WO2023021495A2 - Aggregation induced emissive fluorophores (aiegens) interconnected through nonconjugated spacer, process for preparation and applications thereof - Google Patents

Abstract

An aggregation induced emissive fluorophore (AIEgen) interconnected through nonconjugated spacer having a structural formula selected from: formulas (1), (2), or (3) wherein, (a); and X is (b), (c), (d), (e), (f), (g) or (h). The present disclosure further provides a process for preparing the AIEgen interconnected through nonconjugated spacer and applications thereof.

Description

AGGREGATION INDUCED EMISSIVE FLUOROPHORES (AIEGENS) INTERCONNECTED THROUGH NONCONJUGATED SPACER, PROCESS FOR PREPARATION AND APPLICATIONS THEREOF

FIELD OF THE INVENTION

[0001] The present disclosure generally relates to the field of organic solid-state fluorescent materials and particularly relates to an aggregation induced emissive fluorophores (AIEgens). Specific embodiments relate to aggregation induced emissive fluorophores (AIEgens) interconnected through a nonconjugated spacer, process for preparation and applications thereof.

BACKGROUND OF THE INVENTION

[0002] Background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0003] High sensitivity and great applicability of the fluorescence phenomenon has made it an inevitable research tool in the modern scientific fields of chemistry, biology, materials science, biomedical science, and their interfaces. Organic fluorescence materials have tremendous applications in advanced tools and techniques such as sensors, optical switches, optical displays, information processing and anti-counterfeiting. A large number of organic materials such as fluorescent dyes and fluorescent proteins, have been extensively studied for the purpose of FR/NIR fluorescence imaging. Fluorescent dyes and fluorescent proteins, however, suffer from limited molar absorptivity and low photobleaching thresholds. This has greatly limited their application. Recently, organic solid-state fluorescent materials have emerged as potential candidates for diverse optoelectronic and bio-medical applications, specifically organic fluorophores have been considered interesting candidates to explore the cellular structures and biochemical processes. The fluorophores with red/NIR emission have found application in diagnostic imaging as well as image-guided photodynamic therapy.

[0004] The discovery of aggregation induced emission (AIE) phenomena in non-planar propeller systems has opened new dimension in developing solid-state fluorescent materials. Organic fluorophores with aggregation-induced emission characteristics (AIEgens) have been developed in an unprecedented manner and applied in the biomedical field including specific imaging such as mitochondrial membrane, lipids and tissue regeneration over the past decade owing to their solid-state emissive properties, superior fluorescence quantum yields, photosensitivities, as well as their “turn-on” characteristics in their aggregate states. However, precise control of organic π-conjugated molecules fluorescence remains a great challenge and depend on the molecular structure and organization. The strong solution fluorescence of aromatic π-conjugated molecules often turn-out to be very weak or non- fluorescence in solid state due to aggregation caused quenching effect. To improve the solid- state fluorescence efficiency, obtain tunable fluorescence and smart fluorescence character, structural tailoring and iterative synthetic methodologies are adopted, which are cumbersome and involve multi-step synthesis. The free intramolecular rotation and conformational flexibility often result into very weak or non-fluorescence fluorescence materials.

[0005] Therefore, there remains an unmet need to avoids multi-step structural tailoring and develop AIEgens by a strategy involving a simple synthetic process that can provide on- toxic AIEgens with tunable fluorescence, desired solid state fluorescence efficiency and that can yield NIR emissive fluorophore suitable for live cell imaging and other applications including for diagnostic and therapeutic purposes.

OBJECTIVE OF THE INVENTION

[0006] An objective of the present invention is to provide an aggregation induced emissive fluorophores (AIEgens) with desired solid state fluorescence efficiency and that can yield NIR emissive fluorophore.

[0007] Another objective of the present invention is to provide a strategy for a simple synthetic process that can provide an aggregation induced emissive fluorophores (AIEgens) with tunable fluorescence, desired solid state fluorescence efficiency and that can yield NIR emissive fluorophore.

SUMMARY OF THE INVENTION

[0008] The present invention in general relate to aggregation induced emissive fluorophores (AIEgens) interconnected through nonconjugated spacer.

[0009] In an aspect the present disclosure provides aggregation induced emissive fluorophores (AIEgens) interconnected through nonconjugated spacer and further condensed through one or more acceptor unit. [0010] In one aspect, the present disclosure provides an aggregation induced emissive fluorophore (AIEgen) interconnected through nonconjugated spacer having a structural formula selected from:

or a pharmaceutically acceptable salt thereof; wherein,

[0011] In one aspect, the present disclosure provides aggregation induced emissive fluorophore (AIEgen) interconnected through nonconjugated spacer having a structural formula selected from: or a pharmaceutically acceptable salt thereof; wherein

[0012] In a specific aspect, the AIEgens comprise two triphenylamine based donor-π acceptor (D-π-A), said AIEgens being interlinked with a non-conjugate spacer, the spacer being xylene isomer selected from ortho-, meta- or para- isomer and triphenylaminemoiety being optionally substituted with one or more acceptor unit(s) “X”. Such two individual fluorophores interconnected through a nonconjugated spacer impose structural rigidification and enhance the fluorescence efficiency and acceptor unit variation further condense the structure and modulate tunable fluorescence.

[0013] In another aspect, the present disclosure provides a process for preparing aggregation induced emissive fluorophores (AIEgens) interconnected through nonconjugated spacer, the process comprising the steps of a) reacting a fluorophore with a nonconjugated spacer to provide π acceptor (D-π-A), AIEgens interlinked with a nonconjugated spacer; and b) condensing AIEgens by substituting the fluorophore moiety with one or more acceptor unit(s).

[0014] In one specific aspect, the present disclosure provides a process for preparing aggregation induced emissive fluorophores (AIEgens) interconnected through nonconjugated spacer, the process comprising the steps of a) reacting 4-(diphenylamino)-2-hydroxy benzaldehyde with dibromo xylene to provide interlinked ortho-, meta- or para-triphenylamine aldehyde(TPA D-π-A) isomer; b) providing a solution of an interlinked TPAD-π-A isomer; and c) adding an acceptor to the solution obtained in step (b) and reacting under suitable condition to provide the isomer of an interlinked TPA D-π-A substituted with acceptors.

[0015] In another aspect, the present disclosure provides a polymer composite comprising isomer of an interlinked TPAD-π-A substituted with acceptors.

[0016] In further aspects, the present disclosure provides use of isomer of an interlinked TPA D-π-A substituted with acceptors or polymer composite comprising the same in external stimuli-induced fluorescence switching, latent fingerprinting, bioimaging live cell imaging and other such applications.

[0017] Various objects, features, aspects and advantages of the inventive subject matter will become more apparent from the following detailed description of preferred embodiments.

BRIEF DESCRIPTION OF FIGURES

[0018] The foregoing will be apparent from the following more particular description of example embodiments of the invention, as illustrated in the accompanying drawings.

[0019] FIG. 1A illustrates a synthetic route for preparation of isomers 1a, 2a, 3a, 1e, 2e, 3e, 1f, 2f, 3f and 3 g of an interlinked TP A D-π-A substituted with acceptor “x” .

[0020] FIG. IB illustrates a synthetic route for preparation of isomers lb, 2b, 3b, 1c, 2c, 3 c, 1d, 2d, and 3d of an interlinked TP AD-π-A substituted with acceptor “x” .

[0021] FIG. 2 illustrates molecular structure of 1-3 isomers of an interlinked TPA D-π - A with different acceptors in the crystal lattice obtained from single crystal structural analysis. C (grey), H (white), O (red) and N (blue).

[0022] FIG. 3A illustrates Molecular packing in the crystal lattice of isomers of an interlinked TPA D-π-A with different acceptors (a) la, (b) lb, (c) 1c and (d) If.

