CN1557955A - Diazocines compound and method for preparing it using slime bacteria fermentation and extraction method - Google Patents
Diazocines compound and method for preparing it using slime bacteria fermentation and extraction method Download PDFInfo
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- CN1557955A CN1557955A CNA2004100234725A CN200410023472A CN1557955A CN 1557955 A CN1557955 A CN 1557955A CN A2004100234725 A CNA2004100234725 A CN A2004100234725A CN 200410023472 A CN200410023472 A CN 200410023472A CN 1557955 A CN1557955 A CN 1557955A
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Abstract
The present invention relates to one new kind of diazocine compound. The diazocine compound is separated from the fermented liquid of myxobactin sorangus, has molecular expression of C28H20N4O2S2, and is named 2, 6-bis-(6-mercapto-3-pyridyl)-4, 8-bis-(4-phenol)-[1, 5]- diazocine chemically. The present invention also relates to the method of extracting the compound from the fermented liquid. The compound of the present invention may be used in preparing antitumor and antifungal medicine.
Description
(1) technical field
The method that the present invention relates to a kind of diazocine compounds and utilize the slime bacteria fermentation and extract, relate in particular to a kind of novel diazocine compounds 2,6-two-(6-sulfydryl-3-pyridine)-4,8-two-(4-phenol)-[1,5]-diazocine, and the method for utilizing the bacterium fermentation of slime bacteria heap capsule and this compound of separation and Extraction.
(2) background technology
Slime bacteria is the most high prokaryotic organism monoid, has complicated many cells behavior and form and takes place, and occupies critical role in cytodifferentiation, growth and organic evolution research.In recent years, slime bacteria can produce the microbe groups that enriches secondary metabolites as a class and comes into one's own day by day.By the quantity of finding biologically active substance, slime bacteria only comes after actinomycetes and the genus bacillus in prokaryotic organism.From slime bacteria, find about more than 600 kinds of biologically active substances at present, accounted for 3.5% of microbe-derived sum.It needs to be noted that the repetition probability of screening new biological activity material is more and more higher from microorganisms such as actinomycetes at present, and slime bacteria has special advantages in this respect.See that with regard to present circumstances 55% bacterial strain can produce biologically active substance in molten bacterial flora slime bacteria, and fibrinolysin group slime bacteria mainly is a sorangium cellulosum even up to 95%.This screening for new texture or new role matrix material provides a good quasi-microorganism resource.
The biologically active substance that slime bacteria produces is of a great variety, and exposure level is on many levels, and the mechanism of action is also varied.According to the difference of the mechanism of action, mainly be divided into following a few class: respiration inhibition, nucleic acid is synthetic to be suppressed, and protein synthesis suppresses, and interferencing protein phosphorylation system disturbs the metal ion transportation, acts on lipid metabolism, disturbs the carbohydrate utilization and acts on cell walls.The meta-bolites of heap capsule bacterium, significant feature to as if eukaryote, not only to various fungies, and tumour cell also had general activity.Wherein, reports such as He Fule separated the 16 membered macrolide compounds ebormycine that make new advances from sorangium cellulosum in 1993, be considered to the substitute of at present successful anti-cancer medicine paclitaxel and enter clinical experimental stage, caused people's extensive concern.
Recently there are some researches show, diazocine and derivative thereof can play restraining effect to the anti-apoptosis function of Bcl-2, thereby the apoptosis of triggering tumor cell reaches effect [the Istvan J Enyedy et al. of kill tumor cell, J Med Chem2001 (44), 4313-4324.].Therefore, the development of novel diazocine and derivative thereof, exploitation, utilization have great importance to the research and development and the application of clinical medicine.
The diazocine compounds 2 that the present invention relates to, 6-two-(6-sulfydryl-3-pyridine)-4,8-two-(4-phenol)-[1,5]-diazocine not only structurally is brand-new, and this compounds is studied seldom in organism, obtain this compounds by the slime bacteria fermentation, and utilize slime bacteria heap capsule bacterium to ferment and do not appear in the newspapers by the method for this compound of fermented liquid separation and Extraction.
