Compositions and methods for liquid phase oligonucleotide synthesis

Abstract

Embodiments of the present application relate to polymers as polymer multivalent hubs for liquid phase oligonucleotide synthesis. Methods of preparing oligonucleotides by liquid-phase oligonucleotide synthesis using these polymers are also provided.

Description

Background Art Technical Field

[0001] This disclosure relates to methods and compositions for the liquid-phase synthesis of oligonucleotides using polymers with side poly(ethylene glycol) arms. Related technical description

[0002] Oligonucleotide-based drugs have become a powerful weapon in the treatment of a variety of diseases. Currently, the demand for oligonucleotides can be met through conventional solid-phase oligonucleotide synthesis (SPOS). SPOS offers certain advantages, such as simple product isolation and the use of an anhydrous synthetic environment. However, for oligonucleotide sequences, the overall yield of SPOS is typically low after multiple steps, and the costs of reagents, solid supports, and waste disposal are high. Furthermore, SPOS can lead to oligonucleotide sequence mismatches, making purification difficult. The ever-increasing demand for metric tons of oligonucleotides far exceeds the production capacity of solid-phase oligonucleotide synthesis.

[0003] Liquid-phase oligonucleotide synthesis (LPOS) is a technique that has the potential to provide the required production capacity. One of the main advantages of LPOS over SPOS is the absence of heterogeneity in the steps, i.e., the absence of insoluble solid supports. The soluble scaffolds or supports used in LPOS allow the synthetic steps to proceed in the liquid phase and improve kinetics.

[0004] Polyethylene glycol (PEG) is one of the most widely used soluble polymer carriers and has attracted considerable attention. (Current Protocol in Nucleic Acid Chemistry (2019) 77, e82). Firstly, the PEG process is low-cost. Secondly, due to its high coupling efficiency and the possibility of using convergent synthesis, the PEG platform is highly advantageous for large-scale production. Therefore, further exploration of PEG-based platforms holds great promise for the development of LPOS (Liquid Polymer Portfolio Synthesis). Summary of the Invention

[0005] Some aspects of this disclosure relate to polymers for the synthesis of liquid-phase oligonucleotides having the structure of formula (I): in: R is H, or an unsubstituted or substituted C1-C6 alkyl group; W is C1-C 20 Alkylene, 2-20 heteroalkylene, or bond; Q is L 1 For C1-C 20Alkylene, 2-20-membered heteroalkylene, optionally substituted phenylene, optionally substituted 5-6-membered heteroarylene, optionally substituted 3-10-membered heterocyclic group or optionally substituted C3-C 10 Cycloalkylene, or C1-C 20 Alkylene, or 2-20-membered heteroalkylene, wherein one or more methylene repeating units are each independently substituted by a group selected from the group consisting of: optionally substituted phenylene, optionally substituted 5-6-membered heteroarylene, optionally substituted 3-10-membered heterocyclic group, optionally substituted C3-C 10 Cycloalkylene compounds, -C(=O)-, -CH=CH-, and -C≡C-; R 1 and R 2 Each independently of -OR 3 or -NR 4a R 4b ; R 3 Protected by H, C1-C6 alkyl, hydroxyl groups or R 4a and R 4b Each is H, optionally substituted C1-C6 alkyl, -C(=O)(C1-C6 alkyl), -C(=O)phenyl, amino protecting group or or R 4a and R 4b Together they form a divalent amino protecting group; L 2a and L 2b Each independently is C1-C 20 Alkylene, 2-20-membered heteroalkylene, optionally substituted phenylene, optionally substituted 5-6-membered heteroarylene, optionally substituted 3-10-membered heterocyclic group or optionally substituted C3-C 10 Cycloalkylene, or C1-C 20 Alkylene or 2-20-membered heteroalkylene, wherein one or more methylene repeating units are each independently substituted by a group selected from the group consisting of: optionally substituted phenylene, optionally substituted 5-6-membered heteroarylene, optionally substituted 3-10-membered heterocyclic group, optionally substituted C3-C 10 Cycloalkylene compounds, -C(=O)-, -CH=CH-, and -C≡C-; R 5a 、R 5b 、R 6a and R 6b Each is independently H, -OR 7 or -NR 8a R 8b ; R 7 Each is independently protected by H, C1-C6 alkyl, hydroxyl groups or R 8a and R 8b Each is independently H, optionally substituted C1-C6 alkyl, -C(=O)(C1-C6 alkyl), -C(=O)phenyl, amino protecting group or or R 8a and R 8b Together they form a divalent amino protecting group; L 3a and L 3b Each independently is C1-C 20 Alkylene, 2-20-membered heteroalkylene, optionally substituted phenylene, optionally substituted 5-6-membered heteroarylene, optionally substituted 3-10-membered heterocyclic group or optionally substituted C3-C 10 Cycloalkylene, or C1-C 20 Alkylene or 2-20-membered heteroalkylene, wherein one or more methylene repeating units are each independently substituted by a group selected from the group consisting of: optionally substituted phenylene, optionally substituted 5-6-membered heteroarylene, optionally substituted 3-10-membered heterocyclic group, optionally substituted C3-C 10 Cycloalkylene compounds, -C(=O)-, -CH=CH-, and -C≡C-; R 9a 、R 9b 、R 10a and R 10b Each is independently H, -OR 11 or -NR 12a R 12b ; R 11 Each is independently protected by H, C1-C6 alkyl, hydroxyl groups or R 12a and R 12b Each is independently H, optionally substituted C1-C6 alkyl, -C(=O)(C1-C6 alkyl), -C(=O)phenyl, amino protecting group or or R 12a and R 12b Together they form a divalent amino protecting group; L 4a and L 4b Each independently is C1-C 20 Alkylene, 2-20-membered heteroalkylene, optionally substituted phenylene, optionally substituted 5-6-membered heteroarylene, optionally substituted 3-10-membered heterocyclic group or optionally substituted C3-C 10 Cycloalkylene, or C1-C 20Alkylene or 2-20-membered heteroalkylene, wherein one or more methylene repeating units are each independently substituted by a group selected from the group consisting of: optionally substituted phenylene, optionally substituted 5-6-membered heteroarylene, optionally substituted 3-10-membered heterocyclic group, optionally substituted C3-C 10 Cycloalkylene compounds, -C(=O)-, -CH=CH-, and -C≡C-; R 13a 、R 13b 、R 14a and R 14b Each can be independently H, -OH, a protected hydroxyl group, -NH (optionally substituted C1-C6 alkyl), -NH2, or a protected amino group; m1, m2, m3, m4, m5, m6, and m7 are each independently 0 or 1; and j is an integer between 15 and 1500.

[0006] In some embodiments of the polymer of formula (I), R is -CH3. In some embodiments, R is H. In some embodiments, W is -CH2CH2NH-. In other embodiments, W is -CH2CH2O-. In some embodiments, W is a C2-C6 alkylene group, such as -CH2CH2-. In other embodiments, W is a bond.

[0007] In some embodiments of the polymer of formula (I), L 1 It is a C2-C6 alkylene group. In other embodiments, L 1 It is a 3-12 or 3-6 heteroalkylene group containing one, two or three heteroatoms selected from N, O or S. In other embodiments, the heteroalkylene group contains one or two nitrogen atoms.

[0008] In some embodiments of the polymer of formula (I), R 1 It is -OH or a protected hydroxyl group. In other embodiments, R 1 It is -NH2, -NH (optionally substituted C1-C6 alkyl), -NHC(=O)CH3(-NHAc), or a protected amino group. In other embodiments, R 1 yes In some embodiments of the polymer of formula (I), R 2 It is -OH or a protected hydroxyl group. In other embodiments, R 2 It is -NH2, -NH (optionally substituted C1-C6 alkyl), -NHC(=O)CH3, or a protected amino group. In other embodiments, R 2 yes In some implementations, L 2a and L 2bEach is independently a C2-C6 alkylene group. In other embodiments, L 2a and L 2b Each is independently an optionally substituted phenylene. In some embodiments, R 5a 、R 5b 、R 6a and R 6b Each is independently H, -OH, a protected hydroxyl group, -NH2, -NH (optionally substituted C1-C6 alkyl), -NHC(=O)CH3, or a protected amino group. In some other embodiments, m2 and m3 are each 1. In other embodiments, m2 is 0 and m3 is 1. In other embodiments, m2 is 1 and m3 is 0. In other embodiments, both m2 and m3 are 0.

[0009] In some embodiments of the polymer of formula (I), Q is selected from the group consisting of:

[0010] In some embodiments of the polymer of formula (I), each R 3 Independently, it is either an H or hydroxyl protecting group, each R 4a and R 4b Independently, it is H, an optionally substituted C1-C6 alkyl group, a -C(=O)CH3 or amino protecting group. As in some other embodiments, each R... 4a For H, each R 4b Independently protected by H, -C(=O) (C1-C6 alkyl), -C(=O) phenyl, amino, or Or -NHR 4b Hydrogen is absent in R, and R 4b It is a divalent amino protecting group. In some such embodiments, R 3 for R 4a is H, and R 4b for In other embodiments, R 3 H, R 4a is H, and R 4b for or R 4a and R 4b Each is H, and R 3 for In some other embodiments, m2 and m3 are each 1. In other embodiments, m2 is 0 and m3 is 1. In other embodiments, m2 is 1 and m3 is 0. In other embodiments, both m2 and m3 are 0.

[0011] In some other embodiments of the polymer of formula (I) or (I'), Q is selected from the group consisting of: In some of these implementations, each R 3 Independently protected by H or hydroxyl groups. In some other embodiments, each R... 3 for In some other embodiments, R 3 One of them is H, and the other is R. 3 for In some other implementations, m2 is 1. In other implementations, m2 is 0.

[0012] In some embodiments of the polymer of formula (I) or any of the substructures described herein, L 2a and L 2b Each independently is C1-C 10 Alkylene or 2-10 heteroalkylene; R 5a 、R 5b 、R 6a and R 6b Each is independently H, -OR 7 or -NR 8a R 8b ; R 7 Each is independently protected by H, C1-C6 alkyl, hydroxyl groups or And R 8a and R 8b Each of the following is independently H, an optionally substituted C1-C6 alkyl group, -C(=O)(C1-C6 alkyl), -C(=O)phenyl, an amino protecting group, or In some implementations, L 2a It is a C2-C6 alkylene linkage. In other embodiments, L 2a It is a 3-12 or 3-6-membered heteroalkylene linkage containing 1, 2, or 3 heteroatoms selected from O, S, or N. In other embodiments, the heteroalkylene contains 1 or 2 nitrogen atoms. In some embodiments, L 2b It is a C2-C6 alkylene linkage. In other embodiments, L 2b It is a 2-6 membered heteroalkylene linkage containing 1, 2, or 3 heteroatoms selected from O, S, or N. In other embodiments, the heteroalkylene contains 1 or 2 nitrogen atoms. In other embodiments, L 2a and L 2b Each is independently an optionally substituted phenylene; or a C2-C6 alkylene or a 3-12 heteroalkylene, wherein one of the methylene units is optionally substituted with a substituted phenylene. In some embodiments, each R 8a is H, and R 5a 、R 5b 、R6a and R 6b Each is independently H, -OR 7 or -NHR 8b Each R 8b Each is independently H, -C(=O) (C1-C6 alkyl), -C(=O) phenyl, amino protecting group or Or -NHR 8b Hydrogen is absent in R, and R 8b It is a divalent amino protecting group. In some embodiments, each R 5a 、R 5b 、R 6a and R 6b Each of these can be independently H, -OH, a protected hydroxyl group, -NH2, -NH (optionally substituted C1-C6 alkyl), -NHC(=O)CH3, or a protected amino group. In some other embodiments, R 5a 、R 5b 、R 6a and R 6b Each independently of -OR 7 or -NR 8a R 8b Among them, R 7 At least one of them is And R 8a and R 8b At least one of them is In some other implementations, R 7 Each independently And R 8a and R 8b Each independently In some specific implementations, R 8a H, R 8b It is protected by H or an amino group (e.g., Ac), and R 5a 、R 5b 、R 6a and R 6b Each is independently -NH2, -NH (optionally substituted C1-C6 alkyl), or -NHC(=O)CH3. In other embodiments, R 5a 、R 5b 、R 6a and R 6b At least one of them is H. In other embodiments, R 7 is H, and R 5a 、R 5b 、R 6a and R 6b Each is -OH. In other embodiments, R 7 yes In other embodiments, R8a It is H and R 8b yes In some other embodiments, m4 and m5 are each 1. In other embodiments, m4 is 0 and m5 is 1. In other embodiments, m4 is 1 and m5 is 0. In other embodiments, both m4 and m5 are 0. In some other embodiments, L 3a and L 3b Each independently is C1-C 10 Alkylene or 2-10 heteroalkylene; R 9a 、R 9b 、R 10a and R 10b Each is independently H, -OR 11 or -NR 12a R 12b ; R 11 Each is independently protected by H, C1-C6 alkyl, hydroxyl groups or And R 12a and R 12b Each of the following is independently H, an optionally substituted C1-C6 alkyl group, -C(=O)(C1-C6 alkyl), -C(=O)phenyl, an amino protecting group, or In some other embodiments, m6 and m7 are each 1. In other embodiments, m6 is 0 and m7 is 1. In other embodiments, m6 is 1 and m7 is 0. In other embodiments, both m6 and m7 are 0. In some other embodiments, L 3a It is a C2-C6 alkylene group. In other embodiments, L 3a It is a 3-12 or 3-6-membered heteroalkylene group containing one, two, or three heteroatoms selected from O, S, or N. In other embodiments, the heteroalkylene group contains one or two nitrogen atoms. In some embodiments, L 3b It is a C2-C6 alkylene linkage. In other embodiments, L 3b It is a 3-12 or 3-6-membered heteroalkylene group, containing 1, 2, or 3 heteroatoms selected from O, S, or N. In other embodiments, the heteroalkylene group contains 1 or 2 nitrogen atoms. In other embodiments, L 3a and L 3b Each is independently an optionally substituted phenylene; or a C2-C6 alkylene or a 3-12 heteroalkylene, wherein one of the methylene units is optionally substituted with a substituted phenylene. In some other embodiments, each R 12a is H, and R 9a 、R 9b 、R 10a and R 10b Each is independently H, -OR 11 or -NHR 12b Each R12b Independently protected by H, -C(=O) (C1-C6 alkyl), -C(=O) phenyl, amino, or Or -NHR 12b Hydrogen is absent in R, and R 12b It is a divalent amino protecting group. In some specific embodiments, R 12a H and R 12b It is protected by H or an amino group (e.g., Ac), and R 9a 、R 9b 、R 10a and R 10b Each is independently -NH2, NH (optionally substituted C1-C6 alkyl) or -NHC(=O)CH3. In other embodiments, R 11 is H, and R 9a 、R 9b 、R 10a and R 10b Each is -OH. In other embodiments, R 9a 、R 9b 、R 10a and R 10b At least one of them is H. In other embodiments, R 11 for In other embodiments, R 12a is H, and R 12b for In some implementations, L 4a It is a C2-C6 alkylene group. In other embodiments, L 4a It is a 3-12 or 3-6-membered heteroalkylene group containing one, two, or three heteroatoms selected from O, S, or N. In other embodiments, the heteroalkylene group contains one or two nitrogen atoms. In some embodiments, L 4b It is a C2-C6 alkylene group. In other embodiments, L 4b It is a 3-12 or 3-6-membered heteroalkylene linkage containing one, two, or three heteroatoms selected from O, S, or N. In other embodiments, the heteroalkylene contains one or two nitrogen atoms. In other embodiments, L 4a and L 4b Each is independently an optionally substituted phenylene. In some such embodiments, R 13a 、R 13b 、R 14a and R 14b Independently, it is H, a hydroxyl group, or a protected hydroxyl group. In some other embodiments, R 13a 、R 13b 、R 14a and R 14b Each is independently H, -NH 2、-NH (optionally substituted C1-C6 alkyl) or protected amino group. In other embodiments, R 13a 、R 13b 、R 14a and R 14b At least one of them is H.

[0013] In some embodiments, the polymer of formula (I) has the structure of formula (Ia):

[0014] In other embodiments, the polymer of formula (I) or (Ia) has the structure of formula (Ib), (Ib-1), (Ic), or (Ic-1): In some such implementations, R 5b and R 6b Each is independently -NH2 or -NHAc. In other embodiments, R 5b and R 6b Each is -OH. In some other embodiments, R... 5b and R 6b At least one of them is H. In other embodiments, the structure of formula (Ib-1) also has the structure of formula (Ib-2) or (Ib-3): In other embodiments, the structure of formula (Ib-1) also has the structure of formula (Ib-4):

[0015] In some embodiments, the polymer of formula (I) has the structure of formula (Id), (Ie), or (If):

[0016] In some embodiments of polymers of formula (I) or any of its substructures, j is an integer from about 50 to 1000, from about 100 to about 800, from about 200 to about 700, from about 300 to about 600, or from about 400 to about 500. In some embodiments, the average molecular weight of the polymer is from about 5 kDa to about 100 kDa, from about 10 kDa to about 75 kDa, from about 12.5 kDa to about 50 kDa, or from about 15 kDa to about 30 kDa. In some specific embodiments, the molecular weight of the polymer is about 20 kDa.

[0017] Another aspect of this disclosure relates to polymers for the synthesis of liquid-phase oligonucleotides having the structure of formula (II): in: A is selected from the following group: carbon atom, C2-C 20 Alkylene, 2-20-membered heteroalkylene, phenylene, 5-10-membered heteroarylene, C 5-10 Cycloalkylene and 5-10 membered heterocyclic alkylene; Each R 15 Independently selected from the group consisting of: -OC1-C6 alkyl, -OC(O)(C1-C6 alkyl), -OC(O) phenyl, -NHC(O)(C1-C6 alkyl), -NHC(O) phenyl, -NHC(O) phenylene-acetoxymethyl, protected hydroxyl or protected amino; Each R 16 Independently -OH, -NH2, or -NH (optionally substituted C1-C6 alkyl); q 1 and q 2 Each is an independent integer between 10 and 500; and s and t are each independent integers from 1 to 4, where s+t is equal to or greater than 2.

[0018] In some embodiments of the polymer of formula (II), s+t is 4. In some specific embodiments, s is 2 and t is 2. In some embodiments, the polymer of formula (II) has the structure of (IIa):

[0019] In some embodiments, A is a carbon atom or a phenylene oxide. In some specific embodiments, A is a carbon atom.

