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WO2008139204A2 - Polymères imprimés à groupes fonctionnels - Google Patents

Polymères imprimés à groupes fonctionnels Download PDF

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Publication number
WO2008139204A2
WO2008139204A2 PCT/GB2008/001681 GB2008001681W WO2008139204A2 WO 2008139204 A2 WO2008139204 A2 WO 2008139204A2 GB 2008001681 W GB2008001681 W GB 2008001681W WO 2008139204 A2 WO2008139204 A2 WO 2008139204A2
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WIPO (PCT)
Prior art keywords
group
polymer
functional group
functional monomer
phosphate
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PCT/GB2008/001681
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English (en)
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WO2008139204A3 (fr
Inventor
Ruediger Woscholski
Sally Louise Ewen
Joachim H. G. Steinke
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Imperial Innovations Ltd
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Publication of WO2008139204A2 publication Critical patent/WO2008139204A2/fr
Publication of WO2008139204A3 publication Critical patent/WO2008139204A3/fr

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
    • B01J20/26—Synthetic macromolecular compounds
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 and B01D15/30 - B01D15/36, e.g. affinity, ligand exchange or chiral chromatography
    • B01D15/3852—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 and B01D15/30 - B01D15/36, e.g. affinity, ligand exchange or chiral chromatography using imprinted phases or molecular recognition
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
    • B01J20/26—Synthetic macromolecular compounds
    • B01J20/268—Polymers created by use of a template, e.g. molecularly imprinted polymers
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6842—Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins
    • 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

  • the present invention relates to a novel class of imprinted polymers, termed 'functional group imprinted polymers' (FIPs), which selectively recognise molecules due to the presence of one or several predetermined functional groups, irrespective of the type, class, substructure or size of the molecule.
  • FEPs functional group imprinted polymers'
  • polymeric matrices containing recognition sites of pre- determined specificity for a target molecule.
  • These polymeric matrices can involve covalent or non-covalent linkage of monomers or a hybrid network thereof.
  • these polymeric matrices comprise polymerisable functional monomers which have functionality enabling them to interact with a target molecule.
  • the polymerisable functional monomers are allowed to form a complex with a template molecule and then are co-polymerised with a cross-linker in the presence of a porogenic solvent.
  • MIPs molecularly imprinted polymers
  • MIPs have been developed that bind specifically to cholesterol, L-menthol, the pesticide bentazone, the barbiturate cyclobarbital, or the herbicide 2,4-dichlorophenoxyacetic acid) or a class of molecules (for example, ⁇ -lactam antibiotics).
  • Binding selectivity of known MIP technology is represented in Figure 2.
  • Molecular imprinting has received considerable attention over the last few decades, and the technology has been successfully applied to a broad range of areas including: biosensors; solid-phase extraction; immunoassays; and chiral separation.
  • MIPs have been prepared for the selective recognition of a protein.
  • Exemplary approaches to achieve selective MIP formation include employing a combination of metal complex binding sites and substrate inhibitors as template, post-functionalisation and interpenetrating network generation, application of biomolecule substructures (e.g. consensus DNA sequences) as a partial molecular imprint approach to biomolecule recognition and the inclusion of MIPs as part of a polymer composite to facilitate imprinting of large target substrates.
  • biomolecule substructures e.g. consensus DNA sequences
  • MIP technology is tailored to producing MIPs that bind specifically to a particular target molecule, analogously to antibodies.
  • MIPs are unable to discriminate between substrates based solely on the nature of the functional groups present.
  • MIPs have been produced according to the 'epitope approach' methodology. These MIPs do exhibit a certain degree of substrate promiscuity, since they are designed to recognise molecules bearing the same structural motif.
  • MIPs as described above are not designed to, and cannot, recognise substrates based on the functional groups alone.
  • proteomics An exemplary application requiring recognition on the basis of the presence of a functional group lies within the field of proteomics. It is well known that protein phosphorylation is an important post-translational modification that underlies the regulation of signal transduction pathways involved in all cellular processes. Phosphoproteomics, a subdiscipline of proteomics, focusses on the analysis of phosphorylated proteins and it is widely accepted that determination of the phosphoproteome is an enormously challenging task.
