WO2020147738A1

WO2020147738A1 - Fluorescent compounds with wide color tunability and aggregation-induced emission characteristics

Info

Publication number: WO2020147738A1
Application number: PCT/CN2020/072183
Authority: WO
Inventors: Benzhong Tang; Wenhan XU; Dong Wang
Original assignee: The Hong Kong University Of Science And Technology
Priority date: 2019-01-16
Filing date: 2020-01-15
Publication date: 2020-07-23
Also published as: CN113498411A

Abstract

The present subject matter relates to fluorescent compounds that have aggregation-induced emission (AIE) characteristics and tunable emission colors. The emission range of these compounds covers the whole visible region and extends to the near infrared (NIR) area. The compounds can be utilized as bio-probes for lipid droplet (LD) -specific imaging with excellent image contrast to the cell background. Additionally, the high brightness and homology of these compounds endow them with excellent performance for visualizing cell fusion. Further, upon exposure to white light irradiation, the compounds can generate reactive oxygen species (ROS) with high efficiency. As such, the compounds can be effective in photodynamic ablation of cancer cells.

Description

Fluorescent Compounds with Wide Color Tunability and Aggregation-Induced Emission Characteristics

CROSS-REFERENCE

The present application claims priority to provisional United States Patent Application No. 62/918,110, filed January 16, 2019, which was filed by the inventors hereof and is incorporated herein by reference in its entirety.

FIELD

The present subject matter relates generally to a series of fluorescent compounds with aggregation-induced emission characteristics and near infrared absorption and their applications in bioimaging and phototheranostics.

BACKGROUND

The exploration of fluorescent materials and technologies has opened new avenues to scientific advancement, societal development and public health. Recently, fluorescence-related research has even received Nobel Prize recognition. Fluorescent bio-materials offer researchers a powerful platform for analytical sensing and optical imaging and have proven extremely useful for biological visualizations, clinical diagnosis and disease treatment by virtue of their non-invasion, in situ workability, excellent accuracy, superb sensitivity and simple operation. Although many types of fluorophores have been commercialized for biological applications, conventional technologies are still far from ideal, mainly due to the following limitations: 1) inherent fluorescence quenching upon aggregate formation due to intermolecular π-π stacking and other nonradiative pathways, also known as aggregation-caused quenching (ACQ) ; 2) the difficulty of widely tuning emission colors by simple modification of molecular structures; and 3) complicated and laborious syntheses of fluorophores.

As an anti-ACQ phenomenon, aggregation-induced emission (AIE) was coined in 2001 by Professor Benzhong Tang’s group. AIE refers to the unique phenomenon of non-emission or weak emission of compounds in the molecularly dissolved state and intense emission in the aggregated state, owing to the restriction of the intramolecular motions (RIM) . Remarkably, the AIE principle has triggered state-of-the-art developments in an array of biological fields, ranging from bioimaging, biosensing, stimuli-responsive systems, therapeutics, and theranostics. Various impressive advantages of AIE luminogens (AIEgens) include, for example, high photobleaching threshold, high signal-to-noise ratio for imaging, excellent tolerance for any concentration, large Stokes shift, turn-on feature for detecting analytes, and efficient photosensitizing ability. Although numerous conventional AIEgens have been constructed on the basis of different structural motifs, including tetraphenylethene, hexaphenylsilole, tetraphenylpyrazine and distyrylanthracene, emission of these AIE systems cannot be arbitrarily tuned to provide emission in each color of visible light and the near-infrared (NIR) region. Considering the great significance of tunable fluorescent systems in the application of multi-target sensing, optoelectronic devices and full-color bio-imaging, the development of an AIE system exhibiting wide color tunability is highly desired and remains a challenging task.

Compared with inorganic complexes and quantum dots, organic fluorophores are advantageous for bio-imaging, diagnosis and therapy, due to their good bio-compatibility, tunable molecular structures and chemical compositions at will, and scalable synthesis.

Accordingly, AIEgens with both AIE attributes and emission color tunability across a wide wavelength range are highly desirable.

SUMMARY

The present subject matter relates to fluorescent compounds that have aggregation-induced emission (AIE) characteristics and tunable emission colors. The emission range covers the whole visible region and extends to the near infrared (NIR) area. The compounds can be utilized as bio-probes for lipid droplet (LD) -specific imaging with excellent image contrast to the cell background and higher photostability than commercial LD-staining fluorophores. Additionally, these compounds have high brightness and homology, which endow them with excellent performance for visualizing cell fusion. Further, upon exposure to white light irradiation, the compounds can generate reactive oxygen species (ROS) with high efficiency. As such, the compounds can be effective in photodynamic ablation of cancer cells.

In an embodiment, the fluorescent compounds are TPA-thiophene building block-based AIEgens that can be facilely prepared by simple synthetic protocols. The fluorescent compounds show high fluorescence quantum yields, e.g., up to about 40.79%in solid state.

In an embodiment, the fluorescent compound has a backbone structural formula selected from the group consisting of:

wherein each R is independently selected from the group consisting of H, alkyl, unsaturated alkyl, heteroalkyl, alkoxy, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkyl-NCS, alkyl-N ₃, and alkyl-NH ₂, wherein R is substituted or unsubstituted;

wherein Y is selected from the group consisting of alkyl optionally substituted by one or more cyano groups, alkenyl optionally substituted by one or more cyano groups, optionally substituted phenyl, and optionally substituted heteroaryl; and

wherein when Y is an optionally substituted heteroaryl, Y is not

wherein Y is alkyl, alkenyl,

wherein each Y is unsubstituted or substituted with one or more groups selected from the group consisting of C=O, one or more cyano groups, and alkyl or alkenyl substituted with one or more cyano groups.

In an embodiment, the compound has a backbone structural formula selected from the group consisting of:

wherein each R is independently selected from the group consisting of H, alkyl, unsaturated alkyl, heteroalkyl, alkoxy, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkyl-NCS, alkyl-N ₃, and alkyl-NH ₂, wherein R is substituted or unsubstituted; and

wherein X is selected from the group consisting of phenyl, heteroaryl, and C=C.

In an embodiment, the compound comprises at least one compound selected from the group consisting of:

In an embodiment, a method of cellular imaging can include contacting a target cell with one or more fluorescent compounds; and identifying a cellular target of interest using an imaging method, the one or more fluorescent compounds comprising a compound having the following backbone structural formula:

wherein Y is selected from the group consisting of alkyl optionally substituted by one or more cyano groups, alkenyl optionally substituted by one or more cyano groups, oxygen, hydrogen, optionally substituted phenyl, and optionally substituted heteroaryl.

In an embodiment, one or more of the fluorescent compounds can have a backbone structural formula selected from the group consisting of:

wherein X is selected from the group consisting of phenyl, heteroaryl, and C=C.

In an embodiment, the fluorescent compound comprises at least one compound selected from the group consisting of:

BRIEF DESCRIPTION OF DRAWINGS

Various embodiments will now be described in detail with reference to the accompanying drawings.

Fig. 1A depicts a single crystal structure of TTG.

Fig. 1B depicts a side view of the crystal structure of TTG.

Fig. 1C depicts various inter-and intramolecular interactions in crystals of TTG.

Fig. 2A depicts normalized absorption spectra of TTV, TTB, TTG, TTY, TTO, TTR, TTDR and TTNIR in ACN solution.

Fig. 2B depicts PL spectra of TTY (1×10 ^-5 M) in ACN/water mixtures with different water fraction (f _w) ; λ _ex: 410 nm.

Fig. 2C depicts the plot of the emission maximum and the relative emission intensity (I/I ₀) versus the composition of the aqueous mixture of TTV, TTB, TTG, TTY, TTO, TTR, TTDR and TTNIR.

Fig. 2D depicts normalized PL spectra of TTV (λ _ex: 417 nm) , TTB (λ _ex: 489 nm) , TTG (λ _ex: 539 nm) , TTY (λ _ex: 583 nm) , TTO (λ _ex: 603 nm) , TTR (λ _ex: 659 nm) , TTDR (λ _ex: 684 nm) , and TTNIR (λ _ex: 706 nm) in the solid state.