[0023] FIG. 3B illustrates Molecular packing in the crystal lattice of isomers of an interlinked TPA D-π-A with different acceptors (a) 2a, (b) 2band (c) 2c.

[0024] FIG. 3C illustrates Molecular packing in the crystal lattice of isomers of an interlinked TPA D-π-A with different acceptors (a) 2d, (b) 2eand (c) 2f.

[0025] FIG. 3D illustrates Molecular packing in the crystal lattice of isomers of an interlinked TPA D-π-A with different acceptors (a) 3a, (b) 3cand (c) 3e.

[0026] FIG 4A illustrates intermolecular interactions in the crystal lattice of isomers of an interlinked TPA D-π-A with different acceptors(a) lb, (b) If, (c) 2a and (d) 2b. C (grey), H (white), O (red) and N (blue). Dotted lines indicate the hydrogen bonding and C-H...π interactions in A.

[0027] FIG 4B illustrates intermolecular interactions in the crystal lattice of isomers of an interlinked TPA D-π-A with different acceptors (a) 2d, (b) 2f, (c) 3a (d) 3cand (e) 3e. C (grey), H (white), O (red) and N (blue). Dotted lines indicate the hydrogen bonding and C- H.. ,π interactions in A. [0028] FIG.5 illustrates solid state fluorescence spectra of the (a) ortho, (b) meta and (c) para-isomers an interlinked TPA D-π-A with different acceptors. Digital fluorescence images of the compounds are shown at the top of every graph (λexc = 370 nm (for graph) and 365 nm (for digital images).

[0029] FIG.6 illustrates concentration dependent fluorescence tuning from blue to NIR of isomer 1g of an interlinked TPA D-π-A (a) 1g, (b) 2g and (c) 3g. Corresponding digital fluorescence images are also shown that also clearly supported the fluorescence tuning (λexc = 370 nm (for graph) and 365 nm (for digital images).

[0030] FIG.7 illustrates concentration dependent fluorescence tuning of the interlinked TPA D-π-A isomer Igthat has been transformed into poly(methyl methacrylate) PMMA polymer matrix that also showed clear tuning of fluorescence. Digital images and spectra of 1g (10^-7 to 10^-2 M) in PMMA polymer thin films. λ_exc = 365 nm (for digital images) and 370 nm (for spectra).

[0031] FIG.8 A illustrates spectra of mechanofluorochromic(MFA) studies showing effect of mechanical crushing and heating induced reversible fluorescence switching of isomers of an interlinked TPAD-π-A with different acceptors(a) 1a, (b) 1b and (c) 1c.

[0032] FIG.8B illustrates spectra of mechanofluorochromic (MFA) studies showing effect of mechanical crushing and heating induced reversible fluorescence switching of isomers of an interlinked TPAD-π-A with different acceptors(a) 1f, (b) 2a and (c) 2d.

[0033] FIG.8B illustrates spectra of mechanofluorochromic (MFA) studies showing effect of mechanical crushing and heating induced reversible fluorescence switching of isomers of an interlinked TPAD-π-A with different acceptors(a)2e, (b) 2f and (c) 3 a.

[0034] FIG.8C illustrates spectra of mechanofluorochromic (MFC) studies showing effect of mechanical crushing and heating induced reversible fluorescence switching of isomers of an interlinked TPA D-π-A with different acceptors(a)3c, (b) 3d, (c) 3f, (d) 3band (d) 3e.

[0035] FIG. 9 illustrates digital fluorescent LFPs images of isomers of an interlinked TPA D-π-A with different acceptors (a) by dip printing on glass plate, coin and plastic sheet surfaces using (i) 1b, (ii) 1c and (iii) 1e and (b) by powder-dusting method using 1b, 1c and le on glass surface. [0036] FIG.10 illustrates bioimaging of uropathogenic Escherichia coli. 0.5 OD live bacterial cells stained with 0.2 mM fluorophores, and imaged using green and red filter with isomers of an interlinked TPAD-π-A with different acceptors(a) la, (b) 1b and (c) 1e.

[0037] FIG.11 illustrates in vitro toxicity assay. The viability of the cells after exposing to 0.2 mM interlinked TPA D-π-A with different acceptorsla, 1b and 1e was assessed by bacterial colony count assay and reported in colony forming units (cfu/mL). Unexposed E. coli serves as a control. While compared to control, no significant difference in CFU of the group treated with interlinked TPA D-π-A with different acceptors. Experiments were done in triplicate, mean ± SD.

[0038] FIG. 12 illustrates (i) in vitro toxicity assay: (A) Control, untreated RBC, (B) treated with isomer 1g of an interlinked TPA D-π-A (1mg/mL), and (C) Control, treated with 0.1 % Triton X-100. The morphology of RBC was assessed in phase contrast microscopy and (ii) biocompatibility assay: (A) Liver carboxylesterase activity of zebrafish, α- carboxylesterase (black bar) and P-carboxylesterase (grey bar), (B) Brain acetylcholinesterase activity of zebrafish (blue bar): Experiments were done in triplicates and the values are expressed as mean ± SD of 5 determinations. One way ANOVA shows no significant (ns) between the means of control and 1g.

[0039] FIG. 13 illustrates biocompatibility assay. (A) Liver carboxylesterase activity of zebrafish, α-carboxylesterase (black bar) and β-carboxylesterase (white cross bar), (B) Brain acetylcholinesterase activity of zebrafish. Experiments are done in triplicates and the values are expressed as mean±SDof3 determinations.

DETAILED DESCRIPTION OF THE INVENTION

[0040] The following is a full description of the disclosure's embodiments. The embodiments are described in such a way that the disclosure is clearly communicated. The level of detail provided, on the other hand, is not meant to limit the expected variations of embodiments; rather, it is designed to include all modifications, equivalents, and alternatives that come within the spirit and scope of the current disclosure as defined by the attached claims. Unless the context indicates otherwise, the term "comprise" and variants such as "comprises" and "comprising" throughout the specification are to be read in an open, inclusive meaning, that is, as "including, but not limited to." [0041] When "one embodiment" or "an embodiment" is used in this specification, it signifies that a particular feature, structure, or characteristic described in conjunction with the embodiment is present in at least one embodiment. As a result, the expressions "in one embodiment" and "in an embodiment" that appear throughout this specification do not necessarily refer to the same embodiment. Furthermore, in one or more embodiments, the specific features, structures, or qualities may be combined in any way that is appropriate.

[0042] Unless the content clearly demands otherwise, the singular terms "a," "an," and "the" include plural referents in this specification and the appended claims. Unless the content explicitly mandates differently, the term "or" is normally used in its broad definition, which includes "and/or."

[0043] In some embodiments, the numbers expressing quantities of ingredients, properties such as concentration, reaction conditions, and so forth, used to describe and claim certain embodiments of the invention are to be understood as being modified in some instances by the term “about.” Accordingly, in some embodiments, the numerical parameters set forth in the written description are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be constructed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable.

[0044] The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein.

[0045] All processes described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention. [0046] The headings and abstract of the invention provided herein are for convenience only and do not interpret the scope or meaning of the embodiments.

[0047] The following discussion provides many example embodiments of the inventive subject matter. Although each embodiment represents a single combination of inventive elements, the inventive subject matter is considered to include all possible combinations of the disclosed elements. Thus if one embodiment comprises elements A, B, and C, and a second embodiment comprises elements B and D, then the inventive subject matter is also considered to include other remaining combinations of A, B, C, or D, even if not explicitly disclosed.

[0048] All publications herein are incorporated by reference to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Where a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.

