Oligonucleotides, reagents, and their preparation

Abstract

This disclosure describes novel reagents and processes for preparing oligonucleotides having two or more nucleotides. In one embodiment, the reagents are represented by Formula I' or B: TIFF2023551647000536.tif54119

Description

[Technical Field]

[0001] Related applications This application claims the benefit as of the filing date of U.S. Provisional Application No. 63 / 112,281 (filed November 11, 2020) under Section 119(e) of the U.S. Patent Act. The entire contents of that document are incorporated herein by reference.

[0002] The present invention relates to oligonucleotides, reagents, and methods for preparing oligonucleotides. [Background technology]

[0003] Oligonucleotides are short DNA or RNA oligomers that can be chemically synthesized for a wide range of applications. Recent advances in the use of synthetic oligonucleotides as therapeutic agents have increased the demand for synthesis methods that can produce oligonucleotides efficiently, with high purity, and in large quantities.

[0004] Traditionally, oligonucleotides have been synthesized using solid-phase automated synthesizers employing phosphoramidite chemistry, and are limited to scales of less than 2 moles. Thus, solid-phase synthesis is insufficient for producing the materials necessary for the clinical development and commercialization of oligonucleotide drugs for a wide range of indications. In addition, solid-phase synthesis often requires the use of excessive reagents, resulting in increased costs associated with the production of target oligonucleotides.

[0005] Therefore, there is a need for novel reagents and robust methods for synthesizing oligonucleotides suitable for large-scale, highly efficient, and high-purity manufacturing processes. [Overview of the project]

[0006] One aspect of this disclosure relates to compounds of formula I' or B. [ka] or a salt thereof, ring A, A 1 , A 2 , A3 , R1, R2, P1, Y, e, and f relate to a compound as defined below.

[0007] One aspect of the present disclosure relates to a nucleotide or oligonucleotide represented by formula III or IIIP Chemical formula or a salt thereof, wherein R 31 , R 32 , R 34 , R 35 , R 36 , q, X, and Z relate to a nucleotide or oligonucleotide as defined below.

[0008] One aspect of the present disclosure relates to a nucleotide or oligonucleotide represented by formula III' or IIIP' Chemical formula or a salt thereof, wherein R 31 , R 32 , R 34 , R 35 , R 36 , q, Q, X, Chemical formula and Z relate to a nucleotide or oligonucleotide as defined below.

[0009] One aspect of the present disclosure relates to a process for preparing an oligonucleotide fragment of formula (V) Chemical formula or a salt thereof, comprising: 1) deprotecting a compound of formula (VA) Chemical formula or a salt thereof to obtain a compound of formula (VB) Chemical formula or a salt thereof; 2) reacting a compound of formula (VB) or a salt thereof with a compound of formula (VC)

Chemical Formula

Chemical Formula

Chemical Formula

Chemical Formula

[0010] One aspect of the present disclosure provides a process for preparing an oligonucleotide fragment of formula (V')

Chemical Formula

Chemical Formula

[0011] One aspect of this disclosure relates to an oligonucleotide fragment of formula (V-C1) or (V-C2). [ka] or a process for preparing the salt thereof, 1) Compound of formula (VB)

Chem.

Chem.

[0012] One aspect of the present disclosure provides an oligonucleotide fragment of formula (V-C1) or (V-C2)

Chem.

Chem.

Chem.

Chem.

Chem.

[0013] One aspect of this disclosure is an oligonucleotide fragment of formula (VBZ) [ka] or a process for preparing the salt thereof, 1) Compound of formula (VBZ-1) [ka] or its salt, compound of formula (VBZ-2) [ka] Alternatively, by reacting it with its salt, the compound of formula (VBZ-3) can be obtained. [ka] or the step of forming a salt thereof, 3) The step of sulfiding or oxidizing a compound of formula (VBZ-3) or a salt thereof with a sulfidating or oxidizing agent to form a compound of formula (VBZ) or a salt thereof, R 31 , R 32 , R 34 , R 35 , R 36 q, X, and Z are defined below in relation to the process.

[0014] One aspect of this disclosure is an oligonucleotide fragment of formula (V) [ka] or a process for preparing the salt thereof, a) Nucleotides of formula (V-1) [ka] or its salt, Oligonucleotide fragment of formula (V-2) [ka] or a salt thereof, Coupling in solution yields the oligonucleotide fragment of formula (V-3). [ka] or the step of forming a salt thereof, b) Sulfurize or oxidize the oligonucleotide of formula (V-3) or a salt thereof to obtain the oligonucleotide of formula (V). [ka] or its salt The step of forming R 31 , R 32 , R 34 , R 35 , R 36 , R 37a , R 37b q, X, and Z are defined below in relation to the process.

[0015] One aspect of this disclosure is an oligonucleotide fragment of formula (V*). [ka] or a process for preparing the salt thereof, a) Nucleotides of formula (V-1) [ka] or its salt, Oligonucleotide fragment of formula (V-2') [ka] or its salt, Coupling in solution yields the oligonucleotide fragment of formula (V-3'). [ka] or the step of forming a salt thereof, b) The step of sulfiding or oxidizing an oligonucleotide of formula (V-3') or a salt thereof to form an oligonucleotide of formula (V*) or a salt thereof, R 31 , R 32 , R 34 , R 35 , R 36 q, X, and Z are defined below in relation to the process.

[0016] One aspect of the present disclosure relates to a target oligonucleotide of formula (VI) or (VI-1). [ka] or a process for preparing the salt thereof, a) Oligonucleotide fragment of formula (F1) or (F1-1) [ka] or its salt, Oligonucleotide fragment of formula (F2) [ka] or its salt, Coupling in solution to oligonucleotide fragments of formula (F3) or (F3-1) [ka] or to form a salt thereof, b) Sulfurizing or oxidizing an oligonucleotide fragment or salt of formula (F3) or (F3-1) to form an oligonucleotide or salt of formula (VI) or (VI-1), R 31 , R 32 , R 34 , R 35 , R 36 , R 37a , R 37b o, p, Q, X, [ka] And Z are defined below, relating to the process.

[0017] One aspect of the present disclosure relates to a target oligonucleotide of formula (VI') or (VI'-1). [ka] or a process for preparing the salt thereof, a) Oligonucleotide fragment of formula (F1) or (F1-1) [ka] or a salt thereof, a fragment of oligonucleotide formula (F2') [ka] Alternatively, a salt thereof can be coupled in solution with an oligonucleotide fragment of formula (F3') or (F3'-1). [ka] or to form a salt thereof, b) Sulfurizing or oxidizing an oligonucleotide fragment or salt of formula (F3') or (F3'-1) to form an oligonucleotide or salt of formula (VI') or (VI'-1), R 31 , R 32 , R 34 , R 35 , R 36 , R 37a , R 37b o, p, Q, X, [ka] And Z are defined below, relating to the process. [Brief explanation of the drawing]

[0018] [Figure 1] This figure shows a retrosynthesis scheme for preparing oligonucleotide I. [Figure 2]This figure shows a synthesis scheme for preparing oligonucleotide fragment A. [Figure 3] This figure shows the synthesis scheme for preparing oligonucleotide fragment B from reagent M19. [Figure 4] This figure shows a synthesis scheme for preparing oligonucleotide fragment C. [Figure 5] This figure shows a synthesis scheme for preparing oligonucleotide fragment D. [Figure 6] This figure shows a synthesis scheme for preparing oligonucleotide fragment E. [Figure 7] This figure shows the synthesis scheme for preparing oligonucleotide fragment F. [Figure 8] This figure shows a synthesis scheme for preparing oligonucleotide fragment J. [Figure 9] This figure shows the synthesis scheme for preparing oligonucleotide fragment K. [Figure 10] This figure shows a synthesis scheme for preparing oligonucleotide fragments O. [Figure 11] This figure shows the synthesis scheme for preparing oligonucleotide fragment B from reagent M40. [Figure 12] This figure shows the reaction products and by-products of a one-pot procedure for preparing a P=O bond. [Figure 13] This figure shows a synthesis scheme for the large-scale preparation of oligonucleotide I. [Modes for carrying out the invention]

[0019] This disclosure describes reagents for accelerating the preparation of oligonucleotides, particularly on a large scale. The synthesis process based on the reagents of this disclosure enables the production of highly purified, large-scale protected target oligonucleotides without the need for chromatographic purification from an assembly of oligonucleotide fragments. Furthermore, the protected target oligonucleotides can be selectively and easily deprotected based on the state disclosed. Following deprotection and standard downstream purification, highly purified ASO oligonucleotides suitable for therapeutic applications are obtained. Therefore, the novel reagents and synthesis process of this disclosure offer significant advantages over conventional preparations of oligonucleotides.

[0020] definition The term “nucleic acid base” refers to the heterocyclic base portion of a nucleoside. Nucleic acid bases may be naturally occurring or modified. In some embodiments, a nucleic acid base may include any atom or group of atoms that can hydrogen-bond to a nucleic acid base of another nucleic acid. In particular, nucleic acid bases are heterocyclic bases, typically purines and pyrimidines. In addition to “unmodified” or “natural” nucleic acid bases such as the purine nucleic acid bases adenine (A) and guanine (G), and the pyrimidine nucleic acid bases thymine (T), cytosine (C), and uracil (U), many modified nucleic acid bases or nucleic acid base mimics known to those skilled in the art can be incorporated into compounds synthesized by the methods described herein. In some embodiments, the modified nucleic acid base is a nucleic acid base that is structurally quite similar to the parent nucleic acid base, such as 7-deazapurine, 5-methylcytosine, or G-clamp. In some embodiments, the nucleic acid base mimic includes more complex structures, such as the tricyclic phenoxazine nucleic acid base mimic. Methods for preparing the aforementioned modified nucleic acid bases are well known to those skilled in the art.

[0021] The term "nucleoside" refers to a compound containing a heterocyclic base moiety and a sugar moiety that can be modified at the 2'-terminus.

[0022] The term "nucleotide" refers to a nucleoside containing a phosphate, thiophosphate, or dithiophosphate linking group.

[0023] The term "oligonucleotide" refers to a compound containing multiple bound nucleosides. In some embodiments, one or more of the nucleosides are modified. In some embodiments, the oligonucleotide contains one or more ribonucleosides (RNA) and / or deoxyribonucleosides (DNA).

[0024] As used herein, “target oligonucleotide” refers to an oligonucleotide product that can be prepared based on the reagents and processes of this disclosure. In some embodiments, the target oligonucleotide comprises at least 10 or at least 15 nucleotides. In some embodiments, the target oligonucleotide has 10-500, 15-500, 15-200, 15-100, 15-50, 15-40, 15-30, or 16-30 nucleotides.

[0025] As used herein, “oligonucleotide fragment” refers to a short oligonucleotide assembled to form a target oligonucleotide. In some embodiments, the oligonucleotide fragment has 3 to 10, 3 to 8, 3 to 6, or 4 to 6 nucleotides. In some embodiments, the oligonucleotide fragment has 4 or 5 nucleotides.

[0026] As used herein, the term “alkyl” refers to a fully saturated branched or unbranched hydrocarbon moiety. In some embodiments, alkyls include 1 to 30 carbon atoms, 1 to 20 carbon atoms, 1 to 16 carbon atoms, 1 to 10 carbon atoms, 1 to 6 carbon atoms, or 1 to 4 carbon atoms. In some embodiments, alkyls include 6 to 20 carbon atoms. Typical examples of alkyls include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, or n-decyl.

[0027] As used herein, “carbocyclyl” refers to a saturated or unsaturated monocyclic, bicyclic, or tricyclic (e.g., condensed, cross-linked, or spirocyclic) ring system having 4 to 12 ring members, all of which are carbon atoms. The term “carbocyclyl” encompasses cycloalkyl groups, cycloalkenyl groups, and aromatic groups (i.e., aryl groups). “Cycloalkyl” refers to a fully saturated monocyclic hydrocarbon group of 3 to 7 carbon atoms, including cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cyclopentyl. “Cycloalkylenyl” refers to an unsaturated, non-aromatic monocyclic hydrocarbon group of 3 to 7 carbon atoms, including cyclopentenyl, cyclohexenyl, and cyclopentenyl.

[0028] The term "aryl" refers to a monocyclic, bicyclic, or tricyclic aromatic hydrocarbon group having 6 to 14 carbon atoms in its ring portion. In one embodiment, the term aryl refers to monocyclic and bicyclic aromatic hydrocarbon groups having 6 to 10 carbon atoms. Typical examples of aryl groups include phenyl, naphthyl, fluorenyl, and anthracenyl.

[0029] The term "aryl" also refers to a bicyclic or tricyclic group in which at least one ring is aromatic and fused to one or more non-aromatic hydrocarbon rings. Non-limiting examples include tetrahydronaphthalene, dihydronaphthalenyl, and indanyl.

[0030] As used herein, the term "bridged ring system" refers to a ring system having a carbocyclyl or heterocyclyl ring, wherein two non-adjacent atoms of the ring are linked (bridged) by one or more (preferably 1 to 3) atoms selected from C, N, O, or S. A bridged ring system may have 6 to 7 ring members.

[0031] As used herein, the term "spiro-ring system" refers to a ring system having two rings, each independently selected from a carbocyclyl or heterocyclyl, where the two cyclic structures share a single ring atom. A spiro-ring system has 5 to 7 ring members.

[0032] As used herein, the term “heterocyclyl” means a saturated or unsaturated monocyclic or bicyclic (e.g., bridging or spirocyclic) ring system having 3 to 7 ring members, or 3 to 6 ring members, or 5 to 7 ring members, of which at least one is a heteroatom, and up to four (e.g., 1, 2, 3, or 4) may be heteroatoms, the heteroatoms being independently selected from O, S, and N, C being oxidizable (e.g., C(O)), N being oxidizable (e.g., N(O)) or quaternizable, and S being optionally oxidizable to sulfoxides and sulfones. Examples of unsaturated heterocycles include heteroaryl rings. As used herein, the term “heteroaryl” refers to an aromatic 5- or 6-membered monocyclic ring system having 1 to 4 heteroatoms independently selected from O, S, and N, where N can be oxidized (e.g., N(O)) or quaternized, and S can optionally be oxidized to sulfoxides and sulfones. In one embodiment, the heterocyclyl is a 3- to 7-membered saturated monocyclic ring, a 3- to 6-membered saturated monocyclic ring, or a 5- to 7-membered saturated monocyclic ring. In another embodiment, the heterocyclyl is a 3- to 7-membered monocyclic ring, a 3- to 6-membered monocyclic ring, or a 5- to 7-membered monocyclic ring. The heterocyclyl group can be bonded to a heteroatom or a carbon atom.Examples of heterocyclyls include azilidinyl, oxyranil, thyranil, oxazilidinyl, dioxyranil, azetidinil, oxetanil, thietanil, pyrrolidinil, tetrahydrofuranil, thioranil, imidazolidinil, pyrazolidinil, oxazolidinil, isoxazolidinil, thiazolinidyl, isothiazolidinil, dioxolanil, dithioranil, oxathioranil, piperidinil, tetrahydropyranil, thianil, piperazinil, morpholinil, thiomorpholinil, dioxanil, dithianil, trioxanil, trithianil, azepanil, oxepanil, thiepanil, dihydrofuranil, imidazolinil, dihydropyranil, and he Examples of teloaryl rings include azilinyl, oxylenyl, thirenyl, diazilinyl, azetyl, oxetyl, thiethyl, pyrrolyl, furanil, thiophenyl (or thienyl), imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, furazanil, oxadiazolyl, thiadiazolyl, dithiazolyl, triazolyl, tetrazolyl, pyridinyl, pyranyl, thiopyranil, pyrazinyl, pyrimidinyl, pyridazinyl, oxazinyl, thiadinyl, dioxynyl, dithiinyl, oxathianyl, triazinyl, tetradinyl, azepinyl, oxepinyl, thiepinyl, diazepinyl, and thiazepinyl. Examples of bicyclic heterocyclic systems include 3-azabicyclo[3.1.0]hexanyl, 3-azabicyclo[3.1.1]heptanyl, 2-azaspiro[3.3]heptanyl, 2-oxa-6-azaspiro[3.3]heptanyl, and 5-azaspiro[2.3]hexanyl.

[0033] "Halogen" or "halo" may be fluoro, chloro, bromo, or iodine.

[0034] As used herein, "hydroxyl protecting group" refers to a group suitable for protecting a hydroxyl group (-OH) from reaction with other reagents. Examples of hydroxyl protecting groups can be found in Greene, TW et al., Protective Groups in Organic Synthesis, 4th Ed., John Wiley and Sons (2007).

[0035] In some embodiments, the hydroxyl protecting group can be selected from, for example, acetyl (Ac); benzoyl (Bz); benzyl (Bn); β-methoxyethoxymethyl ether (MEM); methoxymethyl ether (MOM); methoxytrityl [(4-methoxyphenyl)diphenylmethyl, MMT]; 4,4′-dimethoxytrityl (DMT); methoxyethyl (MOE); p-methoxybenzyl ether (PMB); methylthiomethyl ether; pivaloyl (Piv); tetrahydropyranyl (THP); tetrahydrofuran (THF); silyl ethers (for example, without limitation, trimethylsilyl (TMS), tert-butyldiphenylsilyl (TBDPS), tert-butoxydiphenylsilyl (TBoDPS), triphenylsilyl (TPS), tert-butyldimethylsilyl (TBDMS), tri-isopropylsilyloxymethyl (TOM), and triisopropylsilyl (TIPS) ether); methyl ethers, and ethoxyethyl ethers (EE).

[0036] In some embodiments, the hydroxyl protecting group protects the 3'-hydroxyl of the nucleoside (referred to as the 3'-hydroxyl protecting group). In some embodiments, the 3'-hydroxyl protecting group is a silyl hydroxyl protecting group, such as trimethylsilyl, triethylsilyl, triisopropylsilyl, dimethylisopropylsilyl, diethylisopropylsilyl, dimethyltexylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, trybenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, diphenylmethylsilyl, di-t-butylmethylsilyltri(trimethylsilyl)silyl, t-butylmethoxyphenylsilyl, and t-butoxydiphenylsilyl. In some embodiments, the 3'-hydroxyl protecting group is TBDPS. In some embodiments, the 3'-hydroxyl protecting group is a large hydrophobic protecting group (LHPG), such as those described herein.

[0037] The suffix "yl" added to the end of a chemical name indicates that the part of the name is attached to the molecule at one point. The subscript "ene" added to the end of a chemical name indicates that the part of the name is attached to the molecule at two points.

[0038] In some embodiments, the hydroxyl protecting group protects the 5'-hydroxyl group of the nucleoside (referred to as the 5'-hydroxyl protecting group). Typical 5'-hydroxyl groups include those described herein (for example, R in any aspect or embodiment). 35 This includes, but is not limited to, the following: In certain embodiments, the 5'-hydroxyl protecting group is an acid-unstable 4,4'-dimethoxytrityl (or bis-(4-methoxyphenyl)phenylmethyl) (DMT or DMTr) protecting group. In some embodiments, the 5'-hydroxyl protecting group is a large hydrophobic protecting group (LHPG), such as those described herein.

[0039] As used herein, “selective precipitation” refers to a purification method that separates a desired product from one or more impurities in a solution by adding a solution to a solvent that precipitates the product, while leaving one or more impurities in the solution. Alternatively, the product can be precipitated by adding a solvent to a solution containing the crude product and one or more impurities. In some embodiments, the desired compound or oligonucleotide of the disclosure comprises a hydrophobic group (e.g., a hydrophobic 3'-hydroxyl protecting group or a hydrophobic 5'-hydroxyl protecting group (e.g., the LHPG group described herein)), and the desired oligonucleotide is precipitated by adding a polar solvent (e.g., CH3CN) to a solution containing the compound or oligonucleotide and one or more impurities. In one embodiment, the desired compound or oligonucleotide of the present disclosure is cosolvent or solvent mixture (e.g., heptane, tert-butyl methyl ether (TBME or MBTE), heptane / MBTE mixture (e.g., a heptane / MBTE mixture where the volume ratio of heptane to MBTE is in the range of 20:1 to 1:20, 9:1 to 1:9, or 4:1 to 1:4, or a heptane / MBTE mixture where the volume ratio of heptane to MBTE is 9:1, 4:1, 2:1, 1:1, 2:5, 1:2, 1:4, or 1:9) The product can be purified by adding it to a solution containing the crude product and one or more impurities in an organic solvent (e.g., dichloromethane (DCM) or ethyl acetate (Â)) and precipitating and separating the product. Alternatively, the solution containing the crude product and one or more impurities can be added to a non-polar or low-polarity solvent or solvent mixture to precipitate and separate the product. A suitable co-solvent can be determined based on the hydrophobicity of the product. In some embodiments, the co-solvent is less polar than the organic solvent in which the product is dissolved.

[0040] As used herein, “extraction” refers to a purification method that separates a desired product from one or more impurities in a solution by contacting the solution with a solvent in which the product is soluble (while one or more impurities are insoluble). Alternatively, a solution containing the product and one or more impurities can be contacted with a solvent in which one or more impurities are soluble (while the product is insoluble). In some embodiments, a solution containing the product and one or more impurities in an organic solvent (e.g., DCM, siRNA, or THF) or a mixture of organic solvents (e.g., a solution of the reaction mixture or crude product) can be contacted (extracted or washed) with water or an aqueous solution (e.g., NaHCO3 / H2O solution or NaCl / H2O solution) to remove hydrophilic impurities.

[0041] As used herein, the term "base" refers to a hydroxide ion (OH) in an aqueous solution. - This refers to a substance that can produce or donate a non-bonding electron pair. Typical bases include, but are not limited to, alkaline hydroxides, alkaline earth hydroxides, alkylamines (e.g., tert-butylamine, sec-butylamine, trimethylamine, triethylamine, diisopropylethylamine, 2-methylpropane-2-amine), 8-diazabicyclo[5.4.0]undeca-7-ene (DBU), imidazole, N-methylimidazole, pyridine, and 3-picoline. As used herein, the term “salt” refers to an organic or inorganic salt of a compound, nucleotide, or oligonucleotide described herein. In some embodiments, the salt is a pharmaceutically acceptable salt. The phrase “pharmaceutically acceptable” means that the substance or composition must be chemically and / or toxicologically compatible with the other components of the formulation and / or the mammal being treated therewith. In some embodiments, the salt of a compound, nucleotide, or oligonucleotide described herein is a sodium salt, a potassium salt, or an ammonium salt. In one embodiment, the salt is a sodium salt or an ammonium salt.

[0042] 1. Reagents In a first aspect, the present disclosure provides a reagent for facilitating the synthesis of oligonucleotides. In one embodiment, the reagent of the present disclosure functions as a protecting group that protects the 3'-hydroxyl group of a nucleotide / oligonucleotide fragment. In another embodiment, the nucleotide, oligonucleotide fragment, or target oligonucleotide protected by the reagent of the present disclosure can be selectively precipitated from the reaction mixture. Thus, the nucleotide, oligonucleotide fragment, or target oligonucleotide can be readily recovered by filtration without chromatography.

[0043] In the first embodiment of the first aspect, the present disclosure relates to compounds of formula I' or B. [ka] or a salt thereof A 1 , A 2 , and A 3 One of them is Y A And the others are H, [ka] It is either a single bond or a double bond. Y A is Y-(CH2) a1 CH2O(CH2) a2 - and a1 and a2 are each independently 0 or integers from 1 to 10. Ring A is a 5-6 member heteroaryl having 1-3 heteroatoms independently selected from phenyl, an 8-10 member bicyclic aryl, oxygen, nitrogen, and sulfur, or a 7-10 member bicyclic heteroaryl having 1-4 heteroatoms independently selected from oxygen, nitrogen, and sulfur. Y stands for H, Halogen, OR 1A , NR 2A R 3A , SR 4A , CR 5A R 6A R 7A , or a hydrophobic group comprising one or more aliphatic hydrocarbon groups having 10 or more carbon atoms, R1A , R 2A , R 3A , R 4A , R 5A , R 6A , and R 7A are each independently C 1-6 alkyl, C 1-6 alkenyl, C 1-6 alkynyl, phenyl, OR 8A , -OC(O)R 8A , -C(O)OR 8A , NR 8A R 9A , -NR 8A COR 9A , -CONR 8A R 9A , 3- to 7-membered saturated or partially unsaturated monocyclic carbocyclyl, or 3- to 7-membered saturated or partially unsaturated monocyclic heterocyclyl having 1 to 2 heteroatoms independently selected from oxygen, nitrogen and sulfur; R 8A and R 9A for each occurrence are independently H or C 1-6 alkyl, P1 is NO2 or a silyl hydroxyl protecting group, R1 and R2 are independently H, C 1-6 alkyl, or phenyl, wherein C 1-6 alkyl and phenyl are optionally substituted with 1 to 3 R3, R3 is C 1-30 alkoxy, e is an integer from 0 to 6, f is an integer from 0 to 6, there is provided the compound, or a salt thereof.

[0044] In a second embodiment of the first aspect, according to the present disclosure, there is provided the compound of formula I' ## STRUCTURE ## , or a salt thereof, Ring A is a 5-6 membered heteroaryl having 1-3 heteroatoms independently selected from phenyl, an 8-10 membered bicyclic aryl, oxygen, nitrogen, and sulfur, or a 7-10 membered bicyclic heteroaryl having 1-4 heteroatoms selected from oxygen, nitrogen, and sulfur. Y stands for H, Halogen, OR 1A , NR 2A R 3A , SR 4A , CR 5A R 6A R 7A , or a hydrophobic group comprising one or more aliphatic hydrocarbon groups having 10 or more carbon atoms, R 1A , R 2A , R 3A , R 4A , R 5A , R 6A , and R 7A Independently, C 1-6 Alkyl, C 1-6 Alkenil, C 1-6 Alkinyl, phenyl, OR 8A -OC(O)R 8A , -C(O)OR 8A , NR 8A R 9A , -NR 8A COR 9A ,-CONR 8A R 9A , a 3-7 member saturated or partially unsaturated monocyclic carbocyclyl, or a 3-7 member saturated or partially unsaturated monocyclic heterocyclyl having 1-2 heteroatoms selected from oxygen, nitrogen, and sulfur, R 8A and R 9A Each instance, independently, H or C 1-6 It is alkyl, P1 is NO2 or a silylhydroxyl protecting group. R1 and R2 are independent of H and C 1-6 Alkyl or phenyl, C 1-6 Alkyl and phenyl groups can be optionally substituted with R3 groups 1-3. R3 is C 1-30 It is an alkoxy, e is an integer from 0 to 6. A compound or a salt thereof is provided, where f is an integer from 0 to 6.

[0045] In a third embodiment, the present disclosure relates to the compound of formula B. [ka] Or a salt thereof is provided. The remaining variables in formula B are described in the first embodiment.

[0046] In the fourth embodiment, the present disclosure relates to compounds of formula B-1 or B-2. [ka] Or a salt thereof is provided. The remaining variables in formula B are described in the third embodiment.

[0047] In a fifth embodiment, the present disclosure provides a compound or salt of formula I' or B, wherein Y is a hydrophobic group comprising one or more aliphatic hydrocarbon groups having 10 or more carbon atoms. The remaining variables in formula I' or B are described in the first, second, third, or fourth embodiment.

[0048] In the sixth embodiment, the present disclosure provides compounds of formula I' or salts thereof, wherein ring A is phenyl or naphthalenyl. The remaining variables in formula I' are described in the second and / or fifth embodiments.

[0049] In the seventh embodiment, the present disclosure provides compounds of formula I' or B or salts thereof, wherein P1 is a silylhydroxyl protecting group selected from the following: [ka] [ka] R5, R6, and R7 are independent of H and C respectively.1-30 Alkyl, or C 1-30 It is an alkoxy. The remaining parts of the variables in formula I' or B are described in any one of the first to sixth embodiments.

[0050] In the eighth embodiment, the present disclosure provides a compound of formula I' or B or a salt thereof, wherein P1 is selected from the group consisting of -O-TBDMS, -O-TIPS, -O-TBDPS, -O-TBoDPS, and -O-TBDAS. [ka] The remaining variables in Equation I' are described in one of the first to seventh embodiments.

[0051] In the ninth embodiment, the present disclosure relates to compounds of formula I or Ia. [ka] or a salt thereof P1 is provided as a compound or salt selected from the group consisting of -O-TBDPS, -O-TBoDPS, and -O-TBDAS. [ka] The remaining variables in formula I or Ia are described in the first, second, and / or fifth to eighth embodiments.

[0052] In the tenth embodiment, the present disclosure provides a compound or salt of formula I', B, or I, where Y is represented by formula A, [ka] Here, [ka] This represents the connection point to Y, W is represented by formulas A1, A2, A2-1, A2-2, A3, A3-1, or A3-2, [ka] Here, [ka] This indicates the point where W and V are connected. Each R w These are independently aliphatic hydrocarbon groups having 10 or more carbon atoms, k is an integer from 1 to 5. V is bond, oxygen, C 1-20 Alkylene, C 1-6 Alkynylene, -C(=O)-, ***-C(=O)-O-**, ***-OC(=O)-**, [ka] Alternatively, a 5-7 membered heteroaryl having 1-3 heteroatoms selected from oxygen, nitrogen, and sulfur, wherein the heteroaryl is optionally substituted with 1-3 R8 atoms. [ka] The symbol indicates the point where V and U connect, and R8 is H or C 1-30 It is alkyl, U represents bond, oxygen, and C 1-20 A 5-7 membered heterocyclyl having 1-3 heteroatoms selected from alkylene, carbonyl, ***-OC(=O)-**, oxygen, nitrogen, and sulfur; a 5-7 membered heteroaryl having 1-3 heteroatoms selected from oxygen, nitrogen, and sulfur, which is optionally substituted with 1-3 R8s; or a group represented by formula A4, A5, or A6. [ka] U1 is C 1-6 Alkylene, C 1-6Compounds or salts thereof are provided, which are 5-7 membered heterocyclyls having 1-3 heteroatoms selected from alkylene oxy, oxygen, nitrogen, and sulfur, or 5-7 membered heteroaryls having 1-3 heteroatoms selected from oxygen, nitrogen, and sulfur. The remaining variables in formulas I, Ia, B, or I' are described in any one of the first to ninth embodiments.

[0053] In the eleventh embodiment, the present disclosure provides a compound or salt of formula I', B, or formula I, wherein the TBDAS group is [ka] A compound or a salt thereof is provided, where s is an integer from 1 to 30. The remaining parts of the variables in formulas I, Ia, B, or I' are described in any one of embodiments 7 to 10.

[0054] In the twelfth embodiment, the present disclosure provides compounds or salts thereof of formula I', B, or formula I or Ia, wherein P1 is -O-TBDPS. The remaining variables in formula I, Ia, or formula I' or B are described in any one of the first to eleventh embodiments.

[0055] In the thirteenth embodiment, the present disclosure provides a compound or salt of formula I, Ia, B, or I', where W is represented by formula A1, [ka] R w C n H 2n+1 A compound or a salt thereof is provided, where n is an integer from 1 to 30. The remaining parts of the variables in formulas I, B, or I' are described in the tenth embodiment.

[0056] In the fourteenth embodiment, the present disclosure provides a compound of formula I, Ia, B, or I' or a salt thereof, R w C 12 H25 , C 18 H 37 , C 20 H 41 , C 22 H 45 , C 24 H 49 , C 26 H 53 , and C 28 H 57 A compound or a salt thereof, selected from the group consisting of the following, is provided. The remaining parts of the variables in formula I, IaB, or I' are described in any one of the 10th to 13th embodiments.

[0057] In the 15th embodiment, the present disclosure provides for a compound or salt of formula I, Ia, B, or I', where V is a bond, CH2, CH2CH2, C(=O), ***-C(=O)-O-**, or [ka] A compound or a salt thereof is provided. The remaining variables in formulas I, Ia, B, or I' are described in any one of the 10th to 14th embodiments.

[0058] In the sixteenth embodiment, the present disclosure provides for a compound or salt of formula I, Ia, B, or I', where U is a bond, CH2, CH2CH2, carbonyl, triazolylene, piperazine, [ka] A compound or a salt thereof is provided. The remaining parts of the variables in formula I, Ia, B, or I' are described in any one of the 10th to 15th embodiments.

