阅读说明：本技术 一种三异丙基硅乙炔修饰的脱氧胞苷亚磷酰胺单体及其制备方法与应用 (Tri-isopropyl silaacetylene modified deoxycytidine phosphoramidite monomer and preparation method and application thereof ) 是由 *** 董田田 于 2020-06-30 设计创作，主要内容包括：本发明公开了一种三异丙基硅乙炔修饰的脱氧胞苷亚磷酰胺单体及其制备方法与应用。本发明三异丙基硅乙炔修饰的脱氧胞苷亚磷酰胺单体的化学结构式为：<Image he="661" wi="408" file="DDA0002562166010000011.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>式Ⅰ中,DMTr-为4,4’-二甲氧基三苯甲基,N(i-Pr)<Sub>2</Sub>-为N,N-二异丙基胺基。其制备方法是将5-I-脱氧胞苷中5’端基保护,然后对其氨基进行保护,再将碱基5号位引入三异丙基硅乙炔进行修饰,然后将其脱氧胞苷引入亚磷酰胺得到三异丙基硅乙炔修饰的脱氧胞苷亚磷酰胺单体。本发明所述式Ⅰ所示的三异丙基硅乙炔修饰的脱氧胞苷亚磷酰胺单体应用于制备寡核苷酸中。本发明一种寡核苷酸可由包括上述式Ⅰ所示的三异丙基硅乙炔修饰的脱氧胞苷亚磷酰胺单体通过DNA固相合成法制成。本发明制备方法简单,能用于制备稳态与瞬态红外光谱结构探针。(The invention discloses a triisopropyl silaacetylene modified deoxycytidine phosphoramidite monomer and a preparation method and application thereof. The chemical structural formula of the deoxycytidine phosphoramidite monomer modified by triisopropyl-silacetylene is as follows: in the formula I, DMTr-is 4, 4' -dimethoxytrityl, N (i-Pr) 2 -is N, N-diisopropylamino. The preparation method comprises the steps of protecting the 5' end group in the 5-I-deoxycytidine, then protecting the amino group, introducing triisopropylsilacetylene into the 5 th position of the basic group for modification, and then introducing the deoxycytidine into phosphoramidite to obtain triisopropylsilacetylene modified deoxycytidine phosphoramidite monomer. The invention relates to application of a triisopropylsilacetylene modified deoxycytidine phosphoramidite monomer shown in formula I in preparation of oligonucleotides. The oligonucleotide can be prepared from the triisopropylsilacetylene modified deoxycytidine phosphoramidite monomer shown in the formula I by a DNA solid phase synthesis method. The preparation method is simple and can be used for preparing probes with stable and transient infrared spectrum structures.)

1. A triisopropylsilaacetylene modified deoxycytidine phosphoramidite monomer has a chemical structural formula as follows:

in the formula I, DMTr-is 4, 4' -dimethoxytrityl, N (i-Pr)₂-is N, N-diisopropylamino.

2. A method for preparing a deoxycytidine phosphoramidite monomer modified with triisopropylsilacetylene as shown in claim 1, which comprises: the synthesis method comprises the following steps:

(1) reacting 5-iodine-2 ' -deoxycytidine with 4,4 ' -dimethoxytrityl chloride in an organic solvent in an anhydrous and oxygen-free environment under the protection of inert gas and in the presence of a catalyst a to obtain 5-iododeoxycytidine with 5 ' end group protection shown in a formula II;

(2) carrying out amidation reaction on the 5 '-iodo deoxycytidine protected by the 5' end group shown in the formula II and acetic anhydride to obtain 5-I-deoxycytidine protected by amino shown in the formula III;

(3) performing Sonogashira coupling reaction on the amino-protected 5-I-deoxycytidine shown in the formula III and triisopropylsilacetylene to obtain base No. 5 triisopropylsilacetylene modified deoxycytidine shown in the formula IV;

(4) under the anhydrous and anaerobic environment, and in the presence of the inert gas protection and the catalyst b, the base No. 5 triisopropylsilacetylene modified deoxycytidine shown in the formula IV undergoes a phosphoramidite reaction to obtain the triisopropylsilacetylene modified deoxycytidine phosphoramidite monomer shown in the formula I.

