Non-peptide small molecule condensates and preparation and use thereof

Abstract

The present application belongs to the technical field of condensate, and particularly relates to a non-peptide small molecule condensate and preparation and application thereof. The present application provides a non-peptide small molecule condensate which is prepared by taking a small molecule shown in formula I as a precursor, wherein Y and Z are each independently NH, O or S, R = or X = O or S; R1 and R2 are each independently selected from H, C 3‑20 alkyl, -C 1‑8 alkylene-phenyl, -C 1‑8 alkylene-5-10-membered heteroaryl, -C 1‑8 alkylene-S-C 1‑8 alkyl, -C 1‑8 alkylene-SH, -C 1‑8 alkylene-O-C 1‑8 alkyl or -C 1‑8 alkylene-NH-C 1‑8 alkyl; and R1 and R2 are not H at the same time. The obtained condensate not only has good spontaneous non-classical fluorescence characteristics, but also has the abilities of drug loading and controllable release, and shows excellent resistance to enzymatic hydrolysis and good biocompatibility. Formula I

Description

Technical Field

[0001] This invention belongs to the field of condensate technology, specifically relating to a non-peptide small molecule condensate and its preparation and application. Background Technology

[0002] Condensates are a type of membrane-free compartmentalized structure formed by molecules through liquid-liquid phase separation. They can effectively encapsulate various active substances such as nucleic acids, proteins, and small molecule drugs, and are therefore considered a highly promising biomimetic drug delivery platform.

[0003] Most reported condensate systems rely on multi-component complexes or macromolecules (such as peptides, proteins, or nucleic acids). Their complex composition and multiple non-covalent interactions not only increase the difficulty of system regulation but also make it difficult to deeply elucidate the mechanisms of phase separation behavior. Although peptide-based systems have been extensively studied, their stability in physiological environments is limited, and they are susceptible to enzymatic degradation, thus restricting their potential for long-term in vivo effects.

[0004] On the other hand, existing condensates generally lack endogenous visualization capabilities. To achieve imaging and dynamic tracking, it is often necessary to introduce exogenous fluorescent molecules or perform additional chemical modifications. This process not only increases the complexity of the system but may also interfere with intramolecular interactions within the condensate, thus adversely affecting phase separation behavior. In biomedical applications, such exogenous labeling is more likely to affect the condensate's encapsulation efficiency, distribution behavior, and release kinetics of drug molecules, thereby weakening its performance as a delivery carrier. Furthermore, the introduction of exogenous molecules may trigger non-specific biological interface interactions or potential toxic side effects, limiting its safety for in vivo application. Summary of the Invention

[0005] To address the shortcomings and challenges of the existing technologies, this invention provides a non-peptide small molecule condensate. This condensate uses a small molecule compound of Formula I as a precursor, and is formed as a single-component non-peptide small molecule condensate through liquid-liquid phase separation. The resulting condensate not only possesses unique atypical dual-emission fluorescence characteristics and supramolecular chirality, enabling efficient drug molecule encapsulation and controlled release, but also exhibits excellent resistance to enzymatic degradation and good biocompatibility. Therefore, the non-peptide small molecule condensate obtained by this invention has broad application prospects in biomedical fields such as fluorescence imaging materials, drug delivery carriers, cell tracing, and anti-tumor therapy.

[0006] The technical solution of the present invention:

[0007] The first technical problem to be solved by this invention is to provide a non-peptide small molecule condensate, which is obtained from the small molecule shown in Formula I as a precursor.

[0008]

[0009] Formula I

[0010] In Equation I, Y and Z are independently NH, O, or S, and R = or X = O or S; R1 and R2 are independently selected from: H and C. 3-20 Alkyl, -C 1-8 alkylene-phenyl, -C 1-8 alkylene-5-10 heteroaryl, -C 1-8 Alkylene-SC 1-8 Alkyl, -C 1-8 Alkylene -SH, -C 1-8 Alkylene-OC 1-8 Alkyl or -C 1-8 Alkylene-NH-C 1-8 Alkyl group; the 5-10-membered heteroaryl group contains 1, 2, 3, or 4 heteroatoms selected from N, O, and S; the 5-10-membered heteroaryl group is unsubstituted or C-substituted. 1-8 Alkyl or C 1-8 Alkyl substitution; and R1 and R2 are not both H.

[0011] Furthermore, R1 and R2 are each independently selected from: H and C. 3-10 Alkyl, -C 1-6 alkylene-phenyl, -C 1-6 alkylene-5-membered heteroarylphenyl, -C 1-6 Alkylene-SC 1-6 Alkyl, -C 1-6 Alkylene -SH, -C 1-6 Alkylene-OC 1-6 Alkyl or -C 1-6 Alkylene-NH-C 1-6 Alkyl group; the 5-membered heteroaryl group is selected from pyrrole, furanyl, thiophene, pyrazolyl, imidazolyl, thiazolyl, isothiazolyl, oxazolyl, or isoxazolyl; the 5-membered heteroarylphenyl group is unsubstituted or C-substituted. 1-4 Alkyl or C 1-4 Alkyl substitution; and R1 and R2 are not both H.

[0012] Furthermore, R1 and R2 are each independently selected from: H and C. 3-6 Alkyl, -C 1-3 alkylene-phenyl, -C 1-3 alkylene-indolyl, -C 1-3 Alkylene-SC 1-3 Alkyl, -C 1-3 Alkylene -SH, -C 1-3 Alkylene-OC 1-3 Alkyl or -C1-3 Alkylene-NH-C 1-3 Alkyl group; the indole group is unsubstituted or substituted with methoxy or ethoxy groups; and R1 and R2 are not both H.

[0013] Furthermore, the small molecule represented by Formula I is selected from substances with the following structures:

[0014] , , , , , , , , , or .

[0015] Furthermore, the non-peptide small molecule condensate has two independent fluorescence emission centers, meaning that the resulting condensate exhibits two independent fluorescence emission peaks in the fluorescence intensity-wavelength test, located in different wavelength regions, indicating the existence of two independent fluorescence emission centers in the system; as in the embodiments of the present invention. Figure 4 The obtained D-PME condensates shown in the figure exhibit two independent fluorescence emission centers at approximately 440 nm and 570 nm, while the obtained LMC condensates exhibit two independent fluorescence emission centers at approximately 360 nm and 554 nm.

[0016] Furthermore, the non-peptide small molecule condensate possesses supramolecular chirality and circularly polarized luminescence properties.

[0017] The second technical problem to be solved by the present invention is to provide a method for preparing the above-mentioned non-peptide small molecule condensates, wherein the preparation method is as follows: the small molecules shown in Formula I are spontaneously prepared into condensates by liquid-liquid phase separation.

[0018] Furthermore, the preparation method specifically involves placing the small molecule shown in I in a buffer solution, adjusting the pH of the buffer solution to ≤6, and observing liquid-liquid phase separation. The resulting turbidity indicates that the non-peptide small molecule aggregate has been obtained.

[0019] Furthermore, in the above preparation method, the buffer solution is HEPES buffer, phosphate buffer, acetate buffer, or Tris buffer; the pH value of the buffer solution is 4 to 6, and the concentration of the buffer solution is 5 mM to 30 mM.

[0020] Furthermore, in the above preparation method, an acid is used to adjust the pH value of the buffer solution, wherein the acid is HCl and the concentration of the acid is 0.1 M to 1 M.

[0021] The third technical problem to be solved by the present invention is to indicate the use of the non-peptide small molecule condensates in fluorescent materials or biomedical materials.

