Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/531—Production of immunochemical test materials
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2600/00—Assays involving molecular imprinted polymers/polymers created around a molecular template

Definitions

• COBALTs compounds, herein designated COBALTs, or
• Covalent-Binding Antibody-Like Trapping or -Trap characterized by specific binding to a target with antibody or antibody-like affinity for a desired, preselected, target substance (a small molecule; a macromolecule such as a protein, a carbohydrate, a nucleic acid, etc.; a cell; a viral particle; etc.) and which contain chemical groups that allow these COBALTs to form strong, specific bonds, such as irreversible covalent bonds, with the target substance for which the COBALT was specifically designed.
• target substance a small molecule; a macromolecule such as a protein, a carbohydrate, a nucleic acid, etc.; a cell; a viral particle; etc.
• antibodies have a number of drawbacks compared to synthetic compounds including: the difficulty of optimization or modification via chemical modification; the necessity of their being maintained at low temperatures; their short shelf life (subject to thermal and microbial degradation); bio-contamination; high production costs.
• the genetic limitations of the animals used may restrict the binding site variability. This last restriction may be overcome by using phage-display and other genetic engineered methods for antibody production but here, too, it has not always been possible to raise effective antibodies for every desired target material.
• the present invention overcomes the deficiencies of the background art by providing a variety of substances which have an affinity for a desired, preselected, target substance (a small molecule; a macromolecule such as a protein, a carbohydrate, a nucleic acid, etc.; a cell; a viral particle; etc.) and which contain chemical groups that allow these substances to form strong bonds, such as irreversible covalent bonds, with the desired target substance.
• a desired, preselected, target substance a small molecule; a macromolecule such as a protein, a carbohydrate, a nucleic acid, etc.; a cell; a viral particle; etc.
• COBALTs Covalent-Binding Antibody-Like Trapping or -Trap
• these substances according to the present invention preferably feature a mechanism in which a covalent bond forms specifically between the desired target and the COBALT substance
• the letter "T” of COBALT may also stand for "tagging” or “tag”, as the covalent bond may cause binding between the desired target and the COBALT substance to be irreversible.
• the present invention optionally and more preferably includes a method wherein a target species (as above) is chosen and then, by synthetic chemical procedures and modifications, novel substances (COBALTs) are obtained or selected from combinatorial libraries that exhibit selective and covalent binding to the preselected target species.
• COBALTs novel substances
• the COBALT substances themselves may optionally be categorized according to different types of structures, including but not limited to, molecularly imprinted polymers, cyclodextrins, triazines and peptides, which may be selected from these combinatorial libraries of chemically reactive substances that can covalently react with a target substance.
• a molecularly imprinted polymer (MIP), as discussed in greater detail below, is typically synthesized in the presence of the target molecule, and hence is designed to bind specifically to that target molecule.
• the peptide derivative may include at least one of cyclic, linear and modified peptides and derivatives thereof.
• the COBALT substances of the present invention are preferably designed to be highly specific, and to bind to the target substance with a high degree of specificity, regardless of the category or type of COBALT substance which is used.
• the uses of the COBALTs include diagnostic, analytical, therapeutic and industrial applications.
• the binding mechanism between the COBALT and the target substance may occur as follows.
• An initial non-covalent complex may be pictured as forming rapidly between the COBALT and the target; other substances, different from the target substance, may be expected to either not form such complexes or alternatively to form complexes with a much shorter lifetime (in other words, their stability will be much lower) than that of the COBALT-target substance complex.
• the orientation of the two components, the COBALT and the target substance, in the complex is such that a chemical reaction can rapidly ensue to produce a new substance having a strong, covalent bond (or bonds) between the two initial components.
• Complexes formed by the COBALT and substances other than the target substance may either not have the orientation or lifetime required for covalent bond formation or the reaction rate would be expected to be far less than that between the COBALT and the desired target substance.
• Use of the term complex does not rule out the possibility of more than one complex between the COBALT and the desired target substance. Indeed, for some of the applications described herein, there may be many different complexes formed, but the overall result is that the COBALTs are expected to display a preferred affinity for the desired target substance, relative to other materials, and to preferentially react chemically with the desired target substance.
• Examples of situations where the chemically reactive, COBALT, approach may have important therapeutic and other applications include increased binding of a target molecule compared to conventional, noncovalently binding agents.
