WO2019227527A1 - Fluorescently labeled amino acid, preparation method therefor, and use thereof - Google Patents

Abstract

Disclosed is a fluorescently labeled amino acid having a structure as shown in Formula I. The amino acid molecule forms a stable covalent bond with a fluorescent dye molecule represented by Formula Q, and has high stability in serum and other detection environments. The amino acid has high biocompatibility, and is applicable to the detection of biomolecules, such as proteins and polypeptides, inside and outside cells. Due to a large Stokes shift of the fluorescent dye molecule, the fluorescently labeled amino acid has the advantages of high fluorescence stability, high fluorescence quantum yields, and achieves high signal-to-noise ratios in imaging results. Further disclosed is a method for preparing the fluorescently labeled amino acid. The method has mild reaction conditions and high reaction selectivity, is simple to execute, and can be used to prepare a fluorescently labeled amino acid in high yields.

Description

Fluorescently labeled amino acid 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 amino acid 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.

As one of the three material foundations that make up the life system, protein is involved in almost every link of life activities and plays a decisive role in the birth, growth and reproduction of life. The occurrence of many diseases is also closely related to the mutation of peptides and proteins in the body. Therefore, selecting proteins, peptides and amino acid residues that are closely related to major diseases as targets, and developing highly selective and sensitive detection methods, revealing the mysteries of life, early diagnosis of diseases and screening of drugs have great significance. At present, the detection methods of diagnostic peptides and proteins mainly include immunolabeling, isotope labeling, and fluorescence detection. Fluorescence detection methods have the advantages of high sensitivity, good selectivity, wide dynamic response range, and can be detected in vivo. Widely used in detection. For example, a liquid-phase chip is coupled to nucleic acid probe molecules on the surface of fluorescent microspheres. Fluorescent microspheres are encoded in micron-sized polymer microspheres with fluorescent materials with different combinations of light emission wavelengths and intensity levels, and have different fluorescent signal codes. Sphere; the surface of the fluorescent microsphere can be coupled with a probe molecule (such as a specific antibody) that binds to the target molecule (such as an antigen), and a reporter molecule (such as a specific antibody) labeled with another fluorescent signal is added, This constitutes the reaction system of the liquid chip. The target antigen to be detected can be combined with the probe molecule and the reporter molecule at the same time, the qualitative analysis of the target antigen can be realized by the fluorescent signal of the fluorescent microsphere, and the quantitative / semi-quantitative detection of the target antigen can be realized by the fluorescent intensity of the reporter molecule.

The liquid-phase chip can be used for multi-sample detection at the same time, thereby realizing multiple quantitative analysis of multiple antigen molecules in one 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 amino acid having a structure represented by Formula I:

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

R ₂ is any amino acid side chain group,

X is halogen.

Preferably, the above-mentioned fluorescently labeled amino acid has a structure represented by Formula II:

Wherein, R _{1 is} selected from one of an alkyl group, a cycloalkyl group, an aryl group and a heterocyclic group, and R ₂ is any amino acid side chain group.

Preferably, in the above-mentioned fluorescently labeled amino acid, the R _{2 is} selected from -H, -CH ₃ , -CH (CH ₃ ) ₂ , -CH ₂ CH (CH ₃ ) ₂ , -CH (CH ₃ ) CH ₂ CH ₃

-CH ₂ COOH,

-CH ₂ C (O) NH ₂ , -CH ₂ CH ₂ COOH, -CH ₂ CH ₂ CH ₂ CH ₂ NH ₂ , -CH ₂ CH ₂ C (O) NH ₂ , -CH ₂ CH ₂ SCH ₃ ,- CH ₂ CH ₂ CH ₂ NHCH (NH) NH ₂ , -CH ₂ OH, -CH (OH) CH ₃ and -CH ₂ SH.

