Methods for preparing oligonucleotides

Abstract

A method for preparing oligonucleotides is disclosed. The method includes: (a) converting a compound having formula X-1 into a compound having formula X-2: wherein R 10 These are residues of oligonucleotides (e.g., phosphorylated diamine morpholine oligomers); R 11 The protecting group is an amine; wherein the compound having formula X-1 is not bound to a solid support; and; (b) optionally, the protecting group in the compound having formula X-2 is removed to obtain the oligonucleotide. The synthetic method described herein is advantageous in many respects, including but not limited to improving the yield and purity of the target phosphoryldiamine morpholino oligomer while reducing 4-nitrostyrene adduct impurities.

Description

Technical Field

[0001] This disclosure generally relates to methods for preparing oligonucleotides, such as phosphoryldiamine morpholino oligomers (PMOs). Background Technology

[0002] Phosphoryl diamine morpholino oligomers (or PMOs) are nucleic acid analogs that bind tightly and sequence-specifically to complementary RNA and can be used to regulate protein synthesis and thus gene expression. These oligomers consist of a base-pairing recognition moiety (heterocyclic base) supported by a morpholino backbone system. The morpholino subunits used to synthesize such oligomers can be readily prepared from the corresponding ribonucleotides, which are readily available and inexpensive precursors.

[0003] During such synthesis, as in conventional oligonucleotide synthesis, functional groups on heterocyclic bases are typically masked to prevent interference with the synthetic transformation.

[0004] It is known that the unprotected guanine subunit of O6 causes side reactions during the oligomerization stage. For example, the O6 oxygen can react with the activated subunit during the coupling step to form O6 phosphorylated or derived substances, and during the final cleavage of the base protecting group with ammonia, ammonia can react at C6 to displace these substances, giving diaminopurine derivatives. Such impurities are difficult to remove by chromatography and lead to significant yield losses.

[0005] Various protection schemes have been proposed in this field to reduce side reactions at the unprotected guanine O6 position in conventional oligonucleotide synthesis. However, these schemes are largely unsuccessful when applied to the synthesis of phosphorylated diamine morpholino oligomers. Therefore, improved methods are needed to increase the yield and purity of phosphorylated diamine morpholino oligomer synthesis, particularly when using G-morpholino subunits.

[0006] Summarize

[0007] Due to the particular challenges of morpholinochemistry, the base protecting group must meet several requirements. The protecting group should be readily introduced onto the heterocyclic moiety and subsequently stable to subunit activation and purification conditions, as well as solid-phase synthesis. The protecting group should not react with the morpholinoamine moiety of the growing chain and should allow the activated morpholino subunit to couple cleanly with the growing oligomer chain. The protecting group should preferably be cleaved by ammonia without introducing new impurities. Finally, the protecting group should yield a crystalline subunit derivative to avoid the need for chromatographic purification prior to activation.

[0008] As described in US 2009 / 0131624 A1, the 4-nitrophenylethyl (NPE) group at the O6 position does not adequately meet these criteria. The NPE group is cleaved via a β-elimination mechanism using a basic reagent. These conditions tend to generate the reactive byproduct 4-nitrostyrene, which can then react with the reactive site on the oligomer. Although various scavengers (e.g., thiols and 1,3-dicarbonyl compounds) have been introduced into the deprotection mixture in an attempt to prevent the oligomer from capturing the byproduct, none have been entirely successful in eliminating this internal reflux problem. Even after purification, the oligomer prepared with this subunit is yellow. In various embodiments, this disclosure provides methods and processes that can be used to address one or more of these problems.

[0009] In one aspect, this disclosure provides a method for preparing oligonucleotides such as phosphoryldiamine morpholino oligomers (“PMO”).

[0010] In another aspect, this disclosure provides methods and compositions for preparing solid-phase supported phosphoryldiamine morpholino oligonucleotides.

[0011] The method for synthesizing PMO described herein is advantageous in many respects, including but not limited to improving the yield and purity of the target phosphoryldiamine morpholino oligomer while reducing 4-nitrostyrene adduct impurities.

[0012] These and other objects and features of this disclosure will become more apparent when reading the following detailed description of its implementation. Detailed Implementation

[0013] Exemplary Oligonucleotide Synthesis Method

[0014] This disclosure provides a method for preparing oligonucleotides, the method comprising:

[0015] (a) Converting a compound having formula X-1 into a compound having formula X-2:

[0016]

[0017] in:

[0018] R 10 These are residues of the starting oligonucleotide (e.g., phosphorylated diamine morpholine oligomer);

[0019] R 11 It is an amine protecting group;

[0020] Preferably, the compound having formula X-1 does not bind to the solid support; and

[0021] (b) Optionally, the protecting group in the compound having formula X-2 is removed to obtain the oligonucleotide.

[0022] Those skilled in the art should understand that R in equations X-1 and X-2 10 These can be the same or different, depending on whether the residues of the starting oligonucleotide (e.g., phosphorylated diamine morpholino oligomer) in Formula X-1 change under conditions requiring the removal of the NPE group. For example, in some embodiments, no other protecting groups are deprotected except for the NPE group shown in Formula X-1, then R in Formula X-1 and X-2... 10 The same can be used. In some embodiments, R in equation X-2 10 R can be expressed in expression X-1 10 The unprotected version.

[0023] There are no particular limitations on the oligonucleotides (and starting oligonucleotides). In some embodiments, the oligonucleotide is a phosphorylated diamine morpholino oligomer. Typically, the oligonucleotide contains a target base sequence for sequence-specific binding to the target nucleic acid. When hybridization occurs in an antiparallel configuration, the target sequence and the target sequence are described as “complementary” to each other. The target sequence may have “close” or “substantially” complementarity with the target sequence and still function for the purposes of the methods described herein, i.e., remain “complementary.” Preferably, the oligonucleotide analog compound used in the methods described herein has at most one mismatch / 10 nucleotides with the target sequence, and preferably at most one mismatch out of 20 nucleotides. Alternatively, the antisense oligomer used has at least 80%, at least 90%, or at least 95% sequence homology with the exemplary target sequence as specified herein. For the purpose of complementary binding to the RNA target and as discussed below, guanine bases may be complementary to cytosine or uracil RNA bases.

[0024] Various amine protecting groups can be used as R 11 Typically, R 11 The amine protecting group can be removed by treatment with NH3. For example, in some embodiments, R 11 It is an acyl group, i.e., R B -C(=O)-, where R B It can be, for example, hydrogen, alkyl, aryl, cycloalkyl, or heteroaryl, each of which is optionally substituted. In some embodiments, R 11 It is -C(=O)-R B , where R B C is an optional substitute 1-6 Alkyl, for example, C 1-6 Alkyl (e.g., isopropyl), aryl-substituted C 1-6 Alkyl (e.g., benzyl) or aryloxy-substituted C1-6 Alkyl group. In some particular embodiments, R 11 It can be -C(=O)-R B , where R B It 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 It is isopropyl.

[0025] In some preferred embodiments, the compound having formula X-1 is not bound to the solid support. It is not desirable to be bound by theory, but it is believed that the deprotection of the NPE group of formula X-1 can be controlled without binding to the solid support, such that the 4-nitrostyrene byproduct reacts preferentially with the scavenger in the reaction medium compared to the compound having formula X-2. However, in some embodiments, the compound having formula X-1 can also be bound to the solid support, and the reaction can be controlled, for example, by adding an excess of scavenger, such that the 4-nitrostyrene byproduct reacts preferentially with the scavenger in the reaction medium compared to the compound having formula X-2.

[0026] There are no particular limitations on the conditions under which a compound having formula X-1 is converted into a compound having formula X-2. However, in some preferred embodiments, the conversion involves adding (preferably in solution) a compound having formula X-1 to a mixture comprising a basic reagent (e.g., as described herein) and a scavenger capable of reacting with a compound having formula X-3 below:

[0027]

[0028] The basic reagent is typically a basic organic amine. For example, in some embodiments, the basic reagent is a basic organic amine having a pKa of about 9 or higher in water, such as about 9-15, about 10-14, about 12, about 13, or about 14. In some embodiments, the basic reagent is a basic cyclic amine. In some embodiments, the basic reagent is 1,8-diazabicyclo[5.4.0]undec-7-ene (“DBU”) or 1,5-diazabicyclo[4.3.0]non-5-ene (“DBN”). The basic reagent is typically added in excess, for example, the molar ratio of the basic reagent to one or more NPE groups of a compound having formula X-1 is typically greater than 1:1, such as 1.2:1, 1.5:1, 2:1, 5:1, or 10:1, or any range between the listed values, such as 1:1 to 10:1.

[0029] The scavenger is not particularly limited, as long as it can react with the byproduct having formula X-3. Typically, the scavenger has a -SH or 1,3-dicarbonyl moiety. In some embodiments, the scavenger can be a compound having formula X-4:

[0030]

[0031] in:

[0032] q is 0, 1, or 2; and

[0033] R A Each time it appears, it is an independently arbitrarily substituted C. 1-6 Alkyl (e.g., methyl).

[0034] In some embodiments, q is 0. In some embodiments, q is 1, and R A C is an optional substitute 1-6 Alkyl groups, such as methyl groups. R A It can be attached to any available site, provided that the compound having formula X-4 can act as a scavenger for the compound having formula X-3. In some particular embodiments, the scavenger is thymine or a derivative thereof.

[0035] Scavengers are typically used in excess. For example, the molar ratio of the scavenger to one or more NPE groups of a compound having 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 listed values, such as 1:1 to 10:1. In cases where the compound having formula X-1 has one or more bases that can react with 4-nitrostyrene, the amount of scavenger may be further increased, for example, to a molar ratio of 50:1 (or more) to one or more NPE groups of the compound having formula X-1.

[0036] Transforming Equation X-1 into Equation X-2 typically involves using one or more solvents. A variety of solvents are suitable. Non-limiting useful solvents include 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.

[0037] In some embodiments, the addition of the compound having formula X-1 to the mixture of the basic reagent and the scavenger can be controlled such that the amount of the oligonucleotide NPE adduct is minimized, for example, less than 10%, such as less than 5%.

