CN111729661B

CN111729661B - Chromatography media for separating boron-containing materials

Info

Publication number: CN111729661B
Application number: CN202010526191.0A
Authority: CN
Inventors: 胡新妹; 杨克; 张伟; 汝炜; 熊志承; 毛慧明
Original assignee: Suzhou Saifen Technology Co ltd
Current assignee: Suzhou Saifen Technology Co ltd
Priority date: 2020-06-09
Filing date: 2020-06-09
Publication date: 2023-05-05
Anticipated expiration: 2040-06-09
Also published as: CN111729661A

Abstract

The invention relates to the field of chromatographic media, in particular toIn particular, it relates to a chromatographic medium for separating boron-containing materials. The chromatographic medium comprises a matrix support and grafted chains grafted to the matrix support; the general formula of the grafting chain is MA-R ₁ ‑X ₁ ‑R ₂ Or (b)

Shown; wherein MA is a group that can be linked to other chemical functional groups, either covalently or non-covalently; r is R ₁ 、R ₂ And R is ₃ Independently selected from a hydrogen atom, a saturated hydrocarbon group, an unsaturated hydrocarbon group or a saturated hydrocarbon group containing a non-electronegative heteroatom, and wherein at least one of the hydrocarbon groups has a cis-ortho-polyhydroxy which is a linear or cyclic skeleton structure; x is X ₁ And X ₂ Divalent and trivalent heteroatoms, respectively, of the non-electronegative heteroatoms; r in the graft chain ₁ 、R ₂ And R is ₃ The sum of the number of carbon atoms carried thereon>2.

Description

Chromatography media for separating boron-containing materials

Technical Field

The invention relates to the field of chromatographic media, in particular to a chromatographic medium for separating boron-containing substances.

Background

Borax is often used as a medicine in ancient times in China. There are many descriptions of their properties in TCM: borax, also known as Borax, is sweet and salty in taste, and enters lung and stomach meridians. Has effects of clearing heat and detoxicating, detumescence and antisepsis, clearing lung-heat and eliminating phlegm, and can be used for treating laryngopharynx swelling and pain, aphtha, conjunctival congestion and swelling and pain, nebula and cough due to phlegm heat. Boron atoms have unique properties that differ from atoms commonly carried in other drug molecules. The boron atom centre can be from sp ² The hybridized triangle plane becomes sp ³ The hybridized tetrahedra, boron-containing compound increases the diversity of the molecular structure of the drug. The compounds become new application in drug development by virtue of brand new structure and special action mechanismAnd (3) diameter. With the development of boron chemistry, more and more organoboron compounds having better bioactivity have been synthesized successively in recent years. They have good effects in anti-tumor, anti-infection, and inhibition of thrombin and dipeptidyl peptidase activities.

Many kinds of boron-containing medicaments are industrialized, and the commercial value is high. Including Tavaborole (sold under the trade name Kerydine), cliborole (sold under the trade name Eucrisa), VABOMERE compound injection, cyclic adenosine monophosphate boric acid complex, borazine derivatives, boric acid-containing reagents and oligonucleotides, bortezomib (sold under the trade name Velcade), el Sha Zuo meters (Lxazomib, sold under the trade name Ninlar), and el Sha Zuo meters (Ixazomib, sold under the trade name Ninlar).

Among them, tavaborol and cliborol are new molecular entities approved by the U.S. FDA as external use in 2014 and 2016, respectively, from Anacor corporation for the treatment of onychomycosis (onychomycosis/nail) and allergic dermatitis (eczema), respectively. Vabomee consisted of Meropenem (an antibacterial agent) and vabrbactam (a drug resistant agent that can inhibit certain bacteria) was approved by the us FDA in 2017 for treatment of adult complex urinary tract infections. Vaborbactam is a cyclic boronic acid pharmacophore beta-lactamase inhibitor, vabomere can cope with infections with gram-negative bacteria that produce beta-lactamase. All three drugs were the first organic borates. Bortezomib and exenatide Sha Zuomi are also an organoboron drug containing a dipeptidyl group. Bortezomib was marketed in 2003 for the treatment of multiple myeloma as a proteasome inhibitor; as a first oral drug for the treatment of multiple myeloma, sha Zuomi was marketed in 2015 as a second generation proteasome inhibitor.

As the medicine, the most critical point of quality control is the control of medicine purity, so the separation and purification of the medicine in production are key procedures, and the separation efficiency and the process not only affect the medicine quality but also affect the medicine production cost. Currently, the production and purification methods of these boron-containing drug molecules are generally crystallization purification and reverse phase chromatography purification. The advantage of such purification methods is that they are simple to operate, but suffer from several serious drawbacks: a large amount of organic solvents are often needed in the purification process, and the organic solvents can have potential safety hazards such as fire and explosion in the use process; the organic solvent is easy to remain in the medicine and has a certain harm to human body; the subsequent treatment of the organic solvent and the reverse phase chromatography silica gel medium can cause huge damage to the environment; the method has low biocompatibility and is easy to cause denaturation and inactivation of biological medicines.

