Materials and methods for performing separation based on halogen bonding

Abstract

The present invention relates to a novel stationary phase having functional groups containing halogen-substituted aromatic rings. Target molecules can interact with this stationary phase through halogen bonding. The stationary phase is suitable for SPE or chromatographic separation.

Description

Technical Field

[0001] The present invention relates to a novel stationary phase having a functional group containing a halogen-substituted aromatic ring. A target molecule can interact with this stationary phase by halogen bonding. The stationary phase is suitable for SPE and chromatographic separation.

Background Art

[0002] Background of the Invention Halogen bonding refers to the non-covalent interaction of a halogen atom X in some molecules, RX (Lewis acid), with a negative site such as a lone pair of electrons of a Lewis base on another neutral or anionic Lewis base. X can be chlorine, bromine or iodine. Halogen bonding can be explained by the presence of a region of positive electrostatic potential, an α-hole, at the outermost part of the surface of the halogen at the center of the RX axis. The strength of the interaction increases in the order of base, bromine, iodine, and can be further modified by modifying the residue R of the halogen. Typically, the strength of the halogen bonding interaction is 10 - 180 kJ / mol and is thus comparable to the strength of hydrogen bonding. This can be seen, for example, from DOI: 10.2021 / acs.chem.rev.5b00484.

[0003] Halogen bonding is currently being utilized in the control of crystal chemistry of organic compounds, in supramolecular chemistry, in catalysis, and in molecular recognition. In addition, halogen bonding is applied to separation applications. Xiao Qing Yan et al., Analytica Chimica Acta 753 (2012) 48-56 discloses the solid phase extraction of perfluorinated iodoalkanes. P. Peluso et al., Journal of Chromatography A, 1467(2016)228-238 describes the chromatographic enantiomeric separation of polyhalogenated 4,4'-bipyridine of atropisomers.

[0004] It has now been found that the principle of halogen bonding can be used not only to separate halogens containing the molecule R-X that forms the Lewis acid part of the bond, but also to separate Lewis bases. The inventors have found that by immobilizing a predetermined molecule R-X on a solid phase, a wide variety of target molecules containing a Lewis base moiety can interact with the immobilized R-X. This interaction can establish a new chromatographic separation principle and provide an additional degree of freedom in chromatographic separation. It has even been shown that proteins can be separated based on halogen bonding.

Summary of the Invention

[0005] Brief Description of the Invention Thus, the present invention is a stationary phase comprising a substrate and at least one type of functional group, whereby the functional group is ---N(R’2) + -ArX, where R’ is H or C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkynyl, C6 aryl, preferably methyl, ethyl, propyl, isopropyl, and ArX is

Chemical formula

[0006] In a preferred embodiment, the positively charged heterocyclic aromatic group having at least one Cl, Br or I residue has the following structure: [Chemical formula] X is, independently of one another, H, I, Br or Cl, and X' is, independently of one another, H, I, Br, Cl, F, NO2, or another electron-withdrawing group, or C1-C10 alkyl, C1-C10 alkenyl, C1-C10 alkynyl, C6-C12 aryl, or an electron-donating group, whereby at least one X or X' is Cl, Br or I, preferably at least one X is Cl, Br, or I, has one of.

[0007] The wavy lines in formula III and in the following formula indicate the connection to the substrate. This may be a single bond, or preferably a linker or another structure, for example part of a tentacle. [Chemical formula] R is H, C1-C10 alkyl, aryl, C1-C10 alkenyl, C1-C10 alkynyl, or an electron-withdrawing or electron-donating group, Y is, independently of one another, N or P, X is H, I, Br or Cl, and X' is, independently of one another, H, I, Br, Cl, F, NO2, or another electron-withdrawing group, or C1-C10 alkyl, C1-C10 alkenyl, C1-C10 alkynyl, C6-C12 aryl, or an electron-donating group, whereby at least one of X or X' is Cl, Br or I, and preferably X is Cl, Br, or I.

[0008] Examples of possible structures according to formula IV are as follows:

Chemical formula

[0009] Another possible structure is formula V,

Chemical formula

[0010] Examples of possible structures according to formula V are as follows:

Chemical formula

[0011] The same structure with S instead of O is possible. Other possible structures are as follows:

Chem.

[0012] In a preferred embodiment, the residue or substituent of the functional group contains only Cl, Br and / or I and H, C1-C6 alkyl and / or one or more electron-withdrawing groups. In a preferred embodiment, the functional group contains only one heterocyclic ring. In another preferred embodiment, the heterocyclic aromatic group has one type of heteroatom. In a preferred embodiment, the heteroatom in the heterocyclic ring is an N atom. In another preferred embodiment, the aromatic ring of the functional group is substituted with one or two iodine atoms.

[0013] The most preferred structure of the functional group is as follows:

Chem.

[0014]

Chemical formula

[0015]

Chemical formula

[0016]

Chemical formula

[0017] Formula XIII (R 3Also preferably a functional group according to Structure VI, VII, VIII and / or IX (wherein monomers according to Structure VI, VII, VIII and / or IX are used) in the grafting process described below, also called a tentacle, attached to a solid support via a polymer chain, with a functional group according to Structure VI, VII, VIII or IX, most preferably VII being highly preferred. Optionally, in addition, a polymer chain having two or more different functional groups, at least one of which is selected from Structures VI, VII, VIII and / or IX, is produced, with other R 3 Other monomers according to Formula III containing 3 can be used.

[0018] When the functional group has one or more positive charges, there are negative counterions. The type of counterion is not important. Suitable counterions are halide ions, acetate, nitrate, triflate, and the like. In a preferred embodiment, the functional group is covalently bonded to the substrate. In one embodiment, the substrate is a bead or a membrane. In one embodiment, the functional group is bonded to the substrate via a linker. In one embodiment, the functional group, also called a tentacle, grafted onto the substrate is included in the polymer chain. In a preferred embodiment, the substrate is an organic polymer, preferably a polyethylene-based polymer or a polyvinyl ether-based polymer.

[0019] The present invention is further directed to a separation device comprising the stationary phase of the present invention. In a preferred embodiment, the separation device is a chromatography column comprising the stationary phase of the present invention. The present invention is further directed to a process for the separation of at least one target molecule from at least one other compound, whereby the stationary phase according to the present invention as described above, preferably that present in the separation device, is contacted with a liquid comprising the target molecule and at least one other compound, and whereby the target molecule exhibits a binding to the stationary phase that is different from the binding of the other compound, for example, interacting with the stationary phase stronger or weaker than the other compound.

[0020] In a preferred embodiment, the more the compound can donate electrons, the stronger it binds to the stationary phase. In a preferred embodiment, the process is a process for chromatographic separation of at least one other compound from a target molecule, whereby the stationary phase according to the invention as described above is present in a chromatography column, and a liquid containing the target molecule and at least one other compound flows through the column, whereby the target molecule and other compounds present in the liquid are eluted from the column according to their interaction with the stationary phase. In a preferred embodiment, the target molecule is a protein.

[0021] In a preferred embodiment, the process is carried out by filling a stationary phase according to the invention and an aqueous loading buffer of a predetermined pH and a predetermined ionic strength, and eluting the target molecule with an aqueous elution buffer having the same pH but a different ionic strength, typically a higher ionic strength. In another embodiment, the process is carried out by filling a stationary phase according to the invention and an aqueous loading buffer of a predetermined pH and a predetermined ionic strength, and eluting the target molecule with an aqueous buffer of a different pH and / or a different ionic strength. In another embodiment, the process is carried out by filling a stationary phase according to the invention and an organic packing medium, and eluting the target molecule using the same medium or a more polar medium, for example using gradient elution. The organic packing medium is a liquid containing no more than 10% water, for example, methanol, ethanol, acetonitrile, THF, heptane, toluene, etc. or mixtures thereof.

[0022] The present invention is further directed to the chromatographic separation of proteins having different pl (isoelectric point) and / or different carboxylic acid contents by halogen bonding, preferably using the stationary phase according to the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0023]

Figure 1

Figure 2

[0024] Before describing the present invention in detail, it should be understood that such is an invention that is not limited to specific compositions or process steps, as such can vary. As used in this specification and the appended claims, it should be noted that the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to "a ligand" includes multiple ligands, a reference to "an antibody" includes multiple antibodies, and the like. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art related to the present invention. The following terms are defined for the purposes of the present invention as described herein.

