CN110627852B

CN110627852B - Biotin-labeled naringin, preparation method and application thereof

Info

Publication number: CN110627852B
Application number: CN201910968369.4A
Authority: CN
Inventors: 马兆成; 李明; 程丽萍
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2019-10-12
Filing date: 2019-10-12
Publication date: 2021-11-23
Anticipated expiration: 2039-10-12
Also published as: CN110627852A

Abstract

The invention belongs to the technical field of organic synthesis and pharmaceutical chemistry, and particularly relates to biotin-labeled naringin, a preparation method and application thereof. The structural formula of the naringin probe marked by the biotin prepared by the invention is shown as the formula (I), and the compound has the advantages of stable structure, simple synthesis and anti-inflammatory activity. Pharmacological experiments show that the biotin-labeled naringin probe has better anti-inflammatory effect than single naringin and has the value of developing anti-inflammatory drugs; on the other hand, the biotin-labeled naringin can be used as a molecular probe to probe the drug target of naringin, and has important significance in probing the action mechanism of naringin in the inflammation process.

Description

Biotin-labeled naringin, preparation method and application thereof

Technical Field

The invention belongs to the technical field of organic synthesis and pharmaceutical chemistry, and particularly relates to biotin-labeled naringin, a preparation method and application thereof.

Background

Biotin, also known as vitamin H and coenzyme R, is a water-soluble vitamin and also belongs to the vitamin B group. Biotin is widely present in various organisms in the natural world, is an essential element for human and animal health maintenance, and is thus named. Avidin is a glycoprotein that binds specifically to biotin. Avidin and biotin can be combined with protein and fluorescein molecules without affecting the biological activity of the latter, and are ideal labeling agents.

Naringin belongs to flavanone compounds, and is a natural product with biological activity and pharmacological action widely existing in citrus fruits. Has the capacity of resisting oxidation and removing free radicals, and participates in the regulation process of various anti-inflammatory mechanisms, so that the naringin has good anti-inflammatory efficacy. In addition, naringin also has anticancer, mutation resisting, antiallergic, analgesic, blood pressure lowering and cardiovascular and cerebrovascular disease preventing and treating effects, and can be widely used in food, medicine and cosmetic fields.

The drug target is a biological macromolecule or a specific biological macromolecule structural domain which plays a therapeutic role and realizes the clinical curative effect of the drug. It enables the drug to exert its expected biological activity, and the protein is the most important drug action target and is the executor of life activities. Therefore, the identification of drug targets is a key scientific problem in drug development, and is also the most challenging leading edge scientific problem in chemical biology, particularly in chemogenomics, and thus has prompted the development of chemical protein mass spectrometry (CCCP) centered on small molecule compounds. When the CCCP technology is applied to drug target protein identification, drug molecules are linked with fluorescent groups or biotin molecules through chemical modification, and a chemical probe capable of being directly and covalently bound with potential target proteins is constructed. After the chemical probe is incubated with cell lysate or whole protein extract, the protein complex covalently bound with the probe is separated and enriched by gel electrophoresis or affinity chromatography technology and then identified.

At present, good anti-inflammatory function of naringin is widely concerned, but the drug target of naringin is still unclear, and the naringin is rarely reported as an anti-inflammatory drug. In order to further find an anti-inflammatory drug target of naringin and clarify the anti-inflammatory mechanism of naringin, the preparation of a naringin probe molecule containing biotin is particularly urgent.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides biotin-labeled naringin, a preparation method and application thereof. The invention takes sugar hydroxyl in a naringin structure as a reaction site for acetalation modification, and is connected with biotin modified by a carboxyl terminal to obtain a compound containing biotin and naringin, and experiments prove that the compound still has anti-inflammatory activity, so that a biotin-labeled naringin probe molecule is obtained.

The invention is realized in such a way that the structural formula of the biotin-labeled naringin is shown as the formula (I).