  • MS mass spectrometry
  • IMAC Immobilised Metal Ion Affinity Chromatography
  • TiO 2 titanium dioxide affinity chromatography
  • IMAC is not necessarily bind all phosphorylated species present in a sample. For example, multiply-phosphorylated species bind with greater affinity than singly-phosphorylated species, and indeed the latter may often not even be retained on a column. Furthermore, IMAC is not selective for phosphorylated residues (the acidic residues aspartate and glutamate are also bound, as are histidine residues).
  • Chemical derivatisation is another method of phosphopeptide enrichment. This method is generally not favoured by biologists since it involves a number of synthetic steps, with the implications of incomplete reactions, side-reactions etc.
  • Example of proposed derivatisation methods have included: reaction of phosphate esters with ⁇ - diazo substituted resins (with prior protection of carboxylate-bearing residues); base- catalysed ⁇ -elimination of phosphorylated serine or threonine residues to form a Michael Acceptor with subsequent reaction with a nucleophile to effect linkage to a biotinylated affinity tag; and a multi-step protocol involving conversion of a phosphate ester to a phosphoramidate and subsequent derivatisation.
  • the invention provides an imprinted polymer comprising a porous polymeric network formed from a functional monomer comprising a chemical moiety capable of interacting with a pre-determined functional group and a crosslinker, wherein the imprinted polymer is capable of selectively recognising and binding to a plurality of molecules which possess the pre-determined functional group, wherein apart from possessing the pre-determined functional group, the plurality of molecules may differ in at least one of molecular class, structure, substructure or size.
  • the polymeric network of the imprinted polymer is arranged such that the chemical moieties of the functional monomer form recognition sites capable of binding selectively to the predetermined functional group. Preferably, this binding is highly selective for the pre-determined functional group.
  • FIPs functional group imprinted polymers'
  • FIPs are designed to recognise, with minimal structural discrimination, compounds exhibiting one or more specific predetermined functional groups whilst at the same time exhibiting selectivity over all compounds with deviating functional group compositions. Discrimination by compound type, class, substructure or size is suppressed as part of the unique recognition and selection process exhibited by FIPs of the invention. This is attributable to the choice of template molecule and porogenic solvent used when forming the polymer, choice of functional monomer, type of polymer backbone and polymer network structure.
  • the porous polymeric network formed from the functional monomer, linked by a crosslinker may be covalent, non-covalent or a hybrid covalent/non-covalent network.
  • the porous polymeric network is a. macroporous polymeric network.
  • a macroporous network is taken to be a network comprising pores with a mean diameter of > 50nm.
  • the porous network may however comprise a distribution of pore sizes and some pores of diameter less than 50nm may be present.
  • the presence of a macroporous structure is important in enabling accessibility of all sizes of analyte, thereby enabling the imprinted polymer of the invention to be selective on the basis of the presence or absence of the pre-determined function group, but not on the basis of molecular size.
  • a macroporous polymeric network will enable access to recognition sites within the polymeric network for molecules of any size able to permeate the macroporous network.
  • Molecules possessing the pre-determined functional group will bind selectively to the recognition sites, whereas those not possessing the predetermined functional group will not bind and will diffuse out of the polymeric network.
  • the inventors have determined that it is possible to achieve sufficient macroporosity to negate size discrimination whilst also achieving high selectivity for a pre-determined functional group.
  • the pre-determined functional group is selected from the group including, but not limited to: acetal; acid chloride; aldehyde; alkene; alkyne; amide; amidine; amine; ammonium; azide; azo; carbonate; carboxylic acid; carboxylate; ester; ether; guanidine; guanidinium; hydroxyl; isocyanate; isothiocyanate; imidazole; imide; imine; ketal; ketone; nitrate; nitrile; nitro; nitroso; phosphate; phosphate ester; peroxy; phosphine; phosphonate; phosphono; phosphonium; pyridyl; sulphate; sulfonate; sulfone; sulfonic acid; thiol; thiourea; thiourethane; thiouronium; urea; urethane; and uronium.
