Summary of the invention
In view of this, the technical problem to be solved in the present invention is the catalyzer being to provide a kind of asymmetric heterocyclic ketone Aldol reaction.Another technical problem that will solve of the present invention is to provide a kind of reaction method of catalyzer of asymmetric heterocyclic ketone Aldol reaction.
The present invention is solved the problems of the technologies described above by following technical proposals:
A catalyzer for asymmetric heterocyclic ketone Aldol reaction, is characterized in that, containing caricin.
A reaction method for the catalyzer of asymmetric heterocyclic ketone Aldol reaction, its step comprises:
(1) in reaction vessel, aromatic aldehyde phenyl ring being connected with electron-withdrawing substituent and heterocyclic ketone is added, the mol ratio of aromatic aldehyde and heterocyclic ketone is 1: (0.5-5), solubilizing agent again, and papain enzyme catalyst, caricin is 25-200mg/ (0.25mmol heterocyclic ketone);
(2) temperature of reaction 15-40 DEG C, stirring reaction 12-144 hour is controlled;
(3) by reacting liquid filtering, filter cake extraction agent washs, merging filtrate and washings, then adds deionized water dilution, extracts with extraction agent;
(4) by after the extraction liquid anhydrous sodium sulfate drying of extraction, then carry out underpressure distillation desolventizing, obtain product.
In order to obtain better technique effect, heterocyclic ketone described in step (1) is one or more combinations in piperidone, pyrone, thiapyrones; In step (1), the aromatic aldehyde described phenyl ring being connected with electron-withdrawing substituent is one or more combinations in 4-fluorobenzaldehyde, 3-fluorobenzaldehyde, 2-fluorobenzaldehyde, 4-chlorobenzaldehyde, 3-chlorobenzaldehyde, 2-chlorobenzaldehyde, 4-bromobenzaldehyde, 3-bromobenzaldehyde, 2-bromobenzaldehyde, 4-nitrobenzaldehyde, 3-nitrobenzaldehyde, 2-nitrobenzaldehyde, 4-cyanobenzaldehyde, 3-cyanobenzaldehyde, 2-cyanobenzaldehyde; Organic solvent in step (1) is one or more combinations in tetrahydrofuran (THF), DMF, acetonitrile, methyl-sulphoxide, Virahol, Isosorbide-5-Nitrae-dioxane, chloroform; In step (1), described solvent is the mixed solvent that deionized water and organic solvent form, V (H
2o)/V (H
2o+ organic solvent) volume ratio is (0-3)/10; Solvent is (0.5-5) mL/ (0.25mmol heterocyclic ketone); In step (1), the mol ratio of aromatic aldehyde and heterocyclic ketone is preferably 1: 0.5, caricin is preferably 100mg/ (0.25mmol heterocyclic ketone), solvent is preferably 1mL/ (0.25mmol heterocyclic ketone), solvent is the mixed solvent that deionized water and organic solvent form, V (H
2o)/V (H
2o+ organic solvent)=2.0: 10; In step (2), temperature of reaction is preferably 35 DEG C, and the reaction times is preferably 120 hours; In step (3), described extraction agent is methylene dichloride or ethyl acetate; After step (4), the mixing solutions of described product flash column chromatography eluent to be volume ratio be sherwood oil and ethyl acetate, obtains the productive rate of 37%.
Late Cambrian caricin of the present invention can as the catalyzer of asymmetric heterocyclic ketone Aldol reaction, particularly be applicable to aromatic aldehyde or the asymmetric heterocyclic ketone Aldol between assorted aromatic aldehyde and heterocyclic ketone reacts, there is the advantages such as catalytic activity is good, enantioselectivity is high, economy, environmental protection, not only widen the range of application of caricin, and provide a kind of excellent biological enzyme agent for asymmetric heterocyclic ketone Aldol reacts, have broad application prospects in organic synthesis field.
