Synthesis and Characterization of a Nitrophenol Disubstituted BODIPY Halochromic Probe

Synthesis and Characterization of a Nitrophenol Disubstituted BODIPY Halochromic Probe | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Synthesis and Characterization of a Nitrophenol Disubstituted BODIPY Halochromic Probe Riccardo Ossana, Elise Michel, Rachel Meallet, Gilles Clavier This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7459615/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 10 Nov, 2025 Read the published version in Photochemical & Photobiological Sciences → Version 1 posted 9 You are reading this latest preprint version Abstract We report the synthesis and characterization of a (3,5)-nitrophenol substituted BODIPY dye bearing a clickable handle. The dye was initially studied in dichloromethane, where it absorbs and emits in the red (666 nm and 687 nm respectively). Stepwise deprotonation of the phenols was achieved by addition of an organic base giving three species: 4-H₂ (λ abs = 666 nm, λ em = 687 nm), 4-H⁻ (λ abs = 730 nm, non-fluorescent), and 4²⁻ (λ abs = 769 nm, λ em = 844 nm), enabling an on-off-on pH sensing behavior Spectral deconvolution, DFT and TDDFT studies were carried out to support the experimental observations. Solvatochromic studies in 19 solvents show four distinct behaviors: J-aggregate formation in apolar alkanes (λabs/λem = 749 nm), conventional BODIPY response in low-polarity solvents, coexistence of diphenol and mono-deprotonated forms in moderately polar solvents, and dominance of the phenolate in polar solvents (λ abs = 831 nm in N,N dimethylformamide). Analysis using the Catalan scale indicates deprotonation is promoted by solvent dipolarity, acidity, and basicity, while the diphenol form is mainly sensitive to polarizability. These properties make such BODIPY a versatile probe for pH and microenvironment sensing, with potential applications in photoacoustic imaging and cellular studies. Solvatochromism BODIPY Photophysics Halochromic probe Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1 Introduction Measurement of pH by spectroscopic techniques is well established for both imaging and sensing applications.[ 1 ] Compared to traditional electrochemical or colorimetric methods, spectroscopic measurements offer several decisive advantages, including their generally non-destructive character, high sensitivity, chemical specificity, and compatibility with microscopic or macroscopic imaging modalities. In addition, the availability of a broad palette of molecular indicator dyes In addition, the availability of a broad palette of molecular indicator dyes [ 1 – 3 ]. Through molecular design, the spectral properties (absorption/emission maxima) as well as the acid–base equilibrium constants (pKa) of dyes can be finely tuned, thus expanding their applicability across biological, chemical, and material sciences. Most conventional pH-sensitive dyes are designed to operate within the physiological range, with pKa values close to neutral pH, making them well suited for biological applications. Nevertheless, other research fields often require pH probes adapted to non-physiological conditions. For instance, indicators with higher pKa values have been employed to investigate the alkalization phenomena occurring during the aging of materials such as concrete [ 4 ] whereas dyes with lower pKa values are valuable tools for probing acidic microenvironments, including the tumor extracellular matrix or intracellular organelles in cancerous cells [ 5 ]. The ability to shift the pKa window of a chromophore by rational design is therefore a central asset in the development of functional molecular probes. Beyond their intrinsic acid–base sensitivity, halochromic and pH-fluorochromic dyes also provide a powerful means of probing solvent properties. Their spectral signatures are influenced not only by protonation equilibria but also by solvation dynamics, polarity, and polarizability of the medium. When multiple protonation states coexist, each form may respond differently to specific solvent parameters, which makes these systems particularly informative. Proper deconvolution of their spectral responses through appropriate solvent models allows simultaneous exploration of both protonation behavior and solvent–solute interactions, offering a dual analytical window into chemical environments. Within this context, BODIPY (boron-dipyrromethene) dyes have attracted long-standing interest owing to their unique combination of advantageous properties. Their modular synthesis enables fine-tuning of absorption and emission wavelengths, spanning from the visible region (≈ 500 nm) to the near-infrared (NIR-II) domain [ 13 , 14 ], while maintaining generally high fluorescence quantum yields and excellent photostability [ 6 – 10 ]. These features have led to their widespread use in the design of sensors for a wide variety of analytes [ 11 , 12 ]. Numerous examples of pH-sensitive BODIPY derivatives have been reported, [ 15 , 16 ] relying on two main design strategies: (i) the incorporation of a non-conjugated pH-sensitive substituent, capable of modulating fluorescence via photoinduced electron transfer (PET), or (ii) the introduction of a conjugated substituent that directly perturbs the absorption and emission maxima, thus generating distinct colorimetric and fluorimetric responses. The choice of pH-sensitive groups is often restricted to aniline- or phenol-based moieties, conferring selective responses to acids or bases, respectively. The pKa of phenol-based substituents, typically in the range 7–9, can be significantly lowered by introducing electron-withdrawing groups on the aromatic ring. For example, O’Shea demonstrated that a 3-nitro-4-phenolic substituent grafted onto an azaBODIPY could reduce the pKa to around 5, making it relevant for acidic biological environments. [ 17 ] In the framework of our research on the development of new photoacoustic contrast agents derived from BODIPY scaffolds, we sought to design a pH-sensitive BODIPY dye with a pKa near 5. For this purpose, we selected the 3-nitro-4-phenolic moiety as the acid–base responsive group, and opted for the conjugated design strategy to maximize spectral separation between the protonated and deprotonated forms, while targeting absorption in the red-to-NIR region. The dye was further engineered with a clickable functionality, enabling future conjugation to nanocarrier systems for drug delivery applications. Prior to such applications, however, a systematic characterization of its intrinsic physicochemical and photophysical properties was required. Surprisingly, during preliminary solvent screening, we observed that the phenolate form could be stabilized in certain solvents upon simple dissolution, even in the absence of added base. In this article, we report the synthesis of this novel BODIPY derivative, followed by a detailed study of its acid–base and solvatochromic behavior. By combining steady-state spectroscopy, density functional theory (DFT) calculations, and quantitative solvent parameter analysis, we aim to elucidate the interplay between protonation equilibria, electronic structure, and solvation effects. This comprehensive approach provides insights not only into the dye’s suitability as a pH probe but also into its broader potential as a solvatochromic sensor and as a building block for further advanced photoacoustic and nanocarrier-based applications. 2 Experimental Section 2.1 Materials and Instruments All reagents used for this study were purchased from the commercial supplier Sigma-Aldrich (France) or TCI (France) and used as received. Spectroscopic experiments were conducted using spectroscopic grade solvents from Sigma-Aldrich or Carlo Erba. Milli-Q water (Merck-Millipore, France) was used throughout. Solvents for synthesis were treated with a MBraun solvent purification system (SPS-800) prior to their use. Monitoring of the reaction was carried out by thin layer chromatography (TLC). Crudes were prepared for chromatographic purification by solid deposition on pre-packed RediSep™ (Teledyne, France) silica columns and purified by Reveleris® X2 flash chromatography. NMR spectra were recorded using a JEOL ECS 400 MHz spectrometer. 1 H NMR and 13 C NMR spectra were referenced to tetramethylsilane (TMS, 0 ppm), and to the central peak of the residual solvent multiplet (CDCl 3 : 77.16 ppm) respectively. Multiplicities are designated as follows: s = singlet, d = doublet, t = triplet, m = multiplet. High-resolution mass spectrometry data (HRMS) were obtained from the SAMM–IPSIT laboratory at Gif-sur-Yvette (France). Mass spectra were obtained using an LTQ-Orbitrap Velos Pro (ThermoFisher) in infusion mode. Samples were analyzed in acetonitrile (ACN) using positive electrospray ionization (ESI+) Fourier-transform mass spectrometry (FTMS). Absorption spectra were recorded on a Cary 4000 using pure solvent as reference. Corrected fluorescence spectra were obtained on a Fluorolog 3 equipped with a double monochromator in excitation and emission in a 90° configuration. All solutions were prepared at a concentration suitable to have an absorbance lower than 0.1 at the maximum absorption band (approximately 1.5×10 − 6 mol.L − 1 ) and the spectra recorded in 10 mm path quartz cell. Titration was done by adding a concentrated solution of base (initially 1.5×10 − 4 mol.L − 1 and 1.0×10 − 2 mol.L − 1 for the final points) to limit the dilution effect (BODIPY was diluted 1.3 times in the final solution). The analysis of the titrations was done with the Specfit software (Spectrum Software Associates, version 2‑11 C, Chapel Hill, NC, 1998) that takes into account the dilution factor. 2.2 Quantum chemistry All compounds were studied by DFT and TDDFT. The geometry of the ground state was optimized at B3LYP/3-31g(d) in vacuo and the absorption properties at the PBE0 / 6-311 + g(d,p) level of theory with the IEFPCM solvent model (dichloromethane, DCM). Geometry optimisation in the ground state was followed by a frequency calculation to confirm that a true minimum was obtained. TDDFT calculation was done on the 12 lowest singlet states. Calculations were done with Gaussian 16 (Revision B.01). [ 18 ] Results were analysed with GaussView 6.0, Mercury 4.2.0 and Multiwfn 3.7. [ 19 ] 2.3 Synthesis and Characterization BODIPY 1 In a three necked round bottom flask charged with 100 mL of dichloromethane (DCM), were dissolved kryptopyrrole (1.0 g, 8.18 mmol, 2 eq.), perfluorobenzaldehyde (0.8 g, 4.06 mmol, 1 eq.) and few drops of trifluoroacetic acid (TFA). When the aldehyde was consumed (as monitored by TLC), the oxidation reagent chloranil (0.998 mg, 4.06 mmol, 1 eq.) was quickly added. After 5 min, diisopropylethylamine (4.1 g, 32.2 mmol, 7.2 eq.) was added. After 15 min, the boron trifluoride diethyl etherate (7.2 g, 44.6 mmol, 11 eq.) was dissolved. Purification was performed on column chromatography (silica gel, petroleum ether PE/DCM with increasing ratio of PE and DCM (70/30, v/v) as eluent. The orange, fluorescent fraction was isolated, affording 0.600 g of 1 (1.21 mmol) as a gold solid. Yield: 26%. 1H-NMR (400 MHz, CDCl 3 ) δ 2.54 (s, 6H), 2.34 (q, J = 7.5 Hz, 4H), 1.51 (s, 6H), 1.02 (t, J = 7.6 Hz, 6H). BODIPY 2 BODIPY 1 (1.25 g, 2.77 mmol, 1.0 eq.) was dissolved in N,N -dimethylformamide (DMF, 50 mL). 2-Mercaptoethanol (1.00 g, 12.56 mmol, 6.0 equiv) and potassium carbonate (0.47 g, 3.40 mmol, 1.6 equiv) were added, and the solution was stirred for 30 min at room temperature. Water (50 mL) was then poured to the solution, and the aqueous phase was extracted with diethyl ether (3 × 50 mL). The organic phases were combined, washed with water (2 × 50 mL), dried over magnesium sulfate, and filtered. The solvent was evaporated under reduced pressure, and the product was purified by flash chromatography (PE/DCM, 0–100%) to obtain 1.287g (2.43 mmol) of BODIPY 2 . Yield 87%. 1 H-NMR (400 MHz, CDCl 3 ) δ 3.76 (t, J = 6.0 Hz, 2H), 3.19 (t, J = 5.7 Hz, 2H), 2.54 (s, 6H), 2.33 (q, J = 7.6 Hz, 4H), 2.01 (t, 1H), 1.51 (s, 6H), 1.01 (t, J = 7.6 Hz, 6H). Propargyloxy acetic acid 5 To a solution of propargyl alcohol (1.14g, 20.3 mmol) in tetrahydrofuran (THF, 1.2 M sol.) was added NaH (1.37g, 34.5 mmol, 1.7 eq.) portion-wise at 0°C. After addition, the mixture was stirred at 0°C for 1 h then tert-butyl 2-bromoacetate (4 g, 20.3 mmol, 1 eq.) was added to the mixture at 0°C. The mixture was stirred at 15°C for 12 h and then the reaction was stopped by addition of HCl (aq.) 37% (2 mL). Water was added (50 mL) extracted with ethyl acetate (EA, 2x 50 mL) and the combined organic layers, were washed with brine (100mL), dried over anhydrous Na 2 SO 4 , filtered and concentrated in vacuo to a brown oil. The crude residue was purified by column chromatography (PE/EA 0 to 100%) recovering 0.78 g (7.89 mmol) of 5 as a colorless oil. Yield 40%. 1 H-NMR (400 MHz, CDCl 3 ) δ 4.26 (d, J = 2.7 Hz, 2H), 4.21 (s, 2H), 2.45 (t, J = 2.3 Hz, 1H); 13 C-NMR (101 MHz, CDCl 3 ) δ 175.4, 78.1, 76.2, 65.7, 58.5. BODIPY 3 (Propargyloxy)acetic acid 5 (0.125 g, 5.7 mmol, 2.4 eq.) and SOCl 2 (1.695 g, 14.3 mmol, 2.5 eq.) were dissolved in DCM (5 mL). The mixture was stirred at room temperature for 30 minutes, then the solvent was evaporated and the crude, containing acyl chloride 6 , was dried in vacuum for 4 hours to ensure complete SOCl 2 evaporation. The residue was diluted in THF (30 mL), then triethylamine (TEA, 0.245 g, 2.43 mmol, 1.1 eq.) and BODIPY 2 (0.245 g, 2.43 mmol, 1 eq.) were added. The reaction mixture was stirred for 15 hours at 40°C, after which water (50 mL) was added, and the mixture was transfered to a separatory funnel and extracted with EA (50 mL), the organic phase was recovered and washed with brine (50 mL) and dried over sodium sulphate. The crude was purified by column chromatography (silica gel, PE/EA gradient 0 to 30%) isolate 0.570 g (0.91 mmol) of BODIPY 3 as a golden solid. Yield 38%. 