(3) summary of the invention
The purpose of this invention is to provide a kind of brand-new diazocine compounds 2,6-two-(6-sulfydryl-3-pyridine)-4,8-two-(4-phenol)-[1,5]-diazocine, and provide a kind of and utilize the fermentation of slime bacteria heap capsule bacterium and by the method for this compound of fermented liquid separation and Extraction.
Technical scheme of the present invention realizes by the following method:
A kind of novel diazocine compounds is characterized in that, it is to separate in the fermented liquid by slime bacteria heap capsule bacterium to make, and molecular formula is C
28H
20N
4O
2S
2, chemical name is 2,6-two-(6-sulfydryl-3-pyridine)-4, and 8-two-(4-phenol)-[1,5]-diazocine,
English name is 2,6-bis-(6-mercapto-pyridin-3-yl)-4,8-bis-(phenol-4-yl)-[1,5]-diazocine.
Chemical structure is as follows:
Above-claimed cpd is a white needle-like crystals, dissolves in methyl alcohol, is insoluble in water.Utilize fourier transformation ion cyclotron resonance (ICR) high resolution mass spec (FT-ICRMS) to measure, use electrospray ionization source (ESI) source to obtain 531.1011[M+Na]
+Mass spectra peak, the calibration molecule formula is C
28H
20N
4O
2S
2Na.Utilize methyl alcohol to measure the uv atlas of compound, use the KBr pressed disc method to measure the infrared spectrum of compound, the results are shown in subordinate list 1 as solvent.With CD
3OD is a solvent, uses 600MHz NMR instrument to measure compound respectively
1H-NMR and
13The C-NMR spectrogram the results are shown in subordinate list 2, strengthens spectrum (DEPT, Fig. 2 .) in conjunction with the undistorted polarization transfer of measuring, and the single quantum coherent spectrum of heteronuclear (HSQC, Fig. 3 .) relevant spectrum spectrograms such as (HMBC, Fig. 4 .) with the heteronuclear multikey has been identified the structure of this compound.
Table 1. 2,6-two-(6-sulfydryl-3-pyridine)-4,8-two-(4-phenol)-[1,5]-diazocine
Ultraviolet and ir data table
Spectrum project absorption peak
UV spectrum 248nm (ε
Max1117), 304nm (ε
Max458)
1243
cm-1，1268
cm-1，1451
cm-1，1459
cm-1，1517
cm-1，
Infrared spectra
1601
cm-1，1631
cm-1，2404
cm-1，3219
cm-1
Table 2.2,6-two-(6-sulfydryl-3-pyridine)-4,8-two-(4-phenol)-[1,5]-diazocine
1H-NMR and
13The C-NMR data sheet
No.
1H-NMR
13C-NMR
2 158.5
3 6.84(1H，t) 102.0
4 163.6
6 158.5
7 6.84(1H，t) 102.0
8 163.6
9 124.6
10 8.12(1H，s) 153.3
12 176.8
13 6.93(1H，dd) 115.3
14 8.05(1H，d) 127.2
16 124.6
17 8.12(1H，s) 153.3
19 176.8
20 6.93(1H，dd) 115.3
21 8.05(1H，d) 127.2
23 123.0
24 7.36(1H，tt) 130.1
25 6.84(1H，tt) 114.9
26 157.4
27 6.84(1H，tt) 114.9
28 7.36(1H，tt) 130.1
30 123.0
31 7.36(1H，tt) 130.1
32 6.84(1H，tt) 114.9
33 157.4
34 6.84(1H，tt) 114.9
35 7.36(1H，tt) 130.1
The present invention utilizes the fermentation of slime bacteria heap capsule bacterium to obtain 2,6-two-(6-sulfydryl-3-pyridine)-4, and the method for 8-two-(4-phenol)-[1,5]-diazocine, its basic step is:
(1) preparation of ferment-seeded: will produce bacterium sorangium cellulosum (Sorangium cellulosum ATCC 25569) and be seeded on the solid slant culture base, cultivate after 3-7 days for 28-32 ℃, be inoculated in liquid seed culture medium,
Cultivated 3-7 days, and obtained 1-8 * 10 for 28-32 ℃
9The somatic cells of/ml.
