N-acyl heterocyclic compounds, preparation method thereof, lysine probe and application

Abstract

The application relates to an N-acyl heterocyclic compound, a preparation method thereof, a lysine probe and application. The N-acyl heterocyclic compound has the structural characteristics shown in the following formula (I). The N-acyl heterocyclic compound can be used as a lysine probe, has good stability and adjustable reactivity, and widens the coverage of proteomics.

Description

Technical Field

[0001] This application relates to the field of bioanalytical technology, and in particular to an N-acyl heterocyclic compound and its preparation method, a lysine probe and its application. Background Technology

[0002] The development of most major diseases is essentially the result of abnormal biomolecular function, disordered signaling pathways, and imbalanced microenvironments. Chemical proteomics, as a powerful tool for drug target development and identification, is widely used in scientific research and drug development. Small molecule compound probes can accurately identify disease-related specific targets, abnormal metabolites, and pathological microenvironments, providing a core means for in-depth analysis of disease mechanisms and solving the pain points of traditional research, which struggles to distinguish between normal and pathological molecules and cannot dynamically track disease progression.

[0003] Lysine comprises 5.7% of the human proteome, nearly three times that of cysteine ​​(only 2%), and its nucleophilic ε-amino group makes it a viable target for covalent drug development. Furthermore, lysine is widely distributed across druggable regions, with approximately 75% of key metabolite binding sites (such as ADP / ATP, NAD(P)H, and SAM) containing at least one lysine residue. These characteristics collectively make lysine a focal point for next-generation covalent drug development.

[0004] However, while some lysine probes are currently commercialized, they still suffer from poor stability, easy hydrolysis in acid-base systems, and poor reaction selectivity. More fundamentally, the interactions between electrophiles and the lysine proteome that have been systematically evaluated so far result in very few well-characterized and modifiable lysine residues available for covalent drug development. Summary of the Invention

[0005] Based on this, this application provides an N-acyl heterocyclic compound, its preparation method, a lysine probe, and its application. This N-acyl heterocyclic compound can serve as a lysine probe, exhibiting good stability and tunable reactivity, and broadening the coverage of the proteome.

[0006] A first aspect of this application provides an N-acyl heterocyclic compound having the structural features shown in formula (I):

[0007] (I),

[0008] Where n is an integer from 1 to 5;

[0009] X1 is a carbonyl group; X2 is -S-, -S(O)2- or a carbonyl group;

[0010] Ring A and X3 satisfy one of the following conditions:

[0011] (1) Ring A does not exist, X3 is -S- or -CH2-;

[0012] (2) Ring A is a C6~C10 aromatic ring, and X3 is C.

[0013] In one embodiment, the N-acyl heterocyclic compound has the structural features shown in formula (I-1) or formula (I-2):

[0014] Formula (I-1),

[0015] Formula (I-2).

[0016] In one embodiment, n is 2 or 3.

[0017] In one embodiment, the N-acyl heterocyclic compound is selected from the following compounds:

[0018] .

[0019] A second aspect of this application provides a method for preparing the N-acyl heterocyclic compound described in the first aspect, comprising the following steps:

[0020] Compound 1 and compound 2 were subjected to an amidation reaction;

[0021] The structure of compound 1 is as follows:

[0022] ;

[0023] The structure of compound 2 is as follows:

[0024] , where R is a hydroxyl group or a halogen.

[0025] In one embodiment, the amidation reaction has one of the following characteristics:

[0026] (1) R is a hydroxyl group, and the amidation reaction is carried out in the presence of a condensing agent and a nucleophilic catalyst, wherein the condensing agent includes a carbodiimide condensing agent and the nucleophilic catalyst includes N,N-dimethylaminopyridine;

[0027] (2) R is a halogen, and the amidation reaction is carried out in the presence of an acid-binding agent, which includes triethylamine.

[0028] A third aspect of this application provides a lysine probe comprising one or more of the N-acyl heterocyclic compounds described in the first aspect.

