A novel type of fluorescent boronic acid that shows large fluorescence intensity changes upon binding with a carbohydrate in aqueous solution at physiological pH

In this paper we report 8-quinolineboronic acid as a novel type of fluorescent probe for carbohydrates. This boronic acid responds to the binding of a carbohydrate with over 40-fold increases in fluorescence intensity and shows optimal fluorescence change at physiological pH in aqueous solution.     

Regulating the fluorescence intensity of an anthracene boronic acid system: a B-N bond or a hydrolysis mechanism?

An anthracene-based fluorescent boronic acid system developed by the Shinkai group has been widely used for the preparation of fluorescent sensors for carbohydrates. Such application is based on the significant fluorescence intensity increase of this system upon binding with a carbohydrate. The mechanism through which this fluorescence intensity change happens was originally proposed to go through a B-N bond formation mechanism, which masks the nitrogen lone pair electrons. However, our own fluorescence studies suggest a possible alternative mechanism for the fluorescence change upon the formation of a boronic acid (1a) complex with diols. In this new proposed mechanism, complex formation induces solvolysis, which results in the protonation of the amine nitrogen if the reactions are carried out in a protic solvent such as water. This protonation prevents the photoinduced electron transfer, resulting in reduced quenching of the anthracene fluorescence. Such a solvolysis mechanism is supported by evidence from various types of experiments and theoretical calculations.     

Effect of xenon on the excited States of phototropic receptor flavin in corn seedlings

The chemically inert, water-soluble heavy atom gas, xenon, at millimolar concentrations specifically quenches the triplet excited state of flavin in solution without quenching the flavin singlet excited state. The preferential quenching of the flavin triplet over the singlet excited state by Xe has been established by showing that the flavin triplet-sensitized photooxidation of NADH is inhibited while the fluorescence intensity and lifetime of flavin are not affected by Xe.     

Influence of alkyl group on amide nitrogen atom on fluorescence quenching of tyrosine amide and N-acetyltyrosine amide

The steady-state and time-resolved fluorescence spectroscopy was applied to determine the influence of an alkyl substituent(s) (methyl or ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, or t-butyl) on amide nitrogen atom on photophysical properties of tyrosine and N-acetyltyrosine amides in water. Generally, the amide group strongly quenches the fluorescence of tyrosine, however, the size and number of substituents on amide nitrogen atom modify the quenching process only in small degree. The fluorescence intensity decays of all amides studied are bi-exponential. The contribution of both components (alphai) to the fluorescence decay undergoes irregular change. An introduction of alkyl substituent on amide nitrogen atom causes an increase of the fluorescence lifetime of tyrosine derivative compared to the unsubstituted amide for both N-acetyltyrosine and tyrosine with the protonated amino group. Calculated, basing on the fluorescence quantum yield (QY) and average lifetime, the radiative rate constants (kf) are similar, which indicates that the substituent(s) does not have substantial influence on radiative process of the deactivation of the excited state of the phenol chromophore for all compounds studied regardless the amino group status as well as the number and type of substituent (linear or branched). The comparison of the ground-state rotamer populations of tyrosine amides and N-acetyltyrosine amides with different alkyl substituent on amide nitrogen atom obtained from 1H NMR with the value of pre-exponential factors indicates that not the rotamer populations, but specific hydration of a whole molecule of the amino acid including chromophore and amino acid moiety, seems to be the main reason of the heterogenous fluorescence intensity decay of tyrosine derivatives.     

Evaluation of the energetic position of the lowest excited singlet state of beta-carotene by NEXAFS and photoemission spectroscopy.

In carotenoids the lowest energetic optical transition belonging to the pi-electron system is forbidden by symmetry, therefore the energetic position of the S(1) (2(1)A(g)) level can hardly be assessed by optical spectroscopy. We introduce a novel experimental approach: For molecules with pi-electron systems the transition C1s-->2p(pi*) from inner-atomic to the lowest unoccupied molecular orbital (LUMO) appears in X-ray absorption near edge spectra (NEXAFS) as an intense, sharp peak a few eV below the carbon K-edge. Whereas the peak position reflects the energy of the first excited singlet state in relation to the ionization potential of the molecule, intensity and width of the transition depend on hybridization and bonding partners of the selected atom. Complementary information can be obtained from ultraviolet photoelectron spectroscopy (UPS): At the low binding energy site of the spectrum a peak related to the highest occupied molecular orbital (HOMO) appears. We have measured NEXAFS and UPS of beta-carotene. Based on these measurements and quantum chemical calculations the HOMO and LUMO energies can be derived.     

