WO2006095739A1

WO2006095739A1 - Process for deblocking the 2'-hydroxyl groups of ribonucleosides

Info

Publication number: WO2006095739A1
Application number: PCT/JP2006/304399
Authority: WO
Inventors: Mitsuo Sekine; Hisao Saneyoshi; Kohji Seio
Original assignee: Tokyo Institute Of Technology
Priority date: 2005-03-09
Filing date: 2006-03-07
Publication date: 2006-09-14
Also published as: JP2006248929A; JP4797156B2

Abstract

A process for deblocking the 2'-hydroxyl groups of ribonucleosides, characterized by reacting a ribonucleoside derivative represented by the general formula (I) wherein the 2'-hydroxyl group is blocked: [Chemical formula 1] (I) [wherein X is hydrogen or the like; Y is hydrogen or the like; and B is a nucleic acid base residue] with a fluorine-containing compound or a basic compound to convert the derivative into a ribonucleoside represented by the general formula (II) or the like: [Chemical formula 2] (II) [wherein X, Y and B are each as defined above].

Description

Specification

Method for deprotection of ribonucleoside 2, hydroxyl group

Technical field

The present invention relates to a method for deprotecting the 2 ′ hydroxyl group of a ribonucleoside protected with a cyanoethyl group.

Background art

[0002] Chemically synthesized RNA is useful as a functional nucleic acid such as ribozyme, aptamer, siRNA, antisense nucleic acid, and nucleic acid pharmaceutical material (Nechiya Review Drug Discovery No. 3 318 2004). To chemically synthesize RNA, appropriate protection of the 2 'hydroxyl group and deprotection after completion of synthesis are important in the synthesis process.

[0003] The RNA synthesis method currently in widespread use uses a quaternary butyldimethylsilyl group (TBDMS group) as a protecting group for the 2 'hydroxyl group (Journal of American Chemical Society, Reference No. 99, 7741 1977). . However, this protecting group does not have sufficient chemical stability. After introducing the TBDMS group into the 2 'hydroxyl group, if the 3' hydroxyl group is released, the TBDMS group is transferred from the 2-position to the 3-position, It is known to be a mixture of 2'- TBDMS nucleosides and 3,1 T BDMS nucleosides (Nucleic Acids Research No. 18 5433 1990). In addition, since the TBDMS group is a bulky protecting group, the reaction that forms an internucleotide bond between the adjacent 3 'hydroxyl group and the 5' hydroxyl group of other nucleoside residues via a phosphorus atom is steric. And has problems such as a decrease in the yield of the internucleotide binding reaction and an increase in the required reaction time (Journal of American Chemical Society, Cited Reference No. 109, 7845 1987).

[0004] On the other hand, a triisopropyl silyloxymethyl group (Helvetica 84 No. 3773 2001) has been reported as a protecting group that can overcome these problems. Although this protecting group is said to overcome the above-mentioned TB DMS group rearrangement problem and steric hindrance problem, a method for selectively introducing the protecting group into the 2 ′ hydroxyl group has been developed! Therefore, at the stage of synthesizing the 2′-protected nucleoside, the 3′-protected nucleoside produced as a by-product must be separated by complicated column chromatography. In addition, the introduction of protecting groups using alkyl tin compounds is toxic. Concerns about residual metals in addition to the properties are expected.

[0005] In addition, as a protecting group developed after the development of the TBDMS group, bis (2cetoxyethoxy) methyl group (Journal of American Chemical Society No. 120 1182 0 1998), 1- (2 cyanoethoxy) ethyl group ( Tetrahedron Letters 45 No. 9529 2004) have been reported, but both have a structure with two substituents on the carbon atom directly connected to the 2 ′ hydroxyl group, and are expected to have high steric hindrance. It is necessary to synthesize reagents separately, and the biggest problem is that there are many synthesis steps.

[0006] Meanwhile, the present inventors have previously reported a 2'-O cyanoethyl nucleoside in which a cyanoethyl group is introduced into the 2 'hydroxyl group of a ribonucleoside (Non-patent Document 1).

