Fluorescein-labeled NRH derivative as well as synthesis and application thereof

Abstract

The invention discloses a fluorescein-labeled NRH derivative as well as synthesis and application thereof, the chemical structural formula of the fluorescein-labeled NRH derivative is as follows: # imgabs0 #, in the formula, R1 is an NRH substituent group, and R2 is a fluorophore. The fluorescein-labeled NRH derivative is applied to compound labeling and tracking. According to the fluorescein-labeled NRH derivative disclosed by the invention, alanine is introduced between the NRH fragment and the fluorescein fragment for color development, and the newly formed molecule does not open the NRH molecular structure and does not open the fluorescein structure, so that the structures of the NRH fragment and the fluorescein fragment are completely preserved; the alanine can form a stable ester bond with a substrate NRH; after the alanine is introduced, the flexibility of the system is increased, and the solubility and the stability of the whole structure can be improved; an amido bond is formed between fluorescein and alanine, so that the stability is good, and the application prospect is great.

Description

Technical Field

[0001] The present invention relates to the technical field of chemical synthesis and molecular modification, and relates to a fluorescein-labeled NRH derivative, synthesis and application thereof, and in particular to a compound capable of tracing the position of NRH in cells and having good stability, a synthesis method thereof and application thereof. Background Art

[0002] Nicotinamide riboside hydrogenated, referred to as NRH, has a molecular structure of It is a naturally occurring light yellow organic compound that can be found in small amounts in the metabolites of bacteria such as Corynebacterium glutamicum and Lactobacillus, but the content is very low, less than 0.1%.

[0003] NRH is currently mainly used as an NAD+ supplement and has been widely used in the fields of biology and medical testing. However, the labeling and tracking of NRH in the human body is still in the exploratory stage. If researchers can complete the tracking of NRH in the human body by labeling NRH, this can provide a more accurate basis for the study of the mechanism of action of NRH. Therefore, how to achieve the labeling and tracking of NRH in the human body is a difficult problem that currently plagues the study of the mechanism of action of NRH.

[0004] Therefore, it is of great practical significance to develop a compound that can trace the location of NRH in cells. Summary of the invention

[0005] Since the prior art has the above-mentioned defects, the present invention provides.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] A fluorescein-labeled NRH derivative, the chemical structure of which is as follows:

[0008] ;

[0009] In the formula, R1 is the NRH substituent (functional group), and R2 is the fluorescent group. The introduction of the fluorescent group is to increase the colorimetric tracing function, so other fluorescent substances with a carboxyl structure should be possible, such as 5-carboxyfluorescein or 6-carboxyfluorescein.

[0010] The reasons why the present invention does not directly connect NRH with fluorescein are as follows: 1) The activity of the primary alcohol hydroxyl group in NRH is low. Whether it is directly subjected to alcohol-acid condensation to form an ester with fluorescein or esterification reaction with the NHS active ester of fluorescein, the entire reaction system is very complicated and the effect is not good; 2) Fluorescently, it contains two -OH groups, and the connection site in the substrate NRH is also a hydroxyl group, which will affect the reaction. Fluorescently, it forms an ester by itself or condenses with the generated target product to form an ester group; 3) Even if the target product is generated, it may be more unstable due to its greater molecular rigidity, and some organic bases will be added to our reaction, and the product will decompose under such weak alkaline conditions; 4) In the reaction of removing the acetone fork (hydroxyl protecting group on the NRH structure), dihydropyridine will oxidize to pyridine, so it needs to be reduced to dihydropyridine again under alkaline conditions, and such alkaline conditions will also have the risk of product decomposition.

[0011] The present invention introduces a small linker (alanine) between the NRH fragment and the fluorescein fragment used for color development to connect the two together. The reasons for choosing alanine are as follows: 1) the newly formed molecule does not open the NRH molecular structure, nor does it open the fluorescein structure, and the structures of the two are completely preserved; 2) alanine can form a stable ester bond with the substrate NRH; 3) if the NRH substrate and fluorescein are directly linked through an ester group, the rigidity of the structure may be relatively large and the stability is poor. After the introduction of the small linker (alanine), the flexibility of the system is increased, which will improve the solubility and stability of the overall structure; 4) an amide bond is formed between fluorescein and alanine, which is very stable, and under the condition of forming an amide bond, the phenolic hydroxyl group on the fluorescein will not affect the reaction; 5) alanine and fluorescein have a stable ester group, but can be dissociated in a slightly stronger alkaline solution, and subsequent laboratories can also perform dissociation as needed.

