Far-red pentamethine cyanine dyes as fluorescent probes for the detection of serum albumins.

Benzothiazole based cyanine dyes with bridged groups in the pentamethine chain were studied as potential far-red fluorescent probes for protein detection. Spectral-luminescent properties were characterized for unbound dyes and in the presence of serum albumins (bovine (BSA), human (HSA), equine (ESA)), and globular proteins (ß-lactoglobulin, ovalbumin). We have observed that the addition of albumins leads to a significant increase in dyes fluorescence intensity. However, the fluorescent response of dyes in the presence of other globular proteins was notably lower. The value of fluorescence quantum yield for dye bearing a sulfonate group complexed with HSA amounted to 42% compared with 0.2% for the free dye. The detection limit of HSA by this dye was greater than 0.004 mg ml-1 which indicates the high sensitivity of dye to low HSA concentrations. Modelling of structure of the dyes complexes with albumin molecules was performed by molecular docking. According to these data, dyes could bind to up to five sites on the HSA molecule; the most preferable are the haemin-binding site in subdomain IB and the dye-binding site in the pocket between subdomains IA, IIA and IIIA. This work confirms that pentamethine cyanine dyes could be proposed as powerful far-red fluorescent probes applicable for highly sensitive detection of albumins.

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.

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 2-8nm and a length of up to 5µm, 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 4-12nm. 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 100nm 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.

Development of a quantitative structure activity relations (QSAR) model to guide the design of fluorescent dyes for detecting amyloid fibrils.

Quantitative structure activity relationship (QSAR) studies were performed on a set of polymethine compounds to develop new fluorescent probes for detecting amyloid fibrils. Two different approaches were evaluated for developing a predictive model: part least squares (PLS) regression and an artificial neural network (ANN). A set of 60 relevant molecular descriptors were selected by performing principal component analysis on more than 1600 calculated molecular descriptors. Through QSAR analysis, two predictive models were developed. The final versions produced an average prediction accuracy of 72.5 and 84.2% for the linear PLS and the non-linear ANN procedures, respectively. A test of the ANN model was performed by using it to predict the activity, i.e., staining or non-staining of amyloid fibrils, using 320 compounds. The five candidates whose greatest activities were selected by the ANN model underwent confirmation of their predicted properties by empirical testing. The results indicated that the ANN model potentially is useful for facilitating prediction of activity of untested compounds as dyes for detecting amyloid fibrils.

Tri- and pentamethine cyanine dyes for fluorescent detection of α-synuclein oligomeric aggregates.

The pathogenesis of Parkinson's disease that is the second most common neurodegenerative disease is associated with formation of different aggregates of α-synuclein (ASN), namely oligomers and amyloid fibrils. Current research is aimed on the design of fluorescent dyes for the detection of oligomeric aggregates, which are considered to be toxic and morbific spices. Fluorescent properties of series of benzothiazole trimethine and pentamethine cyanines were characterized in free state and in presence of monomeric, oligomeric and fibrilar ASN. The dyes with wide aromatic systems and bulky phenyl and alkyl substituents that are potentially able to interact with hydrophobic regions of oligomeric aggregates were selected for the studies. For majority of studied dyes noticeable changes in fluorescence characteristics were shown in the presence of fibrillar or oligomeric ASN, while the dyes slightly responded on the presence of monomeric protein. For pentamethine cyanine SL-631 and trimethine cyanine SH-299 certain specificity to oligomeric aggregates over fibrils was observed. Using these dyes at 10(-6) M concentration permits the detection of oligomeric ASN in the concentrations range of at least 0.2-2 microM. Pentamethine cyanine SL-631 is proposed as dye for fluorescent detection of oligomeric aggregates of ASN, while trimethine cyanine SH-299 is shown to be a sensitive probe both on oligomeric and fibrillar ASN. It is proposed that wide aromatic system of SL-631 pentamethine dye molecule could better fix on the less dense and structured oligomeric formation, while less bulky and more "crescent-shape" molecule of trimethine dye SH-299 could easier enter into the groove of beta-pleated structure.

Novel fluorescent trimethine cyanine dye 7519 for amyloid fibril inhibition assay.

