WO1998016830A2 - Systeme de criblage par gouttelettes - Google Patents

Systeme de criblage par gouttelettes Download PDF

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Publication number
WO1998016830A2
WO1998016830A2 PCT/US1997/019110 US9719110W WO9816830A2 WO 1998016830 A2 WO1998016830 A2 WO 1998016830A2 US 9719110 W US9719110 W US 9719110W WO 9816830 A2 WO9816830 A2 WO 9816830A2
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Prior art keywords
chemical compounds
collection
droplets
compound
composition
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Application number
PCT/US1997/019110
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English (en)
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WO1998016830A3 (fr
WO1998016830A9 (fr
Inventor
Stuart L. Schreiber
Matthew D. Shair
Allen J. Borchardt
Angie J. You
Jing Huang
Mike Foley
Derek Tan
George Whitesides
Rebecca J. Jackman
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The President And Fellows Of Harvard College
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Priority to AU52391/98A priority Critical patent/AU5239198A/en
Publication of WO1998016830A2 publication Critical patent/WO1998016830A2/fr
Publication of WO1998016830A3 publication Critical patent/WO1998016830A3/fr
Publication of WO1998016830A9 publication Critical patent/WO1998016830A9/fr

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    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B80/00—Linkers or spacers specially adapted for combinatorial chemistry or libraries, e.g. traceless linkers or safety-catch linkers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J19/0046—Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00279—Features relating to reactor vessels
    • B01J2219/00306—Reactor vessels in a multiple arrangement
    • B01J2219/00313—Reactor vessels in a multiple arrangement the reactor vessels being formed by arrays of wells in blocks
    • B01J2219/00315—Microtiter plates
    • B01J2219/00317—Microwell devices, i.e. having large numbers of wells
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00351—Means for dispensing and evacuation of reagents
    • B01J2219/00364—Pipettes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00351—Means for dispensing and evacuation of reagents
    • B01J2219/00364—Pipettes
    • B01J2219/00367—Pipettes capillary
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00497—Features relating to the solid phase supports
    • B01J2219/00511—Walls of reactor vessels
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00497—Features relating to the solid phase supports
    • B01J2219/00527—Sheets
    • B01J2219/00531—Sheets essentially square
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00585—Parallel processes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00596—Solid-phase processes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B01J2219/0061—The surface being organic
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B01J2219/00612—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports the surface being inorganic
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B01J2219/00614—Delimitation of the attachment areas
    • B01J2219/00621—Delimitation of the attachment areas by physical means, e.g. trenches, raised areas
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B01J2219/00623—Immobilisation or binding
    • B01J2219/0063—Other, e.g. van der Waals forces, hydrogen bonding
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00639—Making arrays on substantially continuous surfaces the compounds being trapped in or bound to a porous medium
    • B01J2219/00644—Making arrays on substantially continuous surfaces the compounds being trapped in or bound to a porous medium the porous medium being present in discrete locations, e.g. gel pads
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00646—Making arrays on substantially continuous surfaces the compounds being bound to beads immobilised on the solid supports
    • B01J2219/0065—Making arrays on substantially continuous surfaces the compounds being bound to beads immobilised on the solid supports by the use of liquid beads
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00659—Two-dimensional arrays
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/0068—Means for controlling the apparatus of the process
    • B01J2219/00702—Processes involving means for analysing and characterising the products
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B60/00—Apparatus specially adapted for use in combinatorial chemistry or with libraries
    • C40B60/14—Apparatus specially adapted for use in combinatorial chemistry or with libraries for creating libraries

Definitions

  • the present invention provides a system for simultaneously screening a large number of compounds to identify those that have desirable chemical or biological activities.
  • individual test compounds are isolated in droplets of liquid within which their activities are studied. Multiple droplets are displayed simultaneously on a single surface without risk of confusion because the separate identity of each droplet is maintained and diffusion of test compounds from one droplet to another is avoided.
  • these goals are accomplished through reliance on droplet surface tension; in other embodiments, the droplets are localized in micro- wells that retain droplet integrity.
  • One aspect of the present invention is a delivery device that deposits compound-containing liquid droplets on a display surface. Another aspect of the invention is the display surface containing such liquid droplets. Yet another aspect of the invention is a method for assaying chemical or biological activities of test compounds by introducing them into individual liquid droplets, displaying the droplets on a surface, and detecting a pre-determined activity within the displayed droplets.
  • Figure 1 presents schematic representations of certain embodiments of the droplet assay system of the present invention.
  • Figure IA shows a "stochastic" embodiment of the invention;
  • Figure IB shows an "arrayed” embodiment.
  • Figure 2 depicts two embodiments (shown in Panels A and B) of the inventive droplet assay system that allow in vivo assays under changing culture conditions.
  • Figure 3 presents structures of several different severable linkers by which test compounds may be attached to supports.
  • Figure 4 presents a preferred embodiment of the assay system of the present invention, utilizing test compounds attached to a support by means of a photosensitive link.
  • Figure 5 depicts an in vivo assay according to the present invention.
  • Figure 6 presents a representative synthesis of a photocleavable linker.
  • Figure 7 depicts a reaction scheme used to prepare a shikimic acid-based combinatorial library suitable for use in accordance with the present invention.
  • Figure 8 shows certain solid phase reactions utilized in preparing the above-mentioned shikimic acid-based combinatorial library.
  • Figures 9-12 show certain reactions to which tetracyclic templates were subjected in preparing the above-mentioned shikimic acid-based combinatorial library.
  • Figure 13 shows an alternative scheme for preparing a shikimic acid-based combinatorial library for use in accordance with the present invention.
  • Figure 14-15 show representative monomers for use in functionalizing templates in preparing shikimic acid-based combinatorial libraries.
  • Figure 16 depicts example members of shikimic acid-based combinatorial libraries.
  • Figure 17 shows linkage of shikimic acid library members to FK506.
  • Figure 18 shows synthesis of a dioxalane-based combinatorial library for use in accordance with the present invention.
  • Panel A shows the overall reaction scheme;
  • Panel B shows representative monomers used in the reaction scheme;
  • Panel C shows representative library members.
  • Figure 19 shows synthesis of a pyridinium salt-based combinatorial library.
  • Figure 20 shows representative pyridine nuclei, bromoketones, and dienophiles.
  • Figure 21 presents a more detailed reaction scheme for preparation of the above-mentioned pyridinium salt-based combinatorial library.
  • Figure 22 shows representative monomers (Panel A) used in preparation of the above-mentioned pyridinium salt-based combinatorial library, and also shows (Panel B) representative members of that library.
  • Figure 23 depicts the preparation of a rapamycin-bound photocleavable resin for use in the practice of the present invention.
  • Figure 24 presents a particular embodiment of a spray gun for use in the practice of the present invention.
  • Figure 25 depicts the introduction of support-bound test compounds into liquid droplets containing yeast cells.
  • Figure 26 depicts the use of the inventive assay system to detect inhibition of yeast cell growth by rapamycin.
  • Figure 27 shows results of a droplet assay to detect inhibition of yeast cell growth by rapamycin.
  • Figure 28 depicts a preferred "arrayed" assay embodiment of the present invention.
  • Figure 29 shows a scanned image of arrayed droplets.
  • Figure 30 shows the structure of a test compound utilized in one embodiment of the present invention.
  • Figure 31 shows the results of an inventive assay detecting activity of the compound depicted in Figure 30.
  • Figure 32 depicts a transcriptional activation dimerization read-out assay for use in the practice of the present invention.
  • Figure 33 depicts the use of a transcriptional activation dimerization assay in a droplet assay of the present invention.
  • Figure 34 presents a traditional reverse two hybrid transcriptional activation system.
  • Figure 35 depicts an improved reverse two hybrid system developed in the present invention.
  • Figure 36 depicts a particular embodiment of the inventive improved reverse two hybrid system.
  • Panel A shows the yeast strain constructed;
  • Panel B depicts use of the system.
  • Figure 37 shows results achieved with the inventive reverse two hybrid system.
  • Figure 38 depicts a three-hybrid transcriptional activation dimerization assay for use in the practice of the present invention.
  • Figure 39 depicts a dual library three-hybrid transcriptional activation dimerization assay for use in the practice of the present invention.
