PROMPT AND DELAYED FLUORESCENCE OF SOME DNA ADSORBATES

Abstract— The absorption and prompt fluorescence spectra of twelve cationic compounds in the free and bound states were surveyed, and four compounds selected for detailed study, viz: proflavine, benzoflavine, acridine orange and thioflavine T. The absorption spectra, prompt fluorescence spectra and delayed fluorescence and phosphorescence spectra of these four compounds in fluid solutions in the free and bound states were measured. The shifts in the promp fluorescence bands of the diaminoacridines on adsorption were found to differ significantly from those of the other compounds, and the behaviour of acridine orange was found to be different from that of the other two diaminoacridines. All three diaminoacridines gave E -type delayed fluorescence in the free and bound states. The ordered structure of the strongly binding adsorption sites in native DNA is associated with a higher intensity of delayed fluorescence than is observed from the diaminoacridines in the free state or adsorbed on denatured DNA. Quenching experiments with proflavine indicated that, in the weakly binding sites, its triplet is easily quenched by unadsorbed quenchers, but in the strongly binding sites its triplet is quenched only with great difficulty.     

Qualitative and quantitative characterization of the arsenic‐binding behaviour of sulfur‐containing peptides and proteins by the coupling of reversed phase liquid chromatography to electrospray ionization mass spectrometry

Phenylarsenic‐substituted cysteine‐containing peptides and proteins were completely differentiated from their unbound original forms by the coupling of reversed phase liquid chromatography with electrospray ionization mass spectrometry. The analysis of biomolecules possessing structure‐stabilizing disulfide bridges after reduction provides new insights into requirements concerning the accessibility of cysteine residues for reducing agents as well as for arsenic compounds in a spatial protein structure. Complementary binding studies performed using direct ESI‐MS without chromatographic coupling in different solvent systems demonstrated that more than one binding site were activated for aprotinin and lysozyme in denaturing solvents because of a stronger defolding. From the intensities of the different charge states occurring in the mass spectra as well as from the LC elution behaviour, it can be deduced that the folding state of the arsenic‐bound protein species resembles the native, oxidized conformation. In contrast, although the milk protein α‐lactalbumin has several disulfide bridges, only one phenylarsenic moiety was bound under strongly denaturing conditions. Because of the charge state distribution in the ESI mass spectra, a conformational change to a molten globule structure is assumed. For the second considered milk protein ß‐lactoglobulin, a noncovalent interaction with phenylarsine oxide was detected. In general, smaller apparent binding constants for the condensation reactions of the biomolecules with phenylarsine oxide leading to covalent arsenic–sulfur bindings were determined from direct injection ESI‐MS measurements than from LC‐ESI‐MS coupling. The following order of binding affinities for one phenylarsenic group can be assumed from both ESI‐MS and LC‐ESI‐MS: nonapeptide vasopressin > nonapeptide vasotocin > lysozyme > aprotinin > α‐lactalbumin > thioredoxin. Kinetic investigations by LC‐ESI‐MS yielded a partial reaction order of 2 for vasopressin, Lys and α‐lactalbumin and corresponding half‐lives of 0.93, 2.56 and 123.5 min, respectively. Copyright © 2012 John Wiley & Sons, Ltd.     

Excited state proton transfer reaction of two new intramolecularly hydrogen bonded Schiff bases at room temperature and 77K.