Disclosure of Invention

The invention aims to fill the blank of the prior 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, comprising a matrix support and grafted chains grafted to the matrix support; another object of the present invention is to provide a method for preparing the above affinity chromatography medium; the affinity chromatography medium is used as the analysis application of a chromatographic column; the application of the affinity chromatography medium in industrial separation and purification. The chromatographic medium prepared by the method has high separation efficiency, good separation effect and low comprehensive cost, and is suitable for industrialized large-scale separation and purification of boron-containing small molecules, oligonucleotides, polypeptides, proteins and the like.

In particular, the chromatographic medium for separating boron-containing substances provided by the invention comprises a matrix support and a grafting chain grafted on the matrix support;

the general formula of the grafting chain is MA-R ₁ -X ₁ -R ₂ Or (b)

Shown;

wherein MA is a group that can be linked to other chemical functional groups, either covalently or non-covalently;

R ₁ 、R ₂ and R is ₃ Independently selected from a hydrogen atom, a saturated hydrocarbon group, an unsaturated hydrocarbon group or a saturated hydrocarbon group containing a non-electronegative heteroatom, and wherein at least one of the hydrocarbon groups has a cis-ortho-polyhydroxy which is a linear or cyclic skeleton structure;

X ₁ and X ₂ Divalent and trivalent heteroatoms, respectively, of the non-electronegative heteroatoms;

r in the graft chain ₁ 、R ₂ And R is ₃ The sum of the number of carbon atoms carried thereon>2.

The invention also relates to a chromatographic separation device comprising a chromatographic medium as described above.

According to a further aspect of the invention, the invention also relates to the use of a chromatography medium as described above, or a chromatography column as described above, for separating or enriching a boron-containing substance from a liquid medium.

The beneficial effects of the invention are as follows:

1) Has specific binding action on boron-containing molecules;

2) The conditions of separation and purification are mild/simple, the biocompatibility is high, and the biological activity of the purified molecules can be better maintained;

3) The purification chromatographic medium and the chromatographic column can be repeatedly used after being cleaned;

4) The use of large amounts of organic reagents is not required;

5) The generated waste liquid is less, the treatment is easy, and the environmental pollution is reduced.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.

FIG. 1 is a scanning electron microscope image of a medium in accordance with one embodiment of the present invention;

FIG. 2 is a graph of the particle size distribution of a chromatographic medium in one embodiment of the invention;

FIG. 3 is a chromatogram showing the separation of boron-containing molecules from boron-free molecules by a chromatographic medium according to one embodiment of the invention;

FIG. 4 is a graph showing adsorption of boron-containing molecules by a chromatographic medium according to a comparative example of the present invention;

FIG. 5 is a chromatogram of the separation and purification of a bortezomib sample by a chromatographic medium in one embodiment of the invention;

FIG. 6 is a separation chromatogram of a chromatographic medium versus a bortezomib sample in a control embodiment of the present invention.

Detailed Description

Reference now will be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. Indeed, it will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the scope or spirit of the invention. For example, features illustrated or described as part of one embodiment can be used on another embodiment to yield still a further embodiment.

Accordingly, it is intended that the present invention cover such modifications and variations as fall within the scope of the appended claims and their equivalents. Other objects, features and aspects of the present invention will be disclosed in or be apparent from the following detailed description. It is to be understood by one of ordinary skill in the art that the present discussion is a description of exemplary embodiments only, and is not intended as limiting the broader aspects of the present invention.

The present invention relates to a chromatographic medium for separating boron-containing substances, said chromatographic medium comprising a matrix support and polymer chains grafted to said matrix support;

The general formula of the polymer chain is MA-R ₁ -X ₁ -R ₂ Or (b)

Shown;

R ₁ 、R ₂ and R is ₃ Independently selected from hydrogen atom, saturated hydrocarbon group, unsaturated hydrocarbon group or saturated hydrocarbon group containing non-electronegative hetero atom, and at least one of them has cis-ortho polyhydroxy group, cis-ortho polyhydroxy groupIs a straight chain framework structure or a ring chain framework structure;

The separation principle of the invention is based on the rapid stable bond bonding between boric acid and cis diol to form a borate ester, the covalent B-O bond of which is stable, but the formation of B-O bond is reversible under certain conditions or under the action of certain external stimuli. The principle has wider application in boric acid affinity chromatography, namely, benzene boric acid is grafted on a matrix skeleton to affinity adsorb molecules with cis-diol bonds. The present invention innovatively exploits a reverse application based on the principle of affinity adsorption of boron-containing molecules by bonding cis-ortho polyhydroxy groups to a matrix backbone. The invention skillfully passes the group with cis-ortho polyhydroxy through a non-carbon X (X) ₁ Or X ₂ ) The heteroatom is bonded to the matrix backbone; the flexibility of the ortho-polyhydroxy (active group) is increased by the insertion of a spacer arm with a proper length into the X heteroatom, so that the ortho-polyhydroxy (active group) is more easily combined with boron atoms in space to form effective coordination; on one hand, the lone pair electron on the hetero atom X can form an intramolecular hydrogen bond with the hydroxyl in the active group, and the ortho-polyhydroxy forms a cis-form favorable for combining with the boron atom; the lone pair electron on the X heteroatom can interact with the boron atom on the other hand, and pull the molecule containing the boron atom to the periphery of the group with cis-ortho polyhydroxy; the combination of the multiple actions allows the ortho-polyhydroxy groups to more readily sterically and conformationally associate with boron atoms to form effective coordination.

