Preparation method of acetylated anthocyanin monomer

Abstract

The invention relates to the technical field of oriented synthesis of anthocyanin monomers, and particularly discloses a preparation method of an acetylated anthocyanin monomer. The preparation method comprises the following steps: firstly, synthesizing an intermediate as shown in a formula (2) by adopting acetophenone analogue, imidazole and triisopropyl chlorosilane; then reacting with elemental iodine and beta-D-glucose-2, 3, 4, 6-tetraacetate in sequence, and synthesizing an intermediate of a formula (5) through acylation; and reacting the intermediate shown in the formula (5) with 2-hydroxy-4, 6-diacetylbenzaldehyde under the conditions of low temperature and acidic atmosphere, diluting with an acidic organic solvent, and carrying out reduced pressure distillation, separation and purification to obtain the acetylated anthocyanin monomer. According to the preparation method provided by the invention, acetylation directional modification can be carried out on the position 2 of glucoside, and subsequent dealkalization operation is combined, so that the synthesis quality and efficiency of the acetylated anthocyanin monomer are remarkably improved. According to the method, a controllable way is provided for anthocyanin synthesis, the yield of anthocyanin is increased, and the problems that an existing synthesis way is uncontrollable and the yield is low are solved.

Description

Technical Field

[0001] The present application relates to the technical field of organic synthesis, and in particular to a method for preparing acetylated anthocyanin monomers. Background Art

[0002] Anthocyanins, as a natural water-soluble pigment, are widely distributed in the plant kingdom, giving fruits, flowers and other plants colorful and bright colors. In addition to its color-developing function, anthocyanins also have significant antioxidant, anti-inflammatory, anti-cancer and anti-obesity biological activities, showing great application potential in the fields of food, medicine and cosmetics. However, anthocyanins face two major obstacles in practical applications: one is the low bioavailability, and the other is the relative stability of anthocyanins when facing adverse environmental conditions, such as pH changes, temperature changes, enzymatic hydrolysis, light and the presence of oxygen. This seriously limits its wide application in the food, medicine, cosmetics and other industries.

[0003] Anthocyanidin glycosylation mainly refers to the phenomenon that the -OH on the glycoside is acylated and esterified by various organic acids. Studies have shown that anthocyanins usually have low lipophilicity due to the presence of more hydrophilic hydroxyl groups, and acylation can effectively enhance their lipophilicity. In addition, acylation can also improve the stability of anthocyanidins in high temperature environments. At present, most acylated anthocyanidins are synthesized by extraction from natural sources or enzymatic acylation with organic acids. Although a few studies have reported the use of chemical synthesis to synthesize acylated anthocyanins using anthocyanins as reactants, such as Cruz et al., who isolated malvidin-3-O-glucoside (M3G) from young grape wine and stirred it with stearyl chloride at room temperature in anhydrous acetonitrile as the reaction medium overnight to obtain fatty acid derivatives of M3G; Fei et al., who used solid phase grafting to react maleic anhydride with blueberry anthocyanins and 4-dimethylaminopyridine in a 50 °C oven under solvent-free conditions to obtain acylated anthocyanins; Wang et al., who also used solid phase reaction method to react natural blueberry anthocyanins with different fatty acids at 50 °C for 12 hours in a polytetrafluoroethylene reactor to synthesize three acylated anthocyanins. However, there are still few reports on the chemical total synthesis of acylated anthocyanins using acetophenone (B ring) and o-hydroxybenzaldehyde (A ring) as starting materials. Acylated malvidin-3-O-glucoside (M3G) and acylated cyanidin-3-O-glucoside (C3G) are widely present and representative anthocyanins in nature. C3G is widely distributed in various fruits and flowers such as grapes, blueberries, cherries, etc., showing different colors from red to purple. Under the catalysis of acyltransferase, M3G can form a variety of acylated forms, such as malvidin-3-O-(6”-acetyl)-glucoside, malvidin-3-O-(6”-coumaryl)-glucoside, and malvidin-3-O-(6”-caffeoyl)-glucoside. Previous studies have shown that M3G is the main anthocyanin in red wine, among which malvidin-3-O-(6”-acetyl)-glucoside is the most abundant. Structurally, the molecular structures of C3G and M3G are quite representative. Both contain an acetophenone skeleton and have different substituents on the B ring. The B ring structure of C3G is relatively simple, containing only two hydroxyl groups, which helps to precisely control the site and chemical environment during acylation modification. In contrast, the B ring of M3G contains a methoxy group and a hydroxyl group, which makes it show unique reactivity and site selectivity differences during the acylation process. This structural difference provides a good opportunity to explore the effects of acetyl grafting on different anthocyanins.

