CN111411137A

CN111411137A - Method for synthesizing 8-methoxycoumarin-3-carboxylic acid-6' -O-D-glucose ester on line under catalysis of lipase

Info

Publication number: CN111411137A
Application number: CN202010132118.5A
Authority: CN
Inventors: 罗锡平; 杜理华; 陈平峰
Original assignee: Zhejiang A&F University ZAFU
Current assignee: Zhejiang A&F University ZAFU
Priority date: 2020-02-29
Filing date: 2020-02-29
Publication date: 2020-07-14
Anticipated expiration: 2040-02-29
Also published as: CN111411137B

Abstract

A method for synthesizing 8-methoxycoumarin-3-carboxylic acid-6 '-O-D-glucose ester on line under lipase catalysis comprises the steps of uniformly filling L ipozyme RM IM in a reaction channel of a microfluidic channel reactor, dissolving 8-methoxycoumarin-3-carboxylic acid methyl ester and D-glucose respectively with reaction solvents, collecting the dissolved 8-methoxycoumarin-3-carboxylic acid methyl ester and the dissolved D-glucose respectively through a first injector and a second injector and injecting the dissolved 8-methoxycoumarin-3-carboxylic acid methyl ester and the dissolved D-glucose into a reaction channel through pipelines, reacting the mixture in the reaction channel for 10-60 min under the control of the reaction temperature of 30-60 ℃, collecting reaction liquid flowing out of the reaction channel on line through a product collector, and carrying out aftertreatment to obtain the 8-methoxycoumarin-3-carboxylic acid-6' -O-D-glucose ester (I).

Description

Method for synthesizing 8-methoxycoumarin-3-carboxylic acid-6' -O-D-glucose ester on line under catalysis of lipase

Technical Field

The invention relates to a method for synthesizing 8-methoxycoumarin-3-carboxylic acid-6' -O-D-glucose ester on line by lipase catalysis.

Background

Coumarin is a basic structural subunit of various plant secondary metabolites and microbial metabolites, and has a series of attractive biological activities, including antibacterial, anticoagulant, antiviral, antitubercular, antioxidant and antitumor activities. Several natural synthetic drugs containing coumarin scaffolds have been clinically applied, such as warfarin, which is used as an anticoagulant of vitamin K antagonist and has been widely applied in thrombus treatment, and the commonly used antibiotics, i.e., the armillarisin a and the novobiocin, both contain coumarin substructure units. In addition, due to its parent structure, coumarin has been widely used in the fields of specific fluorescent probes, dyes, fluorescence imaging, and the like. Among them, the synthesis of coumarin derivatives containing sugar branches has attracted considerable attention in organic and medical research and development. The sugar-containing coumarins are interesting in that these compounds have good water solubility. These results improve the physicochemical, biopharmaceutical and pharmacokinetic properties of the drug. Studies by solson et al showed that glycosylation of warfarin caused a shift from anticoagulant to anticancer activity, and they demonstrated that glycosylation of warfarin could show 70-fold higher anticancer activity than the original compound. This study clearly demonstrated that additional sugars are critical to alter the mechanism of action and efficacy of the coumarin parent drug. Over the past few years, several studies on sugar-containing coumarins have been reported. A series of glycosyl coumarin carbonic anhydrase IX and XII inhibitors are synthesized by Spilant et al, and have strong inhibitory effect on the growth of primary breast tumors. In 2016, Nilsson et al reported a selective galactocoumarin derivative galectin-3 inhibitor that showed similar effects to known non-selective galectin-1/galectin-3 inhibitors in a mouse model of bleomycin-induced pulmonary fibrosis.

The construction of sugar-containing derivatives can be achieved by basic synthetic methods, the most common synthetic strategy being chemical methods. By "protection" or "protection deprotection" steps, specific reactive hydroxyl groups on the sugar are selectively synthesized. Visible light has also been recently reported as a glycosylation catalyst, however, most of the schemes for photoinduced glycosylation require transition metal catalysts in combination with expensive additives or oxidizing agents to react. Thus, the introduction of sugars chemically is still limited by the disadvantages of poor regio-and stereoselectivity, lengthy functional group protection and deprotection.

