Lipophilic oligonucleotide conjugate, and preparation method therefor and use thereof

Abstract

The present invention provides a lipophilic oligonucleotide conjugate, and a preparation method therefor and a use thereof. The lipophilic oligonucleotide conjugate comprises an oligonucleotide and a lipophilic group linked to the oligonucleotide. The present invention effectively improves skin permeability and cell uptake efficiency by means of systematic optimization of a strategy of conjugating small nucleic acids with lipids, and regulating the type, structure, and linking mode of a hydrophobic group. Compared with small nucleic acids not conjugated with lipids, the present conjugate significantly improves the transdermal delivery performance thereof in the skin while maintaining the biological activity thereof, and exhibits obvious advantages particularly in trans-stratum corneum transport and gene‑silencing efficiency. The platform technology provides a new solution for a use of small nucleic acid drugs in the treatment of skin diseases, and in the fields of aesthetic medicine and cosmetics.

Description

Lipophilic oligonucleotide conjugates, their preparation methods and applications Technical Field

[0001] This invention belongs to the field of oligonucleotide transdermal delivery, and more specifically, relates to lipophilic oligonucleotide conjugates, their preparation methods, and applications. Background Technology

[0002] Small nucleic acid therapies, due to their high specificity and well-defined targets, have shown great potential in gene regulation, genetic disease treatment, and RNA interference (RNAi). However, small nucleotides themselves possess strong hydrophilicity, low serum stability, and poor cell membrane permeability, severely limiting their efficacy in vivo. Furthermore, since cell membranes are primarily composed of a hydrophobic phospholipid bilayer, transmembrane delivery of small nucleotides faces significant challenges in vivo.

[0003] To overcome these obstacles, researchers have developed various chemical modification and delivery strategies aimed at improving the cellular uptake efficiency and in vivo stability of small nucleic acids. Among them, lipid conjugation, as a highly efficient delivery method, has made positive progress in both systemic and local drug delivery in recent years. By chemically conjugating small nucleic acids with hydrophobic lipid molecules (such as cholesterol, saturated fatty acids, or phospholipids), not only can their interaction with the cell membrane be enhanced, but their binding with plasma lipoproteins (such as LDL or HDL) can also be promoted, achieving selective transport to target tissues. For example, cholesterol-modified small nucleic acids can target tissues expressing LDL receptors, such as the liver, fat, myocardium, and skeletal muscle, by binding to apolipoproteins ApoE or ApoB-100, and then enter the cell through lipoprotein-mediated endocytosis.

[0004] Furthermore, lipid conjugation can significantly enhance the residence time of small nucleic acid molecules near the cell membrane. Lipid groups can insert into the phospholipid bilayer to form an anchoring effect, increasing their concentration in the local environment and thus promoting passive uptake and internalization by cells. Compared with traditional carriers, lipid conjugation is structurally simple, has low immunogenicity, and is highly controllable, avoiding the toxicity and complex manufacturing processes that may arise from macromolecular carriers. Therefore, it is considered a promising small nucleic acid delivery strategy. Previous studies have shown that different lipid groups (such as cholesterol, eicosenoic acid, stearic acid, etc.) have a significant impact on the cellular uptake and in vivo distribution of small nucleic acids, and their delivery efficiency is also regulated by the connective arm structure, conjugation site, and conjugation method.

[0005] Despite this, small nucleic acids still face numerous challenges in skin delivery. The stratum corneum, as a natural barrier, possesses a highly hydrophobic and dense structure, significantly hindering the transdermal diffusion of hydrophilic macromolecules. Simultaneously, the presence of nucleases in the epidermis degrades unmodified small nucleic acids, further affecting their stability and bioavailability. Therefore, overcoming the stratum corneum barrier, prolonging the residence time of nucleic acids in the skin, and promoting their effective entry into target cells are core challenges in the design of current skin delivery systems. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention systematically optimizes the small nucleic acid-lipid conjugation strategy, regulating the type, structure, and connection method of hydrophobic groups to effectively enhance skin permeability and cellular uptake efficiency. Compared to small nucleic acids without lipid conjugation, this technology significantly improves transdermal delivery performance in the skin while maintaining its biological activity, particularly demonstrating significant advantages in cross-stratum corneum transport and gene silencing efficiency. This platform technology provides a new solution for small nucleic acid drugs in the treatment of skin diseases and in the fields of medical aesthetics and cosmetics.

[0007] Therefore, this invention provides a class of lipophilic oligonucleotide conjugates and their application in therapeutic oligonucleotide delivery, enabling efficient transdermal or intradermal delivery of functional oligonucleotides. The conjugates consist of a lipophilic group covalently coupled to an oligonucleotide, wherein the lipophilic group includes, but is not limited to, cholesterol, long-chain fatty acids, or other hydrophobic molecules that can bind to cell membrane phospholipid structures, as well as linking groups for connecting the hydrophobic molecules and the oligonucleotide. Through structural optimization and combinatorial design, this invention has screened and constructed a variety of lipid-oligonucleotide conjugate structures. These conjugates exhibit excellent performance in terms of cellular uptake efficiency and target gene silencing ability, providing strong support for the transdermal or intradermal delivery of small nucleic acid drugs and achieving excellent therapeutic effects.

[0008] In one aspect, the present invention provides a lipophilic oligonucleotide conjugate comprising an oligonucleotide and a lipophilic group linked to said oligonucleotide.

[0009] In some embodiments, the lipophilic group includes a lipophilic moiety capable of binding to the phospholipid structure of the cell membrane, particularly a lipid-soluble group capable of penetrating the skin barrier. Examples of the lipophilic moiety include, but are not limited to, groups derived from the following compounds: lipid-soluble alcohols or thiols, such as cholesterol and its derivatives (e.g., choline, cholesterol, cholesteryldiene), bile acids (e.g., cholic acid, deoxycholic acid, dehydrocholic acid), sterols, steroids; fatty acids (e.g., long-chain fatty acids, branched-chain fatty acids, saturated or unsaturated fatty acids); phospholipids (e.g., phosphatidylcholine, phosphatidylethanolamine); glycolipids; synthetic polymer-modified lipids (e.g., polyethylene glycol-lipid derivatives); and ceramides.

[0010] In some embodiments, the lipophilic group further includes a linker group for connecting the lipophilic portion capable of binding to the cell membrane phospholipid structure and the oligonucleotide. The linker group can be a group having a urethane bond, ester bond, thioether bond, carbon-carbon bond, ether bond, thioether bond, phosphate ester bond, or sulfonamide bond, or a combination thereof. By using the linker group, the embedding of the hydrophobic molecular group capable of binding to the cell membrane phospholipid structure in the phospholipid bilayer can be optimized, enhancing affinity for biological membranes and significantly improving transdermal delivery efficiency. The connection methods between the linker group and the hydrophobic molecular group capable of binding to the cell membrane phospholipid structure include: urethane bonds, providing stable polar linkage; ester bonds, readily biodegradable and suitable for controlled release; carbon-carbon bonds, imparting high stability and chemical inertness; ether bonds, enhancing lipophilicity and chemical stability; amide bonds, widely used in drug design due to their strong bond energy; thioether bonds, increasing hydrophobicity and maintaining stability; phosphate ester bonds, highly compatible with the phospholipid bilayer; and sulfonamide bonds, introducing additional polarity-regulating molecular properties. These diverse connection methods can be flexibly selected according to specific application needs, thereby optimizing the performance of molecules in transdermal delivery systems.

[0011] In a further embodiment, the lipophilic group is selected from the following groups:

[0012] in This indicates the location where the oligonucleotide was introduced.

[0013] In some embodiments, the oligonucleotide can be single-stranded, double-stranded, or other structures. The oligonucleotide can also be based on DNA, RNA, or modified nucleoside units (such as phosphate-thioester modified oligonucleotides). For example, suitable types of oligonucleotides include antisense oligonucleotides (ASO) or small interfering RNA (siRNA).

[0014] In some embodiments, each nucleotide in the oligonucleotides of the present invention is independently a modified or unmodified nucleotide. In some embodiments, the oligonucleotides of the present invention may comprise at least one modified nucleotide and / or at least one interstrand modification.

[0015] In some embodiments, all the nucleotides of the oligonucleotide of the present invention are modified nucleotides.

[0016] In some preferred embodiments, the oligonucleotides of the present invention are siRNAs, for example, siRNAs targeting tyrosinase (TYR) or matrix metalloproteinase 1 (MMP1). In some embodiments, each nucleotide in the siRNA of the present invention is independently modified or unmodified. In some embodiments, the siRNA of the present invention may contain at least one modified nucleotide and / or at least one interstrand modification. In some embodiments, all nucleotides on the sense strand are modified nucleotides. In some embodiments, all nucleotides on the antisense strand are modified nucleotides. In some embodiments, substantially all nucleotides on both the sense and antisense strands are modified nucleotides. In some embodiments, all nucleotides on both the sense and antisense strands are modified nucleotides.

[0017] The modified nucleotides include, but are not limited to, nucleotides modified with 2'-F, 2'-OME, 2'-MOE, GNA, UNA, PNA, 5'-VP, etc.

[0018] 2'-F, 2'-OME, and 2'-MOE modifications refer to the substitution of the 2'-hydroxyl group at the furanyl ring of the nucleotide by fluorine (F), methoxy (OMe), or methoxyethoxy (MOE), respectively.

[0019] GNA modification refers to glycerol nucleotides, which are composed of glycerol (a pentose sugar in DNA and RNA) linked by phosphate ester bonds.

[0020] UNA modification refers to open-ring nucleotides. UNA lacks the C2' and C3' chemical bonds of the RNA ribose ring, but when bound to the RNA double-strand structure, it can still mimic unmodified RNA.

[0021] PNA modification refers to peptide nucleic acids, a class of DNA analogs in which the polypeptide backbone replaces the phosphate backbone.

[0022] 5'-VP modification refers to the modification of the 5' end of siRNA via vinyl-phosphate.

[0023] The interchain modifications mentioned include, but are not limited to, thiophosphate (PS) modification, methylphosphonate (MP) modification, and methoxypropylphosphonate (MOP) modification. Thiophosphate modification refers to the substitution of hydroxyl groups on the phosphate backbone with mercapto groups, MP modification refers to the substitution of hydroxyl groups on the phosphate backbone with methyl groups, and MOP modification refers to the substitution of hydroxyl groups on the phosphate backbone with methoxypropyl groups.

[0024] In one implementation, the sense strand of the siRNA targeting TYR is UmGfCmCfAmAfUmUfUmCfAmGfCmUfUmUfAmGfAm, and the antisense strand is UmCfUmAfAmAfGmCfUmGfAmAfAmUfUmGfGmCfAmdTsdT.

[0025] In one embodiment, the sense strand of the MMP1-targeting siRNA is GmsAmsGmGmAmAmAfAfCfAmCmUmGmGmAmAmAmAmAm, and the antisense strand is UmsUfsUmUmUfCfCfAmGmUmGmUmUmUmUmCfCmUmCmsAmsGm. In some embodiments, the lipophilic group may be conjugated to the 5′ and / or 3′ ends of the oligonucleotide, or bound to the oligonucleotide via a specific hydrocarbon chain or linker.

[0026] In another aspect, the present invention provides a method for improving the transdermal and / or intradermal delivery capability of oligonucleotides, the method comprising introducing the aforementioned lipophilic group into the oligonucleotide to prepare the aforementioned lipophilic oligonucleotide conjugate. The lipophilic oligonucleotide conjugate exhibits significantly higher transdermal and / or intradermal delivery capability compared to oligonucleotides without the introduced lipophilic group, and can enter skin cells via transdermal or intradermal delivery, achieving significantly superior therapeutic or cosmetic effects. The lipophilic group and the oligonucleotide are described as above.

[0027] In another aspect, the present invention provides a method for delivering oligonucleotides into skin cells, the method comprising: introducing a lipophilic group into the oligonucleotide to prepare a lipophilic oligonucleotide conjugate, wherein the oligonucleotide and the lipophilic group are described as above, and delivering the lipophilic oligonucleotide conjugate into skin cells by transdermal delivery or intradermal delivery.