(2) liquid fermenting: the somatic cells that step (1) is prepared, by with the fermentating liquid volume ratio be that 0.5 ~ 1.5: 10 inoculum size is inoculated in the automatic fermentor tank, culture temperature is 30 ℃, mixing speed is 30-60 rev/min, cultivates 5-9 days.
(3) the solid slant culture based formulas of above-mentioned steps (1) use is:
KNO
30.5-1.5g/L; K
2HPO
42H
2O 0.5-1.0g/L; MgSO
47H
2O 1.0-2.0g/L; CaCl
2H
2O1.0-2.0g/L; FeCl
36HO 0.1-0.5g/L; PH 6.5-7.5; Agar 15g/L.
The liquid seed culture medium prescription that above-mentioned steps (1) is used is:
1.0-2.0% glucose, 0.2-1.0% soy peptone, 0.1-0.5% MgSO
4H
2O, 0.1-0.5%CaCl
2H
2O, pH 7-7.5.
The liquid fermentation medium prescription that above-mentioned steps (2) is used is:
The 0.5-2.5% yam starch, 0.1-1.5% yeast powder, 0.1-0.5% MgSO
4H
2O, 0.1-0.5%CaCl
2H
2O, 0.001-0.01% FeCl
3, 1-5% macroporous adsorbent resin, pH are 6.5-8.
Obtain 2 in the above-mentioned slime bacteria fermentation that utilizes, 6-two-(6-sulfydryl-3-pyridine)-4, in the liquid fermentation medium prescription of the method for 8-two-(4-phenol)-[1,5]-diazocine, the macroporous adsorbent resin of interpolation is meant Amberlite XAD-16 macroporous adsorbent resin.
The invention still further relates to separation and Extraction 2 from slime bacteria heap capsule fermented liquid, 6-two-(6-sulfydryl-3-pyridine)-4, the method for 8-two-(4-phenol)-[1,5]-diazocine the steps include:
(1) adopts filtration or centrifugation method, from fermented liquid, collect and the separation macroporous adsorbent resin.
(2) stepwise solvent extraction: use methyl alcohol respectively, methylene dichloride and normal hexane carry out three step extraction treatment.
(3) silica gel column chromatography: the 3rd step extract is carried out silicagel column low pressure chromatography, utilize methyl alcohol-methylene dichloride to carry out gradient elution, elution time is 130-155min, obtains the component of purification by silica gel column chromatography.
(4) sieve chromatography: the component of purification by silica gel column chromatography heavily is dissolved in 2-4 times of volumes methanol, carries out dextrane gel (Sephadex LH-20) molecular sieve column chromatography and separate, moving phase is methyl alcohol or methylene dichloride.
(5) high performance liquid phase separates: when utilizing high performance liquid phase to separate this compound, use reversed-phase column, moving phase is the first alcohol and water, can obtain 2 of purifying after the collection, 6-two-(6-sulfydryl-3-pyridine)-4,8-two-(4-phenol)-[1,5]-diazocine.
Wherein, the concrete grammar of above-mentioned steps (2) stepwise solvent extraction is:
(1) use the methyl alcohol of 10-20 times of volume that resin compound is extracted, be incubated 30-50 ℃, 24-48 hour, the centrifugal resin of removing obtained first part's extract.
(2) with 40 ℃ of evaporated in vacuo of methyl alcohol of first part's extract, pulverize, use the methylene dichloride of 10-20 times of volume to carry out the extraction of second step, be incubated 20-30 ℃, 24-48 hour, the centrifugal precipitation of removing obtained the second section extract.
(3) with 30 ℃ of evaporated in vacuo of methylene dichloride of second section extract, pulverize, use the normal hexane of 10-20 times of volume to carry out the extraction of the 3rd step, be incubated 20-30 ℃, 24-48 hour, centrifugal collecting precipitation used normal hexane flushing precipitation, obtains the third part extract.
Wherein, the gradient process can be in above-mentioned steps (3) silica gel column chromatography: keep 100%CH
2Cl
230min changes into 80%CH through 60min then
2Cl
2And 20%CH
3OH keeps 120min; The detection wavelength is 254nm,, sensitivity is 0.2A.
