Chiral morpholino phosphorus oxychloride monomer, its preparation method and application

Abstract

The application provides a chiral morpholino phosphorus oxychloride monomer and a preparation method and application thereof, and the structure of the monomer is shown in formula I or II: wherein X represents an oxygen atom or a sulfur atom; B represents a base; R 1 is selected from an optionally substituted secondary substituted amine group, an optionally substituted alkoxy group, an optionally substituted phenoxy group, a guanidino group, and a linked nitrogen forming an optionally substituted heterocyclic ring; R 2 is selected from an optionally substituted or unsubstituted tertiary alkyl group, an optionally substituted or unsubstituted benzyl group, an optionally substituted sulfonyl group, and an optionally substituted acyl group. The application can obtain the chiral morpholino phosphorus oxychloride monomer in a stereochemically pure or substantially pure form by using an efficient catalytic asymmetric desymmetrization strategy. And the corresponding morpholino oligonucleotide chain can be synthesized based on the monomer, thereby providing a powerful synthesis platform for developing a new generation of morpholino oligonucleotide drugs with precise stereostructure and optimized performance.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical and pharmaceutical intermediate synthesis technology, specifically relating to a chiral morpholine phosphoryl chloride monomer, its preparation method, and its application. Background Technology

[0002] The synthesis of diastereomeric phosphoric acid oligonucleotides is considerably complex due to the presence of chiral phosphorus bonds. The presence of chiral phosphorus presents significant challenges to the synthesis of nucleotides involving the linkage of a series of phosphoric acid diamines. However, to date, there has been a lack of efficient, simple, stereochemically pure, and universally applicable methods capable of stereoselectively forming phosphoric acid diamine bonds. For example, in... Figure 1 As illustrated in the diagram, most reported methods for synthesizing long-chain PMOs currently utilize the stereochemically uncontrolled coupling of racemic monomers to prepare oligonucleotides and sequences of the desired lengths, resulting in a heterogeneous mixture of many diastereomers. Theoretically, the number of diastereomers is 2-1. n Where n is the number of nucleotides linked to form the oligonucleotide. The formation of a large number of diastereomers poses a significant challenge to subsequent separation techniques. A highly effective method to address this problem is to use chiral pure PMO monomers to synthesize phosphoridamide morpholino oligonucleotide chains with the desired specific conformational sequence via stereospecific substitution reactions.

[0003] There are currently two reported strategies for synthesizing stereopure monomers: (1) Direct separation of racemic monomers (Assignee: Eisai, Management Co., Ltd. Chiralreagents for preparation and HPLC separation of morpholino nucleosides in synthesis of homogeneous DNA. WO2017 / 024264 A2); (2) Chiral pure PMO monomers were synthesized by resolving the racemic cofactor and then carrying out a multi-step reaction (CH Wook, et al. Crystalline monomers for preparing antisense oligonucleotides and methods of their preparation and use. WO 2024 / 010870 A2).

[0004] Both strategies have drawbacks: lengthy synthetic routes (e.g., the OTP strategy requires 3-4 steps), necessitate the use of stoichiometric chiral reagents and chromatographic separation of diastereomers, which are time-consuming, labor-intensive, and wasteful of solvents. They may also limit the scale of chiral pure monomer preparation. In contrast, using a catalytic strategy to directly obtain monomers with the desired configuration eliminates all prosthetic group-related operations. It allows for the selection of specific diastereomers before synthesis, and the subsequent synthesis of the selected diastereomers in stereochemically pure or substantially pure form, which is highly valuable. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a chiral morpholine oligophosphoryl chloride monomer, its preparation method, and its applications.

[0006] The objective of this invention is achieved through the following technical solution: In a first aspect, the present invention provides a chiral morpholine phosphoryl chloride monomer, the structure of which is shown in formula I or II below: Where X represents an oxygen atom or a sulfur atom; B represents a base; R 1 It is selected from any one of the following: a secondary substituted amino group, a heterocycle formed by a nitrogen linkage, an substituted alkoxy group, a substituted phenoxy group, and a guanidinyl group; R 2 It is selected from any one of the following: optionally substituted or unsubstituted tertiary alkyl, optionally substituted or unsubstituted benzyl, optionally substituted sulfonyl, optionally substituted acyl.

[0007] Preferably, the base is selected from any one of substituted or unsubstituted uracil, thymine, cytosine, adenine, and guanine.

[0008] Preferably, R 1 In this process, the nitrogen linked to form an optionally substituted heterocycle, including pyrrolidine, piperazine, or morpholine.

[0009] Preferably, R 2Selected from any one of optionally substituted or unsubstituted tertiary alkyl groups, optionally substituted or unsubstituted benzyl groups, optionally substituted sulfonyl groups, and optionally substituted acyl groups; specifically, the substituted or unsubstituted tertiary alkyl groups include, but are not limited to, tert-butyl, triphenylmethyl (Tr), and substituted triphenylmethyl groups (e.g., MMTr). (p-Methoxyphenyldiphenylmethyl), DMTr (dimethoxyphenyldiphenylmethyl); the optionally substituted or unsubstituted benzyl groups include, but are not limited to, benzyl (Bn), 4-methoxybenzyl (PMB), 3,4-dimethoxybenzyl (DMB), diphenylmethyl (Dpm), 4-methoxybenzyl, 2,4-dimethoxybenzyl (DMB), 2,4,6-trimethoxybenzyl (TMB), 9-anthrabenzyl, 2,4-dichlorobenzyl, 1-naphthylbenzyl, 2-naphthylbenzyl, 7-quinolinebenzyl; the optionally substituted sulfonyl groups include, but are not limited to, 2-nitrobenzenesulfonyl, p-toluenesulfonyl; the optionally substituted acyl groups include, but are not limited to, benzyloxycarbonyl (CBz), tert-butoxycarbonyl (Boc), phenoxyacetyl (Pac), 9-fluorenemethoxycarbonyl (Fmoc).

[0010] Preferably, the structure of the monomer is specifically shown in formulas I-1, I-2, I-3, I-4, II-1, II-2, II-3, or II-4: Among them, R 3 Selected from H, optionally substituted alkyl or alkoxy groups, and optionally substituted halogens; R 4 R 5 R 6 and R 7 Each is selected from -H, -C(O)R 10 and -C(O)OR 10 Any of the following, where R 10 It is alkyl or aryl; R 8 R 9 Each is selected from any one of optionally substituted alkyl, cyanoethyl, acyl, carbonate, carbamate, optionally substituted benzyl, 4-pentanoyloxybenzyl and silyl.

[0011] Secondly, the present invention provides a method for preparing a chiral morpholine phosphoryl chloride monomer, comprising the following steps: Under the conditions of organic solvent, base, chiral ligand and copper catalyst, the compound with the structure shown in Formula III is reacted with the compound with the structure shown in Formula IV to generate a chiral morpholinophosphoryl chloride monomer. The structures shown in Equation III and Equation IV are as follows: ; In Formula III, X represents an oxygen atom or a sulfur atom; R 1 It is selected from any one of the following: a optionally substituted secondary substituted amino group, a optionally substituted heterocycle formed by the nitrogen attached thereto, an optionally substituted alkoxy group, an optionally substituted phenoxy group, and a guanidinyl group; In formula IV, B represents a base; R 2 It is selected from any one of the optional substituted or unsubstituted tertiary alkyl, optional substituted benzyl, optional substituted sulfonyl, and optional substituted acyl.

[0012] Preferably, the organic solvent is selected from at least one of tetrahydrofuran, 2-methyltetrahydrofuran, ethyl acetate, ethylene glycol dimethyl ether, and diethyl ether.

[0013] Preferably, the base is selected from at least one of triethylamine, tributylamine, diisopropylethylamine, and 1,2,2,6,6-pentamethylpiperidine.

[0014] Preferably, the copper catalyst is selected from at least one of copper hexafluoroacetylacetonate, copper acetate, copper chloride, copper bromide, and cuprous bromide.

[0015] Preferably, the chiral ligand is selected from pyridine imidazole ligands.

[0016] Preferably, the structure of the pyridine imidazole ligand is as follows: （R） -L、 （S） -L、( S -L2、( S -L3、( S -L4、( S -L5、( S -L6、( S -L7、( S As shown in -L10: .

[0017] Preferably, the molar ratio of the compound with the structure shown in Formula III to the compound with the structure shown in Formula IV is 3:1.

[0018] Preferably, the molar ratio of the copper catalyst to the morpholine nucleoside represented by Formula IV is 0.01-0.1:1.

[0019] Preferably, the molar ratio of the base to the compound with the structure shown in Formula IV is 1-5:1.

[0020] Preferably, the molar ratio of the chiral ligand to the morpholinonucleotide represented by Formula IV is 0.02-0.15:1.

[0021] Preferably, the reaction temperature is -30℃ to -50℃, and the reaction time is 4 to 24 hours.

[0022] Thirdly, the present invention provides the application of a chiral morpholinophosphoryl chloride monomer in the synthesis of morpholino oligonucleotide chains.

[0023] Fourthly, the present invention provides a method for preparing morpholine oligonucleotide chains, comprising the following steps: S1. Deprotect the protecting group Tr on the linker derived from aminomethyl polystyrene resin to obtain a free secondary amine; S2. The free secondary amine is coupled with the chiral morpholinophosphoryl chloride monomer to obtain the morpholino oligonucleotide dimer; S3. After capping with a capping reagent, remove the protecting group on the morpholine oligonucleotide dimer to obtain the deprotected morpholine oligonucleotide dimer. S4. Repeat steps S2 and S3 for the next round of coupling, end-capping and deprotection steps until the target oligomer crude product is obtained. S5. After purifying the crude target oligomer, morpholine oligonucleotide chains are separated.