[0059] In the seventeenth embodiment, the present disclosure provides a compound or salt of formula I, Ia, B, or I' in which UV is [ka] R8 is selected from the group consisting of H or C 1-6A compound or a salt thereof that is alkyl is provided. The remaining parts of the variables in formula I, B, or I' are described in any one of the 10th to 16th embodiments.

[0060] In the eighteenth embodiment, the present disclosure provides a compound of formula I or Ia or formula I' or B or a salt thereof, wherein Y is [ka] Selected from the group consisting of, R8 is H or C 1-6 It is alkyl, A compound or a salt thereof is provided in which m is an integer from 1 to 5. The remaining parts of the variables in formulas I', B, or I or Ia are described in any one of the first to twelfth embodiments.

[0061] In the 19th embodiment, the present disclosure provides a compound or salt of formula I or Ia or formula I', wherein R1 and R2 are independently H or CH3. The remaining variables in formula I or Ia or formula I' are described in the first, second, and / or fifth to eighteenth embodiments. In certain embodiments, both R1 and R2 are H. In other particular embodiments, both R1 and R2 are CH3.

[0062] In the 20th embodiment, the present disclosure provides a compound or salt of formula I' or formula B, wherein e is 0, 1, or 2 and f is 0, 1, or 2. The remaining variables in formula I' are described in the first, second, third, and / or fifth to 19th embodiments.

[0063] In the 21st embodiment, the present disclosure provides a compound or salt of formula I, Ia, I', or B, where R8 is H or C 1-4 A compound or a salt thereof that is alkyl is provided. The remaining parts of the variables in formula I, Ia, I', or B are described in any one of the 10th to 20th embodiments.

[0064] In the 22nd embodiment, the present disclosure relates to compounds of formula II or IIa. [ka] or a salt thereof t is an integer between 10 and 30. [ka] teeth, [ka] R8 is selected from the group consisting of H or C 1-6 A compound that is alkyl or a salt thereof is provided.

[0065] In the 23rd embodiment, the present disclosure provides that [ka] A compound of formula II or a salt thereof, selected from the group consisting of the following, is provided.

[0066] In the 24th embodiment, the present disclosure relates to the following compounds [ka] Or the salt is provided.

[0067] In the 25th embodiment, the present disclosure provides that [ka] A compound or a salt thereof selected from one of the above is provided. The remaining parts of the variables in the above formula are described in the first embodiment. In some embodiments, a1 and a2 are integers from 1 to 6, 1 to 5, or 1 to 4, respectively.

[0068] In the 26th embodiment, the present disclosure provides the compounds shown in Table 1 and prepared in the examples, both in their neutral form and as salts thereof. [Table 1]

[0069] 2.3'-Protected nucleotides or oligonucleotides In a second aspect, the disclosure describes nucleotides or oligonucleotides protected by a 3'-hydroxyl protecting group as described herein. In one embodiment, the 3'-hydroxyl protecting group is derived from the aforementioned reagent. In another embodiment, the protected nucleotide or oligonucleotide is separated by selective precipitation. In another embodiment, the protected nucleotide or oligonucleotide is soluble in nonpolar organic solvents such as dichloromethane but precipitates in polar organic solvents such as acetonitrile.

[0070] In the 27th embodiment, the present disclosure provides for nucleotides or oligonucleotides represented by formula III or IIIP. [ka] or a salt thereof Here, R 31 Each instance is independently a nucleic acid base, and the NH2 of the nucleic acid base is optionally protected by an amine protecting group, if present. R 32 Each time it appears, C 1-6 H, halo, OH, and C are optionally substituted by alkoxys. 1-6 Independently selected from the group consisting of alkoxys, the OH group is optionally protected by a hydroxyl protecting group. R 34 Each instance is, independently, either H or R 32 The alkoxy group forms a ring, R 35 It is a hydroxyl protecting group, R 36 Each instance, independently, H and C 1-6 Alkyl alkyl group, C 2-6The group is an alkenyl group, a phenyl group, or a benzyl group, which can be optionally substituted with -CN, -NO2, or a halogen, respectively. R 36 teeth, [ka] And, q is an integer between 1 and 20. In each instance, X is either O or S independently. Z is given by equation I * or a group represented by B*, [ka] Here, [ka] This represents a connection point to Z, A 1 , A 2 , and A 3 One of them is Y A And the others are H, [ka] It is either a single bond or a double bond. Y A is Y-(CH2) a1 CH2O(CH2) a2 - and a1 and a2 are each independently 0 or integers from 1 to 10. Ring A is a 5-6 member heteroaryl having 1-3 heteroatoms independently selected from phenyl, an 8-10 member bicyclic aryl, oxygen, nitrogen, and sulfur, or a 7-10 member bicyclic heteroaryl having 1-4 heteroatoms independently selected from oxygen, nitrogen, and sulfur. Y stands for H, Halogen, OR 1A , NR 2A R 3A , SR 4A , CR 5A R 6A R 7A, or a hydrophobic group comprising one or more aliphatic hydrocarbon groups having 10 or more carbon atoms, R 1A , R 2A , R 3A , R 4A , R 5A , R 6A , and R 7A Each is independent of C 1-6 Alkyl, C 1-6 Alkenil, C 1-6 Alkinyl, phenyl, OR 8A -OC(O)R 8A , -C(O)OR 8A , NR 8A R 9A , -NR 8A COR 9A ,-CONR 8A R 9A , a 3-7 member saturated or partially unsaturated monocyclic carbocyclyl, or a 3-7 member saturated or partially unsaturated monocyclic heterocyclyl having 1-2 heteroatoms independently selected from oxygen, nitrogen, and sulfur, R 8A and R 9A Each instance, independently, H or C 1-6 It is alkyl, P1 is NO2 or a silylhydroxyl protecting group. R1 and R2 are independent of H and C 1-6 Alkyl or phenyl, C 1-6 Alkyl and phenyl groups can be optionally substituted with R3 groups 1-3. R3 is C 1-30 It is an alkoxy, e is an integer from 0 to 6. A nucleotide or oligonucleotide is provided, where f is an integer between 0 and 6.

[0071] In the 28th embodiment, the nucleotide or oligonucleotide represented by formula III' or IIIP' in this disclosure is used. [ka] or a salt thereof Q is a hydroxyl protecting group, [ka] This is a nucleic acid base containing an NH2 group modified by Z, R 31 Each instance is independently a nucleic acid base, and the NH2 of the nucleic acid base is optionally protected by an amine protecting group, if present. R 32 Each time it appears, C 1-6 H, halo, OH, and C are optionally substituted by alkoxys. 1-6 Independently selected from the group consisting of alkoxys, the OH group is optionally protected by a hydroxyl protecting group. R 34 Each instance is, independently, either H or R 32 The alkoxy group forms a ring, R 35 It is a hydroxyl protecting group, R 36 Each instance, independently, H and C 1-6 Alkyl alkyl group, C 2-6 The group is an alkenyl group, a phenyl group, or a benzyl group, which can be optionally substituted with -CN, -NO2, or a halogen, respectively. R 36 teeth, [ka] And, q is an integer between 1 and 20. In each instance, X is either O or S independently. Z is given by equation I * or B * It is a base represented by, [ka] Here, [ka] This represents a connection point to Z, A 1 , A 2 , and A 3One of them is Y A And the others are H, [ka] It is either a single bond or a double bond. Y A is Y-(CH2) a1 CH2O(CH2) a2 - and a1 and a2 are each independently 0 or integers from 1 to 10. Ring A is a 5-6 member heteroaryl having 1-3 heteroatoms independently selected from phenyl, an 8-10 member bicyclic aryl, oxygen, nitrogen, and sulfur, or a 7-10 member bicyclic heteroaryl having 1-4 heteroatoms independently selected from oxygen, nitrogen, and sulfur. Y stands for H, Halogen, OR 1A , NR 2A R 3A , SR 4A , CR 5A R 6A R 7A , or a hydrophobic group comprising one or more aliphatic hydrocarbon groups having 10 or more carbon atoms, R 1A , R 2A , R 3A , R 4A , R 5A , R 6A , and R 7A Each is independent of C 1-6 Alkyl, C 1-6 Alkenil, C 1-6 Alkinyl, phenyl, OR 8A -OC(O)R 8A , -C(O)OR 8A , NR 8A R 9A , -NR 8A COR 9A ,-CONR 8A R 9A , a 3-7 member saturated or partially unsaturated monocyclic carbocyclyl, or a 3-7 member saturated or partially unsaturated monocyclic heterocyclyl having 1-2 heteroatoms independently selected from oxygen, nitrogen, and sulfur, R 8A and R 9AEach instance, independently, H or C 1-6 It is alkyl, P1 is NO2 or a silylhydroxyl protecting group. R1 and R2 are independent of H and C 1-6 Alkyl or phenyl, C 1-6 Alkyl and phenyl groups can be optionally substituted with R3 groups 1-3. R3 is C 1-30 It is an alkoxy, e is an integer from 0 to 6. A nucleotide or oligonucleotide is provided, where f is an integer between 0 and 6.

[0072] In one embodiment, R 32 The hydroxyl protecting group is a silyl protecting group. In some embodiments, the silyl protecting group is selected from the group consisting of trimethylsilyl, triethylsilyl, triisopropylsilyl, dimethylisopropylsilyl, diethylisopropylsilyl, dimethyltexylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, trynzylsilyl, tri-p-xylylsilyl, triphenylsilyl, diphenylmethylsilyl, di-t-butylmethylsilyltri(trimethylsilyl)silyl, t-butylmethoxyphenylsilyl, and t-butoxydiphenylsilyl.

[0073] In the 29th embodiment, the present disclosure provides a nucleotide or oligonucleotide or salt thereof represented by formula III, III', IIIP, or IIIP', where Z is formula I * A nucleotide or oligonucleotide or a salt thereof, which is a group represented by , is provided. [ka] The remaining variables in formulas III, III', IIIP, or IIIP' are described in the 27th and / or 28th embodiments.

[0074] In the 30th embodiment, the present disclosure provides a nucleotide or oligonucleotide or salt thereof represented by formula III, III', IIIP, or IIIP', where Z is formula B * A nucleotide or oligonucleotide or a salt thereof, which is a group represented by , is provided. [ka] The remaining variables in formulas III, III', IIIP, or IIIP' are described in the 27th and / or 28th embodiments.

[0075] In the 31st embodiment, the present disclosure provides a nucleotide or oligonucleotide or salt thereof represented by formula III, III', IIIP, or IIIP', where Z is formula B-1 * Or B-2 * A nucleotide or oligonucleotide or a salt thereof, which is a group represented by , is provided. [ka] The remaining variables in formulas III, III', IIIP, or IIIP' are described in the 27th and / or 28th embodiments.

[0076] In the 32nd embodiment, the present disclosure provides a nucleotide or oligonucleotide or a salt thereof represented by formula III, III', IIIP, or IIIP', wherein Y is a hydrophobic group comprising one or more aliphatic hydrocarbon groups having 10 or more carbon atoms. The remaining variables in formula III, III', IIIP, or IIIP' are described in the 27th and / or 28th embodiments.

[0077] In the 33rd embodiment, the present disclosure provides nucleotides or oligonucleotides or salts thereof represented by formula III, III', IIIP, or IIIP', wherein ring A is phenyl or naphthalenyl. The remaining variables in formula III, III', IIIP, or IIIP' are described in the 27th, 28th, and / or 32nd embodiments.

[0078] In the 34th embodiment, the present disclosure provides for a nucleotide or oligonucleotide or salt thereof represented by formula III, III', IIIP, or IIIP', wherein P1 is [ka] A silylhydroxyl protecting group selected from, [ka] R5, R6, and R7 are independent of H and C respectively. 1-30 Alkyl, or C 1-30 A nucleotide or oligonucleotide or a salt thereof that is an alkoxy is provided. The remaining variables in formulas III, III', IIIP, or IIIP' are described in any one of embodiments 27 to 33.

[0079] In the 35th embodiment, the present disclosure provides a nucleotide or oligonucleotide or a salt thereof represented by formula III, III', IIIP, or IIIP', wherein P1 is selected from the group consisting of -O-TBDMS, -O-TIPS, -O-TBDPS, -O-TBoDPS, and -O-TBDAS. [ka] The remaining variables in equations III, III', IIIP, or IIIP' are described in any one of embodiments 27 through 34.

[0080] In the 36th embodiment, the present disclosure provides a nucleotide or oligonucleotide or salt thereof represented by formula III, III', IIIP, or IIIP', where Z is formula I** or Ia ** [ka] A nucleotide or oligonucleotide or a salt thereof is provided, where P1 is selected from the group consisting of -O-TBDPS, -O-TBoDPS, and -O-TBDAS, and is represented by the group or a salt thereof. [ka] The remaining variables in equations III, III', IIIP, or IIIP' are described in any one of embodiments 27 through 35.

[0081] In the 37th embodiment, the present disclosure provides a nucleotide or oligonucleotide or salt thereof represented by formula III, III', IIIP, or IIIP', where Y is formula A [ka] Represented by, [ka] This represents the connection point to Y, W is represented by formulas A1, A2, A2-1, A2-2, A3, A3-1, or A3-2, [ka] Here, [ka] This indicates the point where W and V are connected. Each R w These are independently aliphatic hydrocarbon groups having 10 or more carbon atoms, k is an integer from 1 to 5. V is bond, oxygen, C 1-20 Alkylene, C 1-6 Alkynylene, -C(=O)-, ***-C(=O)-O-**, ***-OC(=O)-**, [ka] Alternatively, a 5-7 membered heteroaryl having 1-3 heteroatoms selected from oxygen, nitrogen, and sulfur, wherein the heteroaryl is optionally substituted with 1-3 R8 atoms. [ka] The symbol indicates the point where V and U connect, and R8 is H or C 1-30 It is alkyl, U represents bond, oxygen, and C 1-20 A 5-7 membered heterocyclyl having 1-3 heteroatoms selected from alkylene, carbonyl, ***-OC(=O)-**, oxygen, nitrogen, and sulfur; a 5-7 membered heteroaryl having 1-3 heteroatoms selected from oxygen, nitrogen, and sulfur, which is optionally substituted with 1-3 R8s; or a group represented by formula A4, A5, or A6. [ka] U1 is C 1-6 Alkylene, C 1-6 Nucleotides or oligonucleotides or salts thereof are provided, which are nucleotides or oligonucleotides or salts thereof

[0082] In the 38th embodiment, the present disclosure provides for a nucleotide or oligonucleotide or salt thereof represented by formula III, III', IIIP, or IIIP', wherein the TBDAS group is [ka] A nucleotide or oligonucleotide or a salt thereof is provided, where s is an integer from 1 to 30. The remaining parts of the variables in formulas III, III', IIIP, or IIIP' are described in any one of embodiments 34 to 37.

[0083] In the 39th embodiment, the present disclosure provides a nucleotide or oligonucleotide or a salt thereof represented by formula III, III', IIIP, or IIIP', where P1 is TBDPS. The remaining variables in formula III, III', IIIP, or IIIP' are described in any one of the 27th to 37th embodiments.

[0084] In the 40th embodiment, the present disclosure provides a nucleotide or oligonucleotide or salt thereof represented by formula III, III', IIIP, or IIIP', where W is formula A1 [ka] Represented by R w C n H 2n+1 A nucleotide or oligonucleotide or a salt thereof is provided, where n is an integer from 1 to 30. The remaining parts of the variables in formulas III, III', IIIP, or IIIP' are described in the 37th embodiment.

[0085] In the 41st embodiment, the present disclosure provides for a nucleotide or oligonucleotide or salt thereof represented by formula III, III', IIIP, or IIIP', where R w C 12 H 25 , C18 H 37 , C 20 H 41 , C 22 H 45 , C 24 H 49 , C 26 H 53 , and C 28 H 57 A nucleotide or oligonucleotide or a salt thereof, selected from the group consisting of the following, is provided. The remaining parts of the variables in formula III, III', IIIP, or IIIP' are described in the 37th and / or 40th embodiment.

[0086] In the 42nd embodiment, the present disclosure provides a nucleotide or oligonucleotide or salt thereof represented by formula III, III', IIIP, or IIIP', where V is a bond, CH2, CH2CH2, C(=O)-, ***-C(=O)-O-**, or [ka] A nucleotide or oligonucleotide or a salt thereof is provided. The remaining variables in formulas III, III', IIIP, or IIIP' are described in any one of embodiments 37 to 41.

[0087] In the 43rd embodiment, the present disclosure provides a nucleotide or oligonucleotide or salt thereof represented by formula III, III', IIIP, or IIIP', where U is a bond, CH2, CH2CH2, carbonyl, triazolylene, piperazine, [ka] A nucleotide or oligonucleotide or a salt thereof is provided. The remaining variables in formulas III, III', IIIP, or IIIP' are described in any one of embodiments 37 to 42.

[0088] In the 44th embodiment, the present disclosure provides for a nucleotide or oligonucleotide or salt thereof represented by formula III, III', IIIP, or IIIP', wherein UV is [ka] R8 is selected from the group consisting of H or C 1-6 Alkyl nucleotides or oligonucleotides or salts thereof are provided. The remaining variables in formulas III, III', IIIP, or IIIP' are described in any one of embodiments 37 to 41.

[0089] In the 45th embodiment, the present disclosure provides a nucleotide or oligonucleotide represented by formula III, III', IIIP, or IIIP', wherein Y is [ka] Selected from the group consisting of, R8 is H or C 1-6 It is alkyl, A nucleotide or oligonucleotide or a salt thereof is provided, where m is an integer from 1 to 5. The remaining variables in equations III, III', IIIP, or IIIP' are described in any one of embodiments 27 through 39.

[0090] In the 46th embodiment, the present disclosure provides a nucleotide or oligonucleotide or a salt thereof represented by formula III, III', IIIP, or IIIP', wherein R1 and R2 are independently H or CH3. The remaining variables in formula III, III', IIIP, or IIIP' are described in any one of embodiments 27 to 45. In a particular embodiment, both R1 and R2 are H. In another particular embodiment, both R1 and R2 are CH3.

[0091] In the 47th embodiment, the present disclosure provides a nucleotide or oligonucleotide or a salt thereof represented by formula III, III', IIIP, or IIIP', where e is 0, 1, or 2 and f is 0, 1, or 2. The remaining variables in formula III, III', IIIP, or IIIP' are described in any one of embodiments 27 to 46.

[0092] In the 48th embodiment, the present disclosure provides a nucleotide or oligonucleotide or salt thereof represented by formula III, III', IIIP, or IIIP', where R8 is H or C 1-4 Alkyl nucleotides or oligonucleotides or salts thereof are provided. The remaining variables in formula III or IIIP are described in the 37th embodiment. In one embodiment, R8 is H or methyl.

[0093] In the 49th embodiment, the present disclosure provides a nucleotide or oligonucleotide or salt thereof represented by formula III, III', IIIP, or IIIP', where Z is formula II* or IIa. * [ka] Represented by, t is an integer between 10 and 30. [ka] teeth, [ka] R8 is selected from the group consisting of H or C 1-6 Alkyl nucleotides or oligonucleotides or salts thereof are provided. The remaining variables in formulas III, III', IIIP, or IIIP' are described in the 27th and / or 28th embodiments.

[0094] In the 50th embodiment, the present disclosure provides for a nucleotide or oligonucleotide or salt thereof represented by formula III, III', IIIP, or IIIP', where Z is [ka] A nucleotide or oligonucleotide or a salt thereof is provided. The remaining variables in formulas III, III', IIIP, or IIIP' are described in the 49th embodiment.

[0095] In the 51st embodiment, the present disclosure provides for a nucleotide or oligonucleotide or salt thereof represented by formula III, III', IIIP, or IIIP', where Z is [ka] A nucleotide or oligonucleotide or a salt thereof is provided. The remaining variables in formulas III, III', IIIP, or IIIP' are described in the 27th and / or 28th embodiments.

[0096] In the 52nd embodiment, the present disclosure provides for a nucleotide or oligonucleotide or salt thereof represented by formula III, III', IIIP, or IIIP', where Z is [ka] A nucleotide or oligonucleotide or a salt thereof is provided. The remaining variables in equations III, III', IIIP, or IIIP' are described in the 27th and / or 28th embodiments. In some embodiments, a1 and a2 are integers between 1 and 6, 1 and 5, or 1 and 4, respectively.

[0097] In the 53rd embodiment, the present disclosure provides a nucleotide or oligonucleotide or salt thereof represented by formula III, III', IIIP, or IIIP', where X is S, and the phosphorothiolate group has the following S-configuration: [ka] or The following R-configuration is observed: [ka] Here, [ka] This indicates a connection point to the 3'-OH group, [ka] This provides a nucleotide or oligonucleotide or a salt thereof that exhibits a connection point to a 5'-OH group. The remaining parts of the variables in formulas III, III', IIIP, or IIIP' are described in any one of embodiments 27 to 52.

[0098] In one embodiment, the present disclosure provides for a nucleotide or oligonucleotide or salt thereof represented by formula III, III', IIIP, or IIIP', where R 31 Each instance of provides a nucleotide or oligonucleotide or a salt thereof, which is adenine (A), guanine (G), thymine (T), cytosine (C), or uracil (U). The remaining parts of the variables in formulas III, III', IIIP, or IIIP' are described in any one of embodiments 27 to 53.

[0099] In one embodiment, the present disclosure provides for a nucleotide or oligonucleotide or salt thereof represented by formula III, III', IIIP, or IIIP', where R 32Each occurrence independently provides a nucleotide or oligonucleotide or a salt thereof, which is H, F, Cl, Br, I, or -OCH2CH2OMe. The remaining parts of the variables in formulas III, III', IIIP, or IIIP' are described in any one of embodiments 27 to 53. In certain embodiments, R 32 Each instance is independently either H or -OCH2CH2OMe.

[0100] In one embodiment, the present disclosure provides for a nucleotide or oligonucleotide or salt thereof represented by formula III, III', IIIP, or IIIP', where R 34 A nucleotide or oligonucleotide or a salt thereof is provided, where is H. The remaining parts of the variables in formulas III, III', IIIP, or IIIP' are described in any one of embodiments 27 to 53.

[0101] In one embodiment, the present disclosure provides for a nucleotide or oligonucleotide or salt thereof represented by formula III, III', IIIP, or IIIP', where R 35 A nucleotide or oligonucleotide or a salt thereof is provided, wherein is 4,4'-dimethoxytrityl. The remaining parts of the variables in formulas III, III', IIIP, or IIIP' are described in any one of embodiments 27 to 53.

[0102] In one embodiment, the present disclosure provides for a nucleotide or oligonucleotide or salt thereof represented by formula III, III', IIIP, or IIIP', where R 36 A nucleotide or oligonucleotide or a salt thereof is provided, which is -CH2CH2CN. The remaining parts of the variables in formulas III, III', IIIP, or IIIP' are described in any one of embodiments 27 to 53.

[0103] In one embodiment, the present disclosure provides for a nucleotide or oligonucleotide or salt thereof represented by formula III, III', IIIP, or IIIP', where R 32 A nucleotide or oligonucleotide or a salt thereof is provided, which is -OCH2CH2OMe. The remaining parts of the variables in formulas III, III', IIIP, or IIIP' are described in any one of embodiments 27 to 53.

[0104] 3. Process for preparing oligonucleotide fragments In a third aspect, the disclosure describes a process for preparing oligonucleotide fragments having a hydroxyl protecting group at the 3'-terminus (e.g., a hydrophobic hydroxyl protecting group) (where the fragment has a hydrophobic hydroxyl protecting group, it may be referred to herein as a “3'-fragment”) or having an amino protecting group on the nucleic acid base (where the nucleic acid base contains an NH2 group). These may be referred to herein as “nucleic acid base SiLHPG fragment”. Surprisingly, it has been found that the methods of the disclosure for synthesizing 3'-fragments or nucleic acid base SiLHPG fragments can be used to prepare oligonucleotide fragments having 3 to 20 (e.g., 3 to 10, 3 to 8, 3 to 5, or 4 to 5) nucleotides with high purity without chromatographic purification. In some embodiments, a hydrophobic 3'-hydroxyl protecting group is used to facilitate the separation of the oligonucleotide fragment product by selective precipitation. In some embodiments, a hydrophobic amino protecting group is used to facilitate the separation of the oligonucleotide fragment product by selective precipitation. In some embodiments, the liquid-phase process includes (1) a 5'-OH deprotection step, (2) a coupling step, and (3) an oxidation or sulfidation step, and steps (1), (2), and (3) are repeated until a desired number of nucleotides are joined together to form a 3'-oligonucleotide fragment.

[0105] In the 54th embodiment, the present disclosure provides an oligonucleotide fragment of formula (V) [ka] or a process for preparing the salt thereof, 1) Compound of formula (VA) [ka] Alternatively, the salt can be deprotected to obtain the compound of formula (VB). [ka] or the step of forming a salt thereof, 2) A compound of formula (VB) or a salt thereof, and a compound of formula (VC) [ka] Alternatively, react it with its salt to form the compound of formula (VD). [ka] or the step of forming a salt thereof, 3) A compound of formula (VD) or a salt thereof is sulfurized or oxidized with a sulfurizing or oxidizing agent to obtain a compound of formula (VE). [ka] or the step of forming a salt thereof, 4) Deprotect the compound of formula (VE) or its salt to obtain the compound of formula (VF). [ka] or the step of forming a salt thereof, 5) When q is 2 or greater, the step of starting with a compound of formula (VF) and repeating steps 2), 3), and 4) q-2 times, followed by steps 2) and 3) to obtain a fragment of formula (V) or a salt thereof, R 31 Each instance is independently a nucleic acid base, and the NH2 of the nucleic acid base is protected by an amine protecting group, if present. R 32 Each time it appears, C 1-6H, halo, OH, and C are optionally substituted by alkoxys. 1-6 Independently selected from the group consisting of alkoxys, the OH group is optionally protected by a hydroxyl protecting group. R 34 Each instance is, independently, either H or R 32 The alkoxy group forms a ring, R 35 It is a hydroxyl protecting group, R 36 Each instance, independently, C 1-6 Alkyl alkyl group, C 2-6 The group is an alkenyl group, a phenyl group, or a benzyl group, which can be optionally substituted with -CN, -NO2, or a halogen, respectively. R 36 teeth [ka] And, R 37a and R 37b Independently, C 1-6 It is alkyl, q is an integer between 1 and 20. In each instance, X is either O or S independently. Z is given by equation I * or a group represented by B*, [ka] Here, [ka] This represents a connection point to Z, A 1 , A 2 , and A 3 One of them is Y A And the others are H, [ka] It is either a single bond or a double bond. Y A is Y-(CH2) a1CH2O(CH2) a2 - and a1 and a2 are each independently 0 or integers from 1 to 10. Ring A is a 5-6 member heteroaryl having 1-3 heteroatoms independently selected from phenyl, an 8-10 member bicyclic aryl, oxygen, nitrogen, and sulfur, or a 7-10 member bicyclic heteroaryl having 1-4 heteroatoms independently selected from oxygen, nitrogen, and sulfur. Y stands for H, Halogen, OR 1A , NR 2A R 3A , SR 4A , CR 5A R 6A R 7A , or a hydrophobic group comprising one or more aliphatic hydrocarbon groups having 10 or more carbon atoms, R 1A , R 2A , R 3A , R 4A , R 5A , R 6A , and R 7A Each is independent of C 1-6 Alkyl, C 1-6 Alkenil, C 1-6 Alkinyl, phenyl, OR 8A -OC(O)R 8A , -C(O)OR 8A , NR 8A R 9A , -NR 8A COR 9A ,-CONR 8A R 9A , a 3-7 member saturated or partially unsaturated monocyclic carbocyclyl, or a 3-7 member saturated or partially unsaturated monocyclic heterocyclyl having 1-2 heteroatoms independently selected from oxygen, nitrogen, and sulfur, R 8A and R 9A Each instance, independently, H or C 1-6 It is alkyl, P1 is NO2 or a silylhydroxyl protecting group. R1 and R2 are independent of H and C 1-6 Alkyl or phenyl, C 1-6 Alkyl and phenyl groups can be optionally substituted with R3 groups 1-3. R3 is C 1-30 It is an alkoxy, e is an integer from 0 to 6. A process is provided where f is an integer between 0 and 6.

[0106] In the 55th embodiment, the present disclosure provides an oligonucleotide fragment of formula (V') [ka] or a process for preparing the salt thereof, 1) Compound of formula (VA) [ka] Alternatively, the salt can be deprotected to obtain the compound of formula (VB). [ka] or the step of forming a salt thereof, 2) A compound of formula (VB) or a salt thereof, and a compound of formula (VC') [ka] Alternatively, react it with its salt to form the compound of formula (VD'). [ka] or the step of forming a salt thereof, 3) The compound of formula (VD') or its salt is sulfurized or oxidized with a sulfidating or oxidizing agent to obtain the compound of formula (VE'). [ka] or the step of forming a salt thereof, 4) Deprotect the compound of formula (VE') or its salt to obtain the compound of formula (VF'). [ka] or the step of forming a salt thereof, 5) When q is 2 or greater, the step of starting with a compound of formula (VF') and repeating steps 2), 3), and 4) q-2 times, followed by steps 2) and 3) to obtain a fragment of formula (V') or a salt thereof, R 31 , R 32 , R 34 , R 35 In the 54th embodiment, a process is provided in which q, X, and Z are as previously described for formula (V).

[0107] In the 56th embodiment, the present disclosure provides an oligonucleotide fragment of formula (V-C1) or (V-C2). [ka] or a process for preparing the salt thereof, 1) Compound of formula (VB) [ka] or a salt thereof, of the compound of formula (V-CR1) or (V-CR2). [ka] The step of reacting the salt thereof with a base to form a compound of formula (V-C1) or (V-C2), R 31 , R 32 , R 34 , R 35 , R 36 A process is provided in the 54th embodiment in which q, X, and Z are as previously described for formula (V). The reaction of formula (VB) with (V-CR1) forms a compound of formula (V-C1), and the reaction of formula (VB) with (V-CR2) forms a compound of formula (V-C2).

[0108] In the 57th embodiment, the present disclosure relates to oligonucleotide fragments of formula (V-C1) or (V-C2). [ka] or a process for preparing the salt thereof, 1) Compound of formula (VB) [ka] Alternatively, a salt thereof can be used as a reagent of formula (VR1) or (VR2). [ka] Reacting with it to form a compound of formula (V-CR3) or (V-CR4) [ka] or the step of forming a salt thereof, 2) A compound of formula (V-CR3) or (V-CR4) or a salt thereof, and a compound of formula (VG) [ka] The step of reacting the salt or a base with the salt or a base to form a compound of formula (V-C1) or (V-C2), 31 , R 32 , R 34 , R 35 , R 36 A process is provided in the 54th embodiment in which q, X, and Z are as previously described for formula (V). Reaction of reagent (VR1) with the compound of formula (VB) forms the compound of formula (V-CR3), which reacts with the compound of formula (VG) to form the compound of formula (V-C1). Reaction of reagent (VR2) with the compound of formula (VB) forms the compound of formula (V-CR4), which reacts with the compound of formula (VG) to form the compound of formula (V-C2).

[0109] In the 58th embodiment, the present disclosure relates to an oligonucleotide fragment of formula (VBZ). [ka] or a process for preparing the salt thereof, 1) Compound of formula (VBZ-1) [ka] or its salt, compound of formula (VBZ-2) [ka] Alternatively, by reacting it with its salt, the compound of formula (VBZ-3) can be obtained. [ka] or the step of forming a salt thereof, 2) The step of sulfiding or oxidizing a compound of formula (VBZ-3) or a salt thereof with a sulfidating or oxidizing agent to form a compound of formula (VBZ) or a salt thereof, Here, Q is a hydroxyl protecting group, [ka] This is a nucleic acid base containing an NH2 group modified by Z, and R 31 , R 32 , R 34 , R 35 , R 36 , R 37a , R 37b In the 54th embodiment, a process is provided in which q, X, and Z are as previously described for formula (V).

[0110] In the 59th embodiment, the present disclosure provides a process for preparing an oligonucleotide fragment of formula (VBZ) or a salt thereof as described in the 58th embodiment, wherein the compound of formula VBZ-1 is 1) Compound of formula (VBZ-4) [ka] Alternatively, reacting its salt with Z-OH yields the compound of formula VBZ-5. [ka] or to form a salt thereof, 2) A process is provided for preparing the compound by deprotecting the compound of formula (VBZ-5) to form the compound of formula (VBZ-1).

