Oligonucleotide synthesis process of chimeric PMO structure

Abstract

A PMO molecular structural formula adopted in the oligonucleotide synthesis process of the chimeric PMO structure is # imgabs0 #. According to the oligonucleotide synthesis process of the chimeric PMO structure, through specific limitation of the PMO molecular structure, synthesis of nucleotide (phosphate) amide in solid-phase synthesis of oligonucleotide is carried out in the direction from 5'to 3 ', that is, in each cycle period, the synthesis time of the oligonucleotide is shortened, and the yield of the oligonucleotide is increased. The O5'or O6 'position of the nucleotide (phosphoryl) amide monomer is firstly subjected to a coupling reaction, then a protecting group is removed at the O3' or N3 'position, active hydroxyl or amino / amido is released, and the next cycle is carried out; in addition, the PMO molecule adopts DMTr as a protecting group which is consistent with protecting groups of other nucleotides, so that the conversion rate of each coupling reaction step can be conveniently detected on line by using chromaticity in the coupling reaction process.

Description

Technical Field

[0001] The invention relates to the technical field of solid phase oligonucleic acid synthesis, and in particular to a process for synthesizing oligonucleic acid with a chimeric PMO structure. Background Art

[0002] Phosphorodiamidate morpholino oligonucleotide (PMO) is a new generation of chemical modification of nucleic acid drugs. PMO uses a morpholine ring instead of a deoxyribose or ribose structure and a phosphorodiamidate bridge as a connecting unit. The electrically neutral morpholine phosphorodiamidate replaces the negatively charged phosphate group of PMO, showing high binding affinity and strong stability against enzymatic hydrolysis. As a structural unit for chemical modification of nucleic acid drugs, PMO has great potential for drug development.

[0003] At present, the PMO protecting group is trityl Tr, and the maximum absorption wavelength of the removed protecting group Tr cation is about 400-435nm. This type of nucleotide unit is coupled on a solid phase synthesizer to prepare a nucleic acid molecule of the target sequence. Other nucleotides commonly use the protecting group dimethoxytrityl DMTr, and the maximum absorption wavelength of the cation is about 490-515nm. There are differences in the absorption coefficients of the two protecting groups of trityl Tr and dimethoxytrityl DMTr at specific wavelengths. The coupling efficiency of each step will be inconsistent when using light of a certain wavelength for colorimetric detection; in addition, the traditional solid phase synthesis of nucleotide phosphoamidophosphite, the synthesis of oligonucleotides is carried out along the 3' to 5' direction, and only morpholine rings can be used in the synthetic sequence instead of deoxyribose structures, and the coexistence of morpholine rings and deoxyribose structures cannot be achieved.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] The object of the present invention is to provide a process for synthesizing an oligonucleic acid with a chimeric PMO structure. The synthesis process of the present invention is carried out along the 5' to 3' direction in the solid phase synthesis of oligonucleic acid by specifically limiting the molecular structure of the PMO. That is, in each cycle, the O5' or O6' position of the nucleotide (hypo)phosphoamide monomer is first subjected to a coupling reaction, and then the protecting group is deprotected at the O3' or N3' position to release the active hydroxyl group or amino group / amine group to enter the next cycle; and the PMO molecule of the present application adopts DMTr as a protecting group, which is consistent with the protecting groups of other nucleotides, so that the conversion rate of each step of the coupling reaction can be detected online by colorimetry.

[0006] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0007] The first aspect of the present invention provides a chimeric PMO structure oligonucleic acid synthesis process, wherein the PMO molecular structure used in the chimeric PMO structure oligonucleic acid synthesis process is shown in Formula I:

[0008]

[0009] In Formula I, Base is adenine (A), guanine (G), cytosine (C), thymine (T), uracil (U) or hypoxanthine (I) containing a protecting group;

[0010] R1 and R2 are independently chlorine, bromine, fluorine, hydroxy, alkoxy, alkenyloxy, amino / amine, alkylamino, alkenylamino, cycloalkoxy, cycloalkylamino or alkoxyalkoxy;

[0011] R3 is hydrogen, fluorine, methoxy, alkoxy, alkenyloxy, silyloxymethyleneoxy or alkoxyalkoxy;

[0012] R4 is trityl (Tr), (4-methoxyphenyl)diphenylmethyl (MMTr), 4,4'-dimethoxytrityl (DMTr) or 4,4',4"-trimethoxyphenyl (TMTr).

[0013] Preferably, the protecting group is selected from one of 4,4'-dimethoxytrityl, trityl, allyl, tert-butyldimethylsilyl and tert-butyldiphenylsilyl.

[0014] Preferably, the PMO molecular structure is

[0015] Preferably, the nucleotide monomers used in the oligonucleic acid synthesis process of the chimeric PMO structure include at least one of Formula II to Formula V;

[0016]

[0017] In formula II, Base is adenine, guanine, cytosine, thymine, uracil or hypoxanthine containing a protecting group;

[0018] PG is trityl (Tr), (4-methoxyphenyl) benzhydryl (MMTr), 4,4'-dimethoxytrityl (DMTr), 4,4',4"-trimethoxyphenyl (TMTr), allyl (Allyl), allyloxycarbonyl (Alloc), or trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), tert-butyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS);

[0019] X1 and X2 are independently hydrogen, fluorine, methoxy, alkoxy, alkenyloxy, silyloxymethyleneoxy or alkoxyalkoxy;

[0020] LG1 is a secondary amine, fluorine, chlorine or bromine;

[0021] LG2 is cyanoethyl, cyanopropyl, 3-cyanoisobutyl, (CH3)2CHCH2CN, phenethyl or allyl;

[0022]

[0023] In formula III, Base is adenine, guanine, cytosine, thymine, uracil or hypoxanthine containing a protecting group;

[0024] PG is trityl, (4-methoxyphenyl)benzhydryl, 4,4'-dimethoxytrityl, 4,4',4"-trimethoxytrityl, allyl, allyloxycarbonyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl or tert-butyldiphenylsilyl;

[0025] R1, R2 and R3 are alkyl, alkenyl, cycloalkyl or aromatic groups;

[0026] LG1 is a secondary amine, fluorine, chlorine or bromine;

[0027] LG2 is cyanoethyl, cyanopropyl, 3-cyanoisobutyl, (CH3)2CHCH2CN, phenethyl or allyl;

[0028]

[0029] In formula IV, Base is adenine, guanine, cytosine, thymine, uracil or hypoxanthine containing a protecting group;

[0030] PG is trityl, (4-methoxyphenyl)benzhydryl, 4,4'-dimethoxytrityl, 4,4',4"-trimethoxytrityl, allyl, allyloxycarbonyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl or tert-butyldiphenylsilyl;

[0031] R1 and R2 are alkyl, alkenyl, cycloalkyl or aromatic

[0032] R3 and R4 are independently hydrogen, fluorine, methoxy, alkoxy, alkenyloxy, silyloxymethyleneoxy or alkoxyalkoxy;

[0033] X is fluorine, chlorine, bromine, hydroxy, alkoxy, alkenyloxy, amino, alkylamino, cycloalkyloxy or cycloalkylamino;

[0034]

[0035] In Formula V, Base is adenine, guanine, cytosine, thymine, uracil or hypoxanthine containing a protecting group;

[0036] PG is trityl, (4-methoxyphenyl)benzhydryl, 4,4'-dimethoxytrityl, 4,4',4"-trimethoxytrityl, allyl, allyloxycarbonyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl or tert-butyldiphenylsilyl;

[0037] R1, R2, R3, R4 and R5 are alkyl, alkenyl, cycloalkyl or aromatic groups respectively;

[0038] X is fluorine, chlorine, bromine, hydroxy, alkoxy, alkenyloxy, amino, alkylamino, cycloalkyloxy or cycloalkylamino.

[0039] Preferably, the protecting group is selected from one of 4,4'-dimethoxytrityl, trityl, allyl, tert-butyldimethylsilyl and tert-butyldiphenylsilyl.

