Liquid-phase process for preparing oligonucleotides

Abstract

The present invention relates to a liquid-phase process for preparing oligonucleotides. More particularly, the present invention relates to a membrane filtration-assisted, liquid-phase process for preparing oligonucleotides, in which one or more building blocks used to grow an oligonucleotide comprises a nucleobase protected by a nucleobase protecting group (NPG), wherein NPG comprises a group Q. The inventors have determined that the use of building blocks in which the nucleobase comprises a nucleobase protecting group (NPG) notably reduces instances of inefficient membrane separation and / or fouling as part of a membrane-assisted LPOS technique.

Description

LIQUID-PHASE PROCESS FOR PREPARING OLIGONUCLEOTIDES INTRODUCTION

[0001] The present invention relates to a liquid-phase process for preparing oligonucleotides. More particularly, the present invention relates to a membrane filtration-assisted, liquid-phase process for preparing oligonucleotides. BACKGROUND OF THE INVENTION

[0002] Oligonucleotides (oligos) are short sequence-defined polymers of nucleotides (a.k.a. nucleoside phosphates). The structure of oligos typically consists of a ribose phosphate backbone, usually linking the 3’- and 5’-oxygen substituents of the ribose sugar via a phosphodiester unit, and each ribose ring has an aromatic nucleobase bound to the 1’-C of ribose. The combination of ribose sugar (or other analogue discussed below) and nucleobase is called a nucleoside, and the nucleobase varies from one monomer unit to the next in natural and medicinal sequences. It is the nucleobase sequence that confers target specificity on oligos.

[0003] Oligonucleotide-based drugs are now being advanced as a new generation of therapeutics, functioning at the protein expression level, and have recently been validated as a new pharmaceutical modality for treating a wide range of serious or life-threatening indications. Oligo drugs differ chemically from natural oligos in that they bear chemical modifications at multiple sites, principally to increase stability in the body and to improve targeting. However, similarly to natural oligos, it is the precise sequence of nucleobase side-chains that defines the oligonucleotide’s pharmaceutical function.

[0004] In pharmaceutical oligos the modifications commonly include replacing oxygen with sulfur on the phosphate backbone, placing substituents on the 2’-C of ribose (e.g. MeO, F, methoxyethyloxy (OMoe)), constraining the configuration of the ribose sugar with extra rings of atoms, methylating or fluorinating nucleobases, and replacing ribose with another heterocycle, such as morpholine. Even so, drugs containing such modifications are still recognisable as oligonucleotides.

[0005] For decades oligos have been prepared using solid-phase oligonucleotide synthesis (SPOS) wherein a growing oligo is tethered to an insoluble solid support and grown by flowing reactive nucleoside phosphoramidite building blocks over the insoluble solid support. This cyclic process consists of three principal chemical steps: 1) extension of an exposed chain terminal OH by the building block then, 2) oxidation of, or sulfur transfer to the internucleotide linkage, and finally, 3) removal of a temporary chain terminal protecting group to expose a new OH group.The cycle is repeated to build up the desired oligo sequence one monomer (or other building block) at a time. Additionally, most oligo synthesis protocols include an additional capping step to prevent any unextended chain termini from participating in further cycles, thus reducing the number of chomatographically similar impurities to the full-length product (FLP). However, capping is only partially effective and can introduce impurities itself.

[0006] Canonical commercial nucleotide building blocks or monomers are protected as follows: The phosphorus bearing moiety is a highly reactive P(III) phosphoramidite, usually bound to the ribose 3’-O of the ribose ring, and is protected with a permanent 2-cyanoethyl ester which is base labile; the nucleobase is usually permanently protected on any reactive NH (and occasionally O) with ammonolytically sensitive (acyl, amidine) groups; the 5’-O of ribose is temporarily protected with a mild acid labile protecting group, almost universally 4,4’-dimethoxytriphenylmethyl (a.k.a. dimethoxytrityl, Dmtr).

[0007] Although the method has been the industry standard for many years, there are a number of drawbacks associated with SPOS. In particular, an excess of nucleotide building block is required to drive the reaction to completion, which raises the cost of SPOS considerably. Moreover, scaling up of SPOS reactions is limited to producing only ~ 15kg of final fully deprotected and purified oligo per batch. This is highly undesirable for pharmaceutical preparation if tonnes of oligo per annum were required for a major medical indication (e.g. cardiovascular disease).

[0008] One alternative strategy to SPOS which aims to address the scale-up and economic challenges is liquid phase oligonucleotide synthesis (LPOS). Liquid phase reactions and liquid phase material handling are established technologies that can be performed at the multi-tonne scale, making LPOS a strong candidate for oligo preparation at scale. A typical approach to LPOS is to carry out sequential coupling reactions, adding monomers or multi-monomer oligomers (a.k.a. fragments or building blocks) to a growing oligo in solution in a stepwise fashion, and then to use a suitable separation technology (Molina et al.), such as precipitation (Creuse et al., Zhou et al.) or liquid extraction (de Koning et al.) to separate unreacted monomers or fragments from the growing oligo. LPOS has an inherent advantage over SPOS in that the entire reaction solution can be observed and sampled in real time.

[0009] Membrane-assisted LPOS (Gaffney et al., Kim et al.) is unique amongst oligo synthesis strategies in that the growing oligo remains in a single solution at all times. After the step of coupling a building block onto a growing oligo, membrane filtration (e.g., US 8,664,357, US 9,127,123, US 10,239,996, etc.) is used to separate the unreacted building block and any reaction debris from the growing oligo, without the need for phase change or phase separation.

[0010] During LPOS a high concentration of growing oligo is preferred to ensure that the critical chain extension step of the oligo synthesis cycle is rapid and complete. High concentration also favours efficient coupling because a lower excess of building block is required to force completion. Furthermore, it is easier to exclude moisture which consumes building block because solvent, being the most abundant species present, is usually the largest source of water.

[0011] However, in the case of membrane-assisted LPOS, solutes can become concentrated at the membrane interface (known as “concentration polarisation”) during the pressure-driven process of diafiltration. Such concentration polarisation can lead to increases in viscosity and / or aggregation, thereby engendering inefficient membrane separation and / or fouling.

[0012] Accordingly, there remains a need for an improved membrane-assisted LPOS technique.

[0013] The present invention was devised with the foregoing in mind. SUMMARY OF THE INVENTION

[0014] According to a first aspect of the present invention there is provided a liquid-phase process for preparing an oligonucleotide, the process comprising growing an oligonucleotide by performing one or more chain extension cycles, in which each chain extension cycle comprises a step of coupling a monomeric or oligomeric building block to a chain extension site of a growing oligonucleotide, wherein in at least one chain extension cycle, the building block comprises a nucleobase protected by a nucleobase protecting group (NPG), wherein NPG comprises a group Q of formula I:(I) wherein n is 1-9 and R1is H or methyl, and wherein in at least one of the chain extension cycles in which the building block comprises NPG, one or more membrane filtration steps is used to isolate the growing oligonucleotide (e.g., from uncoupled building blocks and / or reaction debris). DETAILED DESCRIPTION OF THE INVENTION

[0015] Throughout the entirety of the description and claims of this specification, where subject matter is described herein using the term “comprise” (or “comprises” or “comprising”), the samesubject matter instead described using the term “consist of” (or “consists of” or “consisting of”) or “consist essentially of” (or “consists essentially of” or “consisting essentially of”) is also contemplated.

[0016] Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

[0017] Features described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and / or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive. The invention is not restricted to the details of any of the specific embodiments recited herein. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

[0018] Through rigorous investigations, the inventors have devised the liquid-phase process for preparing an oligonucleotide according to the first aspect, which addresses the aforementioned disadvantages associated with LPOS techniques. In particular, the inventors have determined that the use of building blocks in which the nucleobase comprises a nucleobase protecting group (NPG) notably reduces instances of inefficient membrane separation and / or fouling as part of a membrane-assisted LPOS technique. Without wishing to be bound by theory, it is believed that the presence of group Q within the NPG-containing, growing oligonucleotide mitigates problems, such as increases in viscosity and / or aggregation, that would otherwise occur during membrane filtration as a result of concentration polarisation factors. The fact that group Q is present on one or more of the incoming building blocks (as opposed to being tethered to only one end of the growing oligo) allows for greater flexibility in preventing the emergence of any such issues throughout the entirety of the stepwise growth process. Indeed, it allows the frequency of the group Q to be varied in order to complement a growing oligo’s tendency to give rise to concentration polarisation issues at all stages of its preparation, irrespective of its overall length. Although each NPG may be relatively small, the collective effect on the whole oligo is cumulative. This is in direct contradistinction to similar groups appended to one end of an oligonucleotide chain, the effects of which will be large in the early stages of synthesis (when the intermediate oligo is short), but diminish as the oligo grows and eventually comes to dominate the physical properties of the construct at sufficient length. Using NPG as described herein, the degree ofproperty modulation can be adjusted such that it is proportional to the length of the oligo. Once the desired length of the growing oligo has been reached, all NPGs can be straightforwardly cleaved from their respective nucleobases, thereby leaving the FLP. The inventors have therefore found that the process of the invention allows for the straightforward preparation of oligos of increased length (e.g. ≥8 nucleotides in length) by a plurality of sequential coupling reactions in certain industry-favoured solvents (e.g., acetonitrile).

[0019] The process of the invention is conducted in solution. It will therefore be understood that the growing oligonucleotide remains in a dissolved state during chain extension cycles, including when it is isolated by membrane filtration.

[0020] Oligonucleotides will be familiar to those skilled in the art and will be understood to comprise a plurality of nucleotides linked (i.e., coupled) to one another to form a nucleotide sequence. As described herein, oligonucleotides are prepared by the stepwise addition of monomeric or oligomeric building blocks to a growing oligonucleotide, with each addition being referred to as a coupling reaction.

[0021] Each building block used in the process suitably comprises: (i) at least one nucleosidic moiety (Nuc), (ii) a reactive terminal (RT) for coupling the building block to the chain extension site of the growing oligonucleotide, and (iii) a temporary protecting group (TPG), said temporary protecting group being cleavable, after coupling of the building block to a chain extension site of a growing oligonucleotide, to expose a new chain extension site of the growing oligonucleotide.

[0022] The building blocks used in the process can be monomeric or oligomeric (e.g., 2-8 mer), wherein mer refers to a nucleosidic moiety. Therefore, a monomeric building block comprises one nucleosidic moiety (e.g., ribose coupled to a nucleobase) to which RT and TPG are covalently bound. An oligomeric building block comprises two or more nucleosidic moieties (e.g., ribose coupled to a nucleobase), each pair of adjacent nucleosidic moieties being linked by an internucleoside linkage (also known as an internucleotide linkage), with RT being covalently bound to one terminal nucleosidic moiety and TPG being covalently bound to the other terminal nucleosidic moiety. It is well known that ribose may be deoxygenated at the 2’ carbon. Additionally, as herein, those of ordinary skill in the art will appreciate that synthetic nucleosides may contain various structural modifications, for example substitution at the 2’ carbon of ribose (e.g., with fluoro, methoxy or methoxyethyloxy), fusing one or more additional rings to ribose to constrain its configuration, bridging the ribose to constrain its configuration (e.g., as in Locked Nucleic Acid) and / or substitution on the nucleobase (e.g., with methyl or fluoro). Similarly, those of ordinary skill in the art will be familiar with variations of ribonucleosides comprising alternativebackbone-forming heterocyclic moieties, such as in morpholine nucleosides, as well as the manner in which they are coupled to form oligos. It will be understood that such modifications and variations are embraced herein. It will also be understood that in oligomeric building blocks, the two or more nucleosidic moieties are not necessarily identical. For example, the two or more nucleosidic moieties may differ in terms of the nature of the nucleobases and / or structural modifications on ribose.