1 H-NMR (400 MHz, CDCl 3 ) δ 4.26–4.22 (d + t overlapping signals, 4H), 4.17 (s, 2H), 3.20 (t, J = 6.6 Hz, 2H), 2.47 (s, 6H), 2.42 (t, 1H), 2.26 (q, J = 7.5 Hz, 4H), 1.45 (s, 6H), 0.94 (t, J = 7.6 Hz, 7H); 13 C-NMR (101 MHz, CDCl 3 ) δ 169.6, 156.1, 136.8, 134.0, 130.2, 78.3, 75.9, 66.0, 62.4, 58.5, 32.9, 29.8, 17.2, 14.7, 12.9, 11.0, 1.1; HRMS (ESI) m/z [M + H] + calculated for C30H31BF6N2O3S: 624.2053; Found: 624.2026. BODIPY 4 In a 25 mL round bottom flask BODIPY 3 (0.570 g, 0.91 mmol), 3-nitro-4-phenol-benzaldehyde (0.456 g, 0.91 mmol, 1 eq.) and piperidine (0.078 g, 2.73 mmol, 3 eq.) added as a 0,01% v/v toluene solution, were dissolved in toluene (10 mL). After setting up a Dean-Stark apparatus, the reaction mixture was stirred at 160°C for 17h (TLC PE\EA 30%, Rf 0.70). It was then brought to room temperature and toluene was evaporated. The residue was redissolved in DCM (50 mL) and transfered to a separatory funnel. The organic phase was washed with water (50 mL), then the aqueous phase was extracted with DCM (2x50 mL). Recollected organic phases were washed with brine (100 mL) and dried over sodium sulphate. The solvent was evaporated, and the crude was purified by column chromatography (Silica gel, eluent toluene/acetone 4%). The isolated product BODIPY 4 is a dark green solid (0.270 g, 0.29 mmol). Yield: 31%. 1 H-NMR (400 MHz, DMF-d7) δ 8.21 (d, J = 1.8 Hz, 2H), 7.87 (t, J = 5.3 Hz, 3H), 7.53 (dd, 4H), 7.20 (d, J = 8.7 Hz, 2H), 4.24 (d + t overlapping, Jd = 3.0 Hz, 4H), 4.15 (s, 2H), 3.36 (t + t overlapping, Jt = 3.0 Hz, Jt’ = 7.3 Hz, 3H), 1.60 (s, 6H), 1.04 (t, J = 7.3 Hz, 4H) (-CH 2 CH 3 covered by DMF-d7 quintet); 13 C-NMR (101 MHz, DMF-d7) δ 169.9, 154.2, 151.7, 138.7, 137.5, 135.8, 133.0, 132.8, 129.1, 128.6, 125.3, 120.7, 118.9, 79.6, 77.4, 66.2, 62.9, 58.1, 33.3, 32.1, 22.8, 18.3, 18.1, 14.0, 10.6; HRMS (ESI) m/z [M + H] + calculated for C44H37BF6N4O9S: 922.2278; Found: 921.2203. 3 Results and Discussion 3.1 Synthesis The fluorophore (3,5)-bis-(3-nitrophen-4-ol) substituted BODIPY 4 was obtained after a 4 steps reaction sequence, shown in Scheme 1 . The BODIPY scaffold was synthesized by dissolving in dichoromethane two equivalents of 2,4-dimethyl-3-ethylpyrrole (kryptopyrrole) and pentafluorobenzaldehyde and following a well-known three steps one-pot protocol.[ 20 – 22 ] The details are reported in the experimental section. In order to add the clickable handle it was decided to leverage the high reactivity to nucleophilic substitution of the para position on the pentafluorophenyl ring.[ 23 ] Hence, 1 was dissolved in DMF with an excess of mercaptoethanol and potassium carbonate, affording BODIPY 2 with a yield of 87%. The free hydroxyl was functionalized with a short, clickable linker whose synthesis was achieved by adapting a published protocol.[ 24 ] Tert-butyl-bromo acetate was stirred with propargyl alcohol in THF in the presence of sodium hydride to afford propargyloxy acetic acid (compound 5 ) in 40% yield. Compound 5 was then converted into its more reactive acyl chloride derivative (compound 6 ) by reaction with thionyl chloride in dry DCM for 30 minutes. The solvent was evaporated, and the residue was dried in-vacuo for about 3 hours to ensure the complete removal of unreacted thionylchloride. The intermediate acyl chloride 6 was immediately redissolved in dry THF under inert atmosphere (Ar) and a dry THF solution of N,N,N -triethylamine and BODIPY 2 was injected through the rubber stopper under stirring. After 24 hours, the solvent was evaporated, and the reaction mixture was purified to isolate BODIPY 3 with 38% yield. The desired compound 4 was obtained through the functionalization of positions 3- and 5- of BODIPY 3 by Knoevenagel condensation with the 3-nitro-4-phenol aldehyde in the presence of piperidine under azeotropic distillation in toluene for 17 hours. After removal of the solvent, purification of the crude material by standard phase column chromatography proved to be particularly challenging due to heavy tailing stemming from the products low solubility in most solvents used for elution. BODIPY 4 is insoluble or very poorly soluble in polar protic solvents such as water, methanol, ethanol or isopropanol as well as non-polar solvents like petroleum ether, hexane, and heptane. In polar aprotic, non-chlorinated solvents such as acetone, it is only partially soluble and tends to form suspensions. Good solubility was observed only in a limited set of solvents, including DCM and toluene. Eventually, BODIPY 4 was isolated in 38% yield by using an isocratic mixture of toluene and acetone (4%) as mobile phase. All compounds have been characterized by 1 H and 13 C NMR and unreported compounds by HRMS (spectra in Fig. S1 to S13 in supplementary information). 3.2 DFT Study Prior to the photophysical studies, BODIPY 4 was investigated by quantum-chemical calculations to gain insight into its electronic properties. All three possible forms of the BODIPY were studied in their ground state, namely: diphenol form ( 4-H 2 ), mono deprotonated ( 4-H − ) and fully deprotonated ( 4 2− ). The clickable side chain was omitted as it was expected to have minimal impact on the photophysical properties and was replaced with a fluorine atom. The BODIPY core is completely planar in all cases and the styryl substituents are slightly twisted: 22.2° for 4-H 2 , 15.7° for 4-H 2− , and 9.5° (phenolate) and 15.6° (phenol) for 4-H − . This last result reflects the non-symmetric nature of the mono deprotonated BODIPY, where the substituent with the stronger donor character is less twisted. The pentafluorophenyl moiety is highly twisted (83 to 86°) relative to the BODIPY core because of the steric hindrance and should have minimal impact on the photophysical properties. Frontiers molecular orbitals have also been studied ( Fig. S14 in Supplementary Information). Highest occupied molecular orbital (HOMO) of 4-H 2 is delocalized over the BODIPY and styryl moieties, while its lowest unoccupied molecular orbital (LUMO) is more localized on the BODIPY core, in accordance with the acceptor nature of the BODIPY core. The molecular orbitals of 4 2− are similarly distributed, however the electronic density of the HOMO is more concentrated on the two nitrophenolates, reflecting their stronger electron rich nature compared to the phenol counterpart. In the case of 4-H − the unsymmetrical nature can be observed in the HOMO, which is localized mostly on the BODIPY moiety and the phenolate branch. Contrary to expectations, the LUMO is localized on the nitrophenol group, whereas the LUMO + 1 involves both the BODIPY core and the phenolate group. TDDFT with solvent effect (DCM) analysis provided insights into the nature of the major electronic transitions (see Supplementary Informations for details). In all cases an intense low energy transition of charge transfer character from the phenol or phenolate moieties towards the BODIPY is observed ( Fig. S15 in Supplementary Information). Stepwise deprotonation yields a red shift of the transition from 631 nm for 4-H 2 to 720 nm for 4-H − and 783 nm for 4 2− . Notably, the lower energy transition of 4-H − is a pure charge transfer HOMO-LUMO transition localized at 785 nm with a very weak oscillator strength (f = 0.08). The presence of this transition should lead a quenching of the BODIPY fluorescence. 3.3 Photophysical Properties in dichloromethane First, BODIPY 4 was studied in dichloromethane, which is an apolar aprotic solvent and poor hydrogen bond acceptor. It is thus expected that only the diphenolate form 4-H 2 will be present. BODIPY 4 exhibits three absorption bands between 300 and 800 nm. The main (lowest energy) transition, the S0→S1 transition, is centered at 666 nm, with a vibrionic shoulder appearing at 620 nm. Below 500 nm are found two transitions involving higher energy levels. Specifically, the second, broad band is centered at 445 nm and corresponds to the typical S 0 →S 2 transition of BODIPYs. The third band at 357 nm (shoulder at 345 nm) arises from a π–π* transition localized on the styryl moiety, as demonstrated by calculations for similar compounds. A molar absorption coefficient of 66 000 L·mol − 1 ·cm − 1 was determined for the main transition. BODIPY 4 is fluorescent and emits at 685 nm in dichoromethane, with a Stokes shift of 319 cm − 1 . Such a small value is typical for this class of dyes. The associated fluorescence quantum yield is 0.08, indicative of weak fluorescence. When compared to literature values the data collected for BODIPY 4 reveal that the nitrophenol substituent is a weak donor, since the photophysical properties in dichoromethane lies in between a phenyl (λ abs = 661 nm and λ em = 675 nm) and a phenol (λ abs = 677 nm and λ em = 700 nm). [ 20 ] BODIPY 4 was then titrated with the non-nucleophilic base 1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU) in dichoromethane to gain insight in the photophysical properties of the basic forms. The absorption spectra (Fig. 1 , left) show a gradual decrease of the 666 nm band corresponding to 4-H 2 (blue spectrum in Fig. 1 ) in favor of a red shifted band at 730nm (green spectrum in Fig. 1 ). This second band is maximum for 7.5 equivalents of added base. Upon further addition of DBU, a third band appears at 769 nm (red spectrum in Fig. 1 ) whose intensity starts to level off at 100 equivalents of base. The behavior of BODIPY 4 upon titration with DBU seems to indicate a stepwise deprotonation in dichoromethane with the intermediate spectrum corresponding to 4-H − and the final one to 4 2− . These absorption data were further analyzed using the SPECFIT software, which performs global analysis of equilibrium systems with nonlinear regression modeling by the Levenberg-Marquardt method. Several models were tested but the only one that resulted in a satisfying fit is the one corresponding to the stepwise deprotonation of 4 , to the phenolate first ( 4-H − ), and then to the bisphenolate ( 4 2− ). The calculated spectra (Fig. 2 left) are in good agreement with the experimental ones, and the distribution diagram shows the rapid deprotonation of 4-H 2 to 4-H − , followed by the formation of 4 2− which becomes predominant above 15 equivalents of base. The main absorption bands become broader upon deprotonation as seen by their Full Width at Half-Maximum (FWHM): 1030 cm − 1 for 4-H 2 , 1373 cm − 1 for 4-H − and 1478 cm − 1 for 4 2− . The enlargement can be attributed to the stronger charge transfer (ICT) character of the transition upon deprotonation and formation of the stronger electron donor phenolate. The calculated associated equilibrium constants for the two successive processes are log(K 1 ) = 5.21 and log(K 2 ) = 4.79. The second deprotonation is slightly disfavoured, likely due to electrostatic repulsions but does not require harsh conditions to occur. This behavior is consistent with the expected high basicity of the nitrophenolate. The titration was also monitored by fluorescence emission (Fig. 3 ). Two excitation wavelengths were selected: 610 nm, were 4-H 2 is the predominant absorbing specie and 380 nm where all three species absorb light with a rather close efficiency. The initial fluorescence band is found at 684 nm (left, λ ex = 610 nm) and 687 nm (λ ex = 380 nm). The difference probably comes from the fact that large bandpass (8.5 nm) was necessary to record the spectra due to the low fluorescence efficiency of BODIPY 4 . Upon addition of DBU the band gradually disappears and a new very red-shifted one appears at 844 nm. In both experiments, the red shifted band appears more intense than the initial one. The difference is more pronounced in the experiment where the excitation was set at 380 nm where all three species absorb light with close efficiency. However, it is difficult to conclude that the second species emits light more efficiently because the emission appears at the limit of the detector sensitivity, where efficiency is very low and the correction factor plays an important role. The analysis of the data where not as straight forward as for the absorption. Two models where tested: one where the singly deprotonated form 4-H − is considered as a dark specie as the calculation hinted and the doubly protonated form 4 2− as an emissive one together with the initial 4-H 2 .In the second model the intermediate form 4-H − was omitted. The second model seemed less realistic even though the error on the association constant was lower. In the first model, analysis of the experiment with excitation at 610 nm gave better results (lower error) that the other model, in which the equilibrium constant for the first deprotonation K 1 was very small (log(K1) = 2.57) and determined with an error of 50%. With excitation at 610 nm the fitted equilibrium constants were close to the one calculated for the absorption spectra (log(K 1 ) = 5.38 and log(K 2 ) = 4.79). The fluorescence thus probes the same stepwise deprotonation process as the absorption, but with a dark intermediate 4-H − specie and only the extreme ones ( 4-H 2 and 4 2− ) are emissive. This study allowed us to better understand the behavior of BODIPY 4 under basic conditions in dichoromethane. It undergoes two successive deprotonation processes giving a singly deprotonated intermediate followed by the doubly deprotonated one. Each specie has its own photophysical signature: 4-H 2 absorbs at 666 nm and emit at 685 nm, 4-H − absorbs at 730 nm and is not fluorescent and 4 2− absorbs at 769 nm and emits at 844 nm. The second deprotonation is slightly less favorable than the first but still occurs under reasonable conditions (requiring about a tenfold excess of base). 3.4 Solvatochromic Properties The photophysical properties of BODIPY 4 where then studied in a broad range of solvents. BODIPY are usually weakly sensitive to solvent effect apart from its fluorescence quantum yield that usually drops dramatically in polar solvents.