In some embodiments, the non-electronegative heteroatom is strongly electronegative.

In some embodiments, the non-electronegative heteroatoms each have a bowing electronegativity value of greater than 2.0, preferably greater than 2.1, more preferably greater than 2.5, and even more preferably greater than 2.9.

In some embodiments, the non-electronegative heteroatoms are selected from one or more of O, S, N, P.

In some embodiments, the saturated hydrocarbon group containing a non-electronegative heteroatom may be a polyamine, an oligoimine, a polyether, an oligoether, a polythioether, an oligosulfide; more preferred are polyamines and oligoimines, such as: ethylenediamine, diethylenetriamine, dipropylenetriamine, triethylenetetramine, linear and branched oligoethyleneimines.

In some embodiments, X ₁ Selected from O or S atoms.

In some embodiments, X ₂ Selected from N or P atoms.

The number of cis-ortho-polyhydroxy groups is greater than 2, such as 3, 4, 5, 6, 7, 8, 9, 10 or more.

In some embodiments, R in the grafted chain ₁ 、R ₂ And R is ₃ The sum of the numbers of carbon atoms is 3 to 40, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13, 14, 15, 20, 25, 30, 35 can be selected.

The saturated or unsaturated hydrocarbon radical may be C _x -C _y An alkyl group. The term "C _x -C _y Alkyl "refers to an alkyl group having from x to y (inclusive of x and y) carbon atoms in a branched or unbranched hydrocarbon group. By way of illustration, but not limitation, the term "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. The term "C ₁ -C ₄ N-alkyl "refers to a straight-chain hydrocarbon moiety having from 1 to 4 carbon atoms, including methyl, ethyl, n-propyl, and n-butyl. C (C) _x -C _y Where x may be from 1 to 10 and y is from 2 to 20. Each alkyl, cycloalkyl group may be optionally substituted such as, but not limited to, those specified herein. In some embodiments, the groups are mono-or di-substituted. In some embodiments, alkyl is C ₁ -C ₃ 、C ₁ -C ₄ 、C ₁ -C ₆ 、C ₄ -C ₆ Or C ₁ -C ₁₀ An alkyl group.

In some embodiments, the cyclic chain backbone structure may also be cycloalkyl. Such as, but not limited to, cyclopropylmethyl, dimethylcyclopropyl, cyclopropylethyl, cyclopropylpropyl, cyclopropylbutyl, cyclobutylmethyl, cyclobutylethyl, cyclobutylpropyl, cyclopentylmethyl, cyclopentylethyl, cyclopentylpropyl, cyclohexylmethyl, cyclohexylethyl, and cyclohexylpropyl.

Each alkyl, cycloalkyl may be optionally substituted or modifiable such as, but not limited to, those indicated herein. In some embodiments, the groups are mono-or di-substituted.

In some embodiments, the cyclic backbone structure is a monosaccharide, disaccharide, or polysaccharide;

in some embodiments, the cyclic backbone structure is preferably mannose, fructose, galactose, mannose-containing disaccharides, and mannose-containing oligosaccharides.

The matrix support comprises sites MA which may be chemically modified. May be covalently or non-covalently linked to other chemical functional groups. Such modification sites include, but are not limited to, the following chemical functionalities: MA is selected from at least one of epoxy, amino, aldehyde, hydroxyl, carboxyl, oxo and thiol groups. The modification site typically also comprises derivative groups having these corresponding chemical functional groups, for example: amino derivatives, thiol derivatives, aldehyde derivatives, formyl derivatives, biotin derivatives, alkyne derivatives, hydroxy derivatives, activated hydroxy or derivatives, carboxylate derivatives, activated carbonates, activated esters, NHS esters (succinimidyl), NHS carbonates (succinimidyl), imidyl esters or derivatives, cyanobromide derivatives, maleimide derivatives, haloacetyl derivatives, iodoacetamide/iodoacetyl derivatives, epoxy derivatives, streptavidin derivatives, tresyl derivatives, diene/conjugated diene derivatives (Diels-Alder type reactions), alkene derivatives, substituted phosphate derivatives, bromohydrin/halohydrin, substituted disulfides, pyridyl-disulfide derivatives, azolide, hydride derivatives, halogenated benzene derivatives, nucleoside derivatives, branched/multifunctional linker molecules, dendrimers, derivatives, nucleoside derivatives, or any combination thereof.

In some embodiments, the density of the surface chemical modification sites of these chromatographic media is optimized. For example, >0.1mmol/ml of medium, preferably >0.5mmol/ml of medium, more preferably >1.0mmol/ml of medium, more preferably >2.0mmol/ml of medium.

In some embodiments, R ₁ 、R ₂ And R is ₃ At least one of which carries a cis-ortho-polyol; preferably cis-o-diol; more preferably terminal cis-vicinal diols;

in some embodiments, R ₁ 、R ₂ And R is ₃ With catechol, e.g., catechol homologs.

In some embodiments, the matrix support comprises any one of borosilicate glass, agar, agarose derivatives, magnetic beads, silica, titania, alginate, cellulose derivatives, dextran, starch, cyclodextrin, chitosan, carrageenan, guar gum, gum arabic, gum ghatti, gum tragacanth, karaya gum, locust bean gum, xanthan gum, pectin, mucin, liver thioesters, and gelatin, silicon, ceramic, glass, polyurethane, polystyrene divinylbenzene, polymethyl methacrylate, polyacrylamide, polyethylene glycol terephthalate, polyvinyl acetate, polyethylene, polypropylene, polyvinyl chloride, polyvinylpyrrolidone, or copolymers formed from any of several.