[0012] As the preferred technical solution:

[0013] The chemical structure of the fluorescein-labeled NRH derivative described above is as follows:

[0014] .

[0015] The present invention also provides a method for preparing a fluorescein-labeled NRH derivative as described above, comprising the following steps:

[0016] (1) R2-COOH reacts with compound 7B to obtain intermediate 7, wherein the structural formula of compound 7B is:

[0017] ;

[0018] (2) Nicotinamide ribose chloride reacts with 2,2-dimethoxypropane to prepare intermediate 2;

[0019] (3) Intermediate 2 reacts with sodium bicarbonate and ethyl acetate to obtain intermediate 3;

[0020] (4) Intermediate 3 reacts with intermediate 4 to obtain intermediate 5, wherein the structural formula of intermediate 4 is:

[0021] ;

[0022] Of course, conventional amino acids can also be used to replace intermediate 4;

[0023] (5) Intermediate 5 reacts with dioxane hydrochloride to obtain intermediate 6, and the Boc protecting group of alanine is removed under acidic conditions, which will not affect the stability of the ester group;

[0024] (6) Intermediate 6 reacts with intermediate 7 to prepare a fluorescein-labeled NRH derivative.

[0025] The preparation method has simple process and relatively mild reaction conditions and has good application prospects.

[0026] As the preferred technical solution:

[0027] In the method described above, the reaction in step (1) is carried out in an organic solvent under the catalysis of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and the reaction conditions are 25° C. and 1 h;

[0028] The reaction in step (2) is carried out in a sulfuric acid-acetonitrile mixture under the reaction conditions of 25° C. and 5 h;

[0029] The reaction in step (3) is carried out in an aqueous solution under the protection of a nitrogen atmosphere, and the reaction conditions are 0° C. and 4 h.

[0030] In the method described above, the reaction in step (4) is carried out in an organic solvent under nitrogen atmosphere protection and 4-dimethylaminopyridine and N,N-dicyclohexylcarboximide are also added. The reaction conditions are 25° C. and 12 h.

[0031] In the method described above, the reaction in step (5) is carried out in an organic solvent at 25° C. for 4 h.

[0032] In the method described above, the reaction in step (6) is carried out in an organic solvent and N,N-diisopropylethylamine is added. The reaction conditions are 25° C. and 2 h.

[0033] In addition, the present invention also provides the use of the above-mentioned fluorescein-labeled NRH derivatives in compound labeling and tracking.

[0034] The above technical solution is only a feasible technical solution of the present invention, and the protection scope of the present invention is not limited thereto. Those skilled in the art can reasonably adjust the specific design according to actual needs.

[0035] The above invention has the following advantages or beneficial effects:

[0036] (1) The fluorescein-labeled NRH derivative of the present invention introduces a small linker (alanine) between the NRH fragment and the fluorescein fragment used for color development. The newly formed molecule does not open the NRH molecular structure or the fluorescein structure, and the structures of both are completely preserved;

[0037] (2) In the fluorescein-labeled NRH derivative of the present invention, alanine can form a stable ester bond with the substrate NRH;

[0038] (3) After the introduction of a small linker (alanine) into the fluorescein-labeled NRH derivative of the present invention, the flexibility of the system is increased, which will improve the solubility and stability of the overall structure;

[0039] (4) In the fluorescein-labeled NRH derivative of the present invention, an amide bond is formed between fluorescein and alanine, which is very stable. Under the conditions for forming an amide bond, the phenolic hydroxyl group on fluorescein will not affect the reaction, and alanine and fluorescein form a stable ester group.