[0004] Although past studies have used 1D and 2D NMR techniques to characterize the position of acyl substitution on glycosides, the acylation site is determined only based on the chemical shift generated by 1D NMR, and no specific coupling information between the carbonyl carbon and the proton is given, which has certain limitations. Although some researchers have tried to use two-dimensional nuclear magnetic resonance (2D NMR) techniques, such as heteronuclear multiple bond correlation spectroscopy (HMBC) and homonuclear correlation spectroscopy (COSY) to determine the acylation site, in actual operation, these methods often have difficulty in observing obvious coupling signals in the HMBC and COSY spectra, resulting in the acylation site cannot be further clarified. Therefore, there is an urgent need to explore a method for synthesizing acetylated anthocyanin monomers with a controllable synthetic pathway. Summary of the invention

[0005] In view of this, the purpose of the present application is to provide a method for preparing acetylated anthocyanin monomers, so as to solve the problems of uncontrollable synthesis routes and low yields in existing ones.

[0006] In order to achieve the above technical objectives, the present application provides a method for preparing acetylated anthocyanin monomers, comprising the following steps:

[0007] Step S1, placing an acetophenone analogue of formula (1), imidazole and triisopropylsilyl chloride in DMF for mixed reaction to obtain an intermediate of formula (2);

[0008] Step S2, mixing the intermediate of formula (2) and elemental iodine in an organic solvent for reaction to obtain the intermediate of formula (3);

[0009] Step S3, mixing the intermediate of formula (3) and β-D-glucose-2,3,4,6-tetraacetate in an organic solvent for reaction to obtain the intermediate of formula (4);

[0010] Step S4, subjecting the intermediate of formula (4) to acetylation reaction to obtain the intermediate of formula (5);

[0011] Step S5, introducing an acidic gas under low temperature conditions, and mixing the intermediate of formula (5) and 2-hydroxy-4,6-diacetylbenzaldehyde in a solvent for reaction until the acidic gas reaches saturation, then heating the reaction, and the reaction is completed, followed by dilution with an acidic organic solvent, vacuum distillation, separation and purification, to obtain an acetylated anthocyanin monomer of formula (6);

[0012] .

[0013] Furthermore, when the acetophenone analogue of formula (1) is 3,4-dihydroxyacetophenone, the molar mass ratio of the intermediate of formula (3) to β-D-glucose-2,3,4,6-tetraacetate is 1:1.5.

[0014] Furthermore, when the acetophenone analogue of formula (1) is 4-hydroxy-3,5-dimethoxyacetophenone, the molar mass ratio of the intermediate of formula (3) to β-D-glucose-2,3,4,6-tetraacetate is 1:2.5.

[0015] Furthermore, in step S5, the low temperature condition is set to -20°C; then the temperature is raised to 0°C, and the reaction is kept at 0°C for 40 to 50 hours.

[0016] Furthermore, the acidic gas is one of hydrochloric acid gas and hydrogen fluoride.

[0017] Furthermore, the acidic organic solvent is acidic methanol.

[0018] Furthermore, in step S5, a liquid chromatograph is used for separation and purification, and a C18 reverse phase column is selected as the chromatographic column.

[0019] Furthermore, the parameters for separation and purification are as follows: the mobile phase A of the medium pressure separation chromatography is 0.1% trifluoroacetic acid aqueous solution, the mobile phase B is 100% methanol solution, the flow rate is set to 30 mL / min, the monitoring wavelength in the program is 280 nm, the detection wavelength and the collection wavelength are 520 nm, and the instrument collection threshold is 900 mAU; the gradient program of the mobile phase setting is: 0-10 min, 6% B; 10-20 min, 6-15% B; 20-50 min, 15-35% B; 50-200 min, 35-42% B; 200-215 min, 42-44% B; 215-225 min, 44-81% B; 225-230, 81-100% B; 230-260 min, 100% B; 260-265 min, 100-50% B; 265-275 min, 50% B; 275~280 min, 50~6% B; 280~285 min, 6% B.

[0020] The present application provides an acetylated anthocyanin monomer obtained by the above-mentioned preparation method, wherein the acetylated anthocyanin monomer comprises one of acylated malvidin-3-O-glucoside and acylated cyanidin-3-O-glucoside.

[0021] In summary, the present application provides an acetylated anthocyanin monomer, firstly using an acetophenone analogue of formula (1), imidazole and triisopropylsilyl chloride to synthesize an intermediate of formula (2); then reacting with β-D-glucose-2,3,4,6-tetraacetate and elemental iodine to synthesize an intermediate of formula (5) through acylation; then, under low temperature and acidic atmosphere conditions, reacting the intermediate of formula (5) with 2-hydroxy-4,6-diacetylbenzaldehyde in a specific molar mass ratio, diluting with an acidic organic solvent, distilling under reduced pressure, separating and purifying, the acetylated anthocyanin monomer is obtained. The preparation method provided in the present application can carry out acetylation-directed modification at the 2nd position of the glucoside to improve the efficiency of acetylation; and combined with the debasing operation, a large number of acetyl groups are retained, so that in the subsequent cyclization reaction, most of the acetyl groups retained in the early stage can be smoothly removed, thereby efficiently forming a large number of new anthocyanin monomers. This process not only significantly improves the synthesis rate of anthocyanins, but also opens up a new controllable path for the synthesis of acylated anthocyanins, completely solving the industry problems of uncontrollable and low-yield existing synthesis paths.

