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Chromophore-modified deoxynucleoside phosphoramidite monomer compound, preparation method therefor and application thereof

A technology of deoxynucleoside phosphoramidite and deoxynucleoside, which is applied to chromophore-modified deoxynucleoside phosphoramidite monomer compounds and the fields of preparation and application thereof, and can solve the problem that single-base mutation cannot be effectively distinguished, and there is no single-base mutation. Generality, tedious chemical synthesis of pyrene-modified oligonucleotides, etc., to achieve the effects of excellent properties, significant fluorescence enhancement, and strong recognition ability

Inactive Publication Date: 2016-02-24
PEKING UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For the existing pyrene-modified nucleic acid probes, most of them cannot effectively distinguish single-base mutations
For example, when using a click reaction at the 2' position of the sugar ring and using a triazole as a connecting arm to connect a pyrene fluorophore, it contains the site to be detected M (A) and the mutation sites MM (T), MM (G) and MM After the sequence of (C) is combined with the probe, there is almost no obvious difference
[0008] Also, most pyrene-modified oligonucleotides are chemically synthesized tediously.
When connecting pyrene fluorophores to bases, it is often necessary to consider the protection and removal of amino groups on the bases. At the same time, the strategy of linking pyrene fluorophores to purine bases and pyrimidine bases is often not universal; When modifying the ring, the sugar ring itself does not contain groups that can be further linked to fluorophores, often requiring special design of synthetic strategies

Method used

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  • Chromophore-modified deoxynucleoside phosphoramidite monomer compound, preparation method therefor and application thereof
  • Chromophore-modified deoxynucleoside phosphoramidite monomer compound, preparation method therefor and application thereof
  • Chromophore-modified deoxynucleoside phosphoramidite monomer compound, preparation method therefor and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Synthesis of embodiment 1 phosphoric acid position pyrene butanol modified phosphoramidite monomer

[0057] 1. Preparation of Compound PB-0

[0058] 2M concentration of borane dimethyl sulfide tetrahydrofuran solution 3mL, ice bath cooled to zero; pyrene butyric acid (560mg, 1.94mmoL, purchased from Bailingwei company) was dissolved in freshly distilled anhydrous THF solution 5mL. Under stirring in an ice bath, the tetrahydrofuran solution of pyrenebutyric acid was added dropwise to the borane solution, and stirred overnight at room temperature. The reaction mixture was stirred in an ice bath, and methanol was added dropwise to quench the reaction until no bubbles were generated, and the stirring was continued for 2 hours. The reaction system was concentrated to a small amount under reduced pressure, and 30 mL of ethyl acetate was added; the mixed solution was washed with saturated sodium carbonate solution (30 mL×3). The organic phase was collected, dried over anhydr...

Embodiment 2

[0064] Synthesis of embodiment 2 phosphoric acid position pyrene methanol modified phosphoramidite monomer

[0065] 1. Preparation of compound PM-1

[0066]Under nitrogen protection, bis-diisopropylphosphorus amidochloride (293mg, 1.1mmoL) was dissolved in 5mL of anhydrous tetrahydrofuran, and 1mL of triethylamine was added, and cooled to 0°C in an ice bath. Pyrenemethanol (230mg, 1.0mmoL) was dissolved in 1mL of anhydrous tetrahydrofuran under nitrogen protection, added dropwise to the former solution, and stirred overnight at room temperature. After the reaction was completed, the insoluble matter was quickly filtered off, and the filtrate was quickly spin-dried to obtain a light yellow solid. It was directly used in the next reaction without further processing. 31 PNMR (162MHz, CDCl 3 )δ=121.8.

[0067] 2. Preparation of Compound PM-2

[0068] Under nitrogen, DMT-protected deoxythymidine (350 mg, 0.64 mmoL) and tetrazolium (80 mg, 1.1 mmoL) were dissolved in 1.5 mL of ...

Embodiment 3

[0070] Synthesis of embodiment 3 phosphoric acid position perylene propanol modified phosphoramidite monomer

[0071] Synthesis of Compound Perylene-1

[0072] Under nitrogen protection, bis-diisopropylphosphorus amidochloride (293mg, 1.1mmoL) was dissolved in 5mL of anhydrous tetrahydrofuran, and 1mL of triethylamine was added, and cooled to 0°C in an ice bath. Perylene propanol (310 mg, 1.0 mmoL) was dissolved in 1 mL of anhydrous tetrahydrofuran under nitrogen protection, added dropwise to the former solution, and stirred overnight at room temperature. After the reaction was completed, the insoluble matter was quickly filtered off, and the filtrate was quickly spin-dried to obtain a yellow solid. It was directly used in the next reaction without further processing.

[0073] Synthesis of Compound Perylene-dC

[0074] Under nitrogen, DMT-protected deoxycytidine (412 mg, 0.65 mmoL) and tetrazolium (80 mg, 1.1 mmoL) were dissolved in 1.5 mL of anhydrous dichloromethane. All...

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Abstract

The invention discloses a chromophore-modified deoxynucleoside phosphoramidite monomer compound, a preparation method therefor and an application thereof. The preparation method comprises the steps of: connecting chromophores such as pyrene, perylene or naphthalene carboxamide with bis(diisopropylamino) chlorophosphine to obtain a phosphorous intermediate; and reacting the phosphorous intermediate with DMT-protected deoxynucleoside to obtain a chromophore-modified deoxynucleoside phosphoramidite monomer compound. By virtue of solid-phase synthesis of DNA, the compound is inserted into oligonucleotide at a fixed point to obtain a chromophore-modified fluorescent oligonucleotide probe with a stable double-chain structure. The fluorescent oligonucleotide probe is free of fluorescence-emission, and only being combined with a perfectly matching target chain, the fluorescence can be enhanced by 23.5 times, and the response speed is fast. Mismatched bases are obviously identified with nearly no fluorescence-emission, so that single base mismatch can be obviously identified. The compound can be applied to single base mutation analysis of a gene and detection of a PCR reaction process and the like, and is wide in application prospect in aspects of single base polymorphism detection and nucleic acid detection in a biochemical sample and the like.

Description

technical field [0001] The present invention relates to a phosphoramidite monomer compound, in particular to a deoxynucleoside phosphoramidite monomer compound modified with a chromophore at the phosphoric acid position, and the present invention also relates to a deoxynucleoside phosphoramidite monomer compound modified with a chromophore The preparation method and the prepared oligonucleotide probe and its application in single base polymorphism detection and target gene or RNA detection belong to the field of functionalized oligonucleotides. Background technique [0002] Nucleic acid probes can specifically bind to DNA, RNA, or proteins, and have a wide range of applications in the fields of chemistry, biology, and medical research, such as nucleic acid nanoparticles, biochips, mRNA imaging, genetic screening, and single-base polymorphism ( SNP) detection, etc. Among them, molecular beacons (MBs) are most widely used in these fields. Traditionally, molecular beacons are...

Claims

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Application Information

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IPC IPC(8): C07H19/10C07H1/00C07H21/04C07F9/22C07F9/576C09K11/06C12Q1/68C12N15/11
CPCC07F9/22C07F9/5765C07H1/00C07H19/10C07H21/04C09K11/06C09K2211/1011C09K2211/1029C09K2211/1044C09K2211/1088C12Q1/6827C12Q2563/107
Inventor 汤新景李鹏飞王志轩
Owner PEKING UNIV
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