Catabolism of arylboronic acids by Arthrobacter nicotinovorans strain PBA

Arthrobacter sp. strain PBA metabolized phenylboronic acid to phenol. The oxygen atom in phenol was shown to be derived from the atmosphere using (18)O(2). 1-Naphthalene-, 2-naphthalene-, 3-cyanophenyl-, 2,5-fluorophenyl-, and 3-thiophene-boronic acids were also transformed to monooxygenated products. The oxygen atom in the product was bonded to the ring carbon atom originally bearing the boronic acid substituent with all the substrates tested.     

Catabolism of Arylboronic Acids by Arthrobacter nicotinovorans Strain PBA

Arthrobacter sp. strain PBA metabolized phenylboronic acid to phenol. The oxygen atom in phenol was shown to be derived from the atmosphere using ¹⁸O₂. 1-Naphthalene-, 2-naphthalene-, 3-cyanophenyl-, 2,5-fluorophenyl-, and 3-thiophene-boronic acids were also transformed to monooxygenated products. The oxygen atom in the product was bonded to the ring carbon atom originally bearing the boronic acid substituent with all the substrates tested.     

Synthesis and carbohydrate binding studies of fluorescent α-amidoboronic acids and the corresponding bisboronic acids

Fluorescent boronic acids are very useful for the design and synthesis of carbohydrate sensors. In an earlier communication, we first described the effort of developing water soluble fluorescent α-amidoboronic acids, which change fluorescence upon sugar binding. In this report, we describe a general method of functionalizing such boronic acids and their applications in the preparation of bis-α-amidoboronic acids with significantly enhanced binding for oligosaccharides as compared to their monoboronic acid counterparts. The advantages of good water solubility, easy modification to generate diversity, and modularity in synthesis will make α-amidoboronic acids very useful building blocks for future synthesis of boronic acid-based fluorescent sensors.     

Photochemistry and photophysics of the endoperoxide of mesodiphenylhelianthrene: a contribution to the localization of the S1(π*σ*) state of aromatic endoperoxides

The endoperoxide of mesodiphenylhelianthrene MDHPO has been studied in detail with respect to fluorescence and photo-induced rearrangement. MDHPO proved to be non-fluorescent, although its absorption spectrum is dominated at the low energy side by a strong ππ* band with a maximum at 429.5 nm. Irradiation of that band effects rearrangement to the corresponding diepoxide MDHDO, a reaction typical for S(1)(π*σ*) excited endoperoxides (EPOs). The absorption spectrum of the product MDHDO is blue shifted by only 3.5 nm. MDHDO has the same extended planar aromatic system like its precursor MDHPO, but MDHDO fluoresces strongly. These results set the excitation energy of the S(1)(π*σ*) state of MDHPO to ≤23?000 cm(-1), which is considered to be a generally realistic value of the S(1)(π*σ*) state energy of aromatic EPOs. The main reaction of S(1)(π*σ*) excited MDHPO is, however, chemical deactivation to ground state MDHPO via an oxygen biradical. The sequence of O-O bond opening and closing is the general way of repopulation of the S(0) state of aromatic EPOs from S(1)(π*σ*) excited states.     

The development of photometric sensors for boronic acids

Boronic acids bind certain 1,2- and 1,3-diols with high affinity through reversible formation of boronate esters. They have been utilized as the recognition moiety for artificial receptors, particularly receptors for carbohydrates that have cis-diol moieties. Therefore, sensors for boronic acids could serve as universal reporters for monitoring boronate formation. This paper reports the design and synthesis of a series of photometric chemosensors for phenylboronic acid using diethanolamine as the recognition moiety. Diethanolamine, which binds strongly to boronic acids, has been linked to three different types of optical reporters. A photoinduced electron transfer system based on the anthracene fluorophore has been used to create sensors that show up to a fivefold increase in fluorescent intensity in the presence of millimolar concentrations of phenylboronic acid. Sensor designs based on the restriction of free rotation of extended pi systems and on the perturbed electronic properties of azo dyes are also included. This work demonstrates that sensors based on several different designs can be used for the detection of boronic acids.     

Photochemistry of 2-acyloxycarbazoles. A potential tool in the synthesis of carbazole alkaloids.

