GLYCOSIDE COMPOUND, AMIDITE COMPOUND, AND PRODUCTION METHOD FOR POLYNUCLEOTIDE USING SAID COMPOUNDS

Abstract

The present invention provides an amidite compound capable of improving a yield and a purity of a polyoligonucleotide, a glycoside compound as an intermediate thereof, and a production method for a polynucleotide using the amidite compound. The present invention also provides an amidite compound of formula (1) capable of improving a yield and a purity of a polyoligonucleotide, a glycoside compound of formula (10) (in formulae (10) and (1), B.sup.a, R.sup.a, R.sup.b, R.sup.c, G.sup.1, G.sup.2, and G.sup.3 are as defined in the description, and R is represented by the following formulae), and a production method for a polynucleotide using the amidite compound.

Claims

1. An amidite compound represented by formula (1): ##STR00046## wherein R represents a group represented by a formula: ##STR00047## wherein: R.sup.a and R.sup.b are identical to or different from each other and each represents a methyl group, an ethyl group, or a hydrogen atom, provided that R.sup.a and R.sup.b do not represent a hydrogen atom at the same time; and R.sup.c represents a phenyl group, a C1 to C10 alkyl group, or a benzyl group which may be substituted with a halogen atom, a methyl group, a nitro group, a methoxy group, or a trifluoromethyl group, B.sup.a represents a group having an optionally-protected nucleobase structure, G.sup.1 and G.sup.2 are identical to or different from each other and each represents a protecting group of a hydroxyl group, and G.sup.3 is identical to or different from each other and each represents an alkyl group.

2. The amidite compound according to claim 1, wherein R.sup.a represents a methyl group, and R.sup.b represents a hydrogen atom.

3. The amidite compound according to claim 1, wherein R.sup.a and R.sup.b both represent a methyl group.

4. The amidite compound according to claim 1, wherein G.sup.1 represents the following group: ##STR00048## wherein R.sup.1, R.sup.2, and R.sup.3 are identical to or different from each other and each represents hydrogen or an alkoxy group.

5. The amidite compound according to claim 1, wherein G.sup.2 represents the following group ##STR00049##

6. The amidite compound according to claim 1, wherein G.sup.3 represents an isopropyl group.

7. The amidite compound according to claim 1, wherein R.sup.c represents a phenyl group or a tolyl group.

8. A production method for a compound containing a polynucleotide structure represented by formula (2), the method comprising a step of using the amidite compound according to claim 1 for a solid-phase synthesis reaction: ##STR00050## wherein B.sup.a is identical to or different from each other and each represents a group having an optionally-protected nucleobase structure, X represents an oxygen atom or a sulfur atom, and m represents a positive integer.

9. The production method according to claim 8, wherein the compound containing a polynucleotide structure of formula (2) is formed by reacting a compound having an oligonucleotide structure represented by formula (3), formed in the solid-phase synthesis reaction using the amidite compound, with a tetraalkylammonium fluoride: ##STR00051## wherein B.sup.a is identical to or different from each other and each represents a group having an optionally-protected nucleobase structure, X represents an oxygen atom or a sulfur atom, and R is identical to or different from each other and each represents a group represented by a formula: ##STR00052## wherein: R.sup.a and R.sup.b are identical to or different from each other and each represents a methyl group, an ethyl group, or a hydrogen atom, provided that R.sup.a and R.sup.b do not represent a hydrogen atom at the same time; and R.sup.c represents a phenyl group, a C1 to C10 alkyl group, or a benzyl group which may be substituted with a hydrogen atom, a halogen atom, a methyl group, a nitro group, a methoxy group, or a trifluoromethyl group.

10. The production method according to claim 9, wherein R.sup.a represents a methyl group, and R.sup.b represents a hydrogen atom.

11. An ether compound represented by formula (4): ##STR00053## wherein: R.sup.a and R.sup.b are identical to or different from each other and each represents a methyl group, an ethyl group, or a hydrogen atom, provided that R.sup.a and R.sup.b do not represent a hydrogen atom at the same time; R.sup.c represents a phenyl group, a C1 to C10 alkyl group, or a benzyl group which may be substituted with a halogen atom, a methyl group, a nitro group, a methoxy group, or a trifluoromethyl group; and R.sup.d represents C1 to C10 alkyl or a phenyl group.

