CN118791536B - A method for removing impurities from a mixture - Google Patents
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- CN118791536B CN118791536B CN202411287594.9A CN202411287594A CN118791536B CN 118791536 B CN118791536 B CN 118791536B CN 202411287594 A CN202411287594 A CN 202411287594A CN 118791536 B CN118791536 B CN 118791536B
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- C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
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- C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
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- C07H19/207—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids the phosphoric or polyphosphoric acids being esterified by a further hydroxylic compound, e.g. flavine adenine dinucleotide or nicotinamide-adenine dinucleotide
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Abstract
本公开涉及一种去除混合物中杂质的方法，所述混合物含有主要成分和杂质，所述杂质为含有二酰亚胺基团的化合物，该方法包括在酸碱反应的条件下，将所述混合物与有机强碱接触，其中，所述主要成分不与所述有机强碱反应。该方法操作简单，显著降低杂质含量，避免亚磷酰胺基团水解。The present disclosure relates to a method for removing impurities from a mixture, wherein the mixture contains a main component and an impurity, wherein the impurity is a compound containing an imide group, and the method comprises contacting the mixture with an organic strong base under acid-base reaction conditions, wherein the main component does not react with the organic strong base. The method is simple to operate, significantly reduces the impurity content, and avoids hydrolysis of the phosphoramidite group.
Description
Technical Field
The present disclosure is in the field of chemistry, and in particular relates to a method for removing impurities from a mixture.
Background
In the fields of chemical industry, pharmacy and the like, the removal of impurities in a mixture is a key step for ensuring the quality and purity of a product. Conventional impurity removal methods include purification methods such as washing, extraction, column chromatography, etc., which, although having many advantages, do not show desired effects in treating a compound containing a specific functional group such as a diimine group, for example, require a plurality of purification steps, which not only increase production costs but may also result in loss of the target product, and furthermore, the increase of steps requires the use of a large amount of organic solvents, which is contrary to the current trend of green chemistry and sustainable development.
CN 101580528a discloses a purification method of cytidine (deoxy) and its derivatives, which comprises the steps of firstly loading an aqueous solution containing cytidine (deoxy) and its derivatives to an alkaline resin, and then obtaining high purity cytidine (deoxy) and its derivatives through a water elution step, wherein impurities in the aqueous solution include uridine (deoxy) and its derivatives. Although this method is simple to operate, it is not applicable to a compound having a group which is easily hydrolyzed as a main component.
Disclosure of Invention
The present inventors have found in oligonucleotide drug studies that it is difficult to remove the imide group-containing impurities in nucleoside monomers or intermediates of nucleoside monomers by conventional purification methods such as washing, extraction, column chromatography, etc. The impurities containing the diimide group are critical impurities, which affect the purity of the oligonucleotide drug and need to be removed from the nucleoside monomer or the intermediate of the nucleoside monomer.
The present disclosure provides a method for removing impurities from a mixture, the mixture comprising a major component and impurities, the impurities being imide group-containing compounds, the method comprising contacting the mixture with a strong organic base under acid-base reaction conditions, wherein the major component is not reactive with the strong organic base.
In some embodiments, the method further comprises adding a non-polar solvent after contacting the mixture with the organic strong base.
In some embodiments, the method further comprises separating a major component from the mixture after the acid-base reaction.
Advantageous effects
The method for removing impurities in the mixture has the advantages of (1) preventing the problem that main components are hydrolyzed in the purification process due to containing groups which are easy to hydrolyze, such as phosphoramidite groups, (2) simplifying the purification operation and being suitable for large-scale amplification on the premise of not influencing the product quality, and (3) effectively removing the impurities.
Incorporated by reference
All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.
Detailed Description
The following describes specific embodiments of the present disclosure in detail. It should be understood that the detailed description and specific examples, while indicating and illustrating the disclosure, are not intended to limit the disclosure.
Those skilled in the art will appreciate that for any group comprising one or more substituents, these groups are not intended to introduce any substitution or pattern of substitution that is sterically impractical, synthetically infeasible, and/or inherently unstable.