[0020] In some other embodiments of the polymer of formula (II), A is C6 to C6. 12 Alkylene. In some such embodiments, s+t is 8. In some specific embodiments, s and t are each 4. In other embodiments, s is 5 and t is 3, or s is 3 and t is 5. In some embodiments, the polymer of formula (II) has the structure of formula (IIb):

[0021] In some embodiments, polymers of formula (II) (including formulas (IIa) and (IIb)) each q 1 Independently, it is about 20 to about 150, about 40 to about 100, or about 50 to about 75. In some embodiments, each q 2 Independently, the molecular weight is about 30 to about 150, about 40 to about 100, or about 50 to about 75. In some embodiments, the average molecular weight of the polymer is about 2 kDa to about 60 kDa, about 5 kDa to about 50 kDa, about 10 kDa to about 50 kDa, about 15 kDa to about 30 kDa, or about 20 kDa.

[0022] Another aspect of this disclosure relates to a method for preparing oligonucleotides via liquid-phase oligonucleotide synthesis, including: The polymer described herein is dissolved in a first solvent to form a reaction matrix; and The polymer is reacted with one or more nucleoside analogs to form a first bioconjugate containing a structure of formula (III): in: B 1 It is a nitrogen-containing base; G 1 It is a 5' hydroxyl-blocking group; X is O or NR 20 ; R 20 It is an H or C1-C6 alkyl group; R a It is -H, -OH, halogen, -O-(C1-C6 alkyl), -O-(C1-C6 haloalkyl), or -OY, where Y is a 2' hydroxyl protecting group; and L 5 It is a cleavable heteroalkylene linkage in which one or more carbon atoms are replaced by O, S, N, C (=O) or C (=S).

[0023] In some embodiments of the method described herein, the structure of equation (III) is also represented by equation (IIIa):

[0024] In some implementations, B 1 Independently, it may be an optionally protected adenine, an optionally protected denitroadenine, an optionally protected cytosine, an optionally protected guanine, an optionally protected denitroguanine, an optionally protected thymine, or an optionally protected uracil. In some embodiments, B 1 yes where R x It is hydrogen, an unsubstituted or substituted C1-C6 alkyl or amino protecting group, or -NHR. x Hydrogen is absent in R x It is a divalent amino protecting group. In some embodiments, G 1 The protecting group is a triphenylmethyl hydroxyl group selected from the group consisting of (4-methoxyphenyl)diphenylmethyl, bis(4-methoxyphenyl)phenylmethyl, tris(4-methoxyphenyl)methyl, 9-phenylxanth-9-yl, and 9-(4-methoxyphenyl)xanth-9-yl. In some specific embodiments, G 1 It is bis(4-methoxyphenyl)phenylmethyl.

[0025] In some embodiments of the methods described herein, the average molecular weight of the polymer is about 10 kDa to about 50 kDa, about 15 kDa to about 30 kDa, or about 20 kDa.

[0026] In some embodiments, the method described herein further includes: removing the 5' hydroxyl blocking group (G 1 The process involves forming a 5' unblocked first bioconjugate and separating the 5' unblocked first bioconjugate. In some embodiments, the separation of the 5' unblocked first bioconjugate is achieved by precipitation, dialysis, or filtration. In some specific embodiments, the separation of the 5' unblocked first bioconjugate is achieved by precipitation. In some of these specific embodiments, precipitation is achieved in diethyl ether. In other such specific embodiments, precipitation is achieved in isopropanol.

[0027] In some embodiments, the method described herein further includes: (a) Reacting the 5' unclosed first bioconjugate with one or more nucleoside phosphoramidide analogs in a second solvent to form a second bioconjugate containing the (IV) structure: in: G 2 It is a 5' hydroxyl-blocking group; B 2 It is a nitrogenous base; and R e It is a phosphite protecting group; (b) The phosphorous portion in oxidized form (IV); (c) Removal of the 5' blocking group G 2 To form a second bioconjugate with an inclusive (IV') structure at the 5' unclosed position: Where Z is O or S; and (d) Separate the 5' unsealed second bioconjugate.

[0028] In some embodiments, the structure of equation (IV) is further represented by (IVa) and the structure of equation (IV') is further represented by (IV'a):

[0029] In some embodiments of the method described herein, the method further includes blocking the unreacted 5' hydroxyl group in the unblocked first bioconjugate prior to step (b). In some embodiments, B 2Independently, it may be an optionally protected adenine, an optionally protected denitroadenine, an optionally protected cytosine, an optionally protected guanine, an optionally protected denitroguanine, an optionally protected thymine, or an optionally protected uracil. In some embodiments, B2 is... where R x It is hydrogen, an unsubstituted or substituted C1-C6 alkyl or amino protecting group, or -NHR. x Hydrogen is absent in R x G2 is a divalent amino protecting group. In some embodiments, G2 is a triphenylmethyl hydroxy protecting group selected from the group consisting of (4-methoxyphenyl)diphenylmethyl, bis(4-methoxyphenyl)phenylmethyl, tris(4-methoxyphenyl)methyl, 9-phenylxanth-9-yl, and 9-(4-methoxyphenyl)xanth-9-yl. In some specific embodiments, G... 2 It is bis(4-methoxyphenyl)phenylmethyl.

[0030] In some embodiments, separation of the 5' unblocked second bioconjugate is achieved by precipitation, filtration, or dialysis. In some preferred embodiments, separation of the 5' unblocked second bioconjugate is achieved by precipitation. In some preferred embodiments, precipitation is carried out in diethyl ether. In other preferred embodiments, precipitation is carried out in isopropanol.

[0031] In some embodiments of the method described herein, steps (a)-(d) are repeated multiple times until one or more oligonucleotides of the desired length are synthesized. In some embodiments, steps (a)-(d) of the method described herein are repeated for at least about 10 cycles. In some embodiments, the method described herein further includes the removal of oligonucleotides from the polymer.

[0032] In some embodiments, the first solvent and the second solvent comprise one or more aprotic polar solvents or combinations thereof. In some embodiments, one or more aprotic polar solvents include acetonitrile, tetrahydrofuran (THF), dimethylformamide (DMF), dimethyl sulfoxide (DMSO), dichloromethane (DCM), sulfolane, or combinations thereof. In some specific embodiments, one or more aprotic polar solvents are acetonitrile.

[0033] Another aspect of this disclosure relates to oligonucleotides prepared by the methods described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is an HPLC chromatogram of an 8dT oligonucleotide conjugate synthesized using a polymer (1a) with an average molecular weight of about 20 kDa via the liquid phase method disclosed herein, according to embodiments of this application.

[0035] Figure 2 This is an HPLC chromatogram of 8dT oligonucleotide synthesized using polymer (2) by the liquid phase method disclosed herein according to an embodiment of this application.

[0036] Figure 3 The image shows the HPLC chromatogram of dT-dT-dA-dA-dG-dG-dC-dC oligonucleotides synthesized using a polymer (1a) with an average molecular weight of about 20 kDa according to embodiments of this application via the liquid phase method disclosed herein.

[0037] Figure 4 This is an HPLC chromatogram of an 8dT oligonucleotide conjugate synthesized using a polymer (1b) with an average molecular weight of 10 kDa via the liquid phase method disclosed herein, according to embodiments of this application.

[0038] Figure 5 The image shows the HPLC chromatogram of the dT-mU-mU-mU-mU-mU-mU-mU-mU oligonucleotide conjugate synthesized using a polymer (3a) with an average molecular weight of 10 kDa through the liquid phase method disclosed herein, according to embodiments of this application.

[0039] Figure 6A This is an HPLC chromatogram of a 9dT oligonucleotide conjugate synthesized using a polymer (3b) with an average molecular weight of 5 kDa via the liquid phase method disclosed herein, according to embodiments of this application. Figure 6B This is an HPLC chromatogram of an oligonucleotide conjugate synthesized using a polymer (3b) with an average molecular weight of 5 kDa via the liquid phase method disclosed herein, according to embodiments of this application.

[0040] Figure 7 The HPLC chromatogram is of the dT(S)dT(S)dT(S)dT(S)dT(S)dT(S)dT(S)dT(S)dT oligonucleotide synthesized using the liquid phase method disclosed herein with a polymer (3b) having an average molecular weight of 5 kDa, according to the embodiments of this application.

[0041] Figure 8 The HPLC chromatogram is of the dTdTdT(S)dT(S)dT(S)dT(S)dT(S)dT(S)dT oligonucleotide synthesized using the liquid phase method disclosed herein with a polymer (1b) having an average molecular weight of 10 kDa, according to the embodiments of this application.

[0042] Figure 9A The HPLC chromatogram is of a 7dT(S)dTdT-OH oligonucleotide conjugate synthesized using a polymer (1c) with an average molecular weight of 5 kDa and m-CPBA as an oxidant according to embodiments of this application. Figure 9BThe HPLC chromatogram is of a 7dT(S)dTdT-OH oligonucleotide conjugate synthesized according to embodiments of this application using a polymer (1c) with an average molecular weight of 5 kDa and tBuOOH as an oxidant via the liquid phase method disclosed herein.

[0043] Figure 10 This is an HPLC chromatogram of dT-8mU(VP)mU oligonucleotide conjugate synthesized using a polymer (1b) with an average molecular weight of 10kDa via the liquid phase method disclosed herein, according to embodiments of this application.

[0044] Figure 11 This is an HPLC chromatogram of an 8dT-VPmA oligonucleotide conjugate synthesized using a polymer (1c) with an average molecular weight of 5 kDa via the liquid phase method disclosed herein, according to embodiments of this application.

[0045] Figure 12 The HPLC chromatogram is of the dTdTdT(S)dT(S)dT(S)dT(S)dT(S)dT(S)dT oligonucleotide synthesized using a polymer (3b) with an average molecular weight of 5 kDa according to the embodiments of this application via the liquid phase method disclosed herein.

[0046] Figure 13A This is an HPLC chromatogram of polymer (4)-8dT-oligonucleotide synthesized by the liquid phase method disclosed herein according to the embodiments of this application.

[0047] Figure 13B This is an HPLC chromatogram of polymer (6)-8dT-oligonucleotide synthesized by the liquid phase method disclosed herein according to the embodiments of this application.

[0048] Figure 13C This is an HPLC chromatogram of polymer (7)-8dT-oligonucleotide synthesized by the liquid phase method disclosed herein according to the embodiments of this application. DETAILED DESCRIPTION

[0049] Solid-phase oligonucleotide synthesis enables oligonucleotide synthesis at a solid-support-liquid interface. The solid support is insoluble in the liquid medium (e.g., organic solvent). Examples of solid supports include controlled-pore glass (CPG) and porous cross-linked polystyrene particles. In contrast, liquid-phase oligonucleotide synthesis (LPOS) relies on a soluble organic compound as a support (hub) for oligonucleotide synthesis in solution. Typically, conventional LPOS utilizes a soluble support having one or more functional groups as an anchor to bind and synthesize oligonucleotides. Embodiments of this disclosure relate to a method for liquid-phase oligonucleotide synthesis using a soluble polymer having multiple functional groups as an anchor for oligonucleotide synthesis. For example, the polymers described herein may contain, for example, reactive amino groups, which are capable of efficiently binding nucleosides or nucleotide analogs and offer higher yields compared to known liquid-phase and solid-phase oligonucleotide synthesis. The polymers described herein include one or more polyethylene groups whose length can be controlled to effectively reduce impurity trapping and nonspecific adsorption in the polymer, thereby improving the performance of liquid-phase oligonucleotide synthesis using these polymers. The methods described herein are suitable for oligonucleotide synthesis at the kilogram level, exhibiting good loading capacity and oligonucleotide yield. definition

[0050] The chapter titles used in this document are for organizational purposes only and should not be construed as limiting the content.

[0051] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Unless otherwise stated, all patents, applications, published applications, and other publications cited herein are incorporated herein by reference in their entirety. If a term has multiple definitions herein, the definition in this section shall prevail unless otherwise stated. Unless the context clearly requires otherwise, the singular forms “a,” “an,” and “the” used in the specification and appended claims include the plural forms. Unless otherwise stated, conventional methods of mass spectrometry, NMR, HPLC, protein chemistry, biochemistry, recombinant DNA techniques, and pharmacology are employed. Unless otherwise stated, the use of “or” or “and” means “and / or.” Furthermore, the use of the term “comprising” and other forms (e.g., “including,” “containing,” and “having”) is not restrictive. As used herein, whether in transitional phrases or in the body of claims, the terms “comprising” and “including” shall be interpreted as having an open meaning. That is, these terms shall be interpreted synonymously with the phrases “having at least” or “at least comprising.” When used in the context of a process / method / procedure, the term "comprising" means that the process / method / procedure includes at least the stated step, but may include other steps. When used in the context of a compound, composition, or apparatus, the term "comprising" means that the compound, composition, or apparatus includes at least the stated feature or component, but may also include other features or components. "Constitutes of" means including and limited to the contents listed in the phrase "composes of". The reference to "substantially (or substantially) constitutes" means including any element listed in the phrase and also including other elements that do not interfere with or contribute to the activity or effect of the listed elements.

[0052] As used in this article, the term "average molecular weight" refers to the weight-average molecular weight (Mw) of a sample population composed of polymer species with multiple molecular weights. This quantity is defined by the following formula: Where n i M represents the number of molecules of substance i. i This represents the molecular weight of the i-th substance. Unless otherwise stated, the term "molecular weight" as used herein refers to weight-average molecular weight.

[0053] As used herein, the term "polymer" traditionally refers to a large molecule composed of smaller monomers or oligomeric subunits covalently linked together to form a chain. A "homopolymer" is a polymer consisting of only one repeating monomer unit. A "copolymer" is a polymer consisting of two or more repeating monomer units. Linear polymers consist of monomer subunits linked together in a continuous length to form a polymer chain. Branched polymers are similar to linear polymers but have side chains extending from various branch points along the main polymer line. Star polymers are similar to branched polymers, except that multiple side branches radiate from a single branch point, resulting in a star-shaped or spoke-like appearance.

[0054] As used herein, “alkyl” refers to a straight or branched hydrocarbon chain containing a fully saturated (without double or triple bonds) hydrocarbon group. An alkyl group can have 1 to 20 carbon atoms (numerical ranges appearing herein, such as “1 to 20”, refer to integers within a given range; for example, “1 to 20 carbon atoms” means that an alkyl group can consist of 1, 2, 3, etc., up to and including 20 carbon atoms, but this definition also covers the occurrence of the term “alkyl” without a specified numerical range). An alkyl group can also be a medium-sized alkyl group having 1 to 10 carbon atoms. An alkyl group can also be a lower alkyl group having 1 to 6 carbon atoms. The alkyl group of a compound can be specified as “C1-C4 alkyl” or a similar designation. By way of example only, “C1-C4 alkyl” means that the alkyl chain has 1 to 4 carbon atoms, i.e., the alkyl chain is selected from methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl. Typical alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl (straight-chain or branched), and hexyl (straight-chain or branched). Alkyl groups can be substituted or unsubstituted.

[0055] As used herein, "alkenyl" refers to a straight-chain or branched hydrocarbon chain containing one or more double bonds. Alkenyl groups can have 2 to 20 carbon atoms. For example, "C2-C6 alkenyl" indicates that the alkenyl chain contains 2-6 carbon atoms, meaning the alkenyl chain is selected from the group consisting of: vinyl, propen-1-yl, propen-2-yl, propen-3-yl, buten-1-yl, buten-2-yl, buten-3-yl, buten-4-yl, 1-methyl-propen-1-yl, 2-methyl-propen-1-yl, 1-ethyl-vinyl-1-yl, 2-methyl-propen-3-yl, but-1,3-dienyl, but-1,2-dienyl, and but-1,2-dien-4-yl. Typical alkenyl groups include, but are not limited to, vinyl, propenyl, butenyl, pentenyl, and hexenyl. Alkenyl groups can be substituted or unsubstituted.

[0056] As used herein, "alkynyl" refers to a straight-chain or branched hydrocarbon chain containing one or more triple bonds. Alkynyl groups can have 2-20 carbon atoms. For example, "C..." 2- "C4 ynyl" indicates that the ynyl chain contains 2-6 carbon atoms, meaning the ynyl chain is selected from the following group: ethynyl, propyn-1-yl, propyn-2-yl, butyn-1-yl, butyn-3-yl, butyn-4-yl, and 2-butynyl. Typical ynyl groups include, but are not limited to, ethynyl, propynyl, butynyl, pentynyl, and hexynyl. The ynyl group can be substituted or unsubstituted.

[0057] As used herein, “cycloalkyl” refers to a fully saturated (without double or triple bonds) monocyclic or polycyclic hydrocarbon ring system. When two or more rings are involved, they may be fused, bridged, or spiro-linked. The term “fused” as used herein refers to two rings having two atoms and a common bond. The term “bridged cycloalkyl” as used herein refers to a compound in which the cycloalkyl group contains a bond connecting one or more atoms that are not adjacent. The term “spiro-linked” as used herein refers to two rings having a common atom and not connected by a bridging bond. Cycloalkyl groups may contain 3-10 atoms in one or more rings, 3-8 atoms in one or more rings, or 3-6 atoms in one or more rings. Cycloalkyl groups may be unsubstituted or substituted. Examples of monocyclic cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Examples of bicyclic fused cycloalkyl groups are decahydronaphthalenyl, phenalenyl, and tetradecahydroanthracenyl; examples of bicyclic bridged cycloalkyl groups are bicyclic [1.1.1]pentyl, adamantyl, and norbornanyl; examples of bicyclic spiroalkyl groups include spiro[3.3]heptane and spiro[4.5]decane.

[0058] As used herein, “carbocyclic group” refers to a non-aromatic monocyclic or polycyclic hydrocarbon ring system. As described herein, when two or more rings are involved, the rings may be fused, bridged, or spirocyclic. A carbocyclic group may contain 3-30 atoms in one or more rings, 3-20 atoms in one or more rings, 3-10 atoms in one or more rings, 3-8 atoms in one or more rings, or 3-6 atoms in one or more rings. A carbocyclic group may be unsubstituted or substituted. Examples of carbocyclic groups include, but are not limited to, cycloalkyl groups as defined herein and the non-aromatic portions of 1,2,3,4-tetrahydronaphthalene, 2,3-dihydro-1H-indene, 5,6,7,8-tetrahydroquinoline, and 6,7-dihydro-5H-cyclopenta[b]pyridine.

[0059] As used herein, "aryl" refers to a carbocyclic (all-carbon) monocyclic or polycyclic aromatic ring system (including fused ring systems where two carbon rings share a chemical bond) that has a fully delocalized π-electron system in all rings. The number of carbon atoms in an aryl group can vary. For example, an aryl group can be a C6 aryl group or a C6 aryl group. 10 Aryl groups. Examples of aryl groups include, but are not limited to, benzene and naphthalene. Aryl groups can be substituted or unsubstituted.

