Microwave synthesis and fluorescence properties of homo- and heterodimeric monomethine cyanine dyes TOTO and their precursors

ABSTRACT A series of monomeric and dimeric cyanine dyes belonging to the thiazole orange family have been prepared via an improved synthetic procedure, by the reaction of the monomethine dye containing an iodoalkyl group with tertiary diamine linkers under microwave irradiation. The effects of microwave power and irradiation time on yield were examined. The electronic absorption and steady-state fluorescence spectra of prepared dyes have been investigated. Fluorescence properties indicate significance in singlet oxygen sensitization and make the present compounds potential candidates in the area of photodynamic therapy. GRAPHICAL ABSTRACT

According to the classical method, bis-intercalating ТОТО families were synthesized by the reaction of the monomethine dye containing a haloalkyl group with tertiary diamine linkers in DMF for 12 h as minimum (5,(28)(29)(30)(31)(32)(33). In some cases, the reaction required long time and may exceed 3 days followed by the addition of methanol and keeping of the reaction mixture at 0°C and in this case, the yield of the product does not exceed 25% (3). Thus, the problem of improvement of methods for their synthesis seems to be urgent. Therefore, we report a rapid and more efficient method to synthesize these types of very important dyes by using microwave irradiation. The reported method achieves the Greenness approach in the manuscript in addition to the approach that it does not require additional recrystallization/purification steps, which are often necessary and can be demanding for these types of reactions. Microwave irradiation presents a powerful tool toward organic reactions and is known as environmentally benign method, which offers several advantages, including shorter reaction times, cleaner reaction profiles and simple experimental/product isolation procedures (34)(35)(36).
In addition to this developed synthetic method, the electronic absorption and steady-state fluorescence spectra were also reported.

Measurements
Melting points were taken on a XT-4 micromelting apparatus and are uncorrected. IR spectra were recorded with PERKIN ELMER MODEL 1720 FTIR spectrometer. 1 HNMR and 13 C-NMR spectra were measured with a Varian EM 390 and Bruker AC-250 spectrometers respectively. The chemical shifts in ppm are expressed in the δ scale using tetramethylsilane (Me 4 Si) as internal standard. Coupling constants are given in Hz. Fast Atom Bombardment Mass Spectrometry [FAB-MS] were recorded in a Micromass Autospec M, operating at 70 eV, using a matrix of 3-nitrobenzyl alcohol. UV-VIS absorption spectra were recorded on a Shimadzu UV-1700 UV-VIS spectrometer. Fluorescence spectra were recorded on a Hitachi F-4500 Spectrofluorimeter. TLC was performed on Merck silica gel 60-F 254 precoated plastic plates.

Synthetic procedure
Monomethine cyanine dyes 1 a-f were prepared in good yields via microwave-assisted solvent-free method according to our previously reported method (37).
A series of mono-intercalators dicationic thiazole orange (TO) 2 a-f and bis-intercalating tetra cationic (TOTO) 3 a-h dyes were successfully synthesized with high yields of 75-90% within 78-110 min using modified microwave irradiation at 70-120 watts in the presence of DMF and few drops of triethyl amine as a base (Schemes 1, 2).
It is necessary to emphasize that the dyes 2 a, 2 b, 2 f and 3 a were previously synthesized by classical methods according to literatures (28,29) and were used for other purposes.
The chemical structures of prepared (TOTO) 3 a-h dyes and their corresponding starting materials are listed ( Table 1).
All microwave reactions were conducted using Start S Milestone S/N 129802 microwave apparatus.
2.2.1. General procedure for preparation of dicationic cyanine diamino derivatives dyes (2 a-f ) A mixture of equivalent amount of monomethine cyanine dyes 1 (1 mmol) and corresponding diamino linker L (1 mmol) in the presence of DMF (20 mL) and few drops of triethylamine is subjected to microwave irradiation with stirring for proper time and power. The reaction progress was monitored by TLC (eluent, Pet. ether : ethyl acetate, 3 : 1). The precipitates that formed were filtered off, washed with CH 2 Cl 2 and dried at 60°C. The details of reaction conditions and yields are provided in Table 2 and the optimizing process for experimental conditions of dye 2 d is listed in Table 3.