• the irreversible chemical reaction can eventually tag or trap selectively essentially all targets that initially bind, whereas with conventional, noncovalent binding, once the equilibrium constant is reached, no additional target molecules may be trapped.
• Illustrative potential applications include assay detection at low concentrations of the target analyte or the more effective therapeutic action of a COBALT allowing lower dosages of more effective drugs.
• a compound for specifically binding a molecular structure comprising a selective and chemically reactive compound with an enhanced apparent affinity constant.
• the compound is an antibody mimic.
• the compound is a molecularly imprinted polymer (MIP), the MIP being modified through chemical activation in order to react covalently with the molecular structure.
• MIP molecularly imprinted polymer
• the target substance is a chemically reactive substance and the COBALT is designed or selected so as to selectively react with the target.
• An important implementation of the present invention involves MIPs designed to react covalently with reactive organophosphate toxins (OP-agents) such as DFP, soman, sarin, VX, etc.
• OP-agents reactive organophosphate toxins
• this COBALT which is preferably an MIP
• the MIP is chemically modified by including an isocyanate, or isothiocyanate, functional group.
• the MIP is chemically modified by including an isocyanate, or isothiocyanate functional group and the molecular structure is a steroid. More preferably, the steroid is cholesterol or a bile acid.
• the MIP contains a nucleophile such as at least one of an oxime, a hydroxylamine, a hydrazine, a phenol and a 2-iodosobenzoic acid, and so forth, for specific and tight binding to organophosphates.
• the MIP contains two or more boronic acids or two or more aldehyde functions for specific and tight binding to carbohydrates.
• COBALT may contain, preferably as part of the MIP implementation, include but are not limited to chloromethylphenyl and 2,5-diketo- N-phenyltriazoline or any other triazine related functional group; functional group includes at least one of an alpha-halomethyl ether, wherein a halogen moiety may be fluoro, chloro, bromo or iodo; a beta-haloethyl ether, wherein a halogen moiety may be chloro, bromo or iodo; and a halomethylaryl, wherein a halogen moiety may be fluoro, chloro, bromo, or iodo; or carboxylic acid chloride or an activated carboxylic acid (e.g., 4-nitrophenyl ester, N- hydroxysuccinimide ester, pentafluorophenyl ester, etc.).
• an activated carboxylic acid e.g., 4-nitrophenyl ester
• the COBALT compound is a cyclodextrin derivative which has been chemically modified to react covalently with the target molecular structure.
• this COBALT which is preferably a cyclodextrin, and more preferably is an alpha-, beta-, or gamma-cyclodextrin, includes at least one or more amino groups (replacing one or more of the hydroxyl groups).
• the COBALT compound is a triazine derivative being chemically modified to react covalently with the target molecular structure.
• this COBALT is preferably a triazine, and more preferably is a derivative of 2,4,6-trichloro-l,3,5- triazine (cyanuric chloride) wherein one or more of the chloro groups are replaced by alcohols, phenols or preferably by amine-containing derivatives, as described, for example, by R-X Li, V. Dowd, D.J. Stewart, S.J. Burton and C.R. Lowe (1998) Nature Biotechnology 16, 190-195, and references therein.
• cyanuric chloride 2,4,6-trichloro-l,3,5- triazine
• the COBALT compound is a peptide r derivative thereof being chemically modified to react covalently with the target molecular structure.
• the peptide derivative is at least one of cyclic, linear and modified peptides and derivatives thereof.
• the COBALT compound is an antibody or antibody fragment or derivative thereof, being chemically modified to react covalently with the target molecular structure.
• the chemical modification to the cyclodextrin, triazine, peptide or antibody includes introduction of one or more isothiocyanate groups.
• cyclodextrin, triazine, peptide or antibody include but are not limited to chloromethylphenyl and 2,5-diketo-N-phenyltriazoline or any other triazine related functional group; functional group includes at least one of an alpha-halomethyl ether, wherein a halogen moiety may be fluoro, chloro, bromo or iodo; a beta-haloethyl ether, wherein a halogen moiety may be chloro, bromo or iodo; and a halomethylaryl, wherein a halogen moiety may be fluoro, chloro, bromo, or iodo; or carboxylic acid chloride or an activated carboxylic acid (e.g., 4-nitrophenyl ester, N-hydroxysuccinimide ester, pentafluorophenyl ester, etc.).