[0060] As used herein, a “heteroaryl” is a monocyclic or polycyclic aromatic ring system (a ring system with a fully delocalized π-electron system) containing one or more heteroatoms (e.g., 1, 2, or 3 heteroatoms), that is, elements other than carbon, including but not limited to nitrogen, oxygen, and sulfur. The number of atoms in one or more rings of a heteroaryl group can vary. For example, a heteroaryl group may contain 5-10 atoms in one or more rings, 6-10 atoms in one or more rings, or 5-6 atoms in one or more rings, such as nine carbon atoms and one heteroatom; eight carbon atoms and two heteroatoms; seven carbon atoms and three heteroatoms; eight carbon atoms and one heteroatom; seven carbon atoms and two heteroatoms; six carbon atoms and three heteroatoms; five carbon atoms and four heteroatoms; five carbon atoms and one heteroatom; four carbon atoms and two heteroatoms; three carbon atoms and three heteroatoms; four carbon atoms and one heteroatom; three carbon atoms and two heteroatoms; or two carbon atoms and three heteroatoms. Furthermore, the term "heteroaryl" includes fused ring systems in which two rings (such as at least one aryl ring and at least one heteroaryl ring, or at least two heteroaryl rings) share at least one chemical bond. Examples of heteroaryl rings include, but are not limited to, furan, furazolidone, thiophene, benzothiophene, phthalazine, pyrrole, oxazole, benzoxazole, 1,2,3-oxadiazole, 1,2,4-oxadiazole, thiazole, 1,2,3-thiadiazole, 1,2,4-thiadiazole, benzothiazole, imidazole, benzimidazole, indole, indazole, pyrazole, benzopyrazole, isoxazole, benzoisoxazole, isothiazole, triazole, benzotriazole, thiadiazole, tetrazolium, pyridine, pyridazine, pyrimidine, pyrazine, purine, pteridine, quinoline, isoquinoline, quinazoline, quinoxaline, borazine, and triazine. Heteroaryl groups can be substituted or unsubstituted.

[0061] As used herein, a "heterocyclic group" refers to a ternary, quaternary, pentaneary, hexanal, septaneary, octaneary, nonanal, or decacyclic monocyclic, bicyclic, or tricyclic ring system in which a carbon atom, together with 1 to 5 heteroatoms, constitutes the ring system. However, the heterocycle may optionally contain one or more unsaturated bonds positioned such that no fully delocalized π-electron system exists in any of the rings (i.e., the heterocyclic group is not aromatic). Heteroatoms are elements other than carbon, including but not limited to oxygen, sulfur, and nitrogen. Heterocycles may further contain one or more carbonyl functional groups, thus including oxocyclic systems such as lactams, lactones, and cyclic carbamates. When composed of two or more rings, the rings may be fused, bridged, or spirocoupled together. As used herein, the term "fused" refers to two rings having two atoms and a common bond. As used herein, the term "bridged heterocyclic group" refers to a compound in which the heterocyclic group contains a bond connecting one or more atoms that are not adjacent to each other. As used herein, the term "spirocyclic" refers to two rings that share a common atom and are not connected by a bridging link. Heterocyclic groups may contain 3-10 atoms in one or more rings, 3-8 atoms in one or more rings, 3-6 atoms in one or more rings, or 5-6 atoms in one or more rings. Examples include five carbon atoms and one heteroatom; four carbon atoms and two heteroatoms; three carbon atoms and three heteroatoms; four carbon atoms and one heteroatom; three carbon atoms and two heteroatoms; two carbon atoms and three heteroatoms; one carbon atom and four heteroatoms; three carbon atoms and one heteroatom; or two carbon atoms and one heteroatom. Additionally, any nitrogen atom in the heterocyclic group may be quaternized. Heterocyclic groups can be connected to the rest of the molecule via a carbon atom (C-linked) or via a heteroatom (such as a nitrogen atom (N-linked)) in the heterocyclic group. Heterocyclic groups can be unsubstituted or substituted.Examples of such "heterocyclic" groups include, but are not limited to, aziridine, ethylene oxide, thiacyclopropane, aziridine, oxacyclobutane, 1,3-dioxin, 1,3-dioxane, 1,4-dioxane, 1,2-dioxopentane, 1,3-dioxopentane, 1,4-dioxopentane, 1,3-oxathion, 1,4-oxathionine, 1,3-oxathionecyclopentane, 1,3-dithionecyclopentane, 1,3-dithionecyclopentane, 1,4-oxathionecyclohexane, tetrahydro-1,4-thiazine, 2H-1,2-oxazine, maleimide, succinimide, barbituric acid, thiobarbituric acid, etc. Dioxopiperazine, hydantoin, dihydrouracil, trioxane, hexahydro-1,3,5-triazine, imidazoline, imidazoline, isoxazoline, isoxazoline, oxazoline, oxazoline, oxazolidinone, thiazoline, thiazoline, morpholine, ethylene oxide, piperidine N-oxide, piperidine, piperazine, pyrrolidine, aziridine heptane, pyrrolidone, pyrrolidone, 4-piperidinone, pyrzoline, pyrrolidine, 2-oxopyrrolidine, tetrahydropyran, 4H-pyran, tetrahydrothiaranol, thiomorpholine sulfoxide, thiomorpholine sulfone and their benzo[a]-fused analogues (e.g., benzimidazolinone, tetrahydroquinoline and / or 3,4-methylenedioxyphenyl). Examples of spirocycloheterocyclic groups include 2-azaspiro[3.3]heptane, 2-oxaspiro[3.3]heptane, 2-oxa-6-azaspiro[3.3]heptane, 2,6-diazaspiro[3.3]heptane, 2-oxaspiro[3.4]octane and 2-azaspiro[3.4]octane.

[0062] As used in this article, "alkylene" refers to a fully saturated bimolecular chemical group, either branched or straight-chain, containing only carbon and hydrogen, and connected to the rest of the molecule via two connection points. For example, "C..." 1- C 10 "alkylene" indicates that the alkylene chain contains 1-10 carbon atoms. Non-limiting examples include ethylene (-CH2CH2-), propylene (-CH2CH2CH2-), butylene (-CH2CH2CH2CH2-), and pentylene (-CH2CH2CH2CH2CH2-).

[0063] As used in this article, "alkenyl" refers to a straight-chain or branched bimolecular chemical group that contains only carbon and hydrogen and at least one carbon-carbon double bond connecting the rest of the molecule through two connection points. The alkenyl group can be designated as "C". 2- C 10 "Alkenyl" or similar names. For example only, "C..." 2- C 10 "Subalkenyl" indicates that there are 2-10 carbon atoms in the subalkenyl chain.

[0064] As used in this article, "ethynyl group" refers to a straight-chain or branched bimacro chemical group containing only carbon and hydrogen and at least one carbon-carbon triple bond connecting the rest of the molecule through two bonding sites. The ethynyl group can be designated as "C". 2- C 10 "Alynyl" or similar names. For example, "C 2- C 10 "Imyynyl" indicates that there are 2-10 carbon atoms in the isynyl chain.

[0065] As used herein, “heteroalkylene” means an alkylene group containing one or more heteroatoms in its carbon backbone as defined herein (i.e., an alkylene group in which one or more carbon atoms are substituted by heteroatoms (e.g., nitrogen, oxygen, or sulfur atoms)). For example, -CH2- can be substituted by -O-, -S-, or -NH-, or -CH(R)- can be substituted by -N(R)-. Heteroalkylene groups include, but are not limited to, ethers, thioethers, amino-alkylene, and alkylene-amino-alkylene moieties. In some embodiments, a heteroalkylene group may comprise one, two, three, four, or five -CH2CH2O- units. Alternatively and / or additionally, one or more carbon atoms (e.g., -CH2-) may also be oxo-substituted (=O) to form a carbonyl-C(=O)-, or substituted (=S) to form a thiocarbonyl-C(=S)-.

[0066] As used herein, "aralkyl" and "(aryl)alkyl" refer to an aryl group as defined above, linked by an alkylene group as a substituent as described above. The alkylene and aryl groups of an aralkyl group may be substituted or unsubstituted. Examples include, but are not limited to, benzyl, 2-phenylalkyl, 3-phenylalkyl, and naphthylalkyl. In some embodiments, the alkylene group is an unsubstituted straight chain containing 1, 2, 3, 4, 5, or 6 methylene units.

[0067] As used herein, “heteroarylalkyl” and “(heteroaryl)alkyl” refer to a heteroaryl group as defined above, linked by an alkylene group as a substituent as described above. The alkylene and heteroaryl groups of a heteroarylalkyl group may be substituted or unsubstituted. Examples include, but are not limited to, 2-thienylalkyl, 3-thienylalkyl, furanylalkyl, thienylalkyl, pyrroliylalkyl, pyridylalkyl, isoxazolylalkyl, and imidazolylalkyl, and their benzo-fused analogs. In some embodiments, the alkylene group is an unsubstituted straight chain containing 1, 2, 3, 4, 5, or 6 methylene units.

[0068] As used herein, “(heterocyclic)alkyl” refers to a heterocyclic or heterocyclic group as defined above, linked by an alkylene group as a substituent. The alkylene and heterocyclic groups of a (heterocyclic)alkyl group may be substituted or unsubstituted. Examples include, but are not limited to, (tetrahydro-2H-pyran-4-yl)methyl, (piperidin-4-yl)ethyl, (piperidin-4-yl)propyl, (tetrahydro-2H-thiaran-4-yl)methyl, and (1,3-thiazin-4-yl)methyl. In some embodiments, the alkylene group is an unsubstituted straight chain containing 1, 2, 3, 4, 5, or 6 methylene units.

[0069] As used herein, “cycloalkylalkyl” and “(cycloalkyl)alkyl” refer to a cycloalkyl group (as defined herein) linked via an alkylene group as a substituent. Examples include, but are not limited to, cyclopropylmethyl, cyclobutylmethyl, cyclopentylethyl, and cyclohexylpropyl. In some embodiments, the alkylene group is an unsubstituted straight chain containing 1, 2, 3, 4, 5, or 6 methylene units.

[0070] As used in this article, "alkoxy" refers to the formula -OR, where R is an alkyl group as defined above, such as "C". 1-9 "Alkoxy" includes, but is not limited to, methoxy, ethoxy, n-propoxy, 1-methylethoxy (isopropoxy), n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, etc.

[0071] As used herein, "haloalkyl" refers to an alkyl group in which one or more hydrogen atoms are substituted by a halogen (e.g., monohaloalkyl, dihaloalkyl, and trihaloalkyl). Such groups include, but are not limited to, chloromethyl, fluoromethyl, difluoromethyl, trifluoromethyl, and 1-chloro-2-fluoromethyl, 2-fluoroisobutyl. Haloalkyl groups may be substituted or unsubstituted.

[0072] As used herein, "haloalkoxy" refers to an alkoxy group in which one or more hydrogen atoms are substituted by a halogen (e.g., monohaloalkoxy, dihaloalkoxy, and trihaloalkoxy). Such groups include, but are not limited to, chloromethoxy, fluoromethoxy, difluoromethoxy, trifluoromethoxy, and 1-chloro-2-fluoromethoxy, 2-fluoroisobutoxy. Haloalkoxy groups may be substituted or unsubstituted.

[0073] As used herein, “amino” refers to the -NH2 group. The term “monosubstituted amino group” as used herein refers to an amino (-NH2) group in which one hydrogen atom is replaced by a substituent. The term “disubstituted amino group” as used herein refers to an amino (-NH2) group in which both hydrogen atoms are replaced by substituents. As used herein, the term “optionally substituted amino” refers to -NR A R B Group, wherein R A and R BIndependently, it is hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, heterocyclic, aralkyl, or heterocyclic (alkyl), as defined herein.

[0074] As used herein, "alkylamino" or "(alkyl)amino" refers to -NR A R B Group, wherein R A and R B It is hydrogen or an alkyl group as defined above, and R A and R B At least one of them is an alkyl group. The alkyl portion of an (alkyl)amine includes, for example, C1-C6 alkyl groups.

[0075] As used herein, “aminoalkyl” or “(amino)alkyl” means one or more hydrogen atoms surrounded by an amino group or “-NR” as defined herein. A R B "The alkyl group is replaced by a group. The alkyl part of an aminoalkyl group includes, for example, C1-C6 alkyl groups."

[0076] As used in this article, the term "halogen atom" or "halogen" refers to any radioactive stable atom in column 7 of the periodic table (such as fluorine, chlorine, bromine, and iodine).

[0077] As used herein, “alkoxyalkyl” or “(alkoxy)alkyl” refers to an alkoxy group linked by an alkylene group, such as C2-C8 alkoxyalkyl or (C1-C6 alkoxy)C1-C6 alkyl, for example –(CH2). 1-3 -OCH3.

[0078] As used herein, “O-alkoxyalkyl” or “-O-(alkoxy)alkyl” refers to an alkoxy group linked by an –O-(alkylene) group, such as –O-(C1-C6 alkoxy)C1-C6 alkyl, for example –O-(CH2). 1-3 -OCH3.

[0079] As used in this article, "aryloxy group" and "arylthio group" refer to RO- and RS-, where R is an aryl group as defined above, such as, but not limited to, phenyl. Both aryloxy and arylthio groups can be substituted or unsubstituted.

[0080] The "thio" group refers to the "SR" group, where R can be hydrogen, alkyl, alkenyl, alkynyl, carbocyclic, aryl, heteroaryl, heterocyclic, aralkyl, or heterocyclic (alkyl), as defined above. The thio group can be substituted or unsubstituted.

[0081] The "thionyl" group refers to the "S(=O)R" group, where R can be the same as the thio group as defined. The thionyl group can be substituted or unsubstituted.

[0082] The "sulfonyl" group refers to the "SO2R" group, where R can be the same as the thio group as defined. The sulfonyl group can be substituted or unsubstituted.

[0083] The “O-carboxyl” group refers to the “RC(=O)O-” group, where R can be hydrogen, alkyl, alkenyl, alkynyl, carbocyclic, aryl, heteroaryl, heterocyclic, aralkyl, or heterocyclic (alkyl), as defined herein. The O-carboxyl group can be substituted or unsubstituted.

[0084] The terms "ester" and "C-carboxyl" refer to the "C(=O)OR" group, where R can be the same as defined for O-carboxyl. Esters or C-carboxyl groups can be substituted or unsubstituted.

[0085] The "trihalomethanesulfonyl" group refers to the "X3CSO2-" group, where X is a halogen.

[0086] The “trihalomethanesulfonamide” group refers to the “X3CS(O)2N(R)-” group, where X is a halogen and R is hydrogen, alkyl, alkenyl, alkynyl, carbocyclic, aryl, heteroaryl, heterocyclic, aralkyl or heterocyclic (alkyl), as defined above.

[0087] The "thiol" group refers to the "-SH" group.

[0088] The "S-sulfonamide group" refers to "-SO2N(R)". A R B )" group, wherein R A and R B It can be hydrogen, alkyl, alkenyl, alkynyl, carbocyclic, aryl, heteroaryl, heterocyclic, aralkyl, or heterocyclic (alkyl) as defined herein. The S-sulfonamide group can be substituted or unsubstituted.

[0089] The "N-sulfonamide group" refers to "RSO2N(R A )-” groups, wherein R and R A It can be hydrogen, alkyl, alkenyl, alkynyl, carbocyclic, aryl, heteroaryl, heterocyclic, aralkyl, or heterocyclic (alkyl) as defined herein. The N-sulfonamide group can be substituted or unsubstituted.

[0090] The "O-carbamoyl" group refers to "-OC(=O)N(R"). A R B )" group, wherein R A and R B It can be the same as defined by the S-sulfonamide group. The O-carbamoyl group can be substituted or unsubstituted.

[0091] The "N-carbamoyl" group refers to "ROC(=O)N(R A)-” groups, wherein R and R A It can be the same as defined regarding the N-sulfonamide group. The N-carbamoyl group can be substituted or unsubstituted.

[0092] The "O-thiocarbamoyl" group refers to "-OC(=S)-N(R)". A R B )" group, wherein R A and R B It can be the same as defined by the S-sulfonamide group. The O-thiocarbamoyl group can be substituted or unsubstituted.

[0093] The "N-thiocarbamoyl" group refers to "ROC(=S)N(R A )-” groups, wherein R and R A It can be the same as defined by the N-sulfonamide group. The N-thiocarbamoyl group can be substituted or unsubstituted.

[0094] The "C-amide group" refers to "-C(=O)N(R"). A R B )" group, wherein R A and R B It can be the same as defined by the S-sulfonamide group. The C-amide group can be substituted or unsubstituted.

[0095] The "N-amide group" refers to "RC(=O)N(R A )-” groups, wherein R and R A It can be the same as defined by the N-sulfonamide group. The N-amide group can be substituted or unsubstituted.

[0096] When the number of substituents is not specified (e.g., haloalkyl), one or more substituents may be present. For example, "haloalkyl" may include one or more of the same or different halogens.

[0097] It should be understood that in any compound described herein having one or more chiral centers, unless the absolute stereochemistry is explicitly specified, each center may independently have an R-configuration or an S-configuration or a mixture thereof. Therefore, the compounds provided herein may be enantiomerically pure, enantiomerically enriched, or mixtures of stereoisomers, and include all diastereomeric and enantiomeric forms. Furthermore, it should be understood that in any compound described herein having one or more double bonds that produce geometrical isomers that can be defined as E or Z, each double bond may independently be E or Z or a mixture thereof. Stereoisomers can be obtained, if desired, by methods such as stereoselective synthesis and / or separation of stereoisomers by chiral chromatography. Likewise, it should be understood that all tautomeric forms are intended to be included in any compound described.

[0098] Whenever a substituent is described as bimolecular (i.e., having two points of attachment to the rest of the molecule), it should be understood that, unless otherwise stated, the substituent can be configured in any direction. Thus, for example, it may be depicted as -AE- or The substituents include those oriented such that A is attached to the leftmost connection point of the molecule, and those attached to the rightmost connection point of the molecule. Additionally, if the group or substituent is described as... And when L is defined as a bond or does not exist; such groups or substituents are equivalent to

[0099] It should be understood that when the compounds disclosed herein have unfilled valences, those valences will be filled with hydrogen and / or deuterium.

[0100] It should be understood that the compounds described herein may be labeled by isotopic labeling or by other means, including but not limited to the use of chromophores or fluorescent moieties, bioluminescent labeling, or chemiluminescent labeling. Substitution with an isotope (such as deuterium) can provide certain therapeutic advantages resulting from increased metabolic stability (e.g., prolonged half-life in vivo or reduced dose requirement). The chemical elements represented in the compound structure may include any isotope of said element. For example, in the compound structure, a hydrogen atom may be explicitly disclosed or understood to be present in the compound. At any position in a compound where a hydrogen atom may be present, the hydrogen atom may be any isotope of hydrogen, including but not limited to hydrogen-1 (protium), hydrogen-2 (deuterium), and hydrogen-3 (tritium). Therefore, unless the context clearly specifies otherwise, the compounds referred to herein include all potential isotopic forms.

