CN103897039A - Ramoplanin derivative and preparation method and use thereof - Google Patents
Ramoplanin derivative and preparation method and use thereof Download PDFInfo
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Abstract
The invention provides a ramoplanin derivative shown in a formula (I) and a preparation method and use thereof in preparation of anti-infection drugs, especially anti-serious infection drugs. The formula (I) is shown in the specification.
Description
Technical field
The invention belongs to genetically engineered and medical technical field, be specifically related to ramoplanin derivative shown in formula (I) and preparation method thereof with its preparation anti-infective, the especially purposes in the medicine of severe infections.
Background technology
Ramoplanin (ramoplanin) is from the fermented liquid of actinoplanes Actinoplanes sp.ATCC 33076, to separate a kind of lipopeptide antibiotic obtaining, to methicillin-resistant staphylococcus aureus (methicillin-resistant Stap hylococcus aureus, MRSA), Vancomycin-resistant Enterococcus (vancomycin-resistant Enterococcus faecium, VRE) and the gram-positive microorganism of some anti-erythromycin or Ampicillin Trihydrate have remarkable bacteriostatic activity.With other glycopeptide antibiotics vancomycins and teicoplanin comparison, ramoplanin has higher inhibition activity [Cavalleri etc., 1984 to most of Gram-positive pathogenic bacteria; Pallanza etc., 1984; Kettenring etc., 1989].In June, 2000, the oral and local administration preparation of ramoplanin has started III phase clinical study at US and European, is used for the treatment of the blood infection causing due to VRE.
The cyclic peptide skeleton that the molecular structure core of ramoplanin is made up of 17 amino-acid residues, wherein has 9 L-type amino acid and 7 D type amino acid.On this skeleton, be connected with two D-MANNOSE molecules and different two unsaturated fatty acids side chain molecules.According to the difference of fatty acid side chain, be divided into A1, A2, tri-components of A3, in its natural product, A2 component accounts for approximately 80%.Aspect the structure of modification and structure activity study of ramoplanin, existing document is mainly to obtain its analogue based on synthetic and semisynthetic method, and studies on this basis [McCafferty etc., 2002] such as its anti-microbial activity and functional groups.
Ramoplanin A2 component structure
According to the molecular structure feature of ramoplanin, mainly can range 3 kinds to its structure of modification: the first is the transformation to core cyclic peptide skeleton, comprise that linearizing, lactone bond replace with amido linkage, amino acid whose replacement etc., as its 4 or 10 s' Orn is replaced with to Ala, the bacteriostatic activity of the analogue obtaining or reservation (4 replacements) or reduction (10 replacements) [Cudic etc., 2002]; The second is the ramoplanin aglycone that hydrolysis obtains desugar, and it is active quite front with hydrolysis, and therefore the effect of two mannose groups in ramoplanin molecule waits research [Ciabatti etc., 1996; Cudic etc., 2002]; The third is the transformation to fatty acid side chain; comprise chemical reduction, the replacement of side chain etc. of two keys on side chain; as degrading by Edman, Ciabatti etc. obtains exceeding with the method for acidylate the analogue that 130 kinds of side chains are replaced; and the activity of part of compounds [Ciabatti etc., 2007] are carried out testing.
According to literature survey, the 2Z that not yet withs a hook at the end, the semi-synthetic report of the ramoplanin analogue that the side chain of 4E-diene structure extends.Therefore, contriver produces bacterium by engineered method transformation ramoplanin, obtains to have more length fatty acids side chain and retain its 2Z, the ramoplanin analogue of 4E-diene structure.
Summary of the invention
An object of the present invention is to provide novel lipopeptide derivative, relate in particular to ramoplanin derivative, through preliminary MIC test, confirming has stronger fungicidal activity to resistance staphylococcus and Vancomycin-resistant Enterococcus (VRE).
The present invention is to provide as shown in the formula the ramoplanin derivative shown in (I): its molecular formula is C
121h
158clN
21o
40, absolute molecular weight is 2580.07, relative molecular weight is 2582.12.It is characterized by the cyclic peptide that 17 natural amino acids form, on L-Hpg amino acid phenyl ring hydroxyl, connect two seminoses and the 11 carbon longer chain fatty acids that are connected on L-Asn amino acid; Than the difference of Ramoplanin A2 component be branched fatty acid many two carbon.This structure is that reported first of the present invention separates and obtains, the preliminary resistance faecalis activity of killing is better than Ramoplanin A2 component, and there is obvious difference with known vancomycin compound, can kill the bacterial strain to drug resistance of vancomycin, promise to be the best substitute antibiotics of rear this type of resistant organism of drug resistance of vancomycin enhancing future.
