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WO2025106093A1 - Procédés de purification d'oligonucléotides produisant des oligonucléotides purifiés ayant des groupes phosphate à extrémité 5' terminale - Google Patents

Procédés de purification d'oligonucléotides produisant des oligonucléotides purifiés ayant des groupes phosphate à extrémité 5' terminale Download PDF

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WO2025106093A1
WO2025106093A1 PCT/US2023/081448 US2023081448W WO2025106093A1 WO 2025106093 A1 WO2025106093 A1 WO 2025106093A1 US 2023081448 W US2023081448 W US 2023081448W WO 2025106093 A1 WO2025106093 A1 WO 2025106093A1
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oligonucleotide
affinity
tag
support
phosphate
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Joel Myerson
Daniel Ryan
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Agilent Technologies Inc
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Agilent Technologies Inc
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    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C07H1/06—Separation; Purification
    • 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
    • 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
    • C07H21/04—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 deoxyribosyl as saccharide radical

Definitions

  • the present disclosure provides purification methods for oligonucleotides yielding oligonucleotides with 5 '-terminal phosphate groups. Also disclosed are general methods of purification of oligonucleotides that is well suited for synthetic RNA, particularly RNA made using the “TC-RNA” chemistry.
  • Standard methods for purification of synthetic oligonucleotides include ion exchange and reverse phase chromatography, polyacrylamide gel purification, and what is commonly known as “DMT-on” or “trityl-on” purification.
  • chromatographic and gel-based methods become less efficient for longer oligonucleotides because it becomes more difficult to resolve the shorter failure sequences from the desired full-length sequence as the length increases.
  • Trityl-on purification enhances the chromatographic retention of the full-length material because the extremely hydrophobic dimethoxytriphenylmethyl group remains on the 5'- hydroxyl of the synthetic oligonucleotide.
  • Trityl-on purifications while simple to implement, must be followed by removal of the DMT group under acidic conditions in order to isolate the final 5'- hydroxyl product. While DNA is reasonably stable to short exposure to acidic conditions, RNA can undergo strand scission and isomerization. The isomerization, in which the normal 3'-5' linkage is changed to a 2'-5' linkage is particularly difficult to detect but will result in RNA that does not have the correct backbone configuration. Improved methods of purification of oligonucleotides that is well suited for synthetic RNA, particularly RNA made using the “TC-RNA” chemistry (Dellinger et al., 201 1) would be desirable.
  • oligonucleotide may comprise one or more nucleobase exocyclic amino-protecting groups, one or more phosphate triester protecting groups, or one or more 2'-hydroxyl protecting groups; reacting the free 5 '-hydroxyl group of the support-bound oligonucleotide with a phosphoramidite comprising a substituent containing a cleavable linker and a protected hydroxyl group, wherein reacting forms an extended 5 '-phosphoester support-bound oligonucleotide; deprotecting the protected hydroxyl group of the 5 '
  • the oligonucleotide under suitable conditions, and eluting the formed 5'-phosphate oligonucleotide off the column or affinity capture support, thereby obtaining the purified 5 '-phosphate oligonucleotide; or b) eluting the washed captured affinity -tagged 5 '-phosphoester oligonucleotide from the chromatographic column or the affinity capture support and then cleaving the cleavable linker off the eluted washed affinity-tagged 5 '-phosphoester oligonucleotide to obtain the purified 5 '-phosphate oligonucleotide, wherein the purified 5'- phosphate oligonucleotide may comprise one or more nucleobase exocyclic amino-protecting groups, one or more phosphate triester protecting groups, one or more 2'-hydroxyl protecting groups or a combination thereof.
  • the oligonucleotide may comprise one or more nucleobase ex
  • the oligonucleotide of the support-bound oligonucleotide does not comprise any nucleobase exocyclic amino-protecting groups, any phosphate triester protecting groups, or any 2'-hydroxyl protecting groups.
  • the oligonucleotide of the support-bound oligonucleotide comprises at least one nucleobase exocyclic amino-protecting group, phosphate triester protecting group, or 2'-hydroxyl protecting group.
  • the oligonucleotide of the support-bound oligonucleotide may comprise one or more 2'-hydroxyl protecting groups.
  • all the 2'-hydroxyl groups of the oligonucleotide of the support-bound oligonucleotide are protected with a 2'-hydroxyl protecting group.
  • the 2'-hydroxyl protecting group is a thionocarbamate (TC), bis(2-acetoxyethoxy)methyl (ACE) protecting group, /-butyldimethylsilyl (TBDMS), triisopropylsilyloxymethyl (TOM), pivaloyloxymethyl (PivOM), or 2- cyanoethoxymethyl (OEM).
  • TC thionocarbamate
  • ACE bis(2-acetoxyethoxy)methyl
  • TDMS /-butyldimethylsilyl
  • TOM triisopropylsilyloxymethyl
  • PivOM pivaloyloxymethyl
  • OFEM 2- cyanoethoxymethyl
  • the oligonucleotide of the support-bound oligonucleotide comprises one or more phosphate triester protecting groups. In some of these embodiments, all the phosphate esters of the oligonucleotide of the supportbound oligonucleotide are protected with a phosphate triester protecting group.
  • the phosphate triester protecting group is cyanoethyl.
  • the support-bound oligonucleotide is prepared via solid-phase oligonucleotide synthesis. In some embodiments, the support-bound oligonucleotide is prepared using a phosphoramidite-based method. In some embodiments, the support-bound oligonucleotide is prepared using a synthesis method comprising a support-bound nucleoside having a 5'-DMT protecting group.
  • the support-bound oligonucleotide comprises an oligoribonucleotide (RNA).
  • RNA comprises at least 70 nucleotides.
  • the phosphoramidite is compound I, II, 6, 7, 7b, 8 or 9.
  • the phosphoramidite may be compound I.
  • the phosphoramidite is compound 8.
  • the phosphoramidite is compound 9.
  • the cleavable linker comprises a photocleavable moiety, a disulfide group, an abasic nucleotide or a moiety that is cleavable under enzymatic conditions.
  • the affinity tag comprises one or more of the following: a fluorous tag, a hydrophobic tag, a biotin tag, a glutathione tag, a maltose tag, an arylboronic acid tag, a poly-histidine peptide tag, a poly-sulfhydryl tag, a cyclodextrin tag, an adamantane tag, a polyamine tag, a maleimide tag, an alkyne tag, an azido tag, a hydrazide tag, an amino tag, a diol tag, a thiol tag, or any combination thereof.
  • the affinity tag is a fluorous or a hydrophobic tag with a c Log P value of at least 3, wherein the cLog P value is the calculated ratio of solubility of the affinity tag in octanol and water.
  • the affinity tag is a fluorosubstituted alkyl, a fluorosubstituted alkenyl, a fluorosubstituted alkynyl, or a fluorosubstituted carbocyclyl.
  • the reagent comprising the affinity tag is a phosphoramidite.
  • the reagent comprising the affinity tag is compound III, IV, V, 12-(4,4'-Dimethoxytrityloxy)dodecyl-l-[(2-cyanoethyl)- (N,N-diisopropyl)]-phosphoramidite, or [l-N-(4,4'-dimethoxytrityl)-biotinyl-6-amino- ethoxyethyl]-2-cyanoethyl-(N,N-diisopropyl)-phosphoramidite.
  • the reagent comprising the affinity tag is compound Ill, IV, or V.
  • the reagent comprising the affinity tag is compound III.
  • the reagent comprising the affinity tag is 12-(4,4'-Dimethoxytrityloxy)dodecyl-l-[(2-cyanoethyl)-(N,N- diisopropy 1) ] -phosphoramidite.
  • the deprotecting of a 2'-hydroxyl is performed after obtaining the purified 5'-phosphate oligonucleotide.
  • the cleavage of the 3'-hydroxyl or 3'-phosphate of the 5 '-phosphate affinity-tagged oligonucleotide from the synthesis solid support and the deprotection of the phosphate triester protecting groups and optionally the nucleobase exocyclic amino protecting groups is performed in a single reaction.
  • the methods further comprise the deprotection of the 2'-hydroxyl groups in the single reaction.
  • FIG. 1 shows the process of using an affinity tag or purification handle to purify and isolate a 5 '-phosphate oligonucleotide. It depicts the attachment of an affinity tag to the 5 '-terminal phosphate of an oligonucleotide through a cleavable linker. The phosphate and tag remain attached after the oligonucleotide is deprotected and cleaved off of the synthesis resin with deprotection and cleavage reagents. The oligonucleotide is then trapped on the affinity resin or other means, and capped oligos and side products are removed by washing.
  • Cleavage of the affinity tag through the cleavable linker leaves the phosphate on the oligonucleotide and the affinity tag on the resin, yielding upon elution the purified 5 '-phosphate oligonucleotide in solution.
  • elution of the affinity tagged 5'-phosphate oligonucleotide is performed prior cleavage of the cleavable linker comprising the affinity tag giving rise to the purified 5'-phosphate oligonucleotide.
