Rational design of the benzothiazole-based fluorescent scaffold for tunable emission

The 2-(2-hydroxyphenyl)-benzothiazole (HBT) fluorophore has attracted considerable attention due to its excited-state intramolecular proton transfer (ESIPT) based emission and its large Stokes shift. However, this fluorophore possesses several disadvantages including low quantum yield and short emission in the blue range. In this study, by coupling HBT at the ortho-, meta-, and para-positions to the hydroxyl group with different heterocycles to extend the conjugation system, we have successfully obtained new fluorophores with tunable emissions both in solution and in the solid-state (409–652?nm). Notably, all of the derivatives demonstrated improved quantum yields compared with the parent HBT structure. Moreover, selected compounds have been shown to shine brightly in live cells, indicating promising potential for bioimaging.     

Substituent Modulation from ESIPT to ICT Emission in Benzoimidazolphenyl-methanones Derivatives: Synthesis,Photophysical and DFT Study

Design and synthesis of five new derivatives of benzophenone based imidazole dyes is presented. Synthesized dyes were well characterized by ¹H NMR, ¹³C NMR, FT-IR and mass analysis. Dyes contain a secondary acceptor, ESIPT core and different donors forming (D-ESIPT core-A) as basic skeleton in order to study both ESIPT and ICT systematically in this same class of dyes. Dyes without a donor substituent showed ESIPT emission while dyes with a substituted strong donor showed intramolecular charge transfer (ICT) emission. Moreover emission properties of methoxy analogue dyes has been studied to further confirm non-ESIPT emission in dyes without donors and ICT emission in strong donor substituted dyes. All dyes exhibited long range emissions from 392 to 567 nm. Dyes exhibiting ESIPT emission showed negative solvatochromism while ICT emission exhibiting dyes shows positive solvatochromism. ICT and ESIPT characteristics are well correlated with polarity functions plots and Mulliken–Hush analysis. Experimental observations are well supported by TD–DFT and computed energies. The electrophilicity index has been calculated to get details of the stabilities of possible tautomers.     

Fluorescent Probes Based on Charge and Proton Transfer for Probing Biomolecular Environment

Fluorescent probes for sensing fundamental properties of biomolecular environment, such as polarity and hydration, help to study assembly of lipids into biomembranes, sensing interactions of biomolecules and imaging physiological state of the cells. Here, we summarize major efforts in the development of probes based on two photophysical mechanisms: (i) an excited-state intramolecular charge transfer (ICT), which is represented by fluorescent solvatochromic dyes that shift their emission band maximum as a function of environment polarity and hydration; (ii) excited-state intramolecular proton transfer (ESIPT), with particular focus on 5-membered cyclic systems, represented by 3-hydroxyflavones, because they exhibit dual emission sensitive to the environment. For both ICT and ESIPT dyes, the design of the probes and their biological applications are summarized. Thus, dyes bearing amphiphilic anchors target lipid membranes and report their lipid organization, while targeting ligands direct them to specific organelles for sensing their local environment. The labels, amino acid and nucleic acid analogues inserted into biomolecules enable monitoring their interactions with membranes, proteins and nucleic acids. While ICT probes are relatively simple and robust environment-sensitive probes, ESIPT probes feature high information content due their dual emission. They constitute a powerful toolbox for addressing multitude of biological questions.     

A General Strategy for Through-Bond Energy Transfer Fluorescence Probes Combining Intramolecular Charge Transfer: A Silyl Ether System for Endogenous Peroxynitrite Sensing

A general strategy is reported for developing through-bond energy transfer (TBET) fluorescence probes by combining intramolecular charge transfer (ICT). The strategy uses a coplanar donor-π-bridge-acceptor system (SiOPh-PyOH) without spirolactam. The off-on switch of TBET and ICT is controlled by coplanar structure changes in the sensing process instead of spirolactam ring-opening in traditional TBET probes. DFT calculations showed that the energy and charge transfers from SiOPh to PyOH are prohibited. Since the SiOPh has no fluorescence, the probe SiOPh-PyOH shows fluorescence properties similar to that of pyrene. After sensing ONOO⁻, the silyl ether is removed and the probe changes into ⁻OPh-PyO⁻. Electron-donating ICT from ⁻OPh to PyO⁻ induces a large redshift of emission to 594 nm (179 nm shift). TBET from ⁻OPh to PyO⁻ ensures the probe exhibits a large pseudo-Stokes shift of 213 nm. Furthermore, the probe was successfully used in endogenous ONOO⁻ detection. This study offers a new strategy for the construction of TBET probes emitting in the red region without spirolactam ring-opening, a new ONOO⁻ sensing system using silyl ether as a reaction site, and a method for the deprotection of silyl ethers with ONOOH under mild conditions.     

