes with BF₄⁻ counter anions are paramagnetic, high-spin systems in the solid state with μ_eff at high temperatures of 5.2 (1, S = 2), 5.4 (2, S = 2), 4.9 (3, S = 3/2) and 4.6 (4, S = 3/2) BM, respectively. Comparisons of bond lengths from the metal centre to the Tm^R nitrogen donors, and from the metal centre to the acetonitrile nitrogen donors indicate that the neutral tripodal ligands appear to be more weakly coordinated to the metal centre than are the acetonitrile ligands. Reactions of these tripodal complexes with bidentate phosphine ligands, such as 1,2-diphosphinoethane or 1,2-bis(diallylphosphino)ethane leads to displacement of the tripodal ligand, or to the formation of more thermally stable bis-ligand complexes M(Tm^R)₂ (R = 3,5-dimethyl).     

Single-electron photoreduction of the PM intermediate of cytochrome c oxidase

The P_M → F transition of the catalytic cycle of cytochrome c oxidase from bovine heart was investigated using single-electron photoreduction and monitoring the subsequent events using spectroscopic and electometric techniques. The P_M state of the oxidase was generated by exposing the oxidized enzyme to CO plus O₂. Photoreduction results in rapid electron transfer from heme a to oxoferryl heme a₃ with a time constant of about 0.3 ms, as indicated by transients at 605 nm and 580 nm. This rate is ∼ 5-fold more rapid than the rate of electron transfer from heme a to heme a₃ in the F → O transition, but is significantly slower than formation of the F state from the P_R intermediate in the reaction of the fully reduced enzyme with O₂ to form state F (70–90 μs). The ∼ 0.3 ms P_M → F transition is coincident with a rapid photonic phase of transmembrane voltage generation, but a significant part of the voltage associated with the P_M → F transition is generated much later, with a time constant of 1.3 ms. In addition, the P_M → F transition of the R. sphaeroides oxidase was also measured and also was shown to have two phases of electrogenic proton transfer, with τ values of 0.18 and 0.85 ms.     

Green biosynthesis of silver nanoparticles from Annona squamosa leaf extract and its in vitro cytotoxic effect on MCF-7 cells

The biological method for the synthesis of silver nanoparticles (AgNPs) using Annona squamosa leaf extract and its cytotoxicity against MCF-7 cells are reported. The synthesized AgNPs using A. squamosa leaf extract was determined by UV–visible spectroscopy and it was further characterized by FT-IR, X-ray diffraction (XRD), Transmission electron microscopy (TEM), Zeta potential and energy dispersive spectrometric (EDS) analysis. The UV–visible spectrum showed an absorption peak at 444 nm which reflects surface plasmon resonance (SPR) of AgNPs. TEM photography showed biosynthesized AgNPs were predominantly spherical in shape with an average size ranging from 20 to 100 nm. The Zeta potential value of ?37 mV revealed the stability of biosynthesized AgNPs. Furthermore, the green synthesized AgNPs exhibited a dose-dependent cytotoxicity against human breast cancer cell (MCF-7) and normal breast epithelial cells (HBL-100) and the inhibitory concentration (IC₅₀) were found to be 50 μg/mL, 30 μg/mL, and 80 μg/mL, 60 μg/ml for AgNPs against MCF-7 and normal HBL-100 cells at 24 h and 48 h incubation respectively. An induction of apoptosis was evidenced by (AO/EtBr) and DAPI staining. Application of such eco-friendly nanoparticles makes this method potentially exciting for the large scale synthesis of nanoparticles.     

Fungal surface protein mediated one-pot synthesis of stable and hemocompatible gold nanoparticles

Despite their large secretome and wide applications in bioprocesses, fungi remain underexplored in metal nanoparticles (MNP) biosynthesis. Previous studies have shown that cell surface proteins of Rhizopus oryzae play a crucial role in biomineralization of Au(III) to produce gold nanoparticles (AuNPs). Therefore, it is hypothesized that purified cell surface protein may produce in vitro AuNPs with narrow size distribution for biomedical and biocatalytic applications. However, different protein extraction methods might affect protein stability and the AuNP biosynthesis process. Herein, we have explored the extraction of cell surface proteins from R. oryzae using common detergents and reducing agent (sodium dodecyl sulfate (SDS) Triton X-100, and 1,4-dithiothreitol (DTT)) and their effect on the size and shape of the biosynthetic AuNPs. The surface proteins extracted with reducing agent (DTT) and non-ionic detergent (Triton X-100) produce spherical AuNPs with a mean particle size of 16 ± 7 nm, and 19 ± 4 nm, respectively, while the AuNPs produced by the surface protein extracted by ionic detergent (SDS) are flower-like AuNPs with broader size distribution of 43 ± 19 nm. This synthetic approach does not require use of any harsh chemicals, multistep preparation and separation process, favouring environmental sustainability. The biosynthetic AuNPs thus formed, are stable in different physiological buffers and hemocompatible, making them suitable for biomedical applications.     