Two new orthohydroxy Schiff bases, 7-phenylsalicylidene benzylamine (PSBA) and 7-ethylsalicylideneaniline (ESA) have been synthesized. The excited state intramolecular proton transfer (ESIPT) and the structure of PSBA and ESA in its crystalline form and in the solvents n-hexane, n-heptane and 1,4-dioxane have been investigated by means of absorption, emission and nanosecond spectroscopy at room temperature and 77K. One ground state species has been detected both in neutral and basic solutions of both PSBA and ESA: the cis-enol form with an intramolecular hydrogen bond. The ESIPT and formation of keto tautomer are evidenced by a large Stokes shifted emission (approximately 12000 cm(-1)) at room temperature only in the case of ESA. On the other hand the keto tautomer is the predominant species at 77K in a solid matrix and as a solid sample at room temperature both in the case of ESA and PSBA. In the case of both ESA and PSBA the more intense, higher energy emission is due to the species which has not undergone ESIPT and attributed mainly due to cis-enol form. The trans-enol form is also observed by changing the excitation wavelength. Both the compounds are found to undergo a structural change to a zwitterionic and intermolecular hydrogen bonded form in the presence of a strong base like triethylamine. From the nanosecond measurements and quantum yield of fluorescence we have estimated the decay rates of proton transfer reaction in the case of PSBA. Our theoretical calculation at the AM1 level of approximation shows that the ground singlet state has a rather large activation barrier both in the case of PSBA and ESA. The barrier height is much lower on the corresponding excited singlet surface only in the case of ESA. The process is predicted to be endothermic in the ground state and exotherrmic in the excited singlet state.     

Fiber-optic biosensors based on fluorescence energy transfer

A new optical homogeneous biochemical method for the assay of glucose has been developed, based on fluorescence energy transfer between a glucose analog, dextran labeled with fluorescein isothiocyanate (FITC-dextran), and a glucose-receptor protein, Rhodamine-labeled Concanavalin A (Rh-ConA). When FITC-dextran binds to Rh-ConA in solution, and is light-activated, the FITC label transfers its absorbed energy to the Rhodamine label, which then emits light according to its own characteristic fluorescence spectrum. When glucose is added to this solution, the FITC fluorescence intensity increases as FITC-dextran is released from the Rh-ConA and is replaced by glucose. Thus it is possible to determine glucose concentrations directly from the level of FITC fluorescence.     

Multifunctional nanostructures based on inorganic nanoparticles and oligothiophenes and their exploitation for cellular studies

The combination of materials that possess different properties (such as, for instance, fluorescence and magnetism) into one single object of nanoscale size represents an attractive challenge for biotechnology, especially for their potential relevance in biomedical applications. We report here the preparation of novel bifunctional conjugates based on the linkage of inorganic nanoparticles to organic oligothiophene fluorophores (OTFs). In comparison to the organic dyes commonly used in bioimaging and more similarly to colloidal quantum dots, OTFs have broad optical absorption spectra, and therefore OTF fluorophores emitting at different colors can be excited with a single excitation source, allowing for easier multiplexing analysis. In this work we show the preparation of OTF-nanoparticle conjugates based on gold and iron oxide nanoparticles and their characterization using different techniques such as gel electrophoresis, photoluminescence spectroscopy, dynamic light scattering, and so on. In addition, by performing an in vitro study on human tumor cells we show that OTF-nanoparticle conjugates emitting at different colors can be used for multiplexing detection. Also, in the case of iron oxide-OTF conjugates, once uptaken by the cells, we show that they preserve both their fluorescent and their magnetic properties.     

A facile synthesis of bifunctional phospholipids for biomimetic membrane engineering

We report a facile synthesis of bifunctional phospholipid conjugates by acylation of N-protected lyso-phosphatidylethanolamine with 12-acryloxy-1-dodecanoic acid and followed with deprotection and conjugation with biotin, FITC, Texas Red, or EMC groups. The lipid conjugates can be used to generate a multifunctional substrate-supported phospholipid membrane via bioconjugation reaction to biotin or covalent attachment to EMC at their hydrophilic terminus. In addition, conjugation to fluorophores, FITC or Texas Red, provides a convenient mechanism to monitor lipid membrane formation and stability. Significantly, in situ photopolymerization of the acrylate group at the end of one of two hydrophobic alkyl chains stabilizes the phospholipid membrane.     

Exhaustive search of the configurational space of heat-shock protein 90 with its inhibitor by multicanonical molecular dynamics based dynamic docking

Multicanonical molecular dynamics based dynamic docking was used to exhaustively search the configurational space of an inhibitor binding to the N-terminal domain of heat-shock protein 90 (Hsp90). The obtained structures at 300 K cover a wide structural ensemble, with the top two clusters ranked by their free energy coinciding with the native binding site. The representative structure of the most stable cluster reproduced the experimental binding configuration, but an interesting conformational change in Hsp90 could be observed. The combined effects of solvation and ligand binding shift the equilibrium from a preferred loop-in conformation in the unbound state to an α-helical one in the bound state for the flexible lid region of Hsp90. Thus, our dynamic docking method is effective at predicting the native binding site while exhaustively sampling a wide configurational space, modulating the protein structure upon binding.     

