Biomaterials Science This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online PAPER View Journal | View Issue Efficient blue light emitting materials based on m-carborane–anthracene dyads. Structure, Open Access Article. Published on 02 October 2019. Downloaded on 7/14/2021 4:40:33 PM. Cite this: Biomater. Sci., 2019, 7, 5324 photophysics and bioimaging studies† Mahdi Chaari, a Zsolt Kelemen, a Duane Choquesillo-Lazarte, b Nerea Gaztelumendi, c Francesc Teixidor, a Clara Viñas, a Carme Nogués *c and Rosario Núñez *a Efficient monosubstitution of the non-iodinated, mono-iodinated and di-iodinated m-carborane cluster at one Ccluster has led to the preparation of three single organic molecule-carborane dyads (4–6), which exhibited exceptional fluorescence properties with quantum yield values of 100% in solution, for all of them, with maxima around 415 nm, which correspond to the locally excited state (LE) emission. These results suggest that simply linking the m-carborane fragment to one anthracene unit through a CH2 spacer produces a significant enhancement of the fluorescence in the final fluorophore, probably due to the free rotation of the anthracene linked to the Ccluster. Besides, the presence of one or two iodine atoms linked to boron atoms does not cause any influence on the photophysical properties of the dyads, as it is confirmed by TD-DFT calculations. Notably, the three conjugates show good fluorescence efficiency in the aggregate state with quantum yields in the range of 19–23%, which could be ascribed to the presence of CH2, particularly for 4, and the iodine atoms in 5 and 6, which prevent π–π stacking. All these results indicate that our dyads are extremely good emitters in solution while maintaining the emission properties in the aggregate state. Crystal packing, fingerprint plot analysis, and TD-DFT calculations for the three Received 10th June 2019, compounds support these results. Confocal microscopy studies show that 6 is the best-internalized com- Accepted 29th September 2019 pound by HeLa cells via endocytosis, although 4 and 5 also presented a high fluorescence intensity emis- DOI: 10.1039/c9bm00903e sion. Moreover, due to the blue emission, this compound is an excellent candidate to be applied as a flu- rsc.li/biomaterials-science orescent dye in bioimaging studies. Introduction organic light-emitting diodes (OLEDs), and for sensing metal ions, pH, small organic molecules, etc.2,3 On the other hand, Anthracene is a π-conjugated system widely investigated in the excimer emission from anthracene is a well reported both chemistry and materials science, due to its significant phenomenon, which is of special interest in photochemistry, photophysical, photochemical and chemical properties.1 supramolecular chemistry, photobiology and polymer science.4 Owing to their excellent luminescence properties, anthracene The excimer formation of anthracene is from its excited dimer, derivatives have potential applications in organic functional which has four possible structures, such as normal dimer, materials, such as organic field effect transistors (OFETs) and twisted dimer, offset dimer and T-shaped dimer.5 Usually, planar aromatic molecules can easily form an excimer in con- centrated solutions or in the solid state because of the inter- a Institut de Ciència de Materials de Barcelona (ICMAB-CSIC), Campus U.A.B., molecular π–π interactions, which play a crucial role in 08193 Bellaterra, Barcelona, Spain. E-mail: rosario@icmab.es; excimer formation. Usually, the excimer exhibits red-shifted, Fax: +34 93 580 5729; Tel: +34 93 580 1853 b broadened and structureless fluorescence emission with Laboratorio de Estudios Cristalográficos, IACT-CSIC, Avda. de las Palmeras 4, 18100 – Armilla, Granada, Spain regard to that of its monomer. c Departament de Biologia Cellular, Fisiologia i Immunologia. Universitat Autònoma Carborane clusters are fascinating chemical species that de Barcelona, Campus U.A.B., 08193 Bellaterra, Barcelona, Spain. have been the subject of intense and attractive research over E-mail: Carme.Nogues@uab.cat the last six decades.6 They are very robust compounds charac- † Electronic supplementary information (ESI) available: 1H and 11B{1H} NMR terized by a unique three-dimensional (3D) σ-delocalization,7 spectra, computational details, crystallographic data and Hirshfeld surface ana- lyses for all the compounds. CCDC 1907569–1907571. For ESI and crystallo- high thermal and chemical stability,8 hydrophobicity, meta- graphic data in CIF or other electronic format see DOI: 10.1039/c9bm00903e bolic inertness and low toxicity in biological systems.6d–f,9 5324 | Biomater. Sci., 2019, 7, 5324–5337 This journal is © The Royal Society of Chemistry 2019 View Article Online Biomaterials Science Paper Carboranes show high versatility toward functionalization, tion and the solid state. The crystal structures of the syn- which makes them suitable building blocks easily linkable to thesized compounds have been established by X-ray diffraction molecular and polymeric π-conjugated systems.10 One of the analysis. Photophysical analysis in solution and the aggregate main features of the o-carborane cluster is its electron-accept- state have been performed and complemented with TD-DFT This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. ing ability through substitution at the Ccluster (Cc),11 when it is theoretical calculations to establish a meaningful structure– linked to an aryl donor group, and subsequently accepts an photophysical property relationship. A comparison between excited charge via intramolecular charge transfer (ICT) from the current dyads and related triads is also reported. Owing to the donor group. These ICT states are influenced by the Cc–Cc our interest in the biological applications of fluorescent car- bond vibration,12 which can vibrate and relax non-radiatively borane derivatives, the cytotoxicity and internalization of the or radiatively giving a red-shift emission.13,14 Moreover, these dyads and their cellular localization in HeLa cancer cells are Open Access Article. Published on 02 