Materials and methods for separations based on halogen bonding

Abstract

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

Description

Technical Field

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

[0002] 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 Lewis base, an otherwise neutral or anionic Lewis base with a lone pair of electrons. X may be chlorine, bromine or iodine. Halogen bonding can be explained by the presence of a positive electrostatic potential region, i.e., a σ-hole, centered on the RX axis on the outermost part of the halogen surface. From chlorine to bromine to iodine, the intensity of the interaction increases and can be further modified by modifying the residue R of the halogen. Typically, the interaction strength in halogen bonding is between 10 and 180 kJ / mol, and is therefore comparable to the strength of a hydrogen bridge. This can be seen, for example, from DOI: 10.2021 / acs.chem.rev.5b00484.

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

[0004] It has now been found that the principle of halogen bonding can be used not only to separate halogen-containing molecules RX that form the Lewis acid portion of the bond, but also to separate Lewis bases. The inventors have found that by fixing certain molecules RX on a solid phase, a wide range of target molecules containing Lewis base portions will be able to interact with the fixed RX. Through this interaction, new chromatographic separation principles can be established, thereby providing additional degrees of freedom for chromatographic separation. It can be shown that even proteins can be separated based on halogen bonding. Summary of the invention

[0005] The present invention therefore relates to a stationary phase comprising a base material and at least one type of functional groups, wherein the functional groups are

[0006] ---N(R'2) +-ArX, wherein R' is H or C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkynyl, C6 aryl, preferably methyl, ethyl, propyl, isopropyl, and ArX is

[0007] I or II

[0008] in

[0009] L is the same as --N(R'2) + Key

[0010] X and X' are independently H, I, Br or Cl or another electron withdrawing group such as F or NO2 or C1-C10 alkyl, C1-C10 alkenyl, C1-C10 alkynyl, C6-C12 aryl or an electron donating group,

[0011] R is H or C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkynyl, C6 aryl

[0012] Y is independently C or N

[0013] Wherein in formula I, at least one X or X' is Cl, Br or I, preferably one or two of X are Cl, Br or I,

[0014] Wherein at least one L is present in Formula II and

[0015] wherein at least one Y is C, and wherein for each Y that is N, there is no L / R / X at that N;

[0016] Alternatively, the functional group is a positively charged heterocyclic aromatic group carrying at least one Cl, Br or I residue.

[0017] In a preferred embodiment, the positively charged heterocyclic aromatic group carrying at least one Cl, Br or I residue has one of the following structures:

[0018] III

[0019] Wherein X is H, I, Br or Cl, X' is independently 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 electron donating group, wherein at least one X or X' is Cl, Br or I, preferably at least one X is Cl, Br or I.

[0020] The serpentine line in formula III and in the following formulae shows the connection to the base material. This may be a single bond or preferably a linking group or another structure, for example part of an antenna.

[0021] IV

[0022] wherein R is H, C1-C10 alkyl, aryl, C1-C10 alkenyl, C1-C10 alkynyl, or an electron withdrawing group or an electron donating group,

[0023] Y is independently N or P,

[0024] X is H, I, Br or Cl, and X' is independently H, I, Br, Cl, F, NO2 or another electron-withdrawing group or a C1-C10 alkyl group, a C1-C10 alkenyl group, a C1-C10 alkynyl group, a C6-C12 aryl group or an electron-donating group, wherein at least one X or X' is Cl, Br or I, preferably X is Cl, Br or I.

[0025] Examples of possible structures according to formula IV are:

[0026]

[0027] Another possible structure is similar to Formula V,

[0028] V

[0029] Where one atom A of the aromatic ring (not the one providing the connection to the base material) is O + or S + , and the other ring atoms A are C-X', wherein X' are independently H, a C1 to C6 alkyl group, I, Br, Cl or another electron withdrawing group or a C1-C10 alkyl group, a C1-C10 alkenyl group, a C1-C10 alkynyl group, a C6-C12 aryl group or an electron donating group, wherein at least one X' is Cl, Br or I.

[0030] Examples of possible structures according to formula V are:

[0031]

[0032] wherein X is independently H, a C1 to C6 alkyl group, I, Br or Cl, and X' is independently H, a C1 to C6 alkyl group, I, Br, Cl or another electron withdrawing group or a C1-C10 alkyl group, a C1-C10 alkenyl group, a C1-C10 alkynyl group, a C6-C12 aryl group or an electron donating group, wherein at least one X or X' is Cl, Br or I, preferably X is Cl, Br or I.

[0033] The same structure is possible with S replacing O.

[0034] Other possible structures are:

[0035]

[0036] wherein X is independently H, a C1 to C6 alkyl group, I, Br or Cl, X' and X" are independently H, I, Br, Cl or another electron withdrawing group or a C1-C10 alkyl, C1-C10 alkenyl, C1-C10 alkynyl, C6-C12 aryl 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 X or X' is Cl, Br or I, preferably X is Cl, Br or I.

[0037] In a preferred embodiment, the residue or substituent of the functional group comprises only Cl, Br and / or I and H, C1-C6 alkyl and / or one or more electron withdrawing groups.

[0038] In a preferred embodiment, the functional group has only one heterocyclic ring.

[0039] In another preferred embodiment, the heterocyclic aromatic group has one type of heteroatom.

[0040] In a preferred embodiment, the heteroatom in the heterocycle is a N atom.

[0041] In another preferred embodiment, the aromatic ring of the functional group is substituted with one or two iodine atoms.

[0042] The most preferred functional group structure is:

[0043] VI

[0044] Wherein R' is H or C1-C6 alkyl, preferably methyl, ethyl, propyl, isopropyl,

[0045] EW is independently H, CH3 or an electron withdrawing group, preferably H, CH3, F, Cl, NO2

[0046] And Hal are independently H, CH 3 , Cl, Br or I, preferably Br or I, wherein at least one Hal is Cl, Br or I.

[0047] VII

[0048] Wherein EW is independently H, CH3, an electron withdrawing group, preferably H, F, Cl, NO2

[0049] And Hal are independently H, CH 3 , Cl, Br or I, preferably Br or I, wherein at least one Hal is Cl, Br or I.

[0050] VIII

[0051] Wherein R' is H or C1-C6 alkyl, preferably methyl, ethyl, propyl, isopropyl,

[0052] EW is independently H, CH3 or an electron withdrawing group, preferably H, F, Cl, NO2

[0053] And Hal is Cl, Br or I, preferably Br or I.

[0054]

[0055] One of the R / L is H or C1-C6 alkyl, preferably methyl, ethyl, propyl, isopropyl and the other R / L is a connection to the base material,

[0056] EW is H, CH3 or an electron-withdrawing group, preferably F, Cl, NO2

[0057] And Hal is Cl, Br or I, preferably Br or I.

[0058] Very preferred are functional groups according to structures VI, VII, VIII or IX, most preferably VII, which are linked to the solid support via polymer chains (also called antennae) by a grafting process as described below using monomers according to formula XIII, wherein R 3 is a functional group according to structures VI, VII, VIII and / or IX. Optionally, other R 3 of other monomers according to formula III, thereby generating a polymer chain having two or more different functional groups, at least one functional group of which is selected from structures VI, VII, VIII and / or IX.

[0059] In the case where the functional group has one or more positive charges, a negative counterion is present. The type of counterion is not important. Suitable counterions are halides, acetate, nitrate, triflate, and the like.

[0060] In a preferred embodiment, the functional groups are covalently bonded to the base material.

[0061] In one embodiment, the base material is a bead or a film.

[0062] In one embodiment, the functional group is bonded to the base material via a linking group.

[0063] In one embodiment, the functional groups are contained in polymer chains, also referred to as tentacles, which are grafted to the base material.

[0064] In a preferred embodiment, the base material is an organic polymer, preferably a polystyrene-based or polyvinyl ether-based polymer.

[0065] The invention also relates to a separation device comprising the stationary phase according to the invention.

[0066] In a preferred embodiment, the separation device is a chromatography column comprising the stationary phase of the invention.

