CN104711235B - A kind of aromatic ring list oxygenation expandase and its application in oxepin 2 (3H) ketone compounds are prepared - Google Patents
A kind of aromatic ring list oxygenation expandase and its application in oxepin 2 (3H) ketone compounds are prepared Download PDFInfo
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- CN104711235B CN104711235B CN201510033023.7A CN201510033023A CN104711235B CN 104711235 B CN104711235 B CN 104711235B CN 201510033023 A CN201510033023 A CN 201510033023A CN 104711235 B CN104711235 B CN 104711235B
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- aromatic ring
- expandase
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Abstract
The present invention relates to a kind of aromatic ring list oxygenation expandase from fungi, its preparation method and for the oxidation of phenols aromatic ring to prepare the application of oxepin ketone compounds.Described aromatic ring list oxygenation expandase is to be prepared the mycelia homogenate supernatant of the brown tough lead fungi (Boreostereum vibrans) of lid of fungi by steps such as gel filtration, centrifugal concentratings；Biocatalyst is used as by the use of aromatic ring list oxygenation expandase, catalysis phenols aromatic ring carries out single oxygenation ring expansion oxidation generation oxepin 2 (3H) ketone compounds under mild conditions, for this kind of rare compound synthesis provides a kind of mild condition, it is environmentally friendly, need to a step catalytic reaction new way.
Description
Technical field：
The invention belongs to biological organic synthesis field, and in particular to a kind of aromatic ring list oxygenation expandase, its preparation method and
Its application in oxepin -2 (3H) -one class compound is prepared.
Background technology：
Oxepin ketone construction unit is widely present in a variety of natural products, and these natural products often have multiple
Miscellaneous structure and the physiologically active of wide spectrum, are the important foundations of new drug development.In the various of oxepin ketone compounds
In structure type, (3H) the ketone construction unit of oxepin -2 is then very rare, only occurs in 1,5-
Secovibralactone (1), RetipolideC, a few natural products such as D (2,3) and are obtained by chemical modification
In natural products derivative (4,5).Due to the ring strain and alkene that heptatomic ring in oxepin -2 (3H) ketone structure is larger
The presence of alcohol of formula, is still the work of a very challenging property by this kind of compound of chemical synthesis, at present relevant this kind of chemical combination
The synthetic method report of thing is few, and Enrique Aguilar et al. report the ring using transition metal gold catalysis cyclopropyl alkynes
Change isomerization high yield build oxepin -2 (3H) ketone method (Fern á ndez-Garc í a et al.,
Regioselective synthesis of oxepinones and azepinones by gold-catalyzed
cycloisomerization of functionalyzed cyclopropyl alkynes.Chem.Commun.2013,
11185-11187), but be due to that substrate synthetic route is longer, prepare complicated, expensive catalyst, and operation require it is strict etc. because
Element causes the application of this method to be very restricted.Because (3H) assimilation of oxepin -2 compound source is rare, to it
Further investigation and the work further developed are difficult to carry out.Prepare at present mainly based on chemical synthesis, and biology enzyme system
Standby oxepin -2 (3H) -one is at home and abroad not reported.
The content of the invention：
It is an object of the invention to provide a kind of aromatic ring list oxygenation expandase, its preparation method and its oxepane three is being prepared
Application in alkene -2 (3H) -one class compound.The present invention makees catalyst with the aromatic ring list oxygenation expandase from the brown tough lead fungi of lid,
Single step reaction is only needed to prepare oxepin -2 (3H) -one class compound by phenols aromatic compound catalysis.The present invention
Method high catalytic efficiency, selectivity is strong, reaction condition is gentle, environmentally friendly, and method is simple and direct, and enzyme reaction is easy to operate.Its
Aromatic ring list oxygenation expandase activity improves more than 70 times.
In order to realize the above-mentioned purpose of the present invention, the invention provides following technical scheme：
Aromatic ring list oxygenation expandase, it is prepared by following methods：By the brown tough lead fungi (Boreostereum of lid of fungi
Vibrans mycelia homogenate supernatant) is prepared by gel filtration, centrifugal concentrating step.
Aromatic ring list oxygenation expandase, it is prepared by following methods：The tough lead fungi mycelia of brown lid obtained by fermented and cultured
It is homogenized in buffer solution, homogenate obtains supernatant after centrifugation, supernatant addition Sephadex G-25 gel columns is coagulated
Glue is filtered, and is collected first eluting peak and is concentrated.
