WO2019227526A1

WO2019227526A1 - Fluorescently labelled nucleotide and preparation method and use thereof

Info

Publication number: WO2019227526A1
Application number: PCT/CN2018/090779
Authority: WO
Inventors: 车团结
Original assignee: 苏州百源基因技术有限公司
Priority date: 2018-05-29
Filing date: 2018-06-12
Publication date: 2019-12-05
Also published as: CN108752406A

Abstract

Provided is a fluorescently labelled nucleotide having a structure represented by formula I. The fluorescently labelled nucleotide is linked by a covalent bond between deoxyribonucleoside triphosphate and a fluorescent dye molecule and is suitable for detection of nucleic acid molecules inside and outside the cells. The fluorescently labelled nucleotide is prepared by click chemistry.

Description

Fluorescently labeled nucleotide and preparation method and application thereof

Technical field

The invention belongs to the technical field of medical biological detection, and particularly relates to a fluorescently labeled nucleotide and a preparation method and application thereof.

Background technique

Fluorescent labeling technology refers to labeling a substance capable of emitting fluorescence by covalent bonding or physical adsorption on a certain group of the molecule under study, and using its fluorescence characteristics to reflect the information of the research object. After using fluorescent labeling reagents to be adsorbed or covalently bound to the research object (nucleic acid, protein, peptide, etc.), their fluorescence characteristics change, which reflects the information about the performance of the research object. With the continuous development of modern medicine and biological technology, the discovery of new fluorescently labeled dyes and the application of various advanced fluorescence detection technologies and instruments, such as flow cytometry (FCM), laser scanning confocal microscope (LSCM), etc., serve as A non-radioactive labeling technology. Fluorescent labeling has the characteristics of simple operation, high stability, high sensitivity, and good selectivity. It can be widely used in the detection of intracellular and extracellular substances, the imaging of tissue and living animal markers, drug analysis, pathological model research, and Early diagnosis of diseases plays an important role in the field of biomedical research.

Nucleic acid is the genetic material of life and plays an important role in life activities. Nucleic acid detection has important applications in the fields of disease diagnosis, environmental microorganism identification, and aptamers. Non-radioactive detection of nucleic acids using fluorescent markers is an important technology in molecular biology and is widely used in biological detection technology fields such as DNA sequencing, in situ hybridization, and liquid-phase chips. The use of multiple fluorescent labels to detect one or more target nucleic acid molecules has important application significance. For example, liquid phase chips are coupled to nucleic acid probe molecules on the surface of fluorescent microspheres. Fluorescent microspheres are in micron-scale polymer microspheres. Spheres with different fluorescent signal codes formed by loading fluorescent materials with different combinations of light emission wavelengths and intensity levels; on the other hand, by fluorescently labeling the target nucleic acid molecule to be detected, a fluorescent reporter molecule is labeled on the target nucleic acid molecule; a nucleic acid probe After the molecule is hybridized with the target nucleic acid molecule to be detected, the fluorescent signal of the fluorescent microsphere is used to realize the qualitative analysis of the target nucleic acid molecule, and the fluorescent intensity of the reporter molecule is used to realize the quantitative / semi-quantitative detection of the target nucleic acid molecule.

The liquid phase chip can be used for multiple sample detection at the same time, thereby achieving multiple quantitative analysis of multiple nucleic acid molecules in a sample. However, the current liquid crystal chip technology still has the following defects: 1. The Stokes shift of the fluorescent dye generally does not exceed 30nm, the fluorescent dye is easy to quench, the signal is unstable, and it is not conducive to distinguishing between different fluorescent signals; 2 In the liquid phase chip, fluorescent microspheres need to be prepared in advance, and then the probe molecules are coupled with the fluorescent microspheres, which leads to complicated manufacturing steps of the liquid phase chip. In addition, due to the limitation of the types of fluorescent signals in the fluorescent microspheres, currently only the Fluorescent labeling of about 100 probe molecules.

Summary of the Invention

Therefore, the technical problem to be solved by the present invention is to overcome the defects that the fluorescently labeled probe molecules in the prior art have unstable fluorescent signals, are unable to effectively distinguish different fluorescent signals, and have a small number of fluorescent signal types of labeled probe molecules.

To this end, the present invention provides the following technical solutions:

In a first aspect, the present invention provides a fluorescently labeled nucleotide having a structure represented by Formula I:

Wherein W is dNTP, said dNTP is selected from one of dUTP, dATP, dGTP, dCTP and dTTP,

X is halogen,

R _{1 is} selected from one of an alkyl group, a cycloalkyl group, an aryl group, and a heterocyclic group.

Preferably, in the above fluorescently labeled nucleotide, X is Br.

Preferably, the above-mentioned fluorescently labeled nucleotides have a structure represented by Formula II:

Further preferably, the structure of the above-mentioned fluorescently labeled nucleotide is as shown in Formula A to Formula H:

In a second aspect, the present invention provides a method for preparing the above-mentioned fluorescently labeled nucleotides, including the following steps:

S1. The compound dN-I and 1,6-heptadiyne are subjected to a cross-coupling reaction under the action of a catalyst to obtain a compound dN-P, and then the compound dN-P and tri-n-butylamine pyrophosphate and 2- The reaction occurs under the combined action of chloro-4H-1,3,2-benzodioxan-4-one to obtain the compound dNTP-P;

Among them, dN-I is selected from the chemical structure shown below:

dN-P is selected from the chemical structure shown below:

dNTP-P is selected from the chemical structure shown below:

S2. Compound of the formula Q and 2-azidoethylamine

A condensation reaction occurs to prepare the compound QN ₃ ,

The structure of compound Q is shown below:

The structure of compound QN ₃ is shown below:

S3. The compound QN _{3 is} subjected to a click chemical reaction with the compound dNTP-P to obtain a fluorescently labeled nucleotide having a structure represented by Formula I.