  • Figure 40 depicts a dual library screen in accordance with the present invention.
  • Figure 41 depicts a translocation dimerization assay for use in the practice of the present invention.
  • Figure 42 depicts an apoptosis dimerization assay for use in the practice of the present invention.
  • the present invention provides a system in which test compounds are introduced into and assayed within individual liquid droplets.
  • the test compounds and liquid are combined together and are deposited onto a display surface in a manner that produces liquid droplets containing a limited number of test compounds per droplet.
  • Figure IA depicts one preferred embodiment of the invention, in which the test compounds 100 and liquid 200 are combined together within a delivery device 300 that is used to deposit droplets 500 onto a display surface 400 in a "stochastic" manner.
  • the term “stochastic” describes a display process in which the positioning of the resultant droplets is not controlled, so that droplets are displayed substantially randomly on the display surface.
  • Figure IB presents an alternative preferred embodiment, in which the droplets 500 are "arrayed" on the display surface 400.
  • each droplet 500 has substantially the same volume, and the droplets 500 are arranged on the display surface 400 in a pre-determined pattern.
  • test compound is preferably provided in a manner that guarantees that multiple molecules of the compound are introduced into each droplet that receives a compound.
  • the test compounds are provided in association with a support to which multiple molecules are directly or indirectly attached. Any form of packaging that bundles multiple test compound molecules together in a manner that allows simultaneous introduction of all of the molecules into a single liquid droplet qualifies as a "support" as that term is used in the present application.
  • Preferred support materials include solid polymer materials such as, for example, polydextran, sephadex, polystyrene, polyethylene glycol, polyacrylamide, cellulose, and combinations thereof. Glass, latex, acrylic, or ceramic supports may also be employed, as may any of a variety of encapsulation matrices.
  • the supports are combined with the liquid and support-containing liquid droplets are deposited on the display surface.
  • the system is designed so that each droplet receives either no supports or a limited number of supports.
  • the droplets that receive a support receive, on average, only one support.
  • Test compounds may be attached to the support by any available mechanism.
  • a test compound may be covalently linked with the support, or may be associated with the support through a binding interaction (e.g., by means of ionic, electrostatic, and/or hydrogen-bonding interactions).
  • test compounds may be attached to another molecule that is in turn attached to the support. In certain preferred embodiments of the invention described in the Examples, approximately 100 pmol of compound were loaded onto each bead support.
  • test compounds are reversibly associated with the support so that, after a support is introduced into a droplet, test compound(s) attached to the support can be released from the support and assayed.
  • test compound(s) attached to the support can be released from the support and assayed.
  • Any severable linkage may be employed to attach test compounds to beads.
  • a wide variety of chemical compounds that act as severable linkers sensitive to cleavage by, for example, exposure to acids, bases, or electromagnetic radiation of appropriate wavelength are known in the art (for review, see, for example, Fruchtel et al., Angew. Chem. Int. Ed. Engl. 35: 17, 1996, Tables 2 and 3 of which are incorporated herein by reference).
  • Figure 2 presents the chemical structures of an array of several such severable chemical linkers.
  • Severable linkage may alternatively be accomplished by linking the test compound molecules to an agent, such as a protein or polypeptide, that is sensitive to cleavage by a known enzyme or chemical cleavage agent (see, for example, glutathione-S-transferase [GST] fusion system available from Pharmacia).
  • an agent such as a protein or polypeptide
  • GST glutathione-S-transferase
  • a wide variety of chemical (e.g., cyanogen bromide) and enzymatic (e.g., trypsin, chymotrypsin, carboxypeptidase Y, precursor protein processing enzymes, etc.), protein cleavage agents with specific recognition sites are known in the art (see, for example, Hermodson, Methods in Protein Sequencing Analysis, ed.
  • test compound molecules may be linked to a nucleic acid molecule that contains a cleavage site for a restriction endonuclease or other nucleic acid cleaving agent (e.g., a ribozyme).
  • Test compounds may also be attached by means of, for example, a disulfate linker that will be cleaved by exposure to reducing conditions (e.g., the interior of a cell).
  • Severable linkage may also be accomplished if the association between the test compound and the support to can be competed out by exposure to a competitive agent.
  • test compounds fused to GST will bind to a solid support to which glutathione is attached, and this binding can be competed by free glutathione.
  • Preferred severable linkages are those that allow the extent of compound release to be controlled by exposure to varying degrees of release signal, and therefore allow control of the concentration of active test compound in the droplet.
  • the extent of severance of photo-cleavable linkages can generally be varied by altering the time of exposure to radiation of the appropriate wavelength (see, for example, Example 6).
  • the extent of severance of competable attachments can be adjusted by altering the concentration of competitive agent. It will be appreciated that the concentration of test compound achieved in any particular droplet will depend both on the extent of release of compound in the droplet and on the volume of the droplet.
  • one advantage of certain "arrayed" embodiments of the present invention is that arrayed droplets typically have substantially identical volumes. Under such circumstances, exposure of each droplet to the same extent of compound release (e.g., to radiation applied for the same amount of time) produces the same concentration of test compound in each droplet, allowing straightforward side-by-side comparisons.
  • each support have approximately the same number of molecules attached to it, so that every droplet that receives a support receives approximately the same number of test compound molecules.
  • the support is in the shape of a bead, it is generally desirable for all support beads to be substantially the same size so that approximately uniform loading can be readily accomplished.
  • a single test compound be associated with each support. In such cases, introduction of one support into one droplet allows analysis of the activity of the particular test compound associated with the support. It is appreciated that in some instances it will be desirable to introduce more than one test compound into a single droplet at the same time.
  • test compounds to be screened it will sometimes be desirable to assay for synergistic, cooperative, or competitive interactions between or among test compounds.
  • collection of test compounds to be screened is very large, it may be desirable to assay sub-collections of test compounds together, detect those sub- collections that include one or more test compounds having the desired activity, and then to screen the sub-collections to identify the individual desirable test compounds within the sub-collections.
  • Approaches that allow introduction of multiple test compounds into a single liquid droplet include, for example, attaching multiple test compounds to a single support or introducing multiple supports, each of which contains only one attached test compound, into a single droplet.
  • the number of test molecules introduced into a droplet may depend on the extent to which test molecules were loaded on the support (i.e., the number of molecules initially attached to the support), the concentration of supports in the liquid/support mixture, the extent of release of test compound molecules from the support, and the size of the liquid droplets.
  • the size of the liquid droplets is adjusted by altering the rate at which the liquid is ejected from the delivery device, the extent of air flow within the device, the size of the aperture through which the droplets are delivered, the surface tension of the liquid and/or the viscosity of the liquid.
  • the size of the well or pocket determines the size of the droplet.
  • Liquid viscosity may be altered, for example, by selecting a liquid that inherently has the desired viscosity or by providing an additive that alters the liquid viscosity.
  • Available liquids and additives are known in the art. It will be appreciated that liquid viscosity can affect not only droplet size but also, and perhaps more importantly, the ease with which supports are delivered to liquid droplets. Effective delivery generally requires that the supports be maintained in a substantially uniform suspension in the liquid; liquid viscosity should preferably be selected to assist in the accomplishment of this goal. Of course, the importance of liquid viscosity in maintaining support suspension is reduced when the delivery device is designed to maintain effective suspension itself (e.g., by incorporating a mixing device).
  • Liquid viscosity may also affect evaporation.
  • Preferred droplet sizes are determined by the display and read-out systems employed. Particularly preferred are approximately 50 nL or 100 nL (volume) droplets.
  • the number of supports introduced per droplet can readily be set by dilution of the support/liquid mixture.
  • the surface on which the droplets are displayed is selected so that droplets maintain their discrete form when located on it and test compounds do not diffuse from one droplet to another through the support.
  • the surface is selected and/or utilized so that each droplet is also substantially immobilized on the surface.
  • a display surface containing wells or pockets is utilized, so that droplet discreteness is ensured and diffusion problems are substantially avoided.
  • Preferred surfaces for use with aqueous solutions include plastic, glass, and membranes, including polymer membranes.
  • Preferred polymer membranes include, for example, membranes formed from polymethyl methacrylate, polyurethane, or polydimethyl siloxane (PDMS).