[0007] Non-Patent Document 1: Nucleic Acid Research Symposium Series 48, 13-14 pages 2004

Disclosure of the invention

Problems to be solved by the invention

[0008] As a protecting group for the 2 'hydroxyl group of ribonucleoside during RNA synthesis, 1) it can be introduced using an inexpensive raw material, and 2) it is selected as a 2' hydroxyl group using an appropriately protected nucleoside. 3) Having only one substituent on the carbon directly linked to the 2 'hydroxyl group to reduce steric hindrance, 4) RNA synthesis using phosphoramidite method 5) Stable to the chemical reaction used in each process. 5) At the final stage of RNA synthesis, the base part, sugar part, phosphodiester part, etc. of the synthesized RNA are removed without significant damage. Conditions such as being able to be protected are required. The previously reported 2′-O cyanoethyl nucleoside cyanoethyl group had no established deprotection method satisfying the conditions of force 5) satisfying the above conditions 1) to 4). An object of the present invention is to provide a means for removing a cyanoethyl group from a 2 ′ O cyanonucleoside without greatly damaging the base part, sugar part, phosphodiester part and the like of RNA.

Means for solving the problem

[0009] As a result of intensive studies to solve the above-mentioned problems, the present inventor has found that tetra (n-butyl) ammonium full-fluid is required for deprotection of RNA containing 2'-O cyanoyl nucleoside. It has been found that deprotection without damaging the base portion, sugar portion, etc. of RNA can be achieved by using an olido and the present invention has been completed.

That is, the present invention provides the following (1) to (4).

[0011] (1) General formula (I):

[0012] [Chemical 1] N,

[Wherein, X represents a hydrogen atom or a hydroxyl-protecting group, Y represents a hydrogen atom, a hydroxyl-protecting group, or a general formula (III):

[Chemical 2]

R ¹

R

(Where

R represents the same or different alkyl group, or a group in which R ¹ and R are bonded to each other to form a ring which may contain a hetero atom, and R ³ represents a protecting group for a phosphate group. ) And B represents a nucleobase residue. ]

A ribonucleoside derivative protected with a 2 ′ hydroxyl group or an RNA derivative containing this ribonucleoside derivative as a constituent unit is reacted with a fluorine-containing compound or a basic compound to give a general formula (II):

[0014] [Chemical 3]

[Wherein, X, Y and B are as defined above. ]

Or a RNA containing the ribonucleoside as a structural unit. 2) A method for deprotecting a hydroxyl group.

[0015] (2) The method for deprotecting a 2 ′ hydroxyl group according to (1), wherein the compound power is a tetraalkylammonium fluoride containing fluorine.

[0016] (3) The method for deprotecting a 2 ′ hydroxyl group according to (2), which is tetraalkylammonium fluoride tetra (n-butyl) ammonium fluoride.

[0017] (4) The method for deprotecting a 2 ′ hydroxyl group according to (1), wherein the basic compound is ammonia, a primary amine, a secondary amine, a tertiary amine, or an amidine compound.

The invention's effect

[0018] According to the present invention, RNA having a hydroxyl group protected with a cyanoethyl group that does not damage the base part, sugar part, phosphodiester part, etc. of RNA can be deprotected. This enables efficient RNA synthesis.

Brief Description of Drawings

FIG. 1 is a diagram showing reverse phase HPLC profiles of 2′-O-cyanethylated uridylate dimer (A), uridylate dimer (B), and uridin (C).

FIG. 2 is a graph showing the relationship between the protecting group of the 2′-hydroxyl group of phosphoramidite and the phosphate ester formation reaction rate.

[Figure 3] Reversed phase HPLC profile of uridine 12mer (Purified Ucel2mer) with protecting group (A) and reverse phase HPLC profile of uridine 12mer (U12mer) without protecting group (B) ).

FIG. 4 is a diagram showing the ion exchange HPLC profile of purified RNA oligomer (GACUGACUGACU). FIG. 5 is a diagram showing an ion exchange HPLC profile of a purified RNA oligomer (uridine 30-mer).

