KR20160125772A - A method of preparing L-nucleic acid derivatives comprising nanofiltration - Google Patents
A method of preparing L-nucleic acid derivatives comprising nanofiltration Download PDFInfo
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- KR20160125772A KR20160125772A KR1020150056668A KR20150056668A KR20160125772A KR 20160125772 A KR20160125772 A KR 20160125772A KR 1020150056668 A KR1020150056668 A KR 1020150056668A KR 20150056668 A KR20150056668 A KR 20150056668A KR 20160125772 A KR20160125772 A KR 20160125772A
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- nucleic acid
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H9/00—Compounds containing a hetero ring sharing at least two hetero atoms with a saccharide radical
- C07H9/06—Compounds containing a hetero ring sharing at least two hetero atoms with a saccharide radical the hetero ring containing nitrogen as ring hetero atoms
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
Abstract
The present invention relates to a process for preparing an L-nucleic acid derivative using an L-arabinaminooxyoxazoline derivative and a process for purifying a high-purity L-nucleic acid derivative using a nanofiltration process. The nano-filtration method can be introduced into the purification step of the L-nucleic acid derivative to purify the high-purity L-nucleic acid derivative in a short time while greatly reducing the generation of the waste solution.
Description
The present invention relates to a method for producing an L-nucleic acid derivative comprising the step of purifying using a nanofiltration membrane.
Naturally occurring nucleic acids are components of DNA and RNA, and their skeletons are widely used as medicines. Naturally occurring nucleic acids are in a D-configuration, and so far, D-derivatives have been used as pharmaceuticals. However, recently, their usefulness as non-natural L-nucleic acid derivatives, particularly L-thymidine, has attracted attention.
In order to prepare the L-nucleic acid derivative, the L-form sugar, which is the basic skeleton, is required as a raw material. However, the L-form sugar or its derivative is not present in nature, and L-arabinose, which is an L- A method of using L-arabinose as a raw material for an existing L-type is known as a general method for synthesizing an L-nucleic acid derivative.
The L-nucleic acid derivative is prepared by forming an intermediate by L-arabinose adduct of L-nucleic acid derivative with an alkali metal carbonate to finally form an L-nucleic acid derivative .
In the process for preparing the L-nucleic acid derivative, a large amount of metal salt is generated in an intermediate cyclization reaction, and since water is used as a reaction solvent, both the reaction intermediate having high solubility in water and the final product are dissolved in water. Lt; / RTI > of the L-nucleic acid derivative, a purification process is essential. After completion of the reaction, various steps for removing the metal salt and the unreacted material are required. In the conventional purification method, water as a reaction solvent is removed in a vacuum state at a temperature of 50 ° C to concentrate the L-nucleic acid derivative, The metal salt and the unreacted material are first extracted and removed using an excess solvent such as ethanol, the solvent is removed again in a vacuum state at a temperature of 50 ° C, and the above process is repeated to extract and remove the metal salt and the unreacted material Method.
The purification method for obtaining the high-purity L-nucleic acid derivative takes a long time in the process of concentrating the L-nucleic acid derivative solution, eluting the product from which the inorganic salt has been removed, and concentrating it in the vacuum. In addition, in the process of removing the solvent of the L-nucleic acid derivative solution by evaporation, the L-nucleic acid derivative is decomposed by the temperature rise to generate a large amount of loss, and hydrolysis by-products, which are main reaction products, are produced, Is formed in an increased amount. Thus, according to the conventional purification method, it takes a long time to purify L-nucleic acid derivatives of high purity, and the generation of waste liquid is greatly increased due to the use of excessive amount of purified water and solvent, There is a problem that the property is extremely deteriorated.
On the other hand, a technique of using a separation membrane as one of the methods applied to a purification process is known. Membrane (membrane) is a phase separating two three-dimensional homogeneous phases. Membrane filtration column is a method of separating substances in molecular state by using this membrane. The filtration membrane is largely divided into reverse osmosis, nanofiltration, ultrafiltration or microfiltration according to the pore size or separation phenomenon of the membrane. Among them, the nanofiltration Is a method of passing a predetermined substance through a nanofiltration membrane having a nanoscale pore and separating it into a component having a size smaller than that of the air and a component having a larger size.
Nanofiltration membranes are used to remove multi-valent ions and low-molecular organic substances from water. Nanofiltration using the nanofiltration membranes is widely used in dairy technology (refer to Patent Publication No. 2005-12242), waste solution regeneration treatment technology (Patent Publication No. 1999-66922 (WO 98/15581 A1) or refining technology for lithium reuse (WO 98/15581 A1), sugar-making technology (U.S. Patent No. 6,406,546), technology for removing viruses from proteins (European Patent Publication No. 0979237B1), carbohydrate purification technology 59385A1), and the like.
