Connecting unit for chemical modification of nucleic acid end group, solid-phase carrier and preparation method of solid-phase carrier

Abstract

The embodiment of the invention provides a connecting unit for chemical modification of a nucleic acid terminal group, a solid-phase carrier and a preparation method of the solid-phase carrier, the chemical modification comprises N-acetylgalactosamine, and the method comprises the following steps: mixing L-deoxyuridine with iodine, and carrying out iodine substitution reaction to obtain 8-iodine-L-deoxyuridine; carrying out triphenyl methylation on the 8-iodine-L-deoxyuridine to protect 5 '-terminal hydroxyl, carrying out palladium catalytic coupling reaction to obtain 8-alkenyl-L-deoxyuridine ester compounds, and hydrolyzing to obtain unsaturated carboxyl modified L-deoxyuridine derivatives; the unsaturated carboxyl modified L-deoxyuridine derivative and N-acetylgalactosamine are subjected to condensation, and the connecting unit for nucleic acid end group chemical modification is obtained. The connection unit obtained by the method has better enzymolysis resistance, and the synthesis method is simple and efficient.

Description

Technical Field

[0001] This disclosure relates to the field of biotechnology, and in particular to a linker unit, a solid-phase support, and a method for preparing the same for chemical modification of nucleic acid terminal groups. Background Technology

[0002] N-Acetyl-D-galactosamine (GalNAc) is a monosaccharide that binds specifically to the asialoglycoprotein receptor (ASGPR) on the liver surface, making it an important targeting molecule for hepatic drug delivery and used in various liver-targeting drugs. In recent years, small interface RNA (siRNA) drugs have been developed, covalently linking the N-acetyl-galactosamine unit to the nucleic acid drug, which improves the targeting and delivery efficiency of nucleic acid drugs to the liver, achieving promising results.

[0003] There are three main ways to covalently link N-acetylgalactose units to nucleic acid drugs: to the 3' end, 5' end, and the middle of the nucleic acid chain. Linking different numbers of N-acetylgalactose units at different positions on the nucleic acid produces different effects. Developing N-acetylgalactose units with different linkage structures has significant market value for developing novel small nucleic acid drug delivery systems.

[0004] However, the current N-acetylgalactose unit modification for the synthesis of nucleic acid drugs, the simplification and reproducibility of the preparation process of solid-phase carriers still need improvement, and the resistance of the linker and solid-phase carrier to enzymatic degradation during the preparation of nucleic acid substances also needs improvement. Summary of the Invention

[0005] This disclosure provides a solid-phase support for N-acetylgalactosamine unit modification and its preparation method, in order to solve or alleviate one or more technical problems in the related art: This application proposes a linker unit for modifying the 3' end of nucleic acid with N-acetylgalactosamine, as well as a solid-phase support and its preparation method. The linker uses a linker arm containing a non-natural L-configuration deoxynucleosyl compound to link N-acetylgalactosamine, which has good resistance to enzymatic degradation and the synthesis method is simple and efficient.

[0006] As a first aspect of the present disclosure, the present disclosure provides a method for preparing a linker unit for chemical modification of nucleic acid terminal groups, the chemical modification including N-acetylgalactosamine, the method comprising: L-deoxyuridine is mixed with iodine to undergo an iodine substitution reaction to give 8-iodo-L-deoxyuridine; The 8-iodo-L-deoxyuridine was triphenylmethylated to protect the 5' hydroxyl group, and then subjected to palladium-catalyzed coupling reaction to obtain 8-alkenyl-L-deoxyuridine ester compounds, which were then hydrolyzed to obtain unsaturated carboxyl-modified L-deoxyuridine derivatives. The unsaturated carboxyl-modified L-deoxyuridine derivative is condensed with N-acetylgalactosamine to obtain the linker unit used for nucleic acid terminal chemical modification.

[0007] This method can easily obtain the linker unit of N-acetylgalactosamine-L-deoxyuridine derivatives, and the L-configuration of the deoxynucleoside is beneficial to improving the resistance to enzymatic degradation.

[0008] According to an embodiment of the present invention, obtaining the 8-iodo-L-deoxyuridine comprises: dissolving L-deoxyuridine in a solvent, adding silver nitrate at room temperature, adding dropwise a 1,4-dioxane solution of iodine, reacting at 8-15 degrees Celsius for 1 hour, and then stirring overnight at 25-35 degrees Celsius.

[0009] According to embodiments of the present invention, obtaining the unsaturated carboxyl-modified L-deoxyuridine derivative comprises: The 8-iodo-L-deoxyuridine was dissolved in anhydrous pyridine, and a pyridine solution of 4,4-dimethoxytriphenylchloromethane was added dropwise. The mixture was stirred at 25-35 degrees Celsius for 10-20 hours, and methanol was added and stirring was continued for 1 hour. After concentration to remove the solvent, the resulting oily product was dissolved in ethyl acetate, washed with saturated ammonium chloride and saturated brine, dried, and the solvent was removed by rotary evaporation after filtering to obtain a yellow waxy solid. This solid was dissolved in anhydrous tetrahydrofuran, and anhydrous triethylamine, ethyl acrylate, and tetra(triphenylphosphine)palladium were added. The mixture was heated to 70 degrees Celsius under nitrogen protection and reacted for 10-15 hours to obtain the intermediate product. The intermediate product was dissolved in methanol, and an aqueous solution of lithium hydroxide was added. The mixture was heated to 35-45°C under nitrogen protection and reacted for 10-15 hours. The mixture was then adjusted to neutral by adding a saturated ammonium chloride solution. The solvent was removed by concentration, and the organic phase was separated and washed with saturated brine and dried with anhydrous sodium sulfate. Triethylamine was added and dissolved in ethyl acetate. The mixture was then added dropwise to n-heptane at 0°C with stirring. After stirring, the mixture was filtered to obtain the final product.

