Unveiling Structural Motions of a Highly Fluorescent Superphotoacid

Nov 17, 2017 - Superphotoacidity involves ultrafast proton motions implicated in numerous chemical and biological processes. We used conformational lo...
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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 J. Phys. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.jpclett.7b02661 • Publication Date (Web): 17 Nov 2017 Downloaded from http://pubs.acs.org on November 20, 2017

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The Journal of Physical Chemistry Letters

Unveiling Structural Motions of a Highly Fluorescent Superphotoacid by Locking and Fluorinating the GFP Chromophore in Solution Cheng Chen,† Weimin Liu,†,Á 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,

USA ‡

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, USA Á

Present address: School of Physical Science and Technology, ShanghaiTech University, Pudong,

Shanghai 201210, P. R. China. 2

Summer program participant (high school student) from Woodward Academy, 1662 W Rugby

Ave, College Park, Georgia 30337, USA. ^

To whom correspondence may be addressed. E-mail: [email protected] or

[email protected].



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ABSTRACT. Super-photoacidity 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 analog: p-HO-3,5-diFBDI: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 structural basis for super-photoacidity. We reveal a multistep ESPT reaction from diF to methanol with an initial proton dissociation on the ~600 fs timescale that forms a charge-separated state, stabilized by solvation, and followed by a diffusion-controlled proton transfer on the ~350 ps timescale. 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.

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Green 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 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 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 ability to undergo ESPT in non-aqueous 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-6-hydroxyquinolinium is controlled by solvent motions which minimize the intermolecular dipole interaction.14 Notably, the photoacidity in homologs can be enhanced by the strategic 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 excited-state structural motions of a novel fluorescent superphotoacid as a functional analog to 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-(4-hydroxybenzylidene)-1,2dimethyl-1H-imidazol-5(4H)-one (p-HBDI, 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.



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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 the restrictive β-barrel of GFP.1,21 Recently, Solntsev and co-workers reported the synthesis and study of a GFP-chromophore analog (Z)-4-(2-(difluoroboryl)-4-hydroxybenzylidene)-1,2-dimethyl-1H-imidazol-5(4H)-one (pHOBDI-BF2) and the phenolic and imidazolinone rings are irreversibly locked by a BF2 group. This fully planar structure suppresses photoisomerization, enables intermolecular 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-HOBDIBF2 and simultaneously achieves high QY and fast ESPT outside a restraining protein matrix. It is also soluble in most polar and medium-polar solvents which is an appealing property (SI Text).

Scheme 1. Structures of the GFP-derived HBDI chromophore analogs and engineered superphotoacids. 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 (