Massively parallel synthesis of biopolymeric arrays

Abstract

Methods for fabricating dense arrays of polymeric molecules in a highly multiplexed manner are provided using semiconductor-processing-derived lithographic methods. Advantageously, the methods are adaptable to the synthesis of a variety of polymeric compounds. For example, arrays of branched peptides and polymers joined by peptide bonds may be fabricated in a highly multiplexed manner.

1 . A method for making an array of polymers comprising, 
 attaching to a substrate surface a first molecule capable of forming a peptide bond wherein the molecule contains a protecting group that prevents the formation of a peptide bond,    depositing a photosensitive layer over the substrate surface wherein the photosensitive layer contains a photo-active compound that upon activation generates a second compound capable of causing the removal of the protecting group,    exposing a portion of the substrate surface to ultraviolet radiation wherein ultraviolet radiation exposure causes the removal of protecting groups,    removing the photosensitive layer, and    coupling a second molecule capable of forming two or more peptide bonds, wherein the molecule contains two or more different protecting groups capable of preventing the formation of a peptide bond, to the first molecule capable of forming a peptide bond that has been deprotected.    
   
   
       2 . The method according to  claim 1  also including heating the substrate after exposing a portion of the substrate surface to ultraviolet radiation.  
   
   
       3 . The method according to  claim 1  also including capping unreacted peptide bond-forming sites on the first molecule capable of forming a peptide bond after coupling the second molecule capable of forming a peptide bond.  
   
   
       4 . The method according to  claim 1  wherein attaching is accomplished through the formation of a peptide bond.  
   
   
       5 . The method according to  claim 1  wherein the second compound capable of causing the removal of the protecting group is a photogenerated acid or base.  
   
   
       6 . The method according to  claim 1  wherein the second compound capable of causing the removal of the protecting group is a photogenerated acid and the photo-active compound is selected from the group consisting of sulfonium salts, halonium salts, and polonium salts.  
   
   
       7 . The method according to  claim 1  wherein the substrate surface to which the first molecule capable of forming a peptide bond is attached is an amino-functionalized SiO 2  surface.  
   
   
       8 . The method according to  claim 7  wherein the substrate is comprised of silicon having a layer of SiO 2  on the surface.  
   
   
       9 . The method according to  claim 1  wherein the photosensitive layer comprises a polymer, a photo-active compound, and a solvent.  
   
   
       10 . The method according to  claim 1  wherein the photosensitive layer additionally includes a photosensitizer.  
   
   
       11 . The method according to  claim 10  wherein the photosensitizer is selected from the group consisting of benzophenones, thioxanthenones, anthraquinone, fluorenone, acetophenone, and perylene.  
   
   
       12 . The method of  claim 1  wherein one or more of the molecules capable of forming a peptide bond are selected from the group consisting of natural and unnatural amino acids.  
   
   
       13 . The method according to  claim 1  additionally including 
 depositing a photosensitive layer over the substrate surface wherein the photosensitive layer contains a photo-active compound that upon activation generates a second compound capable of causing the removal of one of the two protecting groups,    exposing a portion of the substrate surface to ultraviolet radiation wherein ultraviolet radiation exposure causes the removal of one of the two protecting groups,    removing the photosensitive layer, and    coupling a third molecule capable of forming a peptide bond, wherein the molecule contains a protecting group capable of preventing the formation of a peptide bond, to the second molecule capable of forming a peptide bond that has been deprotected.    
   
   
       14 . The method according to  claim 13 , wherein the elements of  claim 13  are repeated to form a branched peptide attached to the substrate surface having a length from about 4 peptide bonds to about 25 peptide bonds.  
   
   
       15 . The method of  claim 1  wherein a molecule capable of forming a peptide bond is a molecule selected from the group consisting of aryl acetylenes, polyethyleneglycols, nascent polypeptides, diamines, diacids, peptides, and combinations thereof.  
   
   
       16 . The method of  claim 1  wherein a feature size of the array is less than 100 μm 2 .  
   
   
       17 . The method of  claim 1  wherein the array contains 1,000 to 10,000 features.  
   
   
       18 . The method of  claim 1  wherein a protecting group is selected from the group consisting of t-butoxycarbonyl, benzyloxycarbonyl, or 9-fluorenylmethoxycarbonyl.  
   
   
       19 . A method for making an array of biopolymers comprising, 
 attaching to the substrate surface a first molecule capable of forming a peptide bond wherein the molecule contains a photo-removable protecting group that prevents the formation of a peptide bond,    exposing a portion of the substrate surface to radiation wherein radiation exposure causes the removal of the protecting group,    coupling a second molecule capable of forming two or more peptide bonds, wherein the molecule contains two or more different photo-removable protecting groups capable of preventing the formation of a peptide bond, to the first molecule capable of forming a peptide bond that has been deprotected.    
   
   
       20 . The method according to  claim 19  wherein attaching is accomplished through the formation of a peptide bond.  
   
   
       21 . The method according to  claim 19  wherein the substrate surface to which the first molecule capable of forming a peptide bond is attached is an amino-functionalized SiO 2  surface.  
   
   
       22 . The method according to  claim 19  or  21  wherein the substrate is comprised of silicon having a layer of SiO 2  on the surface.  
   
   
       23 . The method of  claim 19  wherein a feature size of the array is less than 100 μm 2 .  
   
   
       24 . The method of  claim 19  wherein the array contains 1,000 to 10,000 features.  
   