[0049] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description that follows, and the embodiments described herein, is provided by way of illustration of an example, or examples, of particular embodiments of the principles and aspects of the present disclosure. These examples are provided for the purposes of explanation, and not of limitation, of those principles and of the disclosure.

[0050] It should also be appreciated that the present invention can be implemented in numerous ways, including as a system, a method or a device. In this specification, these implementations, or any other form that the invention may take, may be referred to as processes. In general, the order of the steps of the disclosed processes may be altered within the scope of the invention.

[0051] Various terms as used herein are shown below. To the extent a term used in a claim is not defined below, it should be given the broadest definition persons in the pertinent art have given that term as reflected in printed publications and issued patents at the time of filing.

[0052] As used herein, terms “interconnect” and “interlinked” as used interchangeably to denote that the aggregation induced emissive fluorophores (AIEgens) is interconnected through or interlinked by nonconjugated spacer.

[0053] As used herein “Aggregation-induced emission” refers to a property in which a fluorophore, when dispersed, for example in organic solvent, emits little or no light. Upon aggregation of fluorophore molecules, however, for example in the solid state or in water due to the hydrophobicity of the fluorophore, light emission from the fluorophore is significantly enhanced.

[0054] The phrases “aggregation induced emissive fluorophores (AIEgens) interconnected through nonconjugated spacer”, “interlinked isomer/isomers”, “interlinked fluorophore isomer/isomers” are used interchangeably and mean the invented AIEgens interconnected through nonconjugated spacer of the present invention.

[0055] The present invention relates to aggregation induced emissive fluorophores (AIEgens) interconnected through nonconjugated spacer, the process for preparation and applications thereof.

[0056] In an embodiment, the present disclosure provides aggregation induced emissive fluorophores (AIEgens) interconnected through nonconjugated spacer and further condensed through one or more acceptor unit.

[0057] In one embodiment, the present disclosure provides an aggregation induced emissive fluorophore (AIEgen)interconnected through nonconjugated spacer having a structural formula selected from:

or a pharmaceutically acceptable salt thereof; wherein,

[0058] In one embodiment, the present disclosure provides an aggregation induced emissive fluorophore (AIEgen) interconnected through nonconjugated spacer having a structural formula selected from:

or a pharmaceutically acceptable salt thereof; wherein

[0059] In a specific embodiment, the AIEgens comprise two triphenylamine based donor-π acceptor (D-π-A), said AIEgens being interlinked with a non-conjugate spacer, the spacer being xylene isomer selected from ortho-, meta- or para- isomer and triphenylamine moiety being optionally substituted with one or more acceptor unit(s) “X” selected from without limitation

[0060] In accordance with the present disclosure, two individual fluorophores when interconnected through a nonconjugated spacer impose structural rigidification and enhance the fluorescence efficiency and acceptor unit variation further condense the structure and modulate tunable fluorescence.

[0061] The interlinked fluorophores with acceptor units in accordance with the present disclosure provide concentration dependent tunable fluorescence from blue (457 nm) to NIR (720 nm). The interlinked isomers provide tunable blue to NIR emission upon increasing concentration from 10^-7 M to 10^-2 M.

[0062] In another embodiment, the present disclosure provides a process for preparing aggregation induced emissive fluorophores (AIEgens) interconnected through nonconjugated spacer, the process comprising the steps of: a) reacting a fluorophore with a nonconjugated spacer to provide π acceptor (D-π-A), AIEgens interlinked with a nonconjugated spacer; and b) condensing AIEgens by substituting the fluorophore moiety with one or more acceptor unit(s).

[0063] In one specific embodiment, the present disclosure provides a process for preparing aggregation induced emissive fluorophores (AIEgens) interconnected through nonconjugated spacer, the process comprising the steps of: a) reacting 4-(diphenylamino)-2-hydroxy benzaldehyde with an ortho-, para-or meta- isomer of dibromo xylene to provide interlinked ortho-, meta- or para-triphenylamine aldehyde(TPAD-π-A) isomer; b) providing a solution of the interlinked TPAD-π-A isomer; and c) adding an acceptor to the solution obtained in step (b) and reacting under suitable condition to provide the isomer of an interlinked TPA D-π-A substituted with acceptors.

[0064] In one specific embodiment, the present disclosure provides a A process for preparing aggregation induced emissive fluorophores (AIEgens) interconnected through nonconjugated spacer having a structural formula selected from:

a) reacting 4-(diphenylamino)-2-hydroxy benzaldehyde with an ortho-, para-or meta- isomer of dibromo xylene to provide interlinked ortho-, meta- or para-tri phenyl amine aldehyde(TPAD-π-A) isomer; b) providing a solution of the interlinked TPAD-π-A isomer; and c) adding an acceptor selected from selected from without limitation

and reacting under suitable condition to provide the isomer of an interlinked TPA D-π-A substituted with acceptors. [0065] The solvent that can be used for providing the solution of the interlinked TPA D-π-A isomer will depend upon the acceptor unit(s).

[0066] In certain embodiments, the solvent can be organic polar solvent like C1-C6 alcohol, for example ethanol.

[0067] In certain embodiments, the solvent can be an acid for example, glacial acetic acid either alone or in combination with solvent such as ammonium acetate.

[0068] The suitable condition for the process can be a room temperature to a temperature up to 95 °C, preferable 85 °C.

[0069] The process can be carried out for a persion of about 1 h to about 8 h, preferably from about 2 h to about 4 h.

[0070] In another aspect, the present disclosure provides a polymer composite comprising isomer of an interlinked TPA D-π-A substituted with acceptors. The polymer can be polymethylmethacrylate.

[0071] In certain embodiments, the present disclosure provides use of isomer of an interlinked TPA D-π-A substituted with acceptors or polymer composite comprising the same in external stimuli-induced fluorescence switching, latent fingerprinting, bioimaging, live cell imaging and other such applications.

[0072] While the foregoing describes various embodiments of the disclosure, other and further embodiments of the disclosure may be devised without departing from the basic scope thereof. The scope of the invention is determined by the claims that follow. The invention is not limited to the described embodiments, versions or examples, which are included to enable a person having ordinary skill in the art to make and use the invention when combined with information and knowledge available to the person having ordinary skill in the art.

EXAMPLES

[0073] The present invention is further explained in the form of the following examples. However, it is to be understood that the following examples are merely illustrative and are not to be taken as limitations upon the scope of the invention.

Example 1

Synthesis and characterization of aggregation induced emissive fluorophores (AIEgens) interconnected through nonconjugated spacer and condensed through acceptor units A. Synthesis

Triphenylamine, dibromoxylene isomers, malanonitrile, cyanoacetamide, cyanoacetic acid, ethyl cyanoacetate, Meldrum’s acid, N-methylbarbituric acid, 1,3-indandione and solvents were purchased from Sigma- 111 Aldrich or Merck India and used without further purification.

[0074] 4-(diphenylamino)-2-hydroxy-benzaldehyde was synthesized by following the reported procedure (Hariharan, P. S.; Anthony, S. P. Selective Turn-on Fluorescencefor Zn²⁺ and Zn ²⁺ + Cd ²⁺ Metal Ions by Single Schiff Base Chemosensor. Anal. Chim. Acta 2014, 848, 74-79).

[0075] The aggregation induced emissive fluorophores (AIEgens) interconnected through nonconjugated spacer that is interlinked triphenylamine (TPA) D-π-A fluorophores were synthesized by following procedure:

[0076] To the stirred solution of 4-(diphenylamino)-2-hydroxybenzaldehyde 117 (1.0 equiv) in DMF were added dibromo-o-xylene/dibromo-m- xylene/dibromo-p-xylene (0.5 equiv) and potassium carbonate (5 119 equiv), and the mixture wasstirred for 10 min. Then the reaction 120 mixture was heated at 100 °C for 2 h. After completion of the reaction, the reaction mixture was brought to room temperature and slowly added to cold water. The product that formed was filtered and washed with water

[0077] Aldehyde unit of the interlinked TPA D-π-A fluorophoremolecules were further condensed with different acceptor units “X” as follows as to obtain the target nonconjugate spacerinterconnected AIEgensisomeric compounds.

