CN107384993A - A kind of method of lipase-catalyzed online synthesis 5 '-O- acetyl -5-methyl-uridin - Google Patents
A kind of method of lipase-catalyzed online synthesis 5 '-O- acetyl -5-methyl-uridin Download PDFInfo
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- CN107384993A CN107384993A CN201610327830.4A CN201610327830A CN107384993A CN 107384993 A CN107384993 A CN 107384993A CN 201610327830 A CN201610327830 A CN 201610327830A CN 107384993 A CN107384993 A CN 107384993A
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Abstract
The invention discloses a kind of method of the lipase-catalyzed online methyluridine of synthesis 5'O acetyl 5.Methods described includes：Using volume ratio as 1:8~16 dimethyl sulfoxide and tert-pentyl alcohol is reaction dissolvent, using mol ratio as 1:5~13 5 methyluridines and vinyl acetate are raw material; using 0.5~1.0g Lipozymes TLIM as catalyst; raw material and reaction dissolvent are placed in syringe; Lipozyme TLIM is uniformly filled in the reaction channel of microfluidic channel reactor; raw material and reaction dissolvent is continuously passed through in reaction channel under the promotion of syringe pump and carry out acylation reaction; the reaction channel internal diameter of the microfluidic channel reactor is 0.8~2.4mm, a length of 0.5~1.0m of reaction channel；It is 15~50 DEG C to control esterification reaction temperature, and reaction time of esterification is 20~35min, collects reaction solution online by product collector, reaction solution obtains the methyluridine of 5'O acetyl 5 through conventional post processing.The present invention has the advantages of reaction time is short, selectivity is high and yield is high.
Description
(1) technical field
The present invention relates to a kind of method of lipase-catalyzed online controllable selectivity synthesis 5'-O- acetyl -5-methyl-uridin.
(2) background technology
Nucleoside medicine occupies an important position in the treatment of viral disease.The antiviral agent clinically used at present
In thing, nucleoside medicine proportion is up to more than 60%.Most nucleoside class compound is polyol, exist polarity compared with
It is high, intestinal permeability is relatively low, it is fat-soluble poor, toxic side effect is big and oral administration biaavailability is relatively low the defects of.Ucleosides chemical combination
Its is fat-soluble by after being esterified and modifying, can strengthening for thing, improves pharmacological activity, improves its oral administration biaavailability.In commonization
In method esterification process, multiple hydroxyls are likely to participate in esterification, and product is the mixture of monoesters and polyester, thus needs through " base
Three steps of group's protection --- esterification --- deprotection group " could obtain the product of single position esterification.And enzyme has to substrate
Good selectivity and selectivity, selectively some hydroxyl of nucleosides can be esterified, reaction selectivity is higher, reduces
The difficulty of product later separation, therefore biocatalysis technology plays more and more important angle in the esterification of nucleoside compound
Color.
It is micro-fluidic learn (Microfluidics) be in micron scale construction manipulation nanoliter to picoliters volume fluid technology with
Science, it is the new cross discipline to emerge rapidly nearly ten years.Currently, the development of micro-fluidic has surmounted originally main significantly
For the purpose of analytical chemistry service, and turn into whole chemistry subject, life science, instrumental science or even information science new one
Take turns the important technological platform of innovation research.
The text that a first piece synthesizes compound in micro-fluidic chip microreactor has been delivered from Harrison seminars in 1997
After offering, micro-fluidic chip reactor has been successfully used to a variety of organic synthesis, and illustrates the prospect of being widely applied.With
The development of microring array, micro-reacting tcchnology in micro-fluidic chip, synthetic reaction is carried out in the chips and has become micro-fluidic chip neck
One of the study hotspot in domain.