Formula (I)
Another object of the present invention is to provide the preparation method of the ramoplanin derivative shown in above-mentioned formula (I), comprises the following steps:
(1) respectively ramoplanin is produced to ramo orf24-orf25-orf26 and the orf26 gene knockout of bacterium actinoplanes Actinoplanes sp.ATCC 33076, obtain actinoplanes mutant strain mPHX9-96-6 and mPHX10-12-6.
(2) and respectively build containing grace and draw rhzomorph to produce the end orf45-orf46 gene of bacterium fungicidal streptomycete Streptomycesfungicidicus ATCC 21013 and recombinant plasmid pPHX9-88-6 and the pPHX10-23-1 of end orf45, and be transferred in intestinal bacteria ET12567.
(3), by conjugal transfer between the genus between intestinal bacteria and actinoplanes, respectively recombinant plasmid pPHX9-88-6 and pPHX10-23-1 are imported in actinoplanes mutant strain mPHX9-96-6 and mPHX10-12-6.The mutant strain called after mPHX10-43-2 that recombinant plasmid pPHX9-88-6 imports, the mutant strain called after mPHX10-43-3 that recombinant plasmid pPHX10-23-1 imports.
(4) the actinoplanes mutant strain mPHX10-43-2 and the mPHX10-43-3 that utilize step (1)-(3) gene engineering method to build ferment, regulate pH to 3.0-4.0, centrifugal collection wet thallus, by prepare the crude extract that contains the ramoplanin derivative shown in formula (I) with 3-6 times of solvent extraction, and be concentrated into the 1/5-1/10 of original volume, obtain concentrated solution;
(5) by the pH regulator of above-mentioned concentrated solution to 7.0-8.0, by macroporous adsorbent resin, use elutriant wash-out, obtain the crude product that contains the ramoplanin derivative shown in formula (I);
(6) through mesolow or the described crude product of high pressure purification procedures (5) of C-18, C-8 or polymkeric substance chromatogram, make the ramoplanin derivative shown in formula (I).
According to the present invention, one preferred embodiment, the slant culture based formulas that fermentation is used in step (4) is (g/L): extractum carnis (traditional Chinese medicines) 3.0, peptone (trypton) 10.0, Semen Maydis powder 24.0, glucose 1.0, agar 22.0,7.0,121 ℃ of sterilizing 30min of pH before sterilizing.Cultivate after 7d for 30 ℃, access in right amount in seed culture medium shaking flask by transfering loop scraping.Seed culture based formulas is (g/L): extractum carnis 3.0, and peptone 5.0, yeast powder 5.0, oatmeal 30.0, calcium carbonate 4.0, tap water preparation, with NaOH or the HCl tune pH to 7.0 of 2mol/L, 121 ℃ of sterilizing 30min.Seed culture medium loading amount is 100mL/750mL, and in 28 ℃, the rotary shaking table of 250rpm is cultivated 72h.Fermentative medium formula is (g/L): soybean oil 10.0, and glucose 60.0, analysis for soybean powder 30.0, calcium carbonate 10.0, L-Leu 7.0, tap water preparation, with NaOH or the HCl tune pH to 7.0 of 2mol/L, 121 ℃ of sterilizing 30min.Inoculum size 6%, is seeded in the stainless steel fermentor tank of loading amount 15L/30L, in 25 ℃, stirs aerated culture 6d, moisturizing in fermenting process, benefit sugar.
According to the present invention another preferred embodiment, the acid that regulates pH to use in step (4) is oxalic acid, hydrochloric acid or sulfuric acid; The solvent using is ethanol or acetone, and usage quantity is 3-6 times of wet thallus volume.
According to the present invention, one preferred embodiment; the resin that the macroporous adsorbent resin using in step (5) is common middle polarity; preferably HZ-803, the HZ-806 of Shanghai Hua Zhen resin company, the NM100 of Suzhou Na Wei scientific & technical corporation, the XAD-4 of Rhom and Hass.
According to the present invention another preferred embodiment, the eluent using in step (5) is respectively the aqueous solution containing 30% ethanol, containing the aqueous solution of 50% ethanol, containing the aqueous solution of 70% ethanol with containing the aqueous solution of the pH 3.0-4.0 of 70% ethanol.
According to the present invention one preferred embodiment, in step (6), mesolow moving phase used is ammonium acetate solution (4g/L): methyl alcohol=75-10: 25-90 (v/v).High pressure moving phase used is ammonium acetate solution (4g/L): methyl alcohol=40-60: 60-40 (v/v).
According to the present invention one preferred embodiment, step (5) also comprises carries out desalination to crude product with 0.2-0.45um millipore filtration.
According to the present invention one preferred embodiment, the HPLC purity of the ramoplanin derivative shown in the formula (I) that step (6) obtains is more than 95% (Area Ratio).
