Methods and processes for attaching compounds to matrices

Abstract

The present invention describes extremely rapid and efficient methods for the attachment of chemical moieties to matrices by the use of microwave technology. The methods of the invention can be applied in a variety of ways for the preparation of different types of matrices for a variety of applications including but not limited to the functionalization of various solid supports, and matrices in the form of powder, beads, sheets, and other suitable surfaces for use in applications including but not limited to oligonucleotide synthesis, peptide synthesis, environmental clean up (removal of toxic materials), immunoassays, affinity chromatography, combinatorial chemistry, microarrays, proteomics and medical diagnostics.

1 . A method for attaching a chemical moiety to a matrix comprising the steps of: 
 (a) contacting the matrix with a reagent capable of adding a nucleophilic group;    (b) exposing the reaction mixture of step (a) to microwave radiation thereby resulting in a functionalized matrix;    (c) contacting the functionalized matrix of step (b) with a reagent capable of forming an ester or amide bond with the matrix and further comprising a free carboxyl termini on the matrix;    (d) exposing the reaction mixture of step (c) to microwave radiation thereby forming a mono-ester or mono-amide linkage with the matrix comprising a free carboxyl termini on the matrix; and    (e) coupling the carboxylated matrix of step (d) with the chemical moiety via a reative region of the chemical moiety capable of reacting with the carboxylated matrix thereby resulting in a matrix functionalized with the chemical moiety.    
   
   
       2 . The method of  claim 1  wherein the contacting of steps (a) and (c) are carried out in the presence of a solvent having a dielectric constant.  
   
   
       3 . The method of  claim 1  wherein the matrix is selected from the group consisting of: controlled pore glass; glass beads; glass powders; silica gels; alumina; 
 substituted or unstubstituted polystyrene; polyethylene glycol; cellulose, ceramics, zeolite, clay, titanium (Ti), Carbon, silicon (Si), and gold.    
   
   
       4 . The method of  claim 1  wherein the chemical moiety is selected from the group consisting of: modified and unmodified nucleotides and nucleosides; DNA; 
 RNA; amino acids; peptides; proteins; synthetic block polymers; small molecules; and organometallic synthesis reagents.    
   
   
       5 . A method for preparing a functionalized matrix for oligonucleotide synthesis comprising the steps of: 
 (a) contacting the matrix with a reagent capable of adding an amino functional group to the matrix;    (b) exposing the reaction mixture of step (a) to microwave radiation thereby resulting in an amino-functionalized matrix;    (c) contacting the amino-functionalized matrix of step (b) with a succinylating reagent capable of chemically succinylating the matrix;    (d) exposing the reaction mixture of step (c) to microwave radiation thereby resulting in a succinylated matrix; and    (e) coupling the succinylated matrix with a nucleoside capable of reacting with the succinylated matrix thereby forming a functionalized matrix suitable for further use in the synthesis of oligonucleotides.    
   
   
       6 . The method of  claim 5  wherein the contacting of step (a) is carried out in the presence of a solvent having a dielectric constant.  
   
   
       7 . The method of  claim 5  wherein the reagent of step (a) capable of adding an amino functional group is an aminoalkylsilane.  
   
   
       8 . The method of  claim 6  wherein the solvent is dimethylformamide, dimethyl acetamide, N,N-dialkyl formamides and acetamides, N-methyl pyrrolidone, and DMSO.  
   
   
       9 . The method of  claim 5  wherein the succinylating reagent is a substituted or unsubstituted dicarboxylic acid or their corresponding anhydrides, or any reagent capable of forming a mono-ester linkage with the matrix and having a free carboxyl termini.  
   
   
       10 . The method of  claim 9  wherein the succinylating reagent is succinic anhydride.  
   
   
       11 . The method of  claim 5  further comprising the step of recovering excess nucleoside generated in the coupling step (e) by aqueous work up of the filtrate.  
   