  • the pre-determined functional group is a phosphate or phosphate ester functional group. More preferably, the pre-determined functional group is the phosphate monoester functional group.
  • the functional monomer comprises a polymerisable group and one or more chemical moieties capable of interacting with the pre-determined functional group, the one or more chemical moieties being selected from the following: acetal; acid chloride; aldehyde; alkene; alkyne; amide; amidine; amidinium; amine; polyamine; ammonium; azide; azo; carbonate; carboxylic acid; carboxylate; cyclodextrin; ester; ether; guanidine; guanidinium; hydroxyl; isocyanate; isothiocyanate; imidazole; imide; imine; ketal; ketone; nitrate; nitrile; nitro; nitroso; phosphate; phosphate ester; peroxy; phosphine; phosphonate; phosphono; phosphonium; pyridyl; polypyrrole; porphyrin; sulphate; sulfonate;
  • the functional monomer is capable of binding strongly to a phosphate or phosphate monoester functional group and comprises one or more chemical moieties selected from amide, amidine, amidinium, carboxylic acid, polyamine, guanidinium, guanidine, polypyrrole, porphyrin, urea, urethane, thiourea, thiourethane and thiouronium as well as metal pyridine dipicolyl amines, metal cyclens (3, 4 and 5), metal coordination enes and metal guanidine chelates.
  • chemical moieties selected from amide, amidine, amidinium, carboxylic acid, polyamine, guanidinium, guanidine, polypyrrole, porphyrin, urea, urethane, thiourea, thiourethane and thiouronium as well as metal pyridine dipicolyl amines, metal cyclens (3, 4 and 5), metal coordination enes and metal guanidine
  • the functional monomer comprises a thiouronium group, bis(pyridine-2-ylmethyl)amino, (diacetic acid)amino, l,2-bis(o-aminophenoxy)ethane -NJsfJV' JV'-tetraace ⁇ c acid.
  • (diacetic acid)amino is l-(o- aminophenoxy)methane- ⁇ N-diacetic acid.
  • the thiouronium group has the formula -SC(NHR 2 )(NHR 3 ), wherein R 2 and R 3 are each independently selected from hydrogen, Ci -6 alkyl or 5- or 6-membered aryl or heteroaryl.
  • a chelated metal ion is present where the functional monomer comprises a metal pyridine dipicolyl amine, a metal cyclen (3, 4 and 5), a metal coordination ene, metal guanidine chelate, bis(pyridine-2-ylmethyl)amino, a (diacetic acid)amino group (for example l-(o-aminophenoxy)methane-7V,./V'-diacetic acid) or l,2-bis(o- aminophenoxy)ethane -N,N,N',N'-tet ⁇ aace ⁇ c acid.
  • the functional monomer comprises a metal pyridine dipicolyl amine, a metal cyclen (3, 4 and 5), a metal coordination ene, metal guanidine chelate, bis(pyridine-2-ylmethyl)amino, a (diacetic acid)amino group (for example l-(o-aminophenoxy)methane-7V,./V
  • the chelated metal ion is Al 3+ , Fe 3+ , Ca 2+ , Zr 4+ , Ni 2+ , Co 2+ , Cu 2+ , Mn 2+/3+ , Mg 2+ , Ca 2+ , Zn 2+ or Ga 3+ .
  • the metal ion is Zn 2+ and for a (diacetic acid)amino group or 1 ,2-bis(o-aminophenoxy)ethane acid the metal ion is Ga 3+ .
