Membrane filtration assisted solution phase oligonucleotide synthesis

Abstract

The invention relates to a membrane filtration assisted method for preparing oligonucleotides by solution phase synthesis. In particular, the present invention relates to a solution phase process for preparing oligonucleotides in which membrane filtration (e.g., diafiltration) is used to purify and / or isolate oligonucleotides during stepwise growth of oligonucleotides.

Description

Technical Field

[0001] The present invention relates to a membrane filtration assisted method for preparing oligonucleotides by solution phase synthesis. In particular, the present invention relates to a solution phase method for preparing oligonucleotides, wherein membrane filtration (e.g., diafiltration) is used to purify and / or isolate oligonucleotides during the stepwise growth of the oligonucleotides. Background Art

[0002] Oligonucleotide-based drugs have previously been proposed as a new generation of therapeutic agents that act at the protein expression level and have recently been validated as a new drug form for treating a variety of serious or life-threatening indications. Oligonucleotides are formed from a phosphoribose monomer backbone, each monomer having a variable nucleobase side chain; the building block units of phosphoribose that bind to the nucleobases constitute nucleotides. The precise sequence of nucleotides determines the biological function of the oligonucleotide.

[0003] For many years, oligonucleotides have been prepared using solid-phase oligonucleotide synthesis (SPOS), in which the growing oligonucleotide is tethered to an insoluble solid support and grows by flowing nucleotide building blocks over the insoluble solid support. Although this method has become the industry standard, SPOS has many disadvantages. In particular, to drive the reaction to completion, an excess of nucleotide building blocks is typically required, which significantly increases the cost of SPOS. In addition, the scale-up of SPOS reactions is limited to producing only about 15 kg of oligonucleotides per batch. This is particularly undesirable for use as a drug form, where for major medical indications (e.g., cardiovascular disease), several metric tons of oligonucleotides would be required annually.

[0004] One alternative strategy to SPOS that aims to address these scale-up and economic challenges is liquid-phase oligonucleotide synthesis (LPOS). In fact, liquid-phase reactions and liquid-phase material handling are well-established technologies that can be carried out on a metric ton scale, which makes LPOS a strong candidate for large-scale oligonucleotide preparation. A typical method of LPOS is to perform sequential coupling reactions to add monomers or multimonomer oligomers (fragments) stepwise to the oligonucleotide growing in solution, and then use a suitable separation technique, such as membrane filtration, to separate unreacted monomers or fragments from the growing oligonucleotide. 1-10

[0005] After the step of coupling a monomer or fragment to the growing oligonucleotide, membrane filtration can be used to separate unreacted monomers / fragments and any reaction debris from the growing oligonucleotide. Thus, the use of membrane filtration is well-suited for LPOS, where a thorough purification step is preferably carried out after each coupling step. Membrane filtration is particularly useful when the process of preparing oligonucleotides is carried out in a single organic solvent system (e.g., acetonitrile).

[0006] Although this technique has addressed many challenges faced by SPOS, there are still some issues with using membrane filtration separation in LPOS. Most notably, when preparing long oligonucleotides (i.e., length ≥ 8 nucleotides) formed by multiple coupling reactions, as the length of the oligonucleotide increases, membrane filtration purification becomes increasingly difficult. In such cases, the viscosity of the solution may increase to the extent that the membrane flux drops to an unacceptably low level, and the retention rate of the growing oligonucleotide may decrease due to concentration polarization, making it difficult and inefficient (if not impossible) to separate the growing oligonucleotide, thus endangering any further coupling reactions. Additionally, the increase in viscosity significantly increases the likelihood of membrane fouling, thus requiring a complete membrane replacement or even the selection of a new, more porous membrane. To date, attempts to solve this problem have focused on using an excessive amount of solvent to allow additional coupling steps. However, even with the addition of fresh solvent, the membrane may still continue to foul during subsequent filtration steps. Therefore, there is a need to improve the membrane filtration-assisted method for preparing oligonucleotides in solution, particularly in cases where the length of the target oligonucleotide increases.

[0007] The present invention has been designed in view of the foregoing. Summary of the Invention

[0008] According to a first aspect of the present invention, there is provided a solution-phase method for preparing an oligonucleotide, the method comprising the steps of:

[0009] growing the oligonucleotide by performing one or more sequential coupling reactions, each sequential coupling reaction increasing the length of the growing oligonucleotide by at least one nucleotide,

[0010] wherein the growing oligonucleotide comprises at least one protected uracil nucleobase and / or at least one protected thymine nucleobase, and

[0011] wherein the step of growing the oligonucleotide comprises one or more membrane filtration steps to separate the growing oligonucleotide.

[0012] According to a second aspect of the present invention, there is provided an oligonucleotide obtained directly or obtainable by the method of the first aspect. Detailed Description

[0013] Throughout the specification and claims of this application, when the term "comprise" (or "comprises" or "comprising") is used to describe a subject matter herein, it is also contemplated to use the term "consist of" (or "consists of" or "consisting of") or "consist essentially of" (or "consists essentially of" or "consisting essentially of") to describe the same subject matter.

[0014] Throughout the specification and claims of this application, the singular covers the plural unless the context otherwise requires. In particular, in the case of using an indefinite article, the specification shall be understood to contemplate both the plural and the singular unless the context otherwise requires.

[0015] 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 therewith. 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, unless at least some of such features and / or steps are mutually exclusive combinations. The invention is not limited to the details of any particular embodiment described herein. The invention extends to any novel one or any novel combination of the features disclosed in this application (including any appended claims, abstract and drawings), or to any novel one or any novel combination of the steps of any method or process disclosed.

[0016] As described above, in a first aspect, the present invention provides a solution-phase method for preparing oligonucleotides, the method comprising the following steps:

[0017] Growing the oligonucleotide by performing one or more sequential coupling reactions, each sequential coupling reaction increasing the length of the growing oligonucleotide by at least one nucleotide,

[0018] wherein the growing oligonucleotide comprises at least one protected uracil nucleobase and / or at least one protected thymine nucleobase, and

[0019] wherein the step of growing the oligonucleotide comprises one or more membrane filtration steps to separate the growing oligonucleotide.

[0020] Through rigorous research, the present inventors have designed a significantly improved membrane filtration-assisted method for the preparation of oligonucleotides by solution-phase synthesis. In particular, the present inventors have found that the presence of at least one protected uracil nucleobase and / or at least one protected thymine nucleobase on the growing oligonucleotide can significantly reduce the viscosity of the oligonucleotide solution. The surprising viscosity reduction imparted by at least one protected uracil nucleobase and / or at least one protected thymine nucleobase alleviates the drawbacks typically associated with conventional membrane filtration-assisted LPOS techniques, including membrane fouling and poor membrane flux. In fact, the present inventors have surprisingly found that the solution-phase method of the present invention is capable of directly preparing oligonucleotides of increasing length (e.g., length ≥ 8 nucleotides) through multiple sequential coupling reactions in certain industrially favored solvents.

[0021] Oligonucleotides are familiar to those skilled in the art and will be understood to comprise a plurality of nucleotides that are interconnected (i.e., coupled) to form a nucleotide sequence. As described herein, oligonucleotides are prepared by the stepwise addition of monomers, dimers, and / or oligonucleotide building blocks to the growing oligonucleotide, with each addition being referred to as a coupling reaction. In the context of the present invention, the term "oligonucleotide" refers to the nucleotide sequence after the final coupling reaction (sometimes referred to industrially as the full-length product), while the term "growing oligonucleotide" refers to the nucleotide sequence up to and during the final coupling reaction. In its simplest sense, a "growing oligonucleotide" can be a single nucleotide.

[0022] The growing oligonucleotide can be a single growing oligonucleotide or a plurality (e.g., 2 - 12) of growing oligonucleotides. Suitably, the growing oligonucleotide is 2 - 10 growing oligonucleotides. More suitably, the growing oligonucleotide is 2 - 8 growing oligonucleotides. Most suitably, the growing oligonucleotide is 3 - 4 growing oligonucleotides. Thus, the step of growing the oligonucleotide can include growing a plurality (e.g., 2 - 12) of oligonucleotides. Suitably, the step of growing the oligonucleotide includes growing 2 - 10 oligonucleotides. More suitably, the step of growing the oligonucleotide includes growing 2 - 8 oligonucleotides. Most suitably, the step of growing the oligonucleotide includes growing 3 - 4 oligonucleotides. It should be understood that in embodiments where the growing oligonucleotide is a plurality of growing oligonucleotides, each growing oligonucleotide has the same nucleotide sequence.

[0023] The molecular weight of each oligonucleotide after it is prepared (i.e., grown into the full-length product) can be ≥ 1000 Da. Suitably, the molecular weight of each oligonucleotide is ≥ 2000 Da. More suitably, the molecular weight of each oligonucleotide is ≥ 3000 Da. Even more suitably, the molecular weight of each oligonucleotide is ≥ 5000 Da.

[0024] The growing oligonucleotide comprises at least one protected uracil nucleobase and / or at least one protected thymine nucleobase. The term "protected" in the context of the present invention is to be understood to mean that the nucleobase comprises a protecting group (i.e., at least one protecting group). Thus, the growing oligonucleotide comprises: (i) at least one protected uracil nucleobase, wherein the protected uracil nucleobase comprises a protecting group; and / or (ii) at least one protected thymine nucleobase, wherein the protected thymine nucleobase comprises a protecting group. Protecting groups are familiar to those skilled in the art and are to be understood as organic moieties that are attached to a functional group to block the reactivity of the functional group.

[0025] The position of the protecting group depends on the nature of the protected nucleobase. As is familiar to those skilled in the art, the uracil nucleobase adopts the following configuration in the oligonucleotide

[0026]

[0027] where represents the point of attachment to the growing oligonucleotide.

[0028] The thymine nucleobase adopts the following configuration in the oligonucleotide:

[0029]

[0030] where represents the point of attachment to the growing oligonucleotide.

[0031] Suitably, in all protected uracil and thymine nucleobases as defined herein, represents the point of attachment to the pentose sugar (e.g., ribose or deoxyribose) in the growing oligonucleotide. Some examples of protected uracil and protected thymine attached to the pentose sugar are shown below:

[0032]

[0033] where represents the point of attachment to the remainder of the growing oligonucleotide and R is H, OH, OMe or F.

[0034] As described above, protected uracil nucleobases and protected thymine nucleobases can be attached to the growing oligonucleotide at the N1 position (e.g., uridine and thymidine). In such embodiments, the protecting group can be located at the N3 position or the O4 position (i.e., the oxygen atom bonded to C4). Thus, when at least one protected uracil nucleobase contains a protecting group and is attached to the growing oligonucleotide at the N1 position, the protecting group is located at the N3 position or the O4 position. Similarly, when at least one protected thymine nucleobase contains a protecting group and is attached to the growing oligonucleotide at the N1 position, the protecting group is located at the N3 position or the O4 position. Suitably, in the above two embodiments, the protecting group is located at the N3 position.

[0035] The nature of the protecting group will depend on whether the protecting group is located at the N3 position or the O4 position of the uracil nucleobase and / or thymine nucleobase. For example, certain protecting groups may initially be located at the O4 position (kinetic product), but may subsequently migrate to the N3 position (thermodynamic product).

[0036] Alternatively, the protected uracil nucleobase can be attached to the growing oligonucleotide at the C5 position (e.g., pseudouridine). In such embodiments, the protecting group can be located at the N3 position or the O2 position (i.e., the oxygen atom bonded to C2). Thus, when at least one protected uracil nucleobase contains a protecting group and is attached to the growing oligonucleotide at the C5 position, the protecting group is located at the N3 position or the O2 position. In this embodiment, an additional protecting group can also be located at the N1 position.