12. The ether compound according to claim 11, wherein R.sup.a represents a methyl group, R.sup.b represents a hydrogen atom, and R.sup.c represents a phenyl group or a tolyl group.

13. A production method for an ether compound represented by formula (4), the method comprising step a of reacting a 2-hydroxyalkylsulfone represented by formula (5) with a bisthioether compound represented by formula (12) in a solvent in presence of a halogenating agent and an acid: ##STR00054## wherein: R.sup.a and R.sup.h are identical to or different from each other and each represents a methyl group, an ethyl group, or a hydrogen atom, provided that R.sup.a and R.sup.b do not represent a hydrogen atom at the same time; and R.sup.c represents a phenyl group, a C1 to C10 alkyl group, or a benzyl group which may be substituted with a halogen atom, a methyl group, a nitro group, a methoxy group, or a trifluoromethyl group,
(R.sup.dSCH.sub.2).sub.2O   (12) wherein R.sup.d represents C1 to C10 alkyl or a phenyl group, ##STR00055## wherein R.sup.a, R.sup.b, R.sup.c, and R.sup.d are as defined above.

14. The production method according to claim 13, wherein R.sup.a represents a methyl group or an ethyl group, R.sup.b represents a hydrogen atom, and R.sup.c represents a phenyl group or a tolyl group.

15. The production method according to claim 13, wherein R.sup.a represents a methyl group, R.sup.b represents a hydrogen atom, and R.sup.c represents a phenyl group or a tolyl group.

16. A production method for a compound represented by formula (8), the method comprising reacting a compound represented by formula (7) with a compound represented by formula (4) in presence of a halogenating agent: ##STR00056## wherein B.sup.a represents a compound having an optionally-protected nucleobase structure, and G.sup.4 represents a protecting group of a hydroxyl group, ##STR00057## wherein R.sup.a and R.sup.b are identical to or different from each other and each represents a methyl group, an ethyl group, or a hydrogen atom, provided that R.sup.a and R.sup.b do not represent a hydrogen atom at the same time, R.sup.c represents a phenyl group, a C1 to C10 alkyl group, or a benzyl group which may be substituted with a halogen atom, a methyl group, a nitro group, a methoxy group, or a trifluoromethyl group, and R.sup.d represents C1 to C10 alkyl or a phenyl group, ##STR00058## wherein B.sup.a, R.sup.a, R.sup.b, and R.sup.c are as defined above, and G.sup.4 represents a protecting group of a hydroxyl group.

17. A production method for the compound of formula (1) according to claim 1, the method comprising the steps of: further deprotecting the compound of formula (8) to obtain a compound represented by formula (9); selectively protecting a hydroxyl group at 5′ of the compound of formula (9) to obtain a compound represented by formula (10); and reacting the compound of formula (10) with a phosphordiamidite represented by formula (11): ##STR00059## wherein B.sup.a represents a compound having an optionally-protected nucleobase structure, R.sup.a and R.sup.b are identical to or different from each other and each represents a methyl group, an ethyl group, or a hydrogen atom, provided that R.sup.a and R.sup.b do not represent a hydrogen atom at the same time, R.sup.c represents a phenyl group, a C1 to C10 alkyl group, or a benzyl group which may be substituted with a halogen atom, a methyl group, a nitro group, a methoxy group, or a trifluoromethyl group, ##STR00060## wherein B.sup.a, R.sup.a, R.sup.b, and R.sup.c are as defined above, and G.sup.1 represents a protecting group of a hydroxyl group, ##STR00061## wherein G.sup.2 represents a protecting group of a hydroxyl group, and G.sup.3 is identical to or different from each other and each represents an alkyl group.

18. The production method according to claim 16, wherein G.sup.4 has a G.sup.4-1 or G.sup.4-2 structure; ##STR00062##

19. A compound represented by formula (8): ##STR00063## wherein B.sup.a represents a compound having an optionally-protected nucleobase structure, R.sup.a and R.sup.b are identical to or different from each other and each represents a methyl group, an ethyl group, or a hydrogen atom, provided that R.sup.a and R.sup.b do not represent a hydrogen atom at the same time, R.sup.c represents a phenyl group, a C1 to C10 alkyl group, or a benzyl group which may be substituted with a halogen atom, a methyl group, a nitro group, a methoxy group, or a trifluoromethyl group, and G.sup.4 represents a protecting group of a hydroxyl group.

