Large scale photolithographic solid phase synthesis of an array of polymers

• US 5,405,783 A
• Filed: 03/12/1992
• Issued: 04/11/1995
• Est. Priority Date: 06/07/1989
• Status: Expired due to Term

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Affymax Incorporated
• Inventors: Pirrung, Michael C., Fodor, Stephen P. A., Stryer, Lubert, Read, J. Leighton
• Primary Examiner(s): Kepplinger, Esther M.
• Assistant Examiner(s): Green, Lora M.
• Application Number:
  US07/850,356
• Time in Patent Office: 1,125 Days
• Field of Search: 436/518, 436/527, 436/528, 436/807, 530/333-337, 435/7.92, 435/7.94, 435/7.95, 435/961, 435/968, 435/973, 427/2, 422/131-134, 422/141-142, 422/190, 536/25.3, 536/25.31, 536/126
• US Class Current: 436/518
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/00315 Microtiter plates
• CPC Class Codes: B01J 2219/00432 Photolithographic masks
• CPC Class Codes: B01J 2219/00434 Liquid crystal masks
• CPC Class Codes: B01J 2219/00436 Maskless processes
• CPC Class Codes: B01J 2219/00459 Beads
• CPC Class Codes: B01J 2219/00468 by manipulation of individual beads
• CPC Class Codes: B01J 2219/00475 Sheets
• CPC Class Codes: B01J 2219/005 Beads
• CPC Class Codes: B01J 2219/00527 Sheets
• CPC Class Codes: B01J 2219/00529 DNA chips
• CPC Class Codes: B01J 2219/00531 essentially square
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/0059 Sequential processes
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00605 the compounds being directly bound or immobilised to solid supports
• CPC Class Codes: B01J 2219/00608 DNA chips
• CPC Class Codes: B01J 2219/0061 The surface being organic
• CPC Class Codes: B01J 2219/00612 the surface being inorganic
• CPC Class Codes: B01J 2219/00617 by chemical means
• CPC Class Codes: B01J 2219/00619 : using hydrophilic or hydrophobic regions
• CPC Class Codes: B01J 2219/00621 : by physical means, e.g. trenches, raised areas
• CPC Class Codes: B01J 2219/00626 : Covalent
• CPC Class Codes: B01J 2219/00637 : by coating it with another layer
• CPC Class Codes: B01J 2219/00639 : the compounds being trapped in or bound to a porous medium
• CPC Class Codes: B01J 2219/00648 : by the use of solid beads
• CPC Class Codes: B01J 2219/00659 : Two-dimensional arrays
• CPC Class Codes: B01J 2219/00689 : using computers
• CPC Class Codes: B01J 2219/00695 : Synthesis control routines, e.g. using computer programs
• CPC Class Codes: B01J 2219/00711 : Light-directed synthesis
• CPC Class Codes: B01J 2219/00722 : Nucleotides
• CPC Class Codes: B01J 2219/00725 : Peptides
• CPC Class Codes: B01L 7/52 : with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples amplification or hybridisation processes per se C12Q1/68; controlling sequential reactions for synthesis B01J19/0046
• CPC Class Codes: B82Y 10/00 : Nanotechnology for information processing, storage or transmission, e.g. quantum computing or single electron logic
• CPC Class Codes: B82Y 30/00 : Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: C07B 2200/11 : Compounds covalently bound to a solid support
• CPC Class Codes: C07C 229/14 : to carbon atoms of carbon skeletons containing rings
• CPC Class Codes: C07C 229/16 : to carbon atoms of hydrocarbon radicals substituted by amino or carboxyl groups, e.g. ethylenediamine-tetra-acetic acid, iminodiacetic acids
• CPC Class Codes: C07D 263/44 : Two oxygen atoms
• CPC Class Codes: C07D 317/62 : with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to atoms of the carbocyclic ring
• CPC Class Codes: C07H 19/04 : Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
• CPC Class Codes: C07H 19/10 : with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 21/00 : Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C07K 1/04 : on carriers C07K1/003, C07K1/006 take precedence
• CPC Class Codes: C07K 1/042 : characterised by the nature of the carrier
• CPC Class Codes: C07K 1/045 : using devices to improve synthesis, e.g. reactors, special vessels
• CPC Class Codes: C07K 1/047 : Simultaneous synthesis of different peptide species; Peptide libraries
• CPC Class Codes: C07K 1/062 : for alpha- or omega-carboxy functions
• CPC Class Codes: C07K 17/06 : attached to the carrier via a bridging agent
• CPC Class Codes: C07K 17/14 : Peptides being immobilised on, or in, an inorganic carrier
• CPC Class Codes: C07K 7/06 : having 5 to 11 amino acids
• CPC Class Codes: C08G 69/00 : Macromolecular compounds obtained by reactions forming a carboxylic amide link in the main chain of the macromolecule products obtained from isocyanates or isothiocyanates C08G18/00; polyamide-imides C08G73/14
• CPC Class Codes: C12Q 1/6809 : Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6816 : characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6837 : using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6874 : involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C40B 40/06 : Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 40/10 : Libraries containing peptides or polypeptides, or derivatives thereof
• CPC Class Codes: C40B 60/14 : for creating libraries
• CPC Class Codes: C40B 80/00 : Linkers or spacers specially adapted for combinatorial chemistry or libraries, e.g. traceless linkers or safety-catch linkers
• CPC Class Codes: G01N 15/1433 : using image recognition
• CPC Class Codes: G01N 21/253 : for batch operation, i.e. multisample apparatus analytical automats G01N35/00
• CPC Class Codes: G01N 21/6428 : Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 21/6452 : Individual samples arranged in a regular 2D-array, e.g. multiwell plates
• CPC Class Codes: G03F 7/00 : Photomechanical, e.g. photolithographic, production of textured or patterned surfaces, e.g. printing surfaces; Materials therefor, e.g. comprising photoresists; Apparatus specially adapted therefor using photoresist structures for special production processes, see the relevant places, e.g. B44C, H01L, e.g. H01L21/00, H05K
• CPC Class Codes: G03F 7/26 : Processing photosensitive materials; Apparatus therefor G03F7/12 - G03F7/24 take precedence
• CPC Class Codes: G03F 7/265 : Selective reaction with inorganic or organometallic reagents after image-wise exposure, e.g. silylation
• CPC Class Codes: G03F 7/38 : Treatment before imagewise removal, e.g. prebaking G03F7/265 takes precedence
• CPC Class Codes: G11C 13/0014 : comprising cells based on organic memory material
• CPC Class Codes: G11C 13/0019 : comprising bio-molecules
• CPC Class Codes: H01L 25/50 : Multistep manufacturing processes of assemblies consisting of devices, each device being of a type provided for in group H01L27/00 or H01L29/00 H01L21/50 takes precedence
• CPC Class Codes: H01L 2924/00 : Indexing scheme for arrangements or methods for connecting or disconnecting semiconductor or solid-state bodies as covered by H01L24/00
• CPC Class Codes: H01L 2924/0002 : Not covered by any one of groups H01L24/00, H01L24/00 and H01L2224/00
• CPC Class Codes: Y02P 20/55 : Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
• CPC Class Codes: Y10S 435/961 : including a step of forming, releasing, or exposing the antigen or forming the hapten-immunogenic carrier complex or the antigen per se
• CPC Class Codes: Y10S 435/968 : High energy substrates, e.g. fluorescent, chemiluminescent, radioactive
• CPC Class Codes: Y10S 435/969 : Multiple layering of reactants
• CPC Class Codes: Y10S 435/973 : Simultaneous determination of more than one analyte
• CPC Class Codes: Y10S 436/807 : Apparatus included in process claim, e.g. physical support structures
• CPC Class Codes: Y10S 436/809 : Multifield plates or multicontainer arrays
• CPC Class Codes: Y10T 428/31971 : Of carbohydrate
• CPC Class Codes: Y10T 436/2575 : Volumetric liquid transfer

