German Edition: DOI: 10.1002/ange.201800772Hydrogen BondingInternational Edition: DOI: 10.1002/anie.201800772

An Easily Accessible Ionic Aggregation-Induced Emission Luminogenwith Hydrogen-Bonding-Switchable Emission and Wash-Free ImagingAbilityYuncong Chen, Weijie Zhang, Zheng Zhao, Yuanjing Cai, Junyi Gong, Ryan T. K. Kwok,Jacky W. Y. Lam, Herman H. Y. Sung, Ian D. Williams, and Ben Zhong Tang*

Abstract: Ionic fluorophores are powerful tools for the studyof environmental science and bio-imaging. However, tradi-tional ionic dyes usually require long synthetic steps and sufferfrom a quenching effect caused by aggregation. A water-soluble ionic aggregation-induced emission luminogen calledDBTA is presented, which is readily accessed by a one-stepreaction. The switchable emission manipulated by hydrogenbonding provided solid evidence for the restriction of intra-molecular motions as the mechanism of aggregation-inducedemission. DBTA can not only differentiate solvents withdifferent H-bond donor acidities but also capable of wash-free imaging in living HeLa cells and fish larva.

Fluorescent materials have been widely used in many fieldsincluding light-emitting devices, environmental detection,chemical sensing, biological imaging, and medical diagno-sis.[1–3] Because of their high sensitivity, noninvasiveness, andgood spatial–temporal resolution, fluorescent molecules areexcellent imaging candidates for monitoring various physio-logical processes.[4–6] However, most of the luminescent dyesare inherently hydrophobic, which hinders their practicalapplications in environmental water quality monitoring and inbiological systems since water is the predominant component.To improve the water compatibility of the fluorophores,

synthetic chemists need to modify the fluorescent moleculeswith highly polar or charged groups.

Great efforts have been devoted in this field and a numberof inherently ionic or charge modified fluorophores areavailable. These fluorophores have gained much attentionand have been successfully applied to various fields such asmaterial science,[7] DNA detection,[8] targeted imaging,[9] andtheranostics.[10] However, chemical modification usuallyrequires multiple synthetic steps, which is time consumingand will lower the total yield of the target fluorophores.Moreover, the functional charged group or the inherentlyionic feature endow the resulting fluorescent molecule withhigh polarity, which will lead to sophisticated and tediouspurification procedures. Therefore, to save time, labor,energy, and economic cost, the development of ionic fluo-rophores with high efficiency and easy purification is in highdemand.

On the other hand, most of ionic fluorescent moleculescontains planar aromatic rings as the luminescent core parts,which are somewhat hydrophobic and tend to aggregate whenthe concentration is high. These traditional fluorophoresusually suffer from aggregation caused quenching (ACQ).Aggregation-induced emission luminogens (AIEgens) showweak or negligible emission in the solution state but exhibitintense emission in aggregates or the solid state, whichprovides an excellent strategy to solve the ACQ problem.[11]

Owing to their unique feature, AIEgens have emerged asa novel class of luminescent material with wide applicationfields.[12–14] Experimentalists and theoreticians have devotedgreat efforts on unveiling the photophysical processes of theAIE phenomenon.[15–18] Although restriction of intramolecu-lar motions (RIM) have been proposed as the generalmechanism for AIE phenomenon,[19,20] direct experimentalevidence is still needed to support this mechanism.

Herein, we report a water-soluble ionic AIEgen, DBTA,which was readily synthesized by a novel one-step reactionwith simple purification procedure. DBTA showed aggrega-tion-induced emission (AIE) and intramolecular hydrogen-bonding-induced emission (IHBIE). In aprotic solvents, twointramolecular hydrogen bonds (intra-HBs) were formed andserved as noncovalent conformational locks (NCLs)[21, 22] torestrict intramolecular motions, resulting a strong fluores-cence. However, in highly protic solvents, the intra-HBs ofDBTA were undermined and a dramatic decrement offluorescence intensity was observed due to the large non-radiative decay of the flexible conformation. This feature ofDBTA not only provided a direct evidence to validate the

[*] Dr. Y. Chen, Prof. B. Z. TangHKUST Shenzhen Research InstituteNo. 9 Yuexing 1st RD, South Area, Hi-tech Park NanshanShenzhen 518057 (China)E-mail: tangbenz@ust.hk