20. A compound represented by formula (9): ##STR00064## wherein B.sup.a represents a compound having an optionally-protected nucleobase structure, R.sup.a and R.sup.b are identical to or different from each other and each represents a methyl group, an ethyl group, or a hydrogen atom, provided that R.sup.a and R.sup.b do not represent a hydrogen atom at the same time, R.sup.c represents a phenyl group, a C1 to C10 alkyl group, or a benzyl group which may be substituted with a halogen atom, a methyl group, a nitro group, a methoxy group, or a trifluoromethyl group.

21. A compound represented by formula (10): ##STR00065## wherein B.sup.a represents a compound having an optionally-protected nucleobase structure, R.sup.a and R.sup.b are identical to or different from each other and each represents a methyl group, an ethyl group, or a hydrogen atom, provided that R.sup.a and R.sup.b do not represent a hydrogen atom at the same time, R.sup.c represents a phenyl group, a C1 to C10 alkyl group, or a benzyl group which may be substituted with a halogen atom, a methyl group, a nitro group, a methoxy group, or a trifluoromethyl group, and G.sup.1 represents a protecting group of a hydroxyl group.

22. (canceled)

Description

EXAMPLES

[0215] Hereinafter, Examples will be described to explain the present invention in more detail. However, the present invention is not limited to these Examples and so on.

[0216] The following abbreviations are used herein.

[0217] TPM=(1-(4-methylbenzenesulfonyl)propan-2-yl)oxy)methoxy)methyl group; A=adenine, G=guanine, C=cytosine, U=uracil.

Production of TPM amidite U

Production Example 1

1) Production of TPM Reagent (TPMR)

[Chemical Formula 41]

[0218] ##STR00041##

[0219] Bis(methylthiomethyl)ether (5.9 g, 0.043 mol) was dissolved in an anhydrous tetrahydrofuran (THF) (60 mL), Molecular Sieves 4A (5.9 g) was added thereto, and the mixture was stirred for 10 minutes. After the mixture was cooled to −50° C., N-iodosuccinimide (NIS) (11.5 g, 1.19 eq.) and then trifluoromethanesulfonic acid (TfOH) (0.11 mL, 0.030 eq.) were added. An acetonitrile (20 mL) solution of 1-(4-methylbenzenesulfonyl)propan-2-ol (10 g, 1.09 eq.) (manufactured by ENAMINE Ltd.) was dropped to the mixture, and the mixture was stirred at −50 to −45° C. for 4 hours. After triethylamine (4.0 mL) was dropped to a reaction solution, the temperature was raised to −30 to −20° C., and then the reaction solution was added to a solution including a sodium thiosulfate pentahydrate (17.1 g), a sodium hydrogen carbonate (6.0 g), and water (130 mL) which had been pre-cooled to 5 to 10° C. in an ice bath. Ethyl acetate (42 mL) was added to the mixture, and the mixture was stirred at 10 to 15° C. for 30 minutes, and then was filtered through celite (5.9 g). A filtrate was separated, and then an organic layer was washed with a 20% brine (24 mL). The organic layer was dried using an anhydrous magnesium sulfate (3 g), and then a solvent was distilled off under reduced pressure (bath temperature: 40° C.). A residue was purified by silica gel chromatography (hexane/ethyl acetate=3/1, silica gel 207 mL) to obtain 3.1 g of yellow oily matter.

[0220] As a result of purity analysis by GC/FID, the purity was 91%.

[0221] The purification was performed by silica gel chromatography again (hexane/ethyl acetate=8/1, silica gel 150 mL) to obtain 3.1 g of TPMR with a purity of 98.2% and 1.5 g of TPMR with a purity of 97.8%. In the subsequent reactions, both were mixed and used.

