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-acetylgalactosamine, a small molecule that binds specifically to desialyl glycoprotein on the liver surface, is an important target molecule for hepatic drug delivery. In recent years, small-interface RNA (siRNA) drugs have been developed that covalently link N-acetylgalactose units to nucleic acid drugs, improving the targeting and delivery efficiency of nucleic acid drugs to the liver and achieving good 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-acetylgalactose 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 chemical modification of nucleic acid 3' ends, including N-acetylgalactose trimethylamine, as well as a solid-phase support and its preparation method. The linker uses a linker arm containing a non-natural L-configuration deoxyribonucleotide to connect N-acetylgalactose trimethylamine, which has good resistance to enzymatic hydrolysis and the synthesis method is simple and efficient. Multidentate / multi-forked N-acetylgalactosamine units bind more efficiently to asialoglycoprotein receptors (ASGPRs). Specifically, the binding affinity of tripentate / triflecked N-acetylgalactosamine units to ASGPRs is approximately 440 times greater than that of bidentate / biflecked N-acetylgalactosamine units, and the binding affinity of bidentate / biflecked N-acetylgalactosamine units to ASGPRs is approximately 440 times greater than that of monodentate / monoflecked N-acetylgalactosamine units. In other words, the binding affinity of tripentate / triflecked N-acetylgalactosamine units to ASGPRs is approximately 195,000 times greater than that of monodentate / monoflecked N-acetylgalactosamine units to ASGPRs. Therefore, the more N-acetylgalactosamine units there are, the stronger the binding affinity to ASGPRs. However, the synthesis difficulty of N-acetylgalactose increases with the increase of its number of teeth / forks. Therefore, the organic molecule of tridentate / trifleshed N-acetylgalactose unit is a structure that achieves a better balance between activity and cost.

[0006] As a first aspect of the present disclosure, this disclosure provides a method for preparing a linker unit for chemical modification of nucleic acid terminal groups, the chemical modification including N-acetylgalactosyltriphenylamine, the method comprising: Iodine-substituted deoxyuridine is obtained by iodine substitution at C8 of L-deoxyuridine. The iodine-substituted deoxyuridine was protected with a 5' hydroxyl group and then coupled and hydrolyzed to obtain an acrylic acid-modified L-deoxyuridine derivative. The L-deoxyuridine derivative is condensed with N-acetylgalactosyltriphenylamine to obtain the linker unit used for chemical modification of nucleic acid terminals.

[0007] This method can easily obtain linker units with L-deoxyuridine derivatives, whose L-configuration deoxynucleosides are advantageous for enhancing resistance to enzymatic degradation.

[0008] According to an embodiment of the present invention, obtaining the iodine-substituted deoxyuridine comprises: adding a solution of 1,4-dioxane with iodine dropwise to a solution containing L-deoxyuridine and silver nitrate at 5-15°C, mixing thoroughly, and then heating to 25-35°C and reacting for 10-20 hours.

[0009] According to embodiments of the present invention, obtaining the acrylic acid-modified L-deoxyuridine derivative comprises: The iodine-substituted deoxyuridine solution was added to a pyridine solution of 4,4-dimethoxytriphenylchloromethane to protect the 5'-terminal hydroxyl group. The iodine-substituted deoxyuridine with the 5'-terminal hydroxyl group protected was then coupled with ethyl acrylate under tetrakis(triphenylphosphine)palladium catalysis to introduce an acrylate group at C8 to obtain an intermediate. The intermediate is hydrolyzed in an aqueous solution of lithium hydroxide under an inert atmosphere at a temperature of 35-45°C for 10-15 hours.

[0010] According to an embodiment of the present invention, the condensation of the L-deoxyuridine derivative with N-acetylgalactosyltriphenylamine comprises: The L-deoxyuridine derivative was reacted with N-acetylgalactosyltriphenylamine under an inert atmosphere and with HATU coupling agent for 10-15 hours.

[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 n is an integer from 1 to 20.

[0012] 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.

