JPS60152495A - Preparation of protected nucleoside - Google Patents

Abstract

PURPOSE:To obtain the titled compound having protected functional groups in high purity efficiently by easy operation, by reacting a raw material nucleoside with a protecting agent in the presence of a hydrophilic solvent, and mixing the reaction solution with water to precipitate and separate selectively the reaction product. CONSTITUTION:(A) A raw material nucleoside is reacted with (B) a protecting agent or (C) a deprotecting agent to protect a nucleoside having at least partially protected functional groups. In the process, the total amount of the component (A) and further the total amount of the component (B) which is a water-insoluble substance are converted into a protected nucleoside in the presence of a hydrophilic solvent. The reaction solution is then mixed with water to precipitate and separate selectively the reaction product, and if necessary the resultant reaction product is dehydrated to afford the aimed compound having at least partially protected functional groups.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing a nucleoside in which at least a portion of the functional group is protected by a protecting group. 0 Regarding methods In recent years, chemical synthesis of DNA and RNA has become popular.

There is an increasing demand for protected nucleosides, in which some or all of the functional groups present in nucleosides are protected with protecting groups, as raw materials.0 Such protected nucleosides generally start from natural nucleosides in which functional groups are not protected. The specific method is ■
A method of directly protecting a natural nucleoside, a method of further protecting a partially protected nucleoside with another protecting agent, and a method of partially deprotecting a protected nucleoside are known.

In the case of these methods, 5 unreacted raw materials, protective agents, by-products, etc. coexist in the reaction system, so a step of separating the target product from the reaction solution is essential. A method (for example, Methods
inFfnzymolθg7 VOL, 65, page 610, published in 1980) 2) In hydrophobic solvents such as chloroform and dichloromethane@
A method in which the dissolved mixture is adsorbed on a silica gel adsorbent through a column, developed with an eluent such as chloroform-methanol, dichloromethane-methanol, etc., and fractionated by separation (for example, the above-mentioned literature) 3) Acetone-water , a mixture dissolved in an aqueous mixed solvent such as acetonitrile-water is passed through a column to be adsorbed on an alkylsilanized silica gel adsorbent, and then the mixed solvent is developed,
Methods of elution and fractionation as an eluent
in lcn*ymology VOL, 6B, page 90, published in 1979), and 1) above.
, 2), and 3) have been reported.

However, method 1) has drawbacks such as insufficient extraction effect, coexistence of a hydrophilic solvent may cause emulsion, making separation difficult, and a large amount of solvent must be removed after extraction.

In addition, methods 2) and 6 have the disadvantage that they are complicated to operate, are likely to cause decomposition of the target product during the operation, and require a step to recover the target product from a dilute fractionated solution. The inventors of the present invention have carried out intensive studies to improve the shortcomings of conventional methods and to develop a method that can efficiently produce a large amount of protected nucleosides with simple operations and with high purity. They discovered that it is extremely effective to combine specific reaction conditions and specific purification steps, leading to the completion of the present invention ◇ Thus, according to the present invention, raw material nucleoside (-1) and protecting agent (bl or When producing a nucleoside (6) with at least a part of the functional group protected by a deprotecting agent (clk reaction), the production process is carried out in the presence of a hydrophilic solvent (
-) When substantially the entire amount of the component and (b) component is a water-insoluble substance, the step of converting the substantially entire amount of the component (bl) into the component (11) + 11, mixing the reaction solution of the step with water and (a step (2) of selectively precipitating and separating the dl component and, if necessary, a step (3) of dehydrating the 41 component). ◇In the present invention, a reaction between the (-1 component and the (tl) component) or a reaction between the (-1 component and the (C1 component) is performed in the first step, and at least one of the functional groups is A partially protected nucleoside (d) is synthesized.

The (-) component to be subjected to the reaction is capable of producing a nucleoside with a part or all of the functional groups protected by protection or deprotection, and is used for the reaction with the (b) component. ) components include unprotected or partially protected nucleosides, while (C
The (,) component used in the reaction with the () component includes a fully protected nucleoside or a partially protected nucleoside having a protecting group at position 2 or more.

