CN107988277B - Method for synthesizing S-benzylpalmitic acid thioester on line under catalysis of lipase - Google Patents
Method for synthesizing S-benzylpalmitic acid thioester on line under catalysis of lipase Download PDFInfo
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Abstract
The invention discloses a method for synthesizing S-benzyl palmitate thioester on line by lipase catalysis, which comprises the following steps: taking dimethyl sulfoxide as a reaction solvent, taking benzyl mercaptan and ethylene palmitate with a molar ratio of 1: 0.5-6 as raw materials, taking lipase Lipozyme TL IM as a catalyst, placing the raw materials and the reaction solvent in an injector, uniformly filling the lipase Lipozyme TL IM in a reaction channel of a microfluidic channel reactor, and continuously introducing the raw materials and the reaction solvent into the reaction channel reactor under the driving of an injection pump for carrying out acylation reaction, wherein the inner diameter of the reaction channel of the microfluidic channel reactor is 0.8-2.4 mm, and the length of the reaction channel is 0.5-1.0 m; and (3) controlling the acylation reaction temperature to be 20-60 ℃, carrying out acylation reaction for 20-40 min, collecting reaction liquid on line through a product collector, and carrying out conventional post-treatment on the reaction liquid to obtain the S-benzyl palmitate thioester. The invention has the advantages of short reaction time, high selectivity and high yield.
Description
(I) technical field
The invention relates to a method for synthesizing S-benzyl palmitate thioester on line by lipase catalysis.
(II) background of the invention
Many synthetic or natural sulfur-containing organic compounds are biologically active. The thioester compound has pharmacological activities of oxidation resistance, antibiosis, tumor resistance and the like due to the unique chemical structure, and is an important intermediate in organic synthesis and chemical biology. Meanwhile, the thioester structure also has the function of protecting unstable thiol functional groups, increases the pharmaceutical activity of the thioester structure, covers the smell of SH bonds, and is widely applied to the industries of food, medicine, pesticide and cosmetics.
The synthesis of thioester compounds generally adopts an esterification method, which mainly uses strong acids such as sulfuric acid, benzenesulfonic acid and the like as catalysts, and the catalysts have strong corrosivity, harsh requirements on equipment and serious environmental pollution. In addition, the thioester compounds are prepared by adopting the triflate or transition metal catalysis, and the reaction has the defects of difficult preparation of transition metal complexes, high price, easy loss, difficult recovery, generation of substances harmful to the environment and the like. With the increasingly prominent problem of environmental pollution, the development of a green and efficient synthesis method which has less harm to human health and environment is urgently required.
Enzyme-catalyzed reactions are an important area of green chemistry research. Enzymatic reactions have attracted considerable attention in organic synthesis due to their mild reaction conditions, high selectivity and broad substrate range. However, the enzymatic reaction usually requires a long reaction time, and the restriction of a reaction medium on substrate dissolution and enzyme activity inhibition exists for a specific substrate, so that the development of the synthesis of an enzymatic thioester compound based on a microfluidic reaction technology on the basis of a biocatalytic reaction becomes a research hotspot of the people.
The microfluidic technology is an emerging technology in the 20 th century since 90 th, is a technology which is mainly characterized in that the operation of reactants is performed by using a micron-scale space as an order of magnitude, and operation units such as preparation, reaction, separation, detection and the like of a sample can be integrated on a tiny chip, and a network is formed by microchannels, so that a controllable fluid penetrates through the whole system, and multiple functions of a conventional biochemical laboratory are replaced. The micro-fluidic technology is widely applied in the fields of biology, chemistry, medicine and the like.
In 1995, microsystems were first applied to chemical and biological reactions in a laboratory of meeinz, western germany, which can be regarded as the beginning of the widespread application of microsystems. The first international conference on microtechnology was held in 1997 (IMRET 1). So far, the microfluidic chip reactor has been successfully used for various organic synthesis reactions and shows wide application prospects. With the development of micro-mixing and micro-reaction technology in the microfluidic chip, the synthesis reaction in the chip has become one of the research hotspots in the field of microfluidic chips.
Compared with the conventional chemical reactor, the microchannel has micron-sized inner diameter scale and ultra-large specific surface area, the diffusion distance of the microchannel is much shorter, and the mass transfer speed is high; the heat transfer efficiency of the reactor is much higher than that of a conventional chemical reactor, and the reactor can be applied to reactions with violent exotherms or high selectivity. The reaction conditions such as reactant proportion, temperature, reaction time, flow rate and the like are easy to control, and side reactions are less; the method needs little reactant, can reduce the consumption of expensive, toxic and harmful reactants, generates little environmental pollutants in the reaction process, and is a technology for synthesizing and researching new substances with environmental friendliness.
