Supramolecular Fluorescence Resonance Energy Transfer in Nucleobase-Modified Fluorogenic RNA Aptamers

RNA aptamers form compact tertiary structures and bind their ligands in specific binding sites. Fluorescence-based strategies reveal information on structure and dynamics of RNA aptamers. Herein, we report the incorporation of the universal emissive nucleobase analog 4-cyanoindole into the fluorogenic RNA aptamer Chili, and its application as a donor for supramolecular FRET to the bound ligands DMHBI⁺ or DMHBO⁺. The photophysical properties of the new nucleobase–ligand-FRET pair revealed structural restraints for the overall RNA aptamer organization and identified nucleotide positions suitable for FRET-based readout of ligand binding. This strategy is generally suitable for binding-site mapping and may also be applied for responsive aptamer devices.     

Supramolecular Fluorescence Resonance Energy Transfer in Nucleobase‐Modified Fluorogenic RNA Aptamers

RNA aptamers form compact tertiary structures and bind their ligands in specific binding sites. Fluorescence‐based strategies reveal information on structure and dynamics of RNA aptamers. Herein, we report the incorporation of the universal emissive nucleobase analog 4‐cyanoindole into the fluorogenic RNA aptamer Chili, and its application as a donor for supramolecular FRET to the bound ligands DMHBI⁺ or DMHBO⁺. The photophysical properties of the new nucleobase–ligand‐FRET pair revealed structural restraints for the overall RNA aptamer organization and identified nucleotide positions suitable for FRET‐based readout of ligand binding. This strategy is generally suitable for binding‐site mapping and may also be applied for responsive aptamer devices.     

Na+ Selective Fluorescent Tools Based on Fluorescence Intensity Enhancements,Lifetime Changes,and on a Ratiometric Response

Over the years, we developed highly selective fluorescent probes for K⁺ in water, which show K⁺-induced fluorescence intensity enhancements, lifetime changes, or a ratiometric behavior at two emission wavelengths (cf. Scheme 1, K1 – K4 ). In this paper, we introduce selective fluorescent probes for Na⁺ in water, which also show Na⁺ induced signal changes, which are analyzed by diverse fluorescence techniques. Initially, we synthesized the fluorescent probes 2 , 4 , 5 , 6 and 10 for a fluorescence analysis by intensity enhancements at one wavelength by varying the Na⁺ responsive ionophore unit and the fluorophore moiety to adjust different K_d values for an intra- or extracellular Na⁺ analysis. Thus, we found that 2 , 4 and 5 are Na⁺ selective fluorescent tools, which are able to measure physiologically important Na⁺ levels at wavelengths higher than 500 nm. Secondly, we developed the fluorescent probes 7 and 8 to analyze precise Na⁺ levels by fluorescence lifetime changes. Herein, only 8 (K_d=106 mm ) is a capable fluorescent tool to measure Na⁺ levels in blood samples by lifetime changes. Finally, the fluorescent probe 9 was designed to show a Na⁺ induced ratiometric fluorescence behavior at two emission wavelengths. As desired, 9 (K_d=78 mm ) showed a ratiometric fluorescence response towards Na⁺ ions and is a suitable tool to measure physiologically relevant Na⁺ levels by the intensity change of two emission wavelengths at 404 nm and 492 nm.     

ANALYSIS OF MICROSECOND FLUORESCENCE YIELD AND DELAYED LIGHT EMISSION CHANGES AFTER A SINGLE FLASH IN PEA CHLOROPLASTS: EFFECTS OF MONO- AND DIVALENT CATIONS

