CN103333936A - Phloroglucinol glucoside derivatives and preparation method thereof - Google Patents
Phloroglucinol glucoside derivatives and preparation method thereof Download PDFInfo
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- CN103333936A CN103333936A CN2013102564019A CN201310256401A CN103333936A CN 103333936 A CN103333936 A CN 103333936A CN 2013102564019 A CN2013102564019 A CN 2013102564019A CN 201310256401 A CN201310256401 A CN 201310256401A CN 103333936 A CN103333936 A CN 103333936A
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Abstract
The invention relates to phloroglucinol glucoside derivatives and a preparation method thereof. The preparation method is characterized in that galactosylated modification is carried out on phloroglucinol by using phloroglucinol as a raw material, a low-cost lactose substrate as a galactosyl donor and glycosidase modified by molecules originated from microorganisms as a tool enzyme so as to synthesize multiple phloroglucinol galactoside derivatives. Compared with the raw material phloroglucinol, the synthesized phloroglucinol galactoside derivatives have the advantages that the synthesized phloroglucinol galactoside derivatives contain galactosyl, the water solubility and the stability are improved, the application range is widened, and the synthesized phloroglucinol galactoside derivatives can serve as new chemical synthesis intermediates to be used for deriving and synthesizing new high value-added compounds.
Description
Technical field
The present invention relates to Phloroglucinol glycosides derivatives and preparation method thereof, particularly a kind of method of utilizing beta-galactosidase enzymes mutant enzyme method for synthesizing phloroglucinol galactoside belongs to sugared field of engineering technology.
Technical background
Phloroglucinol (1,3,5-trihydroxybenzene) is a kind of good pharmaceutical prod, early oneself be widely used in antibiotic, suppress aspects such as spleen tyrosine kinase, apple are anticorrosion.This compound also becomes the pure flat sliding flesh spasmolytic of the non-opium poppy bases of the non-coromegine of clinical novel close flesh in recent years, and its injection liquid has obtained clinical application widely in France and other European countries, has become the choice drug for the treatment of spasmic pain.In addition, Phloroglucinol still is important fine chemical product, can be used as that the synthetic intermediate of medicine is anticancer for the preparation of flavones, isoflavones etc., anti-cardiovascular disease class medicine and anti-immune deficiency virus (HIV) new drug etc.Yet also there is the shortcoming of phenolic compound in Phloroglucinol, as character such as poorly water-soluble, instability and photodissociation.
According to the literature, the glycosylation modified energy of phenolic compound increases water-soluble and stable, improves pharmacological property and curative effect.Still the bibliographical information that does not have at present the glycosylation modified Phloroglucinol of enzyme process.
Summary of the invention
The present invention is directed to the deficiencies in the prior art, Phloroglucinol glycosides derivatives and preparation method thereof is provided.
The preparation method of Phloroglucinol galactoside comprises the steps:
(1) adopting phosphoric acid buffer preparation lactose concn is 0.1M～0.3M, and Phloroglucinol concentration is 0.05M～0.2M, and the beta-galactosidase enzymes addition of aminoacid sequence shown in SEQ ID NO.1 is the reaction system of 2 μ g～10 μ g/mL;
(2) step (1) is made reaction system and in 37～45 ℃ of water-baths, reacted 20～60 minutes, boil termination reaction, 10000～12000 rev/mins centrifugal 20 minutes, get supernatant liquor;
(3) supernatant liquor that step (2) is made after separation, drying, makes the Phloroglucinol galactoside.
Preferred according to the present invention, it is ACE06986.1 that the middle beta-galactosidase enzymes of aminoacid sequence shown in SEQ ID NO.1 of described step (1) selected the GenBank accession number for use, and the 980th tryptophane replaced with phenylalanine, makes.Beta-galactosidase enzymes after the replacement can be according to delivering document [Synthesis of galactosyl sucralose by β-galactosidase from Lactobacillus bulgaricus L3, Food Chem, 2012,134:269-275] described method prepares zymoprotein, and above-mentioned beta-galactosidase enzymes has remarkable commentaries on classics glycosyl activity to Phloroglucinol.