[0022] Furthermore, the non-peptide small molecule condensates are used as fluorescent imaging materials, drug carrier materials, cell tracking materials, or anti-tumor materials.

[0023] The beneficial effects of this invention are:

[0024] (1) The non-peptide small molecule condensates provided by this invention are prepared using small molecule compounds represented by Formula I as precursors. Compared with the polypeptide molecules or complex macromolecular complex systems commonly used in the prior art, the condensates of this invention have significant advantages such as well-defined structure, simple composition, concise synthesis route, and mild preparation conditions. This system avoids the inherent defect of peptide materials being easily degraded by enzymes in physiological environments, thereby significantly improving the long-lasting effect and biosafety of the condensates.

[0025] (2) The non-peptide small molecule condensates obtained in this invention exhibit dual-emission fluorescence characteristics generated by two independent luminescent centers. Compared with a single-emission system, this dual-emission behavior can provide multi-channel fluorescence signal output and can be used for ratiometric fluorescence analysis, thereby improving signal resolution and detection accuracy. Based on the above characteristics, the condensates can achieve in-situ visualization observation and are suitable for cell imaging and monitoring of related biological processes.

[0026] (3) The non-peptide small molecule condensates obtained in this invention can be ordered to stack during the formation process, forming an aggregated structure with supramolecular chirality, and can further transfer chiral information to the excited state, thereby exhibiting circularly polarized luminescence characteristics, which has application potential in circularly polarized fluorescence imaging and chiral information recognition.

[0027] (4) The non-peptide small molecule condensates obtained in this invention have a microenvironment constructed by hydrophobic interactions and hydrogen bonding, which is conducive to the enrichment and encapsulation of guest molecules. By regulating the small molecule structure or external conditions, the drug loading performance and release behavior can be adjusted, thereby endowing the system with good drug delivery capabilities. The condensates have inherent fluorescence properties, which can be used to monitor the drug release process in real time and realize visual analysis, thus possessing potential application value for integrated diagnosis and treatment.

[0028] It is evident that the non-peptide small molecule condensates obtained in this invention have promising applications in biomedical fields such as drug delivery, enabling efficient drug encapsulation and controlled release. Based on their fluorescence properties and structural advantages, these condensates can be used as fluorescence imaging materials, drug delivery carriers, and cell tracking materials, and show promise in anti-tumor therapy. They also have application potential in circularly polarized fluorescence imaging and chiral information recognition. Attached Figure Description

[0029] Figure 1 Comparative Example 1 GMA ( Figure 1 A) and Example 2 D-PME ( Figure 1 Figure B) shows the fluorescence performance results.

[0030] Figure 2 : Comparative Example 1 GMA failed to form aggregates in different pH buffers and Example 1 D-ILME and Example 2 D-PME formed aggregates in different pH buffers.

[0031] Figure 3 Optical microscope images of the aggregates obtained in Comparative Example 5 and Examples 1-5: A: LSA, B: D-ILME, C: D-PME, D: D-MME, E: LMA, F: LMC.

[0032] Figure 4 Example 2: D-PME-A diagram ( Figure 4 A), the fluorescence performance results of the aggregates prepared in Example 5 LMC-B ( Figure 4 B).

[0033] Figure 5 Example 1: D-ILME ( Figure 5 A), Example 2 D-PME ( Figure 5 B), Example 4 LMA ( Figure 5 C) Fluorescence test results of the obtained condensate using a two-dimensional excitation-emission matrix.

[0034] Figure 6 Example 2: Condensates prepared by D-PME in a bright field ( Figure 6 B) and confocal plots at different excitation wavelengths (405 nm and 488 nm, respectively). Figure 6 C and D), Confocal plot of Comparative Example 1 (GMA) in bright field ( Figure 6 A).

[0035] Figure 7 Chirality test results of aggregates prepared by adjusting pH to ~6 in 1 mL 5 mM HEPES buffer with 10 mg D-PME in Example 2 and LMC in Example 5.

[0036] Figure 8 Example 2: The circular polarization luminescence asymmetry factor test results of condensates prepared by adjusting the pH to ~6 in 1 mL 5 mM HEPES buffer with 10 mg D-PME.

[0037] Figure 9Example 2: Drug release kinetics of levofloxacin encapsulated in aggregates prepared by adjusting the pH to ~6 in 1 mL 5 mM HEPES buffer at pH=8 and pH=6.

[0038] Figure 10 Figure 1 shows the CCK-8 assay results of aggregates prepared from all small molecules in Examples 1-5 at pH ~6 in 1 mL of 5 mM HEPES buffer at 10 mg.

[0039] Figure 11 Example 1: D-ILME, Example 2: D-PME, Example 3: D-MME, Example 4: LMA, Example 5: LMC, Comparative Example 5: The condensates prepared by LSA were incubated in a protease environment at 37 °C. The result is a graph showing the change in residual rate over time. Detailed Implementation

[0040] This invention provides a non-peptide small molecule condensate, which is prepared from the small molecule represented by Formula I as a precursor.

[0041]

[0042] Formula I

[0043] In Equation I, Y and Z are independently NH, O, or S, and R = or X = O or S; R1 and R2 are independently selected from: H and C. 3-20 Alkyl, -C 1-8 alkylene-phenyl, -C 1-8 alkylene-5-10 heteroaryl, -C 1-8 Alkylene-SC 1-8 Alkyl, -C 1-8 Alkylene -SH, -C 1-8 Alkylene-OC 1-8 Alkyl or -C 1-8 Alkylene-NH-C 1-8 Alkyl group; the 5-10 heteroaryl group contains 1, 2, 3 or 4 heteroatoms selected from N, O and S; the 5-10 heteroaryl group is unsubstituted or C-substituted. 1-8 Alkyl or C 1-8 Alkyl substitution; and R1 and R2 are not both H.

[0044] This invention utilizes small molecules with specific structures to prepare a non-peptide small molecule condensate through a simple method. The resulting condensate has a simple and well-defined structure, is easy to synthesize, resistant to enzymatic hydrolysis, and exhibits good biocompatibility, as well as supramolecular chiral properties. Simultaneously, the condensate possesses dual-emission fluorescence characteristics generated by two independent luminescent centers, providing multi-channel fluorescence signal output and suitable for ratiometric fluorescence analysis, thereby improving signal resolution and detection accuracy. It is suitable for in-situ imaging and dynamic behavior monitoring of intracellular condensates. Furthermore, the microenvironment constructed by hydrophobic interactions and hydrogen bonding within the resulting condensate is conducive to the enrichment and encapsulation of guest molecules, thereby achieving efficient drug encapsulation and controlled release. Therefore, the non-peptide small molecule condensate can be used as a fluorescence imaging material, drug delivery carrier, or cell tracking material, and has application potential in biomedical fields such as anti-tumor therapy.

[0045] The following embodiments are given to specifically describe the present invention. However, it should be noted that the following embodiments are only used to further illustrate the present invention and should not be construed as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made to the present invention by those skilled in the art based on the content of the present invention are still within the scope of protection of the present invention.

[0046] All raw materials used in this invention are provided by the Exploration Reagent Platform and can be used without further purification. The solvents, catalysts, and desalting agents used in this invention are all redistilled.