[0029] A fourth aspect of this application provides the use of the N-acyl heterocyclic compounds described in the first aspect as lysine probes.

[0030] A fifth aspect of this application provides the application of the N-acyl heterocyclic compounds described in the first aspect in bioanalysis.

[0031] In one embodiment, the bioanalysis includes chemical proteomics analysis.

[0032] The aforementioned N-acyl heterocyclic compounds, through appropriate structural design, can be used as lysine probes, exhibiting higher stability and tunable reactivity, and possessing broad proteome coverage, thus expanding the range of lysine-targeting sites and facilitating the research and development of more druggable sites. Furthermore, the aforementioned N-acyl heterocyclic compounds have the following advantages:

[0033] (1) It has high nucleophilic specificity for lysine and tunable reactivity, enabling real-time and controllable manipulation of the modification process of lung cancer proteins. It can accurately distinguish target residues from dense protozoan backgrounds, significantly reducing non-specific off-target labeling and achieving highly selective and highly reactive covalent modification of lysine sites in lung cancer.

[0034] (2) It can more comprehensively reveal and identify new druggable sites in the lung cancer proteome, which is beneficial to the development of next-generation lung cancer lysine-targeted covalent drugs. Attached Figure Description

[0035] Figure 1 Gel imaging images of compounds BIT-1 to BIT-5 and the commercially available lysine reactive electrophile NHS.

[0036] Figure 2 A comparison of lysine modification selectivity between the BIT-1 probe and commercially available STP and NHS probes.

[0037] Figure 3 A heatmap for detecting the amino acid selectivity of BIT-1-derived modifications using an AZ-UV tagging workflow.

[0038] Figure 4 Scatter plot of the BIT-1 probe for the labelable sites of lysine.

[0039] Figure 5 The image shows the pChem analysis results of the BIT-1 probe. Detailed Implementation

[0040] The following detailed description, in conjunction with specific embodiments, illustrates the N-acyl heterocyclic compounds, their preparation methods, lysine probes, and applications of this application. This application can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a more thorough and complete understanding of the disclosure of this application.

[0041] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0042] In this article, "one or more" refers to any one, two or more of the listed items.

[0043] In this application, terms such as "first aspect," "second aspect," "third aspect," and "fourth aspect" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, terms such as "first," "second," "third," and "fourth" serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0044] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0045] In this application, numerical ranges are referred to as continuous unless otherwise specified, and include the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe a feature or characteristic, the ranges may be merged. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

[0046] Unless otherwise specified, the percentage content mentioned in this application refers to mass percentage for solid-liquid mixtures and solid-phase-solid mixtures, and volume percentage for liquid-phase-liquid mixtures.

[0047] Unless otherwise specified, all percentage concentrations mentioned in this application refer to the final concentration. The final concentration refers to the proportion of the added component in the system after the addition of that component.

[0048] Unless otherwise specified, the temperature parameters in this application may be either constant temperature processing or processing within a certain temperature range. The constant temperature processing allows for temperature fluctuations within the precision range controlled by the instrument.

[0049] In this application, room temperature generally refers to 4℃~30℃, and preferably 20±5℃.

[0050] In this application, the term "aromatic ring" refers to an aromatic hydrocarbon group derived from an aromatic ring compound by removing two hydrogen atoms (fused with adjacent rings). This can be a monocyclic aromatic ring, a fused-ring aromatic ring, or a polycyclic aromatic ring. For example, "C6-C10 aromatic ring" refers to an aromatic ring containing 6 to 10 carbon atoms, and each occurrence can be independently of a C6, C7, C8, C9, or C10 aromatic ring. Suitable examples include, but are not limited to, benzene, naphthalene, and their derivatives.

[0051] In this application, the term "carbonyl" refers to .