Dr. Y. Chen, Dr. W. Zhang, Dr. Z. Zhao, Dr. Y. Cai, J. Gong,Dr. R. T. K. Kwok, Dr. J. W. Y. Lam, Dr. H. H. Y. Sung,Prof. I. D. Williams, Prof. B. Z. TangDepartment of Chemistry, Hong Kong Branch of Chinese NationalEngineering Research Center for Tissue Restoration and Recon-struction, Division of Biomedical Engineering, Institute for AdvancedStudy and State Key Laboratory of Molecular Neuroscience, TheHong Kong University of Science and TechnologyClear Water Bay, Kowloon, Hong Kong (China)

Prof. B. Z. TangNSFC Center for Luminescence from Molecular Aggregates, SCUT-HKUST Joint Research Institute, State Key Laboratory of LuminescentMaterials and Devices, South China University of TechnologyGuangzhou 510640 (China)

Supporting information (including experimental details, character-ization, theoretical calculation methods, and fluorescence imagingdetails) and the ORCID identification number(s) for the author(s) ofthis article can be found under:https://doi.org/10.1002/anie.201800772.

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RIM mechanism, but also endowed DBTA with the potentialapplication to differentiate solvents with different H-bondingdonor acidity (solvent acidity). Moreover, wash-free imagingwas realized in living HeLa cells and fish larva using DBTA.

DBTA was synthesized by a novel and simple one-stepreaction with a decent yield of 74%, and its chemicalstructure was fully characterized by 1H NMR, 13C NMR,HRMS, and confirmed by X-ray single-crystal analysis(Figure 1; see also the Supporting Information). At room

temperature, benzothiazole-2-yl-acetonitrile (BTA) was suc-cessively treated with sodium hydride and trimethylsilylchloride (TMS-Cl) in THF to generate 2-(benzo[d]thiazol-2-yl)-2-(trimethylsilyl)acetonitrile, which was further deproton-ated by the excess of sodium hydride.[23, 24] The resulting a-silylcarbanion underwent an intermolecular nucleophilicattack to the cyano carbon of its neutral counterpart. Aftera series of rearrangements and another intramolecularnucleophilic reaction, a new six member ring was formed(Supporting Information, Scheme S1). Pure DBTA could beobtained by simple filtration with a decent yield. This novelreaction exhibits superior advantage of one step reaction atroom temperature and simple purification process, no heatingor cooling bath is needed. This makes it highly attractive interms of saving time, labor, energy, and economy forachieving an ionic fluorophore. ESI-MS assay was performedto capture the stable reaction intermediates (SupportingInformation, Figure S4), two peaks with m/z of 173.00 and491.25 were collected after 2 min of TMS-Cl addition, whichcan be assigned as the anion form of BTA and compounds 5–9(share the same m/z ; Supporting Information, Scheme S1),respectively. Single-crystal analysis showed two intramolecu-lar H-bonds, including an interesting sulfur centered H-bond,which is generally regarded as weak H-bond and usuallyfound in sulfur containing proteins.

The intensive green emission of DBTA crystal encour-aged us to study its photophysical properties (Figure 2).Interestingly, 20 mm of DBTA in pure methanol (a goodsolvent of DBTA) only showed weak blue emission. How-

ever, with the increasing amount of poor solvent THF, thefluorescence intensity at 480 nm gradually enhanced and anobvious red-shift (to 515 nm) was observed at high THF ratioover 90 % (Figure 2A,B,E). The green emission in mixturesolution with high THF ratio is very similar to the crystalemission, suggesting that DBTA exhibited AIE property. TheDLS data showed that the DBTA aggregates were quiteuniform with average size of about 87 nm in methanol/THFmixture containing 99 % of THF (Supporting Information,Figure S5). The emission of DBTA was almost completelyquenched in pure water solution. With the increment ofDMSO ratio over 60%, the emission intensity drasticallyincreased with no emission wavelength change. The emissionintensity in pure DMSO was about 450-fold greater than thatin pure H2O (Figure 2C,D,F). The quenching of PL intensityof DBTA in highly protic solvents such as methanol and watersuggested that the emission of DBTA could be switched bymanipulating H-bonding.

