Excited-state Deprotonation Research Articles

A cyanide selective colorimetric and turn-on fluorescent probe in solution and on test strips and its live cell imaging

The naphthol derivative (E)-N′-((2-hydroxynaphthalen-1-yl)methylene)-1H-indole-2-carbohydrazide (1) has been synthesized and characterized. Sensor 1 binds selectively with CN− ion in DMSO/H2O (7:3v/v) mixed solvent medium with significant change in its UV–vis spectrum and detection limit of 1.2μM. Sensor 1 shows dual emission bands at 335nm and 460nm due to naphthalene and excited state deprotonation of naphthalene OH group, respectively. Upon addition of CN− ion to 1, fluorescence is turned on for the emission band at 460nm with slight red-shift and fluorescence enhancement of 5.5 times. 1 undergoes binding followed by deprotonation of OH on titration with CN− ion, as supported by 1H NMR titration. Solution of 1 in DMSO/H2O (7:3v/v) exhibits considerable change in colour from colourless to yellow in visible light and green in long wavelength UV light (350nm) upon addition of CN− ion selectively. Same visual colour change of 1 is observed on Whatman filter paper testing strips when it is impregnated on testing strips and immersed in CN− ion solution (DMSO/H2O, 7:3v/v). 1 is used as a live cell imaging reagent for intra-cellular detection of CN− ion in A549 cells.

Reactions of 5-methylcytosine cation radicals in DNA and model systems: Thermal deprotonation from the 5-methyl group vs. excited state deprotonation from sugar

Purpose: To study the formation and subsequent reactions of the 5-methyl-2′-deoxycytidine cation radical (5-Me-2′-dC•+) in nucleosides and DNA-oligomers and compare to one-electron oxidized thymidine.Materials and methods: Employing electron spin resonance (ESR), cation radical formation and its reactions were investigated in 5-Me-2′-dC, thymidine (Thd) and their derivatives, in fully double-stranded (ds) d[GC*GC*GC*GC*]2 and in the 5-Me-C/A mismatched, d[GGAC*AAGC:CCTAATCG], where C* = 5-Me-C.Results: We report 5-Me-2′-dC•+ production by one-electron oxidation of 5-Me-2′-dC by Cl2•− via annealing in the dark at 155 K. Progressive annealing of 5-Me-2′-dC•+ at 155 K produces the allylic radical (C-CH2•). However, photoexcitation of 5-Me-2′-dC•+ by 405 nm laser or by photoflood lamp leads to only C3′• formation. Photoexcitation of N3-deprotonated thyminyl radical in Thd and its 5′-nucleotides leads to C3′• formation but not in 3′-TMP which resulted in the allylic radical (U-CH2•) and C5′• production. For excited 5-Me-2′,3′-ddC•+, absence of the 3′-OH group does not prevent C3′• formation. For d[GC*GC*GC*GC*]2 and d[GGAC*AAGC:CCTAATCG], intra-base paired proton transferred form of G cation radical (G(N1-H)•: C(+ H+)) is found with no observable 5-Me-2′-dC•+ formation. Photoexcitation of (G(N1-H)•:C(+ H+)) in d[GC*GC*GC*GC*]2 produced only C1′• and not the expected photoproducts from 5-Me-2′-dC•+. However, photoexcitation of (G(N1-H)•:C(+ H+)) in d[GGAC*AAGC:CCTAATCG] led to C5′• and C1′• formation.Conclusions: C-CH2• formation from 5-Me-2′-dC•+ occurs via ground state deprotonation from C5-methyl group on the base. In the excited 5-Me-2′-dC•+ and 5-Me-2′,3′-ddC•+, spin and charge localization at C3′ followed by deprotonation leads to C3′• formation. Thus, deprotonation from C3′ in the excited cation radical is kinetically controlled and sugar C-H bond energies are not the only controlling factors in these deprotonations.