[0038] In some embodiments, the method further includes treating a compound having formula X-2 with NH3 to partially or completely remove the protecting group in formula X-2. For example, in some embodiments, R 11 It is C 1-6 Alkyl-C(=O)-, after treatment with NH3, remove R. 11The group provides the deprotected base G.

[0039] This document describes an exemplary procedure for converting a compound having formula X-1 into formula X-2, from which those skilled in the art can readily adapt the embodiments disclosed herein.

[0040] Exemplary PMO Synthesis Method

[0041] In some specific embodiments, this disclosure provides a method for preparing PMO. In some embodiments, the method includes:

[0042] (a) Converting a compound having formula X-5 into a compound having formula X-6:

[0043]

[0044] in:

[0045] m1 and m2 are independent integers from 0 to 50 (e.g., 0 to 30);

[0046] R 11 It is an amine protecting group;

[0047] Each time a base appears, it is independently selected from G (guanine), C (cytosine), A (adenine), U (uracil), and T (thymine), their modified analogs, and their protected derivatives, provided that when the base in formula X-5 is a protected base, the corresponding base in formula X-6 may be the same protected base or the corresponding partially or completely unprotected base;

[0048] in:

[0049] T 1 It is a suitable 5' terminal group (e.g., a short peptide, an optionally substituted alkylamino group, an optionally substituted heterocyclic group, etc.); and

[0050] T 2 Suitable 3' terminal groups, such as hydrogen or protecting groups (e.g., acyl groups, triphenylmethyl groups, etc.); and

[0051] (b) Optionally, the protecting group in the compound having formula X-6 is partially or completely removed to obtain the oligonucleotide.

[0052] The oligonucleotide of formula X-5 or X-6 may 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, such as 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or any range between the listed values, such as 10-40, 15-30, etc.

[0053] The bases in formula X-5 or X-6 may be independently selected from G (guanine), C (cytosine), A (adenine), U (uracil), and T (thymine), their modified analogs, and their protected derivatives. As used herein, modified analogs include those non-standard bases such as 5-methylcytosine, inosine (I), and 7-deazo-G bases. In some embodiments, the bases in formula X-5 or X-6 may be independently selected from...

[0054]

[0055] In some embodiments, when the base in formula X-5 is a G monomer unit, it can be NPEG:

[0056] Where R 11 Defined herein. In some embodiments, all G monomer units in Formula X-5 may be NPEG.

[0057] Oligonucleotides (such as PMO) can have different ends at the 5' or 3' end. For the synthetic methods described herein, the precision of such ends is not critical and any of those known ends suitable for PMO can be included.

[0058] In some embodiments, T in formula X-5 or X-6 1 It can be an alkylamine with optional substitution, for example, -N(C 1-6 Alkyl)(C 1-6 Alkyl), wherein two C 1-6 The alkyl groups may be the same or different, and each of them may optionally be substituted with an amide, for example. In some embodiments, T in formula X-5 or X-6 1 It can be an alkylamine substituted with an amide (e.g., -C(O)NH2), for example,

[0059] In some embodiments, T in formula X-5 or X-6 1 It can be an optionally substituted heterocycle, such as an optionally substituted 4-7 membered heterocycle having one or two independently selected cyclic heteroatoms chosen from N, O, and S, such as an optionally substituted piperazine ring, for example... Where R CIt is an acyl group, acyloxy group, or peptide residue.

[0060] In some embodiments, T in formula X-5 or X-6 2 It could be hydrogen.

[0061] In some embodiments, T in formula X-5 or X-6 2 It can be a triphenylmethyl or methoxy-substituted triphenylmethyl group (e.g., MMT, DMT, etc.).

[0062] In some embodiments, T in formula X-5 or X-6 2 It can be an acyl group (e.g., an acetyl group).

[0063] The PMO of formula X-5 or X-6 can have any sequence, but it preferably contains a targeting base sequence for sequence-specific binding of the target nucleic acid.

[0064] Various amine protecting groups can be used as R in formula X-5 or X-6. 11 Typically, R 11 The amine protecting group can be removed by treatment with NH3. For example, in some embodiments, R 11 It is an acyl group, i.e., R B -C(=O)-, where R B It can be, for example, hydrogen, alkyl, aryl, cycloalkyl, or heteroaryl, each of which is optionally substituted. In some embodiments, R 11 It is -C(=O)-R B , where R B C is an optional substitute 1-6 Alkyl, for example, C 1-6 Alkyl (e.g., isopropyl), aryl-substituted C 1-6 Alkyl (e.g., benzyl) or aryloxy-substituted C 1-6 Alkyl group. In some particular embodiments, R 11 It can be -C(=O)-R B , where R B It 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 It is isopropyl.

[0065] In some preferred embodiments, the compound having formula X-5 is not bound to the solid support.

[0066] There are no particular limitations on the conditions under which a compound having formula X-5 is converted into a compound having formula X-6. However, in some preferred embodiments, the conversion involves adding (preferably in solution) a compound having formula X-5 to a mixture comprising a basic reagent (e.g., as described herein) and a scavenger capable of reacting with a compound having formula X-3 below:

[0067]

[0068] The basic reagent is typically a basic organic amine. For example, in some embodiments, the basic reagent is a basic organic amine having a pKa of about 9 or higher in water, such as about 9-15, about 10-14, about 12, about 13, or about 14. In some embodiments, the basic reagent is a basic cyclic amine. In some embodiments, the basic reagent is 1,8-diazabicyclo[5.4.0]undec-7-ene (“DBU”) or 1,5-diazabicyclo[4.3.0]non-5-ene (“DBN”). The basic reagent is typically added in excess, for example, the molar ratio of the basic reagent to one or more NPE groups of a compound having 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 listed values, such as 1:1 to 10:1.

[0069] The scavenger is not particularly limited, as long as it can react with the byproduct having formula X-3. Typically, the scavenger has a -SH or 1,3-dicarbonyl moiety. In some embodiments, the scavenger can be a compound having formula X-4:

[0070]

[0071] in:

[0072] q is 0, 1, or 2; and

[0073] R A Each time it appears, it is an independently arbitrarily substituted C. 1-6 Alkyl (e.g., methyl).

[0074] In some embodiments, q is 0. In some embodiments, q is 1, and R A C is an optional substitute 1-6 Alkyl groups, such as methyl groups. R A It can be attached to any available site, provided that the compound having formula X-4 can act as a scavenger for the compound having formula X-3. In some particular embodiments, the scavenger is thymine or a derivative thereof.

[0075] Scavengers are typically used in excess. For example, the molar ratio of the scavenger to one or more NPE groups of a compound having 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 listed values, such as 1:1 to 10:1. In cases where the compound having formula X-5 has one or more bases that can react with 4-nitrostyrene, the amount of scavenger may be further increased, for example, to a molar ratio of 50:1 (or more) to one or more NPE groups of the compound having formula X-5.

[0076] Transforming Formula X-5 into Formula X-6 typically involves the use of one or more solvents. A variety of solvents are suitable. Non-limiting useful solvents include 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.

[0077] In some embodiments, the addition of the compound having formula X-5 to the mixture of the basic reagent and the scavenger can be controlled such that the amount of the oligonucleotide NPE adduct is minimized, for example, less than 10%, such as less than 5%.

[0078] In some embodiments, the method further includes treating a compound having formula X-6 with NH3 to partially or completely remove the protecting group in formula X-6. For example, in some embodiments, R 11 It is C 1-6 Alkyl-C(=O)-, after treatment with NH3, remove R. 11 The group provides the deprotected base G.

[0079] In view of this disclosure, those skilled in the art can readily prepare compounds having formula X-5. For example, in some embodiments, compounds having formula X-5 can be prepared by a method comprising (e.g., using NH3) cleaving a solid support from an oligonucleotide having formula X-7:

[0080]

[0081] in

[0082] SS stands for solid support, such as polystyrene solid support.

[0083] L 1 It is a connector, such as a sarcosine-based connector, for example The nitrogen end is connected to the phosphorus atom, and the carbonyl end forms an amide bond with the solid support.

[0084] m1, m2 and R in equation X-7 11The corresponding group is the same as that in formula X-5.

[0085] Each time a base appears, it is independently selected from G (guanine), C (cytosine), A (adenine), U (uracil), and T (thymine), their analogues, and their protected derivatives, provided that when the 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 completely unprotected base; and

[0086] T 2 It is a suitable 3' terminal group, such as hydrogen or a protecting group (e.g., acyl group, triphenylmethyl).

[0087] In some embodiments, SS is a solid support having a -CH2-NH2 group, such as a polystyrene solid support, and may alternatively be referred to as SS-CH2-NH2.

[0088] In some embodiments, L 1 It is a sarcosine-based linker, for example The nitrogen end is connected to the phosphorus atom, and the carbonyl end forms an amide bond with the solid support.

[0089] In some embodiments, the bases in formula X-7 may be independently selected from...

[0090]

[0091] In some embodiments, T in equation X-7 2 It could be hydrogen.

[0092] In some embodiments, T in equation X-7 2 It can be a triphenylmethyl or methoxy-substituted triphenylmethyl group (e.g., MMT, DMT, etc.).

[0093] In some embodiments, T in equation X-7 2 It can be an acyl group (e.g., an acetyl group).

[0094] In view of this disclosure, those skilled in the art can prepare compounds having formula X-7. Exemplary procedures are also described in the Examples section of this document.

[0095] This document describes an exemplary procedure for converting a compound having formula X-5 into formula X-6, from which those skilled in the art can readily adapt embodiments of this disclosure.

[0096] It should be noted that compounds having formula X-5, X-6, or X-7 as defined herein are also novel compositions of this disclosure. Additionally, any oligonucleotide produced by the methods described herein is also a novel composition of this disclosure. In some embodiments, this disclosure further provides a pharmaceutical composition comprising an oligonucleotide produced by the methods described herein.

