CN111455005B - Method for synthesizing coumarin-3-carboxylic acid-6' -O-D-sucrose ester derivative on line enzymatically based on flow chemistry - Google Patents
Method for synthesizing coumarin-3-carboxylic acid-6' -O-D-sucrose ester derivative on line enzymatically based on flow chemistry Download PDFInfo
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Abstract
A method for synthesizing coumarin-3-carboxylic acid-6' -O-D-sucrose ester derivatives based on flow chemistry online enzymatic: uniformly filling lipase RM IM in a reaction channel of a microfluidic channel reactor, dissolving coumarin-3-carboxylic acid methyl ester derivatives and D-sucrose respectively by using a reaction solvent, respectively injecting the mixture into a pipeline through a first injector and a second injector for integration, then entering the reaction channel for reaction, controlling the reaction temperature to be 30-60 ℃, continuously flowing mixed solution 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 aftertreatment to obtain the coumarin-3-carboxylic acid-6' -O-D-sucrose ester derivatives; the method has the advantages of short reaction time, high yield and good selectivity.
Description
Technical Field
The invention relates to a method for synthesizing coumarin-3-carboxylic acid-6' -O-D-sucrose ester derivatives based on flow chemistry on-line enzymatic.
Background
Coumarin is a basic structural subunit of a variety of plant secondary and microbial metabolites, and has a range of attractive biological activities including antibacterial, anticoagulant, antiviral, antitubercular, antioxidant, and antitumor activities. Several natural synthetic drugs containing coumarin scaffolds, such as warfarin as an anticoagulant for vitamin K antagonists, have been used clinically and widely in thrombosis treatment, and the common antibiotics, armillaria mellea a and novobiocin, both contain coumarin subunits. In addition, due to the parent structure of coumarin, it is widely used in the fields of specific fluorescent probes, dyes, fluorescent imaging, and the like. Among them, the synthesis of coumarin derivatives containing saccharide branches has attracted considerable attention in organic and pharmaceutical research and development. The interesting point of the coumarin containing saccharides is that these compounds have good water solubility. These results can improve the physicochemical, biopharmaceutical and pharmacokinetic properties of the drug. The study by sorsen 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 demonstrates that additional sugars are critical to altering the mechanism of action and potency of coumarin parent drugs. Over the past several years, several studies have been reported on coumarin containing sugars. Su Pulan et al synthesized a series of glycosyl coumarin carbonic anhydrase IX and XII inhibitors, which have strong inhibition effect on the growth of primary breast tumors. In 2016, nilsson et al reported a selective galectin-3 inhibitor, a coumarin derivative, which showed similar effects to the known non-selective galectin-1/galectin-3 inhibitor 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. The specific active hydroxyl groups on the saccharides are selectively synthesized by a "protection" or "protection deprotection" step. Visible light has also recently been reported as a glycosylation catalyst, however, most schemes for light-induced glycosylation require a transition metal catalyst in combination with expensive additives or oxidants to perform the reaction. Thus, the introduction of sugar chemically is still limited by the disadvantages of poor regio-and stereoselectivity, lengthy protection and deprotection of functional groups.
Biocatalysts have attracted considerable attention from chemists and biochemists as an efficient green bioconversion tool in organic synthesis. Particularly, the catalytic compounding in biocatalysis, namely, the old enzyme is utilized to form a new bond and follow a new path, so that the method has extremely and rapidly expanded. Some enzymes, such as engineered C-glycosyltransferase (micgtb-gagm), have been used in the synthesis of coumarin C-glycosides, where both C-glucosides synthesized have strong SGLT2 inhibitory activity. The enzyme-catalyzed reaction is relatively mild, green, but requires a long reaction time (typically as long as 24 hours or more) to achieve the desired result, and some rely on expensive enzymes. In recent years, continuous flow microreactors have become an effective way to shorten reaction time and increase yield by enzyme coupling.
Modern synthetic chemistry faces challenges in providing society with high performance, environmental protection, low cost, safety, and atomic efficiency valuable products. In this regard, continuous flow microreactor technology (MRT) is becoming increasingly popular as a replacement for traditional batch chemical synthesis. In particular, with respect to the 12-element principle of green chemistry, MRT can play a major role in improving chemical processes. The high surface to volume ratio of the microreactor means results in better heat exchange and efficient mixing, thus increasing the reaction efficiency. Furthermore, MRT systems have at the beginning of science involved reaction scales that allow efficient on-demand production of compounds in compact, reconfigurable equipment. In this case, "outwardly expanded" or "upwardly numbered" 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. At the same time, when the continuous flow column is filled with heterogeneous catalyst, separation of catalyst and product is very easy. In order to explore new, eco-friendly and efficient schemes for sugar-containing coumarins, and as part of our ongoing research on the development of new sugar-containing drugs, we have found a method for lipase-catalyzed on-line synthesis of 6-methylcoumarin-3-carboxylic acid-6 '-O-D-sucrose esters in microchannel reactors, aiming at finding an efficient and environmentally friendly on-line controllable selective synthesis method for 6-methylcoumarin-3-carboxylic acid-6' -O-D-sucrose esters.
Disclosure of Invention
The invention aims to provide a novel process method for synthesizing coumarin-3-carboxylic acid-6' -O-D-sucrose ester derivatives on line by lipase catalysis in a microfluidic channel reactor, which has the advantages of short reaction time, high yield and good selectivity.
