key: cord-0746092-i45uqj0y
authors: Li, Yonghao; Dahal, Dipendra; Abeywickrama, Chathura S.; Pang, Yi
title: Progress in Tuning Emission of the Excited-State Intramolecular Proton Transfer (ESIPT)-Based Fluorescent Probes
date: 2021-03-04
journal: ACS Omega
DOI: 10.1021/acsomega.0c06252
sha: e0f233242a7463cc45bac169f143e8ecf0f2a94c
doc_id: 746092
cord_uid: i45uqj0y

[Image: see text] In this review, we will summarize our recent progress in the design and application of novel organic sensors with emission in the near-infrared region (600–900 nm). By coupling different functional groups with excited-state intramolecular proton transfer (ESIPT) segments, new probes are developed to achieve a large Stokes shift, high sensitivity, and selectivity and to tune the emission toward the near-infrared region. The developed probes exhibit attractive optical properties for bioimaging and environmental science applications. In addition, we further discuss the photophysical properties of ESIPT dyes and how their fluorescence could be affected by structural/environmental factors, which should be considered during the development of robust ESIPT-based fluorescence probes. Their potential applications as imaging reagents are illustrated for intracellular membranes, mitochondria, lysosomes, and some biomolecules.

Fluorescence organic dyes have been widely used in biology, pharmacology, and environmental science as selective sensors for specific analytes for more than a century. 1 The novel fluorescent dyes with excellent photostability, high fluorescence quantum yield, high sensitivity, and selectivity are desirable for medical diagnostics, cell biology applications, and drug delivery in recent years, especially after the global pandemic. 2 The species to be detected can react with sensors 3, 4 or interact through host−guest interaction 5 and then give a fluorescence signal. Some molecular probes that emit in the near-infrared (NIR) region are ideal to be developed for analytical and biological applications such as in cell imaging. In comparison with commercial dyes that emit in the UV/vis range, NIR dyes have advantages of deep tissue penetration, less tissue damage, and minimal interference caused by tissue absorption and autofluorescence. 6−10 Donor−acceptor-involved fluorescence mechanisms for certain dyes have been well-investigated over the past decade. Typically, there are three well-known mechanisms which include PET (photoinduced electron transfer), FRET (Forster resonance energy transfer), and ICT (internal charge transfer). 1 Traditional dyes based on these three mechanisms always have a noticeable restriction. Their characteristic small Stokes shift (Δλ < 25 nm) causes self-quenching and impedes their application in species detection, as the detected signals may not accurately reflect the analyte concentrations. 11, 12 Excited-state intramolecular proton transfer (ESIPT), discovered by Weller in the 1950s, 13 involves a four-level photochemical process, which leads to low energy fluorescence with a large Stokes shift and enhanced quantum yield. 1, 14 The molecules typically contain a hydrogen bond donor (−OH) and exist in an enol form in the ground state. When the enol tautomer 1 is excited upon photon absorption, the hydrogen atom is transferred to the −CN− group (a proton acceptor), resulting in the excited keto tautomer 2 that gives longwavelength emission (Scheme 1). 15 2-(2′-Hydroxyphenyl)benzoxazole (HBO, X = O in 1) and 2-(2′-hydroxyphenyl)benzothiazole (HBT, X = S in 1) and their derivatives are the most common ESIPT chromophores. 1 However, the emission of such designs is often limited to blue, green, and orange-red colors.

The proton transfer from the phenol to the −CN− group can be viewed simply as an acid−base reaction, as the phenolic proton is known to become more acidic in the excited state (pK a ∼ 5.7). 16 The acidity of the phenolic proton could be influenced by a suitable electron-withdrawing or electrondonating substituent, which could actively perturb the proton transfer process. 7−10 2. ESIPT-BASED FLUORESCENT PROBES AND THEIR APPLICATIONS In the effort of seeking ESIPT fluorophores with NIR emission and large Stokes shifts, our group has recently synthesized a series of Zinhbo compounds, 5 in which a strong e-donating group (i.e., −OH) is placed at the para-position of the phenol group that is involved for ESIPT (Scheme 2). Interestingly, its zinc complex (i.e., "Zinhbo-Zn 2+ ") gives NIR emission while retaining the dual emission feature of ESIPT fluorophores (with λ em at ∼536 and 750 nm from its enol and keto tautomers, respectively). These compounds have been demonstrated to be useful for intracellular sensing of Zn 2+ cations via live cell fluorescence imaging.

An alternative strategy to tune the optical absorption/ emission is attaching an electron-withdrawing group to ESIPT chromophores. Among very limited examples is HBO derivative 3, in which a strong electron-withdrawing group is attached to the benzoxazole segment. 17 Compound 3 is reported to exhibit λ abs ≈ 370 nm, which is only slightly affected by solvent polarity. Whereas its enol form gives emission at λ em ≈ 430 nm, its keto form gives solventdependent emission (λ em ≈ 525 nm in cyclohexane and ∼600 nm in ether). Interestingly, the ESIPT enables a strong donor− acceptor interaction, as shown by the arrows in 4 (Scheme 3).

