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WO2021080021A1 - Procédé de production d'oligonucléotide - Google Patents

Procédé de production d'oligonucléotide Download PDF

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Publication number
WO2021080021A1
WO2021080021A1 PCT/JP2020/040096 JP2020040096W WO2021080021A1 WO 2021080021 A1 WO2021080021 A1 WO 2021080021A1 JP 2020040096 W JP2020040096 W JP 2020040096W WO 2021080021 A1 WO2021080021 A1 WO 2021080021A1
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Prior art keywords
group
phenyl
formula
activator
independently
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PCT/JP2020/040096
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English (en)
Japanese (ja)
Inventor
正史 岩本
正典 片岡
智▲祥▼ 飯田
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Natias Inc
Nitto Denko Corp
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Natias Inc
Nitto Denko Corp
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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
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the present invention relates to a method for producing an oligonucleotide and an activator for producing an oligonucleotide.
  • oligonucleotides with the desired sequence are typically obtained by sequentially adding appropriately protected nucleosides one by one, with each nucleoside being added in at least three steps. That is, it requires a coupling step, an oxidation or sulfurization step, and a deprotection step.
  • Patent Documents 1 and 2 describe a method for obtaining an oligonucleotide by reacting a nucleoside in which a hydroxyl group at the 3'- or 5'-position is protected with a nucleoside phosphoramidite using tetrazole as an activator. Has been done.
  • a nucleoside in which the hydroxyl groups at the 3'- and 5'-positions are unprotected can be used in the method, the deprotection step becomes unnecessary, and the amount of reagents used is reduced, time and labor. It is expected that the cost will be significantly reduced.
  • An object of the present invention is to provide a method for producing an oligonucleotide and an activator for producing the oligonucleotide.
  • the present inventors are working diligently on a method capable of efficiently producing a desired oligonucleotide, and even when using a nucleoside in which both the 3'hydroxyl group and the 5'hydroxyl group are unprotected, the nucleoside is used.
  • a method for producing an oligonucleotide A nucleoside phosphoramidite in which a protecting group is bonded to a hydroxyl group at the 5'position and a nucleoside in which the hydroxyl groups at the 3'and 5'positions are unprotected are expressed in the formula (I) :.
  • R 1 , R 2 and R 3 are independently H, F, Cl, Br, CN, NO 2 , methoxy, cyclobutadiene, adamantan, linear or branched saturated or unsaturated C 1-22.
  • R 1 , R 2 and R 3 are independently H, F, Cl, Br, CN, NO 2 , methoxy, methyl, i-propyl, t-butyl, cyclobutadiene, adamantane, and optionally. Selected from the group consisting of phenyl, naphthyl, and anthracenyl which may be substituted with, or R 2 and R 3 together form a phenyl ring with two carbon atoms on the imidazole ring to which they are attached. The method according to [1], which is formed.
  • R 1 is the formula (II): During the ceremony R 4 and R 5 are independently selected from the group consisting of H, F, Cl, Br, CN, NO 2 , methyl, t-butyl, methoxy, i-propyl and adamantane, respectively. The method according to [1] or [2].
  • R 1 is phenyl and R 2 and R 3 are H.
  • B R 1 is t-butyl and R 2 and R 3 are H.
  • C R 1 is phenyl, and R 2 and R 3 together form a phenyl ring with two carbon atoms on the imidazole ring to which they are attached.
  • D R 1 is t-butyl, and R 2 and R 3 together form a phenyl ring with two carbon atoms on the imidazole ring to which they are attached.
  • E R 1 is phenyl and R 2 and R 3 are CN.
  • F R 1 is t-butyl and R 2 and R 3 are CN.
  • R 1 is Br, and R 2 and R 3 are CN.
  • H R 1 is 1-naphthyl and R 2 and R 3 are H.
  • I R 1 is 9-anthrasenyl and R 2 and R 3 are H.
  • J R 1 is Br, and R 2 and R 3 are H.
  • K R 1 is 2-chlorophenyl and R 2 and R 3 are H.
  • L R 1 is 2,6-dichlorophenyl and R 2 and R 3 are H.
  • M is pentafluorophenyl and R 2 and R 3 are H.
  • N R 1 is 2-chlorophenyl and R 2 and R 3 are phenyl.
  • O R 1 , R 2 and R 3 are Br.
  • R 1 is Br
  • R 2 is NO 2
  • R 3 is H
  • R 1 is 1-adamantyl
  • R 2 and R 3 are H
  • R 1 is Br
  • R 2 and R 3 are together and they are Forming a phenyl ring with two carbon atoms on the imidazole ring to be bonded
  • [5] The method according to any one of [1] to [4], wherein the activator forms a salt with an acid.
  • the acid is trifluoromethanesulfonic acid.
