CN106636248A - Method for preparing crizotinib intermediate by using carbonyl reductase - Google Patents
Method for preparing crizotinib intermediate by using carbonyl reductase Download PDFInfo
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- CN106636248A CN106636248A CN201611193868.3A CN201611193868A CN106636248A CN 106636248 A CN106636248 A CN 106636248A CN 201611193868 A CN201611193868 A CN 201611193868A CN 106636248 A CN106636248 A CN 106636248A
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- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/16—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing two or more hetero rings
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Abstract
The invention discloses a method for preparing a crizotinib intermediate by using carbonyl reductase. The method comprises the following steps: by taking 2',6'-dichloro-3'-fluoroacetophenone as a substrate and phosphate buffer as a reaction medium, adding the carbonyl reductase, a cofactor and a hydrogen donor to form a biological catalysis reaction system; performing a biological catalysis reaction to generate (S)-2',6'-dichloro-3'- fluorobenzene ethanol. In the method, the novel carbonyl reductase is used for catalyzing a reduction reaction, so that the method has the advantages of small using amount of a biological catalyst, mild reaction conditions, short reaction time, high yield, high optical purity and the like.
Description
Technical field
The present invention relates to biological pharmacy technical field, more particularly to a kind of to prepare gram azoles in the middle of Buddhist nun using carbonyl reductase
The method of body.
Background technology
Sai Kerui (Xalkori, gram azoles replaces Buddhist nun) is that the ATP of the suppression Met/ALK/ROS developed by Pfizer is competitive
Mutiple Targets kinases inhibitor.Confirm that gram azoles replaces Buddhist nun in the tumor patient of ALK, ROS and MET abnormal kinase respectively
There is notable clinical efficacy to human body.Xalkori is one of most quick medicine in tumour medicine research and development history, and 2011 in the U.S.
Create much of a stir behind city.
Gram azoles is as follows for the chemical structural formula of Buddhist nun：
(S) -2 ', 6 '-two chloro- 3 '-fluorobenzene ethanol are gram azoles for an important intermediate during Buddhist nun's chemical synthesis.But
At present main based on chemical synthesis, but the method complex steps, product yield is low, and pollution is big, is unsuitable for large-scale production
(US7858643B2).At present (S) -2 ', the biological preparation method of 6 '-two chloro- 3 '-fluorobenzene ethanol, need to make in catalytic process
Regenerating coenzyme is carried out with the coenzyme NAD P of costliness and using GDH, is unfavorable for the cheap preparation of the intermediate.
The content of the invention
It is an object of the invention to solve deficiencies of the prior art, there is provided a kind of to be prepared using carbonyl reductase
Gram azoles for Buddhist nun's intermediate method, method is simple, and product yield is high, low production cost, environmental protection, so as to meet gram azoles for Buddhist nun
Large-scale industrial production require.
The technical solution adopted for the present invention to solve the technical problems is：
A kind of utilization carbonyl reductase prepares method of gram azoles for Buddhist nun's intermediate, is with 2 ', 6 '-two chloro- 3 '-fluoro acetophenones
Substrate, phosphate buffer is reaction medium, adds carbonyl reductase, co-factor and hydrogen donor to form biocatalytic reaction system,
Generation biocatalytic reaction generates (S) -2 ', 6 '-two chloro- 3 '-fluorobenzene ethanol.
Preferably, the amino acid sequence of the carbonyl reductase is as shown in SEQ ID NO.1, the carbonyl reductase
Encoding gene is as shown in SEQ ID NO.2.
Preferably, the co-factor is NAD+。
Preferably, the hydrogen donor is isopropanol.
Preferably, the consumption of 2 ', 6 '-two chloro- 3 '-fluoro acetophenones is 50-100g in every liter of biocatalytic reaction system.
Preferably, the consumption of carbonyl reductase is 3-10g in every liter of biocatalytic reaction system.
Preferably, the consumption of co-factor is 0.05-0.1g in every liter of biocatalytic reaction system.
Preferably, the consumption of hydrogen donor is 50-100mL in every liter of biocatalytic reaction system.
Preferably, the pH of the phosphate buffer is 6.5-7.5.
Preferably, the temperature of the biocatalytic reaction is 25-35 DEG C, reaction time 8-12h.
The invention has the beneficial effects as follows：The present invention adopts new carbonyl reductase catalytic reduction reaction, urges with biology
The advantage such as agent usage amount is little, reaction condition is gentle, the reaction time is short, high income, optical purity are good.With the chemistry of prior art
Method is compared, and economy is high, pollute little, with important industrial application value.