Some enzymes, such as engineered C-glycosyltransferase (micgtb-gag), have been applied to the synthesis of coumarin C-glycosides, wherein the two synthesized C-glucosides have strong SG L T2 inhibitory activity.

Modern synthetic chemistry faces the challenge of providing high performance, environmentally friendly, low cost, safe and atom efficient valuable products to society. In this regard, continuous-flow microreactor technology (MRT) is becoming increasingly popular as an alternative to traditional batch chemical synthesis. In particular, MRT may play a major role in improving chemical processes in terms of the 12-item principle of green chemistry. The high surface to volume ratio of the microreactor means leads to better heat exchange and efficient mixing, thereby increasing the efficiency of the reaction. In addition, MRT systems included a scale of reaction at the beginning of science, allowing efficient on-demand generation of compounds in compact, reconfigurable equipment. In this case, "scale out" or "numbering up" refers to a continuous flow system array operating in parallel to meet the desired output. Flow chemistry, particularly catalyst/substrate conditions in continuous flow systems, can improve reactivity and selectivity. Meanwhile, when a heterogeneous catalyst is filled in the continuous flow column, the separation of the catalyst and the product is very easy. In order to explore a new, eco-friendly and efficient scheme of sugar-containing coumarin and serve as a part of the ongoing development and research of novel sugar-containing drugs, the research finds a method for synthesizing 8-methoxycoumarin-3-carboxylic acid-6 '-O-D-glucose ester on line under catalysis of lipase in a microchannel reactor, and aims to find an efficient and environment-friendly method for synthesizing 8-methoxycoumarin-3-carboxylic acid-6' -O-D-glucose ester on line in a controllable and selective manner.

Disclosure of Invention

The invention aims to provide a novel process method for synthesizing 8-methoxycoumarin-3-carboxylic acid-6' -O-D-glucose ester on line by lipase catalysis in a microfluidic channel reactor, and the method has the advantages of short reaction time, high yield and good selectivity.

The technical scheme of the invention is as follows:

a method for synthesizing 8-methoxycoumarin-3-carboxylic acid-6' -O-D-glucose ester on line under catalysis of lipase comprises the following steps:

uniformly filling L ipozyme RM IM (catalyst) in a reaction channel of a microfluidic channel reactor, dissolving 8-methoxycoumarin-3-carboxylic acid methyl ester and D-glucose with reaction solvents respectively, then injecting the dissolved methyl ester and D-glucose into a pipeline through a first injector and a second injector respectively for gathering, then allowing the mixture to enter the reaction channel for reaction, controlling the reaction temperature to be 30-60 ℃ (preferably 35 ℃), controlling the continuous flow reaction time of the mixed solution in the reaction channel to be 10-60 min (preferably 40min), collecting the reaction solution flowing out of the reaction channel on line through a product collector, and performing post-treatment to obtain a product 8-methoxycoumarin-3-carboxylic acid-6' -O-D-glucose ester (I);

the reaction solvent is a mixed solvent of dimethyl sulfoxide and tertiary amyl alcohol, wherein the volume ratio of the dimethyl sulfoxide to the tertiary amyl alcohol is 1: 8-20, preferably 1: 18;

in the mixed liquid entering the reaction channel, the mass ratio of the 8-methoxycoumarin-3-carboxylic acid methyl ester to the D-glucose is 1: 0.2 to 3, preferably 1: 0.25, the specific operation can be as follows: after the 8-methoxycoumarin-3-carboxylic acid methyl ester and the D-glucose are respectively dissolved by using a reaction solvent, the mass concentration ratio of the obtained 8-methoxycoumarin-3-carboxylic acid methyl ester solution to the D-glucose solution is 1: 0.2 to 3, preferably 1: 0.25; when the 8-methoxycoumarin-3-carboxylic acid methyl ester solution and the D-glucose solution are injected through the first injector and the second injector respectively, the flow rates are the same;

