Liquid phase process for preparing oligonucleotides

Abstract

Membrane filtration assisted liquid phase processes for preparing oligonucleotides are described. The method solves the disadvantages associated with the preparation, purification and storage of nucleoside phosphoramidite. The method of the invention allows high purity supported oligonucleotides to grow from their constituent nucleosides in a stepwise manner, using simple nucleosides or short chain oligonucleotides as monomeric building blocks that are sequentially coupled to the supported growth oligonucleotides.

Description

Technical Field

[0001] This invention relates to a liquid-phase method for preparing oligonucleotides. More particularly, this invention relates to a membrane filtration-assisted liquid-phase method for preparing oligonucleotides. Background Technology

[0002] Oligonucleotides are short, sequence-defined polymers of nucleotides (also known as nucleoside phosphate groups). The structure of an oligonucleotide typically consists of a ribose-phosphate backbone, usually linked by phosphodiester units to the 3'- and 5'-oxy substituents of the ribose sugar, and each ribose ring has an aromatic nucleobase linked to the 1'-C of the ribose sugar. The combination of ribose and nucleobase is called a nucleoside, and the nucleobase varies depending on the monomer unit in both natural and pharmaceutical sequences. The nucleobase sequence gives oligonucleotides target specificity.

[0003] Oligonucleotide-based drugs have now been proposed as a new generation of therapeutic agents that act at the protein expression level and have recently been validated as novel drug forms for treating a variety of serious or life-threatening indications. Oligonucleotide drugs differ from natural oligonucleotides in their chemical structure in that they undergo chemical modifications at multiple sites, primarily to increase stability in vivo and improve targeting. However, similar to natural oligonucleotides, the precise sequence of the nucleobase side chains determines the drug function of the oligonucleotide.

[0004] In pharmaceutical oligonucleotides, modifications typically include: replacing oxygen on the phosphate backbone with sulfur; placing a substituent (e.g., MeO, F, methoxyethoxy (Moe)) at the 2'-C of the ribose; restricting the configuration of the ribose with an additional atomic ring; methylating or fluorinating the nucleobases; and replacing the ribose with another heterocycle (e.g., morpholine). Even so, drugs containing such modifications can still be considered oligonucleotides.

[0005] For many years, oligonucleotides have been prepared using solid-phase oligonucleotide synthesis (SPOS), in which growing oligonucleotides are bound to an insoluble solid support and grown by passing reactive nucleoside phosphoramide building blocks (4) through the insoluble solid support, such as... Figure 1 As shown. Although this method has been an industry standard for decades, SPOS has many drawbacks. In particular, an excess of phosphorus amide is typically required to drive the reaction to completion (4). On a small scale, excesses of tens of equivalents are common, thus significantly increasing the cost of SPOS. Furthermore, the scale-up of SPOS reactions is limited to producing only ~15 kg of oligonucleotides per batch. This is particularly undesirable for their use in pharmaceutical forms, where several metric tons of oligonucleotides would be needed annually for major medical indications such as cardiovascular diseases.

[0006] One SPOS alternative strategy designed to address these scaling and economic challenges is liquid-phase oligonucleotide synthesis (LPOS). Indeed, liquid-phase reactions and liquid-phase material handling are well-established technologies that can be performed on a multi-tonnage scale, making LPOS a strong candidate for large-scale oligonucleotide production. A typical approach to LPOS involves sequential coupling reactions, adding monomeric or multi-monomer oligomers (fragments) in a stepwise manner to oligonucleotides growing in solution, followed by the separation of unreacted monomers or fragments from the grown oligonucleotides using suitable separation techniques, such as membrane filtration (US8,664,357; US9,127,123; US10,239,996; EP3347402; Gaffney et al.; Kim et al.; So et al.; Yeo et al.; Dong et al.; WO2016 / 188835 A1).

[0007] Following the step of conjugating monomers or fragments to the growing oligonucleotides, membrane filtration can be used to separate unreacted monomers / fragments and any reactive debris from the growing oligonucleotides (Gaffney et al.; Kim et al.). Therefore, the use of membrane filtration is well-suited for LPOS, where a thorough purification step is crucial after each conjugation step.

[0008] However, oligonucleotide production is extremely expensive in both the traditional SPOS and LPOS development fields compared to typical small molecule drugs. The largest portion of the cost of producing oligonucleotides via SPOS comes from nucleoside phosphoramide (4), the amount of solvent required, solid support, and the final purification of the crude oligonucleotides (Andrews et al.; Scozzari et al.). All else being equal, significantly reducing the cost of any one of these factors would significantly improve the economics of oligonucleotide production.

[0009] Although membrane-assisted LPOS can be considered a more scalable alternative to SPOS, they are still limited by the need for large quantities of expensive nucleoside phosphoramide (4).

[0010] The Dmtr-nucleoside phosphoramidamide (4) used in the production of SPOS oligonucleotide drugs is prepared from the corresponding 5'-Dmtr-3'-hydroxynucleoside (1), which is linked with all the key features required for synthesis (except for the phosphorus moiety), including: (i) a key temporary 5'-O-(4,4'-dimethoxytriphenylmethyl) (Dmtr) protecting group, which is removed under weakly acidic conditions at the end of each chain extension cycle; (ii) a permanent protecting group that is removed only during the final overall deprotection process; (iii) ribose or other related sugar-like substances; and (iv) nucleobases.

[0011] There are multiple pathways for preparing 5'-Dmtr-nucleosides from a series of relatively simple precursors. Where possible, intermediates in the synthetic pathway are isolated by crystallization or precipitation (assuming purification is indeed necessary) to minimize cost and maximize scalability. Typically, the basic nucleoside (which includes the desired permanent modification of the ribose and nucleobase) is constructed first, then the nucleobase is protected, and finally the Dmtr group is attached (EP0813539 B1; EP1612213 B1; Legorburu et al.; Liu et al.).

[0012] Still refer to Figure 1 5'-Dmtr-nucleoside can be converted to 3'-phosphoramide (Nielsen et al.; Pedersen et al.; Sanghvi et al.; Xie et al.) or nucleoside-OP(OPG)(NR2) by reacting with chlorophosphine [(PGO)PCl(NR2)] (3) or bis-amidite [(PGO)P(NR2)2] (2), where PG (e.g. Figure 1 The Cne group in the phosphate triester is a permanent protective group that will be carried throughout the oligonucleotide synthesis. The phosphorus in the resulting phosphoramidite building block (4) is in the P(III) oxidation state and is sensitive to oxidation (including oxygen in the air) for inactivation. If phosphoramidite is exposed to any acid, it becomes extremely sensitive to hydrolysis and loses the 5'-O-Dmtr group. Due to these sensitivities, commercial phosphoramidite is typically refrigerated and transported via the cold chain.

[0013] Before phosphoramide can be used for oligonucleotide synthesis, it must first be purified. The main purpose is to remove any impurities that may participate in the chain elongation cycle and reduce the purity of the final oligonucleotide, i.e., critical impurities. The most likely critical impurity from phosphoramide synthesis is an excess of phosphoramidating reagent (e.g., (2) or (3)), which, if present in phosphoramide (4), will permanently block the grown oligonucleotide sequence.

[0014] Unlike Dmtr-nucleoside (1) and its precursors, phosphoramidite building blocks (4) are generally not purifiable by crystallization or precipitation. While they sometimes form solids rather than colloids, these methods tend to be nonreproducible to one of the two phosphoramidite diastereomers and do not guarantee the removal of critical impurities. This can then cause a deviation in the diastereomer ratio of the resulting bonds when phosphoramidite building blocks rich in one diastereomer are subsequently used for oligonucleotide chain extension; this is particularly important if the phosphite intermediate is subsequently converted to a thiophosphate analog.

[0015] Therefore, Dmtr-nucleoside phosphoramidite building blocks (4) are almost always purified by normal-phase silica gel column chromatography, typically using an anhydrous solvent gradient containing small amounts of organic bases (e.g., ethyl acetate and heptane) to ensure hydrolytic stability. Chromatography is not a popular process-scale purification technique because: (i) it is difficult to scale up; (ii) it requires large amounts of flammable solvents; (iii) it is time-consuming and requires careful monitoring; (iv) large fraction volumes must be screened and selected, combined, and evaporated to dryness; and (v) it generates a lot of solid waste (silica gel column). Attempts have been made to simplify the purification of nucleoside phosphoramidites without chromatography (US7,960,542 B2), but none have replaced this technique. Therefore, the complexity of chromatographic purification methods significantly increases the final cost of nucleoside phosphoramidites. In fact, it is estimated that chromatographic purification methods make phosphoramidites (4) 3 to 10 times more expensive than their direct nucleoside precursors (1).

[0016] This invention was designed with the foregoing in mind. Summary of the Invention

[0017] According to a first aspect of the present invention, a liquid-phase method for forming oligonucleotides is provided, the method comprising the following steps:

[0018] a) Provides compound of formula I:

[0019]

[0020] I

[0021] Where A is a nucleoside or oligonucleotide, and T... A Z is the reactive end of a nucleoside or oligonucleotide, and Z is the soluble synthetic support to which the nucleoside or oligonucleotide is attached.