[0022] Compared with the prior art, the present application fundamentally improves the synthesis quality and efficiency of acetylated anthocyanin monomers through precise acetylation-directed modification and unique de-alkalization operation, simplifies the preparation process, and reduces unnecessary steps and time costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0024] Figure 1 A schematic diagram of the synthesis process of acetylated anthocyanins provided in the examples of the present application;

[0025] Figure 2 COSY and HMBC spectra of acetylated anthocyanins provided in the examples of the present application;

[0026] Figure 3 This is a thermal degradation curve of anthocyanins and acetylated anthocyanins in the examples of the present application under high temperature conditions. DETAILED DESCRIPTION

[0027] The technical solutions of the embodiments of the present application will be described clearly and completely below in conjunction with the accompanying drawings. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the specification of the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection requested by the present application.

[0028] The sources of raw materials in the present invention are as follows:

[0029] 3′,4′-Dihydroxyacetophenone, 4′-hydroxy-3′,5′-dimethoxyacetophenone, triisopropylsilyl chloride, β-D-glucose pentaacetate, and hydrazine acetate were purchased from Bide Pharmatech Ltd., Shanghai, China;

[0030] Imidazole, iodine, triethylamine, and acetyl chloride were purchased from Macklin (Shanghai, China); sodium hydride was purchased from Aladdin (Shanghai, China);

[0031] Tetrabutylammonium fluoride was purchased from Heowns Biochem LLC (Tianjin, China);

[0032] 2,4,6-Trihydroxybenzaldehyde was purchased from Bide (Shanghai, China);

[0033] Dimethylaminopyridine (DMAP) was purchased from Meryer (Meryer Chemical Technology Co., Ltd., Shanghai, China);

[0034] Acetic anhydride, hydrogen chloride (95.0-98.0%), sulfuric acid (95.0-98.0%), and hydrogen peroxide were purchased from a chemical reagent factory (Guangzhou, China).

[0035] The following compounds were used as solvents: N,N-dimethylformamide (AR grade), dichloromethane (AR grade), methanol (AR grade), tetrahydrofuran (AR grade) and ethyl acetate (AR grade) were purchased from Guanghua Sci-Tech (Guangdong, China). All low-temperature reactions were performed using a XHDHJF-4002 low-temperature constant-temperature stirring reaction bath from Xiaohan (Shanghai, China). The hydrogen chloride connection device required for the cyclization reaction was homemade in the laboratory. All intermediates were purified by 300-mesh silica gel powder column chromatography from Haiyang Chemical (Qingdao, China). The final product was purified using an EZ PLUS 2000 medium-pressure preparative liquid chromatograph purchased from Lisure Science (Suzhou, China). The 1H and 13C NMR spectral data were recorded by Bruker Avance III (300 MHz) or Bruker AVANCE NEO 600M, and the 2D NMR spectral data COSY and HMBC were recorded by Bruker AVANCE NEO 600M.

[0036] The sources of all raw materials in the present invention are not particularly limited and can be purchased on the market or prepared according to conventional methods known to those skilled in the art.

[0037] See also Figure 1 The present invention provides a method for preparing an acetylated anthocyanin monomer, comprising the following steps:

[0038] Step S1, placing an acetophenone analogue of formula (1), imidazole and triisopropylsilyl chloride in DMF for mixed reaction to obtain an intermediate of formula (2);

[0039] Step S2, mixing the intermediate of formula (2) and elemental iodine in an organic solvent for reaction to obtain the intermediate of formula (3);

[0040] Step S3, mixing the intermediate of formula (3) and β-D-glucose-2,3,4,6-tetraacetate in an organic solvent for reaction to obtain the intermediate of formula (4);

[0041] Step S4, subjecting the intermediate of formula (4) to acetylation reaction to obtain the intermediate of formula (5);

[0042] Step S5, introducing an acidic gas under low temperature conditions, and mixing the intermediate of formula (5) and 2-hydroxy-4,6-diacetylbenzaldehyde in a solvent for reaction until the acidic gas reaches saturation, then heating the reaction, and the reaction is completed, followed by dilution with an acidic organic solvent, vacuum distillation, separation and purification, to obtain an acetylated anthocyanin monomer of formula (6);

[0043] It should be noted that in order to optimize the glycosylation modification process of the anthocyanin B ring, we first used triisopropylsilyl chloride (TIPSCl) to protect the hydroxyl group on the acetophenone analog of formula (1). Compared with other hydroxyl protecting groups, TIPSCl has mild reaction conditions, easy removal, and resistance to strong acids and bases, which makes it easier to replace the acetyl group as a protecting group in the subsequent synthesis process and improve the synthesis yield of the acetylated anthocyanin monomer. In the glycosylation step S2, a selective iodination halogen substitution method is used to achieve specific site modification of the B ring. This selective iodination technology has been shown in our previous work to be able to effectively replace the methyl group without affecting other positions and avoid the formation of by-products (see CN116410220A). β-D-glucose tetraacetate was prepared using β-D-glucose pentaacetate as the glycosylation reagent, mainly because β-D-glucose pentaacetate has a lower cost and all hydroxyl groups are protected by acetyl groups. This design not only helps to conveniently remove part of the acetyl protection in the subsequent synthesis process, but also increases the yield of the target acetylated anthocyanins.

