Chemical modification method for irreversible aldehyde-amine condensation coupling reaction based on aromatic ring triazole formaldehyde and application of chemical modification method

Abstract

The invention discloses a chemical modification method for irreversible aldehyde amine condensation coupling reaction based on aromatic ring triazole formaldehyde and application of the chemical modification method, and belongs to the technical field of chemical pharmacy and biology. Aromatic ring triazole formaldehyde with a novel structure is adopted in the condensation coupling reaction, the aromatic ring triazole formaldehyde can react with various molecules containing primary amino groups in a broad spectrum mode, the reaction is efficient, high in selectivity, environmentally friendly and completely biocompatible, and the formed 1, 2, 3-triazole product is stable. The reaction is successfully applied to primary amine modification of biological macromolecules such as drug molecules, polypeptides, proteins, antibodies, DNA and the like, and has great commercial value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of pharmaceutical and biotechnology, and specifically discloses a kind of aromatic ring and triazole aldehyde and primary amine condensation coupling reaction, and its application in drug molecules, polypeptide, protein, antibody, DNA directed chemical modification and marking etc. BACKGROUND

[0002] 1,2,3-triazole compounds have excellent properties and rich biological activity, not only in the field of organic chemistry, in the field of pharmaceutical chemistry also plays an important role. 1,2,3-triazole group is extremely stable under acidic and basic hydrolysis conditions and redox environment, has high aromatic stability, is a key structural unit in a variety of bioactive molecules, a large number of literatures show that its derivatives have a wide range of biological activities, including anticancer, antibacterial, antifungal, antitumor, anti-inflammatory, antituberculosis, anticonvulsant, antimalarial, antiviral, antioxidant activity. As recently reported potential DNA intercalator I 1 , and the compound II 2 and compound III 3 with very good anticancer activity. There are a variety of 1,2,3-triazole-containing drug molecules on the market, including antibiotics cefatrizine (Cefatrizine), oral EGFR / HER2 tyrosine kinase inhibitor mubritinib (Mubritinib), anti-infective drug tazobactam (Tazobactam), tumor drug carboxyamidotriazole (Carboxyamidotriazole) and the like.

[0003]

[0004] 1,2,3-triazole structure in drug and bioactive molecules reflects various effects: first, as a bioisostere, it can replace amide bonds and ester bonds, such as amide bond substitutes can be used to stabilize the bioactive linear peptide 4 ; second, by introducing different substituents at the N1 and C4 positions of the triazole, the structure-activity relationship is regulated, especially by hybridizing 1,2,3-triazole with other active groups to produce dual or multiple pharmacological activities, such as 1,2,3-triazole-sugar molecule hybrid exhibits better anti-leukemia effect 5 ; third, by connecting 1,2,3-triazole to cleavable groups, targeted release can be achieved, such as click chemistry prodrugs; fourth, the ability of targeted binding is enhanced, 1,2,3-triazole can act as a hydrogen bond acceptor and donor, participating in non-covalent binding with biological targets; fifth, triazole can coordinate with metal ions, suitable for designing metal enzyme inhibitors, such as HIV integrase inhibitors; and so on. In summary, 1,2,3-triazole modules have broad prospects in the development of new drugs.

[0005] The most classic synthesis of 1,2,3-triazoles is the copper-catalyzed azide-alkyne cycloaddition (CuAAC) click chemistry method, which was honored by the Nobel Prize in Chemistry in 2022. Researchers in the fields of medicinal chemistry and biochemistry have synthesized a large number of 1,4-disubstituted 1,2,3-triazoles using this method. Although this method is very efficient, it has limitations in modifying biomolecules. First, the reaction requires a Cu(I) catalyst, which has potential toxicity to biomolecules, and a tedious purification step is required to remove residual copper. In addition, due to the difficulty of copper catalysts penetrating cell membranes, their application in living cells is limited. Second, the reaction requires the modification of molecules to alkyne or azide in advance. For macromolecules such as proteins or nucleic acids, the introduction of alkyne or azide groups may greatly reduce the reaction efficiency due to steric hindrance, and other functional groups in the biomolecule may undergo side reactions during the introduction process, resulting in changes in the product and reducing the final modification effect.

[0006] The development of modern biological therapies requires powerful tools to precisely design complex molecular structures. Chemical modification of biomolecules is an important means. Chemical modification not only improves the function of biomolecules, but also endows them with new functions. Chemical modification of polypeptides or proteins is the main target of biomolecule chemical modification, and has played an important role in the fields of life science, medicine, biomaterials, etc. Among them, efficient methods for cross-linking biomolecules (including native biomolecules and synthetic drugs and probes) are the basis for constructing multifunctional bioconjugates.

[0007] Despite decades of development, practical cross-linking strategies remain limited, mainly relying on reactions with highly active but scarce thiol groups, and often using time-consuming and laborious step-by-step operations. If the scope is expanded to less reactive but more abundant primary amino groups (mainly referring to lysine side chains in proteins and polypeptides), the functionality of the biological coupling toolbox can be significantly enhanced; if the coupling technology developed uses 1,2,3-triazoles as linking groups, it must endow drug molecules or biomolecules with more functions. However, truly biocompatible lysine-targeted coupling technologies are still very scarce.

[0008] REFERENCES 1. Chowdhary, S. et al. Isatin-Indoloquinoxaline Click Adducts With a Potential to Overcome Platinum-Based Drug-Resistance in Ovarian Cancer [J]. Bioorganic Chemistry 2023, 142: 106953. 2.Tyagi, R. et al. Efficient Synthesis of Indole-Chalcones Based Glycohybrids and Their Anticancer Activity [J]. Bioorganic & Medicinal Chemistry 2024, 109: 117778. 3.Bakale, R. D. et al. Design, Synthesis and Molecular Docking Study of Novel Quinoline-Triazole Molecular Hybrids as Anticancer Agents [J]. Journal of Molecular Structure. 2025, 1321: 140072. 4.Re nik LM. et al. 1,4-Disubstituted 1,2,3-Triazoles as Amide Bond Surrogates for the Stabilisation of Linear Peptides with Biological Activity[J]. Molecules. 2020, 25(16): 3576. 5.Amdouni, H. et al. In Vitro and in Vivo Evaluation of Fully Substituted (5-(3-Ethoxy-3-oxopropynyl)-4-(ethoxycarbonyl)-1,2,3-triazolyl-glycosides as Original Nucleoside Analogues to Circumvent Resistance in Myeloid Malignancies [J]. J. Med. Chem. 2017, 60(4): 1523. SUMMARY

[0009] In order to make up for the deficiency and blank of the existing biological molecule coupling, modification and cross-linking technology, and fully utilize the lysine residues existing in the biological molecules, the application provides a kind of novel aromatic ring triazole formaldehyde and an aldehyde amine condensation coupling reaction based on the triazole formaldehyde, the reaction is very efficient, high selectivity, and completely biocompatible. The reaction provides an effective, convenient and widely applicable coupling method for the modification of drug molecules, polypeptides, proteins and other complex functional molecules.

[0010] The application provides a chemical modification method based on irreversible aldehyde amine condensation coupling reaction of aromatic ring triazole formaldehyde, wherein the structural formula of the aromatic ring triazole formaldehyde is shown as formula I or formula II, the amine is any molecule containing NH2 group, including alkyl primary amine, heteroalkyl primary amine, cycloalkyl primary amine, aryl primary amine, heteroaryl primary amine, and drug small molecules, polypeptides, proteins, antibodies, DNA and the like containing NH2 group; the aromatic ring triazole formaldehyde can be dehydrated and condensed with various primary amine compounds under mild conditions and rearranged into 1,4-disubstituted-1,2,3-triazole.

[0011] The structural general formula of the aromatic ring triazole formaldehyde 1 is , wherein R 1 , R 2 , R 4 are each independently selected from any one of hydrogen, linear or branched saturated or unsaturated C1-C7 alkyl, C3-C5 cycloalkyl, linear or branched saturated or unsaturated C1-C7 alkoxy, linear or branched saturated or unsaturated C2-C7 heteroalkyl, halogen, aryl, heteroaryl, hydroxyl, mercapto, aryl-substituted mercapto, carboxyl, ester group (-COOR', R' is saturated C1-C4 alkyl); R 3 is hydrogen, linear or branched saturated or unsaturated C1-C 10 alkyl, C3-C7 cycloalkyl, linear or branched saturated or unsaturated C1-C 10 alkoxy, linear or branched saturated or unsaturated C2-C7 heteroalkyl, halogen, aryl, heteroaryl, hydroxyl, mercapto, aryl-substituted mercapto, carboxyl, ester group (-COOR', R' is saturated C1-C4 alkyl), alkyne, biotin-labeled alkyne (Biotin-alkyne), dansyl-labeled alkyne (Dansyl-alkyne).

[0012] The structural general formula of the aromatic ring triazole formaldehyde 2 is , wherein Z is C or N; R 5 , R 6 , R 8 , R 9 , R 10each independently selected from any one of hydrogen, linear or branched, saturated or unsaturated C1-C7alkyl, C3-C5cycloalkyl, linear or branched, saturated or unsaturated C1-C7alkoxy, linear or branched, saturated or unsaturated C2-C7heteroalkyl, halogen, aryl, heteroaryl, hydroxyl, thiol, aryl-substituted thiol, carboxyl, ester (-COOR', R' being a saturated C1-C4alkyl), or R 9 absent; R 7 is hydrogen, linear or branched, saturated or unsaturated C1-C 10 alkyl, C3-C7cycloalkyl, linear or branched, saturated or unsaturated C1-C 10 alkoxy, linear or branched, saturated or unsaturated C2-C7heteroalkyl, halogen, aryl, heteroaryl, hydroxyl, thiol, aryl-substituted thiol, carboxyl, ester (-COOR', R' being a saturated C1-C4alkyl), alkyne, biotin-labeled alkyne (Biotin-alkyne), dansyl-labeled alkyne (Dansyl-alkyne).