[0025] As used herein, the term "target molecule" refers to any molecule, substance or compound that is isolated, separated or purified from one or more other components, and as an example, refers to impurities in a sample. In a manufacturing and / or purification process, the target molecule typically exists in a liquid. The liquid may be water, a buffer, ethanol, acetonitrile, a non-aqueous organic solvent such as heptane, or any mixture of the specified liquids. In addition to the target molecule, the liquid may contain one or more impurities. The liquid may also be referred to as a sample. The composition of the liquid may change during manufacturing and / or purification depending on the process steps being performed. After a chromatographic step, the liquid typically contains more of other solvents than before because of the eluent used in the chromatographic step. Examples of target molecules are low molecular weight molecules such as drugs having a molecular weight of approximately 2000 g / mol or less. The target molecule may be a high molecular weight compound such as a protein, for example an antibody.

[0026] The term "antibody" refers to a protein having the ability to specifically bind to an antigen. "Antibody" or "IgG" further refers to a polypeptide substantially encoded by an immunoglobulin gene(s) that specifically binds to and recognizes an analyte (antigen). The recognized immunoglobulin genes include kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as numerous immunoglobulin variable region genes. The light chain is classified as either gamma or lambda. The heavy chain is classified as kappa, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE, respectively. An exemplary immunoglobulin (antibody) structural unit is composed of two pairs of polypeptide chains, each pair having one "light chain" (about 25 kD) and one "heavy chain" (about 50 - 70 kD), and the chains are stabilized, for example, by interchain disulfide bonds. The N-terminus of each chain defines a variable region of about 100 - 110 or more amino acids involved in antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains, respectively.

[0027] Antibodies can be monoclonal or polyclonal and can exist in monomeric or polymeric forms, such as IgM antibodies present in pentameric form, and / or IgA antibodies present in monomeric, dimeric, or multimeric forms. Antibodies also include bispecific antibodies (e.g., bispecific antibodies) and antibody fragments as long as they retain or are modified to include a ligand-specific binding domain. The term "fragment" refers to a part or portion of an antibody or antibody chain that contains fewer amino acid residues than an intact or full antibody or antibody chain. Fragments can be obtained via chemical or enzymatic treatment of an intact or full antibody or antibody chain. Fragments can also be obtained by recombinant means. When produced recombinantly, fragments can be expressed alone or as part of a larger protein referred to as a fusion protein. Exemplary fragments include Fab, Fab’, F(ab’)2, Fc, and / or Fv fragments. Exemplary fusion proteins include Fc fusion proteins. According to the present invention, fusion proteins are also encompassed by the term "antibody". In some embodiments, the antibody is a protein, e.g., an immunoglobulin, containing an Fc region.

[0028] As used herein and unless stated otherwise, the term "sample" refers to any composition or mixture containing a target molecule. The sample may be derived from a biological source or other source. Biological sources include eukaryotic sources such as animals or humans. The sample may also include diluents, buffers, detergents, and contaminants, etc. that are found to be mixed with the target molecule.

[0029] As used herein, the terms "impurity" or "contaminant" refer to any foreign or undesirable molecule, including biological macromolecules such as additives that may be present in a sample containing a target molecule separated from DNA, RNA, one or more host cell proteins, nucleic acids, endotoxins, lipids, impurities of synthetic origin, and one or more foreign or undesirable molecules.

[0030] The terms "purify", "separate", or "isolate" used interchangeably herein refer to increasing the purity of a target molecule by separating the target molecule from a composition or sample containing the target molecule and one or more other components, such as impurities. Typically, the purity of the target molecule is increased by removing at least one impurity (completely or partially) from the composition.

[0031] The term "chromatography" refers to any type of technique for separating a target analyte (e.g., a target molecule) from other molecules present in a mixture. Generally, the target molecule is separated from other molecules as a result of differences in the rate of movement of the individual molecules of the mixture through a stationary phase affected by the mobile phase, or in binding and elution processes. Examples of chromatographic separation processes are reverse phase chromatography, ion exchange chromatography, size exclusion chromatography, affinity chromatography, hydrophobic interaction chromatography, and mixed mode chromatography. The chromatographic processes of the present invention are based on halogen bonding and any one or more additional separation processes mentioned above.

[0032] A "buffer" is a solution that resists changes in pH due to the action of acid-base conjugate components. Various buffers can be employed depending on the desired pH of the buffer, as described, for example, in Buffers. A Guide for the Preparation and Use of Buffers in Biological Systems, Gueffroy, D., ed. Calbiochem Corporation (1975). Non-limiting examples of buffers include MES, MOPS, MOPSO, Tris, HEPES, phosphate, acetate, citrate, succinate, glycine, and ammonium buffers, and combinations thereof. An aqueous buffer is a buffer in which the solvent contains more than 90%, preferably 100%, water.

[0033] The term "stationary phase" refers to any type of adsorbent, matrix, resin, or solid phase in a separation process that separates a target molecule from other molecules present in a mixture. Generally, the target molecule is separated from other molecules as a result of differences in the rate at which the individual molecules of the mixture bind to the stationary phase and / or the rate at which they move through the stationary phase under the influence of the mobile phase. The stationary phase can be placed in a column or a cartridge. The stationary phase according to the present invention contains at least one type of functional group.

[0034] A "functional group" is a ligand that attaches to the substrate of the stationary phase and determines the binding characteristics of the stationary phase. Examples of "functional groups" include, but are not limited to, ion exchange groups, hydrophobic interaction groups, hydrophilic interaction groups, thiophilic interaction groups, metal affinity groups, affinity groups, bioaffinity groups, and mixed mode groups (combinations of the foregoing). The stationary phase according to the present invention contains at least a halogen-bonding functional group. They may contain one or more additional other groups as listed above. Often, one functional group has more than one binding characteristic.

[0035] When "packing" a chromatography column in the binding and elution modes, a buffer is used to "pack" a sample or composition containing a target molecule and one or more impurities onto the chromatography column. The buffer has a conductivity and / or pH such that the target molecule binds to the stationary phase while ideally all impurities do not bind and flow through the column. Separation of the bound target molecule from one or more impurities can be additionally performed using changes in conductivity and / or pH such that the target molecule is washed or eluted before or after one or more impurities.

[0036] Typically, the buffer in which a sample is packed onto the stationary phase is referred to as a loading buffer or sample buffer. When "packing" a chromatography column to "flow" a target molecule, a buffer is used to "pack" a sample or composition containing a target molecule and one or more impurities onto the chromatography column. The buffer has a conductivity and / or pH such that the target molecule binds to the stationary phase and flows through the column while ideally all impurities do not bind to the column.

[0037] The term "equilibration" refers to the use of a buffer used to equilibrate the stationary phase prior to loading of the target molecule. Typically, the loading buffer is used for equilibration. "Washing" or "washing" the stationary phase means passing or bypassing a suitable liquid, such as a buffer, through the stationary phase. Typically, washing is used to remove weakly bound contaminants from the stationary phase in the binding / elution mode prior to elution of the target molecule, or to remove target molecules that do not bind or bind weakly after loading. In this case, typically, the wash buffer and the loading buffer are the same. If a virus inactivation buffer is used, it is used to inactivate any existing virus prior to elution of the target molecule. In this case, typically, the virus inactivation buffer contains a detergent(s) or may have different properties (pH / conductivity / salt or their amounts) and is thus different from the loading buffer.

[0038] Washing can also be used to remove contaminants from the stationary phase after elution of the target molecule. This is done by passing or flushing an appropriate liquid, such as a buffer, through the stationary phase after elution of the target molecule. In this case, typically the wash buffer is different from the loading buffer. It may contain a detergent(s) or may have different properties (pH / conductivity / salt or their amounts). The wash buffer can be, for example, an acidic buffer.

[0039] "Eluting" a molecule (e.g., a target molecule or an impurity) from the stationary phase means removing the molecule therefrom. Elution may be done directly in flow-through mode when the target molecule is eluted using the solvent front of the loading buffer, or by changing the solution conditions such that a buffer different from the loading buffer competes with the target molecule for the ligand on the stationary phase. A non-limiting example is eluting a molecule from an ion exchange resin by changing the ionic strength of the buffer surrounding the ion exchange material such that the buffer competes with the molecule for the charged sites on the ion exchange material.