October 2019. Downloaded on 7/14/2021 4:40:33 PM. systems that show low or null fluorescence efficiency in solu- also discussed. tion usually exhibit aggregation-induced emission (AIE) in the solid state, due to the restriction of the molecular motion, which leads to a significant increase in the emission quantum Results and discussion yield.15 Over the last years, we have designed carborane-containing Synthesis and characterization of anthracenyl- single molecules, whose emission in solution depends criti- monosubstituted m-carborane derivatives 4–6 cally on the cluster isomerism (o- or m-) and the fluorescence The synthesis of compounds 4–6 was achieved by nucleophilic efficiency can be tailored by changing the substituent at the substitution at one Cc atoms,20 following a similar procedure adjacent Cc.8,16 It was observed that molecules showing moder- to that used for the previous o- and m-carborane ate emission in solution were able to exhibit good fluorescence derivatives.16,17,19 Compound 4 was obtained by the nucleophi- efficiency in the solid state.17 In this way, and depending on lic substitution of 1,7-closo-C2B10H12 (1), whereas 5 and 6 were the targeted application, we are capable of preparing highly obtained from the respective mono- and di-iodinated deriva- fluorescent materials in both states. Noticeably, m-carborane tives, 9-I-1,7-closo-C2B10H11 (2) and 9,10-I2-1,7-closo-C2B10H10 has been demonstrated to be a perfect platform to boost the (3), which were previously prepared by electrophilic substi- photoluminescence properties of organic π-conjugated systems tution at the B atoms.21 The key step for the preparation of linked to it, both in solution and the solid state.16,17 compounds is the monolithiation of starting compounds 1–3, Some examples of anthracene-o-carborane dyads and triads which was efficiently performed using THF as a solvent under where the anthracene is directly linked to the Cc of the o-car- diluted conditions at 0 °C; then the reaction of the corres- borane or through a π-conjugated spacer between both frag- ponding monolithium salts with one equiv. of 9-chloromethyl ments have been reported.18 It is well described that o-carbor- anthracene at reflux overnight gave compounds 4, 5 and 6, ane acts as a strong electron-withdrawing unit in the excited respectively, in 52, 54 and 41% yields, respectively (Scheme 1). state of these conjugated systems causing a rapid ICT from the The nucleophilic substitution reactions were monitored by 11B anthracene to the o-carborane, with a subsequent quenching {1H} NMR following the appreciable changes in the boron reso- of the locally excited state (LE) fluorescence emission in solu- nance distributions. Although for m-carborane the monosub- tion. Moreover, these dyads have shown highly-efficient solid- stitution at the Ccluster is relatively easier than for o-carborane, state emission, as well as mechanochromic and thermochro- in both cases a mixture of mono- and di-substituted species is mic luminescence properties.18 Noticeably, m-carborane– always obtained, even on controlling the reaction conditions. anthracene triads obtained by linking two anthracene units to Proof of this is that only a few examples of mono-substituted the m-carborane fragment exhibited a significant increase in derivatives from m-carborane have been reported in the more than two-fold in the intrinsic fluorescence quantum literature.22 yield of the anthracene in solution as well as a red-shift of the The structures of 4–6 were established on the basis of IR, 1 emission maximum and moderate quantum efficiencies in the H, 13C{1H} and 11B{1H} spectroscopy and elemental analysis aggregate state.19 Nevertheless, the differences in the ϕF values and all of them were confirmed by X-ray diffraction analysis. obtained for different triads in the aggregate state were attribu- On the one hand, IR spectra show typical ν(B–H) strong bands ted to the arrangement of dimers in the solid state structures; for closo clusters between 2556 and 2609 cm−1. On the other it was concluded that the presence of a large number of BH⋯I hand, 1H NMR spectra of 4–6 display one new singlet at contacts leads to a less delocalized system for the diiodo around δ = 4.30 ppm due to the Cc–CH2 protons. Then, com- derivative, and therefore a lower quantum yield value. pounds 4–6 show a Cc–H single resonance in the region at We present here a set of three m-carborane–anthracene 2.75–2.92 ppm, which is slightly shifted upfield (approximately dyads efficiently synthesized by Cc monosubstitution of the 0.2 ppm) with regard to starting compounds 1–3. Due to the m-carborane with one anthracene group through a CH2 unit. mono and diiodination of the m-carborane cluster, the 11B{1H} Iodination of one and two B atoms is also performed to NMR spectra for the three compounds are different; m-carbor- produce B–I vertices. These systems were designed to enhance ane derivative 4 shows resonances in the typical closo region, the photoluminescence (PL) properties with regard to pre- from δ −3.66 to −15.58 ppm, with the general pattern viously reported m-carborane–anthracene triads, both in solu- 2 : 4 : 2 : 2; mono-iodinated compound 5 shows resonances in This journal is © The Royal Society of Chemistry 2019 Biomater. Sci., 2019, 7, 5324–5337 | 5325 View Article Online Paper Biomaterials Science This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. Open Access Article. Published on 02 October 2019. Downloaded on 7/14/2021 4:40:33 PM. Scheme 1 Procedure to obtain compounds 4–6. the range from δ −2.65 to −23.87 ppm, with the cular structures for 4–6 were established by single crystal X-ray 1 : 4 : 1 : 1 : 1 : 1 : 1 pattern, whereas di-iodinated compound 6 diffraction (Fig. 1) and are in agreement with the NMR data shows resonances in the range from δ −1.62 to −21.15 ppm, (vide supra). Experimental crystal data and structure refinement with the 1 : 1 : 2 : 2 : 2 : 2 pattern. Moreover, it is remarkable parameters for all structures reported in this work are listed in that the two B–H resonances at high frequency for all the Table 1. Whereas