[ 25 , 26 ] However in our case we reasoned that the charge transfer nature of the main transition and the presence of a moiety that can be easily deprotonated could by influenced by the solvent nature. BODIPY 4 has thus been studied in 19 solvents from apolar to polar (protic and non protic) at a constant concentration of 1.55 µmol/L and four distinct behaviors could be found. The photophysical data are summarized in Table 1 , and the spectra in each solvent are provided in the supplementary information ( Fig. S16 to S36 ). Table 1 Spectroscopic properties of BODIPY 4 in several solvents solvent λ abs (nm) λ fluo (nm) \(\:\varvec{\delta\:}\stackrel{-}{\varvec{\nu\:}}\) (cm − 1 ) a 1,1,2,2-tetrachloroethane 671 692 452 n -hexane 749 676 / 749 chloroform 667 688 458 n -pentane 749 675 / 748 2,2,2-trifluoroethanol 655 676 474 toluene 668 688 435 Dichloromethane 666 687 459 mesitylene 668 689 456 benzonitrile 670 693 495 acetonitrile 660 / 751 682 489 propylene carbonate 667 / 758 687 436 1,4-dioxane 667 687 436 acetone 661 684 509 ethyl acetate 661 / 721 681 444 methanol 702 685 / 776 tetrahydrofuran 666 685 416 tetrahydrofuran + water b 666 685 416 N,N -dimethylformamide 831 - dimethyl sulfoxide 826 702 ethanol 715 687 / 790 c a Stokes shift calculated on the phenol band; b 30 µL of water added; c shoulder. In the apolar n -pentane and n -hexane BODIPY 4 formed J aggregates (Fig. 4 ) as clearly evidenced by the thin, red-shifted absorption band (λ abs = 749 nm in both solvent). A shoulder in the blue region corresponds likely to the non-aggregated form. The fluorescence spectra display two bands: one at 675 nm and one at 749 nm. The high energy one is clearly coming from the non-aggregated form while the low energy one is typical of J-aggregates of BODIPY (thin emission band with no Stokes shift). [ 27 ] While BODIPY are known to form aggregates in polar solvents [ 28 ] it is less common to observe such behavior in apolar solvents at such a low concentration. The aggregation likely comes from the presence of the nitro-phenol groups, whose polarity and ability to form hydrogen bonds promote the formation of well-organized aggregates. In solvents of low to medium polarity solvents, the main absorption band is only slightly affected, shifting from 655 nm in 2,2,2-trifluoroethanol to 671 nm in 1,1,2,2-tetrachloroethane (Fig. 5 ). Similarly, the fluorescence band is relatively insensitive to the nature of the solvent and shits from 676 in 2,2,2-trifluoroethanol to 693 in benzonitrile. The Stokes shift are small (between 400 and 500 cm − 1 ). These results are in line with the expected solvatochromic behavior of BODIPY, usually weakly sensitive to solvent properties. Unexpected results occurred in more polar solvents. In three solvents (acetonitrile, propylene carbonate and ethyl acetate) the absorption spectra display a red-shifted band in addition to the expected BODIPY one around 665 nm (Fig. 6 ). The new band appears between 720 and 760 nm. Its position and relative intensity are solvent dependent. There is also a small band above 500nm that is easily identifiable in propylene carbonate and could also be seen in the phenolate spectrum obtained in DCM (Fig. 2 ). This result indicates that in these solvents there is an equilibrium between the phenol 4-H 2 form and the phenolate 4-H − one. The fluorescence spectra only display the phenol band around 685 nm, which is reasonable considering that the the 4-H − is not fluorescent. Finally in very polar solvents (dimethylsulfoxide, N,N -dimethylformamide, ethanol and methanol) the main absorption band is located between 700 and 820 nm (Fig. 7 ). In the alcohols, since the main emission band was found at higher energy (685 nm and 687 nm for methanol and ethanol respectively ( Fig. S31 and S36 in Supplementary Information), the nature of the absorbing species was attributed to the phenolate form. In dimethylsulfoxide and N,N -dimethylformamide the absorption spectra clearly display the characteristic bands of the phenolate and the fluorescence of the phenol BODIPY is hardly seen in former and quenched in the later. In the alcoholic solvents a second band can be seen at lower energy. However, its origin is uncertain since it appears at the limit of the detection capacity of the apparatus used and the correction is quite large above 750 nm, sometimes creating artefacts. Further analysis would be needed to confirm the occurrence of an emission in that range. To confirm that the appearance of the phenolate band was due to solvent properties rather than trace water, 30 µL of water were added to the THF solution, in which the phenolate band was not observed. The absorption and emission spectra before and after water addition were found identical ( Fig. S32 and S33 ) ruling out the possible role of residual water in promoting the formation of the phenolate in the polar solvents. To the best of our knowledge, the occurrence of the absorption band of the phenolate form for phenol-substituted BODIPY is not reported in the literature. Examples of pH sensitive BODIPYs were designed with halogenated phenols (Chloride or Fluoride) in order to bring the phenol’s pKa close to 7.[ 29 ] The nitrophenol group was introduced by O’Shea on an aza-BODIPY to lower the pKa of the phenol even further than that (down to ~ 5). The increased acidity of the nitrophenol group is likely explains why we observe the appearance of the phenolate band in polar solvents, which will be discussed further later. Solvent-dependent spectral shifts can be analyzed through various approaches that encompass empirical single-parameter solvent scales and multi-parameters one. We used four scales to analyze our results: two single parameter (Lippert-Mataga and E T (30)) and two multi-parameters (Kamlet–Taft and the generalized solvent scale proposed by Catalan). We analyzed the variations of the absorption and emission maxima as well as the Stokes shift. The single parameters scales completely failed to model the solvent effect (a discussion is included in Supplementary Information). The Kamlet–Taft gave good results only in the case of the phenolate absorption band but failed on all other properties. The Catalan scale offers the advantage of splitting the solvent properties in four distinct parameters: SA, SB, SP and SdP which characterize the solvent acidity, basicity, polarizability and dipolarity, respectively.[ 30 ] Previous analysis of BODIPY derivatives with this scale proved to work well for us [ 31 ] and others.[ 25 , 26 , 32 ] Table 2 Estimated coefficients (y 0 , a SP , b SdP , c SA and d SB ), their standard errors and correlation coefficients (r 2 ) for the multiple linear regression analyses of \(\:\stackrel{-}{\nu\:}\) abs of the phenol and phenolate bands, \(\:\stackrel{-}{\nu\:}\) em of the phenol and Stokes shift ( \(\:{\Delta\:}\stackrel{-}{\nu\:}\) ) as a function of the Catalan solvent scale. Catalan y 0 a SP b SdP c SA d sb r 2 \(\:\stackrel{-}{\nu\:}\) abs (phenol) 15925 ± 97 -1221 ± 114 36 ± 22 -23 ± 41 -75 ± 43 0.98 \(\:\stackrel{-}{\nu\:}\) abs (phenolate) 18576 ± 694 -1351 ± 1644 -3915 ± 713 2529 ± 488 -2661 ± 639 0.99 \(\:\stackrel{-}{\nu\:}\) em (phenol) 15743 ± 104 -1499 ± 147 -16 ± 41 -135 ± 59 -223 ± 52 0.93 \(\:{\Delta\:}\stackrel{-}{\nu\:}\) 530 ± 132 -119 ± 156 51 ± 30 -39 ± 56 -56 ± 59 0.35 The Stokes shift of the phenol transition appears to be difficult to fit with any model, probably because of the small variations observed in the solvents studied, while the Catalán scale fares well with the absorption and emission maxima. The electronic transition of the phenol form 4-H 2 appears to be mostly dependent on the solvent polarizability (SP) which was already observed for other BODIPY and notably derivatives substituted with a chlorophenol or a fluorophenol.[ 25 , 33 ] The phenol form of BODIPY 4 has solvatochromic properties that are similar to related BODIPY. On the other hand, the phenolate absorption is much more sensitive to the solvents: the band shifts from 702 nm in methanol to 831 nm in N,N -dimethylformamide. The analysis of this solvent dependence with the Catalan scale gave an excellent correlation (r 2 = 0.99) and reveals that in that case the band position strongly depends on the solvent dipolarity (SP) as well as solvent acidity (SA) and basicity (SB). This is in line with the fact that the solvent must interact with the proton of a phenol of the solvent to form the polar phenolate. The ability of the solvent to give and accept hydrogen bonds is crucial in that regard. The appearance of the nitrophenolate band necessitate a polar solvent that can give and/or accept hydrogen bonds. 4 Conclusion BODIPY 4 , bearing two nitrophenol substituents, was successfully synthesized and thoroughly characterized. The diphenol form, 4-H ₂ , exhibits absorption and emission in the red region (λ abs = 666 nm, λ em = 687 nm), providing a strong baseline for studying its photophysical behavior. Its response to basic conditions in dichloromethane revealed a stepwise deprotonation process, leading to the singly deprotonated 4-H ⁻ (λ abs = 730 nm, non-emissive) and the doubly deprotonated 4² ⁻ (λ abs = 769 nm, λ em = 844 nm). The distinct absorption and emission signatures of these species enable BODIPY 4 to function as a sensitive on-off-on pH fluorescent sensor, where only the fully protonated and fully deprotonated forms are emissive. This properties could be exploited in fluorescence microscopy or photoacoustic imaging with spectral unmixing to probe media of different pH (on-going experiments). The solvatochromic behavior of BODIPY 4 is particularly noteworthy, as it exhibits four distinct responses depending on solvent polarity: 1. Apolar solvents ( n -pentane, n -hexane): spontaneous formation of J-aggregates at micromolar concentrations, evidenced by characteristic red-shifted absorption and emission bands. 2. Low-polarity solvents: conventional BODIPY behavior, with minimal shifts in absorption and emission. 3. Moderately polar solvents: presence of an equilibrium between diphenol and mono-deprotonated forms, allowing both species to be detected spectroscopically. 4. Highly polar solvents (dimethylsulfoxide, N,N -dimethylformamide, alcohols): dominance of the mono-deprotonated phenolate, with diphenol detectable only through residual fluorescence Analysis using the Catalan solvent scale revealed that the deprotonation of the phenol groups is strongly influenced by solvent dipolarity, acidity, and basicity, while the diphenol form itself is primarily sensitive to solvent polarizability. This insight underscores the intricate interplay between solvent environment and the electronic structure of BODIPY derivatives. Overall, the study demonstrates that BODIPY 4 is a versatile photophysical probe capable of reporting on both pH and local solvent environment, combining stepwise deprotonation, dual fluorescence response, and solvent-sensitive spectral shifts. These properties could be particularly valuable in biological imaging, enabling the differentiation of cell compartments and environments of varying polarity. Further research is ongoing to explore applications in live-cell imaging and photoacoustic sensing, leveraging the unique photophysical and solvatochromic features of this nitrophenol-substituted BODIPY. Declarations Conflict of interests The authors declare no competing interests. Supplementary Information The online version contains supplementary material available at Acknowledgements The University Paris-Saclay, ENS Paris-Saclay and the CNRS are acknowledged for funding. RM additionally thanks the Région Ile-de-France, DIM NanoK and LabEx CHARMMMAT for financial support. Author contribution GC and RM designed the research, RO, EM and GC performed the experiments and analyzed the data, RM, RO and GC wrote and reviewed the manuscript. Funding This project has received financial support from the French National Research Agency under the program ANR-21-CE09-0024-01. References J. Han and K. Burgess. Fluorescent Indicators for Intracellular pH. Chem. Rev. 110, 2709–2728 L. 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Chem. 183, 190–197 Scheme 1 Scheme 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files 2025ppsSIGClavier.docx Scheme1.png Scheme 1 Synthesis of the probe BODIPY 4. Reaction Conditions: (a) I) NaH (1.7 eq.), THF, 0°C to r.t., 24h; II) NaOH (aq.) in MeOH (50%), r.t. 24h, yield 40%; (b) SOCl 2 , DCM, r.t., 30’; (c) I) TFA, DCM, r.t., 180 min; II) Chloranil (1 eq.), DCM, rt, 5 min; III) DIPEA (7 eq.), BF 3 Et 2 O (11 eq.), DCM, r.t., yield 26%; (d) Mercaptoethanol (6 eq.), K 2 CO 3 (1.6 eq.), DMF, r.t., 30’, yield 87%; (e) TEA (1.1 eq.), THF, 40°C, 15h; (f) 3-Nitro-4-hydroxy-benzaldehyde (1 eq.), piperidine (3 eq.), toluene, 150 °C, Dean-Stark, 24h, yield 38%. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7459615","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":508202512,"identity":"36480c0e-b844-4d96-8b2a-d2071f674b07","order_by":0,"name":"Riccardo Ossana","email":"","orcid":"","institution":"University of Paris-Saclay","correspondingAuthor":false,"prefix":"","firstName":"Riccardo","middleName":"","lastName":"Ossana","suffix":""},{"id":508202515,"identity":"8b12e49c-764c-43aa-9629-b524047bf196","order_by":1,"name":"Elise Michel","email":"","orcid":"","institution":"École Normale Supérieure Paris-Saclay","correspondingAuthor":false,"prefix":"","firstName":"Elise","middleName":"","lastName":"Michel","suffix":""},{"id":508202516,"identity":"69a5df41-9c46-49e3-b17a-3c54a79903d4","order_by":2,"name":"Rachel Meallet","email":"","orcid":"","institution":"University of Paris-Saclay","correspondingAuthor":false,"prefix":"","firstName":"Rachel","middleName":"","lastName":"Meallet","suffix":""},{"id":508202517,"identity":"5f09e709-ff40-4969-b6f1-07fb723be817","order_by":3,"name":"Gilles Clavier","email":"data:image/png;base64,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","orcid":"","institution":"École Normale Supérieure Paris-Saclay","correspondingAuthor":true,"prefix":"","firstName":"Gilles","middleName":"","lastName":"Clavier","suffix":""}],"badges":[],"createdAt":"2025-08-26 06:53:39","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7459615/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7459615/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s43630-025-00806-8","type":"published","date":"2025-11-10T15:57:46+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":90527063,"identity":"2b018632-6c05-4f05-abf3-8334053b2ad2","added_by":"auto","created_at":"2025-09-03 17:14:25","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":98958,"visible":true,"origin":"","legend":"\u003cp\u003eAbsorption spectra of BODIPY \u003cstrong\u003e4\u003c/strong\u003e (initial concentration 1.55 µmol/L) upon incremental addition of DBU (from 0 to 140 equivalents) in dichoromethane (left). Plot of the absorption values as a function of DBU equivalents for the three maxima observed (right).