WO2021248302A1 - 用于分离含硼物质的层析介质 - Google Patents
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- WO2021248302A1 WO2021248302A1 PCT/CN2020/095070 CN2020095070W WO2021248302A1 WO 2021248302 A1 WO2021248302 A1 WO 2021248302A1 CN 2020095070 W CN2020095070 W CN 2020095070W WO 2021248302 A1 WO2021248302 A1 WO 2021248302A1
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
- B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
- B01J20/286—Phases chemically bonded to a substrate, e.g. to silica or to polymers
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/60—Construction of the column
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- the present invention relates to the field of chromatographic media, and in particular to a chromatographic medium for separating boron-containing substances.
- Borax was often used for medicinal purposes in ancient my country. There are many descriptions of its medicinal properties in Chinese medicine: Borax, also known as moonstone, is sweet and salty in taste, cool in nature, and belongs to the lung and stomach meridians. It has the effects of clearing heat and detoxification, reducing swelling and antiseptic, clearing lungs and resolving phlegm. It is used for sore throat, sore mouth and tongue, red eyes, swelling and pain, phlegm, phlegm-heat, cough, etc. Boron atoms have unique properties that are different from those often found in other drug molecules. The boron atom center can change from a sp 2 hybridized triangular plane to a sp 3 hybridized tetrahedron.
- the boron-containing compound increases the diversity of the drug molecular structure. Such compounds have become a new approach for drug research and development with their brand-new structure and special mechanism of action. With the development of boron chemistry, more and more organoboron compounds with better biological activity have been synthesized in recent years. They have good effects in anti-tumor, anti-infection, and inhibition of thrombin and dipeptidyl peptidase activity.
- boron-containing drugs Many types have been industrialized and have high commercial value. Including Tavaborole (trade name Kerydin), Crisaborole (trade name Eucrisa), VABOMERE compound injection, cyclic adenosine phosphate boric acid complex, boron-containing acridine derivatives, boric acid-containing Reagents and oligonucleotides, Bortezomib (Bortezomib, trade name Velcade), Ixazomib (Lxazomib, trade name Ninlaro), Ixazomib (Ixazomib, trade name Ninlaro).
- Tavaborrol and Crimborrol are new molecular entities approved by the US FDA in 2014 and 2016 for external use from Anacor. They are used to treat onychomycosis (grey toes/nails) and allergic dermatitis (eczema). ).
- VABOMERE is composed of Meropenem (meropenem, an antibacterial drug) and Vaborbactam (which can inhibit the resistance of certain bacteria). In 2017, it was approved by the US FDA for the treatment of adult complicated urinary tract infections. Vaborbactam is a cyclic boronic acid pharmacophore ⁇ -lactamase inhibitor, VABOMERE can deal with the infection of gram-negative bacteria that produce ⁇ -lactamase.
- the three drugs are the first organic borate esters.
- Bortezomib and ixazomib are also organoboron drugs containing dipeptidyl groups. Bortezomib was marketed in 2003 for the treatment of multiple myeloma as a proteasome inhibitor; ixazomib was marketed in 2015 as the first oral drug for the treatment of multiple myeloma and is a second-generation proteasome inhibitor .
- the separation and purification of drugs in production is a key process.
- the separation efficiency and process not only affect the quality of the drug but also the production cost of the drug.
- the production and purification methods of these boron-containing drug molecules are generally crystallization purification and reversed-phase chromatography purification.
- the purpose of the present invention is to fill the gap in the existing purification technology and provide an affinity chromatography medium for separating or enriching boron-containing small molecules, oligonucleotides, polypeptides and proteins;
- the present invention relates to a chromatographic medium for separating boron-containing substances.
- the chromatographic medium includes a matrix support and a graft chain grafted on the matrix support; another object of the present invention is to provide the above-mentioned affinity And the preparation method of the chromatography medium; the above affinity chromatography medium is used as the analytical application of the chromatographic column; the above affinity chromatography medium is used in the industrial separation and purification.
- the chromatographic medium prepared in this way has high separation efficiency, good separation effect, and low overall cost, and is suitable for industrial large-scale separation and purification of boron-containing small molecules, oligonucleotides, polypeptides and proteins.
- the chromatographic medium for separating boron-containing substances provided by the present invention includes a matrix support and a graft chain grafted on the matrix support;
- the general formula of the graft chain is MA-R 1 -X 1 -R 2 or Shown
- MA is a group that can be covalently or non-covalently bonded to other chemical functional groups
- R 1 , R 2 and R 3 are independently selected from hydrogen atoms, saturated hydrocarbon groups, unsaturated hydrocarbon groups or saturated hydrocarbon groups containing non-carbon electronegative heteroatoms, and at least one of them has a cis ortho position polyhydroxyl group, cis ortho position
- the position polyhydroxyl group is a straight chain skeleton structure or a cyclic chain skeleton structure;
- X 1 and X 2 are the divalent and trivalent heteroatoms in the non-carbon electronegative heteroatoms, respectively;
- the present invention also relates to a chromatographic separation device, which contains the chromatographic medium as described above.
- the present invention also relates to the chromatographic medium as described above, or the use of the chromatographic column as described above for separating or enriching boron-containing substances from a liquid medium.
- Figure 1 is a scanning electron micrograph of a chromatographic medium in an embodiment of the present invention.
- Figure 2 is a particle size distribution diagram of a chromatographic medium in an embodiment of the present invention.
- FIG. 3 is a chromatogram of the separation effect of a chromatographic medium on a boron-containing molecule and a boron-free molecule in an embodiment of the present invention
- Fig. 5 is a chromatogram of separation and purification of a bortezomib sample by a chromatographic medium in an embodiment of the present invention
- Fig. 6 is a chromatogram of the separation chromatogram of a bortezomib sample by a chromatographic medium in a comparative example of the present invention.
- the present invention relates to a chromatographic medium for separating boron-containing substances.
- the chromatographic medium includes a matrix support and a polymer chain grafted on the matrix support;
- the general formula of the polymer chain is MA-R 1 -X 1 -R 2 or Shown
- MA is a group that can be covalently or non-covalently bonded to other chemical functional groups
- R 1 , R 2 and R 3 are independently selected from hydrogen atoms, saturated hydrocarbon groups, unsaturated hydrocarbon groups or saturated hydrocarbon groups containing non-carbon electronegative heteroatoms, and at least one of them has a cis ortho position polyhydroxyl group, cis ortho position
- the position polyhydroxyl group is a straight chain skeleton structure or a cyclic chain skeleton structure;
- X 1 and X 2 are the divalent and trivalent heteroatoms in the non-carbon electronegative heteroatoms, respectively;
- the separation principle of the present invention is based on the rapid and stable bond combination between boric acid and cis-diol to form borate.
- the covalent BO bond of borate is very stable, but under certain conditions or under certain external stimuli, BO The bond formation is reversible.
- This principle has a wide range of applications in boric acid affinity chromatography, that is, phenylboronic acid is attached to the matrix framework to affinity adsorb molecules with cis-diol bonds.
- the present invention innovatively develops a reverse application based on the above principle, that is, a group with a cis-ortho-position polyhydroxyl group is bonded to the matrix framework to affinity adsorb boron-containing molecules.
- the present invention cleverly bonds the group with cis-ortho-position polyhydroxyl group to the matrix skeleton through a non-carbon X (X 1 or X 2 ) heteroatom with electronegativity; through this X heteroatom, a suitable length of Spacer is used to increase the flexibility of the ortho-position polyhydroxyl (active group), so that the ortho-position polyhydroxyl (active group) is more likely to combine with the boron atom in space to form an effective coordination; the lone pair on the X heteroatom
- electrons can form intramolecular hydrogen bonds with the hydroxyl groups in the active group, and the ortho-position polyhydroxyl groups can form a cis conformation that is conducive to the bonding of boron atoms; on the other hand, the lone pair of electrons on the X heteroatom can interact with the boron atom.
- the non-carbon electronegative heteroatom is strongly electronegative.
- the Pauling electronegativity values of the non-carbon electronegative heteroatoms are all greater than 2.0, preferably greater than 2.1, more preferably greater than 2.5, and more preferably greater than 2.9.
- the non-carbon electronegative heteroatom is selected from one or more of O, S, N, and P.
- the saturated hydrocarbon group containing non-carbon electronegative heteroatoms may be polyamines, oligomeric imines, polyethers, oligopolyethers, polysulfides, oligomeric sulfides; more preferably, polyamines And oligomeric imines, such as ethylene diamine, diethylene triamine, dipropylene triamine, triethylene tetramine, linear and branched oligoethylene imines.