[0010] According to an embodiment of the present invention, the condensation of the unsaturated carboxyl-modified L-deoxyuridine derivative with N-acetylgalactosamine comprises: The unsaturated carboxyl-modified L-deoxyuridine derivative and N-acetylgalactosamine were dissolved in dichloromethane, and triethylamine and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate were added. The mixture was stirred at 25-35 degrees Celsius for 10-15 hours under nitrogen protection. The product was separated by 200-300 mesh neutral silica gel column chromatography, with a mixed solvent of dichloromethane and methanol as the eluent.

[0011] In another aspect of this application, a linker unit for chemical modification of nucleic acid terminals is proposed. The linker unit is prepared using the method described above and comprises: , Where AcO is acetoxy, NHAc is acetamino, and m and n are independent integers from 1 to 17.

[0012] According to an embodiment of the present invention, the connection unit includes: .

[0013] In another aspect of this application, a solid support is proposed, the surface of which is covalently modified with linking units for chemical modification of nucleic acid end groups, the linking units being prepared using the method described above, or being the linking units described above.

[0014] According to an embodiment of the present invention, the solid support comprises glass microspheres and a modifier modified on the glass microspheres by amino groups, the modifier comprising: , Where AcO is acetoxy, NHAc is acetamino, and m and n are independent integers from 1 to 17. X includes CH2, (CH2)2, (CH2)3, (CH2)4, (CH2)5, (CH2)6, (CH2)7, (CH2)8, (CH2)9, and (CH2) 10 At least one of (CH2OCH2) and (CH2CH2OCH2CH2).

[0015] In another aspect of this application, a method for preparing the aforementioned solid support is provided, comprising: The linker unit prepared by the method described above was dissolved in anhydrous pyridine, and a solution of dianhydride was added dropwise under ice bath stirring. The mixture was heated to 25-35 degrees Celsius and stirred for 12 hours to obtain the intermediate product. The intermediate product was dissolved in anhydrous dimethyl sulfoxide, and amino-modified porous glass microspheres were added. N,N-carbonyldiimidazole, anhydrous pyridine and 4,4-dimethylaminopyridine were added, and the mixture was stirred at 25-35 degrees Celsius for 24 hours.

[0016] According to an embodiment of the present invention, the pore size of the porous glass microspheres is 500-1000 angstroms. Attached Figure Description

[0017] In the accompanying drawings, unless otherwise specified, the same reference numerals throughout the various drawings denote the same or similar parts or elements. These drawings are not necessarily drawn to scale. It should be understood that these drawings depict only some embodiments according to this disclosure and should not be construed as limiting the scope of this disclosure.

[0018] Figure 1 This is a schematic diagram of a method for preparing a connecting unit according to an embodiment of the present disclosure; Figure 2 The 1H NMR spectrum of compound 2 in the method for preparing the linker unit according to an embodiment of this disclosure; Figure 3 Electrospray ionization mass spectrometry of compound 2 in the method for preparing the linker unit according to embodiments of this disclosure; Figure 4 The 1H NMR spectrum of compound 3 in the method for preparing the linker unit according to an embodiment of the present disclosure; Figure 5 Electrospray ionization mass spectrometry of compound 3 in the method for preparing the linker unit according to embodiments of this disclosure; Figure 6 The 1H NMR spectrum of compound 4 in the method for preparing the linker unit according to an embodiment of the present disclosure; Figure 7 Electrospray ionization mass spectrometry of compound 4 in the method for preparing the linker unit according to embodiments of this disclosure; Figure 8 The matrix-assisted laser ionization desorption / resorption mass spectrum of the sequence obtained according to Example 2 of this disclosure; Figure 9 The graph shows the test results of the anti-enzyme digestion performance according to an embodiment of this disclosure. Detailed Implementation

[0019] The embodiments of the technical solution of this application will now be described in detail with reference to the accompanying drawings. These embodiments are only used to more clearly illustrate the technical solution of this application and are therefore merely examples, and should not be used to limit the scope of protection of this application.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains; the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the application; the terms “comprising” and “having”, and any variations thereof, in the specification, claims, and foregoing description of the drawings are intended to cover non-exclusive inclusion.

[0021] In this document, the term "embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this application. The appearance of this phrase in various places throughout the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment mutually exclusive with other embodiments. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0022] In the description of the embodiments in this application, the term "and / or" is merely a description of the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, and B existing alone. Additionally, the character " / " in this document generally indicates that the preceding and following related objects have an "or" relationship.

[0023] In the description of the embodiments of this application, the term "multiple" refers to two or more (including two), similarly, "multiple sets" refers to two or more (including two sets), and "multiple pieces" refers to two or more (including two pieces).

