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. More specifically, the present invention relates to a membrane filtration-assisted, liquid-phase process for preparing oligonucleotides including photolabile protecting groups (PPG) and a soluble synthesis support.

Description

LIQUID-PHASE PROCESS FOR PREPARING OLIGONUCLEOTIDESINTRODUCTION

[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 are short sequence-defined polymers of nucleotides (i.e., nucleoside phosphates). The structure of oligonucleotides 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 oligonucleotides.

[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. Oligonucleotide-based drugs differ chemically from natural oligonucleotides in that they bear chemical modifications at multiple sites, principally to increase stability in the body and to improve targeting. However, similarly to natural oligonucleotides, it is the precise sequence of nucleobase side-chains that defines the oligonucleotide’s pharmaceutical function.

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

[0005] For decades oligonucleotides have been prepared using solid-phase oligonucleotide synthesis (SPOS) wherein a growing oligonucleotide is tethered to an insoluble solid support and grown by flowing reactive nucleotidic 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, 2) oxidation of, or sulfur transfer to the internucleotide linkage, and 3) removal of a temporary chain terminal protecting group to expose a new OH group (see steps 1 , 2 and 4 in figure 1). The cycle is repeated to build up the desired oligonucleotide sequence one building block at a time. The efficiency of each cycle is important to ensure a high purity of the crude full-length product (FLP) oligonucleotide. For example, with a 99.5% cycle efficiency, after20 cycles the resultant oligonucleotide 21-mer will have a purity of 90.5%. By contrast a 98% cycle efficiency, after 20 cycles, will result in a purity of 66.8%. In addition to the three main steps outlined above, most oligonucleotide synthesis protocols also include a capping step (see step 3 in figure 1) to prevent any unextended chain termini from participating in further cycles, thus reducing the number of chromatographically similar impurities to the FLP oligonucleotide. However, capping is only partially effective and can introduce impurities itself.

[0006] The nucleotidic building block used in the chain extension cycle is typically protected to avoid uncontrolled polymerisation. Protection of the building block is often as follows: The phosphorus bearing moiety is a highly reactive P(lll) phosphoramidite, usually bound to the ribose 3’-O, 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 (e.g., acyl or amidine) or base-labile (e.g., beta-eliminating) groups; and the 5’-0 of ribose is temporarily protected with an acid-labile protecting group, almost universally 4,4’-dimethoxytriphenylmethyl (i.e., dimethoxytrityl, Dmtr).

[0007] Although SPOS has been the industry standard for many years, there are a number of drawbacks associated with this method. In particular, an excess of nucleotidic 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 oligonucleotide per batch. This is highly undesirable for pharmaceutical preparation if tonnes of oligonucleotide per annum are required for a major medical indication (e.g., cardiovascular disease). In addition to this, during synthesis side reactions are prevalent, contributing to a drop in purity of the FLP oligonucleotide, thereby contributing to yield loss.

[0008] An alternative strategy to SPOS which aims to address these 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 oligonucleotide preparation at scale. A typical approach to LPOS is to carry out sequential coupling reactions, adding building blocks (i.e., monomers or multi-monomer oligomers) to a growing oligonucleotide in solution in a stepwise fashion, and then to use a suitable separation technology (A. G. Molina et al), such as membrane filtration (A.G. Livingston et al, P.R.J. Gaffney et al, J.F. Kim et al, So Su et al and Yeo J et al), precipitation (G. Creusen et al and X. Zhou et al), or liquid extraction (M. C. de Koning et al) to separate unreacted building blocks and reaction debris from the growing oligonucleotide.

[0009] Membrane-assisted LPOS is unique amongst oligonucleotide synthesis strategies in that it requires the solution of growing oligonucleotide to be contacted with a membrane separator. The entire volume of reaction solution can be circulated past the membrane under pressure multiple times to effect efficient purification. This is an inherent advantage to membrane- assisted LPOS as the entire reaction solution can be observed and sampled in real time, unlikein SPOS where the only species that can be analysed practically during the growth process are those that wash off the support bed, not the actual growing oligonucleotide.

[0010] In spite of their numerous advantages relative to SPOS, the solution-phase nature of membrane-assisted LPOS techniques often makes them more susceptible to side reactions and the formation of unwanted byproducts, which can accumulate with each chain extension cycle, thereby compromising the isolation, purity and yield of the final oligonucleotide.

[0011] The present invention was devised with the foregoing in mind.SUMMARY OF THE INVENTION

[0012] 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, wherein each chain extension cycle comprises coupling a monomeric or oligomeric building block to a chain extension site of a growing oligonucleotide, wherein each building block comprises: at least one nucleosidic moiety (Nuc); a reactive terminal (RT) for coupling the building block to the chain extension site of the growing oligonucleotide; and 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; wherein in at least one chain extension cycle: the temporary protecting group (TPG) is a photolabile protecting group (PPG); and membrane filtration is performed to isolate the growing oligonucleotide (e.g., from uncoupled building blocks and / or reaction debris).

[0013] According to a second aspect of the present invention there is provided a liquid-phase oligonucleotide synthesis (LPOS) process, wherein a photolabile protecting group (PPG) is used to protect the chain extension site of a growing oligonucleotide, the growing oligonucleotide being isolated by membrane filtration.

[0014] According to a third aspect of the present invention there is provided an oligonucleotide obtained, directly obtained or obtainable by the process of the first or second aspect.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 same subject 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 membrane-assisted liquid-phase process for preparing an oligonucleotide according to the first and second aspects, which addresses the aforementioned disadvantages associated with conventional LPOS techniques. The inventors have found that using photolabile protecting groups presents several advantages in the context of membrane-assisted LPOS. The use of strongly acidic reagents to cleave acid-labile protecting groups (e.g., Dmtr - the most commonly used temporary protecting group in oligonucleotide synthesis) is prone to initiating side reactions by protonating weakly basic heteroatoms on the oligonucleotide. For example, oligonucleotides can undergo depurination as a consequence of acid-induced protonation of 7-N of either adenine or guanine. By reducing the reliance on acid-based deprotection, incidence of these side reactions and generation of their resulting byproducts is considerably reduced, thereby facilitating membrane separation of the growing oligonucleotide, and improving purity and yield. Furthermore, in the membrane-assisted process of the invention, photodebris (such as the cleaved PPG) can be removed from the reaction medium as soon as it is generated, thereby preventing it from continuing to absorb light, which would otherwise compromise the efficiency of the intended, light-induced deprotection step. Reducing the use of strongly acidic reagents in membrane- assisted LPOS also means that the membrane itself is not subjected to significant and regular fluctuations in pH, which may have a deleterious effect on membrane performance.

[0019] The following paragraphs provide further discussion of the features of the first and second aspects of the invention.

[0020] The process of the invention is a liquid-phase process and is therefore conducted in solution. It will be understood that the growing oligonucleotide remains in a dissolved state during chain extension cycles, including when it is isolated by membrane filtration.

[0021] 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. In the context of the present invention, the term “oligonucleotide” (i.e., used in isolation) refers to the sequence of nucleotides after the final coupling reaction (sometimes termed the full-length product (FLP) in industry), whereas the term “growing oligonucleotide” refers to the sequence of nucleotides up to (and during) the final coupling reaction. In its simplest sense, the “growing oligonucleotide” may be a single nucleotide.

[0022] Those of ordinary skill in the art will be familiar with the concept of a chain extension cycle in the context of oligonucleotide 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(lll) state to its P(V) state), and a deprotection step (to expose a new reactive terminal on the growing oligonucleotide), with purification / isolation techniques being employed at one or more instances during each cycle.

[0023] The building blocks used in the process can be monomeric or oligomeric (e.g., 2-8 mer), wherein mer refers to a nucleosidic moiety (i.e., a 2-mer is a building block comprising two nucleosidic moieties). Therefore, a monomeric building block comprises one nucleosidic moiety, Nuc (e.g., ribose coupled to a nucleobase), a reactive terminal, RT, and a temporary protecting group, TPG. In monomeric building blocks, RT and TPG may be covalently bound to Nuc. An oligomeric building block comprises two or more nucleosidic moieties, Nuc (e.g., ribose coupled to a nucleobase), each pair of adjacent nucleosidic moieties being linked by an internucleoside linkage, a reactive terminal, RT, and a temporary protecting group, TPG. In oligomeric building blocks, RT may be covalently bound to one terminal nucleosidic moiety and TPG may be covalently bound to the other terminal nucleosidic moiety.

[0024] 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 alternative backboneforming heterocyclic moieties, such as in morpholine nucleosides, as well as the manner in which they are coupled to form oligonucleotides. It will be understood that such modifications andvariations 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.

[0025] In most instances, each building block independently comprises 1-3 nucleosidic moieties. Most often, each building block is monomeric (i.e. , comprises 1 nucleosidic moiety).

[0026] Each building block may have a molecular weight of >200 Da. Suitably, each building block has a molecular weight of >400 Da. More Suitably, each building block has a molecular weight of >600 Da.

[0027] Each building block typically has a structure according to formula I:I whereinNuc is a nucleosidic moiety;RT is the reactive terminal, which is covalently bound to Nuc;TPG is the temporary protecting group, which is covalently bound to Nuc;Lpis an internucleoside linkage; andV is 0-10 (e.g., 0-3).

[0028] In embodiments wherein the building block is monomeric (i.e., v is 0), RT and TPG are covalently bound to the same (and only) nucleosidic moiety. In embodiments wherein the building block is oligomeric (i.e., v is 1-10), RT is covalently bound to one terminal nucleosidic moiety and TPG is covalently bound to the other terminal nucleosidic moiety.

[0029] 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(lll) 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 oligonucleotide has been reached (e.g., during global deprotection, as described herein).

[0030] In most instances, v is 0, 1 or 2. Most often, v is 0.

[0031] RT may be covalently bound at the 5’ terminal of the nucleosidic moiety to which it is attached. Alternatively, RT may be covalently bound at the 3’ terminal of the nucleosidic moiety to which it is attached. TPG may be covalently bound at the 5’ terminal of the nucleosidic moiety to which it is attached. Alternatively, TPG may be covalently bound at the 3’ terminal of thenucleosidic moiety to which it is attached. Thus, in some embodiments RT is covalently bound at the 5’ terminal of the nucleosidic moiety to which it is attached, and TPG is covalently bound at the 3’ terminal of the nucleosidic moiety to which it is attached. In alternative embodiments, RT is covalently bound at the 3’ terminal of the nucleosidic moiety to which it is attached, and TPG is covalently bound at the 5’ terminal of the nucleosidic moiety to which it is attached. In the aforementioned 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) and include the oxygen atoms located at the 3’ and 5’ terminals (termed OA / Bherein).

[0032] The building blocks of formula I may each have a structure according to formula la:RT— OA-NUC LP-NUC -OB-TPG ' 'V la whereinOAis the oxygen located at the 5’ terminal carbon of Nuc, and OBis the oxygen located at the 3’ terminal carbon of Nuc; orOAis the oxygen located at the 3’ terminal carbon of Nuc, and OBis the oxygen located at the 5’ terminal carbon of Nuc; andRT, Nuc, TPG, Lpand v are as defined herein.

[0033] For the avoidance of doubt, it will be understood that in sub-formulae la-lg described herein, the 3’ and 5’ terminal oxygen atoms present within the nucleosidic moieties (Nuc) to which RT and TPG are attached in formula I have been depicted (as OAand OB). Therefore, it will be understood that the moieties “RT-OA-Nuc-“ and “-Nuc-OB-TPG” in sub-formula la are respectively analogous to moieties “RT-Nuc-“ and “-Nuc-TPG” in formula I.

[0034] For building blocks of formula la, 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).

[0035] Most Suitably in compounds of formula la-lg, OAis the oxygen located at the 5’ terminal carbon of Nuc, and OBis the oxygen located at the 3’ terminal carbon of Nuc.

[0036] 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.

[0037] In compounds of formula I (and any sub-formulae thereof, e.g., la-lg) RT may have a structure according to formula II:II whereinY is a leaving group;PG is a protecting group; and denotes the point of attachment to the nucleosidic moiety (Nuc) to which RT is attached (e.g., via OAin formula la).

[0038] Y may be -NR2, 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. More suitably, each R is independently selected from methyl, ethyl, propyl (e.g., n-propyl or iso-propyl) or butyl (e.g., n- butyl, sec-butyl, iso-butyl or tert-butyl), 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.

[0039] 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.

[0040] In many instances, RT has the following structure:wherein' / v o / ' denotes the point of attachment to the nucleosidic moiety (Nuc) to which RT is attached (e.g., via OAin formula la).

[0041] Thus, the building blocks of formula I may each have a structure according to formula lb:wherein OA, Nuc, Lp, v, OBand TPG are as defined herein.

[0042] Most suitably in compounds of formula lb, Nuc comprises a ribosidic core (notwithstanding the aforementioned modifications that may occur at the 2’ carbon) and v is 0. In such embodiments, OAis suitably the oxygen located at the 5’ terminal carbon of Nuc, and OBis suitably the oxygen located at the 3’ terminal carbon of Nuc.

[0043] 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. Indeed, those of ordinary skill in the art will appreciate that the chain extension site of the growing oligonucleotide is the portion of the growing oligonucleotide to which a building block would be coupled to extend the length of the oligonucleotide chain as part of an LPOS process. 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.

[0044] In compounds of formula I (and any sub-formulae thereof, e.g., la-lg), each nucleosidic moiety (Nuc) may have a structure according to formula III:whereinZ is a nucleobase, optionally protected by a protecting group;Rxis selected from H, OH, F, O-tert-butyldimethylsilyl (OTbdms), methoxy, O-methoxyethyl (OMoe), O-propargyl, NH2 and N3, in which case both are absent, or Rxis O and both are present;1and2independently denotes the points of attachment to RT (e.g., via 0Ain formulae la-lg), TPG (e.g., via OBin formulae la-lg) or LP (as appropriate). < / VW'

[0045] 1suitably denotes the point of attachment to OA. In such embodiments, OAis suitably the oxygen located at the 5’ terminal carbon of Nuc.

[0046] 2suitably denotes the point of attachment to OB. In such embodiments, OBis suitably the oxygen located at the 3’ terminal carbon of Nuc.

[0047] Z may be adenine, guanine, cytosine, thymine or uracil, any of which being optionally protected by a protecting group. Protecting groups will be familiar to those skilled in the art and will be understood to mean an organic moiety attached to a functional group in order to block the reactivity of said functional group. Each protecting group may independently be an acid-labile protecting group, a base-labile protecting group, an-ammonia labile protecting group, an oximate- labile protecting group, an oxidatively-labile protecting group, a hydrogenolytically-labile protecting group or a transition metal catalysed cleavage protecting group. Suitably, each protecting group is independently selected from the group consisting of iso-butyryl (Ibu), 2,4,6- trimethylphenyl, 2-nitrophenyl, 2,4-dimethylphenyl, toluyl, 2-(4-nitrophenyl)ethyl, 2-(4- cyanophenyl)ethyl, allyl, benzoyl (Bz), 2,4-dimethylbenzoyl, tert-Bu benzoyl (tert-BuBz), acetyl (Ac), anisoyl (An), 4-chlorobenzoyl, diphenylcarbamoyl, butylthiocarbonyl, 2-nitrophenylsulfenyl, 2,4-dinitrophenylsulfenyl, 2-nitro-4-toluylsulfenyl, and triphenylmethylsulfenyl. Suitably, each protecting group is independently selected from the group consisting of iso-butyryl (Ibu), benzoyl (Bz), 2,4-dimethylbenzoyl, tert-Bu benzoyl (tert-BuBz), acetyl (Ac) and anisoyl (An).