The present disclosure provides a method for removing impurities from a mixture, the mixture comprising a major component and impurities, the impurities being imide group-containing compounds, the method comprising contacting the mixture with a strong organic base under acid-base reaction conditions, wherein the major component is not reactive with the strong organic base.
Any suitable acid-base reaction in the art may be used in the acid-base reactions of the present disclosure, which in some embodiments are strong base weak acid-base reactions. In some embodiments, the strong base is an organic strong base. In some embodiments, an acid in an acid-base reaction refers to a compound that contains one or more electron withdrawing groups, resulting in the compound being acidic. In some embodiments, the acid-base reaction is a reaction of a strong organic base with a compound containing a diimide group. The hydrogen atoms in the compound containing the diimide group are weak acid and can react with organic strong base to form strong base weak acid reaction. In some embodiments, the impurity is one or more compounds containing a diimide group. In some embodiments, the impurity is one or more compounds containing a diimide group and reacts in contact with the strong organic base, the principal component not reacting with the strong organic base.
Those skilled in the art are aware of various acid-base reaction conditions, and any conditions that allow the acid-base reaction to occur after contacting the diimide group-containing compound in the mixture mentioned in this disclosure with an organic strong base can be used in this disclosure. In some embodiments, the solvent used in the acid-base reaction conditions is a polar organic solvent. The conditions of the acid-base reaction include the temperature, time, and ratio of the amounts of the reactants to each other and the solvent when the acid-base reaction is performed. In some embodiments, the acid-base reaction conditions include a contact temperature of 5-35 ℃, a contact time of 10-60min, a weight ratio of the mixture to the organic strong base of 1:0.02-0.5, and a weight-to-volume ratio of the mixture to the polar organic solvent of 1:0.5-8 g/ml. In some embodiments, the acid-base reaction conditions include a contact temperature of 15-30 ℃, a contact time of 20-40min, a weight ratio of the mixture to the organic strong base of 1:0.05-0.20, and a weight-to-volume ratio of the mixture to the polar organic solvent of 1:0.8-6g/ml.
In some embodiments, the organic strong base is an organic strong base capable of performing the acid base reaction of the present disclosure, or the organic strong base is an alkoxide selected from the group consisting of metal salts of alcohols, or the alkoxide is an alkali metal alkoxide. Considering the cost of the materials, in some embodiments, the alkali metal alkoxide is at least one of sodium methoxide, potassium methoxide, sodium ethoxide, potassium tert-butoxide, or sodium tert-butoxide, for example, at least one of potassium tert-butoxide, sodium tert-butoxide, or sodium methoxide.
In some embodiments, the polar organic solvent is selected from at least one of esters, halogenated hydrocarbons, nitriles, and aromatic hydrocarbons. In some embodiments, the esters are fatty acid esters, or the fatty acid esters are C1-C6 saturated fatty acid esters, or C1-C6 saturated fatty acid esters are the C1-C6 saturated fatty acid alkyl esters, or the C1-C6 saturated fatty acid alkyl esters are selected from at least one of ethyl acetate, isopropyl acetate, or n-butyl acetate, the halogenated hydrocarbon is selected from at least one of chloroform, methylene chloride, or 1, 2-dichloroethane, the nitriles are selected from acetonitrile or propionitrile, the aromatic hydrocarbons are selected from substituted benzene, or the substituted benzene is selected from alkyl-substituted benzene, or the alkyl-substituted benzene is selected from C1-C6 alkyl-substituted benzene, or C1-C3 alkyl-substituted benzene, or the C1-C3 alkyl-substituted benzene is selected from at least one of toluene, xylene, or ethylbenzene. In some embodiments, the polar organic solvent is selected from at least one of ethyl acetate, isopropyl acetate, n-butyl acetate, or toluene.