BEST MODE FOR CARRYING OUT THE INVENTION

[0020] Hereinafter, the present invention will be described in detail.

[0021] The method for deprotecting the 2 'hydroxyl group of the present invention is represented by the general formula (I):

[0022] [Chemical 4]

[0023] [Chemical 5]

Or a RNA containing this ribonucleoside as a structural unit.

[0024] X in the general formulas (I) and (II) represents a hydrogen atom or a protecting group for a hydroxyl group. As the protecting group for the hydroxyl group, a general protecting group used for protecting the 5′-hydroxyl group or the 3′-hydroxyl group of the nucleoside can be used. For example, a silyl group which may have a substituent (for example, tert-butyl diphenyl). -Rusilyl group), 4-methoxytrityl group, 4,4'-dimethoxytrityl group and the like can be used. X may be combined with Y to form a protecting group. Examples of such protecting groups include 1, 1, 3, 3-tetraisopropyldisiloxa-lide. And cyclic silyl groups such as di (t-butyl) silane diyl.

[0025] Y in the general formulas (I) and (II) represents a hydrogen atom, a hydroxyl-protecting group, or the general formula (III): [0026] [Chemical Formula 6] D ₂

"(I I I)

(Where

R ² represents the same or different alkyl group, or a group in which R ¹ and R ² are bonded to each other to form a ring which may contain a hetero atom, and R ³ represents a protecting group for a phosphate group. ) Represents a group represented by As the hydroxyl protecting group, the same protecting groups as described above can be used. In general formula (III), R ² includes an isopropyl group, and R ³ includes, but is not limited to, a 2-cyanoethyl group.

[0027] B in the general formulas (I) and (Π) represents a nucleobase residue. Here, “nucleobase” refers to all nucleobases found in naturally occurring nucleic acids such as chromosomal DNA, plasmid DNA, messenger RNA, ribosomal RNA, transfer RNA, and small nuclear RNA of organisms. And all heteroaromatic rings optionally having substituents that can be used for nucleic acid synthesis. Representative nucleobases include, but are not limited to, adenine, guanine, cytosine, uracil, thymine and the like. Nucleobases also include those having a protecting group.

[0028] The "RNA derivative containing a ribonucleoside derivative with a protected 2 'hydroxyl group represented by general formula (I) as a constituent unit" is a formula obtained by removing X and Y in general formula (I). It means that the residue of the represented nucleoside derivative is contained in the RNA derivative. “RNA containing a ribonucleoside represented by the general formula (II) as a structural unit” has the same meaning.

[0029] The fluorine-containing compound is not particularly limited as long as it can remove the cyanoethyl group. Tetramethylammonium fluoride, tetraethylammonium fluoride, tetra (n-butyl) ammonium- Examples include tetraalkyl ammonium fluoride such as um fluoride. Tetraalkyl ammonium fluoride contains tetra (n —Butyl) ammonium fluoride (TBAF) is particularly preferred.

[0030] The amount of the fluorine-containing compound used in the reaction is not particularly limited. For example, in the case of tetraalkylammonium fluoride, a ribonucleoside derivative represented by the general formula (I), etc.

It is preferable to use about 1 to 3000 moles per mole!

[0031] The basic compound is not particularly limited as long as the cyanoethyl group can be eliminated, and examples thereof include ammonia, primary amine, secondary amine, tertiary amine, amidine compound, and the like. . Of these compounds, ammonia is preferred.

[0032] The amount of the basic compound used in the reaction is also not particularly limited. For example, in the case of ammonia, about 1 to 3000 moles with respect to 1 mole of the ribonucleoside derivative represented by the general formula (I), etc. It is preferred to use.