Specific examples of such (-1 component) include compounds in the following categories. Namely, as specific examples of sugars constituting nucleosides, 2'-thioxy+7bose type or lipose type furanose is the most preferred. Commonly used include arabinose 3/f oxyribose, 21.
Other furanoses such as 31-dideoxyribose, derivatives such as 5'-70 rhodemexyribose and 3'-aminoribose may be used, and pyranose types such as mafnose and glucose may also be used.

Specific examples of bases bonded to the 1'-position of Kinuta include common nucleobases such as adenine, guanine, thymine, /tokun, tsu2syl, inosine, 5-methyl7tosine, 5-hydroxymethylcytosine, etc. In addition, 8-bromoadenine, 5-fluorouracil, 6-thioguanine, and 6-mercaptopurine.

Various derivatives such as 5-nitrouracil and etenocytidine, 5-azaortic acid and 6-azauridine.

Examples include nitrogen-containing heterocyclic bases having similar structures such as 6-azacytidine, pyridone, and penzimidazole.

Furthermore, any commonly used preservation agent can be used to protect the functional groups present in the ears and the base.

For example, protective groups for amino groups include acetyl group, imbutyryl group, benzoyl group, anisoyl group, naphthoyl group, 0-phthaloyl group, etc. Generally, when the base is a cutidyl group or an adenyl group, a benzoyl group is used.

In the case of a guanyl group, an isobutyryl group is preferred.

Examples of protecting groups for hydroxyl groups include those forming a carboxylic acid ester structure such as acetyl, imbutyryl, benzoyl, anisoyl, naphthoyl, monomethoxytrityl, dimethoxytrityl,
There are those that form an ether structure such as tolyldiphenylmethyl group, 0-methoxyphenylnaphthylphenitetrahydropyranyl group, methoxytetrahydropyranyl group, etc. For protection at the 3'-position, a carboxylic acid ester type is used. Ether structures are preferred for protection at the 5'-position.

Furthermore, examples of the protecting group for the adjacent diol include those that form an orthoformate structure and those that form a ketal structure.

On the other hand, the component (1) to be subjected to the reaction is a drug for protecting functional groups such as hydroxyl groups and amino groups present in the component (,). Substances are exemplified. These substances are usually supplied in the form of acid halides, acid anhydrides, alkyl halides, allyl halides, dihydrobilane, etc. Component (C) to be subjected to the reaction is It is a drug that eliminates some of the

For example, bases such as ammonia, sodium hydroxide, potassium hydroxide, etc. are used to remove imbutyryl groups and benzoyl groups that protect d-amino groups, and monomethquine trityl groups and dimethoxytrityl groups that protect hydroxyl groups. For removal of such substances, acids such as acetic acid, acetic acid, benzene, sulfonic acid, and zinc bromide can be used.

In the present invention, it is necessary to consume substantially the entire amount of component (a) by reacting the (al component and (bl component) or the (a) component and (C) component in a hydrophilic solvent. 0 Also, in the case of (b+ acid component) being non-water-bathable, it is also necessary to consume substantially the entire amount of this component.

Here, substantially the entire amount can be taken, and although it is not necessarily uniform depending on the purity required for the target product, 1 usually L95 mol head or more, preferably 99 mol head or more, more preferably B9q,
The higher the conversion rate, the higher the conversion rate, the higher the purity of the target product.

The hydrophilic solvent used in the reaction may be any solvent as long as it is inert to the reaction and can homogenize the system. Among them, those that are completely compatible with water at 25 volumes or more are preferred, and benzene, toluene, etc. Suitable solvents are those that are also compatible with aromatic solvents such as, cyclene, and the like.

Specific examples of such hydrophilic solvents include methanol, ethanol, inglobanol, ethylene glycol monoethyl ether, tetoxyhydrofurfuryl almal, ethylene glycol monoacetate, acetol,
Diacetone alcohol, cyanoethanol, 2-aminoethanol.

