CN113150041A - Preparation method of thiooligonucleotide - Google Patents
Preparation method of thiooligonucleotide Download PDFInfo
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Abstract
The invention provides a preparation method of thiooligonucleotide, which adopts a solid phase phosphoramidite triester method to sequentially connect nucleotide monomers from a 3 'end to a 5' end according to a set base sequence, and finally forms oligonucleotide fragments connected by 3', 5' -phosphorothioate diester bonds, wherein cross-linked polystyrene beads are used as a solid phase carrier. The preparation method provided by the invention is used for improving the existing synthesis process aiming at the characteristics of the thiooligonucleotide YK102, can be used for obviously improving the yield of YK102, and provides technical support for preparing antisense oligonucleotide for treating tumor diseases with high yield.
Description
Technical Field
The invention relates to the technical field of nucleic acid synthesis, in particular to a preparation method of sulfo-oligonucleotide.
Background
Oligonucleotides are a generic term for short-chain nucleotides with only less than 30 bases (including nucleotides in deoxyribonucleic acid DNA or ribonucleic acid RNA), and have positive significance in treating diseases such as cancer and the like because of their easy docking with their complementary chains, inhibition of RNA fragments and prevention of translation into protein.
Antisense oligonucleotides (ASO) are one of the methods for targeted modulation of IGF1R signaling pathway, and can complementarily bind to a specific sequence of target mRNA to form a double strand, thereby activating RNase H to degrade mRNA orPrevent the initiation of protein translation, and inhibit the expression of target gene, so far, two antisense nucleic acid drugs have been approved to be on the market internationally (Vitravene)TMAnd
)。
for the synthesis of nucleic acid drugs, DNA synthesis developed by holarana (Khorana) in the 50-60 s was performed in solution, the method of liquid phase synthesis was long in cycle, low in coupling yield, the intermediate product was purified after each coupling, and the synthesis method was complicated. In the 70 s, Itakura developed solid phase synthesis of DNA using solid phase synthesis techniques for polypeptides. Solid phase synthesis is that the first nucleotide at the 3 'end of the DNA chain to be synthesized is directionally connected with a solid phase carrier of a high molecular material (the 3' end is connected to the solid phase carrier); removing the protecting group at the 5' end of the nucleotide by using a deprotection agent, activating the second nucleotide to be connected by using tetrazole, and then coupling the 3' end phosphate group with the 5' end hydroxyl group of the first nucleotide to form a phosphate (or phosphite) triester bond between the two connected nucleotides. The solid-phase synthesis method has the advantages of high coupling efficiency, easy separation and purification of the synthesized product, high final yield, and very simplified and easily mastered whole operation process.
The solid phase synthesis methods are classified into a phosphotriester method and a phosphite triester method. The phosphotriester process was developed from the liquid-phase triester process, and each coupling reaction time as long as 15 minutes to 2 hours could not satisfy the requirement of rapid mass synthesis, and the ligation yield could only reach 95%. The phosphite triester method is classified into a chlorophosphite triester method and a diisopropylamide phosphite triester method according to the difference in reactive groups to which the phosphite groups are bonded. Due to the instability of the chlorophosphite derivative, the use of the trichlorochlorophosphite method is limited. The diisopropylphosphoramidite process, also known as the phosphoramidite process or the phosphotriester process, is the more modern of the phosphotriester processes. The diisopropylamine combines with the phosphorous acid groups to form a diisopropylphosphoramidite compound. This functional group does not react with a hydroxyl group itself, but is converted under mild acid conditions, such as in the presence of tetrazole, into an activated intermediate of amidinotetrazole, which reacts with the 5' -hydroxyl group of another nucleotide with a relatively high coupling efficiency.
Very few literature reports on the synthesis process of YK102 exist, and the existing synthesis method has the problem of low final product yield in the process of preparing the product. Therefore, the synthesis process thereof needs to be studied intensively.