[0052] Some embodiments of this application provide an N-acyl heterocyclic compound having the structural features shown in formula (I):

[0053] (I),

[0054] Where n is an integer from 1 to 5;

[0055] X1 is a carbonyl group; X2 is -S-, -S(O)2- or a carbonyl group;

[0056] Ring A and X3 satisfy one of the following conditions:

[0057] (1) Ring A does not exist, X3 is -S- or -CH2-;

[0058] (2) Ring A is a C6~C10 aromatic ring, and X3 is C.

[0059] In some embodiments, the N-acyl heterocyclic compound has the structural features shown in formula (I-1) or formula (I-2):

[0060] Formula (I-1),

[0061] Formula (I-2).

[0062] Specifically, the value of n includes, but is not limited to: 1, 2, 3, 4, 5, or any two of the aforementioned values. Further, n can be 2 or 3.

[0063] Without limitation, the N-acyl heterocyclic compounds are selected from the following compounds:

[0064] .

[0065] Other embodiments of this application provide a method for preparing the N-acyl heterocyclic compound as described above, comprising the following steps:

[0066] Compound 1 and compound 2 were subjected to an amidation reaction;

[0067] The structure of compound 1 is as follows:

[0068] ;

[0069] The structure of compound 2 is as follows:

[0070] , where R is a hydroxyl group or a halogen.

[0071] Furthermore, when R is a hydroxyl group, the amidation reaction is carried out in the presence of a condensing agent and a nucleophilic catalyst, wherein the condensing agent includes a carbodiimide condensing agent and the nucleophilic catalyst includes N,N-dimethylaminopyridine. Without limitation, the carbodiimide condensing agent includes 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide salt.

[0072] Furthermore, when R is a halogen, the amidation reaction is carried out in the presence of an acid-binding agent, which includes triethylamine.

[0073] In other embodiments of this application, a lysine probe is provided, comprising one or more of the N-acyl heterocyclic compounds described above.

[0074] Other embodiments of this application provide the use of N-acyl heterocyclic compounds as described above as lysine probes.

[0075] Other embodiments of this application provide the application of the N-acyl heterocyclic compounds described above in bioanalysis. Further, the bioanalysis includes chemical proteomics analysis.

[0076] For experimental parameters not specified in the following specific embodiments, please refer to the guidelines given in this application document first, or refer to experimental manuals or other experimental methods known in the art, or refer to the experimental conditions recommended by the manufacturer.

[0077] The raw materials and reagents involved in the following specific embodiments can be obtained commercially or prepared by those skilled in the art using known methods.

[0078] Example 1

[0079] This example describes the preparation of compounds BIT-1 to BIT-5.

[0080]

[0081] Preparation of compound BIT-1:

[0082]

[0083] In a 100 mL single-necked round-bottom flask equipped with a magnetic stirrer, 1,2-benzisothiazolinone (BIT, 3.0 mmol, 1.0 equivalent), pentylene acid (3.6 mmol, 1.2 equivalent), and N,N-dimethylaminopyridine (DMAP, 0.15 mmol, 0.05 equivalent) were added. Dichloromethane (CH₂Cl₂, 3 mL, 1.0 M relative to BIT) was added to dissolve the reactants, and the reaction mixture was cooled in an ice bath. Then, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl, 3.6 mmol, 1.2 equivalent) was added. After the addition was complete, the reaction mixture was allowed to reach room temperature and stirred continuously for 10 h. The reaction progress was monitored by thin-layer chromatography (TLC). After the reaction was complete, the solvent was removed under reduced pressure. The resulting residue was milled with water, and the precipitated solid was collected by filtration. The obtained solid was washed successively with saturated sodium bicarbonate (NaHCO3) solution (10 mL × 3) and water (30 mL). The crude product was dissolved in dichloromethane, dried over anhydrous magnesium sulfate (MgSO4), filtered, and concentrated under reduced pressure to obtain compound BIT-1. High-performance liquid chromatography (HPLC) analysis showed a purity greater than 95%. 1 H NMR (600 MHz, DMSO) δ 7.95 (t, J = 8.1 Hz,2H), 7.80 (t, J = 7.7 Hz, 1H), 7.48 (t, J = 7.5 Hz, 1H), 3.31 (t, J = 7.3 Hz,2H), 2.85 (t, J = 2.6 Hz, 1H), 2.54 (td, J = 7.3, 2.6 Hz, 2H). 13 C NMR (151MHz, DMSO) δ 171.72 (s), 163.61 (s), 141.38 (s), 135.07 (s), 127.47 (s), 126.62 (s), 125.54 (s), 122.58 (s), 83.68 (s), 72.12 (s), 36.63 (s), 13.45(s). HRMS (ESI) calcd. for C 12 H9NO2S 232.0356 [M + H] +, found 232.0359.