[0040] (5) The preparation method of the fluorescein-labeled NRH derivative of the present invention has a simple process and relatively mild reaction conditions, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] The present invention and its features, appearance and advantages will become more apparent by reading the detailed description of non-limiting embodiments with reference to the following drawings. The same reference numerals indicate the same parts in all the drawings. The drawings are not drawn to scale, and emphasis is placed on illustrating the subject matter of the present invention.

[0042] Figure 1 This is a schematic diagram of the test of fluorescein-labeled NRH derivatives on the basilar membrane of the cochlea, where the rows from top to bottom are 0.5h, 2h, 6h, 12h, and 24h, and the columns from left to right are fluorescein-labeled NRH derivatives showing their localization into the basilar membrane, MyosinVIIa showing the location of basilar membrane hair cells, DAPI showing the location of cell nuclei, and overlays. The green in the figure corresponds to the fluorescein-labeled NRH derivatives, the red corresponds to MyosinVIIa, and the gray corresponds to DAPI;

[0043] Figure 2This is a schematic diagram of the test of fluorescein-labeled NRH derivatives in the HEI-OC1 cell line, where the rows from top to bottom are 2h and 24h, respectively. HEI-OC1 cells were incubated with fluorescein-labeled NRH derivatives for 24h, and green fluorescent protein-labeled NRH was still visible in the cells. From left to right, the fluorescein-labeled NRH derivatives show their localization into the basement membrane, DAPI shows the position of the cell nucleus, and the overlay image. The green in the figure corresponds to the fluorescein-labeled NRH derivatives, and the blue corresponds to DAPI. DETAILED DESCRIPTION

[0044] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, but they are not intended to limit the present invention.

[0045] Example 1

[0046] A method for preparing a fluorescein-labeled NRH derivative, the preparation route of which is shown in the following formula:

[0047]

[0048] The following steps are involved:

[0049] Step 1: Dissolve intermediate 7A (8 g, 21.26 mmol, 1 eq) and 7B (3.18 g, 27.64 mmol, 1.3 eq) in N,N-dimethylformamide (60 mL), add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (5.30 g, 27.64 mmol, 1.3 eq), and stir the reaction solution at 25°C for 1 hour. Pour the reaction solution into ice water (300 mL), and a red-brown solid precipitates. Filter, collect the solid, and dry to obtain a red-brown solid product intermediate 7 (10 g, 21.12 mmol, yield 99.37%).

[0050] Step 2: Sulfuric acid (4.49 g, 45.75 mmol, 2.44 mL, 0.7 eq) was added to acetonitrile (250 mL) at 0°C. After stirring for 5 minutes, 2,2-dimethoxypropane (68.07 g, 653.60 mmol, 80.08 mL, 10 eq) and intermediate 1 (19 g, 65.36 mmol, 1 eq) were added at 0°C, and the reaction solution was stirred at 25°C for 5 hours. The reaction solution was cooled to 0°C, solid sodium carbonate was added to quench the reaction, and then stirred at 0°C for 1 hour. 5 ml of water was slowly added, and the solid was filtered out when the pH value of the reaction solution was 6-7. The filtrate was concentrated under reduced pressure, and the concentrate was purified by column chromatography (silica, dichloromethane / methanol = 1 / 0 to 9 / 1) to obtain a white solid product intermediate 2 (8.4 g, hydrochloride, 25.32 mmol, yield 38.74%).

[0051] Step 3: Dissolve intermediate 2 (8.4 g, 25.32 mmol, 1 eq, HCl) and sodium bicarbonate (19.14 g, 227.87 mmol, 8.87 mL, 9 eq) in water (30 mL), slowly add hydrosulfite (8.82 g, 50.64 mmol, 11.02 mL, 2 eq) at 0°C, and replace with nitrogen three times. The reaction solution was stirred at 0°C for 5 minutes, ethyl acetate (60 mL) was added, and then stirred at 0°C for 4 hours. After the reaction was completed, the reaction solution was extracted with 30 mL of ethyl acetate and 30 mL of water, and the combined organic phase was washed with water (40 mL * 3), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a light yellow solid product intermediate 3 (4.3 g, 13.64 mmol, yield 53.88%, purity 94%).