[0023] In most instances, each building block comprises 1-3 nucleosidic moieties. Most often, each building block is monomeric.

[0024] Those of ordinary skill in the art will be familiar with the concept of a chain extension cycles in the context of oligo synthesis. Typically, each cycle will comprise a coupling step (to add a building block to a reactive terminal of a growing oligonucleotide), an oxidation step (to convert phosphorus from its P(III) state to its P(V) state, and a deprotection step (to expose a reactive terminal on the growing oligonucleotide), with purification / isolation techniques being employed at one or more instances during each cycle.

[0025] In modern SPOS using phosphoramidites, oligos are almost always prepared with protecting groups on the exocyclic amino groups of the nucleobases to prevent branching side- reactions during phosphoramidite coupling reactions, and these are readily commercially available. Most commonly, acyl groups fulfil this role with acetyl or benzoyl on cytosine 4-N, benzoyl on adenine 4-N and isobutyryl on guanine 2-N. Additional protection can be installed on the reactive guanine 6-O, such as an aryl ether, which is essential to prevent side-reactions in H-phosphonate or phosphotriester approaches to oligo synthesis. Additional protection can also be appended to the relatively reactive uracil and thymine 3-N, most commonly as a benzoyl or anisoyl group, although this is not required for phosphoramidites. Alternative protection can be installed on uracil and thymine on the 4-O instead of the 3-N, like on guanine 6-O most commonly as an aryl ether, and again most developed for oligo synthesis by H-phosphonate or phosphotriester approaches. Accordingly, those of ordinary skill in the art of oligo synthesis will be readily familiar with the types of protecting groups suitable for protecting nucleobases, as well as the N or O atoms of those nucleobases to which said protecting groups are typically attached. The following illustrates the positions on adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U) where protecting groups (denoted RPG) are typically applied:Patterns of nucleobase protection

[0026] In at least one chain extension cycle of the process of the invention, the building block comprises a nucleobase protected by a nucleobase protecting group (NPG). The presence of group Q within NPG advantageously improves the physical properties of the building block(s), which are then cumulatively transferred to the growing oligo during the coupling step.

[0027] Suitably, in at least one of the chain extension cycles in which the building block comprises a nucleobase protected by NPG, the nucleobase is adenine, guanine or cytosine. It has been determined that A, G and C nucleobases protected with NPG are less likely to generate unwanted side products upon deprotection than NPG-protected uracil and NPG-protected thymine. In some instances, all A-, G- and C-containing building blocks comprises NPG and / or there are no instances of NPG-protected uracil or NPG-protected thymine. Most suitably, all A-, G- and C-containing building blocks comprises NPG and there are no instances of NPG- protected uracil and NPG-protected thymine.

[0028] In the group Q of formula I, n may vary depending on the nature of NPG. In some instances, n is suitably 1-7, more suitably 1-5, and even more suitably 2-3. In other instances, n may be 1 or 2.

[0029] Each NPG used in the process may independently have a structure according to formula IIa, IIb or IIc:wherein represents the point of attachment to the N or O atom (more typically the N atom) of the nucleobase, L1is selected from absent, –O–, –CH2–, –OCH2– and –CH2O–, L2is selected from absent and phenylene, and L3is selected from absent and –OCH2–, with the proviso that at least one of L1, L2and L3is not absent;wherein represents the point of attachment to the O atom of the nucleobase, and m is 0, 1 or 2; (IIIc) wherein represents the point of attachment to the N atom of the nucleobase, RAis selected from (1-6C)alkyl and Q, RBis selected from (1-6C)alkyl and –(CH2)p–Q, in which p is 1 or 2, and RCis (1-6C)alkyl, with the proviso that at least one of RAand RBis not (1-6C)alkyl.

[0030] Each NPG used in the process may independently have a structure according to any one of the following:, ,, and , wherein represents the point of attachment to the N or O atom (as appropriate) of the nucleobase, each k is independently 1-4 (e.g., 1-2), and Q1and Q2are as defined hereinbefore for Q.

[0031] In Q1, n is suitably 1-8. More suitably, n is 2-6. Even more suitably, n is 2-3.

[0032] In Q2, n is suitably 1-8. More suitably, n is 1-6. Even more suitably, n is 1-3. Most suitably, n is 1.

[0033] Suitably, in at least one chain extension cycle, the building block comprises a nucleobase protected by a NPG of type IIa-4. Q2may have any of the aforementioned definitions for Q or Q2.

[0034] More suitably, in at least one chain extension cycle, the building block comprises a nucleobase protected by a NPG having the following structure:

[0035] In IIa-4-p, Q2may have any of the aforementioned definitions for Q or Q2. Suitably, n is 1-8. More suitably, n is 1-8. Even more suitably, n is 1-3. Most suitably, n is 1.

[0036] In at least one of the chain extension cycles, the building block comprises a nucleobase protected by NPG. Suitably, in at least 20% of the chain extension cycles (to the nearest whole number), the building block comprises a nucleobase protected by NPG. For example, in the preparation of a 20-mer oligo by 19 chain extension cycles, each one involving a monomeric building block, in four or more of the chain extension cycles the nucleobase of the building block is protected by NPG. More suitably, in at least 30% of the chain extension cycles (to the nearest whole number), the building block comprises a nucleobase protected by NPG. Even more suitably, in at least 50% of the chain extension cycles (to the nearest whole number), the building block comprises a nucleobase protected by NPG. Yet even more suitably, in at least 75% of the chain extension cycles (to the nearest whole number), the building block comprises a nucleobase protected by NPG. Still even more suitably, in at least 90% of the chain extension cycles (to the nearest whole number), the building block comprises a nucleobase protected by NPG.

[0037] In some instances, in each one of the chain extension cycles, the building block comprises a nucleobase protected by NPG.

[0038] In at least one of the chain extension cycles in which the building block comprises NPG, membrane filtration is performed to isolate the growing oligonucleotide (e.g., from uncoupled building blocks and / or reaction debris). The person of ordinary skill in the art will be familiar with reaction debris typically generated as part of an LPOS process. Such debris will be taken to include by-products, cleaved temporary protecting groups (TPG) and excess reagents. Suitably, in at least 20% of the chain extension cycles (to the nearest whole number) in which the building block comprises NPG, membrane filtration is performed to isolate the growing oligonucleotide. For example, where 8 chain extension cycles within a process use a building block comprising NPG, membrane filtration is performed to isolate the growing oligonucleotide in 2 of those chain extension cycles. Suitably, in at least 50% of the chain extension cycles (to the nearest whole number), membrane filtration is performed to isolate the growing oligonucleotide. More suitably, in at least 80% of the chain extension cycles (to the nearest whole number) in which the building block comprises NPG, membrane filtration is performed to isolate the growing oligonucleotide. Most suitably, in each one of the chain extension cycles in which the building block comprises NPG, membrane filtration is performed to isolate the growing oligonucleotide.

[0039] It will be understood that membrane filtration may be used to isolate the growing oligonucleotide in a given chain extension cycle even when the building block used in that chain extension cycle does comprise NPG. Suitably, membrane filtration is used to isolate the growing oligonucleotide in at least 75% (to the nearest whole number) of all chain extension cycles (i.e.,irrespective of the nature of the building block). More suitably. membrane filtration is used to isolate the growing oligonucleotide in at least 90% (to the nearest whole number) of all chain extension cycles (i.e., irrespective of the nature of the building block). Most suitably, membrane filtration is used to isolate the growing oligonucleotide in all chain extension cycles (i.e., irrespective of the nature of the building block).

[0040] Where membrane filtration is used as part of a chain extension cycle, it is suitably used in two discrete steps. For example, a first membrane filtration step separates the NPG-containing oligonucleotide from excess, unreacted building block, while a second membrane filtration step separates cleaved temporary protecting group (TPG) from the NPG-containing oligonucleotide.

[0041] The building blocks may each have a structure according to formula III:wherein RT is a reactive terminal for coupling the building block to the chain extension site of the growing oligonucleotide, TPG is a temporary protecting group that is cleavable, after coupling of the building block to a chain extension site of the growing oligonucleotide, to expose a new chain extension site of the growing oligonucleotide, Lpis an internucleoside linkage, and v is 0-10 (e.g., 0-3).

[0042] Those of ordinary skill in the art of oligonucleotide preparation will be familiar with internucleoside linkages. In particular, it will be understood that such linkages comprise phosphorus in the P(III) or P(V) state. Exemplary phosphorus-containing internucleosidic linkages include a phosphodiester linkage, a methyl phosphonate linkage, a phosphorothioate linkage, a boranophosphate linkage, a phosphoramidate, or a mesyl phosphoramidate linkage. It will be understood that such linkages may comprise protecting groups to protect reactive moieties, where such protecting groups are only removed once the target length of the oligo has been reached (e.g., during global deprotection, as described herein).

[0043] In most instances, v is 0, 1 or 2. Most often, v is 0.

[0044] RT may be disposed at the 5’ carbon of the nucleosidic moiety to which it is attached, and TPG disposed at the 3’ carbon of the nucleosidic moiety to which it is attached. Alternatively, RT may be disposed at the 3’ carbon of the nucleosidic moiety to which it is attached, and TPGdisposed at the 5’ carbon of the nucleosidic moiety to which it is attached. In both instances, it will be appreciated that the nucleosidic moieties to which RT and TPG are attached comprise a ribosidic core (notwithstanding the aforementioned modifications that may occur at the 2’ carbon).

[0045] The building blocks of formula III may each have a structure according to formula IIIa:wherein OAis the oxygen atom located at the 5’ carbon of the nucleosidic moiety to which it is attached, and OBis the oxygen atom located at the 3’ carbon of the nucleosidic moiety to which it is attached, or OAis the oxygen atom located at the 3’ carbon of the nucleosidic moiety to which it is attached, and OBis the oxygen atom located at the 5’ carbon of the nucleosidic moiety to which it is attached.

[0046] For building blocks of formula IIIa, it will be appreciated that the nucleosidic moieties to which RT and TPG are attached comprise a ribosidic core (notwithstanding the aforementioned modifications that may occur at the 2’ carbon).

[0047] A variety of different RT groups will be familiar to those of ordinary skill in the art. For example, RT may be a phosphoramidite, a phosphate monoester, a phosphate diester, an H- phosphonate, a cyclic thiophosphate or a cyclic dithiophosphate triester moiety.

[0048] The nature of the chain extension site of the growing oligonucleotide will be apparent to those of ordinary skill in the art of oligonucleotide synthesis. Solely for the sake of illustration, where RT is a phosphoramidite located at the 5’ terminal of a building block, the chain extension site may be a hydroxyl group located at the 3’ terminal of the growing oligonucleotide.

[0049] RT may have a structure according to formula IV:(IV) whereinLG is a leaving group; PG is a protecting group; and represents the point of attachment to the nucleosidic moiety (Nuc) to which RT is attached (e.g., via OAin formula IIIa).

[0050] LG may be any suitable leaving group. In most instances, LG is a tertiary amino group, i.e., –NR2. Suitably, each R is independently selected from (1-6C)alkyl, or both R groups are linked such that when taken in combination with the nitrogen atom to which they are attached, they collectively form a 5- to 7-membered heterocycle. More suitably, each R is independently selected from (1-4C)alkyl, or both R groups are linked such that when taken in combination with the nitrogen atom to which they are attached, they collectively form a 5-membered heterocycle. Even more suitably, each R is independently selected from methyl, ethyl and isopropyl, or both R groups are linked such that when taken in combination with the nitrogen atom to which they are attached, they collectively form a pyrrolidinyl group. Most suitably, each R is isopropyl..