A further object of the present invention is to provide the application of the ramoplanin derivative shown in above-mentioned formula (I) on the medicine of the serious gram positive bacteria infection of preparation treatment, and especially treatment is or/and the purposes in the medicine of assisting therapy endocarditis, meningitis, pneumonia, septicemia, acute and chronic osteomyelitis, burn infection, skin or soft tissue purulent infection.
A further object of the present invention is to provide the pharmaceutical composition of the invention described above compound that contains significant quantity, and pharmaceutical composition of the present invention can also contain carrier well known in the art, as vehicle, and thinner, flavouring agent, sweeting agent etc.
Accompanying drawing explanation
Figure 1A is according to the HPLC collection of illustrative plates of the patent Ramoplanin A2 reference substance that openly prepared by the embodiment 2 of CN 101024661B.
Figure 1B is the structural formula HPLC collection of illustrative plates of the ramoplanin derivative shown in the formula (I) prepared of embodiment 5.
Fig. 2 A is the mass spectrum MS collection of illustrative plates of the ramoplanin derivative shown in the formula (I) prepared of embodiment 5.
Fig. 2 B is the partial approach figure of Fig. 2 A.
Fig. 3 is the high resolution mass spectrum collection of illustrative plates of the ramoplanin derivative shown in the formula (I) prepared of embodiment 5.
Fig. 4 is the H of the ramoplanin derivative (Fig. 4 B) shown in embodiment 5 Ramoplanin A2 (Fig. 4 A) and the formula (I) prepared
1nMR trace analysis.
Fig. 5 is the C of the ramoplanin derivative (Fig. 5 B) shown in embodiment 5 Ramoplanin A2 (Fig. 5 A) and the formula (I) prepared
13nMR trace analysis.
Fig. 6 is two-dimensional spectrum H-HCOSY (Fig. 6 A), HSQC (Fig. 6 B) and the HMBC (Fig. 6 C) of the ramoplanin derivative shown in the formula (I) prepared of embodiment 5.
Embodiment
Below in conjunction with specific embodiment, the invention will be further described.Should understand.Following examples are only for the present invention is described but not for limiting scope of the present invention.
The solvent ratio using in following examples is volume ratio; And the purity content ratio of sample for this reason sample HPLC area account for the Area Ratio of all these wavelength absorption materials.
Embodiment 1: the actinoplanes mutant strain Actinoplanes sp.mPHX10-43-2 that builds the ramoplanin derivative shown in presentation (I).
(1) ramoplanin produces the structure of ramo orf26 gene knock-out bacterial strain in bacterium
Adopt the method for double exchange homologous recombination to build ramo orf26 gene knock-out bacterial strain.Take the genomic dna of actinoplanes Actinoplanes sp.ATCC 33076 as template, adopt primer 5 '-TAAAAGCTTTGCCCGGACAGCACCCATC-3 ' and primer 5 '-TAAGAATTCCTGCACGCCGAGCTCGAGC-3 ' pcr amplification to the about 2.2kb product of left arm, adopt primer 5 '-TAAGAATTCCGCCAGGTAGAGCTCGAC-3 ' and primer 5 '-TAATCTAGAACCGTTGTAGCCGAACACC-3 ' pcr amplification to the about 2kb product of right arm, left arm and right arm respectively flush end are connected in the StuI otch in the multiple clone site of carrier pANT841.Left arm is cut out with endonuclease HindIII and EcoRI from the plasmid building, right arm is cut out from the plasmid building with endonuclease EcoRI and XbaI, obtained fragment is connected in the pKC1139 carrier with HindIII and XbaI double digestion together.The plasmid pPHX9-53-1 that the orf26 gene that structure is obtained knocks out with frame is transferred in intestinal bacteria ET12567, by conjugal transfer between the genus between intestinal bacteria and actinoplanes, recombinant plasmid is imported in actinoplanes Actinoplanes sp.SIPI-A.2001, after 37 ℃ of integration, lax cultivation induces it that double exchange occurs, the mutant strain obtaining, with antibiotic-free flat board with containing Am microbiotic plate screening, obtains Am responsive type bacterial strain.Recycling PCR checking, checking primer is 5 '-CAGCGGGCGTACGGTCATCG-3 ' and 5 '-ATGGTCATCGACGCCGCCAC-3 ', gene knockout mutant strain called after mPHX9-96-6.
(2) enramycin produces the clone of end orf45 gene and the structure of heterogenous expression plasmid in bacterium
Take the genomic dna of fungicidal streptomycete Streptomyces fungicidicus ATCC 21013 as template, adopt primer 5 '-TTTTCTAGACATCAGCGGCCGTCCTGGGAG-3 ' and primer 5 '-TTTGAGCTCCCGTTGATGGTCAGCGAGC-3 ' pcr amplification to the 3.8kb product that comprises endorf45 gene, flush end is connected in the StuI otch in the multiple clone site of carrier pANT841.End orf45 gene is cut out with endonuclease SacI and XbaI from the plasmid building, erythromycin promotor is cut out with endonuclease EcoRI and SacI from plasmid pLL6212, obtain about 450bp erythromycin promoter fragment, obtained fragment is connected in the pSET152 carrier with EcoRI and XbaI double digestion together, obtains heterogenous expression plasmid pPHX9-88-6.
