Fluorescence of cis-1-amino-2-(3-indolyl)cyclohexane-1-carboxylic acid: a single tryptophan chi(1) rotamer model

A constrained derivative, cis-1-amino-2-(3-indolyl)cyclohexane-1-carboxylic acid, cis-W3, was designed to test the rotamer model of tryptophan photophysics. The conformational constraint enforces a single chi(1) conformation, analogous to the chi(1) = 60 degrees rotamer of tryptophan. The side-chain torsion angles in the X-ray structure of cis-W3 were chi(1) = 58.5 degrees and chi(2) = -88.7 degrees. Molecular mechanics calculations suggested two chi(2) rotamers for cis-W3 in solution, -100 degrees and 80 degrees, analogous to the chi(2) = +/-90 degrees rotamers of tryptophan. The fluorescence decay of the cis-W3 zwitterion was biexponential with lifetimes of 3.1 and 0.3 ns at 25 degrees C. The relative amplitudes of the lifetime components match the chi(2) rotamer populations predicted by molecular mechanics. The longer lifetime represents the major chi(2) = -100 degrees rotamer. The shorter lifetime represents the minor chi(2) = 80 degrees rotamer having the ammonium group closer to C4 of the indole ring (labeled C5 in the cis-W3 X-ray structure). Intramolecular excited-state proton transfer occurs at indole C4 in the tryptophan zwitterion (Saito, I.; Sugiyama, H.; Yamamoto, A.; Muramatsu, S.; Matsuura,T. J. Am. Chem. Soc. 1984, 106, 4286-4287). Photochemical isotope exchange experiments showed that H-D exchange occurs exclusively at C5 in the cis-W3 zwitterion, consistent with the presence of the chi(2) = 80 degrees rotamer in solution. The rates of two nonradiative processes, excited-state proton and electron transfer, were measured for individual chi(2) rotamers. The excited-state proton-transfer rate was determined from H-D exchange and fluorescence lifetime data. The excited-state electron-transfer rate was determined from the temperature dependence of the fluorescence lifetime. The major quenching process in the -100 degrees rotamer is electron transfer from the excited indole to carboxylate. Electron transfer also occurs in the 80 degrees rotamer, but the major quenching process is intramolecular proton transfer. Both quenching processes are suppressed by deprotonation of the amino group. The results for cis-W3 provide compelling evidence that the complex fluorescence decay of the tryptophan zwitterion originates in ground-state heterogeneity with the different lifetimes primarily reflecting different intramolecular excited-state proton- and electron-transfer rates in various rotamers.     

2',7'-Difluorofluorescein excited-state proton reactions: correlation between time-resolved emission and steady-state fluorescence intensity

The presence of excited-state buffer-mediated proton exchange reactions influences the steady-state fluorescence signals from dyes in solution. Since biomolecules in general have some chemical groups that can act as proton acceptors/donors and are usually dissolved in buffer solutions which can also behave as appropriate proton acceptors/donors, the excited-state proton exchange reactions may result in distorted steady-state fluorescence signals. In a previous paper (J. Phys. Chem. A 2005, 109, 734-747), we evaluated kinetic and other pertinent parameters for the excited-state proton reactions of the prototropic forms of 2',7'-difluorofluorescein (Oregon Green 488, OG488), recording a fluorescence decay surface at different pH values and acetate buffer concentrations, analyzed by means of global compartmental analysis. In this article we use the rate constants and the corrected pre-exponential factors from the previously recorded fluorescence decay traces to simulate the decay times and associated pre-exponentials at different acetate buffer concentrations and constant pH and compare these theoretically calculated values with new experimental data. We also calculate the steady-state fluorescence intensity vs pH and vs acetate buffer concentration (at constant pH) and compare these calculated emission values with the experimental data previously published. The agreement between the experimental and simulated data is excellent.     