[0067] The present invention also relates to a method for separating a target molecule from at least one other compound, wherein a stationary phase according to the invention as described above, preferably present in a separation device, is contacted with a liquid comprising the target molecule and at least one other compound, and wherein the target molecule exhibits a binding to the stationary phase that is different from the binding of the other compounds, for example its interaction with the stationary phase is stronger or weaker than that of the other compounds.

[0068] In a preferred embodiment, the more electron donating a compound is, the more strongly it binds to the stationary phase.

[0069] In a preferred embodiment, the method is a method for chromatographically separating a target molecule from at least one other compound, wherein the stationary phase according to the present invention as described above is present in a chromatographic column, and a liquid containing the target molecule and at least one other compound is passed through the column, whereby the target molecule and other compounds present in the liquid are eluted from the column depending on their interaction with the stationary phase.

[0070] In a preferred embodiment, the target molecule is a protein.

[0071] In a preferred embodiment, the method is carried out by loading the stationary phase according to the present invention with an aqueous loading buffer of a certain pH and a certain ionic strength, and eluting the target molecule with an aqueous elution buffer having the same pH but another ionic strength, wherein the other ionic strength is generally a higher ionic strength.

[0072] In another embodiment, the method is performed by loading the stationary phase according to the invention with an aqueous loading buffer of a certain pH and a certain ionic strength, and eluting the target molecule with an aqueous buffer of another pH and / or another ionic strength.

[0073] In another embodiment, the method is carried out by loading the stationary phase according to the invention with an organic loading medium and eluting the target molecule with the same medium or a more polar medium, for example, using a gradient elution. The organic loading medium is an organic liquid that does not contain more than 10% water, for example, methanol, ethanol, acetonitrile, THF, heptane, toluene, etc. or a mixture thereof.

[0074] The invention also relates to the chromatographic separation of two proteins having different pi (isoelectric points) and / or different carboxylic acid contents by halogen bonding, preferably using a stationary phase according to the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0075] Figure 1 and 2 Chromatograms performed with the stationary phase according to the invention and other stationary phases are shown. Further details can be found in Application Example 2.1. DETAILED DESCRIPTION

[0076] Before describing the present invention in detail, it should be understood that the present invention is not limited to specific compositions or process steps, as these may vary. It must be noted that as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a ligand" includes a plurality of ligands, and reference to "an antibody" includes a plurality of antibodies, etc.

[0077] 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 to which the invention relates.For the purposes of the present invention as described herein, the following terms are defined.

[0078] As used herein, the term "target molecule" refers to any molecule, substance or compound that should be separated, isolated or purified from one or more other components in a sample, such as impurities. In the production and / or purification process, the target molecule is usually present in a liquid. The liquid can be water, a buffer, a non-aqueous organic solvent such as ethanol, acetonitrile, heptane or any mixture of the above-mentioned 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. Depending on the process steps performed, the composition of the liquid may change in the production and / or purification process. After the chromatographic step, due to the eluent used in the chromatographic step, the liquid usually contains other solvents different from before. Examples of target molecules include low molecular weight molecules, such as drugs with a molecular weight of about or less than 2000g / mol. The target molecule can also be a high molecular weight compound, such as a protein, such as an antibody.

[0079] The term "antibody" refers to a protein with the ability to specifically bind to an antigen. "Antibody" or "IgG" also refers to a polypeptide substantially encoded by one or more immunoglobulin genes or fragments thereof, which specifically binds and recognizes an analyte (antigen). Recognized immunoglobulin genes include kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as numerous immunoglobulin variable region genes. Light chains are classified as kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE, respectively.

[0080] An exemplary immunoglobulin (antibody) structural unit consists of two pairs of polypeptide chains, each pair having one "light" chain (about 25 kD) and one "heavy" chain (about 50-70 kD), which are stabilized, for example, by interchain disulfide bonds. The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids that is primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains, respectively.

[0081] Antibodies can be monoclonal or polyclonal and can exist in monomeric or polymeric form, for example, IgM antibodies in pentamer form and / or IgA antibodies in monomeric, dimer or polymeric form. Antibodies can also include multispecific antibodies (e.g., bispecific antibodies) and antibody fragments, as long as they retain or are modified to contain ligand-specific binding domains. The term "fragment" refers to a portion or part of an antibody or antibody chain that contains fewer amino acid residues than a complete or complete antibody or antibody chain. Fragments can be obtained via chemical or enzymatic treatment of a complete or complete antibody or antibody chain. Fragments can also be obtained by recombinant means. When recombinantly produced, fragments can be expressed alone or as part of a larger protein called 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 in the term "antibody".

[0082] In some embodiments, the antibody is a protein, such as an immunoglobulin, that contains an Fc region.

[0083] As used herein, unless otherwise indicated, the term "sample" refers to any composition or mixture containing a target molecule. The sample may be derived from a biological or other source. Biological sources include eukaryotic sources, such as animals or humans. The sample may also contain diluents, buffers, detergents, and contaminants found mixed with the target molecule.

[0084] As used herein, the term "impurity" or "contaminant" refers to any foreign or unwanted molecule, including biological macromolecules such as DNA, RNA, one or more host cell proteins, nucleic acids, endotoxins, lipids, impurities of synthetic origin, and one or more additives, which may be present in a sample containing a target molecule to be separated from one or more foreign or unwanted molecules.

[0085] The terms "purify", "isolate" or "separate" as used interchangeably herein refer to increasing the purity of a target molecule by separating the target molecule from a composition or sample comprising the target molecule and one or more other components, such as impurities. Typically, the purity of the target molecule is increased by removing (completely or partially) at least one impurity from the composition.

[0086] The term "chromatography" refers to any type of technique by which an analyte of interest (e.g., a target molecule) is separated from other molecules present in a mixture. Typically, the target molecule is separated from other molecules due to the difference in the rate at which individual molecules in the mixture migrate through the stationary phase under the influence of the mobile phase or in the binding and elution process. Examples of chromatographic separation methods include reverse phase chromatography, ion exchange chromatography, size exclusion chromatography, affinity chromatography, hydrophobic interaction chromatography, and mixed mode chromatography. Chromatography of the present invention is based on halogen bonding and optionally one or more other separation methods mentioned above.

[0087] A "buffer" is a solution that resists pH changes through the action of its acid-base conjugate components. Various buffers are described in Buffers. A Guide for the Preparation and Use of Buffers in Biological Systems, Gueffroy, D., ed. Calbiochem Corporation (1975), which can be used, for example, depending on the desired buffer pH. Non-limiting examples of buffers include MES, MOPS, MOPSO, Tris, HEPES, phosphate, acetate, citrate, succinate, glycine, and ammonium buffers and combinations of these. An aqueous buffer is a buffer whose solvent contains more than 90%, preferably 100%, water.

[0088] The term "stationary phase" refers to any type of sorbent, matrix, resin or solid phase that separates a target molecule from other molecules present in a mixture during a separation process. Typically, the target molecule is separated from other molecules due to differences in the rates at which individual molecules in the mixture bind to the stationary phase and / or migrate through the stationary phase under the influence of a mobile phase. The stationary phase can be placed in a column or cartridge. The stationary phase according to the present invention comprises at least one type of functional group.

[0089] "Functional group" is a part that is connected to the base material of the stationary phase and determines the binding properties of the stationary phase. Examples of "functional group" include, but are not limited to, ion exchange groups, hydrophobic interaction groups, hydrophilic interaction groups, sulfur affinity interaction groups, metal affinity groups, affinity groups, bioaffinity groups and mixed mode groups (aforesaid combination). The stationary phase according to the present invention at least comprises a functional group for halogen bonding. They may also comprise one or more other groups as listed above in addition. Typically, a functional group has more than one binding property.

[0090] When "loading" a chromatographic column in bind and elute mode, a buffer is used to load a sample or composition containing a target molecule and one or more impurities onto the chromatographic column. The buffer has a conductivity and / or pH that allows the target molecule to bind to the stationary phase while ideally all impurities are not bound and flow through the column. Separation of the bound target molecule from the one or more impurities can be accomplished by altering the conductivity and / or pH so that the target molecule is washed out or eluted before or after the one or more impurities.