[0084] Preparation of compound BIT-2:

[0085]

[0086] At 0°C, hexano-5-acetyl chloride (10.0 mmol) was added dropwise via syringe to a solution of saccharin (1.83 g, 10.0 mmol, 1.0 equivalent) and triethylamine (NEt3, 1.4 mL, 10.0 mmol, 1.0 equivalent) in dimethylacetamide (DMA, 15.0 mL). After stirring the mixture for 1 hour, it was slowly poured into water (100.0 mL) at 0°C. The resulting pale yellow precipitate was collected by filtration and washed with cold water (0-5°C, 200.0 mL). The resulting solid was dissolved in dichloromethane (DCM), further diluted with DCM, and washed twice with saturated brine (15.0 mL each time). The organic layer was dried over anhydrous sodium sulfate (Na2SO4) and then concentrated under vacuum to obtain the crude product. The crude product was washed with petroleum ether and ethyl acetate, dried, and the compound BIT-2 was obtained. The purity was greater than 95% as determined by high-performance liquid chromatography (HPLC). 1 H NMR (600 MHz, DMSO) δ 8.33 (d, J= 7.7 Hz, 1H), 8.20 (d, J = 7.6 Hz, 1H), 8.14 (t, J = 7.4 Hz, 1H), 8.06 (t, J= 7.5 Hz, 1H), 3.15 (t, J = 7.2 Hz, 2H), 2.85 (s, 1H), 2.29 (t, J = 6.5 Hz, 2H), 1.83 (dd, J = 14.2, 7.1 Hz, 2H). 13 C NMR (151 MHz, DMSO) δ 171.70 (s), 157.83 (s), 137.58 (s), 136.11 (s), 126.58 (s), 125.22 (s), 121.93 (s), 84.13(s), 72.40 (s), 36.71 (s), 22.54 (s), 17.44 (s). HRMS (ESI) calcd. forC 13 H 11 NO4S 278.0415 [M + H] +, found 278.0425.

[0087] Preparation of compound BIT-3:

[0088]

[0089] At 0 °C, hexyl-5-acetyl chloride (10.0 mmol) was added dropwise via syringe to a solution of 1H-pyrrole-2,5-dione (980 mg, 10.0 mmol, 1.0 equivalent) and triethylamine (NEt3, 1.4 mL, 10.0 mmol, 1.0 equivalent) in dimethylacetamide (DMA, 15.0 mL). After stirring the mixture for 1 hour, it was slowly poured into water (100.0 mL) at 0 °C. The precipitated pale yellow precipitate was collected by filtration and washed with cold water (0-5 °C, 200.0 mL). The resulting solid was dissolved in dichloromethane (DCM), further diluted with DCM, and washed twice with saturated brine (15.0 mL each time). The organic layer was dried over anhydrous sodium sulfate (Na2SO4) and then concentrated under vacuum to obtain the crude product. The crude product was washed with petroleum ether and ethyl acetate, dried, and the compound BIT-3 was obtained. High-performance liquid chromatography (HPLC) was used to detect a purity greater than 95%. 1 H NMR (600 MHz, CDCl3) δ3.02 (t, J = 7.2 Hz, 2H), 2.81 (s,4H), 2.30 (td, J = 6.9, 2.5 Hz, 2H), 2.00(t, J = 2.5 Hz, 1H), 1.91 (dd, J = 14.1, 7.1 Hz, 2H). 13 C NMR (151 MHz, CDCl3)δ 174.41 (s), 172.34 (s), 83.09 (s), 69.50 (s), 37.53 (s), 28.49 (s), 22.40(s), 17.53 (s). HRMS (ESI) calcd. for C 10 H 11 NO3 194.0743 [M + H] +, found194.0742.