Femtosecond S3-->S1 conversion and structural reorganisation of trans-3-phenylprop-2-enaldehyde and derivatives in solution.

Photoinduced excited-state relaxation of trans-3-phenylprop-2-enaldehyde (cinnamaldehyde) and three derivatives was studied in hexane and acetonitrile with the pump-supercontinuum-probe technique. Transient spectra were measured with 50 fs resolution in the range 260-660 nm after S3<--S0 excitation at 288 nm. The early spectra reveal an ultrafast, 0.1-0.5 ps, interchange of intensity between two excited-state absorption (ESA) bands followed by a approximately 3 ps decay of ESA. This behaviour is interpreted with the help of semiempirical calculations, which indicate that the sub-picosecond evolution is consistent with S3-->S1 excited-state relaxation while the picosecond decay should be due to a structural intramolecular rearrangement. The latter may consist of a twist of the phenyl ring and rotation around the C==C bond to a perpendicular conformation which corresponds to the global energy minimum in the S1 state and serves as source for trans and cis isomers in the ground state.     

Structural Characterization of a Zinc High‐affinity Binding Site in Rhodopsin†

For the first time to our knowledge, X‐ray absorption spectroscopy (XAS) has been used to investigate the environment of putative Zn²⁺ binding sites in rhodopsin. We studied native purified nondeionized rhodopsin without any further addition of Zn²⁺, as well as with 1.5 mol of Zn²⁺—as zinc chloride—per mole of protein. Three different binding sites in rhodopsin were considered based on computational chemistry studies, and a quantitative analysis of the XAS signal was performed by fitting the experimental data to their simulated XAS spectra. Our results demonstrate that Zn²⁺ is intrinsically bound to rhodopsin and are compatible with the existence of an octahedral coordination involving six oxygen atoms in the first shell (average Zn‐O distance of 2.08 Å), and with a second coordination shell containing one or two phosphorus or sulfur atoms at an average distance of 2.81 Å.     

Unraveling the absorption spectra of alkali metal atoms attached to helium nanodroplets

The absorption spectra of the first electronic exited state of alkali metal atoms on helium nanodroplets formed of both 4He and 3He isotopes were studied experimentally as well as theoretically. In the experimental part new data on the 2p<--2s transition of lithium on 3He nanodroplets are presented. The absorption spectrum changes drastically when compared to 4He droplets, in contrast to sodium where only marginal differences were observed in former studies. To explain these large differences and to answer some still open questions concerning the interaction of alkali metal atoms with helium nanodroplets, a model calculation was performed. New helium density profiles as well as a refined model allowed us to achieve good agreement with the experimental findings. For the first time the red-shifted intensities in the lithium and sodium spectra are explained in terms of enhanced binding configurations in the excited state displaced spatially from the ground state configurations.     

Hydrogen Bonding Effects on the Photophysical Properties of 2,3-dihydro-3-keto-1H-pyrido[3,2,1-kl]phenothiazine

Time-dependent density functional theory (TDDFT) and femtosecond transient absorption spectroscopy were used to investigate the photophysical properties of 2,3-dihydro-3-keto-1H-pyrido[3,2,1-kl]phenothiazine (PTZ4) and 3-keto-1H-pyrido[3,2,1-kl]phenothiazine (PTZ5). The calculated results obtained from TDDFT suggest that the red-shifts of the absorption spectra of these two fluorophores in methanol are due to the formation of hydrogen-bonded complexes at the ground state. Four conformers of PTZ4 were obtained by TDDFT. The two fluorescence peaks of PTZ4 in tetrahydrofuran (THF) came from the ICT states of the four conformers. The fluorescence of PTZ4 in THF showed a dependence on the excitation wavelength because of butterfly bending. The excited state dynamics of PTZ4 in THF and methanol were obtained by transient absorption spectroscopy. The lifetime of the excited PTZ4 in methanol was 53.8 ps, and its relaxation from the LE state to the ICT state was completed within several picoseconds. The short lifetime of excited PTZ4 in methanol was due to the formation of out-of-plane model hydrogen bonds between PTZ4 and methanol at the excited state.     