[0044] In some embodiments, the acetophenone analog of formula (1) includes one of 3,4-dihydroxyacetophenone and 4-hydroxy-3,5-dimethoxyacetophenone.

[0045] It should be noted that 3,4-dihydroxyacetophenone and 4-hydroxy-3,5-dimethoxyacetophenone were used as basic raw materials, respectively, acetophenone analogs of formula (1), to carry out a series of final synthesis of the following acetylated anthocyanins: C3AG and M3AG. Among them, C3AG can smoothly follow our previous synthesis scheme in the B-ring glycosylation synthesis process and show a higher yield.

[0046] Preferably, when the acetophenone analog of formula (1) is 3,4-dihydroxyacetophenone, the molar mass ratio of the intermediate of formula (3) to β-D-glucose-2,3,4,6-tetraacetate is 1:1.5; when the acetophenone analog of formula (1) is 4-hydroxy-3,5-dimethoxyacetophenone, the molar mass ratio of the intermediate of formula (3) to β-D-glucose-2,3,4,6-tetraacetate is 1:2.5.

[0047] It should be noted that in the synthesis process of the B-ring glycosylation of M3AG, due to the large steric hindrance of the B-ring of M3AG, the synthesis route suitable for C3AG can hardly see the generation of product points in the synthesis application of M3AG. Therefore, the feed ratio of the glycosylation reaction of M3AG was optimized, and the molar mass ratio of the intermediate of formula (3) to β-D-glucose-2,3,4,6-tetraacetate was adjusted to 1:2.5.

[0048] In some embodiments, in the operation involved in step S5, the low temperature condition is set to -20°C, then the temperature is raised to 0°C, and the temperature is maintained for a reaction time of 40 to 50 hours.

[0049] It should be noted that the use of the disclosed reaction temperature (low temperature of -20 °C, followed by heating to room temperature) will lead to a decrease in the solubility of hydrogen chloride gas, and higher temperatures will catalyze side reactions, which will increase the amount of acidic reagents used. Therefore, this scheme adjusts the subsequent heating temperature to 0 °C to reduce the formation of by-products and save the amount of acidic gas used.

[0050] In some embodiments, the acidic gas is one of hydrochloric acid gas and hydrogen fluoride.

[0051] In some embodiments, the organic solvent is ethyl acetate.

[0052] In some embodiments, the acidic organic solvent is acidic methanol.

[0053] In some embodiments, in step S5, a liquid chromatograph is used for separation and purification, and a C18 reverse phase column is used as the chromatographic column.

[0054] Preferably, the parameters for separation and purification are: the mobile phase A of the medium pressure separation chromatography is 0.1% trifluoroacetic acid aqueous solution, the mobile phase B is 100% methanol solution, the flow rate is set to 30 mL / min, the monitoring wavelength in the program is 280 nm, the detection wavelength and the collection wavelength are 520 nm, and the instrument collection threshold is 900 mAU; the gradient program of the mobile phase setting is: 0-10 min, 6% B; 10-20 min, 6-15% B; 20-50 min, 15-35% B; 50-200 min, 35-42% B; 200-215 min, 42-44% B; 215-225 min, 44-81% B; 225-230, 81-100% B; 230-260 min, 100% B; 260-265 min, 100-50% B; 265-275 min, 50% B; 275~280 min, 50~6% B; 280~285 min, 6% B.

[0055] The present application provides an acetylated anthocyanin monomer obtained by the above-mentioned preparation method, wherein the acetylated anthocyanin monomer includes one of acylated malvidin-3-O-glucoside (M3AG) and acylated cyanidin-3-O-glucoside (C3AG).

[0056] The applicant further provides the following reference specific embodiments to describe the present invention. It should be noted that these embodiments are merely illustrative and do not limit the present invention in any way.

[0057] Example 1

[0058] This embodiment provides a method for preparing an acetylated anthocyanin monomer (C3AG), comprising the following steps:

[0059] Step S1, 6.57 mmol of 3,4-dihydroxyacetophenone was dissolved in 15 mL of DMF, 16.75 mmol of imidazole and 16.42 mmol of triisopropylsilyl chloride were added in sequence, and the mixture was stirred at room temperature for 2 h to obtain a colorless oily substance - 3,4-ditriisopropylsilylacetophenone (i.e., intermediate R1'=OH, R2'=H of formula (2)), with a yield of 99.1%;