[0078] Nonconjugate spacer interconnected AIEgensisomeric compoundsa, e, f and g were prepared as per synthetic route in FIG. 1A: The interlinked TPA D-π-A fluorophore(1 mmol) was dissolved in ethanol (10 ml). Malononitrile, N-methyl barbituric acid, Meldrum’s acid and 1,3-indandione(1.3 mmol) acceptors (a, e, f and g respectively) were added individually into separate ethanol solutions of interlinked TPA D-π-A fluorophoreand stirred at room temperature for 2 h.

[0079] Nonconjugate spacer interconnected AIEgens isomeric compounds b, c, and dwere prepared as per synthetic route in FIG. IB: The interlinked TPA D-π-A fluorophore(1 mmol) was dissolved in glacial acetic acid (10 ml). Cyanoacetamide, cyanoacetic acid and ethyl cyanoacetate 1.5 mmol) acceptors (b, c and d) were added individually into separate glacial acetic acid solution of interlinked TPA D-π-A fluorophore along with ammonium acetate (150 mg) and heated at 80 °C for 4 h.

[0080] After completion of the reaction, the mixture obtained above were poured individually into ice-water. The formed precipitate was filtered, washed by distilled water, and dried under vacuum. Each of the isomer of an interlinked TPA D-π-A with different acceptors (a, b, c, d, e, f and g) that is compounds 1a, 1b, 1c, 1d, 1e, 1f, 1g, 2a, 2b, 2c, 2d, 2e, 2f, 1g, 3a, 3b, 3c, 3d, 3e, 3f and 3g was further purified by recrystallization and confirmed by NMR and Single Crystal X-ray structural analysis.

B. Characterization

1. NMR la : Yield: 82%. ¹H NMR (600 MHz, CDCl₃) δ 8.11-8.09 (d, 2H), 7.67 (s, 2H), 7.35-7.30 (m, 10H), 7.27 (d, 1H), 7.23-7.21 (m, 4H), 7.19-7.15 (m, 7H), 7.12-7.10 (2H),6.52-6.50(dd, 2H), 6.22 (d, 2H) 4.79 (t, 4H). ¹³C NMR (150 MHz, CDCl₃) δ 159.86, 155.37, 151.63, 145.01, 136.11, 130.24, 130.02, 129.55, 127.88, 126.97, 126.83, 126.33, 114.81, 112.94, 112.01, 101.20, 73.69, 70.48. C₅₂H₃₆N₆O₂ (776.29): calculated. C 80.39, H 4.67, N 10.82; found C 80.28, H 4.75, N 10.72. lb : Yield: 72%. ¹H NMR (600 MHz, CDCl₃) δ 8.58 (t, 2H), 8.14-8.13 (d, 2H), 7.28-7.23 (m, 10H), 7.15-7.13 (m, 7H), 7.09-7.08 (m, 9H), 6.52-6.50 (d, 2H), 6.22-6.21(d, 2H), 5.57 (s, 2H), 4.75 (q, 4H). ¹³C NMR (150 MHz, DMSO d₆) δ 163.68, 159.08, 153.40, 145.65, 144.45, 134.70, 130.47, 129.79, 127.06, 126.09, 112.96, 111.47, 102.28, 100.51, 79.71, 68.02. C₅₂H₄₀N₆O₄ (812.31): calculated. C 76.83, H 4.96, N 10.34; found C 76.91, H 4.87, N 10.39.

1c : Yield: 78%. ¹H NMR (600 MHz, CDCl₃) δ 8.53(t, 2H), 8.28-8.26(d, 2H), 7.28-7.25(m, 10H), 7.20-7.19 (q, 2H), 7.15-7.12 (m, 4H), 7.09-7.08 (m, 8H), 6.53-6.51 (dd, 2H), 6.22 (d, 2H), 4.75 (t, 4H), 4.30-4.27 (h, 4H), 1.34-1.31 (m, 6H). ¹³C NMR (150 MHz, CDCl₃) δ 163.95, 159.61, 154.10, 147.50, 145.48, 134.03, 130.62, 129.87,128.88, 128.77, 126.77, 125.72, 117.46, 113.09, 112.11, 101.83, 96.17, 68.25, 62.03, 14.32. C₅₂H₄₀N₄O₆ (870.34): calculated. C 77.22, H 5.32, N 6.43; found C 77.30, H 5.39, N 6.51. Id : Yield: 70%. ¹H NMR (600 MHz, CDCl₃) 10.10 (s, 2H), 8.54 (s, 2H), 8.27-8.24 (d, 2H), 7.43 (s, 6H), 7.31-7.26(m, 5H), 7.20-7.14 (m, 4H), 7.10-7.07 (d, 8H), 6.55-6.51 (dd, 2H),

6.20-6.19(d, 2H), 4.81-4.74 (t, 5H). ¹³C NMR (150 MHz, DMSO d₆) δ 164.89, 159.72, 154.08, 146.45, 145.71, 145.37, 134.74, 130.52, 130.05, 129.22, 127.28, 126.43, 118.0, 112.39,111.27, 101.54, 96.97, 78.05, 68.39. C₅₂H₃₈N₄O₆ (814.28): calculated. C 76.64, H

4.70, N 6.88; found C 76.59, H 4.81, N 6.71. le : Yield: 79%. ¹H NMR (600 MHz, CDCl₃) 8.40-8.38 (d, 2H), 7.29-7.25 (m,10H), 7.20- 7.18 (m, 4H), 7.16-7.14 (m,4H), 7.08-7.07 (m, 8H), 8.47-8.45 (dd, 2H), 6.29-6.28 (d, 2H), 4.99-4.94 (t,4H), 1.72 (s, 12H). ¹³C NMR (150 MHz, CDCl₃) δ 164.83, 161.79, 161.51, 155.40, 151.40, 145.03, 135.85, 135.55, 129.92, 127.60, 126.99, 126.15, 113.97, 111.14, 107.35, 103.67, 101.33, 70.08, 27.44. C₅₈H₄₈N₂O₁₀ (932.33): calculated. C 74.66, H 5.19, N 3.00; found C 74.56, H 5.24, N 3.08.

If : Yield: 81%. ¹H NMR (600 MHz, CDCl₃) δ 8.91-8.87 (s, 2H), 8.57-8.55 (d, 2H), 7.28- 7.24 (m, 10H), 7.19-7.18 (m, 2H), 7.15-7.12 (m, 4H), 7.12-7.10 (m, 7H), 6.45-6.44 (dd, 2H),

6.21-6.20 (d, 2H), 4.86-4.81 (t, 4H), 3.37-3.31 (q, 12H). ¹³C NMR (150 MHz, CDCl₃) δ 163.60, 161.73, 161.45, 155.14, 152.16, 151.87, 145.34, 135.99, 134.15, 129.85, 128.90,

128.70, 126.93,125.85, 114.74, 111.56, 110.78, 101.07,68.43, 28.95, 28.33. C₅₈H4₈N₆O₈ (956.35): calculated. C 72.79, H 5.06, N 8.78; found C 72.71, H 5.13, N 8.81.