Alcohols such as acetone, methyl ethyl ketone, etc.; ethers such as tetrahydrofuran, dioxane, dioxolane, morpholine, etc.; amines such as propylamine, butylamine, pyridine, picoline, pyrimidine; acetonitrile, propionitrile, etc. Nitriles such as; acetic acid,
Carboxylic acids such as propionic acid and butyric acid; amides such as dimethylformamide, dimethylacetamide, N-methylpyrrolidone, etc.; dimethylsulfonate, diethylsulfoxide, tetrahydrothiophene-1
, 1-dioxide, etc., and may be used by appropriately mixing them as necessary.

Among them, those with high solubility of the Al component and product, neutrality, stability, low toxicity, and low price are preferred, and those with a boiling point range of about 40 to 120 C are particularly preferred from the viewpoint of ease of handling. Specific examples of such preferred solvents include ethanol, acetone, methyl ethyl ketone,
Examples include tetrahydrofuran, dioxane, pyridine, and picoline.

(The reaction between component -1 and component (b) is usually 1 to 5 moles of (bl component, preferably 11 to 15 moles of bl component, per 1 mole of al component)
100 to 100 parts by weight of both components
It is carried out in the presence of 2000 parts of a hydrophilic solvent.

The reaction temperature varies depending on the type of component (b); for example, when a fluorothermic reaction occurs with acid halides, acid anhydrides, etc., the reaction temperature is kept at room temperature or below, preferably below freezing point (b+acid component gradually increases). The reaction is generally carried out at room temperature when component (b) causes an endothermic reaction such as an alkyl halide or allylnol 2ide. Time is 5-180
minutes, preferably 10 to 30 minutes.

(,), (b) If it is necessary to consume substantially all of both components, the ratio of each component to be added may be strictly controlled, or a nitrogen-containing base such as pyridine may be used as an activator. Alternatively, it is necessary to take appropriate measures such as setting the reaction temperature high.

On the other hand, the reaction between component (, 1 and component (C) is (a) component 1
Usually per mole (1 to 5 moles of C1 component, preferably 2 to 3 moles)
This is done by using moles. The amount of the hydrophilic solvent to be used may be appropriately selected within the range that allows the formation of a uniform solution of component (a), but it is usually used at a ratio of (a + acid component 1 to 20 times the weight).The reaction conditions are the usual method. For example, it is carried out at a temperature of -10 to SOC for several minutes to several hours.

For each component (al, ('b), and (c), in addition to the specific examples above, see "Nucleic Acid Organic Chemistry" (Ikehara et al.,
All compounds disclosed in Kagaku Doujin (Published in 1979) that meet the definition of the present invention are included.The reaction solution thus obtained in the first step is
Selective precipitation of the desired partially protected nucleoside compound (dl) is achieved by optionally removing a portion of the hydrophilic solvent and then mixing with water in a second step. The amount of water is sufficient to precipitate the compound (61), and it is usually at least 2 times the volume of the reaction solution, preferably 3 It is 200 times the volume, more preferably 5 to 100 times the volume, and the optimum value can be easily determined by conducting a simple preliminary experiment depending on the composition of the reaction solution.

In addition, when mixing water, the ionic strength in the system can be increased by coexisting an inert salt with compound (d).
This treatment promotes the precipitation power of compound (41) and improves the recovery rate, but if the ionic strength is increased too much, compound (41)
6) shows a tendency to decrease in purity, so its strength is 5M.
/J3 or less, particularly α1 to 2M/no, is preferable. Specific examples of such salts include, for example, sodium chloride,
Neutral salts such as ammonium chloride, sodium sulfate, sodium phosphorus rR2, dipotassium hydrogen phosphate,
hydrogen carbonate), acid salts of polybasic acids such as triethylamine hydrogen carbonate, mixtures of acids and bases appropriately combined to have a pH buffering effect (e.g. a combination of acetic acid and potassium hydroxide, hydrogen carbonate, etc.) (a combination of IJum and sodium hydroxide, etc.) etc.

