CN107384991B

CN107384991B - Method for synthesizing 5' -O-ethylene adipamide uridine on line by lipase catalysis

Info

Publication number: CN107384991B
Application number: CN201610326588.9A
Authority: CN
Inventors: 杜理华; 成柄灼; 徐亮亮; 罗锡平
Original assignee: Zhejiang University of Technology ZJUT
Current assignee: Zhejiang University of Technology ZJUT
Priority date: 2016-05-17
Filing date: 2016-05-17
Publication date: 2020-11-13
Anticipated expiration: 2036-05-17
Also published as: CN107384991A

Abstract

The invention discloses a method for synthesizing 5' -O-ethylene uridine adipamide on line by lipase catalysis. The method comprises the following steps: taking dimethyl sulfoxide and tert-amyl alcohol in a volume ratio of 1: 8-16 as reaction solvents, uridine and divinyl adipate in a molar ratio of 1: 5-13 as raw materials, taking 0.5-1.0 g of lipase TLIM as a catalyst, placing the raw materials and the reaction solvents in an injector, uniformly filling the lipase TLIM in a reaction channel of a microfluidic channel reactor, and continuously introducing the raw materials and the reaction solvents into a reaction channel device under the driving of an injection pump for acylation reaction, wherein the inner diameter of the reaction channel for microfluidic channel reaction is 0.8-2.4 mm, and the length of the reaction channel is 0.5-1.0 m; controlling the esterification reaction temperature to be 15-50 ℃, controlling the esterification reaction time to be 20-35 min, collecting the reaction liquid on line through a product collector, and carrying out conventional post-treatment on the reaction liquid to obtain the 5' -O-ethylene adipamide uridine. The invention has the advantages of short reaction time, high selectivity and high yield.

Description

Method for synthesizing 5' -O-ethylene adipamide uridine on line by lipase catalysis

(I) technical field

The invention relates to a method for synthesizing 5' -O-ethylene adipamide uridine in an online controllable and selective manner under catalysis of lipase.

(II) background of the invention

Nucleoside drugs play an important role in the treatment of viral diseases. In the antiviral drugs used clinically at present, the nucleoside drugs account for more than 60 percent of the total weight. Most nucleoside compounds are polyhydroxy compounds, and have the defects of higher polarity, lower intestinal permeability, poor fat solubility, large toxic and side effects, lower oral bioavailability and the like. After the nucleoside compound is modified by esterification, the lipid solubility of the nucleoside compound can be enhanced, the pharmacological activity is improved, and the oral bioavailability of the nucleoside compound is improved. In the conventional chemical esterification process, a plurality of hydroxyl groups are likely to participate in esterification, and the product is a mixture of monoester and polyester, so that three steps of 'group protection-esterification-deprotection group' are needed to obtain a product with single-position esterification. The enzyme has good specificity and selectivity to a substrate, can selectively esterify a certain hydroxyl of the nucleoside, has higher reaction selectivity, and reduces the difficulty of subsequent separation of products, so that the biocatalysis technology plays an increasingly important role in the esterification of nucleoside compounds.

Microfluidics (Microfluidics) is a technology and science for manipulating nanoliter to picoliter volumes of fluid in micron-scale structures, a new interdisciplinary discipline that has grown up rapidly in the last decade. Currently, the development of microfluidics has greatly surpassed the original purpose of mainly serving analytical chemistry, and is becoming an important technical platform for a new round of innovative research in the whole chemical discipline, life science, instrument science and even information science.

Since the first literature on the synthesis of compounds in microfluidic chip microreactors was published by Harrison topic group 1997, microfluidic chip reactors have been successfully used for a variety of organic synthesis reactions and show broad 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 reactor not only greatly shortens the diffusion distance between reactants, but also has high mass transfer speed; 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.