(3) structure of heterogenous expression bacterial strain
Recombinant plasmid pPHX9-88-6 for heterogenous expression is transferred to intestinal bacteria ET12567.By conjugal transfer between the genus between intestinal bacteria and actinoplanes, recombinant plasmid pPHX9-88-6 is imported in actinoplanes mutant strain mPHX9-96-6, obtain the mutant strain with A Baila mycin resistance, utilize PCR checking genotype.The mutant strain called after mPHX10-43-2 that recombinant plasmid pPHX9-88-6 imports.
Embodiment 2: the actinoplanes mutant strain Actinoplanes sp.mPHX10-43-3 that builds the ramoplanin derivative shown in presentation (I).
(1) ramoplanin produces the structure of ramo orf24-orf25-orf26 gene knock-out bacterial strain in bacterium
Adopt the method for double exchange homologous recombination to build ramo orf24-orf25-orf26 gene region knock-out bacterial strain.Take the genomic dna of actinoplanes Actinoplanes sp.ATCC 33076 as template, adopt primer 5 '-TAAAAGCTTCGCGGACGATCCGGTAGAC-3 ' and primer 5 '-TAAGAATTCCGGTACACCGTGCTGCTGG-3 ' pcr amplification to the about 2.2kb product of left arm, flush end is connected in the StuI otch in the multiple clone site of carrier pANT841.Left arm is cut out with endonuclease HindIII and EcoRI from the plasmid building, and right arm is identical with the right arm in orf26 gene knockout, and obtained left and right arms fragment is connected in the pKC1139 carrier with HindIII and XbaI double digestion together.The plasmid pPHX9-60-4 of the ramo orf24-orf25-orf26 gene knockout that structure is obtained is transferred in intestinal bacteria ET12567, by conjugal transfer between the genus between intestinal bacteria and actinoplanes, recombinant plasmid is imported in actinoplanes Actinoplanes sp.SIPI-A.2001, after 37 ℃ of integration, lax cultivation induces it that double exchange occurs, the mutant strain obtaining, with antibiotic-free flat board with containing Am microbiotic plate screening, obtains Am responsive type bacterial strain.Recycling PCR checking, checking primer is 5 '-GGAGCTGACGGGCGCTCAGG-3 ' and 5 '-ATGGTCATCGACGCCGCCAC-3 ', gene knockout mutant strain called after mPHX10-12-6.
(2) enramycin produces the clone of end orf45-orf46 gene and the structure of heterogenous expression plasmid in bacterium
Take the genomic dna of fungicidal streptomycete Streptomyces fungicidicusATCC 21013 as template, adopt primer 5 '-TTTTCTAGAGTCCGCTCAGCCGGGCACGTG-3 ' and primer 5 '-GGCTCGCGACGACGCTGCGGACAC-3 ' pcr amplification to the 1.8kb product that comprises end orf46 gene, flush end is connected in the StuI otch in the multiple clone site of carrier pANT841.Above-mentioned 1.8kb fragment is cut out with endonuclease NruI and XbaI from the plasmid building, plasmid pPHX9-88-6 is cut and obtains large fragment with endonuclease NruI and XbaI, two fragments are connected and obtain heterogenous expression plasmid pPHX10-23-1.
(3) structure of heterogenous expression bacterial strain
Recombinant plasmid pPHX10-23-1 for heterogenous expression is transferred to intestinal bacteria ET12567.By conjugal transfer between the genus between intestinal bacteria and actinoplanes, recombinant plasmid and pPHX10-23-1 are imported in actinoplanes mutant strain mPHX10-12-6, obtain the mutant strain with A Baila mycin resistance, utilize PCR checking genotype.The mutant strain called after mPHX10-43-3 that recombinant plasmid pPHX10-23-1 imports.
Embodiment 3: slant culture, the liquid seeds of actinoplanes mutant strain Actinoplanes sp.mPHX10-43-2 and mPHX10-43-3 are cultivated and liquid fermentation tank fermentation culture
Two plant mutant strain bacterium liquid are passed after slant culture 30 ℃ of 7d, access in right amount in seed culture medium shaking flask by transfering loop scraping.Seed culture medium shaking flask loading amount is 100mL/750mL, and in 28 ℃, the rotary shaking table of 250rpm is cultivated 72h.The liquid of cultivation is pressed to seed inoculum size 6%, be seeded in the stainless steel fermentor tank of loading amount 15L/30L, in 25 ℃, stir aerated culture 6d, moisturizing in fermenting process, benefit sugar.
