Method for constructing high-brightness non-conjugated luminescent molecules based on diethylenetriamine and application of high-brightness non-conjugated luminescent molecules

Abstract

The invention discloses a method for constructing a high-brightness non-conjugated luminescent molecule based on diethylenetriamine and application of the high-brightness non-conjugated luminescent molecule. According to the method, diethylenetriamine is taken as a core, and luminous micromolecules with an adjustable structure are efficiently synthesized through two-step reaction (p-toluenesulfonyl esterification and amine substitution / ion exchange). The obtained molecules present strong blue fluorescence at 430-440 nm, the absolute fluorescence quantum yield is up to 75%, and the molecules have significant aggregation-induced emission (AIE) characteristics. Experiments show that the smaller the ring structure is or the shorter the chain length is, the higher the fluorescence efficiency is, and the stability of molecules is excellent in the pH range of 3-10 and 0-60 DEG C. A biocompatibility test shows that the 24-hour survival rate gt to HeLa and MKN-45 cells is shown; and the method is successfully applied to imaging of living zebrafish. The invention breaks through the limitation that the traditional luminescent material depends on a conjugated structure, provides a low-cost and high-performance non-conjugated fluorescent molecule construction strategy, and has wide application prospects in the fields of biological imaging, optical sensing and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of organic luminescent materials, and in particular relates to a method and application of constructing high-brightness non-conjugated luminescent molecules based on diethylenetriamine. Background Art

[0002] Light plays a vital role in the development of human civilization, particularly in the process of modernization. The development of luminescent materials has made significant contributions to modern scientific and technological advancements and lifestyle changes. Therefore, the development of novel luminescent materials is of great research significance. Due to their unique photophysical properties, organic luminescent materials have been widely used in optoelectronic devices, biochemical sensing, photodynamic therapy, drug delivery, and bioimaging.

[0003] Traditional organic light-emitting materials typically exhibit efficient luminescence in dilute solutions, but their luminescence efficiency diminishes or even disappears completely in concentrated solutions or when aggregated. This phenomenon, known as aggregation-induced quenching (ACQ), significantly limits the widespread application of these materials. In contrast to ACQ, aggregation-induced emission (AIE) was first proposed by Tang et al. in 2001. These compounds are non-luminescent in dilute solutions but exhibit strong fluorescence in aggregated states. Aggregation-induced emission compounds have two luminescence mechanisms: active intramolecular motion in the isolated state, fully dissipating excited-state energy and generating fluorescence; and a distorted three-dimensional structure in the aggregated state restricts the rotation of the aromatic rings, effectively preventing π-π stacking interactions and resulting in significant fluorescence emission. However, for both ACQ and AIE, large conjugated aromatic rings or significant π-conjugated units are considered essential for achieving efficient luminescence.

[0004] In recent years, research has revealed that a large number of natural and synthetic small molecules, macromolecules, and some supramolecules that lack aromatic ring structures can also emit intrinsic fluorescence or phosphorescence. This phenomenon overcomes the challenge of requiring traditional luminophores for efficient luminescence and is therefore also known as non-traditional luminescence (NTL). Compared with traditional organic light sources, NTLs offer advantages such as abundant raw materials, simple preparation, low cost, and environmental friendliness. Research has found that non-traditional luminogens (NTLs) typically contain only non-conventional chromophores (NCCs), such as heteroatoms with lone pairs (N, O, S, P, etc.) or unsaturated bonds such as C=C, C=O, C≡N, and S=O. Consequently, the photoluminescence mechanism of NTLs has attracted widespread attention. Tang et al. and Yuan et al. proposed the "clusteroluminescence (CL)" and "clusterization-triggered emission (CTE)" mechanisms, respectively.

[0005] The key to both mechanisms lies in the overlapping or sharing of electron clouds of non-conventional chromophores, which leads to spatial conjugation within or between molecules, forming extended n-n, n-π, and π-π conjugations. This lowers energy levels, promotes electronic transitions, and leads to photoluminescence emission. However, luminescence from non-conjugated compounds is not a new phenomenon. As early as 1605, Francis Bacon documented the mechanoluminescence of sugars in his book "Advancement of Learning." However, the connection between their luminescence mechanism and non-conventional luminescence was not fully appreciated. In 2007, the research group of Academician Tang Benzhong reported that non-aromatic poly[(maleic anhydride)-alt-(vinyl acetate)] exhibited blue fluorescence in colloidal suspensions, but not in dilute solutions. This phenomenon, along with efficient room-temperature phosphorescence, also occurs in natural compounds and polymers, such as starch, cellulose, fetal bovine serum albumin, and some other carbohydrates. In 2022, Chu et al. synthesized 24 non-conjugated aliphatic polyesters with adjustable colors and tunable efficiency through the copolymerization of six epoxides and four anhydrides. Figure 12As shown, experimental and computational results indicate that, at the primary structural level, CL efficiency can be significantly improved without changing the wavelength by adjusting the side chain length to balance structural flexibility and rigidity. However, when the monomer is switched from succinic anhydride to trans-maleic anhydride (MA), cis-maleic anhydride, and citric anhydride (CA), the secondary structure of these polyesters changes from helical to straight sheets and folds due to increased n-π* interactions, accompanied by a gradual red shift of the CL from 460 to 570 nm. Subsequently, single-molecule white light emission based on short-wavelength overlapping CL was achieved in CA-based polyesters with a CIE coordination of (0.30, 0.32). This work not only provides further insights into the emission mechanism of CL but also offers a new strategy for manipulating CL properties by regulating the hierarchical structure of CLgens.

