Aflatoxin templates, molecularly imprinted polymers, and methods of making and using the same

Abstract

Molecularly imprinted polymers (MIPs) are materials exhibiting molecular recognition of a target molecule. MIPs are synthesized in the presence of an aflatoxin template, a mimic to the targeted molecule, used as an imprint that is further washed away with suitable solvent after completion of the polymerization process, leaving a cavity in the polymer of the same stereochemistry, functionality and morphology to the template. When the MIP encounters an aflatoxin, the molecule is bound in the cavity with a receptor-like affinity.

What is claimed is: 
     
         1 . An aflatoxin template having a Formula (I): 
       
         
           
           
               
               
           
         
         wherein R 1  is selected from H, C 1-6  alkyl, substituted C 1-6  alkyl, and a halo substituted C 1-6  alkyl; 
         R 2  is selected from halo, C 1-6  alkyl, substituted C 1-6  alkyl, a halo substituted C 1-6  alkyl, CH 2 C(O)OR′, and CH(C(O)OR′) 2 ; wherein R′ is selected from H, C 1-6  alkyl, and substituted C 1-6  alkyl; and 
         R 3  is selected from H, C 1-6  alkoxy, and substituted C 1-6  alkyl. 
       
     
     
         2 . The aflatoxin template of  claim 1 , wherein R′ further comprises substituents selected from a group consisting of halo, hydroxy and alkoxy. 
     
     
         3 . The aflatoxin template of  claim 1 , that is an isolated compound that has a Formula of: 
       
         
           
           
               
               
           
         
       
     
     
         4 . The aflatoxin template of  claim 1 , that is an isolated compound having a formula selected from the group consisting of: 
       
         
           
           
               
               
           
         
         and combinations thereof. 
       
     
     
         5 . A method of synthesis of an aflatoxin template having a Formula (I) comprising:
 reacting 3,5-dimethoxy phenol with ethyl 4-chloroacetoacetate in acid to form 4-(2-chloroethyl)-5,7-dimethoxy coumarin.   
     
     
         6 . A method of synthesis of an aflatoxin template comprising:
 suspending a monoacid according to a Formula of:   
       
         
           
           
               
               
           
         
         in polyphosphoric acid and heating to at least 50° C.; 
         cooling the reaction mixture below 50° C. and adding an aqueous solution to obtain an aflatoxin template according to a Formula of: 
       
       
         
           
           
               
               
           
         
       
     
     
         7 . The method of  claim 6 , wherein the monoacid is provided by suspending a diacid according to a Formula of: 
       
         
           
           
               
               
           
         
         in a solvent and heating to at least 100 to 140° C. 
       
     
     
         8 . A method of synthesis of an aflatoxin template compound comprising:
 deprotecting diethyl intermediate 2-((5,7-dimethoxy-2-oxo-2H-chromen-4-yl)methyl)malonate to form a diacid analog; and   precipitating the diacid analog to isolate the aflatoxin template according to a Formula of:   
       
         
           
           
               
               
           
         
       
     
     
         9 . A method of synthesis of  claim 8 , wherein the diethyl 2-((5,7-dimethoxy-2-oxo-2H-chromen-4-yl)methyl)malonate is prepared by a method comprising:
 combining 4-(2-chloroethyl)-5,7-dimethoxy coumarin with diethyl malonate, potassium iodide, and a crown ether in a polar solvent to form a mixture; and   adding potassium butoxide to the mixture to form diethyl 2-((5,7-dimethoxy-2-oxo-2H-chromen-4-yl)methyl)malonate.   
     