[0037] In embodiments where the protected uracil nucleobase is attached to the growing oligonucleotide at the C5 position, the protecting group can be located at the N1 position or the O2 position (i.e., the oxygen atom bonded to C2). Thus, when at least one protected uracil nucleobase contains a protecting group and is attached to the growing oligonucleotide at the C5 position, the protecting group is located at the N1 position or the O2 position. In this embodiment, an additional protecting group can also be located at the N3 position.

[0038] As discussed above, the inventors have found that the protecting groups of protected uracil nucleobases and / or protected thymine nucleobases surprisingly play an important role in reducing the solution viscosity during membrane filtration. In particular, by reducing the solution viscosity, membrane fouling and the reduction of membrane flux can be alleviated. This avoids the need to replace the membrane or select a looser membrane during the purification and separation process, which can be expensive and time-consuming. In addition, the use of excessive amounts of solvent to reduce the concentration of the building blocks is avoided.

[0039] Each protecting group can independently be an acid-labile protecting group, a base-labile protecting group, an ammonia-labile protecting group, an oxime-labile protecting group, an oxidation-labile protecting group, a hydrogenolysis-labile protecting group, or a transition metal-catalyzed cleavage protecting group. Suitably, each protecting group is independently selected from the group consisting of: 2,4,6-trimethylphenyl, 2-nitrophenyl, 2,4-dimethylphenyl, toluyl, 2-(4-nitrophenyl)ethyl, 2-(4-cyanophenyl)ethyl, allyl, benzoyl (Bz), 2,4-dimethylbenzoyl, tert-butylbenzoyl (tert-BuBz), acetyl (Ac), anisoyl (An), 4-chlorobenzoyl, diphenylcarbamoyl, butylthiocarbonyl, 2-nitrophenylsulfenyl, 2,4-dinitrophenylsulfenyl, 2-nitro-4-toluylsulfenyl, and triphenylmethylsulfenyl. The protecting group can be located at the above-mentioned positions on the protected uracil nucleobase and / or thymine nucleobase. Suitably, each protecting group is independently selected from the group consisting of benzoyl (Bz), 2,4-dimethylbenzoyl, tert-butylbenzoyl (tert-BuBz), and anisoyl (An).

[0040] It should be understood that in embodiments where the growing oligonucleotide contains more than one protected uracil nucleobase, each uracil nucleobase can independently be attached to the growing oligonucleotide at a different position (i.e., one protected uracil nucleobase at the N1 position and another protected uracil nucleobase at the C5 position).

[0041] In the step of growing the oligonucleotide, the oligonucleotide is grown by performing one or more sequential coupling reactions, each sequential coupling reaction increasing the length of the growing oligonucleotide by at least one nucleotide. The coupling reaction can be monomeric, dimeric, or oligomeric in nature. For example, the coupling reaction can include adding a monomer building block to each growing oligonucleotide (i.e., in a single coupling reaction, the length of the growing oligonucleotide is increased by one nucleotide). Alternatively, the coupling reaction can include adding a dimeric building block (i.e., two pre-coupled nucleotides) to each growing oligonucleotide (i.e., in a single coupling reaction, the length of the growing oligonucleotide is increased by two nucleotides). Alternatively, the coupling reaction can include adding an oligomeric building block (i.e., three or more pre-coupled nucleotides) to each growing oligonucleotide (i.e., in a single coupling reaction, the length of the growing oligonucleotide is increased by three or more nucleotides).

[0042] Each sequential coupling reaction will be independent of one another. Thus, the step of growing an oligonucleotide by performing one or more sequential coupling reactions can include a coupling reaction that increases the length of the growing oligonucleotide by one nucleotide and a coupling reaction that increases the length of the growing oligonucleotide by two nucleotides. The step of growing an oligonucleotide by performing one or more sequential coupling reactions can include a coupling reaction that increases the length of the growing oligonucleotide by one nucleotide and a coupling reaction that increases the length of the growing oligonucleotide by three or more nucleotides. The step of growing an oligonucleotide by performing one or more sequential coupling reactions can also include a coupling reaction that increases the length of the growing oligonucleotide by two nucleotides and a coupling reaction that increases the length of the growing oligonucleotide by three or more nucleotides.

[0043] In its simplest sense, the step of growing an oligonucleotide can include only a single coupling reaction, such as a coupling reaction between an initial monomer unit (i.e., the growing oligonucleotide) and an additional monomer, dimer, or oligomer building block (i.e., a single nucleotide, two pre-coupled nucleotides, or three or more nucleotides, respectively). The step of growing an oligonucleotide can include performing two or more sequential coupling reactions. Suitably, the step of growing an oligonucleotide includes performing three or more sequential coupling reactions. More suitably, the step of growing an oligonucleotide includes performing four or more sequential coupling reactions. Even more suitably, the step of growing an oligonucleotide includes performing six or more sequential coupling reactions. More suitably, the step of growing an oligonucleotide includes performing ten or more sequential coupling reactions. Most suitably, the step of growing an oligonucleotide includes performing fifteen or more sequential coupling reactions.

[0044] The step of growing an oligonucleotide can include multiple sequential coupling reactions, where each sequential coupling reaction increases the length of the growing oligonucleotide by one nucleotide. Suitably, the multiple sequential coupling reactions are two or more sequential coupling reactions. More suitably, the multiple sequential coupling reactions are three or more sequential coupling reactions. More suitably, the multiple sequential coupling reactions are four or more sequential coupling reactions. Even more suitably, the multiple sequential coupling reactions are six or more sequential coupling reactions. Even more suitably, the multiple sequential coupling reactions are ten or more sequential coupling reactions. Most suitably, the multiple sequential coupling reactions are fifteen or more sequential coupling reactions.

[0045] Each coupling reaction typically involves reacting the free (unprotected) end of the growing oligonucleotide with the reactive end of a monomer, dimer, or oligomer building block to be coupled, and then deprotecting the end of the newly coupled monomer, dimer, or oligomer building block to generate a new free (unprotected) end (in preparation for subsequent coupling reactions). Those skilled in the art will be familiar with the protecting groups used to prevent uncontrolled polymer chain extension in the liquid-phase synthesis of oligonucleotides, and the ways in which they can be removed.

[0046] The reaction site of a monomer, dimer, or oligomer building block (i.e., a single nucleotide, two pre-coupled nucleotides, or three or more nucleotides, respectively) to be coupled to the distal end of the growing oligonucleotide chain can consist of: a phosphoramidite, a phosphomonoester, a phosphodiester, an H-phosphonate, a cyclic phosphorothioate or cyclic dithiophosphotriester moiety, or any other phosphorus-containing precursor of a nucleotide internucleotide bond, or any other substance that results in an analogue of a nucleotide internucleotide bond, which is well known to those skilled in the chemical synthesis of oligonucleotides. 12

[0047] The step of growing the oligonucleotide can be carried out in at least one organic solvent. Suitably, the step of growing the oligonucleotide is carried out in acetonitrile, optionally mixed with another organic solvent. More suitably, the step of growing the oligonucleotide is carried out in acetonitrile mixed with sulfolane. In such an embodiment, the ratio of acetonitrile to sulfolane can be 4:1 v / v. Alternatively, in some embodiments, the step of growing the oligonucleotide is carried out in acetonitrile (i.e., pure acetonitrile). Acetonitrile is the solvent of choice in the industry for coupling nucleotides to prepare oligonucleotides. Most suitably, the step of growing the oligonucleotide is carried out in pure acetonitrile or a mixture of acetonitrile and sulfolane (e.g., 4:1 v / v).

[0048] In some embodiments, the step of growing the oligonucleotide comprises two or more sequential coupling reactions and is carried out in at least one organic solvent. In some embodiments, the step of growing the oligonucleotide comprises four or more sequential coupling reactions and is carried out in acetonitrile, optionally mixed with another organic solvent. In some embodiments, the step of growing the oligonucleotide comprises six or more sequential coupling reactions and is carried out in pure acetonitrile or a mixture of acetonitrile and sulfolane (e.g., 4:1 v / v).

[0049] The steps for growing oligonucleotides include one or more membrane filtration steps to separate the growing oligonucleotides. Suitably, the membrane filtration is membrane diafiltration. Most suitably, the membrane filtration is organic solvent nanofiltration (OSN) or ultrafiltration (UF). Membrane filtration can be carried out to separate the growing oligonucleotides from reaction by-products formed as part of the coupling reaction (e.g., protecting groups cleaved from the ends of the growing oligonucleotides) or excess reagents used as part of the coupling reaction (e.g., excess monomers, dimers, or oligomeric building blocks to be coupled). During the steps of growing oligonucleotides, the growing oligonucleotides, excess reagents, and reaction by-products remain in solution.

[0050] One or two membrane filtrations can be carried out for a given coupling reaction. The first filtration can include separating the growing oligonucleotides from reaction by-products formed as part of the coupling reaction (e.g., protecting groups cleaved from the ends of the growing oligonucleotides). The second filtration can include separating the growing oligonucleotides from excess reagents used as part of the coupling reaction (e.g., excess monomers, dimers, or oligomeric building blocks to be coupled). Suitably, two membrane filtrations are carried out for each coupling reaction.

[0051] Membrane filtration need not be carried out as part of each sequential coupling reaction carried out in the steps of growing oligonucleotides. For example, if the steps of growing oligonucleotides include 3 sequential coupling reactions, membrane filtration can be carried out as part of only 1 or 2 of these reactions. However, in some embodiments, membrane filtration is carried out as part of each sequential coupling reaction carried out in the steps of growing oligonucleotides.

[0052] The steps of growing oligonucleotides and the one or more membrane filtration steps to separate the growing oligonucleotides can be carried out in the same solvent. Suitably, the steps of growing oligonucleotides and the one or more membrane filtration steps are carried out in at least one organic solvent. More suitably, the steps of growing oligonucleotides and the one or more membrane filtration steps are carried out in acetonitrile optionally mixed with another organic solvent. More suitably, the steps of growing oligonucleotides and the one or more membrane filtration steps are carried out in acetonitrile mixed with sulfolane. In such an embodiment, the ratio of acetonitrile to sulfolane can be 4:1 v / v. Additionally, in some embodiments, the steps of growing oligonucleotides and the one or more membrane filtration steps are carried out in pure acetonitrile. Most suitably, the steps of growing oligonucleotides and the one or more membrane filtration steps to separate the growing oligonucleotides are carried out in pure acetonitrile or a mixture of acetonitrile and sulfolane (e.g., 4:1 v / v).

[0053] Suitable membranes for the one or more membrane filtration steps to separate the growing oligonucleotides include polymer membranes, ceramic membranes, and mixed polymer / inorganic membranes. The membrane rejection rate Ri is a commonly known term to those skilled in the art and is defined as:

[0054]

[0055] where C P,i = the concentration of substance i in the permeate, the permeate being the liquid that has passed through the membrane, C R,i = the concentration of substance i in the retentate, the retentate being the liquid that has not passed through the membrane. It should be understood that the membrane is suitable for the present invention if:

[0056] R (生长的寡核苷酸) >R (至少一种反应副产物或试剂)

[0057] Typically, in one or more membrane filtration steps, a crude mixture containing the growing oligonucleotide is pressed against a size-selective solvent-stable membrane. Here, the soluble synthetic support serves more than just as a passive solubilization aid. During the process of diafiltration (separating solutes by permeating a solution through a selective membrane), the solutes that exhibit membrane rejection accumulate on the retentate side at the interface between the bulk solution and the membrane. The soluble synthetic support is designed to have the highest possible, preferably 100%, membrane retention rate. The excess reagents (such as nucleotides) used in the coupling reaction typically have the second highest molecular weight, such that they typically have the second highest retention rate. It is important to remove all excess reagents before starting the next coupling reaction to prevent the free hydroxyl groups of the retained nucleotides from providing sites for the growth of unwanted truncated oligonucleotide contaminants.