[0090] Preparation of compound BIT-4:

[0091]

[0092] At 0°C, hexano-5-acetyl chloride (10.0 mmol) was added dropwise via syringe to a solution of 2,4-thiazolidinedione (1.15 mg, 10.0 mmol, 1.0 equivalent) and triethylamine (NEt3, 1.4 mL, 10.0 mmol, 1.0 equivalent) in dimethylacetamide (DMA, 15.0 mL). After stirring the mixture for 1 hour, it was slowly poured into water (100.0 mL) at 0°C. The resulting pale yellow precipitate was collected by filtration and washed with cold water (0-5°C, 200.0 mL). The resulting solid was dissolved in dichloromethane (DCM), further diluted with DCM, and washed twice with saturated brine (15.0 mL each time). The organic layer was dried over anhydrous sodium sulfate (Na2SO4) and then concentrated under vacuum to obtain the crude product. The crude product was washed with petroleum ether and ethyl acetate, dried, and the compound BIT-4 was obtained. The purity was greater than 95% by high-performance liquid chromatography (HPLC). 1 H NMR (600 MHz, CDCl3) δ4.03 (s, 2H), 3.05 (t, J = 7.2 Hz, 2H), 2.31 (td, J = 6.9, 2.6 Hz, 2H), 2.00(t, J = 2.6 Hz, 1H), 1.95 (p, J = 7.0 Hz, 2H). 13 C NMR (151 MHz, CDCl3) δ171.51 (s), 169.06 (s), 82.75 (s), 69.73 (s), 37.76 (s), 33.87 (s), 22.56(s), 17.51 ​​(s). HRMS (ESI) calcd. for C9H9NO3S 212.0301 [M + H] +, found212.0302.

[0093] Preparation of compound BIT-5 (comparative compound):

[0094]

[0095] At 0 °C, hexano-5-acetyl chloride (10.0 mmol) was added dropwise via syringe to a solution of benzisothiazoline-1,1-dioxide (1.68 mg, 10.0 mmol, 1.0 equivalent) and triethylamine (NEt3, 1.4 mL, 10.0 mmol, 1.0 equivalent) in dimethylacetamide (DMA, 15.0 mL). After stirring the mixture for 1 hour, it was slowly poured into water (100.0 mL) at 0 °C. The precipitate was collected by filtration and washed with cold water (0-5 °C, 200.0 mL). The resulting solid was dissolved in dichloromethane (DCM), further diluted with DCM, and washed twice with saturated brine (15.0 mL each time). The organic layer was dried over anhydrous sodium sulfate (Na2SO4) and then concentrated under vacuum to obtain the crude product. The crude product was washed with petroleum ether and ethyl acetate, dried, and the compound BIT-5 was obtained. High-performance liquid chromatography (HPLC) was used to detect a purity greater than 95%. 1 H NMR (600 MHz, CDCl3) δ 7.83 (d, J = 7.9 Hz, 1H), 7.73 – 7.66 (m, 1H), 7.60 (d, J = 7.8 Hz, 1H), 7.49 (d, J = 7.8 Hz, 1H), 4.93 (s, 1H), 3.09 (s, 1H), 2.36 (td, J = 7.0,2.6 Hz, 1H), 2.08 – 1.94 (m, 2H). 13 C NMR (151 MHz, CDCl3) δ 170.32 (s), 134.37 (s), 134.12 (s), 130.78 (s), 129.72 (s), 125.03 (s), 121.86 (s), 83.18(s), 69.41 (s), 47.40 (s), 33.98 (s), 23.05 (s), 17.82 (s). HRMS (ESI) calcd.for C 13 H 13 NO3S 264.0621 [M + H] +, found 264.0611.