Preferably, in the above preparation method, the compound of the structure represented by formula Q is prepared by the following steps:

(1) Preparation of intermediate I'-1

Phenylhydrazine was added to glacial acetic acid, stirred, and 3-methyl-2-butanone was slowly added dropwise. After the dropwise addition was completed, the mixture was heated to 60-65 ° C, reacted for 3-4 hours, extracted, concentrated, and purified to obtain intermediate I. '-1;

Wherein, the molar ratio of phenylhydrazine to 3-methyl-2-butanone is 1: (1.0-1.2);

(2) Preparation of intermediate I'-2

Add intermediate I'-1 and 1,2-dibromoethylene to toluene, protect with nitrogen, heat under reflux for 16-18 hours, cool, and precipitate a solid, namely intermediate I'-2;

Wherein, the molar ratio of the intermediate I'-1 and 1,2-dibromoethylene is 1: (1.5-2.0);

(3) Preparation of intermediate I'-4

Add dry N, N-dimethylformamide to dry dichloromethane, add dichloromethane solution of phosphorus oxychloride under ice bath, stir, add cyclohexanone, remove the ice bath, heat to reflux 2 -3 hours, pour the reaction solution into crushed ice and let it stand overnight to precipitate a solid, namely intermediate I'-4;

Wherein, the molar ratio of cyclohexanone, N, N-dimethylformamide and phosphorus oxychloride is 1: (1.0-1.1): (1.0-1.05);

(6) Preparation of intermediate I'-5

Add intermediate I'-2 and intermediate I'-4 to the mixed solution of n-butanol and toluene, heat to reflux for 2-3 hours, precipitate a solid, and filter to obtain intermediate I'-5;

(7) Preparation of compound Q

Intermediate I'-5 and Br-R ₁ -COOH undergo an amino substitution reaction, and NaOH is added during the reaction to produce compound Q;

The synthetic route is as follows:

Further preferably, the above-mentioned preparation method:

In the step (1), the molar ratio of the phenylhydrazine to the 3-methyl-2-butanone is 1: (1.0-1.2);

In the step (2), the molar ratio of the intermediate I'-1 and 1,2-dibromoethylene is 1: (1.5-2.0);

In the step (3), the molar ratio of the cyclohexanone, N, N-dimethylformamide, and phosphorus oxychloride is 1: (1.0-1.1): (1.0-1.05).

Preferably, in the above preparation method, in step S3, the molar ratio of the compound I′-N ₃ and the compound dNTP-P is 1: (1 to 1.2).

In a third aspect, the present invention provides the use of the above-mentioned fluorescently labeled nucleotides in the preparation of a fluorescent probe.

In a fourth aspect, the present invention provides the use of the above-mentioned fluorescently labeled nucleotides in gene sequencing, in situ hybridization detection, Northern blot, Southern blot, or liquid-phase chip.

The technical solution of the present invention has the following advantages:

1. A fluorescently labeled nucleotide provided by the present invention, having a structure represented by Formula I. The compound of the structure represented by Formula I is formed by the covalent combination of deoxyribonucleoside triphosphate (dNTP) and a fluorescent dye represented by Formula Q. The Stokes shift of the fluorescent dye molecule represented by Formula Q is large and the fluorescent signal is stable The fluorescence quantum yield is high, which can be effectively distinguished from the emitted fluorescence of other fluorescent dye molecules, and has the advantage of high signal-to-noise ratio in fluorescence imaging.

The fluorescently labeled nucleotides represented by Formula I are highly stable in detection environments such as serum and can be retained in cells for a long time. At the same time, fluorescently labeled nucleotides have low cytotoxicity and high biocompatibility, and are suitable for use in It is widely used in the detection of biologically active molecules inside and outside cells, imaging of tissue and live animal markers, drug analysis, pathological model research, and early diagnosis of diseases.

Fluorescently labeled nucleotides can be directly used in the synthesis of nucleic acid molecules. Since the dye molecules are directly covalently coupled to the nucleotides, the synthesized nucleic acids can emit fluorescence without the need for fluorescent coupling modification. Needles are used for the detection of target biomolecules (proteins, DNA or RNA, etc.) inside or outside cells or in vivo. The above fluorescent probe has the advantages of high fluorescence quantum yield and stable fluorescence performance. At the same time, when synthesizing the fluorescent probe, it can flexibly introduce the required number of the above-mentioned fluorescently labeled nucleotides at specific positions of the nucleic acid molecular chain. On the one hand, the biological activity of the synthesized nucleic acid molecules with fluorescent properties can be guaranteed, and on the other hand, the intensity of the fluorescent signal can be controlled by introducing a specific number of fluorescent dye molecules.

