A method for the synthesis of thiol-reactive nucleotide derivatives

Abstract

The application belongs to the technical field of organic synthesis, and provides a synthesis method of a thiol reactive nucleotide derivative. The application provides a preparation method of KBC-C2, which comprises the following steps: mixing a first compound shown in KBC-C1, trimethylsilazide, boron trifluoride ether and a first solvent, performing an azide substitution reaction, and obtaining a second compound shown in KBC-C2. The application uses BF3Et2O as a catalyst, and pure beta configuration product KBC-C2 can be obtained without alpha configuration product. Therefore, the method of the application is beneficial to the synthesis of beta configuration TReND molecules.

Description

Technical Field

[0001] This invention relates to the field of organic chemistry, and more particularly to a method for synthesizing a thiol-reactive nucleotide derivative. Background Technology

[0002] Bacterial pathogens cause millions of deaths globally each year. However, antibiotic development struggles to address the ever-emerging resistance. Therefore, understanding the molecular basis of bacterial infection is crucial in combating pathogenic diseases. Against this backdrop, the discovery of a post-translational modification activity—adenylation—of Fic (filamentation induced by cAMP) enzymes has provided new insights into the molecular basis of bacterial infection. Adenylation refers to the covalent attachment of an adenosine monophosphate (AMP) molecule to a hydroxyl-containing amino acid residue (tyrosine, threonine, or serine) on a protein. Pathogenic bacteria release Fic enzymes into the eukaryotic host during infection. Fic enzymes disrupt vital intracellular signaling networks by adenylating key regulatory target proteins. Fic enzymes contain a unique catalytic amino acid sequence motif (Fic motif: HPFx(D / E)GNGR), facilitating their identification through bioinformatics analysis. Consequently, more than 8,000 Fic enzymes are currently known.

[0003] However, protein targets cannot be predicted based on sequence or structural comparisons. Therefore, only a few Fic enzyme targets have been identified to date based on credible inference. Standard methods have proven unsuitable for detecting and identifying AMP-modified proteins from complex proteomes. Therefore, a specific and universally applicable strategy is urgently needed to identify Fic enzyme targets.

[0004] Existing technology has established an activity-based protein profiling (ABPP) system based on TReND (thiol-reactive nucleotide derivatives). This system utilizes the specific introduction of a cysteine ​​mutant into the Fic enzyme, where its thiol group undergoes a nucleophilic reaction with the chloroacetamide group of TReND to form a binary complex, thereby capturing the substrate and forming a low-affinity enzyme-substrate complex. This universal technique will provide a powerful approach for identifying the target proteins of Fic enzymes, potentially becoming fundamental to overcoming bacterial infections.

[0005] However, in the existing publicly available methods for synthesizing TReND (as shown in the formula below), the intermediate KBC-C2 exists in two configurations, α and β, with a ratio of 1:3. Since natural adenosine triphosphate (ATP) is generally β-configured, the intermediate KBC-C2 used to prepare TReND must be β-configured to efficiently generate the final product with the correct configuration.

[0006] TReND, KBC-C2.

[0007] In TReND, n=1, 2 or 3 correspond to TReND1, TReND2 or TReND3 respectively. Summary of the Invention

[0008] In view of this, the object of the present invention is to provide a method for synthesizing KBC-C2 and its thiol-reactive nucleotide derivatives. The method of the present invention can obtain pure β-configuration KBC-C2, thereby facilitating the preparation of pure β-configuration thiol-reactive nucleotide derivatives.

[0009] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for preparing KBC-C2, comprising the following steps: The first compound shown in KBC-C1, trimethylsilyl azide, boron trifluoride diethyl ether and the first solvent were mixed and subjected to an azide substitution reaction to obtain the second compound shown in KBC-C2. KBC-C1, KBC-C2.

[0010] Preferably, the molar ratio of the first compound to trimethylsilyl azide is 1:1.1~1.3, and the molar ratio of the first compound to boron trifluoride diethyl ether is 1:1.1~1.3; The azide substitution reaction is carried out at a temperature of 0-30°C for 3-5 hours.

[0011] This invention also provides a method for synthesizing a thiol-reactive nucleotide derivative, wherein the thiol-reactive nucleotide derivative has the structure shown in Formula 1: Formula 1; When n is 1 in Equation 1, the synthesis method includes the following steps: Step 1: Prepare the second compound shown in KBC-C2 according to the method described in claim 1 or 2; Step 2: The second compound, sodium methoxide, and the second solvent are mixed and subjected to a deprotection / ester exchange reaction to obtain the third compound shown in KBC-C3; KBC-C3; Step 3: Mix the third compound, the fourth compound shown in KBC-C4, 5-ethylthiotetrazole, tert-butylhydrogen peroxide and the third solvent, and carry out the first coupling-oxidation reaction to obtain the compound shown in KBC-C5A; KBC-C4, KBC-C5A; Step 4: Mix the compound shown in KBC-C5A, piperidine, and the third solvent to carry out the first Fm group removal reaction to obtain the fifth compound shown in KBC-C5; the molar ratio of the third compound and piperidine is 1:1~1.6, and the time of the first Fm group removal reaction is 10~30 min; KBC-C5; Step 5: Mix the fifth compound, the fourth compound shown in KBC-C4, 5-ethylthiotetrazole, tert-butylhydrogen peroxide and the third solvent to carry out the second coupling-oxidation reaction to obtain the compound shown in KBC-C6A; KBC-C6A; Step 6: Mix the compound represented by formula KBC-C6A, piperidine, and the third solvent to carry out the second Fm group removal reaction to obtain the sixth compound represented by KBC-C6; the molar ratio of the fifth compound to piperidine is 1:0.3~0.8, and the time of the second Fm group removal reaction is 10~30 min; KBC-C6; Step 7: Mix the sixth compound, the fourth compound shown in formula KBC-C4, 5-ethylthiotetrazole, tert-butylhydrogen peroxide and the third solvent, and carry out the third coupling-oxidation reaction to obtain the compound shown in KBC-C7A. KBC-C7A; Step 8-1: Mix the compound represented by formula KBC-C7A, piperidine, and the third solvent to carry out the third Fm group removal reaction to obtain the seventh compound represented by KBC-C7; the molar ratio of the sixth compound to piperidine is 1:0.3~0.8, and the time of the third Fm group removal reaction is 10~30 min; KBC-C7; Step 9-1: Mix the seventh compound, the alkynyl chloride shown in formula KBC-C8-1, copper sulfate, sodium ascorbate and the fourth solvent, and perform the first click reaction and the first ion exchange reaction in sequence to obtain the thiol reactive nucleotide derivative; KBC-C8-1; When n is 2 or 3 in Equation 1, replace steps 8-1 and 9-1 with: Step 8-2: Mix the compound represented by formula KBC-C7A, piperidine, and the third solvent, and carry out the fourth Fm group removal reaction to obtain the compound represented by formula KBC-C7B; the molar ratio of the sixth compound and piperidine is 1:0.3~0.8, and the time of the fourth Fm group removal reaction is 5 min; Formula KBC-C7B; Step 9-2: Mix the compound shown in KBC-C7B, the alkynyl chloride shown in KBC-C8-2, copper sulfate, sodium ascorbate and the fourth solvent, and carry out the second click reaction to obtain the tenth compound shown in KBC-C10; KBC-C8-2, KBC-C10; Step 10-2: The tenth compound is subjected to a fifth Fm group removal reaction and a second ion exchange reaction in a third solvent containing triethylamine to obtain the thiol-reactive nucleotide derivative.

[0012] This invention provides a method for preparing KBC-C2. The method uses boron trifluoride diethyl ether as a catalyst to prepare a second compound, KBC-C2, from a first compound, KBC-C1, such that the second compound is β-configured. Then, the second compound is used as a raw material to prepare thiol-reactive nucleotide derivatives, such that the thiol-reactive nucleotide derivatives are also β-configured.

[0013] The present invention also provides a method for synthesizing a thiol-reactive nucleotide derivative.

[0014] The synthesis method provided by this invention has the following advantages: (1) Natural ATP is in the β configuration, therefore the intermediate product in the synthesis must be in the β configuration to efficiently generate the final product with the correct configuration. When KBC-C1 is used as a raw material, different ratios of isomers can be obtained under different conditions: Existing literature reports that using KBC-C1 as a raw material, in TMSN3 (1.1 eq), SnCl4 (1 eq), DCM, 0℃-room temperature, and 12 h of reaction, the ratio of α configuration product to β configuration product is 1:3; while the method of the present invention, using new reaction conditions: TMSN3 (1.1~1.3 eq), BF3Et2O (1.1~1.3 eq), DCM, 0℃-room temperature, 3~5 h, can obtain the pure β configuration product KBC-C2, without the α configuration product. Therefore, the method of the present invention is beneficial for the synthesis of β configuration TReND molecules.

[0015] Second, this invention uses the phosphoramide method to controllably introduce different numbers of phosphate groups. This not only solves the problem of difficulty in introducing monophosphorylation into phosphorus oxychloride; at the same time, this step can obtain mono / di / triphosphorylated intermediates, as well as the final product with a chloroacetamide structure.

