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WO2025133559A1 - Procédé en phase liquide pour la préparation d'oligonucléotides - Google Patents

Procédé en phase liquide pour la préparation d'oligonucléotides Download PDF

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Publication number
WO2025133559A1
WO2025133559A1 PCT/GB2024/051523 GB2024051523W WO2025133559A1 WO 2025133559 A1 WO2025133559 A1 WO 2025133559A1 GB 2024051523 W GB2024051523 W GB 2024051523W WO 2025133559 A1 WO2025133559 A1 WO 2025133559A1
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chain extension
building block
npg
oligonucleotide
nucleobase
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Andrew Guy Livingston
Piers Robert James Gaffney
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Exactmer Ltd
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Exactmer Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/02—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical

Definitions

  • the present invention relates to a liquid-phase process for preparing oligonucleotides. More particularly, the present invention relates to a membrane filtration-assisted, liquid-phase process for preparing oligonucleotides. BACKGROUND OF THE INVENTION [0002] Oligonucleotides (oligos) are short sequence-defined polymers of nucleotides (a.k.a. nucleoside phosphates).
  • oligos typically consists of a ribose phosphate backbone, usually linking the 3’- and 5’-oxygen substituents of the ribose sugar via a phosphodiester unit, and each ribose ring has an aromatic nucleobase bound to the 1’-C of ribose.
  • the combination of ribose sugar (or other analogue discussed below) and nucleobase is called a nucleoside, and the nucleobase varies from one monomer unit to the next in natural and medicinal sequences. It is the nucleobase sequence that confers target specificity on oligos.
  • oligos have been prepared using solid-phase oligonucleotide synthesis (SPOS) wherein a growing oligo is tethered to an insoluble solid support and grown by flowing reactive nucleoside phosphoramidite building blocks over the insoluble solid support.
  • SPOS solid-phase oligonucleotide synthesis
  • This cyclic process consists of three principal chemical steps: 1) extension of an exposed chain terminal OH by the building block then, 2) oxidation of, or sulfur transfer to the internucleotide linkage, and finally, 3) removal of a temporary chain terminal protecting group to expose a new OH group.
  • the cycle is repeated to build up the desired oligo sequence one monomer (or other building block) at a time.
  • most oligo synthesis protocols include an additional capping step to prevent any unextended chain termini from participating in further cycles, thus reducing the number of chomatographically similar impurities to the full-length product (FLP).
  • capping is only partially effective and can introduce impurities itself.
  • the phosphorus bearing moiety is a highly reactive P(III) phosphoramidite, usually bound to the ribose 3’-O of the ribose ring, and is protected with a permanent 2-cyanoethyl ester which is base labile; the nucleobase is usually permanently protected on any reactive NH (and occasionally O) with ammonolytically sensitive (acyl, amidine) groups; the 5’-O of ribose is temporarily protected with a mild acid labile protecting group, almost universally 4,4’-dimethoxytriphenylmethyl (a.k.a. dimethoxytrityl, Dmtr).
  • Liquid phase reactions and liquid phase material handling are established technologies that can be performed at the multi-tonne scale, making LPOS a strong candidate for oligo preparation at scale.
  • a typical approach to LPOS is to carry out sequential coupling reactions, adding monomers or multi-monomer oligomers (a.k.a. fragments or building blocks) to a growing oligo in solution in a stepwise fashion, and then to use a suitable separation technology (Molina et al.), such as precipitation (Creuse et al., Zhou et al.) or liquid extraction (de Koning et al.) to separate unreacted monomers or fragments from the growing oligo.
  • Molina et al. such as precipitation (Creuse et al., Zhou et al.) or liquid extraction (de Koning et al.)
  • LPOS has an inherent advantage over SPOS in that the entire reaction solution can be observed and sampled in real time.
  • Membrane-assisted LPOS (Gaffney et al., Kim et al.) is unique amongst oligo synthesis strategies in that the growing oligo remains in a single solution at all times.
  • membrane filtration e.g., US 8,664,357, US 9,127,123, US 10,239,996, etc. is used to separate the unreacted building block and any reaction debris from the growing oligo, without the need for phase change or phase separation.
  • a liquid-phase process for preparing an oligonucleotide comprising growing an oligonucleotide by performing one or more chain extension cycles, in which each chain extension cycle comprises a step of coupling a monomeric or oligomeric building block to a chain extension site of a growing oligonucleotide, wherein in at least one chain extension cycle, the building block comprises a nucleobase protected by a nucleobase protecting group (NPG), wherein NPG comprises a group Q of formula I: (I) wherein n is 1-9 and R 1 is H or methyl, and wherein in at least one of the chain extension cycles in which the building block comprises NPG, one or more membrane filtration steps is used to isolate the growing
  • the inventors have devised the liquid-phase process for preparing an oligonucleotide according to the first aspect, which addresses the aforementioned disadvantages associated with LPOS techniques.
  • the use of building blocks in which the nucleobase comprises a nucleobase protecting group (NPG) notably reduces instances of inefficient membrane separation and/or fouling as part of a membrane-assisted LPOS technique.