[0048] Rxis most suitably H, OH, F, methoxy or O-methoxyethyl (OMoe).

[0049] In some embodiments, Z is adenine, guanine, cytosine, thymine or uracil, any of which being optionally protected by Ibu, Bz, 2,4-dimethylbenzoyl, tert-BuBz, Ac and An, and Rxis H, OH, F, methoxy or OMoe.

[0050] TPG will be understood to be a protecting group used to prevent uncontrolled chain extension during a chain extension cycle. In at least one chain extension cycle TPG is a photolabile protecting group (PPG). However, in instances wherein TPG is not a PPG (e.g., in a second chain extension cycle), the TPG may be acid-labile protecting group, non-limiting examples of which include dimethoxytrityl (Dmtr), 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. Therefore, the TPG may be cleavable by treatment with acid to expose a new chain extension site of the growing oligonucleotide.

[0051] In any chain extension cycle wherein the TPG is a PPG, the PPG is cleavable, after coupling of the building block to a chain extension site of a growing oligonucleotide, to expose anew chain extension site of the growing oligonucleotide. PPGs are cleavable once exposed to light and will be familiar to those of ordinary skill in the art, as well as the conditions under which they can be cleaved. For example, the PPG may be cleavable, after coupling of the building block, to expose a new chain extension site of the growing oligonucleotide when irradiated at a wavelength of >350 nm (e.g., 365 nm). PPGs which are cleavable when irradiated at a wavelength of >350 nm are preferred as this irradiation causes little or no photo-induced damage to the rest of the growing oligonucleotide.

[0052] It will be understood that in any chain extension cycle wherein the TPG is a PPG, the building block may have a structure according to formula I (and any sub-formulae thereof, e.g., la or lb), wherein the TPG group is replaced by a PPG group.

[0053] Each PPG may independently have a structure according to formula IV:whereinL is absent or a linker group selected from -CRaRb-, -C(O)-O-CRaRb- or -C(O)-O-, wherein Raand Rb are each independently selected from H or (1 -3C)alkyl;R1is selected from H, (1-3C)alkyl or -C(O)-aryl; n is 1-4; each R2is independently selected from -(1-3C)alkyl, -NO2, -OMe, -C(O)-aryl or -SPh; or when two R2groups are present on adjacent carbon atoms in the phenyl ring, both R2groups are linked such that they form a fused 5- to -7-membered heterocycle; and ww denotes the point of attachment to a chain extension site.

[0054] It will be understood that where a TPG of a compound of formula I (or a sub-formula thereof) is a PPG, that PPG may have a structure according to formula IV (or a sub-formula thereof), with ww denoting the point of attachment of the PPG to the compound of formula I (or a sub- formula thereof).

[0055] suitably denotes the point of attachment to OBin formula la or lb, wherein OBis the oxygen located at the 3’ terminal carbon of Nuc. In such embodiments, Nuc comprises a ribosidic core (notwithstanding the aforementioned modifications that may occur at the 2’ carbon) and v may be 0.

[0056] Suitably, L is absent or a linker group selected from -CRaRb-, -C(O)-O-CRaRb- or -C(O)- O-, wherein Raand Rb are each independently selected from H or methyl. More suitably, L isabsent or a linker group selected from -CH2-, -C(O)-O-CH2- or -C(O)-O-. Most suitably, L is - C(O)-O-CH2- or -C(O)-O-.

[0057] Suitably, R1is selected from H, methyl or -C(O)-phenyl. Most suitably, R1is methyl.

[0058] Suitably, n is 1 , 2 or 3. Most suitably n is 1 or 3.

[0059] Suitably, each R2is independently selected from -Me, -Et, -NO2, -OMe, -C(O)-Ph or - SPh; or when two R2groups are present on adjacent carbon atoms in the phenyl ring, both R2groups are linked such that they form a fused 5-membered oxygen-containing heterocycle. More suitably, each R2is independently selected from -Et, -NO2, -OMe, -C(O)-Ph or -SPh; or when two R2groups are present on adjacent carbon atoms in the phenyl ring, both R2groups are linked such that they form a fused dioxolane (e.g., a 1 ,3-dioxolane). Most suitably, each R2is independently selected from -NO2 or -OMe.

[0060] In some embodiments, L is absent or a linker group selected from -CH2-, -C(O)-O-CH2- or -C(O)-O-, R1is selected from H, methyl or -C(O)-phenyl and n is 1 , 2 or 3. In such embodiments, it may be that each R2is independently selected from -Et, -NO2, -OMe, -C(O)-Ph or -SPh; or when two R2groups are present on adjacent carbon atoms in the phenyl ring, both R2groups are linked such that they form a fused dioxolane (e.g., a 1 ,3-dioxolane). In some embodiments, L is -C(O)-O-CH2- or -C(O)-O-, R1is methyl and n is 1 or 3. In such embodiments, it may be that each R2is independently selected from -NO2 or -OMe.

[0061] PPGs of formula IV may have a structure according to formula IVa:IVa whereinR1Ais H or methyl;L is -C(O)-O-CH2- or -C(O)-O-;R^ is -OMe or absent;R2Bis -OMe or absent; or, R2Aand R2Bare each O and are linked to one another by a -CH2- group (i.e., resulting in a 1 ,3-benzodioxole ring system); and is as described hereinbefore.

[0062] Each PPG may independently have any one of the following structures:2-nitrobenzyl 1 -(2-nitrophenyl)ethyl 2 -nitrophenethyl 2-(2-nitrophenyl)propyl2-(2-nitrophenyl)propoxycarbonyl (Nppoc) dimethoxybenzoin carbonate (Dmboc)(a-methyl-2-nitropiperonyl)oxycarbonyl (Me-Npoc) benzoyl-2-(2-nitrophenyl)propoxycarbonyl (Bz-Nppoc)thiophenyl-2-(2-nitrophenyl)propoxycarbonyl (SPh-Nppoc) methyl-6-nitroveratryloxycarbonyl (MeNvOC) wherein v / wcr is as described hereinbefore (e.g., as denoting the point of attachment to the nucleosidic moiety (Nuc) to which PPG is attached, such as via OBin formula la or lb).

[0063] Suitably, each PPG is Nppoc, Bz-Nppoc or MeNvOC:2-(2-nitrophenyl)propoxycarbonyl (Nppoc) benzoyl-2-(2-nitrophenyl)propoxycarbonyl (Bz-Nppoc)methyl-6-nitroveratryloxycarbonyl (MeNvOC)

[0064] More suitably, each PPG is Nppoc or MeNvOC:2-(2-nitrophenyl)propoxycarbonyl (Nppoc) methyl-6-nitroveratryloxycarbonyl (MeNvOC)

[0065] In particular instancesl, each PPG is MeNvOC. The inventors have determined thatMeNvOC, once cleaved, exhibits little to no aggregation, thus making it particularly suitable for removal by membrane filtration.

[0066] The building blocks of formula I used in a chain extension cycle wherein the TPG is a PPG may therefore each have a structure according to formula Ic:Ic wherein RT, OA, Nuc, Lp, v and OBare as defined herein.

[0067] In some instances, the building blocks of formula I used in a chain extension cycle wherein the TPG is a PPG may each have a structure according to formula Id:wherein OA, Nuc, L, v and OBare as defined herein.

[0068] It may also be that the building blocks of formula I used in a chain extension cycle wherein the TPG is a PPG each have a structure according to formula le:wherein Y, PG, OA, Nuc, Lp, v, OB, L, R1, R2and n are as defined herein.

[0069] The building blocks of formula I used in a chain extension cycle wherein the TPG is a PPG may each have a structure according to formula If:wherein RT, OA, Nuc, Lp, v and OBare as defined herein.

[0070] In some instances, the building blocks of formula I used in a chain extension cycle wherein the TPG is a PPG may each have a structure according to formula Ig:ig wherein 0A, Nuc, Lp, v and 0Bare as defined herein.

[0071] 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 three or more chain extension cycles. More suitably, the one or more chain extension cycles is four or more chain extension cycles. Even more suitably, the one or more chain extension cycles is five or more chain extension cycles. Yet even more suitably, the one or more chain extension cycles is eight or more chain extension cycles. Yet still even more suitably, the one or more chain extension cycles is ten or more chain extension cycles. Most suitably, the one or more chain extension cycles is fifteen or more chain extension cycles.

[0072] In at least one of the chain extension cycles, the TPG is a PPG. Suitably, in at least 20% of the chain extension cycles (to the nearest whole number), the TPG is a PPG. 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 TPG on the building block is a PPG. More suitably, in at least 30% of the chain extension cycles (to the nearest whole number), the TPG on the building block is a PPG. Even more suitably, in at least 50% of the chain extension cycles (to the nearest whole number), the TPG on the building block is a PPG. Yet even more suitably, in at least 75% of the chain extension cycles (to the nearest whole number), the TPG on the building block is a PPG. Still even more suitably, in at least 90% of the chain extension cycles (to the nearest whole number), the TPG on the building block is a PPG.

[0073] In some instances, in each one of the chain extension cycles (i.e., 100% of the chain extension cycles), the TPG on the building block is a PPG.

[0074] In at least one of the chain extension cycles, the TPG is a PPG and 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 TPGs and excess reagents. Suitably, in at least 20% of the chain extension cycles (to the nearest whole number) in which the TPG on the building block is a PPG, membrane filtration is performedto isolate the growing oligonucleotide. For example, where in 8 chain extension cycles within a process the TPG on the building block is a PPG, 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) in which the TPG on the building block is a PPG, 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 TPG on the building block is a PPG, membrane filtration is performed to isolate the growing oligonucleotide.

[0075] Most suitably, in each one of the chain extension cycles in which the TPG on the building block is a PPG, membrane filtration is performed to isolate the growing oligonucleotide.

[0076] It will be understood that membrane filtration may be used to isolate the growing oligonucleotide in a given chain extension cycle even when the TPG on the building block is not a PPG. Suitably, membrane filtration is used to isolate the growing oligonucleotide in at least 50% (to the nearest whole number) of all chain extension cycles (i.e., irrespective of the nature of the TPG). More 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 TPG). Even 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 TPG).

[0077] Most suitably, membrane filtration is used to isolate the growing oligonucleotide in all chain extension cycles (i.e., irrespective of the nature of the TPG).

[0078] In some embodiments, in each one of the chain extension cycles, the TPG on the building block is a PPG and membrane filtration is used to isolate the growing oligonucleotide.

[0079] 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 growing oligonucleotide from excess, unreacted building block, while a second membrane filtration step separates cleaved TPGs or PPGs from the growing oligonucleotide.

[0080] Each chain extension cycle suitably comprises a step of cleaving the TPG 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. In any chain extension cycle wherein the TPG is a PPG, the chain extension cycle suitably comprises a step of cleaving the PPG 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 PPGs and how both are cleaved.

[0081] Suitably, in any chain extension cycle wherein the TPG is a PPG, the PPG is cleaved by irradiating the growing oligonucleotide at a wavelength of >350 nm (e.g., 365 nm).

[0082] Membrane filtration may be performed on multiple occasions within a single chain extension cycle. Suitably, membrane filtration is performed after coupling of the building block to a chain extension site of a growing oligonucleotide. Suitably, membrane filtration is performed after a step of cleaving the TPG or PPG. Most suitably, membrane filtration is performed before and after a step of cleaving the TPG or PPG, with the first membrane filtration being performed after coupling of the building block to a chain extension site of a growing oligonucleotide.

[0083] In any chain extension cycle wherein the TPG is a PPG, the reaction debris, or photodebris,, can inhibit the step of cleaving PPGs by absorbing light itself, often more strongly than the PPG itself. Membrane filtration to remove the photodebris therefore improves the efficiency of cleaving the PPGs. In some embodiments, membrane filtration is performed simultaneously with (i.e. , at the same time as) a step of cleaving the TPG or PPG.

[0084] In some embodiments, in at least one chain extension cycle where the TPG is a PPG, the steps of: (i) cleaving the PPG 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, and (ii) membrane filtration, are performed simultaneously. As discussed hereinbefore, the cleaved photodebris can be readily removed by membrane filtration, thereby improving the efficiency of the deprotection (cleaving) step. Suitably, in at least 20% of the chain extension cycles (to the nearest whole number) where the TPG is a PPG, the steps of: (i) cleaving the PPG 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, and (ii) membrane filtration, are performed simultaneously. For example, in the preparation of a 5-mer oligo by 4 chain extension cycles, each one involving a monomeric building block, in one or more of the chain extension cycles in which the TPG is a PPG, the steps of cleaving the PPG 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 and membrane filtration are performed simultaneously. More suitably, in at least 40% of the chain extension cycles (to the nearest whole number) where the TPG is a PPG, the steps of: (i) cleaving the PPG 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, and (ii) membrane filtration, are performed simultaneously. Even more suitably, in at least 60% of the chain extension cycles (to the nearest whole number) where the TPG is a PPG, the steps of: (i) cleaving the PPG 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, and (ii) membrane filtration, are performed simultaneously. Yet even more suitably, in at least 80% of the chain extension cycles (to the nearest whole number) where the TPG is a PPG, the steps of: (i) cleaving the PPG 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, and (ii) membrane filtration, are performed simultaneously. Still even moresuitably, in at least 90% of the chain extension cycles (to the nearest whole number) where the TPG is a PPG, the steps of: (i) cleaving the PPG 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, and (ii) membrane filtration, are performed simultaneously.

[0085] In some embodiments, in each one of the chain extension cycles (i.e. , 100% of the chain extension cycles) where the TPG is a PPG, the steps of: (i) cleaving the PPG 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, and (ii) membrane filtration, are performed simultaneously.

[0086] In the process according to the first aspect of the present invention, each chain extension cycle suitably comprises the following steps:1. coupling a monomeric or oligomeric building block to a chain extension site of a growing oligonucleotide;2. membrane filtration to isolate the growing oligonucleotide (e.g., from uncoupled building blocks);3. cleaving the TPG or PPG; and4. membrane filtration to isolate the growing oligonucleotide (e.g., from reaction debris).

[0087] In the aforementioned embodiment, step 3. suitably involves cleaving a PPG.

[0088] Membrane filtration is particularly well suited for isolating the growing oligonucleotide from uncoupled building blocks and reaction debris. In any chain extension cycle wherein the TPG is a PPG, the reaction debris is often photodebris, which can provide a competing reaction to the step of cleaving PPGs by absorbing light itself. Membrane filtration to remove the photodebris therefore improves the efficiency of cleaving the PPGs. This is augmented in embodiments wherein steps 3. and 4. occur simultaneously. Suitably, the step of cleaving the TPG 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 and membrane filtration are performed simultaneously. Most suitably, the step of cleaving the PPG 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 and membrane filtration are performed simultaneously.