- X 1 is selected from O or S atoms.
- X 2 is selected from N or P atoms.
- the number of cis-vicinal polyhydroxyl groups is greater than 2, for example, 3, 4, 5, 6, 7, 8, 9, 10 or more.
- the total number of carbon atoms on R 1 , R 2 and R 3 in the graft chain is 3-40, such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35 can also be selected.
- the saturated hydrocarbon group or the unsaturated hydrocarbon group may be a C x -C y alkyl group.
- C x -C y alkyl group refers to an alkyl group having carbon atoms from x to y (including x and y) in a branched or unbranched hydrocarbon group.
- C 1 -C 4 alkyl refers to a straight or branched hydrocarbon moiety having from 1 to 4 carbon atoms, including methyl, ethyl, n-propyl, isopropyl , N-butyl, isobutyl, sec-butyl and tert-butyl.
- C 1 -C 4 n-alkyl refers to a linear hydrocarbon moiety having from 1 to 4 carbon atoms, including methyl, ethyl, n-propyl, and n-butyl.
- x can be from 1 to 10 and y is from 2 to 20.
- Each alkyl group and cycloalkyl group can be optionally substituted such as, but not limited to, as indicated herein.
- the group is mono- or di-substituted.
- the alkyl group is a C 1 -C 3 , C 1 -C 4 , C 1 -C 6 , C 4 -C 6 or C 1 -C 10 alkyl.
- the cyclic chain skeleton structure may also be a cycloalkyl group.
- a cycloalkyl group such as but not limited to cyclopropylmethyl, dimethylcyclopropyl, cyclopropylethyl, cyclopropylpropyl, cyclopropylbutyl, cyclobutylmethyl, cyclobutylethyl, cyclobutylpropyl , Cyclopentylmethyl, cyclopentylethyl, cyclopentylpropyl, cyclohexylmethyl, cyclohexylethyl, and cyclohexylpropyl.
- Each alkyl group and cycloalkyl group can be optionally substituted or modifiable, such as but not limited to those specified in the text.
- the group is mono- or di-substituted.
- the cyclic chain backbone structure is monosaccharide, disaccharide or polysaccharide
- the cyclic chain backbone structure is preferably mannose, fructose, galactose, mannose-containing disaccharides, and mannose-containing oligosaccharides.
- the matrix support contains sites MA that can be chemically modified. It can be connected to other chemical functional groups by covalent or non-covalent bonds. These modification sites include but are not limited to the following chemical functional groups: MA is selected from at least one of epoxy group, amino group, aldehyde, hydroxyl group, carboxyl group, oxo group and thiol group.
- the modification site usually also contains derivative groups with these corresponding chemical functional groups, such as: amino derivatives, thiol derivatives, aldehyde derivatives, formyl derivatives, biotin derivatives, alkyne derivatives, hydroxyl derivatives, Activated hydroxyl or derivative, carboxylate derivative, activated carboxylate derivative, activated carbonate, activated ester, NHS ester (succinimidyl), NHS carbonate (succinimide) Group), imino ester or derivative, cyano bromide derivative, maleimide derivative, haloacetyl derivative, iodoacetamide/iodoacetyl derivative, epoxy derivative, streptavidin Harmonic derivatives, Tresyl derivatives, diene/conjugated diene derivatives (Diels-Alder type reaction), olefin derivatives, substituted phosphate derivatives, bromohydrins/halohydrins, substituted disulfides , Pyridyl-dis
- the density of chemical modification sites on the surface of these chromatographic media can be optimized.
- >0.1mmol/ml medium preferably >0.5mmol/ml medium, more preferably >1.0mmol/ml medium, more preferably >2.0mmol/ml medium.
- At least one of R 1 , R 2 and R 3 has a cis vicinal polyol; preferably a cis vicinal diol; more preferably a terminal cis vicinal diol;
- At least one of R 1 , R 2, and R 3 carries catechol, such as a catechol homolog.
- the matrix support includes borosilicate glass, agar, agarose, agarose derivatives, magnetic beads, silicon dioxide, titanium dioxide, alginate, cellulose, cellulose derivatives, and cellulose derivatives. Glycan, starch, cyclodextrin, chitosan, carrageenan, guar gum, acacia, Indian gum, tragacanth, karaya, locust bean gum, xanthan gum, pectin, mucin, Liver thioesters and gelatin, silicon, ceramics, glass, polyurethane, polystyrene, polystyrene divinylbenzene, polymethyl methacrylate, polyacrylamide, polyethylene glycol terephthalate, polyacetic acid Any one of vinyl ester, polyethylene, polypropylene, polyvinyl chloride, polyvinylpyrrolidone, or a copolymer formed by any of them.
- the base support can be selected from a series of options according to the application of suitable shape, form, material and modification.
- the surface of the base support may be substantially flat or planar. It can also be round or other shapes.
- the shape of the surface of the base support includes, but is not limited to, holes, depressions, columns, ridges, channels, membranes or the like.
- the base support is preferably a particulate material having a substantially flat surface.
- the shape of the particulate matter is preferably spherical or substantially spherical.
- the matrix support is granular, and the particle size ranges from 1 ⁇ m to 500 ⁇ m.
- the particle size ranges from 1 ⁇ m to 300 ⁇ m;
- the particle size ranges from 2 ⁇ m to 200 ⁇ m.
- the matrix support may be non-porous, or include one or more pores.
- it may be a porous resin.
- porous resins may include: other agarose-based resins (for example, Sepharose Fast Flow series (GE)), polymethacrylate: (Sepax PGMA series), polystyrene divinyl benzene (Sepax PolyRP and Sepax Proteomix series), silica (Sepax Silica series), controllable pore glass, dextran derivatives, polyacrylamide derivatives, and/or other polymers; or any combination thereof.
- the non-porous resin can be prepared from the same matrix material as the above porous resin.
- the pore size is
- the pore size is
- the pore size is
- non-porous means that the matrix support has no obvious measurable pores, such as pore size
- chromatography media examples include but are not limited to the listed types:
- Chromatography media that have been covalently attached to other groups: including but not limited to -OH (hydroxyl), -CHO (aldehyde group), IEX (anion, cation exchange group), HIC (hydrophobic group), IMAC (metal chelating group), Affinity (affinity group), Hydrophilic-Hydrophobic balanced (hydrophilic and hydrophobic group), Mixed-mode (mixed group).
- groups including but not limited to -OH (hydroxyl), -CHO (aldehyde group), IEX (anion, cation exchange group), HIC (hydrophobic group), IMAC (metal chelating group), Affinity (affinity group), Hydrophilic-Hydrophobic balanced (hydrophilic and hydrophobic group), Mixed-mode (mixed group).
- chromatographic separation devices can be developed using the chromatographic medium containing the above-mentioned chromatographic medium.
- chromatographic separation devices including but not limited to the following types: SPE solid phase extraction columns, centrifuge tubes with separation membranes, magnetic beads, separation membranes (membranes), rapid detection bio-chips (bio-chips), kits, Fiber bundle column, monolithic column and conventional analytical grade or preparative grade chromatography column.
- the chromatographic separation device is preferably column-shaped and consists of a number of necessary components: the material of the chromatographic medium, the particle size and pore size of the chromatographic medium, the matrix material of the column, the size of the column, and the method of loading the column.
- the chromatographic column packed by using the chromatographic medium of the present invention can have a variety of physical forms, and is composed of a combination of different necessary components of the chromatographic column.
- Chromatographic media with a particle size of less than 15 ⁇ m are usually packed with constant flow and constant pressure methods;
- Chromatographic media with a particle size greater than 15 ⁇ m should be packed in a constant flow or variable flow method according to the column bed and the pressure resistance of the chromatographic medium;
- Homogenate water, saline, water containing organic phase or buffer salt, etc., 20-80% (v:v) solid content
- Chromatographic column material stainless steel, glass, plastic, SPE tube
- Chromatographic column cleaning conditions long-term use of the chromatographic column will adsorb some impurities that are difficult to clean. These impurities will affect the performance of the chromatographic column, and these impurities need to be cleaned regularly. Different impurity cleaning methods are different. Generally, 0.5M HCl or 0.5-1.0M NaOH is used for impurities, and the impurities with strong hydrophobic binding can be cleaned with 0.1-1% Tween or Triton X-100. Alternatively, the chromatographic column can be flushed with 0.5M NaOH, and the chromatographic column can be stored in a 20% ethanol aqueous solution at room temperature.
