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.

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.

Uptake of Chlorin e6 Photosensitizer by Polystyrene-Diphenyloxazole-Poly(N-Isopropylacrylamide) Hybrid Nanosystem Studied by Electronic Excitation Energy Transfer.

Polystyrene (PS)-diphenyloxazole (PPO) nanoparticles with attached cross-linked poly-N-isopropylacrylamide (PNIPAM) chains were obtained resulting in PS-PPO-PNIPAM hybrid nanosystems (NS). Fluorescence spectra of chlorin e6 added to PS-PPO-PNIPAM hybrid NS revealed electronic excitation energy transfer (EEET) from PS matrix and encapsulated PPO to chlorin e6. EEET efficiency increased strongly during 1 h after chlorin e6 addition, indicating that uptake of chlorin e6 by PNIPAM part of hybrid NS still proceeds during this time. Heating of PS-PPO-PNIPAM-chlorin e6 NS from 21 to 39 °C results in an enhancement of EEET efficiency; this is consistent with PNIPAM conformation transition that reduces the distance between PS-PPO donors and chlorin e6 acceptors. Meanwhile, a relatively small part of chlorin e6 present in the solution is bound by PNIPAM; thus, further studies in this direction are necessary.

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.

Nonradiative deactivation of the electronic excitation energy in cyanine dyes: influence of binding to DNA.

The processes of nonradiative deactivation of electronic excitation energy in cyanine dyes determine their quantum yield. Because of that, the study of the influence of cyanines binding to DNA on these processes can provide information on the causes leading to the cyanines fluorescence intensity enhancement in the presence of DNA. In the presented paper, the activation energies of nonradiative degradation of electronic excitation, quantum yields and rate constants of nonradiative transitions of several cyanines in free state and in the presence of DNA were established and compared. The mechanisms of nonradiative deactivation of dye excitation energy were discussed.

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: XXVI. Intercalation of the trimethine cyanine dye cyan 2 into double-stranded DNA: study by spectral luminescence methods.

The interaction between double-stranded (ds) DNA and the cyanine dye Cyan 2 has been studied with spectral luminescence methods. Binding constant values have been determined by fluorescence titration and dye distribution in the two-phase system ethyl acetate-water (3.6 x 10(4) and 1.5 x 10(4) M(-1), respectively). Cyan 2 exhibits a small specificity for guanine-cytosine (GC) sequences in total DNA and synthetic polydeoxynucleotides poly(dA/dT) and poly(dGdC/dGdC). The DNA complexes with Cyan 2 are stable at high-ionic strength solution when NaCl is added. The dye molecule complexed with DNA is apparently shielded from the anionic quencher--iodide ion. The negative linear dichroism of the visible absorption band of aligned Cyan 2-DNA complexes indicates that the bound dye lies almost perpendicularly to the DNA helix axis. The linear dichroism of the absorption band at 260 nm suggests a considerable change in the DNA B-form. The results are consistent with an intercalative binding interaction between Cyan 2 and ds DNA.

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.

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.

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.