Disclosure of Invention
In order to overcome the technical problems, the invention provides a preparation method of thiooligonucleotide, and the method can obviously improve the yield of the final product.
The preparation method of the thiooligonucleotide adopts a solid phase phosphoramidite triester method to sequentially connect nucleotide monomers from a 3 'end to a 5' end according to a set base sequence, and finally forms oligonucleotide fragments connected by 3', 5' -phosphorothioate diester bonds, wherein cross-linked polystyrene beads are used as a solid phase carrier.
The inventor finds out through a great deal of practice that the initial nucleotide is more favorably immobilized on the surface of the specific solid phase carrier through the coupling mode of the functional groups, thereby being favorable for improving the yield of the final product.
In some specific embodiments, the ammonolysis reagent used in the ammonolysis step of the solid phase phosphite triester method is ammonia water with a concentration of 25-28%, the amount of the ammonia water is 0.5-2.5 times of the total weight of the nucleotide monomers, and the ammonolysis temperature is 58-90 ℃.
The inventor further carries out a great deal of research on the preparation method of the thiooligonucleotide, finds that the ammonolysis conditions, particularly the dosage of the ammonolysis reagent and the ammonolysis temperature obviously influence the yield of the final product, and experiments show that the ammonolysis conditions can obviously improve the yield of the final product. And the ammonolysis conditions are matched with the solid phase carrier, so that the final product can be removed from the surface of the solid phase carrier more conveniently, and the yield of the final product is improved.
In order to further improve the yield of the final product, the invention also optimizes the ammonolysis conditions, wherein the dosage of the ammonia water is 0.8-1.6 times of the total weight of the nucleotide monomers, and the reaction temperature is 60-75 ℃.
According to some embodiments of the invention, the thioacid oligonucleotide has a sequence length of 8 to 30 bases, preferably 15 to 30 bases, more preferably 20 bases, and the preferred sequence is 5'-TCCTCCGGAGCCAGACTTCA-3' (SEQ ID NO: 1).
Chinese patent CN1184316C discloses an antisense oligonucleotide for treating tumor diseases, wherein the base sequence of one product is: TCCTCCGGAGCCAGACTTCA A derivative of a sulfur atom substituted for the oxygen atom bearing the double bond in the phosphate of the oligonucleotide chain has the formula C192H245O99N72S19P19The chemical name is: thymidylate nucleoside- (3'→ 5' O, O-thiophosphate) -2 '-deoxycytidine- (3' → 5'O, O-thiophosphate) -2' -deoxyguanosine- (3'→ 5' O, o-phosphorothioate) -2' -deoxyadenosine- (3' → 5' O, O-phosphorothioate) -2' -deoxyguanosine- (3' → 5' O, O-phosphorothioate) -2' -deoxycytidine- (3' → 5' O, O-phosphorothioate) -2' -deoxyadenosine- (3' → 5' O, O-phosphorothioate) -2' -deoxyguanosine- (3' → 5' O, o-phosphorothioate) -2' -deoxycytidine (3' → 5' O, O-phosphorothioate) -thymidylate (3' → 5' O, O-phosphorothioate) -2' -deoxycytidine (3' → 5' O, O-phosphorothioate) -2' -deoxyadenosine (named YK 102).
Research finds that YK102 can resist nuclease, so that the action time of YK102 in vivo is prolonged, YK102 is an antisense nucleic acid medicament which is developed by taking IGF1R as a target spot and has the effect of obviously inhibiting tumor proliferation, is different from the molecular targeted medicament used for liver cancer treatment at present, is used as a brand new gene targeted treatment medicament, and has the characteristics of clear action target spot, easiness in design and capability of treating diseases fundamentally. Compared with the traditional cytotoxic chemotherapy medicament, the targeted therapy medicament has strong specificity, small adverse reaction and more definite curative effect.