[0111] In the 60th embodiment, the present disclosure provides a process for preparing oligonucleotide fragments or salts thereof of formula (V), (V'), (V-C1), (V-C2), or (VBZ) as described in embodiments 54 to 59, wherein Y is a hydrophobic group comprising one or more aliphatic hydrocarbon groups having 10 or more carbon atoms. The remaining variables in formula (V), (V'), (V-C1), (V-C2), or (VBZ) are described in any one of embodiments 54 to 59.

[0112] In the 61st embodiment, the present disclosure provides a process for preparing oligonucleotide fragments or salts thereof of formula (V), (V'), (V-C1), (V-C2), or (VBZ) as described in the 54th to 59th embodiments, the process being one that does not use chromatography to purify the reaction product of any one of steps 1), 2), 3), and 4).

[0113] In the 62nd embodiment, the present disclosure provides a process for preparing oligonucleotide fragments or salts thereof of formula (V), (V'), (V-C1), (V-C2), or (VBZ) as described in embodiments 54 to 59, wherein the process involves purifying any one of the reaction products from steps 1), 2), 3), and 4) by selective precipitation. In some embodiments, selective precipitation of any one of the reaction products or salts thereof from steps 1), 2), 3), and 4) can be achieved by adding acetonitrile to a solution of the crude product in DCM. Alternatively, the desired product can be precipitated and separated by adding acetonitrile to a solution of the crude product.

[0114] In some embodiments, one of the reaction products or salts from steps 1), 2), 3), and 4) is added to selective precipitation, and the solution containing one of the reaction products or salts from steps 1), 2), 3), and 4) in an organic solvent (MBTE, SiO, heptane / MBTE mixture, DCM, etc.) is purified by extraction with an aqueous solution (e.g., NaHCO3 / H2O or NaCl / H2O). In some embodiments, the extraction is performed before selective precipitation. Alternatively, the extraction is performed after selective precipitation. In some embodiments, selective precipitation of one of the reaction products or salts from steps 1), 2), 3), and 4) can be achieved by adding heptane or a heptane / MBTE mixture to a DCM or SiO solution of the crude product. Alternatively, the solution of the crude product can be added to heptane or a heptane / MBTE mixture to precipitate and separate the desired product. A heptane / MBTE mixture can be used with an appropriate volume ratio (e.g., the volume ratios described herein).

[0115] In the 63rd embodiment, the present disclosure provides an oligonucleotide fragment of formula (V) [ka] or a process for preparing the salt thereof, a) Nucleotides of formula (V-1) [ka] or its salt, Oligonucleotide fragment of formula (V-2) [ka] or its salt, Coupling in solution yields the oligonucleotide fragment of formula (V-3). [ka] or the step of forming a salt thereof, b) Sulfurize or oxidize the oligonucleotide of formula (V-3) or a salt thereof to obtain the oligonucleotide of formula (V). [ka] or a step of forming a salt thereof, Here, R 31 Each instance is independently a nucleic acid base, and the NH2 of the nucleic acid base is protected by an amine protecting group, if present. R 32 Each time it appears, C 1-6 H, halo, OH, and C are optionally substituted by alkoxys. 1-6 Independently selected from the group consisting of alkoxys, the OH group is optionally protected by a hydroxyl protecting group. R 34 Each instance is, independently, either H or R 32 The alkoxy group forms a ring, R 35 It is a hydroxyl protecting group, R 36 Each instance, independently, C 1-6 Alkyl alkyl group, C 2-6 The group is an alkenyl group, a phenyl group, or a benzyl group, which can be optionally substituted with -CN, -NO2, or a halogen, respectively. R 36 teeth, [ka] And, R 37a and R 37b Independently, C 1-6 It is alkyl, q is an integer between 1 and 20. In each instance, X is either O or S independently. Z is given by equation I * or B * It is a base represented by, [ka] Here, [ka] This represents a connection point to Z, A 1 , A 2 , and A 3 One of them is Y A And the others are H, [ka] It is either a single bond or a double bond. Y A is Y-(CH2) a1 CH2O(CH2) a2 - and a1 and a2 are each independently 0 or integers from 1 to 10. Ring A is a 5-6 membered heteroaryl having 1-3 heteroatoms independently selected from phenyl, an 8-10 membered bicyclic aryl, oxygen, nitrogen, and sulfur, or a 7-10 membered bicyclic heteroaryl having 1-4 heteroatoms selected from oxygen, nitrogen, and sulfur. Y stands for H, Halogen, OR 1A , NR 2A R 3A , SR 4A , CR 5A R 6A R 7A , or a hydrophobic group comprising one or more aliphatic hydrocarbon groups having 10 or more carbon atoms, R 1A , R 2A , R 3A , R 4A , R 5A , R 6A , and R 7A Independently, C 1-6 Alkyl, C 1-6 Alkenil, C 1-6 Alkinyl, phenyl, OR 8A -OC(O)R 8A , -C(O)OR 8A , NR 8A R 9A , -NR 8A COR 9A ,-CONR 8A R 9A, a 3-7 member saturated or partially unsaturated monocyclic carbocyclyl, or a 3-7 member saturated or partially unsaturated monocyclic heterocyclyl having 1-2 heteroatoms selected from oxygen, nitrogen, and sulfur, R 8A and R 9A Each instance, independently, H or C 1-6 It is alkyl, P1 is NO2 or a silylhydroxyl protecting group. R1 and R2 are independent of H and C 1-6 Alkyl or phenyl, C 1-6 Alkyl and phenyl groups can be optionally substituted with R3 groups 1-3. R3 is C 1-30 It is an alkoxy, e is an integer from 0 to 6. A process is provided where f is an integer between 0 and 6.

[0116] In the 64th embodiment, the present disclosure provides an oligonucleotide fragment of formula (V*) [ka] or a process for preparing the salt thereof, a) Nucleotides of formula (V-1) [ka] or its salt, Oligonucleotide fragment of formula (V-2') [ka] or its salt, Coupling in solution yields the oligonucleotide fragment of formula (V-3'). [ka] or the step of forming a salt thereof, b) The step of sulfiding or oxidizing an oligonucleotide of formula (V-3') or a salt thereof to form an oligonucleotide of formula (V*) or a salt thereof, R31 , R 32 , R 34 , R 35 , R 36 , R 37a , R 37b In the 63rd embodiment, a process is provided in which q, X, and Z are as previously described for formula (V).

[0117] In the 65th embodiment, the present disclosure provides a process for preparing an oligonucleotide fragment or salt thereof of formula (V) or (V*) as described in the 63rd or 64th embodiment, wherein Y is a hydrophobic group comprising one or more aliphatic hydrocarbon groups having 10 or more carbon atoms.

[0118] In the 66th embodiment, the present disclosure provides a process for preparing an oligonucleotide fragment or salt of formula (V) as described in the 54th or 63rd embodiment, comprising deprotecting the fragment of formula (V) to obtain a deprotected fragment of formula (VH). [ka] A process is provided which further includes forming a salt thereof.

[0119] In the 67th embodiment, the present disclosure provides a process for preparing an oligonucleotide fragment or salt thereof of formula (V') as described in the 55th embodiment, comprising deprotecting the fragment of formula (V') to obtain a deprotected fragment of formula (VH'). [ka] A process is provided which further includes forming a salt thereof.

[0120] In the 68th embodiment, the present disclosure provides a process for preparing oligonucleotide fragments or salts of formula (V-C1) or (V-C2) as described in the 56th or 57th embodiment, comprising deprotecting the fragment of formula (V-C1) or (V-C2) to obtain a deprotected fragment of formula (V-C3) or (V-C4). [ka] or its salt, [ka] A process is provided which further includes forming a salt thereof.

[0121] In the 69th embodiment, the present disclosure provides a process for preparing an oligonucleotide fragment or salt of formula (VBZ) as described in the 58th embodiment, comprising deprotecting the fragment of formula (VBZ) to obtain a deprotected fragment of formula (VBZ-6). [ka] A process is provided which further includes forming a salt thereof.

[0122] In the 70th embodiment, the present disclosure provides a process for preparing an oligonucleotide fragment or salt of formula (V*) as described in the 64th embodiment, comprising deprotecting the fragment of formula (V*) to obtain a deprotected fragment of formula (V*-1). [ka] A process is provided which further includes forming a salt thereof.

[0123] In the 71st embodiment, the present disclosure provides a process for preparing oligonucleotide fragments or salts of formula (V), (V'), (V-C1), (V-C2), (VBZ), or (V*) as described in embodiments 54 to 64, comprising desilylation of the fragments of formula (V), (V'), (V-C1), (V-C2), (VBZ), or (V*) to obtain fragments of formula (VJ), (VJ'), (V-C5), (V-C6), (VBZ-7), or (V*-2), respectively. [ka] or its salt, [ka] or its salt, [ka] or its salt, [ka] or its salt, [ka] or its salt, [ka] A process is provided which further comprises forming a salt thereof. In one embodiment, when Q and P1 in formula (VBZ) are the same, a desilylation reaction is performed to form formula (VBZ-7'). [ka] The compound is formed.

[0124] In the 72nd embodiment, the present disclosure provides a process for preparing fragments of formula (VJ), (VJ'), (V-C5), (V-C6), (VBZ-7), or (V*-2) as described in any one of the 71st embodiments, wherein the desilylation reaction is carried out by reacting a compound of formula (V), (V'), (V-C1), (V-C2), (VBZ), or (V*) with HF in the presence of a base.

[0125] In the 73rd embodiment, the present disclosure provides a process as described in the 72nd embodiment, wherein the base is imidazole or pyridine, and the imidazole or pyridine is optionally substituted. In one embodiment, pyridine and / or imidazole is a halogen, C 1-6 Alkyl, C 1-6 Alkoxy, -OH, and C 1-6 Each of the 1-3 substituents selected from the haloalkyl group is independently substituted.

[0126] In the 74th embodiment, the present disclosure provides a process described in the 73rd embodiment, wherein the desilylation reaction is carried out by reacting a compound of formula (V), (V'), (V-C1), (V-C2), (VBZ), or (V*) with HF in the presence of pyridine and imidazole.

[0127] In the 75th embodiment, the present disclosure provides a process as described in the 74th embodiment, wherein the molar ratio of imidazole to HF is in the range of 0.5:1 to 10:1.

[0128] In the 76th embodiment, the present disclosure provides a process as described in the 75th embodiment, wherein the molar ratio of imidazole to HF is in the range of 1.1:1 to 5:1.

[0129] In the 77th embodiment, the present disclosure provides a process as described in the 76th embodiment, wherein the molar ratio of imidazole to HF is in the range of 2:1.

[0130] In the 78th embodiment, the present disclosure provides a process as described in the 74th to 77th embodiments, wherein the molar ratio of pyridine to HF is in the range of 100:1 to 1:1.

[0131] In the 79th embodiment, the present disclosure provides a process as described in the 74th to 77th embodiments, wherein the molar ratio of pyridine to HF is in the range of 1:1.

[0132] In the 80th embodiment, the present disclosure provides a process described in any one of the 54th to 71st embodiments, wherein the fragments for formulas (V), (V'), (V-C1), (V-C2), (VBZ), (V*), (VH), (VH'), (V-C3), (V-C4), (VBZ-6), (V*-1), (VJ), (VJ'), (V-C5), (V-C6), (VBZ-7), (VBZ-7'), or (V*-2) are not purified by chromatography.

[0133] In the 81st embodiment, the present disclosure provides a process for purifying fragments of formulas (V), (V'), (V-C1), (V-C2), (VBZ), (V*), (VH), (VH'), (V-C3), (V-C4), (VBZ-6), (V*-1), (VJ), (VJ'), (V-C5), (V-C6), (VBZ-7), (VBZ-7'), or (V*-2) by selective precipitation and / or extraction.

[0134] In the 82nd embodiment, the present disclosure provides a process described in any one of the 54th to 81st embodiments, wherein q is 2 to 5.

[0135] In the 83rd embodiment, the present disclosure provides a process described in any one of the 82nd embodiments, wherein q is 4.

[0136] In one embodiment, for the process described in the third aspect or any embodiment described herein (for example, embodiments 65 to 75), the variable R 31 , R 32 , R 34 , R 35 , R 36 q, and / or Z are described in the second aspect or any one of the embodiments described herein (for example, embodiments 27 to 33).

[0137] In some embodiments, for the process described in the third aspect or any one of the embodiments described therein (e.g., embodiments 54 to 83), the 5'-OH deprotection step is a detritylation method for removing the 5'-trityl group. It has been found that when the detritylation reaction is carried out under anhydrous or substantially anhydrous conditions, a significant reduction in side reactions (e.g., deamination of nucleic acid bases cytosine or 5-methylcytosine or their derivatives, which are widely used in oligonucleotide synthesis) can be achieved. This detritylation method also includes the addition of a cation scavenger to accelerate the completion of the reaction. As a result, a high-purity product can be obtained without the need for chromatography (e.g., column chromatography). The water level in the detritylation reaction can be controlled by using a drying agent (e.g., molecular sieves), azeotropic distillation, or other suitable methods known in the art. Alternatively, the solvent, acid, and other reagents used in the detritylation reaction, the substrate to be subjected to the detritylation reaction, and the reaction vessel can be dried to a residual water level before use in the detritylation reaction.

[0138] In one embodiment, for the process described in the third embodiment or any one of the embodiments described herein (for example, embodiments 54 to 83), R 36 It is one of the following: [ka] See Nat Biotechnol. 2017 Sep;35(9):845-851; J.Org.Chem. 1999,64,7515-7522; Biopolymers (Peptide Science), 2001,60,3. These are incorporated herein by reference, respectively.

[0139] In some embodiments, the 5'-OH deprotection (or detritylation) reaction is carried out in the presence of a desiccant for the process described in the third aspect or any one of the embodiments described therein (for example, embodiments 54 to 83). Any suitable desiccant can be used in the deprotection reaction. In some embodiments, the desiccant is selected from calcium chloride, potassium chloride, sodium sulfate, calcium sulfate, magnesium sulfate, and molecular sieves.

[0140] In one embodiment, the desiccant is a molecular sieve for the process described in the third embodiment or any one of the embodiments described herein (for example, embodiments 54 to 83).

[0141] In one embodiment, the molecular sieve size is 3 Å or 4 Å for the process described in the third aspect or any one of the embodiments described therein (for example, embodiments 54 to 83). In one embodiment, the molecular sieve size is 3 Å.

[0142] In one embodiment, for the process described in the third aspect or any one of the embodiments described therein (for example, embodiments 54 to 83), an anhydrous or substantially anhydrous solution for the deprotection reaction is obtained by removing water using azeotropic distillation before the deprotection reaction.

[0143] Alternatively, the solvent, acid or acidic solution, other reagents or solutions including the reagent to be used in the detritylation reaction, the substrate or substrate solution to be subjected to the detritylation reaction, and the reaction vessel can be dried separately or combined before the detritylation reaction.

[0144] In one embodiment, for the process described in the third aspect or any one of the embodiments described therein (for example, embodiments 54 to 83), the deprotection reaction is carried out in the presence of a scavenger selected from cationic scavengers including -SH groups, silane scavengers (e.g., HSiPh3, HSiBu3, triisopropylsilane, etc.), siloxanes, polystyrene, furan, pyrrole, and indole.

[0145] In one embodiment, the deprotection reaction is carried out in the presence of a scavenger selected from 1-dodecanethiol, cyclohexanethiol, 1-octanthiol, triisopropylsilane, indole, 2,3-dimethylfuran, diphenylsilane, 2-mercaptoimidazole, diphenylmethylsilane, phenylsilane, 5-methoxyindole, methylphenylsilane, chlorodimethylsilane, 1,1,3,3-tetramethyldisiloxane, 1-thioglycerol, triphenylsilane, tert-butyldimethylsilane, butylsilane, methyldiethoxysilane, 1,1,3,3,5,5-hexamethyltrisiloxane, hexylsilane, (mercaptomethyl)polystyrene, or dimethylphenylsilane.

[0146] In one embodiment, the cation scavenger is a compound of the formula RSH, where R is an alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl group, each of which can be optionally substituted.

[0147] In one embodiment, the cation scavenger is CH3(CH2)5SH, CH3(CH2) 11 SH, cyclohexanethiol (CySH), or CH3CH2OC(=O)CH2CH2SH.

[0148] In one embodiment, for the process described in the third embodiment or any one of the embodiments described herein (for example, embodiments 54 to 83), R 35 This is a 4,4'-dimethoxytrityl (DMT) group.

[0149] In some embodiments, the deprotection reaction is carried out by reacting a compound of formula (VA) with a detritylated reagent, relative to the process described in the third aspect or any one of the embodiments described herein (for example, embodiments 54 to 83). Any suitable detritylated reagent can be used.

[0150] In one embodiment, the detritylation reagent is a strong organic acid.

[0151] In one embodiment, the detritylation reagent is selected from CF3COOH, CCl3COOH, CHCl2COOH, CH2ClCOOH, H3PO4, methanesulfonic acid (MSA), benzenesulfonic acid (BSA), CClF2COOH, CHF2COOH, PhSO2H (phenylsulfinic acid), etc. In a preferred embodiment, the detritylation reagent is CH2ClCOOH. In another specific embodiment, the detritylation reagent is CF3COOH. In yet another specific embodiment, the detritylation reagent is CHCl2COOH.

[0152] In one embodiment, the detritylation reagent is citric acid. In another embodiment, the detritylation reagent is a saturated citric acid solution.

[0153] In some embodiments, the coupling reaction of step 2) can be carried out in the presence of an activator described herein (for example, the activator described in Embodiment 39) for the process described in the third embodiment or any one of the embodiments described herein (for example, Embodiments 54 to 83). In some embodiments, the activator is 4,5-dicyanoimidazole (DCI) or 5-ethylthio-1H-tetrazole (ETT).

[0154] In some embodiments, for the process described in the third aspect or any one of the embodiments described herein (for example, embodiments 54 to 83), the sulfurization reaction in step 3) is carried out using a sulfurizing agent such as 3-amino-1,2,4-dithiazole-5-thione (xanthan hydride or ADTT), 3-(N,N-dimethylamino-methylidene)amino)-3H-1,2,4-dithiazole (DDTT), phenylacetyl disulfide (PADS), 3H-1,2-benzodithiol-3-one 1,1-dioxide (Buechage reagent), or phenyl-3H-1,2,4-dithiazole-3-one (POS). In certain embodiments, the sulfurizing agent is DDTT. In certain embodiments, the sulfurizing agent is xanthan hydride. In some embodiments, the sulfurization reaction is carried out in the presence of a base as described herein. In some embodiments, the base is pyridine or imidazole. In one embodiment, the sulfurization reaction in step 3) is carried out in the presence of DDTT and 4,5-dicyanoimidazole (DCI).

[0155] In some embodiments, for the process described in the third aspect or any one of the embodiments described therein (for example, embodiments 54 to 83), the oxidation reaction in step 3) is carried out by using a standard oxidizing agent known in the literature. Typical oxidizing agents include, but are not limited to, tert-butyl hydroperoxide (t-BuOOH), (1S)-(+)-(10-camphorsulfonyl)oxaziridine (CSO), (1R)-(-)-(10-camphorsulfonyl)oxaziridine (an enantiomer of CSO), I2, and iodine-pyridine-hydroxylizer solutions. In certain embodiments, the oxidizing agent is t-BuOOH.

[0156] In some embodiments, the coupling / oxidation / detritylation step is carried out in a one-pot reaction for the process described in the third embodiment or any one of the embodiments described therein (for example, embodiments 54 to 83). In some embodiments, the oxidizing agent in the one-pot reaction is BPO or tBuOOH. [ka]

[0157] 4. Process for preparing target oligonucleotides In a fourth aspect, the disclosure describes a process for preparing a target oligonucleotide, wherein the target oligonucleotide is assembled in a 3'-terminal to 5'-terminal direction (3'-5' direction). The process of the disclosure has been demonstrated to be successfully used for the large-scale synthesis of target oligonucleotides. In addition, high-purity, protected target oligonucleotides can be obtained by the method of the disclosure without chromatographic purification.

[0158] In some embodiments, the process described herein involves synthesizing a target oligonucleotide by stepwise adding oligonucleotide fragments to a liquid (solution) phase. For example, pentamer and tetramer fragments are first coupled to synthesize a necaper fragment, which is then further reacted with another pentamer fragment to synthesize a decamer oligonucleotide. The decamer oligonucleotide can be further coupled with other fragments until a target oligonucleotide of the desired length is obtained. In some embodiments, a pentamer fragment having a 3'-hydrophobic hydroxyl protecting group (3'-LHPG) (3'-terminal fragment) or an amino protecting group in the nucleic acid base (when the nucleic acid base contains an NH2 group; hereafter referred to as "nucleic acid base LHPG fragment") is first coupled with a pentamer fragment to form a decamer fragment having a 3'-LHPG group or a nucleic acid base LHPG group, which is then further reacted with a tetramer fragment to form a decamer fragment, which is then coupled with another tetramer fragment to form a target 18-mer oligonucleotide. In one embodiment, a 3'-terminal fragment having n nucleotides (e.g., a pentamer fragment) is synthesized by coupling a single nucleotide having a 3'-LHPG group with a fragment having n-1 nucleotides (e.g., a tetramer fragment). In another embodiment, a nucleic acid base LHPG fragment having n nucleotides (e.g., a pentamer fragment) is synthesized by coupling a single nucleotide having an LHPG group in the nucleic acid base with a fragment having n-1 nucleotides (e.g., a tetramer fragment).

[0159] In the 84th embodiment, the present disclosure provides an oligonucleotide of formula (VI) or (VI-1). [ka] or a process for preparing the salt thereof, a) Oligonucleotide fragment of formula (F1) or (F1-1) [ka] or its salt, Oligonucleotide fragment of formula (F2) [ka] or its salt, Coupling in solution to oligonucleotide fragments of formula (F3) or (F3-1) [ka] or to form a salt thereof, b) sulfurizing or oxidizing an oligonucleotide fragment or salt of formula (F3) or (F3-1) to form an oligonucleotide or salt of formula (VI) or (VI-1), Here, Q is a hydroxyl protecting group, [ka] This is a nucleic acid base containing an NH2 group modified by Z, R 31 Each instance is independently a nucleic acid base, and the NH2 of the nucleic acid base is protected by an amine protecting group, if present. R 32 Each time it appears, C 1-6 H, halo, OH, and C are optionally substituted by alkoxys. 1-6 Independently selected from the group consisting of alkoxys, the OH group is optionally protected by a hydroxyl protecting group. R 34 Each instance is, independently, either H or R 32 The alkoxy group forms a ring, R 35 It is a hydroxyl protecting group, R 36 Each instance, independently, C 1-6 Alkyl alkyl group, C 2-6 The group is an alkenyl group, a phenyl group, or a benzyl group, which can be optionally substituted with -CN, -NO2, or a halogen, respectively. R 36 teeth, [ka] And, R 37a and R 37b Independently, C 1-6 It is alkyl, p is an integer between 2 and 20. o is an integer between 1 and 200. In each instance, X is either O or S independently. Z is given by equation I * or B * It is a base represented by, [ka] Here, [ka] This represents a connection point to Z, A 1 , A 2 , and A 3 One of them is Y A And the others are H, [ka] It is either a single bond or a double bond. Y A is Y-(CH2) a1 CH2O(CH2) a2 - and a1 and a2 are each independently 0 or integers from 1 to 10. Ring A is a 5-6 member heteroaryl having 1-3 heteroatoms independently selected from phenyl, an 8-10 member bicyclic aryl, oxygen, nitrogen, and sulfur, or a 7-10 member bicyclic heteroaryl having 1-4 heteroatoms independently selected from oxygen, nitrogen, and sulfur. Y stands for H, Halogen, OR 1A , NR 2A R 3A , SR 4A , CR 5A R 6A R 7A, or a hydrophobic group comprising one or more aliphatic hydrocarbon groups having 10 or more carbon atoms, R 1A , R 2A , R 3A , R 4A , R 5A , R 6A , and R 7A Each is independent of C 1-6 Alkyl, C 1-6 Alkenil, C 1-6 Alkinyl, phenyl, OR 8A -OC(O)R 8A , -C(O)OR 8A , NR 8A R 9A , -NR 8A COR 9A ,-CONR 8A R 9A , a 3-7 member saturated or partially unsaturated monocyclic carbocyclyl, or a 3-7 member saturated or partially unsaturated monocyclic heterocyclyl having 1-2 heteroatoms independently selected from oxygen, nitrogen, and sulfur, R 8A and R 9A Each instance, independently, H or C 1-6 It is alkyl, P1 is NO2 or a silylhydroxyl protecting group. R1 and R2 are independent of H and C 1-6 Alkyl or phenyl, C 1-6 Alkyl and phenyl groups can be optionally substituted with R3 groups 1-3. R3 is C 1-30 It is an alkoxy, e is an integer from 0 to 6. A process is provided where f is an integer between 0 and 6.

[0160] In the 85th embodiment, the present disclosure provides an oligonucleotide of formula (VI') or (VI'-1). [ka] or a process for preparing the salt thereof, a) Oligonucleotide fragment of formula (F1) or (F1-1) [ka] or a salt thereof, a fragment of oligonucleotide formula (F2') [ka] Alternatively, a salt thereof can be coupled in solution with an oligonucleotide fragment of formula (F3') or (F3'-1). [ka] or to form a salt thereof, b) sulfurizing or oxidizing an oligonucleotide fragment or salt of formula (F3') or (F3'-1) to form an oligonucleotide or salt of formula (VI') or (VI'-1), Q, [ka] R 31 , R 32 , R 34 , R 35 , R 36 , R 37a , and R 37b In the 84th embodiment, a process is provided in which p, o, X, and Z are as previously described for formula (VI) or (VI-1).

[0161] In the 86th embodiment, the present disclosure provides a process for preparing oligonucleotides of formula (VI), (VI'), (VI-1), or (VI'-1) as described in the 84th or 85th embodiment, wherein Y is a hydrophobic group comprising one or more aliphatic hydrocarbon groups having 10 or more carbon atoms.

[0162] In the 87th embodiment, the present disclosure provides a process for preparing oligonucleotides of formula (VI), (VI'), (VI-1), or (VI'-1) as described in the 84th or 85th embodiment, comprising deprotecting the oligonucleotide of formula (VI), (VI'), (VI-1), or (VI'-1) to obtain oligonucleotides of formula (VII), (VII-1), (VII'), or (VII'-1). [ka] [ka] A process is provided which further includes step c) forming a salt thereof.

[0163] In the 88th embodiment, the present disclosure provides a process for preparing oligonucleotides of formula (VII), (VII-1), (VII'), or (VII'-1) as described in the 87th embodiment, further comprising starting with an oligonucleotide of formula (VII), (VII-1), (VII'), or (VII'-1), repeating steps a), b), and c) 1 to 10 times, followed by step a), and b), to form a target oligonucleotide of a desired length.

[0164] In the 89th embodiment, the present disclosure provides a process described in the 88th embodiment, further comprising repeating steps a), b), and c) one to three times, followed by steps a) and b), to form a target oligonucleotide of a desired length.

[0165] In the 90th embodiment, the present disclosure provides a process as described in the 84th to 89th embodiments, wherein o is an integer from 2 to 20.

[0166] In the 91st embodiment, the present disclosure provides a process as described in the 90th embodiment, wherein o is an integer from 2 to 5.

[0167] In the 92nd embodiment, the present disclosure provides the process described in the 91st embodiment, wherein o is 4.

[0168] In the 93rd embodiment, the process described in the Third or Fourth Embodiment (for example, any one of Embodiments 54 to 92) is described herein, wherein Z is Formula I * A process that is the basis represented by is provided. [ka]

[0169] In the 94th embodiment, the process described in the Third or Fourth Embodiment (for example, any one of Embodiments 54 to 92) is described herein, wherein Z is the formula B * A process that is the basis represented by is provided. [ka]

[0170] In the 95th embodiment, the process described in the Third or Fourth Embodiment (for example, any one of Embodiments 54 to 92) is described herein, wherein Z is formula B-1 * Or B-2 * A process that is the basis represented by is provided. [ka]

[0171] In the 96th embodiment, the present disclosure provides a process described in a third or fourth embodiment (for example, any one of embodiments 54 to 92) wherein ring A is phenyl or naphthalenyl.

[0172] In the 97th embodiment, the process described in the third or fourth embodiment (for example, any one of embodiments 54 to 96) is described herein, wherein P1 is [ka] A silyl hydroxyl protecting group selected from, where represents the connection point to P1, and R5, R6, and R7 are independently H, C 1-30 Alkyl, or C 1-30 A process that is alkoxy is provided.

[0173] In the 98th embodiment, the present disclosure provides a process described in a third or fourth embodiment (for example, any one of embodiments 54 to 97), wherein P1 is a process selected from the group consisting of -O-TBDMS, -O-TIPS, -O-TBDPS, -O-TBoDPS, and -O-TBDAS. [ka]

[0174] In the 99th embodiment, the process described in the Third or Fourth Embodiment (for example, any one of Embodiments 54 to 93) is described herein, wherein Z is Formula I ** or Ia ** [ka] or a group represented by a salt thereof, P1 is -O-TBDPS, -O-TBoDPS, and -O-TBDAS [ka] Selected from the group consisting of, R5, R6, and R7 are each independently H, C 1-30 Alkyl, or C 1-30 A process that is alkoxy is provided.

[0175] In the 100th embodiment, the process described in the present disclosure in a third or fourth embodiment (for example, any one of embodiments 54 to 99), wherein Y is formula A [ka] Represented by, Here, [ka] This represents the connection point to Y, W is a formula A1, A2, A2-1, A2-2, A3, A3-1, or A3-2 [ka] Represented by, Here, [ka] This indicates the point where W and V are connected. Each R w These are independently aliphatic hydrocarbon groups having 10 or more carbon atoms, k is an integer from 1 to 5. V is bond, oxygen, C 1-20 Alkylene, C 1-6 Alkynylene, -C(=O)-, ***-C(=O)-O-**, ***-OC(=O)-**, [ka] Alternatively, a 5-7 membered heteroaryl having 1-3 heteroatoms selected from oxygen, nitrogen, and sulfur, wherein the heteroaryl is optionally substituted with 1-3 R8 atoms. [ka] The symbol indicates the point where V and U connect, and R8 is H or C 1-30 It is alkyl, U represents bond, oxygen, and C 1-20A 5-7 member heterocycline having 1-3 heteroatoms selected from alkylene, carbonyl, ***-OC(=O)-**, oxygen, nitrogen, and sulfur; a 5-7 member heteroaryl having 1-3 heteroatoms selected from oxygen, nitrogen, and sulfur, which is optionally substituted with 1-3 R8s; or formulas A4, A5, or A6 [ka] It is a base represented by, U1 is C 1-6 Alkylene, C 1-6 A process is provided in which the cyclyl is a 5-7 member having 1-3 heteroatoms selected from alkylene oxy, oxygen, nitrogen, and sulfur, or a 5-7 member heteroaryl having 1-3 heteroatoms selected from oxygen, nitrogen, and sulfur.

[0176] In the 101st embodiment, the process described in any one of the 97th to 100th embodiments of this disclosure, wherein the TBDAS group is [ka] And, A process is provided where s is an integer between 1 and 30.

[0177] In the 102nd embodiment, the present disclosure provides a process as described in embodiments 54 to 100, wherein P1 is TBDPS.

[0178] In the 103rd embodiment, the process described in the 100th to 102nd embodiments is reproduced herein, wherein W is the formula A1 [ka] Represented by, R w C n H 2n+1 And, A process is provided where n is an integer between 1 and 30.

[0179] In the 104th embodiment, the present disclosure provides a process described in the 100th to 103rd embodiments, R w C 12 H 25 , C 18 H 37 , C 20 H 41 , C 22 H 45 , C 24 H 49 , C 26 H 53 , and C 28 H 57 A process is provided that is selected from a group consisting of the following.

[0180] In the 105th embodiment, the process described in the 100th to 104th embodiments of this disclosure is described, where V is a bond, CH2, CH2CH2, C(=O)-, ***-C(=O)-O-**, or [ka] A process is provided that is as follows.

