Sergiy M. Yarmoluk

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.

Synthesis and biological evaluation of substituted (thieno[2,3-d]pyrimidin-4-ylthio)carboxylic acids as inhibitors of human protein kinase CK2

A novel series of substituted (thieno[2,3-d]pyrimidin-4-ylthio)carboxylic acids has been synthesized and tested in vitro towards human protein kinase CK2. It was revealed that the most active compounds inhibiting CK2 are 3-{[5-(4-methylphenyl)thieno[2,3-d]pyrimidin-4-yl]thio}propanoic acid and 3-{[5-(4-ethoxyphenyl)thieno[2,3-d]pyrimidin-4-yl]thio}propanoic acid (IC(50) values are 0.1 μM and 0.125 μM, respectively). Structure-activity relationships of 28 tested thienopyrimidine derivatives have been studied and binding mode of this chemical class has been predicted. Evaluation of the inhibitors on seven protein kinases revealed considerable selectivity towards CK2.

Identification of 3H-Naphtho(1,2,3-de)quinoline-2,7-diones as Inhibitors of Apoptosis Signal-Regulating Kinase 1 (ASK1)

Apoptosis signal-regulating kinase 1 (ASK1) has recently emerged as an attractive therapeutic target for the treatment of cardiac and neurodegenerative disorders. The selective inhibitors of ASK1 may become important compounds for the development of clinical agents. We have identified the ASK1 inhibitor among 3H-naphtho[1,2,3-de]quinoline-2,7-diones using receptor-based virtual screening. In vitro kinase assay revealed that ethyl 2,7-dioxo-2,7-dihydro-3H-naphtho[1,2,3-de]quinoline-1-carboxylate (NQDI-1) inhibited ASK1 with a K(i) of 500 nM. The competitive character of inhibition is demonstrated in Lineweaver-Burk plots. In our preliminary selectivity study this compound exhibited strong specific inhibitory activity toward ASK1.

A Monomethine Cyanine Dye Cyan 40 for Two-photon–excited Fluorescence Detection of Nucleic Acids and Their Visualization in Live Cells¶

Abstract Monomethine cyanine dye 4-((1-methylbenzothiazolyliliden-2)methyl)-1,2,6-trimethylpyridinium perchlorate (Cyan 40) was investigated as a two-photon–excited fluorescence probe for nucleic acids (NA). Cyan 40 has been shown to demonstrate efficient two-photon–excited fluorescence in the presence of NA in vitro in contrast to solutions without NA. Two-photon confocal laser scanning microscopy (TPCLSM) and two-photon laser scanning microspectrofluorometry were used to check the possibility of using Cyan 40 as two-photon–excited fluorescence label for NA in living cells. Study of dye effect on viability of cells was also carried out. We ascertained that Cyan 40 is a cell-permeant dye, manifesting efficient two-photon–excited fluorescence when bound to NA in living cells, without any significant influence on viability of cells. TPCLSM images obtained from stained cells indicate preferential RNA staining by Cyan 40 compared with DNA.

Interaction of cyanine dyes with nucleic acids. XVII. Towards an aggregation of cyanine dyes in solutions as a factor facilitating nucleic acid detection

Spectral properties of newly synthesized cyanine dyes, namely 1-[6-(4-[6-[2,6-dimethyl-4-(3-methyl-2,3-dihydro-1,3-benzothiazol- 2-ylidenmethyl)-1-pyridiniumyl]hexanoyl]piperazino)-6- oxohexyl]-2,6-dimethyl-4-(3-ethyl-2,3-dihydro-1,3-benzothiazol+ ++-2-ylidenmethyl)pyridinium (K-6) (bichromophoric dye) and 1-[5-di(3-[5-[2,6-dimethyl-4-(3-methyl-2,3-dihydro-1,3-benzothiazol++ +-2-ylidenmethyl)-1-pyridiniumyl]pentylcarboxamido]pro pyl) carbamoylpentyl]-2,6-dimethyl-4-(3-methyl-2,3-dihydro-1,3-benzo thiazol-2-ylidenmethyl) pyridinium (K-T) (trichromophoric dye) in solutions in the presence of and without deoxyribonucleic acid (DNA) were studied within a wide concentration range. It has been established that absorption, as well as fluorescence of investigated dye solutions, without DNA are mainly determined by H-aggregates of dye molecules. On the contrary, the fluorescence of dye solutions in the presence of DNA gives an intrinsic dye molecular fluorescence. H-aggregates are broken because of binding dye molecules with DNA. It has been suggested that both K-T and K-6 molecules bind mainly with DNA via the interaction of two chromophores. As the ratio of the number of dye molecules to that of DNA base pairs increases with an increase in dye concentration, a formation of dye molecule H-aggregates on DNA molecules are observed. Such aggregates have a different structure than those formed in the solutions without DNA. On the grounds of the data obtained, it is concluded that it is possible to use a dye aggregation capable of obtaining higher values for fluorescence enhancement of the DNA stains.

Interaction of cyanine dyes with nucleic acids. XII.β-substituted carbocyanines as possible fluorescent probes for nucleic acids detection

Results of investigations of fluorescent properties of a beta-substituted carbocyanine and its complexes with nucleic acids in comparison with those for the unsubstituted dye are presented. Carbocyanine substituted in polymethine chain has shown promising properties for use as a fluorescent probe in homogeneous systems of nucleic acids detection.

Symmetric cyanine dyes for detecting nucleic acids

A novel approach to the design of sensitive fluorescent probes for nucleic acids detection is proposed. Suitable modifications of tri- and pentamethine cyanine dyes in the polymethine chain and/or in the heterocyclic residues can result in a significant decrease in unbound dye fluorescence intensity and an increase in dye emission intensity in the presence of DNA compared to the unsubstituted dye. The sharp enhancement in the fluorescence intensity upon dye interaction with double-stranded DNA permits the application of the modified tri- and pentamethine dyes as fluorescent probes in double-stranded DNA detection in homogeneous assays.

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.

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.

Interaction of the iron(II) cage complexes with proteins: protein fluorescence quenching study.

Interaction of the iron(II) mono- and bis-clathrochelates with bovine serum albumin (BSA), β-lactoglobulin, lysozyme and insulin was studied by the steady-state and time-resolved fluorescent spectroscopies. These cage complexes do not make significant impact on fluorescent properties of β-lactoglobulin, lysozyme and insulin. At the same time, the monoclathrochelates strongly quench a fluorescence intensity of BSA and substantially decrease its excited state lifetime due to their binding to this protein. This occurs due to the excitation energy transfer from a tryptophan residue to a cage molecule or/and to the change of the tryptophan nearest environment caused by either clathrochelate binding or an alteration of the BSA conformation. The effect of the iron(II) bis-clathrochelate on BSA fluorescence is much weaker as compared to its monomacrobicyclic analogs as a result of an increase in its size.