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

S1. Dissolve intermediate 1 in an organic solvent, stir after adding the first catalyst, then mix with intermediate 2 and triethylamine and stir for 4-6 hours; wash the reaction solution, dry, concentrate, and recrystallize to obtain an intermediate 3;

S2. Intermediate 3 is dissolved in anhydrous ether, a second catalyst is added under an ice bath, and the reaction is performed at room temperature for 0.5 to 2 hours. Ethyl acetate is further added to adjust the pH to 6.5 to 7.5, filtered, dried, and concentrated to obtain intermediate 4 ;

S3. Intermediate 4 is dissolved in anhydrous THF, and triethylamine and methanesulfonyl chloride are continuously added. After stirring for 3 to 5 hours, the reaction solution is washed, dried, and concentrated to obtain intermediate 5;

S4. Dissolve the fluorescent dye represented by formula Q in DMF, add an alkali metal salt, stir, add intermediate 5, and react overnight; then pour the reaction solution into ethyl acetate, wash the reaction solution, concentrate, and recrystallize to obtain Intermediate 6;

S5. Add intermediate 6 to toluene, stir well, add silica gel, heat to reflux, filter after the reaction, wash the silica gel, and evaporate the filtrate to obtain the fluorescently labeled amino acid represented by formula I;

The reaction route is as follows:

Preferably, in the above preparation method, the intermediate 1 is prepared by the following steps:

The amino acid represented by formula B is dissolved in an alkaline solution, and the amino acid is dissolved in an alkaline solution. An anhydrous THF solution of di-tert-butyl carbonate is gradually added dropwise thereto, and then stirred in an ice bath and room temperature in order to control the reaction process. Carried out in an alkaline environment;

After the reaction, the THF was removed, the reaction solution was washed, dried, and concentrated to obtain the intermediate 1;

The reaction route is as follows:

Preferably, in the above preparation method, the organic solvent is dichloromethane, and the first catalyst is 1-hydroxybenzotriazole and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide. A mixture of amine hydrochloride, the second catalyst is lithium tetrahydroaluminum.

Preferably, in the above preparation method, the fluorescent dye represented by the 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);

(6) Preparation of intermediate I'-5

Add intermediate I'-2 and intermediate I'-4 to a mixed solution of n-butanol and toluene, heat and 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 -OH undergo an amino substitution reaction, and NaOH is added during the reaction to produce compound Q;

The synthetic route is as follows:

Preferably, the above 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).

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

Preferably, in the above application, the fluorescent probe is a polypeptide fluorescent probe and / or a protein fluorescent probe.

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

1. A fluorescently labeled amino acid provided by the present invention, having a structure represented by Formula I. The compound of the structure shown by Formula I is formed by the covalent combination of any amino acid molecule with the fluorescent dye molecule shown by Formula Q, wherein the Stokes shift of the fluorescent dye molecule shown by Formula Q is large, the fluorescent signal is stable, and the fluorescent quantum yield is High rate, can be effectively distinguished from the emission fluorescence of other fluorescent dye molecules, has the advantage of high signal-to-noise ratio in fluorescence imaging.

The fluorescently labeled amino acid represented by Formula I has high stability in the detection environment such as serum and can be retained in cells for a long time. At the same time, the fluorescently labeled amino acid has low cytotoxicity and high biocompatibility, and is suitable for being widely used in cells. Detection of internal and external bioactive molecules, imaging of tissue and live animal markers, drug analysis, pathological model research, and early diagnosis of diseases.

Fluorescently labeled amino acids can be directly used in the synthesis of polypeptides or proteins. Since the dye molecule is directly covalently coupled to the amino acid, the synthesized polypeptide or protein does not need to be modified by fluorescent coupling, and can emit fluorescence as a fluorescent probe. For the detection of target antigen molecules (such as viruses, cytokines, hormones, enzymes, disease markers, etc.) inside or outside cells or in vivo. The above-mentioned 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 amino acids into specific positions of the protein or polypeptide. On the one hand, the biological activity of the synthesized polypeptide or protein molecules with fluorescent properties can be guaranteed; on the other hand, a specific number of fluorescent dye molecules can be introduced to control the fluorescence signal intensity.

When the fluorescently labeled amino acid is applied to a liquid crystal chip, the probe molecule synthesized with the fluorescently labeled amino acid does not need to be coupled with a fluorescent microsphere, which simplifies the preparation process of the liquid crystal chip; and the liquid crystal chip is in the near infrared region. A new fluorescent 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 a fluorescently-labeled amino acid provided by the present invention, the raw materials required for synthesis are readily available, the reaction conditions are mild, the operation is simple, the reaction selectivity is high, and the fluorescently-labeled amino acid prepared by the reaction has 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 II-1 in Example 1;

2 is a 1H NMR spectrum of a compound II-2 in Example 2;

3 is a 1H NMR spectrum of a compound Q-1 in Example 3;