According to some embodiments of the invention, the solid phase phosphoramidite triester process comprises:
removing the DMT protecting group of the C5' -hydroxyl of the initial nucleotide, wherein the initial nucleotide is pre-connected and loaded on a solid phase carrier;
repeatedly adding C3 '-hydroxyl to modify through phosphoramidite, coupling C5' -hydroxyl by a nucleotide monomer protected by DMT, oxidizing, protecting hydroxyl, and removing DMT protection, and sequentially connecting the nucleotide monomer to the nucleotide of the solid phase carrier;
and carrying out ammonolysis on the synthetic product to obtain the thiooligonucleotide.
In some specific embodiments, the nucleotide is formulated with acetonitrile into a nucleotide solution prior to use;
and/or, after each step of DMT protecting group removal, coupling, oxidation and hydroxyl protection is finished, washing with acetonitrile;
and/or said acetonitrile has a water content limited to 0-50ppm, preferably 5-25ppm
Researches find that the water content of acetonitrile used as a solvent in the preparation method has certain influence on the yield of a final product, and the water content of the acetonitrile is controlled within a certain limit, so that the side reaction can be effectively inhibited, and the reaction yield is improved.
The nucleotide monomer refers to a nucleotide monomer with a C3 '-hydroxyl modified by phosphoramidite and a C5' -hydroxyl protected by DMT, and specifically comprises DMT-dA (bz) phosphoramidite monomer (CAS:98796-53-3), DMT-dC (bz) phosphoramidite monomer (CAS:102212-98-6), DMT-dG (ib) phosphoramidite monomer (CAS:93183-15-4) and DMT-dT phosphoramidite monomer (CAS: 98796-51-1).
The deprotection reagent used in the DMT protection removing step is 1-5 w/v% of a toluene solution of dichloroacetic acid, a dichloromethane solution of dichloroacetic acid, a methanol solution of dichloroacetic acid, a toluene solution of trichloroacetic acid, a dichloromethane solution of trichloroacetic acid or a methanol solution of trichloroacetic acid, and preferably a toluene solution of 3 w/v% of dichloroacetic acid. Wherein w/v% refers to the weight to volume ratio, i.e., the weight (g) of solute in 100mL of solution.
The coupling step uses 5-ethylthio tetrazole as an activator.
Or; in the oxidation step, ((E) -N, N-dimethyl-N' - (3-thio-3H-1, 2, 4-dithiothiazol-5-yl) formamidine or hydrogenated yellow element is used as an oxidizing agent.
Or; acetic anhydride is used as a protecting agent in the step of protecting the hydroxyl.
The invention has the following beneficial effects:
the preparation method provided by the invention is used for improving the existing synthesis process aiming at the characteristics of the thiooligonucleotide YK102, can be used for obviously improving the yield of YK102, and provides technical support for preparing antisense oligonucleotide for treating tumor diseases with high yield.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
The following examples will use the following instruments and reagents: an Oligopilot 400 synthesizer of GE company, USA, wherein a solid phase carrier is a cross-linked polystyrene bead (cross-linked polystyrene bead) connected with an initial nucleotide which has a base A and a C5' -hydroxyl group protected by DMT;
four nucleotide solutions were prepared separately with acetonitrile:
DMT-da (bz) phosphoramidite monomer + acetonitrile 0.33kg +1.36 kg;
DMT-dc (bz) phosphoramidite monomer + acetonitrile 0.44kg +1.81 kg;
DMT-dg (ib) phosphoramidite monomer + acetonitrile 0.28kg +1.16 kg;
DMT-dT phosphoramidite monomer + acetonitrile 0.25kg +1.17 kg.
Example 1
This example provides a method for preparing thiooligonucleotide YK102 (a thiooligonucleotide product as described in the summary of the invention), wherein acetonitrile contains 10ppm of water, comprising:
(1) deprotection of the amino acid
DMT protecting groups were removed using 3 w/v% dichloroacetic acid in toluene (mass to volume ratio: 8%) as a deprotecting agent, followed by washing with acetonitrile.