compound 4 crystallizes in the triclinic P1 ˉ anthracene derivatives 4–6 were shifted to downfield at around space group, compound 5 crystallizes in the monoclinic P21/n 1.5 ppm and the ranges between them are larger with regard space group and compound 6 crystallizes in the orthorhombic to 1–3 (see the ESI†). As for the previously reported iodinated Ima2 space group. The molecular structures for all these com- derivatives,23 the B–I are indubitably identified as the highest pounds show typical icosahedral geometry with very similar field resonances in the range from δ −21.15 to −23.87 ppm, bond distances and angles, which are also similar to those in which remain as a singlet in the 11B NMR. The 13C{1H} NMR anthracenyl-disubstituted m-carborane compounds.19 spectra of 4–6 show one resonance at around δ = 55.10 ppm In all structures, the m-carborane moiety is linked to one that is assigned to the Cc–CH2 carbons, which is shifted down- anthracene unit through a methylene spacer (–CH2–). As field (approximately 1 to 1.6 ppm) in 5 and 6 compared to 4. shown in Fig. 1, the rotation of the anthracene rings through In contrast, one resonance at around δ = 33.88 ppm, which is the (H)(H)C–C(anthracene) bond allows more or less similar slightly shifted upfield in 5 and 6 compared to 4, is assigned conformations in the solid state. Conformations found in the to the Cc–H carbons. All compounds show aromatic reso- solid state and torsion angles are given in Fig. 1 caption. nances in the range from δ 124.17 to 131.53 ppm. Elemental The solid-state structures in 4–6 are mainly dominated by analyses also confirmed the stoichiometry of all these com- intermolecular C–H⋯anthracenyl (A) interactions (Fig. S1† pounds (see the Experimental section). and Table 2). Thus, extensive Cc–H⋯A interactions are found in all molecules, mainly between the Cc–H and B–H hydrogen X-ray structural analysis atoms in 4 and 6, and C–H⋯π in 5 (Fig. S1† and Table 2). Single crystals suitable for X-ray structural determination of Moreover, interactions between –CH2– hydrogen atoms and compounds 4, 5 and 6 were obtained by slow evaporation from aromatic Canthracene atoms are found in 4. Other weak B–H⋯I–B a mixture of chloroform/n-heptane (9 : 1), chloroform or di- contact in 6 is listed in Table 2. From the crystal packing of 4 chloromethane at room temperature, respectively. The mole- (Fig. 1 and Fig. S1†), we can see that there are no π–π stacking 5326 | Biomater. Sci., 2019, 7, 5324–5337 This journal is © The Royal Society of Chemistry 2019 View Article Online Biomaterials Science Paper This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. Open Access Article. Published on 02 October 2019. Downloaded on 7/14/2021 4:40:33 PM. Fig. 1 (Left column) Molecular structures of 4, 5 and 6. Torsion angles: 4 (C14C13C1B2, 37.7(2)°), 5 (C14C13C1B2, 41.1(4)°), and 6 (C14C13C1B3, 29°); (right column) projections showing the organization of four molecules of the compounds in the solid state. Color code: B pink; C grey; H white; and I violet. Table 1 Crystal data and refinement details for structures of com- Table 2 Geometrical parameters of weak D–H⋯A (A = C, H, I) contacts pounds 4–6 (Å, °), involved in the supramolecular construction in 4–6. For C–H⋯π contacts, geometries are given with respect to the aromatic centroid M 4 5 6 or one of the ring carbons Empirical formula C17H22B10 C17H21B10I C17H20B10I2 Compounds D–H⋯A d(H⋯A) ∠(DHA) ∠(HHB) Formula weight 334.44 460.34 586.23 Crystal system Triclinic Monoclinic Orthorhombic 4 C(13)–H(13B)⋯C(18) i 2.828 122.5 — Space group P1ˉ P21/n Ima2 C(13)–H(13B)⋯C(19)i 2.910 139.3 — Temperature/K 293 293 298 C(7)–H(7)⋯H(3)–B(3)ii 2.484 120.5 166.6 Wavelength/Å 0.71073 0.71073 0.71073 a/Å 6.9712(5) 9.3190(8) 13.801(3) 5 C(7)–H(7)⋯Miii 2.669 150.3 — b/Å 11.7604(7) 11.7553(6) 25.643(5) C(17)–H(17)⋯H(11)–B 2.307 131.8 153.1 c/Å 12.9797(6) 18.5808(12) 6.8626(13) (11)iv α/° 107.511(2) 90 90 β/° 102.996(3) 94.913 90 6 C(7)–H(7)⋯H(5)–B(5)v 2.364 119.9 118 γ/° 102.125(3) 90 90 C(7)–H(7)⋯I(1)–B(9)v 3.103 135.3 — Volume/Å3 943.63(10) 2028.0(2) 2428.7(8) B(4)–H(4)⋯I(1)–B(9)vi 3.215 171.3 — Z 2 4 4 Density (calculated)/ 1.177 1.508 1.603 Symmetry codes (i) 1 + x, y, z (ii) 1 − x, 1 − y, 2 − z (iii) 1 − x, 1 − y, Mg m−3 1 − z (iv) 0.5 − x, −0.5 + y, 0.5 − z (v) x, y, 1 + z (vi) 1 − x, 1 − y, z. F(000) 348 904 1112 Theta range for data 1.903 to 2.052 to 2.803 to collection/° 27.483 27.495 25.014 Absorption coefficient/ 0.058 1.579 2.591 between two anthracenes, because the presence of the CH2 mm−1 spacer, from which one hydrogen atom interacts with an aro- Goodness-of-fit on F2 1.030 1.123 1.038 matic Canthracene (2.82 Å), avoids the π–π packing from inter- R1 [I > 2sigma(I)] 0.0577 0.0459 0.0405 wR2 [I > 2sigma(I)] 0.1464 0.0991 0.0766 molecular anthracene groups that could allow the excimer for- R1 (all data) 0.0874 0.0634 0.0603 mation or even a quenching of the fluorescence. In com- wR2 (all data) 0.1624 0.1073 0.0791 pounds 5 and 6, the iodine atoms dominate the crystal CCDC 1907569 (4), 1907570 (5) and 1907571 (6)† contain the packing (Fig. 1 and Fig. S1†), obviously preventing π–π stacking supplementary crystallographic data for this paper. between anthracene rings. This journal is © The Royal Society of Chemistry 2019 Biomater. Sci., 2019, 7, 5324–5337 | 5327 View Article Online Paper Biomaterials Science From the fingerprint plot analysis, it can be found that value goes in parallel with the number of iodine atoms in the H⋯H contacts comprise nearly 78% of the total Hirshfeld molecules. surface area for the non-iodinated compound 4 and it Fluorescence emission spectra of 4–6 were also measured decreases to 65% and 52% for the iodinated compounds 5 and in THF as well as in the aggregate state (THF/H2O = 1/99 (v/v), This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. 