The technical scheme of the invention is as follows:
a method for the on-line enzymatic synthesis of coumarin-3-carboxylic acid-6' -O-D-sucrose ester derivatives based on flow chemistry, which comprises the steps of:
uniformly filling lipase RM IM (catalyst) into a reaction channel of a microfluidic channel reactor, dissolving coumarin-3-carboxylic acid methyl ester derivative and D-sucrose respectively by using a reaction solvent, respectively injecting the mixture into a pipeline through a first injector and a second injector for integration, then entering the reaction channel for reaction, controlling the reaction temperature to be 30-60 ℃ (preferably 35 ℃), controlling the continuous flowing reaction time of mixed solution in the reaction channel to be 10-60 min (preferably 40 min), collecting the reaction solution flowing out of the reaction channel on line through a product collector, and performing aftertreatment to obtain a product coumarin-3-carboxylic acid-6' -O-D-sucrose ester derivative (I);
the reaction solvent is a mixed solvent of dimethyl sulfoxide and tertiary amyl alcohol, wherein the volume ratio of dimethyl sulfoxide to tertiary amyl alcohol is 1:8 to 20, preferably 1:18;
the ratio of the mass of coumarin-3-carboxylic acid methyl ester derivative to D-sucrose in the mixed solution entering the reaction channel is 1:0.2 to 3, preferably 1:0.25, the specific operation can be as follows: after the coumarin-3-carboxylic acid methyl ester derivative and the D-sucrose are respectively dissolved by using a reaction solvent, the mass concentration ratio of substances of the coumarin-3-carboxylic acid methyl ester derivative solution and the D-sucrose solution is 1:0.2 to 3, preferably 1:0.25; the coumarin-3-carboxylic acid methyl ester derivative solution and the D-sucrose solution are respectively injected by a first injector and a second injector, and the flow rates are the same;
the lipase RM IM is commercially available, for example from Novozymes corporation, which is a preparation of 1, 3-position specific, food grade lipase (EC 3.1.1.3) on granular silica gel prepared from microorganisms produced by submerged fermentation with a genetically modified Aspergillus oryzae (Aspergillus oryzae) microorganism, obtained from Rhizomucor miehei; the lipase RM IM can be obtained by directly and uniformly fixing a granular catalyst in a reaction channel through a physical method; the catalyst is added in an amount of 0.025-0.05 g/mL based on the volume of the reaction medium within the maximum limit of the reaction channel capable of accommodating the filled catalyst;
the post-treatment method comprises the following steps: the solvent was removed by distillation under reduced pressure from the reaction mixture, and the mixture was subjected to column chromatography on a silica gel column, and the column was packed with 200-300 mesh silica gel by wet method, with the volume ratio of dichloromethane and methanol=10: 1.5, collecting the eluent containing the target compound by TLC tracking the elution process, evaporating the solvent and drying to obtain the product coumarin-3-carboxylic acid-6' -O-D-sucrose ester derivative (I);
the synthesis method adopts a microfluidic channel reactor, and the microfluidic channel reactor comprises the following steps: a first syringe, a second syringe, a reaction channel, and a product collector; the first injector and the second injector are connected with the inlet of the reaction channel through Y-shaped or T-shaped pipelines, and the product collector is connected with the outlet of the reaction channel through a pipeline;
further, the method comprises the steps of,
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 specification of the first injector is consistent with that of the second injector;
the microfluidic channel reactor also comprises an incubator, wherein the reaction channel is arranged in the incubator, so that the reaction temperature can be effectively controlled, and the incubator can be selected according to the reaction temperature requirement, such as a water bath incubator and the like;
the material of the reaction channel is not limited, and green and environment-friendly materials such as silicone tubes are recommended; the shape of the reaction channel is preferably curved, so that the reaction liquid can pass through the reaction channel stably at a constant speed.
Compared with the prior art, the invention has the beneficial effects that:
the method utilizes lipase to catalyze and synthesize coumarin-3-carboxylic acid-6' -O-D-sucrose ester derivatives on line in the microfluidic channel reactor, so that the method not only greatly shortens the reaction time, but also has high conversion rate and selectivity; meanwhile, the economic lipase RM IM is utilized for the first time to catalyze the reaction of coumarin-3-carboxylic acid methyl ester derivative and D-sucrose, 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 according to an embodiment of the present invention.
In the figure, a 1-first syringe, a 2-second syringe, a 3-reaction channel, a 4-product collector and a 5-water bath incubator.
Detailed Description
The invention is further illustrated by the following examples, but the scope of the invention is not limited thereto:
the structure of the microfluidic channel reactor used in the embodiment of the present invention is shown in fig. 1, and includes an injection pump, a first injector 1, a second injector 2, a reaction channel 3, a water bath incubator 5 (only a schematic plan view thereof is shown), and a product collector 4; the first injector 1 and the second injector 2 are arranged in an injection pump and are connected with the inlet of a reaction channel 3 through a Y-shaped interface, the reaction channel 3 is arranged in a water bath constant temperature box 5, the reaction temperature is controlled through the water bath constant temperature box 5, the inner diameter of the reaction channel 3 is 2.0mm, the tube length is 1.0m, and the outlet of the reaction channel 3 is connected with a product collector 4 through an interface.
Example 1: synthesis of 6-methylcoumarin-3-carboxylic acid-6' -O-D-sucrose ester
The apparatus is described with reference to fig. 1: methyl 6-methylcoumarin-3-carboxylate (2.0 mmol) was dissolved in 0.52mL of dimethyl sulfoxide and 9.48mL of t-amyl alcohol, and D-sucrose (0.5 mmol) was dissolved in 0.52mL of dimethyl sulfoxide and 9.48mL of t-amyl alcohol, and then separately filled into 10mL syringes for use. Will be0.87g lipase RM IM is uniformly filled in the reaction channel, and under the pushing of PHD 2000 injection pump, the two reaction solutions are respectively filled in a volume of 7.8 mu L.min -1 The reaction is carried out by the flow rate of the reaction liquid entering the reaction channel through the Y joint, the temperature of the reactor is controlled to be 35 ℃ through a water bath constant temperature box, the reaction liquid flows in the reaction channel for 40min, and the reaction result is tracked and detected through thin layer chromatography TLC.
Collecting reaction liquid on line through a product collector, distilling under reduced pressure to remove solvent, loading into a column by using 200-300 mesh silica gel wet method, eluting with dichloromethane: methanol=10:2, column height of 35cm, column diameter of 4.5cm, dissolving sample with a small amount of eluting reagent, loading onto the column by wet method, and collecting eluent with flow rate of 2mL.min -1 Simultaneously, TLC tracks the elution progress, and the obtained eluates containing single products are combined and evaporated to dryness to obtain white solid, so as to obtain 6-methylcoumarin-3-carboxylic acid-6 '-O-D-sucrose ester, and HPLC (high performance liquid chromatography) detects that the conversion rate of the 6-methylcoumarin-3-carboxylic acid-6' -O-D-sucrose ester is 45%, and the selectivity is 99%.