In some embodiments, the impurity is a compound of formula (I):
(I)
Wherein R 1 is hydrogen, a hydroxyl protecting group or a structure shown as a formula (R1-1),
Formula (R1-1)
Wherein, Represents a radical connection site, B1 is the same as or different from B'1, each independently selected from substituted or unsubstituted-C1-C5 alkyl, and B2 is selected from one of-C1-C5 alkylene-CN;
r 2 is selected from hydrogen, -C1-C5 alkyl, halogen;
R 3 is selected from hydrogen, hydroxy, -O-C1-C5 alkyl, halogen, -O-hydroxy protecting group or is a structure represented by formula (R3-1):
Formula (R3-1),
Wherein, Represents a radical connection site, B3 is the same as or different from B'3, each independently selected from substituted or unsubstituted-C1-C5 alkyl, and B4 is selected from one of-C1-C5 alkylene-CN;
X is hydrogen, halogen, -O- (C1-C5 alkyl), -O- (C1-C5 alkylene) -CN or-O-tert-butyldimethylsilyl.
In some embodiments, the hydroxy protecting group is selected from trityl, 4-methoxytrityl, or 4,4' -dimethoxytrityl.
In some embodiments, B1 is the same as B'1, each independently selected from methyl, ethyl, propyl, or isopropyl.
In some embodiments, B2 is selected from cyanoethyl (-CH 2CH2 CN).
In some embodiments, the structure of formula (R1-1-1) is a structure of formula (R1-1-1):
Formula (R1-1-1).
In some embodiments, R1 is selected from hydrogen, trityl, 4-methoxytrityl, 4' -dimethoxytrityl, or a structure represented by formula (R1-1-1).
In some embodiments, -C1-C5 alkyl is selected from methyl, ethyl, propyl, or isopropyl.
In some embodiments, the halogen is selected from fluorine, chlorine, bromine, or iodine.
In some embodiments, R2 is selected from hydrogen, methyl, ethyl, fluoro, or chloro.
In some embodiments, B3 is the same as B'3, each independently selected from methyl, ethyl, propyl, or isopropyl.
In some embodiments, B4 is selected from cyanoethyl (-CH 2CH2 CN).
In some embodiments, the structure of formula (R3-1) is a structure of formula (R3-1-1):
Formula (R3-1-1).
In some embodiments, R3 is selected from hydrogen, hydroxy, -O-trityl, -O-4-methoxytrityl, -O-4,4' -dimethoxytrityl, or a structure of formula (R1-1-1).
In some embodiments, -O- (C1-C5 alkyl) is selected from-O-methyl, -O-ethyl, -O-propyl, or-O-isopropyl.
In some embodiments, -O- (C1-C5 alkylene) -O- (C1-C5 alkyl) is selected from the group consisting of-O-methylene-O-methyl, -O-methylene-O-ethyl, -O-methylene-O-propyl, -O-methylene-O-isopropyl, -O-ethylene-O-methyl, -O-ethylene-O-ethyl, -O-ethylene-O-propyl, -O-ethylene-O-isopropyl, -O-propylene-methyl, or-O-propylene-O-ethyl.
In some embodiments, -O- (C1-C5 alkylene) -CN is selected from-O-CH 2CH2 -CN or-O-CH 2 -CN.
In some embodiments, X is selected from fluorine, chlorine, bromine, -O-methyl, -O-ethyl, -O-propyl, -O-methylene-O-methyl, -O-methylene-O-ethyl, -O-methylene-O-propyl, -O-methylene-O-isopropyl, -O-ethylene-O-methyl, -O-ethylene-O-ethyl, -O-ethylene-O-propyl, -O-ethylene-O-isopropyl, -O-propylene-methyl, or-O-propylene-O-ethyl.
In some embodiments, R 1 is hydrogen or a hydroxy protecting group, R 2 is hydrogen or-C1-C5 alkyl, R 3 is hydrogen, hydroxy, -O-C1-C5 alkyl, -O-hydroxy protecting group, or a structure of formula (3-1), X is hydrogen, halogen, or-O- (C1-C5 alkylene) -O- (C1-C5 alkyl).
In some embodiments, R 1 is trityl, 4-methoxytrityl, or 4,4' -dimethoxytrityl, R 2 is hydrogen, methyl, ethyl, propyl, or isopropyl, R 3 is a structure represented by formula (3-1-1), X is fluoro, chloro, -O-methyl, -O-ethyl, -O-propyl, -O-methylene-O-methyl, -O-methylene-O-ethyl, -O-methylene-O-propyl, -O-methylene-O-isopropyl, -O-ethylene-O-methyl, -O-ethylene-O-ethyl, -O-ethylene-O-propyl, -O-ethylene-O-isopropyl, -O-propylene-methyl, or-O-propylene-O-ethyl.