[0097] In another aspect, a method is provided for deprotecting the 4-nitrophenylethyl (NPE) group from a base-protected phosphoryldiamine morpholine oligomer, the method comprising treating the base-protected phosphoryldiamine morpholine oligomer in the presence of an alkaline reagent and a scavenging agent (such as those having a 1,3-dicarbonyl moiety). The PMO preferably contains a targeting base sequence for sequence-specific binding to a target nucleic acid.

[0098] In some embodiments disclosed herein, the method includes adding a base-protected phosphoryl diamine morpholine oligomer to a mixture of an alkaline reagent and a scavenging agent.

[0099] In some embodiments of this disclosure, the base-protected phosphoryl diamine morpholine oligomer is, for example, a compound having formula X-5 as described in this disclosure.

[0100] There are no particular limitations on the basic reagent used for deprotecting NPE groups. However, such basic reagents, for example, do not exhibit significant reactivity with the PMO skeleton. Basic reagents typically have pKa suitable for removing NPE groups from protected bases such as protected guanine. In this disclosure, the basic reagent can be an organic basic reagent, such as a basic organic amine having a pKa of about 9 or higher in water, such as about 9-15, about 10-14, about 12, about 13, or about 14. In some embodiments, the basic 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 mixtures thereof, for example, DBU.

[0101] The scavenging agents used in the methods herein include any of those known in the art that can react with 4-nitrostyrene generated by the deprotection of one or more NPE groups. In some embodiments, the scavenging agent may have a -SH group or a 1,3-dicarbonyl moiety, for example, as described herein (e.g., compounds having formula X-4 as described in this disclosure). In some preferred embodiments of this disclosure, the scavenging agent may be a 1,3-dicarbonyl compound, such as thymine, diethyl malonate, or mixtures thereof, for example, thymine.

[0102] In one embodiment, the molar ratio of the basic reagent to one or more NPE groups in the base-protected phosphoryl diamine morpholine oligomer can be greater than 1:1, such as 1:1-10:1, 1.2-10:1, or 1.5:1-10:1. In one embodiment, the amount of the basic reagent is in excess relative to one or more NPE groups.

[0103] In one embodiment, the scavenger is typically used in excess. For example, the molar ratio of the scavenger to one or more NPE groups in the base-protected phosphoryldiamine morpholine 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 listed values, such as 1:1 to 10:1. In cases where the base-protected phosphoryldiamine morpholine oligomer has one or more bases that can react with 4-nitrostyrene, the amount of scavenger may be further increased, for example, to a molar ratio of 50:1 (or more) to one or more NPE groups of the base-protected phosphoryldiamine morpholine oligomer.

[0104] In this disclosure, the method for deprotecting NPE groups can be carried out in the presence of a solvent. The solvent can be any solvent used in the art for deprotecting NPE groups. In this disclosure, the solvent can be selected from polar aprotic solvents, such as DMF, DMA, DMI, NMP, etc.

[0105] In some embodiments disclosed herein, a method for deprotecting NPE groups from base-protected phosphoryldiamine morpholine oligomers includes adding a mixture of base-protected phosphoryldiamine morpholine oligomers in a first solvent to a mixture of an alkaline reagent and a scavenging agent 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 phosphoryldiamine morpholine oligomers in the first solvent, the concentration of the base-protected phosphoryldiamine morpholine oligomers may be from 0.001M to 0.1M, such as 0.01M to 0.02M. In the mixture of the alkaline reagent and the scavenging agent in the second solvent, the concentration of the alkaline reagent may be from 0.1M to 5M, such as 0.5M to 1.0M; and the concentration of the scavenging agent may be from 0.1M to 5M, such as 0.5M-1.5M, for example 0.8M.

[0106] In some embodiments disclosed herein, a method for deprotecting NPE groups from base-protected phosphoryldiamine morpholine oligomers includes adding a mixture of base-protected phosphoryldiamine morpholine oligomers in a first solvent to a mixture of an alkaline reagent and a scavenging agent 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 phosphoryldiamine morpholine oligomers in the first solvent, the concentration of the base-protected phosphoryldiamine morpholine oligomers may be from 0.001M to 0.1M, such as 0.01M to 0.02M. In the mixture of the alkaline reagent and the scavenging agent in the second solvent, the concentration of the alkaline reagent may be from 0.1M to 5M, such as 0.5M to 1.0M; and the concentration of the scavenging agent may be from 0.1M to 5M, such as 0.5M-1.5M, for example 0.8M. In one embodiment, the addition rate can be 1-10 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.

[0107] In some embodiments, this disclosure provides a method for preparing phosphorylated diamine morpholine oligomers, the method comprising the method described above for deprotecting one or more NPE groups from base-protected phosphorylated diamine morpholine oligomers.

[0108] As described herein, the synthesis of the oligomers is typically carried out on a supporting medium. Generally, a first synthon (e.g., a monomer, such as a morpholino subunit) is first attached to the supporting medium, and then the oligomer is synthesized by sequentially coupling the subunits to the synthon bound to the support. This iterative extension ultimately forms the final oligomeric compound. Suitable supporting media can be soluble or insoluble, or can have variable solubility in different solvents to allow the polymer bound to the growing support to be in or out of solution as needed. Conventional supporting media are largely insoluble and are typically placed in a reaction vessel while reagents and solvents react with and / or wash the growth chain until the oligomer reaches the target length, after which it is cleaved from the support: and, if necessary, further processed to yield the final polymeric compound. Recent methods have introduced soluble supports, including soluble polymer supports, to allow precipitation and dissolution of the iteratively synthesized product at desired points in the synthesis.

[0109] In some embodiments, the morpholino group is conjugated to a "tail" portion at the 5' or 3' end of the oligomer to increase its stability and / or solubility. Exemplary tails include short peptides, optionally substituted alkylamino groups, optionally substituted heterocyclic groups, acyl groups, triphenylmethyl groups, etc. In one embodiment, a suitable 5' terminal group is, for example, a short peptide, optionally substituted alkylamino groups, optionally substituted heterocyclic groups, etc. (e.g., -C(O)NH2-substituted alkylamines, e.g., ...). In another embodiment, a suitable 3' terminal group is, for example, hydrogen or a protecting group (e.g., an acyl group, triphenylmethyl, etc.).

[0110] In some embodiments, this disclosure provides a method for preparing phosphorylated diamine morpholine oligomers, the method comprising:

[0111] (a) Reaction of a solid-supported morpholino subunit with a base-protected morpholino subunit monomer, the solid-supported morpholino subunit having an unprotected cyclic nitrogen, and the base-protected morpholino subunit monomer having a protected cyclic nitrogen and an activated phosphoramide group on the 5'-external carbon.

[0112] This forms a phosphoryl diamine bond between the 5'-external carbon and the unprotected nitrogen;

[0113] (b) to deprotect the protected nitrogen to form unprotected nitrogen;

[0114] (c) Repeating steps (a) and (b) once or more with a separately protected morpholino subunit monomer to obtain a base-protected morpholino-modified solid support; and

[0115] (d) Performing three steps of cleavage and deprotection to obtain the phosphoryl diamine morpholine oligomer; the three steps of cleavage and deprotection include the method described above for deprotecting the NPE group from the base-protected phosphoryl diamine morpholine oligomer;

[0116] At least one of the base-protected morpholino derivative monomers is a protected guanine morpholino compound with structure (M):

[0117]

[0118] R 1 It is a chlorophosphoramide group;

[0119] R 2 It is a lower alkyl group, a monocyclic arylmethyl group, or a monocyclic (aryloxy)methyl group;

[0120] R 3 It is a triarylmethyl group.

[0121] The triarylmethyl protecting group (R) used for morpholine cyclic nitrogen.3 Examples of triphenylmethyl (triphenylmethyl), 4-methyltriphenylmethyl, 4,4'-dimethyltriphenylmethyl, 4,4',4”-trimethyltriphenylmethyl, monomethoxytriphenylmethyl (e.g., 4-methoxytriphenylmethyl) or dimethoxytriphenylmethyl (e.g., 4,4'-dimethoxytriphenylmethyl).

[0122] R 1 It can be -OP(=O)-N(CH3)2Cl.

[0123] R 2 It can be benzyl or -CH(CH3)2.

[0124] In one aspect, this article provides a method for preparing a compound having formula (II), the method comprising contacting compound (E) with a deblocking agent to obtain a compound having formula (II);

[0125]

[0126] Where SS is the supporting medium, and Z1 is... The oxygen terminus is connected to a sarcosine unit, where m is 1, 2, 3, 4, or 5, and R... 4 It is Tr (triphenylmethyl) or a derivative thereof, such as Tr (triphenylmethyl), MMTr (p-methoxyphenyl diphenylmethyl), or DMTr (di-(p-methoxyphenyl)phenylmethyl). In a preferred embodiment, m is 3, and R 4 It is Tr (triphenylmethyl).

[0127] In one embodiment, the method for preparing a compound having formula (II) further includes the step of contacting the deblocked compound with a neutralizing agent.

[0128] On the other hand, this article provides a method for preparing a compound having formula (III), the method comprising coupling a compound having formula (II) with a compound having formula (G) to obtain a compound having formula (III).

[0129]

[0130] The bases are selected from any protected nucleic acid bases, such as PC, T, PA, P5mC, U, I, PG, DPG, or NPEG; G (guanine), C (cytosine), A (adenine), U (uracil), and T (thymine), their modified analogs, and their protected derivatives, such as any protected nucleic acid bases, such as PC, T, PA, P5mC, U, I, PG, DPG, or NPEG;

[0131]

[0132] SS, Z1, R 1 R 2 and R 3 The definition is as described in this disclosure.

[0133] In one embodiment, the method for preparing a compound having formula (III) further includes contacting a compound having formula (II) with a capping agent.

[0134] In another embodiment, the compound having formula (II) is obtained by the method as defined above.