6, respectively. The H⋯C contacts contribute around 13–19% 1 × 10−5 M for 4–6). All of them show similar vibronic emission to the total Hirshfeld surface area for the three compounds. spectra in solution, with maxima at around λem = 415 nm The contribution of H⋯I interactions varies from 15% to 28% (Fig. 2a), which can be assigned to the LE emission of the for 5 to 6, respectively (see more details in Fig. S2 in the ESI†). anthracene moiety. As for the previous compounds reported by DFT-calculations were carried out (see more details in the us,19 the similarity between the spectra of 4–6 in solution and ESI†) and different rotamers were investigated for 4–6 (Fig. S3 that of anthracene (λem = 420 nm) suggests that small elec- Open Access Article. Published on 02 October 2019. Downloaded on 7/14/2021 4:40:33 PM. in the ESI†). The structure of the most stable rotamer is identi- tronic interactions between the anthracene units take place, cal to the rotamer obtained from the crystal structures. It and there is no influence of the iodine atom. Remarkably, should be noted that the energy differences between the rota- compounds 4–6 exhibit extraordinary high fluorescence mers are tiny and probably the rotational barrier is small, thus quantum yield values (ϕF) in solution that are around 100%. in solution there is not preferred orientation. Table 3 summarises the photophysical data for all the com- pounds. It is worth noting that monosubstituted anthracene Photophysical properties and TD-DFT calculations derivatives 4–6 exhibit significant higher fluorescence The photophysical properties of compounds 4–6 were deter- quantum yields in solution than the previously reported di- mined by UV-Vis absorption and fluorescence spectroscopy in substituted m-carborane that contains two anthracene units, THF solutions (Table 3). Electronic properties of 4–6 in the which exhibited ϕF values in the range of 63–66%.19 ground state were assessed by UV-Vis absorption measure- To gain more insight into the photophysical behaviour of ments (∼10−5 M, Fig. 2). All compounds exhibit similar absorp- 4–6 the Kohn–Sham orbitals and TD-DFT spectra were calcu- tion spectra showing vibrational structures assigned to the lated at the B3LYP/6-31G* (for the iodine atom LANL2DZ basis π–π* transition band of the anthracene moiety, with a sharp set) level of theory. The B3LYP functional with 6-31G* basis set band around 258 nm and four peaks at around 334, 350, 369 was successfully applied earlier for the investigation of related and 389 nm. The molar absorption coefficients (ε) are slightly carborane based fluorophores.19,24 As it was noted the energy different for the three compounds, being in the range from difference between the different rotamers is small and presum- 102 × 102 to 112 × 102 M−1 cm−1, where a slight increase in the ably all of them occur in solution; thus the frontier orbitals Table 3 Photophysical data for compounds 4–6 THF THF/water (1/99) ε/105 Brightness 104 Stokes Stokes Compounds λabs (nm) (M−1 cm−1) λem (nm) ϕF a (M−1 cm−1) shift (nm) λabs (nm) λem (nm) ϕF a shift (nm) 4 369 0.102 414 1 1.02 45 372 457 0.23 85 5 369 0.106 415 0.95 1.01 46 373 459 0.19 86 6 369 0.112 415 1 1.12 46 375 456 0.2 81 a Reference compound quinine sulfate (0.5 M H2SO4, ϕF = 0.54). Fig. 2 Absorption and emission spectra of 4 (red), 5 (green) and 6 (blue) in THF solutions (a) and aggregates (THF/H2O, v/v = 1/99) (b). AU: arbitrary units. 5328 | Biomater. Sci., 2019, 7, 5324–5337 This journal is © The Royal Society of Chemistry 2019 View Article Online Biomaterials Science Paper sition exhibits an excellent numerical agreement with the observed spectra (Table S1 in the ESI†). Simple TD-DFT calcu- lations could not describe the vibrational fine structure, which is specific for a rigid aromatic system such as anthracene. The This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. calculated excitation refers to the transition from the ground state to the lowest vibrational level of the excited state, which corresponds to the band around 390 nm in the experimental spectra (calculated 389 nm). After investigating the influence of the iodine atoms, it could be established that they have no impact on the shape and the energy of the frontier orbitals, so Open Access Article. Published on 02 October 2019. Downloaded on 7/14/2021 4:40:33 PM. they do not change the HOMO–LUMO transitions. On the other hand, the lone pairs of the iodine atoms do participate in the HOMO−1 and HOMO−2 for 5 and HOMO−X (X = 1–4) for 6. TD-DFT calculations demonstrate that the transitions from the lone pairs of the iodine atom(s) to the π* orbitals of the anthra- cene unit exist, but the probability of these transitions is low (the calculated oscillator strength is below 0.001); thus they could be not observed in the experimental spectra. Finally, we investigated the PL behaviour in the aggregate state (THF/H2O = 1/99 (v/v)); emission spectra of 4–6 are shown in Fig. 2b. The PL spectra for these compounds are very similar, showing non-vibronic structures and a maximum emission at around 456–459 nm (Table 3), which are about 40 nm red-shifted with respect to the THF solutions. Consequently, larger Stokes shifts are also observed. In general, all compounds were fluorescent in the aggregate state with ϕF values in the range of 19–23%. Nevertheless, a decrease in the quantum efficiency compared to the respective ones in solution (95–100%) is clearly observed, while maintain- ing the fluorescence properties. Investigating the literature data, similar emission spectra (with non-vibronic structures and maximum at around 450–460 nm) have been observed for anthracene excimers.4c,d,25 Our DFT calculations (see more details in the ESI, Fig. S4†) do not rule out the possible for- mation of excimers, but at this point it is difficult to establish a relationship between the emission spectra and the structure of our compounds, as all of them show similar emission pro- perties although they have very different packing in the solid state. Biological assays Fig. 3 Some important Kohn–Sham molecular orbitals of 4-cryst, 5- Fluorescence imaging techniques have been used to visualize cryst and 6-cryst and their energies in eV and at B3LYP/6-31G* level of bio-components and bio-processes by transforming the chemi- theory. The order and the energy level of the orbitals are not strongly