Fluorescence spectroscopy was used to study the ability of dye 7519 to follow the transition of monomeric insulin into fibrils and applicability of the dye to the insulin aggregation inhibition assay. The commercially available classic amyloid stain, thioflavin T, was used as the reference dye. For selecting potential inhibitors, the QSAR approach was applied. Dye 7519 appeared to be suitable for monitoring insulin aggregation into fibrils in vitro. The properties of the dye allowed us to test it as a potential probe in the screening assay of potential inhibitors of insulin fibrillization. One hundred forty-four flavonoids were tested as potential inhibitors of amyloid fibril formation using the quantitative structure activity relationship approach. Among them, 10 candidates with high indexes of inhibition were selected for tests in vitro using dye 7519 and the reference amyloid dye thioflavine T. Using dye 7519 fluorescence, we found that two compounds had inhibitory effects on insulin amyloid formation. These results agree with inhibition data using the thioflavine T assay. Our studies demonstrated that the fluorescent cyanine dye 7519 is a sensitive probe for quantitative detection of insulin amyloid formation and can be applied to screen agents capable of affecting aggregation of amyloid proteins.

Explorations of the application of cyanine dyes for quantitative alpha-synuclein detection.

We examined the practical aspects of using fluorescent mono (T-284) and trimethinecyanine (SH-516) dyes for detecting and quantifying fibrillar alpha-synuclein (ASN). We studied the interaction of cyanine dyes with fibrillar proteins using fluorescence spectroscopy and atomic force microscopy. The commercially available classic amyloid stain thioflavin T (Thio T) was used as the reference dye. T-284 and SH-516 dyes can be used for fluorometric quantification of fibrillar wild-type ASN at concentrations of approximately 1.5-20 microg/ml. Both dyes appeared suitable for step-wise monitoring of ASN variants (wild-type and mutants A30P and A53T) aggregation into fibrils in vitro, demonstrating good reproducibility, exceeding that for the commonly used Thio T. Our assay may be used for screening in vitro of agents capable of affecting the aggregation of ASN. In addition, T-284 and SH-516 cyanine dyes were shown to recognize amyloid proteins of various amino acid compositions selectively. T-284 demonstrated particular sensitivity to wild-type and A53T ASN, while for SH 516, the fluorescence response to fibrillar proteins was nearly the same except for lysozymes. T-284 and SH-516 cyanine dyes are sensitive and specific fluorescent probes for monitoring ASN fibril formation process in vitro, quantification of fibrillar ASN in solution, and fluorescent detection of various fibrillar protein species.

The mechanism of benzothiazole styrylcyanine dyes binding with dsDNA: studies by spectral-luminescent methods.

In the presented work studies of the interaction mode of monomer and two homodimer benzothiazole styryl dyes containing spermine-like linkage/tail group with the double stranded (ds) DNA are reported. For these dyes, equilibrium constant of dye binding to DNA (K(b)), as well as the number of dsDNA base pairs occupied by one bound dye molecule (n) were determined. The data obtained show that the presence of spermine-like group containing quaternary nitrogen (Bos-5) results in increase of K(b) value as compared to this of unsubstituted analogue (Sbt). Besides, for the dimer dyes containing benzothiazole styryl chromophores, the K(b) value is either five times higher (DBos-13) or almost the same (DBsu-10) as compared to this of corresponding monomer Sbt, depending on the position in the benzothiazole ring where the linker is attached. Moreover, the n values for both dimers are significantly different as well, pointing to the bis-intercalative binding mechanism for DBos-13 and for the groove-binding one for DBsu-10. The conclusion about the dimer dyes-dsDNA binding mechanisms is also supported by the study of the fluorescent response of these dyes on the presence of AT- and GC-containing polynucleotides.

Specific fluorescent detection of fibrillar alpha-synuclein using mono- and trimethine cyanine dyes.

With the aim of searching of novel amyloid-specific fluorescent probes the ability of series of mono- and trimethine cyanines based on benzothiazole, pyridine and quinoline heterocycle end groups to recognize fibrillar formations of alpha-synuclein (ASN) was studied. For the first time it was revealed that monomethine cyanines can specifically increase their fluorescence in aggregated ASN presence. Dialkylamino-substituted monomethine cyanine T-284 and meso-ethyl-substituted trimethine cyanine SH-516 demonstrated the higher emission intensity and selectivity to aggregated ASN than classic amyloid stain Thioflavin T, and could be proposed as novel efficient fluorescent probes for fibrillar ASN detection. Studies of structure-function dependences have shown that incorporation of amino- or diethylamino- substituents into the 6-position of the benzothiazole heterocycle yields in a appearance of a selective fluorescent response to fibrillar alpha-synuclein presence. Performed calculations of molecular dimensions of studied cyanine dyes gave us the possibility to presume, that dyes bind with their long axes parallel to the fibril axis via insertion into the neat rows (so called 'channels') running along fibril.

Cyanine dye-protein interactions: looking for fluorescent probes for amyloid structures.