- the chromatographic column is stored in a 20% ethanol aqueous solution at room temperature.
- the chromatographic medium is stored in an aqueous solution containing 20% ethanol at 2-8°C.
- the present invention also relates to the chromatographic medium as described above, or the use of the chromatographic separation device as described above for separating or enriching boron-containing substances from a liquid medium.
- the removal of boron-containing substance pairs includes: separation and purification between boron-containing molecules and non-boron molecules (affinity chromatography) and separation and purification between different boron-containing molecules (not limited to the use of affinity chromatography media).
- the chromatographic medium adsorbs the boron-containing substance under alkaline conditions, and elutes the boron-containing substance under acidic conditions.
- the pH under alkaline conditions may be >7.0, preferably pH>7.5, more preferably pH>8.0.
- the pH under acidic conditions may be ⁇ 7.0, preferably pH ⁇ 6.0, and more preferably pH ⁇ 5.0.
- a carbohydrate-containing mobile phase (such as sorbitol) can also be used to elute the boron-containing material.
- the boron-containing substance includes all of a sudden:
- BNCT Boron compound carriers for neutron capture tumor therapy (Boron Neutron Capture Therapy, BNCT), such as boron-containing acridine derivatives;
- the chemical reaction is preferably used for the reaction of solid surfaces or polymers and soluble boron-containing reagents.
- boron compound used in the boron selection reaction for molecular modular synthesis is well known to those skilled in the art, such as “Liang Xu, et al., Chem. Soc. Rev., 2015, 44(24): 8848-8858 "And “Raphael Oeschger, et al., Science, 2020, 368, 736-741” and other articles described in the boron compound.
- Boron-containing intermediates used in organic synthesis such as organic halides containing masked borane groups and di- or poly-boron compounds; boron selective reactions refer to chemical transformations in which two or more boranes are present in the reactants Groups, and distinguish their reactivity; therefore, the more reactive boryl groups can be selectively converted, while the relatively inert boryl groups remain intact.
- Inert borane groups can usually be used under different reaction conditions and converted into other functional groups, so they can provide a continuous and flexible synthesis method for complex molecules.
- porous polymethacrylate microspheres produced by Suzhou Saifen Technology Co., Ltd. (the particle size is 30 ⁇ m, and the pore size is Item No. 281030952), its surface contains a lot of epoxy groups.
- the chromatographic medium prepared in this embodiment contains a cis-diol structure that has an adsorption effect on boron-containing molecules under alkaline conditions, and the sample molecules are eluted under acidic conditions.
- the scanning electron microscope image of the embodiment is shown in Fig. 1, and the particle size distribution is shown in Fig. 2.
- porous polymethacrylate microspheres produced by Suzhou Saifen Technology Co., Ltd. (the particle size is 30 ⁇ m, and the pore size is Item No. 281030952), its surface contains a lot of epoxy groups.
- porous polymethacrylate microspheres produced by Suzhou Saifen Technology Co., Ltd. (the particle size is 30 ⁇ m, and the pore size is Item No. 2813030502), its surface contains a lot of epoxy groups.
- porous polymethacrylate microspheres produced by Suzhou Saifen Technology Co., Ltd. (the particle size is 30 ⁇ m, and the pore size is Item No. 281030502), its surface contains a lot of epoxy groups.
- porous polymethacrylate microspheres (with a particle size of 60 ⁇ m and a pore size of 60 ⁇ m) produced by Suzhou Saifen Technology Co., Ltd. are used Item No. 281060952), its surface contains a lot of epoxy groups.
- porous polymethacrylate microspheres (with a particle size of 60 ⁇ m and a pore size of 60 ⁇ m) produced by Suzhou Saifen Technology Co., Ltd. are used Item No. 281060952), its surface contains a lot of epoxy groups.
- porous polymethacrylate microspheres (with a particle size of 10 ⁇ m and a pore size of 10 ⁇ m) produced by Suzhou Saifen Technology Co., Ltd. are used Item No. 281010952), its surface contains a lot of epoxy groups.
- porous polymethacrylate microspheres (with a particle size of 10 ⁇ m and a pore size of 10 ⁇ m) produced by Suzhou Saifen Technology Co., Ltd. are used Item No. 281010952), its surface contains a lot of epoxy groups.
- This example is a comparative example.
- the chromatographic medium has common cis-diol groups on the surface, but has not been further modified.
- Adopt porous polymethacrylate microspheres produced by Suzhou Saifen Technology Co., Ltd. (particle size is 30 ⁇ m, pore size is Product number: 281030952), its surface contains a large number of epoxy groups, and the epoxy groups are ring-opened to form a cis-diol group.
- the porous polystyrene divinylbenzene produced by Suzhou Saifen Technology Co., Ltd. (the particle size is 15 ⁇ m, and the pore size is Item No.: 221715952), its surface contains a lot of epoxy groups.
- the porous polystyrene divinylbenzene produced by Suzhou Saifen Technology Co., Ltd. (the particle size is 15 ⁇ m, and the pore size is Item No.: 221715952), its surface contains a lot of epoxy groups.
- a non-porous polystyrene divinylbenzene (particle size of 5 ⁇ m, non-porous, article number: 221605002) produced by Suzhou Saifen Technology Co., Ltd. is used, and its surface contains a large amount of epoxy groups.
- a non-porous polystyrene divinylbenzene (particle size of 5 ⁇ m, non-porous, article number: 221605002) produced by Suzhou Saifen Technology Co., Ltd. is used, and its surface contains a large amount of epoxy groups.
- a non-porous polystyrene divinylbenzene (particle size of 10 ⁇ m, non-porous, article number: 22161002) produced by Suzhou Saifen Technology Co., Ltd. is used, and its surface contains a large amount of epoxy groups.
- a non-porous polystyrene divinylbenzene (particle size of 10 ⁇ m, non-porous, article number: 22161002) produced by Suzhou Saifen Technology Co., Ltd. is used, and its surface contains a large amount of epoxy groups.
- porous polymethacrylate microspheres produced by Suzhou Saifen Technology Co., Ltd. (the particle size is 30 ⁇ m, and the pore size is Item No. 281030952), its surface contains a lot of epoxy groups.
- porous polymethacrylate microspheres produced by Suzhou Saifen Technology Co., Ltd. (the particle size is 30 ⁇ m, and the pore size is Item No. 281030952), its surface contains a lot of epoxy groups.
- porous polymethacrylate microspheres produced by Suzhou Saifen Technology Co., Ltd. (the particle size is 30 ⁇ m, and the pore size is Item No.: 280130950), its surface contains a lot of hydroxyl groups.
- porous polymethacrylate microspheres produced by Suzhou Saifen Technology Co., Ltd. (the particle size is 30 ⁇ m, and the pore size is Item No.: 280130950), its surface contains a lot of hydroxyl groups.
- porous polymethacrylate microspheres produced by Suzhou Saifen Technology Co., Ltd. (the particle size is 30 ⁇ m, and the pore size is Item No. 281030952), its surface contains a lot of epoxy groups.
- porous polymethacrylate microspheres produced by Suzhou Saifen Technology Co., Ltd. (the particle size is 30 ⁇ m, and the pore size is Item No.: 280130950), its surface contains a lot of hydroxyl groups.
- Example 1 and Example 9 were respectively packed into a 4.6*50mm stainless steel chromatographic column, and the affinity chromatographic medium modified by the method of the present invention was compared with the ordinary diol with adjacent diol.
- the adsorption and desorption of boron-containing molecules by the chromatography medium were respectively packed into a 4.6*50mm stainless steel chromatographic column, and the affinity chromatographic medium modified by the method of the present invention was compared with the ordinary diol with adjacent diol. The adsorption and desorption of boron-containing molecules by the chromatography medium.
- the selected samples are: phenylboronic acid (boron-containing molecule); ribonuclease A (boron-free molecule)
- Example 1 The separation effect of affinity chromatography medium (affinity chromatography medium modified by the present invention) on phenylboronic acid and ribonuclease A is shown ( Figure 3):
- the chromatographic medium synthesized in Example 1 has a strong adsorption effect on phenylboronic acid under alkaline conditions.
- the phenylboronic acid molecule binds firmly to the chromatography medium, and the phenylboronic acid can only be eluted under acidic conditions.
- the chromatographic medium synthesized in Example 1 has no adsorption effect on ribonuclease A under alkaline conditions. Ribonuclease A is directly carried out by the alkaline mobile phase.