[0060] Step S2, 10.75 mmol of the above intermediates were dissolved in 40 mL of methanol, and then 5.35 mmol of iodine, 6.45 mmol of 30% aqueous hydrogen peroxide solution and 1 mmol of concentrated sulfuric acid were added, and the mixture was stirred at 60°C for 3 h to obtain a yellow oil, α-iodo-3,4-ditriisopropylsilylacetophenone (i.e., intermediate R1'=OTIPS, R2'=H of formula (3)), with a yield of 76.05% (the subsequent separation, dilution and purification of the compound can refer to the synthesis steps of formula 3 shown in Example 1 in CN116410220A);

[0061] Step S3, dissolving 4.82 mmol of β-D-glucose-2,3,4,6-tetraacetate in 5 mL of dichloromethane, stirring and reacting at 0°C for 30 minutes to obtain a mixed solution 1; taking 7.24 mmol of α-iodo-3,4-ditriisopropylsilylacetophenone and α-iodo-3,5-dimethoxy-4-triisopropylsilylacetophenone, respectively, and dissolving them in 5 mL of dichloromethane to obtain a mixed solution 2; slowly adding the mixed solution 1 to the mixed solution 2, and after the addition is completed, slowly raising the reaction temperature to room temperature, stirring and reacting for 3 hours to obtain an unacylated intermediate of formula (4), which is a yellow oil (i.e., intermediate of formula (4) R1'=OTIPS, R2'=H), with a yield of 34.23% (the subsequent separation, dilution and purification of the compound can refer to the synthesis steps of formula 6 shown in Example 1 in CN116410220A);

[0062] Step S4, taking 4.85 mmol of the unacylated intermediate product and putting it into 14.55 mmol of n-tetrabutylammonium fluoride, and stirring the reaction at room temperature for 10 min to obtain a reaction mixture; the reaction mixture was treated with sodium sulfate to remove water, and then distilled under reduced pressure, dissolved in 20 mL of dichloromethane, and 14.55 mmol of acetyl chloride and 19.4 mmol of triethylamine were added in sequence, and stirred at 0°C for 2 h to obtain the intermediate of formula (5), α-O-tetraacetyl-β-D-glucoside oxy-3,4-ditriisopropylsilyl acetophenone (i.e., intermediate of formula (5) R1"=OAc, R2"=H), which was a white solid with a yield of 59.58 % (the subsequent separation, dilution and purification of the compound can refer to the synthesis steps of Formula 7 in Example 1 of CN116410220A, which differs from the above in that when the acetophenone analogue of Formula (1) is 3,4-dihydroxyacetophenone, the molar mass ratio of the intermediate of Formula (3) to β-D-glucose-2,3,4,6-tetraacetate is 1:1.5);

[0063] Step S5, 1.37 mmol of the intermediate of formula (5) and 1.64 mmol of compound 3 were dissolved in ethyl acetate at -20 °C and stirred to disperse, and then hydrogen chloride gas was introduced until saturated, and then the temperature was raised to 0 °C and stirred to react for 40 h. After the reaction was completed, 40 mL of acidic methanol (containing 0.1% HCl) was added for dilution and then distilled under reduced pressure. The separated product was separated and purified using the following C18 reverse phase column to obtain acetylated anthocyanin C3AG (i.e., intermediate of formula (6) R1" = OAc, R2" = H), which was a black-red solid with a yield of 47.44% (the subsequent separation, dilution and purification of the compound can refer to the synthesis steps of Example 1 Formula 10 in CN116410220A, which differs from it in that no alkalization operation was performed).

[0064] The preparation process of some intermediates is shown in Chinese patent CN116410220A, as shown below:

[0065] β-D-glucose pentaacetate (see CN116410220A, Example 1, Formula 4); β-D-glucose-2,3,4,6-tetraacetate (see CN116410220A, Example 1, Formula 5, synthesis steps); 2-hydroxy-4,6-diacetylbenzaldehyde preparation ( Figure 1 Compound 4): (see the synthesis steps of Formula 9 described in Example 1 of CN116410220A); and the following compounds are all separated, diluted and purified after synthesis, which is the same as the treatment method after synthesis of the compounds in CN116410220A.

[0066] The present application also provides a method for purifying acetylated anthocyanin monomers, comprising the following steps:

[0067] The crude products of C3AG and M3AG prepared above were purified by C18 medium pressure separation chromatography column, the mobile phase A of the medium pressure separation chromatography was 0.1% trifluoroacetic acid aqueous solution, the mobile phase B was 100% methanol solution, the flow rate was set to 30 mL / min, the monitoring wavelength in the program was 280 nm, the detection wavelength and the collection wavelength were 520 nm, and the instrument collection threshold was 900 mAU; the mobile phase gradient program was: 0-10 min, 6% B; 10-20 min, 6-15% B; 20-50 min, 15-35% B; 50-200 min, 35-42% B; 200-215 min, 42-44% B; 215-225 min, 44-81% B; 225-230, 81-100% B; 230-260min, 100% B; 260-265 min, 100-50% B; 265~275 min, 50% B; 275~280 min, 50~6% B; 280~285 min, 6% B.

