Dipolar 3-methoxychromones as bright and highly solvatochromic fluorescent dyes

Herein, three environment-sensitive (solvatochromic) fluorescent dyes presenting a strong electron acceptor 3-methoxychromone unit and varied electron donor 2-aryl were developed. All three dyes showed remarkable polarity-dependent shifts of the emission maximum, which increase with extension of the dye conjugation. For the 3-methoxychromone bearing a 7-(diethylamino)-9,9-dimethylfluoren-2-yl donor group the difference between the excited and the ground state dipole moments, estimated from the Lippert-Mataga expression, reached 20 D, which is among the largest reported for neutral dipolar fluorophores. Moreover, the new dyes are characterized by significant two-photon absorption cross-section (up to 450 GM) and large fluorescence quantum yields. The strong decrease in the fluorescence quantum yields of the dyes in polar protic solvents was observed together with the increase in the non-radiative deactivation rates, which can originate from twisted intramolecular charge transfer and intermolecular proton transfer phenomena. In comparison to the parent 3-hydroxychromone derivatives, the new dyes presented significantly improved photostability, which confirms that photodegradation of 3-hydroxychromones occurs from a product of the excited-state intramolecular proton transfer (phototautomer). Finally, an application of the new dyes for probing local binding site polarity of serum albumin was shown. This new class of fluorescent dyes may serve as attractive building blocks for future molecular sensors utilizing environment-sensitive fluorophores.     

Excited-state intramolecular proton transfer distinguishes microenvironments in single- and double-stranded DNA

Herein, the efficient interaction of an environment-sensitive fluorophore that undergoes excited-state intramolecular proton transfer (ESIPT) with DNA has been realized by conjugation of a 3-hydroxychromone (3HC) with polycationic spermine. On binding to a double-stranded DNA (dsDNA), the ratio of the two emission bands of the 3HC conjugates changes up to 16-fold, so that emission of the ESIPT product increases dramatically. This suggests an efficient screening of the 3HC fluorophore from the water molecules in the DNA complex, which is probably realized by its intercalation into dsDNA. In sharp contrast, the 3HC conjugates show only moderate changes in the dual emission on binding to a single-stranded DNA (ssDNA), indicating a much higher fluorophore exposure to water at the binding site. Thus, the 3-hydroxychromone fluorophore being conjugated to spermine discriminates the binding of this polycation to dsDNA from that to ssDNA. Consequently, ESIPT-based dyes are promising for monitoring the interaction of polycationic molecules with DNA and probing the microenvironment of their DNA binding sites.     

H-bond cooperativity: polarisation effects on secondary amides

Formation of a H-bond with an amide carbonyl oxygen atom increases the strength of subsequent H-bonds formed by the amide NH, due to polarisation of the bond. The magnitude of this effect has been quantified by measuring association constants for the formation of 1 : 1 complexes of 2-hydroxylbenzamides with tri-n-butyl phosphine oxide. In 2-hydroxybenzamides, there is an intramolecular H-bond between the phenol OH group and the carbonyl oxygen atom. Comparison of the association constants measured for compounds with and without the 2-hydroxy group allows direct quantification of the effect of the intramolecular H-bond on the H-bond donor properties of the amide NH group. Substituents were used to modulate the strength of the intramolecular and intermolecular H-bonds. The presence of an intramolecular H-bond increases the strength of the intermolecular H-bond by more than one order of magnitude in n-octane solution. The increase in the H-bond donor parameter used to describe the amide NH group is directly proportional to the H-bond donor parameter of the phenol OH group that makes the intramolecular H-bond. These polarisation effects will lead to substantial cooperativity in complex systems that feature networks of non-covalent interactions, and the measurements described here provide a quantitative basis for understanding such phenomena.

Formation of an intramolecular phenol-amide H-bond leads to a dramatic increase in the H-bond donor strength of the amide NH group. Polarisation of the amide group is directly proportional to the polarity of the phenol H-bond donor.     