When the fluorescently labeled nucleotide is applied to a liquid phase chip, the probe molecule synthesized with the fluorescently labeled nucleotide does not need to be coupled with a fluorescent microsphere, which simplifies the preparation process of the liquid phase chip; In the near-infrared region, a new fluorescence detection signal with stable signal and high discrimination is provided, and the luminescence form of the probe molecule is increased.

2. The method for preparing fluorescently labeled nucleotides provided by the present invention uses a click chemical reaction to achieve the covalent bonding of dNTPs and fluorescent dye molecules. Raw materials required for synthesis are readily available, mild reaction conditions, simple operation, and selectivity of the reaction. High, fluorescently labeled nucleotides prepared by the reaction have high fluorescence yield, fluorescence stability, and biological activity.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly explain the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the specific embodiments or the description of the prior art will be briefly introduced below. Obviously, the appended descriptions in the following description The drawings are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without paying creative labor.

1 is a 1H NMR spectrum of a compound represented by Formula Q-1 in Example 1;

2 is a 13C NMR spectrum of a compound represented by Formula A in Example 1;

3 is a 13C NMR spectrum of a compound represented by Formula C in Example 1;

4 is a histogram of the effect of the compound of formula A prepared in Experimental Example 1 of the present invention on the cell survival rate of HEK-293T cells at different concentrations;

5 is a histogram of the effect of the compound of formula C prepared in Experimental Example 2 of the present invention on the cell survival rate of HEK-293T cells under different concentrations;

6 is a graph of chemical purity of the compound shown by Formula A prepared in Experimental Example 1 of the present invention incubated at different times in PBS or serum;

FIG. 7 is a chemical purity chart of the compound represented by Formula C prepared in Experimental Example 2 of the present invention incubated in PBS or serum at different times;

FIG. 8 shows the results of detection of fluorescence intensity of fluorescently labeled nucleotides in HEK-293T cells.

Detailed ways

The technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are part of the present invention, but not all of them. Based on the embodiments of the present invention, all other embodiments obtained by a person of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention. In addition, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

The basic chemical raw materials such as the reagents used in the embodiments of the present invention can be purchased in the domestic chemical product market, or can be customized at the relevant intermediate preparation factories.

Nuclear magnetic resonance instrument (Bruker DRX-500), high performance liquid chromatography (Waters 2445), gamma counter (Perkin-Elmer 1470), elemental analyzer (Perkin-Elmer 240C), enzyme-linked immunoassay (Bio-Rad) High-speed centrifuge (American Beckman Coulter J2-HS). All the cells involved in the following examples were purchased from the Institute of Cells, Shanghai Institutes for Biological Sciences.

Example 1

This embodiment provides a fluorescently labeled nucleotide having a molecular structure represented by the following formula A:

The synthetic route of the fluorescently labeled nucleotide shown in Formula A is:

The method for preparing a fluorescently labeled nucleotide represented by formula A includes the following steps:

S1. Synthesis of compound dUTP-P

The synthetic route of compound dUTP-P is shown below:

The steps are:

(1) Synthesis of dU-P: Compound dU-I (0.7 mmol, 247 mg) was added to a single-necked flask, and then 9.7 mg of cuprous iodide (CuI) and 20.3 mg of tetrakis (triphenylphosphine) palladium ( Pd (PPh ₃ ) ₄ ) was added to the reaction flask, evacuated, protected by nitrogen, wrapped in aluminum foil, added with 2.3 ml of DMF, stirred to dissolve, added 0.2 ml of TEA, weighed 1,6-heptadiyne (193.2 mg, 2.1 mmol) with After DMF was dissolved, it was added to the above reaction flask, stirred at room temperature, and reacted overnight. After the reaction was completed, the solvent was evaporated to dryness under reduced pressure, and was directly separated by column chromatography to obtain 151 mg, which was dU-P with a yield of 68%.

(2) Synthesis of dU-F ₃ : Add 60 ml of methanol to a single-necked flask, stir in an ice-water bath, add propynylamine (60 mmol, 3.3042 g), and slowly add methyl trifluoroacetate (86.7 mmol, 11.0957 g), the ice-water bath was removed after 10 minutes, and the reaction was carried out at room temperature for 24 hours. Distillation under reduced pressure (51 ° C, 280Pa) to obtain trifluoroethylpropynamine

23.53g;

Add dU-I (0.7 mmol, 247 mg) to a single-necked flask, weigh 9.7 mg CuI and 20.3 mg Pd (PPh ₃ ) ₄ into the reaction flask, vacuum, nitrogen protection, wrapped in aluminum foil, add 2.3 ml DMF, stir Dissolve, add 0.2ml TEA, weigh trifluoroethylpropynamine (254mg, 1.7mmol), dissolve it in DMF, add to the above reaction flask, stir at room temperature, and react overnight. After the reaction is completed, the solvent is evaporated to dryness under reduced pressure and separated by direct column chromatography. Dichloromethane and methanol are mixed at a volume ratio of 20: 1 as the eluent. Finally, dU-F _{3 is prepared} . DU-F ₃ has the following properties:示结构: Display structure:

