An alkynyl-modified nad + Analogues, methods of preparation, uses thereof

Abstract

The application discloses an alkyne-modified NAD + Analog and a preparation method and application thereof, and belongs to the technical field of medicines. The nicotinamide adenine dinucleotide is modified by an alkyne group, and a terminal alkyne group is introduced as a biological orthogonal reaction functional group, so that a functional NAD + Analog for analyzing ADP-ribosylation regulation processes and PAR-dependent biological processes, and for analyzing the pathogenesis of PAR-related diseases such as amyotrophic lateral sclerosis and Parkinson's disease, and further discovering new treatment targets.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, and particularly relates to an alkyne-modified NAD. + Analogs, their preparation methods, and applications. Background Technology

[0002] The PAR signaling pathway includes PARylation, specifically the poly-ADP ribosome signaling pathway, which uses NAD+. + Using these as raw materials, post-translational modifications of proteins covalently modified with ADP-ribose chains (PAR) are performed. ADP-ribosylation is a key protein post-translational modification (PTM) catalyzed by enzymes of the poly-ADP-ribose polymerase (PARPs) family. Poly-ADP-riboglycosidase (PARG) can degrade PAR chains through both endo- and exo-glycosidase activities.

[0003] Intracellular PAR chains consist of 2 to 200 ADP-ribose units and can serve as a multivalent interaction platform. Through non-covalent interactions, they can specifically recognize proteins and other macromolecules, thereby initiating downstream signaling cascade reactions and regulating core life processes such as DNA damage repair, chromatin remodeling, transcriptional regulation, apoptosis, and cell division.

[0004] It is noteworthy that PAR signaling pathway dysregulation is closely associated with a variety of human diseases. Abnormally elevated PAR levels have been detected in the motor neurons of patients with amyotrophic lateral sclerosis (ALS) and in the cerebrospinal fluid of patients with Parkinson's disease. Increasing research confirms that PAR can induce liquid-liquid phase separation (LLPS) in vitro and in vivo with key neuropathological proteins such as TDP-43, hnRNPA1, FUS, and α-synuclein, suggesting a direct regulatory role in the pathogenesis of neurodegenerative diseases. Furthermore, using advanced chemistry and proteomics technologies, a large number of PAR-binding "reader" proteins have been identified, implying the existence of a broad and incompletely understood PAR-dependent biological regulatory network within cells. Given the extensive physiological and pathological significance of the PAR signaling pathway, systematically elucidating the biological processes of PAR regulation will not only help elucidate the fundamental molecular mechanisms of cellular regulation but also uncover novel therapeutic targets for PAR-related diseases.

[0005] Mediating NAD + The molecular mechanism of ADP-ribonucleoside covalent transfer to protein substrates has driven the development of various NAD+ pathways. + The development of analogues aims to explore the biological functions and mechanisms of ADP-ribosylation. Currently, various NAD⁺ analogues have been developed by modifying the adenine or ribose sites of natural NAD⁺ with alkynyl, azide, biotin, and fluorescent groups. +Derivatives can be used to label and identify ADP-ribosylated substrates, visualize intracellular ADP-ribosylation modification processes, and track the dynamic turnover of PAR chains at DNA damage sites. Simultaneously, chemically or enzymatically synthesized NAD+ with azide / alkynyl groups can be used. + Analogs, after generating PAR chains through intracellular or extracellular reactions, can be coupled with biotin or photocrosslinking groups, serving as powerful research tools for identifying PAR-binding proteins.

[0006] Although chemical probes and synthetic PAR tools have helped map ADP-ribosylated proteins and their interacting proteins, the PAR-dependent biological processes mediated by PAR readers and PAR interactions still lack systematic research compared to the extensive involvement of PAR hydration in physiological and pathological processes. To fill this research gap, it is urgent to develop new tools and technologies to deeply elucidate PAR-dependent biological processes and subsequently identify targets for PAR-related diseases such as amyotrophic lateral sclerosis (ALS) and Parkinson's disease. Summary of the Invention

[0007] The purpose of this invention is to provide a structurally stable functional NAD. + Analogs are used to study the regulation of ADP-ribosylation and PAR-dependent biological processes, and to analyze the pathogenesis of PAR-related diseases such as amyotrophic lateral sclerosis and Parkinson's disease, thereby discovering new therapeutic targets.

[0008] Specifically, firstly, this invention provides a functional NAD for resolving PAR dependency processes. + The analogue is an alkyne-modified nicotinamide adenine dinucleotide, wherein a terminal alkyne group is introduced at a specific site as a bioorthogonal functional group. The alkyne group is specifically modified at the 4th position of the nicotinamide ribose, and its chemical formula is: .

[0009] This invention also provides the above-mentioned alkynyl-modified NAD + The preparation methods for analogues specifically include the following preparation routes: .

[0010] Furthermore, the intermediate 12 synthesizes NAD. + The reaction buffer for the analogue contains dithiothreitol and adenine nucleoside triphosphate, and the reaction terminator is trichloroacetic acid; the reaction solvent for the synthesis of intermediate 12 from intermediate 11 is trimethyl phosphate, and the quencher is water; the reaction temperature for the synthesis of intermediate 11 from intermediate 10 is -20℃ to 30℃, and the catalyst for the reaction is trimethylsilyl trifluoromethanesulfonate.

[0011] Furthermore, the reaction temperature for synthesizing intermediate 7 from intermediate 6 is 0 ℃-30 ℃, the reaction solvent is methanol, and the reaction reagent is dimethyl (1-diazo-2-oxopropyl)phosphonate.

[0012] Furthermore, the reaction temperature for intermediate 5 to synthesize intermediate 6 is 0 ℃-30 ℃, the oxidant is Dys-Martin oxidant, and the quencher is Na2S2O3; the reaction temperature for intermediate 7 to synthesize intermediate 8 is -78 ℃-30 ℃, the reaction solvent is dichloromethane, and the quencher is pyridine and methanol; the reaction system for intermediate 8 to synthesize intermediate 9 includes tetrabutylammonium fluoride, pyridine, and acetic anhydride, the reaction solvent is tetrahydrofuran, and the quencher is methanol; the reaction system for intermediate 9 to synthesize intermediate 10 includes acetic anhydride and acetic acid, and the quencher is methanol.

[0013] Further, the solvent for synthesizing intermediate 2 from intermediate 1 is an acetic acid solution, and the quencher is a saturated NaHCO3 solution; the solvent for synthesizing intermediate 3 from intermediate 2 is a mixed solution of dichloromethane and water, and the quencher is ethanol; the solvent for synthesizing intermediate 4 from intermediate 3 is a mixed solution composed of 1,4-dioxane, 37% formaldehyde solution, NaOH solution, and water; the reaction temperature for synthesizing intermediate 5 from intermediate 4 is -78 ℃ to 30 ℃, the solvent is tetrahydrofuran, and the quencher is a saturated NH4Cl solution.

[0014] The present invention also provides the use of the above-mentioned alkyne-modified NAD⁺ analogues in the preparation of reagents or kits for studying ADP-ribosylation and PAR-dependent processes.