[0047] Comparative Example 1: Synthesis of GMA (AMA)

[0048] This comparative example provides a single-component symmetrical carboxylic acid compound (GMA) with a urea group. It is mainly prepared by a one-pot process using an amino component (component A) with the participation of an activator (component M) to produce an ester compound, followed by deprotection to obtain the carboxylic acid compound. The amino component is glycine methyl ester, and the activator is N,N'-carbonyldiimidazole. The specific preparation steps are as follows:

[0049] 1) Weigh 2.51 g of glycine methyl ester hydrochloride into a reaction flask, perform three suction and three traverse, and add dichloromethane (40 mL) and N,N-diisopropylethylamine (2.91 g) under an inert gas atmosphere for premixing. Lower the temperature to 0°C and maintain a constant stirring speed under normal pressure until completely dissolved.

[0050] 2) Weigh 1.82 g of N,N'-carbonyldiimidazole at a molar ratio of A:M of 2:1. Add the mixture to the reaction flask at 0 °C under an inert gas atmosphere and react for 30 min, then react at room temperature for 12 h.

[0051] 3) After the reaction is complete, wash the product sequentially with 1 M hydrochloric acid, deionized water, saturated sodium bicarbonate, and saturated sodium chloride solution, and then evaporate the solvent to obtain the crude product.

[0052] 4) The crude product was recrystallized multiple times and dried under vacuum to obtain symmetrical ester compounds;

[0053] 5) Dissolve the product in 5 mL of tetrahydrofuran, add 5 mL of 1 M NaOH aqueous solution, and react at room temperature for 24 h; after evaporating the tetrahydrofuran in the system to dryness, adjust the pH to 2; add 5 mL of dichloromethane and stir to dissolve, then transfer to a separatory funnel to separate the layers; extract the aqueous phase twice more with the same solvent; combine all organic phases, wash once with saturated brine, add 2 g of anhydrous sodium sulfate and let stand to dry for 15 min, filter out the desiccant, and evaporate the filtrate to dryness to obtain GMA, a symmetrical carboxylic acid compound with carbonate groups.

[0054] 1 H NMR (400 MHz, DMSO-d6, 25 °C): δ 13.03 ppm (CH2-CO-OH), 6.46 ppm (CH2-NH-CO), 4.05 ppm (CO-CH2-NH).

[0055] AMA was prepared by replacing glycine methyl ester hydrochloride (2.51 g) in Comparative Example 1 with alanine methyl ester (2.83 g) using the same method.

[0056]

[0057] 1 H NMR (400 MHz, DMSO-d6, 25 °C): δ 12.56 ppm (CH2-CO-OH), 6.46 ppm(CH2-NH-CO), 4.39 ppm (CO-CH(CH3)-NH), 1.43 ppm (CO-CH(CH3)-NH).

[0058] Comparative Example 2: Synthesis of DAA

[0059] This comparative example provides a symmetrical tetracarboxylic acid compound (DAA) with a sulfonamide group, mainly obtained by deprotection of an intermediate product obtained from an amino component (component A) via a one-pot process in the presence of an activator (component M); the amino component is selected as diethyl aspartate, and the activator is selected as N,N'-thiodiimidazole; the specific preparation steps are as follows:

[0060] 1) Prepare the raw materials according to the formula. Weigh out diethyl aspartate (4.5134 g) and add it to the reaction flask. Pull the flask three times and open the valve three times. Under an inert gas atmosphere, add dichloromethane (40 mL) and N,N-diisopropylethylamine (2.585 g) for premixing. Lower the temperature to 0℃ and maintain a constant stirring speed under normal pressure until completely dissolved.

[0061] 2) Weigh N,N'-thiodiimidazole (1.982 g) according to the molar ratio of A:M of 2:1. Under an inert gas atmosphere, add component M to the reaction flask and react with A for 30 min at 0 °C. Then slowly restore to room temperature and react for 12 h.

[0062] 3) After the reaction was completed, the organic phase was washed successively with 1 M hydrochloric acid, deionized water, saturated sodium bicarbonate and saturated sodium chloride solution. After drying with anhydrous magnesium sulfate, the solvent was evaporated to obtain the crude product of ester-protected symmetrical sulfonamide intermediate.

[0063] 4) Dissolve the above crude product in a mixed solvent of tetrahydrofuran and water (5 mL), add (8 mL) of lithium hydroxide aqueous solution, and stir the reaction thoroughly at room temperature until the ester group is completely hydrolyzed into carboxylate.

[0064] 5) After the reaction is complete, remove the organic solvent by rotary evaporation under reduced pressure, cool the remaining aqueous solution in an ice bath, and slowly add hydrochloric acid to adjust the pH of the system to about 2, so that the free tetracarboxylic acid product precipitates out.

[0065] 6) The precipitated solid was filtered, the filter cake was washed with a small amount of ice water, and dried under vacuum to obtain DAA, a symmetrical tetracarboxylic acid compound with sulfonamide groups.

[0066]

[0067] 1 H NMR (400 MHz, DMSO-d6, 25 °C): δ 13.89 ppm (CH-CO-OH), 12.57 ppm (CH2-CO-OH), 7.34 ppm (SOO-NH-CH), 3.82 ppm (NH-CH-CO), 2.90;2.65 ppm (CO-CH2-CH).

[0068] Comparative Example 3: Synthesis of LME

[0069] This comparative example provides a single-component symmetrical ester compound (LME) with a urea group, mainly produced by a one-pot method from an amino component (component A) with the participation of an activator (component M); the amino component is selected as leucine methyl ester, and the activator is selected as N,N'-carbonyldiimidazole; the specific preparation steps are as follows:

[0070] 1) Prepare the raw materials according to the formula. Weigh 4.360 g of leucine methyl ester and add it to the reaction flask. Pull the flask three times and then add dichloromethane (60 ml) and N,N-diisopropylethylamine (3.231 g) under an inert gas atmosphere for premixing. Lower the temperature to 0°C and maintain a constant stirring speed under normal pressure until completely dissolved.

[0071] 2) Weigh 2.025 g of N,N'-carbonyldiimidazole at a molar ratio of A:M of 2:1. Under an inert gas atmosphere, add component M to the reaction flask and react with A for 30 min at 0 °C, then react at room temperature for 12 h.

[0072] 3) After the reaction is complete, wash the product sequentially with 1M hydrochloric acid, deionized water, saturated sodium bicarbonate and saturated sodium chloride solution, and evaporate the solvent to obtain the crude product.

[0073] 4) The crude product was recrystallized multiple times and dried under vacuum to obtain LME, a symmetrical ester compound with urea groups.

[0074]

[0075] 1 H NMR (400 MHz, DMSO-d6, 25 ℃): δ 6.32 ppm (CH-NH-CO), 4.14 ppm (CH-CH-NH), 3.60 ppm (CO-O-CH3), 1.60 ppm (CH2-CH-CH3), 1.43 ppm (CH-CH2-CH), 0.88ppm (CH2-CH-CH3).

[0076] Comparative Example 4: Synthesis of ELS

[0077] This comparative example provides an asymmetric carboxylic acid ester compound (ELS), mainly obtained by ring-opening esterification reaction of a hydroxyl component (component A) and an acid anhydride component (component B) in the presence of a catalyst (component M); the hydroxyl component is selected as ethyl lactate, the acid anhydride component is selected as succinic anhydride, and the catalyst is selected as 4-dimethylaminopyridine; the specific preparation steps are as follows:

[0078] 1) Prepare the raw materials according to the formula. Weigh ethyl lactate (1.181 g) and add it to the reaction flask. Pull the flask three times and open the valve three times. Add anhydrous dichloromethane (20 mL) under an inert gas atmosphere for premixing. Keep the stirring constant until it is completely dissolved.

