Synthesis , Absorption and Fluorescence Spectral Characteristics of Trinucleus Dimethine Cyanine Dyes as Fluorescent Probes for DNA Detection

Neste trabalho descreve-se a preparação de seis corantes cianina dimetina trinucleares com núcleos de piridina, obtidos a partir da condensação de iodeto de trimetilpiridínio com aldeídos aromáticos heterocíclicos. As propriedades de absorção e fluorescência dos corantes foram estudadas em solventes com polaridades diferentes. O deslocamento hipsocrômico dos máximos de absorção dos corantes foi observado com o aumento da polaridade do solvente. Foram estudadas as propriedades fluorescentes dos corantes em solução e na presença de DNA. Foi observado um aumento significativo do rendimento quântico de fluorescência na presença de DNA para quatro corantes.. Em particular, um dos corantes emite fluorescência fraca no tampão Tris-HCl, apresentando contudo fluorescência intensa na presença de DNA.


Introduction
[8][9][10] From the applications of cyanine dyes it is known that the optical properties of cyanine dyes are often influenced by their conjugated systems and the solution environment.Trinucleus dimethine cyanine dyes are large conjugated systems, which contain three heteroaromatic rings joined by two vinyl chains.However the synthesis of trinucleus dimethine cyanine dyes and their applications as fluorescent probe were rarely reported. 11,12][15][16][17] In this paper, six trinucleus dimethine cyanine dyes with pyridine nucleus, four dyes of which were novel, were synthesized in water or ethanol via the condensation of appropriate heteroaromatic aldehydes with quaternary salts of heterocyclic compounds containing active methyl groups.The influence of different solvents on spectral properties of the trinucleus dimethine cyanine dyes was studied by UV-Vis and fluorescence spectroscopy.A further objective of this study was to investigate the fluorescence spectral properties of prepared trinucleus dimethine cyanine dyes both in solution and in the DNA presence, exploring their future applications as fluorescence probe.

General
All reagents were obtained from commercial sources and used without further purification.All chemicals were of analytical grade.Melting points were taken on a XT-4 micromelting apparatus and uncorrected.IR spectra in cm -1 were recorded on Shimadzu IRPrestige-21 spectrometer and Bruker Equiox-55 spectrometer. 1 H NMR spectra were recorded at 400 MHz on a Varian Inova-400 spectrometer and chemical shifts were reported relative to internal Me 4 Si.

13
C NMR spectra were recorded at 100 MHz on a Varian Inova-400 spectrometer and chemical shifts were reported relative to internal Me 4 Si.Elemental analysis was performed with Vario EL-III instrument.The electron impact (EI) mass spectra were recorded at 70 eV with a GCMS-QP2010 system equipped with the solid sample direct insertion probe.The absorption spectra were recorded on a Shimadzu UV-1700 UV-Vis spectrometer.Fluorescence measurements were carried out on a Hitachi F-4500 spectrofluorimeter.

Measurements of the spectral properties of the dyes in different solvents
The dye stock solutions (5.0×10 -4 mol L -1 in dimethylsulfoxide (DMSO)) were diluted with different solvents and resulted in working solutions of dyes (2.0×10 -5 mol L -1 ).The absorption spectra were examined at room temperature in different solvents and recorded using 1 cm quartz cells on a Shimadzu UV-1700 UV-Vis spectrometer.Fluorescence measurements were carried out at room temperature on a Hitachi F-4500 spectrofluorimeter in 1 cm quartz cells.Fluorescence emission was excited at the maximum of the absorption.The absorption and fluorescence spectral data were listed in Table 1.

Measurements of spectral properties of the dyes in the presence of DNA
Dye stock solutions (2.0×10 -4 mol L -1 ) were prepared by dissolving the dyes in DMSO and further diluted with Tris-HCl buffer (pH 7.4) to result in working solutions of dyes (1.0×10 -5 mol L -1 ).Stock solution of DNA was prepared by dissolving salmon sperm DNA in 0.05 mol L -1 Tris-HCl buffer.The concentration of DNA in stock solution was 3.1×10 -3 mol L -1 base pairs (bp).The fluorescence of dyes in the presence of DNA was tested by adding 0.5 mL DNA solution (3.1×10 -3 mol L -1 bp) into 1.0 mL 1.0×10 -5 mol L -1 working solutions of dyes.All working solutions were prepared immediately before the experiment.
The absorption spectra were examined at room temperature in different solvents and recorded using 1 cm quartz cells on a Shimadzu UV-1700 UV-Vis spectrometer.Fluorescence measurements were carried out at room temperature on a Hitachi F-4500 spectrofluorimeter in 1 cm quartz cells.Fluorescence emission was excited at the maximum of the fluorescence excitation spectrum.The absorption and fluorescence spectral date were listed in Table 2 and Table 3.