The nuclear magnetic characterization results were as follows:
1 H NMR(DMSO-d 6 ,500MHz,δ,ppm)8.66(s,1H,H-4),7.72(d,J＝2.1Hz,1H,H-5),7.56(dd,J＝8.5,2.1Hz,1H,H-7),7.34(d,J＝8.4Hz,1H,H-8),5.23(d,J＝3.7Hz,1H,C1'-H),5.19(d,J＝6.0Hz,1H,C4'-OH),5.14(dd,J＝10.0,6.0Hz,2H,C2'-OH,C3'-OH),4.96(d,J＝4.8Hz,1H,C3”-OH),4.86(t,J＝6.3Hz,1H,C1”-OH),4.69(d,J＝7.9Hz,1H,C4”-OH),4.49-4.44(m,1H,C6'-Ha),4.42(t,J＝5.5Hz,1H,C6”-OH),4.27(dd,J＝11.8,5.9Hz,1H,C6'-Hb),4.07(ddd,J＝10.1,5.8,1.8Hz,1H,C5'-H),3.91(t,J＝8.1Hz,1H,C3”-H),3.79(td,J＝7.9,5.8Hz,1H,C5”-H),3.62-3.51(m,3H,C4”-H,C6”-Ha,C6”-Hb),3.49-3.45(m,1H,C3'-H),3.45-3.42(m,2H,C1”-Ha,C1”-Hb),3.31–3.21(m,2H,C2'-H,C4'-H),2.37(s,3H,H-12). 13 C NMR(126MHz,DMSO)δ162.35(C-11),156.17(C-2),152.76(C-9),148.77(C-4),135.52(C-6),134.19(C-7),129.97(C-5),117.53(C-10),117.19(C-8),115.95(C-3),104.04(C-2”),91.67(C-1'),82.55(C-5”),76.90(C-3”),74.44(C-4”),72.71(C3'),71.54(C-2'),70.17(C-5'),70.08(C-4'),65.00(C-6'),62.51,(C-1”)62.18(C-6”),20.23(C-12).
examples 2 to 8
The volume ratio of the reaction medium DMSO to the tertiary amyl alcohol in the microfluidic channel reactor was changed, the substrate ratio of the 6-methylcoumarin-3-carboxylic acid methyl ester to the D-sucrose was 2:1 (1.0 mmol:0.5 mmol), the temperature was controlled to be 50 ℃, the reaction time was 30min, the other same as in example 1, and the reaction results are shown in Table 1:
TABLE 1 influence of the volume ratio of DMSO to t-amyl alcohol in the reaction Medium on the reaction
| Examples | DMSO: tert-amyl alcohol | Conversion [%] | Selectivity [%] |
| 2 | 1:8 | n.d. | / |
| 3 | 1:10 | 15％ | / |
| 4 | 1:12 | 20％ | 98％ |
| 5 | 1:14 | 24％ | 99％ |
| 6 | 1:16 | 29％ | 99％ |
| 7 | 1:18 | 32％ | 99％ |
| 8 | 1:20 | 31％ | 99％ |
The results in Table 1 show that when the substrate molar ratio of reactant methyl 6-methylcoumarin-3-carboxylate to D-sucrose is 2:1, the flow rate is 10.4. Mu.L.min -1 The reaction time is 30min, when 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, and when the volume ratio of the DMSO in the reaction medium to the tertiary amyl alcohol is 1:18, the conversion rate of the reaction is optimal, and at the moment, if the volume ratio of the tertiary amyl alcohol is continuously increased, the dissolution amount of sugar in the reaction medium is reduced, so that the conversion rate of the reaction is reduced. The optimal reaction medium volume ratio for this reaction in the microfluidic microchannel reactor of the invention is thus DMSO: t-amyl alcohol=1:18.
Examples 9 to 15
The molar ratio of 6-methylcoumarin-3-carboxylic acid methyl ester to D-sucrose in the microfluidic microchannel reactor was changed, the reactor temperature was controlled at 50deg.C, the reaction time was 30min, and the results are shown in Table 2, except for example 1:
TABLE 2 influence of the ratio of the amounts of methyl 6-methylcoumarin-3-carboxylate and D-sucrose substrate substances on the reaction
The results in Table 2 show that the volume ratio of the reaction medium DMSO to t-amyl alcohol is 1:18, the flow rate is 10.4. Mu.L.min -1 The reaction time is 30min, the reaction conversion rate is increased along with the increase of the reactant methyl 6-methylcoumarin-3-carboxylate at the reaction temperature of 50 ℃, and the reaction conversion rate is optimal when the substrate ratio is 4:1, and the reaction conversion rate is reduced if the use amount of the reactant methyl 6-methylcoumarin-3-carboxylate is continuously increased. The optimal substrate molar ratio for this reaction in the microfluidic microchannel reactor of the invention is thus 6-methylcoumarin-3-carboxylic acid methyl ester to D-sucrose=4:1.
Examples 16 to 22
The temperature of the microfluidic channel reactor was varied and the reaction time was controlled to 30min, otherwise the same as in example 1, and the reaction results are shown in table 3:
table 3: influence of temperature on the reaction
| Examples | Temperature [ DEGC] | Conversion [%] | Selectivity [%] |
| 16 | 30℃ | 42％ | 98％ |
| 17 | 35℃ | 43％ | 99％ |
| 18 | 40℃ | 42％ | 99％ |
| 19 | 45℃ | 40％ | 98％ |
| 20 | 50℃ | 38％ | 97％ |
| 21 | 55℃ | 33％ | 94％ |
| 22 | 60℃ | 30％ | 90％ |
The results in Table 3 show that when the reaction mediumThe volume ratio of DMSO to tertiary amyl alcohol is 1:18, the molar ratio of 6-methylcoumarin-3-carboxylic acid methyl ester to D-sucrose is 4:1, and the flow rate is 10.4 mu L.min -1 When the reaction time is 30min, the conversion rate of the reaction is optimal when the reaction temperature is 35 ℃, and the activity of the enzyme is affected by the temperature which is too high or too low. The optimum reaction temperature for this reaction in the microfluidic microchannel reactor of the invention is 35 ℃.