[0091] The buffer in which the sample is loaded onto the stationary phase is often called the loading buffer or sample buffer.

[0092] When "loading" a chromatographic column to "flow through" a target molecule, a buffer is used to load a sample or composition comprising a target molecule and one or more impurities onto the chromatographic column. The buffer has a conductivity and / or pH such that the target molecule does not bind to the stationary phase and flows through the column while ideally all impurities bind to the column.

[0093] The term "equilibration" refers to the use of a buffer to equilibrate the stationary phase prior to loading the target molecule. Typically, a loading buffer is used to perform the equilibration.

[0094] "Washing" a stationary phase refers to passing a suitable liquid, such as a buffer, through or over the stationary phase. Washing is typically used to remove weakly bound contaminants from the stationary phase in a bind / elute mode before eluting the target molecule, or to remove unbound or weakly bound target molecules after loading.

[0095] In this case, typically, the wash buffer and the loading buffer are the same. In the case of using a virus inactivation buffer, it is used to inactivate certain viruses present before eluting the target molecule. In this case, typically, the virus inactivation buffer is different from the loading buffer because it may contain one or more detergents or have different properties (pH / conductivity / salt and its amount).

[0096] Washing can also be used to remove contaminants from the stationary phase after the target molecule is eluted. This is accomplished by passing a suitable liquid, such as a buffer, through or over the stationary phase after the elution of the target molecule. In this case, typically, the wash buffer is different from the loading buffer. It may contain one or more detergents or have different properties (pH / conductivity / salt and amount thereof). The wash buffer may, for example, be an acidic buffer.

[0097] "Eluting" a molecule (e.g., a target molecule or an impurity) from a stationary phase refers to removing the molecule therefrom. Elution can be performed directly in flow-through mode when the target molecule is eluted with a solvent front of a loading buffer, or by changing the solution conditions so that a buffer different from the loading buffer competes with the target molecule for ligand sites 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 so that the buffer competes with the molecule for charged sites on the ion exchange material.

[0098] As used interchangeably herein, the terms "flow-through," "flow-through mode," and "flow-through operation" refer to a separation technique in which at least one target molecule contained in a sample along with one or more impurities is expected to flow through a chromatographic stationary phase, which typically binds the one or more impurities, wherein the target molecule typically does not bind (i.e., flows through) and is eluted from the stationary phase with a loading buffer.

[0099] As used herein, the terms "bind and elute mode" and "bind and elute method" refer to a separation technique in which at least one target molecule contained in a sample binds to a suitable stationary phase and is subsequently eluted with a buffer different from the loading buffer.

[0100] Solid phase extraction (SPE) is a sample preparation method in which compounds dissolved or suspended in a liquid mixture are separated from other compounds depending on their physical and chemical properties. As a result, the target molecule or unwanted impurities in the sample are retained on the stationary phase. The fraction that passes through the stationary phase is collected or discarded, 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 can be removed from the stationary phase for collection in an additional step in which the stationary phase is rinsed with a suitable eluent.

[0101] Particle size is determined by laser diffraction, preferably using a Master Sizer from Malvern.

[0102] Pore ​​size was determined by reverse phase SEC. Particle size was determined by sieving.

[0103] Electron-withdrawing groups include hydrogen and atoms or groups of atoms with electron-withdrawing induction and / or mesoscopic effects and higher electronegativity than hydrogen. Exemplary electron-withdrawing groups include H, I, Br, Cl, F, CO2H, NO2, CN.

[0104] Electron donating groups include atoms or groups of atoms that have electron donating induction and / or mesotropic effects and are less electronegative than hydrogen. Exemplary electron donating groups include -OH, O-alkyl.

[0105] Alkyl or alkyl group denotes a straight-chain or branched alkyl group having generally 1 to 20 C atoms or having the number of C atoms as indicated, for example methyl, ethyl, isopropyl, propyl, butyl, sec-butyl or tert-butyl, furthermore 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, preferably the alkyl group has 1 to 10 C atoms.

[0106] Alkenyl or alkenyl radical is a straight-chain or branched alkenyl radical having generally 2 to 20 C atoms, wherein in addition, a plurality of double bonds may be present, for example allyl, 2- or 3-butenyl, isobutenyl, sec-butenyl, furthermore, 4-pentenyl, isopentenyl, hexenyl, heptenyl, octenyl, -C9H 17 , -C 10 H 19 To -C 20 H 39 ; preferably allyl, 2- or 3-butenyl, isobutenyl, sec-butenyl.

[0107] Aryl or aryl group denotes an aryl group generally having 6 to 12 C atoms, for example phenyl, naphthyl or anthracenyl, which may be unsubstituted or substituted by Hal, NH2, NAlk2, NHalkyl, NO2, CN, SO3H or Oalkyl. The substitution may occur one or more times, preferably once, by the indicated substituents.

[0108] The invention provides a new type of stationary phase, which can be used as a separation material for chromatography or solid phase extraction, also referred to as resin or matrix. The stationary phase according to the present invention comprises a base material and a functional group preferably covalently attached to the base material. It has been found that the principle of halogen bonding can be used to separate the target molecule from other compounds in a mixture, based on their different combinations with a stationary phase, wherein the stationary phase comprises a Cl, Br or preferably I functional group connected to an aromatic ring, wherein the aromatic ring is positively charged due to a positive charge directly adjacent to the ring or due to a positive charge in the ring. It has been found that the combination of a positive charge and a halogen atom attached to an aromatic ring structure can produce an effective σ hole at the halogen atom, which enables effective halogen bonding with a Lewis base.

[0109] The base material may consist of irregularly shaped or spherical particles, and the particle size may be between 2 and 1000 µm. The preferred particle size is between 3 and 300 µm.

[0110] In particular, the base material may be in the form of non-porous, core-shell or preferably porous particles. The pore size may be between 2 and 300 nm. Preferably the pore size is between 5 and 200 nm.

[0111] The base material can also be in the form of a membrane, a fiber, a hollow fiber, a coating, a filter, a capillary, a surface or a monolithic molding. The monolithic molding is preferably a porous three-dimensional body, for example in the form of a cylinder. Preferably, the base material is a porous bead or a membrane. The base material can also be manufactured by additive manufacturing such as 3D printing.

[0112] In one embodiment, the base material is an inorganic material, for example made of metal oxides such as SiO2, Al2O3, titanium dioxide, zirconium dioxide. It can also be made of other silica-based materials, such as controlled pore glass.

[0113] The base material may also be an inorganic-organic hybrid material or any other combination of organic and inorganic materials.

[0114] In another embodiment, the base material is made of a natural polymer, preferably in the form of porous beads, such as agarose-based polysaccharides, cellulose, cellulose derivatives and dextran-based polymers. Natural polymer beads are, for example, Sepharose. ® or Sephadex ®In an alternative embodiment, the base material is made of a synthetic polymer, preferably in the form of porous beads or membranes, consisting of a crosslinked synthetic polymer, such as styrene or styrene derivatives, divinylbenzene, acrylamide, acrylate, methacrylate, vinyl ester, vinylamide, etc. Preferred are polymers based on copolymers of polystyrene, polyvinyl alcohol or (meth)acrylate derivatives with comonomers having aliphatic hydroxyl groups. Polymers based on a certain structure type such as, for example, polystyrene or polyvinyl ether are polymers comprising said structures. They may also contain other structures, for example resulting from the copolymerization of two different monomers.