The matrix support may be selected from a range of options for the appropriate shape, form, material and variation depending on the application. The substrate support surface may be substantially flat or planar. Or may be circular or the like. The shape of the surface of the base support includes, but is not limited to, holes, depressions, pillars, ridges, channels, membranes, or the like. The matrix support is preferably a particulate material having a substantially planar surface. The shape of the particles is preferably spherical or substantially spherical.

In some embodiments, the matrix support is particulate with a particle size in the range of 1 μm to 500 μm.

In some embodiments, the particle size ranges from 1 μm to 300 μm;

in some embodiments, the particle size ranges from 2 μm to 200 μm.

Further, the matrix support may be non-porous or include one or more pores. In particular, a porous resin may be used, and examples of the porous resin may include: other agarose-based resins (e.g., sepharose Fast Flow series (GE)), polymethacrylates: (Sepax PGMA series), polystyrene divinylbenzene (Sepax polypp and Sepax Proteomix series), silica (Sepax Silica series), controlled pore glass, dextran derivatives, polyacrylamide derivatives, and/or other polymers; or any combination thereof. The non-porous resin may be prepared for the same matrix material as the above porous resin.

In some embodiments, the pore size is

In some embodiments, the pore size is

In some embodiments, the pore size is

In the present invention, "nonporous" means that the matrix support has no appreciable pores, e.g., pore size

Further, various types of chromatographic media products are exemplified by Siracer, inc., including but not limited to the listed types:

(a) Chromatography media to which epoxy groups have been covalently attached: polar MC-Epoxy (porous polydisperse polymethacrylate series), monomix MC-Epoxy (porous monodisperse polymethacrylate series), proteomix Epoxy (nonporous monodisperse crosslinked polystyrene series), proteomix POR-Epoxy (porous monodisperse crosslinked polystyrene series).

(b) Chromatography media to which other groups have been covalently attached: 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), hydrohil-Hydrophobic balanced (Hydrophilic hydrophobic amphoteric group), mixed-mode (Mixed-action group).

The present invention can develop various chromatographic separation devices using a chromatographic medium containing the above-described substances. Chromatographic separation devices are of a wide variety including, but not limited to, the following: SPE solid phase extraction column, centrifuge tube with separation membrane, magnetic beads, separation membrane (membranes), rapid detection biochip (bio-chips), kit, fiber bundle column, monolithic column and conventional analytical or preparative chromatographic column.

The chromatographic separation device is preferably cylindrical and consists of a plurality of essential components: chromatography medium material, particle size and pore size of the chromatography medium, matrix material of the column, size of the column, and column packing method. The chromatographic column packed with the chromatographic medium of the present invention may have several physical forms and consists of different chromatographic column components.

The method for installing the column comprises the following steps: constant current/variable current/constant voltage/multi-level pressure regulation/DAC.

Different column packing methods are selected according to the performances such as the medium particle size, the pore diameter and the like and the specification of the chromatographic column.

The chromatographic medium with the grain diameter smaller than 15 mu m is packed by a multi-purpose constant-current and constant-pressure method;

the chromatographic medium with the grain diameter larger than 15 mu m is packed by a constant-current or variable-current method according to the pressure resistance of the bed and the chromatographic medium;

the large-size chromatographic column is assembled by multi-stage pressure regulation or DAC method.

Homogenate: water, brine, water containing organic phase or buffer salt, etc., 20-80% (v: v) solid content

Selecting proper homogenate mobile phase and homogenate volume according to chromatographic column specification and chromatographic medium performance

Chromatographic column material: stainless steel, glass, plastic, SPE tube

The column tubes of different materials have different pressure resistance, and when the chromatographic column is filled, the control pressure is not only controlled to exceed the highest pressure resistance of the filling material, but also controlled to not exceed the highest pressure resistance of the column tube material.

Chromatographic column washing conditions: the chromatographic column has long service life, and can adsorb some impurities which are difficult to clean, and the impurities can affect the performance of the chromatographic column, so that the impurities need to be cleaned periodically. Different impurity cleaning methods are different, and common impurities are 0.5M HCl or 0.5-1.0M NaOH, and impurities with strong hydrophobicity can be cleaned by using 0.1-1% Tween or Triton X-100. Alternatively, the column may be rinsed with 0.5M NaOH and stored in 20% aqueous ethanol at room temperature.

The chromatographic column preservation method comprises the following steps:

the column was stored in an aqueous solution containing 20% ethanol at room temperature.

The chromatographic medium is preserved in water solution containing 20% ethanol at 2-8deg.C.

According to a further aspect of the present invention, the present invention also relates to the use of a chromatographic medium as described above, or a chromatographic separation device as described above, for separating or enriching a boron-containing material from a liquid medium.

The boron-containing material pair removal includes: separation and purification between boron-containing molecules and boron-free molecules (affinity chromatography) and separation and purification between different boron-containing molecules (not limited to methods of use of affinity chromatography media).