  • NPG nucleobase protecting group
  • the presence of group Q within the NPG-containing, growing oligonucleotide mitigates problems, such as increases in viscosity and/or aggregation, that would otherwise occur during membrane filtration as a result of concentration polarisation factors.
  • group Q is present on one or more of the incoming building blocks (as opposed to being tethered to only one end of the growing oligo) allows for greater flexibility in preventing the emergence of any such issues throughout the entirety of the stepwise growth process. Indeed, it allows the frequency of the group Q to be varied in order to complement a growing oligo’s tendency to give rise to concentration polarisation issues at all stages of its preparation, irrespective of its overall length. Although each NPG may be relatively small, the collective effect on the whole oligo is cumulative.
  • 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.
  • 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.
  • Each building block used in the process suitably comprises: (i) at least one nucleosidic moiety (Nuc), (ii) a reactive terminal (RT) for coupling the building block to the chain extension site of the growing oligonucleotide, and (iii) a temporary protecting group (TPG), said temporary protecting group being cleavable, after coupling of the building block to a chain extension site of a growing oligonucleotide, to expose a new chain extension site of the growing oligonucleotide.
  • Nuc nucleosidic moiety
  • RT reactive terminal
  • TPG temporary protecting group
  • the building blocks used in the process can be monomeric or oligomeric (e.g., 2-8 mer), wherein mer refers to a nucleosidic moiety. Therefore, a monomeric building block comprises one nucleosidic moiety (e.g., ribose coupled to a nucleobase) to which RT and TPG are covalently bound.
  • a monomeric building block comprises one nucleosidic moiety (e.g., ribose coupled to a nucleobase) to which RT and TPG are covalently bound.
  • An oligomeric building block comprises two or more nucleosidic moieties (e.g., ribose coupled to a nucleobase), each pair of adjacent nucleosidic moieties being linked by an internucleoside linkage (also known as an internucleotide linkage), with RT being covalently bound to one terminal nucleosidic moiety and TPG being covalently bound to the other terminal nucleosidic moiety.
  • ribose may be deoxygenated at the 2’ carbon.
  • 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).
  • 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
  • substitution on the nucleobase e.g., with methyl or fluoro
  • each building block comprises 1-3 nucleosidic moieties. Most often, each building block is monomeric.
  • each cycle will comprise a coupling step (to add a building block to a reactive terminal of a growing oligonucleotide), an oxidation step (to convert phosphorus from its P(III) state to its P(V) state, and a deprotection step (to expose a reactive terminal on the growing oligonucleotide), with purification/isolation techniques being employed at one or more instances during each cycle.
  • oligos are almost always prepared with protecting groups on the exocyclic amino groups of the nucleobases to prevent branching side- reactions during phosphoramidite coupling reactions, and these are readily commercially available.
  • acyl groups fulfil this role with acetyl or benzoyl on cytosine 4-N, benzoyl on adenine 4-N and isobutyryl on guanine 2-N.
  • Additional protection can be installed on the reactive guanine 6-O, such as an aryl ether, which is essential to prevent side-reactions in H-phosphonate or phosphotriester approaches to oligo synthesis.
  • Additional protection can also be appended to the relatively reactive uracil and thymine 3-N, most commonly as a benzoyl or anisoyl group, although this is not required for phosphoramidites.
  • Alternative protection can be installed on uracil and thymine on the 4-O instead of the 3-N, like on guanine 6-O most commonly as an aryl ether, and again most developed for oligo synthesis by H-phosphonate or phosphotriester approaches. Accordingly, those of ordinary skill in the art of oligo synthesis will be readily familiar with the types of protecting groups suitable for protecting nucleobases, as well as the N or O atoms of those nucleobases to which said protecting groups are typically attached.
  • the building block comprises a nucleobase protected by a nucleobase protecting group (NPG).
  • NPG nucleobase protecting group
  • the nucleobase is adenine, guanine or cytosine. It has been determined that A, G and C nucleobases protected with NPG are less likely to generate unwanted side products upon deprotection than NPG-protected uracil and NPG-protected thymine. In some instances, all A-, G- and C-containing building blocks comprises NPG and/or there are no instances of NPG-protected uracil or NPG-protected thymine.
  • n may vary depending on the nature of NPG. In some instances, n is suitably 1-7, more suitably 1-5, and even more suitably 2-3. In other instances, n may be 1 or 2.
  • Each NPG used in the process may independently have a structure according to formula IIa, IIb or IIc: wherein represents the point of attachment to the N or O atom (more typically the N atom) of the nucleobase, L 1 is selected from absent, –O–, –CH 2 –, –OCH 2 – and –CH 2 O–, L 2 is selected from absent and phenylene, and L 3 is selected from absent and –OCH 2 –, with the proviso that at least one of L 1 , L 2 and L 3 is not absent; wherein represents the point of attachment to the O atom of the nucleobase, and m is 0, 1 or 2; (IIIc) wherein represents the point of attachment to the N atom of the nucleobase, R A is selected from (1-6C)alkyl and Q, R B is selected from (1-6C)alkyl and –(CH2)p–Q, in which p is 1 or 2, and R C is (1-6C)alkyl, with the provis
  • Each NPG used in the process may independently have a structure according to any one of the following: , , , and , wherein represents the point of attachment to the N or O atom (as appropriate) of the nucleobase, each k is independently 1-4 (e.g., 1-2), and Q 1 and Q 2 are as defined hereinbefore for Q.