the lipase L ipozyme RM IM is commercially available, for example, from Novozymes (Novozymes), a 1, 3-position specific, food grade lipase (EC 3.1.1.3) on granular silica gel preparation prepared from microorganisms, produced by submerged fermentation using a genetically modified Aspergillus oryzae (Aspergillus oryzae) microorganism, obtained from Rhizomucor miehei, the lipase L ipozyme RM IM can physically fix the catalyst in the reaction channel uniformly, the catalyst is added in an amount of 0.025 to 0.05g/m L based on the volume of the reaction medium to the maximum extent that the reaction channel can accommodate the catalyst;

the post-treatment method comprises the steps of distilling the obtained reaction liquid under reduced pressure to remove the solvent, carrying out silica gel column chromatography separation, carrying out wet column packing by using 200-mesh 300-mesh silica gel, taking a mixed liquid with the volume ratio of dichloromethane to methanol being 10:1.5 as an eluent, tracking the elution process by T L C, collecting the eluent containing the target compound, distilling off the solvent and drying to obtain a product of 8-methoxycoumarin-3-carboxylic acid-6' -O-D-glucose ester (I);

the synthesis method adopts a microfluidic channel reactor, and the microfluidic channel reactor comprises the following steps: the device comprises a first injector, a second injector, a reaction channel and a product collector; the first injector and the second injector are connected with the inlet of the reaction channel through a Y-shaped or T-shaped pipeline, and the product collector is connected with the outlet of the reaction channel through a pipeline;

further, in the present invention,

the inner diameter of the reaction channel is 0.8-2.4 mm, and the length of the reaction channel is 0.5-1.0 m;

the first injector and the second injector are arranged in the injection pump and are synchronously pushed by the injection pump, and the specifications of the first injector and the second injector are consistent;

the microfluidic channel reactor also comprises a thermostat, the reaction channel is arranged in the thermostat so as to effectively control the reaction temperature, and the thermostat can be selected automatically according to the reaction temperature requirement, such as a water bath thermostat and the like;

the material of the reaction channel is not limited, and green and environment-friendly materials such as a silicone tube are recommended; the shape of the reaction channel is preferably curved, so that the reaction liquid can be ensured to stably pass through at a constant speed.

Compared with the prior art, the invention has the beneficial effects that:

the method for synthesizing the 8-methoxycoumarin-3-carboxylic acid-6' -O-D-glucose ester on line in the microfluidic channel reactor by using lipase catalysis not only greatly shortens the reaction time, but also has high conversion rate and selectivity, and simultaneously catalyzes the reaction of the 8-methoxycoumarin-3-carboxylic acid methyl ester and D-glucose by using the L ipozyme RM IM of the economic lipase for the first time, so that the reaction cost is reduced, and the method has the advantages of economy and high efficiency.

Drawings

Fig. 1 is a schematic structural diagram of a microfluidic channel reactor used in an embodiment of the present invention.

In the figure, 1-first injector, 2-second injector, 3-reaction channel, 4-product collector, 5-water bath incubator.

Detailed Description

The invention is further illustrated by the following specific examples, without limiting the scope of the invention thereto:

referring to fig. 1, a microfluidic channel reactor according to an embodiment of the present invention includes a syringe pump, a first syringe 1, a second syringe 2, a reaction channel 3, a water bath incubator 5 (only a schematic plan view thereof is shown), and a product collector 4; first syringe 1 and second syringe 2 are installed in the syringe pump, through a Y type interface and 3 entry linkage of reaction channel, reaction channel 3 is arranged in water bath thermostated container 5, through 5 control reaction temperatures of water bath thermostated container, 2.0mm of internal diameter of reaction channel 3, the pipe length 1.0m, 3 exports of reaction channel are connected with product collector 4 through an interface.

Example 1: synthesis of 8-methoxycoumarin-3-carboxylic acid-6' -O-D-glucose ester

Referring to the device in FIG. 1, 8-methoxycoumarin-3-carboxylic acid methyl ester (2.0mmol) is dissolved in 0.52m L dimethyl sulfoxide and 9.48m L tert-amyl alcohol, and D-glucose (0.5mmol) is dissolved in 0.52m L dimethyl sulfoxide and 9.48m L tert-amyl alcohol, and then the solution is respectively filled in 10m L syringes for standby application, 0.87g lipase L ipozyme RM IM is uniformly filled in a reaction channel, and two reaction solutions are respectively 7.8 mu L min under the push of a PHD 2000 syringe pump^-1The flow rate of the reaction solution enters a reaction channel through a Y joint for reaction, the temperature of the reactor is controlled at 35 ℃ through a water bath thermostat, the reaction solution continuously flows in the reaction channel for reaction for 40min, and the reaction result is tracked and detected through thin-layer chromatography T L C.