  • affinity pair refers to a pair of molecules or substances that associate with one another through a strong and/or specific biologic or chemical affinity interaction such as that between antigen and antibody, enzyme and substrate, or receptor and ligand or hydrophobic compound and hydrophobic resin and the like. Any of these substances, covalently linked to an insoluble support or immobilized in a gel, may serve as the sorbent allowing the interacting substance to be isolated from relatively impure samples.
  • affinity capture The technique of using this kind of associative interaction of the affinity pairs along with the use of insoluble support or gel, to separate, isolate or purify a substance is named affinity capture.
  • This technique usually implies retaining the desired affinity-tagged biomolecule on the insoluble support or gel through the associative interaction with the other member of the affinity pair that is linked to the insoluble support, and subsequently eluting the impurities or undesired byproducts that are not tagged in a liquid phase.
  • the affinity capture may be performed by filtration of the insoluble material and separation from the filtrate followed by elution or cleavage of the desired purified biomolecule from the insoluble support.
  • affinity pairs include biotin/avidin, biotin/streptavidin, glutathione/GST, maltose/MBP (Maltose Binding Protein), diols/arylboronic acids, nickel or cobalt/histidines or thiols, cucurbiturils/adamantane, cyclodextrin/adamantane, fluoroalky 1/fluoroalkyl, and hydrophobic moieties/Cis resin.
  • affinity tag may refer to one member of an affinity pair or to a moiety that has an intrinsic property (such as hydrophobicity, hydrophilicity, polarity, charges, fluorophilicity, etc.) that allows isolation or separation of a target molecule (e.g., an oligonucleotide) linked to the affinity tag using the intrinsic property.
  • An affinity pair comprises an affinity tag and a recognition moiety that has a specific binding capability to the affinity tag.
  • the affinity tag may be present in the target molecule (e.g. , as a substituent) or attached to the target molecule via a linker (e.g. , an orthoester linker).
  • An affinity tag may also be covalently captured on a solid support.
  • an affinity tag examples include a fluorous affinity tag, a hydrophobic tag, a biotin tag, a cyclodextrin tag, an adamantane tag, a maltose, or a polyamine tag, charged tag etc.
  • An affinity tag may also be a chemical functional group, optionally protected, which reacts selectively with a specific chemical functional group on a molecule which imparts an intrinsic property (such as hydrophobicity, hydrophilicity, polarity, charges, fluorophilicity, etc.) that allows isolation or separation of the newly linked target molecule, or which reacts selectively with a specific chemical functional group attached to a solid phase or affinity capture medium.
  • functional chemical tags include maleimide tag, alkyne tag, azido tag, hydrazide tag, amino tag, a diol tag, and a thiol tag.
  • deprotected means fully or partially deprotected.
  • recognition moiety refers to the second member of an affinity pair, which interacts specifically with the affinity tag.
  • hydrophobic tag refers to a hydrophobic substituent or a combination of hydrophobic substituents that are carbon rich.
  • the hydrophobicity of a substituent can be determined, measured or calculated through the value of its partition coefficient (log P).
  • the partition coefficient (log P) of a substance defines the ratio of its solubility in two immiscible solvents, normally octanol: water. When this value is calculated rather than measured, it is called c Log P.
  • a hydrophobic tag has a c Log P of at least 3 or a combination of two, three or four “partial hydrophobic tags” has a collective value of c Log P of at least 3.
  • hydrophobic tags include C6-C24 alkyl, C4-C24 alkenyl, C4-C24 alkynyl, carbocycles and aryls, trityls, lipids, steroids, adamantane. Fluoro substituents or fluorosubstituted groups can be used to increase the hydrophobicity of a hydrophobic tag.
  • a hydrophobic tag comprises a least six carbon atoms.
  • An orthoester linker that comprises a hydrophobic tag includes an orthoester linker with one, two, three or four hydrophobic tags, each located on one of its four variable R groups.
  • a hydrophobic orthoester linker also includes an orthoester linker with “partial hydrophobic tags” (i.e. with less than six carbons each) but when combined together comprise at least six carbons.
  • fluorous tag refers to a perfluorinated or fluorinated substituent or to a combination of perfluorinated or fluorinated substituents for example a fluorosubstituted C1-C24 alkyl, a fluorosubstituted C2-C24 alkenyl, a fluorosubstituted C2-C24 alkynyl, a fluorosubstituted carbocyclyl and a fluorosubstituted aryl.
  • a fluorous tag comprises a least seven fluorine atoms and three carbon atoms.
  • a “fluorous tag” can act as a "hydrophobic tag” and can be isolated using a hydrophobic medium rather than a fluorous medium.
  • chromatographic method refers to a method of separating one or more compounds involving the use of a stationary phase and a mobile phase or eluent that moves through or across the stationary phase.
  • chromatographic methods include fluorous affinity purification and high performance liquid chromatography.
  • nucleotide or “nucleotide moiety” refers to a sub-unit of a nucleic acid (whether DNA or RNA or analogue thereof) which includes a phosphate group, a sugar group and a heterocyclic base, as well as analogs of such sub-units.
  • Other groups e.g. , protecting groups
  • nucleoside or “nucleoside moiety” references a nucleic acid subunit including a sugar group and a heterocyclic base, as well as analogs of such sub-units.
  • Other groups e.g., protecting groups
  • nucleoside residue refers to a molecule having a sugar group and a nitrogen containing base (as in a nucleoside) as a portion of a larger molecule, such as in a polynucleotide, oligonucleotide, or nucleoside phosphoramidite.
  • nucleotide monomer refers to a molecule which has not been incorporated in a larger oligo- or poly-nucleotide chain and which corresponds to a single nucleotide sub-unit; nucleotide monomers may also have activating or protecting groups, if such groups are necessary for the intended use of the nucleotide monomer.
  • nucleoside and nucleotide are intended to include those moieties that contain not only the known purine and pyrimidine bases, e.g., adenine (A), thymine (T), cytosine (C), guanine (G), or uracil (U), but also other heterocyclic bases that have been modified. Such modifications include methylated purines or pyrimidines, acylated purines or pyrimidines, alkylated riboses or other heterocycles.
  • Such modifications include, e.g., diaminopurine and its derivatives, inosine and its derivatives, alkylated purines or pyrimidines, acylated purines or pyrimidines, thiolated purines or pyrimidines, and the like, or the addition of a protecting group such as acetyl, difluoroacetyl, trifluoroacetyl, isobutyryl, benzoyl, 9-fluorenylmethoxycarbonyl, phen oxy acetyl, dimethylformamidine, dibutylformamidine, ?V,7V-diphenyl carbamate, or the like.
  • a protecting group such as acetyl, difluoroacetyl, trifluoroacetyl, isobutyryl, benzoyl, 9-fluorenylmethoxycarbonyl, phen oxy acetyl, dimethylformamidine, dibutylformam
  • the purine or pyrimidine base may also be an analog of the foregoing; suitable analogs will be known to those skilled in the art and are described in the pertinent texts and literature. Common analogs include, but are not limited to, 1- methyladenine, 2-methyladenine, Ari-methyladenine, N6-isopentyladenine, 2- melhyllhio-Ari-isopentyladenine, A' /V-dimelhyladenine.
  • nucleoside and nucleotide include those moieties that contain not only conventional ribose and deoxyribose sugars, but other sugars as well. Modified nucleosides or nucleotides also include modifications on the sugar moiety, e.g., wherein one or more of the hydroxyl groups are replaced with halogen atoms or aliphatic groups, or are functionalized as ethers, amines, or the like.
  • Analogues refer to molecules having structural features that are recognized in the literature as being mimetics, derivatives, having analogous structures, or other like terms, and include, for example, polynucleotides incorporating non-natural (not usually occurring in nature) nucleotides, unnatural nucleotide mimetics such as 2% modified nucleosides, peptide nucleic acids, oligomeric nucleoside phosphonates, and any polynucleotide that has added substituent groups, such as protecting groups or linking groups.
  • an “internucleotide bond” or “nucleotide bond” refers to a chemical linkage between two nucleoside moieties, such as the phosphodiester linkage in nucleic acids found in nature, or linkages well known from the art of synthesis of nucleic acids and nucleic acid analogues.
  • An intemucleotide bond may include a phospho or phosphite group, and may include linkages where one or more oxygen atoms of the phospho or phosphite group are either modified with a substituent or replaced with another atom, e.g., a sulfur atom, or the nitrogen atom of a mono- or di-alkyl amino group.
  • a “group” includes both substituted and unsubstituted forms.
  • Substituents of interest include one or more lower alkyl, amino, imino, amido, alkylamino, arylamino, alkoxy, aryloxy, thio, alkylthio, arylthio, or aryl, or alkyl; aryl, alkoxy, thioalkyl, hydroxyl, amino, amido, sulfonyl, thio, mercapto, imino, halo, cyano, nitro, nitroso, azido, carboxy, sulfide, sulfone, sulfoxy, phosphoryl, silyl, silyloxy, and boronyl, or optionally substituted on one or more available carbon atoms with a nonhydrocarbyl substituent such as cyano, nitro, halogen, hydroxyl, sulfonic acid, sulfate,
  • any substituents are chosen so as not to substantially adversely affect reaction yield (for example, not lower it by more than 20% (or 10%, or 5%, or 1 %) of the yield otherwise obtained without a particular substituent or substituent combination).