3. The production method according to claim 1 or 2, characterized in that: in the step (1), the catalyst a is 4-dimethylaminopyridine;

an acid-binding agent is also added in the reaction, and the acid-binding agent is triethylamine;

the molar ratio of the 5-iodo-2 '-deoxycytidine, the triethylamine, the 4-dimethylaminopyridine to the 4, 4' -dimethoxytrityl chloride is 1: 1.5-2.5: 0.2-0.5: 1.5-2.5;

the reaction is carried out in a solvent, wherein the solvent is pyridine;

the reaction temperature is room temperature, and the reaction time is 3-6 h;

the inert gas comprises at least one of nitrogen, argon and helium; and/or

In the step (2), the molar ratio of the 5' -end group protected 5-iodo deoxycytidine shown in the formula II to acetic anhydride is 1: 2-3;

the temperature of the amidation reaction is room temperature, and the reaction time is 20-30 h;

the amidation reaction is carried out in the presence of an organic solvent comprising N' N-dimethylformamide and/or pyridine.

4. The production method according to claim 2 or 3, characterized in that: in the step (3), the molar ratio of the amino-protected 5-I-deoxycytidine shown in the formula III to the triisopropylsilacetylene is 1: 2-4;

the temperature of the Sonogashira coupling reaction is room temperature, and the reaction time is 24-36 h;

the Sonogashira coupling reaction is carried out in the presence of a catalyst c comprising cuprous iodide and tetrakis- (triphenylphosphine) -palladium;

an acid-binding agent is also added in the Sonogashira coupling reaction, and the acid-binding agent is triethylamine; and/or

In the step (3), the Sonogashira coupling reaction is carried out in the presence of an organic solvent, wherein the organic solvent comprises N' N-dimethylformamide;

the amino-protected 5-I-deoxycytidine shown in the formula III, cuprous iodide, tetrakis- (triphenylphosphine) -palladium, triethylamine and triisopropylsilacetylene are in a molar ratio of 1: 0.1-0.3: 0.1-0.25: 2-4.

5. The production method according to any one of claims 2 to 4, characterized in that: in the step (4), 2-cyanoethyl N, N-diisopropyl phosphoramidite chloride is adopted for the phosphoramidite reaction;

the catalyst b comprises N, N-diisopropylethylamine;

the molar ratio of base No. 5 triisopropylsilylacetylene modified deoxycytidine, N-diisopropylethylamine and 2-cyanoethyl N, N-diisopropyl phosphoramidite shown in the formula IV is 1: 2-3: 1.7-2;

the temperature of the phosphoramidite reaction is-15-10 ℃, and the time is 3-5 h;

the phosphoramidite reaction is carried out in the presence of an organic solvent, including methylene chloride.

6. Use of triisopropylsilacetylene-modified deoxycytidine phosphoramidite monomers as described in claim 1 for the preparation of oligonucleotides.

7. An oligonucleotide, characterized in that: the oligonucleotide is prepared from a deoxycytidine phosphoramidite monomer modified by triisopropylsilacetylene shown as a formula I in claim 1 by a DNA solid phase synthesis method;

the number of the triisopropylsilacetylene-modified deoxycytidine formed by the triisopropylsilacetylene-modified deoxycytidine phosphoramidite monomer shown in the formula I in the oligonucleotide is 1 or more;

the triisopropylsilacetylene-modified deoxycytidine is at an arbitrary position in the oligonucleotide.

8. Use of the oligonucleotide of claim 7 in the preparation of probes of both steady state and transient infrared spectroscopic structures.

9. A triisopropylsilacetylene modified deoxycytidine has a chemical structural formula as follows:

。

10. a method for preparing triisopropylsilacetylene-modified deoxycytidine represented by the formula v as set forth in claim 9, comprising the steps of:

performing Sonogashira coupling reaction on the 5-iodine-2' -deoxycytidine and the triisopropylsilylacetylene to obtain base No. 5 triisopropylsilylacetylene modified deoxycytidine shown in the formula V;

specifically, the molar ratio of the 5-iodine-2' -deoxycytidine to the triisopropylsilacetylene is 1: 2-4;

the temperature of the Sonogashira coupling reaction is room temperature, and the reaction time is 24-36 h;

the Sonogashira coupling reaction is carried out in the presence of the catalyst c comprising cuprous iodide and tetrakis- (triphenylphosphine) -palladium;

the acid-binding agent is also added in the Sonogashira coupling reaction, and the acid-binding agent is triethylamine; and/or

The Sonogashira coupling reaction is carried out in the presence of an organic solvent comprising N' N-dimethylformamide;

the molar ratio of the 5-iodine-2' -deoxycytidine, cuprous iodide, tetrakis- (triphenylphosphine) -palladium, triethylamine and triisopropylsilacetylene is 1: 0.1-0.3: 0.1-0.25: 2-4.