[0013] .

[0014] In particular, the aldehyde-amine condensation coupling reaction process provided by the present application is as follows: a certain proportion of aromatic ring-fused triazole aldehyde 1 or 2 is dissolved in a solvent, 1 to 2 equivalents of acetic acid (AcOH) or diisopropylethylamine (DIPEA) is added or not added, stirred at a certain temperature, or placed in a shaker for a certain time, TLC or LC-MS is used to monitor the reaction, the solvent is removed under reduced pressure, and the residue is purified by silica gel column chromatography or C18 preparative HPLC to obtain the corresponding 1,4-disubstituted-1,2,3-triazole 4 or 5. For the reaction of proteins, antibodies and DNA with aromatic ring-fused triazole aldehyde, ESI-QTOF mass spectrometry is used for monitoring.

[0015] The above reaction process is summarized in the following equations (1)-(2):

[0016] Most of the aromatic ring-fused triazole aldehydes 1 or 2 can be conveniently prepared from the corresponding 2-methyl-substituted aromatic ring compounds S1. First, S1 reacts with diethyl carbonate to obtain compound S2, which is diazotized to obtain S3, and then reduced by DIBAL-H and oxidized by DMP to obtain the aromatic ring-fused triazole aldehyde 1 or 2, as shown in equation (3). The structures of the remaining special substituents can be prepared by referring to the relevant literature.

[0017]

[0018] The structural formula of the primary amine 3 is RNH2, and R can be any group, that is, the primary amine 3 is any molecule containing an NH2 group, including alkyl primary amine, heteroalkyl primary amine, cycloalkyl primary amine, aryl primary amine, heteroaryl primary amine, a small molecule drug containing an NH2 group, a polypeptide, a protein, an antibody, DNA, etc.

[0019] The molar ratio of the aromatic ring triazolyl aldehyde 1 or 2 to the primary amine 3 in the reaction process is 1:0.8 1.2. The solvent includes commonly used alcohol solvents (methanol, ethanol, isopropanol, butanol, trifluoroethanol, etc.), water, acetic acid, a buffer solution (PBS), commonly used polar solvents (DMF, DMSO, DMA, N-methyl pyrrolidone, diethyl ether, tetrahydrofuran, dioxane, acetonitrile, etc.), commonly used non-polar solvents (dichloromethane, dichloroethane, toluene, chlorobenzene, etc.), and a mixed system of the above solvents; the reaction time is 10 minutes 12 hours. The reaction temperature is 0 80 degrees Celsius.

[0020] The two novel aromatic ring triazolyl aldehydes provided by the application can be efficiently dehydrated and condensed with various primary amine compounds and rearranged into 1,4-disubstituted-1,2,3-triazole 4 or 5 under mild conditions, the reaction has complete specificity for the primary amine group, and the biomolecule does not need to be modified in advance. The reaction does not require additional additives, and the only byproduct of the reaction is water, which is green and environmentally friendly. The reaction solvent has extremely wide applicability, and there is almost no limitation on the selection of the solvent, which significantly improves the flexibility, operability and biological compatibility of the reaction.

[0021] Further, the generated 1,4-disubstituted-1,2,3-triazole product is stable. Essentially, the application converts the reversible aldehyde amine condensation into an irreversible aldehyde amine coupling reaction.

[0022] The beneficial effects of the present application are: the technology disclosed in the present application successfully modifies small molecule drugs or drug intermediates containing NH2 groups, such as Cefalexin, Amlodipine, Alogliptin, Afatinib intermediates, Glucosamine, which are all efficiently reacted with methylpyridinotriazole aldehyde to obtain 1,2,3 triazole linked modified products with high yield. Furthermore, the technology is effectively applied to the primary amine modification of polypeptides, proteins, antibodies and DNA and other biological macromolecules. For example, the targeted chemical modification of various polypeptide lysine residues is realized, including active peptide Angstrom 6, beta-interferon specific peptide segment, rat neuromedin U (Neuromedin U(rat)), 3 to 23 peptides such as the peptide hormone Spexin, which are all efficiently reacted with various pyridinotriazole aldehydes to obtain the corresponding primary amine group coupling products; the primary amine group of the peptide drug Atisiban and the two primary amine groups in Bacitracin are all successfully modified by methylpyridinotriazole aldehyde. It is particularly worth pointing out that when modifying polypeptides and proteins, the method not only has excellent primary amine selectivity and efficiency, but also exhibits the rare ability to completely avoid the interference of thiol groups. For example, the lysine residue in Amyloid P Component(27-38) amide is modified by propargyloxy substituted quinolinotriazole aldehyde, and the cysteine residue is further labeled with a fluorescent probe 4-bromomethyl-7-methoxycoumarin; the lysine residue in Glucoprotein(33-41) is modified by methylpyridinotriazole aldehyde, and the cysteine residue is further labeled with Biotin-Maleimide. For example, lysozyme protein, RNaseA and green fluorescent protein sfGFP are all reacted with various different substituted pyridinotriazole aldehydes, and the products of the directed chemical modification of the lysine residues in the proteins are all successfully obtained. For example, the antibody Trastuzumab is reacted with propargyloxy substituted quinolinotriazole aldehyde, and the product of the primary amine labeling is efficiently and selectively obtained. For the DNA fragment DNA Headpiece containing amino groups, it is also successfully reacted with dansyl labeled alkynylpyridinotriazole aldehyde to obtain the product of the primary amine modification and fluorescent labeling. Therefore, the technology disclosed in the present application has great application prospects and commercial value in the fields of medicinal chemistry and directed chemical modification of biological molecules. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1Reaction equation (A) and product mass spectrum (B) for NH2 modification in lysozyme protein.

[0024] Figure 2 Reaction equation (A) and product mass spectrum (B) for NH2 modification in ribonuclease A protein.

[0025] Figure 3 Reaction equation (A) and product mass spectrum (B) for NH2 modification in green fluorescent protein.

[0026] Figure 4 Reaction equation (A) and product mass spectrum (B) for NH2 modification in trastuzumab.

[0027] Figure 5 Reaction equation (A) and product liquid chromatogram (B) for NH2 modification in DNA head fragment. DETAILED DESCRIPTION

[0028] The application will be further described below in conjunction with specific examples, which are intended to illustrate the application rather than further limit it.

[0029] The primary amine small molecules used in the application are commercially purchased or synthesized according to related literatures; the small molecule drugs (or drug intermediates), medicinal polypeptides (or active peptides), proteins, antibodies and DNA are commercially purchased; part of the polypeptides are synthesized by using Rink resin through conventional solid-phase synthesis (SPPS), as shown in equation (5), wherein AA1 to AAn represent the peptide chain, n represents the length of the peptide chain, and n is any integer in 4-25. Those skilled in the art can select other suitable polypeptide synthesis methods according to the synthesis requirements.

[0030]

[0031] i) Fmoc protecting group removal; ii) amino acid condensation; iii) cleavage of Rink resin

[0032] Example 1

[0033] First, pyridotriazole aldehyde 1a is prepared:

[0034] S2a is synthesized according to the literature method (Firth, J. D., O'Brien, P. & Ferris, L. Revisiting the sparteine surrogate: development of a resolution route to the (-)-sparteine surrogate [J]. Org. Biomol. Chem.2014, 12: 9357-9365.).

[0035] S3a reference method synthesis (Rawat, D. & Adimurthy, S. Transannulation of pyridotriazoles with naphthoquinones and indoles: synthesis of benzo[ f ]pyrido[1,2-a]indoles and indolizino[3,2-b]indoles [J]. Adv. Synth. Catal. 2022, 364: 71-76. Or Yan, W. et al. Cp RhIII-catalyzed cascade annulation ofarylimidates with pyridotriazoles toward isoquinolin-3-ol derivatives [J]. J. Org. Chem. 2022, 87: 10858-10868.

[0036] S3a (21 mmol, 1 equiv) was dissolved in dichloromethane, cooled to -30°C, and a solution of DIBAL-H in toluene (1.5 M in toluene, 52.5 mmol, 2.5 equiv) was added dropwise. After the addition was complete, the reaction was stirred at room temperature for 5 hours. After quenching with water and NaOH solution, the mixture was filtered, the filtrate was concentrated, and the concentrate was dissolved in dichloromethane and placed in an ice water bath. DMP (Dess-Martin Periodinace) was added in portions, and the reaction was stirred at room temperature for 2 hours. Then, saturated NaHCO3 solution was added to quench the reaction, and dichloromethane was extracted. The organic phase was dried and concentrated, and the concentrate was purified by silica gel column chromatography to obtain pyridotriazole aldehyde 1a, a white solid, with a total yield of about 60% for the four steps: 1 H NMR (400 MHz, CDCl3) δ 10.33 (s, 1H), 8.88 – 8.86 (m, 1H), 8.37 – 8.34 (m, 1H), 7.67 – 7.63 (m, 1H), 7.26 – 7.22 (m, 1H). 13C NMR (101 MHz, CDCl3) δ 185.07, 137.79, 133.48, 130.98, 126.00, 119.53,117.47. HRMS-ESI ( m / z C7H5N3NaO + [M+Na] + Calculated value: 170.0325, measured value: 170.0330.