[0024] Compared with the prior art, the present invention has the following beneficial effects: 1. The chiral morpholinophosphoryl chloride monomer prepared by this invention can exist stably in solvents or environments and can undergo subsequent substitution and chain extension. The protecting group has a certain degree of stability and exists stably under coupling conditions, while also having the characteristic of easy removal. It can achieve efficient chain extension through DNA / peptide synthesizers and / or solid-phase synthesis methods, and is especially suitable for the preparation of oligonucleotide chains. It is particularly suitable for constructing morpholino-based oligonucleotide (PMO or TMO) chains.

[0025] 2. This invention provides a novel method for preparing chiral morpholinophosphoryl chloride monomers, which can obtain stereochemically pure or substantially pure forms of chiral morpholinophosphoryl chloride monomers. The method employs mild reaction conditions, avoiding the risk of racemization caused by strong bases / high temperatures. The "two-way control" characteristic of the copper catalyst used in this method allows for the separate acquisition of Rp / Sp monomers simply by changing the configuration of the chiral ligands. The monomers with all four bases can achieve a dr ratio of >80:20, and the highest can reach >99:1 dr, which is crucial for drug development requiring structure-activity relationship studies. Traditional chromatographic separation often yields less than 30%, while the monomer yield of the method of this invention is between 50-93%, demonstrating significant scale-up potential.

[0026] 3. The preparation method of this invention utilizes a highly efficient catalytic asymmetric desymmetry strategy, completely eliminating the reliance on traditional chiral cofactors or chromatographic resolution. With its simple steps, high atom economy, excellent stereoselectivity, and broad substrate applicability, it enables the flexible and scalable preparation of chiral pure morpholine monomers with various configurations, bases, and skeletons (PMO / TMO, etc.). This provides a powerful synthetic platform for developing next-generation morpholine oligonucleotide drugs with precise stereostructures and optimized performance.

[0027] 4. Traditional phosphoramide imine ester routes cannot synthesize thio analogs, but the preparation method of this invention achieves stereo-oriented preparation of thio monomers for the first time through a protecting group strategy, which provides the possibility for the development of novel antisense drugs. Attached Figure Description

[0028] Figure 1 This demonstrates the preparation route of the chiral pure phosphoryl diamine morpholino oligonucleotide monomer reported in existing literature.

[0029] Figure 2 The structures of the chiral morpholine phosphoryl chloride monomers prepared in Example 14 of this invention are shown.

[0030] Figure 3 This invention demonstrates the preparation routes of the PMO / TMO chains in Application Examples 1 and 2.

[0031] Figure 4 The HPLC chromatograms of crude casimersen prepared in Example 3 of this invention and the HPLC chromatograms of purified casimersen are shown. Figure 5 The MS spectrum of casimersen prepared in Application Example 3 of this invention is shown.

[0032] Figure 6 The MALDI-TOF image of casimersen prepared in Application Example 3 of this invention is shown. Detailed Implementation

[0033] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention. These all fall within the scope of protection of the present invention.

[0034] Chiral morpholinophosphoryl chloride monomer In a specific embodiment of the present invention, a chiral morpholine phosphoryl chloride monomer with the structure shown in Formula I or II is provided: In formula I or II, X represents an oxygen atom or a sulfur atom.

[0035] In formula I or II, B represents a base, including but not limited to thymine, cytosine, adenine, and guanine.

[0036] In formula I or II, R 1 The group is selected from any of the following: optionally substituted secondary substituted amino groups, optionally substituted heterocycles formed by the linked nitrogen, optionally substituted alkoxy groups, optionally substituted phenoxy groups, and guanidinyl groups; specifically, the optionally substituted secondary substituted amino groups can be, for example, dimethylamine, diethylamine, diisopropylamine, dibenzylamine, N-methylbenzylamine, bis(2-chloroethyl)amine, or diethyl iminodiacetic acid; the optionally substituted heterocycles formed by the linked nitrogen can be, for example, pyrrolidine, piperazine, morpholine, or piperidine; the optionally substituted alkoxy groups can be, for example, methoxy, ethoxy, isopropoxy, phenylethoxy, or p-nitrophenylethyl; the optionally substituted phenoxy groups can be, for example, 2-nitrophenoxy, 2-fluorophenoxy, 2-chlorophenoxy, p-nitrophenoxy, 4-methoxyphenoxy, or 3-nitrophenoxy.

[0037] In formula I or II, R 2 Selected from any one of optionally substituted or unsubstituted tertiary alkyl groups, optionally substituted or unsubstituted benzyl groups, optionally substituted sulfonyl groups, and optionally substituted acyl groups; specifically, the substituted or unsubstituted tertiary alkyl groups include, but are not limited to, tert-butyl, triphenylmethyl (Tr), substituted triphenylmethyl groups (e.g., MMTr (p-methoxyphenyldiphenylmethyl), DMTr), etc. (Dimethoxyphenyldiphenylmethyl); the optionally substituted or unsubstituted benzyl group includes, but is not limited to, benzyl (Bn), 4-methoxybenzyl (PMB), 3,4-dimethoxybenzyl (DMB), diphenylmethyl (Dpm), 4-methoxybenzyl, 2,4-dimethoxybenzyl (DMB), 2,4,6-trimethoxybenzyl (TMB), 9-anthracene benzyl, 2,4-dichlorobenzyl, 1-naphthylbenzyl, 2-naphthylbenzyl, 7-quinoline benzyl; the optionally substituted sulfonyl group includes, but is not limited to, 2-nitrobenzenesulfonyl, p-toluenesulfonyl; the optionally substituted acyl group includes, but is not limited to, benzyloxycarbonyl (CBz), tert-butoxycarbonyl (Boc), phenoxyacetyl (Pac), 9-fluorenemethoxycarbonyl (Fmoc).

[0038] In one specific embodiment, the chiral morpholine phosphoroyl chloride monomer structure shown in Formula I is specifically shown in Formulas I-1, I-2, I-3, and I-4, and the chiral morpholine phosphoroyl chloride monomer structure shown in Formula II is specifically shown in Formulas II-1, II-2, II-3, and II-4.

[0039] Furthermore, in Equations I-1 and II-1, R 3The halogen is selected from H, substituted alkyl or alkoxy, or optionally substituted halogens; specifically, the optionally substituted alkyl or alkoxy groups include, but are not limited to, methyl, cyano, and methoxy groups; the optionally substituted halogens include, but are not limited to, fluorine (F), chlorine (Cl), bromine (Br), and iodine (I).

[0040] Furthermore, in equations I-2 and II-2, R 4 R 5 Each is selected from -H, -C(O)R 10 and -C(O)OR 10 Any of the following, where R 10 It is alkyl or aryl.

[0041] Furthermore, in Equations I-3 and II-3, R 6 R 7 Each is selected from -H, -C(O)R 10 and -C(O)OR 10 Any of the following, where R 10 It is alkyl or aryl.

[0042] Furthermore, in Equations I-4 and II-4, R 8 R 9 Each is selected from any one of optionally substituted alkyl, cyanoethyl, acyl, carbonate, carbamate, optionally substituted benzyl, 4-pentanoyloxybenzyl and silyl.

[0043] Monomer preparation method The compounds described herein can be prepared in a variety of ways known to those skilled in the art of organic synthesis or in variations thereof as understood by those skilled in the art. The compounds described herein can be prepared from readily available starting materials. Optimal reaction conditions may vary depending on the specific reactants or solvents used, but such conditions can be determined by those skilled in the art. Variations of the compounds described herein include the addition, reduction, or removal of various components as described for each compound. Similarly, the chirality of a molecule can be altered when one or more chiral centers are present. Furthermore, the synthesis of compounds may involve the protection and deprotection of various chemical groups. The use of protection and deprotection, and the selection of appropriate protecting groups, can be determined by those skilled in the art. The chemical properties of protecting groups can be found, for example, in Wuts and Greene, Protective Groups in Organic Synthesis, 4th Edition, Wiley & Sons, 2006, the entire contents of which are incorporated herein by reference. Starting materials and reagents used to prepare the disclosed compounds and compositions are available from commercial suppliers such as Adamas, Bide, Leyan, Aldrich Chemical Co. (Milwaukee, WI), Acros Organics (Morris Plains, NJ), Fisher Scientific (Pittsburgh, PA), Sigma (St. Louis, MO), Pfizer (New York, NY), or Merck (Whitehouse Station, NJ). Other materials, such as the drug delivery systems disclosed herein, are available from commercial sources.

[0044] The reaction that produces the compounds described in this invention can be carried out in a solvent, which can be selected by those skilled in the art of organic synthesis. Under the conditions of the reaction (i.e., temperature and time), the solvent may not react substantially with the starting materials (reactants), intermediates, or products. The reaction can be carried out in one solvent or a mixture of more than one solvent. The formation of products or intermediates can be monitored according to any suitable method known in the art. For example, product formation can be achieved by spectroscopic means such as nuclear magnetic resonance spectroscopy (e.g., 1 H or 29 P) or can be monitored by chromatographic methods such as high performance liquid chromatography (HPLC) or thin layer chromatography.

[0045] In a specific embodiment of the present invention, a method for preparing the chiral morpholine phosphoryl chloride monomer represented by Formula I or II is provided, the reaction formula of which is as follows: Specifically, the following steps are included: In the presence of an organic solvent, a base, a chiral ligand, and a copper catalyst, the compound shown in Formula IV is reacted with the compound shown in Formula III to generate a chiral morpholinophosphoryl chloride monomer.

[0046] In one specific embodiment, in the compound represented by Formula III, X represents an oxygen atom or a sulfur atom. R 1 The group is selected from any of the following: optionally substituted secondary substituted amino groups, optionally substituted heterocycles formed by the linked nitrogen, optionally substituted alkoxy groups, optionally substituted phenoxy groups, and guanidinyl groups; specifically, the optionally substituted secondary substituted amino groups can be, for example, dimethylamine, diethylamine, diisopropylamine, dibenzylamine, N-methylbenzylamine, bis(2-chloroethyl)amine, or diethyl iminodiacetic acid; the optionally substituted heterocycles formed by the linked nitrogen can be, for example, pyrrolidine, piperazine, morpholine, or piperidine; the optionally substituted alkoxy groups can be, for example, methoxy, ethoxy, isopropoxy, phenylethoxy, or p-nitrophenylethyl; the optionally substituted phenoxy groups can be, for example, 2-nitrophenoxy, 2-fluorophenoxy, 2-chlorophenoxy, p-nitrophenoxy, 4-methoxyphenoxy, or 3-nitrophenoxy.