[0079] 2) Weigh succinic anhydride (1.201 g) and add it to the above reaction system, then add 4-dimethylaminopyridine (0.122 g), and stir the reaction at room temperature for 12 h;

[0080] 3) After the reaction was completed, the organic solvent was removed by rotary evaporation under reduced pressure to obtain the crude product. The crude product was dissolved in an appropriate amount of ethyl acetate, and the organic phase was washed successively with a small amount of 1 M hydrochloric acid, deionized water and saturated sodium chloride solution. Then anhydrous sodium sulfate was added for drying, filtered and concentrated under reduced pressure. Then purified by silica gel column chromatography and dried under vacuum to obtain the asymmetric carboxylic acid ester compound ELS.

[0081]

[0082] 1 H NMR (400 MHz, DMSO-d6, 25 ℃): δ 12.18 ppm (OH-CO-CH2), 5.63 ppm(O-CH(CH3)-CO), 2.71 ppm (CH2-CH2-CO(OH)), 2.52 ppm (CH2-CH2-CO), 2.49 ppm(CH3-CH2-CO), 1.44 ppm (O-CH(CH3)-CO), 1.06 ppm (CH3-CH2-CO).

[0083] 10 mg of the small molecule solids obtained in Comparative Examples 1-4 were dissolved in 1 mL of 5 mM HEPES buffer (pH ~10). The pH of the system was adjusted each time with 10 μL of 0.1 M hydrochloric acid solution. As the pH decreased to 7 or below, no liquid-liquid phase separation occurred in any of the systems; that is, the small molecules obtained in Comparative Examples 1-4 could not be converted into aggregates.

[0084] Synthesis of LSA in Comparative Example 5

[0085] This comparative example provides a single-component asymmetric dicarboxylic acid compound (LSA) with an amide bond, mainly obtained by a stepwise one-pot method using a carboxylic acid component (component A) and an amino component (component B) in the presence of an activator (component M) to obtain an intermediate product, followed by deprotection. The carboxylic acid component is selected as monomethyl succinate, the amino component is selected as methyl leucine, and the activator is selected as N,N'-carbonyldiimidazole. The specific preparation steps are as follows:

[0086] 1) Prepare the raw materials according to the formula. Weigh monomethyl succinate (2.641 g) and add it to the reaction flask. Pull the flask three times and then add dichloromethane (40 mL) under an inert gas atmosphere for premixing. Cool the system to 0 °C and keep stirring at a constant pressure until completely dissolved.

[0087] 2) Weigh 4.053 g of N,N'-carbonyldiimidazole at a molar ratio of A:M of 1:1, and add it in batches to the reaction flask at 0 °C under an inert gas atmosphere, controlling the feeding rate and keeping the mixture stirred for 60 min.

[0088] 3) Subsequently, an equimolar amount of leucine methyl ester hydrochloride (4.542 g) dissolved in dichloromethane was slowly added to the above system, along with N,N-diisopropylethylamine (3.23 g) to promote amino group release. After the addition was complete, the mixture was allowed to rise naturally to room temperature and the reaction was continued for 12 h to obtain an asymmetric ester intermediate with an amide bond.

[0089] 4) After the reaction was completed, the organic phase was washed successively with 1 M hydrochloric acid, deionized water, saturated sodium bicarbonate solution and saturated sodium chloride solution. The solvent was removed by rotary evaporation under reduced pressure to obtain the crude product. The crude product was purified by recrystallization multiple times and dried under vacuum to obtain an asymmetric ester compound with amide bonds.

[0090] 5) Dissolve the above product in tetrahydrofuran (5 mL), add 1 M NaOH aqueous solution (5 mL), and react at room temperature for 24 h. After the reaction is complete, remove the tetrahydrofuran from the system under reduced pressure and adjust the pH of the system to approximately 2. Add 10 mL of dichloromethane and stir to dissolve, then transfer to a separatory funnel to separate the layers. Extract the aqueous phase twice more with 10 mL of the same solvent. Combine the organic phases, wash once with saturated brine, add anhydrous sodium sulfate and dry for 15 min. After filtering out the desiccant, evaporate the filtrate under reduced pressure to dryness to obtain the asymmetric dicarboxylic acid target product LSA with amide bonds.

[0091]

[0092] 1 H NMR (400 MHz, DMSO-d6, 25 °C): δ 12.66 ppm (OH-CO-CH), 12.18 ppm(OH-CO-CH2), 8.32 ppm (CO-NH-CH), 4.55 ppm (NH-CH-CO), 2.60 ppm (CH2-CH2-CO(OH)), 2.34 ppm (CH2-CH2-CO), 1.75 ppm (CH-CH2-CH(CH3)2), 1.49 ppm (CH2-CH(CH3)-CH3), 0.91 ppm (CH2-CH(CH3)-CH3).

[0093] 10 mg of the small molecule solid obtained in Comparative Example 5 was dissolved in 1 mL of 5 mM HEPES buffer (pH ~10). The pH of the system was adjusted each time with 10 μL of 0.1 M hydrochloric acid solution. As the pH decreased to 7 or below, liquid-liquid phase separation (LLPS) immediately occurred, and the solution immediately became turbid, indicating the formation of aggregates. However, the resulting aggregates are prone to structural damage and dissociation under enzymatic action. Figure 11 It has poor resistance to enzymatic hydrolysis, meaning it is easily recognized and hydrolyzed by proteases and is difficult to remain stable in the physiological environment for a long time.

[0094] Example 1 Synthesis of small molecule D-ILME

[0095] This embodiment provides a single-component symmetrical acid compound (D-ILME) with a urea group, mainly obtained by deprotection of an intermediate product obtained from an amino component (component A) in a one-pot process with the participation of an activator (component M); wherein the amino component is selected as isoleucine methyl ester, and the activator is selected as N,N'-carbonyldiimidazole; the specific preparation steps are as follows:

[0096] 1) Weigh isoleucine methyl ester hydrochloride (3.6332 g) and add it to the reaction flask. After three suctions and three tracings, add dichloromethane (40 ml) and N,N-diisopropylethylamine (2.60 g) under an inert gas atmosphere for premixing. Lower the temperature to 0°C and maintain a constant stirring speed under normal pressure until completely dissolved.

[0097] 2) Weigh 1.6215 g of N,N'-carbonyldiimidazole at a molar ratio of A:M of 2:1. Add the mixture to the reaction flask at 0 °C under an inert gas atmosphere and react for 30 min, then react at room temperature for 12 h.

[0098] 3) After the reaction is complete, wash the product sequentially with 1 M hydrochloric acid, deionized water, saturated sodium bicarbonate, and saturated sodium chloride solution, and then evaporate the solvent to obtain the crude product.

[0099] 4) The crude product was recrystallized multiple times and dried under vacuum to obtain symmetrical ester compounds with urea groups;

[0100] 5) Dissolve the product in tetrahydrofuran (5 mL), add 1 M NaOH aqueous solution (5 mL), and react at room temperature for 24 h; after evaporating the tetrahydrofuran in the system, adjust the acidity; add 10 mL of dichloromethane and stir to dissolve, then transfer to a separatory funnel to separate the layers; extract the aqueous phase twice more with 10 mL of the same solvent; combine all organic phases, wash once with saturated brine, add 2 g of anhydrous sodium sulfate and let stand to dry for 15 min, filter out the desiccant, and evaporate the filtrate to dryness to obtain the symmetrical carboxylic acid compound D-ILME with urea group.