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7459615/v1/b74150d288200cd3dae1beae.png"},{"id":90527064,"identity":"edba2b72-c33f-4110-a55a-551e56afae5a","added_by":"auto","created_at":"2025-09-03 17:14:25","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":64789,"visible":true,"origin":"","legend":"\u003cp\u003eCalculated absorption spectra of BODIPY to \u003cstrong\u003e4-H\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e (blue), \u003cstrong\u003e4-H\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003c/sup\u003e (green) and \u003cstrong\u003e4\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e2-\u003c/strong\u003e\u003c/sup\u003e (red) after data treatments (left) and their distribution diagram (right).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7459615/v1/653f05e9c4cf90286020788f.png"},{"id":90527066,"identity":"0f447c12-81b8-4e9f-9217-eca27084f6c9","added_by":"auto","created_at":"2025-09-03 17:14:25","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":86346,"visible":true,"origin":"","legend":"\u003cp\u003eFluorescence emission spectra of BODIPY \u003cstrong\u003e4\u003c/strong\u003e (initial concentration 1.55 µmol/L) upon stepwise addition of DBU (from 0 to 140 equivalents) in dichoromethane: left λ\u003csub\u003eex\u003c/sub\u003e=610 nm and right λ\u003csub\u003eex\u003c/sub\u003e=380 nm.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7459615/v1/e2a5e352148e57e7f852cc61.png"},{"id":90527942,"identity":"f7900122-d594-4344-9971-559487de00db","added_by":"auto","created_at":"2025-09-03 17:30:25","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":62953,"visible":true,"origin":"","legend":"\u003cp\u003eAbsorption (left) and fluorescence emission spectra (right, λ\u003csub\u003eex\u003c/sub\u003e=380 nm) of BODIPY \u003cstrong\u003e4\u003c/strong\u003e (concentration 1.55 µmol/L) in \u003cem\u003en\u003c/em\u003e-pentane (red) and \u003cem\u003en\u003c/em\u003e-hexane (blue).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7459615/v1/197dfbd624ea0410653d495f.png"},{"id":90527436,"identity":"46c538fb-8ac0-4ba9-8434-eebf36ac31e3","added_by":"auto","created_at":"2025-09-03 17:22:25","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":104262,"visible":true,"origin":"","legend":"\u003cp\u003eAbsorption (left) and fluorescence emission spectra (right, λ\u003csub\u003eex\u003c/sub\u003e=380 nm) of BODIPY \u003cstrong\u003e4\u003c/strong\u003e (concentration 1.55 µmol/L) in low to medium polarity solvents.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7459615/v1/95e0acde4f57d139e6b5f16b.png"},{"id":90527067,"identity":"60bead33-83e5-4c94-a77e-76c36ed8ffa1","added_by":"auto","created_at":"2025-09-03 17:14:25","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":72424,"visible":true,"origin":"","legend":"\u003cp\u003eAbsorption (left) and fluorescence emission spectra (right, λ\u003csub\u003eex\u003c/sub\u003e=380 nm) of BODIPY \u003cstrong\u003e4\u003c/strong\u003e (concentration 1.55 µmol/L) in acetonitrile (blue), propylene carbonate (red) and ethyl acetate (green).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7459615/v1/b64d077250a53303fee2b3f7.png"},{"id":90527073,"identity":"523cf4e8-0a28-4015-8c5b-101077495c9c","added_by":"auto","created_at":"2025-09-03 17:14:25","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":83079,"visible":true,"origin":"","legend":"\u003cp\u003eAbsorption (left) and fluorescence emission spectra (right, λ\u003csub\u003eex\u003c/sub\u003e=380 nm) of BODIPY \u003cstrong\u003e4\u003c/strong\u003e (concentration 1.55 µmol/L) in dimethylsulfoxide (DMSO, blue), \u003cem\u003eN,N\u003c/em\u003e-dimethylformamide (DMF, purple), ethanol (green) and methanol (red). BODIPY \u003cstrong\u003e4\u003c/strong\u003e is not fluorescent in \u003cem\u003eN,N\u003c/em\u003e‑dimethylformamide.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7459615/v1/c52dcd44e8879dfa21993d70.png"},{"id":96105342,"identity":"d2ca7d7e-c8b6-4d1a-8ad5-ee47d88827df","added_by":"auto","created_at":"2025-11-17 16:11:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1346523,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7459615/v1/7cf634f0-b798-4795-abd4-f76d0d3f6e98.pdf"},{"id":90527095,"identity":"40aa587e-9972-4047-97e5-f35e0a903799","added_by":"auto","created_at":"2025-09-03 17:14:26","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":19467085,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"2025ppsSIGClavier.docx","url":"https://assets-eu.researchsquare.com/files/rs-7459615/v1/8e718ffee6cb2f65e17bfed1.docx"},{"id":90527941,"identity":"9e7ca9ac-850c-460b-b4a0-a5cc52c9fae9","added_by":"auto","created_at":"2025-09-03 17:30:25","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":88890,"visible":true,"origin":"","legend":"\u003cp\u003eScheme 1 Synthesis of the probe BODIPY 4. Reaction Conditions: (a) I) NaH (1.7 eq.), THF, 0°C to r.t., 24h; II) NaOH\u003csub\u003e(aq.)\u003c/sub\u003e in MeOH (50%), r.t. 24h, yield 40%; (b) SOCl\u003csub\u003e2\u003c/sub\u003e, DCM, r.t., 30’; (c) I) TFA, DCM, r.t., 180 min; II) Chloranil (1 eq.), DCM, rt, 5 min; III) DIPEA (7 eq.), BF\u003csub\u003e3\u003c/sub\u003eEt\u003csub\u003e2\u003c/sub\u003eO (11 eq.), DCM, r.t., yield 26%; (d) Mercaptoethanol (6 eq.), K\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e (1.6 eq.), DMF, r.t., 30’, yield 87%; (e) TEA (1.1 eq.), THF, 40°C, 15h; (f) 3-Nitro-4-hydroxy-benzaldehyde (1 eq.), piperidine (3 eq.), toluene, 150 °C, Dean-Stark, 24h, yield 38%.\u003c/p\u003e","description":"","filename":"Scheme1.png","url":"https://assets-eu.researchsquare.com/files/rs-7459615/v1/5fe479f8e77c17eddb9cc69b.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Synthesis and Characterization of a Nitrophenol Disubstituted BODIPY Halochromic Probe","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eMeasurement of pH by spectroscopic techniques is well established for both imaging and sensing applications.[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] Compared to traditional electrochemical or colorimetric methods, spectroscopic measurements offer several decisive advantages, including their generally non-destructive character, high sensitivity, chemical specificity, and compatibility with microscopic or macroscopic imaging modalities. In addition, the availability of a broad palette of molecular indicator dyes In addition, the availability of a broad palette of molecular indicator dyes [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Through molecular design, the spectral properties (absorption/emission maxima) as well as the acid\u0026ndash;base equilibrium constants (pKa) of dyes can be finely tuned, thus expanding their applicability across biological, chemical, and material sciences.\u003c/p\u003e\u003cp\u003eMost conventional pH-sensitive dyes are designed to operate within the physiological range, with pKa values close to neutral pH, making them well suited for biological applications. Nevertheless, other research fields often require pH probes adapted to non-physiological conditions. For instance, indicators with higher pKa values have been employed to investigate the alkalization phenomena occurring during the aging of materials such as concrete [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] whereas dyes with lower pKa values are valuable tools for probing acidic microenvironments, including the tumor extracellular matrix or intracellular organelles in cancerous cells [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The ability to shift the pKa window of a chromophore by rational design is therefore a central asset in the development of functional molecular probes.\u003c/p\u003e\u003cp\u003eBeyond their intrinsic acid\u0026ndash;base sensitivity, halochromic and pH-fluorochromic dyes also provide a powerful means of probing solvent properties. Their spectral signatures are influenced not only by protonation equilibria but also by solvation dynamics, polarity, and polarizability of the medium. When multiple protonation states coexist, each form may respond differently to specific solvent parameters, which makes these systems particularly informative. Proper deconvolution of their spectral responses through appropriate solvent models allows simultaneous exploration of both protonation behavior and solvent\u0026ndash;solute interactions, offering a dual analytical window into chemical environments.\u003c/p\u003e\u003cp\u003eWithin this context, BODIPY (boron-dipyrromethene) dyes have attracted long-standing interest owing to their unique combination of advantageous properties. Their modular synthesis enables fine-tuning of absorption and emission wavelengths, spanning from the visible region (\u0026asymp;\u0026thinsp;500 nm) to the near-infrared (NIR-II) domain [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], while maintaining generally high fluorescence quantum yields and excellent photostability [\u003cspan additionalcitationids=\"CR7 CR8 CR9\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. These features have led to their widespread use in the design of sensors for a wide variety of analytes [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Numerous examples of pH-sensitive BODIPY derivatives have been reported, [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] relying on two main design strategies: (i) the incorporation of a non-conjugated pH-sensitive substituent, capable of modulating fluorescence via photoinduced electron transfer (PET), or (ii) the introduction of a conjugated substituent that directly perturbs the absorption and emission maxima, thus generating distinct colorimetric and fluorimetric responses. The choice of pH-sensitive groups is often restricted to aniline- or phenol-based moieties, conferring selective responses to acids or bases, respectively. The pKa of phenol-based substituents, typically in the range 7\u0026ndash;9, can be significantly lowered by introducing electron-withdrawing groups on the aromatic ring. For example, O\u0026rsquo;Shea demonstrated that a 3-nitro-4-phenolic substituent grafted onto an azaBODIPY could reduce the pKa to around 5, making it relevant for acidic biological environments. [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/p\u003e\u003cp\u003eIn the framework of our research on the development of new photoacoustic contrast agents derived from BODIPY scaffolds, we sought to design a pH-sensitive BODIPY dye with a pKa near 5. For this purpose, we selected the 3-nitro-4-phenolic moiety as the acid\u0026ndash;base responsive group, and opted for the conjugated design strategy to maximize spectral separation between the protonated and deprotonated forms, while targeting absorption in the red-to-NIR region. The dye was further engineered with a clickable functionality, enabling future conjugation to nanocarrier systems for drug delivery applications. Prior to such applications, however, a systematic characterization of its intrinsic physicochemical and photophysical properties was required. Surprisingly, during preliminary solvent screening, we observed that the phenolate form could be stabilized in certain solvents upon simple dissolution, even in the absence of added base.\u003c/p\u003e\u003cp\u003eIn this article, we report the synthesis of this novel BODIPY derivative, followed by a detailed study of its acid\u0026ndash;base and solvatochromic behavior. By combining steady-state spectroscopy, density functional theory (DFT) calculations, and quantitative solvent parameter analysis, we aim to elucidate the interplay between protonation equilibria, electronic structure, and solvation effects. This comprehensive approach provides insights not only into the dye\u0026rsquo;s suitability as a pH probe but also into its broader potential as a solvatochromic sensor and as a building block for further advanced photoacoustic and nanocarrier-based applications.\u003c/p\u003e"},{"header":"2 Experimental Section","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Materials and Instruments\u003c/h2\u003e\u003cp\u003eAll reagents used for this study were purchased from the commercial supplier Sigma-Aldrich (France) or TCI (France) and used as received. Spectroscopic experiments were conducted using spectroscopic grade solvents from Sigma-Aldrich or Carlo Erba. Milli-Q water (Merck-Millipore, France) was used throughout. Solvents for synthesis were treated with a MBraun solvent purification system (SPS-800) prior to their use. Monitoring of the reaction was carried out by thin layer chromatography (TLC). Crudes were prepared for chromatographic purification by solid deposition on pre-packed RediSep\u0026trade; (Teledyne, France) silica columns and purified by Reveleris\u0026reg; X2 flash chromatography.\u003c/p\u003e\u003cp\u003eNMR spectra were recorded using a JEOL ECS 400 MHz spectrometer. \u003csup\u003e1\u003c/sup\u003eH NMR and \u003csup\u003e13\u003c/sup\u003eC NMR spectra were referenced to tetramethylsilane (TMS, 0 ppm), and to the central peak of the residual solvent multiplet (CDCl\u003csub\u003e3\u003c/sub\u003e: 77.16 ppm) respectively. Multiplicities are designated as follows: s\u0026thinsp;=\u0026thinsp;singlet, d\u0026thinsp;=\u0026thinsp;doublet, t\u0026thinsp;=\u0026thinsp;triplet, m\u0026thinsp;=\u0026thinsp;multiplet. High-resolution mass spectrometry data (HRMS) were obtained from the SAMM\u0026ndash;IPSIT laboratory at Gif-sur-Yvette (France). Mass spectra were obtained using an LTQ-Orbitrap Velos Pro (ThermoFisher) in infusion mode. Samples were analyzed in acetonitrile (ACN) using positive electrospray ionization (ESI+) Fourier-transform mass spectrometry (FTMS).