[0037] The reaction between pyridotriazole formaldehyde 1a and benzylamine was then investigated: A certain proportion of pyridotriazole formaldehyde 1a and benzylamine 2a were dissolved in 2 mL of solvent at room temperature, with or without additives, and the reaction was stirred at room temperature and monitored by TLC. After 1a or 2a disappeared, the reaction solution was directly concentrated and purified by silica gel column chromatography to obtain the coupling product 3aa. The conditions investigated are shown in Table 1. Table 1 Screening of reaction conditions between pyridotriazole carbaldehyde 1a and benzylamine

[0038]

[0039] The NMR data for coupling product 3aa are as follows: 1 H NMR (400 MHz, CDCl3) δ 8.52 (d, J = 4.7Hz, 1H), 8.17 (d, J = 8.0 Hz, 1H), 8.04 (s, 1H), 7.75 (td, J = 7.8, 1.5 Hz,1H), 7.43 – 7.30 (m, 5H), 7.23 – 7.16 (m, 1H), 5.57 (s, 2H). 13 C NMR (101 MHz, CDCl3) δ 150.23, 149.37, 148.75, 136.96, 134.35, 129.21, 128.89, 128.36, 122.91, 121.95, 120.26, 54.41.

[0040] Example 2

[0041] Pyridazine formaldehyde 1a (0.25 mmol, 1.25 equiv) and ( S)-2-Aminophenylpropanol 2b (0.20 mmol, 1.0 equiv) was dissolved in 2 mL of 95% ethanol at room temperature and stirred for 2 hours at room temperature. After concentration, the coupled product 3ab was purified by silica gel column chromatography as a yellow oil in 91% yield. 1 H NMR (400 MHz, CDCl3) δ 8.32 (d, J = 4.8 Hz, 1H), 8.15 (s, 1H), 7.96 (d, J = 8.0, 1H), 7.68 (t, J = 7.8 Hz, 1H),7.20 – 7.07 (m, 6H), 4.84 – 4.78 (m, 1H), 4.13 – 4.00 (m, 2H), 3.31 – 3.25(m, 2H). 13 C NMR (101 MHz, CDCl3) δ 149.76, 148.87, 146.86, 137.29, 136.54,129.00, 128.76, 127.03, 123.17, 122.86, 120.34, 65.55, 63.96, 37.40. HRMS-ESI( m / z ) C 16 H 17 N4O + [M+H] + : calculated 281.1397, found 281.1406.

[0042] Example 3

[0043] Pyridinotriazole aldehyde 1a (0.24 mmol, 1.2 equiv) and 2-aminoethanol 2c (0.20 mmol, 1.0 equiv) were dissolved in 2 mL of dimethyl sulfoxide at room temperature and stirred for 30 minutes at room temperature. After adding water, ethyl acetate was extracted, and the extract was dried and concentrated to give the coupled product 3ac as a colorless oil in 92% yield. 1 H NMR (400 MHz,CDCl3) δ 8.43 (ddd, J = 4.9, 1.8, 0.9 Hz, 1H), 8.29 (s, 1H), 8.04 (dt, J =8.0, 1.1 Hz, 1H), 7.74 (td,J 7.7, 1.8 Hz, 1H), 7.20 (ddd, J 7.6, 4.9,1.2 Hz, 1H), 4.54 (t, J 4.9 Hz, 2H), 4.12 (t, J 4.9 Hz, 2H). 13 C NMR (101 MHz, CDCl3) δ 149.94, 149.03, 147.56, 137.42, 123.65, 123.02, 120.51, 61.18,53.16.

[0044] Example 4

[0045] Pyridinotriazole aldehyde 1a (0.20 mmol, 1.0 equiv) and aniline 2d (0.24 mmol, 1.2 equiv) were dissolved in 2 mL 95% ethanol at room temperature, acetic acid (0.20 mmol, 1.0 equiv) was added, and the reaction was stirred at room temperature for 10 minutes. After concentration, the coupled product 3ad was purified by silica gel column chromatography to obtain a white solid with a yield of 96%. 1 H NMR (400 MHz,CDCl3) δ 8.62 – 8.58 (m, 2H), 8.24 (dd, J 7.9, 1.1 Hz, 1H), 7.85 - 7.75 (m,3H), 7.56 – 7.49 (m, 2H), 7.47 - 7.40 (m, 1H), 7.26 - 7.21 (m, 1H). 13 C NMR(101 MHz, CDCl3) δ 149.98, 149.53, 148.98, 137.04, 129.86, 128.90, 123.14,120.47, 120.45, 119.97.HRMS-ESI ( m / z ) C 13 H 10 N4Na + [M+Na] + : calculated 245.0798, found 245.0798.

[0046] Example 5

[0047] Pyridinotriazole aldehyde 1a (0.24 mmol, 1.2 equiv) and 4-fluoroaniline 2e (0.20 mmol, 1.0 equiv) were dissolved in 2 mL of dimethyl sulfoxide at room temperature, and the reaction was stirred at room temperature for 30 min. After adding water, ethyl acetate was extracted, and the extract was dried and concentrated, and then purified by silica gel column chromatography to obtain the coupling product 3ae as a dark blue solid in a yield of 86%. 1 H NMR (400 MHz, CDCl3) δ 8.65 – 8.61 (m, 1H), 8.60 (s, 1H), 8.25 (d, J = 7.9 Hz, 1H), 7.87 –7.75 (m, 3H), 7.32 – 7.20 (m, 3H). 13 C NMR (101 MHz, CDCl3) δ 162.50 (d, J =249.4 Hz), 149.75, 149.46, 148.97, 137.18, 133.26 (d, J = 3.2 Hz), 123.26,122.43 (d, J = 8.8 Hz), 120.54, 120.20, 116.84 (d, J = 23.4 Hz)。

[0048] Example 6

[0049] Reference Example 1 was synthesized according to the method of Reference Example 1, using 2,6-dimethylpyridine instead of 2-methylpyridine S1a in Example 1. White solid: 1 H NMR (400 MHz, CDCl3) δ 10.29 (s, 1H), 8.17(d, J = 8.8 Hz, 1H), 7.55 (t, J = 7.8 Hz, 1H), 7.02 (d, J = 7.1 Hz, 1H), 2.94(s, 3H). 13 C NMR (101 MHz, CDCl3) δ 185.15, 138.04, 137.21, 133.65, 131.10,116.75, 116.54, 17.45. HRMS-ESI (m / z) C8H7N3NaO+ [M+Na] + Calculated value: 184.0481, measured value: 184.0483.

[0050] 7-methylpyridotriazole formaldehyde 1b (0.24 mmol, 1.2 equiv) and ( S 2-Aminophenylpropanol 2b (0.20 mmol, 1.0 equiv) was dissolved in 2 mL of dimethyl sulfoxide at room temperature, and the reaction was stirred at room temperature for 30 minutes. After adding water, the mixture was extracted with ethyl acetate, and the extract was dried, concentrated, and purified by silica gel column chromatography to give the coupling product 3bb, a colorless oil with a yield of 89%. 1 H NMR (400 MHz, CDCl3) δ 8.14 (s, 1H), 7.76 (d, J = 7.8 Hz, 1H), 7.56 (t, J =7.8 Hz, 1H), 7.23 – 7.10 (m, 3H), 7.07 (d, J = 7.1 Hz, 2H), 6.99 (d, J = 7.7Hz, 1H), 4.82 – 4.73 (m, 1H), 4.10 (dd, J = 11.8, 6.9 Hz, 1H), 3.99 (dd, J =11.8, 3.5 Hz, 1H), 3.35 – 3.19 (m, 2H), 2.44 (s, 3H). 13 C NMR (101 MHz, CDCl3) δ 157.84, 149.24, 147.16, 137.25, 136.64, 129.00, 128.72, 126.96, 123.06,122.51, 117.39, 65.53, 63.67, 37.40, 24.23. HRMS-ESI ( m / z C 17 H 19 N4O + [M+H] + Calculated value: 295.1553, measured value: 295.1559.

[0051] Example 7

[0052] Following the method of Example 1, 7-chloropyridinium triazole formaldehyde 1c was synthesized by replacing 2-methylpyridine S1a in Example 1 with 2-chloro-6-methylpyridine. The resulting product was a white solid. 1 H NMR (400 MHz, CDCl3) δ 10.00 (s, 1H), 8.18 (s, 1H), 7.61 (t, J = 7.9 Hz, 1H), 7.18 (s, 1H). 13 C NMR (101 MHz, DMSO- d 6) δ 192.20, 170.48, 139.74, 128.99, 120.48. HRMS-ESI ( m / z C7H4ClN3NaO + [M+Na] + Calculated value: 203.9935, measured value: 203.9937.

[0053] 7-chloropyridazine formaldehyde 1c (0.24 mmol, 1.2 equiv) and ( S 2-Aminophenylpropanol 2b (0.20 mmol, 1.0 equiv) was dissolved in 2 mL of dimethyl sulfoxide at room temperature, and the reaction was stirred at room temperature for 2 hours. After adding water, the mixture was extracted with ethyl acetate, and the extract was dried, concentrated, and purified by silica gel column chromatography to give the coupling product 3cb, a yellow oil, with a yield of 93%. 1 H NMR (400 MHz, CDCl3) δ 8.06 (s, 1H), 7.85 (d, J = 7.7 Hz, 1H), 7.66 (t, J =7.8 Hz, 1H), 7.25 – 7.13 (m, 4H), 7.12 – 7.01 (m, 2H), 4.83 – 4.71 (m, 1H), 4.20 (dd, J = 12.0, 7.1 Hz, 1H), 4.07 (dd, J = 12.0, 3.3 Hz, 1H), 3.39 – 3.23(m, 2H). 13 C NMR (101 MHz, CDCl3) δ150.70, 150.24, 145.99, 139.56, 136.37, 128.94, 128.81, 127.11, 123.74, 123.29, 118.28, 65.94, 64.05, 37.29. HRMS-ESI ( m / z ) C 16 H 16 ClN4O + [M+H] + : calculated 315.1007, found 315.1013.