[0101] It should be understood that the methods and formulations described herein include the use of crystalline forms, amorphous phases, and / or pharmaceutically acceptable salts, solvates, hydrates, and conformational isomers of the compounds of preferred embodiments, as well as the use of metabolites and active metabolites having the same type of activity as these compounds. A conformer is a conformational isomer structure. Conformational isomerism refers to the phenomenon where the structures are identical but the conformations (conformations) of atoms around spin bonds differ. In specific embodiments, the compounds described herein are present in a solvated form in a pharmaceutically acceptable solvent (such as water, ethanol, etc.). In other embodiments, the compounds described herein are present in a non-solvated form. Solvates contain stoichiometric or non-stoichiometric amounts of solvent, which can form with pharmaceutically acceptable solvents (such as water, ethanol, etc.) during crystallization. When the solvent is water, a hydrate is formed, or when the solvent is an alcohol, an alcohol is formed. Furthermore, the compounds provided herein can exist in both non-solvated and solvated forms. Generally, for the purposes of the methods and compounds provided herein, a solvated form is considered equivalent to a non-solvated form. Other forms of the compound of the preferred embodiments may be provided, including amorphous, ground, and nanoparticle forms.

[0102] Similarly, it is understood that the compounds described herein (such as those of preferred embodiments) include any form of the compounds described herein (e.g., pharmaceutically acceptable salts, crystalline forms, amorphous forms, solvated forms, enantiomeric forms, isomeric forms, etc.).

[0103] As used herein, any abbreviations for protecting groups, amino acids and other compounds, unless otherwise stated, are in accordance with their common usage, recognized abbreviations, or the IUPAC-IUB Biochemical Nomenclature Committee (see Biochem. 11:942-944 (1972)).

[0104] As used herein, the terms “protecting group” and “multiple protecting groups” refer to any atom or group of atoms added to a molecule to prevent undesirable chemical reactions with existing groups in the molecule. Examples of protecting groups are described in T.W. Greene and P.G. W. Uts, *Protective Groups in Organic Synthesis*, 3rd ed., John Wiley & Sons, 1999, and in J.F. W. McOmie, *Protective Groups in Organic Chemistry*, Plenum Press, 1973, and are incorporated herein by reference only for the purpose of disclosing suitable protecting groups. Protecting groups are chosen in a manner that allows them to be stable to certain reaction conditions and to be removed at a convenient stage using methods known in the art. A non-limiting list of protecting groups includes benzyl (Bn); substituted benzyl; alkyl carbonyl (e.g., tert-butoxycarbonyl (BOC), acetyl (i.e., -C(=O)CH3 or Ac) or isobutyryl (iBu); arylalkyl carbonyl (e.g., benzyloxycarbonyl or benzoyl (i.e., -C(=O)Ph or Bz)); substituted methyl ether (e.g., methoxymethyl ether (MOM)); substituted ethyl ether (e.g., methoxyethyl ether (MOE); substituted benzyl ether; tetrahydropyranyl ether; silyl ether (e.g., trimethylsilyl (TMS), triethylsilyl, triisopropylsilyl), tert-butyldimethylsilyl (TBDMS), triisopropylsilyloxy methyl ester (TOM or tert-butyldiphenylsilyl); ester (e.g., benzoate); carbonate (e.g., methoxymethyl carbonate); sulfonate (e.g., toluenesulfonate or methanesulfonate); acyclic ketal (e.g., dimethyl acetal); cyclic ketal (e.g., 1,3-dioxane or 1,3-dioxolane); acyclic acetal; cyclic acetal; acyclic hemiacetal; cyclic hemiacetal; cyclic dithioketal (e.g., 1,3-dithiane or 1,3-dithiopentane); and triarylmethyl groups (e.g., triphenylmethyl; monomethoxytriphenylmethyl (MMTr); 4,4'-dimethoxytriphenylmethyl (DMTr); or 4,4',4”-trimethoxytriphenylmethyl (TMTr)).

[0105] As used herein, the term “leaving group” means any atom or part that can be substituted by another atom or part in a chemical reaction. More specifically, in some embodiments, “leaving group” means an atom or part that is substituted in a nucleophilic substitution reaction. In some embodiments, “leaving group” is any atom or part that is a conjugate base of a strong acid. Examples of suitable leaving groups include, but are not limited to, toluenesulfonates and halogens. Non-limiting features and examples of leaving groups can be found, for example, in *Organic Chemistry*, 2nd edition, Francis Carey (1992), pp. 328–331; *Introduction to Organic Chemistry*, 2nd edition, Andrew Streitwieser and Clayton Heathcock (1981), pp. 169–171; *Organic Chemistry*, 5th edition, John McMurry (2000), pp. 398 and 408; all of these are incorporated herein by reference only for the purpose of disclosing features and examples of leaving groups.

[0106] The term "pharmaceutically acceptable salt" as used herein is a broad term and should be given its common and customary meaning (and not limited to a particular or customary meaning) to those skilled in the art, and "salt" means, but is not limited to, a salt of a compound that will not cause significant irritation to the organism to which it is given and will not eliminate the biological activity and properties of the compound. In some embodiments, the salt is an acid addition salt of the compound. Pharmaceutical salts can be obtained by reacting the compound with inorganic acids such as hydrohalic acids (e.g., hydrochloric acid or hydrobromic acid), sulfuric acid, nitric acid, and phosphoric acid. Pharmaceutical salts can also be obtained by reacting the compound with an organic acid, such as aliphatic or aromatic carboxylic acids or sulfonic acids, for example, formic acid, acetic acid (AcOH), propionic acid, glycolic acid, pyruvic acid, malonic acid, maleic acid, fumaric acid, trifluoroacetic acid (TFA), benzoic acid, cinnamic acid, mandelic acid, succinic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, nicotinic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, stearic acid, mucoconic acid, butyric acid, phenylacetic acid, phenylbutyric acid, valproic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 2-naphthalenesulfonic acid, or naphthalenesulfonic acid. Pharmaceutical salts can also be obtained by reacting compounds with bases to form salts, such as ammonium salts, alkali metal salts (such as lithium, sodium, or potassium salts), alkaline earth metal salts (such as calcium, magnesium, or aluminum salts), organic bases (such as dicyclohexylamine, N-methyl-D-glucosamine, tri(hydroxymethyl)methylamine, (C1-C7 alkyl)amines, cyclohexylamine, dicyclohexylamine, triethanolamine, ethylenediamine, ethanolamine, diethanolamine, triethanolamine, tromethamine), and salts with amino acids (such as arginine and lysine); or inorganic bases (such as aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, sodium hydroxide, etc.).

[0107] As used herein, a “nucleotide” comprises a nitrogenous heterocyclic base, a sugar, and one or more phosphate groups. They are the monomeric units of a nucleic acid sequence. In RNA, the sugar is ribose, and in DNA, the sugar is deoxyribose, which lacks the hydroxyl group found in ribose. The nitrogenous heterocyclic base can be a purine or pyrimidine base. Purine bases include adenine (A) and guanine (G), and their modified derivatives or analogs (such as deoxypurine). Pyrimidine bases include cytosine (C), thymine (T), and uracil (U), and their modified derivatives or analogs. The C-1 atom of the deoxyribose is bonded to the N-1 of a pyrimidine or the N-9 of a purine.

[0108] As used herein, a "nucleoside" is structurally similar to a nucleotide but lacks a phosphate moiety. An example of a nucleoside analog is one in which the label is linked to a base and the sugar molecule does not have a phosphate group attached. The term "nucleoside" is used herein in its ordinary sense as understood by those skilled in the art. Examples include, but are not limited to, ribonucleotides containing a ribose moiety and deoxyribonucleotides containing a deoxyribose moiety. A modified pentose moiety is a pentose moiety in which an oxygen atom has been substituted by a carbon atom and / or a carbon atom has been substituted by a sulfur or oxygen atom. A "nucleoside" is a monomer that may have substituted bases and / or sugar moieties. Additionally, nucleosides can be incorporated into larger DNA and / or RNA polymers and oligomers.

[0109] The term "purine base" is used herein in its ordinary sense as understood by those skilled in the art and includes its tautomers. Similarly, the term "pyrimidine base" is used herein in its ordinary sense as understood by those skilled in the art and includes its tautomers. A non-limiting list of optionally substituted purine bases includes purine, denitropurine, 7-denitropurine, adenine, 7-denitroadenine, guanine, 7-denitroguanine, hypoxanthine, xanthine, tetraxanthine, 7-alkylguanine (e.g., 7-methylguanine), theobromine, caffeine, uric acid, and isoguanine. Examples of pyrimidine bases include, but are not limited to, cytosine, thymine, uracil, 5,6-dihydrouracil, and 5-alkylcytosine (e.g., 5-methylcytosine).

[0110] As used herein, “derivative” or “analyte” means a synthetic nucleoside or nucleotide derivative having a modified base moiety and / or a modified sugar moiety. Such derivatives and analogs are discussed, for example, in Scheit, Nucleotide Analogs (John Wiley & Son, 1980) and Uhlman et al., Chemical Reviews 90:543-584, 1990. Nucleotide analogs may also contain modified phosphodiester bonds, including thiophosphate, dithiophosphate, alkylphosphonate, aminophosphate, phosphoramide, and aminophosphate bonds. The terms “derivative” and “analyte” as used herein are used interchangeably and are covered by the terms “nucleotide” and “nucleoside” as defined herein.

[0111] As used herein, the term "phosphate / ester" is used in its ordinary sense as understood by those skilled in the art, and includes its protonated form (e.g., As used herein, the terms “monophosphate,” “diphosphate,” and “triphosphate” are used in their ordinary sense as understood by those skilled in the art, and include the protonated form.

[0112] As used herein, “loading capacity” or “loading” means mmol or μmol of nucleosides bound to the polymer described herein per gram of polymer (i.e., mmol / g). Polymers for Liquid-Phase Oligonucleotide Synthesis

[0113] Several aspects of this application relate to polymers for liquid-phase synthesis. In some embodiments, liquid-phase synthesis includes liquid-phase oligonucleotide synthesis, liquid-phase peptide synthesis, liquid-phase polynucleotide (i.e., nucleic acid) synthesis, or liquid-phase small molecule synthesis. In some embodiments, the polymer comprises or is a polymer for liquid-phase oligonucleotide synthesis. The polymer may include poly(ethylene glycol) (PEG) side chain arms having reactive groups, including but not limited to amines, alcohols, azides, or alkynyl groups or combinations thereof, which allow reaction with nucleosides or nucleotide analogs. The average molecular weight of the polymer can be controlled by controlling the length of the PEG side chain arms. By adjusting the length of the PEG arms, impurity trapping can be reduced, thereby improving the performance of these structures in liquid-phase oligomerization. Polymers of formula (I) or (I′)

[0114] Some embodiments of this disclosure relate to polymers for liquid-phase oligonucleotide synthesis having the structure of formula (I): in: R is H, or an unsubstituted or substituted C1-C6 alkyl group; W is C1-C 20 Alkylene, 2-20 heteroalkylene, or bond; Q is L 1 For C1-C 20 Alkylene, 2-20-membered heteroalkylene, optionally substituted phenylene, optionally substituted 5-6-membered heteroarylene, optionally substituted 3-10-membered heterocyclic group or optionally substituted C3-C 10 Cycloalkylene, or C1-C 20 Alkylene, or 2-20-membered heteroalkylene, wherein one or more methylene repeating units are each independently substituted by a group selected from the group consisting of: optionally substituted phenylene, optionally substituted 5-6-membered heteroarylene, optionally substituted 3-10-membered heterocyclic group, optionally substituted C3-C 10 Cycloalkylene compounds, -C(=O)-, -CH=CH-, and -C≡C-; R 1 and R 2 Each independently of -OR 3 or -NR 4a R 4b ; R 3Protected by H, C1-C6 alkyl, hydroxyl groups or R 4a and R 4b Each is H, optionally substituted C1-C6 alkyl, -C(=O)(C1-C6 alkyl), -C(=O)phenyl, amino protecting group or or R 4a and R 4b Together they form a divalent amino protecting group; L 2a and L 2b Each independently is C1-C 20 Alkylene, 2-20-membered heteroalkylene, optionally substituted phenylene, optionally substituted 5-6-membered heteroarylene, optionally substituted 3-10-membered heterocyclic group or optionally substituted C3-C 10 Cycloalkylene, or C1-C 20 Alkylene or 2-20-membered heteroalkylene, wherein one or more methylene repeating units are each independently substituted by a group selected from the group consisting of: optionally substituted phenylene, optionally substituted 5-6-membered heteroarylene, optionally substituted 3-10-membered heterocyclic group, optionally substituted C3-C 10 Cycloalkylene compounds, -C(=O)-, -CH=CH-, and -C≡C-; R 5a 、R 5b 、R 6a and R 6b Each is independently H, -OR 7 or -NR 8a R 8b ; R 7 Each is independently protected by H, C1-C6 alkyl, hydroxyl groups or R 8a and R 8b Each is independently H, optionally substituted C1-C6 alkyl, -C(=O)(C1-C6 alkyl), -C(=O)phenyl, amino protecting group or or R 8a and R 8b Together they form a divalent amino protecting group; L 3a and L 3b Each independently is C1-C 20 Alkylene, 2-20-membered heteroalkylene, optionally substituted phenylene, optionally substituted 5-6-membered heteroarylene, optionally substituted 3-10-membered heterocyclic group or optionally substituted C3-C 10 Cycloalkylene, or C1-C 20Alkylene or 2-20-membered heteroalkylene, wherein one or more methylene repeating units are each independently substituted by a group selected from the group consisting of: optionally substituted phenylene, optionally substituted 5-6-membered heteroarylene, optionally substituted 3-10-membered heterocyclic group, optionally substituted C3-C 10 Cycloalkylene compounds, -C(=O)-, -CH=CH-, and -C≡C-; R 9a 、R 9b 、R 10a and R 10b Each is independently H, -OR 11 or -NR 12a R 12b ; R 11 Each is independently protected by H, C1-C6 alkyl, hydroxyl groups or R 12a and R 12b Each is independently H, optionally substituted C1-C6 alkyl, -C(=O)(C1-C6 alkyl), -C(=O)phenyl, amino protecting group or or R 12a and R 12b Together they form a divalent amino protecting group; L 4a and L 4b Each independently is C1-C 20 Alkylene, 2-20-membered heteroalkylene, optionally substituted phenylene, optionally substituted 5-6-membered heteroarylene, optionally substituted 3-10-membered heterocyclic group or optionally substituted C3-C 10 Cycloalkylene, or C1-C 20 Alkylene or 2-20-membered heteroalkylene, wherein one or more methylene repeating units are each independently substituted by a group selected from the group consisting of: optionally substituted phenylene, optionally substituted 5-6-membered heteroarylene, optionally substituted 3-10-membered heterocyclic group, optionally substituted C3-C 10 Cycloalkylene compounds, -C(=O)-, -CH=CH-, and -C≡C-; R 13a 、R 13b 、R 14a and R 14b Each can be independently H, -OH, protected hydroxyl, -NH2, -NH (optionally substituted C1-C6 alkyl) or protected amino; m1, m2, m3, m4, m5, m6, and m7 are each independently 0 or 1; and j is an integer between 15 and 1500. In some implementations, each R... 4a For H, each R 4bIndependently protected by H, -C(=O) (C1-C6 alkyl), -C(=O) phenyl, amino, or Or -NHR 4b Hydrogen is absent in R 4b It is a divalent amino protecting group; each R 8a H, R 5a 、R 5b 、R 6a and R 6b Each is H, -OR independently 7 or -NHR 8b Each R 8b Independently, it is H, -C(=O) (C1-C6 alkyl), -C(=O) phenyl, amino protecting group or Or -NHR 8b Hydrogen is absent in R 8b It is a divalent amino protecting group; each R 12a H, R 9a 、R 9b 、R 10a and 10b Each is H, -OR independently 11 or -NHR 12b Each R 12b Independently protected by H, -C(=O) (C1-C6 alkyl), -C(=O) phenyl, amino, or Or -NHR 12b Hydrogen is absent in R 12b It is a divalent amino protecting group.

[0115] In some embodiments of the polymer of formula (I), R is an unsubstituted C1-C6 alkyl group (e.g., -CH3). In other embodiments, R is a substituted C1-C6 alkyl group (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, which is independently substituted with an azide or alkynyl group). In other embodiments, R is H. In some embodiments, W is -CH2CH2NH-. In other embodiments, W is -CH2CH2O-. In some embodiments, W is a C2-C6 alkylene group, such as -CH2CH2-. In still other embodiments, W is a bond.

[0116] In some embodiments of the polymers described herein, the polymer of formula (I) can also be represented by the structure of formula (I′): in: R is a C1–C6 alkyl group; W is either -NH- or -O-; Q is L 1 For C1-C10 Alkylene linkage or 2-10 heteroalkylene linkage; R 1 and R 2 Each independently of -OR 3 or -NR 4a R 4b ; R 3 Protected by H, C1-C6 alkyl, hydroxyl groups or R 4a and R 4b Each is H, optionally substituted C1-C6 alkyl, -C(=O)(C1-C6 alkyl), -C(=O)phenyl, amino protecting group or or R 4a and R 4b Together they form a divalent amino protecting group; L 2a and L 2b Each independently is C1-C 10 Alkylene linkage or 2-10 heteroalkylene linkage; R 5a 、R 5b 、R 6a and R 6b Each independently is hydrogen, -OR 7 or -NR 8a R 8b ; R 7 Each is independently protected by H, C1-C6 alkyl, hydroxyl groups or R 8a and R 8b Each is independently H, optionally substituted C1-C6 alkyl, -C(=O)(C1-C6 alkyl), -C(=O)phenyl, amino protecting group or or R 8a and R 8b Together they form a divalent amino protecting group; L 3a and L 3b Each independently is C1-C 10 Alkylene linkage or 2-10 heteroalkylene linkage; R 9a 、R 9b 、R 10a and R 10b Each independently of -OR 11 or -NR 12a R 12b ; R 11Each is independently protected by H, C1-C6 alkyl, hydroxyl groups or R 12a and R 12b Each is independently H, optionally substituted C1-C6 alkyl, -C(=O)(C1-C6 alkyl), -C(=O)phenyl, amino protecting group or or R 12a and R 12b Together they form a divalent amino protecting group; L 4a and L 4b Each independently is C1-C 10 Alkylene linkage or 2-10 heteroalkylene linkage; R 13a 、R 13b 、R 14a and R 14b Each is independently H, -OH, a protected hydroxyl group, -NH2, -NH (optionally substituted C1-C6 alkyl), or a protected amino group; and j is an integer between 15 and 1500. In some implementations, each R... 4a For H, each R 4b Independently protected by H, -C(=O) (C1-C6 alkyl), -C(=O) phenyl, amino, or Or -NHR 4b Hydrogen is absent in R 4b It is a divalent amino protecting group; each R 8a H, R 5a 、R 5b 、R 6a and R 6b Each is H, -OR independently 7 or -NHR 8b Each R 8b Independently, it is H, -C(=O) (C1-C6 alkyl), -C(=O) phenyl, amino protecting group or Or -NHR 8b Hydrogen is absent in R 8b It is a divalent amino protecting group; each R 12a H, R 9a 、R 9b 、R 10a and R 10b Each is H, -OR independently 11 or -NHR 12b Each R 12b Independently protected by H, -C(=O) (C1-C6 alkyl), -C(=O) phenyl, amino or Or -NHR 12bHydrogen is absent in R 12b It is a divalent amino protecting group. In some embodiments of the polymer of formula (I′), R is methyl. In one embodiment, W is -NH-. In some embodiments, W is -O-.