Excited-state proton transfer of 3-hydroxybenzoic acid and 4-hydroxybenzoic acid

The excited-state proton transfer of 3-hydroxybenzoic acid and 4-hydroxybenzoic acid was studied by time-resolved laser-induced fluorescence spectroscopy with ultra-short laser pulses. The excited-state reactions were identified in aqueous media as a function of the pH value. Apart from the well-known inversion of the ordinary dissociation properties of these compounds, new species were found which exist only in the excited-state resulting from a temporal and reversible annihilation of the aromatic bond system. These species and their reaction mechanisms were detected by their absorption and fluorescence spectra.     

Excited-state proton-relay dynamics of 7-hydroxyquinoline controlled by solvent reorganization in room temperature ionic liquids

The excited-state triple proton relay of 7-hydroxyquinoline (7HQ) along a hydrogen-bonded methanol chain in room temperature ionic liquids (RTILs) has been investigated using picosecond time-resolved fluorescence spectroscopy. The rate constant of the proton relay in a methanol-added RTIL is found to be slower by an order of magnitude than that in bulk methanol and to have unity in its kinetic isotope effect. These suggest that the excited-state tautomerization dynamics of 7HQ in methanol-added RTILs is mainly controlled by the solvent reorganization dynamics to form a cyclically hydrogen-bonded complex of 7HQ·(CH(3)OH)(2) upon absorption of a photon due to high viscosity values of RTILs. Because the cyclic complex of 7HQ·(CH(3)OH)(2) at the ground state is unstable in RTILs, the collision-induced slow formation of the cyclic complex should take place upon excitation prior to undergoing subsequent intrinsic proton transfer rapidly.     

Proton translocation and electronic relaxation along a hydrogen-bonded molecular wire in a 6-hydroxyquinoline/acetic acid complex

A hydrogen-bonded network formed between 6-hydroxyquinoline (6-HQ) and acetic acid (AcOH) has been characterized using a time-resolved fluorescence technique. In the bridged hydrogen-bonded complex of cis-6-HQ and AcOH, an excited-state reaction proceeds via proton transfer along the hydrogen bond, resulting in a keto-tautomer (within approximately 200 ps) that exhibits large Stokes-shifted fluorescence. The unbridged complex also undergoes excited-state proton transfer, but the Stokes shift is rather smaller.     

A New Series of Proton/Charge Transfer Molecules: Synthesis and Spectral Studies of 2-(5-Aryl-1-carbomethoxy-1H-pyrazol-3-yl)phenols

Abstract. The carbomethoxyhydrazone of 2'-hydroxyacetophenone was trilithiated with excess lithium diisopropylamide and C-acylated with a variety of benzoate esters followed by acid cyclization of the intermediates to 2-(5-aryl-l-car-bomethoxy-lHpyrazol-3-yl)phenols [3-(2-hydroxyphen-yl)-lH-pyrazoles]. The products were characterized by Fourier transform-IR, ^lH NMR, ¹³C NMR, UV-visible absorption and fluorescence. All the derivatives in n -heptane have an absorption maximum at ˜304 nm and an extremely weak (φ_r= 10 ⁴) fluorescence with maxima in the range of 335–460 nm. The broad range of fluorescence maxima and fluorescence quantum yields is attributed to varying contributions of charge transfer that are dependent on both the identity of the substituent and solvent polarity. A phenomenally large Stokes-shifted fluorescence maximum at 620 nm was observed for 2-(l-car-bomethoxy-5-[4-dimethylaminophenyl]-lff-pyrazol-3-y1)phenol in n-heptane and attributed to excited-state intramolecular proton transfer. As a result, competitive excited-state proton/charge transfer properties have been observed in the pyrazoles studied, of which the spectral properties can be fine tuned by substituent as well as solvent effects.     