Examples 23 to 27
The reaction time of the microfluidic channel reactor was varied, and the reaction results are shown in table 4, except for example 1:
table 4: effect of reaction time on reaction
| Examples | Time [ min] | Conversion [%] | Selectivity [%] |
| 23 | 10 | 28％ | 99％ |
| 24 | 20 | 37％ | 99％ |
| 25 | 30 | 43％ | 99％ |
| 1 | 40 | 45％ | 99％ |
| 26 | 50 | 42％ | 98％ |
| 27 | 60 | 34％ | 95％ |
The results in Table 4 show that when the volume ratio of DMSO to t-amyl alcohol is 1:18, the substrate molar ratio of methyl 6-methylcoumarin-3-carboxylate to D-sucrose is 4:1, and the reaction temperatures are 35 ℃, the flow rate is 7.8. Mu.L.min -1 When the reaction time is up to 40min, the reaction conversion rate can reach 45%, and if the reaction time is prolonged, the reaction conversion rate is reduced. Thus, the optimal reaction time for this reaction in a microfluidic channel reactor was 40min.
Comparative examples 1 to 3
The catalysts in the microfluidic microchannel reactor were changed to porcine pancreatic lipase PPL (comparative example 1), lipase Novozym435 (comparative example 2), and subtilisin (comparative example 3), respectively, and the results are shown in table 5.
Table 5: influence of different enzymes on reaction conversion and selectivity
The results in Table 5 show that for the regioselective transesterification of enzymatic methyl 6-methylcoumarin-3-carboxylate with D-sucrose in a microfluidic reactor, different enzymes have a very pronounced effect on the reaction. The porcine pancreatic lipase PPL is utilized for catalytic reaction, and the conversion rate is 5%; catalyzing the reaction by using bacillus subtilis alkaline protease, wherein the conversion rate is 0%; the reaction was catalyzed with Novozym435 with 17% conversion. From the results in Table 5, the most efficient catalyst for the regioselective transesterification of enzymatic 6-methylcoumarin-3-carboxylic acid methyl ester with D-sucrose in a microfluidic reactor was lipase RM IM with a conversion of 45% and a selectivity of 99%.
Example 28: synthesis of 6-chlorocoumarin-3-carboxylic acid-6' -O-D-sucrose ester
The apparatus is described with reference to fig. 1: methyl 6-chlorocoumarin-3-carboxylate (2.0 mmol) was dissolved in 0.52mL of dimethyl sulfoxide and 9.48mL of t-amyl alcohol, and D-sucrose (0.5 mmol) was dissolved in 0.52mL of dimethyl sulfoxide and 9.48mL of t-amyl alcohol, and then separately filled into 10mL syringes for use. Uniformly filling 0.87g lipase RM IM in the reaction channel, and respectively mixing the two reaction solutions at a concentration of 7.8 μL.min under the drive of PHD 2000 injection pump -1 The reaction is carried out by the flow rate of the reaction liquid entering the reaction channel through the Y joint, the temperature of the reactor is controlled to be 35 ℃ through a water bath constant temperature box, the reaction liquid flows in the reaction channel for 40min, and the reaction result is tracked and detected through thin layer chromatography TLC.
Collecting reaction liquid on line through a product collector, distilling under reduced pressure to remove solvent, loading into a column by using 200-300 mesh silica gel wet method, eluting with dichloromethane: methanol=10:2, column height of 35cm, column diameter of 4.5cm, dissolving sample with a small amount of eluting reagent, loading onto the column by wet method, and collecting eluent with flow rate of 2mL.min -1 While TLC tracks the progress of elution, the resulting washes containing a single productThe removal of the liquid and the evaporation to dryness are combined to obtain white solid, and the conversion rate of the 6-chlorocoumarin-3-carboxylic acid-6' -O-D-sucrose ester is 47 percent and the selectivity is 99 percent by HPLC detection.
The nuclear magnetic characterization results were as follows:
1 H NMR(DMSO-d 6 ,500MHz,δ,ppm)8.70(s,1H,H-4),8.07(d,J＝2.6Hz,1H,H-5),7.77(dd,J＝8.9,2.6Hz,1H,H-7),7.48(d,J＝8.8Hz,1H,H-8),5.21(d,J＝3.7Hz,1H,C1'-OH),5.16(d,J＝5.9Hz,1H,C4'-OH),5.12(d,J＝5.6Hz,1H,C3'-OH),5.09(d,J＝6.3Hz,1H,C2'-OH),4.93(d,J＝4.8Hz,1H,C3”-OH),4.83(t,J＝6.3Hz,1H,C1”-OH),4.68(d,J＝7.8Hz,1H,C4”-OH),4.47(dd,J＝11.8,1.8Hz,1H,C6'-Ha),4.41(dd,J＝6.0,4.9Hz,1H,C6”-OH),4.27(dd,J＝11.8,6.1Hz,1H,C6”-Hb),4.07(ddd,J＝10.1,6.0,1.9Hz,1H,C5'-H),3.90(t,J＝8.1Hz,1H,C3”-H),3.78(td,J＝8.0,5.9Hz,1H,C5”-H),3.59–3.50(m,3H,C4”-H,C6”-Ha,C6”-Hb),3.48–3.44(m,1H,C3'-H),3.40(d,J＝6.3Hz,2H,C1”-Ha,C1”-Hb),3.29–3.18(m,2H,C2”-H,C4'-H). 13 C NMR(126MHz,DMSO)δ161.98(C-11),155.53(C-2),153.17(C-9),147.56(C-4),133.87(C-7),129.29(C-5),128.45(C-6),119.16(C-10),118.37(C-8),118.16(C-3),103.95(C-2”),91.53(C-1'),82.45(C-5”),76.78(C-3”),74.38(C-4”),72.69(C-3'),71.48(C-2'),70.13(C-5'),70.02(C-4'),65.16(C-6'),62.47(C-1”),62.17(C-6”).
