CN108341827A - A method of preparing bacteripheophytin a and bacteripheophytin a esters - Google Patents
A method of preparing bacteripheophytin a and bacteripheophytin a esters Download PDFInfo
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- CN108341827A CN108341827A CN201810104433.XA CN201810104433A CN108341827A CN 108341827 A CN108341827 A CN 108341827A CN 201810104433 A CN201810104433 A CN 201810104433A CN 108341827 A CN108341827 A CN 108341827A
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Abstract
The present invention relates to a kind of methods preparing bacteripheophytin a esters.This method is from Fermentation by Photosynthetic Bacteria liquid, by being collected to thalline after separation of solid and liquid, then the antibiotics sensitivity test in thalline is extracted using aqueous acetone solution, after the processing of extracting solution tune acid, 95.0% or more bacteripheophytin a is can be obtained by solvent extraction, crystallization.First using a kind of above-mentioned bacteripheophytin a of solvent dissolving that can be dissolved bacteripheophytin a again can be miscible with alcohol, then sulfuric acid alcoholic solution is added to be esterified under heating conditions, esterification reaction solution obtains bacteripheophytin a ester crude products by post-processing, and gained bacteripheophytin a ester crude products are directly over the sterling that crystallization can be obtained 95.0% or more.
Description
Technical field
The invention belongs to technological field of biochemistry, and in particular to a kind of to prepare bacteripheophytin a and de-magging bacterium
The method of chlorophyll a ester.
Background technology
Photodynamic therapy (Photodynamic Therapy, PDT) is to carry out medical diagnosis on disease using photodynamic effect and control
A kind of new technology treated, exposure basis is photodynamic effect.This is a kind of light for the adjoint biological effect for having oxygen molecule to participate in
Sensitized reaction.Its process is that the laser irradiation of specific wavelength makes the photosensitizer of tissue resorption be excited, and excitation state is photosensitive
Agent generates highly active singlet oxygen, oxygen occurs for singlet oxygen and adjacent large biological molecule again energy transmission to the oxygen of surrounding
Change reaction, and then leads to cell damage or even death.Compared with common chemotherapy and radiotherapy, photosensitizer has more preferable
Selectivity the life quality of subject therefore can be improved.PDT is considered as since the 1980s in cancer
A kind of new method of most foreground in disease treatment.
After photosensitizing agents, due to the presence of photosensitive side reaction, need certain to be protected from light the time after treatment.
In addition, if absorbing wavelength ＜ 700nm, tissue penetration depths will be limited, therefore PDT treatment Deep Lesions or larger swollen of knurl
Need to have have the photosensitizer that long wavelength absorbs in infrared/near-infrared region when tumor.The characteristic absorption peak of chlorophyll a is located at
430nm~450nm and 630nm~650nm；The characteristic absorption peak of chlorophyll b is located at 450nm~480nm and 650nm~680nm；
And the characteristic absorption of antibiotics sensitivity test (bacteriochlorophyll a, Bchl a) is located at 350nm~400nm and 750nm
~800nm is not absorbed in the visible light region of 400nm~700nm.If by Bchl a class photosensitizers for tumour diagnosis and
Treatment, so that it may it uses the near infrared ray of 750nm or more as treatment light source, considerably increases penetration capacity in the tissue, it can be straight
Connect the diagnosing and treating for in-vivo tissue.Meanwhile reducing the visible light photosensitized reaction after treatment.Therefore, Bchl a classes light
Quick dose has the superiority of bigger than plant chlorophyll class photosensitizer in the fields PDT.
Although Bchl a have good photosensitive activity, its stability is bad, and dissolubility is also poor.Therefore,
It can not be developed directly as photosensitizer.The These characteristics of directed toward bacteria chlorophyll a (Bchl a), improve its stability and
Deliquescent derivative achieves greater advance.Currently, phase II clinical trials are being carried out from the WST11 of Bchl a,
WST-09 is carrying out phase III clinical trial.Their Bchl a raw materials are by the thiophilic small red oomycetes (Rhodovolum that ferments
Sulfidophilum, WO 00/33833), then pass through what extraction purification obtained.Except Rhodovolum Sulfidophilum
Outside, the photosynthetic bacteria of production Bchl a also have hydrogenlike silicon ion (Rhodopseudomonas sphaeroides,
J.Org.Chem.1980,45,1969-1974), (Rhodopseudomonas plustris, Shanxi are big for Rhodopseudomonas palustris
Learn journal (natural science edition) .2005,28 (2):169-172) and Rhodopseudomonas rutila (Rhodopseudomonas
Rutila, Acta Pharmaceutica Sinica .2005,40 (10):920-923) etc..