Photophysics of Hypericin and Hypocrellin A in Complex with Subcellular Components: Interactions with Human Serum Albumin

Time-resolved fluorescence and absorption measurements are performed on hypericin complexed with human serum albumin, HSA (1:4, 1:1 and approximately 5:1 hypericin: HSA complexes). Detailed comparisons with hypocrellin A/HSA complexes (1:4 and 1:1) are made. Our results are consistent with the conclusions of previous studies indicating that hypericin binds to HSA by means of a specific hydrogen-bonded interaction between its carbonyl oxygen and the N1-H of the tryptophan residue in the IIA subdomain of HSA. (They also indicate that some hypericin binds nonspecifically to the surface of the protein.) A single-exponential rotational diffusion time of 31 ns is measured for hypericin bound to HSA, indicating that it is very rigidly held. Energy transfer from the tryptophan residue of HSA to hypericin is very efficient and is characterized by a critical distance of 94 A, from which we estimate a time constant for energy transfer of approximately 3 x 10(-15) s. Although it is tightly bound to HSA, hypericin is still capable of executing excited-state intramolecular proton (or hydrogen atom) transfer in the approximately 5:1 complex, albeit to a lesser extent than when it is free in solution. It appears that the proton transfer process is completely impeded in the 1:1 complex. The implications of these results for hypericin (and hypocrellin A) are discussed in terms of the mechanism of intramolecular excited-state proton transfer, the mode of binding to HSA and the light-induced antiviral and antitumor activity.     

Interactions of pyridinecarboxylic acid chelators with brain metal ions: Cu(II), Zn(II), and Al(III)

Abstract  

The interactions of Cu(II), Zn(II), and Al(III) with 1,6-dimethyl-4-hydroxy-3-pyridinecarboxylic acid (DQ716) and 2,6-dimethyl-3-hydroxy-4-pyridinecarboxylic acid (DT726), possible chelating agents in Alzheimer’s disease, were investigated in aqueous solution. The proton dissociation constants of the ligands, the stability constants, and the coordination modes of the metal complexes formed were determined by pH-potentiometric, UV–vis spectrophotometric, and ¹H NMR methods. The nitrogen of the pyridine ring changes the proton affinity of the carboxylate and phenolate moieties and these pyridine derivatives form stronger complexes with Cu(II), Zn(II), and Al(III) than salicylic acid. Interactions of the ligands with human serum albumin as their potential transporter in blood were investigated at physiological pH through ultrafiltration by UV–vis and fluorescence spectroscopy.     

First fluorescence spectroscopic investigation of Am(III) complexation with an organic carboxylic ligand, pyromellitic acid

For the first time Am(III) complexation with a small organic ligand could be identified and characterized with time-resolved laser-induced fluorescence spectroscopy (TRLFS) at room temperature and trace metal concentration. With pyromellitic acid (1,2,4,5-benzene-tetracarboxylic acid, BTC) as ligand spectroscopic characteristics for the Am-BTC complex system were determined at pH 5.0, an ionic strength of 0.1 M (NaClO4) and room temperature. The fluorescence lifetimes were determined to be 23.2±2.2 ns for Am3+(aq) and 27.2±1.2 ns for the Am-BTC 1:1 complex; the emission maximum for the 5D1-(7)F1 transition is 691 nm for both species. The complex stability constant for the Am-BTC 1:1 complex was calculated to be logβ110=5.42±0.16.     

Multiband fluorescence spectral properties of QMOM

The spectral characteristics of the 1-methyl-2-(4-methoxyphenyl)-3-hydroxy-4(1H)-quinolone (QMOM) dye with dual fluorescence in acetonitrile were studied under selective excitation in a wide temperature range. This dye is a structural analog of 3-hydroxyflavone and exhibits excited-state proton transfer, which forms a fluorescent tautomeric form, while the solution is characterized by dual fluorescence. The thermal behavior of the relative band intensities revealed the kinetic character of the proton transfer. The third form showed itself as a maximum between the bands of the normal and tautomeric forms upon excitation in several regions of the absorption spectrum and became dominant in solution at 60–80°C. The characteristics of the third form were studied. Additional experiments showed that this was possibly the anionic form of the dye.     

