Efficient two-step synthesis of water soluble BODIPY–TREN chemosensors for copper(ii) ions

Molecular Design and Synthesis, Dep Celestijnenlaan 200f – bus 02404, 3001 kuleuven.be Instituto de Ciencia Molecular, Departamen Valencia, C/Catedrático José Beltrán 2, 4 enrique.garcia-es@uv.es † Electronic supplementary information (E characterization data, absorption and em and cell permeability study. See DOI: 10.1 ‡ Current address: Institute of Nan “Demokritos”, Terma Patr. Gregoriou & Attikis, Greece. Cite this: RSC Adv., 2017, 7, 3066


Introduction
BODIPY dyes (4,4-diuoro-4-bora-3a,4a-diaza-s-indacenes, also known as boron dipyrrins) 1 are an important class of uorophores with many excellent characteristics, such as a bright uorescence in the visible spectral range and a good robustness towards light and chemicals. 2 Another major attraction of these dyes is their rich functionalization chemistry, 3 allowing practically unlimited structural modication which leads to sophisticated dyes with ne-tuned chemical and spectroscopic properties. 4 Recently, several C-H functionalization strategies for boron dipyrrins have been reported. 5-7 Particularly promising in this regard are the nucleophilic substitution of hydrogen 6 and the radical functionalization reactions 7 developed in our group. Their mild reaction conditions and broad substrate scopes make these protocols useful strategies to efficiently functionalize BODIPY dyes. These excellent properties and functionalization potential contribute to the growing importance of boron dipyrrins, as is clearly illustrated by the numerous applications being reported for these dyes, including their use as chemosensors. 8 Fluorescent chemosensors for the recognition and measurement of transition metal ions are very sensitive and indispensable tools in chemistry, life science and biotechnology. 9 For instance, the design of Cu(II) sensors has received much attention 10,11 because of the importance of Cu(II) in environmental and biological systems. In fact, Cu(II) is the third most abundant transition metal in the human body, 10 is required as a cofactor for many fundamental biological processes, 12 is associated with neurodegenerative diseases, such as Alzheimer's disease, 10,12,13 and has been identied as an environmental pollutant. 11 Recently, several BODIPY based Cu(II) sensors have been reported. 14 However, their synthesis required a long multi-step protocol and/or unstable intermediates. Moreover, most BODIPY based Cu(II) sensors are poorly water-soluble and are thus oen characterized in non-aqueous systems or in mixtures of organic solvents with water. However, for many applications, water solubility is an important property of Cu(II) sensors.
The synthesis of new highly uorescent BODIPY Cu(II) sensors might be improved by using the efficient C-H functionalization protocols developed for boron dipyrrins. Herein, we report the synthesis of two simple Cu(II) sensors based on a tris(2-aminoethyl)amine (TREN) 15 ligand as the receptor moiety starting from a standard 8-aryl-BODIPY dye 1 using the C-H functionalization reactions recently developed by our group. 6,7 The interaction of these sensors with protons and transition metal ions and their ability to permeate the cell membrane were investigated and their high selectivity for Cu(II) was demonstrated.

Synthesis
The desired BODIPY-TREN conjugates were synthesized starting from an accessible 8-aryl-BODIPY dye 1. 2,6b The TREN ligand could be directly incorporated onto its 3-position using an oxidative nucleophilic substitution of hydrogen 6a in DMF under an oxygen atmosphere at room temperature. Unfortunately, the formed product turned out to be unstable and decomposed during work up. This might be caused by the two remaining primary amines of the formed BODIPY-TREN conjugate, as these might react further at the free 5-position. Blocking the 5-position with a substituent should prevent this side reaction, allowing the isolation of the desired BODIPY-TREN conjugate (Scheme 1). A phenyl or a cyclohexyl substituent can easily be placed on the 5position using the radical functionalization reactions developed in our group. 7 The phenyl group was introduced via a ferrocene catalyzed radical arylation 7a with benzenediazonium tetra-uoroborate forming 5-phenyl-BODIPY 2 in a good yield of 58%. 5-Cyclohexyl-BODIPY 3, on the other hand, was synthesized in an excellent yield of 77% via an oxidative radical alkylation 7b with potassium cyclohexyltriuoroborate. Both 5-substituted dyes were successfully converted to the corresponding stable BODIPY-TREN conjugates 4 and 5 in a yield of about 60% using the oxidative nucleophilic substitution of hydrogen reaction with an excess of the TREN polyamine.