Abstract. New results are presented on the effects of mono- and divalent cations on concurrent changes in the microsecond yields and kinetics of chlorophyll a fluorescence and delayed light emission, and the light saturation curve for the latter at 100 μs, following a 10 ns flash at 337 nm. (1) The fluorescence yield increases exponentially from 3 to 30 μs (lifetime, τ, 6.4 ± 0.6/μs), and decays biphasically between 50 and 800μs. (2) The delayed light emission decays biphasically with two exponential phases: fast phase, T= 7–10μs, and slow phase, T= 33–40μs. (3) The light saturation curve for 100μs delayed light emission is satisfactorily represented by a one-hit Poisson saturation curve. (4) Addition of 5 mM NaCl to salt-depleted chloroplasts decreases (by as much as 40%) the yields of μs fluorescence and delayed light emission, and the subsequent addition of 5mM MgCl₂ increases the yields (≤2 × over samples with only NaCl). (5) The fluorescence yield rise and delayed light emission decay kinetics are independent of low concentrations of cations. The lifetime of the fast phase of fluorescence decay changes from ?90μs to ?160μs, when Na⁺ or Na⁺+ Mg²⁺ are added. Based on a detailed analysis presented in this paper, the following conclusions regarding the effects of low concentrations (few mM) of mono-and divalent cations in sucrose-washed chloroplasts at room temperature are made: (a) Na⁺ decreases (?6%) and Mg²⁺ increases (? 20% compared with the Na⁺ sample) the sensitization of photosystem II photochemistry: this effect is small, but significant. (b) Na⁺ increases and Mg²⁺ decreases the efficiency for radiationless transitions in singlet excited Chl a in the antenna and closed reaction center of PS II; this includes non-radiative energy transfer to PS I, intramolecular intersystem crossing and internal conversion. The ratio of the sum of the rate constants for radiationless transitions to that for fluorescence increases by ? 2-fold upon the addition of Na⁺, and is completely reversed by the addition of Mg²⁺. (c) The rate constant for the re-oxidation of Q^- decreases (about 50%) in the presence of Na⁺ or Na⁺+ Mg²⁺. These conclusions imply that cations produce multiple changes in the primary photoprocesses of PS II at physiological temperatures. It is proposed that these changes are mutually independent and can co-exist.     

Mapping the Complete Photocycle that Powers a Large Stokes Shift Red Fluorescent Protein

Large Stokes shift (LSS) red fluorescent proteins (RFPs) are highly desirable for bioimaging advances. The RFP mKeima, with coexisting cis- and trans-isomers, holds significance as an archetypal system for LSS emission due to excited-state proton transfer (ESPT), yet the mechanisms remain elusive. We implemented femtosecond stimulated Raman spectroscopy (FSRS) and various time-resolved electronic spectroscopies, aided by quantum calculations, to dissect the cis- and trans-mKeima photocycle from ESPT, isomerization, to ground-state proton transfer in solution. This work manifests the power of FSRS with global analysis to resolve Raman fingerprints of intermediate states. Importantly, the deprotonated trans-isomer governs LSS emission at 620 nm, while the deprotonated cis-isomer's 520 nm emission is weak due to an ultrafast cis-to-trans isomerization. Complementary spectroscopic techniques as a table-top toolset are thus essential to study photochemistry in physiological environments.     

An orange fluorescent protein with a large Stokes shift for single-excitation multicolor FCCS and FRET imaging

Multicolor imaging based on genetically encoded fluorescent proteins (FPs) is a powerful approach to study several dynamic processes in a live cell. We report a monomeric orange FP with a large Stokes shift (LSS), called LSSmOrange (excitation/emission at 437/572 nm), which fills up an existing spectral gap between the green-yellow and red LSSFPs. Brightness of LSSmOrange is five-fold larger than that of the brightest red LSSFP and similar to the green-yellow LSSFPs. LSSmOrange allows numerous multicolor applications using a single-excitation wavelength that was not possible before. Using LSSmOrange we developed four-color single-laser fluorescence cross-correlation spectroscopy, solely based on FPs. The quadruple cross-correlation combined with photon counting histogram techniques allowed quantitative single-molecule analysis of particles labeled with four FPs. LSSmOrange was further applied to simultaneously image two F?rster resonance energy transfer pairs, one of which is the commonly used CFP-YFP pair, with a single-excitation laser line. The combination of LSSmOrange-mKate2 and CFP-YFP biosensors enabled imaging of apoptotic activity and calcium fluctuations in real time. The LSSmOrange mutagenesis, low-temperature, and isotope effect studies revealed a proton relay for the excited-state proton transfer responsible for the LSS phenotype.     

Synthesis and optical properties of cis‐dibenzothiazolyldibenzo‐24‐crown‐8

New crown ether carrying two fluorionophores of cis‐dibenzothiazolyldibenzo‐24‐crown‐8 was synthesized from cis‐diformyldibenzo‐24‐crown‐8 and 2‐aminobenzenethiol. The binding behavior and the optical properties of the crown ether were examined through UV‐visible spectroscopy and fluorescence spectroscopy. When complexed with Na⁺, K⁺, Rb⁺, and Cs⁺ ions, it led to intramolecular charge transfer and caused the changes of the fluorescence spectra. The protonation of the crown ether was also studied. With protonation using CF₃COOH, the absorption bands and the fluorescence spectroscopy changed, the maximal fluorescence wavelengths red shifted and the fluorescence intensity with the maximum at 433 nm enhanced strongly. J. Heterocyclic Chem., (2011).     