[0054] Example 8

[0055] The method of Reference Example 1 was followed using methyl 2-methylisonicotinate in place of 2-methylpyridine S1a in Example 1 to synthesize 5-methoxycarbonylpyridotriazolyl aldehyde 1d as a white solid: 1 H NMR (400 MHz, CDCl3) δ 10.38 (s, 1H), 9.01 – 8.99 (m, 1H), 8.89 (dd, J = 7.2, 1.1 Hz, 1H), 7.79 (dd, J = 7.2, 1.7 Hz, 1H), 4.02 (s, 3H). 13 C NMR (101 MHz, CDCl3) δ 184.91, 163.88, 139.33, 132.76, 132.19, 125.85, 121.64, 116.66, 53.27. HRMS-ESI ( m / z ) C9H7N3O3Na + [M+Na] + : calculated 228.0379, found 228.0376.

[0056] 5-methoxycarbonylpyridotriazolyl aldehyde 1d (0.20 mmol, 1.0 equiv) and 4-chloroaniline 2f (0.24 mmol, 1.2 equiv) were dissolved in 2 mL of 95% ethanol and placed in an ice water bath (0 °C). Acetic acid (0.20 mmol, 1.0 equiv) was added and the reaction was stirred at 0 °C for 1 h. After concentration, the coupled product 3df was purified by silica gel column chromatography to give a white solid with a yield of 90%. 1 H NMR (400 MHz, CDCl3)δ 8.80 - 8.71 (m, 2H), 8.59 (s, 1H), 7.82(dd, J = 5.0, 1.6 Hz, 1H), 7.77 (d, J = 8.8 Hz, 2H), 7.53 (d, J = 8.8 Hz,2H), 3.99 (s, 3H). 13 C NMR (101 MHz, CDCl3) δ 165.44, 150.84, 150.38, 148.56,138.52, 135.37, 134.83, 130.07, 122.32, 121.65, 120.24, 119.76, 52.83. HRMS-ESI ( m / z ) C 15 H 11 ClN4O2Na + [M+Na] + : calculated 337.0463, found 337.0467.

[0057] Example 9

[0058] The method of Reference Example 1 was followed using 6-chloro-2-methylquinoline instead of 2-methylpyridine S1a in Example 1 to synthesize 7-chloroquinolinetrizolecarboxaldehyde 1e as a white solid: 1 H NMR (400 MHz, CDCl3) δ 10.43 (s, 1H), 8.82(d, J = 8.9 Hz, 1H), 8.22 (d, J = 9.3 Hz, 1H), 7.96 (s, 1H), 7.88-7.78 (m,2H). 13 C NMR (101 MHz, CDCl3) δ 184.56, 138.38, 133.05, 130.74, 129.68, 128.65,126.92, 124.51, 117.26, 115.77. HRMS-ESI ( m / z ) calcd for C1MR (400 MHz, CDCl3)δ 10.43 (s, 1H), 8.82 (d, J = 8.9 Hz, 1H), 8.22 (d,J = 9.3 Hz, 1H), 7.96 (s,1H), 7.88-7.78 (m, 2H). 13 C NMR (101 MHz, CDCl3) δ 184.56, 138.38, 133.05,130.74, 129.68, 128.65, 126.92, 124.51, 117.26, 115.77. HRMS-ESI ( m / z ) calcdfor C 11 H7ClN3O + [M+H] + Calculated value: 232.0272, measured value: 232.0281.

[0059] 7-Chloroquinolinetriazole formaldehyde 1e (0.24 mmol, 1.2 equiv) and ( S 2-Aminophenylpropanol 2b (0.20 mmol, 1.0 equiv) was dissolved in 2 mL of dimethyl sulfoxide at room temperature, and the reaction was stirred at room temperature for 2 hours. After adding water, the mixture was extracted with ethyl acetate, and the extract was dried, concentrated, and purified by silica gel column chromatography to give the coupling product 3eb, a brown solid, in 93% yield. 1 HNMR (400 MHz, CDCl3) δ 8.15 (s, 1H), 7.99 (d, J = 8.6 Hz, 1H), 7.93 (d, J =8.6 Hz, 1H), 7.60 (d, J = 2.3 Hz, 1H), 7.53 (d, J = 9.0 Hz, 1H), 7.43 (dd, J = 9.0, 2.3 Hz, 1H), 7.23 – 7.13 (m, 3H), 7.11 – 7.05 (m, 2H), 4.86 – 4.76 (m,1H), 4.54 (br, 1H), 4.23 (dd, J = 12.1, 7.4 Hz, 1H), 4.09 (dd, J = 11.9, 3.6Hz, 1H), 3.41 – 3.22 (m, 2H). 13 C NMR (101 MHz, CDCl3) δ149.85, 146.85,145.75, 136.46, 135.90, 132.02, 130.49, 129.93, 128.96, 128.80, 127.98,127.07, 126.23, 124.17, 119.05, 66.22, 64.11, 37.20. HRMS-ESI ( m / z C 20 H 18 ClN4O + [M+H] + Calculated value: 365.1164, measured value: 365.1165.

[0060] Example 10

[0061] Following the method of Example 1, 4-methylquinoxalanoic acid triazole formaldehyde 1f was synthesized by replacing 2-methylpyridine S1a in Example 1 with 2,3-dimethylquinoxalanoic acid, resulting in a bright yellow solid. 1 H NMR (400 MHz, CDCl3) δ 10.47 (s, 1H), 8.75 – 8.70 (m, 1H), 8.20 – 8.10 (m, 1H), 7.84 – 7.76 (m, 2H), 3.21 (s, 3H). 13 C NMR (101 MHz, CDCl3) δ 184.90, 153.15, 140.71, 137.02, 133.82, 130.44,129.92, 129.47, 125.28, 116.02, 25.82. HRMS-ESI ( m / z C 11 H8N4NaO + [M+Na] + Calculated value: 235.0590, measured value: 235.0593.

[0062] 4-methylquinoxalanotriazole formaldehyde 1f (0.24 mmol, 1.2 equiv) and ( S 2-Aminophenylpropanol 2b (0.20 mmol, 1.0 equiv) was dissolved in 2 mL of dimethyl sulfoxide at room temperature, and the reaction was stirred at room temperature for 2 hours. After adding water, the mixture was extracted with ethyl acetate, and the extract was dried, concentrated, and purified by silica gel column chromatography to give the coupling product 3fb as a gray solid with a yield of 84%. 1HNMR (400 MHz, CDCl3) δ 8.18 (s, 1H), 7.95 (d, J = 8.0 Hz, 1H), 7.89 (d, J =7.9 Hz, 1H), 7.71 – 7.60 (m, 2H), 7.27 – 7.17 (m, 3H), 7.14 (d, J = 7.2 Hz,2H), 4.91 – 4.81 (m, 1H), 4.22 – 4.09 (m, 2H), 3.35 (d, J = 7.5 Hz, 2H), 3.04(s, 3H). 13 C NMR (101 MHz, CDCl3) δ 152.54, 140.93, 140.40, 136.34, 129.92,129.33, 129.04, 128.87, 128.65, 128.25, 127.22, 125.92, 65.39, 63.72, 37.64,25.10. HRMS-ESI (m / z) C 20 H 20 N5O + [M+H] + : Calculated 346.1662, Found 346.1657.

[0063] Modification of small molecule drug or drug intermediate NH2(Example 11 15)

[0064] Example 11

[0065] Cefalexin (0.20 mmol, 1.0 equiv) and 2-methylpyridinotriazole aldehyde 1e (0.24 mmol, 1.2 equiv) were dissolved in 2 mL dimethyl sulfoxide at room temperature, and the reaction was stirred at room temperature for 2 hours. After adding water, ethyl acetate was extracted, and the extract was dried and concentrated, and then purified by silica gel column chromatography to obtain Cefalexin-NH2 modification product Cefalexin-1b, yellow oil, yield 92%. 1 H NMR (400 MHz, DMSO- d 6 ) δ 9.75 (d, J= 8.0 Hz, 1H), 8.26(s, 1H), 7.87 – 7.63 (m, 2H), 7.51-7.28 (m, 5H), 7.15 (d, J = 7.6 Hz, 1H),6.84 (s, 1H), 5.63 (dd, J = 8.0, 4.6 Hz, 1H), 5.01 (d, J = 4.6 Hz, 1H), 3.45(d, J = 17.9 Hz, 1H), 3.20 (d, J = 18.0 Hz, 1H), 2.42 (s, 3H), 1.94 (s, 3H). 13 C NMR (101 MHz, DMSO- d 6) δ 167.58, 164.89, 163.41, 158.54, 149.30, 147.83,137.92, 134.69, 129.81, 129.59, 128.60, 128.55, 122.99, 122.90, 117.03,65.49, 59.03, 57.25, 29.27, 24.50, 19.81. HRMS-ESI ( m / z ) C 24 H 22 N6NaO4S + [M+Na] + : calculated 513.1315, found 513.1322.