  • n is suitably 1-8. More suitably, n is 2-6. Even more suitably, n is 2-3.
  • Q 2 n is suitably 1-8. More suitably, n is 1-6. Even more suitably, n is 1-3. Most suitably, n is 1.
  • the building block comprises a nucleobase protected by a NPG of type IIa-4.
  • Q 2 may have any of the aforementioned definitions for Q or Q 2 .
  • the building block comprises a nucleobase protected by a NPG having the following structure:
  • Q 2 may have any of the aforementioned definitions for Q or Q 2 .
  • n is 1-8. More suitably, n is 1-8. Even more suitably, n is 1-3. Most suitably, n is 1.
  • the building block comprises a nucleobase protected by NPG.
  • the building block comprises a nucleobase protected by NPG.
  • NPG nucleobase protected by NPG.
  • the building block comprises a nucleobase protected by NPG.
  • the building block comprises a nucleobase protected by NPG.
  • the building block comprises a nucleobase protected by NPG. Still even more suitably, in at least 90% of the chain extension cycles (to the nearest whole number), the building block comprises a nucleobase protected by NPG. [0037] In some instances, in each one of the chain extension cycles, the building block comprises a nucleobase protected by NPG. [0038] In at least one of the chain extension cycles in which the building block comprises NPG, membrane filtration is performed to isolate the growing oligonucleotide (e.g., from uncoupled building blocks and/or reaction debris). The person of ordinary skill in the art will be familiar with reaction debris typically generated as part of an LPOS process.
  • Such debris will be taken to include by-products, cleaved temporary protecting groups (TPG) and excess reagents.
  • TPG temporary protecting groups
  • membrane filtration is performed to isolate the growing oligonucleotide in at least 20% of the chain extension cycles (to the nearest whole number) in which the building block comprises NPG.
  • membrane filtration is performed to isolate the growing oligonucleotide in 2 of those chain extension cycles.
  • membrane filtration is performed to isolate the growing oligonucleotide in at least 50% of the chain extension cycles (to the nearest whole number).
  • membrane filtration is performed to isolate the growing oligonucleotide. More suitably, in each one of the chain extension cycles in which the building block comprises NPG, membrane filtration is performed to isolate the growing oligonucleotide. [0039] It will be understood that membrane filtration may be used to isolate the growing oligonucleotide in a given chain extension cycle even when the building block used in that chain extension cycle does comprise NPG. Suitably, membrane filtration is used to isolate the growing oligonucleotide in at least 75% (to the nearest whole number) of all chain extension cycles (i.e., irrespective of the nature of the building block).
  • membrane filtration is used to isolate the growing oligonucleotide in at least 90% (to the nearest whole number) of all chain extension cycles (i.e., irrespective of the nature of the building block). Most suitably, membrane filtration is used to isolate the growing oligonucleotide in all chain extension cycles (i.e., irrespective of the nature of the building block). [0040] Where membrane filtration is used as part of a chain extension cycle, it is suitably used in two discrete steps.
  • a first membrane filtration step separates the NPG-containing oligonucleotide from excess, unreacted building block, while a second membrane filtration step separates cleaved temporary protecting group (TPG) from the NPG-containing oligonucleotide.
  • TPG temporary protecting group
  • the building blocks may each have a structure according to formula III: wherein RT is a reactive terminal for coupling the building block to the chain extension site of the growing oligonucleotide, TPG is a temporary protecting group that is cleavable, after coupling of the building block to a chain extension site of the growing oligonucleotide, to expose a new chain extension site of the growing oligonucleotide, L p is an internucleoside linkage, and v is 0-10 (e.g., 0-3).
  • RT is a reactive terminal for coupling the building block to the chain extension site of the growing oligonucleotide
  • TPG is a temporary protecting group that is cleavable, after coupling of the building block to a chain extension site of the growing oligonucleotide, to expose a new chain extension site of the growing oligonucleotide
  • L p is an internucleoside linkage
  • v is 0-10 (e
  • linkages comprise phosphorus in the P(III) or P(V) state.
  • exemplary phosphorus-containing internucleosidic linkages include a phosphodiester linkage, a methyl phosphonate linkage, a phosphorothioate linkage, a boranophosphate linkage, a phosphoramidate, or a mesyl phosphoramidate linkage.
  • linkages may comprise protecting groups to protect reactive moieties, where such protecting groups are only removed once the target length of the oligo has been reached (e.g., during global deprotection, as described herein).
  • v is 0, 1 or 2. Most often, v is 0.
  • RT may be disposed at the 5’ carbon of the nucleosidic moiety to which it is attached, and TPG disposed at the 3’ carbon of the nucleosidic moiety to which it is attached.
  • RT may be disposed at the 3’ carbon of the nucleosidic moiety to which it is attached, and TPG disposed at the 5’ carbon of the nucleosidic moiety to which it is attached.
  • 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).
