Method and apparatus for combinatorial chemistry

• US 7,179,591 B2
• Filed: 05/16/2001
• Issued: 02/20/2007
• Est. Priority Date: 05/05/1999
• Status: Expired due to Term

• Current Assignee: Robert S. Foote
• Original Assignee: Robert S. Foote
• Inventors: Foote, Robert S.
• Primary Examiner(s): Shibuya; Mark L.
• Assistant Examiner(s): Lundgren; Jeffrey S.
• Application Number:
  US09/859,028
• Publication Number:
  US 20010029028A1
• Time in Patent Office: 2,106 Days
• Field of Search: 435/7.1
• US Class Current: 435/6.11
• 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 2219/00286 Reactor vessels with top and bottom openings
• CPC Class Codes: B01J 2219/00306 Reactor vessels in a multiple arrangement
• CPC Class Codes: B01J 2219/00369 in multiple or parallel arrangements
• CPC Class Codes: B01J 2219/00427 using masks
• CPC Class Codes: B01J 2219/00432 Photolithographic masks
• CPC Class Codes: B01J 2219/00511 Walls of reactor vessels
• CPC Class Codes: B01J 2219/0052 in the shape of elongated tubes
• CPC Class Codes: B01J 2219/00522 in a multiple parallel arrangement
• CPC Class Codes: B01J 2219/00524 in the shape of fiber bundles
• 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 2219/0061 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 2219/00626 Covalent
• CPC Class Codes: B01J 2219/00637 by coating it with another layer
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00664 : Three-dimensional arrays
• CPC Class Codes: B01J 2219/00675 : In-situ synthesis on the substrate
• CPC Class Codes: B01J 2219/00711 : Light-directed synthesis
• CPC Class Codes: B01J 2219/0072 : Organic compounds
• CPC Class Codes: B01J 2219/00722 : Nucleotides
• CPC Class Codes: B01J 2219/00725 : Peptides
• CPC Class Codes: B01J 2219/00731 : Saccharides
• CPC Class Codes: B82Y 30/00 : Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: C07H 19/06 : Pyrimidine radicals
• 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: 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 40/12 : Libraries containing saccharides or polysaccharides, or derivatives thereof
• CPC Class Codes: C40B 60/14 : for creating libraries

Abstract

A method and apparatus are provided for performing light-directed reactions in spatially addressable channels within a plurality of channels. One aspect of the invention employs photoactivatable reagents in solutions disposed into spatially addressable flow streams to control the parallel synthesis of molecules immobilized within the channels. The reagents may be photoactivated within a subset of channels at the site of immobilized substrate molecules or at a light-addressable site upstream from the substrate molecules. The method and apparatus of the invention find particularly utility in the synthesis of biopolymer arrays, e.g., oligonucleotides, peptides and carbohydrates, and in the combinatorial synthesis of small molecule arrays for drug discovery.

Claims

1. A method for the synthesis of combinatorial libraries of oligonucleotide arrays comprising:

(a) providing a housing containing a unitary synthesis block, said unitary synthesis block including an array of microchannels enclosed within the unitary synthesis block having a first end and a second end of the microchannels, wherein each of the microchannels has an inner microchannel surface, the inner surface of each of the microchannels containing immobilized substrate molecules comprised of oligomeric or polymeric molecules thereon wherein the termini of said substrate molecules have protecting groups coupled thereto;

(b) providing a light source and a mask for selectively illuminating microchannels in the array;

(c) flowing a photoactivatable reagent through the array of microchannels from the first end to the second end while selectively illuminating at least a portion of the microchannels from an end thereof, to provide photoactivated reagent in the selectively illuminated microchannels;

(d) removing protecting groups from the termini of the substrate molecules using the photoactivated reagent thereby forming deprotected substrate molecules in the selectively illuminated microchannels;

(e) reacting the deprotected substrate molecules with one or more nucleoside monomers under conditions effective for coupling the nucleoside monomers with the deprotected substrate molecules; and

(f) repeating steps (c) through (e) until a desired oligonucleotide array is formed within the microchannels.