[0101]

[0102] 1H NMR (400 MHz, DMSO-d6, 25 °C): δ 12.39 ppm (CH-CO-OH), 6.46 ppm(CH-NH-CO), 4.25 ppm (CO-CH-NH), 2.50 ppm (CH-CH(CH3)-CH2) 1.55 ppm (CH-CH2-CH3), 1.11 ppm (CH-CH(CH3)-CH2), 0.99 ppm (CH-CH2-CH3).

[0103] After replacing the above isoleucine methyl ester hydrochloride with valine ethyl ester hydrochloride (3.3526 g) and leucine methyl ester hydrochloride (3.6332 g), D-VME and D-LME were prepared respectively by the same method.

[0104]

[0105] 1 H NMR (400 MHz, DMSO-d6, 25 °C): δ 12.39 ppm (CH-CO-OH), 6.41 ppm (CH-NH-CO), 4.25 ppm (CO-CH-NH), 1.90 ppm (NH-CH-CH(CH3)2), 0.98 ppm (NH-CH-CH(CH3)2).

[0106]

[0107] 1 H NMR (400 MHz, DMSO-d6, 25 ℃): δ 12.66 ppm (CH-CO-OH), 6.43 ppm(CH-NH-CO), 4.55 ppm (CO-CH-NH), 1.75 ppm (CH-CH2-CH(CH3)2), 1.49 ppm (CH-CH2-CH(CH3)2)), 0.90 ppm (CH-CH2-CH(CH3)2).

[0108] Example 2 Synthesis of small molecule D-PME

[0109] This embodiment provides a single-component symmetrical acid compound (D-PME) with a urea group, mainly obtained by deprotection of an intermediate product obtained from an amino component (component A) in a one-pot process with the participation of an activator (component M); the amino component is selected as phenylalanine methyl ester, and the activator is selected as N,N'-carbonyldiimidazole; the specific preparation steps are as follows:

[0110] 1) Weigh 4.3136 g of phenylalanine methyl ester hydrochloride into a reaction flask, perform three suction and three-way mixing, and add dichloromethane (40 ml) and N,N-diisopropylethylamine (2.60 g) under an inert gas atmosphere for premixing. Lower the temperature to 0°C and maintain a constant stirring speed under normal pressure until completely dissolved.

[0111] 2) Weigh 1.6215 g of N,N'-carbonyldiimidazole at a molar ratio of A:M of 2:1. Add the mixture to the reaction flask at 0 °C under an inert gas atmosphere and react for 30 min, then react at room temperature for 12 h.

[0112] 3) After the reaction is complete, wash the product sequentially with 1 M hydrochloric acid, deionized water, saturated sodium bicarbonate, and saturated sodium chloride solution, and then evaporate the solvent to obtain the crude product.

[0113] 4) The crude product was recrystallized multiple times and dried under vacuum to obtain symmetrical ester compounds with urea groups.

[0114] 5) Dissolve the product in tetrahydrofuran (5 mL), add 1 M NaOH aqueous solution (5 mL), and react at room temperature for 24 h; after evaporating the tetrahydrofuran in the system to dryness, adjust the acidity; add 10 mL dichloromethane and stir to dissolve, then transfer to a separatory funnel to separate the layers; the aqueous phase is extracted twice more with 10 mL of the same solvent; combine all organic phases, wash once with saturated brine, add 2 g anhydrous sodium sulfate and let stand to dry for 15 min, filter out the desiccant, and evaporate the filtrate to dryness to obtain the symmetrical carboxylic acid compound D-PME with urea group.

[0115]

[0116] 1 H NMR (400 MHz, DMSO-d6, 25 ℃): δ 12.89 ppm (CH-CO-OH), 7.23;7.18ppm(C6H5-CH2-CH), 6.46 ppm (CH-NH-CO), 3.12;2.86 ppm (C6H5-CH2-CH).

[0117] Example 3 Synthesis of D-MME

[0118] This embodiment provides a single-component urea-based small molecule (D-MME), mainly prepared by a self-condensation reaction of an amino acid component in the presence of an activator; the amino acid component is selected as methionine; the activator is selected as N,N'-carbonyldiimidazole; the specific preparation steps are as follows:

[0119] 1) Weigh methionine (3.483 g) into a reaction flask, perform a three-way evacuation, add dichloromethane (40 mL) and N,N-diisopropylethylamine (3.0 g) under an inert gas atmosphere for premixing, and cool the system to 0 °C;

[0120] 2) Weigh N,N'-carbonyldiimidazole (2.025 g) according to the molar ratio of A:M of 2:1, and add it in batches to the reaction flask at 0 °C under an inert gas atmosphere. Control the feeding rate and keep stirring, so that the system reacts at 0 °C for 1 h, and then naturally rises to room temperature to continue the reaction for 12 h to generate the intermediate product.

[0121] 3) After the reaction is complete, remove the solvent under reduced pressure, add an appropriate amount of dichloromethane to dissolve the residue, transfer it to a separatory funnel, and wash the organic phase successively with dilute hydrochloric acid, deionized water and saturated sodium chloride solution to remove unreacted raw materials and by-products.

[0122] 4) After merging the organic phases, dry with anhydrous sodium sulfate, filter out the desiccant, and remove the solvent by rotary evaporation under reduced pressure to obtain the crude product; after recrystallization purification and vacuum drying, the symmetrical acid compound D-MME with urea groups is obtained.

[0123]

[0124] 1 H NMR (400 MHz, DMSO-d6, 25 ℃): δ 12.66 ppm (CH-CO-OH), 6.46 ppm (CH-NH-CO), 4.55 ppm (CO-CH-NH), 2.60 ppm (S-CH2-CH2), 2.05 ppm (S-CH2-CH2), 2.07 ppm (CH2-S-CH3).

[0125] Example 4 Synthesis of LMA

[0126] This embodiment provides a single-component symmetrical acid compound (LMA) with a sulfonyl diamine bond, which is mainly obtained by deprotection of an intermediate product obtained by a one-pot process from an amino component (component A) in the presence of an activator (component M); wherein the amino component is selected as isoleucine methyl ester, and the activator is selected as sulfonyl diimidazole.

[0127] The specific preparation steps are as follows:

[0128] 1) Weigh isoleucine methyl ester hydrochloride (3.6332 g) and add it to the reaction flask. With the flask purging three times and the flask purging three times, add dichloromethane (40 mL) and N,N-diisopropylethylamine (2.60 g) under an inert gas atmosphere for premixing. Lower the temperature to 0 °C and keep stirring at a constant pressure until completely dissolved.

[0129] 2) Weigh 2.828 g of sulfonyl diimidazole at a molar ratio of A:M of 2:1 and add it to the reaction flask at 0 °C under an inert gas atmosphere. After maintaining the low temperature reaction for 30 min, allow it to rise naturally to room temperature and continue the reaction for 12 h to obtain a symmetrical ester intermediate with sulfonyl diamine bonds.

[0130] 3) After the reaction was completed, the organic phase was washed successively with 1 M hydrochloric acid, deionized water, saturated sodium bicarbonate solution and saturated sodium chloride solution, and the solvent was evaporated to obtain the crude product; the crude product was purified by recrystallization multiple times and dried under vacuum to obtain a symmetrical ester intermediate with sulfonyl diamine bonds;

[0131] 4) Dissolve the above intermediate in tetrahydrofuran (5 mL), add 1 M NaOH aqueous solution (5 mL), and react at room temperature for 24 h to hydrolyze the methyl ester group;

[0132] 5) After the reaction is complete, evaporate the tetrahydrofuran in the system to dryness and adjust the system to acidity; add 10 mL of dichloromethane and stir to dissolve, then transfer to a separatory funnel to separate the layers; extract the aqueous phase twice more with 10 mL of the same solvent.

