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Phosphoramidite and its preparation method and use

A technology of phosphoramidite and diisopropyl phosphoramidite, which is applied in the field of phosphoramidite and its preparation, can solve the problems of cumbersome synthesis steps, high price and difficult separation and purification of photodegradable reagents, and achieve good results. Effects of photoresponsiveness, good market value, and high photoresponse efficiency

Active Publication Date: 2021-02-02
SHANGHAI ZHAOWEI TECH DEV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the photodegradable reagents currently commercialized and used in literature patents have problems such as cumbersome synthesis steps, difficult separation and purification, and high prices, which limit their further applications.

Method used

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  • Phosphoramidite and its preparation method and use
  • Phosphoramidite and its preparation method and use
  • Phosphoramidite and its preparation method and use

Examples

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preparation example Construction

[0038] Wherein the etherification reaction in the preparation method of the present invention is under the above reaction conditions, so that 4,4'-dimethoxytriphenylchloromethane can be preferentially combined with benzyl alcohol in 5-hydroxyl-2-nitrobenzyl alcohol The middle hydroxyl group reacts while avoiding the etherification of the phenolic hydroxyl groups on the remaining benzene rings. This operation is carried out at 0°C, which can also avoid the possible decomposition of 4,4'-dimethoxytriphenyl at higher temperatures, which helps to simplify subsequent purification steps and reduce operating costs.

[0039] In an optional embodiment: the purification treatment specifically includes the following steps: taking the crude product and dissolving it in ethyl acetate, washing with water, drying, filtering, and rotary evaporation to remove the organic solvent, and then dissolving the product in ethyl acetate , added dropwise into cooled n-hexane, filtered and dried to obtai...

Embodiment 1

[0058] A preparation method of phosphoramidite, comprising the following steps:

[0059] Step 1: Preparation of 5-hydroxy-2-nitrobenzyl-(4,4'-dimethoxytriphenyl) ether: Dissolve 1.7 g of 5-hydroxy-2-nitrobenzyl alcohol in 100 ml of Add dropwise to 30 ml of anhydrous pyridine dissolved in 4.0 g of 4,4'-dimethoxytriphenylchloromethane under ice-bath stirring, and stir the reaction solution at 0°C after the addition is complete for 1 hour, returned to room temperature and stirred overnight; after the reaction was completed, the organic phase was rotary evaporated, and the crude product was dissolved in 30 ml of ethyl acetate, washed twice with 50 ml of water, and the organic phase was dried with anhydrous sodium sulfate; filtered After rotary evaporation to remove the organic solvent, the obtained dark yellow oily liquid was dissolved in 5 ml of ethyl acetate and added dropwise to 100 ml of n-hexane cooled to 0°C to obtain a yellow precipitate, which was filtered and dried in vac...

Embodiment 2

[0068] A method for preparing deoxyribonucleic acid containing a photosensitive unit, comprising the following steps:

[0069] Step 1: Take 600mg of 2-nitrobenzyl-(4,4'-dimethoxytriphenyl)ether-5-(2-O-cyanoethyl-N,N-dimethoxy) prepared in Example 1 Isopropyl phosphoramidite) was dissolved in 10 milliliters of anhydrous acetonitrile, and transferred to the reagent bottle labeled "5" of the ABI3400 DNA synthesizer under nitrogen protection, defined as X, and the DNA sequence was input, using the synthesis mode of DMT retention, Perform synthesis on the order of 1 μmol;

[0070] Step 2: After the synthesis is completed, take out the solid-phase carrier, disperse it in 1 ml of 23% ammonia water, heat it in an airtight manner at 55°C for 2 hours, centrifuge to remove the insoluble matter, and concentrate the solution to obtain the crude product; pass the nucleic acid containing the photosensitive unit of the target product through High-performance liquid phase separation, and stru...

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Abstract

The present invention proposes a phosphoramidite and its preparation method and use. The phosphoramidite includes 5-hydroxy-2-nitrobenzyl alcohol as a starting material, and obtains 5-hydroxy-2-nitrobenzyl alcohol through etherification reaction. After nitrobenzyl-(4,4'-dimethoxytriphenyl) ether, then prepare 2-nitrobenzyl-(4,4'-dimethoxytriphenyl) through esterification base) ether‑5‑(2‑O‑cyanoethyl‑N,N‑diisopropylphosphoramidite). Among them, phosphoramidites can be used to prepare nucleic acids containing photosensitive units. The phosphoramidite of the present invention exhibits good stability at room temperature and under light-shielding conditions, has an exquisite structure, simple synthesis, high photoresponse efficiency, and can be directly used for functional modification in nucleic acid solid-phase synthesis. The introduction of photosensitive units at any position in the sequence provides a more concise and efficient option for the functionalization of nucleic acids.

Description

technical field [0001] The invention belongs to the technical field of chemical synthesis, and in particular relates to a phosphoramidite and its preparation method and application. Background technique [0002] With the development of biotechnology, the functionalization of natural biomacromolecules is an important means in the fields of bio-based material design, gene detection and gene therapy. In the research field of polyribonucleic acid (RNA) and polydeoxyribonucleic acid (DNA), the introduction of non-enzymatic cleavage linking unit is the basic design for aptamer screening, gene therapy, protein engineering and biomaterial design. The controllable cutting of nucleic acid sequences has important theoretical significance and application value in the fields of molecular biology, highly sensitive detection technology, protein engineering, and the construction of new biological materials. In recent years, people have developed a variety of linking reagents that can be de...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07F9/24C07H1/00C07H21/02C07H21/04
CPCC07F9/242C07H1/00C07H21/02C07H21/04
Inventor 孙亚伟吕环芳夏宁远王栋王继乾徐海
Owner SHANGHAI ZHAOWEI TECH DEV
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