Among these, compounds that have pH buffering effects in the pH range where dl is stable will receive awards.

The method of mixing the reaction solution and water is not particularly limited and may be a method of adding water to one reaction solution or a method of adding the reaction solution to water. It may be selected as appropriate depending on the composition of
40C5 Preferably at about 0 to 3 aC [L5 to 180 minutes, preferably by stirring for 2 to 30 minutes. The pH of the system can be selected as appropriate, but it is usually 5 p.
Maintaining the stability of compound (4) within the range of H2 to 11,
and a pH that increases the solubility of other components in the mixed solvent.
It is appropriate to select the area. Further, the mixing device is not particularly limited, and either a batch system or a continuous system can be used.

In the present invention, by performing such a simple operation, the compound (dl) can be recovered in the form of a viscous candy or powder.Also, if necessary, the recovered compound (fdl) can be rehydrated in a hydrophilic solvent. The operation of dissolving compound #41 and mixing it with water can be repeated two or more times, thereby further increasing the purity of compound #41.Especially when all salts are present in the mixed solvent, Since salt is likely to be entrained in the recovered material + al, it is preferable to increase the salt removal rate by repeatedly treating with a mixed solvent that does not contain salt.

The thus obtained compound (dl) usually contains 10 to 40 parts by weight of water, but if it is necessary to remove water, it can be dehydrated by an appropriate method in the third step. Specifics of such drying method Take Hiro for example.

For example, t-1'n-hebutane, toluene 1. There are methods such as dehydration as an azeotrope using a solvent such as pyridine or dioxane, drying under reduced pressure, and drying after dissolving in a solvent and dehydrating using a dehydrating agent.

Thus, according to the present invention, by combining the reaction conditions in the first step and the purification means in the second step, it is possible to achieve higher yields and higher purity with much simpler operations compared to conventional methods. Compound (dl) can be obtained.

The present invention will be explained in more detail with reference to Examples below.

Example l N6-penzoyltaoxyadenosine C (a+sei 135
3i (1wo/) was dissolved in pyridine 700& and after azeotropic dehydration, pyridine 2-1jK was dissolved and stirred while dimethoxytrityl chloride [component (b)) 559i (1mo'!
) was added and reacted for 30 minutes at room temperature (Step 1). Transfer 40 μl of the reaction solution to a normal phase silica gel thin layer chromatography plate.
chloroform/methanol (2/1, V/
V) Developed with a mixed solvent.

Since the presence of N6-benzoyldeoxyadenokune was no longer recognized by an ultraviolet (260 nm) detector, 1M sodium bicarbonate water bath solution 352 was gradually added under water cooling, and after stirring at room temperature for 10 minutes, the formed precipitate was left behind. The precipitate was then removed with an incline (permeation m2-1>). The precipitate was dissolved in 70% of acetone and 10% of water was added with stirring at room temperature to obtain a precipitate again (Step 22).

Next, when the precipitate was vacuum dried (Step 3), its weight was 624 fP? there were. In addition, 10 μf of the precipitate was adsorbed onto a normal-phase silica gel thin layer chromatography plate, developed with a chloroform/methanol-10/1 (V/V) mixed solvent, and detected with a 260 nm ultraviolet detector, and one spot was found. In addition, using a normal phase 7 silica gel column and a reverse phase octadecyl silica gel column, chloroform/methanol/pyridine (so/11
0. o s ) and acetone/water/pyridine (7/x1
0. o 1) When eluted with a mixed solvent and analyzed with an R detector and a UV detector (260 Roughy), the purity was 9B, 9
%Met. The yield of 5'-dimethoxytrityl-N6-penzoyldeoxy/adenosine was 933%.