[0040] Preferably, the nucleotide monomers include

[0041]

[0042]

[0043] In the nucleotide monomer, R is hydrogen, fluorine, methoxy, alkoxy, alkenyloxy, silyloxymethyleneoxy or alkoxyalkoxy.

[0044] Preferably, the nucleotide monomer is

[0045] The preparation method comprises the following steps: linking a pentavalent phosphorus compound at the O5' position; connecting a protective group PG at the O3' position, wherein the protective group PG is (4-methoxyphenyl)diphenylmethyl, triphenylmethyl, 4,4'-dimethoxytrityl, 4,4',4"-trimethoxytrityl, allyl, allyloxycarbonyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl or tert-butyldiphenylsilyl;

[0046] In the nucleotide monomer, R is hydrogen, fluorine, methoxy, alkoxy, alkenyloxy, silyloxymethyleneoxy or alkoxyalkoxy.

[0047] The second aspect of the present invention provides a chimeric PMO structured oligonucleotide, wherein the chimeric PMO structured oligonucleotide comprises a structure shown in Formula VI or VII;

[0048]

[0049] In Formula VI and Formula VII, Base is adenine, guanine, cytosine, thymine, uracil or hypoxanthine containing a protecting group;

[0050] R1 and R2 are independently alkyl, alkenyl or cycloalkyl;

[0051] X1 and X2 are independently hydrogen, fluorine, methoxy, alkoxy, alkenyloxy, silyloxymethyleneoxy or alkoxyalkoxy.

[0052] Compared with the prior art, the beneficial effects of the present invention include at least:

[0053] The present invention specifically defines the PMO molecular structure, and the synthesis of nucleotide (hypo)phosphoamide in solid phase oligonucleic acid synthesis is carried out along the 5' to 3' direction, that is, in each cycle, the O5' or O6' position of the nucleotide (hypo)phosphoamide monomer is first subjected to coupling reaction, and then the protecting group is deprotected at the O3' or N3' position to release the active hydroxyl or amino / amine group to enter the next cycle; and the PMO molecule of the present application adopts DMTr as the protecting group, which is consistent with the protecting groups of other nucleotides, so that the conversion rate of each step of the coupling reaction can be detected online by colorimetry. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following is a brief introduction to the drawings required for the specific embodiments or the description of the prior art. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the elements or parts are not necessarily drawn according to the actual scale.

[0055] Figure 1 is a flow chart of the solid phase synthesis of oligonucleic acids of the present invention;

[0056] Figure 2 The structural diagram of the solid phase synthesis nucleic acid sequence TTTApTT of the present invention and the coupling efficiency of each step;

[0057] Figure 3 The structural diagram of the solid phase synthesis nucleic acid sequence TTTGpTT of the present invention and the coupling efficiency of each step;

[0058] Figure 4 The structural diagram of the solid phase synthesis nucleic acid sequence TTTCpTT of the present invention and the coupling efficiency of each step;

[0059] Figure 5 The structural diagram of the solid phase synthesized nucleic acid sequence TTTUpTT of the present invention and the coupling efficiency of each step;

[0060] Figure 6 The structural diagram of the solid phase synthesized nucleic acid sequence TTTpTpTT of the present invention and the coupling efficiency of each step;

[0061] Figure 7 The structural diagram of the solid phase synthesis nucleic acid sequence TTTIpTT of the present invention and the coupling efficiency of each step;

[0062] Figure 8 The structural diagram of the solid phase synthesis nucleic acid sequence UUUApUU of the present invention and the coupling efficiency of each step;

[0063] Fig. 9 The structural diagram of the solid phase synthesis nucleic acid sequence UUUGpUU of the present invention and the coupling efficiency of each step;

[0064] Fig.10 The structural diagram of the solid phase synthesis nucleic acid sequence UUUCpUU of the present invention and the coupling efficiency of each step;

[0065] Fig.11 The structural diagram of the solid phase synthesis nucleic acid sequence UUUUpUpU of the present invention and the coupling efficiency of each step;

[0066] Fig.12 The structural diagram of the solid phase synthesized nucleic acid sequence UUUTpUU of the present invention and the coupling efficiency of each step;

[0067] Fig.13 The diagram shows the structure of the solid phase synthesized nucleic acid sequence UUUIpUU of the present invention and the coupling efficiency of each step. DETAILED DESCRIPTION

[0068] The following embodiments of the technical solution of the present invention are described in detail in conjunction with the embodiments. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and are therefore only used as examples, and cannot be used to limit the scope of protection of the present invention.

[0069] It should be noted that, unless otherwise specified, the technical terms or scientific terms used in this application should have the common meanings understood by those skilled in the art to which the invention belongs.

[0070] The substances corresponding to the abbreviations of letters in the present invention are as follows:

[0071] PMO: phosphorodiamidate morpholinooligonucleotide;

[0072] DBU: 1,8-diazobispiro[5.4.0]undec-7-ene;

[0073] CPG: controlled pore glass;

[0074] PS: polystyrene; DMI: 1,3-dimethyl-2-imidazolidinone;

[0075] DIEA: N,N-diisopropylethylamine; NMP: N-methylpyrrolidone;

[0076] Alkyl: alkyl; Bz: benzoyl;

[0077] Allyl: allyl; DMTr: dimethoxytrityl;

[0078] Alloc: allyloxycarbonyl; MMTr: methoxytrityl;

[0079] Ph: aromatic group; DMAP: 4-dimethylaminopyridine;

[0080] TBDMS: tert-butyldimethylsilyl; THF: tetrahydrofuran;

[0081] TBDPS: tert-butyldiphenylsilyl; TFA: trifluoroacetic acid;

[0082] Tr: trityl; TFE: 2,2,2-trifluoroethanol;

[0083] DCM: dichloromethane; iPrOH: isopropanol;

[0084] DMF: N,N-dimethylformamide; TLC: thin layer chromatography;

[0085] A:Adenine; Ap:APMO;

[0086] G: Guanine; Gp: G PMO;

[0087] C:Cytosine; Cp:C PMO;

[0088] T:Thymine; Up:UPMO;

[0089] U:Uracil; Tp:T PMO;

[0090] I: hypoxanthine; Ip: IPMO.

[0091] Example 1

[0092] This embodiment is a PMO molecule for solid phase synthesis of oligonucleic acid, and the PMO molecule structure is

[0093] The synthetic route of the above PMO molecule is:

[0094]

[0095] Specific synthesis methods include:

[0096] 1.1. Synthesis of N3'-DMTr-O6'-TBDPS-N6-Bz-A-PMO;

[0097] Synthesis method 1:

[0098] The preparation and synthesis of O6′-TBDPS-N6-Bz-A-PMO monomer refer to (Journal of Medicinal Chemistry, 2015, vol. 58, #18, p. 7349-7369).

[0099] 5.93 g of O6'-TBDPS-N6-Bz-A-PMO (10 mmol, 1.0 eq.), 3.73 g of 4,4'-dimethoxytrityl chloride (DMTrCl, 11 mmol, 1.1 eq.), and 0.12 g of DMAP (1 mmol, 0.1 eq.) were stirred and mixed in 30 ml of dry dichloromethane, and the temperature was controlled at 0-5°C. Under nitrogen protection, 2.78 ml of triethylamine (20 mmol, 1.0 eq.) was added dropwise to the reaction solution, and the mixture was stirred and slowly heated to room temperature for more than 6 hours. According to TLC detection, the PMO monomer reaction was complete. 50 ml of saturated sodium bicarbonate aqueous solution was added, the organic layer was separated, and the aqueous layer was extracted twice with 30 ml of dichloromethane. The mixed organic layer was washed with saturated brine, dried over solid sodium sulfate, and concentrated. 7.70 g of the target product N3'-DMTr-O6'-TBDPS-N6-Bz-A-PMO (purity above 98%, yield 86%) was obtained by silica gel column chromatography.

[0100] Synthesis method 2:

[0101] The preparation and synthesis of O6′-TBDPS-A-PMO monomers refer to the literature (Tetrahedron Letters, 2011, vol. 52, #1, p. 34–37).

