Classifications

• • 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
  • 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
  • C07H1/00—Processes for the preparation of sugar derivatives
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H1/00—Processes for the preparation of sugar derivatives
  • C07H1/02—Phosphorylation
• • 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 invention relates to a method for producing oligonucleotides.
• the phosphoramidite method is widely used in the chemical synthesis of nucleic acids such as DNA oligonucleotides and RNA oligonucleotides.
• oligonucleotides are typically synthesized by the sequential addition of nucleoside phosphoramidites to nucleosides, nucleotides or oligonucleotides in the presence of a suitable activating agent.
• the nucleoside phosphoramidite is generally used in an amount 1.5 to 10.0 times the theoretical amount. Nucleoside phosphoramidites are expensive among synthetic raw materials, so if the amount of nucleoside phosphoramidites used can be reduced, a significant reduction in production costs is expected.
• Patent Document 1 describes that the amount of the activating agent to be used is suitably 1 to 20 times the amount of the nucleoside derivative, and preferably 1 to 10 times the molar amount.
• the amount of the activating agent to be used is suitably 1 to 20 times the amount of the nucleoside derivative, and preferably 1 to 10 times the molar amount.
• the purpose of the present invention is to provide a method of suppressing the residual amount of nucleoside phosphoramidites in a reaction vessel and reducing the amount of nucleoside phosphoramidites used in a method of producing oligonucleotides.
• the present inventors have been intensively researching a method for producing oligonucleotides, and have found that by increasing the amount of the activating agent used, the amount of nucleoside phosphoramidite remaining in the reaction vessel in the synthesis process can be suppressed, and the nucleoside We have found that the amount of phosphoramidite used can be reduced. As a result of further research based on such knowledge, the present invention was completed.
• the present invention relates to the following.
• a method for producing an oligonucleotide wherein, in the presence of an activating agent, a , the step of binding a nucleoside phosphoramidite, wherein the amount of the activating agent used in the step is 10.0 to 15.0 times equivalent to the amount of the nucleoside phosphoramidite used in the step.
• the activator is selected from the group consisting of 4,5-dicyanoimidazole, 5-(ethylthio)-1H-tetrazole, 5-(benzylthio)-1H-tetrazole and saccharin 1-methylimidazole, [1 ] The method according to any one of [3]. [5] The method according to any one of [1] to [4], wherein the temperature of the solution in the step is 0 to 20°C.
• the nucleoside phosphoramidite remains in the reaction vessel due to the unintentional removal of the 2-cyanoethyl (CNET) protecting group of the phosphate moiety of the oligonucleotide.
• CNET 2-cyanoethyl
• a nucleoside phosphoramidite binds to the formed hydroxyl group in the coupling step, resulting in a chain elongation reaction (reaction with the 5' or 3' terminal hydroxyl group of the nucleoside supported directly or indirectly on the carrier)
• a chain elongation reaction reaction with the 5' or 3' terminal hydroxyl group of the nucleoside supported directly or indirectly on the carrier.
• FIG. 1 shows the in-column residual ratio and purity in Comparative Example 1, Example 1, Example 2 and Example 3.
• FIG. 2 shows the in-column residual ratio and purity in Comparative Example 2, Example 4, Example 5 and Example 6.
• 3 shows the in-column retention rate and purity in Comparative Example 3 and Example 7.
• FIG. 4 shows the in-column retention rate and purity in Comparative Example 4, Example 8 and Example 9.
• oligonucleotides are produced by the so-called phosphoramidite method, in which nucleotide addition is performed by condensation reaction of nucleoside phosphoramidites with nucleosides, nucleotides or oligonucleotides in the presence of a suitable activating agent. performed using
• the method for producing an oligonucleotide includes, for example, (a) removing the protecting group from the protected nucleoside directly or indirectly carried on the carrier and having the protecting group bound to the hydroxyl group, thiol group or amino group at the 3′- or 5′-position (deprotection ), (b) in the presence of an activating agent, a nucleoside phosphoramidite is attached to the hydroxyl group, thiol group or amino group at the 3'- or 5'-position of the nucleoside supported directly or indirectly on the carrier from which the protective group has been removed; A step of coupling with (c) sulfurating or oxidizing the bond formed by step (b); and (d) unbound 3′ or 5′ hydroxyl group, thiol in the nucleoside supported directly or indirectly on the support. capping the group or amino group; may include
• the method for producing an oligonucleotide may include further steps in addition to the above (a) to (d).
• a method for producing an oligonucleotide comprises: in the presence of an activating agent, a hydroxyl group, a thiol group or an amino The step of attaching a nucleoside phosphoramidite to the group is included.
• a nucleoside refers to a compound in which a nucleoside base and a sugar are linked, and may be naturally occurring nucleosides such as adenosine, thymidine, guanosine, cytidine, uridine, or modified nucleosides.