[0028] The method of the present invention for improving the transdermal delivery capability (i.e., improving skin permeability) and / or intradermal delivery capability of oligonucleotides includes the steps of conjugating a lipophilic group to the 5′ and / or 3′ end of the oligonucleotide, or binding it to the oligonucleotide backbone via a specific hydrocarbon chain or linker. In some embodiments, if the lipophilic moiety contained in the lipophilic group is an alcohol, such as a sterol, the lipophilic moiety can be linked to a hydrocarbon group having a suitable terminal functional group via etherification to form an ether-linked intermediate. This ether-linked intermediate can be further transformed to form a reagent suitable for solid-phase nucleic acid synthesis, such as a phosphoramide intermediate, which can be used for phosphorylation modification at the 5′-hydroxyl position of the nucleotide, or for amidation modification of the support terminal group, to improve the coupling efficiency of nucleic acid synthesis. In some embodiments, the lipophilic moiety can also be linked to a linker having a suitable terminal functional group via an amide or the like, further linked to a solid-phase support (such as an aminomethyl resin), and then further linked to the 3′ position of the oligonucleotide via solid-phase synthesis.

[0029] The lipophilic oligonucleotide conjugation strategy provided by this invention shows significant improvements in skin permeability, cellular uptake, stability, and gene silencing, and has broad potential for therapeutic applications.

[0030] In another aspect, the present invention provides a composition comprising the above-described lipophilic oligonucleotide conjugate and optionally a carrier acceptable in cosmetics or pharmaceuticals. Specifically, the composition is a transdermal delivery drug or cosmetic product, or an intradermal delivery drug or cosmetic product. The carrier refers to any carrier, diluent, or excipient that is compatible with the said lipophilic oligonucleotide conjugate and other ingredients and is harmless to the user. Excipients include microcrystalline cellulose, magnesium stearate, calcium stearate, any acceptable sugar (e.g., mannose, xylitol), and oil-based excipients.

[0031] In some embodiments, the composition may also include other whitening active ingredients. These whitening active ingredients refer to active ingredients that help reduce or slow down skin pigmentation, achieving a skin whitening effect; they can also be called whitening agents. Examples include hydroquinone, arbutin, vitamin C (i.e., ascorbic acid or vitamin C) and its derivatives (vitamin C fatty acid esters (3-o-ethyl ascorbic acid, ascorbate glucoside) and vitamin C phosphate salts (magnesium ascorbate phosphate), etc.), retinoic acid, tranexamic acid, azelaic acid, kojic acid, nicotinamide, glycyrrhizin, phenylethyl resorcinol, tranexamic acid, potassium methoxysalicylate, styrax, bisabolol, ellagic acid, and whitening plant extracts, but are not limited to these. Whitening plant extracts mainly include oil-soluble licorice extract, mulberry leaf extract, mulberry extract, hydrangea extract, safflower extract, and chamomile extract, but are not limited to these.

[0032] In some embodiments, the composition may also include a delivery carrier (e.g., liposomes, carbomer gels, lipid nanoparticles, hyaluronic acid gels, etc.).

[0033] The compositions described herein may be provided in any cosmetic and / or dermatologically suitable form (e.g., emulsion, cream, mousse, gel, foam, lotion, mask, ointment, balm, solution, serum, spray, face mask, etc.).

[0034] The actual dose level of the lipophilic oligonucleotide conjugate in the composition can be varied, and the amount of the lipophilic oligonucleotide conjugate provides satisfactory results for a particular user without producing toxicity or side effects.

[0035] In another aspect, the present invention provides the use of the above-mentioned lipophilic oligonucleotide conjugates in the preparation of transdermal delivery small nucleic acid drugs.

[0036] In some embodiments, the transdermal delivery small nucleic acid drug can be used in the treatment of skin diseases and in the field of medical aesthetics and cosmetics, such as reducing melanin and increasing collagen. Attached Figure Description

[0037] Figure 1 shows the inhibition of mTYR mRNA expression in the melanocyte B16 cell line by the siRNA-lipophilic conjugate of the present invention.

[0038] Figure 2 shows the inhibition of hTYR mRNA expression in the human melanocyte cell line MNT1 by the siRNA-lipophilic conjugate of the present invention.

[0039] Figure 3 shows the inhibition of melanin content in the melanocyte cell line B16 by the siRNA-lipophilic conjugate of the present invention.

[0040] Figure 4 shows the inhibition of hMMP1 mRNA expression in the human fibrosarcoma cell line HT1080 by the siRNA-lipophilic conjugate of the present invention.

[0041] Figure 5 shows that the siRNA-lipophilic conjugate of the present invention can significantly affect the expression levels of matrix metalloproteinase 1 (MMP1) and type I and type III collagen (COL I and COL III) in mouse primary skin fibroblasts.

[0042] Figure 6 shows the Masson staining results of paraffin sections of skin tissue treated with the siRNA-lipophilic conjugate of the present invention (including application and injection). Detailed Implementation

[0043] This invention provides a lipophilic oligonucleotide conjugate designed to enhance the cell membrane affinity of oligonucleotides and significantly improve their ability to penetrate the stratum corneum of the skin, thereby optimizing their application in dermal drug delivery or topical therapy. By introducing a suitable hydrophobic lipophilic conjugate into the oligonucleotide, the conjugate can effectively intercalate into the phospholipid bilayer, improving the membrane permeability of the oligonucleotide, thereby enhancing delivery efficiency and stability in biological barrier environments. Furthermore, this lipophilic conjugate can reduce the degradation of oligonucleotides by nucleases, improve metabolic stability in vivo, and prolong their biological half-life.

[0044] Furthermore, this invention optimizes the length of the linker between the oligonucleotide and the lipophilic group to further improve its lipophilic properties. By rationally adjusting the length, flexibility, and hydrophobicity of the linker, it is made easier to embed into the phospholipid bilayer, and the polar groups that may affect membrane binding ability are reduced, thereby improving the transdermal delivery efficiency of the oligonucleotide. The optimized linker structure not only improves the lipophilicity of the oligonucleotide but also reduces its impact on the oligonucleotide's activity. Thus, the delivery efficiency is improved without sacrificing the efficacy of the oligonucleotide drug. In addition, by optimizing the chemical stability of the linker, the degradation mediated by esterases or phosphatases in vivo can be effectively reduced, further improving the bioavailability of nucleic acid drugs.

[0045] The lipophilic portion can possess groups capable of binding to membrane phospholipids, particularly lipid-soluble groups that can embed into cell membranes or penetrate the skin barrier, thereby enhancing the membrane permeability and skin permeability of oligonucleotides. These lipophilic groups include, but are not limited to, cholesterol and its derivatives (such as cholene, cholesterol, and cholesteryldiene), bile acids (such as cholic acid, deoxycholic acid, and dehydrocholic acid), sterols, steroids, and other lipid-soluble alcohols or thiols. Furthermore, these lipophilic groups also include, but are not limited to, fatty acids (such as long-chain fatty acids, branched-chain fatty acids, and saturated or unsaturated fatty acids), phospholipids (such as phosphatidylcholine and phosphatidylethanolamine), glycolipids and synthetic polymer-modified lipids (such as polyethylene glycol-lipid derivatives), ceramides, etc. These groups not only enhance the lipophilicity of oligonucleotides, improving their ability to penetrate the skin barrier and cell membrane, but also promote cellular uptake efficiency through interactions with cell membrane lipids and, to some extent, protect oligonucleotides from nuclease degradation. In addition, certain specific lipophilic groups, such as ceramides, can enhance the permeability of the stratum corneum, thereby further improving the transdermal absorption of oligonucleotides.

[0046] The present invention will be described in detail below by way of examples. However, the examples provided herein are for illustrative purposes only and are not intended to limit the invention.

[0047] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0048] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0049] Example 1: Preparation of delivery ligand Chol 01-CPG

[0050] 1.1 Preparation of Compound 2

[0051] N-Benzyloxycarbonyl-O-tert-butyl-L-serine (compound 1, 4.78 g, 20 mmol, 1.0 equivalent) and N,N-diisopropylethylamine (DIPEA, 10.4 mL, 60 mmol, 3.0 equivalent) were dissolved in anhydrous dichloromethane (DCM, 40 mL). Under ice bath stirring, a dichloromethane solution (48 mL) of 4,4′-dimethoxytriphenylchloromethane (DMTrCl, 4.73 g, 24 mmol, 1.2 equivalent) was added dropwise. After the addition was complete, the reaction was brought to room temperature and stirred overnight. The reaction was quenched with water. The reaction system was extracted three times with dichloromethane, and the combined organic phases were washed once with saturated brine. After drying with anhydrous sodium sulfate, the mixture was filtered, concentrated by vacuum distillation, and purified by silica gel column chromatography (dichloromethane / methanol = 100:1-10:1, with 0.5% triethylamine added to each of mobile phases A and B, and gradient elution). After drying under vacuum, compound 2 was obtained as a brown oily substance with a mass of 5.33 g and a yield of 49%. The product was identified by LC-MS: m / z: C 32 H 31 NO7, [M+1] + Theoretical value: 541.21, Actual value: 541.20. 1 H NMR (500MHz, DMSO) 87.42-7.16 (m, 14H), 6.97 (brs, 1H), 6.90-6.76 (m, 4H), 5.05 (q, J = 12.7Hz, 2H), 4.03 (brs, 1H), 3.72 (s, 6H), 3.27-3.15 (m, 2H).

[0052] 1.2 Preparation of Compound 3

[0053] Compound 2 (4.51 g, 8.34 mmol, 1.0 equivalence) and 4-hydroxypiperidine hydrochloride (1.15 g, 8.34 mmol, 1.0 equivalence) were dissolved in anhydrous N,N-dimethylformamide (DMF, 16 mL). 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU, 4.12 g, 10.84 mmol, 1.3 equivalence) was added, and the reaction system was purged under a nitrogen atmosphere using a vacuum pump. Then, N,N-diisopropylethylamine (5.8 mL, 33.34 mmol, 3.0 equivalence) was added with stirring in an ice bath. The mixture was stirred overnight at room temperature, and the reaction was quenched with water. The reaction solution was extracted three times with dichloromethane. The combined organic phases were washed once each with saturated sodium bicarbonate and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel rapid column chromatography (dichloromethane / methanol = 100:1-10:1, with 0.5% triethylamine added to each of mobile phases A and B, gradient elution). After drying under vacuum, compound 3 was obtained as a pale yellow, foamy solid with a mass of 3.95 g and a yield of 76%. The product was identified by LC-MS: m / z: C 37 H 40 N₂O₇, [M+1] + Theoretical value: 625.28, Actual value: 625.28. 1 H NMR (500MHz, DMSO) δ7.42-7.10 (m, 15H), 6.95-6.75 (m, 4H), 5.07 (td, J=14.4, 7.5Hz, 2H), 4.73 (dd, J=15 .3, 5.7Hz, 2H), 3.73 (s, 6H), 3.70-3.54 (m, 5H), 3.27-3.15 (m, 2H), 2.50-2.45 (m, 2H), 1.73-1.51 (m, 2H).

[0054] 1.3 Preparation of Compound 4

[0055] Compound 3 (2.24 g, 3.59 mmol) and palladium / carbon (Pd / C, 224 mg, 10% palladium loading) were added to anhydrous methanol (MeOH, 10 mL), and the reaction mixture was then purged under a hydrogen atmosphere using a vacuum pump. The mixture was stirred overnight at room temperature. The palladium / carbon was then removed by diatomaceous earth filtration, followed by vacuum distillation and concentration. The product was dried under vacuum to give compound 4, a light brown oil with a mass of 1.76 g and a yield of approximately 100%. This product could be used directly in the next reaction without further purification. The product was identified by LC-MS: MS m / z: C 29 H 34 N₂O₅, [M+1] +Theoretical value: 491.25, Actual value: 491.24.