[0058] To obtain the highest possible oligonucleotide purity (i.e., after full growth), the highest possible coupling efficiency is desirable. Given that the bimolecular reaction rate between the hydroxyl terminus of the growing oligonucleotide and the nucleotide building block is approximately proportional to the concentrations of the two substances, the highest practical concentrations of the growing oligonucleotide and the building block should be achieved to allow the process to achieve near-quantitative conversion. Since high concentrations of the building block can be used to drive the reaction closer to 100% completion, a large excess of the building block might seem advantageous. However, for economic oligonucleotide synthesis (e.g., nucleotide building blocks are very expensive) and to minimize the amount of debris that must be removed by diafiltration, a low excess of the building block is preferred and thus highly desirable. In addition, a low excess of the building block minimizes the amount of diafiltration solvent required to achieve the specified purity of the oligonucleotide; typically, an initial concentration of 0.1% of the residual building block (= 0.001 x 0.5 equivalents) is acceptable.

[0059] As a compromise between these two extremes, oligonucleotides grown at concentrations of 4 to 40 mM are ideal for rapid coupling with an economically excess amount of 1 - 2 (e.g., 1.5) equivalents of oligonucleotide (e.g., phosphoramidite) building blocks in acetonitrile / acetonitrile mixed with sulfolane. The LPOS phosphoramidite chain extension reaction can be initiated with common activators such as ethanethiotetrazole (ETT) or dicyanoimidazole (DCI) and carried out for 5 to 20 minutes. The reaction can be quenched with a small excess of alcohol or water and then oxidized (using reagents such as camphorsulfonyl oxaziridine (CSO), cumenyl hydroperoxide, or tert - butyl hydroperoxide) or sulfur transfer (using reagents such as phenylacetyl disulfide (PADS), or xanthogenic acid hydride (XH), or 3 - phenyl - 1,2,4 - dithiazolin - 5 - one (POS)), after which the crude mixture can be purified by OSN.

[0060] The membrane(s) that can be used in one or more membrane filtration steps can be formed from any polymeric or ceramic material that provides a separation layer capable of preferentially separating the growing oligonucleotide from at least one reaction by - product or reagent used in the step of growing the oligonucleotide. In other words, the membrane will exhibit a higher rejection rate for the growing oligonucleotide than for the reaction by - product or reagent. Suitably, the membrane is formed from or comprises a polymeric material suitable for making microfiltration membranes, ultrafiltration membranes, nanofiltration membranes, or reverse osmosis membranes, including polyethylene, polypropylene, polytetrafluoroethylene (PTFE), polyvinylidene fluoride (PVDF), polysulfone, polyethersulfone, polyacrylonitrile, polyamide, polyester, polyimide, polyetherimide, cellulose acetate, polyaniline, polypyrrole, polybenzimidazole, polyetheretherketone (PEEK), and mixtures thereof. The membrane can be made by any technique known in the art, including sintering, stretching, track etching, template leaching, interfacial polymerization, or phase inversion. The membrane can be composite in nature (e.g., thin - film composite membrane) and / or can be cross - linked or treated to improve their stability in the solvents used. PCT / GB2007 / 050218, PCT / GB2015 / 050179, and US10,913,033 describe membranes that can be used in one or more membrane filtration steps. The membrane will be stable in the organic solvent system used in the step of growing the oligonucleotide and in the one or more membrane filtration steps for separating the growing oligonucleotide.

[0061] Suitably, one or more membrane filtration steps are carried out using a cross - linked polybenzimidazole membrane (e.g., a skinned asymmetric cross - linked polybenzimidazole membrane) or a polyetheretherketone membrane.

[0062] In some embodiments, each coupling reaction is subjected to one or two membrane filtrations, and is carried out using a crosslinked polybenzimidazole membrane or a polyetheretherketone membrane. In some embodiments, each coupling reaction is subjected to two membrane filtrations, and is carried out using a crosslinked polybenzimidazole membrane.

[0063] In the step of growing the oligonucleotide, the growing oligonucleotide may be attached at one end to a soluble synthetic support. The nature of the attachment between the growing oligonucleotide and the soluble synthetic support may be direct or indirect (e.g., via a linker). Various soluble synthetic supports capable of dissolving the oligonucleotide during growth may be used. The soluble synthetic support may comprise a central hub and one or more solubility enhancing polymers, each polymer being attached to the central hub. The nature of the attachment between each solubility enhancing polymer and the central hub may be direct or indirect (e.g., via a linker). Each growing oligonucleotide may be attached directly or indirectly (e.g., via a linker) at one end to the central hub, to a solubility enhancing polymer, or to any linker that may connect the central hub to the solubility enhancing polymer. Suitably, each growing oligonucleotide is attached directly or indirectly (e.g., via a linker) at one end to a solubility enhancing polymer. The growing oligonucleotide attached to the soluble synthetic support is herein referred to as the support - growing oligonucleotide.

[0064] In certain embodiments, one or more solubility enhancing polymers are each attached to the central hub, and each growing oligonucleotide is attached at one end to a solubility enhancing polymer. Suitably, within each soluble synthetic support molecule, the number of solubility enhancing polymers is equal to the number of growing oligonucleotides.

[0065] One or more solubility enhancing polymers may be a single solubility enhancing polymer or multiple (e.g., 2 - 12) solubility enhancing polymers (i.e., each soluble synthetic support molecule may comprise multiple solubility enhancing polymers). Suitably, one or more solubility enhancing polymers are 2 - 10 solubility enhancing polymers. More suitably, one or more solubility enhancing polymers are 2 - 8 solubility enhancing polymers. Most suitably, one or more solubility enhancing polymers are 3 - 4 solubility enhancing polymers.

[0066] In some embodiments, the growing oligonucleotide is attached at one end to a soluble synthetic support, where the soluble synthetic support comprises a central hub and a plurality (e.g., 2 - 12) of solubility enhancing polymers, each polymer being attached to the central hub. In some embodiments, the growing oligonucleotide is attached at one end to a soluble synthetic support, where the soluble synthetic support comprises a central hub and 2 - 8 solubility enhancing polymers, each polymer being attached to the central hub. In some embodiments, the growing oligonucleotide is attached at one end to a soluble synthetic support, where the soluble synthetic support comprises a central hub and 3 - 4 solubility enhancing polymers, each polymer being attached to the central hub.

[0067] In some embodiments, the growing oligonucleotide is attached at one end to a soluble synthetic support, where the soluble synthetic support comprises a central hub and a plurality (e.g., 2 - 12) of solubility enhancing polymers, each polymer being attached to the central hub, and where the number of solubility enhancing polymers is equal to the number of growing oligonucleotides. In some embodiments, the growing oligonucleotide is attached at one end to a soluble synthetic support, where the soluble synthetic support comprises a central hub and 2 - 8 solubility enhancing polymers, each polymer being attached to the central hub, and where the number of solubility enhancing polymers is equal to the number of growing oligonucleotides. In some embodiments, the growing oligonucleotide is attached at one end to a soluble synthetic support, where the soluble synthetic support comprises a central hub and 3 - 4 solubility enhancing polymers, each polymer being attached to the central hub, and where the number of solubility enhancing polymers is equal to the number of growing oligonucleotides.

[0068] When each growing oligonucleotide is attached to a solubility enhancing polymer via a linker, a series of chemistries can be used to construct such attachment. For example, if the solubility enhancing polymer terminates with a hydroxyl functional group, this can be esterified with nucleoside succinate. If the solubility enhancing polymer terminates with an amino functional group, it can be directly condensed with nucleoside succinate to form a succinate - amide. Other linkages may have higher stability; for example, PEG - amine can react with Fmoc - sarcosine or Boc - sarcosine and then deprotected to leave a secondary N - methyl polyethylene glycol chain terminus. Scheme 1 below illustrates various suitable linkers:

[0069]

[0070] Scheme 1 - Linkers suitable for attaching growing oligonucleotides to solubility enhancing polymers

[0071] Alternatively, when the liquid-phase method involves preparing oligonucleotides by phosphoramidite chemistry, PEG-amine can react with one of the "universal" linkers. Universal linkers are desirable because the support can be directly loaded with the selected nucleoside phosphoramidites (but releases the hydroxyl-terminated oligonucleotide during the overall deprotection process) without the need for separate nucleoside succinate building blocks. After loading onto the soluble synthetic support, the temporary hydroxyl protecting group (usually Dmtr) is deblocked and ready to participate in the oligonucleotide chain elongation cycle.

[0072] Suitably, each growing oligonucleotide is attached at one end to a solubility-enhancing polymer via a linker with a molecular weight < 600 Da. More suitably, the linker has a molecular weight < 300 Da.

[0073] The central hub can take various forms. The central hub can be an atom (e.g., N or C) or an organic moiety (e.g., a benzene ring) to which one or more solubility-enhancing polymers are directly or indirectly attached. Suitably, the central hub of each soluble synthetic support has a molecular weight < 1500 Da. Most suitably, the central hub of each soluble synthetic support has a molecular weight < 300 Da (e.g., a carbon atom).

[0074] One or more solubility-enhancing polymers are optionally selected from the group consisting of: polyalkylene glycols, polyesters, polyamides, vinyl polymers, diene polymers, polyalkyleneimines, polyamidoamines, and polysiloxanes. Examples of solubility-enhancing polymers include polyethylene glycol (PEG), polypropylene glycol (PPG), polybutylene glycol, polydimethylsiloxane (PDMS), polybutadiene, polyisoprene, polystyrene, nylon, polyethyleneimine (PEI), polypropyleneimine, poly(L-lysine) (PLL), polymethyl methacrylate (PMMA), polyvinyl benzoic acid, polyhydroxystyrene, N-substituted glycine, and lactide-glycolide copolymers (PLGA). Suitably, one or more solubility-enhancing polymers are selected from the group consisting of: polyalkylene glycols (e.g., PEG), polyesters (e.g., lactide-glycolide copolymers), and polysiloxanes (e.g., PDMS). Even more suitably, one or more solubility-enhancing polymers are polyalkylene glycols. In a particular embodiment, one or more solubility-enhancing polymers are PEG. PEG is highly soluble in acetonitrile, which is an industrially favored solvent for coupling nucleotides to prepare oligonucleotides.

[0075] In one embodiment, each growing oligonucleotide is attached at one end to a solubility enhancing polymer via a linker having a molecular weight < 600 Da, and one or more solubility enhancing polymers are PEG. In one embodiment, each growing oligonucleotide is attached at one end to a solubility enhancing polymer via a linker having a molecular weight < 300 Da, and one or more solubility enhancing polymers are PEG.

[0076] One or more solubility enhancing polymers can be PEG derivatives such as polypropylene glycol, polyetheramine (e.g. or ), or (H2NCHMeCH2(OCHMeCH2) x (OCH2CH2) y OMe), which are commercially available in a range of lengths (x + y small to large) and hydrophobicities (x / y large = hydrophobic; x / y small = hydrophilic).

[0077] In one embodiment, the central hub of each soluble synthetic support has a molecular weight < 1500 Da, and one or more solubility enhancing polymers are PEG. In one embodiment, the central hub of each soluble synthetic support has a molecular weight < 300 Da (e.g. carbon atoms), and one or more solubility enhancing polymers are PEG.