At present, most domestic and foreign scholars research enzymatic synthesis of nucleoside acylation reaction in organic medium, but the method mostly uses acylase for catalysis, which usually needs longer reaction time (12-24h) and has low conversion rate and selectivity of reaction, so that the method for synthesizing 5 '-O-ethylene adipamide on line by lipase catalysis in a microchannel reactor is researched, and the aim is to find an efficient and environment-friendly method for synthesizing 5' -O-ethylene adipamide on line in a controllable and selective manner.

Disclosure of the invention

The technical problem to be solved by the invention is to provide a novel process for synthesizing 5' -O-ethylene uridine adipate on line by lipase catalysis 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 5' -O-ethylene adipamide on line by lipase catalysis adopts a microfluidic channel reactor, the microfluidic channel reactor comprises an injection pump, an injector, a reaction channel and a product collector, the injector is installed in the injection pump and is connected with an inlet of the reaction channel through an interface, the product collector is connected with an outlet of the reaction channel through an interface, 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: dimethyl sulfoxide (DMSO) and tert-amyl alcohol in a volume ratio of 1: 8-16 are used as reaction solvents, uridine and divinyl adipate in a molar ratio of 1: 5-13 are used as raw materials, 0.5-1.0 g of lipase TLIM is used as a catalyst, the raw materials and the reaction solvents are placed in an injector, the lipase Lipozyme TLIM is uniformly filled in a reaction channel, the raw materials and the reaction solvents are continuously introduced into the reaction channel under the driving of an injection pump for acylation reaction, the concentration of uridine in the reaction system is 0.03-0.07 mmol/mL, the acylation reaction temperature is controlled to be 15-50 ℃, the acylation reaction time is 20-35 min, the reaction liquid is collected on line through a product collector, and the reaction liquid is subjected to conventional post-treatment to obtain 5' -O-ethyleneadipyl uridine.

In the microfluidic channel reactor adopted by the invention, the number of the injectors can be one or more, and is determined according to specific reaction requirements. For example, when two syringes are used, a T-or Y-port can be used to introduce different reactants from two inlets into a common reaction channel, and the probability of reactant molecular contact and collision increases through the microchannel, allowing the two reactant streams to mix and react in the common reaction channel.

The microfluidic channel reactor also comprises a thermostat, and the reaction channel is arranged in the thermostat so as to effectively control the reaction temperature. 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 implementation process of the invention, the uridine is dissolved by DMSO, and then the tert-amyl alcohol is added to a certain volume and is filled in an injector for standby; then dissolving divinyl adipate to a certain volume by using tertiary amyl alcohol, and filling the divinyl adipate into another syringe for later use; finally, the raw materials and the reaction solvent are led into the reaction channel to carry out the reaction under the driving of a syringe pump (such as a PD 1200 syringe pump).

In the present invention, the lipase Lipozyme TLIM is a preparation prepared from microorganisms, using a commercial product manufactured by Novozymes (Novozymes), which is a 1, 3-site specific, food-grade lipase (EC3.1.1.3) on granular silica gel. It is obtained from Thermomyces lanuginosus and produced by submerged fermentation using a genetically modified Aspergillus oryzae microorganism.

Further, the volume ratio of dimethyl sulfoxide to tert-amyl alcohol in the reaction solvent is preferably 1: 12-1: 16, and preferably 1: 14.

Further, the molar ratio of uridine to divinyl adipate is preferably 1:7 to 13, more preferably 1:9 to 11, and most preferably 1: 9.

Further, the concentration of uridine in the reaction system is preferably 0.04 to 0.06mmol/mL, and most preferably 0.05 mmol/mL.

Further, the acylation reaction temperature is preferably 20-40 ℃, and most preferably 30 ℃.

Further, the acylation reaction time is preferably 25-35 min, and most preferably 30 min.

The reaction product of the invention can be collected on line, and the obtained reaction solution can be processed by a conventional post-treatment method to obtain the 5' -O-ethylene adipamide uridine. The conventional post-treatment method may be: and distilling the obtained reaction solution under reduced pressure to remove the solvent, filling the reaction solution into a column by using a 200-mesh 300-mesh silica gel wet method, dissolving a sample by using a small amount of elution reagent, then loading 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 eluent containing a single product, and evaporating to dryness to obtain a white solid, namely the 5' -O-ethylene adipamide uridine.