\u003c/p\u003e\u003cp\u003eAbsorption spectra were recorded on a Cary 4000 using pure solvent as reference. Corrected fluorescence spectra were obtained on a Fluorolog 3 equipped with a double monochromator in excitation and emission in a 90\u0026deg; configuration. All solutions were prepared at a concentration suitable to have an absorbance lower than 0.1 at the maximum absorption band (approximately 1.5\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e mol.L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and the spectra recorded in 10 mm path quartz cell. Titration was done by adding a concentrated solution of base (initially 1.5\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e mol.L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 1.0\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e mol.L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for the final points) to limit the dilution effect (BODIPY was diluted 1.3 times in the final solution). The analysis of the titrations was done with the Specfit software (Spectrum Software Associates, version 2‑11 C, Chapel Hill, NC, 1998) that takes into account the dilution factor.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Quantum chemistry\u003c/h2\u003e\u003cp\u003eAll compounds were studied by DFT and TDDFT. The geometry of the ground state was optimized at B3LYP/3-31g(d) in vacuo and the absorption properties at the PBE0 / 6-311\u0026thinsp;+\u0026thinsp;g(d,p) level of theory with the IEFPCM solvent model (dichloromethane, DCM). Geometry optimisation in the ground state was followed by a frequency calculation to confirm that a true minimum was obtained. TDDFT calculation was done on the 12 lowest singlet states. Calculations were done with Gaussian 16 (Revision B.01). [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] Results were analysed with GaussView 6.0, Mercury 4.2.0 and Multiwfn 3.7. [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Synthesis and Characterization\u003c/h2\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eBODIPY 1\u003c/b\u003e In a three necked round bottom flask charged with 100 mL of dichloromethane (DCM), were dissolved kryptopyrrole (1.0 g, 8.18 mmol, 2 eq.), perfluorobenzaldehyde (0.8 g, 4.06 mmol, 1 eq.) and few drops of trifluoroacetic acid (TFA). When the aldehyde was consumed (as monitored by TLC), the oxidation reagent chloranil (0.998 mg, 4.06 mmol, 1 eq.) was quickly added. After 5 min, diisopropylethylamine (4.1 g, 32.2 mmol, 7.2 eq.) was added. After 15 min, the boron trifluoride diethyl etherate (7.2 g, 44.6 mmol, 11 eq.) was dissolved. Purification was performed on column chromatography (silica gel, petroleum ether PE/DCM with increasing ratio of PE and DCM (70/30, v/v) as eluent. The orange, fluorescent fraction was isolated, affording 0.600 g of \u003cb\u003e1\u003c/b\u003e (1.21 mmol) as a gold solid. Yield: 26%. 1H-NMR (400 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) δ 2.54 (s, 6H), 2.34 (q, J\u0026thinsp;=\u0026thinsp;7.5 Hz, 4H), 1.51 (s, 6H), 1.02 (t, J\u0026thinsp;=\u0026thinsp;7.6 Hz, 6H).\u003c/p\u003e\u003cp\u003e\u003cb\u003eBODIPY 2\u003c/b\u003e BODIPY \u003cb\u003e1\u003c/b\u003e (1.25 g, 2.77 mmol, 1.0 eq.) was dissolved in \u003cem\u003eN,N\u003c/em\u003e-dimethylformamide (DMF, 50 mL). 2-Mercaptoethanol (1.00 g, 12.56 mmol, 6.0 equiv) and potassium carbonate (0.47 g, 3.40 mmol, 1.6 equiv) were added, and the solution was stirred for 30 min at room temperature. Water (50 mL) was then poured to the solution, and the aqueous phase was extracted with diethyl ether (3 \u0026times; 50 mL). The organic phases were combined, washed with water (2 \u0026times; 50 mL), dried over magnesium sulfate, and filtered. The solvent was evaporated under reduced pressure, and the product was purified by flash chromatography (PE/DCM, 0\u0026ndash;100%) to obtain 1.287g (2.43 mmol) of BODIPY \u003cb\u003e2\u003c/b\u003e. Yield 87%. \u003csup\u003e1\u003c/sup\u003eH-NMR (400 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) δ 3.76 (t, J\u0026thinsp;=\u0026thinsp;6.0 Hz, 2H), 3.19 (t, J\u0026thinsp;=\u0026thinsp;5.7 Hz, 2H), 2.54 (s, 6H), 2.33 (q, J\u0026thinsp;=\u0026thinsp;7.6 Hz, 4H), 2.01 (t, 1H), 1.51 (s, 6H), 1.01 (t, J\u0026thinsp;=\u0026thinsp;7.6 Hz, 6H).\u003c/p\u003e\u003cp\u003e\u003cb\u003ePropargyloxy acetic acid 5\u003c/b\u003e To a solution of propargyl alcohol (1.14g, 20.3 mmol) in tetrahydrofuran (THF, 1.2 M sol.) was added NaH (1.37g, 34.5 mmol, 1.7 eq.) portion-wise at 0\u0026deg;C. After addition, the mixture was stirred at 0\u0026deg;C for 1 h then tert-butyl 2-bromoacetate (4 g, 20.3 mmol, 1 eq.) was added to the mixture at 0\u0026deg;C. The mixture was stirred at 15\u0026deg;C for 12 h and then the reaction was stopped by addition of HCl\u003csub\u003e(aq.)\u003c/sub\u003e 37% (2 mL). Water was added (50 mL) extracted with ethyl acetate (EA, 2x 50 mL) and the combined organic layers, were washed with brine (100mL), dried over anhydrous Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e, filtered and concentrated \u003cem\u003ein vacuo\u003c/em\u003e to a brown oil. The crude residue was purified by column chromatography (PE/EA 0 to 100%) recovering 0.78 g (7.89 mmol) of \u003cb\u003e5\u003c/b\u003e as a colorless oil. Yield 40%. \u003csup\u003e1\u003c/sup\u003eH-NMR (400 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) δ 4.26 (d, J\u0026thinsp;=\u0026thinsp;2.7 Hz, 2H), 4.21 (s, 2H), 2.45 (t, J\u0026thinsp;=\u0026thinsp;2.3 Hz, 1H); \u003csup\u003e13\u003c/sup\u003eC-NMR (101 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) δ 175.4, 78.1, 76.2, 65.7, 58.5.\u003c/p\u003e\u003cp\u003e\u003cb\u003eBODIPY 3\u003c/b\u003e (Propargyloxy)acetic acid \u003cb\u003e5\u003c/b\u003e (0.125 g, 5.7 mmol, 2.4 eq.) and SOCl\u003csub\u003e2\u003c/sub\u003e (1.695 g, 14.3 mmol, 2.5 eq.) were dissolved in DCM (5 mL). The mixture was stirred at room temperature for 30 minutes, then the solvent was evaporated and the crude, containing acyl chloride \u003cb\u003e6\u003c/b\u003e, was dried in vacuum for 4 hours to ensure complete SOCl\u003csub\u003e2\u003c/sub\u003e evaporation. The residue was diluted in THF (30 mL), then triethylamine (TEA, 0.245 g, 2.43 mmol, 1.1 eq.) and BODIPY \u003cb\u003e2\u003c/b\u003e (0.245 g, 2.43 mmol, 1 eq.) were added. The reaction mixture was stirred for 15 hours at 40\u0026deg;C, after which water (50 mL) was added, and the mixture was transfered to a separatory funnel and extracted with EA (50 mL), the organic phase was recovered and washed with brine (50 mL) and dried over sodium sulphate. The crude was purified by column chromatography (silica gel, PE/EA gradient 0 to 30%) isolate 0.570 g (0.91 mmol) of BODIPY \u003cb\u003e3\u003c/b\u003e as a golden solid. Yield 38%. \u003csup\u003e1\u003c/sup\u003eH-NMR (400 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) δ 4.26\u0026ndash;4.22 (d\u0026thinsp;+\u0026thinsp;t overlapping signals, 4H), 4.17 (s, 2H), 3.20 (t, J\u0026thinsp;=\u0026thinsp;6.6 Hz, 2H), 2.47 (s, 6H), 2.42 (t, 1H), 2.26 (q, J\u0026thinsp;=\u0026thinsp;7.5 Hz, 4H), 1.45 (s, 6H), 0.94 (t, J\u0026thinsp;=\u0026thinsp;7.6 Hz, 7H); \u003csup\u003e13\u003c/sup\u003eC-NMR (101 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) δ 169.6, 156.1, 136.8, 134.0, 130.2, 78.3, 75.9, 66.0, 62.4, 58.5, 32.9, 29.8, 17.2, 14.7, 12.9, 11.0, 1.1; HRMS (ESI) m/z [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e calculated for C30H31BF6N2O3S: 624.2053; Found: 624.2026.\u003c/p\u003e\u003cp\u003e\u003cb\u003eBODIPY 4\u003c/b\u003e In a 25 mL round bottom flask BODIPY \u003cb\u003e3\u003c/b\u003e (0.570 g, 0.91 mmol), 3-nitro-4-phenol-benzaldehyde (0.456 g, 0.91 mmol, 1 eq.) and piperidine (0.078 g, 2.73 mmol, 3 eq.) added as a 0,01% v/v toluene solution, were dissolved in toluene (10 mL). After setting up a Dean-Stark apparatus, the reaction mixture was stirred at 160\u0026deg;C for 17h (TLC PE\\EA 30%, Rf 0.70). It was then brought to room temperature and toluene was evaporated. The residue was redissolved in DCM (50 mL) and transfered to a separatory funnel. The organic phase was washed with water (50 mL), then the aqueous phase was extracted with DCM (2x50 mL). Recollected organic phases were washed with brine (100 mL) and dried over sodium sulphate. The solvent was evaporated, and the crude was purified by column chromatography (Silica gel, eluent toluene/acetone 4%). The isolated product BODIPY \u003cb\u003e4\u003c/b\u003e is a dark green solid (0.270 g, 0.29 mmol). Yield: 31%. \u003csup\u003e1\u003c/sup\u003eH-NMR (400 MHz, DMF-d7) δ 8.21 (d, J\u0026thinsp;=\u0026thinsp;1.8 Hz, 2H), 7.87 (t, J\u0026thinsp;=\u0026thinsp;5.3 Hz, 3H), 7.53 (dd, 4H), 7.20 (d, J\u0026thinsp;=\u0026thinsp;8.7 Hz, 2H), 4.24 (d\u0026thinsp;+\u0026thinsp;t overlapping, Jd\u0026thinsp;=\u0026thinsp;3.0 Hz, 4H), 4.15 (s, 2H), 3.36 (t\u0026thinsp;+\u0026thinsp;t overlapping, Jt\u0026thinsp;=\u0026thinsp;3.0 Hz, Jt\u0026rsquo; = 7.3 Hz, 3H), 1.60 (s, 6H), 1.04 (t, J\u0026thinsp;=\u0026thinsp;7.3 Hz, 4H) (-CH\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e3\u003c/sub\u003e covered by DMF-d7 quintet); \u003csup\u003e13\u003c/sup\u003eC-NMR (101 MHz, DMF-d7) δ 169.9, 154.2, 151.7, 138.7, 137.5, 135.8, 133.0, 132.8, 129.1, 128.6, 125.3, 120.7, 118.9, 79.6, 77.4, 66.2, 62.9, 58.1, 33.3, 32.1, 22.8, 18.3, 18.1, 14.0, 10.6; HRMS (ESI) m/z [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e calculated for C44H37BF6N4O9S: 922.2278; Found: 921.2203.\u003c/p\u003e\u003c/div\u003e"},{"header":"3 Results and Discussion","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Synthesis\u003c/h2\u003e\u003cp\u003eThe fluorophore (3,5)-bis-(3-nitrophen-4-ol) substituted BODIPY \u003cb\u003e4\u003c/b\u003e was obtained after a 4 steps reaction sequence, shown in Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The BODIPY scaffold was synthesized by dissolving in dichoromethane two equivalents of 2,4-dimethyl-3-ethylpyrrole (kryptopyrrole) and pentafluorobenzaldehyde and following a well-known three steps one-pot protocol.[\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] The details are reported in the experimental section.\u003c/p\u003e\u003cp\u003eIn order to add the clickable handle it was decided to leverage the high reactivity to nucleophilic substitution of the para position on the pentafluorophenyl ring.[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] Hence, \u003cb\u003e1\u003c/b\u003e was dissolved in DMF with an excess of mercaptoethanol and potassium carbonate, affording BODIPY \u003cb\u003e2\u003c/b\u003e with a yield of 87%. The free hydroxyl was functionalized with a short, clickable linker whose synthesis was achieved by adapting a published protocol.[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] Tert-butyl-bromo acetate was stirred with propargyl alcohol in THF in the presence of sodium hydride to afford propargyloxy acetic acid (compound \u003cb\u003e5\u003c/b\u003e) in 40% yield. Compound \u003cb\u003e5\u003c/b\u003e was then converted into its more reactive acyl chloride derivative (compound \u003cb\u003e6\u003c/b\u003e) by reaction with thionyl chloride in dry DCM for 30 minutes. The solvent was evaporated, and the residue was dried \u003cem\u003ein-vacuo\u003c/em\u003e for about 3 hours to ensure the complete removal of unreacted thionylchloride. The intermediate acyl chloride \u003cb\u003e6\u003c/b\u003e was immediately redissolved in dry THF under inert atmosphere (Ar) and a dry THF solution of \u003cem\u003eN,N,N\u003c/em\u003e-triethylamine and BODIPY \u003cb\u003e2\u003c/b\u003e was injected through the rubber stopper under stirring. After 24 hours, the solvent was evaporated, and the reaction mixture was purified to isolate BODIPY \u003cb\u003e3\u003c/b\u003e with 38% yield.\u003c/p\u003e\u003cp\u003eThe desired compound \u003cb\u003e4\u003c/b\u003e was obtained through the functionalization of positions 3- and 5- of BODIPY \u003cb\u003e3\u003c/b\u003e by Knoevenagel condensation with the 3-nitro-4-phenol aldehyde in the presence of piperidine under azeotropic distillation in toluene for 17 hours. After removal of the solvent, purification of the crude material by standard phase column chromatography proved to be particularly challenging due to heavy tailing stemming from the products low solubility in most solvents used for elution.\u003c/p\u003e\u003cp\u003eBODIPY \u003cb\u003e4\u003c/b\u003e is insoluble or very poorly soluble in polar protic solvents such as water, methanol, ethanol or isopropanol as well as non-polar solvents like petroleum ether, hexane, and heptane. In polar aprotic, non-chlorinated solvents such as acetone, it is only partially soluble and tends to form suspensions. Good solubility was observed only in a limited set of solvents, including DCM and toluene. Eventually, BODIPY \u003cb\u003e4\u003c/b\u003e was isolated in 38% yield by using an isocratic mixture of toluene and acetone (4%) as mobile phase. All compounds have been characterized by \u003csup\u003e1\u003c/sup\u003eH and \u003csup\u003e13\u003c/sup\u003eC NMR and unreported compounds by HRMS (spectra in Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e to S13 in supplementary information).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e3.2 DFT Study\u003c/h2\u003e\u003cp\u003ePrior to the photophysical studies, BODIPY \u003cb\u003e4\u003c/b\u003e was investigated by quantum-chemical calculations to gain insight into its electronic properties. All three possible forms of the BODIPY were studied in their ground state, namely: diphenol form (\u003cb\u003e4-H\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e), mono deprotonated (\u003cb\u003e4-H\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;\u003c/b\u003e\u003c/sup\u003e) and fully deprotonated (\u003cb\u003e4\u003c/b\u003e\u003csup\u003e\u003cb\u003e2\u0026minus;\u003c/b\u003e\u003c/sup\u003e). The clickable side chain was omitted as it was expected to have minimal impact on the photophysical properties and was replaced with a fluorine atom.