In some embodiments, the impurity of formula (I) is selected from:、 And At least one of them.
In some embodiments, the principal component is a nucleoside monomer that is a nucleoside monomer of formula (II):
the compound of formula (II),
B is a non-imide nucleotide base, a non-imide nucleotide base analog or a protected non-imide nucleotide base. In some embodiments, the "non-imide nucleotide base" refers to a natural nucleotide base, adenine (a), guanine (G), and cytosine (C), respectively. In some embodiments, the "non-imide nucleotide base analog" refers to an analog of a natural nucleotide base, a chemical species that has the function of replacing a nucleobase in a nucleotide. In some embodiments, the nucleotide base analog is selected from nucleotide bases with a substituent group, which refers to a "substituted" group at any atom on the nucleotide base analog group, but without an imide group. In some embodiments, the non-imide nucleotide base analog is selected from a substituted purine, a substituted pyrimidine, or a deazapurine. In some embodiments, a substituted purine or substituted pyrimidine refers to the substitution of a hydrogen atom on an aromatic ring with a halogen, amino, methoxy, or C 1-C3 alkyl group. In some embodiments, a substituted purine or substituted pyrimidine refers to an oxygen on an aromatic ring substituted with a sulfur atom. In some embodiments, the nucleotide base analog is selected from the group consisting of 2-amino adenine, 7-deazaguanine, 2-amino-7-deazaadenine, 8-azaadenine, 5-azacytosine, 6-methyladenine 2-amino-6-chloropurine, 2-amino-6-iodopurine, 2-chloroadenine, 2-fluoroadenine, isoguanine, 7-methylguanine, 7-deazaadenine, 6-thioguanine, 5-fluorocytosine, or 5-methylcytosine of one of the unnatural nucleotide base residues. The selection ranges and definitions of R1, R3 and X are as described above for R1, R3 and X.
In some embodiments, the non-imide nucleotide base may be: Or (b) 。
In some embodiments, the protected non-imide nucleotide base refers to a non-imide nucleotide base protected by a protecting group that is a protecting group capable of protecting an amino group, which may be:
(C(ac)) (A (bz)) or (5-Me-C(bz)),
Wherein ac represents acetyl, bz represents benzoyl, and Me represents methyl.
In some embodiments, the nucleoside monomer of formula (II) is selected from:、 And At least one of them.
In some embodiments, the impurity is selected from、AndAt least one of (a) and (b);
The main component is selected from 、AndAt least one of them.
The person skilled in the art is aware of the methods of preparation of the mixtures mentioned in the present disclosure, which can be carried out, for example, with reference to WO2009/143369, non-patent document 1(Andrew McPherson, Daniel Capaldi, Lijian Chen, An Improved Process for the Manufacture of 5'-O-(4,4'-Dimethoxytrityl)-N2-isobutyryl-2'-O-(2-methoxyethyl)guanosine, Organic Process Research&Development, 2020, 11, 2583-2590 and non-patent document 2(Chaoyu Xie, Michael A. Staszak, John T, etc, Nucleosidic Phosphoramidite Synthesis via Phosphitylation: Activator Selection and Process Development,2005, 9, 730-737).
In some embodiments, the method further comprises adding a non-polar solvent after contacting the mixture with the organic strong base. In some embodiments, the method further comprises adding a nonpolar solvent after the mixture is contacted with the organic strong base to perform an acid-base reaction. In some embodiments, the method further comprises adding a non-polar solvent after contacting the mixture with the organic strong base for 10-60 minutes. In some embodiments, the nonpolar solvent is added followed by stirring for 2-30 minutes, or 5-20 minutes. In some embodiments, the non-polar solvent is selected from at least one of alkanes or cycloalkanes. In some embodiments, the alkane is at least one of n-pentane, isopentane, hexane, or heptane, and the cycloalkane is selected from cyclohexane and/or cyclopentane. In some embodiments, the non-polar solvent is selected from at least one of n-hexane, n-heptane, cyclohexane, or cyclopentane.