[0078] The total molecular weight of one or more solubility enhancing polymers present within each molecule of the soluble synthetic support is ≥ 1000 Da (e.g., ≥ 2000 Da). Suitably, the total molecular weight of one or more solubility enhancing polymers present within each molecule of the soluble synthetic support is ≥ 4000 Da. As used herein, the molecular weight of a given solubility enhancing polymer refers to the mass of the polymeric (i.e., repeating) portion of the polymer. For example, for a PEG solubility enhancing polymer that is attached at one end to a central hub and at the other end to a growing oligonucleotide, the molecular weight of the solubility enhancing polymer is the mass of all -[CH2CH2O]- repeating units. For illustrative purposes, each molecule of the soluble synthetic support can contain a carbon atom (as the central hub) that is directly or indirectly attached to 4 PEG polymers (used as solubility enhancing polymers), where the molecular weight of each PEG polymer is 2500 Da (about 57 repeating -[CH2CH2O]- units) (i.e., a 10 kDa 4 - arm PEG - star support). Suitably, the total molecular weight of one or more solubility enhancing polymers present within each molecule of the soluble synthetic support is ≥ 8000 Da. More suitably, the total molecular weight of one or more solubility enhancing polymers present within each molecule of the soluble synthetic support is ≥ 9000 Da. More suitably, the total molecular weight of one or more solubility enhancing polymers present within each molecule of the soluble synthetic support is ≥ 9500 Da. Most suitably, the total molecular weight of one or more solubility enhancing polymers present within each molecule of the soluble synthetic support is ≥ 10,000 Da. Alternatively, the total molecular weight of one or more solubility enhancing polymers present within each molecule of the soluble synthetic support is ≥ 15,000 Da. Suitably, the total molecular weight of one or more solubility enhancing polymers present within each molecule of the soluble synthetic support is ≥ 20,000 Da. More suitably, the total molecular weight of one or more solubility enhancing polymers present within each molecule of the soluble synthetic support is ≥ 30,000 Da.

[0079] In one embodiment, one or more solubility enhancing polymers are PEG and the total molecular weight of one or more solubility enhancing polymers present within each molecule of the soluble synthetic support is ≥ 9000 Da. In one embodiment, one or more solubility enhancing polymers are PEG and the total molecular weight of one or more solubility enhancing polymers present within each molecule of the soluble synthetic support is ≥ 20,000 Da.

[0080] One or more solubility enhancing polymers can be a single solubility enhancing polymer or multiple (e.g., 2 - 12) solubility enhancing polymers, each having a molecular weight ≥ 1000 Da. Suitably, each solubility enhancing polymer has a molecular weight ≥ 2000 Da. More suitably, each solubility enhancing polymer has a molecular weight ≥ 2250 Da. Alternatively, each solubility enhancing polymer has a molecular weight ≥ 4000 Da. Suitably, each solubility enhancing polymer has a molecular weight ≥ 8000 Da. More suitably, each solubility enhancing polymer has a molecular weight ≥ 10,000 Da.

[0081] One or more solubility enhancing polymers can be 2 - 10 solubility enhancing polymers, each having a molecular weight ≥ 1000 Da, or each having a molecular weight ≥ 2000 Da, or each having a molecular weight ≥ 2250 Da, or each having a molecular weight ≥ 4000 Da, or each having a molecular weight ≥ 8000 Da, or each having a molecular weight ≥ 10,000 Da.

[0082] One or more solubility enhancing polymers can be 2 - 8 solubility enhancing polymers, each having a molecular weight ≥ 1000 Da, or each having a molecular weight ≥ 2000 Da, or each having a molecular weight ≥ 2250 Da, or each having a molecular weight ≥ 4000 Da, or each having a molecular weight ≥ 8000 Da, or each having a molecular weight ≥ 10,000 Da.

[0083] One or more solubility enhancing polymers can be 3 - 4 solubility enhancing polymers, each having a molecular weight ≥ 1000 Da, or each having a molecular weight ≥ 2000 Da, or each having a molecular weight ≥ 2250 Da, or each having a molecular weight ≥ 4000 Da, or each having a molecular weight ≥ 8000 Da, or each having a molecular weight ≥ 10,000 Da.

[0084] Each molecule of the soluble synthetic support can suitably have the structure according to Formula I:

[0085]

[0086] where

[0087] X represents a central hub (e.g., a carbon atom);

[0088] SEP represents a solubility enhancing polymer (e.g., PEG);

[0089] L is absent or is a linker (e.g., an organic moiety having a molecular weight < 600 Da or < 300 Da); and

[0090] n is 2 - 12 (e.g., 2 - 10, 2 - 8 or 3 - 4).

[0091] Each growing oligonucleotide can be attached at one end to L (if present), to SEP, or to X. Suitably, each growing oligonucleotide is attached at one end to L.

[0092] In one embodiment, one or more solubility enhancing polymers are 2 - 8 PEG polymers, each having a molecular weight of ≥1000 Da. In one embodiment, one or more solubility enhancing polymers are 3 - 4 PEG polymers, each having a molecular weight of ≥2250 Da (e.g., ≥2500 Da). In one embodiment, one or more solubility enhancing polymers are 2 - 8 PEG polymers, each having a molecular weight of ≥8000 Da.

[0093] In one embodiment, each molecule of the soluble synthetic support comprises 4 PEG polymers, each having a molecular weight of 2300 - 2800 Da. Suitably, each molecule of the soluble synthetic support comprises 4 growing oligonucleotides, each oligonucleotide being attached at one end to a PEG polymer. For example, the soluble synthetic support can be a 10 kDa 4 - arm PEG - star, a term used herein to denote a support comprising 4 PEG chains, each 2500 Da, radiating from a central hub carbon atom.

[0094] In one embodiment, each molecule of the soluble synthetic support comprises 4 PEG polymers, each having a molecular weight of 4000 - 6000 Da. Suitably, each molecule of the soluble synthetic support comprises 4 oligonucleotides, each oligonucleotide being attached at one end to a PEG polymer. For example, the soluble synthetic support can be a 20 kDa 4 - arm PEG - star, a term used herein to denote a support comprising 4 PEG chains, each 4000 - 6000 Da, radiating from a central hub carbon atom.

[0095] In one embodiment, each molecule of the soluble synthetic support comprises 4 PEG polymers, each having a molecular weight of 8000 - 12,000 Da. Suitably, each molecule of the soluble synthetic support comprises 4 growing oligonucleotides, each oligonucleotide being attached at one end to a PEG polymer. For example, the soluble synthetic support can be a 40 kDa 4 - arm PEG - star, a term used herein to denote a support comprising 4 PEG chains, each 10 kDa, radiating from a central hub carbon atom.

[0096] The method may also include one or more deprotection steps. In particular, the method may include deprotecting a protected uracil nucleobase and / or a protected thymine nucleobase. It should be understood that deprotection refers to removing the protecting groups from the protected uracil and / or protected thymine of the growing oligonucleotide. Any suitable deprotection technique may be used, such as ammonolysis, selective oxidation, selective reduction, oximation displacement, cleavage of the protecting group with an organometallic catalyst, cleavage of the protecting group with fluoride, cleavage of the protecting group with an acid, cleavage of the protecting group with a base, or a combination of one or more of these deprotection techniques. Deprotecting the protected uracil nucleobase and / or thymine nucleobase may occur during and / or after the step of growing the oligonucleotide. Most suitably, deprotecting the protected uracil nucleobase and / or thymine nucleobase occurs after the step of growing the oligonucleotide (i.e., deprotecting the fully grown oligonucleotide).

[0097] In some embodiments, deprotecting the protected uracil nucleobase and / or thymine nucleobase occurs after the step of growing the oligonucleotide, and each resulting oligonucleotide (i.e., deprotected) has a molecular weight of ≥2000 Da. In some embodiments, deprotecting the protected uracil nucleobase and / or thymine nucleobase occurs after the step of growing the oligonucleotide, and each oligonucleotide (i.e., deprotected) has a molecular weight of ≥5000 Da.

[0098] One or more deprotection steps may not occur until after the final coupling reaction. In such embodiments, the oligonucleotide (after full growth) may be protected (i.e., contain protecting groups). Accordingly, the present invention also relates to a liquid phase method for preparing an oligonucleotide, wherein the oligonucleotide contains at least one protected uracil nucleobase and / or at least one protected thymine nucleobase.

[0099] An oligonucleotide (after full growth) may contain uracil nucleobases, where at least 10% of the uracil nucleobases are protected uracil nucleobases. Protected uracil nucleobases are defined anywhere herein. Suitably, the oligonucleotide contains uracil nucleobases, where at least 30% of the uracil nucleobases are protected uracil nucleobases. More suitably, the oligonucleotide contains uracil nucleobases, where at least 50% of the uracil nucleobases are protected uracil nucleobases. Even more suitably, the oligonucleotide contains uracil nucleobases, where at least 70% of the uracil nucleobases are protected uracil nucleobases. More suitably, the oligonucleotide contains uracil nucleobases, where at least 90% of the uracil nucleobases are protected uracil nucleobases. Even more suitably, the oligonucleotide contains uracil nucleobases, where at least 95% of the uracil nucleobases are protected uracil nucleobases. Even more suitably, the oligonucleotide contains uracil nucleobases, where at least 99% of the uracil nucleobases are protected uracil nucleobases. Most suitably, the oligonucleotide contains at least one uracil nucleobase, where 100% (i.e., all) of the uracil nucleobases are protected uracil nucleobases.

[0100] An oligonucleotide (after full growth) may contain thymine nucleobases, where at least 10% of the thymine nucleobases are protected thymine nucleobases. Protected thymine nucleobases are defined anywhere herein. Suitably, the oligonucleotide contains thymine nucleobases, where at least 30% of the thymine nucleobases are protected thymine nucleobases. More suitably, the oligonucleotide contains thymine nucleobases, where at least 50% of the thymine nucleobases are protected thymine nucleobases. Even more suitably, the oligonucleotide contains thymine nucleobases, where at least 70% of the thymine nucleobases are protected thymine nucleobases. More suitably, the oligonucleotide contains thymine nucleobases, where at least 90% of the thymine nucleobases are protected thymine nucleobases. Even more suitably, the oligonucleotide contains thymine nucleobases, where at least 95% of the thymine nucleobases are protected thymine nucleobases. Even more suitably, the oligonucleotide contains thymine nucleobases, where at least 99% of the thymine nucleobases are protected thymine nucleobases. Most suitably, the oligonucleotide contains at least one thymine nucleobase, where 100% (i.e., all) of the thymine nucleobases are protected thymine nucleobases.

[0101] In one embodiment, the oligonucleotide (after full growth) contains thymine nucleobases and uracil nucleobases, wherein at least 10% of the thymine nucleobases are protected thymine nucleobases, and at least 10% of the uracil nucleobases are protected uracil nucleobases. Suitably, the oligonucleotide contains thymine nucleobases and uracil nucleobases, wherein at least 30% of the thymine nucleobases are protected thymine nucleobases, and at least 30% of the uracil nucleobases are protected uracil nucleobases. More suitably, the oligonucleotide contains thymine nucleobases and uracil nucleobases, wherein at least 50% of the thymine nucleobases are protected thymine nucleobases, and at least 50% of the uracil nucleobases are protected uracil nucleobases. Even more suitably, the oligonucleotide contains thymine nucleobases and uracil nucleobases, wherein at least 70% of the thymine nucleobases are protected thymine nucleobases, and at least 70% of the uracil nucleobases are protected uracil nucleobases. More suitably, the oligonucleotide contains thymine nucleobases and uracil nucleobases, wherein at least 90% of the thymine nucleobases are protected thymine nucleobases, and at least 90% of the uracil nucleobases are protected uracil nucleobases. Even more suitably, the oligonucleotide contains thymine nucleobases and uracil nucleobases, wherein at least 95% of the thymine nucleobases are protected thymine nucleobases, and at least 95% of the uracil nucleobases are protected uracil nucleobases. Even more suitably, the oligonucleotide contains thymine nucleobases and uracil nucleobases, wherein at least 99% of the thymine nucleobases are protected thymine nucleobases, and at least 99% of the uracil nucleobases are protected uracil nucleobases. Most suitably, the oligonucleotide contains at least one thymine nucleobase and at least one uracil nucleobase, wherein 100% (i.e., all) of the thymine nucleobases are protected thymine nucleobases, and 100% (i.e., all) of the uracil nucleobases are protected uracil nucleobases.