[0006] In 2023, He et al. successfully designed and synthesized a new class of hyperbranched polyborosiloxanes (P1-P4). The general design strategy is as follows Figure 13 As shown, they exhibit different fluorescence colors in their aggregated states, such as green, yellow, brownish-yellow, and even red. This is due to the fact that as the electron density on the monomeric diol increases, the optimal emission wavelengths of the P1-P4 polymers gradually red-shift to 510, 570, 575, and 640 nm, respectively. In particular, P4 exhibits not only red emission but also delayed fluorescence, with a lifetime of 9.73 μs and a minimum critical cluster concentration of 1.76 mg / mL. These experimental and theoretical results demonstrate that the synergistic effect of electron delocalization induced by the dual heteroatoms and the steric n…n interaction effectively enhance the red delayed fluorescence. Furthermore, these hyperbranched polyborosiloxanes not only exhibit excitation-dependent emission properties, but also simultaneously enhance the fluorescence intensity and red-shift the emission wavelength simply by increasing their concentration, making them suitable for dual information encryption. This study provides a general design strategy for developing unconventional fluorescent polymers with delayed fluorescence and long-wavelength emission. Most reported unconventional luminescent compounds are high-molecular-weight compounds, and even small molecules are rarely reported, primarily due to the lack of universal synthetic methods.

[0007] In summary, traditional organic light-emitting materials usually rely on large π-conjugated structures to achieve efficient luminescence, but are prone to fluorescence quenching (ACQ effect) in the aggregated state. In recent years, non-traditional light-emitting (NTL) materials have attracted widespread attention due to their characteristics of not containing aromatic rings but still being able to emit light efficiently. However, existing NTL materials are mostly polymers or complex supramolecular structures, and small molecule NTL materials have been rarely reported due to limited synthesis methods. The present invention uses diethylenetriamine (DETA) as the core "magic wand" to design and synthesize a series of non-conjugated small molecule light-emitting materials with adjustable structure and high quantum yield, which solves the problem of fluorescence quenching of traditional light-emitting materials in the aggregated state and expands their application in biomedicine and optical materials. Summary of the Invention

[0008] The purpose of the present invention is to provide a method and application of constructing high-brightness non-conjugated luminescent molecules based on diethylenetriamine.

[0009] 1. Method for constructing high-brightness non-conjugated luminescent molecules based on diethylenetriamine (1) Synthesis of sulfonyl intermediates: p-Toluenesulfonyl chloride dissolved in dichloromethane is mixed with a dichloromethane solution containing an alcohol compound and triethylamine. The reaction system is stirred at 0-5°C for 10-15 hours, and then water is added to quench the reaction. The sulfonyl intermediate is obtained by separation, extraction, washing, drying, vacuum concentration, and column chromatography purification. The alcohol compound is cyclohexanol, cyclopentanol, cyclobutanol, cyclopropylmethanol, ethanol, 1-butanol, or 1-hexanol. The molar ratio of p-toluenesulfonyl chloride to the alcohol compound is 1:1-1:2; the molar ratio of the alcohol compound to triethylamine is 1:3-1:5.

[0010] (2) The sulfonyl intermediate was dissolved in dry NMP, KI and diethylenetriamine were added, and the mixture was stirred at 40-60°C for 4-6 hours. After the reaction solution was cooled, it was precipitated with methyl tert-butyl ether and freeze-dried for 5-8 hours. The product was dissolved in acetonitrile, sodium tetrafluoroborate was added, and the mixture was stirred for 20-25 hours. The non-conjugated luminescent molecule was obtained by filtration and concentration. The structural formula of the non-conjugated luminescent molecule is: , R is selected from the following groups: cyclohexylmethyl, cyclopentylmethyl, cyclobutylmethyl, cyclopropylmethyl, ethyl, butyl or hexyl.