Compared with conventional chemical reactor, micro passage reaction, which not only has, makes the diffusion length between reactant contract significantly
It is short, and mass transfer velocity is fast；The easy control of reaction conditions such as reactant ratio, temperature, reaction time and flow velocity, side reaction compared with
It is few；Need reactant dosage little, can not only reduce the dosage of expensive, poisonous adverse reaction thing, caused ring in course of reaction
Border pollutant is also few, is a kind of environment-friendly, study on the synthesis novel substance technology.
At present, more domestic and foreign scholars grind the Enzyme catalyzed synthesis of nucleosides acylation reaction in organic media
Study carefully, but this method is catalyzed from acylase more, generally requires longer reaction time (12-24h), and the conversion reacted
Rate and selectivity be not high, therefore we have studied lipase-catalyzed online synthesis 5'-O- acetyl -5- methyl in micro passage reaction
The method of uridine, it is intended to find a kind of online controllable selectivity synthesis side of 5'-O- acetyl -5-methyl-uridin of high-efficiency environment friendly
Method.
(3) content of the invention
The technical problem to be solved in the present invention is to provide lipase-catalyzed online synthesis in a kind of microfluidic channel reactor
The new technology of 5'-O- acetyl -5-methyl-uridin, there is the advantages of reaction time is short, yield is high, selectivity is good.
In order to solve the above technical problems, the present invention adopts the following technical scheme that：
A kind of method of lipase-catalyzed online synthesis 5'-O- acetyl -5-methyl-uridin, methods described is using micro-fluidic logical
Road reactor, described microfluidic channel reactor include syringe pump, syringe, reaction channel and product collector, the note
Emitter is installed in syringe pump, is connected by an interface with reaction channel entrance, the product collector passes through an interface
Export and connect with reaction channel, the reaction channel internal diameter is 0.8~2.4mm, a length of 0.5~1.0m of reaction channel；The side
Method includes：Using volume ratio as 1:8~16 dimethyl sulfoxide (DMSO) and tert-pentyl alcohol is reaction dissolvent, using mol ratio as 1:5~13
5-methyl-uridin and vinyl acetate be raw material, using 0.5~1.0g Lipozymes TLIM as catalyst, by raw material
It is placed in reaction dissolvent in syringe, Lipozyme TLIM is uniformly filled in reaction channel, in pushes away for syringe pump
Make raw material and reaction dissolvent continuously be passed through in reaction channel under dynamic to carry out acylation reaction, make reaction system (raw material+reaction dissolvent)
The concentration of middle 5-methyl-uridin is 0.03~0.07mmol/mL, and it is 15~50 DEG C to control acylation reaction temperature, the acylation reaction time
For 20~35min, reaction solution is collected by product collector online, reaction solution obtains 5'-O- acetyl -5- first through conventional post processing
Base uridine.
In the microfluidic channel reactor that the present invention uses, the syringe number can be one or more, depending on specific
Depending on reaction requirement.For example when using two syringes, T-shaped or Y type interfaces can be used different reactants is entered from two
Mouth introduces, and confluxes into public reaction channel, by the contact of middle reactant molecule and the collision probability increase of microchannel, makes two
Stock reaction liquid stream is mixed and reacted in public reaction channel.
Described microfluidic channel reactor also includes insulating box, and described reaction channel is placed in insulating box, can with this
With effective controlling reaction temperature.Described insulating box can voluntarily select according to reaction temperature requirement, such as constant temperature water box etc..
The present invention is unlimited for the material of reaction channel, it is recommended to use green, the material of environmental protection, such as silicone tube；For
The shape of reaction channel is preferably shaped form, it is ensured that reaction solution stably passes through.
The present invention first dissolves 5-methyl-uridin in implementation process with DMSO, is further continued for plus tert-pentyl alcohol is to certain volume, fill
It is standby in syringe；Then vinyl acetate is dissolved to certain volume with tert-pentyl alcohol, loaded on standby in another syringe；Finally
Raw material and reaction dissolvent is set to be passed through in reaction channel and be reacted under syringe pump (such as the syringe pumps of PD 1200) promotion.