The preparation method of described aromatic ring list oxygenation expandase, the tough lead fungi mycelia of brown lid obtained by fermented and cultured is in buffering
It is homogenized in liquid, homogenate obtains supernatant after centrifugation, supernatant is added into Sephadex G-25 gel columns carries out gel mistake
Filter, collects first eluting peak and concentrates.
The preparation method of oxepin -2 (3H) -one class compound, makees using described aromatic ring list oxygenation expandase
For biocatalyst, phenols aromatic compound is catalyzed under mild conditions and carries out single oxygenation ring expansion oxidation generation oxepane three
Alkene -2 (3H) -one class compound.
The preparation method of oxepin -2 (3H) -one class compound, makees using described aromatic ring list oxygenation expandase
For biocatalyst, using phenols aromatic compound as substrate, cosolvent is DMSO, in 28 DEG C of waters bath with thermostatic control, by single oxygenation
Ring expansion oxidation generation oxepin -2 (3H) -one class compound, prepared by enzyme and the pH of cushioning fluid of enzymatic is 7.5
(Hepes,25mM)。
Application of the described aromatic ring list oxygenation expandase in oxepin -2 (3H) -one class compound is prepared, should
With described aromatic ring list oxygenation expandase as biocatalyst, phenols aromatic compound is catalyzed under mild conditions and carries out list
Oxygenation ring expansion oxidation generation oxepin -2 (3H) -one class compound.
Application of the described aromatic ring list oxygenation expandase in oxepin -2 (3H) -one class compound is prepared, should
With described aromatic ring list oxygenation expandase as biocatalyst, using phenols aromatic compound as substrate, cosolvent is DMSO,
In 28 DEG C of waters bath with thermostatic control, by single oxygenation ring expansion oxidation generation oxepin -2 (3H) -one class compound, enzyme is prepared and enzyme
The pH of cushioning fluid of catalysis is 7.5 (Hepes, 25mM).
The tough lead fungi mycelia of brown lid obtained by fermented and cultured is homogenized in buffer solution, and homogenate obtains supernatant after centrifugation
Liquid, adds Sephadex G-25 gel columns by supernatant and carries out gel filtration, collects first eluting peak and concentration is made slightly
Enzyme.It is substrate with compound 6 or 8, cosolvent is DMSO, in 28 DEG C of waters bath with thermostatic control, and oxepane three can be made using the enzyme
Alkene -2 (3H) -one class compound.Prepared by the enzyme by optimization and the pH of cushioning fluid of enzymatic is 7.5 (Hepes, 25mM).
Compared with prior art, the present invention possesses following excellent benefit：
1. compared with general chemical catalysis, reaction of the invention has high catalytic efficiency, selectivity is strong, reaction condition temperature
With, it is environmentally friendly the advantages of.
2. compared with the thick enzyme of supernatant, obtained thick enzyme is handled by Sephadex G-25 gel filtrations, its aromatic ring list adds
Oxygen expandase activity improves more than 70 times.
3. thick enzyme preparation method is simple and direct, enzyme reaction is easy to operate.
Brief description of the drawings：
Fig. 1 is the structure of oxepane alkene -2 (3H) -one class compound；
Fig. 2 is that aromatic ring list oxygenation ring expansion enzymatic prepares oxepin -2 (3H) -one class compound 1 and 7；
Fig. 3 is conversion (control) of the thick enzyme of supernatant to compound 6；
Fig. 4 is conversion of the thick enzyme to compound 6 after gel filtration is handled；
Fig. 5 is the influence that different buffer solutions form 7 to aromatic ring list oxygenation ring expansion enzymatic 6.
Embodiment：
Below in conjunction with the accompanying drawings, the essentiality content of the present invention is described further with specific examples below, but not
The present invention is limited with this.
In following embodiments, no specified otherwise is conventional method.
In following embodiments, the percentage composition is weight/mass percentage composition unless otherwise specified.
Embodiment 1：
The preparation of aromatic ring list oxygenation expandase：
The brown tough lead fungi mycelia of lid of 10g is added in the buffer solution (pH7.5Hepes, 25mM) of 10mL precoolings to be homogenized, it is even
Slurries 9000rpm at 4 DEG C is centrifuged 15 minutes, is taken supernatant, is obtained volume about 10mL thick enzyme, and wherein 5mL is used to implement
Example 2, remaining 5mL loadings Sephadex G-25 gel columns are eluted, UV-detector with 25mM pH7.5Hepes buffer solutions
280nm detect eluent, collect first eluting peak and at 4 DEG C 6000rpm ultrafiltration centrifugal concentrating to starting supernatant phase
Same volume 5mL, for embodiment 3.