• functional group includes at least one of an alpha-halomethyl ether, wherein
• the chemical modification to the cyclodextrins includes two or more boronic acids or two or more aldehyde functions for specific and tight binding to carbohydrates.
• a combinatorial library of compounds containing chemically reactive groups screened for selectivity and chemical reaction with the molecular structure as a target, for creating the compound of the present invention is provided.
• a compound for specifically reacting at any site on a target molecule such that these sites are not limited to the conventional active site or ligand binding site of the target.
• the term 'antibody mimic' includes but is not limited to, any synthetic substance such as an MIP or a triazine derivative, or a derivative of a natural product such as a cyclodextrin or a peptide, which has been designed or selected so as to display selective affinity for a given target structure.
• FIG. 1 is a schematic flow chart, showing illustrative preparation of "chemically reactive" molecularly imprinted polymers (MlP-based COBALT) for the selective covalent binding of hydroxyl-containing target substances, ROH;
• FIG. 2 shows a schematic depiction of the preparation of a conventional cholesterol- binding, amino-containing MIP (MS50);
• FIG. 3 shows a schematic view of the two MIPs, one binding non-covalently and one binding covalently, the latter being a COBALT;
• FIG. 4 shows the calibration curve for cholesterol
• FIGS. 5 and 6 show the Scatchard and binding isotherm plots respectively for the third Example
• FIG. 7 is related to the preparation of crMIP - MS71;
• FIG. 8 shows the IR Spectrum of MS71 (when the maximum conversion into NCO is reached);
• FIG. 9 describes the overall approach for developing MIP -based COBALTS for the binding of toxic organophosphates, in which functional monomers, A, are polymerized with a large excess of crosslinker, porogen, etc, to create an MIP, B (MIP-B), that is hydrolyzed to remove the phosphonate, leaving behind complementary cavities, containing a nucleophile, X, in MIP-C, that selectively reacts covalently with DFP to form D (MIP-D).
• MIP-B MIP, B
• X nucleophile
• MIP-C selectively reacts covalently with DFP to form D
• the nucleophile, X, in MIP-C differs from the functional group Z in the monomer as well as Y following reaction with DFP.
• FIG. 10 depicts representative structures of synthesized functional monomers used for the DFP-binding MIPs that were prepared
• FIG. 11 shows the synthetic scheme used for the preparation of the 4- vinylbenzaldehyde oxime phosphate and phosphonate functional monomers 3, 9, and 10;
• FIG. 12 shows a calibration curve for converting percent BChe inhibition to DPFP concentration
• FIG. 13 shows a calibration curve for DCP concentration
• Rl, R2 phenyl, substituted phenyl, naphthyl, substituted naphthyl, etc
• the present invention is of a variety of substances which have an affinity for a desired, preselected, target substance (a small molecule; a macromolecule such as a protein, a carbohydrate, a nucleic acid, etc.; a cell; a viral particle; etc.) and which contain chemical groups that allow these substances to form strong bonds, such as irreversible covalent bonds, with the desired target substance, which as previously described may be termed COBALTs, Covalent-Binding Antibody-Like Trapping or -Trap.
• a desired, preselected, target substance a small molecule; a macromolecule such as a protein, a carbohydrate, a nucleic acid, etc.; a cell; a viral particle; etc.
• COBALTs Covalent-Binding Antibody-Like Trapping or -Trap.
• the present invention includes a mechanism wherein a target species (as above) is chosen and then, by synthetic chemical procedures and modifications, novel substances (COBALTs) are obtained that exhibit selective and covalent binding to the preselected target species.
• COBALTs novel substances
• the uses of the COBALTs include diagnostic, analytical, therapeutic and industrial applications.
• the binding mechanism between the COBALT and the target substance may occur as follows.
• An initial non-covalent complex may be pictured as forming rapidly between the COBALT and the target; other substances, different from the target substance, may be expected to either not form such complexes or alternatively to form complexes with a much shorter lifetime (in other words, their stability will be much lower) than that of the COBALT-target substance complex.
• the orientation of the two components, the COBALT and the target substance, in the complex is such that a chemical reaction can rapidly ensue to produce a new substance having a strong, covalent bond (or bonds) between the two initial components.
• Complexes formed by the COBALT and substances other than the target substance may either not have the orientation or lifetime required for covalent bond formation or the reaction rate would be expected to be far less than that between the COBALT and the desired target substance.