[0068] Example 2

[0069] This embodiment provides a method for preparing an acetylated anthocyanin monomer (M3AG), comprising the following steps:

[0070] Step S1, 15.8 mmol of 4-hydroxy-3,5-dimethoxyacetophenone was dissolved in 30 mL of DMF, 20.06 mmol of imidazole and 20.54 mmol of triisopropylsilyl chloride were added, and the mixture was stirred at room temperature for 2 h to obtain a colorless oily substance 3,5-dimethoxy-4-triisopropylsilylacetophenone (i.e., intermediate R1'=R2'=OCH 3 ), the yield is 98.2%;

[0071] Step S2, 13.07 mmol of the above intermediate was dissolved in 40 mL of methanol, and then 6.54 mmol of iodine, 7.85 mmol of 30% aqueous hydrogen peroxide and 1.31 mmol of concentrated sulfuric acid were added, and the mixture was stirred at 60 °C for 3 h to obtain α-iodo-3,5-dimethoxy-4-triisopropylsilylacetophenone (i.e., intermediate R1'=R2'=OCH 3 ), the substance is a yellow oil, and the yield is 72.48% (the subsequent separation, dilution and purification of the compound can refer to the synthesis steps of Formula 3 shown in Example 1 in CN116410220A);

[0072] Step S3, dissolve 8.34 mmol of β-D-glucose-2,3,4,6-tetraacetate in 5 mL of dichloromethane, and stir the mixture for 30 minutes at 0°C to obtain a mixed solution 1; take 3.33 mmol of α-iodo-3,4-ditriisopropylsilylacetophenone and α-iodo-3,5-dimethoxy-4-triisopropylsilylacetophenone and dissolve them in 5 mL of dichloromethane to obtain a mixed solution 2; slowly add the mixed solution 1 to the mixed solution 2, and after the addition is completed, slowly raise the reaction temperature to room temperature, stir the mixture for 3 hours, and obtain the unacylated intermediate product (i.e., the intermediate of formula (4) R1'=R2'=OCH 3 ), the intermediate product is a yellow oil with a yield of 41.0% (the subsequent separation, dilution and purification of the compound can refer to the synthesis steps of Formula 6 shown in Example 1 in CN116410220A);

[0073] Step S4, taking 0.98 mmol of the unacylated intermediate product and putting it into 1.96 mmol of n-tetrabutylammonium fluoride, stirring and reacting at room temperature for 10 min to obtain a reaction mixture; treating the reaction mixture with sodium sulfate to remove water, distilling it under reduced pressure, dissolving it in 20 mL of dichloromethane, adding 2.94 mmol of acetyl chloride and 0.49 mmol of triethylamine in turn, stirring and reacting at 0°C for 2 h to obtain the intermediate of formula (5), α-O-tetraacetyl-β-D-glucosideoxy-3,5-dimethoxy-4-triisopropylsilylacetophenone (i.e., the intermediate of formula (5) R1"=R2"=OCH 3 ), the substance is a white solid with a yield of 83.6% (the subsequent separation, dilution and purification of the compound can refer to the synthesis steps of Example 1 Formula 7 in CN116410220A. When the acetophenone analogue of formula (1) is 4-hydroxy-3,5-dimethoxyacetophenone, the molar mass ratio of the intermediate of formula (3) to β-D-glucose-2,3,4,6-tetraacetate is 1:2.5);

[0074] Step S5, 1.99 mmol of the intermediate of formula (5) and 2.19 mmol of compound 3 were dissolved in ethyl acetate at -20 °C and stirred to disperse, and then hydrogen chloride gas was introduced until saturation, and then the temperature was raised to 0 °C and stirred to react for 40 h. After the reaction was completed, 40 mL of acidic methanol (containing 0.1% HCl) was added for dilution and then distilled under reduced pressure. The separated product was separated and purified using the following C18 reverse phase column to obtain acetylated anthocyanin M3AG (i.e., the intermediate of formula (6) R1"=R2"=OCH 3), the substance is a black-red solid with a yield of 12.5% ​​(the subsequent separation, dilution and purification of the compound can be found in the synthesis steps of Example 1 Formula 10 in CN116410220A, the difference being that no alkalization operation is performed).

[0075] Characterization 1: Purity analysis

[0076] The purity of the acetylated anthocyanins prepared in Example 1 was analyzed by HPLC. The parameters of HPLC liquid chromatography were set as follows: mobile phase A was 2% formic acid water, mobile phase B was 100% acetonitrile, flow rate was 1 mL / min, injection volume was 10 μL, column oven temperature was 30 °C, detection wavelengths were 254 nm, 280 nm, 500 nm, 520 nm. The elution program was: 0-30 min, 6-38% B; 30-35 min, 38-95% B; 35-40 min, 95% B; 40-41 min, 95-6% B; 41-44 min, 6% B. Electron spray ionization (ESI) source, interface temperature of 300 °C, interface voltage of 4.0 kV, heating block temperature of 400 °C, positive and negative ion scanning modes, scanning range of 100~1000 m / z: nitrogen was used as nebulizer gas, drying gas and heating gas, argon was used as collision gas, with nebulizer gas flow rate of 3.0 L / min, drying gas flow rate of 10.0 L / min, heating gas flow rate of 10.0 L / min, and collision chamber pressure of 230 kPa.