[0056] 1.4 Preparation of Compound 6

[0057] Aminotetraethylene glycol carboxyethyl (purchased from Shanghai Bied Pharmaceutical Technology Co., Ltd., 597 mg, 2.25 mmol, 1.0 equivalent) was dissolved in anhydrous pyridine. After cooling in an ice bath, trimethylchlorosilane (TMSCl, 978 mg, 9.00 mmol, 4.0 equivalent) was added. After stirring in an ice bath for 1 hour, cholesterol formyl chloride (compound 5, 505 mg, 1.12 mmol, 0.5 equivalent) was added. After reacting for 1 hour, another portion of cholesterol formyl chloride (505 g, 1.12 mmol, 0.5 equivalent) was added. The reaction was brought to room temperature and then continued at room temperature for 3 hours. The reaction was then cooled again in an ice bath, and the pH was adjusted to 5 by adding 1 N dilute hydrochloric acid. The mixture was extracted three times with dichloromethane. The combined organic phases were washed once each with 1 N dilute hydrochloric acid and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated by vacuum distillation. The residue was purified by silica gel rapid column chromatography (dichloromethane / methanol = 100:1-20:1 gradient elution) to give compound 6, 1.3 g, in 85% yield. The product was identified by LC-MS: MS m / z: C 39 H 67 NO.8, [M+H] + Theoretical value: 678.50, Actual value: 678.49.

[0058] 1.5 Preparation of Compound 7

[0059] Compound 6 (678 mg, 1.00 mmol, 1.0 equivalence) and compound 4 (491 mg, 1.00 mmol, 1.0 equivalence) were dissolved in anhydrous N,N-dimethylformamide (5 mL). 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU, 494 mg, 1.30 mmol, 1.3 equivalence) was added, and the reaction system was purged under a nitrogen atmosphere using a vacuum pump. Then, N,N-diisopropylethylamine (522 μL, 3.0 mmol, 3.0 equivalence) was added with stirring in an ice bath. The mixture was stirred overnight at room temperature, and the reaction was quenched with water. The reaction solution was extracted three times with dichloromethane. The combined organic phases were washed once each with saturated sodium bicarbonate and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel rapid column chromatography (dichloromethane / methanol = 100:1-10:1, with 0.5% triethylamine added to each of mobile phases A and B, and gradient elution). After drying under vacuum, compound 7 was obtained as a pale yellow, foamy solid with a mass of 962 mg, yielding 84%. The product was identified by LC-MS: MS m / z: C 68 H 99 N3O 12 [M+H] + Theoretical value: 1150.73, Actual value: 1150.72.

[0060] 1.6 Preparation of Compound 8

[0061] Compound 7 (575 mg, 0.5 mmol, 1.0 equivalence), succinic anhydride (150 mg, 1.5 mmol, 3.0 equivalence), and 4-dimethylaminopyridine (DMAP, 183 mg, 1.5 mmol, 3.0 equivalence) were dissolved in anhydrous dichloromethane (4 mL). The reaction system was then purged under a nitrogen atmosphere using a vacuum pump. N,N-diisopropylethylamine (260 μL, 1.5 mmol, 3.0 equivalence) was added with stirring in an ice bath. The reaction was brought to room temperature and stirred overnight. After complete reaction as detected by LC-MS, the reaction was quenched with water. The reaction system was extracted three times with dichloromethane. The combined organic phases were washed once each with saturated ammonium chloride solution and saturated brine solution, dried over anhydrous sodium sulfate, filtered, concentrated by vacuum distillation, and purified by silica gel rapid column chromatography (dichloromethane / methanol = 100:1-10:1, with 0.5% triethylamine added to each of mobile phases A and B, gradient elution). After drying under vacuum, compound 8 was given as a pale yellow foamy solid with a mass of 420 mg and a yield of 67%. The product was identified by LC-MS: MS m / z: C 72 H 103 N3O 15 [M+1] +Theoretical value: 1250.75, Actual value: 1250.73.

[0062] 1.7 Preparation of Compound 9

[0063] Aminosilanized microporous glass spheres

[0064] Compound 8 (100 mg, 0.08 mmol, 1.0 equivalent) and benzotriazole-N,N,N′,N′-tetramethylurea hexafluorophosphate (HBTU, 36.4 mg, 0.10 mmol, 1.3 equivalent) were dissolved in anhydrous N,N-dimethylformamide (5 mL), followed by the addition of N,N-diisopropylethylamine (56 μL, 0.32 mmol, 4.0 equation). After shaking for 15 minutes, aminosilanized microporous glass beads (CPG, purchased from Hebei Dinaxingke Biotechnology Co., Ltd.) were added. The mixture was prepared by shaking for 24 hours (80 μmol / g, 1.0 g). After the reaction was complete, the mixture was filtered. The CPG was washed three times each with N,N-dimethylformamide, dichloromethane / methanol (v / v, 9:1), and dichloromethane, and then dried under vacuum. Acetic anhydride / pyridine (v / v, 1:3, 6 mL) was added to the CPG filter cake, and the mixture was shaken for 30 minutes to cap the unlinked amino groups with acetyl groups. The mixture was filtered again, and the CPG filter cake was washed three times each with dichloromethane / methanol (v / v, 9:1) and dichloromethane, and then dried under vacuum. Product compound 9 was obtained, which is CPG linked to ligand Chol 01, with a yield of 1.0 g. The loading amount was determined to be 54.1 μmol / g by detecting the absorbance of the 4,4′-dimethoxytriphenylmethyl group (DMTr).

[0065] Example 2: Preparation of compound Chol O2-CPG

[0066] 2.1 Preparation of Compound 10

[0067] 6-Aminohexanoic acid (0.66 g, 5.00 mmol) was dissolved in anhydrous pyridine (25 mL). After cooling in an ice bath, trimethylchlorosilane (2.17 g, 20.00 mmol, 4.0 equivalence) was added. After stirring in an ice bath for 1 hour, cholesterol formyl chloride (compound 5, 1.13 g, 2.50 mmol, 2.5 equivalence) was added. After reacting for 1 hour, another portion of cholesterol formyl chloride (1.13 g, 2.50 mmol, 2.5 equivalence) was added. The reaction was brought to room temperature and then continued at room temperature for 3 hours. The reaction was then cooled again in an ice bath, and the pH was adjusted to 5 by adding 1 N dilute hydrochloric acid. The mixture was extracted three times with dichloromethane. The combined organic phases were washed once each with 1 N dilute hydrochloric acid and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated by vacuum distillation. The residue was purified by silica gel rapid column chromatography (dichloromethane / methanol = 100:1-20:1, gradient elution) to give compound 10 as a white solid, with a mass of 2.20 g and a yield of 81%. The product was identified by LC-MS: MS m / z: C 34 H 57 NO4, [M+Na] + Theoretical value: 566.42, Actual value: 566.41.

[0068] 2.2 Preparation of Compound 11

[0069] Compound 10 (544 mg, 1.00 mmol) and compound 4 (491 mg, 1.00 mmol, 1.0 equivalence) were dissolved in anhydrous N,N-dimethylformamide (5 mL), and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU, 494 mg, 1.30 mmol, 1.3 equivalence) was added. The reaction system was then purged under a nitrogen atmosphere using a vacuum pump. N,N-diisopropylethylamine (522 μL, 3.0 mmol, 3.0 equivalence) was then added with stirring in an ice bath. The mixture was stirred overnight at room temperature, and the reaction was quenched with water. The reaction solution was extracted three times with dichloromethane. The combined organic phases were washed once each with saturated sodium bicarbonate and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel rapid column chromatography (dichloromethane / methanol = 100:1-10:1, with 0.5% triethylamine added to each of mobile phases A and B, gradient elution). After drying under vacuum, compound 11 was obtained as a pale yellow, foamy solid with a mass of 681 mg, yielding 67%. The product was identified by LC-MS: MS m / z: C 63 H 89 N3O8, [M+Na] + Theoretical value: 1038.65, Actual value: 1038.64.

[0070] 2.3 Preparation of Compound 12

[0071] Compound 11 (1.0 g, 1.00 mmol), succinic anhydride (300 mg, 3.00 mmol, 3.0 equivalence) and 4-dimethylaminopyridine (366 mg, 3.00 mmol, 3.0 equivalence) were dissolved in anhydrous dichloromethane (10 mL). The reaction system was then purged under a nitrogen atmosphere using a vacuum pump. N,N-diisopropylethylamine (869 μL, 5.00 mmol, 5.0 equivalence) was added with stirring in an ice bath. The reaction was brought to room temperature and stirred overnight. After complete reaction as detected by LC-MS, the reaction was quenched with water. The reaction system was extracted three times with dichloromethane. The combined organic phases were washed once each with saturated ammonium chloride solution and saturated brine solution, dried over anhydrous sodium sulfate, filtered, concentrated by vacuum distillation, and purified by silica gel rapid column chromatography (dichloromethane / methanol = 100:1-10:1, with 0.5% triethylamine added to each of mobile phases A and B, gradient elution). After drying under vacuum, compound 12 was obtained as a pale yellow, foamy solid with a mass of 910 mg, yielding approximately 82%. The product was identified by LC-MS: MS m / z: C 67 H 93 N3O 11 [M+1] + Theoretical value: 1116.69, Actual value: 1116.65.

[0072] 2.4 Preparation of Compound 13

[0073] Compound 12 (89 mg, 0.08 mmol, 1.0 equivalence) and HBTU (36 mg, 0.10 mmol, 1.2 equivalence) were dissolved in anhydrous N,N-dimethylformamide (5 mL), followed by the addition of N,N-diisopropylethylamine (56 μL, 0.32 mmol, 4.0 equivalence). After shaking for 15 minutes, aminosilanized microporous glass beads (CPG, purchased from Hebei Dinaxingke Biotechnology Co., Ltd.) were added. The mixture was prepared by shaking for 24 hours (80 μmol / g, 1.0 g). After the reaction was complete, the mixture was filtered. The CPG was washed three times each with N,N-dimethylformamide, dichloromethane / methanol (v / v, 9:1), and dichloromethane, and then dried under vacuum. Acetic anhydride / pyridine (v / v, 1:3, 8 mL) was then added to the CPG filter cake, and the mixture was shaken for 30 minutes to cap the unlinked amino groups with acetyl groups. The mixture was filtered again, and the CPG filter cake was washed three times each with dichloromethane / methanol (v / v, 9:1) and dichloromethane, and then dried under vacuum. Product compound 13 was obtained, which is CPG linked to ligand Chol O2. The yield was 1.0 g, and the loading was determined to be 58.9 μmol / g by detecting the absorbance of the 4,4′-dimethoxytriphenylmethyl group (DMTr).

[0074] Example 3: Preparation of compound Chol O3-CPG

[0075] 3.1 Preparation of Compound 14

[0076] 3-Amino-1,2-propanediol (455 mg, 5.00 mmol, 1.0 equivalence) was dissolved in anhydrous pyridine (5 mL). After cooling in an ice bath, cholesterol formyl chloride (compound 5, 2.24 g, 5.00 mmol, 1.0 equivalence) was dissolved in anhydrous dichloromethane (5 mL) and then added dropwise to the reaction mixture. After the addition was complete, the mixture was brought to room temperature and stirred for 16 hours. The reaction solvent was evaporated to dryness, and the residue was purified by rapid silica gel column chromatography (petroleum ether / ethyl acetate = 100:1-10:1, gradient elution) to give compound 14 as a white solid with a mass of 2.1 g and a yield of approximately 83%. The product was identified by LC-MS: MS m / z: C 31 H 53 NO4, [M+1] + Theoretical value: 504.41, Actual value: 504.40.