Through the preliminary screening of substratum, determine that the slant culture based formulas using is (g/L): extractum carnis (traditional Chinese medicines) 3.0, peptone (trypton) 10.0, Semen Maydis powder 24.0, glucose 1.0, agar 22.0,7.0,121 ℃ of sterilizing 30min of pH before sterilizing.Through the preliminary screening of substratum, determine that the seed culture based formulas using is (g/L): extractum carnis 3.0, peptone 5.0, yeast powder 5.0, oatmeal 30.0, calcium carbonate 4.0, tap water preparation, with NaOH or the HCl tune pH to 7.0 of 2mol/L, 121 ℃ of sterilizing 30min.Through the preliminary screening of substratum, determine that the fermentative medium formula using is (g/L): soybean oil 10.0, glucose 60.0, analysis for soybean powder 30.0, calcium carbonate 10.0, L-Leu 7.0, tap water preparation, with NaOH or the HCl tune pH to 7.0 of 2mol/L, 121 ℃ of sterilizing 30min.
Embodiment 4: the HPLC testing conditions of the ramoplanin derivative shown in formula (I) and Ramoplanin A2 is set up
The HPLC testing conditions of taking into account after resolution and efficiency optimization is:
Chromatographic system: Agilent 1100
Chromatographic column: Agilent ZORBAX SB-C185 μ m 4.6 × 250mm
Moving phase: 4g/L ammonium acetate solution: acetonitrile=64: 36
Column temperature: 30 ℃
Flow velocity: 0.8ml/min
Sample size: 20 μ L
Detect wavelength: 254nm
Embodiment 5: the separation and purification of the ramoplanin derivative shown in formula (I)
Adjust pH to 4.0 to collect wet thallus 1.8L after the centrifugal 30min of 4000rpm with oxalic acid the fermented liquid 12.8L after embodiment 3, by thalline, with after 6L ethanol ultrasonic agitation extracting 12h, recentrifuge is collected supernatant liquor.Supernatant concentration is adjusted to the NM100 macroporous adsorptive resins that 4: 1 column volumes of aspect ratio that pH to 7.5 rear upper prop rinsed in advance with 800ml20% ethanol are in advance 400ml to 2L, and respectively rinse 1000ml with 30% aqueous ethanolic solution, 50% aqueous ethanolic solution and 70% aqueous ethanolic solution successively, the pH 4.0 finally regulating with hydrochloric acid is containing the aqueous solution wash-out of 70% ethanol, substep is collected, HPLC detects to get wherein and merges containing the higher receiving flask of the ramoplanin derivative component shown in formula (I), and reduced vacuum is concentrated into 100ml.
Match point Flash mesolow chromatographic column by above-mentioned 100ml crude product upper prop containing 80g C-8 reversed-phase bonded silica, with water (A): the aqueous solution and organic phase (B) containing 4g/L ammonium acetate: methyl alcohol gradient elution, be down to 10% from A content from 75%, B content rises to 90% from 25%, flow velocity 40ml/min, detect wavelength 254nm and 268nm, gradient flush time is 30min.Collect B content 70%-90% wherein and have the cut of uv-absorbing, and detect to get wherein with HPLC and merge higher than 88% receiving flask containing the ramoplanin derivative component purity shown in formula (I), reduced vacuum is concentrated into 50ml.
Above-mentioned concentrated solution 50ml upper prop is received to the Flash mesolow chromatographic column of micro-polymer packing containing 40g Suzhou, with water (A): the aqueous solution and organic phase (B) containing 4g/L ammonium acetate: methyl alcohol gradient elution, be down to 10% from A content from 75%, B content rises to 90% from 25%, flow velocity 40ml/min, detect wavelength 254nm and 268nm, gradient flush time is 30min.Collect B content 70%-85% wherein and have the cut of uv-absorbing, and detect to get wherein with HPLC and merge higher than 95% receiving flask containing the ramoplanin derivative component purity shown in formula (I), 35 degrees Celsius of reduced vacuum are concentrated except methyl alcohol.And by after concentrated solution freeze-drying, the ramoplanin derivative 18.5mg shown in acquisition formula (I).
Embodiment 6: the separation and purification of the ramoplanin derivative shown in formula (I)
Adjust pH to 4.0 to collect wet thallus 2.1L after the centrifugal 30min of 4000rpm with sulfuric acid the fermented liquid 13.5L after embodiment 3, by thalline, with after 6L acetone ultrasonic agitation extracting 12h, recentrifuge is collected supernatant liquor.Supernatant concentration is adjusted to the XAD-4 macroporous adsorptive resins that 4: 1 column volumes of aspect ratio that pH to 7.5 rear upper prop rinsed in advance with 800ml20% ethanol are in advance 400ml to 2L, and respectively rinse 1000ml with 30% aqueous ethanolic solution, 50% aqueous ethanolic solution and 70% aqueous ethanolic solution successively, the 70% aqueous ethanolic solution wash-out of the pH 3.5 finally regulating with hydrochloric acid, substep is collected, HPLC detects to get wherein and merges containing the higher receiving flask of the ramoplanin derivative component shown in formula (I), and reduced vacuum is concentrated into 100ml.