[0117] In some embodiments of polymers of formula (I) or (I′), L 1 It is a C2-C6 alkylene group. In some embodiments of the polymer of formula (I), L 1 It is a 3-12, 3-8, 3-6, or 4-6 heteroalkylene group containing one, two, or three heteroatoms selected from N, O, S, C (=O), or C (=S). In other embodiments, the heteroalkylene group contains one or two nitrogen atoms.

[0118] In some other embodiments of the polymer of formula (I) or (I′), Q is: In some of these implementations, each R 3 Independently protected by H or hydroxyl groups. In some embodiments, each R... 4a It is H. In some implementations, each R 4a and R 4b It is H, optionally substituted C1-C6 alkyl, -C(=O)CH3, or an amino protecting group. In some embodiments of the polymer of formula (I), R 3 One is H, and the other is R. 3 for In some implementations, R 4a For H, and R 4b for In other embodiments, R 3 H, R 4a For H, and R 4b for Or R 4a H, R 4b For H, and R 3 for In other embodiments, R 3 Each independently In other embodiments, R 4a and R 4b Each independently In some other embodiments, m2 and m3 are each 1. In other embodiments, m2 is 0 and m3 is 1. In other embodiments, m2 is 1 and m3 is 0. In other embodiments, both m2 and m3 are 0. In some embodiments of the polymer of formula (I′), R 3 One is H, and the other is R. 3 for In some implementations, R 4s For H, and R 4b for In other embodiments, R 3 H, R 4a is H, and R 4b for Or R 4a H, R 4b is H, and R 3 for In other embodiments, R 3 Each independently In other embodiments, R 4a and R 4b Each independently

[0119] In some other embodiments of the polymer of formula (I) or (I'), Q is selected from the group consisting of: In some of these implementations, each R 3 Independently protected by H or hydroxyl groups. In some other embodiments, each R... 3 for In some other embodiments, R 3 One of them is H, and the other is R. 3 for In some other implementations, m2 is 1. In other implementations, m2 is 0.

[0120] In some embodiments of polymers of formula (I) or (I′), L 2a and L 2b Each independently is C1-C 10 An alkylene linkage or a 2-10 heteroalkylene linkage. In some other embodiments, L 2a and L 2b Each is independently a 3-12, 3-8, 3-6, or 4-6 heteroalkylene group, containing one, two, or three heteroatoms selected from N, O, S, C (=O), or C (=S). In other embodiments, the heteroalkylene group contains one or two nitrogen atoms. In other embodiments of the polymer of formula (I), L 2a and L 2b Each is independently an optionally substituted phenylene; or a C2-C6 alkylene or a 3-12 heteroalkylene, wherein one of the methylene units is optionally substituted with a substituted phenylene.

[0121] In some embodiments of the polymer of formula (I), R 5a 、R 5b 、R 6a and R6b Each is independently hydrogen, -OH, a protected hydroxyl group, -NH2, -NH (optionally substituted C1-C6 alkyl), -NHC(=O)CH3, or a protected amino group. In some such embodiments, R 5a 、R 5b 、R 6a and R 6b At least one of them is H. In some embodiments of the polymer of formula (I′), R 5a 、R 5b 、R 6a and R 6b Each is independently -OH, a protected hydroxyl group, -NH2, -NH (optionally substituted C1-C6 alkyl), -NHC(=O)CH3, or a protected amino group (e.g., -NHAC). In some embodiments of polymers of formula (I) or (I′), R 5a 、R 5b 、R 6a and R 6b Each is a hydroxyl group or a protected hydroxyl group. In other embodiments, R 5a 、R 5b 、R 6a and R 6b Each is -NH2, -NH (optionally substituted C1-C6 alkyl), or a protected amino group (e.g., -NHAC). In other embodiments of the polymer of formula (I), R 7 At least one of them is In other embodiments, R 8a and R 8b At least one of them is In other embodiments, R 7 Each independently In other embodiments, each R 8a is H, and R 8b Each independently In some other embodiments, m4 and m5 are both 1. In other embodiments, m4 is 0 and m5 is 1. In other embodiments, m4 is 1 and m5 is 0. In other embodiments, m4 and m5 are both 0. In other embodiments of the polymer of formula (I) or formula (I′), R 5a 、R 5b 、R 6a and R 6b At least one of them is independently -OR 7 And R 7 At least one of them is In other embodiments, R 5a 、R 5b 、R 6a and R 6bAt least one of them is independently -NR 8a R 8b And R 8a and R 8b At least one of them is In other embodiments, R 5a 、R 5b 、R 6a and R 6b Each independently of -OR 7 And R 7 Each independently In other embodiments, each R 8a H and R 5a 、R 5b 、R 6a and R 6b Each of them is independently -NHR 8b And each R 8b Independently In some such embodiments of polymers of formula (I) or (I′), L 3a It is a C2-C6 alkylene group. In other embodiments, L 3a It is a 3-12, 3-8, 3-6, or 4-6-membered heteroalkylene group, containing one, two, or three heteroatoms selected from N, O, S, C (=O), or C (=S). In other embodiments, the heteroalkylene group contains one or two nitrogen atoms. In some embodiments, L... 3b It is a C2-C6 alkylene group. In other embodiments, L 3b It is a 3-12, 3-8, 3-6, or 4-6-membered heteroalkylene group containing one, two, or three heteroatoms selected from N, O, S, C (=O), or C (=S). In other embodiments, the heteroalkylene group contains one or two nitrogen atoms. In other embodiments of the polymer of formula (I), L 3a and L 3b Each is independently an optionally substituted phenylene; or a C2-C6 alkylene or a 3- to 12-membered heteroalkylene, wherein one of the methylene units is optionally substituted with a substituted phenylene. In some embodiments of the polymer of formula (I), R 9a 、R 9b 、R 10a and R 10b Each is independently H, -OH, a protected hydroxyl group, -NH2, -NH (optionally substituted C1-C6 alkyl), -NHC(=O)CH3, or a protected amino group (e.g., -NHAC). In some such embodiments, R 9a 、R 9b 、R 10a and R 10b At least one of them is H. In some embodiments of the polymer of formula (I′), R9a 、R 9b 、R 10a and R 10b Each is independently -OH, a protected hydroxyl group, -NH2, -NH (optionally substituted C1-C6 alkyl), -NHC(=O)CH3, or a protected amino group. In some embodiments of polymers of formula (I) or (I′), R 9a 、R 9b 、R 10a and R 10b Each is a hydroxyl group or a protected hydroxyl group. In other embodiments, R 9a 、R 9b 、R 10a and R 10b Each is -NH2, -NH (optionally substituted C1-C6 alkyl), or a protected amino group (e.g., -NHAC). In other embodiments of polymers of formula (I) or (I′), R 9a 、R 9b 、R 10a and R 10b At least one of them is independently -OR 11 And R 11 At least one of them is In other embodiments, R 9a 、R 9b 、R 10a and R 10b At least one of them is independently -NR 12a R 12b , and R 12a and R 12b At least one of them is In other embodiments, R 9a 、R 9b 、R 10a and R 10b Each independently of -OR 11 And R 11 Each independently In other embodiments, R 12a Each is H and R 9a 、R 9b 、R 10a and R 10b Each is -NHR 12b And R 12b Each independently In some other embodiments, m6 and m7 are each 1. In other embodiments, m6 is 0 and m7 is 1. In other embodiments, m6 is 1 and m7 is 0. In other embodiments, both m6 and m7 are 0. In other embodiments of the polymer of formula (I′), R 11 At least one of them is In other embodiments, R 12a and R 12b At least one of them is In other embodiments, R 11 Each independently In other embodiments, R 12a Each is H and R 12b Each independently In some such embodiments of polymers of formula (I) or (I′), L 4a It is a C2-C6 alkylene group. In other embodiments, L 4a It is a 3-12, 3-8, 3-6, or 4-6-membered heteroalkylene group, containing one, two, or three heteroatoms selected from N, O, S, C (=O), or C (=S). In other embodiments, the heteroalkylene group contains one or two nitrogen atoms. In some embodiments, L... 4b It is a C2-C6 alkylene group. In other embodiments, L 4b It is a 3-12, 3-8, 3-6, or 4-6-membered heteroalkylene group containing one, two, or three heteroatoms selected from N, O, S, C (=O), or C (=S). In other embodiments, the heteroalkylene group contains one or two nitrogen atoms. In other embodiments of the polymer of formula (I), L 4a and L 4b Each is independently an optionally substituted phenylene; or a C2-C6 alkylene or a 3-12 heteroalkylene, wherein one of the methylene units is optionally substituted with a substituted phenylene. In some such embodiments, R 13a 、R 13b 、R 14a and R 14b Each is independently H, -OH, or a protected hydroxyl group, provided that the polymer contains at least one, two, three, four, five, or six terminal hydroxyl or protected hydroxyl functional groups. In some other embodiments, R 13a 、R 13b 、R 14a and R 14b Each of the following is independently H, -NH2, -NH (optionally substituted C1-C6 alkyl) or a protected amino group, provided that the polymer contains at least one, two, three, four, five or six terminal amino groups, -NH (optionally substituted C1-C6 alkyl) or protected amino functional groups (e.g., -NHAC).

[0122] In some embodiments, the polymer of formula (I) or (I′) has the structure of formula (Ia):

[0123] In some embodiments, the polymer of formula (I) or (I′) has the structure of formula (Ia'):

[0124] In some other embodiments, the polymer of formula (I) or (I') has the structure of formula (Ib), (Ib-1), (Ic), or (Ic-1):

[0125] In some embodiments, the polymer of formula (I) has the following structure:

[0126] In some embodiments, the polymer of formula (I) has the following structure:

[0127] In some implementations, L 2b It is a C2-C6 alkylene group. In other embodiments, L 2b It is a 3-12 or 4-8-membered heteroalkylene group containing one or more oxygen or nitrogen atoms. In other embodiments, L 2b The substituted phenylene oxide is used. In other embodiments, L... 2b It is a C2-C6 alkylene group, wherein one of the methylene units is replaced by an optionally substituted ring structure (e.g., an optionally substituted phenylene) as described herein. In other embodiments, L 2b It is a 3-12 or 4-8-membered heteroalkylene group containing one or more oxygen or nitrogen atoms, wherein one methylene unit is substituted with an optionally substituted cyclic structure (e.g., optionally substituted phenylene) as described herein. In some such embodiments, R 5b and R 6b Each is independently -NH2 or -NHAC. In other such embodiments, R 5b and R 6b Each is either -OH or a protected hydroxyl group. In other embodiments, R 5b and R 6b At least one of them is -OR 7 , and R 7 for In other embodiments, R 5b and R 6b Each of them is In some implementations, m4 is 1. In other implementations, m4 is 0. In other implementations, R 5b and R 6b At least one of them is -NHR 8 , and R 8 for In other embodiments, R 5b and R 6b Each of them is In some implementations, m5 is 1. In other implementations, m5 is 0. In other implementations, R 5b and R 6b At least one of them is H. In some such embodiments, L 3a and L 3b Each of them is a C2-C6 alkylene linker. In other embodiments, L 3a and L 3b Each is a 3-12 or 4-8-membered heteroalkylene linker containing one or more oxygen or nitrogen atoms. In other embodiments, R 9a 、R 9b 、R 10a and R 10b At least one of them is H. In other embodiments, R 9a 、R 9b 、R 10a and R 10b At least one of them is -OR 11 , and R 11 for In other embodiments, R 9a 、R 9b 、R 10a and R 10b Each for themselves In some implementations, m6 is 1. In other implementations, m6 is 0. In some of these implementations, R 13a and R 14a Each is -OH. In other embodiments, R... 13a and R 14a At least one of them is H. In other embodiments, R 9a 、R 9b 、R 10a and R 10b At least one of them is -NHR 12 , and R 12 for In other embodiments, R 9a 、R 9b 、R 10a and R 10b Each for themselves In some implementations, m7 is 1. In other implementations, m7 is 0. In some such implementations, R 13b and R 14b Each is independently -NH2 or -NHAC. In other embodiments, R 13b and R 14bAt least one of them is H. In some other embodiments, the structure of formula (Ib-1) has the structure of formula (Ib-2) or (Ib-3): In some other embodiments, the structure of formula (Ib-1) has the structure of formula (Ib-4): In some other embodiments, the structure of formula (Ib-1) has the structure of formula (Ib-5): The structure of formula (Ib-1) has the same structure as formula (Ib-6):

[0128] In some embodiments of polymers of formula (I), (I′), (Ia), (Ia'), (Ib), (Ib-1), (Ib-2), (Ib-3), (Ib-4), (Ib-5), (Ib-6), (Ic), (Ic-1), (Id), (Ie), (If), (Ig), (Ih), (Ij), (Ik), or (Il), j is about 50 to about 1000, about 200 to about 800, or about 300 to about 600. In some implementations, j is approximately 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, or within a range defined by any of the above values. In some embodiments, the polymers of formula (I), (Ia), (Ia'), (Ib), (Ib-1), (Ib-2), (Ib-3), (Ib-4), (Ib-5), (Ib-6), (Ic), (Ic-1), (Id), (Ie), (If), (Ig), (Ih), (Ij), (Ik), or (Il) have an average molecular weight of about 2 kDa to about 60 kDa. For example, polymers of formulas (I), (Ia), (Ia'), (Ib), (Ib-1), (Ib-2), (Ib-3), (Ib-4), (Ib-5), (Ib-6), (Ic), (Ic-1), (Id), (Ie), (If), (Ig), (Ih), (Ij), (Ik), or (Il) have average molecular weights of approximately 2 kDa, 5 kDa, 10 kDa, 15 kDa, 16 kDa, 17 kDa, 18 kDa, etc. 19kDa, 20kDa, 21kDa, 22kDa, 23kDa, 24kDa, 25kDa, 26kDa, 27kDa, 28kDa, 29kDa, 30kDa, 32kDa, 34kDa, 36kDa, 38kDa, 40kDa, 42kDa, 44kDa, 46kDa, 48kDa, 50kDa, 52kDa, 54kDa, 58kDa, or 60kDa, or within the range defined by any of the aforementioned values. For example, polymers of formula (I), (Ia), (Ia'), (Ib), (Ib-1), (Ib-2), (Ib-3), (Ib-4), (Ib-5), (Ib-6), (Ic), (Ic-1), (Id), (Ie), (If), (Ig), (Ih), (Ij), (Ik) or (Il) have an average molecular weight of about 2 to about 60 kDa, about 5 to about 50 kDa, about 5 to about 30 kDa, about 10 to about 40 kDa, about 15 to 30 kDa, or about 15 to 25 kDa. Formula (II) polymer

[0129] In some embodiments of the polymer described herein, the polymer is a compound having the structure of formula (II): Where: A is selected from the following group: carbon atom, C2-C 20 Alkylene; 2-20-membered heteroalkylene, phenylene, 5-10-membered heteroarylene, C 5-10 Cycloalkylene and 5-10 membered heterocyclic alkylene; each R 15 Independently selected from the following group - OC 1- C6 alkyl. -OC(O)(C 1- C6 alkyl), -OC(O)phenyl, -NHC(O)(C 1- C6 alkyl), -NHC(O)phenyl, -NHC(O)phenylene-acetoxymethyl Protected hydroxyl groups or protected amino groups; each R 16 Independently -OH or -NH2; q 1 and q 2 Each is an independent integer from 10 to 500; and s and t are each independent integers from 1 to 4, provided that s + t equals or is greater than 2. In some of these embodiments, A is a carbon atom. In other such embodiments, A is a phenylene oxide. In still other such embodiments, A is a C2-C atom. 20 Alkylene.

[0130] In some embodiments of the polymer of formula (II), s+t is 4. In other embodiments, s+t is 8. In some embodiments, s+t is 5, 6, or 7. In some specific embodiments, s is 2 and t is 2, or one of s and t is 1, and the other s and t is 3. In other specific embodiments, s is 4 and t is 4. In other specific embodiments, one of s and t is 5, and the other s and t is 3. In one embodiment, s is 5 and t is 3. In another embodiment, s is 3 and t is 1. In another embodiment, s is 3 and t is 3. In yet another embodiment, s is 3 and t is 5. In an additional embodiment, s is 2 and t is 4, or s is 4 and t is 2. In another additional embodiment, s is 2 and t is 5, or s is 5 and t is 2, or s is 3 and t is 4, or s is 4 and t is 3.

[0131] In some embodiments, the polymer of formula (II) has the structure of formula (IIa): In some of these embodiments, A is a carbon atom. In other embodiments, A is a phenylene oxide. For example, polymers of formula (IIa) can also be represented by formulas (IIa-1) or (IIa-2):

[0132] In some embodiments, the polymer of formula (II) has the structure of formula (IIa'): Among them, each R 30 C can be substituted independently. 1-6 Alkyl group. In some of these embodiments, A is a carbon atom. In other embodiments, A is a phenylene group. For example, polymers of formula (IIa') can also be represented by formula (IIa'-1) or (IIa'-2):

[0133] Alternative embodiments of the polymers (IIa'), (IIa'-1), and (IIa'-2) may include polymers having one or three free amino groups and three or one protected amino group (e.g., acetyl or AC groups). Other common amino protecting groups, such as Bz, may be used in addition to the AC group. In some other embodiments, A is C2-C. 20 Alkylene, and the polymer of formula (II) has the structure of formula (IIb'): It has eight PEG side arms. Alternative embodiments of the polymer of formula (IIb') may include polymers having three, four, five, six, or seven PEG side arms, wherein at least one PEG side arm has a free amino group. In some other embodiments, at least one PEG side arm has a protected amino group that cannot be used as an anchor for liquid-phase synthesis. Alternative embodiments of the polymer of formula (IIb') may also include polymers having two, four, or five free amino groups and one, two, three, or four protected amino groups (e.g., acetyl or AC groups). In addition to AC groups, other common amino protecting groups, such as Bz, may be used. In another embodiment, A is a 3-20, 5-15, or 6-12-membered heteroalkylene group containing one or more O, N, S, C (=O), or C (=S).