A Chimeric DNA/RNA Antiparallel Quadruplex with Improved Stability

Nucleic acid quadruplexes are proposed to play a role in the regulation of gene expression, are often present in aptamers selected for specific binding functions and have potential applications in medicine and biotechnology. Therefore, understanding their structure and thermodynamic properties and designing highly stable quadruplexes is desirable for a variety of applications. Here, we evaluate DNA→RNA substitutions in the context of a monomolecular, antiparallel quadruplex, the thrombin-binding aptamer (TBA, GGTTGGTGTGGTTGG) in the presence of either K⁺ or Sr²⁺. TBA predominantly folds into a chair-type configuration containing two G-tetrads, with G residues in both syn and anti conformation. All chimeras with DNA→RNA substitutions (G→g) at G residues requiring the syn conformation demonstrated strong destabilization. In contrast, G→g substitutions at Gs with anti conformation increased stability without affecting the monomolecular chair-type topology. None of the DNA→RNA substitutions in loop positions affected the quadruplex topology; however, these substitutions varied widely in their stabilizing or destabilizing effects in an unpredictable manner. This analysis allowed us to design a chimeric DNA/RNA TBA construct that demonstrated substantially improved stability relative to the all-DNA construct. These results have implications for a variety of quadruplex-based applications including for the design of dynamic nanomachines.     

A Facile Molecular Machine: Optically Triggered Counterion Migration by Charge Transfer of Linear Donor‐π‐Acceptor Phosphonium Fluorophores

The D‐π‐A type phosphonium salts in which electron acceptor (A=‐⁺PR₃) and donor (D=‐NPh₂) groups are linked by polarizable π‐conjugated spacers show intense fluorescence that is classically ascribed to excited‐state intramolecular charge transfer (ICT). Unexpectedly, salts with π=‐(C₆H₄)_n‐ and ‐(C₁₀H₆C₆H₄)‐ exhibit an unusual dual emission (F₁ and F₂ bands) in weakly polar or nonpolar solvents. Time‐resolved fluorescence studies show a successive temporal evolution from the F₁ to F₂ emission, which can be rationalized by an ICT‐driven counterion migration. Upon optically induced ICT, the counterions move from ‐⁺PR₃ to ‐NPh₂ and back in the ground state, thus achieving an ion‐transfer cycle. Increasing the solvent polarity makes the solvent stabilization dominant, and virtually stops the ion migration. Providing that either D or A has ionic character (by static ion‐pair stabilization), the ICT‐induced counterion migration should not be uncommon in weakly polar to nonpolar media, thereby providing a facile avenue for mimicking a photoinduced molecular machine‐like motion.     

Intense Red-Blue Luminescence Based on Superfine Control of Metal–Metal Interactions for Self-Assembled Platinum(II) Complexes

A series of assembled Pt^II complexes comprising N-heterocyclic carbene and cyanide ligands was constructed using different substituent groups, [Pt(CN)₂(R-impy)] (R-impyH⁺=1-alkyl-3-(2-pyridyl)-1H-imidazolium, R=Me ( Pt-Me ), Et ( Pt-Et ), ⁱPr ( Pt- ⁱ Pr ), and ^tBu ( Pt- ^t Bu )). All the complexes exhibited highly efficient photoluminescence with an emission quantum yield of 0.51–0.81 in the solid state at room temperature, originating from the triplet metal-metal-to-ligand charge transfer (³MMLCT) state. Their emission colors cover the entire visible region from red for Pt-Me to blue for Pt- ^t Bu . Importantly, Pt- ^t Bu is the first example that exhibits blue ³MMLCT emission. The ³MMLCT emission was proved and characterized based on the temperature dependences of the crystal structures and emission properties. The wide-range color tuning of luminescence using the ³MMLCT emission presents a new strategy of superfine control of the emission color.     

