JPS61291595A - Production of 2'-0-methylated rna - Google Patents

Abstract

PURPOSE:To obtain the titled nucleic acid probe to form stable complex with RNA or DNA, by bonding successively 5'-OH and 3'-OH of riboside such as 2'-O-methyluridine, etc., through phosphoric acid and removing the protecting group. CONSTITUTION:5'-OH and 3'-OH of a riboside selected from 2'-O-methyluridine, 2'-O-methyl-N<4>-protected cytidine, 2'-O-methyl-N<6>-protected-adenosine and 2'-O- methyl-N<2>-protected guanosine are successively bonded (condensed) through phosphoric acid by phosphoric acid triester method or phosphorus acid triester method in such a way that preset sequence (e.g., sequence of partial structure of messenger RNA, etc.) order is obtained and the protecting group is removed to give the aimed compound. The condensation reaction is carried out on a solid-phase carrier such as silica gel, polystyrene, porous glass, etc., and the reaction product is separated from the carrier after the condensation reaction is over, isolated and purified by column chromatography, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application This invention relates to 2'-0-methylated RNA (hereinafter referred to as "methyl RNA J"). Methyl RNA is a type of RNA or DNA that has a complementary base sequence. Forms a stable complex and becomes an excellent glove when selecting the desired RNA or chromosomal gene segment from Messenger RNA or chromosomal genes 〇 Conventional technology Messenger RNA or chromosomal genes from the desired RNA
Conventionally, DNA has been used as a glove for selecting A or chromosomal gene divisions. However, globe DNA
The stability of the complex between DNA and RNA or DNA that is complementary to it is not necessarily sufficient; it may dissociate even under mild conditions, or the base sequence of one part of the globe DNA and the target RNA or DNA may be partially different from that of the other. If they are physically different, they may not form a complex. Furthermore, conventional natural probes have had problems such as being subject to cycleolysis by nucleolytic enzymes. In addition, 2'-0-methylated RNA is
It has great advantages when used as gloves because it does not serve as a substrate for A-degrading enzyme and exhibits resistance to nuclease, phosphogesterase, and phosphorylase.

Problems to be Solved by the Invention It is therefore an object of the present invention to obtain nucleic acid globes that form stable complexes with RNA or DNA and to find a method for producing them.

Means for Solving the Problems In order to solve the above-mentioned problems, the present inventors, as a result of various studies, found that 2'-0-methyluridine, 2'-O-methyl-N
4-protected-cytidine, 2'-0-methyl-N6-protected-
Adenosine and 2'-0-methyl-N2-protected-guanosine are selected by adding phosphoric acid to the 5'-hydroxyl and 3'-hydroxyl groups of the cytosine in a predetermined arrangement order. 2′-〇- having a predetermined base sequence by sequentially bonding through
We succeeded in producing methylated RNA. Furthermore, it has been found that the obtained 2'-0-methylated RNA undergoes a hybridization reaction with DNA and RNA having complementary base sequences to sufficiently achieve the above-mentioned purpose.

Process for producing 2'-〇-methyluridine Uridine is protected with a protecting group that can simultaneously protect the 713' and 5' hydroxyl groups, such as the 1,1,3,3-tetraisoglovir sidilyl group, and then the 2-sil N3 The 2' hydroxyl group is protected with an acyl group such as benzoyl, and the 2' hydroxyl group is methylated using methyl iodide and silver oxide in an organic solvent such as benzene to protect the 3',5'-0-protection@ 2'-0-
Methyluridine can be obtained.

When performing methylation using methyl iodide and silver oxide,
As a protecting group for 5' hydroxyl group, dimethoxytrityl group,
Using a protecting group such as a monomethoxytrityl group, trityl group, trityloxyacetyl group, reprinyl group, dibromomethylbenzoyl group or tert-butyldimethylsilyl group, the 3' hydroxyl group can be converted to a tert-butyldimethylsilyl group, ortho-nitrobenzyl group, etc. Methylation can also be performed after protection with

Alternatively, only the 5' hydroxyl group is protected with the above protecting group, the 2' hydroxyl group is methylated using stannous chloride and diazomethane, and the 3' hydroxyl group is methylated using stannous chloride and diazomethane.
It can also be separated from the body. 1,1,3.3-tetrainglobyldisilyl group or tert-butyldimethylsilyl group is reacted with n-tetrabutylammonium fluoride or 0.5N hydrogen chloride in an organic solvent such as dioxane or tetrahydrofuran. The protecting group can be removed by performing Furthermore, the acyl group in the base part is released under basic conditions such as aqueous ammonia! I, and the 2'-0-methyl group is stable under these deprotection reaction conditions.

