Pyrene-BODIPY-substituted novel water-soluble cyclotriphosphazenes: synthesis, characterization, and photophysical properties

In the present work, pyrene-boron-dipyrromethene (BODIPY)-substituted novel water-soluble cyclotriphosphazene derivatives (6 and 7) were synthesized by click reactions between a cyclotriphosphazene derivative with a hydrophilic glycol side group (2) and BODIPYs (4 and 5). All of the new compounds (2, 6, and 7) were characterized by Fourier-transform infrared and nuclear magnetic resonance spectroscopy, as well as mass spectrometry and elemental analysis. The photophysical properties of the BODIPY-substituted cyclotriphosphazenes (6 and 7) were investigated by UV-Vis and fluorescence emission spectroscopy in water and water/solvent mixtures. It was found that the target compounds were soluble in water and could be potential candidates as water-soluble fluorescent dyes for the desired applications.


Introduction
Water-soluble fluorescent compounds are generally used to define and label targets in aqueous systems [1].
Compared to other fluorescent dyes, boron-dipyrromethene (BODIPY) dyes have special features, like strong absorption in the visible region, narrow emission bands, high quantum yields, and high stability [2][3][4][5]. BODIPY dyes mainly have good solubility in organic solvents, but not in water [6]. On the other hand, a great deal of applications, such as fluorescence imaging and metal ion detection, are done in aqueous media and are frequently inhibited by the weak solubility of these dyes in water [7][8][9][10][11]. In general, there are 2 options to overcome this problem: one is linkage of water-soluble groups to the BODIPY core [12] and the other is linkage of the BODIPY dyes to a water-soluble compound [13,14].
Recently, there has been substantial interest in cyclophosphazenes because they not only have an extensive stability range but they can also ensure excellent photophysical properties in combination with suitable fluorescent dyes, such as BODIPYs [15][16][17][18][19] and pyrene derivatives [20][21][22]. Hexachlorocyclotriphosphazene is a versatile starting scaffold for the synthesis of new compounds, as the chlorine groups attached to the phosphorus atoms are easily substituted by various nucleophiles to form reactive cyclotriphosphazenes. According to the literature, phosphazene compounds generally have good solubility in organic solvents. In order to make these compounds water-soluble, hydrophilic groups should be present in the molecular structure. There are several examples of water-soluble phosphazenes in the literature, but to the best of our knowledge, most of them are focused on polyphosphazenes [23][24][25][26][27][28]. Thus, the design and synthesis of water-soluble cyclotriphosphazene compounds is particularly important for studies in aqueous media.
This study aimed at the synthesis of water-soluble novel cyclotriphosphazene compounds (6 and 7) with fluorescent properties. For this purpose, mono-and distyryl pyrene-substituted BODIPYs (4 and 5) were used to introduce fluorescent properties to the cyclotriphosphazene core and diethylene glycol methyl ether (DEGME) was used to make these compounds water-soluble. Novel cyclotriphosphazene compounds (6 and 7) (Figure 1) were defined using EA, FTIR, MS (Figure 2), and 1 H, 13 C, and 31 P NMR ( Figure   3) spectroscopy. Photophysical properties of these cyclotriphosphazenes were investigated by UV-Vis and fluorescence spectroscopy and compared with their precursor BODIPYs in water and different water-miscible solvents.

Results and discussion 2.1. Synthesis and structural characterization of the compounds
Water-soluble novel cyclotriphosphazene compounds 6 and 7 were prepared according to the Scheme. First, hexachlorocyclotriphosphazene was allowed to react with DEGME at a ratio of 1:5 and compound 1 was obtained. Next, compound 2 was prepared by treating 2-azido-1-ethanol with compound 1. Pyrene-substituted BODIPY compounds 4 and 5 were also prepared according to the Scheme. Finally, cyclotriphosphazene compound 2 underwent click reactions with BODIPYs (4 and 5) in the presence of CuBr and pentamethyldiethylenetriamine (PMDETA), and novel cyclotriphosphazene compounds 6 and 7 were obtained, as shown in Figure 1. Column chromatography was used for purification of the products. The synthesized compounds were characterized by EA, FTIR, MS, and 1 H, 13 C, and 31 P NMR spectroscopy. Mass spectra of compounds 6 and 7 are given in Figure 2. In both spectra, molecular ion peaks were related to the calculated values. 31 P NMR spectra of  compounds 6 and 7 were shown as the AB 2 spin system, owing to the different environments of the 2 different phosphorus atoms on the cyclotriphosphazene ring ( Figure 3). However, it was not possible to calculate the coupling constants due to the complexity of the spectra.