Wherein, moving phase is methyl alcohol in above-mentioned steps (4) sieve chromatography, and flow velocity is 0.8ml/min, detects wavelength 254nm, and elution time is 170-190min.
Wherein, above-mentioned steps (5) high performance liquid phase uses C18,10 μ m, the anti-phase preparative column of 250 * 20mm Shim-packPREP-ODS in separating.
Creativeness of the present invention is, finds and differentiated a kind of novel diazocine compounds, and the fermentation preparation and the separation purification method of this compound are provided, for the exploitation of this application of compound and slime bacteria resource lays a solid foundation.
Utilize method of the present invention, 2,6-two-(6-sulfydryl-3-pyridine)-4,8-two-(4-phenol)-[1,5]-diazocine can reach the output of 2mg/L in fermented liquid, and the compound purity that final purifying obtains is more than 99%.
The diazocine compounds 2 that the present invention relates to, 6-two-(6-sulfydryl-3-pyridine)-4,8-two-(4-phenol)-[1,5]-diazocine can be used for preparing antitumor and antifungal drug.Be used for preparation treatment liver cancer for exploitation, cervical cancer, kidney and leukemic medicine, and the people is treated in preparation, animal and plant fungi infestation medicine is significant.
(4) description of drawings
Fig. 1 .2,6-two-(6-sulfydryl-3-pyridine)-4, the chemical structure and the ordination number of 8-two-(4-phenol)-[1,5]-diazocine
Fig. 2 .2,6-two-(6-sulfydryl-3-pyridine)-4, the DEPT spectrum of 8-two-(4-phenol)-[1,5]-diazocine
Fig. 3 .2,6-two-(6-sulfydryl-3-pyridine)-4, the hsqc spectrum of 8-two-(4-phenol)-[1,5]-diazocine
Fig. 4 .2,6-two-(6-sulfydryl-3-pyridine)-4, the long-range C-H of 8-two-(4-phenol)-[1, the 5]-diazocine synoptic diagram (drawing) of being correlated with by HMBC spectrum
(5) embodiment
Embodiment 1
How to realize utilizing the slime bacteria fermentation to obtain 2,6-two-(6-sulfydryl-3-pyridine)-4,8-two-(4-phenol)-[1,5]-diazocine below by embodiment 1 explanation.
(1) preparation of ferment-seeded: an inoculation shovel sorangium cellulosum (Sorangium cellulosum ATCC 25569) is seeded in (KNO on the solid slant culture base
31.0g/L; K
2HPO
42H
2O 0.5g/L; MgSO
47H
2O2.0g/L; CaCl
2H
2O 2.0g/L; FeCl
36HO 0.5g/L; PH7.5; Agar 15g/L), cultivate after 3 days, be inoculated in 100ml liquid seed culture medium (2.0% glucose, 0.2% soy peptone, 0.5%MgSO for 32 ℃
4H
2O, 0.5%CaCl
2H
2O, pH7.2), cultivated 5 days, and obtained 5 * 10 for 30 ℃
9The somatic cells of/ml.
(2) liquid fermenting: the liquid fermentation medium of use consists of: 1.5% yam starch, 0.8% yeast powder, 0.5%MgSO
4H
2O, 0.5%CaCl
2H
2O, 0.005%FeCl
3, 3%Amberl ite XAD-16, pH are 7.5.The fermentation operation method is: the somatic cells for preparing is inoculated by the inoculum size of 4L contained in the 40L fermention medium, fermenting container is the automatic fermentor tank of 50L, and culture temperature is 30 ℃, and mixing speed is 45 commentaries on classics/min, cultivates 7 days.
(3) extraction of metabolite and analysis: the Amberlite XAD-16 resin in the fermented liquid is taken out, add 10 times of volumes methanol lixiviates, thereby obtain to contain 2,6-two-(6-sulfydryl-3-pyridine)-4, the tunning of 8-two-(4-phenol)-[1,5]-diazocine.
How to realize that separation and purification obtains 2 from the slime bacteria fermented liquid, 6-two-(6-sulfydryl-3-pyridine)-4,8-two-(4-phenol)-[1,5]-diazocine below by embodiment 2 explanations.