[0115] In another preferred embodiment, the base material is a material based on polyvinyl ether, in particular a copolymer formed by copolymerization of at least one compound from groups a) and b), wherein

[0116] a) at least one hydrophilic substituted alkyl vinyl ether of formula IX

[0117] IX

[0118] Wherein R1, R2, R3 can be independently H or C1 to C6 alkyl, preferably H or -CH3,

[0119] R4 is a group having at least one hydroxyl group

[0120] and

[0121] b) at least one crosslinking agent according to formula X and / or XI and / or XII,

[0122] X

[0123] wherein X is a divalent alkyl radical having 2 to 5 C atoms, preferably 2 or 3 C atoms, wherein one or more non-adjacent methylene radicals not immediately adjacent to N may be replaced by O, C=O, S, S=O, SO2, NH, NOH or N, and one or more H atoms of the methylene radicals may be replaced independently of one another by hydroxyl radicals, C1-C6-alkyl, halogen, NH2, C5-C10-aryl, NH-(C1-C8)-alkyl, N-(C1-C8)-alkyl2, C1-C6-alkoxy or C1-C6-alkyl-OH, and

[0124]

[0125]

[0126] Wherein Y1 and Y2 in formula XI and XII are independently

[0127] C1 to C10 alkyl or cycloalkyl, wherein one or more non-adjacent methylene groups or methylene groups not immediately adjacent 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 be replaced independently of one another by hydroxyl groups, C1-C6-alkyl, halogen, NH2, C5-C10-aryl, NH(C1-C8)alkyl, N(C1-C8)alkyl2, C1-C6-alkoxy or C1-C6-alkyl-OH,

[0128] or C6 to C18 aryl, wherein one or more H in the aryl system may be substituted independently of one another by a hydroxyl group, a C1-C6-alkyl group, a halogen, NH2, NH(C1-C8)alkyl, N(C1-C8)alkyl2, a C1-C6-alkoxy group or a C1-C6-alkyl-OH group, and

[0129] A is a divalent alkyl group having 2 to 5 C atoms, preferably 2 or 3 C atoms, wherein one or more non-adjacent methylene groups or methylene groups not adjacent to N can be replaced by O, C=O, S, S=O, SO2, NH, NOH or N and one or more H of the methylene groups can be replaced independently of each other by hydroxyl groups, C1-C6-alkyl, halogen, NH2, C5-C10-aryl, NH(C1-C8)alkyl, N(C1-C8)alkyl2, C1-C6-alkoxy or C1-C6-alkyl-OH.

[0130] R4 in formula IX is generally an alkyl group, alicyclic group or an aryl group carrying at least one hydroxyl group.

[0131] In a very preferred embodiment, the matrix is ​​formed by copolymerization of a hydrophilic substituted alkyl vinyl ether selected from 1,4-butanediol monovinyl ether, 1,5-pentanediol monovinyl ether, diethylene glycol monovinyl ether or cyclohexanedimethanol monovinyl ether with divinylethylene urea (1,3-divinylimidazolin-2-one) as a crosslinker.

[0132] An example of a suitable commercially available vinyl ether-based base material is Eshmuno® (Merck KGaA, Germany).

[0133] The functional group as defined above is attached to the base material. This can be accomplished via non-covalent or preferably covalent attachment. Covalent attachment can be accomplished, for example, by bonding the functional group directly to a suitable residue on the base material such as OH, NH2, carboxyl, phenol, anhydride, aldehyde, epoxide or sulfhydryl, etc.

[0134] Functional groups can also be attached via suitable linking groups. Suitable linking groups have structures such as

[0135] --X--Z--Y--, wherein X and Y are the first and second reactive or activatable groups, and X and Y can be independently selected from hydroxyl, amino, thiol, carboxyl, oxirane, formyl, halogen, isocyanato and chlorosulfonyl moieties; Z can be selected from groups such as

[0136] (a) C1-C15-alkyl,

[0137] (b) Aryl

[0138] (c) a C1-C10 alkylaryl group,

[0139] (d) C1-C10 alkylarylC1-6 alkyl, wherein 1, 2 or more carbon atoms of the alkyl group may be replaced by oxygen, sulfur or nitrogen, and wherein the aryl group includes but is not limited to phenyl, naphthyl, pyridyl or thienyl, and wherein one or more carbon atoms may be substituted by OH or C to C6 alkyl; and

[0140] (e) peptides of 2 to 10 amino acids, including, but not limited to, L- and D-forms of amino acids including glycine, alanine, valine, leucine, isoleucine, serine, threonine, aspartic acid, asparagine, glutamic acid, glutamine, lysine, hydroxylysine, histidine, arginine, phenylalanine, tyrosine, tryptophan, cysteine, methionine, ornithine, β-alanine, homoserine, homotyrosine, homophenylalanine, and citrulline.

[0141] Other suitable spacers may include, but are not limited to, p-benzoquinone, bis-(diazobenzidine), 3,6-bis-(mercurymethyl)dioxane, diethylene oxide, cyanuric chloride, p,p'-difluoro-m,m'-dicyclohexylcarbodiimide, dinitrophenyl sulfone, dimethyl adipimidate, dimethyl suberimidate, divinyl sulfone, N,N'-ethylene-bis-(iodoacetamide), glutaraldehyde, hexamethylenebis-(maleimide), hexamethylene diisocyanate, N,N'-1,3-phenylene-bis-(maleimide), phenol-2,4-disulfonyl chloride, o-dianisidine tetrazolate, toluene diisocyanate, Woodward's reagent K, water-soluble carbodiimide, 6-aminohexanoic acid, hexamethylenediamine, 1,7-diamino-4-aza-heptane (3,3'-diamino-dipropylamine), and amino acids or peptides.

[0142] The stationary phase according to the invention may also be produced by polymerizing monomers comprising functional groups and polymerizable moieties. Examples of stationary phases produced by polymerization of suitable monomers are stationary phases based on polystyrene, polymethacrylamide or polyacrylamide produced by polymerizing suitable styrene or acryl monomers.

[0143] In another embodiment, the stationary phase can be produced by grafting functional groups onto a base material.

[0144] In the case of "grafting onto", the polymer chains must first be formed from the monomers and bound to the surface of the base material in a second step. In the case of "grafting from", the polymerization reaction is initiated on the surface of the base material and the grafted polymer is built directly from the individual monomers.

[0145] The "grafting from" method is preferred, and variants in which only a small amount of by-products (such as non-covalently bonded polymers) are formed, which must be separated off, are particularly preferred. Methods using controlled radical polymerization, such as atom transfer radical polymerization (ATRP), are suitable. Here, in a first step, the initiator groups are covalently bonded to the surface of the base material with the desired density. The initiator groups can, for example, be halides bonded via ester functions, as in 2-bromo-2-methylpropionate. The graft polymerization is carried out in the presence of a copper (I) salt in a second step.

[0146] A highly preferred one-step graft autopolymerization reaction suitable for producing the stationary phase of the present invention can be initiated from cerium (IV) on a hydroxyl-containing support without activating the support.

[0147] This cerium (IV) initiated grafting is preferably carried out according to EP 0 337 144 or US 5,453,186. The chains produced are linked to the base material via monomer units. For this purpose, the base material according to the invention is suspended in a solution of monomers, preferably in an aqueous solution. The grafting of the polymer material is achieved in a conventional redox polymerization process that does not include oxygen. The polymerization catalyst used is cerium (IV) ions, because the catalyst forms free radical sites on the surface of the base material, from which the graft polymerization of the monomers is initiated. The reaction is usually carried out in a dilute mineral acid. In order to carry out this graft polymerization, the acid is usually used in an aqueous solution in a concentration range of 1 to 0.00001 mol / l, preferably 0.1 to 0.001. Very particularly preferably, dilute nitric acid is used, which is used in a concentration of 0.1 to 0.001 mol / l.

[0148] To prepare the separation material according to the invention, the monomers are usually added to the base material in excess. Typically, 0.05 to 100 mol of total monomers are employed per liter of deposited polymer material, preferably 0.05-25 mol / l are employed.

[0149] The polymerization is terminated by a termination reaction involving a cerium salt. The (average) chain length can therefore be influenced by the concentration ratio of base material, initiator and monomer. Furthermore, homogeneous monomers or mixtures of different monomers can be employed; in the latter case, graft copolymers will be formed.