Fig. 2E depicts fluorescence photographs of TTV, TTB, TTG, TTY, TTO, TTR, TTDR and TTNIR (from left to right) in ACN/water mixtures with 95%water fractions (upper) and in the solid state (below) taken under 365 nm UV irradiation.

Fig. 2F depicts fluorescence decay curves of TTV, TTB, TTG, TTY, TTO, TTR, TTDR and TTNIR in the solid state.

Fig. 3 depicts molecular orbital amplitude plots of HOMO and LUMO energy levels of TTV, TTB, TTG, TTY, TTO, TTR, TTDR and TTNIR calculated at B3LYP/6-31+G (d) level based on the geometries optimized at TD-B3LYP/6-31+G (d) level.

Fig. 4A depicts a single crystal structure of TTY.

Fig. 4B depicts a side view of the crystal structure of TTY.

Fig. 4C depicts various inter-and intramolecular interactions in crystals of TTY.

Fig. 5A depicts a single crystal structure of TTDR.

Fig. 5B depicts a side view of the crystal structure of TTDR.

Fig. 5C depicts various inter-and intramolecular interactions in crystals of TTDR.

Fig. 6A depicts PL spectra of TTV in different solvents for solvatochromic effect evaluation.

Fig. 6B depicts PL spectra of TTG in different solvents for solvatochromic effect evaluation.

Fig. 7A depicts confocal images of living NCM460cells after incubation with TTNIR (1 μM) for 20 min. λ _ex: 488 nm.

Fig. 7B depicts confocal images of living DLD1 cells after incubation with TTNIR (1 μM) for 20 min. λ _ex: 488 nm.

Fig. 7C depicts confocal images of living SW480 cells after incubation with TTNIR (1 μM) for 20 min. λ _ex: 488 nm.

Fig. 7D depicts confocal images of living SW620 cells after incubation with TTNIR (1 μM) for 20 min. λ _ex: 488 nm.

Fig. 7E depicts confocal images of living COS-7 cells after incubation with TTNIR (1 μM) for 20 min. λ _ex: 488 nm.

Fig. 8A depicts colocalization bright-field imaging of COS-7 cells stained with (5 μm) TTV (excited with a 405 nm laser (14%laser power) and emission collected with 415-550 nm filter) .

Fig. 8B depicts colocalization confocal imaging of COS-7 cells stained with (5 μm) TTV (excited with a 405 nm laser (14%laser power) and emission collected with 415-550 nm filter) .

Fig. 8C depicts colocalization confocal imaging of COS-7 cells stained with (5 μm) Nile Red (excited with a 514 nm laser (14%laser power) and emission collected with 580-620 nm filter) .

Fig. 8D depicts a merged image of the images provided in Figs. 8B and 8C.

Fig. 9A depicts colocalization bright-field imaging of COS-7 cells stained with (5 μm) TTB (excited with a 405 nm laser (40%laser power) and emission collected with 415-550 nm filter) .

Fig. 9B depicts colocalization confocal imaging of COS-7 cells stained with (5 μm) TTB (excited with a 405 nm laser (40%laser power) and emission collected with 415-550 nm filter) .

Fig. 9C depicts colocalization confocal imaging of COS-7 cells stained with (5 μm) Nile Red (excited with a 514 nm laser (6.5%laser power) and emission collected with 580-620 nm filter) .

Fig. 9D depicts merged images of the images provided in Figs. 9B and 9C.

Fig. 10A depicts colocalization bright-field imaging of COS-7 cells stained with (5 μm) TTG (excited with a 405 nm laser (0.2%laser power) and emission collected with 480-545 nm filter) .

Fig. 10B depicts colocalization confocal imaging of COS-7 cells stained with (5 μm) TTG (excited with a 405 nm laser (0.2%laser power) and emission collected with 480-545 nm filter) .

Fig. 10C depicts colocalization confocal imaging of COS-7 cells stained with (5 μm) Nile Red (excited with a 514 nm laser (6.5%laser power) and emission collected with 580-630 nm filter) .

Fig. 10D depicts merged images of the images provided in Figs. 10B and 10C.

Fig. 11A depicts colocalization bright-field imaging of COS-7 cells stained with TTY (5 μm) (excited with a 405 nm laser (0.2%laser power) and emission collected with 490-625 nm filter) .

Fig. 11B depicts colocalization confocal imaging of COS-7 cells stained with TTY (5 μm) (excited with a 405 nm laser (0.2%laser power) and emission collected with 490-625 nm filter) .

Fig. 11C depicts colocalization confocal imaging of COS-7 cells stained with Nile Red (5 μm) (excited with a 514 nm laser (6.5%laser power) and emission collected with 580-630 nm filter) .

Fig. 11D depicts merged images of the images provided in Figs. 11B and 11C.

Fig. 12A depicts colocalization bright-field imaging of COS-7 cells stained with TTO (5 μm) (excited with a 488 nm laser (8%laser power) and emission collected with 560-650 nm filter) .

Fig. 12B depicts colocalization confocal imaging of COS-7 cells stained with TTO (5 μm) (excited with a 514 nm laser (6.5%laser power) and emission collected with 580-630 nm filter) .

Fig. 12C depicts colocalization confocal imaging of COS-7 cells stained with BODIPY493/503 Green (5 μm) (excited with a 488 nm laser (2.8%laser power) and emission collected with 500-540 nm filter) .

Fig. 12D depicts merged images of the images provided in Figs. 12B and 12C.

Fig. 13A depicts colocalization bright-field imaging of COS-7 cells stained with TTR (5 μm) (excited with a 488 nm laser (8%laser power) and emission collected with 560-740 nm filter) .

Fig. 13B depicts colocalization confocal imaging of COS-7 cells stained with TTR (5 μm) (excited with a 488 nm laser (2.8%laser power) and emission collected with 500-540 nm filter) .

Fig. 13C depicts colocalization confocal imaging of COS-7 cells stained with BODIPY493/503 Green.

Fig. 13D depicts merged images of the images provided in Figs. 13B and 13C.

Fig. 14A depicts colocalization bright-field imaging of COS-7 cells stained with TTDR (5 μm) (excited with a 488 nm laser (0.1%laser power) and emission collected with 570-740 nm filter) .

Fig. 14B depicts colocalization confocal imaging of COS-7 cells stained with TTDR (5 μm) (excited with a 488 nm laser (2.8%laser power) and emission collected with 500-540 nm filter) .

Fig. 14C depicts colocalization confocal imaging of COS-7 cells stained with BODIPY493/503 Green (5 μm) (excited with a 488 nm laser (2.8%laser power) and emission collected with 500-540 nm filter) .

Fig. 14D depicts merged images of the images provided in Figs. 14B and 14C.

Fig. 15A depicts colocalization bright-field imaging of COS-7 cells stained with TTNIR (5 μm) (excited with a 560 nm laser (0.1%laser power) and emission collected with 570-740 nm filter) .

Fig. 15B depicts colocalization confocal imaging of COS-7 cells stained with TTNIR (5 μm) (excited with a 560 nm laser (0.1%laser power) and emission collected with 570-740 nm filter) .

Fig. 15C depicts colocalization confocal imaging of COS-7 cells stained with BODIPY493/503 Green (5 μm) (excited with a 488 nm laser (2.8%laser power) and emission collected with 500-540 nm filter) .

Fig. 15D depicts merged images of the images provided in Figs. 15B and 15C.

Fig. 16A depicts colocalization bright-field imaging of HeLa cells stained with TTNIR.

Fig. 16B depicts colocalization confocal imaging of HeLa cells stained with TTNIR.

Fig. 16C depicts colocalization confocal imaging of HeLa cells stained with BODIPY493/503 Green.

Fig. 16D depicts merged images of the images provided in Figs. 16B and 16C.

Fig. 16E depicts confocal image of HeLa cells stained with TTNIR before laser irradiation.

Fig. 16F depicts confocal image of HeLa cells stained with TTNIR after laser irradiation.

