Materials and methods for performing separations based on boron clusters - Patents.com

Abstract

The present invention relates to new stationary phases carrying boron clusters. Target molecules can interact with this stationary phase depending on the type and substituents of the cluster. The stationary phases are suitable for SPE and chromatographic separations.

Description

[Technical field]

[0001] The present invention relates to new stationary phases carrying boron clusters. Target molecules can interact with this stationary phase depending on the type and substituents of the cluster. The stationary phases are suitable for SPE and chromatographic separations. [Background technology]

[0002] 2. Background of the Invention Boron clusters are polyhedral boron derivatives that possess unique properties. Among the types of boron clusters, closo-boranes (especially the 10 and 12 vertex cages), closo-carboranes (especially the 12 vertex cages), metallacarboranes, and selected nido-boranes are of particular interest. These compounds exhibit many advantageous properties in comparison with their related organic counterparts, namely, for example, [closo-B 12 H 12 ] 2- , [Closo-1-CB 11 H 11 ] - , and Closo-C2B 10 H 12 and metallabisdicarbolide [3,3'-M(1,2-C2B9H 11 )2] - (M=Co 3+ , Fe 3+ etc.) are unique chemical, electrochemical, and thermal stability. Boron clusters exhibit both electron-withdrawing and electron-donating properties depending on the type of cluster and the substituents attached to the carbon or boron atoms. The tunable molecular weight and geometry combined with tunable hydrophilicity allows for fine tuning of the properties of materials containing such boron clusters.

[0003] Boron clusters are of interest for applications in biology and medicine, for example for boron neutron capture therapy (BNCT) for the treatment of cancer. The development of novel bioactive molecules and potential drugs containing boron clusters is another topic of actual research investigation in this emerging area.

[0004] It has been reported that many polyhedral boranes are essentially non-toxic due to their inertness towards biochemical processes (Pleshek, J. Potential Applications of the Boron Cluster Compounds. Chem. Rev. 1992, 92, 269-278.). For boron enrichment in tumor cells, icosahedral boron clusters are highly promising due to their high number of boron atoms per molecule. In this context, current research investigations are focused on the use of icosahedral closo-boron clusters, for example carba-closo-dodecaborane, carba-closo-dodecaborate anion or closo-dodecaborate anion.

[0005] The weakly acidic CH unit(s) of carba-closo-dodecaborane and carba-closo-dodecaborate anions can be deprotonated and thus offer relatively facile substitution chemistry and the potential for further derivatization (Grimes, RN Carboranes; 3 ed.; Academic Press / Elsevier Inc.: London, UK, 2016.).

[0006] A series of adenosine derivatives modified with either boron clusters or substituted phenyls were synthesized and their physicochemical and biological properties were compared. It is noteworthy that a positive effect of boron clusters was detected. This result is an example and shows that boron clusters are a useful tool for tuning the physical properties of biomolecules. It provides a means for the modification of nucleoside structures or other biomolecules with the aim of developing novel drugs.

[0007] Carborane clusters are of particular interest as substituents and ligands in supramolecular chemistry due to their particular properties. Carba-closo-dodecaborane and related icosahedral boron clusters are commonly applied as bulky pharmacophores, e.g., in biologically active molecules for the replacement of various hydrophobic units. The introduction of 12-vertex carboranes into biologically active molecules often increases their in vivo stability and bioavailability, and enhances the interaction between drugs and receptors due to the hydrophobic properties of these boron clusters. Carboranes are extremely stable against enzymatic degradation. As a result, the use of boron clusters as building blocks for new drugs has grown in recent years.

[0008] The interactions of selected boron clusters and their derivatives with serum albumin, the most abundant protein in mammalian blood, have been reported (Goszczynski, TM; Fink, K.; Kowalski, K.; Lesnikowski, ZJ; Boratynski, J. Interactions of Boron Clusters and their Derivatives with Serum Albumin. Sci. Rep. 2017, 7, 9800.). The results of this study demonstrate that metallacarboranes strongly interact with albumin. The observed strength of interaction of boron clusters with albumin follows the following order: metallacarboranes [M(C2B9H 11 )2] - > Carborane (C2B 10 H 12 )>>Dodecaborate anion [B 12 H 12 ] 2- The metallacarboranes interact specifically with the binding cavity of albumin and nonspecifically with the protein surface. The authors highlight the importance of this finding for the development of new bioactive compounds containing boron clusters.

[0009] The examples described above demonstrate the versatility of application of boron clusters in various fields. However, the application of boron clusters in chromatographic materials for the separation and / or purification of biomolecules remains unknown, with potential for high selectivity towards and purification of selected biopharmaceutical molecules. Summary of the Invention

[0010] BRIEF DESCRIPTION OF THE PRESENT DISCLOSURE The present invention is therefore directed to the application of boron clusters in stationary phases for the separation and / or purification of target molecules, particularly in solid phase extraction (SPE) and chromatographic separations. These separation materials have the potential for high selectivity for and purification of target molecules, e.g. biomolecules.

[0011] Boron clusters, which can vary in cluster size, geometry, charge, atomic composition (all boron, or cages with heteroatoms), and substituents attached to the cluster atoms, are attached to a particle (substrate), preferably via a linker (Figure 1).

[0012] Furthermore, the present invention is directed to stationary phases comprising boron clusters, separation devices comprising the stationary phases, and processes for the separation of a target molecule from at least one other compound using the stationary phases or separation devices. [Brief description of the drawings]

[0013] [Figure 1] FIG. 1 shows that the boron clusters are attached to the particles via linkers. [Diagram 2] FIG. 2 shows the substitution of a boron cluster. [Diagram 3] FIG. 3 shows the substitution of a boron cluster. [Figure 4] FIG. 4 shows the substitution of boron clusters. [Diagram 5] FIG. 5 shows the substitution of a boron cluster. [Figure 6] FIG. 6 shows the substitution of a boron cluster. [Figure 7] FIG. 7 shows the substitution of a boron cluster. [Figure 8] FIG. 8 shows the results of the analysis for selected pH and conductivity conditions. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] Detailed Description of the Invention The present invention refers to a stationary phase comprising a substrate and at least one boron cluster, where the boron cluster contains only main group elements of the periodic table.

[0015] Before describing the invention in detail, it is to be understood that the invention is not limited to particular compositions or process steps, as such may vary. It should 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, and so forth.

[0016] 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 this invention pertains. For purposes of the present invention as described herein, the following terms are defined.

[0017] According to the invention, the boron clusters are polyhedral boron derivatives (Beckett, MA; Brellochs, B.; Chizhevsky, IT; Damhus, T.; Hellwich, K.-H.; Kennedy, JD; Laitinen, R.; Powell, WH; Rabinovich, D.; Vinias, C.; Yerin, A. Nomenclature for boranes and related species (IUPAC Recommendations 2019). Pure Appl. Chem. 2020, 92, 355-381.). The boron clusters according to the invention and their nomenclature are published by Beckett et al. 2020. In particular, the boron clusters are all boron cross cages {cross-B n} and other geometric shapes such as nido-, arachno-, hypho-clusters. The boron clusters are connected to suitable substrates, shown as "particles" in Figures 2-7, preferably via linkers. The boron clusters according to the invention can be uncharged or charged, preferably the boron clusters have a charge of -1 or -2. The charge of the boron clusters is typically controlled by variations in the type of cluster, by the introduction of heteroatoms (main group elements or transition metals) into the boron cage, or by the introduction of charged substituents on the boron cluster, e.g. NH3 + The substitution of boron clusters is controlled by the introduction of X5 to X in Figures 2 to 7. 11 and X 17 As shown in the figure.

[0018] One or more boron atoms can be replaced by heteroatoms. The replacement of one or more boron atoms in a boron cluster with heteroatoms leads to other boron cage-based end groups with tunable properties. Preferably, the heteroatom is carbon (carborane), as exemplified by the one-carbon boron cluster shown in Figure 3 and the two-carbon boron cluster depicted in Figure 4 (Grimes, RN Carboranes; 3rd ed.; Academic Press / Elsevier Inc.: London, UK, 2016.).

[0019] In addition to boron clusters containing one or two carbon atoms, related heteroatom clusters with other typical atoms are part of the present invention. These heteroatoms include, for example, tin, lead, silicon, and phosphorus. The positions of the heteroatoms depend on the type of cluster (size and geometric shape) and the type and number of heteroatoms. Typical positions are shown in Figures 3 and 4. However, other positions are also possible.

[0020] Conjunct-boron clusters represent a further class of boron clusters according to the invention. These conjunct-clusters are represented by the formula {closo-B 21 These include all-boron cages, such as the} cluster, and clusters with heteroatoms, e.g. carbon.

[0021] Metallacarboranes are a further type of polyhedral boron cluster. These clusters can differ in (i) the (transition) metal atom(s), (ii) additional heteroatoms (=non-boron atoms), (iii) cluster size (number of cluster atoms), (iv) charge, (v) additional ligands of the metal atom (e.g. cyclopentadienyl, Cp), and (vi) substituents attached to the cluster. Figure 6 depicts examples of metal carboranes attached to particles, and Figure 7 shows more specific examples based on the metals Cu, Co, and Ni.

[0022] Among the types of boron clusters, closo-boranes (especially the 12-vertex cage), closo-carboranes (especially the 12-vertex cage), and metallacarboranes are of particular interest. These compounds exhibit many advantageous properties in comparison with their organic counterparts, namely, for example, [closo-B 12 H 12 ] 2- , [Closo-1-CB 11 H 11 ] - , and Closo-C2B 10 H 12 and metallabisdicarbolide [3,3'-M(1,2-C2B9H 11 )2] - (M=Co 3+ , Fe 3+ etc.) their unique chemical, electrochemical, and thermal stability. Boron clusters exhibit both electron-withdrawing and electron-donating properties, depending on the type of cluster and the substituents attached to the carbon or boron atoms.

[0023] One aspect of the invention is a stationary phase comprising a substrate and at least one boron cluster.

[0024] For the avoidance of doubt, the term boron clusters refers to discrete polyhedral boron molecules, as described above, and does not incorporate boron polymers.

[0025] In a preferred embodiment, the boron cluster contains only typical elements of the periodic table. A boron cluster containing only typical elements excludes boron clusters containing at least one transition metal element, such as Cu, Co or Ni. Typical elements include elements of groups 1 and 2 (s block) and groups 13-18 (p block) of the periodic table.

[0026] The term "the boron cluster contains only typical elements of the periodic table" can be replaced by "the boron cluster only contains typical elements of the periodic table" or "the boron cluster consists of typical elements of the periodic table".

[0027] The stationary phase comprising the substrate and at least one boron cluster exhibits very good separation properties, especially with the binding proteins as shown in Example 2. The stationary phase comprising the boron cluster, in which the boron cluster contains only the main elements of the periodic table, has advantageous separation properties compared to the stationary phase in which the boron cluster contains at least one transition element, for example Cu, Co or Ni. It can be shown that the elution of the bound target molecule is relatively better with the boron cluster containing only the main elements of the periodic table.

[0028] Examples 2.1-2.4 show separation experiments using separation materials with boron clusters containing only main group elements of the periodic table. The concentrations of bound protein in Runs 1-3 are generally similar across the different separation materials. Example 2.5 shows the separation of boron clusters containing the transition element cobalt (parent COSAN cluster [3,3'-Co(1,2-C2B9H 11 )2] - Separation experiments using separation materials bearing boron clusters (derivatives of boron clusters) are shown. It can be seen that the concentration of bound protein decreases by more than 50% from run 1 to run 2. This indicates that the interaction of target molecules, especially proteins, with boron clusters containing only main group elements of the periodic table is easier to control and is beneficial for the separation of target molecules.

[0029] A similar trend is observed for separation materials having boron clusters with a charge of -1 or -2, preferably -2.

[0030] In a further embodiment, the boron cluster has a cluster type of closo, nido, arachno, hypho, hypercloso, conjunct. Preferably, the cluster type is selected from the group consisting of closo, nido, arachno, hypho, hypercloso, conjunct, more preferably closo, nido or conjunct, most preferably closo. In a further embodiment, the boron cluster has a cluster type of conjunct. In a further embodiment, the boron cluster has a cluster type of {closo-B 21} cluster.

[0031] In further embodiments, the boron clusters have a cluster size of 6, 7, 8, 9, 10, 11, or 12 atoms per cluster. In further embodiments, the boron clusters have a cluster size of 12 atoms or less.

[0032] In a further embodiment, the boron cluster contains one, two or more atoms that are not boron atoms. Preferably, at least one of the non-boron atoms is selected from the group consisting of carbon, sulfur, nitrogen, germanium, tin, lead and phosphorus, more preferably carbon.

[0033] In a further embodiment, the boron cluster is uncharged or has a charge of -1 or -2, preferably the boron cluster has a charge of -1 or -2, more preferably the boron cluster has a charge of -2.

[0034] In a further embodiment, the boron cluster is a metallacarborane.Preferably, the at least one transition metal atom is selected from the group consisting of Cu, Co, Fe and Ni.

[0035] The boron cluster is optionally substituted by one or more covalently bonded substituents, preferably further functional groups according to the definition below. In a further embodiment, one or more hydrogen atoms of the boron cluster are replaced by a covalently bonded substituent.

[0036] Preferably, the covalently bonded substituent is (1) Halogen, (2) Pseudohalogens, (3)-NR 1 R 2 , Here, R 1 and R 2 represent, independently of each other, H, alkyl, aryl, heteroaryl or heteroalkyl, (4)-N + R 1 R 2 R 3 , Here, R 1 , R 2 and R 3 represent, independently of each other, H, alkyl, aryl, heteroaryl or heteroalkyl, (5) Hydroxy, (6)-OR 4 , Here, R 4 represents alkyl, aryl, heteroaryl or heteroalkyl, (7)-O + R 4 R 5 or -S + R 4 R 5 , Here, R 4 and R 5 represent, independently of each other, alkyl, aryl, heteroaryl or heteroalkyl, (8) alkyl, (9) cycloalkyl, (10) (Per)fluoroalkyl, (11) alkenyl, (12) alkynyl, (13) aryl, (14) heteroaryl, (15)-C(=O)OR 6 , Here, R 6 represents H, alkyl, aryl, heteroalkyl, heteroaryl or boranyl; (16)-C(O)R 6 , Here, R 6 represents H, alkyl, aryl, heteroalkyl, heteroaryl or boranyl; (17) Acid derivatives such as acid amides, imides, ureas, thioureas, guanidine and their derivatives; (18)-SR 7 , Here, R 7 represents H, alkyl or aryl, (19)-SSR 8 , Here, R 8 represents alkyl, aryl or boranyl; (20)-SeR 9 , Here, R 9 represents alkyl, aryl or heteroaryl; (21)-TeR 9 , Here, R 9 represents alkyl, aryl or heteroaryl; (22)-C(S)R 10 , Here, R 10 represents alkyl or aryl; (23)-BR 11 R 12 , Here, R 11 and R 12 represent, independently of each other, alkyl, aryl or heteroaryl, (24)-B - R 13 R 14 R 15 Here, R 13 , R 14 and R 15 represent, independently of each other, H, alkyl, aryl, heteroaryl or heteroalkyl, (25) guanidine, (26) sugar derivatives, (27)-P(O)(OR 16 )R 17 , Here, R 16 and R 17 represent, independently of each other, H, alkyl, aryl, heteroaryl or heteroalkyl; R 17 may be a fluorinated alkyl, aryl or heteroaryl; and (28)-SiR 18 R 19 R 20 and -Ge 18 R 19 R 20 Here, R 18 , R 19 and R 20 represent, independently of each other, H, alkyl, aryl, heteroaryl, heteroalkyl, hydroxy or alkoxy, The selection is made from a list consisting of:

[0037] More preferably, the covalently bonded substituents are halogen, hydroxy, cyano, carboxy, amino and -N + R 1 R 2 R 3 where R 1 , R 2 and R 3 represent, independently of each other, H, alkyl, aryl, heteroaryl, or heteroalkyl.

[0038] In one embodiment, the at least one boron cluster is selected from the group consisting of the boron clusters in Table 1. [Table 1-1] [Table 1-2] [Table 1-3]

[0039] In one embodiment, at least one boron cluster is selected from the group consisting of parent boron clusters in Table 2, where only hydrogen substituents are attached to the cluster, one of which is formally replaced by a linker to allow attachment to the particle. [Table 2]

[0040] The boron clusters of the present invention are capable of directly binding target molecules. Optionally, functional groups with specific binding properties are attached to the boron clusters. Thus, the materials and stationary phases of the present invention are particularly flexible and tunable to accommodate different separation processes.

[0041] As used herein, the term "target molecule" refers to any molecule, substance or compound that is to be isolated, separated or purified from one or more other components, e.g. impurities, in a sample. In a manufacturing and / or purification process, the target molecule is typically present in a liquid. The liquid may be water, a buffer, a non-aqueous organic solvent such as ethanol, acetonitrile, heptane, or any mixture of the named liquids. The liquid may contain one or more impurities in addition to the target molecule. The liquid may also be called a sample. The composition of the liquid may change during manufacturing and / or purification depending on the process steps performed. After a chromatography step, the liquid typically contains a different solvent than before due to the eluent used in the chromatography step.