The preparation method of the biotin-labeled naringin comprises the following steps:

step 1: taking sugar hydroxyl in a naringin molecular structure as a reaction site, and carrying out acetalation modification to obtain acetalation-modified naringin;

step 2: taking terminal carboxyl in a biotin molecular structure as a reaction site, and carrying out esterification modification to obtain terminal alkyne containing biotin fragments;

and step 3: performing Sonogashira coupling reaction on the acetalized and modified naringin prepared in the step 1 and the terminal alkyne containing the biotin fragment prepared in the step 2 to obtain the compound of claim 1.

Further, in the step 1, naringin, 4-toluenesulfonamide and p-bromobenzaldehyde dimethyl acetal are mixed and dissolved in an organic solvent, and react for 12 to 24 hours at room temperature, the organic solvent is removed by reduced pressure distillation, and the residue is recrystallized to obtain the acetalization modified naringin with the structural formula shown in formula (II).

And further, in the step 2, biotin, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, 3-butyn-1-ol and 4-dimethylaminopyridine are mixed and dissolved in an organic solvent, an esterification reaction is carried out for 36-72 hours at room temperature, the organic solvent is removed by reduced pressure distillation, and the end-group alkyne containing the biotin fragment is obtained after drying, wherein the structural formula is shown as a formula (III).

Further, in the step 3, under the protection of nitrogen, naringin, dichloro-bis (triphenylphosphine palladium), cuprous iodide, triethylamine, end group alkyne containing biotin fragment and tetrahydrofuran which are subjected to acetalation modification are mixed and dissolved in an organic solvent, and react for 6-12 hours at the temperature of 60-80 ℃; removing the organic solvent by rotary evaporation, dissolving the residue with methanol, precipitating solid, filtering, and purifying by silica gel column chromatography to obtain biotin-labeled naringin of the structural formula of claim 1.

Further, in the step 1, the molar ratio of the naringin to the 4-toluenesulfonamide to the dimethyl p-bromobenzaldehyde formaldehyde acetal is 25-45: 1: 50-80 parts; the mass volume ratio of naringin to organic solvent is 1: 5-10, i.e. the volume of organic solvent per gram of naringin is 5-10 ml.

Further, in step 2, the molar ratio of biotin, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, 3-butyn-1-ol and 4-dimethylaminopyridine is as follows: 1: 3-8: 1-4: 0.2 to 2; the mass-volume ratio of the biotin to the organic solvent is 1: 25-40, namely the volume of the organic solvent is 25-40 ml per gram of biotin; the separating eluent adopted by the silica gel column chromatography is a mixed solution of ethyl acetate and methanol, wherein the volume ratio of ethyl acetate to methanol is 20-30: 1-2.

Further, the molar ratio of the acetalized naringin, dichloro-bis (triphenylphosphine-palladium), cuprous iodide and the end-group alkyne containing biotin fragment in step 3 is as follows: 1: 0.01-0.08: 0.05-0.12: 1-2; the mass-to-volume ratio of compound 2 to THF is 1: 15-20, i.e. the volume of THF per gram of compound 2 is 15-20 ml.

Further, the organic solvent is N, N-dimethylformamide.

The application of the biotin-labeled naringin in preparing anti-inflammatory drugs or researching naringin drug targets is disclosed.

In summary, the advantages and positive effects of the invention are:

(1) the invention provides a compound shown in formula (I), which is obtained by a classical Sonogashira coupling reaction of bromophenyl-substituted naringin and terminal alkyne containing biotin fragment, and is a biotin-labeled naringin molecular probe.

(2) The preparation method of the biotin-labeled naringin molecular probe provided by the invention comprises the steps of firstly carrying out acetalation modification on sugar hydroxyl in a naringin structure as a reaction site, then carrying out esterification modification on a carboxyl terminal of biotin to obtain terminal alkyne containing a biotin fragment, and then carrying out Sonogashira coupling reaction on the naringin subjected to acetalation modification and the terminal alkyne containing the biotin fragment to obtain the naringin molecular probe. The preparation method is simple, and the purity of the product is up to more than 95 percent after further purification by preparative liquid chromatography.