2'-0-methyluridine containing impurities can be purified by recrystallization from a mixed solvent of methanol and ethyl acetate.

The amino group at the N4 position of cytidine is protected with an acyl group such as anisoyl group or benzoyl group, and the 2'-〇-
2'-0-Methyl to N-protected-cytidine can be produced by a method similar to that for producing methyluridine. Also, 2'
If the -O-methyl-N-protected cytidine contains impurities, it can be purified by recrystallization in ethanol.

Preparation of 2'-0-methyl-N6 protected-a≠nosine 6-chloro-9-(β-D-ly?72nosyl) f-phosphorus
For example, 3
', 5' hydroxyl group 1.1, 3.3. 2'-O- with tetraisoglopylsilyl group protected, 2' hydroxyl group methylated, 6-position diol group replaced with diamino group by treatment with ammonia, 3'+5' hydroxyl group protected Methyladenosine can be produced.

Next, the amino group at the 6-position was protected with a more acyl group of the benzoyl group, and the 3', 5' -0-LL3+3+tetrainglobil disilyl group was converted to 7. By deprotecting with n-butylammonium chloride, 2'-0-methyl-N-protected adenosine can be produced. If the 2'-0-methyl-N-protected adenosine contains impurities, it can be purified by silica dal chromatography.

The amino group at the 2-position of guanosine is protected with an acyl group such as imbutyryl group, the 5' hydroxyl group is protected with a protecting group as described in the section of the general method for producing 2'-0-methyluridine, and 2'-0-methylation with tin and diazomethane, separation of the 3'-0-methylated product by silica dal chromatography, and removal of the 5'-0-protecting group. It is possible to produce '-O-methyl-N protected guanosine. If the 2'-O-methyl-N-protected guanosine contains impurities, it can be purified by silica gluchromatography.

For example, a method for producing 2'-〇-methyl oligonucleotide having a sequence similar to the partial structure of Metsenger RNA that corresponds to the amino acid sequence of the target protein uses phosphate The triester method and the phosphite triester method (hereinafter referred to as the phosphite method) are currently known, and any of these methods may be used. Therefore, the protected nucleotides that can be used as raw materials for each method are the aforementioned 2'
It can be produced by protecting the 5' hydroxyl group of an -O-methyl-N protected nucleotide with a protecting group such as dimethoxytrityl group or monomethoxytrityl group, and phosphorylating the 3' hydroxyl group. If phosphorylation is performed using trivalent phosphorus, such as orthochlorophenyl phosphoside diazolide, as a phosphorylating agent, a raw material for the phosphoric acid triester process can be provided. Phosphorylation using isoglobylaminomethoxychlorophosphine or the like can provide a raw material for the phosphite method. In addition, in the predetermined sequence, the nucleotide located at the 3' end is 5'-0-protected-N, protected-2
′-〇-Methyl nucleotide with a radical such as succinic acid? If it is bonded to a resin insoluble in organic solvents such as silica glue, polystyrene, or porous glass via an acid group, the condensation reaction can be carried out on a solid support. In the condensation process, the chain length can be extended by using a condensing agent such as arenesulfonylnitrotriazole in the IJ ester method, and by activating the phosphorus atom using tetrazole in the phosphite method. . In this case, a 2'-0-methyl oligonucleotide having an arbitrary sequence can be produced by sequentially condensing the necessary bases to a predetermined sequence.

The chain lengthening reaction is completed and fully protected on a solid phase support.
By treating the 2'-0-methyl oligonucleotide with ammonia, the 9N acyl group and phosphoric acid can be deprotected and the 2'-0-methyl oligonucleotide can be cut from the resin at the same time. Furthermore, in the case of synthesis by the phosphotriester method, the yield of deprotection can be improved by treating with 2-pyridine aldoxime/tetramethylguanidine before treatment with ammonia.

On the other hand, this is not necessary when synthesized by the phosphite method.