Absorption and emission spectroscopic characterization of lumichrome in aqueous solutions

The spectroscopic behavior of lumichrome (7,8-dimethyl-alloxazine, LC) in aqueous solutions in a pH range from -1.08 to 14.6 is studied. Absorption spectra, fluorescence quantum distributions, quantum yields, and lifetimes are determined. The ionization stage of ground-state LC changes with rising pH from the cationic form (LCH(2)(+)) to the neutral form (LCH) with a mid-point pH of pK(c) ≈ -0.53, and to the anionic form (LC(-)) with a mid-point pH of pK(a) ≈ 12.5. Above pH 7 a partial ground-state tautomerization of LCH to 7,8-dimethyl-isoalloxazine (IAH) occurs by N1-N10 intra-molecular proton transfer. For pH > pK(a) ≈ 12.5 LCH and IAH change to the anionic forms LC(-) and IA(-), and above pH 14 LC(-) tautomerizes completely to IA(-). In the excited state some neutral lumichrome (LCH*) converts to cationic lumichrome (LCH(2)(+)) at low pH by proton transfer from H(3)O(+) to LCH*. No photoinduced excited-state tautomerization of lumichrome was observed. LCH for pH > 3 and IAH are reasonably fluorescent. The fluorescence efficiencies of LC(-) and IA(-) are lower than those of LCH and IAH. The fluorescence of LCH(2)(+) is strongly quenched likely by intra-molecular diabatic charge transfer and excited-state relaxation by potential surface touching with the ground state.     

Photophysics of the fluorescent pH indicator BCECF

The photophysical behavior of BCECF [2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein]--currently the most widely used fluorescent pH indicator for near-neutral intracellular pH measurements--has been explored by using absorption and steady-state and time-resolved fluorescence measurements. The influence of ionic strength as well as total buffer concentration on the absorbance and steady-state fluorescence has been investigated. The apparent acidity constant of the pH indicator determined by absorbance and fluorescence titration is dependent on the added buffer and salt concentrations. A semiempirical model is proposed to rationalize the variations in the apparent pKa values. The excited-state proton exchange of BCECF at physiological pH becomes reversible upon addition of phosphate buffer, inducing a pH-dependent change of the fluorescence decay times. Fluorescence decay traces collected as a function of total buffer concentration and pH were analyzed by global compartmental analysis yielding the following values of the rate constants describing excited-state dynamics of BCECF: k01 = 3.4 x 10(8) s(-1), k02 = 2.6 x 10(8) s(-1), k21 approximately 1 x 10(6) M(-1) s(-1), k12(B) = 1.4 x 10(8) M(-1) s(-1), and k21(B) = 4.3 x 10(7) M(-1) s(-1).     

Speciation, luminescence, and alkaline fluorescence quenching of 4-(2-methylbutyl)aminodipicolinic acid (H2MEBADPA)

4-(2-Methylbutyl)aminodipicolinic acid (H(2)MEBADPA) has been synthesized and fully characterized in terms of aqueous phase protonation constants (pK(a)'s) and photophysical measurements. The pK(a)'s were determined by spectrophotometric titrations, utilizing a fully sealed titration system. Photophysical measurements consisted of room temperature fluorescence and frozen solution phosphorescence as well as quantum yield determinations at various pH, which showed that only fully deprotonated MEBADPA(2-) is appreciably emissive. The fluorescence of MEBADPA(2-) has been determined to be quenched by hydroxide and methoxide anions, most likely through base-catalyzed excited-state tautomerism or proton transfer. This quenching phenomenon has been quantitatively explored through steady-state and time-resolved fluorescence measurements. Utilizing the determined pK(a)s and quenching constants, the fluorescent intensity of MEBADPA(2-) has been successfully modeled as a function of pH.     