[0133] 6) After merging the organic phases, wash once with saturated brine, add anhydrous sodium sulfate and dry for 15 min. After filtering out the desiccant, evaporate the filtrate under reduced pressure to dryness to obtain LMA, a symmetrical carboxylic acid compound with sulfonyl diamine bonds.

[0134]

[0135] 1 H NMR (400 MHz, DMSO-d6, 25 ℃): δ 12.39 ppm (CH2-CO-OH), 7.34 ppm(SOO-NH-CH), 3.40 ppm (NH-CH-CO), 1.75 ppm (CH-CH2-CH(CH3)2), 1.49 ppm (CH-CH2-CH(CH3)2)), 0.90 ppm (CH-CH2-CH(CH3)2).

[0136] Example 5 Synthesis of LMC

[0137] This embodiment provides a single-component asymmetric acid compound (LMC) with a thiocarbamate bond, which is mainly obtained by deprotection of an intermediate product obtained by a stepwise one-pot method with the participation of a thiol component (component A) and an amino component (component B) in the presence of an activator (component M); wherein the thiol component is selected from methyl mercaptoacetate, the amino component is selected from methyl leucine, and the activator is selected from N,N'-carbonyldiimidazole.

[0138] The specific preparation steps are as follows:

[0139] 1) Weigh methyl mercaptoacetate (component A, 2.6535 g) into a reaction flask, perform three suction and three tracing operations, add dichloromethane (40 mL) under an inert gas atmosphere for premixing, cool the system to 0 ℃, and keep stirring at constant pressure until completely dissolved;

[0140] 2) Weigh N,N'-carbonyldiimidazole (CDI, 4.0538 g) according to the molar ratio of A:M of 1:1, and add it in batches to the reaction flask at 0 °C under an inert gas atmosphere. Control the feeding rate and keep stirring so that the mercapto group reacts with the activator for 60 min to generate a sulfur-containing activated intermediate.

[0141] 3) Subsequently, an equimolar amount of leucine methyl ester (component B, 4.5415 g) dissolved in dichloromethane was slowly added to the above system, along with N,N-diisopropylethylamine (3.23 g) to promote amino release. After the addition was complete, the mixture was allowed to rise naturally to room temperature and the reaction was continued for 12 h to obtain an asymmetric ester intermediate with a thiocarbamate bond.

[0142] 4) After the reaction is complete, the organic phase is washed successively with 1 M hydrochloric acid, deionized water, saturated sodium bicarbonate solution and saturated sodium chloride solution, and the solvent is evaporated to dryness to obtain the crude product;

[0143] 5) The crude product was purified by recrystallization multiple times and dried under vacuum to obtain an asymmetric ester compound with thiocarbamate bonds.

[0144] 6) Dissolve the above product in tetrahydrofuran (5 mL), add 1 M NaOH aqueous solution (5 mL), and react at room temperature for 24 h to hydrolyze the methyl ester group; after the reaction is complete, evaporate the tetrahydrofuran in the system to dryness and adjust the system to acidity; if no solid precipitates or a viscous oily substance precipitates, add 10 mL of dichloromethane and stir to dissolve, transfer to a separatory funnel for separation; the aqueous phase is then extracted twice more with 10 mL of the same solvent;

[0145] 7) After combining the organic phases, wash once with saturated brine, add anhydrous sodium sulfate and dry for 15 min. After filtering out the desiccant, evaporate the filtrate under reduced pressure to dryness to obtain LMC, an asymmetric carboxylic acid compound with thiocarbamate bonds.

[0146]

[0147] 1 H NMR (400 MHz, DMSO-d6, 25 °C): δ 12.80 ppm (S-CH2-CO-OH), 12.66 ppm (NH-CH2-CO(OH)), 8.18 ppm (CH2-NH-CO), 4.55 ppm (NH-CH-CO), 3.96 ppm (S-CH2-CO), 1.75 ppm (CH-CH2-CH(CH3)2), 1.49 ppm (CH-CH2-CH(CH3)2)), 0.90 ppm (CH-CH2-CH(CH3)2.

[0148] Examples 1-5: Preparation of condensates from small molecules:

[0149] 10 mg of all the small molecule solids obtained in Examples 1-5 were dissolved in 1 mL of 5 mM HEPES buffer (pH ~10). The pH of the system was adjusted each time with 10 μL of 0.1 M hydrochloric acid solution. As the pH decreased to 7 or below, liquid-liquid phase separation (LLPS) immediately occurred, and the solution immediately became turbid; this proves that these small molecules can form the non-peptide small molecule aggregates.

[0150] Performance testing:

[0151] 1. Fluorescence properties of small molecules

[0152] Fluorescence tests were performed on the small molecules obtained from the comparative examples and the examples. The test method was as follows: GMA from Comparative Example 1 and D-PME from Example 2 were dissolved in DMF solution and tested at the same concentration (10). -5 Fluorescence testing was performed under conditions of M, by adjusting different excitation wavelengths (λ). ex The effect of fluorescence on fluorescence emission behavior was investigated. Fluorescence measurements were performed on an F-4600FL spectrophotometer (Hitachi, Ltd., Japan) with an excitation and emission slit of 5 nm. Emission spectra were collected in the excitation wavelength range of 360–640 nm, corresponding to 375–700 nm.

[0153] Figure 1The fluorescence emission spectra of Comparative Example 1 GMA and Example 2 D-PME at different excitation wavelengths are shown. Figure 1 (A) It can be seen that: Comparative Example 1 GMA is not λ ex Under these conditions, its emission peak (λ) em The α-rays mainly appear between 380 nm and 440 nm, exhibiting a single luminescent center characteristic. In contrast, the D-PME in Example 2 shows different λ values. ex The main emission peak is concentrated around 408 nm, while a long-wavelength emission peak appears at about 560 nm, indicating that there are two independent emission centers in the system. Figure 1 B).

[0154] 2. The process of condensation formation

[0155] Figure 2 These are photographs showing the changes in the state of the systems (Comparative Example 1 GMA, Example 1 D-ILME, and Example 2 D-PME) in 5 mM HEPES buffer as the pH was gradually adjusted with 0.1 M HCl. Figure 2 It was found that all samples (10 mg / mL) were completely soluble within a pH range of 9. As the pH was gradually decreased by adding 0.1 M HCl (10 μL each time), slight turbidity began to appear in the solutions of Example 1 (D-ILME) and Example 2 (D-PME) when the pH dropped to approximately 7. Further decreasing the pH to approximately 6 resulted in significant turbidity, exhibiting typical liquid-liquid phase separation, indicating the formation of aggregates. In contrast, the GMA in Comparative Example 1 remained clear under the same conditions, even when the pH was decreased to approximately 5, and no significant phase separation was observed.

[0156] 3. Morphological characterization of condensates under an optical microscope

[0157] Aggregates prepared by adjusting the pH of Comparative Example 5 LSA, Example 1 D-ILME, Example 2 D-PME, Example 3 D-MME, Example 4 LMA, and Example 5 LMC to approximately pH 6 in 5 mM HEPES buffer were dropped onto a glass slide, sealed with a coverslip, and incubated at 4 °C for 12 h. Subsequently, their morphology was observed using an optical microscope (Olympus, Japan), and the results are as follows. Figure 3 As shown. By Figure 3(AF) shows that: Comparative Example 5 LSA, Example 1 D-ILME, Example 2 D-PME, Example 3 D-MME, Example 4 LMA, and Example 5 LMC all formed typical spherical droplets with a size of approximately 1-10 μm in 5 mM HEPES buffer. The resulting droplets exhibited obvious liquid-like behavior, such as aggregation, fusion, and deformation, indicating that they possess liquid-liquid phase separation (LLPS) characteristics; the dashed circles indicate the droplet aggregation regions.