[0011] The molar ratio of the sulfonyl intermediate to diethylenetriamine is 1:3-1:4; the molar ratio of the sulfonyl intermediate to KI is 1:0.08-1:0.1.

[0012] The synthetic route of non-conjugated luminescent molecules is as follows: 2. Performance Study of Non-conjugated Luminescent Molecules 1. Investigation of solvent effects UV-visible absorption and fluorescence spectroscopy were used to study the optical properties of seven diethylenetriamine luminescent small molecules. Figure 1 As shown, the broad, intense absorption band between 200 and 300 nm corresponds to the n-n transition of N-related surface states or defect bonds. The optimal excitation wavelength for the seven diethylenetriamine luminescent small molecules is 360 nm, and the optimal emission wavelength is roughly around 433 nm. Changes in the side group structure of these seven diethylenetriamine luminescent small molecules do not result in red- or blue-shifted fluorescence emission wavelengths, indicating that the wavelength is not affected by their structure and that the primary chromophore is diethylenetriamine.

[0013] like Figure 2 , the seven diethylenetriamine luminescent small molecules are soluble in methanol (MeOH), ethanol (EtOH), dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF) and acetonitrile (ACN) organic solvents. Figure 2 As shown in the figure, fluorescence measurements were performed under the same external conditions. It was found that when DMSO was used as a solvent, the fluorescence intensity was weaker and the peak shape was wider. Ethanol and methanol both had a quenching effect, and therefore could not be considered the optimal solvent. By comparison, H2O not only had the highest fluorescence intensity but also had a better peak shape, making it the optimal solvent.

[0014] 2. Effects of pH and temperature on fluorescence properties Because the application environments of the seven diethylenetriamine luminescent small molecules are generally relatively complex and pH changes occur during different physiological processes, measuring the pH and temperature of the fluorescent probe molecules is crucial. Next, we tested the fluorescence spectra of the seven diethylenetriamine luminescent small molecules in H2O at different pH values.

[0015] like Figure 3 (a, b) The fluorescence intensities of all seven diethylenetriamine luminescent molecules are relatively stable within the pH range of 3-10. However, when the pH increases to 11 and 12, the dimer fluorescence intensity decreases sharply due to the disruption of the aggregation state in the strong alkaline solution. Apparently, increasing the NaOH concentration to a certain level disrupts the intermolecular hydrogen bonds, causing a change in the initial aggregation state of the seven diethylenetriamine luminescent small molecules.

[0016] like Figure 3 As shown in Figures (c, d), the fluorescence intensity of aqueous solutions of seven diethylenetriamine luminescent small molecules increases linearly when the temperature is cooled from 60°C to 0°C. This is attributed to the restriction of thermally activated intramolecular motion (vibration and rotation) during the cooling process. The decrease in solution temperature helps reduce the non-radiative deactivation of excitons. The restriction of molecular motion at low temperatures leads to the aggregation of imino and amino groups, and strengthens intermolecular hydrogen bonding, thereby enhancing fluorescence emission.

[0017] 3. AIE Characteristic Study Since our previous research group used diethylenetriamine to construct poly(β-CD dimer), three non-aromatic β-CD dimers, and β-CD-based NLPs nanoparticles, all of which exhibited AIE, we investigated the AIE properties of seven diethylenetriamine luminescent small molecules from two perspectives: poor solvents and varying concentrations. We first tested the AIE luminescence of the fluorescent probes using fluorescence spectroscopy. Since the seven diethylenetriamine luminescent small molecules are highly water-soluble but poorly soluble in THF, we then tested the AIE effect in a mixture of the good solvent H2O and the poor solvent THF.

[0018] Figure 4 (ag) are the fluorescence spectra of seven diethylenetriamine luminescent small molecules. As can be seen from the figure, the increase in fluorescence intensity of the seven diethylenetriamine luminescent small molecule solutions is not linearly proportional to the increase in water volume fraction. When dissolved in the benign solvent H2O, the molecules are dispersed, resulting in weak fluorescence. With the addition of the poor solvent water, the fluorescence continues to increase. This is primarily due to the increased aggregation caused by the addition of the poor solvent, which restricts intramolecular motion and rotation, and emits energy as fluorescence, resulting in strong fluorescence sensitization of the system, indicating that the seven diethylenetriamine luminescent small molecules exhibit typical AIE properties. The AIE properties of the seven diethylenetriamine luminescent small molecules were then investigated to determine how different concentrations affect fluorescence emission intensity.

[0019] like Figure 5 As shown in the figure, with the continuous increase of the concentration of the seven diethylenetriamine luminescent small molecules, the fluorescence intensity is significantly enhanced. This is mainly because with the increase of the probe concentration, the aggregation degree is significantly enhanced, the intramolecular motion is restricted, and the excited state energy is attenuated through the radiation channel, thereby causing fluorescence enhancement, indicating that the seven diethylenetriamine luminescent small molecules have typical AIE characteristics.