1g : Yield: 60%. ¹H NMR (600 MHz, CDCl₃) δ 8.3 (s, 2H), 7.89-7.86 (m, 4H), 7.70-7.68 (m, 4H), 7.29-7.27 (m, 3H), 7.25-7.24 (m, 10H), 7.23-7.22 (t, 2H), 7.14-7.08 (m, 11H), 6.49-6.47 (dd, 2H), 6.24-6.23 (d, 2H), 4.92-4.84 (p, 4H). ¹³C NMR (150 MHz, CDCl₃) δ 187.20, 161.84, 161.29, 154.68, 145.81, 140.44, 135.39, 134.27, 134.22, 131.73, 129.92, 129.76, 129.03, 128.66, 126.86, 125.65, 125.36, 118.0, 111.95, 102.23, 68.14. C64H44N2O6 (936.32): calculated. C 82.03, H 4.73, N 2.99; found C 82.11, H 4.79, N 2.84.

2a : Yield: 84%. ¹H NMR (600 MHz, CDCl₃) δ 8.16-8.14 (d, 2H), 8.07 (s, 2H), 7.35-7.33 (m,10H), 7.25-7.20 (m, 6H), 7.15-7.13(m, 8H), 6.53-6.51(q, 2H), 6.37-6.36 (d, 2H), 4.84 (t, 4H). ¹³C NMR (150 MHz, CDCl₃) δ 159.86, 155.37, 151.63, 145.01, 136.11, 130.24, 130.02, 129.55, 127.88, 126.97, 126.83, 126.33, 116.13, 114.81, 112.94, 112.01, 101.20, 73.69, 70.48. C₅₂H₃₆N₆O₂ (776.29): calculated. C 80.39, H 4.67, N 10.82; found C 80.41, H 4.70, N 10.86. 2b : Yield: 77%. ¹H NMR (600 MHz, CDCl₃) δ 8.76 (t, 2H), 8.16-8.14 (d, 2H), 7.29-7.25 (m, 13H), 7.15-7.13 (q, 5H), 7.10-7.07 (m, 6H), 6.54-6.52 (dd, 2H), 6.38-6.37 (d, 2H), 6.20 (s, 2H), 5.60 (S, 2H), 4.89 (t, 4H). ¹³C NMR (150 MHz, DMSO d⁶) δ 163.77, 159.47, 153.26, 145.68, 144.98, 137.06, 130.46, 129.89, 127.36, 126.90, 126.01, 118.29, 113.31, 117.73, 103.07, 100.93, 70.26. C52H40N6O4 (812.31): calculated. C 76.83, H 4.96, N 10.34; found C 76.91, H 4.87, N 10.39.

2c : Yield: 72%. ¹H NMR (600 MHz, CDCl₃) δ 8.70 (s, 1H), 8.29-8.28 (d, 2H), 7.31-7.25 (m, 9H), 7.20-7.18 (d, 2H), 7.16-7.13 (t, 4H), 7.10-7.09 (m, 9H),7.02 (s, 1H), 6.55-6.53 (dd, 2H), 6.39-6.38 (d, 2H), 4.93-4.88 (s, 4H), 4.32-4.29 (q, 4H), 1.35-1.33 (t, 6H). ¹³C NMR (300 MHz, CDCl₃) δ 164.08, 160.01, 154.15, 148.03, 145.53, 136.58, 130.62, 129.82, 129.33, 126.89, 126.68, 125.65, 117.51, 113.39, 112.33, 102.46, 96.21, 70.25, 62.03, 14.34. C₅₆H₄₆N₄O₆ (870.34): calculated. C 77.22, H 5.32, N 6.43; found C 77.10, H 5.29, N 6.31.

2d : Yield: 60%. ¹H NMR (600 MHz, DMSO d⁶) δ 10.11 (s, 1H), 8.47 (s, 2H), 8.08-8.07 (d, 2H), 7.35-7.28 (m, 9H), 7.18-7.12 (q, 7H), 7.09-7.08(d, 8 H), 6.36-6.35 (m, 4H), 4.95-4.94 (t, 4H), 3.29 (1H). ¹³C NMR (150 MHz, DMSO d⁶) δ 164.96, 159.92, 154.00, 146.89, 145.37, 137.01, 130.50, 130.15, 129.56, 127.36, 127.12, 126.35, 117.96, 112.68, 111.48, 102.38, 97.43, 70.23. C₅₂H₃₈N₄O₆ (814.28): calculated. C 76.64, H 4.70, N 6.88; found C 76.75, H 4.61, N 6.92.

2e : Yield: 81%. ¹H NMR (600 MHz, CDCl₃) δ 8.93 (s, 2H), 8.38-8.36 (d, 2H), 7.31-7.29 (m, 8H), 7.25 (s, 4H), 7.21-7.16 (m, 4H), 7.12-7.07 (m, 8H), 6.48-6.46 (dd, 2H), 6.32 (d, 2H), 4.91-4.89 (d, 4H), 1.70(s, 12H). ¹³C NMR (150 MHz, CDCl₃) δ 164.73, 161.92, 161.49, 155.44, 151.43, 145.99, 145.18, 135.48, 129.99, 129.34, 126.94, 126.57, 126.03, 125.85, 125.30, 114.04, 111.37, 107.44, 103.64, 101.52, 70.34, 31.0, 27.41. C₅₈H₄₈N₂O₁₀ (932.33): calculated. C 74.66, H 5.19, N 3.00; found C 74.76, H 5.09, N 3.12.

2f : Yield: 76%. ¹H NMR (600 MHz, CDCl₃) δ 9.08-9.04(t, 2H), 8.57-8.56 (q, 1H), 7.30-7.25 (m, 10H), 7.20-7.16 (m, 7H), 7.13-7.09 (m, 8H), 6.52-6.48 (d, 2H), 6.37-6.33 (s, 2H), 4.96- 4.91 (q, 4H), 3.37-3.31 (m, 12H). ¹³C NMR (150 MHz, CDCl₃) δ 163.70, 162.10, 161.50, 155.23, 152.71, 151.88, 145.34, 136.68, 135.98, 129.82, 129.24, 126.88, 126.66, 126.52, 125.86, 125.59, 125.26, 115.02, 111.68, 111.02, 101.58, 70.35, 28.89, 28.31. C₅₈H₄₈N₆O₈ (956.35): calculated. C 72.79, H 5.06, N 8.78; found C 72.85, H 5.15, N 8.65. 2g : Yield: 62%. ¹H NMR (600 MHz, CDCl₃ ) δ 8.34 (s, 2H), 7.89-7.86 (m, 4H), 7.70- 7.68 (m, 4H), 7.27-7.25 (m, 10H), 7.25-7.22(m, 4H), 7.14-7.08 (m, 12H), 6.49-6.47 (dd, 2H), 6.24-6.23 (d, 2H), 4.92-4.84 (p, 4H). ¹³C NMR (150 MHz, DMSOd₆) δ 191.55, 163.94, 161.62, 154.86, 145.92, 145.42, 136.10, 134.45, 134.26, 129.71, 126.74, 126.61, 126.52, 125.64, 125.24, 124.10, 122.62, 122.56, 115.76, 111.67, 69.88. C₆₄H₄₄N₂O₆ (936.32): calculated. C 82.03, H 4.73, N 2.99; found C 82.15, H 4.69, N 2.92.

3a : Yield: 83%. ¹H NMR (600 MHz, CDCl₃) δ 8.16-8.13 (d, 2H), 8.09-8.07 (t, 2H), 7.37- 7.30 (m, 9H), 7.25-7.18 (m, 7H), 7.16-7.11 (m, 8H), 6.53-6.51 (dd, 2H), 6.37-6.36 (d, 2H), 4.87-4.82 (t, 4H). ¹³C NMR (150 MHz, CDCl₃) δ 162.08, 159.77, 155.30, 154.76, 151.61, 145.96, 144.93, 137.76, 135.35, 130.26, 130.02, 129.80, 128.06, 127.83, 127.79, 126.96, 126.60, 126.36,125.35, 116.12, 114.82, 112.95, 111.92, 101.21, 98.16, 70.20. C₅₂H₃₆N₆O₂ (776.29): calculated. C 80.39, H 4.67, N 10.82; found C 80.32, H 4.72, N 10.76.

