CN113544137A - Separation and purification method of vancomycin analogue - Google Patents
Separation and purification method of vancomycin analogue Download PDFInfo
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Abstract
A separation and purification method of vancomycin analogue (I) uses crude vancomycin as raw material, firstly uses polymer matrix chromatographic packing for chromatographic enrichment, and then uses reversed phase silica gel matrix chromatographic packing for chromatographic enrichment to obtain vancomycin analogue with purity greater than 80%.
Description
The invention belongs to the field of biological pharmacy, and particularly relates to a method for separating and preparing a vancomycin structural analogue with purity of more than 80% by using a vancomycin crude product as a raw material.
Since the 40 th century of 20 th, penicillin has been used clinically, the number of anti-infective drugs has reached thousands, and hundreds of them have been used clinically. In 2006, 500 strong anti-infective drugs are sold globally at the merchantable level, and the number of the anti-infective drugs reaches 77, and the anti-infective drugs are the first drugs in 19 major classes of drugs.
Vancomycin (Vancomycin) is a natural amphoglycopeptide antibiotic extracted from fermentation broth of actinomycetes, such as nocardia orientalis, and functions by binding with dipeptide residue of D-alanyl-D-alanine (D-Ala-D-Ala) which is a bacterial cell wall precursor, inhibiting the synthesis of peptidoglycan, thereby inhibiting the synthesis of bacterial cell walls; in addition, the permeability of cell membranes and RNA synthesis can be altered to achieve desired pharmacological effects.
With the large-scale use of antibiotics, bacteria gradually evolve resistance to drugs; in particular, the use of large amounts of beta-lactam antibiotics has led to a growing prevalence of infections caused by methicillin-resistant staphylococcus aureus (MRSA). Under the circumstances, vancomycin hydrochloride becomes the first choice drug for clinical treatment of severe infectious diseases caused by MRSA, and has been known as "the last line of defense of human against intractable drug-resistant strains" and "Wang brand antibiotic" by international antibiotic experts for a long time in the past. Vancomycin hydrochloride is mainly suitable for primary treatment of severe or severe infection caused by staphylococcus resistant to beta-lactamine antibiotics, and also for treatment of patients allergic to penicillin or having no effect by using penicillin and cephalosporin; vancomycin hydrochloride alone or in combination with other aminoglycosides can be used to treat endocarditis caused by staphylococci, streptococci, enterococci or diphtheria.
Since the clinical large-scale application of vancomycin hydrochloride, it has been widely used in the treatment of bacterial infections as a last means of resisting gram-positive bacteria for the last decades; however, since vancomycin-resistant enterococci (VRE) were first detected clinically in 1986, vancomycin-resistance of enterococci has become increasingly prevalent (> 20%) and has begun to spread to organisms other than humans. Recently, many reports have shown that foreign countries have detected a new methicillin-resistant Staphylococcus aureus (VRSA) with high resistance to vancomycin in anti-infective therapy for hemodialysis patients, which causes panic in the medical world, and the medical industry has put the work of finding and modifying antibiotics on a very urgent schedule, and there is an urgent need to develop a second generation glycopeptide antibiotic specific for drug-resistant pathogenic strains.
The vancomycin molecule consists of two basic structures, a glycosyl moiety (α -o-vancosamine- β -o-glucose) and a central heptapeptide core of the peptidyl moiety, the structure of which determines its instability. During the past decades, some important advances have been made in the strategy of making synthetically active vancomycin analogs using structural modifications. Several compounds chemically modified with different natural vancomycin analogs are known to enter clinical trials, and some modified compounds, such as: oritavan, Dalbavancin and Telavancin preliminarily show good bacteriostatic action on vancomycin sensitive bacteria and drug-resistant bacteria (MRSA, VRSA and VRA). The structural modification of vancomycin by combining the international development experience of compounds with biological activity and utilizing the modern biotechnology and chemical synthesis technology is one of the research directions, and the other research mode is to screen from the source and screen new compounds with better pharmacological action aiming at vancomycin analogues.
Patent CN 101724645A discloses a method for obtaining vancomycin analogue-chlorine-free vancomycin by genetically engineering a vancomycin production strain.
Patent CN 101724644A discloses a method for obtaining vancomycin analogue-norvancomycin by genetically engineering a vancomycin production strain.