[0181] In the 106th embodiment, the present disclosure relates to the process described in embodiments 54 to 100, wherein Y is [ka] Selected from the group consisting of, R8 is H or C 1-6 It is alkyl, A process is provided where m is an integer between 1 and 5.

[0182] In the 107th embodiment, the present disclosure provides a process as described in a third or fourth embodiment (for example, embodiments 54 to 106), wherein R1 and R2 are independently H or CH3. In a particular embodiment, both R1 and R2 are H. In another particular embodiment, both R1 and R2 are CH3.

[0183] In the 108th embodiment, the present disclosure provides a process as described in a third or fourth embodiment (for example, embodiments 54 to 107), wherein e is 0, 1, or 2 and f is 0, 1, or 2.

[0184] In the 109th embodiment, the present disclosure provides a process as described in the third or fourth embodiment (for example, embodiments 54 to 108), wherein e is 1 and f is 1.

[0185] In the 110th embodiment, the present disclosure provides a process as described in the third or fourth embodiment (for example, embodiments 54 to 108) where e is 0 and f is 1, or e is 1 and f is 0.

[0186] In the 111th embodiment, the process described in the Third or Fourth Embodiment (for example, Embodiments 54 to 110) is described herein, wherein R8 is H or C 1-4 A process that is alkyl is provided.

[0187] In the 112th embodiment, the process described in the third or fourth embodiment (for example, embodiments 54 to 111) is described herein, wherein Z is formula II * or IIa * [ka] Represented by, t is an integer between 10 and 30. [ka] teeth, [ka] R8 is selected from the group consisting of H or C 1-6 A process that is alkyl is provided.

[0188] In the 113th embodiment, the process described in the third or fourth embodiment (for example, embodiments 54 to 112) is described herein, where Z is [ka] A process is provided that is as follows.

[0189] In the 114th embodiment, the process described in the third or fourth embodiment (for example, embodiments 54 to 93) is described herein, where Z is [ka] The process.

[0190] In the 115th embodiment, the process described in the third or fourth embodiment (for example, embodiments 54 to 93) of this disclosure, wherein Z is [ka] The process.

[0191] In the 116th embodiment, the present disclosure provides a nucleotide or oligonucleotide or process as described in the 27th to 53rd embodiments or as described in the 54th to 115th embodiments, wherein all P=X groups in the nucleotide or oligonucleotide are P=S.

[0192] In the 117th embodiment, the present disclosure provides a nucleotide or oligonucleotide or process as described in the 27th to 53rd embodiments or as described in the 54th to 115th embodiments, wherein all P=X groups in the nucleotide or oligonucleotide are P=O.

[0193] In the 118th embodiment, the present disclosure provides a nucleotide or oligonucleotide or process as described in the 27th to 53rd embodiments or as described in the 54th to 115th embodiments, wherein 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or more than 90% of the P=X groups in the compound or oligonucleotide are P=S.

[0194] In the 119th embodiment, the present disclosure provides a nucleotide or oligonucleotide or process as described in the 27th to 53rd embodiments or as described in the 54th to 115th embodiments, wherein 10 to 90%, 20 to 80%, 30 to 70%, or 40 to 60% of the P=X groups in the compound or oligonucleotide are P=S.

[0195] In the 120th embodiment, the present disclosure provides a nucleotide or oligonucleotide or process as described in the embodiments 27 to 53 or as described in the embodiments 54 to 116, wherein the nucleic acid base is selected from the group consisting of cytosine, guanine, adenine, thymine, uracil, hypoxanthine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine, and 5-hydroxymethylcytosine, and the NH2 group of the nucleic acid base is protected, if present, by PhCO-, CH3CO-, iPrCO-, Me2N-CH=, or Me2N-CMe=.

[0196] In the 121st embodiment, the present disclosure provides a nucleotide or oligonucleotide or process as described in the 27th to 53rd embodiments or as described in the 54th to 115th embodiments, wherein the nucleic acid base is selected from the group consisting of cytosine, guanine, adenine, thymine, uracil, and 5-methylcytosine, and the NH2 group of the nucleic acid base is protected, if present, by PhCO-, CH3CO-, iPrCO-, Me2N-CH=, or Me2N-CMe=.

[0197] In the 122nd embodiment, the present disclosure provides for a nucleotide or oligonucleotide as described in the 27th to 53rd embodiments or a process as described in the 54th to 121st embodiments, Each R 32 C 1-4 H, F, and C are optionally substituted by alkoxys. 1-4 Independently selected from the group consisting of alkoxys, Each R 34 Independently, H or R 2 The alkoxy group forms a ring, and the ring has 1-3 C 1-4 A 5- or 6-membered ring that can be optionally substituted with an alkyl group. Each R 35 This is a 4,4'-dimethoxytirtyl group, R 36 It is -CH2CH2CN, R 37a and R 37b Independently, C 1-4 A alkyl nucleotide or oligonucleotide or process is provided.

[0198] In the 123rd embodiment, the present disclosure provides for a nucleotide or oligonucleotide as described in the 27th to 53rd embodiments or a process as described in the 54th to 121st embodiments, Each R 32 These were independently selected from the group consisting of H, F, -OCH3, -OCH2CH2OCH3, and -OTBDMS. Each R 34 Independently, H or R 32 A nucleotide or oligonucleotide or process is provided, which forms a ring with an alkoxy group, and the ring is a five-membered ring.

[0199] In the 124th embodiment, the present disclosure provides for a nucleotide or oligonucleotide as described in embodiments 27 to 53 or a process as described in embodiments 54 to 121, where each R 34 Independently, H or R 32 A nucleotide or oligonucleotide or process is provided, which forms a -CH2-O- with an alkoxy group.

[0200] In the 125th embodiment, the present disclosure provides for a nucleotide or oligonucleotide as described in the 27th to 53rd embodiments or a process as described in the 54th to 121st embodiments, Each R 32 It is independently selected from H or -OCH2CH2OMe, Each R 34 H is H, Each R 35 This is a 4,4'-dimethoxytirtyl group, R 36 It is -CH2CH2CN, R 37a and R 37b Both are provided as nucleotides or oligonucleotides or processes, which are -CH(CH3)2.

[0201] In the 126th embodiment, the present disclosure provides a process as described in the 55th, 64th, or 85th embodiment, wherein the salt of the compound of formula (VD'), (V-2'), or (F2') is selected from trimethylamine salts, triethylamine salts, and triisopropylamine salts.

[0202] In the 127th embodiment, the present disclosure provides a process as described in the 126th embodiment, wherein the salt of the compound of formula (VD'), (V-2'), or (F2') is a triethylamine salt.

[0203] In the 128th embodiment, the present disclosure provides for a nucleotide or oligonucleotide described in a second embodiment (for example, the 28th embodiment) or a process described in a third or fourth embodiment (for example, any one of the 58th, 59th, 69th, and 71st to 92nd embodiments), [ka] A nucleotide or oligonucleotide or process is provided, which is adenine, cytosine, or guanine.

[0204] In the 129th embodiment, the present disclosure provides a nucleotide or oligonucleotide or process as described in a second embodiment (for example, the 28th embodiment) or a third or fourth embodiment (for example, any one of the 58th, 59th, 69th, and 71st to 92nd embodiments), wherein Q is a silyl protecting group.

[0205] In the 130th embodiment, the present disclosure provides a nucleotide or oligonucleotide or process as described in a second embodiment (for example, embodiment 28) or a third or fourth embodiment (for example, any one of embodiments 58, 59, 69, and 71 to 92), wherein Q is selected from the group consisting of trimethylsilyl, triethylsilyl, triisopropylsilyl, dimethylisopropylsilyl, diethylisopropylsilyl, dimethyltexylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, trybendylsilyl, tri-p-xylylsilyl, triphenylsilyl, diphenylmethylsilyl, di-t-butylmethylsilyltri(trimethylsilyl)silyl, t-butylmethoxyphenylsilyl, and t-butoxydiphenylsilyl.

[0206] In the 131st embodiment, the present disclosure provides a nucleotide or oligonucleotide or process as described in a second embodiment (e.g., the 28th embodiment) or a third or fourth embodiment (e.g., any one of the 58th, 59th, 69th, and 71st to 92nd embodiments), wherein Q is t-butyldiphenylsilyl.

[0207] In one embodiment, for the process described in the fourth aspect or any embodiment described therein (for example, embodiments 84 to 115), the variable R 31 , R 32 , R 34 , R 35 , R 36 q, and / or Z are described in the second aspect or any one of the embodiments described herein (for example, embodiments 27 to 53).

[0208] In one embodiment, for the process described in the fourth aspect or any embodiment described therein (for example, embodiments 84 to 115), the 5'-OH deprotection (or detritylation) step, the coupling step, and the oxidation or sulfidation step are carried out under the conditions described in the third aspect or any one of the embodiments described therein (for example, embodiments 54 to 83).

[0209] In some embodiments, if X is S for a nucleotide or oligonucleotide described in the second embodiment or any embodiment described therein, or for a process described in the third or fourth embodiment or any embodiment described therein, the phosphorothiolate group may have an S-configuration, an R-configuration, or a mixture thereof (e.g., a racemic mixture). [Examples]

[0210] abbreviation ACN = Acetonitrile Calcd = Calculated value DBU=8-diazabicyclo[5.4.0]undeca-7-ene DCA = CHCl2COOH or dichloroacetic acid DCM = Dichloromethane DDTT = 3-(N,N-dimethylamino-methylidene)amino)-3H-1,2,4-dithiazole DCI = 4,5-dicyanoimidazole DIEA = N,N-diisopropylethylamine DMT or DMTr = 4,4'-dimethoxytrityl or bis-(4-methoxyphenyl)phenylmethyl DMSO = Dimethyl sulfoxide methoxy or EA = ethyl acetate ETT = 5-ethylthio-1H-tetrazol h or hr = time HBTU = 3-[bis(dimethylamino)methylumyl]-3H-benzotriazole-1-oxidehexafluorophosphate HOBt = Hydroxybenzotriazole imid = imidazole IPAC = Isopropylacetate iPrOH = Isopropyl alcohol MOE = Methoxyethyl MS = Molecular Sieve MTBE or TBME = Methyl tert-butyl ether NMI = N-methylimidazole TBS = tert-butyldimethylsilyl Py = Pyridine RBF = Round-bottom flask RT=retention time TBAF = Tetra-n-butylammonium fluoride TBuAA = Tributylamine Acetate TBDPSCl=tert-butyl(chloro)diphenylsilane TCA = Trichloroacetic Acid TEA = Triethylamine TEAB = Tetraethylammonium bromide TFA = Trifluoroacetic acid THF = Tetrahydrofuran TLC = Thin-Layer Calculation Tol = Tol

[0211] Example 1. Synthesis of compound M19 a. Synthesis scheme for compound M19 [ka] [ka]

[0212] b. Procedure for the synthesis of compound M19

[0213] General procedure for the preparation of compound 2 [ka]

[0214] A mixture of compound 1 (1.20 kg, 6.52 mol, 1.00 equivalent) and K2CO3 (5.40 kg, 39.1 mol, 6.00 equivalent) in DMF (12 L) was to be mixed with 1-bromooctadecane (8.69 kg, 26.1 mol, 4.00 equivalent) in one addition under N2 conditions at 25°C. The mixture was stirred at 90°C for 16 hours. TLC (dichloromethane / methanol = 5 / 1, starting material, R) was performed. f =0.52, product, R f A reading of 0.88 indicated that no starting material was detected. 18 L of H2O was added, the mixture was cooled to 25°C, filtered, and washed with 7 L of H2O and 10 L of acetone. The solid was recrystallized at 55°C for 1 hour using 30 L of n-heptane. The mixture was cooled to 25°C, filtered, and the solid was washed with 5 L of n-heptane. Compound 2 (8.60 kg, crude) was obtained as a white solid.

[0215] General procedure for the preparation of compound 3 [ka]

[0216] A mixture of compound 2 (3.00 kg, 3.19 mol, 1.00 equivalent) in EtOH (15 L) was to be suddenly mixed with a solution of KOH (268 g, 4.78 mol, 1.50 equivalent) in H2O (3 L) at 25°C under N2 conditions. The mixture was stirred at 80°C for 16 hours. TLC (petroleum ether / ethyl acetate = 5 / 1, starting material R) was used. f =0.33, product R f A result of 0.86 indicated that no starting material was detected. The pH was adjusted to 2-3 with 2N HCl (6 L), cooled to 25°C, and poured into 75 L of H2O. The solid was filtered and washed with 20 L of H2O and 10 L of acetone. It was dried in an oven at 50°C for 24 hours. It was dissolved in 28 L of DCM and triturated at 40°C for 1 hour. It was cooled to 25°C. It was filtered and washed with 40 L of MeOH. It was dried in an oven at 50°C for 48 hours. Compound 3 (4.20 kg, 4.53 mol, 71.1% yield) was obtained as a white solid. 1H NMR: 400 MHz CDCl37.33 (s, 2H), 4.06-4.01 (m, 6H), 1.84-1.76 (m, 6H), 1.50-1.26 (m, 6H), 1.26 (m, 86H), 0.90-0.87 (t, J = 6.8 Hz, 9H).

[0217] General procedure for the preparation of compound 4 [ka]

[0218] A mixture of compound 3 (4.00 kg, 4.31 mol, 1.00 equivalent), EDCI (1.65 kg, 8.62 mol, 2.00 equivalent), and DMAP (105 g, 862 mmol, 0.200 equivalent) in DCM (28 L) was mixed with tert-butylpiperazine-1-carboxylate (1.04 kg, 5.61 mol, 1.30 equivalent) at 25°C. The mixture was stirred at 25°C under N2 for 16 hours. TLC (dichloromethane / methanol = 20 / 1, starting material R) was performed. f =0.32, product R f A reading of 0.53 indicated that no starting material was detected. The mixture was poured into 50 L of MeOH, filtered, and the cake was washed with 30 L of MeOH. Compound 4 (4.62 kg, 4.22 mol, 97.7% yield) was obtained as a white solid. 1 H NMR: 400 MHz CDCl36.57 (s, 2H), 3.97-3.94 (m, 6H), 3.57-3.29 (m, 8H), 1.82-1.71 (m, 6H), 1.47 (m, 16H), 1.25 (m, 16H), 0.89-0.86 (t, J = 6.8 Hz, 9H).

[0219] General procedure for the preparation of compound 5 [ka]

[0220] To a mixture of compound 4 (1.50 kg, 1.37 mol, 1.00 equivalent) in DCM (10 L), 4N HCl (4 M, 3.42 L, 10.0 equivalent) in EtOH was added all at once under N2 conditions at 25°C. The mixture was stirred at 25°C for 16 hours. TLC (DCM / MeOH = 20 / 1, product R) was performed. f =0.74, starting material R f The value (=0.18) indicated the disappearance of compound 4. The solid was filtered and washed with 5 L of EtOH. Compound 5 (3.80 kg, 3.68 mol, 89.6% yield, HCl salt form) was obtained as a white solid.

[0221] General procedure for the preparation of compound 7 [ka]

[0222] A mixture of compound 6 (500 g, 3.01 mol, 1.00 equivalent) and CH2O (181 g, 6.02 mol, 2.00 equivalent) was to which oleum (825 mL) was added all at once under N2 conditions at 25°C. The mixture was stirred at 140°C for 15 hours. The exhaust gas absorbent was packed with a 10% NaOH aqueous solution. HPLC (starting material: RT=2.73 min; product: RT=2.84 min) showed that compound 6 had disappeared. The mixture was cooled to 25°C and quenched with 3300 mL of H2O. It was filtered and washed with H2O until the pH was 3-4. It was recrystallized at 80°C with 3200 mL of DMF. It was filtered and washed with 2 L of EtOH. It was dried under vacuum. Compound 7 (1.60 kg, crude) was obtained as a gray solid. A mixture of compound 7 (1.60 kg, 8.98 mol, 1.00 equivalent) in DMF (3200 mL) was stirred at 80°C for 1 hour. It was slowly cooled to 25°C over 16 hours. The mixture was filtered and washed with 500 mL of EtOH. It was dried under vacuum. Compound 7 (620 g, 3.48 mol, 38.7% yield) was obtained as a bright orange solid.

[0223] Alternatively, compound 7 was prepared by the following procedure. [ka]

[0224] Compound 6A (1.50 kg, 9.43 mol, 1.00 equivalent) was added all at once to a 50% H2SO4 aqueous solution (15.0 L) at 25°C under N2. The mixture was stirred at 120°C for 16 hours. LC-MS (ET49477-3-P1A2, product: RT=0.597 min) showed that the starting material was completely consumed. The mixture was cooled to 25°C. It was poured into H2O (ice, 15.0 L), filtered, and the solid was washed with H2O (2.00 L x 5). The filtered cake was dried in a vacuum oven (55°C for 48 hours). Compound 7 (2.50 kg, 14.0 mol, 74% yield, 98.8% purity) was obtained as a white solid. ESI + :MS:C9H6O4(M+H) + Calculated value: 178.0, measured value: 178.1. 1 H NMR (400 MHz, CDCl3) δ 8.19 (s, 1H), 8.09-8.07 (d, J = 8.0 Hz, 1H), 7.93-7.91 (d, J = 8.0 Hz, 1H), 5.45 (s, 2H). 13 C{ 1 H} NMR (100 MHz, CDCl3) δ 169.9, 166.5, 147.6, 135.7, 129.8, 128.5, 125.1, 124.0, 70.1.

[0225] General procedure for the preparation of compound M-17 [ka]

[0226] To a mixture of compound 5 (3.60 kg, 3.49 mol, 1.00 equivalent, HCl) and compound 7 (683 g, 3.84 mol, 1.10 equivalent) in DCM (24 L), DMAP (852 g, 6.98 mol, 2.00 equivalent) and EDCI (1.34 kg, 6.98 mol, 2.00 equivalent) were added in one go under N2 at 25°C. The mixture was stirred at 25°C for 2 hours. TLC (DCM / MeOH = 20 / 1, starting material R) was performed. f =0.62, product R f A result of 0) indicated that the starting material had been completely consumed. The mixture was poured into EtOH (50 L), filtered, and washed with EtOH (20 L). Compound M-17 (3.80 kg, 3.29 mol, 94.3% yield) was obtained as a white solid. 1 H NMR: 400 MHz CDCl37.99-7.97 (d, J = 6.8 Hz, 1H), 7.56-7.53 (m, 2H), 6.59 (s, 2H), 5.36 (s, 2H), 3.97-3.94 (t, J = 6.4 Hz, 6H), 3.78-3.44 (m, 8H), 1.82-1.71 (m, 7H), 1.46-1.42 (m, 7H), 1.30-1.26 (m, 93H), 0.89-0.86 (t, J = 6.8 Hz, 9H)

[0227] General procedure for the preparation of compound M-18 [ka]

[0228] A solution of LiOH·H2O (175g, 4.16 mol, 1.30 equivalents) in H2O (4000mL) was added all at once to a mixture of compound M-17 in THF (20L) under N2 conditions at 25°C. The mixture was stirred at 25°C for 3 hours. TLC (DCM / MeOH = 20 / 1, starting material R) was used. f =0.46, product R fA pH of 0.05 indicated that the starting material had been completely consumed. The solution was concentrated and diluted with 40 L of H2O, and the pH was adjusted to 4-5 with 1 N HCl (10 L). The solution was filtered and washed with 45 L of H2O until the pH reached 6-7. It was then washed with 4 L of ACN. Compound M-18 (3.90 kg, crude) was obtained as a white solid. 1 H NMR: ET29928-65-P1A1 400 MHz CDCl38.05-8.00 (m, 1H), 7.57-7.54 (m, 1H), 7.38-7.36 (m, 1H), 6.59 (s, 2H), 6.6 (s, 2H), 4.79 (s, 2H), 3.97-3.94 (m, 8H), 3.81-3.45 (m, 8H), 1.82-1.77 (m, 4H), 1.45 (m, 7H), 1.29-1.25 (m, 90H), 0.89-0.86 (t, J = 7.2 Hz, 9H).

[0229] General procedure for the preparation of compound M-19-A [ka]

[0230] To a mixture of compound M-18 (1.70 kg, 1.45 mol, 1.00 equivalent) in DCM (20 L), imidazole (986 g, 14.5 mol, 10.0 equivalent) and TBDPSCl (3.98 kg, 14.5 mol, 3.72 L, 10.0 equivalent) were added in one step under N2 conditions at 25°C. The mixture was stirred at 25°C for 2 hours. TLC (DCM / MeOH = 10 / 1, starting material R) was performed. f =0.18, product R f A ratio of 0.92 indicated that the starting material was completely consumed. The reaction proceeded with ET29928-70. The organic layer was washed with H2O (15 L x 2), separated, dried over anhydrous Na2SO4, and concentrated. Compound M-19-A (5.80 kg, crude) was obtained as a white solid.

[0231] General procedure for the preparation of compound SiLHPG M19 [ka]

[0232] A mixture of compound M-19-A (2000 g, 485 mmol, 1.00 equivalent) in THF (16 L) was simultaneously added to H2O (6000 mL) and a solution of K2CO3 (87.1 g, 630 mmol, 1.30 equivalent) in MeOH (2000 mL) under N2 conditions at 25°C. The mixture was stirred at 25°C for 16 hours. TLC (PE / EA = 2 / 1, starting material R) was performed. f =0.43, product R f A result of (=0) indicated that the starting material was completely consumed. The solution was concentrated, diluted with 10 L of H2O, pH adjusted to 5 with 1 M KHSO4, extracted with DCM (10 L x 2), and dried over anhydrous Na2SO4. The solution was concentrated to approximately 5 L, poured into 10 L of MeOH, filtered, and washed with MeOH (5 L x 4) to remove the TBDPS byproduct. The solution was dissolved in DCM (5 L), added dropwise to MeCN (10 L), filtered, washed with MeCN (2 L x 4), dissolved in DCM (12 L), filtered through a silica gel pad, and washed with DCM / dimethyl = 1 / 1 (10 L). Compound SiLHPG M19 (835 g, 591 mmol, 48.8% yield) was obtained as a white solid. 1 H NMR: 400 MHz CDCl3δ 8.10-8.08 (d, J = 8.0 Hz, 1H), 7.99 (s, 1H), 7.66-7.64 (m, 4H), 7.43-7.32 (m, 7H), 6.59 (s, 2H), 5.21 (s, 2H), 3.99-3.93 (m, 6H), 3.78-3.44 (m, 8H), 1.82-1.75 (m, 6H), 1.47-1.42 (m, 7H), 1.31-1.27 (m, 93H), 1.12 (s, 10H),0.90-0.87 (t, J = 6.8 Hz, 9H).

[0233] General procedure for the preparation of compound M-18A [ka] A mixture of compound M-17 (2.00 kg, 1.73 mol, 1.00 equivalent) in THF (20.0 L) was to be suddenly mixed with a solution of LiOH·H2O (94.4 g, 2.25 mol, 1.30 equivalent) in H2O (4000 mL) at 25°C under N2 conditions. The mixture was stirred at 25°C for 3 hours. TLC (dichloromethane / methanol = 20 / 1, starting material R) was performed. f =0.5, product R f A reading of 0.1) indicated that the starting material was completely consumed. The mixture was poured into ACN (20.0 L) and filtered. After collecting the combined solid, it was dried under vacuum oven (50°C, 10 days). The reaction was carried out in parallel in five batches. Compound M-18A (10.8 kg, 9.15 mol, 106% yield, 87.4% purity) was obtained as a white solid. HRMS:C 74 H 129 N2O8(M-Li+2H) + Calculated value for this: 1173.9671, measured value: 1173.9755. 1 1H NMR: (Li salt neutralized with free acid for 1H-NMR). 400 MHz, CDCl3δ 8.05-8.00 (m, 1H), 7.57-7.54 (m, 1H), 7.38-7.36 (m, 1H), 6.59 (s, 2H), 6.6 (s, 2H), 4.79 (s, 2H), 3.97-3.94 (m, 8H), 3.81-3.45 (m, 8H), 1.82-1.77 (m, 4H), 1.45 (m, 7H), 1.29-1.25 (m, 90H), 0.89-0.86 (t, J = 7.2 Hz, 9H).

[0234] Alternatively, compound M19 was prepared by the following procedure. [ka]

[0235] To a mixture of compound M-18A (3.00 kg, 2.54 mol, 1.00 equivalent) in THF (24.0 L), imidazole (1.21 kg, 17.80 mol, 7.0 equivalent) and TBDPSCl (4.19 kg, 15.3 mol, 3.92 L, 6.0 equivalent) were added in five separate additions under N2 conditions at 25°C. The mixture was stirred at 25°C for 16 hours. HPLC (starting material t) was performed. R =5.92 min, product t R The reaction time of 9.32 minutes indicated that approximately 5% of the starting material remained. The reaction mixture was diluted with DCM (20.0 L) and washed with H2O (20.0 L x 2). Next, the organic layer was dried over anhydrous Na2SO4, filtered, concentrated to approximately 7 L, poured into 15 L of MeOH, filtered, and washed with MeOH (5 L x 4) to remove the TBDPS byproduct. Dissolved in DCM (7 L), added dropwise to MeCN (15 L), filtered, washed with MeCN (5 L x 4), and the filtered cakes were combined and dried under an oven (50°C, 72 hours). Compound SiLHPG M19 (13.6 kg, crude, 89% purity, 5% M-17, 3% M-18) was obtained as a white solid. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 1 / 10 to 4 / 1) (with 10% DCM added to the PE eluent). The reaction was carried out in parallel in three batches. The concentrate and compound SiLHPG M19 (7.20 kg, 5.09 mol, 66.5% yield, 95.08% purity) were obtained as a white solid. HRMS:C 90 H 147 N2O8Si(M+H) + Calculated value for this: 1412.0848, measured value: 1412.0908. 1H NMR (400 MHz, CDCl3) δ 8.10-8.08 (d, J = 8.0 Hz, 1H), 7.99 (s, 1H), 7.66-7.64 (m, 4H), 7.43-7.32 (m, 7H), 6.59 (s, 2H), 5.21 (s, 2H), 3.99-3.93 (m, 6H), 3.78-3.44 (m, 8H), 1.82-1.75 (m, 6H), 1.47-1.42 (m, 7H), 1.31-1.27 (m, 90H), 1.12 (s, 10H),0.90-0.87 (t, J = 6.8 Hz, 9H). 13 C{1H} NMR (100 MHz, CDCl3) δ 170.8, 169.9, 153.3, 144.7, 139.8, 139.4, 135.4, 134.8, 133.1, 132.1, 129.9, 129.6, 127.9, 127.7, 125.6, 125.1, 105.7, 73.6, 69.3, 64.0, 60.4, 47.5, 42.3, 31.9, 30.3, 29.4-29.7,26.9, 26.1, 22.7, 14.1.

[0236] Example 2. Synthesis of Compound M22 a. Synthesis scheme for compound M22 [ka]

[0237] b. Procedure for the synthesis of compound M22

[0238] General procedure for the preparation of compound 3 [ka]

[0239] A mixture of compound 1 (24.00 g, 113 mmol), compound 2 (44.2 g, 451 mmol, 62.4 mL), CuI (6.45 g, 33.8 mmol), Pd(PPh3)4 (6.51 g, 5.64 mmol), and TEA (5.70 g, 56.3 mmol, 7.83 mL) in DMF (144 mL) was degassed, purged three times with N2, and then stirred at 65°C for 24 hours under an N2 atmosphere. The desired product was detected in TLC (petroleum ether / ethyl acetate = 10 / 1, product: RT = 0.43). The filtrate was diluted with SiO2 (600 mL) and washed with brine (450 mL x 3). The organic layer was dried over anhydrous Na2SO4 (45.0 g), filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 100 / 1 to 5 / 1). Compound 3 (15.0 g, 57.8% yield) was obtained as a brown solid.

[0240] General procedure for the preparation of compound 4 [ka]

[0241] To a solution of compound 3 (15.0 g, 65.1 mmol) in THF (90.0 mL), TBAF (1.00 M, 65.1 mmol, 65.1 mL) was added. The mixture was stirred at 0°C for 20 minutes. The desired product was detected by LC-MS (ET25847-215-P1B, RT=1.446) and TLC (petroleum ether / ethyl acetate=5 / 1, product: Rf=0.43). The reaction mixture was diluted with DCM (300 mL) and washed with brine (300 mL x 3). The combined organic layers were dried over anhydrous Na2SO4 (30.0 g), filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate=30 / 1~5 / 1). Compound 4 (3.20 g, 31.1% yield) was obtained as a yellow solid. 1H NMR: 400 MHz CDCl37.84-7.86 (d, J = 8.0 Hz, 1H), 7.53-7.63 (m, 2H), 5.29 (s, 2H), 0.28 (s, 9H).

[0242] General procedure for the preparation of compound 6 [ka]

[0243] To a solution of compound 5 (48.0 g, 51.0 mmol) in THF (288 mL), LiAlH4 (3.18 g, 83.6 mmol) was added at 0°C. The mixture was stirred at 25°C for 16 hours. TLC (DCM / MeOH = 5 / 1, product: R) f A reading of 0.80 indicated that compound 5 was completely consumed and a new spot was formed. The reaction mixture was quenched by adding Na2SO4·10H2O (90.0 g) and then filtered. The filtrate was concentrated. The residue was diluted with DCM (900 mL), washed with water (450 mL x 2) and brine (600 mL), dried over anhydrous Na2SO4 (90.0 g), filtered, and concentrated under reduced pressure to obtain the residue. Compound 6 (40.0 g, crude) was obtained as a white solid. 1 H NMR: 400 MHz CDCl36.57 (s, 2H), 4.60-4.61 (d, J = 4.0 Hz, 2H), 3.93-4.00 (m, 6H), 1.71-1.84 (m, 6H), 1.27-1.31 (m, 90H), 0.87-0.91 (t, J = 6.4 Hz, 9H).

[0244] General procedure for the preparation of compound 7 [ka]

[0245] To a solution of compound 6 (40.0 g, 43.8 mmol) in DCM (240 mL), TMSBr (8.04 g, 52.5 mmol, 6.82 mL) was added at 0°C and the mixture was stirred for 1 hour. The mixture was then stirred at 25°C for 3 hours. TLC (DCM / MeOH = 20 / 1, product: Rf = 0.95) showed that compound 6 was completely consumed and a new spot was formed. The reaction mixture was combined and concentrated under reduced pressure to remove the solvent. The residue was dissolved in DCM (200 mL) and triturated with ACN (1.00 L). The solid was washed with ACN (200 mL x 3) and filtered. It was then concentrated. Compound 7 (42.0 g, 98.2% yield) was obtained as a pale yellow solid. 1 H NMR: 400 MHz CDCl36.58 (s, 2H), 4.44 (s, 2H), 3.93-3.99 (m, 6H), 1.72-1.84 (m, 6H), 1.27-1.47 (m, 90H), 0.87-0.91 (t, J = 6.4 Hz, 9H).

[0246] General procedure for the preparation of compound 8 [ka]

[0247] To a solution of compound 7 (42.0 g, 43.0 mmol) in DMF (252 mL) and THF (200 mL), NaN3 (4.20 g, 64.5 mmol) in H2O (36.0 mL) was added. The mixture was stirred at 40°C for 12 hours. TLC (petroleum ether / ethyl acetate = 10 / 1, product: RT = 0.66) showed that compound 7 was completely consumed and a new spot was formed. The reaction mixture was diluted with DCM (300 mL) and washed with brine (450 mL x 3). The combined organic layers were dried over anhydrous Na2SO4 (30.0 g), filtered, and concentrated under reduced pressure to obtain the residue. Compound 8 (40.0 g, crude) was obtained as a white solid. 1H NMR: 400 MHz CDCl36.49 (s, 2H), 4.25 (s, 2H), 3.94-4.00 (m, 6H), 1.75-1.84 (m, 6H), 1.27-1.49 (m, 90H), 0.87-0.91 (t, J = 6.4 Hz, 9H).

[0248] General procedure for the preparation of compound 9 [ka]

[0249] A mixture of compound 9 (2.70 g, 2.88 mmol), compound 4 (682 mg, 4.32 mmol), sodium ascorbate (570 mg, 2.88 mmol), and CuSO4·5H2O (360 mg, 1.44 mmol) in THF (16.2 mL) and H2O (5.40 mL) was degassed, purged three times with N2, and then stirred at 70°C for 12 hours under an N2 atmosphere. The desired product was obtained by TLC (petroleum ether / ethyl acetate = 3 / 1, R f It was detected in (=0.22). Next, it was filtered and concentrated under reduced pressure to remove the solvent. The residue was dissolved in DCM (60.0 mL) and triturated with MeOH (600 mL). Compound 9 (3.00 g, crude) was obtained as a yellow solid.