cal and biological information into detectable signals. dependent on the orientation of the anthracene unit (different rotamers). Recently, we have reported that carborane–BODIPY dyads were easily internalized by HeLa cells, those bearing m-carborane clusters being the ones that show the best fluorescence charac- teristics for cell imaging purposes.26 Owing to our interest to and TD-DFT were calculated for all rotamers (Fig. S3 in the use fluorescent carborane derivatives for biological purposes, ESI†). The orientation of the anthracene unit does not have a herein we have analysed the cytotoxicity and the potential use significant effect on the shape and the energy of the frontier of compounds 4–6 as fluorescent probes for biological orbitals, and as a consequence on the calculated transitions in systems. the TD-DFT calculations (Fig. 3); the HOMO and LUMO are Cytotoxicity was analyzed by the Alamar Blue assay after localized at the anthracene unit in all the investigated rota- HeLa cell incubation with each compound at 10 µM for 24 h. mers of 4–6 and TD-DFT calculations confirm the π–π* Only viable cells can reduce resazurin (the active compound of (HOMO–LUMO) transitions for 4–6. The energy of this tran- Alamar Blue) into resorufin, a measurable fluorescent com- This journal is © The Royal Society of Chemistry 2019 Biomater. Sci., 2019, 7, 5324–5337 | 5329 View Article Online Paper Biomaterials Science pound whose quantity correlates with the amount of living the compounds, a greyscale (Fig. 4b) and a colour palette scale cells. None of the compounds tested were cytotoxic for HeLa (Fig. 4c) were also used. In the greyscale, black and white cells; in fact, an increase in cell proliferation was observed for colours indicate, respectively, the minimum and maximum all three compounds (Fig. S5a†). However, optical images of fluorescence intensity, whereas the different grey colours rep- This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. the same cultures after compound removal demonstrated that resent intermediate intensities (Fig. 4b). In the colour palette the cell density was comparable to that of the control cultures scale (Fig. 4c), the intermediate intensities between the black (Fig. S5b†). This means that the compounds may increase the and white ends are transformed into different colours (blue, metabolic activity of the cells, but not their proliferation, or red, orange and yellow) to easily visualize the differences in generate some kind of interference with the Alamar Blue tech- the fluorescence intensity. Notably, the fluorescence emission nique, as has already been reported with both resazurin and of the di-iodo derivative 6 was much greater than the mono- Open Access Article. Published on 02 October 2019. Downloaded on 7/14/2021 4:40:33 PM. MTT assays.27,28 iodo and non-iodine derivatives 5 and 4, respectively, which Cell internalization of the compounds was assessed by con- suggests that the presence of two iodo groups leads to a more focal microscopy. Although the laser used was in the limit of efficient transport across the plasma membrane and a better the compound absorption range (300–400 nm), all compounds cellular uptake of the m-carborane anthracene dyads by HeLa were successfully detected inside the cells, showing a fluo- cells. rescence spotted pattern in the cytosol (Fig. 4a). To better The effect of iodine on the cell membrane transportability appreciate the differences in the fluorescence intensity among of fluorescent polymers has been recently reported, demon- Fig. 4 Cellular uptake of compounds 4, 5 and 6 by confocal microscopy. (a) Fluorescence intensity shown with a false colour assigned accordingly with the emission wavelength of the fluorochromes used. Plasma membrane (red); compounds (blue); blebs of the plasma membrane (yellow arrows). (b) Fluorescence intensity in a greyscale. (c) Fluorescence intensity transformed into a colour palette scale, in which blue and white colours represent the lowest and highest fluorescence intensity, respectively. Scale bar: 50 µm. 5330 | Biomater. Sci., 2019, 7, 5324–5337 This journal is © The Royal Society of Chemistry 2019 View Article Online Biomaterials Science Paper strating that the introduction of iodo groups is able to enhance the cellular internalization of fluorescent probes, which could provide a novel strategy to improve the cellular uptake of organic molecules for drug delivery and cell track- This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. ing.29 It was suggested that the nature of halogen bonding plays a crucial role in plasma membrane transport in mamma- lian cells. Moreover, the influence of iodine on cell biological effects was previously discussed by our group for the case of the anionic boron cluster [3,3-Co(C2B9H11)2]− (COSAN) deriva- tives, where it was demonstrated that the cellular uptake for Open Access Article. Published on 02 October 2019. Downloaded on 7/14/2021 4:40:33 PM. [3,3′-Co(8-I-C2B9H10)2]− (I2-COSAN) was higher than that for COSAN.30 In this case, the lipophilicity of I2-COSAN was approximately 3.5 times greater than that of COSAN, which suggests a higher affinity for the lipid environment.31 This higher lipophilicity is also consistent with the results obtained on artificial membranes, where the rate-limiting factor for COSAN and I2-COSAN translocation is due to partitioning between the lipid and water phase.30 The slower permeation rate seen for I2-COSAN could be due to its greater affinity for lipids.31 Altogether, the increase of lipophilicity with the iodo content and the improvement of cellular internalization would explain why compound 6 is better internalized by cells than Fig. 5 Cellular uptake of 6 by confocal microscopy at different temp- compounds 4 and 5. eratures. Internalization is considerable at 37 °C (a and b) but inefficient Regarding the laser used to excite the compounds, when at 4 °C (c and d). Upper panel: Fluorescence intensity in a greyscale. Lower panel: Fluorescence intensity shown as a colour palette scale. living cells were illuminated for a few seconds with this laser, Scale bar: 