[0016] Third, this invention utilizes the phosphorous amide method, allowing the intermediates KBC-C5A, KBC-C6A, and KBC-C7A, containing phosphate groups, to be purified through simple column chromatography. Furthermore, after removing the Fm protecting group with piperidine, the monophosphate intermediate KBC-C5 can be directly purified by precipitation and crystallization to obtain a pure β-configuration solid. While the more polar polyphosphate intermediates KBC-C6 / C7 precipitate as an oil phase, precipitation and washing with a poor solvent (diethyl ether) effectively remove a large amount of organic impurities from the system, significantly improving the purity of the crude product and thus greatly reducing the difficulty of subsequent reversed-phase chromatography separation. Overall, this strategy is simpler and more efficient than traditional methods for separating nucleotide analogs (multiple complex HPLC separation methods). Attached Figure Description

[0017] Figure 1 For KBC-C2 in Example 1 1 H NMR spectrum; Figure 2 For KBC-C2 in Example 1 13 C NMR spectrum; Figure 3 For example, KBC-C3 in Example 1 1 H NMR spectrum; Figure 4 For example, KBC-C5 in Example 1 1 H NMR spectrum; Figure 5 For example, KBC-C5 in Example 1 31P NMR spectrum; Figure 6 For example, KBC-C6 in Example 1 1 H NMR spectrum; Figure 7 For example, KBC-C6 in Example 1 31 P NMR spectrum; Figure 8 For example, KBC-C7 in Example 1 1 H NMR spectrum; Figure 9 For example, KBC-C7 in Example 1 31 P NMR spectrum; Figure 10 For TReND1 in Example 1 1 H NMR spectrum; Figure 11 For TReND1 in Example 1 31 P NMR spectrum; Figure 12 For example, KBC-C7B in Example 2 1 H NMR spectrum; Figure 13 For example, KBC-C7B in Example 2 13 C NMR spectrum; Figure 14 For example, KBC-C7B in Example 2 31 P NMR spectrum Figure 15 For example, KBC-C10-1 in Example 2 1 H NMR spectrum; Figure 16 For example, KBC-C10-1 in Example 2 31 P NMR spectrum; Figure 17 For TReND2 in Example 2 1 H NMR spectrum; Figure 18 For TReND2 in Example 2 31 P NMR spectrum; Figure 19 For example, KBC-C10-2 in Example 3 1 H NMR spectrum; Figure 20 For example, KBC-C10-2 in Example 3 31 P NMR spectrum; Figure 21 For example, KBC-C10-2 in Example 3 13 C NMR spectrum; Figure 22 For TReND3 in Example 3 1 H NMR spectrum; Figure 23 For TReND3 in Example 3 31 P NMR spectrum; Figure 24 For the KBC-C2 prepared in Comparative Example 1 1 H NMR spectrum. Detailed Implementation

[0018] This invention discloses a method for preparing KBC-C2, comprising the following steps: The first compound shown in KBC-C1, trimethylsilyl azide (TMSN3), boron trifluoride diethyl ether (BF3·Et2O) and the first solvent were mixed and subjected to an azide substitution reaction to obtain the second compound shown in KBC-C2. KBC-C1, KBC-C2.

[0019] Unless otherwise specified, the raw materials used in this invention are preferably commercially available products.

[0020] In this invention, the first solvent preferably comprises dichloromethane (DCM). In this invention, the molar ratio of the first compound to trimethylsilyl azide is preferably 1:1.1~1.3, more preferably 1:1.2. In this invention, the molar ratio of the first compound to boron trifluoride diethyl ether is preferably 1:1.1~1.3, more preferably 1:1.2. In this invention, the temperature of the azide substitution reaction is preferably 0~30°C, more preferably room temperature; the time is preferably 3~5 h, more preferably 4 h; the azide substitution reaction is preferably carried out under stirring. In this invention, the specific process of the azide substitution reaction preferably includes: dissolving the first compound in the first solvent to obtain a first compound solution; adding boron trifluoride diethyl ether to the first compound solution and stirring; then adding trimethylsilyl azide to carry out the azide substitution reaction. In this invention, the stirring time is preferably 5 min. In this invention, during the azide substitution reaction, the first compound is preferably monitored by TLC until it is completely consumed. Following the azide substitution reaction, the present invention preferably further includes a first post-treatment, which preferably includes the following steps: quenching the azide substitution reaction with a saturated sodium bicarbonate solution, followed by extraction with dichloromethane (CH2Cl2) to collect the organic layer; sequentially washing, drying, filtering, and vacuum concentrating the organic layer to obtain a crude product; purifying the crude product using rapid column chromatography, concentrating the desired fraction to obtain the second compound. In the present invention, the washing agent preferably includes brine, and the drying is preferably sodium sulfate drying.

[0021] In this invention, under the promotion of Lewis acid BF3·Et2O, trimethylsilazine (TMSN3) converts the active site in the first compound into an azide-substituted product, generating a second compound with a pure β configuration; the -N3 functional group is introduced to prepare for the subsequent click reaction.

[0022] This invention also provides a method for synthesizing a thiol-reactive nucleotide derivative, wherein the thiol-reactive nucleotide derivative has the structure shown in Formula 1: Formula 1; When n is 1 in Equation 1, the synthesis method includes the following steps: Step 1: Prepare the second compound shown in KBC-C2 according to the preparation method described in the above technical solution; Step 2: The second compound, sodium methoxide, and the second solvent are mixed and subjected to a deprotection / ester exchange reaction to obtain the third compound shown in KBC-C3; KBC-C3; Step 3: Mix the third compound, the fourth compound shown in KBC-C4, 5-ethylthiotetrazole, tert-butylhydrogen peroxide and the third solvent, and carry out the first coupling-oxidation reaction to obtain the compound shown in KBC-C5A; KBC-C4, KBC-C5A; Step 4: Mix the compound shown in KBC-C5A, piperidine, and the third solvent to carry out the first Fm group removal reaction to obtain the fifth compound shown in KBC-C5; the molar ratio of the third compound and piperidine is 1:1~1.6, and the time of the first Fm group removal reaction is 10~30 min; KBC-C5; Step 5: Mix the fifth compound, the fourth compound shown in KBC-C4, 5-ethylthiotetrazole, tert-butylhydrogen peroxide and the third solvent to carry out the second coupling-oxidation reaction to obtain the compound shown in KBC-C6A; KBC-C6A; Step 6: Mix the compound represented by formula KBC-C6A, piperidine, and the third solvent to carry out the second Fm group removal reaction to obtain the sixth compound represented by KBC-C6; the molar ratio of the fifth compound to piperidine is 1:0.3~0.8, and the time of the second Fm group removal reaction is 10~30 min; KBC-C6; Step 7: Mix the sixth compound, the fourth compound shown in formula KBC-C4, 5-ethylthiotetrazole, tert-butylhydrogen peroxide and the third solvent, and carry out the third coupling-oxidation reaction to obtain the compound shown in KBC-C7A. KBC-C7A; Step 8-1: Mix the compound represented by formula KBC-C7A, piperidine, and the third solvent to carry out the third Fm group removal reaction to obtain the seventh compound represented by formula KBC-C7; the molar ratio of the sixth compound to piperidine is 1:0.3~0.8, and the time of the third Fm group removal reaction is 10~30 min; KBC-C7; Step 9-1: Mix the seventh compound, the alkynyl chloride shown in formula KBC-C8-1, copper sulfate, sodium ascorbate and the fourth solvent, and perform the first click reaction and the first ion exchange reaction in sequence to obtain the thiol reactive nucleotide derivative shown in formula TReND; KBC-C8-1; When n is 2 or 3 in Equation 1, replace steps 8-1 and 9-1 with: Step 8-2: Mix the compound represented by formula KBC-C7A, piperidine, and the third solvent, and carry out the fourth Fm group removal reaction to obtain the compound represented by formula KBC-C7B; the molar ratio of the sixth compound and piperidine is 1:0.3~0.8, and the time of the fourth Fm group removal reaction is 5 min; Formula KBC-C7B; Step 9-2: Mix the compound shown in KBC-C7B, the alkynyl chloride shown in KBC-C8-2, copper sulfate, sodium ascorbate and the fourth solvent, and carry out the second click reaction to obtain the tenth compound shown in KBC-C10; KBC-C8, KBC-C10; Step 10-2: The tenth compound is subjected to a fifth Fm group removal reaction and a second ion exchange reaction in a third solvent containing triethylamine to obtain the thiol-reactive nucleotide derivative.

[0023] The present invention first describes the preparation method of the thiol-reactive nucleotide derivative in Formula 1, where n is 1.

[0024] Step 1: The second compound represented by KBC-C2 is prepared according to the method described in the above technical solution. In this invention, the preparation method and parameters for the second compound represented by KBC-C2 are consistent with the above technical solution and will not be repeated here.