Properties of 2,6-diamino-8-azapurine, a highly fluorescent purine analog and its N-alkyl derivatives: Tautomerism and excited-state proton transfer reactions

Abstract Fluorescence emission properties of 2,6-diamino-8-azapurine (2-amino-8-azaadenine) in aqueous medium were examined. The title compound exhibits strong fluorescence centered at 365 nm, with quantum yield 0.40 and bi-exponential decay with lifetimes of ∼7.5 and ca. 0.2 ns. The spectral properties of 2,6-diamino-8-azaapurine are very similar to those of its 9-alkoxyphosphonate, suggesting that the protomer responsible for the emission of the former must be identified as N(9)H form. In acidic media (pH ∼ 2.5), fluorescence of the title compound is shifted to 410 nm, and is very similar to that of the neutral form of the N8-methyl derivative, the latter emitting at 410 nm with yield 0.85 and lifetime 12.7 ns. The N8-methyl derivative in the moderately acidic media undergoes rapid excited-state deprotonation, with p K * calculated to be as low as −2, compared to p K a 4.8 in the ground state, and resulting in the presence of the 410 nm fluorescence band well below the ground-state p K a . We therefore propose that the 410 nm band observed in the fluorescence of 2,6-diamino-8-azapurine cation is the consequence of (a) shift in tautomerism upon protonation from N(9)H toward N(8)H form and (b) rapid excited-state deprotonation of the latter. This conclusion is supported by the study of solvent, isotope and buffer concentration effects.

A guanidine derivative of naphthalimide with excited-state deprotonation coupled intramolecular charge transfer properties and its application

A new fluorophore based on guanidine substituted 1,8-naphthalimide was synthesized and characterized. In aqueous solution, the guanidine group undergoes deprotonation/protonation with a pKa of ∼8.5 in the ground-state and ∼0.9 in the excited-state. The emission of its protonated and deprotonated forms exhibits a large Stokes shift (Ex/Em: 350/460 nm and 400/580 nm) due to the excited-state intramolecular charge transfer (ICT) process. The protonated form of this fluorophore exhibits dual fluorescence emission (Em: 460 and 580 nm; Ex: 350 nm) that is contributed to by an excited-state deprotonation coupled ICT process. The emission properties of this fluorophore are strongly dependent on the solvent environment, which make it possible to tune the luminescence of the materials made using this fluorophore. The absorption and emission spectra of this fluorophore respond to fluoride ions ratiometrically, showing the potential application as a fluoride ion sensor.

Intraprotein Formation of a Long Wavelength Absorbing Complex and Inhibition of Excited-State Deprotonation in a Chiral Hydroxybiphenyl

The two enantiomers of 2-(2-hydroxybiphenyl-4-yl)propanoic acid ((S)- and (R)-BPOH) have been selected as probes for human serum albumin (HSA). Photophysical characterization in the absence of protein led to emission maxima, singlet energies, quantum yields, and fluorescence lifetime values of 332 nm, 91 kcal mol(-1), and 0.28 and 1.8 ns for BPOH or 414 nm, 79 kcal mol(-1), and 0.31 and 3.3 ns for the corresponding phenolate BPO(-); the pK(a)* was found to be 1.17. In the presence of HSA, a light-absorbing ground-state complex (S)-BPOH@HSA was detected (maximum at ca. 300 nm) whose intensity increased with increasing protein concentration. The fluorescence spectra of (S)-BPOH in PBS, after addition of HSA, revealed a progressive diminution of the phenolate band, indicating that excited-state deprotonation is disfavored within the hydrophobic protein cavities. A similar trend was observed for (R)-BPOH, but the extent of deprotonation was significantly lower for this enantiomer. Addition of increasing amounts of the site II displacement probe (S)-ibuprofen ((S)-IBP) to BPOH@HSA led to a significant decrease of the absorption maximum at ca. 300 nm and to a recovery of the phenolate emission band at ca. 410 nm, which were again configuration dependent. The transient absorption spectrum of (S)-BPOH consisted on a broad band centered at 380 nm, attributed to the first triplet excited state. A dramatic enhancement of the triplet lifetimes within HSA was observed (19.0 μs within protein versus 1.3 μs in bulk PBS), although no stereodifferentiation was noticed in this case.