[0047] In one specific embodiment, in the compound represented by Formula IV, B represents a base, including but not limited to thymine, cytosine, adenine, and guanine. Further, the B is selected from the following groups: , , , R in this context 3 The halogen is selected from H, substituted alkyl or alkoxy groups, or optionally substituted halogens; specifically, the optionally substituted alkyl or alkoxy groups include, but are not limited to, methyl, cyano, and methoxy groups; the optionally substituted halogens include, but are not limited to, fluorine (F), chlorine (Cl), bromine (Br), and iodine (I). R is selected from H, substituted alkyl or alkoxy groups, or optionally substituted halogens; specifically, the optionally substituted alkyl or alkoxy groups include, but are not limited to, methyl, cyano, and methoxy groups; the optionally substituted halogens include, but are not limited to, fluorine (F), chlorine (Cl), bromine (Br), and iodine (I). 4 R 5 R 6 R 7 Each is selected from -H, -C(O)R 10 and -C(O)OR 10 Any of the following, where R 10 It is alkyl or aryl. Wherein R... 8 R 9 Each is selected from any one of optionally substituted alkyl, cyanoethyl, acyl, carbonate, carbamate, optionally substituted benzyl, 4-pentanoyloxybenzyl and silyl.

[0048] In one specific embodiment, in the compound represented by Formula IV, R 2 Selected from any one of optionally substituted or unsubstituted tertiary alkyl, optionally substituted benzyl, optionally substituted sulfonyl, optionally substituted acyl; specifically, R2 Selected from any one of optionally substituted or unsubstituted tertiary alkyl groups, optionally substituted or unsubstituted benzyl groups, optionally substituted sulfonyl groups, and optionally substituted acyl groups; specifically, the substituted or unsubstituted tertiary alkyl groups include, but are not limited to, tert-butyl, triphenylmethyl (Tr), substituted triphenylmethyl groups (e.g., MMTr (p-methoxyphenyldiphenylmethyl), DMTr), etc. (Dimethoxyphenyldiphenylmethyl); the optionally substituted or unsubstituted benzyl group includes, but is not limited to, benzyl (Bn), 4-methoxybenzyl (PMB), 3,4-dimethoxybenzyl (DMB), diphenylmethyl (Dpm), 4-methoxybenzyl, 2,4-dimethoxybenzyl (DMB), 2,4,6-trimethoxybenzyl (TMB), 9-anthracene benzyl, 2,4-dichlorobenzyl, 1-naphthylbenzyl, 2-naphthylbenzyl, 7-quinoline benzyl; the optionally substituted sulfonyl group includes, but is not limited to, 2-nitrobenzenesulfonyl, p-toluenesulfonyl; the optionally substituted acyl group includes, but is not limited to, benzyloxycarbonyl (CBz), tert-butoxycarbonyl (Boc), phenoxyacetyl (Pac), 9-fluorenemethoxycarbonyl (Fmoc).

[0049] In one specific embodiment, the organic solvent is selected from at least one of tetrahydrofuran, 2-methyltetrahydrofuran, ethylene glycol dimethyl ether, and ethyl acetate.

[0050] In one specific embodiment, the base is selected from at least one of triethylamine, tributylamine, diisopropylethylamine, and 1,2,2,6,6-pentamethylpiperidine. The molar ratio of the base to the morpholinonucleotide represented by Formula IV is 1:1 to 5:1.

[0051] In one specific embodiment, the copper catalyst is selected from at least one of copper hexafluoroacetylacetonate, copper acetate, copper chloride, copper bromide, and cuprous bromide. The molar ratio of the catalyst to the morpholine nucleoside represented by Formula IV is 0.02:1-0.01.

[0052] In one specific embodiment, the chiral ligand is selected from pyridine imidazole ligands, and specifically, the structure of the pyridine imidazole ligand is as follows: （R） -L、 （S） -L、( S -L2、( S -L3、( S -L4、( S -L5、( S -L6、( S -L7、( S As shown in L10: .

[0053] The ligand （R） -L、 （S）-L is prepared by the following method: Step 1: Take a dried 250 mL flask and add 80 mL of THF, 5-trifluoromethyl-2-pyridinecarboxylic acid (18 mmol, 1.0 equiv.), (R / S)-tert-leucine (18 mmol, 1.0 equiv.), and TBTU (18 mmol, 1.0 equiv.), respectively. Add 5 mL of NEt3 and stir for 24 h. Remove the solvent under reduced pressure. Dissolve the residue in DCM (3 × 20 mL), wash with 1 M HCl solution, combine the organic layers, wash with brine, dry with anhydrous Na2SO4, filter, and concentrate. Purify the residue by silica gel column chromatography.

[0054] Step 2: Dissolve (R / S)-5-trifluoromethyl-N-(1-hydroxy-3,3-dimethylbutane-2-yl)pyrcolinamide (5.0 mmol, 1.0 equiv.) in chloroform (5.0 mL, 1.0 M). Under N2 protection, add 0.4 mL of thionyl chloride (5.5 mmol, 1.1 equiv.) dropwise at room temperature and reflux for 2 hours. After cooling to room temperature, add phosphorus pentachloride (5.5 mmol, 1.1 equiv.) at room temperature and reflux the resulting suspension overnight. Cool the solution to 0 °C and add dropwise a chloroform solution of the desired 3,5-di-tert-butylaniline (6.0 mmol, 1.2 equiv.) and triethylamine (15 mmol). Stir the mixture at 0 °C for 30 min and then reflux for 12 hours. After removing volatiles, an aqueous solution of NaOH (20% w / v, 10 mL) was added to the residue. The mixture was extracted with dichloromethane and washed with saturated brine. The combined organic layers were washed with brine, dried over anhydrous Na₂SO₄, filtered, and concentrated. The residue was purified by silica gel column chromatography to obtain the ligand ( S )-L / ( R )-L (that is, corresponding to (in the following embodiments) S )-L1、( R )-L1).

[0055] ( S )-L / ( R )-L : 1 H NMR (500 MHz, Chloroform- d ) δ 8.80 – 8.75 (m, 1H), 7.94(dd, J = 8.2, 2.4 Hz, 1H), 7.80 (d,J = 8.4 Hz, 1H), 7.01 (t, J = 1.8 Hz, 1H), 6.48(d, J = 1.8 Hz, 2H), 4.16 – 4.07 (m, 2H), 3.88 – 3.79 (m, 1H), 1.14 (s, 18H), 1.04 (s, 9H) ppm. 13 C NMR (126 MHz, Chloroform- d ) δ159.8, 154.9, 151.2, 146.2(q, J = 3.8 Hz), 141.5, 133.6 (q, J = 3.2 Hz), 126.7 (q, J = 33.1 Hz), 123.2 (q, J =273.4 Hz), 123.9, 117.5, 116.4, 74.9, 54.6, 34.7, 34.3, 31.2, 26.1 ppm. 19 F NMR (471 MHz, Chloroform- d ) δ -62.69 ppm.HRMS (ESI) calcd for C 27 H 37 FN3[M+H] + 460.2934; 460.2936 was found. The molar ratio of the chiral ligand to the morpholinonucleotide shown in Formula IV is 0.02:1-0.15:1.

[0056] In one specific embodiment, the reaction temperature is -40℃ to -50℃, and the reaction time is 4 to 24 hours. For example, the reaction temperature can be any temperature value or a range formed by any two of the following: -40℃, -41℃, -42℃, -43℃, -44℃, -45℃, -46℃, -47℃, -48℃, -49℃, and -50℃. The reaction time can be any time or a range formed by any two of the following: 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, and 24 hours.

[0057] Based on the above preparation method, different morpholine nucleosides can be used to react and obtain the corresponding chiral morpholine phosphoryl chloride monomers, which will not be listed one by one in the embodiments of the present invention.

[0058] Monolithic application In a specific embodiment of the present invention, an application of the chiral morpholine phosphoryl chloride monomer represented by Formula I or II is provided. This application includes the preparation of oligonucleotide chains, particularly for the synthesis of phosphoryldiamine morpholine oligonucleotide (PMO) chains and their thiophosphoramide morpholine oligonucleotide (TMO) chains.

[0059] In one specific embodiment, a method for preparing stereochemically pure oligonucleotide dimers by activating the stereoselective coupling of monomers is further provided.

[0060] In one specific embodiment, substantially diastereomeric oligonucleotide chains prepared by stereoselective coupling of activated monomers are further provided. For example, the appropriately protected chiral pure morpholinophosphoryl chloride monomer has been successfully used for chain extension in solid-phase synthesis to obtain chiral pure forms of drugs for treating Duchenne muscular dystrophy, such as casimersen and golodirsen.