5'-〇-dimethoxytrityl-2'-〇-methyl oligonucleotide (or 5'-0-lythyl derivative-2'-〇-methyl oligonucleotide) excised from the resin by the above method can be separated from by-products that do not have a dimethoxytrityl group (or 5'-0-trityl derivative) in 5'-0 by column chromatography using reversed-phase silica gel.

The 5'-〇-dimethoxytrityl-2'-〇-methyl oligonucleotide (or i!5'
-0-trityl derivative oligonucleotide) under acidic conditions.
0-trityl derivative) and completely remove the protecting group by column chromatography using anion exchange resin or reversed-phase silica gel or electrophoresis on polyacrylamide gel denatured with urea or formamide, etc. The resulting 2'-0-methyl oligonucleotide can be isolated and purified.

Example 2'-0-Methyluridine was prepared by the route shown in Reaction Scheme 1.

10.9 of uridine was dissolved in 801 nt of pyridine, and while stirring under water cooling, 1,3-dichloro-1,1°3 was added to the solution.
．． 3-tetraisoglobildisiloxane 14-(1,1
equivalent amount) was added, the mixture was warmed to room temperature, and stirring was continued for 140 minutes. Next, this was cooled on ice and 40% of water was added to stop the reaction, and the mixture was separated into layers with chloroform and water, and the chloroform layer was washed with water. The chloroform layer was removed and the solvent was distilled off, and the resulting mixture was subjected to column chromatography packed with silica fyC-200,5ooI and 2.5% methanol-
Both fractions eluted with 97.5% chloroform were collected, the solvent was distilled off, and pure 1b was obtained at 18.71%! , with a yield of 93.6%.

3',5'-0-tetraisoglovirsicilyuridine (Ib) UV spectrum (methanol): λmax2
61 nm+λmin229nm (basic): λmax
261 n1llTλmin242nm'H-NMR
: δppm 8.82 (s+, l, 3-H) 7.7
0(d,l,6-H,J=8.06Hz)5.73
(d, 2, 5-H, 1'-H, J=6.84Hz
)4.15 (m + 512'-Hl 3'-HI
4'-H15'-H) 3.14(br, 1.2'-0H
) 1.1 (m, 28, TIPDS group) Compound 1b15JF was dissolved in 150 d of dimethylacetamide, 5.55 d (1,3 equivalents) of triethylamine was added thereto, cooled with water, and benzoyl chloride 3.94+d (1,1 equivalents) was dissolved. ) was added dropwise to the mixture, and after returning to room temperature, the mixture was stirred for 180 minutes. Next, the reaction mixture was concentrated and separated into layers with ethyl acetate and water, and the ethyl acetate layer was washed with water, dried over anhydrous sodium sulfate, and concentrated to dryness. The residue is silica gel C-300, 350g
Separated and purified by column chromatography packed with 0
．． 2.1 Pure fractions eluted with ethyl acetate-99.8% chloroform mixture were collected, the solvent was distilled off, and compound 1c was extracted from 11.6. F, obtained in a yield of 63.996.

C UV spectrum (methano-#): λmax 252 nm,
λmin221nm (basic): λmax252nm
, λmin223nm 1H-NMR (CDC6,):
δppm 7.94 (d, 1.6-H, J=6.
35Hz) 7.84-7.49 (m, 5.N-benzo(ru group) 5.81 (d, 2.5-H, 1'-H, J=
8.3Hz) 4.18(m, 5 +2'-Hl3'-
H, 4'-H, 5'-H) 2.85 (br, 1.2'
-0H) 1.1 (m, 28, TIPDS group) Compound I
e 7.3511 was dissolved in benzene 70d and Ag2O
After adding 8.26g (2.g equivalent), ultrasonication was applied for several tens of minutes, and CH3119.36m (25g amount) was added and heated at 40°C.
The mixture was stirred overnight. After filtering the reaction mixture, the filtrate was concentrated, separated with chloroform and water, the chloroform layer was washed with water, and anhydrous sulfuric acid was added. After drying over IJum, the solvent was distilled off, and the resulting residue was purified with silica y/I/c- Perform column chromatography packed with 200° 20ON and 0.5 timethanol-9.
Collect the pure fractions eluted with 9.51% chloroform,
The solvent was distilled off to obtain 6.34.9 (yield: 84.3) of compound 1d.