Fig. 16G depicts confocal image of HeLa cells stained with BODIPY 493/503 Green before laser irradiation.

Fig. 16H depicts confocal image of HeLa cells stained with BODIPY 493/503 Green before laser irradiation.

Fig. 16I depicts amount of fluorescence lost in HeLa cells stained with TTNIR BODIPY 493/503 Green with the laser irradiation scans.

Fig. 17A depicts confocal images of HeLa cells stained with TTV (Concentration: AIEgen (1 μM) ) .

Fig. 17B depicts confocal images of HeLa cells stained with TTB (Concentration: AIEgen (1 μM) ) .

Fig. 17C depicts confocal images of HeLa cells stained with TTG (Concentration: AIEgen (1 μM) ) .

Fig. 17D depicts confocal images of HeLa cells stained with TTY (Concentration: AIEgen (1 μM) ) .

Fig. 17E depicts confocal images of HeLa cells stained with TTO (Concentration: AIEgen (1 μM) ) .

Fig. 17F depicts confocal images of HeLa cells stained with TTR (Concentration: AIEgen (1 μM) ) .

Fig. 17G depicts confocal images of HeLa cells stained with TTDR (Concentration: AIEgen (1 μM) ) .

Fig. 17H depicts confocal images of HeLa cells stained with TTNIR (Concentration: AIEgen (1 μM) ) .

Figs. 18A depicts a confocal image of cell fusion of COS-7 cells induced by 50%polyethylene glycol (PEG) and stained with (500 nM) TTG and (2 μM) TTNIR, and visualized through fluorescence imaging of TTG and TTNIR (for TTG: λex: 405 nm (1%laser power) , λem: 425-540 nm) ; for TTNIR (λex: 560 nm (6.5%laser power) , λem: 600-740 nm) .

Fig. 18B depicts a bright-field image of cell fusion of COS-7 cells induced by 50%polyethylene glycol (PEG) and stained with (500 nM) TTG and (2 μM) TTNIR (for TTG: λex: 405 nm (1%laser power) , λem: 425-540 nm) ; for TTNIR (λex: 560 nm (6.5%laser power) , λem: 600-740 nm) .

Fig. 18C depicts merged images of panels depicted in Fig. 18A and Fig. 18B.

Fig. 18D depicts confocal image of cell fusion of COS-7 cells induced by 50%polyethylene glycol (PEG) and stained with (500 nM) TTG, (2 μM) TTNIR and Hoechst 33258 (for TTG: λex: 405 nm (1%laser power) , λem: 425-540 nm) ; for TTNIR (λex: 560 nm (6.5%laser power) , λem: 600-740 nm; for (2.5 μM) Hoechst 33258, λex: 405 nm (3.5%laser power) emission: 425-540 nm) .

Fig. 18E depicts bright-field image of mixed cells respectively stained with (500 nM) TTG, TTNIR and Hoechst 33258.

Fig. 18F depicts merged images of panels depicted in Fig. 18D and Fig. 18E (scale bar =20 μm) .

Fig. 19A depicts relative change in fluorescent intensity (I/I0) at 534 nm of H2DCF-DA, TTNIR, and mixtures of TTNIR and H2DCF-DA in PBS upon white light irradiation for different time (Concentrations: 10 μM (TTNIR) and 5 μM (H2DCF-DA) ) .

Fig. 19B depicts cell viability of HeLa cells stained with different concentrations of TTNIR in the absence or presence of white light irradiation.

DETAILED DESCRIPTION

The following definitions are provided for the purpose of understanding the present subject matter and for constructing the appended patent claims.

Definitions

It should be understood that the drawings described above or below are for illustration purposes only. The drawings are not necessarily to scale, with emphasis generally being placed upon illustrating the principles of the present teachings. The drawings are not intended to limit the scope of the present teachings in any way.

Throughout the application, where compositions are described as having, including, or comprising specific components, or where processes are described as having, including, or comprising specific process steps, it is contemplated that compositions of the present teachings can also consist essentially of, or consist of, the recited components, and that the processes of the present teachings can also consist essentially of, or consist of, the recited process steps.

In the application, where an element or component is said to be included in and/or selected from a list of recited elements or components, it should be understood that the element or component can be any one of the recited elements or components, or the element or component can be selected from a group consisting of two or more of the recited elements or components. Further, it should be understood that elements and/or features of a composition, an apparatus, or a method described herein can be combined in a variety of ways without departing from the spirit and scope of the present teachings, whether explicit or implicit herein

The use of the terms "include, " "includes" , "including, " "have, " "has, " or "having" should be generally understood as open-ended and non-limiting unless specifically stated otherwise.

The use of the singular herein includes the plural (and vice versa) unless specifically stated otherwise.

It should be understood that the order of steps or order for performing certain actions is immaterial so long as the present teachings remain operable. Moreover, two or more steps or actions may be conducted simultaneously.

The term “λ _ex” as used herein refers to excitation wavelength.

The phrase “aggregation caused quenching” or “ACQ” as used herein refers to the phenomenon wherein the aggregation of π-conjugated fluorophores significantly decreases the fluorescence intensity of the fluorophores. The aggregate formation is said to “quench” light emission of the fluorophores.

The phrase “aggregation induced emission” or “AIE” as used herein refers to the phenomenon manifested by compounds exhibiting significant enhancement of light-emission upon aggregation in the amorphous or crystalline (solid) states whereas they exhibit weak or almost no emission in dilute solutions.

“Emission intensity” as used herein refers to the magnitude of fluorescence/phosphorescence normally obtained from a fluorescence spectrometer or fluorescence microscopy measurement; “fluorophore” or “fluorogen” as used herein refers to a molecule which exhibits fluorescence; “luminogen” or “luminophore” as used herein refers to a molecule which exhibits luminescence; and “AIEgen” as used herein refers to a molecule exhibiting AIE characteristics.

As used herein, “halo” or “halogen” refers to fluoro, chloro, bromo, and iodo.

As used herein, “alkyl” refers to a straight-chain or branched saturated hydrocarbon group. Examples of alkyl groups include methyl (Me) , ethyl (Et) , propyl (e.g., n-propyl and z'-propyl) , butyl (e.g., n-butyl, z'-butyl, sec-butyl, tert-butyl) , pentyl groups (e.g., n-pentyl, z'-pentyl, -pentyl) , hexyl groups, and the like. In various embodiments, an alkyl group can have 1 to 40 carbon atoms (i.e., C1-40 alkyl group) , for example, 1-30 carbon atoms (i.e., C1-30 alkyl group) . In some embodiments, an alkyl group can have 1 to 6 carbon atoms, and can be referred to as a “lower alkyl group” . Examples of lower alkyl groups include methyl, ethyl, propyl (e.g., n-propyl and z'-propyl) , and butyl groups (e.g., n-butyl, z'-butyl, sec-butyl, tert-butyl) . In some embodiments, alkyl groups can be substituted as described herein. An alkyl group is generally not substituted with another alkyl group, an alkenyl group, or an alkynyl group.

As used herein, “alkenyl” refers to a straight-chain or branched alkyl group having one or more carbon-carbon double bonds. Examples of alkenyl groups include ethenyl, propenyl, butenyl, pentenyl, hexenyl, butadienyl, pentadienyl, hexadienyl groups, and the like. The one or more carbon-carbon double bonds can be internal (such as in 2-butene) or terminal (such as in 1-butene) . In various embodiments, an alkenyl group can have 2 to 40 carbon atoms (i.e., C2-40 alkenyl group) , for example, 2 to 20 carbon atoms (i.e., C2-20 alkenyl group) . In some embodiments, alkenyl groups can be substituted as described herein. An alkenyl group is generally not substituted with another alkenyl group, an alkyl group, or an alkynyl group.

As used herein, “heteroatom” refers to an atom of any element other than carbon or hydrogen and includes, for example, nitrogen, oxygen, silicon, sulfur, phosphorus, and selenium.