- the chromatographic medium synthesized in Example 1 can completely separate phenylboronic acid from ribonuclease.
- the purity of phenylboronic acid is more than 95%, and the yield is more than 90%.
- Example 9 (comparative example) chromatographic medium (ordinary chromatographic medium with diol group): the adsorption result of p-phenylboronic acid shows ( Figure 4):
- Example 9 The chromatographic medium synthesized in Example 9 (comparative example) has an adsorption effect on phenylboronic acid under alkaline conditions. But it has already begun to be eluted under alkaline conditions.
- Example 9 The chromatographic medium synthesized in Example 9 (comparative example) has weaker adsorption of boron-containing molecules than the chromatographic medium of Example 1.
- the chromatographic medium synthesized by the method of Example 1 and Example 9 was packed into a 4.6*50mm stainless steel chromatographic column, and the chromatographic column was used to separate the target molecule from the crude product containing impurities. Test the adsorption effect of the chromatography medium on the bortezomib molecule.
- the specific separation conditions are as follows:
- Sample sample solution containing bortezomib molecule
- Loading solution adjust the pH value of the sample solution to 8.0
- Example 1 shows the separation effect of the affinity chromatography medium (affinity chromatography medium modified by the present invention) on the bortezomib molecule and its impurities (Figure 5):
- the chromatographic medium synthesized in Example 1 has a strong adsorption effect on the bortezomib molecule under alkaline conditions.
- the bortezomib molecule binds firmly to the chromatographic medium, and it can only be eluted under acidic conditions.
- the chromatographic medium synthesized in Example 1 has no adsorption effect on most of the impurities in the sample under alkaline conditions. Impurities are directly carried out by the alkaline mobile phase.
- the chromatographic medium synthesized in Example 1 can separate the bortezomib molecule and its impurities.
- the purity of bortezomib is more than 95%, and the yield is more than 90%.
- Example 9 The chromatographic medium synthesized in Example 9 (comparative example) has an adsorption effect on bortezomib under alkaline conditions. But it has already begun to be eluted under alkaline conditions.
- Example 9 The chromatographic medium synthesized in Example 9 (comparative example) has weaker adsorption effect on bortezomib than the chromatographic medium of Example 1.
- Example 1 The chromatographic medium in Example 1 has multiple synergistic effects to make it easier for the ortho-position polyhydroxyl to combine with the boron atom in space and conformation to form an effective coordination, and the purity and yield of bortezomib are high;
- Example 9 (comparative example)
- the surface of the chromatographic medium has only ordinary cis-diol groups, which are not easy to combine with boron atoms to form effective coordination.
- the results of the separation and purification experiment are worse than those of Example 1.
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- Solid-Sorbent Or Filter-Aiding Compositions (AREA)
Abstract
一种用于分离含硼物质的层析介质,所述层析介质包括基体支持物和接枝于所述基体支持物上的接枝链;所述接枝链的通式为MA-R 1-X 1-R 2或式(I)所示;其中MA为可以共价或非共价键与其它化学官能团连接的基团;R 1、R 2和R 3独立的选自氢原子、饱和烃基、不饱和烃基或含非碳电负性杂原子的饱和烃基,并且其中至少一个带有顺式邻位多羟基,顺式邻位多羟基为直链骨架结构或环链骨架结构;X 1和X 2分别为所述非碳电负性杂原子中二价和三价的杂原子;所述接枝链中R 1、R 2和R 3上带有的碳原子数量之和>2个。
Description
本发明涉及层析介质领域,具体而言,涉及一种用于分离含硼物质的层析介质。