[0158] 4. Fluorescence properties of aggregates

[0159] Aggregates prepared from small molecule D-PME (Example 2) and small molecule LMC (Example 5) in 5 mM HEPES buffer at a pH adjusted to approximately 6 were subjected to fluorescence testing at different excitation wavelengths. The fluorescence was measured by adjusting different excitation wavelengths (λ). ex The excitation slits were 300-520 nm, and the corresponding emission spectra were collected from 315-700 nm to investigate the effect of excitation conditions on luminescence behavior. Fluorescence measurements were performed on an F-4600 fluorescence spectrophotometer (Hitachi, Japan), with both excitation and emission slits at 5 nm. Results are as follows: Figure 4 As shown, by Figure 4 It can be seen that the condensate prepared by D-PME (abbreviated as D-PME condensate) exhibits two independent luminescent centers at approximately 440 nm and 570 nm. Figure 4 A), LMC condensates exhibit two independent luminescent centers at approximately 360 nm and 554 nm. Figure 4 B).

[0160] The present invention also performed two-dimensional excitation-emission matrix (EEM) fluorescence tests on the aggregates prepared under the same conditions (5 mM HEPES buffer, 10 mg / mL, pH ~ 6) of Example 1 D-ILME, Example 2 D-PME, and Example 4 LMA: excitation wavelength (λ) ex The range is 320-700 nm, and the emission wavelength (λ) em The excitation range was 400-700 nm. EEM spectra were constructed based on the emission spectra under different excitation conditions. Fluorescence measurements were performed on an F-4600FL spectrophotometer (Hitachi, Ltd., Japan), with an excitation and emission slit width of 5 nm. The results are as follows: Figure 5 As shown, by Figure 5 It can be seen that the condensates prepared by D-ILME in Example 1, D-PME in Example 2, and LMA in Example 4 all exhibit the characteristic of having two independent luminescent centers: the luminescent centers of the condensate prepared by D-ILME in Example 1 are at 440 nm and 640 nm, respectively. Figure 5A); Example 2: The luminescent centers of the condensates prepared by D-PME were at 440 nm and 570 nm, respectively ( Figure 5 B); In Example 4, the luminescent centers of the condensates prepared by LMA were at 410 nm and 620 nm, respectively ( Figure 5 C).

[0161] Morphological characterization of D-PME from Example 2 and GMA from Comparative Example 1 was performed using a confocal laser scanning microscope (CLSM), and fluorescence imaging analysis was further performed on the aggregates obtained from D-PME in Example 2. Specifically, D-PME from Example 2 and GMA from Comparative Example 1 were dissolved in 5 mM HEPES buffer to prepare solutions with a concentration of 10 mg / mL, and the pH was adjusted to ~6 by adding 0.1M HCl dropwise. Subsequently, approximately 10 μL of each sample was added to a glass slide, covered with a coverslip, and incubated at 4 °C for 12 h. The samples were placed under a confocal laser scanning microscope (CLSM, Olympus FV1000 / Nikon A1RMP, Japan). Initial observation was performed in bright field mode, and then the D-PME aggregates were switched to fluorescence mode for imaging analysis.

[0162] Figure 6 (A) is the GMA image of Comparative Example 1 under bright field, where no obvious droplet structure was observed; Figure 6 (B) is an image of the condensate prepared by D-PME in Example 2 under bright field, where spherical droplets can be clearly observed. Furthermore, under excitation conditions of 405 nm and 488 nm, the condensate prepared by D-PME emitted blue light, respectively. Figure 6 C) and green fluorescence ( Figure 6 D).

[0163] 5. Chirality of condensates

[0164] The chirality of the aggregates prepared by D-PME in Example 2 and LMC in Example 5 was characterized by circular dichroism (CD). The two small molecules were dissolved in 5 mM HEPES buffer to prepare 10 mg / mL solutions, and aggregates were prepared by adjusting the pH to ~6. Subsequently, the analyses were performed at room temperature using a J-1500-150 circular dichroism spectrometer (JASCO Corporation, Japan), with a scanning range of 190-300 nm. The molar ellipticity [θ] was calculated using the following formula: [θ] = (θ × 100 × M w ) / (C×l), where θ is obtained from a CD spectrometer, M w Where C is the molecular weight, C is the sample concentration, and l is the optical path length of the cuvette; the results are as follows Figure 7 As shown, by Figure 7It can be seen that the aggregates obtained by D-PME in Example 2 and the aggregates obtained by LMC in Example 5 both have obvious chiral structures.

[0165] The chiral luminescence properties of the condensates prepared by D-PME in Example 2 were characterized using circularly polarized emission spectroscopy (CPL). Small molecule D-PME was dissolved in 5 mM HEPES buffer to prepare a 10 mg / mL solution, and condensates were prepared by adjusting the pH to approximately 6. The samples were then detected at room temperature using an OLIS CPL Solo spectrometer with an excitation wavelength of 340 nm and a digital integration time (DIT) of 0.2 s. The CPL asymmetry factor as a function of wavelength was recorded within the emission wavelength range. The results are as follows: Figure 8 As shown, the condensate prepared by D-PME in Example 2 exhibits a significant circularly polarized luminescence response in the wavelength range of approximately 360-550 nm. Its CPL asymmetry factor is generally positive, reaching a maximum value near approximately 460 nm, with a peak value of about 0.05. This result indicates that the D-PME condensate not only emits light but also exhibits significant chiral selectivity in its luminescence process, i.e., there is a significant difference in the intensity of left- and right-circularly polarized luminescence. The circularly polarized luminescence asymmetry factor is an important parameter for measuring the chiral luminescence capability of a system; a larger value indicates a stronger selectivity for left- and right-circularly polarized light. This demonstrates that the condensate possesses strong circularly polarized luminescence properties. Therefore, the above results show that the condensate formed by D-PME in Example 2 not only has a significant chiral structure but also that this chirality is further reflected in its luminescence behavior, proving the existence of a supramolecular chiral structure within it.

[0166] 6. Drug release properties of aggregates

[0167] This invention uses D-PME from Example 2 as a model to investigate the encapsulation and release behavior of the agglomerates prepared therefrom on the loading of levofloxacin. D-PME was dissolved in 5 mM HEPES buffer to prepare a 10 mg / mL solution, and the pH was adjusted to approximately 6 to form agglomerates. Subsequently, levofloxacin was dissolved in PBS solution and mixed with the agglomerates at a volume ratio of 1:9 (drug solution: agglomerate) to obtain drug-loaded agglomerates. 0.5 mL of the drug-loaded agglomerate was placed in a 1000 MWCO dialysis bag, sealed, and then placed in a centrifuge tube containing 4.5 mL of release medium (internal / external volume ratio 1:9). The release medium was HEPES buffer preheated to 37 °C (adjusted to pH 6 and pH 8, respectively). The system was shaken at 37 °C and 100 rpm. At different time points, 0.2 mL of the external solution was taken, and the absorbance was measured at 330 nm using a UV-Vis spectrophotometer to calculate the drug release. An equal volume of fresh buffer was added after each sampling to maintain a constant system volume. Release curves were plotted based on the cumulative release at each time point, and the results are shown below. Figure 9 As shown. By Figure 9 It can be seen that the aggregates prepared by D-PME in Example 2 showed obvious pH-responsive release behavior for levofloxacin: almost no drug was released at pH=6, indicating that the aggregates could effectively encapsulate and stably retain the drug; while the release rate was significantly increased at pH=8, indicating that a slightly alkaline environment can trigger the drug release process.