[0150] The monomers advantageously used for preparing the separation material according to the invention are those according to formula XIII

[0151] XIII

[0152] Where R 1 , R 2 and Y are independently H or CH3, preferably H.

[0153] R 3 is the functional group according to the present invention or a chemical structure comprising the functional group of the present invention.

[0154] The separation materials according to the invention preferably contain only tentacle-like linear polymer structures grafted onto a base material, which are built up from monomers according to formula XIII. Preferably, they contain linear polymers built up from only one type of monomer according to formula XIII.

[0155] But it is also possible to build up linear polymers by copolymerization of two or more different monomers according to formula XIII. It is also possible to build up linear polymers by copolymerization of one or more different monomers according to formula XIII with one or more other polymerizable monomers such as other acrylamides, methacrylates, acrylates, methacrylates, etc., which are functionalized, for example, with ionic, hydrophilic or hydrophobic groups.

[0156] The same applies to stationary phases according to the invention made by different methods. They may contain one, two or more functional groups as described above for halogen bonding and other functionalities in addition to the functional groups described for halogen bonding. This may be, for example, ionic, hydrophilic or hydrophobic groups. By producing stationary phases with two different functionalities, a mixed mode material with separation properties produced by two or several types of functions is obtained, wherein in the case of the present invention, at least one functional group is suitable for halogen bonding.

[0157] The present invention also relates to a separation device comprising a stationary phase according to the present invention. The device can be used, for example, for solid phase extraction or for chromatographic applications. In any case, it comprises means for holding the stationary phase. The stationary phase may be surrounded by or connected to the device. In one embodiment, the device comprises a housing with an inlet and an outlet. In another embodiment, it is a plate or a pin, and the stationary phase is connected to one side thereof. In another embodiment, it is a filter comprising the stationary phase. In a preferred embodiment, the device is a chromatographic column comprising the above-mentioned stationary phase according to the present invention. Chromatographic columns are known to those skilled in the art. They generally comprise a cylindrical tube or cartridge filled with the stationary phase and a filter and / or a means for fixing the stationary phase in the tube or cartridge and optionally a connector for delivering a solvent to and from the tube or cartridge. The size of the chromatographic column varies according to the application, for example, analysis or preparation.

[0158] The stationary phase according to the invention may also be described as a base material provided with separation effectors, wherein at least one separation effector comprises a functional group as defined above. They may be used for the selective, partially selective or non-selective binding or adsorption of one or more target molecules with the purpose of separation from a sample liquid, or for the selective, partially selective or non-selective binding or adsorption of one or more secondary components with the purpose of separation from a matrix, separation, enrichment and / or depletion of biopolymers from natural sources, separation, enrichment and / or depletion of biopolymers from recombinant sources, separation, enrichment and / or depletion of proteins and peptides, separation, enrichment and / or depletion of monoclonal and polyclonal antibodies, separation, enrichment and / or depletion of viruses, separation, enrichment and / or depletion of host cell proteins, separation, enrichment and / or depletion of ADCs, separation, enrichment and / or depletion of alkaloids, lipids (such as di- or tri-glycerides), carbohydrates, nucleic acids or other biomolecules.

[0159] The target molecule is separated from at least one or more other substances from the sample, wherein the sample comprising the target molecule is a liquid or is dissolved in a liquid, and the liquid is contacted with the stationary phase according to the present invention. The contact time is usually in the range of 30 seconds to 24 hours. It will be advantageous to work according to the principle of liquid chromatography, and the approach is to pass the liquid through the chromatographic column containing the stationary phase according to the present invention. The liquid can only pass through the column by its gravity, or it can be pumped through by means of a pump. An alternative method is batch chromatography, wherein as long as the target molecule needs to be able to be combined with the stationary phase, the stationary phase is mixed with the liquid by stirring or shaking. It is equally possible to work according to the principle of a chromatographic fluidized bed, and the approach is to introduce the liquid to be separated into, for example, a suspension comprising the stationary phase, wherein the separation material is selected so that it is suitable for the desired separation due to its high density and / or magnetic core.

[0160] If the chromatographic process is run in a binding and elution mode, the target molecule will bind to the stationary phase according to the present invention. Optionally, the stationary phase may be subsequently washed with a wash buffer solution, which preferably has the same ionic strength and the same pH as the liquid that contacts the target molecule with the stationary phase. The wash buffer solution will remove all substances that are not bound to the stationary phase. Further washing steps may then be carried out with other suitable buffer solutions without desorbing the target molecule. The desorption of the bound target molecule is usually 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. The target molecule can therefore be obtained in the eluent in a purified and concentrated form. After desorbing from the stationary phase, the target molecule has a purity of 70% to 99%, preferably 85% to 99%, particularly preferably 90%-99%.

[0161] However, if the chromatography process is run in flow-through mode, the target molecule remains in the liquid phase, but other accompanying substances bind to the stationary phase. The target molecule is then obtained directly by collecting the flow-through column eluate.

[0162] The stationary phases according to the invention can be used in many different applications. They can be used, for example, for hydrophilic and hydrophobic separations and for aqueous and non-aqueous separations.

[0163] Proteins such as insulin can be suitably purified, for example, on a stationary phase comprising a hydrophilic base material to which the functional groups are bonded. Preferably, the functional groups are grafted onto the base material. Example 2.1 shows a stationary phase suitable for this application. Chromatographic separation is then preferably performed by using an aqueous buffer system at a neutral pH and by increasing the ionic strength of the buffer used for elution (e.g., by adding sodium chloride).

[0164] The chromatographic column of the present invention can also be used to separate two proteins with different pI (isoelectric points) and / or different carboxylic acid contents by halogen bonding. Carboxylic acids, especially those exposed on the surface of proteins, can interact with the stationary phase through halogen bonding. Proteins with a higher content of carboxylic acid groups that can interact with the stationary phase will bind more strongly to the stationary phase and require a higher conductivity to elute. This is also shown in Example 2.2.

[0165] However, stationary phases based on hydrophilic base materials to which the functional groups are bonded (preferably by grafting) can also be used to separate target molecules in organic solvent systems. In this case, loading and elution can be performed using the same solvent or using a solvent gradient by slowly increasing the amount of a more polar solvent than the loading solvent.

[0166] The stationary phase according to the present invention may also comprise a hydrophobic base material, such as polystyrene. The functional group may be connected to the polystyrene base material, for example, via a suitable linking group. Such a stationary phase may be suitably applied to the separation of low molecular weight compounds using an organic solvent for loading and elution.

[0167] Stationary phase and method according to the present invention provide a new and widely applicable chromatographic separation principle. The use of a functional group comprising at least one Cl, Br or I combined with a positively charged or chargeable aromatic ring structure provides a wide range of ligands that can be adjusted for a specific separation task. For example, the selection of halogen can affect the intensity of interaction with the target molecule. Generally, iodine shows the strongest interaction, followed by Br, and the interaction of the functional group substituted by chlorine is the least strong.

[0168] In addition, by selecting certain aromatic ring structures or residues, the mode of interaction can also be changed. For example, usually, the electron-withdrawing residue at the aromatic ring structure can expand the halogen binding effect, while the electron-donating or alkyl group can reduce the halogen bonding strength. The halogen bonding strength of the functional group can be adjusted according to the target molecule and the separation to be performed. For example, it can be started from a structure that only comprises a Cl, Br or I residue and does not comprise other residues except H. If the intensity is to be expanded, the type of halogen atom can be replaced by Br or I and / or the number of halogen atoms can be increased and / or other electron-withdrawing residues can be inserted. On the other hand, if the halogen bonding strength is to be reduced, the type of halogen atom can be replaced by Br or Cl and / or an electron-donating group, for example, an O-alkyl group or an alkyl group can be inserted.

[0169] In one embodiment, the halogen binding properties of the stationary phase according to the present invention can be turned on and off. When using a stationary phase comprising a functional group as shown in Formula I or II, the stationary phase can be "activated" under low pH conditions by protonating the nitrogen atom of the aniline-like substance and containing a positive charge adjacent to the aromatic ring. As long as the nitrogen atom adjacent to the aromatic ring is protonated, it acts as an electron-withdrawing group, thereby supporting the σ hole at the halogen atom at the adjacent aromatic ring and the target molecule bonded via the halogen. Then the stationary phase can be "deactivated" to perform elution by simply raising the pH and eliminating its electron-withdrawing function by deprotonating the N atom.