  • the oligonucleotide is of formula (III): Has a structure represented by The nucleoside phosphoramidite in which a protecting group is bonded to the hydroxyl group at the 5'position has the formula (IV): Has a structure represented by The nucleosides in which the hydroxyl groups at the 3'and 5'positions are unprotected have the formula (V) :.
  • R 6 is a protecting group
  • R 9 is an independently substituted or unsubstituted aliphatic group, substituted or unsubstituted aromatic group
  • Each X is independently one of a shared electron pair, O or S
  • Y is each independently one of H, NHR 10 , halogen, CN, CF 3 , or hydroxyl group protected by an acyl protecting group, an ether protecting group or a silyl protecting group
  • Each of R 10 is independently any of -H, a substituted or unsubstituted aliphatic group, a substituted or unsubstituted aromatic group, a substituted or unsubstituted aralkyl group, or a protecting group.
  • Z independently forms a ZZ bond with H, alkyl, O-alkyl, N-alkyl, halogen, or said Y; and n is an integer greater than or equal to 0 and less than or equal to 23; The method according to any one of [1] to [6].
  • Nucleoside phosphoramidite has the formula (VI): Has a structure represented by A step of producing an oligonucleotide by the method according to [7]. The method.
  • R 1 and R 3 are each independently, H, F, Cl, Br , CN, NO 2, methoxy, cyclobutadiene, adamantane, -XR A, optionally substituted with a group containing a hetero atom Selected from the group consisting of saturated or unsaturated C 1-22 alkyl groups, which may be linear or branched, and aromatic rings, which may be optionally substituted, where X is a heteroatom.
  • RA is a linear or branched saturated or unsaturated C 1-22 alkyl group, or R 2 and R 3 are phenyl together with two carbon atoms on the imidazole ring to which they are attached. Forming a ring, An activator represented by. [10] R 1 , R 2 and R 3 are independently H, F, Cl, Br, CN, NO 2 , methoxy, methyl, i-propyl, t-butyl, cyclobutadiene, adamantane, and optionally.
  • R 1 is the equation (II): During the ceremony R 4 and R 5 are independently selected from the group consisting of H, F, Cl, Br, CN, methyl, t-butyl, methoxy, i-propyl and adamantane, respectively. The activator according to [9] or [10]. [12] (a) R 1 is phenyl and R 2 and R 3 are H. (B) R 1 is t-butyl and R 2 and R 3 are H. (C) R 1 is phenyl, and R 2 and R 3 together form a phenyl ring with two carbon atoms on the imidazole ring to which they are attached.
  • R 1 is t-butyl, and R 2 and R 3 together form a phenyl ring with two carbon atoms on the imidazole ring to which they are attached.
  • R 1 is phenyl and R 2 and R 3 are CN.
  • R 1 is t-butyl and R 2 and R 3 are CN.
  • R 1 is Br, and R 2 and R 3 are CN.
  • H R 1 is 1-naphthyl and R 2 and R 3 are H.
  • R 1 is 9-anthrasenyl and R 2 and R 3 are H.
  • R 1 is Br, and R 2 and R 3 are H.
  • R 1 is 2-chlorophenyl and R 2 and R 3 are H.
  • L R 1 is 2,6-dichlorophenyl and R 2 and R 3 are H.
  • M R 1 is pentafluorophenyl and R 2 and R 3 are H.
  • N R 1 is 2-chlorophenyl and R 2 and R 3 are phenyl.
  • O R 1 , R 2 and R 3 are Br.
  • P R 1 is Br, R 2 is NO 2 , and R 3 is H.
  • R 1 is 1-adamantyl
  • R 2 and R 3 are H
  • R 1 is Br
  • R 2 and R 3 are together and they are Forming a phenyl ring with two carbon atoms on the imidazole ring to be bonded
  • the activator according to [9] or [10] which is selected from the group consisting of.
  • the activator according to [13], wherein the acid is trifluoromethanesulfonic acid.
  • the activator of the present invention By using the activator of the present invention, even when a nucleoside in which the hydroxyl groups at the 3'- and 5'positions are unprotected can be used, the 5'hydroxyl group of the nucleoside can be selectively reacted, which is desired. Oligonucleotides can be efficiently produced. Therefore, by using the activator of the present invention, it becomes possible to use a nucleoside in which the hydroxyl groups at the 3'- and 5'positions are unprotected, and the deprotection step becomes unnecessary.
  • the present invention is a method for producing an oligonucleotide, in which a nucleoside phosphoramidite in which a protecting group is bonded to a hydroxyl group at the 5'position and a nucleoside in which the hydroxyl groups at the 3'and 5'positions are unprotected.
  • the present invention relates to the above method, which comprises a step of reacting with and in the presence of an activator.
  • an oligonucleotide is a so-called phosphorotide in which nucleotides are added by a condensation reaction between a nucleoside phosphoramidite and a nucleoside in solution or on a solid support in the presence of a suitable activator. It is performed using the amidite method.