[0024] Unless otherwise stated, the terms used in this application have their common meanings as commonly understood by those skilled in the art. Unless otherwise stated, the values ​​of the parameters mentioned in this application can be measured using various measurement methods commonly used in the art (e.g., they can be tested according to the methods given in the embodiments of this application).

[0025] In a first aspect of this application, a method for preparing linker units for chemical modification of nucleic acid terminal groups is proposed. The chemical modification includes N-acetylgalactosamine, and the method comprises: L-deoxyuridine is mixed with iodine to undergo an iodine substitution reaction to give 8-iodo-L-deoxyuridine; The 8-iodo-L-deoxyuridine was triphenylmethylated to protect the 5' hydroxyl group, and then subjected to palladium-catalyzed coupling reaction to obtain 8-alkenyl-L-deoxyuridine ester compounds, which were then hydrolyzed to obtain unsaturated carboxyl-modified L-deoxyuridine derivatives. The unsaturated carboxyl-modified L-deoxyuridine derivative is condensed with N-acetylgalactosamine to obtain the linker unit used for nucleic acid terminal chemical modification.

[0026] This method can easily obtain the linker unit of N-acetylgalactosamine-L-deoxyuridine derivatives, whose L-configuration deoxynucleoside structure is beneficial for enhancing resistance to enzymatic degradation. The natural D-configuration deoxyuridine is shown in Formula 1 below: Formula 1 The non-natural L-configuration deoxyuridine and the structure shown in Formula 1 are enantiomers: Formula 2 As mentioned earlier, N-acetylgalactosamine exhibits high specificity for recognizing desialyl glycoproteins on the liver surface, making it an important targeting molecule in the design of liver-targeted nucleic acid drugs. When developing targeting structures containing N-acetylgalactosamine, designing the linker unit connecting N-acetylgalactosamine and the nucleic acid drug moiety is a crucial component in improving the overall efficacy and stability of the nucleic acid drug. L-configured deoxyuridines are enantiomers of natural D-configured deoxyuridines and are mirror images of natural nucleosides. L-configured nucleosides, nucleotides, and nucleic acid structures have the same molecular weight, the same thermodynamic properties, and completely opposite chiral signals as their natural D-configured counterparts. However, because L-configured nucleosides are largely unrecognized by natural nucleases, their interaction with nucleases is weak, and they are almost never degraded by nucleases, thus exhibiting better resistance to enzymatic degradation. Linking the N-acetylgalactose structure to an L-configuration nucleoside via chemical modification yields a linker unit. This unit is then linked to a porous glass microsphere carrier, resulting in a class of solid-phase synthetic carriers suitable for nucleic acid 3' modification. The nucleic acid sequence of L-configuration uracil linked with N-acetylgalactose exhibits good targeting and resistance to enzyme cleavage.

[0027] The synthesis of this method can be... Figure 1 As shown, the synthesis conditions for each synthesis step can be: a) Iodine substitution reaction: iodine, silver nitrate, 1,4-dioxane, 10 degrees Celsius; b) Triphenylmethylation: 5'-(4,4-dimethoxytriphenylmethyl)-L-deoxyuridine, pyridine, 0°C to room temperature; Palladium-catalyzed coupling: tetra(triphenylphosphine)palladium, ethyl acrylate, tetrahydrofuran, triethylamine, 70 degrees Celsius; Hydrolysis: Lithium hydroxide, water, methanol, 40 degrees Celsius; c) Condensation: 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, triethylamine, dichloromethane, room temperature.

[0028] According to an embodiment of the present invention, obtaining the 8-iodo-L-deoxyuridine comprises: dissolving L-deoxyuridine in a solvent, adding silver nitrate at room temperature, adding dropwise a solution of iodine in 1,4-dioxane, reacting at 8-15 degrees Celsius for 1 hour, and then stirring overnight at 25-35 degrees Celsius. This allows an iodine atom to be introduced into the C8 position of L-deoxyuridine, providing an active site for the subsequent coupling reaction.

[0029] According to an embodiment of the present invention, obtaining the unsaturated carboxyl-modified L-deoxyuridine derivative comprises: dissolving the 8-iodo-L-deoxyuridine in anhydrous pyridine, adding dropwise a pyridine solution of 4,4-dimethoxytriphenylchloromethane, stirring at 25-35 degrees Celsius for 10-20 hours, adding methanol and continuing stirring for 1 hour, concentrating to remove the solvent, dissolving the resulting oily product in ethyl acetate, washing with saturated ammonium chloride and saturated brine, drying, filtering off the desiccant, and then rotary evaporating to remove the solvent to obtain a yellow waxy solid, which is soluble in anhydrous tetrahydrofuran. Anhydrous triethylamine, ethyl acrylate, and tetra(triphenylphosphine)palladium were added, and the mixture was heated to 70°C under nitrogen protection and reacted for 10-15 hours to obtain the intermediate product. The intermediate product was dissolved in methanol, and an aqueous solution of lithium hydroxide was added. The mixture was heated to 35-45°C under nitrogen protection and reacted for 10-15 hours. The mixture was adjusted to neutrality with saturated ammonium chloride solution, concentrated to remove the solvent, separated the organic phase, washed with saturated brine, dried over anhydrous sodium sulfate, and triethylamine was added and dissolved in ethyl acetate. The mixture was then added dropwise to n-heptane at 0°C with stirring, and filtered to obtain the final product.

