CN112209919B - Compound with flavone as mother nucleus and preparation method and application thereof - Google Patents

Compound with flavone as mother nucleus and preparation method and application thereof Download PDF

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CN112209919B
CN112209919B CN202011126670.XA CN202011126670A CN112209919B CN 112209919 B CN112209919 B CN 112209919B CN 202011126670 A CN202011126670 A CN 202011126670A CN 112209919 B CN112209919 B CN 112209919B
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flavone
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虞沂
曹应龙
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Hubei Carbon Yuan Materia Medica Biotechnology Co ltd
Wuhan University WHU
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    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
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    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks

Abstract

The invention discloses a compound taking flavone as a parent nucleus, a preparation method and application thereof. The preparation method of the compound taking flavone as a parent nucleus comprises the following steps: (1) Get K 2 CO 3 Adding the flavone derivative and the alkyne derivative into a solvent, mixing, adding the flavonoid compound, adjusting the pH value of a reaction system after the reaction in the dark, and concentrating to obtain a flavone derivative extract; (2) And (2) taking the flavone derivative extract obtained in the step (1), extracting, distilling under reduced pressure to recover the solvent and purifying to obtain the compound taking the flavone as the parent nucleus. The preparation method is simple, raw materials are easy to obtain, the yield of the target product is high, and the by-product is low. The compound taking flavone as a parent nucleus has important significance for screening key enzymes of biosynthesis pathways of plant or microbial flavonoid secondary metabolites as a photoaffinity fluorescent probe.

Description

Compound with flavone as mother nucleus and preparation method and application thereof
Technical Field
The invention belongs to the field of chemical synthesis, and particularly relates to a compound taking flavone as a parent nucleus, and a preparation method and application thereof.
Background
The flavonoids compounds are important plant polyphenol compounds, are widely distributed in the plant world, and are main pigment components of plant petals, fruit peels, leaves and other parts. In plants, flavone has extremely important physiological activity, and can protect plants from being damaged by ultraviolet rays and phytotoxins; or endowing the plant petals with colorful colors to promote the flowering plants to complete the pollination process; can also be used as a natural plant pesticide to protect plants from insects and microorganisms; it can also be used as a signal molecule, which is beneficial to the root system to fix the microorganism in the nitrogen fixation plant; meanwhile, the frost resistance and drought resistance of the plant can be improved. In the research history of natural plant products, flavonoids are found to have pharmacological activities such as oxidation resistance, microorganism resistance, inflammation resistance, cancer resistance, mutagenesis resistance and the like. Besides medicinal use, anthocyanin in flavonoid compounds is widely used as a natural pigment and a pigment additive in the aspects of food industry and cosmetics. A great amount of natural products take flavone as a mother nucleus for post-modification, such as glycosylation, methylation, isopentenyl and the like, and different from a microbial natural product research method, most of plant biosynthetic genes are not distributed in clusters but dispersed on a genome, the synthetic genes are difficult to detect by utilizing the traditional sequence homology analysis, and how to find specific synthetase in plants is a key point for solving the natural product biosynthetic pathway.
The photoaffinity fluorescent probe is a fluorescent molecule which has characteristic fluorescence in ultraviolet-visible-near infrared and the fluorescence property of which can be sensitively changed along with the change of the property of the environment. The photoaffinity labeling technology combines the advantages of modern molecular biology, cell biology, organic chemistry and other subjects, uses a synthesized photosensitive small molecular compound as a tool probe, and generates a high-activity intermediate through the decomposition of the synthesized photosensitive small molecular compound under the irradiation of light with specific wavelength, and forms specific irreversible covalent bonding with a receptor active site. The probe is one of core tools for researching the interaction between the ligand and the receptor on a molecular level, and has a great promoting effect on the elucidation of the mechanism of the interaction between the ligand and the receptor and the discovery of a drug precursor, so that the finding of a new photoaffinity fluorescent probe is of great significance in the finding of specific synthetase in plants.
Disclosure of Invention
Aiming at the defects and shortcomings of the prior art, the invention mainly aims to provide a compound taking flavone as a parent nucleus.
The invention also aims to provide a preparation method of the compound taking flavone as a parent nucleus.
The invention also aims to provide application of the compounds taking flavone as a parent nucleus.