[0020] 4. Optical properties In order to verify whether the other synthesized compounds also have luminescent properties, all the dried compounds were taken at C = 1 × 10 -3 mol / L H2O solution to measure its fluorescence emission spectrum, such as Figure 6 As shown in the figure, the seven diethylenetriamine luminescent small molecules containing atypical chromophores synthesized by us all have good water solubility, and their aqueous solutions emit strong blue fluorescence.

[0021] like Figure 6 As shown, the maximum excitation wavelength is 360 nm, the maximum emission wavelength is 435 nm, and the luminescent small molecule solution exhibits strong fluorescence emission. Figure 6 The photos of the various solutions under UV light clearly demonstrate the distinct differences in fluorescence intensity among the seven diethylenetriamine luminescent small molecule solutions. It can be seen that the maximum fluorescence emission and excitation wavelengths of the seven diethylenetriamine luminescent small molecules are essentially identical for solutions of the same concentration; only the fluorescence intensity varies. The smaller the ring structure and the shorter the chain length, the stronger the fluorescence intensity, indicating a stronger steric effect, weakening the strength of the ionic bonds between the small molecules and reducing the rigidity of the luminescent cluster conformation. In other words, by modifying the side group structures of these seven diethylenetriamine luminescent small molecules, their fluorescence emission wavelengths do not undergo red or blue shifts, and the luminescence colors do not differ significantly, further verifying that the diethylenetriamine structure is adjustable and redesignable.

[0022] Fluorescence lifetime measurements were performed using standard time-correlated single-photon counting methods. Excitation light was provided by a portable diode laser (EPL-375, Edinburgh Instruments). The laser beam was directed into the sample, and fluorescence was detected at the corresponding sample emission maximum. The bandwidth of excitation and emission was less than 2 nm. The fluorescence lifetimes of seven diethylenetriamine luminescent small molecules are shown in Table 1. Figure 7 As shown in Figure 2, the calculated average fluorescence lifetimes of the seven diethylenetriamine luminescent small molecules are all approximately 10 ns.

[0023] We also used a steady-state / transient fluorescence spectrometer equipped with an integrating sphere to study the absolute PLQY values ​​and time-resolved PL decay curves of seven diethylenetriamine luminescent small molecules. Figure 6 As shown in the small figure on the right and Table 1, the absolute fluorescence quantum yields of the seven diethylenetriamine luminescent small molecules reached a combined 75% at the maximum excitation wavelength. For cyclic diethylenetriamine luminescent small molecules, the smaller the ring structure, the greater the QY; for chain diethylenetriamine luminescent small molecules, the shorter the chain length, the greater the QY.

[0024] 3. Cytotoxicity Testing and Fluorescence Imaging of Non-conjugated Luminescent Small Molecules Low cytotoxicity is one of the most critical requirements for multifunctional biomaterials with bioimaging capabilities. Therefore, in this study, the traditional MTT assay was used to test the cytotoxicity of the samples. First, HeLa and MKN-45 cells were transferred to a centrifuge tube and centrifuged at 1000 rpm for 7 minutes. Culture medium was then added to a cell concentration of 10^4 cells / mL. Next, 200 μL of sample per well was added to a 96-well plate, and PBS buffer was added to all edge wells of the plate. The plate was then incubated in an incubator for 24 hours. After 24 hours, the culture medium and PBS buffer were aspirated, and samples of varying concentrations were added to each well. After another 24 hours, the culture medium and PBS buffer were again aspirated, and samples of varying concentrations were added to each well. After another 24 hours, 20 μL of MTT solution was added to each well, and incubation continued for another 4 hours. The culture medium was then aspirated, and 180 μL of DMSO was added to each well. Crystals were dissolved by shaking for 10 minutes. The absorbance was then measured using an RT 6100 microplate reader and repeated three times to calculate cell viability. Cell viability was calculated using the following formula: Cell viability (%) = ODr / ODc × 100% (ODr is the absorbance measured in the experimental well, and ODc is the absorbance measured in the control well).

[0025] To expand the applicability of the probe, the ability of seven diethylenetriamine luminescent small molecules to directly enter HeLa, MKN-45 cells, and living zebrafish was evaluated by fluorescence microscopy. The cytotoxicity of the seven diethylenetriamine luminescent small molecules was first evaluated using the MTT assay. HeLa and MKN-45 cells were tested, with solutions of the seven diethylenetriamine luminescent small molecules at concentrations of 0 μg / mL, 10 μg / mL, 20 μg / mL, 40 μg / mL, 80 μg / mL, and 100 μg / mL added to the cells and incubated for 24 hours.

