WO2002001223A1

WO2002001223A1 - Substructure approach to molecularly imprinted polymers with high selectivity for folic acid and analogues

Info

Publication number: WO2002001223A1
Application number: PCT/SE2001/001483
Authority: WO
Inventors: Börje SELLERGREN; Milena Quaglia; Karine Chenon
Original assignee: MIP Technologies AB
Current assignee: MIP Technologies AB
Priority date: 2000-06-28
Filing date: 2001-06-28
Publication date: 2002-01-03
Anticipated expiration: 2002-12-28
Also published as: AU2001267970A1; SE0002463D0

Abstract

The invention refers to templates and functional monomers that can be used to generate molecularly imprinted polymers for the selective recognition of folic acid and its analogues.

Description

SUBSTRUCTURE APPROACH TO MOLECULARLY IMPRINTED POLYMERS WITH HIGH SELECTIVITY FOR FOLIC ACID AND ANALOGUES

In the fields of medical-, dietary-, environmental- and chemical sciences there is an increasing need for the selective separations of specific substances in complex mixtures of related substances. The end goal can be the preparative isolation of a certain compound or compounds, their selective removal from various environments or measurements of their concentration. Molecularly imprinted polymers (MIPs) exhibit often a high selectivity towards their substrate in analogy the antibody-antigen complementarit . ^ The technique shows promise in chiral separations of for example amino acid derivatives, peptides, phosphonates, aminoalcohols and beta-blocking compounds, affinity chromatography of nucleotides and the DNA-bases as well as substitute for antibodies in immunoassays or extractions of commercial drugs.² Molecular imprinting (MI) consists of the following key steps (Figure 1) : (1) Functional monomers are allowed to interact reversibly with a template molecule in solution. (2) The hereby formed template assemblies are compolymerized with a crosslinking monomer resulting in a crosslinked network polymer. (3) The template is displaced and the materials can be used for selective molecular recognition of the corresponding compound. If these are crushed and sieved they can be packed in a chromatographic column and used for chromatographic separation of the template from structurally related analogs. Analytical as well as preparative applications are here possible. Preparative applications can be separation of a compound from a complex mixture of structurally related compounds and isolation of the compound. This can be through an affinity chromatographic procedure where pH, ion strength or solvent gradients can be used in order to control the strength of interaction with the stationary phase. The separation can target enantiomers or diastereomers in a mixture of enantiomers or diastereomers of one or many compounds. Analytical applications can in addition to the above mentioned separations be: competetitive binding assays, chemical sensors or selective sample enrichments .¹

Currently the most widely applied technique to generate molecularly imprinted binding sites is represented by the noncovalent route.3 This makes use of noncovalent self-assembly of the template with functional monomers prior to polymerization, free radical polymerization with a crosslinking monomer and then template extraction followed by rebinding by noncovalent inter- actions. Although the preparation of a MIP by this method is technically simple it relies on the success of stabilisation of the relatively weak interactions between the template and the functional monomers. Stable monomer-template assemblies will in turn lead to a larger concentration of high affinity binding sites in the resulting polymer. The materials can be synthesized in any standard equipped laboratory in a relatively short time and some of the MIPs exhibit binding affinities and selectivities in the order of those exhibited by antibodies towards their antigens. Most MIPs are synthesized by free radical polymerization of functional monounsaturated (vinylic, acrylic, methacrylic) monomers and an excess of crosslinking di- or tri- unsaturated (vinylic, acrylic, methacrylic) monomers resulting in porous organic network materials. These polymerizations have the advantage of being relatively robust allowing polymers to be prepared in high yield using different solvents (aqueous or organic) and at different temperatures. This is necessary in view of the varying solubilities of the template molecules.

The most successful noncovalent imprinting systems are based on commodity acrylic or methacrylic monomers, such as methacrylic acid (MAA) , crosslinked with ethyleneglycol dimethacrylate (EDMA) . Initially, derivatives of amino acid enantiomers were used as templates for the preparation of imprinted stationary phases for chiral separations (MICSPs) but this system has proven generally applicable to the imprinting of templates allowing hydrogen bonding or electrostatic interactions to develop with MAA.⁴ The procedure has been applied to the imprinting with L-phenylalanine anilide (L-PA) . In the first step, the template (L-PA) , the functional monomer (MAA) and the crosslinking monomer (EDMA) are dissolved in a poorly hydrogen bonding solvent (diluent) of low to medium polarity. The free radical polymerization is then initiated with an azo initiator, commonly azo-N,N' -bis-isobutyronitrile (AIBN) either by photochemical homolysis below room temperature or thermochemically at 60°C or higher. In the final step, the resultant polymer is crushed by mortar and pestle or in a ball mill, extracted by a Soxhlet apparatus, and sieved to a particle size suitable for chromatographic

(25-38 μm) or batch (150-250 μm) applications. The polymers are then evaluated as stationary phases in chromatography by comparing the retention time or capacity factor (k' ) of the template with that of structurally related analogs.