[0022] b) Modifying compound I to form compound II:

[0023]

[0024] II

[0025] Where A and Z are as defined with respect to Formula I, X is a tertiary amino group, and PG is a protecting group;

[0026] c) Separation of compound II by membrane filtration;

[0027] d) Provides compounds of formula III:

[0028]

[0029] III

[0030] Where B is a nucleoside or oligonucleotide, and T...B1 It is the reactive terminus of a nucleoside or oligonucleotide, T PG It is the protected end of a nucleoside or oligonucleotide;

[0031] e) Reacting compound II with compound III to form compound IV:

[0032]

[0033] IV

[0034] Where A and Z are as defined with respect to Equation I, PG is as defined with respect to Equation II, and B and T PG As defined with respect to Equation III. Detailed Implementation

[0035] Throughout the description and claims of this specification, where the term “comprise” (or “comprises” or “comprising”) is used to describe the subject matter herein, it is also contemplated that the terms “consist of” (or “consists of” or “consisting of”) or “consist essentially of” (or “consistses essentially of” or “consisting essentially of”) be used instead to describe the same subject matter.

[0036] Throughout the description and claims of this specification, the singular encompasses the plural unless the context requires otherwise. In particular, where the indefinite article is used, the specification should be understood to consider both the plural and singular unless the context requires otherwise.

[0037] Features described in connection with a particular aspect, embodiment, or example of the invention should be understood to be applicable to any other aspect, embodiment, or example described herein, unless incompatible with it. All features disclosed in this specification (including any appended claims, abstract, and drawings) and / or all steps of any method or process so disclosed may be combined in any combination, except where at least some of such features and / or steps are mutually exclusive combinations. The invention is not limited to the details of any specific embodiment described herein. The invention extends to any novel one or any novel combination of features disclosed in this specification (including any appended claims, abstract, and drawings), or to any novel one or any novel combination of steps of any method or process so disclosed.

[0038] Through rigorous research, the inventors have devised a liquid-phase method for forming oligonucleotides according to the first aspect, which overcomes the aforementioned drawbacks associated with the preparation, purification, and storage of nucleoside phosphoramide (4). The method of the present invention allows for the stepwise growth of high-purity supporting oligonucleotides from their constituent nucleosides using simple nucleosides or short-chain oligonucleotides (instead of nucleoside / oligonucleotide phosphoramide, e.g., (4)) as monomer building blocks sequentially coupled to the supporting oligonucleotides. The method of the present invention may be referred to herein as a reverse phosphoramide coupling method, in contrast to the conventional forward phosphoramide coupling method of LPOS.

[0039] It should be understood that the term "nucleoside" as used herein refers to a building block used to form an oligonucleotide and typically contains nucleobases coupled to ribose, a heterocyclic moiety forming the backbone. In such a backbone-forming heterocyclic moiety, oxygen atoms at the 3' and 5' carbon atoms typically act as reactive ends through which nucleosides can couple to each other. It is well known that ribose can be deoxygenated at the 2' carbon. Furthermore, as previously described, those skilled in the art will understand that synthetic nucleosides can contain a variety of structural modifications, such as substitutions at the 2' carbon of the ribose (e.g., with fluorine, methoxy, or methoxyethoxy), fusion of one or more additional rings to the ribose to restrict its configuration, bridging of the ribose to restrict its configuration (e.g., as in locked nucleic acids), and / or substitutions at the nucleobases (e.g., with methyl or fluorine). Similarly, those skilled in the art will be familiar with variations of ribonucleotides that include alternative backbone-forming heterocyclic moieties, such as in morpholine nucleosides, and the ways in which they couple to form oligonucleotides. It should be understood that such modifications and variations are covered in this article.

[0040] It should be understood that, as used herein, the term "oligonucleotide" refers to a chain of two or more nucleosides, wherein each pair of adjacent nucleosides in the chain is linked to each other by a phosphorus-containing linker (e.g., a phosphodiester bond, a methylphosphonate bond, a thiophosphate bond, a boron phosphate bond, an aminophosphate bond, or a mesylate phosphoramidate linkage), for example, at the 3' and 5' positions of its ribose moiety. Generally, as used herein, the term oligonucleotide refers to at least 5 linked nucleosides or at least 10 linked nucleosides.

[0041] This method can be carried out in any suitable solvent. Suitably, the method is carried out in acetonitrile or a mixture of solvents containing acetonitrile.

[0042] In compounds of formula I, A can bind to Z at any chemically feasible position. In many cases, Z is attached to the heterocycle of A that forms the backbone, for example, at the 3' or 5' end of the ribose. Suitablely, Z is attached to A at the 3' end of the ribose.

[0043] Typically, T A Z is located at the opposite end of A. For example, when A is a nucleoside, Z can be attached to the 3' end of the ribose, while T... A Located at the 5' end of the ribose, and vice versa. When A is an oligonucleotide, T... A Z and T are located on the opposite distal nucleotide. For example, Z can be attached to the 3' end of the ribose of a distal nucleotide, while T... A Located at the 5' end of the ribose of another distal nucleoside, and vice versa.

[0044] In many cases, T A It is located at the 5' end of the nucleoside or oligonucleotide A, and Z is located at the 3' end of the nucleoside or oligonucleotide A.

[0045] Compounds of formula I can have structures according to formula Ia:

[0046]

[0047] Ia

[0048] in

[0049] -O 5A -A'-O 3A - is a nucleoside or oligonucleotide, in which O 5A It is oxygen located at the terminal 5' carbon and O 3A It is oxygen located at the terminal 3' carbon;

[0050] T A It is hydrogen; and

[0051] Z is a soluble synthetic support.

[0052] Therefore, it should be understood that the group -O in formula Ia 5A -A'-O 3A - It can be equivalent to group A in formula I.

[0053] In step b), compound I is modified to obtain compound II. The modification of compound I can be described as phosphoramidation. Those skilled in the art will be familiar with the phosphoramidation of nucleosides.

[0054] X can be a group -NR2, wherein each R is independently selected from (1-6C) alkyl groups, or two R groups are linked such that when combined with the nitrogen atom to which they are attached, they together form a 5- to 7-membered heterocycle. Suitably, each R is independently selected from (1-4C) alkyl groups, or two R groups are linked such that when combined with the nitrogen atom to which they are attached, they together form a 5-membered heterocycle. More suitably, each R is independently selected from methyl, ethyl, and isopropyl, or two R groups are linked such that when combined with the nitrogen atom to which they are attached, they together form a pyrrolidinyl group. Most suitably, each R is isopropyl.

[0055] Those skilled in the art will be familiar with a variety of protecting groups suitable for use as PGs. A PG can be a base-labile protecting group. Examples of PGs include methyl, isopropyl, tert-butyl, benzyl, allyl, phenyl, 2-chlorophenyl, 4-chlorophenyl, 3',5'-dimethoxybenzoyl, p-hydroxybenzoylmethyl, cyanoethyl, 9-fluorenylmethyl, 2-(trimethylsilyl)ethyl, 2-(methylsulfonyl)ethyl, 2-(benzenesulfonyl)ethyl, 4-nitrophenylethyl, and 2,2,2-trichloroethyl. Suitably, a PG is selected from the group consisting of cyanoethyl, 2-chlorophenyl, 4-chlorophenyl, 2,2,2-trichloroethyl, and methyl. Most preferably, a PG is cyanoethyl.

[0056] Step b) may include reacting compound I with compound A:

[0057]

[0058] A

[0059] Wherein PG and X are as defined for compounds of formula II and Y is a halogen or has any of the definitions of X described above;

[0060] When Y is not a halogen, step b) is carried out in the presence of the first phosphorous amide activator.

[0061] Compounds of formula A can be described as phosphoramidizing agents, i.e., agents used to convert nucleosides into nucleoside phosphorimides. Those skilled in the art will be familiar with suitable agents for phosphoramidizing nucleosides and their methods of use. For example, those skilled in the art will understand that compounds of formula A, where Y is a halogen, are typically used in the presence of a base (e.g., Et3N or DIPEA).

[0062] Y can be either Cl or any of the definitions of X described above (e.g., -N( i Pr)2).

[0063] Examples of particularly suitable compounds of formula A include:

[0064] ;and .

[0065] Those skilled in the art will be familiar with phosphorus amide activators. In particular, it should be understood that the first phosphorus amide activator can react with compound A to form compound A':

[0066]

[0067] A'

[0068] Where X and PG are as defined for compounds of formula II, and

[0069] LG 1 It is a leaving group;

[0070] Furthermore, compound A' can react with compound I to form compound II.

[0071] In many cases, LG 1 It is a nitrogen-containing heteroaryl group. Suitablely, LG 1 It is imidazole, pyridinyl, or tetrazolyl. Specific, non-limiting examples of the first phosphoramidite activator are:

[0072] ; ;and .

[0073] The first phosphorus amide activator can be represented as A1 in this paper.

[0074] Step b) is performed appropriately using an excess of compound A. This ensures that compound I is converted to compound II to the greatest extent possible and avoids sequence deletion errors in the prepared oligonucleotide structure.

[0075] When compound I is compound Ia, compound II can be compound IIa:

[0076]

[0077] IIa

[0078] Where PG and X are as defined in Equation II and -O 5A -A'-O 3A - and Z are as defined with respect to equation Ia.

[0079] In step c), compound II is separated by membrane filtration (e.g., membrane permeation). Compound II can be separated from one or more reactants used in step b). Such reactants may include compound I, compound A, a first phosphorus amide activator, and / or compound A'. It is important to remove or significantly reduce the amount of residual compound A before coupling step d) because for every mole of residual compound A, up to two moles of compound III may be undesirably consumed in the formation of unwanted symmetrical dimers.

[0080] The membrane used in step c) is suitably an organic solvent-resistant membrane. For example, compounds of formula II can be separated by organic solvent nanofiltration. More preferably, the membrane is insoluble in one or more solvents (e.g., acetonitrile or a mixture of solvents containing acetonitrile) used in step b). In such cases, compounds of formula II can be prepared and separated in the same one or more solvents.