4 is a histogram of the effect of the compound of formula II-1 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 II-2 prepared in Experimental Example 2 of the present invention on the cell survival rate of HEK-293T cells at different concentrations;

FIG. 6 is a chemical purity chart of the compound shown in Formula II-1 prepared in Experimental Example 1 of the present invention incubated in PBS or serum at different times;

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

FIG. 8 is a result of detecting fluorescence intensity of a fluorescently labeled amino acid 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-Elmer1470), elemental analyzer (Perkin-Elmer 240C), enzyme-linked immunoassay (Bio-Rad), High-speed centrifuge (Beckman Coulter J2-HS, USA). 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 amino acid having a molecular structure represented by the following formula II-1:

The synthetic route of the fluorescently labeled amino acid shown in Formula II-1 is shown below:

The method for preparing a fluorescently labeled amino acid represented by Formula II-1 includes the following steps:

1. Preparation of intermediate 1-1:

Dissolve 14.9 g of methionine (compound B-1) in 110 mL of a 1M sodium hydroxide solution, and slowly add 50 mL of an anhydrous THF (tetrahydrofuran) solution containing 2211 g of di-tert-butyl carbonate in an ice bath, while using a 1 M sodium hydroxide solution Control the pH of the reaction solution at about 9 and complete the dropwise addition in about 1 h. Stir for 1 h under ice bath conditions, remove the ice bath, and stir at room temperature for 15 h. The whole process keeps the system pH around 9. After the reaction is completed, the THF is distilled off, washed with petroleum ether (100mL × 3), the aqueous phase is adjusted to pH 2 ~ 3 with saturated citric acid solution, and extracted with ethyl acetate (100mL × 3). Water magnesium sulfate was dried overnight and concentrated to give 20.5 g of a pale yellow oil with a yield of 87%.

The structure of intermediate 1-1 is shown below:

2. Preparation of Intermediate 2:

In an ice bath, 9.62 mL of acetyl chloride was added dropwise to 100 mL of anhydrous methanol, and the ice bath was stirred for 15 minutes. 6.4 g of p-aminobenzoic acid was added, and the ice bath was stirred for 1 hour and refluxed for 2 hours. Concentration gave a white solid, which was recrystallized from methanol-ether to give 6.88 g of white powder with a yield of 95%.

The structure of intermediate 2 is shown below:

3. Preparation of intermediate 3-1:

2.7 g of intermediate 1-1 was dissolved in 100 mL of dichloromethane, and 1.5 g of 1-hydroxybenzotriazole (HOBT) and 2.1 g of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide were added. Amine hydrochloride (EDCI), stirred at room temperature for 15 min, added 1.7 g of intermediate 2 and 2.1 mL of triethylamine, and stirred at room temperature for 6 hours. The reaction solution was washed with saturated citric acid (50 mL × 3), saturated sodium bicarbonate (50 mL × 3), and saturated brine (50 mL × 1). Anhydrous magnesium sulfate was dried overnight. Concentrated and recrystallized from ethyl acetate-n-hexane to obtain 3.1 g of a white solid with a yield of 80% and a melting point of 100-102 ° C.

The structure of intermediate 3-1 is shown below:

4. Preparation of intermediate 4-1:

0.76 g of Intermediate 3-1 was dissolved in 20 mL of anhydrous ether, and 0.23 g of lithium aluminum tetrahydroxide was added in portions in an ice-salt bath, and reacted at room temperature for 1 hour. Under an ice bath, 3 mL of ethyl acetate was added to the reaction solution, and the pH was adjusted to 7 with 2M hydrochloric acid. After filtration, the organic phase was washed with saturated brine (20 mL × 3), and dried over anhydrous magnesium sulfate overnight. It was concentrated and separated by combiflash chromatography to obtain 0.34 g of colorless oil with a yield of 48%.

The structure of intermediate 4-1 is shown below:

5. Preparation of intermediate 5-1:

0.26 g of intermediate 4-1 was dissolved in 40 mL of anhydrous THF, 0.3 mL of triethylamine and 0.18 mL of methanesulfonyl chloride were added under an ice bath, and the reaction was stirred below 5 ° C. The reaction was completed after 4 hours. The reaction solution was washed with 0.5 M hydrochloric acid (20 mL × 3), saturated brine (20 mL × 1), and dried over anhydrous magnesium sulfate. Concentration yielded 0.28 g of a yellow oil with a crude yield of 80%. It was used directly in the next reaction without purification.