[0042] Examples of target molecules are low molecular weight molecules such as drugs with molecular weights around or below 2000 g / mol.Target molecules can also be high molecular weight compounds such as proteins, for example antibodies.Further examples of target molecules are biopolymers of natural origin, biopolymers of recombinant origin, proteins and peptides, monoclonal and polyclonal antibodies, viruses such as lentiviruses, adenoviruses, adeno-associated viruses, measles viruses, virus-like particles, exosomes, host cell proteins, ADCs, lipids such as alkaloids, diglycerides or triglycerides, carbohydrates, nucleic acids such as mRNA or plasmid DNA.Preferred target molecules are proteins, for example antibodies.

[0043] A "protein" is defined herein as a polymer of amino acids linked together by peptide bonds to form a polypeptide. Preferably, the chain length is sufficient to produce at least a detectable tertiary structure. Proteins may be naturally occurring or non-naturally occurring, synthetic, or semi-synthetic. The term "protein" is understood to extend to peptides, oligopeptides, polypeptides, and also any therapeutic protein as defined below.

[0044] The term "therapeutic protein" as used herein refers to any protein or polypeptide administered to a subject with the aim of treating or preventing a disease or condition. In particular, the subject may be a mammal or a human. Therapeutic proteins are administered for different purposes, such as replacing missing or abnormal proteins, enhancing existing pathways, providing new functions or activities, interfering with molecules or organisms, and delivering other compounds or proteins, such as radionuclides, cytotoxic drugs, or effector proteins. Therapeutic proteins encompass antibodies, antibody-based drugs, Fc fusion proteins, anticoagulants, blood factors, bone morphogenetic proteins, recombinant protein scaffolds, enzymes, growth factors, hormones, interferons, interleukins, antibody drug conjugates (ADCs), and thrombolytic drugs. Therapeutic proteins can be naturally occurring or recombinant proteins. Their sequences can be natural or modified.

[0045] The term "antibody" refers to a protein capable of specifically binding to an antigen. "Antibody" or "IgG" further refers to a polypeptide substantially encoded by an immunoglobulin gene or genes, or fragments thereof, which specifically binds and recognizes an analyte (antigen). Recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, and define the immunoglobulin classes, IgG, IgM, IgA, IgD, and IgE, respectively.

[0046] 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), which 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 primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains, respectively.

[0047] Antibodies can be monoclonal or polyclonal and can exist in monomeric or polymeric form, for example, IgM antibodies existing in pentameric form and / or IgA antibodies existing in monomeric, dimeric or multimeric form. Antibodies can also include multispecific antibodies (e.g., bispecific antibodies) and antibody fragments, so long as they retain or are modified to include a ligand-specific binding domain. The term "fragment" refers to a portion or part of an antibody or antibody chain that contains fewer amino acid residues than an intact or complete antibody or antibody chain. Fragments can be obtained via chemical or enzymatic treatment of an intact or complete 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, 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 by the term "antibody". In some embodiments, an antibody is a protein that contains an Fc region, such as an immunoglobulin.

[0048] As used herein, and unless otherwise stated, the term "sample" refers to any composition or mixture that contains a target molecule. Samples may be derived from biological or other sources. Biological sources include eukaryotic sources such as animals or humans. Samples may also include diluents, buffers, detergents, and contaminants that are found mixed with the target molecule, and the like.

[0049] The term "impurity" or "contaminant" as used herein refers to any foreign or deleterious 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 that may be present in a sample containing a target molecule to be separated from one or more foreign or deleterious molecules.

[0050] The terms "purify," "separate," or "isolate," as used interchangeably herein, refer to increasing the purity of a target molecule by separating the target molecule from a composition or sample that contains the target molecule and one or more other components, such as impurities. Typically, the purity of a target molecule is increased by removing (completely or partially) at least one impurity from the composition.

[0051] The term "chromatography" refers to any type of technique that separates an analyte of interest (e.g., a target molecule) from other molecules present in a mixture. Most often, the target molecule is separated from other molecules as a result of differences in the speed at which individual molecules of the mixture move through a stationary phase under the influence of a mobile phase, or in a binding and elution process. Examples of chromatographic separation processes include reversed-phase chromatography, ion-exchange chromatography, size-exclusion chromatography, affinity chromatography, hydrophobic interaction chromatography, and mixed-mode chromatography. The chromatographic process of the present invention is based on the interaction with boron clusters, and optionally, in addition, is based on one or more of the other separation processes mentioned above.

[0052] A "buffer" is a solution that resists changes in pH by the action of its acid-base conjugate components. Depending on, for example, the desired pH of the buffer, various buffers that are typically employed are described 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 whose solvent comprises more than 90%, preferably 100%, water.

[0053] The term "stationary phase" refers to any type of adsorbent, matrix, resin or solid phase that separates a target molecule from other molecules present in a mixture in a separation process. Most often, the target molecule is separated from other molecules as a result of the difference in the rate at which individual molecules of the mixture bind to the stationary phase and / or move through the stationary phase under the influence of the mobile phase. The stationary phase is typically contained in a column or cartridge. The stationary phase according to the present invention comprises a substrate and at least one boron cluster.

[0054] A "functional group" is a ligand that is attached to the substrate of the stationary phase and determines the binding properties 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 phases according to the present invention contain at least one boron cluster as a functional group. It is contemplated that they may additionally contain one or more other functional groups as listed above. These other functional groups are typically either attached to the boron cluster or attached to the substrate independently of the boron cluster. Often, one functional group or one boron cluster has more than one binding property.

[0055] When "loading" a chromatography column in a bind and elute mode, a sample or composition containing a target molecule and one or more impurities is loaded onto the chromatography column using a buffer. The buffer has a conductivity and / or pH such that the target molecule binds to the stationary phase, while ideally all impurities pass through the column unbound. Separation of the bound target molecule from the one or more impurities can be further performed using a change in conductivity and / or pH, such that the target molecule is washed or eluted before or after the one or more impurities.

[0056] Typically, the buffer in which the sample is loaded onto the stationary phase is called the loading buffer or sample buffer. When "loading" a chromatography column to allow the target molecule to "flow through", a sample or composition containing the target molecule and one or more impurities is loaded onto the chromatography column using a buffer having 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.

[0057] 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 for equilibration.

[0058] "Wash" or "washing" means passing a suitable liquid, e.g., a buffer, through or over the stationary phase. Typically, washing is used to remove weakly bound contaminants from the stationary phase before eluting the target molecules in bind / elute mode, or to remove unbound or weakly bound target molecules after loading.

[0059] In this case, typically the washing buffer and the loading buffer are the same. If a virus inactivation buffer is used, it is used to inactivate any existing viruses before eluting the target molecule. In this case, typically the virus inactivation buffer is different from the loading buffer because it may contain detergents or have different properties (pH / conductivity / salts and their amounts).

[0060] Washing can also be used to remove contaminants from the stationary phase after elution of the target molecule. This is done by passing a suitable liquid, e.g. a buffer, through or over the stationary phase after elution of the target molecule. In this case, typically the washing buffer is different from the loading buffer. It may contain a surfactant / surfactants or have different properties (pH / conductivity / salt and their amount). The washing buffer can be, for example, an acidic buffer.

[0061] "Eluting" a molecule (e.g., a target molecule or an impurity) from a stationary phase means removing the molecule therefrom. Elution can occur directly in flow-through mode, when the target molecule is eluted with a solvent in front of the loading buffer, or by changing the solution conditions so that a buffer different from the loading buffer competes with the molecule of interest for the 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 the charged sites on the ion exchange material.

[0062] The terms "flow-through process", "flow-through mode", and "flow-through operation", as used interchangeably herein, are intended to mean that at least one target molecule contained in a sample along with one or more impurities passes through a chromatographic stationary phase that generally binds the one or more impurities, where the target molecule generally does not bind (i.e., flows through) and elutes from the stationary phase with the loading buffer.

[0063] The terms "bind and elute mode" and "bind and elute process" as used herein 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.

[0064] Solid phase extraction (SPE) is a sample preparation method 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 molecules or undesired impurities in the sample are retained on the stationary phase. The portion that passes through the stationary phase is either collected or discarded depending on whether it contains the target molecules or undesired impurities. If the portion retained on the stationary phase includes the target molecules, they can then be removed from the stationary phase for recovery in an additional step of rinsing the stationary phase with an appropriate eluent.

[0065] According to the present invention, particle size is determined by laser diffraction, preferably using a Malvern Master Sizer, and pore size is determined by inverse SEC.

[0066] Electron-withdrawing groups include atoms or groups of atoms that have an electron-withdrawing inductive and / or mesomeric effect and are typically more electronegative than hydrogen. Exemplary electron-withdrawing groups are I, Br, Cl, F, CO2H, NO2, CN. Electron donating groups include atoms or groups of atoms that have an electron donating inductive and / or mesomeric effect. Exemplary electron donating groups are -OH, O-alkyl, -NH2, -N-alkyl, -N(alkyl)2.

[0067] Alkyl or alkyl group typically represents a linear or branched alkyl group having 1 to 20 C atoms or the number of C atoms as indicated, for example methyl, ethyl, isopropyl, propyl, butyl, sec-butyl or tert-butyl, furthermore 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-tetracecyl, n-pentadecyl, n-hexadecyl, n-heptadecyl, n-octadecyl, n-nonadecyl or n-eicosyl, preferably the alkyl group has 1 to 10 C atoms. C1 to C4 alkyl, C1 to C6 alkyl, C1 to C8 alkyl, C1 to C 10 Alkyl and C1-C 15 Alkyl represents a straight-chain or branched alkyl group having the number of C atoms as indicated.

[0068] Alkoxy or alkoxy groups in the context of the present invention are typically linear or branched alkoxy radicals having 1 to 20 C atoms or with the number of C atoms as indicated, for example C1-C4 alkoxy is a linear or branched alkoxy radical having 1 to 4 carbon atoms. Preferred examples include methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy, iso-butoxy, sec-butoxy and tert-butoxy.

[0069] Cycloalkyl or cycloalkyl group represents a saturated hydrocarbon ring containing 3, 4, 5, 6, 7, 8 or more carbon atoms or having the number of carbon atoms as indicated. Said cycloalkyl group is, for example, a monocyclic hydrocarbon ring, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl or cyclooctyl, a bicyclic hydrocarbon ring, such as bicyclo[4.2.0]octyl or octahydropentalenyl, or bridged or caged saturated ring groups such as norborane or adamantyl, and cubane, preferably cyclohexyl and adamantyl.

[0070] Heteroalkyl or heteroalkyl group denotes a straight or branched alkyl group as defined above, in which the alkyl group contains one or more different heteroatoms from the N, O and S series.

[0071] A (per)fluoroalkyl, (per)fluoroalkenyl or (per)fluoroaryl group represents an aliphatic and / or aromatic group with fluorine substituents. In a fully fluorinated group, all hydrogen atoms are replaced by fluorine. In a partially fluorinated group, one, two or more hydrogen atoms are replaced by fluorine. Examples are fully fluorinated substituents such as -CF3, -C2F5, -C6F5, -CF=CF2. An example of a partially fluorinated group is -CH2CF 3、 -C6H3(CF3)2.

[0072] Alkenyl or alkenyl group typically denotes a straight-chain or branched alkenyl having 2 to 20 C atoms or the number of C atoms as indicated, where in addition multiple double bonds may be present, such as, for example, allyl, 2- or 3-butenyl, isobutenyl, sec-butenyl, furthermore 4-pentenyl, iso-pentenyl, hexenyl, heptenyl, octenyl, -CH 17 , -C 10 H 19 ~-C 20 H 39 and preferably allyl, 2- or 3-butenyl, iso-butenyl, sec-butenyl.

[0073] Alkynyl or alkynyl group represents a straight-chain or branched alkynyl radical, typically having 2 to 20 C atoms or the number of C atoms as indicated and at least one triple bond. Those alkynyl groups are straight-chain or branched alkynyl radicals having 2 to 4 carbon atoms [(C2-C4)-alkynyl], examples being ethynyl, prop-1-yn-1-yl, prop-2-yn-1-yl (propargyl), but-1-yn-1-yl, but-2-yn-1-yl, but-3-yn-1-yl and but-3-yn-2-yl.

[0074] Aryl or aryl group typically represents an aryl group having 6, 7, 8, 9, 10, 11, 12 or more C atoms or the number of C atoms as indicated, for example phenyl, naphthyl or anthracenyl. 10 Aryl or C6-C 18 Aryl represents an aryl group having the number of C atoms as indicated. The aryl group may be unsubstituted or substituted, for example, by halogen, NH2, NAlk2, NH alkyl, NO2, CN, SO3H or O alkyl. Substitution may occur once or as many times as indicated by the number of substituents, preferably once.

[0075] Heteroaryl or heteroaryl group denotes a monovalent, monocyclic, bicyclic or tricyclic aromatic ring having 5, 6, 8, 9, 10, 11, 12, 13 or 14 ring atoms ("5- to 14-membered heteroaryl" group), in particular 5, 6, 9 or 10 ring atoms, or the number of C atoms as indicated, which contains at least one ring heteroatom from the N, O and / or S series and optionally 1, 2 or 3 further ring heteroatoms, which are bonded via a ring carbon atom or, optionally, via a ring nitrogen atom (if permitted by the valences). The heteroaryl group may be, for example, a 5-membered heteroaryl group, such as thienyl, furanyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiadiazolyl or tetrazolyl; or a 6-membered heteroaryl group, such as, for example, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl or triazinyl; or a tricyclic heteroaryl group, such as, for example, carbazolyl, acridinyl or phenazinyl. or a 9-membered ring heteroaryl group, such as, for example, benzofuranyl, benzothienyl, benzoxazolyl, benzoisoxazolyl, benzimidazolyl, benzothiazolyl, benzotriazolyl, indazolyl, indolyl, isoindolyl, indolizinyl or purinyl; or a 10-membered ring heteroaryl group, such as, for example, quinolinyl, quinazolinyl, isoquinolinyl, cinnolinyl, phthalazinyl, quinoxalinyl or pteridinyl.In general, unless otherwise stated, a heteroaryl or heteroarylene group also includes all possible isomeric forms thereof, for example, tautomers, and positional isomers with respect to the point of connection with the rest of the molecule.Preferred heteroaryl groups are imidazolyl, pyridyl, furanyl, thienyl, substituted pyridyl and morpholyl.

[0076] Halogen refers to a moiety selected from the list including fluorine, chlorine, bromine, or iodine. Pseudohalogen or pseudohalogen group refers to a moiety that is a polyatomic analogue of halogen, such as cyano, isocyano, thiocyanato, cyanate, or isocyanate. A sugar derivative refers to a carbohydrate, especially a mono- or disaccharide, such as glucose, fructose, galactose, sucrose or lactose.

[0077] A substrate is a material to which the boron clusters are attached. In accordance with the present invention, the term "substrate" is used interchangeably with "support", "support material", "carrier" or "carrier material".

[0078] The substrate may consist of irregularly shaped or spherical particles, the particle size of which is preferably between 2 and 1000 μm, with particle sizes between 3 and 300 μm being preferred. The substrate may be in the form of, inter alia, non-porous, core-shell or, preferably, porous particles. The pore size is preferably between 2 and 300 nm. Pore sizes between 5 and 200 nm are preferred.

[0079] The substrate may also be in the form of a membrane, fiber, hollow fiber, coating, filter, capillary, surface, monolith or monolithic molding. Preferably, the substrate is a bead or membrane, preferably a porous bead or membrane. It is also contemplated that the substrate may be produced by additive manufacturing, such as 3D printing.

[0080] Membranes as substrates can be distinguished from particle-based chromatography by the fact that the interaction between the solute, e.g., the target nucleic acid or contaminants, and the matrix does not occur in the dead-end pores of the particle, but mainly in the through-pores of the membrane. Exemplary membrane types are flat sheet systems, stacks of membranes, microporous polymer sheets with integrated cellulose, polystyrene or silica-based membranes, as well as radial flow cartridges, hollow fiber modules and hydrogel membranes. Preferred are hydrogel membranes. Such membranes include a membrane support and a hydrogel formed within the pores of the support. The membrane support provides mechanical strength to the hydrogel. The hydrogel determines the properties of the final product, such as pore size and binding chemistry.

[0081] The membrane support may consist of any porous membrane, such as a polymeric membrane, a ceramic-based membrane, a woven or non-woven fibrous material, etc. Suitable polymeric materials for the membrane support are preferably cellulose or cellulose derivatives, as well as other, preferably inert, polymers, such as polyethylene, polypropylene, polybutylene terephthalate or polyvinylidene difluoride.

[0082] Particularly preferred are membranes made from an assembly containing an inert, flexible fibrous reticulate support, such as Natrix® Q chromatography membranes, Merck KGaA, Germany, which contain porous polyacrylamide hydrogels with quaternary ammonium groups (strong anion exchange groups) inside and around the fibrous reticulate support.

[0083] Monoliths and monolithic articles are three-dimensional bodies that have through-holes, such as interconnected channels, that allow liquid to flow from one side of the monolith through the monolith to the other side of the monolith.

[0084] The mobile phase flows through these through-pores, so that the molecules to be separated are transported by convection rather than diffusion. Monolithic adsorbents, due to their structure, exhibit separation efficiency and dynamic capacity that are independent of flow rate. Monoliths are typically formed in situ from reactant solutions and can have any shape or constrained geometry, typically frit-free structures, which ensures convenience of operation. Preferably, monolithic materials have a dual porous structure, mesopores and macropores. The micron-sized macropores are through-pores and ensure fast dynamic transport and low back pressure in applications; the mesopores contribute to sufficient surface area and thus high loading capacity.