[0170] Coulombic interactions with the stationary phase can also be suppressed by using high concentrations of salts such as sodium chloride.

[0171] Without further elaboration, it is believed that one skilled in the art can, using the preceding description, fully utilize the present invention.The preferred specific embodiments and examples are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.

[0172] The entire disclosure of all applications, patents and publications cited above and below and the corresponding European patent application EP19205109.2 filed on October 24, 2019 is incorporated herein by reference. Example

[0173] The following examples represent practical applications of the present invention.

[0174] 1. Synthesis

[0175] N Synthesis of -(2-aminoethyl)-pyridinium chloride hydrochloride

[0176]

[0177] A round-bottom flask was charged 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 was stirred and heated to reflux for 14 h. The clear yellow solution was washed with toluene (2×25 mL), n-pentane (20 mL) and isopropanol (3×50 mL) and evaporated to dryness in vacuo. The product was obtained as a colorless solid. N -(2-Aminoethyl)-pyridinium chloride hydrochloride.

[0178] Yield: 5.54 g (28.4 mmol, 82%).

[0179] 1 H-NMR (D2O, 300 MHz): δ = 3.80 (m, 2H, H2N-CH2), 5.08 (m, 2H, H2C-N 吡啶 ),8.24 (m, 2H, Meta -H), 8.71 (m, 1H, Counterpoint -H), 9.03 ppm (m, 2H, Ortho -H).

[0180] Mp.:199 – 202℃.

[0181] MS (ESI) +: 123.0 (100 %) [M] + , 281.1 (22 %), 2[M] + Cl - .

[0182] N Synthesis of -(2-aminoethyl)-3-bromopyridinium chloride hydrochloride

[0183]

[0184] 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) was stirred and heated to reflux for two weeks. The precipitate was filtered and washed with ethanol (5 x 5 mL). N -(2-aminoethyl)-3-bromopyridinium chloride hydrochloride. The filtrate was partially evaporated and heated to reflux again for one week. The resulting colorless solid was washed with ethanol (5 x 5 mL), evaporated to dryness in vacuo and added to the previous product.

[0185] Yield: 41.2 g (211 mmol, 81%).

[0186] 1 H-NMR (D2O, 300 MHz): δ = 3.68 (t, 2H, J = 7 Hz, H2N-CH2), 4.95 (t,2H, J = 7 Hz, H2C-N 吡啶 ), 8.03 (m, 1H, Meta -H), 8.79 (m, 1H, Counterpoint -H), 8.94 (m,1H, Ortho -CH-CH-) 9.25 ppm (m, 1H, Ortho -CH-CBr-).

[0187] Elemental analysis: Calculated: N 10.26 %, C 30.69 %, H 4.05 %, Measured: N 10.34 %, C 30.76%, H 3.99 %.

[0188] Mp.:237℃

[0189] MS (ESI) +: 202.9 (100 %) [M] + , 157.8 (6 %) [3-Bromopyridinium] + .

[0190] N Synthesis of -(2-aminoethyl)-3-iodopyridinium chloride hydrochloride

[0191]

[0192] 2-Chloroethylamine hydrochloride (30.0 g, 259 mmol, 1 eq.) and 3-iodopyridine (57.2 g, 279 mmol, 1.1 eq.) were dissolved in ethanol (100 mL), stirred and heated to reflux for one week. The precipitate was filtered and washed with ethanol (6 x 20 mL). N -(2-aminoethyl)-3-bromopyridinium chloride hydrochloride. The filtrate was partially evaporated and heated to reflux again for one week. The resulting colorless solid was washed with ethanol (4×5 mL), evaporated to dryness in vacuo and added to the previous product. This procedure was repeated until the total yield was 76%.

[0193] Yield: 68.2 g (213 mmol, 76%).

[0194] Mp.:268.2℃.

[0195] Elemental analysis: Calculated: N 8.73%, C 26.19%, H 3.45%, Measured: N 8.82%, C 26.24%, H 3.41%.

[0196] MS (ESI) +: 248.9 (100 %) [M] + .

[0197] 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 吡啶 ), 7.84 (t, J = 6 Hz 1H, Meta -H), 8.91 (m, 2H, Counterpoint -H, adjacent Bit -CH-CH-), 9.28 ppm (s, 1H, Ortho -CH-CBr-).

[0198] Synthesis of 1-(2-Methacryloylaminoethyl)-pyridinium chloride

[0199]

[0200] Will N -(2-aminoethyl)-pyridinium chloride hydrochloride (38.6 g, 147 mmol) was dissolved in water (50 mL), followed by the addition of dichloromethane (200 mL), tributylamine (54.7 g, 295 mmol) and methacrylic anhydride (24.8 g, 161 mmol). The reaction was stirred at room temperature for 18 hours and then filtered. 1 H-NMR spectroscopy was used to check the remaining N -(2-aminoethyl)-pyridinium chloride. If any remain, separate the aqueous layer and mix again with dichloromethane, and add tributylamine and methacrylic anhydride according to the remaining educt.

[0201] The aqueous phase was separated and washed with dichloromethane (2 x 50 mL). 1 The yields were determined by H-NMR spectroscopy in aqueous solution with tetraphenylphosphonium chloride.

[0202] Yield: 26.2 g (116 mmol, 71%)

[0203] MS (ESI) +: 191.0 (100 %) [M] + , 234.1 (28 %) [1-(2-((2-methacrylamidoethyl)amino)ethyl)pyridinium] + .

[0204] 1 H-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 = 8Hz, Counterpoint -H), 8.81 (d, 2H, J = 6 Hz, Ortho -H).

[0205] Synthesis of 1-(2-Methacryloylaminoethyl)-3-bromopyridinium chloride

[0206]

[0207] Will N -(2-aminoethyl)-3-bromopyridinium chloride hydrochloride (3.09 g, 13.0 mmol) was dissolved in water (5 mL), followed by the addition of dichloromethane (50 mL), tributylamine (3.38 g, 14.2 mmol) and methacrylic anhydride (2.24 g, 14.2 mmol). The reaction was stirred at room temperature for 19 hours and then filtered. 1 H-NMR spectroscopy was used to check the remaining N -(2-aminoethyl)-3-bromopyridinium chloride. If there is any residue, separate the aqueous layer and mix it with dichloromethane again, add tributylamine and methacrylic anhydride according to the remaining educt. After stirring at room temperature for six hours, separate the aqueous phase, wash with dichloromethane (3×30 mL) and evaporate to dryness in vacuo. Obtain 3-bromo-1-(2-methacrylamidoethyl)-pyridinium chloride as a brown glass.

[0208] Yield: 2.77 g (9.02 mmol, 70%).

[0209] MS (ESI) +: 217.0 (100 %) [M] + , 575.0 (6 %) 2[M] + Cl - .

[0210] 1 H-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, Counterpoint -H), 8.88 (m, 1H, N-CH-CH), 9.20 (s, 1H, N-CH-CBr).

[0211] Synthesis of 3-iodo-1-(2-methacryloylaminoethyl)-pyridinium chloride

[0212]

[0213] Will N -(2-aminoethyl)-3-iodopyridinium chloride hydrochloride (32.0 g, 99.7 mmol) was dissolved in water (50 mL), followed by the addition of dichloromethane (200 mL), tributylamine (37.0 g, 200 mmol) and methacrylic anhydride (17.8 g, 116 mmol). The reaction was stirred at room temperature for 17 hours and then filtered. 1 H-NMR spectroscopy was used to check the remaining N -(2-aminoethyl)-3-iodopyridinium chloride. If any remain, separate the aqueous layer and mix again with dichloromethane, and add tributylamine and methacrylic anhydride according to the remaining educt.