The purpose of the invention is realized by the following technical scheme:
a compound taking flavone as a parent nucleus has one of the structures shown in the following general formula:
Figure BDA0002733831270000021
wherein n =1 to 10; r is 1 、R 2 And R 3 Independently of one another, from hydrogen, a hydrocarbon radical having from 1 to 10 carbon atoms and one of the following sugar-based structures:
Figure BDA0002733831270000022
preferably, the compound taking flavone as a parent nucleus has one of the following structures:
Figure BDA0002733831270000031
the preparation method of the compound taking flavone as the parent nucleus comprises the following steps:
(1) Get K 2 CO 3 Adding the flavone derivative and the alkyne derivative into a solvent, mixing, adding the flavonoid compound, adjusting the pH value of a reaction system after the reaction in the dark, and concentrating to obtain a flavone derivative extract;
(2) And (2) taking the flavone derivative extract obtained in the step (1), extracting, distilling under reduced pressure to recover the solvent and purifying to obtain the compound taking the flavone as the parent nucleus.
Preferably, the solvent in step (1) is dimethyl sulfoxide (DMSO).
Preferably, K is as described in step (1) 2 CO 3 And the molar ratio of the alkyne derivative to the flavonoid compound is 1-5: 0.1 to 10:0.1 to 5.
Preferably, the concentration of the flavonoid compound in the step (1) in the solvent is 10-50 mol/L.
Preferably, the reaction time in the step (1) is 10 to 30 hours.
Preferably, the alkyne derivative in step (1) has the following structural formula:
Figure BDA0002733831270000032
wherein R is 5 Is halogen, n =1 to 10.
Preferably, the alkyne derivative in step (1) has the following structure:
Figure BDA0002733831270000033
preferably, the flavonoid compound in the step (1) has the following structure:
Figure BDA0002733831270000034
wherein R is 4 Is hydrogen, hydroxy or hydrocarbyl; r 1 、R 2 And R 3 Independently of one another, selected from hydrogen, alkyl groups having 1 to 10 carbon atoms and one of the following structures:
Figure BDA0002733831270000041
preferably, the flavonoid compound has one of the following structures:
Figure BDA0002733831270000042
preferably, the pH of the reaction system is adjusted to 4 to 9 in the step (1).
Preferably, the solvent is recovered by extraction and vacuum distillation in step (2) in a manner that: suspending the flavone derivative extract with water, extracting with ethyl acetate, collecting the upper ethyl acetate layer until the water layer is nearly colorless, and recovering the ethyl acetate layer under reduced pressure to obtain ethyl acetate part; by silica gel column chromatography, the eluent with the petroleum ether-ethyl acetate system in the volume ratio of (1:0), (100.
Preferably, the purification mode of step (2) is: separating by column chromatography, eluting with methanol, detecting flavonoids by thin layer chromatography, and detecting absorption at 254nm with ultraviolet spectrophotometer; heating with methanol sulfate solution to develop red color, and collecting by stages; and then carrying out primary purification on the gel separation part by using ODS reverse column chromatography elution, carrying out HPLC half preparation, eluting by using acetonitrile aqueous solution as an eluent, and finishing the purification.
Preferably, the column chromatography filler is one or more than two of silica gel, diatomite, alumina, adsorption resin, dextran gel and ODS.
The compounds taking flavone as a parent nucleus are used as a photoaffinity fluorescent probe in the acquisition of key enzymes in the biosynthesis pathway of plant or microbial flavonoid secondary metabolites.
Compared with the prior art, the invention has the following advantages and beneficial effects:
(1) The invention discloses a compound taking flavone as a parent nucleus, which has important significance as a photoaffinity fluorescent probe for screening key enzyme of a biosynthesis pathway of a plant or microbial flavonoid secondary metabolite.
(2) The preparation method of the compound taking flavone as the parent nucleus is simple, the raw materials are easy to obtain, the yield of the target product is high, and the by-product is low.
Drawings
FIG. 1 shows kaempferol-tag-1 1 H NMR spectrum.
FIG. 2 shows kaempferol-tag-1 13 C NMR spectrum.
FIG. 3 is the HSQC spectrum of kaempferol-tag-1.
FIG. 4 is an HMBC map of kaempferol-tag-1.
FIG. 5 is a graph showing the results of the experiment in example 3, wherein the left graph shows a Coomassie Blue staining pattern and the right graph shows a fluorescence scan.
FIG. 6 is a graph showing the results of the experiment in example 4, wherein the left graph shows the Coomassie Blue staining pattern and the right graph shows the fluorescence scanning pattern.