[0135] On the other hand, this article provides a method for preparing compounds having formula (IV), the method comprising the following sequential steps:

[0136] (i) Couple a compound having formula (II) with a compound having formula (G) to obtain a compound having formula (III);

[0137] (ii) Perform n-1 iterations of the following sequential steps:

[0138] (ii-1) Contact the product obtained in the immediately preceding step with the deblocking agent; and

[0139] (ii-2) Couple the compound obtained by the preceding step with the compound having formula (G) to form the compound having formula (IV);

[0140]

[0141] At least one base may be NPEG;

[0142]

[0143] Among them, SS, Z1, R 1 R 2 and R 3 The definition is as described in this disclosure; n is an integer from 10 to 40, for example, n is an integer from 20 to 30, such as 25;

[0144] Each time a base appears, it is independently selected from optional protected nucleic acid bases, such as PC, T, PA, P5mC, U, I, PG, DPG, or NPEG; G (guanine), C (cytosine), A (adenine), U (uracil), and T (thymine), their modified analogs, and their protected derivatives, such as optional protected nucleic acid bases, such as PC, T, PA, P5mC, U, I, PG, DPG, or NPEG.

[0145] In one embodiment, step (ii-1) further includes contacting the deblocking compound with a neutralizing agent.

[0146] In another embodiment, step (ii-2) further includes contacting the compound obtained by the immediately preceding step with the capping agent.

[0147] In another aspect, this article provides a method for preparing a compound having formula (V), the method comprising contacting a compound having formula (IV) with a deblocking agent to obtain a compound having formula (V);

[0148]

[0149] Among them, SS, Z1, base, R 3 The definitions of n are as described in this disclosure.

[0150] In one embodiment, the method for preparing a compound having formula (V) further includes the step of contacting the deblocked compound with a neutralizing agent.

[0151] In another aspect, a method for preparing a compound having formula (VI) is provided, the method comprising contacting a compound having formula (V) with a cleaving agent to obtain a compound having formula (VI).

[0152]

[0153] Among them, SS, Z1, base, R 3 The definitions of n are as described in this disclosure.

[0154] On the other hand, this article provides a method for preparing phosphoryldiamine morpholine oligomers, the method comprising:

[0155] (a) Contacting a compound having formula (VI) with a deprotecting agent; and

[0156] (b) Optionally, the compound obtained by the immediately preceding step is subjected to ammonolysis to obtain a phosphoryl diamine morpholine oligomer;

[0157]

[0158] The definitions of bases and n are as described in this disclosure.

[0159] In one embodiment, each method (step) is performed in the presence of at least one solvent.

[0160] In another embodiment, the neutralizing agent is in a solution containing dichloromethane and isopropanol.

[0161] In yet another embodiment, the neutralizing agent is a monoalkyl, dialkyl, or trialkylamine.

[0162] In another embodiment, the neutralizing agent is N,N-diisopropylethylamine.

[0163] In a preferred embodiment, the neutralizing agent used in each method (step) is 5% diisopropylethylamine in 25% isopropanol / dichloromethane.

[0164] In another embodiment, the compound having formula (G) is in a solution containing ethylmorpholine and dimethylimidazolinone.

[0165] In another embodiment, the compound having formula (G) is selected from PMO-NPEG monomers, PMO-PA monomers, PMO-PC monomers, and PMO-T monomers:

[0166]

[0167] In some embodiments, the methods disclosed herein can be performed in continuous flow or discontinuous flow modes known in the art. In some embodiments, the methods disclosed herein can be performed in a custom-designed peptide batch reactor.

[0168] In another aspect, a method for preparing compound (E) is provided, the method comprising contacting compound (D) with compound (S) to obtain compound (E);

[0169]

[0170] Among them, SS, Z1 and R 4 The definition is as defined in this disclosure.

[0171] In a method for preparing compound (E), compound (S) may need to be activated before use, wherein the activation of compound (S) may include the following steps: suspending compound (S) in a solvent and swelling it, then removing the solvent and washing it in turn with a mixture of a chlorinated hydrocarbon solvent and a base in the solvent.

[0172] In the activation of compound (S), the solvent used to suspend compound (S) can be selected from polar aprotic solvents, such as alkylketone solvents, for example, NMP. The amount of solvent is not particularly limited. The volume-to-mass ratio of solvent to compound (S) can be 10 mL / g to 30 mL / g. The solvent removal operation can be filtration. The chlorinated hydrocarbon solvent can be DCM. The base in the mixture can be an organic base, such as 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). In the mixture, the mass percentage of the base can be 1% to 10%, for example, 5%, where % represents the percentage of the base's mass in the total mass of the mixture.

[0173] The method for preparing compound (E) can be carried out in a solvent. The solvent can be selected from polar aprotic solvents, such as alkyl ketone solvents, amide solvents, or mixtures thereof, for example, NMP, DMI, DMF, or mixtures thereof. There is no particular limitation on the amount of solvent. The volume-mass ratio of solvent to compound (S) can be 10 mL / g to 30 mL / g.

[0174] In the method for preparing compound (E), the molar ratio of compound (D) to compound (S) can be 1:1 to 1:3.

[0175] In the method for preparing compound (E), the reaction temperature can be between 20°C and 50°C, for example, 40°C to 45°C. The progress of the reaction can be monitored using conventional detection methods in the art (such as TLC, HPLC, GC, or NMR). The disappearance of compound (D) is generally considered the completion of the reaction. The reaction time can be 24–48 hours.

[0176] In a preferred embodiment, the method for preparing compound (E) preferably includes adding a solution of compound (D) in a solvent to a suspension of compound (S) in a solvent to carry out a reaction.

[0177] In the method for preparing compound (E), the post-treatment can be a conventional post-treatment used in the art for such reactions. In this disclosure, the post-treatment preferably includes filtering the resulting mixture, then washing the filter cake with a solvent (such as an alkane solvent, a chlorinated hydrocarbon solvent, or a combination thereof; the alkane solvent may be NMP; the chlorinated hydrocarbon solvent may be DCM; when the solvent is a combination of an alkane solvent and a chlorinated hydrocarbon solvent, the volume ratio may be 1:1 to 1:10), followed by sequentially adding NEM (0.2-1g) to a solvent (such as an alkane solvent, for example, NMP; the volume-mass ratio of solvent to compound (S) may be 50 mL / g to 200 mL / g). The compound (S) is prepared by mixing a solution of B2O (0.2-1.0 M) in a solvent (such as an alkane solvent, e.g., NMP; the volume-mass ratio of solvent to compound (S) can be 50 mL / g-200 mL / g) and performing an end-capping reaction. After the end-capping reaction is completed, the resulting mixture is filtered and washed with a solvent (such as a chlorinated hydrocarbon solvent, e.g., DCM; the volume-mass ratio of solvent to compound (S) can be 100 mL / g-300 mL / g), and then dried to obtain compound (E).

[0178] In the preferred embodiment disclosed herein, compound (S) is an aminomethyl polystyrene resin available from Xi'an Lanxiao Technology Co., Ltd.

[0179] In a preferred embodiment of this disclosure, the method for preparing compound (E) may further include a method for preparing compound (D), which includes 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);

[0180]

[0181] Among them, Z1 and R 4 The definition is as defined in this disclosure.

[0182] In the method for preparing compound (D), the catalyst is a conventional catalyst used in the art for such reactions. In this disclosure, the catalyst may be DMAP. The molar ratio of the catalyst to compound (C) may be 0.01:1 to 0.5:1, such as 0.33:1.

[0183] In the method for preparing compound (D), the base is a conventional base used in the art for such reactions. In this disclosure, the base may be an organic base, such as an organic amine, for example, DIPEA. The amount of base is a conventional amount used in the art for such reactions. In this disclosure, the molar ratio of base to compound (C) may be 1:-3:1, such as 2.5:1.

[0184] In the method for preparing compound (D), the condensing agent is a conventional condensing agent used in the art for such reactions. In this disclosure, the condensing agent may be EDCI, DCC, DIC, or a mixture thereof. The amount of condensing agent is a conventional amount used in the art for such reactions. In this disclosure, the molar ratio of the condensing agent to compound (C) may be 1:1 to 2:1, such as 1.1:1.

[0185] In the method for preparing compound (D), the molar ratio of compound (C) to compound (SM4) can be 1:1 to 1:2, such as 1:1.02.

[0186] In the method for preparing compound (D), the solvent is a conventional solvent used in the art for such reactions. In this disclosure, the solvent may be a chlorinated hydrocarbon solvent, such as DCM. The volume-to-mass ratio of the solvent to compound (C) may be 10 mL / g to 20 mL / g, such as 10 mL / g.

[0187] In the method used to prepare compound (D), the reaction temperature can be between 20°C and 30°C. The progress of the reaction can be monitored using conventional detection methods in the art, such as TLC, HPLC, GC, or NMR. The disappearance of compound (C) is generally considered as the completion of the reaction.

[0188] In a preferred embodiment, the method for preparing compound (D) includes 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.

[0189] In the method for preparing compound (D), the post-treatment can be a conventional post-treatment used in the art for such reactions. In this disclosure, the post-treatment preferably includes washing the reaction mixture sequentially with citric acid (e.g., a 10% citric acid solution) and brine, and concentrating the organic layer to dryness to obtain compound (D).

[0190] In a preferred embodiment of this disclosure, the method for preparing compound (E) may further include a method for preparing compound (C), which includes contacting compound (B) with compound (SM3) in a solvent to obtain compound (C);

[0191]

[0192] Where m is an integer from 1 to 5, R 4 The definition is as defined in this disclosure.

[0193] In the method for preparing compound (C), the molar ratio of compound (B) to compound (SM3) can be 1:1 to 1:2, such as 1:2.

[0194] In the method for preparing compound (C), the solvent can be a conventional solvent used in the art for such reactions. In this disclosure, the solvent can be an ether solvent, such as THF. The volume-to-mass ratio of the solvent to compound (B) can be 10-20 mL / g, such as 10 mL / g.

[0195] In the method used to prepare compound (C), the reaction temperature can be between 20°C and 55°C. The progress of the reaction can be monitored using conventional detection methods in the art, such as TLC, HPLC, GC, or NMR. The disappearance of compound (B) is generally considered the completion of the reaction.