Collecting reaction liquid on line through a product collector, distilling under reduced pressure to remove a solvent, filling the reaction liquid into a column by using a 200-mesh 300-mesh silica gel wet method, dissolving a sample in a small amount of an elution reagent, namely dichloromethane and methanol at a ratio of 10:1.5, wherein the column height is 35cm, the column diameter is 4.5cm, filling the sample into the column by using the wet method, and collecting the eluent at a flow rate of 2m L & min^-1And simultaneously, T L C tracks the elution process, the obtained eluents containing single products are merged and evaporated to dryness to obtain a light yellow solid, so that 8-methoxycoumarin-3-carboxylic acid-6 '-O-D-glucose ester is obtained, the conversion rate of the 8-methoxycoumarin-3-carboxylic acid-6' -O-D-glucose ester is 63 percent by HP L C detection, and the selectivity is 99 percent.

The nuclear magnetic characterization results were as follows:

¹H NMR(DMSO-d₆,500MHz,,ppm)8.67(s,0.5H,H-4ofβ-D-glucose),8.65(s,0.5H,H-4ofα-D-glucose),7.45-7.39(m,2H,H-5,H-7),7.33(t,J＝7.9Hz,1H,H-6),6.72(s,0.5H,C1′-OH ofβ-D-glucose),6.39(s,0.5H,C1′-OH ofα-D-glucose)5.35-4.78(m,3H,C1′-H of D-glucose,C4′-OH ofD-glucose,C2′-OH ofβ-D-glucose),4.70-4.47(m,1.5H,C3′-OH ofα-D-glucose,C6′-Ha of D-glucose),4.36(d,J＝7.7Hz,0.5H,C3′-OH ofβ-D-glucose),4.30(td,J＝12.1,6.2Hz,1H,C6′-Hb of D-glucose),3.92(s,3H,H-12),3.91(s,0.5H,C5′-H ofα-D-glucose),3.51-3.47(m,1H,C5′-H ofβ-D-glucose,C3′-Hofα-D-glucose),3.24-3.18(m,2H,C2′-H of D-glucose,C3′-H ofβ-D-glucose,C4′-H ofα-D-glucose),2.97(t,J＝8.2Hz,0.5H,C4′-H ofβ-D-glucose).¹³C NMR(126MHz,DMSO)162.53,162.49(C-11),155.67(C-2),148.93,148.81(C-8),146.26(C-9),143.92(C-4),124.84(C-6),121.16(C-5),118.23(C-3),117.79,117.72(C-7),116.51(C-10),97.02(C1′ofβ-D-glucose),92.41(C1′ofα-D-glucose),76.46(C3′ofβ-D-glucose),74.72(C2′ofβ-D-glucose),73.50(C5′ofβ-D-glucose),72.91(C3′ofα-D-glucose),72.18(C2′ofα-D-glucose),70.59(C4′ofα-D-glucose),70.19(C4′ofβ-D-glucose),69.21(C5′ofα-D-glucose),65.26(C6′of D-glucose),56.21,56.17(C-12).

examples 2 to 8

The volume ratio of the reaction medium DMSO to the tertiary amyl alcohol in the microfluidic channel reactor is changed, the substrate ratio of the 8-methoxycoumarin-3-carboxylic acid methyl ester to the D-glucose is 2:1(1.0 mmol: 0.5mmol), the temperature is controlled to be 50 ℃, the reaction time is 30min, and the reaction results are shown in the table 1 as other examples:

TABLE 1 influence of the volume ratio of DMSO to tert-amyl alcohol in the reaction Medium on the reaction

Examples	DMSO, DMSO: tert-amyl alcohol	Conversion [% ]]	Selectivity [% ]]
				2	1:8	n.d.	/
3	1:10	15％	99％
				4	1:12	26％	99％
5	1:14	34％	99％
				6	1:16	39％	99％
7	1:18	44％	99％
				8	1:20	42％	99％