d UV light (methanol): λmax 252 nm,
λmin221nm (basic): λmax252nme
λmin225nm' H-NMR (CDC13):
δppm8.03 (d, 1t6-H, J=8.3Hz)
7.99-7.50 (m+5, N5-benzoyl
group) 5.80 (d, 2, 5-H, l'-H, J
=7.08Hz)4.05 (m, 5, 2'-H
, 3'-H, 4'-H, 5'-H) 3.60 (s 1
1.2'-0CHs ) 1.1 (m, 28.TIP
DS group) Next, this was dissolved in 90 ml of dioxane, and concentrated aqueous ammonia was added thereto, followed by stirring at room temperature for 4 hours and 30 minutes. The reaction mixture was concentrated, separated into layers with chloroform and water, and the chloroform layer was washed with water and then with a 0.5% aqueous sodium bicarbonate solution, dried over anhydrous sulfuric acid, and the solvent was distilled off. The obtained residue was treated with silica gel C-20.
The pure fraction eluted with 1-thimethanol-99-thichloroform was collected and the solvent was distilled off, and compound Ie
．． It was obtained in a yield of 98.9° and 95.2%.

s σV absorption quictor (methanol): λmax261n
m+λmin222nm (basic):λmax260n
162.89 g of m+λmin 242 nm compound was dissolved in 30 g of dioxane, and 1 N hydrochloric acid was added to this,
After stirring at room temperature for 3 hours and 30 minutes, Dowex lX2 (b
icarbonate form). This was filtered, the resin was washed with methanol-water (1:10), and the washing liquid and filtrate were combined and the solvent was distilled off. The obtained residue was dissolved in 3001 nt of methanol-water (1:10), washed with chloroform, the chloroform layer was back-extracted with water, the aqueous layers were combined and concentrated to dryness, then methanol was added and the solvent was distilled off. The obtained residue was dissolved while heating by adding 20 t of methanol/80 ml of ethyl acetate, filtered out while hot, and the filtrate was allowed to cool to give acicular crystals of 2'-0-methyluridine (Ir). ! i' was obtained in a yield of 87.9%.

If UV spectrum (methanol): λmax 261 nm,
λmin230nm (basic): λmax260nms
λml n 242nm elemental analysis value Measured value 2C, 46
, 59:H, 5, 39:N, 11.11 theoretical value (C, o
H14N206):C,46,51:H,5,46:N
, 10.84Mass spectrum m/e 258
(M”), 147 (S?-S) melting point 156-15
9°C 1H-NMR (DMSO-d6) δppm 11.3
6 (a, 1, N'-H) 7.93 (d, 1.6
-H, J=8.3Hz) 5.86 (d, l,
1'-H, J=5.12Hz)5.66(d,1.5
-H,J-8,06Hz)4.12(m,1.2'-H
) 3.80 (m, 4 *3'-H+4'-H+5'-H)
3.36 (s + m, 2'-OCHs), 3
As a method for removing the '15' silyl group, the conditions used in the production of 2'-0-methyl-N6 penzoyladenosine, that is, the method using tetra-n-2 thylammonium heptadide, can also be applied. .

Reaction formula 1% Formula% BT, = benzoyl group 2'-O-methyl-N4-benzoylcytidine production 2
'-O-methyl-N4-benzoylcytidine has the reaction formula 2
- The literature (T, 5asalci) produced by the following route shown in
+Y, Mizuno + Chem, Pharm,
Bull r vol. 15, p. 894, 1967) using cytidine selectively benzoylated at the N-4 position as a raw material, and according to the method for producing 2'-0-methyluridine (
However, benzoylation at the N-3 position is omitted)2'-0-
Methyl-N4-benzoylcytidine Ile was prepared.

1e α spectrum (methanol): λmax 259 nm + 303
nm *λmin284nm (basic): λmax 317 nm +λmin26
0nm (acidic): λmax 257 nm + 31
7 nm *λmin282nm Melting point 178-180°C Mag II Su-I'cutor 361 (M+), 2
16(B+2)1H-NMR (DMSO-d6): δ
ppm 11.22 (s, 1.N'-H) 8.55 (
d, 1, 6-H, J=7.57Hz) 7.98
~7.47 (m, 5, N'-benzoyl group)
5.89 (d, 1, 1'-H, J=2.2Hz
)3.48 (s, 3, 2” 0CH5)Ue
(3',5'-0-TIPDS-N'-Pencyi'/fgin) UV absorption spectrum (methanol): λmax 259 nm * 303
nm +λmin284nm (basic): λmax 317 nm +λmin26
0nm (acidic): λmax 258nm, 315nm
Iλmin282nm ffd (2'-0-mef+3'+5'-0-TIP
DS-N'-PyCy) stone absorption spectrum (methanol): λwax 259 nm 1303
nm.