[0077] 3.2 Preparation of Compound 15

[0078] Compound 14 (2.1 g, 4.17 mmol, 1.0 equivalence) was dissolved in anhydrous pyridine (15 mL). Under ice bath stirring, a solution of 4,4′-dimethoxytriphenylchloromethane (1.69 g, 5.00 mmol, 1.2 equivalence) in dichloromethane (10 mL) was added dropwise. After the addition was complete, the reaction was brought to room temperature and stirred overnight. The reaction was quenched with water. The reaction mixture was extracted three times with dichloromethane. The combined organic phases were washed once with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated by vacuum distillation, and purified by rapid silica gel column chromatography (dichloromethane / methanol = 100:1-10:1, with 0.5% triethylamine added to each of mobile phases A and B, gradient elution). After drying under vacuum, compound 15 was obtained as a light brown, foamy solid, weighing 3.0 g, with a yield of 90%. The product was identified by LC-MS: m / z: C 52 H 71 NO6, [M+1] + Theoretical value: 806.54, Actual value: 806.52.

[0079] 3.3 Preparation of Compound 16

[0080] Compound 15 (3.0 g, 3.72 mmol), succinic anhydride (1.1 g, 11.17 mmol, 3.0 equivalence) and 4-dimethylaminopyridine (1.36 g, 11.17 mmol, 3.0 equivalence) were dissolved in anhydrous dichloromethane (4 mL). The reaction system was then purged under a nitrogen atmosphere using a vacuum pump. N,N-diisopropylethylamine (1.95 mL, 11.17 mmol, 3 equivalence) was added with stirring in an ice bath. The reaction was brought to room temperature and stirred overnight. After complete reaction as detected by LC-MS, the reaction was quenched with water. The reaction system was extracted three times with dichloromethane. The combined organic phases were washed once each with saturated ammonium chloride solution and saturated brine solution, dried over anhydrous sodium sulfate, filtered, concentrated by vacuum distillation, and purified by silica gel rapid column chromatography (dichloromethane / methanol = 100:1-10:1, with 0.5% triethylamine added to each of mobile phases A and B, gradient elution). After drying under vacuum, compound 16 was obtained as a white, foamy solid with a mass of 2.8 g and a yield of approximately 83%. The product was identified by LC-MS: MS m / z: C 56 H 75 NO9, [M+1] + Theoretical value: 906.55, Actual value: 906.53.

[0081] 3.4 Preparation of Compound 17

[0082] Compound 16 (100 mg, 0.11 mmol, 1.0 equivalence) and HBTU (50 mg, 0.13 mmol, 1.2 equivalence) were dissolved in anhydrous N,N-dimethylformamide (5 mL), followed by the addition of N,N-diisopropylethylamine (77 μL, 0.44 mmol, 4.0 equivalence). After shaking for 15 minutes, aminosilanized microporous glass beads (CPG, purchased from Hebei Dinaxingke Biotechnology Co., Ltd.) were added. The product compound 17, CPG with 80 μmol / g (1.37 g), was reacted with shaking for 24 hours. After the reaction was complete, the mixture was filtered. The CPG was washed three times each with N,N-dimethylformamide, dichloromethane / methanol (v / v, 9:1), and dichloromethane, and then dried under vacuum. Acetic anhydride / pyridine (v / v, 1:3, 8 mL) was added to the CPG filter cake, and the mixture was shaken for 30 minutes to cap the unlinked amino groups with acetyl groups. The mixture was filtered again, and the CPG filter cake was washed three times each with dichloromethane / methanol (v / v, 9:1) and dichloromethane, and then dried under vacuum. The product compound 17 was obtained, which is CPG linked to the Chol 03 ligand. The yield was 1.38 g, and the loading was 57.1 μmol / g as determined by the absorbance of the 4,4′-dimethoxytriphenylmethyl group (DMTr).

[0083] Example 4: Preparation of compound Chol O4-CPG

[0084] 4.1 Preparation of Compound 18

[0085] 3-Aminopropionic acid (3.0 g, 34.00 mmol) was dissolved in dioxane (20 mL), sodium carbonate (11.0 g, 104.00 mmol, 3.05 equivalence) and water (60 mL) was added, and the mixture was cooled to 0 °C. Benzyl chloroformate (6.0 mL, 41.00 mmol, 1.20 equivalence) was then added. The reaction mixture was stirred overnight at room temperature. After washing with n-hexane, the aqueous phase was adjusted to pH 1 and extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated by vacuum distillation to give compound 18 as a colorless oil with a mass of 6.3 g and a yield of approximately 83%. The product was identified by LC-MS: MS m / z: C 11 H 13 NNaO4, [M+Na] + Theoretical value: 246.07, Actual value: 246.03.

[0086] 4.2 Preparation of Compound 19

[0087] Compound 18 (1.10 g, 4.93 mmol, 1.0 equivalence) and HBTU (2.24 g, 5.92 mmol, 1.2 equivalence) were dissolved in anhydrous N,N-dimethylformamide (15 mL), followed by the addition of N,N-diisopropylethylamine (3.4 mL, 19.72 mmol, 4.0 equivalence). After stirring for 30 min, 3-amino-1,2-propanediol (583 mg, 6.41 mmol, 1.3 equivalence) was added, and the mixture was stirred overnight at room temperature. The reaction was then quenched with water. The reaction solution was extracted three times with dichloromethane. The combined organic phases were washed once with saturated sodium bicarbonate and once with saturated brine, respectively. After drying with anhydrous sodium sulfate and filtration, the solution was concentrated under reduced pressure and purified by silica gel rapid column chromatography (dichloromethane / methanol = 100:1-10:1, gradient elution). After drying under vacuum, compound 19 was given as a white solid with a mass of 890 mg, yielding 61%. The product was identified by LC-MS: MS m / z: C 14 H 21 N₂O₅, [M+H] + Theoretical value: 297.14, Actual value: 297.16.

[0088] 4.3 Preparation of Compound 20

[0089] Compound 19 (500 mg, 1.69 mmol, 1.0 equivalence) was dissolved in anhydrous pyridine (5 mL). Under ice bath stirring, a solution of 4,4′-dimethoxytriphenylchloromethane (685 mg, 2.03 mmol, 1.2 equivalence) in dichloromethane (2 mL) was added dropwise. After the addition was complete, the reaction was brought to room temperature and stirred overnight. The reaction was quenched with water. The reaction mixture was extracted three times with dichloromethane. The combined organic phases were washed once with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated by vacuum distillation, and purified by rapid silica gel column chromatography (dichloromethane / methanol = 100:1-10:1, with 0.5% triethylamine added to each of mobile phases A and B, gradient elution). After drying under vacuum, compound 20 was obtained as a light brown, foamy solid with a mass of 808 mg, yielding 80%. The product was identified by LC-MS: m / z: C 35 H 39 N₂O₇, [M+1] + Theoretical value: 599.28, Actual value: 599.16.

[0090] 4.4 Preparation of Compound 21

[0091] Compound 20 (808 mg, 1.35 mmol) and palladium / carbon (Pd / C, 81 mg, 10% palladium loading) were added to anhydrous methanol (MeOH, 5 mL), and the reaction mixture was then purged under a hydrogen atmosphere using a vacuum pump. The mixture was stirred overnight at room temperature. The palladium / carbon was then removed by diatomaceous earth filtration, followed by vacuum distillation and concentration. The product was dried under vacuum to obtain compound 21 as a colorless oil with a mass of 614 mg, yielding approximately 98%. This product could be used directly in the next reaction without further purification. The product was identified by LC-MS: MS m / z: C 27 H 33 N₂O₃, [M+1] + Theoretical value: 465.23, Actual value: 465.14.

[0092] 4.5 Preparation of Compound 22

[0093] Compound 21 (614 mg, 1.32 mmol, 1.0 equivalence) was dissolved in anhydrous pyridine (5 mL). After cooling in an ice bath, cholesterol formyl chloride (compound 5, 593 mg, 1.32 mmol, 1.0 equivalence) was dissolved in anhydrous dichloromethane (2 mL) and then added dropwise to the reaction mixture. After the addition was complete, the mixture was brought to room temperature and stirred for 16 hours. The reaction solvent was evaporated to dryness, and the residue was purified by silica gel rapid column chromatography (petroleum ether / ethyl acetate = 100:1-10:1, gradient elution) to give compound 22 as a white solid with a mass of 937 mg, yielding approximately 81%. The product was identified by LC-MS: MS m / z: C 55 H 77 N₂O₇, [M+1] + Theoretical value: 877.56, Actual value: 877.38.

[0094] 4.6 Preparation of Compound 23

[0095] Compound 22 (512 mg, 0.58 mmol), succinic anhydride (175 mg, 1.75 mmol, 3.0 equivalence) and 4-dimethylaminopyridine (212 mg, 1.74 mmol, 3.0 equivalence) were dissolved in anhydrous dichloromethane (4 mL). The reaction system was then purged under a nitrogen atmosphere using a vacuum pump. N,N-diisopropylethylamine (300 μL, 1.74 mmol, 3 equivalence) was added with stirring in an ice bath. The reaction was brought to room temperature and stirred overnight. After complete reaction as detected by LC-MS, the reaction was quenched with water. The reaction system was extracted three times with dichloromethane. The combined organic phases were washed once each with saturated ammonium chloride solution and saturated brine solution, dried over anhydrous sodium sulfate, filtered, concentrated by vacuum distillation, and purified by silica gel rapid column chromatography (dichloromethane / methanol = 100:1-10:1, with 0.5% triethylamine added to each of mobile phases A and B, gradient elution). After drying under vacuum, compound 23 was obtained as a white foamy solid with a mass of 350 mg and a yield of approximately 62%. The product was identified by LC-MS: MS m / z: C 59 H 81 N2O 10 [M+1] + Theoretical value: 977.58, Actual value: 977.51.

[0096] 4.7 Preparation of Compound 24

[0097] Compound 23 (100 mg, 0.10 mmol, 1.0 equivalence) and HBTU (45 mg, 0.12 mmol, 1.2 equivalence) were dissolved in anhydrous N,N-dimethylformamide (3 mL), followed by the addition of N,N-diisopropylethylamine (70 μL, 0.40 mmol, 4.0 equivalence). After shaking for 15 minutes, aminosilanized microporous glass beads (CPG, purchased from Hebei Dinaxingke Biotechnology Co., Ltd.) were added. The product compound 24, consisting of Chol O4 ligand-linked CPG, was reacted with N,N-dimethylformamide, dichloromethane / methanol (v / v, 9:1), and dichloromethane three times each, and then dried under vacuum. Acetic anhydride / pyridine (v / v, 1:3, 8 mL) was added to the CPG filter cake, and the mixture was shaken for 30 minutes to cap unlinked amino groups with acetyl groups. The mixture was then filtered again, and the CPG filter cake was washed three times each with dichloromethane / methanol (v / v, 9:1) and dichloromethane, and then dried completely under vacuum. This yielded product compound 24, which is CPG linked to the Chol O4 ligand. The yield was 1.23 g, and the loading was determined to be 41.1 μmol / g by detecting the absorbance of the 4,4′-dimethoxytriphenylmethyl group (DMTr).

[0098] Example 5: Preparation of compound Chol 05-CPG

[0099] 5.1 Preparation of Compound 25

[0100] 5-Aminovaleric acid (3.5 g, 30.00 mmol) was dissolved in dioxane (20 mL), sodium carbonate (9.7 g, 91.50 mmol, 3.05 equivalence) and water (60 mL) was added, and the mixture was cooled to 0 °C. Benzyl chloroformate (5.0 mL, 36.00 mmol, 1.20 equivalence) was then added. The reaction mixture was stirred overnight at room temperature. After washing with n-hexane, the aqueous phase was adjusted to pH 1 and extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated by vacuum distillation to give compound 25 as a colorless solid with a mass of 6.1 g and a yield of approximately 81%. The product was identified by LC-MS: MS m / z: C 13 H 17 NNaO4, [M+Na] + Theoretical value: 274.10, Actual value: 274.08.