[0015] The present invention also provides a method for screening candidate compounds in vitro for non-therapeutic purposes, comprising the following steps: (a) The alkynyl-modified NAD as described in claim 1 + The analogue, PARP enzyme, substrate protein, and test compound are contacted in an in vitro system; (b) Determine whether the test compound has PARP inhibitory activity by detecting the ADP-ribosylation level on the substrate protein; The ADP-ribosylation level is quantitatively detected by coupling the alkyne group with an azide-labeled detection probe via a click chemistry reaction. Beneficial effects

[0016] This invention is the first to achieve site-directed alkyne modification at the 4-position of nicotinamide ribose while completely preserving NAD. + The molecule originally contained all hydroxyl groups, making this analogue similar to natural NAD. + The spatial structures are highly homologous. Furthermore, the terminal alkyne groups have minimal steric hindrance and will not interfere with NAD under physiological conditions. +The process of recognizing and binding with substrate proteins preserves natural NAD to the greatest extent. + Its ability to bind to proteins and its biological activity allow it to precisely replace natural NAD. + Conduct various biological studies related to PAR.

[0017] Specifically, it has the following technical advantages and beneficial effects: 1. Structural advantages: Site-directed 4-position alkyne modification preserves natural NAD. + The structure is entirely composed of hydroxyl groups, exhibiting a highly biomimetic spatial structure with no significant steric hindrance, thus not affecting the natural interaction between the molecule and the protein.

[0018] 2. Technical versatility: It can simultaneously realize five major functions: enzyme activity detection, affinity comparison, interaction protein identification, cell process tracing, and disease target screening, and is suitable for in vitro molecular experiments and cellular mechanism research.

[0019] 3. High specificity and sensitivity: Based on the specific reaction of bioorthogonal click chemistry, the non-specific binding is low. Combined with mass spectrometry and MST quantitative technology, the detection and screening results are accurate and reliable.

[0020] 4. Wide range of applications: It can be used to analyze the regulatory mechanism of ADP-ribosylation, and at the same time provide a new technical platform for target discovery and drug development in neurodegenerative diseases. Attached Figure Description

[0021] Figure 1 The compound’s substrate activity on PARP enzymes and the interaction between the generated PAR chain and the substrate protein.

[0022] Figure 2 A comparison of the binding affinity of PAR chains generated from compounds to their substrate proteins with that of natural PAR chains.

[0023] Figure 3 Part II. Comparison of the binding affinity of PAR chains generated from compounds to their substrate proteins with that of natural PAR chains.

[0024] Figure 4 Immunoblotting results of PARylation activity in cells under stress conditions.

[0025] Figure 5 Confocal imaging results of PARylation activity in cells under stress conditions.

[0026] Figure 6 The intensity changes of K48 ubiquitinated protein and PAR, and the co-localization of FUS protein and PAR.

[0027] Figure 7 Workflow for using compounds to identify PARdU protein.

[0028] Figure 8 Results of compound screening and identification of PARdU candidate proteins. Detailed Implementation

[0029] To facilitate understanding of the present invention, a more complete description will be given below with reference to the accompanying drawings. The drawings illustrate preferred embodiments of the invention. However, the invention 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 the invention. The invention will be further described below with reference to the accompanying drawings and specific embodiments. Unless otherwise specified, the materials, reagents, etc., used in the following embodiments are commercially available.

[0030] The general methods for synthesizing compounds are as follows:

[0031] Example 1: 1-[(2R,3R,4S,5R)-5-({({[(2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl]methoxy}(hydroxy)phosphoryl)oxyphosphoryl}oxy)methyl]-5-ethynyl-3,4-dihydroxytetrahydrofuran-2-yl-3-carbamoylpyridinium (compound NR-4′-a-NAD) + Preparation of ).

[0032]

[0033] (1) Preparation of (3aR,6R,6aR)-6-benzyloxy-5-((R)-2,2-dimethyl-1,3-dioxolane-4-yl)-2,2-dimethyltetrahydrofurano[2,3-d][1,3]dioxane (intermediate 1).

[0034] (3aR,6R,6aR)-6-benzyloxy-5-((R)-2,2-dimethyl-1,3-dioxolane-4-yl)-2,2-dimethyltetrahydrofurano[2,3-d][1,3]dioxane (10 g, 38.42 mmol), BrBn (9.12 mL, 76.84 mmol, 2.5 eq), and NaH (3.2 g, 80.68 mmol, 2.1 eq, 60% mineral oil dispersion) were dissolved in anhydrous THF (40 mL) and reacted at room temperature for 2 hours. The reaction was quenched with saturated NaHCO3 solution (20 mL), extracted with EA and water, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography to give a colorless solid, intermediate 1 (12.1 g, 90% yield).

[0035] 1H NMR (400 MHz, CDCl3): d 7.43-7.28 (m, 5H, ArH), 5.75 (d, 1H, J =3.6 Hz, CH), 4.77 (d, 1H, J = 11.6 Hz, CH2), 4.60-4.56 (m, 2H, CH2+CH ), 4.37(td, 1H, J = 7.2, 3.2 Hz, CH), 4.14 (dd, 1H, J = 8.8, 3.2 Hz, CH), 4.04-3.94(m, 2H, CH2), 3.88 (dd, 1H, J = 8.8, 4.4 Hz, CH), 1.59 (s, 3H, CH3), 1.38 (s,3H, CH3), 1.36 (s, 3H, CH3), 1.35 (s, 3H, CH3). 13C NMR (151 MHz, CDCl3): d137.6, 128.5, 128.3, 128.1, 113.0, 109.7, 104.0, 78.2, 77.9, 77.6, 74.9,72.3, 65.1, 26.9, 26.7, 26.3, 25.2.

[0036] (2) Preparation of (3aR,6R,6aR)-6-benzyloxy-5-((R)-2,2-dimethyl-1,3-dioxolane-4-yl)-2,2-dimethyltetrahydrofurano[2,3-d][1,3]dioxane (intermediate 2).

[0037] Intermediate 1 (12 g, 34.6 mmol) was dissolved in 35 mL of 85% acetic acid solution and reacted at 42 °C for 24 h. The reaction was quenched with 20 mL of saturated NaHCO3 solution, extracted with EA and water, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography to give intermediate 2 (8.58 g, 80% yield) as a colorless oil.

[0038] 1 H NMR (300 MHz, CDCl3): d 7.40-7.27 (m, 5H, ArH), 5.73 (d, 1H, J =3.6 Hz, CH), 4.74 (d, 1H, J = 11.4 Hz, CH2), 4.58-4.51 (m, 2H, CH+CH2), 4.07(dd, 1H, J= 9.0, 3.3 Hz, CH2), 4.00-3.90 (m, 2H, CH2+CH), 3.65-3.63 (m, 2H,2CH), 1.57 (s, 3H, CH3), 1.34 (s, 3H, CH3). 13 C NMR (151 MHz, CDCl3): δ 137.0,128.6, 128.30, 128.27, 113.2, 104.2, 79.1, 77.4, 77.0, 72.2, 71.1, 63.1,26.8, 26.6.