[0030] Thus, a carboxylic acid-containing linking group can be introduced at the C8 position of L-deoxyuridine, while retaining the 5'-O-MMTr protecting group.

[0031] According to an embodiment of the present invention, the condensation of the unsaturated carboxyl-modified L-deoxyuridine derivative with N-acetylgalactosamine comprises: dissolving the unsaturated carboxyl-modified L-deoxyuridine derivative and N-acetylgalactosamine in dichloromethane, adding triethylamine and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, and stirring the reaction at 25-35 degrees Celsius for 10-15 hours under nitrogen protection. The product is then separated by 200-300 mesh neutral silica gel column chromatography, with a mixed solvent of dichloromethane and methanol as the eluent.

[0032] 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU) is a potent condensing agent that promotes the formation of amide bonds between carboxylic acids and amines, thus allowing for the relatively simple preparation of GalNAc-L-deoxyuridine conjugates.

[0033] In another aspect of this application, a linker unit for chemical modification of nucleic acid terminals is proposed. The linker unit is prepared using the method described above and comprises: , Where AcO is acetoxy, NHAc is acetamino, and m and n are independent integers from 1 to 17.

[0034] According to an embodiment of the present invention, the connection unit includes: .

[0035] In another aspect of this application, a solid support is proposed, the surface of which is covalently modified with linking units for chemical modification of nucleic acid end groups, the linking units being prepared using the method described above, or being the linking units described above.

[0036] According to an embodiment of the present invention, the solid support comprises glass microspheres and a modifier modified on the glass microspheres by amino groups, the modifier comprising: , Where AcO is acetoxy, NHAc is acetamino, and m and n are independent integers from 1 to 17. X includes CH2, (CH2)2, (CH2)3, (CH2)4, (CH2)5, (CH2)6, (CH2)7, (CH2)8, (CH2)9, and (CH2) 10 At least one of (CH2OCH2) and (CH2CH2OCH2CH2).

[0037] According to embodiments of this application, the porous glass microspheres can be selected from commonly used glass microspheres in the art, or selected according to the specific requirements of the application scenario of the solid-phase support. For example, the pore size of the porous glass microspheres is 500-1000 angstroms.

[0038] According to embodiments of this application, the solid support may have the following structure: .

[0039] The surface of porous glass microspheres can be modified with amino groups, which can be linked to the aforementioned linking units via hydroxyl groups in a five-membered ring to form a solid-phase support modified with N-acetylgalactosamine and L-deoxyuridine.

[0040] For example, specifically, the solid support may have the following structure: .

[0041] In another aspect of this application, a method for preparing the aforementioned solid support is provided, comprising: The linker unit prepared by the method described above was dissolved in anhydrous pyridine, and a solution of dianhydride was added dropwise under ice bath stirring. The mixture was heated to 25-35 degrees Celsius and stirred for 12 hours to obtain the intermediate product. The intermediate product was dissolved in anhydrous dimethyl sulfoxide, and amino-modified porous glass microspheres were added, along with N,N-carbonyldiimidazole, anhydrous pyridine, and 4,4-dimethylaminopyridine. The mixture was stirred at 25-35 degrees Celsius for 24 hours. This yielded a solid-phase support with the aforementioned connecting units.

[0042] Specifically, the diacid anhydride can be succinic anhydride, which can be determined based on the structure of the X group in the aforementioned solid support.

[0043] This solid-phase support can be used to perform nucleic acid synthesis using the DMT-off method.

[0044] The foregoing content will now be described in detail with reference to specific embodiments.

[0045] Instruments and reagents: The high-performance liquid chromatography (HPLC) system used was a Waters 2695. Mobile phase A was chromatographic grade acetonitrile, and mobile phase B was a 10% deionized aqueous solution of triethylamine acetate. The analytical column was a C18 Waters X-Bridge with a particle size of 5 μm and dimensions of 250 mm × 4.6 mm. The flow rate was 0.2 mL / min, and the column temperature was 25 degrees Celsius.

[0046] The structure and purity of organic molecules can be determined by proton nuclear magnetic resonance (NMR) spectroscopy. 1 H-NMR, carbon spectrum ( 13 The DNA structure and photodegradation process were determined by C-NMR and electrospray ionization (ESI-MS), and the reaction efficiency was determined by matrix-assisted laser ionization desorption-time-of-flight mass spectrometry (MALDI-TOF).

[0047] The NMR spectrometer used was a Bruker AMX 400 Spectrometer (400 MHz), and the solvents used were deuterated chloroform (CDCl3) and deuterated dimethyl sulfoxide (d... 6 -DMSO), with TMS internal standard; The ESI mass spectrometer was an Agilent 6510 Q-TOF, and the detection mode was anion mode. The MALDI-TOF mass spectrometer is a Shimadazu Biotech Axima Performance model, with cation and anion detection modes.