[0089] In other instances, each chain extension cycle comprises the following steps:1. coupling a monomeric or oligomeric building block to a chain extension site of a growing oligonucleotide;2. cleaving the TPG or PPG; and3. membrane filtration to isolate the growing oligonucleotide (e.g., from uncoupled building blocks and reaction debris).

[0090] In such instances, each chain extension cycle may involve only a single membrane filtration step. Where the TPG is a PPG, steps 2 and 3 are suitably performed simultaneously. As described hereinbefore, removing cleaved photodebris at the same time as conducting the photolytic deprotection improved the efficiency of the latter.

[0091] 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 forms a P(lll) 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).

[0092] Each chain extension cycle suitably comprises a step of converting one of more P(lll) 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(lll) phosphite triester linkages to a P(V) phosphotriester linkage (e.g., by oxidation or sulfur transfer) is conducted before a step of cleaving the TPG or PPG to expose a new chain extension site of the growing oligonucleotide.

[0093] 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, cleaving of protecting groups 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, cleaving of protecting groups and membrane filtration) are performed in the same solvent (e.g., acetonitrile).

[0094] The membrane filtration steps forming part of the process are suitably conducted by membrane diafiltration. In membrane diafiltration, the crude mixture comprising the growingoligonucleotide 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).

[0095] Suitable membranes for use in isolating the growing oligonucleotide include polymeric membranes, ceramic membranes, and mixed polymeric / inorganic membranes. Membrane rejection Rj is a common term known by those skilled in the art and is defined as: eq. (1)where CP = concentration of species i in the permeate, permeate being the liquid which has passed through the membrane, and CR = 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 (Product)> (reactants), where product is the growing oligonucleotide.

[0096] 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 TiC>2 or ZrC>2. 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.

[0097] Most suitably, the membrane is a crosslinked polybenzimidazole membrane (e.g., an integrally skinned, asymmetric, crosslinked polybenzimidazole membrane) or a polyetheretherketone membrane.

[0098] 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 protecting groups (e.g., cyanoethyl as PG in formulae lb, Id, le, Ig and II) from internucleoside linkages present throughout the oligonucleotide, and / or may result in removal of all protecting groups, including protecting groups 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.

[0099] 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 synthesis support 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).

[0100] 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).

[0101] The soluble synthesis support may have a structure according to formula VV in whichV is an organic branch point,W is a polymeric chain,' / vw' represents a point of attachment to the growing oligonucleotide, and m is 1-8.

[0102] 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.

[0103] 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.

[0104] m may, for example, be 1-6. In many instances, m is 3-4.

[0105] In some embodiment, m is 8.

[0106] 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.

[0107] A particularly suitable example of a soluble synthesis support is:in which ww' 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).

[0108] Thus, it may be that the soluble synthesis support and growing oligonucleotide have the following structure:wherein Rxand Z are as defined herein; and' / vw' denotes the point of attachment to a monomeric or oligomeric building block.

[0109] 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 10 - 100 (e.g., 25 - 75).

[0110] Thus, it may be that the soluble synthesis support and growing oligonucleotide have the following structure:wherein Rxand Z are as defined herein; and' / VW' denotes the point of attachment to a monomeric or oligomeric building block.

[0111] Each oligonucleotide, once prepared (i.e., grown to FLP), 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.

[0112] 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.

[0113] 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'-0-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 K. 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'-0-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, a-L-LNA, p-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, PMOPIus, PMO- X); and their derivatives.

[0114] 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 its derivatives, 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. 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.

[0115] 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.

[0116] 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.

[0117] The processes of the first and second aspects may be conducted in a closed-loop reactor.

[0118] 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 or second aspect.