[0040] As used interchangeably herein, the terms "flow-through process", "flow-through mode", and "flow-through operation" refer to a separation technique in which at least one target molecule contained in a sample together with one or more impurities is intended to flow through a chromatographic stationary phase that binds one or more impurities, where the target molecule generally does not bind (i.e., flows through), and is eluted from the stationary phase using a loading buffer. As used herein, the terms "bind and elute mode" and "bind and elute process" refer to a separation technique in which at least one target molecule contained in a sample binds to a suitable stationary phase and is eluted using a buffer different from the loading buffer.

[0041] Solid-phase extraction (SPE) is a method for preparing a sample in which compounds dissolved or suspended in a liquid mixture are separated from other compounds according to their physical and chemical properties. The result is that either the target molecule or the unwanted impurities in the sample are retained on the stationary phase. The fraction passing through the stationary phase is collected or disposed of depending on whether it contains the target molecule or the unwanted impurities. If the fraction retained on the stationary phase contains the target molecules, they are then removed from the stationary phase for collection in an additional step, where the stationary phase is rinsed with a suitable eluent.

[0042] Particle size is determined by laser diffraction, preferably by using a Malvern ‘Master Sizer. Pore size is determined by inverse SEC. Particle size is determined by sieving. An electron-withdrawing group includes a hydrogen, and an atom or group of atoms having an electron-withdrawing inductive effect and / or a mesomeric effect, and being more electronegative than hydrogen. Exemplary electron-withdrawing groups are H, I, Br, Cl, F, CO2H, NO2, CN. An electron-donating group includes an atom or group of atoms having an electron-donating inductive effect and / or a mesomeric effect, and being less electronegative than hydrogen. Exemplary electron-donating groups are -OH, O-alkyl.

[0043] An alkyl or alkyl group is a straight-chain or branched alkyl group typically having 1 to 20 carbon atoms or having the number of carbon atoms as indicated, for example, methyl, ethyl, isopropyl, propyl, butyl, sec-butyl or tert-butyl, and also pentyl, 1-, 2- or 3-methylbutyl, 1,1-, 1,2- or 2,2-dimethylpropyl, 1-ethylpropyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl or n-dodecyl, n-tridecyl, n-tetradecyl, n-pentadecyl, n-hexadecyl, n-heptadecyl, n-octadecyl, n-nonadecyl or n-eicosyl, and preferably, the alkyl group has 1 to 10 carbon atoms.

[0044] An alkenyl or alkenyl group is a straight-chain or branched alkenyl group typically having 2 to 20 carbon atoms, where in addition a plurality of double bonds may be present, for example, allyl, 2- or 3-butenyl, isobutenyl, sec-butenyl, and also 4-pentenyl, isopentenyl, hexenyl, heptenyl, octenyl, -C9H 17 、-C 10 H 19 ~-C 20 H 39 ; preferably allyl, 2- or 3-butenyl, isobutenyl, sec-butenyl. An aryl or aryl group is an aryl group typically having 6 to 12 carbon atoms, for example, phenyl, naphthyl or anthracenyl, which may be unsubstituted or substituted by Hal, NH2, NAlk2, NH alkyl, NO2, CN, SO3H or O alkyl. The substitution may be carried out one or more times, preferably once, by the indicated substituents.

[0045] The present invention provides a new class of stationary phases that can be used as separation materials, also referred to as resins or matrices, for chromatography or for solid-phase extraction. The stationary phases according to the present invention include a substrate and preferably functional groups covalently attached to the substrate. The principle of halogen bonding is based on different bonds to a stationary phase having Cl, Br or preferably I connected to an aromatic ring, whereby the aromatic ring contains a functional group that is positively charged either due to a positive charge directly adjacent to the ring or due to a positive charge within the ring. It has been found that it can be used to separate target molecules from other compounds in a mixture. The positive charge combined with the halogen atom linked to the aromatic ring structure results in an effective sigma hole at the halogen atom that enables effective halogen bonding to a Lewis base.

[0046] The substrate may consist of regularly shaped particles or spherical particles whose particle size can be between 2 and 1000 μm. A particle size between 3 and 300 μm is preferred. The substrate may, inter alia, be in the form of a non-porous core-shell or preferably a porous particle. The pore size can be between 2 and 300 nm. A pore size between 5 and 200 nm is preferred. The substrate may likewise also be in the form of a membrane, fiber, hollow fiber, coating, filter, capillary, surface or monolithic body. The monolithic body is preferably in the form of a porous three-dimensional body, for example a cylindrical form. Preferably, the substrate is a porous bead or membrane. The substrate may also be made by additional fabrication such as 3D printing.

[0047] In one aspect, the substrate is made of an inorganic material, such as metal oxides such as SiO2, Al2O3, titanium dioxide, zirconium dioxide, etc. It may also be made of other silica-based materials such as glass with controlled pores. The substrate may be an inorganic-organic hybrid material or any other combination of organic and inorganic materials. In another aspect, the substrate is made from natural polymers, preferably in the form of porous beads, for example, polysaccharides based on agarose, cellulose, cellulose derivatives and polymers based on dextran. Natural polymer beads are of the type known, for example, as Sepharose® or Sephadex®. In an alternative aspect, the substrate is a synthetic polymer, preferably a cross-linked synthetic polymer in the form of porous beads or membranes, for example, those made from styrene or styrene derivatives, divinylbenzene, acrylamide, acrylate esters, methacrylate esters, vinyl esters, vinylamides and the like. Polymers based on copolymers of polystyrene, polyvinyl alcohol or (meth)acrylate derivatives and comonomers having aliphatic hydroxyl groups are preferred. Polymers based on a given type of structure, for example, polymers based on polystyrene or polyvinyl ether polymers, are polymers containing such structures. They may also include those resulting from the copolymerization of other structures, for example, those from the copolymerization of two different monomers.

[0048] In another preferred aspect, the substrate is a copolymer formed by copolymerization of a polyvinyl ether-based material, in particular at least one compound from groups a) and b), a) at least one hydrophilic substituted alkyl vinyl ether represented by formula IX,

Chemical formula

[0049] b) at least one cross-linking agent conforming to formula X and / or XI and / or XII,

Chemical formula

[0050]

Chemical formula

Chemical formula

[0051] Here, Y1 and Y2 in formulas XI and XII are, independently of one another, C1-C10 alkyl or cycloalkyl, where one or more non-adjacent methylene groups or methylene groups not positioned in close proximity to N may be replaced by O, C=O, S, S=O, SO2, NH, NOH or N, and one or more H of the methylene groups are, independently of one another, a hydroxyl group, C1-C6-alkyl, halogen, NH2, C5-C10-aryl, NH(C1-C8)alkyl, N(C1-C8)alkyl 2、 may also be substituted by C1-C6-alkoxy or C1-C6-alkyl-OH, or, C6-C18 aryl, where one or more H in the aryl system are, independently of one another, a hydroxyl group, C1-C6-alkyl, halogen, NH2, NH(C1-C8)alkyl, N(C1-C8)alkyl 2、 may also be substituted by C1-C6-alkoxy or C1-C6-alkyl-OH, and

[0052] A is a divalent alkyl radical having 2 to 5 C atoms, preferably 2 or 3 C atoms, where one or more non-adjacent methylene groups or methylene groups not positioned in proximity to N may be replaced by O, C=O, S, S=O, SO2, NH, NOH or N, and one or more H of the methylene groups may, independently of one another, be substituted by a hydroxyl group, C1-C6-alkyl, halogen, NH2, C5-C10-aryl, NH(C1-C8)alkyl, N(C1-C8)alkyl 2、 and may also be substituted by C1-C6-alkoxy or C1-C6-alkyl-OH. R4 in formula IX is typically an alkyl radical, a cycloaliphatic radical or an aryl radical having at least one hydroxyl group.