• modified nucleosides include, but are not limited to, those in which the hydroxyl group at the 3'- or 5'-position of the nucleoside is substituted with a thiol group or an amino group.
• Nucleoside bases may be naturally occurring bases such as adenine, guanine, cytosine, thymine, and uracil, or modified nucleoside bases.
• the sugar moieties of the nucleosides may be naturally occurring deoxyribose or ribose, and may have the D or L configuration.
• a nucleotide refers to a compound in which a nucleoside base, a sugar and a phosphate are linked, and includes adenosine triphosphate, thymidine triphosphate, guanosine triphosphate, cytidine triphosphate, and uridine triphosphate. It may be a naturally occurring nucleotide or a modified nucleotide.
• the nucleoside base portion of the nucleotide may be naturally occurring bases such as adenine, guanine, cytosine, thymine, and uracil, or modified nucleoside bases.
• the sugar moieties of the nucleosides may be naturally occurring deoxyribose or ribose, and may have the D or L configuration.
• Phosphate moieties can be, for example, phosphorothioates, phosphorodithioates, methylphosphonates, and methyl phosphates.
• an oligonucleotide refers to a compound having a structure in which nucleoside bases, sugars and phosphoric acids are linked by phosphodiester bonds, and naturally occurring oligonucleotides such as 2'-deoxyribonucleic acid (hereinafter referred to as "DNA”). and ribonucleic acids (hereinafter "RNA"), and nucleic acids containing modified sugar moieties, modified phosphate moieties, or modified nucleobases. Modifications to the sugar moiety include replacing the ribose ring with a hexose, cyclopentyl, or cyclohexyl ring.
• the D-ribose ring of naturally occurring nucleic acids may be replaced with an L-ribose ring, or the ⁇ -anomer of naturally occurring nucleic acids may be replaced with an ⁇ -anomer.
• Oligonucleotides may also contain one or more non-basic moieties. Modified phosphate moieties include phosphorothioates, phosphorodithioates, methylphosphonates, and methyl phosphates. Such nucleic acid analogues are known to those of skill in the art.
• Oligonucleotides comprising mixtures of two or more of the above are, for example, mixtures of deoxyribonucleosides and ribonucleosides, particularly deoxyribonucleosides and 2'-O-methyl or 2'-O-methyl or 2'-O-methoxyethyl ribonucleosides such as ribonucleosides.
• - can be produced from oligonucleotides comprising mixtures of substituted ribonucleosides; Examples of oligonucleotides comprising mixtures of nucleosides include ribozymes.
• nucleoside phosphoramidites refer to nucleosides derivatized with amidites.
• the nucleoside phosphoramidite is obtained by phosphoramidating either the 3′-hydroxyl group or the 5′-hydroxyl group of the nucleoside, and a protecting group is attached to the other. Amiditation is carried out by, for example, using 1H-tetrazole as an activating agent and reacting an appropriately protected nucleoside with 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite. be able to.
• Nucleoside phosphoramidites can be monomeric or oligomeric such as 2-24mers.
• an activator refers to an agent that activates nucleoside phosphoramidites, is used to react with nucleosides, nucleotides or oligonucleotides, and is also called an activator or a coupling agent.
• activators commonly used in the phosphoramidite method can be used.
• Activators used in the present invention include, but are not limited to, 4,5-dicyanoimidazole, 5-(ethylthio)-1H-tetrazole, 5-(benzylthio)-1H-tetrazole and saccharin 1-methyl Examples include imidazole, preferably 4,5-dicyanoimidazole.
• nucleoside directly supported on a carrier refers to a nucleoside portion of a compound in which a nucleoside or nucleotide is bound to the reaction point of the carrier (a compound portion in which a nucleoside base and a sugar are bound), and " A nucleoside indirectly supported on a carrier means a nucleoside moiety (a compound moiety formed by binding a nucleoside base and a sugar) in which a nucleotide is bound to the reactive site of the carrier via a compound such as a polynucleotide. Point.
• a nucleoside phosphoramidite is added to the hydroxyl group, thiol group or amino group at the 3'- or 5'-position of a nucleoside supported directly or indirectly on a carrier in the presence of an activating agent.
• the amount of the activating agent used in the binding step is, for example, 10.0 to 15.0 times the amount of the nucleoside phosphoramidite used in the step.
• a nucleoside phosphoramidite is added to the hydroxyl group, thiol group or amino group at the 3'- or 5'-position of a nucleoside supported directly or indirectly on a carrier in the presence of an activating agent.
• the amount of the activating agent used in the binding step is not particularly limited, but is preferably, for example, 10.0 to 25.0 equivalents of the supported nucleoside.
