Synthesis and Characterization of Heptamethine Cyanine Dyes

A series of meso-chlorine substituted heptamethine-cyanine dyes and related heptamethine cyanine dyes unsubstituted on the vinyl chain were synthesized by condensation of 2,3,3-trimethyl indolenium salts with reactive intermediates, 2-chloro-formyl-3-hydroxy-methylenecyclohexene or N-[5-(phenylamino)-2,4-phentadienylidene] aniline monohydro-c hloride. The structures of the dyes were characterized by virtue of 1HNMR, 13C-NMR, IR, MS, etc. The reaction conditions and physicochemical properties of the dyes were extensively explored with classical photo-physical techniques


Introduction
The indotricarbocyanine dyes, due to their relative stability, high molar extinction coefficient, high fluorescent intensity etc., have a wide application in various fields, such as near-infrared laser dyes [1], fluorescent labelling agents for proteins [2][3], fluorescent tags in DNA sequencing [4], immunoassay [5], and flow cytometry [6], as well as sensitizers for various silver halide emulsions [7] and optical recording medium [8].These applications of indotricarbocyanine dyes take advantage of the property that indocyanine dyes are more stable to light and heat than other cyanine dyes containing the same chromophore and non-indolenine ring, e.g.benzoxazole, benzothiazole and thiazoline.It is known that incorporating more methine groups in the ring can increase the stability of the tricarbocyanine.The physicochemical properties of various indotricarbocyanines containing methine groups in different bridge rings have been studied in several patents [9].The majority of them are unsubstituted on the indolenine ring, and the systematic investigation of tricarbocyanine dyes carrying different substituents on indolenine ring has not yet been observed.In this paper, we report the synthesis of a series of indotricarbocyanine dyes, investigate both the synthetic conditions and the physicochemical properties and also show the correlation of the structure of the dyes with their properties.

Results and Discussion
The structures of the dyes studied in this paper are shown in scheme 1.They were synthesized, as outlined in Schemes 1 and 2, according to a general route for synthesis of heptamethine cyanine dyes [10].

Synthesis of cyanine dyes
A substituted hydrazine, formed by diazotisation of βnaphthyl-amine or p-substituted anilines with NaNO 2 then reduction of the diazonium salt with stannous chloride or sodium sulfate, is condensed with methyl isopropyl ketone in the Fischer indole synthesis [11].
Methylation with methyl iodide gives substituted indolinium salts, the purity of the salts recrystallized from ethanol was examined by elemental analysis.The reaction conditions are collated in Table 1.Furthermore, the indolinium salts react with 2-chloro-formyl-3-hydroxymethylenecyclohexene in a mixture of glacial acetic acid and acetic anhydride to produce the cyanine dyes 5a−5f, in which odd-numbered carbon chain is cyclic.The reaction generally requires 15−60 min for completion.The crude reaction products were purified by washing with cooled acetone and alcohol, recrystallizing from ethyl alcohol or methanol.A high yield was obtained with a ratio of indolinium : intermediate : sodium acetate = 2 : 1 : 2, and a ratio of glacial acetic acid and acetic anhydride = 4:3.It was found (see Table 2) that by using the mixture as solvent and sodium acetate as reaction catalyst the reaction with indolinium iodide (4a, 4b and 4d) afforded the dye 5a, dye 5b and dye 5d in excellent yields (entries 1, 2 and 4), but the dye 5e in low yield (36%).The results clearly indicate that a nitro group on the indolenine ring renders a decreased reactivity of the heterocycle 5e.
The indolenium salts react with N-[5-(phenylamino)-2,4-phentadienylidene] aniline monohy-drochloride in a mixture of pyridine, acetic anhydride and N,N-dimethylformamide to give dye 5g and dye 5h, in which the vinyl chain is unsubstituted.The synthetic conditions and elemental analysis data of the dyes purified are shown in Table 2 together with melting points.