  • substituents are chosen so as to be chemically compatible with the other groups present and to avoid side reactions known to those skilled in the art.
  • an alcohol would not be substituted with a lithium group, as the hydroxide of the alcohol and the lithium group are incompatible and would react with each other.
  • each substituent may include up to 40, 35, 30, 25, 20, 18, 16, 14, 12, 11, 10, 9, 8, 7, 6, 5, 4 or 3 carbon atoms.
  • the total number of carbon atoms in all the substituents for any group is, in certain embodiments, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 18, 16, 14, 12, 11, 10, 9, 8, 7, 6, 5, 4 or 3 or less.
  • heterocycle refers to fully saturated or partially or completely unsaturated cyclic groups having at least one heteroatom in at least one carbon atom-containing ring, including aromatic (“heteroaryl”) or nonaromatic (for example, 3 to 13 member monocyclic, 7 to 17 member bicyclic, or 10 to 20 member tricyclic ring systems).
  • aromatic for example, 3 to 13 member monocyclic, 7 to 17 member bicyclic, or 10 to 20 member tricyclic ring systems.
  • Each ring of the heterocyclic group containing a heteroatom may have 1, 2, 3, or 4 heteroatoms selected from nitrogen atoms, oxygen atoms and/or sulfur atoms, where the nitrogen and sulfur heteroatoms may optionally be oxidized, and the nitrogen heteroatoms may optionally be quaternized.
  • the heterocyclic group may be attached at any heteroatom or carbon atom of the ring or ring system.
  • the rings of multi-ring heterocycles may be fused, bridged and/or joined through one or more spiro unions.
  • Nitrogen-containing bases are examples of heterocycles.
  • Other examples include piperidinyl, morpholinyl and pyrrolidinyl.
  • substituted heterocycle refers to heterocycle, heterocyclic, and heterocyclo groups substituted with one or more groups preferably selected from alkyl, substituted alkyl, alkenyl, oxo, aryl, substituted aryl, heterocyclo, substituted heterocyclo, carbocyclo (optionally substituted), halo, hydroxy, alkoxy (optionally substituted), aryloxy (optionally substituted), alkanoyl (optionally substituted), aroyl (optionally substituted), alkylester (optionally substituted), arylester (optionally substituted), cyano, nitro, amido, amino, substituted amino, lactam, urea, urethane, sulfonyl, and the like, where optionally one or more pair of substituents together with the atoms to which they are bonded form
  • protecting group refers to a species which prevents a portion of a molecule from undergoing a specific chemical reaction, but which is removable from the molecule following completion of that reaction.
  • a “protecting group” is used in the conventional chemical sense as a group which reversibly renders unreactive a functional group under certain conditions of a desired reaction, as taught, for example, in Greene, et al., “Protective Groups in Organic Synthesis,” John Wiley and Sons, Second Edition, 1991. After the desired reaction, protecting groups may be removed to deprotect the protected functional group. All protecting groups should be removable (and hence, labile) under conditions which do not degrade a substantial proportion of the molecules being synthesized.
  • a “capping group” binds to a segment of a molecule to prevent any further chemical transformation of that segment and may or may not be removable under conditions appropriate for the rest of the synthesis.
  • the capping group is meant to be present at least until the synthesis of the full-length sequence of the oligonucleotide is achieved. It should be noted that the functionality protected by the protecting group may or may not be a part of what is referred to as the protecting group.
  • a “hydroxyl protecting group” or “O-protecting group” refers to a protecting group where the protected group is a hydroxyl.
  • a “reactive-site hydroxyl” is the terminal 5'-hydroxyl during 3'-5' polynucleotide synthesis, or the 3'-hydroxyl during 5'-3' polynucleotide synthesis.
  • a “free reactive-site hydroxyl” is a reactive-site hydroxyl that is available to react to form an internucleotide bond (e.g., with a phosphoramidite functional group) during polynucleotide synthesis.
  • TC-RNA chemistry refers to the method of synthesizing RNA in which the nucleotide building blocks (i.e., the ribonucleotide phosphoramidites) have a 2'- hydroxyl protecting group that comprises a thionocarbamate group (2'-TC) as described in U.S. Patent 9,067,961, U.S. Patent 9,273,086 and U.S. Patent 9,896,472.
  • the preferred 2'-TC thionocarbamate protecting group is 1 , 1 -dioxo-26-thiomorpholine-4- carbothioate.
  • TC-RNA refers to the RNA product synthesized by TC-RNA chemistry that still have protecting groups either on the 2'-hydroxyl groups, and/or on the nucleobases and/or on the phosphate internucleotide linkages.
  • deprotecting simultaneously refers to a process which aims at removing different protecting groups in the same process and performed substantially concurrently or concurrently. However, as used herein, this term does not imply that the deprotection of the different protecting groups occur at exactly the same time or with the same rate or same kinetics.
  • a “phospho” group includes a phosphodiester, phosphotriester, and H- phosphonate groups.
  • a chemical moiety other than a substituted 5-membered sugar ring may be attached to an O atom of the phospho or phosphite group which links between the sugar ring and the P atom.
  • the term “phosphoramidite group” refers to a group comprising the structure — P — (OR ] )(NR 2 R 3 ), wherein each of R 1 , R 2 , and R 3 is independently a hydrocarbyl, substituted hydrocarbyl, heterocycle, substituted heterocycle, aryl or substituted aryl.
  • R 1 , R 2 , and R 3 may be selected from lower alkyls, lower aryls, and substituted lower alkyls and lower aryls (preferably substituted with structures containing up to 18, 16, 14, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2 carbons).
  • R 1 is 2-cyanoethyl or methyl, and either or both of R 2 and R 3 is isopropyl.
  • R 2 and R 3 can optionally be connected in a cyclic ring structure.
  • alkyl refers to a saturated straight chain, branched or cyclic hydrocarbon group of 1 to 24, typically 1- 12, carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, pentyl, cyclopentyl, isopentyl, neopentyl, hexyl, isohexyl, cyclohexyl, 3 -methylpentyl, 2,2-dimethylbutyl, and 2,3-dimethylbutyl.
  • lower alkyl intends an alkyl group of one to six carbon atoms, and includes, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, pentyl, cyclopentyl, isopentyl, neopentyl, hexyl, isohexyl, cyclohexyl, 3-methylpentyl, 2,2-dimethylbutyl, and 2,3-dimethylbutyl.
  • cycloalkyl refers to cyclic alkyl groups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl.
  • alkyl includes “modified alkyl”, which references an alkyl group having from one to twenty-four carbon atoms, and further having additional groups, such as one or more linkages selected from ether-, thio-, amino-, phospho-, oxo-, ester-, and amido-, and/or being substituted with one or more additional groups including lower alkyl, aryl, alkoxy, thioalkyl, hydroxyl, amino, sulfonyl, thio, mercapto, imino, halo, cyano, nitro, nitroso, azide, carboxy, sulfide, sulfone, sulfoxy, phosphoryl, silyl, silyloxy, and boronyl.
  • additional groups such as one or more linkages selected from ether-, thio-, amino-, phospho-, oxo-, ester-, and amido-, and/or being substituted with one or more additional groups including lower
  • lower alkyl includes “modified lower alkyl”, which references a group having from one to six carbon atoms and further having additional groups, such as one or more linkages selected from ether-, thio-, phospho-, keto-, ester-, and amido-, and/or being substituted with one or more groups including lower alkyl; aryl, alkoxy, thioalkyl, hydroxyl, amino, sulfonyl, thio, mercapto, imino, halo, cyano, nitro, nitroso, azide, carboxy, sulfide, sulfone, sulfoxy, phosphoryl, silyl, silyloxy, and boronyl.
  • alkenyl refers to a branched, unbranched or cyclic hydrocarbon group of 2 to 24, typically 2 to 12, carbon atoms containing at least one double bond, such as ethenyl, vinyl, allyl, octenyl, decenyl, and the like, with cyclic versions having at least 5 carbon atoms.
  • lower alkenyl intends an alkenyl group of two to eight carbon atoms, and specifically includes vinyl and allyl.
  • cycloalkenyl refers to cyclic alkenyl groups. Such groups will have at least 5 carbon atoms.
  • alkynyl refers to a branched or unbranched hydrocarbon group of 2 to 24carbon atoms, typically 2 to 12 such atoms, containing at least one triple bond, such as acetylenyl, ethynyl, n-propynyl, isopropynyl, n-butynyl, isobutynyl, t-butynyl, octynyl, decynyl and the like.
  • lower alkynyl intends an alkynyl group of two to eight carbon atoms, and includes, for example, acetylenyl and propynyl, and the term “cycloalkynyl” refers to cyclic alkynyl groups.
  • hydrocarbyl refers to alkyl, alkenyl or alkynyl.
  • substituted hydrocarbyl refers to hydrocarbyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone.
