Processes for preparing oligonucleotides

Abstract

To provide a method for preparing oligonucleotides that can improve yields and purities of target phosphorodiamidate morpholino oligomers and reduce 4-nitrostyrene adduct impurities.SOLUTION: The process comprises: (a) converting a compound of Formula X-1 into a compound of Formula X-2: where R10 is a residue of an oligonucleotide (e.g., a phosphorodiamidate morpholino oligomer); R11 is an amine protecting group; wherein the compound of Formula X-1 is not bound to a solid support; and (b) optionally removing protecting groups in the compound of Formula X-2 to obtain the olignucleotide.SELECTED DRAWING: None

Description

Detailed Description of the Invention

[0001] [Technical field] The present disclosure relates generally to methods for preparing oligonucleotides, such as phosphorodiamidate morpholino oligomers (PMOs).

[0002] [background] Phosphorodiamidate morpholino oligomers (PMOs) are nucleic acid analogs that bind tightly to complementary RNA in a sequence-specific manner, making them useful for regulating protein synthesis and, therefore, gene expression. These oligomers consist of base-pair recognition moieties (heterocyclic bases) supported by a morpholino backbone system. The morpholino subunits used in the synthesis of these oligomers can be readily prepared from the corresponding ribonucleosides, which are readily available and inexpensive precursors.

[0003] During such synthesis, as in conventional oligonucleotide synthesis, functional groups on the heterocyclic bases are usually masked so as not to interfere with synthetic transformations.

[0004] O6-unprotected guanine subunits are known to undergo side reactions during the oligomerization stage. For example, the O6 oxygen can react with activated subunits during the coupling step to form O6-phosphorylated or derivative species. During the final cleavage of the base protecting group with ammonia, ammonia reacts at C6 to displace these species and give diaminopurine derivatives. Such impurities are difficult to remove by chromatography, resulting in significant yield reductions.

[0005] In conventional oligonucleotide synthesis, various protection schemes have been proposed in the art to reduce side reactions at the unprotected O6 position of guanine. However, when these protocols are applied to phosphorodiamidate morpholino oligomer synthesis, they are largely unsuccessful. Therefore, improved methods are needed to increase yield and purity in phosphorodiamidate morpholino oligomer synthesis, especially when using G morpholino subunits.

[0006] [overview] A unique challenge of morpholino chemistry is that the base protecting group must satisfy several requirements. The protecting group must be easily installed on the heterocyclic moiety and, once installed, be stable to subunit activation, purification conditions, and solid-phase synthesis. The protecting group must not be reactive with the morpholino amine moiety of the growing chain, allowing clean coupling of the activated morpholino subunit to the growing oligomer chain. The protecting group must be cleaved, preferably with ammonia, without introducing new impurities. Finally, it must lead to crystalline subunit derivatives, eliminating the need for chromatographic purification prior to activation.

[0007] As described in U.S. Patent Application Publication No. 2009 / 0131624, the 4-nitrophenethyl (NPE) group at the O6-position does not fully meet these criteria. The NPE group is cleaved by alkaline reagents via a β-elimination mechanism. These conditions tend to generate the reactive by-product 4-nitrostyrene, which can react with the reactive sites on the oligomer. Various scavengers (e.g., thiols and 1,3-dicarbonyl compounds) were introduced into the deprotection mixture to prevent the by-product from being trapped by the oligomer, but none completely solved the internal recycling problem. Even after purification, oligomers prepared using this subunit retained a yellowish color. In various embodiments, the present disclosure provides methods and processes useful for solving one or more of these problems.

[0008] In one embodiment, the present disclosure provides methods for preparing oligonucleotides, such as phosphorodiamidate morpholino oligomers ("PMOs").

[0009] In a further aspect, the present disclosure provides methods and compositions useful for preparing solid-phase supported phosphorodiamidate morpholino oligonucleotides.

[0010] The methods for synthesizing PMOs described herein are advantageous in many aspects, including, but not limited to, improved yield and purity of the desired phosphorodiamidate morpholino oligomers and reduced 4-nitrostyrene adduct impurities.

[0011] These and other objects and features of the present disclosure will become more fully apparent from the following detailed description of the disclosure.

[0012] [Detailed disclosure description] Exemplary Methods of Oligonucleotide Synthesis The present disclosure provides: (a) converting a compound of formula X-1 into a compound of formula X-2: [ka] (In the formula, R 10 is a residue of the starting oligonucleotide (e.g., a phosphorodiamidate morpholino oligomer); R 11 is an amine protecting group; Preferably, the compound of formula X-1 is not bound to a solid support. (b) optionally removing the protecting group in the compound of formula X-2 to obtain an oligonucleotide; The present invention provides a method for preparing an oligonucleotide, comprising:

[0013] In formulas X-1 and X-2, R 10 It will be apparent to one skilled in the art that R may be the same or different, depending on whether the residues of the starting oligonucleotide (e.g., phosphorodiamidate morpholino oligomer) in formula X-1 are altered under the conditions under which the NPE group is removed. For example, in some embodiments, if no other protecting groups are deprotected other than the NPE group shown in formula X-1, then R 10 may be the same in formulas X-1 and X-2. In some embodiments, R in formula X-2 10 is R in formula X-1 10It may represent the deprotected version of

[0014] The oligonucleotide (and starting oligonucleotide) is not particularly limited. In some embodiments, the oligonucleotide is a phosphorodiamidate morpholino oligomer. Typically, the oligonucleotide comprises a targeting base sequence for sequence-specific binding to a target nucleic acid. A target sequence and a targeting sequence are described as "complementary" to each other when hybridization occurs in an antiparallel configuration. A targeting sequence may have "near" or "substantial" complementarity to a target sequence and still function, i.e., remain "complementary," for the purposes of the methods described herein. Preferably, the oligonucleotide analogs employed in the methods described herein have a maximum of one mismatch with the target sequence every 10 nucleotides, preferably a maximum of one mismatch every 20 nucleotides. Alternatively, the antisense oligomers employed have at least 80% sequence identity, at least 90% sequence identity, or at least 95% sequence identity with the exemplary targeting sequences specified herein. For complementary binding to an RNA target, and as discussed below, guanine bases may be complementary to either cytosine or uracil RNA bases.

[0015] R 11 A variety of amine protecting groups can be used as R 11 is an amine protecting group that can be removed by treatment with NH. For example, in some embodiments, R 11 is an acyl group, i.e., R B -C(=O)-, where R B may be, for example, hydrogen, alkyl, aryl, cycloalkyl, heteroaryl, each of which may be optionally substituted. In some embodiments, R 11 is -C(=O)-R B where R B may be substituted C 1~6 Alkyl, e.g., C 1~6alkyl (e.g., isopropyl), aryl-substituted C 1~6 Alkyl (e.g., benzyl), or aryloxy-substituted C 1~6 In some particular embodiments, R 11 is -C(=O)-R B where R B is C 1~6 Alkyl (e.g., isopropyl), aryl (e.g., phenyl) or aryl-substituted C 1~6 alkyl (e.g., benzyl), preferably R B is isopropyl.

[0016] In some preferred embodiments, the compound of formula X-1 is not bound to a solid support. Without wishing to be bound by theory, it is believed that the deprotection of the NPE group of formula X-1 can be controlled without being bound to a solid support, thereby allowing the 4-nitrostyrene by-product to react preferentially with a scavenger in the reaction medium over the compound of formula X-2. However, in some embodiments, the compound of formula X-1 may be bound to a solid support, and the reaction can be controlled by, for example, adding an excess amount of a scavenger, thereby allowing the 4-nitrostyrene by-product to react preferentially with a scavenger in the reaction medium over the compound of formula X-2.

[0017] The conditions for converting the compound of formula X-1 to the compound of formula X-2 are not particularly limited. However, in some preferred embodiments, the converting step comprises adding the compound of formula X-1, preferably in solution, to a mixture containing an alkaline reagent (e.g., as described herein) and a scavenger capable of reacting with the compound of formula X-3. [ka]

[0018] The alkaline reagent is typically a basic organic amine. For example, in some embodiments, the alkaline reagent is a basic organic amine having a pKa in water of about 9 or greater, e.g., about 9-15, about 10-14, about 12, about 13, or about 14. In some embodiments, the alkaline reagent is a basic cyclic amine. In some embodiments, the alkaline reagent is 1,8-diazabicyclo[5.4.0]undec-7-ene ("DBU") or 1,5-diazabicyclo[4.3.0]non-5-ene ("DBN").

[0019] The alkaline reagent is typically added in excess, e.g., the molar ratio of alkaline reagent to NPE group(s) of the compound of formula X-1 is typically greater than 1:1, e.g., 1.2:1, 1.5:1, 2:1, 5:1, or 10:1, or any range between the recited values, e.g., 1:1 to 10:1.

[0020] The scavenger is not particularly limited as long as it can react with the by-product of formula X-3. Typically, the scavenger has an -SH or 1,3-dicarbonyl moiety. In some embodiments, the scavenger can be a compound of formula X-4: [ka] (In the formula: q is 0, 1, or 2; R A Each occurrence may be independently substituted C 1~6 alkyl (e.g., methyl).

[0021] In some embodiments, q is 0. In some embodiments, q is 1 and R A may be substituted C 1~6 If the compound of formula X-4 can act as a scavenger for the compound of formula X-3, then R Acan be attached at any available position. In certain embodiments, the capture agent is thymine or a derivative thereof. In certain embodiments, the capture agent is thymine.

[0022] The scavenger is also typically used in excess. For example, the molar ratio of scavenger to NPE group(s) of the compound of formula X-1 is typically greater than 1:1, such as 1.2:1, 1.5:1, 2:1, 5:1, 10:1, or any range between the recited values, such as 1:1 to 10:1. When the compound of formula X-1 has one or more bases capable of reacting with 4-nitrostyrene, the amount of scavenger can be further increased, for example, to 50:1 (or more) relative to the NPE group(s) of the compound of formula X-1.