Description of the drawings
Fig. 1 is the expression plasmid pET26-CM3 of the restructuring carbonyl reductase that the present invention builds；
Fig. 2 is that the SDS-PAGE of restructuring carbonyl reductase after e. coli bl21 (DE3)/pET26-CM3 abduction deliverings is electric
Swimming figure；Wherein M is Protein Marker product, No. 1 be it is broken after supernatant, No. 2 is restructuring carbonyl reductase after purification.
Fig. 3 is substrate and product HPLC liquid phase figure in course of reaction.(A) the liquid phase figure of 0h is reacted；(B) liquid after 6h is reacted
Phasor.
Specific embodiment
Below by specific embodiment, technical scheme is described in further detail.
In the present invention, if not refering in particular to, the raw material for being adopted and equipment etc. are commercially available or commonly used in the art.
Method in following embodiments, if no special instructions, is the conventional method of this area.
The process route of the present invention is as follows：
Embodiment 1：
(1) synthesis of carbonyl reduction enzyme coding gene：According to the original gene sequence (genebank of carbonyl reductase CM3
Sequence ID:ABB91667.1), codon is carried out to original gene sequence with table according to e. coli codon analysis excellent
Change and carry out the fully synthetic of genes of interest, gene chemical synthesis commission Shanghai JaRa bio tech ltd is carried out, the coding of acquisition
As shown in SEQ ID NO.2, the carbonyl reduction enzyme amino acid sequence after translation is as shown in SEQ ID NO.1 for gene order.Synthesis
Respectively band is with NdeI and XhoI restriction enzyme sites and is connected on pET26b (+) at encoding gene two ends afterwards, obtains recombinant expressed load
Body pET26b-CM3 (see Fig. 1).
(2) expression of carbonyl reductase：
E. coli bl21 (DE3) competent cell is prepared, by recombinant expression carrier pET26b-CM3 by heat-shock transformed
Method goes in expression bacterial strain BL21 (DE3) and is applied to (10g/L peptones, 0.5g/L yeast on the LB flat boards containing 50 μ g/mL
Powder, 10g/L sodium chloride, 20g/L agar, pH7.0), 37 DEG C of incubated overnights.Next day, picking single bacterium colony is linked into single bacterium colony
In LB nutrient solutions of the 5mL containing kanamycins, 37 DEG C, overnight, next day takes 1mL bacterium solutions and is added to containing kanamycins shaken cultivation
In 100mL TB culture mediums (12g/L peptones, 24g/L dusty yeasts, 4mL/L glycerine, 0.1M phosphate buffers, pH 7.0), 37
Shaken cultivation is to OD at DEG C600To 3.0, then add IPTG to final concentration of 0.1mM, Fiber differentiation is overnight at 25 DEG C.Fig. 2 says
Bright restructuring carbonyl reductase obtains correct expression in Escherichia coli.
(3) clasmatosis：Collects thalline after 5000g centrifugation 10min, with 0.1M phosphate buffers (pH7.0) buffer solution weight
Outstanding thalline, gained liquid is crude enzyme liquid after ultrasonication.
(4) biocatalytic reaction：The chloro- 3 '-fluoro acetophenone of 5g substrates 2 ', 6 '-two is added to containing 85mL phosphoric acid buffers
In the 250mL reactors of liquid (0.1M, pH7.0), subsequently, 15mL is contained into 5mL isopropanols, 10mg NAD+, 500mg carbonyls are also
The 0.1M phosphate buffers (pH7.0) of protoenzyme are added in the reactor.The magnetic agitation reaction at 30 DEG C, and adjust reaction with alkali
PH value so as to maintain 7.0 or so.Consumption and the generation situation of product using TLC qualitative detection substrate.
HPLC detection substrates consume completely (about 8-10 hours), plus NaCl is to saturation, are extracted with ethyl acetate 3 times, merge organic phase extraction
Take.Anhydrous sodium sulfate eliminating water, suction filtration, reduced pressure concentration obtains oily liquids, and chiral ee values are more than 99, and yield is 90.5%.Fig. 3 says
Bright to generate product with catalytic substrate using the restructuring carbonyl reductase, product peak is significantly increased after 6h, and substrate peak reduces.