ZL99816058.X discloses one kind and is carried from photosynthetic bacteria Rhodovolum Sulfidophilum lyophilized cells
The method for taking Bchla.This extracting method is the current universal method for preparing Bchl a.Its by solvent from freeze-drying photosynthetic bacteria
After extracting Bchl a in thalline, by the Bchl a of DEAE- agarose gel column chromatographies isolated 90% or more.However,
Bchl a are very unstable, and being easy to de-magging under the conditions ofs acid and heating etc. forms bacteripheophytin.Moreover, this method
It needs by DEAE- agarose gel column chromatographies, technics comparing is complicated, and purification time is longer.In general, work done in the manner of a certain author is starting material
The chemical substance of material should be the substance after isolating and purifying, and impurities should not be the source of major impurity in bulk pharmaceutical chemicals.Therefore,
For synthesizing the derivative of Bchl a, Bchl a are not an ideal starting material.In addition, bacteripheophytin methyl esters one
Sour a (Bpheid a) is unstable, the selection that hardly possible purifying is not very good.Bchl a catabolite bacteripheophytin a (Bphe
A), bacteripheophytin a esters especially bacteripheophytin a methyl esters (Mbp a) is relatively stablized, if it is possible to solve
Preparation problem is produced, they will be the ideal starting material for synthesizing Bchl a derivatives.
Bchl a catabolite Bphe a are required for just obtaining by column chromatography more high-purity in now disclosed method
The Bphe a of degree.In addition, bacteriochlorophyll is more more unstable than chlorophyll, therefore, the esterification of bacteripheophytin a esters is not
The esterification process of conventional chlorophyll can be used.According to the literature, Kozyrev, AN etc. use the methyl esters of conventional chlorophyll
The yield of change method, obtained antibiotics sensitivity test methyl esters is less than 27%, although using Methy esterification by diazomethane yield up to 78%
(J.Org.Chem., 2006,71,1949-1960), still, in industrialized production, diazomethane is forbidden to use.Bphe
A is easy slowly oxidation in alcoholic solution, and therefore, carrying out esterification reaction of organic acid to it using sulfuric acid alcoholic solution must quickly complete, no
Then easy to produce side reaction, especially oxidation reaction etc..In addition, since the polarity of Bphe a is smaller, in sulfuric acid alcoholic solution
Solvability is limited, is reacted completely if insoluble, and the reaction time can extend, can be because being aoxidized or other side reaction
Generate a large amount of by-products.To sum up, the production technology of Bphe a and bacteripheophytin a esters is all to be improved.
Invention content
Complicated for the starting material technology of preparing of existing Bchl a derivatives, purity is difficult the problems such as raising.The present invention
Be designed to provide a kind of method preparing high-purity Bchl a catabolite Bphe a and bacteripheophytin a esters.This
Invention technology of preparing is simple, and the Bphe a and bacteripheophytin a esters purity being prepared are synthesis 95.0% or more
The ideal starting material of Bchl a derivatives.
The present invention relates to a kind of method preparing bacteripheophytin a esters, this method can prepare bacteripheophytin a
Methyl esters (Mbp a) and bacteripheophytin a ethyl esters (Ebp a), the described method comprises the following steps：
(a) using it is a kind of can dissolve bacteripheophytin a again can with alcohol it is miscible solvent dissolving de-magging bacterium leaf it is green
Then plain a is added sulfuric acid alcoholic solution and is esterified under heating conditions, is concentrated to dryness to obtain de-magging after esterification reaction solution is post-treated
Antibiotics sensitivity test ester crude product；
(b) bacteripheophytin a esters crude product obtains the sterling of bacteripheophytin a esters by crystallization.
Preferably, a kind of in the step (a) can dissolve the solvent that bacteripheophytin a again can be miscible with alcohol and be
The mixture of chloroform, dichloromethane or both, preferably chloroform, described one kind can dissolve bacteripheophytin a again can be with
Additive amount (the unit of the miscible solvent of alcohol：Ml) it is bacteripheophytin a (units：G) 10-20 times.