[0023] The conversion of Formula X-1 to Formula X-2 typically uses one or more solvents. A variety of solvents are suitable. Useful solvents include, but are not limited to, any of those described herein. For example, in some embodiments, the solvent may be an aprotic polar solvent, such as DMF, DMA, DMI, NMP, etc. In some preferred embodiments, the solvent may be NMP.

[0024] In some embodiments, the addition of the compound of formula X-1 to the mixture of alkaline reagent and scavenger can be controlled to minimize the amount of NPE adduct on the oligonucleotide, e.g., less than 10%, e.g., less than 5%.

[0025] In some embodiments, the method further comprises treating the compound of formula X-2 with NH to partially or completely remove the protecting group of formula X-2. For example, in some embodiments, R 11 is C 1~6 alkyl-C(=O)-, which when treated with NH3, gives R 11 The group is removed to form the deprotected base G.

[0026] Exemplary procedures for converting compounds of formula X-1 to compounds of formula X-2 are described herein and can be readily adapted by one of skill in the art to embodiments of the present disclosure.

[0027] Exemplary Methods for PMO Synthesis In some particular embodiments, the present disclosure provides methods for preparing a PMO. In some embodiments, the methods include: (a) converting a compound of formula X-5 to a compound of formula X-6: [ka] (In the formula: m1 and m2 are independently an integer of 0 to 50 (e.g., 0 to 30); R 11 is an amine protecting group; Base, for each occurrence, is independently a base selected from G (guanine), C (cytosine), A (adenine), U (uracil), and T (thymine), modified analogs thereof, and protected derivatives thereof, provided that when Base in Formula X-5 is a protected base, the corresponding Base in Formula X-6 can be the same protected base or a corresponding partially or fully deprotected base; During the ceremony: T 1 is a suitable 5'-terminal group (e.g., a short peptide, an optionally substituted alkylamino group, an optionally substituted heterocyclic group, etc.); and T 2 is a suitable 3' terminal group, such as hydrogen or a protecting group (e.g., acyl, trityl, etc.); (b) optionally partially or completely removing the protecting groups in the compound of formula X-6 to obtain an oligonucleotide; Includes:

[0028] Oligonucleotides of formula X-5 or X-6 can have different numbers of G monomers at different positions in the sequence. In some embodiments, m1 is 0. In some embodiments, m2 is 0. In some embodiments, neither m1 nor m2 is 0. In some embodiments, the sum of m1 and m2 is between 5 and 50, e.g., 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or any range between the recited values, e.g., 10-40, 15-30.

[0029] The Bases in Formula X-5 or X-6 can be independently selected from G (guanine), C (cytosine), A (adenine), U (uracil), and T (thymine), their modified analogs, and their protected derivatives. Modified analogs used herein include non-standard bases, such as 5-methylcytosine, inosine (I), and 7-deaza-G bases. In some embodiments, the Bases in Formula X-5 or X-6 can be independently selected from the following: [ka]

[0030] In some embodiments, when Base in formula X-5 is a G monomer unit, it can be NPEG: [ka] (In the formula, R 11 is defined herein. In some embodiments, all of the G monomer units of formula X-5 can be NPEG.

[0031] Oligonucleotides, such as PMOs, can have a variety of termini at the 5' and 3' ends. The exact identity of such termini is not critical to the synthetic methods described herein and can include any of the known termini suitable for PMOs.

[0032] In some embodiments, T in Formula X-5 or Formula X-61 is an optionally substituted alkylamine, for example, —N(C 1~6 Alkyl)(C 1~6 alkyl), in which case two C 1~6 The alkyls may be the same or different, and each of these may be substituted, for example, by amide. In some embodiments, the T 1 is an alkylamine substituted with an amide (e.g., —C(O)NH2), e.g., [ka] It may be.

[0033] In some embodiments, T in formula X-5 or X-6 1 represents an optionally substituted heterocycle, for example, an optionally substituted 4- to 7-membered heterocycle having 1 or 2 ring heteroatoms independently selected from N, O, and S, such as an optionally substituted piperidine ring, for example, [ka] (In the formula, R C is an acyl group, an acyloxy group, or a peptide residue. It may be.

[0034] In some embodiments, T in formula X-5 or X-6 2 may be hydrogen.

[0035] In some embodiments, T in formula X-5 or X-6 2 may be a trityl group or a methoxy-substituted trityl group (eg, MMT, DMT, etc.).

[0036] In some embodiments, T in formula X-5 or X-6 2 may be an acyl group (e.g., acetyl).

[0037] The PMO of formula X-5 or X-6 may have any sequence, and preferably includes a targeting base sequence for sequence-specific binding to a target nucleic acid.

[0038] R in Formula X-5 or X-6 11 A variety of amine protecting groups can be used as R 11 is an amine protecting group that can be removed by treatment with NH. For example, in some embodiments, R 11 is an acyl group, i.e., R B -C(=O)-, where R B may be, for example, hydrogen, alkyl, aryl, cycloalkyl, heteroaryl, each of which may be optionally substituted. In some embodiments, R 11 is -C(=O)-R B where R B may be substituted C 1~6 Alkyl, e.g., C 1~6 C substituted with alkyl (e.g., isopropyl), aryl 1~6 C substituted with alkyl (e.g., benzyl) or aryloxy 1~6 In some particular embodiments, R 11 is -C(=O)-R B where R B is C 1~6 Alkyl (e.g., isopropyl), aryl (e.g., phenyl) or aryl-substituted C 1~6 alkyl (e.g., benzyl), preferably R B is isopropyl.

[0039] In some preferred embodiments, the compound of formula X-5 is not bound to a solid support.

[0040] The conditions for converting the compound of formula X-5 to the compound of formula X-6 are not particularly limited. However, in some preferred embodiments, the converting step comprises adding the compound of formula X-5, preferably in solution, to a mixture containing an alkaline reagent (e.g., as described herein) and a scavenger capable of reacting with the compound of formula X-3: [ka]

[0041] The alkaline reagent is typically a basic organic amine. For example, in some embodiments, the alkaline reagent is a basic organic amine having a pKa in water of about 9 or greater, e.g., about 9-15, about 10-14, about 12, about 13, or about 14. In some embodiments, the alkaline reagent is a basic cyclic amine. In some embodiments, the alkaline reagent is 1,8-diazabicyclo[5.4.0]undec-7-ene ("DBU") or 1,5-diazabicyclo[4.3.0]non-5-ene ("DBN"). The alkaline reagent is typically added in excess; for example, the molar ratio of alkaline reagent to NPE group(s) of the compound of Formula X-5 is typically greater than 1:1, e.g., 1.2:1, 1.5:1, 2:1, 5:1, 10:1, or any range between the recited values, e.g., 1:1 to 10:1.

[0042] The scavenger is not particularly limited as long as it can react with the by-product of formula X-3. Typically, the scavenger has an -SH moiety or a 1,3-dicarbonyl moiety. In some embodiments, the scavenger can be a compound of formula X-4: [ka] (In the formula: q is 0, 1, or 2; R A Each occurrence may be independently substituted C 1~6 alkyl (e.g., methyl).

[0043] In some embodiments, q is 0. In some embodiments, q is 1 and R A may be substituted C 1~6 If the compound of formula X-4 can act as a scavenger for the compound of formula X-3, then R A can be attached at any available position. In certain embodiments, the capture agent is thymine or a derivative thereof. In certain embodiments, the capture agent is thymine.

[0044] Typically, the scavenger is also used in excess. For example, the molar ratio of the scavenger to the NPE group(s) of the compound of formula X-5 is typically greater than 1:1, such as 1.2:1, 1.5:1, 2:1, 5:1, 10:1, or any range between the recited values, such as 1:1 to 10:1. When the compound of formula X-5 has one or more bases capable of reacting with 4-nitrostyrene, the amount of scavenger can be further increased, for example, to 50:1 (or more) relative to the NPE group(s) of the compound of formula X-5.

[0045] The conversion of Formula X-5 to Formula X-6 typically uses one or more solvents. A variety of solvents are suitable. Useful solvents include, but are not limited to, any of those described herein. For example, in some embodiments, the solvent may be an aprotic polar solvent, such as DMF, DMA, DMI, NMP, etc. In some preferred embodiments, the solvent may be NMP.

[0046] In some embodiments, the addition of the compound of formula X-5 to the mixture of alkaline reagent and scavenger can be controlled to minimize the amount of NPE adduct on the oligonucleotide, e.g., less than 10%, e.g., less than 5%.

[0047] In some embodiments, the method further comprises treating the compound of formula X-6 with NH to partially or completely remove the protecting group of formula X-6. For example, in some embodiments, R 11 is C 1~6alkyl-C(=O)-, which when treated with NH3, gives R 11 The group is removed to form the deprotected base G.

[0048] Compounds of formula X-5 can be readily prepared by one of ordinary skill in the art in light of the present disclosure. For example, in some embodiments, compounds of formula X-5 can be prepared from an oligonucleotide of formula X-7 by a method comprising cleaving the solid support with, for example, NH: [ka] (In the formula: SS is a solid support, e.g., a polystyrene solid support; L 1 is a linker, e.g., a sarcosine-based linker, e.g., [ka] wherein the nitrogen terminus is bonded to the phosphorus atom and the carbonyl terminus forms an amide bond with the solid support; m1, m2, and R in Formula X-7 11 is the same as the corresponding group in formula X-5, Base, for each occurrence, is independently a base selected from G (guanine), C (cytosine), A (adenine), U (uracil), and T (thymine), their analogs, and protected derivatives thereof, provided that when Base in Formula X-7 is a protected base, the corresponding base in Formula X-5 can be the same protected base or a corresponding partially or fully deprotected base; and T 2 is a suitable 3' terminal group, such as hydrogen or a protecting group (eg, acyl group, trityl).

