Synthesizing and screening molecular diversity

• US 5,639,603 A
• Filed: 11/02/1993
• Issued: 06/17/1997
• Est. Priority Date: 09/18/1991
• Status: Expired due to Fees

• Current Assignee: Affymax Incorporated, NEXUS Biosystems, Inc. (Azenta, Inc.)
• Original Assignee: Affymax Incorporated
• Inventors: Gallop, Mark A., Barrett, Ronald W., Dower, William J., Needels, Michael C.
• Primary Examiner(s): Fleisher, Mindy
• Assistant Examiner(s): KETTER, JAMES S
• Application Number:
  US08/146,886
• Time in Patent Office: 1,323 Days
• Field of Search: 536/25.3, 530/334, 530/335, 435/6
• US Class Current: 506/28
• 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 19/0093 Microreactors, e.g. miniaturised or microfabricated reactors laboratory containers with capillary fluid transport in microfabricated channels or chambers B01L3/5027
• CPC Class Codes: B01J 2219/00286 Reactor vessels with top and bottom openings
• CPC Class Codes: B01J 2219/00308 interchangeably mounted in racks or blocks
• CPC Class Codes: B01J 2219/00313 the reactor vessels being formed by arrays of wells in blocks
• CPC Class Codes: B01J 2219/00412 In multiple arrangements
• CPC Class Codes: B01J 2219/00418 using pressure
• CPC Class Codes: B01J 2219/00423 using filtration, e.g. through porous frits
• CPC Class Codes: B01J 2219/00466 in a slurry
• CPC Class Codes: B01J 2219/00484 by shaking, vibrating or oscillating of the reaction vessels
• CPC Class Codes: B01J 2219/00495 Means for heating or cooling the reaction vessels
• CPC Class Codes: B01J 2219/005 Beads
• CPC Class Codes: B01J 2219/00502 Particles of irregular geometry
• CPC Class Codes: B01J 2219/00536 in the shape of disks
• CPC Class Codes: B01J 2219/00542 Alphanumeric characters
• CPC Class Codes: B01J 2219/00545 Colours
• CPC Class Codes: B01J 2219/00547 Bar codes
• CPC Class Codes: B01J 2219/00554 Physical means
• CPC Class Codes: B01J 2219/00563 Magnetic means
• CPC Class Codes: B01J 2219/00565 Electromagnetic means
• CPC Class Codes: B01J 2219/00572 : Chemical means
• CPC Class Codes: B01J 2219/00574 : radioactive
• CPC Class Codes: B01J 2219/00576 : fluorophore
• CPC Class Codes: B01J 2219/00585 : Parallel processes
• CPC Class Codes: B01J 2219/0059 : Sequential processes
• CPC Class Codes: B01J 2219/00592 : Split-and-pool, mix-and-divide processes
• CPC Class Codes: B01J 2219/00596 : Solid-phase processes
• 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 2219/00698 : Measurement and control of process parameters
• 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: C07B 2200/11 : Compounds covalently bound to a solid support
• CPC Class Codes: C07H 19/10 : with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/14 : Pyrrolo-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: C07K 1/047 : Simultaneous synthesis of different peptide species; Peptide libraries
• 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 general stochastic method for synthesizing compounds can be used to generate large collections of tagged compounds that can be screened to identify and isolate compounds with useful properties.

Claims

1. A method of screening a tagged library of diverse compounds, wherein said library comprises a plurality of different members, each member comprising:

a solid support;

multiple copies of a compound bound to each of said supports, wherein the compound bound to one of said supports is different from the compound bound to selected other solid supports, and wherein said compound comprises a peptide; and

one or more identifier tags bound to each of said solid supports, wherein said tag identifies the compound bound to said solid support or identifies a reaction said solid support has experienced, and wherein said tag is an oligonucleotide or a fluorescent tag, said method comprising the steps of;

a) cleaving at least a portion of said compounds from said solid supports to yield a collection of untagged soluble compounds wherein said tags remain bound to said solid supports;

b) incubating said collection of untagged soluble compound s with a receptor under conditions conducive to binding of a ligand to said receptor; and

c. c) determining whether any compounds of said collection have bound to the receptor.

2. The method of claim 1 wherein said solid support is a bead 50 to 500 microns in diameter.

3. The method of claim 1, wherein said identifier tag is covalently attached to a first linker and said first linker is attached to said solid support and not to said compound.

4. The method of claim 1, whrein all of said compounds on said solid supports are cleaved prior to said incubation step.

5. The method of claim 1, further comprising the step of deducing the structure of the compounds that have bound to the receptor by examining the tags associated with the receptor-bound compounds.

6. The method of claim 1, wherein said receptor is a mixture of receptors and wherein each receptor bears a receptor-identifier tag that identifies the receptor.

7. The method of claim 1, wherein said receptor is immobilized on a second solid support.

8. The method of claim 7, wherein said second solid support is a surface or portion of the surface of a small individual compartment or well.

9. The method of claim 1, wherein said compound is attached to said solid support by a cleavable linker.

10. The method of claim 9, wherein said cleavable linker is a mixture of cleavable linkers.

11. The method of claim 10, wherein only a portion of said compounds on said solid supports are cleaved prior to said incubation step.

12. In a method of synthesizing a synthetic peptide library comprising a plurality of different members, each member comprising a peptide composed of a sequence of amino acid monomers linked to a bead to which bead is also linked one or more oligonucleotide identifier tags identifying the sequence of monomers in said peptide, wherein said amino acid monomers are protected with Fmoc and piperidine is used to remove the Fmoc protecting group, the improvement comprising effecting Fmoc removal by treatment with 5 to 15% piperidine for 5 to 60 minutes or 15 to 30% piperidine for 1 to 30 minutes.

13. The improvement of claim 12, wherein said bead is about 10 μ

m in diameter and composed of a macroporous styrene-divinylbenzene copolymer derivatized with a dodecylamine linker.

14. The improvement of claim 12, wherein said amino acid monomers have side ^t Bu side chain protecting groups, trifluoroacetic acid is used to remove said ^t Bu side chain protecting groups, and said oligonucleotide tags comprise 7-deaza-2'"'"'-deoxyadenosine.