[0051] Those of ordinary skill in the art will be familiar with a variety of protecting groups suitable for use as PG. PG may be a base-labile protecting group. Examples of PG include methyl, isopropyl, tert-butyl, benzyl, allyl, phenyl, 2-chlorophenyl, 4-chlorophenyl, 3’5’-dimethoxybenzoin, p-hydroxyphenacyl, cyanoethyl, 9-fluorenylmethyl, 2-(trimethylsilyl)ethyl, 2-(methylsulfonyl)ethyl, 2-(benzenesulfonyl)ethyl, 4-nitrophenethyl, and 2,2,2-trichloroethyl. Suitably, PG is selected from the group consisting of cyanoethyl, 2-chlorophenyl, 4-chlorophenyl, 2,2,2-trichloroethyl and methyl. Most suitably, PG is cyanoethyl.

[0052] In many instances, RT has a structure according to the following:where represents the point of attachment to the nucleosidic moiety (Nuc) to which RT is attached (e.g., via OAin formula IIIa).

[0053] TPG will be understood to be a protecting group used to prevent uncontrolled chain extension during a chain extension cycle. Those of ordinary skill in the art will be readily familiar with protecting groups suitable for this purpose, as well as the conditions under which they can be cleaved. In many instances, TPG is an acid-labile protecting group, non-limiting examples of which include dimethoxytrityl (Dmtr / DMT), tert-butyl (tBu), tert-butyl oxycarbonyl (Boc), mono-methoxytriphenyl (Mmtr), triphenyl methyl (Tr), pentamethyl dihydrobenzofuran sufonyl (Pbf), Tetrahydropyranyl (Thp), tetrahydrofuranyl (Thf), para-methoxybenzyl (Pmb) and 2,4- dimethoxybenzyl. Most suitably, TPG is dimethoxytrityl bound to the 3’ or 5’ oxygen (as appropriate) of the nucleosidic moiety to which it is attached.

[0054] The building blocks of formula III may each have a structure according to formula IIIb:wherein OAis the oxygen atom located at the 5’ carbon of the nucleosidic moiety to which it is attached, and OBis the oxygen atom located at the 3’ carbon of the nucleosidic moiety to which it is attached, or OAis the oxygen atom located at the 3’ carbon of the nucleosidic moiety to which it is attached, and OBis the oxygen atom located at the 5’ carbon of the nucleosidic moiety to which it is attached.

[0055] In building blocks of formula IIIb, TPG is suitably dimethoxytrityl.

[0056] In many instances, in formula III, IIIa or IIIb, each nucleosidic moiety (Nuc) has a structure according to formula V:(V) wherein Z is a nucleobase, optionally protected by a protecting group, wherein said protecting group may be NPG;Rxis selected from H, OH, F, O-tert-butyldimethylsilyl (OTbdms), methoxy, O-methoxyethyl (OMoe), O-propargyl, NH2and N3, in which case both are absent, or Rxis O and both are present; 1 and 2 independently represent points of attachment to RT, TPG, OA, OBor LP (as appropriate).

[0057] In formula V, Rxis most suitably H or OH.

[0058] The process of the invention comprises growing an oligonucleotide by performing one or more chain extension cycles. Suitably, the one or more chain extension cycles is five or more chain extension cycles. More suitably, the one or more chain extension cycles is eight or more chain extension cycles. Even more suitably, the one or more chain extension cycles is ten or more chain extension cycles. Yet even more suitably, the one or more chain extension cycles is twelve or more chain extension cycles. Most suitably, the one or more chain extension cycles is fifteen or more chain extension cycles.

[0059] Each chain extension cycle suitably comprises a step of cleaving the temporary protecting group (TPG, e.g., dimethoxytrityl) after coupling of the building block to a chain extension site of the growing oligonucleotide to expose a new chain extension site of the growing oligonucleotide. As described hereinbefore, those of ordinary skill in the art of oligonucleotide synthesis will be readily familiar with exemplary TPGs and how they are cleaved.

[0060] Within each chain extension cycle, the step of coupling the monomeric or oligomeric building block to the chain extension site of the growing oligonucleotide suitably form a P(III) phosphite triester linkage between the coupled building block and growing oligonucleotide. Such coupling reactions can be initiated with common activators, such as ethylthiotetrazole (ETT) or dicyanoimidazole (DCI).

[0061] Each chain extension cycle suitably comprises a step of converting one of more P(III) phosphite triester linkages to a P(V) phosphotriester linkage (e.g., by oxidation or sulfur transfer) after coupling of the building block to a chain extension site of the growing oligonucleotide. Such a step may be described generically herein as an oxidation step. Suitable reagents for performing this step will be familiar to those of ordinary skill in the art, and include camphorsulfonyl oxaziridine (CSO), cumenyl hydroperoxide, tert-butyl hydroperoxide, phenylacetyl disulfide (PADS), xanthane hydride (XH) and or 3-phenyl 1,2,4-dithiazoline-5-one (POS). As part of this step, the oxidation product (i.e., the compound comprising one or more P(V) phosphotriester linkages) may be isolated by membrane filtration. In most instances, the step of converting one of more P(III) phosphite triester linkages to a P(V) phosphotriester linkage (e.g., by oxidation or sulfur transfer) is conducted before a step of cleaving the temporary protecting group (TPG, e.g., dimethoxytrityl) to expose a new chain extension site of the growing oligonucleotide.

[0062] Where membrane filtration is used as part of a chain extension cycle, it is suitably used in two discrete steps. A first membrane filtration step may be conducted after coupling of the building block to a chain extension site of the growing oligonucleotide and before a step of cleaving the temporary protecting group (TPG, e.g., dimethoxytrityl, Dmtr) to expose a new chain extension site of the growing oligonucleotide. A second membrane filtration step may be conducted after a step of cleaving the temporary protecting group (TPG, e.g., dimethoxytrityl) to expose a new chain extension site of the growing oligonucleotide.

[0063] The process may be performed in any suitable solvent. Suitably, the process is performed in acetonitrile, or in a solvent mixture comprising acetonitrile (e.g., acetonitrile mixed with sulfolane). Acetonitrile is the solvent favoured by industry for coupling nucleotides to prepare oligonucleotides More suitably, all steps of a given chain extension cycle (e.g., coupling, oxidation, deprotection and membrane filtration) are performed in the same solvent (e.g., acetonitrile). Even more suitably, all chain extension cycles and their associated steps (e.g., coupling, oxidation, deprotection and membrane filtration) are performed in the same solvent (e.g., acetonitrile).

[0064] The membrane filtration steps forming part of the process are suitably conducted by membrane diafiltration. In membrane diafiltration, the crude mixture comprising the growing oligonucleotide is pressurised against a size-selective membrane. More suitably, the membrane filtration steps are conducted by organic solvent nanofiltration (OSN) or ultrafiltration (UF). The membrane is suitably insoluble in the solvent(s) used in the step of coupling the monomeric or oligomeric building block to the chain extension site of the growing oligonucleotide (e.g., acetonitrile).

[0065] Suitable membranes for use in isolating the growing oligonucleotide include polymeric membranes, ceramic membranes, and mixed polymeric / inorganic membranes. Membrane rejection Ri is a common term known by those skilled in the art and is defined as:eq. (1) Ri where CP,i = concentration of species i in the permeate, permeate being the liquid which has passed through the membrane, and CR,i = concentration of species i in the retentate, retentate being the liquid which has not passed through the membrane. It will be appreciated that a membrane is suitable for the invention if R(product)>R(reactants), where product is the growingoligonucleotide.

[0066] The membrane may be formed from any polymeric or ceramic material which provides a separating layer capable of preferentially separating the growing oligonucleotide from uncoupled building blocks and / or reaction debris. In other words, the membrane will exhibit a rejection for the growing oligonucleotide that is greater than the rejection for the uncoupled building blocks and / or reaction debris. Suitably, the membrane is formed from or comprises a polymeric material suitable for fabricating microfiltration, ultrafiltration, nanofiltration or reverse osmosis membranes, including polyethylene, polypropylene, polytetrafluoroethylene (PTFE), polyvinylidene difluoride (PVDF), polysulfone, polyethersulfone, polyacrylonitrile, polyamide, polyester, polyimide, polyetherimide, cellulose acetate, polyaniline, polypyrrole, polybenzimidazole (PBI), polyetheretherketone (PEEK) and mixtures thereof. Ceramic membranes may be made from TiO2or ZrO2. The membranes can be made by any technique known in the art, including sintering, stretching, track etching, template leaching, interfacial polymerisation or phase inversion. Membranes may be composite in nature (e.g., a thin film composite membrane) and / or be crosslinked or treated so as to improve their stability in certain solvents. PCT / GB2007 / 050218 and PCT / GB2015 / 050179 describe membranes which may be suitable for use as part of the invention. US 10,913,033 describes a membrane that is particularly suitable for use as part of the invention.

[0067] Most suitably, the membrane is a crosslinked polybenzimidazole membrane (e.g., an integrally skinned, asymmetric, crosslinked polybenzimidazole membrane) or a polyetheretherketone membrane.

[0068] The process may further comprises one or more global deprotection steps. Such step(s) may be conducted once a target length of the oligonucleotide has been reached. The global deprotection step(s) may result in removal of all instances of PG (e.g., cyanoethyl) from internucleoside linkages present throughout the oligonucleotide, and / or may result in removal of all protecting groups, including NPG, present on nucleobases throughout the oligonucleotide. Those of ordinary skill in the art will be familiar with techniques by which such global deprotection can be achieved.

[0069] The growing oligonucleotide may be attached (e.g., at the 3’ or 5’ terminal opposite to the chain extension site) to a soluble synthesis support. Such soluble synthesis supports may be used to confer additional solubility to the growing oligonucleotide, and / or to increase its molecular bulk relative to building blocks / reaction debris in order to facilitate membrane filtration. For example, the soluble synthesis support may be a polymer (e.g., a star polymer), a dendrimer, a dendron, a hyperbranched polymer, or an organic / inorganic material, including nanoparticles, fullerenes and 2-D materials such as graphene and boron nitride. Suitably, the soluble synthesissupport is polymeric and is formed from poly(alkylene glycols), polyesters, polyamides, vinyl polymers, diene polymers, poly(alkylene imines), poly(amidoamines) and polysiloxanes. More suitably, the soluble synthesis support is formed from poly(ethylene glycol).

[0070] In some instances, the soluble synthesis support is a star polymer comprising a central branch point and m number of radiating polymeric arms, wherein polymeric arm is attached to a growing oligonucleotide, and wherein m is 3 or more. In such instances, it will be understood that the process can be used to grow m number of identical oligonucleotide chains. Suitably, each polymeric arm is selected from the group consisting of poly(alkylene glycols), polyesters, polyamides, vinyl polymers, diene polymers, poly(alkylene imines), poly(amidoamines) and polysiloxanes. More suitably, each polymeric arm is poly(ethylene glycol).

[0071] The soluble synthesis support may have a structure according to formula VIin which V is an organic branch point, W is a polymeric chain, represents a point of attachment to the growing oligonucleotide, and m is 1-8.

[0072] V will be understood to refer to a polyfunctional organic “hub” having a plurality of terminals to which polymeric chain(s), W, are attached. In many instances, V comprises fewer than 50 atoms in total, more suitably fewer than 20 atoms in total.

[0073] Each W is a polymeric chain (e.g., polyethylene glycol) having a molecular weight (Mw) of 500 – 20,000 Da. More suitably, each W is a polymeric chain having a molecular weight (Mw) of 2000 – 15,000 Da. Most suitably, each W is a polymeric chain having a molecular weight (Mw) of 8000 – 12,000 Da.

[0074] m may, for example, be 1-6. In many instances, m is 3-4.

[0075] It will be understood that each W may be attached directly to the growing oligonucleotide, or attached indirectly to the growing oligonucleotide via a linking moiety. A typical linking moiety will comprise fewer than 50 atoms in total, more suitably fewer than 30 atoms in total.