As a result of intensive researches to develop a method for producing a high purity L-nucleic acid derivative from an L-arabinaminooxoxazoline derivative, the present inventors have found that when L-arabinaminooxazoline derivative is reacted with an alkali metal salt in a solution state, Nucleic acid derivatives are prepared and the resulting metal salts and unreacted materials can be easily removed by purification using a nanofiltration membrane. Thus, it has been confirmed that a high purity L-nucleic acid derivative can be purified in a short time while reducing the generation of a waste solution, .
According to an aspect of the present invention,
A first step of reacting an L-arabinaminooxazoline derivative represented by the following formula (1) with an alkali metal carbonate in a solution state to prepare an L-nucleic acid derivative represented by formula (2); And a second step of purifying the L-nucleic acid derivative solution prepared in the previous step using a nanofiltration membrane.
[Chemical Formula 1]
(2)
In this formula,
R 1 is a C 1-6 alkyl group;
R 2 and R 3 are each independently a hydroxyl group or a C 1-6 hydroxyalkyl group; And
X is bromine, chlorine, mesylate, acetate, p-toluenesulfonyloxy or methanesulfonyloxy.
Hereinafter, the present invention will be described in detail.
In order to prepare an L-type nucleic acid derivative, a L-type sugar, which is a basic skeleton, is required as a raw material. L-arabinose is generally used as an industrially available raw material for an L- L-nucleic acid derivative is prepared by synthesizing a corresponding L-type portion from os through a multistage process. For example, in the case of the L-nucleic acid derivative L-FMAU (1- (2'-deoxy-2'-fluoro-β-L-arabinofuranosyl) thymine), the L- , 5-di-O-benzoyl-1-bromo-2-deoxy-2-fluoro-β-L-arabinofuranose and reacting with silylated thymine followed by deprotection. Nucleosides and Nucleotides: 18 (2) (1999) 187 (1999) 187 (1999) 187. The entire process for the preparation of L-nucleic acid derivatives involves a total of 14 processes, which take a long time to process and include industrially unsafe processes such as chromic acid oxidation. -195].
Thus, the inventors of the present invention have developed a method for producing L-nucleic acid derivatives using L-arabinose as a raw material by a simple process, and have succeeded in using L-arabinaminooxyoxazoline derivatives which can be easily derived from L-arabinose An L-nucleic acid derivative, L-2,2'-anhydro-1- (β-L-arabinofuranosyl) thymine can be synthesized through a cyclization reaction and an isomerization reaction.
On the other hand, in order to obtain a high-purity L-nucleic acid derivative, a process of purifying inorganic salts or organic impurities produced during the reaction is required. In order to elute the L-nucleic acid derivative dissolved in the aqueous solution as the reaction solvent, a purification method using a resin such as a cation-exchange resin or a non-ion-adsorbing resin has been developed. This is because a solution in which the L- A separate solvent is required to elute the L-nucleic acid derivative from the resin and the time for adsorption and elution.
Therefore, the present invention is based on the first finding that a purification method using a nanofiltration membrane as a method for obtaining a high-purity L-nucleic acid derivative in a short time while reducing the generation of a waste solution, and an economical and efficient purification using the nanofiltration method are possible .
An inorganic salt having a pore size smaller than that of the nanofiltration membrane or an organic impurity having a molecular weight of 250 or less is removed through the nanofiltration membrane by applying the nanofiltration method to the L-nucleic acid derivative solution produced during the production of the L-nucleic acid derivative, L-nucleic acid derivatives larger than the size remain behind the membrane and are separated from impurities. Therefore, it is possible to easily purify the L-nucleic acid derivative of high purity from the L-nucleic acid derivative solution, and the amount of the solvent used during the purification process can be significantly reduced as compared with the prior art.
In addition, when the nanofiltration membrane is used, the temperature during the purification process can be adjusted to room temperature (25 ° C) or less, and the amount of the hydrolysis by-products generated in the conventional purification process conducted under a vacuum of 50 ° C can be significantly reduced.
The L-nucleic acid derivative solution to be purified in the present invention can be prepared by the first step.
The alkali metal carbonate may be potassium carbonate (K 2 CO 3 ), sodium carbonate (Na 2 CO 3 ), sodium hydrogencarbonate (NaHCO 3 ), potassium hydrogencarbonate (KHCO 3 ), or a mixture thereof.
R 2 constituting the L-arabinaminooxyoxazoline derivative may be a hydroxymethyl group and R 3 may be a hydroxyl group.