[0013] According to an embodiment of the present invention, the solid support comprises: Where AcO represents acetoxy, NHAc represents acetamino, and n is an integer from 1 to 20. X includes O, S, NHCH3, and NHCH2CH. 3， NHCH(CH3) 2， CH2CH2 is at least one of them.

[0014] According to embodiments of this application, X includes NHCH3 and NHCH2CH. 3， NHCH(CH3) 2， At least one of CH2CH2.

[0015] In another aspect of this application, a method for preparing a solid support is proposed, which involves carboxylating the connecting units prepared by the method described above and mixing them with amino-modified porous glass microspheres for reaction.

[0016] According to an embodiment of the present invention, the pore size of the porous glass microspheres is [value missing]. 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 the embodiments of this disclosure; Figure 10 This is a diagram of a nucleic acid structure prepared on a solid-phase support according to an embodiment of the present 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 is proposed for chemically modifying a linker unit at the end of a nucleic acid cell, the chemical modification comprising N-acetylgalactosyltriphenylamine, the method comprising: Iodine-substituted deoxyuridine is obtained by iodine substitution at C8 of L-deoxyuridine. The iodine-substituted deoxyuridine was protected with a 5' hydroxyl group and then coupled and hydrolyzed to obtain an acrylic acid-modified L-deoxyuridine derivative. The L-deoxyuridine derivative is condensed with N-acetylgalactosyltriphenylamine to obtain the linker unit used for chemical modification of nucleic acid terminals.

[0026] This method allows for the simple acquisition of the linker unit, whose L-configuration deoxynucleoside structure is advantageous 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 is well known to those skilled in the art, in the development of targeting structures containing N-acetylgalactose, the linker unit connecting N-acetylgalactosamine and the nucleic acid drug is an important component for improving the overall efficacy and stability of the nucleic acid drug. The aforementioned L-configuration is an enantiomer of the D-configuration deoxyuridine, which is a natural uridine acid and therefore has poor resistance to enzymatic cleavage. The L-configuration, however, is a mirror image of the D-configuration. Nucleosides, nucleotides, and nucleic acids with this configuration (i.e., the L-configuration) have the same molecular weight, the same thermodynamic properties, and completely opposite chiral signals as the natural nucleic acid structure. Therefore, L-configuration nucleosides are largely unrecognized by natural nucleases, resulting in weak interaction with nucleases and almost no degradation by them, thus exhibiting better resistance to enzymatic degradation. Linking the N-acetylgalactose structure to the L-configuration nucleoside through chemical modification yields the linker unit. Connecting this structure to a porous glass microsphere carrier provides a type of solid-phase synthetic carrier suitable for nucleic acid 3' modification. Therefore, the nucleic acid sequence obtained based on this linker unit exhibited good targeting and resistance to enzyme digestion.

[0027] The synthesis of this method can be... Figure 1 As shown, the synthesis steps for each synthesis step can be: a) Iodine substitution reaction; substitution of the H atom on the 8th carbon atom yields iodine-substituted deoxyuridine; b) Hydroxyl protection, palladium-catalyzed coupling and hydrolysis: to obtain L-deoxyuridine with hydroxyl passivation and modified with acrylic acid; c) Condensation: Condensation with N-acetylgalactosyltriphenylamine yields the aforementioned linker unit.

[0028] According to an embodiment of the present invention, obtaining the iodine-substituted deoxyuridine comprises: adding a solution of 1,4-dioxane iodine dropwise to a solution containing L-deoxyuridine and silver nitrate at 5-15°C, mixing thoroughly, and then heating to 25-35°C for 10-20 hours. 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 embodiments of the present invention, obtaining the acrylic acid-modified L-deoxyuridine derivative comprises: The iodine-substituted deoxyuridine solution was added to a pyridine solution of 4,4-dimethoxytriphenylchloromethane to protect the 5'-terminal hydroxyl group. The iodine-substituted deoxyuridine with the 5'-terminal hydroxyl group protected was then coupled with ethyl acrylate under tetrakis(triphenylphosphine)palladium catalysis to introduce an acrylate group at C8 to obtain an intermediate. The intermediate is hydrolyzed in an aqueous solution of lithium hydroxide under an inert atmosphere at a temperature of 35-45°C for 10-15 hours.