   
       25 . The method according to  claim 19  also including 
 exposing a portion of the substrate surface to radiation wherein radiation exposure causes the removal of one of the two protecting groups that is photo-removable, and    coupling a third molecule capable of forming a peptide bond, wherein the molecule contains a protecting group capable of preventing the formation of a peptide bond, to the second molecule capable of forming two different peptide bond that has been deprotected at one peptide-bond forming site.    
   
   
       26 . The method according to  claim 25 , wherein the elements of  claim 25  are repeated to form a branched peptide attached to the substrate surface having a length from about 4 peptide bonds to about 25 peptide bonds.  
   
   
       27 . The method of  claim 19  wherein a molecule capable of forming a peptide bond is a spacer molecule selected from the group consisting of aryl acetylenes, polyethyleneglycols, nascent polypeptides, diamines, diacids, peptides, and combinations thereof.  
   
   
       28 . The method of  claim 19  wherein a feature size of the array is less than 100 μm 2 .  
   
   
       29 . The method of  claim 19  wherein the array contains 1,000 to 10,000 features.  
   
   
       30 . The method of claims  1 ,  13 ,  19 , or  25  additionally including the application of microwave energy to the substrate while exposing a portion of the substrate surface to radiation wherein radiation exposure causes the removal of protecting groups.  
   
   
       31 . A method for making an array of polymers comprising, 
 attaching to a substrate surface a first molecule capable of forming a peptide bond wherein the molecule contains a protecting group that prevents the formation of a peptide bond,    depositing a photosensitive layer over the substrate surface wherein the photosensitive layer contains a photo-active compound that upon activation generates a second compound capable of causing the removal of the protecting group,    exposing a portion of the substrate surface to both ultraviolet and microwave radiation wherein ultraviolet radiation exposure causes the removal of protecting groups,    removing the photosensitive layer, and    coupling a second molecule capable of forming a peptide bond, wherein the molecule contains a protecting group that prevents the formation of a peptide bond, to the first molecule capable of forming a peptide bond that has been deprotected.    
   
   
       32 . The method according to  claim 31  also including baking the substrate after exposing a portion of the substrate surface to ultraviolet radiation.  
   
   
       33 . The method according to  claim 31  also including capping unreacted peptide bond-forming sites on the first molecule capable of forming a peptide bond after coupling the second molecule capable of forming a peptide bond.  
   
   
       34 . The method according to  claim 31  wherein attaching is accomplished through the formation of a peptide bond.  
   
   
       35 . The method according to  claim 31  wherein the second compound capable of causing the removal of the protecting group is a photogenerated acid or base.  
   
   
       36 . The method according to  claim 31  wherein the second compound capable of causing the removal of the protecting group is a photogenerated acid and the photo-active compound is selected from the group consisting of sulfonium salts, halonium salts, and polonium salts.  
   
   
       37 . The method according to  claim 31  wherein the substrate surface to which the first molecule capable of forming a peptide bond is attached is an amino-functionalized SiO 2  surface.  
   
   
       39 . The method according to  claim 31  wherein the photosensitive layer comprises a polymer, a photo-active compound, and a solvent.  
   
   
       40 . The method according to  claim 31  wherein the photosensitive layer additionally includes a photosensitizer.  
   
   
       41 . The method according to  claim 40  wherein the photosensitizer is selected from the group consisting of benzophenones, thioxanthenones, anthraquinone, fluorenone, acetophenone, and perylene.  
   
   
       42 . The method of  claim 40  wherein a molecule capable of forming a peptide bond is a spacer molecule selected from the group consisting of aryl acetylenes, polyethyleneglycols, nascent polypeptides, diamines, diacids, peptides, and combinations thereof.  
   
   
       43 . The method of  claim 42  wherein the protecting group is t-butoxycarbonyl, benzyloxycarbonyl, or 9-fluorenylmethoxycarbonyl.  
   
   
       44 . An array of peptides on a substrate containing 1,000 to 10,000 features wherein the features contain peptides of known sequence and at least one of the features contains branched peptides of known sequence.  
   
   
       45 . The array of  claim 44  wherein the substrate is silicon or silicon having a SiO 2  layer.  
   
   
       46 . The array of  claim 44  wherein a feature size of the array is less than 100 μm 2 .  
   
   
       47 . A method of synthesizing an array of biomolecules on a solid support comprising: 
 exposing the substrate to microwave radiation to cause localized heating of the substrate during at least one of the synthesis reactions that create the biomolecule on the substrate surface.    
   
   
       48 . The method of  claim 47  wherein the biomolecule is a peptide or a peptidemimetic.  
   
   
       49 . The method of  claim 47  wherein the biomolecule is a polynucleotide.  
   
   
       50 . The method of  claim 47  wherein the substrate is comprised of silicon.  
   
   
       51 . The method of  claim 50  wherein the substrate is silicon having a SiO 2  layer.  
   
   
       52 . The method of  claim 47  or  50  wherein the array is one of a plurality of arrays formed on a silicon substrate.  
   
   
       53 . The method of  claim 47  wherein the at least one synthesis reaction that creates the biomolecule on the surface of the substrate is the deprotection of a polymer-bond forming site on a biopolymer chain.  
   
   
       54 . The method of  claim 53  wherein the at least one synthesis reaction that creates the biomolecule on the surface of the substrate is the deprotection of an amino acid peptide-bond forming group through the photogeneration of an acid or a base in a photoresist.