     
         10 . A method of synthesis of an aflatoxin template having a Formula (I) comprising:
 deprotecting diethyl 2-((5,7-dimethoxy-2-oxo-2H-chromen-4-yl)methyl)malonate to form a diacid analog, and precipitating the diacid analog of Formula:   
       
         
           
           
               
               
           
         
         Suspending the diacid analog in a solvent, heating to at least 100 to 140° C., and precipitating the monoacid of Formula: 
       
       
         
           
           
               
               
           
         
         Suspending the monoacid in an acid and heating to at least 50° C., cooling the reaction mixture to below 50° C., and adding aqueous solution to obtain a compound of Formula: 
       
       
         
           
           
               
               
           
         
       
     
     
         11 . A molecularly imprinted polymer intermediate comprising a complex of a crosslinked polymer made from a monomer and an aflatoxin template having Formula (I): 
       
         
           
           
               
               
           
         
         wherein R 1  is selected from H, C 1-6  alkyl, substituted C 1-6  alkyl, and a halo substituted C 1-6  alkyl; 
         R 2  is selected from halo, C 1-6  alkyl, substituted C 1-6  alkyl, a halo substituted C 1-6  alkyl, CH 2 C(O)OR′, and CH(C(O)OR′) 2 ; wherein R′ is selected from H, C 1-6  alkyl, and substituted C 1-6  alkyl; and 
         R 3  is selected from H, C 1-6  alkoxy, and substituted C 1-6  alkyl, or 
         wherein R 1  together with R 2  form a C 4-7  cycloalkyl ring, a halo substituted C 4-7  cycloalkyl ring, an oxo substituted C 4-7  cycloalkyl ring, C 4-7  cycloalkoxy ring a hydroxy substituted C 4-7  cycloalkyl ring and a carboxylic group substituted C 4-7  cycloalkyl; and R 3  is selected from H, C 1-6  alkoxy, and substituted C 1-6  alkyl. 
       
     
     
         12 . The molecularly imprinted polymer intermediate of  claim 11 , wherein the aflatoxin template to monomer ratio is from about 100:1 to 1:100. 
     
     
         13 . The molecularly imprinted polymer intermediate of  claim 11 , wherein the monomer to crosslinker ratio is from about 1:4.1 to 1:10. 
     
     
         14 . The molecularly imprinted polymer intermediate of  claim 11 , wherein the aflatoxin template of Formula (I) is selected from the group consisting of 4-(2-chloroethyl)-5,7-dimethoxy coumarin, 5,7-dimethoxycyclo pentenon[2,3-c]coumarin, and combinations thereof. 
     
     
         15 . A molecularly imprinted polymer comprising a crosslinked polymer made from a monomer, wherein the polymer has a plurality of cavities, wherein at least one of the cavities was made using the aflatoxin template having Formula (I): 
       
         
           
           
               
               
           
         
         wherein R 1  is selected from H, C 1-6  alkyl, substituted C 1-6  alkyl, and a halo substituted C 1-6  alkyl; 
         R 2  is selected from halo, C 1-6  alkyl, substituted C 1-6  alkyl, a halo substituted C 1-6  alkyl, CH 2 C(O)OR′, and CH(C(O)OR′) 2 ; wherein R′ is selected from H, C 1-6  alkyl, and substituted C 1-6  alkyl; and R 3  is selected from H, C 1-6  alkoxy, and substituted C 1-6  alkyl, or 
         wherein R 1  together with R 2  form a C 4-7  cycloalkyl ring, a halo substituted C 4-7  cycloalkyl ring, an oxo substituted C 4-7  cycloalkyl ring, C 4-7  cycloalkoxy ring a hydroxy substituted C 4-7  cycloalkyl ring and a carboxylic group substituted C 4-7  cycloalkyl; and R 3  is selected from H, C 1-6  alkoxy, and substituted C 1-6  alkyl. 
       
     
     
         16 . The molecularly imprinted polymer of  claim 15 , wherein aflatoxin template is 4-(2-chloroethyl)-5,7-dimethoxy coumarin. 
     
     
         17 . The molecularly imprinted polymer of  claim 15 , wherein aflatoxin template compound is 5,7-dimethoxycyclo pentenon[2,3-c]coumarin. 
     
     
         18 . The molecularly imprinted polymer of  claim 15 , wherein the monomer is methacrylic acid, 2-vinylpyridine, 2-hydroxyethylmethacrylate and combinations thereof. 
     