Therefore, DBTA might have the potential application ofdifferentiating solvents with different hydrogen bonding

Figure 1. Synthetic procedure of DBTA (top), bright field (bottom left),and fluorescence (bottom right) images of DBTA crystals. Single-crystal structure of DBTA with two intramolecular hydrogen bonds(bottom middle), solvent methanol, and counteranion Cl@ wereomitted for clarity.

Figure 2. A) Fluorescence spectra of DBTA (20 mm) in MeOH/THFmixture with different THF fraction, excitation 415 nm. B) PL intensitychanges of I/I0 at 515 nm in (A). C) Fluorescence spectra of DBTA(20 mm) in H2O/DMSO mixture with different DMSO fraction, excita-tion 415 nm. D) PL intensity changes of I/I0 at 480 nm in (C).E) Photograph of DBTA (20 mm) in MeOH/THF mixture with differentTHF fraction under excitation of a UV lamp at 365 nm. F) Photographof DBTA (20 mm) in H2O/DMSO mixture with different DMSO fractionunder excitation of a UV lamp at 365 nm.

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donor acidities. H2O and five alcohols with different alkylchain lengths were chosen as the analytes (SupportingInformation, Table S1). The absorption and PL spectra ofDBTA (20 mm) in these solvents were collected (Figure 3A).

In H2O solution with the strongest H-bond donor acidity,DBTA showed a strong absorption peak at about 330 nm withalmost no absorption over 400 nm. Meanwhile, the fluores-cence of DBTA in water was negligible. With the decreasingof the HB donor acidity from methanol to n-pentanol, theabsorbance at about 330 nm gradually decreased and a newbroad absorption band at about 415 nm appeared distinctly,concomitant with a dramatic enhancement of the fluores-cence at 480 nm. The results could be rationalized by the on–off switching of intramolecular hydrogen bonds (intra-HBs)in different solvents (Figure 3 B). In aprotic solvents orsolvents with weak HB donor acidity, DBTA tended toadopt a near-planar conformation with two intra-HBs, whichserve as the NCLs to restrict the intramolecular motions.However, the intra-HBs in DBTA were compromised instrongly protic solvents and the benzothiazol ring should bemore twisted. The loss of NCLs and twisted structure couldpromote the intramolecular motions of DBTA to quench thefluorescence. Therefore, DBTA showed IHBIE characteristic

and was capable of distinguishing protic solvents with minorstructural variation. Moreover, the HBs manipulated on-offPL switching behavior provided strong evidence for the RIMmechanism. Similar intra-HB induced fluorescence quench-ing was found in PRODAN derivatives.[25] However, besidesH-bonding, the fluorescence of PRODAN systems wassignificantly affected by solvent polarity.

Theoretical calculations were carried out to investigatethe absorption difference between two forms of DBTA instrongly protic solvents and aprotic solvents. Highly twistedstructure with a perpendicular benzothiazol ring was chosenas the model for the simulation of DBTA in methanol, whilethe crystal structure was chosen as the model for simulation ofDBTA in DMSO. Frontier molecular orbitals (FMOs) of thetwo structures were calculated (Figure 3C). For the twistedstructure in methanol, the electron cloud density of HOMOand LUMO were mainly localized on the newly formed 3-imino-3H-benzo[4,5]thiazolo[3,2-a]pyridin-1-amine core,indicating a classic p–p* transition. For the near-planarstructure in DMSO, the HOMO and LUMO electronclouds were delocalized in the whole molecule with obviousp–p* transition and intramolecular charge transfer (ICT)characteristics. The simulated absorption maxima were about310 nm and about 370 nm for the vertical and planarconformation, respectively (Supporting Information, Fig-ure S6). The theoretically calculated red-shift of the planarconformation compare to the highly twisted conformation inthe absorption spectra was in good accordance with theexperimental data. Therefore, the absorption over 400 nmand the PL intensity at 480 nm were determined by the ratiosof near-planar conformation of DBTA in various solvents.