[0052] Step 4: Dissolve intermediate 4 (3.5 g, 18.50 mmol, 1 eq) and 4-dimethylaminopyridine (225.99 mg, 1.85 mmol, 0.1 eq) in dichloromethane (60 mL). Add intermediate 3 (5.48 g, 18.50 mmol, 1 eq) under nitrogen atmosphere. Cool the reaction solution to 0°C, add N,N-dicyclohexylcarboximide (4.01 g, 19.42 mmol, 3.93 mL, 1.05 eq), stir at 0°C for 5 minutes, then heat to 25°C and stir for 12 hours. After the reaction was completed, the reaction solution was filtered, the filtrate was concentrated under reduced pressure, and the concentrate was purified by column chromatography (silica, dichloromethane / methanol = 1 / 0 to 10 / 1) to obtain a yellow solid product intermediate 5 (6 g, 12.83 mmol, yield 69.38%).

[0053] Step 5: Dissolve intermediate 5 (6 g, 12.83 mmol, 1 eq) in dichloromethane (50 mL), add dioxane hydrochloride solution (2 M, 32.08 mL, 5 eq), and stir the reaction solution at 25°C for 4 hours. After the reaction, the reaction solution was concentrated under reduced pressure to obtain a light yellow solid product intermediate 6 (4.6 g, 6.20 mmol, yield 48.28%).

[0054] Step 6: Intermediate 6 (900 mg, 1.21 mmol, 1 eq, hydrochloride) and intermediate 7 (573.85 mg, 1.21 mmol, 1 eq) were dissolved in N,N-dimethylformamide (12 mL), and N,N-diisopropylethylamine (470.00 mg, 3.64 mmol, 633.43 μL, 3 eq, pH=~7) was added. The reaction solution was stirred at 25°C for 2 hours. After the reaction was completed, the reaction solution was concentrated under reduced pressure, and the concentrate was purified by column chromatography (silica, dichloromethane / methanol = 1 / 0 to 9 / 1) and high performance liquid chromatography (Phenomenex Luna C18 column, 10um silica, 40mm diameter, 100mm length); (using a decreasing polarity mixture of water (containing 0.225% formic acid) and acetonitrile (12%~42%) as the eluent) to obtain a red-brown solid product Target (910 mg, 1.25 mmol, yield 20.58%, purity 94%).

[0055] The hydrogen nuclear magnetic resonance test data or other relevant data of the intermediate and Target are as follows:

[0056] H NMR analysis of compound 5 1 H NMR (400 MHz, DMSO-d6) δ = 7.38 (d, J = 7.2Hz, 1H), 6.99 (s, 1H), 6.74 (s, 2H), 6.12 - 6.01 (m, 1H), 4.91 (d, J = 4.4Hz, 1H), 4.75 - 4.68 (m, 2H), 4.61 (dd, J = 3.7, 6.6 Hz, 1H), 4.23 - 4.16 (m,2H), 4.10 - 4.02 (m, 2H), 2.93 (s, 2H), 1.48 (s, 3H), 1.38 (s, 9H), 1.28 (s,3H), 1.24 (d, J = 7.2 Hz, 3H)

[0057] The prepared fluorescein-labeled NRH derivative was tested, and the specific test operation was as follows:

[0058] 1. Inner ear basement membrane explant experiment: The experimental animals were randomly divided into 3 groups, including: The basement membrane of newborn mice 3 days after birth was dissected out in ice PBS under sterile conditions and placed in culture medium. The ambient temperature was controlled at 37°C and the culture environment contained 10% CO2. After 24 hours, fluorescein-labeled NRH derivatives were added to the culture medium. After 0.5h, 2h, 6h, 12h, and 24h of co-culture, the tissue samples were taken out and fixed with 4% paraformaldehyde for 1 hour. Immunofluorescence staining was performed with MyosinVIIa antibody, and the slices were sealed with DAPI-containing sealing solution and photographed and observed using a laser confocal microscope.

[0059] 2. HEI-OC1 cell experiment: Mouse cochlear hair cell-like cells (HEI-OC1 cells) were cultured in a medium containing 10% FBS, the ambient temperature was controlled at 33°C, and the culture environment contained 10% CO2. Fluorescein-labeled NRH derivatives were added to the culture medium, and the cell samples were taken out after 2h and 24h of co-culture, fixed with 4% paraformaldehyde for 10 minutes, and then sealed with a sealing solution containing DAPI, and then photographed and observed using a laser confocal microscope.