[0061] Example 1 This embodiment provides an efficient method for preparing chiral morpholinophosphoryl chloride monomers. The chiral morpholinophosphoryl chloride monomer is (9H-fluorene-9-yl)methyl(2S,6R)-2-((((R)-chloro(dimethylamino)phosphoryl)oxy)methyl)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)morpholino-4-carboxylate (compound I-1a), the structure of which is shown below: The specific preparation steps of compound I-1a are as follows: Add the ligand ( ) to an 8 mL reaction flask. S The following solutions were prepared: L1 (6.9 mg, 0.015 mmol), copper hexafluoroacetylacetonate (4.8 mg, 0.01 mmol), Fmoc-protected thymidine morpholino-CH2OH (46.4 mg, 0.1 mmol), ultra-dry tetrahydrofuran (0.2 mL), and triethylamine (48.0 μL, 0.3 mmol). The reaction apparatus was sealed and stirred at -40°C for half an hour. Then, a solution of dimethylamine phosphoryl dichloride (55.8 mg, 0.3 mmol) in tetrahydrofuran (0.1 mL), pre-cooled to -40°C, was added to the above-stirred solution, and the reaction was allowed to proceed for 4 hours. The resulting reaction solution was subjected to column chromatography (DCM: MeOH = 20:1, R...). f = 0.50) to isolate a white solid intermediate product, namely compound I-1a (50.7 mg, yield 86%). 1H NMR (500 MHz, Chloroform-d) δ 8.73 (s, 1H), 7.77 (dd, J = 7.6, 3.9 Hz, 2H), 7.56 (dd, J = 10.1, 7.4Hz, 2H), 7.49 – 7.27 (m, 5H), 5.84 – 5.55 (m, 1H), 4.74 – 4.16 (m, 6H), 4.14 – 3.94 (m, 1H), 3.87 – 3.61 (m, 1H), 3.00 – 2.77 (m, 2H), 2.74 (d, J = 13.9Hz, 6H), 1.96 (s, 3H) ppm. 13 C NMR (126 MHz, Chloroform-d) δ 163.3, 154.7,149.7, 143.6, 141.3, 135.2, 127.9, 127.3 (d, J = 4.6 Hz), 125.0, 120.1,111.5, 79.0, 74.2, 66.9, 47.1, 44.0, 36.8 (d, J = 2.8 Hz), 12.6 ppm. 31 P NMR (202 MHz, Chloroform-d) δ 18.42 – 17.59 ppm. HPLC analysis yielded 99:1 dr (CHIRALPAKAD-H, 40% iPrOH / hexanes, 1.0 mL / min, T = 40 ℃, 254 nm, t R (main) = 19.8 min, t R (times) = 29.6 min), dr = 99:1. Example 2 This embodiment provides an efficient method for preparing chiral morpholinophosphoryl chloride monomers. The chiral morpholinophosphoryl chloride monomer is (9H-fluorene-9-yl)methyl(2S,6R)-2-((((S)-chloro(dimethylamino)phosphoryl)oxo)methyl)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)morpholin-4-carboxylate (compound II-1a), the structure of which is shown below: The specific preparation steps for compound II-1a are as follows: Add the ligand ( ) to an 8 mL reaction flask. RThe following solutions were prepared: L1 (6.9 mg, 0.015 mmol), copper hexafluoroacetylacetonate (4.8 mg, 0.01 mmol), Fmoc-protected thymidine morpholino-CH2OH (46.4 mg, 0.1 mmol), ultra-dry tetrahydrofuran (0.2 mL), and triethylamine (48.0 μL, 0.3 mmol). The reaction apparatus was sealed and stirred at -40°C for half an hour. Then, a solution of dimethylamine phosphoryl dichloride (55.8 mg, 0.3 mmol) in tetrahydrofuran (0.1 mL), pre-cooled to -40°C, was added to the above-stirred solution, and the reaction was allowed to proceed for 4 hours. The resulting reaction solution was subjected to column chromatography (DCM: MeOH = 20:1, R...). f = 0.50) to isolate a white solid intermediate product, namely compound II-1a (53.6 mg, yield 91%). 1 H NMR (500 MHz, Chloroform-d) δ 8.86 (s, 1H), 7.77 (dd, J = 10.1, 2.4 Hz, 2H), 7.55 (t, J =8.7 Hz, 2H), 7.41 (t, J = 7.5 Hz, 2H), 7.34 (q, J = 6.9 Hz, 2H), 7.30 – 7.17(m, 1H), 5.81 – 5.52 (m, 1H), 4.76 – 4.18 (m, 6H), 4.16 – 3.95 (m, 1H), 3.84 – 3.57 (m, 1H), 2.92 – 2.76 (m, 2H), 2.73 (dd, J = 14.0, 1.9 Hz, 10H), 1.95(s, 3H) ppm. 13 C NMR (126 MHz, Chloroform-d) δ 163.3, 154.7, 149.7, 143.7,143.6, 141.3, 134.8, 127.9, 127.3 (d, J = 3.2 Hz), 125.0, 120.1, 111.5, 79.1,74.2, 68.3, 66.5, 47.3, 43.9, 36.7 (d, J = 3.2 Hz), 12.6 ppm. 31 P NMR (202 MHz, Chloroform-d) δ 18.63 ppm. HPLC analysis yielded 99:1 dr (CHIRALPAK AD-H, 40% iPrOH / hexanes, 1.0 mL / min, T = 40 ℃, 254 nm, tR (times) = 19.8 min, t R (Main) = 29.6 min), dr = 99:1. Example 3 This embodiment provides an efficient method for preparing chiral morpholinophosphoryl chloride monomers. The chiral morpholinophosphoryl chloride monomer is (9H-fluorene-9-yl)methyl(2S,6R)-2-((((R)-chloro(dimethylamino)phosphoryl)oxo)methyl)-6-(4-decanoamido-2-oxopyrimidin-1(2H)-yl)morpholino-4-carboxylate (compound I-2a), the structure of which is shown below: The specific preparation steps of compound I-2a are as follows: Add the ligand ( ) to an 8 mL reaction flask. S The following solutions were prepared: L1 (6.9 mg, 0.015 mmol), copper hexafluoroacetylacetonate (4.8 mg, 0.01 mmol), Fmoc-protected cytidine morpholino-CH2OH (60.2 mg, 0.1 mmol), ultra-dry tetrahydrofuran (0.2 mL), and triethylamine (48.0 μL, 0.3 mmol). The reaction apparatus was sealed and stirred at -40°C for half an hour. Then, a solution of dimethylamine phosphoryl dichloride (55.8 mg, 0.3 mmol) in tetrahydrofuran (0.1 mL), pre-cooled to -40°C, was added to the above-prepared solution, and the reaction was allowed to proceed for 4 hours. The resulting reaction solution was subjected to column chromatography (DCM: MeOH = 20:1, R...). f = 0.50) to isolate a white solid intermediate product, namely compound I-2a (63.3 mg, yield 87%). 1 H NMR (500 MHz, Chloroform- d ) δ 10.15 (s, 1H), 8.19 – 7.86 (m, 1H), 7.74 (d, J = 7.8 Hz, 2H), 7.70 – 7.46(m, 3H), 7.34 (dt, J = 25.3, 7.4 Hz, 4H), 5.98 – 5.64 (m, 1H), 4.72 – 4.21 (m,6H), 4.18 – 3.68 (m, 2H), 3.04 – 2.76 (m, 2H), 2.72 (d, J= 13.9 Hz, 6H), 2.62– 2.45 (m, 2H), 1.73 – 1.55 (m, 2H), 1.46 – 1.04 (m, 12H), 0.86 (t, J = 6.9 Hz, 3H) ppm. 13 C NMR (126 MHz, Chloroform- d ) δ 174.4, 162.9, 154.7, 144.3, 143.6,141.2, 127.8, 127.2, 125.2, 120.0, 80.9, 74.3, 68.6, 67.1, 46.9, 43.6, 37.5,36.7 (d, J = 2.8 Hz), 31.8, 31.2, 29.4, 29.3, 29.1, 24.8, 22.6, 14.1 ppm. 31 P NMR (202 MHz, Chloroform- d δ 17.97 ppm. HPLC analysis yielded a concentration of 99:1 dr (CHIRALPAK AD-H, 10% iPrOH / hexanes, 1.0 mL / min, T = 40 ℃, 254 nm, t). R (main) = 70.8 min, t R (times) = 86.3 min), dr = 99:1. Example 4 This embodiment provides an efficient method for preparing chiral morpholinophosphoryl chloride monomers. The chiral morpholinophosphoryl chloride monomer is (9H-fluoren-9-yl)methyl(2S,6R)-2-((((S)-chloro(dimethylamino)phosphoryl)oxy)methyl)-6-(4-decanoamido-2-oxopyrimidin-1(2H)-yl)morpholino-4-carboxylic acid ester (compound II-2a), the structure of which is shown below: The specific preparation steps of compound II-2a are as follows: Add the ligand ( ) to an 8 mL reaction flask. RThe following solutions were prepared: L1 (6.9 mg, 0.015 mmol), copper hexafluoroacetylacetonate (4.8 mg, 0.01 mmol), Fmoc-protected cytidine morpholino-CH2OH (60.2 mg, 0.1 mmol), ultra-dry tetrahydrofuran (0.2 mL), and triethylamine (48.0 μL, 0.3 mmol). The reaction apparatus was sealed and stirred at -40°C for half an hour. Then, a solution of dimethylamine phosphoryl dichloride (55.8 mg, 0.3 mmol) in tetrahydrofuran (0.1 mL), pre-cooled to -40°C, was added to the above-prepared solution, and the reaction was allowed to proceed for 4 hours. The resulting reaction solution was subjected to column chromatography (DCM: MeOH = 20:1, R...). f = 0.50) to isolate a white solid intermediate product, namely compound II-2a (57.6 mg, yield 79%). 