  • compound activity can be analyzed using any of a variety of "read-out” assays (see below).
  • read-out assay may well influence the choice of materials employed in the display surface. For example, where the read-out assay involves detection of a fluorescent molecule that is only detectable after excitation, it may be desirable to use a display surface that is transparent to the excitation (and/or emission) radiation; where the read-out assay utilizes living cells, the display material must be compatible with continued viability.
  • the read-out assay involves detection of a fluorescent molecule that is only detectable after excitation
  • Other relevant considerations will be apparent to those of ordinary skill in the art.
  • the system is designed (e.g. , liquid and/or display surface are selected, conditions of reaction are controlled) to minimize droplet evaporation.
  • certain preferred embodiments of the invention utilize plastic petri dishes as display surfaces. Such petri dishes can readily be wrapped, or can be placed in a humid environment, to avoid evaporation.
  • FIG. 1 depicts a "replica-plating" embodiment of the invention, in which cell-containing droplets 500 are deposited on a display surface 400 comprising a movable membrane. The membrane is placed on a first growth medium 550 for a period of time and then is transferred to a second growth medium 560. As is known in the art, such procedures are particularly useful with, for example, yeast or bacterial cells.
  • Figure 2 presents an embodiment of the inventive droplet assay system in which changes to the growth medium can be accomplished without concern over diffusion among droplets.
  • cell-containing droplets 500 are arrayed in wells 580 on a first display surface 400a.
  • the first display surface 400a is overlayed with a second display surface 400b to which cells in the droplets 500, and the droplets 500 themselves, adhere (e.g., a fibronectin-coated glass coverslip if the cells are fibroblasts).
  • the second display surface 400b containing the droplets 500 and cells, can then be exposed to treatments that will modify the medium composition in the droplets 500.
  • it may be overlayed on a third display surface, which third display surface has wells substantially identical to those on the first display surface except that the wells on the third display surface contain one or more chemicals that alter the composition of the droplet medium when the droplets are introduced into the wells.
  • the read-out assays identify those droplets that contain test compounds displaying the desired activity.
  • the test compounds present in those droplets are then identified, for example by being isolated from the droplet or by other means.
  • each support contains information about the chemical structure of the compound with which it was associated, so that the compound can be identified by isolation and analysis of the support (see below for further discussion).
  • a second round of screening is typically performed to identify which one of the compounds (or which combination of compounds) within the droplet actually has the activity.
  • the Delivery Device Any device capable of depositing liquid droplets on a display surface may be utilized in the practice of the present invention. The characteristics and mechanics of the device will vary depending upon the desired method of delivery.
  • the delivery device of the present invention may be constructed and arranged to deposit drops in series (i.e., one at a time) or simultaneously, or merely to deliver liquid and test compounds to the display surface in a manner that allows subsequent formation and/ or arrangement of droplets.
  • the delivery device has a spray-type action.
  • the delivery device deposits a single sample of liquid/ test compound suspension on the display surface, and the droplets are distributed on the surface through a wetting/dewetting procedure (see Example 7).
  • any device in which liquid passes through an opening can be engineered to deposit droplets.
  • any known spray device such as an atomizer, a spray gun, a syringe, or any sort of squirt bottle, may be utilized.
  • an automatic or manual pipetter may be used.
  • a multi-tip manual pipetter can be employed to simultaneously deposit a large number of droplets in a defined array on a surface; a single-tip pipetter may be used when the droplets are to be distributed by a mechanism separate from their delivery (e.g., in the embodiment depicted in Figure 28).
  • the Library The present invention can be used to screen virtually any collection of test compounds. So long as the test compounds can be introduced into liquid droplets and assayed as described herein, they can be utilized in the practice of the present invention.
  • the support contain information identifying the compound with which it is associated so that isolation and analysis of the support constitutes identification of the compound (see below for further discussion of desirable means of identification). It is also preferred, as discussed above, that the association between the compound and the support be severable so that the compound can be released from the support after being introduced into the droplet. Severable attachment of the compound to the support provides the additional advantage that the concentration of active test compound in the droplet can be varied by altering the extent of exposure of the droplet to the agent responsible for disrupting the compound-support attachment (unless, of course, the compound is active without being released from the support).
  • Figure 3 depicts examples of a variety of known severable linkers that can be used to attach test compounds to supports according to the present invention.
  • Test compounds whether or not attached to a support, may be linked to another molecule or material.
  • a support may be linked to another molecule or material.
  • each member compound in a combinatorial library is functionalized with a common molecule.
  • the common molecule is then utilized in the read-out assay that is used to evaluate the test compounds (see below).
  • the assay system of the present invention is particularly suitable for analyzing synthetic chemical libraries produced by combinatorial synthesis.
  • Many examples of such libraries are known in the art, as are methods for producing them, such as "split and pool” or "parallel" solid phase synthesis (see, for example, Furka et al., Abstr. Mth Congr. Biochem. , Prague, Czechoslovakia, 5:47, 1988; Furka et al., Int. J. Pept. Protein Res. 37:487, 1991; Lorn et al., Nature 354:82, 1991, each of which is incorporated herein by reference; see also Fruchtel et al., Angew. Chem. Int. Ed. Engl. 35:17, 1996, incorporated herein by reference, and references cited therein).
  • These techniques allow for the generation of large libraries of compounds, each of which is attached (in multiple copies) to a single support.
  • test compound (or compounds) attached to a specific bead can be determined through a process of deconvolution (see, for example, Houghton et al., Biotechnique 13:901, 1992; Houghten et al., Nature 354:84, 1991, each of which is incorporated herein by reference).
  • deconvolution can be a laborious process.
  • preferred combinatorial libraries for use in the present invention are "encoded" or "tagged" libraries, in which each compound is attached to a support that also contains an identifier tag defining the compound (Ni et al., Met. Enzymol.
  • tags are typically molecules that can be cleaved from the bead and that, when analyzed, allow the synthetic history of the test compound attached to the bead to be retraced.
  • an encoded combinatorial library in which the compounds lOOa-n are attached to beads 600 through a photocleavable linker 800 is utilized.
  • Each bead 600 is labeled with a tag 900a-n that identifies the bound compound lOOa-n.
  • the photocleavable linker 800 is UV-sensitive.
  • the concentration of test compound released in the droplet can be controlled by controlling the time of exposure to UV radiation. For example, as presented in Example 6, exposure to 15 seconds of radiation at 365 nm releases enough test compound to give a concentration in the range of approximately 50-100 nM in a 100 nL droplet.
  • the amount of compound released in any particular experiment will depend on the efficiency of bead loading and the extent of bead functionalization.
  • Read-out assays for use in accordance with the present invention analyze chemical or biological activities of test compounds.
  • Read-out assays can be designed to test in vitro or in vivo activities. Both kinds of assays are illustrated in the Examples. Many preferred embodiments of the invention utilize read-out assays that test in vivo activities.
  • Figure 5 illustrates one preferred embodiment of the invention, utilizing an in vivo read-out assay.
  • test compounds 100 are detachably linked to beads 600 and are mixed with cells 700 in a liquid 200 that is selected to be compatible with cell viability.
  • the mixture is sprayed out of a delivery device 300 so that liquid droplets 500 are stochastically deposited on a display surface 400.
  • each liquid droplet 500 contains about 1-100,000 cells, preferably about 10-10,000 cells.
  • the number of cells per droplet is dependent on the droplet size and the concentration of cells in the original mixture and is therefore readily adjustable.
  • test compounds 100 are released from the beads 600 so that they may enter the cells and their effects on the cells can be detected.
  • system depicted in Figure 5 will only identify cell-permeable compounds and compounds that exert their effects externally to cells.
  • In vitro assay systems can be utilized to detect other classes of compounds (see, for example, Examples 15 and 17).
  • many, if not most, pharmaceutical applications involve extracellular administration of active agents it is anticipated that many of the most useful biologically active compounds identified with the present system will be cell- permeable compounds or compounds that act outside of cells.
  • Preferred read-out assays for use in detecting compounds with biological activity according to the present invention include assays that detect cell growth; cell death (or absence of growth); changes in cell morphology; changes in expression or localization of a colored, fluorescent, or otherwise detectable marker; etc.