10 µm. bubbles began to appear in the plasma membrane almost immediately (Fig. 4a, yellow arrows). The energy of the 405 nm laser is very potent and, probably, it causes cell damage in living cells visualized as plasma membrane blebs. Although protein that facilitates iron uptake in cells, is internalized by these compounds would not be advisable for in vivo studies, endocytosis. Therefore, we analysed the cellular uptake of they could be appropriate dyes for fixed cells. Transferrin-Alexa fluor 488 (Tf-A488) at 37 and 4 °C as a control On the other hand, the spotted fluorescence pattern of the of ATP-dependent transport inhibition at low temperature. compounds within the cell resembles that of the endosomes/ Under standard conditions, this protein binds to its receptor lysosomes. Compound 6, the best internalized, was chosen to located in the plasma membrane and is internalized by endocy- analyse whether the compound internalization was done tosis showing the typical spotted pattern (Fig. 6a and b). through plasma membrane diffusion or by a mechanism of However, when the same experiment was performed at 4 °C, the endocytosis, an energy-dependent (ATP consuming) process in amount of Tf-A488 within the cells decreased considerably, which extracellular molecules are internalized through early- being mainly located at the plasma membrane (Fig. 6c and d). endosome vesicles which mature to late-endosomes and This suggests that Tf-A488 can couple with its plasma mem- finally into lysosomes. For this purpose, the cells were incu- brane receptor, but the lack of ATP production prevents its bated for 4 h with compound 6 either under standard con- internalization. To demonstrate that the low temperature did ditions (37 °C) or at 4 °C, a temperature in which energy- not promote cell death (which could also explain the low intern- dependent processes are arrested. Then, the fluorescence alization), one of the cultures were kept at 4 °C for 3 h and then emission was assessed by confocal microscopy. At 37 °C a fluo- at 37 °C for 1 h, and in this case, the endocytosis was recovered rescent spotted pattern typical of endosome/lysosome labelling (Fig. 6e and f). The obtained results showed that cellular uptake was observed, indicating that 6 was, probably, inside some was considerably ineffective in all cultures incubated at 4 °C kinds of vesicles (Fig. 5a and b). At 4 °C the spotted pattern (compound 6 or Tf-A488), suggesting that endocytosis is the was also present, but the fluorescence intensity obtained was major pathway for the internalization of 6, but other mecha- very low (Fig. 5c and d). The stability of compound 6 at 4 °C nisms of transport could also be involved, since a low signal was also studied to rule out that the low intensity of the signal was detected within the cells at 4 °C. observed was due to a decrease in the fluorescence emission at Finally, to confirm that compound 6 is internalized via this temperature (Fig. S6 and S7†). endocytosis, colocalization experiments were performed, The spotted pattern and the internalization arrest observed specifically using early-endosome and lysosome markers at 4 °C suggested that compound 6 enters the cell through (Fig. 7). As expected, 6 is internalized via endocytosis. After 4 h endocytosis. In order to find this, new experiments were per- of incubation 88.19% of the compound is colocalized with formed. It is well-known that Transferrin (Tf ), an iron binding lysosomes, while only 22.16% with endosomes, which means This journal is © The Royal Society of Chemistry 2019 Biomater. Sci., 2019, 7, 5324–5337 | 5331 View Article Online Paper Biomaterials Science This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. Open Access Article. Published on 02 October 2019. Downloaded on 7/14/2021 4:40:33 PM. Fig. 6 Cellular uptake of Tf-A488 by confocal microscopy at different temperatures. Internalization is considerable at 37 °C (a and b) but inefficient at 4 °C (c and d). Internalization can be recovered switching the temperature to standard conditions (e and f ). Upper panel: Fluorescence intensity in a greyscale. Lower panel: Fluorescence intensity transformed into a colour palette scale. Scale bar: 10 µm. Fig. 7 Colocalization of compound 6 with endosomes (a) and lysosomes (b). Compound 6 in green. Endosome/lysosome in red. Merged images showing colocalization in orange. Scale bar: 10 µm. Inset scale bar: 1 µm. that after 4 h most of the products had already reached the Conclusions lysosomes. Again, false colours were assigned to fluorescence intensity to better visualize the organelles and the compound. A set of three m-carborane–anthracene dyads has been success- In this case, green colour was assigned to compound 6 and fully synthesized by the monosubstitution of the m-carborane red colour to organelles to obtain an orange colour when colo- at one Ccluster; moreover, one or two hydrogens from the B–H calization occurs. groups have been substituted by iodine atoms. Remarkably, by 5332 | Biomater. Sci., 2019, 7, 5324–5337 This journal is © The Royal Society of Chemistry 2019 View Article Online Biomaterials Science Paper linking m-carborane to the well-known anthracene dye pro- Olympus) was used to analyze the cell internalization. The duces an exceptional enhancement of the fluorescence pro- images obtained were processed with ImageJ software. perties in the final molecule, with fluorescence quantum yield values near 100% in solution for all of them. Materials This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. Noticeably, these values are much higher than those pre- All reactions were performed under an atmosphere of dinitro- viously obtained for disubstituted m-carborane–anthracene gen employing standard Schlenk techniques. Tetrahydrofuran triads, and clearly, the presence of the iodo units does not was purchased from Merck and distilled from sodium benzo- have any influence on the PL properties of compounds. phenone prior to use. Commercial grade diethyl ether, hexane, These results indicate that our dyads are extremely good petroleum ether, n-heptane, chloroform and dichloromethane emitters in solution due to the free rotation of the anthra- were used without further purification. 