Embodiment 2：
The thick enzyme of supernatant is directly used as biocatalyst：
Take the supernatant 1mL in above-described embodiment 1, add 1mM compounds 6 (auxiliary agent is DMSO) and 3mM NADPH, three
Repeat, 4h is reacted in 28 DEG C of waters bath with thermostatic control, each reaction adds 0.3 μ g internal standard compound 4-hydroxy base benzophenone, uses acetic acid
Ethyl ester is extracted three times, is concentrated into 0.1mL, and quasi-molecular ion peak [M+Na] is detected through liquid chromatography mass spectrometric+M/z 203, as a result such as Fig. 3 institutes
Show, oxepin -2 (3H) the -one class compound 7 of generation is few, the ratio between with interior target peak area, three repetitions are averaged
It is worth for 0.014.
Embodiment 3：
The thick enzyme obtained after gel filtration processing is used as biocatalyst：
The thick enzyme 1mL obtained after taking being handled through gel filtration in above-described embodiment 1, adding 1mM compounds 6, (auxiliary agent is
DMSO) and 3mM NADPH, three repetitions, compare to boil the thick enzyme of inactivation, 4h is reacted in 28 DEG C of waters bath with thermostatic control, it is each anti-
0.3 μ g internal standard compound 4-hydroxy base benzophenone should be added, is extracted with ethyl acetate three times, 0.1mL is concentrated into, through liquid chromatography mass spectrometric
Detect quasi-molecular ion peak [M+Na]+M/z 203, as a result as shown in figure 4, oxepin -2 (3H) -one class of generation
Extremely significantly, the ratio between with interior target peak area, the average value of three repetitions is 1.042 to compound 7, thicker than supernatant in embodiment 2
The enzymatic activity of enzyme improves more than 70 times.
Embodiment 4：
Different buffer solutions form 7 influence to aromatic ring list oxygenation ring expansion enzymatic 6：
It is 25mM but three kinds of different buffer solutions of pH value using concentration, is pH7.5Hepes (ethoxy pyrroles respectively
Pyridine ethyl sulfonic acid), pH6.4Mes (morpholine ethyl sulfonic acid) and pH 5.3Mes.Take above-mentioned three kinds of buffer solution 2mL respectively with 2g mycelia by
The thick enzyme 2mL of gel filtration is prepared according to above-described embodiment 1, then catalytic reaction is carried out according to above-described embodiment 3, through liquid chromatography mass spectrometric
Detect quasi-molecular ion peak [M+Na]+M/z 203, as a result as shown in figure 5, the oxa- ring generated in pH7.5Hepes buffer solutions
Heptantriene -2 (3H) -one class compound 7 extremely significantly, reaches 1.15, in pH6.4Mes buffer solutions with the ratio between interior target peak area
The ratio between 7 and interior target peak area of middle generation are 0.22, and product 7 is not detected in pH5.3Mes buffer solutions.It is therefore preferable that
PH7.5Hepes (25mM) buffer solution is used for enzyme and prepared and enzymatic.
Embodiment 5：
Conversion to compound 8：
The thick enzyme 1mL obtained after taking being handled through gel filtration in above-described embodiment 1, adding 1mM compounds 8, (auxiliary agent is
DMSO) and 3mM NADPH, compare to boil the thick enzyme of inactivation, react 4h in 28 DEG C of waters bath with thermostatic control, be extracted with ethyl acetate three
It is secondary, 0.1mL is concentrated into, quasi-molecular ion peak [M+Na] is detected through liquid chromatography mass spectrometric+M/z 231, the oxepin -2 of generation
(3H) -one class compound 1 is extremely notable.
The testing conditions of above-described embodiment 2,3,4 and 5 are：Instrument is Agilent Technologies companies of the U.S.
1290/6530UPLC-Q-TOF liquid chromatograph-mass spectrometers.Mass Spectrometry Conditions：Ionization mode is double source ESI；Energy 3500V；
Mass range 50-500.Liquid phase chromatogram condition：ZORBAX Eclips Plus C18Rapid Resolution HD posts (50mm
×2.1mm×1.8μm)；Column flow is 0.3mL/min；The μ L of sample size 1.0；Compound 7 and standard items use 25% methanol 75%
Water carries out chromatographic elution；Compound 1 and standard items carry out chromatographic elution using the water of 43% methanol 57%；Sample and standard items are simultaneously
Detection.