  • the oligonucleotide refers to a compound having a structure in which a nucleoside base, a sugar, and a phosphoric acid are linked by a phosphodiester bond, and a naturally occurring oligonucleotide, for example, 2'-deoxyribonucleic acid (hereinafter, "DNA”).
  • DNA 2'-deoxyribonucleic acid
  • RNA ribonucleic acid
  • RNA nucleic acid containing a modified sugar moiety, a modified phosphate moiety, or a modified nucleobase. Modifications to the sugar moiety include replacing the ribose ring with a hexose, cyclopentyl, or cyclohexyl ring.
  • the D-ribose ring of a naturally occurring nucleic acid may be replaced with an L-ribose ring, or the ⁇ -anomer of a naturally occurring nucleic acid may be replaced with an ⁇ -anomer.
  • Oligonucleotides may also contain one or more non-basic moieties. Modified phosphate moieties include phosphorothioate, phosphorodithioate, methylphosphonate, and methyl phosphate. Such nucleic acid analogs are known to those of skill in the art.
  • Oligonucleotides comprising the above two or more mixtures include, for example, mixtures of deoxyribo and ribonucleosides, particularly 2'-O such as deoxyribonucleoside and 2'-O-methyl or 2'-O-methoxyethylribonucleoside.
  • Examples of oligonucleotides comprising a mixture of nucleosides include ribozymes.
  • the oligonucleotides are of formula (III) :. It has a structure represented by.
  • the nucleoside phosphoramidite refers to a nucleoside derivatized with amidite.
  • the 3'hydroxyl group of the nucleoside is phosphoramidite-ized, and a protecting group is bonded to the 5'hydroxyl group.
  • Amiditeization is carried out, for example, by reacting a properly protected nucleoside with 2-cyanoethyl-N, N, N', N'-tetraisopropylphosphorodiamidite using 1H-tetrazole as an activator. be able to.
  • the nucleoside phosphoramidite may be a monomer or an oligomer such as 2 mer to 24 mer.
  • the nucleoside phosphoramidite is of formula (IV): It has a structure represented by.
  • the nucleotide refers to a compound in which a nucleoside base, a sugar, and phosphoric acid are bound, and such as adenosine triphosphate, thymidin triphosphate, guanosine triphosphate, citidine triphosphate, and uridine triphosphate. It may be a naturally occurring nucleotide or a modified nucleotide.
  • the nucleoside base portion of the nucleotide may be a naturally occurring base such as adenine, guanine, cytosine, thymine, and uracil, or a modified nucleoside base.
  • the sugar moiety of the nucleoside may be naturally occurring deoxyribose or ribose, and may be in the D or L configuration.
  • the phosphoric acid moiety may be, for example, phosphorothioate, phosphorodithioate, methylphosphonate, and methyl phosphate.
  • the nucleoside refers to a compound in which a nucleoside base and a sugar are bound, and may be a naturally occurring nucleoside such as adenosine, thymidine, guanosine, cytidine, or uridine, or a modified nucleoside.
  • the nucleoside base may be a naturally occurring base such as adenine, guanine, cytosine, thymine, and uracil, or a modified nucleoside base.
  • the sugar moiety of the nucleoside may be naturally occurring deoxyribose or ribose, and may be in the D or L configuration.
  • the nucleoside in which the hydroxyl groups at the 3'-position and the 5'-position are unprotected refers to a nucleoside in which the hydroxyl groups at the 3'-position and the 5'-position are not protected by any protecting group.
  • the nucleosides in which the hydroxyl groups at the 3'and 5'positions are unprotected are of formula (V): It has a structure represented by.
  • B is an independently protected or unprotected nucleoside base.
  • Nucleoside bases include, but are not limited to, naturally occurring bases such as, for example, adenine, guanine, cytosine, thymine, and uracil, 7-deazaguanine, 7-deaza-8-azaguanine, 5-propynylcytocin, 5 -Propinyl uracil, 7-deazaadenine, 7-deaza-8-azaadenine, 7-daza-6-oxopurine, 6-oxopurine, 3-deazaadenosin, 2-oxo-5-methylpyrimidine, 2-oxo-4-methylthio -5-Methylpyrimidine, 2-thiocarbonyl-4-oxo-5-methylpyrimidine, 4-oxo-5-methylpyrimidine, 2-aminopurine, 5-fluorouracil, 2,6-diaminopurine, 8-amin
  • the nucleoside base protected by a protecting group is a nucleoside base in which the reactive functional group of the base is protected.
  • the nucleoside base has an amine group that can be protected with an amine protecting group, such as an amide or carbamate.
  • an amine protecting group such as an amide or carbamate.