[0222] .sup.1H—NMR (CDCl.sub.3): δ7.79(d,2H)7.36(d,2H)4.76(s,2H)4.63(d,2H)4.27(m,1H)3.44(dd, 1H) 3.14 (dd, 1H) 2.45 (s, 3H) 2.13 (s, 3H) 1.32 (d, 3H)

Production Example 2

2) Production of TPM-U-2

[Chemical Formula 42]

[0223] ##STR00042##

[0224] Anhydrous toluene (16.5 mL, 5 vol/wt) was added to U-1 (3.3 g, 6.78 mmol) , the mixture was concentrated under reduced pressure to 3 vol/wt, anhydrous toluene (6.6 mL, 2.0 vol/wt) was further added thereto, and the mixture was concentrated under reduced pressure to 3 vol/wt. Anhydrous tetrahydrofuran (6.6 mL, 2.0 vol/wt) was added, the mixture was cooled to around −55° C., then PMMR (3.09 g, 10.17 mmol, 1.5 eq.) was dropped thereto, and the mixture was washed with 2 mL of THF. NIS (2.06 g, 9.15 mmol, 1.35 eq.) was added to the mixture, and TfOH (0.72 mL, 8.14 mmol, 1.2 eq.) was dropped at −55 to −45° C. The mixture was stirred at −55 to −45° C. for 1 hour, and the reaction solution was added to a mixed liquid including a sodium thiosulfate pentahydrate (3.3 g), a sodium hydrogen carbonate (1.12 g), water (22 mL) , and a toluene which had been cooled in an ice bath. The mixture was stirred in the ice bath for 30 minutes and separated. A solution including a sodium thiosulfate pentahydrate (1.65 g) , a sodium hydrogen carbonate (0.6 g) , and water (11 mL) was added to an organic layer, and the mixture was stirred at room temperature for 15 minutes and then separated. The organic layer was dried with a sodium sulfate (1 g) and then concentrated to dryness under reduced pressure. A residue was purified by silica gel column chromatography (hexane/ethyl acetate=3/1, silica gel 250 mL) to obtain colorless clear glassy solid TPM-U-2 (5.3 g).

Production Example 3

3) Production of TPM-U-3

[Chemical Formula 43]

[0225] ##STR00043##

[0226] After TPM-U-2 (5.3 g, 7.13 mmol) was dissolved in an acetone (10 mL), triethylamine hydrotrifluoride (1.3 mL, 7.84 mmol) was added, and the mixture was stirred at room temperature for 2 hours. Methyl tert-butyl ether (MTBE) (53 mL) was added to the reaction solution, the mixture was stirred for 30 minutes, and then a MTBE layer was removed by decantation. This procedure was repeated 3 times, and then the residue was dried and solidified under reduced pressure to obtain white amorphous TPM-U-3 (3.5 g) .

Production Example 4

4) Production of TPM-U-4

[Chemical Formula 44]

[0227] ##STR00044##

[0228] Pyridine (10.5 mL, 3 vol/wt) was added to TPM-U-3 (3.5 g, 7.00 mmol) , azeotropic dehydration was conducted twice, then pyridine (7.0 mL, 2 vol/wt), toluene (17.5 mL, 5 vol/wt), and acetonitrile (7.0 mL, 2 vol/wt) were added thereto, and the mixture was cooled to around 0° C. 4,4T-dimethoxytrityl chloride (2.85 g, 8.40 mmol, 1.2 eq.) was added thereto. The mixture was stirred at room temperature for 6 hours, methanol (1.75 mL, 0.5 vol/wt) was added, and the mixture was stirred for 10 minutes. A solution including a sodium hydrogen carbonate (0.53 g) and water (10.5 mL) was added, and the mixture was stirred for 15 minutes and separated (this procedure was further repeated once more). A solution including a sodium chloride (1.05 g) and water (10.5 mL) was added to an organic layer, and the mixture was stirred for 15 minutes and then separated. The organic layer was dried with a sodium sulfate (1 g) and then concentrated under reduced pressure. A residue was purified by silica gel column chromatography (hexane/ethyl acetate=1/1 to ethyl acetate only, silica gel 280 mL) to obtain 3.8 g of white amorphous TPM-U-4 (yield: 670).