In the present invention, described Lipozyme TLIM believes the business of (Novozymes) company production using Novi
Product, it is a kind of by microorganism preparation, the system of 1,3 position-specifics, food-grade lipase (EC3.1.1.3) on particle silica gel
Agent.It is obtained from Thermomyceslanuginosus, with a kind of gene-modified aspergillus oryzae (Aspergillusoryzae)
Microorganism is by submerged fermentation production.
Further, the volume ratio of dimethyl sulfoxide and tert-pentyl alcohol is preferably 1 in the reaction dissolvent:12~1:16, be preferably
1:14。
Further, the mol ratio of the 5-methyl-uridin and vinyl acetate is preferably 1:7~13, more preferably 1：9~11,
Most preferably 1:9.
Further, the concentration of 5-methyl-uridin is preferably 0.04~0.06mmol/mL in reaction system, is most preferably
0.05mmol/mL。
Further, the acylation reaction temperature is preferably 20~40 DEG C, most preferably 30 DEG C.
Further, the acylation reaction time is preferably 25~35min, most preferably 30min.
The reaction product of the present invention can be collected online, and gained reaction solution be able to can be obtained by conventional post-processing approach
5'-O- acetyl -5-methyl-uridin.The conventional post-processing approach can be：Gained reaction solution, which is evaporated under reduced pressure, removes solvent, uses
200-300 mesh silica gel wet method dress posts, elution reagent is ethyl acetate:Methanol=20:1, after sample is dissolved with a small amount of elution reagent
Wet method upper props, eluent, while TLC tracking elution processes are collected, the obtained eluent containing single product is merged and is evaporated,
White solid, as 5'-O- acetyl -5-methyl-uridin can be obtained.
Compared with prior art, beneficial effects of the present invention are：The present invention utilizes fat in microfluidic channel reactor
Enzymatic synthesizes 5'-O- acetyl -5-methyl-uridin online, and the method not only significantly shortens the reaction time, and with high
Conversion ratio and selectivity；Reduced anti-using economic Lipozyme TLIM catalysis nucleosides esterifications first simultaneously
Cost is answered, the advantage with economical and efficient.
(4) illustrate
Fig. 1 is the structural representation for the microfluidic channel reactor that the embodiment of the present invention uses.
(5) embodiment
Protection scope of the present invention is described further with specific embodiment below, but protection scope of the present invention is unlimited
In this：
The structural reference Fig. 1 for the microfluidic channel reactor that the embodiment of the present invention uses, including a syringe pump (do not show
Show), two syringes 1 and 2, reaction channel 3, constant temperature water box (5, only show its floor map) and product collector 4；Two
Individual syringe 1 and 2 is installed in syringe pump, is connected by a Y types interface with the entrance of reaction channel 3, the reaction channel 3 is put
In constant temperature water box 5, by the controlling reaction temperature of constant temperature water box 5, the internal diameter 2.0mm of described reaction channel 3, pipe range
1m, the outlet of reaction channel 3 are connected by an interface with product collector 4.
Embodiment 1：The synthesis of 5'-O- acetyl -5-methyl-uridin
Device is with reference to figure 1：5-methyl-uridin (1.0mmol) is dissolved in 1.33mLDMSO and 8.67mL tert-pentyl alcohols,
Vinyl acetate (9.0mmol, 2.3mL) is dissolved in 10mL tert-pentyl alcohols, is then loaded on respectively standby in 10mL syringes.
0.87g Lipozymes TLIM is uniformly filled in reaction channel, under the promotion of the syringe pumps of PD 1200, two-way reaction solution
Respectively with 10.4 μ Lmin-1Flow velocity by " Y " joint enter reaction channel in reacted, controlled by constant temperature water box
For temperature of reactor at 30 DEG C, reaction solution continuous flowing reactive 30min, reaction result in reaction channel pass through thin-layer chromatography TLC
Tracing detection.