λmin284nm (basic): λmax 317 nm aλmin26
0 nm (acidic): λmax 258 nm, 31 nm
.

λmin282nm'H-NMR (CDC1,): δppm8.40 (d, 1, 6-H, J=7.
33) 7.88-7.50 (m, 5, N'-benzoyl group) 4.25-3.79 (m, 5, 2'-
H, 3', -H, 4'-H, 5' -H) 3.74 (s
, 3.2'-0-CH3) 1.1 (m, 28
, TIPDS group) Reaction formula 2 2'-O-methyl-N6-penzoyladenosine was prepared by nine routes shown in Reaction formula 3.

Using 6-chloro-9-(β-D-IJ&7lanosyl)purine 6.211, 3'+-5'-0-tetra Isoprobyl disilylated product (compound m-b>9.45
g, with a yield of 82.4%.

b ■ Absorption spectrum (methanol) (acidic, basic, neutral): λmax 264 nm + λmin 226n
m1H-NMR (CDCl2): δppm 8.70
(s, 1.8-H)8.32(s, 1.2-H
) 6.07 (s, 1, 1'-H) 5.02 (m
, 1.2'-H) 4.61 (m, 1, 3'-H) 4.11 (
m, 2.4'-H) 3.26 (m, 1, 5'-H) 1.12 (pin,
1.2'-0H) 1.1 (m, 28, TIPDSi group) Next, this compound (I[Ib) 9.42 li, and Ag2゜20
．． 100- of CH3I was added to 7 g (5 equivalents), stirred at 40°C for 50 minutes, filtered, and then concentrated to dryness.

The obtained residue was dissolved in chloroform, washed with a 0.5% aqueous sodium thiosulfate solution and then with water, and the chloroform layer was dried over anhydrous sodium sulfate, concentrated, and packed with silica gel C-200,400,9 into nine columns. Separation and purification was performed using chromatography, and the pure fraction eluted with 1-thimethanol-99-thichloroform was collected, the solvent was distilled off, and the compound mc was 6.41! ! Obtained in a yield of (66.1 h).

α sictor (methanol) (neutral, basic, acidic): λmax 264 nm + λmin 230 nm1
H-NMR (CDC23): δppm 8.73 (s
, 1, 8-H) 8.45 (s, 1.2-H) 6.10 (s, 1, 1'-H) 4.70 (m
, 1, 2'-H) 4.09 (rn+4+3'-
H, 4'-H.

5'-H) 3.72 (s, 3, 2'0-OH5) 1.1
(m, 28, TIPDS group) Next, this compound (Illc) 4.451 was dissolved in 30 g of dichloromethane, placed in a sealed tube, added with 301 nl of cooled liquid ammonia, sealed, and stirred at room temperature for 24 hours. Next, 200 d of dichloromethane was added and dissolved in the reaction solution from which ammonia had been distilled off under reduced pressure. After washing with a dilute aqueous sodium bicarbonate solution and then with water, the dichloromethane layer was dried with anhydrous sulfuric acid. The solvent was distilled off to remove a residual residue. Silica gel C-200,1
The product was separated and purified by column chromatography packed with 20F, and the pure fraction eluted with 1.5% methanol-98.5% chloroform was collected and the solvent was distilled off. The obtained good compound II[d was recrystallized from a small amount of methanol to finally obtain 2.67 g with a yield of 66.0%.

11d Takumi absorption spectrum (M・OH): (neutral, basic): λmax 259 nm, λmi
n233nm (acidic): λmax 257 nm +
λmin 234 nmMass spectrum m/e
523 (M+), 480 (M-43)'H-NMR (
CDC/=, ): δppm8.33 (s, 1
, 8-H) 8.13 (s, 1.2-H) 6.03 (s, 1, 1'-H) 5.71 (s,
2,6-NH2) 4.70 (m, 1, 2'-H) 4.11 (m
, 4.3'-H, 4'-H.