(1) collection and separation macroporous adsorbent resin from fermented liquid can adopt and filter or centrifugation method.
(2) stepwise solvent extraction:
1. use the methyl alcohol of 20 times of volumes that resin compound is extracted, be incubated 45 ℃, 48 hours, the centrifugal resin of removing obtained first part's extract.
2. with 40 ℃ of evaporated in vacuo of methyl alcohol of first part's extract, pulverize, use the methylene dichloride of 20 times of volumes to carry out the extraction of second step, be incubated 25 ℃, 48 hours, the centrifugal precipitation of removing obtained the second section extract.
3. with 30 ℃ of evaporated in vacuo of methylene dichloride of second section extract, pulverize, use the normal hexane of 20 times of volumes to carry out the extraction of the 3rd step, be incubated 20 ℃, 48 hours, centrifugal collecting precipitation used normal hexane flushing precipitation, and precipitation is as the third part extract.
(3) silica gel column chromatography: the third part extract is carried out silicagel column low pressure chromatography, utilize methyl alcohol-methylene dichloride to carry out gradient elution, obtain partially purified silica gel column chromatography component.The gradient process can be: keep 100%CH
2Cl
230min changes into 80%CH through 60min then
2Cl
2And 20%CH
3OH keeps 120min.The detection wavelength is 254nm,, sensitivity is 0.2A.
(4) sieve chromatography: the silica gel column chromatography component heavily is dissolved in 2 times of volumes methanol, carries out dextrane gel (Sephadex LH-20) molecular sieve column chromatography according to the volume of column volume 3.5% as silica gel column chromatography application of sample amount and separate.Moving phase is methyl alcohol or methylene dichloride, flow velocity be 0.5 column volume/hour, detect wavelength 254nm.
(5) high performance liquid phase separates: when utilizing high performance liquid phase to separate this compound, use anti-phase preparative column (C18,10 μ m, 250 * 20mm), moving phase is the first alcohol and water.Can obtain 2 of purifying after the collection, 6-two-(6-sulfydryl-3-pyridine)-4,8-two-(4-phenol)-[1,5]-diazocine compound.
Embodiment 3
How to realize utilizing stepwise solvent extraction initial gross separation 2,6-two-(6-sulfydryl-3-pyridine)-4,8-two-(4-phenol)-[1,5]-diazocine below by embodiment 3 explanations.
(1) the 1L resin compound is cleaned 3 times with 5L distilled water, be positioned over 45 ℃ of complete dryinies then, remove moisture.
(2) dried 1L resin compound is transferred in the 50L port grinding bottle, smashed to pieces the bulk particle gently, add 20L analytical pure methyl alcohol, and slowly stir, resin can be suspended in the methyl alcohol comparatively uniformly,, be incubated 40 ℃, 48 hours extracting.March into the arena during the course and vibrate, shake up resin.Cross and filter to remove resin, obtain about 19L first part extract.
(3) 19L first part extract is divided transfer in the 3L round-bottomed flask for 20 times, be placed on the Rotary Evaporators, be heated to 40 ℃, the methyl alcohol evaporated in vacuo.The 0.2L solid matter that obtains is pulverized with mortar, be transferred in the 10L port grinding bottle, add 4L analytical pure methylene dichloride, and slowly stir, solid particulate can be suspended in the methylene dichloride comparatively uniformly,, be incubated 27 ℃, 48 hours extracting.March into the arena during the course and vibrate, shake up solution.1000rpm, the centrifugal precipitation of removing of 20min obtains the second section extract.Obtain about 3.5L second section extract.
(4) 3.5L second section extract is divided transfer in the 3L round-bottomed flask for 4 times, be placed on the Rotary Evaporators, be heated to 30 ℃, the methylene dichloride evaporated in vacuo.The 0.1L solid matter that obtains is pulverized with mortar, be transferred in the 5L port grinding bottle, add 2L analytical pure normal hexane, and slowly stir, solid particulate can be suspended in the normal hexane comparatively uniformly,, be incubated 20 ℃, 48 hours extracting.2000rpm, the 20min centrifugal collecting precipitation.Then, precipitation is transferred on the funnel, slowly added 0.2L analytical pure normal hexane flushing precipitation, drying precipitated, obtain the third part extract.