[0025] After obtaining the second compound, the present invention proceeds to step 2: mixing the second compound, sodium methoxide, and a second solvent to carry out a deprotection / ester exchange reaction, yielding the third compound shown in KBC-C3. In the present invention, the second solvent preferably includes methanol (MeOH). In the present invention, the molar ratio of the second compound to sodium methoxide is preferably 1:0.4~0.6, more preferably 1:0.5. In the present invention, the temperature of the deprotection / ester exchange reaction is preferably 10~30°C, more preferably room temperature, i.e., neither additional heating nor cooling is required; the time of the deprotection / ester exchange reaction is preferably 8~12 hours; the deprotection / ester exchange reaction is preferably carried out under stirring. In the present invention, the specific process of the deprotection / ester exchange reaction preferably includes the following steps: adding sodium methoxide to the second solvent containing the second compound under stirring to carry out the deprotection / ester exchange reaction. In the present invention, during the deprotection / ester exchange reaction, the second compound is preferably detected by TLC, and the deprotection / ester exchange reaction is considered complete when it is completely consumed. Following the deprotection / ester exchange reaction, the present invention preferably further includes a second post-treatment, which preferably includes: neutralizing the reaction mixture obtained from the deprotection / ester exchange reaction to pH 7 using IR 120 resin, filtering to remove solids, concentrating the obtained filtrate under vacuum to obtain a crude product; purifying the crude product by rapid column chromatography, concentrating the desired components, and obtaining the third compound.

[0026] In this invention, a mild deprotection / ester exchange is performed using sodium methoxide to expose the desired hydroxyl sites, yielding a third compound.

[0027] After obtaining the third compound, the present invention proceeds to step 3: the third compound, the fourth compound shown in KBC-C4, 5-ethylthiotetrazole (ETT), tert-butyl hydroperoxide (t-BuOOH), and the third solvent are mixed to perform a first coupling-oxidation reaction to obtain the compound shown in KBC-C5A. In the present invention, the third solvent preferably includes dimethylformamide (DMF), more preferably dried dimethylformamide. In the present invention, the molar ratio of the third compound to the fourth compound is preferably 1:1.1 to 1.3, more preferably 1:1.2. In the present invention, the molar ratio of the third compound to 5-ethylthiotetrazole is preferably 1:1.4 to 1.6, more preferably 1:1.5. In the present invention, the molar ratio of the third compound to tert-butyl hydroperoxide is preferably 1:4 to 6, more preferably 1:5; the tert-butyl hydroperoxide is preferably used in the form of a tert-butyl hydroperoxide solution, the solvent of the tert-butyl hydroperoxide solution is preferably decane, and the concentration is preferably 5.5 mol / L. In this invention, the preferred process for mixing the third compound, the fourth compound shown in KBC-C4, 5-ethylthiotetrazole (ETT), tert-butyl hydroperoxide (t-BuOOH), and a third solvent to carry out the first coupling-oxidation reaction includes the following steps: dissolving the third compound in the third solvent to obtain a third compound solution; dissolving the fourth compound in the third solvent to obtain a fourth compound solution; sequentially adding ETT and the fourth compound solution to the third compound solution to carry out the first coupling reaction; and then adding a tert-butyl hydroperoxide solution to carry out the first oxidation reaction. In this invention, the addition temperature of the ETT and fourth compound solution is preferably 0~30℃, specifically preferably 0℃ or room temperature. In this invention, the addition temperature of the tert-butyl hydroperoxide solution is preferably 0~30℃. In this invention, the temperature of the first coupling reaction is preferably 0~30℃, and the time is preferably 30~60 min, more preferably 45 min; the first coupling reaction is preferably carried out under stirring conditions. In this invention, the temperature of the first oxidation reaction is preferably 0~30℃, and the time is preferably 30~60 min, more preferably 45 min; the first oxidation reaction is preferably carried out under stirring conditions. In this invention, the time of the first coupling-oxidation reaction is preferably 1~2 h, specifically preferably 1.5 h; the first coupling-oxidation reaction specifically refers to the total time of the first coupling reaction and the first oxidation reaction. After the first coupling-oxidation reaction is completed, this invention preferably includes a third post-treatment, which preferably includes the following steps: quenching the first coupling-oxidation reaction with sodium bicarbonate aqueous solution, separating the layers, and collecting the organic layer; washing, drying, filtering, and vacuum concentrating the organic layer sequentially to obtain a crude product; purifying the crude product using rapid column chromatography, concentrating the desired fraction, and obtaining the compound shown in KBC-C5A.In this invention, the washing preferably includes washing with sodium bicarbonate solution and washing with brine in sequence; the drying is preferably drying with Na2SO4.

[0028] In this invention: under the activation of ETT, the hydroxyl group of the third compound is coupled with the fourth compound to generate an Fm-protected phosphite intermediate; subsequently, the intermediate is oxidized by tert-butyl peroxide to an Fm-protected phosphate intermediate KBC-C5A.

[0029] After obtaining the compound shown in KBC-C5A, the present invention proceeds to step 4: mixing the compound shown in KBC-C5A, piperidine, and a third solvent to perform a first Fm group removal reaction, thereby obtaining the fifth compound shown in KBC-C5A. In this invention, the third solvent is consistent with the third solvent described in the above technical solution, and will not be repeated here. In this invention, the molar ratio of the third compound and piperidine used to prepare the compound shown in formula KBC-C5A is 1:1 to 1.6, specifically preferably 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, or 1:1.6; the volume ratio of piperidine to the third solvent is preferably 1:19. In this invention, the temperature of the first Fm group removal reaction is preferably 10 to 30°C, more preferably room temperature, i.e., neither additional heating nor additional cooling is required; the time of the first Fm group removal reaction is preferably 10 to 30 min, more preferably 10 min, 15 min, 20 min, 25 min, or 30 min. In this invention, the specific process of the first Fm group removal reaction preferably includes the following steps: dissolving the compound represented by KBC-C5A in a third solvent, then adding piperidine to carry out the first Fm group removal reaction. In this invention, during the first Fm group removal reaction, the target product will precipitate out as the reaction proceeds. After the first Fm group removal reaction is completed, this invention preferably further includes a fourth post-treatment, which preferably includes the following steps: adding diethyl ether to the obtained first Fm group removal reaction system, then separating the solid, dissolving the solid in water, and then freeze-drying the solid with acetonitrile to obtain the fifth compound.

[0030] In this invention, piperidine can selectively remove the Fm group to generate the fifth compound shown in formula KBC-C5, thus preparing for the next step of phosphorylation coupling.

[0031] After obtaining the fifth compound, the present invention proceeds to step 5: the fifth compound, the fourth compound shown in KBC-C4, 5-ethylthiotetrazole, tert-butyl hydroperoxide, and the third solvent are mixed to carry out a second coupling-oxidation reaction to obtain the compound shown in KBC-C6A. In the present invention, the third solvent is preferably the same as the third solvent described in the above technical solution, and will not be repeated here. In the present invention, the molar ratio of the fifth compound to the fourth compound is preferably 1:1.1~1.3, more preferably 1:1.2. In the present invention, the molar ratio of the fifth compound to 5-ethylthiotetrazole is preferably 1:1.4~1.6, more preferably 1:1.5. In the present invention, the molar ratio of the fifth compound to tert-butyl hydroperoxide is preferably 1:4~6, more preferably 1:5; the tert-butyl hydroperoxide is preferably used in the form of a tert-butyl hydroperoxide solution, the solvent of the tert-butyl hydroperoxide solution is preferably decane, and the concentration is preferably 5.5 mol / L. In this invention, the preferred process for carrying out the second coupling-oxidation reaction by mixing the fifth compound, the fourth compound shown in KBC-C4, 5-ethylthiotetrazole, tert-butyl hydrogen peroxide, and a third solvent includes the following steps: dissolving the fifth and fourth compounds in the third solvent, then adding ETT to carry out the second coupling reaction, and then adding a tert-butyl hydrogen peroxide solution to carry out the second oxidation reaction. In this invention, the addition temperature of the ETT is preferably 0-30°C. In this invention, the temperature of the second coupling reaction is preferably 0-30°C; the time is preferably 30-60 min, more preferably 45 min; the second coupling reaction is preferably carried out under stirring. In this invention, the addition temperature of the tert-butyl hydrogen peroxide solution is preferably 0-30°C; the temperature of the second oxidation reaction is preferably 0-30°C; the time is preferably 30-60 min, more preferably 45 min; the second oxidation reaction is preferably carried out under stirring. In this invention, the second coupling-oxidation reaction time is preferably 1-2 hours, specifically 1.5 hours; the second coupling-oxidation reaction specifically refers to the total time of the second coupling reaction and the second oxidation reaction. After the second coupling-oxidation reaction is completed, this invention preferably includes a fifth post-treatment, which preferably includes the following steps: adding diethyl ether to the second coupling-oxidation reaction system, and then performing solid-liquid separation to obtain a solid as the compound shown in KBC-C6A.

[0032] In this invention, the second coupling-oxidation reaction links another equivalent amount of the fourth compound to the fifth compound, yielding a compound with a further extended phosphate skeleton intermediate formula KBC-C6A.