Preferably, the alcohol in the step (a) in sulfuric acid alcoholic solution is methanol or ethyl alcohol, the sulfuric acid alcoholic solution is body
Sulfuric acid alcoholic solution of the product than 5-15%, preferably 10% sulfuric acid alcoholic solution, the additive amount (unit of the alcohol sulphate：Ml it is) de-
Magnesium antibiotics sensitivity test (unit：G) 40-60 times.
In the present invention, the sulfuric acid alcoholic solution of the volume ratio 5-15% refers to the volume of the volume and sulfuric acid alcoholic solution of sulfuric acid
Than the sulfuric acid is the concentrated sulfuric acid.
Preferably, the heating and temperature control being esterified in the step (a) is at 40-55 DEG C.
Preferably, post-processing step in the step (a) is to be diluted with water in esterification reaction solution, then use chloroform or
Person's dichloromethane extracts, and gained chloroform or dichloromethane mutually first use sodium bicarbonate aqueous solution and water washing respectively, then with anhydrous
Sodium sulphate is dried, and the washing times of the sodium bicarbonate aqueous solution and water are preferably each twice.
As further preferred technical solution, the amount being diluted with water in the post-processing in the step (a) is esterification
1-2 times (volume ratio) of liquid product, the chloroform or dichloromethane extract addition (unit：Ml) it is bacteripheophytin
A (units：G) 10-40 times, the sodium bicarbonate aqueous solution is preferably 10% sodium bicarbonate aqueous solution, and dosage is extraction institute
Obtaining chloroform, either the dosage of 1-2 times of volume (volume ratio) described water washing of dichloromethane phase is to extract gained chloroform or two
The 1-2 times of volume (volume ratio) of chloromethanes phase.
Preferably, in the step (a), in order to reduce the by-product of oxidation reaction generation, this reaction can be in indifferent gas
It is carried out under conditions of body protection, the inert gas is preferably nitrogen.
Response situation is supervised preferably, silica gel thin-layer chromatography (TLC) can be used in the reaction in above-mentioned steps (a)
It surveys, wherein TLC developing solvent systems are n-hexane:Acetone=2:1.
Preferably, the preferred C of recrystallisation solvent that the crystallization in the step (b) uses1-C7Halogenated alkyl object and C1-C10
Hydrocarbon compound mixed solvent, the further preferably mixed solvent of chloroform and normal heptane, wherein the in the mixed solvent
Chloroform and normal heptane volume ratio are preferably 3:7.
As further preferred technical solution, the crystallisation step in the step (b) is first by bacteripheophytin a esters
Crude product (unit：G) 3 times of volume (units are dissolved in：Ml) in chloroform, 7 times of volume (units are added after filtering：Ml) normal heptane mixes
It is transferred in 4 DEG C of refrigerators and is stood still for crystals for 24 hours after closing uniformly.
In a preferred embodiment, Bphe a are prepared into bacteripheophytin a methyl esters by the present invention：Bphe a are first used
Chloroform dissolves, and the methanolic solution for being then added 10% carries out esterification under 45 DEG C of heating conditions, and esterification reaction of organic acid liquid adds
Chloroform extraction is added after water dilution, chloroform is mutually dried with after 10% sodium bicarbonate and water washing with anhydrous sodium sulfate respectively, then
It is concentrated in vacuo to dry up to Mbp a crude products；By Mbp a crude products with first being dissolved by heating with chloroform, normal heptane (chloroform is then added:
Normal heptane=3:7) it is down to room temperature, is then transferred in 4 DEG C of refrigerators and crystallizes up to Mbp a sterlings.
Preferably, the method for preparing bacteripheophytin a in the present invention includes the following steps：
(1) zymotic fluid containing antibiotics sensitivity test is prepared in Fermentation by Photosynthetic Bacteria；
(2) zymotic fluid obtains the mycelium containing antibiotics sensitivity test after separation of solid and liquid；
(3) mycelium containing antibiotics sensitivity test is extracted using aqueous acetone solution, filters to obtain extracting solution；
(4) organic phase is obtained by extraction with organic solvent after the extracting solution tune acid containing antibiotics sensitivity test；Organic phase is concentrated into
Concentrate is obtained after dry；
(5) by concentrate obtained by step (4) again by crystallizing up to bacteripheophytin a.
Preferably, photosynthetic bacteria in the step (1) be selected from Rhodovolum Sulfidophilum,
Rhodopseudomonas sphaeroides、Rhodopseudomonas plustris、Rhodopseudomonas
rutila。
Preferably, separation of solid and liquid in the step (2) is to be centrifuged after plate-frame filtering or flocculating setting after flocculating setting.