1 H NMR (500 MHz, Chloroform- d ) δ 9.00 (s, 1H), 8.03 – 7.81 (m, 1H), 7.76 (d, J = 7.3 Hz, 2H),7.65 – 7.45 (m, 3H), 7.39 (t, J = 7.4 Hz, 2H), 7.34 (q, J = 7.2 Hz, 6H), 5.83 (s,1H), 4.77 – 4.36 (m, 3H), 4.34 – 4.08 (m, 5H), 4.08 – 3.74 (m, 1H), 2.88 (s,1H), 2.75 (s, 3H), 2.72 (s, 3H), 2.47 (t, J = 7.5 Hz, 2H), 1.67 (p, J = 7.4 Hz,2H), 1.35 – 1.22 (m, 12H), 0.87 (t, J = 7.0 Hz, 3H) ppm. 13 C NMR (126 MHz, Chloroform- d ) δ 162.6, 154.7, 154.3, 143.9, 143.7, 141.3, 127.8, 127.3 (d, J =5.4 Hz), 125.2, 120.0 (d, J = 3.7 Hz), 97.1, 80.9, 74.5, 68.5, 66.5, 47.2,43.8, 37.8, 36.8 (d,J = 3.2 Hz), 31.9, 29.4, 29.3, 29.3, 29.1, 24.8, 22.7,14.1 ppm. 31 P NMR (162 MHz, Chloroform- d δ 18.71 ppm. HPLC analysis yielded a concentration of 99:1 dr (CHIRALPAK AD-H, 10% iPrOH / hexanes, 1.0 mL / min, T = 40 ℃, 254 nm, t). R (times) = 75.5 min, t R (Main) = 87.8 min), dr = 99:1. Example 5 This embodiment provides an efficient method for preparing chiral morpholinophosphoryl chloride monomers. The chiral morpholinophosphoryl chloride monomer is (9H-fluorene-9-yl)methyl(2R,6S)-2-(6-amino-9H-purine-9-yl)-6-((((R)-chloro(dimethylamino)phosphoryl)oxy)methyl)morpholin-4-carboxylate (compound I-3a), and its structure is shown below: The specific preparation steps of compound I-3a are as follows: Add the ligand ( ) to an 8 mL reaction flask. S The following solutions were prepared: L1 (6.9 mg, 0.015 mmol), copper hexafluoroacetylacetonate (4.8 mg, 0.01 mmol), Fmoc-protected adenosylmorpholino-CH2OH (47.2 mg, 0.1 mmol), ultra-dry tetrahydrofuran (0.4 mL), and triethylamine (48.0 μL, 0.3 mmol). The reaction apparatus was sealed and stirred at -40°C for half an hour. Then, a solution of dimethylaminophosphoryl dichloride (55.8 mg, 0.3 mmol) in tetrahydrofuran (0.1 mL), pre-cooled to -40°C, was added to the above-stirred solution, and the reaction was allowed to proceed for 16 hours. The resulting reaction solution was subjected to column chromatography (DCM: MeOH = 20:1, R...). f = 0.50) to isolate a white solid intermediate product, namely compound I-3a (42.5 mg, yield 71%). 1 H NMR (500 MHz, Chloroform- d) δ 8.42 (s, 1H), 8.10 (s, 1H), 7.87 – 7.65 (m, 2H), 7.62 – 7.53(m, 2H), 7.49 – 7.28 (m, 4H), 5.93 (s, 2H), 5.75 (d, J = 8.1 Hz, 1H), 4.80 –4.36 (m, 3H), 4.33 – 3.98 (m, 4H), 3.84 (s, 1H), 3.35 – 3.23 (m, 1H), 2.94(t, J = 12.3 Hz, 1H), 2.72 (d, J = 13.9 Hz, 6H) ppm. 13 C NMR (126 MHz, Chloroform- d ) δ 155.2, 154.8, 152.4, 149.3, 143.6, 141.4, 138.4, 127.9, 127.2, 124.9,120.1 (d, J = 2.3 Hz), 79.3, 74.2, 68.0, 66.5, 60.4, 53.2, 47.3, 44.1, 36.8,36.7, 29.3 ppm. 31 P NMR (202 MHz, Chloroform- d δ 18.41 ppm. HPLC analysis yielded a concentration of 99:1 d.r. (CHIRALPAK AD-H, 40% iPrOH / hexanes, 1.0 mL / min, T = 40 ℃, 254 nm, t). R (main) = 25.3 min, t R (Main) = 29.4 min), dr = 99:1. Example 6 This embodiment provides an efficient method for preparing chiral morpholinophosphoryl chloride monomers. The chiral morpholinophosphoryl chloride monomer is (9H-fluorene-9-yl)methyl(2R,6S)-2-(6-amino-9H-purin-9-yl)-6-((((S)-chloro(dimethylamino)phosphoryl)oxy)methyl)morpholin-4-carboxylate (compound II-3a), and its structure is shown below: The specific preparation steps for compound II-3a are as follows: Add the ligand ( ) to an 8 mL reaction flask.R The following solutions were prepared: L1 (6.9 mg, 0.015 mmol), copper hexafluoroacetylacetonate (4.8 mg, 0.01 mmol), Fmoc-protected adenosylmorpholino-CH2OH (47.2 mg, 0.1 mmol), ultra-dry tetrahydrofuran (0.2 mL), and triethylamine (48.0 μL, 0.3 mmol). The reaction apparatus was sealed and stirred at -40°C for half an hour. Then, a solution of dimethylaminophosphoryl dichloride (55.8 mg, 0.3 mmol) in tetrahydrofuran (0.1 mL), pre-cooled to -40°C, was added to the above-stirred solution, and the reaction was allowed to proceed for 16 hours. The resulting reaction solution was subjected to column chromatography (DCM: MeOH = 20:1, R...). f = 0.50) to isolate a white solid intermediate product, namely compound II-3a (44.9 mg, yield 75%). 1 H NMR (500 MHz, Chloroform-d) δ 8.42 (s, 1H), 8.12 (s, 1H), 7.86 – 7.66 (m, 2H), 7.59 (t, J =8.5 Hz, 2H), 7.48 – 7.31 (m, 4H), 6.79 – 6.45 (m, 2H), 5.85 – 5.71 (m, 1H), 4.78 – 4.39 (m, 3H), 4.36 – 4.01 (m, 4H), 4.01 – 3.76 (m, 1H), 3.31 (t, J =11.7 Hz, 1H), 2.94 (dd, J = 13.5, 10.9 Hz, 1H), 2.72 (d, J = 13.9 Hz, 6H). 13 CNMR (126 MHz, Chloroform-d) δ 156.0, 154.8, 153.1, 143.6, 141.3, 138.4,127.9, 127.2, 125.0, 120.1 (d, J = 2.3 Hz), 79.4, 74.1, 68.1, 66.4, 47.2,44.1, 36.7 (d, J = 2.8 Hz), 31.2. 31 P NMR (202 MHz, Chloroform-d) δ 18.50. HPLC analysis yielded a 99:1 dr (CHIRALPAK AD-H, 40% iPrOH / hexanes, 1.0 mL / min, T = 40℃, 254 nm, t R (times) = 25.0 min, tR (Main) = 28.8 min), dr = 99:1. Example 7 This embodiment provides an efficient method for preparing chiral morpholinophosphoryl chloride monomers. The chiral morpholinophosphoryl chloride monomer is (9H-fluoren-9-yl)methyl(2S,6R)-2-((((R)-chloro(dimethylamino)phosphoryl)oxy)methyl)-6-(2-isobutamido-6-(4-nitrophenylethoxy)-9H-purin-9-yl)morpholin-4-carboxylate (compound I-4a), the structure of which is shown below: The specific preparation steps for compound I-4a are as follows: Add the ligand ( ) to an 8 mL reaction flask. S The following solutions were prepared: L1 (6.9 mg, 0.015 mmol), copper hexafluoroacetylacetonate (4.8 mg, 0.01 mmol), Fmoc-protected guanylate morpholino-CH2OH (70.7 mg, 0.1 mmol), ultra-dry tetrahydrofuran (0.4 mL), and triethylamine (48.0 μL, 0.3 mmol). The reaction apparatus was sealed and stirred at -40°C for half an hour. Then, a solution of dimethylamine phosphoryl dichloride (55.8 mg, 0.3 mmol) in tetrahydrofuran (0.1 mL), pre-cooled to -40°C, was added to the above-stirred solution, and the reaction was allowed to proceed for 4 hours. The resulting reaction solution was subjected to column chromatography (DCM: MeOH = 20:1, R...). f = 0.50) to isolate a white solid intermediate product, namely compound I-4a (77.5 mg, yield 93%). 1 H NMR (500 MHz, Chloroform- d ) δ 8.16 (dd, J = 8.7, 2.6 Hz, 2H), 8.02 – 7.71 (m, 4H), 7.57 (dd, J = 11.6, 7.5Hz, 2H), 7.51 (d, J = 8.5 Hz, 2H), 7.47 – 7.28 (m, 4H), 5.65 (dd, J = 10.2, 3.1Hz, 1H), 4.83 (t, J = 6.6 Hz, 2H), 4.71 – 3.68 (m, 10H), 3.37 – 3.21 (m, 2H), 2.92 (t, J = 12.4 Hz, 1H), 2.71 (d,J = 13.9 Hz, 6H), 1.30 – 1.17 (m, 6H) ppm. 13 CNMR (126 MHz, Chloroform- d ) δ 160.7, 154.8, 152.3, 152.1, 146.9, 145.7,141.3, 139.2, 130.0, 127.9, 127.2, 124.9, 123.8, 120.1, 117.9, 79.4, 74.1,67.0, 66.4 (d, J = 5.5 Hz), 53.5, 47.3, 44.5, 36.7 (d, J = 3.2 Hz), 35.9, 35.1,31.1, 30.9, 19.3 ppm. 31 P NMR (202 MHz, Chloroform- d δ 18.47 ppm. HPLC analysis yielded a concentration of 99:1 dr (CHIRALPAK AD-H, 40% iPrOH / hexanes, 1.0 mL / min, T = 40 ℃, 254 nm, t). R (times) = 16.1 min, t R (Main) = 19.6 min), dr = 99:1. Example 8 This embodiment provides an efficient method for preparing chiral morpholinophosphoryl chloride monomers. The chiral morpholinophosphoryl chloride monomer is (9H-fluoren-9-yl)methyl(2S,6R)-2-((((S)-chloro(dimethylamino)phosphoryl)oxy)methyl)-6-(2-isobutamido-6-(4-nitrophenylethoxy)-9H-purin-9-yl)morpholino-4-carboxylic acid ester (compound II-4a), the structure of which is shown below: The specific preparation steps for compound II-4a are as follows: Add the ligand ( ) to an 8 mL reaction flask. RThe following solutions were prepared: L1 (6.9 mg, 0.015 mmol), copper hexafluoroacetylacetonate (4.8 mg, 0.01 mmol), Fmoc-protected guanylate morpholino-CH2OH (70.8 mg, 0.1 mmol), ultra-dry tetrahydrofuran (0.2 mL), and triethylamine (48.0 μL, 0.3 mmol). The reaction apparatus was sealed and stirred at -40°C for half an hour. Then, a solution of dimethylamine phosphoryl dichloride (55.8 mg, 0.3 mmol) in tetrahydrofuran (0.1 mL), pre-cooled to -40°C, was added to the above-stirred solution, and the reaction was allowed to proceed for 4 hours. The resulting reaction solution was subjected to column chromatography (DCM: MeOH = 20:1, R...). f = 0.50) to isolate a white solid intermediate product, namely compound II-4a (78.6 mg, yield 75%). 