examples 29 to 35
The volume ratio of the reaction medium DMSO to t-amyl alcohol in the microfluidic channel reactor was changed, the substrate ratio of 6-chlorocoumarin-3-carboxylic acid methyl ester to D-sucrose was 2:1 (1.0 mmol:0.5 mmol), the temperature was controlled to 50 ℃, the reaction time was 30min, the other same as in example 28, and the reaction results are shown in Table 6:
TABLE 6 influence of the volume ratio of DMSO to t-amyl alcohol in the reaction Medium on the reaction
| Examples | DMSO: tert-amyl alcohol | Conversion [%] | Selectivity [%] |
| 29 | 1:8 | n.d. | / |
| 30 | 1:10 | 10％ | 98％ |
| 31 | 1:12 | 19％ | 98％ |
| 32 | 1:14 | 23％ | 99％ |
| 33 | 1:16 | 28％ | 99％ |
| 34 | 1:18 | 31％ | 99％ |
| 35 | 1:20 | 30％ | 99％ |
The results in Table 6 show that when the substrate molar ratio of reactant methyl 6-chlorocoumarin-3-carboxylate to D-sucrose is 2:1, the flow rate is 10.4. Mu.L.min -1 The reaction time is 30min, when 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, and when the volume ratio of the DMSO in the reaction medium to the tertiary amyl alcohol is 1:18, the conversion rate of the reaction is optimal, and at the moment, if the volume ratio of the tertiary amyl alcohol is continuously increased, the dissolution amount of sugar in the reaction medium is reduced, so that the conversion rate of the reaction is reduced. The optimal reaction medium volume ratio for this reaction in the microfluidic microchannel reactor of the invention is thus DMSO: t-amyl alcohol=1:18.
Examples 36 to 42
The molar ratio of 6-chlorocoumarin-3-carboxylic acid methyl ester to D-sucrose substrate in the microfluidic microchannel reactor was changed, the reactor temperature was controlled at 50℃and the reaction time was 30min, and the results were as shown in Table 7, except for example 28:
TABLE 7 influence of the ratio of the amounts of methyl 6-chlorocoumarin-3-carboxylate and D-sucrose substrate materials on the reaction
| Examples | 6-chlorocoumarin-3-carboxylic acid methyl ester: d-sucrose | Conversion [%] | Selectivity [%] |
| 36 | 5:1 | 37％ | 98％ |
| 37 | 4:1 | 38％ | 99％ |
| 38 | 3:1 | 36％ | 99％ |
| 39 | 2:1 | 31％ | 98％ |
| 40 | 1:1 | 26％ | 98％ |
| 41 | 1:2 | 24％ | 97％ |
| 42 | 1:3 | 20％ | 97％ |
The results in Table 7 show that the volume ratio of the reaction medium DMSO to t-amyl alcohol is 1:18, the flow rate is 10.4. Mu.L.min -1 The reaction time is 30min, the reaction conversion rate is increased along with the increase of the reactant methyl 6-chlorocoumarin-3-carboxylate at the reaction temperature of 50 ℃, and the reaction conversion rate is optimal when the substrate ratio is 4:1, and the reaction conversion rate is reduced if the consumption of the reactant methyl 6-chlorocoumarin-3-carboxylate is continuously increased. The optimal substrate molar ratio for this reaction in the microfluidic microchannel reactor of the invention is thus 6-chlorocoumarin-3-carboxylic acid methyl ester to D-sucrose=4:1.
Examples 43 to 49
The temperature of the microfluidic channel reactor was varied and the reaction time was controlled to 30min, and the reaction results were as shown in table 8, except for example 28:
table 8: influence of temperature on the reaction
The results in Table 8 show that when the volume ratio of DMSO to t-amyl alcohol in the reaction medium is 1:18, the molar ratio of methyl 6-chlorocoumarin-3-carboxylate to D-sucrose is 4:1, and the flow rate is 10.4. Mu.L.min -1 When the reaction time is 30min, the conversion rate of the reaction is optimal when the reaction temperature is 35 ℃, and the activity of the enzyme is affected by the temperature which is too high or too low. The optimum reaction temperature for this reaction in the microfluidic microchannel reactor of the invention is 35 ℃.
Examples 50 to 54
The reaction time of the microfluidic channel reactor was varied, and the reaction results are shown in Table 9, except for example 28:
table 9: effect of reaction time on reaction
| Examples | Time [ min] | Conversion [%] | Selectivity [%] |
| 50 | 10 | 36％ | 99％ |
| 51 | 20 | 38％ | 99％ |
| 52 | 30 | 44％ | 99％ |
| 28 | 40 | 47％ | 99％ |
| 53 | 50 | 46％ | 98％ |
| 54 | 60 | 42％ | 94％ |
The results in Table 9 show that when the volume ratio of DMSO to t-amyl alcohol is 1:18, the substrate molar ratio of methyl 6-chlorocoumarin-3-carboxylate to D-sucrose is 4:1, and the reaction temperatures are 35 ℃, the flow rate is 7.8. Mu.L.min -1 When the reaction time is up to 40min, the reaction conversion rate can reach 47%, and if the reaction time is prolonged, the reaction conversion rate is reduced. Thus, the optimal reaction time for this reaction in a microfluidic channel reactor was 40min.
Comparative examples 4 to 6
The catalysts in the microfluidic microchannel reactor were changed to porcine pancreatic lipase PPL (comparative example 4), lipase Novozym435 (comparative example 5), and subtilisin (comparative example 6), respectively, and the results are shown in Table 10.
Table 10: influence of different enzymes on reaction conversion and selectivity
| Comparative example | Enzyme source | Conversion [%] | Selectivity [%] |
| 4 | PPL | 8％ | 74％ |
| 5 | Novozym 435 | 13％ | 63％ |
| 6 | Bacillus subtilis alkaline protease | 0 | 0 |
| Example 28 | Lipozyme RM IM | 47％ | 99％ |
The results in Table 10 show that for the regioselective transesterification of methyl 6-chlorocoumarin-3-carboxylate with D-sucrose in a microfluidic reactor, different enzymes have a very pronounced effect on the reaction. The porcine pancreatic lipase PPL is utilized for catalytic reaction, and the conversion rate is 8%; catalyzing the reaction by using bacillus subtilis alkaline protease, wherein the conversion rate is 0%; the reaction was catalyzed with Novozym435 with a conversion of 13%. From the results in table 10, the most efficient catalyst for the regioselective transesterification of enzymatic 6-chlorocoumarin-3-carboxylic acid methyl ester with D-sucrose in a microfluidic reactor was lipase RM IM with a conversion of 47% and a selectivity of 99%.