Preferred according to the present invention, the phosphoric acid buffer in the described step (1) is concentration 10～100mM, the buffer solution of potassium phosphate of pH6～8; Further preferred according to the present invention, the damping fluid in the described step (1) is 50mM, the potassium phosphate buffer of pH7.0.
Preferred according to the present invention, the reaction conditions in the described step (2) is 45 ℃ of water-baths 45 minutes.
Preferred according to the present invention, the termination reaction condition of boiling in the described step (2) is 100 ℃ and boiled 5 minutes.
Preferred according to the present invention, the separation in the described step (3) adopts preparation type thin layer chromatography board to separate, model Silica gel60F254 (Merck, Germany).
Preferred according to the present invention, the drying in the described step (3) is lyophilize.
Further preferred according to the present invention, comprise also in the described step (3) that the thin-layer chromatography when separating detects, merge the step of the identical product of migration distance.
The step that above-mentioned thin-layer chromatography detects is as follows:
In developing agent, launch behind the thin layer chromatography board point sample, behind the spray painting developer, made sugared spot colour developing in 5 minutes in 120 ℃ of bakings.
Above-mentioned developing agent is by propyl carbinol, dehydrated alcohol and water 5:3:2 mixed preparing by volume; Developer is that volume percent is that 20% sulfuric acid and concentration are 3 of 0.5wt%, the solution of 5-orcin.
Beneficial effect
The present invention is raw material with the Phloroglucinol, be the galactosyl donor with cheap lactose substrate, adopt the beta-galactosidase enzymes method for synthesizing phloroglucinol galactoside through sudden change, product is compared with its stock yard benzenetriol, contain galactosyl, increased water-solublely and stable, enlarged range of application, this compounds self can be used as new chemosynthesis intermediate in order to the synthetic new high added value compound of deriving, and has potential using value at chemical industry and pharmacy industry.
Description of drawings
Fig. 1 is the mass spectrum of Phloroglucinol glycosides derivatives 1;
Fig. 2 is the mass spectrum of Phloroglucinol glycosides derivatives 2;
Fig. 3 is the mass spectrum of Phloroglucinol glycosides derivatives 3;
Fig. 4 is the hydrogen spectrum of Phloroglucinol glycosides derivatives 2.
Embodiment
The present invention will be further described below in conjunction with embodiment, but institute of the present invention protection domain is not limited thereto.
Embodiment 1
The Phloroglucinol glycosides derivatives, preparation process is as follows:
1. the preparation of beta-galactosidase enzymes
Synthetic GenBank accession number is the beta-galactosidase gene sequence (proteins encoded GenBank accession number is ACE06986.1) of EU734748.1, be connected on pET-21b (+) plasmid, transformed into escherichia coli BL21 (DE3), extract recombinant plasmid then, be template with the plasmid, adopt sudden change test kit (full formula gold Easy Mutagenesis System, Beijing) in this enzyme gene order, to introduce sudden change by PCR;
Upstream primer is: 5 '-CGGGGATGACTCC
TTTGGGCAGAAGGTCCA-3 '; SEQ ID NO.3
Downstream primer is: 5 '-
AAAGGAGTCATCCCCGCCGACCCCCATCTG-3 '; SEQ ID NO.4
TTT coding phenylalanine replaces with phenylalanine by PCR with 980 tryptophanes in the primer;
The pcr amplification condition is: 95 ℃ of pre-sex change 5 minutes; (72 ℃ were extended 7 minutes for 95 ℃ of sex change 30 seconds, 55 ℃ of annealing 30 seconds to react 20 circulations; 72 ℃ were extended 10 minutes after 20 loop ends.
The PCR product after Dpn I enzyme (TaKaRa) is handled, transformed into escherichia coli BL21 (DE3), the beta-galactosidase enzymes mutant enzyme gene of acquisition is through order-checking, nucleotide sequence is shown in SEQ ID NO.2, its amino acid sequence coded is shown in SEQ ID NO.1.