[0081] Suitable membranes for separating compounds of type II (or any other compounds described herein loaded on type Z) include polymer membranes, ceramic membranes, and hybrid polymeric / inorganic membranes. Membrane rejection ratio R i These are commonly used terms known to those skilled in the art, and are defined as:

[0082] x 100% equation (1)

[0083] Where C P,i = The concentration of substance i in the permeate, where the permeate is the liquid that has already penetrated the membrane, C R,i = The concentration of substance i in the retentate, where the retentate is a liquid that has not passed through the membrane. It should be understood that the membrane is suitable for this invention if R （产物） >R （反应物） The product is a compound of formula II or any other compound supported on Z as described herein.

[0084] The membrane can be formed of any polymeric or ceramic material, providing a separation layer capable of preferentially separating compounds of Formula II from one or more reactants used in step b). In other words, the membrane will exhibit a greater rejection rate for compounds of Formula II (or any other compounds loaded on Z as described herein) than for the reactants used in step b. Suitably, the membrane is formed of or contains a polymeric material suitable for manufacturing microfiltration, ultrafiltration, nanofiltration, or reverse osmosis membranes, including polyethylene, polypropylene, polytetrafluoroethylene (PTFE), polyvinylidene fluoride (PVDF), polysulfone, polyethersulfone, polyacrylonitrile, polyamide, polyester, polyimide, polyetherimide, cellulose acetate, polyaniline, polypyrrole, polybenzimidazole (PBI), polyetheretherketone (PEEK), and mixtures thereof. The membrane can be manufactured by any technique known in the art, including sintering, stretching, track etching, template leaching, interfacial polymerization, or phase inversion. Membranes may be composite (e.g., thin-film composite membranes) and / or cross-linked or treated to improve their stability in specific solvents. PCT / GB2007 / 050218 and PCT / GB2015 / 050179 describe membranes suitable for use as part of this invention. US 10,913,033 describes a membrane particularly suitable for use as part of this invention.

[0085] Most preferably, the membrane is a cross-linked polybenzimidazole membrane (e.g., an asymmetric cross-linked polybenzimidazole membrane with a skinned structure).

[0086] In compounds of formula III, T PG and T B1 Located at the opposite end of B. For example, when B is a nucleoside, T B1 It can be located at the 3' end of the ribose, while T PG Located at the 5' end of the ribose, and vice versa. When B is an oligonucleotide, T... B1 and T PG Located on the opposite distal nucleoside. For example, T B1 It can be attached to the 3' end of the ribose of a distal nucleoside, while T PG Located at the 5' end of the ribose of another distal nucleoside, and vice versa.

[0087] In many cases, T B1 Located at the 3' end of nucleosides or oligonucleotides B, while T PG It is located at the 5' end of a nucleoside or oligonucleotide B.

[0088] Given T PG Those skilled in the art will be familiar with a variety of protective groups used to prevent uncontrolled chain elongation during oligonucleotide preparation. Typically, T... PGIt is a protective group that binds to the 5' oxygen of B. In many cases, T PG It is a protective group that is unstable in acids, and non-limiting examples include dimethoxytriphenylmethyl (Dmtr / DMT), tert-butyl (tBu), tert-butoxycarbonyl (Boc), monomethoxytriphenyl (Mmtr), triphenylmethyl (Tr), pentamethyldihydrobenzofuransulfonyl (Pbf), tetrahydropyranyl (Thp), tetrahydrofuranyl (Thf), p-methoxybenzyl (Pmb), and 2,4-dimethoxybenzyl. Most preferably, T PG It is a dimethoxytriphenylmethyl group that is bound to the 5' oxygen of B.

[0089] In many cases, compounds of formula III can have a structure according to formula IIIa:

[0090]

[0091] in

[0092] -O 5B -B'-O 3B - is a nucleoside or oligonucleotide, in which O 5B It is oxygen located at the terminal 5' carbon and O 3B It is oxygen located at the terminal 3' carbon;

[0093] T B1 It is hydrogen; and

[0094] T PG It is a protective group (e.g., DMT).

[0095] Therefore, it should be understood that the group -O in formula IIIa 5B -B'-O 3B - It can be equivalent to group B in formula III.

[0096] In step e), the compound of formula II reacts with the compound of formula III to give the compound of formula IV. Those skilled in the art will be able to select suitable conditions for the coupling reaction in step e).

[0097] Step e) is suitably carried out in the presence of a second phosphorus amide activator. Those skilled in the art will be familiar with phosphorus amide activators. In particular, it should be understood that the second phosphorus amide activator can react with the compound of formula II to form the compound of formula II':

[0098]

[0099] II'

[0100] Z, A, and PG are as defined for compounds of formula II;

[0101] LG 2 It is a leaving group;

[0102] Furthermore, the compound of formula II' can react with the compound of formula III to form the compound of formula IV.

[0103] In many cases, LG 2 It is a nitrogen-containing heteroaryl group. Suitablely, LG 2 It is an imidazole group or a tetrazolium group. Specific, non-limiting examples of the second phosphorus amide activator are tetrazolium, 5-(ethylthio)-1H-tetrazole, 4,5-dicyanimidazolium, and 5-(benzylthio)-1H-tetrazole. The second phosphorus amide activator may be referred to herein as A2.

[0104] When compound I is a compound of formula Ia and compound III is a compound of formula IIIa, compound IV can be a compound of formula IVa:

[0105]

[0106] Where PG is defined as in Equation II, -O 5A -A'-O 3A - and Z are as defined with respect to equation Ia and -O 5B -B'-O 3B - and TPG as defined with respect to equation IIIa.

[0107] Step e) is suitably performed under anhydrous conditions. At this point in the oligonucleotide preparation process, the presence of water can affect the purity and yield of the product. The presence of water during 5'-phosphonamide activation can lead to the formation of 5'-H-phosphonate diesters. H-phosphonates can be converted to the corresponding thiophosphate diesters using existing sulfur transfer agents, such as hydrogenated flavin (XH) or polyorganosulfonamides (PolyOrg Sulfa, POS), which can block the oligonucleotide in which they reside, resulting in truncated impurities.

[0108] Compared to conventional forward phosphoramidite coupling methods, the method of the present invention is significantly less prone to unwanted side reactions. It is well known that in SPOS, the presence of a high concentration of acidic phosphoramidite activator during the coupling reaction can lead to a low level of 5'-O-detriphenylmethylation. The resulting newly exposed 5'-OH is then extended, resulting in an n+1 defect, where two nucleoside building blocks are added to one chain during the same coupling reaction. These are among the most difficult impurities to isolate from the final full-length oligonucleotide. The method of the present invention prevents such impurities. Isolation of the Formula II compound (e.g., from the residual Formula A compound) in step c) means that even if unwanted T occurs during step d), PG Deprotection (e.g., 5'-O-detriphenylmethylation) will not result in an n+1 defect.

[0109] Z is a synthesis support soluble in one or more solvents used in the methods of the present invention. Z can be used to retain the grown oligonucleotide chains in solution and / or to impart molecular volume to facilitate their separation by membrane filtration. For example, Z can be one or more of the following: branch point molecules, polymers, dendritic polymers, dendrons, hyperbranched polymers, or organic / inorganic materials, including nanoparticles, fullerenes, and 2-D materials (e.g., graphene and boron nitride).

[0110] In many cases, Z is a polymer-soluble synthetic support (e.g., polyethylene glycol). The molecular weight of Z (M...) w It can be 500-50,000 Da.

[0111] Z can be a compound of formula B:

[0112]

[0113] B

[0114] in

[0115] V is an organic branch point.

[0116] W is a cluster chain.

[0117] Indicates the point connected to A, and

[0118] n is 1-8.

[0119] V will be understood as a multifunctional organic "hub" with multiple ends, to which one or more polymer chains W are connected. In many cases, V contains fewer than 50 atoms in total, more appropriately fewer than 20 atoms in total.

[0120] Each W represents the molecular weight (M). w The polymer chain is 500-20,000 Da (e.g., polyethylene glycol). More preferably, each W is a molecular weight (M). w The W represents a polymer chain with a molecular weight of 2000-15000 Da. More appropriately, each W represents the molecular weight (M). w () is a polymer chain of 8000-12000Da.

[0121] For example, n can be 1-6. In many cases, n is 3-4.

[0122] Each W binds to a nucleoside or oligonucleotide A as defined herein. Therefore, it should be understood that when Z is a compound of formula B, the process of forming an oligonucleotide produces n identical oligonucleotide chains.

[0123] It should be understood that each W can be directly connected to A, or indirectly connected to A through a connecting portion. A typical connecting portion will contain fewer than 50 atoms in total, more appropriately fewer than 30 atoms in total.

[0124] A particularly suitable example of a compound of formula B is:

[0125]

[0126] in This represents a point directly connected to A (e.g., the 3' oxygen directly connected to A) and m is 100-500 (e.g., 200-300).

[0127] The method may also include a step of converting one or more P(III) phosphite-triester bonds to P(V) phosphotriester bonds, for example by oxidation or sulfur transfer. Such a step may generally be described herein as an oxidation step. Suitable reagents for carrying out this step will be familiar to those skilled in the art. As part of this step, the oxidation product (i.e., a compound containing one or more P(V) phosphotriester bonds) may be separated by membrane filtration. Membrane filtration may be performed as described above with respect to step c) (e.g., Z-tethered oligonucleotides are collected in the retentate, and oxidation step reactants and / or byproducts are collected in the permeate).

[0128] The method may further include the step of contacting the oligonucleotide with a deprotecting agent to form a compound of formula V:

[0129]

[0130] V

[0131] in

[0132] Z and A are as defined with respect to equation I;

[0133] PG is as defined with respect to Equation II;

[0134] Q is selected from non-existent, O, or S; and

[0135] T B2 It is the reactive end of B (e.g., the 5' end).