     
         19 . The molecularly imprinted polymer of  claim 15 , wherein the crosslinker is ethylene glycol dimethacrylate. 
     
     
         20 . The molecularly imprinted polymer of  claim 15 , wherein the aflatoxin template to monomer ratio is from about 100:1 to 1:100. 
     
     
         21 . The molecularly imprinted polymer of  claim 15 , wherein the monomer to crosslinker ratio is from about 1:4.1 to 1:10. 
     
     
         22 . A method of making a molecularly imprinted polymer comprising the steps of:
 providing an aflatoxin template having Formula (I):   
       
         
           
           
               
               
           
         
         wherein R 1  is selected from H, C 1-6  alkyl, substituted C 1-6  alkyl, and a halo substituted C 1-6  alkyl; 
         R 2  is selected from halo, C 1-6  alkyl, substituted C 1-6  alkyl, a halo substituted C 1-6  alkyl, CH 2 C(O)OR′, and CH(C(O)OR′) 2 ; wherein R′ is selected from H, C 1-6  alkyl, and substituted C 1-6  alkyl; and R 3  is selected from H, C 1-6  alkoxy, and substituted C 1-6  alkyl, or 
         wherein R 1  together with R 2  form a C 4-7  cycloalkyl ring, a halo substituted C 4-7  cycloalkyl ring, an oxo substituted C 4-7  cycloalkyl ring, C 4-7  cycloalkoxy ring a hydroxy substituted C 4-7  cycloalkyl ring and a carboxylic group substituted C 4-7  cycloalkyl; and R 3  is selected from H, C 1-6  alkoxy, and substituted C 1-6  alkyl; 
         combining the aflatoxin template with at least one monomer and one or more crosslinkers; 
         polymerizing the monomer and the one or more crosslinkers to form a molecularly imprinted polymer intermediate; and 
         removing the aflatoxin template from the molecularly imprinted polymer intermediate to form a molecularly imprinted polymer. 
       
     
     
         23 . The method of  claim 22 , wherein the combining of aflatoxin template with at least one monomer and one or more crosslinkers comprises mixing the monomer and the crosslinker in a solution of one or more organic solvents. 
     
     
         24 . The method of  claim 23 , wherein one or more solvents are selected from the group consisting of acetonitrile, toluene, cyclohexane, polyvinyl alcohol in water solution, and a mixture of two or more of acetonitrile, toluene, cyclohexane, polyvinyl alcohol in water solution. 
     
     
         25 . The method of  claim 22 , wherein the method further comprises adding an initiator. 
     
     
         26 . The method of  claim 25 , wherein the initiator is azo(bis)-isobutyronitrile (AIBN), wherein free radicals are formed by thermal decomposition of AIBN acting as an initiator. 
     
     
         27 . The method of  claim 26 , wherein said polymerization is initiated by forming free radicals in an organic solvent at a temperature between 55 and 110° C. 
     
     
         28 . The method of  claim 22 , wherein the aflatoxin template comprises 5,7-dimethoxy-cyclopentenon [2,3-c]coumarin, 4-(2-chloroethyl)-5,7-dimethoxy coumarin, or combinations thereof. 
     
     
         29 . The method of  claim 22 , wherein the removal of the aflatoxin template from the molecularly imprinted polymer intermediate comprises washing the molecularly imprinted polymer intermediate with a solvent. 
     
     
         30 . The method of  claim 34 , wherein said organic solvent is selected from the group of ethyl alcohol, methyl alcohol, acetonitrile, toluene, and a mixture of thereof. 
     
     
         31 . The method of  claim 22 , wherein said molecularly imprinted polymer is dried after said one or more washes. 
     
     
         32 . The molecularly imprinted polymer prepared by the process of  claim 22 . 
     