Amphiphilic Fluorescence Resonance Energy-Transfer Dyes: Synthesis,Fluorescence, and Aggregation Behavior in Water

Amphiphilic pyrene/perylene bis-chromophore dyes were synthesized from unsymmetrically substituted perylene bisimide dyes, which were obtained through three synthetic methods. The optical and aggregation behaviors of these functional dyes were studied by means of UV/Vis absorption and fluorescence spectroscopy, dynamic light scattering, and TEM. These dyes are highly fluorescent and cover the whole visible-light region. A donor/acceptor dye displays intramolecular fluorescence resonance energy transfer (FRET), with a high efficiency of up to 96.4 % from pyrene to perylene bisimide chromophores, which leads to a high fluorescence color sensitivity to environmental polarity. Under a λ=365 nm UV lamp, the light-emitting colors of the donor/acceptor dye change from green to yellow with increasing solvent polarity, which demonstrates application potential as a new class of FERT probes. The donor/acceptor dye in water was assembled into hollow vesicles with a narrow size distribution. The bilayer structure of the vesicular wall was directly observed by means of TEM. These vesicular aggregates in water are fluorescent at λ=650–850 nm within the near-infrared region.     

Electronic properties of multifurcated bent hydrogen bonds CH3...Y and CH2...Y

H-bonding angle angleYHX has an important effect on the electronic properties of the H-bond Y...HX, such as intra- and intermolecular hyperconjugations and rehybridization, and topological properties of electron density. We studied the multifurcated bent H-bonds of the proton donors H3CZ (Z = F, Cl, Br), H2CO and H2CF2 with the proton acceptors Cl(-) and Br(-) at the four high levels of theory: MP2/6-311++G(d,p), MP2/6-311++G(2df,2p), MP2/6-311++G(3df,3pd) and QCISD/6-311++G(d,p), and found that they are all blue-shifted. These complexes have large interaction energies, 7-12 kcal mol(-1), and large blue shifts, delta r(HC) = -0.0025 --0.006 A and delta v(HC) = 30-90 cm(-1). The natural bond orbital analysis shows that the blue shifts of these H-bonds Y...HnCZ are mainly caused by three factors: rehybridization; indirect intermolecular hyperconjugation n(Y) -->sigma*(CZ), in that the electron density from n(Y) of the proton acceptor is transferred not to sigma*(CH), but to sigma*(CZ) of the donor; intramolecular hyperconjugation n(Z) -->sigma*(CH), in that the electron density in sigma*(CH) comes back to n(Z) of the donor such that the occupancy in sigma*(CH) decreases. The topological properties of the electron density of the bifurcated H-bonds Y...H2CZ are similar to those of the usual linear H-bonds, there is a bond critical point between Y and each hydrogen, and a ring critical point inside the tetragon YHCH. However, the topological properties of electron density of the trifurcated H-bonds Y...H3CZ are essentially different from those of linear H-bonds, in that the intermolecular bond critical point, which represents a closed-shell interaction, is not between Y and hydrogen, but between Y and carbon.     

7-(2-Methoxycarbonylvinyl)-3-hydroxychromones: new dyes with red shifted dual emission

7-(2-Methoxycarbonylvinyl)-3-hydroxychromones have been synthesized using Heck coupling reaction from the corresponding 7-bromo-3-hydroxychromones. Introduction of the electron acceptor (2-methoxycarbonylvinyl) group at 7-position of 3-hydroxychromone results in a 30-40 nm red shift in absorption and >50 nm red shifts of both bands in emission. This derivatization allowed us to develop dyes with absorption maxima reaching 480 nm and dual emission in the red region of the spectrum. In comparison to the parent dyes, 7-acryl-3-hydroxychromones demonstrate significantly stronger solvatochromism. This is due to the acceptor group at 7-position, which increases the transfer character of the excited state of the dyes. The new dyes are highly prospective for the development of new fluorescent probes in biological research.     

Ultrasensitive fluorescent probe for the hydrophobic range of solvent polarities

The new 3-hydroxychromone derivative 2-(6-diethylaminobenzo[b]furan-2-yl)-3-hydroxychromone (FA) displays a dramatic solvent-dependent transformation of fluorescence spectra in the range of low-polarity solvents. The two well-separated emission bands change their relative intensities so that the short-wavelength band being of a very low intensity in hexane becomes dominant in the more polar ethyl acetate and trichloromethane. We suggest the participation in this effect of excited-state intramolecular proton transfer, which is characteristic for other 3-hydroxychromone and 3-hydroxyflavone derivatives, in the range of solvents of much higher polarities. Because of these unique properties, a number of spectroscopic parameters (positions of absorption and two fluorescence maxima, the ratio of their intensities and the fluorescence quantum yield) can be measured in this solvent range with multiparametric analysis of the data. In terms of solvent polarity, the shifts in both emission bands and their intensity ratio demonstrate a good correlation with empirical polarity scales E_T^N, P_y and SPP, while the absorption spectra reveal some deviations for the tested oxygen-containing solvent molecules. A good cross-correlation is observed between fluorescence spectral shifts and the ratio of band intensities. The latter provides the means for a dramatic amplification of solvent response. Thus, a new approach for ultrasensitive scaling and probing the solvent polarity in the low-polararity range can be suggested. It involves very simple ratiometric measurements at two emission bands and can be posed for a variety of applications. We present examples of these applications for distinguishing of polarities between methylated benzene derivatives, for quantitative assay of polar impurities in low-polar solvents and for detection of the changes of solvent polarity as a function of temperature.     

