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Oligonucleotide manufacturing method

a production method and technology of oligonucleotides, applied in the field of oligonucleotide production methods, can solve the problems of inferior preservation stability of 2-cyanoethyl-n,n-diisopropylchlorophosphoramidite, safety problems in deprotection use of hydrazine, etc., and achieve the effect of producing more stably and efficiently

Inactive Publication Date: 2018-10-11
AJINOMOTO CO INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides a method for preparing a phosphitylating agent and producing phosphoramidited oligonucleotide in a stable and efficient way. This method suppresses the falling off of a protecting group on phosphoric acid during phosphorylation reaction, resulting in more stable production of oligonucleotide. Additionally, the method allows for the elimination of a silyl-protecting group without falling off of a cyanoethyl-protecting group on phosphoric acid between nucleotides, which facilitates more efficient production of oligonucleotide and fragment condensation using the same. The technical effects include improved stability and efficiency of phosphoramidited oligonucleotide production.

Problems solved by technology

The deprotection requires use of hydrazine having safety problems.
However, cleavage of phosphate and elimination of cyanoethyl as a protecting group pose problems in the reaction process.
While a protective group, which is difficult to be eliminated, is used in patent document 1, the protecting group is special and cannot be utilized directly to conventional reaction conditions and the like.
However, 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite is inferior in preservation stability and the market value is high.
The reactivity of the agent itself is poor, and an activator is required for the phosphitylation of nucleoside.
In addition, when triethylamine is used for desilylation of the 3′-terminal of oligonucleic acid having a cyanoethyl-protected phosphoric acid moiety, problems such as falling off of the cyanoethyl protecting group can be mentioned occur.

Method used

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  • Oligonucleotide manufacturing method
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Examples

Experimental program
Comparison scheme
Effect test

example 4

t of Decyanoethylaed Specimen Using DBU

[0202]DMTr-d[TGiBuCCO-TOP]-TBDMS (0.0043 g, 0.00179 mmol) obtained in the same manner as in Example 2 was dissolved in tetrahydrofuran (0.15 mL). 1,8-Diazabicyclo[5.4.0]undec-7-ene (hereinafter DBU) (2.0 μL, 0.0133 mmol) was added and mixed. Decyanoethylation was performed at 23° C. for 5 min. To the reaction mixture was added acetonitrile (1.0 mL) and the mixture was centrifuged at 10,000G, 4° C. for 5 min. The supernatant was removed, the remaining was dispersed in acetonitrile (1.0 mL) and similarly centrifuged again. Whole precipitate was dried in vacuo to give 0.0017 g in weight. When 31P{1H}NMR(CDCl3) of the resultant product was measured, all peaks appeared in 56.5-56.8 ppm, and it was confirmed that a decyanoethylated form shows 31P{1H}NMR signals in a region remarkably different from that of a cyanoethyl-protected form.

example 5

Various Fluoride Ion Sources Using Desilylation

(1) Study Using 3HF-TEA

[0203](DMTr-d[TGiBuCCO-TOP]-TBDMS (0.0239 g, 0.00998 mmol) obtained in the same manner as in Example 2 was dissolved in dichloromethane (0.3 mL) and cooled to 15° C. A solution of 3HF-TEA (29.0 mg, 0.180 mmol) in THF (0.60 mL) was added thereto and they were mixed. Desilylation was performed at 15° C. for 24 hr. The reaction mixture was divided into 4 by about 200 μL, acetonitrile (1.0 mL) was added to each, and the mixture was centrifuged at 10,000G, 4° C. for 5 min. The supernatant was removed, the remaining was dispersed in acetonitrile (1.0 mL) and similarly centrifuged again. Whole precipitate was dried in vacuo to give 0.0230 g in weight. When 31P{1H}NMR(CDCl3) of the resultant product was measured, all peaks were observed in 65.8-68.1 ppm corresponding to a cyanoethyl-protected form, and a signal in 60 ppm or below corresponding to a decyanoethylated form was not observed. In the measurement by LC-TOF MS, c...

example 21

Activation and Phosphitylation Using 4,5-dichloroimidazole

(1) Determination of pKa by Titration of 4,5-DICHLOROIMIDAZOLE

[0321]4,5-Dichloroimidazole (0.2396 g, 1.749 mmol) was dissolved in ion exchange water deaerated by boiling to 50.0 mL (0.0350 M). This solution (20 mL) was titrated at 25° C. with 0.10 M NaOH.

[0322]By non-log linearization plot of titration results (Benet, L. Z.; Goyan, J. E. J. Pharm. Sci. 1967, 56, 665-680.), pKa of dichloroimidazole in water at 25° C. was determined to be 9.09.

(2) Diamidite Activation and Phosphitylation Using 4,5-dichloroimidazole

[0323]2-Cyanoethyl-N,N,N′,N′-tetraisopropylphosphordiamidite (0.120 g, 0.40 mmol) was dissolved in dry toluene:cyclohexane=1:1 (v / v) solvent (1.5 mL). Thereto was added 4,5-dichloroimidazole (0.138 g, 1.00 mmol) and the mixture was stirred at under dry argon for 17 hr to prepare a solution containing a phosphitylating agent. The total amount of this solution was filtered through a 0.45 μm membrane filter. N-methylmorp...

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Abstract

The present invention aims to provide a more stable and efficient method for producing oligonucleotide, particularly, oligonucleotide having various functional groups linked to the 3′-terminal and the like. Efficient production of oligonucleotide becomes possible by a production method of an oligonucleotide represented by the formula (Ia-2) (each symbol is as defined in the DESCRIPTION) and having a functional group at the 3′-terminal, the method including a step of subjecting an oligonucleic acid with 3′-terminal protected by a silyl-protecting group to 3′-terminal-selective deprotection under desilylation conditions that do not affect protecting groups other than the silyl group, subjecting same to phosphitylation conditions with a phosphoramidite reagent that do not affect protecting groups on the oligonucleic acid to give a 3′-terminal-phosphoramidited oligonucleotide represented by the formula (Ia-1) (each symbol is as defined in the DESCRIPTION), and linking a functional group to the 3′-terminal of the 3′-terminal phosphoramidited oligonucleotide directly or via a linker, and the like.

Description

CROSS REFERENCES TO RELATED APPLICATIONS[0001]This application is a continuation of International Patent Application No. PCT / JP2016 / 088580, filed on Dec. 22, 2016, and claims priority to Japanese Patent Application No. 2015-250665, filed on Dec. 22, 2015, all of which are incorporated herein by reference in their entireties.BACKGROUND OF THE INVENTIONField of the Invention[0002]The present invention relates to a production method of an oligonucleotide. More particularly, the present invention relates to a method for producing an oligonucleotide by utilizing a silyl-protecting group, a method for producing an oligonucleotide by utilizing a phosphitylating agent, and a method for producing an oligonucleotide by fragment condensation and the like.Discussion of the Background[0003]The synthesis method of oligonucleotide includes a phosphate triester method, an H-phosphonate method, a phosphoramidite method and the like, and solid phase synthesis (solid phase method) using a phosphoramid...

Claims

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Application Information

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IPC IPC(8): C07H21/04
CPCC07H21/04Y02P20/55
Inventor YAMASHITA, KENHIRAI, KUNIHIROKATAYAMA, SATOSHIICHIMARU, TAISUKETAKAHASHI, DAISUKEHIROSE, NAOKO
Owner AJINOMOTO CO INC
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