Technical Field

The invention relates to a triisopropylsilaacetylene modified deoxycytidine phosphoramidite monomer and a preparation method and application thereof, belonging to the technical field of chemical synthesis of nucleotide.

Background

In recent years, with the development of chemical synthesis technology of nucleotides, a series of chemically modified nucleotide monomers and oligonucleotide derivatives have been reported in academia and industry. The chemical modification of nucleic acid mainly includes modification of phosphate group, base and sugar ring of nucleic acid and substitution of non-natural monomer for natural monomer, and mainly includes a method of direct chemical modification of oligonucleotide fragment by introducing nucleotide monomer derivative into oligonucleotide fragment; the introduced modifying group can be used as various spectral probes and has very important function on the research of the structure and the function of the biological genetic material. However, a few types of modifying groups have been reported as infrared spectroscopic probes for nucleic acids, particularly those having a modifying group (typically an acetylenic group) that does not overlap with the infrared absorption peak of nucleic acids, and this has been done with very little research and has not been reported so far in conjunction with steady-state and transient infrared spectroscopic methods.

On the other hand, with the development of "click chemistry", the research of "click chemistry" on nucleic acids has become an important field. The common click chemical reaction is that azide reacts with terminal alkyne to generate triazole, the reaction product is single, stable and free of chemical toxicity, and the triazole product has a strong fluorescence effect. Nucleic acid modification by 'click chemistry' of azide and terminal alkyne can be used for labeling, fixing, metalizing, crosslinking, artificial synthesis and the like of oligonucleotide, and has strong functions. Therefore, the method has important significance for efficiently and quickly obtaining the alkynyl-modified oligonucleotide. In addition, the ethynyl group on the base is susceptible to hydrolysis during solid phase synthesis of DNA, which also increases the difficulty of isolation of the oligonucleotide fragment.

Disclosure of Invention

The invention aims to provide a triisopropylsilaacetylene modified deoxycytidine phosphoramidite monomer and a preparation method and application thereof. The invention modifies the basic group of a nucleic acid monomer by triisopropylsilacetylene, is used for introducing a phosphoramidite monomer into an oligonucleotide segment, leads the specific oligonucleotide segment to contain an alkynyl group modified by triisopropylsilyl, becomes an infrared spectrum probe with obvious infrared activity, and provides an exogenous spectrum probe with characteristic infrared absorption peak for researching the structural dynamics of a special site of DNA by using steady-state and transient infrared spectrum means.

The invention provides a triisopropyl silaacetylene modified deoxycytidine phosphoramidite monomer, which has the chemical structural formula as follows:

in the formula I, DMTr-is 4, 4' -dimethoxytrityl, N (i-Pr)₂-is N, N-diisopropylamino.

The invention also provides a preparation method of the triisopropylsilacetylene modified deoxycytidine phosphoramidite monomer shown in the formula I, which comprises the following steps:

(1) reacting 5-iodine-2 ' -deoxycytidine with 4,4 ' -dimethoxytrityl chloride in an organic solvent in an anhydrous and oxygen-free environment under the protection of inert gas and in the presence of a catalyst a to obtain 5-iododeoxycytidine with 5 ' end group protection shown in a formula II;

(2) carrying out amidation reaction on the 5 '-iodo deoxycytidine protected by the 5' end group shown in the formula II and acetic anhydride to obtain 5-I-deoxycytidine protected by amino shown in the formula III;

(3) performing Sonogashira coupling reaction on the amino-protected 5-I-deoxycytidine shown in the formula III and triisopropylsilacetylene to obtain base No. 5 triisopropylsilacetylene modified deoxycytidine shown in the formula IV;

(4) under the anhydrous and anaerobic environment, and in the presence of the inert gas protection and the catalyst b, the base No. 5 triisopropylsilacetylene modified deoxycytidine shown in the formula IV undergoes a phosphoramidite reaction to obtain the triisopropylsilacetylene modified deoxycytidine phosphoramidite monomer shown in the formula I.