(3) Experiments prove that the biotin-labeled naringin molecule provided by the invention has an effective anti-inflammatory effect. Lipopolysaccharide (LPS) is adopted to induce macrophage of a mouse to generate an inflammatory response, and the anti-inflammatory effect of the compound is proved by researching the expression quantity of cyclooxygenase (COX-2) on the mRNA level. The compound of the invention not only has obvious inhibition effect on the inflammation generated by mouse macrophage induced by LPS, but also has better anti-inflammatory effect compared with the single naringin used on an inflammatory cell model.

(4) The naringin is marked by biotin, so that the naringin can be used as a molecular probe for researching the target of an anti-inflammatory drug of the naringin, the action target of the naringin is explored, the anti-inflammatory mechanism of the naringin can be more clearly understood, and a safer and more efficient anti-inflammatory method can be found. On the other hand, the biotin-labeled naringin probe also has an anti-inflammatory effect, has a better anti-inflammatory effect than naringin, and can be developed into an anti-inflammatory drug.

The invention also provides application of the synthesized biotin-labeled naringin compound, which can be used for preparing anti-inflammatory drugs and can also be used as a biotin-labeled naringin molecular probe because the compound still has anti-inflammatory activity after being connected with avidin. The biotin-labeled naringin probe constructed by the invention can find target protein directly and potentially covalently bound with the probe through avidin specifically bound with biotin, and lays a solid foundation for researching anti-inflammatory mechanism of naringin.

Drawings

FIG. 1 is a diagram of the reaction process for the synthesis of Biotin-Naringin according to the present invention;

FIG. 2 is a drawing of Compound 1¹H NMR spectrum;

FIG. 3 is a drawing of Compound 2¹H NMR spectrum;

FIG. 4 is a sample of biotin-labeled naringin¹H NMR spectrum;

FIG. 5 is a graph of absorption peaks of biotin-labeled naringin after preparative liquid chromatography purification;

FIG. 6 shows the quantitative expression of biotin-labeled naringin synthesized in the examples of the present invention and inflammatory factors in cells treated with naringin (A: COX-2, B: iNOS, C: IL-1. beta., and D: IL-6). Results data are presented as mean ± standard deviation; different lower case letters indicate significant differences (P <0.05) between treatments, and the inclusion of the same letter indicates insignificant differences;

FIG. 7 is an immunoprecipitation assay protocol;

FIG. 8 shows the results of SDS-PAGE protein gel silver staining; the arrow marks the band as a protein capable of specifically binding to naringin.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples, and the equipment and reagents used in the examples and test examples are commercially available without specific reference. The specific embodiments described herein are merely illustrative of the invention and are not intended to be limiting.

The invention discloses a biotin-labeled naringin, a preparation method and application thereof. The synthetic reaction process of Biotin-labeled Naringin (Biotin-Naringin) is shown in figure 1, and the specific temperature range of the room temperature is 10-35 ℃. The principle process is as follows: taking sugar hydroxyl in a naringin molecular structure as a reaction site, and carrying out acetalation modification to obtain acetalation-modified naringin; taking terminal carboxyl in a biotin molecular structure as a reaction site, and carrying out esterification modification to obtain terminal alkyne containing biotin fragments; and carrying out Sonogashira coupling reaction on the acetalized and modified naringin and the end-group alkyne containing the biotin fragment to obtain a biotin-labeled naringin probe compound.

See in particular the examples below.

Example 1 preparation of Biotin-labeled Naringin

(1) In a round-bottomed flask, naringin (2.6 g), 4-toluenesulfonamide (PTSA) (30 mg) and N, N-Dimethylformamide (DMF) (15 mL) as an organic solvent were added and dissolved by stirring. 2.6g of p-bromobenzaldehyde dimethyl acetal is added to react for 12 to 24 hours, 20 hours in the embodiment, at the temperature of 35 ℃. In other embodiments, the amounts of the materials used in this step may satisfy the following conditions: the molar ratio of naringin, PTSA and dimethyl p-bromobenzaldehyde acetal is 25-45: 1: 50-80 parts; the mass volume ratio of naringin to organic solvent is 1: 5-10, i.e. the volume of organic solvent per gram of naringin is 5-10 ml.