Synthesis
The trinucleus dimethine cyanine dye was synthesized via the reaction of heteroaromatic aldehydes with the quaternary ammonium salt of 1,2,4-trimethyl pyridine quaternary salt or 1,2,6-trimethyl pyridine quaternary salt (Scheme 1).In all cases investigated, we found that the formation reactions of trinucleus dimethine cyanine dyes with pyridine nucleus proceeded efficiently under catalysis of piperidine or NaOH, and the better and purer yields were obtained in ethanol for 3a-3d and in water for 3e-3f.For example, the yield of 3d could reach 86% in ethanol, and 65% in water. 12The yield of 3f could reach 83% in water, but in ethanol 3f could hardly be obtained.Because in organic solvents furaldehyde reacted with quaternary salt so quickly that the reaction could not be controlled, and there would be serious side reactions.In the synthesis of dyes 3a-3f , the required reaction time of heterocyclic aromatic aldehydes with trimethylpyridinium iodides was furaldehyde (30 min) < 1-methylpyrrole-2-carboxaldehyde (20 h) < indole-3-carboxaldehyde (24 h), thereby the reaction activity of heterocyclic aromatic aldehydes was furaldehyde > 1-methylpyrrole-2-carboxaldehyde > indole-3-carboxaldehyde. From the structure it was also suggested that the electron-withdrawing ability of the group attached to aldehyde group was =C−O > =C−N > =C−C=, leading to the positive charge density of carbon in aldehyde group was furaldehyde > 1-methylpyrrole-2-carboxaldehyde > indole-3-carboxaldehyde.The larger the positive charge density of carbon in aldehyde group is, the higher the reaction activity of heterocyclic aromatic aldehyde with trimethylpyridinium iodides is.

Spectral properties of the dyes in different solvents
Figure 1 gives the absorption maxima (λ max ) of six trinucleus dimethine cyanine dyes in different solvents (their dielectric constant: CHCl 3 4.9, EtOH 24.6, MeOH 32.6, dimethylformamide 38.3, H 2 O 78.4).From Figure 1 it could be found that the λ max of 3a, 3b, 3c and 3d was longer than the λ max of 3e and 3f.The reason suggested here was that dyes 3a-3f were all D-π-A molecules, in dyes 3a-3d the electron donor was N of indole or pyrrole ring, and the electron donor was O of furan ring in dyes 3e-3f, and the electron acceptor of six dyes was all N + of pyridinium.The stronger was electron-donating capability of electron donor, the longer was the λ max of dyes under the same electron-withdrawing capability of electron acceptor.The electron-donating capability of N situated indole or pyrrole ring was stronger than that of O situated furan ring, so the λ max of 3a-3d was longer than the λ max of 3e-3f.It could be also found that with the increasing of solvent polarity the λ max of the dyes decreased.The effect of the solvent polarity on the absorption maximum could be illustrated by interactions between the dye molecules and the solvents, as the interactions made the ground state of dye more stable by forming hydrogen bonds. 18,19e absorption, excitation maxima (λ ex ), fluorescence maxima emission (λ em ) of the dyes are summarized in Table 1.The dyes exhibited fluorescence properties at room temperature.Their fluorescence maxima were located at 502-565 nm.Compared with the absorption maxima of the dyes, the emission spectra were shifted to the red by 69-140 nm (Stokes shift).The stokes shift of six dyes were all large, this might be attributed to an excited-state intramolecular charge transfer between the donor and acceptor in the dyes.Large stokes shift could help to reduce self-quenching and measurement error by excitation light and scattered light. 20The fluorescence quantum yield of six dyes was in the region 0.0001-0.0781 in different solvents.From Table 1 it could be found that the order of fluorescence quantum yield was 3a, 3b > 3c, 3d.The possible reason was that the conjugated system of 3a, 3b was larger than that of 3c, 3d.We also found the order of fluorescence quantum yield was 3a > 3b and 3c > 3d.The cause lay in the fact that two D-π-A system in 3b and 3d was in opposite orientation, which made their conjugated system became small.It could be found that the fluorescence performance 3e and 3f was complex, because oxygen of furan ring was of both electron-donating and electron-acceptor effect.And there was also influence of solvents on the fluorescence quantum yield of these dyes.Further investigation of the influence of solvents on the fluorescence quantum yield of the dyes is in progressing.