1,7-closo-C2B10H12 (1) Open Access Article. Published on 02 October 2019. Downloaded on 7/14/2021 4:40:33 PM. cene linked to the Cc, as it is confirmed by TD-DFT calcu- was supplied from KatChem Ltd (Prague) and used as received. lations. Notably, these dyads are also good emitters in the Compounds 9-I-1,7-dicarba-closo-dodecaborane (2) and 9,10-I2- aggregate state, which could be ascribed to the presence of 1,7-dicarba-closo-dodecaborane (3) were synthesized according CH2, particularly for 4, and the iodo units in 5 and 6, which to the literature.21 n-BuLi solution (1.6 M in hexane) was pur- prevent π–π stacking. Crystal packing, fingerprint plot ana- chased from Aldrich and 9-(chloromethyl)anthracene was pur- lysis, and TD-DFT calculations for the three compounds chased from Alfa Aesar. Cell Mask Orange was purchased from support this hypothesis. Among the different compounds Thermo Fisher. tested, compound 6 is the best-internalized compound by HeLa cells, the endocytic process being the main pathway X-ray single-crystal structure determination observed to cross the plasma cell membrane. The high fluo- Measured crystals were prepared under inert conditions rescence intensity emitted, as well as the blue color emission, immersed in perfluoropolyether as protecting oil for manipu- make this compound a potential agent to be applied in bio- lation. Suitable crystals were mounted on MiTeGen imaging studies with fixed cells. Micromounts™ and these samples were used for data collec- tion. Data were collected with a Bruker D8 Venture diffract- ometer. The data were processed with the APEX3 suite [Bruker, Bruker AXS Inc. V2016.1, 2016, Madison, Wisconsin, USA]. The Experimental structures were solved by direct methods,34 which revealed the position of all non-hydrogen atoms. These atoms were refined Instrumentation on F2 by a full-matrix least-squares procedure using anisotropic Elemental analyses were performed using a Carlo Erba displacement parameters. All hydrogen atoms were located in EA1108 microanalyzer. ATR-IR spectra were recorded on a difference Fourier maps and included as fixed contributions high-resolution JASCO FT/IR-4700 spectrometer. The 1H NMR riding on attached atoms with isotropic thermal displacement (300.13 MHz), 11B{1H} (96.29 MHz) and 13C{1H} NMR parameters 1.2 times those of the respective atom. (75.47 MHz) spectra were recorded on a Bruker ARX 300 Crystallographic data for the structure of compounds 4, 5 and spectrometer. All NMR spectra were recorded in CDCl3 solu- 6 have been deposited with the Cambridge Crystallographic tions at 25 °C. Chemical shift values for 11B{1H} NMR spectra Data Center as supplementary publication no. CCDC were referenced to external BF3·OEt2, and those for 1H and 13C 1907569–1907571.† Geometric calculations and molecular {1H} NMR were referenced to SiMe4 (TMS). Chemical shifts are graphics were performed with Mercury.35 Additional crystal reported in units of parts per million downfield from the refer- data are shown in Table 1. ence, and all coupling constants are reported in hertz. UV-Vis spectra were recorded using a VARIANT Cary 5 UV-Vis-NIR Biological assays spectrophotometer, using spectroscopic grade THF (Sigma- Compounds 4–6 were dissolved and filtered following the Aldrich), in a normal quartz cuvette having 1 cm path length, same protocol as that in a previous work,26 obtaining a final for different solutions for each compound in the range of 10−4 compound concentration of 10 µM and 0.5% of DMSO (Sigma/ to 10−5 M in order to calculate the molar extinction coeffi- Aldrich).26 HeLa cells, which were routinely cultured under cients (ε). The fluorescence emission spectra and excitation standard conditions, were used to analyse the cytotoxicity, the spectra for all samples were recorded using a Varian Cary cellular uptake and the internalization pathway.26,27 Eclipse fluorescence spectrometer. No fluorescent contami- nants were detected on excitation in the wavelength region of Cytotoxicity experimental interest. The fluorescence quantum yields were The Alamar Blue assay (Invitrogen) was used to assess the cyto- determined by a “single point method” and repeated three toxicity of the compounds. To perform these experiments, 6 × times with a similar optical density for reproducibility,32 104 cells per ml were seeded into 24 well plates and the day against quinine sulfate in 0.5 M aqueous sulfuric acid with after they were incubated with each compound at 10 µM for ϕF = 0.54 as a standard.33 For the suspensions in THF/water 24 h. Then, the compounds were removed and the cells were (1/99, v/v) the refractive index was assumed to be that of pure rinsed with 500 µl of HBSS and incubated with a fresh water (1.33). Confocal laser scanning microscopy (CLSM, medium containing 10% of Alamar Blue for 3 h at 37 °C pro- This journal is © The Royal Society of Chemistry 2019 Biomater. Sci., 2019, 7, 5324–5337 | 5333 View Article Online Paper Biomaterials Science tected from light. Finally, the medium with Alamar Blue was acquired with a Leica SP5 CLSM. Endosome and lysosome collected and the fluorescence intensity was measured with a colocalization with the product was quantified by Imaris soft- Cary Eclipse Fluorescence Spectrophotometer (λex = 560; λem = ware. Fluorescence settings: product 6 (λex = 405 nm; λem = 590; Agilent). Experiments were performed in triplicate and 423–516 nm); early endosomes (λex = 561 nm; λem = This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. the results were normalized with control cultures (non-treated 581–717 nm,); and lysosomes (λex = 561 nm; λem = cells cultured under the same conditions without any com- 581–717 nm). pounds and considered as 100% of cell viability). Synthesis of 4 Cellular uptake A dry 250 mL round-bottomed flask equipped with a magnetic HeLa cells (2 × 105 cells) were seeded into glass bottom culture stirring bar was charged under nitrogen with a solution of 1,7- Open Access Article. Published on 02 October 2019. Downloaded