Patent CN 101397333a discloses a method for obtaining vancomycin analogue-dehydroxy vancomycin by a vancomycin production strain.
The reaction pathway for the production of several analogs of Vancomycin, including the compounds to which this patent relates, is described in the Vancomycin: Structure and Transformation to CDP-I, Constance M.Harris, J.Am.chem.Soc, Vol.105, No.23,6915-Achain 6922.
The inventor of the application finds a method for separating and preparing a vancomycin analogue with the purity of more than 80% by using a vancomycin crude product as a raw material when purifying the vancomycin generated by fermenting the thallerion arboricola thalli. After the compound is prepared, structural identification is carried out on the compound, and the compound is confirmed to be a vancomycin analogue.
Disclosure of Invention
The invention discloses a preparation method of a vancomycin analogue shown as the following formula (I):
whereas vancomycin structural formula (II) is as follows, the two differ only in the red box:
the invention discloses a method for separating and purifying vancomycin analogue with purity more than 80% as shown in formula (I) by taking a crude vancomycin as a raw material, wherein the separation and purification method comprises the following steps: a) first chromatographic enrichment of vancomycin analogs: taking a crude product containing vancomycin analogue as a raw material, carrying out first chromatographic enrichment by using a polymer matrix chromatographic filler, isocratic elution or gradient elution, and collecting first chromatographic enrichment liquid with the purity of more than 35% in a segmented manner; wherein the polymer matrix chromatographic filler is polystyrene/divinylbenzene copolymer UniPS, UniPSA (both UniPS and UniPSA are polystyrene/divinylbenzene matrixes, and synthesis parameters are different during bonding to cause difference of two hydrophilicities and polarities), or polyacrylate and divinylbenzene mixed copolymer UniPSN, and the mobile phase is methanol aqueous solution, ethanol aqueous solution or acetonitrile aqueous solution; b) second chromatographic purification of vancomycin analogue: concentrating the first chromatographic enrichment solution with the purity of more than 35 percent obtained in the step a) under reduced pressure to remove a solvent, carrying out second chromatographic purification by using a reversed-phase silica matrix chromatographic filler, adjusting the pH value to acidity by using phosphoric acid, carrying out isocratic elution or gradient elution, and collecting the second chromatographic enrichment solution with the purity of more than 65 percent in sections; wherein the reversed phase silica gel matrix chromatographic filler is octyl silica gel (C8) or octadecyl silica gel (C18), the mobile phase is methanol-buffer solution, ethanol-buffer solution or acetonitrile-buffer solution, and the buffer solution is triethylamine-water solution; c) third chromatographic purification of vancomycin analogs: concentrating the second chromatographic enrichment solution with the purity of more than 65 percent obtained in the step b) under reduced pressure to remove the solvent, then carrying out third chromatographic purification by using reversed-phase silica matrix chromatographic packing, isocratic elution or gradient elution, and collecting the third chromatographic enrichment solution with the purity of more than 80 percent in sections; wherein the reversed phase silica gel matrix chromatographic filler is octyl silica gel (C8) or octadecyl silica gel (C18), the mobile phase is methanol-buffer solution, ethanol-buffer solution or acetonitrile-buffer solution, and the buffer solution is formic acid-water solution, acetic acid-water solution or trifluoroacetic acid-water solution; and d) carrying out reduced pressure concentration on the third chromatographic enrichment solution with the purity of more than 80% to remove the solvent, and freeze-drying to obtain vancomycin analogue dry powder.
In a preferred embodiment of the separation and purification method of the present invention, in step a), the polymer matrix chromatographic packing is polystyrene/divinylbenzene UniPS, or UniPSA, or polyacrylic acid UniPSN from nano-micro corporation, and the packing has a particle size of 5 μm, or 10 μm, or 30 μm, preferably 10 μm; the pore size of the filler is 100A, or 300A, or 500A, preferably the pore size is 300A.
In a preferred embodiment of the separation and purification method of the present invention, in step a), when the chromatography mobile phase is an aqueous methanol solution, the methanol ratio is 25% to 45% (by volume), preferably 30% to 40% (by volume), and more preferably 33% to 38% (by volume); when the chromatography mobile phase is ethanol water, the ethanol proportion is 40-60% (volume ratio), preferably 45-55% (volume ratio), more preferably 48-53% (volume ratio); when the mobile phase for chromatography is an aqueous acetonitrile solution, the acetonitrile proportion is 15% to 35% (by volume), preferably 20% to 30% (by volume), more preferably 23% to 28% (by volume).