[0250] General procedure for the preparation of compound 10 [ka]

[0251] To a solution of compound 9 (3.00 g, 2.74 mmol) in THF (18.0 mL), NaOH (438 mg, 11.0 mmol) in H2O (3.60 mL) was added. The mixture was stirred at 60°C for 3 hours. TLC (petroleum ether / ethyl acetate = 2 / 1, product: R) fA value of 0.04 indicated that compound 9 was completely consumed and a new spot was formed. This reactant was combined with reactant (ET258474-259). The reaction mixture was concentrated under reduced pressure to remove THF. The residue was diluted with H2O (300 mL). The solution was adjusted to pH approximately 2 with HCl (1.00 M), filtered to pH approximately 7 with H2O, and concentrated under reduced pressure to obtain the residue. This was then washed with ACN (100 mL). Compound 10 (4.80 g, crude) was obtained as a pale yellow solid.

[0252] General procedure for the preparation of compound 11 [ka]

[0253] To a solution of compound 10 (4.80 g, 4.32 mmol) in DCM (28.8 mL), TBDPSCl (2.96 g, 10.8 mmol, 2.77 mL) and Im (880 mg, 12.9 mmol) were added. The mixture was stirred at 40°C for 3 hours. TLC (petroleum ether / ethyl acetate = 2 / 1, product: R) f A reading of 0.94 indicated that compound 10 was completely consumed and a new spot was formed. The reaction mixture was quenched by adding NaHCO3 (150 mL) and extracted with DCM (300 mL). The combined organic layers were washed with brine (300 mL x 2), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The crude product was redissolved in DCM (90.0 mL) and added dropwise to MeOH (600 mL) with vigorous stirring. The desired product was precipitated, filtered, and concentrated under reduced pressure to obtain the residue. This was then washed with MeOH (300 mL x 3). Compound 11 (6.05 g, crude) was obtained as a white solid.

[0254] General procedure for the preparation of compound M22 [ka]

[0255] To a solution of compound 11 (6.00 g, 3.76 mmol) in THF (36.0 mL) and MeOH (6.00 mL), K2CO3 (1.30 g, 9.42 mmol) in H2O (12 mL) was added. The mixture was stirred at 25°C for 12 hours. TLC (petroleum ether / ethyl acetate = 2 / 1, product: R f A pH of 0.43 indicated that compound 11 had been completely consumed, forming a new spot. The reactants were combined and concentrated under reduced pressure. The reaction mixture was quenched by adding brine (200 mL) and adjusted to pH approximately 2 with aqueous KHSO4 solution (200 mL, 1.00 M). It was then extracted with DCM (300 mL), washed with brine (100 mL x 2), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The crude product was redissolved in DCM (50.0 mL) and added dropwise to MeOH (300 mL) with vigorous stirring. The desired product was precipitated and filtered. Compound SiLHPG M22 (4.5 g, 88.2% yield) was obtained as a white solid. 1 H NMR: 400 MHz CDCl38.17 (s, 1H), 8.12-8.10 (d, J = 8.0 Hz, 1H), 7.94-7.92 (d, J = 8.0 Hz, 1H), 7.72-7.64 (m, 5H), 7.39-7.30 (m, 6H), 6.52 (s, 2H), 5.47 (s, 2H), 5.18 (s, 2H), 3.97-3.92 (m, 6H), 1.82-1.74 (m, 6H), 1.47-1.42 (m, 6H), 1.31-1.26 (m, 84H), 1.11 (s, 9H), 0.90-0.87 (t, J = 6.8 Hz, 9H).

[0256] Example 3. Synthesis of Compound M36 a. Synthesis scheme for compound M36 [ka]

[0257] b. Procedure for the synthesis of compound M36

[0258] General procedure for the preparation of compound M20 [ka]

[0259] Compound 1 (70 g, 328 mmol, 1.00 equivalent), tert-butylpiperazine-1-carboxylate (61.2 g, 328 mmol, 1 equivalent), K3PO4 (139 g, 657.19 mmol, 2 equivalents), and Tol. (700 mL) were added to a three-necked round-bottom flask packed with N2. The mixture was purged and degassed three times with N2. Next, RuPhos (15.3 g, 32.8 mmol, 0.1 equivalent) and Pd2(dba)3 (10.71 g, 16.43 mmol, 0.05 equivalent) were added to the solution. The reaction mixture was purged and degassed three times with N2 and heated to 100°C. It was stirred at 100°C for 16 hours. The reaction mixture turned black. TLC (petroleum ether / ethyl acetate = 2 / 1, starting material Rf = 0.70, product Rf = 0.30) showed that the starting material was consumed and new spots were formed. The reaction mixture was cooled to 20°C. Next, it was filtered to remove the solid and washed twice with ethyl acetate (1000 mL and 500 mL). The organic layers were combined and concentrated to obtain a crude solid. The solid was stirred with a solution of MTBE:DCM (800 mL, V / V = 10 / 1) for 16 hours. Next, it was filtered to obtain a solid, which was dried under an oil pump. M20 was obtained as an off-white solid (64 g, 201.03 mmol, 61.18% yield). 1 H NMR: 400 MHz CDCl37.76 (d, J = 8.8 Hz 1H), 7.01 (dd, J =2.0Hz, J =8.8Hz, 1H), 6.80 (s, 1H), 5.21 (s, 2H), 3.59-3.62(m, 4H), 3.35-3.38 (m, 4H), 1.49 (s, 9H).

[0260] General procedure for the preparation of compound M25 [ka]

[0261] To a solution of M-20 (30.0 g, 1.0 equivalent) in DCM (60 mL), HCl / MeOH (150 ml, 6.37 equivalents, 4 M) was added. The mixture was stirred at 25°C for 16 hours. TLC (dichloromethane:methanol = 20:1, Rf = 0.0) indicated that the reaction product M20 was completely consumed. The reaction mixture was concentrated under reduced pressure to remove MeOH and DCM. The crude product was washed with DCM (200 ml). Next, it was filtered and concentrated under reduced pressure to obtain compound M-35 (27 g, crude) as a white solid. 1 H NMR: 400 MHz DMSO-d69.43 (s, 1H), 7.66 (d, J = 8.4Hz 1H), 7.13-7.18 (m, 2H), 5.28(s, 2H), 3.60-3.63(m, 4H), 3.16-3.19 (m, 4H).

[0262] General procedure for the preparation of compound M31

[0263] Compound M31 was prepared using the same procedure as that used to prepare compound 2 in Example 1.

[0264] General procedure for the preparation of compound M32

[0265] Compound M32 was prepared using the same procedure as for preparing compound 3 in Example 1.

[0266] General procedure for the preparation of compound M33 [ka]

[0267] To a solution of compound M-32 (80.0 g, 1.0 equivalent) in DCM (50 mL), DMAP (21.1 g, 2.0 equivalents), M-25 (28.4 g, 1.3 equivalents), and EDCI (33.1 g, 2.0 equivalents) were added. The mixture was stirred at 25°C for 16 hours. TLC (dichloromethane:methanol = 20:1, Rf = 0.60) indicated that product M-32 was completely consumed. The reaction mixture was quenched by adding NaHCO3 (800 mL) and extracted with DCM (800 mL x 3). The combined organic layers were washed with brine (800 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain the crude product. This was redissolved in DCM (160 mL) and added dropwise to ACN (4800 mL) with vigorous stirring. The solid was recovered by filtration and dried under reduced pressure to obtain compound M-33 (96.0 g, 85.1 mmol, 98.69% yield) as a white solid. 1 H NMR: 400 MHz CDCl37.78 (d, J = 8.4Hz 1H), 7.78 (d, J = 8.4Hz 1H), 6.82 (s, 1H), 6.63(s, 2H), 5.22(s, 2H), 3.97(t, J = 6.4Hz 1H), 3.41-3.98 (m, 8H), 1.75-1.84 (m, 6H), 1.26-1.48 (m, 96H), 0.88(t, J = 6.4Hz 9H).

[0268] General procedure for the preparation of compound M34 [ka] To a solution of M-33 (116 g, 1.0 equivalent) in THF (1160 mL), NaOH (20.56 g, 5.0 equivalents) was added. The mixture was stirred at 60°C for 12 hours. TLC (dichloromethane:methanol = 20:1, Rf = 0.50) showed that reactant 1 was completely consumed. The reaction mixture was concentrated under reduced pressure to remove THF. The residue was diluted with 1.5 L of H2O. The solution was adjusted to pH 5-6 with HCl (1 M), filtered, and concentrated under reduced pressure to obtain the residue. The crude product was evaporated six times with DCM, THF, and ACN. Compound M-34 (87 g, 75.93 mmol, 73.82% yield) was obtained as a pale yellow solid. 1 H NMR: 400 MHz CDCl38.01 (d, J = 8.4Hz 1H), 7.22 (s, 1H), 6.85 (s, 1H),6.75 (d, J = 8.8Hz, 1H), 6.68(s, 2H), 4.74(s, 2H), 3.97,\(t, J = 6.4Hz, 6H), 3.41-3.98 (m, 8H), 1.70-1.81 (m, 6H), 1.26-1.48 (m, 96H), 0.83(t, J = 6.4Hz, 9H),

[0269] General procedure for the preparation of compound M35 [ka]

[0270] To a solution of compound M-34 (87 g, 1.0 equivalent) in DCM (870 mL), imidazole (14.96 g, 3.0 equivalents) and TBDPSCl (41.5 mL, 2.2 equivalents) were added. The mixture was stirred at 25°C for 16 hours. TLC (dichloromethane:methanol = 20:1, Rf = 0.90) indicated that product M-34 was completely consumed. The reaction mixture was quenched by adding 3600 mL of NaHCO3 and extracted with DCM (700 mL x 3). The combined organic layers were washed with brine (500 mL x 2), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain the crude product. This was redissolved in DCM (800 mL) and added dropwise to ACN (2.5 L) with vigorous stirring. The solid was filtered and concentrated under reduced pressure to obtain compound M-35 (118 g, crude) as a yellow solid. HPLC showed that the starting material was completely consumed.

[0271] General procedure for the preparation of compound M36 [ka] A solution of compound M-35 (115 g, 1.0 equivalent) in THF (345 mL) and MeOH (920 mL) was mixed with a solution of K2CO3 (24.72 g, 2.5 equivalents) in H2O (345 mL). The mixture was stirred at 25°C for 16 hours. TLC (dichloromethane:methanol = 20:1, Rf = 0.43) showed that compound M-35 was completely consumed. The reaction mixture was concentrated under reduced pressure, and one-quarter of the solvent was removed. The residue was diluted with 450 mL of brine and extracted by DCM (500 mL x 4). The combined organic layers were washed with brine (500 mL x 3), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography (SiO2, dichloromethane / ethyl acetate = 1 / 0~10 / 1). Compound M-36 (25g, 93% purity) was obtained as a white solid. 1H NMR: 400 MHz CDCl37.99 (d, J = 8.4Hz, 1H), 7.67-7.69 (m, 4H), 7.33-7.40 (m, 7H), 6.75 (dd, J = 2.0Hz, J = 8.8Hz, 1H), 6.63(s, 2H), 5.18(s, 2H), 3.97-4.00,(m, 6H), 3.38-3.98 (m, 8H), 1.70-1.81 (m, 6H), 1.26-1.48 (m, 90H), 0.90(s, 9H), 0.83(t, J = 6.4Hz, 9H).

[0272] Example 4. Synthesis of compound M40 a. M40 synthesis scheme [ka]

[0273] b. Procedure for the synthesis of compound M40

[0274] General procedure for the preparation of compound M37 [ka]

[0275] To a mixture of compound 2-methyl-1,4-benzenedicarboxylic acid (1.1 equivalents) and compound 5 (1.10 equivalents) in DCM (7V), DMAP (2.00 equivalents) and EDCI (2.00 equivalents) were added in one step under N2 conditions at 25°C. The mixture was stirred at 25°C for 2 hours. TLC showed that the starting materials were completely consumed. The reaction mixture was poured into EtOH (50V), filtered, and washed with EtOH (10V). Compound M37 was obtained as a white solid.

[0276] General procedure for the preparation of compound M38 [ka]

[0277] A mixture of compound M37 (1.0 equivalent) and AIBN (4.0 equivalents) in DCM (10V) was mixed with NBS (2.0 equivalents) under N2 conditions at 25°C. The mixture was refluxed for 4 hours. TLC showed that the starting materials were completely consumed. After cooling, the mixture was washed with a saturated aqueous NaHCO3 solution. The mixture was dried over MgSO4, and the filtrate was concentrated to obtain the residue. This was precipitated in ACN to obtain a white solid M38. See JOC, 2008, 73, 9125-9128.

[0278] General procedure for the preparation of compound M39 [ka]

[0279] To a solution of compound M38 (1.0 equivalent) in DCM (10V), AgNO2 (3.0 equivalents) was added at 25°C under N2. The mixture was stirred for 2 hours. TLC showed that the starting material was completely consumed. The mixture was washed with saturated NaHCO3 aqueous solution. The mixture was dried over MgSO4, and the filtrate was concentrated to obtain the residue. This was precipitated in ACN to obtain the white solid M39. See Synthesis 1980, 814-815.

[0280] General procedure for the preparation of compound M40 [ka]

[0281] A solution of compound M39 (1.0 equivalent) in THF (10V) was slowly added in one go to a solution of 1.0 M NaOH (5.0 equivalents) under N2 at 25°C. The mixture was stirred at 25°C for 3 hours. TLC showed that the starting material was completely consumed. The solution was concentrated and diluted with H2O (50V), and the pH was adjusted to 4-5 with 1N HCl. The solution was filtered and washed with H2O until the pH was 6-7. It was then washed with ACN. Compound M40 was obtained as a white solid in 72% yield and 90% purity.

[0282] Example 5. Synthesis of Compound M50 a. Synthesis scheme for compound M50 [ka]

[0283] b. Procedure for the synthesis of compound M50

[0284] General procedure for the preparation of compound M-50-A [ka]

[0285] To a solution of compound M-18 (2.0 g, 1.70 mmol, 1.00 equivalent) in THF (16.0 mL), imidazole (579.99 mg, 8.52 mmol, 5.00 equivalent) and TBSCl (1.28 g, 8.52 mmol, 1.04 mL, 5.00 equivalent) were added. The mixture was stirred at 25°C for 2 hours. HPLC showed that the starting material was completely consumed. The residue was diluted with H2O (50 mL) and extracted by DCM (2 x 30 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. Compound M-50-A (2.39 g, crude) was obtained as a white solid.

[0286] General procedure for the preparation of compound M-50 [ka]

[0287] A solution of compound M-50-A (2.39 g, 1.70 mmol, 1.00 equivalent) in THF (19.0 mL) and MeOH (2.30 mL) was mixed with a solution of K2CO3 (305.44 mg, 2.21 mmol, 1.30 equivalent) in H2O (7.0 mL). The mixture was stirred at 25°C for 3 hours. TLC (DCM / MeOH = 20 / 1, starting material Rf = 0.50, product Rf = 0.30) indicated that the starting material was completely consumed. The pH was adjusted to 5 with 1 M KHSO4 (5 mL). The residue was diluted with H2O (50 mL) and extracted with DCM (2 x 30 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The crude product was triturated with ACN (30V, 70.0mL) at 25°C for 30 minutes. It was filtered and concentrated. Compound M-50 (1.00g, 93.8% purity, 45.67% yield) was obtained as a white solid.

[0288] Example 6. Synthesis of Compound M60 a. Synthesis scheme for compound M60 [ka]

[0289] b. Procedure for the synthesis of compound M60

[0290] General procedure for the preparation of compound M-60-A [ka]

[0291] To a solution of compound M-18 (1.0 g, 0.85 mmol, 1.00 equivalent) in DCM (8.0 mL), imidazole (579.99 mg, 8.52 mmol, 10.0 equivalent) and TIPSCl (1.64 g, 8.52 mmol, 10.00 equivalent) were added. The mixture was stirred at 25°C for 8 hours. HPLC showed that the starting material was completely consumed. The residue was diluted with H2O (50 mL) and extracted by DCM (2 x 30 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. Compound M-60-A (1.27 g, crude) was obtained as a white solid.

[0292] General procedure for the preparation of compound M-60 [ka]

[0293] A solution of compound M-60-A (1.27 g, 0.85 mmol, 1.00 equivalent) in THF (10.4 mL) and MeOH (1.30 mL) was mixed with a solution of K2CO3 (153.5 mg, 1.11 mmol, 1.30 equivalent) in H2O (4.0 mL). The mixture was stirred at 25°C for 3 hours. The pH was adjusted to 5 with 1 M KHSO4 (3 mL). The residue was diluted with H2O (50 mL) and extracted with DCM (2 x 30 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The crude product was triturated with ACN (30 V, 70.0 mL) at 25°C for 30 minutes. Filtered and concentrated to obtain compound M-60 (0.90 g, 92% purity, 72.7% yield) as a white solid. 83 H 149 N2O8Si + [M+H + Calculated mass value for ]: 1330.1, measured value: 1330.4.

[0294] Example 7. Synthesis of oligonucleotide fragment A from reagent M19 a. Synthesis scheme for oligonucleotide fragment A

[0295] Fragment A was synthesized according to the synthesis scheme shown in Figure 2.

[0296] b. Procedure for the synthesis of oligonucleotide fragment A from M19

[0297] General procedure for the preparation of compound M19-fragment IU-DMTr [ka]

[0298] To a solution of M19 (20.00 g, 14.16 mmol, 1.00 equivalent) and dU (13.14 g, 21.24 mmol, 1.50 equivalent) in DCM (200 mL), DMAP (3.46 g, 28.32 mmol, 2.00 equivalent) and EDCI (5.43 g, 28.32 mmol, 2.00 equivalent) were added. The mixture was stirred at 25°C for 16 hours. TLC (dichloromethane:methanol = 15:1, product, Rf = 0.70) showed that the reaction was complete and a new spot had formed. According to TLC, the reaction was clean. The reaction mixture was washed with NaHCO3 (5% aqueous solution, 100 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to a residue of 3V. The crude product was added dropwise to MeOH (600 ml, 30V) with vigorous stirring. The desired product precipitated. Compound M19-fragmentIU-DMTr (27.00 g, 13.31 mmol, 94.54% yield) was obtained as a white solid.

[0299] General procedure for the preparation of compound M19-fragment IU [ka]

[0300] To a solution of M19-fragment IU-DMTr (27.0 g, 13.31 mmol, 1.00 equivalent) in DCM (170 mL), dodecane-1-thiol (8.15 g, 39.93 mmol, 9.64 mL, 3.00 equivalent) and TFA (7.59 g, 66.55 mmol, 4.92 mL, 5.00 equivalent) were added at 0°C. The mixture was stirred at 0°C for 1 hour. TLC (dichloromethane:methanol = 15:1, product, Rf = 0.54) showed that the reactants were completely consumed and a new spot was formed. According to TLC, the reaction was clean. The reaction mixture was quenched by adding NaHCO3 (100 mL of 5% aqueous solution) and then extracted with DCM (100 mL). The combined organic layers were washed with brine (100 mL x 2), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The crude product was redissolved in DCM (30 mL) and added dropwise to MeOH / ACN (3:1, 450 mL) with vigorous stirring. The desired product precipitated, yielding compound M19-fragment IU (15.00 g, 8.77 mmol, 65.51% yield, and 98.5% purity) as a white solid.

[0301] General procedure for the preparation of compound M19-fragment I-UC [ka]

[0302] To a solution of M19-fragment IU (15.00 g, 8.76 mmol, 1.00 equivalent) and dC (12.12 g, 13.14 mmol, 1.50 equivalent) in Tol. / IPAC=3:1 (120 mL), 3A MS (7.50 g) was added and the mixture was stirred for 1 hour. Next, DCI (2.07 g, 17.55 mmol, 2.00 equivalent) was added to the mixture. The mixture was stirred at 30°C for 1 hour. TLC (Dichloromethane:Methanol=20:1, Product:R) fThe reaction (=0.53) showed that the reactant M19-fragment IU was completely consumed and a new spot was formed. According to TLC, the reaction was clean. HX (2.64 g, 17.55 mmol, 2.00 equivalents) was added to the crude product. After stirring the mixture at 30°C for 0.5 hours, dodecane-1-thiol (5.31 g, 26.31 mmol, 6.30 mL, 3.00 equivalents) and TFA (15.00 g, 134.49 mmol, 9.75 mL, 15.00 equivalents) were added to the mixture at 0°C. The mixture was stirred at 0°C for 1 hour. TLC (dichloromethane:methanol = 20:1, product:R f A reaction of 0.43) indicated that the reactants were completely consumed and a new spot was formed. According to TLC, the reaction was clean. The reaction mixture was diluted to pH 7 with NMI (175.2 mmol, 14 mL, 20.00 equivalents), filtered, and concentrated under reduced pressure to obtain the residue. The filtrate was added dropwise to ACN (800 ml), allowed to precipitate for 0.5 hours, and filtered through a 9 cm Buchner funnel for 3.5 hours to obtain 14.3 g (98% yield, 97.7% purity) as a white solid.

[0303] General procedure for the preparation of compound M19-fragment I-UCC [ka]

[0304] To a solution of M19-fragment I-UC (19.00 g, 8.40 mmol, 1.00 equivalent) and dC (11.55 g, 12.60 mmol, 1.50 equivalent) in Tol. / IPAC=3:1 (150 mL), 7.5 g of 3A molecular sieve was added. The mixture was stirred for 1 hour. Subsequently, DCI (1.97 g, 16.80 mmol, 2.00 equivalent) was added. Next, it was stirred at 30°C for 1 hour. TLC (dichloromethane:methanol=20:1, product:Rf=0.53) showed that compound M19-fragment I-UC was completely consumed and a new spot was formed. According to TLC, the reaction was clean. HX (2.51 g, 16.80 mmol, 2.00 equivalent) was added to the mixture. The mixture was stirred at 30°C for 0.5 hours. Next, dodecane-1-thiol (5.04 g, 25.20 mmol, 5.94 mL, 3.00 equivalents) and TFA (14.16 g, 126 mmol, 9.21 mL, 15.00 equivalents) were added at 0°C. The mixture was stirred at 0°C for 1 hour. TLC (dichloromethane:methanol = 15:1, product: Rf = 0.43) showed that the reaction was complete and a new spot had formed. According to TLC, the reaction was clean. NMI (168 mmol, 14.5 mL, 20.00 equivalents) was added to the reaction mixture. The mixture was added dropwise to ACN (800 mL). The solid was allowed to precipitate for 0.5 hours and filtered through a 15 cm Buchner funnel for 2.0 hours to obtain 15.4 g (94% yield, 95.0% purity) as a white solid.

[0305] General procedure for the preparation of compound M19-fragment I-UCCC [ka]

[0306] To a solution of M19-fragment I-UCC (5.00 g, 1.78 mmol, 1.00 equivalent) and dC (2.46 g, 2.67 mmol, 1.50 equivalent) in Tol. / ACN=3:1 (40 mL), 3A MS (2.0 g) was added. The mixture was stirred at 30°C for 1 hour. Subsequently, DCI (420.05 mg, 3.56 mmol, 2.00 equivalent) was added. The mixture was stirred at 30°C for 1 hour. TLC (dichloromethane:methanol=15:1, product:R) was performed. f The reaction was complete and a new spot was formed (=0.53). According to TLC, the reaction was clean. To the mixture, tert-butyl hydroperoxide (320.5 mg, 3.56 mmol, 2.00 equivalents) was added. The mixture was stirred at 30°C for 0.5 hours. Subsequently, dodecane-1-thiol (1.08 g, 5.34 mmol, 1.28 mL, 3.00 equivalents) and TFA (3.04 g, 26.68 mmol, 1.98 mL, 15.00 equivalents) were added at 0°C. The mixture was stirred at 0°C for 1 hour. TLC (dichloromethane:methanol = 20:1, product:R f A reading of 0.43 indicated that the reaction was complete and a new spot had formed. The reaction mixture was quenched by adding 100 mL of NaHCO3 (2%) and 2 equivalents of Na2SO3 at 0°C, filtered, and extracted with DCM (80 mL x 3). The combined organic layers were washed with brine (100 mL x 2), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. This was added dropwise to ACN (200 mL) with vigorous stirring. The desired product precipitated. The cake was washed with ACN (30 mL x 2) and filtered through a 7 cm Buchner funnel for 1.5 hours to obtain 5.00 g (84% yield, 90.9% purity) as a white solid.

[0307] General procedure for preparing oligonucleotide fragment A [ka]

[0308] To a solution of M19-fragment I-UCCC (4.00 g, 1.20 mmol, 1.00 equivalent) and dA (1.67 g, 1.79 mmol, 1.50 equivalent) in Tol. / ACN=3:1 (40 mL), 2.0 g of 3A MS was added. The mixture was stirred at 30°C for 1 hour. Subsequently, DCI (282.36 mg, 2.39 mmol, 2.00 equivalent) was added. The mixture was stirred at 30°C for 1 hour. TLC (dichloromethane:methanol=20:1, product:R) was performed. f The reaction was complete and a new spot was formed (=0.53). According to TLC, the reaction was clean. Next, glucose (0.5 equivalents) was added. HX (358.99 mg, 2.39 mmol, 2.00 equivalents) was added to the mixture. The mixture was stirred at 30°C for 0.5 hours. Next, dodecane-1-thiol (725.66 mg, 3.59 mmol, 0.86 mL, 3.00 equivalents) and TFA (2.04 g, 17.93 mmol, 1.33 mL, 15.00 equivalents) were added at 0°C. The mixture was stirred at 0°C for 1 hour. TLC (dichloromethane:methanol = 15:1, product:R f A reading of 0.43 indicated that the reaction was complete and a new spot had formed. The reaction mixture was quenched by adding 80 mL of 2% NaHCO3 at 0°C, filtered, and extracted with DCM (50 mL x 3). The combined organic layers were washed with brine (80 mL x 2), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. This was added dropwise to ACN (200 mL) with vigorous stirring. The desired product precipitated. The cake was washed with ACN (30 mL x 2) and filtered through a 7 cm Buchner funnel for 1.5 hours to obtain 4.20 g (85% yield, 86.4% purity) of fragment A as a white solid.

[0309] Example 8. Synthesis of oligonucleotide fragment B from reagent M19 a. Synthesis scheme for oligonucleotide fragment B

[0310] Fragment B was synthesized according to the synthesis scheme shown in Figure 3.

[0311] b. Procedure for the synthesis of oligonucleotide fragment B from M19

[0312] General procedure for the preparation of compound M19-fragment III-C-DMTr [ka]

[0313] To a solution of M19 (20.00 g, 14.16 mmol, 1.00 equivalent) and dC (13.76 g, 21.24 mmol, 1.50 equivalent) in DCM (200 mL), DMAP (1.73 g, 14.16 mmol, 1.00 equivalent) and EDCI (4.08 g, 21.24 mmol, 1.50 equivalent) were added. The mixture was stirred at 30°C for 12 hours. TLC (dichloromethane:methanol = 10:1, product, Rf = 0.70) showed that the reaction was complete and a new spot had formed. According to TLC, the reaction was clean. The reaction mixture was washed with NaHCO3 (300 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to a residue of 5V. This was added dropwise to ACN / MeOH (3:1, 1000 ml, 50V) with vigorous stirring. The desired product precipitated. Compound M19-fragmentIII-C-DMTr (28.90 g, 14.15 mmol, 99.94% yield) was obtained as a white solid.

[0314] General procedure for the preparation of compound M19-fragment III-C [ka]

[0315] To a solution of M19-fragment III-C-DMTr (28.0 g, 13.72 mmol, 1.00 equivalent) in DCM (250 mL), dodecane-1-thiol (8.32 g, 41.12 mmol, 9.84 mL, 3.00 equivalent) and TFA (7.80 g, 68.56 mmol, 5.08 mL, 5.00 equivalent) were added. The mixture was stirred at 0°C for 1 hour. TLC (dichloromethane:methanol = 10:1, product, Rf = 0.54) showed that the reaction was complete and a new spot had formed. According to TLC, the reaction was clean. The reaction mixture was diluted with NMI (9.00 g, 109.72 mmol, 8.76 mL, 8.00 equivalent). The crude product was added dropwise to ACN (900 ml, 30V) with vigorous stirring. The desired product precipitated. Compound M19-fragment III-C (23.00 g, 13.22 mmol, 96.42% yield) was obtained as a white solid.

[0316] General procedure for the preparation of compound M19-fragment III-CT [ka]

[0317] To a solution of M19-fragment III-C (16.00 g, 9.20 mmol, 1.00 equivalent) and dT (10.28 g, 13.80 mmol, 1.50 equivalent) in Tol. / ACN=3:1 (140 mL), 7.00 g of 3A MS was added and the mixture was stirred for 1 hour. DCI (2.17 g, 18.40 mmol, 2.00 equivalent) was added to the mixture. The mixture was stirred at 25°C for 1 hour. TLC (dichloromethane:methanol=20:1, product:Rf=0.53) showed that the reaction was complete and a new spot had formed. According to TLC, the reaction was clean. HX (2.77 g, 18.42 mmol, 2.00 equivalent) was added to the reaction mixture. The mixture was stirred at 30°C for 0.5 hours. Next, dodecane-1-thiol (5.61 g, 27.70 mmol, 6.63 mL, 3.00 equivalents) and TFA (10.53 g, 92.33 mmol, 6.84 mL, 10.00 equivalents) were added at 0°C. The mixture was stirred at 0°C for 0.5 hours. TLC (dichloromethane:methanol = 20:1, product: Rf = 0.45) showed that the reaction was complete and a new spot had formed. According to TLC, the reaction was clean. The reaction mixture was diluted with NMI (11.37 g, 138.50 mmol, 11.04 mL, 15.00 equivalents). The crude product was added dropwise to ACN (500 ml, 30 V), allowed to precipitate for 0.5 hours, and filtered for 1.5 hours using a 15 cm Buchner funnel to obtain 14.0 g (93.30% yield, 97.28% purity) of M19-fragment III-CT as a white solid.

[0318] General procedure for the preparation of compound M19-fragment III-CTT [ka]

[0319] To a solution of M19-fragment III-CT (15.00 g, 7.10 mmol, 1.00 equivalent) and dT (7.93 g, 10.65 mmol, 1.50 equivalent) in Tol. / ACN=3:1 (120 mL), 3A molecular sieve (2.00 g) was added. The mixture was stirred for 1 hour. Subsequently, DCI (1.68 g, 14.20 mmol, 2.00 equivalent) was added. The mixture was stirred at 30°C for 1 hour. TLC (dichloromethane:methanol=20:1, product: Rf=0.42) showed that the reaction was complete and a new spot had formed. According to TLC, the reaction was clean. HX (2.16 g, 14.37 mmol, 2.00 equivalent) was added to the crude product. The mixture was stirred at 30°C for 0.5 hours, after which dodecane-1-thiol (4.37 g, 21.57 mmol, 5.17 mL, 3.00 equivalents) and TFA (8.20 g, 71.90 mmol, 5.32 mL, 10.00 equivalents) were added at 0°C. The mixture was stirred at 0°C for 0.5 hours. TLC (dichloromethane:methanol = 10:1, product: Rf = 0.54) showed that the reaction was complete and a new spot had formed. According to TLC, the reaction was clean. NMI (8.86 g, 107.85 mmol, 8.60 mL, 15.00 equivalents) was added to the reaction mixture. The mixture was added dropwise to ACN (200 mL, 30 V), allowed to precipitate for 0.5 hours, and filtered through a 7 cm Buchner funnel for 1.0 hour to obtain 5.76 g (96.60% yield, 93.99% purity) of M19-fragment III-CTT as a white solid.