[0026] Depend on Figure 8 (ag) It can be seen that the cell survival rates of the seven diethylenetriamine luminescent small molecules were maintained above 90%. Even after adding 100 μg / mL sample, they still had a high cell survival rate, indicating that the cytotoxicity was low and bioimaging research could be carried out.

[0027] The seven prepared fluorescent small molecules were applied to live zebrafish imaging, such as Figure 9 As shown, the imaging results show that seven fluorescent small molecules enter the living zebrafish through endocytosis and emit light, and exhibit dual-color fluorescence and controllable fluorescence signals by changing the excitation wavelengths of the green and red emission channels.

[0028] In summary, this study used diethylenetriamine (DTA) as a "magic wand" to design and synthesize seven clustered luminescent small molecules by regulating ring size and side chain length. Nuclear magnetic resonance (NMR) characterization confirmed the successful preparation of the target compounds, with their maximum excitation wavelengths all centered at 360 nm and emission wavelengths concentrated at 433 nm, exhibiting blue fluorescence in aqueous solution. Fluorescence spectra, measured by solvent effects, revealed typical aggregation-induced emission (AIE) properties, with fluorescence intensity increasing with smaller ring size and shorter chain length. Calculated absolute fluorescence quantum yields reached 75% overall. For cyclic DTA luminescent small molecules, smaller ring size correlated with higher QY, while for chain DTA luminescent small molecules, shorter chain length correlated with higher QY. Biocompatibility assessments revealed that this series of molecules exhibited a 24-hour viability exceeding 90% for HeLa and MKN-45 cells, and were successfully applied to live zebrafish imaging. This work, through structure-performance regulation, enabled the construction of high-quantum-yield, low-cytotoxic AIE fluorescent probes, providing a novel molecular tool for bioimaging applications.

[0029] Compared with the prior art, the present invention has the following beneficial effects: 1. Innovative molecular design Breaking through the limitations of traditional conjugated structures, for the first time, using diethylenetriamine (DETA) as the core "magic wand of light", seven structurally adjustable non-conjugated small molecule luminescent materials were constructed through modular synthesis, solving the technical bottleneck that most existing non-traditional luminescent materials (NTLs) are polymers or complex supramolecular structures.

[0030] A creative "ring size / chain length-fluorescence intensity" regulation strategy was proposed: the smaller the ring structure (cyclopropylmethyl>cyclobutylmethyl>cyclopentylmethyl>cyclohexylmethyl) or the shorter the chain structure (ethyl>butyl>hexyl), the higher the fluorescence quantum yield (up to 75%), providing clear guidance for performance optimization.

[0031] 2.Excellent optical performance High brightness and stability: All derivatives exhibit strong blue fluorescence at 430-440 nm, with an absolute fluorescence quantum yield of up to 75%, far exceeding most reported non-conjugated luminescent materials (usually <50%).

[0032] Significant aggregation-induced emission (AIE) properties: Typical AIE effects were confirmed through water / THF mixed solvent system and concentration gradient experiments. The fluorescence intensity of the aggregated state increased by 5-8 times compared with the dispersed state, overcoming the aggregation quenching problem of traditional ACQ materials.

[0033] Wide range of environmental adaptability: The fluorescence remains stable in the pH range of 3-10 and 0-60℃, meeting the application requirements of complex physiological environments.

[0034] 3. Simple and efficient synthesis method The two-step synthesis route (p-toluenesulfonyl esterification → amine substitution / ion exchange) can achieve a total yield of 60-75%, which is 3-4 steps shorter than the existing NTL material synthesis route.

[0035] A one-pot post-processing process (methyl tert-butyl ether precipitation / freeze-drying combined) avoids traditional column chromatography purification and is suitable for gram-scale preparation.

[0036] The raw materials are cheap and readily available (diethylenetriamine, alcohols, etc. are bulk chemicals), and the cost of single-batch synthesis is reduced by about 40%.

[0037] 4. Outstanding biocompatibility Low cytotoxicity: MTT assays confirmed that even at a high concentration of 100 μg / mL, the 24-hour viability of HeLa and MKN-45 cells was still >90%, significantly superior to traditional fluorescent probes (usually 70-80% viability).

[0038] The successful imaging of living zebrafish confirmed its ability to penetrate biological barriers and tissue distribution characteristics, laying the foundation for subsequent integrated diagnosis and treatment applications.

[0039] 5. Broad application prospects Biomedical field: It can be used as a new generation of fluorescent markers for organelle tracking, tumor targeted imaging, etc. Its non-conjugated structure avoids the problem of phototoxicity.

[0040] Optical materials: Through side chain structure regulation, precise design of luminescence color / efficiency can be achieved, which is suitable for the development of devices such as OLEDs and fluorescent sensors.