The results in Table 1 show that when the substrate molar ratio of the reactant methyl 8-methoxycoumarin-3-carboxylate to D-glucose is 2:1, the flow rate is 10.4. mu. L. multidot.min^-1On the contraryThe reaction time is 30min, the reaction temperature is 50 ℃, the conversion rate of the reaction is increased along with the increase of the volume ratio of the tertiary amyl alcohol in the reaction medium, the conversion rate of the reaction is optimal when the volume ratio of the DMSO in the reaction medium to the tertiary amyl alcohol is 1:18, and at the moment, if the volume ratio of the tertiary amyl alcohol is continuously increased, the dissolving amount of the sugar in the reaction medium is reduced, so that the conversion rate of the reaction is reduced. Therefore, the optimal volume ratio of the reaction medium in the microfluidic microchannel reactor is DMSO to tert-amyl alcohol is 1: 18.

Examples 9 to 15

The substrate molar ratio of 8-methoxycoumarin-3-carboxylic acid methyl ester to D-glucose in the microfluidic microchannel reactor is changed, the temperature of the reactor is controlled at 50 ℃, the reaction time is controlled at 30min, the method is otherwise the same as in example 1, and the results are shown in Table 2:

TABLE 2 influence of the ratio of the amounts of methyl 8-methoxycoumarin-3-carboxylate and D-glucose substrate material on the reaction

The results in Table 2 show that the volume ratio of DMSO to t-amyl alcohol in the reaction medium is 1:18, and the flow rate is 10.4. mu. L. min^-1When the reaction time is 30min, the reaction temperature is 50 ℃, the conversion rate of the reaction is increased along with the increase of the reactant 8-methoxycoumarin-3-carboxylic acid methyl ester, and when the substrate ratio is 4:1, the conversion rate of the reaction is optimal, and at the moment, if the dosage of the reactant 8-methoxycoumarin-3-carboxylic acid methyl ester is continuously increased, the conversion rate of the reaction is reduced. Thus, the optimal substrate molar ratio of the reaction in the microfluidic microchannel reactor of the invention is 8-methoxycoumarin-3-carboxylic acid methyl ester to D-glucose ═ 4: 1.

Examples 16 to 22

The temperature of the microfluidic channel reactor was changed, the reaction time was controlled to 30min, and the reaction results are shown in table 3 as in example 1:

table 3: influence of temperature on the reaction

Examples	Temperature [ deg.C ]]	Conversion [% ]]	Selectivity [% ]]
				16	30℃	55％	98％
17	35℃	59％	99％
				18	40℃	57％	99％
19	45℃	53％	98％
				20	50℃	50％	97％
21	55℃	46％	94％
				22	60℃	42％	90％

The results in Table 3 show that when the volume ratio of DMSO to t-amyl alcohol in the reaction medium is 1:18, the substrate molar ratio of methyl 8-methoxycoumarin-3-carboxylate to D-glucose is 4:1, and the flow rate is 10.4. mu. L. min^-1When the reaction time is 30min, the conversion rate of the reaction is optimal when the reaction temperature is 35 ℃, and the enzyme activity is influenced by the temperature which is too high or too low. Therefore, the optimal reaction temperature for the reaction in the microfluidic microchannel reactor of the invention is 35 ℃.

Examples 23 to 27

The reaction time of the microfluidic channel reactor was changed, and the reaction results are shown in Table 4 as in example 1:

table 4: influence of reaction time on the reaction

Examples	Time [ min ]]	Conversion [% ]]	Selectivity [% ]]
				23	10	41％	99％
24	20	51％	99％
				25	30	59％	99％
1	40	63％	99％
				26	50	61％	98％
27	60	57％	94％

The results in Table 4 show that the flow rate is 7.8. mu. L. min when the reaction medium DMSO/t-amyl alcohol volume ratio is 1:18, the substrate molar ratio of 8-methoxycoumarin-3-carboxylic acid methyl ester to D-glucose is 4:1, and the reaction temperature is 35 ℃^-1When the reaction time is up to 40min, the reaction conversion rate can reach 63%, and at this time, if the reaction time is continued to be prolonged, the reaction conversion rate is reduced. Thus, the optimal reaction time for the reaction in the microfluidic channel reactor was 40 min.