[0085] Suitable organic polymers are polymethacrylates, polyacrylamides, polystyrenes, polyurethanes, and the like, such as poly(methacrylic acid-ethylene dimethacrylate), poly(glycidyl methacrylate-ethylene dimethacrylate) or poly(acrylamide-vinylpyridine-N,N'-methylenebisacrylamide). Inorganic monoliths can be made of silica or other inorganic oxides. Preferably, they are made of silica. Monoliths can be made of organic, inorganic, or organic / inorganic hybrid materials. Preferred are organic polymer-based monoliths.

[0086] In one embodiment, the substrate is in the form of a porous particle. The particle can be made of inorganic or organic materials or polymers. In one embodiment, the substrate is an inorganic material, for example made of metal oxides such as SiO2, Al2O3, titanium dioxide, zirconium dioxide, or other silica-based materials such as controlled pore glasses.

[0087] The substrate may also be an inorganic-organic hybrid material or any other combination of organic and inorganic materials. The substrate may also be an organic material or a polymer. In one embodiment, the substrate 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, of the type known as Sepharose® or Sephadex®.

[0088] In one embodiment, the substrate is made of synthetic polymers, preferably in the form of porous beads or membranes, and comprises cross-linked synthetic polymers such as styrene or styrene derivatives, divinylbenzene, acrylamide, acrylic acid esters, methacrylic acid esters, vinyl esters, vinyl amides, etc. Preferred are polymers based on polystyrene, polyvinyl alcohol, or copolymers of (meth)acrylate derivatives and comonomers bearing aliphatic hydroxyl groups. Polymers based on a certain type of structure, such as polystyrene or polyvinyl ether, are polymers that contain said structure. They may also contain other structures, such as resulting from the copolymerization of two different monomers.

[0089] In another preferred embodiment, the substrate is a polyvinyl ether-based material, in particular a copolymer formed by copolymerization of at least one compound from groups a) and b), where group a) is at least one alkyl vinyl ether of formula (I), [ka] During the ceremony, R1, R2, R3 independently of one another represent H or C1-C6 alkyl, preferably H or -CH3; and R4 represents a substituent having at least one hydroxyl group; And Group b) is at least one crosslinker according to formula (II) and / or (III) and / or (IV), [ka] During the ceremony, X represents a divalent alkyl group having 2 to 5 C atoms, preferably 2 or 3 C atoms, in which one or more methylene groups that are not adjacent and are not located in the direct vicinity of 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 groups may be, independently of one another, hydroxyl, C1-C6 alkyl, halogen, NH2, C6-C 10 Aryl, NH-(C1-C8)-alkyl, N-(C1-C8)-alkyl 2, may be substituted by C1-C6-alkoxy or C1-C6-alkyl-OH; [ka] During the ceremony In the formulas (III) and (IV), Y1 and Y2 are each independently selected from C1 to C 10 represents an alkyl or cycloalkyl, in which one or more non-adjacent methylene groups or methylene groups that are not located in the direct vicinity of an N may be replaced by O, C=O, S, S=O, SO2, NH, NOH or N, and one or more H of a methylene group may be, independently of one another, hydroxyl, C1-C6 alkyl, halogen, NH2, C6-C 10 -Aryl, NH(C1-C8)alkyl, N(C1-C8)alkyl2, C1-C6-alkoxy or C1-C6-alkyl-OH or C6-C 18 aryl, where one or more H in the aryl system may be replaced, independently of one another, by hydroxyl groups, C1-C6-alkyl, halogen, NH2, NH(C1-C8)alkyl, N(C1-C8)alkyl2, C1-C6-alkoxy or C1-C6-alkyl-OH, and A is a divalent alkyl group having 2 to 5 C atoms, preferably 2 or 3 C atoms, in which one or more non-adjacent methylene groups or methylene groups not located in the direct vicinity of 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, independently of one another, a hydroxyl group, a C1-C6 alkyl, a halogen, NH2, a C6-C6 alkyl group, a C1-C6 alkyl group ... 10 Aryl, NH(C1-C8) alkyl, N(C1-C8) alkyl 2, It may be substituted by C1-C6 alkoxy or C1-C6 alkyl-OH.

[0090] R4 in formula (I) is typically an alkyl, cycloalkyl or aryl bearing at least one hydroxyl group.

[0091] In a highly preferred embodiment, the matrix is ​​formed by copolymerization of an alkyl vinyl ether, selected from the group consisting of 1,4-butanediol monovinyl ether, 1,5-pentanediol monovinyl ether, diethylene glycol monovinyl ether or cyclohexanedimethanol monovinyl ether, and divinylethyleneurea (1,3-divinylimidazolin-2-one) as a crosslinker.

[0092] An example of a suitable commercially available vinyl ether-based substrate is Eshmuno®, Merck KGaA, Germany. Further examples of suitable commercially available matrices are Macro-Prep CM Resin, Fractogel® 2, and Sepharose®.

[0093] The boron clusters are attached to the substrate as defined above. The boron clusters can be non-covalently or covalently bound to the substrate, preferably the boron clusters are covalently bound to the substrate. In one embodiment, at least one boron cluster is directly bonded to the substrate. Covalent bonding can be achieved by directly bonding the boron cluster to a suitable moiety on the substrate, such as, for example, OH, NH, carboxyl, phenol, anhydride, aldehyde, epoxide, or thiol.

[0094] In a preferred embodiment, at least one boron cluster is attached to the substrate via a linker. According to the present invention, a linker is a chemical group that connects the boron cluster and the substrate. The chemical group can contain a chemical moiety of the substrate and / or the boron cluster, such as amine, hydroxy, epoxy or carboxy. Any linker suitable for connecting the substrate and the boron cluster can be used.

[0095] In one embodiment, the linker has the structure of formula (V): -XZY- (V) During the ceremony, X and Y represent first and second reactive or activating groups, preferably X and Y are independently selected from the moiety consisting of hydroxy, amino, thiol, carboxy, oxiranyl, formyl, halo, isocyanato, and chlorosulfonyl; and Z is (a) C1~C 15 Alkyl, (b) aryl, (c) C1~C 10 Alkyl-aryl, (d) C1~C 10 alkyl-aryl-C1-C6 alkyl, where one or more of the carbon atoms of the alkyl may be replaced by oxygen, sulfur or nitrogen, and where aryl includes, but is not limited to, phenyl, naphthyl, pyridyl or thienyl, and where one or more of the carbon atoms may be replaced by OH or C1-C6 alkyl; (e) Peptides of 2 to 10 amino acids, including L- and D-forms of amino acids, including, but not limited to, glycine, alanine, valine, leucine, isoleucine, serine, threonine, aspartic acid, asparagine, glutamic acid, glutamine, lysine, hydroxylysine, histidine, arginine, phenylalanine, tyrosine, tryptophan, cysteine, methionine, ornithine, beta-alanine, homoserine, homotyrosine, homophenylalanine, and citrulline. is selected from the group consisting of:

[0096] Other linkers include, but are not limited to, p-benzoquinone, bis-(diazobenzidine), 3,6-bis-(mercurimethyl)dioxane, bisoxylane, cyanuric chloride, dicyclohexylcarbodiimide, dinitrophenylsulfone, dimethyladipimidate, dimethylsuberimidate, divinylsulfone, N,N'-ethylene-bis-(iodoacetamide), glutaraldehyde, hexamethylenebis(maleimide), These may include hexamethylene diisocyanate, N,N'-1,3-phenylene bis-(maleimide), phenol-2,4-disulfonyl chloride, tetraazotized o-dianisidine, toluene diisocyanate, Woodward K reagent, water soluble carbodiimides, 6-aminohexanoic acid, hexamethylenediamine, 1,7-diamino-4-aza-heptane (3,3'-diamino-dipropylamine), and amino acids or peptides.

[0097] In one embodiment, the structure of the linker is -X m -(CH-A) n -X-(CHA-CHB)- and During the ceremony, X represents O, NR' or S, where R' represents H or C1-C4 alkyl; A represents H or C1-C4 alkyl; B represents H or OH; n represents an integer from 0 to 12, m represents the integer 1, or represents the integer 0 when n represents the integer 0;

[0098] In one embodiment, the structure of the linker is -D x -(CHA-CHB) y -XZ m -(CHA-CH2) n -O- and During the ceremony, X represents O or NR' or, when Z represents CH2 or m represents the integer 0, X represents S; D represents O, NR' or S; Z represents CH2 or C=O; A, B, and R' each independently represent H or C1-C4 alkyl; n represents an integer from 1 to 12, m represents an integer of 0 or 1; x represents the integer 1, or if y represents the integer 0, then x represents the integer 0; y represents an integer from 0 to 4.

[0099] In one embodiment, the structure of the linker is -D x -(CHA-CHB) y -[O k -(CHA-CHB)] z -XZ m -(CHY-CH2) n -O- and During the ceremony, X represents O or NR' or, when Z represents CH2 or m represents the integer 0, X represents S; D represents O, NR' or S; Z represents CH2 or C=O; Y is H, C1-C4 alkyl, -(CH2-CH2) p represents C(O)OH, A, B, and R' each independently represent H or C1-C4 alkyl; n represents an integer from 1 to 12, m, k, and p each independently represent an integer 0 or 1; x represents the integer 1, or if y represents the integer 0, then x represents the integer 0; y represents an integer from 0 to 4, and z represents an integer from 1 to 4.

[0100] In one embodiment, the structure of the linker is as a structure selected from the group consisting of: -CH(OH)CH2-NH-, -O-(CH2)2-(C=O)-NH-, -O-(CH2)3-(C=O)-NH-(CH2)2-O-(CH2)2-O-, -O-(CH2)2-(C=O)NH-(CH2)4-O-, -O-(CH2)2-(C=O)NH-(CH2)2-O-(CH2)2-O- and -O-(CH2)2-O-(CH2)2-NH-(C=O)-(CH2)2-O-.

[0101] The stationary phase according to the present invention comprises a substrate and at least one boron cluster as defined above. The stationary phase may comprise further functional groups in addition to the at least one boron cluster. This may be, for example, ionic, hydrophilic or hydrophobic groups. By producing a stationary phase with two different functionalities, a mixed-mode material is obtained with separation properties resulting from both or multiple types of functionality, which in the present case is due to at least one functional group being a boron cluster.

[0102] The stationary phase according to the invention can also be described as a substrate provided with separation effectors, at least one of which is a boron cluster as defined above.

[0103] In a preferred embodiment, the stationary phase comprises a substrate formed by copolymerization of an alkyl vinyl ether selected from the group of 1,4-butanediol monovinyl ether, 1,5-pentanediol monovinyl ether, diethylene glycol monovinyl ether or cyclohexane dimethanol monovinyl ether and divinylethylene urea (1,3-divinylimidazolin-2-one) as a crosslinker. More preferably, the substrate is a suitable commercially available vinyl ether-based substrate, for example Eshmuno®, Merck KGaA, Germany.

[0104] In a preferred embodiment, the stationary phase is Closo-B 12 X 11 , Closo-B 10 X9, Nido-C2B9X 11 or 3,3'-Co(1,2-C2B9X 10 )(1',2'-C2B9X 11 ) where X is a suitable substituent, for example, closo-B 12 H 11 , Closo-B 10 H9, Nido-C2B9H 11 or 3,3'-Co(1,2-C2B9H 10 )(1',2'-C2B9H 11 ) is hydrogen as in closo-B 12 X 11 For example, Closo-B 12 H 11 It is.

[0105] According to the present invention, the term "group density" of the stationary phase refers to the density of functional groups for the attachment of boron clusters on the substrate, e.g., the COO of the substrate starting material expressed in μeq / g. - Refers to base density. According to the present invention, the term "ligand density" of the stationary phase refers to the density of boron clusters on the substrate expressed in μeq / g.

[0106] In a preferred embodiment, the stationary phase has a group density of 300 to 2400 microequivalents per gram (μeq / g), preferably 700 to 1500 μeq / g, more preferably 1000 μeq / g. In a preferred embodiment, the stationary phase has a ligand density of 140-1500 microequivalents per gram (μeq / g), preferably 500-1000 μeq / g, more preferably 500-700 μeq / g, and most preferably 621 μeq / g. Increasing the group or ligand density increases the amount of target molecules that bind and elute.

[0107] In a preferred embodiment, the stationary phase comprises irregularly shaped or spherical particles, the particle size of which is between 2 and 1000 μm. Preferably, the particle size is between 3 and 600 μm, 3 and 300 μm, 20 and 150 μm, more preferably between 20 and 100 μm. The particles may be in the form of non-porous, core-shell, or preferably porous particles. The pore size of the particles is preferably between 2 and 300 nm. Preferably, the pore size is between 5 and 200 nm, more preferably between 40 and 110 nm. In one embodiment the stationary phase is a combination of the preferred embodiments of the stationary phase as described above which may be combined.

[0108] The stationary phase according to the present invention can be made by different processes. At least one boron cluster is either directly attached to the substrate or via a linker as described above. Covalent attachment can be performed, for example, by direct attachment via a suitable residue on the substrate. Suitable residues are known to those skilled in the art, for example OH, NH2, carboxyl, phenol, anhydride, aldehyde, epoxide or thiol. Exemplary syntheses are described in the literature, for example in Graalfs, H.; Graft Copolymer for Cation-exchange Chromatography; WO2008145270.

[0109] It is also possible to produce stationary phases according to the invention by polymerizing monomers comprising a boron cluster 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.

[0110] In the case of "grafting to", polymer chains must first be formed from the monomers and in a second step are attached to the surface of the substrate. In the case of "grafting from", the polymerization reaction is initiated on the surface of the substrate and the graft polymer is built up directly from the individual monomers.

[0111] The "grafting from" method is preferred, and particularly preferred are variants in which only a small number of by-products, such as non-covalently bound polymers, are formed, which must be separated. For example, processes involving controlled free radical polymerization, such as the method of atom transfer free radical polymerization (ATRP), are suitable. Here, initiator groups are covalently bound to the surface of the substrate with the desired density in a first step. The initiator groups are, for example, halides bound via ester functional groups, as in 2-bromo-2-methylpropionic acid ester. The graft polymerization is carried out in a second step in the presence of copper(I) salts.

[0112] In one embodiment the stationary phase is produced by grafting boron clusters onto a substrate, in a further embodiment the boron clusters are contained in polymer chains, also called tentacles, which are grafted onto the substrate.

[0113] Depending on the process conditions, grafting with a monomer results in the coupling of one or more monomers in a polymerization reaction to form a polymer chain. The polymer chain can contain multiple monomers and can contain multiple boron clusters. Throughout the experimental part, e.g. the examples in sections 1.7, 1.8 and 1.10, for simplicity, the products resulting from such grafting processes are shown as products obtained by coupling of only one monomer. It is emphasized that the products extend to stationary phases resulting from the coupling of one monomer with multiple monomers, whereas the coupling of multiple monomers results in polymer chains that can contain more than one boron cluster.

[0114] As an example, Example 1.8.3 is depicted by formula (VI) below. [ka]

[0115] Eshmuno® カルボキシ The starting material was Eshmuno® HPLC as exemplified in Section 1.8.1. ヒドロキシ and acrylic acid. The grafting reaction with the monomeric acrylic acid can result in the coupling of one acrylic acid or multiple acrylic acids in a polymerization reaction to form a polymer chain. The polymer chain contains two or more monomers with respective carboxy groups. Each carboxy group can be coupled with an amine derivative of an adamantane or closo-boron cluster under the protocols exemplified in Sections 1.8.2-1.8.13. Hence, the polymer chain can contain multiple boron clusters.

[0116] The following formula (VII) illustrates a possible chemical structure for Example 1.8.3. [ka] Wherein, n represents an integer of 0, 1, 2 or more. Preferably, n represents an integer from 0 to 10. Formula (VII) is illustrated with one boron cluster, but it is emphasized that each of the carboxy groups can be coupled with a boron cluster. In accordance with the present invention, the illustration of Example 1.8.3 with formula (VI) extends to all chemical structures as mentioned above. This applies equally to all examples in sections 1.8 and 1.10.

[0117] As an example, Example 1.7.3 is depicted by formula (VIII) below. [ka] Formula (VIII) can be prepared by the process of Eshmuno®, as exemplified in Section 1.7.1. ヒドロキシ and a monomer that is an allylic derivative of an acrylic acid or a boron cluster (a boron-containing monomer). The grafting reaction can result in the coupling of one boron-containing monomer or multiple boron-containing monomers in a polymerization reaction to form a polymer chain. The polymer chain contains two or more monomers with their own boron cluster. Thus, the polymer chain contains multiple boron clusters.

[0118] The following formula (IX) illustrates a possible chemical structure for Example 1.7.3. [ka] Wherein n represents an integer of 1, 2 or more. Preferably, n represents an integer of 1 to 10, more preferably 1 to 5. In accordance with the present invention, the illustration of Example 1.7.3 using formula (VIII) extends to all chemical structures as mentioned above. This applies equally to all examples in section 1.7.

[0119] The highly preferred one-step grafting from polymerization reaction suitable for the preparation of the stationary phase of the present invention can be initiated by cerium(IV) on a hydroxyl-containing support without the need to activate the support.