[0041] Environmental monitoring field: pH / temperature response characteristics make it suitable for the development of microenvironmental probes.

[0042] 6. Breakthrough in theoretical mechanisms Experiments confirmed that the luminescence originated from the nn* transition of the DETA core, and for the first time proposed a "spatial electron cloud overlap-molecular rigidification" synergistic effect model: small rings / short chains enhance steric hindrance → restrict intramolecular rotation → promote cluster luminescence, providing a new perspective for the study of NTL mechanism. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 (ag) UV absorption, fluorescence excitation, and emission spectra of seven diethylenetriamine luminescent small molecules.

[0044] Figure 2 (ag) Fluorescence spectra of seven diethylenetriamine luminescent small molecules in different solvents.

[0045] Figure 3 (a, b) Cyclic and chain structures in different pH solutions (1×10 -4 mol / L); (c, d) The temperature of the ring and chain decreased from 60 ℃ to 0 ℃ (1×10 -4 mol / L) of fluorescence intensity changes.

[0046] Figure 4 (ag) Fluorescence spectra of seven diethylenetriamine luminescent small molecules at different concentrations.

[0047] Figure 5 (ag) Fluorescence spectra of seven diethylenetriamine luminescent small molecules in water fractions with different volumes.

[0048] Figure 6 (a) At the same concentration (1× 10 –3 M) Fluorescence emission spectra of four cyclic diethylenetriamine luminescent small molecules; (b) The same concentration (1× 10 –3 M) Fluorescence emission spectra of three chain-like diethylenetriamine luminescent small molecules.

[0049] Figure 7(a) Fluorescence lifetimes of four cyclic diethylenetriamine luminescent small molecules; (b) Fluorescence lifetimes of three chain diethylenetriamine luminescent small molecules.

[0050] Figure 8 Toxicity test of seven diethylenetriamine luminescent small molecules in HeLa and MKN-45 cells at different concentrations for 24 h.

[0051] Figure 9 In vivo zebrafish imaging experiments.

[0052] Figure 10 H NMR spectrum of Chm-DETA.

[0053] Figure 11 C NMR spectrum of Chm-DETA.

[0054] Figure 12 Chemical structures, photophysical data, and images taken under 365 nm UV light for copolymers of six epoxides and four cyclic anhydrides. (A) P1-P6, copolymerized with SA (epoxides); (B) P7-P12, trans-isomers, copolymerized with MA (epoxides); (C) P13-P18, cis-isomers, copolymerized with MA (epoxides); and (D) P13-P24, copolymerized with CA (epoxides). λem: maximum PL.

[0055] Figure 13 Schematic diagram of the intrachain and steric interactions of P1-P4. DETAILED DESCRIPTION

[0056] The method for constructing high-brightness non-conjugated luminescent molecules based on diethylenetriamine of the present invention is described in detail below through specific embodiments.

[0057] Example 1 Synthesis of Chm-DETA (1) Synthesis of Chm-Ots p-Toluenesulfonyl chloride (3.81 g, 20 mmol) and DCM (50 ml) were mixed and stirred at 0°C. A mixture of cyclohexanemethanol (2.28 g, 20 m mol) and triethylamine (5.6 ml, 40 m mol) was added to DCM (15 ml) and slowly added dropwise via syringe. The mixture was then stirred at 0°C for 12 h. Water (50 ml) was added to quench the reaction. The layers were separated, and the aqueous layer was extracted with DCM (3 × 50 ml). The organic phase was washed sequentially with saturated NaCl solution (50 ml), saturated NaHCO3 solution (50 ml), and aqueous HCl (3 M, 50 ml), dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. Purification by column chromatography (n-hexane:ethyl acetate = 30:1) afforded Chm-OTs as a white oil in a yield of 76.82%.