[0049] In some embodiments, SS is a solid support, such as a polystyrene solid support, having -CH2-NH2 groups, alternatively SS-CH2-NH2.

[0050] In some embodiments, L 1 is a sarcosine-based linker, e.g. [ka] (wherein the nitrogen terminus is bonded to the phosphorus atom and the carbonyl terminus forms an amide bond with the solid support).

[0051] In some embodiments, Base in formula X-7 can be independently selected from the following: [ka]

[0052] In some embodiments, T in formula X-7 2 may be hydrogen.

[0053] In some embodiments, T in formula X-7 2 may be a trityl or methoxy-substituted trityl group (e.g., MMT, DMT, etc.).

[0054] In some embodiments, T in formula X-7 2 may be an acyl group (e.g., acetyl).

[0055] Compounds of formula X-7 can be prepared by one of ordinary skill in the art in light of the present disclosure. Exemplary procedures are also described in the Examples section herein.

[0056] Exemplary procedures for converting compounds of formula X-5 to compounds of formula X-6 are described herein and can be readily adapted by one of skill in the art from the description to embodiments of the present disclosure.

[0057] It should be noted that compounds of formula X-5, X-6, or X-7, as defined herein, are also novel compositions of the present disclosure. In addition, any oligonucleotide produced by the methods described herein is also a novel composition of the present disclosure. In some embodiments, the present disclosure further provides a pharmaceutical composition comprising an oligonucleotide produced by the methods described herein.

[0058] In a further aspect, a method for deprotecting a 4-nitrophenethyl (NPE) group from a base-protected phosphorodiamidate morpholino oligomer is provided, comprising treating the base-protected phosphorodiamidate morpholino oligomer in the presence of an alkaline reagent and a scavenger, such as one having a 1,3-dicarbonyl moiety. The PMO preferably contains a targeting base sequence for sequence-specific binding to a target nucleic acid.

[0059] In some embodiments of the present disclosure, the method comprises adding a base-protected phosphorodiamidate morpholino oligomer to a mixture of an alkaline reagent and a scavenger.

[0060] In some embodiments of the present disclosure, the base-protected phosphorodiamidate morpholino oligomer is, for example, a compound of formula X-5, as described in the present disclosure.

[0061] The alkaline reagent for deprotecting the NPE group is not particularly limited. However, such alkaline reagents do not have significant reactivity with, for example, the backbone of the PMO. The alkaline reagent typically has a pKa suitable for removing the NPE group from a protected base, such as a protected guanine. In the present disclosure, the alkaline reagent may be an organic alkaline reagent, such as a basic organic amine having a pKa of about 9 or higher in water, e.g., about 9-15, about 10-14, about 12, about 13, or about 14. In some embodiments, the alkaline reagent is a basic cyclic amine, such as DBU (1,8-diazabicyclo[5,4,0]undec-7-ene), DBN (1,5-diazabicyclo[4.3.0]non-5-ene), DABCO (1,4-diazabicyclo[2.2.2]octane), or a mixture thereof, such as DBU.

[0062] The scavenger used in the methods herein can be any known in the art that can react with 4-nitrostyrene generated by deprotection of the NPE group(s). In some embodiments, the scavenger can have, for example, an -SH group or a 1,3-dicarbonyl moiety as described herein (e.g., a compound of formula X-4 as described in this disclosure). In some preferred embodiments of the present disclosure, the scavenger can be a 1,3-dicarbonyl compound, such as thymine, diethyl malonate, or a mixture thereof, such as thymine.

[0063] In one embodiment, the molar ratio of the alkaline reagent to the NPE group(s) in the base-protected phosphorodiamidate morpholino oligomer is greater than 1:1, and can be, for example, 1:1 to 10:1, 1.2 to 10:1, or 1.5:1 to 10:1. In one embodiment, the amount of alkaline reagent is in excess of the NPE group(s).

[0064] In one embodiment, the scavenger may be used in excess. For example, the molar ratio of scavenger to NPE group(s) in the base-protected phosphorodiamidate morpholino oligomer is typically greater than 1:1, such as 1.2:1, 1.5:1, 2:1, 5:1, 10:1, or any range between the recited values, such as 1:1 to 10:1. When the base-protected phosphorodiamidate morpholino oligomer has one or more bases capable of reacting with 4-nitrostyrene, the amount of scavenger can be further increased, for example, up to 50:1 (or more) relative to the NPE group(s) of the base-protected phosphorodiamidate morpholino oligomer.

[0065] In the present disclosure, the process of deprotecting NPE group may be carried out in the presence of a solvent. The solvent may be any solvent used in the art for deprotecting NPE group. In the present disclosure, the solvent may be selected from polar aprotic solvents such as DMF, DMA, DMI, NMP, etc.

[0066] In some embodiments of the present disclosure, the process of deprotecting the NPE group from a base-protected phosphorodiamidate morpholino oligomer comprises adding a mixture of the base-protected phosphorodiamidate morpholino oligomer in a first solvent to a mixture of an alkaline reagent and a scavenger in a second solvent. The first and second solvents may be the same or different. In some embodiments, the first and second solvents may be selected from polar aprotic solvents such as DMF, DMA, DMI, NMP, etc. In the mixture of base-protected phosphorodiamidate morpholino oligomers in the first solvent, the concentration of the base-protected phosphorodiamidate morpholino oligomer may be 0.001 M to 0.1 M, for example, 0.01 M to 0.02 M. In the mixture of alkaline reagent and scavenger in the second solvent, the concentration of the alkaline reagent may be 0.1M to 5M, for example, 0.5M to 1.0M; the concentration of the scavenger may be 0.1M to 5M, for example, 0.5M to 1.5M, for example, 0.8M.

[0067] In some embodiments of the present disclosure, the process of deprotecting the NPE group from a base-protected phosphorodiamidate morpholino oligomer comprises adding a mixture of the base-protected phosphorodiamidate morpholino oligomer in a first solvent to a mixture of an alkaline reagent and a scavenger in a second solvent. The first and second solvents may be the same or different. In some embodiments, the first and second solvents may be selected from polar aprotic solvents such as DMF, DMA, DMI, NMP, etc. In the mixture of base-protected phosphorodiamidate morpholino oligomers in the first solvent, the concentration of the base-protected phosphorodiamidate morpholino oligomer may be 0.001 M to 0.1 M, for example, 0.01 M to 0.02 M. In the mixture of alkaline reagent and scavenger in the second solvent, the concentration of the alkaline reagent can be 0.1 M to 5 M, for example, 0.5 M to 1.0 M; and the concentration of the scavenger can be 0.1 M to 5 M, for example, 0.5 M to 1.5 M, for example, 0.8 M. In one embodiment, the rate of addition can be 1 to 10 mL / min, for example, 2 mL / min, 3 mL / min, 4 mL / min, 5 mL / min, 6 mL / min, 7 mL / min, 8 mL / min, or 9 mL / min.

[0068] In some embodiments, the present disclosure provides a method for preparing a phosphorodiamidate morpholino oligomer, comprising deprotecting the NPE group(s) from a base-protected phosphorodiamidate morpholino oligomer, as described hereinabove.

[0069] The synthesis of oligomers herein is generally carried out on a support medium, as described herein. Typically, oligomers are synthesized by first attaching a first starting material (e.g., a monomer, e.g., a morpholino subunit) to a support medium, followed by sequentially coupling subunits to the support-bound starting material. This repeated extension produces the final oligomeric compound. Suitable support media can be soluble or insoluble and may have varying solubilities in different solvents, allowing the growing support-bound polymer to be in or out of solution as desired. Traditional support media are often insoluble and are typically placed in a reaction vessel, where reagents and solvents react with the growing chain and / or wash the growing chain until the oligomer reaches the desired length, after which it is cleaved from the support and, if necessary, further processed to produce the final polymeric compound. A more recent approach has been to introduce a soluble support containing a soluble polymeric support, allowing the repeated precipitation and dissolution of the synthesized product at any point during the synthesis.

[0070] In certain embodiments, the morpholino is conjugated to a "tail" moiety at the 5' or 3' end of the oligomer to enhance its stability and / or solubility. Exemplary tails include short peptides, optionally substituted alkylamino groups, optionally substituted heterocyclic groups, etc., acyl groups, trityl, etc. In one embodiment, a suitable 5' terminal group is, for example, an alkylamine substituted with a short peptide, an optionally substituted alkylamino group, an optionally substituted heterocyclic group, etc. (e.g., -C(O)NH2), e.g., [ka] In another embodiment, a suitable 3' terminal group is, for example, hydrogen or a protecting group (e.g., acyl group, trityl, etc.).

[0071] In some embodiments, the present disclosure provides a method for preparing a phosphorodiamidate morpholino oligomer, comprising: (a) reacting a solid-phase supported morpholino subunit having an unprotected ring nitrogen with a base-protected morpholino subunit monomer having a protected ring nitrogen and an activated phosphoramidate group on the 5'-exocyclic carbon; thereby forming a phosphorodiamidate bond between the 5'-exocyclic carbon and the unprotected nitrogen; (b) deprotecting the protected nitrogen to form an unprotected nitrogen; (c) repeating steps (a) and (b) one or more times with additional base-protected morpholino subunit monomers to obtain a base-protected morpholino-modified solid support; and (d) performing three steps of cleavage and deprotection to obtain a phosphorodiamidate morpholino oligomer; the three steps of cleavage and deprotection include deprotecting the NPE group from the base-protected phosphorodiamidate morpholino oligomer as described above; Including, wherein at least one of the base-protected morpholino subunit monomers is a protected guanine morpholino compound having the structure (M): [ka] (In the formula, R 1 is a chlorophosphoramidate group; R 2 is a lower alkyl, a monocyclic arylmethyl group, or a monocyclic (aryloxy)methyl; R 3 is a triarylmethyl group).