Detailed Description
Example 1
A preparation method of a compound taking flavone as a parent nucleus comprises the following steps:
(1) 1mol of K is added into the reaction vessel 2 CO 3 10mL of DMSO and 0.2mol of Iodo Diazirine-alkyne (3- (3-alkyne-1-butyl) -3- (2-iodoethyl) -3H-bisaziridine, shanghai Bian, medical science and technology Limited), mixing, adding 0.1mol of kaempferol to perform a light-shielding reaction for 18 hours, adjusting the pH of the system to =5 by using hydrochloric acid after the reaction is finished, and concentrating to obtain a flavone derivative extract;
(2) Suspending the flavone derivative extract with water, extracting with ethyl acetate, collecting the upper ethyl acetate layer until the water layer is nearly colorless, and recovering ethyl acetate layer under reduced pressure to obtain ethyl acetate part; eluting with a petroleum ether-ethyl acetate system at a volume ratio of (1:0), (100; separating by gel column chromatography, eluting with methanol, detecting flavone derivatives by thin layer chromatography, and detecting absorption at 254nm with ultraviolet spectrophotometer; using color reagent methanol sulfate test solution to show red color, and collecting by sections; then carrying out primary purification on the gel separation part by using ODS reverse column chromatography elution; and (3) performing HPLC half preparation, and eluting by using 45% acetonitrile water as an eluent to obtain the compound taking flavone as a parent nucleus.
Weighing the compound which is prepared in the example 1 and takes flavone as a parent nucleus, carrying out nuclear magnetic resonance detection, wherein a byproduct exists but the content is lower and the compound is not separated, and obtaining 2 target products through detection, wherein the target products are respectively marked as kaempferol-tag-1 and kaempferol-tag-2 (the ratio of the kaempferol-tag-1 to the kaempferol-tag-2 in the product is 2:1). TABLE 1 of kaempferol-tag-1 and kaempferol-tag-2 1 H NMR (400 Hz) and 13 c NMR (100 MHz) data.
From kaempferol-tag-1, kaempferol-tag-2 1 H NMR (400 Hz) and 13 c NMR (100 MHz) data gave kaempferol-tag-1 and kaempferol-tag-2The structural formula is as follows:
Figure BDA0002733831270000061
FIGS. 1 to 4 are respectively kaempferol-tag-1 1 H NMR、 13 C NMR, HSQC, and HMBC.
The main 2DNMR related structure schematic diagram of kaempferol-tag-1 (kaempferol-3-O-Diazirine-alkyne) is as follows, wherein the solid line is related: HMBC.
Figure BDA0002733831270000062
Scheme for correlative structure of kaempferol-tag-1D NMR
The structure diagram related to the kaempferol-tag-1D NMR can be obtained: h-1' is related to C-3 with HMBC, so it can be judged that the hydrogen of the hydroxyl group at the 3-position is replaced by the Diazirine-alkyne group.
TABLE 1 of kaempferol-tag-1 and kaempferol-tag-2 1 H NMR and 13 c NMR data
Figure BDA0002733831270000071
Example 2
A preparation method of a compound taking flavone as a parent nucleus comprises the following preparation steps:
(1) 1mol of K is added to the reaction vessel 2 CO 3 Mixing 10mL of DMSO and 0.5mol of Diazirine-alkyne (3- (3-alkyne-1-butyl) -3- (2-iodoethyl) -3H-bisaziridine, shanghai Bigdi medical science and technology Co., ltd.), adding 0.2mol of icariin to carry out a light-shielding reaction for 18 hours, terminating the reaction, adjusting the pH of the system to =5 by using HCl, and concentrating to obtain a flavone derivative extract;
(2) Suspending the flavone derivative extract with water, extracting with ethyl acetate, collecting the upper ethyl acetate layer until the water layer is nearly colorless, and recovering ethyl acetate layer under reduced pressure to obtain ethyl acetate part; passing through a silica gel column layerEluting sequentially by using a petroleum ether-ethyl acetate system with a volume ratio of (1:0), (100; separating by gel column chromatography, eluting with methanol, detecting flavonoids by thin layer chromatography, and detecting absorption at 254nm with ultraviolet spectrophotometer; using color reagent methanol sulfate test solution to develop red color, and collecting by sections; then carrying out primary purification on the gel separation part by using ODS reverse column chromatography elution; and (3) performing HPLC half-preparation, and eluting by using 45% acetonitrile water as an eluent to obtain the compound with flavone as a parent nucleus. Denoted icariin-tag (icariin-5-O-Diazirine-alkyne), which is useful for treating prostate cancer 1 H NMR (400 Hz) and 13 the C NMR (100 MHz) data are shown in Table 2.