     
         33 . A method of sequestering an aflatoxin comprising:
 providing a molecularly imprinted polymer having a plurality of cavities, wherein at least one of the cavities is made using the aflatoxin template having Formula (I):   
       
         
           
           
               
               
           
         
         
           wherein R 1  is selected from H, C 1-6  alkyl, substituted C 1-6  alkyl, and a halo substituted C 1-6  alkyl; 
           R 2  is selected from halo, C 1-6  alkyl, substituted C 1-6  alkyl, a halo substituted C 1-6  alkyl, CH 2 C(O)OR′, and CH(C(O)OR′) 2 ; wherein R′ is selected from H, C 1-6  alkyl, and substituted C 1-6  alkyl; and R 3  is selected from H, C 1-6  alkoxy, and substituted C 1-6  alkyl, or 
           wherein R 1  together with R 2  form a C 4-7  cycloalkyl ring, a halo substituted C 4-7  cycloalkyl ring, an oxo substituted C 4-7  cycloalkyl ring, C 4-7  cycloalkoxy ring a hydroxy substituted C 4-7  cycloalkyl ring and a carboxylic group substituted C 4-7  cycloalkyl; and R 3  is selected from H, C 1-6  alkoxy, and substituted C 1-6  alkyl; 
         
         providing a material, wherein the material optionally contains an aflatoxin; and 
         contacting the molecularly imprinted polymer with the material. 
       
     
     
         34 . The method of  claim 33 , wherein the material is a liquid, a solid, or a gas. 
     
     
         35 . The method of  claim 34 , wherein the material is selected from the group consisting of soil, a spice, a beverage, a foodstuff, an animal feed, a pharmaceutical composition, a nutraceutical composition, and a cosmetic composition. 
     
     
         36 . The method of  claim 35 , wherein the material is milk. 
     
     
         37 . The method of  claim 33 , wherein the molecularly imprinted polymer is contacted with the material for at least 1 second. 
     
     
         38 . The method of  claim 33 , further comprising separating the molecularly imprinted material from the material. 
     
     
         39 . The method of  claim 33 , wherein the separation of the molecularly imprinted polymer comprises separating by filtration or by centrifugation. 
     
     
         40 . The method of  claim 33 , further comprising detecting an amount of aflatoxin complexed with the molecularly imprinted polymer, detecting the amount of aflatoxin in the material after contact with the molecularly imprinted polymer, or both. 
     
     
         41 . A method of sequestering an aflatoxin comprising steps of:
 a) providing a molecularly imprinted polymer having a plurality of cavities, wherein at least one cavity was made using an aflatoxin template having a Formula (I):   
       
         
           
           
               
               
           
         
         R 1  is selected from H, C 1-6  alkyl, substituted C 1-6  alkyl, and a halo substituted C 1-6  alkyl; 
         R 2  is selected from halo, C 1-6  alkyl, substituted C 1-6  alkyl, a halo substituted C 1-6  alkyl, CH 2 C(O)OR′, and CH(C(O)OR′) 2 ; wherein R′ is selected from H, C 1-6  alkyl, and substituted C 1-6  alkyl; and R 3  is selected from H, C 1-6  alkoxy, and substituted C 1-6  alkyl, or 
         wherein R 1  together with R 2  form a C 4-7  cycloalkyl ring, a halo substituted C 4-7  cycloalkyl ring, an oxo substituted C 4-7  cycloalkyl ring, C 4-7  cycloalkoxy ring a hydroxy substituted C 4-7  cycloalkyl ring and a carboxylic group substituted C 4-7  cycloalkyl; and R 3  is selected from H, C 1-6  alkoxy, and substituted C 1-6  alkyl; 
         b) providing a material containing an aflatoxin; and 
         c) contacting the molecularly imprinted polymer with the material, wherein the molecularly imprinted polymer sequesters at least 40 percent of the aflatoxin by weight per unit of the material. 
       
     
     
         42 . The method of  claim 41 , wherein the material is a liquid and the molecularly imprinted polymer sequesters at least 40 percent of the weight of aflatoxin per volume of the material.