[0039] (3) Preparation of (3aR,5S,6R,6aR)-6-benzyloxy-2,2-dimethyltetrahydrofurano[2,3-d][1,3]dioxane-5-carboxaldehyde (intermediate 3).

[0040] Intermediate 2 (8 g, 25.8 mmol) and NaIO4 (6.62 g, 31 mmol, 1.2 eq) were dissolved in a mixed solution of DCM (15 mL) and water (25 mL). After reacting at 30 °C for 1.5 hours, EtOH (15 mL) was added to quench the reaction. The mixture was filtered to remove salts, extracted with DCM and water, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography to obtain a colorless oily substance, intermediate 3 (6.3 g, 88% yield).

[0041] 1 H NMR (400 MHz, CDCl3): d 9.61 (d, 1H, J = 2.0 Hz, CHO), 7.37-7.35(m, 5H, ArH), 5.81 (d, 1H, J = 3.2 Hz, CH), 4.75 (d, 1H, J = 12.0 Hz, CH2), 4.63 (d, 1H, J = 12.0 Hz, CH2), 4.60-4.58 (m, 1H, CH), 4.48 (dd, 1H, J = 9.6,2.0 Hz, CH2), 3.85 (dd, 1H, J = 9.6, 4.0 Hz, CH2), 1.60 (s, 3H, CH3), 1.37 (s, 3H, CH3). 13C NMR (151 MHz, CDCl3): d 198.5, 136.9, 128.7, 128.3, 128.2, 114.0, 104.8, 82.4, 78.1, 77.9, 72.5, 27.0, 26.7.

[0042] (4) Preparation of ((3aR,6S,6aR)-6-benzyloxy-2,2-dimethyltetrahydrofurano[2,3-d][1,3]dioxane-5,5-diyl)diethanol (intermediate 4).

[0043] Intermediate 3 (7.7 g, 27.7 mmol) was dissolved in a mixture of 1,4-dioxane (19 mL), 37% formaldehyde solution (15 mL), 1N NaOH solution (50 mL), and water (50 mL), and reacted at room temperature for 3 days. Extraction was performed with EA and water, followed by drying to anhydrous sodium sulfate, filtration, and concentration under reduced pressure. The solution was then subjected to silica gel column chromatography to give a white solid, intermediate 4 (8.58 g, 80% yield).

[0044] 1 H NMR (400 MHz, CDCl3): d 7.38-7.33 (m, 5H, ArH), 5.76 (d, 1H, J =4.0 Hz, CH), 4.80 (d, 1H, J = 11.6 Hz, CH2), 4.64 (dd, 1H, J = 5.2, 4.0 Hz,CH), 4.56 (d, 1H, J = 11.6 Hz, CH2), 4.21 (d, 1H, J = 5.2 Hz, CH), 3.96-3.84(m, 2H, CH2+CH2), 3.78 (d, 1H, J = 12.0 Hz, CH2), 3.54 (d, 1H, J = 9.6 Hz, 1H,CH2), 1.63 (s, 3H, CH3), 1.33 (s, 3H, CH3). 13 C NMR (151 MHz, CDCl3): d 137.4,128.7, 128.3, 127.9, 113.6, 104.5, 86.5, 78.54, 78.46, 72.8, 64.3, 63.3,26.7, 26.0.

[0045] (5) Preparation of ((3aR,5R,6S,6aR)-6-benzyloxy-5-((tert-butyldiphenylsiloxy)methyl)-2,2-dimethyltetrahydrofurano[2,3-d][1,3]dioxacyclopenten-5-yl)methanol (intermediate 5).

[0046] At -78 °C, t-BuLi (7 mL, 17.6 mmol, 1.05 eq) was added to a THF solution of intermediate 4 (5.21 g, 16.8 mmol). The reaction was carried out at -78 °C for 30 min, then increased to room temperature and reacted for 1.5 h. The reaction was quenched with saturated NH4Cl solution, extracted with EA and water, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography to give a colorless oil, intermediate 5 (3 g, 33% yield).

[0047] 1 H NMR (300 MHz, CDCl3): d 7.67-7.60 (m, 4H, ArH), 7.46-7.32 (m, 11H, ArH), 5.84 (d, 1H, J = 3.9 Hz, CH), 4.84 (d, 1H, J = 11.7 Hz, CH2), 4.71 (dd, 1H, J = 5.4, 3.9 Hz, CH), 4.53 (d, 1H, J = 11.7 Hz, CH2), 4.46 (d, 1H, J =5.4 Hz, CH), 3.89 (d, 1H, J = 12.0 Hz, CH2), 3.82 (d, 1H, J = 12.0 Hz, CH2), 3.78 (d, 1H, J = 10.8 Hz, CH2), 3.70 (d, 1H, J = 10.8 Hz, CH2), 1.67 (s, 3H,CH3), 1.39 (s, 3H, CH3), 1.01 (s, 9H, 3CH3). 13C NMR (151 MHz, CDCl3): d 137.5,135.74, 135.69, 133.4, 133.2, 129.9, 129.8, 128.7, 128.2, 127.91, 127.86,127.82, 113.9, 104.7, 87.6, 79.3, 78.2, 72.7, 65.6, 63.3, 27.02, 26.97, 26.4,19.4.

[0048] (6) Preparation of (3aR,5R,6S,6aR)-6-benzyloxy-5-((tert-butyldiphenylsiloxy)methyl)-2,2-dimethyltetrahydrofurano[2,3-d][1,3]dioxane-5-carboxaldehyde (intermediate 6).

[0049] At 0 °C, Dysmart oxidant (5.4 g, 12.8 mmol, 2.0 eq) was added to a DCM (15 mL) solution of intermediate 5 (3.5 g, 6.38 mmol). The reaction solution was slowly heated to room temperature and reacted for 2 hours. The reaction was quenched with saturated Na2S2O3 (10 mL), filtered through diatomaceous earth, extracted with DCM and water, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography to give a colorless solid, intermediate 6 (2.5 g, 73% yield).

[0050] 1 H NMR (300 MHz, CDCl3): d 9.92 (s, 1H, OCH), 7.64-7.59 (m, 4H, ArH), 7.44-7.33 (m, 11H, ArH), 5.89 (d, 1H, J = 3.3 Hz, CH), 4.76 (d, 1H, J = 12.0Hz, CH2), 4.69-4.66 (m, 1H, CH), 4.64 (d, 1H, J = 12.0 Hz, CH2), 4.56 (d, 1H, J = 4.5 Hz, CH), 3.90 (d, 1H, J = 11.4 Hz, CH2), 3.81 (d, 1H, J = 11.4 Hz, CH2), 1.64 (s, 3H, CH3), 1.38 (s, 3H, CH3), 1.00 (s, 9H, 3CH3). 13C NMR (151MHz, CDCl3): d 200.3, 137.1, 135.7, 135.6, 133.0, 132.7, 130.0, 129.9, 128.6,128.2, 128.0, 127.92, 127.89, 114.3, 105.1, 90.8, 79.3, 78.7, 72.9, 63.2,26.9, 26.8, 26.3, 19.3. HRMS (ESI) for C 32 H 38 NaO6Si (M+Na) + : Calcd.: 569.2330Da; Obs.: 569.2349 Da.