[0101] 5.2 Preparation of Compound 26

[0102] Compound 25 (2.5 g, 10.0 mmol, 1.0 equivalence) and HBTU (4.6 g, 12.00 mmol, 1.2 equivalence) were dissolved in anhydrous N,N-dimethylformamide (30 mL), followed by the addition of N,N-diisopropylethylamine (7 mL, 40.00 mmol, 4.0 equivalence). After stirring for 30 minutes, 3-amino-1,2-propanediol (1.18 g, 13.00 mmol, 1.3 equivalence) was added, and the mixture was stirred overnight at room temperature. The reaction was then quenched with water. The reaction solution was extracted three times with dichloromethane. The combined organic phases were washed once with saturated sodium bicarbonate and once with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography (dichloromethane / methanol = 100:1-10:1, gradient elution). After drying under vacuum, compound 26 was given as a white solid with a mass of 2.3 g and a yield of 71%. The product was identified by LC-MS: MS m / z: C 16 H 25 N₂O₅, [M+H] + Theoretical value: 324.17, Actual value: 324.14.

[0103] 5.3 Preparation of Compound 27

[0104] Compound 26 (2.0 g, 6.17 mmol, 1.0 equivalence) was dissolved in anhydrous pyridine (30 mL). Under ice bath stirring, a solution of 4,4′-dimethoxytriphenylchloromethane (2.51 g, 7.40 mmol, 1.2 equivalence) in dichloromethane (15 mL) was added dropwise. After the addition was complete, the reaction was brought to room temperature and stirred overnight. The reaction was quenched with water. The reaction mixture was extracted three times with dichloromethane. The combined organic phases were washed once with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated by vacuum distillation, and purified by rapid silica gel column chromatography (dichloromethane / methanol = 100:1-10:1, with 0.5% triethylamine added to each of mobile phases A and B, gradient elution). After drying under vacuum, compound 27 was obtained as a brown, foamy solid with a mass of 3.4 g and a yield of 88%. The product was identified by LC-MS: m / z: C 37 H 43 N₂O₇, [M+1] + Theoretical value: 627.30, Actual value: 627.18.

[0105] 5.4 Preparation of Compound 28

[0106] Compound 27 (3.0 g, 4.8 mmol) and palladium / carbon (Pd / C, 300 mg, 10% palladium loading) were added to anhydrous methanol (20 mL), and the reaction mixture was then purged under a hydrogen atmosphere using a vacuum pump. The mixture was stirred overnight at room temperature. The palladium / carbon was then removed by diatomaceous earth filtration, followed by vacuum distillation and concentration. The product was dried under vacuum to obtain compound 28 as a colorless oil, weighing 2.3 g, with a yield of approximately 96%. This product could be used directly in the next reaction without further purification. The product was identified by LC-MS: MS m / z: C 29 H 36 N₂NaO₅, [M+Na] + Theoretical value: 515.25, Actual value: 515.20.

[0107] 5.5 Preparation of Compound 29

[0108] Compound 28 (2.3 g, 4.67 mmol, 1.0 equivalence) was dissolved in anhydrous pyridine (20 mL). After cooling in an ice bath, cholesterol formyl chloride (compound 5, 2.1 g, 4.67 mmol, 1.0 equivalence) was dissolved in anhydrous dichloromethane (10 mL) and then added dropwise to the reaction mixture. After the addition was complete, the mixture was brought to room temperature and stirred for 16 hours. The reaction solvent was evaporated to dryness, and the residue was purified by silica gel rapid column chromatography (petroleum ether / ethyl acetate = 100:1-10:1, gradient elution) to give compound 29 as a white foamy solid with a mass of 3.7 g and a yield of approximately 89%. The product was identified by LC-MS: MS m / z: C57 H 81 N₂O₇, [M+1] + Theoretical value: 905.60, Actual value: 905.44.

[0109] 5.6 Preparation of Compound 30

[0110] Compound 29 (1.0 g, 1.11 mmol), succinic anhydride (333 mg, 3.33 mmol, 3.0 equivalence) and 4-dimethylaminopyridine (406 mg, 3.33 mmol, 3.0 equivalence) were dissolved in anhydrous dichloromethane (10 mL). The reaction system was then purged under a nitrogen atmosphere using a vacuum pump. N,N-diisopropylethylamine (580 μL, 3.33 mmol, 3 equivalence) was added with stirring in an ice bath. The reaction was brought to room temperature and stirred overnight. After complete reaction as detected by LC-MS, the reaction was quenched with water. The reaction system was extracted three times with dichloromethane. The combined organic phases were washed once each with saturated ammonium chloride solution and saturated brine solution, dried over anhydrous sodium sulfate, filtered, concentrated by vacuum distillation, and purified by silica gel rapid column chromatography (dichloromethane / methanol = 100:1-10:1, with 0.5% triethylamine added to each of mobile phases A and B, gradient elution). After drying under vacuum, compound 30 was obtained as a white foamy solid with a mass of 713 mg, yielding approximately 64%. The product was identified by LC-MS: MS m / z: C 61 H 84 N2O 10 [M+1] + Theoretical value: 1005.61, Actual value: 1005.63.

[0111] 5.7 Preparation of Compound 31

[0112] Compound 30 (100 mg, 0.1 mmol, 1.0 equivalence) and HBTU (45 mg, 0.12 mmol, 1.2 equivalence) were dissolved in anhydrous N,N-dimethylformamide (3 mL), followed by the addition of N,N-diisopropylethylamine (70 μL, 0.40 mmol, 4.0 equivalence). After shaking for 15 minutes, aminosilanized microporous glass beads (CPG, purchased from Hebei Dinaxingke Biotechnology Co., Ltd.) were added. The product compound 31, CPG with 80 μmol / g (1.25 g), was reacted with shaking for 24 hours. After the reaction was complete, the mixture was filtered. The CPG was washed three times each with N,N-dimethylformamide, dichloromethane / methanol (v / v, 9:1), and dichloromethane, and then dried under vacuum. Acetic anhydride / pyridine (v / v, 1:3, 8 mL) was added to the CPG filter cake, and the mixture was shaken for 30 minutes to cap the unlinked amino groups with acetyl groups. The mixture was filtered again, and the CPG filter cake was washed three times each with dichloromethane / methanol (v / v, 9:1) and dichloromethane, and then dried under vacuum. The product compound 31 was obtained, which is CPG linked to the Chol 05 ligand. The yield was 1.25 g, and the loading was determined to be 46.2 μmol / g by detecting the absorbance of the 4,4′-dimethoxytriphenylmethyl group (DMTr).

[0113] Example 6: Preparation of compound Chol 06-CPG

[0114] 6.1 Preparation of Compound 32

[0115] 7-Aminoheptanoic acid (5.0 g, 34.43 mmol) was dissolved in dioxane (35 mL), sodium carbonate (11.13 g, 105.03 mmol, 3.05 equivalence) and water (60 mL) was added, and the mixture was cooled to 0 °C. Benzyl chloroformate (5.8 mL, 41.32 mmol, 1.20 equivalence) was then added. The reaction mixture was stirred overnight at room temperature. After washing with n-hexane, the aqueous phase was adjusted to pH 1 and extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated by vacuum distillation to give compound 32 as a colorless solid with a mass of 7.4 g and a yield of approximately 77%. The product was identified by LC-MS: MS m / z: C 15 H 21 NNaO4, [M+Na] + Theoretical value: 302.14, Actual value: 302.22.

[0116] 6.2 Preparation of Compound 33

[0117] Compound 32 (5.0 g, 17.92 mmol, 1.0 equivalence) and HBTU (8.15 g, 21.50 mmol, 1.2 equivalence) were dissolved in anhydrous N,N-dimethylformamide (36 mL), followed by the addition of N,N-diisopropylethylamine (12.5 mL, 71.68 mmol, 4.0 equivalence). After stirring for 30 minutes, 3-amino-1,2-propanediol (2.12 g, 23.30 mmol, 1.3 equivalence) was added, and the mixture was stirred overnight at room temperature. The reaction was then quenched with water. The reaction solution was extracted three times with dichloromethane. The combined organic phases were washed once with saturated sodium bicarbonate and once with saturated brine, respectively. After drying with anhydrous sodium sulfate and filtration, the solution was concentrated under reduced pressure and purified by silica gel column chromatography (dichloromethane / methanol = 100:1-10:1, gradient elution). After drying under vacuum, compound 33 was given as a white solid with a mass of 5.2 g and a yield of 82%. The product was identified by LC-MS: MS m / z: C 18 H 29 N₂O₅, [M+H] + Theoretical value: 353.20, Actual value: 353.10.

[0118] 6.3 Preparation of Compound 34

[0119] Compound 33 (5.0 g, 14.20 mmol, 1.0 equivalence) was dissolved in anhydrous pyridine (50 mL). Under ice bath stirring, a solution of 4,4′-dimethoxytriphenylchloromethane (5.8 g, 17.04 mmol, 1.2 equivalence) in dichloromethane (30 mL) was added dropwise. After the addition was complete, the reaction was brought to room temperature and stirred overnight. The reaction was quenched with water. The reaction mixture was extracted three times with dichloromethane. The combined organic phases were washed once with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated by vacuum distillation, and purified by rapid silica gel column chromatography (dichloromethane / methanol = 100:1-10:1, with 0.5% triethylamine added to each of mobile phases A and B, gradient elution). After drying under vacuum, compound 34 was obtained as a reddish-brown foamy solid with a mass of 8.4 g and a yield of 91%. The product was identified by LC-MS: m / z: C 39 H 47 N₂O₇, [M+1] + Theoretical value: 655.33, Actual value: 655.33.

[0120] 6.4 Preparation of Compound 35

[0121] Compound 34 (8.0 g, 12.23 mmol) and palladium / carbon (Pd / C, 800 mg, 10% palladium loading) were added to anhydrous methanol (50 mL), and the reaction mixture was then purged under a hydrogen atmosphere using a vacuum pump. The mixture was stirred overnight at room temperature. The palladium / carbon was then removed by diatomaceous earth filtration, followed by vacuum distillation and concentration. The product was dried under vacuum to obtain compound 35 as a reddish-brown, foamy solid, weighing 6.0 g, with a yield of approximately 95%. This product could be used directly in the next reaction without further purification. The product was identified by LC-MS: MS m / z: C 31 H 40 N₂NaO₅, [M+Na] + Theoretical value: 543.28, Actual value: 543.22.

[0122] 6.5 Preparation of Compound 36

[0123] Compound 35 (3.1 g, 5.96 mmol, 1.0 equivalence) was dissolved in anhydrous pyridine (25 mL). After cooling in an ice bath, cholesterol formyl chloride (compound 5, 2.67 g, 5.96 mmol, 1.0 equivalence) was dissolved in anhydrous dichloromethane (15 mL) and then added dropwise to the reaction mixture. After the addition was complete, the mixture was brought to room temperature and stirred for 16 hours. The reaction solvent was evaporated to dryness, and the residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 100:1-10:1, gradient elution) to give compound 36 as a brown, foamy solid with a mass of 4.2 g and a yield of approximately 76%. The product was identified by LC-MS: MS m / z: C 59 H 85 N₂O₇, [M+1] + Theoretical value: 933.63, Actual value: 933.61.

[0124] 6.6 Preparation of Compound 37

[0125] Compound 36 (2.1 g, 2.25 mmol), succinic anhydride (676 mg, 6.76 mmol, 3.0 equivalence) and 4-dimethylaminopyridine (825 mg, 6.76 mmol, 3.0 equivalence) were dissolved in anhydrous dichloromethane (25 mL). The reaction system was then purged under a nitrogen atmosphere using a vacuum pump. N,N-diisopropylethylamine (1.2 mL, 6.76 mmol, 3 equivalence) was added with stirring in an ice bath. The reaction was brought to room temperature and stirred overnight. After complete reaction as detected by LC-MS, the reaction was quenched with water. The reaction system was extracted three times with dichloromethane. The combined organic phases were washed once each with saturated ammonium chloride solution and saturated brine solution, dried over anhydrous sodium sulfate, filtered, concentrated by vacuum distillation, and purified by silica gel rapid column chromatography (dichloromethane / methanol = 100:1-10:1, with 0.5% triethylamine added to each of mobile phases A and B, gradient elution). After drying under vacuum, compound 37 was obtained as a white foamy solid with a mass of 1.70 g and a yield of approximately 73%. The product was identified by LC-MS: MS m / z: C 63 H 89 N2O 10 [M+1] + Theoretical value: 1033.64, Actual value: 1033.75.