Reaction solution is collected by product collector online, is evaporated under reduced pressure and removes solvent, is filled with 200-300 mesh silica gel wet method
Post, elution reagent are ethyl acetate:Methanol=20:1, pillar height 35cm, column diameter 4.5cm, sample are dissolved with a small amount of elution reagent
Wet method upper prop afterwards, eluent collect flow velocity 2mLmin-1, while TLC tracking elution processes, it will obtain containing single product
Eluent, which merges, to be evaporated, and obtains white solid, obtains 5'-O- acetyl -5-methyl-uridin, HPLC detection 5-methyl-uridin conversion ratios
80%, selectivity 98%.
Nuclear-magnetism characterization result is as follows：
1H-NMR(DMSO-d6,δ,ppm)：11.36(s,H3), 7.43 (d, J=1Hz, H6), 5.77 (d, 1H, J=5Hz,
H1'), 5.43 (d, 1H, J=5.5Hz, 3'-OH), 5.27 (d, 1H, J=5.5Hz, 2'-OH), 4.23 (m, 2H, H2'+H3'),
4.07(m,1H,H4'),3.96(m,2H,H5'),2.06(s,3H,H2″), 1.80 (d, 3H, J=0.5Hz, 5-CH 3).
13C NMR(DMSO-d6,ppm)：170.20(C1″),163.78(C4),150.76(C2),136.58(C6),109.73
(C5),88.39(C1'),81.04(C4'),72.49(C3'),69.88(C2'),63.87(C5'),20.65(C2″),12.09(5-CH3).
Embodiment 2-6
Change organic solvent volume ratio in microfluidic channel reactor, control temperature as 50 DEG C, other are with embodiment 1, instead
Answer result as shown in table 1：
Table 1:Organic solvent compares the influence of reaction
The result of table 1 shows, when flow velocity is 10.4 μ Lmin-1, the reaction time is 30min, and reaction temperature is 50
DEG C, reactant 5-methyl-uridin and vinyl acetate mol ratio are 1:9,5-methyl-uridin concentration is in reaction system
During 0.05mmol/mL, conversion ratio increases with organic solvent volume in reactor than increase, works as DMSO:Tert-pentyl alcohol volume ratio reaches
To 1:Reach optimal when 14, being further continued for increase volume ratio will cause reactant dissolving not exclusively to reduce conversion ratio.So this
Optimal organic solvent volume ratio is 1 in micro-fluidic micro passage reaction in invention:14.
Embodiment 7-11
Change the substrate mol ratio of 5-methyl-uridin and vinyl acetate in micro-fluidic micro passage reaction, control temperature 50
DEG C, other are with embodiment 1, as a result as shown in table 2：
Table 2:5-methyl-uridin and influence of the vinyl acetate substrate mol ratio to reaction
The result of table 2 shows, when flow velocity is 10.4 μ Lmin-1, the reaction time is 30min, and reaction temperature is 50
DEG C, organic solvent DMSO in reactor:Tert-pentyl alcohol volume ratio is 1:14,5-methyl-uridin concentration is in reaction system
During 0.05mmol/mL, with the increase of reactant vinyl acetate, the conversion ratio of reaction also increases as, and works as 5-methyl-uridin
It is 1 with vinyl acetate substrate mol ratio:When 9, the conversion ratio of reaction is optimal, so micro-fluidic microchannel plate in the present invention
It is 1 to answer optimal substrate mol ratio in device:9.
Embodiment 12-15
Change the temperature of microfluidic channel reactor, with embodiment 1, reaction result is as shown in table 3 for other：
Table 3：Influence of the temperature to reaction
The result of table 3 shows, when flow velocity is 10.4 μ Lmin-1, the reaction time is 30min, organic solvent in reactor
DMSO:Tert-pentyl alcohol volume ratio is 1:14, reactant 5-methyl-uridin and vinyl acetate mol ratio are 1:9, reaction system
When middle 5-methyl-uridin concentration is 0.05mmol/mL, when reaction temperature is in 30 DEG C, the conversion ratio of reaction is optimal, temperature
Or the Tai Gao or too low activity that will all influence enzyme.So optimum temperature is 30 DEG C in micro-fluidic micro passage reaction in the present invention.
