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
  • C07H21/04—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
• • 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
  • C07H21/02—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
• • 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 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 of 1.5-10.0 relative to 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 each reaction temperature is about 0°C to about 27°C, and Patent Document 2 describes that the reaction temperature is 0°C to 100°C. Preferably, 20 to 50 ° C. is more preferable, and 20 to 30 ° C. is particularly preferable. A method for obtaining oligonucleotides of good purity while reducing the .sup.2 is not described.
• 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 in the course of intensive research on a method for producing oligonucleotides, have found that the residual amount of nucleoside phosphoramidites in the reaction vessel in the synthesis process is reduced by lowering the temperature of the solution in the process of producing oligonucleotides. It was found that the amount of nucleoside phosphoramidite used could be reduced. As a result of further research based on such knowledge, the present invention was completed.
• a method for 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. capping the group or amino group; including The above method, wherein the temperature of the solution in any one of steps (a) to (d) is 0 to 20°C.
• 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].
• oligonucleotides By lowering the temperature of the solution in each step in the production of oligonucleotides, it is possible to suppress the residual amount of nucleoside phosphoramidite in the reaction vessel in the synthesis step, and the use of nucleoside phosphoramidite, which is an expensive material. Oligonucleotides can be obtained with good purity while reducing the amount.
• 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
• the detachment of the CNET protecting group of the nucleotide is suppressed during the process, and binding to the hydroxyl group of the oligonucleotide formed by the detachment of the CNET protecting group. It is thought that the amount of nucleoside phosphoramidite consumed by the method is reduced, and the reduction in the amount of nucleoside phosphoramidite used is realized.
• FIG. 1 shows the in-column retention rate and purity in Comparative Example 1, Example 1, Example 2, Example 3 and Example 4.
• FIG. 2 shows the in-column residual ratio and purity in Comparative Example 2, 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, nucleosides or oligonucleotides in the presence of a suitable activating agent.
• 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) binding (coupling) a nucleoside phosphoramidite 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 protecting group has been removed; process, (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.
• a method for producing an oligonucleotide comprises: (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
• 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)
• nucleoside indirectly supported on a carrier refers to a nucleoside moiety (a compound moiety in which a nucleoside base and a sugar are bonded) in which a nucleotide is bound to the reaction site of the carrier via a compound such as a polynucleotide.
• 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 20°C, preferably 5 to 20°C. Specifically, 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15° C., 16° C., 17° C., 18° C., 19° C., 20° C., and may be within a range between any two of these numbers.
• 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 the activating agent used in step (b) is not particularly limited, but is, for example, 2.0 to 15.0 times the amount of the nucleoside phosphoramidite used in the step.
• Equivalent weight is preferred. Specifically, 2.0-fold equivalent, 2.5-fold equivalent, 3.0-fold equivalent, 3.5-fold equivalent, 4.0-fold equivalent, 4.5-fold equivalent, 5.0-fold equivalent, 5.5-fold equivalent double equivalent, 6.0 fold equivalent, 6.5 fold equivalent, 7.0 fold equivalent, 7.5 fold equivalent, 8.0 fold equivalent, 8.5 fold equivalent, 9.0 fold equivalent, 9.5 fold equivalent , 10.0-fold equivalent, 10.5-fold equivalent, 11.0-fold equivalent, 11.5-fold equivalent, 12.0-fold equivalent, 12.5-fold equivalent, 13.0-fold equivalent, 13.5-fold equivalent, 14 0 equivalents, 14.5 equivalents, 15.0 equivalents, and may be within a range between any two of these numbers.
• the amount of the activating agent used in step (b) is not particularly limited, but is preferably, for example, 10.0 to 25.0 equivalents of the supported nucleoside. Specifically, 10.0-fold equivalent, 10.5-fold equivalent, 11.0-fold equivalent, 11.5-fold equivalent, 12.0-fold equivalent, 12.5-fold equivalent, 13.0-fold equivalent, 13.5-fold equivalent double equivalent, 14.0 fold equivalent, 14.5 fold equivalent, 15.0 fold equivalent, 15.5 fold equivalent, 16.0 fold equivalent, 16.5 fold equivalent, 17.0 fold equivalent, 17.5 fold equivalent , 18.0-fold equivalent, 18.5-fold equivalent, 19.0-fold equivalent, 19.5-fold equivalent, 20.0-fold equivalent, 20.5-fold equivalent, 21.0-fold equivalent, 21.5-fold equivalent, 22 0-fold equivalent, 22.5-fold equivalent, 23.0-fold equivalent, 23.5-fold equivalent, 24.0-fold equivalent, 24.5-fold equivalent, 25.0-fold equivalent, any two of these numerical values It may be within a range between two.
• the amount of the nucleoside phosphoramidite used in step (b) is not particularly limited, but is preferably, for example, 1.0 to 2.0 equivalents of the supported nucleoside. Specifically, 1.0-fold equivalent, 1.1-fold equivalent, 1.2-fold equivalent, 1.3-fold equivalent, 1.4-fold equivalent, 1.5-fold equivalent, 1.6-fold equivalent, 1.7-fold equivalent double equivalents, 1.8 fold equivalents, 1.9 fold equivalents, 2.0 fold equivalents, and may be within a range between any two of these numbers.
• 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.
• 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 4 show the residual ratios of Examples 1 to 4 when the residual ratio in the column of Comparative Example 1 is 1, and the values of Examples 5 to 6 show Comparative Example 2.
• the residual ratio in the column is 1, the residual ratios of Examples 5 and 6 are shown. 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.
• Example 1 shows the residual ratio in the column and the purity in Example 4 at 15°C. When the temperature of the liquid supplied was 20°C or lower, the residual rate in the column decreased. The purity of the synthesized DNA oligonucleotides showed the highest value when the supply liquid temperature was 15° C., and the purity of the DNA oligonucleotides synthesized in Example 4 showed even higher values.
• Purity is shown in FIG. As shown in FIG. 2, when the temperature of the supplied solution was 20° C. or lower, the residual rate in the column decreased and the purity of the synthesized DNA oligonucleotide showed a high value. In Example 6, the purity of the synthesized DNA oligonucleotide showed even higher values.
• 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. m/z 303 Mobile phase A: 0.1% formic acid aqueous solution Mobile phase B: Acetonitrile Column temperature: 40°C
• 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).
• Results Table 2 shows the results.
• the value of Example 7 shows the residual ratio in Example 7 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.
• Example 7 using 2.0 equivalents of nucleoside phosphoramidite, 3.3 times the amount of activating agent relative to the amount of nucleoside phosphoramidite, and feed temperatures of 15° C. and 22.5° C., respectively, and FIG. 3 shows the in-column retention rate and purity in Comparative Example 3.
• the residual ratio of the synthesized RNA oligonucleotides in the column decreased to 16.7% when the supply liquid temperature was 15°C, and the purity of the RNA oligonucleotides was improved.
• Example 9 Column residual rate and Purity is shown in FIG. In FIG. 4 as well, when the supply liquid temperature was lowered to 20° C. or lower, the residual rate in the column decreased and the purity of the synthesized RNA oligonucleotide showed a high value. In Example 9, the purity of the synthesized RNA oligonucleotide showed even higher values. 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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