[0119] The following numbered statements 1 to 146 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, wherein each chain extension cycle comprises coupling a monomeric or oligomeric building block to a chain extension site of a growing oligonucleotide, wherein each building block comprises: at least one nucleosidic moiety (Nuc); a reactive terminal (RT) for coupling the building block to the chain extension site of the growing oligonucleotide; and 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; wherein in at least one chain extension cycle: the temporary protecting group (TPG) is a photolabile protecting group (PPG); and membrane filtration is performed to isolate the growing oligonucleotide (e.g., from uncoupled building blocks and / or reaction debris).2. The process of statement 1 , wherein each building block independently comprises 1-3 nucleosidic moieties.3. The process of statement 1 or 2, wherein each building block comprises 1 nucleosidic moiety.4. The process of statement 1 , 2 or 3, wherein each building block has a molecular weight of >200 Da.5. The process of any one of the preceding statements, wherein each building block has a molecular weight of >400 Da.6. The process of any one of the preceding statements, wherein each building block has a molecular weight of >600 Da.7. The process of any one of the preceding statements, wherein each building block has a structure according to formula I:I whereinNuc is a nucleosidic moiety;RT is the reactive terminal, which is covalently bound to Nuc;TPG is the temporary protecting group, which is covalently bound to Nuc;Lpis an internucleoside linkage; andV is 0-10 (e.g., 0-3).8. The process of statement 7, wherein v is 0, 1 or 2.9. The process of statement 7 or 8, wherein v is 0.10. The process of statement 7, 8 or 9, wherein RT is covalently bound at the 5’ terminal of the nucleosidic moiety to which it is attached.11. The process of statement 7, 8 or 9, wherein RT is covalently bound at the 3’ terminal of the nucleosidic moiety to which it is attached.12. The process of any one of statements 7-11, wherein TPG is covalently bound at the 5’ terminal of the nucleosidic moiety to which it is attached.13. The process of any one of statements 7-11, wherein TPG is covalently bound at the 3’ terminal of the nucleosidic moiety to which it is attached.14. The process of any one of statements 7-13, wherein the building blocks of formula I each have a structure according to formula la:RT— 0A-Nuc4Lp-Nuc4-0B-TPG ' 'V la wherein0Ais the oxygen located at the 5’ terminal carbon of Nuc, and OBis the oxygen located at the 3’ terminal carbon of Nuc; orOAis the oxygen located at the 3’ terminal carbon of Nuc, and OBis the oxygen located at the 5’ terminal carbon of Nuc; andRT, Nuc, TPG, Lpand v are as defined in any one of statements 7-13.15. The process of statement 14, wherein OAis the oxygen located at the 5’ terminal carbon of Nuc, and OBis the oxygen located at the 3’ terminal carbon of Nuc.16. The process of any one of the preceding statements, wherein RT has a structure according to formula II:II whereinY is a leaving group;PG is a protecting group; and denotes the point of attachment to the nucleosidic moiety (Nuc) to which RT is attached (e.g., via OAin formula la).17. The process of statement 16, wherein Y is -NR2, 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.18. The process of statement 17, wherein each R is independently selected from methyl, ethyl, propyl (e.g., n-propyl or iso-propyl) or butyl (e.g., n-butyl, sec-butyl, iso-butyl or tert-butyl), 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.19. The process of statement 17 or 18, 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.20. The process of statement 17, 18 or 19, wherein each R is isopropyl.21. The process of any one of statements 16-20, wherein PG is a base-labile protecting group.22. The process of any one of statements 16-21 , wherein PG is selected from the group consisting of 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.23. The process of any one of statements 16-22, wherein PG is selected from the group consisting of cyanoethyl, 2-chlorophenyl, 4-chlorophenyl, 2,2,2-trichloroethyl and methyl.24. The process of any one of statements 16-23, wherein PG is cyanoethyl.25. The process of any one of the preceding statements, wherein RT has the following structure:wherein denotes the point of attachment to the nucleosidic moiety (Nuc) to which RT is attached (e.g., via OAin formula la).26. The process of any one of statements 7-25, wherein the building blocks of formula I each have a structure according to formula lb:wherein OA, Nuc, Lp, v, OBand TPG are as defined in any one of statements 7-25.l. The process of any one of the preceding statements, wherein each nucleosidic moiety (Nuc) comprises a ribosidic core (notwithstanding the aforementioned modifications that may occur at the 2’ carbon).28. The process of any one of the preceding statements, wherein each nucleosidic moiety (Nuc) has a structure according to formula III:III whereinZ is a nucleobase, optionally protected by a protecting group;Rxis selected from H, OH, F, O-tert-butyldimethylsilyl (OTbdms), methoxy, O-methoxyethyl (OMoe), O-propargyl, NH2 and N3, in which case bothare absent, or Rxis O and both are present;1and2independently denotes the points of attachment to RT (e.g., via OAin formulae la-le), TPG (e.g., via OBin formulae la-le) or LP (as appropriate)., / VW*29. The process of statement 28, wherein1denotes the point of attachment to OA.JWV'30. The process of statement 28 or 29, wherein2denotes the point of attachment to OB.31. The process of statement 28, 29 or 30, wherein Z is adenine, guanine, cytosine, thymine or uracil, any of which being optionally protected by a protecting group.32. The process of statement 31, wherein the protecting group is selected from the group consisting of an acid-labile protecting group, a base-labile protecting group, an-ammonia labile protecting group, an oximate-labile protecting group, an oxidatively-labile protecting group, a hydrogenolytically-labile protecting group or a transition metal catalysed cleavage protecting group.33. The process of statement 31 or 32, wherein the protecting group is independently selected from the group consisting of iso-butyryl (Ibu), 2,4,6-trimethylphenyl, 2-nitrophenyl, 2,4- dimethylphenyl, toluyl, 2-(4-nitrophenyl)ethyl, 2-(4-cyanophenyl)ethyl, allyl, benzoyl (Bz), 2,4- dimethylbenzoyl, tert-Bu benzoyl (tert-BuBz), acetyl (Ac), anisoyl (An), 4-chlorobenzoyl, diphenylcarbamoyl, butylthiocarbonyl, 2-nitrophenylsulfenyl, 2,4-dinitrophenylsulfenyl, 2-nitro-4- toluylsulfenyl, and triphenylmethylsulfenyl.34. The process of statement 31 , 32 or 33, wherein the protecting group is independently selected from the group consisting of iso-butyryl (Ibu), benzoyl (Bz), 2,4-dimethylbenzoyl, tert-Bu benzoyl (tert-BuBz), acetyl (Ac) and anisoyl (An).35. The process of any one of statements 28-34, wherein Rxis H, OH, F, methoxy or O- methoxyethyl (OMoe).36. The process of any one of the preceding statements, wherein when TPG is not a PPG, it is an acid-labile protecting group.37. The process of statement 37, wherein each acid-labile TPG is independently selected from the group consisting of dimethoxytrityl (Dmtr), 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.38. The process of any one of the preceding statements, wherein each PPG independently has a structure according to formula IV:whereinL is absent or a linker group selected from -CRaRb-, -C(O)-O-CRaRb- or -C(O)-O-, wherein RaandRb are each independently selected from H or (1 -3C)alkyl;R1is selected from H, (1-3C)alkyl or -C(O)-aryl; n is 1-4;each R2is independently selected from -(1-3C)alkyl, -NO2, -OMe, -C(O)-aryl or -SPh; or when two R2groups are present on adjacent carbon atoms in the phenyl ring, both R2groups are linked such that they form a fused 5- to -7-membered heterocycle; and' rw' denotes the point of attachment to a chain extension site (e.g., the point of attachment to the nucleosidic moiety (Nuc) to which PPG is attached, such as via OBin formula la or lb).39. The process of statement 38, wherein denotes the point of attachment to OBin formula la or lb, wherein OBis the oxygen located at the 3’ terminal carbon of Nuc.40. The process of statement 38 or 39, wherein L is absent or a linker group selected from -CRaRb-, -C(O)-O-CRaRb- or -C(O)-O-, wherein Raand Rb are each independently selected from H or methyl.41. The process of statement 38, 39 or 40, wherein L is absent or a linker group selected from -CH2-, -C(O)-O-CH2- or -C(O)-O-.42. The process of statement 38, 39 or 40, wherein L is absent or a linker group selected from -C(O)-O-CH2- or -C(O)-O-.43. The process of any one of statements 38-42, wherein L is -C(O)-O-CH2-.44. The process of any one of statements 38-43, wherein R1is selected from H, methyl or - C(O)-phenyl.45. The process of any one of statements 38-44, wherein R1is methyl.46. The process of any one of statements 38-45, wherein n is 1 , 2 or 3.47. The process of any one of statements 38-46, wherein n is 1 or 3.48. The process of any one of statements 38-47, wherein each R2is independently selected from -Me, -Et, -NO2, -OMe, -C(O)-Ph or -SPh; or when two R2groups are present on adjacent carbon atoms in the phenyl ring, both R2groups are linked such that they form a fused 5- membered oxygen-containing heterocycle.49. The process of any one of statements 38-48, wherein each R2is independently selected from -Et, -NO2, -OMe, -C(O)-Ph or -SPh; or when two R2groups are present on adjacent carbonatoms in the phenyl ring, both R2groups are linked such that they form a fused dioxolane (e.g., a 1,3-dioxolane).50. The process of any one of statements 38-49, wherein each R2is independently selected from -NO2 or -OMe.51. The process of statement 38 or 39, wherein L is -C(O)-O-CH2- or -C(O)-O-, R1is H, methyl or -C(O)-phenyl, and each R2is independently -NO2 or -OMe.52. The process of statement 38 or 39, wherein L is -C(O)-O-CH2- or -C(O)-O-, n is 1 or 3, R1is methyl, and each R2is independently -NO2 or -OMe.53. The process of statement 38 or 39, wherein each PPG independently has a structure according to formula IVa:IVa whereinR1Ais H or methyl;L is -C(O)-O-CH2- or -C(O)-O-;R^ is -OMe or absent;R2Bis -OMe or absent; or, R2Aand R2Bare each O and are linked to one another by a -CH2- group (i.e., resulting in a 1,3-benzodioxole ring system); and« / W' is as described in statement 38 or 39.54. The process of any one of the preceding statements, wherein each PPG independently has any one of the following structures:2-(2-nitrophenyl)propoxycarbonyl (Nppoc) dimethoxybenzoin carbonate (Dmboc)(a-methyl-2-nitropiperonyl)oxycarbonyl (Me-Npoc) benzoyl-2-(2-nitrophenyl)propoxycarbonyl (Bz-Nppoc)thiophenyl-2-(2-nitrophenyl)propoxycarbonyl (SPh-Nppoc) methyl-6-nitroveratryloxycarbonyl (MeNvOC) wherein is as described in statement 38 or 39.55. The process of any one of the preceding statements, wherein each PPG is independently Nppoc, Bz-Nppoc or MeNvOC:2-(2-nitrophenyl)propoxycarbonyl (Nppoc) benzoyl-2-(2-nitrophenyl)propoxycarbonyl (Bz-Nppoc)methyl-6-nitroveratryloxycarbonyl (MeNvOC)56. The process of any one of the preceding statements, wherein each PPG is Nppoc or MeNvOC:2-(2-nitrophenyl)propoxycarbonyl (Nppoc) methyl-6-nitroveratryloxycarbonyl (MeNvOC)57. The process of any one of statements 7-56, wherein the building blocks of formula I used in a chain extension cycle wherein the TPG is a PPG each have a structure according to formula Ic:Ic wherein RT, OA, Nuc, Lp, v and OBare as defined in any one of statements 7-56.58. The process of any one of statements 7-57, wherein the building blocks of formula I used in a chain extension cycle wherein the TPG is a PPG each have a structure according to formula Id:Id wherein OA, Nuc, L, v and OBare as defined in any one of statements 7-56.59. The process of any one of statements 7-56, wherein the building blocks of formula I used in a chain extension cycle wherein the TPG is a PPG each have a structure according to formula le:le wherein Y, PG, OA, Nuc, Lp, v, OB, L, R1, R2and n are as defined in any one of statements 7-56.60. The process of any one of statements 7-56, wherein the building blocks of formula I used in a chain extension cycle wherein the TPG is a PPG each have a structure according to formula If:If wherein RT, OA, Nuc, Lp, v and OBare as defined in any one of statements 7-56.61. The process of any one of statements 7-56, wherein the building blocks of formula I used in a chain extension cycle wherein the TPG is a PPG each have a structure according to formula Ig:wherein OA, Nuc, Lp, v and OBare as defined in any one of statements 7-5662. The process of any one of the preceding statements, wherein the one or more chain extension cycles is three or more chain extension cycles.63. The process of any one of the preceding statements, wherein the one or more chain extension cycles is four or more chain extension cycles.64. The process of any one of the preceding statements, wherein the one or more chain extension cycles is five or more chain extension cycles.65. The process of any one of the preceding statements, wherein the one or more chain extension cycles is eight or more chain extension cycles.66. The process of any one of the preceding statements, wherein the one or more chain extension cycles is ten or more chain extension cycles.67. The process of any one of the preceding statements, wherein the one or more chain extension cycles is fifteen or more chain extension cycles.68. The process of any one of the preceding statements, wherein in at least 20% of the chain extension cycles (to the nearest whole number), the TPG is a PPG.69. The process of any one of the preceding statements, wherein in at least 30% of the chain extension cycles (to the nearest whole number), the TPG is a PPG.70. The process of any one of the preceding statements, wherein in at least 50% of the chain extension cycles (to the nearest whole number), the TPG is a PPG.71. The process of any one of the preceding statements, wherein in at least 75% of the chain extension cycles (to the nearest whole number), the TPG is a PPG.72. The process of any one of the preceding statements, wherein in at least 90% of the chain extension cycles (to the nearest whole number), the TPG is a PPG.73. The process of any one of the preceding statements, wherein in each one of the chain extension cycles (i.e. , 100% of the chain extension cycles), the TPG is a PPG.74. The process of any one of the preceding statements, wherein in at least 20% of the chain extension cycles (to the nearest whole number) in which the TPG is a PPG, membrane filtration is performed to isolate the growing oligonucleotide.75. The process of any one of the preceding statements, wherein in at least 50% of the chain extension cycles (to the nearest whole number) in which the TPG is a PPG, membrane filtration is performed to isolate the growing oligonucleotide.76. The process of any one of the preceding statements, wherein in at least 80% of the chain extension cycles (to the nearest whole number) in which the TPG is a PPG, membrane filtration is performed to isolate the growing oligonucleotide.77. The process of any one of the preceding statements, wherein in each one of the chain extension cycles in which the TPG is a PPG, membrane filtration is performed to isolate the growing oligonucleotide.78. The process of any one of the preceding statements, wherein membrane filtration is used to isolate the growing oligonucleotide in at least 50% (to the nearest whole number) of all chain extension cycles (i.e., irrespective of the nature of the TPG).79. The process of 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 (i.e. , irrespective of the nature of the TPG).80. The process of 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 (i.e., irrespective of the nature of the TPG).81. The process of any one of the preceding statements, wherein membrane filtration is used to isolate the growing oligonucleotide in all chain extension cycles (i.e., irrespective of the nature of the TPG).82. The process of any one of the preceding statements, wherein in each one of the chain extension cycles, the TPG is a PPG and membrane filtration is used to isolate the growing oligonucleotide.83. The process of any one of the preceding statements, wherein at least one chain extension cycle comprises a step of cleaving the TPG 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.84. The process of any one of the preceding statements, wherein each chain extension cycle comprises a step of cleaving the TPG 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.85. The process of any one of the preceding statements wherein in any chain extension cycle wherein the TPG is a PPG, at least one of the chain extension cycles comprises a step of cleaving the PPG 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.86. The process of any one of the preceding statements wherein in any chain extension cycle wherein the TPG is a PPG, each chain extension cycle comprises a step of cleaving the PPG 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.87. The process of statement 85 or 86, the PPG is cleaved by irradiating the growing oligonucleotide at a wavelength of >350 nm (e.g., 365 nm).88. The process of any one of the preceding statements, wherein membrane filtration is performed on multiple occasions within a single chain extension cycle.89. The process of any one of the preceding statements, wherein membrane filtration is performed after coupling of the building block to a chain extension site of a growing oligonucleotide.90. The process of any one of statements 83-87, wherein membrane filtration is performed after a step of cleaving the TPG or PPG.91. The process of statement 90, wherein membrane filtration is performed before and after a step of cleaving the TPG or PPG, with the first membrane filtration being performed after coupling of the building block to a chain extension site of a growing oligonucleotide.92. The process of any one of the preceding statements, wherein membrane filtration is performed simultaneously with (i.e. , at the same time as) a step of cleaving the TPG or PPG.93. The process of any one of the preceding statements, wherein in at least one chain extension cycle where the TPG is a PPG, the step of cleaving the PPG 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 and membrane filtration are performed simultaneously.94. The process of any one of the preceding statements, wherein in at least 20% of the chain extension cycles where the TPG is a PPG, the step of cleaving the PPG 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 and membrane filtration are performed simultaneously.95. The process of any one of the preceding statements, wherein in at least 40% of the chain extension cycles where the TPG is a PPG, the step of cleaving the PPG 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 and membrane filtration are performed simultaneously.96. The process of any one of the preceding statements, wherein in at least 60% of the chain extension cycles where the TPG is a PPG, the step of cleaving the PPG 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 and membrane filtration are performed simultaneously.97. The process of any one of the preceding statements, wherein in at least 80% of the chain extension cycles where the TPG is a PPG, the step of cleaving the PPG 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 and membrane filtration are performed simultaneously.98. The process of any one of the preceding statements, wherein in at least 90% of the chain extension cycles where the TPG is a PPG, the step of cleaving the PPG 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 and membrane filtration are performed simultaneously.99. The process of any one of the preceding statements, wherein in all chain extension cycles where the TPG is a PPG, the step of cleaving the PPG 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 and membrane filtration are performed simultaneously.100. The process of any one of the preceding statements, wherein each chain extension cycle comprises the following steps:1. coupling a monomeric or oligomeric building block to a chain extension site of a growing oligonucleotide;2. membrane filtration to isolate the growing oligonucleotide (e.g., from uncoupled building blocks);3. cleaving the TPG or PPG; and4. membrane filtration to isolate the growing oligonucleotide (e.g., from reaction debris).101. The process of statement 100, wherein step 3. involves cleaving a PPG.102. The process of statement 100 or 101 , wherein the step of cleaving the TPG 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 and membrane filtration are performed simultaneously.103. The process of statement 100, 101 or 102, wherein the step of cleaving the PPG 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 and membrane filtration are performed simultaneously.104. The process of any one of statements 1 to 99, wherein each chain extension cycle comprises the following steps:1. coupling a monomeric or oligomeric building block to a chain extension site of a growing oligonucleotide;2. cleaving the TPG or PPG; and3. membrane filtration to isolate the growing oligonucleotide (e.g., from uncoupled building blocks and reaction debris).105. The process of statement 104, wherein steps 2 and 3 are performed simultaneously.106. The process of any one of the preceding statements, wherein each chain extension cycle comprises a step of converting one of more P(lll) 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.107. The process of 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).108. The process of any one of the preceding statements, wherein all steps of a given chain extension cycle (e.g., coupling, oxidation, cleaving of protecting groups and membrane filtration) are performed in the same solvent (e.g., acetonitrile).109. The process of any one of the preceding statements, wherein all chain extension cycles and their associated steps (e.g., coupling, oxidation, cleaving of protecting groups and membrane filtration) are performed in the same solvent (e.g., acetonitrile).110. The process of any one of the preceding statements, wherein the membrane filtration steps forming part of the process are conducted by membrane diafiltration.111. The process of any one of the preceding statements, wherein the membrane filtration steps are conducted by organic solvent nanofiltration (OSN) or ultrafiltration (UF).112. The process of any one of the preceding statements, wherein the membranes for use in isolating the growing oligonucleotide include polymeric membranes, ceramic membranes, and mixed polymeric / inorganic membranes.113. The process of statement 112, wherein 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 TiC>2 or ZrC>2.114. The process of statement 112 or 113, wherein the membrane is a crosslinked polybenzimidazole membrane (e.g., an integrally skinned, asymmetric, crosslinked polybenzimidazole membrane) or a polyetheretherketone membrane.115. The process of any one of the preceding statements further comprising one or more global deprotection steps.116. The process of any one of the preceding statements, wherein the growing oligonucleotide is attached (e.g., at the 3’ or 5’ terminal opposite to the chain extension site) to a soluble synthesis support.117. The process of statement 116, 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.118. The process of statement 116 or 117, wherein the soluble synthesis support is polymeric and is formed from poly(alkylene glycols), polyesters, polyamides, vinyl polymers, diene polymers, poly(alkylene imines), poly(amidoamines) and polysiloxanes.119. The process of statement 116, 117 or 118, wherein the soluble synthesis support is formed from poly(ethylene glycol).120. The process of any one of statements 116-119, wherein the soluble synthesis support is a star polymer comprising a central branch point and m number of radiating polymeric arms, wherein each polymeric arm is attached to a growing oligonucleotide, and wherein m is 3 or more.121. The process of statement 120, wherein 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.122. The process of statement 120 or 121, wherein each polymeric arm is poly(ethylene glycol).123. The process of any one of statements 116-122, wherein the soluble synthesis support has a structure according to formula VV in whichV is an organic branch point,W is a polymeric chain,' / VW' represents a point of attachment to the growing oligonucleotide, and m is 1-8.124. The process of statement 123, wherein V comprises fewer than 50 atoms in total.125. The process of statement 123 or 124, wherein V comprises fewer than 20 atoms in total.126. The process of statement 123, 124 or 125, wherein each W is a polymeric chain (e.g., polyethylene glycol) having a molecular weight (Mw) of 500 - 20,000 Da.127. The process of any one of statements 123-126, wherein each W is a polymeric chain having a molecular weight (Mw) of 2000 - 15,000 Da.128. The process of any one of statements 123-127, wherein each W is a polymeric chain having a molecular weight (Mw) of 8000 - 12,000 Da.129. The process of any one of statements 123-128, wherein m is 1-6.129. The process of any one of statements 123-128, wherein m is 3-4.130. The process of any one of statements 123-128, wherein m is 8.131. The process of any one of statements 123-130, wherein each W is attached directly to the growing oligonucleotide, or attached indirectly to the growing oligonucleotide via a linking moiety.132. The process of statement 131, wherein the linking moiety comprises fewer than 50 atoms in total.133. The process of statement 131 or 132, wherein the linking moiety comprises fewer than 30 atoms in total.134. The process of any one of statements 116-133, 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).135. The process of any one of statements 116-133, wherein the soluble synthesis support and growing oligonucleotide have the following structure:wherein Rxand Z are as defined in any one of statements 28 and 31-35; and ' / vw' denotes the point of attachment to a monomeric or oligomeric building block.136. The process of any one of statements 116-133, wherein the soluble synthesis support is:in which ww' represents the point of direct attachment to the growing oligonucleotide (e.g., at the 3’ oxygen) and q is 10 - 100 (e.g., 25 - 75).137. The process of any one of statements 116-133, wherein the soluble synthesis support and growing oligonucleotide have the following structure:wherein Rxand Z are as defined in any one of statements 28 and 31-35; and ww' denotes the point of attachment to a monomeric or oligomeric building block.138. A liquid-phase oligonucleotide synthesis (LPOS) process, wherein a photolabile protecting group (PPG) is used to protect the chain extension site of a growing oligonucleotide, the growing oligonucleotide being isolated by membrane filtration.139. The process of statement 138, wherein the PPG, chain extension site, growing oligonucleotide, membrane filtration and / or membrane are as defined in any one of statements 1 to 137.140. The process of statement 138 or 139, wherein during chain extension of the growing oligonucleotide, photolytic cleavage of the PPG is conducted simultaneously with membrane filtration to isolate the growing oligonucleotide from the cleaved PPG.141 . The process of any one of the preceding statements, wherein each oligonucleotide, once prepared (i.e., grown to FLP), has a molecular weight of >1000 Da.142. The process of any one of the preceding statements, wherein each oligonucleotide has a molecular weight of >2000 Da.143. The process of any one of the preceding statements, wherein each oligonucleotide has a molecular weight of >3000 Da.144. The process of any one of the preceding statements, wherein each oligonucleotide has a molecular weight of >5000 Da.145. The process of any one of the preceding statements, wherein the process is conducted in a closed-loop reactor.146. An oligonucleotide obtained, directly obtained or obtainable by the process of any one of the preceding statements.EXAMPLES