Compared with the prior art, the invention has the beneficial effects that: the invention utilizes lipase to catalyze and synthesize 5' -O-ethylene adipamide uridine on line in a microfluidic channel reactor, and the method not only greatly shortens the reaction time, but also has high conversion rate and selectivity; meanwhile, the economic lipase Lipozyme TLIM is used for catalyzing the nucleoside esterification reaction 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 5' -O-ethylene adipamide uridine

The device is shown in figure 1: uridine (1.0mmol) was dissolved in 1.33mL of LDMSO and 8.67mL of t-amyl alcohol, and divinyl adipate (9.0mmol) was dissolved in 10mL of t-amyl alcohol and each was filled in a 10mL syringe for use. 0.87g of lipase Lipozyme TLIM is evenly filled in the reaction channel, and the two paths of reaction liquid are respectively driven by a PD 1200 injection pump to be 10.4 mu L/min^-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 30 ℃ through a water bath thermostat, the reaction solution continuously flows in the reaction channel for reaction for 30min, 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 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 ethyl acetate and methanol, wherein the ratio of the elution reagent to the methanol is 40:1, the column height is 35cm, the column diameter is 4.5cm, and then filling the sample into the column by using the wet method, wherein the flow rate of eluent collection is 2 mL/min^-1And simultaneously tracking the elution process by TLC (thin layer chromatography), merging obtained eluents containing single products and evaporating to dryness to obtain a white solid, obtaining 5' -O-ethylene adipamide uridine, and detecting the conversion rate of uridine by HPLC (high performance liquid chromatography) and the selectivity of uridine is 98%.

The nuclear magnetic characterization results were as follows:

¹H-NMR(DMSO-d₆,,ppm)：11.36(s,H₃),7.61(d,J＝9Hz,H₆),7.22(dd,1H,J＝6.5Hz,J＝14Hz,＝CH-O),5.75(d,1H,J＝4.5Hz,H_1'),5.66(d,1H,J＝9Hz,H₅),5.48(d,1H,J＝5.5Hz,3'-OH),5.28(d,1H,J＝5.5Hz,2'-OH),4.90(dd,1H,J＝1.5Hz,J＝14Hz,CH＝C-O),4.65(dd,1H,J＝1.5Hz,J＝6Hz,CH＝C-O),4.24(m,2H,H_2'+H_3'),4.07(m,1H,H_4'),3.98(m,2H,H_5'),2.45(t,2H,J＝7Hz,H_2”),2.38(t,2H,J＝7Hz,H_5”),1.57(m,4H,H_4”+H_3”).

¹³C NMR(DMSO-d₆,ppm)：172.24(C_1”),170.27(C_6”),163.63(C₄),150.60(C₂),141.24(C＝C-O),140.76(C₆),102.02(C₅),98.09(C＝C-O),88.76(C_1'),81.04(C_4'),72.70(C_3'),69.77(C_2'),63.51(C_5'),32.92(C_5”),32.66(C_2”),23.67(C_4”),23.42(C_3”).

examples 2 to 6

The volume ratio of the organic solvent in the microfluidic channel reactor was changed, the temperature was controlled to 50 ℃, and the reaction results are shown in table 1 as in example 1:

TABLE 1 influence of organic solvent ratio on the reaction

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 ℃, the molar ratio of uridine to divinyl adipate serving as reactants is 1:9, the conversion rate is increased along with the increase of the volume ratio of an organic solvent in a reactor when the concentration of uridine in a reaction system is 0.05mmol/mL, the optimal conversion rate is reached when the volume ratio of DMSO to tert-amyl alcohol reaches 1:14, and the incomplete dissolution of the reactants is caused and the conversion rate is reduced by continuously increasing the volume ratio. Therefore, the optimal volume ratio of the organic solvent in the microfluidic microchannel reactor is 1: 14.

