1. SYNTHESIS AND PROPERTIES OF NOVEL ASYMMETRIC MONOMETHINE CYANINE DYES AS NON-COVALENT LABELS FOR NUCLEIC ACIDS a University of Sofia, Faculty of Chemistry, 1, James Bourchier Ave., 1164 Sofia, Bulgaria e-mail: toddel@chem.uni-sofia.bg; ngadjev@chem.uni-sofia.bgtoddel@chem.uni-sofia.bgngadjev@chem.uni-sofia.bg b Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria e-mail: iltim@orgchm.bas.bgiltim@orgchm.bas.bg c Institute of Molecular Biology, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria e-mail: vmaximova@yahoo.comvmaximova@yahoo.com d University of Crete, Department of Chemistry, Heraklion, 71 409 Crete, Greece e-mail: kater@chemistry.uoc.grkater@chemistry.uoc.gr INTRODUCTION In recent years, there is a growing interest in the research on bioapplications of fluorescent dyes. We have been investigating [1-4] novel representatives of monomethine cyanine dyes as non-covalently binding nucleic acid fluorogenic probes. In our previous works [1-4], we studied monomethine cyanine dyes based mainly on the Thiazole Orange (TO) and Oxazole Yellow (YO) chromophores. In this report we present the results of the synthesis of novel momomethine cyanines (with different chromophores than TO and YO) and their properties as non-covalent nucleic acid probes. RESULTS AND DISCUSSION We synthesized the intermediates by known methods involving the condensation of acetylacetone with 2-amino-6- substituted-benzothiazoles [5,6], 1, with 2-aminopyridine [7], 2, and with 2-cyanomethylbenzothiazole [8], 3 (Scheme 1). Hartmann and Zhou [9] reported that mono-condensation dyes could be obtained using compounds 1a-d and that the 2-methyl group is the most reactive one (Scheme 2). When an excess of the reagents 4a, b was used in the condensation reaction with 1a, the bis-condensation products 6a and 6b were obtained (Scheme 3). By condensation of 2 with 2-methylthio-4-methyloxazolo[4,5-b]pyridinium methosulfate 7 and 2-methylthio-3- methylbenzothiazolium methosulfate 4a, the dyes 8a and 8b were prepared (Scheme 4). Finally the dye 10 was synthesized by reaction of 7 with 1a (Scheme 6). The synthesis of dyes 9a-c was performed by reaction of 1-cyano-2,4-dimethylbenzothiazolo[3,2-a]pyridin-5-um perchlorate 3 with 7 and 4a, b (Scheme 5). Table 1. Absorption maxima λ max (nm), molar absorptivity ε (l.mol -1.cm -1 ), fluorescence maxima F (nm) of the studied dyes in TE buffer (dye concentration 1x10 -6 M) as well as of the complexes with ds DNA and ss DNA; ( / ) – no fluorescence. Dye Absorption maxima and molar absorptivities Free Dye Dye + dsDNA a Dye + ssDNA b Fluorescence enhancement №λ max (ε l.mol –1.cm –1 ) F F F dye+dsDNAdye+ssDNA 5a449sh, 475(77000, 135000)630499497360x180x 5b434sh, 452(90000, 124000)54548148050x35x 5c450, 474(83200, 139000)49549949720x10x 5d429, 453(102130, 144300)51848047940x20x 5e448sh, 474(71400, 118700)599498496250x160x 5f434, 448(101100, 135400)482//// 5g451, 476(74000, 116000)500499498100x50x 5h434sh, 451(109200, 124300)490480 20x30x 6a449sh, 475(106000, 186000)586498497100x 6b414, 500(89500, 89500)62054453830x13x 8a484(114300)///// 8b460(73100)511//// 9a440sh, 461(-, 129300)5125155145x2x 9b471sh, 496(85400, 134000)52552152020x 9c469sh, 500(69000, 194000)49549349230x50x 10455sh, 495(62500, 158300)51952152010x15x a Fish sperm ds DNA at a concentration of 2x10 –6 M; b Fish sperm ss DNA at a concentration of 2x10 –6 M Todor G. Deligeorgiev a, Nikolai I. Gadjev a, Iliana I. Timcheva b, Vera A. Maximova c, Haralambos E. Katerinopoulos d The longest wavelength absorption maxima of the studied asymmetric monomethine cyanine dyes in TE buffer (10 mM Tris-HCl, pH 7.0, 1 mM EDTA) at room temperature are in the region 450-500 nm (Table 1). The corres- ponding molar absorptivities are between 70000 and 200000 l.mol –1.cm –1. Most of the dyes have very high molar absorptivities, usually over 100000 l.mol –1.cm –1. Both the intensity and the position of the longest wavelength absorption maxima of the investigated dyes remain unchanged after binding to nucleic acids. The investigated dyes have low fluorescence in their free form but some of them become strongly fluorescent after binding to DNA (Table 1). The fluorescence maxima of the complexes are in the range 500 and 550 nm. It was found that the complexes of compound 5f as well as of compounds 8a and 8b with ds DNA and ss DNA do not fluoresce. In some cases the fluorescence intensity of the dye – DNA complexes is 100-300 fold higher than those of the free dyes. The same holds especially for some representatives of the studied compounds 5a, 5e, 5g, and 6a. A coincidence of the fluorescence maxima of the complexes with both ds DNA and ss DNA has been observed. As a rule the fluorescence intensity after binding to ds DNA is higher compared to that in the presence of ss DNA. The detection minimum using dye 5a was 100 ng ds DNA in aqueous solution. EXPERIMENTAL Absorption spectra were scanned on a Specord M40 (Carl Zeiss, Jena) UV-VIS spectrophotometer and the cor- rected fluorescence spectra (excitation at 460 nm) were obtained on a Perkin Elmer MPF44 spectrofluorimeter. The emission spectra were corrected using a standard Tungsten lamp, while the excitation spectra were corrected with Rhodamine B. Stock solutions were prepared by dissolving 1 mM of each dye in 1 ml DMSO and subsequent dilution with TE buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA) to a final concentration of 1x10 –7 M. The fish sperm ds DNA was purchased from Sigma (USA). The ss DNA was obtained after thermal denaturation of ds DNA. REFERENCES 1.T. Deligeorgiev, N. Gadjev, Il. Timtcheva, V. Maximova Dyes and Pigments, 2003, 57(2) 161-164. 2.T. Deligeorgiev, Il. Timtcheva, V. Maximova, N. Gadjev, K-H. Drexhage, J. of Fluorescence, 2002, 12(2) 225-229 3.J. Bunkenborg, N. Gadjev, T. Deligeorgiev, J. P. Jacobsen, Bioconjugate Chem., 2000, 11, 861-867. 4.Iliana I. Timcheva, Vera A. Maximova, Todor G. Deligeorgiev, Daphinka A. Zaneva, Ivan G. Ivanov, J. Photochem. Photobiol. A: Chemistry, 2000, 130, 7-11 5.S.I. Shul’ga, V.A. Chuiguk, Ukr. Khim. Zh., 1973, 39(11), 1151-1155 6.S.I. Shul’ga, V.A. Chuiguk, Ukr. Khim. Zh., 1970, 36, 483-485. 7.A.M. Khmaruk, Y.M. Volovenko, V.A. Chuiguk, Ukr. Khim. Zh., 1972, 38(3), 262-264 8.V.A. Chuiguk, Y.M. Volovenko, Khim. Geterotskl. Soedin. 1975, (11), 530-532. 9.H. Hartmann, Z. Zhou, J. prakt. Chem., 2000, 342, 249-255