As further preferred technical solution, the solid-liquid separation step in the step (2) is as follows：It is added into zymotic fluid
Acid stands acidification sedimentation one day or more to realize the flocculating setting of thalline after adjusting pH to 2-5；In order to reduce sedimentation time and
Enhance the effect of sedimentation, a preferred embodiment is that flocculant, such as chitosan is added when adjusting pH value；Zymotic fluid after sedimentation can
Selection be added diatomite stir evenly, the filter residue containing thalline is then obtained by plate-frame filtering, alternatively by directly from
Zymotic fluid after heart sedimentation obtains thalline.
Preferably, the separation of solid and liquid in the step (2) is ceramic membrane filter, the aperture of the ceramic membrane is 100-
500nm, preferably 200nm.
Preferably, the volume content of acetone is 70%-80% in aqueous acetone solution in the step (3), described third
The additive amount of ketone aqueous solution is 1/3-1 times (volume ratio) of fermentating liquid volume in step (1), can extract carrot less in this way
The impurity such as element.Due to there are part Bchl a to have changed into Bphe a in aqueous acetone solution extracting solution, carried to reduce Bphe a
It takes in liquid and is precipitated, the risk for reducing extract yield may be selected that C is added before filtration1-C10Alkane derivative.
Preferably, the tune acid pH that aqueous acetone solution extracting solution described in step (4) carries out de-magnesium reaction is preferably 2-5,
Wherein, the adoptable solvent of the tune acid is the aqueous solution of hydrochloric acid, acetic acid, phosphoric acid, sulfuric acid or above-mentioned acid.
Preferably, the organic solvent that the organic solvent extraction described in step (4) uses is C1-C10Alkanes chemical combination
Object, polarity becomes smaller after Bchl a de-maggings, uses C1-C10Hydrocarbon compound extracted, obtained C1-C10Hydrocarbon compound
It is mutually concentrated to dryness up to Bphe a crude products；Above-mentioned C1-C10Hydrocarbon compound be selected from petroleum ether, n-hexane, hexamethylene, normal heptane
In one or more, further preferably normal heptane.
As further preferred technical solution, the dosage of organic solvent is whole in step (4) the organic solvent extraction
The 2-10% (volume ratio) of a extraction solution system, the extraction times are preferably 2 times.
Preferably, the recrystallisation solvent in step (5) is preferably C1-C10Alkane derivative and acetonitrile mixing it is molten
Agent crystallizes Bphe a, above-mentioned C1-C10Hydrocarbon compound in petroleum ether, n-hexane, hexamethylene, normal heptane
It is one or more, further preferably normal heptane.
Preferably, recrystallisation solvent is more preferably the mixed solvent of normal heptane and acetonitrile, volume ratio in step (5)
Preferably 1:2.
As further preferred technical solution, the crystallisation step in the step (5) is by concentrate obtained by step (4)
(unit：G) 3 times of volume (units are directly dissolved in：Ml the in the mixed solvent of normal heptane and acetonitrile), wherein normal heptane and acetonitrile
Volume ratio be 1:2, after filtering, it is transferred in 4 DEG C of refrigerators and stands still for crystals for 24 hours.
95.0% or more Bphe a, Mbp a and Ebp a can not only be prepared in the method for the present invention well, to close
Ideal starting material is provided at Bchla analog derivatives, and relative to the existing side for preparing Bphe a, Mbp a and Ebp a
It is higher need not to carry out cumbersome column chromatography, simple for process, time-consuming short and yield for method.
Specific implementation mode
Fermentation by Photosynthetic Bacteria liquid in the embodiment of the present invention, using from hydrogenlike silicon ion (Rhodobacter
Sphaeroides, ATCC17023, the bacterial strain are bought from ATCC), bibliography " G Cohen-Bazire, WR Sistrom, RY
Stanier.Kinetic studies of pigment synthesis by non-sulfur purple
Side in bacteria.Journal of Cellular and Comparative Physiology, 1957,49,25-68. "
Method ferments to obtain.
The analysis method of antibiotics sensitivity test and bacteripheophytin a：Chromatographic column, Yi Lite ODS2-C18 (4.6 ×
250mm, 5 μm)；Column temperature, 25 DEG C；Ultraviolet detection wavelength, 358nm；Flow velocity, 1.0mL/min；Mobile phase, methanol:Isopropanol=9:
1 (volume ratio).