[0033] To obtain the compound represented by KBC-C6A, the present invention proceeds to step 6: mixing the compound represented by KBC-C6A, piperidine, and a third solvent, and performing a second Fm group removal reaction to obtain the sixth compound represented by KBC-C6. In this invention, the type of the third solvent is preferably the same as described above, and will not be repeated here. In this invention, the molar ratio of the fifth compound and piperidine used to prepare the compound represented by KBC-C6A is preferably 1:0.3~0.8, specifically preferably 1:0.3, 1:0.35, 1:0.36, 1:0.4, 1:0.45, 1:0.46, 1:0.5, 1:0.6, 1:0.63, 1:0.7, 1:0.72, or 1:0.8; the volume ratio of piperidine to the third solvent is preferably 1:19. In this invention, the temperature of the second Fm group removal reaction is preferably 10-30°C, more preferably room temperature, i.e., no additional heating or cooling is required; the time of the second Fm group removal reaction is preferably 10-30 min, specifically preferably 10 min or 15 min; the second Fm group removal reaction is preferably carried out under stirring conditions. In this invention, the specific process of the second Fm group removal reaction preferably includes the following steps: dissolving the compound shown in KBC-C6A in a third solvent, then adding piperidine to carry out the second Fm group removal reaction. In this invention, during the second Fm group removal reaction, LCMS is preferably used for monitoring until the Fm-protected diphosphate ester of the intermediate completely disappears. After the second Fm group removal reaction is completed, the present invention preferably further includes a sixth post-treatment, which preferably includes the following steps: adding diethyl ether to the reaction solution obtained from the second Fm group removal reaction, and then centrifuging to obtain an oily substance; dissolving the oily substance in water, purifying it by reversed-phase column chromatography using a C18 column, collecting the desired components, and then sequentially performing vacuum concentration and freeze-drying on the components to obtain the sixth compound represented by formula KBC-C6; the vacuum concentration temperature is preferably 30°C.

[0034] In this invention, the second Fm group removal reaction can remove the Fm group, preparing for the synthesis of the triphosphate skeleton.

[0035] After obtaining the sixth compound, the present invention proceeds to step 7: the sixth compound, the fourth compound represented by formula KBC-C4, 5-ethylthiotetrazole, tert-butyl hydroperoxide, and the third solvent are mixed to perform a third coupling-oxidation reaction to obtain the compound represented by KBC-C7A. In the present invention, the third solvent is preferably the same as the third solvent described in the above technical solution, and will not be repeated here. In the present invention, the molar ratio of the sixth compound to the fourth compound is preferably 1:1.1~1.3, more preferably 1:1.2. In the present invention, the molar ratio of the sixth compound to 5-ethylthiotetrazole is preferably 1:1.4~1.6, more preferably 1:1.5. In the present invention, the molar ratio of the sixth compound to tert-butyl hydroperoxide is preferably 1:4~6, more preferably 1:5; the tert-butyl hydroperoxide is preferably used in the form of a tert-butyl hydroperoxide solution, the concentration of the tert-butyl hydroperoxide solution is preferably 5.5 mol / L, and the solvent is preferably decane. In this invention, the process of mixing the sixth compound, the fourth compound represented by formula KBC-C4, 5-ethylthiotetrazole, tert-butyl hydrogen peroxide, and a third solvent to carry out a third coupling-oxidation reaction preferably includes the following steps: dissolving the sixth and fourth compounds in the third solvent, adding ETT, and then carrying out a third coupling reaction; then adding a tert-butyl hydrogen peroxide solution to carry out a third oxidation reaction. In this invention, the addition temperature of the ETT is preferably 0-30°C. In this invention, the addition temperature of the tert-butyl hydrogen peroxide solution is preferably 0-30°C. In this invention, the temperature of the third coupling reaction is preferably 0-30°C, and the time is preferably 30-60 min, more preferably 45 min; the third coupling reaction is preferably carried out under stirring. In this invention, the temperature of the third oxidation reaction is preferably 0-30°C, and the time is preferably 30-60 min, more preferably 45 min; the third oxidation reaction is preferably carried out under stirring. In this invention, the time for the third coupling-oxidation reaction is preferably 1 to 2 hours, and more preferably 1.5 hours; the third coupling-oxidation reaction specifically refers to the total time of the third coupling reaction and the third oxidation reaction.

[0036] After the third coupling-oxidation reaction is completed, the present invention preferably includes a seventh post-treatment, which preferably includes the following steps: adding diethyl ether to the obtained third coupling-oxidation reaction solution, centrifuging to separate and collecting the compound shown in solid KBC-C7A.

[0037] In this invention, the third coupling-oxidation reaction couples the sixth compound with the fourth compound again to obtain the compound shown in the further extended triphosphate intermediate KBC-C7A.

[0038] After obtaining the compound represented by KBC-C7A, the present invention proceeds to step 8-1: the compound represented by KBC-C7A, piperidine, and a third solvent are mixed, and a third Fm group removal reaction is carried out to obtain the seventh compound represented by KBC-C7. In the present invention, the third solvent is preferably the same as the third solvent described in the above technical solution, and will not be repeated here. In the present invention, the molar ratio of the sixth compound and piperidine used to prepare the compound represented by KBC-C7A is preferably 1:0.3~0.8, specifically preferably 1:0.3, 1:0.35, 1:0.36, 1:0.4, 1:0.45, 1:0.46, 1:0.5, 1:0.6, 1:0.63, 1:0.7, 1:0.72, or 1:0.8; the volume ratio of piperidine to the third solvent is preferably 1:19, that is, the volume content of piperidine in the third solvent is preferably 5%. In the present invention, the third solvent preferably includes dimethylformamide (DMF). In this invention, the temperature of the third Fm group removal reaction is preferably 10-30°C, more preferably room temperature, i.e., no additional heating or cooling is required; the time of the third Fm group removal reaction is 10-30 min, specifically preferably 10 min or 15 min; the third Fm group removal reaction is preferably carried out under stirring conditions. In this invention, the specific process of the third Fm group removal reaction preferably includes the following steps: dissolving the compound represented by formula KBC-C7A in a third solvent, then adding piperidine to carry out the third Fm group removal reaction. In this invention, during the third Fm group removal reaction, LCMS is preferably used for monitoring until the Fm-protected phosphate in the intermediate has completely disappeared. After the third Fm group removal reaction is completed, the present invention preferably further includes an eighth post-treatment, which preferably includes the following steps: adding diethyl ether to the reaction solution for the third Fm group removal, and then centrifuging to obtain an oily substance; dissolving the oily substance in water, purifying it by reversed-phase column chromatography using a C18 column, collecting the relevant components, and sequentially performing vacuum concentration and freeze-drying to obtain the seventh compound represented by formula KBC-C7; the vacuum concentration temperature is preferably 30°C.

[0039] In this invention, the third Fm group removal reaction selectively deprotects / removes Fm to obtain a triphosphate skeleton, yielding the seventh compound represented by formula KBC-C7.

[0040] After obtaining the seventh compound shown in KBC-C7, the present invention proceeds to step 9-1: the seventh compound, the alkynyl chloride shown in formula KBC-C8-1, copper sulfate, sodium ascorbate, and the fourth solvent are mixed, and a first click reaction and a first ion exchange reaction are carried out sequentially to obtain the thiol-reactive nucleotide derivative. In the present invention, the molar ratio of the seventh compound to the alkynyl chloride is preferably 1:1.2 to 1.6, more preferably 1:1.5. In the present invention, the molar ratio of the seventh compound to copper sulfate is preferably 1:0.1 to 0.3, specifically preferably 1:0.2. In the present invention, the molar ratio of the seventh compound to sodium ascorbate is preferably 1:0.3 to 0.5, more preferably 1:0.4. In the present invention, the fourth solvent preferably comprises acetone and water, and the volume ratio of acetone to water is preferably 8:2. In this invention, the temperature of the first click chemistry is preferably 10~30℃, more preferably room temperature, i.e., no additional heating or cooling is required; the time is preferably 0.5~2h, specifically 0.5h, 1h, 1.5h, or 2h; the first click reaction is preferably carried out under stirring. In this invention, the process of the first click reaction preferably includes the following steps: dissolving the seventh compound in the fourth solvent, then adding alkynyl chloride, copper sulfate, and sodium ascorbate to carry out the first click reaction. In this invention, the process of the first click reaction is preferably monitored by LCMS until the seventh compound disappears. After the first click reaction is completed, this invention preferably also includes a ninth post-processing, which preferably includes the following steps: evaporating the solvent in the first click reaction solution, then performing first reversed-phase column chromatography purification using C18 AQ column chromatography to obtain the relevant components, and then concentrating them under vacuum to obtain the first click product.

[0041] In this invention, the resin used in the first ion exchange reaction is preferably Na. + Resin. In this invention, the temperature of the first ion exchange reaction is preferably room temperature, and the time is preferably 1-3 hours, more preferably 2 hours; the first ion exchange reaction is preferably carried out under stirring conditions. In this invention, the specific process of the first ion exchange reaction preferably includes the following steps: dissolving the first click product in water, and then adding Na... + The resin undergoes a first ion exchange reaction. After the first ion exchange reaction, the present invention preferably further includes: filtration to remove Na. + The resin was used to remove water from the filtrate under vacuum, followed by a second reversed-phase column chromatography purification using a C18 AQ column to obtain a second purified product. The second purified product was then dissolved in water and passed through a column packed with Na... + The resin column is used to collect the eluent; after evaporating to remove the solvent from the eluent, it is freeze-dried to obtain the thiol-reactive nucleotide derivative.

[0042] In this invention, under the catalysis of Cu(I) (generated in situ from CuSO4 / sodium ascorbate), azide (-N3) undergoes a 1,3-dipolar cycloaddition (click reaction) with an alkyne group to form a triazole linker, while simultaneously generating the corresponding sodium triphosphate form, to obtain the final product, the thiol-reactive nucleotide derivative.

