WO1998035923A1 - Procede permettant de creer de la diversite moleculaire - Google Patents

Procede permettant de creer de la diversite moleculaire Download PDF

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Publication number
WO1998035923A1
WO1998035923A1 PCT/US1998/002812 US9802812W WO9835923A1 WO 1998035923 A1 WO1998035923 A1 WO 1998035923A1 US 9802812 W US9802812 W US 9802812W WO 9835923 A1 WO9835923 A1 WO 9835923A1
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Prior art keywords
molecules
library
tool
molecule
core
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PCT/US1998/002812
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English (en)
Inventor
Julius Rebek, Jr.
Thomas Carell
Edward A. Wintner
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Massachusetts Institute Of Technology
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Priority to AU61638/98A priority Critical patent/AU6163898A/en
Publication of WO1998035923A1 publication Critical patent/WO1998035923A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/78—Ring systems having three or more relevant rings
    • C07D311/80—Dibenzopyrans; Hydrogenated dibenzopyrans
    • C07D311/82—Xanthenes
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/08—Liquid phase synthesis, i.e. wherein all library building blocks are in liquid phase or in solution during library creation; Particular methods of cleavage from the liquid support

Definitions

  • This invention relates to Methods for generating combinatorial libraries and to the libraries produced thereby.
  • the libraries are useful for identifying pharmaceutical and agricultural lead compounds.
  • Recombinant molecular libraries are generated by inserting random segments of nucleic acid into a vector, such as a phage, and allowing the vector to replicate, transcribe and express the inserted sequence.
  • a vector such as a phage
  • the nucleic acid is inserted into the phage genome in a manner which permits expression of the inserted material on the phage surface (see, e.g., Pavia, M. , et al., Biooro. i Mad. Cham. Ltr «. 3 tm . ⁇ a7- 396 (1993) and references cited therein; Devlin, J. , et al., Science 2A2-404-406 (1990)).
  • the virus particles are screened for the presence of a lead compound by contacting the particles with an immobilized ligate and isolating the virus particles which express a ligand that binds to the immobilized ligate.
  • recombinantly-produced library for drug discovery is the ability to clone and amplify the nucleic acids encoding the lead compounds which are identified during the screening process, unfortunately, recombinant libraries inherently are limited in diversity to linear polymers (e.g., oligonucleotides, peptide ⁇ ) formed of naturally-occurring monomers (e.g., nucleotides, L-amino acids). Typically, these naturally-occurring polymers exhibit metabolic instability and poor absorption properties vivo. Accordingly, pharmaceutical lead compounds that are isolated from recombinant libraries frequently require substantial modification to obtain a clinically useful drug.
  • linear polymers e.g., oligonucleotides, peptide ⁇
  • naturally-occurring monomers e.g., nucleotides, L-amino acids
  • Houghten et al. rBioTechni ⁇ ues 4f6):522-528 (1986) and Nature 354:84-86 (1991)) described a method for generating peptide libraries in which small amounts of a resin support were encapsulated in a plurality of porous polypropylene bags. By sequentially immersing the bags in solutions of individual amino acids under conditions for forming a peptide linkage, Houghten et al. generated a collection of bags, each of which contained a unique resin- immobilized peptide.
  • the present invention overcomes the limitations of the prior art by providing a simple, solution phase process for generating tens of thousands of molecularly diverse molecules.
  • the library molecules have structures that are not recognized by degradative enzymes i XiXS, a feature which contributes to the metabolic stability and improved absorption properties of these molecules i vivo.
  • the conformation e.g., linear, spherical, disc-like
  • a method for forming a combinatorial library includes (a) admixing a plurality of core molecules having at least one reactive center with a plurality of different tool molecules, each having at least one functional group to form a reaction mixture, and (b) reacting the reactive centers of the core molecules with the functional groups of the tool molecules to form a plurality of library molecules.
  • the combinatorial library is screened for the presence of molecules having biological activity, for example, by identifying library molecules which modulate the biological activity of a ligate (i.e., a molecule which is capable of specifically recognizing and associating with a ligand) .
  • the core molecule primarily serves as a scaffold to which different tool molecules can be attached at a fixed spatial orientation relative to one another.
  • the instant invention provides a simple, one-step process for creating a combinatorial library of vast molecular diversity.
  • Complementary pairs of reactive centers and functional groups are selected so that the reactive centers are capable of reacting with the functional groups to form the library molecules.
  • the reactive center is an acid halide
  • the functional group is an amine
  • the library molecule is a non-naturally occurring molecule in which the core molecule and the tool molecule(s) are linked via an amide bond.
  • Alternative complementary pairs of reactive centers and functional groups are familiar to those skilled in the art.