Comparative examples 1 to 3

The results are shown in Table 5, except that the catalysts in the microfluidic microchannel reactor were changed to porcine pancreatic lipase PP L (comparative example 1), lipase Novozym435 (comparative example 2), and subtilisin (comparative example 3), respectively, and the other examples are the same as those in example 1.

Table 5: effect of different enzymes on reaction conversion and selectivity

Comparative example	Enzyme source	Conversion [% ]]	Selectivity [% ]]
				1	PPL	18％	64％
2	Novozym 435	33％	73％
				3	Bacillus subtilis alkaline protease	0	0
Example 1	Lipozyme RM IM	63％	99％

The results in Table 5 show that different enzymes have a very significant effect on the reaction for the regioselective transesterification of the enzymatic 8-methoxycoumarin-3-carboxylic acid methyl ester with D-glucose in the microfluidic reactor the conversion rate was 18% with porcine pancreatic lipase PP L, 0% with Bacillus subtilis alkaline protease and 33% with Novozym 435. from the results in Table 5, the most efficient catalyst for the regioselective transesterification of the enzymatic 8-methoxycoumarin-3-carboxylic acid methyl ester with D-glucose in the microfluidic reactor was the lipase L ipozyme RM, with a conversion rate of 63% and a selectivity of 99%.

Comparative examples 4 to 6

The reaction raw materials in the microfluidic microchannel reactor were changed, the acyl donor was changed to 8-methoxycoumarin-3-carboxylic acid tert-butyl ester (comparative example 4), the acyl acceptor was changed to D-galactose (comparative example 5), the acyl donor and the acyl acceptor were changed simultaneously (comparative example 6), and the results are shown in table 6 in the same manner as in example 1.

TABLE 6 Effect of different enzymes on reaction conversion and selectivity

Comparative example	Acyl donors	Acyl acceptors	Conversion [% ]]	Selectivity [% ]]
					4	8-Methoxycoumarin-3-carboxylic acid tert-butyl ester	D-glucose	13％	73％
5	8-Methoxycoumarin-3-carboxylic acid methyl ester	D-galactose	15％	58％
					6	8-Methoxycoumarin-3-carboxylic acid tert-butyl ester	D-galactose	0	0
Example 1	8-Methoxycoumarin-3-carboxylic acid methyl ester	D-glucose	63％	99％

The results in table 6 show that for the regioselective transesterification synthesis of the enzymatic 8-methoxycoumarin-3-carboxylic acid sugar ester in a microfluidic reactor, different substrates have a very significant effect on the reaction. 8-methoxy coumarin-3-carboxylic acid tert-butyl ester is used as an acyl donor, D-glucose is used as an acyl acceptor, and the conversion rate is 13%; 8-methoxy coumarin-3-methyl carboxylate is used as an acyl donor, D-galactose is used as an acyl acceptor, and the conversion rate is 15%; 8-methoxy coumarin-3-carboxylic acid tert-butyl ester is used as an acyl donor, D-galactose is used as an acyl acceptor, and the conversion rate is 0%. From the results in Table 6, it can be seen that 8-methoxycoumarin-3-carboxylic acid tert-butyl ester is not an effective acyl donor and D-galactose is not an effective acyl acceptor for the regioselective transesterification synthesis of the enzymatic 8-methoxycoumarin-3-carboxylic acid sugar ester in the microfluidic reactor.

Application example 1

The size of the inhibition zone of 8-methoxycoumarin-3-carboxylic acid-6' -O-D-glucose ester on staphylococcus aureus is determined by Oxford cup method, and 100 mu L of test bacterial liquid (the concentration of the bacterial liquid is 1 × 10) is added on a nutrient agar plate⁷CFU/m L), uniformly coating the bacterial liquid by using an aseptic coater, placing 3 Oxford cups at equal intervals on the surface of the culture medium, slightly pressing to contact the Oxford cups with the culture medium, adding 8-methoxycoumarin-3-carboxylic acid-6' -O-D-glucose ester 200 mu L (the concentration is 1g/m L) into the Oxford cups, repeating the steps for 3 times, placing the Oxford cups in a water-proof constant temperature incubator at the temperature of 28 ℃ for culturing for 24 hours, and observing the result.