However, the compound exhibits low fluorescence quantum yield (φ fl = 2.6 × 10 −3 in cyclohexane) and a radiative lifetime of ∼0.725 ns in nonpolar solvent with the absence of keto emission.

It is assumed that the electronic impact can be achieved by introducing a substituent on the phenol ring of the HBO unit. One example is 5 (Scheme 4), which is reported to exhibit two absorption bands (λ abs = 382 and 458 nm) and very weak fluorescence (λ em ≈ 510 nm) in DMSO/H 2 O solution (8:2, v/ v) at 25°C. 4 It appears that the classical e-withdrawing groups may play a very limited role in tuning the emission toward longer wavelength.

Another strategy is to couple the traditional cyanine dyes with ESIPT chromophores. As a typical class of dyes, cyanines possess significant advantages including high fluorescence quantum yields and tunable emission, 18 which have made them popular in various applications for decades. However, conventional cyanine dyes display small Stokes shifts, e.g., often less than 30 nm. 19 It can be assumed that the combination of ESIPT chromophore with cyanine structures could lead to attractive new dyes that exhibit long-wavelength emission (of cyanines), while still giving ESIPT emission with a large Stokes shifts. As illustrated in Scheme 5, a cyanine terminal group (Y + ) is attached to the ortho-position of the central phenol ring. In the ground state of 6a, the ESIPT and cyanine fragments share the central phenol ring. However, the benzoxazole (part of the ESIPT unit) has a poor electronic connection with the cyanine terminal group (−CHCH−Y + ), as they are placed at 2.6-position of the central phenol. The electronic connection between the two groups is greatly enhanced in the excited keto state 6b, thereby generating ESIPT emission with a large Stokes shift. The presence of an ewithdrawing group could also enhance the acidity of the phenolic proton, thereby increasing the ESIPT emission.

Recently, Paul et al. reported that 7a was used for hydrogen sulfide detection. 20 A later study from our group 10 suggests that the absorption peaks at ∼428 and 570 nm could be attributed to the neutral form Ar−OH and its anionic form Ar−O − , respectively. By comparison with its model compound (10) , the study revealed that the absorption, λ abs , of 8 is basically determined by the cyanine fragment ( Figure 1 ). However, the presence of the ESIPT unit in 8 causes a significant bathochromic shift in emission, by about ∼100 nm in comparison to that with 10. 8 Strong emission from only the keto tautomer of 8 is observed (Figure 1 ), in contrast to traditional ESIPT dyes that give emission from both enol and keto tautomers. 21 It appears The synthesis of additional examples allows us to further evaluate the properties of ESIPT−cyanine dyes. As shown in Table 1 , the absorption λ abs values change with the type of the e-withdrawing groups (compare 11, 12, and 9), consistent with the assumption that the dye's conjugation length is basically defined by the cyanine segment. 10 Temperature Effect on Emission. As seen from 6, the ESIPT−cyanine dyes include an ESIPT unit and a cyanine segment. Once the ESIPT occurs in the excited state (upon photon absorption), it enables a strong donor−acceptor interaction (shown in 6b). Low-temperature fluorescence has been used to evaluate the impact of the cyanine segment. 7, 8 As illustrated in Figure 2a , fluorescence at 712 nm can be observed from the ethanol solution of 8 at room temperature. As the temperature is decreased by cooling the solution with liquid nitrogen, the fluorescence signal is increased significantly, and the emission peak is blue-shifted to 635 nm. The spectral blue shift occurs in a narrow range of temperature from −100 to −125°C, as the solution is frozen to prevent intramolecular charge transfer. 8 It should be noted that the keto emission predominates at low temperature (Figure 2b ), as reported from HBO 1 in MeOH solution in a previous study. 15 Since ESIPT will not be restricted by low temperature (as observed from spectra of 1), the emission peak at 635 nm, observed from low-temperature fluorescence spectra of 8, is likely from the keto tautomer, and the observed spectral blue shift can be attributed mainly to ICT.