(2) Coupling of
Coupling of acetonitrile solution of each nucleotide monomer was performed using 5-ethylthiotetrazole as an activator, followed by rinsing with acetonitrile.
(3) Oxidation by oxygen
The oxidation was carried out using elemental hydrogen yellow as an oxidizing agent, followed by rinsing with acetonitrile.
(4) Protection of hydroxyl groups
Acetic anhydride was used as the hydroxyl protecting reagent for hydroxyl protection, followed by rinsing with acetonitrile.
And (4) circulating according to the set sequence to obtain a fully protected product.
(5) The last nucleotide was deprotected from DMT using 3 w/v% dichloroacetic acid in toluene (mass to volume ratio: 8%) as a deprotection reagent, followed by washing with acetonitrile.
(6) Ammonolysis
Transferring the solid phase carrier to a reaction kettle, adding concentrated ammonia water (25-28%, 1.21kg), maintaining ammonolysis at 60 ℃ for 13h, cooling the system to room temperature, transferring the mixture to a pressure filtration tank, leaching with a mixed solution of purified water and ethanol, and combining the filtrate 1.65kg and 1.8L. The filtrate had a YK102 content of 87.9% by HPLC.
Example 2
This example provides a method for preparing thiooligonucleotide YK102 (a thiooligonucleotide product as described in the summary of the invention), wherein acetonitrile contains 40ppm water, comprising:
(1) deprotection of the amino acid
DMT protecting groups were removed using 3 w/v% dichloroacetic acid in toluene (mass to volume ratio: 8%) as a deprotecting agent, followed by washing with acetonitrile.
(2) Coupling of
Coupling of acetonitrile solution of each nucleotide monomer was performed using 5-ethylthiotetrazole as an activator, followed by rinsing with acetonitrile.
(3) Oxidation by oxygen
The oxidation was carried out using elemental hydrogen yellow as an oxidizing agent, followed by rinsing with acetonitrile.
(4) Protection of hydroxyl groups
Acetic anhydride was used as the hydroxyl protecting reagent for hydroxyl protection, followed by rinsing with acetonitrile.
And (4) circulating according to the set sequence to obtain a fully protected product.
(5) The last nucleotide was deprotected from DMT using 3 w/v% dichloroacetic acid in toluene (mass to volume ratio: 8%) as a deprotection reagent, followed by washing with acetonitrile.
(6) Ammonolysis
Transferring the solid phase carrier to a reaction kettle, adding concentrated ammonia water (25-28%, 1.8kg), maintaining ammonolysis at 85 ℃ for 3h, cooling the system to room temperature, transferring the mixture to a pressure filtration tank, leaching with a mixed solution of purified water and ethanol, and combining 2.38kg of filtrate and 2.68L of filtrate. The YK102 content was found to be 83.5% by HPLC.
Example 3
This example provides a method for preparing thiooligonucleotide YK102 (a thiooligonucleotide product as described in the summary of the invention), wherein acetonitrile contains 20ppm of water, comprising:
(1) deprotection of the amino acid
DMT protecting groups were removed using 3 w/v% dichloroacetic acid in toluene (mass to volume ratio: 8%) as a deprotecting agent, followed by washing with acetonitrile.
(2) Coupling of
Coupling of acetonitrile solution of each nucleotide monomer was performed using 5-ethylthiotetrazole as an activator, followed by rinsing with acetonitrile.
(3) Oxidation by oxygen
The oxidation was carried out using elemental hydrogen yellow as an oxidizing agent, followed by rinsing with acetonitrile.
(4) Protection of hydroxyl groups
Acetic anhydride was used as the hydroxyl protecting reagent for hydroxyl protection, followed by rinsing with acetonitrile.
And (4) circulating according to the set sequence to obtain a fully protected product. .