Cross-strand split tetra-Cys motifs as structure sensors in a beta-sheet protein

We have designed "split tetra-Cys motifs" that bind the biarsenical fluorescein dye 4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein (FlAsH) across strands of a model beta-rich protein. Our strategy was to divide the linear FlAsH binding tetra-Cys sequence such that dye could be fully liganded only when the strands were arranged in space correctly by native protein conformational proximities. We introduced pairs of alternating cysteines on adjacent beta strands of cellular retinoic acid binding protein to create FlAsH binding sites in the native structure. Selective labeling occurred both in vitro and in vivo relative to sites with fewer than four Cys or with inappropriate geometry. Interestingly, two of the split tetra-Cys motif-carrying proteins bound FlAsH whether native or urea unfolded, while one was capable of binding FlAsH only when native. This latter design exemplifies the potential of split motifs as structure sensors.     

Intramolecular proton-transfer processes starting at higher excited states: a fluorescence study on 2-butylamino-6-methyl-4-nitropyridine N-oxide in nonpolar solutions

This article describes the exceptional photophysics of 2-butylamino-6-methyl-4-nitropyridine N-oxide (2B6M). It is known from the literature that this compound may undergo excited-state intra- or intermolecular proton-transfer reactions. In nonpolar solvents, 2B6M exhibits an unusual fluorescence behavior: there is a substantial difference between the relative band intensities of the excitation and absorption spectra. Furthermore, in emission two bands are observed, and their relative intensities depend on the excitation wavelength, thus violating the Kasha-Vavilov rule. It is the objective of this research to interpret these results. For this purpose, steady-state fluorescence excitation and emission spectra in the liquid state were recorded and quantum yields were determined for the two types of emission. In addition, absorption spectra were measured at room temperature and under low-temperature conditions. Finally, fluorescence lifetimes of the emitting species were determined using the time-correlated single photon counting technique. The results suggest that in the liquid state only one (monomeric) ground state species dominates, which can emit via two different pathways (from the normal and the tautomeric excited state). The excitation spectra point at two different internal proton-transfer processes, one starting at the S1 state and one starting at the S2 state. On the basis of the measured lifetimes and fluorescence quantum yields, a kinetic scheme was completed that can quantitatively explain the observations.     

Decoupling fluorescence and photochromism in bifunctional azo derivatives for bulk emissive structures

Bifunctional molecules that combine independent push-pull fluorophores and azo photochromes have been synthesized to create fluorescent structures upon light-induced migration in neat thin films. Their photochromic and emissive properties have been systematically investigated and interpreted in light of those of the corresponding model compounds. Fluorescence lifetimes and photoisomerization and fluorescence quantum yields have been determined in toluene solution. Kinetic analyses of the femtosecond transient absorption spectra reveal that the fluorophores evolve in a few picoseconds into a distorted intramolecular charge-transfer excited state, strongly stabilized in energy. Radiative relaxation to the ground state occurred competitively with the energy-transfer process to the azo moiety. Introduction of a 10??-long rigid and nonconjugated bridge between the photoactive units efficiently inhibits the energy transfer while it imparts enhanced free volume, which favors photoactivated molecular migration in the solid state.     

A transient absorption study of allophycocyanin

Transient dynamics of allophycocyanin trimers and monomers are observed by using the pump-probe, transient absorption technique. The origin of spectral components of the transient absorption spectra is discussed in terms of both kinetics and spectroscopy. We find that the energy gap between the ground and excited states of the unexcited subunit of allophycocyanin monomer decreases via an interaction with another excited subunit. For allophycocyanin trimer, we find that the fast dynamics results from the fast internal conversion and the first excited state is the only one electronic state which can trap the final population.     