[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 L-deoxyuridine derivative with N-acetylgalactosyltriphenylamine comprises: The L-deoxyuridine derivative was reacted with N-acetylgalactosyltriphenylamine under an inert atmosphere and with HATU coupling agent for 10-15 hours. The HATU coupling agent was 2-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethylurea hexafluorophosphate, which promotes the formation of amide bonds between the carboxylic acid and the amine, thus allowing for a relatively simple acquisition of the aforementioned linking unit.

[0032] 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 n is an integer from 1 to 20.

[0033] 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.

[0034] According to an embodiment of the present invention, the solid support comprises: Where AcO is acetoxy, NHAc is acetamino, n is an integer from 1 to 20, and X includes O, S, NHCH3, and NHCH2CH 3， NHCH(CH3) 2， At least one of CH2CH2.

[0035] According to embodiments of this application, X includes NHCH3 and NHCH2CH.3， NHCH(CH3) 2， At least one of CH2CH2.

[0036] 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.

[0037] According to embodiments of this application, the solid support may have the following structure:

[0038] 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.

[0039] In another aspect of this application, a method for preparing the aforementioned solid support is proposed, comprising: carboxylating the connecting unit prepared by the aforementioned method and mixing it with amino-modified porous glass microspheres for reaction.

[0040] This solid-phase support can be used to perform nucleic acid synthesis using the DMT-off method.

[0041] In some embodiments, nucleic acids synthesized using this solid-phase support may have the following structure: Where n can be 1-17, Y can be CH3O, F, CH3OCH2CH2O, CH2CH2O-3' (locked nucleic acid), S-configuration CH(CH3)CH2O-3' (S-cET), etc., and B can include guanine, adenine, uracil, thymine, cytosine, 5-methylcytosine, pseudouridine, 6-methyladenine, etc.

[0042] Specifically, the nucleic acids obtained using the aforementioned solid-phase carrier can be: .

[0043] For example, the aforementioned solid-phase support can be used to synthesize, such as Figure 10 The nucleic acid shown is modified with N-acetylgalactosyltriphenylamine.

[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 chromatographically pure 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 ˚C.

[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: Connection Unit Synthesis Step a: 2.28 g of L-deoxyguanosine was dissolved in 150 mL of 1,4-dioxane. 3.4 g of silver nitrate was added at room temperature. The flask was placed in a 10 °C water bath and stirred. 5.1 g of iodine was dissolved in 30 mL of 1,4-dioxane and added dropwise to the above solution through a dropping funnel. The reaction was carried out at 10 °C for 1 hour, then brought to room temperature and stirred overnight. 20 g of diatomaceous earth was added to the reaction solution, and the mixture was filtered. The filtrate was washed with 100 mL of saturated sodium bicarbonate and 150 mL of saturated sodium thiosulfate, then washed with 100 mL of saturated brine. Anhydrous sodium sulfate was added for drying. The drying agent was filtered off, and the filtrate was concentrated to 15 mL. The mixture was placed in a 4 °C refrigerator for 12 hours, filtered, and the filter cake was collected to give compound 2, a white solid of 3.1 g, with a yield of 88%. The 1H NMR spectrum of the obtained compound is shown below. Figure 2 The peak position information is shown below: 1H-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 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 3 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 added dropwise to 120 mL of n-heptane at 0°C with stirring. After stirring for 30 minutes, the mixture was filtered. The filter cake was recrystallized from 20 mL of methyl tert-butyl ether to give the target product. 2.7 g of a pale yellow solid was obtained, with a yield of 45%.