Embodiment 16-18
Change the reaction time of microfluidic channel reactor, with embodiment 1, reaction result is as shown in table 4 for other：
Table 4：Influence of the reaction time to reaction
The result of table 4 shows, as organic solvent DMSO in reactor:Tert-pentyl alcohol volume ratio is 1:14, reactant 5- first
Base uridine and vinyl acetate mol ratio are 1:9, reaction temperature is 30 DEG C, and 5-methyl-uridin concentration is in reaction system
During 0.05mmol/mL, when reacted between when be 30min, reaction conversion ratio reaches optimal 80%, with miniflow in the present invention
It is 30min to control optimum reacting time in micro passage reaction.
Embodiment 19-22
Change the concentration of microfluidic channel reactant, with embodiment 1, reaction result is as shown in table 5 for other：
Table 5：Influence of the reactant concentration to reaction
The result of table 5 shows, as organic solvent DMSO in reactor:Tert-pentyl alcohol volume ratio is 1:14, reactant 5- first
Base uridine and vinyl acetate mol ratio are 1:9, reaction temperature is 30 DEG C, and the reaction time is 30min, 5-methyl-uridin
When concentration is 0.05mmol/mL, reaction conversion ratio is up to 80%, so in the present invention in micro-fluidic micro passage reaction most
Good reactant concentration is 0.05mmol/mL.
Comparative example 1-3
Change the catalyst in micro-fluidic micro passage reaction, be changed to Lipozyme RM IM (comparative examples respectively
1), lipase Novozym 435 (comparative example 2), bacillus alkaline protease (comparative example 3), other are with embodiment 1, as a result
As shown in table 6.
Table 6：Influence of the different enzymes to reaction conversion ratio and selectivity
The result of table 6 shows, is esterified instead for the regioselectivity of enzymatic 5-methyl-uridin in microfluidic channel reactor
For answering, different enzymes has fairly obvious influence to reaction.Utilize Lipozyme RMIM catalytic reactions, 5'-O-
The conversion ratio of acetyl -5-methyl-uridin is 40%.And it is catalyzed the reaction, 5'-O- acetyl -5- using bacillus alkaline protease
The conversion ratio of methyluridine is only 5%.In terms of the result of table 6, for enzymatic 5-methyl-uridin in microfluidic channel reactor
For regioselectivity esterification, maximally effective catalyst is Lipozyme TLIM, the conversion ratio of 5-methyl-uridin
For 80%, selectivity is 98%.
Comparative example 4-5
Change different types of uridine compound in micro-fluidic micro passage reaction, by the 5-methyl-uridin of embodiment 1
1mmol be changed to respectively guanosine 1mmol (comparative example 4), 3'- BrdUs 1mmol (comparative example 5), other with embodiment 1, as a result
As shown in table 7.
Table 7：Influence of the different nucleoside compounds to reaction
The result of table 7 shows, is esterified for the regioselectivity of enzymatic nucleoside compound in microfluidic channel reactor
For reaction, different nucleoside compounds has different reaction results.3'- BrdUs under the same reaction conditions,
Conversion ratio is only 17%, and reaction is relatively difficult.The reaction result of guanosine is also undesirable, and conversion ratio is only 23%.From the result of table 7
See, for the regioselectivity esterification of enzymatic nucleoside compound in microfluidic channel reactor, 5-methyl-uridin
More satisfactory reaction result can be obtained, the conversion ratio of reaction can reach 80%, and selectivity is 98%.