[0126] 6.7 Preparation of Compound 38

[0127] Compound 37 (103 mg, 0.1 mmol, 1.0 equivalence) and HBTU (45 mg, 0.12 mmol, 1.2 equivalence) were dissolved in anhydrous N,N-dimethylformamide (3 mL), followed by the addition of N,N-diisopropylethylamine (70 μL, 0.40 mmol, 4.0 equivalence). After shaking for 15 minutes, aminosilanized microporous glass beads (CPG, purchased from Hebei Dinaxingke Biotechnology Co., Ltd.) were added. The product compound 17, CPG with 80 μmol / g (1.25 g), was reacted with shaking for 24 hours. After the reaction was complete, the mixture was filtered. The CPG was washed three times each with N,N-dimethylformamide, dichloromethane / methanol (v / v, 9:1), and dichloromethane, and then dried under vacuum. Acetic anhydride / pyridine (v / v, 1:3, 8 mL) was added to the CPG filter cake, and the mixture was shaken for 30 minutes to cap the unlinked amino groups with acetyl groups. The mixture was filtered again, and the CPG filter cake was washed three times each with dichloromethane / methanol (v / v, 9:1) and dichloromethane, and then dried under vacuum. This yielded product compound 17, which is CPG linked to the Chol 06 ligand. The yield was 1.24 g, and the loading was determined to be 51.2 μmol / g by detecting the absorbance of the 4,4′-dimethoxytriphenylmethyl group (DMTr).

[0128] Example 7 Preparation of compound Lp 01 CPG

[0129] 7.1 Preparation of Compound 39

[0130] Compound 4 (1.00 g, 2.04 mmol, 1.0 equivalence), palmitic acid (0.58 g, 2.24 mmol, 1.1 equivalence), and HATU (1.01 g, 2.65 mmol, 1.3 equivalence) were dissolved in anhydrous N,N-dimethylformamide (10 mL). The reaction system was then purged under a nitrogen atmosphere using a vacuum pump. N,N-diisopropylethylamine (DIPEA, 1.06 mL, 6.12 mmol, 3.0 equivalence) was added under ice bath conditions, and the mixture was then heated to room temperature and stirred overnight. Then, ethyl acetate (50 mL) was added to dilute the reaction mixture. The combined organic phases were washed once each with saturated sodium bicarbonate, water, and saturated brine. After drying with anhydrous sodium sulfate and filtration, the mixture was concentrated by vacuum distillation and purified by silica gel rapid column chromatography (dichloromethane / methanol = 100:1-10:1, with 0.5% triethylamine added to each of mobile phases A and B, and gradient elution). After drying under vacuum, compound 39 was obtained as a reddish-brown oil with a mass of 1.20 g and a yield of 81%.

[0131] 7.2 Preparation of Compound 40

[0132] Compound 39 (0.61 g, 0.84 mmol, 1.0 equivalence), succinic anhydride (251 mg, 2.51 mmol, 3.0 equivalence), and 4-dimethylaminopyridine (307 mg, 2.52 mmol, 3.0 equivalence) were dissolved in anhydrous dichloromethane (5 mL). The reaction system was then purged under a nitrogen atmosphere using a vacuum pump. DIPEA (200 μL, 2.52 mmol, 3.0 equivalence) was added with stirring in an ice bath. The reaction was brought to room temperature and stirred overnight. After complete reaction as detected by LC-MS, the reaction was quenched with water. The reaction system was extracted three times with dichloromethane. The combined organic phases were washed once each with saturated ammonium chloride solution and saturated brine solution, dried over anhydrous sodium sulfate, filtered, concentrated by vacuum distillation, and dried under vacuum to obtain compound 40 as a colorless oil with a mass of 0.58 g, yielding approximately 83%.

[0133] 7.3 Preparation of Compound 41

[0134] Compound 40 (290 mg, 0.35 mmol, 1.0 equivalence) and HBTU (159 mg, 0.42 mmol, 1.2 equivalence) were dissolved in anhydrous N,N-dimethylformamide (10 mL), followed by the addition of molecular sieve (20 mg) and N,N-diisopropylethylamine (243 μL, 1.40 mmol, 4.0 equivalence). After shaking for 15 minutes, aminosilanized microporous glass beads (CPG, purchased from Hebei Dinaxingke Biotechnology Co., Ltd.) were added. The CPG filter cake was prepared by shaking for 24 hours (80 μmol / g, 4.38 g) and then filtered. The cake was washed three times each with N,N-dimethylformamide, dichloromethane / methanol (v / v, 9:1), and dichloromethane, and then dried under vacuum. Acetic anhydride / pyridine (v / v, 1:3, 8 mL) was then added to the CPG filter cake, and the mixture was shaken for 30 minutes to cap unlinked amino groups with acetyl groups. The mixture was filtered again. The CPG filter cake was washed three times each with dichloromethane / methanol (v / v, 9:1) and dichloromethane, and then dried under vacuum to obtain compound 41, which is the Lp 01 ligand-linked CPG. The yield was 4.40 g, and the loading was determined to be 37.1 μmol / g by detecting the absorbance of the 4,4′-dimethoxytriphenylmethyl group (DMTr).

[0135] Example 8: Preparation of compound Lp01 phosphorous amide

[0136] Compound 39 (0.73 g, 1.00 mmol, 1.0 equivalence) and 4,5-dicyanimidazolium (DCI, 30 mg, 0.25 mmol, 0.25 equivalence) were dissolved in anhydrous dichloromethane (5 mL). The reaction system was purged under a nitrogen atmosphere by a vacuum pump. Bis(diisopropylamino)(2-cyanoethoxy)phosphine (392 mg, 1.30 mmol, 1.3 equivalence) was added dropwise to the reaction system with a syringe while stirring in an ice bath. The reaction was then brought to room temperature and stirred for 4 hours. After the reaction was complete as detected by TLC, dichloromethane was added to dilute the reaction mixture. The mixture was washed once with saturated sodium bicarbonate solution and once with saturated brine. After drying with anhydrous sodium sulfate and filtering, the mixture was concentrated by vacuum distillation. The residue was purified by rapid silica gel column chromatography (petroleum ether / ethyl acetate = 10:1-2:1 gradient elution) to give compound 42, a colorless oil, which is Lp1 phosphorous amide. This compound can be used for the solid-phase synthesis of oligonucleotides, with a mass of 0.61 g and a yield of 65%. The product was identified by LC-MS: MS m / z: C 54 H 81 N4O7P, [M+1] + Theoretical value: 929.59, Actual value: 929.61.

[0137] Example 9: Preparation of compound Lp02 (ceramide)-CPG

[0138] 9.1 Preparation of Compound 43

[0139] Ceramide TIC-001 (purchased from Takasago Fragrance Industry Co., Ltd., 0.5 g, 0.88 mmol) was dissolved in anhydrous pyridine (2.5 mL). The reaction system was then purged under nitrogen atmosphere using a vacuum pump. 4,4′-bismethoxytriphenylmethylchloro (448 mg, 1.32 mmol, 1.5 equivalence) was added with stirring in an ice bath, and the mixture was heated to room temperature and stirred overnight. The pyridine was then removed by vacuum distillation, dissolved in dichloromethane, washed once with saturated sodium bicarbonate and once with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated by vacuum distillation to obtain crude product 43. This product did not require further purification and could be used for the next reaction. The product was identified by LC-MS: MS m / z: C 57 H 91 NNaO5, [M+Na] + Theoretical value: 892.68, Actual value: 892.90.

[0140] 9.2 Preparation of Compound 44

[0141] Compound 43 (470 mg, 0.54 mmol), succinic anhydride (108 mg, 1.08 mmol, 2.0 equivalence) and 4-dimethylaminopyridine (198 mg, 1.62 mmol, 3.0 equivalence) were dissolved in anhydrous dichloromethane (5 mL). The reaction system was then purged under a nitrogen atmosphere using a vacuum pump. N,N-diisopropylethylamine (0.5 mL, 2.70 mmol, 5.0 equivalence) was added with stirring in an ice bath. After the reaction was heated to room temperature and stirred overnight, LC-MS was performed to confirm complete reaction. The reaction was then quenched with water. The reaction system was extracted three times with dichloromethane. The combined organic phases were washed once each with saturated ammonium chloride solution and saturated brine solution, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by rapid silica gel column chromatography (dichloromethane / methanol = 100:1-10:1, with 0.5% triethylamine added to each of mobile phases A and B, gradient elution). After drying under vacuum, compound 44 was obtained as a white, foamy solid with a mass of 251 mg, yielding 48%. The product was identified by LC-MS: MS m / z: C 61 H 95 NNaO8, [M+Na] + Theoretical value: 992.69, Actual value: 992.75.

[0142] 9.3 Preparation of Compound 45

[0143] Compound 44 (213 mg, 0.22 mmol) and HBTU (126 mg, 0.33 mmol, 1.5 equivalences) were dissolved in anhydrous N,N-dimethylformamide (10 mL), followed by the addition of triethylamine (50 μL, 0.44 mmol, 2.0 equivalences). After shaking for 30 minutes, aminosilanized CPG (purchased from Hebei Dinaxingke Biotechnology Co., Ltd.) was added. The sample was loaded with 80 μmol / g (2.75 g), and the reaction was shaken for 24 hours. After the reaction was complete, the mixture was filtered, and the CPG was washed three times each with N,N-dimethylformamide and dichloromethane, and then dried under vacuum. Then, Cap A (3 mL) and Cap B (3 mL) reagents were added to the CPG filter cake, and the mixture was shaken for 2 hours to cap the unconnected amino groups with acetyl groups. The mixture was filtered again, and the CPG filter cake was washed three times each with N,N-dimethylformamide and dichloromethane, and then dried completely under vacuum. The product SJXA-CPG was obtained. The yield was 2.66 g, and the loading of SJXA-CPG was determined to be 33.64 μmol / g by detecting the absorbance of the 4,4′-dimethoxytriphenylmethyl group (DMTr).

[0144] Example 10 Synthesis of siRNA-lipophilic conjugates

[0145] Single-stranded RNA (sense and antisense strands) was synthesized on a solid-phase support using an LK-48E synthesizer (Jiangsu Lingkun Biotechnology Co., Ltd.). Oligonucleotides were synthesized using a phosphoramide chemical method at scales of 500 nmol or 1 μmol.

[0146] For the positive chain, the solid-phase supports used included the cholesterol-loaded CPG (Chol O1, Chol O2, Chol O3, Chol O4, Chol O5, Chol O6) and fatty acid-loaded CPG (LpO1, LpO2) synthesized in the above examples. Both supports were synthesized in the laboratory. Oligonucleotide chains without conjugated lipophilic conjugates were synthesized using UnyLinker (purchased from Hebei Dinaxingke Biotechnology Co., Ltd.).

[0147] Nucleoside monomers were sequentially linked in the 3′ to 5′ direction according to the nucleotide types and sequence of the oligonucleotides in the conjugate. The linkage of each nucleoside monomer required the same cyclic steps, including deprotection, coupling, capping, and oxidation or sulfidation. The nucleoside monomers were prepared with a 0.10 mol / L acetonitrile solution, and molecular sieves were added to remove trace amounts of water. In the deprotection step, a 3% trichloroacetic acid / dichloromethane solution was used as the deprotecting agent. In the coupling step, 5-ethylthio-1H-tetrazole was used as the coupling agent, dissolved in anhydrous acetonitrile at a concentration of 0.5 mol / L. In the capping step, acetic anhydride-acetonitrile solution (Cap A, v / v 1:5) and N-methylimidazolium / pyridine / acetonitrile solution (Cap B, v / v 2:3:5) were used as capping reagents. In the oxidation step, the oxidant is a 0.02 mol / L solution of iodine in pyridine / water (volume ratio 90 / 10); in the thiolation step, the reagent used is a 0.10 mol / L solution of 5-imino-1,2,4-dithiazolidin-3-thione / pyridine.