  • the functional monomer comprises the following structure:
  • A comprises (CH 2 ) n , wherein n is 0 or 1 ;
  • B comprises (a) a thiouronium group of formula -SC(NHR 2 )(NHR 3 ), bis(pyridine-2- ylmethyl)amino optionally substituted on one or both pyridine rings with Y, a (diacetic acid)amino group (for example l-(o-aminophenoxy)methane-NJV-diacetic acid), 1 ,2-bis(o-aminophenoxy)ethane-AUV,./V v JV -tetraacetic acid, or an analogue, derivative or ring-fused variant thereof; (b) a ditopic thiouronium group of formula:
  • R 2 and R 3 are each independently selected from hydrogen, Ci -6 alkyl (preferably methyl) optionally substituted with Y, Ci -6 haloalkyl (preferably CF 3 or CH 2 CF 3 ), optionally substituted 5- or 6-membered aryl, heteroaryl (preferably phenyl), alkylaryl (preferably benzyl) or alkylheteroaryl; and R 4 is Ci -6 alkyl (preferably methyl) or
  • X comprises hydrogen or a polymerisable group
  • each occurrence of Y independently comprises hydrogen or a polymerisable group; provided that at least one occurrence of X or Y must be a polymerisable group.
  • V is -(CH 2 )- W-(CH 2 )-, wherein W is 1-, 3-, 5- substituted phenyl.
  • the preferred option for group B is option (a) as set out above.
  • the polymerisable group is a group that can be polymerised by free radical chemistry, for example an acrylate, methacrylate, acrylamide, methacrylamide, or a vinyl group.
  • the polymerisable group may be a group that is polymerisable by ring-opening metathesis polymerisation, for example a norbornene or other strained cyclic alkene derivative.
  • the polymerisable group comprises a vinyl moiety, such that where X comprises a polymerisable group and n is 0, the functional monomer comprises a styrene group and where n is 1 the functional monomer comprises an alpha-methyl styrene group.
  • X is para to -A-B.
  • X is hydrogen
  • R 1 is hydrogen
  • n is 1
  • B is -SC(NHR 2 )(NHR 3 ) wherein R 2 is CH 2 CHCH 2 and R 3 is benzyl.
  • the functional monomer comprises one of the following structures:
  • the functional monomer has the structure:
  • the crosslinker is water-soluble.
  • the crosslinker comprises an acrylate, a methacrylate, an acrylamide, a methacrylamide, a styrene, a polyethylene glycol (PEG) derivative bearing a polymerisable group at each end of the PEG chain or a 1,2-disubstituted alkene such as a maleimide or a vinyl ether.
  • the cross linker is ethylene glycol dimethacrylate, a polyethylene glycol derivative of formula: wherein Z is a polymerisable group
  • the polyethylene glycol derivative is a poly(ethylene glycol) diacrylate, a poly(ethylene glycol) dimethacrylate, a poly(ethylene glycol) diacrylamide or a poly(ethylene glycol) dimethacrylamide.
  • Z is a norbenene.
  • the functional monomer is custom-made.
  • the polymer is formed from a functional monomer and at least one co-monomer.
  • the co-monomer may be a second functional monomer as defined above or it may be any other monomer comprising a polymerisable group.
  • the polymer comprises two or more types of functional monomer, each being capable of interacting with a different pre- determined functional group.
  • the polymer is capable of selectively recognising a molecule which possesses one or more of a pre-determined selection of two or more functional groups.
  • the polymer comprises two or more types of crosslinker as defined above.
  • the present invention provides a method of preparing an imprinted polymer which is capable of selectively recognising and binding to a molecule which possesses a pre-determined functional group, wherein the imprinted polymer is capable of selectively recognising and binding to a plurality of molecules which possess the pre-determined functional group, wherein the plurality of molecules may differ in at least one of molecular type, structure, substructure or size, the method comprising the steps of: a) mixing a template molecule possessing the pre-determined functional group and a functional monomer comprising a chemical moiety capable of interacting with the pre-determined functional group and allowing complex formation; b) providing a porogenic solvent and a cross-linker to the mixture provided in step a) and allowing polymerisation to occur; and c) removing the template molecule thereby providing the imprinted polymer.
  • step a) the template molecule undergoes a self-assembly process with one or more functional monomers that will constitute a functional group's binding site within the imprinted polymer.