  • the building blocks of formula III may each have a structure according to formula IIIa: wherein O A is the oxygen atom located at the 5’ carbon of the nucleosidic moiety to which it is attached, and O B is the oxygen atom located at the 3’ carbon of the nucleosidic moiety to which it is attached, or O A is the oxygen atom located at the 3’ carbon of the nucleosidic moiety to which it is attached, and O B is the oxygen atom located at the 5’ carbon of the nucleosidic moiety to which it is attached.
  • RT may be a phosphoramidite, a phosphate monoester, a phosphate diester, an H- phosphonate, a cyclic thiophosphate or a cyclic dithiophosphate triester moiety.
  • RT may have a structure according to formula IV: (IV) wherein LG is a leaving group; PG is a protecting group; and represents the point of attachment to the nucleosidic moiety (Nuc) to which RT is attached (e.g., via O A in formula IIIa).
  • LG may be any suitable leaving group.
  • LG is a tertiary amino group, i.e., –NR 2 .
  • each R is independently selected from (1-6C)alkyl, or both R groups are linked such that when taken in combination with the nitrogen atom to which they are attached, they collectively form a 5- to 7-membered heterocycle. More suitably, each R is independently selected from (1-4C)alkyl, or both R groups are linked such that when taken in combination with the nitrogen atom to which they are attached, they collectively form a 5-membered heterocycle.
  • 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..
  • 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.
  • PG is selected from the group consisting of cyanoethyl, 2-chlorophenyl, 4-chlorophenyl, 2,2,2-trichloroethyl and methyl. Most suitably, PG is cyanoethyl.
  • RT has a structure according to the following: where represents the point of attachment to the nucleosidic moiety (Nuc) to which RT is attached (e.g., via O A in formula IIIa).
  • TPG will be understood to be a protecting group used to prevent uncontrolled chain extension during a chain extension cycle. Those of ordinary skill in the art will be readily familiar with protecting groups suitable for this purpose, as well as the conditions under which they can be cleaved.
  • TPG is an acid-labile protecting group, non-limiting examples of which include dimethoxytrityl (Dmtr/DMT), tert-butyl (tBu), tert-butyl oxycarbonyl (Boc), mono- methoxytriphenyl (Mmtr), triphenyl methyl (Tr), pentamethyl dihydrobenzofuran sufonyl (Pbf), Tetrahydropyranyl (Thp), tetrahydrofuranyl (Thf), para-methoxybenzyl (Pmb) and 2,4- dimethoxybenzyl.
  • Dmtr/DMT dimethoxytrityl
  • tBu tert-butyl
  • Boc tert-butyl oxycarbonyl
  • Mmtr mono- methoxytriphenyl
  • Tr triphenyl methyl
  • Pbf pentamethyl dihydrobenzofuran sufonyl
  • Thp Te
  • TPG is dimethoxytrityl bound to the 3’ or 5’ oxygen (as appropriate) of the nucleosidic moiety to which it is attached.
  • the building blocks of formula III may each have a structure according to formula IIIb: wherein O A is the oxygen atom located at the 5’ carbon of the nucleosidic moiety to which it is attached, and O B is the oxygen atom located at the 3’ carbon of the nucleosidic moiety to which it is attached, or O A is the oxygen atom located at the 3’ carbon of the nucleosidic moiety to which it is attached, and O B is the oxygen atom located at the 5’ carbon of the nucleosidic moiety to which it is attached.
  • TPG is suitably dimethoxytrityl.
  • each nucleosidic moiety (Nuc) has a structure according to formula V: (V) wherein Z is a nucleobase, optionally protected by a protecting group, wherein said protecting group may be NPG; R x is selected from H, OH, F, O-tert-butyldimethylsilyl (OTbdms), methoxy, O-methoxyethyl (OMoe), O-propargyl, NH 2 and N 3 , in which case both are absent, or R x is O and both are present; 1 and 2 independently represent points of attachment to RT, TPG, O A , O B or LP (as appropriate).
  • R x is most suitably H or OH.
  • the process of the invention comprises growing an oligonucleotide by performing one or more chain extension cycles.
  • the one or more chain extension cycles is five or more chain extension cycles. More suitably, the one or more chain extension cycles is eight or more chain extension cycles. Even more suitably, the one or more chain extension cycles is ten or more chain extension cycles. Yet even more suitably, the one or more chain extension cycles is twelve or more chain extension cycles. Most suitably, the one or more chain extension cycles is fifteen or more chain extension cycles.
  • Each chain extension cycle suitably comprises a step of cleaving the temporary protecting group (TPG, e.g., dimethoxytrityl) after coupling of the building block to a chain extension site of the growing oligonucleotide to expose a new chain extension site of the growing oligonucleotide.
  • TPG temporary protecting group
  • the step of coupling the monomeric or oligomeric building block to the chain extension site of the growing oligonucleotide suitably form a P(III) phosphite triester linkage between the coupled building block and growing oligonucleotide.
  • Such coupling reactions can be initiated with common activators, such as ethylthiotetrazole (ETT) or dicyanoimidazole (DCI).
  • Each chain extension cycle suitably comprises a step of converting one of more P(III) phosphite triester linkages to a P(V) phosphotriester linkage (e.g., by oxidation or sulfur transfer) after coupling of the building block to a chain extension site of the growing oligonucleotide.