(3) Synthesis of dUTP-P: Weigh 51 mg of compound dU-P, 150 mg (0.32 mmol) of tri-n-butylamine pyrophosphate, and 2-chloro-4H-1,3,2-benzodioxo-4 respectively. -66 mg (0.32 mmol) of ketone were placed in three reaction tubes. Dissolve tri-n-butylamine pyrophosphate in 0.5 mL of anhydrous DMF, add 0.6 mL of freshly distilled tri-n-butylamine, and stir for half an hour. Dissolve 2-chloro-4H-1,3,2-benzodioxan-4-one in 0.5 mL of anhydrous DMF, add the above tri-n-butylamine pyrophosphate solution through a syringe under vigorous stirring, and stir for half an hour . This mixture was then poured into compound dU-F ₃ and stirred for 1.5 h. 5 mL of a 3 wt% iodine solution was added. After 15 min, 4 mL of water was added and stirred for 2 h. Add 0.5mL of 3M NaCl solution, and then add 30mL of absolute ethanol, freeze at -20 ° C overnight, and centrifuge (3200r / min, 25 ° C) for 20min. The supernatant was decanted to obtain a precipitate, and the solvent was dried off. Then add TEAB solution and concentrated ammonia water in order, and stir overnight at room temperature. The solvent was evaporated under reduced pressure and a white solid appeared. Analysis was performed by analytical HPLC, conditions: column: C18, 10 μm, 4.6 × 250 mm; flow rate: 1 mL / min; mobile phase: 20 mM triethylamine acetate and CH 3 CH 2 OH, gradient washing, 0 to 20% ethanol (35 min); UV detector: 254nm. A product peak was formed at t = 16.5 min. 22 mg of product was isolated by preparative HPLC, which was dUTP-P.

S2. Synthesis of compound QN ₃ -1

The synthetic route of compound QN ₃ -1 is shown below:

The steps are:

1. Synthesis of compound Q-1:

Its preparation method is as follows:

(1) Preparation of intermediate I'-1

Phenylhydrazine was added to glacial acetic acid, stirred, and 3-methyl-2-butanone was slowly added dropwise. After the dropwise addition was completed, the mixture was heated to 62.5 ° C, reacted for 3-4 hours, extracted, concentrated, and purified to obtain intermediate I'- 1;

Wherein, the molar ratio of phenylhydrazine to 3-methyl-2-butanone is 1: 1.1;

(2) Preparation of intermediate I'-2

Add intermediate I'-1 and 1,2-dibromoethylene to toluene, protect with nitrogen, heat and reflux for 17 hours, cool, and precipitate a solid, namely intermediate I'-2;

Wherein, the molar ratio of intermediates 1-1 and 1,2-dibromoethylene is 1: 1.75;

(3) Preparation of intermediate I'-4

Add dry N, N-dimethylformamide to dry dichloromethane, add dichloromethane solution of phosphorus oxychloride in an ice bath, stir, add cyclohexanone, remove the ice bath, and heat to reflux for 2.5 Hour, pour the reaction solution into crushed ice and let it stand overnight to precipitate a solid, namely intermediate I'-4;

The molar ratio of cyclohexanone, N, N-dimethylformamide and phosphorus oxychloride is 1: 1.05: 1.025;

(4) Preparation of intermediate I'-5

Intermediate I'-2 and Intermediate I'-4 were added to a mixed solution of n-butanol and toluene, and heated under reflux for 2.5 hours to precipitate a solid, and filtered to obtain Intermediate I'-5.

(5) Preparation of compound Q-1

Intermediate I'-5 is used as a raw material to perform a conventional amino substitution reaction. Take intermediate I'-5 and add bromoacetic acid for reaction, and add NaOH to obtain the desired compound Q-1. The NMR spectrum of compound Q-1 is shown in FIG. 1.

Elemental analysis calculated value: C ₃₆ H ₃₈ Br ₂ N ₃ O ₂ ⁺

Mass spectrum (MS +): 702.13 (M +)

m / z: 704.13 (100.0%), 702.13 (51.3%), 706.13 (49.0%), 705.13 (40.0%), 703.14 (20.2%), 707.13 (19.6%), 706.14 (8.0%), 704.14 (4.1%) ), 708.14 (3.8%), 707.14 (1.3%), 705.14 (1.1%).

Elemental analysis: C, 61.37; H, 5.44; Br, 22.68; N, 5.96; O, 4.54.

2. Under basic conditions, take compound Q-1 and 2-azidoethylamine with terminal amino group.

Amidation reaction is performed to obtain compound QN ₃ -1. The specific steps are:

In a single-necked flask, weigh 0.1 mmol of the compound Q-1 and dissolve it in 7.8 ml of DMF. Continue to add N-methylmorpholine (180 μL, 1.8 mmol), 2- (7-azobenzotrisine) while stirring in an ice-water bath. Azazole) -N, N, N ', N'-tetramethylurea hexafluorophosphate (0.57g, 1.5mmol) was activated for 40min, then 2-azidoethylamine (1.8mmol) was added and stirred for 1h, then the temperature was raised to room temperature After 8 hours of reaction, stop the reaction, add an appropriate amount of dichloromethane for extraction, wash twice with water, twice with saturated NaCl solution, and distill the organic phase under reduced pressure to obtain a pale yellow solid. Column chromatography gives QN ₃ -1-1, yield 91%. The structure of compound QN ₃ -1 is as follows:

S3. Synthesis of fluorescently labeled nucleotides represented by formula A

In two-necked flasks, equivalent dUTP-P and QN ₃ -1 were dissolved in an appropriate amount of THF, so that the concentration of both compounds was 11.8 mmol / ml. The system was purged with nitrogen three times, and the reaction was carried out under the protection of nitrogen. 0.7 times equivalent of anhydrous copper sulfate solids and 2.1 times equivalent of sodium ascorbate were mixed, evacuated, and deionized water was added to shake to obtain a yellow suspension, which was then injected into the reaction system and stirred at room temperature for 36h. The solvent was removed by rotary evaporation, and the HPLC separation was performed to obtain a fluorescently labeled nucleotide represented by Formula A with a yield of 61%.