[0102] 4.89 g of O6'-TBDPS-A-PMO (10 mmol, 1.0 eq.), 3.73 g of 4,4'-dimethoxytrityl chloride (DMTrCl, 11 mmol, 1.1 eq.), and 0.12 g of DMAP (1 mmol, 0.1 eq.) were stirred and mixed in 20 ml of dry DMF, and the temperature was controlled at 0-5°C. Under nitrogen protection, 2.78 ml of triethylamine (20 mmol, 1.0 eq.) was added dropwise to the reaction solution, and stirring was continued, and the reaction was slowly heated to room temperature for more than 6 hours. TLC detection showed that the PMO monomer reaction was complete. Most of the solvent was removed under reduced pressure, 50 ml of saturated sodium bicarbonate aqueous solution was added to the residue, and extracted three times with 30 ml of dichloromethane. The organic layer was washed with saturated brine, dried over solid sodium sulfate, and concentrated, and 5.98 g of the intermediate was obtained by silica gel column chromatography;

[0103] The intermediate was dissolved in 30 ml of dry dichloromethane, and 2.11 ml of triethylamine (15.2 mmol, 2.0 eq.) was slowly added to the reaction solution, and the temperature was controlled at 0-5°C. Under nitrogen protection, 1.44 g of benzoyl chloride (10.27 mmol, 1.35 eq.) was stirred and slowly reacted at room temperature for more than 4 hours. TLC detection showed that the PMO intermediate was completely reacted. 50 ml of saturated sodium bicarbonate aqueous solution was added, the organic layer was separated, and the aqueous layer was extracted twice with 30 ml of dichloromethane. The mixed organic layer was washed with saturated brine, dried over solid sodium sulfate, and concentrated. 5.38 g of the target product N3'-DMTr-O6'-TBDPS-N6-Bz-A-PMO (purity above 98%, yield of two-step reaction 60.1%) was obtained by silica gel column chromatography.

[0104] 1.2. Synthesis of N3'-DMTr-N6-Bz-A-PMO:

[0105] 4.48 g N3'-DMTr-O6'-TBDPS-N6-Bz-A-PMO (5 mmol, 1.0 eq.) was dissolved in 40 ml tetrahydrofuran and stirred to control the temperature between 0-5°C. Under nitrogen protection, 10 ml tetrabutylammonium fluoride tetrahydrofuran solution (TBAF, 1.0 mol / L) was slowly added dropwise to the mixed solution. The mixture was slowly warmed to room temperature and stirred overnight. TLC detection showed that the starting material completely disappeared. The solvent was removed under reduced pressure, 50 ml saturated sodium bicarbonate aqueous solution was added to the residue, and the mixture was extracted three times with 30 ml dichloromethane. The mixed organic layer was washed with saturated brine, dried over solid sodium sulfate, and concentrated. 2.51 g solid N3'-DMTr-N6-Bz-A-PMO (purity above 98%, yield 76%) was obtained by silica gel column chromatography.

[0106] 1.3. Synthesis of N3'-DMTr-N6-Bz-A-PMO phosphoramidate:

[0107] Under nitrogen protection, 263 mg of N3'-DMTr-N6-Bz-A-PMO (0.4 mmol, 1.0 eq.) was added to a dry mixed solvent (3 ml of dichloromethane and 3 ml of acetonitrile), the temperature was controlled at 0°C, 70 mg of lithium bromide (0.8 mmol, 2.0 eq.) was added, and the mixture was stirred for 5 minutes. 0.12 ml of DBU (0.8 mmol, 2.0 eq.) and 0.08 ml of NN-dimethylphosphoamino dichloride (0.6 mmol, 1.5 eq.) dissolved in 0.5 ml of dry dichloromethane were added in sequence. Under nitrogen protection, the mixed solution was stirred at 0°C for 40 minutes. The reaction solvent was removed under reduced pressure, 30 ml of ethyl acetate was added to the residue, and after washing with 10 ml of cold water, the organic phase was dried over sodium sulfate and concentrated under reduced pressure, separated by silica gel chromatography, and the mobile phase was selected as a mixed solvent of dichloromethane / acetone / methanol to obtain 216 mg of the target product (purity above 99%, yield 69%).

[0108] Example 2

[0109] This embodiment is a PMO molecule for solid phase synthesis of oligonucleic acid, and the PMO molecule structure is

[0110] The synthetic route of the above PMO molecule is:

[0111]

[0112] Specific synthesis methods include:

[0113] 1.1. Synthesis of N3'-DMTr-O6'-TBDPS--N2-iBu-G-PMO:

[0114] The preparation and synthesis of O6'-TBDPS-G-PMO monomer refer to the literature (Nucleosides, Nucleotides & Nucleic Acids, 31(11), 763–782).

[0115] 5.05 g of O6'-TBDPS-G-PMO (10 mmol, 1.0 eq.), 3.73 g of 4,4'-dimethoxytrityl chloride (DMTrCl, 11 mmol, 1.1 eq.), and 0.12 g of DMAP (1 mmol, 0.1 eq.) were stirred and mixed in 20 ml of dry DMF, and the temperature was controlled at 0-5°C. Under nitrogen protection, 2.78 ml of triethylamine (20 mmol, 1.0 eq.) was added dropwise to the reaction solution, and stirring was continued. The reaction was slowly heated to room temperature for more than 6 hours. TLC detection showed that the PMO monomer reaction was complete. Most of the solvent was removed under reduced pressure, and 50 ml of saturated sodium bicarbonate aqueous solution was added to the residue, and extracted three times with 30 ml of dichloromethane. The mixed organic layer was washed with saturated brine, dried over solid sodium sulfate, concentrated, and silica gel column chromatography was performed to obtain the intermediate.

[0116] The intermediate was dissolved in 30 ml of dry pyridine and stirred, and 0.10 g of DMAP was added, and the temperature was controlled at 0-5°C. Under nitrogen protection, 1.28 g of isobutyryl chloride (12 mmol, 1.2 eq.) was slowly added, and stirring was continued. The reaction was slowly allowed to rise to room temperature overnight. TLC detection showed that the PMO intermediate was completely reacted. The organic solvent was removed by distillation under reduced pressure, and 50 ml of saturated sodium bicarbonate aqueous solution was added to the residue. The organic layer was separated, and the aqueous layer was extracted twice with 50 ml of dichloromethane. The mixed organic layer was washed with saturated brine, dried over solid sodium sulfate, and concentrated. 4.93 g of the target product N3'-DMTr-O6'-TBDPS-N2-iBu-G-PMO (purity above 98%, yield of two-step reaction 56%) was obtained by silica gel column chromatography.

[0117] 1.2. Synthesis of N3'-DMTr-N2-iBu-G-PMO:

[0118] 4.39 g of N3'-DMTr-O6'-TBDPS-N2-iBu-G-PMO (5 mmol, 1.0 eq.) was dissolved in 40 ml of tetrahydrofuran and stirred to control the temperature between 0-5°C. Under nitrogen protection, 10 ml of tetrabutylammonium fluoride tetrahydrofuran solution (TBAF, 1.0 mol / L) was slowly added dropwise to the mixed solution. The mixture was slowly warmed to room temperature and stirred overnight. TLC detection showed that the starting material completely disappeared. The solvent was removed under reduced pressure, 50 ml of saturated sodium bicarbonate aqueous solution was added to the residue, and the mixture was extracted three times with 30 ml of dichloromethane. The mixed organic layer was washed with saturated brine, dried over solid sodium sulfate, and concentrated. 2.63 g of solid N3'-DMTr-N2-iBu-G-PMO (purity above 98%, yield 82%) was obtained by silica gel column chromatography.