After completion of the reaction, DMF was distilled off under reduced pressure, and the residue was recrystallized from methanol to obtain compound 1 as a white solid with a yield of 40%. The obtained white solid compound 1 was subjected to nuclear magnetic analysis,¹the H NMR spectrum is shown in FIG. 2, and the structural formula is as follows:

(2) to a Schlenk flask were added biotin (1.0 g), 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI) (3.8 g) and DMF (30 mL), and the mixture was dissolved with stirring. Under the protection of nitrogen, 0.9mL of 3-butyn-1-ol and 0.49g of 4-Dimethylaminopyridine (DMAP) are added in sequence, and the mixture is reacted at room temperature for 36-72h, in the example, 48 h. DMF was evaporated under reduced pressure and the residue was dissolved in ethyl acetate, washed successively with 1mol/L hydrochloric acid (3 × 20mL), sodium bicarbonate solution (3 × 15mL) and saturated sodium chloride solution (2 × 20mL), dried over anhydrous magnesium sulfate, rotary evaporated to remove the solution and chromatographed over silica gel column [ eluent: a ═ V (ethyl acetate): V (methanol) ═ 25:1]Purification gave compound 2 as a white solid in 66% yield. The obtained white compound 2 was subjected to nuclear magnetic analysis,¹the HNMR map is shown in FIG. 3, and the structural formula is as follows:

in other embodiments, the amounts of the materials used in this step may satisfy the following conditions: the molar ratio of biotin, EDCI, 3-butyn-1-ol and DMAP is: 1: 3-8: 1-4: 0.2 to 2; the mass-volume ratio of biotin to DMF is 1: 25-40; the volume of the organic solvent corresponding to each gram of biotin is 25-40 ml; the separating eluent adopted by the silica gel column chromatography is a mixed solution of ethyl acetate and methanol, wherein the volume ratio of ethyl acetate to methanol is 20-30: 1-2.

(3) 0.75g of compound 1, 28mg of dichlorobis (triphenylphosphine palladium) and 15mg of cuprous iodide were added to a Schlenk flask, and 8mL of anhydrous DMF was added under nitrogen protection, and the mixture was stirred to dissolve. Vacuumizing, replacing nitrogen, quickly adding 6mL of triethylamine, vacuumizing again when the solution is changed from yellow to reddish brown, and repeatedly replacing nitrogen for multiple times. A THF solution containing 0.35g of Compound 2 (6 mL) was added, the mixture was evacuated, and the nitrogen gas was repeatedly replaced several times, and the reaction was carried out at 60 to 80 ℃ for 6 to 12 hours, in this example, at 80 ℃ for 12 hours. Removing triethylamine and THF by rotary evaporation; the DMF was evaporated under reduced pressure and the residue was taken up in methanol to precipitate a solid. Filtration and washing of the filter cake with methanol until TLC colour reagent shows no product. The filtrate and wash solution were combined and chromatographed over silica gel column [ eluent: a ═ V (ethyl acetate): V (methanol) ═ 10:1]Purifying to obtainThe light yellow solid is Naringin (Biotin-Naringin) marked by Biotin, and the Naringin is further purified by preparative liquid chromatography, wherein the yield of the Naringin marked by the Biotin is 15 percent, and the purity reaches 95 percent. The nuclear magnetic analysis is carried out on the sample,¹the H NMR spectrum is shown in FIG. 4, and the structural formula is as follows:

nuclear magnetic and mass spectrum characterization:¹H NMR(DMSO-d₆,400MHz):δ12.05(br,1H),9.60(s,1H),7.47-7.39(m,4H),7.34-7.31(m,2H),6.80(d,J＝7.6Hz,2H),6.40(s,1H),6.33(s,1H),6.12-6.10(m,2H),6.04and 5.86(s,1H),5.55-5.34(m,4H),5.24-5.11(m,2H),4.57(s,1H),4.27(t,J＝6.4Hz,1H),4.21-4.18(m,3H),4.09-3.94(m,2H),3.76-3.37(m,6H),3.26-3.00(m,3H),2.80-2.73(m,4H),2.56(d,J＝12.8Hz,1H),2.34(t,J＝7.2Hz,2H),1.64-1.25(m,6H),1.23(d,J＝6.0Hz,3H).LCMS:m/z 963.2[M+H]⁺。

FIG. 5 is the absorption peak diagram and purity detection of biotin-labeled naringin after preparative liquid chromatography purification.

