Bioinspired Molecular Lantern: Tuning the Firefly Oxyluciferin Emission with Host−Guest Chemistry Na’il Saleh,* ,† Abdul Rahman Ba Suwaid, †,∥ Ahmad Alhalabi, †,∥ Ahmed Z. A. Abuibaid, †,∥ Oleg V. Maltsev, ‡ Lukas Hintermann, ‡ and Panc ̌ e Naumov* ,§ † Department of Chemistry, College of Science, United Arab Emirates University, P. O. Box 15551, Al Ain, United Arab Emirates ‡ Department Chemie, Technische Universitä t Mü nchen, Lichtenbergstrasse 4, 85748 Garching bei Mü nchen, Germany § New York University Abu Dhabi, P. O. Box 129188, Abu Dhabi, United Arab Emirates * S Supporting Information ABSTRACT: Fireflies generate flashes of visible light via luciferase- catalyzed chemiexcitation of the substrate (luciferin) to the first excited state of the emitter (oxyluciferin). Microenvironment effects are often invoked to explain the effects of the luciferase active pocket on the emission; however, the exceedingly complex spectrochemistry and synthetic burdens have precluded elucidation of the nature of these interactions. To decipher the effects of microenvironment on the light emission, here the hydrophobic interior of cucurbit[7]uril (CB7) is used to mimic the nonpolar active pocket of luciferase. The hydrophobic interior of CB7 induces shifts of the ground-state pK a s by 1.9−2.5 units to higher values. Upon sequestration, the emission maxima of neutral firefly oxyluciferin and its conjugate monodeprotonated base are blue-shifted by 40 and 39 nm, respectively, resulting in visual color changes of the emitted light. ■ INTRODUCTION Fireflies communicate with each other by generating flashes of light in a very efficient two-stage, four-step reaction catalyzed by an enzyme, fire fly luciferase (Luc), whereby the benzothiazolyl-dihydrothiazole carboxylic acid luciferin, LH 2 (“H 2 ” here stands for the two ionizable protons) and oxygen react to give the first excited state of the oxidation product, oxyluciferin (OxyLH 2 ; Scheme 1). 1 The chemiexcited oxy- luciferin deexcites on a nanosecond time scale with emission of a photon of green-yellow light (λ ≈ 560 nm). The high efficiency of this process of generation of cold light 2 has spurred ample experimental 3−12 and computational research ef- forts 13−24 into the photophysics and photochemistry of the emitter. The ongoing research revolves around the yet unresolved chemical form from which oxyluciferin emits light and the unknown mechanism by which some natural and genetically engineered luciferases can generate light of varying colors. 9 The multiple chemical equilibria (dissociation of the two hydroxyl groups and keto−enol tautomerism of the hydroxythiazole, Chart 1) and the chemical instability of OxyLH 2 in basic solutions 25 have precluded direct studies into the chemistry of the firefly emitter in the enzyme. The electronic spectra, distribution with pH, 11 and ground- and excited-state-dissociation constants of the chemical forms of OxyLH 2 in buffered model solutions have only recently been unveiled. 12 Notably, the effects of polarity of the Luc active pocket on OxyLH 2 spectrochemistry are central to one of the theories that is commonly invoked to explain the color variations in the emitted light. 4 Early studies have concluded that the active pocket is of low polarity. 1 Recent results, however, indicate that a single water molecule can affect the wavelength of emitted light 26 and thus the water dynamics could play a critical role in the OxyLH 2 photophysics in vivo. 17,23 Even that the collective interactions with the active pocket of Luc, often referred to as microenvironment ef fects, are undoubtedly relevant to the firefly photochemistry, the true nature of these interactions remains elusive. To investigate the effect of a nonpolar environment within a binding pocket on the emission from firefly oxyluciferin, we resorted to host−guest chemistry as a straightforward and inexpensive approach that bypasses the synthetic burdens associated with purposefully engineered mutant luciferases. Sequestration of fluorophores into macromolecules are known to have multiple benefits, notably prevention of the fluorescence quenching observed with solid OxyLH 2 9 by suppression of aggregation. 27 This supramolecular “isolation” by complexation could enhance the emission from OxyLH 2 , alleviate its instability at high pH by preventing dimerization, 25 and protect its anionic forms from oxidation. 27−29 Macro- molecular hosts such as cucurbiturils (CBs) 30 and cyclodextrins (CDs) 31 appear ideally suited to encage OxyLH 2 and could be considered a mimic of the hydrophobic microenvironment of Luc since they have cavities of variable size and well-defined interactions. Received: June 30, 2016 Revised: July 20, 2016 Published: July 21, 2016 Article pubs.acs.org/JPCB © 2016 American Chemical Society 7671 DOI: 10.1021/acs.jpcb.6b06557 J. Phys. Chem. B 2016, 120, 7671−7680