• Use of the term complex does not rule out the possibility of more than one complex between the COBALT and the desired target substance. Indeed, for some of the applications described herein, there may be many different complexes formed, but the overall result is that the COBALTs are expected to display a preferred affinity for the desired target substance, relative to other materials, and to preferentially react chemically with the desired target substance.
• Examples of situations where the chemically reactive, COBALT, approach may have important therapeutic and other applications include increased binding of a target molecule compared to conventional, noncovalently binding agents.
• the irreversible chemical reaction will eventually trap all targets that initially bind, whereas with conventional, noncovalent binding, once the equilibrium constant is reached, no additional target molecules may be trapped.
• Illustrative potential applications include assay detection at low concentrations of the target analyte or the more effective therapeutic action of a COBALT allowing lower dosages of more effective drugs.
• MIP molecularly imprinted polymer
• the system is somewhat analogous to the irreversible enzyme inhibitors and antibody affinity labeling reagents. Note that substances differing from X to an appreciable extent will tend not to react effectively within the MIP cavity because, even if they enter the MIP cavity, they will not have the orientation necessary for reaction (the stereochemical or the "spatiotemporal" [Khanjin NA, Snyder JP, Menger FM, J. Amer. Chem. Soc. 121 (50): 11831-11846 (1999)] demands are not met).
• the term "enhanced apparent affinity constant" is used herein to describe the ability of the COBALT to bind more of a target substance than a conventional, non-covalently binding substance. Since affinity or binding constants are restricted to equilibrium systems, and since the use of irreversible inhibitors technically does not allow equilibrium to be reached, this definition is a generalization from a classical equilibrium system. However, the definition does permit a quantitative estimate of the improved sequestering of a given target substance to be made. Thus, if a conventional MIP, after 24 hours, or 48 hr, is determined to have bound a given fraction of a target substance, the equilibrium binding constant can be determined for this equilibrium system.
• the COBALTs comprise many different classes of laboratory-synthesized substances and also include chemically modified monoclonal antibodies, as well. In the latter case, an antibody is elicited to an appropriate hapten and then the antibody is chemically "activated” in order to convert it to a COBALT, e.g., amine to isothiocyanate; tyrosine to an o-quinone; thiol to chloromethylthioether; etc.
• the antibody-based COBALT will now complex with the desired target substance and subsequently bind covalently and irreversibly.
• the many substances that are suitable for producing COBALTs include, but are not restricted to: (i) molecularly imprinted polymers (MIPs), which have been prepared in one of the conventional manners reported in the literature, and then chemically converted to an "activated MIP", i.e., a COBALT, which specifically binds to and then reacts, forming a covalent bond with the target substance; (ii) peptides, e.g., cyclic peptides, which have been modified to contain a reactive functional group, such as an isothiocyanate group, so that the peptide will not only bind to a specific target substance but covalently react with that substance; (iii) peptide-derivatives of "platform” molecules, such as cyclodextrins, where the peptide or cyclodextrin has been chemically activated to specifically bind to, and covalently react with, a desired target substance; (iv) non-peptide substances, such as tria
• the target substances are not restricted to any class of material and include but are not limited to, small molecules such as steroids, sugars, lipids; macromolecules such as proteins, carbohydrates and nucleic acids; cell surface substances and receptors; and other molecules of biomedical interest.
• the invention particularly relates to the use of COBALTs as drugs and for detection as well as separation applications.
• Production of the COBALT may include, but is not limited to, the following three approaches, which are given only as non-limiting illustrative examples: i. Creating a compound having selectivity for the target substance (e.g., an MIP) and then 'activating' the compound by introducing a chemically reactive functional group at an appropriate, specific locus on the compound in order for covalent bond reaction to take place when the target molecule is in contact with the compound. Alternatively, if the target substance has a reactive functional group, e.g., an ester, epoxide, disulfide, etc.
• a reactive functional group e.g., an ester, epoxide, disulfide, etc.
• the MIP or other COBALT can optionally be prepared such that an appropriate functional group is present to react with the target.
• the COBALTs may be further "evolved", i.e., chemically changed and further selected in order to obtain improved binding-plus-reacting- substances for the given, desired target material.
• the COBALT has been described as having one reactive group for covalent bond formation with the target substance, but, as illustrated below, two or more activated functional groups may be present on each COBALT.
• boronic acids were used in one of the first examples of an MIP, tighter binding of carbohydrates, sugars, glycolipids, etc. may be achieved using two or more boronic acid derivatives.