[0196] In a preferred embodiment, the method for preparing compound (C) preferably includes adding compound (SM3) to a solution of compound (B) in a solvent to carry out a reaction.

[0197] In the method for preparing compound (C), the post-treatment can be a conventional post-treatment used in the art for such reactions. In this disclosure, the post-treatment includes adjusting the pH of the reaction mixture to about 8.5 with an aqueous solution of NaHCO3 (e.g., 10% aqueous solution of NaHCO3), then 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), then extracting with a chlorinated hydrocarbon solvent (e.g., DCM) and washing with an aqueous solution of Na2SO4 (e.g., 10% aqueous solution of Na2SO4), and concentrating the resulting organic layer to obtain compound (C).

[0198] In a preferred embodiment of this disclosure, the method for preparing compound (E) may further include a method for preparing compound (B), which includes contacting compound (A) with compound (SM2) in a solvent in the presence of a base to obtain compound (B);

[0199]

[0200] Where m is an integer from 1 to 5, R 4 The definition is as defined in this disclosure.

[0201] In the method for preparing compound (B), the base is a conventional base used in the art for such reactions. In this 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, such as 0.01:1.

[0202] In the method for preparing compound (B), the molar ratio of compound (A) to compound (SM2) can be 1:5 to 1:20, such as 1:10.

[0203] In the method for preparing compound (B), the solvent can be a conventional solvent used in the art for such reactions. In this disclosure, the solvent can be an alkyl-ketone solvent; for example, NMP. The amount of solvent is not particularly limited. The volume-to-mass ratio of solvent to compound (A) can be 15 mL / g to 25 mL / g, such as 20 mL / g.

[0204] In the method used to prepare compound (B), the reaction temperature can be between 20°C and 30°C. The progress of the reaction can be monitored using conventional detection methods in the art, such as TLC, HPLC, GC, or NMR. The disappearance of compound (A) is generally considered the completion of the reaction.

[0205] In a preferred embodiment, the method for preparing compound (B) preferably includes adding a base to a solution of compound (SM2) in a solvent under stirring, and then adding compound (A) to carry out the reaction. More preferably, it includes adding a base to a solution of compound (SM2) in a solvent at 20°C-30°C, then stirring the resulting mixture at 20°C-30°C for 10-30 minutes, and then adding compound (A) to carry out the reaction.

[0206] In the method for preparing compound (B), the post-treatment can be a conventional post-treatment used in the art for such reactions. In this disclosure, the post-treatment includes adding water and an organic solvent for extraction (e.g., a chlorinated hydrocarbon solvent, an ether solvent, or a mixture thereof, preferably a mixture of DCM and MTBE) to the reaction mixture, washing the obtained organic layer with brine, and concentrating and purifying (e.g., by silica gel column chromatography) the resulting organic layer to obtain compound (B).

[0207] In a preferred embodiment of this disclosure, the method for preparing compound (E) may further include a method for preparing compound (A), which includes reacting compound (SM1) with R in a solvent in the presence of a base. 4 Cl contact is used to obtain compound (A);

[0208]

[0209] Where R 4 As defined in this disclosure.

[0210] In the method for preparing compound (A), the base is a conventional base used in the art for such reactions. In this disclosure, the base may be an organic base, such as an organic amine, for example, DIPEA. The amount of base is a conventional amount used in the art for such reactions. In this disclosure, the base reacts with R... 4 The molar ratio of Cl can be 1:1 to 3:1, such as 1.5:1.

[0211] In the method for preparing compound (A), compound (SM1) and R 4 The molar ratio of Cl can be 1:1 to 1:3, such as 1:2.

[0212] In the method for preparing compound (A), the solvent is a conventional solvent used in the art for such reactions. In this disclosure, the solvent may be selected from amide solvents, such as amide solvents, alkane-ketone solvents, chlorinated hydrocarbon solvents, or mixtures thereof, for example, DMF, DCM, NMP, or mixtures thereof. The amount of solvent is not particularly limited. In this disclosure, the volume-to-mass ratio of solvent to compound (SM1) may be 10 mL / g to 30 mL / g, such as 10 mL / g.

[0213] In the method used to prepare compound (A), the reaction temperature can be 20°C–25°C. The progress of the reaction can be monitored using conventional detection methods in the art, such as TLC, HPLC, GC, or NMR. The disappearance of compound (SM1) is generally considered the completion of the reaction.

[0214] In a preferred embodiment, the method for preparing compound (A) preferably includes reacting a base with R. 4 Cl is added sequentially to the solution of compound (SM1) in the solvent to carry out the reaction.

[0215] In the method for preparing compound (A), the post-treatment can be a conventional post-treatment used in the art for such reactions. In this disclosure, the post-treatment includes adding water and an organic solvent for extraction (e.g., an ester solvent, preferably EtOAc) to the reaction mixture, washing the obtained organic layer with an aqueous NaCl solution (e.g., a 20% aqueous NaCl solution), and concentrating and purifying (e.g., by silica gel column chromatography) the obtained organic layer to obtain compound (A).

[0216] On another front, compound (E) is provided:

[0217]

[0218] Where Z1 is The oxygen terminus is connected to a sarcosine unit, where m is 1, 2, 3, 4, or 5, and SS and R 4 The definition is as defined in this disclosure.

[0219] In another aspect, the use of the compound (E) as described in this disclosure in the preparation of oligonucleotides, such as phosphoryldiamine morpholino oligomers (PMO) is provided.

[0220] As described in this paper, it was found that guanine monomer units protected by NPE can be used to prepare oligonucleotides in high yields and with high purity. It was also found that deprotection of NPE groups from oligonucleotides bound to non-solid supports can be carried out efficiently with minimal NPE adducts (impurities).

[0221] definition

[0222] 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 pertains.

[0223] It should be understood that wherever embodiments are described herein with the language “comprising,” other similar embodiments described with the terms “containing,” “consisting of,” and / or “substantially consisting of” are also provided. However, when used as transitional phrases in the claims, each phrase should be interpreted separately within the appropriate legal and factual context (e.g., in the claims, the transitional phrase “comprising” is more often considered an open-ended phrase, while “consisting of” is more exclusive, and “substantially consisting of” takes a middle ground).

[0224] As used in this article, the singular forms “a” and “the” include plural referents unless explicitly stated or unambiguously indicated from the context that it is not intended to be so.

[0225] Headings and subheadings are used for convenience and / or formal compliance only, do not limit the subject matter, and are not related to the interpretation of the description of the subject matter. In various embodiments, features described under one heading or subheading of the subject matter disclosure may be combined with features described under other headings or subheadings. Furthermore, not all features under a single heading or subheading are necessarily used together in embodiments.

[0226] When listing a range of values, it is intended to cover every value within that range and its subranges. For example, "C 1–6 "Aims to cover 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 C 2–3 C 3–6 C 3–5 C 3–4 C 4–6 C 4–5 and C5–6 .

[0227] "Morpholine oligomers" refer to polymeric molecules having a backbone of supporting bases capable of hydrogen bonding with typical polynucleotides. These polymers lack the pentose sugar backbone and, more specifically, the ribose backbone typically linked by phosphodiester bonds found in nucleotides and nucleosides, but contain a cyclic nitrogen ring through which coupling occurs. Preferred morpholine oligomers are constructed from "morpholine subunit" structures as shown below, in which these structures are preferably linked together by (thio)phosphoryldiamine bonds, attaching the morpholine nitrogen of one subunit to the 5' exocyclic carbon of the adjacent subunit. Each subunit includes a purine or pyrimidine base pairing moiety, which binds efficiently to the bases in the polynucleotide via base-specific hydrogen bonding.

[0228]

[0229] The "phosphoryldiamine" group comprises phosphorus having two attached oxygen atoms and two attached nitrogen atoms, and may also refer herein to phosphorus having one attached oxygen atom and three attached nitrogen atoms. In the inter-subunit bonds of the oligomers described herein, one nitrogen atom is typically side-attached to the backbone chain, and the second nitrogen atom is a cyclic nitrogen in a morpholine ring structure, as shown in formula (a1) below. Alternatively or additionally, nitrogen may be present at the 5'-external carbon, as shown in formulas (b1) and (c1) below.

[0230]

[0231] Base 1 and base 2 can be the same or different, and their definitions are the same as those of bases as described in this disclosure.

[0232] In the thiophosphoric diamine bond, an oxygen atom (typically the oxygen side-attached to the backbone in the oligomers described herein) is replaced by sulfur.

[0233] In a preferred embodiment, the phosphoryldiamine morpholine oligomer refers to a phosphoryldiamine morpholine oligomer having the following general structure:

[0234]

[0235] "Solid-supported morpholino subunit" can be the first or any subsequent morpholino subunit monomer incorporated into a morpholino oligomer via solid-phase stepwise synthesis as described herein. The subunit is attached to a solid support or an oligomer chain grown on a solid support via its 5' exocyclic carbon. "Base-protected" means that the base-pairing groups (e.g., purine or pyrimidine bases) on the morpholino subunit are protected with protecting groups suitable for preventing reaction or interference of the base-pairing groups during stepwise oligomer synthesis.

[0236] "Activated phosphoramide groups" are typically chlorophosphoramide groups, with the desired substitution at the nitrogen in the final phosphoramide bond of the oligomer. An example is (dimethylamino)chlorophosphoramide, i.e., -OP(=O)(NMe2)Cl.

[0237] "Base-protected" or "base-protected" refers to the protection of the base-pairing groups (e.g., purine or pyrimidine bases) on the morpholino subunit with protecting groups suitable for preventing reaction or interference of the base-pairing groups during stepwise oligomer synthesis. In a preferred embodiment, at least one base-protected morpholino subunit monomer is derived from a protected guanine morpholino compound having the structure (M):

[0238]

[0239] R 1 R 2 and R 3 The definition is as described in this disclosure.