Embodiment 4
How to realize utilizing silica gel column chromatography preliminary purification 2,6-two-(6-sulfydryl-3-pyridine)-4,8-two-(4-phenol)-[1,5]-diazocine below by embodiment 4 explanations.
(1) silica gel column chromatography belongs to the adsorpting column chromatogram method, be utilize each component sorbent material and eluent between the absorption and the difference of desorption ability and reach isolating purpose.Employed sorbent material can be materials such as silica gel or aluminium sesquioxide, and employed eluent can be a methyl alcohol, methylene dichloride, normal hexane, ethyl acetate, acetone and normal butane equal solvent.
(2) preparation of chromatographic column: the diameter of pillar is 10cm, and length is 150cm, and ratio is 1: 15.
(3) preparation of sorbent material: selecting particle diameter is the sorbent material silica gel of 70 μ m, should soak 48 hours with methyl alcohol earlier before silica gel uses, again with 110 ℃ of activation two hours.
(4) wet method dress post: sorbent material is put into small beaker, add methylene dichloride and stirring, add 1/2 volume eluent methylene dichloride in the post in advance.Open piston, slowly add silica gel, up to filling whole Zhi Zhuzi.
(5) application of sample: the solubleness of the isolating sample of column chromatography in eluent is not good owing to carrying out, so adopt the solid-state sample method that goes up.At first sample is dissolved in appropriate solvent (methylene dichloride), adds about 5 times sorbent material silica gel then.Mixture is used the Rotary Evaporators evaporate to dryness down at 30 ~ 40 ℃, then the powder of gained is added to the top of chromatographic column.
(6) wash-out: utilize methyl alcohol-methylene dichloride to rely on gravity to carry out gradient elution, obtain partially purified component.The gradient process can be: keep 100%CH
2Cl
230min changes into 80%CH through 60min then
2Cl
2And 20%CH
3OH keeps 120min.
(7) detection and record: the detection wavelength is 254nm, and detector sensitivity is 0.2A, and registering instrument sensitivity is 200mV, and chart drive speed is 6cm/hr.
(8) collection of separated portion: use automatic Fraction Collector to carry out the collection of sample separation, compound 2,6-two-(6-sulfydryl-3-pyridine)-4, the elution time of 8-two-(4-phenol)-[1,5]-diazocine is 130-155min.
How to realize utilizing sieve chromatography to be further purified 2 6-two-(6-sulfydryl-3-pyridine)-4,8-two-(4-phenol)-[1,5]-diazocine below by embodiment 5 explanations.
(1) basic parameter of Sephadex LH-20: particle diameter is 25 ~ 100 microns; Isolating molecular weight ranges: 100 ~ 4000 (ethanol), 100 ~ 2000 (chloroforms); Get the dried glue of water number: 4ml/mg.
(2) heating method swelling Sephadex LH-20 gel: the gel that suspends in water is warming up to boiling gradually, can finishes swelling through 1 ~ 2 hour.After the cooling, use the methyl alcohol repetitive scrubbing, remove moisture, decompression is bled, and removes bubble.
(3) dress post: in post, add earlier the methyl alcohol of 1/3rd volumes, the scrutiny column base plate, bubble is removed in vibration, under agitation, the gel suspension in the beaker is poured into, near filling.After the gel of 1 ~ 2cm is arranged when sedimentation on the base plate, open the outlet of post, extract methyl alcohol out with medium flow rate.Constantly add gel, methyl alcohol is flowed out naturally,, stop to adorn post when depositional plane during apart from top 3 ~ 5cm.
(4) application of sample amount: sample volume equals 2 ~ 5% of column volume.
(5) wash-out: moving phase is methyl alcohol, and flow velocity is 0.8ml/min.
(6) detection and record: the detection wavelength is 254nm, and detector sensitivity is 0.1A, and registering instrument sensitivity is 200mV, and chart drive speed is 3cm/hr.