[0119] 1.3. Synthesis of N3'-DMTr-N2-iBu-G-PMO phosphoramidate:

[0120] Under nitrogen protection, 256 mg of N3'-DMTr-N6-Bz-A-PMO (0.4 mmol, 1.0 eq.) was added to a dry mixed solvent (3 ml of dichloromethane and 3 ml of acetonitrile), the temperature was controlled at 0°C, 70 mg of lithium bromide (0.8 mmol, 2.0 eq.) was added, and the mixture was stirred for 5 minutes. 0.12 ml of DBU (0.8 mmol, 2.0 eq.) and 0.08 ml of NN-dimethylphosphoamino dichloride (0.6 mmol, 1.5 eq.) dissolved in 0.5 ml of dry dichloromethane were added in sequence. Under nitrogen protection, the mixed solution was stirred at 0°C for 40 minutes. The reaction solvent was removed under reduced pressure, 30 ml of ethyl acetate was added to the residue, and after washing with 10 ml of cold water, the organic phase was dried over sodium sulfate and concentrated under reduced pressure, separated by silica gel chromatography, and the mobile phase was selected as a mixed solvent of dichloromethane / acetone / methanol to obtain 188 mg of the target product (purity above 99%, yield 61%).

[0121] Example 3

[0122] This embodiment is a PMO molecule for solid phase synthesis of oligonucleic acid, and the PMO molecule structure is

[0123] The synthetic route of the above PMO molecule is:

[0124]

[0125] Specific synthesis methods include:

[0126] 1.1. Synthesis of N3'-DMTr-O6'-TBDPS-N2-iBu-O6-NPE-G-PMO:

[0127] 4.39 g of N3'-DMTr-O6'-TBDPS-N2-iBu-G-PMO (5 mmol, 1.0 eq.) was dissolved in 30 ml of dry 1,4-dioxane, and 1.7 ml of trimethylsilyl imidazole (11.5 mmol, 2.3 eq.) was added and stirred for 15 minutes. Under nitrogen protection, 4.33 g of triphenylphosphine (16.5 mmol, 3.3 eq.), 2.76 g of 4-nitrophenylethanol (16.5 mmol, 3.3 eq.) and 3.3 ml of diisopropyl azodicarboxylate (DIAD, 16.5 mmol, 3.3 eq.) were added in sequence. The temperature was controlled at 40°C and the reaction was continued with stirring for 2 hours. According to TLC detection, the starting material completely disappeared. The solvent was removed under reduced pressure, 50 ml of saturated sodium bicarbonate aqueous solution was added to the residue, and extracted three times with 30 ml of dichloromethane. The mixed organic layer was washed with saturated brine, dried over solid sodium sulfate and concentrated. 3.91 g of solid N3'-DMTr-O6'-TBDPS-N2-iBu-O6-NPE-G-PMO (purity above 97%, yield 76%) was obtained by silica gel column chromatography.

[0128] 1.2. Synthesis of N3'-DMTr-N2-iBu-O6-NPE-G-PMO:

[0129] 3.08 g of N3'-DMTr-O6'-TBDPS-N2-iBu-O6-NPE-G-PMO (3 mmol, 1.0 eq.) was dissolved in 30 ml of tetrahydrofuran and stirred to control the temperature between 0-5°C. Under nitrogen protection, 6 ml of tetrabutylammonium fluoride tetrahydrofuran solution (TBAF, 2.0 mol / L) was slowly added dropwise to the mixed solution. The mixture was slowly warmed to room temperature and stirred overnight. TLC detection showed that the starting material completely disappeared. The solvent was removed under reduced pressure, 50 ml of saturated sodium bicarbonate aqueous solution was added to the residue, and the mixture was extracted three times with 30 ml of dichloromethane. The mixed organic layer was washed with saturated brine, dried over solid sodium sulfate, and concentrated. 1.90 g of solid N3'-DMTr-N2-iBu-O6-NPE-G-PMO (purity above 98%, yield 80%) was obtained by silica gel column chromatography.

[0130] 1.3. Synthesis of N3'-DMTr-N2-iBu-O6-NPE-G-PMO phosphoramidate:

[0131] Under nitrogen protection, 316 mg of N3'-DMTr-N6-Bz-A-PMO (0.4 mmol, 1.0 eq.) was added to a dry mixed solvent (3 ml of dichloromethane and 3 ml of acetonitrile), the temperature was controlled at 0°C, 70 mg of lithium bromide (0.8 mmol, 2.0 eq.) was added, and the mixture was stirred for 5 minutes. 0.12 ml of DBU (0.8 mmol, 2.0 eq.) and 0.08 ml of NN-dimethylphosphoamino dichloride (0.6 mmol, 1.5 eq.) dissolved in 0.5 ml of dry dichloromethane were added in sequence. Under nitrogen protection, the mixed solution was stirred at 0°C for 40 minutes. The reaction solvent was removed under reduced pressure, 30 ml of ethyl acetate was added to the residue, and after washing with 10 ml of cold water, the organic phase was dried over sodium sulfate and concentrated under reduced pressure, separated by silica gel chromatography, and the mobile phase was selected as a mixed solvent of dichloromethane / acetone / methanol to obtain 241 mg of the target product (purity above 99%, yield 66%).

[0132] According to the above similar method, the structural formula can be prepared PMO molecules, wherein PG1 is CH3CO, (CH3)2CHCO, CHN(CH3)2, PhCO, PhCH2CO, PhOCH2CO or Alloc; PG2 is Ph(CH2) n , specifically including Bn and p-tolyl pivalate.

[0133] Example 4

[0134] This embodiment is a PMO molecule for solid phase synthesis of oligonucleic acid, and the PMO molecule structure is

[0135] The synthetic route of the above PMO molecule is:

[0136]

[0137] Specific synthesis methods include:

[0138] 1.1. Synthesis of N3'-DMTr-O6'-TBDPS-C-PMO:

[0139] The preparation and synthesis of O6′-TBDPS-C-PMO monomers were based on the literature (Nucleosides, nucleotides and nucleic acids, 2012, vol. 31, #11, p. 763–782).

[0140] 4.65 g O6'-TBDPS-C-PMO (10 mmol, 1.0 eq.), 3.73 g 4,4'-bismethoxytrityl chloride (DMTrCl, 11 mmol, 1.1 eq.), 0.12 g DMAP (1 mmol, 0.1 eq.) were stirred and mixed in 20 ml dry DMF, and the temperature was controlled at 0-5 °C. Under nitrogen protection, 2.78 ml triethylamine (20 mmol, 1.0 eq.) was added dropwise to the reaction solution, and the mixture was stirred and slowly heated to room temperature for more than 6 hours. TLC was used to monitor the reaction progress, and the PMO monomer was completely reacted. Most of the solvent was removed under reduced pressure, and 50 ml saturated sodium bicarbonate aqueous solution was added to the residue, and the mixture was extracted three times with 30 ml dichloromethane. The mixed organic layer was washed with saturated brine, dried over solid sodium sulfate, and concentrated, and 6.05 g N3'-DMTr-O6'-TBDPS-C-PMO (purity above 98%, yield 79%) was obtained by silica gel column chromatography.

[0141] 1.2. Synthesis of N3'-DMTr-O6'-TBDPS-N4-Bz-C-PMO:

[0142] 5.37 g of N3'-DMTr-O6'-TBDPS-C-PMO (7 mmol, 1.0 eq.) was dissolved in 30 ml of dry dichloromethane and stirred to mix. 1.94 ml of triethylamine (14 mmol, 2.0 eq.) was slowly added to the reaction solution, and the temperature was controlled at 0-5°C. Under nitrogen protection, 1.33 g of benzoyl chloride (9.45 mmol, 1.35 eq.) was continued to be stirred and mixed, and slowly reacted at room temperature for more than 4 hours. TLC was used to detect the progress of the reaction, and the PMO intermediate was completely reacted. 50 ml of saturated sodium bicarbonate aqueous solution was added, the organic layer was separated, and the aqueous layer was continuously extracted twice with 30 ml of dichloromethane. The mixed organic layer was washed with saturated brine, dried over solid sodium sulfate, and concentrated. 5.25 g of the target product N3'-DMTr-O6'-TBDPS-N4-Bz-C-PMO (purity above 98%, yield 86%) was obtained by silica gel column chromatography.