Strong solvatochromic fluorescence from the intramolecular charge-transfer state created by excited-state intramolecular proton transfer

In this work, we report a peculiar positive solvatochromism in the keto emission of the acceptor-substituted 2-(2'-hydroxyphenyl)benzoxazoles (HBO), which originates from the excited-state intramolecular proton transfer (ESIPT) followed by the intramolecular charge transfer (ICT) and subsequent solvent relaxation. This transient evolution of enhanced ICT characteristic triggered by ESIPT, which is first observed in this work, is responsible for the novel concept of a fast hyperpolarizability modulator as well as the unique solvatochromic behavior.     

Photobase-Driven Excited-State Intramolecular Proton Transfer (ESIPT) in a Strapped π-Electron System

We report a new design strategy for an excited-state intramolecular proton transfer (ESIPT) fluorophore that can be used in acidic media. A photobasic pyridine-centered donor-acceptor-donor-type fluorophore is combined with a basic trialkylamine “strap”. In the presence of an acid, protonation occurs predominantly at the amine moiety in the ground state. A single-crystal X-ray diffraction analysis confirmed the formation of a pre-organized intramolecular hydrogen-bonded structure between the resulting ammonium moiety and the pyridine ring. Upon excitation, the intramolecular charge-transfer transition increases the basicity of the pyridine moiety in the excited state, resulting in proton transfer from the amine to the pyridine moiety. Consequently, the fluorophore takes on a polymethine-dye character in the ESIPT state, which gives rise to significantly red-shifted emission with an increased fluorescence quantum yield.     

Strategic emission color tuning of highly fluorescent imidazole-based excited-state intramolecular proton transfer molecules

Highly fluorescent molecules harnessing the excited state intramolecular proton transfer (ESIPT) process are promising for a new generation of displays and light sources because they can offer very unique and novel optoelectronic properties which are different from those of conventional fluorescent dyes. To realize innovative ESIPT devices comprising full emission colors over the whole visible region, a molecular design strategy for predictable emission color tuning should be established. Here, we have developed a general strategy for a wide-range spectral tuning of imidazole-based ESIPT materials based on three different strategies--introduction of a nodal plane model, extension of effective conjugation length, and modification of heterocyclic rings. A series of nine ESIPT molecules were designed, synthesized and comprehensively investigated for their characteristic emission properties. All these molecules commonly showed no clear and transparent visible range absorption with no absorption color, but showed different colors of intense photoluminescence over broad visible regions from 450 nm (HPI) to 630 nm (HPNO) depending on their molecular structure. With the aid of density functional theory and time-dependent DFT calculations using M06, wB97XD, and B3LYP parameters with the 6-31G(d,p) basis set, these tuned emission bands of nine emitters were assigned from the stabilized excited state conformations that were derived from modified molecular structures.     

Unraveling solvent-dependent hydrogen bonding interaction and excited-state intramolecular proton transfer behavior for 2-(benzo[d]thiazol-2-yl)-4-(9H-carbazol-9-yl)phenol: A theoretical study

Organic chemosensors with excited-state intramolecular proton transfer (ESIPT) behavior have attracted much attention because it has great potential in a wide range of applications. Considering the paramount behavior of excited-state relaxation, in this work, we mainly focus on deciphering photo-induced hydrogen bonding effects and ESIPT mechanism for the novel 2-(benzo[d]thiazol-2-yl)-4-(9H-carbazol-9-yl)phenol (mCzOH) dye. Considering the effects of different solvents on excited-state dynamics of mCzOH flurophore, we adopt four solvents with different polarities. Analyses of fundamental structural changes, infrared (IR) vibrational spectra, and core valence partition index between S₀ and S₁ state, we confirm hydrogen bond O H···N of mCzOH should be enhanced via photoexcitation. Especially, the increase of solvent polarity could promote hydrogen bonding strengthening degree. Intramolecular charge transfer (ICT) resulting from photoexcitation qualitatively facilitates the ESIPT occurrence to a large extent. For further checking and probing into ESIPT mechanism, via constructing potential energy curves (PECs) in four solvents, we clarify the ESIPT behavior for mCzOH. Most worthy of mention is that polar solvent plays critical roles in lowering potential barrier of ESIPT reaction and in facilitating ESIPT process. We not only clarify the detailed excited-state process, but also present the solvent-polarity-dependent ESIPT mechanism for mCzOH fluorophore.     