[0051] (7) Preparation of (((3aR,5R,6S,6aR)-6-benzyloxy-5-ethynyl-2,2-dimethyltetrahydrofurano[2,3-d][1,3]dioxane-5-yl)methoxy)(tert-butyl)diphenylsilane (intermediate 7).

[0052] At 0 °C, dimethyl 1-diazo-2-oxopropyl)phosphonate (0.7 mL, 4.65 mmol, 1.5 eq) was slowly added dropwise to a MeOH (15 mL) solution of intermediate 6 (1.7 g, 3.1 mmol) and CsCO3 (3 g, 15.6 mmol, 5.0 eq). The reaction mixture was slowly brought to room temperature and reacted for 2 hours. The mixture was concentrated under reduced pressure, slurried with EA, and the combined organic phases were extracted with water. After drying with anhydrous sodium sulfate and filtration, the mixture was concentrated under reduced pressure and subjected to silica gel column chromatography to give a gray oily substance, intermediate 7 (1.1 g, 65% yield).

[0053] 1 H NMR (600 MHz, CDCl3): d 7.63-7.60 (m, 4H, ArH), 7.46-7.28 (m, 11H, ArH), 5.74 (d, 1H, J = 3.6 Hz, CH), 4.86 (d, 1H, J = 12.6 Hz, CH2), 4.77 (d, 1H, J = 12.6 Hz, CH2), 4.63 (t, 1H, J = 4.2 Hz, CH), 4.39 (d, 1H, J = 4.2 Hz, CH), 3.93 (d, 1H, J= 11.4 Hz, CH2), 3.76 (d, H, J = 11.4 Hz, CH2), 2.60 (s,1H, CH), 1.75 (s, 3H, CH3), 1.37 (s, 3H, CH3), 0.96 (s, 9H, 3CH3). 13 C NMR (151MHz, CDCl3): d 137.6, 135.8, 135.6, 133.3, 132.7, 129.9, 129.8, 128.7, 128.2128.1, 127.9, 127.8, 114.1, 104.2, 81.2, 81.0, 78.7, 76.7, 76.2, 72.7, 68.1,66.1, 26.9, 26.3, 25.9, 19.4. HRMS (ESI) for C 33 H 38 NaO5Si (M+Na) + : Calcd.:565.2381 Da; Obs: 565.2349 Da.

[0054] (8) Preparation of (3R,4S,5R)-5-((tert-butyldiphenylsiloxy)methyl)-5-ethynyl-4-hydroxy-2-methoxytetrahydrofuran-3-ylacetate (intermediate 8).

[0055] At -78 °C, BCl3 (23 mL, 23 mmol, 10.0 eq) was added to a DCM solution of intermediate 7 (1.25 g, 2.3 mmol), and the reaction was allowed to proceed for 3 hours. The reaction was quenched with pyridine (10 mL) and methanol (5 mL), and the mixture was directly concentrated under reduced pressure. Pyridine (5 mL) and acetic anhydride (10 mL) were then added, and the mixture was allowed to react overnight at room temperature. The mixture was extracted with EA and CuSO4 solutions, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography to give a colorless oil, intermediate 8 (1 g, 50% yield).

[0056] 1 H NMR (300 MHz, CDCl3): d 7.74-7.70 (m, 4H, ArH), 7.45-7.37 (m, 6H, ArH), 5.73 (d, 1H, J = 4.8 Hz, CH), 5.30 (dd, 1H, J = 4.8, 2.1 Hz, CH), 5.09(d, 1H, J= 2.1 Hz, CH), 3.84 (d, 1H, J = 10.5 Hz, CH2), 3.75 (d, 1H, J =10.5 Hz, CH2), 3.39 (s, 3H, OCH3), 2.55 (s, 1H, CH), 2.12 (s, 3H, CH3), 2.10 (s, 3H, CH3), 1.07 (s, 9H, 3CH3). 13 C NMR (151 MHz, CDCl3): d 169.8, 169.5,135.9, 135.8, 133.0, 132.9, 129.94, 129.85, 127.9, 127.8, 106.1, 81.5, 80.3,75.9, 75.1, 71.5, 67.8, 56.1, 26.8, 20.8, 20.7, 19.4. HRMS (ESI) forC 26 H 32 NaO6Si (M+Na) + : Calcd.: 533.1966 Da; Obs.: 533.1961 Da.

[0057] (9) Preparation of (2R,3S,4R)-2-(acetoxymethyl)-2-ethynyl-5-methoxytetrahydrofuran-3,4-dimethyldiacetate (intermediate 9).

[0058] TBAF (1.6 mL, 1.6 mmol, 2.0 eq) was added to a THF (5 mL) solution of intermediate 8 (400 mg, 0.8 mmol), and the reaction was carried out at room temperature for 5 hours. After the reaction was completed, the mixture was concentrated under reduced pressure, and pyridine (5 mL) and acetic anhydride (2 mL) were added to the residual mixture. The reaction was carried out overnight at room temperature. The reaction was quenched by slow addition of methanol (5 mL) under ice bath, and the mixture was concentrated under reduced pressure. The mixture was extracted with EA and CuSO4 solution, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography to give a colorless oil, intermediate 8 (226 mg, 90% yield).

[0059] 1 H NMR (600 MHz, CDCl3): d 5.34 (d, 1H, J = 6.6 Hz, CH), 5.11 (d, 1H, J = 3.6 Hz, CH), 4.91 (dd, 1H, J= 6.6, 3.6 Hz, CH), 4.40 (d, 1H, J = 11.4Hz, CH2), 4.15 (d, 1H, J = 11.4 Hz, CH2), 3.43 (s, 3H, OCH3), 2.61 (s, 1H,CH), 2.13 (s, 3H, CH3), 2.10 (s, 3H, CH3), 2.06 (s, 3H, CH3). 13 HRMS (ESI) for C 14 H 18 NaO8(M+Na) + : Calcd.: 337.0894 Da;Obs: 337.0872 Da.

[0060] (10) Preparation of (3R,4S,5R)-5-(acetoxymethyl)-5-ethynyltetrahydrofuran-2,3,4-trimethyltriacetate (intermediate 10).

[0061] At 0 °C, concentrated H₂SO₄ (40 μL) was slowly added dropwise to a mixed solution of intermediate 9 (190 mg, 0.34 mmol), acetic anhydride (1 mL), and acetic acid (4 mL), and the reaction was carried out at 0 °C for 1 hour. The reaction was quenched with methanol, and saturated NaHCO₃ solution was added until the pH of the reaction system reached 7. The mixture was extracted with DCM and water, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography to obtain a colorless oily substance, intermediate 8 (81 mg, 70% yield).

