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Tuning the Baird Aromatic Triplet-State Energy of Cyclooctatetraene to Maximize the Self-Healing Mechanism in Organic Fluorophores

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Tuning the Baird Aromatic Triplet-State Energy of Cyclooctatetraene to Maximize the Self-Healing Mechanism in Organic Fluorophores Tuning the Baird aromatic triplet-state energy of cyclooctatetraene to maximize the self-healing mechanism in organic fluorophores Avik K. Patia,b, Ouissam El Bakouric,1, Steffen Jockuschd,1, Zhou Zhoua,1, Roger B. Altmana,b, Gabriel A. Fitzgeralda, Wesley B. Ashere,f,g, Daniel S. Terrya,b, Alessandro Borgiab, Michael D. Holseye,f, Jake E. Batcheldera,b, Chathura Abeywickramab, Brandt Huddlea, Dominic Rufaa, Jonathan A. Javitche,f,g, Henrik Ottossonc, and Scott C. Blancharda,b,2 aDepartment of Physiology and Biophysics, Weill Cornell Medicine, New York, NY 10065; bDepartment of Structural Biology, St. Jude Children’s Research Hospital, Memphis, TN 38105; cÅngström Laboratory, Department of Chemistry, Uppsala University, Uppsala, 751 20 Sweden; dDepartment of Chemistry, Columbia University, New York, NY 10027; eDepartment of Psychiatry, Columbia University, New York, NY 10032; fDepartment of Pharmacology, Columbia University, New York, NY 10032; and gDivision of Molecular Therapeutics, New York State Psychiatric Institute, New York, NY 10032 Edited by Taekjip Ha, Johns Hopkins University, Baltimore, MD, and approved August 17, 2020 (received for review April 7, 2020) Bright, photostable, and nontoxic fluorescent contrast agents are which can generate toxic reactive oxygen species (ROS). ROS include critical for biological imaging. “Self-healing” dyes, in which triplet singlet oxygen, peroxide, and superoxide radicals that lead to chem- states are intramolecularly quenched, enable fluorescence imaging ical destruction of the fluorophore (photobleaching), photocross- by increasing fluorophore brightness and longevity, while simul- linking to elements in the surrounding environment, and other po- taneously reducing the generation of reactive oxygen species that tentially toxic side effects (7–11). promote phototoxicity. Here, we systematically examine the self- Organic fluorophores commonly used in modern imaging ap- healing mechanism in cyanine-class organic fluorophores spanning plications (e.g., rhodamine- and cyanine-class fluorophores) the visible spectrum. We show that the Baird aromatic triplet-state typically exhibit relatively short excited-state lifetimes and low energy of cyclooctatetraene can be physically altered to achieve probabilities of intersystem crossing to triplet and radical states order of magnitude enhancements in fluorophore brightness and (13–15). Nonetheless, triplet and radical states can become sig- signal-to-noise ratio in both the presence and absence of oxygen. nificantly populated at elevated illumination intensities, an issue BIOPHYSICS AND COMPUTATIONAL BIOLOGY We leverage these advances to achieve direct measurements of that is particularly problematic when imaging low-abundance large-scale conformational dynamics within single molecules at species, including single-molecule and superresolution imaging submillisecond resolution using wide-field illumination and camera- applications (1–5, 11). In this regime, increasing illumination based detection methods. These findings demonstrate the capacity to image functionally relevant conformational processes in biological Significance systems in the kilohertz regime at physiological oxygen concentra- tions and shed important light on the multivariate parameters critical Fluorescence imaging, a central pillar of medical and funda- to self-healing organic fluorophore design. mental research, is hampered by stochastic blinking, limited photon budgets, and phototoxicities of organic fluorophores. self-healing fluorophore | photostability | Baird aromaticity | Here, we use quantum chemical calculations and targeted cyclooctatetraene | single-molecule imaging synthetic strategies to tune the Baird aromatic triplet-state energy of cyclooctatetraene to enhance its capacity to medi- – espite remarkable progress (1 5), fluorescence imaging ate intramolecular triplet−triplet energy transfer with distinct Dpursuits, which are a central pillar of both medical and organic fluorophores spanning the visible spectrum. By tuning fundamental research, remain severely hampered by the limited the efficiency of the self-healing mechanism, we generate durability and stochastic blinking of organic fluorophores. Such cyanine-class fluorophore derivatives with improved intrinsic issues reduce photon budgets and signal-to-noise ratios, thereby brightness and photostability, and reduced reactive oxygen compromising the information content of the experiments per- species generation. We leverage these advances to extend the formed. Related considerations limit superresolution imaging time scale of camera-based, single-molecule imaging tech- methods (6), the temporal resolution that can be achieved when niques, including Förster (fluorescence) resonance energy examining biological function, and the interpretation of dynamic transfer methods, to the kilohertz regime in fully oxygenated processes within individual molecules