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Letter pubs.acs.org/JPCL
Unveiling Structural Motions of a Highly Fluorescent Superphotoacid by Locking and Fluorinating the GFP Chromophore in Solution Cheng Chen,† Weimin Liu,†,# Mikhail S. Baranov,‡ Nadezhda S. Baleeva,‡ Ilia V. Yampolsky,‡,§ Liangdong Zhu,† Yanli Wang,† Alexandra Shamir,¶,⊥ Kyril M. Solntsev,*,¶ and Chong Fang*,† †
Department of Chemistry, Oregon State University, 153 Gilbert Hall, Corvallis, Oregon 97331, United States Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya 16/10, 117997 Moscow, Russia § Pirogov Russian National Research Medical University, Ostrovitianov 1, Moscow 117997, Russia ¶ School of Chemistry and Biochemistry, Georgia Institute of Technology, 901 Atlantic Drive, Atlanta, Georgia 30332, United States ‡
S Supporting Information *
ABSTRACT: Superphotoacidity involves ultrafast proton motions implicated in numerous chemical and biological processes. We used conformational locking and strategic addition of electron-withdrawing substituents to synthesize a new GFP chromophore analogue: p-HO-3,5-diF-BDI:BF2 (diF). It is highly fluorescent and exhibits excited-state proton transfer (ESPT) in various solvents, placing it among the strongest photoacids. Tunable femtosecond stimulated Raman spectroscopy with unique resonance conditions and transient absorption are complementarily employed to elucidate the structural basis for superphotoacidity. We reveal a multistep ESPT reaction from diF to methanol with an initial proton dissociation on the ∼600 fs time scale that forms a chargeseparated state, stabilized by solvation, and followed by a diffusion-controlled proton transfer on the ∼350 ps time scale. A ∼1580 cm−1 phenolic ring motion is uncovered to accompany ESPT before 1 ps. This study provides a vivid movie of the photoinduced proton dissociation of a superphotoacid with bright fluorescence, effectively bridging fundamental mechanistic insights to precise control of macroscopic functions. reen fluorescent protein (GFP) has revolutionized molecular and cellular biology for decades. The heart of this biomolecular machine is a three-residue chromophore that responds to UV light as a photoacid. When outside of the protein matrix, the GFP chromophore loses excited-state proton transfer (ESPT) capability and becomes dark, attributed to its ultrafast ring twisting motion.1 What remains unclear is the real-time interplay between structure and function of the chromophore in various environments. Such an understanding will power ways to engineer the chromophore in solution to acquire functionality originally only inside of the protein or develop new capabilities. The photoacidity phenomenon has been known for more than 80 years.2 Such light-controlled molecules have enabled great advances in driving photochemical reactions,3−7 inducing pH changes,8,9 performing photolithography,10 catalyzing reactions, and modifying materials.11 Superphotoacids are molecules with a negative excited-state pKa (pKa*) and the ability to undergo ESPT in nonaqueous solvents. Huppert et al. studied quinone−cyanine photoacids,12 and QCy9 is the strongest photoacid with the same ESPT rate in water, methanol, and ethanol, going beyond the solvent control limit.13 The ESPT rate of a strong photoacid N-methyl-6hydroxyquinolinium is controlled by solvent motions that minimize the intermolecular dipole interaction.14 Notably, the photoacidity in homologues can be enhanced by the strategic
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placement of electron-withdrawing groups (EWGs) at positions of increased electron density in aromatic systems.1,15−17 In this study, we report the chemical synthesis and excitedstate structural motions of a novel fluorescent superphotoacid as a functional analogue to the GFP chromophore. Over the years, many new chromophores have been synthesized to investigate chemical properties and fluorogenic behaviors in the repertoire of GFP and RNA.18−20 In particular, the (Z)-4-(4hydroxybenzylidene)-1,2-dimethyl-1H-imidazol-5(4H)-one (pHBDI, Scheme 1) model chromophore represents the core of wild-type (wt)GFP, but its emission behavior in solution differs dramatically from that in a protein. Due to an efficient photoisomerization-induced deactivation, no ESPT is observed for p-HBDI in solution, and the fluorescence quantum yield (QY) drops by 4 orders of magnitude from that inside of the restrictive β-barrel of GFP.1,21 Recently, Solntsev and co-workers reported the synthesis and study of a GFP-chromophore analogue (Z)-4-(2-(difluoroboryl)-4-hydroxybenzylidene)-1,2-dimethyl-1H-imidazol-5(4H)one (p-HOBDI-BF2), and the phenolic and imidazolinone rings are irreversibly locked by a BF2 group. This fully planar structure suppresses photoisomerization, enables intermolecuReceived: October 9, 2017 Accepted: November 17, 2017 Published: November 17, 2017 5921
DOI: 10.1021/acs.jpclett.7b02661 J. Phys. Chem. Lett. 2017, 8, 5921−5928
Letter
The Journal of Physical Chemistry Letters Scheme 1. Structures of the GFP-Derived HBDI Chromophore Analogues and Engineered Superphotoacids
lar ESPT, and greatly improves the fluorescence QY (e.g., 0.73 in acetonitrile with a lifetime of 3.2 ns).22 The measured pKa* ≈ 0.6 implies its moderate photoacidity, which cannot support ESPT in methanol or other alcohols. In this work, our newly synthesized superphotoacid, p-HO-3,5-diF-BDI:BF2 (abbreviated as diF) incorporates two fluorine atoms as EWGs to the rigid skeleton of p-HOBDI-BF2 and simultaneously achieves high QY and fast ESPT outside of a restraining protein matrix. It is also soluble in most polar and medium-polar solvents, which is an appealing property (see the SI). We exploited tunable femtosecond stimulated Raman spectroscopy (FSRS) to study diF in solution. FSRS is a powerful toolset to resolve atomic motions of chemical and biological systems by simultaneously providing high temporal (