Examples 7 to 11

The substrate molar ratio of uridine to divinyl adipate in the microfluidic microchannel reactor was changed, the temperature was controlled at 50 ℃, and the results are shown in table 2 as in example 1:

TABLE 2 Effect of molar ratio of uridine to divinyl adipate substrates on the reaction

The results in Table 2 show that the flow rate was 10.4. mu.L.min^-1The reaction time is 30min, the reaction temperature is 50 ℃, the volume ratio of organic solvent DMSO to tertiary amyl alcohol in the reactor is 1:14, the conversion rate of the reaction is increased along with the increase of reactant divinyl adipate when the concentration of uridine in the reaction system is 0.05mmol/mL, and the conversion rate of the reaction is optimal when the molar ratio of uridine to divinyl adipate substrates is 1:9, so that the optimal substrate molar ratio in the microfluidic microchannel reactor is 1: 9.

Examples 12 to 15

The temperature of the microfluidic channel reactor was changed, and the reaction results are shown in Table 3 as in example 1:

table 3: influence of temperature on the reaction

The results in Table 3 show that the flow rate was 10.4. mu.L.min^-1The reaction time is 30min, the volume ratio of organic solvent DMSO to tertiary amyl alcohol in the reactor is 1:14, the molar ratio of reactant uridine to divinyl adipate is 1:9, and the concentration of uridine in the reaction system is 0.05mmol/mL, when the reaction temperature is 30 ℃, the conversion rate of the reaction is optimal, 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 30 ℃.

Examples 16 to 18

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

The results in Table 4 show that when the volume ratio of the organic solvent DMSO to the tertiary amyl alcohol in the reactor is 1:14, the molar ratio of the reactant uridine to the adipic acid divinyl ester is 1:9, the reaction temperature is 30 ℃, and the uridine concentration in the reaction system is 0.05mmol/mL, the reaction conversion rate is as high as 93 percent and the conversion is almost completely realized when the reaction time is 30 min. Therefore, the optimal reaction time in the microfluidic microchannel reactor is 30 min.

Examples 19 to 22

The reaction results are shown in Table 5, which are otherwise the same as in example 1, with varying concentrations of the microfluidic channel reactants:

table 5: effect of reactant concentration on the reaction

The results in Table 5 show that when the volume ratio of organic solvent DMSO to tertiary amyl alcohol in the reactor is 1:14, the molar ratio of reactant uridine to divinyl adipate is 1:9, the reaction temperature is 30 ℃, the reaction time is 30min, and the concentration of uridine in the reaction system is 0.05mmol/mL, the reaction conversion rate is as high as 93%, so that the optimal concentration of reactant in the microfluidic microchannel reactor is 0.05 mmol/mL.

Comparative examples 1 to 3

The results are shown in Table 6 with the same procedures as in example 1 except that the catalysts in the microfluidic microchannel reactor were changed to lipase Lipozyme RM IM (comparative example 1), lipase Novozym 435 (comparative example 2) and subtilisin (comparative example 3), respectively.

Table 6: effect of different enzymes on reaction conversion and selectivity

The results in table 6 show that for the regioselective esterification of enzymatic uridine in microfluidic channel reactors, different enzymes have a very significant effect on the reaction. The conversion of 5' -O-ethyleneadipamide was 49% by the lipase Lipozyme RMIM-catalyzed reaction. Whereas the conversion of 5' -O-ethyleneadipamide was only 10% using subtilisin to catalyze the reaction. From the results in table 6, the most efficient catalyst for the regioselective esterification of enzymatic uridine in microfluidic channel reactors was the lipase Lipozyme TLIM with 93% conversion and 98% selectivity for uridine.

Comparative examples 4 to 5

The results of changing different types of uridine compounds in the microfluidic microchannel reactor to 1mmol of uridine in example 1 to 1mmol of guanosine (comparative example 4), 1mmol of 3' -deoxyuridine (comparative example 5), and the same examples as in example 1 are shown in Table 7.