• acetals and ketals have been used in MIPs for binding to diols, but the use of two or more aldehydes has not been reported as for the present invention.
• the present invention may be differentiated from the above examples, in that, in an important and major embodiment of the invention, any site on the target substance is available for targeting by the chemically reactive enhancement is designed.
• This is pronounced of the immune system's approach: antibodies elicited to, say, an enzyme, may bind at any point on the surface of the enzyme, and, when characterized, are found to be specific for a given site on the enzyme, including but not restricted to the enzyme active site.
• the chemically reactive, covalently binding compounds of the present invention have a priori random site- selectivity.
• the present invention is not dependent on the availability of a known or defined ligand binding site, or any ligand binding site at all, which is an essential requirement in all of the above approaches.
• the COBALT approach enables feasible structures having chemically reactive groups, such as an isothiocyanate, to be designed or discovered, for which the desired binding plus covalent bond-forming reaction (one that may react at any site on the target substance) occurs.
• chemically reactive groups such as an isothiocyanate
• the design or discovery of such compounds according to the present invention may exploit any knowledge available regarding the target structure to improve probability of discovering effective binders, the approach of the present invention is primarily and preferably a discovery process which is dependent, as with the antibodies of the immune system, on using a large number and variety of potential binding structures.
• This Example relates to the design and implementation of COBALTs for binding cholesterol and bile acids.
• FIG. 1 shows a schematic outline of the approach for the creation of specific COBALTs.
• the hydroxyl-containing cholesterol and DCA targets are represented by R-OH.
• the ester or carbamate is then polymerized with a large excess of crosslinker in the conventional fashion [G. Wulff and A. Sarhan Angew. Chem. Int. Ed. Engl. 11, 341-(1972); G. Wulff. Angew. Chem. Int. Ed. Engl. 34, 1812-1832 (1995); G.
• the top portion of Figure 1 utilizes polymerization of an ester derivative of ROH to give, after hydrolytic removal of the print or template molecule ROH, carboxylic acid-containing cavities complementary to ROH which are activated to acid chlorides in order to form the covalent product of step e.
• the bottom portion exemplifies polymerization of a carbamate derivative of ROH to afford, following removal of ROH, complementary amine-containing cavities, which are activated to isocyanate (or isothiocyanate, not illustrated here) to form the covalent product, step i.
• step a synthesis of the methacylic ester of ROH (synthesis of the carbamate from 4-vinylbenzeneisocyanate is not illustrated); steps b and f, the ester or carbamate is polymerized with a large excess of crosslinker and a porogen (pore-forming solvent) using an initiater such as AIBN and heat (or irradiation); steps c and g, the solid polymer is ground, sieved and treated with reagents to hydrolzye all ester and carbamate bonds and remove ROH; step d, the acid chloride MlP-based COBALT may be prepared by treating with SOC12 or COC12; step h, the isocyanate (or isothiocyanate) MlP-based COBALT can be prepared by reaction with phosgene, COC12- (or thiophosgene, CSCI2).
• the resulting MIPs may be used directly as conventional binding agents or they may be converted into COBALTs by chemical modification, or "activation", using specific chemical reactions.
• the COBALT approach is illustrated using chemically reactive molecular imprint polymers (MIPs).
• MIPs chemically reactive molecular imprint polymers
• Cholesteryl 4-vinylphenyl carbamate was used as a template monomer; cholesteryl methacrylate was an added functional monomer to create hydrophobic binding and recognition.
• Cholesterol was cleaved from the polymer (MS40, MS41) hydrolytically with the concomitant loss of CO 2 ⁇ resulting in the formation of a conventional MIP (MS50) having a non-covalent (or non-reactive) recognition site, bearing an aminophenyl group, capable of interacting with cholesterol through hydrogen bonding.
• FIG 2 shows a schematic depiction of the preparation of a conventional cholesterol- binding, amino-containing MIP (MS50).
• MS50 cholesterol-binding, amino-containing MIP
• Figure 3 shows a schematic view of the two MIPs, one binding non-covalently and one binding covalently, the latter being a COBALT.
• the isocyanate MIP (MS71) is illustrated; the analogous reaction using thiophosgene afforded the isothiocyanate MIP (MS80).
• the neck of the tube was broken and the polymer monolith was broken into small pieces using a spatula and the broken pieces ground in a mortar.