  • 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).
  • the oxidation product i.e., the compound comprising one or more P(V) phosphotriester linkages
  • the oxidation product may be isolated by membrane filtration.
  • the step of converting one of more P(III) phosphite triester linkages to a P(V) phosphotriester linkage is conducted before a step of cleaving the temporary protecting group (TPG, e.g., dimethoxytrityl) to expose a new chain extension site of the growing oligonucleotide.
  • TPG temporary protecting group
  • membrane filtration is used as part of a chain extension cycle, it is suitably used in two discrete steps.
  • a first membrane filtration step may be conducted after coupling of the building block to a chain extension site of the growing oligonucleotide and before a step of cleaving the temporary protecting group (TPG, e.g., dimethoxytrityl, Dmtr) to expose a new chain extension site of the growing oligonucleotide.
  • a second membrane filtration step may be conducted after a step of cleaving the temporary protecting group (TPG, e.g., dimethoxytrityl) to expose a new chain extension site of the growing oligonucleotide.
  • the process may be performed in any suitable solvent.
  • 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
  • all steps of a given chain extension cycle e.g., coupling, oxidation, deprotection and membrane filtration
  • all chain extension cycles and their associated steps e.g., coupling, oxidation, deprotection and membrane filtration
  • are performed in the same solvent e.g., acetonitrile.
  • the membrane filtration steps forming part of the process are suitably conducted by membrane diafiltration.
  • membrane diafiltration the crude mixture comprising the growing oligonucleotide is pressurised against a size-selective membrane. More suitably, the membrane filtration steps are conducted by organic solvent nanofiltration (OSN) or ultrafiltration (UF).
  • OSN organic solvent nanofiltration
  • UF ultrafiltration
  • 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).
  • Suitable membranes for use in isolating the growing oligonucleotide include polymeric membranes, ceramic membranes, and mixed polymeric/inorganic membranes.
  • 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.
  • 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.
  • 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 TiO 2 or ZrO 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.
  • the membrane is a crosslinked polybenzimidazole membrane (e.g., an integrally skinned, asymmetric, crosslinked polybenzimidazole membrane) or a polyetheretherketone membrane.
  • the process may further comprises one or more global deprotection steps. Such step(s) may be conducted once a target length of the oligonucleotide has been reached.
  • the global deprotection step(s) may result in removal of all instances of PG (e.g., cyanoethyl) from internucleoside linkages present throughout the oligonucleotide, and/or may result in removal of all protecting groups, including NPG, present on nucleobases throughout the oligonucleotide.
  • PG e.g., cyanoethyl
  • NPG all protecting groups
  • 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.
  • 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.
  • 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). [0070] In some instances, the soluble synthesis support is a star polymer comprising a central branch point and m number of radiating polymeric arms, wherein polymeric arm is attached to a growing oligonucleotide, and wherein m is 3 or more. In such instances, it will be understood that the process can be used to grow m number of identical oligonucleotide chains.
  • 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).
  • the soluble synthesis support may have a structure according to formula VI in which V is an organic branch point, W is a polymeric chain, represents a point of attachment to the growing oligonucleotide, and m is 1-8.
  • V will be understood to refer to a polyfunctional organic “hub” having a plurality of terminals to which polymeric chain(s), W, are attached.
  • V comprises fewer than 50 atoms in total, more suitably fewer than 20 atoms in total.
  • Each W is a polymeric chain (e.g., polyethylene glycol) having a molecular weight (M w ) of 500 – 20,000 Da. More suitably, each W is a polymeric chain having a molecular weight (M w ) of 2000 – 15,000 Da. Most suitably, each W is a polymeric chain having a molecular weight (M w ) of 8000 – 12,000 Da.
  • m may, for example, be 1-6. In many instances, m is 3-4.
  • 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.
  • a particularly suitable example of a soluble synthesis support is: in which represents the point of direct attachment to the growing oligonucleotide (e.g., at the 3’ oxygen) and q is 100 – 500 (e.g., 200 – 300).
  • Each oligonucleotide, once prepared (i.e., grown to full length product), may have a molecular weight of ⁇ 1000 Da.
  • 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.
  • 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.
  • Modifications may occur at the 2' position of the sugar moiety.
  • Sugar modifications include a modified version of the ribosyl moiety, such as 2'-O-modified RNA such as 2'-O-alkyl or 2'-O(substituted)alkyl e.g., 2'-O-methyl, 2'-O-(2-cyanoethyl), 2'-O-(2-methoxy)ethyl (2'-MOE), 2'-O-(2-thiomethyl)ethyl, 2'-O-butyryl, 2'-O-propargyl, 2'-O-allyl, 2'-O-(3-amino)propyl, 2'-O-(3- (dimethylamino)propyl), 2'-O-(2-amino)ethyl, 2'-O-(2-(dimethylamino)ethyl); 2'-deoxy (DNA); 2'- O(haloalkoxy)methyl (Arai et al.) e.g., 2'
  • BNA bridged or "bicylic” nucleic acid
  • LNA locked nucleic acid
  • xylo-LNA xylo-LNA
  • ⁇ -L-LNA ⁇ -D-LNA
  • cEt 2'-O,4'-C constrained ethyl
  • cMOEt 2'-O,4'-C constrained methoxyethyl
  • ENA ethylene-bridged nucleic acid
  • UPA unlocked nucleic acid
  • CeNA cyclohexenyl nucleic acid
  • ANA altritol nucleic acid
  • HNA hexitol nucleic acid
  • F-HNA fluorinated HNA
  • p-RNA pyranosyl-RNA
  • p-DNA 3'-deoxypyranosyl-DNA
  • morpholino as e.g.