[0120] The cerium(IV)-initiated grafting is preferably carried out according to EP 0337144 or US 5,453,186. The produced chains are linked to the substrate via monomer units. For this purpose, the substrate according to the invention is suspended in a solution of monomers, preferably in an aqueous solution. The grafting of the polymeric material is effected in the course of a conventional redox polymerization with the exclusion of oxygen. The polymerization catalyst employed is cerium(IV) ions, since this catalyst forms free radical sites on the surface of the substrate, from which the graft polymerization of the monomers begins. The reaction is usually carried out in dilute mineral acid. To carry out the graft polymerization, the acid is usually employed in aqueous solution at a concentration ranging from 1 to 0.00001 mol / l, preferably from 0.1 to 0.001 mol / l. The use of dilute nitric acid, employed at a concentration ranging from 0.1 to 0.001 mol / l, is very particularly preferred.

[0121] For the preparation of the separation material according to the invention, the monomers are usually added in excess to the substrate, typically between 0.05 and 100 mol total monomer per liter of precipitated polymeric material, preferably between 0.05 and 25 mol / l.

[0122] The polymerization is terminated by a termination reaction involving cerium salts. For this reason, the (average) chain length can be influenced by the concentration ratio of substrate, initiator, and monomer. Furthermore, homogeneous monomers or mixtures of different monomers can also be employed, in the latter case forming grafted copolymers.

[0123] Monomers advantageously used for the preparation of the separation material according to the invention are those according to formula (X): CR*R**=CR 1 -(CR2 ) z -YB(X) During the ceremony R*, R**, and R 1 represent, independently of one another, H or CH3, R 2 is selected from the group consisting of H, alkyl, phenyl, cycloalkyl or alkylcycloalkyl, phenylalkyl or alkylphenyl radicals with up to 10 C atoms in the alkyl group, which radicals can be mono- or polysubstituted, preferably by: halogen, alkoxy, cyano, amino, mono- or dialkylamino, trialkylammonium, carboxyl, sulfonyl, acetoxy or acetamino moieties, Cyclic or bicyclic substituents having 5 to 10 C atoms, in which one or more CH or CH2 groups can be replaced by N or NH, N or NH and S, or N or NH and O, Or the structure -(CH2) n -SO2-(CH2) n -S(CH2) n OH sulfonyl sulfide, where n represents an integer from 2 to 6; Y is -C(=O)-X, -OC(=O)(CHR 3 )-, -(CH2) m NH-, -(CH2) m -, where X is -NR 4 -, -NR 4 R 5 -, S.R. 4 , -S- or -O-, where R 4 and R 5 are selected independently from the group consisting of alkyl, phenyl, cycloalkyl or alkylcycloalkyl, phenylalkyl or alkylphenyl radicals having up to 10 C atoms in the alkyl radical, which radicals can be mono- or polysubstituted, preferably by: halogen, alkoxy, cyano, amino, mono- or dialkylamino, trialkylammonium, carboxyl, sulfonyl, acetoxy or acetamino moieties, cyclic or bicyclic substituents having 5 to 10 C atoms, in which one or more CH or CH2 groups can be replaced by N or NH, N or NH and S, or N or NH and O, or the structure -(CH2) n -SO2-(CH2) n -S(CH2) n Sulfonyl sulfides of OH and the substituent R 2 and R 3 may also be H, where R 2 and R 3 are coordinated to one another such that either both substituents are acidic or basic, or one or both of the substituents are neutral, where n represents an integer from 2 to 6; R 3 is selected from H or an alkyl group having up to 5 C atoms, m represents an integer from 1 to 12; z represents an integer from 0 to 6, and B represents a boron cluster according to the invention.

[0124] Preferably, the monomers used for the preparation of the separation material according to the invention are those according to formula (XI): [ka] During the ceremony R 1 , R 2 and R 3 is independently selected from H or CH3, preferably H; Y represents a boron cluster according to the present invention.

[0125] The separation materials according to the present invention preferably contain only tentacle-like linear polymer structures grafted onto the substrate constructed from monomers according to formula (X).Preferably, they contain linear polymers constructed only by one type of monomer according to formula (X).

[0126] However, a linear polymer may also be constructed by copolymerization of two or more different monomers according to formula (X). A linear polymer may also be constructed by copolymerization of one or more different monomers according to formula (X) with one or more other polymerizable monomers such as acrylamides, methacrylates, acrylates, methacrylates functionalized with e.g. ionic, hydrophilic or hydrophobic groups, etc.

[0127] The boron clusters as described above, the stationary phases comprising the boron clusters and the separation devices comprising the stationary phases according to the invention are preferably used for the selective, partially selective or non-selective binding, adsorption or purification of one or more target molecules aimed at their separation from a sample liquid or for the selective, partially selective or non-selective binding or adsorption of one or more secondary components aimed at their separation from a matrix, for the isolation, enrichment and / or depletion of biomolecules from natural sources, the isolation, enrichment and / or depletion of biopolymers from recombinant sources, the isolation, enrichment and / or depletion of proteins and peptides, the isolation, enrichment and / or depletion of monoclonal and polyclonal antibodies, the isolation, enrichment and / or depletion of viruses, the isolation, enrichment and / or depletion of host cell proteins, the isolation, enrichment and / or depletion of ADCs, the isolation, enrichment and / or depletion of lipids such as alkaloids, di- or triglycerides, carbohydrates, nucleic acids or other biomolecules. Preferably, the target molecules are proteins. Preferably, the separation and / or purification of the target molecule is solid phase extraction or chromatographic separation, more preferably chromatographic separation.

[0128] In one embodiment the boron clusters, the stationary phase according to the invention or the separation device according to the invention are used for the separation and / or purification of target molecules, preferably proteins. In one embodiment the separation and / or purification of the target molecule is solid phase extraction or chromatographic separation, preferably chromatographic separation.

[0129] The target molecule is separated from at least one or more other substances from the sample by the fact that the sample containing the target molecule is liquid or dissolved in a liquid and is brought into contact with the stationary phase according to the invention. The contact time usually ranges from 30 seconds to 24 hours. It is advantageous to work according to the principle of liquid chromatography by passing a liquid through a chromatography column containing a stationary phase according to the invention. The liquid can simply pass through the column through its gravity or it can be pumped by means of a pump. An alternative method is batch chromatography, in which the stationary phase is mixed with the liquid by stirring or shaking for as long as necessary to allow the target molecule to bind to the stationary phase. It is also possible to work according to the principle of chromatographic fluidized beds by introducing the liquid to be separated, for example, into a suspension containing the stationary phase, in which the separation material is selected so that it is suitable for the desired separation due to its high density and / or magnetic core.

[0130] When the chromatographic process is carried out in bind and elute mode, the target molecule binds to the stationary phase according to the invention. The stationary phase can optionally be washed afterwards with a wash buffer, which preferably has the same ionic strength and the same pH as the liquid in which the target molecule comes into contact with the stationary phase. The wash buffer removes all substances that are not bound to the stationary phase. A further wash step with another suitable buffer may follow this without desorbing the target molecule. Desorption of the bound target molecule is typically carried out by changing the ionic strength in the eluent and / or by changing the pH of the eluent and / or by changing the solvent. The target molecule can thus be obtained in purified and concentrated form in the eluent. The target molecule usually has a purity of 70% to 99%, preferably 85% to 99%, particularly preferably 90% to 99% after desorption from the stationary phase.

[0131] However, when the chromatographic process is carried out in flow-through mode, the target molecule remains in the liquid phase but other accompanying substances bind to the stationary phase, and the target molecule is then directly obtained by collecting the column eluate in the through-flow.

[0132] The stationary phases according to the invention can be used for many different applications, for example they can be used for hydrophilic and hydrophobic separations, as well as aqueous and non-aqueous separations.

[0133] Without further elaboration, it is believed that one skilled in the art 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 not limitative of the remainder of the disclosure in any way whatsoever.

[0134] The present invention is further directed to a method for the separation of a target molecule from at least one other compound, by contacting a stationary phase according to the present invention as described above, preferably present in a separation device, with a liquid containing the target molecule and at least one other compound, and by the target molecule exhibiting a binding to the stationary phase that is different from the binding of the other compounds, for example by it interacting more strongly or weakly with the stationary phase than the other compounds. This process is preferably used in a process for solid phase extraction or chromatographic separation.

[0135] In another embodiment, the process is a process for chromatographic separation of a target molecule from at least one other compound, by which a stationary phase according to the invention as described above is present in a chromatography column and a liquid comprising the target molecule and 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.

[0136] In another embodiment, the process is carried out by loading a stationary phase according to the invention with an aqueous loading buffer of one pH and one ionic strength, and eluting the target molecule with an aqueous elution buffer of the same pH but another ionic strength, typically a higher ionic strength.

[0137] In another embodiment, the process is carried out by loading the stationary phase according to the invention with an aqueous loading buffer of one pH and one ionic strength and eluting the target molecule with an aqueous buffer of another pH and / or another ionic strength, optionally with further additives.

[0138] In another embodiment the process is carried out by loading the stationary phase according to the invention with an aqueous loading buffer having a pH value in the range of 2 to 11, preferably 5 to 9, more preferably 4.5 to 7.5, most preferably having a pH value of 6 and having a conductivity in the range of 1 to 150 mS / cm, preferably in the range of 2 to 100 mS / cm, and eluting the target molecule with an aqueous buffer of another pH and / or another ionic strength, optionally with further additives.

[0139] In another embodiment, the process is carried out by loading the stationary phase according to the invention with an aqueous loading buffer having a pH value of 6 and eluting the target molecule with an aqueous buffer having a pH value of 10 together with imidazole as further additive, preferably a 3 M solution of imidazole.

[0140] In another embodiment, the process is carried out 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 organic medium, which is an organic liquid containing no more than 10% water, such as methanol, ethanol, acetonitrile, THF, heptane, toluene, etc., or mixtures thereof.

[0141] In another embodiment, the process is carried out by loading an organic loading medium onto the stationary phase according to the present invention and eluting the target molecule with the same medium or a more polar medium, for example using gradient elution. The organic loading medium for this embodiment is a liquid that contains water and an organic solvent, with a maximum of 20% organic solvent. Various organic solvents can be selected, for example methanol, ethanol, acetonitrile, THF, heptane, toluene, etc., or mixtures thereof.

[0142] The present invention is further directed to the chromatographic separation of two proteins having different pI (isoelectric points) and / or different contents of carboxylic acids by means of boron clusters, preferably using a stationary phase according to the present invention.

[0143] The present invention is further directed 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 chromatography applications. In either case, it includes a means for holding the stationary phase. It is envisaged that the stationary phase is enclosed in the device or attached to it.

[0144] In one embodiment, the device comprises a housing with an inlet and an outlet. In another embodiment, it is a plate or pin on one side of which the stationary phase is attached. In another embodiment, it is a filter containing the stationary phase. In a preferred embodiment, the device is a chromatography column comprising the stationary phase described above according to the present invention. Chromatography columns are known to those skilled in the art. They usually comprise a cylindrical tube or cartridge packed with the stationary phase, and a filter, and / or a means for fixing the stationary phase within the tube or cartridge, and optionally a connection for solvent delivery to and from the tube or cartridge. The size of the chromatography column varies depending on the application, for example analytical or preparative. In one embodiment, the column, or generally the separation device, is a single-use device.

[0145] example 1. Synthesis of chromatographic materials 1.1 General protocol for the synthesis of functionalized boron clusters of the general formula: boron-cluster-O-CH2CH2-Y-CH2CH2-NH2 (Y=O or no atom) and the synthesis of the respective tetrabutylammonium salts The transformation of oxonium derivatives of the dodecahydro-closo-dodecaborate anion [B 12 H 12 ] 2- Tetramethylene oxonium derivative of [B 12 H 12 ] 2- as a convenient precursor for the synthesis of functional compounds for boron neutron capture therapy. Polyhedron 2000, 19, 627-632; Ishii, S.; Nakamura, H. Synthesis and biological evaluation of closo-dodecaborate ibuprofen conjugate (DIC) as a new boron agent for neutron capture therapy. J. Organomet. Chem. 2018, 865, 178-182; Kubasov, AS; Matveev, EY; Retivov, VM; Akimov, SS; Razgonyaeva, GA; Polyakova, IN; Votinova, NA; Zhizhin, KY; Kuznetsov, NT Nickel(II) complexes with nitrogen-containing derivatives of the closo-decaborate anion. Russ. Chem. Bull. 2014, 63, [nBu4N][1-O(C2H4)2O-closo-B in ammonia as described in (187-193).12 H 11 The reaction was carried out in the same manner as in reaction 1.

[0146] Here, the dioxane and tetrahydrofuran derivatives of the respective closo-dodecaborate and closo-decaborate anions were suspended in ethanol and mixed with acetonitrile until the boron cluster derivatives were completely dissolved. This solution was then mixed with an aqueous solution of ammonia (25% solution) and stirred at 110° C. for 12 hours. The solvent was removed in vacuum, the residue was dissolved in methanol and mixed with a tetrabutylammonium hydroxide solution (1M in methanol). The solvent was removed under reduced pressure and the residue was taken up in dichloromethane. The organic phase was washed with water and dried over magnesium sulfate. The solvent was removed under reduced pressure and the resulting solid was dried in high vacuum.

[0147] Metathesis to cesium salts: The tetrabutylammonium salt was dissolved in dichloromethane and mixed with a solution of cesium fluoride in methanol. The precipitate was filtered off, washed with dichloromethane and methanol, and dried in high vacuum.

[0148] 1.1.1 Kat2[1-H2N(CH2)2O(CH2)2O-klotho-B 12 H 11 Synthesis of ] (Kat=[nBuN], Cs) [ka] The amounts of chemicals used in these synthesis: Kat=[nBu4N]: 16.2 g (34.4 mmol) of [nBu4N][1-O(C2H4)2O-closo-B 12 H 11 ]; 500 mL of ethanol; 200 mL of a 25% aqueous solution of ammonia; 50 mL of acetonitrile; 100 mL of methanol; 41.2 mL of tetrabutylammonium hydroxide solution (1 M in methanol); 100mL of dichloromethane. [nBu4N]2[1-H2N(CH2)2O(CH2)2O-Closo-B 12 H 11 Yield: 24.7 g (33.9 mmol, 99%), white solid.

[0149] Cs2[1-H2N(CH2)2O(CH2)2O-Clotho-B 12 H 11 ] to 24.7 g (33.9 mmol) of [nBuN]2[1-H2N(CH2)2O(CH2)2O-closo-B dissolved in 100 mL of dichloromethane 12 H 11 ]; 11.4 g (74.5 mmol) CsF in 150 mL of methanol. Wash with 3 x 50 mL dichloromethane and 3 x 50 mL methanol. Yield: 16.1 g (31.6 mmol, 93%), white solid.

[0150] Characterization: [1-H2N(CH2)2O(CH2)2O-Closo-B 12 H 11 ] 2- NMR data for the anion:

number

[0151] 1.1.2. Kat2[1-H2N(CH2)4O-closo-B 12 H11 Synthesis of ] (Kat=[nBuN], Cs) [ka] The amounts of chemicals used in these synthesis: Kat=[nBu4N]: 650mg (1.42mmol) of [nBu4N][1-(CH2)4O-closo-B 12 H 11 ]; 15mL ethanol, 5 mL of a 25% aqueous solution of ammonia; 5 mL of acetonitrile; 10mL of methanol, 3.20 mL of tetrabutylammonium hydroxide solution (1 M in methanol); 25mL of dichloromethane. [nBu4N]2[1-H2N(CH2)4O-klotho-B 12 H 11 Yield: 920 mg (1.29 mmol, 91%), white solid.

[0152] Cs2[1-H2N(CH2)4O-closo-B 12 H 11 ] to 743 mg (1.04 mmol) of [nBu4N]2[1-H2N(CH2)4O-closo-B dissolved in 10 mL of dichloromethane 12 H 11 ]; 347 mg (2.28 mmol) CsF in 15 mL of methanol. Wash with 3 x 5 mL dichloromethane and 3 x 5 mL methanol. Cs2[1-H2N(CH2)4O-closo-B 12 H 11 Yield: 320 mg (0.81 mmol, 77%), white solid.

[0153] Characterization: [1-H2N(CH2)4O-closo-B 12 H 11 ] 2-NMR data for the anion:

number

[0154] 1.1.3. Kat2[2-H2N(CH2)2O(CH2)2O-Clotho-B 10 Synthesis of H9 (Kat=[nBu4N], Cs) [ka] The amounts of chemicals used in these synthesis: Kat=[nBu4N]: 447mg (0.99mmol) of [nBu4N][2-O(C2H4)2O-closo-B 10 H9]; 25mL of ethanol; 10 mL of a 25% aqueous solution of ammonia; 15 mL of acetonitrile, 20mL of methanol, 2.40 mL of tetrabutylammonium hydroxide solution (1 M in methanol); 25mL of dichloromethane. [nBu4N]2[2-H2N(CH2)2O(CH2)2O-Closo-B 10 Yield of H9]: 690 mg (0.97 mmol, 98%), white solid.

[0155] Cs2[2-H2N(CH2)2O(CH2)2O-Closo-B 10 Metathesis to H9]: 690 mg (0.97 mmol) of [nBuN]2[2-HN(CH2)2O(CH2)2O-closo-B dissolved in 10 mL of dichloromethane10 H9]; 230 mg (1.52 mmol) CsF in 15 mL of methanol. Wash with 3 x 5 mL dichloromethane and 3 x 5 mL methanol. Cs2[2-H2N(CH2)2O(CH2)2O-Closo-B 10 Yield of H9]: 200 mg (0.41 mmol, 42%), white solid.