[0066] Example 12

[0067] Amlodipine (0.20 mmol, 1.0 equiv) and 2-methylpyridinotriazole aldehyde 1b (0.24 mmol, 1.2 equiv) were dissolved in 2 mL dimethyl sulfoxide at room temperature, and the reaction was stirred at room temperature for 1 hour. After adding water, ethyl acetate was extracted, and the extract was dried and concentrated, and then purified by silica gel column chromatography to obtain the amlodipine NH2 modified product Amlodipine-1b, white solid, yield 95%. 1 H NMR (300 MHz, CDCl3) δ 8.25 (s, 1H), 7.95 (d, J = 7.8Hz, 1H), 7.64 (t,J = 7.7 Hz, 1H), 7.30 (dd, J = 7.7, 1.8 Hz, 1H), 7.19 (dd, J = 7.8, 1.5 Hz, 1H), 7.13 – 7.04 (m, 2H), 7.04 – 6.95 (m, 2H), 5.35 (s, 1H),4.75 (d, J = 4.4 Hz, 2H), 4.71 – 4.61 (m, 2H), 4.09 – 3.92 (m, 4H), 3.57 (s,3H), 2.54 (s, 3H), 2.25 (s, 3H), 1.15 (t, J = 7.1 Hz, 3H). 13 C NMR (75 MHz,CDCl3) δ 167.98, 167.18, 158.33, 149.34, 148.99, 145.74, 144.50, 144.39,137.20, 132.24, 131.45, 129.18, 127.39, 126.96, 122.73, 122.69, 117.28,103.81, 101.90, 69.14, 68.05, 59.89, 50.78, 50.13, 37.02, 24.50, 19.24,14.26. HRMS-ESI ( m / z ) C 28 H 30 ClN5NaO5 + [M+Na] + : calculated 574.1828, found 574.1832.

[0068] Example 13

[0069] Alogliptin (0.20 mmol, 1.0 equiv) and 2-methylpyridinotriazole aldehyde 1b (0.24 mmol, 1.2 equiv) were dissolved in 2 mL of dimethyl sulfoxide at room temperature, and the reaction was stirred at room temperature for 4 hours. After adding water, ethyl acetate was extracted, and the extract was dried and concentrated, and then purified by silica gel column chromatography to obtain the Alogliptin-NH2 modified product Alogliptin-1b, colorless oil, yield 94%. 1 H NMR (400 MHz, CDCl3) δ8.12 (s, 1H), 7.86 (d, J = 7.8Hz, 1H), 7.67 – 7.57 (m, 2H), 7.50 (t, J = 7.7 Hz, 1H), 7.32 (t, J = 7.6 Hz,1H), 7.13 (d, J = 7.9 Hz, 1H), 7.05 (d, J = 7.6 Hz, 1H), 5.41 (s, 1H), 5.33 –5.17 (m, 2H), 4.70 – 4.57 (m, 1H), 3.45 (dd, J = 11.6, 3.9 Hz, 1H), 3.26 (s,3H), 3.16 – 2.96 (m, 2H), 2.79 – 2.65 (m, 1H), 2.51 (s, 3H), 2.40 – 2.30 (m,1H), 2.14 – 2.01 (m, 1H), 2.01 – 1.92 (m, 1H), 1.90 – 1.75 (m, 1H). 13 C NMR (101 MHz, CDCl3) δ 162.84, 158.89, 158.24, 152.51, 149.24, 148.22, 140.33,137.20, 133.41, 133.23, 128.18, 127.05, 122.66, 120.73, 117.33, 117.30,110.68, 91.48, 56.40, 55.89, 51.92, 46.21, 29.86, 28.06, 24.42, 23.46. HRMS-ESI ( m / z ) C 26 H 26 N8NaO2 + [M+Na] + : calculated 505.2071, found 505.2076.

[0070] Example 14

[0071] Afatinib-Int (0.20 mmol, 1.0 equiv) and 2-methylpyridinotriazole carboxaldehyde 1b (0.24 mmol, 1.2 equiv) were dissolved in 2 mL dimethyl sulfoxide at room temperature, and the reaction was stirred at room temperature for 2 hours. After adding water, ethyl acetate extraction, drying and concentration of the extract, Afatinib-Int-NH2 modification product Afatinib-Int-1b was obtained by silica gel column chromatography purification, white solid, yield 91%. 1 H NMR (400 MHz, CDCl3) δ 8.71 (d, J = 6.7Hz, 2H), 8.59 (s, 1H), 8.41 (s, 1H), 7.96 (dd, J = 6.6, 2.7 Hz, 1H), 7.76 (d, J = 7.8 Hz, 1H), 7.61 (t, J = 7.7 Hz, 1H), 7.53 – 7.46 (m, 1H), 7.33 (s, 1H),7.16 – 7.06 (m, 2H), 5.23 – 5.18 (m, 1H), 4.16 (d, J = 10.7 Hz, 1H), 4.12 –4.02 (m, 2H), 4.01 – 3.92 (m, 1H), 2.52 (s, 3H), 2.45 – 2.33 (m, 1H), 2.30 –2.21 (m, 1H). 13 C NMR (101 MHz, CDCl3) δ 158.41, 157.52, 156.08, 153.46,152.19, 149.66 (d, J = 276.9 Hz), 147.84, 137.00, 135.09 (d, J = 3.4 Hz),126.05, 124.27 (d, J = 5.3 Hz), 122.75, 121.86 (d, J = 6.8 Hz), 119.26,117.01 (d, J = 37.2 Hz), 116.29 (d, J= 21.9 Hz), 109.87, 109.37, 79.53,72.59, 67.27, 32.47, 24.19. HRMS-ESI ( m / z ) C 26 H 21 ClFN7NaO2 + [M+Na] + : calculated 540.1321, found 540.1327.

[0072] Example 15

[0073] Glucosamine (0.20 mmol, 1.0 equiv) and 2-methylpyridinotriazole carboxaldehyde 1b (0.24 mmol, 1.2 equiv) were dissolved in 2 mL dimethylsulfoxide at room temperature, and the reaction was stirred at room temperature for 12 hours. After adding water, ethyl acetate extraction, drying and concentration of the extract, and purification by silica gel column chromatography, the glucosamine-modified product Glucosamine-1b was obtained as a white solid in a yield of 91%. The two end-group isomer nuclear magnetic resonance showed a ratio of / = 1 / 1.8. -isomer: 1 H NMR (400MHz, D2O) δ 8.39 (s, 1H), 7.47 (td, J = 7.8, 2.0 Hz, 1H), 7.41 – 7.32 (m,1H), 6.94 (d, J = 7.7 Hz, 1H), 5.47 (d, J = 3.4 Hz, 1H), 4.47 – 4.33 (m, 1H),4.09 – 4.00 (m, 1H), 3.99 – 3.75 (m, 2H), 3.73 – 3.59 (m, 2H), 2.28 (s, 3H). 13 C NMR (101 MHz, D2O) δ 158.24, 147.19, 146.69, 138.17, 123.50, 122.82,117.86, 90.84, 71.55, 70.22, 69.91, 64.83, 60.44, 22.70. -isomer: 1 H NMR(400 MHz, D2O) δ 8.30 (s, 1H), 7.47 (td,J = 7.8, 2.0 Hz, 1H), 7.41 – 7.32 (m, 1H), 6.94 (d, J = 7.7 Hz, 1H), 5.28 (d, J = 8.3 Hz, 1H), 4.47 – 4.33 (m, 1H), 4.28 – 4.19 (m, 1H), 3.99 – 3.75 (m, 2H), 3.73 – 3.59 (m, 2H), 2.28 (s, 3H). 13 C NMR (101 MHz, D2O) δ 158.28, 147.06, 146.75, 138.17, 123.76, 123.58, 117.91, 94.05, 76.18, 73.39, 69.82, 67.60, 60.55, 22.70. HRMS-ESI ( m / z )C 14 H 18 N4NaO5 + [M+Na] + : calculated 345.1169, found 345.1173.

[0074] Polypeptide and peptide NH2modification (Example 16 30)

[0075] Example 16

[0076] Reaction condition [A]: trifluoroacetate salt of polypeptide substrate Peptide 1 (0.05 mmol, 1.0 equiv) and pyrazolyl aldehyde 1a (0.075 mmol, 1.5 equiv) were dissolved in 2.5 mL DMSO at room temperature, followed by the addition of DIPEA (0.1 mmol, 2.0 equiv), and the reaction was stirred at room temperature for 2 h to obtain the coupling product Peptide 1-1a, with an LC detection yield of >95%. HPLC purification and separation yielded a white powdery solid with a yield of 62%. 1 H NMR (400 MHz, Methanol- d4 ) δ 8.56 (s, 1H), 8.47 (s, 1H), 8.29 (s, 1H), 8.00 (d, J= 8.0 Hz, 1H), 7.97 – 7.83 (m, 5H), 7.58 – 7.48 (m, 2H), 7.39 – 7.30 (m, 1H), 4.51 (d, J = 4.4 Hz,1H), 4.41 – 4.26 (m, 4H), 4.06 – 3.83 (m, 2H), 1.96 – 1.73 (m, 4H), 1.42 –1.27 (m, 5H). HRMS-ESI ( m / z ) C 30 H 35 N8O5 + [M+H] + : Calculated 587.2725, Found 587.2760.

[0077] Reaction condition [B]: 2.5 mL DMSO / pH 7.2 PBS (1 / 4, v / v) mixed solvent instead of DMSO in reaction condition [A], LC yield 83%.

[0078] Reaction condition [C]: 2.5 mL DMSO / pH 7.2 PBS (1 / 1, v / v) mixed solvent instead of DMSO in reaction condition [A], LC yield 86%.