Abstract

A technique for the synthesis of arrays of diverse polymers such as polypeptides and nucleic acids. The technique beneficially utilizes solid-phase chemistry techniques. Preferred embodiments utilize photolabile protecting groups, and photolithography. The technique forms polymers with monomer sequences and locations determined by the order of addition of monomers and the activation patterns formed on the substrate.

Claims

1. A method of forming an array of diverse peptides on a substrate, the surface of said substrate comprising at least first and second regions having peptide molecules thereon, said peptide molecules coupled to a photoremovable protective group at a first functional group, said first functional group capable of binding to a second functional group of selected amino acids which also have a first functional group coupled to a photoremovable protective group, said method comprising the steps of:

removing said photoremovable protective group from said peptide molecules in said first region of said substrate with light to expose said first functional group, but not removing said photoremovable protective group from said peptide molecules in said second region;

contacting said first and second regions of said surface with first selected amino acid molecules to covalently bond said second functional group of said first amino acid molecules to said first functional group of said peptides by means of a peptide bond in said first region, but not said second region;

removing the photoremovable protective group from at least a portion of said first selected amino acid molecules in said first region with light to expose said first functional group on said at least a portion of said first selected amino acid molecules; and

contacting said first and said second regions with second selected amino acid molecules to covalently bond a second functional group of said second selected amino acid molecules to said first functional group of said first selected amino acid molecules by means of a peptide bond, forming peptides in said first region having a different amino acid sequence than peptides in said second region.