In other embodiments, the amounts of the materials used in this step may satisfy the following conditions: the molar ratio of the compound 1, the dichloro-bis (triphenylphosphine palladium), the cuprous iodide and the compound 2 is as follows: 1: 0.01-0.08: 0.05-0.12: 1-2; the mass-to-volume ratio of compound 2 to THF is 1: 15-20, i.e. the volume of THF per gram of compound 2 is 15-20 ml.

Example 2 Biotin-labeled Naringin anti-inflammatory pharmacodynamic study

To examine whether the substance still has anti-inflammatory activity after the biotin labeling of naringin. Lipopolysaccharide (LPS) is used for establishing an inflammation model of mouse macrophage RAW264.7 cells. Inoculating RAW264.7 cells in logarithmic growth phase into a 24-well culture plate, and setting 4 different treatments after adherent growth for one day, namely a blank control group of RPMI-1640 culture medium without fetal bovine serum; LPS (1. mu.g/mL) treated group; 50 μ g/mL of Biotin-labeled Naringin probe (Biotin-Naringin) and lipopolysaccharide LPS (1 μ g/mL); and naringin (50. mu.g/mL) and lipopolysaccharide LPS (1. mu.g/mL). After the medicine is treated for 18h, cell supernatant is removed, Trizol reagent is added, RNA is extracted according to the operation of a kit, after reverse transcription, expression of inflammatory factor cyclooxygenase-2 (cyclooxygenase-2, COX-2), Inducible Nitric Oxide Synthase (iNOS), interleukin-1 beta (interleukin 1 beta, IL-1 beta) and interleukin-6 (interleukin 6, IL-6) at the transcription level is detected by utilizing real-time fluorescence quantitative PCR.

Real-time fluorescent quantitative PCR system and program average reference kit

qPCR SYBR Green Master Mix (Low Rox Plus), primer sequences are as follows:

β-actin,

5'-AGGCTGTGCTGTCCCTGTATGC-3' as shown in SEQ ID NO. 1;

5'-ACCCAAGAAGGAAGGCTGGAAA-3' as shown in SEQ ID NO. 2;

COX-2,

5'-ATCTGGCTTCGGGAGCACAAC-3' as shown in SEQ ID NO. 3;

5'-GAGGCAATGCGGTTCTGATACTG-3' as shown in SEQ ID NO. 4;

iNOS,

5'-GAATCTTGGAGCGAGTTGTGGA-3' as shown in SEQ ID NO. 5;

5'-GTGAGGGCTTGGCTGAGTGAG-3' as shown in SEQ ID NO. 6;

IL-1β,

5'-GTTGACGGACCCCAAAAGAT-3' as shown in SEQ ID NO. 7;

5'-CCTCATCCTGGAAGGTCCAC-3' as shown in SEQ ID NO. 8;

IL-6,

5'-ACAAAGCCAGAGTCCTTCAGA-3' as shown in SEQ ID NO. 9;

5'-TCCTTAGCCACTCCTTCTGT-3' as shown in SEQ ID NO. 10.

The results are shown in figure 6 and the table below, with data expressed as mean ± standard deviation.

	COX-2	iNOS	IL-1β	IL-6
					Control	1.000,d	1.000,c	1.000,d	1.000,c
LPS	29.045±1.065,a	6.404±0.284,a	1863.242±233.402,a	818.109±120.114,a
					LPS+Nar	17.846±2.101,b	4.814±0.306,b	1164.258±287.331,b	438.743±106.417,b
LPS+Bio-Nar	4.045±0.326,c	0.649±0.065,d	634.354±189.242,c	17.321±0.479,c

Different lower case letters indicate significant differences (p <0.05) between treatments, and the inclusion of the same letters indicates no significant differences.