The 13C NMR spectrum of the fluorescently labeled nucleotide shown in Formula A is shown in Figure 2. The calculated value of elemental analysis of the fluorescently labeled nucleotide shown in Formula A: C ₅₅ H ₆₅ Br ₃ N ₉ O ₁₅ P ₃ ⁺

Mass spectrum (MS +): 1421.14 (M +)

m / z: 1423.13 (100.0%), 1425.13 (99.3%), 1426.14 (65.0%), 1424.14 (61.7%), 1421.14 (34.3%), 1427.13 (33.5%), 1425.14 (21.4%), 1427.14 (20.9%) ), 1421.14 (20.8%), 1428.13 (20.1%), 1423.14 (7.8%), 1429.14 (6.5%), 1428.14 (6.0%), 1424.13 (3.3%), 1426.13 (3.3%), 1430.14 (1.8%), 1429.13 (1.6%), 1424.15 (1.2%), 1422.13 (1.1%), 1427.15 (1.0%).

Elemental analysis: C, 46.36; H, 4.60; Br, 16.82; N, 8.85; O, 16.84; P, 6.52.

Example 2

This embodiment provides a fluorescently labeled nucleotide having a molecular structure represented by the following formula C:

The synthetic route of the fluorescently labeled nucleotide shown in Formula C is:

The method for preparing a fluorescently labeled nucleotide represented by formula C includes the following steps:

S1. Synthesis of compound dATP-P

The synthetic route of compound dATP-P is shown below,

The steps are:

(1) Synthesis of dA-P: Add 0.6mmol of compound dA-I to a single-necked flask, weigh 6.8mg of CuI and 26.7mg of Pd (PPh ₃ ) ₄ into the reaction flask, evacuate, protect with nitrogen, aluminum foil Wrap, add 4.3 ml of DMF, stir to dissolve, add 1.3 mmol of TEA, weigh 2.0 mmol of 1,6-heptadiyne, dissolve in DMF, add to the above reaction flask, stir at room temperature, and react overnight. After the reaction is completed, reduce the pressure The solvent was evaporated and dA-P was separated by direct column chromatography with a yield of 62%.

(2) Synthesis of dA-F ₃ : Add 60 ml of methanol to a single-necked flask, stir in an ice-water bath, add propynylamine (60 mmol, 3.3042 g), and slowly add methyl trifluoroacetate (86.7 mmol, 11.0957 g), the ice-water bath was removed after 10 minutes, and the reaction was carried out at room temperature for 24 hours. Distillation under reduced pressure (51 ° C, 280Pa) to obtain trifluoroethylpropynamine

23.53g;

Add 0.5mmol of dA-I to a single-necked flask, weigh 8.9mg of CuI and 22.7mg of Pd (PPh ₃ ) ₄ into the reaction flask, evacuate, protect with nitrogen, cover with aluminum foil, add 2.5ml DMF, stir Dissolve, add 0.23 ml of TEA, weigh 1.8 mmol of trifluoroethylpropynamine, dissolve it in DMF, add to the above reaction flask, stir at room temperature, and react overnight. After the reaction is completed, the solvent is evaporated to dryness under reduced pressure and separated by direct column chromatography. Dichloromethane and methanol are mixed at a volume ratio of 20: 1 as the eluent, and dA-F _{3 is} finally obtained. DA-F ₃ has the following properties:示结构: Display structure:

(3) Synthesis of dATP-P: In the glove box, weigh 53mg of dA-P, 150mg of tri-n-butylamine pyrophosphate, and 70mg of 2-chloro-4H-1,3,2-benzodioxo- 4-ketone was placed in three reaction tubes. Dissolve tri-n-butylamine pyrophosphate in 0.8 mL of anhydrous DMF, add 0.6 mL of freshly distilled tri-n-butylamine, and stir for half an hour. Dissolve 2-chloro-4H-1,3,2-benzodioxan-4-one in 0.8mL of anhydrous DMF, add the above tri-n-butylamine pyrophosphate solution through a syringe under vigorous stirring, and stir for half an hour . This mixture was then poured into compound dA-F ₃ and stirred for 1.5 h. 5.5 mL of a 3 wt% iodine solution was added. After 23 min, 5 mL of water was added and stirred for 2 h. Add 0.5 mL of 3M NaCl solution, then add 30 mL of absolute ethanol, freeze overnight at -20 ° C, and centrifuge (3200 r / min, 25 ° C) for 20 min. The supernatant was decanted to obtain a precipitate, and the solvent was dried off. Then add TEAB solution and concentrated ammonia water in order, and stir overnight at room temperature. The solvent was evaporated under reduced pressure and a white solid appeared. DATP-P was isolated by HPLC in 44% yield.

S2. Synthesis of compound QN ₃ -1

The compound QN ₃ -1 was synthesized in the same manner as in Example 1.