  • Such substituents may include, for example, a hydroxyl, a halogen, a carbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), an alkoxyl, a phosphoryl, aphosphonyl, a phosphinyl, an amino, an amido, an amidine, an imine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, a heterocyclic, an aralkyl, or an aromatic or heteroaromatic moiety.
  • a carbonyl such as a carboxy
  • the moieties substituted on the hydrocarbon chain may themselves be substituted, if appropriate.
  • the substituents of a substituted alkyl may include substituted and unsubstituted forms of amino, azido, imino, amido, phosphoryl (including phosphonate and phosphinate), sulfonyl (including sulfate, sulfonamido, sulfamoyl and sulfonate), and silyl groups, as well as ethers, alkylthios, carbonyls (including ketones, aldehydes, carboxylates, and esters), — CN, and the like.
  • Cycloalkyls may be further substituted with alkyls, alkenyls, alkoxys, alkylthios, aminoalkyls, carbonyl-substituted alkyls, — CN, and the like.
  • alkoxy means an alkyl group linked to oxygen and may be represented by the formula: R — O — , wherein R represents the alkyl group.
  • R represents the alkyl group.
  • An example is the methoxy group CH3O — .
  • lower alkoxy refers to a substituent — O — R wherein R is lower alkyl.
  • thioalkyl refers to a substituent — S — R wherein R is alkyl as defined above.
  • MOE means methoxyethyl and methoxyethylene.
  • a nucleoside with a 2'-MOEgroup has the 2' carbon of the nucleoside (or nucleotide) linked to a O- CH2CH2O-CH3 group.
  • aryl refers to 5-, 6-, and 7-membered single-ring aromatic groups that may include from zero to four heteroatoms, for example, benzene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like.
  • aryl groups having heteroatoms in the ring structure may also be referred to as “aryl heterocycles” or “heteroaromatics.”
  • the term “aryl” also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are shared with an adjoining ring (the rings are “fused rings”) wherein at least one of the rings is aromatic (e.g. , the other cyclic rings may be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, and/or heterocycles).
  • a “lower aryl” contains up to 18 carbons, such as up to 14, 12, 10, 8 or 6 carbons.
  • the aromatic rings may be substituted at one or more ring positions with such substituents as described above for substituted hydrocarbyls, for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamide, ketone, aldehyde, ester, heterocyclic, aromatic or heteroaromatic moieties, — CF3, — CN, or the like.
  • substituents as described above for substituted hydrocarbyls, for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro
  • Linkage refers to an intervening moiety bonded to two other moieties, wherein the two other moieties are linked via the intervening moiety.
  • Typical linkages include ether ( — O — ), oxo ( — C(O) — ), amino ( — NH — ), amido ( — N — C(O) — ), thio ( — S — ), phospho ( — P — ), ester ( — O — C(O) — ).
  • “Functionalized” references a process whereby a material is modified to have a specific moiety bound to the material, e.g., a molecule or substrate is modified to have the specific moiety; the material (e.g., molecule or support) that has been so modified is referred to as a functionalized material (e.g., functionalized molecule or functionalized support).
  • substituted refers to the structure, group, or moiety comprising one or more substituents.
  • first moiety e.g., hydrogen atom
  • Substituent references a moiety that replaces another moiety in a chemical structure.
  • Typical substituents include nonhydrogen atoms (e.g., halogens), functional groups (such as, but not limited to amino, sulfhydryl, carbonyl, hydroxyl, alkoxy, carboxyl, silyl, silyloxy, phosphate and the like), hydrocarbyl groups, and hydrocarbyl groups substituted with one or more heteroatoms.
  • substituents include alkyl, lower alkyl, aryl, aralkyl, lower alkoxy, thioalkyl, hydroxyl, thio, mercapto, amino, imino, halo, cyano, nitro, nitroso, azide, carboxy, sulfide, sulfone, sulfoxy, phosphoryl, silyl, silyloxy, boronyl, and modified lower alkyl.
  • Hyphens, or dashes are used at various points throughout this specification to indicate attachment, e.g., where two named moieties are immediately adjacent to a dash in the text, this indicates that the two named moieties are attached to each other.
  • a series of named moieties with dashes between each of the named moieties in the text indicates the named moieties are attached to each other in the order shown.
  • a single named moiety adjacent to a dash in the text indicates that the named moiety is typically attached to some other, unnamed moiety.
  • the attachment indicated by a dash may be, e.g., a covalent bond between the adjacent named moieties.
  • a moiety may be set forth in the text with or without an adjacent dash, (e.g., amido or amido-, further e.g., alkyl or alkyl-, yet further Lnk, Lnk- or -Lnk-) where the context indicates the moiety is intended to be (or has the potential to be) bound to another moiety; in such cases, the identity of the moiety is denoted by the group name (whether or not there is an adjacent dash in the text).
  • a single moiety may be attached to more than one other moiety (e.g. , where a linkage is intended, such as linking moieties).
  • Dashed lines e.g., - - , >
  • wavy lines e.g., are used throughout the specification and figures adjacent to named groups to indicate attachment to some other, named or unnamed group. Wavy lines are also used to represent a chain of atoms, for example to represent an oligonucleotide chain.
  • “Optional” or “optionally” means that the subsequently described circumstance may or may not occur, so that the description includes instances where the circumstance occurs and instances where it does not.
  • the phrase “optionally substituted” means that a non-hydrogen substituent may or may not be present, and, thus, the description includes structures wherein a non-hydrogen substituent is present and structures wherein a non-hydrogen substituent is not present.
  • a moiety may be described as being present zero or more times: this is equivalent to the moiety being optional and includes embodiments in which the moiety is present and embodiments in which the moiety is not present.
  • adjacent groups described as linked by the optional moiety are linked to each other directly.
  • a moiety may be described as being present if the moiety links two adjacent groups, or not present if a bond links the two adjacent groups: this is equivalent to the moiety being optional and includes embodiments in which the moiety is present and embodiments in which the moiety is not present. If the optional moiety is not present (is present in the structure zero times), adjacent groups described as linked by the optional moiety are linked to each other directly.
  • “Bound” may be used herein to indicate direct or indirect attachment.
  • “bound” (or “bonded”) may refer to the existence of a chemical bond directly joining two moieties or indirectly joining two moieties (e.g. via a linking group or any other intervening portion of the molecule).
  • the chemical bond may be a covalent bond, an ionic bond, a coordination complex, hydrogen bonding, van der Waals interactions, or hydrophobic stacking, or may exhibit characteristics of multiple types of chemical bonds.
  • “bound” includes embodiments where the attachment is direct and also embodiments where the attachment is indirect.
  • “Free,” as used in the context of a moiety that is free, indicates that the moiety is unprotected and available to react or interact with or be contacted by other components of the solution in which the moiety is a part.
  • assessing includes any form of measurement and includes determining if an element is present or not.
  • the terms “determining”, “measuring”, “evaluating”, “assessing” and “assaying” are used interchangeably and may include quantitative and/or qualitative determinations. Assessing may be relative or absolute. “Assessing the presence of’ includes determining the amount of something present and/or determining whether it is present or absent.
  • Isolated or “purified” generally refers to isolation of a molecular species (substance, compound, polynucleotide, protein, polypeptide, polypeptide, chromosome, etc.) such that the molecular species comprises a substantial portion of the sample in which it resides (excluding solvents), i.e., greater than the molecule is typically found in its natural or un-isolated state.
  • a substantial portion of the sample comprises at least about 1%, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 50%, preferably at least about 80%, or more preferably at least about 90% of the sample (excluding solvents).
  • a sample of isolated RNA may comprise at least about 75% total RNA, where percent is calculated in this context as mass (e.g., in micrograms) of total RNA in the sample divided by mass (e.g., in micrograms) of the sum of (total RNA+other constituents in the sample (excluding solvent)).
  • Techniques for purifying polynucleotides and polypeptides of interest are well known in the art and include, for example, gel electrophresis, ion-exchange chromatography, affinity chromatography, flow sorting, and sedimentation according to density.
  • pre-determined refers to a molecular attribute whose identity is known prior to its use.
  • a “pre-determined sequence” is a sequence whose identity is known prior to the use or synthesis of the polynucleotide having the sequence.
  • the molecular attribute may be known by name, sequence, molecular weight, its function, or any other attribute or identifier.
  • Upstream refers to the 5' direction along a polynucleotide, e.g., an RNA molecule. “Downstream” refers to the 3' direction along the polynucleotide. “3'-” and “5'-” have their conventional meaning as known in the art. DETAILED DESCRIPTION OF PARTICULAR EMBODIMENTS
  • Methods and compounds are provided that can be used to purify oligonucleotides such as RNA, DNA or chimera.