Competition between energy and proton transfer in ultrafast excited-state dynamics of an oligomeric fluorescent protein red Kaede

We investigated femtosecond and picosecond time-resolved fluorescence dynamics of a tetrameric fluorescent protein Kaede with a red chromophore (red Kaede) to examine a relationship between the excited-state dynamics and a quaternary structure of the fluorescent protein. Red Kaede was obtained by photoconversion from green Kaede that was cloned from a stony coral Trachyphyllia geoffroyi. In common with other typical fluorescent proteins, a chromophore of red Kaede has two protonation states, the neutral and the anionic forms in equilibrium. Time-resolved fluorescence measurements clarified that excitation of the neutral form gives the anionic excited state with a time constant of 13 ps at pH 7.5. This conversion process was attributed to fluorescence resonance energy transfer (FRET) from the photoexcited neutral form to the ground-state anionic form that is located in an adjacent subunit in the tetramer. The time-resolved fluorescence data measured at different pH revealed that excited-state proton transfer (ESPT) also occurs with a time constant of 300 ps and hence that the FRET and ESPT take place simultaneously in the fluorescent protein as competing processes. The ESPT rate in red Kaede was significantly slower than the rate in Aequorea GFP, which highly likely arises from the different hydrogen bond network around the chromophore.     

Acid-Base Properties of the Ground and Excited States of Ruthenium(II) Tris(4′-methyl-2,2′-bipyridine-4-carboxylic Acid)

The acid dissociation constant, pK_a, for the ground and excited states of ruthenium tris(4′-methyl-2,2′-bipyridine-4-carboxylic acid) complex have been measured. The ground state pK_a obtained from the pH titration curve of the complex absorption at 454 nm was 2.5. The lifetimes of the excited-state for deprotonated and protonated ruthenium complexes are 595 and 150 ns, respectively. The excited-state pK_a* is obtained from the emission titration curve at 630 nm and corrected for the excited-state lifetime to be 4.2. The increase of 1.7 pH units in the acid dissociation constant in the excited-state indicates that the ligand is much more basic in the excited-state. This result confirms the MLCT assignment for the lowest electronic transition of [Ru(mbpyCOOH)₃]²⁺.     

八元瓜环超分子作用下2-(2-氨基-3-吡啶基)苯并咪唑的质子转移

利用荧光发射光谱、 紫外吸收光谱和核磁共振氢谱(¹H NMR)研究了八元瓜环(CB8)与2-(2-氨基-3-吡啶基)苯并咪唑(2-A3PyBI)的超分子相互作用及其对2-A3PyBI分子内质子转移过程的影响. 结果表明, 在水溶液中2-A3PyBI具有双重荧光发射峰, 分别对应其2种质子转移异构体. 固定pH值下的荧光滴定实验表明主客体包合比为1:2, 2-A3PyBI进入CB8空腔后促进了其分子内激发态质子转移过程. 同时, ¹H NMR结果表明2-A3PyBI的苯环部分进入了CB8空腔.     

An ultrafast time-resolved fluorescence spectroscopy system for metal ion complexation studies with organic ligands