[0058] (2) Synthesis of Chm-DETA Next, Chm-OTs (0.4156 g, 1.55 mmol) was dissolved in dry NMP (5 ml), and then solid KI (0.025 g, 0.15 mmol) and diethylenetriamine (0.54 ml, 5 mmol) were slowly added with vigorous stirring. Stirring was continued at 50 ° C for 5 h to obtain a light yellow solution that was cooled to room temperature. It was precipitated with methyl tert-butyl ether and freeze-dried for 6 h. The product was dissolved in acetonitrile, and 5 equivalents of sodium tetrafluoroborate were added. It was stirred at room temperature for 24 h, filtered, and concentrated to obtain a light yellow oil, which is Chm-DETA, with a yield of 74.27%. The molecular structure of Chm-DETA can be seen by 1 H NMR and 13 C NMR ( Figure 9 、 Figure 10 ) was confirmed. An appropriate amount of the dried product was placed in an NMR tube and tested for NMR spectra using deuterated methanol as the solvent. The NMR data are as follows: 1 H NMR (400 MHz, CD3OD- d 4 ) δ 3.43 (d, J = 7.1 Hz, 2H), 2.88 – 2.78 (m,10H), 2.77 – 2.72 (m, 4H), 2.38 – 2.32 (m, 4H), 2.07 – 2.00 (m, 1H). 13 C NMR (151 MHz, Methanol- d 4) δ 176.28, 49.41, 48.05, 48.04, 47.98,47.91, 47.90, 47.87, 47.77, 47.76, 47.63, 47.62, 47.48, 47.47, 47.34, 47.33,47.20, 47.19, 41.21, 39.56, 39.54, 30.30, 28.41, 17.13. Example 2 The synthesis method of Cptm-DETA is the same as that of Chm-DETA in Example 1, except that cyclopentanol is used as the raw material. The reaction ultimately produces a light yellow oily product with a yield of 70.56%. 1 H NMR and 13 C NMR spectrum analysis successfully verified the molecular structure of Cptm-DETA.

[0059] 1 H NMR (400 MHz, CD3OD- d 4 ) δ 3.43 (d, J = 7.1 Hz, 2H), 2.88 – 2.78 (m,8H), 2.77 – 2.72 (m, 4H), 2.38 – 2.32 (m, 4H), 2.07 – 2.00(m, 1H). 13 C NMR (151 MHz, Methanol- d 4 ) δ 176.27, 125.53, 49.41, 48.55, 48.13,47.99, 47.98, 47.85, 47.83, 47.71, 47.69, 47.57, 47.55, 47.43, 47.41, 47.27,46.49, 41.69, 39.71, 39.24, 30.35, 28.47, 28.45, 24.75, 17.15. Example 3 The synthesis method of Cbm-DETA is the same as that of Chm-DETA in Example 1, except that cyclobutanemethanol is used as the raw material. The reaction ultimately produces a light yellow oily product with a yield of 69.23%. 1 H NMR and 13 C NMR spectrum analysis successfully verified the molecular structure of Cbm-DETA. 1 H NMR (400 MHz, CD3OD-d 4 ) δ 3.43 (d, J = 7.1 Hz, 2H), 2.88 – 2.78 (m, 6H), 2.77 – 2.72 (m, 4H), 2.38 – 2.32 (m, 4H), 2.07 – 2.00 (m, 1H). 13 C NMR (151 MHz, Methanol- d 4 ) δ 176.25, 176.23, 128.64, 125.54,117.26,54.17, 49.41, 48.45, 48.38, 48.17, 48.04, 48.02, 47.94, 47.90, 47.88,47.76, 47.73, 47.61, 47.59, 47.46, 47.32, 41.61, 39.68, 30.42, 30.39, 28.58,28.53,28.50, 26.00, 17.17. Example 4 The synthesis method of Cppm-DETA is the same as that of Chm-DETA in Example 1, except that cyclopropylmethanol was used in the synthesis of Cppm-DETA, and a light yellow oil was obtained with a yield of 65.84%. The molecular structure of Cppm-DETA can be determined by 1 HNMR and 13 C NMR confirmed. 1 H NMR (400 MHz, CD3OD- d 4 ) δ 3.43 (d, J = 7.1 Hz, 2H), 2.88– 2.78 (m, 6H), 2.77 – 2.72 (m, 4H), 2.38 – 2.32 (m, 2H), 2.07 – 2.00(m, 1H). 13 C NMR (151 MHz, Methanol- d 4) δ 176.27, 125.53, 49.41, 48.55, 48.04,47.90, 47.76, 47.62, 47.47, 47.57, 47.55, 47.43, 47.41, 47.33, 47.27, 47.19,47.07, 39.38, 30.35, 28.47, 28.45, 24.75, 17.15. Example 5 The synthesis method of EA-DETA is the same as that of Chm-DETA in Example 1, except that ethanol was used in the synthesis of EA-DETA, and a light yellow oil was obtained with a yield of 76.21%. The molecular structure of EA-DETA can be determined by 1 H NMR and 13 C NMR confirmed. 1 H NMR (400 MHz, CD3OD- d 4 ) δ 3.21-3.19 (d, 2H), 2.86 – 2.82 (m, 6H), 2.77 – 2.74 (m, 4H), 2.66 (s, 1H), 2.36 (d, J = 7.2 Hz, 6H), 1.22 – 1.15 (m,2H). 13 C NMR (151 MHz, Methanol- d 4) δ 176.25, 117.29, 49.41, 48.70, 48.18,48.04, 47.90, 47.76, 47.62, 47.47, 47.33, 46.39, 39.76, 30.39, 28.52, 17.17,12.88. Example 6 The synthesis method of BA-DETA is the same as that of Chm-DETA in Example 1, except that 1-butanol was used in the synthesis of BA-DETA, and a light yellow oil was obtained with a yield of 74.26%. The molecular structure of BA-DETA can be determined by 1 H NMR and 13 C NMR confirmed. 1 H NMR (400 MHz, CD3OD- d 4) δ 3.47 – 3.42 (m, 2H), 3.20 (s, 1H), 2.86 – 2.81 (m, 6H), 2.77 – 2.73 (m, 6H), 2.65 (s, 1H), 2.36 (d, J = 7.5 Hz, 6H), 2.07– 1.99 (m, 2H), 1.18 (s, 2H). 13 C NMR (151 MHz, Methanol- d 4 ) δ 176.28, 125.52, 85.58, 55.94, 49.41,48.23, 48.07, 47.97, 47.94, 47.83, 47.79, 47.68, 47.65, 47.54, 47.51, 47.40,47.37, 47.23, 42.88, 41.44, 39.63, 39.61, 36.91, 30.35, 30.32, 28.46, 17.15. Example 7 The synthesis method of HA-DETA is the same as that of Chm-DETA in Example 1, except that 1-hexanol was used in the synthesis of HA-DETA, and a light yellow oil was obtained with a yield of 73.52%. The molecular structure of HA-DETA can be determined by 1 H NMR and 13 C NMR confirmed. 1 H NMR (400 MHz, CD3OD- d 4 ) δ 3.47 – 3.42 (m, 2H), 3.20 (s, 1H), 2.87 – 2.81 (m, 8H), 2.77 – 2.72 (m, 6H), 2.65 (s, 1H), 2.36 (d, J = 7.5 Hz, 6H), 2.07– 1.99 (m, 2H), 1.18 (s, 2H). 13 C NMR (151 MHz, Methanol- d4) d 176.28, 49.41, 48.08, 48.06, 47.94,47.92, 47.80, 47.78, 47.73, 47.66, 47.64, 47.51, 47.50, 47.37, 47.35, 47.23,47.21, 40.27, 40.09, 39.50, 38.96, 30.33, 30.31, 28.43, 28.42, 17.14.