• a nucleoside phosphoramidite is added to the hydroxyl group, thiol group or amino group at the 3'- or 5'-position of a nucleoside supported directly or indirectly on a carrier in the presence of an activating agent.
• the amount of the nucleoside phosphoramidite used in the binding step is not particularly limited, but is preferably, for example, 1.0 to 2.0 equivalents of the supported nucleoside.
• a nucleoside phosphoramidite is added to the hydroxyl group, thiol group or amino group at the 3'- or 5'-position of a nucleoside supported directly or indirectly on a carrier in the presence of an activating agent.
• the temperature of the solution in the bonding step is not particularly limited, but is preferably 0 to 30°C, more preferably 0 to 20°C, and even more preferably 5 to 20°C.
• a method of producing an oligonucleotide comprising: (a) removing the protecting group from the protected nucleoside directly or indirectly carried on the carrier and having the protecting group bound to the hydroxyl group, thiol group or amino group at the 3′- or 5′-position; (b) in the presence of an activating agent, a nucleoside phosphoramidite is attached to the hydroxyl group, thiol group or amino group at the 3'- or 5'-position of the nucleoside supported directly or indirectly on the carrier from which the protective group has been removed; the step of combining with (c) sulfurating or oxidizing the bond formed by step (b); and (d) unbound 3′ or 5′ hydroxyl group, thiol in the nucleoside supported directly or indirectly on the support.
• the temperature of the solution in any one of steps (a) to (d) is 0 to 30 ° C., preferably 0 to 20 ° C., more preferably 5 to 20 ° C. .
• a method of controlling the temperature of the solution As a method of controlling the temperature of the solution, a method of supplying a solution controlled to a desired temperature in advance to each step, and a method of controlling the temperature to a desired temperature by cooling the reaction vessel after supplying the solution in each step. , a method in which a pipe is provided in the reaction vessel and a refrigerant or the like is passed through the pipe to control the temperature to a desired level. Further, in the present invention, the temperature at which the solution controlled to a desired temperature in advance is supplied to each step is referred to as the temperature of the supplied solution.
• the amount of nucleoside phosphoramidite remaining in the reaction vessel refers to the amount of nucleoside phosphoramidite remaining in the reaction vessel in the process of synthesizing oligonucleotides, for example according to the theory explained above.
• the residual amount of the phosphoramidite in the reaction vessel can be obtained, for example, by collecting the waste liquid from the oxidation step or the sulfurization step in each synthesis cycle, and optionally the washing solution after the coupling reaction during the synthesis cycle, and removing the nucleoside phosphoramidite DMTr (4, 4'-dimethoxytriphenylmethyl group)
• the amount of protecting group can be measured by HPLC analysis.
• nucleoside phosphoramidite and 4,5-dicyanoimidazole (DCI) as an activator were added in the amounts shown in Table 1, and the cup was prepared under the conditions shown in Table 1.
• a ring reaction (condensation time: 5 minutes) was carried out. All activators were dissolved in acetonitrile and made up to 0.7M.
• Other synthesis reagents were 3% DCA in toluene as the deprotecting agent, xanthan hydride prepared at 0.2 M in pyridine as the sulfurizing agent, a mixed solution of lutidine, N-methylimidazole and acetic anhydride in acetonitrile as the capping agent.
• TBA in acetonitrile (8:2 ratio) was used as the amine wash reaction.
• a 24-mer DNA oligonucleotide (5′-TCGACGTATTGACGTATTGACGTA-3′, all phosphites sulfurized: SEQ ID NO: 1) was synthesized and the terminal DMTr protecting groups removed. After synthesis, the porous resin beads with attached DNA oligonucleotides were dried. Thereafter, the porous resin beads were immersed in aqueous ammonia, the DNA oligonucleotides were cut out from the porous resin beads, and the basic amino groups were deprotected to obtain a filtrate in which the DNA oligonucleotides were dissolved.
• the residual rate of nucleoside phosphoramidites in the column is obtained by dividing the absolute amount of DMTr in the waste liquid (equivalent to the amount of bound nucleoside phosphoramidites) into the amount of nucleoside phosphoramidites introduced into the reaction (synthesis scale ⁇ nucleoside phosphoramidites equivalent).
• Table 1 shows the results.
• the values of Examples 1 to 3 show the residual ratios of Examples 1 to 3 when the residual ratio in the column of Comparative Example 1 is 1, and the values of Examples 4 to 6 show Comparative Example 2.
• the residual ratios of Examples 4 to 6 are shown when the in-column residual ratio is 1. If the % relative to the comparative example is less than 1, it indicates that the residual amount in the column is suppressed compared to the comparative example.
• FIG. 1 shows the residual ratio in the column and the purity in Example 2 and Example 3 in which the activating agent was used 14 times the amount of the nucleoside phosphoramidite and the feed solution temperature was 15°C.