  • the amine groups of adenin and cytosine are typically protected with a benzoyl protecting group
  • the amine groups of guanine are typically protected with an isobutyryl group, an acetyl group, or a t-butylphenoxyacetyl group. ..
  • other protection schemes may be used.
  • the amine groups of adenine and guanine are protected with phenoxyacetyl groups and the amino groups of cytosine are protected with isobutyryl or acetyl groups.
  • the conditions for removing the protecting group depend on the protecting group used. If an amide protecting group is used, it can be removed by treating the oligonucleotide with a base solution such as concentrated ammonium hydroxide solution, n-methylamine solution, or ammonium hydroxide solution of t-butylamine.
  • R 6 is a protecting group, is preferably an acid labile protecting group or t- butyldimethylsilyl or a trialkylsilyl group such as triisopropylsilyl, .
  • Acid-labile protecting groups are protecting groups that can be removed by contacting the group with a protonic acid or Lewis acid. Acid-labile protecting groups are known to those of skill in the art. Examples of acid-unstable protecting groups include substituted or unsubstituted trityl groups, substituted or unsubstituted tetrahydropyranyl groups, substituted or unsubstituted tetrahydrofuranyl groups, pixyl groups and the like.
  • the trityl group is usually replaced by an electron donating group such as an alkoxy group.
  • R 1 is a substituted or unsubstituted trityl, 9- (phenyl) xanthenyl (hereinafter “Pixyl”) or tetrahydropyranyl (hereinafter “THP”).
  • R 6 is unsubstituted trityl, monoalkoxy trityl, dialkoxy trityl, trialkoxy trityl, a THP or pixyl. Most preferably, R 1 is 4,4'-dimethoxytrityl.
  • R 9 is a substituted or unsubstituted aliphatic group and a substituted or unsubstituted aromatic group, respectively.
  • Substituted or unsubstituted aliphatic groups include, but are not limited to, methyl, ethyl, isopropyl, pyrrolidino and morpholino, with preference given to ethyl, isopropyl and morpholino.
  • Substituted or unsubstituted aromatic groups include, but are not limited to, phenyl, benzyl, toluyl, and aniryl, preferably phenyl and benzyl.
  • R 9 is isopropyl.
  • X is independently one of a shared electron pair, O or S, respectively.
  • Y is independently H, NHR 10 , halogen, CN, CF 3 , or acyl-protecting group, ether-protecting group or silyl-protecting group. It is one of the hydroxyl groups protected by a group.
  • Halogen is, for example, F, Cl, Br, and I.
  • Examples of the acyl-based protecting group include acetyl, benzoyl, pivaloyl and the like.
  • Examples of the ether-based protecting group include benzyl, p-methoxybenzyl (PMB), allyl and the like.
  • Examples of the silyl protecting group include t-butyldimethylsilyl (TBS), t-butyldiphenylsilyl (TBDPS), t-triisopropylsilyl (TIPS), triethylsilyl (TES), trimethylsilyl (TMS) and the like. ..
  • Each of R 10 is independently composed of —H, a substituted or unsubstituted aliphatic group, a substituted or unsubstituted aromatic group, a substituted or unsubstituted aralkyl group, or an acyl group. It is one of the protective groups to be used.
  • Substituted or unsubstituted aliphatic groups include, but are not limited to, methyl, ethyl, allyl, 1-pentenyl, and 2-methoxyethyl, preferably methyl, allyl, and 2-methoxyethyl. is there.
  • Substituted or unsubstituted aromatic groups include, but are not limited to, phenyl, benzyl, naphthyl, 2-pyrenylmethyl, and are preferably phenyl and benzyl.
  • the protecting group is, for example, t-butyldimethylsilyl.
  • Y is H.
  • Z independently forms a ZZ bond with H, alkyl, O-alkyl, N-alkyl, halogen, or Y.
  • Halogen is, for example, F, Cl, Br, and I.
  • n is an integer of 0 or more and 23 or less, for example, 0 or more and 21 or less, 0 or more and 20 or less, 0 or more and 19 or less, 0 or more and 18 or less, 0 or more and 17 or less, 0 or more and 16 or less, 0 or more and 15 or less, 0 or more and 14 or less, 0 or more and 13 or less, 0 or more and 12 or less, 0 or more and 11 or less, 0 or more and 10 or less, 0 or more and 9 or less, 0 or more and 8 or less, 0 or more and 7 or less, 0 or more and 6 or less, 0 or more and 5 or less, 0 or more and 4 or less, 0 or more and 3 or less, 0 or more and 2 or less, 0 or more and 1 or less, or 0.
  • the activator is used for reacting a nucleoside phosphoramidite with a nucleoside, and is also referred to as an activator or a coupling agent.
  • the activator is of formula (I) :.