Photophysical properties of compounds
Photophysical properties of compounds 6 and 7 and their counterpart BODIPYs 4 and 5 were investigated using UV-Vis and fluorescence spectroscopy. Spectroscopic evaluation of the compounds was performed in water and different water-miscible solvents, such as methanol (MeOH), ethanol (EtOH), acetone, tetrahydrofuran (THF), acetonitrile (MeCN), dimethylformamide (DMF), and dimethyl sulfoxide (DMSO). BODIPY compounds 4 and 5 were not soluble in water, but DEGME and pyrene-BODIPY-substituted novel cyclotriphosphazene compounds 6 and 7 were soluble in water. The solubility of compounds 4-7 in water and in THF is given in Figures 4a and  4b, respectively. The absorption and normalized absorption spectra of compounds 6 and 7 in water and watermiscible solvents are given in Figures 5 and 6. The emission and normalized emission spectra of compounds 6 and 7 in water and water-miscible solvents are given in Figures  to changing polarities [29]. However, the fluorescence emission spectra obtained in more polar solvents (water, MeOH, EtOH, MeCN) showed negative solvatochromism. This negative solvatochromism has been observed in previously reported pyrenyl systems and is probably due to the disruption of the π -stacking interactions in more polar solvents [30]. In addition, the absorption and emission spectra of the precursor BODIPYs (4 and 5) in water and water-miscible solvents are demonstrated in Figures 9-12. The absorption and fluorescence spectra of 6 and 7 were similar to the pyrene-substituted BODIPY precursors, since the DEGME moiety did not have any optical properties in the UV-Vis region. It is well known that cyclotriphosphazenes are optically transparent in the UV-Vis region and their photophysical properties can be adjusted according to an attached moiety [31,32]. Absorptions and emissions of compounds 6 and 7 and their precursor BODIPYs (4 and 5) were investigated in THF, which is a good solvent for all compounds, at a concentration of 1 × 10 −6 M. As can be seen in Figures 13 and 14, the difference between the absorption and emission maxima of compounds 4 and 6 was 9 nm. However, the difference between the absorption maxima of compounds 5 and 7 was 30 nm, and the difference between the emission maxima of compounds 5 and 7 was 47 nm. These bathochromic shifts were presumably due to ππ interactions of the pyrene components that were more effective on distyryl (5 and 7) than monostyryl (4 and 6) compounds because of the increased number of pyrene components. The fluorescence quantum yields ( ΦF) of compounds 4-7 in THF were predicted by comparison with rhodamine 6G in water ( Φ F = 0.95) [33] and ZnPC in DMSO ( ΦF = 0.18) [34] as standards for BODIPY derivatives.

Conclusion
In the current study, 2 novel cyclotriphosphazene compounds (6 and 7) were synthesized using click reactions and characterized by FTIR, MS, 1 H, 13 C, and 31 P NMR spectroscopies and EA. The photophysical properties of these compounds (6 and 7) were investigated by UV-Vis and fluorescence spectroscopy and compared with their corresponding BODIPY (4 and 5) moieties. The fluorescence quantum yield ( ΦF) values of novel cyclotriphosphazenes 6 and 7 were determined and their fluorescence lifetimes were directly measured. These novel cyclotriphosphazene compounds had solubility in water and good solubility in water-miscible solvents.

Synthesis of compound 2
2-Azido-1-ethanol (84 mg, 1.5 mmol) in 10 mL of THF was put into a 2-necked round-bottomed flask and cooled in an ice bath under Ar atm. NaH (46 mg, 3 mmol) in 20 mL of THF was added to the flask slowly.
Finally, trimeric compound 1 (500 mg, 1 mmol) in 20 mL of THF was added slowly and stirred for 24 h at room temperature (rt). The reaction mixture was filtered and the solvent was evaporated using a rotary evaporator.

Synthesis of compound 4
A

Synthesis of compound 7
According to the above procedure, compound 7 as a dark-green fraction (60 mg; 25%) was obtained using