[0033] The above reaction is performed in an organic solvent which may contain an aqueous solvent. The volume ratio of the organic solvent and the aqueous solvent is not particularly limited, but is preferably 0: 100 to 100: 0. The organic solvent is not particularly limited as long as it does not inhibit the reaction, and a mixed organic solvent obtained by mixing a plurality of organic solvents that do not inhibit the reaction at an arbitrary volume ratio may be used. As the aqueous solvent, not only water but also any buffer solution can be used, and a mixed buffer solution in which a plurality of buffer solutions are mixed at an arbitrary concentration may be used.

[0034] The reaction temperature is not particularly limited, but is preferably in the range of -78 ° C to 100 ° C.

[0035] A method for synthesizing 2'-O-cyanoethyl nucleoside by introducing a cyanoethyl group into the 2 'hydroxyl group of ribonucleoside is, for example, Nucleic Acid Research Symposium Series No. 48, pages 13-14. 2004 It is described in!

Example

[Example 1] Deprotection of 2-cyanoethyl group

The lyophilized 2,1 O-cyanethylated uridylic acid dimer (0.5 micromolar) was placed in a tetrahydrofuran solution of TBAF (1M, 2001) and dissolved by vortexing. After standing at room temperature for 24 hours, it was diluted with 0.1 M ammonium acetate buffer (3 ml) and the organic solvent was distilled off under reduced pressure. The residue of the reagent was removed from the obtained residue using OASIS MCX Cartridge. The deprotected uridylate dimer is analyzed by reverse phase HPLC and the Thereafter, phosphodiesterase 1 (0.1 U) and alkaline phosphatase (1 U) were added and subjected to enzymatic degradation at 37 ° C. for 24 hours to obtain uridine.

[0037] FIG. 1B shows a reverse-phase HPLC profile of the uridylate dimer. Figures 1A and 1C also show the reversed-phase HPLC profiles of 2, -O-cyanoethylated uridylate dimer and uridine, respectively.

[0038] From the change in the HPLC profile shown in Fig. 1A and Fig. 1B, it was confirmed that the deprotection of the 2-cyanoethyl group was completed under the above conditions.

[0039] Further, as shown in FIG. 1C, only the uridine is observed in the enzymatically decomposed product, confirming that this deprotection is progressing without causing a side reaction in the base part and sugar part. It was done.

Example 2 Effect of 2′-hydroxyl protecting group on phosphate ester formation reaction rate

2, -O-cyanethyl-5, -0- (4,4, -dimethoxytrityl) uridine 3,-(2-cyanethyl N, N-diisopropyl phosphoramidite) [2, -cyanethyl], 2, -0- (t-butyldimethylsilyl) -5′—O— (4,4′-dimethoxytrityl) uridine 3, mono (2-cyanoethyl N, N-diisopropyl phosphoramidite) [2, -tert-butyldimethylsilyl], 2 , —O— (Triisopropylpropylsilylmethyl) -5, —O— (4,4, -dimethoxytrityl) uridine 3, — (2-Cyanethyl N, N-diisopropyl phosphoramidite) [2, —Triilopro [Bilsilyl] Each 0.03 mmol was dissolved in deuterated nitrile (1 mL), and 2, 3, — O— 2— N-triacetyl guanosine (25 mg, 0.06 mmol) was prepared here. . Here, 1H-tetrazole (6 mg, 0.086 mmol) was added and the progress of the reaction was followed by 31P-NMR.

[0041] As a result, with regard to the rate of progress of the reaction, 2'-cyanoethyl (CE) is almost the same as 2'-triylopyl bilyl silyl (TOM) and faster than 2, -t-butyldimethylsilyl (TBDMS). It was found to progress (Figure 2).