[0134] In some embodiments of polymers of formulas (II), (IIa), (IIa'), (IIa-1), (IIa-2), (IIa'-1), (IIa'-2), (IIb), or (IIb'), the polyethylene unit (PEG) q can be controlled. 1 and q 2 The quantity is used to adjust the properties of the polymer. In some embodiments, q 1 and q2 Each is an independent integer between 10 and 500. For example, in some implementations, q 1 and q 2 Each can be 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 2 00, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or 500, or within a range defined by any two of the above values. In some embodiments, each q 1 Independently, it is about 30 to about 150, about 40 to about 100, or about 50 to about 75. In some embodiments, each q 2 Independently, it can be about 30 to about 150, about 40 to about 100, or about 50 to about 75. 1 and q 2 They can have the same value. In some implementations, q 1 and q 2 It can have different values. In some implementations, it is preferable to control q. 1 and q 2 The length of the polymer results in an average molecular weight of about 2 kDa to about 60 kDa, about 5 kDa to about 50 kDa, about 10 kDa to about 50 kDa, about 15 kDa to about 30 kDa, or about 20 kDa.

[0135] In any embodiment of the polymer described herein, when the polymer comprises two or more PEG side chain arms, at least one PEG side chain arm has a reactive end group (e.g., -NH2 or -OH) that can be used as a liquid-phase synthesis anchor. In other embodiments, when the polymer comprises two, three or more PEG side chain arms, at least one PEG side chain arm has a non-reactive end group (e.g., a protected amino or protected hydroxyl group) such that the side chain arm cannot be used as a liquid-phase synthesis anchor. Methods for preparing oligonucleotides via liquid-phase oligonucleotide synthesis (LPOS)

[0136] Another aspect of this application relates to a method for preparing a compound via liquid-phase synthesis. The compound may be an oligonucleotide, peptide, polynucleotide (i.e., nucleic acid), or a small molecule. In some embodiments, the method prepares oligonucleotides via liquid-phase oligonucleotide synthesis.

[0137] In some embodiments of the method described herein, the method includes dissolving the polymer described herein in a first solvent to form a reaction matrix, contacting or otherwise reacting the polymer with one or more nucleoside analogs to form a first bioconjugate containing a structure of formula (III): Among them B 1 It is a nitrogenous base; G 1 It is a 5' hydroxyl blocking group; X is O or NR. 20 ; R a It is -H, -OH, halogen, -O-(C1-C6 alkyl), -O-(C1-C6 haloalkyl), or -OY, where Y is a 2' hydroxyl protecting group; and L 5 It is a cleavable heteroalkylene linker in which one or more carbon atoms are substituted with O, S, N, C (=O), or C (=S). In some such embodiments, the nitrogenous base includes a purine base, a denitropurine base, or a pyrimidine base. In some embodiments, the structure of formula (III) is also represented by formula (IIIa) or (IIIb):

[0138] In some implementations, X is 0. In other implementations, X is NR. 20 , and R 20 For H. In some embodiments, the amide bond -NR 20 -C(=O)- can be formed from the terminal amino group of a polymer reacting with a first nucleoside analog containing a 3'-succinate (which contains a free carboxyl group). In other embodiments, the amide bond -NR 20 -C(=O)- can be formed by a linker that binds to the 3' position of the first nucleoside analog. Other alternative linkers may include hydroquinone-O,O'-diacetic acid (HQDA or Q-linker).

[0139] In some implementations of the method described herein, B 1 Independently, it may be an optionally protected adenine, optionally protected deadenine, optionally protected cytosine, optionally protected guanine, optionally protected deadenine, optionally protected thymine, or optionally protected uracil. In some embodiments, B1 is... where R x It is protected by hydrogen, unsubstituted or substituted C1-C6 alkyl or amino groups, or -NHR. x Hydrogen is absent in R xG1 is a divalent amino protecting group. In some embodiments, G1 is a triphenylmethyl hydroxy protecting group selected from the group consisting of (4-methoxyphenyl)diphenylmethyl, bis(4-methoxyphenyl)phenylmethyl, tris(4-methoxyphenyl)methyl, 9-phenylxanth-9-yl, and 9-(4-methoxyphenyl)xanth-9-yl. In some embodiments, G... 1 It is bis(4-methoxyphenyl)phenylmethyl (DMT).

[0140] In some embodiments, the polymer has an average molecular weight of about 2 kDa to about 1000 kDa, about 5 kDa to about 1000 kDa, or about 10 kDa to about 1000 kDa, or about 20 kDa to about 500 kDa, or about 30 kDa to about 100 kDa, or about 15 kDa to about 30 kDa. In some embodiments, the polymer has an average molecular weight of about 10 kDa to about 100 kDa. Without being bound by any particular theory, a polymer with an average molecular weight of about 20 kDa can provide the optimal balance between reaction yield and product purity.

[0141] In some embodiments of the method described herein, the method further includes: removing the 5' hydroxyl blocking group (G 1 To form a 5' unclosed first bioconjugate; and to separate the 5' unclosed first bioconjugate. In some such embodiments, the separation of the 5' unclosed first bioconjugate is achieved by precipitation, dialysis, or filtration.

[0142] In some embodiments, separation is achieved by precipitation. In some embodiments, precipitation of the 5' unblocked first bioconjugate is accomplished by adding a solution containing the bioconjugate to a solvent comprising pentane, hexane, heptane, dialkyl ethers (e.g., diethyl ether, tert-butyl methyl ether, etc.), toluene, isopropyl acetate, dichloromethane, dimethyl sulfoxide, ethyl acetate, alkanols (e.g., methanol, ethanol, isopropanol), or enols or combinations thereof. In some such embodiments, the solvent is diethyl ether. In other such embodiments, the solvent is isopropanol. In other embodiments, separation is achieved by a filtration step. The filtration step may include dialysis, filtration, nanofiltration, ultrafiltration, or any known filtration technique suitable for this document, or combinations thereof. In some embodiments, the filtration step includes dialysis or filtration. In other embodiments, the filtration step includes the use of a membrane. The membrane may include sodium acetate cellulose, glass fiber, carbon-based polymers, regenerated cellulose, or combinations thereof. In some embodiments, the regenerated cellulose has a static charge. In some embodiments, the regenerated cellulose membrane is negatively charged. In some embodiments, the regenerated cellulose comprises a structure... In some embodiments, the molecular weight cutoff (MWCO) of the regenerated cellulose is about 5 kDa to about 50 kDa, about 6 kDa to about 40 kDa, about 7 kDa to about 30 kDa, or about 8 kDa to about 12 kDa. The regenerated cellulose membrane can retain bioconjugates containing PVH, serving as an alternative to expensive nanofiltration membranes made with polyimide. The negatively charged membrane reduces the non-specific adsorption of negatively charged biomolecules. In some embodiments, the regenerated cellulose is treated in a process including carbon disulfide and aqueous metal hydroxide. In some embodiments, the regenerated cellulose comprises dithioester groups and a metal cation. In some embodiments, the metal cation includes Group 1 metals (i.e., Group IA metals or alkali metals), Group 2 metals (i.e., Group IIA metals or alkaline earth metals), and combinations thereof. In some embodiments, the metal cation includes a sodium cation.

[0143] In some embodiments of the method described herein, the method further includes: (a) reacting the 5' unblocked first bioconjugate with one or more nucleoside phosphoramidide analogs in a second solvent to form a second bioconjugate containing the structure of formula (IV): Among them G 2 B is a 5' hydroxyl-blocked group; 2 It is a nitrogenous base; R e It is a phosphorous acid protecting group; (b) The phosphorous portion in oxidized form (IV); (c) Removal of the 5' blocking group G 2 To form a second bioconjugate with an inclusive (IV') structure at the 5' unclosed position: Where Z is O or S; and (d) Separate the unsealed second bioconjugate at 5'. In some such embodiments, the structure of formula (IV) is also represented by (IVa), and formula (IV') is also represented by formula (IV'a): In some such implementations, R e It is an unsubstituted C1-C6 alkyl or a substituted C1-C6 alkyl. Suitable for use as R e Non-limiting examples of substituted C1-C6 alkyl groups include -CH2CH2CN. In some embodiments, the method further includes blocking the unreacted 5' hydroxyl group in the unblocked first bioconjugate prior to step (b). In some such embodiments, X is NR. 20 , and R 20 It’s H.

[0144] In some implementations of the method described herein, B 2Independently, it may be an optionally protected adenine, an optionally protected denitroadenine, an optionally protected cytosine, an optionally protected guanine, an optionally protected denitroguanine, an optionally protected thymine, or an optionally protected uracil. In some embodiments, B 2 yes where R x It is protected by hydrogen, unsubstituted or substituted C1-C6 alkyl or amino groups, or -NHR. x Hydrogen is absent in R x It is a divalent amino protecting group. In some embodiments of the method described herein, G 2 The protecting group is a triphenylmethyl hydroxyl group selected from the group consisting of (4-methoxyphenyl)diphenylmethyl, bis(4-methoxyphenyl)phenylmethyl, tris(4-methoxyphenyl)methyl, 9-phenylxanth-9-yl, and 9-(4-methoxyphenyl)xanth-9-yl. In some embodiments, G 2 It is bis(4-methoxyphenyl)phenylmethyl (DMT).

[0145] In some embodiments of the method described herein, the method further includes blocking the unreacted 5' hydroxyl group in the unblocked first bioconjugate prior to step (b). In some such embodiments, blocking is carried out by reacting the 5' hydroxyl group with acetic anhydride (Ac2O).

[0146] In some embodiments of the methods described herein, the separation or purification of the 5' unblocked second bioconjugate is achieved by precipitation, filtration, or dialysis. In some embodiments, separation is achieved by precipitation. In some embodiments, precipitation of the 5' unblocked second bioconjugate is achieved by adding a solution containing the bioconjugate to a solvent comprising pentane, hexane, heptane, diethyl ether, tert-butyl methyl ether, toluene, isopropyl acetate, dichloromethane, dimethyl sulfoxide, ethyl acetate, methanol, ethanol, isopropanol, or combinations thereof. In some such embodiments, the solvent is diethyl ether. In other such embodiments, the solvent is isopropanol. In other embodiments, separation / purification is performed using a regenerated cellulose membrane having a molecular weight cutoff (MWCO) of about 5 kDa to about 50 kDa, about 6 kDa to about 40 kDa, about 7 kDa to about 30 kDa, or about 8 kDa to about 12 kDa. In some other embodiments, steps (a)-(d) are repeated multiple cycles until an oligonucleotide of the desired length is synthesized.

[0147] In some embodiments of the method described herein, steps (a)-(d) are repeated multiple times until one or more oligonucleotides of the desired length are synthesized. In some such embodiments, steps (a)-(d) are repeated at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 cycles. In some such embodiments, the synthesized oligonucleotide may contain at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 bases.

[0148] In some embodiments, the loading capacity of the polymer described herein is approximately 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or 500 μmol of nucleotides per gram of polymer, or within the range defined by any two of the above loading capacities.

[0149] In some embodiments of the method described herein, the method further includes the removal of oligonucleotides from the polymer. In some such embodiments, the removal step includes a covalent bond breaking step. In some embodiments, the removal step includes hydrolysis. In some embodiments, the removal includes hydrolysis at a temperature of about 0°C to about 80°C, or about 10°C to about 60°C, or about 15°C to about 30°C. In other embodiments, when the first nucleoside is covalently linked to the polymer via a 3'-succinic acid reaction with an amino group of the polymer, the amide bond formed between the first nucleoside and the polymer can be cleaved by hydrolysis.

[0150] In some embodiments of the methods described herein, the first solvent and the second solvent each comprise one or more aprotic polar solvents or combinations thereof. In some embodiments, the one or more aprotic polar solvents include acetonitrile, tetrahydrofuran (THF), dimethylformamide (DMF), dimethyl sulfoxide (DMSO), dichloromethane (DCM), sulfolane, or combinations thereof. In one embodiment, the first solvent and / or the second solvent comprises acetonitrile. In another embodiment, the first solvent and / or the second solvent comprises a mixture of acetonitrile and sulfolane.

[0151] Other embodiments of this application relate to oligonucleotides prepared by any of the methods described herein. Example

[0152] Some aspects of the above-described embodiments are further disclosed in detail in the following examples, which do not limit the scope of this disclosure in any way. Those skilled in the art will understand that many other embodiments, as described above and in the claims, also fall within the scope of the compositions, kits, and methods of this application. General Procedure for DMT Load Measurement

[0153] The DMT loading (μmol / g) of the 5'-DMT-protected nucleotides of the multivalently coupled center (PVH) was determined using UV / Vis spectrophotometry. Samples were dissolved in acetonitrile (AcN) containing toluenesulfonic acid (TSA). The acid-cleaved DMT protecting group and its loading were quantitatively determined. Measurements were performed using an Agilent 8453 UV-Vis spectrophotometer, Agilent UV-Vis ChemStation software Rev.A. 10.0, and Agilent UV-Vis rectangular cuvettes (10 mm, 3.5 mL, P / N 5061-3387). • A masterbatch of TSA solution in AcN was prepared by dissolving 8.0 g of TSA in 500 mL of HPLC-grade AcN. The resulting solution was stable at ambient temperature for 4 weeks. • Launch the Agilent UV-Vis ChemStation software and select a fixed wavelength of 498nm. • Obtain blank spectra using TSA / AcN solution. • Accurately weigh 20.0-26.0 mg of nucleotide-coupled polymer sample and transfer it to 100 mL LSA / AcN solution. Vortex for 2 minutes, and allow any insoluble substances to settle to the bottom within 10-15 minutes. • Transfer the supernatant to a dry cuvette (Agilent UV-Vis rectangular cuvette, 10 mm, 3.5 mL). • Scan the sample solution and record the absorbance value at 498 nm. DMT loading on the CPG stent is determined by the following formula: • Use 5 digits of the absorbance value at 498nm. • The sample volume is 100 mL. The extinction coefficient ε of DMT is estimated to be 76.5 mL / cm*μmol. • Convert the sample weight from mg to g. Example 1. General procedure for the synthesis of polymer (1a)

[0154] 2 g of commercially available MeO-PEG-amine (MW = 20 kDa, Nanosoft Polymer Inc.), 178 mg of Fmoc-Lys(Fmoc)-OH (AAPPTec), 126 mg of 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylurea hexafluorophosphate (HBTU) (AK Scientific), and 56 mg of diisopropylethylamine (TCI Chemicals) were dissolved in 10 mL of anhydrous acetonitrile (Sigma Aldrich). The reaction mixture was stirred for 2 hours. The mixture was then added to stirred diethyl ether to precipitate the Fmoc-Lys(Fmoc)-OH modified PEG. 20 mL of a 20% piperidine dimethylformamide (DMF) solution was added to the dried precipitate and stirred for 1 hour to remove the Fmoc groups, giving polymer (1a), which was separated by precipitation in diethyl ether to give 1.88 g of polymer (1a) (yield: 93.5%). Example 2. Synthesis of polymer (1a)-DMT-dT-3'-succinate coupling compound

[0155] Polymer (1a) (1.88 g) was dissolved in 6 mL of anhydrous acetonitrile. Separately, 298 mg of DMT-dT-3'-succinic acid TEA salt (Hongene Biotech), 168 mg of HBTU, and 74.2 mg of diisopropylethylamine were dissolved in 5 mL of anhydrous acetonitrile, and the resulting solutions were allowed to stand at room temperature for 15 minutes. The polymer solution and the DMT-dT-3'-succinic acid solution were combined and stirred overnight at room temperature. The mixture was precipitated with diethyl ether to obtain the first dT conjugate containing DMT. While stirring, 1.356 mL of trichloroacetic acid (TCA) (600 mg / mL, in DCM) and 324 mg of triethylsilane (TES) were added to the dried precipitate. After approximately 10 minutes, the reaction mixture was precipitated with diethyl ether to give 2.04 g of polymer (1a)-dT conjugate (yield: 96%). Example 3. Synthesis of polymer (2)-DMT-dT-3'-succinate coupling compound

[0156] Commercially available 4-side-chain PEG (2 g) (with an average molecular weight of approximately 5 kDa for each side chain) was dissolved in 6 mL of anhydrous acetonitrile. In a separate container, 149 mg of DMT-dT-3'-succinic acid TEA salt, 168 mg of HBTU, and 74.2 mg of diisopropylethylamine were combined in 5 mL of anhydrous acetonitrile, mixed, and allowed to stand for 15 minutes. The 4-side-chain PEG solution and the DMT-dT-3'-succinic acid solution were then combined and stirred overnight at room temperature. The mixture was poured into diethyl ether and stirred to precipitate the first dT conjugate with DMT. The dried precipitate was added to 5 mL of nucleotide capping solution (capping reagent A (Sigma Aldrich) and 1-methylimidazole / tetrahydrofuran (Applied Biosystem) in a 1:1 ratio), and the mixture was stirred for 1 hour. The reaction mixture was poured into diethyl ether and stirred to precipitate the polymer (2)-dT conjugate with 5'DMT. 1.356 mL of trichloroacetic acid (TCA) (600 mg / mL, DCM) and 324 mg of triethylsilane (TES) were added to the above precipitate, and the mixture was stirred for 10 minutes. The reaction mixture was poured into stirred diethyl ether, dried under reduced pressure, and precipitated to obtain 2.03 g (98.5% yield) of polymer (2)-dT coupling. Example 4. Direct coupling of eight DMT-dT-3'-succinic acid units to polymer (1a)

[0157] 1.02 g of polymer (1a)-dT coupling (where the average molecular weight of polymer (1a) is approximately 20 kDa) was dissolved in a 100 mL round-bottom flask in a mixture of ETT activator (5-(ethylthio)-1H-tetrazole, 250 mM, dissolved in 4.76 mL anhydrous acetonitrile, Sigma-Aldrich) and 248 mg of DMT-dT phosphorous amide (Hongene Biotech). After constant magnetic stirring for 60 min, 158 mg of m–CPBA (3-chloroperbenzoic acid, Sigma-Aldrich) was added in powder form. After constant stirring for 10 min, 2.04 mL of trichloroacetic acid (TCA, 600 mg / mL, dissolved in DCM) and 0.706 mL of TES were added. After constant stirring for 10 min, the reaction mixture was poured into isopropanol and stirred. The precipitate was washed twice with diethyl ether to give 1.024 g of polymer (1a)-2dT (yield: 97.4%). The above steps were repeated six times to obtain polymer (1a)-8dT, which had a full product purity (FLP) of 82.9% after nucleotide cleavage (yield: 77.2%). HPLC results are as follows. Figure 1 As shown, this represents the relative amounts of oligonucleotides after cleavage from the polymer carrier. Example 5. Direct coupling of eight DMT-dT-3'-succinic acid units to polymer (2)