General procedure for preparation of homodimeric TOTO's analogue dyes (3 a-e )
A mixture of monomethine cyanine dyes 1 (1 mmol) and corresponding diamino linker L (1 mmol) in the presence of DMSO (10 mL) and few drops of triethylamine was subjected to microwave irradiation with stirring for proper time and power. The precipitates that formed were filtered off, washed with hot acetone and dried at 60°C. The details of reaction conditions and yields are provided in Table 4.

Synthetic procedures
There are two main routes to modify molecular biology, especially fluorescence properties and intercalating activity of this class of dyes with DNA. The first is via modification of the tertiary di-amino linkers, and the second by changing the alkyl substituent of benzothiazole nucleus. In this communication, we applied these two improvement strategies. N,N,N',N'-tetramethyl-1,3propanediamine (TMPDA) L A ; 1,2-di(4-pyridyl)-ethane L B ; 4,4-bipyridyl L C ; N,N-dimethylcyclohexanamine L D and N,N-dimethyl-4-pyridinamine L E are five tertiary diamino linkers, which were used to modify new series of monomeric and dimeric thiazole orange (TO & TOTO) improved dyes. In addition, changing the methyl group of benzothiazole nucleus with ethyl and benzyl groups (3 d , 3 e , 3 g ) improved the fluorescence properties of this class of dyes.
Dyes 3 f,g were synthesized by equimolar reaction of monomeric thiazole orange 2 a and monomethine cyanine dyes (1 b & 1 c ). The details of reaction conditions and yields are provided in Tables 2 and 4 and the optimizing process for experimental conditions for dye 2 d is listed in Table 3.
In all investigated cases, we found that (TO) and (TOTO) dyes formation reactions preceded efficiently with high to excellent yield in short reaction time, compared with the classical refluxing method. It could be found that the yield increased obviously with prolonging irradiation time within a certain power until achieving optimized reaction time. It could also be found that the reaction yield decreases under lower power and the reaction time becomes shorter with the increase of microwave power. This indicates that the greater the microwave radiation power, the faster the reaction rate.
The constitution of the prepared compounds was secured by their elemental analysis, UV-VIS absorption spectra, IR, 1 HNMR, 13 CNMR; FAB-MS data. The most characteristic bands of FTIR spectra in KBr appeared at the range 1512-1612 cm −1 for (C=C, C=N).
The 1 H-NMR data are in accordance with the structure of synthesized dyes 2 a-f and 3 a-g . The 1 HNMR spectrum of 3 a measured in DMSO-d 6 as a representative example can be seen in Figure 2.
All 52 protons are displayed. Multiplet peak at δ = 1.32 ppm suggested for the most shielded 4 protons corresponding to 2 CH 2 groups of 1,2-di(4-pyridyl)ethane (linker B), as it is the furthest from any electronegative atoms. The two middle -CH 2 groups in propyl chain displayed as broad multiplet signal at δ = 3.32 ppm. The singlet signal at δ= 3.98 ppm for 6 protons corresponding to 2 N-CH 3 groups, these protons appeared at downfield region as they are deshielded with more electronegative nitrogen atom. The broad multiplet peak at δ = 4.81 ppm for 8 protons corresponding to 4 N + -CH 2groups, as they deshielded with nitrogen atoms. The meso-protons of the methine (2=CH) groups displayed as singlet peak at higher chemical shift 6.86 as they are close to nitrogen atom and also to sulfur. Besides, 28 aromatic protons for phenyl and pyridyl rings displayed at rang δ = 7.19 to 9.05 ppm, depending on the deshielding effect by the neighboring electronegative atoms. All 1 H-NMR spectra of all other synthesized dyes 2 a-f and 3 b-g could be interpreted as described for 3 a .
2 a-f and 3 b-g could be interpreted as described for 3 a . The 13 C-NMR spectrum of 3 a measured in DMSO-d 6 shows all 45 carbons as seen in Figure 3.
All 13 C-NMR spectra of all synthesized 2 a-f (TO) and 3 b-g (TOTO) dyes could be interpreted using the same rules as described for 3 a dye.
The structures of all synthesized dyes were confirmed by 1 H-NMR, 13 C-NMR, FTIR spectra, besides the correct elemental analysis and mass spectrum data, the elemental analysis of synthesized dyes showed correct analytical data.