  • a method for manufacturing a combinatorial library having molecular diversity includes (a) admixing a plurality of core molecules having at least one reactive center with a plurality of different tool molecules to form a reaction mixture, each tool molecule including a first functional group for reacting with the reactive center and a second functional group attached to a removable, fat-soluble protecting group; (b) reacting the reactive centers of the core molecules with the functional groups of the tool molecules to form a pro-library of fat-soluble molecules; and (c) extracting the unreacted core molecules and the unreacted tool molecules from the pro-library molecules.
  • the tool molecule may contain more than two functional groups (e.g., a third functional group, a fourth functional group) , each of which may be attached to a protecting group.
  • the protecting groups are removed from the pro-library of molecules to form a second combinatorial library.
  • an alternative method for manufacturing a combinatorial library includes reacting a plurality of core molecules, each having at least two reactive centers at a fixed spatial orientation relative to one another, with a plurality of different tool molecules, so that the reactive centers of the core molecules react with the functional groups of the tool molecules to form a library of molecules, each containing tool molecules located at a fixed spatial orientation relative to one another.
  • a fat-soluble combinatorial pro-library is provided.
  • the terms “fat-soluble” and “water-soluble 1 express the relative degree of solubility of a compound in an organic (“fat") phase or in an aqueous (“water”) phase.
  • a compound or molecule is said to be “fat-soluble” if it is more soluble in an organic phase than it is in an aqueous phase.
  • the fat-soluble pro-library contains a plurality of non-naturally occurring molecules, each molecule including at least one tool molecule linked to a core molecule.
  • the tool molecules further include a removable (e.g., cl ⁇ avable) protecting group, the inclusion of which group in the non-naturally occurring molecule renders the latter molecule fat-soluble.
  • the fat-soluble pro-library is useful for identifying pharmaceutical and/or agricultural lead compounds which modulate the functional activity of, for example, a membrane-associated biological molecule. Removal of the fat- soluble protecting group from the library molecule (e.g., by cleaving the group from the library molecule) and removal of the cleaved protecting group from the reaction mixture (e.g., by extraction) yields a water- soluble library which also can be screened for the presence of pharmaceutical and/or agricultural lead compounds.
  • a library containing a plurality of structurally-diverse non-naturally occurring molecules includes a first tool molecule and a second tool molecule covalently coupled to a core molecule at a fixed spatial orientation.
  • the first and second tool molecules may be coupled to the core molecule at the same fixed spatial orientation or at a different fixed spatial orientation.
  • Such combinatorial libraries are useful, for example, for determining the optimal spatial orientation of tool molecules relative to on. another in a lead compound or group of lead compounds, e.g., by comparing the biological activities of lead compound, identified in combinatorial libraries which di far fro. on.
  • a kit for forming a combinatorial library i. provided.
  • the kit includes a plurality of core molacules, each having at least one reactive center and instruction, for reacting the core molecule, with a plurality of tool molecule, to form a combinatorial library.
  • the kit further include, the plurality of different tool molecule., each having at l.a.t one functional group.
  • a combinatorial library of non-naturally occurring molecule i. provided.
  • the library include, a plurality of non-naturally occurring molecules, each including et least one tool molecule covalently coupled to a xanthene molecule.
  • the tool molecule are .elected from the group con.i.ting of an amino acid and a nucleo ⁇ ide.
  • Figure 1 hows exemplary core molecules for generating the combinatorial libraries of the invention (in which the structures represent carbon cores except were noted) ;
  • Figure 2 show, exemplary amino acid and other primary amine-containing tool molecule, for generating the combinatorial librarie. of the invention
  • Figure 3 how. exemplary nucleoba.e and modified nucl.oba.e tool molecule, for generating the combinatorial librarie. of the invention
  • Figure 4 chematically illu.trate. the reaction of an acid chloride reactive center with a primary amine functional group to form a library molecule containing an amide linkage;
  • Figure 5 chematically illu.trate. the reaction of an acid chloride reactive center with an alcohol functional group to for. a library molecule containing an e.ter linkage;
  • Figure 6 chematically illu.trate. the reaction of an alkyl chloride reactive center with a primary amine functional group to form a library molecule containing a secondary amine linkage;
  • Figure 7 schematically illu.trate. the reaction of an alkyl chloride reactive center with an alcohol functional group to form a library molecule containing an ether linkage
  • Figure 8 chematically illu.trate. the reaction of an alcohol reactive center with an acid chloride functional group to for. a library molecule containing an ester linkage;
  • Figure 9 chematically illu.trate. the reaction of an alcohol reactive center with an alkyl chloride functional group to form a library molecule containing an ether linkage;
  • Figure 10 chematically illu.trate. the reaction of a primary amine reactive center with an acid chloride functional group to form a library molecule containing an amide linkage?