[0143] 1.3. Synthesis of N3'-DMTr-N4-Bz-C-PMO:

[0144] 4.36 g of N3'-DMTr-O6'-TBDPS-N4-Bz-C-PMO (5 mmol, 1.0 eq.) was dissolved in 40 ml of tetrahydrofuran and stirred to control the temperature between 0-5°C. Under nitrogen protection, 10 ml of tetrabutylammonium fluoride tetrahydrofuran solution (TBAF, 1.0 mol / L) was slowly added dropwise to the mixed solution. The mixture was slowly warmed to room temperature and stirred overnight. TLC detection showed that the starting material completely disappeared. The solvent was removed under reduced pressure, 50 ml of saturated sodium bicarbonate aqueous solution was added to the residue, and the mixture was extracted three times with 30 ml of dichloromethane. The mixed organic layer was washed with saturated brine, dried over solid sodium sulfate, and concentrated. 2.24 g of solid N3'-DMTr-N4-Bz-C-PMO (purity above 98%, yield 71%) was obtained by silica gel column chromatography.

[0145] 1.4. Synthesis of N3'-DMTr-N4-Bz-C-PMO phosphoramidate:

[0146] Under nitrogen protection, 254 mg of N3'-DMTr-N6-Bz-A-PMO (0.4 mmol, 1.0 eq.) was added to a dry mixed solvent (3 ml of dichloromethane and 3 ml of acetonitrile), the temperature was controlled at 0°C, 70 mg of lithium bromide (0.8 mmol, 2.0 eq.) was added, and the mixture was stirred for 5 minutes. 0.12 ml of DBU (0.8 mmol, 2.0 eq.) and 0.08 ml of NN-dimethylphosphoamino dichloride (0.6 mmol, 1.5 eq.) dissolved in 0.5 ml of dry dichloromethane were added in sequence. Under nitrogen protection, the mixed solution was stirred at 0°C for 40 minutes. The reaction solvent was removed under reduced pressure, 30 ml of ethyl acetate was added to the residue, and after washing with 10 ml of cold water, the organic phase was dried over sodium sulfate and concentrated under reduced pressure, separated by silica gel chromatography, and the mobile phase was selected as a mixed solvent of dichloromethane / acetone / methanol to obtain 195 mg of the target product (purity above 99%, yield 64%).

[0147] Example 5

[0148] This embodiment is a PMO molecule for solid phase synthesis of oligonucleic acid, and the PMO molecule structure is

[0149] The synthetic route of the above PMO molecule is:

[0150]

[0151] Specific synthesis methods include:

[0152] 1.1. Synthesis of O6'-TBDPS-U-PMO:

[0153] The synthetic preparation reference of 1-((2R,6S)-6-(hydroxymethyl)morpholin-2-yl)pyrimidine-2,4(1H,3H)-dione (CAS: 109205-43-8) is Ref: Organic Preparations and Procedures International, 2018, vol.50, #3, p.332–342; Helvetica Chimica Acta, 2008, vol.91, #1, p.1–20.

[0154] 2.27 g of 1-((2R,6S)-6-(hydroxymethyl)morpholin-2-yl)pyrimidine-2,4(1H,3H)-dione (10 mmol, 1.0 eq.) and 1.50 g of imidazole (22 mmol, 2.2 eq.) were added to 15 ml of dry DMF and stirred and mixed, and the temperature was controlled at 0-5°C. Under nitrogen protection, 1.88 g of tert-butyldimethylsilyl chloride (TBDMSCl, 12.5 mmol, 1.25 eq.) was added to the reaction solution, and stirring and mixing were continued. The mixture was slowly heated to room temperature for more than 6 hours. TLC was used to monitor the progress of the reaction, and the PMO monomer reaction was complete. Most of the solvent was removed under reduced pressure, and 50 ml of saturated sodium bicarbonate aqueous solution was added to the residue, and the mixture was extracted three times with 50 ml of dichloromethane. The mixed organic layer was washed with saturated brine, dried over solid sodium sulfate, and concentrated. 3.40 g of O6'-TBDPS-U-PMO was obtained by silica gel column chromatography, which was directly used in the next step.

[0155] 1.2. Synthesis of N3'-DMTr-O6'-TBDMS-U-PMO:

[0156] 3.40 g of O6'-TBDPS-U-PMO (10 mmol, 1.0 eq.), 3.73 g of 4,4'-dimethoxytrityl chloride (DMTrCl, 11 mmol, 1.1 eq.), and 0.12 g of DMAP (1 mmol, 0.1 eq.) were stirred and mixed in 20 ml of dry DMF, and the temperature was controlled at 0-5°C. Under nitrogen protection, 2.78 ml of triethylamine (20 mmol, 1.0 eq.) was added dropwise to the reaction solution, and stirring was continued. The reaction was slowly heated to room temperature for more than 6 hours. TLC was used to monitor the reaction progress, and the PMO monomer reaction was complete. Most of the solvent was removed under reduced pressure, and 50 ml of saturated sodium bicarbonate aqueous solution was added to the residue, and extracted three times with 30 ml of dichloromethane. The mixed organic layer was washed with saturated brine, dried over solid sodium sulfate and concentrated. 4.81 g of N3'-DMTr-O6'-TBDMS-U-PMO (purity above 98%, two-step reaction yield 75%) was obtained by silica gel column chromatography.

[0157] 1.3. Synthesis of N3'-DMTr-U-PMO:

[0158] 3.22 g of N3'-DMTr-O6'-TBDMS-U-PMO (5 mmol, 1.0 eq.) was dissolved in 40 ml of tetrahydrofuran and stirred to control the temperature between 0-5°C. Under nitrogen protection, 10 ml of tetrabutylammonium fluoride tetrahydrofuran solution (TBAF, 1.0 mol / L) was slowly added dropwise to the mixed solution. The mixture was slowly warmed to room temperature and stirred overnight. TLC detection showed that the starting material completely disappeared. The solvent was removed under reduced pressure, 50 ml of saturated sodium bicarbonate aqueous solution was added to the residue, and the mixture was extracted three times with 30 ml of dichloromethane. The mixed organic layer was washed with saturated brine, dried over solid sodium sulfate, and concentrated. 1.81 g of solid N3'-DMTr-U-PMO (purity above 98%, yield 68%) was obtained by silica gel column chromatography.

[0159] 1.4. Synthesis of N3'-DMTr-U-PMO phosphoramidate:

[0160] Under nitrogen protection, 265 mg of N3'-DMTr-U-PMO (0.5 mmol, 1.0 eq.) was added to a dry mixed solvent (3 ml of dichloromethane and 3 ml of acetonitrile), the temperature was controlled at 0°C, 88 mg of lithium bromide (1.0 mmol, 2.0 eq.) was added, and the mixture was stirred for 5 minutes. 0.15 ml of DBU (1.0 mmol, 2.0 eq.) and 0.10 ml of NN-dimethylphosphoamino dichloride (0.75 mmol, 1.5 eq.) dissolved in 0.5 ml of dry dichloromethane were added in sequence. Under nitrogen protection, the mixed solution was stirred at 0°C for 40 minutes. The reaction solvent was removed under reduced pressure, 30 ml of ethyl acetate was added to the residue, and after washing with 15 ml of cold water, the organic phase was dried over sodium sulfate and concentrated under reduced pressure, separated by silica gel chromatography, and the mobile phase was selected as a mixed solvent of dichloromethane / acetone / methanol to obtain 201 mg of the target product (purity above 99%, yield 61%).

[0161] Example 6

[0162] This embodiment is a PMO molecule for solid phase synthesis of oligonucleic acid, and the PMO molecule structure is

[0163] The synthetic route of the above PMO molecule is:

[0164]

[0165] Specific synthesis methods include:

[0166] 1.1. Synthesis of O6'-TBDPS-T-PMO:

[0167] Synthesis and preparation of 1-((2R,6S)-6-(hydroxymethyl)morpholin-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (CAS: 179073-10-0) Ref: Beilstein Journal of Organic Chemistry, 2014, vol. 10, p. 1151–1158.