  • This self-assembly process leads to the formation of a template- binding site complex in which the functional monomers are spatially oriented for optimal binding to the pre-determined functional group. Minimisation of influence of substrate size, type etc on binding in the produced imprinted polymer is attributable to the choice of template molecule, functional monomer(s) and polymer backbone.
  • the template molecule allows pre-organisation of binding sites (i.e. functional monomers) in solution in such a way that the structural and electronic features of the pre-determined functional group to be imprinted are recognised and during polymerisation become inscribed into the polymer matrix.
  • binding sites i.e. functional monomers
  • the polymer backbone allows the self-assembly and imprinting process to take place without adverse effect and the polymer backbone is compatible with the solvent or solvent mixtures to be used in the process of applying the FIP to an application.
  • the polymer network forms in such a way that it produces pores and pathways which are large enough to ensure accessibility for all compounds that need to be recognised by the FIP.
  • the functional monomer, crosslinker and porogenic solvent are selected to ensure formation of a macroporous network. This is achieved by tailoring the solubility of the functional monomer and crosslinker in the porogenic solvent as well as by tailoring the structure of the template molecule. With regard to stability, in order to achieve a macroporous structure the functional monomer, the crosslinker and any co-monomers or co-crosslinkers used should be soluble in the porogenic solvent. As the polymerization reaction proceeds, this solubility will be lost and polymer will begin to precipitate.
  • a polymerisation initiator is provided in step b).
  • the polymerization initiator may be a thermal or photochemical initiator or a redox initiator.
  • Azo initiators and peroxides are preferred for thermal and photochemical polymerisation and are chosen to be soluble in the pre-polymerisation mixture with a half-life consistent with the preferred polymerisation temperature (0 0 C to 65°C typically).
  • the initiator is azobisisobutyronitrile.
  • Redox initiators are those commonly used to produce hydrogels and are typically persulfates, in combination with amines or Fe salts.
  • Ring-opening metathesis polymerisation (ROMP) initiation requires specific ROMP catalysts, such as Grubbs and Hoveyda-type catalysts and other functional group tolerant metathesis catalysts known in the literature.
  • the pre-determined functional group is a phosphate group or a phosphate monoester group.
  • the template molecule is phosphate-containing compound of formula (VI):
  • R 5 is a C 1-20 linear, branched, cyclic or polycyclic hydrocarbon, a dendron or dendrimer, a linear or branched polymer chain, a cyclodextrin or a polypeptide chain; and wherein Z + is a positively charged counter ion, for example an ammonium ion of formula N(R 6 ) 4"1" , wherein each occurrence of R 6 is independently a Ci -20 linear, branched or cyclic hydrocarbon or bulky group selected from a dendron or dendrimer, a linear or branched polymer chain, a cyclodextrin and a polypeptide chain.
  • modification of the template molecule with a large sacrificial substituent causes the template molecule itself to act as a pore forming agent.
  • Use of space generating substituents such as dendrons and dendrimers, linear and branched polymer chains, cyclodextrins, phosphorylated proteins even is made on the basis of the maximum size of analyte for which functional group selectivity is required.
  • such modified templates can be removed/degraded to facilitate their complete extraction from the FIP in step c) and maximise the loading level of the FIP at the same time.
  • R 5 is adamantyl
  • the functional monomer is a functional monomer as defined above in respect of the first aspect of the invention.
  • the porogenic solvent is CHCl 3 , water, a water/solvent mixture wherein the solvent is an alcohol, DMF, DMSO, N-Methyl-2-pyrrolidone (NMP), a water-miscible ionic liquid or acetonitrile or an aqueous buffer, for example TRIS, HEPES, with a background electrolytes (typically NaCl).
  • the solvent is an alcohol, DMF, DMSO, N-Methyl-2-pyrrolidone (NMP), a water-miscible ionic liquid or acetonitrile or an aqueous buffer, for example TRIS, HEPES, with a background electrolytes (typically NaCl).
  • porogenic solvent is related to the choice of crosslinker, functional monomer, template molecule, co-monomer and co-crosslinker.