Production Example 5

5) Production of TPM-U-5 (TPM Amidite U)

[Chemical Formula 45]

[0229] ##STR00045##

[0230] TPM-U-4 (4.7 g, 5.85 mmol) was dissolved in an anhydrous acetonitrile (47 mL, 10 vol/wt), then diisopropylammonium tetrazolide (1.10 g, 6.56=01, 1.12 eq.) and Molecular Sieves 4A (0.94 g, 0.2 wt/wt) were added, and the mixture was stirred at room temperature for 30 minutes. 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphordiamidite (Phos reagent) (2.65 g, 8.78 mmol, 1.5 eq.) was added to the solution, and the mixture was stirred at bath temperature of 45° C. for 1.5 hours. The mixture was cooled to room temperature and filtered, and a filtrate was concentrated under reduced pressure. A residue was purified by silica gel column chromatography (hexane/acetone=2/1+5% pyridine, silica gel 380 mL) to obtain 4.75 g of white solid TPM amidite U.

[0231] .sup.31P—NMR (CDCl.sub.3): δ151.89,151.86,150.87

Production Example of Nucleic Acid

[0232] Using the TPM amidite U produced in Production Example 5, a uridine 50-mer represented by the sequence of SEQ ID NO: 1 below was synthesized.

TABLE-US-00001 (SEQ ID NO: 1) 5′-UUUUUUUUUU UUUUUUUUUU UUUUUUUUUU UUUUUUUUUU UUUUUUUUUU-3′

[0233] (wherein U represents uridine monophosphate sodium salt)

[0234] Solid-phase synthesis was performed from the 3′ side toward the 5′ side using NTS M-4MX-E (manufactured by NIHON TECHNO SERVICE CO., LTD.) as a nucleic acid synthesis device. For the synthesis, porous glass was used as a solid support, a high-purity trichloroacetic acid toluene solution was used as a deblocking solution, 5-benzylmercapto-1H-tetrazole was used as a condensing agent, an iodine solution was used as an oxidizing agent, and a phenoxyacetic acid solution and a N-methylimidazole solution were used as a capping solution.

[0235] The purity of an oligonucleotide crude product after the solid-phase synthesis was measured by HPLC. The crude product was separated into each component by HPLC (wavelength 260 nm, column ACQUITY UPLC Oligonucleotide BEH C18, 2.1 mm×100 mm), and the purity of the oligonucleotide was calculated from the obtained area value of a main product relative to the total area value of chromatogram.

Production Example of Nucleic Acid of the Present Invention

Production Example 6

[0236] As a result of synthesizing a uridine 50-mer (molecular weight 15246.53) using the TPM amidite U prepared in Example 5, the OD.sub.260 for 0.173 μmol was 37.21 OD, and the purity was 53.5%. From the OD.sub.260 value, the yield for 1 μmol was calculated as 8603 μg/μmol.

[0237] The results are shown in Table 1 below.

[0238] (OD.sub.260 represents an absorbance of UV 260 nm for 10 mm optical path length in an 1 mL solution (pH=7.5). Since RNA is generally known to have 1 OD=40 μg, the production amount of RNA can be calculated from the absorbance.)

Comparative Production Example of Nucleic Acid

Comparative Example 1

[0239] Solid-phase synthesis was performed in the same manner as in the method described in Production Example 6 by using the uridine EMM amidite described in Example 2 of JP 5554881 B2 to produce a uridine 50-mer, and as a result, the OD.sub.260 for 0.228 μmol was 41.41 OD and the purity thereof was 44.1%. From the OD.sub.260 value, the yield for 1 μmol was calculated as 7264 μg/μmol.

[0240] The results are shown in Table 1 below.

TABLE-US-00002 TABLE 1 Comparative Example 1 Example 1 Purity of uridine 50-mer (%) 53.5 44.1 Yield (μg/μmol) 8603 7264 Yield (%) 56.4 47.6

[0241] As shown in Table 1 above, when the amidite produced in the present invention is used, the uridine 50-mer with high purity can be obtained.

INDUSTRIAL APPLICABILITY

[0242] The present invention provides a sulfone group-containing ether compound which is useful as a protecting group of a hydroxyl group at the 2′ position of an amidite, and an amidite compound having the sulfone group-containing ether moiety. The amidite compound of the present invention is suitable for synthesis of an oligonucleic acid with a high-purity.

FREE TEXT OF SEQUENCE LISTING

[0243] SEQ ID NO: 1 in Sequence Listing represents the base sequence of the uridine 50-mer.