[0136] The steps for forming compound V can generally be described in this paper as T PG Deprotection step. Those skilled in the art will be familiar with suitable reagents for performing this step. As part of this step, after the formation of compound V, compound V can be separated by membrane filtration. Membrane filtration can be performed as described above with respect to step c) (e.g., Z-bound oligonucleotides are collected in the retentate, T...). PGDeprotection step reactants and / or byproducts are collected in the permeate.

[0137] It should be understood that in the absence of Q, the P atom is in the P(III) state, while when Q is O or S, the P atom is in the P(V) state (i.e., after oxidation or sulfur transfer of the P(III) phosphite-triester bond).

[0138] It should be understood that T PG The deprotection step (including any associated membrane filtration) is performed after step e) and before or after the oxidation step (including any associated membrane filtration). In many cases, the oxidation step (including any associated membrane filtration) is performed after step e) and before T. PG Perform this step before the protection removal process.

[0139] Steps a) to e), oxidation steps (including any associated membrane filtration), and T PG The sequence of deprotection steps (including any associated membrane filtration) can be repeated any number of times to grow oligonucleotides with target length support by progressively adding nucleoside-containing building blocks.

[0140] Therefore, in many cases, the method may also include the following steps:

[0141] (i) Provide an oligonucleotide for growth, wherein the oligonucleotide for growth is:

[0142] A) Compound V of formula A, or

[0143] B) A compound of formula V coupled with one or more additional nucleosides, wherein the nucleoside furthest from Z has a reactive end;

[0144] (ii) Modify the grown oligonucleotide to form a compound of formula VI:

[0145]

[0146] VI

[0147] in The bonds with the grown oligonucleotide (e.g., at the reactive end), PG and X are as defined with respect to Formula II;

[0148] (iii) Separation of compound VI by membrane filtration;

[0149] (iv) Provide nucleoside or oligonucleotide building blocks with reactive and protected ends;

[0150] (v) React the isolated compound of formula VI with the reactive end of a nucleoside or oligonucleotide building block to form a chain-extended oligonucleotide having a protected end;

[0151] (vi) Separation of oligonucleotides with extended chains by membrane filtration;

[0152] (vii) Deprotect the protected ends of the isolated strand-extended oligonucleotides and optionally repeat steps (i) to (vii) in sequence.

[0153] For example, if the reactive end of the oligonucleotide (i.e., the oligonucleotide provided in step (i) A) or B) is a 5' end, the reactive end of the nucleoside or oligonucleotide building block provided in step (iv) will be a 3' end; in such a case, the protected end of the nucleoside or oligonucleotide building block provided in step (iv) will be a 5' end. For example, if the reactive end of the oligonucleotide (i.e., the oligonucleotide provided in step (i) A) or B) is a 3' end, the reactive end of the nucleoside or oligonucleotide building block provided in step (iv) will be a 5' end; in such a case, the protected end of the nucleoside or oligonucleotide building block provided in step (iv) will be a 3' end. Those skilled in the art will be familiar with the coupling of ribose-free nucleosides (e.g., morpholinonucleotides).

[0154] The method including steps (i) through (vii) may also include a step of converting one or more P(III) phosphite trimer bonds into P(V) phosphotriester bonds (e.g., by oxidation or sulfur transfer). Most preferably, this is an intermediate step performed between steps (v) and (vi). It should be understood that when step (vii) involves repeating the order of steps (i) through (vii), this includes any intermediate step.

[0155] The method may also include the step of removing the protecting group PG from one or more P(V) phosphotriester bonds. Such a step may refer herein to a bulk deprotection step. The bulk deprotection step can be performed once the oligonucleotide reaches the desired length. As described above, those skilled in the art will be familiar with those groups suitable for use as PG, and the techniques for removing them.

[0156] The method may also include the step of breaking the oligonucleotide from Z. Once the oligonucleotide reaches the desired length, the soluble synthetic support can be removed. Techniques that can achieve this will be familiar to those skilled in the art.

[0157] The method of this invention is particularly suitable for fragment-based oligonucleotide synthesis (e.g., where A and B are both oligonucleotides). The advantage of this method is that the overall yield can be maximized due to its convergence, and the purity of the full-length product is increased because each fragment is more easily prepared with high purity. Typically, before fragment coupling can be performed, two fragments must be prepared and isolated, with all protecting groups intact except for the terminal hydroxyl groups on each fragment to be coupled via phosphodiester. This is almost always carried out in solution, partly because fragment permeation into solid-phase synthesis beads has a slow diffusion constant. One fragment must then be converted to reactive phosphoramidite. This is a significant challenge because the phosphoramidite fragment must be completely purified to remove any phosphoramidizing agents, which, if present during coupling with the other fragment, will successfully compete for and block the hydroxyl fragment. This latter problem can be easily avoided using the membrane-assisted reverse phosphoramidite coupling method of this invention. The hydroxyl terminals of the grown oligonucleotides are first phosphoramidized in solution and purified by membrane filtration. Purification can then continue until the bifunctional P(III) reagent is reduced to a low concentration. Since the reactive oligonucleotide fragments are prepared in solution where the reaction rate of the high molecular weight reactants is not reduced by phase transfer kinetics, it is not necessary to cleave the phosphoramidized fragments from the soluble support. The hydroxyl fragments are then introduced into the reactor, and a second phosphoramidite activator is added to initiate coupling. A more open membrane can be selected for membrane filtration, allowing unsupported fragments to be separated from the linked strands after oxidation or sulfur transfer. This provides an opportunity to recover valuable hydroxyl fragments.

[0158] Another advantage of the reverse phosphoramidization method of the present invention is its full compatibility with existing or forward phosphoramidization methods. Therefore, if a given nucleoside is unavailable, but the corresponding nucleoside phosphoramidization is available, the forward building block can be used to complete the chain extension. The forward and reverse coupling methods can be interchanged multiple times as needed to construct the desired sequence because the overall deprotection step is unaffected by whether the forward or reverse method is used to construct the fully protected full-length product.

[0159] The method of the present invention is suitably carried out in a closed-loop reactor. For example, the reactor may include a reaction vessel having an inlet and an outlet, and a membrane-containing purification vessel having an inlet, a retentate outlet, and a permeate outlet, wherein the outlet of the reaction vessel is in fluid communication with the inlet of the purification vessel, and the retentate outlet of the purification vessel is in fluid communication with the inlet of the reaction vessel. The reaction step (e.g., step b) is carried out in the reaction vessel, after which the reaction medium is fed from the outlet of the reaction vessel to the inlet of the purification vessel for the separation (e.g., in step c) of the desired reaction product (e.g., the grown supporting oligonucleotide) from the retentate. The separated reaction product is then fed from the retentate outlet to the inlet of the reaction vessel, whereby the separated reaction product can participate in the next reaction step (e.g., step d). The reactor may be described herein as a synthesizer.

[0160] Statements numbered 1 to 57 below are not claims, but rather describe specific aspects and embodiments of the invention:

[0161] 1. A liquid-phase method for forming oligonucleotides, the method comprising the following steps:

[0162] a) Provides compound of formula I:

[0163]

[0164] I

[0165] Where A is a nucleoside or oligonucleotide, and T... A Z is the reactive end of the nucleoside or oligonucleotide, and Z is the soluble synthetic support to which the nucleoside or oligonucleotide is attached;

[0166] b) Modify the compound of formula I to form the compound of formula II:

[0167]

[0168] II

[0169] Where A and Z are as defined in Formula I, X is a tertiary amino group, and PG is a protecting group;

[0170] c) Separate the compound of formula II by membrane filtration;

[0171] d) Provides compounds of formula III:

[0172]

[0173] III

[0174] Where B is a nucleoside or oligonucleotide, and T... B1 It is the reactive terminus of the nucleoside or oligonucleotide, T PGIt is the protected end of the nucleoside or oligonucleotide;

[0175] e) React the compound of formula II with the compound of formula III to form the compound of formula IV:

[0176]

[0177] IV

[0178] Where A and Z are as defined with respect to Equation I, PG is as defined with respect to Equation II, and B and T PG As defined with respect to Equation III.

[0179] 2. The liquid-phase method according to statement 1, wherein X is a group -NR2, wherein each R is independently selected from (1-6C) alkyl groups, or two R groups are linked such that when combined with the nitrogen atom to which they are attached, they together form a 5- to 7-membered heterocycle.

[0180] 3. The liquid-phase method according to statement 2, wherein each R is independently selected from (1-4C) alkyl groups, or two R groups are linked such that when combined with the nitrogen atom to which they are linked, they together form a 5-membered heterocycle.

[0181] 4. The liquid-phase method according to statement 2, wherein each R is independently selected from methyl, ethyl and isopropyl, or two R groups are linked such that when combined with the nitrogen atom to which they are linked, they together form a pyrrolidinyl group.

[0182] 5. The liquid-phase method according to statement 2, wherein each R is isopropyl.

[0183] 6. The liquid-phase method according to any one of the foregoing statements, wherein PG is a protective group that is unstable against bases.

[0184] 7. The liquid phase method according to any one of the foregoing statements, wherein PG is selected from the group consisting of: methyl, isopropyl, tert-butyl, benzyl, allyl, phenyl, 2-chlorophenyl, 4-chlorophenyl, 3'-5'-dimethoxybenzoyl, p-hydroxybenzoyl, cyanoethyl, 9-fluorenylmethyl, 2-(trimethylsilyl)ethyl, 2-(methylsulfonyl)ethyl, 2-(benzenesulfonyl)ethyl, 4-nitrophenylethyl, and 2,2,2-trichloroethyl.