2. The method of claim 1 wherein said photoactivated reagent is an acid precursor.

3. The method of claim 1 wherein the photoactivatable reagent is selected from the group consisting of triarylsulfonium hexafluoroantimonates, triarylsulfonium hexafluorophosphates, 2,1,4-diazonaphthoquinone sulfonates, perhalogenated triazines, and a trihaloacetic acid having a photocleavable carboxyl-protecting group selected from the group consisting of α

-and phenyl-substituted 2-nitrobenzyl groups, phenacyl groups, benzoinyl groups, arylazidoalkyl alcohol groups, 2,4-dinotrobenzenesulphenyl groups and N-substituted nitroanilido groups.

4. The method of claim 1 wherein said protecting groups are acid labile protecting groups.

5. The method of claim 1 wherein said protecting groups are selected from the group consisting of dimethoxytrityl, methoxymethyl, t-butoxymethyl, siloxymethyl, tetrahydrofuranyl, 1-ethoxyethyl, monomethoxytrityl, 3-imidazol-1-ylmethyl)bis(4′

4″

-dimethoxyphenyl)methyl, 9-(9-phenyl)xanthenyl, 9-flurenyl methoxycarbonyl and t-butyloxycarbonyl.

6. The method of claim 1 further comprising slicing the synthesis block containing oligonucleotide arrays into relatively thin sections having a thickness ranging from about 1 micron to about 100 microns to provide a plurality of planar sections of oligonucleotide arrays.

7. The method of claim 1 wherein the microchannels contain a solid support material.

8. A method for the synthesizing oligonucleotide arrays comprising the steps of:

(a) providing a housing containing a unitary channel array enclosed within a synthesis block including a plurality of channels, each of the channels having a fluid inlet end, a fluid exit end and an inner surface between the inlet end and the exit end;

(b) attaching substrate molecules to inner surface of the channels so that the substrate molecules have a first end attached to the inner surface of the channels and a second end having protecting groups coupled thereto;

(c) flowing a photoactivatable reagent through the channel array from the inlet end to the exit end of the channels;

(d) exposing at least a portion of the channels to light from an end thereof sufficient to activate the photoactivatable reagent sufficient to remove protecting groups from the second end of the substrate molecules to form deprotected substrate molecules in the light exposed channels;

(e) reacting the deprotected substrate molecules with nucleoside monomers to form oligonucleotide arrays on the inner surface of the channels containing the deprotected substrate molecules; and

(f) repeating steps (c) through (e) to provide a predetermined array of chemical entities in the channel array.

9. The method of claim 8 wherein said activated photoactivatable reagent comprises an acid precursor.

10. The method of claim 8 wherein the photoactivatable reagent is selected from the group consisting of triarylsulfonium hexafluoroantimonates, triarylsulfonium hexafluorophosphates, 2,1,4-diazonaphthoquinone sulfonates, perhalogenated triazines, and a trihaloacetic acid having a photocleavable carboxyl-protecting group selected from the group consisting of α

-and phenyl-substituted 2-nitrobenzyl groups, phenacyl groups, benzoinyl groups, arylazidoalkyl alcohol groups, 2,4-dinotrobenzenesulphenyl groups and N-substituted nitroanilido groups.

11. The method of claim 8 wherein said protecting groups are acid labile protecting groups.

12. The method of claim 8 wherein said protecting groups are selected from the group consisting of dimethoxytrityl, methoxymethyl, t-butoxymethyl, sioxymethyl, tetrahydrofuranyl, 1-ethoxyethyl, monomethoxytrityl, 3-imidazol-1-ylmethyl)bis(4′

4″

-dimethoxyphenyl)methyl, 9-(9-phenyl)xanthenyl, 9-flurenyl methoxycarbonyl and t-butyloxycarbonyl.

13. The method of claim 8 further comprising slicing the microchannel array into relatively thin sections having a thickness ranging from about 1 micron to about 100 microns to provide a plurality of planar sections of oligonucleotide arrays.