Interaction of cyanine dyes with nucleic acids. 6. Synthesis and spectroscopic properties of thiazole orange—amino acids conjugates

The synthesis of amino acid, (L-Trp and L-Tyr) derivatives of thiazole orange, monomethine cyanine dye which significantly increases fluorescence intensity when bound to nucleic acids is described. Ifydroxysuccinimide ester of Cyan 6 (2-[4-N-Carboxyethyl-quinoline)-methyl]-3-methylbenr.thiazole-l,4p-toiu-enes.ulfonati:) was used for the conjugation of dye with amino acids. Interaction of obtained conjugates with nucleic acids was investigated using spectroscopic methods.

Materials and Methods.Tlie TLC was performed on Kicsclgel 60 F 254 plates («Merck», Germany) using CHCU-MeOH-AcOH (8.5:1.0:0.5)system.FAB MS analyses were carried out in the positive mode using mass spectrometer MI-1201E (PO «Elektron», Ukraine) .Spectroscopic measurements.The absorption spectra were recorded on «Specord М-40» (VEB Carl Zei.ss Jena, Germany).Fluorescence spectra were obtained vith fluorescence spectrophotometer Hitachi Mcdel 850 (Japan).Fluorescence was excited with 150W Xe-Iamp emission and measurements were carried out in thermostatable quarts: cell (0.5 * χ (1.5 cm).AU spectra were corrected by multiplying fluorescence intensities measured over an interval of 5 iino by proper correction factor for corresponding wavelengths.In corrected spectra fluorescence intensity values were prop*, rtional to a numbers of photons per unit of wavelength interval.
Preparation of DNA, RNA and dyes stock solutions.Stock solutions of dyes (2 10 3 M) were prepared by dissolving dyes in DMSO.AU dyes were stable under these conditions for several months, whereas in aqueous solutions some dyes gradually lost their fluorescence properties.Working solutions were prepared immediately prior to use.For spectral studies total calf thymus DNA («Sigma») and yeast RNA («Sigma») were used.Nucleic acids stock solutions were prepared in TE buffer (Tris-HCl, 50 mM, EDTA, 10 mM, pH 8.0) in concentration 6 IO" 3 b.p./ml for DNA and 1.2• IO" 2 b. p./ml for RNA.
Absorbance and fluorescent emission spectra.For spectral measurements the complexes of dyes with nucleic acids were obtained in Tris-HCl (50 mM, pH 8.0) buffer by mixing of dye stock solution with DNA or RNA solution.The final concentration of NA was IO" 4 and 2.0• IO" 4 mM respectively.Final dyes concentrations were 0.02 mM.Dye-nucleic acid complexes were prepared with approxymal ratio 1 dye per 20 b. p. of RNA and per 10 b. p. of DNA.For optical measurement of free cyanines the same dyes concentrations were used.
Fluorescence titration oy Cyan 6-Trp and Cyan 6-Tyr were carried out in 0.05 M Tris buffer (pH 8) with total calf thymus DN r A and yeast RNA at 540 nm for DNA and 545 nm for RNA.Fixed concentration of DNA 6• 10"' Bt' and 1.2 • IO 2 M were used for the titration of fixed concentration of dye ( Fig. 3).The dye concentration was 10 5 M. The DNA and RNA concentration was changed from 2.5-10 to IO" 4 from 5• IO" 6 to 2-Ю" 4 respectively.
General procedure for the synthesis cyanine dyeamino acids conjugates.The suet inimidyl ester of Cyan 6 was prepared by dissolving U't dye (5.34 ing, 0.01 mmol) in 1 ml of dry DMFA ai'-then adding N-hydroxysuccinimide (1.15 mg, 0.0 1 mmol) and Fij; 1 Syntlietic scheme of preparation oF dye-jrnino acid conjugates: DCC (4.12 nig, 0.02 mmol) to stirred sol Jtion at 0 °С.Mixture was stirred for 4 h.Then amino acid ester hydrochloride (0.011 mM) and 0.05 ml of dry pyridine w ere added.Reaction mixture was stirred for 2 h at 0 °С and was kept in refrigerator for 18 h.Piirifitation of amino acid-Cyan 6 conjugates was performed on the prepared C3 stationary phase [1 J. Conjugates were recovered from the fractions with a rotary evaporator at 40 °С.
Mcnornethine cyanine dyes seem to possess all characteristics; for qua iititation of unknow nucleic acids in solutions, as low intrinsic fluorescence of unbound probe, large fluorescence enhancement upon probe binding, large linear detection range, and sequence non-specific detection [5 |.
The main research of our laboratory concerns development of sequence specific homogeneous detection of nucleic acids in solution.Recently pyrylium cyanine dye -oligonucleotide conjugate was used for the detection of specific DNA sequences [6 ].Here we describe the preparation of cyanirie dye-amino acid conjugates for the potential fluorescence sequence specific determination of nucleic acids in solution.
Before some synthetic procedures for the covalent labelling of biomolecules with cyanine dyes were described by Waggoner et al. |7].Functional groups in proposed cyanine reagents were sulfhydryl [7}, isothiocyanate [8 ] and succinimidyl esters (9 ].Procedure using active N-hydroxysuccinimide esters of dye seems to be more convenient for their conjugation with α-amino group of amino acids or peptides.Besides we have shown recently that incorporation of carboxylic acids groups into basic cyanine structure of TO did not influence on fluorescence probe properties [10] (Fig. 1).
Succinimidyl ester of dye carboxyl group and methyl esters of hydrochlorides of amino acids in pyridine were used to synthesize amino acid-dye conjugates.Active esters were prepared with use the DCC and hydroxysuccinimide (HONSu) in 1DMFA.
Recently published methods for the HPLC of cyanine dyes use C, g stationary phase [111.Unfortunately, used cyanine dyes are very hydrophobic and positively charged compounds.As a result, they have large retention time on HPLC stationary phases.In or der to overcome these problems TMS-silica (C 3 ) stationary phase prepared according to [1] was successfully used (Fig. 2).Chemical structures of synthesized conjugates were confirmed by fast atom bombardment mass spectrometric (FAB MS) analyses.
Spectroscopic data for two synthesized cyanine conjugates and Cyan 6 (TO analog) are pi rented in Table .1. Dye Cyan 6 with previously investigated spectral properties was included for the comparison [9 ].Absorption spectra of Cyan 6 an its amino acid  derivatives were similar.The wavelengths of absorption maxima ( abs A mix ) of Cyan 6-Trp and Cyan 6-Tyr slightly depended ori solvent.Cyanine dyes showed the red shift <5-7 nm) after going from aqueous buffer 1o less polar DMSO.
The fluorescence of conjugates and free Cyan 6 were very low and fluorescence spectra had no clear maxima.Fluorescence intensity oi" amino acids derivatives were slightly higher as Com 1 Iared to free dye.
The data on the absorbance to і fluorescence knowledge the assistance of Ms. Vladislava Kovalska iri spectral characterisation of dye-amino acid conjugates.We tlianl; Dr. Yury Kovtun for providing us with the sample of cyanine dye Cyan 6.