Effect of Regiochemistry. The e-withdrawing group in 6 is at the ortho-position of the phenol ring. Recently, our group examined the possibility of moving the e-withdrawing group to the para-position of the phenol ring by synthesis of 13. 22 In CH 2 Cl 2 , 13a exhibited the fluorescence λ em ≈ 608 nm (φ fl ≈ 0.17), which was notably blue-shifted in comparison to that with 8. Introduction of the second hydroxy group, as seen in 14, could generate a notable bathochromic shift in λ abs (in CH 2 Cl 2 ). However, the emission of 14 was slightly blueshifted, thus decreasing the Stokes shift. The results clearly illustrate that the optical properties of this system are tunable. At a similar time, Rodembusch et al. 23 reported the synthesis of 13b (with R 2 = −CH 3 and −C 8 H 17 ), which was shown to be a useful pH sensor, although their conclusion is different (in terms of ESIPT event and Stokes shift) (Scheme 6) Biological Cell Studies. Despite interests in using ESIPT for molecular probe design in recent year, 1 little is known about using ESIPT probes for imaging subcellular organelles until recently (in 2017). 8 It should be noted that the phenolic proton becomes acidic, with pK a ≈ 5.7 estimated from 8, as a consequence of attaching the cyanine fragment. The ability of 8 to give a large fluorescence turn on in an acidic environment (pH < 6) leads to its application for fluorescence imaging of intracellular organelles, i.e., lysosomes, which are acidic (pH ≈ 4−5) for optimal function. 24 The most attractive features of the reported ESIPT−cyanine dyes are their potential in molecular imaging, due to their unique photophysical properties (large Stokes shift), excellent biocompatibility, and high cell penetration ability. [8] [9] [10] 22 The fluorescence of 13b (R 2 = −CH 3 ) was sensitive to its environmental pH, with pK a ≈ 6.01. 22 Benefiting from its large fluorescence turn on upon protonation (Ar−O − + H + → ArOH), probe 13b exhibits selectivity toward intracellular lysosomes similar to that of its regioisomer 8. However, the low pK a is not the sole reason for the observed lysosome selectivity, as both 8 (pK a ∼ 5.72, X = S) and 11 (pK a ∼ 8.72, X = O) are targeting lysosomes. 8, 10 The intriguing lysosome selectivity of 11 is clearly evidenced by a co-staining experiment with commercial MitoTracker Green and Lyso-Tracker Red (Figure 3) , and the probe exhibits excellent colocalization with the known LysoTracker ( Figure 3D ,H).

Since ESIPT−cyanine dyes contain two segments, one fundamental question is whether the ESIPT or cyanine segment has a larger impact on the intracellular selectivity. In one experiment, the −OH group in 13b was converted to a −OCH 3 group, and the resulting derivative maintained the lysosome selectivity. 22 The same lysosome selectivity is also observed from 8 (with HBT) and 11 (with HBO). 8, 10 The result suggests that the ESIPT unit might have less influence on the cellular selectivity.

When the benzothiazolium terminal group in 11 was replaced by the indolium group, however, the resulting 7 became the mitochondria-targeting probe. 10 The drastic switch in intracellular selectivity can be seen clearly by the remarkable colocalization of 12 with MitoTracker Green (Figure 4) , in sharp contrast to 11 ( Figure 3D ). The mitochondrial selectivity of 12 can be attributed to its Coulombic attraction toward a negative potential gradient across the mitochondrial matrix. Interestingly, the ESIPT−cyanine dye 9 with a pyridinium group stained the membrane of E. coli cells and does not show any selectivity toward eukaryotic cells in vivo; however, the dye selectively stained neuromast hair cells and supporting cells in zebrafish. 7 Therefore, the cyanine terminal groups play a vital role in tuning the cellular selectivity.

Sensor Application Based on Selective Reactions. The structure of ESIPT−cyanine dyes is well-suitable for conjugated addition (Michael addition reaction), as a vinyl bond is connected to a strong e-withdrawing group (called a Michael acceptor). Such a structural feature can be used for the detection of various Michael donors, such as biologically important bisulfite and thiols. Recently, 7a has been used for hydrogen sulfide detection, 20 and 15 has also been demonstrated as a reversible colorimetric and ratiometric probe for SO 2 /HSO 3 − (Scheme 7) in cancer cells (MCF-7). 3 Similarly, Chen et al. reported the ratiometric fluorescence detection of ROS (reactive oxygen species) such as HOCl/ OCl − in HeLa cells using probe 9. 25 During the process, addition of the ROS to the conjugated π-bond breaks the extended π-conjugation, and the emission is given by only the ESIPT chromophore, which is hypsochromically shifted from the original probe.

While the previous studies have been focusing on using the probes with HBT as the ESIPT unit, our group examined the possible use of 11 for bisulfite detection in NHLF cells as it became available. 10 

We acknowledge support from NIH (Grant No. 1R15GM1264-3801A1) and partial support from Coleman Endowment from The University of Akron.

ESIPT, excited states intramolecular proton transfer; NIR, near-infrared; PET, photoinduced electron transfer; FRET, Forster resonance energy transfer; ICT, internal charge transfer; HBO, 2-(2′-hydroxyphenyl)benzoxazole; HBT, 2-(2′-hydroxyphenyl)benzothiazole; NMR, nuclear magnetic resonance; DCM, dichloromethane; NHLF, normal human lung fibroblast; ROS, reactive oxygen species

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