3b : Yield: 76%. ¹H NMR (600 MHz, DMSO d⁶) δ 8.80-8.77 (d, 2H), 8.24-8.15 (d, 4H), 7.29-7.21 (m, 14H), 7.17-7.12 (m, 4H), 7.11-7.03 (m, 8H), 6.53-6.51 (dd, 2H), 6.35 (d, 2H), 4.97-4.88 (m, 4H). ¹³C NMR (150 MHz, DMSO d⁶) δ 163.82, 159.24, 153.11,145.60, 144.82, 136.39, 130.47, 129.93, 127.98, 126.85, 126.0, 118.28, 113.34, 104.26, 103.11, 101.06, 79.71, 69.80. C₅₂H₄₀N₆O₄ (812.31): calculated. C 76.83, H 4.96, N 10.34; found C 76.75, H 4.90, N 10.40.

3c : Yield: 72%. ¹H NMR (600 MHz, CDCl₃) δ 8.75 (s, 2H), 8.29-8.28 (d, 2H), 7.28-7.25 (m, 8H), 7.18-7.14 (m, 8H), 7.09-7.05 (m, 8H), 6.53-6.52 (dd, 2H), 6.37-6.36 (q, 2H), 4.94 (s, 4H), 4.33-4.30 (q, 4H), 1.36-1.34 (t, 6H). ¹³C NMR (150 MHz, CDCl₃) δ 164.14, 159.89, 154.12, 148.02, 145.41, 135.95, 130.65, 129.85, 129.80, 127.49, 126.72, 126.61, 125.72,117.52, 113.32, 112.13 102.32, 96.14, 69.94, 62.06, 31.01, 14.34. C56H46N4O6 (870.34): calculated. C 77.22, H 5.32, N 6.43; found C 77.19, H 5.38, N 6.51.

3d : Yield: 69%. ¹H NMR (600 MHz, DMSO d⁶) δ 10.19 (s, 1H), 8.54-8.52 (t, 2H), 7.37- 7.30 (m, 10H), 7.20-7.18 (d, 5H), 7.12-7.11 (m, 4H), 7.05 (m, 8H), 6.37 (m, 4H), 5.03 (d, 4H). ¹³C NMR (150 MHz, DMSO d⁶) δ 164.98, 159.89, 154.02, 147.01, 145.31, 136.34, 130.53, 130.20, 128.07, 127.15, 126.38, 117.94, 112.67, 111.47, 102.46, 97.25, 79.71, 69.97. C₅₂H₃₈N₄O₆ (814.28): calculated. C 76.64, H 4.70, N 6.88; found C 76.55, H 4.80, N 6.79. 3e : Yield: 75%. ¹H NMR (600 MHz, CDCl₃) δ 8.98 (s, 2H), 8.40-8.38 (d, 2H), 7.29-7.25 (m, 9H), 7.21-7.14 (m, 9H), 7.08-7.07 (d, 6H), 6.47-6.45 (dd, 2H), 6.29-6.28 (d, 2H), 4.97-4.94 (t, 4H), 1.72 (s, 12H). ¹³C NMR (150 MHz, CDCl₃) δ 164.81, 161.80, 161.49, 155.40,

151.39, 145.04, 135.86, 135.55, 129.92, 127.60, 126.99, 126.63, 126.15, 113.99, 111.14,

107.39, 103.66, 101.35, 70.09, 31.01, 27.44. C₅₈H₄₈N₂O₁₀ (932.33): calculated. C 74.66, H 5.19, N 3.00; found C 74.56, H 5.24, N 3.08.

3f : Yield: 83%. ¹H NMR (600 MHz, CDCl₃) δ 8.58-8.55 (d, 2H), 7.31-7.29 (m, 8H), 7.23- 7.17 (m, 10H), 7.11-7.08 (m, 8H), 6.51-6.47 (dd, 2H), 6.32-6.3 l(d, 2H), 4.98-4.95 (d, 4H), 3.39-3.36 (d, 12H). ¹³C NMR (150 MHz, CDCl₃) δ 163.82, 161.95, 161.50, 155.21, 152.77, 151.90, 145.17, 136.05, 129.87, 127.46, 126.94, 126.62, 125.98, 125.37, 114.89, 111.61, 110.70, 101.31, 70.03, 28.94, 28.35. C₅₈H4₈N₆O₈ (956.35): calculated. C 72.79, H 5.06, N 8.78; found C 72.65, H 5.01, N 8.80.

3g : Yield: 70%. ¹H NMR (600 MHz, CDCl₃ ) δ 8.52 (s, 2H), 7.89-7.88 (t,4H), 7.71- 7.69(t, 4H), 7.30-7.25 (m, 12H), 7.22 (s, 2H), 7.17-7.14 (t, 4H), 7.12-7.11 (d, 8H), 6.58-6.56 (dd, 2H), 6.37-6.36 (d, 2H), 4.99-4.94 (t, 4H). ¹³C NMR (150 MHz, CDCl₃ ) δ 187.67, 162.06, 154.69, 145.82, 136.05, 129.87, 129.73, 127.54, 126.81, 126.53, 125.29, 118.27, 112.16, 102.76, 69.69. C₆₄H₄₄N₂O₆ (936.32): calculated. C 82.03, H 4.73, N 2.99; found C 82.17, H 4.68, N 2.82.

2. Single crystal structural analysis

[0081] The structure of the nonconjugate spacer interconnected AIEgens that is all three isomers of interlinked TP A D-π-A with different acceptors were unambiguously confirmed by single crystal structural analysis (FIG. 2). Molecular packing in the crystal lattice ofall three isomers of interlinked TPA D-π-A with different acceptors indicated tighter molecular packing in ortho-isomers as compared to para-isomers(FIGs. 3 A-3D). Further crystal structure analysis revealed intermolecular interaction and conformational modulation depend on the acceptor unit in the solid state ((FIGs. 4A-4B)

3. Tunable solid state fluorescence

[0082] The interlinking produced strongly enhanced fluorescence in the solid state compared to corresponding single fluorophore unit (Table 1). Table 1. Comparison of two D-π -A interlinked 1-3 isomers fluorescence λ_max and quantum yields ( _f) with singleD-π-A molecule.

exhibited green and yellow fluorescence, respectively (Table 1, FIG. 5). 1d-1f displayed orange-red fluorescence between 594 and 611 nm in the solid-state. 2a showed orange fluorescence at 587 nm whereas 2b, 2c and 2d exhibited greenish yellow, yellow and yellow- orange fluorescence, respectively. 2e and 2f revealed orange-red and red fluorescence. Similarly, 3a showed orange fluorescence and 3b and 3c showed yellow fluorescence. 3d-3f showed orange-red fluorescence between 597 and 614 nm. Ortho-isomers showed relatively higher fluorescence efficiency compared to meta and para-isomers. Further, interlinked fluorophores, 1a-1c, showed enhanced fluorescence intensity compared to the corresponding known single TPA D-π-A compound. It is noted that single 3-methoxy TPA D-π-A derivative with cyanoacetamide was non-fluorescent in solid-state. But all three isomers of interlinked TPA D-π-A with cyanoacetamide acceptor showed strong solid-state fluorescence. Interlinked TPA D-π-A isomers 1g-3g showed weak solid-state fluorescence compared to single fluorophore. In contrast, interlinked isomers 1g-3g revealed broad emission covering blue to NIR region. The interlinked isomer 1g showed three fluorescence peaks (433, 630 and 706 nm) whereas interlinked isomers 2g and 3g exhibited dual fluorescence.