[0185] 8. The liquid phase method according to any one of the foregoing statements, wherein PG is selected from the group consisting of cyanoethyl, 2-chlorophenyl, 4-chlorophenyl, 2,2,2-trichloroethyl and methyl.

[0186] 9. The liquid phase method according to any one of the foregoing statements, wherein PG is cyanoethyl.

[0187] 10. The liquid-phase method according to any one of the foregoing statements, wherein step c) comprises isolating the compound of formula II from one or more reactants used in step b).

[0188] 11. The liquid-phase method according to any one of the foregoing statements, wherein during step c), the compound of formula II is collected in the retentate.

[0189] 12. The liquid-phase method according to any one of the foregoing statements, wherein step c) is carried out in an organic solvent or a mixture of organic solvents.

[0190] 13. The liquid phase method according to any one of the foregoing statements, wherein the membrane is a cross-linked polybenzimidazole membrane.

[0191] 14. The liquid-phase method according to any one of the foregoing statements, wherein step b) comprises reacting the compound of formula I with the compound of formula A:

[0192]

[0193] Wherein PG and X are as defined in any of the foregoing statements, and Y is a halogen or any of the definitions of X as described in any of the foregoing statements;

[0194] When Y is not a halogen, step b) is carried out in the presence of the first phosphorous amide activator.

[0195] 15. The liquid-phase method according to statement 14, wherein Y is Cl or -N ( i Pr)2.

[0196] 16. The liquid-phase method according to statement 14, wherein compound A is:

[0197] ;or .

[0198] 17. The liquid-phase method according to statement 14, 15, or 16, wherein the first phosphoramide activator is capable of reacting with compound A to form compound A':

[0199]

[0200] A'

[0201] Where X and PG are as defined in any of the foregoing statements, and

[0202] LG 1 It is a leaving group;

[0203] Furthermore, the compound of formula A' is capable of reacting with the compound of formula I to form the compound of formula II.

[0204] 18. The liquid phase method according to statement 17, wherein LG 1 It is a nitrogen-containing heteroaryl group (e.g., imidazolyl, pyridinyl, or tetrazolyl).

[0205] 19. The liquid-phase method according to any one of statements 14 to 18, wherein the first phosphoramide activator is:

[0206] ; ;or .

[0207] 20. The liquid-phase method according to any one of statements 14 to 19, wherein the first phosphoramide activator is:

[0208] .

[0209] 21. The liquid-phase method according to any one of the foregoing statements, wherein step e) is carried out in the presence of a second phosphorous amide activator.

[0210] 22. The liquid-phase method according to statement 21, wherein the second phosphoramide activator is capable of reacting with the compound of formula II to form compound II':

[0211]

[0212] II'

[0213] Z, A, and PG are as defined in any of the preceding statements;

[0214] LG 2 It is a leaving group;

[0215] Furthermore, the compound of formula II' is capable of reacting with the compound of formula III to form the compound of formula IV.

[0216] 23. The liquid phase method according to statement 22, wherein LG 2 It is a nitrogen-containing heteroaryl group.

[0217] 24. The liquid phase method according to statement 22, wherein LG 2 It is either imidazole or tetrazolium.

[0218] 25. The liquid phase method according to statement 22, wherein LG 2 It is tetrazolium, 5-(ethio)-1H-tetrazole, 4,5-dicyanimidazolium or 5-(benzylthio)-1H-tetrazole.

[0219] 26. The liquid-phase method according to statement 21, wherein the second phosphoramide activator is a nitrogen heterocyclic compound.

[0220] 27. The liquid-phase method according to statement 21, wherein the second phosphoramide activator is tetrazolium, 5-(ethylthio)-1H-tetrazole, 4,5-dicyanimidazolium or 5-(benzylthio)-1H-tetrazole.

[0221] 28. The liquid phase method according to any one of the foregoing statements, wherein step e) is carried out under anhydrous conditions.

[0222] 29. The liquid-phase method according to any one of the foregoing statements, wherein: (i) when A is a nucleoside, Z is attached to the 3' end of A, and T A Located at the 5' end of A, and vice versa; and (ii) when A is an oligonucleotide, Z is attached to the 3' end of a distal nucleoside of A, while T... A It is located at the 5' end of another distal nucleoside of A, and vice versa.

[0223] 30. The liquid phase method according to any one of the foregoing statements, wherein T A It is located at the 5' end of nucleoside or oligonucleotide A and Z is located at the 3' end of nucleoside or oligonucleotide A.

[0224] 31. The liquid-phase method according to any one of the foregoing statements, wherein the compound of formula I has a structure according to formula Ia:

[0225]

[0226] Ia

[0227] in

[0228] -O 5A -A'-O 3A - is a nucleoside or oligonucleotide, in which O 5A It is oxygen located at the terminal 5' carbon and O 3A It is oxygen located at the terminal 3' carbon;

[0229] T A It is hydrogen; and

[0230] Z is a soluble synthetic support.

[0231] 32. The liquid-phase method according to any one of the foregoing statements, wherein when the compound of formula I is a compound of formula Ia, the compound of formula II may be a compound of formula IIa:

[0232]

[0233] IIa

[0234] Among them, PG, X, and -O 5A -A'-O 3A - and Z are as defined in any of the preceding statements.

[0235] 33. The liquid phase method according to any one of the foregoing statements, wherein: (i) when B is a nucleoside, T B1 Located at the 3' end of B, while T PG Located at the 5' end of B, and vice versa; and (ii) when B is an oligonucleotide, T B1 Located at the 3' end of a distal nucleoside of B, while T PG It is located at the 5' end of another distal nucleotide of B, and vice versa.

[0236] 34. The liquid phase method according to any one of the foregoing statements, wherein T B1 Located at the 3' end of nucleoside or oligonucleotide B and T PG It is located at the 5' end of a nucleoside or oligonucleotide B.

[0237] 35. The liquid phase method according to any one of the foregoing statements, wherein T PG It is a protective group that binds to the 5' oxygen of B.

[0238] 36. The liquid phase method according to any one of the foregoing statements, wherein T PG It is an acid-instable protective group, T PG Non-limiting examples include dimethoxytriphenylmethyl (Dmtr / DMT), tert-butyl (tBu), tert-butoxycarbonyl (Boc), monomethoxytriphenyl (Mmtr), triphenylmethyl (Tr), pentamethyldihydrobenzofuransulfonyl (Pbf), tetrahydropyranyl (Thp), tetrahydrofuranyl (Thf), p-methoxybenzyl (Pmb), and 2,4-dimethoxybenzyl.

[0239] 37. The liquid phase method according to any one of the foregoing statements, wherein T PG It is a dimethoxytriphenylmethyl group that is bound to the 5' oxygen of B.

[0240] 38. The liquid-phase method according to any one of the foregoing statements, wherein the compound of formula III has a structure according to IIIa:

[0241]

[0242] in

[0243] -O 5B -B'-O 3B - is a nucleoside or oligonucleotide, in which O 5BIt is oxygen located at the terminal 5' carbon and O 3B It is oxygen located at the terminal 3' carbon;

[0244] T B1 It is hydrogen; and

[0245] T PG It is a protective group (e.g., DMT).

[0246] 39. The liquid-phase method according to any one of the foregoing statements, wherein when the compound of formula I is a compound of formula Ia and the compound of formula III is a compound of formula IIIa, the compound of formula IV is a compound of formula IVa:

[0247]

[0248] IVa

[0249] Among them, PG, -O 5A -A'-O 3A Z, -O 5B -B'-O 3B -and T PG As defined in any of the preceding statements.

[0250] 40. The liquid-phase method according to any one of the foregoing statements, wherein Z is a polymerizable soluble synthesis support.

[0251] 41. The liquid phase method according to any one of the foregoing statements, wherein the molecular weight of Z (M w The value is 500-50,000 Da.

[0252] 42. The liquid-phase method according to any one of statements 1-40, wherein Z is a compound of formula B:

[0253]

[0254] B

[0255] in

[0256] V is an organic branch point.

[0257] W is a cluster chain.

[0258] Indicates the point connected to A, and

[0259] n is 1-8 (e.g., 1-6).

[0260] 43. The liquid phase method according to statement 42, wherein n is 3-4.

[0261] 44. The liquid-phase method according to statement 42 or 43, wherein V is aliphatic or aromatic.

[0262] 45. The liquid-phase method according to statement 42, 43 or 44, wherein the molecular weight (M) of each W w The value is 500-20,000 Da (e.g., 2000-15,000 Da or 8000-12,000 Da).

[0263] 46. ​​The liquid-phase method according to statement 42, wherein Z is a compound of formula B having the following structure:

[0264]

[0265] in This indicates a point directly connected to A (e.g., the 3' oxygen directly connected to A) and m is 100-500 (e.g., 200-300).

[0266] 47. The liquid-phase method according to any one of the foregoing statements further includes an oxidation step, wherein one or more P(III) phosphite triester bonds are converted to P(V) phosphotriester bonds (e.g., by oxidation or sulfur transfer).

[0267] 48. The liquid-phase method according to statement 47, wherein the product generated by the oxidation step is separated by membrane filtration.

[0268] 49. The liquid phase method according to any one of the foregoing statements, further comprising T PG The deprotection step involves contacting the oligonucleotide with a deprotecting agent to form a compound of formula V:

[0269]

[0270] V

[0271] in

[0272] Z and A are as defined with respect to equation I;

[0273] PG is as defined with respect to Equation II;

[0274] Q is selected from non-existent, O, or S; and

[0275] T B2 It is the reactive end of B (e.g., the 5' end).