In spite of the fact that the MIPs can recognize a large number of structures used as templates, a number of compounds classes are only poorly recognized by polymers prepared using the present imprinting protocols . This is the case of several biomolecules i.e. vitamins, antibiotics, cofactors, proteins, peptides, several of which is soluble only in aqueous solvents, not suited for molecular imprinting with the above protocol . Furthermore the binding strength between the functional monomer and the template is often insufficient leading to a low sample load capacity and a significant nonspecific binding. There is therefore a need for the development of new approaches including the use of specially designed template analogues and functional monomers binding stronger to the template and allowing recognition of new compound classes . For instance monomers designed to bind carboxylic-, phosphoric- or phosphonic- acid templates are needed.

This invention describes the synthesis of a new class of molecularly imprinted polymers capable of recognizing folic acid and its analogues (Figure 2) . For this purpose specially designed templates and functional monomers have been used. In order to synthesise polymers with a high selectivity and affinity for folic acid and its analogues, the poor solubility and stability of these templates require alternative approaches to be developed. Thus substructures of these compounds may be targeted using organic soluble analogues or newly designed monomers . Particularly strong binding was obtained when using methacrylic acid as the functional monomer and organic soluble diaminopteridine analogues as templates (Figure 3) , thus targeting the pteridine ring system.

These analogues can be 2 , 4-diamino-6, 7-diisopropylpteri- dine (DIP) , trimethopriim (TRP) or trimetrexate (TRX) . In a separate approach targeting the glutamic acid substructure of folic acid and analogues thereof, N-Cbz- glutamic acid was used as template in combination with a new class of amidinebased functional monomers (Figure 4) . These approaches resulted in polymers showing high selectivity for the target compounds. The polymers were evaluated by HPLC by injecting the template methotrexate, leucovorine and folic acid. After a detailed mobile phase optimization, systems were found allowing strong and highly selective retentions in aqueous mobile phases .

The invention will now be described in more detail giving a number of nonrestricting examples: Example 1

The polymer is synthesized by free radical polymerization of a mixture of methacrylic acid or other functional monomer, and a crosslinking monomer, that can be ethyleneglycoldimethacrylate or trimethylolpropane- trimethacrylate, in presence of a solvent and a template and an initiator, that can be azobisisobutyronitrile. The template can be 2 , 4-diamino-6, 7-diisopropylpteridine (DIP) , trimethoprim (TRP) or trimetrexate (TRX) . (Figure 3) After polymerization the polymer is freed from the template by a washing procedure and can then be used for selective separations.

Example 2

The polymer is synthesized by free radical polymerization of a mixture of a formamidine or any of the functional monomers in Figure 4, targeting the glutamic acid side chain, and a crosslinking monomer, that can be ethyleneglycoldimethacrylate or trimethylolpropanetrimeth- acrylate, in presence of a solvent and a template and an initiator, that can be azobisisobutyronitrile. The template can be Glutamic acid or an analogue thereof, folic acid, methotrexate or leucovorine or analogues of these. After polymerization the polymer is freed from the template by a washing procedure and can then be used for selective separations. Example 3

The polymer prepared according to Example 1 and 2 can be used for separation of enantiomers or diastereomers of the template or for separation of the template or template analogues from structurally related compounds. This can be done by chromatography, capillary electrophoresis, capillary electrochromatography, batch modes or membrane modes . The polymer can further be used for catalysing chemical reactions such as esterolysis, amidolysis, ester synthesis or amide synthesis or used in chemical sensors . Litterature

1. Bartsch, R.A. & Maeda, M. in ACS Symposium Series 703 (Oxford University Press, Washington, 1998) .

2. Andersson, L.I., Mύller, R. , Vlatakis, G. & Mosbach, K. Proc . Natl . Acad . Sci . U. S . A . 92, 4788-92 (1995) .

3. Sellergren, B., Lepistoe, M. & Mosbach, K. J^". Am . Chem. Soc . 110, 5853-60 (1988) .

4. Sellergren, B. in A practical approach to chiral separation by liquid chromatography (ed. Subramanian, G.) 69-93 (VCH, Weinheim, 1994) .

Claims

1. A polymer selective for folic acid or analogues thereof .

2. A polymer according to claim 1 characterised in that it is synthesised in presence of pteridine analogues .

3. A polymer according to claim 1 characterised in that it is synthesised in presence of glutamic acid or derivatives thereof.

4. A polymer according to claim 1 characterised in that it is synthesised in presence of amidinebased functional monomers.