[0072] Morpholino ring nitrogen (R 3Examples of triarylmethyl protecting groups for ) may be triphenylmethyl (trityl), 4-methyltrityl, 4,4'-dimethyltrityl, 4,4',4"-trimethyltrityl, monomethoxytrityl (e.g., 4-methoxytrityl), or dimethoxytrityl (e.g., 4,4'-dimethoxytrityl).

[0073] R 1 may be -OP(=O)-N(CH3)2Cl.

[0074] R 2 may be benzyl or —CH(CH 3 ) 2 .

[0075] In one embodiment, provided herein is a method for preparing a compound of formula (II), comprising contacting compound (E) with a deblocking agent to obtain a compound of formula (II): [ka] (wherein SS is the support medium and Z1 is [ka] wherein the oxygen terminus is connected to a sarcosine unit, m is 1, 2, 3, 4 or 5, and R 4 is Tr (triphenylmethyl) or a derivative thereof, such as Tr (triphenylmethyl), MMTr (p-methoxyphenyldiphenylmethyl), or DMTr (di-(p-methoxyphenyl)phenylmethyl). In a preferred embodiment, m is 3 and R 4 is Tr (triphenylmethyl).

[0076] In one embodiment, the method for preparing a compound of formula (II) further comprises contacting the deblocked compound with a neutralizing agent.

[0077] In another aspect, provided herein is a process for preparing a compound of formula (III), comprising coupling a compound of formula (II) with a compound of formula (G) to obtain a compound of formula (III). [ka]

[0078] Base is selected from optionally protected nucleobases such as PC, T, PA, P5mC, U, I, PG, DPG, or NPEG; G (guanine), C (cytosine), A (adenine), U (uracil), and T (thymine), modified analogs thereof, and protected derivatives thereof, such as PC, T, PA, P5mC, U, I, PG, DPG, or NPEG. [ka]

[0079] SS, Z1, R 1 , R 2 and R 3 The definitions are as explained in this disclosure.

[0080] In one embodiment, the method for preparing a compound of formula (III) further comprises contacting a compound of formula (II) with a capping agent.

[0081] In another embodiment, the compound of formula (II) is obtained from a process as defined above.

[0082] In another aspect, provided herein is a process for preparing a compound of formula (IV), comprising the following sequential steps: (i) coupling a compound of formula (II) with a compound of formula (G) to obtain a compound of formula (III); (ii) repeating the following sequential steps n-1 times: (ii-1) contacting the product obtained in the immediately preceding step with a deblocking agent; (ii-2) coupling the compound obtained in the immediately preceding step with a compound of formula (G) to form a compound of formula (IV); [ka] wherein optionally at least one of the bases is NPEG; [ka] (In the formula, SS, Z1, R 1 , R 2 and R 3 is defined as described in the present disclosure; and n is an integer of 10 to 40, for example, n is an integer of 20 to 30, for example, 25. Base, for each occurrence, is independently a base selected from an optionally protected nucleobase, e.g., PC, T, PA, P5mC, U, I, PG, DPG, or NPEG; G (guanine), C (cytosine), A (adenine), U (uracil), and T (thymine), modified analogs thereof, and protected derivatives thereof, e.g., an optionally protected nucleobase, e.g., PC, T, PA, P5mC, U, I, PG, DPG, or NPEG.

[0083] In one embodiment, step (ii-1) further comprises contacting the deblocked compound with a neutralizing agent.

[0084] In another embodiment, step (ii-2) further comprises contacting the compound obtained by the immediately preceding step with a capping agent.

[0085] In yet another aspect, provided herein is a process for preparing a compound of formula (V), comprising contacting a compound of formula (IV) with a deblocking agent to obtain a compound of formula (V). [ka] (In the formula, SS, Z1, Base, R3 and n are defined in this disclosure. In one embodiment, the method of preparing a compound of formula (V) further comprises contacting the deblocked compound with a neutralizing agent.

[0086] In yet another aspect, there is provided a method for preparing a compound of formula (VI), comprising contacting a compound of formula (V) with a cleaving agent to obtain a compound of formula (VI); [ka] (In the formula, SS, Z1, Base, R 3 and n are defined in the present disclosure.

[0087] In another embodiment, there is provided a method for preparing a phosphorodiamidate morpholino oligomer, comprising: (a) contacting a compound of formula (VI) with a deprotecting agent; (b) optionally, performing an aminolysis reaction on the compound obtained in the immediately preceding step to obtain a phosphorodiamidate morpholino oligomer; Provided herein are methods comprising: [ka] (wherein the definitions of Base and n are as explained in the present disclosure).

[0088] In one embodiment, each process (step) is carried out in the presence of at least one solvent.

[0089] In another embodiment, the neutralizing agent is present in a solution comprising dichloromethane and isopropyl alcohol.

[0090] In yet another embodiment, the neutralizing agent is a monoalkylamine, a dialkylamine, or a trialkylamine.

[0091] In another embodiment, the neutralizing agent is N,N-diisopropylethylamine.

[0092] In a preferred embodiment, the neutralizing agent used in each step is 5% diisopropylethylamine in 25% isopropanol / dichloromethane.

[0093] In another embodiment, the compound of formula (G) is present in a solution comprising ethylmorpholine and dimethylimidazolidinone.

[0094] In a further embodiment, the compound of formula (G) is selected from PMO-NPEG monomers, PMO-PA monomers, PMO-PC monomers and PMO-T monomers; [ka]

[0095] In some embodiments, the methods of the present disclosure can be carried out in continuous flow or discontinuous flow modes known in the art. In some embodiments, the methods of the present disclosure may be carried out in custom-built peptide batch reactors.

[0096] In yet another aspect, there is provided a method for preparing compound (E), comprising contacting compound (D) with compound (S) to obtain compound (E); [ka] (Wherein SS, Z1 and R 4 are as defined in this disclosure.

[0097] In the process of preparing compound (E), compound (S) may need to be activated before use, and the activation of compound (S) may include suspending compound (S) in a solvent to allow it to swell, followed by removing the solvent and washing the compound (S) sequentially with a mixture of a chlorinated hydrocarbon solvent and a base in the solvent.

[0098] In the activation of compound (S), the solvent for suspending compound (S) can be selected from polar aprotic solvents, such as alkanone solvents, for example, NMP. The amount of solvent is not particularly limited. The volume-mass ratio of the solvent to compound (S) can be 10 mL / g to 30 mL / g. The solvent can be removed by filtration. The chlorinated hydrocarbon solvent can be DCM. The base in the mixture can be an organic base, for example, DIPEA. The solvent in the mixture can be an alcohol solvent (e.g., IPA), a chlorinated hydrocarbon solvent (e.g., DCM), or a combination thereof, more preferably a combination of an alcohol solvent and a chlorinated hydrocarbon solvent (e.g., a combination of IPA and DCM, with a volume ratio of 1:1 to 1:5, for example, 1:3). The mass percentage of the base in the mixture can be 1% to 10%, for example, 5%, where % represents the mass of the base in the total mass of the mixture.

[0099] The process for preparing compound (E) can be carried out in a solvent. The solvent can be selected from polar aprotic solvents, such as alkanone solvents, amide solvents, or mixtures thereof, such as NMP, DMI, DMF, or mixtures thereof. The amount of solvent is not particularly limited. The volume-mass ratio of the solvent to compound (S) can be 10 mL / g to 30 mL / g.

[0100] In the process of preparing the compound (E), the molar ratio of the compound (D) to the compound (S) may be 1:1 to 1:3.

[0101] In the preparation of compound (E), the reaction temperature may be 20 to 50°C, for example, 40 to 45°C. The progress of the reaction may be monitored using a conventional detection method in the art (e.g., TLC, HPLC, GC, or NMR). The disappearance of compound (D) is generally considered to be the completion of the reaction. The reaction time may be 24 to 48 hours.

[0102] In a preferred embodiment, the process for preparing compound (E) preferably comprises adding a solvent solution of compound (D) to a solvent suspension of compound (S) to carry out the reaction.

[0103] In the preparation process of compound (E), the post-treatment may be a conventional post-treatment for such a reaction in the art. In the present disclosure, the post-treatment preferably comprises filtering the obtained mixture, washing the filter cake with a solvent (e.g., an alkanone solvent, a chlorinated hydrocarbon solvent, or a combination thereof; the alkanone solvent may be NMP; the chlorinated hydrocarbon solvent may be DCM; when the solvent is a combination of an alkanone solvent and a chlorinated hydrocarbon solvent, the volume ratio may be 1:1 to 1:10), and then washing the filter cake with a solvent (e.g., an alkanone solvent; for example, NMP; the volume-mass ratio of the solvent to compound (S) is 50 mL) of NEM (0.2 to 1.0 M). A solution of BzO (0.2 to 1.0 M) in a solvent (e.g., an alkanone solvent, e.g., NMP; the volume-to-mass ratio of the solvent to compound (S) may be 50 mL / g to 200 mL / g) is sequentially added to carry out the capping reaction, and after completion of the capping reaction, the resulting mixture is filtered, washed with a solvent (e.g., a chlorinated hydrocarbon solvent, DCM; the volume-to-mass ratio of the solvent to compound (S) may be 100 mL / g to 300 mL / g), and dried to obtain compound (E).

[0104] In a preferred embodiment of the present disclosure, compound (S) is aminomethylpolystyrene resin, which is available from Xi'an Lanxiao Technology Co., Ltd.