Table 7: effect of different nucleosides on the reaction

The results in Table 7 show that different nucleosides have different reaction results for regioselective esterification of enzymatic nucleosides in microfluidic channel reactors. Under the same reaction conditions, the conversion of 3' -deoxyuridine was only 25%, which makes the reaction difficult. The reaction results for guanosine were also not ideal and the conversion was only 33%. From the results in table 7, it can be seen that uridine can achieve the desired reaction results for the regioselective esterification of enzymatic nucleosides in microfluidic channel reactors, with 93% conversion and 98% selectivity.

Claims

1. A method for synthesizing 5' -O-ethylene adipamide on line by lipase catalysis is characterized in that: the method adopts a microfluidic channel reactor, wherein the microfluidic channel reactor comprises an injection pump, an injector, a reaction channel and a product collector, the injector is arranged in the injection pump and is connected with an inlet of the reaction channel through an interface, the product collector is connected with an outlet of the reaction channel through an interface, 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: dimethyl sulfoxide and tert-amyl alcohol in a volume ratio of 1: 8-16 are used as reaction solvents, uridine and divinyl adipate in a molar ratio of 1: 5-13 are used as raw materials, 0.5-1.0 g of lipase TLIM is used as a catalyst, the raw materials and the reaction solvents are placed in an injector, the lipase TLIM is uniformly filled in a reaction channel, the raw materials and the reaction solvents are continuously introduced into the reaction channel under the driving of an injection pump for acylation reaction, the concentration of uridine in the reaction system is 0.03-0.07 mmol/mL, the acylation reaction temperature is controlled to be 15-50 ℃, the acylation reaction time is 20-35 min, the reaction liquids are collected on line through a product collector, and the 5' -O-ethyleneadipamide uridine is obtained after post-treatment of the reaction liquids.

2. The lipase-catalyzed, on-line synthesis of 5' -O-ethylene uridine adipamide according to claim 1, wherein: the method comprises the following steps: dissolving uridine with a certain amount of dimethyl sulfoxide, adding tert-amyl alcohol to a certain volume, and filling into an injector for later use; dissolving divinyl adipate to a certain volume by using tertiary amyl alcohol, and filling the divinyl adipate into another syringe for later use; then the raw materials and the reaction solvent are led into the reaction channel to react under the driving of a syringe pump.

3. The lipase-catalyzed, on-line synthesis of 5' -O-ethylene uridine adipamide according to claim 1, wherein: the microfluidic channel reactor comprises a thermostat, and the reaction channel is arranged in the thermostat.

4. The lipase-catalyzed, on-line synthesis of 5' -O-ethylene uridine adipamide according to claim 2, wherein: the microfluidic channel reactor comprises a thermostat, and the reaction channel is arranged in the thermostat.

5. The method for the lipase-catalyzed online synthesis of 5' -O-ethylene uridine adipamide according to any one of claims 1 to 4, wherein: the volume ratio of dimethyl sulfoxide to tert-amyl alcohol in the reaction system is 1: 12-16, the molar ratio of uridine to divinyl adipate is 1: 7-13, the concentration of uridine in the reaction system is 0.04-0.06 mmol/mL, the acylation reaction temperature is 20-40 ℃, and the acylation reaction time is 25-35 min.

6. The lipase-catalyzed, on-line process for the synthesis of 5' -O-ethylene uridine adipamide according to claim 5, wherein: the molar ratio of uridine to divinyl adipate is 1: 9-11.

7. The lipase-catalyzed, on-line process for the synthesis of 5' -O-ethylene uridine adipamide according to claim 6, wherein: the volume ratio of dimethyl sulfoxide to tert-amyl alcohol in the reaction system is 1:14, the molar ratio of uridine to divinyl adipate is 1:9, the concentration of uridine in the reaction system is 0.05mmol/mL, the acylation reaction temperature is 30 ℃, and the acylation reaction time is 30 min.