The present invention's chemical reagent used is as follows:
Caricin (Papain, >=3unit/mg, article No.: P104243), Shanghai company of Jing Chun biochemical technology limited-liability company buys;
Piperidone;
Pyrone;
Thiapyrones;
Tetrahydrofuran (THF);
DMF;
Acetonitrile;
Methyl-sulphoxide;
Virahol;
Isosorbide-5-Nitrae-dioxane;
Chloroform;
Deionized water;
Phenyl ring being connected with the aromatic aldehyde of electron-withdrawing substituent, is one or more combinations in 4-fluorobenzaldehyde, 3-fluorobenzaldehyde, 2-fluorobenzaldehyde, 4-chlorobenzaldehyde, 3-chlorobenzaldehyde, 2-chlorobenzaldehyde, 4-bromobenzaldehyde, 3-bromobenzaldehyde, 2-bromobenzaldehyde, 4-nitrobenzaldehyde, 3-nitrobenzaldehyde, 2-nitrobenzaldehyde, 4-cyanobenzaldehyde, 3-cyanobenzaldehyde, 2-cyanobenzaldehyde;
The two multiplex vacuum pump of A circulating water type of reaction kit: SHB-III; RE52CS-1 type Rotary Evaporators; ABL-224 type analysis balance; DLSB-5/10 type low-temperature cooling fluid circulating pump; DF-101S type heat collecting type constant-temperature heating magnetic stirring apparatus; DHJF-4002 type cryogenic thermostat stirring reaction is bathed; DHG-9076A type electric heating constant-temperature blowing drying box; KQ250DE type ultrasonic cleaner.
Analytical instrument: tlc (GF254 silica-gel plate) monitors reaction process; ZF-2I type ultraviolet analysis instrument for three purposed; Flash column chromatography adopts 100-200 order silica gel pressured column; Employing Bruker AV-300 type nmr determination proton nmr spectra (
1h NMR) and carbon spectrum (
13c NMR).Adopt HPLC chiral fixed phase method to measure enantiomeric excess value (ee) and the diastereomeric excess value (dr) of product, chiral stationary phase adopts AD-H, OD-H, AS-H and OJ-H chiral column.The productive rate provided in embodiment is the product yield after purifying, and anti represents trans product, and syn represents cis-product.
Embodiment
Comparative example
A kind of reaction method, its step comprises:
(1) in reaction vessel, aromatic aldehyde phenyl ring being connected with electron-withdrawing substituent and heterocyclic ketone is added, the mol ratio of aromatic aldehyde and heterocyclic ketone is 1: (0.5-5), add the mixed solvent 1mL of organic solvent and deionized water again, volume ratio is V (H
2o)/V (H
2o+ organic solvent)=(0-3)/10;
(2) at temperature control (15-40 DEG C) stirring reaction (12-144 hour);
(3) by reacting liquid filtering, filter cake extraction agent (methylene dichloride) washing, merging filtrate and washings, then add deionized water dilution, extract with extraction agent;
(4) by after the extraction liquid anhydrous sodium sulfate drying of extraction, then carry out underpressure distillation desolventizing, do not detect product.Embodiment 1
A catalyzer for asymmetric heterocyclic ketone Aldol reaction, is characterized in that, containing caricin.
Embodiment 2: the impact of solvent
The reaction method of the catalyzer of asymmetric heterocyclic ketone Aldol reaction described in embodiment 1, its step comprises:
(1) in reaction vessel, add the 4-nitrobenzaldehyde of 0.50mmol and the N-tertbutyloxycarbonyl-4-piperidone of 0.25mmol, the mol ratio of 4-nitrobenzaldehyde and N-tertbutyloxycarbonyl-4-piperidone is 1: 0.5, add the mixed solvent that the organic solvent of 1mL and deionized water form again, wherein the volume ratio of deionized water and mixed solvent is 1: 10, and caricin is 50mg;
(2) temperature of reaction 25 DEG C is controlled, stirring reaction 120 hours;
(3) by reacting liquid filtering, filter cake extraction agent washed with dichloromethane, merging filtrate and washings, then add deionized water dilution, use extraction agent dichloromethane extraction;
(4) by after the extraction liquid anhydrous sodium sulfate drying of extraction, then carry out underpressure distillation desolventizing, obtain product.