Inuence of the pH on the spectroscopic properties
Both synthesized BODIPY-TREN conjugates 4 and 5 are soluble in water, hence their spectroscopic properties were determined in this medium (Table 1). Both compounds absorb visible light at a similar wavelength around 495 nm. However, the increased conjugation length in the 5-phenyl compound 4 causes a bathochromic shi in the emission wavelength of 24 nm compared to the 5-cyclohexyl derivative 5, which emits light at 551 nm, resulting in a larger Stokes shi for the 5-phenyl dye 4. Hence, the 5-substituent can be used to ne tune the emission wavelength of the resulting BODIPY-TREN conjugates. The Stokes shis of both compounds are rather large compared to typical boron dipyrrin dyes, although 3-amino-BODIPYs oen display large Stokes shis in polar media. 14f,16 In contrast to the absorption and emission wavelengths, the broadness of the absorption and emission peaks and the uorescence quantum yield of both compounds are very similar. Both dyes have high uorescence quantum yields (around 0.6) at pH 7.4 and pH 5, with the one at pH 7.4 being slightly lower.
The primary amines of the synthesized ligands 4 and 5 are susceptible to protonation, hence the effect of the pH on the emission spectra was studied ( Fig. 1 and 2). Both compounds displayed a decrease in uorescence intensity when going from an acidic to a basic pH, with the largest change in the intensity occurring between pH 8 and pH 10 ( Fig. 2 and S3 †). The emission maxima, on the other hand, were unaffected by the pH ( Fig. 1 and S2 †). The uorescence quenching as the degree of protonation of the ligand decreases, is a phenomenon that has been widely observed in similar compounds 15 and has been attributed to a photoinduced electron transfer (PET) process from the lone pairs of the amines to the excited uorophore. 9 Protonation of the primary amines of the BODIPY-TREN conjugates blocks this PET-mediated quenching and hence leads to uorescent compounds.
Using the pH titration data, two stepwise protonation constants could be calculated (Table 2). However, a third protonation step was not observed in the pH range studied. [3][4][5][6][7][8][9][10][11][12] Scheme 1 Synthesis of BODIPY-TREN conjugates 4 and 5 (Ar ¼ 2,6-dichlorophen-1-yl, TREN ¼ tris(2-aminoethyl)amine). More acidic and basic pH values were not tested because BODIPY dyes are known to decompose in strongly acidic and strongly basic conditions. 17 The two observed protonation steps correspond to the two primary amines of the TREN substituent. Protonation of the amine directly connected to the pyrrole ring is unlikely to occur due to the electron withdrawing nature of the BODIPY core. 2 Furthermore, it is also not likely that the tertiary amine will be protonated before the primary amines as it is well known that tertiary nitrogen atoms are less basic in aqueous solution than primary and secondary ones. 18 The protonation constants for the two synthesized BODIPY-TREN conjugates 4 and 5 (Table 2) are similar to each other and to the constants of other primary amines in general. 18 Based on the determined protonation constants, the distribution diagrams for the different protonated species could be calculated in function of the pH (Fig. 2 and S3 †). Comparing these distribution diagrams with the pH titration curves shows that the decrease in emission intensity, when going from acidic to basic pH, corresponds with a decreasing amount of uorescent protonated species and an increasing amount of the PETquenched non-protonated ligand.

Inuence of the presence of a metal ion on the spectroscopic properties
The spectroscopic characteristics of the synthesized BODIPY-TREN conjugates in water in the presence of two different transition metal ions were studied next. Cu(II) and Zn(II) were chosen as representative examples (Table 1). Adding one equivalent of metal ion to ligands 4 and 5 did not signicantly affect the position of the absorption and emission maxima, the Stokes shis or the broadness of the absorption and emission peaks. However, the presence of a metal ion did affect the intensity of the emission spectra, resulting in lower quantum yields of uorescence. At pH 7.4, addition of Cu(II) almost completely quenched the uorescence of the BODIPY-TREN conjugates. Addition of Zn(II) at the same pH reduced the uorescence quantum yield to a lesser extent. However, the differences in quantum yields of the free ligands and the complexes are not as pronounced at pH 5. At this pH, Cu(II) reduced the uorescence quantum yield from 0.6 to 0.4 while Zn(II) had no noticeable effect.
Repeating the pH titration experiments in the presence of one equivalent of these two metal ions resulted again in a quenching of uorescence at basic pH (Fig. 3). However, the titration curves themselves are different. The largest change in emission intensity in the presence of Cu(II) ions was already observed at a lower pH, between pH 4 and pH 6, in comparison to the free ligands ( Fig. 3 and S6 †). Furthermore, the Cu(II) complexes quenched the uorescence of the BODIPY core stronger than the unprotonated free ligands. This effect, called a chelation enhanced quenching (CHEQ) effect, is oen observed for Cu(II) complexes. 9b In contrast, Zn(II) does not strongly shi the pH range where the largest change in emission intensity occurs ( Fig. 3 and S9 †). Moreover, at basic pH a larger emission intensity was observed for the Zn(II) complexes in comparison to the free ligands. This indicates that the unprotonated free ligands resulted in a stronger quenching of the uorescence than the corresponding Zn(II) complexes. Hence, Zn(II) displayed a chelation enhanced uorescence (CHEF) effect. 9b Based on these titration data and on the calculated protonation constants, the stability constants for the Cu(II) and Zn(II) complexes of the BODIPY-TREN conjugates 4 and 5 could be calculated (Table 2). In the case of Cu(II), two different stability constants were found, one for the mono-(BODIPY-TREN)-   Using these stability constants together with the protonation constants, the distribution diagrams for the different complexes and protonated species in the presence of one equivalent of metal ion could be calculated in function of the pH (Fig. 3, S6 and S9). Comparing these distribution diagrams with the pH titration curves shows again that the decrease in emission intensity, when going from acidic to basic pH, corresponds with a decreasing amount of uorescent protonated species and an increasing amount of (partially) quenched complexes.
Aerwards, the effect of the Cu(II) and Zn(II) concentrations on the emission spectra was investigated at a constant pH of 7.4. Because both BODIPY-TREN conjugates 4 and 5 have the same spectroscopic properties, with the exception of their emission maxima, and the same response to pH and to the presence of Cu(II) and Zn(II), the metal titration experiments were done only for ligand 5 (Fig. 4). Similar to protonation, addition of Cu(II) inuenced only the emission intensity resulting in a weaker uorescence at a higher Cu(II) concentration (Fig. S10A †). Addition of Zn 2+ had the same effect, however, in this case, also a slight bathochromic shi of a few nanometers was observed at higher Zn(II) concentrations (Fig. S10B †). The main difference between the two metal ions is that for Cu(II) the uorescence intensity decreases more strongly and levels out aer addition of about 0.5 equivalents of Cu(II). This intensity for the Zn(II) complex, on the other hand, levels out aer about one equivalent of Zn(II) (Fig. 4).
This difference, in amount of equivalents of metal ion, could be explained if a 2 : 1 stoichiometry for the 5-Cu(II) complex and a 1 : 1 stoichiometry for 5-Zn(II) complex are assumed. This hypothesis was veried, by determining the stoichiometry of the Cu(II) and Zn(II) BODIPY-TREN complexes of ligand 5, using a Job plot (Fig. S11 †). Furthermore, these results agree with the two stability constants for the Cu(II) complex and the one stability constant for the Zn(II) complex that resulted from the calculations from the pH titration experiments (see above).