Claims

1. A method for constructing a high-brightness non-conjugated luminescent molecule based on diethylenetriamine, comprising the following steps: (1) Synthesis of sulfonyl intermediates: p-Toluenesulfonyl chloride dissolved in dichloromethane is mixed with a dichloromethane solution containing an alcohol compound and triethylamine. The reaction system is stirred at 0-5°C for 10-15 hours, and then water is added to quench the reaction. The sulfonyl intermediate is obtained by separation, extraction, washing, drying, vacuum concentration, and column chromatography purification. The alcohol compound is cyclohexanol, cyclopentanol, cyclobutanol, cyclopropylmethanol, ethanol, 1-butanol, or 1-hexanol. (2) The sulfonyl intermediate was dissolved in dry NMP, KI and diethylenetriamine were added, and the mixture was stirred at 40-60°C for 4-6 hours. After the reaction solution was cooled, it was precipitated with methyl tert-butyl ether and freeze-dried for 5-8 hours. The product was dissolved in acetonitrile, sodium tetrafluoroborate was added, and the mixture was stirred for 20-25 hours. The non-conjugated luminescent molecule was obtained by filtration and concentration. The structural formula of the non-conjugated luminescent molecule is: , R is selected from the following groups: cyclohexylmethyl, cyclopentylmethyl, cyclobutylmethyl, cyclopropylmethyl, ethyl, butyl or hexyl.

2. The method for constructing a high-brightness non-conjugated luminescent molecule based on diethylenetriamine according to claim 1, characterized in that: In step (1), the molar ratio of p-toluenesulfonyl chloride to the alcohol compound is 1:1-1:2; the molar ratio of the alcohol compound to triethylamine is 1:3-1:

5.

3. The method for constructing high-brightness non-conjugated luminescent molecules based on diethylenetriamine according to claim 1, characterized in that: In step (2), the molar ratio of the sulfonyl intermediate to diethylenetriamine is 1:3 to 1:4; the molar ratio of the sulfonyl intermediate to KI is 1:0.08 to 1:0.

1.

4. A high-brightness non-conjugated luminescent molecule constructed according to any one of claims 1 to 3, characterized in that: The luminescent molecule has aggregation-induced emission characteristics, exhibits blue fluorescence in an aggregated state or in aqueous solution, has a maximum emission wavelength of 430-440 nm, and an absolute fluorescence quantum yield of up to 75%.

5. Use of the non-conjugated luminescent molecule constructed according to the method of claim 1 in biological imaging.

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