The structure of intermediate 5-1 is shown below:

6. Preparation of intermediate 6-1:

0.25 g of the fluorescent dye represented by formula Q-1 was dissolved in 10 mL of DMF, 0.5 g of anhydrous potassium carbonate was added, and after stirring at room temperature for 15 minutes, 0.5 g of intermediate 5-1 was added and reacted overnight. The reaction solution was poured into 100 mL of ethyl acetate, washed with a saturated potassium carbonate solution (50 mL × 5), and saturated brine (50 mL × 1). Concentrated and recrystallized from ethyl acetate-n-hexane to obtain intermediate 6-1 with a yield of 59%.

The molecular structure of the fluorescent dye is shown below:

The chemical structure of intermediate 6-1 is shown below:

7. Preparation of compound II-1:

6ml of toluene was added to intermediate 6-1, stirred at room temperature for 2h, silica gel was added, and then heated to reflux. After the reaction was completed, the silica gel was filtered, the silica gel was washed, and the filtrate was evaporated to dryness to obtain a fluorescently labeled amino acid represented by Formula II-1. The rate is 92%.

The specific structure of the fluorescently labeled amino acid represented by Formula II-1 is as follows:

The 1H NMR spectrum of the fluorescently labeled amino acid shown in Formula II-1 is shown in Figure 1. The calculated value of elemental analysis of the fluorescently labeled amino acid shown in Formula II-1: C47H54Br3N5O2S +

Mass spectrum (MS +): 989.15 (M +)

m / z: 993.15 (100.0%), 991.15 (98.8%), 994.15 (53.1%), 992.16 (51.0%), 995.15 (36.7%), 989.15 (33.3%), 996.15 (19.1%), 990.16 (17.2%) ), 993.16 (13.7%), 995.16 (13.0%), 991.16 (4.9%), 997.16 (4.4%), 992.15 (3.4%), 994.16 (3.2%), 996.16 (2.4%), 997.14 (1.4%), 997.15 (1.2%).

Elemental analysis: C, 56.86; H, 5.48; Br, 24.15; N, 7.05; O, 3.22; S, 3.23.

Example 2

This embodiment provides a fluorescently labeled amino acid having a molecular structure represented by the following formula II-2:

The synthetic route of the fluorescently labeled amino acid shown in Formula II-2 is shown below:

A method for preparing a fluorescently labeled amino acid represented by Formula II-2 includes the following steps:

1. Preparation of Intermediate 1-2:

Dissolve 12.8g of compound B-2 in 100mL of 1M sodium hydroxide solution, and slowly add 50mL of an anhydrous THF (tetrahydrofuran) solution containing 1890g of di-t-butyl carbonate in an ice bath, while controlling the reaction with 1M sodium hydroxide The pH of the solution was about 9, and the addition was completed in about 1 hour. The solution was stirred for 1 hour under ice bath conditions, the ice bath was removed, and the solution was stirred at room temperature for 15 hours. During the whole process, the pH of the system was maintained at about 9. After the reaction was completed, THF was distilled off, washed with petroleum ether (100 mL × 3), and the aqueous phase was adjusted to a pH of 2 to 3 with a saturated citric acid solution, and extracted with ethyl acetate (100 mL × 3). Anhydrous magnesium sulfate was dried overnight and concentrated to obtain Intermediate 1-2 with a yield of 82%.

The structure of intermediate 1-2 is shown below:

2. Preparation of Intermediate 2:

The intermediate 2 was prepared in the same manner as in Example 1.

3. Preparation of intermediate 3-2:

2.6 g of intermediate 1-2 was dissolved in 100 mL of dichloromethane, and 2.2 g of 1-hydroxybenzotriazole (HOBT) and 3.2 g of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide were added. Amine hydrochloride (EDCI), stirred at room temperature for 25 min, added 2.4 g of intermediate 2 and 1.8 mL of triethylamine, and stirred at room temperature for 4 hours. The reaction solution was washed with saturated citric acid (50 mL × 3), saturated sodium bicarbonate (50 mL × 3), and saturated brine (50 mL × 1). Anhydrous magnesium sulfate was dried overnight. Concentrated and recrystallized from ethyl acetate-n-hexane to obtain intermediate 3-1 with a yield of 83%. The structure of intermediate 3-2 is shown below:

4. Preparation of intermediate 4-2:

0.86 g of Intermediate 3-2 was dissolved in 30 mL of anhydrous ether, and 0.35 g of lithium aluminum tetrahydroxide was added in portions in an ice-salt bath, and reacted at room temperature for 1 hour. Under an ice bath, 5 mL of ethyl acetate was added to the reaction solution, and the pH was adjusted to 6.5 with 2M hydrochloric acid. After filtration, the organic phase was washed with saturated brine (20 mL × 3), and dried over anhydrous magnesium sulfate overnight. It was concentrated and separated by combiflash chromatography to obtain intermediate 4-2 with a yield of 50%.