Fluorescence spectral study for some synthesized dyes
The electronic absorption spectra of the studied cyanine dyes are shown in Figures 4-9. The dyes are generally characterized by very small values of Stoke's shifts between absorption and emission spectral bands indicating that the absorption and emission photons exhibit close frequencies. Other emission broad bands in the near IR spectral range are also obtained that are attributed to phosphorescence and are good indication of triplet state formation. This becomes of great significance in singlet oxygen sensitization and makes the present compounds as potential candidates in the area of photodynamic therapy (PDT) (38)(39)(40).
At higher energies, a second excited electronic state absorption occurs around 290 nm. This second electronic state gives its characteristic fluorescence at around 390 nm. This is yet a peculiar behavior of these compounds since fluorescence dominates the internal conversion (ic) photophysical process.
The electronic absorption spectra of some compounds show two-split absorption peaks which are assigned to the first singlet-state absorption of monomeric and J-aggregates of the dye, which is a common phenomenon of many cyanine dyes (41,42). Cyanine molecules can form aggregates. Depending on the molecular orientation in these aggregate, J-and H-aggregates are formed. In J-aggregate, the molecules are aligned in a head to tail arrangement. In H-aggregate, molecular alignment is sidebyside. J-aggregates are characterized by sharp spectral bands that are red shifted with respect to the monomer and by a strong photoluminescence with almost zero stokes shift (43).

Compound [2 a ]
The electronic absorption spectrum of compound 2 a ( Figure 5) shows two-split absorption peaks at 490 and 505 nm, which are assigned to the first singlet-state absorption of monomeric and J-aggregates of the dye (41,42). These lower energy J-aggregates give a symmetrical fluorescence peak of emission maximum at 548 nm ( Figure 5). The symmetry of this peak, together with the fact that its spectral pattern does not alter upon excitation at 480 nm (absorption of monomeric species) or 510 nm (absorption of J-aggregates), indicates an energy transfer from higher energy monomeric species to lower energy aggregates during excited state lifetime. Like cyanine dyes, the compound is characterized by very small values of Stoke's shifts where absorption and emission photons exhibit close frequencies.
Another emission broad band in the spectral range 700-900 nm is also obtained that is attributed to   phosphorescence and is a good indication of triplet state formation. This becomes of great significance in singlet oxygen sensitization.
At higher energies, a second excited electronic state absorption occurs at 290 nm. This second electronic state gives its characteristic fluorescence at 390 nm. This is yet a peculiar behavior of this compound since fluorescence dominates the internal conversion (ic) photophysical process.

Compound [2 b ]
The electronic absorption spectrum of compound 2 b shows absorption peaks at 510 nm; a first excited electronic state absorption occurs at 510 nm. This first electronic state gives its characteristic fluorescence peak of emission maximum at 542 nm (upon excitation wavelength 480 nm).
Another emission broad band in the spectral range 680-900 nm is also obtained.