[0043] In this invention, the thiol-reactive nucleotide derivative TReND1 has a triphosphate structure with a triazole linker at one end and a chloroacetamide structure at the other end.

[0044] In this invention, the preparation method of the compound when n is 2 or 3 in Formula 1 is described below as follows: replacing steps 8-1 and 9-1 with: Step 8-2: Mix the compound represented by formula KBC-C7A, piperidine, and the third solvent, and carry out the fourth Fm group removal reaction to obtain the compound represented by formula KBC-C7B; the molar ratio of the sixth compound and piperidine is 1:0.3~0.8, and the time of the fourth Fm group removal reaction is 5 min; Formula KBC-C7B; Step 9-2: Mix the compound shown in KBC-C7B, the alkynyl chloride shown in KBC-C8-2, copper sulfate, sodium ascorbate and the fourth solvent, and carry out the second click reaction to obtain the tenth compound shown in KBC-C10; KBC-C8-2, KBC-C10; Step 10-2: The tenth compound is subjected to a fifth Fm group removal reaction and a second ion exchange reaction in a third solvent containing triethylamine to obtain the thiol-reactive nucleotide derivative.

[0045] The preparation method of the compound represented by formula KBC-C7A is the same as the above-described technical solution, and will not be repeated here.

[0046] After obtaining the compound shown in KBC-C7A, the present invention proceeds to step 8-2: the compound shown in KBC-C7A, piperidine, and the third solvent are mixed and a fourth Fm group removal reaction is carried out to obtain the compound shown in KBC-C7B; the molar ratio of the sixth compound and piperidine is 1:0.3~0.8, and the time of the fourth Fm group removal reaction is 5 min.

[0047] In this invention, the type of the third solvent is preferably the same as that described above, and will not be repeated here. In this invention, the molar ratio of the sixth compound and piperidine used to prepare the compound represented by formula KBC-C7A is preferably 1:0.3~0.8, specifically preferably 1:0.3, 1:0.35, 1:0.36, 1:0.4, 1:0.45, 1:0.46, 1:0.5, 1:0.6, 1:0.63, 1:0.7, 1:0.72, or 1:0.8; the volume ratio of piperidine to the third solvent is preferably 1:19. In this invention, the temperature of the fourth Fm group removal reaction is preferably 10~30℃, more preferably room temperature, i.e., neither additional heating nor additional cooling is required; the time is 5 min. After the fourth Fm group removal reaction is completed, the present invention preferably further includes: adding diethyl ether, centrifuging to separate and collecting the solid; dissolving the solid in water, performing reversed-phase column chromatography using a C18 AQ column, concentrating the desired components under vacuum at 30°C, and then freeze-drying to obtain the compound shown in KBC-C7B.

[0048] After obtaining the compound represented by formula KBC-C7B, the present invention proceeds to step 9-2: the compound represented by KBC-C7B, the alkynyl chloride represented by formula KBC-C8-2, copper sulfate, sodium ascorbate and the fourth solvent are mixed and subjected to a second click reaction to obtain the tenth compound represented by KBC-C10.

[0049] In this invention, the molar ratio of the compound represented by formula KBC-C7B to the alkynyl chloride is preferably 1:1 to 1.6, more preferably 1:1, 1:1.1, 1:1.15, 1:1.2, 1:1.3, 1:1.4, 1:1.5, or 1:1.6. In this invention, the molar ratio of the compound represented by formula KBC-C7B to copper sulfate is preferably 1:0.1 to 0.3, more preferably 1:0.15, 1:0.167, or 1:0.2. In this invention, the molar ratio of the compound represented by formula KBC-C7B to sodium ascorbate is preferably 1:0.3 to 0.5, more preferably 1:0.3, 1:0.33, 1:0.38, 1:0.4, 1:0.45, or 1:0.5. In this invention, the temperature of the second click reaction is preferably 10-30°C, more preferably room temperature; the time is preferably 0.5-2 h; and the second click reaction is preferably carried out under stirring. In this invention, the process of the second click reaction preferably includes the following steps: dissolving the seventh compound in a fourth solvent, then adding alkynyl chloride, copper sulfate, and sodium ascorbate to carry out the second click reaction. In this invention, during the second click reaction, the reaction mixture is preferably monitored using LCMS, and the reaction is stopped when the corresponding mass is observed. After the second click reaction is completed, this invention preferably further includes: evaporating the solvent, then purifying the mixture using reversed-phase column chromatography with a C18 AQ column to concentrate the relevant components and obtain the tenth compound represented by formula KBC-C10.

[0050] After obtaining the tenth compound, the present invention proceeds to step 10-2: the tenth compound is subjected to a fifth Fm group removal reaction and a second ion exchange reaction in a third solvent containing triethylamine to obtain the thiol reactive nucleotide derivative.

[0051] In this invention, the third solvent is preferably consistent with the above-described technical solution and will not be repeated here. In this invention, the volume concentration of triethylamine in the third solvent containing triethylamine is preferably 50%; the ratio of the tenth compound to the third solvent containing triethylamine is preferably 0.02~0.03 mmol:0.3 mL, specifically preferably 0.025 mmol:0.3 mL or 0.03 mmol:0.3 mL. In this invention, the temperature of the fifth Fm group removal reaction is preferably 10~30°C, more preferably room temperature, i.e., neither additional heating nor additional cooling is required; the time of the fifth Fm group removal reaction is preferably 8~12 h; the fifth Fm group removal reaction is preferably carried out under stirring conditions. In this invention, the specific process of the fifth Fm group removal reaction preferably includes the following steps: dissolving the tenth compound represented by formula KBC-C10 in a third solvent containing triethylamine to carry out the fifth Fm group removal reaction. In this invention, the process of the fifth Fm group removal reaction is preferably monitored using LCMS. After the fourth Fm group removal reaction is completed, the present invention preferably further includes: adding diethyl ether to the fifth Fm group removal reaction solution, separating the precipitate by centrifugation to obtain a solid; dissolving the solid in water, and purifying it by first reversed-phase column chromatography using a C18 AQ column, collecting the relevant components, and sequentially concentrating and drying them under vacuum to obtain a thiol reactive nucleotide derivative in the form of piperidine salt.

[0052] In this invention, the resin used in the second ion exchange reaction is preferably Na. + Resin; the temperature of the second ion exchange reaction is preferably room temperature, and the time is preferably 2 hours. In this invention, the second ion exchange reaction preferably includes the following steps: dissolving a thiol-reactive nucleotide derivative in the form of a piperidine salt in water, and then adding Na... + The resin undergoes a second ion exchange reaction.

[0053] After the second ion exchange reaction is completed, the present invention preferably further includes: filtering to remove the solid, collecting the liquid, removing the water from the liquid under vacuum, purifying it by second reversed-phase column chromatography using a C18 AQ column, collecting the relevant components and freeze-drying them to obtain the thiol-reactive nucleotide derivative.

[0054] The following detailed description of the synthesis method of the thiol-reactive nucleotide derivatives provided by the present invention, with reference to the embodiments, should not be construed as limiting the scope of protection of the present invention.

[0055] Example 1: Preparation of TReND1 Step 1: At 0 °C, BF3·Et2O (30.162 mmol) was added to a stirred solution of KBC-C1 (8 g, 25.135 mmol) in 80 mL of DCM, and the mixture was stirred for 5 min. Then, TMSN3 (30.162 mmol) was added, and the reaction mixture was stirred at 0 °C to room temperature for 4 h. TLC analysis showed that KBC-C1 was completely consumed. The reaction was quenched with saturated NaHCO3 solution, the product was extracted with CH2Cl2, the organic layer was washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated under vacuum to obtain the crude product. The crude product was purified by rapid column chromatography, and the desired fraction was concentrated to obtain the colorless oily product KBC-C2 (6.3 g, 83.2%).

[0056] KBC-C2 was characterized by 1H NMR and 1C NMR spectra, and the results are as follows: Figure 1 and Figure 2 As shown, the specific information is as follows: 1 H NMR (500 MHz, Chloroform-d) δ 5.36 (d, J = 2.0 Hz, 1H), 5.33 (dd, J =6.8, 4.8 Hz, 1H), 5.13 (dd, J = 4.9, 2.0 Hz, 1H), 4.41 (dd, J = 12.2, 3.2 Hz,1H), 4.38 - 4.32 (m, 1H), 4.15 (dd, J = 12.2, 4.3 Hz, 1H), 2.12 (two singlets, 6H), 2.07 (s, 3H), where 7.28 is the solvent peak of deuterated chloroform; it can be seen that KBC-C2 only has the β configuration and does not contain the α configuration.

[0057] 13 C NMR (126 MHz, CDCl3) δ 170.57, 169.55, 169.41, 92.64, 79.36,74.46, 70.44, 62.99, 20.66, 20.49, 20.44. Step 2: To a stirred solution of KBC-C2 (6.3 g, 20.912 mmol) in methanol (60 mL), NaOMe (0.565 g, 10.456 mmol) was added, and the mixture was stirred overnight at room temperature. TLC analysis showed that KBC-C2 was completely consumed. The reaction mixture was neutralized to pH ~7 with IR 120 resin, filtered, and concentrated under vacuum to obtain a crude product. The crude product was purified by rapid column chromatography, and the desired fraction was concentrated to obtain a colorless oily product, KBC-C3 (2.92 g, 79.7%).