[0156] Characterization: [2-H2N(CH2)2O(CH2)2O-Closo-B 10 H9] 2- NMR data for the anion:

number

[0157] 1.1.4 Kat2[2-H2N(CH2)4O-closo-B 10 Synthesis of H9 (Kat = [nBuN], Cs) [ka] The amounts of chemicals used in these synthesis: Kat=[nBu4N]: 650mg (1.51mmol) of [nBu4N][2-(CH2)4O-closo-B 10 H9]; 15mL ethanol, 5 mL of a 25% aqueous solution of ammonia; 5 mL of acetonitrile; 10mL of methanol, 3.30 mL of tetrabutylammonium hydroxide solution (1 M in methanol); 25mL dichloromethane. [nBu4N]2[1-H2N(CH2)4O-klotho-B 10 Yield of H9]: 966 mg (1.40 mmol, 93%), white solid.

[0158] Cs2[1-H2N(CH2)4O-closo-B 10 Metathesis to H9]: 1.00 g (1.45 mmol) of [nBu4N]2[1-H2N(CH2)4O-closo-B was dissolved in 10 mL of dichloromethane. 10 H9]; 484 mg (3.19 mmol) CsF in 15 mL of methanol. Wash with 3 x 5 mL dichloromethane and 3 x 5 mL methanol. Cs2[1-H2N(CH2)4O-closo-B 10 Yield of H9]: 360 mg (0.76 mmol, 53%), white solid.

[0159] Characterization: [1-H2N(CH2)4O-closo-B 10 H9] 2- NMR data for the anion:

number

[0160] 1.1.5 [3,3'-Co(8-{NH3CH2CH2OCH2CH2O}-1,2-C2B9H 10 )(1',2'-C2B9H 11 Synthesis of [ka] Method A The synthesis was performed in a manner similar to the literature procedure (Kvasnichkova, E.; Masak, J.; Chejka, J.; Mat'atkova, O.; Shicha, V. Preparation, characterization, and the selective antimicrobial activity of N-alkylammonium 8-diethyleneglycol cobalt bis-dicarbollide derivatives. J. Organomet. Chem. 2017, 827, 23-31.). [3,3'-Co(8-{O(CH2CH2)2O}-1,2-C2B9H 10 )(1',2'-C2B9H 11 ) (500 mg, 1.2 mmol) was dissolved in dry THF (20 mL). The orange solution was cooled to 0° C. and gaseous ammonia was bubbled through the solution. After 5 min, the flow of ammonia was stopped and the reaction mixture was stirred for a further 25 min. Afterwards, the solvent was evaporated and the residue was dissolved in diethyl ether and washed with aqueous HCl (3×20 mL, 20% v / v) and water (1×20 mL). The organic layer was separated and the diethyl ether was removed under reduced pressure. [3,3'-Co(8-{NH3CH2CH2OCH2CH2O}-1,2-C2B9H 10 )(1',2'-C2B9H 11 )] yield: 480 mg (1.122 mmol, 92%) orange solid.

[0161] Method B [3,3'-Co(8-{O(CH2CH2)2O}-1,2-C2B9H 10 )(1',2'-C2B9H 11 ) (211 mg, 513.6 μmol) was dissolved in acetonitrile (11 mL) and aqueous ammonia (25%, 8 mL) was added. The orange solution was stirred at 55° C. for 17 h. The solvent was evaporated under reduced pressure. [3,3'-Co(8-{NH3CH2CH2OCH2CH2O}-1,2-C2B9H 10)(1',2'-C2B9H 11 )] yield: 200 mg (467 μmol, 91%) of an orange solid.

[0162] Characterization:

number

[0163] 1.1.6 10-{O(CH2CH2)2O}-nido-7,8-C2B9H 12 Synthesis of [ka] The synthesis was performed according to literature procedures (Rhezachova, P.; Pokorna, J.; Brynda, J.; Kozhishek, M.; Cigler, P.; Lepshik, M.; Fanfrlik, J.; Rhezach, J.; Grantz Shashkova, K.; Sieglova, I.; Pleshek, J.; Sicha, V.; Gruener, B.; Oberwinkler, Sedlachek, J.; Kraeusslich, H.-G.; Hobza, P.; Kral, P.; Konvalinka, J. Design of HIV Protease Inhibitors Based on Inorganic Polyhedral Metallacarboranes. J. Med. Chem. 2009, 52, 7132-7141.).

[0164] [Et3NH][7,8-C2B9H 12 (1.27 g, 5.39 mmol) was suspended in toluene (9 mL) and concentrated sulfuric acid (2.2 mL) was added. The two-phase system was vigorously stirred for 10 min. The toluene layer was separated and the sulfuric acid phase was extracted with another portion of toluene (4 mL). To the collected toluene phase was added dioxane (922.2 μL, 10.78 mmol). The clear solution was stirred at 80° C. for 22 h. Afterwards the solvent was evaporated under reduced pressure. 10-{O(CH2CH2)2O}-nido-7,8-C2B9H 12 Yield: 890 mg (4.04 mmol, 75%) of a white solid.

[0165] Characterization:

number

[0166] 1.1.7 10-(NH3CH2CH2OCH2CH2O)-nido-7,8-C2B9H 11 Synthesis of [ka] The synthesis was performed in a manner similar to literature procedures (Bakardjiev, M.; Anwar, SE; Bavol, D.; Ruozhichkova, Z.; Gruener, B. Focus on Chemistry of the 10-Dioxane-nido-7,8-dicarba-undecahydrido Undecaborate Zwitterion; Exceptionally Easy Abstraction of Hydrogen Bridge and Double-Action Pathways Observed in Ring Cleavage Reactions with OH- as Nucleophile. Molecules 2020, 25, 814.).

[0167] 10-{O(CH2CH2)2O}-nido-7,8-C2B9H 12 (890 mg, 4.04 mmol) was dissolved in dry THF (20 mL) and the solution was cooled to 0° C. Gaseous ammonia was bubbled through the solution for 5 min at 0° C. After the flow of ammonia was stopped, the reaction mixture was stirred at 0° C. for an additional 30 min. The mixture was allowed to warm to room temperature and stirred for 15 min. Degassed H2O (10 mL) was added and the solution was stirred for 10 min. The solvent was evaporated under reduced pressure. 10-(NH3CH2CH2OCH2CH2O)-nido-7,8-C2B9H 11 Yield: 770 mg (3.24 mmol, 80%) of a white solid.

[0168] Characterization:

number

[0169] 1.1.8 7-Ph-10-O(CH2)4O-nido-7,8-C2B9H 10 Synthesis of [ka] K[7-Ph-nido-7,8-C2B9H 10 (501 mg, 2.02 mmol) was suspended in dry toluene (6 mL) and sulfuric acid (0.9 mL) was added. The two-phase system was vigorously stirred for 1 h. The toluene layer was separated and the sulfuric acid phase was extracted with toluene (2 x 2 mL). To the combined toluene phases was added dioxane (537 μL, 6.27 mmol). The clear solution was stirred at 80 °C for 16 h. Afterwards the solvent was evaporated under reduced pressure. The residue was dissolved in CHCl3 and filtered through a short silica column. The product was eluted with CHCl3 and then with CH2Cl2. Yield: 451 mg (1.52 mmol, 75%) of a white solid.

number

[0170] 1,4-Dioxane derivative 7-PH-10-O(CH2)4O-nido-7,8-C2B9H 10 can be converted into the respective ammonia derivative according to Example 1.1. The ammonia derivative can be attached to the polymer particles according to the procedure described under 1.8.12. Alternatively, the 1,4-dioxane derivative 7-Ph-10-O(CH2)4O-nido-7,8-C2B9H10 can be converted into the respective allylamine derivative according to Example 1.4.

[0171] 1.1.9 7-PH-10-O(CH2)2O(CH2)2NH3-nido-7,8-C2B9H 10 Synthesis of [ka] 7-Ph-10-O(CH2)4O-nido-7,8-C2B9H 10(599 mg, 2.02 mmol) was dissolved in dry THF (15 mL) and the solution was cooled to 0° C. Gaseous ammonia was bubbled through the solution. After 5 min, the gas flow was stopped and the cloudy suspension was allowed to warm slowly to room temperature over 20 min. Degassed water (5 mL) was added and the solvent of the resulting yellow solution was evaporated under reduced pressure. Yield: 480 mg (1.53 mmol, 76%).

number

[0172] 1.1.10 7-PH-10-O(CH2)2O(CH2)2NH3-nido-7,8-C2B9H 10 Synthesis of [ka] Na[nido-7,8-C2B9H 12 (709 mg, 4.53 mmol) was suspended in dry toluene (7.5 mL) and sulfuric acid (1.8 mL) was added. The two-phase system was vigorously stirred at room temperature for 45 min. The toluene layer was separated and the sulfuric acid phase was extracted with toluene (2×4 mL). To the combined toluene layers was added dry THF (678 mg, 9.40 mmol). The clear solution was stirred at 80° C. for 21 h. The solvent was evaporated under reduced pressure. The residue was dissolved in CHCl3 and filtered through a short silica column. The product was eluted with CHCl3 and then with CH2Cl2. Yield: 601 mg (2.94 mmol, 65%).

number

[0173] THF derivative 10-O(CH2)4-nido-7,8-C2B9H 11 with ammonia to obtain 10-O(CH2)4NH3-nido-7,8-C2B9H according to Example 1.1.2.11 The ammonia derivative can be attached to the polymer particles according to the procedure described under 1.8.12. Alternatively, the THF derivative 10-O(CH2)4-nido-7,8-C2B9H 11 can be converted to the allylamine derivative according to example 1.4.

[0174] 1.2 Synthesis of acrylamides and N-allylamides containing adamantyl substituents or boron clusters as substituents 1.2.1 Synthesis of N-adamantylacrylamide [ka] The synthesis was carried out according to protocols described in the literature (Sokolov, VB; Aksinenko, AY; Epishina, TA; Goreva, TV; Bachurin, SO Synthetic approaches to conjugation of aminoadamantanes and carbazoles. Russ. Chem. Bull. 2017, 66, 2110-2114; Bayguzina, AR; Lutfullina, AR; Khusnutdinov, RI Synthesis of N-(Adamantan-1-yl)carbamides by Ritter Reaction from Adamantan-1-ol and Nitriles in the Presence of Cu-Catalysts. Russ. J. Org. Chem. 2018, 54, 1127-1133.).

[0175] Amantadine (2.50 g, 16.5 mmol) and triethylamine (5.03 g, 6.70 mL, 49.6 mmol) were dissolved in dichloromethane (100 mL) and cooled to 0° C. Then, chloroacrylic acid (1.79 g, 1.60 mL, 19.8 mmol) was added dropwise. The reaction mixture was stirred at room temperature for 4 hours and the solvent was removed under reduced pressure. The crude product was purified by column chromatography (eluent: diethyl ether / petroleum ether, 4:1+1% triethylamine). Yield: 2.10 g (10.2 mmol, 62%), white solid. Characterization:

number

[0176] 1.2.2 1-H5C3HNC(O)-C 10 H 15 Synthesis of [ka] 1-Adamantanecarboxylic acid (2.00 g, 11.1 mmol) was dissolved in thionyl chloride (50 mL) and heated to 60° C. for 12 h. Then all volatiles were removed under reduced pressure and the residue was dissolved in dichloromethane (50 mL). Allylamine (1.99 g, 2.50 mL, 33.3 mmol) was added dropwise at 0° C. The reaction mixture was stirred at room temperature for 12 h and the solvent was removed under reduced pressure. The crude product was purified by column chromatography (eluent: hexane / ethyl acetate, 2:1). Yield: 1.71 g (7.80 mmol, 70%), white solid.

[0177] Characterization:

number

[0178] 1.2.3 1-H3C2C(O)HN-closo-1,2-C2B 10 H 11 Synthesis of [ka] 1-H2N-closo-1,2-C2B 10 H 11 (2.25 g, 14.1 mmol) was dissolved in diethyl ether (200 mL) and mixed with triethylamine (8.68 g, 9.26 mL, 31.1 mmol). The solution was cooled to 0° C. and chloroacrylic acid (2.31 g, 2.08 mL, 16.9 mmol) was added dropwise. The reaction mixture was stirred at room temperature for 12 hours and the solvent was evaporated under reduced pressure. The crude product was purified by column chromatography (eluent: ethyl acetate / hexane, 1:1+1% triethylamine). Yield: 1.05 g (4.92 mmol, 35%), white solid.

[0179] Characterization:

number

[0180] 1.2.4 1-H5C3HNC(O)-closo-1,7-C2B 10 H 11 Synthesis of [ka] 1-HO(O)C-closo-1,7-C2B 10 H 11(2.70 g, 14.3 mmol) was dissolved in chloroform (150 mL) and mixed with thionyl chloride (2.00 ml, 3.41 g, 28.7 mmol). The reaction mixture was stirred at room temperature for 1 h and cooled to 0° C. Then allylamine (2.37 mmol, 1.80 g, 31.6 mmol) was added. The reaction mixture was stirred at room temperature for another 12 h. The solvent was removed under reduced pressure and the residue was purified by column chromatography (eluent: ethyl acetate / hexane, 1:1+1% triethylamine). Yield: 1.31 g (5.76 mmol, 40%) of a beige solid.

[0181] Characterization:

number

[0182] 1.2.5 1-H5C3HNC(O)-closo-1,12-C2B 10 H 11 Synthesis of [ka] 1-HO(O)C-closo-1,12-C2B 10 H 11(0.50 g, 2.65 mmol) was dissolved in thionyl chloride (20 mL). The reaction mixture was stirred at 80° C. for 12 h, after which all volatiles were removed under vacuum. The residue was dissolved in dichloromethane (20 mL) and mixed with allylamine (0.39 mL, 0.30 g, 5.3 mmol) and triethylamine (0.73 mL, 0.53 g, 5.3 mmol) at 0° C. The reaction mixture was stirred at room temperature for another 12 h. The solvent was removed under reduced pressure and the residue was washed with water (2×30 mL). The remaining solid was dried under high vacuum. Yield: 0.53 g (2.33 mmol, 88%), white solid.

[0183] Characterization:

number

[0184] 1.2.6 K[1-H3C2C(O)HN-closo-CB 11 H 11 Synthesis of [ka] K[1-H2N-closo-CB 11 H 11] (3.00 g, 15.2 mmol) was dissolved in acetonitrile (50 mL) and triethylamine (4.61 g, 6.30 mL, 45.7 mmol) was added. The solution was cooled to 0° C. and chloroacrylic acid (1.65 g, 1.48 mL, 18.3 mmol) was added dropwise. The reaction mixture was stirred at room temperature for 1 h and filtered. The filtrate was evaporated under reduced pressure and the residue was dissolved in 10% hydrochloric acid. The aqueous phase was extracted with diethyl ether (4×50 mL) and the organic phase was dried over potassium carbonate. The volume of the organic phase was reduced to approximately 30 mL. Addition of chloroform (100 mL) resulted in the precipitation of a white solid. It was separated by filtration and dried in high vacuum. Yield: 2.36 g (11.1 mmol, 73%). Characterization:

number

[0185] 1.2.7 K[1-H3C2C(O)HN-12-I-closo-CB 11 H 10 Synthesis of [ka] Cs[1-H2N-12-I-closo-CB 11 H 10] (500 mg, 1.19 mmol) was dissolved in acetonitrile (20 mL) and mixed with triethylamine (730 mg, 0.33 mL, 3.82 mmol). The solution was cooled to 0° C. and chloroacrylic acid (237 mg, 0.21 mL, 2.62 mmol) was added dropwise. The reaction mixture was stirred at room temperature for 1 h and filtered. The filtrate was evaporated under reduced pressure and the residue was dissolved in 10% aqueous HCl. The aqueous phase was extracted with diethyl ether (4×50 mL) and the organic phase was dried over potassium carbonate. After filtration, the volume of the organic phase was reduced to approximately 20 mL. Addition of chloroform (100 mL) resulted in the precipitation of a white solid. The product was isolated by filtration and dried in high vacuum. Yield: 2.36 g 260 mg (0.69 mmol, 58%).

[0186] Characterization:

number

[0187] 1.3 Derivatization of dioxane- and tetrahydrofuran-substituted closo-dodecaborate and closo-decaborate anions using sodium allyl alcoholate General protocol: The respective dioxane and tetrahydrofuran substituted closo-dodecaborate and closo-decaborate anions were dissolved in acetonitrile and mixed with potassium carbonate. Sodium allyl alcoholate was dissolved in acetonitrile and added to the suspension. The reaction mixture was stirred at 60° C. for 12 hours. The suspension was filtered and the solvent was removed under reduced pressure. The residue was dissolved in methanol and mixed with tetrabutylammonium hydroxide solution (1M in methanol). The reaction mixture was evaporated under reduced pressure and the residue was taken up in dichloromethane. The organic phase was washed with water (3×50 mL) and dried over magnesium sulfate. After filtration, the solvent was removed under reduced pressure and the resulting solid was dried in high vacuum.