[0096] Example 2

[0097] This example describes the biological testing of compounds BIT-1 to BIT-5.

[0098] (1) Cell labeling experiment using probe compounds BIT-1~5 (BIT-type probes)

[0099] A549 cells were seeded in 10 cm culture dishes and cultured under the above conditions until 80%–90% confluence. The culture medium was removed, and the cells were washed twice with PBS. After centrifugation at 1000 rpm for 3 min, the supernatant was discarded, and 1 mL of lysis buffer (50 mM HEPES, pH 7.6, 150 mM NaCl, 1% IGEPAL) was added. The suspended cells were sonicated on ice for lysis (5 s on, 3 s off, 12% amplitude, for a total of 3 min). After centrifugation at 14,000 rpm for 15 min at 4 °C, the supernatant containing soluble proteins was collected.

[0100] The obtained proteome was divided into several equal portions, and each portion was incubated with a BIT probe (final concentration 100 μm) at 37 °C for 1 h. Then, TAMRA-N3, TBTA, sodium ascorbate, and CuSO4 (final concentrations 100 μM, 100 μM, 1 mM, and 1 mM, respectively) were added to the mixture, and a click chemistry reaction was initiated by gentle shaking. Subsequently, pre-cooled methanol was added to precipitate the protein. The precipitate was dissolved in 1×SDS loading buffer and heated at 95 °C for 10 min. Samples containing 50 μg of protein from each lane were separated by 12% SDS-PAGE gel electrophoresis, and finally, imaging analysis was performed by in-gel fluorescence scanning.

[0101] Test results are as follows Figure 1 As shown in the figure, in gel imaging, after treating protocellular lysates with the same concentration of probes for 1 hour, the lanes of probes BIT-1, BIT-2, BIT-3, and BIT-4 all showed extremely strong and widely distributed green fluorescent bands, while the lanes of the contrast compound BIT-5 and the commercially available lysine-reactive electrophile NHS showed almost no fluorescence signal (the band brightness was indistinguishable from the background). It is evident that in the complex protozoan environment, the BIT-based probes provided in this application exhibit far superior nucleophilic reactivity and labeling efficiency compared to traditional NHS-activated esters, especially the BIT-1 probe. NHS esters are prone to premature hydrolysis in aqueous solutions, making effective proteome labeling within 1 hour impossible; however, the BIT-based probes provided in this application successfully overcome this hydrolysis defect, achieving highly efficient covalent modification.