The photochemistry of two 2-acyloxycarbazoles, 2-acetyl- and 2-benzoyloxycarbazole, in different solvents has been studied. Irradiation of the 2-acyloxycarbazoles in organic media at 254 or 313 nm yields the [1,3]-migrated photoproducts, 1-acyl-2-hydroxycarbazole, 3-acyl-2-hydroxycarbazole and 2-hydroxycarbazole. The effects of the solvent, the atmosphere and the intensity of the light source on the photochemistry of 2-acyloxycarbazole have been studied. Laser flash photolysis as well as photosensitization experiments were performed in order to determine the photoreactive excited state. Electronic spectra (absorption, fluorescence and phosphorescence emission spectra) of the 2-acyloxycarbazoles have been recorded in homogeneous media at 298 K and in solid matrices at 77 K. The dynamic properties of the lowest singlet excited state in terms of fluorescence lifetime and fluorescence quantum yield have been measured in different organic solvents at room temperature. The photo-Fries rearrangement as a mild and clean one-pot reaction for the preparation of an advanced intermediate precursor in the total synthesis of carbazole alkaloids is described.     

Versatile derivatives of carbohydrate-binding modules for imaging of complex carbohydrates approaching the molecular level of resolution

The innate binding specificity of different carbohydrate-binding modules (CBMs) offers a versatile approach for mapping the chemistry and structure of surfaces that contain complex carbohydrates. We have employed the distinct recognition properties of a double His-tagged recombinant CBM tagged with semiconductor quantum dots for direct imaging of crystalline cellulose at the molecular level of resolution, using transmission and scanning transmission electron microscopy. In addition, three different types of CBMs from families 3, 6, and 20 that exhibit different carbohydrate specificities were each fused with either green fluorescent protein (GFP) or red fluorescent protein (RFP) and employed for double-labeling fluorescence microscopy studies of primary cell walls and various mixtures of complex carbohydrate target molecules. CBM probes can be used for characterizing both native complex carbohydrates and engineered biomaterials.     

Excited state annihilation in the photosynthetic unit

The kinetics of the in vivo fluorescence decays and fluorescence yields, as a function of excitation intensity, have been analysed with a model using excited state annihilation and time-dependent quenching processes. Triplet states, formed in the singlet-singlet annihilation processes, account for additional quenching of singlet states and the persistence of annihilation at longer times than the fluorescence lifetime. Together these processes give a satisfactory account of existing experimental data of the intensity dependence of fluorescence in vivo.     

Development of ratiometric carbohydrate sensor based on boron dipyrromethene (BODIPY) scaffold

We designed a ratiometric carbohydrate sensor consisting of the boron dipyrromethene fluorophore substituted with boronic acid at the 2-position, based upon the strong substituent dependency of the absorbance/fluorescence wavelengths of BODIPY. The substituent is in equilibrium between the boronic acid B(OH)₂ and boronate (B(OH)₃⁻) forms, which have different absorbance/fluorescence wavelengths in the visible region. Reaction of the boronic acid moiety with hydroxy groups of carbohydrate affords a cyclic ester and shifts the equilibrium in favor of the boronate (B(OR)₃⁻) form, resulting in a carbohydrate-concentration-dependent change of the fluorescence ratio. Thus, the sensor, BA-BODIPY, can ratiometrically detect carbohydrate at a pH near the pK_a of cyclic ester formation.     

A quantum chemical investigation of the electronic structure of thionine

We have examined the electronic and molecular structure of 3,7-diaminophenothiazin-5-ium dye (thionine) in the electronic ground state and in the lowest excited states. The electronic structure was calculated using a combination of density functional theory and multi-reference configuration interaction (DFT/MRCI). Equilibrium geometries were optimized employing (time-dependent) density functional theory (B3LYP functional) combined with the TZVP basis set. Solvent effects were estimated using the COSMO model and micro-hydration with up to five explicit water molecules. Our calculated electronic energies are in good agreement with experimental data. We find the lowest excited singlet and triplet states at the ground state geometry to be of π→π* (S(1), S(2), T(1), T(2)) and n→π* (S(3), T(3)) character. This order changes when the molecular structure in the electronically excited states is relaxed. Geometry relaxation has almost no effect on the energy of the S(1) and T(1) states (~0.02 eV). The relaxation effects on the energies of S(2) and T(2) are moderate (0.14-0.20 eV). The very small emission energy results in a very low fluorescence rate. While we were not able to locate the energetic minimum of the S(3) state, we found a non-planar minimum for the T(3) state with an energy which is very close to the energy of the S(1) minimum in the gas phase (0.04 eV above). When hydration effects are taken into account, the n→π* states S(3) and T(3) are strongly blueshifted (0.33 and 0.46 eV), while the π→π* states are only slightly affected (<0.06 eV).     