[0053] In a highly preferred embodiment, the matrix is formed by copolymerization of a hydrophilic substituted alkyl vinyl ether selected from the group of 1,4-butanediol monovinyl ether, 1,5-pentanediol monovinyl ether, diethylene glycol monovinyl ether or cyclohexanedimethanol monovinyl ether and divinyl ethylene urea (1,3-divinylimidazolin-2-one) as crosslinking agents. Examples of suitable commercially available vinyl ether-based substrates are Eshmuno® from Merck KGaA, Germany.

[0054] The functional groups as defined above are attached to the substrate. This can be done via non-covalent or, preferably, covalent attachment. Covalent attachment can be carried out, for example, by directly bonding the functional group to a suitable residue on the substrate such as OH, NH2, carboxyl, phenol, anhydride, aldehyde, epoxide or thiol and the like.

[0055] Attachment of the functional group via a suitable linker is also possible. The structure of a suitable linker is --X--Z--Y--, where X and Y are first and second reactive or activatable groups, and X and Y are each independently selected from moieties such as, for example, hydroxy, amino, thiol, carboxy, oxiranyl, formyl, halo, isocyanate, and chlorosulfonyl; and Z is, for example, (a) C1-C15 alkyl, (b) aryl (c) C1-C10 alkylaryl, (d) C1-C10 alkylaryl, C1-C6 alkyl, where one or more carbon atoms of the alkyl may be replaced by oxygen, sulfur or nitrogen, and where the aryl includes, but is not limited to, phenyl, naphthyl, pyridyl or thienyl, and where one or more carbon atoms may be substituted by OH or C-C6 alkyl; and (e) a peptide of 2-10 amino acids, the amino acids including, but not limited to, glycine, alanine, valine, leucine, isoleucine, serine, threonine, aspartic acid, asparagine, glutamic acid, glutamine, lysine, hydroxy-lysine, histidine, arginine, phenylalanine, tyrosine, tryptophan, cysteine, methionine, ornithine, beta-alanine, homoserine, homotyrosine, homophenylalanine and citrulline, including L- and D-forms of amino acids, and may be selected from groups such as.

[0056] Other suitable spacer groups include, but are not limited to, p-benzoquinone, bis-(diazobenzidine), 3,6-bis-(mercurimethyl) dioxane, bisoxirane, cyanuric chloride, p,p'-difluoro-m,m'-, dicyclohexylcarbodiimide, dinitrophenyl sulfone, dimethyl adipimidate, dimethyl sperimidate, divinyl sulfone, N,N'-ethylene-bis-(iodoacetamide), glutaraldehyde, hexamethylene bis-(maleimide), hexamethylene diisocyanate, N,N'-1,3-phenylene-bis-(maleimide), phenol-2,4-disulfonyl chloride, tetra-azidized o-dianisidine, toluene diisocyanate, Woodward's K reagent, water-soluble carbodiimide, 6-aminohexanoic acid, hexamethylene diamine, 1,7-diamino-4-aza-heptane (3,3'-diamino-dipropylamine), and may include amino acids or peptides.

[0057] It is also possible to produce a stationary phase according to the present invention by polymerizing a monomer containing a functional group and a polymerizable moiety. Examples of stationary phases produced by polymerization of suitable monomers are polystyrene, polymethacrylamide or polyacrylamide-based stationary phases produced by polymerizing suitable styrene or acryloyl monomers.

[0058] In another aspect, the stationary phase can be produced by grafting a functional group onto a substrate. In the case of "grafting", the polymer chain must first be formed from monomers and in a second step be attached to the surface of the substrate. In the case of "undergoing grafting", the polymerization reaction is initiated on the surface of the substrate and the graft polymer is constructed directly from individual monomers. The method of "undergoing grafting" is preferred, and specifically preferred is a variant in which by-products such as non-covalently bound polymers that must be separated are hardly formed. A process using controlled free radical polymerization, for example, methods such as atom transfer radical polymerization (ATRP), is suitable. Here, the initiating group is covalently bound to the surface of the substrate at the desired density in the first step. The initiating group can be, for example, a halide bound via an ester functional group such as 2-bromo-2-methylpropionic acid ester. The graft polymerization is carried out in a second step in the presence of a copper(I) salt.

[0059] A highly preferred one-step grafting from a suitable polymerization reaction for the production of the stationary phase of the present invention can be initiated by cerium(IV) on a support containing hydroxyl without activating the support. This cerium(IV) that initiates grafting is preferably carried out according to EP 0 337 144 or US 5,453,186. The produced chains are linked to the substrate via monomer units. For this purpose, the substrate according to the present invention is suspended in a solution of the monomer, preferably in an aqueous solution. Grafting onto the polymer material occurs during the course of a conventional redox polymerization with oxygen excluded. The catalyst for the polymerization employed is cerium(IV) ions, and this catalyst is for forming free radical sites on the surface of the substrate where the graft polymerization of the monomer is initiated. This reaction is usually carried out in dilute mineral acid. For carrying out this graft polymerization, the acid is usually employed in an aqueous solution at a concentration in the range of 1 to 0.00001 mol / l, preferably 0.1 to 0.001. The use of dilute nitric acid employed at a concentration in the range of 0.1 to 0.001 mol / l is highly specifically preferred.

[0060] For the preparation of the separation material according to the present invention, the monomer is usually added in excess to the substrate. Typically, 0.05 to 100 mol of total monomer per liter of the deposited polymer material is employed, preferably 0.05 to 25 mol / l is employed. The polymerization is terminated by a termination reaction involving a cerium salt. For this reason, the (average) chain length can be affected by the concentration ratios of the substrate, initiator and monomer. Furthermore, either a uniform monomer or a mixture of different monomers can also be employed; in the latter case, a graft copolymer is formed.

[0061] The monomers preferably used for the preparation of the separation materials according to the invention are those according to formula XIII,

Chemical formula

[0062] The separation materials according to the invention preferably contain only a tentacle-like linear polymer structure grafted onto a substrate, constructed from monomers according to formula XIII. Preferably, they contain a linear polymer constructed from only one type of monomer according to formula XIII. However, linear polymers constructed by copolymerization of two or more different monomers according to formula XIII are also possible. Also possible are linear monomers constructed by copolymerization of one or more different monomers according to formula XIII and one or more other polymerizable monomers functionalized with ionic, hydrophilic or hydrophobic groups, such as other acrylamides, methacrylates, acrylates, methacrylates, etc.

[0063] The same is true for the stationary phases according to the invention produced by different processes. They may include one, two or more functional groups for halogen bonding as described above, and additional functional groups near the functional groups for halogen bonding. This may be, for example, an ionic group, a hydrophilic group or a hydrophobic group. By using two different functional groups to generate the stationary phase, a mixed-mode material with separation characteristics resulting from both or several types of functional groups is obtained, whereby, in the case of the present invention, at least one functional group is suitable for halogen bonding.

[0064] The present invention is further directed to a separation device comprising a stationary phase according to the invention. The device can be used, for example, for solid-phase extraction or for chromatographic applications. In either case, it includes means for holding the stationary phase. The stationary phase may be surrounded by or attached to the device. In one aspect, the device includes a housing having an inlet and an outlet. In another aspect, it is a flat plate or a pin with the stationary phase attached to one side. In another aspect, it is a filter comprising the stationary phase. In a preferred aspect, the device is a chromatography column comprising the stationary phase as described above according to the invention. Chromatography columns are known to those skilled in the art. Typically, it includes a cylindrical tube or cartridge filled with the stationary phase, and / or means for attaching the stationary phase in the tube or cartridge, and optionally, connections for delivering the solvent in and out of the tube or cartridge. The size of the chromatography varies depending on the application, for example, for analysis or preparative purposes.

[0065] The stationary phase according to the present invention can also be described as a substrate provided with a separation effector, whereby at least one separation effector contains a functional group as defined above. They are for the purpose of separating one or more target molecules from a sample liquid, selective, partially selective or non-selective binding or adsorption, or for the purpose of separating one or more secondary components from a matrix of secondary components, selective, partially selective or non-selective binding or adsorption, isolation, concentration and / or reduction of biopolymers from natural sources, isolation, concentration and / or reduction of biopolymers from recombinant sources, isolation, concentration and / or reduction of proteins and peptides, isolation, concentration and / or reduction of monoclonal and polyclonal antibodies, isolation, concentration and / or reduction of viruses, isolation, concentration and / or reduction of host cell proteins, isolation, concentration and / or reduction of ADCs, isolation, concentration and / or reduction of lipids such as alkaloids, diglycerides or triglycerides, carbohydrates, nucleic acids or other biomolecules, and can be used for.