6-Hydroxyindole-based borondipyrromethene: Synthesis and spectroscopic studies

A 6-hydroxyindole-based BODIPY, named BODIPY-OH, with distinct spectroscopic characteristics is reported. Through a systematic study of the spectroscopic characteristics of BODIPY-OH and BODIPY-O(-) in various solvents containing an organic base, we found that the light-color of the fluorophore can be tuned over a wide range by changing the polarity of solvent/base combinations. The absorption color of the solution can be tuned over a range of 100 nm and the emission color within a wide range from 571 to 681 nm by simply converting the phenol form of BODIPY-OH to the phenolate form. Fluorescence of BODIPY-O(-) with high quantum yield shows relatively large Stokes shift in solvent/base combinations, which are ascribed to the excited state deprotonation from (BODIPY-OH)* to (BODIPY-O(-))*, followed by emission from the ion form.

Importance of Electrostatic Interactions in The Mobility of Cations in Nafion

A molecular level understanding of the mobility of cations in the Nafion membrane has been attempted, using the excited state proton transfer (ESPT) process in the fluorescent probe Coumarin 102. ESPT is hindered significantly upon decreasing the water content. Using TRANES (time-resolved area normalized emission spectroscopy), the evolution of the ESPT state is clearly observed over hundreds of picoseconds in lower water content, implying that ESPT is hindered even in the nanovolume probed by the dye. Most remarkably, in the partially dried membrane, the predominant fluorescent species is the zwitterionic form, generated by excited state deprotonation of the cationic form. This implies that the molecule loses a proton from its nitrogen center in the excited state, as usual, but cannot recapture it readily at the oxygen center, at low water contents. This phenomenon is rationalized in light of an increased electrostatic attraction that is experienced by cations upon drying.

Fast Excited-State Deactivation in N(5)-Ethyl-4a-hydroxyflavin Pseudobase

We present a study of the excited-state behavior of N(5)-ethyl-4a-hydroxyflavin (Et-FlOH), a model compound for bacterial bioluminescence. Using femtosecond pump-probe spectroscopy, we found that the Et-FlOH excited state exhibits multiexponential dynamics, with the dominant decay component having a 0.5 ps lifetime. Several possible mechanisms for fast excited-state decay in Et-FlOH were considered: (i) excited-state deprotonation of the -OH proton, (ii) thermal deactivation via (1)n,π* → (1)π,π* conical intersection, and (iii) excited-state release of OH(-) ion. These mechanisms were excluded based on transient absorption studies of two model compounds (N(5)-ethyl-4a-methoxyflavin, Et-FlOMe, and N(5)-ethyl-flavinium ion, Et-Fl(+)) and based on the results of time-dependent density functional theory (TD-DFT) calculations of Et-FlOH excited-states. Instead, we propose that the fast decay in Et-FlOH is caused by S(1) → S(0) internal conversion, initiated by the excited-state nitrogen planarization (sp(3) → sp(2) hybridization change at the N(5)-atom of Et-FlOH S(1) state) coupled with out-of-plane distortion of the pyrimidine moiety of flavin.

Antiaromatic Dianions: Dianions of Dixanthylidene by Reduction and Attempted Excited-State Deprotonation

Reduction of dixanthylidene with potassium or lithium resulted in formation of the antiaromatic dianion in high yield. Attempts to form the dianion by excited-state deprotonation of dixanthene with n-butyllithium/TMEDA resulted in formation of the tetraanion from deprotonation ortho to the oxygen. Orientation of the sp(3) hydrogens presumably allows preferential deprotonation of the xanthene rings.