[0158] 2.03 g of polymer (2)-dT conjugate was dissolved in ETT activator (250 mM, dissolved in 9.52 mL anhydrous acetonitrile), and 496 mg of DMT-dT phosphorous amide was added to a 100 mL round-bottom flask equipped with a magnetic stir bar. After stirring at a constant magnetic force for 60 min, 317 mg of mCPBA powder was added. After stirring at a constant magnetic force for 10 min, 4.08 mL LTCA (600 mg / mL, dissolved in DCM) and 1.417 mL of TES were added. After stirring at a constant magnetic force for 10 min, the reaction mixture was poured into isopropanol and stirred. The precipitate was washed twice with diethyl ether to give 1.93 g of polymer (2)-2dT (yield: 90.8%). The above steps were repeated six times to obtain polymer (2)-8dT in 81.6% FLP after nucleotide cleavage (yield: 82.9%). HPLC results are shown below. Figure 2 As shown, this represents the relative amounts of oligonucleotides after cleavage from the polymer carrier. Example 6. Total product purity (%FLP) of mononucleotides and oligonucleotides

[0159] The full product purity (FLP) of dT after cleavage from the polymer support is shown in Table 1. It can be seen that polymers (1a) and (2) both exhibit high nucleotide synthesis efficiency, with FLPs exceeding 80% for lengths up to eight nucleotides. However, FLP cannot be used to accurately calculate coupling efficiency, especially in the case of synthesizing long dT sequences, as small impurities may be captured during precipitation. Table 1. Total product purity (%FLP) of mononucleotides and oligonucleotides after cleavage from polymer support Example 7. Direct coupling of eight DMT-dN-3'-succinic acid units to polymer (1a)

[0160] 0.85 g of polymer (1a)-dT conjugate with an average molecular weight of approximately 20 kDa was dissolved in a mixture of ETT activator (5-(ethylthio)-1H-tetrazole in anhydrous acetonitrile) and DMT-dT phosphorous amide (Hongene Biotech) as described in Example 4 to obtain polymer (1a)-2dT. This process was repeated six times with other nucleotides to prepare the sequence polymer (1a)-dT-dT-dA-dA-dG-dG-dC-dC-OH, which exhibited a full product purity (FLP) of 82% after nucleotide cleavage (yield: 88.9%). HPLC results are as follows. Figure 3 As shown, this represents the relative amounts of oligonucleotides after cleavage from the polymer carrier. Example 8. Direct coupling of eight DMT-dT-3'-succinic acid units to polymer (1b)

[0161] The polymer (1b)-dT coupling with an average molecular weight of approximately 10 kDa was dissolved in a mixture of ETT activator (5-(ethylthio)-1H-tetrazole, 250 mM, dissolved in 4.76 mL anhydrous acetonitrile, Sigma Aldrich) and 248 mg DMT-dT phosphorous amide (Hongene Biotech) in a 100 mL round-bottom flask. After constant magnetic stirring for 60 min, 158 mg m–CPBA (3-chloroperbenzoic acid, Sigma Aldrich) was added in powder form. After constant stirring for 10 min, 2.04 mL trichloroacetic acid (TCA, 600 mg / mL, dissolved in DCM) and 0.706 mL TES were added. After constant stirring for 10 min, the reaction mixture was poured into isopropanol and stirred. The precipitate was washed twice with diethyl ether to give 1.024 g of polymer (1b)-2dT. The above steps were repeated six times to obtain polymer (1b)-8dT, which had a purity of 98.3% (yield: 92.8%) after nucleotide cleavage. HPLC results are shown below. Figure 4 As shown, this represents the relative amounts of oligonucleotides after cleavage from the polymer carrier. Example 9. Direct coupling of DMT-nucleoside-3'-succinic acid units to polymer (1c)

[0162] Polymer (1c)-dU conjugates with an average molecular weight of approximately 5 kDa were dissolved in a mixture of ETT activator (5-(ethylthio)-1H-tetrazole in anhydrous acetonitrile) and DMT-mU (i.e., DNT-2'-OMe-U) or DMT-mA (i.e., DMT-2'-OMe-A) phosphorous acid (Hongene Biotech) as described in Example 4 to obtain polymer (1c)-2mU or polymer (1c)-2mUmA. This process was repeated as needed with other nucleotides (e.g., 2'-OMe-U(mU), 2'-fluoro-U)(fU), 2'-OCH2CH2OCH3-T (moeT)) to prepare the sequence polymer (1c)-mU-mU-mU-mU-mU-mU-mU, which exhibited a full product purity (FLP) of 93.6% after nucleotide cleavage; sequence polymer (1c) -mU-mU-mU-mU-mU-mU-mU-mU-fU-moeT, which exhibits a full product (FLP) purity of 90.4% after nucleotide cleavage; and the sequence polymer (1c)-mU-mA-mA-mA-mA-mA, which exhibits a full product purity of 92.8% after nucleotide cleavage. Example 10. General procedure for synthesizing polymer (3a)

[0163] Aliquots of polymer (1b), Fmoc-Lys(Fmoc)-OH, (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylurea hexafluorophosphate (HBTU), and diisopropylethylamine were dissolved in anhydrous acetonitrile. The reaction mixture was stirred for 2 hours. The mixture was then added to stirred diethyl ether to precipitate the intermediate—the Lys-modified product. 20 mL of 20% piperidine (dissolved in dimethylformamide (DMF)) was added to the dried precipitate and the mixture was stirred for 1 hour to remove the Fmoc groups, yielding polymer (3a), which was separated by precipitation in diethyl ether. Polymer (3b) was prepared by a similar method to that used to prepare polymer (1c) from PEG with a molecular weight of approximately 5 kDa. Example 11. Direct coupling of DMT-nucleoside-3'-succinic acid units to polymer (3a)

[0164] Polymer (3a)-dT couplings with an average molecular weight of approximately 10 kDa were dissolved in a 100 mL round-bottom flask in a mixture of ETT activator (5-(ethylthio)-1H-tetrazole, 250 mM, dissolved in 4.76 mL anhydrous acetonitrile, Sigma Aldrich) and DMT-mU (i.e., DMT-2'-OMe-U) or DMT-U phosphorous acid (Hongene Biotech). After constant magnetic stirring for 60 min, 158 mg m–CPBA (3-chloroperbenzoic acid, Sigma Aldrich) was added in powder form. After constant stirring for 10 min, 2.04 mL trichloroacetic acid (TCA, 600 mg / mL, dissolved in DCM) and 0.706 mL TES were added. After constant stirring for 10 min, the reaction mixture was poured into isopropanol and stirred. The precipitate was washed twice with diethyl ether to obtain polymer (3a). -dT-mU-mU-mU-mU-mU-mU-mU-mU, exhibiting 94% full product purity (FLP) after nucleotide cleavage, such as Figure 5 As shown; and polymer (3a). -dT-mU ... Example 12. Elongation of nucleotide chain polymer (3b)-9dT

[0165] Polymer (3b)-9dT conjugates with an average molecular weight of approximately 5 kDa PEG and a complete product purity (FLP) of 76.1% were prepared using the methods described in Examples 4 and 11. Figure 6A The polymer (3b)-10dT was dissolved in a mixture of ETT activator (5-(ethylthio)-1H-tetrazole in anhydrous acetonitrile) and DMT-dT phosphorous amide (Hongene Biotech), in a manner similar to that described in Examples 4 and 11, to obtain the polymer (3b)-10dT. This procedure was repeated 13 times with other nucleotides to prepare the sequence polymer (3b)-23dT, which exhibited a complete product purity (FLP) of 63.2% after nucleotide cleavage. Figure 6B The coupling reaction utilizes an ACN / DCM mixture to improve polymer solubility. Example 13. Preparation of vulcanized polymer (3b)-dT(S)dT(S)dT(S)dT(S)dT(S)dT(S)dT(S)dT(S)dT

[0166] A polymer (3b)-dT conjugate (223 mg) with an average molecular weight of approximately 5 kDa was dissolved in a mixture of ETT activator (5-(ethylthio)-1H-tetrazole, 250 mM in anhydrous acetonitrile, 2.05 mL, Sigma Aldrich) and 168 mg of DMT-dT phosphorous acid (Hongene Biotech). After shaking for 90 minutes, 37 μL of isopropanol was added. After shaking for 60 minutes, excess xanthane hydride (45 mg) was added, and the mixture was shaken for another 60 minutes. Excess trichloroacetic acid (600 mg / mL DCM, 1.53 mL) and TES (527 μL) were added. After shaking for 10 minutes, the reaction mixture was poured into isopropanol and stirred. The precipitate was washed twice with diethyl ether to give polymer (3b)-dT(S)dT. (S) represents the phosphate thioester bond (P=S) in the oligonucleotide backbone. The above steps were repeated six times to obtain polymer (3b)-dT(S)dT(S)dT(S)dT(S)dT(S)dT(S)dT(S)dT, which exhibited a full product purity (FLP) of 97.0% after nucleotide cleavage. Figure 7 ). Example 14. Preparation of polymer (1b)-dTdTdT(S)dT(S)dT(S)dT(S)dT(S)dT

[0167] A polymer (1b)-3dT conjugate (154 mg) with an average molecular weight of approximately 10 kDa was prepared according to the method described in Example 4 and treated in a manner similar to that described in Example 13 to obtain polymer (1b)-dTdTdT(S)dT. This procedure was repeated four more times to obtain polymer (1b). -dTdTdT(S)dT(S)dT(S)dT(S)dT(S)dT, which exhibits a total product purity (FLP) of 96.3% after nucleotide cleavage. Figure 8 ). Example 15. Study of mixed backbone oligonucleotides

[0168] A polymer (3b)-8dT(S) coupling compound (FLP 97.0%) with a molecular weight of approximately 5 kDa was dissolved in a mixture of ETT activator (5-(ethylthio)-1H-tetrazole in anhydrous acetonitrile) and DMT-dT phosphorous amide (Hongene Biotech) using a method similar to that described in Example 4. However, the reaction was carried out using m-chloroperoxybenzoic acid (m-CPBA) or tBuOOH as an oxidant to obtain the polymer (3b)-7dT(S)dT(O)dT-OH. The HPLC results are shown below. Figure 9A and 9B As shown, m-CPBA acid induces significant desulfurization, but no significant desulfurization is observed when tBuOOH is used as the oxidant. Example 16. Synthesis of polymer (1b)-dT-8mU(VP)mU

[0169] A polymer (1b)-dT-8mU conjugate with a molecular weight of approximately 10 kDa was dissolved in a mixture of ETT activator (5-(ethylthio)-1H-tetrazole in anhydrous acetonitrile) and 3 equivalents of 5'-POM-VP-mU (i.e., 5'-[O,O-bis(neovaleroxymethyl)-vinylphosphonate, 2'-OMe-U-phosphite)) (Hongene Biotech), using a dissolution method similar to that described in Example 4, but without the deblocking step, to obtain polymer (1b)-dT-8mU-VPmU, which exhibited a full product purity (FLP) of 83.0% after nucleotide cleavage. Figure 10 ). MS: 1599[M-2H / 2]-2; 1066[M-3H / 3]-3. Example 17. Synthesis of polymer (1c)-8dT-(VP)mA

[0170] A polymer (1)-8dT (FLP: 86%) conjugate with a molecular weight of approximately 5 kDa was dissolved in a mixture of ETT activator (5-(ethylthio)-1H-tetrazole in anhydrous acetonitrile) and 3 equivalents of 5'-POM-VP-mA (Hongene Biotech). The dissolution method was similar to that described in Example 4, but without the desealing step, to obtain polymer (1c)-8dT-VPmA, which exhibited a full product purity (FLP) of 82.0% after nucleotide cleavage. Figure 11 The results were confirmed by HPLC and LC / MS. Example 18. Synthesis of polymer (1c)-dTdT(S)dT

[0171] Using a method similar to that of Example 15, and employing tBuOOH as an oxidant, a polymer (1c)-dT conjugate with a molecular weight of approximately 5 kDa was dissolved in a mixture of ETT activator (5-(ethylthio)-1H-tetrazole in anhydrous acetonitrile, 319 μL) and 1.03 equivalents of DMT-dT(S)dT-phosphoramide (Hongene Biotech) to obtain a polymer (1c)-dTdT(S)dT that exhibited a total product purity (FLP) of 91.9% after nucleotide cleavage. Figure 12 Even with a slight excess of 1 molar equivalent of DMT-dT(P=S)dT-phosphoramide, the conversion rate was as high as 99.2%. Example 19. Synthesis of DPEG-OH anchors for oligonucleotide synthesis

[0172] Synthesis of polymer (4): Commercially available amine-terminated PEG (600 mg, 0.12 mmol) was mixed with 3-bromo-1-propanol (133.4 mg, 0.96 mmol) and potassium carbonate (132.7 mg, 0.96 mmol) in 5 mL of ACN and stirred at 83 °C for 3 days. The reaction was confirmed to be complete by ninhydrin test. The reaction mixture was filtered, and polymer (4) was obtained by subjecting the reaction mixture to three rounds of MTBE precipitation, wherein DCM was used as the resuspension solvent.

[0173] Synthesis of polymer (5): Commercially available amine-terminated PEG (5 g, 1 mmol) was mixed with 3-bromo-2-(bromomethyl)propionic acid (737.7 mg, 3.0 mmol), azirmonatriazole tetramethylurea hexafluorophosphate (HATU, 1.25 g, 3.3 mmol), and N,N-diisopropylethylamine (DIPEA, 775.4 mg, 6.0 mmol) in 50 mL of DMF and stirred overnight at 33 °C. The reaction was confirmed to be complete by ninhydrin test. The intermediate was obtained by pouring the reaction mixture into cold MBTE and drying the resulting solid under vacuum. The halide was hydrolyzed in an alkaline aqueous solution (pH = 10) at 90 °C for 3 days. After hydrolysis, water was removed by lyophilization. The remaining solid was resuspended in DCM and the supernatant was obtained by filtration. Polymer (5) was obtained by MTBE precipitation.

[0174] Synthesis of polymer (6): Commercially available amine-terminated PEG (5 g, 1 mmol) was stirred overnight at room temperature with 2,4-dibromobutyryl chloride (881 mg, 3 mmol) and DIPEA (775.4 mg, 6.0 mmol) in 50 mL of DMF. An intermediate was obtained by pouring the reaction mixture into cold MBTE and drying the resulting solid under vacuum. The halide was hydrolyzed in an alkaline aqueous solution (pH = 10) at 90 °C for 3 days. After hydrolysis, water was removed by lyophilization. The remaining solid was resuspended in DCM and the supernatant was obtained by filtration. Polymer (6) was obtained by MTBE precipitation. Example 20. Binding of succinic acid dT to DPEG-OH anchors and elongation of nucleotide chains

[0175] Under argon atmosphere, polymers (4), (5), or (6) and 5'-O-(4,4'-dimethoxytriphenylmethyl)-thymidine-3'-O-succinate, triethylamine salt (305.6 mg, 0.41 mmol), were dissolved together with N-methylimidazole (NMI, 67.28 mg, 0.82 mmol) in 3 mL of anhydrous DCM. 2,6-dichlorobenzoyl chloride (DcbCl, 85.81 mg, 0.41 mmol) was added to this solution, and the mixture was stirred for 20 minutes to activate the succinate. The activated succinate solution was then slowly added dropwise to polymers 4, 5, or 6 (0.2 mmol) over 40 minutes, and the reaction was allowed to continue overnight at room temperature. To unblock the triphenylmethyl groups on the bases, trichloroacetic acid (TCA, 600 mg / mL, 7.7 mmol in DCM) was then added to the solution, followed by the addition of triethylsilane (TES, 4.6 mmol) as a scavenger. The product was obtained by two rounds of IPA precipitation and MBTE washing, followed by vacuum drying. Further bases were coupled to polymers (4), (5), or (6)-dT anchors using a procedure similar to that described in Example 4. Oligonucleotides consisting of eight thymidines were synthesized using polymers (4), (5), or (6) as starting materials. The products obtained from polymers (4) and (6) were characterized by HPLC (see [link to relevant documentation]). Figure 13A and Figure 13B The yields and overall purity of the cleaved 8dT oligonucleotides are shown in Table 2 below. Table 2. Summary of oligonucleotide synthesis after polymer support cleavage Example 21. Synthesis of DPEG-OH anchors for oligonucleotide synthesis

[0176] First, 4-acetoxybenzoic acid (216.2 mg, 1.2 mmol) was pre-activated for 10 min in 5 mL of DMF with 1-hydroxybenzotriazole (HOBt, 162.4 mg, 1.2 mmol) and N,N'-diisopropylcarbodiimide (DIC, 166.58 mg, 1.32 mmol). This solution was then added to the DPEG-diamine polymer (1c) (600 mg, 0.24 mmol) and stirred overnight at room temperature. The reaction was stopped by IPA precipitation followed by washing with MTBE. To obtain the final product, 5 mL of a 10% piperidine / DMF solution was added to the precipitate and stirred for 1 h at room temperature. The polymer (7) was obtained by two rounds of MTBE precipitation followed by vacuum drying. Example 22. Coupling of succinic acid dT to DPEG-OH anchor (7) and elongation of nucleotide chains

[0177] To couple the first thymidine base to polymer (7), 5'-O-(4,4'-dimethoxytriphenylmethyl)-thymidine-3'-O-succinate, triethylamine salt (305.6 mg, 0.41 mmol), and N-methylimidazole (NMI, 67.28 mg, 0.82 mmol) were dissolved in 3 mL of anhydrous DCM under argon atmosphere. 2,6-dichlorobenzoyl chloride (DcbCl, 85.81 mg, 0.41 mmol) was added to this solution, and the mixture was stirred for 20 minutes to activate the succinate. The activated succinate solution was then slowly added dropwise to polymer 7 (0.2 mmol) over 40 minutes, and the reaction was allowed to continue overnight at room temperature. To unblock the triphenylmethyl group on the base, trichloroacetic acid (TCA, 600 mg / mL, 7.7 mmol in DCM) was added to the solution, followed by the addition of triethylsilane (TES, 4.6 mmol) as a scavenger. The product was obtained by two rounds of IPA precipitation and MBTE washing, and then vacuum drying was performed.