Reaction equation is:
The screening of the Aldol reaction solvent of table 1 papain enzyme catalysis
Reaction media is that the important factor of carrying out is reacted in impact, therefore we have studied the effect (table 1) of a series of solvent to reaction process.Relative to advantage enzymatic in water medium, in non-aqueous system, enzyme shows wonderful character equally, such as, improve the stability of enzyme, can catalysis non-natural reaction etc.From table, can clearly be seen that the catalytic activity impact of reaction solvent on caricin is very large.We find that enzyme shows good catalytic activity in hydrophilic solvent tetrahydrofuran (THF), acetonitrile, methyl-sulphoxide and DMF.
In addition, in order to prove that caricin has katalysis to this Aldol reaction, we devise a series of Control release.As can be seen from Table 1, when not enzyme-added, this Aldol reaction almost can not be carried out (table 1, sequence number 9).But when the bovine serum albumin (B.S.A.) of non-enzymatic and sodium bicarbonate catalysis, obtain the raceme product of 44% and 45% respectively, prove that non-zymoprotein can react by this Aldol of catalysis to a certain extent, but do not have stereoselectivity, ee value is 0.The ficin enzymic catalytic reaction crossed with Urea treatment still obtains the product of 5%, and does not have stereoselectivity.This three-dimensional structure stereoselectivity to product simultaneously describing enzyme is absolutely necessary.We also use serpin methyl methylthiomethyl sulfoxide (MMTS) treat enzyme, result only observed the product (table 1 of trace in chromatographic sheet, sequence number 11), this eliminates the possibility of the amino acid residue catalytic of enzyme, and the avtive spot further demonstrating ficin serves decisive action to catalysis multifunctionality.Above experimental result has absolutely proved that this asymmetric direct asymmetric Aldol reaction must occur on the special construction of ficin active site.
Embodiment 3: the impact of water content
The reaction method of the catalyzer of asymmetric heterocyclic ketone Aldol reaction described in embodiment 1, its step comprises:
(1) in reaction vessel, add the 4-nitrobenzaldehyde of 0.50mmol and the N-tertbutyloxycarbonyl-4-piperidone of 0.25mmol, the mol ratio of 4-nitrobenzaldehyde and N-tertbutyloxycarbonyl-4-piperidone is 1: 0.5, then adds the organic solvent CH of 0.5mL
3the mixed solvent of CN and deionized water, wherein H
2o/ (H
2o+CH
3cN) volume ratio is 0-3/10, and caricin is 50mg;
(2) temperature of reaction 25 DEG C is controlled, stirring reaction 120 hours;
(3) by reacting liquid filtering, filter cake extraction agent washed with dichloromethane, merging filtrate and washings, then add deionized water dilution, use extraction agent dichloromethane extraction;
(4) by after the extraction liquid anhydrous sodium sulfate drying of extraction, then carry out underpressure distillation desolventizing, obtain product.
Water is considered to play a part lubricant usually in the structure of enzyme, and it can increase the toughness of enzymatic structure, and water suitable in system can improve stability and the catalytic activity of enzyme, therefore, determines that in reaction system, the optimum content of water is extremely important.We devise the optimum moisture content that this Aldol reaction is found in experiment.We allow reaction at different ratios [H
2o/ (H
2o+CH
3cN) carry out in mixed reaction solvent], the results are shown in table 2.As can be seen from table we, along with water content is increased to 0.20 from 0, productive rate and ee value be increase (table 2, sequence number 1-5) constantly.When water-content is 0.20, productive rate and ee value reach best, and now productive rate is 34%.But once water-content is more than 0.20, productive rate and ee value start to reduce.Therefore, the preferred 0.15-0.25 of water content of the present invention, more preferably 0.2.