  • methods of preparing purified 5'-phosphate oligonucleotides comprising: obtaining a support-bound oligonucleotide, wherein the oligonucleotide has a free 5 '-hydroxyl group and is attached through a linker via its 3'- hydroxyl or 3 '-phosphate to a solid support, and wherein the oligonucleotide may comprise one or more nucleobase exocyclic amino-protecting groups, one or more phosphate triester protecting groups, or one or more 2'-hydroxyl protecting groups; reacting the free 5 '-hydroxyl group of the support-bound oligonucleotide with a phosphoramidite comprising a substituent containing a cleavable linker and a protected hydroxyl group, wherein reacting forms an extended
  • the purified 5'-phosphoester oligonucleotide under suitable conditions, and eluting the formed 5'-phosphate oligonucleotide off the column or affinity capture support, thereby obtaining the purified 5 '-phosphate oligonucleotide; or d) eluting the washed captured affinity-tagged 5 '-phosphoester oligonucleotide from the chromatographic column or the affinity capture support and then cleaving the cleavable linker off the eluted washed affinity-tagged 5 '-phosphoester oligonucleotide to obtain the purified 5 '-phosphate oligonucleotide, wherein the purified 5'- phosphate oligonucleotide may comprise one or more nucleobase exocyclic amino-protecting groups, one or more phosphate triester protecting groups, one or more 2'-hydroxyl protecting groups or a combination thereof.
  • the present methods are especially useful for separating full-length oligonucleotides from truncated or failure sequences.
  • the present methods provide high resolution of oligonucleotides of similar size, allowing for rapid separation of oligonucleotides from undesired side-products.
  • the affinity-tag may be cleaved — depending on the affinity tag linking chemistry — under acidic conditions, basic conditions, UV light, reductive conditions or any suitable conditions for releasing the purified oligonucleotide in high yield.
  • the present methods are effective for purifying oligonucleotides prepared by various synthetic methods, including oligonucleotides prepared in a solid-phase oligonucleotide synthesis.
  • An oligonucleotide to be purified may be obtained in some embodiments from a natural source or prepared using a synthetic method. Synthetic oligonucleotides may be prepared using any method known in the art. In some embodiments, an oligonucleotide is prepared via solid-phase oligonucleotide synthesis. In some such embodiments, the synthesis is carried out on a solid support. In some of these embodiments, the solid support is generally held between two filters in columns that enable reagents and solvents to pass through freely.
  • the synthesis is carried out on a planar surface. In some embodiments, the synthesis is carried out on a non-planar surface. In such embodiments, the synthesis is carried out on a substantially solid, substantially smooth surface.
  • substantially solid means that the location(s) on the surface of the support where oligonucleotide synthesis is occurring is resistant to the diffusion, absorption, or permeation of the relevant reagents and chemicals of oligonucleotide synthesis beyond the surface and into the body of the support (in contrast to commercial polymeric oligo synthesizer supports, which permit such diffusion and permeation, such that oligo synthesis occurs in the body of the support).
  • substantially smooth means that the location(s) on the surface of the support where the oligonucleotide synthesis is occurring is at most superficially irregular, such that irregularities, if any, are not of a scale which would substantially affect the rapidity with which reagents can be uniformly applied to, mixed on, or removed from the surface (in contrast to commercial “controlled pore glass” oligo synthesizer supports, which contain pores and irregularities that slow the application and removal of reagents).
  • a substantially solid, substantially smooth surface need not be flat, and would include, for example, flat surface regions, tubes, cylinders, arrays of depressions or wells, and combinations of these elements, as well as other designs presenting surface portions with the abovedescribed attributes.
  • substantially solid, substantially smooth surfaces are surfaces (or portions of surfaces) that can be addressed by an inkjet print head.
  • an oligonucleotide is attached to a solid support such as a controlled-pore glass, or a polymeric support for example a polystyrene (PS) support.
  • a solid support such as a controlled-pore glass, or a polymeric support for example a polystyrene (PS) support.
  • PS polystyrene
  • Suitable solid supports are in some cases polymeric and may have a variety of forms and compositions and derive from naturally occurring materials, naturally occurring materials that have been synthetically modified, or synthetic materials.
  • suitable support materials include, but are not limited to, polysaccharides such as agarose (e.g., available commercially as Sepharose®, from Pharmacia) and dextran (e.g.
  • the oligonucleotide is prepared in solution (Benjamin
  • the oligonucleotide is prepared using a phosphoramidite method.
  • the oligonucleotide synthesis method comprises a support-bound nucleoside having a 5'-DMT protecting group.
  • the oligonucleotide synthesis method comprises a support-bound nucleoside having a 3'-DMT protecting group. In some embodiments, the oligonucleotide synthesis method comprises a support-bound nucleoside having a 5'-silyl protecting group. In some embodiments, the oligonucleotide synthesis method comprises a support-bound nucleoside having an oxidation removable protecting group.
  • the oligonucleotide synthesis comprises the steps of detritylation, coupling of a support bound terminal nucleotide with a nucleoside phosphoramidite monomer, capping unreacted 5'-hydroxyl groups, and phosphoramidite oxidation.
  • the oligonucleotide synthesis is automated.
  • the oligonucleotide is detritylated prior to performing a method of the present invention. [0084] In some embodiments, it is acceptable and even sometimes preferable to have the 5' end of RNA terminated with a phosphate rather than a hydroxy.
  • methods of oligonucleotide purification are provided that are particularly suitable for RNA prepared by standard phosphoramidite chemistry whereby a purification handle or affinity tag is introduced post-synthesis in a two steps process, by adding first a 5'-phospho groupto the 3'-support-bound oligonucleotide, and second by adding the purification handle or affinity tag through a cleavable linker to the 5’- phosphogroup. After affinity purification, the linker may be removed along with the handle or tag, leaving behind a 5'-terminal phosphate on the oligonucleotide.
  • an advantage of these methods is the readily achievable separation of the linker and the handle or tag into separate and distinct coupling moieties.
  • This aspect offers more flexibility in the design and use of the tags, which may be joined to the cleavable linker by standard phosphoramidite chemistry steps on a standard oligonucleotide synthesizer.
  • a different conjugation or coupling chemistry than phosphoramidite chemistry is used.
  • the affinity tag may be added to the cleavable linker through click chemistry, implying the use of suitable reactive groups (RG), in this case an alkyne and azido groups, each present on each partner of the coupling reaction.
  • RG reactive groups
  • squaric acid chemistry is used to conjugate the affinity tag to the cleavable linker using adequate reactive groups (squaric mono- or di-alkylesters and amino/thiol groups).
  • Non-limiting examples of reactive groups include a hydroxyl group, an amino group, a NHS ester (N-hydroxysuccnimide) or other activated ester: pentafluorophenol (PFP), hexafluoroisopropyl alcohol (HFP), 4-nitrophenol, and A-hydroxyphthalimide (NHP), a thiol, a maleimide, a hydrazide, a diol, a protected hydroxyl, a protected sulfhydryl, a protected poly-sulfhydryl, a polyhistidine, a protected amino group, a protected hydrazide group, a protected oxyamine group, a cyclooctyne, a conjugated diene, a C2 alkenyl group, a C2 substituted alkenyl group, a C2 alkynyl group or a C2 substituted alkynyl group.
  • PFP pentafluorophenol
  • HFP he
  • multiple tags can be added onto a linker through a single or multiple conjugation steps.
  • this method is well suited for purification of single guide RNAs, which are generally about 100 nucleotides in length, including those synthesized by means of the TC-RNA chemistry, wherein a 2'-TC (l,l-dioxo-X6- thiomorpholine-4-carbothioate) protective group has been introduced into oligoribonucleotide synthesis in order to simplify the downstream processing of synthetic RNA.
  • the method proceeds as outlined in the scheme shown in FIG. 1.
  • an affinity tag or purification handle is attached through a cleavable linker to the 5'-terminal phosphate of an oligonucleotide (for example a TC-RNA), the 5 '-terminal phosphate being itself installed in a prior step with a phosphoramidite reagent.
  • oligonucleotide e.g., TC-RNA
  • oligonucleotide e.g., TC-RNA
  • 2'- protecting groups may be all removed post-synthesis or partial deprotection may be performed when needed or desired, e.g., all nucleobases and phosphates protecting groups are removed but the 2'-hydroxyl protecting groups such as TBDMS, TOMS or ACE or other groups, remained
  • synthesis resin CPG, polystryrene or else
  • another basic reagent e.g., methylamine, ammonia or other bases for example DBU or other cleavage reagents
  • oligonucleotides oligodeoxyribonucleotides or RNA made with other chemistries than TC-RNA chemistry.
  • the oligonucleotide is then trapped on the affinity resin, chromatography column, or other means, and truncated oligos and side products are removed by washing.
  • the affinity tag is released from the 5 '-phosphate group on the 5'-OH of the oligonucleotide that is adsorbed on the resin or column, and subsequently, the purified 5 '-phosphate terminated oligonucleotide is eluted from the resin and collected.
  • the affinity tagged 5 '-phosphate oligonucleotide is eluted and collected from the affinity resin and then the cleavable linker is cleaved to release the affinity tag from the purified 5 '-phosphate oligonucleotide.
  • affinity-based purification may be achieved using fluorous tags.
  • tags on the linker By using different tags on the linker, alternative approaches such as a bead-based separation could be enabled.