The structure of intermediate 4-2 is shown below:

5. Preparation of intermediate 5-2:

0.32 g of intermediate 4-2 was dissolved in 40 mL of anhydrous THF, 0.35 mL of triethylamine and 0.22 mL of methanesulfonyl chloride were added under an ice bath, and the reaction was stirred below 12 ° C. The reaction was completed after 3 hours. The reaction solution was washed with 0.5 M hydrochloric acid (20 mL × 3), saturated brine (20 mL × 1), and dried over anhydrous magnesium sulfate. The crude product of intermediate 5-2 was concentrated and used directly in the next reaction without purification.

The structure of intermediate 5-2 is shown below:

6. Preparation of intermediate 6-2:

0.33 g of the fluorescent dye represented by formula Q-1 was dissolved in 10 mL of DMF, 0.7 g of anhydrous potassium carbonate was added, and after stirring at room temperature for 15 minutes, 0.8 g of intermediate 5-2 was added and reacted overnight. The reaction solution was poured into 100 mL of ethyl acetate, washed with a saturated potassium carbonate solution (50 mL × 5), and saturated brine (50 mL × 1). Concentrated and recrystallized from ethyl acetate-n-hexane to obtain intermediate 6-2 with a yield of 56%.

The molecular structure of the fluorescent dye is shown below:

The chemical structure of intermediate 6-2 is shown below:

7. Preparation of compound II-2:

6ml of toluene was added to the intermediate 6-2, stirred at room temperature for 2h, silica gel was added, and then heated to reflux. After the reaction was completed, the silica gel was filtered, the silica gel was washed, and the filtrate was evaporated to dryness to obtain a fluorescently labeled amino acid represented by Formula II-2. The rate is 89%.

The specific structure of the fluorescently labeled amino acid represented by Formula II-2 is as follows:

The 1H NMR spectrum of the fluorescently labeled amino acid shown in Formula II-2 is shown in Figure 2. The calculated value of elemental analysis of the fluorescently labeled amino acid shown in Formula II-2: C ₄₈ H ₅₆ Br ₃ N ₅ O ₂ +

Mass spectrum (MS +): 971.20 (M +)

m / z: 973.20 (100.0%), 975.19 (96.8%), 974.20 (52.5%), 976.20 (51.4%), 971.20 (34.1%), 977.19 (32.3%), 978.20 (19.1%), 972.20 (18.6%) ), 975.20 (14.6%), 977.20 (13.6%), 973.21 (4.6%), 979.20 (4.4%), 976.21 (2.3%), 974.19 (1.8%), 976.19 (1.8%).

Elemental analysis: C, 59.15; H, 5.79; Br, 24.59; N, 7.19; O, 3.28.

Example 3

This embodiment provides a fluorescent dye molecule having a structure represented by the following formula 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. Intermediate I'-5 was taken to react with bromomethanol, and NaOH was added to obtain the desired compound Q-1. The 1H NMR spectrum of compound Q-1 is shown in FIG. 3.

Elemental analysis calculated value: C ₃₅ H ₃₈ Br ₂ N ₃ O ⁺

Mass spectrum (MS +): 674.14 (M +)

m / z: 676.14 (100.0%), 674.14 (49.5%), 678.13 (46.8%), 677.14 (36.9%), 675.14 (19.5%), 679.14 (18.1%), 678.14 (7.3%), 680.14 (3.5%) ), 677.13 (1.1%).

Elemental analysis: C, 62.14; H, 5.66; Br, 23.62; N, 6.21; O, 2.37.

Experimental Example 1 Detection of Fluorescent Properties of Fluorescent Dyes

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

3.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 2

As shown in Table 1, the fluorescent quantum yield of the fluorescent dye represented by formula Q-1 is> 85%, and the Stoke shift is large, which is suitable for labeling biomolecules such as amino acids to prepare fluorescent probes to achieve stable fluorescent performance and fluorescent quantum ratio. Detection of nucleic acid molecules with high imaging SNR.