The analysis method of bacteripheophytin a methyl esters：Chromatographic column, Yi Lite ODS2-C18 (4.6 × 250mm, 5 μm)；
Column temperature, 25 DEG C；Ultraviolet detection wavelength, 358nm；Flow velocity, 1.0mL/min；Mobile phase, methanol:Water=9:1 (volume ratio).
The monitoring of esterification reaction of organic acid：Response situation is monitored using silica gel tlc, wherein TLC developing solvent systems are
N-hexane:Acetone=2:1 (volume ratio).
Embodiment 1
60L Fermentation by Photosynthetic Bacteria liquid is stirred, oxalic acid is added, adjusted pH to 2.6, add 3kg diatomite, stirred
30min, standing filter to obtain filter residue afterwards for 24 hours, and filter residue is added 30L aqueous acetone solutions (80%) and impregnates, and third is obtained by filtration after stirring 3h
Ketone extracting solution.Gained acetone extract 28L is 3.6 using 1M salt acid for adjusting pH value, then uses 2L normal heptanes at twice, every time
Normal heptane phase is obtained by extraction in 1L, 50 DEG C of normal heptane phase is concentrated in vacuo to dry to get concentrate 9.1g.
Embodiment 2
60L Fermentation by Photosynthetic Bacteria liquid is stirred, oxalic acid is added, adjust pH to 2.5, chitosan aqueous solution (200g is then added
Chitosan is dissolved in 1% acetic acid water of 1L), 3kg diatomite is added, 30min is stirred, standing filters to obtain filter residue afterwards for 24 hours, filters
Slag is added 30L aqueous acetone solutions (80%) and impregnates, and acetone extract is obtained by filtration after stirring 3h.Gained acetone extract 29L, makes
It is 3.6 with 1M salt acid for adjusting pH value, then uses 2L normal heptanes at twice, normal heptane phase is obtained by extraction in each 1L, by normal heptane phase
50 DEG C be concentrated in vacuo to it is dry to get concentrate 9.8g.
Embodiment 3
60L Fermentation by Photosynthetic Bacteria liquid is stirred, oxalic acid is added, adjust pH to 3.0, chitosan aqueous solution (60g is then added
Chitosan is dissolved in 1% aqueous acetic acid), centrifuge (4000rpm, 15mins) is precipitated, and precipitation is added 80%
Aqueous acetone solution 30L impregnates, and acetone extract is obtained by filtration after stirring 3h.Gained acetone extract 31L, is adjusted using 1M hydrochloric acid
PH value is 3.6, then uses 2L normal heptanes at twice, and normal heptane phase is obtained by extraction in each 1L, by 50 DEG C of vacuum concentrations of normal heptane phase
To dry to get concentrate 10.7g.
Embodiment 4
By 60L Fermentation by Photosynthetic Bacteria liquid by ceramic membrane (0.2m2, 200nm) and filtering and concentrating, return pressure, which controls, to exist
2MPa, at 30 DEG C -40 DEG C, initial filtering velocity is 20L/h for temperature control, is slowed down as volume reduces filtering velocity, when remaining one third
Start slowly water top to be added to wash when volume, until stopping adding water after peritoneal effluent paler colour, is concentrated into minimum reflux volume after 10h, stops
Only filtering releases muddy thalline and ejects total 15L with water, and 20L acetone is added, acetone extract is obtained by filtration after stirring 3h.Institute
Acetone extract 30L (content of acetone 76%) is obtained, the use of 1M salt acid for adjusting pH value is 3.6, then uses 2L normal heptanes at twice, often
Normal heptane phase is obtained by extraction in secondary 1L, and 50 DEG C of normal heptane phase is concentrated in vacuo to dry to get concentrate 11.2g.Gained concentrate 11.2g
Being dissolved in the mixed solvent of 33.6mL normal heptanes and acetonitrile, (its volume ratio is 1:2) it, is transferred in 4 DEG C of refrigerators and stood after filtering
Night, filter-cloth filtering obtain 10.2g bacteripheophytin a, purity 96.3%, yield 75%.
Embodiment 5
10% methanolic solution 50ml is added in 4 gained bacteripheophytin a 1g of Example, and 50 DEG C are stirred at reflux
Reaction.By TLC trace analysis reaction process, after reacting 12h, the reaction was complete yet for detection.It is molten to add 10% methanolic
After liquid 50ml, the reaction was continued 4h, by TLC trace analysis reaction process, it is found that the reaction was complete yet, and impurity becomes more.