[0188] Metathesis to cesium salts: The tetrabutylammonium salt was dissolved in dichloromethane and mixed with a solution of cesium fluoride in methanol. The precipitate was filtered off, washed with dichloromethane and methanol, and dried in high vacuum.

[0189] 1.3.1 Kat2[1-H5C3O(CH2)2O(CH2)2O-Clotho-B 12 H 11 Synthesis of (Kat = [nBuN], Cs) [ka] The amounts of chemicals used in these synthesis: Kat=[nBu4N]: 1.98 g (4.20 mmol) of [nBu4N][1-O(C2H4)2O-closo-B 12 H 11 ]; 5.80 g (42.0 mmol) K2CO3; 0.67 g (8.40 mmol) of sodium allyl alcoholate; 50 mL of acetonitrile, 5.03 mL of tetrabutylammonium hydroxide solution (1 M in methanol); 100mL of dichloromethane. Yield: 2.49 g (3.21 mmol, 77%), beige solid.

[0190] Kat=Metathesis to Cs: 1.95 g (2.53 mmol) of [nBu4N]2[1-H5C3O(CH2)2O(CH2)2O-closo-B dissolved in 20 mL of dichloromethane 12 H 11 ]; 844 mg (5.55 mmol) of CsF dissolved in 30 mL of methanol. Wash with 3 x 10 mL dichloromethane and 3 x 10 mL methanol. Yield: 1.11 g (2.01 mmol, 80%), white solid.

[0191] Characterization: [1-H5C3O(CH2)2O(CH2)2O-Clotho-B 12 H 11 ] 2- NMR data for the anion:

number

[0192] 1.3.2 Kat2[1-H5C3O(CH2)4O-closo-B 12 H11 Synthesis of (Kat = [nBuN], Cs) [ka] The amounts of chemicals used in these synthesis: Kat=[nBu4N]: 1.21g (2.80mmol) of [nBu4N][1-(C2H4)4O-closo-B 12 H 11 ];3.87 g (28.0 mmol) K2CO3; 0.45 g (5.60 mmol) of sodium allyl alcoholate; 50 mL of acetonitrile, 6.02 mL of tetrabutylammonium hydroxide solution (1 M in methanol); 60mL of dichloromethane. Yield: 1.49 g (1.97 mmol, 70%), beige solid.

[0193] Kat=Metathesis to Cs: 1.34 g (1.78 mmol) of [nBu4N]2[1-H5C3O(CH2)4O-closo-B was dissolved in 20 mL of dichloromethane. 12 H 11 ]; 594 mg (3.91 mmol) CsF in 30 mL of methanol. Wash with 3 x 10 mL dichloromethane and 3 x 10 mL methanol. Yield: 460 mg (0.86 mmol, 48%), white solid.

[0194] Characterization: [1-H5C3O(CH2)4O-closo-B 12 H 11 ] 2- NMR data for the anion:

number

[0195] 1.3.3 Kat2[2-H5C3O(CH2)2O(CH2)2O-Clotho-B 10 Synthesis of H9 (Kat = [nBuN], Cs) [ka] The amounts of chemicals used in these synthesis: Kat=[nBu4N]: 650mg (1.45mmol) of [nBu4N][2-O(C2H4)2O-closo-B 10 H9]; 449 mg (3.25 mmol) K2CO3; 157 mg (1.97 mmol) of sodium allyl alcoholate; 50 mL of acetonitrile, 2.05 mL of tetrabutylammonium hydroxide solution (1 M in methanol); 60mL of dichloromethane. Yield: 690 mg (0.92 mmol, 64%), beige solid.

[0196] Kat=Metathesis to Cs: 1.05 g (1.41 mmol) of [nBu4N]2[2-H5C3O(CH2)2O(CH2)2O-closo-B dissolved in 10 mL of dichloromethane 10 H9]; 470 mg (3.09 mmol) CsF in 15 mL of methanol. Wash with 3 x 5 mL dichloromethane and 3 x 5 mL methanol. Yield: 212 mg (0.40 mmol, 28%), white solid.

[0197] Characterization: [2-H5C3O(CH2)2O(CH2)2O-Clotho-B 10 H9] 2- NMR data for the anion:

number

[0198] 1.3.4 Kat2[2-H5C3O(CH2)4O-closo-B 10 Synthesis of H9 (Kat = [nBuN], Cs) [ka] The amounts of chemicals used in these synthesis: Kat=[nBu4N]: 650mg (1.51mmol) of [nBu4N][2-(CH2)4O-closo-B 10 H9]; 1.04 g (7.55 mmol) K2CO3; 266 mg (3.32 mmol) of sodium allyl alcoholate; 50 mL of acetonitrile; 6.02 mL of tetrabutylammonium hydroxide solution (1 M in methanol); 60mL of dichloromethane. Yield: 1.08 g (1.48 mmol, 98%), beige solid.

[0199] Kat=Metathesis to Cs: 940 mg (1.29 mmol) of [nBu4N]2[2-H5C3O(CH2)4O-closo-B dissolved in 10 mL of dichloromethane 10 H9]; 427 mg (2.83 mmol) CsF in 15 mL of methanol. Wash with 3 x 5 mL dichloromethane and 3 x 5 mL methanol. Yield: 377 mg (0.74 mmol, 57%), white solid.

[0200] Characterization: [2-H5C3O(CH2)4O-closo-B 10 H9] 2-2- NMR data for the anion:

number

[0201] 1.4 Derivatization of dioxane- and tetrahydrofuran-substituted closo-dodecaborate and closo-decaborate anions using allylamines A general protocol for the reaction of closo-dodecaborate and closo-decaborate anions with dioxane and tetrahydrofuran derivatives of allylamines. The reactions of closo-dodecaborate and closo-decaborate anions with dioxane and tetrahydrofuran derivatives of allylamines were carried out as described in the literature (Semioshkin, A.; Nizhnik, E.; Godovikov, I.; Starikova, Z.; Bregadze, VI Reactions of oxonium derivatives of [B 12 H 12 ] 2- with amines: Synthesis and structure of novel B 12 -based ammonium salts and amino acid. J. Organomet. Chem. 2007, 692, 4020-4028.) were carried out in a similar manner to the reactions with other amines described. The respective derivative of closo-dodecaborate or closo-decaborate anion was dissolved in allylamine and the reaction mixture was stirred at 60° C. for 12 hours. The excess allylamine was evaporated under reduced pressure. The residue was dissolved in methanol and mixed with tetrabutylammonium hydroxide solution (1M in methanol). The solvent was removed under reduced pressure and the residue was taken up in dichloromethane. The organic phase was washed with water (3×50 mL) and dried over magnesium sulfate. The dichloromethane was evaporated under reduced pressure and the solid obtained was dried in high vacuum.

[0202] 1.4.1 Kat2[1-H5C3HN(CH2)2O(CH2)2O-Closo-B 12 H 11 Synthesis of (Kat = [nBuN], Cs) [ka] The amounts of chemicals used in these synthesis: Kat=[nBu4N]: 1.37 g (2.91 mmol) of [nBu4N][1-O(C2H4)2O-closo-B 12 H 11 ]; 25mL of allylamine, 20mL of methanol, 5.81 mL of tetrabutylammonium hydroxide solution (1 M in methanol); 100mL of dichloromethane. Yield: 1.89 g (2.45 mmol, 85%), beige solid.

[0203] Kat=Metathesis to Cs: 1.66 g (2.16 mmol) of [nBuN]2[1-H5C3HN(CH2)2O(CH2)2O-closo-B was dissolved in 20 mL of dichloromethane. 12 H 11 ]; 679 mg (4.72 mmol) CsF in 30 mL of methanol. Wash with 3 x 10 mL dichloromethane and 3 x 10 mL methanol. Yield: 1.00 g (1.82 mmol, 84%), white solid.

[0204] Characterization: [1-H5C3HN(CH2)2O(CH2)2O-Closo-B 12 H 11 ] 2- NMR data for the anion:

number

[0205] 1.4.2 Kat2[1-H5C3HN(CH2)4O-closo-B 12 H 11 Synthesis of (Kat = [nBuN], Cs) [ka] The amounts of chemicals used in these synthesis: Kat=[nBu4N]: 1.64g (3.79mmol) of [nBu4N][1-(CH2)4O-closo-B 12 H 11 ]; 25 mL of allylamine; 20 mL of methanol; 7.60 mL of tetrabutylammonium hydroxide solution (1 M in methanol); 70mL of dichloromethane. Yield: 2.42 g (3.21 mmol, 85%), beige solid.

[0206] Kat=Metathesis to Cs: 2.27 g (3.01 mmol) of [nBu4N]2[1-H5C3HN(CH2)4O-closo-B was dissolved in 20 mL of dichloromethane. 12 H 11 ]; 1.01 g (6.65 mmol) CsF in 30 mL of methanol. Wash with 3 x 10 mL dichloromethane and 3 x 10 mL methanol. Yield: 0.79 g (1.48 mmol, 39%), white solid.

[0207] Characterization: [1-H5C3HN(CH2)4O-closo-B 12 H 11 ] 2- NMR data for the anion:

number

[0208] 1.4.3 Kat2[2-H5C3HN(CH2)2O(CH2)2O-Closo-B 10 Synthesis of H9 (Kat = [nBuN], Cs) [ka] The amounts of chemicals used in these synthesis: Kat=[nBu4N]: 650mg (1.45mmol) of [nBu4N][2-O(C2H4)2O-closo-B 10 H9]; 25 mL of allylamine; 20 mL of methanol; 1.43 mL of tetrabutylammonium hydroxide solution (1 M in methanol); 70mL of dichloromethane. Yield: 1.06 g (1.42 mmol, 98%), beige solid.

[0209] Kat=Metathesis to Cs: 1.24 g (1.68 mmol) of [nBu4N]2[2-H5C3HN(CH2)2O(CH2)2O-closo-B dissolved in 20 mL of dichloromethane 10 H9]; 561 mg (3.98 mmol) CsF in 30 mL of methanol. Wash with 3 x 5 mL dichloromethane and 3 x 5 mL methanol. Yield: 432 mg (0.82 mmol, 49%), white solid.

[0210] Characterization: [2-H5C3HN(CH2)2O(CH2)2O-Closo-B 10 H9] 2- NMR data for the anion:

number

[0211] 1.4.4 Kat2[2-H5C3HN(CH2)4O-closo-B 10 Synthesis of H9 (Kat = [nBuN], Cs) [ka] The amounts of chemicals used in these synthesis: Kat=[nBu4N]: 650mg (1.51mmol) of [nBu4N][2-(CH2)4O-closo-B 10 H9]; 25mL of allylamine, 20mL of methanol, 1.43 mL of tetrabutylammonium hydroxide solution (1 M in methanol); 70mL of dichloromethane. Yield: 1.10 g (1.50 mmol, 99%), beige solid.

[0212] Kat=Metathesis to Cs: 569 mg (0.78 mmol) of [nBu4N]2[2-H5C3HN(CH2)4O-closo-B dissolved in 20 mL of dichloromethane 10 H9]; 260 mg (1.71 mmol) CsF in 30 mL of methanol. Wash with 3 x 5 mL dichloromethane and 3 x 5 mL methanol. Yield: 176 mg (0.34 mmol, 44%), white solid.

[0213] Characterization: [2-H5C3HN(CH2)4O-closo-B 10 H9] 2- NMR data for the anion:

number

[0214] 1.5 Derivatization of dioxane- and tetrahydrofuran-substituted closo-dodecaborate and closo-decaborate anions using chloroacrylic acid General protocol for the reaction of amino-functionalized closo-dodecaborate and closo-decaborate derivatives with chlorinated acrylic acids: The respective closo-dodecaborate or closo-decaborate derivatives bearing amino functionality were dissolved in acetonitrile and mixed with triethylamine. The solution was cooled to 0° C. and chloroacrylic acid was added dropwise. The reaction was stirred at room temperature for 12 hours, filtered, and the solvent was removed under reduced pressure. The residue was dissolved in dichloromethane and washed with 10% aqueous HCl and water. The organic phase was dried over magnesium sulfate, filtered, and evaporated under reduced pressure. The resulting solid was dried in high vacuum.

[0215] 1.5.1 Kat2[1-H3C2C(O)HN(CH2)2O(CH2)2O-Closo-B 12 H 11 Synthesis of ] (Kat=[nBuN], Cs) [ka] The amounts of chemicals used in these synthesis: Kat=[nBu4N]: 4.22 g (5.77 mmol) of [nBuN]2[1-HN(CH2)2O(CH2)2O-closo-B 12 H 11 ]; 2.37 mL (1.75 g, 17.3 mmol) triethylamine; 0.71 ml (0.78 g, 8.66 mmol) of chlorinated acrylic acid; 20 mL of dichloromethane, 3 x 20 mL of 10% aqueous HCl 2 x 20mL water. Yield: 2.27 g (2.90 mmol, 50%), beige solid.

[0216] Kat=Metathesis to Cs: 2.03 g (2.59 mmol) of [nBuN]2[1-H3C2C(O)HN(CH2)2O(CH2)2O-closo-B dissolved in 20 mL of dichloromethane 12 H 11 ]; 863 mg (5.68 mmol) CsF in 30 mL of methanol. Wash with 3 x 15 mL dichloromethane and 3 x 15 mL methanol. Yield: 1.32 g (2.33 mmol, 90%), white solid.

[0217] Characterization: [1-H3C2C(O)HN(CH2)2O(CH2)2O-Closo-B 12 H 11 ] 2- NMR data for the anion:

number

[0218] 1.5.2 Kat2[1-H3C2C(O)HN(CH2)4O-closo-B 12 H 11 Synthesis of (Kat = [nBuN], Cs) [ka] The amounts of chemicals used in these synthesis: Kat=[nBu4N]: 2.15 g (3.01 mmol) of [nBu4N]2[1-H2N(CH2)4O-closo-B 12 H 11 ]; 1.24 mL (0.91 g, 9.03 mmol) triethylamine; 0.37 ml (0.41 g, 4.52 mmol) of chlorinated acrylic acid; 20 mL of dichloromethane, 3 × 20 mL of 10% aqueous HCl, 2 x 20mL water. Yield: 1.29 g (1.68 mmol, 56%), beige solid.

[0219] Kat=Metathesis to Cs: 1.13 g (1.47 mmol) of [nBu4N]2[1-H3C2C(O)HN(CH2)4O-closo-B was dissolved in 20 mL of dichloromethane. 12 H 11 ]; 492 mg (3.24 mmol) CsF in 30 mL of methanol. Wash with 3 x 10 mL dichloromethane and 3 x 10 mL methanol. Yield: 0.60 g (1.09 mmol, 74%), white solid.

[0220] Characterization: [1-H3C2C(O)HN(CH2)4O-closo-B 12 H 11 ] 2- NMR data for the anion:

number

[0221] 1.6 Eshmuno® Amino-Functionalized Adamantane and Closo-Boron Cluster Derivativesエポキシ General protocol for reactions with materials 1.6.1 Eshmuno® エポキシ Starting materials [ka] Elemental analysis: Found: C, 57.58; H, 8.50; N, 10.51%.

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[0222] Eshmuno® エポキシ Material (5 g, dry, surface epoxy functional group density: 554 μeq g -1 ) was washed with water (2 L). The wet material was transferred into a 500 mL three-neck flask equipped with a KPG stirrer and suspended in water (150 mL). The corresponding amino derivative (5.07 mmol) and lithium bromide (436 mg, 5.07 mmol) were dissolved in water (100 mL) and the solution was diluted with Eshmuno® in water at 60° C. エポキシ The resulting suspension was stirred at 120 rpm for 4 h and filtered. The solid material was washed with water (1 L), hot water (60° C., 500 mL) and dried under high vacuum.

[0223] 1.6.2 Eshmuno® アミノ-アダマンタン [ka] Amount of amino-functionalized adamantane used in this synthesis: 766 mg 1-H2N-C 10 H 15 (5.07mmol). Characterization: Elemental analysis, found: C, 58.50; H, 8.74; N, 10.14%.

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[0224] 1.6.3 Eshmuno® -HN-クロソ-CB11H11 [ka] The K[1-H2N-closo-CB used in this synthesis 11 H 11 ] amount: 1.00 g (5.07 mmol). Characterization: Elemental analysis, found: C, 54.66; H, 8.36; N, 10.21; B, 2.38%.

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[0225] 1.6.4 Eshmuno® -HN-12-I-クロソ-CB11H10 [ka] The Cs[1-H2N-12-I-closo-CB 11 H 10 ] amount: 2.11g (5.07mmol). Characterization: Elemental analysis, found: C, 54.37; H, 8.16; N, 11.35; B, 1.91%.