5'-H) 3.72 (s, 3.2'-OCH3) 1.1
(m, 28.TIPDS group) Next, compound old 2I was dissolved in pyridine 2o-, cooled on ice, and benzoyl chloride
．． Add 57m (1,3fi amount), warm to room temperature,
After stirring for an hour, the reaction solution was ice-cooled and 5rnt of water was added to stop the reaction. Chloroform 150- was added to this, washed with a saturated aqueous sodium bicarbonate solution, then washed with water, the organic layer was dried over anhydrous sodium sulfate, and the solvent was distilled off.The resulting residue was packed with 60 g of silica gel C-200. The pure fractions eluted with a mixed solvent of 1 timethanol and 99% chloroform were collected and concentrated to dryness to obtain 1.11.9 of Compound 11[e]. On the other hand, the N6-dibenzoyl compound eluted first was collected separately, the solvent was distilled off, and the residue was mixed with methanol 9-dibenzoyl.
Add concentrated aqueous ammonia (1-1) dissolved in 30-ml solution and react at room temperature for 10 minutes. The solvent was distilled off. The resulting residue was dissolved in 30-chloroform, washed with a saturated aqueous solution of sodium bicarbonate and then with water, and then diluted with anhydrous sulfuric acid. It was dried with monotrium and concentrated to dryness to obtain compound 1 [[e]. The total yield was 88.8%.

e Takumi absorption spectrum (methanol): λmax279 nm, λmin2
47nm (basic): λmax 307 nm *λm
1 m of tetra-n-butylammonium fluoride (TB, AF) in 260 nm compound II [e 0.911
Add 9 oz/l of tetrahydrofuran solution and stir at room temperature for 50 min.
Stir for a minute.

Add pyridine-methanol-water (volume ratio 3:1) to this reaction solution.
:1) 20W1t was added, and this mixture was added to a beaker containing cation exchange resin Dowex 50Wx2 (pyridinium type) 3-. This was filtered, and the resin was washed with the above-mentioned pyridine-methanol-water, combined with Solution F, and concentrated to dryness. The obtained residue was azeotropically distilled with toluene, then methanol was added to dissolve it, and 7g of Silica Dull C-200 was added. /L/C-200,40,9 was packed in a column, separated and purified by chromatography, and the pure fraction eluted with 2.5-thimethanol-97,5-tichloroform was collected and concentrated to dryness. I[rf 0.51. P, 92.
It was obtained in a yield of 7.

f W absorption spectrum (methanol): λmax 279 nm
+λmin247nm (basic):λmax310nm
+lλmin260nm Reaction formula 3 If TfPDSiCt2-1.3-diqtetraru 1.1.3.
Preparation of 3-tetraisoglobyldisiloxane 2'-O-Methyl-N2-isobutyrylguanosine 2'-O-Methyl-N2-isobutyrylguanosine y was prepared by the conceptual route shown in Reaction Scheme 4. According to a conventional method, combine *5'-0-monomethoxytrityl-N8-isobutyrylguanosine 69 and 5nCt2o, 38Il (
0.2 equivalent) was dissolved in dimethylformamide (480), and a solution of diazomethane in 1,2-dimethoxyethane (25) was added dropwise at 0°C over about 3 hours. After stirring for another 2 hours, concentrated aqueous ammonia was added. Add 0.5 tRt and distill off the solvent.Next, add chloroform to the residue, dissolve and wash with water.The aqueous layer is back-extracted with chloroform.The chloroform layers are combined and washed with water, then diluted with anhydrous sulfuric acid). It was dried and concentrated to dryness.

The residue was separated and purified by column chromatography packed with Silica Dull C-200, 200F, and the 2/, 3/methyl compound mixture (b) eluted with 2-thimethanol-98 dichloroform was collected and concentrated to dryness to form t0 compound (b). was obtained as a mixture with a yield of 3.31.9' and 54.0%. 901H-NMR (CDC1,): 699m 12.0
1 (s, 1.t-H) 7.81 (s, 1.8-H) 4.78-3.61 (m, 6+2'-H, 3'-H, 4
'-H, 5'-H, NLH) 3.78 (s, 3
, 0CH5, MMTr group) 2.96 (a , 3 , 2
'-0CH5) Then this mixture IVb fJtKies
Separate and purify by silica gel chromatography using @Igal 60, collect fractions of 2'-○CI (2'-0CI) eluted with a mixed solvent of chloroform:acetonitrile:methanol=60=24:1 (volume ratio), It was concentrated to dryness. To this was added 40-80 thiacetic acid, and after stirring at 50°C for 1 hour, the solvent was distilled off, and the residue was separated between chloroform and water.
Wash the aqueous layer with loloform. The aqueous layer was concentrated, and the resulting residue was recrystallized from water to give 2'-0-CH.