at the single-molecule scale. physiological buffers. The performance of organic fluorophores in experimental settings is maximized when excitation-induced separation of va- Author contributions: A.K.P., O.E.B., S.J., R.B.A., W.B.A., D.S.T., J.A.J., H.O., and S.C.B. lence electrons leads to reliable and repeated relaxation back to designed research; A.K.P., O.E.B., S.J., R.B.A., G.A.F., W.B.A., A.B., M.D.H., J.E.B., C.A., the ground state. Performance demands are particularly accen- B.H., and D.R. performed research; Z.Z. and D.S.T. contributed new reagents/analytic tools; A.K.P., O.E.B., S.J., R.B.A., G.A.F., W.B.A., D.S.T., A.B., J.A.J., H.O., and S.C.B. analyzed tuated in single-molecule applications, where photon emission- data; and A.K.P., O.E.B., S.J., R.B.A., W.B.A., D.S.T., A.B., J.A.J., H.O., and S.C.B. wrote induced relaxation must occur over millions of cycles, while the paper. nonfluorescent decay pathways must be minimized. The flux of Competing interest statement: S.C.B. and R.B.A. have an equity interest in Lumidyne photon emission in fluorescent species can be severely compro- Technologies. mised when excited-state electrons undergo intersystem crossing This article is a PNAS Direct Submission. to relatively long-lived, nonfluorescing triplet and radical states This open access article is distributed under Creative Commons Attribution-NonCommercial- (Fig. 1A), whose lifetimes (ca. 100 μs to seconds) are orders of NoDerivatives License 4.0 (CC BY-NC-ND). magnitude longer than singlet excited states (ca. 0.001 μsto 1O.E.B., S.J., and Z.Z. contributed equally to this work. 0.1 μs). Correspondingly, triplet states can dramatically reduce 2To whom correspondence may be addressed. Email: [email protected]. the number of detected photons per unit time (brightness). The This article contains supporting information online at https://www.pnas.org/lookup/suppl/ relatively long lifetimes and high energies of triplet and radical doi:10.1073/pnas.2006517117/-/DCSupplemental. states also render them prone to reactions with molecular oxygen, First published September 10, 2020. www.pnas.org/cgi/doi/10.1073/pnas.2006517117 PNAS | September 29, 2020 | vol. 117 | no. 39 | 24305–24315 Downloaded by guest on September 30, 2021 ABCTriplet state (T ) + − Self-healing dyes 1 ROXS ROXS or ROXS SO3 O3S R S1 ISC L R − R T R H R L 1 D or N N 8h H Redox process n L L + R n = 1, Cy3 (step I) R T1vert H H n = 2, Cy5 → D L n = 3, Cy7 8h TS 3 COT TS(S0) H ΔEBS E E TET TS TS ∆ T ROXS Energy D4h D4h Absorption Fluorescence inv L E 1 3 Δ COT H Dye* Triplet-triplet energy Triplet-triplet transfer (TET) (step II) L + Redox process D D ROXS 2d 2d H or − S ROXS 0 Intramolecular collision Singlet ground state (S0) D Potassium borate O (0.5 M, pH 8.5) TSQ HOOC Dye NH2 TSQ-NHS HOOC Dye NH dd H2O, DMSO rt, 2 hrs E F 5 1 10 4 3 2 1,2-DAC(I) DAC O 8 ) 1 − N 1,5-DAC DAC H 6 [1,4-DAC(I)] 1,3-DAC (kJ mol AC Triplet state 4 AC Me-COT Triplet state ring distortion Triplet energy of COT molecules energy of COT 2 Baird aromatic 110 115 120 125 130 135 140 Me-COT Triplet energy of COT molecules G (kJ mol−1) 3 3 3 Me-COT AC DAC −1 ET (kJ mol ) COT 112.3 117.2 131.6 molecules 115.5 144.9 173.8 Dyes 3Cy7 3Cy5 3Cy3 Fig. 1. Strategies for triplet-state quenching of organic fluorophores. (A) Simplified state energy diagram for fluorophore excitation and relaxation pathways including fluorophore’s triplet-state quenching by small-molecule protective agents, such as COT and a combination of reducing and oxidizing + – species (ROXS). R ,R,S0,T1, and ISC indicate radical cation, radical anion, singlet ground state, triplet state, and intersystem crossing of a fluorophore, respectively. The rectangular boxes show the electron and spin configurations for the corresponding states of a fluorophore and a COT molecule. H and L indicate highest occupied and lowest unoccupied molecular orbital, respectively. (B) Representative examples of self-healing organic fluorophores, in which a COT molecule is covalently conjugated to cyanine-class organic fluorophores through a flexible linker (squiggly line). R represents electronic substituents (Top). Schematic shows intramolecular collisions between the triplet state of a fluorophore and the ground state of a COT molecule, facilitating triplet quenching of the fluorophore through TET (Bottom). (C) Schematics of relative energy changes of a COT molecule in S0 and T1 potential energy surfaces (12). ΔEinv, ΔEBS, ΔETS(S0)→T1vert, ET, and TS are ring inversion barrier energy, bond-shifting energy, energy for vertical excitation from the planar singlet (D4h)tothe triplet state, triplet energy, and transition state, respectively. D2d, D4h, and D8h are the Schoenflies point group notations, which collectively represent all symmetry elements within a molecule at a certain structure. While the D2d minimum is puckered, the D4h and D8h transition states are planar and, respec- tively, bond-length alternate and bond-length equalized forms of COT in the S0 state. (D) Generalized synthetic strategy for self-healing fluorophores. (E) Anisotropy of induced current density plot (displayed as clockwise) of an amide substituted COT molecule (AC) in its triplet state (SI Appendix, Supplementary Methods).
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