The studies on the enzymatic synthesis of thioesters are relatively rare, and the synthesis of enzymatic thioesters mostly employs acylases, which requires a long reaction time (48h) and conversion rates for specific substrate reactions are not particularly desirable and the reaction cost is high. In order to develop a new technology for synthesizing efficient, green, economic and environment-friendly thioester compounds, a method for synthesizing S-benzyl palmitate thioester on line under catalysis of lipase in a microchannel reactor is researched, and the new technology for synthesizing the S-benzyl palmitate thioester on line is aimed at being efficient and environment-friendly.
Disclosure of the invention
The technical problem to be solved by the invention is to provide a novel process for synthesizing S-benzylpalmitic acid thioester on line under catalysis of lipase in a microfluidic channel reactor, and the novel process has the advantages of short reaction time, high yield and good selectivity.
In order to solve the technical problems, the invention adopts the following technical scheme:
a method for synthesizing S-benzyl palmitate thioester on line under catalysis of lipase adopts a microfluidic channel reactor, wherein the microfluidic channel reactor comprises an injector, a reaction channel and a product collector which are sequentially connected, the injector is arranged in an injection pump, the injector is connected with an inlet of the reaction channel through a first connecting pipeline, the product collector is connected with an outlet of the reaction channel through the first connecting pipeline, 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 method comprises the following steps: taking dimethyl sulfoxide as a reaction solvent, taking benzyl mercaptan and ethylene palmitate as raw materials, taking lipase lipozyme TL IM as a catalyst, placing the raw materials and the reaction solvent into an injector, uniformly filling the lipase lipozyme TL IM in a reaction channel, continuously introducing the raw materials and the reaction solvent into the reaction channel under the driving of an injection pump for carrying out acylation reaction, controlling the reaction temperature to be 20-60 ℃, the reaction time to be 20-40 min, collecting reaction liquid on line through a product collector, and carrying out post-treatment on the reaction liquid to obtain S-benzyl palmitate thioester; the mass ratio of the benzyl mercaptan to the vinyl palmitate is 1: 0.5-6; the adding amount of the catalyst is 0.025-0.05 g/mL based on the volume of the reaction solvent.
Further, the present invention adopts a microfluidic channel reactor, wherein the number of the injectors can be one or more, depending on the specific reaction requirements. The reaction raw materials of the invention are two, preferably two injectors are used, specifically, the injectors are respectively a first injector and a second injector, the first connecting pipeline is a Y-shaped or T-shaped pipeline, the first injector and the second injector are respectively connected with two interfaces of the Y-shaped or T-shaped pipeline, are connected in parallel through the Y-shaped or T-shaped pipeline and are then connected in series with the reaction channel, and the probability of contact and collision of reactant molecules through the microchannel is increased, so that two reactant liquid flows are mixed and react in the common reaction channel.
Still further, more specifically, the method of the present invention comprises the steps of:
taking benzyl mercaptan and vinyl palmitate with the mass ratio of 1: 0.5-6 as raw materials, taking lipase Lipozyme TLIM as a catalyst, taking dimethyl sulfoxide as a reaction solvent, uniformly filling the lipase Lipozyme TLIM in a reaction channel, dissolving the benzyl mercaptan by the dimethyl sulfoxide, and filling the benzyl mercaptan in a first syringe; dissolving the ethylene palmitate by using dimethyl sulfoxide and filling the ethylene palmitate into a second syringe; then leading the raw materials and a reaction solvent into a reaction channel to react under the synchronous pushing of an injection pump, controlling the reaction temperature to be 20-60 ℃ and the reaction time to be 20-40 min, collecting reaction liquid on line through a product collector, and carrying out post-treatment on the reaction liquid to obtain S-benzyl palmitate thioester; the addition amount of the catalyst is 0.5-1 g.
Furthermore, the microfluidic channel reactor also comprises a thermostat, and the reaction channel is arranged in the thermostat, so that the reaction temperature can be effectively controlled. The constant temperature box can be selected according to the reaction temperature requirement, such as a water bath constant temperature box 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.
In the present invention, the lipase lipozyme tl IM is a preparation prepared from microorganisms, using a food grade lipase (ec3.1.1.3) on granular silica gel, which is a commercial product manufactured by novozymes (novozymes). It is obtained from Thermomyces lanuginosus and produced by submerged fermentation using a genetically modified Aspergillus oryzae microorganism.