Dynamics of ultrafast intramolecular charge transfer with 1-tert-butyl-6-cyano-1,2,3,4-tetrahydroquinoline (NTC6) in n-hexane and acetonitrile

The intramolecular charge transfer (ICT) reaction of 1-tert-butyl-6-cyano-1,2,3,4-tetrahydroquinoline (NTC6) in n-hexane and acetonitrile (MeCN) is investigated by picosecond fluorescence experiments as a function of temperature and by femtosecond transient absorption measurements at room temperature. NTC6 in n-hexane is dual fluorescent from a locally excited (LE) and an ICT state, with a quantum yield ratio Phi'(ICT)/Phi(LE) of 0.35 at +25 degrees C and 0.67 at -95 degrees C, whereas in MeCN mainly an ICT emission is observed. From the temperature dependence of Phi'(ICT)/Phi(LE) for NTC6 in n-hexane, an LE/ICT enthalpy difference DeltaH of -2.4 kJ/mol is determined. For comparison, 1-isopropyl-6-cyano-1,2,3,4-tetrahydroquinoline (NIC6) is also investigated. This molecule does not undergo an ICT reaction, because of its larger energy gap DeltaE(S1,S2). From the molar absorption coefficient epsilonmax of NTC6 as compared with other aminobenzonitriles, a ground-state amino twist angle theta of approximately 22 degrees is deduced. The increase of epsilonmax between n-hexane and MeCN indicates that theta decreases when the solvent polarity becomes larger. Whereas single-exponential LE fluorescence decays are obtained for NIC6 in n-hexane and MeCN, the LE and ICT decays of NTC6 in these solvents are double exponential. For NTC6 in n-hexane at -95 degrees C, with a shortest decay time of 20 ps, the forward (ka=2.5x10(10) s(-1)) and backward (kd=2.7x10(10) s(-1)) rate constants for the LE<-->ICT reaction are determined from the time-resolved LE and ICT fluorescence spectra. For NTC6 in n-hexane and MeCN, the excited-state absorption (ESA) spectrum at 200 fs after excitation is similar to the LE(ESA) spectra of NIC6 and 4-(dimethylamino)benzonitrile (DMABN), showing that LE is the initially excited state for NTC6. These results indicate that the LE states of NTC6, NIC6, and DMABN have a comparable molecular structure. The ICT(ESA) spectrum of NTC6 in n-hexane and MeCN resembles that of DMABN in MeCN, likewise indicating a similar ICT structure for NTC6 and DMABN. From the decay of the LE absorption and the corresponding growing-in for the ICT state of NTC6, it is concluded that the ICT state originates from the LE precursor and is not formed by direct excitation from S0, nor via an S2/ICT conical intersection. The same conclusion was made from the time-resolved (picosecond) fluorescence spectra, where there is no ICT emission at time zero. The decay of the LE(ESA) band of NTC6 in n-hexane occurs with a shortest time tau2 of 2.2 ps. The ICT reaction is much faster (tau2 = 0.82 ps) in the strongly polar MeCN. The absence of excitation wavelength dependence (290 and 266 nm) for the ESA spectra in MeCN also shows that LE is the ICT precursor. With NIC6 in n-hexane and MeCN, a decay or growing-in of the femtosecond ESA spectra is not observed, in line with the absence of an ICT reaction involving an S2/ICT conical intersection.     

Hydrogen bonding and solvent effects on the lowest 1(n, pi*) excitations of triazines in water

Our method for estimating solvent effects on electronic spectra in media with strong solute-solvent interactions is applied here to calculate the absorption and fluorescence solvatochromatic shifts of dilute triazines in water. First, the ab initio CASSCF method is used to estimate the gas-phase electronic excitation properties and state charge distributions; second, Monte Carlo simulations are performed to elucidate liquid structures around the ground and excited state solute; finally, the solvent shift is evaluated based on the gas-phase charge distributions and the explicit solvent structures. For the dilute triazine solutions, simulations predict one linear (different) hydrogen bond attached to each nitrogen atom. Upon the first (1)(n, pi*)electronic excitation one hydrogen bond is completely broken. For the absorption and fluorescence spectra, our calculations demonstrated that the specific solvent-solute interaction, in any electronic state, plays a critical role in the determination of solvent shifts.     