[0276] 50. The liquid-phase method according to statement 49 further includes the step of separating compound V by membrane filtration.

[0277] 51. The liquid-phase method according to any one of statements 47 to 50, wherein the oxidation step is performed after step e), and T PG The deprotection step is performed after the oxidation step.

[0278] 52. The liquid phase method according to any one of the foregoing statements further includes the following step:

[0279] (i) Providing an oligonucleotide for growth, wherein the oligonucleotide for growth is:

[0280] A) Compound V of formula A, or

[0281] B) A compound of formula V coupled with one or more additional nucleosides, wherein the nucleoside furthest from Z has a reactive end;

[0282] (ii) Modify the grown oligonucleotide to form a compound of formula VI:

[0283]

[0284] VI

[0285] in The bonds with the grown oligonucleotide (e.g., at the reactive end), PG and X are as defined with respect to Formula II;

[0286] (iii) Separating the compound of formula VI by membrane filtration;

[0287] (iv) Provide nucleoside or oligonucleotide building blocks with reactive and protected ends;

[0288] (v) React the isolated compound of formula VI with the reactive end of the nucleoside or oligonucleotide building block to form a chain-extended oligonucleotide having a protected end;

[0289] (vi) Separate the extended oligonucleotides by membrane filtration;

[0290] (vii) Deprotect the protected ends of the isolated strand-extended oligonucleotides and optionally repeat steps (i) to (vii) in sequence.

[0291] 53. The liquid-phase method according to statement 52 further includes the step of converting one or more P(III) phosphite trimer bonds into P(V) phosphotriester bonds (e.g., by oxidation or sulfur transfer), optionally, wherein said step is performed between steps (v) and (vi).

[0292] 54. The liquid-phase method according to any one of the foregoing statements further includes the step of removing the protective group PG from one or more P(V) phosphate triester bonds.

[0293] 55. The liquid-phase method according to any one of the foregoing statements further includes the step of cleaving oligonucleotides from Z.

[0294] 56. The liquid-phase method according to any one of the foregoing statements, wherein the method is carried out in acetonitrile or a solvent mixture containing acetonitrile.

[0295] 57. The liquid-phase method according to any one of the foregoing statements, wherein the method is carried out in a closed-loop reactor.

[0296] Example

[0297] One or more embodiments of the present invention will now be described with reference to the accompanying drawings, for illustrative purposes only:

[0298] Figure 1 Oligonucleotide synthesis cycles were performed via a forward phosphorus amide coupling method. The coupling between the 4'-OH group of phosphorus amide was initiated by one of several acidic activators. Similarly, various reagents were available to convert P(III) phosphite trimer to P(V) phosphate (X = O or S) trimer. In SPOS, capping agents were used to block any remaining unreacted 5'-OH group (usually in the form of acetate), but this step could be omitted in LPOS. In SPOS, support washing was used to drive the completion of detriphenylmethylation, but in LPOS, cationic scavengers could be used. In membrane-assisted phosphorus amide LPOS, points were noted in the organic solvent nanofiltration (OSN) cycle.

[0299] Figure 2 LPOS reverse phosphoramidite coupling method. The oxidation and detriphenylmethylation steps are the same as the forward phosphoramidite method for oligonucleotide synthesis. Figure 1 The initial phosphoramidation reaction uses a weakly acidic phosphoramidite activator, A1. After washing away excess phosphoramidite, a coupling reaction initiated by a more acidic phosphoramidite activator, A2, is typically carried out under anhydrous conditions to inhibit the blocking of the growing oligonucleotide chain, initially as an H-phosphonate. Non-limiting examples of activators, phosphate triester protecting groups (PG), and leaving groups (LG) are shown.

[0300] Figure 3 Reverse phosphoramidite coupling of fragments.

[0301] Figure 4 Combination of reverse and forward phosphoramide methods in oligonucleotide chain extension cycles.

[0302] Figure 5Ion-pair reversed-phase UV chromatograms of the homotype mA sequence during synthesis: a) mA3 trimer – indicating the main contaminant is the 5'-thiophosphate dimer, which originates from S-transfer to H-phosphonate, thus indicating significant water ingress; b) mA5 pentamer – the asterisk peak is the 5'-thiophosphate dimer from a) indicating that the error is limited. In the pentamer, thiophosphate end-caps accounted for 13.4% of the total impurities; nx (mainly x=1) accounted for 3.1%; PO accounted for 0.3%; and +Cne accounted for 0.3%.

[0303] Figure 6 According to Example 2, 5'-phosphate was installed via reverse phosphitylation.

[0304] A typical synthesis cycle in a synthesizer consists of three steps (see...). Figure 2 The process involves: 1) phosphoramidation followed by membrane filtration (DF) in an anhydrous organic solvent (also known as organic solvent nanofiltration (OSN; DF0, 4 DV)) to remove excess bisphosphonamide reagent; 2) coupling; and 3) oxidation or sulfur transfer, followed by OSN (DF1, 4 percolation volumes or DV) to remove excess nucleosides, activators, and oxidants – this DF can be omitted; finally, 4) detrimethylation, followed by purification of the crude oligonucleotide via OSN (DF2, 6 DV). The final DF removes any residual detrimethylated nucleosides that would otherwise participate in the next chain elongation cycle.

[0305] The following describes a representative loading and chain extension cycle in a membrane synthesizer. Note: Because the membrane synthesizer is sensitive to fine particulate fouling, all solutions injected into the synthesizer are filtered through a PTFE membrane (0.2 μm or denser).

[0306] Example 1: Homopentamer, entirely composed of thiophosphate esters

[0307] 5-Dmtr-mA Bz 3'-succinate was loaded onto a 10 kDa PEG star and loaded onto a Nanostar synthesizer.

[0308] PEG-10k(Sar-H)4 (11 g, 1.068 mmol) and 5'-Dmtr-mA Bz3'-Triethylammonium succinate (6.2 g, 8 mmol, 8 equivalents) was placed in a round-bottom flask. The starting material was dissolved in MeCN (33 mL), followed by the addition of DCC (1.65 g, 8 mmol, 8 equivalents) and HOBt hydrate (1.081 g, 8 equivalents, 8 mmol). The amidation reaction was monitored by LC-MS; typically, the reaction was stirred overnight to ensure completion. The crude solution was then filtered through a PTFE filter (0.1 μm, Sterlitech pressure filter holder) into the round-bottom flask. The filtrate was transferred through a port using a plastic funnel to the synthesis feed tank, and the flask was rinsed with additional MeCN. After removing a sample from a fully mixed Nanostar synthesizer to determine the residual Dmtr-succinate concentration at the start of DF, the sample was then permeated with MeCN (1 L = 4 DV, DF1). At the end of DF1, another sample was analyzed by LC-MS to measure the fractional decrease in succinic acid concentration and the retention of triphenylmethylnucleoside astrone.

[0309] Detriphenylmethylation reaction

[0310] A solution of the cation scavenger in MeCN (10 mL) (4 equivalents / arm, 16 in total; dodecyl mercaptan can be used for this purpose) was injected into the feed tank through the injection port using a syringe equipped with a disposable PTFE filter disc (0.2 μm). Deprotection was then initiated by injection of TFA (12.5 mL = 5 vol%). The deprotection reaction was monitored by LC-MS; after 10 min, no partially deprotected material was detected. After 20 min, the reaction was quenched with pyridine (25 mL), and the intermediate concentration of deprotected succinate was determined by sampling. Dried MeCN (1.5 L = 6 DV, < 20 ppm water) was permeated (DF2) to remove residual 5'-hydroxy-mA. Bz 3'-Succinate and other detrimethylated fragments. At the end of DF2, the final sample was analyzed by LC-MS to verify complete removal of building block fragments and high retention of nucleoside stars. The next step was only performed if the combined decrease in building block concentration on DF1 and DF2 was >99%; otherwise, DF2 was continued until this condition was met.

[0311] Phosphamide reaction

[0312] Before phosphoramidation, dry the reactor; if neither pressurization nor percolation is performed, ambient moisture will slowly enter the reactor. The reactor moisture content is inferred by measuring the moisture content of the percolate at the end of DF2 using a Karl-Fischer coulometric titrator. A value of <40 ppm water is acceptable for the phosphoramidation step; otherwise, additional DF2 is recommended until this value is reached.

[0313] 1-Methylimidazole (NMI, 0.947 g, 11.53 mmol, 2.7 equivalences / arm) was placed in a 50 mL round-bottom flask and dissolved in anhydrous MeCN (15 mL, < 20 ppm water) under an inert atmosphere. TFA (0.817 mL, MW = 114.02, d = 1.49 g / mL, 10.68 mmol, 2.5 equivalences / arm) was added to this solution, followed by drying with 500 mg MgSO4. The activator solution was then stirred at 25°C for 10 min.

[0314] Bis-amidite N,N,N′,N′-tetraisopropyl phosphorodiamidite (2, 4.07 mL, 12.81 mmol, 3 equivalents / arm) was injected into the synthesizer and circulated for 2 minutes. Then, a pre-formulated NMI.TFA activator solution was injected into the synthesizer through a PTFE filter (0.2 μm).