  • the desired activity to be identified will often determine the cell type employed in the assay.
  • any cell e.g. , bacterial, yeast, human, or animal
  • selections will typically be made based on convenience, availability, and appropriateness to the assay.
  • assays that detect cell growth or cell death may desirably be used to identify antibiotic and/or antifungal agents by performing assays on bacterial and/or fungal (e.g., yeast) cells.
  • the assays are repeated on human and/or animal cells in order to identify those agents that inhibit bacterial and/or fungal cell growth without disrupting human or animal cell viability.
  • detectable genes commonly used in biological assay systems include the green fluorescent protein gene (or fusions therewith), the ⁇ -galactosidase gene, the invertase gene, the luciferase gene, the alkaline phosphatase gene, the horse radish peroxidase gene, etc. Examples of detectable genes commonly used in biological assay systems include the green fluorescent protein gene (or fusions therewith), the ⁇ -galactosidase gene, the invertase gene, the luciferase gene, the alkaline phosphatase gene, the horse radish peroxidase gene, etc. Examples of detectable genes commonly used in biological assay systems include the green fluorescent protein gene (or fusions therewith), the ⁇ -galactosidase gene, the invertase gene, the luciferase gene, the alkaline phosphatase gene, the horse radish peroxidase gene, etc. Examples of detectable genes commonly used in biological assay systems include
  • libraries are assembled on the solid phase (e.g., while attached to a resin), for example using “split and pool” synthesis or “in parallel” synthesis.
  • the library molecules are attached to the resin via a photocleavable linking unit, so that exposure to UV light releases the molecule from the resin.
  • a representative synthesis of a photocleavable linker is shown in Figure 6.
  • FIG. 7 presents the reaction scheme we have utilized to prepare one of the preferred combinatorial libraries of the present invention.
  • This library projects various functionalities in a radial array around a central scaffold experimental details for the preparation of this library are presented in Appendix A. Briefly, enantiomeric templates produced from shikimic acid (1) were converted to solid- phase-bound epoxyol templates (4) . The template was then treated with a variety of nitrones (5), under the influence of an esterification promoter, so that a corresponding tetracyclic system (6) was produced. As can be seen, six alternative tetracyclic templates (6a-e) were made.
  • shikimate-derived radial arrayed libraries can be synthesized by substitution of a nitrone based cycloaddition reaction with azomethane ylide cycloadditions, tributyltin hydride radical cyclizations, or 1,3 dipolar addition reactions to generate templates 2, 3, or 4 respectively ( Figure 13).
  • These templates can be functionalized similarly to templates 6a-e discussed above by employing reactions such as palladium cross- couplings, N-acylating lactone openings, lactone alcohol acylations, epoxide openings and epoxide alcohol acylations.
  • the first template depicted in Figure 7 is identical to template 6e.
  • Figures 14 and 15 show representative monomers that can be used to diversify these templates. Additionally, Figure 16 shows representative library members for each of the different shikimate-derived templates. Those of ordinary skill in the art will appreciate that additional functionalization at any of the reactive sites will yield further diversification (see Appendix B).
  • one of the sites on the tetracyclic system is functionalized with a ligand so that every molecule in the library is attached to the same ligand.
  • a ligand Any desirable ligand may be used; we have performed a linkage to FK506 (see Figure 17).
  • Figure 18 presents the reaction scheme we have utilized to prepare another preferred combinatorial library for use in the present invention; experimental details are presented in Appendix B. This system achieves diversification by condensation of 4 "combinatorializable units" in contrast to the shikimic acid based combinatorial library which diversifies in a radial array.
  • the synthesis of this library begins by selecting the base monomer, (1), which is an epoxide. Representative examples of suitable epoxides are shown in Figure 18B. Conversion of this base monomer to a solid phase bound template (2) was achieved using the coupling agent HATU ([O-(7- azabenzotriazol- 1 -yl)- 1 , 1 , 3 , 3-tetramethy luronium hexafluorophosphate] ) and diisopropylethylamine.
  • HATU [O-(7- azabenzotriazol- 1 -yl)- 1 , 1 , 3 , 3-tetramethy luronium hexafluorophosphate]
  • the epoxide was then subjected to reaction with another "combinatorializable unit", a secondary amine (3) (see Figure 18B for examples of suitable secondary amines) under ytterbium catalysis to produce the diol (4).
  • the diol upon reaction with an aldehyde (5) (see Figure 18B for examples of suitable aldehydes) and trimethylsilyl chloride, results in the acetal (6).
  • the dioxalane (8) is produced.
  • the capping group is preferably an acylating agent (7) (see Figure 18B for examples of suitable agents).
  • Figure 19 presents the reaction scheme we have utilized to prepare yet another combinatorial library appropriate for use in the present invention; experimental details are presented in Appendix C.
  • both a N-alkyl based pyridinium salt library and aracyl based library can be synthesized.
  • the N-alkyl based library can be synthesized from a pyridine nucleus, whose functionality can be varied, upon reaction with a dienophile, to include halogens, alkyl groups, and halides, or a bromoketone. Further reaction with a dienophile yields the N-alkyl based pyridinium salt library.
  • Representative pyridine nuclei, bromoketones and dienophiles are shown in Figure 20, but are in no way limited to the depicted compounds.
  • an acyl based pyridinium salt library can be synthesized using the same pyridine based starting material as shown in Figure 19.
  • a more detailed synthetic pathway of the acyl based pyridinium salt library is shown in Figure 21.
  • the pyridine nucleus undergoes addition of a nucleophile to an acyl pyridinium salt to produce the dihydropyridine product (2) .
  • the acylating agent utilized in this reaction can be varied (using R,) to diversify.
  • the dihydropyridine product (2) can then be combined with dienophiles, preferably maleic anhydride, to produce a Diels Alder cycloadduct represented by (3).
  • the Diels Alder adduct (3) can be reacted with a series of primary amines, of which (4) is representative, in which the anhydride of (3) is converted to the imide (5).
  • the primary amine "combinatorializable unit" can be diversified using R 2 ( Figure 22 A) and R 3 ( Figure 21).
  • the imide (5) is then condensed with various amines (R 4 ), of which (6) is representative, in an ipso substitution reaction to produce (7) .
  • (7) is then exposed to an asymmetric dihydroxylation catalyst to produce diol (8), which can be further diversified by reaction with a series of ketones, of which (9) is representative, under acid catalysis to produce the ketal (10).
  • This Example describes the preparation of a test compound comprising rapamycin linked to a photocleavable resin.
  • a photocleavable resin was prepared by mixing amino resin (100 mg, 0.025 mmol, 0.25 mmol/gm; TentaGel S NH 2 from RAPP polymere, catalog number S 30902) with photolinker (17.0mg, 0.075 mmol), diisopropyl carbodiimide (11.74 ⁇ L, 0.075 mmol), hydroxybenzotriazole (10.13 mg, 0.075 mmol), dimethylformamide (0.5 mL), and methylene chloride (0.5 mL), for 30 min. The reaction product was washed with dimethylformamide (10 mL) and methylene chloride (10 mL).
  • the rapamycin-photocleavable resin was prepared by mixing the photocleavable resin of Figure 23 A (25 mg, 0.00625 mmol) with phosgene in toluene (0.5 mL, 20% by weight) and N,N-dimethylaniline (7.92 ⁇ L, 0.0625 -mmol) for 1.5 hr at room temperature.
  • the reaction product was washed with dry methylene chloride (10 mL), under a nitrogen atmosphere.
  • This Example describes the construction of the spray gun that was employed in Examples 4-6.
  • we prepared the spray gun by inserting a bent 18 gauge disposable syringe needle (1.5 inch) through the side of a pi 000 disposable pipette tip. Nitrogen was flowed in under pressure so that droplets 500 were sprayed onto the display surface 400. It will be recognized that any of a variety of alternative arrangements could equally well be employed. The only structural requirements for a spray gun is that the aperture must be large enough that any support can pass through it.