[Example 3] Synthesis of uridine 12-mer

The DNA / RNA synthesizer 392 from Applied Bio System Inc. was used as an automatic nucleic acid synthesizer. 2, 1-O-Cyanethinoleol 5, -0- (4,4, -dimethoxytritinole) uridine 3, 1 (2-Cyanethyl N, N-diisopropyl phosphoramidite) solution of anhydrous acetonitrile with 0.1M Installed on the above automatic synthesizer and purchased from Glen Reserch In. The target RNA strand was extended to a CPG solid phase carrier to which a lysine residue was bound. As an activation reagent for phosphoramidite, a 0.25 M 5 benzylthio 1H-tetrazole in water-free nitronitrile solution was used, except that the condensation time was changed to 10 minutes. Synthesis was performed according to (tritylon). After completion of the synthesis, the solid support was immersed in a large excess of concentrated ammonium acetate (10: 1, wZw) and left for 1 hour. By passing the solution through a C18-cartridge column to remove by-products, the 2% trifluoroacetic acid aqueous solution was used to remove the cage dimethoxytrityl group, and the target product was eluted with distilled water containing acetonitrile. The resulting solution was lyophilized to dryness. The residue was dissolved in distilled water and purified by ion exchange HPLC (GenPak Fax column, 25 mM sodium phosphate pH 6.0, loaded with 1M NaCl concentration gradient at a rate of 1% for 1 minute) (Purified Ucel2mer). Here 1M tetra heptyl ammonium - Umufuruorido / tetrahydrofuranyl Hmm propylamine (20: l, vZv) a 630 _i u L pressurized tut, allowed to stand for 12 hours at room temperature to remove the 2 'hydroxyl of Shianoechiru group. To the solution was added 0.1 M aqueous ammonium acetate solution and concentrated under reduced pressure. Dissolve the residue in distilled water and pass it through an OASIA MCX cartridge column and then through an H LB cartridge column. After lyophilization, the residue is ion-exchanged HPLC (GenPak Fax column, 25 mM sodium phosphate pH 6.0, 1M NaCl The concentration gradient was loaded at a rate of 1% for 1 minute). (U12mer) ₀

[0043] Purified Ucel2mer and U12mer were analyzed by the same ion exchange HPLC as described above, and it was confirmed that the cyano group of the 2 'hydroxyl group was deprotected (Figs. 3A and B).

[0044] Further, when MALDI-TOF mass spectrometry was performed on Purified Ucel2mer and U12mer, as shown below, the theoretical value and the actual measurement value coincided.

[0045] Purified Ucel2mer

Theoretical value: 4236. 51, actual value: 4238. 41

U12mer

Theoretical value: 3609. 34, measured value: 3609. 72

From this result, it was confirmed that the cyanoethyl group of the 2 ′ hydroxyl group was deprotected.

[Example 4] Synthesis of RNA oligomer GACUGACUGACU

Applied Bio System Inc. DNA / RNA synthesizer 392 is used as an automated nucleic acid synthesizer. I used it. 2, 1 O cyanoethinole 5, -0- (4,4, -dimethoxytritinore) uridine 3, 1 (2 cyanoethyl N, N-diisopropyl phosphoramidite), 4-N-acetyl-2, 1 O cyano etinore 5, 1 O— (4,4, -dimethoxytritinole) cytidine 3, 1 (2 cyanoethyl N, N diisopropyl phosphoramidite) 2, 2, 1 O cyanoethyl 1,5, -0- (4,4′-dimethoxytrityl) 6— N— (N, N dimethylaminomethylene) adenosine 3, 1 (2 cyanoethinole N, N diisopropinorephosphoramidite), 2, 1 O— cyanoethyl-5, -0- (4, 4, dimethoxy (Trityl) 2-N- (N, N dimethylaminomethylene) guanosine 3, 1 (2 cyanoethyl N, N-diisopropyl phosphoramidite) solution of anhydrous acetononitrile at 0.1M, the above automatic synthesizer The target RNA strand was extended to a CPG solid phase carrier attached with a uridine residue purchased from Glen Reserch In. 0.25 M 5 Benzylthio-1H-tetrazole anhydrous acetononitrile solution was used as the phosphoramidite activating reagent, and the condensation time was changed to 10 minutes. Synthesis was performed according to the RNA synthesis protocol (tritylone). After the synthesis, the solid support was immersed in a large excess of concentrated ammonium acetate (10: 1, wZw) and left for 2 hours. The solution was passed through a C18-cartridge column to remove by-products, and then the 2% trifluoroacetic acid aqueous solution was used to remove the dimethoxytrityl group, and the target product was eluted with distilled water containing acetonitrile. The resulting solution was lyophilized to dryness, and 1M tetraptylammonium-fluoride Ztetrahydrofuran-propylamine (20: 1, vZv) was added to the mixture at 630 _i u L and left at room temperature for 12 hours. The cyanoethyl group was removed. To the solution was added 0.1 M aqueous ammonium acetate solution and concentrated under reduced pressure. Dissolve the residue in distilled water and pass it through an OASIA MCX cartridge column and then through an H LB cartridge column. After lyophilization, the residue is ion-exchanged HPLC (GenPak Fax column, 25 mM sodium phosphate pH 6.0, 1M NaCl The concentration gradient was loaded at a rate of 1% for 1 minute.) (Figure 4). The isolation yield was 25%.