To explain the intensive and redshifted emission in thenanoaggregates and crystals, we investigated the detailedpacking modes and intermolecular interaction inside thecrystal structure. DBTA molecules showed offset columnarstacks of antiparallel dimers with short intermolecular stack-ing distances of 3.267 c and 3.216 c, suggesting strongintermolecular interaction inside the dimer. The drivingforce of the dimer formation was attributed to electrostaticinteraction according to the theoretical calculation (Support-ing Information, Figure S7). However, the relatively largedistance (3.605 c) between two dimers indicated negligiblep–p stacking between dimers (Figure 4A–C). Furthermore,the existence of solvent methanol and chloride anion insidethe DBTA crystal showed multiple intermolecular hydrogenbonding (Figure 4D; Supporting Information, Figure S9).Together with the intra-HBs, these NCLs could restrict themolecular motions and suppress the non-radiative decay.Therefore, the red-shifted emission could be attributed to theformation of dimers, while the strong emission was ascribed tothe existence of multiple NCLs and the lack of p–p stackingbetween the DBTA dimers.

Since DBTA is non-fluorescent in H2O solution, it has thepotential application for wash-free imaging, which showsattractive advantages such as simplified imaging procedureand avoid effecting cell morphology during the washingprocess. Laser scanning confocal microscopy (LSCM) wasconducted in HeLa cells using DBTA and a water soluble Cy3derivative (Figure 5). Without washing, the fluorescence

Figure 3. A) Absorption spectra (left) and PL spectra (right) of DBTA(20 mm) in different solvents. B) Illustration of twisted conformation(left) with intermolecular H-bonding and near-planar conformation(right) with intramolecular H-bonding. C) Calculated FMOs and ener-gies of the twisted conformation (left, in methanol) and the near-planar conformation (right, in DMSO).

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image was collected by staining with DBTA for 30 min, whichshowed strong fluorescence in HeLa cells and negligiblebackground fluorescence. In sharp contrast, the fluorescenceimage obtained by incubation with Cy3 dye exhibited strongfluorescence in the whole imaging area. Moreover, thefluorescence intensity remained over 90 % after 30 mincontinuous irradiation, suggesting good photostability ofDBTA (Figure 5E) which is favorable for long-term trackingof living cells. Furthermore, the wash-free imaging ability infish larva was also evaluated. The fluorescence image

collected without washing showed bright blue-greenish fluo-rescence inside the fish larva and almost no backgroundsignal. The results demonstrated that DBTA could easilyenter biological samples including living cells and fish larva,and showed excellent signal to noise ratio without washing theunstained dye. DBTA is much easier to access than theexisting AIE wash-free imaging systems.[26, 27]

In summary, we have developed a novel water-solubleionic AIEgen DBTA by a simple one step reaction witha decent yield. The facile access of DBTA is very attractive interms of saving time, labor, and economy cost. DBTA showedAIE characteristics and intramolecular hydrogen bondinginduced emission. The enhanced emission in aggregates orcrystal was attributed to the formation of intra-HBs andisolated dimers containing multiple NCLs. The intra-HBsacted as NCLs to suppress the non-radiative decay, whichverified the RIM mechanism. The intra-HBs were destroyedand changed to inter-HBs in highly protic solvents, thusswitching off of the fluorescence. Owing to its unique feature,DBTA was able to distinguish solvents with different HBdonor acidities and could serve as a practical fluorescenceimaging agent without washing process.

Acknowledgements

This work has been partially supported by the NationalNatural Science Foundation of China (21788102), theNational Basic Research Program of China (973 Program,2013CB834701 and 2013CB834702), the Research GrantsCouncil of Hong Kong (16308016, 16305015, C2014-15G,N_HKUST604/14 and A-HKUST605/16), the TechnologyPlan of Shenzhen (JCYJ20160229205601482), and the Inno-vation and Technology Commission (ITC-CNERC14SC01and ITCRD/17-9).

Conflict of interest

The authors declare no conflict of interest.

Keywords: aggregation-induced emission · hydrogen bonding ·ionic fluorophores · non-covalent conformational locks ·wash-free imaging

How to cite: Angew. Chem. Int. Ed. 2018, 57, 5011–5015Angew. Chem. 2018, 130, 5105–5109

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Figure 4. Detailed packing mode and intermolecular interactions inthe crystal structure. A) Crystal packing mode from the z direction.B) Enlarged view of the dashed white box in (A). C) Side view from thex-direction in (B). D) Side view from the y direction of (B). Atom colorsin (A), (C), and (D): C gray; N blue, S yellow, O red, H white, Cl green.

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Manuscript received: January 19, 2018Revised manuscript received: March 4, 2018Accepted manuscript online: March 7, 2018Version of record online: March 22, 2018

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