[0315] The phosphoramidation reaction was monitored by LC: 0.1 mL of sample from the synthesizer was added to an excess of a mixture of dicyanimidazole and cyanoethanol (0.2 mL), followed by 2 minutes of addition to an excess of pyridine (0.2 mL) of hydroflavin; this converted the reactive oligonucleotide astroxide 5'-O-phosphamide to a stable dicyanoethanol thiophosphate triester. The treated sample was then diluted with MeCN (0.6 mL) and analyzed by LC: Waters Acquity UPLC, C18 column; solvent A, 50 mM sodium acetate aqueous solution; solvent B, MeCN-MeOH 4:1; temperature 60°C, 20-100% solvent B, duration 6 minutes. After 5 minutes, all phosphoramidation intermediates were consumed, and percolation began after 20 minutes. After anhydrous MeCN (< 10 ppm water; 1 L = 4 DV) has been permeated (DF0), the oligonucleotide star 5'-O-phosphoramide is ready for coupling with the next nucleoside. The water content in the membrane reactor, determined by permeate sampling and KF coulometric analysis, should be < 40 ppm or less for the coupling step.

[0316] Coupling reaction

[0317] 5'-Dmtr-mABz nucleoside (4.4 g, 6.4 mmol, 1.5 equivalences / arm) and 4,5-dicyanimidazole (DCI, 2.018 g, MW = 118.1 Da, 17 mmol, 4 equivalences / arm) were placed in separate round-bottom flasks. The building block was co-evaporated from MeCN (< 15 ppm water, 3 × 100 mL), redissolved in MeCN (20 mL), and injected into the synthesizer through a PTFE filter (0.2 μm). DCI was dissolved in MeCN (20 mL) and also injected into the synthesizer through a PTFE filter (0.2 μm). The reaction was monitored by LC: after 2 minutes, the coupling intermediate was consumed, but the reaction was continued in the synthesizer for 25 minutes to ensure completion. 3-Phenyl 1,2,4-Dithiazolin-5-one (POS, 3.33 g, 17 mmol, 4 equivalences / arm) was added to the synthesizer to obtain a chain-extended trithiophosphate. The crude dimeric star was purified by percolation with MeCN (1 L = 4 DV).

[0318] Repeat the above synthesis cycle in the same manner (changing the building blocks as needed) to extend the oligonucleotide chain to the desired length.

[0319] Protect the whole

[0320] During synthesis, oligonucleotide stars were periodically analyzed by performing bulk deprotection on small samples, and the purity of crude oligonucleotides was estimated by analyzing ion-pair reversed-phase UHPLC. Sample preparation methods are described below:

[0321] A crude HO-oligonucleotide star-shaped solution sample (3 mL, approximately 0.048 mmol oligonucleotides) was removed from the synthesizer and placed in a concentrated ammonia solution (~3 mL) in an ACE pressure tube. Diethylamine (0.1 mL) was added to this solution, the tube was sealed, and the solution was heated overnight at 35°C. The next day, the solution was transferred to a round-bottom flask and evaporated. The aqueous residue was evaporated three times from MeCN, and finally the residue was ground with MeCN. The suspension was transferred to a Falcon tube (~30 mL) and centrifuged (5 min, 6000 rpm). The supernatant was removed, and the precipitate was washed with additional MeCN and centrifuged more than twice. The precipitate was then analyzed by IP-RP on an Agilent UHPLC system.

[0322] Example 2: Installation of 5'-phosphate ester via reverse phosphoramidation

[0323] 5'-phosphatization

[0324] Reference Figure 6 3-Methyl picoline (0.255 mL, 2.6 mmol, 2.62 equivalents / arm) was dissolved in anhydrous MeCN (5 mL, < 20 ppm water) under an inert atmosphere, and methanesulfonic acid (MSA, 0.162 mL, 2.5 mmol, 2.5 equivalents / arm) was added to the solution. Molecular sieves (3 Å, 5 g bag) were added to the solution, and the solution was stirred overnight at 25°C to provide an aqueous solution of the Pic.MSA activator.

[0325] The next day, PEG-10k(Sar-Suc-mU-mGIbu-mU-mU-mU-mU-mCAc-mGIbu)4 (3.15 g, 0.25 mmol) was dissolved in anhydrous MeCN-sulfolane (80:20 v / v, 5 mL) and transferred via syringe through the diaphragm port to the feed tank of a miniature synthesizer (system volume 50 mL). The flask was then rinsed with additional MeCN-sulfolane. Percolation was continued with anhydrous MeCN-sulfolane (80:20 v / v, < 20 ppm water) until the water content of the percolate, as determined by a Karl-Fischer coulometric titrator, was < 30 ppm water.

[0326] N,N,N′,N′-Tetraisopropylphosphorodiamidite (0.953 mL, 3 mmol, 3 equivalences / arm) was injected into the synthesizer and circulated for 2 minutes. Next, a pre-formed Pic.MSA activator solution was injected into the synthesizer through a PTFE filter (0.2 μm). The phosphoramidation reaction was monitored by LC: a sample (0.1 mL) from the synthesizer was added to a solution containing dicyanimidazole (100 mg) and cyanoethanol (0.2 mL), and after 2 minutes, this solution was transferred to another sample tube containing a solution of pyridine (0.2 mL) containing hydroflavin (100 mg); this converted the reactive oligonucleotide astrome 5'-O-phosphoramide to a stable dicyanoethanol thiophosphate triester. The treated sample was then diluted with MeCN (0.6 mL) and analyzed by LC: WatersAcquity UPLC, C18 column; solvent A, 50 mM sodium acetate aqueous solution; solvent B, MeCN-MeOH 4:1; temperature 60°C, 20-100% solvent B, duration 6 minutes. After 30 minutes, all phosphoramidized intermediates were consumed.

[0327] Converted to 5'-phosphate and deprotected overall.

[0328] Ethylene cyanohydrin (410 µL, 6 mmol, 6 DV / arm) was injected into the synthesizer, followed by the addition of dicyanimidazole (DCI, 0.710 g, 6 mmol, 6 DV / arm) dissolved in MeCN (5 mL). After 20 minutes, camphorsulfonazine (CSO, 0.51 g, 2.25 mmol, 9 DV / arm) dissolved in MeCN (10 mL) was added to the synthesizer to convert the chain ends to 5'-dicyanoethyl phosphate triester. The crude phosphorylated octamer star was purified by perfiltration with MeCN-sulfolane 80:20 v / v (200 mL = 4 DV).

[0329] The purified 5-phosphorylated octamer star solution sample (2 mL, approximately 0.01 mmol oligonucleotide) was removed from the synthesizer and placed in concentrated ammonia (~2 mL) in an ACE pressure tube. Diethylamine (120 µL) was added, the tube was sealed, and the mixture was heated overnight at 35 °C. The next day, the solution was transferred to a round-bottom flask and evaporated. The aqueous residue was co-evaporated three times from MeCN, and finally ground with MeCN. The suspension was transferred to a Falcon tube (~30 mL) and centrifuged (5 min, 6000 rpm). The supernatant was removed, and the precipitate was washed with additional MeCN and centrifuged at least twice. The precipitate was then analyzed by analytical ion-pair reversed-phase (IP-RP) UHPLC on an Agilent UHPLC system: mobile phase A: optimal water-MeOH 9:1, 2 μM EDTA, 60 mM HFIP, 6 mM HA; mobile phase B: MeCN:MeOH 1:1, Acquity Premier peptide BEH C18 column, 300 Å, 1.7 µm, 2.1 x 150 mm, column temperature 65°C, detector 260 nm, sample temperature 10°C, method 5-25% B, run time 15 min, 0.4 mL / min, injection volume 2 µL. The desired 5'-phosphorylated octamer eluted at 10.46 min, while the unphosphorylated octamer eluted at 8.10 min.

[0330] Although specific embodiments of the invention have been described herein for purposes of reference and illustration, various modifications will be apparent to those skilled in the art without departing from the scope of the invention as defined by the appended claims.

[0331] References

[0332] US8,664,357

[0333] US9,127,123

[0334] US10,239,996

[0335] EP3347402

[0336] EP0813539 B1

[0337] EP1612213 B1

[0338] WO 2016 / 188835 A1

[0339] US 7,960,542 B2

[0340] P.R.J. Gaffney, J.F. Kim, I.B. Valtcheva, G.D. Williams, M.S. Anson, A.M. Buswell, A.G. Livingston, Liquid-Phase Synthesis of 2'-Methyl-RNA on a Homostar Support through Organic-Solvent Nanofiltration, Chem. Eur. J., 2015, 21, 9535 - 9543

[0341] J.F. Kim, P.R.J. Gaffney, I.B. Valtcheva, G. Williams, A.M. Buswell, M.S. Anson, A.G. Livingston, Organic Solvent Nanofiltration (OSN): A New Technology Platform for Liquid-Phase Oligonucleotide Synthesis (LPOS), Org. Process Res. Dev. 2016, 20, 1439 - 1452

[0342] So Su, Peeva L.G., Tate E.W., Leatherbarrow R.J., Livingston A.G. “Organic Solvent Nanofiltration - A New Paradigm in Peptide Synthesis” Org. Process. Res. & Dev. 14 (2010) pp. 1313 - 132

[0343] Yeo J, Peeva L, Chung S, Gaffney P, Kim D, Luciani C, Tsukanov S,Siebert K, Kopach M, Albericio F, Livingston A “Liquid Phase PeptideSynthesis by One Pot Nanostar Sieving (PEPSTAR)”; Angew.Chem.Int. Ed.2021,60, pp. 7786 - 7795

[0344] Dong R., Liu R., Gaffney P.R.J., Schaepertoens M., Marchetti P.,Williams C.M., Chen R., and Livingston A.G. “Sequence-defined multifunctionalpolyethers via liquid-phase synthesis with molecular sieving” NatureChemistry (2019) 11 pp. 136 - 145

[0345] B. I. Andrews, F. D. Anita, S. B. Brueggemeier, L. J. Diorazio, S. G.Koenig, M. E. Kopach, H. Lee, M. Olbrich, A. L Watson, Sustainabilitychallenges and opportunities in oligonucleotide manufacturing, J. Org.Chem.,2021, Vol. 86, pp. 49 - 61.