(5) The last nucleotide was deprotected from DMT using 3 w/v% dichloroacetic acid in toluene (mass to volume ratio: 8%) as a deprotection reagent, followed by washing with acetonitrile.
(6) Ammonolysis
Transferring the solid phase carrier to a reaction kettle, adding concentrated ammonia water (25-28%, 1.6kg), maintaining ammonolysis at 65 ℃ for 10h, cooling the system to room temperature, transferring the mixture to a pressure filtration tank, leaching with a mixed solution of purified water and ethanol, combining the filtrate 2.16kg and 2.38L, and storing in a tetra-fluoro bucket. The YK102 content was 86.7% by HPLC.
Example 4
This example provides a method for preparing thiooligonucleotide YK102 (a thiooligonucleotide product as described in the summary of the invention) wherein acetonitrile contains 25ppm of water, comprising:
(1) deprotection of the amino acid
DMT protecting groups were removed using 3 w/v% dichloroacetic acid in toluene (mass to volume ratio: 8%) as a deprotecting agent, followed by washing with acetonitrile.
(2) Coupling of
Coupling of acetonitrile solution of each nucleotide monomer was performed using 5-ethylthiotetrazole as an activator, followed by rinsing with acetonitrile.
(3) Oxidation by oxygen
The oxidation was carried out using elemental hydrogen yellow as an oxidizing agent, followed by rinsing with acetonitrile.
(4) Protection of hydroxyl groups
Acetic anhydride was used as the hydroxyl protecting reagent for hydroxyl protection, followed by rinsing with acetonitrile.
And (4) circulating according to the set sequence to obtain a fully protected product.
(5) The last nucleotide was deprotected from DMT using 3 w/v% dichloroacetic acid in toluene (mass to volume ratio: 8%) as a deprotection reagent, followed by washing with acetonitrile.
(6) Ammonolysis
Transferring the solid phase carrier to a reaction kettle, adding concentrated ammonia water (25-28%, 1.7kg), maintaining ammonolysis at 58 ℃ for 13h, cooling the system to room temperature, transferring the mixture to a pressure filtration tank, leaching with a mixed solution of purified water and ethanol, combining the filtrate 2.18kg and 2.41L, and storing in a tetra-fluoro barrel. The YK102 content was 85.6% by HPLC.
Comparative example 1
This comparative example provides a process for the preparation of thiooligonucleotide YK102 (a thiooligonucleotide product as described in the summary of the invention) having an acetonitrile water content of 25ppm, comprising:
(1) deprotection of the amino acid
DMT protecting groups were removed using 3 w/v% dichloroacetic acid in toluene (mass to volume ratio: 8%) as a deprotecting agent, followed by washing with acetonitrile.
(2) Coupling of
Coupling of acetonitrile solution of each nucleotide monomer was performed using 5-ethylthiotetrazole as an activator, followed by rinsing with acetonitrile.
(3) Oxidation by oxygen
The oxidation was carried out using elemental hydrogen yellow as an oxidizing agent, followed by rinsing with acetonitrile.
(4) Protection of hydroxyl groups
Acetic anhydride was used as the hydroxyl protecting reagent for hydroxyl protection, followed by rinsing with acetonitrile.
And (4) circulating according to the set sequence to obtain a fully protected product.
(5) The last nucleotide was deprotected from DMT using 3 w/v% dichloroacetic acid in toluene (mass to volume ratio: 8%) as a deprotection reagent, followed by washing with acetonitrile.
(6) Ammonolysis
Transferring the solid phase carrier to a reaction kettle, adding concentrated ammonia water (25-28%, 0.5kg), maintaining ammonolysis at 70 ℃ for 8h, cooling the system to room temperature, transferring the mixture to a pressure filtration tank, leaching with a mixed solution of purified water and ethanol, combining the filtrate 1.01kg and 1.1L, and storing in a tetra-fluoro barrel. The filtrate was found to contain 45.6% YK102 by HPLC.