We ascertained the ability to detect fibrillar beta-lactoglobulin (BLG) of a series of mono-, tri-, penta-, and heptamethinecyanines based on benzothiazole and benzimidazole heterocycles, and of benzothiazole squaraine. Fluorescence properties of these cyanine dyes were measured in the unbound state and in the presence of monomeric and fibrillar BLG and compared with those for the commercially available benzothiazole dye Thioflavin T. The correlation between the chemical nature of the dye molecules and the ability of dyes to bind aggregated proteins was established. We found that meso-substituted cyanines with amino substituents in heterocycle in contrast to the corresponding unsubstituted dyes have a binding preference to fibrillar BLG and a noticeable fluorescence response in the presence of the aggregated protein. For the squaraines and benzimidazole penthamethinecyanines studied, fluorescence emission increased both in the presence of native and fibrillar protein. The trimethinecyanines T-49 and SH-516 exhibit specifically increased fluorescence in the presence of fibrillar BLG. These dyes demonstrated the same or higher emission intensity and selectivity to aggregated BLG as Thioflavin T, and are proposed for application in selective fluorescent detection of aggregated proteins.

Fluorescence of styryl dyes-DNA complexes induced by single- and two-photon excitation.

The series of novel monomer and homodimer styryl dyes based on (p-dimethylaminostyryl) benzothiazolium residues were synthesized and studied as possible fluorescent probes for nucleic acids detection. Spectral-luminescent and spectral-photometric properties of obtained dyes in the unbound state and in DNA presence were studied. Fluorescence emission induced by two-photon excitation of dye-DNA complexes in aqueous buffer solution was registered. Two-photon absorption cross section values of the studied dyes in DNA presence were evaluated.

Studies of monomeric and homodimeric oxazolo[4,5-b]pyridinium cyanine dyes as fluorescent probes for nucleic acids visualization.

The series of recently synthesized monomeric and homodimeric cyanine dyes based on monomethine cyanine chromophore with oxazolo[4,5-b]pyridinium and quinoline end groups [Vassilev A, Deligeorgiev T, Gadjev N, Drexhage K-H. Synthesis of novel monomeric and homodimeric cyanine dyes based on oxazolo[4,5-b]pyridinium and quinolinium end groups for nucleic acid detection, Dyes Pigm 2005;66:135-142] were studied as possible fluorescent probes for nucleic acids detection. Significant fluorescence enhancement and intensity level (quantum yield up to 0.75) was observed for all the dyes in the presence of DNA. The oxazolo[4,5-b]pyridinium cyanines demonstrated high sensitivity as fluorescent stains for post-electrophoretic visualization of nucleic acids in agarose gels upon both VIS and UV transillumination, and the visualized band contained 0.8 ng of dsDNA.

Fluorescent properties of pentamethine cyanine dyes with cyclopentene and cyclohexene group in presence of biological molecules.

A series of pentamethine cyanine dyes with cyclohexene or cyclopentene group in polymethyne chain, assumed as DNA groove-binders, were studied as fluorescent probes for nucleic acids as well as for native and denatured proteins. It was revealed that the presence of methyl or dimethyl substituent in 5 position of the cyclohexene group hinders the formation of dye-DNA fluorescent complex, while the methyl substituent in 2 position leads to the increasing of the dye-DNA complex fluorescence intensity. The dyes SL-251, SL-1041, and SL-1046 containing methyl group in the 2 position of the cyclic group, are reported as bright DNA-sensitive dyes. The study of the dyes DNA-binding specificity demonstrated significant AT-preference that points to the groove-binding interaction mode. At the same time, the dyes SL-251, SL-377, and SL-957 with the 2-methyl substituted cyclohexene group were shown to be sensitive fluorescent dyes both for nonspecific (in SDS presence) proteins detection and for native BSA.

Studies on the spectral-luminescent properties of the novel homodimer styryl dyes in complexes with DNA.

Series of homodimer styryls containing on (p-dimethylaminostyryl) pyridinium residues that are connected with aliphatic linkage group was synthesized. Spectral luminescent properties of obtained dyes in free state and in nucleic acids presence were studied. It was shown that DNA binding affinity of the novel homodimers exceeds that of parent monomer (p-dimethylaminostyryl)pyridine iodide. For homodimers with the linkage 4-10 carbon atoms preference in binding to DNA than to RNA was observed. It could be concluded that parent monomer has different mechanisms of binding to nucleic acids than corresponding homodimer dye.

Luminescence spectroscopic studies of trimethinecyanines substituted in polymethine chain with nucleic acids and proteins.