In some embodiments, the chromatographic medium adsorbs the boron-containing species under basic conditions and elutes the boron-containing species under acidic conditions.

The pH under alkaline conditions may be >7.0, preferably pH >7.5, more preferably pH >8.0.

The pH of the acidic condition may be <7.0, preferably pH <6.0, more preferably pH <5.0.

In addition, the interaction between the boron-containing material and the cis diol groups is enhanced in the presence of mg2+ ions. A mobile phase containing carbohydrates (e.g., sorbitol) may also be used to elute the boron-containing material.

In some embodiments, the boron-containing species comprises the following:

a) Boron-containing compounds for proteasome inhibitors, such as bortezomib, ezetimibe Sha Zuo meters;

b) Boron-containing antifungal, bacterial, viral organoboronic acid drug molecules such as, for example, tavalborol, cliborol, and Vaborbacam-Meropenem;

c) Boron compound carriers, such as borazine derivatives, for use in neutron capture tumor therapy (Boron Neutron Capture Therapy, BNCT);

d) A boron-containing compound having thrombin inhibitory activity;

e) Boron-containing oligonucleotides, polypeptides, and proteins having therapeutic effects;

f) Boron compounds for use in boron selective reactions for molecular modular synthesis.

g) For recovery of unreacted or excess boron-containing reagent in chemical reactions, preferably for solid surfaces or reactions of polymers and soluble boron-containing reagents.

Boron compounds used in boron selective reactions for molecular modular synthesis are well known to those skilled in the art, for example, "Liang Xu, et al, chem. Soc. Rev.,2015, 44 (24): 8848-8858 "and" Raphael Oeschger, et al, science,2020,368,736-741", and the like. Boron-containing intermediates used in organic synthesis such as organic halides containing masked borane groups and di-or polyborocompounds; boron selective reaction refers to chemical transformations in which two or more borane groups are present in the reactants and their reactivities can be distinguished; thus, the more reactive borane groups may be selectively converted while the relatively inert borane groups remain intact. Inert boranyl groups can generally be used under different reaction conditions and converted to other functional groups, thus providing a continuous and flexible synthetic method for complex molecules.

Embodiments of the present invention will be described in detail below with reference to examples.

Example 1

Amine diol examples, epoxy Access from Medium surface

This example uses porous polymethacrylate microspheres (30 μm particle size, pore size) produced by Suzhou Sedan technology Co., ltd

Cargo number: 281030952 And the surface of which contains a large amount of epoxy groups.

1) Adding 60.0g of 65-70% ethylamine aqueous solution by mass fraction into 100g of chromatographic medium with epoxy groups on the surface, and mechanically stirring to react for 16h at room temperature;

2) After the reaction is finished, filtering the washing medium to be neutral by deionized water; the chromatography medium was then transferred to 300ml of 0.05M hydrochloric acid solution and stirred well. Dipping a small amount of liquid by using pH test paper to detect the pH value less than 3, and filtering and draining after the stirring of the hydrochloric acid solution is continued until the pH value is less than 3 if the solution does not reach the pH value; filtering the washing medium to neutrality by deionized water;

3) The chromatography medium was transferred to 500ml of 0.1M aqueous sodium bicarbonate solution and stirred well. Dipping a small amount of liquid by using pH test paper to detect the pH value of more than 8, and filtering and draining after stirring until the pH value of more than 8 is reached when the stirring is not completed by continuously adding sodium bicarbonate aqueous solution; washing with purified water for several times until pH test paper is neutral, and draining until no filtrate comes out.

4) The medium is added into a reaction device, 150ml of solution containing 50% glycidol and 2.0g of sodium hydroxide is added into the reaction device, the reaction is carried out for 2 hours under the condition of room temperature and light shielding, and after the reaction is finished, the washing medium is filtered by at least 10 times of deionized water in volume.

The chromatographic medium prepared in this example contains a cis diol structure that adsorbs boron-containing molecules under alkaline conditions and the sample molecules are eluted under acidic conditions.

Examples electron microscope scans are shown in fig. 1 and particle size distribution in fig. 2.

Example 2

This example uses porous polymethacrylate microspheres (particle size of Suzhou Sedan technology Co., ltd.)30 μm, pore diameter of

Adding 276g of 30% methylamine water solution with the mass fraction into 100g of chromatographic medium with epoxy groups on the surface, adding into a reaction device, and mechanically stirring at room temperature for reaction for 16h; other steps refer to example 1.

Example 3

Cargo number: 281030502 And the surface of which contains a large amount of epoxy groups.

Example 4

Example 5

This example uses porous polymethacrylate microspheres (60 μm particle size, pore size) produced by Suzhou Sedan technology Co., ltd

Cargo number: 281060952 And the surface of which contains a large amount of epoxy groups.

Adding 60.0g of 65-70% ethylamine aqueous solution by mass fraction into 100g of chromatographic medium with epoxy groups on the surface, adding into a reaction device, and mechanically stirring at room temperature for reaction for 16h; other steps refer to example 1.

Example 6

Example 7

This example uses porous polymethacrylate microspheres (particle size 10 μm, pore size

Cargo number: 281010952 And the surface of which contains a large amount of epoxy groups.

Adding 60.0g of 65-70% ethylamine aqueous solution by mass fraction into 100g of chromatographic medium with epoxy groups on the surface, and mechanically stirring at room temperature for reaction for 16h; other steps refer to example 1.