Wherein, Control represents blank Control group, LPS represents LPS treatment group, LPS + Nar represents Naringin and LPS treatment group, LPS + Bio-Nar represents Biotin-Naringin and LPS treatment group, and different lower case letters represent differences among different groups. The result shows that the mRNA expression levels of COX-2, iNOS, IL-1 beta and IL-6 in macrophages treated by LPS are obviously higher than the mRNA expression levels of various inflammatory factors in a blank control group which is not treated by LPS, and the mRNA expression levels of COX-2, iNOS, IL-1 beta and IL-6 in LPS + Nar and LPS + Bio-Nar treatment groups are all obviously lower than the mRNA expression levels of various inflammatory factors in an LPS treatment group, so that the Biotin-Naringin after Naringin is marked by Biotin still has anti-inflammatory activity, and can be applied to the preparation of anti-inflammatory drugs and used as a probe for exploring Naringin drug targets.

EXAMPLE 3 screening binding proteins to Naringin by immunoprecipitation

In order to screen target protein which is specifically combined with naringin from mouse macrophage, synthetic biotin-labeled naringin is used as a probe, and screening is carried out by using an immunoprecipitation method.

The immunoprecipitation assay protocol is shown in FIG. 7. mu.L of streptavidin-agarose beads were placed in 2 1.5mL centrifuge tubes, 500. mu.L of Phosphate Buffered Saline (PBS) was added to each tube, and the supernatant was centrifuged (14000g, 5 s). Then, 500. mu.L of PBS and 4. mu.L of 1. mu.g/. mu.L of Biotin-Naringin and Biotin solution were added, respectively, and incubated at 4 ℃ for 1 hour with shaking. After the incubation was complete, the supernatant was centrifuged off (14000g, 5 s). Add 500. mu.L PBS and centrifuge wash 3 times (14000g, 5 s). Then, 500. mu.L of 1. mu.g/. mu.L total mouse macrophage protein was added thereto and incubated at 4 ℃ for 1 hour with shaking. After the incubation was complete, the supernatant was centrifuged off (14000g, 5 s). Add 500. mu.L PBS and centrifuge wash 3 times (14000g, 5 s). 60 μ L of 2-fold protein loading buffer 60 μ L was added, and the mixture was boiled in boiling water for 5min, centrifuged to collect the supernatant (12000g, 3min), and subjected to SDS-PAGE, with the results of SDS-PAGE protein gel strips shown in FIG. 8. The differential bands were excised and analyzed by mass spectrometry to identify proteins as shown in table 1.

TABLE 1 LC-MS/MS detection of candidate naringin target proteins

The experimental results show that the naringin labeled by the biotin, which is synthesized by the invention and used as a probe, can find the target protein directly and covalently bound with the probe through the avidin specifically bound with the biotin, thereby laying a solid foundation for researching the anti-inflammatory mechanism of the naringin.

The biotin-labeled naringin probe prepared by the invention has the advantages of stable structure, simple synthesis and anti-inflammatory activity. Pharmacological experiments show that the biotin-labeled naringin probe has the value of developing anti-inflammatory drugs, can be used as a molecular probe to probe the drug target of naringin, and has important significance in probing the action mechanism of naringin in the inflammation process.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

Sequence listing

<110> university of agriculture in Huazhong

<120> naringin marked by biotin, preparation method and application thereof

<160> 10

<170> SIPOSequenceListing 1.0

<210> 1

<211> 22

<212> DNA

<213> Artificial sequence (beta-actin-F)

<400> 1

aggctgtgct gtccctgtat gc 22

<210> 2

<211> 22

<212> DNA

<213> Artificial sequence (beta-actin-R)

<400> 2

acccaagaag gaaggctgga aa 22

<210> 3

<211> 21

<212> DNA

<213> Artificial sequence (COX-2-F)

<400> 3

atctggcttc gggagcacaa c 21

<210> 4

<211> 23

<212> DNA

<213> Artificial sequence (COX-2-R)

<400> 4

gaggcaatgc ggttctgata ctg 23

<210> 5

<211> 22

<212> DNA

<213> Artificial sequence (iNOS-F)

<400> 5

gaatcttgga gcgagttgtg ga 22

<210> 6

<211> 21

<212> DNA

<213> Artificial sequence (iNOS-R)