[0240] There are no particular limitations on the term "nucleic acid base," as long as it can be used for nucleic acid synthesis, and it includes, for example, pyrimidine bases (such as cytosyl, uracil, and thymine groups) and purine bases (such as adenine and guanine groups). "Optionally protected nucleic acid bases" means, for example, that the amino group in the adenine, guanine, or cytosine group of a nucleic acid base containing an amino group can be protected, and preferably that the amino group in the nucleic acid base is protected by a protecting group that is sustainable under the deprotection condition of the nitrogen atom of the morpholine ring in the morpholine nucleotide. There are no particular restrictions on the term "amino protecting group". Specific examples of "amino protecting groups" include neopentanoyl group, neopentanoyloxymethyl group, trifluoroacetyl group, phenoxyacetyl group, 4-isopropylphenoxyacetyl group, 4-tert-butylphenoxyacetyl group, acetyl group, benzoyl group, isobutyryl group, dimethylformamidinyl group, 9-fluorenylmethyloxycarbonyl group, etc. Furthermore, the carbonyl group of nucleic acid bases is optionally protected and can 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-neopentyloxybenzyl alcohol, 4-nitrophenylethyl alcohol, 2-(methylsulfonyl)ethanol, 2-(phenylsulfonyl)ethanol, 2-cyanoethanol, 2-(trimethylsilyl)ethanol, dimethylcarbamoyl chloride, diethylcarbamoyl chloride, ethylphenylcarbamoyl chloride, 1-pyrrolidine carbonyl chloride, 4-morpholine carbonyl chloride, dephenylcarbamoyl chloride, etc. In some cases, it is not necessary to specifically introduce a carbonyl protecting group. In addition to the groups mentioned above, "nucleic acid base" also includes modified nucleic acid bases (e.g., 8-bromoadenine group, 8-bromoguanine group, 5-bromocytosine group, 5-iodocytosine group, 5-bromouracil group, 5-iodouracil group, 5-fluorouracil group, hypoxanthine group, etc.), which are nucleic acid bases that are substituted at any one or more positions by any one to three substituents (e.g., halogen atom, alkyl group, aralkyl group, alkoxy group, acyl group, alkoxyalkyl group, hydroxy group, amino group, monoalkylamino, dialkylamino, carboxyl, cyano, nitro, etc.).

[0241] "Lower alkyl" refers to an alkyl radical with one to six carbon atoms, such as methyl, ethyl, n-butyl, isobutyl, tert-butyl, isopentyl, n-pentyl, and isopentyl. In selected embodiments, the "lower alkyl" group has one to four carbon atoms or 1-2 carbon atoms; i.e., methyl or ethyl.

[0242] The term "support-bonded" refers to a chemical entity covalently bonded to a support medium.

[0243] The term "support medium" refers to any material, including, for example, any particles, beads, or surface, on which oligomers may be attached or synthesized, or which may be modified for attaching or synthesizing oligomers. Representative matrices include, but are not limited to, inorganic and organic supports, such as glass and modified or functionalized glass, plastics (including acrylic, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutene, polyurethane, TEFLON, etc.), polysaccharides, nylon or nitrocellulose, ceramics, resins, silica or silica-based materials (including silicon and modified silicon), carbon, metals, inorganic glasses, plastics, fiber bundles, and a variety of other polymers. For some embodiments, particularly useful support media and solid surfaces are located within a flow cell apparatus. In some embodiments of the methods described herein, the support medium comprises polystyrene having 1% crosslinked divinylbenzene. In other embodiments of the methods described herein, the support medium is aminomethyl polystyrene resin (e.g., purchased from Xi'an Lanxiao Technology Co., Ltd. and loaded at, for example, 1 mmol / g).

[0244] In some embodiments, a representative support medium includes at least one reactive site for attaching or synthesizing oligomers. For example, in some embodiments, the support medium disclosed herein includes one or more terminal amino or hydroxyl groups capable of forming chemical bonds with the subunit to be added or other activated groups to attach or synthesize oligomers.

[0245] The term "flow cell device" refers to a chamber containing a surface (e.g., a solid surface) through which one or more fluid reagents (e.g., liquids or gases) can flow.

[0246] The term "deblocking agent" refers to a composition (e.g., a solution) comprising a chemical acid or combination of chemical acids for removing protecting groups. Exemplary chemical acids used in deblocking agents include haloacids 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 triphenylmethyl groups from, for example, oligomers, support-bound oligomers, support-bound subunits, or other protected nitrogen or oxygen moieties. In another embodiment, the deblocking agent used in each method (step) is a solution comprising 4-cyanopyridine, dichloromethane, trifluoroacetic acid, trifluoroethanol, and water, or a solution comprising 4-cyanopyridine trifluoroacetate, trifluoroethanol, dichloromethane, and ethanol. In a preferred embodiment, the deblocking agent used in each method (step) is 2% 4-cyanopyridine trifluoroacetate (CYTFA) (w / v) in 20% trifluoroethanol / dichloromethane containing 1% ethanol.

[0247] The terms “halogen” and “halogenated” refer to atoms selected from fluorine, chlorine, bromine, and iodine.

[0248] The term "capping agent" refers to an acid anhydride (e.g., benzoic anhydride, acetic anhydride, phenoxyacetic anhydride, etc.) that can be used to block reactive sites, such as those of a supporting medium, from which a chemical bond is formed with an upcoming subunit or other activated group. In embodiments, the capping agent is in a solution comprising ethylmorpholine and methylpyrrolidone. In a preferred embodiment, the capping agent disclosed herein comprises capping A and capping B, wherein capping A is a solution of NEM in NMP, and capping B is a solution of capping agent in NMP.

[0249] The term "cutting agent" refers to a composition (e.g., a liquid solution or gaseous mixture) comprising a chemical base (e.g., ammonia or 1,8-diazabicycloundec-7-ene) or a combination of chemical bases that can be used to cut oligomers, such as support-bound oligomers, from a support medium. In another embodiment, the cutting agent is in a solution comprising N-methyl-2-pyrrolidone.

[0250] The term "deprotectant" refers to a composition (e.g., a liquid solution or gaseous mixture) comprising a chemical base (e.g., ammonia, 1,8-diazabicyclo[5,4,0]undec-7-ene) or a combination of chemical bases that can be used to remove protecting groups. For example, in some embodiments, the deprotectant 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.

[0251] 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, benzyl nitrile, benzyl alcohol, 1-butanol, 2-butanol, isobutanol, 2-butanone, tert-butanol, carbon disulfide, carbon tetrachloride, chlorobenzene, chloroform, cyclohexane, cyclohexanol, cyclohexanone, di-n-butyl phthalate, 1,1-dichloroethane, 1,2-dichloroethane, diethylamine, diethylene glycol, diethylene glycol dimethyl ether, dimethoxyethane, N,N-dimethylaniline, dimethylformamide (DMF), dimethylacetamide (DM...). A) 1,3-Dimethyl-2-imidazolinone (DMI), 1-methyl-2-pyrrolidone (NMP), dimethyl phthalate, dimethyl sulfoxide, dioxane, ethanol, ether, ethyl acetate, ethyl acetoacetate, ethyl benzoate, ethylene glycol, glycerol, heptane, 1-heptanol, hexane, 1-hexanol, methanol, methyl acetate, methyl tert-butyl ether, dichloromethane, 1-octanol, pentane, 1-pentanol, 2-pentanol, 3-pentanol, 2-pentanone, 3-pentanone, 1-propanol, 2-propanol, pyridine, tetrahydrofuran, toluene, water, p-xylene).

[0252] In this disclosure, G, C, A, U, and T are guanine, cytosine, adenine, uracil, and thymine, respectively.

[0253]

[0254] abbreviation

[0255] DMF stands for N,N-dimethylformamide;

[0256] DIPEA stands for N,N-diisopropylethylamine;

[0257] TrCl represents triphenylmethyl chloride;

[0258] TLC stands for Thin-Layer Chromatography;

[0259] EtOAc represents ethyl acetate;

[0260] NaCl represents sodium chloride.

[0261] DBU stands for 1,8-diazabicyclo[5,4,0]undec-7-ene;

[0262] DBN represents 1,5-diazabicyclo[4.3.0]non-5-ene;

[0263] DABCO represents 1,4-diazabicyclo[2.2.2]octane;

[0264] NMP stands for 1-methyl-2-pyrrolidone;

[0265] NPE stands for 4-nitrophenylethyl (NPE) group;

[0266] IPC stands for process control;

[0267] UV stands for ultraviolet light;

[0268] CYTFA stands for 4-cyanopyridine trifluoroacetate;

[0269] Vol. (vol.) represents volume;

[0270] DCM stands for dichloromethane;

[0271] MTBE stands for methyl tert-butyl ether;

[0272] DMAP stands for dimethylaminopyridine;

[0273] EDCI stands for 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride;

[0274] IPA stands for isopropanol;

[0275] NEM stands for N-ethylmorpholine;

[0276] Bz2O represents benzoic anhydride;

[0277] DMI stands for 1,3-dimethyl-2-imidazolinone;

[0278] PMO stands for phosphoryl diamine morpholine oligomer;

[0279] CAP A represents end cap A;

[0280] CAP B stands for end cap B;

[0281] “a M” represents “a mol / L”, where a is a number;

[0282] min represents one minute or more.

[0283] Example

[0284] The following examples further illustrate the invention, but the invention is not limited thereto. If no temperature restriction is specified in the operation of the examples, it means that the operation is performed at room temperature.

[0285] Synthesis of compound (E) in Example 1

[0286] Synthesis route:

[0287]

[0288] Step 1: Synthesis of compound (A)

[0289] To a solution of compound (SM1) (35 g, 0.25 mol, 1 equivalent) in DMF (350 mL), DIPEA (0.75 mol, 3 equivalents) and TrCl (0.5 mol, 2 equivalents) were added sequentially. The reaction mixture was stirred at 20–25 °C until TLC showed complete consumption of compound (SM1). 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 a 20% NaCl aqueous solution to remove DMF. The resulting organic layer was concentrated and purified by silica gel column chromatography to obtain 79 g of 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).