[0214] The aqueous phase was separated, washed with dichloromethane (2 x 100 mL) and evaporated to dryness, leaving 3-iodo-1-(2-methacrylamidoethyl)-pyridinium chloride as a brown solid.

[0215] Yield: 33.76 g (95.7 mmol, 96%).

[0216] Mp.:254.5℃.

[0217] Elemental analysis: Calculated: N 7.94%, C 37.47%, H 4.00%, Measured: N 7.67%, C 36.14%, H 4.35%.

[0218] MS (ESI) +: 317.0 (100 %) [M] + .

[0219] 1 H-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-).

[0220] 13 C{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).

[0221] 1 H, 13 C-HMBC (D2O, 300 / 75 MHz): δ = 4.7 / 61.2 ppm ( J = 147 Hz, NH-CH2-CH2-N).

[0222] Synthesis of N-(3-bromophenyl)ethane-1,2-diamine

[0223]

[0224] 3-Bromoaniline (3.000 g, 25.86 mmol), 2-chloroethane-1-amine hydrochloride (4.786 g, 27.82 mmol) and ethanol (11 mL) were added to a 100 ml disruptor. The reaction mixture was stirred at room temperature for a few minutes. It was then placed in a microwave reaction vessel. The reaction vessel was placed in a microwave and the following method was selected and started: Method: Quick Test; Temperature: 110°C; Power: 300 W; Pressure: x bar; Hold Time: 30 minutes; Stirring: Off; Cooling: Off. After cooling, a new method was selected and started: Method: New Method; Temperature: 120°C; Power: 300 W; Pressure: 8 bar; Hold Time: 30 minutes; Stirring: On (Medium); Cooling: On. After cooling overnight, the pink crystals were filtered, washed with isopropanol and evaporated to dryness in vacuo.

[0225] Yield: 1.221 g (4.24 mmol, 16%).

[0226] Mp.:212.0℃ – 213.0℃

[0227] 1 H-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, Counterpoint -H), 7.07 (m, 1H, between Bit -H).

[0228] Functionalization of beads with 1-(2-methacrylamidoethyl)pyridinium chloride

[0229]

[0230] 1-(2-Methacrylamidoethyl)-pyridinium chloride in aqueous solution (1.29 mol / L, 2.13 mL, 2.75 mmol) was added to a three-necked round-bottom flask and water (15.3 mL) was added. The pH was set to 1.5 with nitric acid. A base material made of hydrophilic polyvinyl ether beads with OH groups, similar to the base material of the commercially available Eshmuno® sorbent (17.0 g, wet) (beads) was added. An initiator solution was 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 were degassed by vacuum and nitrogen. The suspension was heated to 30 ° C and the initiator solution was added as quickly as possible. The mixture was stirred for four hours, then filtered and washed with the following substances:

[0231] 3 x 100 mL demineralized water (ultrapure water / Milli-Q® water) = VE water (deionized water)

[0232] 7 × 100 mL 1 M sulfuric acid, 0.2 M ascorbic acid

[0233] 10×100mL demineralized water

[0234] 2 x 100 mL 1M sodium hydroxide solution

[0235] 2 x 100 mL demineralized water

[0236] Adjust pH to 6.5 - 7.0 with 25% hydrochloric acid

[0237] 1×100mL demineralized water

[0238] 1 x 100 mL ethanol.

[0239] The colorless solid was dried under vacuum

[0240] Yield: 3.38 g.

[0241] Functionalization of beads with 1-(2-methacrylamidoethyl)-3-bromopyridinium chloride

[0242]

[0243] 1-(2-Methacrylamidoethyl)-3-bromopyridinium chloride (838 mg, 2.75 mmol) was added to a three-necked round bottom flask and water (17.5 mL) was added. The pH was set to 1.5 with nitric acid. Beads (17.0 g, wet) were added. An initiator solution was 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 were degassed by vacuum and nitrogen. The suspension was heated to 30 °C and the initiator solution was added as quickly as possible. The mixture was stirred for four hours, then filtered and washed as described above. The resulting colorless solid was dried under vacuum.

[0244] Yield: 3.17 g.

[0245] Functionalization of beads with 1-(2-methacrylamidoethyl)-3-iodopyridinium chloride

[0246]

[0247] 1-(2-Methacrylamidoethyl)-3-iodopyridinium chloride (970 mg, 2.75 mmol) was added to a three-necked round bottom flask and water (17.5 mL) was added. The pH was set to 1.5 with nitric acid. Beads (17.0 g, wet) were added. An initiator solution was 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 were degassed by vacuum and nitrogen. The suspension was heated to 30 °C and the initiator solution was added as quickly as possible. The mixture was stirred for four hours, then filtered and washed as described above. The resulting colorless solid was dried under vacuum.

[0248] Yield: 3.22 g.

[0249] Synthesis of Styrene-Based Stationary Phases

[0250]

[0251] The reactor was charged with VE-water, sodium dodecane-1-sulfonic acid salt (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-ethylene-1-hexanol (hexanole) (90.3 g) and AIBN (1.0 g) was added.

[0252] The stirrer speed was set to 500 rpm for 10 seconds and the resulting emulsion was stirred at 25°C for 30 minutes.

[0253] Water (640 g) was added and the stirrer speed was set to 120 rpm. After the emulsion stabilized, the temperature ramp was started. From 25°C to 72°C in 90 minutes and kept at 72°C for 120 minutes. From 72°C to 82°C in 20 minutes and kept at 82°C for 120 minutes. The temperature was then reduced to 60°C in 30 minutes and kept at this temperature for 12 hours.

[0254] The obtained polymer was washed with VE water and an organic solvent.

[0255] The polymer was then dried in vacuum at 50°C for 24 hours.

[0256] analyze

[0257] Calculated Cl content: 2.33% Cl

[0258] X-ray fluorescence analysis: 2.5% Cl

[0259] Elemental analysis: 0.62% Cl.

[0260] Functional group attachment

[0261] Styrene-based polymers can be functionalized at the chlorine position. To do this, the chlorine is first exchanged for iodine by a Finkelstein reaction and then the functional group is introduced.

[0262]

[0263] 2. Application Examples

[0264] 2.1 Hydrophilic separation of insulin using functionalized beads (with antennae prepared as described above)

[0265] Prepare a chromatographic column with functionalized beads. Prepare a column with beads bearing functional groups functionalized with iodine and beads bearing functional groups without iodine. This way, if the resulting separation is only visible for the stationary phase bearing iodine and not for the otherwise identical stationary phase not functionalized with iodine, it can be ensured that the resulting separation is indeed due to halogen bonding.

[0266] Samples containing insulin and A21-desamidated insulin were processed as follows:

[0267] Condition 1:

[0268] Eluent: (A) 50mM (NH4)2SO4 pH3.5

[0269] (B) 50mM (NH4)2SO4 + 1M NaCl pH3.5

[0270] Gradient: 0-15min 100% (A)

[0271] 30-50min 50% (A) 50% (B)

[0272] Flow rate: 0.5mL / min

[0273] Detection: UV214nm

[0274] Temperature: 25℃

[0275] The results can be found in Figure 1 Seen in.

[0276] The graph shows the chromatogram for the following stationary phases:

[0277] A Beads functionalized with iodine-containing functional groups

[0278] B Beads functionalized with iodine-bearing functional groups (half the amount of functional groups compared to A)

[0279] C Beads functionalized with iodine-free functional groups

[0280] D Beads functionalized with iodine-free functional groups (half the amount of functional groups compared to C)

[0281] E Base Beads – Unfunctionalized

[0282] It can be seen that the graphs A and B using the material with iodine groups show stronger retention compared to the material without iodine. However, insulin and A21-deamidated insulin were not separated.

[0283] Condition 2:

[0284] Eluent: (A) 100mM NH4H2PO4 pH7.3

[0285] (B) 100mM NH4H2PO4 + 1M NaCl pH7,3

[0286] Gradient: 0-15min 100% (A)

[0287] 30-50min 50% (A) 50% (B)

[0288] Flow rate: 0.5mL / min

[0289] Detection: UV214nm

[0290] Temperature: 25℃

[0291] The results can be found in Figure 2 Seen in.