  • Example 5 Detecting Growth Inhibition in Stochastically-Displayed Liquid Droplets/ Absence of Diffusion Between Droplets
  • This Example describes the use of the inventive droplet system to assay the ability of rapamycin to inhibit yeast cell growth. Rapamycin is a known antibiotic whose activity was being assayed only to establish that the inventive assay system could in fact be used to detect growth- inhibiting activity of a test compound. The work described in this Example established that the assay system is effective and, in particular, established that rapamycin did not diffuse across the petri dish from one droplet to others.
  • yeast growth was inhibited in rapamycin-bead-containing droplets yeast growth was not inhibited in droplets that lacked beads (see Panel B) yeast growth was also not inhibited in droplets that were not exposed to UV (Panel A); nor in droplets containing tor yeast cells that are resistant to rapamycin (Panel C).
  • the present Example describes use of a photocleavable linker in a droplet assay of the present invention so that the concentration of test compound in the droplet is varied.
  • Rapamycin-containing beads were prepared and introduced into droplets as described in Example 5.
  • the droplet-containing petri dishes were split into groups and were exposed to UV radiation for different times (1 min, 30 sec, 15 sec). We found that 15 sec of radiation released approximately the minimal amount of rapamycin needed to inhibit yeast cell growth in the droplets.
  • Example 7 Preparation of a Display Surface for an Arrayed Droplet Assay
  • This Example describes the preparation of a display surface containing wells or pockets that can be utilized in certain "arrayed" embodiments of the present assay system.
  • one preferred method of preparing a display surface containing wells of pre-determined configuration and arrangement is by the method of photolithography (see, for example, PCT Application publication number WO 96/29629, incorporated herein by reference).
  • Those of ordinary skill in the art will appreciate that other methods may alternatively be employed and that any method that produces a display surface having wells of appropriate (preferably substantially uniform) size and arrangement is suitable according to the present invention.
  • an acetate layer 710 on which the desired pattern of wells had been printed was placed on a silicon wafer 730 covered with a photoresist 720. Irradiation through the acetate layer created a "master" 740 over which a solution of PDMS monomers was poured. Polymerization of the PDMS produced a display surface 400 having wells 580 that were 20 ⁇ M deep, 1 ⁇ M in diameter, and that were separated from one another by 250 ⁇ M.
  • Test compounds and/or beads were introduced into liquid droplets 500 displayed on the surface 400 by means of a standard wetting/dewetting procedure. Specifically, compounds (and/or beads) were mixed with liquid and the mixture was applied to the surface. The surface 400 was then rotated so that liquid filled the wells and excess liquid was discarded.
  • Figure 29 shows a scanned image, at 50X magnification, of twelve arrayed droplets achieved by this method. Each droplet was 50 nL in volume. On average, each droplet contained 1-2 beads; the number of beads per droplet ranged from 0 to about 5.
  • the average number of beads per droplet (as well as the range in numbers of beads per droplet) can readily be adjusted by varying, for example, droplet size, bead concentration in the pre-droplet suspension, liquid viscosity, etc.
  • wetting/dewetting procedure to distribute droplets has certain advantages in the practice of the present invention. For example, such procedures are very mild, so that the viability of even sensitive cells, such as human stem cells, can be preserved in the procedure, allowing a wide range of read-out assays to be utilized. Of course, other mild procedures, such as manual pipetting (e.g., with a multi-tip pipetter), would likely provide a similar advantage.
  • the present Example describes the use of the arrayed liquid droplet system described in Example 7 to detect growth inhibition by the compound isolated from a combinatorial library.
  • a combinatorial library of 125,000 molecules was prepared by split-and- pool synthesis, and a single compound within that library was identified in prior experiments as having the ability to inhibit the growth of budding yeast cells.
  • the structure of this compound, as well as its photocleavable linkage to a support bead, is depicted in Figure 30.
  • the inhibitory compound (attached to its support bead) was combined with a suspension of budding yeast cells and applied to the display surface by a wetting/dewetting procedure (see Figure 28).
  • Experimental droplets were exposed to 60 seconds of UV radiation, so that the inhibitory compound was released from the beads in those droplets that contained beads, and then were incubated under conditions favorable for cell growth for 24 hours.
  • cells did not grow in irradiated droplets that contained beads; cells grew to high density both in irradiated droplets that lacked beads and in non- irradiated droplets.
  • Example 9 Detecting Ligands (and/or Inhibitors) in an Intracellular Transcriptional Activation Assay Dependent on Dimerization
  • the present Example describes an application of the droplet system of the present invention to detect test compounds that act as ligands in that they bind to intracellular receptors.
  • the term "receptor”, as used herein, refers to any protein to which a ligand binds.
  • the assay described in this Example can also be used to detect compounds that inhibit interactions between ligands and their receptors.
  • the read-out assay utilized in this embodiment of the invention detects the transcriptional activation of a gene required for cell growth.
  • the assay is presented schematically in Figure 32: a binding site 1400 for a known DNA binding entity (e.g. , a protein or protein domain, and anti-DNA antibody, an intercalation compound, a nucleic acid capable of triple helix formation, etc.) is positioned upstream of a reporter gene 1500 that is essential under the conditions of the assay (e.g., a gene required to produce a metabolite that is not present in the growth medium).
  • the DNA binding entity 1600 is fused to a first target receptor 1700, and a second target receptor 1800 is fused to a transcriptional activation domain 1900.
  • the first 1700 and second 1800 target receptors may or may not be the same.
  • the system depicted in Figure 32 is arranged so that in the absence of a ligand, the DNA binding entity 1600 and transcriptional activation domain 1900 do not interact with one another and the gene is not expressed. Provision of a ligand 100 (also called a "dimerizer") that interacts with both the first 1700 and second 1800 target receptors, however, allows the transcriptional activation domain 1900 to be recruited to the DNA binding entity 1600 so that transcription of the gene 1500 is activated.
  • a ligand 100 also called a "dimerizer”
  • Preferred DNA binding entities include the DNA binding domains of helix- turn-helix or homeodomain proteins. Also preferred are the DNA binding domains of hormone receptors. Particularly preferred DNA binding entities include the DNA binding domains of the Gal4, lex A, ⁇ cl, ⁇ l, engrailed proteins. Preferred transcriptional activation domains include those of Gal4 and VP16.
  • the read-out assay described in this Example and depicted in Figure 32 can be employed in cell droplets to detect homo- or hetero-dimerizing ligands, or compounds that interfere with such ligands.
  • FK506 is a known ligand that forms a complex with a first receptor called FKBP12, which complex can then interact with a second receptor, calcineurin A (Liu et al., Cell 66:807, 1991; Griffith et al., Cell 82:507, 1995).
  • FK506 therefore can act as a heterodimerizing ligand by mediating interactions between FKBP12 and calcineurin A (see Ho et al., Nature 382:822, August 29, 1996; Belshaw et al. , Proc. Natl. Acad. Sci. USA 93:4604, 1996, each of which is incorporated herein by reference).
  • a DNA binding moiety that recognizes a site positioned upstream of an essential gene is fused to one of FKBP12 or calcineurin A, and a transcriptional activation domain is fused to the other. Both fusions are expressed in cells.
  • the cells are then mixed with beads to which FK506 is severably attached, and the cells and beads are introduced into liquid droplets that are displayed on a selective growth medium (i.e. , a growth medium on which the essential gene is truly required for cell growth).
  • a selective growth medium i.e. , a growth medium on which the essential gene is truly required for cell growth.
  • FK506 is not the only known heterodimerizing ligand. In fact, there is at least one other known compound (rapamycin) that interacts with FKBP12 and at least one other ligand. Rapamycin competes with FK506 for binding to FKBP12, but does not interact with calcineurin A at all. Instead, rapamycin interacts with the FKBP-rapamycin binding (FRB) domain of FRP/RAFTl (Chen et al., Proc. Natl. Acad. Sci. USA 92;4947, 1995).
  • FRB FKBP-rapamycin binding
  • rapamycin is attached to beads, one of FKBP12 and FRB is attached to a DNA binding entity, and the other is attached to a transcriptional activation domain.
  • Figure 33 A particular version of this embodiment of the invention is depicted in Figure 33.
  • yeast cells are provided that are resistant to rapamycin but that cannot grow on media lacking histidine unless a particular gene 1500 (the HIS gene) is expressed.
  • the HIS gene 1500 is positioned adjacent a binding site 1400 recognized by a fusion protein that contains a DNA binding domain 1600 fused to FKBP12 1700.