Comparative example 2
This comparative example provides a process for the preparation of thiooligonucleotide YK102 (a thiooligonucleotide product as described in the summary of the invention) having an acetonitrile water content of 55ppm, comprising:
(1) deprotection of the amino acid
DMT protecting group was removed using 3 w/v% dichloroacetic acid in toluene (mass to volume ratio: 8%) as a deprotecting agent. Followed by washing with acetonitrile.
(2) Coupling of
Coupling of acetonitrile solution of each nucleotide monomer was performed using 5-ethylthiotetrazole as an activator, followed by rinsing with acetonitrile.
(3) Oxidation by oxygen
The oxidation was carried out using elemental hydrogen yellow as an oxidizing agent, followed by rinsing with acetonitrile.
(4) Protection of hydroxyl groups
Acetic anhydride was used as the hydroxyl protecting reagent for hydroxyl protection, followed by rinsing with acetonitrile.
And (4) circulating according to the set sequence to obtain a fully protected product.
(5) The last nucleotide was deprotected from DMT using 3 w/v% dichloroacetic acid in toluene (mass to volume ratio: 8%) as a deprotection reagent, followed by washing with acetonitrile.
(6) Ammonolysis
Transferring the solid phase carrier to a reaction kettle, adding concentrated ammonia water (25-28%, 1.21kg), maintaining ammonolysis at 55 ℃ for 15-18h, cooling the system to room temperature, transferring the mixture to a pressure filtration tank, leaching with a mixed solution of purified water and ethanol, and combining filtrate 1.64kg and 1.8L. The YK102 content was 28.2% by HPLC.
Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Sequence listing
<110> Beijing Yuekang Kongchuang medicine science and technology GmbH
<120> a method for preparing a thiooligonucleotide
<130> KHP211112909.6
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
tcctccggag ccagacttca 20
Claims (10)
1. A preparation method of thiooligonucleotide is characterized in that nucleotide monomers are sequentially connected from a 3 'end to a 5' end according to a set base sequence by adopting a solid-phase phosphite amide triester method, and finally oligonucleotide fragments connected by 3', 5' -phosphorothioate diester bonds are formed, wherein cross-linked polystyrene beads are used as a solid-phase carrier.
2. The method for preparing thiooligonucleotide according to claim 1, wherein the aminolysis reagent used in the step of aminolysis by solid phase phosphite triester method is ammonia water with concentration of 25-28%, the amount of ammonia water is 0.5-2.5 times of total weight of nucleotide monomers, and the aminolysis temperature is 58-90 ℃.
3. The method for preparing thiooligonucleotide according to claim 2, wherein the amount of ammonia water is 0.8-1.6 times of the total weight of nucleotide monomers, and the reaction temperature is 60-75 ℃.
4. The method of preparing a thiooligonucleotide according to any one of claims 1 to 3, wherein the sequence length of the thiooligonucleotide is 8 to 30 bases, preferably 15 to 30 bases.
5. The method of claim 4, wherein the thioacid oligonucleotide has a sequence length of 20 bases, and preferably has a base sequence of 5'-TCCTCCGGAGCCAGACTTCA-3'.
6. The method of preparing thioacid oligonucleotides according to any one of claims 1 to 5, wherein the solid phase phosphoramidite triester method comprises:
removing the DMT protecting group of the C5' -hydroxyl of the initial nucleotide, wherein the initial nucleotide is pre-connected and loaded on a solid phase carrier;
repeatedly adding a nucleotide monomer with C3 '-hydroxyl group for modification by phosphoramidite, carrying out coupling, oxidation and hydroxyl protection by using the nucleotide monomer with the C5' -hydroxyl group protected by DMT, and removing DMT protecting groups, wherein the nucleotide monomer is sequentially connected to the nucleotide of the solid phase carrier;
and carrying out ammonolysis on the synthetic product to obtain the thiooligonucleotide.