[0062] 1 H NMR (600 MHz, CDCl3): d 5.34 (d, 1H, J = 6.6 Hz, CH), 5.11 (d, 1H, J = 3.6 Hz, CH), 4.91 (dd, 1H, J = 6.6, 3.6 Hz, CH), 4.40 (d, 1H, J = 11.4Hz, CH2), 4.15 (d, 1H, J= 11.4 Hz, CH2), 3.43 (s, 3H, OCH3), 2.61 (s, 1H,CH), 2.13 (s, 3H, CH3), 2.10 (s, 3H, CH3), 2.06 (s, 3H, CH3). 13 HRMS (ESI) for C 14 H 18 NaO8(M+Na) + : Calcd.: 337.0894 Da;Obs: 337.0872 Da.

[0063] (11) Preparation of 3-carbamoyl-1-((2R,3R,4S,5R)-5-ethynyl-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyridinium (intermediate 11).

[0064] At 0 °C, Tmsotf (260 μL, 1.2 mmol, 4.0 eq) was added to a CH3CN solution of intermediate 10 (102 mg, 0.3 mmol) and nicotinamide (73 mg, 0.6 mmol, 2.0 eq), and the reaction was slowly brought to room temperature for 24 hours. The reaction was quenched with MeOH, concentrated under reduced pressure, and then reacted for 2 days at -20 °C with 2 mL of NH3-MeOH solution (7 N in MeOH) slowly added under ice bath. After the reaction was completed, the solution was concentrated under reduced pressure, purified by HPLC, and collected by a fraction collector to give a colorless solid, intermediate 11 (53 mg, yield 63%).

[0065] 1 H NMR (300 MHz, CD3OD): d 9.67 (s, 1H, ArH), 9.39 (d, 1H, J = 6.3 Hz, ArH), 9.04 (dt, 1H, J = 8.1, 1.5 Hz, ArH), 8.28 (dd, 1H, J = 8.1, 6.3 Hz, ArH), 6.24 (d, 1H, J = 5.4 Hz, CH), 4.54 (t, 1H, J= 5.4 Hz, CH), 4.35 (d, 1H, J = 5.1 Hz, CH), 3.40 (d, 1H, J = 12.0 Hz, CH2), 3.89 (d, 1H, J = 12.0Hz, CH2), 3.27 (s, 1H, CH). 13 C NMR (151 MHz, CD3OD): d 165.2, 146.6, 143.8,142.4, 135.9, 129.3, 102.3, 90.3, 80.1, 79.5, 79.4, 73.7 66.9. HRMS (ESI) forC 13 H 16 N2O5 + (M+H) + : Calcd.: 280.1048 Da; Obs.: 280.1026 Da.

[0066] (12) Preparation of ((2R,3S,4R,5R)-5-(3-carbamoylpyridinium-1-yl)-2-ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methylhydrogen phosphate (intermediate 12).

[0067] At 0 °C, P(O)Cl3 (49 µL, 0.54 mmol, 6.0 eq) was added to a 1 mL solution of intermediate 11 (25 mg, 0.09 mmol) in trimethyl phosphate, and the reaction was carried out at 0 °C for 6 hours. The reaction was quenched with water, extracted with DCM, concentrated under reduced pressure, purified by HPLC, and collected by a fraction collector to give a colorless solid, intermediate 12 (17 mg, 54% yield).

[0068] 1 H NMR (300 MHz, D2O): d 9.46 (s, 1H, ArH), 9.26 (d, 1H, J = 6.3 HzArH), 8.99 (dt, 1H, J = 8.1, 1.5 Hz, ArH), 8.29 (dd, 1H, J = 8.1, 6.3 Hz, ArH), 6.29 (d, 1H, J = 5.7 Hz, CH), 4.65 (t, 1H, J = 5.7 Hz, CH), 4.50 (d, 1H,J = 5.4 Hz, CH), 4.32 (dd, 1H, J = 11.7, 4.5 Hz, CH2), 4.20 (dd, 1H, J =11.7, 5.1 Hz, CH2), 3.32 (s, 1H, CH). 13 C NMR (151 MHz, D2O): d 165.8, 146.3,142.7, 140.1, 134.1, 128.7, 99.7, 87.3(d, J = 10.0 Hz), 80.8, 77.2, 76.8,72.1, 67.6(d, J = 4.7 Hz). 31 P NMR (121 MHz, D2O): d -1.0. HRMS (ESI) forC 13 H 16 N₂O₈P (M+H) + : Calcd.: 359.0644 Da; Obs.: 359.0658 Da.

[0069] (13) 1-((2R,3R,4S,5R)-5-({({[(2R,3S,4R,5R)-5-(6-amino-9H-purine-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl]methoxy}(hydroxy)phosphoryl)oxyphosphoryl}oxy)methyl)-5-ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)-3-carbamoylpyridinium (compound NR-4′-a-NAD) + Preparation of ).

[0070] The enzyme NMNAT1 (5 μM) was added to the reaction buffer (28 mL, 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 20 mM MgCl2, 1 mM DTT, 5 mM ATP) of intermediate 12 (10 mg, 0.03 mmol) and reacted at room temperature for 2 hours. The reaction was terminated by adding 10% TCA, centrifuged, filtered through a membrane, purified by HPLC, and collected as a fraction to obtain a colorless solid, NR-4′-a-NAD. + (18 mg, 70% yield).

[0071] 1 H NMR (600 MHz, D2O): d 9.50 (s, 1H, ArH), 9.33 (d, 1H,J = 6.6 Hz, ArH), 9.04 (d, 1H, J = 7.8 Hz, ArH), 8.68 (s, 1H, ArH), 8.51 (s, 1H, ArH), 8.38 (t, 1H, J = 7.2 Hz, ArH), 6.32 (d, 1H, J = 6 Hz, CH), 6.21 (d, 1H, J =5.4 Hz, CH), 4.83-4.79 (m, 1H, CH, overlap with solvent residue peak), 4.74(t, 1H, J = 5.4 Hz, CH), 4.61-4.58 (m, 2H, 2CH), 4.47-4.41 (m, 2H, CH+CH2), 4.37-4.30 (m, 2H, CH2+CH2), 4.29-4.26 (m, 1H, CH2) 3.37 (s, 1H, CH). 13 C NMR (151 MHz, D2O): d 165.6, 150.0, 148.4, 146.4, 144.8, 142.8, 142.6, 140.1,134.1, 128.8,118.7, 99.7, 88.1, 86.9 (d, J = 10.3 Hz), 84.1 (d, J = 8.8 Hz),80.8, 77.0, 76.7, 74.6, 71.7, 70.2, 68.1 (d, J = 5.3 Hz), 65.2 (d, J = 4.7Hz). 31 P NMR (121 MHz, D2O): d -11.2, -12.3. HRMS (ESI) for C 23 H 28 N7NaO 14 P2 + (M+Na) + : Calcd.: 688.1164 Da; Obs.: 688.1189 Da.

[0072] Example 2: Analysis of the substrate activity of the compound on PARP enzyme and the interaction between the generated PAR chain and the substrate protein.

