Very large scale immobilized polymer synthesis

• US 20020155588A1
• Filed: 09/05/2001
• Published: 10/24/2002
• Est. Priority Date: 06/07/1989
• Status: Abandoned Application

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Fodor, Stephen P.A., Pirrung, Michael C., Stryer, Lubert, Read, J. Leighton
• Application Number:
  US09/946,605
• Publication Number:
  US 20020155588A1
• Time in Patent Office: Days
• US Class Current: 435/287.2
• 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 2219/00612 the surface being inorganic
• CPC Class Codes: B01J 2219/00617 by chemical means
• CPC Class Codes: B01J 2219/00626 Covalent
• CPC Class Codes: B01J 2219/00637 : by coating it with another layer
• 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: 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 19/073 : with 2-deoxyribosyl as the saccharide radical
• CPC Class Codes: C07H 19/173 : with 2-deoxyribosyl as the saccharide radical
• CPC Class Codes: C07H 21/04 : with deoxyribosyl as saccharide radical
• 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: 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 15/1475 : using image analysis for extracting features of the particle
• 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: G01N 21/6458 : Fluorescence microscopy fluorescence microscopes per se G02B21/0076 and G02B21/16
• 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 7/0045 : with organic non-macromolecular light-sensitive compounds not otherwise provided for, e.g. dissolution inhibitors
• 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: Y02P 20/55 : Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
• 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

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.

Claims

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.

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.

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.

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.

27. The method as recited in claim 26 wherein said predetermined statistical distribution is a normal distribution.

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 a claim 29 wherein R¹ and R² are each a hydrogen atom.

32. The compound of claim 31 wherein R³ is a hydrogen atom.

33. The compound of claim 31 wherein R³ 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 R¹ and R² are each a hydrogen atom.

36. The compound of claim 35 wherein R³ is a hydrogen atom.

37. The compound of claim 35 wherein R³ is a methyl group.

39. The compound of claim 38 wherein R¹ through R³ are each a hydrogen atom.

40. The compound of claim 39 wherein R⁴ and R⁵ are each a hydrogen atom.

41. The compound of claim 39 wherein R⁴ and R⁵ are each a methyl group.

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.

28. A compound having the formula:

38. A compound having the formula:

42. A compound having the formula:

43. The compound of claim 42 wherein R² and R³ are each a methoxy group.

44. The compound of claim 43 wherein R¹ and R⁴ are each a hydrogen atom.

45. The compound of claim 44 wherein R⁵ is a methyl 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 R¹ and R² are hydrogen.

49. The compound of claim 48 wherein R³ is a methyl group.

46. A compound having the formula:

50. A compound having the formula:

51. The compound of claim 50 wherein X has the following formula:

52. The compound of claim 51 wherein R¹ and R⁴ are each a hydrogen atom, and R² and R³ are each a methoxy group.

53. The compound of claim 52 wherein R⁵ is a methyl group.

54. The compound of claim 51 wherein R² and R³ are substituted oxygen groups that together form a cyclic acetal.

55. The compound of claim 54 wherein R¹ and R⁴ are each a hydrogen atom.

56. The compound of claim 55 wherein R⁵ is a methyl group.