\u003c/p\u003e\u003cp\u003eThe BODIPY core is completely planar in all cases and the styryl substituents are slightly twisted: 22.2\u0026deg; for \u003cb\u003e4-H\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e, 15.7\u0026deg; for \u003cb\u003e4-H\u003c/b\u003e\u003csup\u003e\u003cb\u003e2\u0026minus;\u003c/b\u003e\u003c/sup\u003e, and 9.5\u0026deg; (phenolate) and 15.6\u0026deg; (phenol) for \u003cb\u003e4-H\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;\u003c/b\u003e\u003c/sup\u003e. This last result reflects the non-symmetric nature of the mono deprotonated BODIPY, where the substituent with the stronger donor character is less twisted. The pentafluorophenyl moiety is highly twisted (83 to 86\u0026deg;) relative to the BODIPY core because of the steric hindrance and should have minimal impact on the photophysical properties.\u003c/p\u003e\u003cp\u003eFrontiers molecular orbitals have also been studied (\u003cb\u003eFig. S14\u003c/b\u003e in Supplementary Information). Highest occupied molecular orbital (HOMO) of \u003cb\u003e4-H\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e is delocalized over the BODIPY and styryl moieties, while its lowest unoccupied molecular orbital (LUMO) is more localized on the BODIPY core, in accordance with the acceptor nature of the BODIPY core. The molecular orbitals of \u003cb\u003e4\u003c/b\u003e\u003csup\u003e\u003cb\u003e2\u0026minus;\u003c/b\u003e\u003c/sup\u003e are similarly distributed, however the electronic density of the HOMO is more concentrated on the two nitrophenolates, reflecting their stronger electron rich nature compared to the phenol counterpart. In the case of \u003cb\u003e4-H\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;\u003c/b\u003e\u003c/sup\u003e the unsymmetrical nature can be observed in the HOMO, which is localized mostly on the BODIPY moiety and the phenolate branch. Contrary to expectations, the LUMO is localized on the nitrophenol group, whereas the LUMO\u0026thinsp;+\u0026thinsp;1 involves both the BODIPY core and the phenolate group.\u003c/p\u003e\u003cp\u003eTDDFT with solvent effect (DCM) analysis provided insights into the nature of the major electronic transitions (see \u003cem\u003eSupplementary Informations\u003c/em\u003e for details). In all cases an intense low energy transition of charge transfer character from the phenol or phenolate moieties towards the BODIPY is observed (\u003cb\u003eFig. S15\u003c/b\u003e in Supplementary Information). Stepwise deprotonation yields a red shift of the transition from 631 nm for \u003cb\u003e4-H\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e to 720 nm for \u003cb\u003e4-H\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;\u003c/b\u003e\u003c/sup\u003e and 783 nm for \u003cb\u003e4\u003c/b\u003e\u003csup\u003e\u003cb\u003e2\u0026minus;\u003c/b\u003e\u003c/sup\u003e. Notably, the lower energy transition of \u003cb\u003e4-H\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;\u003c/b\u003e\u003c/sup\u003e is a pure charge transfer HOMO-LUMO transition localized at 785 nm with a very weak oscillator strength (f\u0026thinsp;=\u0026thinsp;0.08). The presence of this transition should lead a quenching of the BODIPY fluorescence.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Photophysical Properties in dichloromethane\u003c/h2\u003e\u003cp\u003eFirst, BODIPY \u003cb\u003e4\u003c/b\u003e was studied in dichloromethane, which is an apolar aprotic solvent and poor hydrogen bond acceptor. It is thus expected that only the diphenolate form \u003cb\u003e4-H\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e will be present. BODIPY \u003cb\u003e4\u003c/b\u003e exhibits three absorption bands between 300 and 800 nm. The main (lowest energy) transition, the S0\u0026rarr;S1 transition, is centered at 666 nm, with a vibrionic shoulder appearing at 620 nm. Below 500 nm are found two transitions involving higher energy levels. Specifically, the second, broad band is centered at 445 nm and corresponds to the typical S\u003csub\u003e0\u003c/sub\u003e\u0026rarr;S\u003csub\u003e2\u003c/sub\u003e transition of BODIPYs. The third band at 357 nm (shoulder at 345 nm) arises from a π\u0026ndash;π* transition localized on the styryl moiety, as demonstrated by calculations for similar compounds. A molar absorption coefficient of 66 000 L\u0026middot;mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u0026middot;cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e was determined for the main transition.\u003c/p\u003e\u003cp\u003eBODIPY \u003cb\u003e4\u003c/b\u003e is fluorescent and emits at 685 nm in dichoromethane, with a Stokes shift of 319 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Such a small value is typical for this class of dyes. The associated fluorescence quantum yield is 0.08, indicative of weak fluorescence. When compared to literature values the data collected for BODIPY \u003cb\u003e4\u003c/b\u003e reveal that the nitrophenol substituent is a weak donor, since the photophysical properties in dichoromethane lies in between a phenyl (λ\u003csub\u003eabs\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;661 nm and λ\u003csub\u003eem\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;675 nm) and a phenol (λ\u003csub\u003eabs\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;677 nm and λ\u003csub\u003eem\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;700 nm). [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/p\u003e\u003cp\u003eBODIPY \u003cb\u003e4\u003c/b\u003e was then titrated with the non-nucleophilic base 1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU) in dichoromethane to gain insight in the photophysical properties of the basic forms. The absorption spectra (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, left) show a gradual decrease of the 666 nm band corresponding to \u003cb\u003e4-H\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e (blue spectrum in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) in favor of a red shifted band at 730nm (green spectrum in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). This second band is maximum for 7.5 equivalents of added base. Upon further addition of DBU, a third band appears at 769 nm (red spectrum in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) whose intensity starts to level off at 100 equivalents of base. The behavior of BODIPY \u003cb\u003e4\u003c/b\u003e upon titration with DBU seems to indicate a stepwise deprotonation in dichoromethane with the intermediate spectrum corresponding to \u003cb\u003e4-H\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;\u003c/b\u003e\u003c/sup\u003e and the final one to \u003cb\u003e4\u003c/b\u003e\u003csup\u003e\u003cb\u003e2\u0026minus;\u003c/b\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThese absorption data were further analyzed using the SPECFIT software, which performs global analysis of equilibrium systems with nonlinear regression modeling by the Levenberg-Marquardt method. Several models were tested but the only one that resulted in a satisfying fit is the one corresponding to the stepwise deprotonation of \u003cb\u003e4\u003c/b\u003e, to the phenolate first (\u003cb\u003e4-H\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;\u003c/b\u003e\u003c/sup\u003e), and then to the bisphenolate (\u003cb\u003e4\u003c/b\u003e\u003csup\u003e\u003cb\u003e2\u0026minus;\u003c/b\u003e\u003c/sup\u003e). The calculated spectra (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e left) are in good agreement with the experimental ones, and the distribution diagram shows the rapid deprotonation of \u003cb\u003e4-H\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e to \u003cb\u003e4-H\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;\u003c/b\u003e\u003c/sup\u003e, followed by the formation of \u003cb\u003e4\u003c/b\u003e\u003csup\u003e\u003cb\u003e2\u0026minus;\u003c/b\u003e\u003c/sup\u003e which becomes predominant above 15 equivalents of base. The main absorption bands become broader upon deprotonation as seen by their Full Width at Half-Maximum (FWHM): 1030 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for \u003cb\u003e4-H\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e, 1373 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for \u003cb\u003e4-H\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;\u003c/b\u003e\u003c/sup\u003e and 1478 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for \u003cb\u003e4\u003c/b\u003e\u003csup\u003e\u003cb\u003e2\u0026minus;\u003c/b\u003e\u003c/sup\u003e. The enlargement can be attributed to the stronger charge transfer (ICT) character of the transition upon deprotonation and formation of the stronger electron donor phenolate. The calculated associated equilibrium constants for the two successive processes are log(K\u003csub\u003e1\u003c/sub\u003e)\u0026thinsp;=\u0026thinsp;5.21 and log(K\u003csub\u003e2\u003c/sub\u003e)\u0026thinsp;=\u0026thinsp;4.79. The second deprotonation is slightly disfavoured, likely due to electrostatic repulsions but does not require harsh conditions to occur. This behavior is consistent with the expected high basicity of the nitrophenolate.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe titration was also monitored by fluorescence emission (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Two excitation wavelengths were selected: 610 nm, were \u003cb\u003e4-H\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e is the predominant absorbing specie and 380 nm where all three species absorb light with a rather close efficiency.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe initial fluorescence band is found at 684 nm (left, λ\u003csub\u003eex\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;610 nm) and 687 nm (λ\u003csub\u003eex\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;380 nm). The difference probably comes from the fact that large bandpass (8.5 nm) was necessary to record the spectra due to the low fluorescence efficiency of BODIPY \u003cb\u003e4\u003c/b\u003e. Upon addition of DBU the band gradually disappears and a new very red-shifted one appears at 844 nm. In both experiments, the red shifted band appears more intense than the initial one. The difference is more pronounced in the experiment where the excitation was set at 380 nm where all three species absorb light with close efficiency. However, it is difficult to conclude that the second species emits light more efficiently because the emission appears at the limit of the detector sensitivity, where efficiency is very low and the correction factor plays an important role.\u003c/p\u003e\u003cp\u003eThe analysis of the data where not as straight forward as for the absorption. Two models where tested: one where the singly deprotonated form \u003cb\u003e4-H\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;\u003c/b\u003e\u003c/sup\u003e is considered as a dark specie as the calculation hinted and the doubly protonated form \u003cb\u003e4\u003c/b\u003e\u003csup\u003e\u003cb\u003e2\u0026minus;\u003c/b\u003e\u003c/sup\u003e as an emissive one together with the initial \u003cb\u003e4-H\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e.In the second model the intermediate form \u003cb\u003e4-H\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;\u003c/b\u003e\u003c/sup\u003e was omitted. The second model seemed less realistic even though the error on the association constant was lower. In the first model, analysis of the experiment with excitation at 610 nm gave better results (lower error) that the other model, in which the equilibrium constant for the first deprotonation K\u003csub\u003e1\u003c/sub\u003e was very small (log(K1)\u0026thinsp;=\u0026thinsp;2.57) and determined with an error of 50%. With excitation at 610 nm the fitted equilibrium constants were close to the one calculated for the absorption spectra (log(K\u003csub\u003e1\u003c/sub\u003e)\u0026thinsp;=\u0026thinsp;5.38 and log(K\u003csub\u003e2\u003c/sub\u003e)\u0026thinsp;=\u0026thinsp;4.79). The fluorescence thus probes the same stepwise deprotonation process as the absorption, but with a dark intermediate \u003cb\u003e4-H\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;\u003c/b\u003e\u003c/sup\u003e specie and only the extreme ones (\u003cb\u003e4-H\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e and \u003cb\u003e4\u003c/b\u003e\u003csup\u003e\u003cb\u003e2\u0026minus;\u003c/b\u003e\u003c/sup\u003e) are emissive.\u003c/p\u003e\u003cp\u003eThis study allowed us to better understand the behavior of BODIPY \u003cb\u003e4\u003c/b\u003e under basic conditions in dichoromethane. It undergoes two successive deprotonation processes giving a singly deprotonated intermediate followed by the doubly deprotonated one. Each specie has its own photophysical signature: \u003cb\u003e4-H\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e absorbs at 666 nm and emit at 685 nm, \u003cb\u003e4-H\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;\u003c/b\u003e\u003c/sup\u003e absorbs at 730 nm and is not fluorescent and \u003cb\u003e4\u003c/b\u003e\u003csup\u003e\u003cb\u003e2\u0026minus;\u003c/b\u003e\u003c/sup\u003e absorbs at 769 nm and emits at 844 nm. The second deprotonation is slightly less favorable than the first but still occurs under reasonable conditions (requiring about a tenfold excess of base).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e3.4 Solvatochromic Properties\u003c/h2\u003e\u003cp\u003eThe photophysical properties of BODIPY \u003cb\u003e4\u003c/b\u003e where then studied in a broad range of solvents. BODIPY are usually weakly sensitive to solvent effect apart from its fluorescence quantum yield that usually drops dramatically in polar solvents.