[0058] KBC-C3 was characterized by 1H NMR spectroscopy, and the results are as follows: Figure 3 As shown, the specific information is as follows: 1 H NMR (500 MHz, Methanol-d4) δ 5.21 (d, J = 1.8 Hz, 1H), 4.07 (dd, J =6.9, 4.7 Hz, 1H), 4.00 (ddd, J = 6.9, 5.7, 3.3 Hz, 1H), 3.84 (dd, J = 4.6, 1.8Hz, 1H), 3.78 (dd, J = 12.0, 3.3 Hz, 1H), 3.63 (dd, J = 12.1, 5.7 Hz, 1H). Steps 3 and 4: Compound KBC-C3 (2.8 g, 15.987 mmol) was dissolved in dry DMF, and ETT (3.122 g, 23.980 mmol) was added at 0 °C, followed by a DMF solution of KBC-C4 (10.007 g, 19.184 mmol). The mixture was stirred for 45 min, and then t-BuOOH solution (5.5 M in decane) (14.534 mL, 79.935 mmol) was added. The mixture was stirred at 0 °C for 45 min. The reaction mixture was quenched with an aqueous sodium bicarbonate solution, and the organic layer was washed with an aqueous sodium bicarbonate and salt solution. The organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by rapid column chromatography, and the desired fraction was concentrated to obtain a colorless oily Fm-protected intermediate.

[0059] The intermediate was dissolved in DMF and piperidine (2.369 mL, 23.980 mmol) was added, with a volume ratio of piperidine to DMF of 1:19. The mixture was stirred at room temperature for 30 min. After the reactants precipitated, diethyl ether was added, the solid was separated, dissolved in water, and freeze-dried with acetonitrile. The desired compound KBC-C5 (3.6 g, 52.9%) was a white solid in the form of a piperidine salt.

[0060] The proton and phosphorus NMR spectra of the obtained KBC-C5 were characterized, and the results are as follows: Figure 4 and Figure 5 As shown, the specific information is as follows: LCMS: m / z - calculated - 255.1, found - (MH) + - 254.5. 1 H NMR (500 MHz, Deuterium Oxide) δ 5.27 (d, J = 2.5 Hz, 1H), 4.18 (t, J = 5.4 Hz, 1H), 4.07 (q, J = 5.2 Hz, 1H), 3.96 (dd, J = 4.8, 2.6 Hz, 1H), 3.84(dt, J = 11.6, 4.7 Hz, 1H), 3.81 - 3.73 (m, 1H), 3.06 (t, J = 5.7 Hz, 8H), 1.68(p, J = 5.8 Hz, 8H), 1.57 (p, J = 6.0 Hz, 4H). 31 P NMR (202 MHz, D2O) δ 3.67. Steps 5 and 6: KBC-C5 (1.20 g, 2.823 mmol) and KBC-C4 (1.77 g, 3.388 mmol) were added to a round-bottom flask containing DMF. Then, ETT (0.55 g, 4.235 mmol) was added at room temperature. The reaction mixture was stirred for 45 min, and then t-BuOOH solution (5.5 M, decane solvent, 2.566 mL, 14.115 mmol) was added at 0 °C. The mixture was stirred again for 45 min. After the addition of diethyl ether, the solid was separated by centrifugation, and the molecular weight of the solid was determined by LC-MS, showing the desired mass of Fm-protected diphosphate.

[0061] The white solid was then dissolved in DMF, and piperidine (0.2 mL, 2.03 mmol) was added, with a piperidine to DMF volume ratio of 1:19. The mixture was stirred for 15 min, and the reaction was monitored by LCMS, showing that the diphosphate protected by intermediate Fm had completely disappeared. Diethyl ether was added, and centrifugation yielded an oily substance. This was dissolved in water, and the desired fraction was concentrated under vacuum at 30 °C using reversed-phase column chromatography on a C18 column, then freeze-dried. The desired compound KBC-C6 (725 mg, 50.81%) was a white solid containing a piperidine salt.

[0062] The proton and phosphorus NMR spectra of the obtained KBC-C6 were characterized, and the results are as follows: Figure 6 and Figure 7 As shown, the specific information is as follows: LCMS - m / z - calculated - 335.1, found - (MH) + - 334.4. 1 H NMR (500 MHz, Deuterium Oxide) δ 5.27 (d, J = 2.3 Hz, 1H), 4.26 (dd, J = 6.2, 4.7 Hz, 1H), 4.14 - 4.03 (m, 2H), 4.03 - 3.93 (m, 2H), 3.07 (t, J = 5.7Hz, 8H), 1.69 (p, J = 5.8 Hz, 8H), 1.58 (q, J = 6.0 Hz, 4H). 31 P NMR (202 MHz, Deuterium Oxide) δ -10.83 (d, J = 20.3 Hz), -11.23 (d, J= 20.4 Hz). Steps 7 and 8: ETT (42.44 mg, 0.326 mmol) was added to a round-bottom flask containing KBC-C6 (110 mg, 0.217 mmol) and KBC-C4 (136 mg, 0.261 mmol) and dissolved in DMF at room temperature. After stirring for 45 min, t-BuOOH solution (5.5 M, decane solvent, 0.198 mL, 1.089 mmol) was added to the reaction mixture at 0 °C, and stirring was continued for another 45 min. After adding diethyl ether, the solid was separated by centrifugation. LC-MS analysis of the molecular weight of the solid showed di-Fm protected triphosphate.

[0063] The white solid was dissolved in DMF, and piperidine (0.01 mL, 0.1 mmol) was added, with a piperidine to DMF volume ratio of 1:19. The reaction was stirred for 15 min, and LC-MS showed that the phosphate protected by intermediate Fm had completely disappeared. Ether was added, and centrifugation yielded an oily substance. This was dissolved in water, and reversed-phase column chromatography was performed using a C18 column. The desired fraction was concentrated under vacuum at 30 °C, then freeze-dried. The desired compound KBC-C7 (70 mg, 48.10%) was a white solid containing a piperidine salt.

[0064] The proton and phosphorus NMR spectra of the obtained KBC-C7 were characterized, and the results are as follows: Figure 8 and Figure 9 As shown, the specific information is as follows: LCMS - m / z - calculated - 415.0, found - (M+H-2HPO3) + - 254.5. 1 H NMR (500 MHz, Deuterium Oxide) δ 5.26 (d, J = 2.4 Hz, 1H), 4.29 (t, J = 5.4 Hz, 1H), 4.13 - 4.07 (m, 2H), 4.07 - 4.00 (m, 1H), 3.95 (dd, J = 5.0, 2.4Hz, 1H), 3.07 (t, J = 5.6 Hz, 12H), 1.69 (t, J = 5.7 Hz, 12H), 1.60 - 1.55 (m,6H). 31 P NMR (203 MHz, Deuterium Oxide) δ -10.46, -11.23, -22.96. Step 9: KBC-C7 (50 mg, 0.0746 mmol) was dissolved in acetone and water (8:2), and then KBC-C8-1 (14.72 mg, 0.1119 mmol), CuSO4 (2.38 mg, 0.0149 mmol), and sodium ascorbate (5.91 mg, 0.0298 mmol) were added. The reaction mixture was stirred at room temperature for 30 min. The reaction mixture was monitored by LC-MS, and the corresponding mass was observed, and the mass of KBC-C7 also disappeared. The reaction was stopped and the solvent was evaporated. Then, reversed-phase column chromatography was performed using a C18 AQ column, and the relevant components were concentrated under vacuum at 30 °C to give 40 mg of TReND1 in the form of piperidine salt.

[0065] 1 H NMR showed the desired peak, but P31 NMR did not show it, so Na NMR was used. + Ion exchange resins convert piperidine salts into sodium salts of the corresponding compounds.

[0066] Dissolve 40 mg of the piperidine salt form of TReND1 in 0.5 mL of water, then add 200 mg of Na. + The resin was stirred at room temperature for 2 hours and then filtered. The reaction mixture was dehydrated under vacuum and subjected to reversed-phase column chromatography using a C18 AQ column to obtain 15 mg of the compound, still containing some piperidine salt. This 15 mg compound was then dissolved again in water and subjected to Na+ chromatography on a small column. + The resin underwent ion exchange. After solvent evaporation and freeze-drying, 6.3 mg of TReND1 in the form of sodium salt with a concentration of 13.78% was obtained.

[0067] The obtained TReND1 was characterized by 1H NMR and 1P NMR spectroscopy, and the results are shown in the figure. Figure 10 and Figure 11 The specific information is as follows: LCMS - m / z - calculated - 546.6, found - m / z- (MH) + - 545.5, (M / 2) + -272. 1H NMR (600 MHz, Deuterium Oxide) δ 8.22 (s, 1H), 6.11 - 6.08 (m,1H), 4.63 (s,1H), 4.50 (d, J = 14.0 Hz, 3H), 4.35 (s, 1H), 4.14 (d, J = 24.5Hz, 4H). 31 P NMR (202 MHz, D2O) δ -10.78, -21.94 (broad peak). In step 8, if KBC-C7 is in its pure form, the final compound (TReND1) will be obtained in its pure form. However, if diphosphate (KBC-C6) is present in the reaction, the corresponding triazole will also be obtained; these are difficult to separate during purification. This is why the reaction schemes for TReND2 and TReND3 are modified as follows.