Body ■c was obtained at 0.49.9.

F/c.

UV absorption spectrum (H2O): λmax 260nm+
λmii227nm (basic): λmax262nm+
λmin237nmMass spectrum k m/e
367 (M), 437,507 (M+zethyryl group) 'H-NMR (DMSO-d6): 699m 8.
26(s, 1.8-H)5.80(d, 1
, 1'-H) 3.33 (s, 3.2'-0CH5)
Reaction formula 4 lVa MMTr: Production of monomethoxytrityl group 2'-〇-methyl-lipo-oligonucleotide 2'
-〇-Methyluridine (Compound If), 2'-〇-Mefpv -N4-benzoylcytidine (Compound Ice)
, 2'-O-methyl-N6-penzoylua f-nosine (
The 5' hydroxyl group of Compound II [f') and 2'-O-methyl-N2-imbutyrylguanosine (compound fVe) was protected with a dimethoxytrityl group (DMT) according to a conventional method, and the 3' hydroxyl group was protected as described in the literature (Nucleic Ac1
ds Re5erch % Volume 8, Section 5461, 1980
An orthochlorophenyl phosphate group was introduced according to the method described in 2010). Literature (Nucleic Ac1ds R5
5archt Volume 8, Section 550-7-5517, 198
76 μmol of 5'-〇-dimethoxytrityl-2'-〇-methyl-N2-isoptyrylguanosynthesis synthesized according to the method described in -0-Methyl nucleotides U, C, C, A, U, U.

It is condensed to the head of C and A to elongate the chain. One cycle of condensation is described in the literature (Proa, Natl, Acacl,
The method described in 5ciUSA, Vol. 81, Section 5956, 1984@, that is, the method shown in Table 1, was carried out at Tomotsushi.

That is, 5'-0-dimethoxytrityl-2'-0-methyl-N! - Bind 76 μmol of isoptyrylguanosyl t
, ets cross-linked polystyrene resin (50 ml) was placed in a reaction vessel equipped with a glass filter and washed three times with 21 nt of a mixed solution of dichloromethane-methanol (volume ratio 7:3). Next, a solution of 2% benzenesulfonic acid in dichloromethane-methanol (volume ratio 7:3) was added twice and shaken for 1 minute. After the reaction, the bath liquid was filtered to remove the resin.
Washed with. Furthermore, it was reacted for 1 minute with the benzenesulfonic acid solution 2-, and then washed twice with the mixture 2- and then three times with pyridine 2-. Next, 0.3-g of pyridine was added, and the pyridine was distilled off under reduced pressure to dry the resin. Next, a pyridine solution of 5'-dimethoxytrityl-2'-0-methyluridine-3'-orthochlorophenyl phosphoric acid (201n910, 3d') was added and the pyridine was distilled off under reduced pressure. - Pyridine solution of nitrotriazole (20 x 70.3 d
) was added, heated to 40° C., and shaken for 20 minutes. The t0 reaction solution was removed from the resin by filtration, and the resin was washed twice with pyridine 2-. Next, 0.1M dimethylaminopyridine solution 1
．． 8- and 0.2-acetic anhydride were added and shaken for 3 minutes to diblock unreacted 5' hydroxyl groups with acetyl groups. After the reaction solution was removed, the resin was washed three times with 2 t and 11 t of pyridine. This series of operations was repeated until the length of the 2'-0-methyl-protected nucleotide (by adding the necessary bases) was extended.

The yield of each condensation reaction was determined by the absorbance at 500 nm after the dimethoxytritanol produced by de-dimethoxytritylation was developed in perchloric acid-ethanol.

The reaction yield was as follows (total yield: 22 cm).

5/N Ho @ (Am Cm Un UmAm Cm C
m UmGm ) 3'47837810294751
0495 (H) Direction of Chain Length Here, the lowercase letter m represents a 2'-0-methyl group, and each base is linked by a 3'-51 bond via orthochlorophenyl phosphoric acid.