2. The method as recited in claim 1 further comprising additional steps of removing and coupling to form at least 100 different peptides on said substrate.

3. The method as recited in claim 1 further comprising additional steps of removing and coupling to form at least 1000 different peptides on said substrate.

4. The method as recited in claim 1 wherein said substrate is glass.

5. The method as recited in claim 1 wherein said light is selected from the group consisting of UV and IR light.

6. The method as recited in claim 1 wherein said first and said second regions individually have an area of less than 1 mm².

7. The method as recited in claim 1 wherein said first and said second regions individually have an area of less than 250,000 square microns.

8. The method as recited in claim 1 wherein the step of removing protective groups with light comprises the step illuminating said substrate through a mask.

9. The method as recited in claim 8 further comprising the step of illuminating said second region, wherein said step of illuminating said second region illuminates a portion of the substrate that was illuminated when said photoremovable protective groups on said peptide molecules in said first region were removed, and does not illuminate a portion of the substrate that was illuminated when said photoremovable protective groups on said peptide molecules in said first region were removed.

10. The method as recited in claim 9 wherein said step of illuminating said second region illuminates a portion of the substrate that was not illuminated when said photoremovable protective groups on said peptide molecules in said first region were removed, and does not illuminate a portion of the substrate that was not illuminated when said photoremovable protective groups on said peptide molecules in said first region were removed.

11. The method as recited in claim 1 wherein said photoremovable protective groups are ortho-nitrobenzyl protecting groups.

12. A method of making an array comprising at least two different peptides immobilized on a substrate, wherein said peptides differ from one another with respect to monomer sequence, by synthesizing said peptides on a surface of said substrate, said surface having functional groups capable of coupling with an amino acid, said functional groups protected by photoremovable protecting groups, said method comprising the steps of:

selectively activating said surface by removing said photoremovable protecting groups from said functional groups with light so that a first pattern of activated and unactivated regions is formed on said surface;

contacting said surface with a first amino acid, said first amino acid having at least a first functional group capable of coupling with said functional groups in said activated regions of said surface and a second functional group capable of coupling with another amino acid, said second functional group protected by a photoremovable protecting group, under conditions such that said first amino acid is coupled to said functional groups in said activated regions by means of a peptide bond;

selectively activating said surface by removing said photoremovable protecting groups from said functional groups with light so that a second pattern of activated and unactivated regions is formed on said surface;

contacting said surface with a second amino acid, said second amino acid having at least a first functional group capable of coupling with said functional groups in said activated regions of said surface and a second functional group capable of coupling with another amino acid, said second functional group protected by a photoremovable protecting group, under conditions such that said second amino acid is coupled to said functional groups in said activated regions by means of a peptide bond; and

repeating said activating and coupling steps until said two different peptides are formed on said surface, so that each different peptide is present at a defined location on said surface and has a defined amino acid sequence, whereby the amino acid sequence and location of each different polymer is defined by the patterns of activated and unactivated regions formed during each activating step and the monomers coupled in each coupling step.

13. The method as recited in claim 12 wherein said step of selectively activating comprises the steps of placing a mask between a light source and said substrate, and exposing said mask to light, said mask comprising first selected regions that transmit at least one selected wavelength of light and second selected regions that do not transmit said selected wavelength of light.

14. The method as recited in claim 13 wherein said mask is placed in direct contact with said substrate.

15. The method as recited in claim 12 wherein said photoremovable protecting groups are ortho-nitrobenzyl protecting groups.

16. The method as recited in claim 15 wherein said ortho-nitrobenzyl protecting groups are selected from the group consisting of nitroveratryloxycarbonyl and nitrobenzyloxycarbonyl.

17. The method as recited in claim 12 wherein said steps of selectively activating said surface exposes parts of said substrate previously exposed to light, and does not expose parts of said substrate previously exposed to light.

18. The method as recited in claim 17 wherein said step of selectively activating said surface exposes parts of said substrate not previously exposed to light, and does not expose parts of said substrate not previously exposed to light.

19. The method as recited in claim 12 wherein said steps are repeated to synthesize an array of more than 100 different polymers.

20. The method as recited in claim 12 wherein said steps are repeated to synthesize an array of more than 1000 different polymers.

21. The method as recited in claim 12 wherein said at least two different polymers each occupy an area on said substrate of less than 1 mm².

22. The method as recited in claim 12 wherein said at least two different polymers each occupy an area on said substrate of less than 1×

10^-1 cm².

23. The method as recited in claim 12 wherein said at least two different polymers each occupy an area on said substrate of less than 1×

10^-2 cm².

24. The method as recited in claim 12 wherein said at least two different polymers each occupy an area on said substrate of less than 1×

10^-4 cm².