[0068] Example 2: Preparation of TReND2 Steps 1-6 are the same as in Example 1; Steps 7 and 8: KBC-C6 (80 mg, 0.158 mmol) was dissolved in DMF, then ETT (30.85 mg, 0.237 mmol) and KBC-C4 (99 mg, 0.189 mmol) solutions were added, and the mixture was stirred for 45 min. Then, t-BuOOH solution (0.79 mmol) was added at room temperature, and the mixture was stirred again for 45 min. Diethyl ether was then added, and the solid was separated by centrifugation. LC-MS analysis showed the corresponding Fm-protected triphosphate.

[0069] The solid was dissolved in DMF, and piperidine (0.01 mL, 0.1 mmol) was added, with a piperidine to DMF volume ratio of 1:19. The mixture was stirred at room temperature for 5 min, and the reaction was monitored by LC-MS. Dry diethyl ether was added, and the precipitate was separated by centrifugation. The solid was dissolved in water, and the relevant components were concentrated under vacuum at 30 °C using reversed-phase column chromatography on a C18 AQ column. After freeze-drying, a white solid KBC-C7B (42 mg, 31.32%) was obtained.

[0070] The obtained KBC-C7B was characterized by 1H NMR, 1C NMR, and 1NMR spectra, and the results are as follows: Figures 12-14 As shown, the specific information is as follows: LCMS–observed-592.2 (MH) + , calculated as -593.35. 1 H NMR (500 MHz, Deuterium Oxide) δ 7.74 - 7.71 (m, 4H), 7.39 - 7.29(m, 4H), 5.15 (d, J = 2.3 Hz, 1H), 4.27-4.19 (m, 3H), 4.11 (dd, J = 6.0, 4.7Hz, 1H), 4.03 - 3.97 (m, 2H), 3.94 - 3.88 (m, 1H), 3.82 (dd, J = 4.7, 2.4 Hz,1H), 2.99 - 2.94 (m, 10H), 1.60 (p, J = 5.8 Hz, 11H), 1.48 (dd, J = 7.1, 4.5Hz, 5H). 13 C NMR (126 MHz, D2O) δ 144.11, 140.85, 127.90, 127.42, 125.61,119.99, 94.33, 82.25, 82.18, 74.45, 70.03, 67.93, 67.89, 65.61, 65.56, 47.97,47.91, 47.11, 44.42, 22.17, 21.45, 18.23. 31 P NMR (202 MHz, Deuterium Oxide) δ -11.35, -23.07. Step 9: KBC-C7B (40 mg, 0.047 mmol) was dissolved in acetone and water (8:2), and then KBC-C8-2 (10.2 mg, 0.070 mmol), CuSO4 (1.50 mg, 0.0094 mmol), and sodium ascorbate (3.72 mg, 0.0188 mmol) were added. The reaction mixture was stirred at room temperature for 30 min. The reaction mixture was monitored by LCMS, and the corresponding masses were observed. The reaction was stopped, the solvent was evaporated, and then the desired fraction was concentrated by reversed-phase column chromatography using a C18 AQ column to give KBC-C10-1 (26 mg, 55.63%) as a white solid.

[0071] The obtained KBC-C10-1 was characterized by 1H NMR and 1NMR spectroscopy, and the results are as follows: Figure 15 and Figure 16 The specific information is as follows: 1 H NMR (500 MHz, Methanol-d4) δ 8.34 (s, 1H), 7.80 (t, J = 9.0 Hz, 4H), 7.38 (t, J = 7.4 Hz, 2H), 7.30 (t, J = 6.8 Hz, 2H), 6.06 (s, 1H), 4.57 (s, 2H), 4.30 (d, J = 21.8 Hz, 4H), 4.07 (s, 2H), 3.68 - 3.63 (m, 1H), 3.54 (d, J = 6.7Hz, 2H), 3.41 - 3.36 (m, 2H), 3.10 (s, 7H), 2.96 (t, J = 6.3 Hz, 2H), 1.75 (s,7H), 1.65 - 1.62 (m, 4H). 31 P NMR (202 MHz, MeOD) δ -11.18, -22.43. Step 10: KBC-C10-1 (25 mg, 0.025 mmol) was dissolved in triethylamine (0.15 mL) and DMF (0.15 mL), and the reaction was stirred at room temperature, monitored by LC-MS. After the reaction was complete, dry diethyl ether was added, and the precipitate was separated by centrifugation. The solid was dissolved in water, and the fraction was concentrated under vacuum at 30 °C using reversed-phase column chromatography on a C18 AQ column. The fraction was then lyophilized to give a white solid, TReND2 piperidine salt (12 mg).

[0072] 1 H NMR showed a relevant peak, but P31 NMR did not. Therefore, Na + Ion exchange resins convert piperidine salts into sodium salts of the corresponding compounds.

[0073] Dissolve the above-mentioned piperidine salt form of TReND2 (12 mg) in 0.3 mL of water, and add 200 mg of Na. + The resin was stirred at room temperature for 2 hours and then filtered. The reaction mixture was dehydrated under vacuum and subjected to reversed-phase column chromatography again using a C18 AQ column. The mixture was then freeze-dried to give sodium TReND2 (4.6 mg, 29.36%) as a white solid.

[0074] The obtained TReND2 was subjected to 1H NMR and 1P NMR spectra, and the results are shown in the figure. Figure 17 and Figure 18 The specific information is as follows: LCMS: Observed- m / z- 559 (MH)+, Calculated- m / z-560. 1 H NMR (500 MHz, Deuterium Oxide) δ 8.12 (s, 1H), 6.06 (d, J = 5.2 Hz, 1H), 4.59 (t, J = 5.1 Hz, 1H), 4.47 (t, J = 4.4 Hz, 1H), 4.32 (d, J = 3.5 Hz, 1H), 4.14 (s, 2H), 4.00 (s, 2H), 3.48 (td, J = 6.5, 2.7 Hz, 2H), 2.92 (t, J =6.7 Hz, 2H). 31 P NMR (202 MHz, D2O) δ -10.34, -11.33, -23.00. Example 3: Preparation of TReND3 Steps 1-8 are the same as in Example 2; Step 9: KBC-C7B (100 mg, 0.117 mmol) was dissolved in acetone and water (8:2), and then KBC-C8-3 (28.1 mg, 0.176 mmol), CuSO4 (3.73 mg, 0.0234 mmol), and sodium ascorbate (9.27 mg, 0.0468 mmol) were added. The reaction mixture was stirred at room temperature for 30 min. The reaction mixture was monitored by LCMS, and the desired mass was observed, and the mass of KBC-C7B also disappeared. The reaction was then stopped, the solvent was evaporated, and the desired fraction was concentrated by reversed-phase column chromatography using a C18 AQ column to give a white solid, KBC-C10-2 (46 mg, 38.98%) in the form of piperidine salt.

[0075] The obtained KBC-C10-2 was characterized by 1H NMR, 1C NMR, and 1P NMR spectra. The results are shown in the figure. Figures 19-21 The specific information is as follows: 11H NMR (500 MHz, Deuterium Oxide) δ 7.78 (s, 1H), 7.70 (dd, J J = 7.6, 3.4 Hz, 2H), 7.61 (dd, J J = 7.6, 3.1 Hz, 2H), 7.32 (td, J J = 7.5, 2.6 Hz, 2H), 7.23 (t, J J = 7.5 Hz, 2H), 5.84 (d, J J = 4.9 Hz, 1H), 4.37 (t, J J = 5.0 Hz, 1H), 4.30 (t, J J = 4.6 Hz, 1H), 4.18 - 4.03 (m, 6H), 3.89 (s, 2H), 3.39 - 3.27 (m, 2H), 3.00 (t, J J = 5.7 Hz, 9H), 2.95 (t, J J = 6.9 Hz, 2H), 2.35 (t, J J = 7.6 Hz, 2H), 1.63 (p, J J = 5.8 Hz, 9H), 1.51 (q, J J = 6.1, 4.3 Hz, 6H). 31 31P NMR (202 MHz, D2O) δ -11.09, -22.71. 13 13C NMR (126 MHz, D2O) δ 169.24, 143.95, 143.92, 140.74, 127.83, 127.28, 127.25, 125.51, 119.93, 92.05, 83.98, 75.16, 70.13, 67.95, 65.06, 47.89, 47.84, 47.15, 46.53, 44.46, 42.16, 39.17, 27.16, 22.18, 21.96, 21.46, 18.24, 8.16. Step 10: KBC-C10-2 (30 mg, 0.0297 mmol) was dissolved in triethylamine (0.15 mL) and DMF (0.15 mL), and the reaction was stirred at room temperature, with the reaction monitored by LC-MS. After the reaction was complete, dry diethyl ether was added, and the precipitate was separated by centrifugation. The solid was dissolved in water, and the desired fraction was concentrated by reversed-phase column chromatography using a C18 AQ column under vacuum at 30 °C. The concentrate was then lyophilized to give a white solid, TReND3 piperidine salt (16 mg).