Comparative Example 1 In the same manner as in Example 1, (, 1, (1, 1, 1) components were reacted, and then 1 reaction solution was poured into OC water El with stirring, and ethyl acetate 10 form was added thereto for 10 minutes. Then, the oil layer was completely stripped, poured into anhydrous sodium sulfate A2 for 9 times, dried in one apparatus, and then the solvent was distilled off.When purifying the residual liquid by normal phase silica gel two-person chromatography, the sample was Because of the large amount, the purification was carried out in 5 batches using a chromatography tube packed with silica 5k (IF).The developing solution for each batch was initially chloroform/pyridine:/=l 0010.1
(V/V) 20°11 then chloroform/methanol/pyridine 1oo/s10. t (v/v/v)20
It took ~251. Fraction 91-e containing the target product was distilled off using an evaporator, and the weight of the product obtained by vacuum drying was 612? Met.

Immediately after the reaction, there was a small spot of the substance from which the dimethoxytrityl group was removed, in addition to the large spot of the component. Since it was not observed, it is presumed that a small amount of deprotected product was generated in the silica column. Purity was analyzed by high performance liquid column chromatography in the same manner as in Example 1.

It was 95.6%. 5'-〇-dimethquine trityl N
The yield of 6-pennoylzooxyadenokune was 90.4%.
Met.

Example 2 2'-deoxyadenosy 72501 (1!IIoe)
f After mixing with pyridine, stir and suspend in 2.5 liters of pyridine, and add O
Add benzoyl chloride 843F (6 mol) dropwise to react while maintaining the temperature at C. ◇ After 40μ7fta of the reaction solution was deposited on a normal phase silica gel thin-phase chromatography plate, react with a chloroform/methanol (10/1 (V/l) mixed bath part-mum agent). After developing and no longer detecting the presence of deoxyanosine using an ultraviolet detector, add 25 g of a 1M aqueous solution of sodium bicarbonate under water cooling, stir, and leave to stand still to completely remove the resulting overhang by tilting. A precipitate of the protected reaction product [component (a)] was obtained.Then, the precipitate was dissolved in a mixed solvent of tetrahydrofuran/ethanol/pyridine-2,13/5, B/24, and 2M caustic soda was added while maintaining the OC. (C) Component] 41/
A mixed solution of 4 J of ethanol was added dropwise. After the dropwise addition, the mixture was returned to room temperature and reacted until no fully protected nucleoside was observed before the reaction by normal phase thin layer chromatography (Step 1). After the reaction, 23 g of 1M aqueous sodium bicarbonate solution and 2 g of salt were added. s kg was added and allowed to stand while cooling to OC to crystallize the precipitate (Step 2).

The precipitate was filtered, washed with water, dehydrated with pyridine, and dried under reduced pressure (Step 3). The yield of N6-benzoyldeoxyadenosine produced was 240f.

The yield based on 2'-deoxyadenosine was 68.1%. The purity measured by ultraviolet absorption method was 98.0%.

The experiment was carried out in the same manner as in Example 1, except for the following. The results are shown in Table 1.

Example 4 An experiment was carried out in the same manner as in Example 1, except that the water bath liquid used in step (2-1) was changed as shown in Table 2. The results are shown in Table 2.

Table 2

Claims (1)

[Scope of Claims] 1. When producing a nucleoside (dl) in which at least a part of the functional group is protected by reacting the raw material nucleoside (-1 with a protecting agent (1,) or a deprotecting agent (C1), The manufacturing process is a process of converting substantially the entire amount of component (Al) and, if component (b) is a water-insoluble substance, substantially the entire amount of component (a) into component (41) in the presence of a hydrophilic bath agent. 1),
A functional method characterized by comprising a step (2) of selectively precipitating and separating the component (4) by mixing the reaction solution of the step with water, and a step (3) of dehydrating the FDL component as necessary. Process for producing a nucleoside in which at least a portion of the group is protected ◇ 2 Production method 06, step (2) according to claim 1, wherein the precipitation separation operation in step (2) is carried out in the presence of a salt. The manufacturing method according to claim 1, wherein the precipitation separation operation in step (2) is repeated two or more times. 4. The method according to claim 3, comprising the step of: