Xanthone-based solvatochromic fluorophores for quantifying micropolarity of protein aggregates

Abstract

Proper three-dimensional structures are essential for maintaining the functionality of proteins and for avoiding pathological consequences of improper folding. Misfolding and aggregation of proteins have been both associated with neurodegenerative disease. Therefore, a variety of fluorogenic tools that respond to both polarity and viscosity have been developed to detect protein aggregation. However, the rational design of highly sensitive fluorophores that respond solely to polarity has remained elusive. In this work, we demonstrate that electron-withdrawing heteroatoms with (d-p)-π* conjugation can stabilize lowest unoccupied molecular orbital (LUMO) energy levels and promote bathochromic shifts. Guided by computational analyses, we have devised a novel series of xanthone-based solvatochromic fluorophores that have rarely been systematically studied. The resulting probes exhibit superior sensitivity to polarity but are insensitive to viscosity. As proof of concept, we have synthesized protein targeting probes for live-cell confocal imaging intended to quantify the polarity of misfolded and aggregated proteins. Interestingly, our results reveal several layers of protein aggregates in a way that we had not anticipated. First, microenvironments with reduced polarity were validated in the misfolding and aggregation of folded globular proteins. Second, granular aggregates of AgHalo displayed a less polar environment than aggregates formed by folded globular protein represented by Htt-polyQ. Third, our studies reveal that granular protein aggregates formed in response to different types of stressors exhibit significant polarity differences. These results show that the solvatochromic fluorophores solely responsive to polarity represent a new class of indicators that can be widely used for detecting protein aggregation in live cells, thus paving the way for elucidating cellular mechanisms of protein aggregation as well as therapeutic approaches to managing intracellular aggregates.

Fig. 1. Rational design of solvatochromic fluorophores to detect protein aggregation in live cells. (A) Polymethine dyes had been previously developed to detect protein aggregation based on viscosity and polarity change. In this work, xanthone-based solvatochromic fluorophores with single-atom substitutions have been designed to quantify micropolarity of protein aggregates in live cells. (B) A single-atom replacement strategy based on TD-DFT optimization is employed for designing solvatochromic fluorophores with high sensitivity to protein polarity.

Fig. 2. Rational design of xanthone derivative probes with sensitivity to polarity. (A) Structures of solvatochromic xanthone derivatives. (B) Emission spectra of MA in different solvents. (C) Polarity sensitivity calculated based on maximal emission over the E_T30 in standard solvents. (D) HOMO and LUMO orbitals of xanthone derivatives. (E) Change in emission maximum of different solvatochromic xanthone derivatives going from hexane to water. (F) Quantitative polarity dependence of solvatochromic xanthone derivatives in protic and aprotic solvents.

Fig. 3. Sulfonyl substituted acridone derivative exhibits improved polarity sensitivity evidenced by bathochromic shifts. (A) The emission maximum of SO₂X in different solvents. (B) Polarity sensitivity of SO₂X calculated based on maximal emission over the E_T30 in standard solvents. (C) TD-DFT calculated fluorescence emission energy of SO₂X in water solvent environment. (D) Electrostatic potential maps based on the electron density of SO₂X in water and vacuum. (E) Calculation showed that with solvent polarity increasement, the emission wavelength of SO₂X underwent bathochromic shifts, accompanied by decreases in the energy band gap and increases in the dipole moment.

Fig. 4. Quantifying the polarity of stress-induced protein aggregates. (A) SO₂X was used to label proteins for the detection of polarity changes in stress-induced protein aggregates. (B) Halo-SO₂X probe was used to visualize stress-induced protein aggregation. K73T was expressed in HEK293T cells, in the presence of 1 μM Halo-SO₂X, followed by treatment with PBS, sorbitol (0.5 M, 5 min), NaCl (1 M, 5 min), nilotinib (50 μM, 18 h). (C and D) Lambda scan imaging to reveal polarity changes in K73T–SO₂X conjugates treated with different stressors as indicated in (B). Scale bar = 10 μm. Error bars in (D) = standard deviation of the maximum emission wavelength.

Fig. 5. Investigation of the micropolarity of protein aggregates resulted from genetic mutations. (A) Structure of SNAP-SO₂X. (B) Granular 110Q–SNAPf aggregates (110Q–SNAPf) was transiently expressed in HEK293T cells with SNAP-SO₂X (0.5 μM) and drug-induced aggregation elimination. (C and D) Lambda scan imaging to reveal whether drugs effectively ameliorated aggregation of 110Q-SNAPf. Scale bar = 10 μm. Error bars in (D) = standard deviation of the maximum emission wavelength.