Experimental example 2 Toxicity detection of fluorescently labeled amino acids

Cytotoxicity experiment

To determine the cytotoxicity of the compound represented by the formula II-1 and the compound represented by the formula II-2 in HEK-293T (human embryonic kidney cells) by MTT experiment, the following steps are included:

(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 II-1 prepared in Example 1 and the compound represented by Formula II-2 prepared in Example 2 are dissolved with dimethyl sulfoxide (DMSO) to prepare a concentration of 0.1 mol / L mother liquor, diluted with DMEM medium to a concentration of 80 μmol / L, 40 μmol / L, 20 μmol / L, 10 μmol / L, and 5 μmol / L, for future use; another DMEM medium was added and diluted with an equal volume of deionized water , Where the concentration of the fluorescently labeled amino acid 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. and incubated for 3h, 6h, 12h and 24h, 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.

The histogram of the effect of the compound represented by Formula II-1 on the survival rate of HEK-293T cells at different drug concentrations and incubation times is shown in Figure 4. The effect of the compound represented by Formula II-2 on the survival rate of HEK-293T cells The histogram is shown in Figure 4. 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 h, the cell survival rate after incubation of the compound represented by Formula II-1 or the compound represented by Formula II-2 Both are greater than 90%, so it can be judged that fluorescently labeled amino acids are safe and low-toxic to HEK-293T cells, and have good biocompatibility.

Experimental example 3 stability experiment of fluorescently labeled amino acids

(1) Take 4 parts of 800 μL of PBS (pH = 7.4) buffer solution and 200 μL of the compound solution of formula II-1 prepared in Example 1 and mix, respectively, and heat at 37 ° C for 0.5h, 1h, 2h, 3h, 4h. 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 II-1: The volume ratio of mobile phase B is from 80: 20 → 80: 20; 3min → 25min, mobile phase II- 1: The volume ratio of mobile phase B is from 80: 20 → 10: 90; 25min → 30min, mobile phase II-1: The volume ratio of mobile phase B is from 10: 90 → 80: 20, and the flow rate of mobile phase is controlled to 1mL / Min, control the column temperature at 25 ° C. Then, 4 parts of 800 μL mouse serum (purchased from Biyuntian) and 200 μL of the compound solution of the formula II-1 prepared in Example 1 were separately 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 10,000 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) TFA) is mobile phase II-1, using CH ₃ CN (containing 0.1% TFA by volume) as mobile phase B, gradient elution is performed according to the following procedure: 0min → 3min, mobile phase A: the volume of mobile phase B The ratio is 80: 20 → 80: 20; 3min → 25min, the volume ratio of mobile phase A: mobile phase B is 80: 20 → 10: 90; 25min → 30min, the volume ratio of mobile phase A: mobile phase B is 10: 90 → 80: 20, control the flow rate of the mobile phase to 1mL / min, and control the column temperature at 25 ℃. FIG. 6 is a chemical purity chart of liquid chromatographic detection of a compound represented by Formula II-1 incubated in PBS or serum at different times.

(2) Using the detection method in step (1), the chemical purity of the compound represented by formula II-2 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 II-1 and the compound represented by Formula II-2 decreased slightly in PBS and serum. And the chemical purity in serum is still greater than 95%. This indicates that the fluorescently labeled amino acids have good stability in vitro, which is beneficial for further experimental studies in vivo.

Experimental Example 4 Fluorescence Intensity Detection Chart of Fluorescently Labeled Amino Acids

(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 II-1 prepared in Example 1 and the formula II-2 prepared in Example 2 to the final concentration of 1 mg / ml diluted with culture medium. The fluorescently labeled nucleotides shown are further cultured in a cell incubator for 24 h;

(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 detection results of the fluorescence intensity of the fluorescently labeled amino acids in HEK-293T cells. From left to right (7A-7C) in the figure, the fluorescence of the fluorescently labeled amino acids shown in Formula II-1 in HEK-293T cells is sequentially shown. Detection results, fluorescence detection results of the fluorescently labeled amino acids shown in Formula II-2 in HEK-293T cells, and DAPI staining results of HEK-293T cells. As can be seen from FIG. 8, in the cells incubated with the fluorescently labeled amino acids shown by Formula II-1 and Formula II-2, obvious fluorescence can be detected, indicating that HEK-293T cells can take in the fluorescently labeled amino acids and the fluorescence The labeled amino acid has a 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 (10)