Prepare the beta-galactosidase enzymes mutant enzyme according to delivering the described method of document [Synthesis of galactosyl sucralose by β-galactosidase from Lactobacillus bulgaricus L3, Food Chem, 2012,134:269-275].Measure the enzyme amount of beta-galactosidase enzymes enzyme liquid with the Xylene Brilliant Cyanine G method.
2. beta-galactosidase enzymes catalyzes and synthesizes the Phloroglucinol galactoside
With pH7.0,50mM potassium phosphate buffer preparation reaction system 50mL, the lactose final concentration is 0.2M, and the Phloroglucinol final concentration is 0.1M, and the addition of enzyme is 4 μ g/mL.After 45 minutes, 100 ℃ were boiled termination reaction 5 minutes 45 ℃ of reactions.
3. the purifying of Phloroglucinol galactoside
Centrifugal 20 minutes of 12000 rev/mins of reaction solutions after will boiling are drawn supernatant liquor, at preparation thin layer chromatography board (PLCSilica gel60F254, Merck) point sample exhibition layer.After the exhibition layer finishes, on chromatoplate, get the wide bar shaped platelet colour developing of 1cm every 10cm, judge the position of target carbohydrate on chromatoplate.Scrape then get chromatoplate not color development area contain the silica gel powder of target glucosides, it is dissolved in the water again, the centrifuging and taking supernatant, the gained powder is Phloroglucinol galactoside derivative after the lyophilize.
Be divided into from obtaining three kinds of different glucosides products, show different migration distances in thin layer chromatography board, be labeled as Phloroglucinol glycosides derivatives 1, Phloroglucinol glycosides derivatives 2, Phloroglucinol glycosides derivatives 3 respectively by the mobility size.
The step that above-mentioned thin-layer chromatography detects is as follows:
The thin layer chromatography board point sample launches in developing agent, behind the spray painting developer, makes sugared spot colour developing in 5 minutes in 120 ℃ of bakings.
Above-mentioned developing agent is by propyl carbinol, dehydrated alcohol and water 5:3:2 mixed preparing by volume; Developer is that volume percent is that 20% sulfuric acid and concentration are 3 of 0.5wt%, the solution of 5-orcin.
4. the evaluation of Phloroglucinol galactoside derivative
Getting above-mentioned three kinds of Phloroglucinol galactoside dilute with waters respectively is that mass percent is 1% solution, carries out mass spectroscopy, and Fig. 1 shows that the feature molecular ion peak (m/z) of Phloroglucinol glycosides derivatives 1 is [M-H]
-287.076, judge that this compound is the Phloroglucinol monoglycosides; Fig. 2 shows that the feature molecular ion peak (m/z) of Phloroglucinol glycosides derivatives 2 is [M-H]
-449.128, judge that this compound is Phloroglucinol disaccharide glycosides; Fig. 3 shows that the feature molecular ion peak (m/z) of Phloroglucinol glycosides derivatives 2 is [M-H]
-449.127, judge that this compound is Phloroglucinol disaccharide glycosides.Get 2 five milligrams of powder of Phloroglucinol glycosides derivatives and be dissolved in deuterium in the water, carry out
1HNMR analyzes, and the result is at chemical shift δ〉the 8ppm zone only observes a free phenyl ring hydroxyl proton signal (as shown in Figure 4), illustrates that two galactosyls of this compound form glycosidic link with two hydroxyls of phenyl ring respectively.Because Phloroglucinol disaccharide glycosides has only two kinds of possible mode of connection, thereby judge that Phloroglucinol glycosides derivatives 3 is that two glycan molecules are directly linked and formed glycosidic link on the phenyl ring.
Phloroglucinol glycosides derivatives 1 structure is as follows:
Phloroglucinol glycosides derivatives 3 structures are as follows:
The used instrument of above-mentioned mass spectroscopy is Tianjin, island LCMS-IT-TOF mass spectrograph (Japan); The used instrument of nmr analysis is the super shielding of AVANCE600 type superconduction fourier transform NMR spectrometer (Switzerland Bruker company).