In a preferred embodiment of the separation and purification method of the present invention, in step b), the chromatographic packing is a reversed phase silica gel matrix chromatographic packing, such as C8 or C18 from nanomicro corporation. The filler particle size is 5 μm, or 10 μm, or 30 μm, preferably 10 μm; the pore size of the filler is 60A, 100A or 120A, and the pore size is preferably 120A.
In a preferred embodiment of the separation and purification method of the present invention, in step b), when the chromatography mobile phase is an aqueous methanol solution, the methanol ratio is 5% to 25% (by volume), preferably 10% to 20% (by volume), and more preferably 12% to 17% (by volume); when the chromatography mobile phase is ethanol water, the ethanol proportion is 10-30% (volume ratio), preferably 15-25% (volume ratio), more preferably 18-23% (volume ratio); when the mobile phase for chromatography is an aqueous acetonitrile solution, the acetonitrile proportion is 5% to 25% (by volume), preferably 10% to 20% (by volume), more preferably 12% to 18% (by volume).
In a preferred embodiment of the separation and purification method of the present invention, in step b), the triethylamine-water solution contains triethylamine in a proportion of 0.1% to 0.3% (by volume), preferably 0.15% to 0.25% (by volume), and more preferably 0.18% to 0.23% (by volume).
In the preferable technical scheme of the separation and purification method, in the step b), the ph is adjusted to be 2.0-4.0, preferably 2.5-3.5, and more preferably 2.8-3.3 by using phosphoric acid.
In a preferred embodiment of the separation and purification method of the present invention, in step C), the chromatographic packing is a reversed phase silica gel matrix chromatographic packing, such as C8 or C18 from nanomicro corporation. The filler particle size is 5 μm, or 10 μm, or 30 μm, preferably 10 μm; the pore size of the filler is 60A, 100A or 120A, and the pore size is preferably 120A.
In a preferred embodiment of the separation and purification method of the present invention, in step c), when the chromatography mobile phase is an aqueous methanol solution, the methanol ratio is 5% to 25% (by volume), preferably 10% to 20% (by volume), and more preferably 12% to 17% (by volume); when the chromatography mobile phase is ethanol water, the ethanol proportion is 10-30% (volume ratio), preferably 15-25% (volume ratio), more preferably 18-23% (volume ratio); when the mobile phase for chromatography is an aqueous acetonitrile solution, the acetonitrile proportion is 5% to 25% (by volume), preferably 10% to 20% (by volume), more preferably 12% to 18% (by volume).
In a preferred embodiment of the separation and purification method of the present invention, in step c), the buffer solution is formic acid-water solution, acetic acid-water solution, or trifluoroacetic acid-water solution, wherein the ratio of the added acid is 0.1-0.3% (volume ratio), preferably 0.15-0.25% (volume ratio), and more preferably 0.18-0.23% (volume ratio).
In a preferred embodiment of the separation and purification method of the present invention, in step d), the temperature is controlled to be 10 to 50 ℃, preferably 20 to 40 ℃, and more preferably 25 to 35 ℃ during the concentration under reduced pressure.
In a preferable technical scheme of the separation and purification method, in the step d), the pressure is controlled to be-0.09 mPa to-0.1 mPa during the reduced pressure concentration.
The method takes the crude vancomycin as a raw material, obtains the vancomycin analogue with the purity of more than 80 percent through separation and purification, carries out structural identification, and lays a foundation for further screening the biological activity or screening the biological activity after carrying out structural modification on the vancomycin analogue. Through subsequent work, a new compound with better drug effect and less adverse reaction can be obtained.
FIG. 1 is a diagram showing the elemental composition of a vancomycin analog obtained according to the present invention;
FIG. 2 shows MS profiles of vancomycin analogs obtained in the present invention;
FIG. 3 shows the spectrum C of vancomycin analogs obtained by the present invention;
FIG. 4 shows scheme H of vancomycin analogs obtained by the present invention;
the invention is further illustrated by the following examples, which are given solely for the purpose of illustration and are not to be construed as limitations of the present invention.