[0073] (1) Experimental method: PARP enzymes inhibit NR-4′-α-NAD + Utilization: Mix 1 μM PARP1 or PARP5a with 1 mM NAD + Or NR-4′-a-NAD + Mix the reaction solution in a reaction buffer (100 mM Tris-HCl (pH 8.0), 50 mM NaCl, 20 mM MgCl2, 0.2 mM DTT, 100 ng / μL single-stranded DNA) to a total volume of 100 μL. The reaction system was then treated with 100 μM olaparib as the negative group and DMSO as the positive group, and reacted overnight at 30 °C. Take 30 μL of the reaction solution and add 15 μL of click chemistry solution (containing 2 mM THPTA, 1 mM CuSO4, 100 μM azide biotin, and 10 mM sodium ascorbate) to a total volume of 45 μL. React at 37 °C for 2 hours, then add loading buffer and boil for 5 minutes to terminate the reaction. The samples were separated by 10% SDS-PAGE gel, transferred to a PVDF membrane, blocked with 5% skim milk powder at room temperature for 2 hours, and then incubated with Streptavidin-HRP (1:10000) at room temperature for 1 hour. The PAR signal was detected by ECL development.

[0074] PARG for NR-4′-a-NAD + Hydrolysis to generate PAR: Take PARP1 or PARP5a (1 mM NAD⁺ and NR-4′-a-NAD) respectively + 30 μL of each click-labeled chemical reaction solution was added, along with 1.2 ng of human PARG, and the mixture was reacted at 30 °C for 2 hours. Samples were then directly subjected to SDS-PARG and Western blotting, followed by detection using Streptavidin-HRP (1:10000). A control group without PARG was also included.

[0075] PAR substrate protein for NR-4′-α-NAD + PAR generation identification: PARP1 or PARP5a (1 mM NAD) respectively + and NR-4′-a-NAD + The reaction solution without click-labeled chemicals was directly subjected to SDS-PAGE, transferred to a membrane, and then blocked. The PVDF membrane was incubated at room temperature for 3 hours with purified macro domains of human Af1521, WWE domains of RNF146, and catalytic domains of PARG. After washing with TBST, the binding protein was detected with anti-His antibody (1:5000), and ECL was used for imaging.

[0076] (2) Experimental results: like Figure 1 As shown, both poly-ADP-ribose polymerase 1 (PARP1) and end-anchored polymerase 1 (PARP5a / TNKS1) can recognize and utilize NR-4′-a-NAD. + The formation of PAR chains, while the disappearance of the signal in the olaparib (PARP inhibitor) treatment group, indicates that NR-4′-a-NAD... + It is an effective substrate for PARP enzymes; poly(ADP-ribose) hydrolase (PARG) can significantly hydrolyze NR-4′-a-NAD. + PAR chains are generated, and substrate proteins specifically bind to NR-4′-a-NAD. + The generation of PAR chains and the disappearance of signals in the olaparib or PARPi (mixed inhibitors, including the PARP inhibitor olaparib and the TNKS inhibitor XAV939) treatment groups indicate that the signal is generated by PAR and that PAR can be recognized by substrate proteins. In summary, the compounds designed in this invention can not only serve as PARP enzyme substrates, but the PAR chains they form also retain the same protein recognition characteristics as natural PAR chains, which can be used for the study of enzyme-substrate interaction regulation during ADP-ribosylation.

[0077] Example 3: Comparison of the binding affinity of the PAR chain generated by the compound to its substrate protein with that of the natural PAR chain.

[0078] (1) Experimental method: Mix 1 μM PARP1 or 10 μM PARP5a with 1 mM NAD + Or NR-4′-a-NAD + The mixture was prepared in a reaction buffer (100 mM Tris-HCl (pH 8.0), 50 mM NaCl, 20 mM MgCl2, 0.2 mM DTT, 100 ng / μL single-stranded DNA) to a total volume of 100 μL. The reaction mixture was then treated with 100 μM olaparib as a negative control and DMSO as a positive control. The reaction was incubated overnight at 30 °C.

[0079] Immunoblot analysis was used to compare substrate-protein binding affinity: PARP1 reaction groups were loaded with 5 μL, 10 μL, 15 μL, 20 μL, and 25 μL respectively (PARP5a reaction groups were loaded with 5 μL, 10 μL, 15 μL, and 20 μL respectively). After separation by 10% SDS-PAGE gel, the samples were transferred to PVDF membranes and blocked with 5% skim milk powder at room temperature for 2 hours. The PVDF membranes were then incubated with the purified macro domain of human Af1521 at room temperature for 3 hours. After washing with TBST, the binding protein was detected using anti-His antibody, and ECL was used for imaging.

[0080] Micro-thermophoresis (MST) was used to determine the dissociation constant: PARP1 or PARP5a reaction solutions were serially diluted 2-fold with PBS, resulting in 16 concentration points. 10 μL of each serially diluted reaction solution was mixed with 10 μL of GST-Af1521 Macro-EGFP (final concentration 0.1 μM) and incubated at room temperature for 15 minutes. MST data were acquired under 20% infrared laser power, and the dissociation constant (Kd) was analyzed and calculated using software.

[0081] (2) Experimental results: like Figure 2 As shown in the figure, the quantitative binding experiment results indicate that the sample loading volume of the reaction solution is highly linearly correlated with the amount of GST-Af1521Macro-EGFP protein bound, and the slopes of the corresponding regression curves are almost identical, indicating that the binding of NR-4′-a-NAD... + The binding efficiency of synthesized PAR to purified PAR recognition protein (GST-Af1521 Macro-EGFP) and natural NAD + The generated PAR values ​​are similar.

[0082] like Figure 3 As shown, the MST detection results indicate that NR-4′-a-NAD... + The dissociation constants of the synthesized PAR are similar to those of the naturally sourced PAR.

[0083] In summary, the above data confirms that NR-4′-a-NAD + Not only can it serve as a highly efficient substrate for PARP1 and PARP5a, but the synthesized PARP also retains the key structural features required for specific and high-affinity recognition by PARP-binding proteins.

[0084] Example 4: Detection of the compound's effect on intracellular PAR-dependent processes.

[0085] (1) Experimental method: As a negative control group, HeLa cells were treated with NR-4′-α-NAD.+ Before treatment, cells were pretreated with DMEM medium containing 10 μM PARPi (10 μM olaparib and 10 μM XAV939) at 37 °C for 2 h, and subsequent treatments were the same as the experimental group. For the experimental group, cells were cultured in DMEM medium containing 0.015% Triton X-100 and 100 μM NR-4′-a-NAD. + Incubate in PBS at 4 °C for 15 min. After discarding the reaction solution, wash twice with PBS, and add 100 μM NR-4′-a-NAD. + The control group was cultured in DMEM at 37 ℃ for 8 h; the blank control group was cultured in DMEM at 37 ℃ for 8 h; after pretreatment, all were treated with 500 μM sodium arsenite at 37 ℃ for 30 min to induce stress response.