  • 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.
  • nucleobase modifications may be selected from the group consisting of 5-methyl pyrimidines, 7-deazaguanosines and abasic nucleotides.
  • the modification may be a 5-methyl cytosine.
  • 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.
  • PS phosphorothioate
  • PS2 phosphorodithioate
  • PACE phosphonoacetate
  • PACE
  • 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.
  • PNA peptide-base nucleic acid
  • POPNA pyrrolidine-based oxy-peptide nucleic acid
  • GAA glycol- or glycerol-based nucleic acid
  • TAA threose-based nucleic acid
  • aTNA acyclic threoninol-based nucleic acid
  • OOMs pyrrolidine-amide oligonucleotides
  • BNA bridged nucleic acid
  • BNA bridged nucleic acid
  • an oligonucleotide obtained, directly obtained or obtainable by the process of the first aspect.
  • the following numbered statements 1 to 79 are not claims, but instead describe particular aspects and embodiments of the invention: 1.
  • a liquid-phase process for preparing an oligonucleotide comprising growing an oligonucleotide by performing one or more chain extension cycles, in which each chain extension cycle comprises a step of coupling a monomeric or oligomeric building block to a chain extension site of a growing oligonucleotide, wherein in at least one chain extension cycle, the building block comprises a nucleobase protected by a nucleobase protecting group (NPG), wherein NPG comprises a group Q of formula I: wherein n is 1-9 and R 1 is H or methyl, and wherein in at least one of the chain extension cycles in which the building block comprises NPG, one or more membrane filtration steps is used to isolate the growing oligonucleotide (e.g., from uncoupled building blocks and/or reaction debris).
  • NPG nucleobase protecting group
  • each monomeric or oligomeric building block comprises: (i) at least one nucleosidic moiety (Nuc), (ii) a reactive terminal (RT) for coupling the building block to the chain extension site of the growing oligonucleotide, and (iii) a temporary protecting group (TPG), said temporary protecting group being cleavable, after coupling of the building block to a chain extension site of a growing oligonucleotide, to expose a new chain extension site of the growing oligonucleotide.
  • Nuc nucleosidic moiety
  • RT reactive terminal
  • TPG temporary protecting group
  • each monomeric or oligomeric building block comprises: one nucleosidic moiety (i.e., the building block is monomeric), or two or more nucleosidic moieties, each pair of adjacent nucleosidic moieties being linked by an internucleoside linkage (e.g., the building block is oligomeric). 4.
  • each monomeric or oligomeric building block comprises 1-3 nucleosidic moieties. 5.
  • each monomeric or oligomeric building block comprises 1 nucleosidic moiety. 6.
  • nucleobase is adenine, guanine or cytosine. 7.
  • n is 1-7.
  • n is 1-5.
  • n is 2-3.
  • 10. The process as defined in any one of statements 1-9, wherein n is 1 or 2 11.
  • each NPG independently has a structure according to formula IIa, IIb or IIc: wherein represents the point of attachment to the N or O atom (more typically the N atom) of the nucleobase, L 1 is selected from absent, –O–, –CH2–, –OCH2– and –CH2O–, L 2 is selected from absent and phenylene, and L 3 is selected from absent and –OCH2–, with the proviso that at least one of L 1 , L 2 and L 3 is not absent; (IIb) wherein represents the point of attachment to the O atom of the nucleobase, and m is 0, 1 or 2; wherein represents the point of attachment to the N atom of the nucleobase, R A is selected from (1-6C)alkyl and Q, R B is selected from (1-6C)alkyl and –(CH2)p–Q, in which p is 1 or 2, and R C is (1-6C)alkyl,
  • each NPG independently has a structure according to any one of the following: , , , and , wherein represents the point of attachment to the N or O atom (as appropriate) of the nucleobase, each k is independently 1-4 (e.g., 1-2), and Q 1 and Q 2 are as defined hereinbefore for Q. 13.
  • Q 2 is 1-8.
  • the building block comprises a nucleobase protected by NPG. 23.
  • the building block comprises a nucleobase protected by NPG.
  • each building blocks has a structure according to formula III: wherein RT is a reactive terminal for coupling the building block to the chain extension site of the growing oligonucleotide, TPG is a temporary protecting group that is cleavable, after coupling of the building block to a chain extension site of the growing oligonucleotide, to expose a new chain extension site of the growing oligonucleotide, L p is an internucleoside linkage, and v is 0-10 (e.g., 0-3). 36. The process as defined in statement 35, wherein v is 0, 1 or 2 (e.g., 0). 37.