Example 8

Adding 165.6g of 30% methylamine water solution with the mass fraction of 100g of chromatographic medium with epoxy groups on the surface, adding a reaction device, and mechanically stirring at room temperature for reaction for 16h; other steps refer to example 1.

Example 9 (comparative example)

This example is a control example, where the chromatography media surface has normal cis diol groups, but is not further modified. Porous polymethacrylate microsphere (particle diameter of 30 μm, pore diameter of 30 μm) produced by Suzhou Siro technical Co., ltd

Cargo number: 281030952 The surface of which contains a plurality of epoxy groups which open to form cis diol groups.

Adding 300ml of 0.5M sulfuric acid into 100g of chromatographic medium with epoxy groups on the surface, adding into a reaction device, and mechanically stirring at 40 ℃ for reaction for 2 hours; and after the reaction is finished, filtering the washing medium to be neutral by deionized water.

Example 10

This example uses porous polystyrene divinylbenzene (particle size 15 μm, pore size

Cargo number: 221715952 And the surface of which contains a large amount of epoxy groups.

Example 11

Example 12

This example uses non-porous polystyrene divinylbenzene (particle size 5 μm, non-porous, cat# 221605002) produced by Sedan technology Co., ltd.) whose surface contains a large number of epoxy groups.

Example 13

Example 14

This example uses non-porous polystyrene divinylbenzene (particle size 10 μm, non-porous, cat# 221610002) produced by Sedan technology Co., ltd.) whose surface contains a large number of epoxy groups.

Example 15

Example 16

Thioether diol examples, epoxy Access from Medium surface

This practice isExamples porous polymethacrylate microspheres (30 μm particle size, pore size) produced by Suzhou Siro technology Co., ltd

Adding 20.0g of chromatographic medium with epoxy groups on the surface into 100ml of 0.1M sodium bicarbonate solution for uniform dispersion, adding 1.30g of 1-thioglycerol, adding into a reaction device, mechanically stirring at room temperature for reaction for 16h, and filtering the washing medium to neutrality by deionized water after the reaction is finished.

Example 17

Ether diol examples, epoxy Access from Medium surface

1) Based on 20.0g of chromatographic medium with epoxy groups on the surface, 50ml of 5M sodium hydroxide aqueous solution is added for uniform dispersion, a reaction device is added, the temperature is raised to 35 ℃, and stirring is kept for 2 hours.

2) 20.0g of glycidol was added thereto, and the reaction was stirred at 35℃for 3 hours. 100ml of purified water is added and stirred for 10min, and after the reaction is finished, the washing medium is filtered by deionized water to be neutral.

Example 18

Polyamine diol examples, access by Medium surface aldehyde groups

Cargo number: 280130950 And the surface of which contains a large number of hydroxyl groups. />

1) 100mL of 0.2M NaIO based on 100.0g of chromatographic medium with hydroxyl groups on the surface ₄ Solution at room temperatureThe reaction was stirred mechanically at 100rpm for 1.5h and after the reaction was completed the microspheres were washed by suction filtration with at least 10 volumes of deionized water.

2) Then 100mL of 0.1M phosphate buffer solution containing 1.52M dipropylene triamine (CAS# 56-18-8, pH 7.2) was added, along with 0.63g NaCNBH ₃ And (3) mechanically stirring at 100rpm for reaction for 5 hours at room temperature, and filtering and washing the microspheres with at least 10 times of deionized water until the pH test paper of the filtrate is neutral, and pumping until no filtrate comes out.

3) The preparation of the chromatographic medium was completed according to step (4) of example 1 by adding 150ml of a solution containing 50% glycidol and 2.0g of sodium bicarbonate to the above medium.

Example 19

Polyamine diol examples, access by Medium surface aldehyde groups

Cargo number: 280130950 And the surface of which contains a large number of hydroxyl groups.

1) 100mL of 0.2M NaIO based on 100.0g of chromatographic medium with hydroxyl groups on the surface ₄ The solution was mechanically stirred at 100rpm for 1.5h at room temperature and after the reaction was completed, the microspheres were washed by suction filtration with at least 10 volumes of deionized water.

2) Then 100mL of 0.1M phosphate buffer solution containing 1.50M triethylenetetramine (CAS# 112-24-3) was added, along with 0.63g of NaCNBH ₃ And (3) mechanically stirring at 100rpm for reaction for 5 hours at room temperature, and filtering and washing the microspheres with at least 10 times of deionized water until the pH test paper of the filtrate is neutral, and pumping until no filtrate comes out.

Example 20

Amine mannitol diol example, epoxy Access from Medium surface

The present embodiment employs SuzhouPorous polymethacrylate microsphere (particle diameter of 30 μm, pore diameter of 30 μm) produced by Sai Ke technology Co., ltd

1) A chromatographic medium having an amine group on the surface was prepared in accordance with steps (1) to (3) in example 1, based on 100g of the chromatographic medium having an epoxy group on the surface.

2) 150ml of N, N-dimethylformamide solution containing 25% of alpha-D-mannopyranose chloride (2, 3,4, 6-tetra-O-acetyl-alpha-D-mannopyranose chloride, CAS# 14257-40-0) and 2.0g of sodium carbonate were added to the above medium, the reaction was mechanically stirred at room temperature under dark conditions overnight, and after the completion of the reaction the medium was washed by suction filtration with at least 10 volumes of deionized water.