[0086] PARylation activity in cells under stress was detected: Proteins were extracted using RIPA complete lysis buffer (1M Tris pH 8.0, 0.5 mM EDTA, 5M NaCl, 1% Triton X-100, 10% sodium deoxycholate, 0.1% SDS, and a 1× protease inhibitor mixture). The supernatant was collected after centrifugation at 12000 rpm for 5 minutes. 25 μL of the supernatant was then subjected to a click chemistry reaction (2 mM MTPTA, 1 mM CuSO4, 100 μM azide biotin, and 10 mM sodium ascorbate) at 37 °C for 2 hours. The reaction was analyzed by Western blotting using Streptavidin-HRP (1:10000) at room temperature for 1 hour, followed by ECL imaging to detect PAR signals.

[0087] To investigate the interaction between parylation and ubiquitination, HeLa cells were treated as described above, and incubated at 37 °C for 2 h with medium containing 10 μM MG132 before sodium arsenite treatment. For the USP2-treated group, the extracted protein lysate was reacted with 4 μM USP2 active domain at 37 °C for 1 h.

[0088] Immunoblot analysis: HeLa cells were treated as described above, blocked with skim milk, and then incubated at room temperature for 1 hour with Streptavidin-HRP (1:10000) and K48 ubiquitin-specific antibody (1:1000), respectively. After washing with TBST, cells were incubated with anti-rabbit secondary antibody (1:5000) for 30 minutes, followed by ECL imaging.

[0089] Confocal imaging analysis: After discarding DMEM, the slides were washed twice with PBS and fixed with 4% paraformaldehyde at room temperature for 20 minutes. After washing twice with PBS, the slides were perforated with 0.2% Triton X-100 at room temperature for 10 minutes. After washing twice with PBS, the slides were blocked with 3% BSA. The slides were incubated overnight at 4 °C with K48 ubiquitin-specific antibody (1:100) or anti-FUS antibody (1:200), washed three times with PBS, and then incubated with Fluor-488-labeled secondary antibody (1:200) at room temperature for 30 minutes. Click chemistry was then performed with TAMRA-biotin at room temperature for 1 hour. Finally, the slides were mounted with DAPI-containing mounting medium, and images were acquired under a laser confocal microscope at wavelengths of 375-435 nm (DAPI), 460-500 nm (Alexa Fluor 488), and 541-551 nm (TAMRA) to analyze the intensity changes of K48 ubiquitinated protein and PAR, as well as the co-localization of FUS protein and PAR.

[0090] (2) Experimental results: Sodium arsenite (NaAsO2) can induce oxidative DNA damage in the cell nucleus, thereby activating PARP1 and PARP2 to initiate DNA repair. Sodium arsenite treatment significantly increases ADP-ribosylation levels in the nucleus and cytoplasm; this effect is inhibited by combining two PARP inhibitors (PARPi, 10 μM olaparib and 10 μM XAV939). Immunoblotting analysis of HeLa cell lysates confirmed that sodium arsenite upregulates the cellular ADP-ribosylation signaling pathway, and this signaling pathway can be blocked by PARP inhibitors. Figure 4 A).

[0091] Given that one of the core functions of ubiquitination is to regulate intracellular protein abundance through proteasome degradation, this study further investigated whether proteins modified by ADP-ribosylation under stress are ubiquitinated and subsequently degraded by the proteasome. Western blot results showed that treatment with the specific proteasome inhibitor MG132 significantly increased the level of poly-ADP-ribosylation (PAR) compared to the untreated control group, suggesting that some proteins modified by ADP-ribosylation can be degraded via the proteasome pathway. Figure 4 B). MG132 treatment can enhance the overall ubiquitination signal; however, in vitro incubation of protein samples with the ubiquitin-specific protease 2 catalytic domain (USP2CD), a broad-spectrum deubiquitinase, significantly weakens the ubiquitination signal. Figure 4 C). It is worth noting that the upregulation effect of ubiquitination signaling was significantly weakened after inhibiting PARP modification with a PARP inhibitor. Figure 4B, C). Confocal microscopy results further corroborate the interaction between PARation and ubiquitination, and changes in the level of PARation modification can directly affect the degree of protein ubiquitination modification. Figure 5 In summary, the above results directly confirm the existence of PAR-dependent ubiquitination (PARdU) under arsenite-induced stress conditions.

[0092] like Figure 6 This study further utilized NR-4′-a-NAD + To investigate the FUS aggregation process mediated by the interaction between PAR and FUS in stressed cells. Sodium arsenite treatment induced the formation of cytoplasmic FUS granules and significantly activated cellular ADP-ribosylation modification (…). Figure 6 A). Under the same stress conditions, simultaneous inhibition of PARP1 and PARP5a enzyme activities significantly reduced NR-4′-a-NAD. + The derived PAR levels significantly inhibited the assembly of FUS particles. Figure 6 A). Importantly, confocal microscopy revealed partial co-location between PAR and FUS punctate agglomerates. Figure 6 (B, C) demonstrates that PAR can directly participate in regulating stress-induced fluid-liquid phase separation (LLPS) of the fluid-structured fuzzy aggregate (FUS). Previous studies have proposed that transient PAR-FUS interactions can initiate FUS condensation without the need for PAR to remain permanently localized within the condensate to maintain structural stability, which is consistent with this conclusion: the vast majority of NR-4′-a-NAD in this study... + The derived PAR is diffusely distributed in the cytoplasm and is not enriched in the punctate aggregates of FUS. Figure 6 A). Confirmation of intracellular NR-4′-a-NAD + Derived PAR can regulate FUS-related stress granules; it also indicates that NR-4′-a-NAD + It is a multifunctional chemical probe for studying intracellular PAR-dependent liquid-liquid phase separation.

[0093] The above results indicate that the compounds of this invention can be used to study the regulation of intracellular ADP-ribosylation processes, accurately tracing the regulatory dynamics of PAR synthesis, hydrolysis, and downstream ubiquitination signals; simultaneously, they can specifically tracing the PAR-dependent process related to FUS, a core pathogenic protein in neurodegenerative diseases, providing a direct experimental tool for target screening of neurodegenerative diseases such as ALS / FTD, and the detected PAR-K48 ubiquitination regulatory axis can serve as a core direction for target screening of neurodegenerative diseases.

[0094] Example 5: Workflow for using the compounds of this invention to identify PARdU protein.

[0095] Figure 7The workflow for identifying PARdU protein by LC-MS / MS specifically includes the identification of NAD+ with terminal alkynes. + The analogue was introduced into HeLa cells; biotin was linked via click chemistry; the target protein was captured using streptavidin-conjugated magnetic beads and anti-ubiquitin antibody-conjugated magnetic beads, respectively; and the identity of the labeled protein was identified by proteomic proteometry.

[0096] Example 6: Compound screening and identification of PARdU candidate proteins.

[0097] (1) Experimental method: HeLa cells were seeded at 0.7 × 10⁶ cells / dish in 6 cm culture dishes with 4 mL of DMEM medium per dish. Each group had 3 replicates, and the cells adhered and grew for 1 day.