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[0226] 1.7 General Procedure for Functionalization of Eshmuno® Materials 1.7.1 Eshmuno® ヒドロキシ Starting materials [ka] Elemental analysis, found: C, 53.47; H, 7.87; N, 10.98%.

number

[0227] The synthesis was carried out under an inert gas atmosphere similar to the synthesis described in the literature (Graalfs, H.; Graft Copolymer for Cation-exchange Chromatography. Merck Patent GmbH, WO2008145270, 2008.). The starting material Eshmuno®, stored under 20% aqueous ethanol, was filtered and washed with distilled water (2 L). Afterwards, Eshmuno® ヒドロキシ The material was stored under water (50 mL) for 1 day. After 24 hours, Eshmuno® ヒドロキシ The material was filtered and transferred into a 500 mL three-neck flask equipped with a KPG stirrer, drip funnel, reflux condenser, and nitrogen gas inlet tube. Acrylic acid or other allylic derivative (5.00 mmol) was dissolved in solvent (180 mL) and added to Eshmuno® ヒドロキシTo the material was added an initiator consisting of cerium(IV) ammonium nitrate (850 mg, 1.55 mmol), concentrated nitric acid (631 mg, 0.417 mL, 10.0 mmol), and water (70 ml) as quickly as possible under vigorous stirring. The suspension was then stirred at 30° C. and 120 rpm for 4 hours. The functionalized Eshmuno® material was filtered and washed with water (3×250 mL), 1 M sulfuric acid and 0.2 M ascorbic acid (8×250 ml), water (3×250 mL), warm water (60° C.; 10×250 ml), water (2×250 mL), 1 M sodium hydroxide solution (2×250 mL), water (2×250 ml), 70% aqueous ethanol (2×250 ml), water (2×250 ml), 20% aqueous ethanol containing 150 mM sodium chloride (2×250 ml), and stored in 20% aqueous ethanol containing 150 mM sodium chloride.

[0228] 1.7.2 Eshmuno® アミドカルボキシ-アダマンチル Synthesis of [ka] Amounts of chemicals used in this synthesis: 1.03 g (5.00 mmol) of 1-H3C2C(O)HN-C 10 H 15 ; 40mL Eshmuno® ヒドロキシ Suspension. Solvent: 105 mL water and 75 mL tert-butanol. Product characterization: Elemental analysis, found: C, 62.22; H, 8.49; N, 10.37%.

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[0229] 1.7.3 Eshmuno® 1-アミドカルボキシ-クロソ-1,2-C2B10H11 Synthesis of [ka] Amounts of chemicals used in this synthesis: 1.13 g (5.00 mmol) of 1-H3C2C(O)HN-closo-1,2-C2B 10 H 11 ; 40mL Eshmuno® ヒドロキシ Suspension. Solvent: 105 mL water and 75 mL tert-butanol. Product characterization: Elemental analysis, found: C, 54.06; H, 8.64; N, 11.66; B, 1.47%.

number

[0230] 1.7.4 Eshmuno® 1-アミドカルボキシ-クロソ-CB11H11 Synthesis of [ka] Amounts of chemicals used in this synthesis: 1.13 g (5.00 mmol) of K[1-H3C2(O)CHN-closo-CB 11 H 11 ]; 40mL Eshmuno® ヒドロキシ Suspension. Solvent: 180mL water. Product characterization: Elemental analysis, found: C, 50.88; H, 7.95; N, 10.24; B, 1.09%.

number

[0231] 1.8 Eshmuno® of Adamantane and Closo-Boron Cluster Derivatives Containing Amino Groups COO Preparation of functionalized Eshmuno® materials by reaction with materials. 1.8.1 Eshmuno® カルボキシ Synthesis of starting materials For the synthesis of COO-containing starting materials, the required amount of acrylic acid is dissolved in water and, if necessary, the pH is adjusted to pH 2.2. The mixture is stirred at a temperature between 0-5° C. to obtain a homogeneous solution. Afterwards, the -OH-containing substrate, for example Eshmuno® particles, is added. Polymerization is initiated by adding cerium(IV) nitrate. The reaction takes place at 30-50° C. for 4 hours. After the polymerization reaction, unreacted components are removed by extensive washing with acidic, basic and solvent mixtures at room temperature or elevated temperature.

[0232] Eshmuno® COO-200 Amounts of chemicals used in this synthesis: 1.19 g (16.5 mmol) acrylic acid; 250 mL of Eshmuno® suspension. Product characterization: Elemental analysis, found: C, 55.89; H, 8.51; N, 11.82%.

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[0233] Eshmuno® COO-700 Amounts of chemicals used in this synthesis: 3.96 g (55.0 mmol) acrylic acid; 250 mL of Eshmuno® suspension. Product characterization: Elemental analysis, found: C, 56.98; H, 8.44; N, 11.73%.

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[0234] Eshmuno® COO-1000 Amounts of chemicals used in this synthesis: 5.94 g (82.5 mmol) acrylic acid; 250 mL of Eshmuno® suspension. Product characterization: Elemental analysis found: C, 56.03; H, 8.25; N, 10.89%.

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[0235] Eshmuno® COO-2400 Amounts of chemicals used in this synthesis: 15.9 g (220.0 mmol) acrylic acid; 250 mL of Eshmuno® suspension. Product characterization: Elemental analysis, found: C, 55.89; H, 8.51; N, 11.82%.

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[0236] The syntheses presented below were carried out under an inert gas atmosphere similar to the syntheses described in the literature (Graalfs, H.; Graft Copolymer for Cation-exchange Chromatography. Merck Patent GmbH, WO2008145270, 2008.).

[0237] General protocol: Eshmuno® stored in 20% aqueous ethanol containing sodium chloride (150 mM) COO The material (300 μeq g-1) was filtered and washed with water (2 L). COO The material (gel height 5 mL) was stored in water (25 mL). After 24 hours, Eshmuno®COO The material was filtered and transferred into a 250 mL round flask. The amine derivative of adamantane or closo-boron cluster was dissolved in water (100 mL) and added to Eshmuno® COO To the material was added 1-ethyl-3-(3-dimethylamino-propyl)carbodiimide (EDC). The suspension was shaken for 17 hours, heated to 60° C., and 1-ethyl-3-(3-dimethylamino-propyl)carbodiimide (EDC) was added. After 3 hours, EDC was added again. The Eshmuno®-functionalized material was filtered, washed with water (4×50 mL), 1 M sodium chloride solution (3×50 mL), 0.1 M sodium phosphate buffer (pH=7) (4×50 mL), 20% aqueous ethanol containing sodium chloride (150 mM) (2×50 mL), and stored in 20% aqueous ethanol containing sodium chloride (150 mM) (150 mM).

[0238] 5mL of Eshmuno® COO Synthesis using gel starting material: 1.8.2 Eshmuno® アミドカルボキシ-アダマンチル Synthesis of [ka] Amounts of chemicals used in this synthesis: 766mg (5.00mmol) of 1-H2N-C 10 H 15 ; 1.00g+1.00g EDC(5.22mmol+5.22mmol). Eshmuno® COO Product characterization: Elemental analysis, found: C, 55.84; H, 8.78; N, 13.59%.

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[0239] 1.8.3 Eshmuno® 1-アミドカルボキシ-クロソ-CB11H11 Synthesis of [ka] Amounts of chemicals used in this synthesis: 1.00 g (5.00 mmol) of K[1-H2N-closo-CB 11 H 11 ]; 1.00g+1.00g EDC(5.22mmol+5.22mmol). Eshmuno® COO Product characterization: Elemental analysis, found: C, 52.76; H, 7.87; N, 11.48; B, 1.34%.

number

[0240] 1.8.4 Eshmuno® アミドカルボキシ-クロソ-B10H9 Synthesis of [ka] Amounts of chemicals used in this synthesis: 2.38 g (5.00 mmol) of Cs2[2-H2N(CH2)2O(CH2)2O-closo-B 10 H9]; 1.00g+1.00g EDC(5.22mmol+5.22mmol). Eshmuno® COO Product characterization: Elemental analysis, found: C, 53.16; H, 8.54; N, 13.32; B, 0.57%.

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[0241] 1.8.5 Eshmuno® アミドカルボキシ-クロソ-B12H11 Synthesis of [ka] Amounts of chemicals used in this synthesis: 3.22g (6.51mmol) of Cs2[1-H2N(CH2)4O-closo-B 12 H 11 ]; 1.30g+1.30g EDC(6.78mmol+6.78mmol), Eshmuno® COO-300 . Product characterization: Elemental analysis, found: C, 53.51; H, 8.63; N, 11.54; B, 1.66%.

number

[0242] 1.8.6 Eshmuno® オキソ-アミドカルボキシ-クロソ-B12H11 Synthesis of [ka] Amounts of chemicals used in this synthesis: 2.50 g (5.00 mmol) of Cs2[1-H2N(CH2)2O(CH2)2O-closo-B 12 H 11 ]; 1.00g+1.00gEDC(5.22mmol+5.22mmol) Eshmuno® COO Product characterization: Elemental analysis, found: C, 54.49; H, 8.54; N, 12.04; B, 0.64%.

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[0243] 50mL of Eshmuno® COO -Synthesis using gel starting material: General protocol: Eshmuno® stored in 20% aqueous ethanol containing sodium chloride (150 mM) COO The material was filtered and washed with water (2 L).COO The material (gel height 50 mL) was stored in water (250 mL) for 24 hours. COO The material was washed with additional water (3×500 mL), filtered, transferred into a 1 L round bottom flask, and suspended in water (100 mL). The amino derivative of adamantane or closo-boron cluster and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) were added to this suspension. The reaction mixture was stirred (115 rpm) at 60° C. for 3 hours. After 3 hours, additional EDC was added and the reaction mixture was stirred at 60° C. and 115 rpm for an additional 17 hours. The Eshmuno®-functionalized material was filtered, washed with water (4×50 mL), 1 M sodium chloride solution (3×50 mL), 0.1 M sodium phosphate buffer (pH=7) (4×50 mL), 20% aqueous ethanol containing sodium chloride (150 mM) (2×50 mL), and stored in 20% aqueous ethanol containing sodium chloride (150 mM).

[0244] 1.8.7 Eshmuno® オキソ-アミドカルボキシ-クロソ-B12H11 Synthesis of [ka] Amounts of chemicals used in this synthesis: 1.25 g (2.41 mmol) of Cs2[1-H2N(CH2)2O(CH2)2O-closo-B 12 H 11 ]; 0.60g+0.60g(1.57mmol+1.57mmol)EDC; 50mL of Eshmuno® COO-200 . Product characterization: Elemental analysis, found: C, 53.80; H, 8.64; N, 11.59; B, 0.50%.

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[0245] 1.8.8 Eshmuno (registered trademark) オキソ-アミドカルボキシ-クロソ-B12H11 Synthesis of [ka] Amounts of chemicals used in this synthesis: 3.32 g (6.51 mmol) of Cs2[1-H2N(CH2)2O(CH2)2O-closo-B 12 H 11 ]; 2.24g+2.24g(5.90mmol+5.90mmol)EDC; 50mL of Eshmuno® COO-700 . Product characterization: Elemental analysis, found: C, 52.99; H, 8.57; N, 11.24; B, 1.33%.

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[0246] 1.8.9 Eshmuno (registered trademark) オキソ-アミドカルボキシ-クロソ-B12H11 Synthesis of [ka] Amounts of chemicals used in this synthesis: 6.56 g (12.9 mmol) of Cs2[1-H2N(CH2)2O(CH2)2O-closo-B 12 H 11 ]; 4.50g+4.50g(11.8mmol+11.8mmol)EDC; 50mL of Eshmuno® COO-1000 . Product characterization: Elemental analysis, found: C, 50.28; H, 8.50; N, 10.71; B, 2.13%.

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[0247] 1.8.10 Eshmuno (registered trademark) オキソ-アミドカルボキシ-クロソ-B12H11 Synthesis of [ka] Amounts of chemicals used in this synthesis: 11.5 g (22.5 mmol) of Cs2[1-H2N(CH2)2O(CH2)2O-closo-B 12 H 11 ]; 7.90g+7.90g(20.6mmol+20.6mmol)EDC; 50mL of Eshmuno® COO-2400 . Product characterization: Elemental analysis, found: C, 49.20; H, 8.53; N, 10.75; B, 2.22%.

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[0248] 1.8.11 Eshmuno (registered trademark) オキソアミドカルボキシ-COSAN Synthesis of [ka]

[0249] The synthesis was carried out using a slight modification of the general method previously described: Eschmuno® COO (6.0 mL, 1000 μeq g -1 ) was washed with water (1 L) and stored in water (40 mL) for 24 h. The particles were washed with water (2 x 500 mL) and transferred into a 250 mL Schlenk flask. [3,3'-Co(8-{NH3CH2CH2OCH2CH2O}-1,2-C2B9H 10 )(1',2'-C2B9H 11) (689 mg, 1.61 mmol) was treated with KOH (90 mg, 1.61 mmol) in a mixture of water (50 mL) and tert-butanol (50 mL). The resulting solution was added to the BDM particles under an inert gas atmosphere. EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) (253 mg, 1.32 mmol) was added and the mixture was shaken on a rotary evaporator (120 rpm) at 60° C. After 3 hours, additional EDC (253 mg, 1.32 mmol) was added and the reaction mixture was shaken on a rotary evaporator (120 rpm) for an additional 21 hours. Eschmuno® COO The material was filtered off. The residue was rinsed with water (2×50 mL) and with aqueous sodium chloride (2×250 mL, 1 M), sodium phosphate buffer solution (3×250 mL, pH=7, 50 mM), and aqueous ethanol solution containing sodium chloride (150 mM) (3×250 mL, 20% v / v). The wet material was stored in an aqueous solution of ethanol containing sodium chloride (150 mM) (20% v / v). Product characterization:

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[0250] 1.8.12 Eshmuno (registered trademark) オキソ-アミドカルボキシ-ニド-C2B9H11 Synthesis of [ka] The synthesis was carried out using a slight modification of the general method previously described: Eschmuno® COO (9.5mL, 1000μeq g -1 ) was washed with water (1 L) and stored in water (40 mL) for 24 h. The particles were washed with water (2 x 500 mL) and transferred into a 250 mL Schlenk flask. 10-(NH3CH2CH2OCH2CH2O)-nido-7,8-C2B9H 11(596 mg, 2.51 mmol) was treated with KOH (140 mg, 2.51 mmol) in water (100 mL). The resulting solution was added to the BDM particles under an inert gas atmosphere. EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) (402.0 mg, 2.09 mmol) was added and the mixture was shaken at 60° C. on a rotary evaporator (120 rpm). After 3 hours, additional EDC (402.0 mg, 2.09 mmol) was added and the reaction mixture was shaken at 60° C. for an additional 21 hours. Eschmuno® COO The material was filtered off. The residue was rinsed with water (2×50 mL) and washed with aqueous sodium chloride (2×250 mL, 1 M), sodium phosphate buffer solution (3×250 mL, pH=7, 50 mM), and aqueous ethanol solution containing sodium chloride (150 mM) (3×250 mL, 20% v / v). The wet material was stored in an aqueous solution of ethanol containing sodium chloride (150 mM) (20% v / v). Product characterization:

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[0251] 1.8.13 Eshmuno (registered trademark) オキソ-アミドカルボキシ-クロソ-B12H11 Synthesis of [ka]

[0252] Eschmuno® COO (11.0 mL, 1000 μeq g -1 ) was washed with water (1 L) and stored in water (40 mL) for 20 h. The particles were washed with water (2 x 500 mL) and transferred into a 250 mL Schlenk flask. Cs2[1-H2N(CH2)2O(CH2)2O-closo-B 12 H 11] (1.35 g, 2.64 mmol) was added. Water (50 mL) and tert-butanol (50 mL) were added to the BDM particles under an inert gas atmosphere. EDC (1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide) (464 mg, 2.42 mmol) was added and the mixture was shaken on a rotary evaporator (120 rpm) at 60° C. After 3 hours, additional EDC (464 mg, 2.42 mmol) was added and the reaction mixture was shaken on a rotary evaporator (120 rpm) for an additional 21 hours. Eschmuno® COO The material was filtered off. The residue was rinsed with water (2×500 mL) and with aqueous sodium chloride (2×250 mL, 1 M), sodium phosphate buffer solution (3×250 mL, pH=7, 50 mM), and aqueous ethanol containing sodium chloride (150 mM) (3×250 mL, 20% v / v). The wet material was stored in an aqueous solution of ethanol containing sodium chloride (150 mM) (20% v / v). Product characterization:

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[0253] 1.9 General Protocol for the Synthesis of Chromatographic Materials by Modification of Macro-Prep CM Resin, Toyopearl® HW-65F, and Sepharose® 4B with Closo-Dodecaborate Anion The starting material (5.00 mL) that settled by gravity was washed with water (500 mL) and stored in water (25 mL) for 24 hours. The material was washed again with water (250 mL). The wet gel was purified by HPLC using the method described above. 12 H 11The mixture was placed in a 100 mL round bottom flask with 1,2-dichlorophenyl ether] and water (50 mL). The reaction mixture was heated to 60° C. and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) was added with shaking. After 3 hours, an additional amount of EDC was added and the suspension was shaken at 60° C. for 17 hours. The material was filtered, washed with water (4×50 mL), 1 M sodium chloride solution (3×50 mL), 0.1 M sodium phosphate buffer (pH=7) (4×50 mL), 20% aqueous ethanol containing sodium chloride (150 mM) (2×50 mL), and stored in 20% aqueous ethanol containing sodium chloride (150 mM).

[0254] Characterization of starting materials: [ka]

[0255] 1.9.1 Macro-Prep® B12 Amounts of chemicals used in this synthesis: 1.24 g (2.42 mmol) of Cs2[1-H2N(CH2)2O(CH2)2O-closo-B 12 H 11 ]; 0.85g+0.85g (2.22mmol+2.22mmol) of EDC. Macro-Prep CM Resin (Macro-Prep CM Support) is a commercially available weak cation exchange support and was obtained from Bio-Rad. Product characterization: Elemental analysis, found: C, 53.06; H, 7.78; N, 1.22; B, 1.10%.