[0077] Characterization results: In the initial study, the classic cyclization reaction was used to synthesize anthocyanins: saturated dry HCl gas was introduced into EtOAc at -20 °C and the temperature was gradually raised to room temperature. The product formation was monitored by HPLC every 12 hours. Figure 1As shown, the C3AG product reached a peak at 36 h and the product peak was obviously observed to gradually decrease at 48 h, so it is speculated that the optimal reaction time for the cyclization reaction is between 36 and 48 h. In the reaction in which potassium hydroxide was subsequently added and reacted at room temperature for 1 h to remove the excess acetyl group (see Comparative Example 1), we observed that due to the introduction of a strong alkaline environment into the sample system, the ester bond of the acetyl group was susceptible to the attack of a strong nucleophilic reagent (OH-), resulting in a hydrolysis reaction, causing the acetyl groups on the glycoside hydroxyl group and the benzene ring to almost completely fall off, and the target monoacetylated anthocyanin could not be obtained. By exploring the reaction conditions, we found that neither shortening the alkalization reaction time nor reducing the KOH equivalent could promote the formation of acetylated anthocyanins, and heating from -20 °C to room temperature would cause the solubility of hydrogen chloride gas to decrease, and higher temperatures would catalyze side reactions, increase the amount of acidic reagents, and reduce the yield. In the present application, the alkalization step is deleted, the reaction temperature is adjusted, the monoacetylated, diacetylated and more acetylated anthocyanin derivatives produced in the cyclization reaction are retained, and these crude products are directly subjected to medium pressure preparative liquid phase separation. HPLC shows that in the separation process, not only C3AG with a purity of 98.53% is obtained, but also 99.12% of C3G entity is separated. When separating M3G and its acylated derivatives, similar results are obtained: M3AG with a purity of 95.28% and M3G entity with a purity of 97.02% are separated; at the same time, diacetylated and triacetylated anthocyanin derivatives with low purity are also obtained, which provides a reference for future research on polyacylated anthocyanin derivatives.

[0078] Representation 2: Positioning Representation

[0079] The structural information of the obtained acetylated anthocyanin monomer was positioned and characterized. The instrument information and parameter details involved are as follows: 1 H NMR, 13 C NMR, HHCOSY, and HMBC NMR spectra were recorded by Bruker AVANCE III (300 MHz), Bruker AVANCE ARX-400 (400 MHz), or Bruker AVANCE NEO 600 M. The purified anthocyanins were dissolved in CD3OD, and the chemical shifts were reported in parts per million (ppm). The residual solvent peaks were CD 3 OD=3.32.

[0080] The characterization results can be found in Figure 2 .in, Figure 2 (a) 1H NMR comparison of C3G and C3AG, with the red dashed box highlighting The signal,shows a clear downfield shift; Figure 2(b) 1H NMR spectra comparison of M3G and M3AG, highlighted by the red dashed box The signal,shows a clear downfield shift; Figure 2 (c) The chemical structure of C3AG and key 2D NMR correlation characterization diagrams; Figure 2 (d) is the chemical structure of M3AG and the key two-dimensional NMR correlation characterization diagram. By analyzing the NMR spectrum, the proton and carbon resonance of C3AG and M3AG can be determined. (δH 5.2 ppm) has a significant downfield shift, coupled with and The key HMBC correlation (δC 170.5 ppm) between Location( Figure 2 a and 2c); Similarly, M3AG, the acetate group is also attached to location, as evidenced by its spectral data ( Figure 2 b and 2d), in summary, it is shown that the preparation method provided in the present application performs acetylation-targeted modification at position 2 of the glucoside.

[0081] Characterization 3: Thermal stability test

[0082] C3G, M3G and acylated C3G and M3G were dissolved in a pH 3.0 buffer solution to prepare 400 μmol·L -1 The solution was incubated at 95 °C. The method of Zhang et al (Food Chemistry, 343) was referenced and optimized. The degradation rate was determined by monitoring the change of peak area over time using HPLC. The first-order degradation kinetics, half-life and degradation activation energy of C3G, M3G and their acylated forms were calculated using the following formula.

[0083] ln(C / C 0 ) = -k × t (1)

[0084] t 1 / 2 = -In(1 / 2) × k -1 (2)

[0085] Among them, C 0 is the initial concentration (based on peak area), C is the concentration at time (t) (based on peak area), k is the reaction rate constant (h -1 ), t 1 / 2 is the half-life, k 0 is the frequency factor (h -1 ).