[0178] Further bases were coupled to the polymer (7)-dT anchor following a procedure similar to that described in Example 4. An oligonucleotide consisting of eight thymidines was synthesized using polymer (7) as the starting material. The final oligonucleotide product was cleaved from the anchor using a 1:1 v / v mixture of NH4OH and methylamine and characterized by HPLC. Figure 13C The recovered products had a total yield of 81.8% and a total purity of 93.2%.

[0179] Compared to LPOS polymer anchors with terminal -NH2 groups, LOPS polymer anchors with terminal hydroxyl groups were observed to have comparable and / or higher loading, FLP, and yield. Furthermore, based on HPLC results, asymmetric polymer anchors (e.g., polymers (4) and (7)) resulted in lower impurity levels and were relatively easier to precipitate during oligonucleotide synthesis.

Claims

1. A polymer for liquid phase oligonucleotide synthesis having a structure of formula (I): in: R is H, or unsubstituted or substituted C1-C6 alkyl; W is C1-C 20 Alkylene, 2-20 membered heteroalkylene or a bond; Q is L 1 C1-C 20 alkylene, 2-20 membered heteroalkylene, optionally substituted phenylene, optionally substituted 5-6 membered heteroarylene, optionally substituted 3-10 membered heterocyclylene or optionally substituted C3-C 10 Cycloalkylene, or C1-C 20 alkylene, or a 2-20 membered heteroalkylene, wherein one or more methylene repeating units are each independently substituted by a group selected from the group consisting of optionally substituted phenylene, optionally substituted 5-6 membered heteroarylene, optionally substituted 3-10 membered heterocyclylene, optionally substituted C3-C 10 Cycloalkylene, -C(=O)-, -CH=CH-, and R 1 and R 2 Each independently is -OR 3 or -N R4a R 4b ; R 3 is H, C1-C6 alkyl, hydroxyl protecting group or R 4a and R 4b Each is independently H, optionally substituted C1-C6 alkyl, -C(=O)(C1-C6 alkyl), -C(=O)phenyl, an amino protecting group or or R 4a and R 4b Together they are a divalent amino protecting group; L 2a and L 2b Each independently is C1-C 20 alkylene, 2-20 membered heteroalkylene, optionally substituted phenylene, optionally substituted 5-6 membered heteroarylene, optionally substituted 3-10 membered heterocyclylene or optionally substituted C3-C 10 Cycloalkylene, or C1-C 20 alkylene or 2-20 membered heteroalkylene, wherein one or more methylene repeating units are each independently substituted by a group selected from the group consisting of optionally substituted phenylene, optionally substituted 5-6 membered heteroarylene, optionally substituted 3-10 membered heterocyclylene, optionally substituted C3-C 10 Cycloalkylene, -C(=O)-, -CH=CH-, and R 5a , R 5b , R 6a and R 6b Each independently is H, -OR 7 or -NR 8a R 8b ; R 7 Each independently represents H, C1-C6 alkyl, hydroxyl protecting group or R 8a and R 8b Each is independently H, optionally substituted C1-C6 alkyl, -C(=O)(C1-C6 alkyl), -C(=O)phenyl, an amino protecting group or or R 8a and R 8b Together they are a divalent amino protecting group; L 3a and L 3b Each independently is C1-C 20 alkylene, 2-20 membered heteroalkylene, optionally substituted phenylene, optionally substituted 5-6 membered heteroarylene, optionally substituted 3-10 membered heterocyclylene or optionally substituted C3-C 10 Cycloalkylene, or C1-C 20 alkylene or 2-20 membered heteroalkylene, wherein one or more methylene repeating units are each independently substituted by a group selected from the group consisting of optionally substituted phenylene, optionally substituted 5-6 membered heteroarylene, optionally substituted 3-10 membered heterocyclylene, optionally substituted C3-C 10 Cycloalkylene, -C(=O)-, -CH=CH-, and R 9a , R 9b , R 10a and R 10b Each independently is H, -OR 11 or -NR 12a R 12b ; R 11 Each independently represents H, C1-C6 alkyl, hydroxyl protecting group or R 12a and R 12b Each is independently H, optionally substituted C1-C6 alkyl, -C(=O)(C1-C6 alkyl), -C(=O)phenyl, an amino protecting group or or R 12a and R 12b Together they are a divalent amino protecting group; L 4a and L 4b Each independently is C1-C 20 alkylene, 2-20 membered heteroalkylene, optionally substituted phenylene, optionally substituted 5-6 membered heteroarylene, optionally substituted 3-10 membered heterocyclylene or optionally substituted C3-C 10 Cycloalkylene, or C1-C 20 alkylene or 2-20 membered heteroalkylene, wherein one or more methylene repeating units are each independently substituted by a group selected from the group consisting of optionally substituted phenylene, optionally substituted 5-6 membered heteroarylene, optionally substituted 3-10 membered heterocyclylene, optionally substituted C3-C 10 Cycloalkylene, -C(=O)-, -CH=CH-, and R 13a , R 13b , R 14a and R 14b Each is independently H, -OH, protected hydroxy, -NH2, -NH(optionally substituted C1-C6 alkyl) or protected amino; m1, m2, m3, m4, m5, m6 and m7 are each independently 0 or 1; and j is an integer from 15 to 1500.

2. The polymer according to claim 1, wherein R is methyl.

3. The polymer of claim 1 or 2, wherein W is -CH2CH2NH-.

4. The polymer of claim 1 or 2, wherein W is -CH2CH2O-.

5. The polymer of claim 1 or 2, wherein W is -CH2CH2-.

6. The polymer according to any one of claims 1 to 5, wherein L 1 It is C2-C 10 Alkylene linking group.

7. The polymer according to any one of claims 1 to 5, wherein L 1 is a 3-12 membered heteroalkylene group containing one, two or three heteroatoms selected from N, O and S.

8. The polymer according to any one of claims 1 to 7, wherein Q is selected from the following group:

9. The polymer according to any one of claims 1 to 8, wherein R 3 Each is independently H or a hydroxyl protecting group.

10. The polymer according to any one of claims 1 to 8, wherein One R 3 is H or a hydroxyl protecting group, and another R 3 for 11. The polymer according to any one of claims 1 to 8, wherein Each R 4a is H, each R 4b are independently H, -C(=O)(C1-C6 alkyl), -C(=O)phenyl, an amino protecting group or or -NHR 4b The hydrogen in R is absent and 4b It is a divalent amino protecting group.

12. The polymer according to claim 11, wherein Each R 4b are independently H, -C(=O)CH3 or an amino protecting group.

13. The polymer according to claim 11, wherein One R 4b is H or an amino protecting group, and another R 4b for 14. The polymer according to claim 11, wherein R 3 Each independently And R 4b Each independently 15. The polymer according to any one of claims 10, 11, 13 and 14, wherein m2 and m3 are each 1.

16. The polymer of any one of claims 10, 11 and 13-15, wherein L 2a and L 2b Each is independently a C2-C6 alkylene group, or a 3-12 membered heteroalkylene group containing one, two or three heteroatoms selected from N, O and S.

17. The polymer of any one of claims 10, 11 and 13-15, wherein L 2a and L 2b Each is independently optionally substituted phenylene; or C2-C6 alkylene or 3-12 membered heteroalkylene in which one methylene unit is substituted by optionally substituted phenylene.

18. The polymer according to any one of claims 1 to 17, wherein Each R 8a is H, and R 5a , R 5b , R 6a and R 6b Each independently is H, -OR 7 or -NHR 8b , each R 8b are independently H, -C(=O)(C1-C6 alkyl), -C(=O)phenyl, an amino protecting group or or -NHR 8b The hydrogen in R is absent and 8b It is a divalent amino protecting group.

19. The polymer of claim 18, wherein R 5a , R 5b , R 6a and R 6b Each is independently H, -OH, protected hydroxy, -NH2, -NHC(=O)CH3 or protected amino.

20. The polymer according to claim 19, wherein R 5a , R 5b , R 6a and R 6b Each is independently a hydroxyl group or a protected hydroxyl group.

21. The polymer of claim 19, wherein R 5a , R 5b , R 6a and R 6b Each is an amino group or a protected amino group.

22. The polymer of claim 18, wherein R 5a , R 5b , R 6a and R 6b At least one of them is independently -OR 7 or -NHR 8b , R 7 At least one of And R 8b At least one of 23. The polymer of claim 18, wherein R 5a , R 5b , R 6a and R 6b Each of the 7 or -NHR 8b , R 7 Each independently And each R 8b Independently 24. The polymer according to claim 22 or 23, wherein m4 and m5 are each 1.

25. The polymer of any one of claims 22 to 24, wherein L 3a and L 3b Each is independently a C2-C6 alkylene group, or a 3-12 membered heteroalkylene group containing one, two or three heteroatoms selected from N, O and S.

26. The polymer of any one of claims 22-24, wherein L 3a and L 3b Each is independently optionally substituted phenylene; or C2-C6 alkylene or 3-12 membered heteroalkylene in which one methylene unit is substituted by optionally substituted phenylene.

27. The polymer of any one of claims 1 to 26, wherein Each R 12a is H, and R 9a , R 9b , R 10a and R 10b Each independently is H, -OR 11 or -NHR 12b , each R 12b are independently H, -C(=O)(C1-C6 alkyl), -C(=O)phenyl, an amino protecting group or or -NHR 12b The hydrogen in R is absent and 12b It is a divalent amino protecting group.

28. The polymer of claim 27, wherein R 9a , R 9b , R 10a and R 10b are each independently H, -OH, protected hydroxyl, -NH 2、 -NHC(=O)CH3 or a protected amino group.

29. The polymer of claim 28, wherein R 9a , R 9b , R 10a and R 10b Each is a hydroxy group or a protected hydroxy group.

30. The polymer of claim 28, wherein R 9a , R 9b , R 10a and R 10b Each is an amino group or a protected amino group.

31. The polymer of claim 27, wherein R 9a , R 9b , R 10a and R 10b At least one of them is independently -OR 11 or -NHR 12b , R 11 At least one of And at least one R 12b for 32. The polymer of claim 27, wherein R 9a , R 9b , R 10a and R 10b Each independently is -OR 11 or -NHR 12b , each R 11 for And each R 12b for 33. The polymer of claim 31 or 32, wherein m6 and m7 are each 1.

34. A polymer according to any one of claims 31 to 33, wherein L 4a and L 4b Each is independently a C2-C6 alkylene group, or a 3-12 membered heteroalkylene group containing one, two or three heteroatoms selected from N, O and S.

35. The polymer of any one of claims 31 to 34, wherein R 13a , R 13b , R 14a and R 14b Each is independently H, -OH, protected hydroxy, -NH2, -NHC(=O)CH3 or protected amino.

36. The polymer of claim 1 having the structure of formula (Ia):

37. The polymer of claim 1 having a structure of Formula (Ib), (Ib-1), (Ic) or (Ic-1):

38. The polymer of claim 37, wherein The structure of formula (Ib-1) has the structure of formula (Ib-2) or (Ib-3):

39. The polymer of claim 37, wherein The structure of formula (Ib-1) has the structure of formula (Ib-4):

40. The polymer of claim 37, wherein R 5b and R 6b Each is independently -NH2 or -NHAc.

41. The polymer of claim 37, wherein R 5b and R 6b Each is -OH.

42. The polymer of claim 1, wherein The structure of formula (I) has the structure of formula (Id), (Ie) or (If):

43. The polymer of any one of claims 1 to 42, wherein j is an integer between 400 and 500.

44. The polymer of any one of claims 1 to 43, wherein The average molecular weight of the polymer is from about 15 kDa to about 30 kDa.

45. The polymer of any one of claims 1 to 44, wherein The average molecular weight of the polymer is about 20 kDa.

46. ​​A polymer for liquid phase oligonucleotide synthesis having a structure of formula (II), in: A is selected from the following group: carbon atom, C2-C 20 alkylene, 2-20 membered heteroalkylene, phenylene, 5-10 membered heteroarylene, C 5-10 Cycloalkylene and 5-10 membered heterocycloalkylene; Each R 15 Independently selected from the group consisting of -OC1-C6 alkyl, -OC(O)(C1-C6 alkyl), -OC(O)phenyl, -NHC(O)(C1-C6 alkyl), -NHC(O)phenyl, -NHC(O)phenylene-acetoxymethyl, protected hydroxyl or protected amino; Each R 16 are independently -OH or -NH2; q 1 and q 2 are each independently an integer from 10 to 500; and s and t are each independently an integer of 1-4, wherein s+t is equal to or greater than 2.

47. The polymer of claim 46, wherein s+t is 4.

48. A polymer according to claim 46 or 47, wherein s is 2 and t is 2.

49. The polymer of any one of claims 46-48, having the structure of formula (IIa):

50. The polymer of any one of claims 46 to 49, wherein A is a carbon atom or a phenylene group.

51. The polymer of claim 46, wherein s+t is 8.

52. The polymer of claim 46 or 47, wherein s is 5 and t is 3.

53. The polymer of any one of claims 46, 51 and 52, having the structure of formula (IIb):

54. The polymer of any one of claims 46 to 53, wherein Each q1 is independently about 30 to about 150, about 40 to about 100, or about 50 to about 75.

55. The polymer of any one of claims 46 to 54, wherein Each q2 is independently about 30 to about 150, about 40 to about 100, or about 50 to about 75.

56. The polymer of any one of claims 46 to 55, wherein The polymer has an average molecular weight of about 10 kDa to about 50 kDa, about 15 kDa to about 30 kDa, or about 20 kDa.

57. A method for preparing an oligonucleotide by liquid phase oligonucleotide synthesis, comprising: dissolving the polymer of any one of claims 1 to 56 in a first solvent to form a reaction matrix; and The polymer is reacted with one or more nucleoside analogs to form a first bioconjugate comprising the structure of formula (III): in: B 1 It is a nitrogenous base; G 1 is a 5' hydroxyl blocking group; X is O or NR 20 ; R 20 is H or C1-C6 alkyl; R a is -H, -OH, halogen, -O-(C1-C6 alkyl), -O-(C1-C6 haloalkyl), or -OY, wherein Y is a 2'hydroxy protecting group; and L 5 is a cleavable heteroalkylene linker, wherein one or more carbon atoms are replaced by O, S, N, C(═O), or C(═S).

58. The method of claim 57, wherein: The structure of formula (III) is also represented by formula (IIIa):

59. The method of claim 57 or 58, wherein: B 1 is independently an optionally protected adenine, an optionally protected deazaadenine, an optionally protected cytosine, an optionally protected guanine, an optionally protected deazaguanine, an optionally protected thymine or an optionally protected uracil.

60. The method of any one of claims 57-59, wherein B 1 yes Where R x is hydrogen, unsubstituted or substituted C1-C6 alkyl or amino protecting group, or -NHR x The hydrogen in R is absent and x It is a divalent amino protecting group.

61. The method of any one of claims 57 to 60, wherein: G 1 It is a trityl-type hydroxy-protecting group selected from the group consisting of (4-methoxyphenyl)diphenylmethyl, bis(4-methoxyphenyl)phenylmethyl, tris(4-methoxyphenyl)methyl, 9-phenylxanthen-9-yl and 9-(4-methoxyphenyl)xanthen-9-yl.

62. The method of claim 38, wherein: G 1 It is bis(4-methoxyphenyl)phenylmethyl.

63. The method of any one of claims 57 to 62, wherein: The polymer has an average molecular weight of about 10 kDa to about 50 kDa, about 15 kDa to about 30 kDa, or about 20 kDa.

64. The method of any one of claims 57-63, further comprising: Removal of 5' hydroxyl blocking group (G 1 ) to form a 5' unblocked first bioconjugate; and The 5' unblocked first bioconjugate was isolated.

65. The method of claim 64, wherein: The separation of the 5' unblocked first bioconjugate is achieved by precipitation, dialysis or filtration.

66. The method of claim 65, wherein: The separation of the 5' unblocked first bioconjugate is achieved by precipitation.

67. The method of claim 66, wherein: The precipitation is effected in diethyl ether or isopropanol.

68. The method of any one of claims 57 to 67, further comprising: (a) reacting the 5' unblocked first bioconjugate with one or more nucleoside phosphoramidite analogs in a second solvent to form a second bioconjugate comprising a structure of formula (IV): in: G 2 is a 5' hydroxyl blocking group; B 2 is a nitrogenous base; and R e is a phosphite protecting group; (b) oxidizing the phosphorous acid moiety in formula (IV); (c) Removal of the 5' blocking group G 2 , to form a 5' unblocked second bioconjugate comprising a structure of formula (IV'): in: Z is O or S; and (d) isolating the 5' unblocked second bioconjugate.

69. The method of claim 68, wherein: The structure of formula (IV) is also represented by formula (IVa), and formula (IV') is also represented by formula (IV'a):

70. The method of claim 68 or 69, further comprising blocking unreacted 5' hydroxyl groups in the 5' unblocked first bioconjugate prior to step (b).

71. The method of any one of claims 68 to 70, wherein: B 2 is independently an optionally protected adenine, an optionally protected deazaadenine, an optionally protected cytosine, an optionally protected guanine, an optionally protected deazaguanine, an optionally protected thymine or an optionally protected uracil.

72. The method of claim 71, wherein: B 2 yes Where R x is hydrogen, unsubstituted or substituted C1-C6 alkyl or amino protecting group, or -NHR x The hydrogen in R is absent and x It is a divalent amino protecting group.

73. The method of any one of claims 68 to 72, wherein: G 2 It is a trityl-type hydroxy-protecting group selected from the group consisting of (4-methoxyphenyl)diphenylmethyl, bis(4-methoxyphenyl)phenylmethyl, tris(4-methoxyphenyl)methyl, 9-phenylxanthen-9-yl and 9-(4-methoxyphenyl)xanthen-9-yl.

74. The method of claim 73, wherein: G 2 It is bis(4-methoxyphenyl)phenylmethyl.

75. The method of any one of claims 68 to 74, wherein: The separation of the 5' unblocked second bioconjugate is achieved by precipitation, filtration or dialysis.

76. The method of any one of claims 68 to 75, wherein: Steps (a)-(d) are repeated for multiple cycles until one or more oligonucleotides of a desired length are synthesized.

77. The method of claim 76, wherein: Steps (a)-(d) are repeated for at least about 10 cycles.

78. The method of claim 76 or 77, further comprising removing the oligonucleotide from the polymer.

79. The method of any one of claims 68 to 78, wherein: The first solvent and the second solvent each include one or more aprotic polar solvents or a combination thereof.

80. The method of claim 79, wherein: The one or more aprotic polar solvents include acetonitrile, tetrahydrofuran (THF), dimethylformamide (DMF), dimethyl sulfoxide (DMSO), dichloromethane (DCM), sulfolane, or a combination thereof.

81. The method of claim 80, wherein: The one or more aprotic polar solvents is acetonitrile.

82. An oligonucleotide prepared by the method of any one of claims 57-81.

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