New sensing mechanisms for design of fluorescent chemosensors emerging in recent years

During the past decade, fluorescent chemosensors have become an important research field of supramolecular chemistry and have attracted great attention because of their simplicity, high selectivity and sensitivity in fluorescent assays. In the design of new fluorescent chemosensors, exploration of new sensing mechanisms between recognition and signal reporting units is of continuing interest. Based on different photophysical processes, conventional sensing mechanisms including photo-induced electron transfer (PET), intramolecular charge transfer (ICT), metal-ligand charge transfer (MLCT), twisted intramolecular charge transfer (TICT), electronic energy transfer (EET), fluorescence resonance energy transfer (FRET), and excimer/exciplex formation have been investigated and reviewed extensively in the literature. This tutorial review will mainly focus on new fluorescent sensing mechanisms that have emerged in the past five years, such as aggregation-induced emission (AIE) and C=N isomerization, which can be ascribed to fluorescence changes via conformational restriction. In addition, excited-state intramolecular proton transfer (ESIPT) has not been well reviewed yet, although a number of chemosensors based on the ESIPT mechanism have been reported. Thus, ESIPT-based chemosensors have been also summarized in this review.     

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.     

Tactfully revealing the working mechanisms on a tetraarylimidazole derivative: AIE characteristic,ESIPT process and ICT effect integrating in one molecule

Recently, a novel tetraarylimidazole derivative 2-(benzo[d]thiazol-2-yl)-4-(4,5-bis(4-methoxyphenyl)-1-phenyl-1H-imidazol-2-yl)-phenol (be called MHBT herein) was architectured by our research group showing the fascinating synergy of aggregation-induced emission (AIE) characteristic, excited-state intramolecular proton transfer (ESIPT) mechanism and intramolecular charge transfer (ICT) effect. Nevertheless, a detailed and reasonable interpretation of its mechanisms both in theory is urgently needed. Consequently, to unveil the working mechanism meticulously, herein, we tactfully applied density functional theory (DFT) and time-dependent density functional theory (TD-DFT) methods to illuminate the underlying mechanisms in different solvent conditions. After optimizing the structures, the geometric parameters of hydrogen bonds (HBs), the infrared (IR) vibrational spectrum, the reduced density gradient (RDG) isosurfaces were calculated in detail, vividly explaining how the enhancement of HBs behaved as the driving force to proceed ESIPT process. Simultaneously, the frontier molecular orbitals (FMOs) combined with the potential energy curves (PECs) were conducted to interpretate the role and character of ICT and ESIPT in molecule MHBT. Further, the PECs of MHBT for dihedral angles in different organic solvents were calculated to compare the dominant torsion degree, rationalizing the AIE phenomenon from the view of the restriction of intramolecular rotation process. This work may well underpin the understanding of the interaction between different mechanisms in fluorescent dyes and thereby provide meaningful guideline for the design and construction of ideal molecules     

Excited state proton transfer and solvent relaxation of a 3-hydroxyflavone probe in lipid bilayers

The photophysics of a ratiometric fluorescent probe, N-[[4'- N, N-diethylamino-3-hydroxy-6-flavonyl]methyl]- N-methyl- N-(3-sulfopropyl)-1-dodecanaminium, inner salt (F2N12S), incorporated into phospholipid unilamellar vesicles is presented. The reconstructed time-resolved emission spectra (TRES) unravels a unique feature in the photophysics of this probe. TRES exhibit signatures of both an excited-state intramolecular proton transfer (ESIPT) and a dynamic Stokes shift associated with solvent relaxation in the lipid bilayer. The ESIPT is fast, being characterized by a risetime of approximately 30-40 ps that provides an equilibrium to be established between the excited normal (N*) and the ESIPT tautomer (T*) on a time scale of 100 ps. On the other hand, the solvent relaxation displays a bimodal decay kinetics with an average relaxation time of approximately 1 ns. The observed slow solvent relaxation dynamics likely embodies a response of nonspecific dipolar solvation coupled with formation of probe-water H-bonds as well as the relocation of the fluorophore in the lipid bilayer. Taking into account that ESIPT and solvent relaxation are governed by different physicochemical properties of the probe microenvironment, the present study provides a physical background for the multiparametric sensing of lipid bilayers using ESIPT based probes.     