• the polymer was Sohxlet-extracted using 60 ml MeOH and dried in vacuo at 70 °C and then weighed.
• the polymer (MS41, l.Og) was suspended in 1M sodium hydroxide in methanol (50 ml) and heated to reflux for 24h. periods. The cooled suspension was neutralized with IN hydrochloric acid, filtered on a sintered glass funnel and washed with water until the washings were neutral, followed by several methanol washings.
• Figures 5 and 6 show the Scatchard and binding isotherm plots respectively for this Example.
• Figure 7 is related to the preparation of crMIP - MS71.
• the suspension was filtered and the polymer was washed with dry acetonitrile (2 x 10 ml) and dried in the oven at 80 °C for 30 min.
• the resulting polymer MS71 was further dried under vacuum for lh and stored in a desiccator.
• the NCO peak %T at 2265 cm-1 reduced to 50% compared to that measured from the reaction mixture.
• the NCO peak disappeared completely.
• Polymer MS50 was converted in the same way to the isothiocyanate MIP, MS80.
• MS71 On standing for 48 hr open to the atmosphere, the decrease of the characteristic NCS peak in the infrared, at 2087 cm '1 , indicated that only about 50% had reacted. When treated with cholesterol as in the case of MS71, it was possible to show significant binding at concentrations where the 'parent' MIP was binding only very small amounts of cholesterol. At an initial cholesterol concentration of 0.005 M, MS80 (20 mg polymer samples were used in all experiments in duplicate) bound 39% more cholesterol than did MS50; at an initial cholesterol concentration of 0.003 M, MS80 bound 48% more cholesterol than did MS50; at an initial cholesterol concentration of 0.001 M, MS80 bound 76% more cholesterol than did MS50.
• DFP was chosen as a representative "OP (organophosphate) agent", illustrating the approach that can be used for sarin, soman, etc. as well as other chemical warfare toxins. Additional “OP agents” are described below to further generalize the method. Briefly, the design was to produce MIPs having complementary cavities for DFP as well as a suitably positioned nucleophile (an active OH group that can react with DFP).
• OP organophosphate
• FIG. 10 depicts representative structures of synthesized functional monomers used for the DFP- binding MIPs that were prepared. Additional functional monomers were prepared for other fluorophosphates and fluorophosponates and representative syntheses are presented below. Each of the functional monomers was characterized (including 1H and 31 P NMR, MS, IR, microanalysis) and tested for thermal and hydrolytic stability for subsequent steps.
• Suspension polymerization droplets of reactants dissolved in toluene suspended in water containing surface active agents were polymerized
• dispersion polymerization also termed precipitation polymerization; solutions of reactants in a solvent - toluene/methanol - were polymerized under rapid stirring
• thermally initiated polymerization and photochemically initiated polymerization methods were also used.
• Divinylbenzene/styrene (DVB/S) and ethylene glycol dimethacrylate/monomethyl acrylate (EGDMA/MMA) crosslinker/monomer mixtures were used. Mixtures of divinylbenzene/styrene/4-vinylpyridine (DVB/S/VP) and other combinations were also used.
• the organic solvent wash after polymerization was analyzed for phosphorus- containing substances ( 31 P-NMR) to indicate the proportion of functional monomer (typically >80%) that had been incorporated into the polymer.
• the MIP polymers were also analyzed for phosphorus by the sensitive ICP method after a weighed sample for fully combusted in oxygen using a Schoeniger flask. The phosphorus content was determined by inductively coupled plasma atomic emission spectrometry (ICP-AES) at 178.200 nm.
• Polymer hydrolysis conditions were varied and optimized for MIPs. Conditions included: (1) aq. KOH/toluene/2-propanol; (2) NH 2 OH-HCl/triethylamine/ toluene/2- propanol; (3) NH 2 OH-HCl/triethylamine/ toluene/2-propanol/DBU; (4) NH 2 OH-HC1/40% aq. KOH/ tetrabutylammonium bromide; (5) NH 2 OH/ toluene/2 -propanol/water. Conditions (4) were generally used. In addition, extensive incubation of 3 under conditions (4) showed no chemical change to the oxime group (e.g., hydrolysis to the aldehyde).