Embodiment 6
4 gained bacteripheophytin a 1g of Example add table after the dissolution solvent dissolving being added in 20ml tables 1
Each 50ml of methanolic solution described in 1 is controlled by temperature described in table 1 and is stirred at reflux reaction, TLC trace analysis reaction process,
Response situation see the table below 1.The concentration and reaction temperature of visible sulfuric acid alcoholic solution are inversely proportional with the reaction time in table 1.Accordingly, methyl esters
Change the methanolic solution that 5-15% may be selected in methanolic solution in reaction, computer heating control temperature range is 40-55 DEG C.
Table 1
| Dissolution solvent | Sulfuric acid alcoholic solution | Reaction temperature (DEG C) | Response situation | Remarks |
| Chloroform | 5% methanolic | 55 | The reaction was complete by 3h | |
| Chloroform | 5% methanolic | 45 | The reaction was complete by 3h | |
| Chloroform | 10% methanolic | 50 | The reaction was complete by 2h | |
| Dichloromethane | 10% methanolic | 40 | The reaction was complete by 2h | |
| Chloroform | 10% methanolic | 30 | The reaction was complete by 4h | |
| Chloroform | 15% methanolic | 45 | The reaction was complete by 2h | |
| Chloroform | 20% methanolic | 30 | The reaction was complete by 2h | Solution colour deepens |
Embodiment 7
10% methanolic solution is added after being dissolved with 20mL chloroforms in 4 gained bacteripheophytin a 1g of Example
40mL, lower 50 DEG C of nitrogen protection are stirred at reflux reaction.Through TLC trace analysis, the reaction was complete after 3h, in reaction solution plus 100mL water
After dilution, add 20mL chloroforms extraction, through TLC analyze upper strata aqueous phase no sample, collect chloroform mutually first with 10% bicarbonate
Sodium solution 50mL is washed at twice, each 25mL, is added deionized water 50mL and is washed at twice, each 25mL, gained chloroform
It is concentrated to dryness up to de-magging bacterium chlorophyllin a methyl esters crude products 1g after being added to the drying of 2g anhydrous sodium sulfates.
Above-mentioned de-magging bacterium chlorophyllin a methyl esters crude product is first used to 50 DEG C of heating for dissolving of 3mL chloroforms, is added after solution filtering
7mL normal heptanes are stood after mixing to room temperature, are then transferred in 4 DEG C of refrigerators and are stood overnight, and it is de- that filter-cloth filtering obtains 512mg
The crystal of magnesium bacterium chlorophyllin a methyl esters, HPLC purity are 97.2%, yield 73%.
Embodiment 8
3000L Fermentation by Photosynthetic Bacteria liquid is transferred in the souring tank of 5T, stirring is added oxalic acid 45kg and adjusts pH to 2.4, then
150kg diatomite is added, stirs 30min, plate-frame filtering after standing for 24 hours, filtrate Clarification Sterile body appears, and 1h filterings are completed, pressure
Thalline is obtained after contracting air blow drying moisture content, and 80% aqueous acetone solution 2000L is added and impregnates, is filtered after stirring 5h, 500L water top is used in combination
It washes to obtain acetone extract 2500L, the use of 1M salt acid for adjusting pH value is 3.0,100L normal heptane single extractions are added and obtain positive heptan
Alkane phase, water phase use the extraction of 100L normal heptanes primary again, and 50 DEG C of normal heptane phase obtained by merging twice is concentrated in vacuo to dry to get concentration
Object 700g.Above-mentioned concentrate is dissolved in the mixed solvent of 2.1L normal heptanes and acetonitrile, and (its volume ratio is 1：2) 4, are transferred to after filtering
It is stood overnight in DEG C refrigerator, filter-cloth filtering obtains 560g bacteripheophytin a, purity 95.7%, yield 82%.
Will above-mentioned bacteripheophytin a 560g dissolved with 9L chloroforms after be added 10% methanolic solution 20L, 50 DEG C
It is stirred at reflux reaction；Through TLC trace analysis, the reaction was complete after 3h, in reaction solution plus after the dilution of 50L water, adds 9L chloroforms extraction
It takes, upper strata aqueous phase no sample is analyzed through TLC, collect chloroform and mutually first washed at twice with 10% sodium bicarbonate solution 25L, every time
12.5L adds deionized water 25L and washs at twice, each 12.5L, after gained chloroform is added to the drying of 1kg anhydrous sodium sulfates
It is concentrated to dryness up to de-magging bacterium chlorophyllin a methyl esters crude products 500g；Above-mentioned de-magging bacterium chlorophyllin a methyl esters crude products are first used into 1.5L
50 DEG C of heating for dissolving of chloroform, are added 3.5L normal heptanes after solution filtering, are stood after mixing to room temperature, be then transferred to 4 DEG C of ice
It is stood overnight in case, filter-cloth filtering obtains the crystal of 273g de-magging bacterium chlorophyllin a methyl esters, and HPLC purity is 96%, yield
It is 69%.