As used herein, “aryl” refers to an aromatic monocyclic hydrocarbon ring system or a polycyclic ring system in which two or more aromatic hydrocarbon rings are fused (i.e., having a bond in common with) together or at least one aromatic monocyclic hydrocarbon ring is fused to one or more cycloalkyl and/or cycloheteroalkyl rings. An aryl group can have 6 to 24 carbon atoms in its ring system (e.g., C6-24 aryl group) , which can include multiple fused rings. In some embodiments, a polycyclic aryl group can have 8 to 24 carbon atoms. Any suitable ring position of the aryl group can be covalently linked to the defined chemical structure. Examples of aryl groups having only aromatic carbocyclic ring (s) include phenyl, 1-naphthyl (bicyclic) , 2-naphthyl (bicyclic) , anthracenyl (tricyclic) , phenanthrenyl (tricyclic) , pentacenyl (pentacyclic) , and like groups. Examples of polycyclic ring systems in which at least one aromatic carbocyclic ring is fused to one or more cycloalkyl and/or cycloheteroalkyl rings include, among others, benzo derivatives of cyclopentane (i.e., an indanyl group, which is a 5, 6-bicyclic cycloalkyl/aromatic ring system) , cyclohexane (i.e., a tetrahydronaphthyl group, which is a 6, 6-bicyclic cycloalkyl/aromatic ring system) , imidazoline (i.e., a benzimidazolinyl group, which is a 5, 6-bicyclic cycloheteroalkyl/aromatic ring system) , and pyran (i.e., a chromenyl group, which is a 6, 6-bicyclic cycloheteroalkyl/aromatic ring system) . Other examples of aryl groups include benzodioxanyl, benzodioxolyl, chromanyl, indolinyl groups, and the like. In some embodiments, aryl groups can be substituted as described herein. In some embodiments, an aryl group can have one or more halogen substituents, and can thus be referred to as a “haloaryl” group. Perhaloaryl groups, i.e., aryl groups where all of the hydrogen atoms are replaced with halogen atoms (e.g., -C ₆F ₅) , are included within the definition of “haloaryl” . In certain embodiments, an aryl group is substituted with another aryl group and can be referred to as a biaryl group. Each of the aryl groups in the biaryl group can be substituted as disclosed herein.

As used herein, “heteroaryl” refers to an aromatic monocyclic ring system containing at least one ring heteroatom selected from oxygen (O) , nitrogen (N) , sulfur (S) , silicon (Si) , and selenium (Se) or a polycyclic ring system where at least one of the rings present in the ring system is aromatic and contains at least one ring heteroatom. Polycyclic heteroaryl groups include those having two or more heteroaryl rings fused together, as well as those having at least one monocyclic heteroaryl ring fused to one or more aromatic carbocyclic rings, non-aromatic carbocyclic rings, and/or non-aromatic cycloheteroalkyl rings. A heteroaryl group, as a whole, can have, for example, 5 to 24 ring atoms and contain 1-5 ring heteroatoms (i.e., 5-20 membered heteroaryl group) . The heteroaryl group can be attached to the defined chemical structure at any heteroatom or carbon atom that results in a stable structure. Generally, heteroaryl rings do not contain O-O, S-S, or S-0 bonds. However, one or more N or S atoms in a heteroaryl group can be oxidized (e.g., pyridine Noxide thiophene S-oxide, thiophene S, S-dioxide) . Examples of heteroaryl groups include, for example, the 5-or 6-membered monocyclic and 5-6 bicyclic ring systems shown below:

where T is O, S, NH, N-alkyl, N-aryl, N- (arylalkyl) (e.g., N-benzyl) , SiH2, SiH (alkyl) , Si (alkyl) 2, SiH (arylalkyl) , Si (arylalkyl) 2, or Si (alkyl) (arylalkyl) . Examples of such heteroaryl rings include pyrrolyl, furyl, thienyl, pyridyl, pyrimidyl, pyridazinyl, pyrazinyl, triazolyl, tetrazolyl, pyrazolyl, imidazolyl, isothiazolyl, thiazolyl, thiadiazolyl, isoxazolyl, oxazolyl, oxadiazolyl, indolyl, isoindolyl, benzofuryl, benzothienyl, quinolyl, 2-methylquinolyl, isoquinolyl, quinoxalyl, quinazolyl, benzotriazolyl, benzimidazolyl, benzothiazolyl, benzisothiazolyl, benzisoxazolyl, benzoxadiazolyl, benzoxazolyl, cinnolinyl, lH-indazolyl, 2H-indazolyl, indolizinyl, isobenzofuyl, naphthyridinyl, phthalazinyl, pteridinyl, purinyl, oxazolopyridinyl, thiazolopyridinyl, imidazopyridinyl, furopyridinyl, thienopyridinyl, pyridopyrimidinyl, pyridopyrazinyl, pyridopyridazinyl, thienothiazolyl, thienoxazolyl, thienoimidazolyl groups, and the like. Further examples of heteroaryl groups include 4, 5, 6, 7-tetrahydroindolyl, tetrahydroquinolinyl, benzothienopyridinyl, benzofuropyridinyl groups, and the like. In some embodiments, heteroaryl groups can be substituted as described herein.

As used herein, a "donor" material refers to an organic material, for example, an organic nanoparticle material, having holes as the majority current or charge carriers.

As used herein, an "acceptor" material refers to an organic material, for example, an organic nanoparticle material, having electrons as the majority current or charge carriers.

As used herein, a "theranostic agent" refers to an organic material, for example, an organic nanoparticle material, having both diagnostic and therapeutic capabilities.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the presently described subject matter pertains.

Where a range of values is provided, for example, concentration ranges, percentage ranges, or ratio ranges, it is understood that each intervening value, to the tenth of the unit of the lower limit, unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the described subject matter. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and such embodiments are also encompassed within the described subject matter, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the described subject matter.

Throughout the application, descriptions of various embodiments use “comprising” language. However, it will be understood by one of skill in the art, that in some specific instances, an embodiment can alternatively be described using the language “consisting essentially of” or “consisting of” .

For purposes of better understanding the present teachings and in no way limiting the scope of the teachings, unless otherwise indicated, all numbers expressing quantities, percentages or proportions, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term “about” . Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. In addition, where the use of the term "about" is before a quantitative value, the present teachings also include the specific quantitative value itself, unless specifically stated otherwise. As used herein, the term "about" refers to a ±10%variation from the nominal value unless otherwise indicated or inferred.

Fluorescent Compounds

The present subject matter relates to fluorescent compounds (also referred to as “AIEgens” herein) that have aggregation-induced emission (AIE) characteristics. Each AIEgen comprises a triphenylamine (TPA) -thiophene building block. The fluorescent compounds have widely tunable emissions, covering the violet, blue, green, yellow, orange, red, deep red and NIR regions. The emission colors can be tuned by simple alteration of HOMO-LUMO energy level by the introduction of electron donor (D) -acceptor (A) substituents. For example, the maximum emission wavelength for TTV, TTB, TTG, TTY, TTO, TTR, TTDR, and TTNIR are, respectively violet (402 nm) , blue (482 nm) , green (531 nm) , yellow (580 nm) , orange (612 nm) , red (649 nm) , deep red (667 nm) , and NIR (724 nm) . Moreover, these AIEgens can be successfully utilized as lipid droplets (LDs) -specific bioprobes in cell imaging, determination of cell fusion, and photodynamic cancer cell ablation.

In an embodiment, a fluorescent compound has a backbone structural formula selected from the group consisting of:

wherein when Y is an optionally substituted heteroaryl, Y is not

wherein Y is alkyl, alkenyl,

wherein X is selected from the group consisting of phenyl, heteroaryl, and C=C.

Each AIEgen can be obtained through a one-or two-step reaction. Exemplary reaction schemes for preparing some of the fluorescent compounds are provided below:

Cell Imaging

One or more of the fluorescent compounds can be contacted with a cell and an imaging method can then be used to visualize a cellular target of interest. The target of interest can be, for example, lipid droplets (LDs) of the cell. The present compounds can be effectively utilized for LDs-specific cell imaging. The present compounds show excellent image contrast to the cell background and higher photostability than commercial LDs-staining fluorophores. The fluorescent compounds can be highly emissive inside cells upon irradiation. The compounds can also or in the alternative exhibit homogeneous dispersion. The imaging method can include, for example, fluorescence microscopy or confocal laser scanning microscopy.