Example 1:
a) first chromatographic enrichment of vancomycin analogs
A vancomycin crude product containing vancomycin analogue (the vancomycin crude product is prepared according to Chinese patent CN 101456903B) is subjected to first chromatographic enrichment by using a polymer matrix chromatographic packing such as UniPS (UniPS), wherein the particle size is 10 μm, the pore diameter is 300A, a mobile phase is a methanol aqueous solution, the methanol proportion is 33% (volume ratio), isocratic elution is carried out, and the vancomycin crude product is collected by stages, and the purity is higher than 35%.
b) Second chromatographic purification of vancomycin analogs
After the first chromatographic enrichment, collecting the part with the purity of more than 35 percent, decompressing and concentrating to remove the solvent, then carrying out the second chromatographic purification by using reversed phase silica gel matrix chromatographic packing, such as C8, the particle size of which is 10 mu m, the pore diameter of which is 100A, wherein the chromatographic mobile phase is ethanol-buffer solution, the proportion of ethanol is 18 percent (volume ratio), the buffer solution is 0.18 percent (volume ratio) triethylamine solution, regulating the PH value to 2.8 by phosphoric acid, isocratic elution and sectional collection, and entering the part with the purity of more than 65 percent into the next step.
c) Third chromatographic purification of vancomycin analogs
After the second chromatographic purification, collecting the part with purity of more than 65%, concentrating under reduced pressure to remove the solvent, performing the third chromatographic purification by using reversed phase silica gel matrix chromatographic packing such as C18 with particle size of 10 μm and pore diameter of 120A, wherein the chromatographic mobile phase is acetonitrile-buffer solution with acetonitrile proportion of 12% (volume ratio), the buffer solution is 0.18% (volume ratio) formic acid aqueous solution, isocratically eluting, collecting by stages, and feeding the part with purity of more than 80% to the next step.
d) After the third chromatographic purification of the vancomycin analogue, collecting the partial liquid with the purity of more than 80 percent, decompressing and concentrating, controlling the pressure at-0.09 mPa to-0.1 mPa, controlling the temperature at 10 ℃, removing the solvent, and freeze-drying to obtain vancomycin analogue dry powder, wherein the purity is 85.1 percent through liquid phase detection, and the product yield is 47.7 percent.
Example 2:
a) first chromatographic enrichment of vancomycin analogs
A vancomycin analogue-containing vancomycin crude product (prepared according to Chinese patent CN 101456903B) is subjected to first chromatographic enrichment by using a polymer matrix chromatographic packing, such as UniPSA, with the particle size of 30 μm and the pore diameter of 100A, wherein a mobile phase is an ethanol water solution, the ethanol proportion is 48% (volume ratio), isocratic elution is carried out, and fractions are collected, wherein the purity is more than 35%.
b) Second chromatographic purification of vancomycin analogs
After the first chromatographic enrichment, collecting the part with the purity of more than 35 percent, decompressing and concentrating to remove the solvent, then carrying out the second chromatographic purification by using reversed phase silica gel matrix chromatographic packing, such as C18, the particle size of which is 10 mu m, the pore diameter of which is 60A, wherein the chromatographic mobile phase is methanol-buffer solution, the methanol proportion is 12 percent (volume ratio), the buffer solution is 0.20 percent (volume ratio) triethylamine solution, the pH value is adjusted to 3.0 by phosphoric acid, isocratic elution and fractional collection, and the part with the purity of more than 65 percent enters the next step.
c) Third chromatographic purification of vancomycin analogs
After the second chromatographic purification, collecting the part with purity of more than 65%, concentrating under reduced pressure to remove solvent, performing third chromatographic purification with reversed phase silica gel matrix chromatographic filler, such as C8, with particle diameter of 30 μm and pore diameter of 120A, wherein the chromatographic mobile phase is ethanol-buffer solution, ethanol proportion is 18% (volume ratio), buffer solution is 0.20% (volume ratio) acetic acid solution, isocratically eluting, collecting by stages, and collecting the part with purity of more than 80% in the next step.
d) After the third chromatographic purification of the vancomycin analogue, collecting the partial liquid with the purity of more than 80 percent, decompressing and concentrating, controlling the pressure at-0.09 mPa to-0.1 mPa, controlling the temperature at 50 ℃, removing the solvent, and freeze-drying to obtain vancomycin analogue dry powder, wherein the purity is 91.5 percent by liquid phase detection, and the product yield is 40.5 percent.