[0066] The target molecule is separated from at least one or more other substances in the sample, whereby the sample containing the target molecule is either liquid or dissolved in a liquid that is brought into contact with the stationary phase according to the present invention. The contact time is generally in the range of 30 seconds to 24 hours. It is advantageous to operate according to the principle of liquid chromatography by passing the liquid through a chromatography column containing the stationary phase of the present invention. The liquid can flow through the column only by its gravity, or it can also be pumped in. An alternative method is batch chromatography, where the stationary phase is mixed with the liquid by stirring or shaking as long as the target molecule needs to bind to the stationary phase. It is also possible to operate according to the principle of chromatographic fluidized bed by introducing the liquid to be separated, for example, into a suspension containing the stationary phase, where the separation material is selected to be suitable for the desired separation by its high density and / or magnetic core.

[0067] When the chromatographic process is carried out in the binding and elution modes, the target molecule binds to the stationary phase according to the present invention. The stationary phase can optionally then be washed with a wash buffer, preferably one having the same ionic strength and the same pH as the liquid with which the target molecule is contacted with the stationary phase. The wash buffer removes all substances that do not bind to the stationary phase. Further washing steps with other suitable buffers may subsequently be carried out without desorbing the target molecule. Desorption of the bound target molecule is carried out by changing the ionic strength in the eluent and / or by changing the pH in the eluent and / or by changing the solvent. Thus, the target molecule can be obtained in a purified or concentrated form in the eluent. The target molecule, after desorption from the stationary phase, generally has a purity of from 70 percent to 90 percent, preferably from 85 percent to 99 percent, and specifically preferably from 90 percent to 99 percent.

[0068] However, when the chromatographic process is carried out in the flow-through mode, the target molecule remains in the liquid phase while other accompanying substances bind to the stationary phase. The target molecule is then obtained directly by collecting the column eluate in the through-flow. The stationary phase according to the present invention can be used for many different applications. They can be used, for example, for hydrophilic and hydrophobic separations as well as for aqueous and non-aqueous separations. Proteins such as insulin can be suitably purified, for example, on a stationary phase comprising a hydrophilic substrate to which functional groups are attached. Preferably, the functional groups are grafted onto the substrate. Example 2.1 shows one suitable stationary phase for this application. The chromatographic separation is then carried out by using an aqueous buffer system at neutral pH and by increasing the ionic strength of the buffer for elution, for example, by adding sodium chloride.

[0069] It is also possible to separate two proteins having different pl (isoelectric points) and / or different contents of carboxylic acids by halogen bonding using the chromatography column of the present invention. Carboxylic acids, especially those exposed on the surface of the protein, can interact with the stationary phase by halogen bonding. Proteins with a higher content of carboxylic acid groups that can interact with the stationary phase bind more strongly to the stationary phase and need to be eluted using a high conductivity. This is also shown in Example 2.2. However, a stationary phase based on a hydrophilic substrate to which the functional group is preferably bonded by grafting can also be used for the separation of target molecules in an organic solvent system. In this case, loading and elution can be carried out using the same solvent or using a solvent gradient by slowly increasing the amount of a more polar solvent compared to the packing solvent. The stationary phase according to the present invention can also include a hydrophobic substrate such as polystyrene. The functional group can be connected to the polystyrene substrate via a suitable linker, for example. Such a stationary phase can be suitably applied to the separation of low molecular weight compounds using an organic solvent for loading and elution.

[0070] The stationary phase and process according to the present invention provide a new chromatographic separation principle that is widely applicable. The use of a functional group containing at least one Cl, Br, or I bonded to a positively charged or chargeable aromatic ring structure provides a wide range of ligands that can be tailored to specific separation tasks. The choice of halogen can affect, for example, the strength of the interaction with the target molecule. Typically, iodine shows the strongest interaction, followed by Br, while chlorine-substituted functional groups are the weakest. In addition, by choosing a given aromatic ring structure or residue, the mode of interaction can also be modified. For example, typically, electron-withdrawing residues in an aromatic ring structure increase the halogen bonding effect, whereby electron-donating groups or alkyl groups decrease the strength of the halogen bond. Depending on the target molecule and the separation being carried out, the strength of the halogen bond of the functional group can be adjusted. Structures containing only one Cl, Br or I residue near H and no other residues can be used to start with. When the strength is increased, the type of halogen atom can be changed to Br or I, and / or the number of halogen atoms can be increased, and / or additional electron-withdrawing residues can be inserted. On the other hand, when the strength of the halogen bond is decreased, the type of halogen bond can be changed to Br or Cl, and / or an electron-donating group, such as an O-alkyl group, or an alkyl group can be inserted.

[0071] In one aspect, the halogen bonding properties of the stationary phase according to the invention can be switched on and off. When using a stationary phase containing a functional group as shown in formula I or II and having a positive charge adjacent to the aromatic ring by protonating the nitrogen atom of an aniline-like species, it is possible to "activate" the stationary phase under low pH conditions. As long as the nitrogen atom adjacent to the aromatic ring is protonated, it acts as an electron-withdrawing group, thereby supporting a sigma hole at the halogen atom positioned adjacent to the aromatic ring, and thus binding the target molecule via a halogen bond. Elution can then be carried out by raising the pH and deprotonating the N atom and thereby eliminating the electron-withdrawing function to "deactivate" the stationary phase.

[0072] It is also possible to suppress the Coulomb interaction of the stationary phase by using a high concentration of a salt such as sodium chloride. Without further elaboration, the skilled artisan can, using the preceding description, utilize the present invention to its fullest extent. Accordingly, the preferred specific embodiments and examples are to be construed as merely illustrative, and in no way limit the remainder of the disclosure herein, whatever the case may be. The entire disclosures of all applications, patents, and publications cited above and below, as well as the corresponding EP application EP19205109.2 filed on October 24, 2019, are hereby incorporated by reference.

[0073] Example The following examples present actual applications of the present invention. Synthesis Synthesis of N-(2-aminoethyl)-pyridinium chloride hydrochloride

Chemical formula

[0074] A round-bottom flask is filled with 2-chloroethylamine hydrochloride (4.04 g, 34.8 mmol, 1 eq.) and pyridine (5.40 mL, 68 mmol, 2 eq.). After the addition of water (14 mL), the two-phase system is stirred and heated to reflux for 14 hours. The clear yellow solution is washed with toluene (2 × 25 mL), n-pentane (20 mL), and isopropyl alcohol (3 × 50 mL), and evaporated to dryness in vacuo. N-(2-aminoethyl)-pyridinium chloride hydrochloride is obtained as a colorless solid. Yield: 5.54 g (28.4 mmol, 82%). 1H-NMR (D2O, 300 MHz): δ = 3.80 (m, 2H, H2N-CH2), 5.08 (m, 2H, H2C-N pyridine ), 8.24 (m, 2H, meta-H), 8.71 (m, 1H, para-H), 9.03 ppm (m, 2H, ortho-H). Mp.: 199~202 °C. MS (ESI) pos.: 123.0 (100 %) [M] + , 281.1 (22 %), 2[M] + Cl-.

[0075] Synthesis of N-(2-aminoethyl)-3-bromopyridinium chloride hydrochloride

Chem.

[0076] A solution of 2-chloroethylamine hydrochloride (30.2 g, 260 mmol, 1 eq.) and 3-bromopyridine (46.0 g, 291 mmol, 1.1 eq.) in ethanol (100 mL) is stirred and heated to reflux for 2 weeks. The precipitate is filtered and washed with ethanol (5 × 5 mL). N-(2-aminoethyl)-3-bromopyridinium chloride hydrochloride is obtained as a colorless solid. The filtrate is partially evaporated and reheated to reflux for 1 week. The resulting colorless solid is washed with ethanol (5 × 5 mL) and evaporated to dryness in vacuo and added to the previous product. Yield: 41.2 g, (211 mmol, 81%). 1 H-NM,R (D2O, 300 MHz): δ = 3.68 (t, 2H, J = 7 Hz, H2N-CH2), 4.95 (t, 2H, J = 7 Hz H2C-N pyridine ), 8.03 (m, 1H, meta-H), 8.79 (m, 1H, para-H), 8.94 (m, 1H, ortho-CH-CH-) 9.25 ppm (m, 1H, ortho-CH-CBr-). Elemental analysis: calc.: N 10.26%, C 30.69%, H 4.05%, measured: N 10.34%, C 30.76%, H 3.99%. Mp.: 237 °C MS (ESI) pos.: 202.9 (100 %) [M] + , 157.8 (6 %) [3-bromopyridinium] + .