Embodiment 9
10% ethanol solution of sulfuric acid is added after being dissolved with 20mL chloroforms in 4 gained bacteripheophytin a 1g of Example
40mL, 50 DEG C are stirred at reflux reaction.Through TLC trace analysis, the reaction was complete after 3h, in reaction solution plus after the dilution of 100mL water, then adds
Enter the extraction of 20mL chloroforms, upper strata aqueous phase no sample is analyzed through TLC, collects chloroform and mutually first divided with 10% sodium bicarbonate solution 50mL
It washs twice, each 25mL, adds deionized water 50mL and wash at twice, each 25mL, it is anhydrous that gained chloroform is added to 2g
It is concentrated to dryness up to de-magging bacterium chlorophyllin a ethyl esters 1g after sodium sulphate drying；
Above-mentioned de-magging bacterium chlorophyllin a ethyl esters are first used to 50 DEG C of heating for dissolving of 3mL chloroforms, are added after solution filtering
7mL normal heptanes are stood after mixing to room temperature, are then transferred in 4 DEG C of refrigerators and are stood overnight, and it is de- that filter-cloth filtering obtains 460mg
The crystal of magnesium bacterium chlorophyllin a ethyl esters, HPLC purity are 97%, yield 64%.
Claims (15)
1. a kind of method preparing bacteripheophytin a esters, which is characterized in that the method includes：
(a) it can dissolve bacteripheophytin a using a kind of and can dissolve bacteripheophytin a with the miscible solvent of alcohol again,
Then sulfuric acid alcoholic solution is added to be esterified under heating conditions, is concentrated to dryness to obtain de-magging bacterium after esterification reaction solution is post-treated
Chlorophyll a ester crude product；
(b) the bacteripheophytin a esters crude product of gained obtains the pure of bacteripheophytin a esters by crystallization in step (a)
Product.
2. according to the method described in claim 1, it is characterized in that, described one kind can dissolve bacteripheophytin a may be used again
The mixture of chloroform, dichloromethane or both, preferably chloroform are selected from the miscible solvent of alcohol.
3. according to the method described in claim 1, it is characterized in that, the alcohol in the sulfuric acid alcoholic solution is methanol or ethyl alcohol；Institute
State the sulfuric acid alcoholic solution that sulfuric acid alcoholic solution is volume ratio 5-15%；Preferably 10% sulfuric acid alcoholic solution.
4. according to the method described in claim 1, it is characterized in that, the heating and temperature control of the esterification in the step (a) exists
40-55℃。
5. according to the method described in claim 1, it is characterized in that, the post-processing step in the step (a) is esterification
It is diluted with water in liquid, then with chloroform, either dichloromethane extraction gained chloroform or dichloromethane mutually first use bicarbonate respectively
Sodium water solution and water washing, then dried with anhydrous sodium sulfate.
6. according to the method described in claim 1, it is characterized in that, the reaction of the step (a) is in inert gas shielding condition
Lower progress, preferably nitrogen.
7. according to the method described in claim 1, it is characterized in that, the recrystallisation solvent that uses of the crystallization in the step (b) for
C1-C7Halogenated alkyl object and C1-C10Hydrocarbon compound mixed solvent；The preferably mixed solvent of chloroform and normal heptane,
Described in the mixed solvent chloroform and normal heptane volume ratio be preferably 3:7.
8. according to any methods of claim 1-7, which is characterized in that the bacteripheophytin a passes through such as lower section
It is prepared by method：
(1) zymotic fluid containing antibiotics sensitivity test is prepared in Fermentation by Photosynthetic Bacteria；
(2) zymotic fluid obtains the mycelium containing antibiotics sensitivity test after separation of solid and liquid；
(3) mycelium containing antibiotics sensitivity test is extracted using aqueous acetone solution, filters to obtain extracting solution；
(4) organic phase is obtained by extraction with organic solvent after the extracting solution tune acid containing antibiotics sensitivity test；After organic phase is concentrated to dryness
Obtain concentrate；
(5) by concentrate obtained by step (4) again by crystallizing up to bacteripheophytin a.