[0076] 1 H NMR showed relevant peaks, but P31 NMR did not. Therefore, it was decided to use Na. + Ion exchange resins convert piperidine salts into sodium salts of the corresponding compounds.

[0077] Dissolve 16 mg of the above-mentioned white solid TReND3 in piperidine salt form in 0.3 mL of water, then add 200 mg of Na. + The resin was stirred at room temperature for 2 hours and then filtered. The reaction mixture was dehydrated under vacuum and subjected to reversed-phase column chromatography again using a C18 AQ column. The fractions were freeze-dried to obtain sodium TReND3 (10 mg, 52.56%) as a white solid.

[0078] The obtained TReND3 was characterized by 1H NMR and 1P NMR spectroscopy, and the results are shown in the figure. Figure 22 and Figure 23 The specific information is as follows: LCMS: Observed- m / z- 575 (M+H) + , Calculated - m / z-574. 1 H NMR (500 MHz, Deuterium Oxide) δ 8.09 (s, 1H), 6.06 (d, J = 3.7 Hz,1H), 4.63 - 4.60 (m, 1H), 4.50 (s, 1H), 4.36 - 4.32 (m, 1H), 4.18 - 4.14 (m,2H), 4.01 (s, 2H), 3.22 (t, J = 6.5 Hz, 2H), 2.72 (t, J = 7.2 Hz, 2H), 1.89 -1.86 (m, 2H). 31 P NMR (202 MHz, D2O) δ -10.51, -22.0 (Broad peak). Comparative Example 1 At 0 °C, SnCl4 (25.1 mmol) was added to a stirred DCM (80 mL) solution of KBC-C1 (8 g, 25.1 mmol) and stirred for 5 min. Then, TMSN3 (27.6 mmol) was added, and the reaction mixture was stirred at room temperature for 12 h. TLC analysis showed that KBC-C1 was completely consumed. The reaction was quenched with saturated NaHCO3 solution, the product was extracted with CH2Cl2, the organic layer was washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated under vacuum to obtain the crude product. The crude product was purified by rapid column chromatography, and the desired fraction was concentrated to obtain the colorless oily product KBC-C2.

[0079] KBC-C2 was characterized by 1H NMR and 1C NMR spectra, and the results are as follows: Figure 24 As shown, the specific information is as follows. From Figure 24 It can be seen that the KBC-C2 prepared in Comparative Example 1 contains α and β configurations in a ratio of 1:3.

[0080] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing KBC-C2, characterized in that, Includes the following steps: The first compound shown in KBC-C1, trimethylsilyl azide, boron trifluoride diethyl ether and the first solvent were mixed and subjected to an azide substitution reaction to obtain the second compound shown in KBC-C2. KBC-C1, KBC-C2。 2. The preparation method according to claim 1, characterized in that, The molar ratio of the first compound to trimethylsilyl azide is 1:1.1~1.3, and the molar ratio of the first compound to boron trifluoride diethyl ether is 1:1.1~1.3; The azide substitution reaction is carried out at a temperature of 0-30℃ for 3-5 hours.

3. A method for synthesizing a thiol-reactive nucleotide derivative, characterized in that, The thiol-reactive nucleotide derivative has the structure shown in Formula 1: Formula 1; When n is 1 in Equation 1, the synthesis method includes the following steps: Step 1: Prepare the second compound shown in KBC-C2 according to the preparation method described in claim 1 or 2; Step 2: The second compound, sodium methoxide, and the second solvent are mixed and subjected to a deprotection / ester exchange reaction to obtain the third compound shown in KBC-C3; KBC-C3; Step 3: Mix the third compound, the fourth compound shown in KBC-C4, 5-ethylthiotetrazole, tert-butylhydrogen peroxide and the third solvent, and carry out the first coupling-oxidation reaction to obtain the compound shown in KBC-C5A; KBC-C4, KBC-C5A; Step 4: Mix the compound shown in KBC-C5A, piperidine, and the third solvent to carry out the first Fm group removal reaction to obtain the fifth compound shown in KBC-C5; the molar ratio of the third compound and piperidine is 1:1~1.6, and the time of the first Fm group removal reaction is 10~30 min; KBC-C5; Step 5: Mix the fifth compound, the fourth compound shown in KBC-C4, 5-ethylthiotetrazole, tert-butylhydrogen peroxide and the third solvent to carry out the second coupling-oxidation reaction to obtain the compound shown in KBC-C6A; KBC-C6A; Step 6: Mix the compound represented by formula KBC-C6A, piperidine, and the third solvent to carry out the second Fm group removal reaction to obtain the sixth compound represented by KBC-C6; the molar ratio of the fifth compound to piperidine is 1:0.3~0.8, and the time of the second Fm group removal reaction is 10~30 min; KBC-C6; Step 7: Mix the sixth compound, the fourth compound shown in formula KBC-C4, 5-ethylthiotetrazole, tert-butylhydrogen peroxide and the third solvent, and carry out the third coupling-oxidation reaction to obtain the compound shown in KBC-C7A. KBC-C7A; Step 8-1: Mix the compound represented by formula KBC-C7A, piperidine, and the third solvent to carry out the third Fm group removal reaction to obtain the seventh compound represented by KBC-C7; the molar ratio of the sixth compound to piperidine is 1:0.3~0.8, and the time of the third Fm group removal reaction is 10~30 min; KBC-C7; Step 9-1: Mix the seventh compound, the alkynyl chloride shown in formula KBC-C8-1, copper sulfate, sodium ascorbate and the fourth solvent, and perform the first click reaction and the first ion exchange reaction in sequence to obtain the thiol reactive nucleotide derivative; KBC-C8-1; When n is 2 or 3 in Equation 1, replace steps 8-1 and 9-1 with: Step 8-2: Mix the compound represented by formula KBC-C7A, piperidine, and the third solvent, and carry out the fourth Fm group removal reaction to obtain the compound represented by formula KBC-C7B; the molar ratio of the sixth compound and piperidine is 1:0.3~0.8, and the time of the fourth Fm group removal reaction is 5 min; Formula KBC-C7B; Step 9-2: Mix the compound shown in KBC-C7B, the alkynyl chloride shown in KBC-C8-2, copper sulfate, sodium ascorbate and the fourth solvent, and carry out the second click reaction to obtain the tenth compound shown in KBC-C10; KBC-C8-2, KBC-C10; Step 10-2: The tenth compound is subjected to a fifth Fm group removal reaction and a second ion exchange reaction in a third solvent containing triethylamine to obtain the thiol-reactive nucleotide derivative.

4. The synthesis method according to claim 3, characterized in that, In step 2, the molar ratio of the second compound to sodium methoxide is 1:0.4~0.6; The deprotection / ester exchange reaction is carried out at a temperature of 10~30℃ for 8~12h.

5. The synthesis method according to claim 3, characterized in that, In step 3, the molar ratio of the third compound to the fourth compound is 1:1.1~1.3, the molar ratio of the third compound to 5-ethylthiotetrazole is 1:1.4~1.6, and the molar ratio of the third compound to tert-butylhydrogen peroxide is 1:4~6; the temperature of the first coupling-oxidation reaction is 0~30℃, and the time is 1~2h. In step 4, the temperature of the first Fm group removal reaction is 10~30℃.

6. The synthesis method according to claim 3, characterized in that, In step 5, the molar ratio of the fifth compound to the fourth compound is 1:1.1~1.3; the molar ratio of the fifth compound to 5-ethylthiotetrazole is 1:1.4~1.6; the molar ratio of the fifth compound to tert-butyl hydroperoxide is 1:4~6; the temperature of the second coupling-oxidation reaction is 0~30℃, and the time is 1~3h. In step 6, the temperature of the second Fm group removal reaction is 10~30℃.

7. The synthesis method according to claim 3, characterized in that, In step 7, the molar ratio of the sixth compound to the fourth compound is 1:1.1~1.3, the molar ratio of the sixth compound to 5-ethylthiotetrazole is 1:1.4~1.6, and the molar ratio of the sixth compound to tert-butyl hydroperoxide is 1:4~6; the temperature of the third coupling-oxidation reaction is 0~30℃, and the time is 1~2h.

8. The synthesis method according to claim 3, characterized in that, In step 9-1, the molar ratio of the seventh compound to the alkynyl chloride is 1:1.2~1.6, the molar ratio of the seventh compound to copper sulfate is 1:0.1~0.3, and the molar ratio of the seventh compound to sodium ascorbate is 1:0.3~0.5; the fourth solvent includes acetone and water, and the volume ratio of acetone to water is 8:2; the temperature of the first click chemistry is 10~30℃, and the time is 0.5~2h.

9. The synthesis method according to claim 3, characterized in that, In step 9-2, the molar ratio of the compound represented by KBC-C7B to the alkynyl chloride is 1:1 to 1.6, the molar ratio of the compound represented by KBC-C7B to copper sulfate is 1:0.1 to 0.3, and the molar ratio of the compound represented by KBC-C7B to sodium ascorbate is 1:0.3 to 0.

5. The temperature of the second click reaction is 10~30℃, and the time is 0.5~2h.

10. The synthesis method according to claim 3, characterized in that, In step 10-2, the volume concentration of triethylamine in the third solvent containing triethylamine is 50%; the temperature of the fifth Fm group removal reaction is 10~30℃ and the time is 10~30min.

Images