3) 300ml of 0.5M sulfuric acid is added, the mixture is added into a reaction device, the mixture is mechanically stirred at 40 ℃ for reaction for 2 hours, and deionized water is used for suction filtration to wash the medium until the reaction is neutral.

Example 21

Polyamine mannediol example, access from Medium surface aldehyde groups

2) Then 100mL of 0.1M phosphate buffer solution containing 1.50M triethylenetetramine (CAS# 112-24-3) was added, along with 0.63g of NaCNBH ₃ Mechanically stirring at 100rpm at room temperature for reaction for 5h, filtering and washing the microspheres with at least 10 times of deionized water until the pH test paper of the filtrate is neutral, and pumping until no filtrate comes out to prepare the microsphere with polyamine groups on the surfaceIs a chromatographic medium of (a).

3) 150ml of N, N-dimethylformamide solution containing 25% of alpha-D-mannopyranose chloride (2, 3,4, 6-tetra-O-acetyl-alpha-D-mannopyranose chloride, CAS# 14257-40-0) and 2.0g of sodium carbonate were added to the above medium, the reaction was mechanically stirred at room temperature under dark conditions overnight, and after the completion of the reaction the medium was washed by suction filtration with at least 10 volumes of deionized water.

4) 300ml of 0.5M sulfuric acid is added, the mixture is added into a reaction device, the mixture is mechanically stirred at 40 ℃ for reaction for 2 hours, and deionized water is used for suction filtration to wash the medium until the reaction is neutral.

Example 22

Separation of phenylboronic acid and ribonuclease A by chromatography medium

The chromatographic media synthesized by the methods of example 1 and example 9 (comparative example) were packed into 4.650 mm stainless steel chromatographic columns, respectively, and the adsorption-desorption conditions of the affinity chromatographic media modified by the method of the invention and the ordinary chromatographic media with vicinal diols on boron-containing molecules were compared.

The selected samples are as follows: phenylboronic acid (boron-containing molecules); ribonuclease A (boron-free molecule)

The specific separation conditions are as follows:

sample: ribonuclease A and phenylboronic acid mixed solution

Equilibrium solution: 0.25M ammonium acetate buffer (pH 8.0)

Eluting solution: 0.1M formic acid solution

The separation process comprises the following steps:

washing the equilibrium chromatographic column with an equilibrium solution;

blank solvent, single-standard ribonuclease A, single-standard phenylboronic acid and mixed-standard ribonuclease A & phenylboronic acid are loaded;

the equilibrium solution continues to rinse to baseline plateau;

eluting the phenylboronic acid adsorbed by the medium by changing the eluting solution;

the column was washed with 0.5M NaOH;

(6) The column was stored in 20% ethanol aqueous solution at room temperature using the completed column.

Separation and purification experiment results:

example 1 affinity chromatography media (affinity chromatography media modified according to the invention) the separation effect of phenylboronic acid and ribonuclease a is shown (fig. 3):

the chromatographic medium synthesized in example 1 has a strong adsorption on phenylboronic acid under alkaline conditions. The phenylboronic acid molecules are firmly combined with the chromatographic medium, and the phenylboronic acid can be eluted only under the acidic condition.

The chromatographic medium synthesized in example 1 had no adsorption of ribonuclease A under alkaline conditions. Ribonuclease A is carried directly out by the alkaline mobile phase.

The chromatographic medium synthesized in example 1 thus makes it possible to completely separate phenylboronic acid from ribonuclease. The purity of phenylboronic acid is more than 95 percent, and the yield is more than 90 percent.

(2) Example 9 (comparative example) chromatography medium (common chromatography medium with diol groups): the adsorption result of p-phenylboronic acid is shown (fig. 4):

the chromatographic medium synthesized in example 9 (comparative example) had an adsorption effect on phenylboronic acid under alkaline conditions. But already started to be eluted under alkaline conditions.

The chromatographic medium synthesized in example 9 (comparative example) had less adsorption to boron-containing molecules than the chromatographic medium of example 1.

Example 23:

separation of boron-containing peptide molecules by chromatography medium

The chromatographic medium synthesized by the methods of example 1 and example 9 (comparative example) was packed into a 4.6 x 50mm stainless steel column, which was used to separate the target molecule from the crude impurity-containing product. The adsorption effect of the chromatographic medium on bortezomib molecules was tested. The specific separation conditions are as follows:

sample: sample solution containing bortezomib molecules

Loading solution: the pH value of the sample solution is adjusted to 8.0

Equilibrium solution: 0.25M ammonium acetate buffer (pH 8.0) +20% methanol

Eluting solution: 0.1M formic acid solution+20% methanol

(1) Example 1 isolation and purification procedure:

flushing and balancing the chromatographic column with balancing solution;

sample solution is loaded;

the equilibrium solution continues to rinse to baseline plateau;

eluting bortezomib molecules adsorbed by the medium by changing the eluting solution;

the column was washed with 0.5M NaOH;

the column was stored in 20% ethanol aqueous solution at room temperature using the completed column.