[0346] A. Scozzari, Oligonucleotides for large market indications, whatneeds to happen or be considered? TKS Webinar Series - Innovations inOligonucleotide Therapeutics Manufacturing, 2021.

[0347] E. Legorburu, CB Reese, Q. Song, Conversion of uridine into 2'-O-(2-methoxyethyl)uridine and 2'-O-(2-methoxyethyl)cytidine, Tetrahedron, 1995, Part 55, Parts 5635-5640.

[0348] P. Liu, A. Sharon, CK Chu, Fluorinated nucleosides; Synthesis andbiological implication, J. Fluorine Chem.

[0349] J. Nielsen, M. Taagaard, JE Marugg, JH van Boom, O. Dahl,Application of 2-cyanoethyl N,N,N',N,-tetraisopropylphosphorodiamidite for insitu preparation of deoxyribonuceoside phosphoramidites and their use inpolymer-supported synthesis of oligonucleotides, Nucl.Acids Res., 1986, No. 14, No. 7391-7403.

[0350] DS Pedersen, C. Rosenbohm, T. Koch, Synthesis, 2002, pp. 802-808.

[0351] YSSanghvi, Z. Guo, HM Pfundheller, A. Converso, Improved Processfor the Preparation of Nucleosidic Phosphoramidites Using a Safer and CheaperActivator, Org.Proc.Res.Dev

[0352] C. Xie, M.l A. Staszak, J. T. Quatroche, C. D. Sturgill, V. V. Khau,M. J. Martinelli, Nucleosidic Phosphoramidite Synthesis via Phosphitylation:Activator Selection and Process Development, Org.Proc.Res.Dev., 2005, Volume 9, pp. 730-737.

[0353] A. F. Sandahl PhD thesis, Method developments in solid-phasechemistry: Easing access to synthetic oligonucleotides and oligo(disulfides),Aarhus University, Denmark, 2020.

Claims

1. A liquid-phase method for forming oligonucleotides, the method comprising the following steps: a) Provides compound of formula I: I Where A is a nucleoside or oligonucleotide, and T... A Z is the reactive end of the nucleoside or oligonucleotide, and Z is the soluble synthetic support to which the nucleoside or oligonucleotide is attached; b) Modify the compound of formula I to form the compound of formula II: II Where A and Z are as defined with respect to Formula I, X is a tertiary amino group, and PG is a protecting group; c) Separate the compound of formula II by membrane filtration; d) Provides compounds of formula III: III Where B is a nucleoside or oligonucleotide, and T... B1 It is the reactive terminus of the nucleoside or oligonucleotide, T PG It is the protected end of the nucleoside or oligonucleotide; e) React the compound of formula II with the compound of formula III to form the compound of formula IV: IV Where A and Z are as defined with respect to Equation I, PG is as defined with respect to Equation II, and B and T PG As defined with respect to Equation III.

2. The liquid-phase method according to claim 1, wherein X is a group -NR2, wherein each R is independently selected from (1-6C) alkyl groups, or two R groups are connected such that when combined with the nitrogen atom to which they are connected, they together form a 5- to 7-membered heterocycle.

3. The liquid phase method according to claim 2, wherein each R is isopropyl.

4. The liquid phase method according to any one of claims 1, 2 or 3, wherein PG is selected from the group consisting of cyanoethyl, 2-chlorophenyl, 4-chlorophenyl, 2,2,2-trichloroethyl and methyl.

5. The liquid-phase method according to any one of the preceding claims, wherein step c) comprises isolating the compound of formula II from one or more reactants used in step b).

6. The liquid-phase method according to any one of the preceding claims, wherein step b) comprises reacting the compound of formula I with the compound of formula A: A Wherein PG and X are as defined in any of the preceding claims, and Y is a halogen or has any of the definitions of X as described in any of the preceding claims; When Y is not a halogen, step b) is carried out in the presence of the first phosphorous amide activator.

7. The liquid-phase method according to claim 6, wherein the first phosphoramide activator is capable of reacting with compound A to form compound A': A’ Wherein X and PG are as defined in any of the preceding claims, and LG 1 It is a leaving group; Furthermore, the compound of formula A' is capable of reacting with the compound of formula I to form the compound of formula II.

8. The liquid phase method according to any one of the preceding claims, wherein step e) is carried out under anhydrous conditions.

9. The liquid phase method according to any one of the preceding claims, wherein (a)T A Located at the 5' end of nucleoside or oligonucleotide A and Z located at the 3' end of nucleoside or oligonucleotide A; and / or (b)T B1 Located at the 3' end of nucleoside or oligonucleotide B and T PG It is located at the 5' end of a nucleoside or oligonucleotide B.

10. The liquid-phase method according to any one of the preceding claims, wherein the compound of formula I has a structure according to formula Ia: Ia in -O 5A -A'-O 3A - is a nucleoside or oligonucleotide, in which O 5A It is oxygen located at the terminal 5' carbon and O 3A It is oxygen located at the terminal 3' carbon; T A It is hydrogen; and Z is a soluble synthetic support.

11. The liquid phase method according to any one of the preceding claims, wherein T PG It is a protective group that binds to the 5' oxygen of B.

12. The liquid phase method according to any one of the preceding claims, wherein T PG It is an acid-instable protective group, and non-limiting examples of such acid-instable protective groups include dimethoxytriphenylmethyl (Dmtr / DMT), tert-butyl (tBu), tert-butoxycarbonyl (Boc), monomethoxytriphenyl (Mmtr), triphenylmethyl (Tr), pentamethyldihydrobenzofuransulfonyl (Pbf), tetrahydropyranyl (Thp), tetrahydrofuranyl (Thf), p-methoxybenzyl (Pmb), and 2,4-dimethoxybenzyl.

13. The liquid-phase method according to any one of the preceding claims, wherein the compound of formula III has a structure according to IIIa: IIIa in -O 5B -B'-O 3B - is a nucleoside or oligonucleotide, in which O 5B It is oxygen located at the terminal 5' carbon and O 3B It is oxygen located at the terminal 3' carbon; T B1 It is hydrogen; and T PG It is a protective group (e.g., DMT).

14. The liquid-phase method according to any one of the preceding claims, wherein step e) is carried out in the presence of a second phosphoramide activator.

15. The liquid-phase method according to claim 14, wherein the second phosphoramide activator is capable of reacting with the compound of formula II to form compound II': II' Z, A, and PG are as defined in any of the preceding claims; LG 2 It is a leaving group; Furthermore, the compound of formula II' is capable of reacting with the compound of formula III to form the compound of formula IV.

16. The liquid phase method according to claim 15, wherein LG 2 It is a nitrogen-containing heteroaryl group.

17. The liquid-phase method according to any one of the preceding claims, wherein Z is a polymerizable soluble synthesis support, optionally wherein the molecular weight of Z (M w ) is 500-50,000 Da.

18. The liquid-phase method according to any one of claims 1-16, wherein Z is a compound of formula B: B in V is an organic branch point. W is a cluster chain. Indicates the point connected to A, and n is 1-8.

19. The liquid phase method according to claim 18, wherein (a) n is 3-4; and / or (b) V is aliphatic or aromatic; and / or (c) Molecular weight of each W (M) w The value is 500-20,000 Da (e.g., 2000-15,000 Da or 8000-12,000 Da).

20. The liquid-phase method according to any one of the preceding claims further comprises an oxidation step, wherein one or more P(III) phosphite triester bonds are converted into P(V) phosphotriester bonds (e.g., by oxidation or sulfur transfer); and wherein the product generated by said oxidation step is separated by membrane filtration.

21. The liquid phase method according to any one of the preceding claims, further comprising T PG The deprotection step involves contacting the oligonucleotide with a deprotecting agent to form a compound of formula V: V in Z and A are as defined with respect to equation I; PG is as defined with respect to Equation II; Q is selected from non-existent, O, or S; and T B2 It is the reactive end of B (e.g., the 5' end); and The method further includes the step of separating the compound of formula V by membrane filtration.

22. The liquid-phase method according to any one of claims 20 to 21, wherein the oxidation step is performed after step e), and T PG The deprotection step is performed after the oxidation step.

23. The liquid phase method according to any one of the preceding claims, further comprising the following steps: (i) Providing an oligonucleotide for growth, wherein the oligonucleotide for growth is: A) Compound V of formula A, or B) A compound of formula V coupled with one or more additional nucleosides, wherein the nucleoside furthest from Z has a reactive end; (ii) Modify the grown oligonucleotide to form a compound of formula VI: VI in The bonds with the grown oligonucleotide (e.g., at the reactive end), PG and X are as defined with respect to Formula II; (iii) Separating the compound of formula VI by membrane filtration; (iv) Provide nucleoside or oligonucleotide building blocks with reactive and protected ends; (v) React the isolated compound of formula VI with the reactive end of the nucleoside or oligonucleotide building block to form a chain-extended oligonucleotide having a protected end; (vi) Separate the extended oligonucleotides by membrane filtration; (vii) Deprotect the protected ends of the isolated strand-extended oligonucleotides and optionally repeat steps (i) to (vii) in sequence.

24. The liquid-phase method of claim 23, further comprising the step of converting one or more P(III) phosphite-triester bonds into P(V) phosphotriester bonds (e.g., by oxidation or sulfur transfer), optionally wherein said step is performed between steps (v) and (vi).

25. The liquid phase method according to any one of the preceding claims, further comprising: (a) The step of removing the protecting group PG from one or more P(V) phosphotriester bonds; and / or (b) The steps of cleaving oligonucleotides from Z.

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