  • the schemes below are shown with DNA but can be extended to use with TC-RNA chemistry or any other RNA synthesis chemistry, using the same principles of affinity separation.
  • the affinity purification may be performed either before or after deprotection of the RNA 2' protecting groups.
  • the oligonucleotide is an oligoribonucleotide (RNA).
  • the oligonucleotide is an oligodeoxyribonucleotide (DNA). In some embodiments, the oligonucleotide is at least 20 nucleotides in length. In some embodiments, the oligonucleotide is at least 50 nucleotides in length. In some embodiments, the oligonucleotide is at least 75 nucleotides in length. In some embodiments, the oligonucleotide is at least 100 nucleotides in length. In some embodiments, the oligonucleotide is at least 125 nucleotides in length. In some embodiments, the oligonucleotide is at least 150 nucleotides in length.
  • DNA oligodeoxyribonucleotide
  • the oligonucleotide is at least 175 nucleotides in length. In some embodiments, the oligonucleotide is at least 200 nucleotides in length. In some embodiments, the oligonucleotide is at least 250 nucleotides in length. In some embodiments, the oligonucleotide is at least 300 nucleotides in length. In some embodiments, the oligonucleotide is from about 20 nucleotides to about 500 nucleotides in length. In some embodiments, the oligonucleotide is from about 40 nucleotides to about 300 nucleotides in length.
  • the support-bound oligonucleotide is first coupled with a photocleavable compound such as
  • Compounds I or II may be used to incorporate a cleavable linker moiety (i.e. a photocleavable linker) on the 5 '-end of the growing oligonucleotide to create an oligonucleotide that is bound to solid support at its opposite 3 '-end, which can be depicted as follows (shown for compound I; the longer squiggly/wavy line represents the oligonucleotide):
  • the cleavable linker is obtained by incorporating a photocleavable group (e.g., a nitrobenzyle moiety) on the phosphoramidite, such as in compounds I and II.
  • the SATE protecting group can be used as a different cleaving group. The SATE was originally developed to come off under biological conditions via esterase activity (for use in prodrugs) and cleaves via an episulfide to give a phosphate. See Perigaud et al., 1993, which is incorporated by reference herein.
  • Disulfides have been shown to also be useful in this regard as well, originally designed to come off under reductase activity. See Perigaud et al., 1993, which is incorporated by reference herein.
  • DTT dithiothreitol
  • TCEP tris(2- carboxyethyl)phosphine
  • DTT dithiothreitol
  • TCEP tris(2- carboxyethyl)phosphine
  • they should be suitable as reductively cleavable phosphoramidite monomers to link the affinity tag to the 5 '-phosphate oligonucleotide. Examples of potentially suitable phosphoramidites containing a cleavable linker are shown below.
  • the R group of the O-R group linked to the phosphorous in compounds 6, 7, 7b and 8 is a standard phosphoramidite protecting group such a 2- cyanoethyl or a methyl group.
  • the R group of the S-R group linked to the sulfur in compounds 7 and 7b is an alkyl group, preferably a Ci-Ce alkyl group.
  • this support-bound oligonucleotide containing the cleavable linker on the 5 '-phosphate group can then be connected to an affinity tag, for example, by first deprotecting the DMT group from the hydroxyl of the photo cleavable linker and then by reacting the oligonucleotide in a second step with a “fluorous” monomer such as the commercially available (Berry & Associates) fluorous trityl containing monomer III: thereby giving rise to the following affinity-tagged 5 '-phosphate support-bound oligonucleotide:
  • this deprotected affinity-tagged oligonucleotide is then eluted off the solid support (e.g., synthesis resin) and trapped on a fluorous column to form an affinity -paired deprotected oligonucleotide for purification.
  • Photocleavage of the affinity-paired deprotected oligonucleotide at 366nm UV light (e.g., in sufficient quantities to expose through the resin) will give the desired 5'-phosphate terminated oligonucleotide.
  • both the fluorous containing byproducts and the photochemical group byproducts will remain attached to the resin.
  • a fluorous tag which does not comprise a nucleotide monomer but rather just a fluorous-rich hydrocarbon chain like the compound of formula IV may be used, wherein R is a standard phosphoramidite protecting group, like a 2-cyanoethyl group, for example:
  • a fluorous tag like the compound of formula V may be used.
  • a suitable reaction scheme to connect the affinity tag to a disulfide cleavable linker involves the following steps: a) oligonucleotide synthesis under standard conditions, with a monomer containing a disulfide cleavable linker (as shown below with compound 6) to create a disulfide containing 5'-phosphate support- bound oligonucleotide:
  • the thiol containing oligonucleotide may be obtained.
  • the cleavable linker contains a blocked abasic nucleotide, which when treated under the appropriate conditions will become an abasic nucleotide. These sites will readily cleave under basic or enzymatic conditions to give rise to the desired oligo containing a 5 '-phosphate.
  • Compound 9 shows a phosphoramidite monomer that can be used for this purpose (available from Glen Research, https://www.glenresearch.eom/media/folio3/productattachments/technical_bulletin/T
  • a suitable reaction scheme using compound 9 would create an oligo containing this monomer, which after extension with a biotinylated phosphoramidite gives a resin bound nucleotide such as
  • Failure sequences are removed by washing the affinity resin containing the captured oligonucleotide.
  • the TBDMS protecting group is removed with fluoride or aqueous acid., giving rise to the abasic oligonucleotide.
  • This abasic oligo is readily cleaved under basic conditions to give the desired 5 ’-phosphate terminated oligonucleotide.
  • Elution of the oligo off of the streptavidin resin can occur after, concomitant with, or before removal of the TBDMS group or cleavage of the oligo via the abasic nucleotide.
  • This approach of using a 5 '-phosphate attached affinity tag is also suitable for DNA synthesis of particularly long oligonucleotides, where the use of ordinary “trityl-on” purification is insufficient for separating unwanted failure sequences from the desired full-length product.
  • the methods provided herein are compatible with any chemistry in which the final cleavage to give the phosphate occurs under chemical conditions which are orthogonal to the phosphoramidite chemistry, the removal of the protecting groups, and oligonucleotide cleavage off of the synthesis resin.
  • a separate affinity monomer unit is also suitable for use as an affinity tag, and in some embodiments, the separate affinity tag is not a fluorous compound, but instead a tag that can be captured via “click” or other chemistry.
  • the chemistry used to attach the affinity tag to the 5'-phosphate moiety can be any known conjugation or coupling chemistry as described previously, including phosphoramidite chemistry, click chemistry, squaric acid chemistry, carbodiimide chemistry (e-g-, dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), ( 1 -Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and other activated esters (such as NHS and other activated esters as described earlier), thiol, maleimide, and such other reactive groups (RG) as were described above, as long as there is a cleavable moiety between the chemistry used to attach the affinity tag to the 5
  • the affinity tag is a fluorous affinity tag.
  • fluorous affinity tags include fluorinated substituents such as fluorosubstituted alkyl, fluorosubstituted heteroalkyl, fluorosubstituted alkenyl, fluorosubstituted alkynyl, fluorosubstituted carbocyclyl, fluorosubstituted heterocyclyl and a fluorosubstituted aryl substituents.
  • an orthoester acts as a linker between a fluorous affinity tag and an oligonucleotide.
  • the orthoester i.e., orthoester linker
  • the fluorous affinity tag directly (as an R group) or indirectly through a linker L and is attached to the oligonucleotide.
  • fluorous affinity purification is used to isolate or purify the deprotected affinity-tagged oligonucleotide.
  • the affinity tag is a hydrophobic tag.
  • hydrophobic affinity tags include molecules that comprise fluorinated groups, trityl, steroid, lipid, long alkyl saturated or unsaturated chains such as octadecyl substituents and carbocyclyl.
  • hydrophobic affinity purification is used to isolate or purify the deprotected affinity-tagged oligonucleotide.
  • the affinity tag is a biotin tag.
  • avidin or streptavidin is attached to a solid phase or embedded in a gel and is used to capture the deprotected affinity-tagged oligonucleotide.
  • the affinity tag is a glutathione tag.
  • glutathione S-tranferase GST is attached to a solid phase or embedded in a gel and is used to capture the deprotected affinity-tagged oligonucleotide.
  • the affinity tag is a maltose tag.
  • maltose binding protein is attached to a solid phase or embedded in a gel and is used to capture the deprotected affinity-tagged oligonucleotide.
  • the affinity tag is an arylboronic acid tag.
  • a diol containing molecule is attached to a solid phase or embedded in a gel and is used to capture the deprotected affinity-tagged oligonucleotide.
  • the affinity tag is a diol-containing compound and the arylboronic acid derivative is linked to the solid support.
  • the affinity tag is a poly-histidine peptide tag.
  • poly-histidine peptide tags include imidazolyl substituents such as imidazole and histidine.
  • immobilized metal affinity chromatography (IMAC) purification is used to isolate or the purify deprotected affinity-tagged oligonucleotide
  • the affinity tag is a poly-sulfhydryl tag.
  • poly-sulfhydryl tags include substituents such as cysteine and dithio threitol.