[0148] Oligonucleotides were cleaved and deprotected at 45°C using 28%–30% concentrated ammonia. Depending on the synthesis scale, CPG was filtered through 0.45 μm syringe filters of varying sizes. Subsequently, the CPG was washed with RNAase-free water, filtered again through a 0.45 μm syringe filter, and combined with the first filtrate. After concentration, the crude oligonucleotides were diluted with RNAase-free water.

[0149] Crude oligonucleotides using AKTA TM Purification was performed using the Pure purification system. Oligonucleotides without lipophilic conjugates were purified using anion exchange (AEX) purification, while oligonucleotides with lipophilic conjugates were purified using reversed-phase (RP) purification. In AEX purification, mobile phase A consisted of 20 mM NaH₂PO₄, 15% acetonitrile, pH 7.4; mobile phase B consisted of 20 mM NaH₂PO₄, 1 M NaBr, 15% acetonitrile, pH 7.4. In RP purification, mobile phase A consisted of 50 mM NaOAc containing 10% acetonitrile, and mobile phase B consisted of 50 mM NaOAc containing 80% acetonitrile. Fractions with a mass purity greater than 85% and a single impurity not exceeding 5% were collected and combined.

[0150] The purified oligonucleotides were desalted using a 3K MWCO centrifugal ultrafiltration tube. They were then washed with RNAase-free water until the conductivity of the filtrate was below 100 μS / cm. After desalting, the concentration of the final desalted oligonucleotides was determined using NanoDrop at an A260 wavelength, and their purity was analyzed by RP-LCMS.

[0151] The sense and antisense strands can be prepared separately using the above method, then mixed and annealed to prepare the lipophilic ligand conjugate of the present invention with siRNA (Table 2). Tables 1 and 2 correspond to the unmodified and modified siRNA sequences, respectively. Typically, the sense and antisense strands are dissolved separately in PBS solution, then the PBS solutions are mixed and annealed. The molar ratio of the sense and antisense strands can be 1–1.2:1. Annealing is performed at 65°C for 10 minutes, followed by slow cooling to room temperature over 40 minutes. The integrity and purity of the duplex are detected by SEC-HPLC.

[0152] Table 1: Unmodified siRNA sequences

[0153] Table 2: Modified siRNA sequences

[0154] Note: Uppercase letters A, C, G, and U represent the base composition of nucleotides; lowercase letter m indicates that the nucleotide adjacent to the left of letter m is a nucleotide modified with 2′-methoxy; lowercase letter f indicates that the nucleotide adjacent to the left of letter f is a nucleotide modified with 2′-fluorine; s indicates that the connection between two adjacent nucleotides is a phosphate thioester group.

[0155] Example 11: Inhibitory effect of TYR siRNA on tyrosinase TYR expression in mouse melanocyte line B16

[0156] The B16 melanocyte cell line (purchased from the Cell Bank of the Chinese Academy of Sciences) was selected for experiments to detect the inhibitory effect of siRNA targeting TYR on tyrosinase (TYR) expression, as shown in Table 2. The experimental procedure was as follows: The B16 melanocyte cell line was digested with trypsin and seeded at an appropriate density into 24-well cell culture plates, with a density of 10 cells per well. 5 Cells were cultured overnight at 37°C and 5% CO2 in DuPont modified Eagle (DMEM) medium containing 10% fetal bovine serum (FBS). The next day, the medium was replaced with serum-free medium, and siRNA targeting TYR (as shown in Table 2) was added to a final concentration of 10 μM, in triplicate. No siRNA was added to the negative control group. Cells were cultured for another 24 hours at 37°C and 5% CO2. After 24 hours, the serum-free medium was replaced with serum-containing medium, and the cells were cultured for another 24 hours at 37°C and 5% CO2. Cells were then harvested, washed with PBS, and RNA was extracted using Trizol (Invitrogen 15596018). Reverse transcription was performed using a Takara PrimeScript kit. TMII. The total RNA extracted was reverse transcribed into cDNA using the 1st Strand cDNA Synthesis Kit (6210A) according to the instructions. Then, a quantitative real-time PCR kit (TB) was used. Premix Ex Taq TM The inhibition rate of TYR mRNA expression was detected using the II, Takara, RR820A method. GAPDH cDNA was used as an internal control in this quantitative real-time PCR method. The PCR reaction conditions were as follows: pre-denaturation at 95℃ for 30 seconds, followed by 40 cycles of 95℃ for 10 seconds and 60℃ for 30 seconds.

[0157] The primer sequences are shown in Table 3.

[0158] Table 3: Sequences of mTYR detection primers

[0159] Data Analysis:

[0160] Using comparison Ct( ΔΔ In the Ct method, siRNA inhibitory activity is measured by the remaining amount of TYR gene expression 2- ΔΔ Ct indicates that ΔΔ Ct = [(Target gene in Ct experimental group - Internal reference in Ct experimental group) - (Target gene in Ct negative control group - Internal reference in Ct negative control group)]. The mRNA inhibition rate is then calculated using the following formula: mRNA inhibition rate = (1 - remaining mTYR gene expression level) × 100%.

[0161] The inhibition of mTYR mRNA expression in the melanocyte B16 cell line by the siRNA conjugates shown in Table 2 is illustrated in Figure 1 and Table 4.

[0162] Table 4

[0163] Example 12: Inhibitory effect of TYR siRNA on tyrosinase TYR expression in human melanocyte cell line MNT1

[0164] Human melanocyte cell line MNT1 (purchased from the Cell Bank of the Chinese Academy of Sciences) was selected for experiments to detect the inhibitory effect of TYR-targeting siRNA on tyrosinase (TYR) expression, as shown in Table 2. The experimental procedure was as follows: MNT1 melanocyte cells were digested with trypsin, adjusted to an appropriate density, and seeded into 24-well cell culture plates at a density of 10 cells per well. 5Cells were cultured overnight at 37°C and 5% CO2 in DuPont modified Eagle (DMEM) medium containing 10% fetal bovine serum (FBS). The next day, the medium was replaced with serum-free medium, and siRNA targeting TYR (as shown in Table 2) was added to a final concentration of 10 μM, in triplicate. No siRNA was added to the negative control group. Cells were cultured for another 24 hours at 37°C and 5% CO2. After 24 hours, the serum-free medium was replaced with serum-containing medium, and the cells were cultured for another 24 hours at 37°C and 5% CO2. Cells were then harvested, washed with PBS, and RNA was extracted using Trizol (Invitrogen 15596018). Reverse transcription was performed using a Takara PrimeScript kit. TM II. The total RNA extracted was reverse transcribed into cDNA using the 1st Strand cDNA Synthesis Kit (6210A) according to the instructions. Then, a quantitative real-time PCR kit (TB) was used. Premix Ex Taq TM The inhibition rate of TYR mRNA expression was detected using the II, Takara, RR820A method. GAPDH cDNA was used as an internal control in this quantitative real-time PCR method. The PCR reaction conditions were as follows: pre-denaturation at 95℃ for 30 seconds, followed by 40 cycles of 95℃ for 10 seconds and 60℃ for 30 seconds.

[0165] The primer sequences are shown in Table 5 as hTYR.

[0166] Table 5: Sequences of hTYR detection primers

[0167] Data Analysis:

[0168] Using comparison Ct( ΔΔ In the Ct method, siRNA inhibitory activity is measured by the remaining amount of TYR gene expression 2- ΔΔ Ct indicates that ΔΔ Ct = [(Target gene in Ct experimental group - Internal reference in Ct experimental group) - (Target gene in Ct negative control group - Internal reference in Ct negative control group)]. The mRNA inhibition rate is then calculated using the following formula: mRNA inhibition rate = (1 - Remaining hTYR gene expression level) × 100%.

[0169] The inhibition of hTYR mRNA expression in the human melanocyte cell line MNT1 by the siRNA conjugates shown in Table 2 is illustrated in Figure 2 and Table 6.

[0170] Table 6

[0171] Example 13: Inhibitory effect of TYR siRNA on melanin content in mouse melanocyte cell line B16

[0172] The B16 melanocyte cell line (purchased from the Cell Bank of the Chinese Academy of Sciences) was selected for the experiment to detect the inhibitory effect of the siRNA targeting TYR shown in Table 2 on melanin content in the cells. The experimental procedure is as follows: The B16 melanocyte cell line (purchased from the Cell Bank of the Chinese Academy of Sciences) was selected for the experiment to detect the inhibitory effect of the siRNA targeting TYR on tyrosinase (TYR) expression shown in Table 2. The experimental procedure is as follows: The B16 melanocyte cell line was digested with trypsin, adjusted to an appropriate density, and seeded into 24-well cell culture plates at a density of 10 cells per well. 5 Cells were cultured overnight at 37°C and 5% CO2 in DuPont modified Eagle (DMEM) medium containing 10% fetal bovine serum (FBS). The next day, the culture medium was replaced with serum-free medium, and siRNA targeting TYR (as shown in Table 2) was added to a final concentration of 10 μM, with three aliquots prepared. No siRNA was added to the negative control group. Cells were cultured for another 24 hours at 37°C and 5% CO2. After 24 hours, the serum-free medium was replaced with serum-containing medium, and the cells were cultured for another 24 hours at 37°C and 5% CO2. Cells were then harvested, with 10⁶ cells counted from each group. After washing with PBS, the precipitate was centrifuged and 120 μL of lysis buffer (1 M sodium hydroxide and 10% DMSO) was added. After lysis at 80°C for 2 hours, the absorbance of the lysis buffer was measured using a microplate reader (OD 400 nm).

[0173] The inhibition of melanin content in the melanocyte cell line B16 by the siRNA conjugates shown in Table 2 is illustrated in Figure 3 and Table 7.

[0174] Table 7

[0175] As shown in Tables 4 and 6-7 and Figures 1-3, under conditions without the use of exogenous liposome transfection reagents, the TYR-targeting siRNA sequences (1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9) of this invention, coupled with lipophilic conjugates, effectively inhibited tyrosinase (TYR) expression in both mouse melanocyte cell line B16 and human melanocyte cell line MNT1, and significantly reduced melanin content in B16 cells. Among them, compounds 1-4, 1-5, 1-6, 1-7, and 1-9 showed superior inhibitory effects.

[0176] Example 14: Inhibitory effect of MMP1 siRNA on matrix metalloproteinase 1 (MMP1) expression in human fibrosarcoma cell line HT1080

[0177] Human fibrosarcoma cell line HT1080 (purchased from the Cell Bank of the Chinese Academy of Sciences) was selected for experiments to detect the inhibitory effect of MMP1-targeting siRNAs (2-1, 2-2, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9) on matrix metalloproteinase 1 (MMP1) expression, as shown in Table 2. The experimental procedure was as follows: HT1080 cells were digested with trypsin and then cultured in DMEM medium containing 2% FBS. 2 × 10⁶ cells were then cultured in the medium. 5 Three copies of siRNA were mixed with each cell and seeded into 12-well cell culture plates. The cells were cultured at 37°C and 5% CO2 for 48 hours. After 48 hours, the cells were harvested, washed with PBS, and RNA was extracted using Trizol (Invitrogen 15596018). Reverse transcription was performed using a Takara PrimeScript kit. TM II. 1st Strand cDNA Synthesis Kit; 6210A) was used to reverse transcribe the extracted total RNA into cDNA according to the instructions, followed by quantitative real-time PCR using a TB (TB) kit. Premix Ex Taq TM MMP1 cDNA was detected using a Takara (RR820A) assay. GAPDH was used as an internal control for quantitative real-time PCR. PCR conditions were as follows: pre-denaturation at 95°C for 30 seconds, followed by 40 cycles of 95°C for 10 seconds and 60°C for 30 seconds.