[0102] (2) BIT-1 quantitative proteomics experiment

[0103] A549 cells were seeded in 10 cm culture dishes and cultured to 80%–90% confluence. The culture medium was removed, and the cells were washed twice with PBS. The sample was centrifuged at 1000 rpm for 3 min to remove the supernatant, followed by the addition of 1 mL of lysis buffer (50 mM HEPES, pH 7.6, 150 mM NaCl, 1% IGEPA). The suspended cells were sonicated on ice (5 s on, 3 s off, 12% amplitude, for a total of 3 min). The cells were then centrifuged at 14,000 rpm for 15 min at 4 °C, and the supernatant containing soluble proteins was collected. The BIT-1 probe (1 mM) was incubated with the A549 cell protein lysis sample at 37 °C for 1 h. In short, the probe-labeled protein was reduced with 10 mM DTT at room temperature for 30 min, followed by alkylation with 40 mM iodoacetamide at room temperature in the dark for 1 h. Next, the protein was precipitated using a methanol-chloroform system (water / methanol / chloroform, 4:4:1, v / v / v), and digested overnight at 37°C using sequencing-grade trypsin (Promega) at an enzyme-to-substrate mass ratio of 1:50 (w / w). The resulting trypsin digest was desalted using an HLB solid-phase extraction column (Waters) and vacuum dried. A copper-catalyzed azido-acetylene cycloaddition reaction (CuAAC) was carried out in a solution containing 30% acetonitrile (ACN, pH approximately 6), with the following reagents added sequentially to the indicated final concentrations: 1 mM UV-cleavable azide-biotin (purchased from 40 mM stock solution), 10 mM sodium ascorbate (purchased from 100 mM stock solution), 1 mM TBTA (purchased from 50 mM stock solution), and 10 mM CuSO4 (purchased from 100 mM stock solution). The sample was rotated at room temperature in the dark for 2 h. Subsequently, the samples were enriched with streptavidin magnetic beads at room temperature for 2 h. The streptavidin magnetic beads were washed twice each with 20 mM sodium acetate (pH 4.5), 50 mM sodium acetate containing 2 M NaCl (pH 4.5), and deionized water to remove nonspecific conjugates. The magnetic beads were resuspended in 25 mM ammonium bicarbonate, transferred to a glass tube (VWR), and irradiated with 365 nm ultraviolet light (UVP, model UVL-28 EL) for 2 h under room temperature and continuous magnetic stirring to cleave the biotinylated peptides. The supernatant was collected, concentrated under vacuum, desalted using an HLB column, and stored at -20 °C for LC-MS / MS analysis.

[0104] A549 cell lysate samples labeled with the BIT-1 probe (1 mM) were enriched. Biotin reagent was introduced for photolysis, and the digested peptide samples were directly injected into a high-resolution liquid chromatography-tandem mass spectrometry (LC-MS / MS) system for DDA mode data acquisition. The raw mass spectrometry data were directly imported into pChem, which can generate all possible mass shifts within a specified range (default ±1000 Da) at the PSM level. Then, all mass shifts were calibrated using calculated systematic errors, and modification candidates were uniformly generated. Specifically, the raw mass spectrometry data were imported into the pChem search software, enabling a "modification-centric" blind search strategy. Without pre-inputting expected modification parameters, it automatically identifies probe-derived modifications (PDMs), locates specific modified amino acid residues, and quantitatively assesses residue selectivity at the whole proteome level.

[0105] Test results are as follows Figures 2-4 As shown. Wherein:

[0106] Figure 2 This is a comparison of the lysine modification selectivity of the BIT-1 probe with commercially available STP and NHS probes. Figure 2 It is known that 95% of the BIT-1-derived modification sites are lysine residues, while the corresponding proportions for commercially available lysine-reactive STP and NHS probes are 78% and 81%, respectively. Therefore, the BIT-based probes provided in this application exhibit significantly higher selectivity for lysine in A549 cell lysates than commercially available STP and NHS probes, demonstrating that BIT-based probes can be used as specific lysine probes in vivo.

[0107] Figure 3 A heatmap was created to detect the amino acid selectivity of BIT-1-derived modifications using an AZ-UV tagging workflow. Figure 3 As can be seen, a deep black band (close to the highest value of 1.0) appears across the horizontal axis in the row for K (lysine), while the gray levels of all other amino acid rows are very light, indicating selectivity close to 0. This demonstrates the high specificity of the BIT probes provided in this application for lysine in A549 cell lysates.

[0108] Figure 4 Scatter plot of the BIT-1 probe for labelable sites of lysine. Figure 4 It is known that the BIT-1 probe can specifically covalently modify 5253 lysine sites in cell lysate. Therefore, the BIT-type probe provided in this application exhibits high reactivity in A549 lung cancer cell lysate and can bind a large number of druggable and non-druggable targets, providing an ideal chemical tool for subsequent research on related targets.