Example 3:
a) first chromatographic enrichment of vancomycin analogs
The vancomycin analogue-containing vancomycin crude product (the vancomycin crude product is prepared according to Chinese patent CN 101456903B) is subjected to first chromatographic enrichment by using a polymer matrix chromatographic packing such as UniPSN, wherein the particle size is 30 mu m, the pore diameter is 300A, the mobile phase is acetonitrile aqueous solution, the acetonitrile proportion is 23% (volume ratio), isocratic elution is carried out, and the vancomycin crude product is collected by sections, and the purity is higher than 35%.
b) Second chromatographic purification of vancomycin analogs
After the first chromatographic enrichment, collecting the part with the purity of more than 35 percent, decompressing and concentrating to remove the solvent, then carrying out the second chromatographic purification by using reversed phase silica gel matrix chromatographic packing, such as C18, the particle size of which is 30 mu m, the pore diameter of which is 120A, wherein the chromatographic mobile phase is acetonitrile-buffer solution, the acetonitrile proportion is 12 percent (volume ratio), the buffer solution is 0.23 percent (volume ratio) triethylamine solution, the pH value is adjusted to 3.3 by phosphoric acid, isocratic elution and fractional collection are carried out, and the part with the purity of more than 65 percent enters the next step.
c) Third chromatographic purification of vancomycin analogs
After the second chromatographic purification, collecting the part with purity of more than 65%, concentrating under reduced pressure to remove solvent, performing third chromatographic purification by using reversed phase silica gel matrix chromatographic filler such as C8 with particle size of 10 μm and pore diameter of 60A, wherein the chromatographic mobile phase is methanol-buffer solution with methanol proportion of 12% (volume ratio) and buffer solution of 0.23% (volume ratio) trifluoroacetic acid solution, isocratically eluting, collecting by stages, and collecting the part with purity of more than 80% in the next step.
d) After the third chromatographic purification of the vancomycin analogue, collecting the partial liquid with the purity of more than 80 percent, decompressing and concentrating, controlling the pressure to be-0.09 mPa to-0.1 mPa, controlling the temperature to be 35 ℃, removing the solvent, and freeze-drying to obtain vancomycin analogue dry powder, wherein the purity is 83.2 percent by liquid phase detection, and the product yield is 51.4 percent.
Example 4:
a) first chromatographic enrichment of vancomycin analogs
A vancomycin crude product containing vancomycin analogue (the vancomycin crude product is prepared according to Chinese patent CN 101456903B) is subjected to first chromatographic enrichment by using a polymer matrix chromatographic packing such as UniPS (UniPS), wherein the particle size is 30 μm, the pore diameter is 100A, a mobile phase is a methanol aqueous solution, the methanol proportion is 34% (volume ratio), isocratic elution is carried out, and the vancomycin crude product is collected by stages, and the purity is higher than 35%.
b) Second chromatographic purification of vancomycin analogs
After the first chromatographic enrichment, collecting the part with the purity of more than 35 percent, decompressing and concentrating to remove the solvent, then carrying out the second chromatographic purification by using reversed phase silica gel matrix chromatographic packing, such as C8, the particle size of which is 10 mu m, the pore diameter of which is 100A, wherein the chromatographic mobile phase is methanol-buffer solution, the methanol proportion is 14 percent (volume ratio), the buffer solution is 0.19 percent (volume ratio) triethylamine solution, the pH value is adjusted to 2.9 by phosphoric acid, isocratic elution and fractional collection are carried out, and the part with the purity of more than 65 percent enters the next step.
c) Third chromatographic purification of vancomycin analogs
After the second chromatographic purification, collecting the part with purity of more than 65%, concentrating under reduced pressure to remove the solvent, performing the third chromatographic purification by using reversed phase silica gel matrix chromatographic packing, such as C18, with particle size of 10 μm and pore diameter of 120A, wherein the chromatographic mobile phase is acetonitrile-buffer solution, the acetonitrile proportion is 14% (volume ratio), the buffer solution is 0.19% (volume ratio) formic acid aqueous solution, isocratically eluting, collecting by stages, and allowing the part with purity of more than 80% to enter the next step.
d) After the third chromatographic purification of the vancomycin analogue, collecting the partial liquid with the purity of more than 80 percent, decompressing and concentrating, controlling the pressure at-0.09 mPa to-0.1 mPa, controlling the temperature at 31 ℃, removing the solvent, and freeze-drying to obtain vancomycin analogue dry powder, wherein the purity is 87.9 percent by liquid phase detection, and the product yield is 42.8 percent.
