Very large scale immobilized polymer synthesis

Abstract

A synthetic strategy for the creation of large scale chemical diversity. Solid-phase chemistry, photolabile protecting groups, and photolithography are used to achieve light-directed spatially-addressable parallel chemical synthesis. Binary masking techniques are utilized in one embodiment. A reactor system, photoremovable protective groups, and improved data collection and handling techniques are also disclosed. A technique for screening linker molecules is also provided.

1 . A reactor system for synthesizing a plurality of polymer sequences on a substrate comprising: 
 a) a reactor for contacting reaction fluids to said substrate;    b) a system for delivering selected reaction fluids to said reactor;    c) a translation stage for moving a mask or substrate from at least a first relative location relative to a second relative location;    d) a light for illuminating said substrate through a mask at selected times; and    e) an appropriately programmed digital computer for selectively directing a flow of fluids from said reactor system, selectively activating said translation stage, and selectively illuminating said substrate so as to form a plurality of diverse polymer sequences on said substrate at predetermined locations.    
     
     
         2 . The reactor system as recited in  claim 1  adapted to provide a plurality of monomers in a reaction fluid to said substrate, said substrate used for an initial screening of polymer sequences.  
     
     
         3 . An ordered method for forming a plurality of polymer sequences by sequential addition of reagents comprising the step of serially protecting and deprotecting portions of said plurality of polymer sequences for addition of other portions of said polymer sequences using a binary synthesis strategy.  
     
     
         4 . The method as recited in  claim 3  wherein said binary synthesis strategy is a binary masking strategy.  
     
     
         5 . The method as recited in  claim 4  wherein said masking strategy in which said masking strategy provides at least two consecutive steps in which a mask factors a previous mask by protecting a portion of a previously illuminated portions to light and exposing a portion of a previously protected portions to light.  
     
     
         6 . The method as recited in  claim 4  in which said masking strategy in which at least two successive steps in said masking strategy illuminate about one half of a region of interest on said substrate.  
     
     
         7 . The method as recited in  claim 4  wherein said masking strategy forms a plurality of polymer sequences on a single substrate.  
     
     
         8 . The method as recited in  claim 4  wherein said masks are arranged in a gray code masking scheme, said gray code masking scheme having one edge illumination on each of a plurality of synthesis sites.  
     
     
         9 . The method as recited in  claim 4  wherein said masking scheme results in a minimum number of masking steps for a number of polymers synthesized.  
     
     
         10 . The method as recited in  claim 4  wherein all possible polymers of length l are formed with a given basis set of monomers.  
     
     
         11 . The method as recited in  claim 4  wherein said masking strategy is developed in an appropriately programmed digital computer inputting at least a desired basis set, and length of polymers.  
     
     
         12 . The method as recited in  claim 4  wherein all possible polymers of a length less than or equal to 1 are formed with a given basis set of monomers.  
     
     
         13 . The method as recited in  claim 4  further comprising the step of forming a portion of said polymers with a non-binary masking scheme.  
     
     
         14 . The method as recited in  claim 10  further comprising the step of outputting a masking strategy.  
     
     
         15 . The method as recited in  claim 10  further comprising the step of outputting a map of synthesized polymers on said substrate.  
     
     
         16 . The method as recited in  claim 15  wherein said map is in the form of  FIG. 10 .  
     
     
         17 . A method of screening a plurality of linker polymers for use in binding affinity studies comprising the steps of: 
 a) forming a plurality of linker polymers on a substrate in selected regions, said linker polymers formed by the steps of recursively: 
 i) on a surface of a substrate, irradiating a portion of said selected regions to remove a protective group; and  
 ii) contacting said surface with a monomer;  
   b) contacting said plurality of linker polymers with a ligand; and    c) contacting said ligand with a labeled receptor.    
     
     
         18 . The method as recited in  claim 17  wherein said ligand is a polypeptide.  
     
     
         19 . The method as recited in  claim 17  wherein said receptor is an antibody.  
     
     
         20 . The method as recited in  claim 17  wherein said monomers added in step ii) are the same in each of said recursive steps, said selected regions comprising linker molecules of different lengths.  
     
     
         21 . The method as recited in  claim 17  wherein said labelled receptor is a fluoresceinated receptor.  
     
     
         22 . A system for determining affinity of a receptor to a ligand comprising: 
 a) means for applying light to a surface of a substrate, said substrate comprising a plurality of ligands at predetermined locations, said means for applying directing light providing simultaneous illumination at a plurality of said predetermined locations; and    b) an array of detectors for detecting light fluoresced at said plurality of predetermined locations.    
     
     
         23 . A system as recited in  claim 22  wherein said means for applying light comprises a point light source and a cylindrical lens for focusing said point light source along a substantially linear path.  
     
     
         24 . A system as recited in  claim 22  wherein said array of detectors comprises a linear array.  
     
     
         25 . A system as recited in  claim 22  wherein said array of detectors comprises a linear CCD array.  
     
     
         26 . In a digital computer, a method of determining the tendency of a receptor to bind to a ligand comprising: 
 a) exposing fluorescently labelled receptors to a substrate, said substrate comprising a plurality of ligands in regions at known locations;    b) at a plurality of data collection points within each of said regions, determining an amount of light fluoresced from said data collection points;    c) removing said data collection points deviating from a preset amount from a predetermined statistical distribution; and    d) determining a relative binding affinity of said receptor to remaining data collection points.    
     
     
         27 . The method as recited in  claim 26  wherein said predetermined statistical distribution is a normal distribution.  
     