   
       12 . The method of  claim 5  wherein the matrix is selected from the group consisting of: controlled pore glass; glass beads; glass powders; silica gels; alumina; 
 substituted or unstubstituted polystyrene; polyethylene glycol; cellulose, ceramics, zeolite, clay, titanium (Ti), Carbon, silicon (Si), and gold.    
   
   
       13 . The method of claim of  claim 5  wherein the matrix is controlled pore glass.  
   
   
       14 . The method of  claim 5  wherein the nucleoside derivative is a 5′-protected nucleoside derivative.  
   
   
       15 . The method of  claim 14  wherein the 5′ nucleoside derivative is 5′-dimethoxytrityl-protected nucleoside with a free 3′ hydroxyl group.  
   
   
       16 . The method of  claim 5  wherein the functionalized nucleoside matrix comprises a loading of nucleoside derivative in the range of about 60-100 micromoles of nucleoside derivative per gram of matrix.  
   
   
       17 . The method of  claim 5  wherein the amino-functionalized matrix of step (b) comprises a loading of amino group in the range of about 60-120 micromole of amino group per gram of matrix.  
   
   
       18 . The method of  claim 5  wherein the contacting of step (a) comprises contacting the matrix with at least two different reagents capable of adding amino functional groups to the matrix.  
   
   
       19 . A method of preparing a functionalized matrix for oligonucleotide synthesis comprising the steps of: 
 (a) contacting the matrix with an aminoalkylsilane reagent in the presence of a solvent having a dielectric constant and exposing the reaction mixture to microwave radiation thereby resulting in an amino-functionalized matrix;    (b) reacting the matrix of step (a) with succinic anhydride in the presence of dimethylformamide and exposing the reaction mixture to microwave radiation thereby forming a succinylated matrix; and    (c) contacting the succinylated matrix of step (b) with a 5′ dimethoxytrityl-protected nucleoside derivative thereby forming a functionalized matrix suitable for further use in oligonucleotide synthesis.    
   
   
       20 . A method of preparing a functionalized matrix for oligonucleotide synthesis comprising the steps of: 
 (a) contacting the matrix with an aminoalkylsilane reagent in the presence of a solvent having a dielectric constant and exposing the reaction mixture to microwave radiation thereby resulting in an amino-functionalized matrix;    (b) reacting the amino-functionalized matrix of step (a) with phenyldiisothiocyanate and exposing the reaction mixture to microwave radiation in the presence of solvent thereby converting the amino groups on the matrix to thiourea groups whereby the matrix is further functionalized with a thioisocyanate terminus;    (c) contacting the matrix of step (b) with polyamidoamine and exposing the reaction mixture to microwave radiation in the presence of a solvent with a dielectric constant thereby resulting in a matrix with multiple amino sites;    (d) reacting the matrix of step (c) with succinic anhydride in the presence of dimethylformamide and exposing the reaction mixture to microwave radiation thereby forming a succinylated matrix;    (e) contacting the succinylated matrix of step (d) with a 5′ dimethoxytrityl-protected nucleoside derivative thereby forming a functionalized matrix suitable for further use in oligonucleotide synthesis.    
   
   
       21 . A method of preparing a functionalized matrix for oligonucleotide synthesis comprising the steps of: 
 (a) contacting native CPG with bifunctional isocyanate or thioisocyanate reagent to generate CPG with a carbamate or thiocarbamate linkage with a terminal isocyanate or thioisocyanate moiety;    (b) contacting the matrix from step (a) with polyamidoamine (PAMAM) to generate CPG with multiple amino groups;    (c) reacting the matrix of step (b) with succinic anhydride in the presence of dimethylformamide and exposing the reaction mixture to microwave radiation thereby forming a succinylated matrix; and    (d) contacting the succinylated matrix of step (c) with a 5′ dimethoxytrityl-protected nucleoside derivative thereby forming a functionalized matrix suitable for further use in oligonucleotide synthesis.    
   