Acid-base properties of the reaction product of sialic acid with fluorogenic reagent, 1,2-diamino-4,5-methylenedioxybenzene (DMB)

N-Acetylneuraminic acid (Neu5Ac) forms the highly fluorophoric quinoxalinone derivative (Q) when treated with 1,2-diamino-4,5-methylenedioxybenzene (DMB). Effects of protonation and deprotonation on the fluorescence of Q were examined at room temperature. The strong fluorescence was found to be caused by the neutral form Q but not the protonated form of its excited state [Q]* and at pH below 1 the emission was completely quenched. The deprotonated singlet form [Q-]* was a less efficient fluorescer than [Q]*.     

Synthesis and spectral properties of a hydrophobic fluorescent probe: 6-propionyl-2-(dimethylamino)naphthalene.

Environmentally sensitive fluorescent probes involve two groups, an electron donor and an electron acceptor, attached to an aromatic ring system, and maximal effects may be expected when these groups are as far apart as feasible. The syntheisis, characterization, and spectroscopic properties of 6-propionyl-2-(dimethylamino)naphthalene (PRODAN), a compound that fulfills these conditions, are described. The maximum of emission is at 401 nm in cyclohexane solution and at 531 nm in water solution, indicating an increase of dipole moment of approximately 20 D units on excitation to the lowest singlet state. The effect of temperature upon the spectral distribution and the bandwidth of fluorescence of PRODAN in 1:1 complexes with albumin shows the existence of a dynamic relaxation process of the protein surroundings within th 2-4 ns of the fluorescence lifetime.     

One- and two-photon excited fluorescence lifetimes and anisotropy decays of green fluorescent proteins

We have used one- (OPE) and two-photon (TPE) excitation with time-correlated single-photon counting techniques to determine time-resolved fluorescence intensity and anisotropy decays of the wild-type Green Fluorescent Protein (GFP) and two red-shifted mutants, S65T-GFP and RSGFP. WT-GFP and S65T-GFP exhibited a predominant approximately 3 ns monoexponential fluorescence decay, whereas for RSGFP the main lifetimes were approximately 1.1 ns (main component) and approximately 3.3 ns. The anisotropy decay of WT-GFP and S65T-GFP was also monoexponential (global rotational correlation time of 16 +/- 1 ns). The approximately 1.1 ns lifetime of RSGFP was associated with a faster rotational depolarization, evaluated as an additional approximately 13 ns component. This feature we attribute tentatively to a greater rotational freedom of the anionic chromophore. With OPE, the initial anisotropy was close to the theoretical limit of 0.4; with TPE it was higher, approaching the TPE theoretical limit of 0.57 for the colinear case. The measured power dependence of the fluorescence signals provided direct evidence for TPE. The general independence of fluorescence decay times, rotation correlation times, and steady-state emission spectra on the excitation mode indicates that the fluorescence originated from the same distinct excited singlet states (A*, I*, B*). However, we observed a relative enhancement of blue fluorescence peaked at approximately 440 nm for TPE compared to OPE, indicating different relative excitation efficiencies. We infer that the two lifetimes of RSGFP represent the deactivation of two substates of the deprotonated intermediate (I*), distinguished by their origin (i.e., from A* or B*) and by nonradiative decay rates reflecting different internal environments of the excited-state chromophore.     

Ab initio study of mechanism of forming a spiro-heterocyclic ring compound with Si and Ge between dimethylsilylene germylidene (Me2Si?=?Ge:) and acetone

The mechanism of the cycloaddition reaction between singlet state dimethylsilylene germylidene (Me(2)Si?=?Ge:) and acetone has been investigated with CCSD(T)//B3LYP/6-31G* method. From the potential energy profile, it could be predicted that the reaction has one dominant reaction pathway. The reaction rules presented are that the two reactants first form a Si-heterocyclic four-membered ring germylene through the [2?+?2] cycloaddition reaction. Because of the 4p unoccupied orbital of Ge atom in the Si-heterocyclic four-membered ring germylene and the π orbital of acetone forming a π→p donor-acceptor bond, the Si-heterocyclic four-membered ring germylene further combines with acetone to form an intermediate. Because the Ge atom in the intermediate happens sp(3) hybridization after transition state, then, the intermediate isomerizes to a spiro-heterocyclic ring compound with Si and Ge via a transition state.