Example 5:
a) first chromatographic enrichment of vancomycin analogs
A vancomycin crude product containing vancomycin analogues (the vancomycin crude product is prepared according to Chinese patent CN 101456903B) is subjected to first chromatographic enrichment by using a polymer matrix chromatographic packing, such as UniPSA, with the particle size of 10 mu m and the pore diameter of 300A, wherein a mobile phase is acetonitrile aqueous solution, the acetonitrile proportion is 26% (volume ratio), isocratic elution is carried out, and fractions are collected, wherein the purity is more than 35%.
b) Second chromatographic purification of vancomycin analogs
After the first chromatographic enrichment, collecting the part with the purity of more than 35 percent, decompressing and concentrating to remove the solvent, then carrying out the second chromatographic purification by using reversed phase silica gel matrix chromatographic packing, such as C18, the particle size of which is 10 mu m, the pore diameter of which is 120A, wherein the chromatographic mobile phase is ethanol-buffer solution, the proportion of ethanol is 51 percent (volume ratio), the buffer solution is 0.21 percent (volume ratio) triethylamine solution, regulating the PH value to 3.1 by phosphoric acid, isocratic elution and sectional collection, and entering the part with the purity of more than 65 percent into the next step.
c) Third chromatographic purification of vancomycin analogs
After the second chromatographic purification, collecting the part with purity of more than 65%, concentrating under reduced pressure to remove the solvent, performing the third chromatographic purification by using reversed phase silica gel matrix chromatographic packing, such as C18, with particle size of 5 μm and pore diameter of 120A, wherein the chromatographic mobile phase is acetonitrile-buffer solution, the acetonitrile proportion is 16% (volume ratio), the buffer solution is 0.21% (volume ratio) acetic acid solution, isocratically eluting, collecting by stages, and allowing the part with purity of more than 80% to enter the next step.
d) After the third chromatographic purification of the vancomycin analogue, collecting the partial liquid with the purity of more than 80 percent, decompressing and concentrating, controlling the pressure at-0.09 mPa to-0.1 mPa, controlling the temperature at 30 ℃, removing the solvent, and freeze-drying to obtain vancomycin analogue dry powder, wherein the purity is 86.1 percent by liquid phase detection, and the product yield is 44.8 percent.
Example 6:
a) first chromatographic enrichment of vancomycin analogs
A vancomycin crude product containing vancomycin analogue (the vancomycin crude product is prepared according to Chinese patent CN 101456903B) is subjected to first chromatographic enrichment by using a polymer matrix chromatographic packing such as UniPSN, wherein the particle size is 10 mu m, the pore diameter is 100A, the mobile phase is ethanol water solution, the ethanol proportion is 51% (volume ratio), isocratic elution is carried out, and the vancomycin crude product is collected by sections, and the purity is more than 35%.
b) Second chromatographic purification of vancomycin analogs
After the first chromatographic enrichment, collecting the part with the purity of more than 35 percent, decompressing and concentrating to remove the solvent, then carrying out the second chromatographic purification by using reversed phase silica gel matrix chromatographic packing, such as C8, the particle size of which is 30 mu m, the pore diameter of which is 120A, wherein the chromatographic mobile phase is methanol-buffer solution, the methanol proportion is 16 percent (volume ratio), the buffer solution is 0.23 percent (volume ratio) triethylamine solution, the pH value is adjusted to 3.2 by phosphoric acid, isocratic elution and fractional collection are carried out, and the part with the purity of more than 65 percent enters the next step.
c) Third chromatographic purification of vancomycin analogs
After the second chromatographic purification, collecting the part with purity of more than 65%, concentrating under reduced pressure to remove solvent, performing third chromatographic purification by using reversed phase silica gel matrix chromatographic filler such as C8 with particle size of 10 μm and pore diameter of 120A, wherein the chromatographic mobile phase is methanol water solution, the methanol proportion is 15% (volume ratio), the buffer solution is 0.2% (volume ratio) trifluoroacetic acid solution, isocratically eluting, collecting by stages, and collecting the part with purity of more than 80% in the next step.
d) After the third chromatographic purification of the vancomycin analogue, collecting liquid with the purity of more than 80 percent, decompressing and concentrating, controlling the pressure at-0.09 mPa to-0.1 mPa, controlling the temperature at 40 ℃, removing the solvent, and freeze-drying to obtain vancomycin analogue dry powder, wherein the purity is 80.3 percent by liquid phase detection, and the product yield is 52.9 percent.