[0079] Example 17

[0080] The polypeptide substrate Peptide 2 (0.05 mmol, 1.0 equiv) and pyridotriazole carboxaldehyde 1a (0.075 mmol, 1.5 equiv) were dissolved in 2.5 mL DMSO at room temperature, followed by the addition of DIPEA (0.1 mmol, 2.0 equiv), and the reaction was stirred at room temperature for 2 h to obtain the coupling product Peptide 2-1a, LC yield 95%. HPLC purification separation gave a white powdery solid with a yield of 61%. HRMS-ESI ( m / z ) C 37 H 45 N 10 O 10 S + [M+H] + : Calculated 821.3036, Found 821.3031.

[0081] Example 18

[0082] The polypeptide substrate Peptide 3 (0.05 mmol, 1.0 equiv) and pyridotriazole carboxaldehyde 1a (0.075 mmol, 1.5 equiv) were dissolved in 2.5 mL DMSO at room temperature, followed by the addition of DIPEA (0.1 mmol, 2.0 equiv), and the reaction was stirred at room temperature for 6 h to give the coupling product Peptide 2-1a, with an LC detection yield of 89%. HPLC purification gave a white powdery solid (HCOOH salt) with a yield of 47%. HRMS-ESI (C m / z ) C 67 H 87 N 16 O 15 + [M+H] + : calc. 1355.6532, found 1355.6546.

[0083] Example 19

[0084] The polypeptide substrate Peptide 4 (0.05 mmol, 1.0 equiv) and 2-methylpyridotriazole carboxaldehyde 1b (0.075 mmol, 1.5 equiv) were dissolved in 2.5 mL DMSO at room temperature, followed by the addition of DIPEA (0.1 mmol, 2.0 equiv), and the reaction was stirred at room temperature for 2 h to give the coupling product Peptide 4-1b, with an LC detection yield of 95%. HPLC purification gave a white powdery solid with a yield of 42%. HRMS-ESI (C m / z ) C 45 H 50 N 11 O8 + [M+H] + : calc. 872.3839, found 872.3875.

[0085] Example 20

[0086] The polypeptide substrate Peptide 5 (0.05 mmol, 1.0 equiv) and pyridotriazole carboxaldehyde 1a (0.075 mmol, 1.5 equiv) were dissolved in 2.5 mL DMSO at room temperature, followed by the addition of DIPEA (0.1 mmol, 2.0 equiv), and the reaction was stirred at room temperature for 2 h to give the coupling product Peptide 5-1a, with an LC detection yield of 94%. HPLC purification gave a white powdery solid (HCOOH salt) with a yield of 40%. HRMS-ESI (m / z) calcd for C m / z ) C 48 H 58 N 15 O 10 + [M+H] + : calcd 1004.4486, found 1004.4493.

[0087] Example 21

[0088] The polypeptide substrate Peptide 6 (0.05 mmol, 1.0 equiv) and 2-methylpyridotriazole carboxaldehyde 1b (0.075 mmol, 1.5 equiv) were dissolved in 2.5 mL DMSO at room temperature, followed by the addition of DIPEA (0.1 mmol, 2.0 equiv), and the reaction was stirred at room temperature for 2 h to give the coupling product Peptide 6-1b, with an LC detection yield of >95%. HPLC purification gave a white powdery solid with a yield of 52%. HRMS-ESI (m / z) calcd for C m / z ) C 30 H 32 N7O5 + [M+H] + : calcd 530.2147, found 530.2163.

[0089] Example 22

[0090] Active peptide Peptide 7 (Angstrom6) (0.05 mmol, 1.0 equiv) and 2-methylpyridazinetrione aldehyde 1b (0.075 mmol, 1.5 equiv) were dissolved in 2.5 mL DMSO at room temperature, followed by the addition of DIPEA (0.1 mmol, 2.0 equiv), and the reaction was stirred at room temperature for 2 h to give the coupling product Peptide 7-1b, with an LC detection yield of >95%. HPLC purification gave a white powdery solid with a yield of 55%. HRMS-ESI (m / z) calculated for C m / z ) C 56 H 72 N 13 O 15 + [M+H] + : calculated 1166.5266, found 1166.5277.

[0091] Example 23

[0092] Active peptide Peptide 7 (Angstrom6) (0.05 mmol, 1.0 equiv) and 2-methylpyridazinetrione aldehyde 1b (0.075 mmol, 1.5 equiv) were dissolved in 2.5 mL DMSO at room temperature, followed by the addition of DIPEA (0.1 mmol, 2.0 equiv), and the reaction was stirred at room temperature for 2 h to give the coupling product Peptide 7-1b, with an LC detection yield of >95%. HPLC purification gave a white powdery solid with a yield of 55%. HRMS-ESI (m / z) calculated for C m / z )C 58 H 77 N 16 O 19 + [M+H] + : calculated 1301.5546, found 1301.5550.

[0093] Example 24

[0094] Peptide 9 (0.05 mmol, 1.0 equiv) and 2-methylpyridinotriazole carboxaldehyde 1b (0.075 mmol, 1.5 equiv) were dissolved in 2.5 mL DMSO at room temperature, followed by the addition of DIPEA (0.1 mmol, 2.0 equiv), and the reaction was stirred at room temperature for 12 h to give the coupling product Peptide 9-1b, with an LC detection yield of 95%. HPLC purification and separation gave a white powdery solid with a yield of 43%. HRMS-ESI (m / z) calculated for C m / z ) C 51 H 73 N 14 O 12 S2 + [M+H] + : calculated 1137.4969, found 1137.4947.

[0095] Example 25

[0096] Peptide 10 (0.05 mmol, 1.0 equiv) and 2-methylpyridinotriazole carboxaldehyde 1b (0.20 mmol, 4.0 equiv) were dissolved in 2.5 mL DMSO at room temperature, followed by the addition of DIPEA (0.1 mmol, 2.0 equiv), and the reaction was stirred at room temperature for 12 h to give the coupling product Peptide 10-1b, with an LC detection yield of 88%. HPLC purification and separation gave a white powdery solid (HCOOH salt) with a yield of 35%. HRMS-ESI (m / z) calculated for C m / z )C 82 H 114 N 23 O 16 S + [M+H] + : calculated 1708.8530, found 1708.8549.

[0097] Example 26

[0098] Reference Example 1 was used as a method, with 2-methyl-8-propargyloxyquinoline instead of 2-methylpyridine S1a in Example 1, to synthesize 9-propargyloxyquinoline triazole carboxaldehyde 1g, a white solid: 1 H NMR (400 MHz, CDCl3) δ 10.43(s, 1H), 8.20 (d,J = 9.2 Hz, 1H), 7.86 (d, J = 9.2 Hz, 1H), 7.69 – 7.55 (m,3H), 5.10 (s, 2H), 2.59 (s, 1H). 13 C NMR (101 MHz, CDCl3) δ 185.81, 148.70,137.47, 133.14, 132.49, 127.81, 127.20, 123.57, 122.15, 116.95, 115.94,77.78, 76.97, 58.01. HRMS-ESI ( m / z ) C 14 H9N3NaO2 + [M+Na] + : calculated 274.0587, found 274.0596.

[0099] Rat Neuromedin U Peptide 11 (0.05 mmol, 1.0 equiv) and 9-vinylquinoline- triazolecarboxaldehyde 1g (0.075 mmol, 1.5 equiv) were dissolved in 2.5 mL DMSO at room temperature, followed by the addition of DIPEA (0.1 mmol, 2.0 equiv) and stirring at room temperature for 2 h to give the coupling product Peptide 11-1g, with an LC detection yield of >95%. HPLC purification and isolation gave a white powdery solid (HCOOH salt) with a yield of 37%. HRMS-ESI ( m / z ) C 140 H 191 N 37 O 33 2+ [M+2H] 2+ : calculated 1459.7214, found 1459.7269.

[0100] Example 27

[0101] Biotin-labeled alkynylpyridinotriazolecarboxaldehyde 1i was first prepared:

[0102] Reference Example 1, 5-bromopyridinotriazolecarboxaldehyde 1h was synthesized by replacing 2-methylpyridine S1a in Example 1 with 4-bromo-2-methylpyridine, a yellow solid: 1 H NMR (400 MHz, CDCl3) δ10.30 (s, 1H), 8.72 (d, J = 7.3 Hz, 1H)., 8.55 (s, 1H), 7.31 (d, J = 7.3 Hz, 1H). 13 C NMR (101MHz, DMSO- d 6) δ 184.87, 134.31, 128.56, 126.64, 121.86, 120.69. HRMS-ESI ( m / z C7H4BrN3NaO + [M+Na] + Calculated value: 247.9430, measured value: 247.9427.

[0103] Biotin substrate S4 was synthesized using the method described in the reference (Wen, L). et al. Chemoenzymatic synthesis of unnatural nucleotide sugars for enzymatic bioorthogonal labeling [J]. ACS Catal. 2018, 8: 7659-7666.