In an embodiment, the target cell can include a fused cell, two of the fluorescent compounds can be contacted with the fused cell, and the cellular target of interest can include lipid droplets derived from multiple parental cells. In an embodiment, two of the AIEgens with different emission ranges can be used to stain two cells. The two cells can be fused and the subsequent fluorescence of two stained nuclei within the fused cell can indicate successful cell fusion. For example, two cells can be respectively stained with TTG and TTNIR and mingled to induce cell fusion. Both green and red fluorescence can be observed within the resulting fused cell, indicating that cell fusion between TTG and TTNIR stained cells successfully occurred.

In an embodiment, a method of cellular imaging can include contacting a target cell with one or more fluorescent compounds; and identifying a cellular target of interest using an imaging method, the one or more fluorescent compounds comprises a compound having the following backbone structural formula:

wherein each R is independently selected from the group consisting of H, alkyl, unsaturated alkyl, heteroalkyl, alkoxy, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkyl-NCS, alkyl-N ₃, and alkyl-NH ₂; and

In an embodiment, one or more of the fluorescent compounds used in the present cellular imaging methods can have a backbone structural formula selected from the group consisting of:

wherein Y is alkyl, alkenyl,

wherein X is selected from the group consisting of phenyl, heteroaryl, and C=C.

In an embodiment, the fluorescent compound used in the present cellular imaging methods comprises at least one compound selected from the group consisting of:

Cancer Therapy

The present compounds can efficiently generate reactive oxygen species (ROS) in vivo when irradiated with visible light. As such, the compounds can be effective in killing cancer cells through image-guided, photodynamic therapy (PDT) processes. PDT is a promising approach to cancer treatment because of the precise controllability, minimal invasive nature, and high spatiotemporal accuracy it offers.

In an embodiment, a method of generating reactive oxygen species can include irradiating one or more of the present compounds with white light. In an embodiment, the method of generating reactive oxygen species can include irradiating the following compound with white light:

A method of killing cancer cells can include contacting a target cancer cell with one or more of the present compounds, imaging the target cancer cell while the one or more compounds contacts the target cancer cell, and subjecting the target cancer cell to white light irradiation while the one or more compounds contacts the target cancer cell. The imaging method can be selected from fluorescence microscopy and confocal laser scanning microscopy. In an embodiment, the target cancer cell can be contacted with the following compound:

with the rest of the above-described procedure being followed.

As described herein, the fluorescent compounds can efficiently generate ROS in cancer cells under white light irradiation to kill the cancer cells. As such, the fluorescent compounds can be successfully used as photosensitizers in photodynamic therapy (PDT) applications.

The present teachings are illustrated by the following examples.

EXAMPLES

Materials and Instruments

Chemicals for synthesis were purchased from Sigma-Aldrich, MERYER or J&K used without further purification. All solvents were purified and dried following standard procedures. 1H spectra were measured on Bruker ARX 400 NMR spectrometers using CD ₂Cl ₂ or CDCl ₃ as the deuterated solvent. Mass spectrometric measurements (HRMS) were performed on a Finnigan MAT TSQ 7000 Mass Spectrometer system operating in matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF) mode. UV-Vis spectra were measured on a Milton Ray Spectronic 3000 array spectrophotometer. Steady-state photoluminescence (PL) spectra were recorded on a PerkinElmer LS 55 spectrophotometer. Fluorescence images of AIEgens in the solid state and the aggregation state were collected on an Olympus BX 41 fluorescence microscope. The cellular fluorescence images were taken using Zeiss laser scanning confocal microscope (LSM7 DUO) and analyzed using ZEN 2009 software (Carl Zeiss) .

For cell imaging and confocal colocalization, the cells (NCM460, DLD1, SW480, and SW620) were seeded and cultured at 37℃ in 35 mm glass-bottomed dishes. After incubation with TTNIR (1 μM) for 20 min, the cells were washed with PBS three times and subjected to imaging analysis using a laser scanning confocal microscope (Zeiss Laser Scanning Confocal Microscope; LSM7 DUO) . The excitation filter was 488 nm and the emission filter was 570-740 nm. For costaining assay, the AIEgen loaded COS-7 cells were subjected to incubation with BODIPY 493/503 Green or Nile red for 20 min. Afterwards, the cells were washed with PBS and then observed with CLSM. The cells were imaged using proper excitation and emission filters for each dye. The colocalization efficiency was analyzed with Olympus FV10-ASW software, in which the calculated Pearson’s coefficient was above 0.90.

For the photostability test, the cells were imaged using a confocal microscope (Zeiss Laser Scanning Confocal Microscope; LSM7 DUO) and analyzed using ZEN 2009 software (Carl Zeiss) . Both TTNIR and BODIPY493/503 Green were excited at 488 nm for one-photon imaging (1%laser power) . The scanning speed was 22.4 s per scan, and the repeated image scans were taken 40 times. The first scan of both TTNIR and BODIPY493/503 Green was set to 100%. Then, the pixel intensity values were averaged and plotted against the scan number. The resulting curve represents the bleaching rate.

For the ROS generation and PDT study, H2DCF-DA was used as the ROS generation indicator. In the experiments, 10 μL of H2DCF-DA of stock solution (1.0 mM) was added to 2 mL of TTNIR suspension, and white light (18 mW/cm ^-2) was employed as the irradiation source. The emission of H2DCF-DA at 534 nm was recorded at various irradiation periods. HeLa cells were seeded in 96-well plates (Costar, IL, USA) at a density of 6000–8000 cells per well. After overnight cell culture, the medium in each well was replaced with 100 mL fresh medium containing different concentrations of TTNIR. After 30 minutes of incubation, the plate containing HeLa cells was exposed to white light (around 18 mW/cm ^-2) for 30 min, and another plate with cells was kept in the dark as the control.

For the cell fusion study, two dishes of COS-7 cells were incubated with TTG and TTNIR for half an hour, separately. After that, the cells were washed with PBS 3 times, collected by adding trypsin, and centrifuged, respectively. Then, the cells were mixed together and incubated for 2 hours in another petri dish with a cover glass. 10 g of polyethylene glycol 3400 were dissolved in 10 mL of Dulbecco's Modified Eagle's medium (DMEM) without FBS. The mixed culture was overlaid for 5 min at 37℃ with 2 mL PEG solution. Then, the PEG solution was diluted with DMEM in gradient, after which the liquid was removed and replaced with DMEM. The obtained mixture was then subjected to image analyses by confocal microscopy.

Example 1

Synthesis of TTV

A mixture of bromide substituted triphenylamine moiety (1.2 mmol) , thiophen-2-ylboronic acid moiety (1 mmol) , THF (20 mL) , K ₂CO ₃ aqueous solution (2 M, 1.6 mL) , and Pd (PPh ₃) ₄ (58 mg, 0.05 mmol) was degassed and charged with N ₂. The mixture was refluxed overnight. The reaction was quenched by the addition of water (30 mL) and extracted with CH ₂Cl ₂ (3 × 30 mL) . The combined organic layer was dried over anhydrous Na ₂SO ₄ and evaporated. The residue was purified by column chromatography over silica gel using petroleum ether to afford the desired product TTV with a yield of 78%.

Compound TTV: ¹H NMR (400 MHz, CD ₂Cl ₂) : 7.60 (d, J = 6.8 Hz, 2H) , 7.41 (d, J = 8 Hz, 2H) , 7.37-7.33 (m, 4H) , 7.13-7.06 (m, 9H) . ¹³C NMR (100 MHz, CDCl ₃) : 147.49, 147.20, 144.26, 129.27, 128.54, 127.95, 126.71, 124.42, 123.98, 123.75, 123.02, 122.21. ESI HRMS: calcd. for C ₂₂H ₁₇NS [M] ⁺: 327.1082, found: 327.1066.

Example 2

Synthesis of TTG

TTG was synthesized according to Scheme 3. The process of synthesizing TTG was similar to TTV except for the change of starting materials.