[0105] In a preferred embodiment of the present disclosure, the process for preparing compound (E) may further include contacting compound (C) with compound (SM4) in a solvent in the presence of a catalyst, a base and a condensing agent to obtain compound (D); [ka] (Wherein Z1 and R 4are as defined in this disclosure. In the preparation of compound (D), the catalyst is a conventional catalyst for such a reaction in the art. In the present disclosure, the catalyst may be DMAP. The molar ratio of the catalyst to compound (C) may be 0.01:1 to 0.5:1, for example, 0.33:1.

[0106] In the preparation process of compound (D), the base is a conventional base for such a reaction in the art. In the present disclosure, the base may be an organic base, for example, an organic amine, for example, DIPEA. The amount of base is a conventional amount for such a reaction in the art. In the present disclosure, the molar ratio of base to compound (C) may be 1 to 3:1, for example, 2.5:1.

[0107] In the preparation of compound (D), the condensing agent is a conventional condensing agent for such a reaction in the art. In the present disclosure, the condensing agent may be EDCI, DCC, DIC, or a mixture thereof. The amount of the condensing agent is a conventional amount for such a reaction in the art. In the present disclosure, the molar ratio of the condensing agent to compound (C) may be 1:1 to 2:1, for example, 1.1:1.

[0108] In the preparation process of compound (D), the molar ratio of compound (C) to compound (SM4) can be 1:1 to 1:2, for example, 1:1.02.

[0109] In the preparation of compound (D), the solvent may be a conventional solvent for such a reaction in the art. In the present disclosure, the solvent may be a chlorinated hydrocarbon solvent, such as DCM. The volume-mass ratio of the solvent to compound (C) may be 10 mL / g to 20 mL / g, for example, 10 mL / g.

[0110] In the preparation of compound (D), the reaction temperature may be 20 to 30°C. The progress of the reaction can be monitored using a conventional detection method in the art (e.g., TLC, HPLC, GC, or NMR). The disappearance of compound (C) is generally considered to be the completion of the reaction.

[0111] In a preferred embodiment, the process for preparing compound (D) comprises adding compound (SM4), a catalyst, a base and a condensing agent to a solution of compound (C) in a solvent to carry out a reaction.

[0112] In the preparation of compound (D), the work-up may be a conventional work-up for such a reaction in the art. In the present disclosure, the work-up preferably includes washing the reaction mixture with citric acid (e.g., 10% citric acid solution) and brine in sequence, followed by concentrating and drying the organic layer to obtain compound (D).

[0113] In a preferred embodiment of the present disclosure, the process for preparing compound (E) may further include a process for preparing compound (C), which comprises contacting compound (B) with compound (SM3) in a solvent to obtain compound (C); [ka] (wherein m is an integer of 1 to 5, and R 4 are as defined in this disclosure.

[0114] In the process of preparing compound (C), the molar ratio of compound (B) to compound (SM3) may be 1:1 to 1:2, for example, 1:2.

[0115] In the preparation of compound (C), the solvent may be a conventional solvent for such a reaction in the art. In the present disclosure, the solvent may be an ether solvent, such as THF. The volume-mass ratio of the solvent to compound (B) may be 10-20 mL / g, for example, 10 mL / g.

[0116] In the preparation of compound (C), the reaction temperature may be 20 to 55°C. The progress of the reaction can be monitored using a detection method commonly used in the art (e.g., TLC, HPLC, GC, or NMR). The disappearance of compound (B) is generally considered to be the completion of the reaction.

[0117] In a preferred embodiment, the process for preparing compound (C) preferably comprises adding compound (SM3) to a solution of compound (B) in a solvent to carry out the reaction.

[0118] In the preparation process of compound (C), the post-treatment may be a conventional post-treatment for such a reaction in the art. In the present disclosure, the post-treatment is carried out by adjusting the pH of the reaction mixture to about 8.5 with an aqueous NaHCO solution (e.g., a 10% aqueous NaHCO solution), adding an ether solvent (e.g., MTBE), adjusting the pH of the resulting aqueous layer to 3-5 with a citric acid solution (20% citric acid solution), extracting with a chlorinated hydrocarbon solvent (e.g., DCM), washing with an aqueous NaSO solution (e.g., a 10% aqueous NaSO solution), and concentrating the resulting organic layer to obtain compound (C).

[0119] In a preferred embodiment of the present disclosure, the process for preparing compound (E) may further comprise a process for preparing compound (B), which comprises contacting compound (A) with compound (SM2) in a solvent in the presence of a base to obtain compound (B): [ka] (wherein m is an integer of 1 to 5, and R 4 are as defined in this disclosure.

[0120] In the preparation of compound (B), the base is a conventional base for such a reaction in the art. In the present disclosure, the base may be a metal hydride, such as NaH. The molar ratio of the base to compound (SM2) may be 0.01:1 to 1:1, for example, 0.01:1.

[0121] In the process of preparing compound (B), the molar ratio of compound (A) to compound (SM2) may be 1:5 to 1:20, for example, 1:10.

[0122] In the preparation process of compound (B), the solvent may be a conventional solvent for such a reaction in the art. In the present disclosure, the solvent may be an alkanone solvent, such as NMP. The amount of the solvent may not be particularly limited. The volume-mass ratio of the solvent to compound (A) may be 15 mL / g to 25 mL / g, for example, 20 mL / g.

[0123] In the preparation of compound (B), the reaction temperature may be 20 to 30°C. The progress of the reaction can be monitored using a conventional detection method in the art (e.g., TLC, HPLC, GC, or NMR). The disappearance of compound (A) is generally considered to be the completion of the reaction.

[0124] In a preferred embodiment, the process for preparing compound (B) preferably comprises adding a base to a solvent solution of compound (SM2) with stirring, followed by adding compound (A) and carrying out a reaction, and more preferably comprises adding a base to a solvent solution of compound (SM2) at 20 to 30°C, stirring the resulting mixture at 20 to 30°C for 10 to 30 minutes, followed by adding compound (A) and carrying out a reaction.

[0125] In the preparation process of compound (B), the post-treatment may be a conventional post-treatment for such a reaction in the art. In the present disclosure, the post-treatment includes adding water and an organic solvent (for example, a chlorinated hydrocarbon solvent, an ether solvent, or a mixture thereof, preferably a mixed solvent of DCM and MTBE) to the reaction mixture for extraction, washing the obtained organic layer with brine, concentrating and purifying the obtained organic layer (for example, by silica gel column) to obtain compound (B).

[0126] In a preferred embodiment of the present disclosure, the process for preparing compound (E) may further comprise a process for preparing compound (A), which comprises reacting compound (SM1) with R 4 Cl to obtain compound (A); [ka] (In the formula, R 4 is as defined in this disclosure.

[0127] In the preparation of compound (A), the base is a conventional base for such a reaction in the art. In the present disclosure, the base may be an organic base, for example, an organic amine, for example, DIPEA. The amount of base is a conventional amount for such a reaction in the art. In the present disclosure, R 4 The molar ratio of base to Cl may be from 1:1 to 3:1, for example, 1.5:1.

[0128] In the preparation process of compound (A), R 4 The molar ratio of compound (SM1) to Cl may be 1:1 to 1:3, for example, 1:2.

[0129] In the preparation process of compound (A), the solvent is a conventional solvent for such a reaction in the art. In the present disclosure, the solvent can be selected from an amide solvent, for example, an amide solvent, an alkanone solvent, a chlorinated hydrocarbon solvent, or a mixture thereof, for example, DMF, DCM, NMP, or a mixture thereof. The amount of the solvent may not be particularly limited. In the present disclosure, the volume-mass ratio of the solvent to compound (SM1) can be 10 mL / g to 30 mL / g, for example, 10 mL / g.

[0130] In the preparation of compound (A), the reaction temperature may be 20 to 25°C. The progress of the reaction can be monitored using a conventional detection method in the art (e.g., TLC, HPLC, GC, or NMR). The disappearance of compound (SM1) is generally considered to be the completion of the reaction.

[0131] In a preferred embodiment, the process for preparing compound (A) comprises reacting a base and R 4 The reaction preferably comprises sequentially adding Cl to a solution of compound (SM1) in a solvent.

[0132] In the preparation process of compound (A), the post-treatment may be a conventional post-treatment for such a reaction in the art. In the present disclosure, the post-treatment includes adding water and an organic solvent (e.g., an ester solvent, preferably EtOAc) for extraction to the reaction mixture, washing the obtained organic layer with an aqueous NaCl solution (e.g., a 20% aqueous NaCl solution), concentrating and purifying the obtained organic layer (e.g., using a silica gel column) to obtain compound (A).

[0133] In yet another embodiment, there is provided compound (E): [ka] (Wherein Z1 is [ka] where the oxygen end is connected to a sarcosine unit, m is 1, 2, 3, 4, or 5, and SS and R 4 are as defined in this disclosure.

[0134] In a further aspect, there is provided the use of compound (E) according to the present disclosure in the preparation of oligonucleotides, such as phosphorodiamidate morpholino oligomers (PMOs).

[0135] As described herein, we have discovered that the use of NPE-protected guanine monomer units allows the preparation of oligonucleotides in high yield and purity, and that deprotection of the NPE group from non-solid support-bound oligonucleotides can be accomplished efficiently with minimal NPE adducts (impurities).

[0136] [Definition] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0137] Where an embodiment is described herein with the word "comprising," it is understood that similar embodiments are also provided that are otherwise described with the terms "containing," "consisting of," and / or "consisting essentially of." However, when used in the claims as transitional phrases, each should be interpreted separately in the appropriate legal and factual context (e.g., in the claims, the transitional phrase "comprising" is considered more open-ended, "consisting of" is more exclusive, and "consisting essentially of" is intermediate ground).