Pyrazolyc 3-hydroxychromones: Regulation of ESIPT reaction by the “flavonol-like” intramolecular hydrogen bonding to carbonyl group oxygen,which dominates over the “alternative” H-bond to heterocyclic nitrogen

Two isomeric pyrazole derivatives of 3-hydroxychromone (3HC) with and without the possibility of the multiple intramolecular hydrogen bonds formation were compared theoretically and experimentally with the aim to find out whether the excited state intramolecular proton transfer (ESIPT) reaction follows the traditional to the most of 3HCs “flavonol-like” direction towards the CO group oxygen or an “alternative” direction towards the heterocyclic nitrogen atom.Quantum-chemical modeling and comparative study of the experimental spectral parameters of the title compounds indicated the preferential realization of “flavonol-like” ESIPT to oxygen channel.The 3HC systems with the “alternative” intramolecular hydrogen bond to nitrogen were characterized as low fluorescent and practically unable to ESIPT with participation of the nitrogen containing heterocyclic unit.     

3-Hydroxybenzo[g]quinolones: dyes with red-shifted absorption and highly resolved dual emission

New derivatives of 3-hydroxyquinolone (3HQ) with a fused benzene ring (3-hydroxybenzo[g]quinolones) have been synthesized. They display a remarkable red shift of their absorption spectrum in comparison with other 3HQ analogs allowing their excitation by common He/Cd and Ar-ion lasers. As a result of their irreversible excited-state intramolecular proton transfer (ESIPT) reaction, they display a dual fluorescence in a series of solvents of varying polarities, starting from toluene to methanol. The dual emission of these dyes correlates well with solvent H-bond basicity, which is connected with the effect of this solvent property on the kinetics of the ESIPT reaction. In addition to their red-shifted absorption and fluorescence, these new derivatives show a larger separation of their two emission bands and a more appropriate range of their intensity ratio than the previously synthesized 3HQs. These properties allow an improved ratiometric evaluation of the local H-bond basicity of unknown environments, which will favor future applications of the new dyes in polymer and biological sciences.     

Solvent influence on excited-state intramolecular proton transfer in 3-hydroxychromone derivatives studied by cryogenic high-resolution fluorescence spectroscopy

High-resolution Shpol'skii spectra (recorded at 10 K in n-octane) of 3-hydroxychromone (3HC) substituted at the 2-position with a furan (3HC-F), a benzofuran (3HC-BF) or a naphthofuran group (3HC-NF) are presented. Being close analogues of 3-hydroxyflavone (3HF), these compounds can undergo excited-state intramolecular proton transfer (ESIPT). Luminescence can occur from the normal N* state (blue) or from the tautomeric T* state (green). Whether blue or green emission is observed is strongly dependent on hydrogen-bonding interactions with the environment. For all three chromones studied, high-resolution emission spectra in the green region (T*-->T) were obtained in pure n-octane, showing four sites with distinct emission bands and detailed vibrational structures, whereas no blue emission was detected. Contrary to the spectra published for 3HF, the emission lines were very narrow (line-broadening effects beyond detection) which implies that the ESIPT rate constants are >10(12) s(-1), at least 25 times lower than for 3HF. In order to study the effects of hydrogen-bonding solvents, four isomers of octanol (1-, 2-, 3- and 4-octanol) were added, forming 1:1 complexes with the 3HC derivatives. For all the combinations considered both blue and additional green emission was observed and in some cases narrow-banded spectra were obtained, mostly in the green. Only for the 3HC-NF/2-octanol complex, narrow-banded emission was found both in the blue and in the green region. It is demonstrated that these emissions come from different configurations of the complex. Possible structures for the two complex species are proposed, supported by semi-empirical calculations on complex formation enthalpies.     