[0104] Biotin substrate S4 (1.38 mmol, 1.2 eq), 5-bromopyridotriazole carbaldehyde 1h (1.15 mmol, 1.0 eq), Pd(PPh3)4 (0.093 mmol, 8 mol%), and CuI (0.093 mmol, 8 mol%) were added sequentially to the reaction flask. After evacuation and nitrogen replacement, THF was added, followed by Et3N (3.45 mmol, 3.0 eq). The reaction flask was heated in a 60 °C metal bath for 12 hours. After cooling, the reaction solution was concentrated and purified by silica gel column chromatography to obtain biotin-labeled alkynylpyridotriazole carbaldehyde 1i, a yellow solid. 1 H NMR (400 MHz, CDCl3) δ 10.31 (s, 1H), 8.80 (d, J = 7.1 Hz,1H), 8.37 (s, 1H), 7.19 (dd, J= 7.1, 1.7 Hz, 1H), 6.89 – 6.75 (m, 2H), 5.90(s, 1H), 4.56 – 4.40 (m, 3H), 4.28 (dd, J = 7.8, 4.5 Hz, 1H), 3.82 – 3.63 (m,4H), 3.58 (t, J = 5.2 Hz, 2H), 3.50 – 3.37 (m, 2H), 3.16 – 3.04 (m, 1H), 2.87(dd, J = 12.8, 4.8 Hz, 1H), 2.72 (d, J = 12.8 Hz, 1H), 2.20 (t, J = 7.5 Hz,2H), 1.78 – 1.54 (m, 4H), 1.47 – 1.32 (m, 2H). 13 C NMR (101 MHz, CDCl3) δ 184.91, 173.41, 164.30, 137.88, 133.02, 125.81, 125.76, 122.26, 119.81,91.84, 83.21, 70.07, 69.89, 69.48, 61.76, 60.25, 58.95, 55.73, 40.57, 39.09,36.04, 28.28, 28.10, 25.64. HRMS-ESI ( m / z ) C 24 H 30 N6O5NaS + [M+Na] + : calculated 537.1891, found 537.1898.

[0105]

[0106] The polypeptide substrate Peptide 12 (0.05 mmol, 1.0 equiv) and biotin-labeled alkynyl pyridotriazole aldehyde 1i (0.075 mmol, 1.5 equiv) were dissolved in 2.5 mL DMSO at room temperature, followed by the addition of DIPEA (0.1 mmol, 2.0 equiv), and the reaction was stirred at room temperature for 2 h to obtain the coupling product Peptide 12-1i, with an LC detection yield of >95%. HPLC purification and separation yielded a white powdery solid with a yield of 40%. HRMS-ESI ( m / z) C 85 H 111 N 20 O 21 S2 + [M+H] + : Calculated 1811.7669, Found 1811.7661.

[0107] Example 28

[0108] First, dansyl-labeled quinoline and triazole aldehyde 1l was prepared:

[0109] Reference Example 1 method, with 8-(4-methoxyphenyl)oxy-2-methylquinoline instead of 2-methylpyridine S1a in Example 1, triazole aldehyde compound 1j was synthesized, white solid: 1 H NMR (400 MHz, CDCl3) δ 10.44 (s, 1H), 8.18 (d, J = 9.1 Hz, 1H), 7.83 (d, J = 9.2 Hz, 1H), 7.62 (d, J = 8.2 Hz, 2H), 7.57 (t, J = 7.9 Hz, 1H), 7.51 (d, J = 7.9 Hz, 1H), 7.42 (d, J = 8.0 Hz, 1H), 7.00 - 6.92 (m, 2H), 5.39 (s, 2H), 3.82 (s, 3H). 13 C NMR (101 MHz, CDCl3) δ 185.72, 159.41, 149.93, 137.36, 133.08, 132.57, 128.70, 128.15, 127.93, 127.14, 123.35, 121.03, 115.79, 115.60, 114.09, 71.77, 55.32. HRMS-ESI ( m / z ) C 19 H 15 N3NaO3 + [M+Na] + : Calculated 356.1006, Found 356.1012.

[0110] To a solution of triazole aldehyde compound 1j (3.1 mmol, 1.0 eq) in DCM was added DDQ (6.5 mmol, 2.1 eq) and the reaction was stirred at room temperature for 6 hours. The reaction was quenched by the addition of saturated NaHC03solution and extracted with dichloromethane. The organic phase was dried and concentrated and purified by column chromatography on silica gel to give 9-hydroxyquinolino triazole aldehyde 1k as a white solid: 1 H NMR (400 MHz, CDCl3) δ 10.43 (d, J = 1.8 Hz, 1H), 10.31 (d, J = 1.8 Hz, 1H), 8.11 (d, J = 9.2 Hz,1H), 7.91 (d, J = 9.5 Hz, 1H), 7.61 (t, J = 8.1 Hz, 1H), 7.46 (dd, J = 10.1,8.1 Hz, 2H). 13 C NMR (101 MHz, CDCl3) δ 185.34, 148.54, 137.87, 133.19,131.63, 129.14, 126.24, 119.22, 118.13, 114.85. HRMS-ESI ( m / z ) C 11 H7N3NaO2 + [M+Na] + : calculated 236.0430, found 236.0429.

[0111] Danisyl chloride S5 (2.82 mmol, 3.0 eq) was dissolved in 5 mL of DCM under nitrogen protection, Et3N (2.82 mmol, 3.0 eq) was added, then 9-hydroxyquinolino triazole aldehyde 1k (0.94 mmol, 1.0 eq) was added. The reaction was stirred at room temperature for 12 hours. The reaction was quenched by the addition of water and extracted with dichloromethane. The organic phase was dried and concentrated and purified by column chromatography on silica gel to give danisyl labeled quinolino triazole aldehyde 11 as a bright yellow solid: 1 H NMR (400 MHz, CDCl3) δ 10.39 (s, 1H),8.66 – 8.64 (m, 1H), 8.56 (d, J8.7 Hz, 1H), 8.21 - 8.16 (m, 2H), 7.85 - 7.83 (m, 2H), 7.63 - 7.46 (m, 3H), 7.30 - 7.24 (m, 1H), 7.22 - 7.17 (m, 1H), 2.90 (s, 6H). 13 C NMR (101 MHz, CDCl3) δ 185.65, 151.92, 139.19, 137.41,133.06, 132.37, 131.77, 131.65, 131.30, 130.07, 129.86, 129.02, 127.84,127.49, 127.11, 125.92, 125.86, 123.00, 119.42, 116.51, 115.69, 45.46. HRMS-ESI ( m / z ) C 23 H 18 N4O4NaS + [M+Na] + : Calculated 469.0941, Found 469.0949.

[0112]

[0113] Peptide 13 (0.05 mmol, 1.0 equiv) and dansyl labeled quinolino triazole aldehyde 1l (0.075 mmol, 1.5 equiv) were dissolved in 2.5 mL DMSO at room temperature, then DIPEA (0.1 mmol, 2.0 equiv) was added, and the reaction was stirred at room temperature for 2 h to obtain the coupling product Peptide 13-1l, with an LC detection yield of 95%. HPLC purification and separation yielded a white powdery solid with a yield of 31%. HRMS-ESI ( m / z ) C 108 H 136 N 24 O 23 S2 2+ [M+2H] 2+ : Calculated 1101.9916, Found 1101.9964.

[0114] Example 29

[0115] Amyloid P Component (27-38) amide Peptide 14 (0.05 mmol, 1.0 equiv) and propargyloxy substituted quinoline triazole aldehyde lg (0.075 mmol, 1.5 equiv) were dissolved in 2.5 mL DMSO at room temperature, followed by the addition of DIPEA (0.1 mmol, 2.0 equiv) and stirring at room temperature for 2 h. Then the fluorescent probe 4-bromomethyl-7-methoxycoumarin FP (0.06 mmol, 1.2 equiv) was added in one pot and the reaction was continued for 2 h to give the further thiol fluorescently labeled product Peptide 14-1i-FP. LC yield 94%. HPLC purification gave white powdery solid (HCOOH salt) in 30% yield. HRMS-ESI (m / z) calcd for C64H73N9O13S [M+H] 2070.9401, found 2070.9407. m / z ) C 104 H 129 N 22 O 22 S + [M+H] + : calculated 2070.9401, found 2070.9407.

[0116] Example 30

[0117] Glucoprotein (33-41) Peptide 15 (0.05 mmol, 1.0 equiv) and methylpyridine triazole aldehyde lb (0.075 mmol, 1.5 equiv) were dissolved in 2.5 mL DMSO at room temperature, followed by the addition of DIPEA (0.1 mmol, 2.0 equiv) and stirring at room temperature for 2 h. Then biotin-maleimide was added in one pot and the reaction was continued for 2 h to give the further thiol biotin labeled product Peptide 15-1b-Biotin. LC yield 90%. HPLC purification gave white powdery solid in 29% yield. HRMS-ESI (m / z) calcd for C42H53N9O7S2 [M+H] 1837.8189, found 1837.8266. m / z ) C 88 H 117 N 20 O 20 S2 + [M+H] + : calculated 1837.8189, found 1837.8266.

[0118] Example 31

[0119] Modification of NH2 in Lysozyme protein, reaction equation is shown in Figure 1 A.

[0120] First, 50 μM of Lysozyme protein was placed in PBS buffer at pH 7.2, then pyridine triazole formaldehyde 1a DMSO solution was added to make its final concentration 2.5 mM. The reaction solution was placed in a 37 ℃ shaking bed, after 4 h of reaction, the reaction was directly monitored by ESI-QTOF mass spectrometry, the mass spectrometry results are shown in Figure 1 B, Lysozyme protein was successfully modified by triazole formaldehyde 1a to obtain Lysozyme-(1a) n , n = 1-4.

[0121] Example 32

[0122] Modification of NH2 in RNase A protein, reaction equation is shown in Figure 2 A.

[0123] First, 50 μM of RNase A protein was placed in PBS buffer at pH 7.2, then biotin-labeled alkyne pyridine triazole formaldehyde 1i DMSO solution was added to make its final concentration 2.5 mM. The reaction solution was placed in a 37 ℃ shaking bed, after 4 h of reaction, the reaction was directly monitored by ESI-QTOF mass spectrometry, the mass spectrometry results are shown in Figure 2 B, RNase A protein was successfully modified by biotin triazole formaldehyde 1i to obtain RNase-(1i) n , n = 1-7.