In some embodiments, the method further comprises separating a major component and impurities from the mixture after the acid-base reaction.
In some embodiments, the method of separating the major component from the mixture after the acid-base reaction is chromatography, filtration or adsorption, preferably chromatography.
In some embodiments, the nonpolar solvent is added after the acid-base reaction, prior to separation, or is added before column chromatography after 10-60 minutes of contacting the mixture with an organic strong base.
Chromatography, filtration and adsorption are common in the art. In some embodiments, the chromatography comprises eluting the mixture after the acid-base reaction with an eluent. In some embodiments, the chromatography is performed in a chromatography column. In some embodiments, the conditions of chromatography may include a chromatography temperature of 5-35 ℃, a weight ratio of mixture to adsorbent of 1:0.5-10, a chromatography pressure of 0.05MPa-0.8MPa, and an amount ratio of adsorbent to eluent of 1:2-6 (g/ml). In some embodiments, the chromatography temperature is 10-30 ℃, the weight ratio of the mixture to the adsorbent is 1:0.8-5, the normal pressure, and the dosage ratio of the adsorbent to the eluent for chromatography is 1:2.5-5.5 (g/ml). The chromatographic column is filled with an adsorbent, and the adsorbent is silica gel and/or alumina. The eluent is an organic solvent capable of eluting the above mixture. In some embodiments, the eluent comprises a non-polar solvent. In some embodiments, the eluent comprises a polar solvent in addition to the non-polar solvent. In some embodiments, a polar solvent is miscible with a non-polar solvent for separation of the major component and impurities. In some embodiments, the eluent comprising a polar solvent and a non-polar solvent has a difference in solubility for the anti-principle component and the impurity. In some embodiments, the principal component and impurities are more easily separated after the acid-base reaction than the principal component and impurities before the acid-base reaction. Compared with the method without acid-base reaction and then column chromatography, the method has the advantages that the content of the obtained main components is further improved, and the impurity is reduced by 6-10 times. In the present disclosure, the solvent used for the acid-base reaction and the column chromatography is an organic solvent free of water, preventing the compound containing a phosphoramidite group from undergoing a hydrolysis reaction. In some embodiments, the volume ratio of polar solvent to non-polar solvent is 1:5 to 10:1, or 1:2 to 8:1, or 1:1 to 5:1. In some embodiments, the polar solvent is the same as the polar organic solvent described above in the ranges and definitions of choice. In some embodiments, the non-polar solvent is the same as the non-polar solvent described above in the ranges and definitions of choice. In some embodiments, the eluent is selected from ethyl acetate and n-hexane, toluene and n-hexane, or ethyl acetate and n-heptane.
Examples
The present invention and its advantageous effects will be described in detail below with reference to specific examples.
The reagents and instrument sources used in the examples below are shown in table 1 below:
TABLE 1 reagents and instrument sources
Example 1
200G of DMT-2' -F-dC (ac) phosphoramidite monomer (prepared in preparation example 10 of WO2009/143369, the content of main component DMT-2' -F-dC (ac) phosphoramidite monomer was 99.2% by HPLC detection, and the content of impurity DMT-2' -F-dU phosphoramidite monomer was 0.3%) was added to 150ml of ethyl acetate at room temperature, and after stirring and dissolution, 16g of potassium t-butoxide was added at room temperature, stirred for 30 minutes, and 100ml of n-hexane was added, and stirred for 10 minutes.
600Ml of a mixed solvent of ethyl acetate and n-hexane (the volume ratio of ethyl acetate to n-hexane is 3:2) is added into 200g of silica gel at room temperature, and the mixture is stirred uniformly, poured into a chromatographic column and naturally settled for 2 hours. The solution after the above reaction was poured into a prepared column for column chromatography under normal pressure, the column was eluted with 800ml of a mixed solvent of the above ethyl acetate and n-hexane, the chromatographic solution was collected, the solvent was distilled off under reduced pressure, and vacuum-dried to obtain 184.6g of a foamed solid (DMT-2 '-F-dC (ac) phosphoramidite monomer content was 99.45% and DMT-2' -F-dU phosphoramidite monomer content was 0.03% by HPLC).