[0120] 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. Oligonucleotide synthesis cycle with nucleotidic building blocks. Coupling between building block, 1 , and the 5’-OH is initiated by one of a wide range of acidic activators. There is also a wide range of reagents to convert the P(lll) phosphite tri-ester to a P(V) phosphate (X = O) or phosphorothioate (X = S) tri-ester. 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. Oligonucleotide synthesis cycle in the 3'^5’ direction with building block, 2a, having photolabile 5’-0 protection (represented by PPG).Fig. 3. Oligonucleotide synthesis cycle in the 5'^3’ direction with building block, 2b, having photolabile 3’-0 protection (represented by PPG).Fig. 4. Schematic of the apparatus for bench-scale LPOS-PPG; liquid phase membrane-assisted oligonucleotide synthesis with photo-cleavable temporary protecting group. R = solvent reservoir;F = glass atmospheric pressure feed tank; P = pump to raise pressure; H = heat exchanger; M = membrane cell; PR = pressure relief valve.Fig. 5. Temporary photolabile protecting groups (PPGs) for iterative oligonucleotide synthesis.Fig. 6. Schematic of the apparatus for larger-scale LPOS-PPG; liquid phase membrane assisted oligonucleotide synthesis with photo-cleavable temporary protecting group. R = solvent reservoir; F = glass atmospheric pressure stirred tank; P = pump to raise pressure; H = heat exchanger; M = membrane cell; PR = pressure relief valve.Fig. 7. LPOS synthesis cycle with photo-cleavage of temporary 3’-protection.Fig. 8. Ion-pair reversed phase (IP-RP) UV chromatogram of3’lnvmUdGdAdTdC5’ pentamer, 5. UV purity 87.1%; deletion errors n-x, mainly x = 1 , account for 3.4% total impurities.Fig. 9. Synthesis of MeNvOC-OPnp, 9, and 3’-0-MeNv0C protected building blocks.Fig. 10.1H-NMR spectrum in CDCh of 3’-MeNvOC-mABz5’-phosphoramidite, 13.Fig. 11.31P-NMR spectrum in CDCh of 3’-MeNvOC-mABz5’-phosphoramidite, 13, with the signals at -150 ppm expanded (inset).Fig. 12. LPOS cycle with photo-cleavage of temporary 3’-O-MeNvOC protection and removal of photo-debris, 16, by diafiltration.Fig. 13. Photo-deprotection of 3’-MeNvOC-(mABz)2-PEG-star: HPLC stack showing fully protected oligo-star at the top, with intermediate time points below, and complete deprotection of all eight 3’-OH of the star at the bottom. Note that all 7 intermediate structures can be detected.Fig. 14. Photo-deprotection of 3’-MeNvOC-(mABz)5-PEG-star: HPLC stack showing fully protected oligo-star at the top, with intermediate time points below, and complete deprotection of all eight 3’-OH of the star at the bottom. Note that all 7 intermediate structures can still be detected.Fig. 15. Ion-pair reversed-phase (IP-RP) UV chromatogram of unpurified, fully deprotected (mA)s pentamer, 17, derived from four cycles of LPOS with 3’-MeNvOC-mABz5’-phosphoramidite, 13, as per Figure 12.Fig. 16. Synthesis of 5’-MeNvOC-mABz3’-phosphoramidite, 21 , and LPOS cycle with photocleavage of temporary 5’-O-MeNvOC protection.Fig. 17.1H-NMR spectrum in CDCh of 5’-MeNvOC-mABz3’-phosphoramidite, 21.Fig. 18.31P-NMR spectrum in CDCh of 5’-MeNvOC-mABz3’-phosphoramidite, 21 , with the signals at -150 ppm expanded (inset).Fig. 19. Ion-pair reversed-phase (IP-RP) UV chromatogram of unpurified, fully deprotected (mA)s pentamer, 17, derived from four cycles of LPOS with 5’-MeNvOC-mABz3’-phosphoramidite, 21 , as per Figure 16.Fig. 20. Conversion of 5’-MeNvOC-3’-Tbdms-mABz, 19, to 3’-Tbdms-mABz, 18, by irradiation at 365 nm in a round-bottomed flask. The concentration of 19 fails to approach zero because the photo-debris, 16, is a strong absorbent of incoming light.Fig. 21. HPLC traces from irradiating of 5’-MeNvOC-3’-Tbdms-mABz, 19, at 365 nm in a round- bottomed flask: Bottom, the crude reaction mixture; top, retentate from diafiltering the reaction mixture. It can be seen that the photo-debris, 16, has a much lower membrane rejection than either the substrate or product because it has almost entirely washed from the system after 7 DV; this is also reflected in the almost complete loss of coloration of the solution after DF.Materials and methods

[0121] A typical cycle in the synthesiser (see figure 7) includes three synthetic steps and two organic solvent nanofiltration (OSN) or diafiltration (DF1 & 2) steps: 1) coupling-, 2) oxidation or sulfur transfer, followed by DF1 , using 4 diavolumes (DV, or system volumes) of solvent to remove excess nucleoside, activator and oxidant - DF1 can be omitted; 3) photocleavage, followed by full purification of the crude oligo-star by OSN in DF2 using 6 DV. The last DF removes all residual unprotected nucleoside that would otherwise participate in the next chain extension cycle.

[0122] The equipment, represented schematically in figure 4: the membrane cell (M, volume 30 mL, MiniMem filtration system from PS Prozesstechnik GmbH) with internal stirring disk; in all cases the membrane is PBI-DBX-Jeffamine-2005, prepared according to Oxley et al., 2022; the glass atmospheric pressure feed tank (F, Duran Pressure Plus bottle (100 mL) low pressure feed tank and photolysis chamber); the solvent reservoir (R, Duran Pressure Plus bottle (1 L) solvent reservoir); the pump (P, Knauer P 4.1S piston pump) to raise the pressure in the membrane cell and provide recirculation; and the lamp (Kessil PR160L lamp set at 370 nm, 100 W intensity). The circulation loop has an estimated volume of 50 mL, maintaining the feed tank at 20 mL.

[0123] Example 1 below describes the PEG-star loading protocol, as well as the procedure repeated for each chain extension cycle in the membrane synthesiser (n.b. membrane synthesisers are sensitive to fouling of the membrane by particulate fines, meaning that all solutions injected into the synthesiser were filtered through PTFE membrane disks (0.2 pm, or tighter)).Example 1 :3’lnvmUdGdAdTdC5’ pentamer, 5, all phosphateLoading of 5’-Dmtr-mU 3’-succinate onto 10 kDa PEG-star in Nanostar synthesiser:

[0124] 4-Arm 10 kDa sarcosine terminated PEG-star (4 in figure 7, PEG-10k(Sar-H)4, MW 10300 Da, 1.25 g, 0.121 mmol) was dissolved in anhydrous MeCN (10 mL) and transferred by syringe to the synthesiser feed tank through a septum port, washing out the flask with further MeCN (5 mL). Next, / V, / V-diisopropylethylamine (DIPEA, 0.254 mL, 1.45 mmol, 12 eq.) was added to the feed tank and, after circulating for 2 min (40 mL / min for all operations), a sample was taken (all analytical samples from synthesiser 50 mL, diluted to a volume of 1.5 mL withMeCN) from the fully mixed synthesiser (t = 0). 5’-Dmtr-mll-3’-succinate (0.924 g, 1 .21 mmol, 10 eq.) dissolved in anhydrous MeCN (3 mL), was added to a round bottom flask containing 1- [bis(dimethylamino)-methylene]-1 H-benzotriazolium 3-oxide tetrafluoroborate (TBTLI, 0.389 g, 1.21 mmol, 10 eq.) under argon and stirred for 30 min at room temperature. The activated 5’- Dmtr-mll-3’-succinate solution was injected into the synthesizer through a 0.2 pm PTFE filter (Sterlitech Pressurised Filtration Holder). The amidation reaction was monitored by LC-MS and appeared complete after 5 min, but was continued for 30 min to ensure completion; a sample was taken to determine the residual Dmtr-succinate concentration at the start of DF1. The membrane was pressurised to 10 bar by slowly closing pressure relief valve (PR), and MeCN was then permeated (200 mL, ~4 diavolumes (DV)). As the circulation loop is airtight, solvent permeation generates a partial vacuum in the feed tank, causing lost solvent to be replaced automatically by fresh solvent drawn in from the reservoir, providing for the conditions of constant volume diafiltration. At the end of DF1 another sample was analysed by LC-MS to measure the fractional drop in the concentration of excess succinate and the rejection of the trityl nucleoside- star.Detritylation reaction:

[0125] Cation trap (2.5 eq. / arm, 10 in total) in MeCN (5 mL) was injected into the feed tank via the injection port using a syringe fitted with a 0.2 pm PTFE filter; the cation trap may be replaced by dodecanethiol. Dmtr deprotection was then initiated by injecting trifluoroacetic acid (TFA, 2.5 mL, 5 vol%). The detritylation reaction was monitored by LC-MS and after 10 min no partially deprotected Nanostar species or residual Dmtr-mU-succinate could be detected. After 20 min the reaction was quenched with pyridine (5 mL) and a sample was taken to determine the intermediate concentration of detritylated succinate. Dry MeCN (< 20 ppm water, 300 mL, ~6 DV) was permeated (DF2), to remove residual 5’-hydroxy-mU 3’-succinate and all other small molecule debris. At the end of DF2 a final sample was analysed by LC-MS to verify complete removal of building block debris and to confirm the high rejection of the 5’-OH-nucleoside-star. Typically, the chain extension was only undertaken if the combined drop in building block concentration over DF1 and DF2 was >99%. DF2 could be continued until this target was met.

[0126] The following two steps, coupling then photocleavage, were repeated four times using the amounts of Nppoc-phosporamidite building block, 3, and diafiltration volumes given in table 1 , but using an otherwise identical procedure, followed by global deprotection to prepare pentamer 5 (shown in figure 8).Coupling reaction:3’-Nppoc-nucleoside 5’-(cyanoethyl)phosphoramidite (3, 0.73 mmol, 1.5 eq. / arm, see table 1) building block and 4,5-dicyanoimidazole (DCI, 230 mg, 1.94 mmol, 4 eq. / arm) were each placed in separate round bottom flasks. The building block was co-evaporated from MeCN (<15 ppm water, 3 x 25 mL), then re-dissolved in MeCN (<30 ppm water, 5 mL) and injected into thesynthesiser, followed by the DCI dissolved in MeCN (5 mL). All components were injected into the synthesiser via a 0.2 pm PTFE filter. The reaction was monitored by LC; after 2 mins the coupling intermediates (1 to 3 arms extended with Nppoc-amidite) appeared to be consumed, but circulation was continued in the synthesiser for 20 min to ensure complete reaction. The reaction was then quenched with 2-cyanoethanol (CneOH, 98 mL, 3 eq. / arm) and circulated for 2 min. Camphorsulfonyl oxaziridine (CSO, 752 mg, 3.27 mmol, 6.8 eq. / arm) dissolved in MeCN (10 mL) was then added to the synthesiser and circulation continued for 1 hr. The oligo-star solution was partially purified by permeating acetonitrile-sulfolane 4:1 v / v (<20 ppm water, 200 mL, ~4 DV) to remove the majority of low molecular weight solutes.Table 1. Chain extension cycle variables.Photocleavage reaction:

[0127] / V-methyl imidazole (NMI, 0.5 mL) was injected into the feed tank via the injection port. A sample (t = 0) was taken to identify the retention time of the starting PEG-1 Ok(0ligo-0-Nppoc)4 Nanostar molecule in the LC trace at 260 nm, and residual 3’-Nppoc-BB 5’- (dicyanoethyl)phosphate debris in negative ionisation mode (ES-). The circulating reaction solution was maintained at 20 °C by means of a heat exchanger (H, figure 4) and chiller circulator. Deprotection was initiated by irradiating the feed tank at 365 nm, whilst maintaining circulation; at this time the feed tank and lamp were covered by a blackout box. The photocleavage reaction was monitored by LC-MS at 30 min intervals, tracking the deprotection of 3’-Nppoc protected building block in both the positive and negative mode ESI. After 3 hr no partially deprotected species could be detected, and a sample was taken to determine the intermediate concentration of deprotected building block. Dry acetonitrile-sulfolane 4:1 v / v (< 20 ppm water, ~6 DV, see table 1) was permeated (DF2), to remove residual building block and other photocleavage debris. At the end of DF2 a final sample was analysed by LC-MS to verify complete removal of building block debris and also the continued high rejection of the oligo-star. Ideally, proceeding to the next step should occur if the combined drop in building block concentration over DF1 and DF2 is >99%; otherwise, DF2 could be continued until this target is met.Global deprotection:

[0128] A sample of full-length HO-oligo-star solution (3 mL, ca. 4.8 mmol oligo) was withdrawn from the synthesizer and placed in an ACE pressure tube in concentrated aq. ammonia (~3 mL). To this solution diethylamine (0.1 mL) was added and the tube was sealed and heated at 35 °C overnight. The next day the 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, then the precipitate washed again and centrifuged twice more with further MeCN. The precipitate was finally analysed by UHPLC using an IP-RP gradient (Figure 8). During an ongoing synthesis, oligo-stars may be periodically analysed using this technique to assess the cumulative purity of the crude oligo.Example 2: Oligo synthesis using 3’-O-MeNvOC amidites and diafiltration of photo-debris Synthesis of MeNvOC-Pnp, 9 (Figure 9):

[0129] 1, Nitration: A solution of 4,5-dimethoxyacetophenone (6, 50.0 g, 0.277 mol) in 17% nitric acid (50 mL) was added dropwise to a solution of sodium nitrite (0.04 eq., 600 mg, 8.7 mmol) in concentrated nitric acid (67%, 400 mL) at 0 °C and the reaction mixture was stirred for 30 min. Iced water (4 x 500 mL) was then added to the reaction, collecting the solids in a glass sinter funnel, and finally rinsing with saturated sodium bicarbonate solution (500 mL) to neutralize any remaining acidity. The crude product was crystallised twice from ethanol, then dried under high vacuum to obtain 4,5-dimethoxy-2-nitroacetophenone (7) as a yellow solid (37.5 g, 75%).

[0130] 1H-NMR (400 MHz, DMSO-d6) 5 = 7.62, 7.21 (2xS, 2x1 H, ArH), 3.93, 3.90 (2xS, 2x3H, OCH3), 2.52 (s, 3H, COCH3) ppm; see Scherz, 2017.

[0131] 13C-NMR (100 MHz, DMSO-d6) 5 = 200.1 , 154.1 , 149.8, 138.7, 133.0, 108.8, 107.0, 56.8, 56.7, 30.5 ppm; see Scherz, 2017.

[0132] 2, Reduction: 4,5-Dimethoxy-2-nitroacetophenone (7, 37.5 g, 166 mmol) was dissolved in a mixture of anhydrous tetrahydrofuran (THF, 380 mL, <40 ppm water) and anhydrous methanol (380 mL, <40 ppm water). The solution was cooled to 0 °C in an ice bath, and sodium borohydride (6.62 g, 174 mmol) was added portion-wise over several min. The reaction mixture was stirred for 5 min at 0 °C, after which the ice bath was removed. Stirring was continued at room temperature for an additional 30 min. The reaction was quenched by the slow addition of 2 M hydrochloric acid (250 mL), followed by stirring for 15 min. The mixture was extracted four times with DCM (4 x 300 mL). The combined organic phases were dried over sodium sulfate (Na2SC>4), then filtered, and the solvent was evaporated under reduced pressure to yield crude 1-(4,5-dimethoxy-2-nitrophenyl) ethanol (methyl nitroveratryl alcohol, MeNv-OH) as a bright yellow solid (8, 37 g, 97%) which was used without further purification.

[0133] 1H-NMR (400 MHz, DMSO-d6) 6 = 7.52, 7.37 (2xS, 2x1H, ArH), 5.28 (m, 1 H, CH3CH), 3.91 , 3.85 (2xs, 2x3H, OCH3), 1.37 (m, 3H, CHCH3) ppm; see Scherz, 2017.

[0134] 13C-NMR (100 MHz, DMSO-d6) 5 = 153.9, 147.9, 139.8, 137.0, 108.6, 107.8, 65.9, 56.6, 56.5, 24.4 ppm; see Scherz, 2017.