[0292] The graph shows the chromatogram for the following stationary phases:

[0293] A Beads functionalized with iodine-containing functional groups

[0294] B Beads functionalized with iodine-bearing functional groups (half the amount of functional groups compared to A)

[0295] C Beads functionalized with iodine-free functional groups

[0296] D Beads functionalized with iodine-free functional groups (half the amount of functional groups compared to C)

[0297] E Base Beads – Unfunctionalized

[0298] It can be seen that the graphs A and B using materials with iodine groups show stronger retention than materials without iodine and they show separation of insulin and A21-desamidated insulin. Materials C, D and E show only very little retention and separation of insulin and A21-desamidated insulin.

[0299] This indicates that the retention and separation of insulin and A21-desamidated insulin is indeed based on halogen bonding. The only difference between the materials used in A / B and C / D is the iodine residue.

[0300] 2.2 Hydrophilic separation of different proteins using functionalized beads (with tentacles prepared as described above)

[0301] Compared with the protein with higher pI (isoelectric point), the protein with lower pI is often more strongly combined with iodine fixation according to the present invention. The protein with lower pI needs stronger ionic strength to elute. In addition, the protein with higher carboxylic acid content is often more strongly combined with fixation. This influence depends on the tertiary structure of the protein, so the exposure of carboxylic acid must also be considered. Therefore, it is possible to easily separate proteins such as carbonic anhydrase and InlB321-GFP.

[0302] Samples containing one of the proteins listed in the table below were treated as follows:

[0303] Eluent: (A) 20 mM tris(hydroxymethyl)aminomethane, pH 8

[0304] (B) 20 mM Tris(hydroxymethyl)aminomethane + 1 M NaCl, pH 8

[0305] Gradient: 0-16mL 100% (A)

[0306] 16-96mL, from 100% (A) to 50% (A) 50% (B)

[0307] 96-120mL 100% (B)

[0308] Flow rate: 4mL / min

[0309] Detection: UV280 / UV260

[0310] Temperature: 4℃

[0311]

[0312] This table shows the names of the tested proteins and their respective values: MW: molecular weight in kDa, pi: isoelectric point, #Carboxy / MW: number of carboxylic acids divided by molecular weight, and conductivity at the largest elution peak.

[0313] The interaction between the functional groups on the stationary phase and the carboxylic acid was demonstrated by the crystal structure obtained from 3-iodo-1-methylpyridinium acetate. Although the sum of the van der Waals radii is 345 pm, the distance between the iodine and oxygen is only 286 pm. Therefore, a binding interaction exists.

[0314] 2.3 Hydrophobic separation of low molecular weight compounds using functionalized beads (prepared as described above)

[0315] The stationary phase is filled into a glass column. The eluent is n-pentane. The column is washed with n-pentane and then the analyte is applied to the column. Detection is performed at the column outlet with the aid of a silica gel TLC plate with a UV indicator and a UV lamp. f Calculated based on the weight of solvent used before compound elution divided by the mass of solvent per column volume.

[0316] The results can be seen in Table 1.

[0317]

[0318] The resin with R = H was used as a reference. Different substances with electron-donating functions were passed through the column with n-pentane as the mobile phase. The results show retention factors, which are not much different between the reference material and the Br-substituted material. However, the retention factor of the I-substituted material shows a significant increase compared to the Br-substituted material (percentage in brackets). Depending on the functional group of the substance, the limit is 25% to 52% higher.

Claims

1. A stationary phase comprising a base material and at least one type of functional group, wherein the functional group is selected from ---N(R'2) + -ArX, wherein R' is H or C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkynyl, C6 aryl, and ArX is I A certain II in L is --N(R'2) + Key X and X' are independently H, I, Br or Cl or another electron withdrawing group selected from F or NO2 or C1-C10 alkyl, C1-C10 alkenyl, C1-C10 alkynyl, C6-C12 aryl or electron donating group, R is H or C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkynyl, C6 aryl Y is independently C or N Wherein in formula I, at least one X or X' is Cl, Br or I, Wherein at least one L is present in Formula II and wherein at least one Y is C, and wherein for each Y that is N, there is no L / R / X at that N; or the functional group is a positively charged heterocyclic aromatic group carrying at least one Cl, Br or I residue; The positively charged heterocyclic aromatic group with at least one Cl, Br or I residue has one of the following structures: III wherein X is independently H, I, Br or Cl, and X' is independently 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 electron donating group, wherein at least one X or X' is Cl, Br or I; IV wherein 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 H, I, Br or Cl, and X' is independently 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 electron donating group, wherein at least one X or X' is Cl, Br or I; V wherein an atom A of the aromatic ring which does not provide a connection to the base material is O + or S + , and the other ring atoms A are C-X', wherein X' are independently H, I, Br, Cl or another electron withdrawing group or C1-C10 alkyl, C1-C10 alkenyl, C1-C10 alkynyl, C6-C12 aryl or electron donating group, wherein at least one X' is Cl, Br or I; or one of the following structures wherein X is independently H, a C1 to C6 alkyl group, I, Br or Cl, and X' and X" are independently H, a C1 to C6 alkyl group, I, Br, Cl or another electron withdrawing or electron donating group, R is H, a C1-C6 alkyl group, a C6 aryl group, a C1-C6 alkenyl group, a C1-C6 alkynyl group or an electron withdrawing or electron donating group, and wherein at least one X or X' is Cl, Br or I.

2. The stationary phase according to claim 1, characterized in that The functional group has a heterocyclic ring.

3. The stationary phase according to claim 1, characterized in that The functional group is selected from one or more of the following WE Wherein R' is H or C1-C6 alkyl, EW is independently H, CH3 or an electron withdrawing group and Hal are independently H, CH3, Cl, Br or I, wherein at least one Hal is Cl, Br or I; VII Where EW is independently H, CH3, electron-withdrawing group and Hal are independently H, CH3, Cl, Br or I, wherein at least one Hal is Cl, Br or I; VIII Wherein R' is H or C1-C6 alkyl, EW is independently H, CH3 or an electron withdrawing group and Hal is Cl, Br or I IX One of the R / L is H or C1-C6 alkyl and the other R / L is a connection to the base material, EW is H, CH3 or an electron-withdrawing group And Hal is Cl, Br or I.

4. The stationary phase according to claim 1, characterized in that The functional groups are covalently bonded to the base material.

5. The stationary phase according to claim 1, characterized in that The base material is a bead or a film.

6. The stationary phase according to claim 1, characterized in that The functional group is bonded to the base material via a linking group.

7. The stationary phase according to claim 1, characterized in that The functional groups are the terminal groups of the polymer chains grafted onto the base material.

8. The stationary phase according to claim 1, characterized in that The base material is an organic polymer.

9. The stationary phase according to claim 3, characterized in that The R' is methyl, ethyl, propyl or isopropyl.

10. A chromatography column comprising a stationary phase according to one of claims 1 to 9.

11. A method for separating a target molecule from at least one other compound, wherein a stationary phase according to one of claims 1 to 9 is contacted with a liquid comprising the target molecule and the 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.

12. The method according to claim 11, characterized in that The more electron donating a compound is, the stronger its binding to the stationary phase is.

13. The method according to claim 11 or 12, characterized in that: The method is a method for chromatographically separating a target molecule from at least one other compound, wherein the stationary phase according to one of claims 1 to 9 is present in a chromatographic column and a liquid containing the target molecule and the at least one other compound is passed through the column, whereby the target molecule and the other compound present in the liquid are eluted from the column depending on their interaction with the stationary phase.

14. The method according to claim 11 or claim 12, characterized in that: The target molecule is a protein.

15. The method according to claim 11 or claim 12, characterized in that The method is performed by loading the stationary phase with an aqueous loading buffer of a certain pH and a certain ionic strength, and eluting the target molecule with an aqueous elution buffer of the same pH but another ionic strength.

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