1. A fluorescently labeled amino acid, characterized in that it has the structure shown in Formula I:

   

   Wherein R _{1 is} selected from the group consisting of alkyl, cycloalkyl, aryl and heterocyclic,

   R ₂ is any amino acid side chain group,

   X is halogen.
2. The fluorescently labeled amino acid according to claim 1, which has a structure represented by Formula II:

   

   Wherein, R _{1 is} selected from one of an alkyl group, a cycloalkyl group, an aryl group and a heterocyclic group, and R ₂ is any amino acid side chain group.
3. The fluorescently labeled amino acid according to claim 1 or 2, wherein the

   R _{2 is} selected from -H, -CH ₃ , -CH (CH ₃ ) ₂ , -CH ₂ CH (CH ₃ ) ₂ , -CH (CH ₃ ) CH ₂ CH ₃ ,

   

   

   -CH ₂ COOH,

   

   -CH ₂ C (O) NH ₂ , -CH ₂ CH ₂ COOH, -CH ₂ CH ₂ CH ₂ CH ₂ NH ₂ , -CH ₂ CH ₂ C (O) NH ₂ , -CH ₂ CH ₂ SCH ₃ ,- CH ₂ CH ₂ CH ₂ NHCH (NH) NH ₂ , -CH ₂ OH, -CH (OH) CH ₃ and -CH ₂ SH.
4. A method for preparing a fluorescently labeled amino acid according to any one of claims 1-3, characterized in that it comprises the following steps:

   S1. Dissolve intermediate 1 in an organic solvent, stir after adding the first catalyst, then mix with intermediate 2 and triethylamine, and stir for 4-6 hours; wash the reaction solution, dry, concentrate, and recrystallize to obtain an intermediate 3;

   S2. Intermediate 3 is dissolved in anhydrous ether, a second catalyst is added under an ice bath, and the reaction is performed at room temperature for 0.5 to 2 hours. Ethyl acetate is further added to adjust the pH to 6.5 to 7.5, filtered, dried, and concentrated to obtain intermediate 4 ;

   S3. Intermediate 4 is dissolved in anhydrous THF, and triethylamine and methanesulfonyl chloride are continuously added. After stirring for 3 to 5 hours, the reaction solution is washed, dried, and concentrated to obtain intermediate 5;

   S4. Dissolve the fluorescent dye represented by formula Q in DMF, add an alkali metal salt, stir, add intermediate 5, and react overnight; then pour the reaction solution into ethyl acetate, wash the reaction solution, concentrate, and recrystallize to obtain Intermediate 6;

   S5. Add intermediate 6 to toluene, stir well, add silica gel, heat to reflux, filter after the reaction, wash the silica gel, and evaporate the filtrate to obtain the fluorescently labeled amino acid represented by formula I;

   The reaction route is as follows:

   

   
5. The method according to claim 4, wherein the intermediate 1 is prepared by the following steps:

   The amino acid represented by formula B is dissolved in an alkaline solution, and the amino acid is dissolved in an alkaline solution. An anhydrous THF solution of di-tert-butyl carbonate is gradually added dropwise thereto, and then stirred in an ice bath and room temperature in order to control the reaction process. Carried out in an alkaline environment;

   After the reaction, the THF was removed, the reaction solution was washed, dried, and concentrated to obtain the intermediate 1;

   The reaction route is as follows:

   
6. The method according to claim 4 or 5, wherein the organic solvent is dichloromethane, and the first catalyst is 1-hydroxybenzotriazole and 1- (3-dimethylaminopropyl). A mixture of 3-ethylcarbodiimide hydrochloride, and the second catalyst is lithium tetrahydroaluminum.
7. The method according to any one of claims 4-6, wherein the fluorescent dye 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 -OH undergo an amino substitution reaction, and NaOH is added during the reaction to produce compound Q;

   The synthetic route is as follows:

   
8. The preparation method according to claim 7, 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).
9. Use of the fluorescently labeled amino acid according to any one of claims 1 to 3 in the preparation of a fluorescent probe.
10. The use according to claim 9, wherein the fluorescent probe is a polypeptide fluorescent probe and / or a protein fluorescent probe.

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