[0109] (3) Evaluation experiment of BIT-1 proteomics based on chemical proteomics mass spectrometry (pChem) search engine

[0110] MS data is directly imported into pChem, which can generate all possible quality offsets within a specified range (default ±1000 Da) at the PSM level. Then, all quality offsets are calibrated using the calculated systematic error, and modification candidates are generated uniformly. Based on light (… 12 The theoretical isotopic mass difference (6.02013233 Da) between C and (C) was used to automatically pair candidate modifications with heavy (C) 13 C6) PDMs. In addition to the other filtering criteria described for pIon, non-PDMs and impractical modifications were further filtered by discarding modification pairs with measured quality differences outside the range of 356.019132 to 6.021132 Da (corresponding to a tolerance of ±166 ppm near the theoretical 6.020132 Da). Specifically, BIT-1 probe-labeled A549 lung cancer cell lysates were enzymatically digested, coupled with isotope-encoded UV-cleavable biotin tags, enriched, and then analyzed by LC-MS / MS.

[0111] pChem analysis results are as follows: Figure 5 As shown, 95% of the BIT-1 probe-derived modification sites were lysine residues. We then performed quantitative LC-MS / MS analysis to map the distribution of BIT-1-labeled lysine residues in the A549 lung cancer proteome, identifying a total of 5253 BIT-1-labeled lysine residue sites in 2024 proteins. This demonstrates that the BIT-based probes provided in this application can specifically identify 5253 BIT-1-labeled lysine residue sites in A549 cells, and that a large number of druggable and non-druggable targets exist. Compared with previously commercialized STP probes, the BIT-1 probes newly identified 4218 specific lysine residue sites, proving the high specificity of the BIT-based probes provided in this application.

[0112] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0113] The embodiments described above are merely illustrative of several implementation methods of this application, intended to facilitate a detailed understanding of the technical solutions of this application, but should not be construed as limiting the scope of protection of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the scope of protection of this application. It should be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided in this application through logical analysis, reasoning, or limited experimentation are all within the scope of protection of the appended claims. Therefore, the scope of protection of this patent application should be determined by the content of the appended claims, and the specification can be used to interpret the content of the claims.

Claims

1. An N-acyl heterocyclic compound, characterized in that, It has the structural features shown in equation (I): （I）， Where n is an integer from 1 to 5; X1 is a carbonyl group; X2 is -S-, -S(O)2- or a carbonyl group; Ring A and X3 satisfy one of the following conditions: (1) Ring A does not exist, X3 is -S- or -CH2-; (2) Ring A is a C6~C10 aromatic ring, and X3 is C.

2. The N-acyl heterocyclic compound according to claim 1, characterized in that, It has the structural features shown in equation (I-1) or equation (I-2) as follows: Equation (I-1), Equation (I-2).

3. The N-acyl heterocyclic compound according to claim 1 or 2, characterized in that, n is 2 or 3.

4. The N-acyl heterocyclic compound according to claim 1 or 2, characterized in that, Selected from the following compounds: 。 5. The method for preparing the N-acyl heterocyclic compound according to any one of claims 1 to 4, characterized in that, Includes the following steps: Compound 1 and compound 2 were subjected to an amidation reaction; The structure of compound 1 is as follows: ； The structure of compound 2 is as follows: , where R is a hydroxyl group or a halogen.

6. The method for preparing the N-acyl heterocyclic compound according to claim 5, characterized in that, The amidation reaction has one of the following characteristics: (1) R is a hydroxyl group, and the amidation reaction is carried out in the presence of a condensing agent and a nucleophilic catalyst, wherein the condensing agent includes a carbodiimide condensing agent and the nucleophilic catalyst includes N,N-dimethylaminopyridine; (2) R is a halogen, and the amidation reaction is carried out in the presence of an acid-binding agent, which includes triethylamine.

7. A lysine probe, characterized in that, It includes one or more of the N-acyl heterocyclic compounds described in any one of claims 1 to 4.

8. The use of the N-acyl heterocyclic compound according to any one of claims 1 to 4 as a lysine probe.

9. The use of the N-acyl heterocyclic compound according to any one of claims 1 to 4 in bioanalysis.

10. The application according to claim 9, characterized in that, The bioanalysis includes chemical proteomics analysis.

Images