  • immobilized metal affinity chromatography (IMAC) purification is used to isolate or purify the deprotected affinity-tagged oligonucleotide.
  • the affinity tag is a maleimide tag.
  • a sulfhydryl containing molecule is attached to a solid phase or embedded in a gel and is used to capture the deprotected affinity- tagged oligonucleotide.
  • the affinity tag is a sulfhydryl-containing compound and the maleimide derivative is linked to the solid support. In another embodiment, the affinity tag is a sulfhydryl-containing compound and an alpha-halocarbonyl derivative is linked to the solid support.
  • the affinity tag is an adamantane tag.
  • a cucurbituril or cyclodextrin is attached to a solid phase or embedded in a gel and is used to capture the deprotected affinity-tagged oligonucleotide.
  • the affinity tag is a cyclodextrin or cucurbituril tag, and adamantane is attached to a solid phase or embedded in a gel for affinity purification.
  • the affinity tag is an azido tag.
  • an alkyne or cyclooctyne is attached to a solid phase or embedded in a gel and is used to capture the deprotected affinity-tagged oligonucleotide.
  • the affinity tag is an alkyne or cyclooctyne tag, and the azido moiety is attached to the solid support or the gel.
  • the affinity tag is cyclooctyne tag.
  • a nitrone is attached to a solid phase or embedded in a gel and is used to capture the deprotected affinity-tagged oligonucleotide.
  • the affinity tag is a nitrone tag and the cyclooctyne moiety is attached to the solid support.
  • the affinity tag is an alkenyl tag.
  • a conjugated diene is attached to a solid phase or embedded in a gel and is used to capture the deprotected affinity-tagged oligonucleotide in a Diels-Alder type reaction ([4+2] cycloaddition).
  • the affinity tag is a conjugated diene and the alkene-containing compound is attached to the solid support or the gel.
  • the affinity tag is an amino tag.
  • an activated ester such as A-hydroxysuccinimide is attached to a solid phase or embedded in a gel and is used to capture the deprotected affinity-tagged oligonucleotide.
  • the affinity tag is a hydrazide or oxy amine tag.
  • an aldehyde or ketone-containing compound is attached to a solid phase or embedded in a gel and is used to capture the deprotected affinity-tagged oligonucleotide.
  • the oligonucleotide comprises a phosphorous protecting group, whereby a phosphorus moiety of an oligonucleotide is attached to a phosphorus protecting group.
  • the phosphorus moiety is a phosphate, phosphoramidite, or a H-phosphonate group.
  • the phosphorus protecting group is a methyl or cyanoethyl group (e.g., beta-cyanoethyl group). The methyl group may be removed using, for example, thiophenol or disodium 2-carbamoyl-2-cyanoethylene-l,l-dithiolate.
  • the cyanoethyl group may be removed using, for example, a non-nucleophilic or hindered amine such as diethylamine, t- butylamine, or l,8-diazabicycloundec-7-ene (DBU).
  • DBU l,8-diazabicycloundec-7-ene
  • the method comprises deprotecting the nucleobase protecting group and optionally the phosphorous protecting group.
  • the oligonucleotide comprises a nucleobase protecting group.
  • Any nucleobase in the oligonucleotide can comprise a nucleobase protecting group.
  • the nucleobase protecting group is acetyl, isobutyryl, benzoyl, or the like.
  • the protected-nucleobase may be N 6 - benzoyl-A, N 6 -isobutyryl-A, N 4 -acetyl-C, N 4 -isobutyryl-C, or N 2 -isobutyryl-G.
  • the nucleobase protecting group is removed by contacting the oligonucleotide to a polyamine.
  • the nucleobase protecting group is removed by contacting the oligonucleotide with a diamine such as 1 ,2-diaminoethane.
  • exposing the oligonucleotide to 1 ,2-diaminoethane for 2 hours at room temperature results in deprotection of the nucleobase.
  • the nucleobase protecting group is phenoxy acetyl, t-butylphenoxyacetyl, dimethylformamidine, dimethylacetamidine, or the like.
  • cleavage of the deprotected affinity-tagged oligonucleotide from the solid support and deprotection of the nucleobase protecting group and optionally the phosphorus protecting group is performed simultaneously and/or in the same reaction.
  • the oligonucleotide is an oligoribonucleotide (RNA) comprising a 2'-hydroxyl protecting group.
  • the 2'-hydroxyl protecting group is a thionocarbamate (TC) protecting group.
  • the 2’-hydroxyl protecting group is a bis(2-acetoxyethoxy)methyl (ACE) protecting group, a t-butyldimethylsilyl (TBDMS) protecting group, a triisopropylsilyloxymethyl (TOM) protecting group, a pivaloyloxymethyl (PivOM) protecting group or a 2-cyanoethoxymethyl (CEM) protecting group.
  • the synthesis of the oligonucleotide is performed on a solid phase (e.g., CPG) that contains the 3 '-terminal nucleotide attached to the solid phase with a base labile linker (such as a succinate linker or Universal Support linker (e.g., Glen Research UnySupport, or a UnyLinker).
  • a base labile linker such as a succinate linker or Universal Support linker (e.g., Glen Research UnySupport, or a UnyLinker).
  • the synthesis cycle includes deprotection of the 5' -OH protecting group (e.g., trityl or DMT), coupling of a phosphoramidite in the presence of an acitvator, capping of the unreacted hydroxyl groups with a base labile protecting group (e.g., acetyl protecting group), and oxidation of the phosphite triester linkage to the phosphate triester linkage.
  • nascently synthesized oligonucleotides, DNA, RNA or modified oligonucleotides are synthesized on a solid support.
  • the 5 '-phosphate affinity-tagged oligonucleotide is adsorbed on a solid support, washed, purified and eluted from the solid support.
  • the affinity tag is then cleaved at the cleavable portion of the linker from the 5'-phosphate oligonucleotide under suitable conditions (e.g., acidic, UV, basic, reductive depending on the cleavage chemistry) only after elution of the tagged oligonucleotide from the column.
  • suitable conditions e.g., acidic, UV, basic, reductive depending on the cleavage chemistry
  • the 5 '-phosphate purified oligonucleotide may be further purified by ultrafiltration or desalting if necessary.
  • the deprotection of the protecting groups is performed with a base or basic reagent (e.g., 1,2-diaminoethane, ammonia, ammonium hydroxide, methylamine and the like) at room temperature or at higher temperature.
  • a base or basic reagent e.g., 1,2-diaminoethane, ammonia, ammonium hydroxide, methylamine and the like
  • synthesis of the oligonucleotide is accomplished on a solid phase with the 3'-terminal nucleotide attached by a non-base labile cleavable linker (e.g., a photocleavable linker).
  • cleavage of the 3 '-end of the oligonucleotide from the solid support leaves a 3 '-hydroxyl at the end.
  • cleavage of the 3'-end of the oligonucleotide from the solid support leaves a 3 '-phosphate at the end.
  • capping of unreacted 5 '-hydroxyl groups on the growing oligonucleotide is accomplished with a non-base labile reagent which upon deprotection leaves something other than a free hydroxyl group on the truncated oligonucleotide sequences, (e.g., capping with UniCap reagent (Glen Research) forming a phosphodi ester after deprotection).
  • a non-base labile reagent which upon deprotection leaves something other than a free hydroxyl group on the truncated oligonucleotide sequences
  • contacting the oligoribonucleotide with a diamine, ammonia, or an alkyl amine results in simultaneous deprotection of the 2'-hydroxyl group and cleavage of the oligoribonucleotide from the solid support.
  • an ACE protecting group is removed by contacting the oligoribonucleotide with an acid.
  • the ACE protecting group and affinity tag linker are removed simultaneously.
  • the 2'-hydroxyl protecting groups are TBDMS or TOM protecting groups and are removed by contacting the oligoribonucleotide with a composition comprising fluoride.
  • the TBDMS or TOM protecting groups may be removed before or after isolating the 5'- phosphate terminated oligonucleotide.
  • 2'-hydroxyl protecting groups can be removed before or after the affinity purification step.
  • the 2'-hydroxyl protecting group is removed prior to isolating the purified 5'-phosphate terminated oligonucleotide. In some embodiments, the 2'-hydroxyl protecting group is removed after isolating the 5'-phosphate terminated oligonucleotide. In some embodiments, the oligonucleotide is treated with 1,2- diaminoethane, resulting in simultaneous removal of the protecting groups from the oligonucleotide and cleavage of the deprotected affinity-tagged oligonucleotide from the solid support.
  • the oligonucleotide comprises a phosphorus protecting group or a nucleobase protecting group. In some embodiments, the oligonucleotide comprises a phosphorus protecting group and a nucleobase protecting group. In some embodiments, the oligonucleotide is an RNA that comprises a phosphorus protecting group, a nucleobase protecting group, and a 2'-hydroxyl protecting group.
  • the 5'-phosphate affinity-tagged support-bound oligonucleotide is treated with 1 ,2-diaminoethane, thereby removing the 2'-OH protecting group (e.g., TC or PivOM) from the oligonucleotide, the nucleobase protecting groups, the phosphate protecting groups and cleaving the affinity-tagged oligonucleotide from the synthesis solid support simultaneously.