[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] However in our case we reasoned that the charge transfer nature of the main transition and the presence of a moiety that can be easily deprotonated could by influenced by the solvent nature. BODIPY \u003cb\u003e4\u003c/b\u003e has thus been studied in 19 solvents from apolar to polar (protic and non protic) at a constant concentration of 1.55 \u0026micro;mol/L and four distinct behaviors could be found. The photophysical data are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, and the spectra in each solvent are provided in the supplementary information (\u003cb\u003eFig. S16\u003c/b\u003e to \u003cb\u003eS36\u003c/b\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eSpectroscopic properties of BODIPY \u003cb\u003e4\u003c/b\u003e in several solvents\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003esolvent\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eλ\u003csub\u003eabs\u003c/sub\u003e (nm)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eλ\u003csub\u003efluo\u003c/sub\u003e (nm)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\varvec{\\delta\\:}\\stackrel{-}{\\varvec{\\nu\\:}}\\)\u003c/span\u003e\u003c/span\u003e (cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1,1,2,2-tetrachloroethane\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e671\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e692\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e452\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003en\u003c/em\u003e-hexane\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e749\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e676 / 749\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003echloroform\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e667\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e688\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e458\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003en\u003c/em\u003e-pentane\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e749\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e675 / 748\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2,2,2-trifluoroethanol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e655\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e676\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e474\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003etoluene\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e668\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e688\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e435\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDichloromethane\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e666\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e687\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e459\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003emesitylene\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e668\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e689\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e456\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ebenzonitrile\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e670\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e693\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e495\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eacetonitrile\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e660 / 751\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e682\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e489\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003epropylene carbonate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e667 / 758\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e687\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e436\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1,4-dioxane\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e667\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e687\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e436\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eacetone\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e661\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e684\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e509\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eethyl acetate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e661 / 721\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e681\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e444\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003emethanol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e702\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e685 / 776\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003etetrahydrofuran\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e666\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e685\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e416\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003etetrahydrofuran\u0026thinsp;+\u0026thinsp;water\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e666\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e685\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e416\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eN,N\u003c/em\u003e-dimethylformamide\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e831\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003edimethyl sulfoxide\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e826\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e702\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eethanol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e715\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e687 / 790\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003csup\u003ea\u003c/sup\u003e Stokes shift calculated on the phenol band; \u003csup\u003eb\u003c/sup\u003e 30 \u0026micro;L of water added; \u003csup\u003ec\u003c/sup\u003e shoulder.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn the apolar \u003cem\u003en\u003c/em\u003e-pentane and \u003cem\u003en\u003c/em\u003e-hexane BODIPY \u003cb\u003e4\u003c/b\u003e formed J aggregates (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) as clearly evidenced by the thin, red-shifted absorption band (λ\u003csub\u003eabs\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;749 nm in both solvent). A shoulder in the blue region corresponds likely to the non-aggregated form. The fluorescence spectra display two bands: one at 675 nm and one at 749 nm. The high energy one is clearly coming from the non-aggregated form while the low energy one is typical of J-aggregates of BODIPY (thin emission band with no Stokes shift). [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] While BODIPY are known to form aggregates in polar solvents [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] it is less common to observe such behavior in apolar solvents at such a low concentration. The aggregation likely comes from the presence of the nitro-phenol groups, whose polarity and ability to form hydrogen bonds promote the formation of well-organized aggregates.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn solvents of low to medium polarity solvents, the main absorption band is only slightly affected, shifting from 655 nm in 2,2,2-trifluoroethanol to 671 nm in 1,1,2,2-tetrachloroethane (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Similarly, the fluorescence band is relatively insensitive to the nature of the solvent and shits from 676 in 2,2,2-trifluoroethanol to 693 in benzonitrile. The Stokes shift are small (between 400 and 500 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). These results are in line with the expected solvatochromic behavior of BODIPY, usually weakly sensitive to solvent properties.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eUnexpected results occurred in more polar solvents. In three solvents (acetonitrile, propylene carbonate and ethyl acetate) the absorption spectra display a red-shifted band in addition to the expected BODIPY one around 665 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The new band appears between 720 and 760 nm. Its position and relative intensity are solvent dependent. There is also a small band above 500nm that is easily identifiable in propylene carbonate and could also be seen in the phenolate spectrum obtained in DCM (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). This result indicates that in these solvents there is an equilibrium between the phenol \u003cb\u003e4-H\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e form and the phenolate \u003cb\u003e4-H\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;\u003c/b\u003e\u003c/sup\u003e one. The fluorescence spectra only display the phenol band around 685 nm, which is reasonable considering that the the \u003cb\u003e4-H\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;\u003c/b\u003e\u003c/sup\u003e is not fluorescent.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFinally in very polar solvents (dimethylsulfoxide, \u003cem\u003eN,N\u003c/em\u003e-dimethylformamide, ethanol and methanol) the main absorption band is located between 700 and 820 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). In the alcohols, since the main emission band was found at higher energy (685 nm and 687 nm for methanol and ethanol respectively (\u003cb\u003eFig. S31\u003c/b\u003e and \u003cb\u003eS36\u003c/b\u003e in Supplementary Information), the nature of the absorbing species was attributed to the phenolate form. In dimethylsulfoxide and \u003cem\u003eN,N\u003c/em\u003e-dimethylformamide the absorption spectra clearly display the characteristic bands of the phenolate and the fluorescence of the phenol BODIPY is hardly seen in former and quenched in the later. In the alcoholic solvents a second band can be seen at lower energy. However, its origin is uncertain since it appears at the limit of the detection capacity of the apparatus used and the correction is quite large above 750 nm, sometimes creating artefacts. Further analysis would be needed to confirm the occurrence of an emission in that range.\u003c/p\u003e\u003cp\u003eTo confirm that the appearance of the phenolate band was due to solvent properties rather than trace water, 30 \u0026micro;L of water were added to the THF solution, in which the phenolate band was not observed. The absorption and emission spectra before and after water addition were found identical (\u003cb\u003eFig. S32\u003c/b\u003e and \u003cb\u003eS33\u003c/b\u003e) ruling out the possible role of residual water in promoting the formation of the phenolate in the polar solvents.\u003c/p\u003e\u003cp\u003eTo the best of our knowledge, the occurrence of the absorption band of the phenolate form for phenol-substituted BODIPY is not reported in the literature. Examples of pH sensitive BODIPYs were designed with halogenated phenols (Chloride or Fluoride) in order to bring the phenol\u0026rsquo;s pKa close to 7.[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] The nitrophenol group was introduced by O\u0026rsquo;Shea on an aza-BODIPY to lower the pKa of the phenol even further than that (down to ~\u0026thinsp;5). The increased acidity of the nitrophenol group is likely explains why we observe the appearance of the phenolate band in polar solvents, which will be discussed further later.\u003c/p\u003e\u003cp\u003eSolvent-dependent spectral shifts can be analyzed through various approaches that encompass empirical single-parameter solvent scales and multi-parameters one. We used four scales to analyze our results: two single parameter (Lippert-Mataga and E\u003csub\u003eT\u003c/sub\u003e(30)) and two multi-parameters (Kamlet\u0026ndash;Taft and the generalized solvent scale proposed by Catalan). We analyzed the variations of the absorption and emission maxima as well as the Stokes shift. The single parameters scales completely failed to model the solvent effect (a discussion is included in Supplementary Information). The Kamlet\u0026ndash;Taft gave good results only in the case of the phenolate absorption band but failed on all other properties.\u003c/p\u003e\u003cp\u003eThe Catalan scale offers the advantage of splitting the solvent properties in four distinct parameters: SA, SB, SP and SdP which characterize the solvent acidity, basicity, polarizability and dipolarity, respectively.[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] Previous analysis of BODIPY derivatives with this scale proved to work well for us [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] and others.[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eEstimated coefficients (y\u003csub\u003e0\u003c/sub\u003e, a\u003csub\u003eSP\u003c/sub\u003e, b\u003csub\u003eSdP\u003c/sub\u003e, c\u003csub\u003eSA\u003c/sub\u003e and d\u003csub\u003eSB\u003c/sub\u003e), their standard errors and correlation coefficients (r\u003csup\u003e2\u003c/sup\u003e) for the multiple linear regression analyses of \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\stackrel{-}{\\nu\\:}\\)\u003c/span\u003e\u003c/span\u003e\u003csub\u003eabs\u003c/sub\u003e of the phenol and phenolate bands, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\stackrel{-}{\\nu\\:}\\)\u003c/span\u003e\u003c/span\u003e\u003csub\u003eem\u003c/sub\u003e of the phenol and Stokes shift (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\Delta\\:}\\stackrel{-}{\\nu\\:}\\)\u003c/span\u003e\u003c/span\u003e) as a function of the Catalan solvent scale.