Above-mentioned 100ml crude product upper prop is received to the Flash mesolow chromatographic column of micro-polymer packing containing 80g Suzhou, with water (A): the aqueous solution and organic phase (B) containing 4g/L ammonium acetate: methyl alcohol gradient elution, be down to 10% from A content from 75%, B content rises to 90% from 25%, flow velocity 40ml/min, detect wavelength 254nm and 268nm, gradient flush time is 30min.Collect B content 70%-90% wherein and have the cut of uv-absorbing, and detect to get wherein with HPLC and merge higher than 88% receiving flask containing the ramoplanin derivative component purity shown in formula (I), reduced vacuum is concentrated into 50ml.
By above-mentioned concentrated solution 50ml several times upper prop containing the C-18 high-pressure chromatographic column of 5 μ m 20 × 250mm, with water (A): the aqueous solution and organic phase (B) containing 4g/L ammonium acetate: methyl alcohol isocratic elution, A content 50%, B content 50%, flow velocity 8ml/min, detect wavelength 254nm, flush time is 50min.Collect the wherein cut containing the ramoplanin derivative component peaks shown in formula (I), 35 degrees Celsius of reduced vacuum are concentrated except methyl alcohol.HPLC detects rear purity higher than 95%, and by after concentrated solution freeze-drying, the ramoplanin derivative 16.5mg shown in acquisition formula (I).
Embodiment 7: the separation and purification of the ramoplanin derivative shown in formula (I)
Adjust pH to 4.0 to collect wet thallus 2.0L after the centrifugal 30min of 4000rpm with hydrochloric acid the fermented liquid 13.2L after embodiment 3, by thalline, with after 8L ethanol ultrasonic agitation extracting 12h, recentrifuge is collected supernatant liquor.Supernatant concentration is adjusted to the HZ-803 macroporous adsorptive resins that 4: 1 column volumes of aspect ratio that pH to 7.5 rear upper prop rinsed in advance with 800ml20% ethanol are in advance 400ml to 2L, and respectively rinse 1000ml with 30% aqueous ethanolic solution, 50% aqueous ethanolic solution and 70% aqueous ethanolic solution successively, the 70% aqueous ethanolic solution wash-out of the pH 3.0 finally regulating with hydrochloric acid, substep is collected, HPLC detects to get wherein and merges containing the higher receiving flask of the ramoplanin derivative component shown in formula (I), and reduced vacuum is concentrated into 100ml.
Match point Flash mesolow chromatographic column by above-mentioned 100ml crude product upper prop containing 80g C-18 reversed-phase bonded silica, with water (A): the aqueous solution and organic phase (B) containing 4g/L ammonium acetate: methyl alcohol gradient elution, A content is down to 10% from 75%, B content rises to 90% from 25%, flow velocity 40ml/min, detect wavelength 254nm and 268nm, gradient flush time is 30min.Collect B content 70%-90% wherein and have the cut of uv-absorbing, and detect to get wherein with HPLC and merge higher than 88% receiving flask containing the ramoplanin derivative component purity shown in formula (I), reduced vacuum is concentrated into 50ml.
By above-mentioned concentrated solution 50ml several times upper prop receive the high-pressure chromatographic column of micro-polymer packing containing 5 μ m 20 × 250mm Suzhou, with water (A): the aqueous solution and organic phase (B) containing 4g/L ammonium acetate: methyl alcohol isocratic elution, A content 50%, B content 50%, flow velocity 8ml/min, detect wavelength 254nm, flush time is 50min.Collect the wherein cut containing the ramoplanin derivative component peaks shown in formula (I), 35 degrees Celsius of reduced vacuum are concentrated except methyl alcohol.HPLC detects rear purity higher than 95%, and by after concentrated solution freeze-drying, the ramoplanin derivative 14.5mg shown in acquisition formula (I).