1 H NMR (400 MHz, Chloroform- d ) δ 8.17 (d, J = 8.5 Hz, 2H), 8.11 – 7.65 (m, 4H), 7.60 – 7.49 (m,4H), 7.47 – 7.28 (m, 4H), 5.64 (dd, J = 10.4, 2.9 Hz, 1H), 4.82 (d, J = 5.9 Hz,2H), 4.71 – 4.35 (m, 3H), 4.33 – 3.98 (m, 4H), 3.97 – 3.69 (m, 1H), 3.40 –3.17 (m, 3H), 3.13 – 2.80 (m, 2H), 2.71 (d, J = 13.9 Hz, 6H), 1.29 – 1.16 (m,6H) ppm. 13 C NMR (126 MHz, Chloroform- d ) δ 160.7, 154.7, 152.3, 152.2, 146.9,145.7, 143.7, 141.3, 139.1, 130.0, 127.9, 127.2, 124.9, 123.8, 120.1, 117.9,79.4, 74.0, 68.0, 67.0, 66.3 (d, J = 5.3 Hz), 47.3, 44.3, 36.7 (d, J= 3.2 Hz),35.9, 35.1, 31.1, 19.3 ppm. 31 P NMR (162 MHz, Chloroform- d δ 18.57 ppm. HPLC analysis showed a concentration of 99:1 dr (CHIRALPAK AD-H, 40% iPrOH / hexanes, 1.0 mL / min, T = 40 ℃, 254 nm, t). R (main) = 18.0 min, t R (times) = 21.6 min), dr = 99:1. Example 9 This embodiment provides an efficient method for preparing chiral morpholinophosphoryl chloride monomers. The chiral morpholinophosphoryl chloride monomer is a chiral (9H-fluoren-9-yl)methyl(2S,6R)-2-((((S)-chloro(4-nitrophenylethoxy)thiophosphoryl)oxy)methyl)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)morpholino-4-carboxylic acid ester (compound I-1b), the structure of which is shown below: The specific preparation steps for compound I-1b are as follows: Add the ligand ( ) to an 8 mL reaction flask. S )-L1 (6.9 mg, 0.015 mmol), copper bromide (4.8 mg, 0.01 mmol), Fmoc-protected thymidine morpholino-CH2OH (46.4 mg, 0.1 mmol), ultradry tetrahydrofuran (0.2 mL), and triethylamine (48.0 μL, 0.3 mmol) were added to the reaction apparatus, which was sealed and stirred at -40 °C for half an hour. Then, a solution of p-nitrophenylethoxythiophosphoric dichloroisocyanurate (89.4 mg, 0.3 mmol) in tetrahydrofuran (0.1 mL) pre-cooled to -40 °C was added to the above-stirred solution, and the reaction was allowed to proceed for 12 hours. The resulting reaction solution was subjected to column chromatography (DCM: MeOH = 20:1, R f = 0.50) to isolate a white solid intermediate product, namely compound I-1b (58.1 mg, yield 78%). 1 H NMR (400 MHz, Chloroform- d ) δ 9.12 (s, 1H), 8.16 (d, J = 8.2 Hz, 2H), 7.76 (dd, J = 7.6, 3.1Hz, 2H), 7.55 (t,J = 7.3 Hz, 2H), 7.44 – 7.27 (m, 7H), 5.67 (s, 1H), 4.46 (dd, J = 10.5, 6.8 Hz, 4H), 4.34 – 4.09 (m, 5H), 3.15 (t, J = 6.6 Hz, 2H), 2.91–2.63(m, 2H), 1.93 (s, 3H) ppm. 13 C NMR (126 MHz, CDCl3) δ 163.5, 154.8, 149.9,147.3, 144.2, 143.6, 141.4, 134.9, 130.0, 128.0, 127.4 (d, J = 3.7 Hz), 125.1,124.0, 120.2 (d, J = 4.1 Hz), 111.6, 79.1, 77.4, 69.3 (d, J = 6.5 Hz), 68.3 (d, J =6.2 Hz), 35.9 (d, J = 8.7 Hz), 12.7 ppm. 31 P NMR (162 MHz, Chloroform- d δ 69.97, 69.70 ppm. HPLC analysis yielded a 99:1 d.r. concentration (CHIRALPAK IE, 55% iPrOH / hexanes, 1.0 mL / min, T = 40 ℃, 254 nm, t). R (times) = 45.2 min, t R (Main) = 49.0 min), dr = 99:1. Example 10 This embodiment provides an efficient method for preparing chiral morpholinophosphoryl chloride monomers. The chiral morpholinophosphoryl chloride monomer is a chiral (9H-fluoren-9-yl)methyl(2S,6R)-2-((((S)-chloro(4-nitrophenylethoxy)thiophosphoryl)oxy)methyl)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)morpholino-4-carboxylic acid ester (compound I-2b), the structure of which is shown below: The specific preparation steps for compound I-2b are as follows: Add the ligand ( ) to an 8 mL reaction flask. S The following solutions were prepared: L1 (6.9 mg, 0.015 mmol), copper bromide (4.8 mg, 0.01 mmol), Fmoc-protected cytidine morpholino-CH2OH (60.2 mg, 0.1 mmol), ultradry tetrahydrofuran (0.2 mL), and triethylamine (48.0 μL, 0.3 mmol). The reaction apparatus was sealed and stirred at -40°C for half an hour. Then, a solution of p-nitrophenylethoxythiophosphoric dichloroisocyanurate (89.4 mg, 0.3 mmol) in tetrahydrofuran (0.1 mL), pre-cooled to -40°C, was added to the above-stirred solution, and the reaction was allowed to proceed for 12 hours. The resulting reaction solution was subjected to column chromatography (DCM: MeOH = 20:1, R...). f = 0.50) to isolate a white solid intermediate product, namely compound I-2b (54.4 mg, yield 63%). 1 H NMR (500 MHz, Chloroform- d ) δ 9.13 (s, 1H), 8.16 (d, J = 8.3 Hz, 2H), 7.99 – 7.82 (m, 1H), 7.75 (d, J = 7.5 Hz, 2H), 7.64 – 7.46 (m, 3H), 7.36 (dt, J = 24.1, 7.5 Hz, 5H), 5.82 (s, 1H), 4.59 (s, 1H), 4.47 (pt, J = 9.7, 6.4 Hz, 4H), 4.38 – 4.15 (m,4H), 3.16 (t, J = 6.6 Hz, 2H), 2.82 –2.78 (m, 2H), 2.49 (t, J = 7.4 Hz, 2H), 1.71– 1.63 (m, 2H), 1.41 – 1.14 (m, 14H), 0.86 (t, J = 6.9 Hz, 3H). 13 C NMR (126 MHz, Chloroform- d ) δ 173.6, 162.6, 154.7, 154.2, 147.2, 144.1, 143.8, 143.7,143.57, 141.3, 129.9, 127.9, 127.3, 123.9, 120.1 (d, J= 4.6 Hz), 97.0, 77.3,69.2 (d, J = 6.7 Hz), 68.2 (d, J = 4.7 Hz), 37.8, 35.9 (d, J = 8.7 Hz), 31.9, 29.7,29.4, 29.3, 29.3, 29.1, 24.8, 22.7, 14.3. ppm. 31 P NMR (162 MHz, Chloroform- d δ 70.02, 69.82 ppm. HPLC analysis yielded a concentration of 98.5:1.5 dr (CHIRALPAK AD-H, 45% iPrOH / hexanes, 1.0 mL / min, T = 40 ℃, 254 nm, t). R (times) = 13.0 min, t R (main) = 15.7 min),dr=98.5:1.5. Example 11 This embodiment provides an efficient method for preparing chiral morpholinophosphoryl chloride monomers. The chiral morpholinophosphoryl chloride monomer is (9H-fluorene-9-yl)methyl(2S,6R)-2-((((R)-chloro(pyrrolidin-1-yl)phosphoryl)oxo)methyl)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)morpholino-4-carboxylic acid ester (compound I-1c), the structure of which is shown below: The specific preparation steps for compound I-1c are as follows: Add the ligand ( ) to an 8 mL reaction flask. S The following solutions were prepared: L1 (6.9 mg, 0.015 mmol), copper hexafluoroacetylacetonate (4.8 mg, 0.01 mmol), Fmoc-protected thymidine morpholino-CH2OH (46.4 mg, 0.1 mmol), ultra-dry tetrahydrofuran (0.2 mL), and triethylamine (48.0 μL, 0.3 mmol). The reaction apparatus was sealed and stirred at -40°C for half an hour. Then, a solution of pyrrolephosphonate dichloride (56.4 mg, 0.3 mmol) in tetrahydrofuran (0.1 mL), pre-cooled to -40°C, was added to the above-prepared solution, and the reaction was allowed to proceed for 4 hours. The resulting reaction solution was subjected to column chromatography (DCM: MeOH = 20:1, R...). f = 0.50) to separate the intermediate product into a white solid, namely compound I-1c (51.7 mg, yield 84%).1 H NMR (500 MHz, Chloroform- d )δ 9.43 (s, 1H), 7.76 (dd, J = 7.6, 4.0 Hz, 2H), 7.55 (t, J = 8.4 Hz, 2H), 7.48 –7.28 (m, 5H), 5.69 (d, J = 36.8 Hz, 1H), 4.52 – 4.31 (m, 2H), 4.30 – 4.20 (m,3H), 4.18 – 3.90 (m, 2H), 3.84 – 3.64 (m, 1H), 3.33 – 3.19 (m, 4H), 2.91 (d, J = 25.5 Hz, 2H), 1.95 (s, 3H), 1.91 – 1.86 (m, 4H) ppm. 13 C NMR (126 MHz, Chloroform- d ) δ 163.3, 154.7, 149.7, 143.5, 141.3, 135.1, 127.9, 127.3 (d, J =3.7 Hz), 125.0, 120.1, 111.5, 79.0, 74.4, 67.1, 66.2 (d, J = 7.8 Hz), 47.2, 44.7, 12.5 ppm. 31 P NMR (202 MHz, Chloroform- d δ 15.2, 14.6 ppm. HPLC analysis showed a concentration >99:1 dr (CHIRALPAK AD-H, 40% iPrOH / hexanes, 1.0 mL / min, T = 40 ℃, 254 nm, t R (times) = 13.0 min, t R (Main) = 17.0 min), dr>99:1. Example 12 This embodiment investigates the effects of different copper catalysts or solvents on the reaction results. This embodiment provides an efficient method for preparing chiral morpholinophosphoryl chloride monomers, wherein the chiral morpholinophosphoryl chloride monomer is ((2S,6R)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-triphenylmethylmorpholin-2-yl)methyl(R)-dimethylphosphoramide chloride. The preparation reaction formula is as follows: The specific preparation steps are basically the same as in Example 2, except that: the Fmoc-protected thymidine morpholino-CH2OH is replaced with the compound of Formula 1 (Tr-protected thymidine morpholino-CH2OH), and the ligand is replaced with ( S The reaction was carried out at -30°C for 16 hours using L2 (structure as shown in Example 13). The types of copper catalysts, molar amounts of compound 2a, and solvents used in each experimental group are shown in Table 1. The yields and dr values ​​of the obtained products are shown in Table 1.