Compound TTG: ¹H NMR (400 MHz, CDCl ₃) : 9.85 (s, 1H) , 7.70 (d, J = 4 Hz, 1H) , 7.52 (d, J = 8.8 Hz, 2H) , 7.31-7.28 (m, 5H) , 7.14-7.05 (m, 8H) . ¹³C NMR (100 MHz, CDCl ₃) : 182.57, 154.55, 149.11, 146.94, 141.28, 137.70, 129.46, 127.22, 126.10, 125.14, 123.85, 122.83, 122.33. ESI HRMS: calcd. for C ₂₃H ₁₇NOS [M] ⁺: 355.1031, found: 355.1030.

Example 3

Synthesis of TTY

TTY was synthesized according to Scheme 3. The process of synthesizing TTY was similar to TTV except for the change of starting materials.

Compound TTY: ¹H NMR (400 MHz, CDCl ₃) : 9.83 (s, 1H) , 7.69 (d, J = 4 Hz, 1H) , 7.46 (d, J = 9.2 Hz, 2H) , 7.25 (d, J = 4.8 Hz, 1H) , 7.10-7.08 (m, 4H) , 6.91-6.85 (m, 6H) , 3.81 (s, 6H) . ¹³C NMR (100 MHz, CDCl ₃) : 182.47, 156.51, 155.06, 149.98, 140.78, 139.85, 137.79, 127.19, 127.11, 124.21, 122.27, 119.23, 114.85, 55.46. ESI HRMS: calcd. for C ₂₅H ₂₁NO ₃S [M] ⁺: 415.1242, found: 415.1248.

Example 4

Synthesis of TTO

TTO was synthesized according to Scheme 3. The process of synthesizing TTO was similar to TTV except for the change of starting materials.

Compound TTO: ¹H NMR (400 MHz, CDCl ₃) : 9.84 (s, 1H) , 7.65 (d, J = 4 Hz, 1H) , 7.39 (d, J = 9.2 Hz, 2H) , 7.30 (d, J = 4 Hz, 1H) , 7.21 (d, J = 4 Hz, 1H) , 7.13-7.07 (m, 5H) , 6.92-6.84 (m, 6H) , 3.81 (s, 6H) . ¹³C NMR (100 MHz, CDCl ₃) : 182.33, 156.24, 148.95, 147.60, 146.72, 141.072, 137.44, 133.48, 127.22, 126.90, 126.46, 125.13, 123.54, 122.60, 119.94, 114.79, 55.47. ESI HRMS: calcd. for C ₂₉H ₂₃NO ₃S ₂ [M] ⁺: 497.1119, found: 497.1127.

Example 5

Synthesis of TTR

TTR was synthesized according to Scheme 3. The process of synthesizing TTR was similar to TTV except for the change of starting materials.

Compound TTR: ¹H NMR (400 MHz, CDCl ₃) : 9.86 (s, 1H) , 7.67 (d, J = 4Hz, 1H) , 7.40 (d, J = 8.8 Hz, 2H) , 7.27 (d, J = 3.2 Hz, 1H) , 7.23 (d, J = 4 Hz, 1H) , 7.16 (d, J = 4 Hz, 1H) , 7.11-6.08 (m, 6H) , 6.92 (d, J = 8.8 Hz, 2H) , 6.86 (d, J = 8.8 Hz, 4H) , 3.82 (s, 6H) . ¹³C NMR (100 MHz, CDCl ₃) : 182.34, 156.13, 148.61, 146.95, 144.74, 141.42, 140.41, 139.56, 137.37, 134.05, 133.93, 126.97, 126.79, 126.31, 125.57, 125.42, 124.05, 123.87, 122.39, 120.17, 114.76, 55.47. ESI HRMS: calcd. for C ₃₁H ₂₁NOS ₃ [M] ⁺: 519.0785, found: 579.0761.

Example 6

Synthesis of TTB

TTB was synthesized according to Scheme 2, and included two steps.

Synthesis of compound 4- (5-bromothiophen-2-yl) -N, N-diphenylaniline: 4-Borate triphenylamine (742 mg, 2.0 mmol) , 2, 5-dibromothiophene (423 mg, 1.8 mmol) , K ₂CO ₃ aqueous solution (2 M, 2.4 mL) , and Pd (PPh ₃) ₄ (116 mg, 0.1 mmol) were placed in a 100 mL two-neck round bottom flask and 30 mL of THF was added as solvent under nitrogen. The mixture was heated to reflux for 10 h, cooled to room temperature, transferred to 40 mL of saturated salt water, extracted with DCM (40 mL × 3) , filtered under reduced pressure, and the crude product was purified by column chromatography (petroleum ether/ethyl acetate = 40/1) to give a pale yellow solid (583 mg, 72%yield) . ¹H NMR (400 MHz, CDCl ₃) δ 7.39 (d, J = 8.7 Hz, 2H) , 7.33–7.26 (m, 4H) , 7.16-7.12 (m, 4H) , 7.11-7.05 (m, 4H) , 7.02 (d, J = 3.8 Hz, 1H) , 6.97 (d, J = 3.8 Hz, 1H) .

Synthesis of compound TTB: Under nitrogen, 4- (5-bromothiophen-2-yl) -N, N-diphenylaniline (406 mg, 1 mmol) , TPE-B (OH) ₂ (451 mg, 1.2 mmol) , Pd (PPh ₃) ₄ (58 mg, 0.05 mmol) , and K ₂CO ₃ aqueous solution (2 M, 0.8 mL) in 20 ml THF were heated to reflux overnight. After cooling to room temperature, the product was extracted with dichloromethane. After removal of the solvent, the crude product was purified on a silica gel column using hexane/ethyl acetate = 20/1 as eluent to give yellow solid. ¹H NMR (400 MHz, CDCl ₃) : 7.47 (d, J = 8.4 Hz, 2H) , 7.37 (d, J = 8.4 Hz, 2H) , 7.30-7.27 (m, 4H) , 7.22 (d, J = 4.0 Hz, 1H) , 7.17 (d, J = 4.0 Hz, 1H) , 7.15-7.0 (m, 25H) . ¹³C NMR (100 MHz, CDCl ₃) : 147.65, 147.44, 143.91, 143.86, 143.79, 143.47, 143.06, 142.69, 141.36, 140.58, 132.47, 132.08, 131.61, 131.54, 129.51, 128.55, 128.03, 127.91, 127.83, 126.76, 126.72, 126.64, 126.54, 124.77, 124.70, 124.04, 123.85, 123.30. ESI HRMS: calcd. for C ₄₈H ₃₅NS [M] ⁺: 657.2490, found: 657.2491.

Example 7

Synthesis of TTDR

TTDR was synthesized according to Scheme 3.

A mixture of TTG (1.0 mmol) and malononitrile (1.1 mmol) in ethanol (3 mL) was heated to reflux for 72 h. After cooling down to room temperature, the solvent was removed under vacuum. Then, water (20 mL) was added into the mixture, which was extracted with CH ₂Cl ₂ (1 mL × 3) . The combined organic phase was dried over Na ₂SO ₄ and filtered; the filtrate was removed under reduced pressure in order to obtain the crude product, which was further purified by silica gel chromatography (petroleum ether/CH ₂Cl ₂ as eluent) to give product with a yield of 49%. ¹H NMR (400 MHz, CDCl ₃) : 7.74 (s, 1H) , 7.68 (d, J = 4 Hz, 1H) , 7.52 (d, J = 8.8 Hz, 2H) , 7.33-7.30 (m, 5H) , 7.16-7.10 (m, 6H) , 7.04 (d, J = 8.8 Hz, 2H) . ¹³C NMR (100 MHz, CDCl ₃) : 157.11, 150.24, 149.91, 146.60, 140.34, 133.06, 129.56, 127.54, 125.48, 124.82, 124.32, 123.25, 121.68, 114.54, 113.66, 75.01. ESI HRMS: calcd. for C ₂₆H ₁₇N ₃S [M] ⁺: 403.1143, found: 403.1131.