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[0256] Toyopearl® HW-65F B12 Amounts of chemicals used in this synthesis: 740mg (1.44mmol) of Cs2[1-H2N(CH2)2O(CH2)2O-closo-B 12 H 11 ]; 0.55g+0.55g (1.32mmol+1.32mmol) of EDC. Toyopearl® HW-65F is a commercially available hydroxylated methacrylate chromatography support and was obtained from Sigma-Aldrich. Product characterization: Elemental analysis, found: C, 52.24; H, 7.80; N, 1.21; B, 0.51%.

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[0257] 1.9.2 CM Sepharose® Fast Flow B12 Amounts of chemicals used in this synthesis: 720 mg (1.40 mmol) of Cs2[1-H2N(CH2)2O(CH2)2O-closo-B 12 H 11 ]; 495mg+495g (1.29mmol+1.29mmol) EDC. CM Sepharose® Fast Flow is a commercially available 6% cross-linked agarose weak cation exchange support and was obtained from Sigma-Aldrich. Product characterization: Elemental analysis, found: C, 44.46; H, 6.97; N, 1.79; B, 1.57%.

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[0258] 1.10 Eshmuno® modified with boron clusters COO One-pot synthesis for chromatography materials based on General protocol: Closo-dodecaborate as the triethylammonium or tetrabutylammonium salt was placed in a 2 L three-neck flask equipped with a reflux condenser, KPG stirrer, nitrogen gas inlet tube, and Na[BF4] and [nBu4N]Br (if triethylammonium closo-dodecaborate was used as the starting material) were added. The mixture was suspended in dioxane (250 mL), HCl (4 M in dioxane) was added, and the suspension was warmed to 120° C. and stirred (120 rpm) for 24 h. -1 The reaction mixture was then cooled to 50° C. and gaseous ammonia was passed through the solution for 15 min. The reaction mixture was warmed again to 120° C. and stirred for 12 h. Excess ammonia was removed by passing nitrogen through the reaction mixture at 80° C. for 60 min. Water (100 ml) and Eshmuno® COO Material (1050μeq g -1 After addition of 100 mL of ethanol (50 mL gel), the pH of the suspension was adjusted to 4.7. EDC was added and the suspension was stirred (100 rpm) at 70° C. for 17 hours, during which an additional amount of EDC was added after 3 hours. The functionalized Eshmuno® material was filtered, washed with water (4×50 ml), 1 M sodium chloride solution (3×50 ml), 0.1 M sodium phosphate buffer (pH=7) (4×50 ml), 20% aqueous ethanol containing sodium chloride (150 mM) (2×50 mL), and stored in 20% aqueous ethanol containing sodium chloride (150 mM).

[0259] 1.10.1 Eshmuno® オキソ-アミドカルボキシ-クロソ-B12H11 One-pot synthesis of [ka] Amounts of chemicals used in this synthesis: 4.36 g (12.6 mmol) of [HNEt3]2[closo-B 12 H 12 ]; 8.93 g (27.7 mmol) of [nBuN]Br; 6.92 g (63.0 mmol) Na[BF4]; 16 mL of HCl (4 M in dioxane); 5.63g + 5.63g EDC (14.7mmol + 14.7mmol). Product characterization: Elemental analysis, found: C, 56.85; H, 8.32; N, 11.98; B, 0.52%.

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[0260] 1.10.2 Eshmuno (registered trademark) オキソ-アミドカルボキシ-クロソ-B12H11 One-pot synthesis of [ka] Amounts of chemicals used in this synthesis: 8.72 g (25.2 mmol) of [HNEt3]2[closo-B 12 H 12 ]; 17.9 g (55.5 mmol) of [nBuN]Br; 13.8 g (130.0 mmol) Na[BF4]; 32 mL of HCl (4 M in dioxane); 5.63g + 5.63g EDC (14.7mmol + 14.7mmol). Product characterization: Elemental analysis, found: C, 56.57; H, 8.37; N, 12.36; B, 0.82%.

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[0261] 1.10.3 Eshmuno (registered trademark) オキソ-アミドカルボキシ-クロソ-B12H11 One-pot synthesis of [ka] Amounts of chemicals used in this synthesis: 7.83 g (12.6 mmol) of [nBu4N]2[closo-B 12 H 12 ]; 6.92 g (63.0 mmol) Na[BF4]; 16 mL of HCl (4 M in dioxane); 5.63g + 5.63g EDC (14.7mmol + 14.7mmol). Instead of gaseous ammonia, an aqueous solution of NH3 (25%) was used. Product characterization: Elemental analysis found: C, 56.94; H, 8.23; N, 12.22; B, 0.63%.

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[0262] 1.10.4 Eshmuno (registered trademark) オキソ-アミドカルボキシ-クロソ-B12H11 One-pot synthesis of [ka] Amounts of chemicals used in this synthesis: 200g (0.32mol) of [nBu4N]2[closo-B 12 H 12 ]; 175 g (63.0 mmol) Na[BF4]; 1.2L of dioxane 239 mL of HCl (4 M in dioxane); 50.0g+50.0g of EDC (0.13mol+0.13mol). 500mL gel (Eshmuno® COO-1050 ) Product characterization: Elemental analysis, found: C, 55.57; H, 8.47; N, 11.77; B, 1.21%.

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[0263] 2. Application Examples 2.1 Use of chromatographic materials (as described above) for the separation of bovine serum albumin (BSA) The boron cluster materials as prepared according to examples 1.8.4, 1.8.5, and 1.8.7-1.8.10 (examples with average particle size between 20-63 μm, average pore size between 40-110 nm, starting material COO-group density between 200-2400, and ligand density between 200-1460 μeq / g) were evaluated for their ability to bind and elute bovine serum albumin (BSA). The boron cluster modified materials were packed into a chromatography column of 5×50 mm dimensions with asymmetry between 0.8-1.2 and >3000 plates / m. After packing the boron cluster modified material, the resulting chromatography column was cleaned with 1 M NaOH solution for 30 min and pre-equilibrated by loading a buffer solution with a pH of 6.0. The buffer solution contained sodium dihydrogen phosphate and NaOH or / and HCl to obtain a pH of 6.0. The same solution was used to dissolve the lyophilized BSA samples to a concentration of 1 mg / ml.

[0264] The solution was loaded onto the prepared chromatography column until a breakthrough value of 10% was reached. These steps and the following steps were performed at a buffer flow rate of 75 cm / h. After loading the BSA, the chromatography column was washed with a solution of pH 6.0 and then eluted using gradient elution with a buffer having a pH of 10.0 and 3M imidazole. The elution buffer was prepared using different salts such as TRIS, imidazole, and NaCl. The conductivity and pH values ​​were tracked during the experimental setup and showed that BSA elution from the column was achieved due to the pH change during the gradient elution.

[0265] The sample eluate was fractionated and the resulting fractions were assessed for the amount of eluted BSA. As shown in Example 1.8.1, Eshmuno® COO, which does not have a boron cluster modification, does not bind BSA under the conditions described above.

[0266] The analytical evaluation of the collected fractions is presented in Table 3, which shows the amount of BSA bound per ml CV (column volume) of boron cluster modified material, ranging from approximately 4 mg for 300 μeq of COO-groups to 67 mg / ml for 1000 μeq of modified material. [Table 3]

[0267] Increasing the group density / ligand density (starting material with a COO-group density of 2400 μeq / g) reduced the binding capacity of BSA, suggesting that the optimal group density for a given modified starting material is within a COO-group density of 700–2400 μeq / g (ligand density between 542–1460 μeq / g). Moreover, the modified material was applied three consecutive times under the same conditions (Run 1-Run 3) and the amount of BSA in the eluted fractions was measured. The amount of eluted BSA was comparable within the runs, which allowed the modified material to be used in multiple runs.

[0268] 2.2 Use of chromatographic materials (as described above) for separation of trypsin under altered pH and conductivity conditions Boron cluster materials as prepared according to examples 1.8.5 and 1.8.8-1.8.10 (examples with average particle size between 20-63 μm, average pore size between 40-110 nm, group density between 400-900 μeq / g, and ligand density between 200-1460 μeq / g) were evaluated for their ability to bind trypsin under varying pH and conductivity conditions. The boron cluster modified materials were cleaned with 1 M NaOH solution for 30 min and then subjected to a vessel containing a corresponding buffer solution of a certain pH and conductivity. The buffer solution contained sodium dihydrogen phosphate, sodium sulfate, and NaOH or / and HCl. The pH of the buffer solution was varied between 4.5-7.5. Amount of Na2SO4: from 0 mM to 900 mM.

[0269] The same solution was used to dissolve the lyophilized trypsin sample to a concentration of 5mg / ml. This solution was loaded onto the preconditioned chromatography resin and incubated for 2 hours under constant agitation. The solution containing the unadsorbed protein residues was then removed from the vessel. The difference in trypsin concentration between the applied and removed solutions corresponded to the amount of trypsin bound to the chromatography material.

[0270] The analytical results for each selected pH and conductivity condition are presented in FIG. 8, which shows static binding capacity values ​​for trypsin bound on different boron cluster modified chromatographic materials (Eshmuno® B12-300 (bottom left), Eshmuno® B12-700 (top left), Eshmuno® B12-1000 (top right), Eshmuno® B12-2400 (bottom right)). FIG. 8 demonstrates strong binding at pH 4.5-6.0 and low Na2SO4 concentrations, as well as binding at high pH values ​​and high NaNa2SO4 concentrations, whereby the conductivity of the solution is approximately 90 mS / cm.

[0271] The amount of bound protein was dependent on the ligand density, with higher ligand density resulting in higher binding capacity, and for all selected boron cluster modified chromatographic materials there remained room for optimal binding.

[0272] 2.3 Use of chromatographic materials prepared in aqueous and organic solvents (as described above) for the separation of bovine serum albumin (BSA) Boron cluster materials as prepared according to Examples 1.8.9 and 1.8.13 (e.g., average particle sizes between 20-63 μm, average pore sizes between 40-110 nm, group densities between 400-900 μeq / g, and ligand densities between 900-1000 μeq / g) were evaluated for their ability to bind and elute bovine serum albumin (BSA). The boron cluster modified material was packed into a chromatographic column of 5 x 50 mm dimensions with an asymmetry between 0.8-1.2 and >3000 plates / m. After packing with the boron cluster modified material, the resulting chromatographic column was cleaned with 1 M NaOH solution for 30 min and pre-equilibrated by loading a buffer solution with a pH of 6.0. The buffer solution contained sodium dihydrogen phosphate and NaOH or / and HCl to obtain a pH of 6.0. The same solution was used to dissolve the lyophilized BSA sample to a concentration of 1 mg / ml.

[0273] The solution was loaded onto the prepared chromatography column until a breakthrough value of 10% was reached. These steps and the following steps were performed at a buffer flow rate of 75 cm / h. After loading the BSA, the chromatography column was washed with a solution of pH 6.0 and then eluted using gradient elution with a buffer having a pH of 10.0 and 3M imidazole. The elution buffer was prepared using different salts such as TRIS, imidazole, and NaCl. The conductivity and pH values ​​were tracked during the experimental setup and showed that BSA elution from the column was achieved due to the pH change during the gradient elution. The sample eluate was fractionated and the resulting fractions were assessed for the amount of eluted BSA.

[0274] The analytical evaluation of the collected fractions is presented in Table 4, which shows the amount of BSA bound per ml CV for boron cluster modified materials prepared in aqueous and organic solvents. [Table 4] Both boron cluster modified materials exhibited binding capacities for BSA ranging from 34 to 46 mg / ml, and this range was maintained in subsequent runs of the materials for at least three runs.

[0275] 2.4 Use of chromatographic material (nido-cluster as described above) for the separation of bovine serum albumin (BSA) Boron cluster materials as prepared according to Example 1.8.12 (e.g., average particle sizes between 20 and 63 μm, average pore sizes between 40 and 110 nm, and group densities between 400 and 900 μeq / g) were evaluated for their ability to bind and elute bovine serum albumin (BSA). The boron cluster modified material was packed into a chromatographic column of 5 x 50 mm dimensions with an asymmetry between 0.8-1.2 and >3000 plates / m. After packing with the boron cluster modified material, the resulting chromatographic column was cleaned with 1 M NaOH solution for 30 min and pre-equilibrated by loading a buffer solution with a pH of 6.0. The buffer solution contained sodium dihydrogen phosphate and NaOH or / and HCl to obtain a pH of 6.0. The same solution was used to dissolve the lyophilized BSA sample to a concentration of 1 mg / ml.

[0276] The solution was loaded onto the prepared chromatography column until a breakthrough value of 10% was reached. These steps and the following steps were performed at a buffer flow rate of 75 cm / h. After loading the BSA, the chromatography column was washed with a solution of pH 6.0 and then eluted using gradient elution with a buffer having a pH of 10.0 and 3M imidazole. The elution buffer was prepared using different salts such as TRIS, imidazole, and NaCl. The conductivity and pH values ​​were tracked during the experimental setup and showed that BSA elution from the column was achieved due to the pH change during the gradient elution. The sample eluate was fractionated and the resulting fractions were assessed for the amount of eluted BSA.

[0277] The assay of the collected fractions is presented in Table 5, which shows the amount of BSA bound per ml CV of boron nido-cluster modified material. [Table 5] Both boron cluster modified materials exhibited binding capacities for BSA ranging from 10 to 25 mg / ml, and this range was maintained in the subsequent performance of the materials for at least three runs.

[0278] 2.5 Use of chromatographic material (COSAN-cluster as described above) for the separation of bovine serum albumin (BSA) Boron cluster material as prepared according to Example 1.8.11 (example average particle size between 20-63 μm, average pore size between 40-110 nm, and group ligand density of 1000 μeq / g) was evaluated for its ability to bind and elute bovine serum albumin (BSA).

[0279] The boron cluster modified material was packed into a chromatographic column of 5 x 50 mm dimensions with an asymmetry between 0.8-1.2 and >3000 plates / m. After packing with the boron cluster modified material, the resulting chromatographic column was cleaned with 1 M NaOH solution for 30 min and pre-equilibrated by loading a buffer solution with a pH of 6.0. The buffer solution contained sodium dihydrogen phosphate and NaOH or / and HCl to obtain a pH of 6.0. The same solution was used to dissolve the lyophilized BSA sample to a concentration of 1 mg / ml.

[0280] The solution was loaded onto the prepared chromatography column until a breakthrough value of 10% was reached. These steps and the following steps were performed at a buffer flow rate of 75 cm / h. After loading the BSA, the chromatography column was washed with a solution of pH 6.0 and then eluted using gradient elution with a buffer having a pH of 10.0 and 3M imidazole. The elution buffer was prepared using different salts such as TRIS, imidazole, and NaCl. The conductivity and pH values ​​were tracked during the experimental setup and showed that BSA elution from the column was achieved due to the pH change during the gradient elution. The sample eluate was fractionated and the resulting fractions were assessed for the amount of eluted BSA.

[0281] The assay of the collected fractions is shown in Table 6, which indicates the amount of BSA bound per mL CV of COSAN-cluster modified material. [Table 6] The boron cluster modified material exhibited binding capacities for BSA ranging from 3 to 9 mg / ml, and this range was maintained in the subsequent performance of the material for at least three runs.

[0282] reference [Table 7-1]

Table 7-2

Claims

1. A stationary phase comprising a substrate and at least one boron cluster, the boron cluster containing only main group elements of the periodic table.

2. 2. The stationary phase according to claim 1, wherein the boron clusters have a cluster type selected from the group consisting of closo, nido, arachno, hypho, hypercloso, conjunct, preferably closo, nido or conjunct.

3. 2. The stationary phase of claim 1, wherein the boron cluster contains at least one atom that is not a boron atom.

4. 2. The stationary phase of claim 1, wherein one or more hydrogen atoms of the boron cluster are replaced by a covalently bonded substituent.

5. 10. The stationary phase of claim 1, wherein at least one boron cluster is non-covalently or covalently bound to the substrate.

6. 10. The stationary phase of claim 1, wherein at least one boron cluster is attached to the substrate via a linker.

7. 10. The stationary phase of claim 1, comprising a polymer chain grafted onto a substrate, the polymer chain comprising at least one boron cluster.

8. 10. The stationary phase of claim 1, wherein the substrate is a bead or a membrane.

9. A separation device comprising a stationary phase according to any one of claims 1 to 8, said separation device being preferably a chromatography column.

10. 1. Use of a boron cluster for the separation and / or purification of a target molecule, wherein the boron cluster contains only main group elements.

11. A process for the separation and / or purification of target molecules using a stationary phase according to any one of claims 1 to 8.

12. 10. A process for the separation of a target molecule from at least one other compound, wherein a 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 differs from the interaction of the other compound.

13. 13. The process of claim 12, wherein the stationary phase is present in a chromatography column, and a liquid containing the target molecule and at least one other compound is passed through the column, and the target molecule and other compounds present in the liquid are eluted from the column in response to their interaction with the stationary phase.

14. 13. The process according to claim 12, carried out by loading the stationary phase with an aqueous loading buffer of one pH and one ionic strength and eluting the target molecule with an aqueous elution buffer of another pH and / or another ionic strength, optionally with further additives.

15. The use according to claim 10, wherein the target molecule is a protein.

16. The process of claim 11, wherein the target molecule is a protein.

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