[0168] 7. Biocompatibility of aggregates

[0169] The cell compatibility of the aggregates of this invention was evaluated using the CCK-8 assay. Fibroblasts were used as model cells to test the aggregates formed by the small molecules (D-VME, D-LME, D-ILME, D-PME, D-MME, LMA, LMC) mentioned in Examples 1-5 in 5 mM HEPES buffer (10 mg / mL, pH ~ 6). The aggregates were numbered 1-7 sequentially, corresponding to... Figure 10 Condensations 1-7 in the text.

[0170] Fibroblasts were distributed at a rate of 1 × 10⁶ cells per well. 4 Cells were seeded at a density in 96-well plates and cultured for 24 h. After discarding the original culture medium, culture medium containing different aggregates was added, and incubation continued for another 24 h. After treatment, 10 μL of CCK-8 reagent was added to each well, and the plates were incubated at 37 °C for 1–2 h. The absorbance at 450 nm was then measured using a microplate reader. Untreated cells were used as a control group. Data from the experimental groups were normalized, and cell viability was calculated. Results are as follows: Figure 10 As shown, by Figure 10 It can be seen that the aggregates 1-7 obtained by the present invention all have good biocompatibility.

[0171] 8. Enzymatic stability of aggregates

[0172] This invention uses the aggregates formed in Example 1 (D-ILME), Example 2 (D-PME), Example 3 (D-MME), Example 4 (LMA), Example 5 (LMC), and Comparative Example 5 (LSA) in 5 mM HEPES buffer (10 mg / mL, pH ~ 6) as models to investigate the enzymatic stability of the obtained aggregates at 37 °C and pH ~ 6. The test method is as follows: neutral protease solution is added to the system to make the final enzyme concentration 0.1 mg / mL (i.e., 10 μL of 10 mg / mL neutral protease stock solution is added to 1 mL of aggregate solution), and incubated at 37 °C; samples are taken at different time points (0, 1, 2, 4, 8, 12, 24 h), and the absorbance change at the characteristic absorption peak of the target compound is measured using a UV-Vis spectrophotometer to calculate its residual rate; the residual rate formula is as follows: Residual rate = ABS t ×100% / ABS0; where ABS0 and ABS t The absorbance values ​​at the initial time and at reaction time t represent the absorbance, respectively. The results are as follows: Figure 10 As shown, by Figure 10 It can be seen that the aggregates formed by D-ILME in Example 1, D-PME in Example 2, D-MME in Example 3, LMA in Example 4, and LMC in Example 5 still maintained a high residual rate under the action of enzymes, indicating that the aggregate structure formed by D-ILME in Example 1, D-PME in Example 2, D-MME in Example 3, LMA in Example 4, and LMC in Example 5 can effectively inhibit enzymatic hydrolysis. The aggregates formed by LSA in Comparative Example 5 gradually degraded over time under the action of enzymes, indicating that they were difficult to resist enzymatic hydrolysis and had significantly poor resistance to enzymatic hydrolysis.

[0173] It should be noted that while the preferred embodiments of the present invention are given in the specification and accompanying drawings, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. These embodiments are not intended to impose additional limitations on the content of the present invention; their purpose is to provide a more thorough and comprehensive understanding of the disclosure of the present invention. Furthermore, the above-described technical features can be combined with each other to form various embodiments not listed above, all of which are considered to be within the scope of the present invention specification. Moreover, those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A non-peptide small molecule condensate, characterized in that, It is obtained from the small molecule shown in Formula I as a precursor. Formula I In Equation I, Y and Z are independently NH, O, or S, and R = or X = O or S; R1 and R2 are each independently selected from: H and C 3-20 Alkyl, -C 1-8 alkylene-phenyl, -C 1-8 alkylene-5-10 heteroaryl, -C 1-8 Alkylene-SC 1-8 Alkyl, -C 1-8 Alkylene -SH, -C 1-8 Alkylene-OC 1-8 Alkyl or -C 1-8 Alkylene-NH-C 1-8 Alkyl group; the 5-10-membered heteroaryl group contains 1, 2, 3, or 4 heteroatoms selected from N, O, and S; the 5-10-membered heteroaryl group is unsubstituted or C-substituted. 1-8 Alkyl or C 1-8 Alkyl substitution; and R1 and R2 are not both H.

2. The non-peptide small molecule condensate according to claim 1, characterized in that, R1 and R2 are each independently selected from: H and C 3-10 Alkyl, -C 1-6 alkylene-phenyl, -C 1-6 alkylene-5-membered heteroarylphenyl, -C 1-6 Alkylene-SC 1-6 Alkyl, -C 1-6 Alkylene -SH, -C 1-6 Alkylene-OC 1-6 Alkyl or -C 1-6 Alkylene-NH-C 1-6 Alkyl group; the 5-membered heteroaryl group is selected from pyrroleyl, furanyl, thiophenyl, pyrazolyl, imidazolyl, thiazolyl, isothiazolyl, oxazolyl, or isoxazolyl; the 5-membered heteroarylphenyl group is unsubstituted or C-substituted. 1-4 Alkyl or C 1-4 Alkyl substitution; and R1 and R2 are not both H.

3. The non-peptide small molecule condensate according to claim 2, characterized in that, R1 and R2 are each independently selected from: H and C 3-6 Alkyl, -C 1-3 alkylene-phenyl, -C 1-3 alkylene-indolyl, -C 1-3 Alkylene-SC 1-3 Alkyl, -C 1-3 Alkylene -SH, -C 1-3 Alkylene-OC 1-3 Alkyl or -C 1-3 Alkylene-NH-C 1-3 Alkyl group; the indole group is unsubstituted or substituted with methoxy or ethoxy groups; and R1 and R2 are not both H.

4. A non-peptide small molecule condensate according to claim 1, characterized in that, The small molecule represented by Formula I is selected from substances with the following structures: , , , , , , , , , or .

5. A non-peptide small molecule condensate according to any one of claims 1 to 4, characterized in that, The non-peptide small molecule condensate has two independent fluorescent luminescent centers.

6. A non-peptide small molecule condensate according to any one of claims 1 to 4, characterized in that, The non-peptide small molecule condensates have circularly polarized luminescence properties or supramolecular chirality.

7. A method for preparing a non-peptide small molecule condensate according to any one of claims 1 to 6, characterized in that, The preparation method is as follows: the small molecules shown in Formula I spontaneously form aggregates through liquid-liquid phase separation.

8. The method for preparing a non-peptide small molecule condensate according to claim 7, characterized in that, The preparation method is as follows: the small molecule is placed in a buffer solution, the pH of the buffer solution is adjusted to ≤6, obvious liquid-liquid phase separation occurs, and the solution becomes turbid, indicating that the non-peptide small molecule aggregate has been prepared; wherein, the buffer solution is HEPES buffer, phosphate buffer, acetate buffer or Tris buffer; the pH of the buffer solution is 4 ~ 6, and the concentration of the buffer solution is 5mM ~ 30 mM.

9. Use of a non-peptide small molecule condensate according to any one of claims 1 to 6 in fluorescent materials or biomedical materials.

10. The use according to claim 9, characterized in that, The non-peptide small molecule condensates are used as fluorescence imaging materials, drug carrier materials, cell tracing materials, or anti-tumor materials.

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