Claims (7)
- A kind of 1. method of lipase-catalyzed online synthesis 5'-O- acetyl -5-methyl-uridin, it is characterised in that：Methods described is adopted With microfluidic channel reactor, described microfluidic channel reactor includes syringe pump, syringe, reaction channel and collection of products Device, the syringe are installed in syringe pump, are connected by an interface with reaction channel entrance, the product collector passes through One interface connects with reaction channel outlet, and the reaction channel internal diameter is 0.8~2.4mm, and reaction channel a length of 0.5~ 1.0m；Methods described includes：Using volume ratio as 1:8~16 dimethyl sulfoxide and tert-pentyl alcohol is reaction dissolvent, using mol ratio as 1:5 ~13 5-methyl-uridin and vinyl acetate are raw material, will using 0.5~1.0g Lipozymes TLIM as catalyst Raw material and reaction dissolvent are placed in syringe, Lipozyme TLIM are uniformly filled in reaction channel, in syringe pump Promotion under make raw material and reaction dissolvent continuously be passed through in reaction channel to carry out acylation reaction, make 5-methyl-uridin in reaction system Concentration be 0.03~0.07mmol/mL, control acylation reaction temperature be 15~50 DEG C, the acylation reaction time is 20~35min, Reaction solution, the post-treated 5'-O- acetyl -5-methyl-uridin obtained shown in formula I of reaction solution are collected by product collector online；
- 2. the method for lipase-catalyzed online synthesis 5'-O- acetyl -5-methyl-uridin as claimed in claim 1, its feature exist In：Described method comprises the following steps：5-methyl-uridin first is dissolved with a certain amount of dimethyl sulfoxide, then adds tert-pentyl alcohol to certain Volume, loaded on standby in syringe；Vinyl acetate is dissolved to certain volume with tert-pentyl alcohol, loaded on standby in another syringe； Then raw material and reaction dissolvent is made to be passed through in reaction channel and be reacted under syringe pump promotion.
- 3. the method for lipase-catalyzed online synthesis 5'-O- acetyl -5-methyl-uridin as claimed in claim 1, its feature exist In：The microfluidic channel reactor includes insulating box, and the reaction channel is placed in insulating box.
- 4. the method for lipase-catalyzed online synthesis 5'-O- acetyl -5-methyl-uridin as claimed in claim 2, its feature exist In：The microfluidic channel reactor includes insulating box, and the reaction channel is placed in insulating box.
- 5. the method for lipase-catalyzed online synthesis 5'-O- acetyl -5-methyl-uridin as described in one of Claims 1 to 4, its It is characterised by：The volume ratio of dimethyl sulfoxide and tert-pentyl alcohol is 1 in the reaction system:12~16, the 5-methyl-uridin and second The mol ratio of vinyl acetate is 1:7~13, the concentration of 5-methyl-uridin is 0.04~0.06mmol/mL in reaction system, described Acylation reaction temperature is 20~40 DEG C, and the acylation reaction time is 25~35min.
- 6. the method for lipase-catalyzed online synthesis 5'-O- acetyl -5-methyl-uridin as claimed in claim 5, its feature exist In：The mol ratio of the 5-methyl-uridin and vinyl acetate is 1:9~11.
- 7. the method for lipase-catalyzed online synthesis 5'-O- acetyl -5-methyl-uridin as claimed in claim 6, its feature exist In：The volume ratio of dimethyl sulfoxide and tert-pentyl alcohol is 1 in the reaction system:14, the 5-methyl-uridin and vinyl acetate Mol ratio is 1:9, the concentration of 5-methyl-uridin is 0.05mmol/mL in reaction system, and the acylation reaction temperature is 30 DEG C, institute It is 30min to state the acylation reaction time.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107955822A (en) * | 2017-12-21 | 2018-04-24 | 浙江工业大学 | A kind of lipase-catalyzed online synthesis S-（4- methyl-benzyls）The method of laurate thioesters |
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Application publication date: 20171124 |