[0048] Example 1: Synthesis of N-acetylgalactose unit of L-configured deoxyuridine Step a: Dissolve 2.28 g of L-deoxyguanosine in 150 mL of 1,4-dioxane. Add 3.4 g of silver nitrate at room temperature. Place the flask in a 10°C water bath and stir. Dissolve 5.1 g of iodine in 30 mL of 1,4-dioxane and add it dropwise to the above solution through a dropping funnel. React at 10°C for 1 hour, then raise to room temperature and stir overnight. Add 20 g of diatomaceous earth to the reaction solution, filter, and wash the filtrate with 100 mL of saturated sodium bicarbonate and 150 mL of saturated sodium thiosulfate, then wash with 100 mL of saturated brine. Dry with anhydrous sodium sulfate. Filter off the drying agent, concentrate the filtrate to 15 mL, place in a 4°C refrigerator for 12 hours, filter, and collect the filter cake to obtain... Figure 1 Compound 2, a white solid, was obtained in a yield of 3.1 g (88%). The proton NMR spectrum of the obtained compound is shown below. Figure 2 The peak position information is shown below: 1 H-NMR (CDCl3) δ ppm: 11.67 (1H, s, NH), 8.42 (1H, s, 6-H), 6.09-6.12(1H, m, 1'-H), 5.25-5.26 (1H, m, 5'-H), 5.15-5.18 (1H, m, 5'-H),4.25-4.26(1H, m, 3'-H), 3.80-3.81 (1H, m, 4'-H), 3.56-3.66 (2H, m, 2'-H), 2.12-2.14(2H, m, 3'-OH+5'-OH). Electrospray ionization mass spectrometry spectrum as shown in the figure Figure 3 As shown, the ESI-MS C9H was obtained through analysis. 11 N₂O₅, theoretical molecular weight 353.9713, measured molecular weight 352.9638 (MH) + .

[0049] Step b: 3.0 g of compound 2 was dissolved in 70 mL of anhydrous pyridine and stirred at room temperature. 3.5 g of 4,4-dimethoxytriphenylchloromethane was dissolved in 30 mL of pyridine solution and added dropwise to the above solution. After the addition was complete, the mixture was stirred at room temperature for 12 hours. After the reaction was complete, 1 mL of methanol was added and the mixture was stirred for another hour. The solvent was removed by concentration, and the oily product was dissolved in 100 mL of ethyl acetate. The product was washed with 100 mL of saturated ammonium chloride and 100 mL of saturated brine, and dried over anhydrous sodium sulfate. After filtering off the drying agent, the solvent was removed by rotary evaporation to obtain a yellow waxy solid. The crude product can be used directly in the next step.

[0050] The crude product was dissolved in 100 mL of anhydrous tetrahydrofuran, and 2 mL of anhydrous triethylamine, 2 mL of ethyl acrylate, and 20 mg of tetra(triphenylphosphine)palladium were added. The atmosphere of the reaction flask was purged with nitrogen three times, and the temperature was raised to 70°C under nitrogen protection, and the reaction was carried out overnight. After the reaction was completed, 10 mL of 200-300 mesh acidic silica gel was added to the system, and the mixture was filtered. The filtrate was evaporated to dryness to obtain a white solid. 10 mL of toluene was added, and the mixture was sonicated and filtered. The filter cake was washed with 5 mL of toluene three times. The crude product was dried and used directly in the next step.