S3. Synthesis of fluorescently labeled nucleotides represented by formula C

In two-necked flasks, equal equivalents of dATP-P and QN ₃ -1 were dissolved in an appropriate amount of THF, so that the concentration of both compounds was 16.7 mmol / ml. The system was purged with nitrogen three times, and the reaction was carried out under the protection of nitrogen. Mix 1.1 times equivalents of anhydrous copper sulfate solids and 2.1 times equivalents of sodium ascorbate, evacuate, add deionized water and shake to obtain a yellow suspension, then inject it into the reaction system and stir at room temperature for 48h. The solvent was removed by rotary evaporation, and the HPLC separation was performed to obtain the fluorescently labeled nucleotide shown in Formula C with a yield of 58%.

The 13C NMR spectrum of the fluorescently labeled nucleotide shown in Formula C is shown in Figure 3. The calculated value of elemental analysis of the fluorescently labeled nucleotide shown in Formula C: C ₅₆ H ₆₄ Br ₃ N ₁₁ O ₁₃ P ₃ +

Mass spectrum (MS +): 1428.14 (M +)

m / z: 1430.14 (100.0%), 1432.14 (99.8%), 1431.15 (63.8%), 1433.14 (63.5%), 1434.14 (36.6%), 1428.14 (34.3%), 1435.14 (21.6%), 1432.15 (21.5%) ), 1429.15 (21.2%), 1434.15 (19.0%), 1430.15 (8.2%), 1436.15 (7.1%), 1433.15 (6.7%), 1435.15 (5.3%), 1431.14 (4.1%), 1437.15 (1.7%), 1436.14 (1.6%), 1429.14 (1.4%).

Elemental analysis: C, 46.98; H, 4.51; Br, 16.74; N, 10.76; O, 14.53; P, 6.49.

Experimental Example 1 Detection of Fluorescent Properties of Fluorescent Dyes

1. Accurately weigh the compound Q-1 to be measured, prepare a solution with a concentration of 1.0 × 10 ^-5 mol / L with a 50% ethanol solution, and measure its fluorescence spectrum to obtain the maximum absorption in the near-infrared spectrum of the compound. Wavelength λ _abs .

2. Use the measured maximum absorption wavelength in the near-infrared spectrum as the excitation wavelength of the fluorescence spectrum to determine the fluorescence spectrum. The test compound was weighed, and a 1.0 × 10 ^-6 mol / L ethanol: water (50:50, v / v) solution was prepared. The emission spectrum was measured, and the Stokes shift was calculated, as shown in Table 1.

3.Measure the molar extinction coefficient of the fluorescent dye

The molar extinction coefficient of the compound was determined by ultraviolet-visible absorption spectrum. The calculation formula is shown in formula (1):

A = εcl Equation (1)

Among them, A represents the absorption intensity, ε is the molar absorption coefficient, c is the concentration of the compound, and l is the thickness of the quartz cell for detection.

4.Measure the fluorescence quantum yield of fluorescent dyes

The fluorescence quantum yield of the fluorescent dye was measured at 20 ° C. Using quinine sulfate (a solvent of 0.1M H ₂ SO ₄ and a quantum yield of 0.56) as a reference, the dilute solution of the fluorescent dye and the reference substance was measured. The fluorescence intensity obtained under the same excitation conditions and the ultraviolet absorption value at the excitation wavelength were used to calculate the fluorescence quantum yield. The product was dissolved in absolute ethanol.

The calculation formula is shown in equation (2):

Among them, Φ is the quantum yield of the test object, and the subscript R represents the reference object. I is the integrated fluorescence intensity and A is the ultraviolet absorption value. η is the refractive index of the solvent. Generally, the absorbances A and A _R are required to be less than 0.1.

Table 1 Spectral properties of the fluorescent dyes described in Example 1

As shown in Table 1, the fluorescent quantum yield of the fluorescent dye represented by Formula Q-1 is> 80%, and the Stoke shift is large, which is suitable for labeling biological molecules such as nucleotides to prepare fluorescent probes to achieve stable fluorescent performance and fluorescent quantum. Detection of nucleic acid molecules with high rate and high imaging signal-to-noise ratio.

Experimental example 2 Toxicity detection of fluorescently labeled nucleotides

The cytotoxicity of a compound represented by the formula A and a compound represented by the formula C in HEK-293T (human embryonic kidney cells) is measured by an MTT experiment, including the following steps:

(1) HEK-293T cells were seeded in a 96-well plate at a density of 5 × 10 ³ cells / 100 μL per well, and the medium was DMEM, and cultured overnight in a constant temperature incubator at 37 ° C. containing 5% CO ₂ ;

(2) The compound represented by Formula A prepared in Example 1 and the compound represented by Formula C prepared in Example 2 were respectively dissolved in dimethyl sulfoxide (DMSO) to prepare a mother liquor having a concentration of 0.1 mol / L. And then dilute with DMEM medium to a concentration of 80 μmol / L, 40 μmol / L, 20 μmol / L, 10 μmol / L, and 5 μmol / L, and reserve for use; another DMEM medium is added and added to the same volume of deionized water for dilution. The concentration of fluorescently labeled nucleotides is 0 μmol / L;

(3) Replace the original medium in the 96-well plate in step (1) with the drug concentration prepared in step (2) above, which is 0 μmol / L, 5 μmol / L, 10 μmol / L, 20 μmol / L, 40 μmol / L And 80 μmol / L DMEM medium, 200 μL per well, and 6 duplicate wells per drug concentration. Subsequently, the 96-well plate was placed in a 5% CO ₂ incubator at 37 ° C. for 3 h, 6 h, 12 h, and 24 h, respectively. After the incubation was completed, 20 μL of MTT (5 mg / mL) was added to each well and the culture was continued for 4 h. After the culture was completed, the medium was aspirated, 150 μL of DMSO was added to each well, and shaken for 10 min on a shaker until the crystals were completely dissolved. Enzyme labeling was used to determine the absorbance of each well at 490nm. The experimental results were the average of at least three independent experiments.