• BICP benzylisopropylchlorophosphate
• MIP92-42 1 g was stirred with 50 ml toluene for 0.5 hr; 5 ml of 2-propanol were then followed by 10 ml of an aqueous NaOH solution (40 g in 100 ml water). While stirring vigorously, 2.5g of solid tetrabutylammonium bromide (same results were obtained using the chloride) were added and the stirred reaction was warmed to 70°C for 12 hr.
• the cooled reaction was filtered on a sintered-glass filter (the filtrate was used to determine phosphorus content), and the polymer washed with water (50 ml), 0.1 N HC1 (50 ml), 1% Na 2 CO 3 solution (50 ml), six portions of 2-propanol (each 50 ml), chloroform (50 ml), three portions of toluene (50 ml), and finally dried at 60°C for 12 hr.
• MIP92-42III is designed to bind diphenylfluorophosphate (DPFP); the unhydrolyzed MIP 92-42 is the control polymer.
• DPFP diphenylfluorophosphate
• the concentrations of DPFP present during various stages of the experiment were determined by measurement of percent inhibition of butyryl choline esterase (BChe) activity.
• a calibration curve was constructed, using fresh solutions of DPFP in isopropyl alcohol, to convert percent BChe inhibition to DPFP concentration.
• BChe activity was measured in units of change in A412 per minute based on the production of thiocholine from the enzymatic hydrolysis of butyryl thiocholine, which created a yellow color in the presence of the colorimetric reagent DTNB.
• Control 1 isopropyl alcohol only 0 0
• Control 1 isopropyl alcohol only 0 0
• COBALT compounds In addition to the previously described illustrative applications of the COBALT compounds according to the present invention, other applications are also possible. These applications may optionally include any application in which highly specific binding to a particular target molecule, followed by the formation of an irreversible covalent bond between the COBALT compound and the target molecule, is both desirable and possible.
• the previous description includes methods for designing and creating these COBALT compounds, which may be used according to the illustrative, exemplary applications given below.
• Bile acid sequestrants A number of polymers, such as cholestyramine, are used as bile acid sequestrants. Their action is based on the presence of strongly basic groups in the polymer (typically, ion exchange resin type of polymers) and they are used for cholesterol lowering and bile-related diseases. These materials are limited because they have limited potency and they also remove (bind) other required substances such as nutrients, drugs, etc.
• COBALTs which bind bile acids and salts do not remove needed nutrients, drugs or other substances and will be more potent.
• the COBALTs can be made so that they are selective to the more hydrophobic bile acids such as deoxycholic acid.
• bile acids and salts serve important functions in the body, such as promoting digestion of fat
• DCA deoxycholic acid
• DAA chenodeoxycholic acid
• LCA lithocholic acid
• these more hydrophobic bile acids are highly significant disease- causing agents.
• COBALTs of the present invention with their irreversible binding provide more efficient removal of bile sequestrants, with more specific binding, than compounds which are known in the art.
• Cyclodextrin-based COBALTS selected from combinatorial libraries
• Illustrative, non-exclusive examples of approaches for obtaining COBALTs based on cyclodeextrins where the covalent bond forming group on the COBALT is an isothiocyanate are shown in Figure 14.
• the beta-cyclodextrin is illustrated but alpha- or gamma- cyclodextrin based combinatorial libraries can also be used.
• the degree of substitution on the cyclodextrin can be varied widely; in Figure 14 one combinatorial library (Comb.Lib.A) is illustrated with one varying substituent, Ri, while the second combinatorial library (Comb.Lib.B) is illustrated with two varying substituents, Riand R 2 .
• the final library is shown as trityl protected structures; when testing for binding the trityl groups are removed, as shown in the upper right hand comer of Figure 14.
• Such COBALTs can be used for irreversible binding to small and molecules, to peptides and proteins, if these targets contain a hydroxyl or amino group.
• a Comb.Lib.B was prepared containing at least 1000 members. The preparation was carried out by reacting one equiv. of ten different R1I substances with tritylated-mono-4-isocyanato-benzyl-beta-cyclodextrin, in ten different tubes, each tube containing a different R1I. Each tube was further divided into ten different tubes and each reacted separately with one equiv. of R2I, again using the same set of ten different substituted benzyl iodides.
• the individual substances (not totally pure materials on the basis of TLC analysis and HPLC of selected wells) were applied at different concentrations (10 microM, 1 microM, and 0.1 microM) to solutions of various proteins, including BSA, lysozyme and purified mouse IgG, and incubated at room temperature for periods of 1 , 8 and 15 hrs. at various pH buffers.

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