A Highly K+‐Selective Two‐Photon Fluorescent Probe

A highly K⁺‐selective two‐photon fluorescent probe for the in vitro monitoring of physiological K⁺ levels in the range of 1–100 mM is reported. The two‐photon excited fluorescence (TPEF) probe shows a fluorescence enhancement (FE) by a factor of about three in the presence of 160 mM K⁺, independently of one‐photon (OP, 430 nm) or two‐photon (TP, 860 nm) excitation and comparable K⁺‐induced FEs in the presence of competitive Na⁺ ions. The estimated dissociation constant (K_d) values in Na⁺‐free solutions (K_d^OP=(28±5) mM and K_d^TP=(36±6) mM ) and in combined K⁺/Na⁺ solutions (K_d^OP=(38±8) mM and K_d^TP=(46±25) mM ) reflecting the high K⁺/Na⁺ selectivity of the fluorescent probe. The TP absorption cross‐section (σ_2PA) of the TPEF probe+160 mM K⁺ is 26 GM at 860 nm. Therefore, the TPEF probe is a suitable tool for the in vitro determination of K⁺.     

Optical Sensing of Cesium Using 1,3-Alternate Calix[4]-mono- and Di(anthrylmethyl)aza-crown-6

We have synthesized two derivatives of alkylanthracene covalently bonded to 1,3-alternate calix[4]aza-crown-6 at the nitrogen position to study the effect of alkali metal ion complexation on the emission properties of anthracene fluorophore. The mono- and dianthryl-substituted probes are weakly fluorescent because their emission is partially quenched by photoinduced electron transfer (PET) from the nitrogen lone pair to the excited singlet state of anthracene. Upon complexation of alkali metal ions (e.g. K⁺, Cs⁺) by the crown moiety, the nitrogen lone pair can no longer participate in the PET process causing an enhancement in the emission of anthracene fluorophore (fluorescent turn on). The maximum fluorescence enhancement observed upon complexation of cesium ions by mono- and dianthryl-substituted calix[4]aza-crown-6 relative to the uncomplexed form was 8.5- and 11.6-fold, respectively.     

Affinity of a new copper(II) complex to DNA/BSA and antioxidant/radical scavenging activities: crystal structure of [Cu(4,7-diphenyl-1,10-phenanthroline)(leucine)(NO3)(H2O)]

A new copper(II) complex, [Cu(Bphen)(Leu)(NO₃)(H₂O)] (Bphen = 4,7-diphenyl-1,10-phenanthroline, leu = L-leucine), has been synthesized and characterized by IR spectroscopy, CHN analysis, and single-crystal X-ray diffraction techniques. The CT-DNA binding properties of the complex have been investigated by both absorption and emission spectroscopy. The binding parameters for the fluorescence Scatchard plot were also determined. Further, the interaction of the complex with bovine serum albumin (BSA) has been investigated using absorption and emission spectroscopy. The thermodynamic parameters, free energy change (ΔG), enthalpy change (ΔH), and entropy change (ΔS), were calculated by the van’t Hoff equation and discussed. The distance between BSA and the complex has been obtained according to fluorescence resonance energy transfer. Conformational changes of BSA have been observed from synchronous fluorescence. Antioxidant and radical scavenging activities of the complex were determined by various in vitro assays such as 1,1-diphenyl-2-picryl-hydrazyl free radicals (DPPH˙), 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) radicals (ABTS˙⁺), and reducing ability determination by H₂O₂ scavenging methods.     

Selective Detection of Hg2+ Ions with Boron Dipyrromethene‐Based Fluorescent Probes Appended with a Bis(1,2,3‐triazole)amino Receptor

By using a copper‐promoted alkyne–azide cycloaddition reaction, two boron dipyrromethene (BODIPY) derivatives bearing a bis(1,2,3‐triazole)amino receptor at the meso position were prepared and characterized. For the analogue with two terminal triethylene glycol chains, the fluorescence emission at 509 nm responded selectively toward Hg²⁺ ions, which greatly increased the fluorescence quantum yield from 0.003 to 0.25 as a result of inhibition of the photoinduced electron transfer (PET) process. By introducing two additional rhodamine moieties at the termini, the resulting conjugate could also detect Hg²⁺ ions in a highly selective manner. Upon excitation at the BODIPY core, the fluorescence emission of rhodamine at 580 nm was observed and the intensity increased substantially upon addition of Hg²⁺ ions due to inhibition of the PET process followed by highly efficient fluorescence resonance energy transfer (FRET) from the BODIPY core to the rhodamine moieties. The Hg²⁺‐responsive fluorescence change of these two probes could be easily seen with the naked eye. The binding stoichiometry between the probes and Hg²⁺ ions in CH₃CN was determined to be 1:2 by Job′s plot analysis and ¹H NMR titration, and the binding constants were found to be (1.2±0.1)×10¹¹ m ^?2 and (1.3±0.3)×10¹⁰ m ^?2, respectively. The overall results suggest that these two BODIPY derivatives can serve as highly selective fluorescent probes for Hg²⁺ ions. The rhodamine derivative makes use of a combined PET‐FRET sensing mechanism which can greatly increase the sensitivity of detection.     