[0138] As used herein, the singular forms "a," "an," and "the" include plural references unless expressly stated or clearly intended from the context.

[0139] Headings and subheadings are used for convenience and / or formal compliance only, do not limit the subject technology, and should not be referenced in connection with interpreting the description of the subject technology. Features described under one heading or subheading of the subject disclosure may be combined with features described under other headings or subheadings in various embodiments. Furthermore, not all features under one heading or subheading may be used together in an embodiment.

[0140] When a range of values ​​is listed, it is intended to encompass each value and subrange within the range. For example, "C 1~6 ” is C1, C2, C3, C4, C5, C6, C 1~6 , C 1~5 , C 1~4 , C 1~3 , C 1~2 , C 2~6 , C 2~5 , C 2~4 , C2~3 , C 3~6 , C 3~5 , C 3~4 , C 4~5 and C 5~6 is intended to encompass:

[0141] A "morpholino oligomer" refers to a polymer having a backbone supporting hydrogen-bondable bases in a typical polynucleotide, lacking a pentose sugar backbone, more specifically a ribose backbone linked by phosphodiester bonds as typically found in nucleotides and nucleosides, but instead containing a ring nitrogen and coupling via the ring nitrogen. Preferred morpholino oligomers are composed of "morpholino subunit" structures, as shown below, which are preferably linked in the oligomer by (thio)phosphorodiamidate linkages, linking the morpholino nitrogen of one subunit to the 5' exocyclic carbon of an adjacent subunit. Each subunit contains a purine or pyrimidine base pair moiety, Base, that is effective for binding to a base in a polynucleotide by base-specific hydrogen bonding. [ka]

[0142] A "phosphorodiamidate" group contains a phosphorus atom bonded to two oxygen atoms and two nitrogen atoms, and is sometimes referred to herein as a phosphorus atom bonded to one oxygen atom and three nitrogen atoms. In the intersubunit linkages of the oligomers described herein, the first nitrogen is typically pendant to the backbone and the second nitrogen is a ring nitrogen morpholino ring structure: as shown in formula (a1) below. Alternatively or additionally, a nitrogen may be present at the 5'-exocyclic carbon, as shown in formulas (b1) and (c1) below. [ka]

[0143] Base1 and Base2 may be the same or different, and have the same definition as the bases described in this disclosure.

[0144] In a thiophosphorodiamidate linkage, one oxygen atom, usually the oxygen pendant to the backbone of the oligomers described herein, is replaced with a sulfur.

[0145] In a preferred embodiment, a phosphorodiamidate morpholino oligomer refers to a phosphorodiamidate morpholino oligomer of the following general structure: [ka]

[0146] A "solid-phase supported morpholino subunit" can be any initial or subsequent morpholino subunit monomer incorporated into a morpholino oligomer by solid-phase stepwise synthesis as described herein. The subunit is attached via its 5' exocyclic carbon to the solid support or to a growing oligomer chain on the solid support. "Base-protected" refers to protecting the base-pairing group, e.g., a purine or pyrimidine base, on the morpholino subunit with a protecting group suitable to prevent reaction or interference of the base-pairing group in the stepwise synthesis of the oligomer.

[0147] An "activated phosphoramidate group" is typically a chlorophosphoramidate group, with a substitution at the nitrogen desired for inclusion in the final phosphoramidate linkage in the oligomer, such as (dimethylamino)chlorophosphoramidate, i.e., -OP(=O)(NMe)Cl.

[0148] "Base-protected" or "base protection" refers to protecting a base-pairing group, e.g., a purine or pyrimidine base, on a morpholino subunit with a protecting group suitable to prevent the base-pairing group from reacting with or interfering with the stepwise synthesis of an oligomer. In a preferred embodiment, at least one of the base-protected morpholino subunit monomers is derived from a protected guanine morpholino compound having the structure (M). [ka]

[0149] R 1 , R 2 and R 3 are defined as described in this disclosure.

[0150] The "nucleobase" is not particularly limited as long as it can be used in the synthesis of nucleic acids, and examples thereof include pyrimidine bases such as cytosyl, uracil, and thyminyl, and purine bases such as adenyl and guanyl. The term "optionally protected nucleobase" refers to, for example, an adenyl, guanyl, or cytosyl group in which the amino group may be protected. It is preferably a nucleobase having an amino group, wherein the amino group is continuously protected by a protecting group under the deprotection conditions of the nitrogen atom of the morpholinonucleotide morpholine ring. The "amino protecting group" is not particularly limited, and specific examples of the "amino protecting group" include pivaloyl, pivaloyloxymethyl, trifluoroacetyl, phenoxyacetyl, 4-isopropylphenoxyacetyl, 4-tert-butylphenoxyacetyl, acetyl, benzoyl, isobutyryl, dimethylformamidinyl, and 9-fluorenylmethyloxycarbonyl groups. In addition, the carbonyl group of the nucleic acid base may be protected, for example, by reacting with phenol, 2,5-dichlorophenol, 3-chlorophenol, 3,5-dichlorophenol, 2-formylphenol, 2-naphthol, 4-methoxyphenol, 4-chlorophenol, 2-nitrophenol, 4-nitrophenol, 4-acetylaminophenol, pentafluorophenol, 4-pivaloyloxybenzyl alcohol, 4-nitrophenethyl alcohol, 2-(methylsulfonyl)ethanol, 2-(phenylsulfonyl)ethanol, 2-cyanoethanol, 2-(trimethylsilyl)ethanol, dimethylcarbamoyl chloride, diethylcarbamoyl chloride, ethylphenylcarbamoyl chloride, 1-pyrrolidinecarbonyl chloride, 4-morpholinecarbonyl chloride, dephenylcarbamoyl chloride, etc. In some cases, it is not necessary to specifically introduce a carbonyl protecting group.In addition to the above groups, further examples include modified nucleic acid bases (e.g., 8-bromoadenyl group, 8-bromoguanyl group, 5-bromocytosyl group, 5-iodocytosyl group, 5-bromouracil group, 5-iodouracil group, 5-fluorouracil group, hypoxanthinyl group, etc.), which are nucleic acid bases substituted at any position with any 1 to 3 substituents (e.g., halogen atoms, alkyl groups, aralkyl groups, alkoxy groups, acyl groups, alkoxyalkyl groups, hydroxy groups, amino groups, monoalkylamino, dialkylamino, carboxy, cyano, nitro, etc.), and these are also encompassed by the term "nucleobase."

[0151] "Lower alkyl" refers to an alkyl group of 1 to 6 carbon atoms, and is exemplified by methyl, ethyl, n-butyl, isobutyl, tert-butyl, isoamyl, n-pentyl, and isopentyl. In selected embodiments, a "lower alkyl" group has 1 to 4 carbon atoms, or 1 to 2 carbon atoms, i.e., methyl or ethyl.

[0152] "Support bound" refers to a chemical entity that is covalently bonded to a support medium.

[0153] The term "support medium" refers to a material, including, for example, particles, beads, surfaces, etc., onto which oligomers can be attached or synthesized, or which can be modified to attach or synthesize oligomers. Representative substrates include inorganic and organic supports, such as glass, modified or functionalized glass, plastics (e.g., acrylics, polystyrene and copolymers of styrene with other materials, polypropylene, polyethylene, polybutylene, polyurethane, Teflon, etc.), polysaccharides, nylon or nitrocellulose, ceramics, resins, silica or silica-based materials, including silicon and modified silicon, carbon, metals, inorganic glass, plastics, fiber optic bundles, and various other polymers. For some embodiments, particularly useful support media and solid surfaces are disposed within a flow cell apparatus. In some embodiments of the methods described herein, the support medium comprises polystyrene cross-linked with 1% divinylbenzene. In another embodiment of the methods described herein, the support medium is aminomethylpolystyrene resin (eg, purchased from Xi'an Lanxiao Technology Co., Ltd., with a loading of, for example, 1 mmol / g).

[0154] In some embodiments, exemplary support media include at least one reactive site for oligomer attachment or synthesis. For example, in some embodiments, support media of the present disclosure include one or more terminal amino or hydroxyl groups that can form chemical bonds with incoming subunits or other activating groups for oligomer attachment or synthesis.

[0155] The term "flow cell device" refers to a chamber that includes a surface (such as a solid surface) through which one or more reagents (such as liquids, gases, etc.) can flow.

[0156] The term "deblocking agent" refers to a composition (e.g., a solution) containing a chemical acid or a combination of chemical acids to remove protecting groups. Exemplary chemical acids used in deblocking agents include halogenated acids, such as chloroacetic acid, dichloroacetic acid, trichloroacetic acid, fluoroacetic acid, difluoroacetic acid, and trifluoroacetic acid. In some embodiments, the deblocking agent removes one or more trityl groups from, for example, an oligomer, a support-bound oligomer, a support-bound subunit, or other protected nitrogen or oxygen moieties. In another embodiment, the deblocking agent used in each process (step) is a solution containing 4-cyanopyridine, dichloromethane, trifluoroacetic acid, trifluoroethanol, and water, or a solution containing 4-cyanopyridinium trifluoroacetic acid, trifluoroethanol, dichloromethane, and ethanol. In a preferred embodiment, the deblocking agent used in each process (step) is 2% 4-cyanopyridinium trifluoroacetate (CYTFA) (w / v) in 20% trifluoroethanol / dichloromethane containing 1% ethanol.

[0157] The terms "halogen" and "halo" refer to an atom selected from fluorine, chlorine, bromine, and iodine.