Example 55: synthesis of 8-methoxycoumarin-3-carboxylic acid-6' -O-D-sucrose ester
The apparatus is described with reference to fig. 1: 8-Methoxycoumarin-3-carboxylic acid methyl ester (2.0 mmol) was dissolved in 0.52mL of dimethyl sulfoxide and 9.48mL of t-amyl alcohol, D-sucrose (0.5 mmol) was dissolved in 0.52mL of dimethyl sulfoxide and 9.48mL of t-amyl alcohol, and then separately charged to 10The syringe was ready for use. Uniformly filling 0.87g lipase RM IM in the reaction channel, and respectively mixing the two reaction solutions at a concentration of 7.8 μL.min under the drive of PHD 2000 injection pump -1 The reaction is carried out by the flow rate of the reaction liquid entering the reaction channel through the Y joint, the temperature of the reactor is controlled to be 35 ℃ through a water bath constant temperature box, the reaction liquid flows in the reaction channel for 40min, and the reaction result is tracked and detected through thin layer chromatography TLC.
Collecting reaction liquid on line through a product collector, distilling under reduced pressure to remove solvent, loading into a column by using 200-300 mesh silica gel wet method, eluting with dichloromethane: methanol=10:2, column height of 35cm, column diameter of 4.5cm, dissolving sample with a small amount of eluting reagent, loading onto the column by wet method, and collecting eluent with flow rate of 2mL.min -1 Simultaneously, TLC tracks the elution progress, the obtained eluates containing single products are combined and evaporated to dryness to obtain light yellow solid, and 8-methoxycoumarin-3-carboxylic acid-6 '-O-D-sucrose ester is obtained, and the conversion rate of the 8-methoxycoumarin-3-carboxylic acid-6' -O-D-sucrose ester is detected by HPLC, wherein the selectivity is 99%.
The nuclear magnetic characterization results were as follows:
1 H NMR(DMSO-d 6 ,500MHz,δ,ppm)8.71(s,1H,H-4),7.51-7.41(m,2H,H-5,H-7),7.38-7.30(m,1H,H-6),5.23(d,J＝3.7Hz,1H,C1'-H),5.14(dt,J＝8.8,6.1Hz,3H,C2'-OH,C3'-OH,C4'-OH),4.94(d,J＝4.6Hz,1H,C3”-OH),4.83(t,J＝6.4Hz,1H,C1”-OH),4.66(d,J＝7.9Hz,1H,C4”-OH),4.48(dd,J＝11.9,2.0Hz,1H,C6'-Ha),4.40(t,J＝4.9Hz,1H,C6”-OH),4.27(dd,J＝11.8,6.0Hz,1H,C6'-Hb),4.07(ddd,J＝10.0,6.0,1.9Hz,1H,C5'-H),3.93(s,3H,H-12),3.90(t,J＝8.1Hz,1H,C3”-H),3.78(td,J＝7.9,5.6Hz,1H,C5”-H),3.61-3.50(m,3H,C4”-H,C6”-Ha,C6”-Hb),3.50-3.45(m,1H,C3'-H),3.42(d,J＝6.1Hz,2H,C1”-Ha,C1”-Hb),3.30-3.20(m,2H,C2'-H,C4'-H). 13 C NMR(126MHz,DMSO)δ162.26(C-11),155.69(C-2),149.05(C-8),146.22(C-9),143.93(C-4),124.73(C-6),121.37(C-5),118.31(C-3),117.42(C-7),116.49(C-10),103.99(C-2”),91.63(C1'),82.50(C-5”),76.86(C-3”),74.41(C-4”),72.66(C-3'),71.50(C-2'),70.17(C-5'),70.06(C-4'),65.05(C-6'),62.51(C-1”),62.14(C-6”),56.21(C-12).
examples 56 to 62
The volume ratio of the reaction medium DMSO to the tertiary amyl alcohol in the microfluidic channel reactor was changed, the substrate ratio of 8-methoxycoumarin-3-carboxylic acid methyl ester to D-sucrose was 2:1 (1.0 mmol:0.5 mmol), the temperature was controlled to be 50 ℃, the reaction time was 30min, the other same as in example 55, and the reaction results are shown in Table 11:
TABLE 11 influence of the volume ratio of DMSO to t-amyl alcohol in the reaction Medium on the reaction
| Examples | DMSO: tert-amyl alcohol | Conversion [%] | Selectivity [%] |
| 56 | 1:8 | n.d. | / |
| 57 | 1:10 | 14％ | / |
| 58 | 1:12 | 23％ | 99％ |
| 59 | 1:14 | 25％ | 99％ |
| 60 | 1:16 | 30％ | 99％ |
| 61 | 1:18 | 31％ | 99％ |
| 62 | 1:20 | 29％ | 99％ |
The results in Table 11 show that when the substrate molar ratio of reactant methyl 8-methoxycoumarin-3-carboxylate to D-sucrose is 2:1, the flow rate is 10.4. Mu.L.min -1 The reaction time is 30min, when 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, and when the volume ratio of the DMSO in the reaction medium to the tertiary amyl alcohol is 1:18, the conversion rate of the reaction is optimal, and at the moment, if the volume ratio of the tertiary amyl alcohol is continuously increased, the dissolution amount of sugar in the reaction medium is reduced, so that the conversion rate of the reaction is reduced. The optimal reaction medium volume ratio for this reaction in the microfluidic microchannel reactor of the invention is thus DMSO: t-amyl alcohol=1:18.
Examples 63 to 69
The molar ratio of 8-methoxycoumarin-3-carboxylic acid methyl ester to D-sucrose substrate in the microfluidic microchannel reactor was changed, the reactor temperature was controlled at 50deg.C, the reaction time was 30min, and the results were as shown in Table 12, except for example 55:
TABLE 12 influence of the ratio of the amounts of methyl 8-methoxycoumarin-3-carboxylate and D-sucrose substrate materials on the reaction
| Examples | 8-methoxycoumarin-3-carboxylic acid methyl ester: d-sucrose | Conversion [%] | Selectivity [%] |
| 63 | 5:1 | 34％ | 98％ |
| 64 | 4:1 | 37％ | 99％ |
| 65 | 3:1 | 35％ | 99％ |
| 66 | 2:1 | 31％ | 98％ |
| 67 | 1:1 | 26％ | 98％ |
| 68 | 1:2 | 24％ | 98％ |
| 69 | 1:3 | 18％ | 98％ |
The results in Table 12 show that the volume ratio of the reaction medium DMSO to t-amyl alcohol is 1:18, the flow rate is 10.4. Mu.L.min -1 The reaction time is 30min, the reaction conversion rate is increased along with the increase of the reactant 8-methoxy coumarin-3-carboxylic acid methyl ester when the reaction temperature is 50 ℃, and the reaction conversion rate is optimal when the substrate ratio is 4:1, and the reaction conversion rate is reduced if the use amount of the reactant 8-methoxy coumarin-3-carboxylic acid methyl ester is continuously increased. The optimal substrate molar ratio for this reaction in the microfluidic microchannel reactor of the invention is thus 8-methoxycoumarin-3-carboxylic acid methyl ester to D-sucrose=4:1.