(5) product identification：Target product is identified using nuclear magnetic resonance 1H NMR, (S) -2 ', 6 '-two chloro- 3 '-fluorine
(d, J=6.8Hz, 3H) 5.58 (q, the J=of benzyl carbinol nuclear magnetic data 1H NMR (400MHz, chloroform-D) δ ppm 1.65
6.9Hz,1H)6.96-7.10(m,1H)7.22-7.36(m,1H)。
Embodiment 2：
The present embodiment and embodiment difference are step (4)：
The chloro- 3 '-fluoro acetophenone of 100g substrates 2 ', 6 '-two is added to containing 850mL phosphate buffers (0.1M, pH7.0)
2.5L reactors in, subsequently, 150mL is contained into 100mL isopropanols, 50mg NAD+, the 0.1M phosphoric acid of 10g carbonyl reductases
Buffer solution (pH7.0) is added in the reactor.The magnetic agitation reaction at 30 DEG C, and adjust the pH value of reaction with alkali so as to tie up
Hold 7.0 or so.Consumption and the generation situation of product using TLC qualitative detection substrate.HPLC detection substrates are complete
Consume, plus NaCl is to saturation, is extracted with ethyl acetate 3 times, merges organic extractant phase.Anhydrous sodium sulfate eliminating water, suction filtration reduces pressure dense
Contracting, obtains oily liquids, and chiral ee values are more than 99, and product yield is 89.5%.
Embodiment 3：
The present embodiment and embodiment difference are step (4)：
The chloro- 3 '-fluoro acetophenone of 80g substrates 2 ', 6 '-two is added to containing 850mL phosphate buffers (0.1M, pH7.0)
2.5L reactors in, subsequently, 150mL is contained into 70mL isopropanols, 80mg NAD+, the 0.1M phosphoric acid of 3g carbonyl reductases delays
Rush liquid (pH7.0) to add in the reactor.The magnetic agitation reaction at 30 DEG C, and adjust the pH value of reaction with alkali so as to maintain
7.0 or so.Consumption and the generation situation of product using TLC qualitative detection substrate.HPLC detection substrates disappear completely
Consumption, plus NaCl is to saturation, is extracted with ethyl acetate 3 times, merges organic extractant phase.Anhydrous sodium sulfate eliminating water, suction filtration reduces pressure dense
Contracting, obtains oily liquids, and chiral ee values are more than 99, and product yield is 88.6%.
Embodiment described above is one kind preferably scheme of the present invention, not makees any pro forma to the present invention
Limit, also have other variants and remodeling on the premise of without departing from the technical scheme described in claim.
SEQUENCE LISTING
<110>Zhejiang Ocean university
<120>A kind of utilization carbonyl reductase prepares method of gram azoles for Buddhist nun's intermediate
<130> 2016.12
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 241
<212> PRT
<213>Artificial sequence
<400> 1
Met Thr Thr Thr Ser Asn Ala Leu Val Thr Gly Gly Ser Arg Gly Ile
1 5 10 15
Gly Ala Ala Ser Ala Ile Lys Leu Ala Gln Glu Gly Tyr Ser Val Thr
20 25 30
Leu Ala Ser Arg Ser Val Asp Lys Leu Asn Glu Val Lys Ala Lys Leu
35 40 45
Pro Ile Val Gln Asp Gly Gln Lys His Tyr Ile Trp Glu Leu Asp Leu
50 55 60
Ala Asp Val Glu Ala Ala Ser Ser Phe Lys Gly Ala Pro Leu Pro Ala
65 70 75 80
Ser Ser Tyr Asp Val Phe Val Ser Asn Ala Gly Val Ala Ala Phe Ser
85 90 95
Pro Thr Ala Asp His Asp Asp Lys Glu Trp Gln Asn Leu Leu Ala Val
100 105 110
Asn Leu Ser Ser Pro Ile Ala Leu Thr Lys Ala Leu Leu Lys Asp Val
115 120 125
Ser Glu Arg Pro Ala Asp Asn Pro Leu Gln Ile Ile Tyr Ile Ser Ser
130 135 140
Val Ala Gly Leu His Gly Ala Ala Gln Val Ala Val Tyr Ser Ala Ser
145 150 155 160
Lys Ala Gly Leu Asp Gly Phe Met Arg Ser Val Ala Arg Glu Val Gly
165 170 175
Pro Lys Gly Ile His Val Asn Ser Ile Asn Pro Gly Tyr Thr Lys Thr
180 185 190