Embodiment 8: the separation and purification of the ramoplanin derivative shown in formula (I)
Adjust pH to 4.0 to collect wet thallus 2.3L after the centrifugal 30min of 4000rpm with hydrochloric acid the fermented liquid 14.2L after embodiment 3, by thalline, with after 10L acetone ultrasonic agitation extracting 12h, recentrifuge is collected supernatant liquor.Supernatant concentration is adjusted to the HZ-806 macroporous adsorptive resins that 4: 1 column volumes of aspect ratio that pH to 7.5 rear upper prop rinsed in advance with 800ml 20% ethanol are in advance 400ml to 2L, and respectively rinse 1000ml with 30% aqueous ethanolic solution, 50% aqueous ethanolic solution and 70% aqueous ethanolic solution successively, the 70% aqueous ethanolic solution wash-out of the pH 3.5 finally regulating with hydrochloric acid, substep is collected, HPLC detects to get wherein and merges containing the higher receiving flask of the ramoplanin derivative component shown in formula (I), and reduced vacuum is concentrated into 100ml.
Match point Flash mesolow chromatographic column by above-mentioned 100ml crude product upper prop containing 80g C-18 reversed-phase bonded silica, with water (A): the aqueous solution and organic phase (B) containing 4g/L ammonium acetate: methyl alcohol gradient elution, A content is down to 10% from 75%, B content rises to 90% from 25%, flow velocity 40ml/min, detect wavelength 254nm and 268nm, gradient flush time is 30min.Collect B content 70%-90% wherein and have the cut of uv-absorbing, and detect to get wherein with HPLC and merge higher than 88% receiving flask containing the ramoplanin derivative component purity shown in formula (I), reduced vacuum is concentrated into 50ml.
By above-mentioned concentrated solution 50ml several times upper prop receive the high-pressure chromatographic column of micro-polymer packing containing 5 μ m 20 × 250mm Suzhou, with water (A): the aqueous solution and organic phase (B) containing 4g/L ammonium acetate: methyl alcohol isocratic elution, A content 60%, B content 40%, flow velocity 8ml/min, detect wavelength 254nm, flush time is 50min.Collect the wherein cut containing the ramoplanin derivative component peaks shown in formula (I), 35 degrees Celsius of reduced vacuum are concentrated except methyl alcohol.HPLC detects rear purity higher than 95%, and by after concentrated solution freeze-drying, the ramoplanin derivative 15.5mg shown in acquisition formula (I).
Embodiment 9: the Structural Identification of the ramoplanin derivative shown in formula (I)
Because ramoplanin compounds molecular weight is large, complex structure, is directly difficult to identify its structure by mass spectrum nuclear magnetic data.Therefore, contriver, by the nuclear magnetic spectrum of the ramoplanin derivative shown in the formula being obtained by embodiment 5 (I) and known compound Ramoplanin A2 component is compared, determines its structure.
First, determine [M+H] of the ramoplanin derivative shown in formula (I) by Q-TOF micro mass spectrograph ESI-MS
+m/z is 2581.08,2582.06,2583.01,2584.04 (70,100,85,75) and [M+H]
2+m/z is 1291.02,1291.52,1292.02,1292.52 as accompanying drawing 2.High resolution mass spectrum [M+H]
2+m/z is 1291.04077, as accompanying drawing 3.
By with the H of Ramoplanin A2 component
1nMR and C
13nMR comparison, it is basically identical with it that its figure composes shape and the chemical shift at peak, and at H
1in NMR, between chemical shift 1.0-2.0, both have nuance, then in conjunction with high resolution mass spectrum, have determined that the structure of ramoplanin derivative of the present invention is suc as formula shown in (I).
Embodiment 10: the minimal inhibitory concentration determination test of the ramoplanin derivative shown in formula (I) and Ramoplanin A2
Under National Committee of Clinical Laboratory Standards (NCCLS) criterion, use the minimal inhibitory concentration (MIC) of the ramoplanin derivative shown in constant diluted liquid culture method (macrondilution broth procedure) mensuration formula (I) for standard faecalis 10007, methicillin-resistant staphylococcus aureus (MRSA43300), Pseudomonas aeruginosa, responsive type enterococcus faecalis and drug-resistant type enterococcus faecalis, and using Ramoplanin A2 as parallel control.Detailed process is as follows:
In Mueller-Hinton nutrient solution by compound sample serial dilution in the aseptic glass test tube of 15 × 100mm, then inoculate every pipe containing 5 × 10
5the bacteria suspension of CFU/ml, is placed in 37 ℃ of overnight incubation by test tube.Determine MIC by range estimation next day, and result is as shown in table 3.Formula (1) compound using obtains after by embodiment 5 separation and purification.
The minimum MIC value of ramoplanin derivative shown in table 1, formula (I) to some bacterial strains
Claims (13)
1. the ramoplanin derivative shown in formula (I):
Formula (I).