The preparation method of Phloroglucinol galactoside, following steps are as follows:
(1) adopting phosphoric acid buffer preparation lactose concn is 0.2M, and Phloroglucinol concentration is 0.05M, and the beta-galactosidase enzymes addition of aminoacid sequence shown in SEQID NO.1 is the reaction system of 10 μ g/mL;
(2) step (1) is made reaction system and in 37 ℃ of water-baths, reacted 20 minutes, boil termination reaction, 12000 rev/mins centrifugal 20 minutes, get supernatant liquor;
(3) purifying of Phloroglucinol galactoside derivative and detection are with embodiment 1.
Claims (10)
1. the preparation method of Phloroglucinol galactoside comprises the steps:
(1) adopting phosphoric acid buffer preparation lactose concn is 0.1M～0.3M, and Phloroglucinol concentration is 0.05M～0.2M, and the beta-galactosidase enzymes addition of aminoacid sequence shown in SEQ ID NO.1 is the reaction system of 2 μ g～10 μ g/mL;
(2) step (1) is made reaction system and in 37～45 ℃ of water-baths, reacted 20～60 minutes, boil termination reaction, 10000～12000 rev/mins centrifugal 20 minutes, get supernatant liquor;
(3) supernatant liquor that step (2) is made after separation, drying, makes the Phloroglucinol galactoside.
2. preparation method as claimed in claim 1 is characterized in that, the phosphoric acid buffer in the described step (1) is concentration 10～100mM, the buffer solution of potassium phosphate of pH6～8.
3. preparation method as claimed in claim 2 is characterized in that, the damping fluid in the described step (1) is 50mM, the potassium phosphate buffer of pH7.0.
4. preparation method as claimed in claim 2 is characterized in that, the reaction conditions in the described step (2) is 45 ℃ of water-baths 45 minutes.
5. preparation method as claimed in claim 2 is characterized in that, the termination reaction condition of boiling in the described step (2) is 100 ℃ and boiled 5 minutes.
6. preparation method as claimed in claim 2 is characterized in that, the separation in the described step (3) adopts preparation type thin layer chromatography board to separate.
7. preparation method as claimed in claim 2 is characterized in that, the drying in the described step (3) is lyophilize.
8. preparation method as claimed in claim 2 is characterized in that, also comprises the step of the product that thin-layer chromatography detects, the merging migration distance is identical when separating in the described step (3).
9. preparation method as claimed in claim 8 is characterized in that, the step that above-mentioned thin-layer chromatography detects is as follows:
In developing agent, launch behind the thin layer chromatography board point sample, behind the spray painting developer, made sugared spot colour developing in 5 minutes in 120 ℃ of bakings.
10. preparation method as claimed in claim 9 is characterized in that, described developing agent is by propyl carbinol, dehydrated alcohol and water 5:3:2 mixed preparing by volume; Developer is that volume percent is that 20% sulfuric acid and concentration are 3 of 0.5wt%, the solution of 5-orcin.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021031594A1 (en) * | 2019-08-21 | 2021-02-25 | 云南巅青生物科技有限公司 | Aromatic ring compound |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000073368A1 (en) * | 1999-06-01 | 2000-12-07 | Solutia Inc. | Composition for use in flexible polyurethane foams |
| CN101312719A (en) * | 2005-09-20 | 2008-11-26 | 瑞沃他生物制药股份公司 | Novel phloroglucinol derivatives having selectin ligand activity |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000073368A1 (en) * | 1999-06-01 | 2000-12-07 | Solutia Inc. | Composition for use in flexible polyurethane foams |
| CN101312719A (en) * | 2005-09-20 | 2008-11-26 | 瑞沃他生物制药股份公司 | Novel phloroglucinol derivatives having selectin ligand activity |
Non-Patent Citations (1)
| Title |
|---|
| LILI LU ET AL: "Synthesis of galactosyl sucralose by β-galactosidase from Lactobacillus bulgaricus L3", 《FOOD CHEMISTRY》, vol. 134, no. 1, 1 September 2012 (2012-09-01) * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021031594A1 (en) * | 2019-08-21 | 2021-02-25 | 云南巅青生物科技有限公司 | Aromatic ring compound |
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