Installation/modulation of the emission response via click reaction

We have demonstrated the installation of a fluorescence property into a nonfluorescent precursor and modulation of an emission response of a pyrene fluorophore via click reaction. The synthesized fluorophores show different solvatochromicity and/or intramolecular charge transfer (ICT) feature as is revealed from the UV-visible, fluorescence photophysical properties of these fluorophores, and DFT/TDDFT calculation. We observed that some of the synthesized fluorophores showed purely ICT character while emission from some of them arose from the LE state. A structureless and solvent polarity-sensitive dual emission behavior was observed for one of the triazolylpyrene fluorophores that contains an electron-donating -NMe(2) substituent (fluorophore, 7a). Conversely, triazolylpyrene with an electron-withdrawing -CN group (fluorophore, 7b) showed a solvent polarity-independent vibronic emission. The effect of ICT on the photophysical properties of these fluorophores was studied by fluorescence emission spectra and DFT/TDDFT calculations. Fluorescence lifetimes were also measured in different solvents. All of our findings revealed the delicate interplay of structure and emission properties and thus having broader general utility. As the CT to LE intensity ratio can be employed as a sensing index, the dual emissive fluorophore can be utilized in designing the molecular recognition system too. We envisage that our investigation is of importance for the development of new fluorophores with predetermined photophysical properties that may find a wide range of applications in chemistry, biology, and material sciences.     

Excited-state intramolecular proton transfer in 2-(2'-arylsulfonamidophenyl)benzimidazole derivatives: the effect of donor and acceptor substituents

A series of water-soluble 2-(2'-arylsulfonamidophenyl)benzimidazole derivatives containing electron-donating and accepting groups attached to various positions of the fluorophore pi-system has been synthesized and characterized in aqueous solution at 0.1 M ionic strength. The measured pK(a)'s for deprotonation of the sulfonamide group of monosubstituted derivatives range between 6.75 and 9.33 and follow closely Hammett's free energy relationship. In neutral aqueous buffer, all compounds undergo efficient excited-state intramolecular proton transfer (ESIPT) to yield a strongly Stokes-shifted fluorescence emission from the phototautomer. Upon deprotonation of the sulfonamide nitrogen at high pH, ESIPT is interrupted to yield a new, blue-shifted emission band. The peak absorption and emission energies were strongly influenced by the nature of the substituents and their attachment positions on the fluorophore pi-system. The fluorescence quantum yield of the ESIPT tautomers revealed a significant correlation with the observed Stokes shifts. The study provides valuable information regarding substituent effects on the photophysical properties of this class of ESIPT fluorophores in an aqueous environment and may offer guidelines for designing emission ratiometric pH or metal-cation sensors for biological applications.     

Molecularly near-infrared fluorescent theranostics for in vivo tracking tumor-specific chemotherapy

Molecularly near-infrared(NIR) theranostics, combining in vivo sensing and tumor-specific therapeutic capability within one molecular system, have received considerable attention in recent years. Compared with the visible fluorescence imaging, NIR imaging(emission wavelength at 650–900 nm) possesses unique advantages including the minimum photodamage to biological samples, deep penetration, and low interference from auto-fluorescence. In over past decades, there has been an explosive development in the design of molecular imaging contrasts and imaging-guided therapeutics. In this review, we have sumarried the strategies of the NIR theranostics for imaging and tumor-specific chemotherapy applications in living systems. It is noted that the molecularly NIR theranostic design strategy could address current challenges of real-time in vivo sense-and-release for the intelligent biosensing and personalized treatment.     

Amphiphilic ESIPT benzoxazole derivatives as prospective fluorescent membrane probes

Fluorescent amphiphilic benzoxazole derivatives were synthesized and used to produce photoactive phosphatidylcholine (PC) liposomes by reserve-phase evaporation. The dyes absorbed in the UV region and were fluorescent in the blue-green region (determined by solvent polarity). The alkyl chain length seemed to play a fundamental role in the photophysics of the benzoxazole fluorophore in reverse liposomes, and despite the same ESIPT core and phospholipid building block, each amphiphilic dye had a particular emission profile related to the dye location in the liposome. The fluorescence emission spectra from dye 5 showed that its fluorophore experienced a polar environment, due to the single normal emission, while dyes 6–7 had (in part) a normal emission, and the main fluorescent band ascribed to the ESIPT emission indicated a more hydrophobic environment. Despite the complex fluorescent profiles, the benzoxazole derivatives could be successfully introduced into the reverse liposome structure due to the interaction between the alkyl chain and PC bilayer.     