[0168] 2.42 g of 1-((2R,6S)-6-(hydroxymethyl)morpholin-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (10 mmol, 1.0 eq.) and 1.50 g of imidazole (22 mmol, 2.2 eq.) were added to 15 ml of dry DMF and stirred and mixed, and the temperature was controlled at 0-5°C. Under nitrogen protection, 1.88 g of tert-butyldimethylsilyl chloride (TBDMSCl, 12.5 mmol, 1.25 eq.) was added to the reaction solution, and stirring and mixing were continued. The mixture was slowly heated to room temperature for more than 6 hours. TLC was used to monitor the progress of the reaction, and the PMO monomer was completely reacted. Most of the solvent was removed under reduced pressure, and 50 ml of saturated sodium bicarbonate aqueous solution was added to the residue, and the mixture was extracted three times with 50 ml of dichloromethane. The mixed organic layer was washed with saturated brine, dried over solid sodium sulfate, and concentrated. 3.55 g of O6'-TBDPS-T-PMO was obtained by silica gel column chromatography, which was directly used in the next step.

[0169] 1.2. Synthesis of N3'-DMTr-O6'-TBDMS-T-PMO:

[0170] 3.55 g of O6'-TBDPS-T-PMO (10 mmol, 1.0 eq.), 3.73 g of 4,4'-dimethoxytrityl chloride (DMTrCl, 11 mmol, 1.1 eq.), and 0.12 g of DMAP (1 mmol, 0.1 eq.) were stirred and mixed in 20 ml of dry DMF, and the temperature was controlled at 0-5°C. Under nitrogen protection, 2.78 ml of triethylamine (20 mmol, 1.0 eq.) was added dropwise to the reaction solution, and stirring was continued. The reaction was slowly heated to room temperature for more than 6 hours. TLC was used to monitor the reaction progress, and the PMO monomer reaction was complete. Most of the solvent was removed under reduced pressure, and 50 ml of saturated sodium bicarbonate aqueous solution was added to the residue, and extracted three times with 30 ml of dichloromethane. The mixed organic layer was washed with saturated brine, dried over solid sodium sulfate and concentrated. 4.67 g of N3'-DMTr-O6'-TBDMS-T-PMO (purity above 98%, two-step reaction yield 75%) was obtained by silica gel column chromatography.

[0171] 1.3. Synthesis of N3'-DMTr-T-PMO:

[0172] 3.29 g of N3'-DMTr-O6'-TBDMS-T-PMO (5 mmol, 1.0 eq.) was dissolved in 40 ml of tetrahydrofuran and stirred to control the temperature between 0-5°C. Under nitrogen protection, 10 ml of tetrabutylammonium fluoride tetrahydrofuran solution (TBAF, 1.0 mol / L) was slowly added dropwise to the mixed solution. The mixture was slowly warmed to room temperature and stirred overnight. TLC detection showed that the starting material completely disappeared. The solvent was removed under reduced pressure, 50 ml of saturated sodium bicarbonate aqueous solution was added to the residue, and the mixture was extracted three times with 30 ml of dichloromethane. The mixed organic layer was washed with saturated brine, dried over solid sodium sulfate, and concentrated. 1.96 g of solid N3'-DMTr-T-PMO (purity above 98%, yield 72%) was obtained by silica gel column chromatography.

[0173] 1.4. Synthesis of N3'-DMTr-T-PMO phosphoramidate:

[0174] Under nitrogen protection, 272 mg of N3'-DMTr-T-PMO (0.5 mmol, 1.0 eq.) was added to a dry mixed solvent (3 ml of dichloromethane and 3 ml of acetonitrile), the temperature was controlled at 0°C, 88 mg of lithium bromide (1.0 mmol, 2.0 eq.) was added, and the mixture was stirred for 5 minutes. 0.15 ml of DBU (1.0 mmol, 2.0 eq.) and 0.10 ml of NN-dimethylphosphoamino dichloride (0.75 mmol, 1.5 eq.) dissolved in 0.5 ml of dry dichloromethane were added in sequence. Under nitrogen protection, the mixed solution was stirred at 0°C for 40 minutes. The reaction solvent was removed under reduced pressure, 30 ml of ethyl acetate was added to the residue, and after washing with 15 ml of cold water, the organic phase was dried over sodium sulfate and concentrated under reduced pressure, separated by silica gel chromatography, and the mobile phase was selected as a mixed solvent of dichloromethane / acetone / methanol to obtain 211 mg of the target product (purity above 99%, yield 63%).

[0175] Example 7

[0176] This embodiment is a PMO molecule for solid phase synthesis of oligonucleic acid, and the PMO molecule structure is

[0177] The synthetic route of the above PMO molecule is:

[0178]

[0179] Specific synthesis methods include:

[0180] 1.1. Synthesis of N3'-DMTr-O6'-TBDPS--I-PMO:

[0181] The preparation and synthesis of O6'-TBDPS-I-PMO monomer refer to the literature (Nucleosides, Nucleotides & Nucleic Acids, 31(11), 763–782).

[0182] 2.94 g O6'-TBDPS-G-PMO (6 mmol, 1.0 eq.), 2.24 g 4,4'-dimethoxytrityl chloride (DMTrCl, 6.6 mmol, 1.1 eq.), 0.07 g DMAP (0.6 mmol, 0.1 eq.) were mixed in 20 ml dry DMF, and the temperature was controlled at 0-5°C. Under nitrogen protection, 1.7 ml triethylamine (12 mmol, 1.0 eq.) was added dropwise to the reaction solution, and stirring and mixing were continued. The mixture was slowly heated to room temperature for more than 6 hours. The reaction progress was detected by TLC, and the PMO monomer reaction was complete. Most of the solvent was removed under reduced pressure, 50 ml saturated sodium bicarbonate aqueous solution was added to the residue, and extracted three times with 30 ml dichloromethane. The mixed organic layer was washed with saturated brine, dried over solid sodium sulfate and concentrated. 3.77 g of the target product N3'-DMTr-O6'-TBDPS-I-PMO (purity above 98%, yield 79%) was obtained by silica gel column chromatography.

[0183] 1.2. Synthesis of N3'-DMTr-I-PMO:

[0184] 3.17 g of N3'-DMTr-O6'-TBDPS-I-PMO (4 mmol, 1.0 eq.) was dissolved in 30 ml of tetrahydrofuran and stirred to control the temperature between 0-5°C. Under nitrogen protection, 8 ml of tetrabutylammonium fluoride tetrahydrofuran solution (TBAF, 1.0 mol / L) was slowly added dropwise to the mixed solution. The mixture was slowly warmed to room temperature and stirred overnight. TLC detection showed that the starting material completely disappeared. The solvent was removed under reduced pressure, 50 ml of saturated sodium bicarbonate aqueous solution was added to the residue, and the mixture was extracted three times with 30 ml of dichloromethane. The mixed organic layer was washed with saturated brine, dried over solid sodium sulfate, and concentrated. 1.72 g of solid N3'-DMTr-I-PMO (purity above 98%, yield 77%) was obtained by silica gel column chromatography.

[0185] 1.3. Synthesis of N3'-DMTr-I-PMO phosphoramidate:

[0186] Under nitrogen protection, 277 mg of N3'-DMTr-I-PMO (0.5 mmol, 1.0 eq.) was added to a dry mixed solvent (3 ml of dichloromethane and 3 ml of acetonitrile), the temperature was controlled at 0°C, 88 mg of lithium bromide (1.0 mmol, 2.0 eq.) was added, and the mixture was stirred for 5 minutes. 0.15 ml of DBU (1.0 mmol, 2.0 eq.) and 0.10 ml of NN-dimethylphosphoamino dichloride (0.75 mmol, 1.5 eq.) dissolved in 0.5 ml of dry dichloromethane were added in sequence. Under nitrogen protection, the mixed solution was stirred at 0°C for 50 minutes. The reaction solvent was removed under reduced pressure, 30 ml of ethyl acetate was added to the residue, and after washing with 15 ml of cold water, the organic phase was dried over sodium sulfate and concentrated under reduced pressure, separated by silica gel chromatography, and the mobile phase was selected as a mixed solvent of dichloromethane / acetone / methanol to obtain 192 mg of the target product (purity above 99%, yield 56%).