  • R 1 , R 2 and R 3 are independently H, F, Cl, Br, CN, NO 2 , methoxy, cyclobutadiene, adamantan, -XR. From the group consisting of A, a linear or branched saturated or unsaturated C 1-22 alkyl group optionally substituted with a heteroatom-containing group, and an optionally substituted aromatic ring.
  • X is a heteroatom
  • RA is a linear or branched saturated or unsaturated C 1-22 alkyl group, or R 2 and R 3 are together, they. Form a phenyl ring with two carbon atoms on the imidazole ring to which the is bonded.
  • X is preferably a heteroatom selected from the group consisting of O, N, S, N, Se and Si, preferably S.
  • the group containing a heteroatom is preferably a CN group.
  • Saturated or unsaturated C 1-22 alkyl groups of linear or branched chains include, but are not limited to, methyl, ethyl, i-propyl, and t-butyl, with preference given to methyl.
  • R 1 , R 2 and R 3 are independently H, F, Cl, Br, CN, NO 2 , methoxy, methyl, i-propyl, t. -Selected from the group consisting of butyl, cyclobutadiene, adamantane, and optionally substituted phenyl, naphthyl, and anthracenyl, or R 2 and R 3 together, the imidazole ring to which they are attached. It forms a phenyl ring with the above two carbon atoms.
  • phenyl examples include, but are not limited to, phenyl, benzyl, naphthyl, and 2-pyrenylmethyl, and preferably phenyl and benzyl.
  • R 1 is Br, phenyl, chlorophenyl, or naphthyl
  • R 2 is H, Br, or NO 2
  • R 3 is H or Br, or R 2. and R 3, taken together, along with the two carbon atoms on the imidazole ring to which they are attached form a phenyl ring.
  • R 1 is the formula (II): Is.
  • R 4 and R 5 each independently consist of H, F, Cl, Br, CN, methyl, t-butyl, methoxy, i-propyl and adamantane. Selected from the group.
  • the activator is represented by structure of formula (I), in formula (I), R 1 is phenyl, R 2 and R 3 is H. In one aspect of the present invention, the activator is represented by structure of formula (I), in formula (I), R 1 is a t- butyl, R 2 and R 3 is H. In one aspect of the invention, the activator is represented by the structure of formula (I), where in formula (I) R 1 is phenyl and R 2 and R 3 are combined and they are bound together. Form a phenyl ring with two carbon atoms on the imidazole ring.
  • the activator is represented by the structure of formula (I), R 1 is t-butyl, and R 2 and R 3 are together on the imidazole ring to which they are attached. Form a phenyl ring with the two carbon atoms of.
  • the activator is represented by the structure of formula (I), R 1 is phenyl, R 2 and R 3 is CN.
  • the activator is represented by the structure of formula (I), where R 1 is t-butyl and R 2 and R 3 are CN.
  • the activator is represented by the structure of formula (I), where R 1 is Br and R 2 and R 3 are CN.
  • the activator is represented by the structure of formula (I), R 1 is a 1-naphthyl, R 2 and R 3 is H. In one aspect of the present invention, the activator is represented by the structure of formula (I), R 1 is a 9-anthracenyl, R 2 and R 3 is H. In one aspect of the present invention, the activator is represented by the structure of formula (I), R 1 is a Br, R 2 and R 3 is H. In one aspect of the present invention, the activator is represented by the structure of formula (I), R 1 is a 2-chlorophenyl, R 2 and R 3 is H.
  • the activator is represented by the structure of formula (I), R 1 is a 2,6-dichlorophenyl, R 2 and R 3 is H. In one aspect of the present invention, the activator is represented by the structure of formula (I), R 1 is a pentafluorophenyl, R 2 and R 3 is H. In one aspect of the invention, the activator is represented by the structure of formula (I), where R 1 is 2-chlorophenyl and R 2 and R 3 are phenyl. In one aspect of the present invention, the activator is represented by the structure of formula (I), R 1, R 2 and R 3 is Br.
  • the activator is represented by the structure of formula (I), where R 1 is Br, R 2 is NO 2 and R 3 is H. In one aspect of the present invention, the activator is represented by the structure of formula (I), R 1 is a 1-adamantyl, R 2 and R 3 is H. In one aspect of the invention, the activator is represented by the structure of formula (I), where R 1 is Br and R 2 and R 3 are together and 2 on the imidazole ring to which they are attached. It forms a phenyl ring with one carbon atom.
  • the activator is 2-phenylimidazole, 2-naphthylimidazole, 2-bromoimidazole, 2- (2-chlorophenyl) -4,5-diphenylimidazole, 2,4,5-tribromo. It is imidazole, 2-bromo-4-nitroimidazole, or 2-bromobenzimidazole.
  • the activator is 2-phenylimidazole, 2-naphthylimidazole, 2,4,5-tribromoimidazole, 2-bromo-4-nitroimidazole, or 2-bromobenzimidazole. ..