Separation and purification experiment results:

example 1 the separation effect of the affinity chromatography medium (modified according to the invention) on bortezomib molecules and impurities is shown (fig. 5):

the chromatographic medium synthesized in example 1 has a strong adsorption effect on bortezomib molecules under alkaline conditions. The bortezomib molecules are firmly combined with the chromatographic medium, and can be eluted only under acidic conditions.

The chromatographic medium synthesized in example 1 had no adsorption of the majority of the impurities in the sample under alkaline conditions. The impurities are carried directly out by the alkaline mobile phase.

The chromatographic medium synthesized in example 1 thus allows to separate the bortezomib molecules and their impurities. The purity of bortezomib is more than 95%, and the yield is more than 90%.

(2) Example 9 (comparative example) isolation procedure:

washing the equilibrium chromatographic column with an equilibrium solution;

sample injection is carried out on the sample solution;

the equilibrium solution continues to rinse to baseline plateau;

the column was washed with 0.5M NaOH;

Separation and purification experiment results:

example 9 (comparative example) chromatographic medium (common chromatographic medium with diol groups): the results of adsorption to bortezomib are shown (fig. 6):

the chromatographic medium synthesized in example 9 (comparative example) had an adsorption effect on bortezomib under alkaline conditions. But already started to be eluted under alkaline conditions.

The chromatographic medium synthesized in example 9 (comparative example) had a weaker adsorption to bortezomib than the chromatographic medium of example 1.

(3) Analysis of bortezomib separation and purification experimental results

The chromatographic medium of example 1 allows sterically and conformationally easier incorporation of the ortho-polyhydroxy groups with the boron atoms to form effective coordination in a variety of synergies, with high purity and yield of bortezomib;

example 9 (comparative example) the chromatography medium surface had only common cis diol groups which did not readily bind to boron atoms to form effective coordination, the results of the separation and purification experiments were worse than those of example 1.

The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.

The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Claims

1. A chromatographic medium for separating boron-containing material, wherein said chromatographic medium comprises a matrix support and grafted chains grafted to said matrix support;

the general formula of the grafting chain is MA-R ₁ -X ₁ -R ₂ Or (b)

Shown;

X ₁ and X ₂ Divalent and trivalent heteroatoms, respectively, of the non-electronegative heteroatoms; r in the graft chain ₁ 、R ₂ And R is ₃ The sum of the number of carbon atoms carried thereon>2.

2. The chromatographic medium of claim 1, wherein the non-electronegative heteroatoms each have a bowin electronegativity number greater than 2.0.

3. The chromatographic medium of claim 2, wherein the non-electronegative heteroatom is selected from one or more of O, S, N, P.

4. The chromatography medium of claim 2, wherein X is ₁ Selected from O or S atoms.

5. The chromatography medium of claim 2, wherein X is ₂ Selected from N or P atoms.

6. The chromatography medium of claim 1, wherein MA is selected from at least one of epoxy, amino, aldehyde, hydroxyl, carboxyl, oxo, and thiol groups.

7. The chromatography medium of claim 1, wherein R ₁ 、R ₂ And R is ₃ At least one of which carries a cis-ortho-polyol.

8. The chromatography medium of claim 1, wherein R ₁ 、R ₂ And R is ₃ At least one of which carries catechol.

9. The chromatography medium of claim 1, wherein the cyclic backbone structure is a monosaccharide, disaccharide, or polysaccharide.

10. The chromatographic medium according to any of claims 1 to 9, wherein the matrix support comprises any of agar, agarose derivatives, magnetic beads, silica, titania, alginate, cellulose derivatives, dextran, starch, cyclodextrin, chitosan, carrageenan, guar gum, gum arabic, gum ghatti, gum tragacanth, karaya gum, locust bean gum, xanthan gum, pectin, mucin, liver thioester and gelatin, ceramic, glass, polyurethane, polystyrene divinylbenzene, polymethyl methacrylate, polyacrylamide, polyethylene glycol terephthalate, polyvinyl acetate, polyethylene, polypropylene, polyvinyl chloride, polyvinylpyrrolidone, or copolymers of any of the several.

11. The chromatographic medium according to any of claims 1 to 9, wherein the matrix support is particulate and has a particle size in the range of 1 μm to 500 μm.

12. The chromatographic medium of any of claims 1-9, wherein the matrix support is non-porous or comprises one or more pores.

13. A chromatographic separation device comprising the chromatographic medium according to any one of claims 1 to 12.

14. Use of a chromatographic medium as claimed in any of claims 1 to 12, or a chromatographic separation device as claimed in claim 13, for separating or enriching a boron-containing material from a liquid medium.

15. The use according to claim 14, wherein the chromatography medium adsorbs the boron-containing material under alkaline conditions and the boron-containing material is eluted under acidic conditions.

16. Use according to claim 15, characterized in that the mobile phase for eluting the boron-containing material contains carbohydrates.

17. Use according to any one of claims 14 to 16, wherein the boron-containing substance comprises the following molecules:

a) Boron-containing compounds for proteasome inhibitors;

b) Boron-containing antifungal, bacterial and viral organoboronic acid drug molecules;

c) Boron compound carrier for neutron capture tumor treatment;

d) A boron-containing compound having thrombin inhibitory activity;

f) Boron compounds for use in boron selective reactions for molecular modular synthesis;

g) For recovery of unreacted or excess boron-containing reagent in chemical reactions.