And (4) judging a result: taking a bacterial growth area which is not visible to naked eyes around the oxford cup as a bacteriostatic circle, measuring the diameter of the bacteriostatic circle by using a ruler, and taking the average value of 3 measurement results as the size of the bacteriostatic circle of the compound on staphylococcus aureus. The diameter of the inhibition zone is expressed by d, and when d is less than 10mm, the inhibition zone is drug resistance (R); when d is more than or equal to 10 and less than or equal to 15, the sensitivity is moderate (I); and when d >15mm, is highly sensitive (S).

TABLE 78 in vitro bacteriostatic test of methoxycoumarin-3-carboxylic acid-6' -O-D-glucose ester against Staphylococcus aureus

Application example	Compound (I)	Average zone diameter/mm	Sensitivity of the device
				1	8-Methoxycoumarin-3-carboxylic acid-6' -O-D-glucose ester	22	S

Table 7 shows that 8-methoxycoumarin-3-carboxylic acid-6' -O-D-glucose ester has good inhibitory action on Staphylococcus aureus, and can be used as an inhibitor of Staphylococcus aureus.

Claims

1. A method for synthesizing 8-methoxycoumarin-3-carboxylic acid-6' -O-D-glucose ester on line by lipase catalysis is characterized by comprising the following steps:

uniformly filling L ipozyme RM IM in a reaction channel of a microfluidic channel reactor, dissolving 8-methoxycoumarin-3-carboxylic acid methyl ester and D-glucose by using reaction solvents respectively, injecting the dissolved methyl ester and D-glucose into a pipeline through a first injector and a second injector respectively for gathering, allowing the mixture to enter the reaction channel for reaction, controlling the reaction temperature to be 30-60 ℃, allowing the mixed solution to continuously flow in the reaction channel for 10-60 min, collecting the reaction solution flowing out of the reaction channel on line through a product collector, and performing post-treatment to obtain a product 8-methoxycoumarin-3-carboxylic acid-6' -O-D-glucose ester (I);

the reaction solvent is a mixed solvent of dimethyl sulfoxide and tertiary amyl alcohol, wherein the volume ratio of the dimethyl sulfoxide to the tertiary amyl alcohol is 1: 8-20;

in the mixed liquid entering the reaction channel, the mass ratio of the 8-methoxycoumarin-3-carboxylic acid methyl ester to the D-glucose is 1: 0.2 to 3;

2. the method for the on-line synthesis of 8-methoxycoumarin-3-carboxylic acid-6' -O-D-glucose ester catalyzed by lipase as claimed in claim 1, wherein the mass concentration ratio of the obtained 8-methoxycoumarin-3-carboxylic acid methyl ester solution to the D-glucose solution is 1: 0.2 to 3; and when the 8-methoxycoumarin-3-carboxylic acid methyl ester solution and the D-glucose solution are injected through the first injector and the second injector respectively, the flow rates are the same.

3. The method for the on-line synthesis of 8-methoxycoumarin-3-carboxylic acid-6' -O-D-glucose ester under the catalysis of lipase as claimed in claim 1, wherein the amount of the catalyst lipase L ipozyme RM IM added is 0.025-0.05 g/m L based on the volume of the reaction medium.

4. The method for the on-line synthesis of 8-methoxycoumarin-3-carboxylic acid-6 '-O-D-glucose ester under the catalysis of lipase as claimed in claim 1, wherein the post-treatment comprises the steps of distilling the obtained reaction solution under reduced pressure to remove the solvent, carrying out silica gel column chromatography, loading the reaction solution into a 200-mesh 300-mesh silica gel wet method, using a mixed solution of dichloromethane and methanol in a volume ratio of 10:1.5 as an eluent, tracking the elution process by T L C, collecting the eluent containing the target compound, evaporating the solvent and drying to obtain the product 8-methoxycoumarin-3-carboxylic acid-6' -O-D-glucose ester (I).