A dedicated spectrofluorimeter using ultrashort laser pulses as an excitation source was developed to measure the fluorescence properties of organic ligands for metal ion complexation with organic ligands. The laser system consists of an oscillator system for generation of femtosecond laser pulses, an amplifier system to increase the pulse energy of the generated pulses to about 2 mJ and an optical parametrical amplifier system to provide tunable laser pulses over a wide wavelength range (280 nm-10 microm). The laser pulses were applied to the sample and the emitted fluorescence was detected using a fast-gating intensified CCD camera-based spectrometer. To verify the performance of the laser, the well-known protonation constant [Pure Appl. Chem. 69 (1997) 329] of 2,3-dihydroxybenzoic acid was determined. The fluorescence lifetime of the excited species was determined as 375+/-32 ps in the pH range from 1.0 to 6.0, having a fluorescence emission maximum at 438 nm. The first protonation constant was determined from fluorescence data as log K(3)=3.17+/-0.05 at an ionic strength of 0.1 M and at 294 K exploiting the Stern-Volmer mechanism. The agreement of the protonation constant with literature data (log K(3)=3.10+/-0.20, I=0.1 M, T=298 K [Bull. Soc. Jpn. 44 (1971) 3459]) demonstrates the excellent performance of our system. Furthermore, we determined the complex formation constant log K(1)=-3.11+/-0.16 by measuring the fluorescence properties of the ligand for the 1:1 uranyldihydroxobenzoate complex in the pH range from 3.0 to 4.5 at ionic strength of 0.1 M and at 294 K. We also determined the complex formation constant via the fluorescence emission of the metal ion uranium(VI). The fluorescence of the uranyl ion is influenced by dynamic quenching of the non-dissociated ligand and by static quenching due to the complex formation. After correction of these effects using the determined fluorescence lifetime, the complex formation constant was calculated to be log K(1)=-3.99+/-0.44. A 1:1 metal:ligand stoichiometry was determined with both measurement methods. However, the difference of the obtained formation constants and the derived standard deviations indicate a superimposition of effects with the excited-state reactions of the ligand.     

Spectrophotometric determination of Fe(III) in alkaline solutions without neutralization

Spectrometric study on the complexation of Fe(III) with an organic reagent obtained by coupling 3-methyl-1-phenyl-5-pyrazolone with diazotized 3-hydroxy-4-amino-benzene sulphonic acid was carried out in alkaline solutions. A 1:2 Fe(III): reagent water soluble complex is formed. The optimum pH is 9.0-11.8. The maximum absorbance of the complex lies at lambda = 560 nm, where the absorbance of the reagent is low. The molar absorptivity is 9000 l.mole(-1).cm(-1) at pH = 11.6. The value of the stability constant determined at 20 +/- 1 degrees C, pH = 11.6 and lambda = 560 nm is 4 x 10(5)M. The Beer-Lambert law is followed for iron concentration in the 0.2-5.0 mug/ml range. The spectrophotometric method was tested on synthetic solutions and thus applied for determination of traces of Fe(III) in several samples of alkaline hydroxides and carbonates without the neutralization of the solutions.     

超分子作用对2-(2-氨基苯基)苯并噻唑分子内质子转移的影响

采用稳态荧光、瞬态荧光及量子化学计算等手段对2-(2-氨基苯基)苯并噻唑(APBT)在不同溶剂中的质子转移进行了研究。结果表明,溶剂的极性及质子化对APBT的质子转移有较大的影响,通过对超分子作用的考察,发现七元瓜环(CB[7])的加入对APBT质子转移起到了一定的抑制作用, APBT与CB[7]能形成化学计量比为1:1的主客体包合物,同时测定了包合物的结合常数等热力学参数。此外,核磁共振氢谱和包合物的理论计算表明APBT分子进入了CB[7]的疏水空腔。     

N-(1-萘基)氨基乙酸的激发态分子内电荷转移和电子转移

氨基酸是蛋白质的基本结构单位 ,水溶液中基态氨基酸分子以内盐形式存在 [1] .由于质子化氨基的正诱导效应 ,羧基的离解常数显著提高 ,如氨基乙酸的羧基离解常数 ( 4.57× 1 0 -3mol/L,2 5℃ [1] )是乙酸 ( 1 .74× 1 0 -5 mol/L[2 ] )的 2 63倍 ;同时内盐结构亦使氨基酸和肽链具有分子内电偶极 ,后者已在肽链上电荷转移过程的调控中发挥重要作用 [3,4] .激发态氨基酸或肽的诱导效应和电偶极性质的研究尚未见报道 ,其主要原因可能是难有合适的激发方式和研究方法 .然而相关研究应予重视 ,因为研究结果将可能为生物酶活性的调节提供一种新的…