• the amount of activating agent used increased, the residual rate of nucleoside phosphoramidite in the column decreased and the purity of the synthesized DNA oligonucleotide increased.
• the residual rate of nucleoside phosphoramidites in the column was further reduced, and the purity of the synthesized DNA oligonucleotides was increased.
• FIG. 2 shows the residual ratio in the column and the purity in Example 5 and Example 6 in which the activating agent was used 14 times the amount of the nucleoside phosphoramidite and the feed solution temperature was 15°C.
• the amount of activating agent used increased, the residual rate of nucleoside phosphoramidite in the column decreased and the purity of the synthesized DNA oligonucleotide increased. Further, by lowering the temperature of the supplied liquid to 15° C. as in Example 6, the residual rate of nucleoside phosphoramidites in the column was further reduced, and the purity of the synthesized DNA oligonucleotides was increased.
• RNA oligonucleotide Porous resin beads (NittoPhase (registered trademark) HL 250-2'OMeA) were placed in a synthesis column (volume 12.6 ml) so that the synthesis scale (total reaction points of beads) was 352 ⁇ mol. , set in AKTAoligopilotplus100 synthesizer (manufactured by Cytiva), 5-(ethylthio)-1H-tetrazole (ETT) as nucleoside phosphoramidite and activating agent, and the amounts shown in Table 2 were added. A coupling reaction (condensation time: 10 minutes) was carried out under the conditions described in .
• a 24mer 2′OMeRNA oligonucleotide (5′-UCGACGUAUUGACGUAUUGACGUA-3′, fully oxidized on phosphite: SEQ ID NO: 2) was synthesized and the terminal DMTr protecting group removed.
• the porous resin beads with attached RNA oligonucleotides were dried. Thereafter, the porous resin beads were immersed in aqueous ammonia, the RNA oligonucleotides were cut out from the porous resin beads, and the basic amino groups were deprotected to obtain a filtrate in which the 2'OMeRNA oligonucleotides were dissolved.
• RNA Oligonucleotide sample filtrate prepared to 5 OD was measured by high performance liquid chromatogram (HPLC) under the following conditions.
• the peak area (%) of the main component was defined as the synthetic purity (Full-length: area %), with the sum of the peak areas from the detection of the main component up to about 10 minutes being 100%.
• Mobile phase A 400 mM HFIP/15 mM TEA aqueous solution
• Mobile phase B Methanol Column temperature: 60°C
• the amount of nucleoside phosphoramidite was estimated from the absolute amount of DMTr protecting groups of the nucleoside phosphoramidite. Hydrolyze the nucleoside phosphoramidite to obtain a DMTr protecting group, dilute appropriately with a 0.1 M p-toluenesulfonic acid monohydrate (pTSA) acetonitrile solution, and then adjust the absolute amount of the DMTr protecting group under the following conditions. was measured by HPLC. Column: Waters, Atlantis T3, 130 ⁇ , 3.0 ⁇ m, 2.1 mm ⁇ 150 mm MS detection: ESI-Posi.
• pTSA p-toluenesulfonic acid monohydrate
• Results Table 2 shows the results.
• the value of Example 7 shows the residual ratio in the column when the residual ratio in the column of Comparative Example 3 is 1
• the values of Examples 8 and 9 show the residual ratio in the column of Comparative Example 4.
• the residual ratios of Examples 8 and 9 are shown when the ratio is set to 1. If the % relative to the comparative example is less than 1, it indicates that the residual amount in the column is suppressed compared to the comparative example.
• the residual rate of the synthesized RNA oligonucleotides in the column decreased to 12.7% when the amount of the activator was 12.0 times the amount of the nucleoside phosphoramidite, and the purity of the RNA oligonucleotides was improved. .
• Comparative Example 4 in which 1.4 equivalents of the nucleoside phosphoramidite was used, the feed solution temperature was 22.5°C, and the activating agent was used at 3.3 times and 12.0 times the amount of the nucleoside phosphoramidite, respectively. and Example 8, and column retention in Example 9 using 1.4 equivalents of nucleoside phosphoramidite, 12 times the amount of activating agent relative to the amount of nucleoside phosphoramidite, and feed liquid temperature of 15 ° C. Percentage and purity are shown in FIG. Also in FIG.
• Example 9 when the amount of the activating agent was increased to 12.0 times the amount of the nucleoside phosphoramidite, the residual rate in the column decreased and the purity of the synthesized RNA oligonucleotide showed a high value.
• the residual ratio of the synthesized RNA oligonucleotide in the column was further reduced, and the purity was even higher. Therefore, compared to Comparative Example 3, even with modified RNA nucleoside phosphoramidites in Example 9, the purity can be improved even if the input amount of RNA nucleoside phosphoramidites is greatly reduced by reducing the residual rate in the column. I found out.

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