硼砂在我国古代常作药用。中医中有很多对其药性的描述:硼砂,又名月石,味甘、咸,性凉,归肺、胃经。具有清热解毒、消肿防腐、清肺化痰的功效,用于咽喉肿痛、口舌生疮、目赤肿痛、翳障、痰热咳嗽等。硼原子具有与其他药物分子中常带有的原子不同的独特性质。硼原子中心可以从sp
2杂化的三角平面变成sp
3杂化的四面体,含硼化合物增加了药物分子结构的多样性。这类化合物以其全新的结构和特殊的作用机制,成为药物研发的新途径。随着硼化学的发展,近年来越来越多具有较好生物活性的有机硼化合物被相继合成。它们在抗肿瘤、抗感染以及抑制凝血酶和二肽基肽酶活性等方面都有较好的效果。
现已有许多种类的含硼药物产业化,商业价值高。包括他伐硼罗(Tavaborole,商品名为Kerydin)、克立硼罗(Crisaborole,商品名为Eucrisa)、VABOMERE复方注射剂、环磷酸腺苷硼酸络合物、含硼吖啶衍生物、含硼酸的试剂和寡核苷酸、硼替佐米(Bortezomib,商品名为Velcade)、依沙佐米(Lxazomib,商品名Ninlaro)、依沙佐米(Ixazomib,商品名Ninlaro)。
其中他伐硼罗和克立硼罗是出自Anacor公司,分别于2014和2016年经美国FDA批准为外用的新分子实体,分别用于治疗甲癣(灰趾/指甲)和过敏性皮炎(湿疹)。VABOMERE由Meropenem(美罗培南,一种抗菌药物)和Vaborbactam(可以抑制某些细菌的耐药性)组成,2017年年经美国FDA批准用于用于成人复杂性尿路感染的治疗。Vaborbactam是一种环状硼酸药效团β-内酰胺酶抑制剂,VABOMERE可以应对产生了β-内酰胺酶的革兰氏阴性菌的感染。三个药物都是首创的有机硼酸酯。而硼替佐米、依沙佐米也是一种含有二肽基的有机硼药物。硼替佐米在2003年上市用于治疗多发性骨髓瘤,为蛋白酶体抑制剂;依沙佐米在2015年上市作为首个口服药物用于治疗多发性骨髓瘤,为第二代蛋白酶体抑 制剂。
作为药物,质量控制的最关键点就是药物纯度的控制,所以药物在生产中的分离纯化是关键工序,分离效率及工艺不仅影响药物质量也影响药物生产成本。目前,这些含硼药物分子的生产提纯方法一般为结晶提纯和反相层析纯化。此类提纯方法的优点是操作简单,却有几个严重的缺点:提纯过程中经常需要用到大量有机溶剂,这些有机溶剂在使用过程中可能存在失火、爆炸等安全隐患;有机溶剂也容易残留在药品中对人体有一定的伤害;后续有机溶剂和反相层析硅胶介质的处理会对环境造成巨大的损害;且此方法的生物相容性低,容易引起生物类药品的变性失活。
发明内容
本发明的目的在于填补现有纯化技术的空白,提供一种分离或富集含硼的小分子、寡核苷酸、多肽和蛋白质的亲和层析介质;
本发明涉及一种用于分离含硼物质的的层析介质,所述层析介质包括基体支持物和接枝于所述基体支持物上的接枝链;本发明另一目的是提供上述亲和层析介质的制备方法;以上亲和层析介质作为色谱柱子的分析应用;以上亲和层析介质在工业分离纯化上面的应用。以此制备的层析介质分离效率高、分离效果好、综合总成本低,适合工业化大规模分离纯化含硼的小分子、寡核苷酸、多肽和蛋白质等。
具体的,本发明所提供的用于分离含硼物质的的层析介质包括基体支持物和接枝于所述基体支持物上的接枝链;
其中MA为可以共价或非共价键与其它化学官能团连接的基团;
R
1、R
2和R
3独立的选自氢原子、饱和烃基、不饱和烃基或含非碳电负性杂原子的饱和烃基,并且其中至少一个带有顺式邻位多羟基,顺式邻位多羟基为直链骨架结构或环链骨架结构;
X
1和X
2分别为所述非碳电负性杂原子中二价和三价的杂原子;
所述接枝链中R
1、R
2和R
3上带有的碳原子数量之和>2个。
本发明还涉及一种层析分离装置,其含有如上所述的层析介质。
根据本发明的再一方面,本发明还涉及如上所述的层析介质,或如上所述的色谱柱用于从液体介质分离或富集含硼物质的用途。
本发明的有益效果为:
1)对含硼分子有特异性结合作用;
2)分离和纯化的条件温和/简单,生物相容性高,可以较好的保持纯化分子的生物活性;
3)纯化层析介质和色谱柱子经过清洗处理后,可以反复多次使用;
4)不需要使用大量有机试剂;
5)产生的废液少,容易处理,减少环境污染。
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案,下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图是本发明的一些实施方式,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1为本发明一个实施例中层析介质的扫描电镜图;
图2为本发明一个实施例中层析介质的粒径分布图;
图3为本发明一个实施例中层析介质对含硼分子与不含硼分子的分离效果色谱图;
图4为本发明一个对照实施例中层析介质对含硼分子的吸附效果色谱图谱;
图5为本发明一个实施例中层析介质对硼替佐米样品的分离纯化色谱图;
图6为本发明一个对照实施例中层析介质对硼替佐米样品的分离色谱图谱。
现将详细地提供本发明实施方式的参考,其一个或多个实例描述于下文。提供每一实例作为解释而非限制本发明。实际上,对本领域技术人员而言,显而易见的是,可以对本发明进行多种修改和变化而不背离本发明的范围或精神。例如,作为一个实施方式的部分而说明或描述的特征可以用于另一实施方式中, 来产生更进一步的实施方式。
因此,旨在本发明覆盖落入所附权利要求的范围及其等同范围中的此类修改和变化。本发明的其它对象、特征和方面公开于以下详细描述中或从中是显而易见的。本领域普通技术人员应理解本讨论仅是示例性实施方式的描述,而非意在限制本发明更广阔的方面。
本发明涉及一种用于分离含硼物质的的层析介质,所述层析介质包括基体支持物和接枝于所述基体支持物上的聚合物链;
其中MA为可以共价或非共价键与其它化学官能团连接的基团;
R
1、R
2和R
3独立的选自氢原子、饱和烃基、不饱和烃基或含非碳电负性杂原子的饱和烃基,并且其中至少一个带有顺式邻位多羟基,顺式邻位多羟基为直链骨架结构或环链骨架结构;
X
1和X
2分别为所述非碳电负性杂原子中二价和三价的杂原子;
所述接枝链中R
1、R
2和R
3上带有的碳原子数量之和>2个。
本发明分离原理是基于硼酸与顺式二醇之间快速稳定的键结合形成硼酸酯,硼酸酯的共价B-O键很稳定,但是在某些条件或者某些外部刺激物作用下,B-O键的形成是可逆的。此原理在硼酸亲和层析上有较广泛的应用,即在基质骨架上接上苯硼酸来亲和吸附带顺式二醇键的分子。本发明创新性地开发了一个是基于以上原理的反向应用,即将带顺式邻位多羟基的基团键合至基质骨架上来亲和吸附含硼的分子。本发明巧妙地将带顺式邻位多羟基的基团通过一个带电负性的非碳X(X
1或X
2)杂原子键合至基质骨架;通过这个X杂原子接入一个合适长度的间隔臂来增加邻位多羟基(活性基团)的柔韧性,以便使邻位多羟基(活性基团)更容易与硼原子在空间上结合形成有效的配位;X杂原子上的孤对电子一方面可以与活性基团中的羟基形成分子内氢键,邻位多羟基形成有利于结合硼原子的顺式构象;X杂原子上的孤对电子另一方面可以与硼原子相互作用,把含硼原子的分子拉近到带顺式邻位多羟基的基团的周围;多种作用协同使邻位多羟基在空间和构象上更容易与硼原子结合形成有效的配位。
在一些实施方式中,所述非碳电负性杂原子带强电负性。
在一些实施方式中,所述非碳电负性杂原子的鲍林电负性数值均大于2.0,优选为大于2.1,进一步优选为大于2.5,更优选为大于2.9。
在一些实施方式中,所述非碳电负性杂原子选自O、S、N、P中的一种或多种。
在一些实施方式中,所述含非碳电负性杂原子的饱和烃基可以为多胺,寡聚亚胺,多醚,寡聚醚,多硫醚,寡聚硫醚;更优选为多胺和寡聚亚胺,例如:乙二胺,二亚乙基三胺,二亚丙基三胺,三亚乙基四胺,直链和带有枝链的寡聚亚乙基亚胺。
在一些实施方式中,X
1选自O或S原子。
在一些实施方式中,X
2选自N或P原子。
顺式邻位多羟基的数目大于2个,例如3、4、5、6、7、8、9、10或更多个。
在一些实施方式中,所述接枝链中R
1、R
2和R
3上带有的碳原子数量之和为3~40个,例如3、4、5、6、7、8、9、10、11、12个,还可以选择13、14、15、20、25、30、35个。
饱和烃基或不饱和烃基可以为C
x-C
y烷基。术语“C
x-C
y烷基”是指在支链或非支链的烃基中具有x到y(包括x和y在内的)的碳原子的烷基。作为说明,但不限制,术语“C
1-C