Example 8

Synthesis of TTNIR

TTNIR was synthesized according to Scheme 3. The process of synthesizing TTNIR was similar to TTDR except for the change of starting materials.

Compounds TTNIR: ¹H NMR (400 MHz, CDCl ₃) : 7.72 (s, 1H) , 7.61 (d, J = 4 Hz, 1H) , 7.41-7.37 (m, 3H) , 7.24 (d, J = 4 Hz, 1H) , 7.16 (d, J = 4 Hz, 1H) , 7.10-7.07 (m, 4H) , 6.91-6.85 (m, 6H) , 3.81 (s, 6H) . ¹³C NMR (100 MHz, CDCl ₃) : 156.37, 149.96, 149.88, 149.29, 148.38, 140.28, 140.06, 132.87, 132.48, 128.53, 127.04.126.54, 124.58, 123.85, 122.92, 119.64, 114.82, 114.45, 113.62, 55.47. ESI HRMS: calcd. for C ₃₂H ₂₃N ₃O ₂S ₂ [M] ⁺: 545.1232, found: 545.1241.

Example 9

Single crystal structure analysis

All compounds included sufficient moieties that freely rotate in the single-molecule state, leading to energy consumption of the excited state through non-radiative pathways. Thus, these compounds were weakly emissive in solution. Aiming to further study and decipher their optical properties in the aggregation state, single crystals of TTG, TTY and TTDR were grown in DCM-MeOH mixtures by slow solvent evaporation. As illustrated in Figs. 1A-1C, 4A-4C, and 5A-5C, the twisted conformation of the TPA segment extends the intermolecular distance

between two parallel planes, remarkably reducing or avoiding the intermolecular π-π interactions and essentially preventing emission quenching in its aggregation state. Moreover, the molecular conformation can be strongly rigidified by abundant intermolecular interactions (such as C-H···O, C-H···C, S···C) , which results in the restriction of molecular motions and is beneficial for enhancing solid state emission efficiency. On the basis of XRD results, it is believed that these synthesized compounds are potentially AIE-active.

Example 10

Photophysical properties: AIE

The UV-vis absorption spectra of TTV, TTB, TTG, TTY, TTO, TTR, TTDR and TTNIR were measured in acetonitrile (ACN) . As shown in Fig. 2A and Table 1, the solution of building block TTV displays a maximum absorption band at 348 nm, and the maximum absorption peaks of these modified compounds range from 383 nm to 512 nm.

Table 1. Optical properties of AIEgens TTV, TTB, TTG, TTY, TTO, TTR, TTDR and TTNIR.

^a) Absorption maximum in ACN solutions; ^b) Emission maximum in ACN (10 μM) ; ^c) Emission maximum in solid state; ^d) Fluorescence quantum yield determined by a calibrated integrating sphere system; ^e) Fluorescence lifetime, measured under ambient conditions.

The gradually red-shifted absorption wavelengths can be attributed to the orderly enhanced D-A effect from TTV to TTNIR. To investigate their AIE features, an ACN/H ₂O mixture with different H ₂O fractions was utilized as a solvent system. It was observed that compounds TTB, TTG, TTY, TTO, TTR, TTDR and TTNIR exhibit typical AIE features (Fig. 2C) . TTY, for example, exhibited almost no fluorescence emission when the H ₂O fraction was below 60%. Upon raising the fraction of water, the PL intensity increased dramatically because of activation of RIM by molecular aggregation and reached its maximum at 90%water fraction. This was 185-fold higher than that which was achieved in ACN solution (Fig. 2B) . Although the fluorescence intensity of TTV is inversely proportional to water fraction, its quantum yield in the solid state (27.5%) is higher than that in the solution state (18.6%) , definitely demonstrating an aggregation-induced emission enhancement (AIEE) attribute. The gradually decreased fluorescence intensity of TTV along with the increased water fraction could be attributed to its twisted intramolecular charge transfer (TICT) feature, which was determined by both the red-shifted emission wavelength and the declined emission efficiency accompanying the raised solvent polarity (Figs. 6A-6B) . As one of the nonradiative pathways for the excited state to relax and deactivate, TICT effect is competitive with AIE properties in determining the PL intensity and efficiency using ACN/H ₂O solution system. In the case of TTV, the AIE feature is strongly depressed by the TICT effect in the nanoaggregation state. As illustrated in Figs. 2D, 2E, and Table 1, these TPA-thiophene building block-based AIEgens emit efficiently in both nanoaggregation and solid states exhibiting relatively high quantum yields ranging from 3.11%to 40.79%. Each maximum emission wavelength accurately peaks in violet (402 nm) , blue (482 nm) , green (531 nm) , yellow (580 nm) , orange (612 nm) , red (649 nm) , deep red (667 nm) and NIR (724 nm) regions, respectively, suggesting the extremely wide emission color tunability, which is ascribed to both of their varied π-conjugation and D-A effect. Additionally, the fluorescence decay curves in the solid state show that their lifetimes range from 0.64 to 3.69 ns (Figs. 2F and Table 1) .

Example 11

Theoretical Calculations

To better understand the optical properties of these AIEgens, density functional theory (DFT) calculations were carried out at B3LYP/6-31+G (d) level with molecular geometries optimized at TD-B3LYP/6-31+G (d) level (Fig. 3) . It was observed that from TTV to TTNIR, the calculated HOMO-LUMO energy gaps are generally decreased, and the results are in good accordance with experimental data of emission maximums. The orderly declined energy gaps are realized through ingenious modification of the TPA-thiophene building block with diverse electron-donating (thienyl or methoxyl groups) units, electron-accepting (aldehyde or cyano groups) units or a π-bridge. Except for TTB, the HOMOs of the rest of the AIEgens are delocalized at the TPA moiety, whereas their LUMOs are distributed on the other side of the structures, demonstrating typical D-A structural features. It has been demonstrated that the separation of HOMO and LUMO distribution is essential to effectively reduce the singlet-triplet energy gap, which facilitates the generation of reactive oxygen species (ROS) , further endowing these AIEgens with prominent potential for photodynamic therapy (PDT) applications. On the contrary, TTB possesses evenly distributed HOMO and LUMO, resulting from its both imperceptible D-A effect and long π-conjugation bridges.

Example 12

Bio-imaging, Visualization of Cell Fusion and Photodynamic Therapy Bioimaging

In the preliminary bioimaging experiment, a cell imaging study was conducted by using HeLa cells as a cell model. Cells were incubated with 1 μM of TTNIR for 20 min. As illustrated in Fig. 16A, bright fluorescence within cells can be observed showing excellent image contrast to the cell background. A co-localization study was further conducted by incubating HeLa cells with TTNIR and BODIPY493/503 Green. The latter dye is a commercially available bioprobe for the LDs, which are ubiquitous lipid-rich spherical organelles and actively involved in various biofunctions, such as signal transduction, lipid metabolism, and protein degradation. The perfect overlap between TTNIR and BODIPY493/503 Green in cell imaging output indicates the excellent LDs-specific targeting capability of TTNIR (Figs. 16B, 16C, and 16D) . Photostability is a key criteria for evaluating the overall stability of photosensitive substances. A continuous scanning method was then utilized to quantitatively study and compare the photostability of TTNIR and BODIPY493/503 Green. As shown in Figs. 16E-16I, after 15 minutes of laser irradiation, the fluorescence intensity of BODIPY493/503 Green encounters discernable decline, whereas TTNIR shows negligible photobleaching, suggesting that the photostability of TTNIR is superior to that of BODIPY493/503 Green.

To further prove its applicability, this staining and imaging strategy using TTNIR was exploited for other cell lines, including NCM460, DLD1, SW480, SW620 and COS-7 (Figs. 7A-7E) . In each case after incubation with TTNIR for 20 min, strong and specific internalization into the LDs were observed. Moreover, other AIEgens including TTV, TTB, TTG, TTY, TTO, TTR and TTDR were also investigated for cell imaging. It was observed that LDs can be clearly visualized with excellent image contrast to the cell background through respective incubation of cells with the AIEgens (Figs. 17A-17H) . Pearson’s correlation coefficients between AIEgens and commercially available LDs-bioprobes were determined to be 90-95%, solidly demonstrating the high specificity of these AIEgens for staining LDs (Figs. 8A-15D) . Their excellent LDs-staining specificity reasonably results from their lipophilic properties, which bring about efficient accumulation of the compounds in the hydrophobic spherical LDs due to the “like-like” interactions. These AIEgens possess various impressive features, such as high brightness, excellent targeting specificities to LDs, extraordinary photostabilities and widely tunable emission colors, making them remarkably important in visualization of biological structures and processes.