Example 7:
and detecting the sample by using UPLC-MS and nuclear magnetic resonance to determine the structure of the sample.
1. UPLC-MS analysis: the instrument comprises the following steps: Q-TOF micro; and (3) testing conditions are as follows: an ES + source.
And (3) testing results:
analysis of Table 1: detecting positive ions of mass spectrum electrospray ionization source (ESI) to determine [ M + H ] of the compound] +The peak was 1431.5046. Determining the elemental composition as C66H 73N 8O 24Cl 2de-NH with vancomycin3The elemental composition of the added H is consistent.
2. Nuclear magnetic spectrum analysis:
testing an instrument: bruker 600 MHz; testing the solvent: DMSO-d 6.
According to13C-NMR spectrum (FIG. 3),1The H-NMR spectrum (FIG. 4) showed that the sample had the following chemical structure of formula (I).
It should be noted that the above summary and the detailed description are intended to demonstrate the practical application of the technical solutions provided by the present invention, and should not be construed as limiting the scope of the present invention. Various modifications, equivalent substitutions, or improvements may be made by those skilled in the art within the spirit and principles of the invention.
Claims (10)
- A method for separating and purifying vancomycin analogue (I), which is characterized by comprising the following steps:a) first chromatographic enrichment of vancomycin analogs:taking a crude product containing vancomycin analogue as a raw material, carrying out first chromatographic enrichment by using a polymer matrix chromatographic filler, isocratic elution or gradient elution, and collecting first chromatographic enrichment liquid with the purity of more than 35% in a segmented manner; wherein the polymer matrix chromatographic filler is polystyrene/divinylbenzene copolymer UniPS, UniPSA or mixed copolymer UniPSN of polyacrylate and divinylbenzene, and the mobile phase is methanol aqueous solution, ethanol aqueous solution or acetonitrile aqueous solution;b) second chromatographic purification of vancomycin analogue:concentrating the first chromatographic enrichment solution with the purity of more than 35 percent obtained in the step a) under reduced pressure to remove a solvent, carrying out second chromatographic purification by using a reversed-phase silica matrix chromatographic filler, adjusting the pH value to acidity by using phosphoric acid, carrying out isocratic elution or gradient elution, and collecting the second chromatographic enrichment solution with the purity of more than 65 percent in sections; wherein the reversed phase silica gel matrix chromatographic filler is octyl silica gel C8 or octadecyl silica gel C18, the mobile phase is methanol-buffer solution, ethanol-buffer solution or acetonitrile-buffer solution, and the buffer solution is triethylamine-water solution;c) third chromatographic purification of vancomycin analogs:concentrating the second chromatographic enrichment solution with the purity of more than 65 percent obtained in the step b) under reduced pressure to remove the solvent, then carrying out third chromatographic purification by using reversed-phase silica matrix chromatographic packing, isocratic elution or gradient elution, and collecting the third chromatographic enrichment solution with the purity of more than 80 percent in sections; wherein the reversed phase silica gel matrix chromatographic filler is octyl silica gel or octadecyl silica gel, the mobile phase is methanol-buffer solution, ethanol-buffer solution or acetonitrile-buffer solution, and the buffer solution is formic acid-water solution, acetic acid-water solution or trifluoroacetic acid-water solution; andd) and (3) carrying out reduced pressure concentration on the third-time chromatographic enrichment solution with the purity of more than 80% to remove the solvent, and freeze-drying to obtain the dry powder of the vancomycin analogue (I).
- The method according to claim 1, characterized in that in step a) the polymer matrix chromatography packing has a particle size of 5 μ ι η, 10 μ ι η or 30 μ ι η; the pore size of the polymeric matrix chromatography packing is 100A, 300A or 500A.