Electronic properties of hydrogen-bonded complexes of benzene(HCN)(1-4): comparison with benzene(H2O)(1-4)

The electronic properties, specifically, the dipole and quadrupole moments and the ionization energies of benzene (Bz) and hydrogen cyanide (HCN), and the respective binding energies, of complexes of Bz(HCN)(1-4), have been studied through MP2 and OVGF calculations. The results are compared with the properties of benzene-water complexes, Bz(H(2)O)(1-4), with the purpose of analyzing the electronic properties of microsolvated benzene, with respect to the strength of the CH/π and OH/π hydrogen-bond (H-bond) interactions. The linear HCN chains have the singular ability to interact with the aromatic ring, preserving the symmetry of the latter. A blue shift of the first vertical ionization energies (IEs) of benzene is observed for the linear Bz(HCN)(1-4) clusters, which increases with the length of the chain. NBO analysis indicates that the increase of the IE with the number of HCN molecules is related to a strengthening of the CH/π H-bond, driven by cooperative effects, increasing the acidity of the hydrogen cyanide H atom involved in the π H-bond. The longer HCN chains (n ≥ 3), however, can bend to form CH/N H-bonds with the Bz H atoms. These cyclic structures are found to be slightly more stable than their linear counterparts. For the nonlinear Bz(HCN)(3-4) and Bz(H(2)O)(2-4) complexes, an increase of the binding energy with the number of solvent molecules and a decrease of the IE of benzene, relative to the values for the Bz(HCN) and Bz(H(2)O) complexes, respectively, are observed. Although a strengthening of the CH/π and OH/π H-bonds, with increasing n, also takes place for the Bz(H(2)O)(2-4) and Bz(HCN)(3-4) nonlinear complexes, Bz proton donor, CH/O, and CH/N interactions are at the origin of this decrease. Thus CH/π and OH/π H-bonds lead to higher IEs of Bz, whereas the weaker CH/N and CH/O H-bond interactions have the opposite effect. The present results emphasize the importance of both aromatic XH/π (X = C, O) and CH/X (X = N, O) interactions for understanding the structure and electronic properties of Bz(HCN)(n) and Bz(H(2)O)(n) complexes.     

Photoinduced intramolecular charge transfer in 9-aminoacridinium derivatives assisted by intramolecular H-bond

Fluorochromic dyes derived from 9-aminoacridinium containing a vinylene function with electron withdrawing groups such as diethyl [(acridinium-9-ylamino)methylene]malonate (I), ethyl [(acridinium-9-ylamino)methylene]cyanoacetate (II), [(acridinium-9-ylamino)methylene]malononitrile (III), are prepared and studied in their monoprotonated form. Absorption spectra of the new dyes are red shifted compared to that of the precursor dye. The observed dual fluorescence and multiexponential decay are ascribed to normal emission from the acridinium chromophore in addition to excited-state intramolecular charge transfer (ESICT) process. However, biexponential decay character is observed only for the dicyano derivative (compound III), whereas for the two other systems, more complex kinetics and a three-component decay is recovered. The analysis of the fluorescence decays in different solvents for the first two compounds reveals two short-lived components in the range of 160-350 ps and 1.1-3.0 ns, related to formation and decay of the ESICT state, plus a third one with decay time of about 9 ns, which is ascribed to the normal emission from the acridinium chromophore as an enol tautomer or as an intramolecular H-bond conformer (closed form tautomer). For the dicyano derivative, in which the absence of carbonyl group precludes the H-bond interaction, the biexponential fitting reveals a slightly fast formation rate of the ESICT state with values on the order of 10(10) s(-1), whereas its decay time is between 0.6 and 3.2 ns, depending on the solvent used.     

Intramolecular hydrogen bonding and cooperative interactions in carbohydrates via the molecular tailoring approach

In spite of many theoretical and experimental attempts for understanding intramolecular hydrogen bonding (H-bonding) in carbohydrates, a direct quantification of individual intramolecular H-bond energies and the cooperativity among the H-bonded networks has not been reported in the literature. The present work attempts, for the first time, a direct estimation of individual intramolecular O-H...O interaction energies in sugar molecules using the recently developed molecular tailoring approach (MTA). The estimated H-bond energies are in the range of 1.2-4.1 kcal mol(-1). It is seen that the OH...O equatorial-equatorial interaction energies lie between 1.8 and 2.5 kcal mol(-1), with axial-equatorial ones being stronger (2.0-3.5 kcal mol(-1)). The strongest bonds are nonvicinal axial-axial H-bonds (3.0-4.1 kcal mol(-1)). This trend in H-bond energies is in agreement with the earlier reports based on the water-water H-bond angle, solvent-accessible surface area (SASA), and (1)H NMR analysis. The contribution to the H-bond energy from the cooperativity is also estimated using MTA. This contribution is seen to be typically between 0.1 and 0.6 kcal mol(-1) when H-bonds are a part of a relatively weak equatorial-equatorial H-bond network and is much higher (0.5-1.1 kcal mol(-1)) when H-bonds participate in an axial-axial H-bond network.     