[0158] The term "capping agent" refers to, for example, an acid anhydride (e.g., benzoic anhydride, acetic anhydride, phenoxyacetic anhydride, etc.) useful for blocking reactive sites on the support medium that form chemical bonds with incoming subunits or other activating groups. In one embodiment, the capping agent comprises ethylmorpholine and methylpyrrolidinone in solution. In a preferred embodiment, the capping agent of the present disclosure comprises Capping A and Capping B, where Capping A is a solution of NEM in NMP and Capping B is a solution of the capping agent in NMP.

[0159] The term "cleaving agent" refers to a composition (e.g., a liquid or gaseous mixture) comprising, for example, a chemical base (e.g., ammonia or 1,8-diazabicycloundec-7-ene) or a combination of chemical bases useful for cleaving a support-bound oligomer from the support medium. In yet another embodiment, the cleaving agent comprises N-methyl-2-pyrrolidone in solution.

[0160] The term "deprotecting agent" refers to a composition (e.g., a liquid or gaseous mixture) containing a chemical base (e.g., ammonia, 1,8-diazabicycloundec-7-ene, or potassium carbonate) or a combination of chemical bases useful for removing protecting groups. For example, a deprotecting agent, in some embodiments, can remove base protection from, for example, a morpholino subunit, a morpholino subunit of a morpholino oligomer, or a support-bound version thereof. In another embodiment, the cleaving agent comprises dithiothreitol and 1,8-diazabicyclo[5,4,0]undec-7-ene.

[0161] The term "solvent" refers to a component of a solution or mixture in which a solute is dissolved. Solvents can be inorganic or organic (e.g., acetic acid, acetone, acetonitrile, acetylacetone, 2-aminoethanol, aniline, anisole, benzene, benzonitrile, benzyl alcohol, 1-butanol, 2-butanol, isobutanol, 2-butanone, tert-butyl alcohol, carbon disulfide, carbon tetrachloride, chlorobenzene, chloroform, cyclohexane, cyclohexanol, cyclohexanone, di-n-butyl phthalate, 1,1-dichloroethane, 1,2-dichloroethane, diethylamine, diethylene glycol, diglyme, dimethoxyethane, N,N-dimethylaniline, dimethylformamide (DMF), dimethylacetamide, etc. dimethylformamide (DMA), 1,3-dimethyl-2-imidazolidinone (DMI), 1-methyl-2-pyrrolidinone (NMP), dimethyl phthalate, dimethyl sulfoxide, dioxane, ethanol, ether, ethyl acetate, ethyl acetoacetate, ethyl benzoate, ethylene glycol, glycerin, heptane, 1-heptanol, hexane, 1-hexanol, methanol, methyl acetate, methyl tert-butyl ether, methylene chloride, 1-octanol, pentane, 1-pentanol, 2-pentanol, 3-pentanol, 2-pentanone, 3-pentanone, 1-propanol, 2-propanol, pyridine, tetrahydrofuran, toluene, water, p-xylene).

[0162] In this disclosure, G, C, A, U, and T are guanine, cytosine, adenine, uracil, and thymine, respectively. [ka]

[0163] Abbreviation DMF stands for N,N-dimethylformamide. DIPEA stands for N,N-diisopropylethylamine. TrCl stands for triphenylmethyl chloride. TLC stands for thin layer chromatography. EtOAc stands for ethyl acetate. NaCl stands for sodium chloride. DBU represents 1,8-diazabicyclo[5,4,0]undec-7-ene. DBN represents 1,5-diazabicyclo[4.3.0]non-5-ene. DABCO stands for 1,4-diazabicyclo[2.2.2]octane. NMP stands for 1-methyl-2-pyrrolidinone. NPE represents a 4-nitrophenethyl (NPE) group. IPC stands for in-process control. UV stands for ultraviolet light. CYTFA stands for 4-cyanopyridinium trifluoroacetate. Vol. (vol.) represents the capacity. DCM stands for dichloromethane. MTBE stands for methyl tert-butyl ether. DMAP stands for dimethylaminopyridine. EDCI stands for 1-ethyl-3-(3-dimethylaminepropyl)carbodiimide hydrochloride. IPA stands for isopropyl alcohol. NEM stands for N-ethylmorpholine. Bz2O represents benzoic anhydride. DMI stands for 1,3-dimethyl-2-imidazolidinone. PMO stands for phosphorodiamidate morpholino oligomer. CAP A stands for Capping A. CAP B stands for capping B. "a M" stands for "a mol / L", where a is a number. min represents minutes. [Example]

[0164] The following examples illustrate the present invention in more detail, but are not intended to limit the scope of the present invention. When no temperature is specified in the operations of the examples, it means that the operations are carried out at room temperature.

[0165] Example 1 Synthesis of Compound (E) Synthetic Route [ka]

[0166] Step 1: Synthesis of compound (A) To a solution of compound (SM1) (35 g, 0.25 mol, 1 eq.) in DMF (350 mL), DIPEA (0.75 mol, 3 eq.) and TrCl (0.5 mol, 2 eq.) were added sequentially. The reaction mixture was stirred at 20-25 °C until TLC showed that compound (SM1) was completely consumed. Water (1400 mL, 40 vol.) and EtOAc (1400 mL, 40 vol.) were added to the reaction mixture. The organic layer was separated and washed with 20% aqueous NaCl to remove DMF. The resulting organic layer was concentrated and purified on a silica gel column to give 79 g of a white powder (compound (A)) in 91% yield. 1 H-NMR (CDCl3) δ: 7.56 (d, 6H), 7.30-7.28(m, 6H), 7.20-7.18(m, 6H), 3.71-3.68(m,3H),2.95(s, 2H), 2.04(s, 3H).

[0167] Step 2: Synthesis of compound (B) To a solution of triglycol (10 eq.) in NMP (500 mL, 10 vol.), 60% NaH (0.1 eq., dissolved in mineral oil) was added at 20-30°C, followed by stirring at 20-30°C for 20 min. Next, compound (A) (50 g, 1 eq.) was added to the reaction mixture at 20-30°C. The reaction mixture was stirred at 20-30°C until most of compound (A) was consumed. After the reaction, DCM / MTBE (3 / 7, 1000 mL, 20 vol.) and water (1000 mL, 20 vol.) were added to the reaction mixture. The organic layer was separated and washed with brine. The resulting organic layer was concentrated to obtain crude compound (B). The crude compound (B) was purified on a silica gel column to give 47 g of product in 70% yield. 1 H-NMR (CDCl3) δ: 7.56 (d, 6H), 7.30-7.28(m, 6H), 7.20-7.18(m, 6H), 4.36-4.34(m,2H),3.75-3.60(m, 8H), 3.60-3.59(m, 2H), 3.05(s, 2H), 2.15(s, 3H).

[0168] Step 3: Synthesis of compound (C) To a solution of compound (B) (47 g, 1 eq.) in THF (470 mL) was added succinic anhydride (2 eq.). The reaction mixture was stirred at 55 °C for 2.0 h until TLC showed that compound (B) was completely consumed. After the reaction, the pH of the reaction mixture was adjusted to approximately 8.5 with 10% aqueous NaHCO3. MTBE (940 mL, 20 vol.) was then added to the mixture, and the aqueous layer was separated. The pH of the resulting aqueous layer was adjusted to 3-5 with 20% citric acid solution, extracted with DCM (940 mL, 20 vol.), and washed with 10% aqueous Na2SO4 (470 mL, 10 vol.) to obtain an organic phase. After concentration, 56 g of a yellow oil was obtained in 98% yield, which was used in the next step without further purification. 1 H-NMR (CDCl3) δ: 7.46 (d, 6H), 7.17-7.21(m, 6H), 7.07-7.10(m, 6H), 4.20-4.24(m,2H),4.17-4.15(m, 2H), 3.65-3.50(m, 8H), 2.96(s, 2H), 2.54-2.52(m, 4H), 2.05(s,3H).

[0169] Step 4: Synthesis of compound (D) To a solution of compound (C) (56 g, 1 eq.) in DCM (560 mL) was added N-hydroxy-5-norbornene-2,3-dicarboxylic imide (HONB, 1.02 eq.), DMAP (0.33 eq.), DIPEA (2.5 eq.), followed by EDCI (1.1 eq.). The reaction mixture was stirred at 20-30 °C until TLC showed that compound (C) was completely consumed. The mixture was washed successively with 10% citric acid solution and brine. The organic layer was concentrated to dryness and carried on to the next step without further purification. 67 g of a foamy solid was obtained in 93% yield. 1 H-NMR (CDCl3) δ: 7.46 (d, 6H), 7.17-7.21(m, 6H), 7.07-7.10(m, 6H), 6.11(d,2H),4.20-4.24(m, 2H), 4.17-4.15(m, 2H), 3.65-3.50(m, 8H), 3.36(s, 2H), 3.23(s,2H),2.96(s, 2H), 2.79(t, 2H), 2.63(t, 2H), 2.05(s, 3H), 1.70(d, 1H), 1.44 (d,1H).

[0170] Step 5 Synthesis of compound (E) Aminomethylpolystyrene resin (10 g, loading 1 mmol / g) (purchased from Xi'an Lanxiao Technology Co., Ltd.) was suspended in NMP (200 mL) and allowed to swell for 1-2 hours. The resin suspension was filtered to remove the NMP and washed sequentially with DCM (200 mL) and 5% DIPEA in IPA / DCM (200 mL, v / v = 1:3). A solution of compound (D) (2.5 eq.) in NMP (10 vol.) was added to a suspension of aminomethylpolystyrene resin (10 g) in NMP (100 mL). The reaction mixture was stirred at 40-45 °C for 24-48 hours. The suspension was filtered and then washed with 100 mL of NMP and 100 mL of DCM. After transferring the wet solid to a reactor, a solution of NEM (0.4 M) in NMP (60 mL) and a solution of BzO (0.4 M) in NMP (60 mL) were added. The remaining amino groups in the resin were capped with BzO. After completion of the capping reaction, the resin was filtered and washed with DCM (100 mL). After drying, the sarcosinate-modified aminomethyl resin was obtained, which was used for the synthesis of phosphorodiamidate morpholino oligomers.