9. according to the method described in claim 8, it is characterized in that, the photosynthetic bacteria in the step (1) is selected from
RhodovolumSulfidophilum、Rhodopseudomonas sphaeroides、Rhodopseudomonas
plustris、Rhodopseudomonas rutila。
10. according to the method described in claim 8, it is characterized in that, the separation of solid and liquid in step (2) is sheet frame after flocculating setting
It is centrifuged after filtering or flocculating setting.
11. according to the method described in claim 8, it is characterized in that, the separation of solid and liquid in step (2) is ceramic membrane filter, institute
The aperture for stating ceramic membrane is 100-500nm, preferably 200nm.
12. according to the method described in claim 8, it is characterized in that, in aqueous acetone solution described in step (3) acetone volume
Content is 70-80%.
13. according to the method described in claim 8, it is characterized in that, it is 2-5 to adjust the pH value range of acid described in step (4).
14. according to the method described in claim 8, it is characterized in that, organic solvent described in step (4) extracts the organic of use
Solvent is C1-C10Alkane derivative, the C1-C10Alkane derivative be selected from petroleum ether, n-hexane, hexamethylene, just
It is one or more in heptane；Preferably normal heptane.
15. according to the method described in claim 8, it is characterized in that, crystallizing the recrystallisation solvent of use described in the step (5)
For C1-C10Alkane derivative and acetonitrile mixed solvent, the C1-C10Hydrocarbon compound be selected from petroleum ether, n-hexane,
It is one or more in hexamethylene, normal heptane, preferably normal heptane；The recrystallisation solvent is preferably the mixing of normal heptane and acetonitrile
Solvent, volume ratio are preferably 1:2.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113845456A (en) * | 2021-08-23 | 2021-12-28 | 山东微研生物科技有限公司 | Process and equipment for filtering and extracting beta-carotene fermentation liquor |
| CN116162092A (en) * | 2023-03-03 | 2023-05-26 | 康俄(上海)医疗科技有限公司 | Preparation method of chlorin e6 triglucamine salt |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1425015A (en) * | 1999-12-01 | 2003-06-18 | 耶达研究及发展有限公司 | Chlorophyll and bacteriochlorophyll esters, and their preparation |
-
2018
- 2018-02-02 CN CN201810104433.XA patent/CN108341827A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1425015A (en) * | 1999-12-01 | 2003-06-18 | 耶达研究及发展有限公司 | Chlorophyll and bacteriochlorophyll esters, and their preparation |
Non-Patent Citations (5)
| Title |
|---|
| ANDREI KOZYREV等: "Synthesis, Photophysical and Electrochemistry of Near-IR Absorbing Bacteriochlorins Related to Bacteriochlorophyll a", 《THE JOURNAL OF ORGANIC CHEMISTRY》 * |
| ANDREI N. KOZYREV 等: "Characterization of porphyrins, chlorins, and bacteriochlorins formed via allomerization of Bacteriochlorophyll a. Synthesis of highly stable bacteriopurpurinimides and their metal complexes", 《THE JOURNAL OF ORGANIC CHEMISTRY》 * |
| HITOSHI TAMIAKI等: "Synthesis and Self-Assembly of Zinc Methyl Bacteriopheophorbide-f and its Homolog", 《TETRAHEDRON》 * |
| 常如波 等: "细菌叶绿素过渡金属配合物的合成和光谱研究", 《山西大学学报（自然科学版）》 * |
| 陈志龙 等: "叶绿素衍生物的合成及其对紫细菌原初光化学特性的影响研究", 《化学学报》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113845456A (en) * | 2021-08-23 | 2021-12-28 | 山东微研生物科技有限公司 | Process and equipment for filtering and extracting beta-carotene fermentation liquor |
| CN113845456B (en) * | 2021-08-23 | 2023-05-09 | 山东微研生物科技有限公司 | Filtering extraction process and equipment for beta-carotene fermentation liquor |
| CN116162092A (en) * | 2023-03-03 | 2023-05-26 | 康俄(上海)医疗科技有限公司 | Preparation method of chlorin e6 triglucamine salt |
| CN116162092B (en) * | 2023-03-03 | 2023-10-10 | 康俄(上海)医疗科技有限公司 | Preparation method of chlorin e6 triglucamine salt |
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