[0051] The proton NMR spectrum of the obtained compound is as follows: 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. 39 H 47 N3O7, theoretical molecular weight 701.3312, measured molecular weight 599.2032 (M-HNEt3) + , 600.2063 (M-NEt3) + . Step c: 0.7 g of compound 3 and 2.1 g of GalNAc triphenylamine were dissolved in 100 mL of dichloromethane. 1 mL of triethylamine and 2.1 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, with dichloromethane:methanol as the eluent at a ratio of 120:1 to 60:1 (volume ratio), yielding 1.11 g of a white, foamy solid (61% yield). 0.8 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 mixture 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 remaining oily substance was dissolved in 100 mL of ethyl acetate, washed with 100 mL of saturated sodium bicarbonate aqueous solution, washed with 100 mL of saturated brine, dried over anhydrous sodium sulfate, and purified by column chromatography on 200-300 mesh silica gel. The eluent was dichloromethane-dichloromethane:methanol = 150:1-100:1, yielding 730 mg of a white foamy solid, with a yield of 32%.

[0052] Nuclear magnetic resonance hydrogen spectrum such as Figure 6 The peak position information is shown below: 1 H-NMR (CDCl3) δ ppm: 7.71-7.81 (9H, NH on amide+6-H), 7.19-7.28 (9H,m, ArH on DMT), 6.85-6.88 (4H, m, ArH on DMT), 6.15-6.19 (1H, m, 1'-H), 5.76(1H,m, alkene), 5.31 (1H, br, 3'-OH), 5.20-5.21 (3H, s, 1-H on galnac),4.97-4.99 (3H, m, H on galnac), 4.48-4.50 (3H, m, H ongalnac), 4.22-4.23 (1H,m,alkene), 3.56-4.03 (36H,m, CH2O+CHO+ MeO on DMT), 3.21-3.44 (9H, m, 3'-H+4'-H+5'-H+CH2N), 3.02-3.06 (12H, m, CH2NH), 2.51 (2H, m, CH+2'-H), 2.29-2.32(6H, m, CH2), 2.11 (12H, s, AcO), 2.03-2.06 (6H, t, CH2COO), 1.99 (12H, s,AcO), 1.89 (9H, s, AcO), 1.78 (9H, s, AcO), 1.46-1.52 (12H, m, CH2on alkylchain). 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 2375.0495, measured molecular weight 1037.9594 (M-DMT+2H) 2+ ,2074.9123 (M-DMT+2H) + . Example 2: Preparation of solid support 300 mg of compound 5 was dissolved in 100 mL of anhydrous dimethyl sulfoxide (DMSO), and 3 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 also added. 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.

[0053] The loading capacity of the solid support obtained in Example 2 was measured: Weigh 15 mg of porous glass microspheres modified with the trifurcation N-acetylgalactose unit of L-configured deoxyuridine and pack them into 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 to obtain 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 a test tube containing L-configured deoxyuridine trimethyl N-acetylgalactose units modified with porous glass microspheres, mix well, let stand for 5 minutes, centrifuge for 10 seconds, and take out the supernatant. The detection wavelength of the UV-Vis spectrophotometer was determined to be 498 nm. The eluent was used as a reference solution, and the absorbance coefficient of the supernatant was measured.

[0054] The formula for calculating load capacity is as follows: Loading capacity (μmol / g) = OD λ498 × Dilution factor × 12 ÷ CPG powder mass (mg) The loading of 500 Å porous glass microspheres was 32 µmol / g, and the loading of 1000 Å porous glass microspheres was 29.5 µmol / g.

[0055] Example 3 Nucleic Acid Sequence Synthesis The sequence information is as follows: 5'C(VP)msGmGmCmCmCmUmAmUmAmUmAmCmX 3' The nucleic acid structure is the positive-positive sequence of siRNA. VP represents vinyl phosphate, m represents methoxy substitution at 2', f represents fluorine substitution at 2', s represents phosphothioester bond, and X represents the structure of this patent application.

[0056] The nucleic acid synthesizer was manufactured by Beijing Haijing Gaochuang Technology Co., Ltd., and its model number is HJ01201202. The nucleic acid synthesis yield was 200 nanomoles. The synthesis column was a porous glass microsphere powder-packed column modified with L-configured deoxyuridine trimethyl N-acetylgalactose units. 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 tetrahydrofuran-water-pyridine 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.

[0057] 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.