Compound [2 e ]
The electronic absorption spectrum of compound 2 e (Figure 7) shows absorption peak at 508 nm; this singlet-state absorption gives a symmetrical fluorescence peak of emission maximum at 544 nm. The spectral pattern does not alter upon excitation at 480 or 508 nm.
Another emission broad band in the spectral range 680-900 nm is also obtained that is attributed to phosphorescence.
At higher energies, a second excited electronic state absorption occurs at 288 nm. This second electronic state gives its characteristic fluorescence at 388 nm ( Figure 7). This is yet a peculiar behavior of this compound since fluorescence dominates the internal conversion (ic) photophysical process.

Compound [3 c ]
The electronic absorption spectrum of compound 3 c (Figure 8) shows absorption peaks at 480 and 505 nm, which are assigned to the first singlet-state absorption of monomeric and J-aggregates of the dye (41)(42). These lower energy J-aggregates give a symmetrical fluorescence peak of emission maximum at 560 nm. The symmetry of this peak together with the fact the its spectral pattern does not alter upon excitation at 480 nm (absorption of H-aggregates) or 505 nm (absorption of monomeric species) indicates an energy transfer from higher energy monomeric species to lower energy aggregates during excited state lifetime.
Another emission broad band in the spectral range 675-900 nm is also obtained that is attributed to phosphorescence.
At higher energies, a second excited electronic state absorption occurs at 289 nm ( Figure 8). This second   electronic state gives its characteristic fluorescence at 385 nm. This is yet a peculiar behavior of this compound since fluorescence dominates the internal conversion (ic) photophysical process.

Compound [3 f ]
The electronic absorption spectrum of compound 3 f (Figure 9) shows absorption peak at 508 nm, a first excited electronic state absorption occurs at 508 nm, which gives its characteristic fluorescence peak of emission maximum at 552 nm upon excitation wavelength 480 nm. Another emission broad band in the spectral range 680-900 nm is also obtained that is attributed to phosphorescence.
At higher energies, a second excited electronic state absorption occurs at 288 nm. This second electronic state gives its characteristic fluorescence at 387 nm ( Figure 9). This is yet a peculiar behavior of this compound since fluorescence dominates the internal conversion (ic) photophysical process.
Another emission broad band in the spectral range 680-900 nm is also obtained that is attributed to phosphorescence.

Compound [3 g ]
The electronic absorption spectrum of compound 3 g (Figure 10) shows absorption peak at 509 nm, a first excited electronic state absorption occurs at 509 nm. This first electronic state gives its characteristic fluorescence peak of emission maximum at 560 nm upon excitation wavelength 480 nm.
At higher energies, a second excited electronic state absorption occurs at 288 nm. This second electronic state gives its characteristic fluorescence at 385 nm.

Conclusions
We have described a rapid and highly efficient method for the synthesis of monomethine cyanine dyes with quinoline nucleus under microwave irradiation.
Both microwave-assisted reactions under solvent-free conditions and microwave-assisted reactions using (organic) solvents were used to synthesize a series of monomeric, homo and hetrodimmeric monomethine cyanine dyes. The Microwave technique showed several advantages such as rapid reactions, high purity of products, less side-products, improved yields and simplified and improved synthetic procedure.
The electronic absorption and steady-state fluorescence spectra of prepared dyes revealed a potential use of these dyes as singlet oxygen sensitizers. The prepared dyes absorb in the region 477-516 nm and their fluorescence emissions are located at 542-900 nm. The dyes strong absorption peak around 500 nm coincides with green laser PDT that applies Ar + laser of λ = 488 nm in PDT treatment (44).

Disclosure statement
No potential conflict of interest was reported by the authors.  spectrometry, environment, multistep synthesis, heterocyclic chemistry, natural product chemistry. He published more than 65 publications in international journals.

Notes on contributors
Mahasen S. Amine is currently Professor Emeritus of Organic Chemistry, Faculty of Science, Chemistry Department Benha University, Egypt. Her research has focused on the organic synthesis, green chemistry, multistep organic synthesis, heterocyclic chemistry, natural product chemistry. She published 61 publications in international journals and works as a reviewer for a number of international indexed journals.