on 7/14/2021 4:40:33 PM. dishes (MatTek) and after 24 h, they were exposed to 1 ml of C2B10H12 (1) (0.600 g, 4.17 mmol) in THF (80 mL) at 0 °C. each compound at 10 µM for 4 h at 37 °C. The cell cultures Then, a solution of 1.6 M n-BuLi in hexanes (2.60 mL, were rinsed twice with sterile phosphate buffered solution 4.17 mmol) was added dropwise to the mixture, which was (PBS from Biowest), and the plasma membrane was stained allowed to stir for 1 h at room temperature and cooled again at with 0.5 µl ml−1 of Cell Mask Orange (Thermo Fisher). Finally, 0 °C. A solution of 9-chloromethyl anthracene (0.964 g, the cells were observed under Confocal Laser Scanning 4.17 mmol) in THF (30 mL) was then added dropwise to the Microscopy (CLSM) (Olympus Fluoview 1000) and the images mixture under vigorous stirring. Then it was stirred for a half were processed by ImageJ/Fiji software. Fluorescence settings: an hour at room temperature and heated to reflux overnight. product 6 (λex = 405 nm; λem = 425–475 nm) and plasma mem- After that, the solvent was removed under vacuum and the brane (λex = 559 nm; λem = 570–625 nm). residue was quenched with H2O (15 mL), transferred to a sep- arating funnel, and extracted with Et2O (3 × 10 mL). The Internalization analysis organic layer was dried over MgSO4 and the volatiles were For each experiment, HeLa cells were seeded into glass bottom reduced under vacuum. The orange oil residue was purified by culture dishes (MatTek) at a density of 2 × 105 cells and main- preparative layer chromatography (dichloromethane/hexane tained under standard culture conditions for 24 h. The cells 10 : 90) to give 4 as a yellowish white solid. Yield: 0.722 g, 52%. were incubated with 1 ml of compound 6 at 10 µM for 4 h at Crystals suitable for X-ray analysis were obtained by slow evap- two different temperatures: 37 °C and 4 °C. Additionally, one oration of a solution of 4 in chloroform/n-heptane. 1H NMR, culture was incubated for 4 h at 37 °C and after removing the δ (ppm): 8.44 (s, 1H, C14H9), 8.19 (d, 3J (H,H) = 9 Hz, 2H, C14H9), compound, it was maintained for an additional 1 h at 4 °C to 8.02 (d, 3J (H,H) = 9 Hz, 2H, C14H9), 7.61–7.55 (m, 2H, C14H9), assess the effect of temperature on the fluorescence intensity. 7.49 (t, 3J (H,H) = 7.5 Hz, 2H, C14H9), 4.29 (s, 2H, CH2), 2.75 (s, As a control of endocytosis, the cells were incubated with 1H, CcH); 11B{1H} NMR, δ (ppm): −3.66 (s, 2B), −10.80 (s, 4B), 20 µg ml−1 of Transferrin-Alexa 488 (Tf-A488 from Life −13.99 (s, 2B), −15.58 (s, 2B); 13C{1H} NMR, δ (ppm): 131.53 (s, Technologies) for 4 h at the same temperatures (37 °C and C14H9), 130.94 (s, C14H9), 129.38 (s, C14H9), 128.33 (s, C14H9), 4 °C). In addition, one culture with Tf-A488 was incubated for 126.35 (s, C14H9), 125.13 (s, C14H9), 124.85 (s, C14H9), 55.10 (s, 3 h at 4 °C and then for 1 h at 37 °C to confirm that endocyto- Cc–H), 33.88 (s, CH2); ATR-IR (cm−1): ν = 3049 (Car–H), 2591, sis restarts when optimal conditions are recovered. In experi- 2577 (B–H), 1625 (CvC); elemental analysis calcd (%) for ments carried out at low temperature, the cells were kept at C17H22B10: C, 61.05; H, 6.63. Found: C, 60.98; H, 6.77. 4 °C during 1 h prior to the addition of 6 or Tf-A488 to arrest their enzymatic activity. Finally, after incubation, the com- Synthesis of 5 pounds were rinsed with PBS and observed under a CLSM The procedure was the same as that for 4, but using a solution (Olympus Fluoview 1000). Non-treated cells were also analysed of 9-I-1,7-C2B10H12 (2) (0.819 g, 3.032 mmol) in THF (70 mL), by confocal microscopy using the same settings as a control 1.6 M n-BuLi in hexanes (1.90 mL, 3.04 mmol) and a solution for autofluorescence (Fig. S6†). of 9-chloromethyl anthracene (0.702 g, 3.034 mmol) in THF (25 mL). After extraction with 3 × 10 mL of brine/CH2Cl2 the Colocalization orange oil was purified by preparative layer chromatography Cells (2 × 105) were seeded into glass bottom culture dishes. (dichloromethane/petroleum ether 20 : 80) to give 5 as a yellow- Two different cell cultures were prepared to label endosomes ish solid. Yield: 0.754 g, 54%. Crystals suitable for X-ray ana- and lysosomes separately. To label endosomes, upon cell lysis were obtained by slow evaporation of a solution of 5 in attachment, 10 µl of CellLight™ Early Endosomes-RFP, chloroform. 1H NMR, δ ( ppm): 8.44 (s, 1H, C14H9), 8.13 (dd, 3 BacMam 2.0 (Invitrogen), was added to the cell culture and the J (H,H) = 9 Hz, 3J (H,H) = 6 Hz, 2H, C14H9), 8.02 (d, 3J (H,H) = 9 cells were maintained overnight under standard culture con- Hz, 2H, C14H9), 7.61–7.56 (m, 2H, C14H9), 7.50 (t, 3J (H,H) = 7.5 ditions. Lysosomes were labelled with LysoTracker Red (1 : 500, Hz, 2H, C14H9), 4.29 (s, 2H, CH2), 2.84 (s, 1H, CcH); 11B{1H} Invitrogen) during 30 min and rinsed prior to observation. NMR, δ ( ppm): −2.65 (s, 1B), −8.93 (s, 4B), −12.82 (s, 1B), Compound 6, at a concentration of 5 µM, was added to the −13.68 (s, 1B), −15.39 (s, 1B), −17.61 (s, 1B), −23.87 (s, 1B); culture and placed at 37 °C. After 4 h of incubation, product 6 13 C{1H} NMR, δ ( ppm): 131.47 (s, C14H9), 130.80 (s, C14H9), was rinsed and replaced with fresh medium. The images were 129.47 (s, C14H9), 128.71 (s, C14H9), 128.62 (s, C14H9), 126.62 5334 | Biomater. Sci., 2019, 7, 5324–5337 This journal is © The Royal Society of Chemistry 2019 View Article Online Biomaterials Science Paper (s, C14H9), 125.20 (s, C14H9), 124.45 (s, C14H9), 56.07 (s, Cc–H), 2 (a) J. Liu, L. Jiang and W. P. Hu, Prog. Chem., 2009, 21, 33.72 (s, CH2); ATR-IR (cm−1): ν = 3048, 2929, 2852 (Car–H), 2568–2577; (b) M. Y. Chen, Y. Zhao, L. J. Yan, S. Yang, 2595, 2572, 2556 (B–H), 1626 (CvC); elemental analysis calcd Y. N. Zhu, I. Murtaza, G. F. He, H. Meng and W. Huang, (%) for C17H21B10I: C, 44.35; H, 4.60. Found: C, 44.42; H, 4.61. Angew. Chem., Int. Ed., 2017, 56, 722–727; (c) Y. Takaki, This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. Y. Wakayama, Y. Ishiguro, R. Hayakawa, M. Yamagishi, Synthesis of 6 T. Okamoto, J. Takeya, K. Yoza and K. Kobayashi, Chem. 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