Fluorescence quenching of naphthols by Cu2+ in micelles

Effect of the micelles of anionic, cationic and non-ionic surfactants on the fluorescence quenching of 1- and 2-naphthols has been studied in the presence of copper ion. The excited state lifetime, dynamic and static quenching constants for these systems have been determined. Fluorescence quenching in water and SDS micelle is due to the collision of the fluorophore with the quencher with a small static component. The negatively charged naphtholate ions in the excited state are quenched with significantly higher rates than the neutral naphthol molecules, which are located further inside the mesophase. CTAB micelle is less effective than the SDS micelle for fluorescence quenching. The effect of CTAB on water-assisted excited-state deprotonation has been investigated in the presence of ZnSO4. For TX-100 micelle there is negligible quenching even at higher concentration of the quencher.

Proton-transfer and hydrogen-bond interactions determine fluorescence quantum yield and photochemical efficiency of bacteriophytochrome

Phytochromes are red-light photoreceptor proteins that regulate a variety of responses and cellular processes in plants, bacteria, and fungi. The phytochrome light activation mechanism involves isomerization around the C15 horizontal lineC16 double bond of an open-chain tetrapyrrole chromophore, resulting in a flip of its D-ring. In an important new development, bacteriophytochrome (Bph) has been engineered for use as a fluorescent marker in mammalian tissues. Here we report that an unusual Bph, RpBphP3 from Rhodopseudomonas palustris, denoted P3, is fluorescent. This Bph modulates synthesis of light-harvesting complex in combination with a second Bph exhibiting classical photochemistry, RpBphP2, denoted P2. We identify the factors that determine the fluorescence and isomerization quantum yields through the application of ultrafast spectroscopy to wild-type and mutants of P2 and P3. The excited-state lifetime of the biliverdin chromophore in P3 was significantly longer at 330-500 ps than in P2 and other classical phytochromes and accompanied by a significantly reduced isomerization quantum yield. H/D exchange reduces the rate of decay from the excited state of biliverdin by a factor of 1.4 and increases the isomerization quantum yield. Comparison of the properties of the P2 and P3 variants shows that the quantum yields of fluorescence and isomerization are determined by excited-state deprotonation of biliverdin at the pyrrole rings, in competition with hydrogen-bond rupture between the D-ring and the apoprotein. This work provides a basis for structure-based conversion of Bph into an efficient near-IR fluorescent marker.

The “Wiggling and Jiggling of Atoms” Leading to Excited‐State Proton Transfer in Green Fluorescent Protein

Shaking off the blues: Femtosecond stimulated Raman spectroscopy reveals vibrational dynamics accompanying excited state deprotonation of the GFP chromophore, which causes a color change from blue to green (see picture).

Highly Sensitive Water-Soluble Fluorescent pH Sensors Based on the 7-Amino-1-methylquinolinium Chromophore

Highly sensitive water-soluble fluorescent pH sensors have been synthesized employing the 7-amino-1-methylquinolinium (7AMQ) chromophore. These compounds, which contained oligomethylene spacers and substituted amine receptor units, were prepared in high yields and purity by a single reaction from the readily available 7-fluoro-1-methylquinolinium iodide. Density functional theory (DFT) and semiempirical INDO/s calculations have been performed to describe the ground state and the locally excited state of the chromophore. The photophysics and the sensor characteristics have been investigated. Dissociation constants pK(A)* ranging from 5.8 to 9.9 have been obtained. An additional quenching process at pH 11, due to excited state deprotonation at N7, has been observed. Fluorescence quantum yields in the protonated "on-state" between 0.75 and 0.85 and fluorescence enhancements (FE) between 2 and 55 were determined. These values are significantly higher than those of molecules based on other CT-chromophores that contain identical spacer-receptor units. The high fluorescence enhancements may be explained by the low rate of fluorescence ( approximately 6 x 10(7) s(-1)) and the high excited state reduction potential ( approximately 1.6 eV) of the 7AMQ chromophore.

