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SYNTHESIS AND PROPERTIES OF NOVEL ASYMMETRIC
MONOMETHINE CYANINE DYES AS NON-COVALENT LABELS
FOR NUCLEIC ACIDS
Todor G. Deligeorgieva, Nikolai I. Gadjeva,
Iliana I. Timchevab, Vera A. Maximovac,
Haralambos E. Katerinopoulosd
aUniversity of Sofia, Faculty of Chemistry, 1,
James Bourchier Ave., 1164 Sofia, Bulgaria
e-mail toddel_at_chem.uni-sofia.bg
ngadjev_at_chem.uni-sofia.bg bInstitute of Organic
Chemistry with Centre of Phytochemistry,
Bulgarian Academy of Sciences, 1113 Sofia,
Bulgaria e-mail iltim_at_orgchm.bas.bg cInstitute
of Molecular Biology, Bulgarian Academy of
Sciences, 1113 Sofia, Bulgaria e-mail
vmaximova_at_yahoo.com dUniversity of Crete,
Department of Chemistry, Heraklion, 71 409 Crete,
Greece e-mail kater_at_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).
Finally the dye 10 was synthesized by reaction of
7 with 1a (Scheme 6).
Table 1. Absorption maxima ?max (nm), molar
absorptivity e (l.mol-1.cm-1), fluorescence
maxima ?F (nm) of the studied dyes in TE buffer
(dye concentration 1x10-6M) as well as of the
complexes with ds DNA and ss DNA ( / ) no
fluorescence.
Dye Absorption maxima and molar absorptivities Free Dye Dye dsDNAa Dye ssDNAb Fluorescence enhancement Fluorescence enhancement
? ?max (e l.mol1.cm1) ?F ?F ?F dyedsDNA dyessDNA
5a 449sh, 475(77000, 135000) 630 499 497 360x 180x
5b 434sh, 452(90000, 124000) 545 481 480 50x 35x
5c 450, 474(83200, 139000) 495 499 497 20x 10x
5d 429, 453(102130, 144300) 518 480 479 40x 20x
5e 448sh, 474(71400, 118700) 599 498 496 250x 160x
5f 434, 448(101100, 135400) 482 / / / /
5g 451, 476(74000, 116000) 500 499 498 100x 50x
5h 434sh, 451(109200, 124300) 490 480 480 20x 30x
6a 449sh, 475(106000, 186000) 586 498 497 100x 100x
6b 414, 500(89500, 89500) 620 544 538 30x 13x
8a 484(114300) / / / / /
8b 460(73100) 511 / / / /
9a 440sh, 461(-, 129300) 512 515 514 5x 2x
9b 471sh, 496(85400, 134000) 525 521 520 20x 20x
9c 469sh, 500(69000, 194000) 495 493 492 30x 50x
10 455sh, 495(62500, 158300) 519 521 520 10x 15x
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).
aFish sperm ds DNA at a concentration of 2x106M
bFish sperm ss DNA at a concentration of 2x106M
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.mol1.cm1. Most of the dyes have very high
molar absorptivities, usually over 100000
l.mol1.cm1. 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.
By condensation of 2 with 2-methylthio-4-methyloxa
zolo4,5-bpyridinium methosulfate 7 and
2-methylthio-3-methylbenzothiazolium methosulfate
4a, the dyes 8a and 8b were prepared (Scheme 4).
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 1x107 M. The fish sperm ds DNA
was purchased from Sigma (USA). The ss DNA was
obtained after thermal denaturation of ds DNA.
The synthesis of dyes 9a-c was performed by
reaction of 1-cyano-2,4-dimethylbenzothiazolo3,2-
apyridin-5-um perchlorate 3 with 7 and 4a, b
(Scheme 5).
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