Next, after the synthesis was completed, the resin was washed with dioxane, and then 2-
1M of pyridine aldoxime tetramethylguanidine
Dioxane solution 0.5 m, dioxane 0.4-1
After adding 0.1% water and shaking overnight at 30°C, the solution was filtered, and the resin was washed three times with 50% pyridine water 2Rt.

After combining the furnace liquid and the washing liquid and distilling off the solvent, the residue was dissolved in 1-pyridine, placed in a sealed tube, added with 10 g of 28 thiammonium water, sealed tightly, and heated at 60° C. for 5 hours. The reaction solution was concentrated to dryness and then mixed with reverse phase silica dal CWaters Bon.
packed with dapack C18 particle size 35-100μ),
Apply to a column with a diameter of 0.7 and a length of 12crR,
Column chromatography was performed using a linear concentration gradient of 5% to 35% acetonitrile as the mobile phase and a 50 mM acetic acid-triethylamine buffer.
The 5' dimethoxytrityl oligonucleotide was separated as determined by absorbance at 54 nm. After distilling off the 38th fraxilon and the minor solvent, an 80% acetic acid aqueous solution was added and the mixture was left at room temperature for 20 minutes. The reaction solution was concentrated to dryness, and further azeotroped with water to remove cyanoacetic acid. The t0 residue was dissolved in water, washed with ethyl acetate, concentrated, and further purified by high performance liquid chromatography to give a solution of sNue.
Elution was carried out using 0.1M acetic acid-triethylamine buffer (pH 7.0) applied with a linear concentration gradient from 13 to 21 as the mobile phase using Leosll 5CIB at a flow rate of 1 to 7 minutes. Collect the main peak and 0.066μ
2'-〇〇H3 oligonucleotide was obtained with a total mo1 yield of 1.1. When the obtained nucleotide was analyzed by high performance liquid chromatography using an anion exchange column (DEAE 2 SW), it was confirmed that it gave a single peak and was highly pure.

To confirm the base sequence, the 5' end of a 2'-0CH5 oligonucleotide 0.050D unit was phosphorylated with 52P using [γ-P]ATP and polynucleotide kinase, and the sample was purified as described in the literature (Proc, Natl+Ac).
ad, 5etUSA Volume 70, Sections 1209-1213,
[1973].

The autoradiography confirmed that the base sequence was correct.

Hybrid formation 2'-OCR, oligonucleotide 5' Am Om
UmUmAm Cm Cm UmQm3' and its complementary sequence deoxyoligonucleotide 5' dCAG
Equal amounts of GTAAGT3' and a concentration of wavelength 260
Dissolve 10 mM sodium cacodylate buffer containing 0.1 M sodium chloride so that the absorbance is 1.0 in the tun, warm it to 60°C, and then let it cool to room temperature. The sample was placed in a cell, and a spectrophotometer (manufactured by BECKMANN, DU-
8B), the temperature was increased from 10℃ to 47℃ every 2 minutes.
The temperature was increased at a rate of .degree. C., and the absorbance at a wavelength of 260 nm at each temperature was measured. A melting curve was obtained by plotting absorbance on the vertical axis and temperature on the horizontal axis, from which the Tm (melting temperature) value was determined to be 34.8°C.

Next, in a similar manner, 2'-〇-methyl oligonucleotide 5' Am Cm Um Un Am Cm Cm
Determine the melting curve using Um Gm 3' and the lyso-oligonucleotide 5'CAGGUAAGU3', which is a base sequence complementary to it. Yes to f oxyoligonucleotide 5' d (ACUUACCU
G) 3' and its complementary base sequence? The Tm value with oligonucleotide 5'CAGGUAGU3' was 41°C.

Patent applicant: Ajinomoto Co., Inc.

Claims (1)

[Claims] 2'-O-methyluridine, 2'-O-methyl-N^4
-Protection-Cytidine, 2'-O-methyl-N^6-Protection-
A liposide selected from adenosine and 2'-O-methyl-N^2-protected-guanosine is sequentially attached to the 5'-hydroxyl group and 3'-hydroxyl group of the liposide via phosphoric acid in a predetermined arrangement order. A method for producing 2'-O-methylated RNA, which comprises binding and removing a protecting group.