  • each building block has a structure according to formula IIIa: wherein O A is the oxygen atom located at the 5’ carbon of the nucleosidic moiety to which it is attached, and O B is the oxygen atom located at the 3’ carbon of the nucleosidic moiety to which it is attached, or O A is the oxygen atom located at the 3’ carbon of the nucleosidic moiety to which it is attached, and O B is the oxygen atom located at the 5’ carbon of the nucleosidic moiety to which it is attached. 38.
  • each RT is independently a phosphoramidite, a phosphate monoester, a phosphate diester, an H- phosphonate, a cyclic thiophosphate or a cyclic dithiophosphate triester moiety.
  • each RT independently has a structure according to formula IV: (IV) wherein LG is a leaving group; PG is a protecting group; and represents the point of attachment to the nucleosidic moiety (Nuc) to which RT is attached (e.g., via O A in formula IIIa).
  • LG is a tertiary amino group, i.e., - NR2.
  • 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.
  • each R is independently selected from (1-4C)alkyl, or both R groups are linked such that when taken in combination with the nitrogen atom to which they are attached, they collectively form a 5-membered heterocycle.
  • 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.
  • each R is isopropyl.
  • PG is a base-labile protecting group.
  • PG is selected from the group consisting of cyanoethyl, 2-chlorophenyl, 4-chlorophenyl, 2,2,2-trichloroethyl and methyl. 47.
  • RT has a structure according to the following: where represents the point of attachment to the nucleosidic moiety (Nuc) to which RT is attached (e.g., via O A in formula IIIa). 48.
  • each TPG is independently selected from the group conssisting of dimethoxytrityl (Dmtr/DMT), tert-butyl (tBu), tert-butyl oxycarbonyl (Boc), mono-methoxytriphenyl (Mmtr), triphenyl methyl (Tr), pentamethyl dihydrobenzofuran sufonyl (Pbf), Tetrahydropyranyl (Thp), tetrahydrofuranyl (Thf), para-methoxybenzyl (Pmb) and 2,4-dimethoxybenzyl. 49.
  • each building block has a structure according to formula IIIb: wherein O A is the oxygen atom located at the 5’ carbon of the nucleosidic moiety to which it is attached, and O B is the oxygen atom located at the 3’ carbon of the nucleosidic moiety to which it is attached, or O A is the oxygen atom located at the 3’ carbon of the nucleosidic moiety to which it is attached, and O B is the oxygen atom located at the 5’ carbon of the nucleosidic moiety to which it is attached. 51.
  • each nucleosidic moiety has a structure according to formula V: wherein Z is a nucleobase, optionally protected by a protecting group, wherein said protecting group may be NPG; R x is selected from H, OH, F, O-tert-butyldimethylsilyl , methoxy, O-methoxyethyl (OMoe), O- and N3, in which case both are absent, or R x is O and both are present; 1 and 2 independently represent points of attachment to RT, TPG, O A , O B or LP (as appropriate). 52.
  • each chain extension cycle comprises a step of cleaving the temporary protecting group (TPG, e.g., dimethoxytrityl) after coupling of the building block to a chain extension site of the growing oligonucleotide to expose a new chain extension site of the growing oligonucleotide.
  • TPG temporary protecting group
  • the step of coupling the monomeric or oligomeric building block to the chain extension site of the growing oligonucleotide form a P(III) phosphite triester linkage between the coupled building block and growing oligonucleotide.
  • the growing oligonucleotide is attached (e.g., at nucleosidic moiety most distal to the chain extension site) to a soluble synthesis support.
  • a 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.
  • 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.
  • each polymeric arm is poly(ethylene glycol).
  • the soluble synthesis support may have a structure according to formula VI in which V is an organic branch point, W is a polymeric chain (e.g., poly(ethylene glycol)), represents a point of attachment to the growing oligonucleotide, and m is 1-8. 75.
  • each W is a polymeric chain (e.g., poly(ethylene glycol)) having a molecular weight (M w ) of 500 – 20,000 Da. 77.
  • M w molecular weight
  • Reagents and conditions i, TsCl, NMI, TEA, DCM, 3 hr; ii, 4-HO-C 6 H 4 - CHO, K 2 CO 3 , DMF, 110 °C, 16 hr; iii, NaOMe, MeOH, DMF 90 min, then water 16 hr; iv, KMnO 4 , Na 2 HPO 4 , water; v, C 2 O 2 Cl 2 , DCM, 30 min; vi, Dmtr-mU, TmsCl, DIPEA, TEA, 90 min, then 13, 40 °C, 16 hr, then water 40 °C, 16 hr; vii, CneOP(NiPr 2 ) 2 , Pyr.TFA, MeCN, 3 hr.
  • Fig.3. UV Chromatogram globally deprotected 3-mer, mU 3 , prepared using mU(mEg 4 An).
  • Fig. 4. UV Chromatogram globally deprotected 6-mer, mU 6 , prepared using all four mU phosphoramidites.
  • Fig.7 1 H NMR of Dmtr-mU(mEg 4 An) cyanoethyl phosphoramidite, 8c, in CDCl 3 .
  • Oxalyl chloride (2 mL) was added and the reaction was stirred for 30 min at room temperature; the reaction was monitored by dissolving samples in MeOH to detect the corresponding methyl ester in HPLC. Once the reaction was complete, the solvent and remaining oxalyl chloride were evaporated under high vacuum, and the crude acyl chloride 6 was used without further purification.