[0135] 3, p-Nitrophenyl carbonate: MeNv-OH (8, 38.21 g, 167 mmol) was dissolved in DCM (800 mL) and cooled to 0 °C. To this solution were added p-nitrophenyl chloroformate (PnpOCOCI, 37.08 g, 184.0 mmol, 1.1 eq.) and / V-methylimidazole (NMI, 20.6 g, 251 mmol, 1.5 eq.), and the reaction was stirred for 2 h. Phosphate buffer (pH 4, 500 mL) was then added to the reaction, the cooling removed, and the mixture was stirred for 30 min. The organic phase was separated, and washed once more with phosphate buffer. The combined aqueous layers were back-extracted with further DCM. The organic phases were combined and dried over Na2SO4. The solvent was removed under reduced pressure to provide a foamy residue. The crude material was re-crystallised from ethyl acetate (700 mL); heating the solution to reflux resulted in a clear solution, which was then allowed to cool slowly to ambient temperature. 1-(4,5- Dimethoxy-2-nitrophenyl)ethyl p-nitrophenyl carbonate was collected as needle-like crystals (9, MeNvOC-OPnp, 25.31 g, 40%).

[0136] 1H-NMR (400 MHz, CDCI3) 5 = 8.19-8.15 (m, 2H, ArH Pnp), 7.53 (s, 1 H, ArH Nv), 7.29- 7.25 (m, 2H, ArH Pnp), 7.05 (s, 1 H, ArH Nv), 6.47 (q, J= 6.4Hz, 1 H, MeCH Nv), 3.95, 3.88 (2xS, 2x3H, OCH3), 1.70 (d, J= 6.4Hz, 3H, CHCH3) ppm; see Scherz, 2017.

[0137] 13C-NMR (100 MHz, DMSO-d6) 5 = 155.5, 153.96, 151.67, 148.61 , 145.62, 140.26, 130.68, 125.77 (2C), 122.96 (2C), 108.97, 107.97, 75.54, 56.77, 56.52, 21.61 ppm; see Scherz, 2017.Synthesis of 2’-O-Me-3’-O-MeNvOC-6-N-Bz-Adenosine 5’-O-phosphoramidite, 13 (Figure 9);

[0138] 1, Attachment of 3’-O-MeNvOC 5’-O-Dimethoxytrityl-3’-O-methyl-6-A / -benzoyl adenosine (10, 5’-Dmtr-mABz, 8.00 g, 11.6 mmol) was evaporated from MeCN (3 x 40 mL) and further dried under high vacuum for 1 hr. The residue was dissolved in anhydrous DCM (20 mL, 12.1 ppm), then solid MeNvOC-OPnp (9, 6.84 g, 17.5 mmol, 1.5 eq.) and dimethylaminopyridine (DMAP, 4.26 g, 34.9 mmol, 3 eq.) were added. The mixture was warmed to 35 °C and stirred for 5 hr, after which LC-MS showed complete reaction. The solution was washed with 1 M sodium phosphate buffer, and the aqueous phases was back-extracted with further DCM. The combined organic layers were dried over Na2SC>4 and concentrated under reduced pressure.

[0139] The same procedure was tested on 5’-Dmtr-mGlbu, 5’-Dmtr-mCBz, 5’-Dmtr-mUBz, and 5’- Dmtr-mU, reacting the 3’-OH of each with MeNvOC-OPnp (9). In all cases there was a cleanreaction to a single product, except the last where a small degree of N-debenzoylation was detected.

[0140] 2, Detritylation: The crude 3’-MeNvOC-5’-Dmtr-mABzwas dissolved in DCM (50 mL), to which were added dodecanethiol (DDT, 34.9 mmol, 3 eq., 8.35 mL) then methanesulfonic acid (MSA, 0.5% v / v = 0.1 mL). The reaction was stirred at 25 °C for 15 min, after which LC-MS showed that it was complete. Saturated sodium hydrogen carbonate (NaHCOs,0.5 mL) was added and, after stirring for 2 min, the DCM phase was separated, dried over Na2SO4, and concentrated under reduced pressure.

[0141] The residue was crystallised from MeCN (300 mL); heating the solution to reflux resulted in a clear solution, which was then allowed to cool slowly to ambient temperature, yielding a first crop of pure 3’-MeNvOC nucleoside as pale yellow needles (11 , 3.5 g, 62.5%). The mother liquor was concentrated, and further purified by reversed phase chromatography: C18 120 g column, eluting with a gradient of water and MeCN-MeOH 4:1 , 20-100%, providing a second crop (1.7 g) of purified product; this gave a combined yield of 3’-MeNvOC-mABznucleoside (11 , 5.2 g) of 92%.

[0142] 1H-NMR (400 MHz, CDCI3) 6 = 8.66, 8.65 (2xS, 2xO.5H, ArH Ado), 8.08, 8.06 (2xS, 2xO.5H, ArH Ado), 7.95-7.93 (m, 2H, ArH Bz), 7.56-7.52 (m, 2H, ArH Bz+Nv), 7.45-7.41 (m, 2H, ArH Bz), 7.05,7.04 (2xS, 2xO.5H, ArH Nv), 6.39, 6.31 (2xq, =6.4 Hz, 2xO.5H, CHMe Nv), 5.86- 5.82 (m, 1 H, T-CH), 5.41-5.38 (m, 1 H, 3’-CH), 4.70, 4.63 (2xdd, J= 7.9 , J= 4.8 Hz, 2xO.5H, 2’- CH), 4.36- 4.27 (2xm, 2xO.5H, 4’-CH), 3.94 -3.86 (m, 6H, OCH3Nv), 3.85- 3.71 (2xm, 2x1H, 5’- CH2), 3.22, 2.95 (2xS, 2x1.5 H, 2’-OCH3), 1.66-1.64 (m, 3H, CH3Nv) ppm.

[0143] 3, Conversion to 5’-Succinate 3’-MeNvOC-mABznucleoside (11 , 2.00 g, 3.30 mmol) was dissolved in pyridine (20 mL), to which solution NMI (0.130 mL, 2 mmol, 0.5 eq.) and Et3N (14 mmol, 1.97 mL, 4.3 eq.) were added. To this mixture was added succinic anhydride (0.658 g, 6.57 mmol, 2.0 eq.) and the reaction was stirred at 25 °C for 18 hr. After this, water (0.2 mL) was added and stirring continued for 20 min. The reaction solvent was evaporated using a rotary evaporator. The resulting crude product was then dispersed in CHCI3 (50 mL) and partitioned with water. The organic phase was separated, and the aqueous phase was back-extracted with CHCI3 (x3). The combined organic layers were washed with triethylammonium phosphate buffer; prepared from 3.4 mL of 85% H3PO4and 10.4 mL Et3N, diluted to 200 mL with water, pH ~7. The organic layer was dried over Na2SO4and evaporated to dryness under reduced pressure. The residue was purified by normal phase chromatography; 60 g silica column, eluting with a gradient of MeOH-CHCI3containing 0.2% triethylamine, 2%-18%. 3’-MeNvOC-mABz5’- succinate (12), triethylammonium salt, was isolated as a pale yellow foam (1.65 g, 75%).

[0144] 1H-NMR (400 MHz, CDCI3) 5 = 8.63 (s, 1 H, ArH Ado), 8.25 (s, 1 H, ArH Ado), 7.96-7.94 (m, 2H, ArH Bz), 7.49-7.43 (m, 2H, ArH Bz), 7.39-7.35 (m, 2H, ArH Bz+Nv), 7.02 (s, 1 H, ArH Nv), 6.35 (q, J= 6.4 Hz, 1 H, ArCHMe Nv), 6.06 (d, =5.4 Hz, 1 H, T-CH), 5.17-5.15 (m, 1 H, 3’-CH),4.66-4.63 (m, 1 H, 2’-CH), 4.37-4.28 (m, 3H, 5’-CH2+4’-CH), 3.94, 3.85 (2xS, 2x3H, OCH3Nv), 3.36 (s, 3H, 2’-OCH3), 2.58-2.44 (m, 4H, CH2CH2Sue), 1.63 (d, =6.40 Hz, 3H, CHCH3Nv) ppm; it is surprising that none of the peaks are resolved into two diastereoisomers.

[0145] 4, Conversion to 5’-phosphoramidite'. 3’-MeNvOC-mABz(11 , 5.00 g, 7.82 mmol) was evaporated from dry MeCN (3 x 100 mL) and further dried under high vacuum. The residue was dissolved in anhydrous DCM (50 mL) and chilled to 4 °C. To the cold solution was slowly added 2-cyanoethyl / V, / V, / V’, / V-tetraisopropylphosphordiamidite (1.5 eq., 11.7 mmol, 3.73 mL) while maintaining the temperature at 4 °C. Solid pyridinium trifluoroacetate (Py-TFA, 1.21 g, 0.8 eq.) was then added; the reaction was stirred at 4 °C for 30 min, and then at 25 °C for 18 hr. The next day triethylamine (0.25 mL) was added, and the crude material was purified by reversed phase chromatography; C18 120 g column, eluting with a gradient of water and MeCN-MeOH 4:1 , 20- 100%. The initial material was further purified by normal phase chromatography; 60 g silica column, eluting with a gradient of DCM-acetone, 2%-10%. This afforded pure 3’-MeNvOC-5’- Dmtr-mABz5’-phosphoramidite as a pale yellow foam (13, 8.45 g, 84%).

[0146] 1 H-NMR (400 MHz, CDCI3; Figure 10) 5 = 8.75, 8.75, 8.71 , 8.71 (4xs, 0.1 H, 0.1 H, 0.4H, 0.4H, ArH Ado), 8.36, 8.35, 8.33 (3xS, 0.4H, 0.1 H, 0.5H ArH Ado), 7.95 (br.d, J= 7.9, 2H, ArH Bz), 7.56-7.52 (m, 2H, ArH Bz+Nv), 7.48-7.44 (m, 2H, ArH Bz), 7.08, 7.06, 7.05 (3xs, 0.8H, 0.1 H, 0.1 H, ArH Nv), 6.41-6.30 (m, 1 H, ArCHMe Nv), 6.21-6.17 (m,1 H, T-CH), 5.35-5.26 (2xm, 2xO.5H, 3’-CH), 4.58-4.52 (m, 0.2H 2’ -CH), 4.43-4.39 (m, 0.8H 2’+ 0.8H 4’ -CH), 4.32-4.29 (m, 0.2H, 4’- CH), 3.98-3.93 (m, 0.4H, 5’-CH2), 3.60-3.51 (2H, CHCH3 / pr), 3.98, 3.97, 3.95, 3.94 (4xs, 0.3H, 0.3H, 1.2H, 1.2H, OCH3NV), 3.89-3.67 (m, 5H, OCH3Nv+OCH2Cne +1.5H 5’-CH2), 3.34, 3.34, 2.97,2.96 (4xs, 3H, 2’ OCH3), 2.67-2.50 (m, 2H, CH2CN Cne), 1.66-1.64 (m, 3H, CHCH3Nv), 1.15-1.07 (m, 12H, CH3 / Pr) ppm.

[0147] 31P-NMR (162MHz, CDCI3; Figure 11) 5 = 149.37 (0.4P), 149.32 (0.1 P), 149.30 (0.1 P), 149.27 (0.4P) ppm.Synthesis of (mA)s pentamer, 17, using 3’-MeNvOC-5’-phosphoramidite, 13 (Figure 12)

[0148] 1, Loading of 20 kDa PEG-star: 8-Arm, methylamino, 20 kDa polyethylene glycol [14, PEG-20k(NMeH)s, 1.00 g, 0.050 mmol] was dissolved in anhydrous MeCN (10 mL) and transferred to the Nanostar synthesiser feed tank via a port using a 12 mL syringe, washing out the flask with further MeCN (5 mL). / V, / V-Diisopropylethylamine (DIPEA, 0.12 mL, 1.0 mmol, 14 eq.) was then added to the synthesiser feed tank and circulated for 2 min; an initial sample (t = 0) was taken from the fully mixed synthesiser. To a solution of 3’-MeNvOC-mABz5’-succinate (13, 0.44 g, 1.0 mmol, 12 eq.) in MeCN (3 mL) in a round bottom flask was added TBTU (0.19 g, 1.0 mmol, 12 eq.) and the solution was stirred for 30 min at room temperature. The solution ofactivated 5’-succinate was then injected into the synthesizer through a 0.2 m PTFE filter (Sterlitech Pressurised Filtration Holder). The amidation reaction was monitored by LC-MS; it typically appeared complete after 5 min, but was allowed to continue for 30 min to ensure completion.

[0149] 2 Photocleavage of 3’-MeNvOC: In addition to the apparatus used in Example 1 , the synthesiser was fitted with a spiral photo-irradiation flow reactor (0.159 cm diameter, 101 cm length FEP tubing ) illuminated with blue LEDs (24 W, 365 nm wavelength, 10.0 mm distance from coil) between the feed tank and pump (Figure 6). A sample was removed from the synthesiser (50 pL, t = 0) and diluted with MeCN (1.5 mL); the retention times of the fully protected (MeNvOC)s-PEG-star and 3’-MeNvOC-mABz5’-succinate (12, or 5’-phosphoramidite, 13) species in the LC-MS chromatogram were identified, analysing at 260 nm. The circulation in the Nanostar synthesiser was thermostatted at 30 °C using the heat exchanger, and diafiltration was commenced using anhydrous acetonitrile-sulfolane (4:1 v / v, < 20 ppm water, 33 cm2, 10 bar).

[0150] MeNvOC deprotection was then initiated by irradiating both the feed tank (Kessil PR160L lamp, 370 nm, 100 W intensity) and the spiral flow reactor. During irradiation 15 DV (600 mL, membrane area 33 cm2, 10 bar) of solvent were diafiltered (DF). The reaction was monitored by LC-MS at 60 min intervals, tracking both the building block and the oligo-star. Initially, residual building block (MeNvOC protected and deprotected) permeated and was washed from the system. As photo-cleavage of the (MeNvOC-oligo)s-PEG-star proceeded, intermediates bearing all the way from seven to one MeNvOC groups could be observed by LC [Figure 13, deprotection of 3’-MeNvOC-(mABz)2-PEG-star; Figure 14, deprotection of 3’-MeNvOC-(mABz)s-PEG-star] to first rise, and then fall to nothing as the deprotection proceeded to completion. Unlike irradiating MeNvOC protected species in a closed flask (see Counter-Example 2), MeNvOC deprotection of the oligo-star in the Nanostar synthesiser proceeds to completion (Figures 12 and 13) because the strongly absorbing photo-debris (16) is continuously removed from the system by membrane permeation. After 5 hr, no partially deprotected species could be detected and the lamp and LEDs were extinguished.

[0151] 3, Coupling reaction: The DF during step 2 ensures that the Nanostar synthesizer has very low moisture content (< 30 ppm). Therefore, coupling can be commenced immediately. 3’- MeNvOC-5’-Dmtr-mABz5’-phosphoramidite (13, 0.500 g, 1.0 mmol, 1.5 eq. / arm) in a round- bottomed flask was evaporated from MeCN (<15 ppm water, 3 x 25 mL), then re-dissolved in MeCN (<30 ppm water, 5 mL) and injected into the synthesiser, followed by DCI (0.140 g, 1.0 mmol, 3 eq. / arm) dissolved in MeCN (5 mL); all components were injected into synthesiser via a 0.2 pm PTFE filter. The reaction was monitored by LC; after 2 min the coupling intermediates were consumed, but circulation was continued for 20 min to ensure complete reaction. CneOH (0.1 mL, 3 eq. / arm) was then added and, after circulating for 2 min, CSO (0.460 g, 2.0 mmol, 5eq. / arm) dissolved in MeCN (10 mL) was then injected into the synthesiser, followed by circulation continued for a further 60 min.