Example 2
100G of DMT-2' -O-Me-A (bz) phosphoramidite monomer (prepared in preparation example 10 of WO2009/143369, the content of the main component DMT-2' -O-Me-A (bz) phosphoramidite monomer was 99.5% as measured by HPLC, and the content of impurity DMT-2' -O-MeU phosphoramidite monomer was 0.3%) was added to 300ml of toluene at room temperature, and after dissolution by stirring, 10g of sodium t-butoxide was added at room temperature, stirred for 30 minutes, 150ml of n-hexane was added, and stirred for 10 minutes.
600ML of a mixed solvent of toluene and n-hexane (the volume ratio of toluene to n-hexane is 2:1) is added into 200g of silica gel at room temperature, the mixture is stirred uniformly, poured into a chromatographic column and naturally settled for 2 hours. The solution after the above reaction was poured into a prepared column for column chromatography under normal pressure, eluting the column with 500ml of a mixed solvent of toluene and n-hexane, collecting the chromatographic solution, evaporating the solvent under reduced pressure, and vacuum-drying to obtain 91.6g of a foamed solid (99.74% of DMT-2'-O-Me-A (bz) phosphoramidite monomer and 0.05% of DMT-2' -O-MeU phosphoramidite monomer by HPLC).
Example 3
100G of DMT-2' -O-MOE-5-meC (bz) phosphoramidite monomer (prepared by reference Andrew McPherson, Daniel Capaldi, Lijian Chen, An Improved Process for the Manufacture of 5'-O-(4,4'-Dimethoxytrityl)-N2-isobutyryl-2'-O-(2 -methoxyethyl) guanosine, Organic Process Research&Development, 2020, 11, 2583-2590;Chaoyu Xie, Michael A. Staszak, John T, etc, Nucleosidic Phosphoramidite Synthesis via Phosphitylation: Activator Selection and Process Development,2005, 9, 730-737), the content of main component DMT-2' -O-MOE-5-meC (bz) phosphoramidite monomer was 99.4% by HPLC detection, the content of impurity DMT-2' -O-MOE-T phosphoramidite monomer was 0.2%) was added to 100ml of ethyl acetate at room temperature, and after stirring and dissolution, 12g of sodium methoxide was added at room temperature, stirring was performed for 30 minutes, and 50ml of n-heptane was further added, followed by stirring for 10 minutes.
At room temperature, 300g of silica gel is added with 900ml of mixed solvent of ethyl acetate and n-heptane (the volume ratio of the ethyl acetate to the n-heptane is 2:1), and the mixture is stirred uniformly, poured into a chromatographic column and naturally settled for more than 2 hours. Pouring the solution after the reaction into a prepared chromatographic column for column chromatography, eluting the chromatographic column by using 400ml of a mixed solvent of ethyl acetate and n-heptane, collecting the chromatographic solution, evaporating the solvent under reduced pressure, and drying in vacuum to obtain 90.3g of foam-like solid. (DMT-2 '-O-MOE-5-meC (bz) phosphoramidite monomer content was 99.57% as measured by HPLC, DMT-2' -O-MOE-T phosphoramidite monomer content was 0.02%).
Comparative example 1
50G of DMT-2' -F-dC (ac) phosphoramidite monomer (prepared in preparation example 10 of WO2009/143369, the content of DMT-2' -F-dC (ac) phosphoramidite monomer as a main component was 99.2% by HPLC detection, and the content of DMT-2' -F-dU phosphoramidite monomer as an impurity was 0.3%) was added to 30ml of ethyl acetate at room temperature, and after stirring and dissolution, 20ml of n-hexane was added thereto, followed by stirring.