The series of symmetrical beta-substituted and alpha,gamma-substituted trimethinecyanine dyes were studied for their absorption and fluorescent characteristics in unbound state and in the presence of nucleic acids and proteins. It was shown that beta-substituted and alpha,gamma-bridged trimethinecyanines containing extended heterocyclic systems or N-phenyl as well as N-cyclohexyl substituents demonstrate increased affinity to proteins. At the same time the presence of both N-phenyl and N-cyclohexyl substituents leads to the decrease of the dye fluorescence intensity in complexes with nucleic acids. For trimethinecyanines similarly to unsymmetrical monomethines the presence of N-omega-hydroxy alkyl substituents results in the increase of fluorescence intensity of dye-DNA complex and the emission decrease of dye-RNA complex.

Interaction of cyanine dyes with nucleic acids: XXXI. Using of polymethine cyanine dyes for the visualization of DNA in agarose gels.

Fifteen polymethine cyanine dyes were studied as fluorescent stains for DNA in electrophoretic gels. Among studied cyanines, two dyes CPent V and CCyan 2-O most effectively visualized covalently closed and linear double-stranded DNA molecules in gels under standard conditions using UV-illumination, green filter and black-and-white photo film. Ethidium bromide was 1.2-1.6 times more effective as compared to cyanine dyes in staining of DNA in the concentration range of 8-18 ng, while studied cyanines were more sensitive to DNA quantity above 50 ng.

Interactions of cyanine dyes with nucleic acids. XXIV. Aggregation of monomethine cyanine dyes in presence of DNA and its manifestation in absorption and fluorescence spectra.

Absorption, fluorescence emission and excitation spectra of benzothiazole cyanine dyes--thiazole orange (TO) and 7-methyl-6-(3-methyl-2,3-dihydro-1,3-benzothiazol-2-ylidenmethyl) [1,3] dioxolo [4',5':4,5] benzo [d] [1,3] thiazolium methylmethosulfate (Cyan 13)--were investigated over a wide concentration range. The dyes form aggregates with a 'sandwich'-like structure in water solution. At low dye to DNA concentrations ratios, Cyan 13 and TO monomers appear to interact with the DNA. On increasing the dye to DNA concentrations ratio, free dye molecules aggregate with the DNA-bound ones. The spectra of the free dye aggregates and the aggregates formed on the DNA, are characterized by an anomalously large (more than 100 nm) Stokes shift. This suggests, that the pi-electron systems of the aggregates undergo substantial changes in excited state, compared to those of the monomers. The formation of aggregates consisting of the free and DNA-bound dye molecules can be explained using the half-intercalation model of the interaction of the cyanine dye monomers with the DNA.

Interaction of cyanine dyes with nucleic acids. XXV. Influence of affinity-modifying groups in the structure of benzothiazol-4-[2,6-dimethylpyridinium] dyes on the spectral properties of the dyes in the presence of nucleic acids.

Novel monomethine pyridinium cyanine dyes of similar structure and containing 'affinity-modifying' groups of different chemical nature were studied by spectral-luminescent methods as possible fluorescent probes for the nucleic acids detection. It was shown that the nature of the functional groups in the dye linker influences the fluorescent properties of the dye-nucleic acids complexes. Incorporation of a hydroxyl group into the linker structure leads to a significant increase in the fluorescence intensity of the dye--double-stranded DNA complexes relative to the parent dye Cyan 40.

Interaction of cyanine dyes with nucleic acids. XVIII. Formation of the carbocyanine dye J-aggregates in nucleic acid grooves.

Spectral properties of carbocyanine dye 3-methyl-2-[3-methyl-2-(3-methyl-2,3-dihydro-1,3-benzothiazole-2-iliden)-1- butenyl]-1,3-benzothiazole-3-il iodide (Cyan betaiPr) in water solution, as well as in the presence of different types of double stranded DNA have been studied. While in water solution of 'free' dye Cyan betaiPr stays mainly in monomeric form, in the presence of DNA the dye molecules form J-aggregates. The molecular structure of these J-aggregates causes the Davydov splitting of their absorption band, corresponding to the first electronic transition. A study of site-specificity showed that in the presence of poly (dA/dT) the majority of Cyan betaiPr molecules form J-aggregates, while in the presence of poly (dGC/dGC) dye molecules stay mainly in monomeric form and in presence of chicken erythrocytes DNA both J-aggregate and monomeric forms of dye are present. We suppose that Cyan betaiPr molecules aggregate in DNA groove, which serves as a template for J-aggregate forming. An increase of ionic strength of solution leads to the release of dye molecules from DNA grooves and prevents J-aggregates formation.