Further, the mass ratio of the benzylmercaptan to the vinyl palmitate is preferably 1:1 to 3, and most preferably 1: 2.
Further, the acylation reaction temperature is preferably 45-55 ℃, and most preferably 50 ℃.
Further, the acylation reaction time is preferably 25-35 min, and most preferably 30 min.
The reaction product can be collected on line, and the obtained reaction solution can be used for obtaining the S-benzyl palmitate thioester by a conventional post-treatment method. The conventional post-treatment method may be: and (3) distilling the obtained reaction liquid under reduced pressure to remove the solvent, filling the reaction liquid into a column by using a 200-mesh 300-mesh silica gel wet method, wherein an elution reagent is petroleum ether and ethyl acetate which are 20:1, dissolving a sample by using a small amount of the elution reagent, then filling the sample into the column by using the wet method, collecting the eluent, tracking the elution process by using TLC (thin layer chromatography), combining the obtained eluents containing the single product, and evaporating to dryness to obtain a white solid, namely the S-benzylpalmitic acid thioester.
Compared with the prior art, the invention has the beneficial effects that:
the S-benzyl palmitate thioester is synthesized on line in the microfluidic channel reactor by using lipase catalysis, and the method not only greatly shortens the reaction time, but also has high conversion rate and selectivity; meanwhile, the thioester acylation reaction is catalyzed by using the economic lipase lipozyme TL IM for the first time, so that the reaction cost is reduced, and the method has the advantages of economy and high efficiency.
(IV) description of the drawings
Fig. 1 is a schematic structural diagram of a microfluidic channel reactor used in an embodiment of the present invention.
(V) detailed description of the preferred embodiments
The scope of the invention is further illustrated by the following examples, but is not limited thereto:
referring to fig. 1, a microfluidic channel reactor used in an embodiment of the present invention includes a syringe pump (not shown), two syringes 1 and 2, a reaction channel 3, a water bath incubator (5, only a schematic plan view thereof is shown), and a product collector 4; two injectors 1 and 2 are installed in the injection pump and are connected with an inlet of a reaction channel 3 through a Y-shaped interface, the reaction channel 3 is arranged in a water bath thermostat 5, the reaction temperature is controlled through the water bath thermostat 5, the inner diameter of the reaction channel 3 is 2.0mm, the length of a tube is 1m, and an outlet of the reaction channel 3 is connected with a product collector 4 through an interface.
Example 1: synthesis of S-benzylpalmitic acid thioester
The device is shown in figure 1: dissolving benzyl mercaptan (1.0mmol) in 10mL of DMSO and dissolving vinyl palmitate (2.0mmol) in 10mL of DMSO, and then respectively filling the materials in a 10mL syringe for standby; 0.87g of lipase lipozyme TL IM is uniformly filled in the reaction channel, and the two paths of reaction liquid are respectively driven by a PHD2000 injection pump to be 10.4 mu L/min-1Is fed through a "Y" junction into a reaction vesselThe reaction is carried out in the reaction channel, the temperature of the reactor is controlled at 50 ℃ by a water bath thermostat, the reaction liquid continuously and continuously reacts for 30min in the reaction channel, and the reaction result is tracked and detected by thin-layer chromatography TLC.
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 petroleum ether and ethyl acetate with the ratio of 20:1, 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 eluent at the flow rate of 2 mL/min-1And simultaneously tracking the elution process by TLC (thin layer chromatography), merging and evaporating the obtained eluent containing the single product to dryness to obtain a white solid, obtaining S-benzyl palmitate thioester, and detecting the conversion rate of benzyl mercaptan by HPLC (high performance liquid chromatography) and the selectivity of the S-benzyl palmitate thioester is 96%.
The nuclear magnetic characterization results were as follows:
1H NMR(500MHz,CDCl3):δ＝7.30(dd,J＝11.2,7.8Hz,5H),4.14(s,2H),2.61-2.54(m,2H),1.66(dd,J＝41.7,34.3Hz,2H),1.47-1.23(m,24H),0.91(t,J＝7.0Hz,3H).13C NMR(125MHz,CDCl3):δ＝198.9,137.8,129.3,129.0,128.8,128.6,127.2,43.9,33.1,31.9,29.7,29.6–29.1,29.0,25.6,22.7,14.1.