7. The method of claim 6, wherein the nucleotide monomers are formulated into a nucleotide solution with acetonitrile prior to use;
and/or after each step of DMT protecting group removal, coupling, oxidation and hydroxyl protection is finished, washing with acetonitrile;
and/or the acetonitrile has a moisture content limited in the range of 0 to 50ppm, preferably 5 to 25 ppm.
8. The method of claim 6 or 7, wherein the nucleotide monomer comprises DMT-dA (bz) phosphoramidite monomer, DMT-dC (bz) phosphoramidite monomer, DMT-dG (ib) phosphoramidite monomer, or DMT-dT phosphoramidite monomer.
9. The method of claim 8, wherein the deprotection reagent used in the DMT protecting group removing step is 1 to 5 w/v% of a toluene solution of dichloroacetic acid, a dichloromethane solution of dichloroacetic acid, a methanol solution of dichloroacetic acid, a toluene solution of trichloroacetic acid, a dichloromethane solution of trichloroacetic acid or a methanol solution of trichloroacetic acid, preferably a toluene solution of 3 w/v% of dichloroacetic acid.
10. The method for preparing thioacid oligonucleotide according to claim 8, wherein, in the coupling step, 5-ethylthio tetrazole is used as an activator;
or; (E) -N, N-dimethyl-N' - (3-thio-3H-1, 2, 4-dithiothiazol-5-yl) formamidine or hydrogenated yellow element is used as an oxidizing agent in the oxidizing step;
or; acetic anhydride is used as a protecting agent in the step of protecting the hydroxyl.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114306370A (en) * | 2021-12-31 | 2022-04-12 | 北京悦康科创医药科技股份有限公司 | Application of antisense oligonucleotide in preparing medicine for treating kidney cancer |
| CN114306369A (en) * | 2021-12-23 | 2022-04-12 | 北京悦康科创医药科技股份有限公司 | Thiooligonucleotide injection and preparation method thereof |
| CN114605481A (en) * | 2022-03-18 | 2022-06-10 | 通用生物(滁州)有限公司 | Ammonolysis method of solid phase carrier in nucleic acid production and ammonolysis kettle used in ammonolysis method |
| CN114957364A (en) * | 2022-05-31 | 2022-08-30 | 湖南大学 | Iodoglycoside base, preparation method thereof and constructed amphiphilic nucleic acid |
| CN115010769A (en) * | 2022-08-04 | 2022-09-06 | 上海百力格生物技术有限公司 | Method for synthesizing long-chain RNA nucleic acid by solid-phase phosphoramidite triester method |
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| CN114306369B (en) * | 2021-12-23 | 2023-12-26 | 北京悦康科创医药科技股份有限公司 | Thio oligonucleotide injection and preparation method thereof |
| CN114306370A (en) * | 2021-12-31 | 2022-04-12 | 北京悦康科创医药科技股份有限公司 | Application of antisense oligonucleotide in preparing medicine for treating kidney cancer |
| CN114605481A (en) * | 2022-03-18 | 2022-06-10 | 通用生物(滁州)有限公司 | Ammonolysis method of solid phase carrier in nucleic acid production and ammonolysis kettle used in ammonolysis method |
| CN114957364A (en) * | 2022-05-31 | 2022-08-30 | 湖南大学 | Iodoglycoside base, preparation method thereof and constructed amphiphilic nucleic acid |
| CN114957364B (en) * | 2022-05-31 | 2024-01-23 | 湖南大学 | Iodine base, preparation method thereof and constructed amphiphilic nucleic acid |
| CN115010769A (en) * | 2022-08-04 | 2022-09-06 | 上海百力格生物技术有限公司 | Method for synthesizing long-chain RNA nucleic acid by solid-phase phosphoramidite triester method |
| CN115010769B (en) * | 2022-08-04 | 2022-11-15 | 上海百力格生物技术有限公司 | Method for synthesizing long-chain RNA nucleic acid by solid-phase phosphoramidite triester method |
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