[0047] Further, when MALDI-TOF mass spectrometry was performed on the synthesized RNA oligomer, the theoretical value (3783. 47) and the measured value (3783. 73) were in agreement.

[Example 5] Synthesis of RNA oligomer uridine 30-mer

Applied Bio System Inc. DNA / RNA synthesizer 392 is used as an automated nucleic acid synthesizer. I used it. 2, 1 O Shianoethinole 5, -0- (4,4, -dimethoxytritinore) uridine 3, 1 (2-cyanethinole N, N-diisopropyl phosphoramidite) uridine residue purchased from Glen Reserch Inc. The target RNA strand was extended to the CPG solid phase carrier to which was bound. As the phosphoramidite activation reagent, 0.25 M 5 benzylthio-1H-tetrazole in anhydrous acetonitrile was used, the condensation time was changed to 10 minutes, and the treatment time of 3% dichloroacetic acid solution for detritylation was increased. The synthesis was carried out according to the RNA synthesis protocol (tritylon) set in advance in the above automatic synthesizer, except that it was extended to 30 seconds. After the synthesis, the solid support was immersed in a large excess of concentrated ammonia / ammonium acetate (10: 1, w / w) and left for 1 hour. By passing the solution through a C18-cartridge column to remove by-products, the 2% trifluoroacetic acid aqueous solution was used to remove the cage dimethoxytrityl group, and the target product was eluted with distilled water containing acetonitrile. The resulting solution was lyophilized to dryness, and 3 mL of 1M tetraptylammonium-fluoride Ztetrahydrofuran propylamine (20: 1, ν Ζν) was added to it and allowed to stand at room temperature for 12 hours. The group was removed. To the solution was added 0.1 M aqueous ammonium acetate solution, and the mixture was concentrated under reduced pressure. Dissolve the residue in distilled water and pass it through an OASIA MCX cartridge column and then through an HLB cartridge column. The concentration gradient was loaded at a rate of 1% for 1 minute). The isolation yield was 15%.

[0049] Further, when MALDI-TOF mass spectrometry was performed on the synthesized RNA oligomer, the theoretical value (9177. 79) and the measured value (9119. 32) were in agreement.

[0050] This specification includes the contents described in the specification and Z or drawings of the Japanese patent application (Japanese Patent Application No. 2005-64883) which is the basis of the priority of the present application. In addition, all publications, patents and patent applications cited in the present invention are incorporated herein by reference as they are.

Claims

The scope of the claims

Formula (I)

[Chemical 2]

R

(Where

[Chemical 3]

[Wherein, X, Y and B are as defined above. ]

[2] The method for deprotecting a 2 ′ hydroxyl group according to claim 1, wherein the fluorine-containing compound is tetraalkylammonium fluoride.

[3] The method for deprotecting a 2 ′ hydroxyl group according to claim 2, which is tetraalkylammonium fluoride tetra (n-butyl) ammonium fluoride.

4. The method for deprotecting a 2 ′ hydroxyl group according to claim 1, wherein the basic compound is ammonia, a primary amine, a secondary amine, a tertiary amine, or an amidine compound.