[0098] NR-4′-a-NAD + Enrichment of labeled proteins: using protein containing 0.015% Triton X-100 and 100 μM NR-4′-α-NAD. + Cells were treated with PBS at 4°C for 15 minutes, followed by treatment with PBS containing 100 μM NR-4′-a-NAD. + Cells were treated with DMEM at 37°C for 8 hours. The experimental group was treated with 10 μM MG132 at 37°C for 2 hours, while the negative control group received no treatment. All cells were then treated with 500 μM sodium arsenite at 37°C for 30 minutes. Protein was extracted using RIPA complete lysis buffer (1M Tris pH 8.0, 0.5 mM EDTA, 5M NaCl, 1% Triton X-100, 10% sodium deoxycholate, 0.1% SDS, and a 1× protease inhibitor mixture). The protein concentration was adjusted to 1 mg / mL, and 50 μL was subjected to click chemistry (2 mM THPTA, 1 mM CuSO4, 100 μM biotin azide, and 10 mM sodium ascorbate) at room temperature for 1 hour. Centrifuge at 12,000 rpm for five minutes using an ultrafiltration tube (molecular weight cutoff of 10 kDa), repeat three times to remove free biotin. Incubate the treated sample with 50 μL of streptavidin-conjugated magnetic beads at room temperature for 2 hours.

[0099] Enrichment of PAR-dependent ubiquitinated proteins: The negative group was pretreated with PARP inhibitors (10 μM olaparib and 10 μM XAV939) at 37 °C for 2 h, while the experimental group was not pretreated. Proteins containing 0.015% Triton X-100 and 100 μM NR-4′-a-NAD were also used. + Cells were treated with PBS at 4 °C for 15 minutes, and then treated with PBS containing 100 μM NR-4′-a-NAD.+ Cells were treated with DMEM at 37°C for 8 hours, followed by treatment with 10 μM MG132 at 37°C for 2 hours, and then treated with 500 μM sodium arsenite for 30 minutes. Protein was extracted using RIPA complete lysis buffer (1M Tris pH 8.0, 0.5 mM EDTA, 5M NaCl, 1% Triton X-100, 10% sodium deoxycholate, 0.1% SDS, 1× protease inhibitor mixture), and the protein concentration was adjusted to 1 mg / mL. 50 μL of the sample was incubated with 50 μL of anti-ubiquitin antibody-conjugated magnetic beads at room temperature for 2 hours.

[0100] Both groups of samples were washed three times each with 1% SDS, 5 M urea, and PBS, and the magnetic beads were separated using a magnetic rack. The samples were divided into two aliquots: one aliquot was boiled for 8 min with 4% SDS loading buffer and then subjected to SDS-PAGE; the other aliquot was used for LC-MS / MS analysis.

[0101] (2) Experimental results: like Figure 8 As shown, using NR-4′-a-NAD + The study successfully identified intracellular proteins that simultaneously undergo ADP-ribosylation and PAR-dependent ubiquitination. Through Venn diagram intersection analysis of the two protein datasets, 38 PARdU candidate proteins were screened. These results demonstrate that the compounds of this invention can efficiently capture PAR substrate proteins. The screened PARdU candidate proteins include PAR-dependent K48 ubiquitination targets associated with neurodegenerative diseases, and can be applied to target screening and identification for neurodegenerative diseases such as ALS / FTD, Alzheimer's disease, and Parkinson's disease. This provides a core candidate target library for mechanistic research in these diseases and also offers potential drug targets for targeted drug development.

[0102] The detailed descriptions listed above are merely specific illustrations of feasible embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any obvious improvements, substitutions, or modifications that can be made by those skilled in the art without departing from the essence of the present invention are within the protection scope of the present invention.

Claims

1. An alkyne-modified NAD + Analogous articles, characterized in that, The NAD + The analogue is a terminal alkyne-modified nicotinamide adenine dinucleotide, wherein the alkyne group is site-specifically modified at the 4th position of the nicotinamide ribose, and its chemical formula is: .

2. An alkyne-modified NAD + The method for preparing analogues is characterized in that, The preparation method includes the following preparation route: 。 3. The alkynyl-modified NAD according to claim 2 + The method for preparing analogues is characterized in that, The intermediate 12 is used to synthesize NAD. + The reaction buffer for the analogue contains dithiothreitol and adenine nucleoside triphosphate, and the reaction terminator is trichloroacetic acid; the reaction solvent for the synthesis of intermediate 12 from intermediate 11 is trimethyl phosphate, and the quencher is water; the reaction temperature for the synthesis of intermediate 11 from intermediate 10 is -20℃ to 30℃, and the catalyst for the reaction is trimethylsilyl trifluoromethanesulfonate.

4. The alkynyl-modified NAD according to claim 2 + The method for preparing analogues is characterized in that, The reaction temperature for synthesizing intermediate 7 from intermediate 6 is 0 ℃-30 ℃, the reaction solvent is methanol, and the reaction reagent is dimethyl (1-diazo-2-oxopropyl)phosphonate.

5. The alkynyl-modified NAD according to claim 2 + The method for preparing analogues is characterized in that, The reaction temperature for the synthesis of intermediate 6 from intermediate 5 is 0 ℃-30 ℃, the oxidant is Dys-Martin oxidant, and the quencher is Na2S2O3; the reaction temperature for the synthesis of intermediate 8 from intermediate 7 is -78 ℃-30 ℃, the reaction solvent is dichloromethane, and the quenchers are pyridine and methanol; the reaction system for the synthesis of intermediate 9 from intermediate 8 includes tetrabutylammonium fluoride, pyridine, and acetic anhydride, the reaction solvent is tetrahydrofuran, and the quencher is methanol; the reaction system for the synthesis of intermediate 10 from intermediate 9 includes acetic anhydride and acetic acid, and the quencher is methanol.

6. The alkynyl-modified NAD according to claim 2 + The method for preparing analogues is characterized in that, The solvent for synthesizing intermediate 2 from intermediate 1 is acetic acid solution, and the quencher is saturated NaHCO3 solution; the solvent for synthesizing intermediate 3 from intermediate 2 is a mixed solution of dichloromethane and water, and the quencher is ethanol; the solvent for synthesizing intermediate 4 from intermediate 3 is a mixed solution composed of 1,4-dioxane, 37% formaldehyde solution, NaOH solution, and water; the reaction temperature for synthesizing intermediate 5 from intermediate 4 is -78 ℃ to 30 ℃, the solvent is tetrahydrofuran, and the quencher is saturated NH4Cl solution.

7. The alkynyl-modified NAD according to claim 1 + Use of analogues in the preparation of reagents or kits for studying ADP-ribosylation and PAR-dependent cellular processes.

8. A method for screening candidate compounds in vitro for non-therapeutic purposes, characterized in that, Includes the following steps: (a) The alkynyl-modified NAD as described in claim 1 + The analogue, PARP enzyme, substrate protein, and test compound are contacted in an in vitro system; (b) Determine whether the test compound has PARP inhibitory activity by detecting the ADP-ribosylation level on the substrate protein; The ADP-ribosylation level is quantitatively detected by coupling the alkyne group with an azide-labeled detection probe via a click chemistry reaction.

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