Cell Fusion

As a common phenomenon in nature, cell fusion is highly associated with many cellular processes, including fertilization, development of placenta, regeneration of skeletal muscle, oncogenesis, aneuploidy, chromosomal instability, and DNA damage. In addition, recent studies show that cell fusion could play a vital role in alternative therapies for restoring organ function through repairing cellular dysfunction. Therefore, the development of effective methods for visualizing cell fusion is of great importance. Encouraged by the excellent cell imaging results and homology of the AIEgens, a straightforward method for visualization of cell fusion outcome was conducted using the combination of TTG and TTNIR as cell imaging agents, due to their minimal overlap of emission range. In this experiment, two sets of cells were respectively stained with TTG and TTNIR, which were then mingled and treated by polyethylene glycol (PEG) to induce cell fusion. As illustrated in Figs. 18A-18F, after treatment by PEG, both green and red fluorescence of lipid droplets were observed within one single cell, suggesting that cell fusion between TTG-and TTNIR-staining cells successfully occured. In addition, the cell fusion outcome was also solidly verified through a commercially available nuclei-staining agent Hoechst 33258. The appearance of two stained nuclei within one single cell (Fig. 18D) indicated that the visualization strategy of cell fusion outcome by using two AIEgens with different emission ranges is definitely reliable. The developed AIEgens have widely tunable emissions and high emission efficiencies and can be useful in the fundamental study of cell fusion.

Photodynamic Therapy

Intense fluorescence in the near-infrared (NIR) region is highly desirable for many clinical processes, due to the salient advantages of deep tissue penetration, minimal photodamage to biological structures, and high image contrast to the physiological background. Moreover, NIR emission is generally realized by intensifying the D-A effect of the structure, resulting in the separation of HOMO and LUMO distribution, as well as the decrease of the singlet-triplet energy gap, thus facilitating the generation efficiency of ROS. Therefore, the AIEgen TTNIR with both bright NIR emission and strong D-A effect is potentially efficient for PDT, which is an extraordinary therapeutic modality, and has captivated much interest for treating various malignant and non-malignant diseases with minimal invasion and precise controllability. In the preliminary test, the ROS generation efficiency of TTNIR was investigated using H2DCF-DA as an indicator, which can emit fluorescence at around 534 nm triggered by ROS. As shown in Fig. 19A, in the presence of TTNIR, the emission of H2DCF-DA was rapidly intensified with the increase of irradiation time using white light as an irradiation source, reaching 36-fold enhancement in 6 min compared to the original emission intensity. On the contrary, the fluorescence intensities of AIEgens or H2DCF-DA alone were very low and remained almost constant under the same irradiation conditions. These results reveal ideal photo-sensitizing properties for ROS generation. Quantitative evaluation of phototherapy effect of TTNIR on HeLa cells was then explored through standard 3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide (MTT) assay. The dose-dependent toxicity study exhibits that there is no obvious cytotoxicity observed for the HeLa cells treated with TTNIR in dark conditions, even with the TTNIR concentration reaching as high as 20 μM (Fig. 19B) . Upon white light exposure, cell viability dropped gradually upon raising the concentration of TTNIR. Only 7%of cell viability remained with utilizing 20 μM of TTNIR, demonstrating almost complete cell apoptosis. Accordingly, TTNIR holds high effectiveness for cancer cell ablation by means of PDT.

The present subject matter being thus described, it will be apparent that the same may be modified or varied in many ways. Such modifications and variations are not to be regarded as a departure from the spirit and scope of the present subject matter, and all such modifications and variations are intended to be included within the scope of the following claims.

Claims

A method of cellular imaging, comprising:

contacting a target cell with one or more fluorescent compounds; and

identifying a cellular target of interest using an imaging method, the one or more fluorescent compounds comprises a compound having the following backbone structural formula:

wherein each R is independently selected from the group consisting of H, alkyl, unsaturated alkyl, heteroalkyl, alkoxy, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkyl-NCS, alkyl-N ₃, and alkyl-NH ₂, wherein R is substituted or unsubstituted; and

wherein Y is selected from the group consisting of alkyl optionally substituted by one or more cyano groups, alkenyl optionally substituted by one or more cyano groups, oxygen, hydrogen, optionally substituted phenyl, and optionally substituted heteroaryl.
The method of claim 1, wherein the compound has a backbone structural formula selected from the group consisting of:

wherein each R is independently selected from the group consisting of H, alkyl, unsaturated alkyl, heteroalkyl, alkoxy, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkyl-NCS, alkyl-N ₃, and alkyl-NH ₂; and

wherein X is selected from the group consisting of phenyl, heteroaryl, and C=C.
The method of claim 2, wherein the compound comprises at least one compound selected from the group consisting of:
The method of claim 1, wherein the cellular target of interest comprises a lipid droplet of the target cell.
The method of claim 1, wherein the target cell comprises a fused cell, two of the compounds are contacted with the target cell and the cellular target of interest comprises lipid droplets derived from multiple parental cells.
The method of claim 1, wherein the imaging method is selected from the group consisting of fluorescence microscopy and confocal laser scanning microscopy.
A fluorescent compound exhibiting aggregation induced emission properties, the compound having the following backbone structural formula:

wherein each R is independently selected from the group consisting of H, alkyl, unsaturated alkyl, heteroalkyl, alkoxy, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkyl-NCS, alkyl-N ₃, and alkyl-NH ₂, wherein R is substituted or unsubstituted;

wherein Y is selected from the group consisting of alkyl optionally substituted by one or more cyano groups, alkenyl optionally substituted by one or more cyano groups, optionally substituted phenyl, and optionally substituted heteroaryl; and

wherein when Y is an optionally substituted heteroaryl, Y is not
The compound of claim 7, wherein the compound has a backbone structural formula selected from the group consisting of:

wherein each R is independently selected from the group consisting of H, alkyl, unsaturated alkyl, heteroalkyl, alkoxy, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkyl-NCS, alkyl-N ₃, and alkyl-NH ₂, wherein R is substituted or unsubstituted; and

wherein X is selected from the group consisting of phenyl, heteroaryl, and C=C.
The compound of claim 8, wherein the compound comprises at least one compound selected from the group consisting of:
A method of cellular imaging, comprising:

contacting a target cell with one or more of the compounds of claim 7; and

identifying a cellular target of interest using an imaging method.
The method of claim 10, wherein the cellular target of interest comprises a lipid droplet of the target cell.
The method of claim 10, wherein the target cell comprises a fused cell, two of the compounds are contacted with the target cell, and the cellular target of interest comprises lipid droplets derived from multiple parental cells.
The method of claim 10, wherein the imaging method is selected from the group consisting of fluorescence microscopy and confocal laser scanning microscopy.
A method of generating reactive oxygen species, comprising irradiating the compound of claim 7 with white light.
The method of claim 14, wherein the compound is TTNIR.
A method of killing cancer cells, comprising:

contacting a target cancer cell with the compound of claim 7;

imaging the target cancer cell while the compound contacts the target cancer cell using an imaging method; and

subjecting the target cancer cell to white light irradiation while the compound is contacting the target cancer cell to kill the target cancer cell.
The method of claim 16, wherein the compound is
The method of claim 16, wherein the imaging method is selected from the group consisting of fluorescence microscopy and confocal laser scanning microscopy.
A fluorescent compound exhibiting aggregation induced emission properties, wherein the compound is selected from the group consisting of:
A method of cellular imaging, comprising:

contacting a target cell with one or more of the compounds of claim 19; and

identifying a cellular target of interest using an imaging method.