Quantitative evaluation of noncovalent chorismate mutase-inhibitor binding by ESI-MS

Electrospray time-of-flight mass spectrometry was used to quantitatively determine the dissociation constant of chorismate mutase and a transition state analogue inhibitor. This system presents a fairly complex stoichiometry because the native protein is a homotrimer with three equal and independent substrate binding sites. We can detect the chorismate mutase trimer as well as chorismate mutase-inhibitor complexes by choosing appropriate conditions in the ESI source. To verify that the protein-inhibitor complexes are specific, titration experiments with different enzyme variants and different inhibitors were performed. A plot of the number of bound inhibitors versus added inhibitor concentration revealed saturation behavior with 3:1 (inhibitor:functional trimer) stoichiometry for the TSA. The soft ESI conditions, the relatively high protein mass of 43.5 kDa, and the low charge state (high m/z) result in broad peaks, a typical problem in analyzing noncovalent protein complexes. Due to the low molecular weight of the TSA (226 Da) the peaks of the free protein and the protein with one, two or three inhibitors bound cannot be clearly resolved. For data analysis, relative peak areas of the deconvoluted spectra of chorismate mutase-inhibitor complexes were obtained by fitting appropriate peak shapes to the signals corresponding to the free enzyme and its complexes with one, two, or three inhibitor molecules. From the relative peak areas we were able to calculate a dissociation constant that agreed well with known solution-phase data. This method may be generally useful for interpreting mass spectra of noncovalent complexes that exhibit broad peaks in the high m/z range.     

Metal-ion sensing fluorophores with large two-photon absorption cross sections: aza-crown ether substituted donor-acceptor-donor distyrylbenzenes

Chromophores based on a donor-acceptor-donor structure possessing a large two-photon absorption cross section and one or two mono-aza-15-crown-5 ether moieties, which can bind metal cations, have been synthesized. The influence of Mg(2+) binding on their one- and two-photon spectroscopic properties has been investigated. Upon binding, the two-photon action cross sections at 810 nm decrease by a factor of up to 50 at high Mg(2+) concentrations and this results in a large contrast in the two-photon excited fluorescence signal between the bound and unbound forms, for excitation in the range of 730 to 860 nm. Experimental and computational results indicate that there is a significant reduction of the electron donating strength of the aza-crown nitrogen atom(s) upon metal ion binding and that this leads to a blue shift in the position as well as a reduction in the strength of the lowest-energy two-photon absorption band. The molecules reported here can serve as models for the design of improved two-photon excitable metal-ion sensing fluorophores.     

Transient excited-state absorption and gain spectroscopy of a two-photon absorbing probe with efficient superfluorescent properties

The synthesis, linear photophysical properties, two-photon absorption (2PA), excited-state transient absorption, and gain spectroscopy of a new fluorene derivative tert-butyl 4,4'-(4,4' (1E,1'E)-2,2'-(9,9-bis(2- (2-ethoxyethoxy)ethyl)-9H-fluorene-2,7-diyl)bis(ethene-2,1-diyl)bis(4,1 phenylene)]dipiperazine-1-carboxylate (1) are reported. The steady-state linear absorption and fluorescence spectra, along with excitation anisotropy, fluorescence lifetimes, and photochemical stability of 1 were investigated in a number of organic solvents at room temperature. The 2PA spectra of 1 with a maximum cross-section of ~ 300 GM were obtained with a 1 kHz femtosecond laser system using open-aperture Z-scan and two-photon-induced fluorescence methods. The transient excited-state absorption (ESA) and gain kinetics of 1 were investigated by a femtosecond pump-probe methodology. Fast relaxation processes (~1-2 ps) in the gain and ESA spectra of 1 were revealed in ACN solution, attributable to symmetry-breaking effects in the first excited state. Efficient superfluorescence properties of 1 were observed in a nonpolar solvent under femtosecond excitation. One- and two-photon fluorescence microscopy imaging of HCT 116 cells incubated with probe 1 was accomplished, suggesting the potential of this new probe in two-photon fluorescence microscopy bioimaging.