[0076] A particularly suitable example of a soluble synthesis support is:in which represents the point of direct attachment to the growing oligonucleotide (e.g., at the 3’ oxygen) and q is 100 – 500 (e.g., 200 – 300).

[0077] Each oligonucleotide, once prepared (i.e., grown to full length product), may have a molecular weight of ≥1000 Da. Suitably, each oligonucleotide has a molecular weight of ≥2000 Da. More suitably, each oligonucleotide has a molecular weight of ≥3000 Da. Even more suitably, each oligonucleotide has a molecular weight of ≥5000 Da.

[0078] As alluded to hereinbefore, various chemical modifications relative to the structure of RNA-based and DNA-based oligonucleotides / building blocks will be apparent to those of ordinary skill in the art and are envisaged herein, particularly in relation to the structure of the backbone, nucleobase and / or sugar moiety.

[0079] Modifications may occur at the 2' position of the sugar moiety. Sugar modifications include a modified version of the ribosyl moiety, such as 2'-O-modified RNA such as 2'-O-alkyl or 2'-O(substituted)alkyl e.g., 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(haloalkoxy)methyl (Arai et al.) e.g., 2'-O-(2-chloroethoxy)methyl (MCEM), 2'-O-(2,2- dichloroethoxy)methyl (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'-halo e.g. 2'-F, FANA (2'-F arabinosyl nucleic acid); carbasugar and azasuar modifications; 3'-O-alkyl e.g. 3'-O-methyl, 3'-O-butyryl, 3'-O-propargyl; and their derivatives. Sugar modifications also include 2'-amino. Other sugar modifications include "bridged" or "bicylic" nucleic acid (BNA), e.g. locked nucleic acid (LNA), xylo-LNA, α-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), tricyclo 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 (as e.g. in PMO, PPMO, PMOPlus, PMO- X); and their derivatives.

[0080] Base modifications include modified versions of the natural purine and pyrimidine bases (e.g. adenine, uracil, guanine, cytosine, and thymine), such as inosine, hypoxanthine, orotic acid, agmatidine, lysidine, 2-thiopyrimidine (e.g. 2-thiouracil, 2-thiothymine), G-clamp and itsderivatives, 5-substituted pyrimidine (e.g. 5-methylcytosine, 5-methyluracil, 5-halouracil, 5- propynyluracil, 5-propynylcytosine, 5-aminomethyl uracil, 5-hydroxymethyl uracil, 5- aminomethylcytosine, 5-hydroxymethylcytosine, Super 5 T), 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 derivatives thereof; N2-cyclopentylguanine (cPent-G), N2-cyclopentyl-2-aminopurine (cPent- AP), and N2-propyl-2-aminopurine (Pr-AP), or derivatives thereof; and degenerate or universal bases, like 2,6-difluorotoluene or absent bases like abasic sites (e.g.1-deoxyribose, 1-deoxy-2- O-methylribose; or pyrrolidine derivatives in which the ring oxygen has been replaced with nitrogen (azaribose)). Examples of derivatives of Super A, Super G and Super T can be found in US6683173. cPent-G, cPent-AP and Pr-AP were shown to reduce immunostimulatory effects when incorporated in siRNA. Alternatively, nucleobase modifications may be selected from the group consisting of 5-methyl pyrimidines, 7-deazaguanosines and abasic nucleotides. Alternatively, the modification may be a 5-methyl cytosine.

[0081] Backbone modifications, i.e., relative to the phosphodiester present in RNA and DNA, include phosphorothioate (PS), phosphorodithioate (PS2), phosphonoacetate (PACE), phosphonoacetamide (PACA), thiophosphonoacetate, thiophosphonoacetamide, phosphorothioate prodrug, H-phosphonate, methylphosphonate, methyl phosphonothioate, methyl phosphate, methyl phosphorothioate, ethyl phosphate, ethyl phosphorothioate, boranophosphate, boranophosphorothioate, methyl boranophosphate, methyl boranophosphorothioate, methyl boranophosphonate, methylboranophosphonothioate, and their derivatives. Another modification includes phosphoramidite, phosphoramidate, N3'~PS' phosphoramidate, phosphordiamidate, phosphorothiodiamidate, sulfamate, dimethylenesulfoxide, sulfonate, triazole, oxalyl, carbamate, methyleneimino (MMI), and thioacetamido nucleic acid (TANA); and their derivatives.

[0082] Other modifications include incorporation of a peptide-base nucleic acid (PNA), boron modified PNA, pyrrolidine-based oxy-peptide nucleic acid (POPNA), glycol- or glycerol-based nucleic acid (GNA), threose-based nucleic acid (TNA), acyclic threoninol-based nucleic acid (aTNA), oligonucleotides with integrated bases and backbones (ONIBs), pyrrolidine-amide oligonucleotides (POMs); a bridged nucleic acid (BNA) such as (5)-cEt-BNA, or a SPIEGELMER.

[0083] According to a second aspect of the present invention, there is provided an oligonucleotide obtained, directly obtained or obtainable by the process of the first aspect.