Proton Transfer and Hydrogen-Bond Interactions Determine the Fluorescence Quantum Yield of Bacteriophytochrome, a Novel Deep-Tissue Fluorescent Probe

Phytochromes are red-light photoreceptors that regulate a variety of responses and cellular processes. The phytochrome light activation mechanism involves isomerization around the C15=C16 double bond of an open-chain tetrapyrrole chromophore, resulting in a flip of its D-ring. In an important new development, bacteriophytochrome (Bph) has been engineered for use as a fluorescent marker in mammalian tissues (Shu et al. Science 2009). Bph fluoresces at ∼720 nm, a wavelength less prone to scattering that can penetrate more deeply into tissue than light emitted by GFP-derived fluorescent proteins. The Bph chromophore biliverdin is a naturally occurring cofactor in mammalian tissue that covalently binds to a conserved cysteine in Bph, and hence BPhs can readily be genetically encoded. BPh photochemistry is thus of considerable significance for biomedical technology. Here we report that an unusual Bph, P3 from Rps. palustris, is highly fluorescent. We identify the factors that determine the fluorescence and isomerization quantum yields of P3 through the application of ultrafast spectroscopy to wild-type and mutants of P3 and a classical Bph, P2. The excited-state lifetime of biliverdin in P3 was significantly longer at 330 - 500 ps than in P2, and accompanied by a significantly reduced isomerization quantum yield. H/D exchange reduces the rate of decay from the biliverdin excite state by a factor of 1.4 and increases the isomerization quantum yield. Comparison of the properties of the P2 and P3 variants in relation to X-ray structures shows that the quantum yields of fluorescence and isomerization are determined by excited-state deprotonation of biliverdin at the pyrrole rings, in competition with hydrogen-bond rupture between the biliverdin D-ring and the apoprotein. This work provides a basis for structure-based conversion of BPh into an efficient near-IR fluorescent marker.

Primary Photoprocesses Involved in the Sensory Protein for the Photophobic Response ofBlepharisma japonicum

We present new femtosecond transient-absorption and picosecond fluorescence experiments performed on OBIP, the oxyblepharismin-binding protein believed to trigger the photophobic response of the ciliate Blepharisma japonicum. The formerly identified heterogeneity of the sample is confirmed and rationalized in terms of two independent populations, called rOBIP and nrOBIP. The rOBIP population undergoes a fast photocycle restoring the initial ground state in less than 500 ps. Intermolecular electron transfer followed by electron recombination is identified as the excited-state decay route. The experimental results support the coexistence of the oxyblepharismin (OxyBP) radical cation signature with a stimulated-emission signal at all times of the evolution of the transient-absorption spectra. This observation is interpreted by an equilibrium being reached between the locally excited state and a charge-transfer state on the ground of a theory developed by Mataga and co-workers to explain the fluorescence quenching of aromatic hydrogen-bonded donor-acceptor pairs in nonpolar solvents. OxyBP is supposed to bind to an as yet unknown electron acceptor by a hydrogen-bond (HB) and the coordinate along which forward and backward electron transfer proceed is assumed to be the shift of the HB proton. The observed kinetic isotope effect supports this interpretation. Protein relaxation is finally proposed to accompany the whole process and give rise to the highly multiexponential observed dynamics. As previously reported, the fast photocycle of rOBIP can be interpreted as an efficient sunscreen mechanism that protects Blepharisma japonicum from continuous irradiation. The nrOBIP population, the transient-absorption of which strongly reminds that of free OxyBP in solution, might be proposed to actually trigger the photophobic response of the organism through excited-state deprotonation of the chromophore occurring in the nanosecond regime. Additional femtosecond transient-absorption spectra of OxyBP and peri-deprotonated OxyBP are also reported and used as a comparison basis to interpret the results on OBIP.

Puerarin as an antioxidant fluorescence probe

Diphenolic isoflavonoid puerarin fluoresces in aqueous solution with maximal intensity at pH 8.5 ( Φ fl = 0.042, τ fl = 1.91 ns). For acidic solutions, weak fluorescence is attributed to fluorescent 7-monophenolate formed via excited-state deprotonation of neutral puerarin. For pH > 8.5, fluorescence decreases monotonically with an unchanged lifetime, suggesting that excited-state acidity of 4′-hydroxyl remains similar to the ground-state one, and that the diphenolate is non-fluorescent. The crucial role of A-ring 7-phenolate for fluorescence of puerarin is substantiated by absence (presence) of fluorescence for the 7-propylpuerarin (4′-propylpuerarin). Puerarin and its derivatives with the unusual properties may be explored to be antioxidant fluorescence probes.