Reagentless Aptamer Based Impedance Biosensor for Monitoring Adenosine

A simple and highly sensitive electrochemical impedance spectroscopy (EIS) biosensor based on nano‐MnO₂ as a platform for the immobilization of the aptamer was developed for the determination of adenosine. In the measurement of adenosine, the change in interfacial electron transfer resistance (R_et) of the biosensor using a redox couple of [Fe(CN)₆]^3?/4? as the probe was monitored. The change of the electron transfer resistance (ΔR_et) of the biosensor was linear with the concentration of adenosine in the range from 1.0 nM to 100 nM. The fabricated sensor was shown to exhibit high sensitivity, desirable selectivity and good stability.     

MODELS FOR BACTERIAL PHOTOSYNTHESIS: ELECTRON TRANSFER FROM PHOTOEXCITED SINGLET BACTERIOPHEOPHYTIN TO METHYL VIOLOGEN AND m-DINITROBENZENE

Abstract. As a model for the primary reactions of photosynthesis, we studied photochemical electron transfer from bacteriopheophytin (BPh) to methyl viologen (MVC1₂) and to m-dinitrobenzene (m-DNB) in solution. Both MVC1₂ and m-DNB cause reductions in the lifetime of the first excited singlet state of BPh (BPh*), in the fluorescence quantum yield, and in the quantum yield of the triplet state, BPh ⁺. The quenching of BPh* probably results from electron transfer, which generates short-lived radical pairs involving the BPh radical cation (BPh⁺) and the reduced form of the quencher. Electron transfer from BPh* is thermodynamically favorable, but that from BPh^T is not. From the magnitude of the quenching, we calculate rate constants for electron transfer in collision complexes formed between BPh* and MVC1₂ or m-DNB. Measurements of the quantum yield of the free BPh⁺ radical indicate that about 3/4 of the [BPh⁺ MV⁺] radical pairs decay by reverse electron transfer, rather than dissociating to give the free radicals. Essentially all of the [BPh⁺m-DNB ⁺] radical pairs must decay by reverse electron transfer, because free BPh⁺ cannot be detected in this case. From these data, we estimate the rate constants for the reverse electron transfer reactions. The higher probability of dissociation in the [BPh⁺ MV⁺] radical pair can be explained by coulombic repulsion. The rate of the primary electron transfer reaction in photosynthetic bacteria is comparable to that of forward electron transfer in the BPh* collision complexes. Reverse electron transfer, however, is at least 10³-times slower in the radical pair formed in the bacterial reaction center than it is in [BPh⁺m-DNB^?], and more than 10⁴-times slower than in [BPh⁺ MV⁺]. The explanation for this dramatic and crucially important difference remains unclear, but several possibilities are discussed.     

The synthesis of novel 4-(3,4-dimethoxyphenyl)chromenone-crown ethers and their cation binding,as determined using fluorescence spectra

o-Dihydroxy-4-(3,4-dimethoxyphenyl)-chromenones (coumarins; 3a,b) were synthesised from 1,2,3-trihydroxy- or 1,2,4-triacetoxybenzenes through a reaction with ethyl 3-(3,4-dimethoxyphenyl)-3-oxopropanoate in H₂SO₄ or CF₃COOH. The chromenone-crown ethers (4a–f) were prepared from the cyclic condensation of o-dihydroxy-4-(3,4-dimethoxyphenyl)chromenones (3a,b) with poly(ethylene glycol) ditosylates, in the presence of CH₃CN/alkali carbonates. The chromatographically purified original chromenone-crown ethers were identified by ¹H NMR, ¹³C NMR, MALDI-TOF mass spectrometry and elemental analysis. The 1:1 binding constants of Li⁺, Na⁺ and K⁺ with the chromenone-crown ethers were estimated in acetonitrile using fluorescence emission spectroscopy. The complexing-enhanced fluorescence spectra and complexing-enhanced quenching fluorescence spectra, along with the cationic recognition rules of the crown ethers allowed the ion binding powers to be determined.     