- The method according to claim 1, wherein in step a), when the mobile phase is an aqueous methanol solution, the methanol proportion is 25-45% (volume ratio); when the mobile phase is ethanol water, the proportion of ethanol is 40-60% (volume ratio); when the mobile phase is acetonitrile water solution, the acetonitrile proportion is 15-35% (volume ratio).
- The method according to claim 1, characterized in that in step b) and step c) the particle size of the reversed phase silica matrix chromatography packing is 5 μ ι η, or 10 μ ι η, or 30 μ ι η, preferably 10 μ ι η; the pore diameter of the reversed phase silica matrix chromatographic packing is 60A, 100A and 120A.
- The method according to claim 1, wherein in step b) and step c), when the mobile phase is an aqueous solution of methanol, the proportion of methanol is 5-25% (volume ratio); when the mobile phase is ethanol water solution, the proportion of ethanol is 10-30% (volume ratio); when the mobile phase is acetonitrile water solution, the acetonitrile proportion is 5-25% (volume ratio).
- The process according to claim 1, wherein in step b) the triethylamine-water solution has a triethylamine ratio of 0.1% to 0.3% (v/v).
- The method according to claim 1, wherein in step b), the pH is adjusted to 2.0 to 4.0 with phosphoric acid.
- The method according to claim 1, wherein in step c), the acid is added to the buffer solution in a proportion of 0.1 to 0.3% (by volume).
- The method as claimed in claim 1, wherein in step d), the temperature of the concentration under reduced pressure is controlled to 10-50 ℃.
- The method according to claim 1, wherein in step d), the pressure of the reduced-pressure concentration is controlled to be-0.09 mPa to-0.1 mPa.
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| CN114405298B (en) * | 2021-12-27 | 2024-05-14 | 苏州艾博生物科技有限公司 | Method for purifying polyethylene glycol conjugated lipids |
| CN114621322A (en) * | 2022-03-28 | 2022-06-14 | 苏州纳微科技股份有限公司 | Separation and purification method of LAGUBIXING |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101397333A (en) * | 2007-09-27 | 2009-04-01 | 浙江医药股份有限公司新昌制药厂 | De-hydroxyl vancomycin, preparation method thereof, medicament composition and use thereof |
| CN101456903A (en) * | 2007-12-14 | 2009-06-17 | 浙江医药股份有限公司新昌制药厂 | Separation and purification method of vancocin |
| CN102050866A (en) * | 2009-11-04 | 2011-05-11 | 上海来益生物药物研究开发中心有限责任公司 | Method for purifying glycopeptide compound |
| CN104610434A (en) * | 2013-11-01 | 2015-05-13 | 浙江医药股份有限公司新昌制药厂 | Separation and purification method of high-purity vancomycin hydrochloride |
| CN106565827A (en) * | 2015-10-12 | 2017-04-19 | 北大方正集团有限公司 | Method for highly purifying glycopeptide compounds |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| SI21924A (en) * | 2004-12-07 | 2006-06-30 | Lek Farmacevtska Druzba D.D. | Pure vancomycin hydrochloride |
| CN101440127B (en) * | 2007-11-19 | 2012-10-17 | 浙江医药股份有限公司新昌制药厂 | Preparation of high-purity vancomycin hydrochloride |
-
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101397333A (en) * | 2007-09-27 | 2009-04-01 | 浙江医药股份有限公司新昌制药厂 | De-hydroxyl vancomycin, preparation method thereof, medicament composition and use thereof |
| CN101456903A (en) * | 2007-12-14 | 2009-06-17 | 浙江医药股份有限公司新昌制药厂 | Separation and purification method of vancocin |
| CN102050866A (en) * | 2009-11-04 | 2011-05-11 | 上海来益生物药物研究开发中心有限责任公司 | Method for purifying glycopeptide compound |
| CN104610434A (en) * | 2013-11-01 | 2015-05-13 | 浙江医药股份有限公司新昌制药厂 | Separation and purification method of high-purity vancomycin hydrochloride |
| CN106565827A (en) * | 2015-10-12 | 2017-04-19 | 北大方正集团有限公司 | Method for highly purifying glycopeptide compounds |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113845573A (en) * | 2021-10-25 | 2021-12-28 | 北大方正集团有限公司 | Preparation method of vancomycin impurity G |
| CN113845573B (en) * | 2021-10-25 | 2024-05-07 | 新方正控股发展有限责任公司 | Preparation method of vancomycin impurity G |
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