[0077] Synthesis of N-(2-aminoethyl)-3-iodopyridinium chloride hydrochloride

Chem.

[0078] 2-Chloroethylamine hydrochloride (30.0 g, 259 mmol, 1 eq.) is dissolved in ethanol (100 mL) using 3-iodopyridine (57.2 g, 279 mmol, 1.1 eq.), stirred, and heated to reflux for 1 week. The precipitate is filtered and washed with ethanol (6 × 20 mL). N-(2-Aminoethyl)-3-bromopyridinium chloride hydrochloride is obtained as a colorless solid. The filtrate is partially evaporated and reheated to reflux for 1 week. The resulting colorless solid is washed with ethanol (4 × 5 mL), evaporated to dryness in vacuo, and added to the previous product. This procedure is repeated until a total yield of 76% is achieved. Yield: 68.2 g, (213 mmol, 76%). Mp.: 268.2 °C Elemental analysis: calc.: N 8.73%, C 26.19%, H 3.45%, measured: N 8.82%, C 26.24%, H 3.41%. MS (ESI) pos.: 248.9 (100 %) [M] + 。 1 H-NMR (D2O, 300 MHz): δ = 3.64 (t, 2H, J = 7 Hz, H2N-CH2), 4.88 (t, 2H, J = 7 Hz, H2C-N pyridine ), 7.84 (t, J = 6 Hz 1H, meta-H), 8.91 (m, 2H, para-H, ortho-CH-CH-), 9.28 ppm (s, 1H, ortho-CH-CBr-).

[0079] Synthesis of 1-(2-Methacrylamidoethyl)-pyridinium chloride

Chemical formula

[0080] N-(2-Aminoethyl)-pyridinium chloride hydrochloride (38.6 g, 147 mmol) is dissolved in water (50 mL), then dichloromethane (200 mL), and tributylamine (54.7 g, 295 mmol) and methacrylic anhydride (24.8 g, 161 mmol) are added. The reaction mixture is stirred at room temperature for 18 h, and then for the remaining N-(2-aminoethyl)-pyridinium chloride 1 confirmation is carried out by 1H-NMR spectroscopy. In this case, the aqueous layer is separated and mixed again using dichloromethane, and tributylamine and methacrylic anhydride are added according to the remaining extract. The aqueous layer is separated and washed with dichloromethane (2 × 50 mL). Due to the polymerization nature of the product, the yield is determined by 1H-NMR spectroscopy in an aqueous solution using 1 tetraphenylphosphonium chloride. Yield: 26.2 g (116 mmol, 71%) MS (ESI) pos.: 191.0 (100 %) [M] + , 234.1 (28 %) [1-(2-((2-Methacrylamidoethyl)amino)ethyl)pyridinium] + . 1 1H-NMR (H2O, 300 MHz): δ = 1.69 (s, 3H, CH3), 3.77 (q, 2H, J = 6 Hz, HN-CH2), 4.72 (t, 2H, J = 5 Hz, HN-CH2-CH2), 5.32 (s, 1H, =CH a ), 5.51 (s, 1H, =CH b ), 8.01 (t, J = 7 Hz, 2H, meta-H), 8.18 (m, 1H, NH), 8.49 (m, 1H, J = 8 Hz, para-H), 8.81 (d, 2H, J = 6 Hz, ortho-H).

[0081] Synthesis of 1-(2-Methacrylamidoethyl)-3-bromopyridinium chloride

Chemical formula

[0082] N-(2-Aminoethyl)-3-bromopyridinium chloride hydrochloride (3.09 g, 13.0 mmol) is dissolved in water (5 mL), then dichloromethane (50 mL), and tributylamine (3.38 g, 14.2 mmol) and methacrylic anhydride (2.24 g, 14.2 mmol) are added. The reaction mixture is stirred at room temperature for 19 h, and then for the remaining N-(2-aminoethyl)-3-bromopyridinium chloride 1 is confirmed by 1H-NMR spectroscopy. In this case, the aqueous layer is separated and mixed again with dichloromethane, and tributylamine and methacrylic anhydride are added according to the remaining extract. After stirring for 6 h at room temperature, the aqueous phase is separated, washed with dichloromethane (3 × 30 mL), and evaporated to dryness in vacuo. 3-Bromo-1-(2-methacrylamidoethyl)-pyridinium chloride is obtained as a brown glass. Yield: 2.77 g (9.02 mmol, 70%). MS (ESI) pos.: 217.0 (100 %) [M] + 、575.0 (6 %) 2[M] + Cl - 。 1 1H-NMR (D2O, 300 MHz): δ = 1.78 (s, 3H, CH3), 3.81 (m, 2H, HN-CH2), XXX (m, 2H, HN-CH2-CH2-), 5.41 (s, 1H, =CH 2(A) ), 5.58 (s, 1H, =CH 2(B) ), 7.97 (m, 1H, meta-H), 8.25 (m, 1H, para-H), 8.88 (m, 1H, N-CH-CH), 9.20 (s, 1H, N-CH-CBr).

[0083] Synthesis of 3-iodo-1-(2-methacrylamidoethyl)-pyridinium chloride

Chemical formula

[0084] N-(2-Aminoethyl)-3-iodopyridinium chloride hydrochloride (32.0 g, 99.7 mmol) is dissolved in water (50 mL), then dichloromethane (200 mL), and tributylamine (37.0 g, 200 mmol) and methacrylic anhydride (17.8 g, 116 mmol) are added. The reaction mixture is stirred at room temperature for 17 h, and then for the remaining N-(2-aminoethyl)-3-iodopyridinium chloride 1 it is confirmed by 1H-NMR spectroscopy. In this case, the aqueous layer is separated and mixed again with dichloromethane, and tributylamine and methacrylic anhydride are added according to the remaining extract. The aqueous phase is separated, washed with dichloromethane (2 × 100 mL), and evaporated to dryness. 3-Iodo-1-(2-methacrylamidoethyl)-pyridinium chloride remains as a brown solid. Yield: 33.76 g (95.7 mmol, 96%). Mp.: 254.5 °C Elemental analysis: calc.: N 7.94%, C 37.47%, H 4.00%, found: N 7.67%, C 36.14%, H 4.35%. MS (ESI) pos.: 317.0 (100 %) [M]+ + 。 1 1H-NMR (D2O, 300 MHz): δ = 1.79 (s, 3H, CH3), 3.80 (t, J = 6 Hz, 2H, HN-NH2), 5.41 (s, 1H, =CH2), 5.57 (s, 1H, =CH2), 7.79 (t, 1H, J = 7 Hz, meta-H), 8.21 (m, 1H, NH), 8.86 (m, 2H, N-CH-CH-CH-), 9.22 ppm (s, 1H, N-CH-CI-). 13C{H}-DEPT135-NMR (D2O, 75 MHz): δ = 17.7 (CH3), 39.8 (-NH-CH2-), 61.2 (-NH-CH2-CH2-), 82.3 (CI), 121.8 (=CH2), 128.5 (meta-C), 138.3 (H3C-C), 143.7 (N-CH-CH-), 150.3 (N-CH-CI-), 154.4 (para-C), 172.2 ppm (C=O). 1 H, 13 C-HMBC (D2O, 300 / 75 MHz): δ = 4.7 / 61.2 ppm (J = 147 Hz, NH-CH2-CH2-N).