[0051] The crude product from the previous step was dissolved in 50 mL of methanol, and a solution of 1.2 g of lithium hydroxide in 30 mL of water was added. The mixture was heated to 40°C under nitrogen protection and reacted overnight. After the reaction was complete, saturated ammonium chloride solution was added to adjust the pH to neutral, and the organic solvent was removed by concentration. The aqueous phase was extracted with ethyl acetate, 20 mL each time, for three extractions. The organic phases were combined, washed with 100 mL of saturated brine, dried over anhydrous sodium sulfate, and 1 mL of triethylamine was added. The crude product was dissolved in 10 mL of ethyl acetate, and while stirring, it was added dropwise to 120 mL of n-heptane at 0°C. After stirring for 30 minutes, the mixture was filtered. The filter cake was recrystallized using 20 mL of methyl tert-butyl ether to obtain the target product (…). Figure 1 Compound 3). 2.7 g of a pale yellow solid, yield 45%. The 1H NMR spectrum of the obtained compound is shown below. Figure 4 The peak position information is shown below: 1 H-NMR (d 6 -DMSO) δ ppm: 11.73 (1H, s, COOH), 8.12 (1H, s, 6-H), 7.38-7.40 (2H, m, 6-H+alkene), 7.18-7.28 (8H, m, ArH on DMT), 6.82-6.88 (5H, m,ArH onDMT), 6.16-6.19 (1H, m, 1'-H), 5.32-5.33 (1H, m, alkene), 4.25-4.28(1H, m, 5'-H), 4.08-4.13 (2H, q, NCH2on TEA), 3.92-3.93(1H, m, 5'-H), 3.70-3.71 (6H, m, MeO on DMT), 3.16-3.27 (2H, m, 3'-H+4'-H), 2.21-2.24 (2H, m, 2'-H), 1.17-1.21 (3H, t, CH3on TEA). Electrospray ionization mass spectrometry spectrum as shown in the figure Figure 5 As shown, the ESI-MS C is obtained through analysis. 39H 47 N3O7, theoretical molecular weight 701.3312, measured molecular weight 599.2032 (M-HNEt3) + , 600.2063 (M-NEt3) + . Step c: 1.4 g of compound 3 and 1.3 g of GalNAc amine were dissolved in 100 mL of dichloromethane. 1 mL of triethylamine and 1.1 g of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate were added, and the mixture was stirred overnight at room temperature under nitrogen protection. After the reaction was complete, 100 mL of saturated sodium bicarbonate was added for washing. The organic phase was washed with saturated brine and dried over anhydrous sodium sulfate. The crude product was separated by column chromatography using 200-300 mesh neutral silica gel. The eluent was dichloromethane:methanol at a ratio of 120:1 to 100:1 (volume ratio), yielding a white, foamy solid, compound 4 (…). Figure 1 (As shown) 1.61 g, yield 73%. The proton NMR spectrum is as follows: Figure 6 The peak position information is shown below: 1H-NMR (CDCl3) δ ppm: 7.75-8.37 (5H, NH on amide+6-H), 6.85-7.38 (14H,m, ArH on DMT+alkene), 6.15-6.18 (1H, m, 1'-H),5.76 (1H, s, 1-H on galnac),5.31 (1H, br, 3'-OH), 5.20-5.21 (1H, m, alkene), 4.94-4.98 (1H, dd, 5'-H), 4.47-4.49 (1H, d, 5'-H), 4.22-4.23 (1H, m, 3'-H), 4.02-4.04 (2H, m, CH2O),3.86-3.90 (2H, m, CHO+CHN ongalnac), 3.70-3.71 (6H, m, MeO on DMT), 3.38-3.43 (1H, m, 4'-H), 3.02-3.22 (4H, m, CHO+CHN ongalnac), 3.02-3.05 (2H, m, CH2O), 2.89-2.91(4H, m, CH2NHCO), 2.28-2.35 (1H, m, 2'-H), 2.15-2.20(1H, m, 2'-H), 2.09 (3H,s, AcO), 2.03-2.06 (2H, t, CH2COO), 1.99 (3H, s, AcO), 1.88 (3H, s, AcO),1.77 (3H, s, AcO), 1.44-4.57 (6H, m, CH2on alkyl chain). Electrospray ionization mass spectrometry spectrum as shown in the figure Figure 7 As shown, the ESI-MS C is obtained through analysis. 55 H 67 N5O 18 Theoretical molecular weight 1085.4481, measured molecular weight 1086.4497 (M+H) + . Example 2: Preparation of porous glass microspheres via the synthetic route of the N-acetylgalactose unit of L-configured deoxyuridine 1.1 g of compound 4 was dissolved in 50 mL of anhydrous pyridine. A solution of 0.1 g of succinic anhydride in 5 mL of anhydrous pyridine was added dropwise with stirring in an ice bath. After the addition was complete, the temperature was allowed to rise naturally to room temperature, and stirring continued for 12 hours. After the reaction was complete, the organic solvent was removed by rotary evaporation. The crude product was dissolved in 100 mL of anhydrous dimethyl sulfoxide (DMSO), and 5 g of amino-modified porous glass microspheres with a pore size of 500 Å were added. 1.6 g of N,N-carbonyldiimidazole, 1 mL of anhydrous pyridine, and 1 g of 4,4-dimethylaminopyridine were added, and the mixture was stirred at room temperature for 24 hours. The mixture was filtered, and the filter cake was washed three times with 20 mL of DMSO each time, followed by five times with 50 mL of anhydrous ethanol each time. The solid was collected and dried under vacuum to obtain the product. The structure of the prepared solid support is shown in the figure below. .

[0052] The loading capacity of the solid support obtained in Example 2 was measured: Weigh 15 mg of porous glass microspheres modified with N-acetylgalactose units of L-deoxyuridine and place them in a 15 mL test tube; weigh 3.55 g of p-toluenesulfonic acid and add it to a clean 250 mL Erlenmeyer flask, dissolve it in anhydrous acetonitrile to a final volume of 200 mL, obtaining a 0.1 mol / L p-toluenesulfonic acid acetonitrile solution; add 1 mL of 0.1 mol / L p-toluenesulfonic acid acetonitrile solution as eluent to the test tube containing the porous glass microspheres modified with N-acetylgalactose units of L-deoxyuridine, mix well, let stand for 5 minutes, centrifuge for 10 seconds, and collect the supernatant; determine the detection wavelength of the UV-Vis spectrophotometer to be 498 nm, use the eluent as a reference solution, and measure the absorbance coefficient of the supernatant.

[0053] The formula for calculating load capacity is as follows: Loading capacity (μmol / g) = OD λ498 × Dilution factor × 12 ÷ CPG powder mass (mg) Table 1 shows the loading capacity of porous glass microspheres with different pore sizes.

[0054] Table 1 Example 3: Synthesis of the N-acetylgalactose unit of L-configured deoxyuridine The sequence information is as follows: 5'AmCmGmGmCmCfUfCfUmUfUmAfCfUfAfGm CfUfAfUf XXX 3' The nucleic acid structure is the positive sequence of siRNA. m represents a methoxy group at 2', f represents a fluorine atom at 2', and X represents the structure of this patent application.

[0055] The nucleic acid synthesizer was manufactured by Beijing Haijing Gaochuang Technology Co., Ltd., and its model number is HJ01201202. The nucleic acid synthesis volume was 200 nanomoles. The synthesis column was a porous glass microsphere powder-packed column modified with N-acetylgalactose units of L-configured deoxyuridine. The concentration of deoxythymidine phosphoramidide monomer was 0.05 mmol / L. The elution acetonitrile contained 17 ppm of water. The deprotection reagent was a 3% (w / v) trichloroacetic acid solution in dichloromethane. The activator was a 0.45 mol / L acetonitrile solution of 5-ethylthiotetrazole. The oxidant was a 0.05 mol / L tetrahydrofuran-water-pyridine mixed solution (with a volume ratio of 90:5:5). End-capping reagent A was a 10% acetic anhydride solution in tetrahydrofuran, and end-capping reagent B was a 5% 1-methylimidazole solution in tetrahydrofuran.