Fluorescent probe based on intramolecular proton transfer for fast ratiometric measurement of cellular transmembrane potential

Development of fast-response potentiometric probes for measuring the transmembrane potential Vm in cell plasma membranes remains a challenge. To overcome the limitations of the classical charge-shift potentiometric probes, we selected a 3-hydroxychromone fluorophore undergoing an excited-state intramolecular proton transfer (ESIPT) reaction that generates a dual emission highly sensitive to electric fields. To achieve the highest sensitivity to the electric field associated to Vm, we modified the fluorophore by adding two rigid legs containing terminal polar sulfonate groups to allow a deep vertical insertion of the fluorophore into the membrane. Fluorescence spectra of the new dye in lipid vesicles and cell membranes confirm the fluorophore location in the hydrophobic region of the membranes. Variation of Vm in lipid vesicles and cell plasma membranes results in a change of the intensity ratio of the two emission bands of the probe. The ratiometric response of the dye in cells is approximately 15% per 100 mV, and is thus quite large in comparison with most single-fluorophore, fast-response probes reported to date. Combined patch-clamp/fluorescence data further show that the ratiometric response of the dye in cells is faster than 1 ms. Analysis of the excitation and emission shifts further suggests that the probe responds to changes in Vm by a mechanism based on electrochromic modulation of its ESIPT reaction. Thus, for the first time, the ESIPT reaction has been successfully applied as a sensing principle for detection of transmembrane potential, allowing to couple classical electrochromic band shifts with changes in the relative intensities of the two well-separated emission bands. The fast two-band ratiometric response as well as the relatively high sensitivity of the new probe are the key features that make it useful for rapid detection of Vm changes in cell suspensions and single cells. Moreover, the new design principles proposed in the present work should allow further improvement of the probe sensitivity.     

Properties of a Ring Critical Pointas Measures of Intramolecular H-Bond Strength

Summary.  The topological parameters derived from the Bader theory such as the electron density and its Laplacian at the ring critical point (RCP) are analysed here as possible measures of the H-bond stength for intramolecular H-bonds. The parameters of RCP correlate well with the other properties of intramolecular H-bonds which are known as good measures of the H-bond strength. The calculations were performed on two samples of compounds with intramolecular H-bonds: the derivatives of malonaldehyde and the derivatives of o-hydroxybenzaldehyde. MP2 and HF calculations were carried out using a 6-311++G^** basis set. E-mail: slagra@krysia.uni.lodz.pl Received February 18, 2002; accepted (revised) May 27, 2002     

Steric control of the excited-state intramolecular proton transfer in 3-hydroxyquinolones: steady-state and time-resolved fluorescence study

3-Hydroxyquinolones (3HQs), similarly to their 3-hydroxychromone analogs, undergo excited state intramolecular proton transfer (ESIPT) resulting in dual emission. In the ground state, 2-phenyl-3HQ derivatives are not flat due to a steric hindrance between the 2-phenyl group and the 3-OH group that participates in the ESIPT reaction. To study the effect of this steric hindrance on the ESIPT reaction, a number of 3HQ derivatives have been synthesized and characterized in different organic solvents by steady-state and time-resolved fluorescence techniques. According to our results, 2-phenyl-3HQ derivatives undergo much faster ESIPT (by nearly 1 order of magnitude) than their 2-methyl-3HQ analogs. Moreover, 1-methyl-2-phenyl-3HQ having a strongly twisted 2-phenyl group undergoes a two- to three-fold slower ESIPT compared to 2-phenyl-3HQ. These results suggest that the flatter conformation of 2-phenyl-3HQ, which allows a close proximity of the 2-phenyl and 3-OH groups, favors a fast ESIPT reaction. The absorption and fluorescence spectra of the 3HQ derivatives additionally confirm that the steric rather than the electronic effect of the 2-phenyl group is responsible for the faster ESIPT reaction. Based on the spectroscopic studies and quantum chemical calculations, we suggest that the 2-phenyl group decreases the rotational freedom of its proximal 3-OH group in the more planar conformation of 2-phenyl-3HQ. As a result, the conformations of 3HQ, where the 3-OH group orients to form an intramolecular H-bond with the 4-carbonyl group, are favored over those with a disrupted intramolecular H-bond. Therefore, the 2-phenyl group sterically favors the intramolecular H-bond and thus accelerates the ESIPT reaction. This conclusion provides a new understanding of the ESIPT process in 3-hydroxyquinolones and related systems and suggests new possibilities for the design of ESIPT based molecular sensors and switchers.     