examples 2 to 6
The ratio of the amounts of the substrate substances of benzylmercaptan and vinyl palmitate in the microfluidic microchannel reactor was varied to control the temperature at 50 ℃ as in example 1, and the results are shown in table 1:
TABLE 1 Effect of the ratio of the amounts of benzylthiol and vinyl palmitate substrate materials on the reaction
| Examples | Benzyl mercaptan vinyl palmitate | Conversion [% ]] | Selectivity [% ]] |
| 2 | 1:0.5 | 25 | 95 |
| 3 | 1:1 | 61 | 95 |
| 1 | 1:2 | 85 | 96 |
| 4 | 1:3 | 79 | 96 |
| 5 | 1:4 | 73 | 94 |
| 6 | 1:5 | 70 | 97 |
The results in Table 1 show that the flow rate was 10.4. mu.L.min-1The reaction time is 30min, the reaction temperature is 50 ℃, DMSO is used as an organic solvent in the reactor, the conversion rate of the reaction is increased along with the increase of a reactant vinyl palmitate, and when the substrate is benzyl mercaptan and ethyl palmitateWhen the alkenyl is 1:2, the conversion rate of the reaction is optimal, so the ratio of the optimal substrate substance in the microfluidic microchannel reactor is 1: 2.
Examples 7 to 10
The temperature of the microfluidic channel reactor was changed, and the reaction results are shown in Table 2 as in example 1:
table 2: influence of temperature on the reaction
| Examples | Temperature [ deg.C ]] | Conversion [% ]] | Selectivity [% ]] |
| 7 | 40 | 60 | 95 |
| 8 | 45 | 76 | 97 |
| 1 | 50 | 85 | 96 |
| 9 | 55 | 80 | 96 |
| 10 | 60 | 75 | 97 |
The results in Table 2 show that the flow rate was 10.4. mu.L.min-1The reaction time is 30min, DMSO is used as an organic solvent in the reactor, the quantity ratio of the reactant benzylmercaptan to the vinyl palmitate substance is 1:2, and when the concentration of the benzylmercaptan in the reaction system is 0.05mmol/mL, the conversion rate of the reaction is optimal when the reaction temperature is 50 ℃, and the activity of the enzyme is influenced by the temperature which is too high or too low. Therefore, the optimal temperature in the microfluidic microchannel reactor is 50 ℃.
Examples 11 to 14
The reaction time of the microfluidic channel reactor was changed, and the reaction results are shown in Table 3 as in example 1:
table 3: influence of reaction time on the reaction
| Examples | Time [ min ]] | Conversion [% ]] | Selectivity [% ]] |
| 11 | 20 | 66 | 97 |
| 12 | 25 | 76 | 98 |
| 1 | 30 | 85 | 96 |
| 13 | 35 | 80 | 94 |
| 14 | 40 | 75 | 98 |
The results in Table 3 show that when DMSO is used as the organic solvent in the reactor, the ratio of the amount of the reactant benzylmercaptan to the amount of the vinyl palmitate substance is 1:2, the reaction temperature is 50 ℃, and when the reaction time is 30min, the reaction conversion rate is 85%. Therefore, the optimal reaction time in the microfluidic microchannel reactor is 30 min.
Comparative examples 1 to 3
The results are shown in table 4 for the same samples as in example 1 except that the catalysts in the microfluidic microchannel reactor were changed to porcine pancreatic lipase PPL (comparative example 1), lipase Novozym 435 (comparative example 2), and subtilisin alkaline protease (comparative example 3), respectively.
Table 4: effect of different enzymes on reaction conversion and selectivity
The results in Table 4 show that for the acylation of enzymatic benzylthiols in microfluidic channel reactors different enzymes have a very significant effect on the reaction. The conversion of S-benzylpalmitate thioester was 22% using subtilisin catalysis. Whereas the conversion of the thioester S-benzylpalmitate was only 10% using Novozym 435 to catalyze the reaction. From the results in table 4, the most effective catalyst for the acylation of enzymatic benzylthiol in microfluidic channel reactors was the lipase lipozyme tl IM with 85% conversion and 96% selectivity of benzylthiol.
Claims (9)
1. A method for synthesizing S-benzyl palmitate thioester on line by lipase catalysis is characterized in that: the method adopts a microfluidic channel reactor, wherein the microfluidic channel reactor comprises an injector, a reaction channel and a product collector which are connected in sequence, the injector is arranged in an injection pump, the injector is connected with an inlet of the reaction channel through a first connecting pipeline, the product collector is connected with an outlet of the reaction channel through the first connecting pipeline, 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 method comprises the following steps: taking dimethyl sulfoxide as a reaction solvent, taking benzyl mercaptan and ethylene palmitate as raw materials, taking lipase Lipozyme TL IM as a catalyst, placing the raw materials and the reaction solvent into an injector, uniformly filling the lipase Lipozyme TL IM in a reaction channel, continuously introducing the raw materials and the reaction solvent into the reaction channel under the driving of an injection pump for carrying out acylation reaction, controlling the reaction temperature to be 20-60 ℃, controlling the reaction time to be 20-40 min, collecting reaction liquid on line through a product collector, and carrying out post-treatment on the reaction liquid to obtain S-benzyl palmitate thioester; the mass ratio of the benzyl mercaptan to the vinyl palmitate is 1: 0.5-6; the adding amount of the catalyst is 0.025-0.05 g/mL based on the volume of the reaction solvent.