Under different drug concentrations and incubation times, the histogram of the survival rate of the compound represented by Formula A on HEK-293T cells is shown in Figure 4, and the histogram of the survival rate of the compound represented by Formula C on HEK-293T cells is shown in Figure 5. With the prolonged action of the drug and the increase of the compound concentration, the cell survival rate does not change significantly, and within 24 hours, the cell survival rate of both the compound represented by Formula A and the compound represented by Formula C is greater than 90% after incubation Therefore, it can be determined that fluorescently labeled nucleotides are safe and low-toxic to HEK-293T cells, and have good biocompatibility.

Experimental Example 3 Stability test of fluorescently labeled nucleotides

(1) Take 4 parts of 800 μL of PBS (pH = 7.4) buffer solution and 200 μL of the compound solution of the formula A prepared in Example 1 and mix, respectively, and heat at 37 ° C. for 0.5 h, 1 h, 2 h, 3 h, and 4 h. Dilute the above-mentioned small amount of solution, and check the stability of the labeled product by radioactive high-performance liquid phase. The conditions of high-performance liquid phase are: water (containing 0.1% TFA by volume) as mobile phase A, and CH ₃ CN (containing 0.1% by volume). % TFA) is mobile phase B, and gradient elution is performed according to the following procedure: 0min → 3min, mobile phase A: the volume ratio of mobile phase B is from 80: 20 → 80: 20; 3min → 25min, mobile phase A: mobile phase The volume ratio of B is from 80: 20 → 10: 90; 25min → 30min, mobile phase A: The volume ratio of mobile phase B is from 10: 90 → 80: 20, the flow rate of the mobile phase is controlled to 1mL / min, and the column temperature is controlled to 25 ℃. Then, 4 parts of 800 μL mouse serum (purchased from Biyuntian) and 200 μL of the labeled product solution of the compound of Formula A prepared in Example 1 were mixed and heated at 37 ° C for 0.5h, 1h, 2h, 3h, and 4h, and then Take 100 μL of the above solution, add 100 μL of acetonitrile, centrifuge at 10000 g for 5 min in a high-speed centrifuge, take the supernatant, and test the stability of the labeled product by high performance liquid chromatography. The conditions of high performance liquid phase: water (containing 0.1% by volume concentration) TFA) is mobile phase A, CH ₃ CN (containing 0.1% TFA by volume) is used as mobile phase B, and gradient elution is performed according to the following procedure: 0min → 3min, mobile phase A: mobile phase B volume ratio is 80: 20 → 80: 20; 3min → 25min, the volume ratio of mobile phase A: mobile phase B is from 80: 20 → 10: 90; 25min → 30min, the volume ratio of mobile phase A: mobile phase B is from 10: 90 → 80:20, the flow rate of the mobile phase was controlled to 1 mL / min, and the column temperature was controlled to 25 ° C. FIG. 6 is a chemical purity chart of liquid chromatographic detection of a compound represented by formula A in PBS or serum at different times.

(2) Using the detection method in step (1), the chemical purity of the compound represented by formula C after incubation in PBS or serum at different times is determined, and the results are shown in FIG. 7.

It can be seen from FIG. 6 and FIG. 7 that with the increase of the incubation time, the radiochemical purity of the compound represented by Formula A and the compound represented by Formula C slightly decreased in PBS and serum. The chemical purity is still greater than 95%. This indicates that fluorescently labeled nucleotides have good stability in vitro, which is beneficial for further experimental studies in vivo.

Experimental Example 4 Fluorescence intensity detection chart of fluorescently labeled nucleotides

(1) HEK-293T cells are cultured to a logarithmic phase in an incubator at 5% CO ₂ and a temperature of 37 ° C;

(2) Inoculate 1 × 10 ⁵ cells / well in a 12-well plate that already contains slides (soak the slides with 75% ethanol for 5 minutes for sterilization, and the slides were purchased from Assitent), and culture overnight;

(3) Aspirate the cell supernatant, and add the fluorescently-labeled nucleotide shown in Formula A prepared in Example 1 and the fluorescent-labeled formula shown in Example 3 to the final concentration of 1 mg / ml diluted with culture medium. Nucleotides, continue to culture in cell incubator for 24h;

(4) Stop the culture, transfer the slides in the well plate to a new 12-well plate, and rinse with PBST (containing 0.1% Tween-20) 3-4 times; add 4% paraformaldehyde to fix for 15min at room temperature, PBST Rinse 3-4 times; add DAPI (purchased from American Cell Signaling) solution at a final concentration of 1 μg / ml and incubate at 37 ° C for 15 min, rinse 3-4 times in PBS; mount, laser confocal microscope (purchased from Olympus, Japan) Companies).