Sono-chemical preparation of cellulose nanocrystals from lignocellulose derived materials

This study examined the production of cellulose nanocrystals from microcrystalline wood cellulose, Avicel and recycled pulp of wood pulp using sono-chemical-assisted hydrolysis. Two hydrolysis systems: deionized water and maleic acid were evaluated. In deionized water, Avicel produced cellulose nanocrystals with average diameter of 21 ± 5 nm (minimum 15 nm and maximum 32 nm). Cellulose nanocrystals from recycled pulp were not distinctively spherical and had an average diameter of 23 ± 4 nm (minimum 14 nm and maximum 32 nm). Maleic acid (50 mM) sono-chemical assisted hydrolysis of Avicel at 15 °C and 90% power output for 9 min produced cellulose nanocrystals which were cylindrical in shape and were of dimensions, length 65 ± 19 nm and width 15 nm.     

Migration mechanism of mesenchymal stem cells studied by QD/NSOM

The migration of mesenchymal stem cells (MSCs) plays a key role in tumor-targeted delivery vehicles and tumor-related stroma formation. However, there so far has been no report on the distribution of cell surface molecules during the VEGF-induced migration of MSCs. Here, we have utilized near-field scanning optical microscopy (NSOM) combined with fluorescent quantum dot (QD)-based nano-technology to capture the functional relationship between CD44 and CD29 adhesion molecules on MSCs and the effect of their spatial rearrangements. Before VEGF-induced migration of MSCs, both CD44 and CD29 formed 200–220 nm nano-domains respectively, with little co-localization between the two types of domains. Surprisingly, the size of the CD44 nano-domain rapidly increased in size to 295 nm and apparently larger aggregates were formed following MSC treatment with VEGF for 10 min, while the area of co-localization increased to 0.327 μm². Compared with CD44, CD29 was activated obviously later, for the fact that CD29 aggregation didn't appear until 30 min after VEGF treatment. Consistently, its co-localization area increased to 0.917 μm². The CD44 and CD29 nano-domains further aggregated into larger nano-domains or even formed micro-domains on the membrane of activated MSCs. The aggregation and co-localization of these molecules promoted FAK formation and cytoskeleton rearrangement. All of the above changes induced by VEGF contributed to MSC migration. Taken together, our data of NSOM-based dual color fluorescent imaging demonstrated for the first time that CD44, together with CD29, involved in VEGF-induced migration of MSCs through the interaction between CD44 and its co-receptor of VEGFR-2.     

Amyloid fibrillation and cytotoxicity of insulin are inhibited by the amphiphilic surfactants

Amyloid fibrils have been associated with at least 25 different degenerative diseases. The 51-residue polypeptide hormone insulin, which is associated with type II diabetes, has been shown to self-assemble to form amyloid fibrils in vitro. With bovine insulin as a model, the research presented here explores the effects of two amphiphilic surfactants (1,2-dihexanoyl-sn-glycero-3-phosphocholine (di-C7-PC) and 1,2-diheptanoyl-sn-glycero-3-phosphocholine (di-C7-PC)) on the in vitro fibrillation process of bovine insulin at pH 2.0 and 55 °C. We demonstrated that insulin fibrillation may be inhibited by both surfactants in a dose-dependent fashion. The best inhibition of fibril formation is observed when insulin is incubated with 4 mM di-C7-PC. Moreover, the addition of either surfactant at the concentrations studied attenuated insulin fibril-induced cytotoxicity in both PC12 and SH-SY5Y cell lines. The results from this work may contribute to the understanding of the molecular factors affecting amyloid fibrillation and the molecular mechanism(s) of the interactions between the membrane and amyloid proteins.     

Synthetic assembly of novel avidin-biotin-GlcNAc (ABG) complex as an attractive bio-probe and its interaction with wheat germ agglutinin (WGA)

A tetravalent GlcNAc pendant glycocluster was constructed with terminal biotin through C₆ linker. To acquire the multivalent carbohydrate-protein interactions, we synthesized a glycopolymer of tetrameric structure using N-acetyl-d-glucosamine (GlcNAc) as the target carbohydrate by the use of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) as coupling reagent, followed by biotin-avidin complexation leading to the formation of glycocluster of avidin-biotin-GlcNAc conjugate (ABG complex). The dynamic light scattering (DLS) system was implied for size detection and to check the binding affinity of GlcNAc conjugate with a WGA lectin we use fluorometric assay by means of specific excitation of tryptophan at λ_ex 295 nm and it was found to be very high K_a ∼ 1.39 × 10⁷ M⁻¹ in case of ABG complex as compared to GlcNAc only K_a ∼ 1.01 × 10⁴ M⁻¹ with the phenomenon proven to be due to glycocluster effect.     

Synthesis and spectral properties of Zr(IV) and Hf(IV) phthalocyanines with β-diketonates as axial ligands

The series of new zirconium(IV) and hafnium(IV) phthalocyanines with various β-dicarbonyl ligands were prepared via direct interaction between di(chloro)zirconium(IV) or hafnium(IV) phthalocyanines and free β-diketones and also with 4-benzoyl-3-methyl-1-phenyl-2-pyrazolin-5-one. The structure of the obtained bis(β-dicarbonilato) zirconium(IV) and hafnium(IV) phthalocyanines was studied by two dimension ¹H NMR spectroscopy (COSY, NOESY, ROESY). Absorption and fluorescence spectroscopic studies have been investigated in various solvents. Analyzed compounds of concentration range below 10^?5 mol/dm³ do not aggregate in the organic solvents. Fluorescence quantum yields (Φ_F) and natural life times (τ) of zirconium phthalocyanine complexes have been calculated in toluene, DMSO and THF.