The L-nucleic acid derivative to be purified may be L-2,2'-anhydro-1- (β-L-arabinofuranosyl) thymine.
The L-nucleic acid derivative solution to be purified may be a solution in which the L-nucleic acid derivative is dissolved in water or a mixed solvent containing water, and the L-nucleic acid derivative in the reaction solution state resulting from the production of the L- Or may be a solution in which the L-nucleic acid derivative produced by recrystallizing the L-nucleic acid derivative in the reaction solution state is dissolved in another solvent.
The L-nucleic acid derivative solution to be purified may be a substance having an L-nucleic acid derivative purity of 10% or less including an inorganic salt such as potassium chloride or potassium bromide and an organic impurity having a molecular weight of 250 or less.
The L-nucleic acid derivative solution to be purified may be a solution in which the L-nucleic acid derivative is dissolved in water or a mixed solvent at a concentration of 1 to 20%, preferably in a concentration of 1 to 10% More preferably in a concentration of 3 to 8%.
If the concentration of the L-nucleic acid derivative solution to be purified is excessively low or high, it is difficult to obtain a high-purity L-nucleic acid derivative or it is difficult to purify the L-nucleic acid derivative solution in a short period of time. In the case of L-nucleic acid derivatives in the reaction solution state obtained in the production of L-nucleic acid derivatives, when the L-nucleic acid derivative does not satisfy the concentration condition, the L-nucleic acid derivative obtained by recrystallizing the L- -Nucleic acid derivative can be dissolved in an appropriate solvent, and a new L-nucleic acid derivative solution corresponding to each of the above-mentioned conditions can be prepared to perform nanofiltration.
The pH of the L-nucleic acid derivative solution to be purified may be 2.0 to 12.0, preferably 4.0 to 9.0. If the pH of the L-nucleic acid derivative solution is too low or too high, the nanofiltration membrane may be damaged during nanofiltration.
The nanofiltration of the L-nucleic acid derivative solution according to the second step can be performed using a tubular nanofiltration membrane having a built-in module. The nanofiltration membrane in the tubular module form can be a thin film having a MgSO4 rejection of greater than 95%, for example, "DK4040F" of the commercially available General Electric (GE).
The pressure of the nanofiltration step is preferably 0.5 to 5.0 MPa, and more preferably 1.0 to 3.0 MPa. When the pressure is 0.5 MPa or less, it may be difficult to purify the L-nucleic acid derivative solution within a short time.
The temperature of the nanofiltration step is preferably 0 to 40 캜, more preferably 5 to 30 캜. If the temperature is outside the above range, the nanofiltration membrane may be damaged.
The concentration of the purified L-nucleic acid derivative solution according to the nanofiltration step may be 3 to 15% by weight, and preferably 6 to 8% by weight.
In the case of purifying L-nucleic acid derivatives using the nanofiltration method according to the present invention, it is possible to remove a large amount of inorganic salts and organic impurities, which are by-products of the synthesis process, without removing the purification step of several steps, The product can be concentrated without further processing.
The method for producing an L-nucleic acid derivative according to the present invention can produce an L-nucleic acid derivative by a simple process using an L-arabinaminooxyoxazoline derivative easily derivable from L-arabinose have.
In addition, the method of purifying L-nucleic acid derivatives by introducing the nanofiltration process can purify L-nucleic acid derivatives of high purity within a short time while greatly reducing the generation of waste liquid, It can contribute to mass productivity and efficiency.
Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are for further illustrating the present invention, and the scope of the present invention is not limited by these examples.
Example 1: Preparation of L-nucleic acid derivative
(1.15 kg) and p-methoxyphenol were dissolved in an aqueous solution of 10 L and cooled to 8-10 [deg.] C in an ice bath. Then, the same equivalent amount of potassium carbonate was added with stirring for 1 hour The mixture was stirred for 4 hours or more. After completion of the reaction, 2 moles of HCl solution was added dropwise while maintaining the temperature at 0 to 4 占 폚, and the solution was deaerated with strong gas evolution. The pH of the resulting solution was approximately 6. The reaction mixture was stirred overnight to obtain an aqueous solution of L-2,2'-anhydro nucleic acid derivative.
The Pd (5%) catalyst in aluminum oxide in an individual vessel was suspended in water under a nitrogen atmosphere and purged with hydrogen for 10 minutes. The suspension was heated to 60-65 [deg.] C for approximately one hour under a hydrogen atmosphere. Then, the hydrogen flow was stopped and purged with nitrogen, and an aqueous solution of L-2,2'-anhydro nucleic acid derivative was added to the suspension while maintaining a temperature higher than 60 ° C. The reaction mixture was purged with hydrogen for another 10 minutes and then purged with nitrogen for another 2 minutes. Additional purging cycles using nitrogen and then hydrogen were performed. After the purged mixture was cooled to room temperature and purged with nitrogen, the pH of the solution was adjusted to approximately 6.5 using 2 mol of aqueous HCl.