[0084] The following numbered statements 1 to 79 are not claims, but instead describe particular aspects and embodiments of the invention:1. A liquid-phase process for preparing an oligonucleotide, the process comprising growing an oligonucleotide by performing one or more chain extension cycles, in which each chain extension cycle comprises a step of coupling a monomeric or oligomeric building block to a chain extension site of a growing oligonucleotide, wherein in at least one chain extension cycle, the building block comprises a nucleobase protected by a nucleobase protecting group (NPG), wherein NPG comprises a group Q of formula I:wherein n is 1-9 and R1is H or methyl, and wherein in at least one of the chain extension cycles in which the building block comprises NPG, one or more membrane filtration steps is used to isolate the growing oligonucleotide (e.g., from uncoupled building blocks and / or reaction debris). 2. The process as defined in statement 1, wherein each monomeric or oligomeric building block comprises: (i) at least one nucleosidic moiety (Nuc), (ii) a reactive terminal (RT) for coupling the building block to the chain extension site of the growing oligonucleotide, and (iii) a temporary protecting group (TPG), said temporary protecting group being cleavable, after coupling of the building block to a chain extension site of a growing oligonucleotide, to expose a new chain extension site of the growing oligonucleotide. 3. The process as defined in statement 2, wherein each monomeric or oligomeric building block comprises: one nucleosidic moiety (i.e., the building block is monomeric), or two or more nucleosidic moieties, each pair of adjacent nucleosidic moieties being linked by an internucleoside linkage (e.g., the building block is oligomeric). 4. The process as defined in statement 2 or 3, wherein each monomeric or oligomeric building block comprises 1-3 nucleosidic moieties.5. The process as defined in statement 4 wherein each monomeric or oligomeric building block comprises 1 nucleosidic moiety. 6. The process as defined in any one of the preceding statements, wherein in at least one of the chain extension cycles in which the building block comprises a nucleobase protected by NPG, the nucleobase is adenine, guanine or cytosine. 7. The process as defined in any one of the preceding statements, wherein n is 1-7. 8. The process as defined in any one of the preceding statements, wherein n is 1-5. 9. The process as defined in any one of the preceding statements, wherein n is 2-3. 10. The process as defined in any one of statements 1-9, wherein n is 1 or 2 11. The process as defined in any one of the preceding statements, wherein each NPG independently has a structure according to formula IIa, IIb or IIc:wherein represents the point of attachment to the N or O atom (more typically the N atom) of the nucleobase, L1is selected from absent, –O–, –CH2–, –OCH2– and –CH2O–, L2is selected from absent and phenylene, and L3is selected from absent and –OCH2–, with the proviso that at least one of L1, L2and L3is not absent;(IIb)wherein represents the point of attachment to the O atom of the nucleobase, and m is 0, 1 or 2;wherein represents the point of attachment to the N atom of the nucleobase, RAis selected from (1-6C)alkyl and Q, RBis selected from (1-6C)alkyl and –(CH2)p–Q, in which p is 1 or 2, and RCis (1-6C)alkyl, with the proviso that at least one of RAand RBis not (1-6C)alkyl. 12. The process as defined in any one of the preceding statements, wherein each NPG independently has a structure according to any one of the following: , ,, and , wherein represents the point of attachment to the N or O atom (as appropriate) of the nucleobase,each k is independently 1-4 (e.g., 1-2), and Q1and Q2are as defined hereinbefore for Q. 13. The process as defined in statement 12, wherein Q1is 1-8. 14. The process as defined in statement 12, wherein Q1is 2-6. 15. The process as defined in statement 12, wherein Q1is 2-3. 16. The process as defined in any one of statement 12-15, wherein Q2is 1-8. 17. The process as defined in any one of statement 12-15, wherein Q2is 1-6. 18. The process as defined in any one of statement 12-15, wherein Q2is 1-3. 19. The process as defined in any one of statement 12-15, wherein Q2is 1. 20. The process as defined in any one of statements 12-19, wherein in at least one chain extension cycle, the building block comprises a nucleobase protected by a NPG of type IIa-4. 21. The process as defined in any one of the preceding statements, wherein in at least one chain extension cycle, the building block comprises a nucleobase protected by a NPG having the following structure:Wherein Q2is as defined in any one of statements 16-19. 22. The process as defined in any one of the preceding statements, wherein in at least 20% of the chain extension cycles (to the nearest whole number), the building block comprises a nucleobase protected by NPG.23. The process as defined in any one of the preceding statements, wherein in at least 50% of the chain extension cycles (to the nearest whole number), the building block comprises a nucleobase protected by NPG. 24. The process as defined in any one of the preceding statements, wherein in at least 75% of the chain extension cycles (to the nearest whole number), the building block comprises a nucleobase protected by NPG. 25. The process as defined in any one of the preceding statements, wherein in at least 90% of the chain extension cycles (to the nearest whole number), the building block comprises a nucleobase protected by NPG. 26. The process as defined in any one of the preceding statements, wherein in each one the chain extension cycles, the building block comprises a nucleobase protected by NPG. 27. The process as defined in any one of the preceding statements, wherein in at least 20% of the chain extension cycles (to the nearest whole number) in which the building block comprises NPG, membrane filtration is performed to isolate the growing oligonucleotide. 28. The process as defined in any one of the preceding statements, wherein in at least 50% of the chain extension cycles (to the nearest whole number) in which the building block comprises NPG, membrane filtration is performed to isolate the growing oligonucleotide. 29. The process as defined in any one of the preceding statements, wherein in at least 80% of the chain extension cycles (to the nearest whole number) in which the building block comprises NPG, membrane filtration is performed to isolate the growing oligonucleotide. 30. The process as defined in any one of the preceding statements, wherein in each one of the chain extension cycles in which the building block comprises NPG, membrane filtration is performed to isolate the growing oligonucleotide. 31. The process as defined in any one of the preceding statements, wherein membrane filtration is used to isolate the growing oligonucleotide in at least 75% (to the nearest whole number) of all chain extension cycles.32. The process as defined in any one of the preceding statements, wherein membrane filtration is used to isolate the growing oligonucleotide in at least 90% (to the nearest whole number) of all chain extension cycles. 33. The process as defined in any one of the preceding statements, wherein membrane filtration is used to isolate the growing oligonucleotide in all chain extension cycles. 34. The process as defined in any one of the preceding statements, wherein each chain extension cycle membrane filtration steps is performed comprises: a first membrane filtration step to separate the NPG-containing oligonucleotide from excess, unreacted building block, and a second membrane filtration step to separate cleaved temporary protecting group from the NPG-containing oligonucleotide. 35. The process as defined in any one of the preceding statements, wherein each building blocks has a structure according to formula III:wherein RT is a reactive terminal for coupling the building block to the chain extension site of the growing oligonucleotide, TPG is a temporary protecting group that is cleavable, after coupling of the building block to a chain extension site of the growing oligonucleotide, to expose a new chain extension site of the growing oligonucleotide, Lpis an internucleoside linkage, and v is 0-10 (e.g., 0-3). 36. The process as defined in statement 35, wherein v is 0, 1 or 2 (e.g., 0). 37. The process as defined in statement 35 or 36, wherein each building block has a structure according to formula IIIa:wherein OAis the oxygen atom located at the 5’ carbon of the nucleosidic moiety to which it is attached, and OBis the oxygen atom located at the 3’ carbon of the nucleosidic moiety to which it is attached, or OAis the oxygen atom located at the 3’ carbon of the nucleosidic moiety to which it is attached, and OBis the oxygen atom located at the 5’ carbon of the nucleosidic moiety to which it is attached. 38. The process as defined in any one of statements 2-37, wherein each RT is independently a phosphoramidite, a phosphate monoester, a phosphate diester, an H- phosphonate, a cyclic thiophosphate or a cyclic dithiophosphate triester moiety. 39. The process as defined in any one of statements 2-38, wherein each RT independently has a structure according to formula IV: (IV) wherein LG is a leaving group; PG is a protecting group; and represents the point of attachment to the nucleosidic moiety (Nuc) to which RT is attached (e.g., via OAin formula IIIa). 40. The process as defined in statement 39, wherein LG is a tertiary amino group, i.e., - NR2. 41. The process as defined in statement 40, wherein each R is independently selected from (1-6C)alkyl, or both R groups are linked such that when taken in combination with the nitrogen atom to which they are attached, they collectively form a 5- to 7-membered heterocycle.42. The process as defined in statement 40, wherein each R is independently selected from (1-4C)alkyl, or both R groups are linked such that when taken in combination with the nitrogen atom to which they are attached, they collectively form a 5-membered heterocycle. 43. The process as defined in statement 40, wherein each R is independently selected from methyl, ethyl and isopropyl, or both R groups are linked such that when taken in combination with the nitrogen atom to which they are attached, they collectively form a pyrrolidinyl group. 44. The process as defined in statement 40, wherein each R is isopropyl. 45. The process as defined in any one of statements 39-44, wherein PG is a base-labile protecting group. 46. The process as defined in any one of statements 39-44, wherein PG is selected from the group consisting of cyanoethyl, 2-chlorophenyl, 4-chlorophenyl, 2,2,2-trichloroethyl and methyl. 47. The process as defined in any one of statements 2-46, wherein RT has a structure according to the following:where represents the point of attachment to the nucleosidic moiety (Nuc) to which RT is attached (e.g., via OAin formula IIIa). 48. The process as defined in any one of statements 2-47, wherein each TPG is independently selected from the group conssisting of dimethoxytrityl (Dmtr / DMT), tert-butyl (tBu), tert-butyl oxycarbonyl (Boc), mono-methoxytriphenyl (Mmtr), triphenyl methyl (Tr), pentamethyl dihydrobenzofuran sufonyl (Pbf), Tetrahydropyranyl (Thp), tetrahydrofuranyl (Thf), para-methoxybenzyl (Pmb) and 2,4-dimethoxybenzyl. 49. The process as defined in any one of statements 2-47, wherein TPG is dimethoxytrityl (Dmtr / DMT).50. The process as defined in any one of statements 35-49, wherein each building block has a structure according to formula IIIb:wherein OAis the oxygen atom located at the 5’ carbon of the nucleosidic moiety to which it is attached, and OBis the oxygen atom located at the 3’ carbon of the nucleosidic moiety to which it is attached, or OAis the oxygen atom located at the 3’ carbon of the nucleosidic moiety to which it is attached, and OBis the oxygen atom located at the 5’ carbon of the nucleosidic moiety to which it is attached. 51. The process as defined in any one of statements 35-50, wherein within formula III, IIIa or IIIb, each nucleosidic moiety (Nuc) has a structure according to formula V:wherein Z is a nucleobase, optionally protected by a protecting group, wherein said protecting group may be NPG; Rxis selected from H, OH, F, O-tert-butyldimethylsilyl , methoxy, O-methoxyethyl (OMoe), O-and N3, in which case both are absent, or Rxis O and both are present;1 and 2 independently represent points of attachment to RT, TPG, OA, OBor LP (as appropriate). 52. The process as defined in statement 51, wherein Rxis H or OH. 53. The process as defined in any one of the preceding statements, wherein the one or more chain extension cycles is five or more chain extension cycles. 54. The process as defined in any one of the preceding statements, wherein the one or more chain extension cycles is eight or more chain extension cycles. 55. The process as defined in any one of the preceding statements, wherein the one or more chain extension cycles is ten or more chain extension cycles. 56. The process as defined in any one of the preceding statements, wherein the one or more chain extension cycles is twelve or more chain extension cycles. 57. The process as defined in any one of the preceding statements, wherein the one or more chain extension cycles is fifteen or more chain extension cycles. 58. The process as defined in any one of the preceding statements, wherein each chain extension cycle comprises a step of cleaving the temporary protecting group (TPG, e.g., dimethoxytrityl) after coupling of the building block to a chain extension site of the growing oligonucleotide to expose a new chain extension site of the growing oligonucleotide. 59. The process as defined in any one of the preceding statements, wherein within each chain extension cycle, the step of coupling the monomeric or oligomeric building block to the chain extension site of the growing oligonucleotide form a P(III) phosphite triester linkage between the coupled building block and growing oligonucleotide. 60. The process as defined in any one of the preceding statements, wherein each chain extension cycle comprises a step of converting one of more P(III) phosphite triester linkages to a P(V) phosphotriester linkage (e.g., by oxidation or sulfur transfer) after coupling of the building block to a chain extension site of the growing oligonucleotide.61. The process as defined in any one of the preceding statements, wherein the process is performed in acetonitrile, or in a solvent mixture comprising acetonitrile (e.g., acetonitrile mixed with sulfolane). 63. The process as defined in statement 61, wherein all steps of a given chain extension cycle (e.g., coupling, oxidation, deprotection and membrane filtration) are performed in the same solvent (e.g., acetonitrile). 63. The process as defined in statement 61, wherein all chain extension cycles and their associated steps (e.g., coupling, oxidation, deprotection and membrane filtration) are performed in the same solvent (e.g., acetonitrile). 64. The process as defined in any one of the preceding statements, wherein membrane filtration steps are conducted by membrane diafiltration. 65. The process as defined in any one of the preceding statements, wherein membrane filtration steps are conducted by organic solvent nanofiltration or ultrafiltration. 66. The process as defined in statement 64 or 65, wherein the membrane is insoluble in the solvent(s) used in the step of coupling the monomeric or oligomeric building block to the chain extension site of the growing oligonucleotide (e.g., acetonitrile). 67. The process as defined in any one statements 64-66, wherein the membrane is polymeric. 68. The process as defined in statement 67, wherein the membrane is a crosslinked polybenzimidazole membrane (e.g., an integrally skinned, asymmetric, crosslinked polybenzimidazole membrane) or a polyetheretherketone membrane. 69. The process as defined in any one of the preceding statements, wherein the process further comprises one or more global deprotection steps. 70. The process as defined in any one of the preceding statements, wherein the growing oligonucleotide is attached (e.g., at nucleosidic moiety most distal to the chain extension site) to a soluble synthesis support.71. The process as defined in statement 68, wherein the soluble synthesis support is a polymer (e.g., a star polymer), a dendrimer, a dendron, a hyperbranched polymer, or an organic / inorganic material, including nanoparticles, fullerenes and 2-D materials such as graphene and boron nitride. 72. The process as defined in statement 70 or 71, wherein the soluble synthesis support is a star polymer comprising a central branch point and m number of radiating polymeric arms, wherein polymeric arm is attached to a growing oligonucleotide, and wherein m is 3 or more. 73. The process as defined in statement 72, wherein each polymeric arm is poly(ethylene glycol). 74. The process as defined in statement 70, wherein the soluble synthesis support may have a structure according to formula VIin which V is an organic branch point, W is a polymeric chain (e.g., poly(ethylene glycol)), represents a point of attachment to the growing oligonucleotide, and m is 1-8. 75. The process as defined in statement 74, wherein m is 3-4. 76. The process as defined in statement 74 or 75, wherein each W is a polymeric chain (e.g., poly(ethylene glycol)) having a molecular weight (Mw) of 500 – 20,000 Da. 77. The process as defined in statement 74, 75 or 76, wherein the soluble synthesis support is:in which represents the point of direct attachment to the growing oligonucleotide (e.g., at the 3’ oxygen) and q is 100 – 500 (e.g., 200 – 300). 78. The process as defined in any one of the preceding statements, wherein all A-, G- and C-containing building blocks comprises NPG and / or none of the building blocks comprise NPG- protected uracil or NPG-protected thymine. 79. The process as defined in any one of the preceding statements, wherein all A-, G- and C-containing building blocks comprises NPG and none of the building blocks comprise NPG- protected uracil and NPG-protected thymine. EXAMPLES

[0085] One or more examples of the invention will now be described, for the purpose of illustration only, with reference to the accompanying figures: Fig.1. Typical membrane-assisted oligo synthesis cycle with phosphoramidite building blocks. In SPOS a capping reagent is used to block any residual unreacted 5’-OH (commonly as an acetate ester), but this step can be omitted in LPOS. Fig.2. Synthesis of a PEG oligomer-bearing phosphoramidite building block, Dmtr-mU(mEg4An) phosphoramidite, 8a-c. Reagents and conditions: i, TsCl, NMI, TEA, DCM, 3 hr; ii, 4-HO-C6H4- CHO, K2CO3, DMF, 110 °C, 16 hr; iii, NaOMe, MeOH, DMF 90 min, then water 16 hr; iv, KMnO4, Na2HPO4, water; v, C2O2Cl2, DCM, 30 min; vi, Dmtr-mU, TmsCl, DIPEA, TEA, 90 min, then 13, 40 °C, 16 hr, then water 40 °C, 16 hr; vii, CneOP(NiPr2)2, Pyr.TFA, MeCN, 3 hr. Fig.3. UV Chromatogram globally deprotected 3-mer, mU3, prepared using mU(mEg4An). Fig. 4. UV Chromatogram globally deprotected 6-mer, mU6, prepared using all four mU phosphoramidites. Fig.51H NMR of mEg4An-OH, 5, in CDCl3. Fig.613C NMR of mEg4An-OH, 5, in CDCl3. Fig.71H NMR of Dmtr-mU(mEg4An) cyanoethyl phosphoramidite, 8c, in CDCl3.Fig.831P NMR of Dmtr-mU(mEg4An) cyanoethyl phosphoramidite, 8c, in CDCl3. Fig.91H NMR of Dmtr-mA(mEg4An) cyanoethyl phosphoramidite, 19, in CDCl3. Fig.1031P NMR of Dmtr-mA(mEg4An) cyanoethyl phosphoramidite, 19, in CDCl3. Example 1: Synthesis of Dmtr-mU(mEg4An) phosphoramidite

[0086] Preparation of mono-tosyl tetragol, TsO-Eg4-OH, 2: Tetraethylene glycol (1, 4.86 g, 22.0 mmol) was dissolved in dry dichloromethane (DCM, 10 mL) under argon. The solution was cooled to 0 °C, and triethylamine (TEA, 1.39 mL, 10 mmol) was added followed by N- methylimidazole (NMI, 0.199 ml, 2.5 mmol), and finally p-toluenesulfonyl chloride (TsCl, 0.953 g, 5 mmol; limiting reagent for steps up to 12). The reaction was stirred at room temperature for 3h, after which the solvent was evaporated. The residue was re-dissolved in DCM and washed with water (x3), then brine. The organic phase was dried over MgSO4, evaporated to dryness, and the crude oil used without further purification.