[0085] Synthesis of N-(3-bromophenyl)ethane-1,2-diamine

Chemical formula

[0086] Provide 3-bromoaniline (3.000 g, 25.86 mmol), 2-chloroethane-1-amine hydrochloride (4.786 g, 27.82 mmol) and ethanol (11 mL) into a 100 mL breaker. Stir the reaction mixture at room temperature for several minutes. Then provide it into a microwave reaction vessel. Place the reaction vessel in the microwave and select and start the following method: Method: Quick test; Temperature: 110 °C; Output: 300 W; Pressure: x bar; Holding time: 30 min; Stirring: Off; Cooling: Off. After cooling, select and start a new method: New method; Temperature: 120 °C; Output: 300 W; Pressure: 8 bar; Holding time: 30 min; Stirring: On (med); Cooling: On. After cooling, filter the pink crystals overnight, wash them with isopropyl alcohol, and evaporate to dryness in vacuo. Yield: 1.221 g (4.24 mmol, 16%). Mp.: 212.0 °C - 213.0 °C 1H-NMR (D2O, 300 MHz): δ = 3.12 (m, 2H, H2N-CH2), 3.38 (m, 2H, HN-CH2), 6.66 (m, 1H, ortho-CH-CH), 6.88 (m, 2H, ortho-CH-CBr, para-H), 7.07 (m, 1H, meta-H).

[0087] Functionalization of beads using 1-(2-methacrylamidoethyl)-pyridinium chloride

Chemical formula

[0088] 1-(2-Methacrylamidoethyl)-pyridinium chloride in an aqueous solution (1.29 mol / L, 2.13 mL, 2.75 mmol) is provided into a three-necked round-bottom flask, and water (15.3 mL) is added. The pH is set to 1.5 using nitric acid. A substrate made of hydrophilic polyvinyl ether beads having OH groups is added, similar to the substrate of the commercially available Eshmuno® adsorbent (17.0 g, wet) (beads). The starting solution is prepared by adding nitric acid (239 μL, 3.85 mmol), water (7 mL), and cerium ammonium nitrate (515 mg, 939 μmol) to a dropping funnel. Both mixtures are degassed by vacuum and nitrogen. The suspension is heated to 30 °C, and the starting solution is added as fast as possible. The mixture is stirred for 4 hours and then filtered and washed as follows: 3 × 100 mL deionized water (ultrapure water / Milli-Q® water) = VE water (VE wasser) 7 × 100 mL 1M sulfuric acid, 0.2M ascorbic acid 10 × 100 mL deionized water 2 × 100 mL 1M sodium hydroxide solution 2 × 100 mL deionized water The pH is adjusted to 6.5 - 7.0 using 25% hydrochloric acid. 1 × 100 mL deionized water 1 × 100 mL ethanol. The resulting colorless solid is dried in vacuo. Yield: 3.38 g

[0089] Functionalization of beads using 1-(2-methacrylamidoethyl)-3-bromopyridinium chloride

Chem.

[0090] 1-(2-Methacrylamidoethyl)-3-bromopyridinium chloride (838 mg, 2.75 mmol) is provided in a three-necked round-bottom flask, and water (17.5 mL) is added. The pH is set to 1.5 using nitric acid. Beads (17.0 g, wet) are added. The starting solution is prepared by adding nitric acid (239 μL, 3.85 mmol), water (7 mL), and cerium ammonium nitrate (515 mg, 939 μmol) to a dropping funnel. Both mixtures are degassed by vacuum and nitrogen. The suspension is heated to 30 °C, and the starting solution is added as fast as possible. The mixture is stirred for 4 hours, then filtered and washed as described above. The resulting colorless solid is dried in vacuo. Yield: 3.17 g

[0091] Functionalization of beads using 1-(2-methacrylamidoethyl)-3-iodopyridinium chloride

Chem.

[0092] 1-(2-Methacrylamidoethyl)-3-iodopyridinium chloride (970 mg, 2.75 mmol) is provided in a three-necked round-bottom flask, and water (17.5 mL) is added. The pH is set to 1.5 using nitric acid. Beads (17.0 g, wet) are added. The starting solution is prepared by adding nitric acid (239 μL, 3.85 mmol), water (7 mL) and cerium ammonium nitrate (515 mg, 939 μmol) to a dropping funnel. Both mixtures are degassed by vacuum and nitrogen. The suspension is heated to 30 °C and the starting solution is added as fast as possible. The mixture is stirred for 4 hours, then filtered and washed as described above. The resulting colorless solid is dried in vacuo. Yield: 3.22 g

[0093] Synthesis of styrene-based stationary phase

Chemical formula

[0094] The reactor is filled with VE-water, sodium dodecane-1-sulfonate (surfactant) and polyvinyl alcohol 40 - 88 (stabilizer), and an organic phase consisting of DVB / ethylstyrene (80 / 20, 83.2 g), EGDMA (41.6 g), 4-vinylbenzyl chloride (13.9 g), toluene (90.3 g), 2-ethylhexan-1-ol (90.3 g) and AIBN (1.0 g) is added. The stirrer speed is set to 500 rpm for 10 sec, and the resulting emulsion is stirred at 25 °C for 30 minutes. Water (640 g) is added and the stirrer speed is set to 120 rpm. After the emulsion is stabilized, the temperature ramp is started. Hold at 25 °C to 72 °C at 90 min for 120 min, and hold at 72 °C for 120 min at 120 min. Hold at 72 °C to 82 °C at 20 min for 120 min, and hold at 82 °C for 120 min. Then, the temperature is decreased to 60 °C at 30 min and held for 12 hours. The resulting polymer is washed with VE water and an organic solvent. Next, the polymer is dried in vacuo at 50 °C for 24 hours. Analysis Calculated Cl content: 2.33% Cl X-ray fluorescence analysis: 2.5% Cl Elemental analysis: 0.62% Cl

[0095] Attachment of functional groups Styrene-based polymers can be functionalized with chlorine. For this purpose, chlorine is first exchanged to iodine by the Finkelstein reaction and then functional groups are induced.

Chemical formula

[0096] 2. Application examples 2.1 Use of functionalized beads (using the tentacles prepared as described above) for the hydrophilic separation of insulin A chromatography column is prepared using the functionalized beads. The column is prepared using beads having functional groups functionalized with iodine and beads having functional groups without iodine. Thereby, it can be confirmed that the resulting separation is truly due to halogen bonding if it is seen only for the stationary phase having iodine and not for the same stationary phase not functionalized with iodine. A sample containing insulin and desamino insulin is processed as follows:

[0097] Condition 1: Eluent: (A) 50 mM (NH4)2SO4 pH 3.5 (B) 50 mM (NH4)2SO4 + 1 M NaCl pH 3.5 Gradient: 0 - 15 min 100% (A) 30 - 50 min 50% (A) 50% (B) Flow rate: 0.5 mL / min Detection: UV 214 nm Temp.: 25 °C

[0098] The results can be seen in Figure 1. The graphs show the chromatograms for the following stationary phases: A Beads functionalized with a functional group having iodine B Beads functionalized with a functional group having iodine (half the amount of functional groups compared to A) C Beads functionalized with a functional group having no iodine D Beads functionalized with a functional group having iodine (half the amount of functional groups compared to C) E Base beads that are not functionalized It can be seen that graphs A and B using materials with iodine groups show stronger retention compared to materials without iodine. However, insulin and A21-desamino insulin are not separated.

[0099] Condition 2: Eluent: (A) 100 mM NH4H2PO4 pH 7.3 (B) 100 mM NH4H2PO4 + 1 M NaCl pH 7.3 Gradient: 0 - 15 min 100% (A) 30 - 50 min 50% (A) 50% (B) Flow rate: 0.5 mL / min Detection: UV 214 nm Temp.: 25 °C

[0100] The results can be seen in Figure 2. The graphs show the chromatograms for the following stationary phases: A Beads functionalized with a functional group having iodine B Beads functionalized with a functional group having iodine (half the amount of functional groups compared to A) C Beads functionalized with a functional group having no iodine D Beads functionalized with a functional group having no iodine (half the amount of functional groups compared to C) E Base beads that are not functionalized Graphs A and B using materials with iodine groups show strong retention compared to materials without iodine and it is found that they show separation of insulin and A21-desamido insulin. Materials C, D, and E show only very little retention and separation of insulin and A21-desamido insulin. This indicates that the retention and separation of insulin and A21-desamido insulin are actually based on halogen bonding. The only difference between the materials used in A / B and C / D is the iodine residue.