[0056] Nucleic acid synthesis was performed using the DMT-off method. After synthesis, the porous glass microsphere carrier was removed and placed in 1 mL of 28% ammonia solution for ammonolysis at 60°C for 2 hours. The ammonolysis solution was then dried and purified by high-performance liquid chromatography.

[0057] Table 2 Where m represents methoxy substitution on 2', f represents fluorine substitution on 2', SS represents the positive sequence, and X is the structure shown in the solid support obtained in Example 2.

[0058] The matrix-assisted laser ionization desorption / resorption spectroscopy (MAD) mass spectrum of the obtained siRNA sense sequence with three GalNac units linked at the 3' end is shown below. Figure 8 As shown.

[0059] The binding efficiency of the nucleic acid sequence obtained in Example 2 above with the anti-desialic acid glycoprotein receptor antibody on hepatocytes was detected: Hepatocytes were added to Stripwell microplates at a density of 200,000 cells / tube / mL, with DuPont modified Eagle medium / 2% bovine serum albumin as the buffer solution, and the plate was incubated at 4°C. GalNAc-modified nucleic acid sequences were added at concentrations of 30, 15, 7.5, 3.8, 1.9, and 0.9 nM. After addition, the plate was gently shaken at 150 rpm for 2 hours at 4°C. The cells were then centrifuged at 50 G for 2 minutes at 4°C, and 750 μL of the supernatant was aspirated. 750 μL of DuPont phosphate buffer containing 2% bovine serum albumin was added. This process was repeated twice. Then, add 750 μL of a 1 / 200 dilution of DuPont modified Eagle medium containing 2% bovine serum albumin and Alexa 647 dye, and incubate at 4°C for 20 minutes. Centrifuge at 50 G for 2 minutes at 4°C, and wash with 750 μL of a 1 / 200 dilution of DuPont modified Eagle medium containing 2% bovine serum albumin and Alexa 647 dye. After centrifugation at 50 G for 2 minutes at 4°C, collect the supernatant, resuspend the hepatocytes in 250 μL of DuPont phosphate buffer containing 2% bovine serum albumin, and measure fluorescence data using a Median Fluorescent Intensity (MFI) flow cytometer. The analysis software used was FlowJo Software and PRISM 4.0 Software. We used the standard L-96 trifid GalNac sequence as a comparison. The Ki value (i.e., inhibition constant) of L-96 was 24.7 ± 3.7, while the Ki value of the sequence obtained in Example 3 was 14.8 ± 2.5, which was better than the inhibition constant of the standard compound L-96, showing higher affinity.

[0060] Anti-enzyme digestion performance test: 100 μL of siRNA sequence with three consecutive GalNac units linked to the 3' junction (as shown in Table 2), at a concentration of 2.0 μM, was prepared in 0.2 M sodium phosphate buffer (pH 7.4). 1.5 μL of Exonuclease III concentrate (5-fold concentration) and 6 μL of sodium phosphate buffer were added. The mixture was incubated at 25°C on a shaker at 150 rpm for the specified time. The reaction was terminated by adding 20 μL of formamide. The proportion of undigested sample was determined using HPLC.

[0061] Test results as follows Figure 9 As shown. With the extension of heat preservation time ( Figure 9 (x-axis in the diagram), the X3 sequence obtained in Example 2 of this application showed higher resistance to enzyme digestion.

[0062] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of this disclosure, "multiple" means two or more, unless otherwise explicitly specified.

[0063] In this disclosure, unless otherwise expressly specified and limited, the terms "installation," "connection," "linking," "fixing," etc., should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral part; they can refer to a mechanical connection, an electrical connection, or a communication connection; they can refer to a direct connection or an indirect connection through an intermediate medium; they can refer to the internal communication of two components or the interaction between two components. Those skilled in the art can understand the specific meaning of the above terms in this disclosure according to the specific circumstances.

[0064] In this disclosure, unless otherwise expressly specified and limited, "above" or "below" a second feature can include direct contact between the first and second features, or contact between the first and second features through another feature between them. Furthermore, "above," "over," and "on top" of a second feature includes the first feature being directly above or diagonally above the second feature, or simply indicates that the first feature is at a higher horizontal level than the second feature. "Below," "below," and "under" a second feature includes the first feature being directly above or diagonally above the second feature, or simply indicates that the first feature is at a lower horizontal level than the second feature. The foregoing disclosure provides many different embodiments or examples for implementing different structures of this disclosure. To simplify this disclosure, components and arrangements of specific examples are described above. Of course, these are merely examples and are not intended to limit this disclosure. Furthermore, reference numerals and / or reference letters may be repeated in different examples of this disclosure; such repetition is for simplification and clarity and does not in itself indicate a relationship between the various embodiments and / or arrangements discussed.