  • the porogen provides solubility for all monomers, crosslinkers and the template molecule and is also compatible with the polymerisation mechanism. During polymerisation, the porogen generates phase separation so that large droplets of porogen are entrapped in a precipitating polymer matrix. These droplets are freed once the network is set and the porous structure has become permanent through cross-linking.
  • the cross-linker is an acrylate, a methacrylate, an acrylamide, a methacrylamide, a styrene, or a 1,2-disubstituted alkenes such as a maleimide or a vinyl ether.
  • the cross linker is ethylene glycol dimethacrylate.
  • the template molecule is removed in step c) by TFA extraction.
  • the present invention provides an imprinted polymer as produced by the second aspect of the invention.
  • the present invention provides a method of separating molecules which possess a pre-determined functional group from molecules which do not possess the pre-determined functional group, wherein the method comprises exposing a mixture of molecules to an imprinted polymer according to the first aspect of the invention or as produced by a method according to the second aspect of the invention.
  • the method is a chromatographic method and the imprinted polymer is used as a chromatographic stationary phase or in a solid-phase extraction mode.
  • all the molecules within the mixture of molecules are soluble in the same solvent, which is preferably an aqueous solvent of high polarity.
  • the method is a method of separating out phosphoproteins, phospholipids and/or phosphorylated metabolites from a biological sample.
  • Useful solvents during the analysis are those which dissolve the analyte and at the same time minimise non-specific interactions between the analyte and the polymer matrix.
  • the present invention provides a functional monomer for use in production of an imprinted polymer, the functional monomer having the structure:
  • A comprises (CH 2 ) n , wherein n is 0 or 1 ;
  • B comprises (a) a thiouronium group of formula -SC(NHR 2 )(NHR 3 ), bis(pyridine-2- ylmethyl)amino optionally substituted on one or both pyridine rings with Y, a (diacetic acid)amino group (for example l-(o-aminophenoxy)methane-N,N'-diacetic acid), l,2-bis(o-aminophenoxy)ethane-A r ,N,N' r A ⁇ '-tetraacetic acid, or an analogue, derivative or ring-fused variant thereof; (b) a ditopic thiouronium group of formula:
  • V disubstituted with (bis(pyridine-2-ylmethyl)amino), wherein V is an alkyl, haloalkyl, aryl, heteroaryl, naphthalene, anthracene or -(CH 2 )-W-(CH 2 )- group, ⁇ herein W is aryl, heteroaryl, naphthalene or anthracene; Ri comprises hydrogen or OR 4 ;
  • R 2 and R 3 are each independently selected from hydrogen, Ci -6 alkyl (preferably methyl) optionally substituted with Y, Ci -6 haloalkyl (preferably CF 3 or CH 2 CF 3 ), optionally substituted 5- or 6-membered aryl or heteroaryl (preferably phenyl); and R 4 is Ci -6 alkyl (preferably methyl) or
  • X comprises hydrogen or a polymerisable group
  • each occurrence of Y independently comprises hydrogen or a polymerisable group; provided that at least one occurrence of X or Y must be a polymerisable group.
  • V is -(CH 2 )- W-(CH 2 )-, wherein W is 1-, 3-, 5- substituted phenyl.
  • the functional monomer is as defined above in respect of the first aspect of the invention.
  • Figure 1 shows a schematic representation of a conventional molecular imprinting protocol. The individual steps shown are i) complexation in solution; ii) polymerisation; and iii) removal of template molecule.
  • Figure 2 shows a schematic representation of selective binding as exhibited by conventional imprinted polymers. As illustrated, the aim of conventional molecular imprinting is to achieve selectivity for one molecule over all other molecules.
  • Figure 3 shows a schematic representation of a general protocol for production of a functional group imprinted polymer according to the present invention. The individual steps shown are i) complexation in solution; ii) polymerisation; and iii) removal of template molecule.
  • FIG. 4 shows a schematic representation of functional group selectivity as exhibited by a functional group imprinted polymer (FIP) of the present invention.