2. The lipase-catalyzed, on-line synthesis of S-benzylpalmitate thioester according to claim 1, wherein: the device comprises a reaction channel, a Y-shaped or T-shaped pipeline, two injectors, a first injector and a second injector, wherein the two injectors are respectively a first injector and a second injector, the first connecting pipeline is a Y-shaped or T-shaped pipeline, the first injector and the second injector are respectively connected with two interfaces of the Y-shaped or T-shaped pipeline, are connected in parallel through the Y-shaped or T-shaped pipeline and are then connected in series with the reaction channel.
3. The lipase-catalyzed, on-line synthesis of S-benzylpalmitate thioester according to claim 2, wherein: the method comprises the following steps: taking benzyl mercaptan and vinyl palmitate with the mass ratio of 1: 0.5-6 as raw materials, taking lipase Lipozyme TL IM as a catalyst, taking dimethyl sulfoxide as a reaction solvent, uniformly filling the lipase Lipozyme TL IM in a reaction channel, dissolving the benzyl mercaptan by the dimethyl sulfoxide, and filling the benzyl mercaptan in a first syringe; dissolving the ethylene palmitate by using dimethyl sulfoxide and filling the ethylene palmitate into a second syringe; then leading the raw materials and a reaction solvent into a reaction channel to react under the synchronous pushing of an injection pump, controlling the reaction temperature to be 20-60 ℃ and the reaction time to be 20-40 min, collecting reaction liquid on line through a product collector, and carrying out post-treatment on the reaction liquid to obtain S-benzyl palmitate thioester; the addition amount of the catalyst is 0.5-1 g.
4. The lipase-catalyzed, on-line synthesis of S-benzylpalmitate thioester according to claim 1, wherein: the microfluidic channel reactor comprises a thermostat, and the reaction channel is arranged in the thermostat.
5. The lipase-catalyzed, on-line synthesis of S-benzylpalmitate thioester according to claim 3, wherein: the microfluidic channel reactor comprises a thermostat, and the reaction channel is arranged in the thermostat.
6. The method for the lipase-catalyzed online synthesis of S-benzylpalmitate thioester according to any one of claims 1 to 5, wherein: the mass ratio of the benzyl mercaptan to the vinyl palmitate is 1: 1-3.
7. The method for the lipase-catalyzed online synthesis of S-benzylpalmitate thioester according to any one of claims 1 to 5, wherein: the acylation reaction temperature is 40-60 ℃, and the acylation reaction time is 25-35 min.
8. The method for the lipase-catalyzed online synthesis of S-benzylpalmitate thioester according to any one of claims 1 to 5, wherein: the mass ratio of benzylmercaptan to vinyl palmitate is 1: 2.
9. The method for the lipase-catalyzed online synthesis of S-benzylpalmitate thioester according to any of claims 1 to 5, wherein the post-treatment method is as follows: and distilling the obtained reaction liquid under reduced pressure to remove the solvent, carrying out chromatographic separation on the obtained crude product by using a silica gel column, carrying out wet column packing by using 200-mesh 300-mesh silica gel, wherein an elution reagent is a mixed solvent of petroleum ether and ethyl acetate which are 20:1, dissolving the crude product by using a small amount of elution reagent, then carrying out wet column packing, collecting eluent, tracking an elution process by TLC (thin layer chromatography), combining the obtained eluents containing the single product, and evaporating to dryness to obtain a white solid, namely the S-benzylpalmitic acid thioester.
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| CN109706194B (en) * | 2018-12-24 | 2021-04-06 | 浙江工业大学 | Method for synthesizing phenethyl alcohol beta-amino alcohol derivatives on line based on mobile chemical enzymatic ammonolysis reaction |
| CN109735582B (en) * | 2018-12-24 | 2022-06-21 | 浙江工业大学 | Method for synthesizing cyclohexanol beta-amino alcohol derivatives on line by lipase catalysis |
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