Prepared from icariin-tag 1 H NMR (400 Hz) and 13 c NMR (100 MHz) data gave the icariin-tag as follows:
Figure BDA0002733831270000081
TABLE 2 icariin-tag of 1 HNMR (400 Hz) and 13 CNMR (100 MHz) data
Figure BDA0002733831270000091
Example 3
Taking two proteins as experimental objects, xylose synthase RmUXS (see the following documents for the preparation method of xylose synthase RmUXS: duan XC, lu AM, gu B, cai ZP, ma HY, wei S, laborda P, liu L, voglmeir J. Functional characterization of the UDP-xylose biosynthesis pathway in Rhodothermus marinus. Applied Microbiol Biotechnol. 2015. Nov;99 (22): 9463-72. Doi. The two proteins are used for testing whether the synthesized affinity fluorescent probe at the early stage has specific recognition.
Dissolving the synthesized probe kaempferol-tag-1 in methanol, wherein the final concentration of mother liquor is 100mM, preparing a kaempferol-tag-1 solution, and mixing 10uL of kaempferol solution with 1mL of RmUXS and AtUGT78D1 protein (the final concentrations of RmUXS and AtUGT78D1 are both 10 mM) respectively to serve as an experimental group; 10uL of kaempferol solution with the concentration of 100mM is mixed with 1mL of AtUGT78D1 and RmUXS protein respectively (the final concentration of RmUXS and the final concentration of AtUGT78D1 are both 10 mM) to serve as a control group, the control group and the experimental group are incubated at 30 ℃ for 1 hour, and radiation (power is 8W) is carried out for 20min under 365nm ultraviolet rays, so that the azide group of the fluorescent affinity probe forms covalent bond with the target protein.
1wt% SDS solution (sodium dodecyl sulfate solution), 0.25uL TAMRA-N were added to each 3 azide (5-carboxytetramethylrhodamine azide, new Biotech Co., ltd., seisan Kai, final concentration 0.1 mM) solution, 0.25uL of CuSO 4 A solution (final concentration of 0.1 mM), 0.25uL of THPTA (tris (3-hydroxypropyltriazolemethyl) amine) solution (final concentration of 0.1 mM) and 0.25uL of sodium ascorbate solution (final concentration of 1 mM) were added to the above experimental group and the control group, and the total volume of the reaction system was 500uL. The target protein and the fluorescein TAMRA-N are reacted by Click reaction 3 and azides are combined to facilitate subsequent observation.
FIG. 5 is a graph showing the results of the experiment in example 3, wherein the left graph is a Coomassie Blue staining graph, and the right graph is a fluorescence scanning graph, wherein 0 represents a control group, 100 represents an experiment group)
As shown in fig. 5, the Coomassie Blue dyeing result chart can obtain: in the experimental group and the control group, bands of two enzymes RmUXS and AtUGT78D1 were clearly visible, indicating that protein expression was successful. Under a fluorescent scan (de-excited with Rhodamine excitation wavelength), no fluorescence was observed for both experimental and control groups for RmUXS. As for the AtUGT78D1, only an experimental group can observe clear fluorescence (indicated by an arrow), so that the judgment is that the kaempferol-tag-1 synthesized by the invention can specifically recognize the glycosyl transferase (namely AtUGT78D 1) capable of catalyzing kaempferol, and the application of the glycosyl transferase to the excavation of the modified key enzyme gene of the flavonoid compound in the secondary metabolite of the plant is feasible.
Example 4
The method comprises the steps of taking traditional Chinese medicine epimedium, using a plant protein extraction kit (Shanghai biological engineering service Co., ltd., model: NO. C500053) to perform plant crude enzyme extraction by referring to an instruction book, and verifying whether kaempferol-tag-1 synthesized in the early stage can accurately identify target protein from plant crude enzyme.
Experimental groups: 10uL of the kaempferol-tag-1 solution (concentration 100 mM) synthesized in example 1 was mixed with 500uL of crude plant enzyme;
control group: 10uL of kaempferol solution (the concentration is 100 mM) is mixed with 500uL of plant crude enzyme,
the experimental group and the control group were incubated at 30 ℃ for 1 hour, respectively, and then irradiated with 365nm ultraviolet radiation (power of 8W) for 20min to form covalent bond between the azide group of kaempferol-tag-1 and the target protein.
