Dense and Acidic Organelle-Targeted Visualization in Living Cells: Application of Viscosity-Responsive Fluorescence Utilizing Restricted Access to Minimum Energy Conical Intersection

Cell-imaging methods with functional fluorescent probes are an indispensable technique to evaluate physical parameters in cellular microenvironments. In particular, molecular rotors, which take advantage of the twisted intramolecular charge transfer (TICT) process, have helped evaluate microviscosity. However, the involvement of charge-separated species in the fluorescence process potentially limits the quantitative evaluation of viscosity. Herein, we developed viscosity-responsive fluorescent probes for cell imaging that are not dependent on the TICT process. We synthesized AnP2-H and AnP2-OEG, both of which contain 9,10-di(piperazinyl)anthracene, based on 9,10-bis(N,N-dialkylamino)anthracene that adopts a nonflat geometry at minimum energy conical intersection. AnP2-H and AnP2-OEG exhibited enhanced fluorescence as the viscosity increased, with sensitivities comparable to those of conventional molecular rotors. In living cell systems, AnP2-OEG showed low cytotoxicity and, reflecting its viscosity-responsive property, allowed specific visualization of dense and acidic organelles such as lysosomes, secretory granules, and melanosomes under washout-free conditions. These results provide a new direction for developing functional fluorescent probes targeting dense organelles.

After the reaction, the reaction mixture was cooled to room temperature and quenched by water (10 mL). The organic layer was separated and the aqueous layer was extracted with toluene (15 mL ×6    Figure S3. HRMS profile of AnP2-H. Figure S4. HRMS profile of AnP2-OEG.

Acid-base titration of AnP2-OEG
AnP2-OEG (20.1 mg) was dissolved in a mixture of dimethyl sulfoxide (DMSO) (15 μL) and milli-Q water (950 μL). The resulting solution was beforehand acidified by HCl aq. (10 μL), and then titrated by an aqueous solution of NaOH (1.0 M). The pH value was monitored by a pH meter. Figure S5. Titration curve of AnP2-OEG at room temperature. The first equivalence point at pH ≈ 4 corresponds to the neutralization of excessively added HCl. respectively.

Viscosity-dependent fluorescence
As described in the main text, the quantum yield Φ and solvent viscosity η follow a power-law relationship that is known as the Förster-Hoffmann equation, S2,S3 where C (C') is a solvent dependent constant and x is a dye dependent constant which is used as an indicator of the sensitivity of the molecule to viscosity.
Using the rate constants for each decay process, fluorescence quantum yield Φ (or fluorescence intensity I) is also described as where A is proportionality constant, kr is radiative decay rate constant, and knr is non-radiative decay rate constant. The viscosity dependence has been discussed mainly associated with the twisted intramolecular charge transfer (TICT) process. The viscosity-dependent fluorescence property originates from the twisting motion of molecule occurring in transition from the LE state to the TICT state, which is affected by the viscosity of the surrounding environment. Namely, the fluorescence is exhibited from the LE state, while the transition to the TICT state causes the non-radiative deactivation depending on the viscosity ( Figure S7). Here, for simplicity, equation (2) assumes the TICT state to be non-fluorescent. S3 However, it can also be applied to dual-emissive molecules. In this case, the ratio of the intensities of the two fluorescent components is used instead of Φ.
Meanwhile, MECI is the lowest crossing point of S0 and S1 potential energy surface. The energy level of MECI dominates non-radiative decay rate of the excited molecules. Here, 9,10bis(N,N-dialkylamino)anthracene undergoes a large conformational change from the fluorescent state to accessing MECI, S4 where the deactivation through MECI is suppressed as the viscosity around the molecule increases.
Based on the above discussion, the decay processes of TICT and MECI systems were schematically shown in Figure S7. In both systems, the first local minimum on the S1 potential surface is involved in the first relaxation (kr and knr). In the case of TICT system ( Figure S8a), the transition to the TICT state, allowing for non-radiative decay, requires conformational changes, thereby resulting in viscosity-dependent non-radiative decay, knr(η). Similarly, in the case of MECI system, conformational changes allow non-radiative decay through MECI, thereby also resulting in viscosity-dependent non-radiative decay, knr(η). Therefore, the viscosity dependence of the fluorescence intensity of the MECI system was also expected to satisfy equation (2). Figure S7. Schematic illustration of the decay process from the photo-excited S1 state of (a) TICT and (b) MECI systems. 2.43, S6 and 0.568 cP, S7 respectively, were used as solvents (5.0 μM at 293 K, λex = 396 nm). e 20 mM CAPS and 50 mM NaCl solution. S11 6. Theoretical study

(TD-)DFT calculation
The theoretical calculation has been carried out to study the reason of different emission wavelength of AnP2 at different pH. The molecular geometries of model compounds, AnP2-Me (deprotonate state) and AnP2-Me-2H (protonate state), in the ground state and the first singlet excited state were optimized at (TD-)ωB97XD/6-31+G(d,p) level with IEF-PCM in water, and the resulting geometries were used for the calculation. Jablonski diagrams of AnP2-Me and AnP2-Me-2H, constructed based on the result of (TD-)DFT calculation were described in Figure S9.
The theoretical calculations revealed that protonation destabilizes the local minima of the S1 state, thereby explaining the spectral blue shift of the emission of AnP2 at low pH of the solution. In addition, TD-DFT calculation showed that the major electronic configuration of S1 state is formed by HOMO-to-LUMO transition, which is attributed to local π-π* transition of the anthracene moiety. The non-bonding orbital of N atoms is at a lower energy level than the HOMO (π) orbital ( Figure S10), suggesting that PET cannot occur in AnP2.

MECI geometry of AnP2-Me
The search of MECI geometry of AnP2-Me was performed at the state-averaged complete active space self-consistent-field theory (CASSCF) level using def2-SVP basis set along with the def2/J auxiliary basis set starting from optimized S1 geometry (BHHLYP/6-31+G(d,p)). In the obtained MECI geometry, the anthracene unit adopted a Dewar-benzene-like non-flat structure, similarly to the previous studies. S8,S9 Figure S11. Searched MECI geometry of AnP2-Me (CASSCF/def2-SVP, def2/J).

Effects of proteins on fluorescence intensity of AnP2-OEG
We evaluated the dependence of emission intensity on the presence of proteins using the cell lysate and Bovine serum albumin (BSA) ( Figure S20), at the concentration range reported for protein-targeting probes (1 μM probe and 10 μM protein). S10 Here, in the case of BSA, 6 mg mL -1 corresponds to 100 μM. In fact, only slight increase in fluorescence enhancement was observed: the intensity remains much weaker than that in 60w% glycerol aqueous solution (ca. 11 cP at 293 K, S5 which is even lower than the reported lysosomal viscosity), suggesting the small influence of proteins on fluorescence enhancement of AnP2-OEG.