  • the cells 700 also contain an FRB(1800)/transcriptional- activation-domain(1900) fusion.
  • Introduction of such cells into liquid droplets containing beads 600 to which rapamycin 100 is or is not severably linked allows identification of those droplets that contain rapamycin-bound beads because the cells in those droplets will be able to grow on media lacking histidine.
  • the assay system can be set up in cells other than yeast cells; both FK506 (see Ho et al., Nature 382:822, August 29, 1996) and rapamycin (Rivera et al. , Nature Medicine, in press) have been demonstrated to act as heterodimerizers in mammalian cells.
  • the assay can be applied to the sorts of combinatorial libraries described herein to identify new compounds capable of directing heterodimerization of any two receptors. That is, when a combinatorial library, instead of FK506 or rapamycin, is attached to the beads, the assay identifies any compounds within the library that direct heterodimerization of whatever receptors have been fused to the DNA binding and activation domains. Similar assays may also be used to identify compounds that direct homodimerization (i.e. , that interact with at least two copies of the same receptor).
  • analogous assays may be used to identify compounds that interfere with any receptor-ligand interaction.
  • the system is set up as described above except that FK506 is provided in active form (not attached to a bead) and a bead-attached combinatorial library is also provided. If the bead in any particular droplet contains a compound that disrupts the ability of FK506 to interact with either FKBP12 or calcineurin A, the cells in that droplet will die.
  • a system can be set up so that expression of an essential gene depends upon an interaction between two components (be they protein-chemical; protein-protein; or chemical-chemical), the present invention provides a system by which compounds that disrupt the interaction can be identified.
  • mammalian cells that express the Fas receptor and are grown in media containing the Fas ligand undergo apoptosis unless they also express the Bcl-2 gene.
  • a transcriptional activation assay analogous to that depicted in Figure 32 can be set up in mammalian cells if the cells are engineered so that expression of Bcl-2 is dependent upon recruitment of a DNA activation domain to a particular DNA site, which recruitment is in turn dependent on the presence of a "dimerizing" compound.
  • transcriptional repression domain e.g., the amino terminal portion of the yeast ⁇ 2 protein
  • transcriptional activation screens will be more popular than transcriptional repression screens. All of the techniques required to create gene fusions and to insert DNA binding sites upstream of reporter genes are known in the art (see, for example, Sambrook et al.
  • Example 10 Detecting Interaction Inhibitors in an Intracellular "Reverse Two- Hybrid" Transcriptional Activation Assay
  • the present Example describes the use of a read-out assay involving a
  • a "reverse two-hybrid" transcriptional activation screen is an assay designed so that interaction between two factors activates transcription of a toxic gene (see, for example, Vidal et al., Proc. Natl. Acad. Sci. USA 93:10315, 1996;
  • a first interaction partner 1700a is linked to a DNA binding entity 1600 that recognizes a regulatory site 1400 operatively linked to a gene 1500a that, when expressed, produces a product that is toxic to the cell under the growth conditions employed.
  • a second interaction partner 1800a is linked to a transcriptional activation domain 1900.
  • the transcriptional activation domain 1900 is recruited to the DNA so that the gene 1500a is activated and the cell dies. In the absence of such association, the cell lives.
  • the reverse two hybrid system was developed for use in identifying mutations in one or both of the first 1700a and second 1800a interaction partners that disrupt their association (Vidal et al., Proc. Natl. Acad. Sci. USA 93: 10321,
  • the inventive droplet assay system provides a format in which a large number of factors can be screened to identify those with an ability to disrupt associations between interaction partners in reverse-two-hybrid systems.
  • the inventive droplet assay system is used to identify small molecule inhibitors of protein-protein interactions, e.g., by screening libraries of chemical compounds (such as combinatorial libraries).
  • the inventive assay system can be used to detect inhibitors that disrupt interactions between any two chemical compounds (e.g., proteins, polypeptides, or nucleic acids) that interact with one another.
  • a yeast strain is constructed in which both a first fusion protein 2000, comprising a DNA binding entity 1600 linked to a first interaction partner 1700a, and a second fusion protein 2100, comprising a second interaction partner 1800a linked to a transcriptional activation domain 1900, are placed under the control of inducible promoters (i.e., are not expressed unless specific, controllable conditions are present).
  • the strain also contains a toxic reporter gene 1500a.
  • the first fusion protein comprises the LexA DNA binding domain linked to the cytoplasmic tail of the type I TGF ⁇ receptor and the second fusion protein comprises FKBP12 linked to the B42 transcriptional activation domain.
  • the genes for these fusion proteins were both placed under control of the GAL1 promoter, so that neither fusion protein is produced when cells are grown in media containing glucose, but both proteins are produced, after about a three hour delay, when cells are introduced into galactose media.
  • Each of these fusion protein genes was introduced on a plasmid into yeast cells (base strain EGY48, available from Roger Brent) that contained an integrated (at its ura3 locus) reporter construct comprising the URA3 gene under control of the SP013 promoter and four upstream LexA sites.
  • yeast cells base strain EGY48, available from Roger Brent
  • an integrated (at its ura3 locus) reporter construct comprising the URA3 gene under control of the SP013 promoter and four upstream LexA sites.
  • the advantage of the strain depicted in Figure 36A for the purposes of identifying molecules that disrupt the interaction between the cytoplasmic tail of the type I TGF/3 receptor and FKBP12 is that there is an approximately three-hour delay after the strain is introduced into galactose media before the first and second fusion proteins are produced. Accordingly, if the cells are exposed to an inhibiting compound at approximately the same time as they exposed to galactose media, the compound has an opportunity to enter the cells where it can block the interaction before the interaction forms.
  • the system may be used to identify compounds that disrupt interactions between cyclin Dl and cdk4. Association of these proteins, which is known to occur in the early Gl phase of the cell cycle, leads to phosphorylation of the retinoblastoma protein (RB) and RB family members, which in turn leads to progression through the cell cycle. Given disruption of this phosphorylation pathway appears to be associated with tumor development (in that many tumors contain mutations of one or more of the genes involved in this pathway, including mutations that result in up-regulation of cyclin Dl), it is likely that compounds that specifically inhibit the cyclin Dl/cdk4 association will be effective anti-tumor agents.
  • RB retinoblastoma protein
  • Example 11 Detecting Ligands in a "Three-Hybrid" Transcriptional Activation Assay This example describes another read-out assay that can be utilized in accordance with the present invention. This assay is related to the transcriptional activation assays described in Example 9, but it encompasses a fundamentally new idea: combinatorial libraries can be prepared as fusions between the combinatorial compounds and a known other compound.
  • This new concept allows the preparation of combinatorial libraries that are linked to a known ligand (see Example 1 for discussion of linkage to FK506.)
  • the receptor for the known ligand and a second receptor whose target is to be identified can then be made into fusions and employed in a transcriptional activation (or transcriptional repression) assay as described in Example 9.
  • Such assays will identify any compound in the combinatorial library that interacts with the second receptor.
  • Figure 38 depicts an example of this type of read-out assay that can be utilized in the practice of the present invention.
  • a library of test compounds 100 is prepared so that each compounds is attached to a bead 600 by means of a severable (in this case, photocleavable) linker 800.
  • Each test compound 100 is also linked to a known ligand 2200. such as FK506 or rapamycin, that interacts with FKBP12. Cleavage of the linker 800 releases the test compound as a test compound/FK506 fusion molecule 2300.
  • the beads 600 are introduced into liquid droplets along with cells 700 that express two fusion proteins: one in which FKBP12 1700 is attached to a DNA binding entity 1600 and one in which a selected test receptor 1800 is attached to a transcriptional activation domain 1900.
  • These cells also contain a "reporter gene” 1500 (typically, as discussed in Example 9, a gene encoding a product that is required for cell growth under the conditions of the assay) and that is not expressed unless the transcriptional activation domain 1900 is recruited to an upstream site 1400 recognized by the DNA binding entity 1600.
  • the test compound/FK506 fusion molecules 2300 are released from the beads and allowed to enter the cells 700. In those droplets that receive a test compound 100 capable of interacting with the test receptor 1800, the reporter gene will be expressed and the cells will grow; cell growth will not occur in other droplets.