Spectral properties of the dyes in the presence of DNA
The spectral properties of the dyes in the presence of DNA are summarized in Table 2 and Table 3.The λ max of DNA-dye solution was situated at 401-458 nm and showed a slight red shift relative to the corresponding maxima of free dyes in buffer.The molar extinction coefficients for dyes 3a-3d were increased in the presence of DNA, and were in the range from 3.2×10 4 to 8.5×10 4 L mol -1 cm -1 .However, for dyes 3e and 3f the values of molar extinction coefficients were unchanged relative to free dyes, which were 3.8×10 4 and 4.2×10 4 L mol -1 cm -1 , respectively.
The fluorescence emission maxima of DNA-dyes were located at 503-555 nm, and showed a slight blue shift relative to free dyes in solution.Stokes shift values for the DNA-dyes were in the range of 97-109 nm.Moreover, the fluorescence intensity of dyes 3a-3c was greatly increased in the presence of DNA.Compared with free dyes, the quantum yields of DNA-dyes were up to 9.5 times for dye 3a, up to 31.6 times for dye 3b (Figure 2) and up to 18.9 times for dye 3c.Specially, the quantum yields of DNA-dye 3a was the highest in six dyes.It was noteworthy that free dye 3d could not be detected significantly fluorescence in buffer, but DNA-dye 3d showed great fluorescence quantum yields.The fluorescence enhancement of trinucleus dimethine cyanine dyes bound to DNA was attributable to the fact that on photoexcitation a lack of free rotation around the internuclear bridge made isomerisation around the C-C bonds of the methine chain difficult; and subsequently nonradiative deactivation of the excited state was not possible, causing the dye to fluoresce. 22,23Upon binding with DNA, 3a-3d maintained their high Stokes shift and showed a red shift, owing to a more efficient ICT in the excited state between the terminal heterocyclic aromatic and the pyridimium group. 24,25

Conclusions
Six trinucleus dimethine cyanine dyes with pyridine nucleus were synthesized and isolated in 69-86% yield with piperidine or NaOH as catalyst.The absorption maxima of the dyes were located at 397-487 nm in different solvents, and with the increase of solvents polarity the maximum absorption wavelength of these dyes had a blue-shift.The fluorescence maxima of the dyes in different solvents were basically located at 502-565 nm, and the fluorescence quantum yield of six dyes was in the region 0.0001-0.0781 in different solvents.
The absorption maxima of the dyes in the presence of DNA were situated at 401-458 nm and showed a slight red shift relative to free dyes.The fluorescence maxima of DNA-dyes were located at 503-555 nm, and showed a slight blue shift relative to free dyes.The fluorescent quantum yields of the DNA-dyes 3a-3c were up to 9.5-31.6times higher than that of free dyes.Specially, free dye 3d emitted weak fluorescence in buffer, but DNA-dye 3d showed great fluorescence quantum yields.Therefore dyes 3a-3d could be proposed as fluorescent dyes for DNA detection.

Figure 1 .
Figure 1.Solvatochromic shifts for the λ max of six trinucleus dimethincyanine dyes in five kinds of solvents.

Figure 2 .
Figure 2. Fluorescence spectra of 3b in buffer, in the presence of different concentration of DNA.

Table 1 .
The absorption and fluorescence spectral characteristics of dyes 3a-3f in different solvents λ max /nm λ ex /nm λ em /nm Stokes shift

Table 2 .
21ectral characteristics of dyes 3a-3f buffer The fluorescence quantum yields of the dyes were determined by the reference standard (rhodamine B Φ F = 0.49 in ethanol at 25 o C).21 a

Table 3 .
21ectral characteristics of dyes 3a-3f in presence of DNA The fluorescence quantum yields of the dyes were determined by the reference standard (rhodamine B Φ F = 0.49 in ethanol at 25 o C).21 a