[0065] The above are merely specific embodiments of this disclosure, but the scope of protection of this disclosure is not limited thereto. Any person skilled in the art can easily conceive of various variations or substitutions within the technical scope disclosed in this disclosure, and these should all be included within the scope of protection of this disclosure. Therefore, the scope of protection of this disclosure should be determined by the scope of the claims.

Claims

1. A method for preparing linker units for chemical modification of nucleic acid terminals, characterized in that, The chemical modification includes N-acetylgalactosamine, and the method includes: L-deoxyuridine is mixed with iodine to undergo an iodine substitution reaction to give 8-iodo-L-deoxyuridine; The 8-iodo-L-deoxyuridine was triphenylmethylated to protect the 5' hydroxyl group, and then subjected to palladium-catalyzed coupling reaction to obtain 8-alkenyl-L-deoxyuridine ester compounds, which were then hydrolyzed to obtain unsaturated carboxyl-modified L-deoxyuridine derivatives. The unsaturated carboxyl-modified L-deoxyuridine derivative is condensed with N-acetylgalactosamine to obtain the linker unit used for nucleic acid terminal chemical modification.

2. The method according to claim 1, characterized in that, Obtaining the 8-iodo-L-deoxyuridine involves: dissolving L-deoxyuridine in a solvent, adding silver nitrate at room temperature, adding dropwise a solution of 1,4-dioxane iodine, reacting at 8-15 degrees Celsius for 1 hour, and then stirring overnight at 25-35 degrees Celsius.

3. The method according to claim 1, characterized in that, Obtaining the unsaturated carboxyl-modified L-deoxyuridine derivative includes: The 8-iodo-L-deoxyuridine was dissolved in anhydrous pyridine, and a pyridine solution of 4,4-dimethoxytriphenylchloromethane was added dropwise. The mixture was stirred at 25-35 degrees Celsius for 10-20 hours, and methanol was added and stirring was continued for 1 hour. After concentration to remove the solvent, the resulting oily product was dissolved in ethyl acetate, washed with saturated ammonium chloride and saturated brine, dried, and the solvent was removed by rotary evaporation after filtering to obtain a yellow waxy solid. This solid was dissolved in anhydrous tetrahydrofuran, and anhydrous triethylamine, ethyl acrylate, and tetra(triphenylphosphine)palladium were added. The mixture was heated to 70 degrees Celsius under nitrogen protection and reacted for 10-15 hours to obtain the intermediate product. The intermediate product was dissolved in methanol, and an aqueous solution of lithium hydroxide was added. The mixture was heated to 35-45 degrees Celsius under nitrogen protection and reacted for 10-15 hours. The mixture was then adjusted to neutral by adding a saturated ammonium chloride solution. The solvent was removed by concentration, and the organic phase was separated and washed with saturated brine and dried with anhydrous sodium sulfate. Triethylamine was added and dissolved in ethyl acetate. The mixture was then added dropwise to n-heptane at 0°C with stirring. After stirring, the mixture was filtered to obtain the final product.

4. The method according to claim 1, characterized in that, The condensation of the unsaturated carboxyl-modified L-deoxyuridine derivative with N-acetylgalactosamine comprises: The unsaturated carboxyl-modified L-deoxyuridine derivative and N-acetylgalactosamine were dissolved in dichloromethane, and triethylamine and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate were added. The mixture was stirred at 25-35 degrees Celsius for 10-15 hours under nitrogen protection. The product was separated by 200-300 mesh neutral silica gel column chromatography, with a mixed solvent of dichloromethane and methanol as the eluent.

5. A linker unit for chemical modification of nucleic acid terminal groups, characterized in that, The connecting unit is prepared using the method described in any one of claims 1-4, and the connecting unit comprises: ， Where AcO is acetoxy, NHAc is acetamino, and m and n are independent integers from 1 to 17.

6. The connecting unit according to claim 5, characterized in that, The connection unit includes: 。 7. A solid-phase support, characterized in that, The surface of the solid support is covalently modified with linker units for nucleic acid end-group chemical modification, wherein the linker units are prepared by the method described in any one of claims 1-4, or are the linker units described in claim 5 or 6.

8. The solid support according to claim 4, characterized in that, The solid support comprises glass microspheres and a modifier on the glass microspheres by amino modification, the modifier comprising: ， Where AcO is acetoxy, NHAc is acetamino, and m and n are independent integers from 1 to 17. X includes CH2, (CH2)2, (CH2)3, (CH2)4, (CH2)5, (CH2)6, (CH2)7, (CH2)8, (CH2)9, and (CH2) 10 At least one of (CH2OCH2) and (CH2 CH2 OCH2 CH2).

9. A method for preparing the solid support according to claim 7 or 8, characterized in that, include: The linker unit prepared by the method according to any one of claims 1-4 is dissolved in anhydrous pyridine, and a solution of dianhydride is added dropwise under ice bath stirring. The mixture is heated to 25-35 degrees Celsius and stirred for 12 hours to obtain the intermediate product. The intermediate product was dissolved in anhydrous dimethyl sulfoxide, and amino-modified porous glass microspheres were added. N,N-carbonyldiimidazole, anhydrous pyridine and 4,4-dimethylaminopyridine were added, and the mixture was stirred at 25-35 degrees Celsius for 24 hours.

10. The method according to claim 9, characterized in that, The porous glass microspheres have a pore size of 500-1000 angstroms.

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