(7) collection of separated portion: use automatic Fraction Collector to carry out the collection of sample separation, compound 2,6-two-(6-sulfydryl-3-pyridine)-4, the elution time of 8-two-(4-phenol)-[1,5]-diazocine is 170-190min.
Embodiment 6
How to realize utilizing high performance liquid chromatography to obtain 2 of complete purifying below by embodiment 6 explanations, 6-two-(6-sulfydryl-3-pyridine)-4,8-two-(4-phenol)-[1,5]-diazocine.
(1) instrument that uses of preparation HPLC is Shimadzu LC-6A system, UV SPD-6AV detector and C-R6A sampler.
(2) the fixedly fragrance C18 of the preparation HPLC post of Shi Yonging (10 μ m), column type is 250 * 20mm Shim-pack PREP-ODS post.
(3) elution speed is 10ml/min, and moving phase consists of mobile phase A (water-0.2% acetic acid) and B (methyl alcohol), and sample size is 100 μ l.
Elution process is: be converted to 40% mobile phase A and 60% Mobile phase B from 100% mobile phase A through the 40min linear gradient, pass through 10min then, linear gradient is converted to 100% Mobile phase B, keeps 10min.
(4) detecting wavelength is 249 nm, uses the FRC-10A automatic collector to collect.
(5) compound 2 of purifying, 6-two-(6-sulfydryl-3-pyridine)-4, the residence time of 8-two-(4-phenol)-[1,5]-diazocine is 34.67min.
Claims (8)
1. a novel diazocine compounds is characterized in that, it is to separate in the fermented liquid by slime bacteria heap capsule bacterium to make, and molecular formula is C
28H
20N
4O
2S
2, name is called 2,6-two-(6-sulfydryl-3-pyridine)-4, and 8-two-(4-phenol)-[1,5]-diazocine, chemical structure is as follows:
2. one kind is utilized the slime bacteria fermentation to obtain the described compound 2 of claim 1,6-two-(6-sulfydryl-3-pyridine)-4, and the method for 8-two-(4-phenol)-[1,5]-diazocine, its basic step is:
(1) preparation of ferment-seeded: will produce bacterium and be seeded on the solid slant culture base, and cultivate after 3-7 days for 28-32 ℃, and be inoculated in liquid seed culture medium, and cultivate 3-7 days, and obtained 1-8 * 10 for 28-32 ℃
9The somatic cells of/ml;
(2) liquid fermenting: the somatic cells that step (1) is prepared, by with the fermentating liquid volume ratio be that 0.5 ~ 1.5: 10 inoculum size is inoculated in the automatic fermentor tank, culture temperature is 30 ℃, mixing speed is 30-60 rev/min, cultivates 5-9 days;
The generation bacterium that above-mentioned steps (1) is used is slime bacteria sorangium cellulosum (Sorangium cellulosum ATCC25569);
The solid slant culture based formulas that above-mentioned steps (1) is used is:
KNO
30.5-1.5g/L; K
2HPO
42H
2O 0.5-1.0g/L; MgSO
47H
2O 1.0-2.0g/L; CaCl
2H
2O1.0-2.0g/L; FeCl
36HO 0.1-0.5g/L; PH6.5-7.5; Agar 15g/L;
The liquid seed culture medium prescription that above-mentioned steps (1) is used is:
1.0-2.0% glucose, 0.2-1.0% soy peptone, 0.1-0.5%MgSO
4H
2O, 0.1-0.5%CaCl
2H
2O, pH7-7.5;
The liquid fermentation medium prescription that above-mentioned steps (2) is used is:
The 0.5-2.5% yam starch, 0.1-1.5% yeast powder, 0.1-0.5%MgSO
4H
2O, 0.1-0.5%CaCl
2H
2O, 0.001-0.01%FeCl
3, 1-5% macroporous adsorbent resin, pH are 6.5-8.
3. as claimed in claim 2ly utilize slime bacteria fermentation to obtain 2,6-two-(6-sulfydryl-3-pyridine)-4,8-two-(4-phenol)-[1,5]-method of diazocine, it is characterized in that the macroporous adsorbent resin that adds in the liquid fermentation medium prescription is meant Amberlite XAD-16 macroporous adsorbent resin.