Photo-induced proton-transfer cycle of 2-naphthol in faujasite zeolitic nanocavities

The excited-state deprotonation and ground-state reprotonation of a 2-naphthol molecule encapsulated in the zeolitic nanocavity of NaX have been studied by measuring static and time-resolved spectra of fluorescence and reflectance. The excited molecule undergoes enol dissociation within 300 ps to form an isolated ion pair, which undergoes geminate recombination in 1200 ps or separation to produce the anionic species of 2-naphtholate on the time scale of 2500 ps. Ground-state reprotonation, controlled by the diffusion rate of a proton, is then followed in 0.8 ms with an activation energy of 13 kJ mol(-1).

Observation of slow charge redistribution preceding excited-state proton transfer

The photoacid 8-hydroxy-N,N,N',N',N',N'-hexamethylpyrene-1,3,6-trisulfonamide (HPTA) and related compounds are used to investigate the steps involved in excited-state deprotonation in polar solvents using pump-probe spectroscopy and time correlated single photon counting fluorescence spectroscopy. The dynamics show a clear two-step process leading to excited-state proton transfer. The first step after electronic excitation is charge redistribution occurring on a tens of picoseconds time scale followed by proton transfer on a nanosecond time scale. The three states observed in the experiments (initial excited state, charge redistributed state, and proton transfer state) are recognized by distinct features in the time dependence of the pump-probe spectrum and fluorescence spectra. In the charge redistributed state, charge density has transferred from the hydroxyl oxygen to the pyrene ring, but the OH sigma bond is still intact. The experiments indicate that the charge redistribution step is controlled by a specific hydrogen bond donation from HPTA to the accepting base molecule. The second step is the full deprotonation of the photoacid. The full deprotonation is clearly marked by the growth of stimulated emission spectral band in the pump-probe spectrum that is identical to the fluorescence spectrum of the anion.

Proton Transport and the Water Environment in Nafion Fuel Cell Membranes and AOT Reverse Micelles

The properties of confined water and diffusive proton-transfer kinetics in the nanoscopic water channels of Nafion fuel cell membranes at various hydration levels are compared to water in a series of well-characterized AOT reverse micelles with known water nanopool sizes using the photoacid pyranine as a molecular probe. The side chains of Nafion are terminated by sulfonate groups with sodium counterions that are arrayed along the water channels. AOT has sulfonate head groups with sodium counterions that form the interface with the reverse micelle's water nanopool. The extent of excited-state deprotonation is observed by steady-state fluorescence measurements. Proton-transfer kinetics and orientational relaxation are measured by time-dependent fluorescence using time-correlated single photon counting. The time dependence of deprotonation is related to diffusive proton transport away from the photoacid. The fluorescence reflecting the long time scale proton transport has an approximately t-0.8 power law decay in contrast to bulk water, which has a t-3/2 power law. For a given hydration level of Nafion, the excited-state proton transfer and the orientational relaxation are similar to those observed for a related size AOT water nanopool. The effective size of the Nafion water channels at various hydration levels are estimated by the known size of the AOT reverse micelles that display the corresponding proton-transfer kinetics and orientational relaxation.

Synthesis of 7-Azaserotonin: Its Photophysical Properties Associated with Excited State Proton Transfer Reaction

We report the synthesis of 3-(2-aminoethyl)-5-ol-1H-pyrrolo[2,3-b]pyridine (7-azaserotonin), which may potentially serve as an agonist or antagonist of serotonin receptors. In alcohols, the solvent (e.g., ethanol) catalyzed proton-transfer reaction takes place for 7-azaserotonin in the excited state, resulting in dual emission. Conversely, excited-state deprotonation takes place in neutral aqueous solution. The unique excitation behavior makes 7-azaserotonin versatile as a potential bioprobe.