What can you learn from a molecular probe? New insights on the behavior of C343 in homogeneous solutions and AOT reverse micelles

The behavior of C343, a common molecular probe utilized in solvation dynamics experiments, was studied in homogeneous media and in aqueous and nonaqueous reverse micelles (RMs). In homogeneous media, the Kamlet and Taft solvatochromic comparison method quantified solute-solvent interactions from the absorption and emission bands showing that the solvatochromic behavior of the dye depends not only on the polarity of the medium but also on the hydrogen-bonding properties of the solvent. Specifically, in the ground state the molecule displays a bathochromic shift with the polarity polarizability (pi) and the H-bond acceptor (beta) ability of the solvents and a hypsochromic shift with the hydrogen donor ability (alpha) of the media. The carboxylic acid group causes C343 to display greater sensitivity to the beta than to the pi polarity parameter; this sensitivity increases in the excited state, while the dependence on alpha vanishes. This demonstrates that C343 forms a stable H-bond complex with solvents with high H-bond acceptor ability (high beta) and low H-bond donor character (low alpha). Spectroscopy in nonpolar solvents reveals J-aggregate formation. With information from the Kamlet-Taft analysis, C343 was used to explore RMs composed of water or polar solvents/sodium 1,4-bis-2-ethylhexylsulfosuccinate (AOT)/isooctane using absorption, emission, and time-resolved spectroscopies. Sequestered polar solvents included ethylene glycol (EG), formamide (FA), N,N-dimethylformamide (DMF), and N,N-dimethylacetamide (DMA). Dissolved in the AOT RM systems at low concentration, C343 exists as a monomer, and when introduced to the RM samples in its protonated form, C343 remains protonated driving it to reside in the interface rather than the water pool. The solvathochromic behavior of the dye depends the specific polar solvent encapsulated in the RMs, revealing different types of interactions between the solvents and the surfactant. EG and water H-bond with the AOT sulfonate group destroying their bulk H-bonded structures. While water remains well segregated from the nonpolar regions, EG appears to penetrate into the oil side of the interface. In aqueous AOT RMs, C343 interacts with neither the sulfonate group nor the water, perhaps because of intramolecular H-bonding in the dye. DMF and DMA interact primarily through dipole-dipole forces, and the strong interactions with AOT sodium counterions destroy their bulk structure. FA also interacts with the Na+ counterions but retains its H-bond network present in bulk solvent. Surprisingly, FA appears to be the only polar solvent other than water forming a "polar-solvent pool" with macroscopic properties similar to the bulk.     

Intriguing H-aggregate and H-dimer formation of coumarin-481 dye in aqueous solution as evidenced from photophysical studies

Photophysical properties of coumarin-481 (C481) dye in aqueous solution show intriguing presence of multiple emitting species. Concentration and wavelength dependent fluorescence decays and time-resolved emission spectra and area-normalized emission spectra suggest the coexistence of dye monomers, dimers, and higher aggregates (mostly trimers) in the solution. Because of the efficient intramolecular charge transfer (ICT) state to twisted intramolecular charge transfer (TICT) state conversion, the dye monomers show very short fluorescence lifetime of ~0.2 ns. Fluorescence lifetimes of dimers (~4.1 ns) and higher aggregates (~1.4 ns) are relatively longer due to steric constrain toward ICT to TICT conversion. Observed results indicate that the emission spectra of the aggregates are substantially blue-shifted compared to monomers, suggesting H-aggregation of the dye in the solution. Temperature-dependent fluorescence decays in water and time-resolved fluorescence results in water-acetonitrile solvent mixtures are also in support of the dye aggregation in the solution. Though dynamic light scattering studies could not recognize the dye aggregates in the solution due to their small size and low concentration, fluorescence up-conversion measurements show a relatively higher decay tail in water than in water-acetonitrile solvent mixture, in agreement with higher dye aggregation in aqueous solution. Time-resolved fluorescence results with structurally related non-TICT dyes, especially those of coumarin-153 dye, are also in accordance with the aggregation behavior of these dyes in aqueous solution. To the best of our knowledge, this is the first report on the aggregation of coumarin dyes in aqueous solution. Present results are important because coumarin dyes are widely used as fluorescent probes in various microheterogeneous systems where water is always a solvent component, and the dye aggregation in these systems, if overlooked, can easily lead to a misinterpretation of the observed results.     