[0152] The synthetic cycle described above, alternating steps 2 and 3, with only a single DF per cycle, was repeated in the same fashion up to (mABz)s pentamer-star, with a final irradiation step to remove the last MeNvOC protecting group, prior to global deprotection. The performance of the synthesiser is reported in Table 2. It is notable that throughout the four phosphoramidite coupling and MeNvOC deprotection cycles there was no change in building block rejection or in the permeate flow. This is in marked contrast to the membrane performance using the same membranes, in the same synthesiser (without irradiation systems), but coupling with 5’-Dmtr- mABz3’-phosphoramidite building blocks and removing the 5’-protecting group with acid (see Counter-Example 1 and Table 3).Table 2. Membrane performance during 3’-MeNvOC synthesis cycles

[0153] 4, Global deprotection: The global deprotection of the pentamer 17 was performed similarly to Example 1 , treating the 5’-HO-pentamer-star with cone, ammonia containing 3 vol% diethylamine. After isolation of the crude (mABz)s pentamer (17), IP-RP LC-MS of the oligo (Figure 15, UV purity 88.1%) demonstrated that the only detectable impurities were n-1 and n+1 related species, from either incomplete coupling, or light-induced impurities in the building block.Counter-Example 1 : Oligo synthesis using 5’-Dmtr amidites

[0154] 1, Loading of 20 kDa PEG-star: PEG-20k(MeNH)s support (6.10 g, 0.3 mmol) was dissolved in anhydrous MeCN (10 mL) and transferred to the synthesiser feed tank via a port using a 12 mL syringe, washing out the flask with further MeCN (5 mL). N,N- Diisopropylethylamine (DIPEA, 0.74 mL, 4.2 mmol, 14 eq.) was then added to the synthesiserfeed tank and circulated for 2 min, when a sample was taken from the fully mixed Nanostar synthesiser (t = 0). In a round bottom flask 5’-Dmtr-mABz5’-succinate (2.03 g, 3.6 mmol, 12 eq.) was dissolved in anhydrous MeCN (6 mL) to which was added TBTLI (1.17 g, 3.6 mmol, 12 eq.) and the solution was stirred for 30 min. The activated succinate solution was then injected into the synthesizer through a 0.2 pm PTFE filter. The amidation reaction was monitored by LC-MS and was typically complete after 5 min, but was left 30 min to ensure completion. After a sample had been taken from the fully mixed Nanostar synthesiser to determine the residual Dmtr- succinate concentration at the start of the first diafiltration (DF1), acetonitrile-sulfolane 4:1 v / v was then permeated (240 mL = 4DV, membrane area 112 cm2, 10 bar). Atthe end of DFI another sample was analysed by LC-MS to measure the fractional drop in the concentration of succinate and the rejection of the trityl nucleoside-star.

[0155] 2, Detritylation of5’-Dmtr: 2,2'-(Ethylenedioxy)diethanethiol (DODT, 6 eq. / arm, 48 eq. in total) was injected into the feed tank via the injection port using a syringe fitted with a 0.2 pm PTFE filter, followed by methanesulfonic acid (MSA, 0.25 mL). The detritylation reaction was monitored by LC-MS and after 10 min neither partially deprotected Nanostar species or residual Dmtr-mABzsuccinate could be detected. After 20 min picoline (0.50 mL) was injected into the feed tank via the injection port and a sample was taken to determine the intermediate concentration of detritylated succinate. Dry MeCN (< 20 ppm water, 360 mL, ~6 DV, membrane area 112 cm2, 10 bar) was permeated (DF2), to remove residual 5’-hydroxy-mABz3’-succinate and all other small molecule debris. At the end of DF2 a final sample was analysed by LC-MS to verify complete removal of building block debris and to confirm the high rejection of the 5’-OH- nucleoside-star. Typically, the chain extension was only undertaken if the combined drop in building block concentration over DF1 and DF2 was >99%. DF2 could be continued until this target was met. Furthermore, the moisture content of the permeate was measured prior to the coupling (Table 3) to ensure that the phosphoramidite

[0156] 3, Coupling reaction: 5’-Dmtr-mABz-3’-phosphoramidite (1, 2.59 g, 2.92 mmol, 1.2 eq. / arm) and DCI (860 mg, 7.3 mmol, 4 eq. / arm) were each placed in separate round bottom flasks. The building block was co-evaporated from MeCN (<15 ppm water, 3 x 25 mL), then redissolved in MeCN (<30 ppm water, 5 mL) and injected into the synthesiser, followed by the DCI dissolved in MeCN (5 mL). All components were injected into the synthesiser via 0.2 pm PTFE filters. The reaction was monitored by LC; after 5 min the coupling intermediates (1 to 7 arms) appeared to be consumed, but circulation was continued in the synthesiser for 20 min to ensure complete reaction. The reaction was then quenched with CneOH (0.5 mL, 3 eq. / arm) and circulated for 2 min. CSC (2.78 g, 12 mmol, 5 eq. / arm) dissolved in MeCN (10 mL) was then added to the synthesiser and circulation continued for 1 hr. The oligo-star solution was partiallypurified by permeating acetonitrile-sulfolane 4:1 (< 20 ppm water, membrane area 33 cm2, 240 mL, ~4DV, 10 bar) to remove the majority of low molecular weight solutes.Table 3. Membrane performance during Dmtr synthesis cyc es

[0157] The preceding two steps, detritylation then coupling, were repeated four times, plus a final detritylation to provide the desired 5’-HO-(mA)s pentamer-star. The membrane performance in each cycle is given in Table 3. It is notable that as the synthesis proceeds, the permeate flow is seen to decrease with each cycle. This indicates a rise in membrane fouling with each chain extension cycle. This contrasts sharply with Example 2 (see Table 2), where the same membranes were used with photolytic cleavage of the terminal protecting group. The permeance starts at a much higher value, the permeance remains remarkably constant from one cycle to the next, and the building block rejection remains so low that none can be detected after DF.

[0158] Global deprotection was conducted similarly to Examples 1 and 2 to provide fully deprotected (mA)s pentamer (17) in 81.6% UV purity. Apart from n-1 (1.2%), the lower purity was associated with late eluters generated during detritylation.Example 3: Oligo synthesis using 5’-O-MeNvOC amidites and diafiltration of photo-debris Synthesis of 2’-O-Me-5’-O-MeNvOC-6-N-Bz-Adenosine 3’-O-phosphoramidite, 18 (Figure 16):

[0159] 1, Silylation'. 5’-Dmtr-mABznucleoside (10, 25.00 g, 36.4 mmol) was evaporated from MeCN (3 x 350 mL) and re-dissolved in dry pyridine (100 mL). To the solution were added imidazole (5.4 g, 80 mmol, 2.2 eq.) and terf-butyldimethylsilyl chloride (Tbdms-CI, 5.40 g, 40 mmol, 1.1 eq.). The reaction mixture was stirred at 25 °C for 18 hr, with progress monitored by LC-MS. Upon completion, methanol (0.5 mL) was added and stirring continued briefly. The solvents were removed by co-evaporation with toluene (3 x 200 mL). The oily residue was redissolved in dichloromethane (DCM) and washed three times with water. The combined aqueouslayers were back-extracted with DCM. The combined organic phases were dried over Na2SO4and evaporated under reduced pressure.

[0160] 2, Detritylation: The crude 3’-Tbdms-5’-Dmtr-mABznucleoside was dissolved in DCM, to which solution were added dodecanethiol (DDT, 26.12 mL, 109 mmol, 3.0 eq.) and methanesulfonic acid (MSA, 0.5 mL). The solution was thermostatted at 25 °C and stirred for 10 min; completion was confirmed by LC-MS. Saturated sodium bicarbonate (2 mL) was added and, after extraction, the DCM phase was dried over Na2SO4and concentrated under vacuum. To the residue were added MeCN and heptane with vigorous mixing; the residual DDT and Dmtr-DDT by-product partitioned into the heptane phase. The MeCN phase was separated and concentrated under reduced pressure. The residue was fractionated by reversed phase chromatography; 300 g C18 column, eluted with water and MeCN-MeOH 4:1 , 20-100%. 3’- Tbdms-mABznucleoside (18) was obtained as a white solid (20.10 g, 80%).

[0161] 1H-NMR (400 MHz, CDCI3) 6 = 8.62 (s, 1 H, ArH Ado), 7.95 (s, 1 H, ArH Ado), 7.88-7.85 (m, 2H, ArH Bz), 7.48-7.43 (m, 1 H, ArH Bz), 7.39-7.34 (m, 2H, ArH Bz), 5.79 (d, J= 7.0 Hz, 1 H, T-CH), 4.46-4.42, (m, 2H, 2’-CH + 3’-CH), 4.07 (br.s, 1 H, 4’-CH), 3.82 (dd, 1 H, J=13.1, 1.8Hz, 5’-CHH) 3.58 (br.d, 1 H, J= 13.2Hz, 5’-CHH), 3.10 (s, 3H, 2’-OCH3), 0.80 (s, 9H, SiCCH3Tbdms), 0.00, 0.02 (2xs, 6H, SiCH3Tbdms) ppm.

[0162] 3, Attachment of 5’-MeNvOC 3’-Tbdms-mABz(18,10.00 g, 20.03 mmol) was dissolved in DCM (50 mL, 12 ppm of water), to which were added MeNvOC-OPnp (9, 15.7 g, 40.06 mmol, 2eq.) and DMAP (7.34 g, 60.09 mmol, 3 eq.), and the reaction was stirred for 3 hr. The solution was then partitioned with 1M sodium phosphate buffer, after which the organic layer was dried over Na2SO4and evaporated to dryness. The residue may be fractionated by reversed phase chromatography; 120 g C18 column, eluting with a gradient of water and MeCN-MeOH 4:1 , 20%- 100%. 5’-MeNvOC-3’-Tbdms-mABz(19) is then obtained as a pale-yellow powder (8.32 g, 81 %).

[0163] 4, Desilylation To a solution of crude 3’-Tbdms-5’-MeNvOC-mABz(19) in MeCN (60 mL) to was added tetraethylammonium fluoride hydrate (TEAF, 12.13 g, 100.15 mmol), and the solution was stirred at room temperature for 3 hr, after which desilylation was complete, monitoring by TLC or LC-MS. The solvent was removed under reduced pressure, the oily residue was re-dissolved in dichloromethane (DCM, 200 mL) and washed three times with water. The combined aqueous layers were back-extracted with DCM. The combined organic phases were dried over Na2SO4and evaporated under reduced pressure. The residue was fractionated by reversed phase chromatography; 120 g C18 column, eluting with a gradient of water and MeCN- MeOH 4:1 , 20%-100%. 5’-MeNvOC-mABz(20) was obtained as a pale-yellow glass (7.55 g, 70%).

[0164] 1H-NMR (400 MHz, CDCh) 6 = 8.69,8.67 (2xS, 2xO.5H ArH Ado), 8.22,8.18 (2xS, 2xO.5H ArH Ado), 7.97-7.95 (m, 2H ArH Bz), 7.56-7.53 (m, 1 H, ArH Bz), 7.49-7.45 (m, 3H, ArH 2xBz+Nv), 7.01 , 6.96 (2xS, 2xO.5H ArH Nv), 6.39-6.31 (m, 1 H, ArCHMe Nv), 6.13, 6.12 (2xd, =2.9 Hz, 2xO.5H, T-CH), 4.49-4.33 (m, 3H, 4’+3’+2’-CH), 4.27-4.23, 4.20-4.16 (2xm, 2x1H, 5’- CH2), 3.90, 3.85, 3.84, 3.83 (4xs, 4x1.5H, OCH3Nv), 3.54, 3.53 (2xS, 2x1.5H, 2’-OCH3), 1.62,1.60 (2xs, 2x1.5H, CHCH3Nv) ppm.

[0165] 5, Conversion to 3’-phosphoramidite'. 5’-MeNvOC-mABznucleoside (20, 10.00 g, 15.65 mmol) was evaporated from MeCN (3 x 200 mL) and then dried under vacuum prior to use. The nucleoside was dissolved in DCM (50 mL) and chilled in an ice bath to 4 °C. Ensuring the low temperature was maintained, 2-cyanoethyl / V, / V, / V’, / V-tetraisopropylphosphordiamidite (7.46 mL, 23.5 mmol, 1.5 eq.) was added. Solid pyridinium trifluoroacetate (Py-TFA, 2.42 g, 12.52 mmol, 0.8 eq.) was then added as a solid, and the reaction was firstly stirred at 4 °C for 30 min, followed by stirring at room temperature for 18 hr. The following day, triethylamine (0.1 mL) was added and the crude was fractionated by reversed phase chromatography; 120 g C18 column, eluting with a gradient of water and acetonitrile-MeOH 4:1 , 20 to 100 %. 5’-MeNvOC-mABz3’- phosphoramidite (21) was isolated as a pale-yellow glass (8.435 g, 84%).

[0166] 1H-NMR (400 MHz, CDCh; Figure 16) 5 = 8.68, 8.67, 8.64, 8.62 (4xs, 0.2H, 0.3H,0.2H, 0.3H ArH Ado), 8.21 , 8.20, 8.15 (3xs, 0.3H, 0.2H, 0.5H ArH Ado), 7.97-7.95 (m, 2H, ArH Bz), 7.56-7.50 (m, 1 H, ArH Bz), 7.48-7.42 (m, 3H, ArH 2xBz+Nv), 7.01 , 7.00, 6.96, 6.94 (4xs, 0.2H, 0.3H, 0.2H, 0.3H, ArH Nv), 6.38-6.33 (m, 1 H, ArCHMe Nv), 6.12, 6.09, 6.06 (3xd, J= 3.9, 4.0, 4.4 Hz, 0.3 H, 0.5H, 0.2H, T-CH), 4.61-4.30 (m, 5H, 5’-CH2+4’+3’+2’-CH), 3.89-3.82 (m, 6H, OCH3 Nv), 3.87-3.73 (m, 2H, OCH2Cne), 3.70-3.52 (m, 2H, NCHMe2), 3.49, 3,45, 3,45, 3.41 (4xS, 0.8H, 0.8H, 0.7H, 0.7H, 2’-OCH3), 2.61-2.53 (m, 2H, CH2CN Cne), 1.61-1.58 (m, 3H, CHCH3Nv), 1.19- 1.07 (m, 12H, NCHCH3) ppm.

[0167] 31P-NMR (162 MHz, CDCh; Figure 18) 5 = 151.16 (0.2P), 150.93 (0.2P), 150.50 (0.3P), 150.43 (0.3P) ppm.Synthesis of (mA)s pentamer, 17, using 5’-MeNvOC-3’-phosphoramidite, 21 (Figure 16).-

[0168] 1, Loading of 20 kDa PEG-star: The loading was conducted similarly to Counter- Example 1 , reacting PEG-20k(MeNH)s (1.25 g, 0.06 mmol) and DIPEA (0.15 mL, 1.0 mmol, 14 eq.) in the Nanostar synthesiser with activated succinate prepared from 5’-Dmtr-mABz-3’- succinate (0.59 g, 1.0 mmol, 12 eq.) and TBTU (0.24 g, 1.0 mmol, 12 eq.) in anhydrous MeCN (3 mL). Acetonitrile-sulfolane 4:1 v / v was permeated (200 mL = 4DV, 10 bar, membrane area 112 cm2, 10 bar, DF1).