Glu Met Thr Ala Gly Ile Glu Ala Leu Pro Asp Leu Pro Ile Lys Gly
195 200 205
Trp Ile Glu Pro Glu Ala Ile Ala Asp Ala Val Leu Phe Leu Ala Lys
210 215 220
Ser Lys Asn Ile Thr Gly Thr Asn Ile Val Val Asp Asn Gly Leu Ile
225 230 235 240
Ala
<210> 2
<211> 726
<212> DNA
<213>Artificial sequence
<400> 2
atgacgacta cttcaaatgc gctcgtcact ggaggcagcc gcggcattgg cgctgcttcc 60
gccattaagc tggctcagga gggctacagt gttacgctgg cctctcgcag tgttgataaa 120
ctgaatgaag taaaggcgaa actcccaatt gtacaggacg ggcagaagca ctacatttgg 180
gaactcgatc tggctgatgt ggaagctgct tcgtcgttca agggtgctcc tttgcctgct 240
agcagctacg acgtcttcgt ttcgaacgcg ggcgtcgctg cgttctcgcc cacagccgac 300
cacgatgata aggagtggca gaacttgctc gccgtgaact tgtcgtcgcc cattgccctc 360
acgaaggccc tcttgaagga cgtctccgaa aggcctgcgg acaatccgtt gcagattatc 420
tacatttcgt cggtggccgg cttgcatggc gccgcgcagg tcgccgtgta cagtgcatct 480
aaggccggtc ttgatggttt tatgcgctcc gtcgcccgtg aggtgggccc gaagggcatc 540
catgtgaact ccatcaaccc cggatacacc aagactgaaa tgaccgcggg cattgaagcc 600
ctgcctgatt tgcctatcaa ggggtggatc gagcccgagg caattgctga cgcggttctg 660
tttctggcaa agtccaagaa tatcaccggc acaaacattg tggtcgacaa tggcttgatt 720
gcttag 726
Claims (10)
1. a kind of utilization carbonyl reductase prepares method of gram azoles for Buddhist nun's intermediate, it is characterised in that with 2 ', 6 '-two chloro- 3 '-
Fluoro acetophenone is substrate, and phosphate buffer is reaction medium, adds carbonyl reductase, co-factor and hydrogen donor to form living things catalysis
, there is biocatalytic reaction and generate (S) -2 ', 6 '-two chloro- 3 '-fluorobenzene ethanol in reaction system.
2. method according to claim 1, it is characterised in that the amino acid sequence of the carbonyl reductase such as SEQ ID
Shown in NO.1, the encoding gene of the carbonyl reductase is as shown in SEQ ID NO.2.
3. method according to claim 1 and 2, it is characterised in that the co-factor is NAD+。
4. method according to claim 1 and 2, it is characterised in that the hydrogen donor is isopropanol.
5. method according to claim 1 and 2, it is characterised in that 2 ', 6 '-two is chloro- in every liter of biocatalytic reaction system
The consumption of 3 '-fluoro acetophenone is 50-100g.
6. method according to claim 1 and 2, it is characterised in that carbonyl reductase in every liter of biocatalytic reaction system
Consumption be 3-10g.
7. method according to claim 1 and 2, it is characterised in that the use of co-factor in every liter of biocatalytic reaction system
Measure as 0.05-0.1g.
8. method according to claim 1 and 2, it is characterised in that the use of hydrogen donor in every liter of biocatalytic reaction system
Measure as 50-100mL.
9. method according to claim 1 and 2, it is characterised in that the pH of the phosphate buffer is 6.5-7.5.
10. method according to claim 1 and 2, it is characterised in that the temperature of the biocatalytic reaction is 25-35 DEG C,
Reaction time 8-12h.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110184245A (en) * | 2019-05-23 | 2019-08-30 | 南京趣酶生物科技有限公司 | Ketoreductase mutant and its in preparation gram azoles for the application in Buddhist nun's chirality alcohol intermediate and the like |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110184245A (en) * | 2019-05-23 | 2019-08-30 | 南京趣酶生物科技有限公司 | Ketoreductase mutant and its in preparation gram azoles for the application in Buddhist nun's chirality alcohol intermediate and the like |
| CN110184245B (en) * | 2019-05-23 | 2023-09-05 | 安徽趣酶生物科技有限公司 | Ketone reductase mutant and application thereof in preparation of crizotinib chiral alcohol intermediate and analogue thereof |
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