The impact that table 2 water content is reacted papain enzyme catalysis Aldol
Embodiment 4: the impact of substrate feed ratio
The reaction method of the catalyzer of asymmetric heterocyclic ketone Aldol reaction described in embodiment 1, its step comprises:
(1) in reaction vessel, add the 4-nitrobenzaldehyde of 0.125-1.25mmol and the N-tertbutyloxycarbonyl-4-piperidone of 0.25mmol, the mol ratio of 4-nitrobenzaldehyde and N-tertbutyloxycarbonyl-4-piperidone is 0.5-5: 1, then adds the organic solvent CH of 2mL
3the mixed solvent of CN and deionized water, wherein H
2o/ (H
2o+CH
3cN) volume ratio is 2/10, and caricin is 50mg;
(2) temperature of reaction 25 DEG C is controlled, stirring reaction 120 hours;
(3) by reacting liquid filtering, filter cake extraction agent washed with dichloromethane, merging filtrate and washings, then add deionized water dilution, use extraction agent dichloromethane extraction;
(4) by after the extraction liquid anhydrous sodium sulfate drying of extraction, then carry out underpressure distillation desolventizing, obtain product.
When the ratio of 4-nitrobenzaldehyde and ketone is 3: 1, obtain the good ee value (table 3 of the highest output, sequence number 4), but productive rate is also more or less the same when feed ratio is 2: 1, ee value is the highest, dr value also comparatively feed ratio be 3: 1 fashions, consider from the angle of Atom economy and reaction result, substrate feed ratio of the present invention (mol ratio of aldehyde/ketone) is preferably 1-3, is more preferably 2.
The impact that table 3 feed ratio reacts papain enzyme catalysis Aldol.
Embodiment 5: the impact of enzyme amount
The reaction method of the catalyzer of asymmetric heterocyclic ketone Aldol reaction described in embodiment 1, its step comprises:
(1) in reaction vessel, add the 4-nitrobenzaldehyde of 0.5mmol and the N-tertbutyloxycarbonyl-4-piperidone of 0.25mmol, the mol ratio of 4-nitrobenzaldehyde and N-tertbutyloxycarbonyl-4-piperidone is 2: 1, then adds the organic solvent CH of 5mL
3the mixed solvent of CN and deionized water, wherein H
2o/ (H
2o+CH
3cN) volume ratio is 2/10, and caricin is 25-200mg;
(2) temperature of reaction 25 DEG C is controlled, stirring reaction 120 hours;
(3) by reacting liquid filtering, filter cake extraction agent washed with dichloromethane, merging filtrate and washings, then add deionized water dilution, use extraction agent dichloromethane extraction;
(4) by after the extraction liquid anhydrous sodium sulfate drying of extraction, then carry out underpressure distillation desolventizing, obtain product.
As can be seen from Table 4, when enzyme carrying capacity is increased to 150mg from 25mg, productive rate continues to raise (table 4, sequence number 1-5), but productive rate is more or less the same during 75mg and 150mg enzyme amount, and during enzyme amount 75mg ee value and dr value the highest, therefore enzyme amount of the present invention is preferably 75-150mg, is more preferably 75mg.
The impact that table 4 enzyme carrying capacity is reacted papain enzyme catalysis Aldol
Embodiment 6: the impact of temperature of reaction
The reaction method of the catalyzer of asymmetric heterocyclic ketone Aldol reaction described in embodiment 1, its step comprises:
(1) in reaction vessel, add the 4-nitrobenzaldehyde of 0.5mmol and the N-tertbutyloxycarbonyl-4-piperidone of 0.25mmol, the mol ratio of 4-nitrobenzaldehyde and N-tertbutyloxycarbonyl-4-piperidone is 2: 1, then adds the organic solvent CH of 3mL
3the mixed solvent of CN and deionized water, wherein H
2o/ (H
2o+CH
3cN) volume ratio is 2/10, and caricin is 75mg;
(2) temperature of reaction 15-45 DEG C is controlled, stirring reaction 120 hours;
(3) by reacting liquid filtering, filter cake extraction agent washed with dichloromethane, merging filtrate and washings, then add deionized water dilution, use extraction agent dichloromethane extraction;
(4) by after the extraction liquid anhydrous sodium sulfate drying of extraction, then carry out underpressure distillation desolventizing, obtain product.
Temperature of reaction is also very large to the activity influence of enzyme, as what display in table 5, when temperature of reaction is 35 DEG C, and productive rate the highest (table 5, sequence number 1-6), and when temperature of reaction is 30 DEG C, ee value is best.Therefore temperature of reaction of the present invention is preferably 30-40 DEG C, is more preferably 30 DEG C.