[0102] After the amount of protected uracil and / or protected thymine nucleobases in the oligonucleotide has been determined (after full growth - after the final coupling reaction), one or more of the protected uracil and / or protected thymine nucleobases of the oligonucleotide can be deprotected. Deprotection is as defined anywhere herein. Suitably, all of the protected uracil and / or protected thymine nucleobases of the oligonucleotide are deprotected.

[0103] The method can further include the step of cleaving the oligonucleotide from the soluble synthesis support after the oligonucleotide has fully grown.

[0104] Compared to RNA- or DNA-based oligonucleotides, the oligonucleotide and / or the growing oligonucleotide can have at least one backbone modification and / or at least one sugar modification and / or at least one base modification.

[0105] The oligonucleotide and / or growing oligonucleotide may contain at least 1 modified nucleotide residue. The modification may be at the 2'-position of the sugar moiety. The sugar modifications in the oligonucleotides and / or growing oligonucleotides described herein may include modified versions of the ribosyl moiety, such as 2'-O-modified RNA, such as 2'-O-alkyl or 2'-O-(substituted)alkyl, such as 2'-O-methyl, 2'-O-(2-cyanoethyl), 2'-O-(2-methoxy)ethyl (2'-MOE), 2'-O-(2-thiomethyl)ethyl, 2'-O-butyryl, 2'-O-propargyl, 2'-O-allyl, 2'-O-(3-amino)propyl, 2'-O-(3-(dimethylamino)propyl), 2'-O-(2-amino)ethyl, 2'-O-(2-(dimethylamino)ethyl); 2'-deoxy (DNA); 2'-O-(haloalkoxymethyl) (Arai K et al. Bioorg. Med. Chem. 2011, 21, 6285) e.g., 2'-O-(2-chloroethoxymethyl) (MCEM), 2'-O-(2,2-dichloroethoxymethyl) (DCEM); 2'-O-alkoxycarbonyl, e.g., 2'-O-[2-(methoxycarbonyl)ethyl] (MOCE), 2'-O-[2-(N-methylcarbamoyl)ethyl] (MCE), 2'-O-[2-(N,N-dimethylcarbamoyl)ethyl] (DCME); 2'-halogen, e.g., 2'-F, FANA (2'-F arabinonucleic acid); carbasugar and azasuarmodifications; 3'-O-alkyl, e.g., 3'-O-methyl, 3'-O-butyryl, 3'-O-propargyl; and derivatives thereof.

[0106] The sugar modification may be selected from the group consisting of: 2'-fluoro (2'-F), 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-MOE), and 2'-amino. Alternatively, the modification may be 2'-O-MOE. Other sugar modifications include "bridged" or "bicyclic" nucleic acids (BNA), such as locked nucleic acid (LNA), xylose-LNA, -L-LNA, β-D-LNA, cET (2'-O,4'-C constrained ethyl) LNA, cMOEt (2'-O,4'-C constrained methoxyethyl) LNA, ethylene-bridged nucleic acid (ENA), tricyclic DNA, unlocked nucleic acid (UNA); cyclohexenyl nucleic acid (CeNA), altritol nucleic acid (ANA), hexitol nucleic acid (HNA), fluorinated HNA (F-HNA), pyranosyl-RNA (p-RNA), 3'-deoxypyranosyl-DNA (p-DNA); morpholino (e.g., in PMO, PPMO, PMOPlus, PMO-X); and derivatives thereof.

[0107] The oligonucleotide and / or growing oligonucleotide may include other modifications such as peptide nucleic acid (PNA), boron-modified PNA, pyrrolidinooxy peptide nucleic acid (POPNA), ethylene glycol or glycerol nucleic acid (GNA), threose nucleic acid (TNA), acyclic threitol nucleic acid (aTNA), oligonucleotides with integrated bases and backbone (ONIBs), pyrrolidine-amide oligonucleotides (POMs); and derivatives thereof.

[0108] The oligonucleotide and / or growing oligonucleotide may comprise phosphorodiamidate morpholino oligomers (PMO), locked nucleic acid (LNA), peptide nucleic acid (PNA), bridged nucleic acid (BNA) such as (5)-cEt-BNA or SPIEGELMER.

[0109] Modifications may also be present in the nucleobases. Base modifications include modified forms of natural purine and pyrimidine bases (e.g., adenine, uracil, guanine, cytosine, and thymine), such as inosine, hypoxanthine, orotic acid, agmatidine, lysidine, 2-thiopyrimidines (e.g., 2-thiouracil, 2-thiothymine), G-clamp and its derivatives, 5-substituted pyrimidines (e.g., 5-methylcytosine, 5-methyluracil, 5-halouracil, 5-propynyluracil, 5-propynylcytosine, 5-aminomethyluracil, 5-hydroxymethyluracil, 5-aminomethylcytosine, 5-hydroxymethylcytosine, Super5T), 2,6-diaminopurine, 7-deazaguanine, 7-deazaadenine, 7-aza-2,6-diaminopurine, 8-aza-7-deazaguanine, 8-aza-7-deazaadenine, 8-aza-7-deaza-2,6-diaminopurine, Super G, Super A, and N4-ethylcytosine or their derivatives; N2-cyclopentylguanine (cPent-G), N2-cyclopentyl-2-aminopurine (cPent-AP), and N2-propyl-2-aminopurine (Pr-AP) or their derivatives; and degenerate or universal bases such as 2,6-difluorotoluene or missing bases such as abasic sites (e.g., 1-deoxyribose, 1-deoxy-2-O-methylribose; or pyrrolidine derivatives where the epoxy is replaced by nitrogen (aza-ribose)). Examples of derivatives of Super A, Super G, and Super T can be found in US6683173. cPent-G, cPent-AP, and Pr-AP show reduced immunostimulatory effects after incorporation into siRNA. 11

[0110] The nucleobase modification can optionally be selected from the group consisting of 5-methylpyrimidine, 7-deazaguanosine, and abasic nucleotides. Alternatively, the modification can be 5-methylcytosine.

[0111] The oligonucleotide and / or the growing oligonucleotide can include backbone modifications, such as modified forms of the phosphodiester present in RNA, such as phosphorothioate (PS), phosphorodithioate (PS2), phosphonoacetate (PACE), phosphonacetamide (PACA), thiophosphonoacetate, thiophosphonacetamide, phosphorothioate prodrug, H-phosphonate, methylphosphonate, methylthiophosphonate, methyl phosphate, methylthiophosphate, ethyl phosphate, ethylthiophosphate, boranophosphate, boranophosphorothioate, methylboranophosphate, methyl boranophosphorothioate, methyl boranophosphonate, methylboranophosphonothioate, and their derivatives. Another modification includes phosphoramidite, aminophosphate, N3'-PS'-aminophosphate, phosphorodiamidate, phosphorothiodiamidate, sulfamate, dimethyl sulfoxide, sulfonate, triazole, oxalyl, carbamate, methyleneimino (MMI), and thioacetamido nucleic acid (TANA); and their derivatives.

[0112] The backbone modification can optionally be selected from the group consisting of: phosphorothioate (PS), aminophosphate (PA), and phosphorodiamidate. The modified oligonucleotide can be a phosphorodiamidate morpholino oligomer (PMO). The PMO has a methylene morpholine ring backbone with phosphorodiamidate linkages. Suitably, the oligonucleotide and / or the growing oligonucleotide can have a phosphorothioate (PS) backbone.

[0113] The oligonucleotide and / or the growing oligonucleotide can contain a combination of two or more of the above-described modifications. Those skilled in the art will understand that there are many synthetic derivatives of oligonucleotides.

[0114] The oligonucleotide and / or the growing oligonucleotide can be a gapmer. The 5' and 3' flanks of the gapmer can comprise or consist of 2'-MOE-modified nucleotides. The spacer segment of the gapmer can comprise or consist of nucleotides having a hydrogen at the 2'-position of the sugar moiety, i.e., DNA-like. For example, the 5' and 3' flanks of the gapmer can consist of 2'-MOE-modified nucleotides, and the spacer segment of the gapmer can consist of nucleotides having a hydrogen at the 2'-position of the sugar moiety (i.e., deoxynucleotides). Alternatively, the 5' and 3' flanks of the gapmer can consist of 2'-MOE-modified nucleotides, and the spacer segment of the gapmer can consist of nucleotides having a hydrogen at the 2'-position of the sugar moiety (i.e., deoxynucleotides), and the linkage between all nucleotides is a phosphorothioate linkage.

[0115] According to a second aspect of the invention, there is provided an oligonucleotide obtainable directly or obtainable by the method of the first aspect.

[0116] The following numbered statements 1 to 62 are not claims but describe particular aspects and embodiments of the invention:

[0117] 1. A solution-phase method for preparing an oligonucleotide, the method comprising the steps of:

[0118] Growing the oligonucleotide by performing one or more sequential coupling reactions, each sequential coupling reaction increasing the length of the growing oligonucleotide by at least one nucleotide,

[0119] wherein the growing oligonucleotide comprises at least one protected uracil nucleobase and / or at least one protected thymine nucleobase, and

[0120] wherein the step of growing the oligonucleotide comprises one or more membrane filtration steps to separate the growing oligonucleotide.

[0121] 2. The solution-phase method according to statement 1, wherein the growing oligonucleotide is a single growing oligonucleotide or multiple (e.g., 2 - 12) growing oligonucleotides.

[0122] 3. The solution-phase method according to statement 1 or 2, wherein the growing oligonucleotide is 2 - 10 growing oligonucleotides.

[0123] 4. The solution-phase method according to statement 1, 2 or 3, wherein the growing oligonucleotide is 2 - 8 growing oligonucleotides.

[0124] 5. The solution-phase method according to any of the preceding statements, wherein the growing oligonucleotide is 3 - 4 growing oligonucleotides.

[0125] 6. The solution-phase method according to any one of the foregoing statements, wherein the molecular weight of each oligonucleotide is ≥ 1000 Da.

[0126] 7. The solution-phase method according to any one of the foregoing statements, wherein the molecular weight of each oligonucleotide is ≥ 2000 Da.

[0127] 8. The solution-phase method according to any one of the foregoing statements, wherein the molecular weight of each oligonucleotide is ≥ 3000 Da.

[0128] 9. The solution-phase method according to any one of the foregoing statements, wherein the molecular weight of each oligonucleotide is ≥ 5000 Da.

[0129] 10. The solution-phase method according to any one of the foregoing statements, wherein the growing oligonucleotide comprises:

[0130] (i) at least one protected uracil nucleobase, wherein the protected uracil nucleobase comprises a protecting group; and / or

[0131] (ii) at least one protected thymine nucleobase, wherein the protected thymine nucleobase comprises a protecting group.

[0132] 11. The solution-phase method according to any one of the foregoing statements, wherein the at least one protected uracil nucleobase comprises a protecting group and is linked to the growing oligonucleotide at the N1 position, and the protecting group is located at the N3 position or the O4 position.

[0133] 12. The solution-phase method according to any one of the foregoing statements, wherein the at least one protected thymine nucleobase comprises a protecting group and is linked to the growing oligonucleotide at the N1 position, and the protecting group is located at the N3 position or the O4 position.

[0134] 13. The solution-phase method according to any one of the foregoing statements, wherein the at least one protected uracil nucleobase comprises a protecting group and is linked to the growing oligonucleotide at the C5 position, and the protecting group is located at the N3 position or the O2 position.

[0135] 14. The solution-phase method according to any one of statements 11, 12 or 13, wherein each protecting group is independently an acid-labile protecting group, a base-labile protecting group, an ammonia-labile protecting group, an oxime-labile protecting group, an oxidation-labile protecting group, a hydrogenolysis-labile protecting group or a transition-metal-catalyzed cleavage protecting group.