[0101] 2.2 Use of functionalized beads (using the tentacles prepared as described above) for hydrophilic separation of different proteins Proteins with a lower pl (isoelectric point) tend to bind more strongly to the iodinated stationary phase according to the present invention than proteins with a higher pl. A stronger ionic strength is required to elute proteins with a lower pl. Also, proteins with a higher content of carboxylic acid tend to bind more strongly to the stationary phase. This effect depends on the tertiary structure of the protein and thus the exposure of carboxylic acid must also be considered. Thus, proteins such as Carboanhydrase and InlB321-GFP can be easily separated. A sample containing one of the proteins listed in the following table is processed as follows:

[0102] Eluent: (A) 20 mM Tris(hydroxymethyl)aminomethane pH 8 (B) 20 mM Tris(hydroxymethyl)aminomethane + 1 M NaCl pH 8 Gradient: 0 - 16 mL 100% (A) 16 - 96 mL 100% (A) - 50% (A) 50% (B) 196 - 120 mL 100% (B) Flow rate: 4 mL / min Detection: UV280 / UV260 Temp.: 4°C

[0103]

Table 1

[0104] This table shows the names of the proteins tested and their individual values. Molecular weight at MW: kDa, isoelectric point: pI, # carboxylic acids divided by the molecular weight: # carboxy / MW, and conductivity of the eluate at the maximum of the elution peak. The interaction between the functional group on the stationary phase and the carboxylic acid was revealed by the crystal structure obtained from 3-iodo-1-methylpyridinium acetate. The sum of the van der Waals radii is 345 pm, but the distance between iodine and oxygen was 286 pm. Therefore, there is a binding interaction.

[0105] 2.3 Use of functionalized beads (prepared as described above) for the hydrophobic separation of low molecular weight compounds Pack the stationary phase into a glass column. The eluent is n-pentane. Wash the column with n-pentane and then apply the sample to the column. Detection is carried out at the column outlet using a silica gel TLC plate equipped with a UV indicator and a UV lamp. R f is calculated based on the weight of the solvent used up to the elution of the compound, divided by the mass of the solvent per column volume. The results can be seen in Table 1.

[0106]

Table 2

[0107] The resin with R = H is treated as a reference. Different substances with an electron-donating function were flowed as the mobile phase with n-pentane. The results show retention coefficients that do not differ greatly between the reference material and the Br-substituted material. However, the retention coefficient of the I-substituted material shows a significant increase in comparison with the Br-substituted material (percentage in parentheses). The restraint rates are 25% and 52% higher, depending on the functional group of the substance.

Claims

1. A stationary phase comprising a substrate and at least one type of functional group, where the functional group is -N(R' ２ ), ＋ -Z, where R' is H or C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkynyl, C6 aryl, and Z is 【Chemical 1】 wherein L is attached to -N(R' ２ ) ＋ and is bonded to X and X' are, independently of one another, H, I, Br or Cl, or another electron-withdrawing group such as F or NO ２ or a C1-C10 alkyl, C1-C10 alkenyl, C1-C10 alkynyl, C6-C12 aryl, or an electron-donating group, R is H, C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkynyl, or C6 aryl; L / R / X represents L, R, or X; Y is independently C or N; wherein in Formula I, at least one of X or X' is Cl, Br, or I; wherein in Formula II, at least one X is present, at least one X is Cl, Br, or I, and at least one L is present, wherein at least one Y is C, and for each Y that is N, L / R / X is absent at this N; or the functional group is a positively charged heterocyclic aromatic group having at least one Cl, Br, or I residue; the positively charged heterocyclic aromatic group having at least one Cl, Br, or I residue has one of the following structures III, IV, and V: 【Chemical 2】 X is, independently of one another, H, I, Br or Cl, and X' is, independently of one another, H, I, Br, Cl, F, NO ２ , or another electron-withdrawing group, or C1-C10 alkyl, C1-C10 alkenyl, C1-C10 alkynyl, C6-C12 aryl, or an electron-donating group, where at least one of X or X' is Cl, Br or I; 【Chemical Formula 3】 R is H, C1-C10 alkyl, aryl, C1-C10 alkenyl, C1-C10 alkynyl, or an electron-withdrawing group or an electron-donating group; Y is independently N or P; X is, independently of one another, H, I, Br or Cl, and X' is, independently of one another, H, I, Br, Cl, F, NO ２ , or another electron-withdrawing group, or C1-C10 alkyl, C1-C10 alkenyl, C1-C10 alkynyl, C6-C12 aryl, or an electron-donating group, where at least one of X or X' is Cl, Br or I; 【Chemical Formula 4】 Here, atom A of aromatic ring 1, which does not provide connection to the substrate, is O ＋ or S ＋ and the other ring atoms A are C-X', where X' is, independently of one another, H, I, Br, Cl, or another electron-withdrawing group, or C1-C10 alkyl, C1-C10 alkenyl, C1-C10 alkynyl, C6-C12 aryl, or an electron-donating group, where at least one X' is Cl, Br or I; or has one of the following structures: 【Chemical Formula 5】 X is independently H, a C1-C6 alkyl group, I, Br, or Cl, and X' and X'' are independently H, a C1-C6 alkyl group, I, Br, Cl, or another electron-withdrawing group or electron-donating group, R is H, C1-C6 alkyl, C6 aryl, C1-C6 alkenyl, C1-C6 alkynyl, or an electron-withdrawing group or an electron-donating group, and wherein at least one of X or X' is Cl, Br, or I; the stationary phase, characterized in that.

2. The stationary phase according to Claim 1, characterized in that the functional group has one heterocyclic ring.

3. The stationary phase according to Claim 1 or 2, characterized in that the functional group is selected from one or more of the following: ​ R' is H or C1-C6 alkyl; EWs are, independently of one another, H, CH ３ , or an electron-withdrawing group, and Hal are, independently of one another, H, CH ３ , Cl, Br or I, where at least one of Hal is Cl, Br or I; 【Chemical Formula 7】 EWs are, independently of each other, H, CH ３ , an electron-withdrawing group, and Hal are, independently of one another, H, CH ３、 Cl, Br or I, where at least one of Hal is Cl, Br or I; [Chemical Formula 8] R' is H or C1-C6 alkyl; EWs are, independently of each other, H, CH ３ or an electron-withdrawing group, and Hal is Cl, Br, or I; 【Chemical Formula 9】 R / L represents R or L; one R / L is H or C1-C6 alkyl, and the other R / L is a connection to the substrate; EW is H, CH ３ or an electron-withdrawing group, and Hal is Cl, Br, or I.

4. The stationary phase according to any one of Claims 1 to 3, characterized in that the functional group is covalently bonded to the substrate.

5. The stationary phase according to any one of claims 1 to 4, wherein the substrate is beads or a film.

6. The stationary phase according to any one of claims 1 to 5, wherein the functional group is bonded to the substrate via a linker.

7. The stationary phase according to any one of claims 1 to 5, wherein the functional group is the end group of a polymer chain grafted onto the substrate.

8. The stationary phase according to any one of claims 1 to 7, wherein the substrate is an organic polymer.

9. A chromatography column comprising the stationary phase according to any one of claims 1 to 8.

10. A process for the separation of at least one target molecule from at least one other compound, wherein the stationary phase according to any one of claims 1 to 8 is contacted with a liquid comprising the target molecule and at least one other compound, and wherein the target molecule exhibits an interaction with the stationary phase that is different from the interaction of the other compound.

11. The process according to claim 10, wherein the more the target molecule can donate electrons, the stronger it binds to the stationary phase.

12. The process is a process for chromatographic separation of at least one target molecule from at least one other compound, wherein the stationary phase according to any one of claims 1 to 8 is present in a chromatography column, and a liquid comprising the target molecule and at least one other compound flows through the column, wherein the target molecule and the other compound present in the liquid are eluted from the column according to their interaction with the stationary phase. The process according to claim 10 or 11, characterized in that

13. The process according to any one of claims 10 to 12, wherein the target molecule is a protein.

14. The process according to any one of claims 10 to 13, characterized in that it is carried out by filling the stationary phase according to any one of claims 1 to 8 and an aqueous loading buffer of a predetermined pH and a predetermined ionic strength, and eluting the target molecule with an aqueous elution buffer of the same pH but a different ionic strength.

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