  • FIP functional group imprinted polymer
  • Figure 5 shows a schematic representation of a protocol for production of a functional group imprinted polymer according to the present invention.
  • Figure 6 shows an experimental design used for comparing selectivity of an imprinted polymer of the invention with that of a conventional MIP.
  • NIP non-imprinted polymer
  • Figure 8 shows the analyte concentration of TBA 1-naphthyl phosphate and TBA 1- naphthyl acetate in (a) NIP and (b) FIP equilibrations.
  • Figures 9 and 10 show the relative values of a phosphorylated substrate and a carboxylated substrate removed during the wash step and extraction step of equilibration experiments.
  • the meanings of terms used herein are explained below, and the present invention is illustrated in detail by reference to the following non-limiting examples.
  • alkyl as used herein means a straight or branched alkyl group, preferably having 1 to 20 carbon atoms, more preferably having 1 to 6 carbons, most preferably being methyl.
  • haloalkyl as used herein means an alkyl group as substituted by one or more halogen (e.g. Cl 1 , Br, F, I).
  • a haloalkyl is a C 1-6 haloalkyl, for example -CF 3 or -CH 2 CF 3 .
  • 5-6 membered aryl as used herein means a monocyclic aryl group constituted by 5 or 6.
  • an aryl group is phenyl.
  • 5-6 membered heteroaryl used herein means a monocyclic heteroaryl group, in which the number of atoms forming the ring is 5 or 6 and wherein one or more ring atom is a heteroatom selected from the group consisting of a nitrogen atom, a sulphur atom and an oxygen atom, for example pyridine.
  • An aryl or heteroaryl as defined above is optionally substituted with one or more substituents preferably selected from methyl, -CF 3 , -CN or NO 2 , halogen (eg. F, Cl, Br or I) or -NH Z .
  • substituents preferably selected from methyl, -CF 3 , -CN or NO 2 , halogen (eg. F, Cl, Br or I) or -NH Z .
  • 'functional group' is used herein in accordance with its standard meaning in the art, i.e. a group of atoms within a molecule responsible for a chemical characteristic of that molecule. Where a molecule is referred to as comprising a functional group, it should be appreciated that the functional group does not necessarily stand alone, but may form part of a larger molecule. In the context of the present application, a 'functional group' may also be referred to as a 'chemical moiety'.
  • the term 'polymerisable group' refers to any functional group which is capable of reacting with another polymerisable group in a polymerisation reaction.
  • a polymerisable group may be one that is capable of undergoing free radical polymerization, for example an acrylate, methacrylate, acrylamide, methacrylamide or a vinyl group.
  • the polymerisable group may be a one that is capable of undergoing ring-opening metathesis polymerisation, for example a norbornene or other strained cyclic alkene derivative.
  • crosslinker' refers to any molecule that is capable of linking polymer chains. Linkage can be by covalent or non-covalent (e.g. ionic) bonding.
  • Reagents The following reagents were purchased from Aldrich Chemical Company, and used without further purification: anhydrous chloroform (>99% contains amylenes as stabilizer); ethylene glycol dimethacrylate (98%); 1-naphthyl phosphate (99%); tetrabutylammonium hydroxide-30-hydrate (98%); 4-vinylbenzyl chloride (90%).
  • 2,2'-Azo-bis-isobutyronitrile (>98%) was purchased from Aldrich Chemical Company and recrystallised from hexanes prior to use.
  • Tetrabutylammonium hydroxide 40 wt.% solution in water was purchased from Acros and used without further purification.
  • l-Methyl-3-phenyl-2 -thiourea was purchased from Fluorochem and used without further purification.
  • 2-Acetonaphthone was purchased from Fluka and used without further purification.
  • Trifluoroacetic acid (98%) was purchased from Apollo Scientific Ltd, and used without further purification.
  • Solvents All solvents (apart from those listed in the preceding paragraph) were obtained from BDH Laboratory Supplies and, unless otherwise specified, used without further purification. All water was purified by simple distillation prior to use.