  • Figure 11 schematically illustrates the reaction of a primary amine reactive center with an alkyl chloride functional group to form a library molecule containing a secondary amine linkage;
  • Figure 12 shows the synthesis and HPLC analysis of a library of 136 theoretical molecules
  • Figure 13 shows the synthesis and HPLC analysis of a library of 1225 theoretical molecules
  • Figure 14 shows the synthesis and HPLC analysis of a library of 10,968 theoretical molecules
  • Figure 15 shows the synthesis and HPLC analysis of a library of 99,141 theoretical molecules
  • Figure 16 shows the synthesis of protecting group-derivatized amino acid tool molecules
  • Figure 17 shows the synthesis of 9 , -dimethylxanthene 2,4,5,7-di- and tetra-acid chloride core molecules.
  • Combinatorial Library refers to a collection of structurally-diverse molecules.
  • the libraries of the invention are said to have molecular diversity.
  • the extent of this diversity is dictated by the number, the nature and the ratio of the reactants (i.e., the core molecules and tool molecules) which react to form the library molecules.
  • the core molecules and tool molecules may be naturally-occurring or non-naturally occurring, the reaction product of the reaction between the core and tool molecules typically is a non- naturally occurring molecule.
  • the libraries of the invention are said to have non-naturally occurring molecular diversity.
  • T,1b Mo l ecul s refers to the plurality of molecules which comprise a combinatorial library.
  • Library molecules are the products of the reaction between the core molecules and the tool molecules (defined below).
  • ffnrf , M ⁇ «cule: refers to a molecule having a rigid or relatively rigid molecular structure (discussed below) and including at least one reactive center for reacting with a functional group of a tool molecule (defined below) .
  • the core molecule serves as a scaffold to which the tool molecules can be linked in a fixed spatial orientation (defined below) relative to one another.
  • core molecules examples include, but are not limited to, chemical compounds (e.g., monocyclic molecules, polycyclic molecules, heterocyclic molecules, aromatic molecules) and biochemical compounds (e.g., adenine, thymine, guanine, cytidine, uracil, ino ⁇ ine, as well as analogs, nucleosides, and nucleotides of the foregoing nucleobases) .
  • biochemical compounds e.g., adenine, thymine, guanine, cytidine, uracil, ino ⁇ ine, as well as analogs, nucleosides, and nucleotides of the foregoing nucleobases.
  • core molecules also include pharmaceutical molecules which have a known biological activity and fragments of such pharmaceutical molecules. Examples of the foregoing categories of chemical compounds are provided in figure 1.
  • the planar aromatic and planar heterocyclic molecules shown in figure 1 exemplify a class of rigid core molecules and the non-aromatic monocyclic and polycyclic molecules shown in the figure exemplify a class of relatively rigid molecules.
  • the core molecule is selected from the group consisting of 9,9-dimethylxanthene-2, ,5,7-tetraacid chloride and 1,3,5,7-cubane tetraacid chloride (see, e.g., A. Bashir-Hashemi , Anoe . Chem. (Intl. Ed. in English) 32:612-613 (1993) for a synthesis procedure for the cubane tetraacid chloride).
  • the core molecule includes two or more reactive centers (described below) for forming a covalent linkage with the functional groups of the tool molecules.
  • the core molecules following formation of the covalent linkage, the core molecules have a sufficiently rigid structure to maintain two or more tool molecules at a fixed spatial orientation relative to one another.
  • Reactive Center refers to a reactive group of the core molecule which is capable of forming a linkage (e.g., a covalent bond) with a complementary functional group (described below) of the tool molecule.
  • the reactive center can be a nucleophile (e.g., an alcohol, an amine, a thiol) or an electrophile (e.g., an acid halide, an alkyl halide).
  • the reactive center is an acid chloride and the functional group of the tool molecule is an amine, an alcohol or a thiol group.
  • reaction mechanisms can be used to form the linkage between the core molecule and the tool molecule(s).
  • palladium can be used to catalyze the reaction between the reactive centers of the core molecules and the functional groups of the tool molecules to form the combinatorial library molecules.
  • Other mechanisms for forming a covalent linkage between the core molecules and tool molecules of the invention are known to those of ordinary skill in the art. (See, e.g., March, J., Advanced Organin chemistry.
  • Tftf? ⁇ Hfr lecule refers to the different molecules having at least one functional group which reacts with the reactive centers of the core molecules to form the combinatorial library molecules.
  • the tool molecules of the invention can be the naturally-occurring L-amino acids or nucleoba ⁇ es (e.g., adenine, guanin ⁇ , thymine, cytidine, uracil).