[0136] 15. The solution-phase method according to any one of statements 11-14, wherein each protecting group is independently selected from the group consisting of 2,4,6-trimethylphenyl, 2-nitrophenyl, 2,4-dimethylphenyl, tolylcarbonyl, 2-(4-nitrophenyl)ethyl, 2-(4-cyanophenyl)ethyl, allyl, benzoyl (Bz), 2,4-dimethylbenzoyl, tert-butylbenzoyl (tert-BuBz) (e.g., p-tert-BuBz), acetyl (Ac), anisoyl (An) (e.g., p-An), 4-chlorobenzoyl, diphenylcarbamoyl, butylthiocarbonyl, 2-nitrobenzenethiol, 2,4-dinitrobenzenethiol, 2-nitro-4-methylbenzenethiol, and triphenylmethylthiol.

[0137] 16. The solution-phase method according to any one of statements 11-15, wherein each protecting group is independently selected from the group consisting of benzoyl (Bz), 2,4-dimethylbenzoyl, tert-butylbenzoyl (tert-BuBz) (e.g., p-tert-BuBz), and anisoyl (An) (e.g., p-An).

[0138] 17. The solution-phase method according to any one of the foregoing statements, wherein the step of growing the oligonucleotide comprises a single coupling reaction.

[0139] 18. The solution-phase method according to any one of the foregoing statements, wherein the step of growing the oligonucleotide comprises performing two or more sequential coupling reactions.

[0140] 19. The solution-phase method according to any one of the foregoing statements, wherein the step of growing the oligonucleotide comprises performing three or more sequential coupling reactions.

[0141] 20. The solution-phase method according to any one of the foregoing statements, wherein the step of growing the oligonucleotide comprises performing four or more sequential coupling reactions.

[0142] 21. The solution-phase method according to any one of the foregoing statements, wherein the step of growing the oligonucleotide comprises performing six or more sequential coupling reactions.

[0143] 22. The solution-phase method according to any one of the foregoing statements, wherein the step of growing the oligonucleotide comprises performing ten or more sequential coupling reactions.

[0144] 23. The solution-phase method according to any one of the foregoing statements, wherein the step of growing the oligonucleotide comprises performing fifteen or more sequential coupling reactions.

[0145] 24. The solution-phase method according to any one of the foregoing statements, wherein each sequential coupling reaction increases the length of the growing oligonucleotide by one nucleotide.

[0146] 25. The solution-phase method according to any one of the foregoing statements, wherein the step of growing the oligonucleotide is carried out in at least one organic solvent.

[0147] 26. The solution-phase method according to any one of the foregoing statements, wherein the step of growing the oligonucleotide is carried out in acetonitrile, optionally mixed with another organic solvent.

[0148] 27. The solution-phase method according to any one of the foregoing statements, wherein the step of growing the oligonucleotide is carried out in a mixture of acetonitrile and sulfolane (e.g., 4:1 v / v).

[0149] 28. The solution-phase method according to any one of statements 1-26, wherein the step of growing the oligonucleotide is carried out in acetonitrile (i.e., pure acetonitrile).

[0150] 29. The solution-phase method according to any one of the foregoing statements, wherein the step of growing the oligonucleotide is carried out in pure acetonitrile or a mixture of acetonitrile and sulfolane (e.g., 4:1 v / v).

[0151] 30. The solution-phase method according to any one of the foregoing statements, wherein the membrane filtration is membrane diafiltration.

[0152] 31. The solution-phase method according to any one of the foregoing statements, wherein the membrane filtration is organic solvent nanofiltration (OSN) or ultrafiltration (UF).

[0153] 32. The solution-phase method according to any one of the foregoing statements, wherein each coupling reaction is subjected to membrane filtration one or two times.

[0154] 33. The solution-phase method according to any one of the foregoing statements, wherein the membrane filtration is carried out as part of each sequential coupling reaction carried out in the step of growing the oligonucleotide.

[0155] 34. The solution-phase method according to any one of the foregoing statements, wherein the step of growing the oligonucleotide and one or more membrane filtration steps for separating the grown oligonucleotide are carried out in the same solvent.

[0156] 35. The solution-phase method according to any one of the foregoing statements, wherein the membrane for carrying out the membrane filtration is formed of or comprises a polymeric material suitable for manufacturing a microfiltration membrane, ultrafiltration membrane, nanofiltration membrane or reverse osmosis membrane, including polyethylene, polypropylene, polytetrafluoroethylene (PTFE), polyvinylidene fluoride (PVDF), polysulfone, polyethersulfone, polyacrylonitrile, polyamide, polyester, polyimide, polyetherimide, cellulose acetate, polyaniline, polypyrrole, polybenzimidazole, polyetheretherketone (PEEK) and mixtures thereof.

[0157] 36. The solution-phase method according to any one of the foregoing statements, wherein the membrane filtration is carried out using a crosslinked polybenzimidazole membrane (e.g., an integrally skinned asymmetric crosslinked polybenzimidazole membrane) or a polyetheretherketone membrane.

[0158] 37. The solution-phase method according to any one of the foregoing statements, wherein the growing oligonucleotide is attached at one end to a soluble synthetic support.

[0159] 38. The solution-phase method according to statement 37, wherein the soluble synthetic support comprises a central hub and one or more solubility enhancing polymers, each of the one or more solubility enhancing polymers being attached to the central hub.

[0160] 39. The solution-phase method according to statement 38, wherein the one or more solubility enhancing polymers are a single solubility enhancing polymer or multiple (e.g., 2 - 12) solubility enhancing polymers.

[0161] 40. The solution-phase method according to statement 38 or 39, wherein the one or more solubility enhancing polymers are 2 - 10 solubility enhancing polymers.

[0162] 41. The solution-phase method according to statement 38, 39 or 40, wherein the one or more solubility enhancing polymers are 2 - 8 solubility enhancing polymers.

[0163] 42. The solution-phase method according to any one of statements 38 - 41, wherein the one or more solubility enhancing polymers are 3 - 4 solubility enhancing polymers.

[0164] 43. The solution-phase method according to any one of statements 38 - 42, wherein the central hub is an atom (e.g., N or C) or an organic moiety (e.g., a benzene ring) to which the one or more solubility enhancing polymers are directly or indirectly attached.

[0165] 44. The solution-phase method according to any one of statements 38 - 43, wherein the central hub of each soluble synthetic support has a molecular weight of < 1500 Da (e.g., < 300 Da).

[0166] 45. The solution-phase method according to any one of statements 38 - 44, wherein the one or more solubility enhancing polymers are selected from the group consisting of polyalkylene glycols, polyesters, polyamides, vinyl polymers, diene polymers, polyalkylimines, polyamidoamines, and polysiloxanes.

[0167] 46. The solution-phase method according to any one of statements 38-45, wherein the one or more solubility enhancing polymers are selected from the group consisting of: polyethylene glycol (PEG), polypropylene glycol (PPG), polybutylene glycol, polydimethylsiloxane (PDMS), polybutadiene, polyisoprene, polystyrene, nylon, polyethyleneimine (PEI), polypropyleneimine, poly(L-lysine) (PLL), polymethyl methacrylate (PMMA), polyvinyl benzoic acid, polyhydroxystyrene, N-substituted glycine, and lactide-glycolide copolymer (PLGA).

[0168] 47. The solution-phase method according to any one of statements 38-46, wherein the one or more solubility enhancing polymers are PEG.

[0169] 48. The solution-phase method according to any one of statements 38-47, wherein the total molecular weight of the one or more solubility enhancing polymers present within each molecule of the soluble synthetic support is ≥1000 Da, ≥2000 Da, ≥4000 Da, ≥8000 Da, ≥9000 Da, ≥9500 Da, ≥10,000 Da, ≥15,000 Da, ≥20,000 Da, or ≥30,000 Da.

[0170] 49. The solution-phase method according to any one of statements 38-48, wherein the molecular weight of each solubility enhancing polymer is ≥1000 Da, ≥2000 Da, ≥2250 Da, ≥4000 Da, ≥8000 Da, or ≥10,000 Da.

[0171] 50. The solution-phase method according to any one of statements 38-49, wherein each molecule of the soluble synthetic support has a structure according to Formula I:

[0172]

[0173] wherein

[0174] X represents a central hub;

[0175] SEP represents a solubility enhancing polymer;

[0176] L is absent or is a linker; and

[0177] n is 2-12 (e.g., 2-10, 2-8, or 3-4).

[0178] 51. The solution-phase method according to any one of the foregoing statements, wherein the method further comprises one or more deprotection steps.

[0179] 52. The solution-phase method according to statement 51, wherein the one or more deprotection steps include selective oxidation, selective reduction, oximation, cleavage of the protecting group with an organometallic catalyst, cleavage of the protecting group with fluoride, cleavage of the protecting group with an acid, cleavage of the protecting group with a base, or a combination thereof.

[0180] 53. The solution-phase method according to statement 51 or 52, wherein the one or more deprotection steps occur during and / or after the step of growing the oligonucleotide.

[0181] 54. The solution-phase method according to statement 51, 52 or 53, wherein the one or more deprotection steps occur after the step of growing the oligonucleotide.

[0182] 55. The solution-phase method according to any one of the foregoing statements, wherein the oligonucleotide comprises at least one protected uracil nucleobase and / or at least one protected thymine nucleobase.

[0183] 56. The solution-phase method according to statement 55, wherein the oligonucleotide comprises uracil nucleobases, wherein at least 10%, 30%, 50%, 70%, 90%, 95%, 99% or 100% of the uracil nucleobases are protected uracil nucleobases.

[0184] 57. The solution-phase method according to statement 55 or 56, wherein the oligonucleotide comprises thymine nucleobases, wherein at least 10%, 30%, 50%, 70%, 90%, 95%, 99% or 100% of the thymine nucleobases are protected thymine nucleobases.

[0185] 58. The solution-phase method according to statement 55, 56 or 57, wherein one or more protected uracil nucleobases and / or protected thymine nucleobases of the oligonucleotide are subjected to a deprotection step.

[0186] 59. The solution-phase method according to any one of statements 55-58, wherein all protected uracil nucleobases and / or protected thymine nucleobases of the oligonucleotide are subjected to a deprotection step.

[0187] 60. The solution-phase method according to statement 58 or 59, wherein the deprotection step includes selective oxidation, selective reduction, oximation, cleavage of the protecting group with an organometallic catalyst, cleavage of the protecting group with fluoride, cleavage of the protecting group with an acid, cleavage of the protecting group with a base, or a combination thereof.

[0188] 61. The solution-phase method according to any one of statements 38-50, wherein the method further comprises a step of cleaving the oligonucleotide from the soluble synthesis support.

[0189] An oligonucleotide obtained directly or obtainable by the solution phase method according to any one of the preceding claims.

[0190] Examples

[0191] One or more examples of the invention will now be described with reference to the accompanying drawings, for illustrative purposes only:

[0192] Figure 1 The protected phosphoramidite structure is shown and its solubility in acetonitrile was evaluated.

[0193] Figure 2 The synthesis of oligonucleotides on 10 kDa polydisperse 4-arm stars and 40 kDa polydisperse 4-arm stars is shown.

[0194] Figure 3 Viscosity measurements of Comparative Example 2 and Examples 2 and 3 at a) 30 °C; and b) 0 °C are shown. Measurements were made starting from HO-pentamer-PEG star 8 during the growth of the oligonucleotide.

[0195] Example 1

[0196] A series of 5'-O-Dmtr-nucleoside-3'-O-(cyanoethyl-N,N-diisopropyl) phosphoramidites ( Figure 1 , Compounds 1-5) were studied to determine their suitability in solution phase oligonucleotide synthesis in acetonitrile. Each compound (approx. 0.5 g) was placed in a flask with a magnetic stir bar and the flask was immersed in an oil bath maintained at 30 °C. Acetonitrile (0.200 mL) was added to the flask and the contents were stirred for 2 min. Thereafter, acetonitrile (0.050 mL) was added again and the contents were stirred for 2 min after each addition. The volume of acetonitrile required to prepare a clear solution was recorded. The solubilities thus determined are recorded in Table 1.