[0087] Preparation of 4-(methyltetragol)benzoic acid, mEg4AnOH, 5: Crude TsO-Eg4-OH (2) from the previous reaction was dissolved in dry DMF (10 mL) under Ar. To this solution was added 4-hydroxybenzalde (0.61 g, 5.0 mmol), followed by K2CO3 (1.1 g, 7.5 mmol). The reaction was stirred at 110 °C overnight. The next day, the reaction was allowed to cool down to room temperature before to be filtered through a sinter funnel. The crude mixture was then transferred to a separatory funnel and the product extracted 3 times with DCM until disappearance of the hydroxy aldehyde 3 peak from the aqueous phase. The organic layer was dried over Na2SO4, and the solvent evaporated.

[0088] To the crude hydroxy aldehyde 3 was added dry DCM (80 mL). The solution was then cooled to 0 °C, and Et3N (31.4 mL, 225 mmol), followed by NMI (4.5 mL, 56 mmol) were added. Finally, TsCl (21.4 g, 112) was added to the reaction in four roughly equal portions, when a cloudy precipitate formed, and the suspension stirred for 90 mins. After this the solvent was stripped off, and the residue co-evaporated (x3) from dry MeCN.

[0089] The residue was kept under high vacuum for 2h, then taken up in dry DMF (50 mL). To this solution was added methanolic NaOMe (25 wt%, 30 ml). After 90 min, the reaction appeared complete by reversed phase analytical chromatography, when water (30 mL) was added and stirring continued overnight. The next day, brine was added and extraction with DCM was repeated (x5) until disappearance of the peak methoxy aldehyde 4 from the aqueous phase. The organic layer was dried over Na2SO4, and the solvent stripped off.

[0090] To the residue was added an aqueous solution of Na2HPO4 (1.34 g made up to 10 mL; add 5 mL, 3.3 mmol). To the briskly stirred solution were added portions saturated aqueousKMnO4(0.5 mL each time, up to 10 portions); once the purple colour had dissipated, the degree of reaction was determined by analytical reversed phase analytical chromatography, and another portion of oxidant was added if required. Once complete conversion of the aldehyde to carboxylic acid 5 had been confirmed, solids were removed by filtration through a glass sinter funnel. To the pale yellow filtrate was added DCA dropwise to pH 3 / 4 resulting in the appearance of a white precipitate. Brine (6 mL) was then added and the aqueous solution was extracted with portions of DCM (x6) until no acid remained in the aqueous phase. The crude material was purified by normal phase chromatography using a 25g cartridge (Sfar Duo, Biotage), eluting with a gradient of MeOH-DCM containing 0.1% TFA, to obtain the desired product (5, 704 mg, C16H24O7= 328.4, 43% w.r.t. TsCl used to make TsO-Eg4-OH 2), Figures 5 and 6.

[0091] Preparation of 4-(methyltetragol)benzoyl chloride, mEg4AnCl, 6: 4- (Methyltetragol)benzoic acid (5, 1.6 g) was dried under vacuum for 30min then dissolved in dry DCM (3 mL) under argon. Oxalyl chloride (2 mL) was added and the reaction was stirred for 30 min at room temperature; the reaction was monitored by dissolving samples in MeOH to detect the corresponding methyl ester in HPLC. Once the reaction was complete, the solvent and remaining oxalyl chloride were evaporated under high vacuum, and the crude acyl chloride 6 was used without further purification.

[0092] Preparation of 5’-O-Dmtr-2’-O-methyl-3-N-[4-(methyltetragoloxy)benzoyl] uridine, 8c: 5’-O-Dmtr-2’-O-methyl uridine (Dmtr-mU-OH, 540 mg, 0.963 mmol) was dissolved in dry pyridine (5 mL) under argon, followed by the addition of diisopropylethylamine (DIPEA, 0.42 mL, 2.41 mmol). The reaction mixture was cooled to 0 °C and chlorotrimethylsilane (TmsCl, 0.5 mL, 3.85 mmol) was added dropwise. The reaction mixture was stirred at room temperature for 90 min, then transferred by syringe into a flask containing the crude mEg4AnCl, prepared as above. The reaction was stirred overnight at 40 °C when the intermediate Dmtr-mU-OTms was seen to be consumed by HPLC analysis. The reaction mixture was then allowed to cool to room temperature and cold water (1 mL) was added dropwise. The reaction mixture was once more stirred overnight at 40 °C to ensure complete deprotection of the 3-'OH. The solvents were evaporated and the residual gummy residue was partitioned between DCM and brine before to be transferred to a separated funnel. The crude mixture was extracted 3 times with DCM, until no product remained in the aqueous phase. The crude mixture was purified by normal phase liquid chromatography. 25 g cartridge (Sfar Duo, Biotage) eluting with a gradient of EtOAc- heptane-isopropanol containing 0.1% TEA. The appropriate fractions were combined and evaporated to dryness to give the desired product (8c, 658 mg, C47H54N2O14 = 870.9, 78%), Figures 7 and 8

[0093] Preparation of 5’-O-Dmtr-2’-O-methyl-3-N-[4-(methyltetragoloxy)benzoyl] uridine 3’-(2-cyanoethyloxy)-N,N-diisopropyl phosphoramidite, 8c: Dmtr-mU(mEg4An) (7a, 385 mg,0.411 mmol) was evaporated from dry MeCN (x3) and held under high vacuum for 1 hr. The residue was dissolved in dry MeCN (5 mL) under argon and 2-cyanoethyl N,N,N’,N’- tetraisopropyl phosphoramidite (0.16 mL, 0.49 mmol, 1.2 eq.) was added, followed by pyridinium trifluoroacetate (95 mg, 0.49 mmol). The reaction was stirred for 3 hr at room temperature. The solvent was then evaporated and the residue loaded directed onto 25 g silica cartridge (Sfar Duo, Biotage) which was eluted with a gradient of EtOAc-heptane containing 0.1% TEA. The appropriate fractions were combined and evaporated to dryness to give the desired product (8c, 388 mg, C56H71N4O15P = 1071.2, 82%).

[0094] The homologous Eg1and Eg2protecting groups, mEg1AnCl and mEg2AnCl, were prepared similarly to mEg4AnCl 6, and reacted with Dmtr-mU as above (Figure 2) to prepare the corresponding phosphoramidites 8a and 8b. Dmtr-mC was also reacted with mEg4AnCl 6 as above to provide Dmtr-mC(mEg4An) phosphoramidite 9.

[0095] The solubility of trityl uridine phosphoramidites 8, with a PEG-oligomer bearing protecting group, were determined in dry acetonitrile, the preferred solvent for oligo synthesis. The solubility of 8c was found to be far higher than other common uridine building blocks, see Table 1. Notably, the related Dmtr-mU(mEg1An) phosphoramidite (8a) with only a single pendant ethylene glycol monomer unit on the nucleobase had a lower solubility than the known anisoyl or benzoyl protected uridine phosphoramidites, demonstrating that >1 monomer unit was required for the effect.Table 1. Solubility of phosphoramidite building blocks in anhydrous acetonitrile. U(Bz) = 3N- benzoyl uridine; U(An) = 3N-anisoyl uridine. Phosphoramidite MW m / g V / mL Solubility Cn / M / g.L-1Dmtr-mU, 14 760.8 0.500 10.00 0.1 0.07 Dmtr-mU(Bz), 15 863.9 0.106 0.08 1.3 1.53 Dmtr-mU(An), 16 894.4 0.102 0.06 1.7 1.90 Dmtr-mU(mEg1An), 8a 983.7 0.051 0.05 1.0 1.09 Dmtr-mU(mEg2An), 8b 983.1 0.25 0.12 2.1 2.12 Dmtr-mU(mEg4An), 8c 1,071.2 0.104 Miscible in all proportions Dmtr-mC(Ac) 801.9 0.202 0.14 1.4 1.80 Dmtr-mC(mEg4An), 9 1,070.2 0.192 0.10 1.9 1.79 Dmtr-mA(Bz) 888.0 0.127 0.05 2.546 2.869 Dmtr-mA(mEg4An), 19 1,094.2 0.128 Miscible in all proportions Example 2: Synthesis of homo-mU6 with protected uridine phosphoramidites

[0096] Loading of Dmtr-mU succinate onto 10 kDa 4-arm PEG: 5’-O-Dmtr-2’-methyl uridine 3’-O-succinate trimethylammonium salt (10, Dmtr-mU-Suc-OH.TEA, 0.91 g, 1.2 mmol, 10 eq.) was dissolved in anhydrous MeCN (5 mL) and premixed with 1-[bis(dimethylamino)methylene]- 1H-benzotriazolium 3-oxide tetrafluoroborate (TBTU, 0.389 g, 1.21 mmol, 10 eq.), stirring for 30 minutes at room temperature. In a separate flask, sarcosine terminated 4-arm 10 kDa PEG star [11, PEG-10k(Sar-H)4, 1.23 g, MW 10.3 kDa, 0.119 mmol) was dissolved in anhydrous MeCN (10 mL, 26.8 ppm water) to which was added diisopropylethyl amine (DIPEA, 0.25 mL, 1.4 mmol, 12 eq.). The solution of PEG-10k(Sar-H)4 was transferred to the activated succinate solution and the amidation reaction was monitored by LC-MS; the reaction appeared complete after 5 minutes, but continued for 1 hr. The solution was then transferred to the feed tank of a Nanostar synthesizer (system volume 50 mL, PBI17-DBX-M2005 membrane), washing out the flask with further MeCN (5 mL). The solution was diafiltered with four system volumes (or diavolumes, DV) of MeCN (= 200 mL). At the end of the end of this first diafiltration (DF1) the fractional drop in the concentration of the Dmtr-succinate was determined by LC-MS, as well as the rejection of the trityl nucleoside-star [12a, PEG-10k(SarSuc-mU-Dmtr)4].

[0097] Detritylation: A solution of cation trap (2.5 eq. per arm; dodecane thiol can be used for this purpose) was injected into the feed tank via the injection port using a syringe fitted with a 0.2 micron PTFE filter, followed by a 1 M solution of methanesulfonic acid (MSA) in in acetonitrile (2.4 eq. per arm). The detritylation reaction was monitored by LC-MS and after 20 min no partially deprotected species could be detected. The reaction was quenched with 3-picoline (0.57 mL). A second diafiltration was undertaken (DF2) with anhydrous MeCN (300 mL = 6DV, <20 ppm water) to remove residual 5’-hydroxy-mU 3’-succinate and other residual reaction debris. At the end of DF2 complete removal of building block debris was confirmed by LC-MS, as well as the continued high rejection of the hydroxy nucleoside-star [12b, PEG-10k(SarSuc-mU-OH)4]. The next cycle of chain extension is only initiated if the combined drop in building block concentration over DF1 and DF2 is >99%, or else DF2 is continued until this condition is satisfied.

[0098] Coupling reaction: For the first synthetic cycle (dimer coupling) Dmtr-mU(mEg4An) phosphoramidite (8c, 0.77 g, 0.7 mmol, 1.5 eq. per arm) and dicyanoimidazole (DCI, 0.23 g, 1.9 mmol, 4 eq. / arm) were each placed in a separate vials. The building block was co-evaporated from anhydrous MeCN (<30 ppm water, 3 x 5 mL). Building block was re-dissolved in anhydrous MeCN (2 mL) and injected into the synthesizer, followed by the DCI dissolved in anhydrous MeCN (2 mL); all components were injected into synthesizer via a 0.2 micron PTFE filter. The reaction was monitored by LC. After 2 minutes the coupling intermediates were consumed, but circulation was continued in the synthesizer for 20 minutes to ensure complete reaction.