[0320] General procedure for the preparation of compound M19-fragment III-CTTU-DMTr [ka]

[0321] To a solution of M19-fragment III-CTT (10.5 g, 4.22 mmol, 1.00 equivalent) and dU (5.19 g, 6.34 mmol, 1.50 equivalent) in Tol. / ACN=3:1 (80 mL), 0.5 g of 3A MS was added. The mixture was stirred at 25°C for 1 hour. Subsequently, DCI (997.52 mg, 8.45 mmol, 2.00 equivalent) was added. The mixture was stirred at 25°C for 1 hour. TLC (dichloromethane:methanol=10:1, product:Rf=0.50) showed that the reaction was complete and a new spot had formed. According to TLC, the reaction was clean. HX (1.74 g, 8.46 mmol, 2.00 equivalent) was added to the crude product. The mixture was stirred at 25°C for 0.5 hours. The reaction mixture was filtered and concentrated under reduced pressure to reduce the residue to 5V. This residue was then added dropwise to ACN (150 mL, 30V) and allowed to precipitate for 0.5 hours. The mixture was then filtered through a 7 cm Buchner funnel for 1.0 hour to obtain 4.22 g of M19-fragmentIII-CTTU-DMTr (92.5% yield, 91.7% purity) as a white solid.

[0322] General procedure for preparing oligonucleotide fragment B [ka]

[0323] To a solution of imidazole (631.13 mg, 9.27 mmol, 20.00 equivalents) in THF (2.00 mL), pyridine hydrogen fluoride (132.51 mg, 4.64 mmol, 120.46 μL, 70% purity, 10.00 equivalents) was added dropwise at 0°C. The mixture was added to a solution of M19-fragment III-CTTU-DMTr (1.50 g, 463.54 μL, 1.00 equivalent) in THF (15.00 mL). The mixture was stirred at 0-20°C for 1 hour. TLC (dichloromethane:ethyl acetate:methanol = 20:10:1, product: Rf = 0.05) showed that the reaction was complete and a new spot had formed. According to TLC, the reaction was clean. The reaction mixture was washed with NaHCO3 (30 mL) and deionized water to pH=7, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude product was dissolved in DCM (3 ml, 2V) and added dropwise to TBME (50 ml) with vigorous stirring. The desired product precipitated. Oligonucleotide II (800 mg, 434.37 umol, 93.71% yield) was obtained as a white solid.

[0324] Example 9. Synthesis of oligonucleotide fragment C a. Synthesis scheme for oligonucleotide fragment C from M22

[0325] Fragment C was synthesized according to the synthesis scheme shown in Figure 4.

[0326] b. Procedure for the synthesis of oligonucleotide fragment C from M22

[0327] General procedure for the preparation of compound M22-fragment IU-DMTr [ka]

[0328] To a solution of M22 (3.10 g, 2.30 mmol) and compound U-DMTr (2.84 g, 4.58 mmol) in DCM (18.0 mL), DMAP (560 mg, 4.58 mmol) and EDCI (879 mg, 4.58 mmol) were added. The mixture was stirred at 25°C for 12 hours. TLC (petroleum ether / ethyl acetate = 1 / 1, product: R) was performed. f A value of 0.34 indicated that M22 had been consumed and one major new spot had been detected. The reaction mixture was concentrated under reduced pressure. The residue was diluted with DCM (100 mL) and washed with saturated NaHCO3 solution (40.0 mL x 3). The combined organic layers were washed with brine (100 mL x 3), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The crude product was redissolved in DCM (60.0 mL) and added dropwise to MeOH / ACN (3 / 1, 300 mL) with vigorous stirring. The desired product was precipitated, filtered, and concentrated under reduced pressure to obtain the residue. M22-fragment IU-DMTr (5.20 g, crude) was obtained as a white solid.

[0329] General procedure for the preparation of compound M22-fragment IU [ka]

[0330] To a solution of M22-fragment IU-DMTr (5.10 g, 2.61 mmol) in DCM (30.0 mL), dodecane-1-thiol (1.58 g, 7.83 mmol, 1.88 mL) and TFA (2.38 g, 20.9 mmol, 1.55 mL) were added. The mixture was stirred at 0°C for 1 hour. TLC (petroleum ether / ethyl acetate = 1 / 1, product: R) fThe result (=0.28) indicated that the M22-fragment IU-DMTr was completely consumed, forming two new spots. The reaction mixture was quenched by adding Py (26.0 equivalents) at 0°C, then diluted with DCM (150 mL) and washed with saturated NaHCO3 aqueous solution (150 mL x 4). The combined organic layers were washed with brine (150 mL x 3), dried over anhydrous Na2SO4 and Na2SO4 (15.0 g), filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography (SiO2, DCM / MeOH = 150 / 1 to 100 / 1). M22-fragment IU (2.90 g, 64.6% yield, 96.0% purity) was obtained as a white solid.

[0331] General procedure for the preparation of compound M22-fragment I-UC [ka]

[0332] To a solution of M22-fragment IU (2.00 g, 1.21 mmol) and C-DMTr (1.67 g, 1.82 mmol) in Tol. (9.60 mL) and IPAC (2.40 mL), DCI (286 mg, 2.42 mmol) and molecular sieve 3A (2.00 g) were added. The mixture was stirred at 30°C for 1 hour. TLC (dichloromethane / methanol = 20 / 1, product: R f The result (=0.43) indicated that the M22-fragment IU was completely consumed and many new spots were formed. Next, xanthan hydride (365 mg, 2.42 mmol) was added to the reaction mixture. The mixture was then stirred at 30°C for 0.5 hours. Next, dodecane-1-thiol (727 mg, 3.60 mmol, 860 uL) and TFA (2.04 g, 18.0 mmol, 1.33 mL) were added. The mixture was stirred at 0°C for 1 hour. TLC (dichloromethane / methanol = 20 / 1, product: R fThe result (=0.34) indicated that M22-fragment I-UC-DMTr was completely consumed and many new spots were formed. The reaction mixture was quenched by adding NMI (20.0 equivalents) and then filtered. It was then triturated with ACN (300 mL) and filtered. M22-fragment I-UC (2.64 g, 95.2% yield, 95.1% purity) was obtained as a white solid.

[0333] General procedure for the preparation of compound M22-fragment I-UCC [ka]

[0334] To a solution of M22-fragment I-UC (2.60 g, 1.18 mmol) and C-DMTr (1.64 g, 1.77 mmol) in Tol. (12.6 mL) and IPAC (3.00 mL), DCI (279 mg, 2.36 mmol) and molecular sieve 3A (2.00 g) were added. The mixture was stirred at 30°C for 1 hour. TLC (dichloromethane / methanol = 20 / 1, product: Rf = 0.48) showed that M22-fragment I-UC was completely consumed and many new spots were formed. Next, xanthan hydride (358 mg, 2.38 mmol) was added to the reaction mixture. The mixture was stirred at 30°C for 0.5 hours. Next, dodecane-1-thiol (716 mg, 3.54 mmol, 846 uL) and TFA (2.02 g, 17.7 mmol, 1.31 mL) were added. The mixture was stirred at 0°C for 1 hour. TLC (dichloromethane / methanol = 20 / 1, product: Rf = 0.41) showed that M22-fragment I-UCC-DMTr was completely consumed and two new spots were formed. The reaction mixture was quenched by adding NMI (20.0 equivalents) and then filtered. Next, it was triturated with ACN (300 mL) and filtered. M22-fragment I-UCC (3.23 g, crude) was obtained as a white solid.

[0335] General procedure for the preparation of compound M22-fragment I-UCCC [ka]

[0336] To a solution of M22-fragment I-UCC (3.20 g, 1.16 mmol) and C-DMTr (1.61 g, 1.74 mmol) in Tol. (15.4 mL) and ACN (3.80 mL), DCI (275 mg, 2.32 mmol) and molecular sieve 3A (1.50 g) were added. The mixture was stirred at 30°C for 1 hour. TLC (dichloromethane / methanol = 20 / 1, twice, R) was performed. f The result (=0.59) indicated that the M22-fragment I-UCC was completely consumed and many new spots were formed. Next, tert-butyl hydroperoxide (5.50 M, 1.18 mmol, 106.85 uL) was added to the reaction mixture. The mixture was stirred at 30°C for 0.5 hours. Next, dodecane-1-thiol (711 mg, 3.52 mmol, 840 uL) and TFA (2.00 g, 17.6 mmol, 1.30 mL) were added. The mixture was stirred at 0°C for 1 hour. TLC (dichloromethane / methanol = 20 / 1, 3 times, product: R f The result (=0.54) indicated that M22-fragment I-UCCC-DMTr was completely consumed and two new spots were formed. The reaction mixture was quenched by adding NMI (20.0 equivalents) and then filtered. It was then triturated with ACN (300 mL) and filtered. The white solid was concentrated under reduced pressure to obtain the residue. M22-fragment I-UCCC (3.60 g, crude) was obtained as a white solid.

[0337] General procedure for preparing oligonucleotide fragment C [ka]

[0338] To a solution of M22-fragment I-UCCC (3.40 g, 1.03 mmol) and A-DMTr (1.45 g, 1.55 mmol) in Tol. (16.0 mL) and ACN (4.00 mL), DCI (244 mg, 2.06 mmol) and molecular sieve 3A (1.00 g) were added. The mixture was stirred at 30°C for 1 hour. TLC (dichloromethane / methanol = 15 / 1, 3 times, R) was performed. f The reaction (=0.47) indicated that M22-fragment I-UCCC was completely consumed and many new spots were formed. Next, xanthan hydride (307 mg, 2.04 mmol) was added to the reaction mixture. The mixture was stirred at 30°C for 0.5 hours. Then, dodecane-1-thiol (615 mg, 3.04 mmol, 727 μL) and TFA (1.73 g, 15.2 mmol, 1.12 mL) were added to the reaction mixture and the mixture was stirred at 0°C for 1 hour. TLC (dichloromethane / methanol = 15 / 1, 3 times, product: R f The result (=0.45) indicated that M22-fragment I-UCCCA-DMTr was completely consumed, forming two new spots. The reaction mixture was quenched by adding NMI (20.0 equivalents) and then filtered. It was then triturated with ACN (600 mL) and filtered again. Oligonucleotide fragment C (3.90 g, 90.3% yield and 84.2% purity) was obtained as a white solid.

[0339] Example 10. Synthesis of oligonucleotide fragment D a. Synthesis scheme for oligonucleotide fragment D

[0340] Fragment D was synthesized according to the synthesis scheme shown in Figure 5.

[0341] b. Procedure for the synthesis of oligonucleotide fragment D

[0342] The procedure for synthesizing oligonucleotide fragment D was the same as that described for the synthesis of oligonucleotide fragment A.

[0343] Example 11. Synthesis of oligonucleotide fragment E a. Synthesis scheme for oligonucleotide fragment E

[0344] Fragment E was synthesized according to the synthesis scheme shown in Figure 6.

[0345] b. Procedure for the synthesis of oligonucleotide fragment E

[0346] General procedure for the preparation of compound E-2 [ka]

[0347] Compound M19-fragment IU-DMTr (2.00 g, 1.17 mmol, 1.00 equivalent), anhydrous DCM (12.0 ml), and CH3CN (4.00 mL) were concentrated three times under reduced pressure to remove water. To the solution of compound M19-fragment IU-DMTr (2.00 g, 1.17 mmol, 1.00 equivalent) in DCM (16 mL), 3A MS (1.60 g) was added in one step under Ar at 25°C and stirred for 0.5 hours. dT-amidite (1.31 g, 1.75 mmol, 1.5 equivalent) and DCI (276 mg, 2.34 mmol, 2.00 equivalent) were added, and the mixture was stirred at 25°C for 1 hour. HPLC showed that the starting materials were completely consumed. DDTT (480 mg, 2.34 mmol, 2.00 equivalents) was added to the reaction mixture. The mixture was stirred at 25°C for 0.5 hours. HPLC showed that the starting material was completely consumed. The crude product was triturated with ACN (160 mL) at 25°C for 1 hour. The mixture was filtered, and the cake was concentrated under vacuum. Compound E-2 (2.3 g, 964 µl, 83.0% yield) was obtained as a white solid. 137 H 198 N7O 22 PSSiNa + [M+Na + Calculated mass value for ]: 2408.4, measured value: 2408.4.

[0348] General procedure for the preparation of compound E-3 [ka]

[0349] In a solution of compound E-2 (2.30 g, 964 umol, 1.00 equivalent) in DCM (20.0 mL), C 12 H 25 SH (585 mg, 2.89 mmol, 693 μL, 3.00 equivalents) was added all at once under N2 conditions at 25°C. TFA (1.32 g, 11.57 mmol, 856.38 μL, 12 equivalents) was added to the solution and the mixture was stirred at 0-5°C for 2 hours. LC-MS showed that the starting materials were completely consumed. NMI (1.19 g, 14.5 mmol, 1.15 mL, 15.0 equivalents) was added to the reaction mixture and stirred at 0-5°C for 0.5 hours. The crude product was triturated with ACN (200 mL) at 25°C for 1 hour. The mixture was filtered and the cake was concentrated under vacuum. Compound E-3 (2.00 g, 960 μL, 99% yield) was obtained as a white solid. 116 H 181 N7O 20 PSSi + [M+H + Calculated mass value for ]: 2083.3, measured value: 2083.3.

[0350] General procedure for the preparation of compound E-4 [ka]

[0351] Compound E-3 (2.00 g, 959 ml, 1.00 equivalent) was concentrated three times under reduced pressure with anhydrous DCM (12.0 ml) and ACN (4.00 mL) to remove water. To the solution of compound E-3 (2.00 g, 959 ml, 1.00 equivalent) in DCM (16 mL), 3A MS (1.60 g) was added all at once under Ar at 25°C and stirred for 0.5 hours. 2'-OMe A amidite (1.28 g, 1.44 mmol, 1.50 equivalent) and DCI (227 mg, 1.92 mmol, 2.00 equivalent) were added, and the mixture was stirred at 25°C for 1 hour. LC-MS showed that the starting materials were completely consumed. DDTT (395 mg, 1.92 mmol, 2.00 equivalents) was added to the reaction mixture, and the mixture was stirred at 25°C for 0.5 hours. LC-MS showed that the starting materials were completely consumed. The crude product was triturated with ACN (160 mL) at 25°C for 1 hour. The mixture was filtered, and the cake was concentrated under vacuum. Compound E-4 (2.11 g, 727 umol, 75.6% yield) was obtained as a white solid. Mass calculation value relative to C158H219N13O28P2S2SiNa+[M+Na+]: 2923.5, measured value: 2924.5.

[0352] General procedure for the preparation of compound E-5 [ka]

[0353] Compound E-4 (2.1 g, 723 umol, 1.00 equivalent) was concentrated three times under reduced pressure with anhydrous DCM (12.0 ml) and ACN (4.00 mL) to remove water. To the solution of compound E-4 (2.1 g, 723 umol, 1.00 equivalent) in DCM (20.0 mL), C was added. 12 H 25SH (439 mg, 2.17 mmol, 520 μL, 3.00 equivalents) was added all at once under N2 conditions at 25°C. TFA (990 mg, 8.68 mmol, 643 μL, 12.0 equivalents) was added to the solution and the mixture was stirred at 0–5°C for 2 hours. LC-MS showed that the starting materials were completely consumed. NMI (891 mg, 10.8 mmol, 865 μL, 15 equivalents) was added to the reaction mixture and stirred at 0–5°C for 0.5 hours. The crude product was triturated with ACN (200 mL) at 25°C for 1 hour. The mixture was filtered and the cake was concentrated under vacuum. Compound E-5 (1.7 g, 653.77 μL, 90.37% yield) was obtained as a white solid. 137 H 202 N 13 O 26 P2S2Si + Calculated mass value for [M+H]: 2599.3, measured value: 2599.4.

[0354] General procedure for the preparation of compound E-6 [ka]

[0355] Compound E-5 (1.00 g, 385 umol, 1.00 equivalent) was concentrated three times under reduced pressure with anhydrous DCM (6.00 ml) and ACN (2.00 mL) to remove water. To the solution of compound E-5 (1.00 g, 385 umol, 1.00 equivalent) in DCM (8.00 mL), 3A MS (1.60 g) was added all at once under Ar at 25°C and stirred for 0.5 hours. 5'-DMTrO-2'-FU amidite (440 mg, 577 umol, 1.50 equivalent) and DCI (90.8 mg, 769 umol, 2.00 equivalent) were added to the mixture and stirred at 25°C for 1 hour. LC-MS showed that the starting materials were completely consumed. DDTT (151 mg, 736 umol, 2.00 equivalents) was added to the reaction mixture, and the mixture was stirred at 25°C for 0.5 hours. LC-MS showed that the starting materials were completely consumed. The crude product was triturated with ACN (80 mL) at 25°C for 1 hour. The mixture was filtered, and the cake was concentrated under vacuum. Compound E-6 (0.9 g, 273 umol, 74.3% yield) was obtained as a white solid. 170 H 233 FN 16 O 34 P3S3Si + Calculated mass value for [M+H]: 3278.5118, measured value: 3278.6255.

[0356] General procedure for preparing fragment E [ka]

[0357] Compound E-6 (800 mg, 244 umol, 1.00 equivalent) was concentrated three times under reduced pressure with anhydrous DCM (6.00 ml) and ACN (2.00 mL) to remove water. C was added to the solution of Compound E-6 (800 mg, 244 umol, 1.00 equivalent) in DCM (8.00 mL). 12 H 25SH (148 mg, 732 µl, 176 µL, 3.00 equivalents) was added all at once under N2 conditions at 25°C. TFA (334 mg, 2.93 mmol, 217 µL, 12.0 equivalents) was added to the solution and the mixture was stirred at 0–5°C for 2 hours. HRMS showed that the starting materials were completely consumed. NMI (300 mg, 3.66 mmol, 292 µL, 15.0 equivalents) was added to the reaction mixture and stirred at 0–5°C for 0.5 hours. The crude product was triturated with ACN (80.0 mL) at 25°C for 1 hour. The mixture was filtered and the cake was concentrated under vacuum. Fragment E (600 mg, 201 µl, 82.6% yield) was obtained as a white solid. C 149 H 214 FN 16 O 32 P3S3Si + HRMS calculated value for [M+H]: 2976.3811, measured value: 2976.3875.

[0358] Example 12. Synthesis of oligonucleotide fragment F a. Synthesis scheme for oligonucleotide fragment F

[0359] Fragment F was synthesized according to the synthesis scheme shown in Figure 7.

[0360] b. Procedure for the synthesis of oligonucleotide fragment F

[0361] General procedure for the preparation of compound F-1 [ka]

[0362] Compound E-1 (0.5 g, 292.31 umol, 1.00 equivalent) was concentrated three times under reduced pressure with anhydrous DCM (4.0 ml) and CH3CN (2.00 mL) to remove water. To the solution of Compound E-1 (0.5 g, 292.31 umol, 1.00 equivalent) in DCM (5.00 mL), 3A MS (0.50 g) was added all at once under Ar at 25°C and stirred for 0.5 hours. LNA-T amidite (451.81 mg, 584.62 umol, 2.00 equivalent) and DCI (75.95 mg, 643.08 umol, 2.20 equivalent) were added, and the mixture was stirred at 25°C for 1 hour. LCMS showed that the starting materials were completely consumed. DDTT (480 mg, 2.34 mmol, 2.00 equivalents) was added to the reaction mixture, and the mixture was stirred at 25°C for 0.5 hours. LC-MS showed that the starting materials were completely consumed. The crude product was triturated with ACN (50 mL) at 25°C for 1 hour. The mixture was filtered, and the cake was concentrated under vacuum. Compound F-1 (2.3 g, 964 umol, 83.0% yield) was obtained as a white solid.

[0363] General procedure for preparing fragment F [ka]

[0364] A solution of compound F-1 (50 mg, 19.23 umol, 1.00 equivalent) saturated with NH3·H2O (2.00 mL) was stirred in a 4 mL sealed tube at 70°C for 16 hours. The reaction mixture was filtered without any purification, and the filtrate was subjected to LC-MS. Fragment F was identified by LC-MS. 45 H 50 N4O 16 PS - [MH + HRMS calculated value for ]: 965.2686, measured value: 965.2846.

[0365] Example 13. Synthesis of oligonucleotide fragment G a. Synthesis scheme for oligonucleotide fragment G

[0366] Fragment G was synthesized according to the synthesis scheme shown below. [ka]

[0367] b. Procedure for the synthesis of oligonucleotide fragment G

[0368] Compound E-1 (500 mg, 292 umol, 1.00 equivalent), anhydrous DCM (4.0 ml), and ACN (2.00 mL) were concentrated three times under reduced pressure to remove water. To the solution of Compound E-1 (500 mg, 292 umol, 1.00 equivalent) in DCM (4.00 mL), 3A MS (500 mg) was added all at once under Ar at 25°C and stirred for 0.5 hours. 2'-OTBS A amidite (578 mg, 585 umol, 2.00 equivalent) and DCI (75.9 mg, 643 umol, 2.20 equivalent) were added to the above mixture and the mixture was stirred at 25°C for 1 hour. LC-MS showed that the starting materials were completely consumed. DDTT (480 mg, 2.34 mmol, 2.00 equivalents) was added to the reaction mixture, and the mixture was stirred at 25°C for 0.5 hours. LC-MS showed that the starting materials were completely consumed. The crude product was triturated with ACN (40 mL) at 25°C for 1 hour. The mixture was filtered, and the cake was concentrated under vacuum. Fragment G (700 mg, 266 umol, 91.1% yield) was obtained as a white solid. Mass calculation value for C150H216N10O22PSSi2+[M+H+]: 2628.5043, measured value: 2628.5959.

[0369] Example 14. Synthesis of oligonucleotide fragment H a. Synthesis scheme for oligonucleotide fragment H

[0370] Fragment H was synthesized according to the synthesis scheme shown below. [ka]

[0371] b. Procedure for the synthesis of oligonucleotide fragment H

[0372] General procedure for the preparation of compound H-1 [ka]

[0373] To a solution of compound M-50 (0.8 g, 621.08 ml, 1.00 equivalent) and 5'-DMTr-Dexoy C-3'-OH (804.57 mg, 1.24 mmol, 2.00 equivalent) in DCM (6.0 mL), DMAP (113.82 mg, 931.62 ml, 1.50 equivalent) was added. The mixture was stirred at 25°C for 0.5 hours, and EDCI (238.12 mg, 1.24 mmol, 2.00 equivalent) was added. The mixture was stirred at 25°C for 3 hours. HPLC showed that the starting material was completely consumed. The residue was diluted with H2O (50 mL) and extracted by DCM (2 x 30 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The crude product was tritulated with EtOH (30V, 30.0mL) at 25°C for 30 minutes. The crude product was tritulated with ACN (30V, 30.0mL) at 25°C for 30 minutes, filtered, and concentrated. Compound H-1 (0.8g, 0.41 mmol, 92.0% purity, 67.17% yield) was obtained as a white solid. 118 H 179 N5O 14 Si2+[M+2H + Calculated mass value for ] / 2: 959.1, measured value: 960.

[0374] General procedure for the preparation of compound H-2 [ka]

[0375] In a solution of compound H-1 (2.0 g, 1.04 mmol, 1.00 equivalent) in DCM (16.0 mL), C 12 H 25SH (274.40 mg, 1.36 mmol, 324.74 μL, 1.30 equivalents) and DCA (1.08 g, 8.34 mmol, 685.20 μL, 8.00 equivalents) were added at 0-5°C. The mixture was stirred at 0-5°C for 2.5 hours, and NMI (856.20 mg, 10.43 mmol, 831.26 μL, 10.00 equivalents) was added. The mixture was stirred at 0-5°C for 0.5 hours. The residue was diluted with NaHCO4 / H2O (50 mL) and extracted with DCM (2 x 30 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The crude product was triturated with ACN (30 V, 60.0 mL) at 25°C for 30 minutes, filtered, and concentrated. Compound H-2 (1.4 g, 713.26 umol, 82.3% purity, 68.4% yield) was obtained as a white solid. 97 H 160 N5O 12 Si + [M+H + Calculated mass value for ]: 1615.1, measured value: 1615.6.

[0376] General procedure for the preparation of compound H-3 [ka]

[0377] Compound H-2 (900 mg, 557 ml, 1.00 equivalent), anhydrous DCM (8.0 ml), and ACN (2.00 mL) were concentrated three times under reduced pressure to remove water. To the solution of Compound H-2 (900 mg, 557 ml, 1.00 equivalent) in DCM (8.00 mL), 3A MS (800 mg) was added all at once under Ar at 25°C and stirred for 0.5 hours. 5'-DMTr-Dexoy T-3'-phosphoramidite (830 mg, 1.11 mmol, 2.00 equivalent) and DCI (197.4 mg, 1.67 mmol, 3.0 equivalent) were added to the above mixture. The mixture was stirred at 25°C for 1 hour. LCMS showed that the starting materials were completely consumed. DDTT (343 mg, 1.67 mmol, 3.00 equivalents) was added to the reaction mixture. The mixture was stirred at 25°C for 0.5 hours. LC-MS showed that the starting materials were completely consumed. The crude product was triturated with ACN (40 mL) at 25°C for 1 hour. The mixture was filtered and the cake was concentrated under vacuum. Compound H-3 (600 mg, 145.85 mmol, 47.3% yield) was obtained as a white solid. 110 H 176 N8O 18 PSSi + [M-DMTr+H + The calculated mass for ] is 1988.2, and the measured mass is 1989.0.

[0378] General procedure for preparing fragment H [ka]

[0379] To a solution of compound H-3 (0.2 g, 87.29 umol, 1.00 equivalent) in THF (1.6 mL), solutions of imidazole (118.86 mg, 1.75 mmol, 20.0 equivalents) and pyridine / hydrofluoride (24.94 mg, 872.95 umol, 70% purity, 10.0 equivalents) in THF (0.6 mL) were added. The mixture was stirred at 0-5°C for 20 hours (compound H-3 was converted to fragment H with >95% conversion). The structure of fragment H was confirmed by LC-MS. 51 H 54 N6O13 PS + [M+H + Calculated mass value for ]: 1021.3, measured value: 1021.4.

[0380] Example 15. Synthesis of oligonucleotide fragment J a. Synthesis scheme for oligonucleotide fragment J

[0381] Fragment J was synthesized according to the synthesis scheme shown in Figure 8.

[0382] b. Procedure for the synthesis of oligonucleotide fragment J

[0383] General procedure for the preparation of compound J-2 [ka]

[0384] Compound H-2 (500 mg, 0.309 mmol, 1.00 equivalent) was concentrated three times under reduced pressure with anhydrous DCM (8.0 ml) and ACN (2.00 mL) to remove water. To the solution of compound H-2 (500 mg, 0.309 mmol, 1.00 equivalent) in DCM (5.00 mL), 3A MS (500 mg) was added all at once under Ar at 25°C and stirred for 0.5 hours. 5'-DMTr-MOE-ACCC-3'-phosphoramidite (compound J-1 (synthesis method is described in WO2020 / 227618, paragraphs

[0332] -

[0333] ; this document is incorporated herein by reference), 1590 mg, 0.62 mmol, 2.00 equivalents) and DCI (110 mg, 0.927 mmol, 3.0 equivalents) were added to the above mixture and the mixture was stirred at 25°C for 1 hour. DDTT (254 mg, 1.23 mmol, 4.00 equivalents) was added to the reaction mixture and the mixture was stirred at 25°C for 0.5 hours. The crude product was triturated with ACN (50 mL) at 25°C for 1 hour. The mixture was filtered and the cake was concentrated under vacuum. Compound J-2 (770 mg, 61% yield) was obtained as a white solid. 210 H 285 N 23 O46 P4S3Si + [M+2H + Calculated mass value for ] / 2: 2057.4, measured value: 2058.0.

[0385] General procedure for preparing fragment J [ka]

[0386] A solution of compound J-2 (50 mg) saturated with NH3·H2O (2.00 mL) was stirred in a 4 mL sealed tube at 65°C for 8 hours. The reaction mixture was filtered without any purification, and the filtrate was subjected to LCMS. Fragment J:C 83 H 109 N 17 O 34 Calculated mass for P4S3[M-2H] / 2: 1053.8, measured mass: 1054.4.

[0387] Example 16. Synthesis of oligonucleotide fragment K a. Synthesis scheme for the mutant fragment K

[0388] Fragment K was synthesized according to the synthesis scheme shown in Figure 9.

[0389] b. Procedure for the synthesis of oligonucleotide fragment K

[0390] General procedure for the preparation of compound K-1 [ka]

[0391] To a solution of compound M-60 (0.9 g, 676 ml, 1.00 equivalent) and 5'-DMTr-Dexoy C-3'-OH (876.5 mg, 1.35 mmol, 2.00 equivalent) in DCM (10.0 mL), DMAP (124 mg, 1.01 mmol, 1.50 equivalent) was added. The mixture was stirred at 25°C for 0.5 hours, and EDCI (260 mg, 1.35 mmol, 2.00 equivalent) was added. The mixture was stirred at 25°C for 3 hours. HPLC showed that the starting material was completely consumed. The residue was diluted with H2O (50 mL) and extracted by DCM (2 x 20 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The crude product was tritulated with EtOH (30V, 30.0mL) at 25°C for 30 minutes. The crude product was tritulated with ACN (30V, 30.0mL) at 25°C for 30 minutes, filtered, and concentrated. Compound K-1 (1.2g, 0.56 mmol, 82.4% yield) was obtained as a white solid. 121 H 184 N5O 14 Si + [M+2H + Calculated mass value for ] / 2: 980.7, measured value: 981.1.

[0392] General procedure for the preparation of compound K-2 [ka]

[0393] In a solution of compound K-1 (2.0 g, 1.04 mmol, 1.00 equivalent) in DCM (16.0 mL), C 12 H 25SH (274.40 mg, 1.36 mmol, 324.74 μL, 1.30 equivalents) and DCA (1.08 g, 8.34 mmol, 685.20 μL, 8.00 equivalents) were added at 0-5°C. The mixture was stirred at 0-5°C for 2.5 hours, and NMI (856.20 mg, 10.43 mmol, 831.26 μL, 10.00 equivalents) was added. The mixture was stirred at 0-5°C for 0.5 hours. The residue was diluted with NaHCO4 / H2O (50 mL) and extracted with DCM (2 x 30 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The crude product was triturated with ACN (30 V, 60.0 mL) at 25°C for 30 minutes, filtered, and concentrated. Compound K-2 (1.4 g, 713.26 umol, 68.4% yield) was obtained as a white solid. 100 H 166 N5O 12 Si + [M+H + Calculated mass value for ]: 1657.2, measured value: 1657.9.

[0394] General procedure for the preparation of compound K-3 [ka]

[0395] Compound K-2 (1.0 g, 603 ml, 1.00 equivalent) was concentrated twice under reduced pressure with anhydrous DCM (8.0 ml) and ACN (2.00 mL) to remove water. To the solution of compound K-2 (1.0 g, 603 ml, 1.00 equivalent) in DCM (8.00 mL), 3A MS (800 mg) was added all at once under Ar at 25°C and stirred for 0.5 hours. 5'-DMTr-Dexoy T-3'-phosphoramidite (898 mg, 1.21 mmol, 2.00 equivalent) and DCI (213.7 mg, 1.81 mmol, 3.0 equivalent) were added to the above mixture. The mixture was stirred at 25°C for 1 hour. LC-MS showed that the starting materials were completely consumed. DDTT (372 mg, 1.81 mmol, 3.00 equivalents) was added to the reaction mixture, and the mixture was stirred at 25°C for 0.5 hours. The crude product was triturated with ACN (40 mL) at 25°C for 1 hour. The mixture was filtered, and the cake was concentrated under vacuum. Compound K-3 (1.20 g, 77.3% yield) was obtained as a white solid. 113 H 182 N8O 18 PSSi + [M-DMTr+H + Calculated mass value for ]: 2031.3, measured value: 2031.0.

[0396] General procedure for preparing fragment K [ka]

[0397] To a solution of compound K-3 (0.2 g, 85.7 ml, 1.00 equivalent) in THF (1.6 mL), solutions of imidazole (116.7 mg, 1.71 mmol, 20.0 equivalents) and pyridine / hydrofluoride (24.5 mg, 857.2 ml, 70% purity, 10.0 equivalents) in THF (0.6 mL) were added. The mixture was stirred at 25°C for 20 hours (compound K-3 was converted to fragment K with >95% conversion). The structure of fragment K was confirmed by LC-MS. 51 H 53 N6NaO 13 PS + [M+Na +Calculated mass value for ]: 1043.3, measured value: 1043.9.