FIG. 8 shows the results of detection of the fluorescence intensity of fluorescently labeled nucleotides in HEK-293T cells. From the figure to the right (FIGS. 8A to 8C), the fluorescently labeled nucleotides shown in Formula A are sequentially represented in HEK-293T cells. The results of fluorescence detection, the results of fluorescence detection of fluorescently labeled nucleotides shown in Formula C in HEK-293T cells, and the results of DAPI staining of HEK-293T cells. As can be seen from FIG. 8, in the cells incubated with the fluorescently labeled nucleotides shown in Formula A and Formula C, obvious fluorescence can be detected, indicating that HEK-293T cells can take up the fluorescently labeled nucleotides, and the fluorescence The labeled nucleotides have higher fluorescence intensity.

Obviously, the foregoing embodiment is merely an example for clear description, and is not a limitation on the implementation manner. For those of ordinary skill in the art, other different forms of changes or modifications can be made on the basis of the above description. There is no need and cannot be exhaustive for all implementations. However, the obvious changes or variations introduced thereby are still within the protection scope created by the present invention.

Claims

A fluorescently labeled nucleotide, characterized in that it has a structure represented by Formula I:

Wherein W is dNTP, said dNTP is selected from one of dUTP, dATP, dGTP, dCTP and dTTP,

X is halogen,

R 1 is selected from one of an alkyl group, a cycloalkyl group, an aryl group, and a heterocyclic group.
The fluorescently labeled nucleotide of claim 1, wherein X is Br.
The fluorescently labeled nucleotide according to claim 1 or 2, characterized in that it has a structure represented by Formula II:
The fluorescently labeled nucleotide according to claim 3, wherein the structure is as shown in Formula A to Formula H:
A method for preparing a fluorescently labeled nucleotide according to claim 1, comprising the following steps:

S1. The compound dN-I and 1,6-heptadiyne are subjected to a cross-coupling reaction under the action of a catalyst to obtain a compound dN-P, and then the compound dN-P and tri-n-butylamine pyrophosphate and 2- The reaction occurs under the combined action of chloro-4H-1,3,2-benzodioxan-4-one to obtain the compound dNTP-P;

Among them, dN-I is selected from the chemical structure shown below:

dN-P is selected from the chemical structure shown below:

dNTP-P is selected from the chemical structure shown below:

S2. Compound of the formula Q and 2-azidoethylamine
A condensation reaction occurs to prepare the compound QN 3 ,

The structure of compound Q is shown below:

The structure of compound QN 3 is shown below:

S3. The compound QN 3 is subjected to a click chemical reaction with the compound dNTP-P to obtain a fluorescently labeled nucleotide having a structure represented by Formula I.
The preparation method according to claim 5, wherein the compound of the structure represented by formula Q is prepared by the following steps:

(1) Preparation of intermediate I'-1

Phenylhydrazine was added to glacial acetic acid, stirred, and 3-methyl-2-butanone was slowly added dropwise. After the dropwise addition was completed, the mixture was heated to 60-65 ° C, reacted for 3-4 hours, extracted, concentrated, and purified to obtain intermediate I. '-1;

Wherein, the molar ratio of phenylhydrazine to 3-methyl-2-butanone is 1: (1.0-1.2);

(2) Preparation of intermediate I'-2

Add intermediate I'-1 and 1,2-dibromoethylene to toluene, protect with nitrogen, heat under reflux for 16-18 hours, cool, and precipitate a solid, namely intermediate I'-2;

Wherein, the molar ratio of the intermediate I'-1 and 1,2-dibromoethylene is 1: (1.5-2.0);

(3) Preparation of intermediate I'-4

Add dry N, N-dimethylformamide to dry dichloromethane, add dichloromethane solution of phosphorus oxychloride in an ice bath, stir, add cyclohexanone, remove the ice bath, and heat to reflux. 2 -3 hours, pour the reaction solution into crushed ice and let it stand overnight to precipitate a solid, namely intermediate I'-4;

Wherein, the molar ratio of cyclohexanone, N, N-dimethylformamide and phosphorus oxychloride is 1: (1.0-1.1): (1.0-1.05);

(4) Preparation of intermediate I'-5

Add intermediate I'-2 and intermediate I'-4 to the mixed solution of n-butanol and toluene, heat to reflux for 2-3 hours, precipitate a solid, and filter to obtain intermediate I'-5;

(5) Preparation of compound Q

Intermediate I'-5 and Br-R 1 -COOH undergo an amino substitution reaction, and NaOH is added during the reaction to produce compound Q;

The synthetic route is as follows:
The preparation method according to claim 6, characterized in that:

In the step (1), the molar ratio of the phenylhydrazine to the 3-methyl-2-butanone is 1: (1.0-1.2);

In the step (2), the molar ratio of the intermediate I'-1 and 1,2-dibromoethylene is 1: (1.5-2.0);

In the step (3), the molar ratio of the cyclohexanone, N, N-dimethylformamide, and phosphorus oxychloride is 1: (1.0-1.1): (1.0-1.05).
The production method according to any one of claims 5-7, wherein said step S3, the molar ratio of Compound I'-N compound with the added dNTP-P 3 is 1: (1 ~ 1.2).
Use of the fluorescently labeled nucleotide according to any one of claims 1 to 4 in the preparation of a fluorescent probe.
Use of the fluorescently labeled nucleotide according to any one of claims 1 to 4 in gene sequencing, in situ hybridization detection, Northern blot, Southern blot, or liquid-phase chip.