[0187] 1.4. Synthesis of N3'-DMTr-T-PMO phosphoramidate:

[0188] Under nitrogen protection, 272 mg of N3'-DMTr-T-PMO (0.5 mmol, 1.0 eq.) was added to a dry mixed solvent (3 ml of dichloromethane and 3 ml of acetonitrile), the temperature was controlled at 0°C, 88 mg of lithium bromide (1.0 mmol, 2.0 eq.) was added, and the mixture was stirred for 5 minutes. 0.15 ml of DBU (1.0 mmol, 2.0 eq.) and 0.10 ml of NN-dimethylphosphoamino dichloride (0.75 mmol, 1.5 eq.) dissolved in 0.5 ml of dry dichloromethane were added in sequence. Under nitrogen protection, the mixed solution was stirred at 0°C for 40 minutes. The reaction solvent was removed under reduced pressure, 30 ml of ethyl acetate was added to the residue, and after washing with 15 ml of cold water, the organic phase was dried over sodium sulfate and concentrated under reduced pressure, separated by silica gel chromatography, and the mobile phase was selected as a mixed solvent of dichloromethane / acetone / methanol to obtain 211 mg of the target product (purity above 99%, yield 63%).

[0189] Example 8

[0190] This embodiment is a nucleotide monomer for solid phase synthesis of oligonucleic acid, and the nucleotide monomer structural formula is

[0191] The above-mentioned nucleotide monomer synthesis route is:

[0192]

[0193] Specific synthesis methods include:

[0194] Under nitrogen protection, 545 mg of O3'-DMTr-T (1.0 mmol, 1.0 eq.) was added to a dry mixed solvent (5 ml of dichloromethane and 5 ml of acetonitrile), the temperature was controlled at 0°C, 174 mg of lithium bromide (2.0 mmol, 2.0 eq.) was added, and the mixture was stirred for 5 minutes. 0.30 ml of DBU (2.0 mmol, 2.0 eq.) and 0.20 ml of NN-dimethylphosphoamino dichloride (1.5 mmol, 1.5 eq.) dissolved in 1 ml of dry dichloromethane were added in sequence. Under nitrogen protection, the mixed solution was stirred at 0°C for 40 minutes. The solvent was removed under reduced pressure, 50 ml of ethyl acetate was added to the residue, and after washing with 30 ml of cold water, the organic phase was dried over sodium sulfate and concentrated under reduced pressure, separated by silica gel chromatography, and the mobile phase was selected as a mixed solvent of dichloromethane / acetone / methanol to obtain 462 mg of the target product (purity above 99%, yield 69%).

[0195] Example 9

[0196] This embodiment is a nucleotide monomer for solid phase synthesis of oligonucleic acid, and the nucleotide monomer structural formula is

[0197] The above-mentioned nucleotide monomer synthesis route is:

[0198]

[0199] Specific synthesis methods include:

[0200] Under nitrogen protection, 561 mg O2'-Me-O3'-DMTr-U (1.0 mmol, 1.0 eq.) was added to a dry mixed solvent (5 ml dichloromethane and 5 ml acetonitrile), the temperature was controlled at 0°C, 174 mg lithium bromide (2.0 mmol, 2.0 eq.) was added, and the mixture was stirred for 5 minutes. 0.30 ml DBU (2.0 mmol, 2.0 eq.) and 0.20 ml NN-dimethylphosphoamino dichloride (1.5 mmol, 1.5 eq.) dissolved in 1 ml dry dichloromethane were added in sequence. Under nitrogen protection, the mixed solution was stirred at 0°C for 40 minutes. The reaction solvent was removed under reduced pressure, 50 ml ethyl acetate was added to the residue, and after washing with 30 ml cold water, the organic phase was dried over sodium sulfate and concentrated under reduced pressure, separated by silica gel chromatography, and the mobile phase was selected as a mixed solvent of dichloromethane / acetone / methanol to obtain 446 mg of the target product (purity above 99%, yield 65%).

[0201] The following nucleotide monomers were prepared by a similar preparation method as above:

[0202]

[0203] In the nucleotide monomer, R is hydrogen, fluorine, methoxy, alkoxy, alkenyloxy, silyloxymethyleneoxy or alkoxyalkoxy, etc.

[0204] Example 10

[0205] This example is a process for synthesizing oligonucleic acids with a chimeric PMO structure. The solution system used by PMO in this process refers to the literature Journal of PLA Pharmacy, Vol. 35, No. 6, 2022, pp. 493-496; the details are as follows:

[0206] DMTr removal solution: 5.355 g of 4-cyanopyridine was added to 100.0 ml of dichloromethane, and then 4.900 g of trifluoroacetic acid was added under stirring at room temperature. After stirring for 1 hour, 4.5 ml of ethanol and 100 ml of trifluoroethanol were added, and the volume was adjusted to 500 ml with dichloromethane to prepare a trifluoroethanol / dichloromethane solution of 4-cyanopyridinium trifluoroacetate (2%) and ethanol (0.9%) as the DMTr removal solution.

[0207] Neutralizing solution: 25.0 ml of diisopropylethylamine and 125.0 ml of isopropanol are made up to 500 ml with dichloromethane, and a solution of diisopropylethylamine (5%) in isopropanol / dichloromethane is prepared as the neutralizing solution.

[0208] Coupling solvent: 2.303 g of N-ethylmorpholine was diluted with 1,3-dimethyl-2-imidazolidinone (DMI) to 50 ml to obtain N-ethylmorpholine DMI solution (0.40 mol / L) as the coupling solvent.

[0209] Lysis solution: Weigh 0.154 g of dithiothreitol, add 5 ml of N-methylpyrrolidone (NMP) to dissolve, add 0.739 g of triethylamine, and make up to 10 ml with NMP to obtain an NMP solution containing 0.10 mol / L dithiothreitol and 0.73 mol / L triethylamine, which is the lysis solution and is prepared before use.

[0210] The steps of removing the DMTr protecting group, coupling, and capping are referred to the literature (Journal of PLA Pharmacy, Vol. 35, No. 6, 2022, pp. 493-496; Nature Communications. 2021 Jul 20; 12(1): 4396). Since conventional nucleotide phosphoamido and PMO phosphoamido use the same protecting group DMTr, the coupling efficiency of each step is determined by measuring the intensity of the removed DMTr by light absorption.

[0211] The oligonucleotide synthesis process of the chimeric PMO structure includes:

[0212] Weigh 0.050 g of the modified solid phase synthesis resin and add it to a solid phase synthesis tube; add 2.0 ml of NMP and swell it at room temperature for 2 hours, then filter and drain the solution. Use 3.0 ml of dichloromethane to wash the resin, and repeat the washing process 3 times to start solid phase synthesis. According to the base sequence of the target sequence, the DMTr removal → neutralization → washing → coupling → neutralization → washing cycle (such as Figure 1 As shown), the nucleotide monomer molecules are coupled one by one. After the last monomer molecule is coupled, the terminal DMTr is removed, and 3.0 ml of p-methoxytrityl chloride blocking solution (0.32 mol / L) is added to the reaction tube. After reacting at 45°C for 90 minutes, the reaction solution is emptied and washed 8 times with 4.0 ml of NMP each time;

[0213] After the main chain solid phase synthesis is completed, 3.0 ml of lysate is added to the solid phase synthesis tube, and the lysate is collected after 30 minutes of reaction. 1.5 ml of lysate is added to the solid phase synthesis tube and reacted for 15 minutes, and the lysate is collected and combined. The lysate is transferred to a 50 ml hydrothermal reactor, 15.0 ml of concentrated ammonia water precooled at -20°C is added, and the reaction is allowed to stand in a 45°C oil bath for 24 hours to remove the base and main chain protecting groups.