At room temperature, 50g of silica gel is added with 150ml of a mixed solvent of ethyl acetate and n-hexane (the volume ratio of ethyl acetate to n-hexane is 3:2), and the mixture is stirred uniformly, poured into a chromatographic column and naturally settled for 2 hours. The solution was poured into a prepared column for column chromatography, the column pressure was set at normal pressure, the column was eluted with 200ml of a mixed solvent of the above ethyl acetate and n-hexane, the column was collected, the solvent was distilled off under reduced pressure, and vacuum-dried to give 45.1g of a foamed solid (DMT-2 '-F-dC (ac) phosphoramidite monomer content was 99.2% and DMT-2' -F-dU phosphoramidite monomer content was 0.3% by HPLC).
Comparative example 2
30G of DMT-2' -F-dC (ac) phosphoramidite monomer (prepared in preparation example 10 of WO2009/143369, the content of DMT-2' -F-dC (ac) phosphoramidite monomer as a main component was 99.2% by HPLC detection, the content of DMT-2' -F-dU phosphoramidite monomer as an impurity was 0.3%) was added to 200ml of toluene at room temperature, and after stirring and dissolution, the mixture was washed with 100ml of DMF/water (DMF: water volume ratio: 1:1) for 3 times, and the upper organic phase was detected. (DMT-2 '-F-dC (ac) phosphoramidite monomer content was 99.2% and DMT-2' -F-dU phosphoramidite monomer content was 0.3% as measured by HPLC).
As can be seen from the above examples and comparative examples, the washing method and the column chromatography which had not been subjected to the acid-base reaction were unchanged in the contents of the main component and the impurity before and after purification (comparative examples 1-2). After the impurity removing method disclosed by the invention, the content of the main component is further improved, for example, the content of the main component in examples 1-3 is respectively improved from 99.2%, 99.5% and 99.4% to 99.45%, 99.74% and 99.57%, the content of the impurity is reduced from 0.3%, 0.3% and 0.2% to 0.03%, 0.05% and 0.02%, and the content of the impurity is reduced by 6-10 times.
Claims (20)
1. A method for removing impurities from a mixture, said mixture comprising a major component and impurities, comprising contacting said mixture with a strong organic base under acid-base reaction conditions, wherein said major component does not react with said strong organic base;
The impurity is a compound shown in a general formula (I):
(I),
the main component is a nucleoside monomer of the general formula (II):
the compound of formula (II),
R 1 is trityl, 4-methoxytrityl or 4,4' -dimethoxytrityl;
R 2 is selected from hydrogen, - (C1-C5 alkyl), halogen;
R 3 is a structure represented by formula (R3-1):
Formula (R3-1),
Wherein, Represents a group connecting site, B3 is the same as or different from B'3, each is independently selected from- (C1-C5 alkyl), and B4 is selected from one of- (C1-C5 alkylene) -CN;
X is hydrogen, halogen, -O- (C1-C5 alkyl), -O- (C1-C5 alkylene) -O- (C1-C5 alkyl) or-O- (C1-C5 alkylene) -CN;
B is: 、 Or (b) ;
The solvent used in the acid-base reaction condition is a polar organic solvent without water;
The method further comprises separating a major component from the mixture after the acid-base reaction;
The method for separating the main component from the mixture after the acid-base reaction is chromatography, the chromatography is carried out in a chromatographic column, the adsorbent filled in the chromatographic column is silica gel and/or alumina,
The eluent used for the chromatography comprises a polar organic solvent and a nonpolar solvent, wherein the volume ratio of the polar organic solvent to the nonpolar solvent is 1:5-10:1.
2. The method of claim 1, wherein the acid-base reaction conditions comprise a contact temperature of 5-35 ℃ and a contact time of 10-60min, wherein the weight ratio of the mixture to the organic strong base is 1:0.02-0.5, and the weight-volume ratio of the mixture to the polar organic solvent is 1:0.5-8 g/ml.
3. The method according to claim 2, wherein the acid-base reaction conditions comprise a contact temperature of 15-30 ℃ and a contact time of 20-40min, the weight ratio of the mixture to the organic strong base is 1:0.05-0.20, and the weight-volume ratio of the mixture to the polar organic solvent is 1:0.8-6 g/ml.
4. The method of claim 1, wherein the strong organic base is at least one of sodium methoxide, potassium methoxide, sodium ethoxide, potassium tert-butoxide, or sodium tert-butoxide.