In the preparation method, in the step (1), the catalyst a is 4-dimethylaminopyridine;

an acid-binding agent is also added in the reaction, and the acid-binding agent is triethylamine; for neutralizing the HCl formed during the reaction; transferring the solution into an ice-water bath when the 4-dimethylaminopyridine is added;

345-iodine-2 '-deoxycytidine, triethylamine, 4-dimethylaminopyridine and the 4, 4' -dimethoxytrityl chloride in a molar ratio of 1: 1.5-2.5: 0.2-0.5: 1.5-2.5, specifically 1:2:0.25:2, 1: 1.5-2: 0.2-0.25: 1.5-2, 1: 2-2.5: 0.25-0.5: 2-2.5;

the reaction is carried out in a solvent, wherein the solvent is pyridine;

the reaction temperature is room temperature, and the reaction time can be 3-6 h, specifically 4h, 3-4 h, 4-6 h or 3.5-5 h;

the inert gas includes at least one of nitrogen, argon, and helium.

In the preparation method, in the step (2), the molar ratio of the 5' -end group-protected 5-iododeoxycytidine shown in the formula II to acetic anhydride can be 1: 2-3, specifically 1:2.5, 1: 2-2.5, 1: 2.5-3, or 1: 2.25-2.75;

the temperature of the amidation reaction is room temperature, and the reaction time can be 20-30 h, specifically 24h, 20-24 h, 24-30 h or 22-27 h;

the amidation reaction is carried out in the presence of an organic solvent comprising N' N-dimethylformamide and/or pyridine.

In the preparation method, in the step (3), the molar ratio of the amino-protected 5-I-deoxycytidine shown in the formula III to the triisopropylsilacetylene is 1: 2-4, specifically 1:2.5, 1: 2.5-4, 1: 2-2.5 or 1: 2.5-3;

the temperature of the Sonogashira coupling reaction is room temperature, the reaction time can be 24-36 h, specifically 24h, 24-27 h or 24-30 h, and the reaction time is properly prolonged to improve the yield;

the Sonogashira coupling reaction is carried out in the presence of a catalyst c comprising cuprous iodide and tetrakis- (triphenylphosphine) -palladium;

and adding an acid-binding agent into the Sonogashira coupling reaction, wherein the acid-binding agent is triethylamine and is used for neutralizing the hydrogen iodide HI generated in the reaction process.

In the above production method, in the step (3), the Sonogashira coupling reaction is carried out in the presence of an organic solvent comprising N' N-dimethylformamide;

the molar ratio of the amino-protected 5-I-deoxycytidine, cuprous iodide, tetrakis- (triphenylphosphine) -palladium, triethylamine and triisopropylsilacetylene shown in the formula III can be 1: 0.1-0.3: 0.1-0.25: 2-4, and specifically can be 1:0.2:0.15:3: 2.5.

In the above preparation method, in the step (4), 2-cyanoethyl N, N-diisopropyl chlorophosphine amide is used for the phosphoramidite reaction;

the catalyst b comprises N, N-diisopropylethylamine;

the molar ratio of the base No. 5 triisopropylsilylacetylene modified deoxycytidine, N-diisopropylethylamine and the 2-cyanoethyl N, N-diisopropyl phosphoroamidite shown in the formula IV can be 1: 2-3: 1.7-2, specifically can be a reaction in an ice water bath, and specifically can be 1:2: 1.7;

the temperature of the phosphoramidite reaction can be-15-10 ℃, and the phosphoramidite reaction is carried out in an ice-water bath for 3-5 hours, specifically 3 hours and 3-4 hours;

the phosphoramidite reaction is carried out in the presence of an organic solvent, including methylene chloride.

In the above preparation method, in the step (1), the post-treatment of the reaction is as follows: repeatedly washing, filtering and drying the mixture by ethyl acetate and sodium bicarbonate water solution;

in step (2), the post-treatment of the amidation reaction is as follows: washing with saturated salt water, and vacuum filtering to remove organic solvent;

in step (3), the post-treatment of the Sonogashira coupling reaction is as follows: extracting with saturated saline and ethyl acetate, and removing the solvent;

in the step (4), the post-treatment of the phosphoramidite reaction is as follows: and (2) separating the triisopropyl silacetylene modified deoxycytidine phosphoramidite monomer shown in the formula I by adopting a silica gel chromatographic column through column chromatography, and carrying out alkali leaching and refrigeration on the silica gel chromatographic column to obtain a target product through rapid separation.

The invention relates to application of a triisopropylsilacetylene modified deoxycytidine phosphoramidite monomer shown in formula I in preparation of oligonucleotides.

The invention provides a raw material for further synthesizing novel oligonucleotide, and the oligonucleotide can be prepared from the triisopropylsilacetylene modified deoxycytidine phosphoramidite monomer shown in the formula I by a DNA solid phase synthesis method.