NMR spectra
Table 3 collates the 1 HNMR spectroscopy data of the synthesized dyes.From the 1 HNMR spectrum of 5d, the signals of the alkyl hydrogen atom are easily recognized, N-CH 3 protons (s, 6H) at δ=3.35 and 3-H (s, 12H) at δ=1.7ppm, the signal of 3-H (t, 4H)at δ=2.7 as well as the signal of 4-H (m, 2H) at δ=1.85,In the vinyl link of the dyes, the signal of 1-H (d, 2H) is at 6.3 ppm, whereas the signal of 2-H (d, 2H) is at 8.2 ppm.It suggests that the electronic charges are distributed alternately on the conjugation link.For the benzene ring protons, there are two signals apparently ascribed to c-H and d-H.As general concept, we assigned the lower field doublet at 7.50 to d-H, δ=7.40 to c-H.The high field singlet at δ =7.8 was assigned to a-H.
In the naphthalene ring For dye 5g, there are eleven 13 CNMR peaks in the sp 2 hybrid range, three peaks in the sp 3  Comparing the δ-values at the same position of hydrogen, it can be seen from Table 3 that the electronwithdrawing substituents on the 5-position of indolenine cycle shifted the δ (ppm) value of the dye to lower field.On the contrary, the electron-donating substituents apparently make the δ (ppm) value of the dyes shift to higher field.

UV spectra
Table 4 shows the electric spectra of the dyes (5) in methanol.The polymethine dyes (dye 5a to dye 5f) containing different substituents exhibit different absorption and fluorescence maxima.And the absorption and emission maxima of dye 5b to dye 5f are at longer wavelength range than that of dye 5a; the bathochromic order induced by the substituents (R) is as follows: It is understandable that the absorption and emission maxima of dye 5b, dye 5c, dye 5d and dye 5f, carrying chloro, methyl, methoxyl and benzo-respectively, increases as the capacity for electron-donating of the substituents (R) increases.Whereas, dye 5e gives an increase in the number of absorption bands accompanied by a red shift of 20 nm.The main cause is that the introduction of a nitro group increases the delocalizing range of the π-electrons in the chromophore of the dyes and decreases the excitation energy of the dyes.As a result, the absorption and emission maxima of dye 5e shows a bathochromic shift compared with dye 5a (R = H), which was also explained in our earlier work for a series of indocarbocyanine dyes [12].The λ max ab −λ max em value of the dyes is 20-25 (nm) (see Table 4).Furthermore, comparing the absorption and emission maxima of dye 5a and dye 5d with that of dye 5g and dye 5h respectively, we found that the absorption and emission maxima of dye 5a and dye 5d are at 32 −40 nm longer wavelength than those of dye 5g and dye 5h.This indicates that the conjugated chain cyclisized and substituted by chloro can cause the electronic energy of the dyes to decrease, and consequently, increase the stability of the dyes.
The snthesis of 5g and 5h will be reported in detail elsewhere.

Experimental
Absorption spectra of dyes in methanol solution were recorded with a Hitachi−557 spectrophoto-meter and a Hitachi UV−8451A spectrophotometer at room temperature (20°C).The experimental results are compiled in Table 1.Fluorescence spectra of the cyanine dyes in methanol solution were recorded with a Rini-MPF-4 spectrophotofluorimeter. 1 H and 13 CNMR spectra were taken with a 300 MHz Varian XL200, using TMS as internal standard and DMSO-d 6 as solvent.Elemental analysis was performed by the Institute of Chemistry, Analytical Laboratory.Melting points were determined on a micro-melting point apparatus and are uncorrected.IR spectra were run in KBr discs on a Perkin-Elmer 683 spectrometer.

Synthesis of indolenium iodide 4
A solution of indolenine and iodomethane in anhydrous trichloromethane was heated for 8-12 h at 90-100°C in a sealed tube.The trichloromethane was removed by evaporation.The solid was washed with water and diethyl ether respectively.and recrystallized from ethanol.The purity of the salts 4 was tested by elemental analysis.The infrared spectroscopy of the salt molecules was also measured.The IR spectra showed typical aromatic absorption (benzen 1600−1610, 1500, 1400, 800 or so,) The main fragment peak value of Mass Spectra are shown in Table 1.which are much correspond to the structures of 4.

General procedure for the preparation of dyes 5a-5f
To a mixture of indolinium salt and 2-chloro -formyl-3hydroxymethylene-cyclohexene sodium acetate was added.The mixture was refluxed under the conditions given in Table 2.The progress of the reaction was monitored by measuring the Vis-near-IR spectroscopy After the mixture was allowed to cool to room temperature, filtered, washed with acetone and a small amount of cooled ethanol.and dried at room temperature to give the crude dye.The analytical samples were obtained by recrystallization twice from methanol.In the case of dye 5e, the crude product was recrystallized from ethanol and acetonitrile.The analytical data and physical properties of dye 5a to dye 5f are given in Tables 2-4.

Table 4 .
The electronic spectra data of the dyes.