[0178] The primer sequences are shown in Table 8.

[0179] Table 8: Sequences of Detection Primers

[0180] Data Analysis:

[0181] Using comparison Ct( ΔΔ In the Ct method, siRNA inhibitory activity is measured by the remaining amount of MMP1 gene expression 2- ΔΔ Ct indicates that ΔΔ Ct = [(Target gene in Ct experimental group - Internal reference in Ct experimental group) - (Target gene in Ct negative control group - Internal reference in Ct negative control group)]. The mRNA inhibition rate is then calculated using the following formula: mRNA inhibition rate = (1 - Remaining MMP1 gene expression level) × 100%.

[0182] The inhibition rates of MMP1 mRNA expression in the human fibrosarcoma cell line HT1080, which targets MMP1, as shown in Table 2 are illustrated in Table 9 and Figure 4.

[0183] Table 9

[0184] Example 15 Effects of siRNA samples 2-4 on matrix metalloproteinase 1 and collagen 1 / 3 content in primary mouse skin fibroblasts

[0185] The effect of MMP1 knockdown on collagen deposition was investigated using primary mouse skin fibroblasts.

[0186] The experimental procedure was as follows: Primary mouse skin fibroblasts (isolated from skin tissue of C57 / BL6 mice purchased from Shanghai Southern Model Biotechnology Co., Ltd.) (F. Boraldi et al., Biomedicines 12, 1586 (2024)) were cultured in an incubator (37℃, 5% CO2) until a sufficient number of cells were obtained. In the UVA modeling group, the culture medium was removed, and the cells were gently rinsed once or twice with D-Hanks solution before UVA irradiation (9 J / cm2). After modeling, the cells in the modeling group and the light-protected group were treated with trypsin and then cultured in complete medium containing 2% serum. In the drug-treated group, 2×10⁶ cells were added to the culture medium. 5 Cells were mixed with 5 μM of double-stranded siRNA numbered 2-4 and seeded in 12-well plates. They were cultured for three days at 37°C and 5% CO2, while the normal control group was cultured under the same conditions in the dark for the same duration. After three days of culture, cells were harvested, and cell protein was extracted according to the manufacturer's protocol (Sangon Biotech, Total Protein Extraction Kit). Protein was quantified, electrophoresed onto a gel, transferred to a membrane, blocked with 5% skim milk for 1 hour, and incubated with primary antibody overnight at 4°C. The membrane incubated with primary antibody was washed three times with TBST for 5 min each time. Subsequently, HRP-labeled secondary antibody was diluted 1:1000 according to the dosage and incubated with the membrane at 37°C for 1 hour. The membrane was washed three times with TBST for 5 min each time. Detection was then performed by chemiluminescence immunoassay. All antibodies used in the experiment are shown in Table 10.

[0187] Table 10

[0188] As shown in Table 10 and Figure 5, the MMP1-targeting siRNAs numbered 2-4 in Table 2 significantly affected the expression levels of matrix metalloproteinase 1 (MMP1) and type I and type III collagen (COL I and COL III) in mouse primary skin fibroblasts. Their gray value percentage relative to β-actin is shown in Table 11.

[0189] Table 11

[0190] As shown in Table 11 and Figure 5, compared with the normal control group, UVA irradiation significantly upregulated the expression of matrix metalloproteinase 1 (MMP1) in primary mouse dermal fibroblasts, while significantly downregulating the expression levels of type I and type III collagen. After treatment with siRNA samples 2-4, the expression level of MMP1 decreased by approximately 45% compared with the model control group, while the expression levels of type I and type III collagen were upregulated by approximately 13-fold and 21-fold, respectively.

[0191] Example 16: Effects of siRNA samples 2-4 on matrix metalloproteinase 1 and type I / III collagen content in mouse skin

[0192] To investigate the effects of siRNA samples 2-4 on skin photoaging-related indicators, 6-week-old BALB / c nude mice (purchased from Shanghai Southern Model Biotechnology Co., Ltd.) were used as experimental animals. A total of 20 animals were randomly divided into five groups: blank control group (N=5), negative control group (N=5), drug treatment group A (topical application, N=5), and drug treatment group B (topical injection, N=5). Except for the blank control group, the mice in the other groups received 150 mJ / cm² treatment. 2 A UVB irradiation-induced photoaging model was established, with irradiation frequency of 5 times per week. Group A received daily topical application of siRNA sample 2-4 (200 μM × 100 μL / mouse) for 6 consecutive weeks; Group B received four injections of siRNA (200 μM × 100 μL / mouse) at the four corners of the irradiated area. After 6 weeks of treatment, mice in each group were euthanized, and their skin surface condition was recorded and photographed to assess the improvement effect on wrinkles and photoaging phenotypes. Subsequently, skin samples were taken from the drug-treated areas, total RNA was extracted, and the expression level of matrix metalloproteinase 1 (MMP1) target genes was detected by qPCR. Paraffin sections and Masson staining were also performed to observe collagen deposition and structural changes in the skin, comprehensively evaluating the biological effects of siRNA sample 2-4 in anti-photoaging.

[0193] Preparation procedure of siRNA sample 2-4 carbomer gel: Weigh 5.0 g of carbomer 940 powder, add 50 mL of RNase-free sterile water, place in a 250 mL beaker, stir well, and let it swell overnight at room temperature. The next day, slowly add triethanolamine dropwise to the swollen carbomer solution while stirring until the pH of the system is adjusted to 7.0. Then, add RNase-free sterile water to make up to a total mass of 100 g to obtain a 5% (w / w) carbomer hydrogel.

[0194] Weigh an appropriate amount of 2-4 lyophilized siRNA sample powder and add RNase-free sterile water to prepare a 200 μM working solution. Mix 5% carbomer hydrogel with the siRNA solution in a controlled ratio to adjust the final drug composition concentration to 100 μM for siRNA and 0.5% (w / v) for carbomer gel. It is recommended to perform the mixing process at 4°C, gently stirring until homogeneous. Store the resulting siRNA gel composition at 4°C for later use.

[0195] Table 12

[0196] As shown in Table 12, the expression level of matrix metalloproteinase 1 (MMP1) in the skin of experimental mice after UVA irradiation was significantly increased compared with the blank control group, with an increase of approximately 7-fold, indicating that photodamage significantly activated molecular pathways related to matrix degradation in the skin. After intervention with MMP1-targeting siRNAs numbered 2-4 (including both topical application and subcutaneous injection), the expression level of MMP1 was significantly downregulated. In particular, the expression level of MMP1 in drug treatment group B (injection group) was significantly reduced, recovering to a level comparable to the blank control group, indicating that this siRNA has a good gene silencing effect in vivo and a significant intervention effect on photoaging-related indicators.

[0197] Meanwhile, Figure 6 shows that UVA irradiation significantly reduced collagen content in mouse skin, manifested as sparse collagen structure and weakened staining in the dermis. Treatment with MMP1-targeting siRNAs numbered 2-4 (including application and injection) significantly increased collagen content in skin tissue, and the collagen fiber distribution became denser, suggesting that this siRNA helps improve UV-induced collagen degradation and has excellent anti-photoaging capabilities.

Claims

1. A lipophilic oligonucleotide conjugate comprising an oligonucleotide and a lipophilic group linked to said oligonucleotide.

2. The lipophilic oligonucleotide conjugate according to claim 1, wherein, The lipophilic groups include lipophilic portions capable of binding to cell membrane phospholipid structures, and in particular, lipid-soluble groups capable of penetrating the skin barrier. Preferably, the lipophilic moiety is selected from groups derived from the following compounds: fat-soluble alcohols or thiols, such as cholesterol and its derivatives (e.g., cholene, cholesterol, cholesteryldiene), bile acids (e.g., cholic acid, deoxycholic acid, dehydrocholic acid), sterols, steroids; fatty acids (e.g., long-chain fatty acids, branched-chain fatty acids, saturated or unsaturated fatty acids); phospholipids (e.g., phosphatidylcholine, phosphatidylethanolamine); glycolipids; synthetic polymer-modified lipids (e.g., polyethylene glycol-lipid derivatives); ceramides, More preferably, the lipophilic group further includes a linking group for linking the lipophilic portion capable of binding to the cell membrane phospholipid structure and the oligonucleotide.

3. The lipophilic oligonucleotide conjugate according to claim 1, wherein, The lipophilic group is selected from the following groups: Chol 01= Chol 0= Chol 03= Chol 04= Chol 05= Chol 06= Lp 01= Lp 0= in This indicates the location where the oligonucleotide was introduced.

4. The lipophilic oligonucleotide conjugate according to any one of claims 1-3, wherein, The oligonucleotides include single-stranded structures, double-stranded structures, and structures based on DNA, RNA, or modified nucleoside units, such as antisense oligonucleotides (ASO) or small interfering RNA (siRNA).

5. The lipophilic oligonucleotide conjugate according to claim 4, wherein, In the oligonucleotide, each nucleotide is independently either modified or unmodified. Preferably, the oligonucleotide comprises at least one modified nucleotide and / or at least one interstrand modification. More preferably, all the nucleotides of the oligonucleotide are modified nucleotides. More preferably, the modified nucleotides include nucleotides modified with 2'-F, 2'-OME, 2'-MOE, GNA, UNA, PNA, or 5'-VP.

6. The lipophilic oligonucleotide conjugate according to claim 4, wherein, The oligonucleotide is siRNA. Preferably, in the siRNA, each nucleotide is independently a modified or unmodified nucleotide. More preferably, the siRNA contains at least one modified nucleotide and / or at least one interstrand modification. Even more preferably, all nucleotides on the sense strand of the siRNA are modified nucleotides, and / or all nucleotides on the antisense strand of the siRNA are modified nucleotides. For example, the siRNA is a siRNA targeting tyrosinase (TYR) or a siRNA targeting matrix metalloproteinase 1 (MMP1). Preferably, the sense strand of the siRNA targeting TYR is UmGfCmCfAmAfUmUfUmCfAmGfCmUfUmUfAmGfAm, and the antisense strand is UmCfUmAfAmAfGmCfUmGfAmAfAmUfUmGfGmCfAmdTsdT; the sense strand of the siRNA targeting MMP1 is GmsAmsGmGmAmAmAfAfCfAmCmUmGmGmAmAmAmAmAmAm, and the antisense strand is UmsUfsUmUmUfCfCfAmGmUmGmUmUmUmCfCmUmCmsAmsGm.

7. The lipophilic oligonucleotide conjugate according to any one of claims 1-6, wherein, The lipophilic group is conjugated to the 5′ and / or 3′ end of the oligonucleotide, or is attached to the oligonucleotide via a hydrocarbon chain or linker.

8. A method for improving the transdermal and / or intradermal delivery capability of oligonucleotides, the method comprising the step of introducing a lipophilic group into the oligonucleotide to prepare a lipophilic oligonucleotide conjugate, wherein, The oligonucleotide and lipophilic group are as described in any one of claims 1-6.

9. A method for delivering oligonucleotides into skin cells, the method comprising: Lipophilic oligonucleotide conjugates are prepared by introducing a lipophilic group into an oligonucleotide, wherein the oligonucleotide and the lipophilic group are as described in any one of claims 1-6. The lipophilic oligonucleotide conjugates are delivered into skin cells via transdermal or intradermal delivery.

10. A composition comprising a lipophilic oligonucleotide conjugate as described in any one of claims 1-6 and optionally a carrier acceptable in cosmetics or pharmaceuticals, particularly, said composition being a transdermal delivery drug or cosmetic, or an intradermal delivery drug or cosmetic.

11. The use of the lipophilic oligonucleotide conjugate as described in any one of claims 1-6 in the preparation of transdermal or intradermal small nucleic acid drugs, preferably, the transdermal small nucleic acid drug is used in the treatment of skin diseases and in the field of medical aesthetics and cosmetics.

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