Selectivity towards Cu(II)
Given the difference in stability constants and stoichiometry of the Cu(II) and Zn(II) complexes of the synthesized BODIPY-TREN conjugates 4 and 5 it might be possible to use these ligands as ion-selective uorescent probes. To this end, the uorescence response of the two synthesized ligands towards an excess of a selection of metal ions was investigated at pH 7.4 and at pH 5 (Fig. 5). At pH 7.4, about half of the tested metal ions caused a reduction in the uorescence intensity (Fig. 5A). Cu(II) had the largest effect on this intensity, almost completely quenching the uorescence of the two BODIPY-TREN conjugates. Co(II), Ni(II), Zn(II) and Hg(II) also signicantly (by about 60%) reduced the emission intensity. Lastly, Cd(II) reduced the uorescence to a lesser extent (by 10-20%). The other metal ions (Na(I), Mn(II), Fe(III) and Pb(II)) did not create a signicant reduction in uorescence intensity. Most ions did not inuence the emission maxima, however complexation to Hg(II) did lead to a bathochromic shi of about 10 nm for both ligands (Fig. S12 †). Moreover, a slight bathochromic shi of a few  nanometers was detected upon addition of an excess of Zn(II) to ligand 5, yet a similar effect was not observed for ligand 4 (Fig. S12 †).
The effect of the presence of an excess of these metal ions is quite different at pH 5. At this pH only Cu(II) resulted in a reduction of the emission intensity, all other tested metal ions le the uorescence of the free ligand intact (Fig. 5B). This is probably because only Cu(II) binds strongly enough with the BODIPY-TREN conjugates 4 and 5 to successfully compete with protons at pH 5. The uorescence quenching of Cu(II) at this pH is not as strong as at pH 7.4, reducing the emission intensity to about 40%, compared to the free ligands, instead of almost completely quenching it. Nonetheless, the good selectivity for Cu(II) ions at pH 5 means that the synthesized BODIPY-TREN conjugates 4 and 5 are promising uorescent sensors for Cu(II).

Cell permeability study
The ability of ligands 4 and 5 to permeate cellular membranes was checked with HeLa cell cultures by ow cytometry and uorescence spectroscopy (Fig. 6). Both compounds were incubated for 24 and 48 hours showing their incorporation in cells as denoted by the observation of the uorescence of the BODIPY-TREN conjugates 4 and 5 in the 550-570 nm range ( Fig. S15-S18 †). On the other hand, the ow cytometry technique reveals a normal cellular growth up to about 30 mM concentrations of the compounds (Fig. S13 and S14 †).

Conclusion
Two simple and novel uorescent sensors for Cu(II) ions based on a BODIPY uorophore and a TREN ligand were synthesized in an efficient way from a standard boron dipyrrin dye using C-H functionalization reactions. The ease and resulting high yields of these reactions demonstrated the utility of nucleophilic substitution of hydrogen and radical reactions in the construction of functionalized BODIPY dyes. The synthesized BODIPY-TREN conjugates are water soluble compounds that can permeate the cell membrane. The interaction of these ligands with different transition metal ions was investigated and it was shown that at pH 5 they possess a high selectivity for Cu(II) ions. Hence, these BODIPY-TREN conjugates are promising uorescent sensors for Cu(II) at this pH.   3070 | RSC Adv., 2017, 7, 3066-3071