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCatalan\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003ey\u003csub\u003e0\u003c/sub\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003ea\u003csub\u003eSP\u003c/sub\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eb\u003csub\u003eSdP\u003c/sub\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003ec\u003csub\u003eSA\u003c/sub\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003ed\u003csub\u003esb\u003c/sub\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003er\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\stackrel{-}{\\nu\\:}\\)\u003c/span\u003e\u003c/span\u003e\u003csub\u003eabs\u003c/sub\u003e (phenol)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e15925\u0026thinsp;\u0026plusmn;\u0026thinsp;97\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e-1221\u0026thinsp;\u0026plusmn;\u0026thinsp;114\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e36\u0026thinsp;\u0026plusmn;\u0026thinsp;22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e-23\u0026thinsp;\u0026plusmn;\u0026thinsp;41\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e-75\u0026thinsp;\u0026plusmn;\u0026thinsp;43\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.98\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\stackrel{-}{\\nu\\:}\\)\u003c/span\u003e\u003c/span\u003e\u003csub\u003eabs\u003c/sub\u003e (phenolate)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e18576\u0026thinsp;\u0026plusmn;\u0026thinsp;694\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e-1351\u0026thinsp;\u0026plusmn;\u0026thinsp;1644\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e-3915\u0026thinsp;\u0026plusmn;\u0026thinsp;713\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e2529\u0026thinsp;\u0026plusmn;\u0026thinsp;488\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e-2661\u0026thinsp;\u0026plusmn;\u0026thinsp;639\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.99\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\stackrel{-}{\\nu\\:}\\)\u003c/span\u003e\u003c/span\u003e\u003csub\u003eem\u003c/sub\u003e (phenol)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e15743\u0026thinsp;\u0026plusmn;\u0026thinsp;104\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e-1499\u0026thinsp;\u0026plusmn;\u0026thinsp;147\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e-16\u0026thinsp;\u0026plusmn;\u0026thinsp;41\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e-135\u0026thinsp;\u0026plusmn;\u0026thinsp;59\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e-223\u0026thinsp;\u0026plusmn;\u0026thinsp;52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.93\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\Delta\\:}\\stackrel{-}{\\nu\\:}\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e530\u0026thinsp;\u0026plusmn;\u0026thinsp;132\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e-119\u0026thinsp;\u0026plusmn;\u0026thinsp;156\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e51\u0026thinsp;\u0026plusmn;\u0026thinsp;30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e-39\u0026thinsp;\u0026plusmn;\u0026thinsp;56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e-56\u0026thinsp;\u0026plusmn;\u0026thinsp;59\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.35\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThe Stokes shift of the phenol transition appears to be difficult to fit with any model, probably because of the small variations observed in the solvents studied, while the Catal\u0026aacute;n scale fares well with the absorption and emission maxima. The electronic transition of the phenol form \u003cb\u003e4-H\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e appears to be mostly dependent on the solvent polarizability (SP) which was already observed for other BODIPY and notably derivatives substituted with a chlorophenol or a fluorophenol.[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] The phenol form of BODIPY \u003cb\u003e4\u003c/b\u003e has solvatochromic properties that are similar to related BODIPY. On the other hand, the phenolate absorption is much more sensitive to the solvents: the band shifts from 702 nm in methanol to 831 nm in \u003cem\u003eN,N\u003c/em\u003e-dimethylformamide. The analysis of this solvent dependence with the Catalan scale gave an excellent correlation (r\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.99) and reveals that in that case the band position strongly depends on the solvent dipolarity (SP) as well as solvent acidity (SA) and basicity (SB). This is in line with the fact that the solvent must interact with the proton of a phenol of the solvent to form the polar phenolate. The ability of the solvent to give and accept hydrogen bonds is crucial in that regard. The appearance of the nitrophenolate band necessitate a polar solvent that can give and/or accept hydrogen bonds.\u003c/p\u003e\u003c/div\u003e"},{"header":"4 Conclusion","content":"\u003cp\u003eBODIPY\u003cstrong\u003e\u0026nbsp;4\u003c/strong\u003e, bearing two nitrophenol substituents, was successfully synthesized and thoroughly characterized. The diphenol form, \u003cstrong\u003e4-H\u003c/strong\u003e\u003cstrong\u003e₂\u003c/strong\u003e, exhibits absorption and emission in the red region (λ\u003csub\u003eabs\u003c/sub\u003e = 666 nm, λ\u003csub\u003eem\u003c/sub\u003e = 687 nm), providing a strong baseline for studying its photophysical behavior. Its response to basic conditions in dichloromethane revealed a stepwise deprotonation process, leading to the singly deprotonated \u003cstrong\u003e4-H\u003c/strong\u003e\u003cstrong\u003e⁻\u003c/strong\u003e (λ\u003csub\u003eabs\u003c/sub\u003e = 730 nm, non-emissive) and the doubly deprotonated \u003cstrong\u003e4²\u003c/strong\u003e\u003cstrong\u003e⁻\u003c/strong\u003e (λ\u003csub\u003eabs\u003c/sub\u003e = 769 nm, λ\u003csub\u003eem\u003c/sub\u003e = 844 nm). The distinct absorption and emission signatures of these species enable BODIPY\u003cstrong\u003e\u0026nbsp;4\u003c/strong\u003e to function as a sensitive on-off-on pH fluorescent sensor, where only the fully protonated and fully deprotonated forms are emissive. This properties could be exploited in fluorescence microscopy or photoacoustic imaging with spectral unmixing to probe media of different pH (on-going experiments).\u003c/p\u003e\n\u003cp\u003eThe solvatochromic behavior of BODIPY\u003cstrong\u003e\u0026nbsp;4\u003c/strong\u003e is particularly noteworthy, as it exhibits four distinct responses depending on solvent polarity:\u003c/p\u003e\n\u003cp\u003e1. Apolar solvents (\u003cem\u003en\u003c/em\u003e-pentane, \u003cem\u003en\u003c/em\u003e-hexane): spontaneous formation of J-aggregates at micromolar concentrations, evidenced by characteristic red-shifted absorption and emission bands.\u003c/p\u003e\n\u003cp\u003e2. Low-polarity solvents: conventional BODIPY behavior, with minimal shifts in absorption and emission.\u003c/p\u003e\n\u003cp\u003e3. Moderately polar solvents: presence of an equilibrium between diphenol and mono-deprotonated forms, allowing both species to be detected spectroscopically.\u003c/p\u003e\n\u003cp\u003e4. Highly polar solvents (dimethylsulfoxide, \u003cem\u003eN,N\u003c/em\u003e-dimethylformamide, alcohols): dominance of the mono-deprotonated phenolate, with diphenol detectable only through residual fluorescence\u003c/p\u003e\n\u003cp\u003eAnalysis using the Catalan solvent scale revealed that the deprotonation of the phenol groups is strongly influenced by solvent dipolarity, acidity, and basicity, while the diphenol form itself is primarily sensitive to solvent polarizability. This insight underscores the intricate interplay between solvent environment and the electronic structure of BODIPY derivatives.\u003c/p\u003e\n\u003cp\u003eOverall, the study demonstrates that \u003cstrong\u003eBODIPY 4\u003c/strong\u003e is a versatile photophysical probe capable of reporting on both pH and local solvent environment, combining stepwise deprotonation, dual fluorescence response, and solvent-sensitive spectral shifts. These properties could be particularly valuable in biological imaging, enabling the differentiation of cell compartments and environments of varying polarity. Further research is ongoing to explore applications in live-cell imaging and photoacoustic sensing, leveraging the unique photophysical and solvatochromic features of this nitrophenol-substituted BODIPY.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch4\u003eConflict of interests\u003c/h4\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Information\u003c/strong\u003e The online version contains supplementary material available at\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e The University Paris-Saclay, ENS Paris-Saclay and the CNRS are acknowledged for funding. RM additionally thanks the R\u0026eacute;gion Ile-de-France, DIM NanoK and LabEx CHARMMMAT for financial support.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution\u003c/strong\u003e GC and RM designed the research, RO, EM and GC performed the experiments and analyzed the data, RM, RO and GC wrote and reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e This project has received financial support from the French National Research Agency under the program ANR-21-CE09-0024-01.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eJ. Han and K. Burgess. Fluorescent Indicators for Intracellular pH. \u003cem\u003eChem. Rev.\u003c/em\u003e 110, 2709\u0026ndash;2728\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eL. Di Costanzo and B. Panunzi. 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Chem.\u003c/em\u003e 183, 190\u0026ndash;197\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Scheme 1","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"photochemical-and-photobiological-sciences","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ppss","sideBox":"Learn more about [Photochemical \u0026 Photobiological Sciences](https://link.springer.com/journal/43630)","snPcode":"43630","submissionUrl":"https://www.editorialmanager.com/ppss/","title":"Photochemical \u0026 Photobiological Sciences","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Solvatochromism, BODIPY, Photophysics, Halochromic probe","lastPublishedDoi":"10.21203/rs.3.rs-7459615/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7459615/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eWe report the synthesis and characterization of a (3,5)-nitrophenol substituted BODIPY dye bearing a clickable handle. The dye was initially studied in dichloromethane, where it absorbs and emits in the red (666 nm and 687 nm respectively). Stepwise deprotonation of the phenols was achieved by addition of an organic base giving three species: \u003cb\u003e4-H₂\u003c/b\u003e (λ\u003csub\u003eabs\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;666 nm, λ\u003csub\u003eem\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;687 nm), \u003cb\u003e4-H⁻\u003c/b\u003e (λ\u003csub\u003eabs\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;730 nm, non-fluorescent), and \u003cb\u003e4\u0026sup2;⁻\u003c/b\u003e (λ\u003csub\u003eabs\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;769 nm, λ\u003csub\u003eem\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;844 nm), enabling an on-off-on pH sensing behavior Spectral deconvolution, DFT and TDDFT studies were carried out to support the experimental observations. Solvatochromic studies in 19 solvents show four distinct behaviors: J-aggregate formation in apolar alkanes (λabs/λem\u0026thinsp;=\u0026thinsp;749 nm), conventional BODIPY response in low-polarity solvents, coexistence of diphenol and mono-deprotonated forms in moderately polar solvents, and dominance of the phenolate in polar solvents (λ\u003csub\u003eabs\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;831 nm in \u003cem\u003eN,N\u003c/em\u003edimethylformamide). Analysis using the Catalan scale indicates deprotonation is promoted by solvent dipolarity, acidity, and basicity, while the diphenol form is mainly sensitive to polarizability. These properties make such BODIPY a versatile probe for pH and microenvironment sensing, with potential applications in photoacoustic imaging and cellular studies.\u003c/p\u003e","manuscriptTitle":"Synthesis and Characterization of a Nitrophenol Disubstituted BODIPY Halochromic Probe","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-03 17:14:20","doi":"10.21203/rs.3.rs-7459615/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-16T10:05:11+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-15T17:15:51+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-04T14:18:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"240802907227911110231139820837184844256","date":"2025-08-26T14:53:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"207117127443787612683371207890325193197","date":"2025-08-26T13:50:19+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-26T13:38:01+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-26T13:33:31+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-26T13:28:33+00:00","index":"","fulltext":""},{"type":"submitted","content":"Photochemical \u0026 Photobiological Sciences","date":"2025-08-26T06:50:33+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"photochemical-and-photobiological-sciences","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ppss","sideBox":"Learn more about [Photochemical \u0026 Photobiological Sciences](https://link.springer.com/journal/43630)","snPcode":"43630","submissionUrl":"https://www.editorialmanager.com/ppss/","title":"Photochemical \u0026 Photobiological Sciences","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"602ca4f5-9f37-4a67-a36c-c384befc47e8","owner":[],"postedDate":"September 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-11-17T16:07:52+00:00","versionOfRecord":{"articleIdentity":"rs-7459615","link":"https://doi.org/10.1007/s43630-025-00806-8","journal":{"identity":"photochemical-and-photobiological-sciences","isVorOnly":false,"title":"Photochemical \u0026 Photobiological Sciences"},"publishedOn":"2025-11-10 15:57:46","publishedOnDateReadable":"November 10th, 2025"},"versionCreatedAt":"2025-09-03 17:14:20","video":"","vorDoi":"10.1007/s43630-025-00806-8","vorDoiUrl":"https://doi.org/10.1007/s43630-025-00806-8","workflowStages":[]},"version":"v1","identity":"rs-7459615","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7459615","identity":"rs-7459615","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}