4烷基”是指具有从1到4的碳原子的直链或支链烃部分,包括甲基、乙基、正丙基、异丙基、正丁基、异丁基、仲丁基以及叔丁基。术语“C
1-C
4正烷基”是指具有从1至4碳原子的直链烃部分,包括甲基、乙基、正丙基以及正丁基。C
x-C
y中x可以从1至10而y是从2至20。每个烷基、环烷基可以为诸如但不限于本文中说指明那样的可选取代的。在一些实施方式中,基团为单或二基取代的。在一些实施方式中,烷基为C
1-C
3、C
1-C
4、C
1-C
6、C
4-C
6或C
1-C
10烷基。
在一些实施方式中,所述环链骨架结构还可以为环烷基。例如但不限于环丙基甲基、二甲基环丙基、环丙基乙基、环丙基丙基、环丙基丁基、环丁基甲基、环丁基乙基、环丁基丙基、环戊基甲基、环戊基乙基、环戊基丙基、环己基甲基、环己基乙基以及环己基丙基。
每个烷基、环烷基可以为诸如但不限于本文中说指明那样的可选取代或可 修饰的。在一些实施方式中,基团为单或二基取代的。
在一些实施方式中,所述环链骨架结构为单糖,二糖或多糖;
在一些实施方式中,所述环链骨架结构优选为甘露糖、果糖、半乳糖、含甘露糖的二糖以及含甘露糖的寡聚糖。
基体支持物包含可被化学修饰的位点MA。可以以共价或非共价键与其它化学官能团连接。这些修饰位点包括但不限于以下化学官能团:MA选自环氧基、氨基、醛、羟基、羧基、氧代基团和硫醇基团中的至少一种。修饰位点通常还包含具有这些相应化学官能团的衍生基团,例如:氨基衍生物,硫醇衍生物,醛衍生物,甲酰基衍生物,生物素衍生物,炔烃衍生物,羟基衍生物,活化的羟基或衍生物,羧酸盐衍生物,活化的羧酸盐衍生物,活化的碳酸盐,活化的酯,NHS酯(琥珀酰亚胺基),NHS碳酸盐(琥珀酰亚胺基),亚氨基酯或衍生物,氰基溴化物衍生物,马来酰亚胺衍生物,卤代乙酰基衍生物,碘乙酰胺/碘乙酰基衍生物,环氧衍生物,链霉亲和素衍生物,Tresyl衍生物,二烯/共轭二烯衍生物(Diels-Alder类型反应),烯烃衍生物,取代的磷酸盐衍生物,溴代醇/卤代醇,取代的二硫化物,吡啶基-二硫化物衍生物,氮内酯,氢化物衍生物,卤代苯衍生物,核苷衍生物,支化/多功能连接分子,树枝状分子及衍生物,核苷衍生物,或其任何组合。
在一些实施方式中,这些层析介质表面化学修饰位点的密度是可优化的。例如,>0.1mmol/ml介质,优选为>0.5mmol/ml介质,进一步优选为>1.0mmol/ml介质,更优选为>2.0mmol/ml介质。
在一些实施方式中,R
1、R
2和R
3中至少一个带有顺式邻多醇;优选为顺式邻二醇;更优选为末端顺式邻二醇;
在一些实施方式中,R
1、R
2和R
3中至少一个带有邻苯二酚,例如儿茶酚同系物。
在一些实施方式中,所述基体支持物包括硼硅酸盐玻璃、琼脂、琼脂糖、琼脂糖衍生物、磁珠、二氧化硅、二氧化钛、藻酸盐、纤维素、纤维素衍生物、葡聚糖、淀粉、环糊精、壳聚糖、角叉菜胶、瓜尔胶、阿拉伯胶、印度胶、黄蓍胶、刺梧桐树胶、槐豆胶、黄原胶、果胶、粘蛋白、肝硫酯和明胶、硅、陶瓷、玻璃、聚氨酯、聚苯乙烯、聚苯乙烯二乙烯基苯、聚甲基丙烯酸甲酯、聚 丙烯酰胺、聚乙二醇对苯二甲酸酯、聚乙酸乙烯酯、聚乙烯、聚丙烯、聚氯乙烯、聚乙烯吡咯烷酮中的任一种,或任意几种形成的共聚物。
基体支持物可以根据应用从一系列选项中选择合适的形状,形式,材料和变型。该基体支持物表面可以是基本平坦的或平面的。也可以是圆形等形状。该基体支持物表面的形状包括但不限于孔,凹陷,柱,脊,通道,膜或类似物。基体支持物优选表面基本平坦的颗粒物。颗粒物的形状优选为球形或者基本球形。
在一些实施方式中,所述基体支持物为颗粒状,颗粒尺寸范围为1μm~500μm。
在一些实施方式中,颗粒尺寸范围为1μm~300μm;
在一些实施方式中,颗粒尺寸范围为2μm~200μm。
进一步的,所述基体支持物可为无孔的,或者包括一个或多个孔隙。特别可以为多孔树脂,多孔树脂的例子可以包括:其他基于琼脂糖的树脂(例如,Sepharose Fast Flow系列(GE)),聚甲基丙烯酸酯:(Sepax PGMA系列),聚苯乙烯二乙烯基苯(Sepax PolyRP和Sepax Proteomix系列),二氧化硅(Sepax Silica系列),可控孔度玻璃,葡聚糖衍生物,聚丙烯酰胺衍生物,和/或其他聚合物;或它们的任意组合。无孔树脂可以为以上多孔树脂相同的基质材料制备。
进一步的,用赛分科技有限公司不同种类层析介质产品举例说明,这些层析介质包括但不限于列举的这些种类:
(a)已共价连接有环氧基团的层析介质:Polar MC-Epoxy(多孔多分散聚甲基丙烯酸酯系列),Monomix MC-Epoxy(多孔单分散聚甲基丙烯酸酯系列),Proteomix Epoxy(无孔单分散交联聚苯乙烯系列),Proteomix POR-Epoxy(多孔单分散交联聚苯乙烯系列)。
(b)已共价连接有其他基团的层析介质:包括但不限于-OH(羟基),-CHO (醛基),IEX(阴离子,阳离子交换基团),HIC(疏水基团),IMAC(金属螯合基团),Affinity(亲和作用基团),Hydrophilic-Hydrophobic balanced(亲水疏水两性基团),Mixed-mode(混合作用基团)。
本发明使用含有如上所述的层析介质可以开发出多种层析分离装置。层析分离装置种类较多,包括但不限于以下种类:SPE固相萃取柱,带分离膜的离心管,磁珠,分离膜(membranes),快速检测生物芯片(bio-chips),试剂盒,纤维束柱,整体柱及常规分析级或制备级层析色谱柱。
层析分离装置优选为柱状,并由多个必要组成要素组成:层析介质材料,层析介质的粒径和孔径,柱子的基质材料,柱子的尺寸,装柱子方法。使用本发明层析介质填装成的色谱柱可以有多种物理形式,由不同的色谱柱必要组成要素组合构成。
装柱子方法:恒流/变流/恒压/多级调压力/DAC。
根据介质粒径、孔径等性能及色谱柱规格选择不同的装柱方法。
粒径小于15μm层析介质多用恒流、恒压方法装柱;
粒径大于15μm层析介质根据柱床及层析介质耐压性能选择恒流或变流方法装柱;
大规格色谱柱多用多级调压力或DAC方法装柱。
匀浆液:水,盐水,含有机相的水或缓冲盐等,20-80%(v:v)固含量
根据色谱柱规格及层析介质性能选择合适的匀浆流动相及匀浆体积
色谱柱子材料:不锈钢,玻璃,塑料,SPE管
不同材质的柱管耐压性能不同,在色谱柱填装时注意控制压力不但不能超过填料的最高耐压,同时不超过柱管材质的最大耐压。
色谱柱子清洗条件:色谱柱使用时间长会吸附一些难以清洗的杂质,这些杂质会影响色谱柱性能,需要定期将这些杂质清洗掉。不同杂质清洗方法不同,一般杂质用0.5M HCl或0.5-1.0M NaOH,强疏水性结合的杂质可用0.1-1%的吐温或Triton X-100清洗。或者可以用0.5M NaOH冲洗色谱柱,并将色谱柱在室温条件下保存于20%乙醇水溶液中。
色谱柱子保存方法:
色谱柱在含20%乙醇水溶液中,室温下保存。
层析介质在含20%乙醇水溶液中,2-8℃保存。
根据本发明的再一方面,本发明还涉及如上所述的层析介质,或如上所述的层析分离装置用于从液体介质分离或富集含硼物质的用途。
含硼物质对移出包括:含硼分子和不含硼分子之间的分离纯化(亲和层析)及不同含硼分子之间的分离纯化(不限于亲和层析介质的使用方法)。
在一些实施方式中,所述层析介质在碱性条件下吸附所述含硼物质,在酸性条件下洗脱所述含硼物质。
碱性条件的pH可以为>7.0,优选为pH>7.5,更优选为pH>8.0。
酸性条件的pH可以为<7.0,优选为pH<6.0,更优选为pH<5.0。
另外在Mg2+离子存在的情况下,含硼物质与顺式二醇基之间的相互作用会增强。含碳水化合物的流动相(例如山梨糖醇)也可以用来洗脱所述含硼物质。
在一些实施方式中,所述含硼物质包括一下子:
a)用于蛋白酶体抑制剂含硼化合物,例如硼替佐米、依沙佐米;
b)含硼的抗真菌、细菌、病毒的有机硼酸类药物分子,例如他伐硼罗、克立硼罗和Vaborbactam-Meropenem;
c)用于中子俘获肿瘤治疗(Boron Neutron Capture Therapy,BNCT)中硼化合物载体,例如含硼吖啶衍生物;
d)具有凝血酶抑制活性的含硼化合物;
e)具有治疗作用的含硼寡核苷酸、多肽、蛋白;
f)用于分子模块化合成的硼选择反应中的硼化合物。
g)用于化学反应中未反应的或者过量的含硼试剂的回收,所述化学反应优选用于固体表面或者聚合物和可溶性含硼试剂反应。