4. Tunable solution fluorescence

[0084] Interlinked TPA D-π-A isomerslg-3g, exhibited solvent polarity dependent strong fluorescence in solution state. All three compounds showed enhanced fluorescence in non-polar solvents compared to polar medium (Figure 6). Further, interlinked isomers 1g-3g showed blue shifted fluorescence in non-polar solvents and red shifted fluorescence in polar medium. 1g in toluene showed blue fluorescence whereas CHCl₃ and DMF solution showed yellow-orange fluorescence. The interlinked 1g-3g isomers not only showed strong fluorescence in solution state and also displayed concentration ( 10^-7 to 10^-2 M) dependent tunable fluorescence between blue and NIR. 10^-7 and 10^-6 M concentration of interlinked isomer 1g in CHCl₃ showed strong blue fluorescence. At 10^-2 M, 1g in CHCl₃ showed NIR emission at 720 nm. Digital fluorescent images further supported the tuning of fluorescence from blue to deep red with increasing concentration.

Example 2

Fabricating tunable fluorescent polymer composite

[0085] In order to fabricate tunable fluorescence in solid state, poly(methyl methacrylate)PMMA polymer (1 wt%) was also dissolved in CHCl₃ along with different concentration of interlinked TPA D-π-A isomerlg (10'⁷ to 10^-2 M). 1-PMMA polymer drop casted film from 10^-7 and 10^-6 M solution showed strongly blue shifted emission at 430 nm along with a weak hump at 538 nm (Figure 7). 1g-PMMAcomposite at 10^-5 M showed relatively strong dual fluorescence at 521 and 554 nm. Further increasing the concentration of interlinked isomer 1g exhibited strong reduction of shorter wavelength fluorescence and red shifting longer wavelength fluorescence. However, interlinked isomer 1g at high concentration exhibited relatively small red shifting compared to interlinked isomer 1g in pure solvent. This may be attributed to hindering strong aggregation of 1g by polymer matrix in the thin film state.

Application of fluorescence materials

Example 3

External stimuli-induced fluorescence switching

[0086] Mechanofluorochromic (MFC) studies of interlinked TPA D-π-A isomers 1-3 with different acceptors provided opportunity to correlate the effect of substituents and isomerism on the stimuli-responsive fluorescence switching. Interestingly, spatial isomerism exhibited significant influence on the MFC (FIGs. 8A-8D). Surprisingly, interlinked ortho- and meta-isomers (1a-f and 2a-f) did not show clear reversible MFC but interlinked para- isomers (3a-f) exhibited reversible MFC. The mechanism for reversible fluorescence switching upon crushing and heating was studied using powder X-ray diffractogram (PXRD) that showed reversible transformation between crystalline and amorphous/partial amorphous state upon crushing and heating.

Example 4

Latent fingerprinting

[0087] The strong tunable AIE of interlinked TPA D-π-A l-3isomers with different acceptors are potential candidates for forensic science and bio-imaging applications. Particularly, obtaining the latent fingerprints (LFP) with more accurate features of ridges and furrows formed on the surfaces (FIG. 9). The use of organic fluorescent materials could provide high contrast LEPs image compared to commonly used metal/magnetic powder in powder-dusting and spraying methods. Interlinked TPA D-π-A isomerslb, 1c and le were used for LFPs applications. The compounds were dissolved in toluene and thumb finger was dipped and printed on different surfaces such as glass, coin and plastic sheet. High contrast LFPs images were clearly visible on all surfaces upon irradiation with UV light. Even rough coin surface also showed only LFPs pattern due to strong fluorescence. The tunable fluorescence of interlinked isomers provided imaging green, yellow and orange with interlinked isomers 1b, 1c and 1e, respectively. The interlinked TPA D-π-A isomerslb, 1c and lewere also used as powder materials in powder-dusting methods on the fingerprints that was placed on the glass plate. LEPs were clearly visible with three different fluorescence colour.

Example 5

Bioimaging

[0088] Interlinked TPA D-π-A isomersla, lb and le (orange, green and red emitting materials) were utilized for live cell bioimaging application. The ability of the fluorophores to image live cells was evaluated by labelling uropathogenic E. coli cells. Healthy cells pre- exposed to the fluorophores were imaged in fluorescence microscopy using green and red filter (FIG.10). The rod shape morphology of E. Coli was clearly visible with labelling with fluorophores that is the interlinked TPA D-π-A isomers with different acceptors. Noteworthy, interlinked isomers 1b and le was selectively fluoresce green and red, respectively. Interestingly, the cells labelled with the interlinked isomer la were imaged with both green and red filter. It is noted that the interlinked isomer la showed dual emission at 556 and 636 nm in solid-state that leads to green and red emission from the live cells. The results clearly indicated the utility of these interlinked fluorophores that is interlinked TPA D-π-A with different acceptors for live cell imaging and the acceptor unit controlled tunable fluorescence offered imaging with different fluorescence colour.

Example 6

Toxicity evaluation of the interlinked TPA D-π-A isomer

[0089] Molecular toxicity is the major factor to be considered for biomedical applications. E. coli was used as a representative model to evaluate the toxicity of the interlinked TPA D-π-A with different acceptors fluorophores (FIGs. 11 and 12). For toxicity experiments, the cells are exposed to the fluorophores for 24 h, and cultured at 37 °C. The viable cells cultured with and without fluorophores forms comparable cfu, suggesting that the reported fluorophores used here for bioimaging are non-toxic. Toxicity studies are basic requirement in modern medicine to recognize a molecule for diagnostics and therapeutic use. Red blood cells are commonly used as an in vitro model to assess the toxicity of various xenobiotics. The morphological changes of RBC in the absence and presence of interlinked TPA D-π-A isomerlg was visualized in phase contrast microscopy (Fig. 12(i)). The morphology of the cells exposed to 1 remains comparable to unexposed control cells, where the shape of RBC remains unaltered with intact membranes. The cells treated with membrane damaging agent, 0.1 % Triton X 100 showed wrinkled cells with uneven membrane surface, suggesting that 1g is not a membrane damaging agent and safer for bioimaging.

[0090] Having observed non-toxicity towards RBC, the toxicity of interlinked isomer 1g in vivoas studied. Further, in vivo toxicity of the fluorophores was evaluated in zebrafish animal model. Zebrafish was selected for this study, because it shares high genetic similarity to humans and also a cost-effective model. The experiments were carried out in compliance with Central Act 26 of the 1982 guidelines). The fish measured 4-5 cm in length and 300 mg weight, irrespective of sex, and were obtained from the local aquarium. Fishes were maintained in aerated glass tanks containing tap water at 25 ± 20C. Fishes were fed adlibitium with a commercial fish diet. The toxicity of the fluorescence molecules was evaluated by injecting 10 μL of 1 mM of fluorescence molecules intra muscularly. The animal behavior and mortality were monitored for 24 h. Fish were observed to be in normal life at 1 mM concentration, the toxicity in the brain and liver was analyzed by evaluating the changes in liver and brain enzymes. Typically, the 10 μL of 1 mM of fluorescence molecules was injected intra muscularly. After 24 h, the fishes were sacrificed, the liver were dissected and homogenized in 20 mM PBS buffer. 50 μL of liver homogenate were incubated with 500μL of α- napththyl acetate or β- naphthyl acetate for 30 min at room temperature. 0.3% of freshly prepared fast blue B in 3.3% of SDS was added to stop the enzymatic action. The mixture was incubated in the dark for 30 min at room temperature. The color developed was measured at 430 nm for α- carboxyl esterase and 588 nm for β carboxyl esterase. The amount of α and β carboxyl esterase was calculated using standard graphs and expressed asμM of α or β naphthol released/mg protein min.