[0099] After 20 minutes, 2-cyanoethanol (CneOH, 0.1 mL, 1.4 mmol, 3 eq. per arm) was added to the feed tank. Camphorsulfonyl oxaziridine (CSO, 0.74 g, 3.2 mmol, 6.75 eq. per arm) dissolved in MeCN (5 mL) was then added to the synthesiser to obtain the chain extended phosphate tri-ester 13a. After 60 minutes of circulation, completion of oxidation was verified by LS-MS and the crude dimer-star was purified by diafiltration with dry acetonitrile (DF1, 4 DV). This was followed by detritylation, quenching with 3-picoline, and DF2 (6 DV), as above, to obtain the hydroxy dimer-star, 13b.

[0100] The synthetic cycle described above was repeated in the same fashion, coupling, then oxidation, then detritylation, changing the building block as required (see Table 2), to elongate the oligo chain up to the desired length.Table 2. Building block used for the synthesis. Stage Building block 1-mer 5'-Dmtr-mU-Suc-OH.TEA, 10 2-mer Dmtr-mU(mEg4An) phosphoramidite, 8c 3-merDmtr-mU(mEg4An) phosphoramidite, 8c4-merDmtr-mU phosphoramidite, 145-merDmtr-mU(Bz) phosphoramidite, 156-merDmtr-mU(An) phosphoramidite, 16

[0101] Membrane performance and rejection data: An overview of the homo-sequence run, with details of diafiltration conditions is provided below in Table 3. Membrane was PBI17-DBX- Jeffamine M2005; all rejections measured at 5 bar, R is defined according to equation 1. Table 3. Diafiltration data for oligo-stars and building block (BB) debris during the homo-mU6synthesis. OligoRejection, RAverageMonomer length HO-oligo- BB debris BB debris permeance star end of DF1 end of DF2 mL / min mU(mEg4An), 8c 2-mer 99.97% 38% 7% 1.3 mU(mEg4An), 8c 3-mer 99.93% 27% 22% 1.1 mU, 14 4-mer 100% 18% n.d. 1.2 mU(Bz), 15 5-mer 99.84 % 27 % n.d. 1.0 mU(An), 16 6-mer 100 % 31 % 6% 1.0

[0102] Global deprotection: During the synthesis, oligo-stars were periodically analysed by global deprotection of a sample of the circulating fluid from the synthesiser. The purities of the crude oligos were estimated by analytical ion-pair reversed phase UHPLC chromatography. A sample of crude HO-oligo-star solution (3 mL, ca. 0.048 mmol oligo) was withdrawn from the synthesizer. The sample was placed in an ACE pressure tube and dissolved in a mixture of concentrated aq. ammonia (3 mL) and diethylamine (3 vol%). The tube was sealed, and the solution was heated at 35 °C overnight with constant stirring. The next day the ammonia solution was transferred to a round bottom flask and evaporated. The aqueous residue was co- evaporated from MeCN three times, and the residue was finally triturated with MeCN. The suspension was transferred to a Falcon tube (~30 mL) and centrifuged (5 min, 6000 rpm). The supernatant was removed, and the precipitate washed and centrifuged twice more with further MeCN. The precipitate was then analysed on Agilent UHPLC by IP-RP.

[0103] Analysis of the 3-mer prepared with Dmtr-mU(mEg4An) phosphoramidite building block 8c is shown in Table 4 and the corresponding chromatogram shown in Figure 3. Analysis of the 6-mer, mU6, prepared using all four mU phosphoramidites is shown in Table 5 and the corresponding chromatogram shown in Figure 4. Table 4. Analysis summary of 3-mer. ^ S6-0093mer^DF2@6DV CGD^ Overall purity^ 80.6% MS purity^ 97.5% UV purity^ 82.7% n-1^ 2.7% n+1^ 0.2% 52 Da loss from FL / Loss of Acrolein 2.3% Total Phosphate Cap / +80Da Ladder 3.0% FLP + 344Da 1.1% FLP + 258Da 5.9% Others^ 2.1% Table 5. Analysis summary of 6-mer. ^ S6-0096mer^DF2@6DV CGD^ Overall purity^ 67.1% MS purity^ 97.6% UV purity^ 68.7% n-1^ 2.2% 52 Da loss from FL / Loss of Acrolein 2.3% Total Phosphate Cap / +80Da Ladder 4.0% FLP + 344Da 0.4% FLP + 258Da 9.8% Others^ 12.6%Example 3: Synthesis of Dmtr-mA(mEg4An) phosphoramidite

[0104] Preparation of 5’-O-Dmtr-2’-O-methyl-6-N-[4-(methyltetragol)benzoyl] adenosine, 18: Dmtr-mA-OH (17, 6.0 g, 10.3 mmol) was dissolved in dry pyridine (14.4 mL) and dry THF (20.4 mL) under argon, followed by the addition of diisopropylethylamine (DIPEA, 8.96 mL, 51.4 mmol). The reaction mixture was cooled to 0 °C and chlorotrimethylsilane (TmsCl, 3.4 mL, 26.8 mmol) was added dropwise. The reaction mixture was stirred at room temperature for 90 min, then transferred by syringe into a flask containing the crude mEg4AnCl (6, 7.1 g, 20.6 mmol). The reaction was stirred overnight at 40 °C; the intermediate Dmtr-mA-Tms was seen to be consumed by HPLC analysis. The reaction mixture was then allowed to cool to room temperature and cold water (10 mL) was added dropwise. The reaction mixture was stirred for a further 1h at room temperature and the solvent was then evaporated. The resulting doubly protected nucleoside was dissolved in MeCN-water (1:1, 100 mL), then transferred into a pressure tube to which conc. ammonia was added (10 mL)*. The reaction was stirred overnight at 35 °C. The next day the solvent was evaporated and the gummy residue was partitioned between DCM and water. The crude mixture was extracted with a saturated solution of NaHPO3(x3), after which the aqueous layers were combined and back-extracted (x3) with DCM until no product remained in the aqueous phase. The organic layers were combined, dried over MgSO4 and the solvent removed giving the desired product (18, 10.4 g, C48H55N5O12 = 893.9).

[0105] * It should be noted that Dmtr-mA(mEg4An)-PO(OCne)2 can be completely deprotected to 5’-Dmtr-mA-3’-phosphate with conc. ammonia-MeCN 1:1 at 35 °C for 16 hr.

[0106] Preparation of 5’-O-Dmtr-2’-O-methyl-6-N-[4-(methyltetragol)benzoyl] adenosine 3’-(2-cyanoethyloxy)-N,N-diisopropyl phosphoramidite, 19: Dmtr-mA(mEg4An) (18, 10.0 g, 11.2 mmol) was evaporated from dry MeCN (x3) and held under high vacuum for 1 hr. The residue was dissolved in dry MeCN (5 mL) under argon and 2-cyanoethyl N,N,N’,N’- tetraisopropyl phosphoramidite (7.1 mL, 22.4 mmol, 2 eq.) was added, followed by pyridinium trifluoroacetate (4.1g , 21.3 mmol). The reaction was stirred for 3 hr at room temperature. The solvent was then evaporated and the residue loaded directly onto silica cartridge (200 g, Sfar Duo, Biotage) which was eluted with a gradient of heptane-acetone containing 0.1% TEA. The appropriate fractions were combined and evaporated to dryness to give the desired product (19, 3.3 g, C57H72N6O13P = 1138.9, 26%), Figures 9 and 10.

[0107] The solubility of trityl adenosine phosphoramidite 19, with a PEG-oligomer bearing protecting group, was determined in dry acetonitrile, when its solubility was found to be far higher than the commonly used benzoyl protected adenosine building block, see Table 1.Example 4: Solubilising a pentamer-star with a single mEg4An group

[0108] Loading of 5’-Dmtr-mU-3’-succinate onto 20 kDa PEG-star: Two separate runs were started. In each case PEG-20k(NMeH)8support (2.49 g, MW 20.6 kDa, 0.12 mmol) was dissolved in anhydrous MeCN (10 mL, 26.8 ppm) to which was added DIPEA (0.29 mL, 1.5 mmol, 14 eq.) and the mixture was stirred at room temperature for 30 minutes.5’-Dmtr-mU-3’-succinate, TEA salt (1.11 g, 1.5 mmol, 12 eq.) was dissolved in anhydrous MeCN (5 mL) to which was added TBTU (0.47 g, 1.5 mmol, 12 eq.) and the solution was stirred for 30 minutes at room temperature. The solution containing the PEG-20k(NMeH)8support was then transferred to the succinate solution. Monitoring the amidation reaction by LC, it appeared complete after 5 minutes but was left for 1 hour to ensure completion. The solution was then transferred into the Nanostar synthesizer feed tank, washing out the flask with further MeCN (5 mL), and MeCN was then permeated (200 mL = 4DV, DF1).

[0109] Detritylation reaction: Into both reactor feed tanks was injected a solution of cation trap (2.5 eq.) via the injection port using a syringe fitted with a 0.2 micron PTFE filter. Dmtr deprotection was then initiated by injecting methanesulfonic acid (MSA, 2.4 eq., 1M solution in acetonitrile, 0.33 mL). The reaction was monitored by LC-MS and after 20 min no partially deprotected species could be detected. The reaction was quenched with 3-picoline (0.65 mL, 4.8 eq.). Dry MeCN (300 mL = 6DV, < 20 ppm water) was permeated (DF2), to remove residual 5’- hydroxy-mU 3’-succinate and other detritylation debris. At the end of DF2 LC-MS confirmed both complete removal of building block debris and the high rejection of the nucleoside-star.

[0110] Coupling reaction: For the dimer couplings, Dmtr-mA(Bz) cyanoethyl phosphoramidite (1.03 g, 1.15 mmol, 2.5 eq. / arm) and Dmtr-mA(mEg4An) cyanoethyl phosphoramidite (19, 1.26 g, 1.15 mmol, 2.5 eq. / arm) were each placed in separate vials. Each building block was co- evaporated from MeCN (<30 ppm water, 3 x 5 mL), then re-dissolved in MeCN (<30 ppm water, 2 mL) and injected into a synthesizer. Into each reactor was then injected DCI (0.34 g, 2.9 mmol, 6 eq. / arm) dissolved in MeCN (2 mL). The reactions were monitored by LC. After 2 minutes the coupling intermediates were consumed, but circulation was continued in the synthesizer for 20 minutes to ensure complete reaction. After 20 minutes, cyanoethanol (0.2 mL, 2.9 mmol, 6 eq. / arm) was added to the feed tank. After 2 minutes, 3-phenyl 1,2,4-dithiazoline-5-one (POS, 0.28 g, 1.45 mmol, 3 eq. / arm) dissolved in MeCN (5 mL) was added to each synthesiser to obtain the chain extended phosphorothioate tri-ester. After 5 minutes of circulation, diafiltration with dry acetonitrile (200 mL = 4 DV) was commenced to purify each Dmtr-dimer-star.

[0111] The synthetic cycle described above was repeated with Dmtr-mG(Ibu) cyanoethyl phosphoramidite in the same fashion to elongate the oligo chain up to 3-mer using dry acetonitrile (<20 ppm). During the trimer detritylation of mU-mA(Bz)-mG(Ibu), the membrane interfacebecame fouled, and a large reduction in permflow had to be alleviated by the addition of 20 vol% sulfolane to the diafiltration solvent. By contrast, detritylation of mU-mA(mEg4An)-mG(Ibu) trimer exhibited no drop in permeate flow despite still being in neat MeCN. The sequence containing mA(mEg4An) was continued as above for two more cycles in neat MeCN, firstly with Dmtr-mU cyanoethyl phosphoramidite, then Dmtr-mG(Ibu) cyanoethyl phosphoramidite, in both cases oxidising with CSO (6.75 eq. per arm). Diafiltration data is shown in Table 6. Table 6. Rejection and permflow data with and without mA(mEg4An) protection Oligo-star DF1 DF1 permflow / Oligo-star DF2 DF2 permflow / rejection / % mL.min-1rejection / % mL.min-1mU-Suc 99.90 99.76 1.6 1.6 98.73 99.58 1.6 1.6 PG Bz mEg4An Bz mEg4An Bz mEg4An Bz mEg4An mA(PG) 99.58 99.64 2.0 1.5 nd 99.46 1.5 1.4 mG(Ibu) 99.85* 99.97 1.8* 1.6 99.97* 99.70 1.0* 1.3 mU 99.81* 99.95 1.5* 1.3 99.98* 99.99 1.0* 1.2

[0112] Only when the pentamer containing mA(mEg4An) at the second position was detritylated did the 3’-mU-PS-mA(mEg4An)-PS-mG(Ibu)-PO-mU-PO-mG(Ibu)-5’ bearing hydroxy-oligo-star start to foul the membrane. This shows that the single mEg4An protecting group on mA provided a substantial beneficial effect for diafiltration of the whole oligo-star.