用于分子模块化合成的硼选择反应中的硼化合物是本领域技术人员所共知的,例如“Liang Xu,et al.,Chem.Soc.Rev.,2015,44(24):8848-8858”和“Raphael Oeschger,et al.,Science,2020,368,736–741”等文章中所记载的硼化合物。有机合成用到的含硼的中间体例如含有被掩蔽的硼烷基和二或多硼化合物的有机卤化物;硼选择性反应是指化学转化,其中反应物中存在两个或多个硼烷基,并且可以区分它们的反应性;因此,反应性更高的硼烷基可以被选择性地转化, 而相对惰性的硼烷基则保持完整。惰性硼烷基通常可在不同的反应条件下使用并转化为其他官能团,因此可为复杂分子提供连续且灵活的合成方法。
下面将结合实施例对本发明的实施方案进行详细描述。
实施例1
胺二醇实施例,由介质表面环氧基接入
1)以100g表面带有环氧基的层析介质计,加入60.0g质量分数为65-70%的乙胺水溶液,加入反应装置在室温下机械搅拌反应16h;
2)反应结束后用去离子水抽滤洗涤介质至中性;然后将层析介质转移至300ml 0.05M盐酸溶液中,充分搅拌。用pH试纸蘸取少量液体检测pH<3,若未达到继续加盐酸溶液搅拌直至pH<3后过滤抽干;用去离子水抽滤洗涤介质至中性;
3)将层析介质转移至500ml 0.1M碳酸氢钠水溶液中,充分搅拌。用pH试纸蘸取少量液体检测pH>8,若未达到继续加碳酸氢钠水溶液搅拌直至pH>8后过滤抽干;用纯化水多次洗涤至pH试纸呈中性,抽干,直至无滤液出来。
4)将上述介质,加入150ml溶液含50%缩水甘油和2.0g氢氧化钠加入反应装置,在室温且避光条件下机械搅拌反应2h,反应结束后用至少10倍体积去离子水抽滤洗涤介质。
本实施例制备的层析介质含有顺式二醇结构在碱性条件下对含硼分子有吸附作用,且在酸性条件下样品分子被洗脱下来。
实施例电镜扫描图如图1所示,粒径分布如图2所示。
实施例2
以100g表面带有环氧基的层析介质计,加入276g质量分数为30%的甲胺水溶液,加入反应装置,在室温下机械搅拌反应16h;其他步骤参考实施例1。
实施例3
以100g表面带有环氧基的层析介质计,加入276g质量分数为30%的甲胺水溶液,加入反应装置,在室温下机械搅拌反应16h;其他步骤参考实施例1。
实施例4
以100g表面带有环氧基的层析介质计,加入276g质量分数为30%的甲胺水溶液,加入反应装置,在室温下机械搅拌反应16h;其他步骤参考实施例1。
实施例5
以100g表面带有环氧基的层析介质计,加入60.0g质量分数为65-70%的乙胺水溶液,加入反应装置,在室温下机械搅拌反应16h;其他步骤参考实施例1。
实施例6
以100g表面带有环氧基的层析介质计,加入276g质量分数为30%的甲胺水溶液,加入反应装置,在室温下机械搅拌反应16h;其他步骤参考实施例1。
实施例7
以100g表面带有环氧基的层析介质计,加入60.0g质量分数为65-70%的乙胺水溶液,在室温下机械搅拌反应16h;其他步骤参考实施例1。
实施例8
以100g表面带有环氧基的层析介质计,加入165.6g质量分数为30%的甲胺水溶液,加入反应装置,在室温下机械搅拌反应16h;其他步骤参考实施例1。
实施例9(对照实施例)
本实施例为对照实施例,层析介质表面有普通的顺式二醇基团,但是未经过进一步修饰。采用苏州赛分科技有限公司生产的多孔聚甲基丙烯酸酯微球(粒径为30μm,孔径为
货号:281030952),其表面含有大量环氧基,环氧基开环形成顺式二醇基团。
以100g表面带有环氧基的层析介质计,加入300ml 0.5M硫酸,加入反应装置,在40℃条件下机械搅拌反应2h;反应结束后用去离子水抽滤洗涤介质至中性。
实施例10
以100g表面带有环氧基的层析介质计,加入60.0g质量分数为65-70%的乙胺水溶液,加入反应装置,在室温下机械搅拌反应16h;其他步骤参考实施例1。
实施例11
以100g表面带有环氧基的层析介质计,加入165.6g质量分数为30%的甲胺水溶液,加入反应装置,在室温下机械搅拌反应16h;其他步骤参考实施例1。
实施例12
本实施例采用苏州赛分科技有限公司生产的无孔聚苯乙烯二乙烯基苯(粒径为5μm,无孔,货号:221605002),其表面含有大量环氧基。
以100g表面带有环氧基的层析介质计,加入60.0g质量分数为65-70%的乙胺水溶液,加入反应装置,在室温下机械搅拌反应16h;其他步骤参考实施例1。
实施例13
本实施例采用苏州赛分科技有限公司生产的无孔聚苯乙烯二乙烯基苯(粒径为5μm,无孔,货号:221605002),其表面含有大量环氧基。
以100g表面带有环氧基的层析介质计,加入165.6g质量分数为30%的甲胺水溶液,加入反应装置,在室温下机械搅拌反应16h;其他步骤参考实施例1。
实施例14
本实施例采用苏州赛分科技有限公司生产的无孔聚苯乙烯二乙烯基苯(粒径为10μm,无孔,货号:221610002),其表面含有大量环氧基。
以100g表面带有环氧基的层析介质计,加入60.0g质量分数为65-70%的乙胺水溶液,加入反应装置,在室温下机械搅拌反应16h;其他步骤参考实施例1。
实施例15
本实施例采用苏州赛分科技有限公司生产的无孔聚苯乙烯二乙烯基苯(粒径为10μm,无孔,货号:221610002),其表面含有大量环氧基。
以100g表面带有环氧基的层析介质计,加入165.6g质量分数为30%的甲胺水溶液,加入反应装置,在室温下机械搅拌反应16h;其他步骤参考实施例1。
实施例16
硫醚二醇实施例,由介质表面环氧基接入
以20.0g表面带有环氧基的层析介质计,加入100ml 0.1M碳酸氢钠溶液中分散均匀,加入1-硫代甘油1.30g,加入反应装置,在室温下机械搅拌反应16h,反应结束后用去离子水抽滤洗涤介质至中性。
实施例17
醚二醇实施例,由介质表面环氧基接入
1)以20.0g表面带有环氧基的层析介质计,加入50ml 5M氢氧化钠水溶液中分散均匀,加入反应装置,升温至35℃,保持搅拌2h。
2)加入缩水甘油20.0g,在35℃温度下搅拌反应3h。加入100ml纯化水搅拌10min,反应结束后用去离子水抽滤洗涤介质至中性。
实施例18
多胺二醇实施例,由介质表面醛基接入
1)以100.0g表面带有羟基的层析介质计,100mL 0.2M NaIO
4溶液,室温下,100rpm机械搅拌反应1.5h,反应结束后用至少10倍体积去离子水抽滤洗涤微球。
2)然后加入100mL 0.1M磷酸盐缓冲溶液,含1.52M的二亚丙基三胺(CAS#56-18-8,pH值为7.2),同时加入0.63g NaCNBH
3,室温下,100rpm机械搅拌反应5h,反应结束后用至少10倍体积去离子水多次抽滤洗涤微球,至滤液pH试纸呈中性,抽干直至无滤液出来。
3)将上述介质,加入150ml溶液含50%缩水甘油和2.0g碳酸氢钠,按照实施例1中步骤(4)完成层析介质的制备。
实施例19
多胺二醇实施例,由介质表面醛基接入
1)以100.0g表面带有羟基的层析介质计,100mL 0.2M NaIO
4溶液,室温下,100rpm机械搅拌反应1.5h,反应结束后用至少10倍体积去离子水抽滤洗涤微球。
2)然后加入100mL 0.1M磷酸盐缓冲溶液,含1.50M的三亚乙基四胺(CAS#112-24-3),同时加入0.63g NaCNBH
3,室温下,100rpm机械搅拌反应5h,反应结束后用至少10倍体积去离子水多次抽滤洗涤微球,至滤液pH试纸呈中性,抽干直至无滤液出来。
3)将上述介质,加入150ml溶液含50%缩水甘油和2.0g碳酸氢钠,按照实施例1中步骤(4)完成层析介质的制备。
实施例20
胺甘露糖二醇实施例,由介质表面环氧基接入
1)以100g表面带有环氧基的层析介质计,按照实施例1中步骤(1)-(3),制备表面带有胺基的层析介质。
2)将上述介质,加入150ml N,N-二甲基甲酰胺溶液含25%α-D-吡喃甘露糖氯化物(2,3,4,6-四-O-乙酰-α-D-吡喃甘露糖氯化物,CAS#14257-40-0)和2.0g碳酸钠,在室温且避光条件下机械搅拌反应过夜,反应结束后用至少10倍体积去离子水抽滤洗涤介质。
3)加入300ml 0.5M硫酸,加入反应装置,在40℃条件下机械搅拌反应2h,反应结束后用去离子水抽滤洗涤介质至中性。
实施例21
多胺甘露糖二醇实施例,由介质表面醛基接入