  • ligands other than FK506 could be fused to the combinatorial library.
  • any ligand whose receptor is known could be utilized so long as the ligand can successfully be linked to the library molecules and its receptor can successfully be linked to the DNA binding entity.
  • the location of the ligand in the fusion is not critical so long as the test compound is released in association with the ligand and the ligand retains its ability to bind its receptor.
  • the assay could be "inverted", so that the known receptor is fused to the transcriptional activation domain instead of to the DNA binding entity.
  • This Example describes a read-out assay for use in the practice of the present invention that is an extension of the assay described in Example 11.
  • the assay is similar to that described in Example 8 except that, instead of being fused to a selected test receptor, the transcriptional activation domain 1900 is fused (via linkage of a cDNA library to a gene encoding the transcriptional actuation domain 1900 to a library 1800 of potential receptors.
  • Use of this read-out assay in the droplet system of the present invention allows simultaneous identification of new receptors and ligands that bind to them.
  • both cells and beads are isolated from droplets in which cells grow.
  • the bead tag is sequenced in order to identify the new test compound, and the particular cDNA fused to the transcriptional activation domain coding sequence is sequenced to identify the new receptor.
  • This embodiment of the invention therefore allows simultaneous screening of both a receptor library and a ligand library and thus dramatically expands the number of useful compounds that can be identified.
  • Example 13 Detecting Compounds that Affect Protein Translocation and/or Subcellular Localization Pathways
  • This Example describes another type of read-out assay that can desirably be employed in the practice of the present invention.
  • the droplet system can be desirably utilized with any of a variety of different kinds of readout assays.
  • any assay that results in a change in cell growth or morphology, or localization of expression of a detectable marker can desirably be utilized.
  • the assay described in the present Example detects subcellular localization of a fluorescent fusion protein within cells.
  • NF B is the prototype of a family of transcriptional regulators (sometimes referred to as "Rel” factors) that are sequestered in the cytoplasm through interaction with an inhibitor (for review, see Baeuerle et al., Cell 87: 13, October 4, 1996, incorporated herein by reference).
  • the inhibitor that interacts with NF/cB is known as I/cB.
  • Exposure of cells to any of a variety of stimuli such as viral infection, tumor necrosis factor [TNF], LPS, INF- ⁇ , oxygen radicals, etc.) initiates a signaling pathway that results in release of NF B from I/cB and translocation of NFcB into the nucleus, where it regulates expression of genes involved in immune regulation and development.
  • stimuli such as viral infection, tumor necrosis factor [TNF], LPS, INF- ⁇ , oxygen radicals, etc.
  • NF cB and other Rel factors in regulation of cellular processes involved in malignant transformation, control of apoptosis, immune function, and embryonic development makes identification of test compounds that enhance or interfere with the pathway leading to NF/cB translocation (or translocation of any other Rel factor) particularly desirable.
  • Figure 41 presents a schematic representation of a droplet screen of the present invention in which the readout assay detects NF/cB translocation.
  • eukaryotic cells preferably mammalian cells such as Jurkat cells, MG63 cells, NIH 3T3 cells, 293 cells, etc.
  • a detectable protein such as green fluorescent protein.
  • TNF an inducing agent
  • the NF/cB/green fluorescent protein fusion is localized in the cytoplasm. Addition of TNF results in translocation of the fusion to the nucleus.
  • the engineered cells are introduced into liquid droplets along with a library of test compounds, as has been described herein.
  • test compounds that stimulate NF/cB translocation are identified by the change in localization of green fluorescent protein
  • test compounds that interfere with NF cB translocation are identified.
  • the present Example describes readout systems that are related to those described in Examples 9 and 11-12 in the sense that they detect compounds that mediate heterodimerization interactions, but differ from the assays described in those prior Examples because the prior assays all detected heterodimerization that resulted in transcriptional activation.
  • the present invention contemplates that similar dimerization schemes can be constructed so that dimerization results in, for example, detectable protein translocation, change in cell morphology, or other phenomenon.
  • a dimerization assay in which dimerization results in apoptosis, is depicted in Figure 42 (see also Belshaw et al., Proc. Natl. Acad. Sci. USA 93:4604, May 1996, incorporated herein by reference).
  • FKBP12 is fused to the cytoplasmic tail of a plasma membrane cellular receptor (i.e., a protein that is present in the plasma membrane of a cell and that, when bound by an extracellular ligand, initiates a signaling pathway within the cell.
  • a plasma membrane cellular receptor i.e., a protein that is present in the plasma membrane of a cell and that, when bound by an extracellular ligand, initiates a signaling pathway within the cell.
  • cellular receptor therefore refers only to proteins that have particular responses and play particular roles in cells, and can be distinguished from the more general use of the term “receptor”, which refers to any protein capable of binding a ligand).
  • test receptor (used in the generic sense) is fused to the intracellular domain of the Fas receptor.
  • some cells will receive a test compound that is capable of interacting with the test receptor.
  • Simultaneous interactions between i) the test receptor and test compound; and ii) FK506 and FKBP12 recruits the Fas receptor to the cellular receptor.
  • multiple copies of FKBP12 are fused to the cellular receptor, so that multiple copies of the Fas receptor are recruited. Dimerization (or multimerization) of the Fas receptor results in production of an apoptotic signal. Accordingly, test compounds that interact with the test receptor are identified because the cells in the droplet that receive them undergo apoptosis, which involves detectable changes in cell morphology.
  • Fas receptor dimerization could be developed, for example, by engineering cells to produce both i) an Fas receptor/FKBP12 fusion and ii) an Fas receptor/target receptor fusion.
  • An FK506-linked combinatorial library could be screened against such cells in a droplet assay of the present invention to identify those compounds that interact with the test receptor because such an interaction would result in Fas dimerization and cell apoptosis. Apoptosis is not induced in the cells in other droplets.
  • test receptor employed in this assay could equally well be a library of possible receptors, so that the assay could be performed as a dual library screen. Other acceptable modifications will be apparent to those of ordinary skill in the art.
  • FKBP12 and the test receptor can be linked to a mitogenic cellular receptor or receptors instead of to the Fas receptor.
  • the present invention can be utilized with a read-out assay in which dimerization results in a detectable change in subcellular localization.
  • cells can be engineered to produce i) a fusion of FKBP12 to a protein or polypeptide that contains a nuclear localization signal; and ii) a fusion of a test receptor to green fluorescent protein.
  • Introduction of such cells into liquid droplets in combination with a library of combinatorial compounds linked to FK506 results in identification of those test compounds that interact with the test receptor (see Figure 42; see also Belshaw et al., Proc. Natl. Acad. Sci USA 93:4604, May 1996, incorporated herein by reference).
  • this dimerization assay for subcellular localization can also be modified in a variety of different ways.
  • the FKBP12 and test receptor fusions can be reversed (that is, FKBP12 can be fused to the green fluorescent protein and the test receptor can be fused to the nuclear localization signal).
  • the assay may be converted into a dual-library screen as described in Example 12. Other desirable modifications will be apparent to those of ordinary skill in the art.
  • Example 15 Detecting Ligand Binding in vitro
  • the droplet assays of the present invention be performed using cellular assays.
  • most of the transcriptional activation-based assays described above can be performed in the absence of cells.
  • in vitro transcription/ translation media can be used.
  • DNA constructs containing the reporter gene and genes encoding any fusion proteins can be introduced into droplets of in vitro transcription/translation media, along with the test compounds, and activation of the reporter gene construct can be detected.
  • the reporter gene encodes a readily detectable protein, such as one that itself is colored (or fluorescent), or that participates in a reaction to produce a colored product.
  • the reporter gene could encode a metal- chelator that competes the metal away from a complex that is fluorescent when it is bound to the metal.
  • expression of the reporter gene would result in removal of the metal from the complex and therefore loss of color from the liquid droplet.
  • Example 10 offers an alternative to the scheme described in Example 10 for using the inventive droplet assay system to identify compounds that interfere with receptor-ligand interactions.
  • ligand refers to any chemical compound, including a protein, polypeptide, or nucleic acid molecule, that interacts with another compound (typically a protein receptor).