[0086] Test results: During the heating process, both C3AG and M3AG underwent thermal degradation, and the color of the samples in pH=3 solution weakened and became colorless. Figure 3 The results show that the acetylated derivatives of C3G, M3G and their acetylated derivatives at 75 °C ( Figure 3 a) and 95 °C ( Figure 3 b) In (C / C 0 ) versus time, where C and C 0 Represent the relative concentration of the samples at different time points and the initial value, respectively, expressed based on the HPLC peak area. Table 1 summarizes the thermal degradation parameters of anthocyanins at 75 ℃ and 95 ℃. From the results, the thermal stability of these compounds decreased with time at high temperature, among which the acetyl-modified compounds (C3AG and M3AG) showed significantly higher thermal stability and longer half-life (t 1 / 2 ) and a lower degradation rate constant K. The half-lives of C3AG and M3AG were 31.63 hours and 31.41 hours, respectively, which were significantly longer than 25.57 hours and 14.04 hours of C3G and M3G. This indicates that acylated anthocyanins have better thermal stability than unmodified anthocyanins to a certain extent.

[0087] Table 1. Reaction rate constants and half-life values ​​of anthocyanins at different temperatures

[0088]

[0089] The above are only preferred embodiments of the present application and are not intended to limit the present invention. Although the present application has been described in detail with reference to the examples, those skilled in the art can still modify the technical solutions recorded in the aforementioned examples or make equivalent substitutions for some of the technical features therein. However, any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present application should be included in the protection scope of the present application.

Claims

1. A method for preparing acetylated anthocyanin monomers, characterized in that: The following steps are involved: Step S1, placing an acetophenone analogue of formula (1), imidazole and triisopropylsilyl chloride in DMF for mixed reaction to obtain an intermediate of formula (2); Step S2, mixing the intermediate of formula (2) and elemental iodine in an organic solvent for reaction to obtain the intermediate of formula (3); Step S3, mixing the intermediate of formula (3) and β-D-glucose-2,3,4,6-tetraacetate in an organic solvent for reaction to obtain the intermediate of formula (4); Step S4, subjecting the intermediate of formula (4) to acetylation reaction to obtain the intermediate of formula (5); Step S5, introducing an acidic gas under low temperature conditions, and mixing the intermediate of formula (5) and 2-hydroxy-4,6-diacetylbenzaldehyde in a solvent for reaction until the acidic gas reaches saturation, then heating the reaction, and the reaction is completed, followed by dilution with an acidic organic solvent, vacuum distillation, separation and purification, to obtain an acetylated anthocyanin monomer of formula (6); 。 2. The method for preparing acetylated anthocyanin monomer according to claim 1, characterized in that: The acetophenone analogue of formula (1) includes one of 3,4-dihydroxyacetophenone and 4-hydroxy-3,5-dimethoxyacetophenone.

3. The method for preparing acetylated anthocyanin monomer according to claim 2, characterized in that: When the acetophenone analogue of formula (1) is 3,4-dihydroxyacetophenone, the molar mass ratio of the intermediate of formula (3) to β-D-glucose-2,3,4,6-tetraacetate is 1:1.

5.

4. The method for preparing acetylated anthocyanin monomer according to claim 2, characterized in that: When the acetophenone analogue of formula (1) is 4-hydroxy-3,5-dimethoxyacetophenone, the molar mass ratio of the intermediate of formula (3) to β-D-glucose-2,3,4,6-tetraacetate is 1:2.

5.

5. The method for preparing acetylated anthocyanin monomer according to claim 1, characterized in that: In the step S5, the low temperature condition is set to -20°C; then the temperature is raised to 0°C, and the reaction is kept at 0°C for 40-50 hours.

6. The method for preparing acetylated anthocyanin monomer according to claim 1, characterized in that: The acidic gas is one of hydrochloric acid gas and hydrogen fluoride.

7. The method for preparing acetylated anthocyanin monomer according to claim 1, characterized in that: The acidic organic solvent is acidic methanol.

8. The method for preparing acetylated anthocyanin monomer according to claim 1, characterized in that: In step S5, a liquid chromatograph is used for separation and purification, and a C18 reverse phase column is used as the chromatographic column.

9. The method for preparing acetylated anthocyanin monomer according to claim 8, characterized in that: The separation and purification parameters are as follows: the mobile phase A of the medium pressure separation chromatography is 0.1% trifluoroacetic acid aqueous solution, the mobile phase B is 100% methanol solution, the flow rate is set to 30 mL / min, the monitoring wavelength in the program is 280 nm, the detection wavelength and the collection wavelength are 520 nm, and the instrument collection threshold is 900 mAU; The gradient program of the mobile phase was as follows: 0-10 min, 6% B; 10-20 min, 6-15% B; 20-50 min, 15-35% B; 50-200 min, 35-42% B; 200~215 min, 42~44% B; 215~225 min, 44~81% B; 225~230, 81~100% B; 230~260 min, 100% B; 260~265 min, 100~50% B; 265~275 min, 50% B; 275~280min, 50~6% B; 280~285 min, 6% B.

10. An acetylated anthocyanin monomer obtained by the preparation method according to any one of claims 1 to 9, characterized in that: The acetylated anthocyanin monomer includes one of acylated malvidin-3-O-glucoside and acylated cyanidin-3-O-glucoside.

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