2. the method for the ramoplanin derivative shown in preparation formula (I), comprises the following steps:
(1) respectively ramoplanin is produced to ramo orf24-orf25-orf26 and the orf26 gene knockout of bacterium actinoplanes Actinoplanes sp.ATCC33076, obtain actinoplanes mutant strain mPHX9-96-6 and mPHX10-12-6;
(2) and respectively build containing grace and draw rhzomorph to produce the end orf45-orf46 gene of bacterium fungicidal streptomycete Streptomycesfungicidicus ATCC 21013 and recombinant plasmid pPHX9-88-6 and the pPHX10-23-1 of end orf45, and be transferred in intestinal bacteria ET12567;
(3) by conjugal transfer between the genus between intestinal bacteria and actinoplanes, respectively recombinant plasmid pPHX9-88-6 and pPHX10-23-1 are imported in actinoplanes mutant strain mPHX9-96-6 and mPHX10-12-6, the mutant strain called after mPHX10-43-2 that recombinant plasmid pPHX9-88-6 imports, the mutant strain called after mPHX10-43-3 that recombinant plasmid pPHX10-23-1 imports;
(4) the actinoplanes mutant strain mPHX10-43-2 and the mPHX10-43-3 that utilize step (1)-(3) gene engineering method to build ferment, regulate pH to 3.0-4.0, centrifugal collection wet thallus, by prepare the crude extract that contains the ramoplanin derivative shown in formula (I) with solvent extraction, and be concentrated into the 1/5-1/10 of original volume, obtain concentrated solution;
(5) by the pH regulator of above-mentioned concentrated solution to 7.0-8.0, by macroporous adsorbent resin, use elutriant wash-out, obtain the crude product that contains the ramoplanin derivative shown in formula (I);
(6) through mesolow or the described crude product of high pressure purification procedures (5) of C-18, C-8 or polymkeric substance chromatograph packing material, make the ramoplanin derivative shown in formula (I).
3. method according to claim 2, is characterized in that the acid that regulates pH to use in step (4) is oxalic acid, hydrochloric acid or sulfuric acid.
4. method according to claim 2, is characterized in that described in step (4), solvent is ethanol or acetone, and usage quantity is 3-6 times of wet thallus volume.
5. method according to claim 2; it is characterized in that the resin that described in step (5), macroporous adsorbent resin is common middle polarity; preferably HZ-803, the HZ-806 of Shanghai Hua Zhen resin company, the NM100 of Suzhou Na Wei scientific & technical corporation or the XAD-4 of Rhom and Hass.
6. method according to claim 2, is characterized in that eluent described in step (5) is respectively the aqueous solution containing 70% ethanol containing the aqueous solution of 30% ethanol, the aqueous solution containing 50% ethanol, the aqueous solution that contains 70% ethanol and pH 3.0-4.0.
7. method according to claim 2, is characterized in that in step (6), mesolow moving phase used is ammonium acetate solution (4g/L): methyl alcohol=75-10: 25-90 (v/v); High pressure moving phase used is ammonium acetate solution (4g/L): methyl alcohol=40-60: 60-40 (v/v).
8. method according to claim 2, it is characterized in that step (5) also comprises carries out desalination to gained crude product with 0.2-0.45um millipore filtration.
9. method according to claim 2, is characterized in that the HPLC purity of the ramoplanin derivative shown in formula (I) that step (6) obtains is more than 95%.
10. the application of the ramoplanin derivative shown in formula (I) on the medicine of the serious gram positive bacteria infection of preparation treatment.
11. application according to claim 10, is characterized in that described infection is selected from endocarditis, meningitis, pneumonia, septicemia, acute and chronic osteomyelitis, burn infection, skin or soft tissue purulent infection.
12. pharmaceutical compositions, it contains ramoplanin derivative and the pharmaceutically acceptable carrier shown in the formula (I) for the treatment of significant quantity.
13. pharmaceutical compositions according to claim 12, is characterized in that described carrier is selected from vehicle, thinner, flavouring agent or sweeting agent.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20060211603A1 (en) * | 2004-08-18 | 2006-09-21 | Vicuron Pharmaceuticals Inc. | Ramoplanin derivatives possessing antibacterial activity |
| CN101838315A (en) * | 2009-03-17 | 2010-09-22 | 上海医药工业研究院 | Method for separating Ramoplanin |
| US20110086388A1 (en) * | 2009-09-22 | 2011-04-14 | Duke University | Chaperone-assisted protein expression and methods of use |
-
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20060211603A1 (en) * | 2004-08-18 | 2006-09-21 | Vicuron Pharmaceuticals Inc. | Ramoplanin derivatives possessing antibacterial activity |
| CN101838315A (en) * | 2009-03-17 | 2010-09-22 | 上海医药工业研究院 | Method for separating Ramoplanin |
| US20110086388A1 (en) * | 2009-09-22 | 2011-04-14 | Duke University | Chaperone-assisted protein expression and methods of use |
Non-Patent Citations (1)
| Title |
|---|
| 苏敏等: "恩拉霉素与雷莫拉宁生物合成研究进展", 《中国抗生素杂志》 * |
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Application publication date: 20140702 |