[0062] Table 1 As shown in Table 1, for the chiral morpholinophosphoryl chloride monomers prepared by different copper catalysts in experimental groups 1-5, the optimal copper catalyst was Cu(hfac)2, followed by Cu(OAc)2, CuBr2, and CuBr, while the worst was CuOTf. For the chiral morpholinophosphoryl chloride monomers prepared by different solvents in experimental groups 6-9, the optimal solvent was THF, followed by 2-Me THF and EtOAc.

[0063] Example 13 This embodiment investigates the effects of different protecting groups and ligands on the reaction results. This embodiment also provides an efficient method for preparing a series of chiral morpholine phosphoryl chloride monomers, the structures of which are shown below: The specific preparation method is basically the same as that of experimental group 5 in Example 12, except that the molar amount of compound 2a used is 0.15 mmol; the different protecting groups used in each experimental group, the cytidine morpholino-CH2OH, and the types of ligands are shown in Table 2. The yield and dr value of the obtained products are shown in Table 2.

[0064] Table 2 The ligand structures used in Table 2 are as follows: ( S -L2( S-L3( S -L4( S -L5( S -L6 ( S -L7( S -L8( S -L9( S -L10 As shown in Table 2, the yields of the corresponding chiral morpholinophosphoryl chloride monomers prepared by experimental groups 10-19 using cytidine morpholino-CH2OH with different protecting groups were above 47%, and the dr ratios were greater than 88:12. Among the chiral morpholinophosphoryl chloride monomers prepared by experimental groups 19-28 using different ligands, except for ( S ) -L8 and ( S Besides L9, the yields of chiral morpholinophosphoryl chloride monomers prepared by other ligands were above 50%, with dr greater than 89:11.

[0065] Example 14 This embodiment uses the preparation method of Example 1 or 2, and by changing the types of compounds with the structure shown in Formula III and the types of compounds with the structure shown in Formula IV, a series of chiral morpholine phosphoryl chloride monomers with different structures were prepared. The specific structures of each monomer are as follows: Figure 2 As shown, (Rp)(I)-1b, (Rp)(I)-2b, and (Rp)(I)-1c are I-1b, I-2b, and I-1c prepared in Examples 10-12 above.

[0066] This embodiment will not provide detailed preparation methods one by one; the structure can be determined by NMR characterization.

[0067] Application Example 1 This application example provides a method for preparing phosphoryldiamine morpholino oligonucleotide (PMO) chains, the preparation route is as follows: Figure 3 As shown, the specific preparation steps are as follows: S1. Deprotect the protecting group Tr on the aminomethyl polystyrene resin to obtain a free secondary amine (steps 1-4 in Table 3). S2. The free secondary amine is coupled with the chiral morpholinophosphoryl chloride monomer prepared in the above examples (based on the base sequence of the target oligomer, the corresponding monomers are selected as: (II)-4a, (II)-1a, (II)-1a, (II)-4a, (II)-2a, (II)-2a, (II)-1a, (II)-2a, (II)-2a, (II)-4a) to obtain morpholino oligonucleotide dimers; S3. After capping with a capping reagent (step 7 in Table 3), remove the protecting group from the morpholine oligonucleotide dimer to obtain the deprotected morpholine oligonucleotide dimer (step 8 and steps 2-4 in Table 3). S4. Repeat steps S2 and S3 for the next round of coupling, end-capping, and deprotection until the crude target oligomer is obtained; (the chain base sequence is: GTTGCCTCCG, SEQ ID NO.1) The specific operating procedures are shown in Table 3 below.

[0068] Table 3 S5. The crude resin carrier containing the target oligomer was treated with lysis buffer (0.5 mL NMP containing 0.1 mol / L 1,4-dithiothreitol (DTT) and 0.73 mol / L NEt3) for 30 minutes. After collecting the protected oligomer solution, the resin was washed twice with 0.3 mL of lysis buffer. The three lysis buffers were combined and transferred to an 8 mL reaction flask. 3.0 mL of 28% ammonia water pre-cooled to -20°C was added. The reaction flask was sealed and kept in a 55°C oil bath for 32 hours to remove the protecting groups.

[0069] S6. Excess NH3 was evaporated using a freeze dryer and then diluted with water. All products were centrifuged using a 3000 MWCO centrifuge filter (12000 rpm, 20 min) and washed four times with water. The residue was collected, and the filter membrane was repeatedly rinsed with 500 µL of water (5 times × 500 µL). All components were combined and the purity of the compounds was determined by HPLC. All compounds were freeze-dried to a white solid and purified by preparative reversed-phase high-performance liquid chromatography (RPHPLC). The final purified product was analyzed by ultra-high performance liquid chromatography (UHPLC) and electrospray ionization mass spectrometry (MS-ESI), confirming the acquisition of chiral pure phosphoryl diamine morpholino oligonucleotide (PMO) chains in 44% yield.

[0070] Application Example 2 This application example provides a method for preparing thiophosphoramide morpholine oligonucleotide (TMO) chains, the preparation route is as follows: Figure 3 As shown, the specific preparation steps are as follows: S1. Deprotect the protecting group Tr on the linker derived from aminomethyl polystyrene resin to obtain a free secondary amine (steps 1-3 in Table 4). S2. The free secondary amine is coupled with the chiral morpholinophosphoryl chloride monomer prepared in the previous example (the corresponding monomers selected according to the base sequence of the target oligomer are: (II)-4b, (II)-3b, (II)-1b, (II)-2b, (II)-2b, (II)-1b, (II)-3b, (II)-4b, (II)-3b, (II)-4b, (II)-1b, (II)-2b) in steps 4-5 of Table 4 to obtain morpholino oligonucleotide dimers; S3. After capping with a capping reagent (step 6 in Table 4), remove the protecting group from the morpholine oligonucleotide dimer to obtain the deprotected morpholine oligonucleotide dimer (step 7 and steps 2-3 in Table 4). S4. Repeat steps S2 and S3 for the next round of coupling, end-capping, and deprotection until the crude target oligomer is obtained; (the chain base sequence is: GATCCTAGAGTC, SEQ ID NO.2) The operating procedure is shown in Table 4 below.

[0071] Table 4 S5. After the cycle is complete, the dried solid support in the synthesis column is treated with 0.5 mL of acetonitrile (CH3CN) solution containing 0.5 M 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) and 0.25 M N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA). The mixture is continuously vortexed for 8 hours. This treatment step is repeated twice, for a total of three times. Subsequently, the support is washed sequentially with dry NMP and DCM, and then dried under vacuum.

[0072] S6. Next, the carrier was transferred to a screw-capped plastic bottle, and 1.5 mL of 28% ammonia solution was added. The mixture was treated at 25°C for 24 hours. The carrier was then removed by filtration and washed with concentrated ammonia. The filtrates were combined and dried under vacuum. The resulting residue was dissolved in water and purified by preparative reversed-phase high-performance liquid chromatography (RP-HPLC). The final product was analyzed by ultra-high-performance liquid chromatography (UHPLC) and electrospray ionization mass spectrometry (MS-ESI), confirming the acquisition of chiral pure thiophosphoramide morpholino oligonucleotide (TMO) chains in 58% yield.