4. described compound 2 of separation and Extraction claim 1 from the slime bacteria fermented liquid, 6-two-(6-sulfydryl-3-pyridine)-4, the method for 8-two-(4-phenol)-[1,5]-diazocine the steps include:
(1) adopts filtration or centrifugation method, from fermented liquid, collect and the separation macroporous adsorbent resin;
(2) stepwise solvent extraction: use methyl alcohol respectively, methylene dichloride and normal hexane carry out three step extraction treatment;
(3) silica gel column chromatography: the 3rd step extract is carried out silicagel column low pressure chromatography, utilize methyl alcohol-methylene dichloride to carry out gradient elution, elution time is 130-155 minute, obtains the component of purification by silica gel column chromatography;
(4) sieve chromatography: the component of purification by silica gel column chromatography heavily is dissolved in 2-4 times of volumes methanol, carries out dextrane gel Sephadex LH-20 molecular sieve column chromatography and separate, moving phase is methyl alcohol or methylene dichloride;
(5) high performance liquid phase separates: when utilizing high performance liquid phase to separate this compound, use reversed-phase column, moving phase is the first alcohol and water, can obtain the described compound 2 of claim 1 of purifying after the collection, 6-two-(6-sulfydryl-3-pyridine)-4,8-two-(4-phenol)-[1,5]-diazocine.
5. as claimed in claim 4 a kind of from the slime bacteria fermented liquid the described compound 2 of separation and Extraction claim 1,6-two-(6-sulfydryl-3-pyridine)-4, the method for 8-two-(4-phenol)-[1,5]-diazocine, it is characterized in that the concrete grammar of step (2) stepwise solvent extraction is:
(1) use the methyl alcohol of 10-20 times of volume that resin compound is extracted, be incubated 30-50 ℃, 24-48 hour, the centrifugal resin of removing obtained first part's extract;
(2) with 40 ℃ of evaporated in vacuo of methyl alcohol of first part's extract, pulverize, use the methylene dichloride of 10-20 times of volume to carry out the extraction of second step, be incubated 20-30 ℃, 24-48 hour, the centrifugal precipitation of removing obtained the second section extract;
(3) with 30 ℃ of evaporated in vacuo of methylene dichloride of second section extract, pulverize, use the normal hexane of 10-20 times of volume to carry out the extraction of the 3rd step, be incubated 20-30 ℃, 24-48 hour, centrifugal collecting precipitation used normal hexane flushing precipitation, obtains the third part extract.
6. as claimed in claim 4 a kind of from the slime bacteria fermented liquid the described compound 2 of separation and Extraction claim 1,6-two-(6-sulfydryl-3-pyridine)-4,8-two-(4-phenol)-[1,5]-method of diazocine, it is characterized in that the gradient process can be in step (3) silica gel column chromatography: keep 100%CH
2Cl
230min changes into 80%CH through 60min then
2Cl
2And 20%CH
3OH keeps 120min; The detection wavelength is 254nm,, sensitivity is 0.2A.
7. as claimed in claim 4 a kind of from the slime bacteria fermented liquid the described compound 2 of separation and Extraction claim 1,6-two-(6-sulfydryl-3-pyridine)-4,8-two-(4-phenol)-[1,5]-method of diazocine, it is characterized in that moving phase is methyl alcohol in step (4) sieve chromatography, detect wavelength 254nm, flow velocity is 0.8ml/min, and elution time is 170-190min.
8. as claimed in claim 4 a kind of from the slime bacteria fermented liquid the described compound 2 of separation and Extraction claim 1,6-two-(6-sulfydryl-3-pyridine)-4,8-two-(4-phenol)-[1,5]-method of diazocine, it is characterized in that, step (5) high performance liquid phase uses C18,10 μ m, the anti-phase preparative column of 250 * 20mm Shim-pack PREP-ODS in separating.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101052666B (en) * | 2004-04-22 | 2011-06-29 | 索维高级聚合物股份有限公司 | Dibenzodiazocine polymers |
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2004
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101052666B (en) * | 2004-04-22 | 2011-06-29 | 索维高级聚合物股份有限公司 | Dibenzodiazocine polymers |
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