Examples 70 to 76
The temperature of the microfluidic channel reactor was varied and the reaction time was controlled to 30min, and the reaction results were as shown in table 13, except for example 55:
table 13: influence of temperature on the reaction
| Examples | Temperature [ DEGC] | Conversion [%] | Selectivity [%] |
| 70 | 30℃ | 40％ | 98％ |
| 71 | 35℃ | 41％ | 99％ |
| 72 | 40℃ | 40％ | 99％ |
| 73 | 45℃ | 39％ | 98％ |
| 74 | 50℃ | 37％ | 97％ |
| 75 | 55℃ | 32％ | 94％ |
| 76 | 60℃ | 28％ | 90％ |
The results in Table 13 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-sucrose is 4:1, and the flow rate is 10.4. Mu.L.min -1 When the reaction time is 30min, the conversion rate of the reaction is optimal when the reaction temperature is 35 ℃, and the activity of the enzyme is affected by the temperature which is too high or too low. The optimum reaction temperature for this reaction in the microfluidic microchannel reactor of the invention is 35 ℃.
Examples 77 to 81
The reaction time of the microfluidic channel reactor was varied, and the reaction results are shown in Table 14, except for example 55:
table 14: effect of reaction time on reaction
| Examples | Time [ min] | Conversion [%] | Selectivity [%] |
| 77 | 10 | 26％ | 99％ |
| 78 | 20 | 35％ | 99％ |
| 79 | 30 | 41％ | 99％ |
| 55 | 40 | 43％ | 99％ |
| 80 | 50 | 40％ | 98％ |
| 81 | 60 | 26％ | 94％ |
The results in Table 14 show that when the volume ratio of DMSO to t-amyl alcohol is 1:18, the substrate molar ratio of methyl 8-methoxycoumarin-3-carboxylate to D-sucrose is 4:1, and the reaction temperatures are 35 ℃, the flow rate is 7.8. Mu.L.min -1 When the reaction time is up to 40min, the reaction conversion rate can reach 43%, and if the reaction time is prolonged, the reaction conversion rate is reduced. Thus, the optimal reaction time for this reaction in a microfluidic channel reactor was 40min.
Comparative examples 7 to 9
The catalysts in the microfluidic microchannel reactor were changed to porcine pancreatic lipase PPL (comparative example 7), lipase Novozym435 (comparative example 8), and subtilisin (comparative example 9), respectively, and the results are shown in Table 15.
Table 15: influence of different enzymes on reaction conversion and selectivity
| Comparative example | Enzyme source | Conversion [%] | Selectivity [%] |
| 7 | PPL | 6％ | 74％ |
| 8 | Novozym 435 | 17％ | 63％ |
| 9 | Bacillus subtilis alkaline protease | 0 | 0 |
| Example 55 | Lipozyme RM IM | 43％ | 99％ |
The results in Table 15 show that for the regioselective transesterification of methyl 8-methoxycoumarin-3-carboxylate with D-sucrose in a microfluidic reactor, different enzymes have a very pronounced effect on the reaction. The porcine pancreatic lipase PPL is utilized for catalytic reaction, and the conversion rate is 6%; catalyzing the reaction by using bacillus subtilis alkaline protease, wherein the conversion rate is 0%; the reaction was catalyzed with Novozym435 with 17% conversion. From the results in table 15, the most efficient catalyst for the regioselective transesterification of enzymatic methyl 8-methoxycoumarin-3-carboxylate with D-sucrose in a microfluidic reactor was lipase RM IM with a conversion of 43% and a selectivity of 99%.
Comparative examples 10 to 12
The reaction materials in the microfluidic microchannel reactor were changed, the acyl donor was changed to tert-butyl 6-methylcoumarin-3-carboxylate (comparative example 10), the acyl acceptor was changed to D-galactose (comparative example 11), and the acyl donor and acyl acceptor (comparative example 12) were simultaneously changed, and the results are shown in table 16, except for example 1.
TABLE 16 influence of different enzymes on reaction conversion and selectivity
| Comparative example | Acyl donor | Acyl acceptors | Conversion [%] | Selectivity [%] |
| 10 | 6-methylcoumarin-3-carboxylic acid tert-butyl ester | D-sucrose | 7％ | 53％ |
| 11 | 6-methylcoumarin-3-carboxylic acid methyl ester | D-galactose | 8％ | 62％ |
| 12 | 6-methylcoumarin-3-carboxylic acid tert-butyl ester | D-galactose | 0 | 0 |
| Example 1 | 6-methylcoumarin-3-carboxylic acid methyl ester | D-sucrose | 45％ | 99％ |
The results in Table 16 show that for the regioselective transesterification synthesis of the enzymatic 6-methylcoumarin-3-carboxylic acid sugar ester in a microfluidic reactor, the different substrates have a very pronounced effect on the reaction. Using 6-methylcoumarin-3-carboxylic acid tert-butyl ester as acyl donor, using D-sucrose as acyl acceptor, and its conversion rate is 7%; using 6-methylcoumarin-3-carboxylic acid methyl ester as acyl donor, and D-galactose as acyl acceptor, the conversion rate is 8%; the conversion rate of the 6-methylcoumarin-3-carboxylic acid tert-butyl ester serving as an acyl donor and the D-galactose serving as an acyl acceptor is 0%. From the results of table 16, it is seen that for the regioselective transesterification synthesis of the enzymatic 6-methylcoumarin-3-carboxylic acid sugar ester in a microfluidic reactor, tert-butyl 6-methylcoumarin-3-carboxylate is not an effective acyl donor and D-galactose is not an effective acyl acceptor.