  • HPLC HPLC was performed using a Laserchrom system equipped with degasser, tertiary gradient pump, variable wavelength UV detector, Phenomenex Synergi 4U max C1212 column and column heater (set to 35° C). Data was processed using Data Apex Clarity Lite software. For all experiments, volume of the injection loop was 20 ⁇ L and the flow rate set to 1 mL/ min. HPLC-grade solvents were used.
  • IR Infrared spectra were recorded on a Satellite FTIR spectrometer (manufactured by Mattson) using KBr plates.
  • MS High resolution mass spectra were recorded by the Imperial College mass spectrometry service using chemical ionisation with ammonium ions (NH 4 + ) or using electrospray mass spectrometry.
  • TLC Pre-coated silica gel F 254 / 366 Merck Kieselgel 60 A plates were used, and visualised using UV light (254 nm).
  • Melting Points Melting points were determined using a Sanyo Gallenkamp hot stage apparatus, and are uncorrected.
  • Elemental analysis was carried out by Stephen Boyer at the London Metropolitan University
  • 1-Adamantyl phosphoric acid was produced according to the teaching of B ⁇ hringer, H.; Vogt, H., Arch. Pharm. (Weinheim). 1977, 310, (11), 894-905.
  • ⁇ phenylisothiouronium chloride (2.0 eq, 0.211 mmol, 67.3 mg) were taken up in anhydrous CHCl 3 (600 ⁇ L).
  • the vial was temporarily sealed under an inert atmosphere using a rubber septum, and ethylene glycol dimethacrylate ⁇ EDMA> (30 eq to tetrabutylammonium 1-adamantyl phosphate, 3.18 mmol, 600 ⁇ L) and azobis- ' c obutyronitrile ⁇ AIBN> (2 mol% to all double bonds, 0.131 mmol, 21.5 mg) were added.
  • the polymer was dried in vacuo and then stirred in 5% v/v TFA in CHCl 3 (150 mL) at rt for 20 h. Following this, the polymer was once again dried in vacuo and washed by means of soxhlet extraction using 5% v/v acetic acid in CHCl 3 (100 mL) over 23 h (oil bath: 90-100°C). Finally, the polymer was further washed by means of soxhlet extraction using CHCl 3 (150 mL) over 67 h (oil bath: 90-100°C). The polymer was then dried in vacuo to afford a free- flowing, off-white solid (390 mg).
  • the protocol for preparing an imprinted polymer (FIP) of the invention is represented schematically in figure 5.
  • the polymer was dried in vacuo and then stirred in 5% v/v TFA in CHCl 3 (150 mL) at it for 20 h. Following this, the polymer was once again dried in vacuo and washed by means of soxhlet extraction using 5% v/v acetic acid in CHCl 3 (100 mL) over 23 h (oil bath: 90- 100° C). Finally, the polymer was further washed by means of soxhlet extraction using CHCl 3 (150 mL) over 67 h (oil bath: 90-100°C). The polymer was then dried in vacuo to afford a free-flowing, off-white solid (401 mg).
  • the bound substrate was then released by stirring the FIP and NIP in 5% v/v TFA in CHCl 3 (150 mL) for 27 h at rt. Afterwards the FIP and NIP particles were isolated via vacuum filtration, and washed through with CHCl 3 (50 mL). The filtrate was then concentrated in vacuo and dried in a vacuum oven (35-45°C, 36 h), and then analysed by HPLC. The relative value of each substrate removed during the substrate extraction step is shown in Figure 10.

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Abstract

La présente invention porte sur une nouvelle classe de polymères imprimés, appelés « polymères imprimés à groupes fonctionnels » (FIP), qui reconnaît sélectivement des molécules en raison de la présence d'un ou plusieurs groupes fonctionnels prédéterminés, indépendamment du type, de la classe, de la sous-structure ou de la dimension de la molécule.
PCT/GB2008/001681 2007-05-15 2008-05-15 Polymères imprimés à groupes fonctionnels WO2008139204A2 (fr)

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