In the present invention, the oligonucleotide is an oligonucleotide containing triisopropylsilacetylene-modified deoxycytidine. The alkynyl C [ identical to ] C contained in the probe can be used as an infrared spectrum probe of the structure and dynamics of a specific site of DNA. It should be noted that C.ident.C stretching vibration, although the absorption frequency does not overlap with the characteristic peak of nucleic acid, is not used as a spectrum probe in the traditional infrared spectroscopy because it usually has only weak infrared activity and low vibration absorption intensity. In the invention, the introduction of triisopropyl silicon group as an alkynyl terminal group modification group greatly enhances the C ≡ C stretching vibration intensity, and makes the vibration frequency and peak width sensitive to chemical structures and solvents, so that the probe becomes a feasible steady-state and transient infrared spectrum structure probe, and provides possibility of description at the chemical bond level and by using an infrared vibration spectrum means for researching the structure dynamics of a DNA special site.

In the oligonucleotide, the number of triisopropylsilacetylene-modified deoxycytidine formed by the triisopropylsilacetylene-modified deoxycytidine phosphoramidite monomer shown in the formula I in the oligonucleotide is 1 or more;

the triisopropylsilacetylene-modified deoxycytidine is at an arbitrary position in the oligonucleotide.

The oligonucleotide provided by the invention is applied to preparation of probes with stable and transient infrared spectrum structures.

In order to prove that an acetylene bond in the triisopropylsilacetylene modified deoxycytidine phosphoramidite monomer shown in the formula I can be used as a probe with an infrared spectrum structure, the invention further provides a base No. 5 triisopropylsilacetylene modified deoxycytidine phosphoramidite monomer without a protecting group in the triisopropylsilacetylene modified deoxycytidine phosphoramidite monomer shown in the formula I, and the chemical structural formula is as follows:

the invention also provides a preparation method of the triisopropylsilacetylene modified deoxycytidine shown in the formula V, which comprises the following steps:

and carrying out Sonogashira coupling reaction on the 5-iodine-2' -deoxycytidine and the triisopropylsilylacetylene to obtain the deoxycytidine modified by the base No. 5 triisopropylsilylacetylene shown in the formula V.

In the preparation method, the molar ratio of the 5-iodo-2' -deoxycytidine to the triisopropylsilacetylene is 1: 2-4, specifically 1:2.5, 1: 2.5-4, 1: 2-2.5 or 1: 2.5-3;

the temperature of the Sonogashira coupling reaction is room temperature, the reaction time can be 24-36 h, specifically 24h, 24-27 h or 24-30 h, and the reaction time is properly prolonged to improve the yield.

In the above preparation method, the Sonogashira coupling reaction is carried out in the presence of a catalyst comprising cuprous iodide and tetrakis- (triphenylphosphine) -palladium;

and adding an acid-binding agent into the Sonogashira coupling reaction, wherein the acid-binding agent is triethylamine and is used for neutralizing the hydrogen iodide HI generated in the reaction process.

In the above preparation method, the Sonogashira coupling reaction is carried out in the presence of an organic solvent, the organic solvent comprising N' N-dimethylformamide;

the molar ratio of the 5-iodine-2' -deoxycytidine, the cuprous iodide, the tetrakis- (triphenylphosphine) -palladium, the triethylamine and the triisopropylsilacetylene can be 1: 0.1-0.3: 0.1-0.25: 2-4, and specifically can be 1:0.2:0.15:3: 2.5.

The invention has the following advantages:

1. 5-triisopropylsilacetylene-deoxycytidine phosphoramidite monomer is used as raw material for DNA solid phase synthesis, and one or more 5-triisopropylsilacetylene-deoxycytidine phosphoramidite monomer or 5-acetylene-deoxycytidine phosphoramidite monomer can be conveniently introduced into any position of oligonucleotide segment to synthesize novel oligonucleotide sequence and probe thereof.

2. The synthesis method has the advantages of mild conditions, simple post-treatment and simple and convenient operation, and is suitable for industrial production.

3. The invention has the advantages that the initial important raw material is 5-I-deoxycytidine (5-iodine-2' -deoxycytidine), the raw material is easy to obtain, and the cost is low.

Drawings

FIG. 1 is a flow chart of the reaction equation of the method for synthesizing a deoxycytidine phosphoramidite monomer modified by triisopropylsilacetylene according to the present invention.

FIG. 2 shows infrared absorption spectra (FTIR spectra) of deoxycytidine and 5-acetylene-deoxycytidine modified with triisopropylsilylacetylene at the base No. 5 in accordance with the present invention.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.