[0058] Table 2 The nucleic acid structure is the positive sequence of siRNA. VP represents vinyl phosphate, m represents methoxy substitution at 2', f represents fluorine substitution at 2', s represents phosphothioester bond, and X is the structure shown in the solid support obtained in Example 2.

[0059] The matrix-assisted laser ionization desorption / resorption mass spectrum of the obtained sequence is as follows: Figure 8 As shown.

[0060] Antibody binding efficiency detection: 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 temperature was controlled 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, respectively. After addition, the plates were 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 and 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's Medium containing 2% bovine serum albumin and Alexa 647 dye, 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's 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. Compared with the standard L-96 trifid GalNac sequence, the Ki value (i.e., inhibition constant) of L-96 is 24.7±3.7, while the Ki value of the sequence obtained in the embodiments of this application is 12.1±3.1, which is better than the inhibition constant of the standard compound L-96, and shows higher affinity.

[0061] Anti-enzyme digestion performance test: 100 μL of siRNA sequence with a 3'-linked triangular GalNac unit (details in Table 1), at a concentration of 2.0 μM, was added to a 0.2 M sodium phosphate buffer solution at pH 7.4. 1.5 μL of Exonuclease III enzyme concentrate at a concentration 5 times the unit concentration and 6 μL of sodium phosphate buffer solution 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.

[0062] Test results as follows Figure 9 As shown. With the extension of heat preservation time ( Figure 9 (xb) The siRNA sequence obtained in Example 2 of this application showed higher resistance to enzyme digestion.

[0063] 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.

[0064] 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.

[0065] 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.

[0066] 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-acetylgalactosyltriphenylamine, and the method includes: Iodine-substituted deoxyuridine is obtained by iodine substitution at C8 of L-deoxyuridine. The iodine-substituted deoxyuridine was protected with a 5' hydroxyl group and then coupled and hydrolyzed to obtain an acrylic acid-modified L-deoxyuridine derivative. The L-deoxyuridine derivative is condensed with N-acetylgalactosyltriphenylamine to obtain the linker unit used for chemical modification of nucleic acid terminals.

2. The method according to claim 1, characterized in that, Obtaining the iodine-substituted deoxyuridine involves: adding a solution of 1,4-dioxane with iodine dropwise to a solution containing L-deoxyuridine and silver nitrate at 5-15°C, mixing thoroughly, and then heating to 25-35°C and reacting for 10-20 hours.

3. The method according to claim 1, characterized in that, The acrylic acid-modified L-deoxyuridine derivatives are obtained by: The iodine-substituted deoxyuridine solution was added to a pyridine solution of 4,4-dimethoxytriphenylchloromethane to protect the 5'-terminal hydroxyl group. The iodine-substituted deoxyuridine with the 5'-terminal hydroxyl group protected was then coupled with ethyl acrylate under tetrakis(triphenylphosphine)palladium catalysis to introduce an acrylate group at C8 to obtain an intermediate. The intermediate is hydrolyzed in an aqueous solution of lithium hydroxide under an inert atmosphere at a temperature of 35-45°C for 10-15 hours.

4. The method according to claim 1, characterized in that, The condensation of the L-deoxyuridine derivative with N-acetylgalactosyltriphenylamine comprises: The L-deoxyuridine derivative was reacted with N-acetylgalactosyltriphenylamine under an inert atmosphere and with HATU coupling agent for 10-15 hours.

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 n is an integer from 1 to 20.

6. 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.

7. The solid support according to claim 6, characterized in that, The solid support includes: Where AcO represents acetoxy, NHAc represents acetamino, and n is an integer from 1 to 20. X includes O, S, NHCH3, and NHCH2CH. 3， NHCH(CH3)2, CH2CH2 At least one of them.

8. The solid support according to claim 6, characterized in that, X includes NHCH3 and NHCH2CH. 3， At least one of NHCH(CH3)2.

9. A method for preparing a solid support, characterized in that, include: The linking unit prepared by the method according to any one of claims 1-4 is carboxylated and then mixed and reacted with amino-modified porous glass microspheres.

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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