[0124] Example 33

[0125] First, the dansyl-labeled alkyne pyridine triazole formaldehyde 1m was synthesized:

[0126] Under nitrogen protection, dansyl chloride S5 (1.8 mmol, 1.3 eq) was dissolved in 5 mL DCM, Et3N (2.8 mmol, 2.0 eq) was added, then compound S6 (1.4 mmol, 1.0 eq) was added. Stir the reaction at room temperature for 12 hours. Quench with water, dichloromethane extraction, the organic phase was dried and concentrated, then purified by silica gel column chromatography to obtain compound S7, light yellow oil: 1 H NMR (400 MHz, CDCl3) δ 8.45 (dt, J = 8.5, 1.1 Hz, 1H), 8.24 (dd, J= 8.7, 1.0 Hz, 1H), 8.16 (dd, J = 7.3, 1.3 Hz, 1H), 7.93 (s, 1H), 7.52 – 7.40(m, 2H), 7.11 (dd, J = 7.6, 0.9 Hz, 1H), 5.61 (t, J = 6.0 Hz, 1H), 4.04 (d, J = 2.4 Hz, 2H), 3.46 – 3.37 (m, 2H), 3.36 – 3.25 (m, 4H), 3.05 – 3.01 (m, 2H),2.86 (s, 3H), 2.80 (s, 3H), 2.40 (t, J = 2.4 Hz, 1H).

[0127] Reference Example 27 was followed, substituting compound S7 for the biotin substrate S4 in Example 27, to synthesize the dansyl-labeled alkynyl pyridinotriazole aldehyde 1m as a brown oil: 1 H NMR (400 MHz, CDCl3) δ 10.28 (s,1H), 8.72 (dd, J = 7.1, 1.0 Hz, 1H), 8.47 (d, J = 8.5 Hz, 1H), 8.31 (s, 1H),8.27 (d, J = 8.6 Hz, 1H), 8.20 (dd, J = 7.2, 1.3 Hz, 1H), 7.55 – 7.42 (m,2H), 7.17– 7.08 (m, 2H), 5.51 (t, J = 6.0 Hz, 1H), 4.36 (s, 2H), 3.61 – 3.52(m, 2H), 3.46 – 3.35 (m, 4H), 3.16 – 3.04 (m, 2H), 2.83 (s, 6H). 13 C NMR (101MHz, CDCl3) δ184.91, 151.95, 137.32, 134.98, 132.95, 130.40, 129.82, 129.58, 129.32, 128.31, 125.86, 125.63, 123.20, 122.11, 119.87, 118.79, 115.15, 91.94, 83.13, 70.04, 69.42, 69.34, 58.95, 45.39, 43.01. HRMS-ESI ( m / z )C 26 H 27 N5NaO5S + [M+Na] + : calculated 544.1625, found 544.1632.

[0128] Modification of NH2 in green fluorescent protein, the reaction equation is shown in Figure 3 A.

[0129] Firstly, 50 μM of green fluorescent protein (sfGFP-WT) was placed in phosphate buffer solution (PBS) at pH 7.2, then the DMSO solution of dansyl-labeled alkyne pyridine and triazole formaldehyde 1m was added to make its final concentration 2.5 mM. The reaction solution was placed in a 37 ℃ shaking bed, after 4 h of reaction, the reaction was directly monitored by ESI-QTOF mass spectrometry, and the mass spectrometry results are shown in Figure 3 B, green fluorescent protein was successfully modified by dansyl triazole formaldehyde 1m to obtain sfGFP-WT-(1m) n , n = 1-9.

[0130] Example 34

[0131] Modification of NH2 in trastuzumab, the reaction equation is shown in Figure 4 A.

[0132] Firstly, PNGase F enzyme was used to deglycosylate the antibody. Then 14 μM of trastuzumab (Trastuzumab) was placed in phosphate buffer solution (PBS) at pH 7.2, then the DMSO solution of propargyloxy-substituted quinoline and triazole formaldehyde 1g was added to make its final concentration 1.4 mM. The reaction solution was placed in a 37 ℃ shaking bed, after 4 h of reaction, TCEP was used to reduce the disulfide bond, and finally the reaction was directly monitored by ESI-QTOF mass spectrometry, and the mass spectrometry results are shown in Figure 4 B, indicating that trastuzumab was successfully modified by quinoline triazole formaldehyde 1g to obtain Trastuzumab-(1g) n , n = 1-22.

[0133] Example 35

[0134] The modification of NH2 in DNA headpiece fragment, the reaction equation is shown in Figure 5 A.

[0135] Firstly, 667 μM of DNA headpiece fragment was placed in water, then the DANS- tagged alkynyl pyridine and triazole carboxaldehyde 1m DMSO solution was added to make the final concentration of 67 mM. The volume ratio of the final solvent was H2O: DMSO = 1:2, the reaction solution was reacted in a shaking bed at 37 ℃ for 12 h, then the reaction was detected by LC-MS, the liquid chromatogram is shown in Figure 5 B, which shows that the DNA headpiece is successfully modified by DANS-triazole carboxaldehyde 1m to obtain DNA headpiece-1m. The LC detection yield is >95%. ESI-QTOF mass spectrometry analysis results: HRMS-ESI ( m / z ) C 180 H 239 N 57 O 105 P 17 S - [M-H] - : calculated 5438.0413, found 5440.5005.

[0136] The above only describes the preferred embodiments of the present application, and several improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A chemical modification method based on the irreversible aldehyde-amine condensation coupling reaction of aryl-triazole formaldehyde, characterized in that, The process includes the following steps: reacting aromatic cyclotriazole formaldehyde with a primary amine dissolved in a solvent to yield 1,4-disubstituted-1,2,3-triazole; wherein: The general structural formula of the aromatic cyclotriazole formaldehyde is: or , where R 1 R 2 R 4 Each is independently selected from any one of hydrogen, straight-chain or branched saturated or unsaturated C1-C7 alkyl, C3-C5 cycloalkyl, straight-chain or branched saturated or unsaturated C1-C7 alkoxy, straight-chain or branched saturated or unsaturated C2-C7 heteroalkyl, halogen, aryl, heteroaryl, hydroxyl, mercapto, aryl-substituted mercapto, carboxyl, ester; R 3 Hydrogen, straight-chain or branched, saturated or unsaturated C1-C 10 Alkyl, C3-C7 cycloalkyl, straight-chain or branched saturated or unsaturated C1-C 10 Any one of the following: alkoxy, straight-chain or branched saturated or unsaturated C2-C7 heteroalkyl, halogen, aryl, heteroaryl, hydroxyl, mercapto, aryl-substituted mercapto, carboxyl, ester, alkynyl, biotin-labeled alkynyl, dansyl-labeled alkynyl. Z is C or N; R 5 R 6 R 8 R 9 R 10 Each is independently selected from any one of hydrogen, straight-chain or branched saturated or unsaturated C1-C7 alkyl, C3-C5 cycloalkyl, straight-chain or branched saturated or unsaturated C1-C7 alkoxy, straight-chain or branched saturated or unsaturated C2-C7 heteroalkyl, halogen, aryl, heteroaryl, hydroxyl, mercapto, aryl-substituted mercapto, carboxyl, ester, or R 9 Does not exist; R 7 Hydrogen, straight-chain or branched, saturated or unsaturated C1-C 10 Alkyl, C3-C7 cycloalkyl, straight-chain or branched saturated or unsaturated C1-C 10 Any one of the following: alkoxy, straight-chain or branched saturated or unsaturated C2-C7 heteroalkyl, halogen, aryl, heteroaryl, hydroxyl, mercapto, aryl-substituted mercapto, carboxyl, ester, alkynyl, biotin-labeled alkynyl, dansyl-labeled alkynyl. The primary amine is any molecule containing an NH2 group, with the molecular formula RNH2; The structural formula of the 1,4-disubstituted-1,2,3-triazole is as follows: or .

2. The chemical modification method based on the irreversible aldehyde-amine condensation coupling reaction of aryl-triazole formaldehyde according to claim 1, characterized in that, The molar ratio of aromatic triazole formaldehyde to primary amine is 1:0.

8. 1.

2.

3. The chemical modification method based on the irreversible aldehyde-amine condensation coupling reaction of aryl-triazole formaldehyde according to claim 1, characterized in that, The solvent includes one or more of methanol, ethanol, isopropanol, butanol, trifluoroethanol, water, acetic acid, PBS buffer solution, DMF, DMSO, DMA, N-methylpyrrolidone, diethyl ether, tetrahydrofuran, dioxane, acetonitrile, dichloromethane, dichloroethane, toluene, and chlorobenzene.

4. The chemical modification method based on the irreversible aldehyde-amine condensation coupling reaction of aryl-triazole formaldehyde according to claim 1, characterized in that, The reaction temperature is 0 80℃.

5. The chemical modification method based on the irreversible aldehyde-amine condensation coupling reaction of aryl-triazole formaldehyde according to claim 1, characterized in that, The reaction time is 10 minutes. 12 hours.

6. The chemical modification method based on the irreversible aldehyde-amine condensation coupling reaction of aryl-triazole formaldehyde according to claim 1, characterized in that, Optionally, the reaction system includes an additive, which is acetic acid or diisopropylethylamine, and the molar ratio of the additive to arylcyclotriazole formaldehyde is 1 to 2:

1.

7. The chemical modification method based on the irreversible aldehyde-amine condensation coupling reaction of aryl-triazole formaldehyde according to claim 1, characterized in that, The reaction was carried out under stirring or shaking conditions; the reaction progress was monitored by TLC, LC-MS or ESI-QTOF mass spectrometry; after the reaction was completed, the mixture was separated and purified.

8. The application of the chemical modification method according to any one of claims 1-7 in the chemical modification of primary amino groups of drug molecules, peptides, proteins, antibodies, and DNA.

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