[0169] 2, Detritylation reaction: A solution of DODT (8.0 eq. / arm, 64 in total) in MeCN (5 mL) was injected into the feed tank via the injection port using a syringe fitted with a 0.2 pm PTFE filter, followed by TFA (2.5 mL). The reaction was monitored by LC-MS and after 10 min no partially deprotected species could be detected. After 20 min the reaction was quenched with pyridine (5 mL) and a sample was taken to determine the intermediate concentration of detritylated succinate. Dry acetonitrile-sulfolane 4:1 (< 20 ppm water, 300 mL = 6DV, 10 bar) was permeated (DF2), to remove residual 3’-succinate and other detritylation debris. At the end of DF2 a final sample was analysed by LC-MS to verify complete removal of building block debris and the high rejection of the nucleoside-star.

[0170] 3, Coupling reaction: 5’-MeNvOC-mABz-3’- phosphoramidite (21 , 0.63 g, 1.0 mmol, 1.5 eq. / arm) and DCI (0.180 g, 1.0 mmol, 3 eq. / arm) were each placed in separate round bottom flasks. The building block was evaporated from MeCN (<15 ppm water, 3x25 mL), then redissolved in MeCN (<30 ppm water, 5 mL) and injected into the synthesiser, followed by the DCI dissolved in MeCN (5 mL); all components were injected into synthesiser via a 0.2 pm PTFE filter. The reaction was monitored by LC; after 2 min the coupling intermediates were consumed, but circulation was continued in the synthesiser for 20 min to ensure complete reaction. The reaction was then quenched with CneOH (0.1 mL, 3 eq. / arm) and circulated for 2 min. CSC (0.57 g, 2.0 mmol, 5 eq. / arm) dissolved in MeCN (10 mL) was then added to the synthesiser, and circulation continued for 60 min to obtain the chain extended phosphate tri-ester.

[0171] 4, Photocleavage of 5’-MeNvOC: Similar apparatus was used here as in Example 2, including a spiral photo-irradiation flow reactor (diameter 0.159 cm, 101 cm length FEP tubing) illuminated with blue LEDs (24W, 365 nm, 10 mm distance from coil) (Figure 6). Each 5’-0 photocleavage was conducted similarly to Example 2, diafiltering with 15 DV (= 750 mL) acetonitrilesulfolane 4:1 v / v (<20 ppm) to wash away debris 16, and the reaction was complete after 5 h. Again, zero drop in membrane permeance was observed throughout the four synthetic cycles using photo-cleavage (Table 4), unlike the typical acidolytic detritylation process demonstrated in Counter-Example 1. The synthetic cycle of coupling and photocleavage described above was repeated three more times in the same fashion to elongate the oligo chain up to fully-protect (mA)5-pentamer-star.Table 4. Membrane performance during 5’-MeNvOC synthesis cycles4, Global deprotection: After the final photo-deprotection a sample was taken from the retentate and subjected to global ammonolytic deprotection as in Examples 1 and 2. The IP-RP chromatogram of the resultant (mA)s pentamer, 17, is shown in Figure 19. The only detectable impurities are again n-1 and n+x (x = 1 ,2,3); the latter were more pronounced in this example due to light sensitivity and exposure during handling of phosphoramidite 21.Counter-Example 2

[0172] 5’-MeNvOC-3’-Tbdms-mABz(19, 150 mg) was dissolved in MeCN-sulfolane 4:1 (30 mL) in a round-bottomed flask and incubated with DCI (60 mg, 3.0 eq.), CneOH (40 pL, 3.0 eq.), and CSO (650 mg, 5.0 eq.), to simulate the contaminants expected from undergoing a phosphoramidite coupling reaction. The mixture was then irradiated with a Kessil PR160L lamp set at 370 nm and 100 W intensity for 2 h, monitoring the reaction by LC-MS. The conversion of 19 to 5’-hydroxy-3’-Tbdms-mABz(18) became slower and slower (Figure 20), as an intense yellow-brown colour developed in the reaction flask, and could not be driven to completion.

[0173] To evaluate whether the photo-debris could be removed by diafiltration, the reaction mixture was transferred to the membrane diafiltration apparatus (MiniMem filtration system, PS Prozesstechnik GmbH, membrane area 33cm2). Diafiltration was carried out in acetonitrilesulfolane 4:1 v / v, permeating 7 DV (105 mL, 10 bar) at room temperature. All solutes were washed through the membrane into the permeate (Figure 21), and the low molecular weight MeNvOC debris, 16, had the lowest rejection.

[0174] 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.REFERENCESA.G. Livingston et al. membrane DF of oligos US 8,664,357A.G. Livingston et al. PEG-stars US 9,127,123A.G. Livingston et al. PEGabet US 10,239,996A.G. Livingston et al PEGabet EP 3347402P.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.9535-9543J.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) 1439-1452So 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. 1313-132Yeo 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.7786-7795PEG and derivatives of PEG have also been synthesised using membrane separation, as described by Dong R., Liu R., Gaffney P.R.J., Schaepertoens M., Marchetti P., Williams C.M., Chen R. and Livingston A.G. “Sequence-defined multifunctional polyethers via liquid-phase synthesis with molecular sieving” Nature Chemistry 11 (2019) pp.136-145Peptide synthesis WO / 2016 / 188835 A15’-NPPOC amidites 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.1105-1108Oligonucleotide matrix synthesis: NPPOC first report M. Beier, J. D. Hohheisel, “Production by quantitative photolithographic synthesis of individually quality checked DNA microarrays”, Nucl. Acids Res. 28 (2000) pp e11BzNPPOC amidites US 7,759,513 B2, Buhler et al. “Photolabile protective groups for improved processes to prepare oligonucleotide arrays”, 2010BzNPPOC amidites US 8,445,734 B2, Buhler et al. “Photolabile protective groups for improved processes to prepare oligonucleotide arrays”, 2013 - cannot see how these two patents differBzNPPOC in MAS N. Kretschy, M. M. Somoza et al. “Next-Generation o-Nitrobenzyl Photolabile Groups for Light-Directed Chemistry and Microarray Synthesis” Angew Chem. Inti. Ed. 54 (2015) pp.8555-8559Maskless Array Synthesis BzNppoc (MAS) 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.6687-6701Reverse BzNPPOC amidites K. Holz, M. M. Somoza et al. “High-Efficiency Reverse (5'— >3') Synthesis of Complex DNA Microarrays” 8 (2018) art. # 15099A. 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 Depurination M. Septak “Kinetic studies on depurination and detritylation of CPG-bound intermediates during oligonucleotide synthesis” Nucl. Acids Res. 24 (1996) 3053-3058LPOS strategies incl. precipitation and extraction A. G. Molina, Y. S. Sanghvi “Liquid-Phase Oligonucleotide Synthesis: Past, Present, and Future Predictions” Current Protocols in Nucleic Acid Chemistry (2019) e82LPOS precipitation G. Creusen, 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. 2087-2110M. 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 Photocleavage on solid support, but grinding to maximise light penetration Y. Bakhatan, M. Hurevich, “The breaking beads approach for photocleavage from solid support” Org. Biomol. Chem. 18 (2020) pp. 4183-4188Large scale photo-reactions L. Buglioni, T. Noel et al. “Technological Innovations in Photochemistry for Organic Synthesis: Flow Chemistry, High-Throughput Experimentation, Scale-up, and Photoelectrochemistry” Chem. Rev. 122 (2022) pp. 2752-2906C. B. Reese, Org. Biomol. Chem., 2005, 3, 3851Baran et al., ACS. Cent. Sci., 2021 , 7, 1473Arai K. et al. Bioorg. Med. Chem. 2011 , 21 , 6285US6683173L. F. Scherz, “Dendronized Polymers with Tailored Intermolecular Interactions: Synthesis and Thermomechanical Characterization”. Thesis, 2017, ETH Zurich.

Claims

CLAIMS1. A liquid-phase process for preparing an oligonucleotide, the process comprising growing an oligonucleotide by performing one or more chain extension cycles, wherein each chain extension cycle comprises coupling a monomeric or oligomeric building block to a chain extension site of a growing oligonucleotide, wherein each building block comprises: at least one nucleosidic moiety (Nuc); a reactive terminal (RT) for coupling the building block to the chain extension site of the growing oligonucleotide; and 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; wherein in at least one chain extension cycle: the temporary protecting group (TPG) is a photolabile protecting group (PPG); and membrane filtration is performed to isolate the growing oligonucleotide (e.g., from uncoupled building blocks and / or reaction debris).

2. The process of claim 1, wherein each building block independently comprises 1-3 nucleosidic moieties.

3. The process of claim 1 or 2, wherein each building block has a structure according to formula I:I whereinNuc is a nucleosidic moiety;RT is the reactive terminal, which is covalently bound to Nuc;TPG is the temporary protecting group, which is covalently bound to Nuc;Lpis an internucleoside linkage; andV is 0-10 (e.g., 0-3).

4. The process of claim 3, wherein v is 0, 1 or 2.

5. The process of claim 3 or 4, wherein the building blocks of formula I each have a structure according to formula la:RT— 0A-Nuc4Lp-Nuc4-0B-TPG' 'V la whereinOAis the oxygen located at the 5’ terminal carbon of Nuc, and OBis the oxygen located at the 3’ terminal carbon of Nuc; orOAis the oxygen located at the 3’ terminal carbon of Nuc, and OBis the oxygen located at the 5’ terminal carbon of Nuc; andRT, Nuc, TPG, Lpand v are as defined in claim 3 or 4.

6. The process of any one of the preceding claims, wherein RT has a structure according to formula II:II whereinY is a leaving group;PG is a protecting group; and ww denotes the point of attachment to the nucleosidic moiety (Nuc) to which RT is attached (e.g., via OAin formula la).

7. The process of claim 6, wherein Y is -NR2, 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.

8. The process of claim 7, wherein each R is isopropyl.

9. The process of claim 6, 7 or 8, wherein PG is a base-labile protecting group.

10. The process of any one of claims 6-9, wherein PG is cyanoethyl.

11. The process of any one of the preceding claims, wherein each nucleosidic moiety (Nuc) has a structure according to formula III:Ill whereinZ is a nucleobase, optionally protected by a protecting group;Rxis selected from H, OH, F, O-tert-butyldimethylsilyl (OTbdms), methoxy, O-methoxyethyl (OMoe), O-propargyl, NH2 and N3, in which case bothare absent, or Rxis O and both are present;1and2independently denotes the points of attachment to RT (e.g., via OAin formulae la-le), TPG (e.g., via OBin formulae la-le) or LP (as appropriate).

12. The process of claim 11 , wherein Rxis H, OH, F, methoxy or O-methoxyethyl (OMoe).

13. The process of any one of the preceding claims, wherein each PPG independently has a structure according to formula IV:whereinL is absent or a linker group selected from -CRaRb-, -C(O)-O-CRaRb- or -C(O)-O-, wherein Raand Rb are each independently selected from H or (1 -3C)alkyl;R1is selected from H, (1-3C)alkyl or -C(O)-aryl; n is 1-4; each R2is independently selected from -(1-3C)alkyl, -NO2, -OMe, -C(O)-aryl or -SPh; or when two R2groups are present on adjacent carbon atoms in the phenyl ring, both R2groups are linked such that they form a fused 5- to -7-membered heterocycle; and denotes the point of attachment to a chain extension site.

14. The process of claim 13, wherein L is absent or a linker group selected from -CH2-, - C(O)-O-CH2- or -C(O)-O-.

15. The process of claim 13 or 14, wherein R1is selected from H, methyl or -C(O)-phenyl.

16. The process of claim 13, 14 or 15, wherein n is 1 , 2 or 3.

17. The process of any one of claims 13-16, wherein each R2is independently selected from -Et, -NO2, -OMe, -C(O)-Ph or-SPh; or when two R2groups are present on adjacent carbon atoms in the phenyl ring, both R2groups are linked such that they form a fused dioxolane (e.g., a 1 ,3- dioxolane).

18. The process of any one of claims 13-17, wherein each PPG independently has a structure according to formula IVa:whereinR1Ais H or methyl;L is -C(O)-O-CH2- or -C(O)-O-;R^ is -OMe or absent;R2Bis -OMe or absent; or, R2Aand R2Bare each O and are linked to one another by a -CH2- group (i.e., resulting in a 1 ,3-benzodioxole ring system); and « / W' is as described in claim 13.

19. The process of any one of claims 1-13, wherein each PPG independently has any one of the following structures:2-nitrobenzyl 1 -(2-nitrophenyl)ethyl 2 -nitrophenethyl 2-(2-nitrophenyl)propyl2-(2-nitrophenyl)propoxycarbonyl (Nppoc) dimethoxybenzoin carbonate (Dmboc)(a-methyl-2-nitropiperonyl)oxycarbonyl (Me-Npoc) benzoyl-2-(2-nitrophenyl)propoxycarbonyl (Bz-Nppoc)thiophenyl-2-(2-nitrophenyl)propoxycarbonyl (SPh-Nppoc) methyl-6-nitroveratryloxycarbonyl (MeNvOC) wherein rwv' is as described in claim 13.

20. The process of any one of the preceding claims, wherein in at least 75% of the chain extension cycles (to the nearest whole number), the TPG is a PPG.

21. The process of any one of the preceding claims, wherein in at least 80% of the chain extension cycles (to the nearest whole number) in which the TPG is a PPG, membrane filtration is performed to isolate the growing oligonucleotide.

22. The process of any one of the preceding claims, wherein in each one of the chain extension cycles, the TPG is a PPG and membrane filtration is used to isolate the growing oligonucleotide.

23. The process of any one of the preceding claims, wherein in any chain extension cycle wherein the TPG is a PPG, each chain extension cycle comprises a step of cleaving the PPG 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.

24. The process of claim 23, wherein the PPG is cleaved by irradiating the growing oligonucleotide at a wavelength of >350 nm (e.g., 365 nm).

25. The process of any one of the preceding claims, wherein in at least one chain extension cycle in which TPG is PPG, the steps of: (i) cleaving the PPG 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, and (ii) membrane filtration, are performed simultaneously.

26. The process of any one of the preceding claims, wherein in all chain extension cycles in which TPG is PPG, the steps of: (i) cleaving the PPG 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, and (ii) membrane filtration, are performed simultaneously.

27. The process of claim 23 or 24, wherein membrane filtration is performed before and after a step of cleaving the TPG or PPG, with the first membrane filtration being performed after coupling of the building block to a chain extension site of a growing oligonucleotide.

28. The process of any one of the preceding claims, wherein the membrane filtration steps are conducted by organic solvent nanofiltration (OSN) or ultrafiltration (UF).

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