     
         28 . A compound having the formula:  
       
         
           
           
               
               
           
         
       
       wherein n=0 or 1; Y is selected from the group consisting of an oxygen of the carboxyl group of a natural or unnatural amino acid, an amino group of a natural or unnatural amino acid, or the C-5′ oxygen group of a natural or unnatural deoxyribonucleic or ribonucleic acid; R 1  and R 2  independently are a hydrogen atom, a lower alkyl, aryl, benzyl, halogen, hydroxyl, alkoxyl, thiol, thioether, amino, nitro, carboxyl, formate, formamido, sulfido, or phosphido group; and R 3  is a alkoxy, alkyl, aryl, hydrogen, or alkenyl group.  
     
     
         29 . The compound of  claim 28  wherein Y is the C-5′ oxygen group of a natural or unnatural deoxyribonucleic or ribonucleic acid.  
     
     
         30 . The compound of  claim 29  wherein n=0.  
     
     
         31 . The compound of  claim 29  wherein R 1  and R 2  are each a hydrogen atom.  
     
     
         32 . The compound of  claim 31  wherein R 3  is a hydrogen atom.  
     
     
         33 . The compound of  claim 31  wherein R 3  is a methyl group.  
     
     
         34 . The compound of  claim 28  wherein Y is an oxygen of the carboxyl group of an amino acid and n=0.  
     
     
         35 . The compound of  claim 34  wherein R 1  and R 2  are each a hydrogen atom.  
     
     
         36 . The compound of  claim 35  wherein R 3  is a hydrogen atom.  
     
     
         37 . The compound of  claim 35  wherein R 3  is a methyl group.  
     
     
         38 . A compound having the formula:  
       
         
           
           
               
               
           
         
       
       wherein n=0 or 1; Y is selected from the group consisting of an amino group of a natural or unnatural amino acid or the C-5′ oxygen group of a natural or unnatural deoxyribonucleic and ribonucleic acid; R 1 , R 2 , and R 3  independently are a hydrogen atom, a lower alkyl, aryl, benzyl, halogen, hydroxyl, alkoxyl, thiol, thioether, amino, nitro, carboxyl, formate, formamido, sulfido or phosphido group; R 4  and R 5  independently are a alkoxy, alkyl, hydrogen, halo, aryl, hydrogen, or alkenyl group.  
     
     
         39 . The compound of  claim 38  wherein R 1  through R 3  are each a hydrogen atom.  
     
     
         40 . The compound of  claim 39  wherein R 4  and R 5  are each a hydrogen atom.  
     
     
         41 . The compound of  claim 39  wherein R 4  and R 5  are each a methyl group.  
     
     
         42 . A compound having the formula:  
       
         
           
           
               
               
           
         
       
       wherein n=0 or 1; Y is a C-5′ oxygen group of a natural or unnatural deoxyribonucleic and ribonucleic acid; R 1  through R 4  independently are a hydrogen atom, a lower alkyl, aryl, benzyl, halogen, hydroxyl, alkoxyl, thiol, thioether, amino, nitro, carboxyl, formate, formamido, sulfido, or phosphido group; and R 5  is a alkoxy, alkyl, aryl, or alkenyl group.  
     
     
         43 . The compound of  claim 42  wherein R 2  and R 3  are each a methoxy group.  
     
     
         44 . The compound of  claim 43  wherein R 1  and R 4  are each a hydrogen atom.  
     
     
         45 . The compound of  claim 44  wherein R 5  is a methyl group.  
     
     
         46 . A compound having the formula:  
       
         
           
           
               
               
           
         
       
       wherein n=0 or 1; Y is an atom to be protected; R 1  and R 2  independently are a hydrogen atom, a lower alkyl, aryl, benzyl, halogen, hydroxyl, alkoxyl, thiol, thioether, amino, nitro, carboxyl, formate, formamido, sulfido, or phosphido group; and R 3  is a alkoxy, alkyl, aryl, or alkenyl group.  
     
     
         47 . The compound of  claim 46  wherein Y is selected from the group consisting of an oxygen of the carboxyl group of a natural or unnatural amino acid, or the C-5′ oxygen group of a natural or unnatural deoxyribonucleic or ribonucleic acid, or the amino group of a natural or unnatural amino acid.  
     
     
         48 . The compound of  claim 47  wherein R 1  and R 2  are hydrogen.  
     
     
         49 . The compound of  claim 48  wherein R 3  is a methyl group.  
     
     
         50 . A compound having the formula:  
       
         
           
           
               
               
           
         
       
       where R is a side chain of a natural or unnatural amino acid and X is a photoremovable protecting group.  
     
     
         51 . The compound of  claim 50  wherein X has the following formula:  
       
         
           
           
               
               
           
         
       
       where R 1 , R 2 , R 3  and R 4  independently are a hydrogen atom, a lower alkyl, aryl, benzyl, halogen, hydroxyl, alkoxyl, thiol, thioether, amino, nitro, carboxyl, formate, formamido or phosphido group, or adjacent substituents are substituted oxygen groups that together form a cyclic acetal or ketal; and R 5  is a hydrogen atom, a alkoxyl, alkyl, hydrogen, halo, aryl, or alkenyl group.  
     
     
         52 . The compound of  claim 51  wherein R 1  and R 4  are each a hydrogen atom, and R 2  and R 3  are each a methoxy group.  
     
     
         53 . The compound of  claim 52  wherein R 5  is a methyl group.  
     
     
         54 . The compound of  claim 51  wherein R 2  and R 3  are substituted oxygen groups that together form a cyclic acetal.  
     
     
         55 . The compound of  claim 54  wherein R 1  and R 4  are each a hydrogen atom.  
     
     
         56 . The compound of  claim 55  wherein R 5  is a methyl group.