   
       22 . A method of preparing a functionalized matrix for oligonucleotide synthesis comprising the steps of: 
 (a) contacting native CPG with at least one activating group selected from p-nitrophenyl, choloroformate, or carbonyldimidazole, to form the corresponding activated groups amenable to further displacement by PAMAM;    (b) reacting the matrix of step (a) with succinic anhydride in the presence of dimethylformamide and exposing the reaction mixture to microwave radiation thereby forming a succinylated matrix; and    (c) contacting the succinylated matrix of step (b) with a 5′ dimethoxytrityl-protected nucleoside derivative thereby forming a functionalized matrix suitable for further use in oligonucleotide synthesis.    
   
   
       23 . A functionalized matrix for oligonucleotide synthesis prepared by a process comprising the steps of: 
 (a) contacting the matrix with a reagent comprising an amino group capable of functionalizing the matrix;    (b) exposing the matrix and the reagent comprising an amino group to microwave radiation thereby resulting in an amino-functionalized matrix;    (c) contacting the amino-functionalized matrix with a succinylating reagent capable of chemically succinylating the matrix;    (d) exposing the amino-functionalized matrix and the succinylating reagent to microwave radiation thereby resulting in a succinylated matrix; and    (e) contacting the succinylated matrix with a nucleoside capable of reacting with the succinylated matrix thereby forming a functionalized matrix suitable for further use in the synthesis of oligonucleotides.    
   
   
       24 . An oligonucleotide attached to the functionalized matrix of  claim 23 .  
   
   
       25 . The method of  claim 1  wherein the reaction mixtures of steps (b) and (d) are exposed to microwave radiation for a total time of at least about 4 minutes  
   
   
       26 . The method of  claim 5  wherein the reaction mixtures of steps (b) and (d) are exposed to microwave radiation for a total time of at least about 4 minutes.  
   
   
       27 . The method of  claim 21  wherein the reaction mixtures of step (a), (b), (c) and (d) are exposed to microwave radiation for a total time of at least about 4 minutes.  
   
   
       28 . The functionalized matrix of  claim 23  wherein the reaction mixtures of steps (b) and (d) are exposed to microwave radiation for a total time of at least about 4 minutes.  
   
   
       29 . A method for attaching a chemical moiety to a matrix comprising the steps of: 
 (a) contacting the matrix with a reagent capable of adding thiol ester, a thioamide a sulfonamide a sulfonate ester a phosphoramide or phosphoric ester on the matrix;    (b) exposing the reaction mixture of step (a) to microwave radiation thereby resulting in a functionalized matrix with at least one free carboxyl termini;    (c) contacting the functionalized matrix of step (b) with a reagent capable of forming an ester or amide bond with the matrix and having a free carboxyl termini on the matrix;    (d) exposing the reaction mixture of step (c) to microwave radiation thereby forming a mono-ester or mono-amide linkage with the matrix having free carboxyl termini; and    (e) contacting the carboxylated matrix of step (d) with the chemical moiety via a functionalized region of the chemical moiety capable of reacting with the carboxylated matrix, thereby resulting in a matrix functionalized with the chemical moiety.    
   
   
       30 . A method for rapid deprotection and cleavage of oligonucleotide assembled on a solid support comprising the steps of: 
 (a) taking up support-bound oligonucleotide in a heavy-walled container with a stopper;    (b) adding alkali such as NaOH of strength <0.2 N, but preferably 0.1 N NaOH in;    (c) exposing the contents to microwave radiation in 10 to 15 second cycles;    (d) maintaining outside temperature of the container at 90 to 95° C. while heating and 75 to 80° C. while cooling during each cycle;    (e) isolating the product by neutralization and filtration; and    (f) separating the support for recycling;    
   
   
       31 . The method of  claim 30  wherein the separating step (f) further comprises employing the recycled support as matrix for rapid attachment of a chemical moiety selected from nucleic acids, proteins, antibodies, carbohydrates and other macromolecules for uses in applications selected from: peptide and carbohydrate synthesis and environmental clean up (removal of toxic materials), RIAs, FIAs, ELISA, and Affinity Chromatography.