[0060] Test results such as Figure 1 , 2 As shown, from Figure 1 It can be seen that after 0.5 hours, a small amount of the fluorescein-labeled NRH derivative enters the inner and outer hair cells, and after 2 hours, a small amount enters the supporting cells outside the outer hair cells. After 6 hours, the hair cells and supporting cells are all marked with the compound. After 12 hours, the compound is still retained in the supporting cells outside the outer hair cells. After 24 hours, the drug is metabolized and excreted from the hair cells. From the above results, it can be seen that the fluorescein-labeled NRH derivative of the present application can be used for compound labeling and tracking. Figure 2 It can be seen that only a small amount of the fluorescein-labeled NRH derivatives entered the HEI-OC1 cells after 2 hours, and after 24 hours, the fluorescein-labeled NRH derivatives could be labeled and retained in the HEI-OC1 cells.

[0061] Those skilled in the art should understand that those skilled in the art can implement variations by combining the prior art and the above embodiments, which will not be described in detail here. Such variations do not affect the essential content of the present invention, and will not be described in detail here.

[0062] The above describes the preferred embodiments of the present invention. It should be understood that the present invention is not limited to the above-mentioned specific embodiments, and the devices and structures that are not described in detail should be understood to be implemented in a common manner in the art; any technician familiar with the art can use the above-disclosed methods and technical contents to make many possible changes and modifications to the technical solutions of the present invention without departing from the scope of the technical solutions of the present invention, or modify them into equivalent embodiments of equivalent changes, which does not affect the essential content of the present invention. Therefore, any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solutions of the present invention are still within the scope of protection of the technical solutions of the present invention.

Claims

1. A fluorescein-labeled NRH derivative, characterized in that: Its chemical structure is as follows: ； In the formula, R1 is an NRH substituent, and R2 is a fluorescent group.

2. A fluorescein-labeled NRH derivative according to claim 1, characterized in that: Its chemical structure is as follows: 。 3. A method for preparing a fluorescein-labeled NRH derivative as claimed in any one of claims 1 to 2, characterized in that: The following steps are involved: (1) R2-COOH reacts with compound 7B to obtain intermediate 7, wherein the structural formula of compound 7B is: ； (2) Nicotinamide ribose chloride reacts with 2,2-dimethoxypropane to prepare intermediate 2; (3) Intermediate 2 reacts with sodium bicarbonate and ethyl acetate to obtain intermediate 3; (4) Intermediate 3 reacts with intermediate 4 to obtain intermediate 5, wherein the structural formula of intermediate 4 is: ； (5) Intermediate 5 reacts with dioxane hydrochloride to obtain intermediate 6; (6) Intermediate 6 reacts with intermediate 7 to prepare a fluorescein-labeled NRH derivative.

4. The method according to claim 3, characterized in that The reaction in step (1) is carried out in an organic solvent under the catalysis of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and the reaction conditions are 25° C. and 1 h; The reaction in step (2) is carried out in a sulfuric acid-acetonitrile mixture under the reaction conditions of 25° C. and 5 h; The reaction in step (3) is carried out in an aqueous solution under the protection of a nitrogen atmosphere, and the reaction conditions are 0° C. and 4 h.

5. The method according to claim 3, characterized in that: The reaction in step (4) is carried out in an organic solvent under nitrogen atmosphere protection and 4-dimethylaminopyridine and N,N-dicyclohexylcarboximide are also added. The reaction conditions are 25° C. and 12 h.

6. The method according to claim 3, characterized in that: The reaction in step (5) is carried out in an organic solvent at 25° C. for 4 h.

7. The method according to claim 3, characterized in that The reaction in step (6) is carried out in an organic solvent and N,N-diisopropylethylamine is added. The reaction conditions are 25° C. and 2 h.

8. Use of the fluorescein-labeled NRH derivative according to any one of claims 1 to 2 in compound labeling and tracking.

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