Reversal of Solvatochromism: A New Strategy to Construct Activatable Two-photon Fluorescent Probes for Sensing

Two-photon (TP) imaging with a donor-acceptor (D?A) type fluorophore is an emerging tool for bioimaging and sensing. However, current TP probes suffer from serious solvatochromic quenching in aqueous solution due to their strong intramolecular charge transfer (ICT) in excited states. In this work, based on solvatochromism reversal, we report a novel strategy to develop TP probes for bioimaging. Specifically, compared with the normal two-photon probes that showed a fluorescence off with ICT suppressed, the novel probes exhibited strong fluorescence in the aqueous solution when their ICT was inhibited. This strategy not only provides a new way for the design of high-performance TP probes, but also expands the biological analysis toolbox for use in living systems.     

Fluorescent sensing of anions based on excited state intramolecular proton transfer in <Emphasis Type="Italic">N</Emphasis>-(3-hydroxy-2-naphthamido)-<Emphasis Type="Italic">N</Emphasis>′-phenylthiourea

A neutral N-amidothiourea-based excited state intramolecular proton transfer (ESIPT) anion receptor bearing an o-hydroxynaphthamide fluorophore and a thiourea binding site, N-(3-hydroxy-2-naphthamide)-N′-phenylthiourea (1a), was designed and synthesized. Fluorescence and absorption response of 1a toward anions were assessed in acetonitrile. IR and NMR experiments indicated that the “OH⋯O=C” intramolecular hydrogen bond (IHB) in 1a was weak so that it only exhibited the short-wavelength normal emission other than ESIPT fluorescence. Due to the high anion binding affinity of the N-amidothiourea binding site and the formation of a hydrogen binding network in the 1a-anion complex, 1a underwent structural change upon anion binding that strengthens the “OH⋯O=C” IHB, leading to the ESIPT and the observation of the long-wavelength ESIPT emission whereas the normal fluorescence is quenched. On the basis of NMR and fluorescence titrations and control experiments with model compounds, a sensing mechanism of the anion-binding-induced ESIPT was proposed. Supported by the National Natural Science Foundation of China (Grant Nos. 20425518, 20675069 & 20835005) and the National Fund for Fostering Talents of Basic Science (Grant No. J0630429)     

Zn2+-triggered excited-state intramolecular proton transfer: a sensitive probe with near-infrared emission from bis(benzoxazole) derivative

Near-infrared (NIR) emission can offer distinct advantages for biological applications. A fluorescent sensor, Zinhbo-1, based on bis(benzoxazole) ligand with 2,2'-dipicolylamine (DPA) as receptor, was synthesized. In aqueous solution, Zinhbo-1 demonstrates high sensitivity and selectivity for sensing Zn(2+) with about 10-fold enhancement and nanomolar sensitivity (K(d) = 0.29 nM). Moreover, sensor Zinhbo-1 can detect Zn(2+) in near-infrared region (over 700 nm) with large Stokes shift (ca. 230 nm) attributing to the Zn(2+)-induced excited state intramolecular proton transfer (ESIPT).     

Excited state intramolecular proton transfer in 2-(2'-arylsulfonamidophenyl)benzimidazole derivatives: insights into the origin of donor substituent-induced emission energy shifts

Donor-substituted 2-(2'-arylsulfonamidophenyl)benzimidazoles undergo efficient excited-state intramolecular proton transfer (ESIPT) upon photoexcitation. The tautomer emission energy depends strongly on the substituent attachment position on the fluorophore pi-system. While substitution with a donor group in the para-position relative to the sulfonamide moiety yields an emission energy that is red-shifted relative to the unsubstituted fluorophore, fluorescence of the meta-substituted derivative appears blue-shifted. To elucidate the origin of the surprisingly divergent emission shifts, we performed detailed photophysical and quantum chemical studies with a series of methoxy- and pyrrole-substituted derivatives. The nature and contribution of solvent-solute interactions on the emission properties were analyzed on the basis of solvatochromic shift data using Onsager's reaction field model, Reichardt's empirical solvent polarity scale ET(30), as well as Kamlet-Abboud-Taft's empirical solvent index. The studies revealed that all ESIPT tautomers emit from a moderately polarized excited-state whose dipole moment is not strongly influenced by the donor-attachment position. Furthermore, the negative solvatochromic shift behavior was most pronounced in protic solvents presumably due to specific hydrogen-bonding interactions. The extrapolated gas-phase emission energies correlated qualitatively well with the trends in Stokes shifts, suggesting that solute-solvent interactions do not play a significant role in explaining the divergent emission energy shifts. Detailed quantum chemical calculations not only confirmed the moderately polarized nature of the ESIPT tautomers but also provided a rational for the observed emission shifts based on the differential change in the HOMO and LUMO energies. The results gained from this study should provide guidelines for tuning the emission properties of this class of ESIPT fluorophores with potential applications in analytical chemistry, biochemistry, or materials science.