The impact that table 5 temperature is reacted papain enzyme catalysis Aldol
Embodiment 7: the impact in reaction times
The reaction method of the catalyzer of asymmetric heterocyclic ketone Aldol reaction described in embodiment 1, its step comprises:
(1) in reaction vessel, add the 4-nitrobenzaldehyde of 0.5mmol and the N-tertbutyloxycarbonyl-4-piperidone of 0.25mmol, the mol ratio of 4-nitrobenzaldehyde and N-tertbutyloxycarbonyl-4-piperidone is 2: 1, then adds the organic solvent CH of 4mL
3the mixed solvent of CN and deionized water, wherein H
2o/ (H
2o+CH
3cN) volume ratio is 2/10, and caricin is 100mg;
(2) temperature of reaction 35 DEG C is controlled, stirring reaction 12-144 hour;
(3) by reacting liquid filtering, filter cake extraction agent washed with dichloromethane, merging filtrate and washings, then add deionized water dilution, use extraction agent dichloromethane extraction;
(4) by after the extraction liquid anhydrous sodium sulfate drying of extraction, then carry out underpressure distillation desolventizing, obtain product.
The present invention has investigated reaction process, and the time curve of whole process as shown in Figure 1.First day continues to increase to the 5th day productive rate, after the 5th day, increases not obvious.Ee value is little with the change in reaction times, and longly there is downtrending in the reaction times.Therefore, the reaction times of the present invention is preferably 120 hours.
Embodiment 8: the versatility that heterocyclic ketone and aromatic aldehyde Aldol react is investigated
The reaction method of the catalyzer of asymmetric heterocyclic ketone Aldol reaction described in embodiment 1, its step comprises:
(1) in reaction vessel, add the aromatic aldehyde of 0.5mmol and the heterocyclic ketone of 0.25mmol, the mol ratio of aromatic aldehyde and heterocyclic ketone is 2: 1, then adds the organic solvent CH of 1mL
3the mixed solvent of CN and deionized water, wherein H
2o/ (H
2o+CH
3cN) volume ratio is 2/10, and caricin is 150mg;
(2) temperature of reaction 35 DEG C is controlled, stirring reaction 120 hours;
(3) by reacting liquid filtering, filter cake extraction agent washed with dichloromethane, merging filtrate and washings, then add deionized water dilution, use extraction agent dichloromethane extraction;
(4) by after the extraction liquid anhydrous sodium sulfate drying of extraction, then carry out underpressure distillation desolventizing, obtain product.
The expansion situation of substrate is as shown in table 6.
Reaction equation is as follows:
The heterocyclic ketone of table 6 papain enzyme catalysis and the expansion situation of aromatic aldehyde Aldol reaction substrate
As can be seen from Table 6, the heterocyclic ketone of papain enzyme catalysis and the Aldol of aromatic aldehyde have been obtained by reacting good productive rate (21-37%) (table 6, sequence number 1-9), we find that on aromatic aldehyde phenyl ring, substituent sterically hindered and electronic effect has a significant impact reaction result.The product that the 2-nitrobenzaldehyde with maximum steric hindrance is obtained by reacting has best ee value, but productive rate even can not obtain along with the increase of steric hindrance reduces rapidly.Such as, thiapyrones and piperidone and the 2-nitrobenzaldehyde with maximum steric hindrance do not react, and the reaction of pyrone and 2-nitrobenzaldehyde increases along with phenyl ring steric hindrance, and productive rate declines and ee value improves (table 6, sequence number 7).In addition, all react better when aromatic aldehyde phenyl ring being connected with electron-withdrawing substituent.And when aromatic aldehyde phenyl ring is connected with electron donating group, have to trace product.This situation may be because electron-withdrawing substituent adds the positive polarity of carbonyl carbon, is more easily subject to the attack of negative charge on the carbon of ketone α position, thereby promotes the carrying out of reaction.We do not obtain the Aldol product of electron-donating group aromatic aldehyde.