Claims (5)
1. aromatic ring list oxygenation expandase, it is characterised in that it is prepared by following methods：The brown Gai Ren obtained by fermented and cultured
Lead fungi (Boreostereum vibrans) mycelia is homogenized in buffer solution, and homogenate obtains supernatant, loading after centrifugation
Sephadex G-25 gel columns, are eluted with 25mM pH7.5Hepes buffer solutions, UV-detector 280nm detection eluents, are received
First eluting peak of collection is simultaneously concentrated.
2. the preparation method of the aromatic ring list oxygenation expandase described in claim 1, it is characterised in that by fermented and cultured obtain it is brown
Cover tough lead fungi mycelia to be homogenized in buffer solution, homogenate obtains supernatant after centrifugation, loading Sephadex G-25 gel columns,
Eluted with 25mM pH7.5Hepes buffer solutions, UV-detector 280nm detection eluents are collected first eluting peak and concentrated.
The preparation method of oxepin -2 3. (3H) -one class compound, it is characterised in that the virtue described in application claim 1
Ring list oxygenation expandase adds 1mM compounds 6 as biocatalyst, and its structural formula is：Auxiliary agent is DMSO,
With 3mM NADPH, three repetitions react 4h in 28 DEG C of waters bath with thermostatic control, and each reaction adds 0.3 μ g internal standard compound 4-hydroxy bases
Benzophenone, is extracted with ethyl acetate three times, is concentrated into 0.1mL, and quasi-molecular ion peak [M+Na] is detected through liquid chromatography mass spectrometric+m/z
203, oxepin -2 (3H) -one class compound 7 is generated, its structural formula is：
The preparation method of oxepin -2 4. (3H) -one class compound, it is characterised in that the virtue described in application claim 1
Ring list oxygenation expandase adds 1mM compounds 8 as biocatalyst, and its structural formula is：Auxiliary agent is DMSO,
With 3mM NADPH, 4h is reacted in 28 DEG C of waters bath with thermostatic control, is extracted with ethyl acetate three times, 0.1mL is concentrated into, through liquid chromatography mass spectrometric
Detect quasi-molecular ion peak [M+Na]+M/z 231, generates oxepin -2 (3H) -one class compound 1, and its structural formula is：
5. the aromatic ring list oxygenation expandase described in claim 1 is in oxepin -2 (3H) -one class compound is prepared
Using, it is characterised in that the aromatic ring list oxygenation expandase described in application claim 1 is aromatic ring with phenols as biocatalyst
Compound 6 or 8 is substrate, and expand oxidation by single oxygenation generates compound 7 or 1 respectively, and cosolvent is DMSO, in 28 DEG C of waters bath with thermostatic control
In, by single oxygenation ring expansion oxidation generation oxepin -2 (3H) -one class compound, enzyme prepares the buffer solution with enzymatic
PH value is 7.5, Hepes, 25mM, and the structural formula of compound 6,7,8,1 is：
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1609786A1 (en) * | 2004-06-25 | 2005-12-28 | Merck Sante | New 5-alkenone-3,3-dimethyl-benzoxepines derivatives useful for the preparation of antidiabetic pentadienoic acids |
| CN1907978A (en) * | 2006-08-17 | 2007-02-07 | 中国科学院昆明植物研究所 | Vibralactone, preparation method and medicinal application thereof |
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2015
- 2015-01-22 CN CN201510033023.7A patent/CN104711235B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1609786A1 (en) * | 2004-06-25 | 2005-12-28 | Merck Sante | New 5-alkenone-3,3-dimethyl-benzoxepines derivatives useful for the preparation of antidiabetic pentadienoic acids |
| CN1907978A (en) * | 2006-08-17 | 2007-02-07 | 中国科学院昆明植物研究所 | Vibralactone, preparation method and medicinal application thereof |
Non-Patent Citations (2)
| Title |
|---|
| Elucidating the Biosynthetic Pathway for Vibralactone: A Pancreatic Lipase Inhibitor with a Fused Bicyclic β-Lactone;Pei-Ji Zhao et al.;《Angew. Chem. Int. Ed.》;20130121;第52卷;第2300页框图2，第2301页左栏第1段 * |
| 中国韧革菌（III）;郭正堂;《植物研究》;19870731;第7卷(第3期);第85-112页 * |
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