[0290] Step 2 Synthesis of compound (B)

[0291] 60% NaH (0.1 equivalent, dissolved in mineral oil) was added to a solution of triethylene glycol (10 equivalents) in NMP (500 mL, 10 vol.) at 20-30°C, and the mixture was stirred at 20-30°C for 20 minutes. Compound (A) (50 g, 1 equivalent) was then 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). Crude compound (B) was purified by silica gel column chromatography to obtain 47 g of product, with a yield of 70%. 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).

[0292] Step 3 Synthesis of compound (C)

[0293] Succinic anhydride (2 equivalents) was added to a solution of compound (B) (47 g, 1 equivalent) in THF (470 mL). The reaction mixture was stirred at 55 °C for 2.0 h until TLC showed complete consumption of compound (B). After the reaction, the pH of the reaction mixture was adjusted to approximately 8.5 with a 10% aqueous solution of NaHCO3. Then, MTBE (940 mL, 20 vol.) was added to the mixture and the aqueous layer was separated. The pH of the resulting aqueous layer was adjusted to 3–5 with a 20% citric acid solution, then extracted with DCM (940 mL, 20 vol.) and washed with a 10% aqueous solution of Na2SO4 (470 mL, 10 vol.) to obtain the organic phase. After concentration, 56 g of a yellow oily substance was obtained in 98% yield, which was used for 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).

[0294] Step 4: Synthesis of compound (D)

[0295] To a solution of compound (C) (56 g, 1 equivalent) in DCM (560 mL), N-hydroxy-5-norbornene-2,3-dicarboxylic acid imide (HONB, 1.02 equivalent), DMAP (0.33 equivalent), and DIPEA (2.5 equivalent) were added, followed by EDCI (1.1 equivalent). The reaction mixture was stirred at 20–30 °C until TLC showed complete consumption of compound (C). The mixture was washed successively with 10% citric acid solution and brine. The organic layer was concentrated to dryness for use in the next step without further purification. 67 g of foamy solid was obtained, with a yield of 93%. 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).

[0296] Step 5: Synthesis of compound (E)

[0297] Aminomethyl polystyrene 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 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 equivalents) in NMP (10 vol.) was added to the suspension of aminomethyl polystyrene 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. The wet cake was transferred to a reactor and subsequently added with NEM (0.4 M) solution in NMP (60 mL) and B2O (0.4 M) solution in NMP (60 mL). The remaining amino groups in the resin were capped with Bz2O. After the capping reaction was complete, the resin was filtered and washed with DCM (100 mL). After drying, sarcosinate-modified aminomethyl resin was obtained, which was used for the synthesis of phosphorylated diamine morpholine oligomers.

[0298] Determining the load size

[0299] Typical procedure:

[0300] The resin loading (number of potentially available reactive sites) was determined by spectroscopic determination. A known weight of dry resin (25 ± 3 mg) was transferred to a silanized 25 mL volumetric flask, and approximately 5 mL of 2% (v / v) trifluoroacetic acid in dichloromethane was added. The contents were mixed by gentle vortexing and then allowed to stand for 30 minutes. The volume was brought to 25 mL with another 2% (v / v) trifluoroacetic acid in dichloromethane, and the contents were thoroughly mixed. Using a positive displacement pipette, an aliquot containing 500 μL of the triphenylmethyl solution was transferred to a 10 mL volumetric flask, and the volume was brought to 10 mL with methanesulfonic acid. The triphenylmethyl cation content in the final solution was measured by UV absorbance at 406 nm, and the resin loading was calculated as triphenylmethyl groups / gram of resin (μmol / g) using compound A as a reference standard. The determination was performed twice, and the average loading was calculated. The results are shown in Table 1.

[0301] Table 1 Loading amount of compound E

[0302]

[0303] Example 2: Synthesis of phosphorylated diamine morpholine oligomers

[0304] Phosphoryldiamine morpholino oligomers were synthesized by assembling phosphoryldiamine morpholino subunits in 25 cycles from sarcosinate-modified aminomethyl resins. The synthetic route for the phosphoryldiamine morpholino oligomers is as follows.

[0305]

[0306]

[0307] The synthesis of phosphoryldiamine morpholino oligomers was carried out manually from a triethylene glycol sarcosinate-modified support via solid-phase synthesis using peptide synthesis equipment for detriphenylmethylation, neutralization, coupling, end-capping, and washing cycles. All reactions were conducted in glass-jacketed columns of 20 mL, 100 mL, 500 mL, and 2 L volumes.

[0308] All solutions are prepared as follows before assembling the monomers onto the solid support.

[0309] Detrimethylated solution: 2% 4-cyanopyridine trifluoroacetate (CYTFA) in 20% trifluoroethanol / dichloromethane (1:4, v / v) with 1% ethanol.

[0310] Neutralization solution: 5% diisopropylethylamine in 25% isopropanol / dichloromethane.

[0311] Coupling solution: 0.36M morpholino derivative solution in DMI and 0.8M N-ethylmorpholine (NEM) in DMI. Simultaneously, the morpholino derivative solution in DMI was treated with molecular sieves for over 12 hours to reduce water content.

[0312] Capping solutions: 0.4M NEM in NMP is used as capping solution A; 0.4M benzoic anhydride or acetic anhydride in NMP is used as capping solution B.

[0313] Table 2. Reaction solutions in the synthesis of phosphorylated diamine morpholine oligomers

[0314]

[0315]

[0316] Sarcosinate-modified aminomethyl polystyrene resin was added to a jacketed column reactor, followed by 15 vol. of 1-methyl-2-pyrrolidone (NMP 15 mL / g resin), and the suspension was allowed to stand for 0.5–1 hour. The NMP was then evacuated, and the resin was washed five times with DCM before detrimethylation. Four reactions were performed to assemble each phosphorylated diamine morpholino oligomer subunit onto the support.

[0317] First, to remove the triphenylmethyl groups from the support, a 2% solution of 4-cyanopyridine trifluoroacetate (CYTFA) (w / v) (2,2,2-trifluoroethanol / DCM 1 / 4 with 1% EtOH) (15-25 vol.) was added to the column reactor. The mixture was bubbled with N2 for 2-5 minutes, then evacuated to remove the solvent. This operation was repeated 5-9 times until IPC showed that all triphenylmethyl groups had been removed (IPC: sample the filtrate and dilute with methanesulfonic acid. UV absorption at 411 nm was measured by UV spectroscopy to check whether the triphenylmethyl groups were completely removed).

[0318] Second, after detrimethylation, 5% DIPEA in the IPA / DCM (1 / 3) mixture is added to the jacketed column to neutralize the resin. Residual CYTFA needs to be completely removed by multiple washes before coupling.

[0319] Third, coupling was performed as follows: a solution of morpholino subunits in DMI and a solution of NEM were added to the reactor, and the reaction mixture was bubbled with N2 at 45°C for 90 minutes. After the morpholino subunits were assembled onto the support, the reaction mixture was evacuated and washed with DCM.

[0320] Finally, the unreacted morpholino subunits on the support are capped to prevent further extension.

[0321] Repeat the four reactions as shown in Table 3 below until the target sequence is complete.

[0322] Table 3 Assembly procedures for phosphorylated diamine morpholine oligomers

[0323]

[0324]

[0325]

[0326] For each coupling reaction, a single synthesis of phosphorylated diamine morpholino oligomers was performed using 250 mg of compound (E) (608 μmol / g loading) and 2.5 equivalents of phosphorylated diamine morpholino oligomers (PMO-NPEG monomer, PMO-PA monomer, PMO-PC monomer, PMO-T monomer, the structures of which are shown in Example 2). After 25 reaction cycles, 2.95 g of wet, base-protected phosphorylated diamine morpholino oligomer-modified solid support was obtained. The base-protected phosphorylated diamine morpholino oligomer-modified solid support was treated with 0.5 M DBU in NMP (20 mL) at 15 °C for 4 h to remove the 4-nitrophenylethyl group, followed by treatment with concentrated ammonium hydroxide to cleave the base-protected phosphorylated diamine morpholino oligomer, and other protecting groups were removed sequentially. 28.8% of 4-nitrostyrene adduct impurities were found (as determined by LC-MS), which significantly reduced the yield of phosphoryldiamine morpholine oligomer synthesis.

[0327] To address the issue of impurities in 4-nitrostyrene adducts, a three-step strategy involving cleavage and deprotection was developed. The base-protected phosphoryldiamine morpholine oligomer was slowly added (5 mL / min) to a solution of DBU and thymine in NMP (DBU concentration: 0.8 M, thymine concentration: 1.0 M). Those skilled in the art will understand that when the target phosphoryldiamine morpholine oligomer sequence contains thymine, the concentration of thymine should be significantly higher than the concentration of thymine in the target phosphoryldiamine morpholine oligomer sequence; for example, the molar ratio of thymine to thymine in the target phosphoryldiamine morpholine oligomer sequence is greater than 10:1. After the three-step cleavage and deprotection process, the 4-nitrostyrene adduct impurities were significantly reduced (less than 5%, determined by LC-MS).

[0328] Three steps to cutting and removing protection

[0329] After solid-phase assembly, a solid support modified with a phosphorylated diamine morpholine oligomer having a 4-nitrophenylethyl group protected on guanine was obtained. Then, three steps of cleavage and deprotection were performed as follows.

[0330] Step (1) First, the base-protected phosphoryl diamine morpholine oligomer crude product is cleaved with concentrated ammonium hydroxide (25%-28% ammonium hydroxide) to obtain an aqueous solution, which is then freeze-dried or concentrated to dryness to produce the base-protected phosphoryl diamine morpholine oligomer crude product.

[0331] Step (2) involves redissolving the crude product from step (1) in NMP (NMP to crude product volume-mass ratio of 10 mL / g) and slowly (5 mL / min) adding it to a DBU / thymine (1.0 M / 0.8 M) solution in NMP (10 Vol. relative to the crude product) to remove 4-nitrophenylethyl groups while minimizing 4-nitrostyrene adduct impurities.