  • the reaction mixture was stirred at room temperature for 90 min, then transferred by syringe into a flask containing the crude mEg4AnCl, prepared as above.
  • the reaction was stirred overnight at 40 °C when the intermediate Dmtr-mU-OTms was seen to be consumed by HPLC analysis.
  • the reaction mixture was then allowed to cool to room temperature and cold water (1 mL) was added dropwise.
  • the reaction mixture was once more stirred overnight at 40 °C to ensure complete deprotection of the 3-'OH.
  • the solvents were evaporated and the residual gummy residue was partitioned between DCM and brine before to be transferred to a separated funnel.
  • the crude mixture was extracted 3 times with DCM, until no product remained in the aqueous phase.
  • the building block was co-evaporated from anhydrous MeCN ( ⁇ 30 ppm water, 3 x 5 mL). Building block was re-dissolved in anhydrous MeCN (2 mL) and injected into the synthesizer, followed by the DCI dissolved in anhydrous MeCN (2 mL); all components were injected into synthesizer via a 0.2 micron PTFE filter. The reaction was monitored by LC. After 2 minutes the coupling intermediates were consumed, but circulation was continued in the synthesizer for 20 minutes to ensure complete reaction.
  • DCI dicyanoimidazole
  • Membrane was PBI17-DBX- Jeffamine M2005; all rejections measured at 5 bar, R is defined according to equation 1.
  • Table 3. Diafiltration data for oligo-stars and building block (BB) debris during the homo-mU 6 synthesis. Oligo Rejection, R Average Monomer length HO-oligo- BB debris BB debris permeance star end of DF1 end of DF2 mL/min mU(mEg4An), 8c 2-mer 99.97% 38% 7% 1.3 mU(mEg 4 An), 8c 3-mer 99.93% 27% 22% 1.1 mU, 14 4-mer 100% 18% n.d. 1.2 mU(Bz), 15 5-mer 99.84 % 27 % n.d.
  • oligo-stars were periodically analysed by global deprotection of a sample of the circulating fluid from the synthesiser. The purities of the crude oligos were estimated by analytical ion-pair reversed phase UHPLC chromatography. A sample of crude HO-oligo-star solution (3 mL, ca. 0.048 mmol oligo) was withdrawn from the synthesizer. The sample was placed in an ACE pressure tube and dissolved in a mixture of concentrated aq. ammonia (3 mL) and diethylamine (3 vol%).
  • reaction mixture was cooled to 0 °C and chlorotrimethylsilane (TmsCl, 3.4 mL, 26.8 mmol) was added dropwise.
  • the reaction mixture was stirred at room temperature for 90 min, then transferred by syringe into a flask containing the crude mEg 4 AnCl (6, 7.1 g, 20.6 mmol).
  • the reaction was stirred overnight at 40 °C; the intermediate Dmtr-mA-Tms was seen to be consumed by HPLC analysis.
  • the reaction mixture was then allowed to cool to room temperature and cold water (10 mL) was added dropwise.
  • the reaction mixture was stirred for a further 1h at room temperature and the solvent was then evaporated.
  • Dmtr-mA(mEg4An)-PO(OCne)2 can be completely deprotected to 5’-Dmtr-mA-3’-phosphate with conc. ammonia-MeCN 1:1 at 35 °C for 16 hr.
  • Example 4 Solubilising a pentamer-star with a single mEg 4 An group [00108] Loading of 5’-Dmtr-mU-3’-succinate onto 20 kDa PEG-star: Two separate runs were started.
  • PEG-20k(NMeH) 8 support (2.49 g, MW 20.6 kDa, 0.12 mmol) was dissolved in anhydrous MeCN (10 mL, 26.8 ppm) to which was added DIPEA (0.29 mL, 1.5 mmol, 14 eq.) and the mixture was stirred at room temperature for 30 minutes.5’-Dmtr-mU-3’-succinate, TEA salt (1.11 g, 1.5 mmol, 12 eq.) was dissolved in anhydrous MeCN (5 mL) to which was added TBTU (0.47 g, 1.5 mmol, 12 eq.) and the solution was stirred for 30 minutes at room temperature.

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Abstract

La présente invention concerne un procédé en phase liquide pour la préparation d'oligonucléotides. Plus particulièrement, la présente invention concerne un procédé en phase liquide assisté par filtration sur membrane pour la préparation d'oligonucléotides, dans lequel un ou plusieurs blocs de construction utilisés pour faire croître un oligonucléotide comprennent une nucléobase protégée par un groupe de protection de nucléobase (NPG), le NPG comprenant un groupe Q. Les inventeurs ont déterminé que l'utilisation de blocs de construction dans lesquels la nucléobase comprend un groupe de protection de nucléobase (NPG) réduit notablement l'occurrence de survenue de séparation et/ou d'encrassement de membrane inefficace dans le cadre d'une technique de LPOS assistée par membrane.
PCT/GB2024/051523 2023-12-20 2024-06-14 Procédé en phase liquide pour la préparation d'oligonucléotides Pending WO2025133559A1 (fr)

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