[0171] Determining the filling amount Typical steps: Resin loading (number of potentially available reactive sites) was determined spectrophotometrically by transferring a known weight of dried resin (25 + 3 mg) to a silanized 25 mL volumetric flask and adding approximately 5 mL of 2% (v / v) trifluoroacetic acid in dichloromethane. The contents were mixed by gentle swirling and allowed to stand for 30 minutes. Additional 2% (v / v) trifluoroacetic acid in dichloromethane was added to bring the volume up to 25 mL, and the contents were mixed thoroughly. Using a positive-displacement pipette, an aliquot (500 μL) of the trityl-containing solution was transferred to a 10 mL volumetric flask and the volume was brought up to 10 mL with methanesulfonic acid. The trityl cation content in the final solution was measured by UV absorbance at 406 nm, and the resin loading was calculated in terms of the amount of trityl groups per gram of resin (μmol / g) using Compound A as a reference. The analysis was performed in duplicate, and the average loading was calculated; the results are shown in Table 1. [Table 1]

[0172] Example 2 Synthesis of phosphorodiamidate morpholino oligomers The synthesis of phosphorodiamidate morpholino oligomers was achieved by assembling 25 cycles of phosphorodiamidate morpholino subunits from sarcosinate-modified aminomethyl resin. The synthetic route for phosphorodiamidate morpholino oligomer synthesis is as follows. [ka]

[0173] The synthesis of phosphorodiamidate morpholino oligomers was carried out manually by solid-phase synthesis from triglycol sarcosinate-modified supports using a peptide synthesizer for cycles of detritylation, neutralization, coupling, capping, and washing. All reactions were carried out in glass-jacketed columns with column volumes of 20 mL, 100 mL, 500 mL, and 2 L.

[0174] Prior to assembling the monomers on the solid support, all solutions were prepared as follows. Detritylation solution: 2% 4-cyanopyridinium trifluoroacetate (CYTFA) (w / v) in 20% trifluoroethanol / dichloromethane (1:4, v / v) containing 1% ethanol. Neutralizing solution: 5% diisopropylethylamine in 25% isopropanol / dichloromethane. Coupling solution: 0.36 M morpholino subunit solution in DMI and 0.8 M N-ethylmorpholine (NEM) in DMI. Meanwhile, the morpholino subunit solution in DMI was treated with molecular sieves for 12 hours or more to reduce the water content. Capping solution: 0.4M NEM in NMP as Capping A; 0.4M benzoic anhydride or acetic anhydride in NMP as Capping B. [Table 2]

[0175] To a jacketed column reactor, sarcosinate-modified aminomethyl polystyrene resin was added, followed by 15 vol. of 1-methyl-2-pyrrolidinone (NMP 15 mL / g resin), and the suspension was allowed to stand for 0.5-1 h. The NMP was then degassed, and the resin was washed five times with DCM and detritylated. Four reactions are carried out to assemble each phosphorodiamidate morpholino oligomer subunit on the support.

[0176] First, to remove the trityl groups on the support, 2% 4-cyanopyridinium trifluoroacetate (CYTFA) (w / v) solution (2,2,2-trifluoroethanol / DCM 1 / 4 and 1% EtOH) (15–25 vol.) was added to the column reactor. The mixture was bubbled with N2 for 2–5 min, and then vacuum degassed to remove the solvent. This procedure was repeated 5–9 times until IPC confirmed that all trityl groups had been removed (IPC: a sample was taken from the filtrate and diluted with methanesulfonic acid. To confirm complete removal of the trityl groups, UV absorption at 411 nm was tested using a UV spectrometer).

[0177] Second, after detritylation, 5% DIPEA in IPA / DCM (1 / 3) was added to the jacketed column to neutralize the resin. Before coupling, residual CYTFA must be thoroughly removed by multiple washes.

[0178] Third, coupling was carried out by adding the morpholino subunit solution and a solution of NEM in DMI to a reactor and bubbling N2 through the reaction for 90 min at 45° C. After assembly of the morpholino subunits on the support, the reaction mixture was degassed and washed with DCM.

[0179] Finally, unreacted morpholino subunits on the support were capped to terminate the elongation.

[0180] The reaction was repeated four times as shown in Table 3 below until the target sequence was completed. [Table 3] [ka]

[0181] For each coupling reaction, 250 mg of compound (E) with a loading of 608 μmol / g and 2.5 eq. of phosphorodiamidate morpholino subunits (PMO-NPEG monomer, PMO-PA monomer, PMO-PC monomer, and PMO-T monomer, the structures of which are shown in Example 2) were used to synthesize a single phosphorodiamidate morpholino oligomer. After 25 cycles of reaction, 2.95 g of wet base-protected phosphorodiamidate morpholino oligomer-modified solid support was obtained. The base-protected phosphorodiamidate morpholino oligomer-modified solid support was treated with 0.5 M DBU in NMP (20 mL) at 15 °C for 4 hours to remove the 4-nitrophenethyl group, and subsequently treated with concentrated ammonium hydroxide to cleave the base-protected phosphorodiamidate morpholino oligomer and remove other protecting groups. An impurity of 28.8% 4-nitrostyrene adduct (measured by LC-MS) was detected, and this material significantly reduced the yield of phosphorodiamidate morpholino oligomer synthesis.

[0182] To solve the problem of the 4-nitrostyrene adduct impurity, a three-step cleavage and deprotection strategy was developed. The base-protected phosphorodiamidate morpholino oligomer was slowly added (5 mL / min) to a solution of DBU and thymine in NMP (the DBU concentration was 0.8 M, and the thymine concentration was 1.0 M). Those skilled in the art will recognize that if the target phosphorodiamidate morpholino oligomer sequence contains thymine, the concentration of thymine will be much greater than the concentration of thymine in the target phosphorodiamidate morpholino oligomer sequence; for example, the molar ratio of thymine to thymine in the target phosphorodiamidate morpholino oligomer sequence will be greater than 10:1. After the three-step cleavage and deprotection, the 4-nitrostyrene adduct impurity was significantly reduced (less than 5% as determined by LC-MS).

[0183] Three steps of cleavage and deprotection After solid-phase assembly, a phosphorodiamidate morpholino oligomer-modified solid support with base protection bearing a 4-nitrophenethyl group on the guanine was obtained, followed by three steps of cleavage and deprotection as follows:

[0184] In step (1), the crude base-protected phosphorodiamidate morpholino oligomer was first cleaved using concentrated ammonium hydroxide (25% to 28% ammonium hydroxide) to obtain an aqueous solution, which was then lyophilized or concentrated to produce the crude base-protected phosphorodiamidate morpholino oligomer.

[0185] In step (2), the crude product from step (1) was redissolved in NMP (the volume-to-mass ratio of NMP to the crude product was 10 mL / g) and slowly added (5 mL / min) to a solution of DBU / thymine (1.0 M / 0.8 M) in NMP (10 volumes relative to the crude product) to remove the 4-nitrophenethyl group while minimizing the impurity of 4-nitrostyrene adduct.

[0186] In step (3), the oligonucleotide obtained after NPE deprotection was treated again with concentrated ammonia hydroxide (25%–28% ammonia hydroxide) to remove any remaining protecting groups, such as isobutyryl, to form the target phosphorodiamidate morpholino oligomer A (PMO-A), whose sequence was 5′-GTT GCC TCC GGT TCT GAA GGT GTT C-3′ (SEQ ID NO: 1). [ka] TIFF2025134950000046.tif94149

[0187] PMO-B, PMO-C, PMO-D and PMO-E were prepared using the same method as that for PMO-A. The nucleotide sequence of PMO-B was 5'-CTC CAA CAT CAA GGA AGA TGG CAT TTC TAG-3' (SEQ ID NO: 2). The base sequence of PMO-C was 5'-CTATATAGTTATTCAACA-3' (SEQ ID NO: 3). The nucleotide sequence of PMO-D was 5'-GGC CAA ACC TCG GCT TAC CTG AAA T -3' (SEQ ID NO: 4). The nucleotide sequence of PMO-E was 5'-CAG CAG CAG CAG CAG CAG-3' (SEQ ID NO: 5).

[0188] Several typical impurities were found during the synthesis of phosphorodiamidate morpholino oligomers, and the results are shown in Table 4. [Table 4]

[0189] FLP purity represents the LC-MS purity of the full-length product. NA, NC, NG, and NT represent the four impurities of N-1. OH represents an impurity due to hydrolysis of phosphoryl demethylamine. N+NPE represents the impurity which is a 4-nitrostyrene adduct. LC-MS: Waters H-Class UPLC, Xevo G2-XS-TOF detector. Crude refers to the unpurified PMO obtained after three steps of cleavage and deprotection. The purified product refers to PMO that has undergone three steps of cleavage and deprotection, followed by purification by ion exchange chromatography.

[0190] It will be understood that the foregoing description of two preferred embodiments is intended to be purely illustrative of the principles of the present invention and not exhaustive; modifications and variations will be apparent to those skilled in the art, and the invention is not intended to be limited except as expressly set forth in the following claims.

Claims

[Claim 1] (a) converting a compound of formula X-1 to a compound of formula X-2 【Chemical 1】 (In the formula: R １０ is a residue of a starting oligonucleotide (e.g., a phosphorodiamidate morpholino oligomer), R １１ is an amine protecting group, wherein the compound of formula X-1 is not bound to a solid support; (b) optionally removing a protecting group in said compound of formula X-2 to obtain an oligonucleotide; A method for preparing an oligonucleotide, comprising:

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