Application examples 1 to 3
Determination of 6-The size of the inhibition zone of methylcoumarin-3-carboxylic acid-6 ' -O-D-sucrose ester, 6-chlorocoumarin-3-carboxylic acid-6 ' -O-D-sucrose ester and 8-methoxycoumarin-3-carboxylic acid-6 ' -O-D-sucrose ester on staphylococcus aureus. 100. Mu.L of test bacteria solution (concentration of bacteria solution 1X 10) was added to the nutrient agar plate 7 CFU/mL), uniformly coating the bacterial liquid by using a sterile coater; placing 9 oxford cups on the surface of the culture medium at equal distance and lightly pressing the oxford cups to enable the oxford cups to be in contact with the culture medium; 200 μl (1 g/mL) of each of the different coumarin-3-carboxylic acid sucrose ester compounds was added to the cup, and 3 replicates of each compound were performed; culturing in a 28 ℃ water-proof constant temperature incubator for 24 hours, and observing the result.
And (3) result judgment: the diameter of the bacteria growth zone is measured by a ruler by taking a bacteria growth zone which is not visible to naked eyes around the oxford cup as the bacteria inhibition zone, and the average value of 3 measurement results is taken as the bacteria inhibition zone of the compound to staphylococcus aureus. The diameter of the inhibition zone is expressed as 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 sensor is moderately sensitive (I); when d >15mm, is highly sensitive (S).
Table 17 in vitro bacteriostasis test of different coumarin-3-carboxylic acid sucrose esters against staphylococcus aureus
| Application example | Compounds of formula (I) | Average inhibition zone diameter/mm | Sensitivity to |
| 1 | 6-methylcoumarin-3-carboxylic acid-6' -O-D-sucrose ester | 23 | |
| 2 | 6-chlorocoumarin-3-carboxylic acid-6' -O-D-sucrose ester | 16 | |
| 3 | 8-Methoxycoumarin-3-carboxylic acid-6' -O-D-sucrose ester | 18 | S |
Table 17 shows that 6-methylcoumarin-3-carboxylic acid-6 ' -O-D-sucrose ester, 6-chlorocoumarin-3-carboxylic acid-6 ' -O-D-sucrose ester and 8-methoxycoumarin-3-carboxylic acid-6 ' -O-D-sucrose ester have good inhibitory effect on Staphylococcus aureus, and can be used as Staphylococcus aureus inhibitor.
Claims (3)
1. A method for the on-line enzymatic synthesis of coumarin-3-carboxylic acid-6' -O-D-sucrose ester derivatives based on flow chemistry, characterized in that it comprises the steps of:
uniformly filling lipase RM IM in a reaction channel of a microfluidic channel reactor, dissolving coumarin-3-carboxylic acid methyl ester derivative and D-sucrose respectively by using a reaction solvent, respectively injecting the mixture into a pipeline through a first injector and a second injector for integration, then entering the reaction channel for reaction, controlling the reaction temperature to be 30-60 ℃, continuously flowing mixed solution 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 aftertreatment to obtain a product coumarin-3-carboxylic acid-6' -O-D-sucrose ester derivative (I);
the coumarin-3-carboxylic acid methyl ester derivative is as follows: methyl 6-methylcoumarin-3-carboxylate, methyl 6-chlorocoumarin-3-carboxylate or methyl 8-methoxycoumarin-3-carboxylate;
the reaction solvent is a mixed solvent of dimethyl sulfoxide and tertiary amyl alcohol, wherein the volume ratio of dimethyl sulfoxide to tertiary amyl alcohol is 1: 8-20;
the ratio of the mass of coumarin-3-carboxylic acid methyl ester derivative to D-sucrose in the mixed solution entering the reaction channel is 1:0.2 to 3;
the post-treatment method comprises the following steps: collecting reaction liquid on line through a product collector, distilling under reduced pressure to remove solvent, loading into a column by using 200-300 mesh silica gel wet method, eluting with dichloromethane: methanol=10:2, column height of 35cm, column diameter of 4.5cm, dissolving sample with eluting reagent, loading onto the column by wet method, and collecting eluent with flow rate of 2mL.min -1 Simultaneously, TLC tracks the elution process, and the obtained eluents containing single products are combined and evaporated to dryness to obtain the coumarin-3-carboxylic acid-6' -O-D-sucrose ester derivative (I);
2. the method for the on-line enzymatic synthesis of coumarin-3-carboxylic acid-6' -O-D-sucrose ester derivatives based on flow chemistry according to claim 1, wherein the ratio of the mass concentrations of the coumarin-3-carboxylic acid methyl ester derivative solution and the D-sucrose solution obtained after dissolution of the coumarin-3-carboxylic acid methyl ester derivative and D-sucrose respectively with the reaction solvent is 1:0.2 to 3; the coumarin-3-carboxylic acid methyl ester derivative solution and the D-sucrose solution have the same flow velocity when being injected by the first injector and the second injector respectively.
3. The method for the on-line enzymatic synthesis of coumarin-3-carboxylic acid-6' -O-D-sucrose ester derivatives based on flow chemistry according to claim 1 wherein the catalyst lipase RM IM is added in an amount of 0.025 to 0.05g/mL based on the volume of the reaction medium.
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Citations (4)
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| CN107418989A (en) * | 2017-08-21 | 2017-12-01 | 浙江工业大学 | A kind of method of lipase-catalyzed online synthesis N (5 sucrose ester valeryl) metoprolol |
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| CN103184256A (en) * | 2011-12-31 | 2013-07-03 | 浙江工业大学 | Method for on-line synthesizing saccharose-6-laurate by lipase catalysis |
| CN103667393A (en) * | 2013-09-02 | 2014-03-26 | 浙江农林大学 | Method for synthesizing 6''-O-palmitoyl-neohesperidin dihydrochalcone ester on line by using lipase as catalyst |
| CN103667394A (en) * | 2013-09-02 | 2014-03-26 | 浙江农林大学 | Method for synthesizing 6''-O-lauroyl-neohesperidin dihydrochalcone ester on line by using lipase as catalyst |
| CN107418989A (en) * | 2017-08-21 | 2017-12-01 | 浙江工业大学 | A kind of method of lipase-catalyzed online synthesis N (5 sucrose ester valeryl) metoprolol |
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