1wt% of SDS solution (sodium dodecyl sulfate solution), 0.25uL of TAMRA-N, respectively 3 Azide (5-carboxytetramethylrhodamine azide, new Biotech Ltd., xianKai) solution (concentration 0.1 mM), 0.25uL of CuSO 4 A solution (concentration: 0.1 mM), a 0.25uLTHPTA (tris (3-hydroxypropyltriazolemethyl) amine) solution (concentration: 0.1 mM) and a 0.25uL sodium ascorbate solution (concentration: 1 mM) were added to the above experimental group and the control group, and the reaction system volume was 500uL. The target protein and the fluorescein TAMRA-N are reacted by Click reaction 3 and azides are combined to facilitate subsequent observation.
FIG. 6 is a graph showing the results of the experiment of example 4, wherein the left graph shows Coomassie Blue staining patterns, the right graph shows fluorescence scanning patterns, and Marker indicates broad molecular weight protein standards.
From the Coomassie Blue staining pattern in fig. 6, it can be derived: the experimental group and the control group both contain a plurality of protein bands, which indicates that the crude enzyme extraction of the plants is successful; as is clear from the fluorescence scan (de-excitation with Rhodamine excitation wavelength), the experimental group had a clear black band under the fluorescence scan, i.e., a protein labeled with kaempferol-tag-1, thereby verifying that kaempferol-tag-1 has the function of recognizing the target protein from crude plant enzymes.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims (10)

1. A compound taking flavone as a parent nucleus is characterized by having one of the structures shown in the following general formula:
Figure DEST_PATH_IMAGE001
Figure 916973DEST_PATH_IMAGE002
Figure DEST_PATH_IMAGE003
wherein n =1 to 10; r 1 、R 2 And R 3 Independently of one another, from hydrogen, a hydrocarbon radical having from 1 to 10 carbon atoms and one of the following structures:
Figure 162010DEST_PATH_IMAGE004
。
2. the compound with flavone as a parent nucleus according to claim 1, wherein the compound with flavone as a parent nucleus has one of the following structures:
Figure 472905DEST_PATH_IMAGE005
Figure DEST_PATH_IMAGE006
Figure 382962DEST_PATH_IMAGE007
。
3. a method for preparing a compound having a flavone as a parent nucleus as claimed in any one of claims 1~2 comprising the steps of:
(1) Get K 2 CO 3 Adding the flavone derivative and the alkyne derivative into a solvent, mixing, adding the flavonoid compound, adjusting the pH value of a reaction system after the reaction in the dark, and concentrating to obtain a flavone derivative extract;
(2) And (2) taking the flavone derivative extract obtained in the step (1), extracting, distilling under reduced pressure to recover the solvent and purifying to obtain the compound taking the flavone as the parent nucleus.
4. The method for preparing a compound taking flavone as a parent nucleus according to claim 3, wherein the solvent in the step (1) is dimethyl sulfoxide; the reaction time in the step (1) is 10 to 30h in a dark place; and (2) adjusting the pH of the reaction system in the step (1) to 4~9.
5. The method for preparing flavone-based compound according to claim 4, wherein K in step (1) 2 CO 3 Alkyne derivative and flavonoid compound at a molar ratio of 1~5:0.1 to 10:0.1 to 5; the concentration of the flavonoid compound in the step (1) in the solvent is 10 to 50mol/L.
6. The method for preparing a compound with flavone as a parent nucleus according to any one of claims 3~5, wherein the alkyne derivative in step (1) has the following general structural formula:
Figure 994071DEST_PATH_IMAGE009
wherein R is 5 Is halogen, n =1 to 10.
7. The method for preparing a class of compounds taking flavone as a parent nucleus according to claim 6, wherein the alkyne derivative in the step (1) has the following structure:
Figure 347692DEST_PATH_IMAGE011
。
8. the method for preparing a compound with flavone as a parent nucleus according to any one of claims 3~5, wherein the flavonoid compound in the step (1) has the following structure:
Figure DEST_PATH_IMAGE012
wherein R is 4 Is hydrogen, hydrocarbyl; r 1 、R 2 And R 3 Independently of one another, from hydrogen, alkyl having 1 to 10 carbon atoms and one of the following structures:
Figure 880305DEST_PATH_IMAGE013
。
9. the method for preparing compounds with flavone as mother nucleus according to claim 8, wherein the flavone compounds have one of the following structures:
Figure DEST_PATH_IMAGE014
。
10. the use of a class of compounds of any one of claims 1~2 having a flavonoid as a parent nucleus as photoaffinity fluorescent probes for obtaining key enzymes in the biosynthetic pathway of secondary metabolites of flavonoids in plants or microorganisms.
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