[0091] Herein, liver enzymes, carboxylesterase (CE) involved in drug detoxification, and brain acetylcholinesterase (ACE) responsible for neurotransmitter acetylcholine break down and other choline esters was used a marker for toxicity. Fish divided into two groups was injected intramuscularly with 10 μL PBS and 10 μL fluorophore (1mM), respectively. The liver and brain were collected from the fish and analyzed for CA and ACE activity. It is noted that the fish injected with PBS and fluorophore showed negligible changes in the CA and ACE activity as can be seen from FIGs.13(A)-(B)that the fluorophore is non-toxic and suitable for further exploration in live cell imaging.

ADVANTAGES OF THE PRESENT INVENTION

[0092] The present disclosure provides a new strategy for providing solid state organic fluorophores with enhanced fluorescence efficiency surprisingly achieved by interlinking the fluorophores using a nonconjugated spacer. Such interlinking of fluorophores with nonconjugated spacer rigidify the fluorophore structure. For example, before interconnecting single fluorophore showed fluorescence efficiency of (absolute quantum yield, f) 23% that was enhanced to 85.5% after interlinking. Further, the interlinking resulted structural rigidification transformed the non-fluorescence compound to strongly fluorescence in the solid state. Before interlinking, single fluorophore with cyanoacetamide acceptor unit was non-fluorescence but after interlinking it showed strongly enhanced fluorescence with all three isomers.

[0093] The present disclosure further provides possibility of controlling fluorescence efficiency by modulating interspace between the fluorophore units. It was unexpectedly found that by changing from ortho-xylene to meta and para-xylene increased the distance between the fluorophores and provided more conformational freedom and resulted decrease of fluorescence efficiency. For example, fluorescence efficiency was decreased from 71.5% (ortho-isomer), 27.3% (meta-isomer) and 7.2% (para-isomer). Hence interspacing distance can be optimized for improving the fluorescence efficiency.

[0094] The solid state fluorescence in accordance with present disclosure can be tuned from green to NIR by integrating different acceptor units with interlinked dialdehyde unit. The fluorescence for example was tuned from 534 nm to 706 nm.

[0095] The interlinking of fluorophores in accordance with present disclosure offers new observation in the fluorescence property. For example, interlinked isomers 1g-3g exhibited relatively strong solution fluorescence compared to solid, whereas the corresponding single fluorophore unit (before interlinking) was non-fluorescence in solution state.

[0096] The interlinked fluorophores with right acceptor units of the present disclosure provide opportunity to realize concentration dependent tunable fluorescence from blue (457 nm) to NIR (720 nm). The interlinked isomers 1g-3g showed tunable blue to NIR emission upon increasing concentration from 10-7 M to 10-2 M.

[0097] The tunable fluorescence was successfully further achieved by transforming the interlinked isomer into polymer composite thin film by dissolving in PMMA polymer.

[0098] The conformational freedom offered by the tunable interlinking spacer produced controllable mechano-responsive reversible fluorescence switching. Ortho-isomer with more rigidification offered little conformational freedom to change upon applying external force such as pressure whereas para-isomer with higher conformational freedom produced clear pressure and heating controlled reversible fluorescence switching.

[0099] The strong and tunable solid state fluorescence of interlinked organic fluorophores of the present disclosure can be used as potential materials for latent finger printing. Particularly, obtaining the latent fingerprints (LFP) with more accurate features of ridges and furrows formed on the surfaces. Organic fluorescent materials could provide high contrast LEPs image compared to commonly used metal/magnetic powder in powder-dusting and spraying methods. The interlinked isomers 1b, 1c and 1e of the present disclosure were found to be useful in the LFPs applications. All three interlinked isomers -ortho, -meta and - para showed high contrast LFPs images on all surfaces upon irradiation with UV light. Even rough coin surface also showed only LFPs pattern due to strong fluorescence. The tunable fluorescence of interlinked isomers provided imaging green, yellow and orange with interlinked isomers 1b, 1c and 1e, respectively.

[00100] The organic fluorescence material that is interlinked isomers in accordance with present disclosureare suitable for application in bioimaging that could be used for diagnostic and therapeutic purpose. The interlinked isomers 1a, 1b and 1e (orange, green and red emitting materials) are found to be suitable for live cell bioimaging application. The ability of the interlinked fluorophore isomer to image live cells when evaluated by labelling uropathogenic E. coli cells, all three interlinked fluorophore isomers are found to be capable of labelling the cell membrane and cytoplasm of E. Coli. The rod shape morphology of E. Coll was clearly visible with labelling with the interlinked fluorophore isomers, thus proving their applicability in various bioimaging.

[00101] Toxicity studies are basic requirement in modem medicine to recognize a molecule for diagnostics and therapeutic use. Both in-vitro and in-vivo studies using red blood cells and E. coli as an in vitro model and zebrafish as a vivo model since ~80 % zebrafish genome shares similarity with humans, the interlinked fluorophore isomers clearly indicated their non-toxic nature thereby proving that interlinked fluorophore isomers are suitable for live cell imaging.

[00102] It should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made without departing from the technical solutions of the present invention.

REFERENCES

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Claims

We Claim:

1. An aggregation induced emissive fluorophore (AIEgen) interconnected through a nonconjugated spacer having a structural formula selected from:

2. An aggregation induced emissive fluorophore (AIEgen) interconnected through a nonconjugated spacer having a structural formula selected from:

3.

The AIEgen interconnected through a nonconjugated spacer according to claim 1 or 2, wherein the AIEgens comprise two triphenylamine based donor-π acceptor (D-π-A), said AIEgens being interlinked with a non-conjugate spacer, the spacer being xylene isomer selected from ortho-, meta- or para- isomer and tri phenyl amine moiety being substituted with one or more acceptor unit(s) “X” selected from without limitation

4. The AIEgen interconnected through a nonconjugated spacer according to claim 1 or 2, wherein the AIEgen interconnected through a nonconjugated spacer provide concentration dependent tunable fluorescence from blue (457 nm) to NIR (720 nm).

5. The AIEgen interconnected through a nonconjugated spacer according to claim 1 or 2, wherein the AIEgen interconnected through a nonconjugated spacer provide tunable blue to NIR emission upon increasing concentration from 10^-7 M to 10^-2 M.

6. A process for preparing aggregation induced emissive fluorophores (AIEgens) interconnected through nonconjugated spacer having a structural formula selected from:

the process comprising the steps of: a) reacting 4-(diphenylamino)-2-hydroxy benzaldehyde with an ortho-, para-or meta- isomer of dibromo xylene to provide interlinked ortho-, meta- or para-triphenylamine aldehyde(TPAD-π-A) isomer; b) providing a solution of the interlinked TPAD-π-A isomer; and adding an acceptor selected from selected from without limitation

the solution obtained in step (b) and reacting under suitable condition to provide the isomer of an interlinked TPA D-π-A substituted with acceptors.

7. The process according to claim 6, wherein the solvent that is C1-C6 alcohol.

8. The process according to claim 7, wherein the solvent that is an acid, preferably glacial acetic acid.

9. The process according to claim 6, wherein the suitable condition is a room temperature to a temperature up to 95 °C.

10. A polymer composite comprising the AIEgen interconnected through a nonconjugated spacer according to any one of claims 1-5, the polymer being polymethylmethacrylate.

11. Use of the AIEgen interconnected through a nonconjugated spacer according to any one of claims 1-5, or a polymer composite according to claim 11 in external stimuli -induced fluorescence switching, latent fingerprinting, bioimaging, or live cell imaging.