[0197] Table 1 - Properties of Compounds 1-5 in Acetonitrile

[0198] Compound No. Base, B Protecting group, PG R Solubility, g / L Solubility, mol / L 1a U None OMe 0.05 0.07 1b U Bz OMe 1.66 1.92 1c U p-tert-BuBz OMe 1.60 1.74 1d U p-An OMe 2.17 2.42 2a T None H 0.86 1.16 2b T Bz H 1.30 1.53 3 C Ac OMe 1.04 1.30 4 A Bz OMe 1.07 1.21 5 G Ibu OMe 1.11 1.28

[0199] Compound 1a is remarkable for its low solubility. While 1a only reaches a solubility of 0.05 M, all other compounds have solubilities greater than 1 M in acetonitrile. After 1a (the unprotected U derivative), the closely related unprotected T derivative (compound 2a) is the least soluble among the remaining building blocks 2 to 5. A series of N3-acylated derivatives were prepared from 5'-Dmtr-mU (Peyrat and Xie, “Synthesis of Thymidine Dimers from 5'-O-Aminothymidine”, Synthesis, 2012, 44, 1718 - 1724) and the intermediate nucleosides were converted into the corresponding phosphoramidites 1b-d using chloro-2-cyanoethoxy-N,N-diisopropylaminophosphine (Sproat et al., “New and Convenient Protection System for Pseudouridine, Highly Suitable for Oligoribonucleotide Synthesis”, J. Chem. Soc., Perkin Trans. 1, 1994, 3423 - 3429). The solubility range of these substances is about 25 to 35 times higher than that of compound 1a with unprotected N3. Even for the more soluble phosphoramidite 2a of unprotected T, protection of N3 (compound 2b) increased the solubility by more than 30%. In theory, the increase in solubility of the pyrimidine building blocks 1 and 2 resulting from N3 protection extends to the protected oligonucleotides containing them.

[0200] Comparative Example 2

[0201] Figure 2 The 21-mer sequence 9 is shown. Due to its unusually high proportion of 2'-methyluridine (mU) residues, this sequence was chosen as a test sequence for the effect of protecting U and T. It was also found that sequence 9 produced a very viscous solution when loaded onto a large soluble PEG-star (see below), which made organic solvent nanofiltration (OSN) impractical as it severely reduced the mass transfer efficiency and was difficult to pump the viscous liquid.

[0202] The soluble synthetic support PEG-40k(SarH)4 (m ~ 255) was condensed with Dmtr-mU succinate (2.5 equivalents per arm) using dicyclohexylcarbodiimide (DCC) and hydroxybenzotriazole (HOBt). The solution was transferred by filtration into a single-stage membrane separation synthesizer equipped with 5 circular PBI16-DBX-M2005 membrane chambers (each 52 cm 2) to reach a final oligonucleotide concentration of 10 mM. The crude material was percolated in pure acetonitrile to remove low MW fragments, with 4 system volumes (or percolation volume, DV) of solvent percolated through, and the system maintained at 30 °C. Then detritylation was carried out using 2.5% trifluoroacetic acid (TFA) and an excess of cation scavenger such as dodecyl mercaptan inside the synthesizer. The detritylation was quenched with an excess of 3-methylpyridine and percolation continued in pure acetonitrile (6DV, final permeation flux 10 mL / min) until no residual succinate building blocks were detected, yielding pure PEG-40k(SarSuc-mU-OH)4,6,B 1 = mU, m~225.

[0203] The loaded nucleoside-star 6 was subjected to one cycle of chain extension: the nucleoside phosphoramidite 5 (R = MeO, 2 equivalents per arm) was added to the synthesizer, activated with DCI, and quenched with CneOH after 20 min. After 2 min, oxidation was initiated with an excess of camphorsulfonyloxaziridine (CSO) in pure acetonitrile, and the reaction proceeded for 1 h. After completion, low MW reagent fragments were removed by percolation (4DV), and the temporary 5'-Dmtr protecting group was deblocked with 2.5% TFA as described above (constant temperature 30 °C). The detritylation was quenched with an excess of 3-methylpyridine and percolation continued (6DV, final permeation flux 10 mL / min) until no building block fragments remained.

[0204] This cycle was repeated, except that only 1.5 equivalents of phosphoramidite were used per cycle from the trimer onwards to the end of the experiment. The reagents were injected into the synthesizer in the order required by the construction sequence 9, and excess reagents and fragments were removed by OSN as described above. For this comparative example, the unprotected mU 1a and dT 2a building blocks were used for the appropriate chain extensions. Starting from the hydroxy-pentamer-PEG-star ( Figure 2 , compound 8, n = 4), the viscosity of the solution was measured at 30 °C (normal synthesizer operating temperature) and 0 °C, and then fresh phosphoramidite was immediately added. Typically, after maximum percolation, this is the point in the cycle with the lowest contaminant concentration.

[0205] As the oligonucleotide length on the PEG-star support increased, the permeation flux decreased slowly (from 14 mL / min at the trimer) and the viscosity increased ( Figure 3)。At the 11-mer-star, both the viscosity of the retentate and the rejection of the building blocks increase, making further processing difficult, although the total permeate flux remains controllable (7.4 mL / min). Therefore, the solvent was changed to acetonitrile-sulfolane 4:1 v / v. Although the mixture of acetonitrile-sulfolane is more viscous than pure acetonitrile, the added sulfolane reduces the overall viscosity of the longer oligonucleotide-PEG-star solution. The chain growth cycle continues, but at the 17-mer-star, the permeation flux drops to an unrealistic level (2.4 mL / min), which is associated with an increase in viscosity. Therefore, the membrane was switched to the more porous PBI14-DBX-M2005, which slightly increased the permeation flux (4.4 mL / min) at the 18-mer, making it difficult to continue running to the full-length 21-mer. For chain growth 20 and 21, hydrogenated xanthogen (XH) dissolved in pyridine was used instead of CSO to effect sulfur transfer, followed by diafiltration after 5 minutes.

[0206] After detritylation of the 21-mer-star, the solvent was changed to acetonitrile-pyridine 9:1 v / v; the oligonucleotide-star is highly soluble in pyridine and a corresponding large decrease in viscosity was observed ( Figure 3 )。Notably, if this full-length 21-mer-PEG-star is dispersed in pure acetonitrile, its viscosity is ca. 530 cP. The final oligonucleotide-star was washed off the synthesizer and the solvent was evaporated under reduced pressure. The crude oligonucleotide-star was deprotected in a mixture of concentrated ammonia and 3 vol% diethylamine (DEA) at 35 °C for 18 h. The solvent was evaporated the next day and then co-evaporated from acetonitrile (x3), and the full-length 21-mer was isolated by trituration with acetonitrile, with a UV purity of 44%.

[0207] Example 2

[0208] The soluble synthesis support PEG-10k(SarH)4 was condensed with Dmtr-mU succinate (2.5 equivalents per arm) using DCC and HOBt. The solution was transferred by filtration to a single-stage membrane separation synthesizer equipped with 5 circular PBI17-DBX-M2005 membrane chambers (each 52 cm 2), reaching a final oligonucleotide concentration of 20 mM - twice that of Comparative Example 2. The crude material was percolated in acetonitrile - sulfolane at 4:1 v / v to remove low MW fragments, percolating 4 DV of solvent with the system maintained at 30 °C. Then detritylation was carried out using 2.5% TFA and an excess of cation scavenger such as dodecyl mercaptan inside the synthesizer. The detritylation was quenched with an excess of 3 - methylpyridine and percolation continued in acetonitrile - sulfolane at 4:1 v / v (6 DV, final flux 19 mL / min) until no residual succinate building blocks were detected, yielding pure PEG - 10k(SarSuc - mU - OH)4,6,B 1 = mU, m ~ 56.

[0209] The loaded nucleoside - star 6 was subjected to one cycle of chain extension: The nucleoside phosphoramidite 5 (R = MeO, 2 equivalents per arm) was added to the synthesizer, activated with DCI, and quenched with CneOH after 20 min. After 2 min, oxidation was initiated with an excess of CSO in pure acetonitrile and the reaction proceeded for 1 hour. After completion, low MW reagent fragments were removed by percolation (4 DV), and the temporary 5'-Dmtr protecting group was deblocked with 2.5% TFA as described above (constant temperature 30 °C). The detritylation was quenched with an excess of 3 - methylpyridine and percolation continued (6 DV, final flux 17 mL / min) until no building block fragments remained, using acetonitrile - sulfolane at 4:1 v / v throughout.

[0210] The cycle was repeated, except that only 1.5 equivalents of phosphoramidite were used per cycle starting from the trimer until the end of the experiment. The reagents were injected into the synthesizer in the order required for construct sequence 9, and excess reagents and fragments were removed by OSN as described above. For this example, N3 - protected mU Bz 1b and dT Bz 2b building blocks were used for the appropriate chain extension. Only at the 14 - mer - star did a decrease in permeation flow rate (5.8 mL / min) and an increase in building block retention become apparent. The reaction continued after changing the membrane disk (8.2 mL / min at 15 - mer, 6.4 mL / min at 21 - mer). Sulfur transfer to the 20 - mer and 21 - mer oligonucleotide - stars was carried out with an acetonitrile solution of 3 - phenyl - 1,2,4 - dithiazol - 5 - one within 5 minutes.

[0211] Starting from the hydroxy - pentamer - PEG - star ( Figure 2 , compound 8, m = 4), the viscosity of the solution was measured at 30 °C (normal synthesizer operating temperature) and 0 °C, and then fresh phosphoramidite was immediately added. Different from Comparative Example 2, a low viscosity was maintained throughout the run, with the viscosity rising slowly and eventually reaching only 10 cP ( Figure 3a). This is surprising since not only is a much smaller solubilizing PEG-star (10 kDa as opposed to 40 kDa in Comparative Example 2) used in this example, but also the oligonucleotide concentration is doubled. In fact, even cooling to 0 °C only results in a small increase in viscosity ( Figure 3 b), which means that if desired, the synthesizer can be cooled below room temperature.

[0212] After detritylation of the 21-mer-star, the solvent was changed to 9:1 v / v acetonitrile - pyridine and the final oligonucleotide-star was washed off the synthesizer. The solvent was then evaporated under reduced pressure. The crude oligonucleotide-star was deprotected in a mixture of concentrated ammonia and 3 vol% DEA at 35 °C for 18 h. The solvent was evaporated the next day and then co-evaporated from acetonitrile (×3), and the full-length 21-mer 9 was isolated by trituration with acetonitrile, with a UV-purity of 64%.

[0213] Example 3

[0214] The soluble synthesis support PEG-10k(SarH)4 was condensed with Dmtr-mU succinate (2.5 equivalents per arm) using DCC and HOBt. The solution was transferred by filtration to a single-stage membrane separation synthesizer equipped with 5 circular PBI17-DBX-M2005 membrane chambers (each 52 cm 2 ), to reach a final oligonucleotide concentration of 20 mM. The crude material was percolated in pure acetonitrile (see 4:1 v / v acetonitrile - sulfolane in Example 2) to remove low MW fragments, percolating 4 DV of solvent, with the system maintained at 30 °C. Then detritylation was carried out using 2.5% TFA and an excess of cation scavenger, such as dodecyl mercaptan, inside the synthesizer. The detritylation was quenched with an excess of 3-methylpyridine and percolation was continued in pure acetonitrile (6 DV, final permeate flux 20 mL / min) until no residual succinate building blocks were detected, to obtain pure PEG-10k(SarSuc-mU-OH)4,6,B 1 = mU, m ∼ 56.