  • 2'-OH protecting group e.g., TC or PivOM
  • the purified 5 '-phosphate terminated oligonucleotide has a purity of at least about 60%.
  • the purified 5'-phosphate terminated oligonucleotide has a purity of at least about 80%, at least about 85%, at least about 90%, at least about 92%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, at least about 99.9%, at least about 99.95%, or at least about 99.99%.
  • the purified 5'-phosphate terminated oligonucleotide comprises one or more impurities at an amount of about 20% or less, about 15% or less, about 10% or less, about 9% or less, about 8% or less, about 7% or less, about 6% or less, about 5% or less, about 4% or less, about 3% or less, about 2% or less, about 1% or less, about 0.5% or less, about 0.1% or less, about 0.05% or less, or about 0.01% or less.
  • the 5 '-phosphate affinity-tagged oligonucleotides of the present disclosure can be purified using any suitable chromatographic method.
  • the oligo can be fully deprotected (DNA or RNA) or partially deprotected RNA still bearing 2'-protecting groups.
  • the 5 '-phosphate affinity-tagged oligonucleotide is purified using fluorous affinity chromatography.
  • the deprotected affinity-tagged oligonucleotide is purified using column chromatography.
  • the deprotected affinity-tagged oligonucleotide is purified using liquid chromatography (e.g., high perfomance liquid chromatography).
  • the deprotected affinity-tagged oligonucleotide is purified using fluorous affinity chromatography.
  • Fluorous affinity chromatography utilizes the concept of fluorophilicity where fluorinated compounds tend to have affinity for other fluorinated compounds.
  • the fluorous column in some embodiments comprises a polymer resin to which fluorinated organic groups are bound.
  • the deprotected affinity-tagged oligonucleotide binds strongly to the column, while the non-labeled impurities (e.g., truncated or failed sequences) do not interact with the column and pass through the column.
  • subsequent washing does not release the deprotected affinity-tagged oligonucleotide from the column due to the strength of fluorophilic interaction with the fluorous column.
  • the affinity -paired deprotected oligonucleotide may be released from the column with a stronger eluant or by cleaving the affinity tag off of the 5 '-phosphate oligonucleotide.
  • a skilled person in the art will adjust the purification conditions to perform the cleavage of the affinity tag prior or post elution of the oligonucleotide of the affinity column depending on the ease of separation, ease of tag cleavage conditions and convenience.
  • the affinity-paired deprotected oligonucleotide is separated from the column or the affinity capture support under mildly acidic conditions, which, for example, breaks interactions between the oligonucleotide and the fluorous column.
  • the deprotected affinity -tagged oligonucleotide is purified using high performance liquid chromatography (HPLC).
  • HPLC high performance liquid chromatography
  • the present method allows for separation of the deprotected affinity- tagged oligonucleotide from impurities of a similar size.
  • the at least one impurity may be of any type.
  • the at least one impurity is a truncated or failure sequence produced during an oligonucleotide synthesis.
  • the deprotected affinity-tagged oligonucleotide is purified using reverse-phase HPLC.
  • the deprotected affinity-tagged oligonucleotide is purified on a reverse-phase column (e.g., Cs or Cis hydrocarbon column). In some embodiments, the deprotected affinity-tagged oligonucleotide is purified on a normal-phase HPLC column. In some embodiments, the deprotected affinity-tagged oligonucleotide is collected from the HPLC system and the affinity tag is cleaved off of the affinity-tagged oligonucleotide to yield the desired purified 5 '-phosphate oligonucleotide,
  • the HPLC system may comprise an injector, a pump, an HPLC column, and a detector.
  • the detector is an absorbance detector (e.g., UV/VIS or PDA), refractive-index detector, scattering detector (e.g. , evaporative or multi-angle), mass spectrometer, conductivity detector, fluorescence detector, chemiluminescence detector, optical rotation detector, or electrochemical detector.
  • the HPLC system is a triple quadrupole LC-MS, Orbitrap LC-MS, Ion Trap LC-MS, or TOF LC-MS.
  • the purification method is partially or fully automated.
  • the deprotected affinity -tagged oligonucleotide is purified using a cartridge comprising a compound that binds an affinity moiety on the affinity -paired deprotected oligonucleotide.
  • the support-bound oligonucleotide is synthesized on a multiplex synthesis platform and the isolation of the purified 5 '-phosphate terminated oligonucleotide is performed in parallel on a multiplex purification platform.
  • the multiplex synthesis or purification platform allows to perform the purification in parallel of 4, 12, 48, 96, 192, or 384 oligonucleotide syntheses.
  • Example 1 Purification of lOOmer oligonucleotide using photocleavable linker and a fluorous resin.
  • Compound C is treated with diaminoethane to remove all of the protecting groups and cleave the oligo nucleotide off of the solid support. See Dellinger et al. , “Streamlined process for the chemical synthesis of RNA using 2'-O-thionocarbamate- protected nucleoside phosphoramidites in the solid phase,” J. Am. Chem. Soc. 133(30) : 11540-56, 2011, which is incorporated herein by reference. After water elution off of the solid support, compound D is obtained, along with any non- fluorous tagged impurities.
  • this mixture of compounds is passed through a column containing a fluorous resin (as described in, e.g., U.S. Patent 11,299,483, which is incorporated herein by reference), trapping the fluorous tagged full-length material and allowing all non- fluorous tagged impurities to pass through.
  • the fluorous tagged material is then eluted off the fluorous resin, as described in U.S. Patent 11,299,483, which is incorporated herein by reference).
  • this solution is then irradiated with 365nm light to cleave the linker, giving rise to the desired 5'-phosphate terminated lOOmer RNA product E.
  • Product E is further purified and desalted via centrifugal ultrafiltration using a 3K molecular weight cutoff filter (available from Amicon).
  • compound F is extended with 12-(4,4'- Dimethoxytrityloxy)dodecyl-l-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (Glen Research), and after oxidation gives compound G.
  • compound G is again extended with 12-(4,4'-Dimethoxytrityloxy)dodecyl-l- [(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, after oxidation forming the doubly tagged compound H.
  • RNA oligonucleotide containing the very hydrophobic tag is retained on the column, while untagged impurities flow through.
  • Oligo J is eluted off of the column and treated with tris(2- carboxyethyl)phosphine (TCEP) to reduce the disulfide bond giving oligo K.
  • TCEP tris(2- carboxyethyl)phosphine
  • Treatment of oligo K with pH 9 buffer gives the desired 5 '-phosphate oligo E via episulfide cleavage. Residual tag molecule is removed by trapping on a cartridge containing a C-
  • Example 3 Purification of lOOmer oligonucleotide using abasic linker and biotin-streptavidin capture
  • compound M is extended with [l-A-(4,4'- dimethoxytrityl)-biotiny]-6-aminoethoxyethy]]-2-cyanoethyl-(7V,jV-diisopropyl)- phosphoramidite (Glen research), and after oxidation, detritylation, deprotection and cleavage from the solid support gives compound N.
  • biotinylated full-length oligo N is bound to a streptavidin resin, and nonbiotinylated impurities are washed off.
  • Treatment of the bound oligo with 80% aqueous acetic acid removes the TBDMS group to give oligo O, followed by alkaline treatment with sodium hydroxide solution gives the desired 5 '-phosphate oligo P via a betaelimination reaction.
  • Compound P is further purified using ultrafiltration or chromatography.

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Abstract

L'invention concerne des procédés de purification d'oligonucléotides permettant de produire un oligonucléotide à extrémité phosphate 5' terminale purifié. L'invention concerne également des procédés de purification d'oligonucléotides qui sont appropriés pour l'ARN synthétique, en particulier l'ARN produit à l'aide de la chimie "TC-ARN". Le procédé de préparation d'un oligonucléotide à extrémité phosphate 5' terminale purifié comprend les étapes consistant à : obtenir un oligonucléotide lié à un support, l'oligonucléotide ayant un groupe hydroxyle 5' libre et étant fixé par l'intermédiaire d'un lieur par l'intermédiaire de son hydroxyle 3' ou phosphate 3' à un support solide; faire réagir le groupe 5' hydroxyle libre de l'oligonucléotide lié à un support avec un phosphoramidite comprenant un substituant contenant un lieur clivable et un groupe hydroxyle protégé, la réaction permettant de former un oligonucléotide lié à un support 5' phosphoester étendu; déprotéger le groupe hydroxyle protégé de l'oligonucléotide lié au support 5' phosphoester, puis le faire réagir avec un réactif comprenant une étiquette d'affinité pour former un oligonucléotide lié à un support 5' phosphoester marqué par affinité, l'étiquette d'affinité étant liée à l'oligonucléotide 5' phosphoester étendu par l'intermédiaire dudit lieur clivable.
PCT/US2023/081448 2023-11-17 2023-11-28 Procédés de purification d'oligonucléotides produisant des oligonucléotides purifiés ayant des groupes phosphate à extrémité 5' terminale Pending WO2025106093A1 (fr)

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