[0073] Application Example 3 This application example provides a method for synthesizing PMO chain casimersen, with the following specific steps: (1) Deprotection of the protecting group Tr on aminomethyl polystyrene resin was performed to obtain a free secondary amine; (2) The free secondary amine obtained in step (1) is reacted with Fmoc-protected dimethylaminomorpholinophosphoryl chloride monomer (2.5 equivalents, the monomers used in sequence being: (I)-2b, (I)-3b, (I)-3b, (I)-1b, (I)-4b, (I)-2b, (I)-2b, (I)-3b, (I)-1b, (I)-2b, (I)-2b, (I)-1b, (I)-4b, (I)-4b, (I)-3b, (I)-4b, (I)-1b (I)-1b, (I)-2b, (I)-2b, (I)-1b, (I)-4b) were reacted / coupled in DMI (1,3-dimethyl-2-imidazolinone, 0.2 M) in the presence of PMP (1,2,2,6,6-pentamethylpiperidine, 10.0 equivalent) to obtain dimers; preferably, the reagent was added in three batches at 2-hour intervals, followed by elution of excess reagent; (3) The free amine on the solid support that did not participate in coupling in step (2) was capped using a (1:1) mixture of 20% Ac2O-NMP and 20% DIPEA (diisopropylethylamine)-NMP; (4) After eluting the capping reagent, Fmoc was removed with 20% piperidine DMF solution; (5) Repeat steps (2)-(4) to unblock / deprotect before the next round of coupling until the crude target oligomer (sequence 5'-CAATGCCATCCTGGAGTTCCTG-3') is obtained; (6) The resin support containing the crude target oligomer was treated for 30 min with a lysis solution consisting of 0.1 mol / L 1,4-dithiothreitol (DTT) and 0.73 mol / L triethylamine in NMP (0.5 mL). After collecting the protected oligomer solution, the resin was washed with two more lysis solutions (0.3 mL), and the three lysis solutions were combined. Concentrated ammonia (3.0 mL, pre-cooled to -20 °C) was added to dissolve the oligomer, and the solution was transferred to an 8 mL reaction flask. The flask was sealed and kept in an oil bath at 55 °C for 32 h to remove the protecting groups. The solution was lyophilized with water, dissolved in a small amount of ultrapure water, and a small amount was sent to LCMS for confirmation. The target oligonucleotide chain was then separated by HPLC and semi-preparative purification.

[0074] The HPLC chromatograms of the crude and purified samples are shown below. Figure 4 As shown, the HPLC purity of PMO chain casimersen before purification was 36.85%, and the purified HPLC showed it to be almost pure. The MS chromatogram of the purified sample is shown below. Figure 5The MALDI-TOF plot shown is as follows ([M+5H]+ m / z 1516.7, found: 1517.8.). Figure 6 As shown (MALDI-TOF Calculated for C) 268 H 425 N 124 O 95 P 22 [M+H] + Exact Mass: is 7581.646 found 7581.823.), confirming the correctness of the molecular weight and sequence of the PMO chain casimersen.

[0075] HPLC analysis was performed under the following conditions, and the coupling efficiency was calculated by integrating the peak area obtained by HPLC from the absorption at UV = 260 nm.

[0076] HPLC conditions: Analytical column: ACQUITY UPLC BEH C18 1.7μm, 2.1×50mm (waters) Temperature: 40℃ Flow rate: 0.3 mL / min Mobile phase A: 20 mM triethylamine acetate buffer solution Mobile phase B: 80% MeCN + 20% 20mM triethylamine acetate buffer solution Gradient: 0-0%B (0-2 min) 0-5%B (2-5 min) 5%B (5-8 min) 5-100%B (8-20 min) 100%B (20-25 min) LC / MS conditions: Analytical column: Agilent HPH C18 (2.5 μm, 4.6 × 50 mm), 40℃ Detection wavelength: 254nm Moving phase A: 20mM AcONH4aq. Moving phase B: MeCN Flow rate: 0.3 mL / min Gradient: 40%–95% B (0–15 min) 95% B (15–24 min) 40% B (24–30 min) Separation by semi-preparative method Separation column: Ulyimate XB-C18 (10 μm, 21.2 × 250 mm), 25℃ Detection wavelengths: 254nm & 280nm Mobile phase A: 50 mM triethylamine carbonate buffer solution. Moving phase B: MeCN Flow rate: 15 mL / min Gradient: 0-5% B (0-10 min) 5-10% B (10-25 min) 10-20% B (25-50 min) 30-30% B (24-30 min) MALDI-TOF conditions: Matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF) data were acquired using a Bruker UltrafleXtreme MALDI-TOF / TOF system. α-cyano-4-hydroxycinnamic acid (CHCA) was used as the matrix, and the solvent system was a 1:1 water-acetonitrile mixture containing 0.1% trifluoroacetic acid.

[0077] It should be noted that, based on the base sequence of the target oligonucleotide chain and using the methods described in the aforementioned application examples, the chiral morpholinophosphoryl chloride monomers of Formula I or Formula II prepared according to the embodiments of the present invention can be successfully used to prepare the respective target oligonucleotide chains. These will not be listed individually in this invention.

[0078] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A chiral morpholine phosphoryl chloride monomer, characterized in that, The structure of the monomer is shown in formula I or II below: ； Where X represents an oxygen atom or a sulfur atom; B represents a base; R 1 It is selected from any one of the following: a secondary substituted amino group, a heterocycle formed by a nitrogen linkage, an substituted alkoxy group, a substituted phenoxy group, and a guanidinyl group; R 2 It is selected from any one of the following: optionally substituted or unsubstituted tertiary alkyl, optionally substituted or unsubstituted benzyl, optionally substituted sulfonyl, optionally substituted acyl.

2. The chiral morpholine phosphoryl chloride monomer according to claim 1, characterized in that, The base is selected from any one of substituted or unsubstituted uracil, thymine, cytosine, adenine, and guanine; R 1 In this configuration, the nitrogen linked to form an optionally substituted heterocycle, including pyrrolidine, piperazine, or morpholine; R 2 In this context, the optional or unsubstituted tertiary alkyl group includes tert-butyl, triphenylmethyl, and substituted triphenylmethyl; the optional substituted benzyl group includes 4-methoxybenzyl, 3,4-dimethoxybenzyl, diphenylmethyl, and 4-methoxybenzyl; the optional substituted sulfonyl group includes 2-nitrobenzenesulfonyl and p-toluenesulfonyl; and the optional substituted acyl group includes benzyloxycarbonyl, tert-butoxycarbonyl, phenoxyacetyl, and 9-fluorenemethoxycarbonyl.

3. The chiral morpholine phosphoryl chloride monomer according to claim 1 or 2, characterized in that, The specific structure of the monomer is shown in formulas I-1, I-2, I-3, I-4, II-1, II-2, II-3, or II-4 as follows: ； Among them, R 3 Selected from H, optionally substituted alkyl or alkoxy groups, and optionally substituted halogens; R 4 R 5 R 6 and R 7 Each is selected from -H, -C(O)R 10 and -C(O)OR 10 Any of the following, where R 10 It is alkyl or aryl; R 8 R 9 Each is selected from any one of optionally substituted alkyl, cyanoethyl, acyl, carbonate, carbamate, optionally substituted benzyl, 4-pentanoyloxybenzyl and silyl.

4. A method for preparing a chiral morpholine phosphoryl chloride monomer according to any one of claims 1-3, characterized in that, Includes the following steps: Under the conditions of organic solvent, base, chiral ligand and copper catalyst, the compound with the structure shown in Formula III is reacted with the compound with the structure shown in Formula IV to generate a chiral morpholinophosphoryl chloride monomer. The structures shown in Equation III and Equation IV are as follows: ； In Formula III, X represents an oxygen atom or a sulfur atom; R 1 It is selected from any one of the following: a optionally substituted secondary substituted amino group, a optionally substituted heterocycle formed by the nitrogen attached thereto, an optionally substituted alkoxy group, an optionally substituted phenoxy group, and a guanidine group; In formula IV, B represents a base; R 2 It is selected from any one of the optional substituted or unsubstituted tertiary alkyl, optional substituted benzyl, optional substituted sulfonyl, and optional substituted acyl.

5. The method for preparing chiral morpholine phosphoryl chloride monomer according to claim 4, characterized in that, The organic solvent is selected from at least one of tetrahydrofuran, 2-methyltetrahydrofuran, ethyl acetate, ethylene glycol dimethyl ether, and diethyl ether; The base is selected from at least one of triethylamine, tributylamine, diisopropylethylamine, and 1,2,2,6,6-pentamethylpiperidine; The copper catalyst is selected from at least one of copper hexafluoroacetylacetonate, copper acetate, copper chloride, copper bromide, and cuprous bromide; The chiral ligand is selected from pyridine imidazole ligands.

6. The method for preparing chiral morpholine phosphoryl chloride monomer according to claim 5, characterized in that, The structure of the pyridimazole ligand is as follows: （R） -L、 （S） -L、( S -L2、( S -L3、( S -L4、( S -L5、( S -L6、( S -L7、( S As shown in -L10: 。 7. The method for preparing chiral morpholine phosphoryl chloride monomer according to claim 4, 5, or 6, characterized in that, The molar ratio of the compound with the structure shown in Formula III to the compound with the structure shown in Formula IV is 3:1; and / or The molar ratio of the copper catalyst to the morpholine nucleoside represented by Formula IV is 0.01-0.1:1; and / or The molar ratio of the base to the compound with the structure shown in Formula IV is 1-5:1; and / or The molar ratio of the chiral ligand to the morpholinonucleotide shown in Formula IV is 0.02-0.15:

1.

8. The method for preparing chiral morpholine phosphoryl chloride monomer according to claim 4, characterized in that, The reaction temperature is -30℃ to -50℃, and the reaction time is 4 to 24 hours.

9. The use of a chiral morpholinophosphoryl chloride monomer according to any one of claims 1-3 or a chiral morpholinophosphoryl chloride monomer prepared by the method according to any one of claims 4-8 in the synthesis of morpholino oligonucleotide chains.

10. A method for preparing a morpholine oligonucleotide chain, characterized in that, Includes the following steps: S1. Deprotect the protecting group Tr on the linker derived from aminomethyl polystyrene resin to obtain a free secondary amine; S2. The free secondary amine is coupled with the chiral morpholinophosphoryl chloride monomer to obtain the morpholino oligonucleotide dimer; S3. After capping with a capping reagent, remove the protecting group on the morpholine oligonucleotide dimer to obtain the deprotected morpholine oligonucleotide dimer. S4. Repeat steps S2 and S3 for the next round of coupling, end-capping and deprotection steps until the target oligomer crude product is obtained. S5. After purifying the crude target oligomer, morpholine oligonucleotide chains are separated.

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