  • the activator forms a salt with an acid.
  • the acid may be any proton acid or Lewis acid, and is not limited to, for example, methanesulfonic acid, toluenesulfonic acid, trifluoromethanesulfonic acid, hexafluorophosphate, tetrafluoride. It may be boron acid or boron trifluoride, preferably trifluoromethanesulfonic acid.
  • the activator is represented by structure of formula (I), in formula (I), R 1 is phenyl, R 2 and R 3 are H, trifluoromethanesulfonic acid And forming salt.
  • the activator is represented by structure of formula (I), in formula (I), R 1 is a t- butyl, R 2 and R 3 are H, trifluoromethane It forms a salt with sulfonic acid.
  • the activator is represented by structure of formula (I), in formula (I), R 1 is phenyl, R 2 and R 3 together, they are bound It forms a phenyl ring with two carbon atoms on the imidazole ring, and forms a salt with trifluoromethanesulfonic acid.
  • the activator is represented by the structure of formula (I), where R 1 is t-butyl and R 2 and R 3 are together on the imidazole ring to which they are attached. It forms a phenyl ring with the two carbon atoms of, and forms a salt with trifluoromethanesulfonic acid.
  • the activator is represented by the structure of formula (I), where R 1 is phenyl and R 2 and R 3 are CN, forming a salt with trifluoromethanesulfonic acid.
  • the activator is represented by the structure of formula (I), where R 1 is t-butyl and R 2 and R 3 are CN, forming a salt with trifluoromethanesulfonic acid.
  • the activator is represented by the structure of formula (I), where R 1 is Br and R 2 and R 3 are CN, forming a salt with trifluoromethanesulfonic acid.
  • the activator is represented by the structure of formula (I), R 1 is a 1-naphthyl, R 2 and R 3 is H, forming a trifluoromethanesulfonic acid salt doing.
  • the activator is represented by the structure of formula (I), where R 1 is 9-anthrasenyl and R 2 and R 3 are H, forming a salt with trifluoromethanesulfonic acid. doing.
  • the activator is represented by the structure of formula (I), where R 1 is Br, R 2 and R 3 are H, forming a salt with trifluoromethanesulfonic acid. There is.
  • the activator is represented by the structure of formula (I), R 1 is a 2-chlorophenyl, R 2 and R 3 is H, forming a trifluoromethanesulfonic acid salt doing. In one aspect of the present invention, the activator is represented by the structure of formula (I), R 1 is a 2,6-dichlorophenyl, R 2 and R 3 are H, trifluoromethanesulfonic acid and salts Is forming. In one aspect of the present invention, the activator is represented by the structure of formula (I), R 1 is a pentafluorophenyl, R 2 and R 3 is H, forming a trifluoromethanesulfonic acid salt doing.
  • the activator is represented by the structure of formula (I), where R 1 is 2-chlorophenyl and R 2 and R 3 are phenyl, forming a salt with trifluoromethanesulfonic acid. ing.
  • the activator is represented by the structure of formula (I), R 1, R 2 and R 3 are Br, to form a trifluoromethanesulfonic acid salt.
  • the activator is represented by the structure of formula (I), where R 1 is Br, R 2 is NO 2 , R 3 is H, and trifluoromethanesulfonic acid. Forming salt.
  • the activator is represented by the structure of formula (I), R 1 is a 1-adamantyl, R 2 and R 3 is H, forming a trifluoromethanesulfonic acid salt doing.
  • the activator is represented by the structure of formula (I), where R 1 is Br and R 2 and R 3 are together and 2 on the imidazole ring to which they are attached. It forms a phenyl ring with one carbon atom and forms a salt with trifluoromethanesulfonic acid.
  • the activator is 2-phenylimidazolium triflate, 2-naphthylimidazolium triflate, 2-bromoimidazolium triflate, 2- (2-chlorophenyl) -4,5-diphenylimidazolium triflate, 2,4,5-Tribromoimidazolium triflate, 2-bromo-4-nitroimidazolium triflate, or 2-bromobenzoimidazolium triflate.
  • the activator is 2-phenylimidazolium triflate, 2-naphthylimidazolium triflate, 2,4,5-tribromoimidazolium triflate, 2-bromo-4-nitroimidazolium triflate, Or 2-bromobenzoimidazolium triflate.
  • the present invention relates to, in one aspect, a method for producing a nucleoside phosphoramidite, which comprises a step of producing an oligonucleotide.
  • the nucleoside phosphoramidite is of formula (VI): It has a structure represented by.
  • B, R 6 , R 7 , R 8 , R 9 , X, Y, R 10 , Z, n are as defined in formula (III), formula (IV) and formula (V). Is.
  • the present invention in one aspect, relates to an activator for producing oligonucleotides.
  • the activator is of formula (I) :. It has a structure represented by. R 1 , R 2 and R 3 are as defined above.