1)以100.0g表面带有羟基的层析介质计,100mL 0.2M NaIO
4溶液,室温下,100rpm机械搅拌反应1.5h,反应结束后用至少10倍体积去离子水抽滤洗涤微球。
2)然后加入100mL 0.1M磷酸盐缓冲溶液,含1.50M的三亚乙基四胺(CAS#112-24-3),同时加入0.63g NaCNBH
3,室温下,100rpm机械搅拌反应5h,反应结束后用至少10倍体积去离子水多次抽滤洗涤微球,至滤液pH试纸呈中性,抽干直至无滤液出来,制备表面带有多胺基的层析介质。
3)将上述介质,加入150ml N,N-二甲基甲酰胺溶液含25%α-D-吡喃甘露糖氯化物(2,3,4,6-四-O-乙酰-α-D-吡喃甘露糖氯化物,CAS#14257-40-0)和2.0g碳酸钠,在室温且避光条件下机械搅拌反应过夜,反应结束后用至少10倍体积去离子水抽滤洗涤介质。
4)加入300ml 0.5M硫酸,加入反应装置,在40℃条件下机械搅拌反应2h,反应结束后用去离子水抽滤洗涤介质至中性。
实施例22
层析介质分离苯硼酸&核糖核酸酶A
将实例1和实施例9(对照实施例)方法合成的层析介质分别填装成4.6*50mm的不锈钢色谱柱,比较经过本发明方法修饰的亲和层析介质与带有邻二醇的普通层析介质对含硼分子的吸附--解吸附情况。
选用的样品为:苯硼酸(含硼的分子);核糖核酸酶A(不含硼的分子)
具体分离条件如下:
样品:核糖核酸酶A与苯硼酸混合液
平衡溶液:0.25M乙酸铵缓冲液(pH 8.0)
洗脱溶液:0.1M甲酸溶液
分离过程:
以平衡溶液冲洗平衡层析色谱柱;
空白溶剂、单标核糖核酸酶A、单标苯硼酸及混标核糖核酸酶A&苯硼酸上 样;
平衡溶液继续冲洗至基线平稳;
换洗脱溶液将介质吸附的苯硼酸洗脱下来;
用0.5M NaOH冲洗色谱柱;
(6)使用完色谱柱将色谱柱保存在20%乙醇水溶液中,室温条件下。
分离纯化实验结果:
实施例1亲和层析介质(经过本发明修饰后的亲和层析介质)对苯硼酸及核糖核酸酶A的分离效果显示(图3):
-实施例1合成的层析介质在碱性条件下对苯硼酸有较强的吸附作用。苯硼酸分子与层析介质结合牢固,需在酸性条件下才能将苯硼酸洗脱。
-实施例1合成的层析介质在碱性条件下对核糖核酸酶A没有吸附作用。核糖核酸酶A被碱性流动相直接带出。
-因此实施例1合成的层析介质可以将苯硼酸与核糖核酸酶完全分开。苯硼酸纯度>95%,收率>90%。
(2)实施例9(对照实施例)层析介质(带有二醇基的普通层析介质):对苯硼酸吸附结果显示(图4):
-实施例9(对照实施例)合成的层析介质对苯硼酸在碱性条件下有吸附作用。但是在碱性条件下已经开始被洗脱。
-实施例9(对照实施例)合成的层析介质对含硼分子吸附作用弱于实施例1的层析介质。
实施例23:
层析介质分离含硼肽类分子
将实例1和实施例9(对照实施例)方法合成的层析介质填装成4.6*50mm的不锈钢色谱柱,用此色谱柱从含杂质的粗品中分离目标分子。测试层析介质对硼替佐米分子的吸附效果。具体分离条件如下:
样品:含硼替佐米分子的样品溶液
上样溶液:将样品溶液pH值调至8.0
平衡溶液:0.25M乙酸铵缓冲液(pH 8.0)+20%甲醇
洗脱溶液:0.1M甲酸溶液+20%甲醇
(1)实施例1分离纯化过程:
以平衡溶液冲洗、平衡层析色谱柱;
样品溶液上样;
平衡溶液继续冲洗至基线平稳;
换洗脱溶液将介质吸附的硼替佐米分子洗脱下来;
用0.5M NaOH冲洗色谱柱;
使用完色谱柱将色谱柱保存在20%乙醇水溶液中,室温条件下。
分离纯化实验结果:
-实施例1亲和层析介质(经过本发明修饰后的亲和层析介质)对硼替佐米分子及其杂质的分离效果显示(图5):
-实施例1合成的层析介质在碱性条件下对硼替佐米分子有较强的吸附作用。硼替佐米分子与层析介质结合牢固,需在酸性条件下才能将其洗脱。
-实施例1合成的层析介质在碱性条件下对大部分样品中杂质没有吸附作用。杂质被碱性流动相直接带出。
-因此实施例1合成的层析介质可以将硼替佐米分子及其杂质分开。硼替佐米纯度>95%,收率>90%。
(2)实施例9(对照实施例)分离过程:
以平衡溶液冲洗平衡层析色谱柱;
样品溶液进样;
平衡溶液继续冲洗至基线平稳;
换洗脱溶液将介质吸附的硼替佐米分子洗脱下来;
用0.5M NaOH冲洗色谱柱;
使用完色谱柱将色谱柱保存在20%乙醇水溶液中,室温条件下。
分离纯化实验结果:
-实施例9(对照实施例)层析介质(带有二醇基的普通层析介质):对硼替佐米吸附结果显示(图6):
-实施例9(对照实施例)合成的层析介质对硼替佐米在碱性条件下有吸附作用。但是在碱性条件下已经开始被洗脱。
-实施例9(对照实施例)合成的层析介质对硼替佐米吸附作用弱于实施例1的层析介质。
(3)硼替佐米分离纯化实验结果分析
-实施例1中的层析介质在多种协同作用使邻位多羟基在空间和构象上更容易与硼原子结合形成有效的配位,硼替佐米的纯度和收率高;
-实施例9(对照实施例)层析介质表面只有普通的顺式二醇基团,该基团不容易与硼原子结合形成有效的配位,分离纯化实验的结果比实施例1的差。
以上所述实施例的各技术特征可以进行任意的组合,为使描述简洁,未对上述实施例中的各个技术特征所有可能的组合都进行描述,然而,只要这些技术特征的组合不存在矛盾,都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。因此,本发明专利的保护范围应以所附权利要求为准。
Claims (13)
- 根据权利要求1所述的层析介质,所述非碳电负性杂原子的鲍林电负性数值均大于2.0;优选的,所述非碳电负性杂原子选自O、S、N、P中的一种或多种;优选的,所述含非碳电负性杂原子的饱和烃基可以为多胺,寡聚亚胺,多醚,寡聚醚,多硫醚,寡聚硫醚;更优选为多胺和寡聚亚胺,例如:乙二胺,二亚乙基三胺,二亚丙基三胺,三亚乙基四胺,直链和带有枝链的寡聚亚乙基亚胺。
- 根据权利要求2所述的层析介质,X 1选自O或S原子。
- 根据权利要求2所述的层析介质,X 2选自N或P原子。
- 根据权利要求1所述的层析介质,MA选自环氧基、氨基、醛、羟基、羧基、氧代基团和硫醇基团中的至少一种。
- 根据权利要求1所述的层析介质,R 1、R 2和R 3中至少一个带有顺式邻多醇;优选为顺式邻二醇;更优选为末端顺式邻二醇;优选R 1、R 2和R 3中至少一个带有邻苯二酚,例如儿茶酚同系物;所述环链骨架结构优选为单糖,二糖或多糖;所述环链骨架结构优选为甘露糖、果糖、半乳糖、含甘露糖的二糖以及含甘露糖的寡聚糖。
- 根据权利要求1~6任一项所述的层析介质,所述基体支持物包括硼硅酸盐玻璃、琼脂、琼脂糖、琼脂糖衍生物、磁珠、二氧化硅、二氧化钛、藻酸盐、 纤维素、纤维素衍生物、葡聚糖、淀粉、环糊精、壳聚糖、角叉菜胶、瓜尔胶、阿拉伯胶、印度胶、黄蓍胶、刺梧桐树胶、槐豆胶、黄原胶、果胶、粘蛋白、肝硫酯和明胶、硅、陶瓷、玻璃、聚氨酯、聚苯乙烯、聚苯乙烯二乙烯基苯、聚甲基丙烯酸甲酯、聚丙烯酰胺、聚乙二醇对苯二甲酸酯、聚乙酸乙烯酯、聚乙烯、聚丙烯、聚氯乙烯、聚乙烯吡咯烷酮中的任一种,或任意几种形成的共聚物。
- 根据权利要求1~6任一项所述的层析介质,所述基体支持物为颗粒状,颗粒尺寸范围为1μm~500μm,优选1μm~300μm;更优选2μm~200μm。
- 层析分离装置,其含有权利要求1~9任一项所述的层析介质。
- 权利要求1~9任一项所述的层析介质,或权利要求10所述的层析分离装置用于从液体介质分离或富集含硼物质的用途。
- 根据权利要求11的用途,所述层析介质在碱性条件下吸附所述含硼物质,在酸性条件下洗脱所述含硼物质;优选的,用于洗脱所述含硼物质的流动相中含有碳水化合物;所述碳水化合物优选为山梨糖醇。
- 根据权利要求11或12所述的用途,所述含硼物质包括一下分子:a)用于蛋白酶体抑制剂含硼化合物,例如硼替佐米、依沙佐米;b)含硼的抗真菌、细菌、病毒的有机硼酸类药物分子,例如他伐硼罗、克立硼罗和Vaborbactam-Meropenem;c)用于中子俘获肿瘤治疗中硼化合物载体,例如含硼吖啶衍生物;d)具有凝血酶抑制活性的含硼化合物;e)具有治疗作用的含硼寡核苷酸、多肽、蛋白;f)用于分子模块化合成的硼选择反应中的硼化合物;g)用于化学反应中未反应的或者过量的含硼试剂的回收,所述化学反应优选用于固体表面或者聚合物和可溶性含硼试剂反应。
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