[0113] While specific embodiments of the invention have been described herein for the purpose of reference and illustration, various modifications will be apparent to a person skilled in the art without departing from the scope of the invention as defined by the appended claims. REFERENCES US 8,664,357 US 9,127,123 US 10,239,996EP 3347402 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.21 (2015) pp.9535-9543 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.20 (2016) 1439−1452 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 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.60 (2021) pp.7786-7795 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 11 (2019) pp.136-145 WO / 2016 / 188835 A1 M. C. Pirrung, L. Wang, M.-P. Montague-Smith “3′-Nitrophenyl-propyloxycarbonyl (NPPOC) Protecting Groups for High-Fidelity Automated 5′^3′ Photochemical DNA Synthesis” Org. Letts. 3 (2001) pp.1105-1108 M. Beier, J. D. Hohheisel, “Production by quantitative photolithographic synthesis of individually quality checked DNA microarrays”, Nucl. Acids Res.28 (2000) pp e11 US 7,759,513 B2 US 8,445,734 B2 N. Kretschy, M. M. Somoza et al. “Next-Generation o-Nitrobenzyl Photolabile Groups for Light- Directed Chemistry and Microarray Synthesis” Angew Chem. Intl. Ed.54 (2015) pp.8555-8559 J. Lietard, M. M. Somoza et al. “Chemical and photochemical error rates inlight-directed synthesis of complex DNA libraries” Nucl. Acids Res.49 (2021) pp.6687-6701 K. Hölz, M. M. Somoza et al. “High-Efficiency Reverse (5′→3′) Synthesis of Complex DNA Microarrays” 8 (2018) art. # 15099 A. Oxley, A. G. Livingston et al. “Graft modification of polybenzimidazole membranes for organic solvent ultrafiltration with scale up to spiral wound modules” J. Mem. Sci.647 (2022) 120199. M. Septak “Kinetic studies on depurination and detritylation of CPG-bound intermediates during oligonucleotide synthesis” Nucl. Acids Res.24 (1996) 3053-3058 A. G. Molina, Y. S. Sanghvi “Liquid-Phase Oligonucleotide Synthesis: Past, Present, and Future Predictions” Current Protocols in Nucleic Acid Chemistry (2019) e82 G. Creuse, A. Walther et al. “Scalable One-Pot-Liquid-Phase Oligonucleotide Synthesis for Model Network Hydrogels” J. Am. Chem. Soc.142 (2020) pp.16610-16621X. Zhou, X. Shi et al. “Development of Kilogram-Scale Convergent Liquid-Phase Synthesis of Oligonucleotides” J. Org. Chem.87 (2022) pp.2087-2110. M. C. de Koning, G. A. van der Marel et al. “Simple and Efficient Solution-Phase Synthesis of Oligonucleotides Using Extractive Work-Up” Org. Proc. Res. & Dev.10 (2006) pp.1238−1245. Y. Bakhatan, M. Hurevich, “The breaking beads approach for photocleavage from solid support” Org. Biomol. Chem.18 (2020) pp.4183-4188. L. Buglioni, T. Noël et al. “Technological Innovations in Photochemistry for Organic Synthesis: Flow Chemistry, High-Throughput Experimentation, Scale-up, and Photoelectrochemistry” Chem. Rev.122 (2022) pp.2752-2906. CB Reese, Org. Biomol. Chem., 2005, 3, 3851 Baran et al., ACS. Cent. Sci., 2021, 7, 1473 Arai et al. Bioorg. Med. Chem.2011, 21, 6285 US 6,683,173

Claims

CLAIMS 1. A liquid-phase process for preparing an oligonucleotide, the process comprising growing an oligonucleotide by performing one or more chain extension cycles, in which each chain extension cycle comprises a step of coupling a monomeric or oligomeric building block to a chain extension site of a growing oligonucleotide, wherein in at least one chain extension cycle, the building block comprises a nucleobase protected by a nucleobase protecting group (NPG), wherein NPG comprises a group Q of formula I:wherein n is 1-9 and R1is H or methyl, and wherein in at least one of the chain extension cycles in which the building block comprises NPG, one or more membrane filtration steps is used to isolate the growing oligonucleotide.

2. The process as claimed in claim 1, wherein each monomeric or oligomeric building block comprises: (i) at least one nucleosidic moiety (Nuc), (ii) a reactive terminal (RT) for coupling the building block to the chain extension site of the growing oligonucleotide, and (iii) a temporary protecting group (TPG), said temporary protecting group being cleavable, after coupling of the building block to a chain extension site of a growing oligonucleotide, to expose a new chain extension site of the growing oligonucleotide.

3. The process as claimed in claim 2, wherein each monomeric or oligomeric building block comprises 1-3 nucleosidic moieties.

4. The process as claimed in any one of the preceding claims, wherein in at least one of the chain extension cycles in which the building block comprises a nucleobase protected by NPG, the nucleobase is adenine, guanine or cytosine.

5. The process as claimed in any one of the preceding claims, wherein n is 2-3.

6. The process as claimed in any one of claims 1-4, wherein n is 1 or 2 7. The process as claimed in any one of the preceding claims, wherein each NPG independently has a structure according to formula IIa, IIb or IIc:wherein represents the point of attachment to the N or O atom (more typically the N atom) of the nucleobase, L1is selected from absent, –O–, –CH2–, –OCH2– and –CH2O–, L2is selected from absent and phenylene, and L3is selected from absent and –OCH2–, with the proviso that at least one of L1, L2and L3is not absent;wherein represents the point of attachment to the O atom of the nucleobase, and m is 0, 1 or 2;(IIIc) wherein represents the point of attachment to the N atom of the nucleobase, RAis selected from (1-6C)alkyl and Q,RBis selected from (1-6C)alkyl and –(CH2)p–Q, in which p is 1 or 2, and RCis (1-6C)alkyl, with the proviso that at least one of RAand RBis not (1-6C)alkyl.

8. The process as claimed in any one of the preceding claims, wherein in at least one chain extension cycle, the building block comprises a nucleobase protected by a NPG having the following structure:wherein Q2is as defined hereinbefore for Q, wherein n is 1 or 2.

9. The process as claimed in any one of the preceding claims, wherein in at least 20% of the chain extension cycles (to the nearest whole number), the building block comprises a nucleobase protected by NPG.

10. The process as claimed in any one of the preceding claims, wherein in at least 50% of the chain extension cycles (to the nearest whole number) in which the building block comprises NPG, membrane filtration is performed to isolate the growing oligonucleotide.

11. The process as claimed in any one of the preceding claims, wherein each chain extension cycle membrane filtration steps is performed comprises: a first membrane filtration step to separate the NPG-containing oligonucleotide from excess, unreacted building block, and a second membrane filtration step to separate cleaved temporary protecting group from the NPG-containing oligonucleotide.

12. The process as claimed in any one of the preceding claims, wherein each building blocks has a structure according to formula III:wherein RT is a reactive terminal for coupling the building block to the chain extension site of the growing oligonucleotide, TPG is a temporary protecting group that is cleavable, after coupling of the building block to a chain extension site of the growing oligonucleotide, to expose a new chain extension site of the growing oligonucleotide, Lp is an internucleoside linkage, and v is 0-10 (e.g., 0-3).

13. The process as claimed in claim 12, wherein each building block has a structure according to formula IIIa: (IIIa) wherein OAis the oxygen atom located at the 5’ carbon of the nucleosidic moiety to which it is attached, and OBis the oxygen atom located at the 3’ carbon of the nucleosidic moiety to which it is attached, or OAis the oxygen atom located at the 3’ carbon of the nucleosidic moiety to which it is attached, and OBis the oxygen atom located at the 5’ carbon of the nucleosidic moiety to which it is attached.

14. The process as claimed in any one of claims 2-13, wherein each RT is independently a phosphoramidite, a phosphate monoester, a phosphate diester, an H-phosphonate, a cyclic thiophosphate or a cyclic dithiophosphate triester moiety.

15. The process as claimed in any one of claims 2-14, wherein each RT independently has a structure according to formula IV:wherein LG is a leaving group; PG is a protecting group; and represents the point of attachment to the nucleosidic moiety (Nuc) to which RT is attached (e.g., via OAin formula IIIa).

16. The process as claimed in any one of claims 2-15, wherein RT has a structure according to the following:where represents the point of attachment to the nucleosidic moiety (Nuc) to which RT is attached (e.g., via OAin formula IIIa).

17. The process as claimed in any one of claims 2-16, wherein each TPG is independently selected from the group consisting of dimethoxytrityl (Dmtr / DMT), tert-butyl (tBu), tert-butyl oxycarbonyl (Boc), mono-methoxytriphenyl (Mmtr), triphenyl methyl (Tr), pentamethyl dihydrobenzofuran sufonyl (Pbf), Tetrahydropyranyl (Thp), tetrahydrofuranyl (Thf), para- methoxybenzyl (Pmb) and 2,4-dimethoxybenzyl.

18. The process as claimed in any one of claims 12-17, wherein within formula III or IIIa, each nucleosidic moiety (Nuc) has a structure according to formula V:wherein Z is a nucleobase, optionally protected by a protecting group, wherein said protecting group may be NPG; Rxis selected from H, OH, F, O-tert-butyldimethylsilyl (OTbdms), methoxy, O-methoxyethyl (OMoe), O-propargyl, NH2and N3, in which caseare absent, or Rxis O and bothpresent; 1 and2independently represent points of attachment to RT, TPG, OA, OBor LP(as appropriate).

19. The process as claimed in any one of the preceding claims, wherein the one or more chain extension cycles is five or more chain extension cycles.

20. The process as claimed in any one of the preceding claims, wherein each chain extension cycle comprises a step of cleaving the temporary protecting group (TPG, e.g., dimethoxytrityl) after coupling of the building block to a chain extension site of the growing oligonucleotide to expose a new chain extension site of the growing oligonucleotide.

21. The process as claimed in any one of the preceding claims, wherein within each chain extension cycle, the step of coupling the monomeric or oligomeric building block to the chain extension site of the growing oligonucleotide form a P(III) phosphite triester linkage between the coupled building block and growing oligonucleotide.

22. The process as claimed in any one of the preceding claims, wherein each chain extension cycle comprises a step of converting one of more P(III) phosphite triester linkages to a P(V) phosphotriester linkage (e.g., by oxidation or sulfur transfer) after coupling of the building block to a chain extension site of the growing oligonucleotide.

23. The process as claimed in any one of the preceding claims, wherein all steps of a given chain extension cycle (e.g., coupling, oxidation, deprotection and membrane filtration) are performed in the same solvent (e.g., acetonitrile or a solvent mixture comprising acetonitrile).

24. The process as claimed in any one of the preceding claims, wherein membrane filtration steps are conducted by membrane diafiltration.

25. The process as claimed in any one of the preceding claims, wherein the growing oligonucleotide is attached (e.g., at nucleosidic moiety most distal to the chain extension site) to a soluble synthesis support.

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