Highly Emissive Diborylphenylene‐Containing Bis(phenylethynyl)benzenes: Structure–Photophysical Property Correlations and Fluoride Ion Sensing

A series of 2,5‐bis(dimesitylboryl)‐1,4‐bis(arylethynyl)benzenes 1 – 6 that contain various p‐substituents on the terminal benzene rings, including NPh₂ ( 1 ), OMe ( 2 ), Me ( 3 ), H ( 4 ), CF₃ ( 5 ), and CN ( 6 ) groups, were synthesized, and the effects of the p‐substituents on the absorption and fluorescence properties were investigated both in solution and in the solid state. Linear relationships were obtained not only between the Hammett σ_p⁺ constants of the p‐substituents and the absorption and fluorescence maxima, quantum yields, and excited‐state dynamics parameters in solution, but also between the σ_p⁺ constants and the fluorescence quantum yields in the solid state. An important finding extracted from these results is that the suppressed fluorescence quenching in the solid state is a common feature for the present laterally boryl‐substituted π‐conjugated skeletons. Hence, the diborylphenylene can serve as a useful core unit to develop highly emissive organic solids. In fact, most of the derivatives showed more intense emission in the solid state than in solution. In addition to these studies, the titration experiment of 1 by the addition of nBu₄NF was conducted, which showed the stepwise bindings of two fluoride ions with high association constants as well as a drastic change in the fluorescence spectra, while constantly maintaining high quantum yields (0.61–0.76), irrespective of the binding modes. This result also demonstrated the potential utility of the present molecules as an efficient fluorescent fluoride ion sensor.     

Synthesis,photophysical, electrochemical,and ion-binding properties of Ru(II) polypyridyl complexes containing benzo-15-crown-5

Two polypyridyl ligands, 5-(4′-ethynylbenzo-15-crown-5)-2,2′-bipyridine (L¹) and 3-bromo-8-(4′-ethynylbenzo-15-crown-5)-1,10-phenanthroline (L²), and their Ru(II) complexes [(bpy)₂RuL](PF₆)₂ have been prepared and characterized. Both complexes exhibit metal-to-ligand charge transfer absorption at around 452 nm and emission at around 640 nm in MeCN solution. Electrochemical studies of the complexes reveal a Ru(II)-centered oxidation at around 1.31 V and three ligand-centered reductions. The binding ability of the complexes with Na⁺ has been investigated by UV/Vis absorption, emission, and electrochemical titrations. Addition of Na⁺ to MeCN solutions of both complexes results in a progressive enhancement of the emission, a red-shift of the UV/Vis absorption, and a progressive cathodic shift of the Ru(II)-centered E _1/2 couple. The stability constants for the 1:1 stoichiometry adducts of the complexes with Na⁺ have been obtained from the UV/Vis absorption titrations.     

Exploring Reversible Quenching of Fluorescence from a Pyrazolo[3,4‐b]quinoline Derivative by Protonation

Pyrazolo[3,4‐b]quinoline derivatives are reported to be highly efficient organic fluorescent materials suitable for applications in light‐emitting devices. Although their fluorescence remains stable in organic solvents or in aqueous solution even in the presence of H₂O, halide salts (LiCl), alkali (NaOH) and weak acid (acetic acid), it suffers an efficient quenching process in the presence of protic acid (HCl) in aqueous or ethanolic solution. This quenching process is accompanied by a change in the UV spectrum, but it is reversible and can be fully recovered. Both steady‐state and transient fluorescence spectra of 1‐phenyl‐3,4‐dimethyl‐1H‐pyrazolo‐[3,4‐b]quinoline (PAQ5) during quenching are measured and analyzed. It is found that a combined dynamic and static quenching mechanism is responsible for the quenching processes. The ground‐state proton‐transfer complex [PAQ5 ??? H⁺] is responsible for static quenching. It changes linearly with proton concentration [H⁺] with a bimolecular association constant K_S=1.95 M ^?1 controlled by the equilibrium dissociation of HCl in ethanol. A dynamic quenching constant K_D=22.4 M ^?1 is obtained by fitting to the Stern–Volmer equation, with a bimolecular dynamic quenching rate constant k_d=1.03×10⁹ s^?1 M ^?1 under ambient conditions. A change in electron distribution is simulated and explains the experiment results.