  • an inhibitory assay of the present invention is to screen for test compounds that disrupt transcriptional activation accomplished in a standard two-hybrid transcriptional regulation system (see, for example, Fields et al. Nature
  • a "standard two-hybrid system” is one in which a DNA binding entity is fused to a first interacting partner and a second interacting partner is fused to a transcriptional activation domain (or, in principle, a transcriptional repression domain. For purposes of simplicity, we explicitly discuss only the use of a transcriptional activation domain but it should be understood that either could be used).
  • a reporter gene whose expression is detectable (e.g., because the cell requires expression to survive under the experimental conditions or because the product is colored or participates in a reaction that gives a colored product, where the term "colored” includes fluorescent) is placed under the control of a regulatory site recognized by the DNA binding domain.
  • the inhibitory assay described herein can be thought of as an alternative version of a "reverse" two-hybrid system (see Example 10). Basically, a combinatorial library is introduced into liquid droplets that also contain cells (or and in vitro transcription/translation system) expressing the two hybrids required for gene activation. Compounds that interfere with the interaction between the first and second interaction partners are detected because the product of the reporter gene is not detectable in the droplets into which they are introduced.
  • an assay can be constructed in which i) the Gal4 DNA binding domain is fused to a peptide that interacts with SH3 domains; ii) An SH3 domain is fused to a transcriptional activation domain; and iii) the herpes virus thymidylate kinase (TK) gene is placed under control of one or more Gal4 binding sites. Expression of the TK gene is toxic to mammalian cells grown in the presence of gangcyclovir.
  • TK herpes virus thymidylate kinase
  • the screen is performed by introducing cells expressing each of the two fusion proteins and carrying the reporter construct into media droplets containing gangcyclovir.
  • Test compounds are also introduced into the droplets. If a droplet receives a test compound that interferes with the SH3/SH3 binding peptide interaction, then cells in that droplet will not express the TK gene and will survive even though there is gancyclovir in the media. Cells in other droplets will die.
  • This assay involves positive rather than negative selection (that is, selection for cell growth rather than cell death), which is often desirable because it allows for isolation and analysis of the growing cells in addition to the test compounds.
  • the system may be desirable to arrange the system so that the SH3/SH3 binding peptide interaction is not established until after the test compounds are delivered to the cells.
  • the assay may be performed so that gangcyclovir is not added to the media until after the test compounds are delivered.
  • the present Example is intended to demonstrate that, in addition to detecting test compounds with desirable biological activities, as is done in the read-out assays described in many of the above Examples, the present invention can be used to detect test compounds with desirable chemical activities.
  • the inventive droplet assay system may be used to detect catalysts, e.g., for hydrolytic processes. So long as one or more of the reactants and products of the reaction is/are detectable (e.g., is fluorescent), the assay system of the present invention may be used to detect compounds that stimulate production of product or use-up of reactant.
  • detectable e.g., is fluorescent
  • Other analytical techniques as are known in the art, may alternatively be used.
  • Methyl (1S,6S)-2R (benzoyl)-7-oxabicycl-[4.1.0Jhept-3-ene-4-carboxylate (lOg, 36.5 mmol) as a solution in THF/H2O (56ml:56ml).
  • the solution was cooled to 0°C and treated with LiOH (3.2g, 76.7mmol) as a solution in H2O (30ml).
  • the solution was stirred for lh. at 0°C and treated with AmberUte IR-120(plus) ion exchange resin until pH 3. The mixture was filtered and concentrated in vacuo.
  • 2-nitrobenzaldehyde (12.21g, 1 eq), malonic acid (8.40g, 1 eq), and ammonium acetate (7.47g, 1.2 eq) were heated in 20 ml EtOH over 30 min to -S0°C. Mixture was held at reflux until gasses ceased to evolve, -30 min, forming a thick precipitate. Cooled mixture was shaken with 30ml further EtOH and filtered with 200ml Et 2 0 wash. Precipitate was shaken with 2 liters MeOH and filtered, giving a precipitate of 2.25g. Precipitate was taken up in 40 ml H20 with 3ml cone. HCI and filtered, and the filtrate adjusted to pH 4.5 with 6 " N KOH to precipitate the producat as a white crystaline solid, 1.93g, 1 1% yield.
  • the reaction was stirred at -20°C 20h- The completed reaction was warmed to 0°C and 30ml each IN HCI and 2N KF were added. Biphasic solution was stirred vigorously 20 min, causing a white precipitate to form. The precipitate was filtered off, 10ml each IN HCI and 2N KF were added, and solution stirred vigorously a further 20 min. The THF layer was separated, the aqueous layer washed with 40ml THF, and the combined organics dried over MgSC .
  • Oxalyl chloride (0.59ml, 1.3 eq) was disolved in 20ml CH 2 O and solution cooled to -60°C under N2. To this was added a solution of 0.81ml DMSO (2.2 eq) in lml total volume CH2CI2 and reaction stirred 2 min at -60°C. A solution of 3-((9-fluorenylmethoxycarbonyl)-amino)- benzylalcohol (1.79g, 1 eq) disolved in 20ml CH 2 CI 2 and minimal DMSO ( ⁇ 2ml) was added dropwise, and the reaction stirred 15 min at -60°C.
  • Oxalyl chloride (0.70ml, 1.7 eq) was disolved in 20ml CH 2 CI 2 and solution cooled to -60°C under N 2 . To this was added 1.02ml DMSO (3.0 eq) and reaction stirred 2 min at -60°C. A chilled solution of 3-((9-fluorenylmemoxyca ⁇ bonyl anti ⁇ o)-4-raethoxy-benzylalcohol (1.72g, 1 eq) disolved in 10ml CH 2 CI 2 was added dropwise, and the reaction stirred 20 min at -60°C, becoming cloudy.
  • Reagents 1. (R)- or (SH ⁇ sH 4 - carb oxy> -hydroxy- 1 ,2-epoxybutane, 1.70 eq in DMF.
  • Resin Tentagel, 1 eq, loaded with 3-Amino-3-(2-nitrophenyl)-propionic acid (Geysen photocleavable linker), coupled to position I epoxy-ol.
  • Reagents 1. Secondary amine of choice, 25 eq in dry THF.
  • Resin Tentagel, 1 eq, loaded with 3-Amino-3-(2-nitrophenyl propionic add (Geysen photocleavable linker), coupled to position I epoxy-ol, epoxide opened by position II amine, cis- diol capped with FMOC protected amino-aldchyde position HI.
  • Free a ine may or may not give a positive ninhydrin test (e.g aniline types will be negative).
  • Resin Tentagel, 1 eq, loaded with 3-Amino-3-(2-nitrophenyl)-propionic acid (Geysen photocleavable linker), coupled to position I epoxy-ol, epoxide opened by position II amine, cis- diol capped with amino-aldehyde position III.
  • Reagents 1. 4-dimethylarninopy ⁇ idine (2 eq) and diisopropylehtylaminc (50 eq) in CH 2 CI 2 .
  • electrophile of choice e.g. acid chloride, (10 eq) in CH 2 CI 2 .
  • Beads of interest (generally 2 x 30 mg for an NMR) are placed in two clear 0.7ml eppendorf tubes and tubes filled with acetonitrile. Tubes are sealed and irradiated lh at 365nm with shaking every 20 min. Mixture is filtered through a pippette w/ cotton plug (acetonitrile wash), and filtrate rotovapped to dryness.
  • the resin was washed successively with CH2O2 (5 x 8ml), THF (5 x 8ml), DMF (5 x 8ml), and isopropanol (5 x 8ml). A small sample was photolytically cleaved from the resin and analyzed by mass spectroscopy. MS (CI, ammonia) 236 (M + H).

Abstract

L'invention concerne un nouveau système permettant d'identifier des composés ayant des activités chimiques ou biologiques désirables. Selon l'invention, on introduit des composés à étudier dans des gouttelettes de liquide et on les crible. Le système est particulièrement utile pour identifier des composés qui agissent par exemple comme catalyseurs ou qui ont des activités biologiques. Dans les modes de réalisation préférés de l'invention, les composés sont criblés in vivo.
PCT/US1997/019110 1996-10-16 1997-10-15 Systeme de criblage par gouttelettes WO1998016830A2 (fr)

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