[0398] Example 17. H-phosphate chemistry for preparing fragment L a. Composition scheme for fragment L

[0399] Fragment L was synthesized according to the synthesis scheme shown below. [ka]

[0400] b. Procedure for the synthesis of oligonucleotide fragment L

[0401] General procedure for preparing fragment L

[0402] A mixture of compound L-1 (0.5 g, 0.70 mmol, 3.00 equivalents) and pivaloyl chloride (85 mg, 0.70 mmol, 3.0 equivalents) in pyridine (5 mL) was stirred at 0°C for 1 hour, and compound E-1 (402 mg, 235.1 mmol, 1.0 equivalent) was added. The reaction mixture was stirred at 0°C for 2.0 hours. A solution of iodine (120 mg, 0.47 mmol, 2.00 equivalents) and pyridine (121 mg, 1.53 mmol, 6.5 equivalents) in THF / H2O (3 mL, 5:1, v / v) was added dropwise to the reaction mixture at 0-5°C and stirred at 0-5°C for 1 hour. A 4 wt% aqueous solution of Na2S2O3 (116 mg, 0.47 mmol, 2.00 equivalents) was added dropwise at 0-5°C and stirred at 15-25°C for 10 minutes. Â (50 mL) was added and the mixture was vigorously stirred for 30 minutes. The top organic layer was separated, washed with 5 wt% NaHCO3 solution (2 x 30 mL) and brine (30 mL), dried over MgSO4, filtered, and concentrated. The crude product was triturated with ACN (40 mL) at 25°C for 1 hour. The mixture was filtered and the cake was concentrated under vacuum. Fragment L (400 mg) was obtained as a white solid. 134 H 195 N6O 23 Calculated mass value for Psi[M] / 2: 1157.6, measured value: 1157.6.

[0403] Example 18. Synthesis of Chiral Oligonucleotide Fragment M a. Synthesis scheme for chiral oligonucleotide fragment M

[0404] The chiral oligonucleotide fragment M was synthesized according to the synthesis scheme shown below. [ka]

[0405] b. Procedure for the synthesis of oligonucleotide fragment M

[0406] General procedure for the preparation of compound M-3 [ka]

[0407] Compound M-1 (134.7 mg, 140.3 µl, 1.00 equivalent) was concentrated twice under reduced pressure with anhydrous DCM (5.0 ml) and ACN (10.00 mL) to remove water. To a solution of compound M-1 (134.7 mg, 140.3 µl, 1.00 equivalent) and compound M-2 (160 mg, 93.5 µmol, 1.0 equivalent) in DCM (2.00 mL), 3A MS (200 mg) was added all at once under Ar at 25°C and stirred for 0.5 hours. BuOK (1.0 M, 281 µL, 3.00 equivalent) was added to the above mixture. The mixture was stirred at 25°C for 1 hour. LCMS showed that the starting material was completely consumed. The mixture was filtered and washed with DCM (0.5 mL). The crude product was triturated with ACN (20 mL) at 25°C for 1 hour. The mixture was filtered, and the cake was concentrated under vacuum. Chiral fragment M (175 mg, 69.9 umol, 74.7% yield) was obtained as a white solid. 120 H 189 N90 22 PSSi + [M-DMTr+H + Calculated mass value for ]: 2200.3, measured value: 2200.1. 31P NMR (162 MHz, CDCl3) δ 58.6 ppm.

[0408] General procedure for preparing fragment M [ka]

[0409] A solution of compound M-3 (30 mg) saturated with NH3·H2O (2.00 mL) was stirred in a 4 mL sealed tube at 65°C for 16 hours. The reaction mixture was filtered without any purification, and the filtrate was subjected to LC-MS. Fragment M was identified by LC-MS. 47 H 56 N7O 16 P - [MH - HRMS calculated value: 1036.3169, measured value: 1036.3502.

[0410] Example 19. Synthesis of Chiral Oligonucleotide Fragment N a. Synthesis scheme for chiral oligonucleotide fragment N

[0411] The chiral oligonucleotide fragment N was synthesized according to the synthesis scheme shown below. [ka]

[0412] General procedure for the preparation of compound N-2 [ka]

[0413] Compound M19-fragment IU (200 mg, 117 µmol, 1.00 equivalent) was concentrated twice under reduced pressure in anhydrous DCM (1.0 ml) and DCM (3.0 mL) to remove water. To a solution of compound M19-fragment IU (200 mg, 117 µmol, 1.00 equivalent) and compound N-1 (68 mg, 152 µmol, 1.30 equivalent) in DCM (2.00 mL), 3A MS (200 mg) was added all at once under Ar at 25°C and stirred for 0.5 hours. DBU (23 mg, 152 µmol, 1.30 equivalent) was added to the above mixture and the mixture was stirred at 25°C for 1 hour. The mixture was filtered and washed with DCM (1.0 mL). The crude product was triturated with ACN (30 mL) at 25°C for 1 hour. The mixture was filtered and the cake was concentrated under vacuum. Compound N-2 (215 mg, 84.6 umol, 84.6% yield) was obtained as a white solid.

[0414] General procedure for preparing fragment N [ka]

[0415] Compound N-2 (109.4 mg, 153 umol, 1.50 equivalents) was concentrated twice under reduced pressure with anhydrous CH3CN (1.0 ml) and DCM (3.0 mL) to remove water. To a solution of compound 5 (109.4 mg, 153 umol, 1.50 equivalents) and 5'-DMTr-MOE G-3'-OH (200 mg, 102.2 umol, 1.0 equivalent) in DCM (2.00 mL), 3A MS (200 mg) was added all at once under Ar at 25°C and stirred for 0.5 hours. BuOK (1.0 M, 307 μL, 3.0 equivalents) was added to the above mixture and the mixture was stirred at 25°C for 1 hour. The mixture was filtered and washed with DCM (1.0 mL). The crude product was triturated with ACN (25 mL) at 25°C for 1 hour. The mixture was filtered and the cake was concentrated under vacuum. Chiral fragment N (175 mg, 54.1 umol, 68.1% yield) was obtained as a white solid. 120 H 189 N90 22 PSSi +[M-DMTr+H + Calculated mass value for ]: 2200.3, measured value: 2200.1. 31 P NMR (162 MHz, CDCl3) δ 58.6 ppm.

[0416] Example 20. Synthesis of oligonucleotide fragment O a. Synthesis scheme for oligonucleotide fragment O

[0417] Oligonucleotide fragment N was synthesized according to the synthesis scheme shown in Figure 10.

[0418] b. Procedure for the synthesis of oligonucleotide fragment O

[0419] General procedure for the preparation of compound O-1 [ka] A solution of 5'-DMTrO-C-OTBDPS-3' (5.0 g, 5.2 mmol, 1.00 equivalent), NH3·H2O (25%, 3.6 g, 26.0 mmol, 5.00 equivalent), and THF (40 mL) was stirred at 20±5°C for 1.0 hour (HLPC showed a 42.8% conversion of 5'-DMTrO-C-OTBDPS-3'). The mixture was concentrated to obtain the crude product residue. This was purified by silica gel chromatography (with 0-10% THF in DCM as the eluent). Compound O-1 was obtained as a pale yellow solid (1.6 g, 36.0% yield). 1H NMR (300 MHz, DMSO-d6) δ 8.02 (s, 1H), 7.90 (d, J = 9.0 Hz, 1 H), 7.64 (d, J = 9.0 Hz, 2 H), 7.58-7.09 (m, 22H), 6.93-6.72 (m, 5H), 5.96 (s, 1H), 4.33 (t, J = 4.5 Hz, 1H), 4.12 (s, 1H), 3.63 (t, J = 3.0 Hz, 1 H), 3.39 (s, 2H), 3.34 (t, J = 3.0 Hz, 2 H), 3.28-3.19 (m, 2H), 3.18 (s, 3H), 3.05-2.93 (m, 1H), 1.39 (s, 3H), 0.94 (s, 9H). 13C{1H} NMR (75 MHz, DMSO-d6) δ 168.4, 165.8, 158.6, 155.3, 114.9, 135.9, 135.7, 135.4, 133.1, 130.2, 128.6, 128.2, 128.1, 127.9, 113.7, 102.0, 87.8, 86.5, 82.5, 81.7, 71.7, 71.4, 69.2, 63.1, 58.6, 55.5, 27.1, 19.3, 13.3. Calculated value for C50H58N3O8Si+ [M+H]+ HRMS: 856.3988, Measured value: 856.4009

[0420] General procedure for the preparation of compound O-2 [ka]

[0421] To a solution of compound O-1 (498 mg, 0.64 mmol, 1.50 equivalents) and compound M19 (600 mg, 0.42 mmol, 1.0 equivalent) in DCM (5.00 mL), DIPEA (186 mg, 3.4 equivalents), HBTU (549 mg, 3.4 equivalents), and HOBT (192 mg, 3.4 equivalents) were added at 25°C and stirred for 4 hours. The crude product was triturated with ACN (40 mL) at 25°C for 1 hour. The mixture was filtered and the cake was concentrated under vacuum. Compound O-2 (700 mg, 0.32 mmol, 76.7% yield) was obtained as a white solid. 137 H 196 N50 13 PSSi2 + [M+H + Calculated mass value for ]: 2175.4363, measured value: 2175.4373.

[0422] General procedure for the preparation of compound O-3 [ka]

[0423] In a solution of compound O-2 (650 mg, 0.298 mmol, 1.00 equivalent) in DCM (6.0 mL), C 12 H 25 SH (180 mg, 3.0 equivalents) and DCA (339 mg, 10.0 equivalents) were added at 0-5°C. The mixture was stirred at 0-5°C for 2.5 hours, and NMI (293.5 mg, 12.00 equivalents) was added. The mixture was stirred at 0-5°C for 0.5 hours. The residue was diluted with NaHCO4 / H2O (50 mL) and extracted with DCM (2 x 30 mL). The crude product was triturated with ACN (40.0 mL) at 25°C for 30 minutes, filtered, and concentrated. Compound O-3 (500 mg, 89% yield) was obtained as a white solid. 116 H 178 N5O 11 Su2 + [M+H + Calculated mass value for ]: 1874.3, measured value: 1874.1.

[0424] General procedure for the preparation of compound O-5 [ka]

[0425] Compound O-3 (0.25 g, 133 umol, 1.00 equivalent), anhydrous DCM (4.0 ml), and ACN (4.00 mL) were concentrated twice under reduced pressure to remove water. To the solution of compound O-3 (0.25 g, 133 umol, 1.00 equivalent) in DCM (4.00 mL), 3A MS (200 mg) was added all at once under Ar at 25°C and stirred for 0.5 hours. Compound O-5 (816 mg, 0.4 mmol, 3.00 equivalent) and DCI (50 mg, 426 umol, 3.2 equivalents) were added to the above mixture. The mixture was stirred at 25°C for 1 hour. DDTT (60 mg, 292 umol, 2.20 equivalents) was added to the reaction mixture and the mixture was stirred at 25°C for 0.5 hours. The crude product was triturated with ACN (40 mL) at 25°C for 1 hour. The mixture was filtered, and the cake was concentrated under vacuum. Compound O-5 (0.25 g, 50% yield) was obtained as a white solid.

[0426] General procedure for preparing fragment O [ka]

[0427] A solution of compound O-5 (20 mg) saturated with NH3·H2O (2.00 mL) was stirred in a 4 mL sealed tube at 65°C for 20 hours. The reaction mixture was filtered without any purification, and the filtrate was subjected to LC-MS. Fragment O was confirmed by LC-MS. 74 H 91 N 12 O 31 P4S4 - [MH - HRMS calculated value for ]: 1895.3752, measured value: 1895.3716.

[0428] Example 21. Synthesis of monomer fragment P a. Synthesis scheme for monomer fragment P [ka]

[0429] b. Procedure for the synthesis of fragment P

[0430] General procedure for the preparation of compound P-5 [ka]

[0431] Compound P-4 (synthesis method described in European Journal of Organic Chemistry (2003), (12), 2327-2335; this document is incorporated herein by reference), 1.0 g, 2.47 mmol, 1.0 equivalent) and 5'-DMTr-MOE C-3'-OH (1.78 g, 2.47 mmol, 1.0 equivalent) were dissolved in DCM (20.0 mL). EDCl (947 mg, 2.0 equivalents) and DMAP (453 mg, 1.5 equivalents) were added under 25°C and stirred for 24 hours. The mixture was concentrated under vacuum, and the residue was purified by silica gel column chromatography (heptane / siRNA 5:1 to 3:1). Compound P-5 was obtained as a white solid (1.7 g, 1.48 mmol, 60% yield). 66 H 69 N30 11 Si + [M+H + Calculated mass value for ]: 1108.4. Measured value: 1108.4.

[0432] General procedure for preparing fragment P [ka]

[0433] To a solution of compound P-5 (0.5 g, 0.45 mmol, 1.00 equivalent) in THF (1.0 mL), solutions of imidazole (614.2 mg, 9.02 mmol, 20.0 equivalents) and pyridine / hydrofluoride (128.9 mg, 4.5 mmol, 70% purity, 10.0 equivalents) in THF (2.0 mL) were added. The mixture was stirred at 25°C for 20 hours (compound P-5 was converted to fragment P with >95% conversion).

[0434] c. Alternative synthesis scheme for monomer fragment P [ka]

[0435] d. Alternative procedure for the synthesis of fragment P

[0436] General procedure for the preparation of compound P-5' [ka]

[0437] To a solution of compound P-4' (1.0 g, 2.92 mmol, 1.0 equivalent) and 5'-DMTr-MOE C-3'-OH (2.10 g, 2.92 mmol, 1.0 equivalent) in DCM (10.0 mL), EDCl (2.80 g, 5.0 equivalents) and DMAP (0.71 g, 2.0 equivalents) were added at 25°C and stirred for 24 hours. The mixture was concentrated under vacuum, and the residue was purified by silica gel column chromatography (heptane / siRNA ratio 5:1 to 3:1). Compound P-5' was obtained as a white solid (1.97 g, 90% yield). Mass calculation value for C61H67N3011Si+[M+H+]: 1046.4, measured value: 1046.4.

[0438] General procedure for preparing fragment P [ka]

[0439] To a solution of compound P-5' (0.3 g, 0.29 mmol, 1.00 equivalent) in THF (1.0 mL), solutions of imidazole (390.2 mg, 5.73 mmol, 20.0 equivalents) and pyridine / hydrofluoride (81.9 mg, 2.9 mmol, 70% purity, 10.0 equivalents) in THF (2.0 mL) were added. The mixture was stirred at 25°C for 20 hours, and compound P-5' was converted to fragment P with approximately 60% conversion.

[0440] e. Alternative synthesis scheme for monomer fragment P [ka]

[0441] General procedure for the preparation of compound P-5* [ka]

[0442] To a solution of compound P-4* (2.12 g, 6.23 mmol, 1.5 equivalents) and 5'-DMTr-MOE C-3'-OH (3.0 g, 4.16 mmol, 1.0 equivalent) in DCM (20.0 mL), EDCl (1.59 g, 2.0 equivalents) and DMAP (1.02 g, 2.0 equivalents) were added at 25°C and the mixture was stirred for 24 hours. The mixture was concentrated under vacuum, and the residue was purified by silica gel column chromatography (heptane / siRNA ratio 5:1 to 3:1). Compound P-5* was obtained as a sticky oil (2.5 g, 93% yield). 61 H 65 N30 11 Si + [M+H + Calculated mass value for ]: 1046.4. Measured value: 1044.4.

[0443] General procedure for preparing fragment P [ka]

[0444] To a solution of compound P-5* (0.15 g, 0.145 mmol, 1.00 equivalent) in THF (1.0 mL), solutions of imidazole (195.1 mg, 2.87 mmol, 20.0 equivalents) and pyridine / hydrofluoride (41 mg, 1.45 mmol, 70% purity, 10.0 equivalents) in THF (2.0 mL) were added. The mixture was stirred at 50°C for 6 hours, and compound P-5* was converted to fragment P with approximately 90% conversion.

[0445] Example 22. Synthesis of oligonucleotide fragment B from M40 a. Synthesis scheme for oligonucleotide fragment B from M40

[0446] Fragment B was synthesized according to the synthesis scheme shown in Figure 11.

[0447] b. Procedure for the synthesis of oligonucleotide fragment B from reagent M40

[0448] The procedure for synthesizing oligonucleotide fragment B from reagent M40 was the same as that described for the synthesis of oligonucleotide fragment B from M19, except for the final step of selective deprotection of M40. The procedure for selective deprotection of M40 is described below.

[0449] Procedure for selective deprotection of M40 [ka]

[0450] Under a hydrogen atmosphere, a mixture of DMTrO-UTTC-OM40 (1.0 equivalent) and palladium carbon (10 wt%) in tetrahydrofuran and methanol (0.05 M, v / v, 3:1) was vigorously stirred at room temperature for 1 hour. The reaction mixture was filtered and concentrated to obtain the residue. This was precipitated in MTBE to obtain the desired product. Fragment B was obtained as a pale yellow solid in 70.5% yield and 85.2% purity.

[0451] Example 23. One-pot procedure for preparing a P=O bond One-pot procedure scheme for preparing aP=O bonds [ka]

[0452] Procedure for preparing a bP=O bond

[0453] Compound 1 (12 g, 10 mmol, 1.00 equivalent), MOE C amidite (11.16 g, 12 mmol, 1.20 equivalent), and 3 Å MS (12.0 g) in CH3CN / DCM (100 mL, v / v=1:3) were stirred at 20-30°C for 1 hour, and DCI (1.94 g, 15 mmol, 1.50 equivalent) was added. The reaction mixture was stirred at 20-30°C for 1.0 hour (HPLC showed reaction conversion >99.5%). H2O (40 mg, 2 mmol, 0.2 equivalent) was added, and the mixture was stirred at 25°C for 30 minutes. NMI (1.35 g, 15 mmol, 1.5 equivalents), BPO (3.89 g, 11 mmol, 1.1 equivalents), and iodine (278 mg, 1 mmol, 0.1 equivalents in 6 mL of DCM) were added to the reaction mixture at 0-5°C and stirred for 1 hour at 0-5°C. Piperazine (652 mg, 7 mmol, 0.7 equivalents) was added and the mixture was stirred at 0-5°C for 30 minutes. Dodecane-1-thiol (6.64 g, 3.0 equivalents) and 3 Å MS (10.0 g) were added and the mixture was stirred at 0-10°C for 60 minutes. TFA (13.7 g, 110 mmol, 11.00 equivalents) was added dropwise at 0-5°C and stirred at 10-20°C for 60 minutes. NMI (9.88 g, 110 mmol, 11.0 equivalents) was added at 0-5°C and stirred for 10 minutes at 0-5°C. The reaction mixture was filtered to remove 3 Å MS and added to 5% NaHCO3 solution (120 mL) with vigorous stirring. HCl (120 mL) and MTBE (120 mL) were added and stirred vigorously for 10 minutes. The organic layer was separated, washed with 5% NaHCO3 aqueous solution (120 mL), H2O (120 mL), and brine (120 mL), dried over MgSO4 (24 g), filtered, and concentrated under reduced pressure. The crude product was dissolved in EtOAC (36 mL) and slowly added to a mixture of heptane / TBME (216 L, 1:1, v / v). The precipitated product was filtered, washed with heptane / TBME (2 x 400 mL, 1:1, v / v), and dried under vacuum at 20-30°C for 16 hours to obtain compound 3 as a white solid (14.3 g, 80.1% yield). 82 H 97 N11O 24 P2SSi + [M+H] +HRMS calculated value for this: 1742.5751, measured value: 1742.5732.

[0454] c. Comparison between different oxidizing reagents

[0455] Several oxidation reagents, including iodine / pyridine, mCPBA, BPO, and tBuOOH, were tested in a one-pot procedure (coupling / oxidation / detritylation) for preparing P=O bonds. Figure 12 shows a scheme illustrating reaction product 3 and by-products 1 and 2. The performance of each oxidation reagent is summarized in Table 2 below. [Table 2]

[0456] The oxidizing reagents BPO and tBuOOH showed superior oxidative performance compared to iodine / pyridine and mCPBA in one-pot procedures for preparing P=O bonds in oligonucleotides. When BPO or tBuOOH were used in the one-pot procedure, both produced fewer byproducts than iodine / pyridine and mCPBA. In addition, the one-pot procedure was unsuccessful with iodine / pyridine because it required a further purification step to remove pyridine before the detritylation step.

[0457] Example 24. Synthesis of oligonucleotide I [ka]

[0458] Step 1: Synthesis of Compound I-3 [ka]

[0459] Compound I-2 (1 equivalent) was added to the first round-bottom flask (RBF) under Ar. This was dried three times by co-depositing with (DCM / ACN=3:1, 4v) at 25-30°C. Next, DCM / ACN=2:1 (6V) was added to the RBF, followed by the addition of 3A MS (5%) at 25-30°C for 1 hour. Next, compound I-1 (1.5 equivalents) was added to the second RBF under Ar and dried three times by co-depositing with (ACN 4v) at 25-30°C. DCM (2V) was added to the second RBF, and the resulting solution in the second RBF was added dropwise to the first RBF at 20-25°C, followed by the addition of DCI (2 equivalents). The resulting mixture was stirred at 25-30°C for 1 hour. A sample was taken for analysis. Next, DDTT (2 equivalents) was added to the reaction mixture. The mixture was stirred at 25-30°C for 0.5 hours. A sample was taken for analysis. Next, the mixture was filtered to remove 3 Å MS and washed with DCM (2V x 2). The resulting solution was slowly added to ACN (50V) at 20°C for 0.5 hours. The solid was recovered by filtration and washed with ACN (5V x 2) to obtain compound I-3 as a white solid.

[0460] Step 2: Detritylation of compound I-3 [ka]

[0461] Compound I-3 (1 equivalent) was added to RBF under Ar, followed by the addition of DCM (7V) under Ar at 0-5°C, and then 3A MS (5%) at 20-25°C for 1 hour. Next, C was added to the mixture. 12 H 25SH (2 equivalents) was added, followed by TCA (10 equivalents) being added dropwise at 0-5°C for 2 hours. A sample was taken for analysis. Py (12 equivalents) was added at 0-5°C. The mixture was filtered to remove 3 Å MS and washed with DCM (2V x 2). The pH was adjusted to 7-8 by adding NaHCO3 (4% wt, 10V). The mixture was then extracted with DCM (2V x 2). The organic layer was dried on anhydrous MgSO4, filtered, and washed with DCM (2V x 2). The filtrate was concentrated to approximately 5V and slowly added to ACN (50V) at 20°C for 0.5 hours. The solid was recovered by filtration and washed with ACN (5V x 2) to obtain compound I-4 as a white solid.

[0462] Step 3: Synthesis of Compound I-6 [ka]

[0463] Compound I-4 (1 equivalent) was added to the first RBF under Ar. It was dried three times by co-depositing with DCM / ACN (3:1, 4v) at 25-30°C. Next, DCM / ACN (2:1, 6v) was added to the RBF, followed by the addition of 3A MS (5%) at 25-30°C for 1 hour. Compound I-5 was added to the second RBF under Ar (1.5 equivalents). The mixture was dried three times by co-depositing with ACN (4v) at 25-30°C, followed by the addition of DCM (2v). The resulting solution in the second RBF was transferred to the first RBF in droplet form at 20-25°C, followed by the addition of DCI (2 equivalents). The resulting mixture was stirred at 25-30°C for 1 hour. A sample was taken for analysis. DDTT (2 equivalents) was added to the RBF. The resulting mixture was stirred at 25-30°C for 0.5 hours. A sample was taken for analysis. Next, the reaction mixture was filtered to remove 3 Å MS and washed with DCM (2V x 2). The resulting solution was slowly added to ACN (50V) at 20°C for 0.5 hours. The solid was recovered by filtration and washed with ACN (5V x 2) to obtain compound I-6 as a white solid.

[0464] Step 4: Detritylation of compound I-6 [ka]

[0465] Compound I-6 (1 equivalent) was added to a round-bottom flask (RBF) under Ar, followed by the addition of DCM (7V) under Ar at 0-5°C, and then 3A MS (5%) at 20-25°C for 1 hour. Next, C was added to the mixture. 12 H 25 SH (3 equivalents) was added, followed by TCA (12 equivalents) being added dropwise at 0-5°C for 2 hours. A sample was taken for analysis. Next, Py (15 equivalents) was added to RBF at 0-5°C. The mixture was filtered to remove 3 Å MS and washed with DCM (2V x 2). The pH was adjusted to 7-8 by adding NaHCO3 (4% wt, 10V). Next, it was extracted with DCM (2V x 2). The organic layer was dried on anhydrous MgSO4, filtered, and washed with DCM (2V x 2). The filtrate was concentrated to approximately 5V and slowly added to ACN (50V) at 20°C for 0.5 hours. The solid was recovered by filtration and washed with ACN (5V x 2) to obtain compound I-7 as a white solid.

[0466] Step 5: Synthesis of Oligonucleotide I [ka]

[0467] Compound I-7 (1 equivalent) was added to the first RBF under Ar. It was dried three times by co-depositing with DCM / ACN (3:1, 4v) at 25-30°C. Next, DCM / ACN (3:1, 10v) was added to the RBF, followed by the addition of 3A MS (5%) at 25-30°C for 1 hour. Compound I-8 was added to the second RBF under Ar (1.7 equivalents). The mixture was dried three times by co-depositing with ACN (4v) at 25-30°C, followed by the addition of DCM (2v). The resulting solution in the second RBF was transferred to the first RBF in droplet form at 20-25°C, and DCI (2.5 equivalents) was added. The resulting mixture was stirred at 25-30°C for 1 hour. A sample was taken for analysis. DDTT (2 equivalents) was added to the RBF. The resulting mixture was stirred at 25-30°C for 0.5 hours. Samples were taken for analysis. Next, the reaction mixture was filtered to remove 3 Å MS and washed with DCM (2V x 2). The resulting solution was slowly added to ACN (50V) at 20°C for 0.5 hours. The solid was recovered by filtration and washed with ACN (5V x 2) to obtain oligonucleotide I as a white solid in 3.1 g (76.4% yield, 69% UV purity).

[0468] Example 25. Expanding the synthesis of oligonucleotide I.

[0469] a. Synthesis scheme for oligonucleotide I

[0470] Oligonucleotide I was synthesized on a 50-gram scale according to the synthesis scheme shown in Figure 13.

[0471] b. Procedure for the synthesis of oligonucleotide I

[0472] General procedure for the preparation of compound I-2 [ka]

[0473] Under an N2 atmosphere, a mixture of fragment 1P (24.0 g, 14.1 mmol), fragment 5 (44.1 g, 17.2 mmol), 3 Å MS (5 g / 100 mL, 12 g), and DCM (240 mL) was stirred at 20-25°C for 1.0 hour. DCI (6.6 g, 56.4 mmol) was added, and the reaction mixture was stirred at 20-30°C for 1.0 hour. DDTT (7.2 g, 35.25 mmol) was added, and the reaction mixture was stirred at 20-25°C for 30 minutes. Dodecane-1-thiol (9.94 g, 49.3 mmol) was added, and the reaction mixture was stirred at 0±5°C for 10 minutes. TFA (14.4 g, 126.9 mmol) was slowly added, and the reaction mixture was stirred at 0±5°C for 1.0 hour. NMI (12.7 g, 155.1 mmol) was added over 10 minutes. The reaction mixture was filtered to remove 3 Å MS, concentrated to approximately 100 mL by rotovap, and added to CH3CN (2.6 L) over 30 minutes with vigorous stirring at 0 ± 5 °C. The precipitated product was filtered, washed with ACN (2 x 100 mL), and dried under vacuum at 20–30 °C for 16 hours to obtain compound I-2 as a white solid (46.7 g, 85.1% yield).

[0474] General procedure for the preparation of compound I-3 [ka]

[0475] Compound I-2 (44.0 g, 11.3 mmol), Compound I-1 (40.8 g, 15.9 mmol), 3 Å MS (44 g), and a mixture of ACN / DCM (440 mL, 1:3, v / v) were stirred for 1.0 hour at 20-25°C under an N2 atmosphere. DCI (6.66 g, 56.5 mmol) was added, and the reaction mixture was stirred for 1.0 hour. DDTT (5.1 g, 34.9 mmol) was added, and the reaction mixture was stirred for 30 minutes at 20-25°C. The reaction mixture was filtered, and the 3 Å MS filtration cake was washed with DCM (2 x 50 mL). The combined filtrate was concentrated to approximately 200 mL on a rotary evaporator and added to ACN (2 L) over 30 minutes with vigorous stirring at 0 ± 5°C. The precipitated product was filtered, washed with ACN (2 x 100 mL), and dried under vacuum at 20-30°C for 12 hours to obtain compound I-3 as a slightly yellow solid (71.4 g, 98.6% yield).

[0476] General procedure for the preparation of compound I-4 [ka]

[0477] A mixture of compound I-3 (69.0 g, 10.8 mmol), 3 Å MS (71.0 g), and DCM (480 mL) was stirred for 1.0 hour at 20-25°C under an N2 atmosphere and cooled to 0±5°C. Dodecane-1-thiol (5.6 g, 27 mmol) was added, and the reaction mixture was stirred at 0°C for 10 minutes. TFA (13.6 g, 118.8 mmol) was slowly added, and the reaction mixture was stirred for 1.5 minutes at 0±5°C. NMI (11.5 g, 140.4 mmol) was added over 10 minutes, the reaction mixture was filtered to remove the 3 Å MS, concentrated to approximately 200 mL on a rotary evaporator, and added to ACN (2.5 L) over 30 minutes with vigorous stirring at 0±5°C. The precipitated product was filtered, washed with ACN (2 x 172 mL), and dried under vacuum at 20-30°C for 14 hours to obtain compound I-4 as a white solid (55.86 g, 85.0% yield).

[0478] General procedure for the preparation of compound I-6 [ka]

[0479] The mixture of compound I-4 (39.9 g, 6.55 mmol), compound I-5 (18.8 g, 9.17 mmol), 3 Å MS (40 g), and ACN / DCM (400 mL, 1:3, v / v) was stirred for 1.0 hour at 20-25°C under an N2 atmosphere. DCI (3.87 g, 32.8 mmol) was added, and the reaction mixture was stirred for 1.0 hour. DDTT (2.96 g, 14.4 mmol) was added, and the reaction mixture was stirred for 30 minutes at 20-25°C. DCM (100 mL) was added, the reaction mixture was filtered, and the 3 Å MS filtration cake was washed with DCM (2 x 30 mL). The combined filtrate was concentrated to approximately 200 mL on a rotary evaporator and slowly added to ACN (1.36 L) over 30 minutes with vigorous stirring at 0 ± 5°C. The precipitated product was filtered, washed with ACN (3 x 100 mL), and dried under vacuum at 20-30°C for 12 hours to obtain compound I-6 as a pale yellow solid (49.8 g, 94.3% yield).

[0480] General procedure for the preparation of compound I-7 [ka]

[0481] A mixture of compound I-6 (50.0 g, 6.20 mmol), 3 Å MS (18.0 g), and DCM (350 mL) was stirred under an N2 atmosphere at 20-25°C for 1.0 hour and then cooled to 0 ± 5°C. Dodecane-1-thiol (3.77 g, 18.6 mmol) was added, and the reaction mixture was stirred at 0°C for 10 minutes. TFA (9.19 g, 80.6 mmol) was slowly added, and the reaction mixture was stirred at 0 ± 5°C for 1.5 hours. NMI (8.14 g, 99.2 mmol) was added over 10 minutes, the reaction mixture was filtered, and the 3 Å MS filtration cake was washed with DCM. The combined filtrate was concentrated to approximately 200 mL on a rotary evaporator and added to ACN (2.65 L) over 30 minutes with vigorous stirring at 0 ± 5°C. The precipitated product was filtered, washed with ACN (3 x 150 mL), and dried under vacuum at 20-30°C for 14 hours to obtain compound I-7 as a white solid (45.9 g, 95.3% yield).