  • reaction between phosphoramidite and nucleoside is carried out in the presence of a suitable solvent.
  • Example 1 Synthesis example of activator 1.1 Preparation of 2-phenylimidazolium triflate A dichloromethane solution (333 mL) of 2-phenylimidazole (24.0 g, 167 mmol) was ice-cooled, and trifluoromethanesulfonic acid (42.8 mL, 167 mmol) was added dropwise. After stirring at room temperature for 30 minutes, the formed white crystals were separated by suction and washed with MTBE (150 mL). The obtained crystals were dried under reduced pressure for 15 hours to obtain 2-phenylimidazolium triflate as white crystals (48.5 g, yield 98.9%).
  • Example 1.2 Preparation of 2-phenylbenzoimidazolium triflate
  • 2-phenylbenzoimidazolium triflate A dichloromethane solution (40 mL) of 2-phenylbenzimidazole (3.88 g, 20.0 mmol) was ice-cooled, and trifluoromethanesulfonic acid (1.75 mL, 20.0 mmol) was added dropwise. After stirring at room temperature for 30 minutes, the reaction solution is concentrated, MTBE-hexane 1: 1 solution (50 mL) is added to the crude product in oil form with stirring, and the white crystals produced are separated by suction and MTBE. It was washed with a 1: 1 solution of -hexane (50 mL). The obtained crystals were dried under reduced pressure for 15 hours to obtain 2-phenylbenzoimidazolium triflate as white crystals (6.80 g, yield 97.4%).
  • Example 2.1 Synthesis of oligonucleotides using 2-phenylimidazolium triflate as an activator 1.0 M dichloromethane-DMF solution (100 ⁇ L) of 2-phenylimidazolium triflate prepared in Example 1.1, of deoxynucleoside A 2.0 M DMF solution (100 ⁇ L) was mixed, and this solution was added dropwise to a 0.5 M dichloromethane solution (100 ⁇ L) of 2-cyanoethyl 5'-O-deoxynucleoside- (N, N'-diisopropyl) phosphoramidite. The reaction was carried out by stirring at room temperature for 30 minutes.
  • the reaction mixture was diluted 500-fold and subjected to HPLC (equipment: UPLCBio manufactured by Waters, column: BHE C18 50 mm, eluent: acetonitrile solution of 0.1 M TEAA buffer over 5 minutes 50 ⁇ 100% linear gradient). It was.
  • the reaction rate is calculated from the HPLC peak area of (target product + impurities + hydrolyzate) ⁇ (target product + impurities + hydrolyzate + remaining raw material) HPLC peak area, and the selectivity is the HPLC peak of the target product.
  • the target substance refers to a compound in which the 5'-position hydroxyl group of the nucleoside is bonded to phosphoramidite, and the impurity is the 3'-position hydroxyl group of the nucleoside to phosphoramidite. Refers to bound compounds; the same shall apply hereinafter).
  • Example 2.2 Synthesis of oligonucleotides using imidazole derivatives as activators Imidazole derivatives (2-naphthylimidazole, 2-bromoimidazole, 2- (2-chlorophenyl) -4,5-diphenylimidazole, 2,4 A 1.0 M dichloromethane-DMF solution (100 ⁇ L) of 5-tribromoimidazole, 2-bromo-4-nitroimidazole, or 2-bromobenzimidazole, a 0.5 M dichloromethane solution of trifluoromethanesulfonic acid (TfOH) (0, 30 or 90 ⁇ L) and a 2.0 M DMF solution of deoxynucleoside (100 ⁇ L) were mixed to obtain a solution of 2-cyanoethyl 5'-O-deoxynucleoside- (N, N'-diisopropyl) phosphoramidite.
  • TfOH trifluoromethanesulfonic acid
  • the reaction was carried out by dropping into a 0.5 M dichloromethane solution (100 ⁇ L) and stirring at room temperature for 30 minutes.
  • the reaction mixture was diluted 500-fold and subjected to HPLC (equipment: UPLCBio manufactured by Waters, column: BHE C18 50 mm, eluent: acetonitrile solution of 0.1 M TEAA buffer over 5 minutes 50 ⁇ 100% linear gradient). It was.
  • the reaction rate is calculated from the HPLC peak area of (target product + impurities + hydrolyzate) ⁇ (target product + impurities + hydrolyzate + remaining raw material) HPLC peak area, and the selectivity is the HPLC peak of the target product.
  • Area Calculated from the HPLC peak area of impurities.
  • Example 2.3 Synthesis of oligonucleotides using 1H-tetrazole (Comparative Example) as an activator 2.5M dichloromethane-DMF solution (100 ⁇ L) of 1H-tetrazole and 2.0M DMF solution (100 ⁇ L) of deoxynucleoside Is added dropwise to a 0.5 M dichloromethane solution (100 ⁇ L) of 2-cyanoethyl 5'-O-deoxynucleoside- (N, N'-diisopropyl) phosphoramidite, and the mixture is stirred at room temperature for 30 minutes. It was reacted by.