[0215] Perform a chain extension cycle on the loaded nucleoside-star 6: Add the nucleoside phosphoramidite 5 (R = MeO, 2 equivalents per arm) to the synthesizer, activate with DCI, and quench with CneOH after 20 min. After 2 min, initiate oxidation with excess CSO in pure acetonitrile and let the reaction proceed for 1 h. After completion, remove the low MW reagent fragments by filtration (4DV), and then deblock the temporary 5'-Dmtr protecting group with 2.5% TFA as described above (constant temperature 30 °C). Quench the detritylation with excess 3-methylpyridine and continue filtration (6DV, final flux 17 mL / min) until no building block fragments remain, using pure acetonitrile throughout.

[0216] Repeat the cycle, except that only 1.5 equivalents of phosphoramidite are used per cycle starting from the trimer until the end of the experiment. Inject the reagents into the synthesizer in the order required by the construction sequence 9, and remove the excess reagents and fragments by OSN as described above. For this example, N3-protected mU Bz 1b and dT Bz 2b building blocks are used for the appropriate chain extension. Only at the 13-mer star, the decline in permeation flux (6.5 mL / min) and the increase in building block retention become obvious. The reaction continues after changing the membrane disk (10 mL / min at the 14-mer and 5.1 mL / min at the 21-mer). Perform sulfur transfer on the 20-mer and 21-mer oligonucleotide-stars with XH in pyridine within 5 min.

[0217] Starting from the hydroxy-pentamer-PEG-star ( Figure 2 , compound 8, m = 4), measure the viscosity of the solution at 30 °C (normal synthesizer operating temperature) and 0 °C, and then immediately add the new phosphoramidite. In the early chain growth cycles, the viscosity is significantly lower than that in a 4:1 acetonitrile-sulfolane solution ( Figure 3 a), but as the length increases, the viscosity begins to approach that of Example 2. When cooled to 0 °C, the viscosity difference between Example 2 and 3 is not obvious ( Figure 3 b).

[0218] After detritylation and filtration of the 21-mer star, wash the final oligonucleotide-star off the synthesizer and evaporate the solvent under reduced pressure. Deprotect the crude oligonucleotide-star in a mixture of concentrated ammonia and 3 vol% DEA at 35 °C for 18 h. Evaporate the solvent the next day and then co-evaporate from acetonitrile (x3), and isolate the full-length 21-mer 9 by trituration with acetonitrile, with a UV purity of 54%.

[0219] Example 4

[0220] Condense the soluble synthetic support PEG-10k(SarH)4 with Dmtr-mA Bz succinate (2.5 equivalents per arm) using DCC and HOBt. Transfer the solution to a single-stage membrane separation synthesizer by filtration. The synthesizer is equipped with 5 circular PBI17-DBX-M2005 membrane chambers (each 52 cm 2 ), achieving a final oligonucleotide concentration of 10 mM. Percolate the crude material in pure acetonitrile to remove low MW fragments, permeating 4 DV of solvent with the system maintained at 30 °C. Then perform detritylation using 2.5% TFA and an excess of cation scavenger such as dodecyl mercaptan inside the synthesizer. Quench the detritylation with an excess of pyridine and continue percolation in pure acetonitrile (6 DV, final permeate flux 20 mL / min) until no residual succinate building blocks are detected, obtaining pure PEG-10k(SarSuc-mABz-OH)4,6,B 1 =A Bz , m~56.

[0221] Then perform chain extension on the loaded nucleoside-star 6: Add the nucleoside phosphoramidite 1b or 1c (B 2 =U Bz or U tBuBz , R = MeO, 2 equivalents per arm) to the synthesizer and activate with DCI. Quench with CneOH after 20 min. Initiate sulfur transfer with XH in an excess of pyridine after 2 min and let the reaction proceed for 20 min. After completion, remove low MW reagent fragments by percolation (4 DV) and deblock the temporary 5'-Dmtr protecting group with 2.5% TFA as described above (constant temperature 30 °C). Quench the detritylation with an excess of 3-methylpyridine and continue percolation (6 DV, final flux 17 mL / min) until no building block fragments remain, using acetonitrile-sulfolane 4:1 v / v throughout.

[0222] Repeat this cycle with phosphoramidite 1b or 1c to construct the sequence mA(mU)7 10, except that only 1.5 equivalents are used per cycle starting from the trimer to the end of the experiment, and remove excess reagents and fragments by OSN as described above. In both cases, the oligonucleotide-star, PEG-10k[mA Bz (mU PG)7]4, PG = Bz or tBuBz, which is kept in solution throughout the synthesis and has a low viscosity. After detritylation and filtration of the octamer-star, the final oligonucleotide-star is washed off the synthesizer and the solvent is evaporated under reduced pressure. The crude oligonucleotide-star is initially treated with DEA-DMF (3:7 v / v) and the solvent is evaporated. The residue is then redissolved in concentrated ammonia and heated in a sealed tube at 55 °C for 18 h. The solvent is evaporated the next day and then co-evaporated from acetonitrile (x3), and the full-length octamer 10 is isolated by trituration with acetonitrile. The octamer prepared with phosphoramidite 1b has a UV-purity of 75%; the octamer prepared with phosphoramidite 1c has a UV-purity of 88%. Bz The octamer prepared with phosphoramidite 1b has a UV-purity of 75%; the octamer prepared with phosphoramidite 1c has a UV-purity of 88%. tBuBz The octamer prepared with phosphoramidite 1c has a UV-purity of 88%.

[0223] 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.

[0224] References

[0225] 1. US8,664,357

[0226] 2. US9,127,123

[0227] 3. US10,239,996

[0228] 4. EP3347402

[0229] 5. 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

[0230] 6. 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

[0231] 7. 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

[0232] 8. Yeo J, Peeva L, Chung S, Gaffney P, Kim D, Luciani C, Tsukanov S, Siebert K, Kopach M, Albericio F, Livingston A “Liquid Phase Peptide Synthesis by One Pot Nanostar Sieving(PEPSTAR)”; Angew. Chem. Int. Ed. 2021, 60, pp7786 - 7795

[0233] 9. PEG and derivatives of PEG have also been synthesised using membrane separation, as described by Dong R., Liu R., Gaffney P.R.J., Schaepertoens M., Marchetti P., Williams C.M., Chen R. and Livingston A.G. “Sequence-defined multifunctional polyethers via liquid-phase synthesis with molecular sieving” Nature Chemistry (2019) 11 pp. 136-145

[0234] 10. WO 2016 / 188835 A1

[0235] 11. Peacock H. et al. J. Am. Chem. Soc. (2011), 133, 9200

[0236] 12. Baran et al. Unlocking P(V): Reagents for chiral phosphorothioate synthesis, Science, Vol 361, 1234-1238. DOI:10.1126 / science.aau3369

Claims

1. A solution-phase method for preparing oligonucleotides, the method comprising the following steps: Growing an oligonucleotide by performing one or more sequential coupling reactions, each sequential coupling reaction increasing the length of the growing oligonucleotide by at least one nucleotide, wherein the growing oligonucleotide comprises at least one protected uracil nucleobase and / or at least one protected thymine nucleobase, and wherein the step of growing the oligonucleotide comprises one or more membrane filtration steps to separate the growing oligonucleotide.

2. The solution-phase method according to claim 1, wherein the molecular weight of the oligonucleotide ≥ 1000 Da.

3. The solution-phase method according to claim 1 or 2, wherein the molecular weight of the oligonucleotide ≥ 5000 Da.

4. The solution-phase method according to claim 1, 2 or 3, wherein the growing oligonucleotide is attached to a soluble synthetic support at one end.

5. The solution-phase method according to claim 4, wherein the soluble synthetic support comprises a central hub and one or more solubility-enhancing polymers, each of the one or more solubility-enhancing polymers being attached to the central hub.

6. The solution-phase method according to claim 5, wherein the one or more solubility-enhancing polymers are selected from the group consisting of: polyalkylene glycols, polyesters, polyamides, vinyl polymers, diene polymers, polyalkyleneimines, polyamidoamines and polysiloxanes.

7. The solution-phase method according to claim 5 or 6, wherein the one or more solubility-enhancing polymers are polyalkylene glycols (e.g., PEG).

8. The solution-phase method according to claim 5, 6 or 7, wherein the total molecular weight of the one or more solubility-enhancing polymers present within each molecule of the soluble synthetic support ≥ 4000 Da.

9. The solution-phase method according to any one of claims 5-8, wherein the total molecular weight of the one or more solubility-enhancing polymers present within each molecule of the soluble synthetic support ≥ 20,000.

10. The solution-phase method according to any one of claims 5-9, wherein the one or more solubility-enhancing polymers are a single solubility-enhancing polymer or multiple (e.g., 2-12) solubility-enhancing polymers.

11. The solution-phase method according to any one of claims 5-10, wherein the molecular weight of each solubility-enhancing polymer ≥ 1000 Da.

12. The solution-phase method according to any one of the preceding claims, wherein the step of growing the oligonucleotide comprises performing two or more sequential coupling reactions.

13. The solution-phase method according to any one of the preceding claims, wherein the step of growing the oligonucleotide comprises performing six or more sequential coupling reactions.

14. The solution-phase method according to any one of the preceding claims, wherein the step of growing the oligonucleotide can be carried out in an organic solvent composition comprising at least one organic solvent.

15. The solution-phase method according to any one of the preceding claims, wherein the step of growing the oligonucleotide is carried out in pure acetonitrile or a mixture of acetonitrile and sulfolane (e.g., 4:1 v / v).

16. The solution-phase method according to any one of the preceding claims, wherein the grown oligonucleotide comprises: at least one protected uracil nucleobase, wherein the protected uracil nucleobase comprises a protecting group; and / or at least one protected thymine nucleobase, wherein the protected thymine nucleobase comprises a protecting group.

17. The solution-phase method according to claim 16, wherein each protecting group is independently an acid-labile protecting group, a base-labile protecting group, an ammonia-labile protecting group, an oxime-labile protecting group, an oxidation-labile protecting group, a hydrogenolysis-labile protecting group, or a transition-metal-catalyzed cleavage protecting group.

18. The solution-phase method according to claim 16 or 17, wherein each protecting group is independently selected from the group consisting of 2,4,6-trimethylphenyl, 2-nitrophenyl, 2,4-dimethylphenyl, tolylcarbonyl, 2-(4-nitrophenyl)ethyl, 2-(4-cyanophenyl)ethyl, allyl, benzoyl (Bz), 2,4-dimethylbenzoyl, tert-butylbenzoyl (tert-BuBz), acetyl (Ac), anisoyl (An), 4-chlorobenzoyl, diphenylcarbamoyl, butylthiocarbonyl, 2-nitrobenzenethiol, 2,4-dinitrobenzenethiol, 2-nitro-4-methylbenzenethiol, and triphenylmethylthiol.

19. The solution-phase method according to any one of the preceding claims, wherein each protecting group is independently selected from the group consisting of benzoyl (Bz), 2,4-dimethylbenzoyl, tert-butylbenzoyl (tert-BuBz), and anisoyl (An).

20. The solution-phase method according to any one of the preceding claims, wherein the step of growing the oligonucleotide comprises a membrane filtration step after each sequential coupling reaction to separate the grown oligonucleotide.

21. The solution-phase method according to any one of the preceding claims, wherein one or more of the membrane filtration steps are carried out using a crosslinked polybenzimidazole membrane or a polyetheretherketone membrane.

22. The solution-phase method according to any one of the preceding claims, wherein the method further comprises one or more deprotection steps.

23. The solution-phase method according to claim 22, wherein the one or more deprotection steps comprise selective oxidation, selective reduction, oximation, cleavage of the protecting group with an organometallic catalyst, cleavage of the protecting group with fluoride, cleavage of the protecting group with an acid, cleavage of the protecting group with a base, or a combination thereof.

24. The solution-phase method according to claim 22 or 23, wherein the one or more deprotection steps occur during and / or after the step of growing the oligonucleotide.

25. An oligonucleotide obtained by the method according to any one of the preceding claims.

Images