Example 2 Purification of L-Nucleic Acid Derivatives
The prepared L-nucleic acid derivative solution was filtered using a module equipped with a nanofiltration membrane having a MgSO 4 rejection of 95% or more. As the nanofiltration membrane, DK4040F manufactured by GE Corporation was used, and a solution of the L-nucleic acid derivative at a concentration of 7 wt% was supplied into the module, followed by nanofiltration.
Specifically, in the nanofiltration process, the L-nucleic acid derivative solution concentrated at 20 to 30 ° C was circulated, and the filtration pressure was 1 to 3 MPa. Next, the nanofiltration was performed on the resultant product in which inorganic salts and organic impurities were removed through the nanofiltration process. The process was performed at a pressure of 0.5 to 3.5 MPa, a temperature of 0 to 40 ° C, 2. ≪ / RTI >
The treatment results of the nanofiltration process are shown in the following table. Table 1 and Table 2 show the treatment results according to the change of the filtration pressure and the filtration time, respectively. The results obtained by increasing the amount of the filtrate by 5 times (50 L) are shown in Table 3.
Also, the solution filtered through the nanofiltration process was collected again, and the filtrate was maintained at a concentration of 7 wt%, which is the concentration of the initial L-nucleic acid derivative solution. Thus, the L-nucleic acid derivative The results for the degree of loss are shown in [Table 4].
As can be seen from the above Tables 1 and 3, the filtration time and the loss of the L-nucleic acid derivative are the least when the filtration pressure is the highest 3 MPa. When the pressure of 3 MPa is maintained, Approximately 90% of impurities and water were removed within 10 minutes of time. In addition, about 90% of impurities and water were removed by filtration of 50 L of the L-nucleic acid derivative solution in which the amount of the L-nucleic acid derivative was increased by 5 times.
In addition, as shown in Table 4, no change in the concentration of the L-nucleic acid derivative was observed even after 12 hours of filtration while maintaining the initial concentration of the L-nucleic acid derivative solution. From the results, There was no loss of the L-nucleic acid derivative due to immersion in the solution.
This is because, in the purification step using the nanofiltration method, the L-nucleic acid derivative solution passes through the nanofiltration membrane, and thus inorganic salts and organic impurities can be removed and a separate solvent or time is not required for eluting the L-nucleic acid derivative solution , And it is possible to concentrate a solution of 80% or more within 10 minutes without applying a separate vacuum concentration process, thereby shortening the time required for concentration.
Claims (11)
A second step of purifying the L-nucleic acid derivative solution prepared in the previous step using a nanofiltration membrane;
[Chemical Formula 1]
(2)
In this formula,
R 1 is a C 1-6 alkyl group;
R 2 and R 3 are each independently a hydroxyl group or a C 1-6 hydroxyalkyl group; And
X is bromine, chlorine, mesylate, acetate, p-toluenesulfonyloxy or methanesulfonyloxy.
Wherein the alkali metal carbonate is selected from the group consisting of potassium carbonate (K 2 CO 3 ), sodium carbonate (Na 2 CO 3 ), sodium hydrogencarbonate (NaHCO 3 ) and potassium hydrogen carbonate (KHCO 3 ) Gt;
R 2 is a hydroxymethyl group, and R 3 is a hydroxyl group.
Wherein the L-nucleic acid derivative is L-2,2'-anhydro-1- (β-L-arabinofuranosyl) thymine.
Wherein the L-nucleic acid derivative solution is an L-nucleic acid derivative wherein the L-nucleic acid derivative is dissolved in a water or a water-containing mixed solvent.
Wherein the L-nucleic acid derivative solution in the second step contains an inorganic salt, an organic impurity, or a combination thereof having a molecular weight of 250 or less.
Wherein the concentration of the L-nucleic acid derivative solution in the second step is 1 to 20 wt%.
And the pH of the L-nucleic acid derivative solution in the second step is 2.0 to 12.0.
Wherein the nanofiltration membrane is a thin film having a MgSO 4 rejection of 95% or more.
Wherein the second step is carried out under the conditions of a pressure of 0.5 to 5.0 MPa, a temperature of 0 to 40 DEG C and a concentration factor of 0 to 2.
And the concentration of the L-nucleic acid derivative solution purified from the second step is 3 to 15% by weight.
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