[0332] Step (3) The oligonucleotides obtained after deprotection of NPE are treated again with concentrated ammonium hydroxide (25%-28% ammonium hydroxide) to remove the remaining protecting groups (such as isobutyryl groups) to form the targeted phosphoryl diamine morpholino oligomer A (PMO-A), whose sequence is: 5'-GTT GCC TCC GGT TCT GAA GGT GTT C-3' (SEQ ID NO:1).

[0333]

[0334]

[0335] PMO-B, PMO-C, PMO-D, and PMO-E were prepared using the same method as that used to prepare PMO-A.

[0336] The base sequence of PMO-B is 5'-CTC CAA CAT CAA GGA AGA TGG CAT TTC TAG-3' (SEQ ID NO:2).

[0337] The base sequence of PMO-C is 5'-CTATATATAGTTATTCAACA-3' (SEQ ID NO:3).

[0338] The base sequence of PMO-D is 5'-GGC CAA ACC TCG GCT TAC CTG AAAT-3' (SEQ ID NO:4).

[0339] The base sequence of PMO-E is 5'-CAG CAG CAG CAG CAG CAG-3' (SEQ ID NO:5).

[0340] Several typical impurities were found during the synthesis of phosphorylated diamine morpholine oligomers, and the results are shown in Table 4.

[0341] Table 4 Typical impurities found in the synthesis of phosphorylated diamine morpholine oligomers

[0342]

[0343]

[0344] FLP purity represents the LC-MS purity of the full-length product.

[0345] NA, NC, NG, and NT represent four impurities of N-1.

[0346] OH represents the impurities resulting from the hydrolysis of phosphoryl demethylamine.

[0347] N+NPE represents 4-nitrostyrene adduct impurities.

[0348] LC-MS: Waters Class H UPLC with Xevo G2-XS-TOF detector.

[0349] Crude product refers to unpurified PMO obtained after the three steps of cleavage and deprotection.

[0350] Purified product refers to PMO obtained after three steps of cutting and deprotection, followed by purification using ion exchange chromatography.

[0351] It should be understood that the above description of the two preferred embodiments is purely intended to illustrate the principles of the invention and not to exhaustively describe it, and variations and modifications will be apparent to those skilled in the art, and the invention is not intended to be limited to anything other than what is expressly set forth in the following claims. SEQUENCE LISTING <110> Changzhou Hequan Pharmaceutical Co., Ltd. Shanghai Wuquan Pharmaceutical R&D Co., Ltd. <120> Methods for preparing oligonucleotides <130> P22118010CP <160> 5 <170> PatentIn version 3.5 <210> 1 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> PMO-A <400> 1 gttgcctccg gttctgaagg tgttc 25 <210> 2 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> PMO‐B <400> 2 ctccaacatc aaggaagatg gcatttctag 30 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PMO‐C <400> 3 ctatatatag ttattcaaca 20 <210> 4 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> PMO‐D <400> 4 ggccaaacct cggcttacct gaaat 25 <210> 5 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> PMO‐E <400> 5 gcagcagc gcagcag 18

Claims

1. A method for preparing oligonucleotides, the method comprising: (a) Converting a compound having formula X-1 into a compound having formula X-2, wherein the conversion comprises adding the compound having formula X-1 to a mixture comprising a basic reagent and a scavenger capable of reacting with a compound having formula X-3: The scavenger is a compound with X-4: in: q is 0, 1, or 2; and R A Each time it appears, it is C independently. 1-6 alkyl; The alkaline reagent is an alkaline organic amine with a pKa of 9 or higher in water; The molar ratio of the basic reagent to one or more NPE groups of the compound having formula X-1 is greater than 1:1; said NPE group is 4-nitrophenylethyl; in: R 10 These are residues of phosphoryl diamine morpholine oligomers; R 11 The amine protecting groups were removed by treatment with NH3; The compound having formula X-1 does not bind to the solid support; and (b) Optionally, the protecting group in the compound having formula X-2 is removed to obtain the oligonucleotide.

2. The method of claim 1, wherein the conversion comprises adding the compound having formula X-1 in solution to a mixture comprising an alkaline reagent and a scavenger capable of reacting with a compound having formula X-3.

3. The method of claim 1, wherein the pKa of the basic organic amine in water is 9-15.

4. The method of claim 1, wherein the basic reagent is a basic cyclic amine.

5. The method of claim 4, wherein the basic reagent is 1,8-diazabicyclo[5.4.0]undec-7-ene or 1,5-diazabicyclo[4.3.0]non-5-ene.

6. The method of claim 1, wherein, R A Each time it appears, it is a methyl group independently.

7. The method of any one of claims 1-6, wherein the scavenging agent is thymine.

8. The method of claim 1, wherein R 11 It is an acyl group.

9. The method of claim 8, wherein R 11 It is -C(=O)-R B , where R B It is C 1-6 alkyl and aryl substituted C 1-6 C substituted with alkyl or aryloxy 1-6 alkyl.

10. The method of claim 9, wherein the aryl-substituted C 1-6 Alkyl groups are benzyl groups.

11. The method of claim 9, wherein C 1-6 The alkyl group is isopropyl.

12. A method for preparing oligonucleotides, the method comprising: (a) Converting a compound having formula X-5 into a compound having formula X-6, wherein the conversion comprises adding the compound having formula X-5 to a mixture comprising a basic reagent and a scavenger capable of reacting with a compound having formula X-3: The alkaline reagent is an alkaline organic amine, which has a pKa of 9 or higher in water. The scavenger is a compound with X-4: in: q is 0, 1, or 2; and R A Each time it appears, it is C independently. 1-6 alkyl; The molar ratio of the basic reagent to one or more NPE groups of the compound having formula X-5 is greater than 1:1; said NPE group is 4-nitrophenylethyl; in: m1 and m2 are independent integers from 0 to 50; R 11 The amine protecting groups were removed by treatment with NH3; Each time a base appears, it is independently selected from guanine, cytosine, adenine, uracil, and thymine, provided that when the base in formula X-5 is a protected base, the corresponding base in formula X-6 is the same protected base or the corresponding partially or completely unprotected base. in: T 1 It is a suitable 5' terminal group; and T 2 It is a suitable 3' terminal group; and (b) Optionally, the protecting group in the compound having formula X-6 is partially or completely removed to obtain the oligonucleotide.

13. The method of claim 12, wherein m1 and m2 are independently integers from 0 to 30.

14. The method of claim 12, wherein T 1 It is a short peptide, an optionally substituted alkylamine group, or an optionally substituted heterocyclic group.

15. The method of claim 12, wherein T 2 It is hydrogen or a protecting group.

16. The method of claim 15, wherein T 2 It is an acyl group or triphenylmethyl.

17. The method of claim 12, wherein the bases in formula X-5 or X-6 are independently selected each time they appear. Where R 11 It is an amine protecting group.

18. The method of any one of claims 12-17, wherein one of m1 and m2 is 0, or neither m1 nor m2 is 0.

19. The method of any one of claims 12-17, wherein the sum of m1 and m2 is between 5 and 50.

20. The method of claim 19, wherein the sum of m1 and m2 is between 10 and 40.

21. The method of any one of claims 12-17, wherein T 1 It is an alkylamine or a heterocyclic compound that is optionally substituted.

22. The method of claim 21, wherein the optionally substituted alkylamine is an amide-substituted alkylamine; Alternatively, the optionally substituted heterocycle is an optionally substituted piperazine ring.

23. The method of claim 22, wherein the optionally substituted alkylamine is a -C(O)NH2-substituted alkylamine; Alternatively, the optionally substituted heterocycle is Where R C It is an acyl group, acyloxy group, or peptide residue.

24. The method of claim 23, wherein the optionally substituted alkylamine is 25. The method of claim 12, wherein T 2 It is hydrogen, triphenylmethyl or acyl group.

26. The method of any one of claims 12-17, wherein T 2 It is an acetyl group.

27. The method of claim 12, wherein the basic organic amine has a pKa of 9-15 in water.

28. The method of claim 12, wherein the basic reagent is a basic cyclic amine.

29. The method of claim 28, wherein the basic reagent is 1,8-diazabicyclo[5.4.0]undec-7-ene or 1,5-diazabicyclo[4.3.0]non-5-ene.

30. The method of claim 12, wherein R A Each time it appears, it is a methyl group independently.

31. The method of claim 30, wherein the scavenger is thymine.

32. The method of claim 12, wherein R 11 It is an acyl group.

33. The method of claim 32, wherein R 11 It is -C(=O)-R B , where R B It is C 1-6 alkyl and aryl substituted C 1-6 C substituted with alkyl or aryloxy 1-6 alkyl.

34. The method of claim 33, wherein the aryl-substituted C 1-6 Alkyl groups are benzyl groups.

35. The method of claim 34, wherein C 1-6 The alkyl group is isopropyl.

36. The method of claim 12, further comprising treating the compound having formula X-6 with NH3 to partially or completely remove the protecting group in formula X-6.

37. The method of claim 12, wherein the compound having formula X-5 is prepared by a method comprising cleaving a solid support from an oligonucleotide having formula X-7: in SS is a solid support; L 1 It's a connector; m1, m2 and R in equation X-7 11 The corresponding group is the same as that in formula X-5. Each time a base appears, it is independently selected from guanine, cytosine, adenine, uracil, and thymine, provided that when the base in formula X-7 is a protected base, the corresponding base in formula X-5 is the same protected base or a corresponding partially or completely unprotected base; and T 2 It is a suitable 3' terminal group.

38. The method of claim 37, wherein the method comprises cleaving a solid support from an oligonucleotide having formula X-7 using NH3.

39. The method of claim 37, wherein SS is a polystyrene solid support.

40. The method of claim 37, wherein L 1 It is a sarcosine-based linker.

41. The method of claim 37, wherein L 1 yes The nitrogen end is connected to the phosphorus atom, and the carbonyl end forms an amide bond with the solid support.

42. The method of claim 37, wherein T 2 It is hydrogen or a protecting group.

43. The method of claim 42, wherein T 2 It is an acyl group or triphenylmethyl.

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