  • the reaction mixture was diluted 500-fold and subjected to HPLC (equipment: UPLCBio manufactured by Waters, column: BHE C18 50 mm, eluent: acetonitrile solution of 0.1 M TEAA buffer over 5 minutes 50 ⁇ 100% linear gradient). It was.
  • the reaction rate is calculated from the HPLC peak area of (target product + impurities + hydrolyzate) ⁇ (target product + impurities + hydrolyzate + remaining raw material) HPLC peak area, and the selectivity is the HPLC peak of the target product.
  • Area Calculated from the HPLC peak area of impurities.
  • Example 2.4 The evaluation results are shown in Table 1.
  • DMTr-A (OCH3) p (OCH2CH2CN) T (OH) (A (OMe) T) No.
  • the activators described in 1 to 8 were used, the reactivity was higher than when 1H-tetrazole was used as the activator.
  • DMTr-A (OH) p (OCH2CH2CN) A (OH) (AA) No.
  • the activators described in 1 to 4 and 6 to 8 were used, the reactivity was higher and the selectivity was also excellent than when 1H-tetrazole was used as the activator. Since these activators have greater steric hindrance than tetrazole, it is considered that they were excellent in selectivity.
  • the reaction solution was added dropwise to pure water (1.2 L), the obtained organic phase was washed with a saturated sodium bicarbonate solution (100 mL x 2 times), the organic phase was transferred to 300 mL Meyer, and then dried over Glauber's salt for 30 minutes.
  • the organic phase was concentrated under reduced pressure to give a crude product (25.7 g).
  • the obtained crude product was dissolved in dichloromethane (260 mL) and then purified by silica gel column chromatography using dichloromethane-methanol as an elution solvent to obtain the desired dimer (Compound 1) (15.9 g, yield). Rate 72.3%).
  • the reaction solution was added dropwise to pure water (1.6 L), the obtained organic phase was washed with a saturated sodium bicarbonate solution (100 mL x 2 times), the organic phase was transferred to 300 mL Meyer, and then dried over Glauber's salt for 30 minutes.
  • the organic phase was concentrated under reduced pressure to give a crude product (25.7 g).
  • the obtained crude product was dissolved in dichloromethane (260 mL) and then purified by silica gel column chromatography using dichloromethane-methanol as an elution solvent to obtain the desired dimer (Compound 1) (12.0 g, yield). Rate 51.8%).
  • Dissolve dAT (Compound 1) (9.90 g, 9.60 mmol) in dichloromethane solution (50 mL) and amidite reagent NCCH 2 CH 2 OP [N (i-C 3 H 7 ) 2 ] 2 (4.00 mL, 12.5 mmol) and 1H-tetrazole (470 mg, 6.70 mmol) were added in two portions and stirred at room temperature for 1 hour.
  • Dichloromethane (50 mL) was added to the reaction solution, and the mixture was purified by silica gel column chromatography using n-hexane-dichloromethane as an elution solvent to obtain the desired phosphoromidite (Compound 2) (9.02 g, yield 76). .4%).
  • the reaction solution was added dropwise to pure water (1.5 L), the obtained organic phase was washed with a saturated sodium bicarbonate solution (100 mL x 2 times), the organic phase was transferred to 300 mL Meyer, and then dried over Glauber's salt for 30 minutes.
  • the organic phase was concentrated under reduced pressure to give a crude product (29.3 g).
  • the obtained crude product was dissolved in dichloromethane (290 mL) and then purified by silica gel column chromatography using dichloromethane-methanol as an elution solvent to obtain the desired dimer (Compound 4) (13.6 g, yield). Rate 53.5%).
  • Table 2 shows the yields of (OCH3) p (OCH2CH2CN) T (OCH2CH2CN) dC (OH) (Compound 3) and DMTr-dG p (OCH2CH2CN) C (OCH2CH2CN) C (OH) (Compound 6).

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Abstract

L'objectif de la présente invention est de fournir un procédé de production d'un oligonucléotide et de fournir un agent d'activation pour produire l'oligonucléotide. Le problème mentionné ci-dessus est résolu par un procédé de production d'un oligonucléotide, ledit procédé comprenant une étape dans laquelle un phosphoramidite nucléosidique ayant un groupe protecteur lié à un groupe hydroxyle en position 5' et un nucléoside ayant un groupe hydroxyle non protégé en position 3' et en position 5' sont mis à réagir en présence d'un agent d'activation représenté par la formule (I).
PCT/JP2020/040096 2019-10-24 2020-10-26 Procédé de production d'oligonucléotide Ceased WO2021080021A1 (fr)

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