[0214] Embodiment 11

[0215] Referring to the method of Example 10, taking the TTTApTT sequence as an example, the PMO molecule (ie, Ap) for solid phase synthesis of oligonucleic acid prepared in Example 1, and the structural formula Solid phase synthesis was performed using nucleotide monomers of, and after Ap coupling, the next round used nucleotide monomers of Example 8;

[0216] The structure of the oligonucleotides synthesized in solid phase and the coupling efficiency of each step are as follows: Figure 2 As shown;

[0217] The above Ap was replaced with Gp in Example 2. The oligonucleotide structure and coupling efficiency of each step were as follows: Figure 3 As shown;

[0218] The above Ap was replaced with Cp in Example 4. The oligonucleotide structure and coupling efficiency of each step were as follows: Figure 4 As shown;

[0219] The above Ap was replaced with Up in Example 5. The oligonucleotide structure and coupling efficiency of each step were as follows: Figure 5 As shown;

[0220] The Ap and the base before AP were replaced with Tp in Example 6. The oligonucleotide structure and coupling efficiency of each step were as follows: Figure 6 As shown;

[0221] The above Ap was replaced with Ip in Example 7. The oligonucleotide structure and coupling efficiency of each step were as follows: Figure 7 shown.

[0222] Example 12

[0223] Referring to the method of Example 10, taking the UUUApUU sequence as an example, the PMO molecule (ie, Ap) for solid phase synthesis of oligonucleic acid prepared in Example 1, and the structural formula Solid phase synthesis was performed with the nucleotide monomers of Example 9, and after Ap coupling, the next round used the nucleotide monomers of Example 9;

[0224] The structure of the oligonucleotides synthesized in solid phase and the coupling efficiency of each step are as follows: Figure 8 As shown;

[0225] The above Ap was replaced with Gp in Example 2. The oligonucleotide structure and coupling efficiency of each step were as follows: Fig. 9 As shown;

[0226] The above Ap was replaced with Cp in Example 4. The oligonucleotide structure and coupling efficiency of each step were as follows: Fig.10 As shown;

[0227] The above Ap and the last base of AP were replaced with Up in Example 5. The oligonucleotide structure and coupling efficiency of each step were as follows: Fig.11 As shown;

[0228] The above Ap was replaced with Tp in Example 6. The oligonucleotide structure and coupling efficiency of each step were as follows: Fig.12 As shown;

[0229] The above Ap was replaced with Ip in Example 7. The oligonucleotide structure and coupling efficiency of each step were as follows: Fig.13 shown.

[0230] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. These modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be included in the scope of the claims and specification of the present invention.

Claims

1. A process for synthesizing oligonucleic acids with a chimeric PMO structure, characterized in that: The PMO molecular structure used in the oligonucleic acid synthesis process of the chimeric PMO structure is shown in Formula I: In Formula I, Base is adenine, guanine, cytosine, thymine, uracil or hypoxanthine containing a protecting group; R1 and R2 are independently chlorine, bromine, fluorine, hydroxy, alkoxy, alkenyloxy, amino / amine, alkylamino / amine, alkenylamino / amine, cycloalkoxy, cycloalkylamino or alkoxyalkoxy; R3 is hydrogen, fluorine, methoxy, alkoxy, alkenyloxy, silyloxymethyleneoxy or alkoxyalkoxy; R4 is trityl, (4-methoxyphenyl)diphenylmethyl, 4,4'-dimethoxytrityl or 4,4',4"-trimethoxytrityl.

2. The oligonucleic acid synthesis process of the chimeric PMO structure according to claim 1, characterized in that: The protecting group is selected from one of 4,4'-dimethoxytrityl, trityl, allyl, tert-butyldimethylsilyl and tert-butyldiphenylsilyl.

3. The oligonucleic acid synthesis process of the chimeric PMO structure according to claim 1, characterized in that: The PMO molecular structure is 4. The oligonucleic acid synthesis process of the chimeric PMO structure according to claim 1, characterized in that: The nucleotide monomers used in the oligonucleic acid synthesis process of the chimeric PMO structure include at least one of Formula II to Formula V; In formula II, Base is adenine, guanine, cytosine, thymine, uracil or hypoxanthine containing a protecting group; PG is trityl, (4-methoxyphenyl)benzhydryl, 4,4'-dimethoxytrityl, 4,4',4"-trimethoxytrityl, allyl, allyloxycarbonyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl or tert-butyldiphenylsilyl; X1 and X2 are independently hydrogen, fluorine, methoxy, alkoxy, alkenyloxy, silyloxymethyleneoxy or alkoxyalkoxy; LG1 is a secondary amine, fluorine, chlorine or bromine; LG2 is cyanoethyl, cyanopropyl, 3-cyanoisobutyl, (CH3)2CHCH2CN, phenethyl or allyl; In formula III, Base is adenine, guanine, cytosine, thymine, uracil or hypoxanthine containing a protecting group; PG is trityl, (4-methoxyphenyl)benzhydryl, 4,4'-dimethoxytrityl, 4,4',4"-trimethoxytrityl, allyl, allyloxycarbonyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl or tert-butyldiphenylsilyl; R1, R2 and R3 are alkyl, alkenyl, cycloalkyl or aromatic groups; LG1 is a secondary amine, fluorine, chlorine or bromine; LG2 is cyanoethyl, cyanopropyl, 3-cyanoisobutyl, (CH3)2CHCH2CN, phenethyl or allyl; In formula IV, Base is adenine, guanine, cytosine, thymine, uracil or hypoxanthine containing a protecting group; PG is trityl, (4-methoxyphenyl)benzhydryl, 4,4'-dimethoxytrityl, 4,4',4"-trimethoxytrityl, allyl, allyloxycarbonyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl or tert-butyldiphenylsilyl; R1 and R2 are alkyl, alkenyl, cycloalkyl or aromatic R3 and R4 are independently hydrogen, fluorine, methoxy, alkoxy, alkenyloxy, silyloxymethyleneoxy or alkoxyalkoxy; X is fluorine, chlorine, bromine, hydroxy, alkoxy, alkenyloxy, amino, alkylamino, cycloalkyloxy or cycloalkylamino; In formula V, Base is adenine, guanine, cytosine, thymine, uracil or hypoxanthine containing a protecting group; PG is trityl, (4-methoxyphenyl)benzhydryl, 4,4'-dimethoxytrityl, 4,4',4"-trimethoxytrityl, allyl, allyloxycarbonyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl or tert-butyldiphenylsilyl; R1, R2, R3, R4 and R5 are alkyl, alkenyl, cycloalkyl or aromatic groups respectively; X is fluorine, chlorine, bromine, hydroxy, alkoxy, alkenyloxy, amino, alkylamino, cycloalkyloxy or cycloalkylamino.

5. The oligonucleic acid synthesis process of the chimeric PMO structure according to claim 4, characterized in that: The protecting group is selected from one of 4,4'-dimethoxytrityl, trityl, allyl, tert-butyldimethylsilyl and tert-butyldiphenylsilyl.

6. The oligonucleic acid synthesis process of the chimeric PMO structure according to claim 4, characterized in that: The nucleotide monomers include In the nucleotide monomer, R is hydrogen, fluorine, methoxy, alkoxy, alkenyloxy, silyloxymethyleneoxy or alkoxyalkoxy.

7. The process for synthesizing oligonucleic acid with a chimeric PMO structure according to claim 4, characterized in that: The nucleotide monomer is The preparation method comprises the following steps: linking a pentavalent phosphorus compound at the O5' position; linking a protective group PG at the O3' position, wherein the protective group PG is (4-methoxyphenyl)diphenylmethyl, triphenylmethyl, 4,4'-dimethoxytrityl, 4,4',4"-trimethoxytrityl, allyl, allyloxycarbonyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl or tert-butyldiphenylsilyl; In the nucleotide monomer, R is hydrogen, fluorine, methoxy, alkoxy, alkenyloxy, silyloxymethyleneoxy or alkoxyalkoxy.

8. An oligonucleic acid with a chimeric PMO structure, characterized in that The chimeric PMO structured oligonucleotide comprises the structure shown in Formula VI or VII; In Formula VI and Formula VII, Base is adenine, guanine, cytosine, thymine, uracil or hypoxanthine containing a protecting group; R1 and R2 are independently alkyl, alkenyl or cycloalkyl; X1 and X2 are independently hydrogen, fluorine, methoxy, alkoxy, alkenyloxy, silyloxymethyleneoxy or alkoxyalkoxy.

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