[0482] General procedure for preparing oligonucleotide I [ka]

[0483] A mixture of compound I-7 (42.0 g, 5.41 mmol), compound I-8 (23.9 g, 9.74 mmol), 3 Å MS (42 g), and DCM / CH3CN (400 mL, 3:1, v / v) was stirred for 1.0 hour at 20-25°C under an N2 atmosphere. DCI (3.2 g, 27.05 mmol) was added, and the reaction mixture was stirred for 1.0 hour. DDTT (2.45 g, 11.9 mmol) was added, and the reaction mixture was stirred for 30 minutes at 20-25°C. DCM (200 mL) was added, the reaction mixture was filtered, and the 3 Å MS filtration cake was washed with DCM (2 x 50 mL). The combined reaction mixture was concentrated to approximately 300 mL on a rotary evaporator and slowly added to ACN (2.50 L) over 30 minutes with vigorous stirring at 0 ± 5°C. The precipitated product was filtered, washed with ACN (2 x 150 mL), and dried under vacuum at 20-30°C for 14 hours to obtain oligonucleotide I as a pale yellow solid (51.6 g, 94.1% yield).

[0484] Characterization of Oligonucleotide I: A mixture of oligonucleotide I (100.0 mg) and 30% NH4OH (2 mL) in a 4 mL pressure flask was stirred at 65°C for 4 hours. The resulting compound was checked by LC-MS. The structure of oligonucleotide I was confirmed by LC-MS. 231 H 318 N 53 O 118 P 17 S 15 / 4 - HRMS calculated value for [M] / 4: 1682.0, measured value: 1682.1.

[0485] The foregoing descriptions of specific embodiments fully illustrate the general nature of the invention, so that others can readily modify and / or adapt such specific embodiments to various uses without excessive experimentation and without departing from the general idea of ​​this disclosure, by applying knowledge within the art. Such adaptations and modifications are therefore intended to be within the meaning and scope of equivalents of the disclosed embodiments, based on the teachings and guidance presented herein. Naturally, the language or terminology used herein is for illustrative purposes only and not limiting, and should be interpreted by those skilled in the art in consideration of the teachings and guidance.

[0486] The scope and breadth of this disclosure should not be limited by any of the typical embodiments described above, but should be defined solely by the following claims and their equivalents.

Claims

1. Equation I or Ia, 【Chemistry 1】 A compound or salt thereof, Y is in equation A 【Chemistry 2】 It is represented as, Here, 【Transformation 3】 This represents the connection point to Y, W is represented by formulas A1, A2, A2-1, A2-2, A3, A3-1, or A3-2, 【Chemistry 4】 Here, 【Transformation 5】 This indicates the point where W and V are connected. Each Rw is independently an aliphatic hydrocarbon group having 10 or more carbon atoms. k is an integer from 1 to 5. V is a bond, oxygen, C 1-20 alkylene, C 1-6 alkynylene, -C(=O)-, ***-C(=O)-O-***, ***-O-C(=O)-***, 【Transformation 6】 or a 5-7 membered heteroaryl having 1-3 heteroatoms selected from oxygen, nitrogen, and sulfur, wherein the heteroaryl is optionally substituted with 1-3 R8s. 【Transformation 7】 The symbol represents the point where V and U are connected, and R8 is H or C1-30 alkyl. U is a 5-7 membered heterocyclyl having 1-3 heteroatoms selected from bond, oxygen, C1-20 alkylene, carbonyl, ***-O-C(=O)-**, oxygen, nitrogen, and sulfur, a 5-7 membered heteroaryl having 1-3 heteroatoms selected from oxygen, nitrogen, and sulfur and optionally substituted with 1-3 R8, or a group represented by formula A4, A5, or A6. 【Transformation 8】 U1 is a 5-7 member heterocyclyl having 1-3 heteroatoms selected from C1-6 alkylene, C1-6 alkylene oxy, oxygen, nitrogen, and sulfur, or a 5-7 member heteroaryl having 1-3 heteroatoms selected from oxygen, nitrogen, and sulfur. P １ NO ２ or a silylhydroxyl protecting group, R １ and R ２ H and C are independent of each other. １－６ Alkyl or phenyl, C １－６ Alkyl and phenyl compounds have 1 to 3 R ３ It can be freely replaced by, R ３ is C １－３０ alkoxy, The compound or a salt thereof.

2. P １ teeth, 【Chemistry 9】 A silylhydroxyl protecting group selected from, 【Chemistry 10】 P １ Represents the connection point to R ５ , R ６ , and R ７ H and C are independent of each other. １－３０ Alkyl, or C １－３０ It is an alkoxy. The compound or a salt thereof according to claim 1.

3. P １ -O-TBDMS, -O-TIPS, -O-TBDPS, -O-TBODPS, and -O-TBDAS: 【Chemistry 11】 Selected from the group consisting of, The compound or a salt thereof according to claim 1.

4. P １ This is selected from the group consisting of -O-TBDPS, -O-TBODPS, and -O-TBDAS. 【Chemistry 12】 R ５ , R ６ , and R ７ H and C are independent of each other. １－３０ Alkyl, or C １－３０ It is an alkoxy. A compound or salt thereof according to any one of claims 1 to 3.

5. TBDAS group 【Chemistry 13】 And s is an integer from 1 to 30. A compound or salt thereof according to any one of claims 1 to 4.

6. P １ The compound or salt thereof according to any one of claims 1 to 5, wherein is -O-TBDPS.

7. i) W is expressed by formula A1, 【Chemistry 14】 R ｗ C ｎ H ２ｎ＋１ And, n is an integer between 10 and 30; ii) R ｗ C １２ H ２５ , C １８ H ３７ , C ２０ H ４１ , C ２２ H ４５ , C ２４ H ４９ , C ２６ H ５３ , and C ２８ H ５７ Selected from the group consisting of; and / or iii) V is a bond, CH ２ ,CH ２ CH ２ , C(=O), ***-C(=O)-O-**, or 【Chemistry 15】 That is, The compound or salt thereof according to claim 5 or 6.

8. U is a bond, CH ２ ,CH ２ CH ２ Carbonyl, triazolylene, piperazinerylene, 【Chemistry 16】 That is, A compound or salt thereof according to any one of claims 5 to 7.

9. U-V is, 【Chemistry 17】 Selected from the group consisting of, R ８ is H or C １－６ It is alkyl. A compound or salt thereof according to any one of claims 5 to 8.

10. Y is, [Chemistry 18] Selected from the group consisting of, R ８ is H or C １－６ It is alkyl, The compound or salt thereof according to any one of claims 1 to 6, wherein m is an integer from 1 to 5.

11. i) R １ and R ２ These are independently H or CH ３ is; and / or ii) R ８ is H or C １－４ It is alkyl. A compound or salt thereof according to any one of claims 1 to 8.

12. A compound or salt thereof according to claim 1, represented by formula II or IIa, 【Chemistry 19】 Here, t is an integer between 10 and 30. 【Chemistry 20】 teeth, 【Chemistry 21】 Selected from the group consisting of R ８ is H or C １－６ It is alkyl; optionally, the compound is 【Chemistry 22】 Selected from the group consisting of or a salt thereof, The compound or a salt thereof.

13. i) The compound is 【Chemistry 23】 or its salt; ii) The compound is given by the following formula: 【Chemistry 24】 It is selected from one of the following, or a salt thereof; or ii) The compound is shown in Table 1 Table 1 A compound or salt thereof The compound according to claim 1.

14. Formulas III, IIIP, III', or IIIP' 【Chemistry 25-1】 【Chemistry 25-2】 A nucleotide or oligonucleotide represented by or a salt thereof, Q is a hydroxyl protecting group, 【Chemistry 26】 NH modified by Z ２ It is a nucleic acid base that contains a group, R ３１ Each instance is independently a nucleic acid base, and the NH of the nucleic acid base ２ If present, it can be optionally protected by an amine protecting group. R ３２ Each time it appears, C １－６ H, halo, OH, and C are optionally substituted by alkoxys. １－６ Independently selected from the group consisting of alkoxys, the OH group is optionally protected by a hydroxyl protecting group. R ３４ Each instance is independently either H or R ３２ The alkoxy group of the above forms a ring, R ３５ It is a hydroxyl protecting group, R ３６ Each instance, H and C appear independently. １－６ alkyl group, C ２－６ These are alkenyl, phenyl, or benzyl groups, respectively, -CN and -NO. ２ , or optionally substituted with halogen, R ３６ teeth, 【Chemistry 27】 And, q is an integer between 1 and 20. Each instance of X is independently either O or S. Z is a base represented by formula I** or Ia**, 【Chemistry 28】 Here, 【Chemistry 29】 represents the connection point to Z, Y is in equation A 【Transformation 30】 It is represented as, Here, 【Chemistry 31】 This represents the connection point to Y, W is represented by formulas A1, A2, A2-1, A2-2, A3, A3-1, or A3-2, 【Chemistry 32】 Here, 【Transformation 33】 This indicates the point where W and V are connected. Each Rw is independently an aliphatic hydrocarbon group having 10 or more carbon atoms. k is an integer from 1 to 5. V is a bond, oxygen, C 1-20 alkylene, C 1-6 alkynylene, -C(=O)-, ***-C(=O)-O-***, ***-O-C(=O)-***, 【Transformation 34】 or a 5-7 membered heteroaryl having 1-3 heteroatoms selected from oxygen, nitrogen, and sulfur, wherein the heteroaryl is optionally substituted with 1-3 R8s. 【Chemistry 35】 The symbol represents the point where V and U are connected, and R8 is H or C1-30 alkyl. U is a 5-7 membered heterocyclyl having 1-3 heteroatoms selected from bond, oxygen, C1-20 alkylene, carbonyl, ***-O-C(=O)-**, oxygen, nitrogen, and sulfur, a 5-7 membered heteroaryl having 1-3 heteroatoms selected from oxygen, nitrogen, and sulfur and optionally substituted with 1-3 R8, or a group represented by formula A4, A5, or A6. 【Transformation 36】 U1 is a 5-7 member heterocyclyl having 1-3 heteroatoms selected from C1-6 alkylene, C1-6 alkylene oxy, oxygen, nitrogen, and sulfur, or a 5-7 member heteroaryl having 1-3 heteroatoms selected from oxygen, nitrogen, and sulfur. P １ NO ２ or a silylhydroxyl protecting group, R １ and R ２ H and C are independent of each other. １－６ Alkyl or phenyl, C １－６ Alkyl and phenyl compounds have 1 to 3 R ３ It can be freely replaced by, R ３ C １－３０ It is an alkoxy. The nucleotide or oligonucleotide or a salt thereof.

15. oligonucleotide fragment of formula (V) or (V') 【Chemistry 37】 or a process for preparing the salt thereof, 1) Compound of formula (VA) 【Transformation 38】 Alternatively, the salt can be deprotected to obtain the compound of formula (VB). 【Chemistry 39】 or the step of forming a salt thereof, 2) The compound of formula (VB) or a salt thereof is used in the compound of formula (VC) or (VC'). 【Chemistry 40】 Alternatively, react with its salt to form compounds of formula (VD) or (VD'). 【Chemistry 41】 or the step of forming a salt thereof, 3) The compound of formula (VD) or (VD') or a salt thereof is sulfurized or oxidized with a sulfurizing or oxidizing agent to obtain the compound of formula (VE) or (VE'). 【Chemistry 42】 or the step of forming a salt thereof, 4) Deprotect the compound of formula (VE) or (VE') or a salt thereof to obtain the compound of formula (VF) or (VF'). 【Chemistry 43】 or the step of forming a salt thereof, 5) When q is 2 or greater, the step of starting with a compound of formula (VF) or (VF') and repeating steps 2), 3), and 4) q-2 times, followed by performing steps 2) and 3) to obtain a fragment or salt of formula (V) or (V'), R ３１ Each instance is independently a nucleic acid base, and the NH of the nucleic acid base ２ If present, it is protected by an amine protecting group. R ３２ Each time it appears, C １－６ H, halo, OH, and C are optionally substituted by alkoxys. １－６ Independently selected from the group consisting of alkoxys, the OH group is optionally protected by a hydroxyl protecting group. R ３４ Each instance is independently either H or R ３２ The alkoxy group of the above forms a ring, R ３５ is a hydroxyl protecting group, and R ３６ Each instance, independently, C １－６ alkyl group, C ２－６ These are alkenyl, phenyl, or benzyl groups, respectively, -CN and -NO. ２ , or optionally substituted with halogen, R ３６ teeth, 【Chemistry 44】 And, R ３７ａ and R ３７ｂ are independently C １－６ alkyl, q is an integer between 1 and 20. Each instance of X is independently either O or S. Z is a base represented by formula I** or Ia**, 【Chemistry 45】 Here, 【Chemistry 46】 This represents a connection point to Z, Y is in equation A 【Chemistry 47】 It is represented as, Here, 【Chemistry 48】 This represents the connection point to Y, W is represented by formulas A1, A2, A2-1, A2-2, A3, A3-1, or A3-2, 【Chemistry 49】 Here, [Transformation 50] This indicates the point where W and V are connected. Each Rw is independently an aliphatic hydrocarbon group having 10 or more carbon atoms. k is an integer from 1 to 5. V is a bond, oxygen, C 1-20 alkylene, C 1-6 alkynylene, -C(=O)-, ***-C(=O)-O-***, ***-O-C(=O)-***, 【Chemistry 51】 or a 5-7 membered heteroaryl having 1-3 heteroatoms selected from oxygen, nitrogen, and sulfur, wherein the heteroaryl is optionally substituted with 1-3 R8s. 【Chemistry 52】 The symbol represents the point where V and U are connected, and R8 is H or C1-30 alkyl. U is a 5-7 membered heterocyclyl having 1-3 heteroatoms selected from bond, oxygen, C1-20 alkylene, carbonyl, ***-O-C(=O)-**, oxygen, nitrogen, and sulfur, a 5-7 membered heteroaryl having 1-3 heteroatoms selected from oxygen, nitrogen, and sulfur and optionally substituted with 1-3 R8, or a group represented by formula A4, A5, or A6. 【Chemistry 53】 U1 is a 5-7 member heterocyclyl having 1-3 heteroatoms selected from C1-6 alkylene, C1-6 alkylene oxy, oxygen, nitrogen, and sulfur, or a 5-7 member heteroaryl having 1-3 heteroatoms selected from oxygen, nitrogen, and sulfur. P １ NO ２ or a silylhydroxyl protecting group, R １ and R ２ H and C are independent of each other. １－６ Alkyl or phenyl, C １－６ Alkyl and phenyl compounds have 1 to 3 R ３ It can be freely replaced by, R ３ C １－３０ It is an alkoxy. The aforementioned process.

16. Oligonucleotide fragments of formula (V-C1) or (V-C2) 【Chemistry 54】 or a process for preparing the salt thereof, 1) Compound of the above formula (VB) 【Transformation 55】 or a salt thereof, of the compound of formula (V-CR1) or (V-CR2) 【Transformation 56】 The step of reacting the salt or a base with the salt or a base to form a compound of formula (V-C1) or (V-C2), R ３１ Each instance is independently a nucleic acid base, and the NH of the nucleic acid base ２ If present, it is protected by an amine protecting group. R ３２ Each time it appears, C １－６ H, halo, OH, and C are optionally substituted by alkoxys. １－６ Independently selected from the group consisting of alkoxys, the OH group is optionally protected by a hydroxyl protecting group. R ３４ Each instance is independently either H or R ３２ The alkoxy group of the above forms a ring, R ３５ It is a hydroxyl protecting group, R ３６ Each instance, independently, C １－６ alkyl group, C ２－６ These are alkenyl, phenyl, or benzyl groups, respectively, -CN and -NO. ２ , or optionally substituted with halogen, R ３６ teeth, 【Chemistry 57】 And, q is an integer between 1 and 20. X is independently O or S in each instance, provided that when X is S, the phosphorothiolate group has an S-configuration, an R-configuration, or a mixture thereof (e.g., a racemic mixture). Z is a base represented by formula I** or Ia**, [Chem. 58] Here, 【Chemistry 59】 This represents a connection point to Z, Y is in equation A 【Transformation 60】 It is represented as, Here, 【Chemistry 61】 This represents the connection point to Y, W is represented by formulas A1, A2, A2-1, A2-2, A3, A3-1, or A3-2, 【Transformation 62】 Here, 【Transformation 63】 This indicates the point where W and V are connected. Each Rw is independently an aliphatic hydrocarbon group having 10 or more carbon atoms. k is an integer from 1 to 5. V is a bond, oxygen, C 1-20 alkylene, C 1-6 alkynylene, -C(=O)-, ***-C(=O)-O-***, ***-O-C(=O)-***, 【Chemistry 64】 or a 5-7 membered heteroaryl having 1-3 heteroatoms selected from oxygen, nitrogen, and sulfur, wherein the heteroaryl is optionally substituted with 1-3 R8s. 【Transformation 65】 The symbol represents the point where V and U are connected, and R8 is H or C1-30 alkyl. U is a 5-7 membered heterocyclyl having 1-3 heteroatoms selected from bond, oxygen, C1-20 alkylene, carbonyl, ***-O-C(=O)-**, oxygen, nitrogen, and sulfur, a 5-7 membered heteroaryl having 1-3 heteroatoms selected from oxygen, nitrogen, and sulfur and optionally substituted with 1-3 R8, or a group represented by formula A4, A5, or A6. [Chem. 66] U1 is a 5-7 member heterocyclyl having 1-3 heteroatoms selected from C1-6 alkylene, C1-6 alkylene oxy, oxygen, nitrogen, and sulfur, or a 5-7 member heteroaryl having 1-3 heteroatoms selected from oxygen, nitrogen, and sulfur. P １ NO ２ or a silylhydroxyl protecting group, R １ and R ２ H and C are independent of each other. １－６ Alkyl or phenyl, C １－６ Alkyl and phenyl compounds have 1 to 3 R ３ It can be freely replaced by, R ３ C １－３０ It is an alkoxy. The aforementioned process.

17. Oligonucleotide fragments of formula (V-C1) or (V-C2) 【Transformation 67】 or a process for preparing the salt thereof, 1) Compound of formula (VB) 【Transformation 68】 Alternatively, a salt thereof can be used as a reagent of formula (VR1) or (VR2). 【Transformation 69】 Reacting with it yields a compound of formula (V-CR3) or (V-CR4). 【Transformation 70】 or the step of forming a salt thereof, 2) A compound of formula (V-CR3) or (V-CR4) or a salt thereof, and a compound of formula (VG) 【Chemistry 71】 The step of reacting the salt or a base with the salt or a base to form a compound of formula (V-C1) or (V-C2), R ３１ Each instance is independently a nucleic acid base, and the NH of the nucleic acid base ２ If present, it is protected by an amine protecting group. R ３２ Each time it appears, C １－６ H, halo, OH, and C are optionally substituted by alkoxys. １－６ Independently selected from the group consisting of alkoxys, the OH group is optionally protected by a hydroxyl protecting group. R ３４ Each instance is independently either H or R ３２ The alkoxy group of the above forms a ring, R ３５ It is a hydroxyl protecting group, R ３６ Each instance, independently, C １－６ alkyl group, C ２－６ These are alkenyl, phenyl, or benzyl groups, respectively, -CN and -NO. ２ , or optionally substituted with halogen, R ３６ teeth, 【Chemistry 72】 And, q is an integer between 1 and 20. X is independently O or S in each instance, provided that when X is S, the phosphorothiolate group has an S-configuration, an R-configuration, or a mixture thereof (e.g., a racemic mixture). Z is a base represented by formula I** or Ia**, 【Transformation 73】 Here, 【Chemistry 74】 This represents a connection point to Z, Y is in equation A 【Chemistry 75】 It is represented as, Here, 【Transformation 76】 This represents the connection point to Y, W is represented by formulas A1, A2, A2-1, A2-2, A3, A3-1, or A3-2, [Chem 77] Here, 【Transformation 78】 This indicates the point where W and V are connected. Each Rw is independently an aliphatic hydrocarbon group having 10 or more carbon atoms. k is an integer from 1 to 5. V is a bond, oxygen, C 1-20 alkylene, C 1-6 alkynylene, -C(=O)-, ***-C(=O)-O-***, ***-O-C(=O)-***, 【Chemistry 79】 or a 5-7 membered heteroaryl having 1-3 heteroatoms selected from oxygen, nitrogen, and sulfur, wherein the heteroaryl is optionally substituted with 1-3 R8s. 【Chemistry 80】 The symbol represents the point where V and U are connected, and R8 is H or C1-30 alkyl. U is a 5-7 membered heterocyclyl having 1-3 heteroatoms selected from bond, oxygen, C1-20 alkylene, carbonyl, ***-O-C(=O)-**, oxygen, nitrogen, and sulfur, a 5-7 membered heteroaryl having 1-3 heteroatoms selected from oxygen, nitrogen, and sulfur and optionally substituted with 1-3 R8, or a group represented by formula A4, A5, or A6. 【Chemistry 81】 U1 is a 5-7 member heterocyclyl having 1-3 heteroatoms selected from C1-6 alkylene, C1-6 alkylene oxy, oxygen, nitrogen, and sulfur, or a 5-7 member heteroaryl having 1-3 heteroatoms selected from oxygen, nitrogen, and sulfur. P １ NO ２ or a silylhydroxyl protecting group, R １ and R ２ H and C are independent of each other. １－６ Alkyl or phenyl, C １－６ Alkyl and phenyl compounds have 1 to 3 R ３ It can be freely replaced by, R ３ C １－３０ It is an alkoxy. The aforementioned process.

18. Oligonucleotide fragment of formula (VBZ) 【Chemistry 82】 or a process for preparing the salt thereof, 1) Compound of formula (VBZ-1) 【Chemistry 83】 or a salt thereof, compound of formula (VBZ-2) 【Chemical 84】 Alternatively, by reacting it with a salt thereof, a compound of formula (VBZ-3) can be obtained. 【Chemical 85】 or the step of forming a salt thereof, 2) The step of sulfiding or oxidizing the compound of formula (VBZ-3) or a salt thereof with a sulfidating or oxidizing agent to form a compound of formula (VBZ) or a salt thereof, Here, Q is a hydroxyl protecting group, [Chem. 86] NH modified by Z ２ It is a nucleic acid base that contains a group, R ３１ Each instance is independently a nucleic acid base, and the NH of the nucleic acid base ２ If present, it is protected by an amine protecting group. R ３２ Each time it appears, C １－６ H, halo, OH, and C are optionally substituted by alkoxys. １－６ Independently selected from the group consisting of alkoxys, the OH group is optionally protected by a hydroxyl protecting group. R ３４ Each instance is independently either H or R ３２ The alkoxy group of the above forms a ring, R ３５ It is a hydroxyl protecting group, R ３６ Each instance, independently, C １－６ alkyl group, C ２－６ These are alkenyl, phenyl, or benzyl groups, respectively, -CN and -NO. ２ , or optionally substituted with halogen, R ３６ teeth, 【Chemistry 87】 And, R ３７ａ and R ３７ｂ Independently, C １－６ It is alkyl, q is an integer between 1 and 20. Each instance of X is independently either O or S. Z is a base represented by formula I** or Ia**, [Chem. 88] Here, [Chem. 89] This represents a connection point to Z, Y is in equation A 【Chemistry 90】 It is represented as, Here, 【Chemistry 91】 This represents the connection point to Y, W is represented by formulas A1, A2, A2-1, A2-2, A3, A3-1, or A3-2, 【Chemistry 92】 Here, 【Chemistry 93】 This indicates the point where W and V are connected. Each Rw is independently an aliphatic hydrocarbon group having 10 or more carbon atoms. k is an integer from 1 to 5. V is a bond, oxygen, C 1-20 alkylene, C 1-6 alkynylene, -C(=O)-, ***-C(=O)-O-***, ***-O-C(=O)-***, 【Chemical 94】 or a 5-7 membered heteroaryl having 1-3 heteroatoms selected from oxygen, nitrogen, and sulfur, wherein the heteroaryl is optionally substituted with 1-3 R8s. 【Chemical 95】 The symbol represents the point where V and U are connected, and R8 is H or C1-30 alkyl. U is a 5-7 membered heterocyclyl having 1-3 heteroatoms selected from bond, oxygen, C1-20 alkylene, carbonyl, ***-O-C(=O)-**, oxygen, nitrogen, and sulfur, a 5-7 membered heteroaryl having 1-3 heteroatoms selected from oxygen, nitrogen, and sulfur and optionally substituted with 1-3 R8, or a group represented by formula A4, A5, or A6. 【Chemistry 96】 U1 is a 5-7 member heterocyclyl having 1-3 heteroatoms selected from C1-6 alkylene, C1-6 alkylene oxy, oxygen, nitrogen, and sulfur, or a 5-7 member heteroaryl having 1-3 heteroatoms selected from oxygen, nitrogen, and sulfur. P １ NO ２ or a silylhydroxyl protecting group, R １ and R ２ H and C are independent of each other. １－６ Alkyl or phenyl, C １－６ Alkyl and phenyl compounds have 1 to 3 R ３ It can be freely replaced by, R ３ C １－３０ It is an alkoxy. The aforementioned process.

19. Oligonucleotide fragment of formula (V) or (V*) 【Chemistry 97】 or a process for preparing the salt thereof, a) Nucleotides of formula (V-1) 【Chem.98】 or its salt, Oligonucleotide fragment of formula (V-2) or (V-2') 【Chem.99】 or its salt, Coupling in solution to oligonucleotide fragments of formula (V-3) or (V-3') 【Chemistry 100】 or the step of forming a salt thereof, b) Sulfurizing or oxidizing the oligonucleotide of formula (V-3) or (V-3') or a salt thereof to obtain the oligonucleotide of formula (V) or (V*). 【Chemistry 101】 or a step of forming a salt thereof, Here, R ３１ Each instance is independently a nucleic acid base, and the NH of the nucleic acid base ２ If present, it is protected by an amine protecting group. R ３２ Each time it appears, C １－６ H, halo, OH, and C are optionally substituted by alkoxys. １－６ Independently selected from the group consisting of alkoxys, the OH group is optionally protected by a hydroxyl protecting group. R ３４ Each instance is independently either H or R ３２ The alkoxy group of the above forms a ring, R ３５ It is a hydroxyl protecting group, R ３６ Each instance, independently, C １－６ alkyl group, C ２－６ These are alkenyl, phenyl, or benzyl groups, respectively, -CN and -NO. ２ , or optionally substituted with halogen, R ３６ teeth, 【Chemical Engineering 102】 And, R ３７ａ and R ３７ｂ Independently, C １－６ It is alkyl, q is an integer between 1 and 20. Each instance of X is independently either O or S. Z is a base represented by formula I** or Ia**, 【Chemistry 103】 Here, 【Chemical 104】 This represents a connection point to Z, Y is, Formula A 【Chemistry 105】 It is represented as, Here, 【Chemistry 106】 This represents the connection point to Y, W is represented by formulas A1, A2, A2-1, A2-2, A3, A3-1, or A3-2, 【Chemistry 107】 Here, 【Chemistry 108】 This indicates the point where W and V are connected. Each Rw is independently an aliphatic hydrocarbon group having 10 or more carbon atoms. k is an integer from 1 to 5. V is a bond, oxygen, C 1-20 alkylene, C 1-6 alkynylene, -C(=O)-, ***-C(=O)-O-***, ***-O-C(=O)-***, 【Chemistry 109】 or a 5-7 membered heteroaryl having 1-3 heteroatoms selected from oxygen, nitrogen, and sulfur, wherein the heteroaryl is optionally substituted with 1-3 R8s. 【Chemical 110】 The symbol represents the point where V and U are connected, and R8 is H or C1-30 alkyl. U is a 5-7 membered heterocyclyl having 1-3 heteroatoms selected from bond, oxygen, C1-20 alkylene, carbonyl, ***-O-C(=O)-**, oxygen, nitrogen, and sulfur, a 5-7 membered heteroaryl having 1-3 heteroatoms selected from oxygen, nitrogen, and sulfur and optionally substituted with 1-3 R8, or a group represented by formula A4, A5, or A6. 【Chemistry 111】 U1 is a 5-7 member heterocyclyl having 1-3 heteroatoms selected from C1-6 alkylene, C1-6 alkylene oxy, oxygen, nitrogen, and sulfur, or a 5-7 member heteroaryl having 1-3 heteroatoms selected from oxygen, nitrogen, and sulfur. P １ NO ２ or a silylhydroxyl protecting group, R １ and R ２ H and C are independent of each other. １－６ Alkyl or phenyl, C １－６ Alkyl and phenyl compounds have 1 to 3 R ３ It can be freely replaced by, R ３ C １－３０ It is an alkoxy. The aforementioned process.

20. oligonucleotides of formula (VI), (VI-1), (VI'), or (VI'-I) 【Chemistry 112-1】 【Chemistry 112-2】 or a process for preparing the salt thereof, a) Oligonucleotide fragment of formula (F1) or (F1-1) 【Chemistry 113】 Alternatively, a salt thereof may be used as an oligonucleotide fragment of formula (F2) or (F2'). 【Chemistry 114】 or its salt, In solution, the oligonucleotide fragments of formula (F3), (F3-1), (F3'), or (F3'-1) are coupled. 【Chemical Engineering 115-1】 【Chemistry 115-2】 or to form a salt thereof, b) Sulfurizing or oxidizing an oligonucleotide fragment or salt of formula (F3), (F3-1), (F3'), or (F3'-1) to form an oligonucleotide or salt of formula (VI), (VI-1), (VI'), or (VI'-1), Here, Q is a hydroxyl protecting group, 【Chemistry 116】 NH modified by Z ２ It is a nucleic acid base that contains a group, R ３１ Each instance is independently a nucleic acid base, and the NH of the nucleic acid base ２ If present, it is protected by an amine protecting group. R ３２ Each time it appears, C １－６ H, halo, OH, and C are optionally substituted by alkoxys. １－６ Independently selected from the group consisting of alkoxys, the OH group is optionally protected by a hydroxyl protecting group. R ３４ Each instance is independently either H or R ３２ The alkoxy group of the above forms a ring, R ３５ It is a hydroxyl protecting group, R ３６ Each instance, independently, C １－６ alkyl group, C ２－６ These are alkenyl, phenyl, or benzyl groups, respectively, -CN and -NO. ２ , or optionally substituted with halogen, R ３６ teeth, 【Chemistry 117】 And, R ３７ａ and R ３７ｂ Independently, C １－６ It is alkyl, p is an integer between 2 and 20. o is an integer between 1 and 200. Each instance of X is independently either O or S. Z is a base represented by formula I** or Ia**, 【Chemistry 118】 Here, 【Chemical 119】 This represents a connection point to Z, Y is in equation A 【Chemical 120】 It is represented as, Here, 【Chemistry 121】 This represents the connection point to Y, W is represented by formulas A1, A2, A2-1, A2-2, A3, A3-1, or A3-2, 【Chemistry 122】 Here, [Formula 123] This indicates the point where W and V are connected. Each Rw is independently an aliphatic hydrocarbon group having 10 or more carbon atoms. k is an integer from 1 to 5. V is a bond, oxygen, C 1-20 alkylene, C 1-6 alkynylene, -C(=O)-, ***-C(=O)-O-***, ***-O-C(=O)-***, 【Chemistry 124】 or a 5-7 membered heteroaryl having 1-3 heteroatoms selected from oxygen, nitrogen, and sulfur, wherein the heteroaryl is optionally substituted with 1-3 R8s. 【Chemistry 125】 The symbol represents the point where V and U are connected, and R8 is H or C1-30 alkyl. U is a 5-7 membered heterocyclyl having 1-3 heteroatoms selected from bond, oxygen, C1-20 alkylene, carbonyl, ***-O-C(=O)-**, oxygen, nitrogen, and sulfur, a 5-7 membered heteroaryl having 1-3 heteroatoms selected from oxygen, nitrogen, and sulfur and optionally substituted with 1-3 R8, or a group represented by formula A4, A5, or A6. 【Chemistry 126】 U1 is a 5-7 member heterocyclyl having 1-3 heteroatoms selected from C1-6 alkylene, C1-6 alkylene oxy, oxygen, nitrogen, and sulfur, or a 5-7 member heteroaryl having 1-3 heteroatoms selected from oxygen, nitrogen, and sulfur. P １ NO ２ or a silylhydroxyl protecting group, R １ and R ２ H and C are independent of each other. １－６ Alkyl or phenyl, C １－６ Alkyl and phenyl compounds have 1 to 3 R ３ It can be freely replaced by, R ３ C １－３０ It is an alkoxy. The aforementioned process.

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