<400> 6

gtgagggctt ggctgagtga g 21

<210> 7

<211> 20

<212> DNA

<213> Artificial sequence (IL-1. beta. -F)

<400> 7

gttgacggac cccaaaagat 20

<210> 8

<211> 20

<212> DNA

<213> Artificial sequence (IL-1. beta. -R)

<400> 8

cctcatcctg gaaggtccac 20

<210> 9

<211> 21

<212> DNA

<213> Artificial sequence (IL-6-F)

<400> 9

acaaagccag agtccttcag a 21

<210> 10

<211> 20

<212> DNA

<213> Artificial sequence (IL-6-R)

<400> 10

tccttagcca ctccttctgt 20

Claims

1. A biotin-labeled naringin has a structural formula:

2. the method of preparing biotin-labeled naringin according to claim 1, comprising the steps of:

and step 3: performing Sonogashira coupling reaction on the acetalized and modified naringin prepared in the step 1 and the terminal alkyne containing the biotin fragment prepared in the step 2 to obtain the compound of claim 1;

3. the method for preparing biotin-labeled naringin according to claim 2, wherein: in the step 1, naringin, 4-toluenesulfonamide and p-bromobenzaldehyde dimethyl acetal are mixed and dissolved in an organic solvent, the mixture reacts for 12 to 24 hours at room temperature, the organic solvent is removed by reduced pressure distillation, and residues are recrystallized to obtain acetalized and modified naringin, wherein the structural formula is as follows:

4. the method for preparing biotin-labeled naringin according to claim 3, wherein: in the step 2, biotin, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, 3-butyn-1-ol and 4-dimethylaminopyridine are mixed and dissolved in an organic solvent, esterification reaction is carried out for 36-72 hours at room temperature, the organic solvent is removed by reduced pressure distillation, and the end group alkyne containing biotin fragments is obtained after drying, wherein the structural formula is as follows:

5. the method for preparing biotin-labeled naringin according to claim 4, wherein: in the step 3, under the protection of nitrogen, naringin, dichloro-bis (triphenylphosphine palladium), cuprous iodide, triethylamine, end group alkyne containing biotin fragment and tetrahydrofuran which are subjected to acetalation modification are mixed and dissolved in an organic solvent, and react for 6 to 12 hours at the temperature of between 60 and 80 ℃; removing the organic solvent by rotary evaporation, dissolving the residue with methanol, precipitating solid, filtering, and purifying by silica gel column chromatography to obtain biotin-labeled naringin of the structural formula of claim 1.

6. The method for preparing biotin-labeled naringin according to claim 3, wherein: in the step 1, the molar ratio of naringin, 4-toluenesulfonamide and p-bromobenzaldehyde dimethyl acetal is 25-45: 1: 50-80 parts; the mass volume ratio of naringin to organic solvent is 1: 5-10.

7. The method for preparing biotin-labeled naringin according to claim 4, wherein: in the step 2, the molar ratio of biotin, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, 3-butyn-1-ol and 4-dimethylaminopyridine is as follows: 1: 3-8: 1-4: 0.2 to 2; the mass-volume ratio of the biotin to the organic solvent is 1: 25-40; the separating eluent adopted by the silica gel column chromatography is a mixed solution of ethyl acetate and methanol, wherein the volume ratio of ethyl acetate to methanol is 20-30: 1-2.

8. The method for preparing biotin-labeled naringin according to claim 5, wherein: in the step 3, the molar ratio of the acetalized and modified naringin, dichloro-bis (triphenylphosphine palladium), cuprous iodide and the end group alkyne containing biotin fragment is as follows: 1: 0.01-0.08: 0.05-0.12: 1-2; the mass-to-volume ratio of compound 2 to THF is 1: 15-20.

9. The method for producing biotin-labeled naringin according to any one of claims 3 to 8, wherein: the organic solvent is N, N-dimethylformamide.

10. The use of biotin-labeled naringin of claim 1 in the preparation of an anti-inflammatory drug or in the preparation of a probe for exploring the target of naringin drugs.