An Expeditious Approach towards the Synthesis and Application of Water-Soluble and Photostable Fluorogenic Chromones for DNA Detection

The intensive research for hybridization probes based on organic molecules with fluorogenic properties is currently attracting particular attention due to their potential to efficiently recognize different DNA conformations and the local environment. However, most established organic chromophores do not meet the requirements of this task, as they do not exhibit good brightness in aqueous buffer media, develop aggregation and/or are not easily conjugated to oligodeoxynucleotides (ODNs) while keeping their photophysics intact. Herein, an important modification strategy was employed for a well-known fluorophore, 2-(4-(diethylamino)phenyl)-3-hydroxychromone (dEAF). Although this push–pull dye absorbs intensively in the visible range and shows emission with large Stokes shifts in all organic solvents, it is strongly quenched in water. This Achilles’ heel prompted us to implement a new strategy to obtain a series of dyes that retain all the photophysical features of dEAF in water, conjugate readily with oligonucleotides, and furthermore demonstrate sensitivity to hydration, thus paving the way for a high-performance fluorogenic DNA hybridization probe.     

Two-way effects between hydrogen bond and intramolecular resonance effect: an ab initio study on complexes of formamide and its derivatives with water

Ab initio calculations up to MP2/aug-cc-pVTZ//MP2/cc-pVTZ level, including natural charge population and natural resonance theory analyses, have been carried out to study the two-way effects between hydrogen bond (H-bond) and the intramolecular resonance effect by using the H-bonded complexes of formamide ( FAO) and its derivatives ( FAXs, X represents the heavy atoms in the substituent groups, CH 2, NH, SiH 2, PH, and S) with water as models. Unlike NH 3 and NH 2CH 3 which prefer being H-bond acceptors ( HA) to form H-bond with water, the amino groups in the six monomers, because of the resonance effect, prefer being H-bond donors ( HD) rather HA. Six monomers can all form HD complexes with water, and only two ( FAC and FASi) with the weakest resonance effect are able to form HA complexes with water. The HD H-bond and resonance effect enhance each other (positive two-way effects) whereas the HA H-bond and resonance effect weaken each other (negative two-way effects). The H-bond energies in the six HD complexes are nearly linearly correlated with the weights of the dipolar resonance in Pauling's model and the N-C bond lengths; the correlation coefficients are 0.91 and 0.93, respectively. The positive two-way effects also happens in FAO-water complex, in which the FAO CO group serves as HA ( HA co ). Interestingly, when the HD and HA co H-bonds are present in FAO H-bond complex simultaneously, the enhancements are much more significant, and the energies of the two types of H-bonds are much larger than those when only one type of H-bond is present, reflecting the cooperative effects. By using the knowledge to the two-way effects, we computationally designed a molecule ( FAO- BH 3 ) to increase H-bond energy. Because of the oxygen lone pair donation to the empty pi orbital of BH 3, FAO- BH 3 has a much stronger resonance effect than FAO. As a result, the H-bond energy (-5.55 kcal/mol) in HD H 2O ... FAO- BH 3 complex is much greater than the -3.30 kcal/mol in the HD H 2O...FAO complex. The two-way effects can be rationalized as follows: the resonance effect leads to intramolecular charge shifts in the monomers which facilitate or prevent the charge donation or acceptation of their H-bond partners. Therefore, the H-bonds are strengthened or weakened. In reverse, the charge donations or acceptations of their H-bond partners facilitate or prevent the intramolecular charge shifts in the monomer moieties, which enhance or weaken the resonance effect. The understanding to the two-way effects may be helpful in drug design and refinement by modulating the H-bond strength and in building empirical H-bond models to study large biological molecules. The study supports Pauling's resonance model.