5. The method of claim 1, wherein the polar organic solvent is selected from at least one of esters, halogenated hydrocarbons, nitriles, and alkyl substituted benzenes.
6. The method of claim 1, wherein the polar organic solvent is selected from at least one of fatty acid esters, chloroform, methylene chloride, 1, 2-dichloroethane, acetonitrile, propionitrile, or C1-C6 alkyl substituted benzene.
7. The process according to claim 1, wherein the polar organic solvent is a C1-C6 saturated fatty acid ester and/or a C1-C3 alkyl substituted benzene.
8. The method of claim 1, wherein the polar organic solvent is at least one of a C1-C6 saturated fatty acid alkyl ester, toluene, xylene, or ethylbenzene.
9. The method of claim 1, wherein the polar organic solvent is at least one of ethyl acetate, isopropyl acetate, or n-butyl acetate.
10. The method of claim 1, wherein R 2 is selected from hydrogen, methyl, ethyl, n-propyl, isopropyl, fluoro, chloro, bromo, or iodo;
Or B3 is the same as B'3 and is selected from methyl, ethyl, n-propyl or isopropyl, B4 is selected from cyanoethyl (-CH 2CH2 CN);
Or X is selected from fluorine, chlorine, -O-methyl, -O-ethyl, -O-n-propyl, -O-isopropyl, -O-methylene-O-methyl, -O-methylene-O-ethyl, -O-methylene-O-n-propyl, -O-methylene-O-isopropyl, -O-ethylene-O-methyl, -O-ethylene-O-ethyl, -O-ethylene-O-n-propyl, -O-ethylene-O-isopropyl, -O-propylene-methyl, -O-propylene-O-ethyl, -O-CH 2CH2 -CN or-O-CH 2 -CN.
11. The method of claim 1, wherein R 2 is selected from hydrogen or- (C1-C5 alkyl) and X is selected from hydrogen, halogen or-O- (C1-C5 alkylene) -O- (C1-C5 alkyl).
12. The method of claim 11, wherein X is selected from-O-methyl, -O-ethyl, -O-n-propyl, -O-isopropyl, -O-methylene-O-methyl, -O-methylene-O-ethyl, -O-methylene-O-n-propyl, -O-methylene-O-isopropyl, -O-ethylene-O-methyl, -O-ethylene-O-ethyl, -O-ethylene-O-n-propyl, -O-ethylene-O-isopropyl, -O-propylene-methyl, or-O-propylene-O-ethyl.
13. The method of claim 11, wherein the impurity of formula (I) is selected from:、 And At least one of them.
14. The method of claim 1, wherein R 2 is selected from hydrogen, methyl, ethyl, n-propyl, isopropyl, X is selected from fluorine, chlorine, -O-methyl, -O-ethyl, -O-n-propyl, -O-isopropyl, -O-methylene-O-methyl, -O-methylene-O-ethyl, -O-methylene-O-n-propyl, -O-methylene-O-isopropyl, -O-ethylene-O-methyl, -O-ethylene-O-ethyl, -O-ethylene-O-n-propyl, -O-ethylene-O-isopropyl, -O-propylene-methyl, or-O-propylene-O-ethyl.
15. The method of claim 14, wherein the nucleoside monomer of formula (II) is selected from the group consisting of:、 And At least one of them.
16. The method of claim 15, wherein the impurity is selected from the group consisting of:、 And At least one of (a) and (b);
The main component is selected from 、AndAt least one of them.
17. The method of claim 1, further comprising adding a non-polar solvent after contacting the mixture with the strong organic base.
18. The method of claim 17, wherein the non-polar solvent is selected from alkanes and/or cycloalkanes.
19. The method of claim 17, wherein the non-polar solvent is selected from at least one of n-pentane, isopentane, hexane, heptane, cyclohexane, and cyclopentane.
20. The method of claim 1, wherein the temperature of the chromatography is 5-35 ℃, the weight ratio of the mixture after the acid-base reaction to the adsorbent is 1:0.5-10, the pressure of the chromatography is 0.05MPa-0.8MPa, and the usage ratio of the adsorbent to the eluent of the chromatography is 1:2-6 g/ml.
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