  • the tool molecules also can be the non-naturally occurring molecules, such as a non-naturally occurring nucleoba ⁇ e, nucleoside or nucleotide analog, a D-amino acid, an L-amino acid analog, a non-naturally occurring carbohydrate (e.g., p ⁇ nto ⁇ e and hexo ⁇ e sugar moieties) and carbohydrate analog.
  • a non-naturally occurring nucleoba ⁇ e, nucleoside or nucleotide analog such as a non-naturally occurring nucleoba ⁇ e, nucleoside or nucleotide analog, a D-amino acid, an L-amino acid analog, a non-naturally occurring carbohydrate (e.g., p ⁇ nto ⁇ e and hexo ⁇ e sugar moieties) and carbohydrate analog.
  • mn ti ⁇ 1 croup refers to a reactive group of the tool molecule which is capable of reacting with the reactive center of a core molecule to form a linkage.
  • the linkage is a covalent bond.
  • the functional group can be a nucleophile (e.g.-, an alcohol, an amine, a thiol) or an electrophile (e.g., an acid halide, an acyl halide).
  • nucleophilic/electrophilic reaction mechanism complementary pairs of reactive centers and functional groups (e.g., a nucleophilic reactive center and an electrophilic functional group, an electrophilic reactive center and a nucleophilic functional group) are selected to form the combinatorial libraries.
  • a nucleophilic reactive center and an electrophilic functional group e.g., an electrophilic reactive center and a nucleophilic functional group
  • other types of tool molecules e.g., non-nucleophilic/non-electrophilic
  • can be reacted with the core molecules depending upon the type of mechanism employed for the coupling reaction discussed below.
  • the tool molecules optionally include more than one functional group, e.g., a first functional group for reacting with the core molecule and a second functional group for reacting with a second molecule, such as a protecting group (described below).
  • F xed Spatial Orientation refers to the placement in space of at least two molecules (attached to the same molecule, such as a core molecule) relative to one another.
  • the core molecules of the invention include two or more reactive centers. The centers are positioned at a fixed spatial orientation relative to one another to permit attachment of two or more tool molecules at a fixed spatial orientation relative to one another.
  • the phrase "fixed spatial orientation” embraces orientations which are entirely restricted in movement, as well as orientations which are partially restricted in movement such as in a library molecule in which the tool molecules on a core molecule are free to move within a limited sphere (e.g., to rotate about the linkage which attaches the tool molecule to the core) .
  • Hon-naturallv occurring molecule refers to a molecule which is not found in nature. Such molecules may be produced by synthetic or recombinant methods.
  • a non-naturally occurring peptide i.e., a peptide which is not found in nature
  • can be prepared using recombinant methods see, e.g., Pavia, M. , et al., Bioorg. & Ned. Chea. Ltrs. 3(3):387-396 (1993) or using chemical synthetic methods (see, e.g., Lam, K. , et al., Nature 354:82-84 (1991)).
  • protecting Group refers to a material which is bound to a functional group or reactive center and which may be selectively removed therefrom to expose the functional group or reactive center in a reactive form.
  • the protecting groups are reversibly attached to the functional groups and can be removed (e.g., chemically or otherwise cleaved) from the functional groups and/or library molecules.
  • the protecting groups include a hydrophobic moiety, which facilitates the separation of the unreacted core molecules and unreacted tool molecules from the combinatorial library molecules (discussed below).
  • Water-soluble/Fat-soluble refers to the relative degree of solubility of a compound in an organic (“fat") phase or in an aqueous (“water”) phase.
  • a compound or molecule is said to be “fat-soluble” if it is more soluble in an organic phase than it is in an aqueous phase.
  • a compound or molecule is said to be “water-soluble” if it is more soluble in an aqueous phase than it is in an organic phase.
  • Bit-logical: ⁇ -active/-inactive refers to the functional activity of a molecule (e.g., ligate or ligand) or a structure (e.g., membrane).
  • a molecule e.g., ligate or ligand
  • a structure e.g., membrane
  • Different categories of ligates exhibit different functional activities.
  • the functional activity of a receptor ligate i. the ability to .pacifically recognize and bind to a ligand.
  • the functional activity of an antibody i. the ability to .pacifically recognize and bind to an antigen, an antigenic determinant, or an epitope.
  • the functional activity of an enzyme i. the ability to catalyze a .p ⁇ cific reaction.
  • the functional activity of a nucleic acid i.
  • a library molecule is said to be biologically active if it ha. the ability to modulate the functional activity of a apecific ligate or class of ligate.. Different type, of assay, are necessary to screen the combinatorial librarie. of the invention for the presence of biologically-active molecules.
  • screening refers to the process by which library molecules are tested for biological activity.
  • the ability to modulate the functional activity of a ligand or ligate can be used as a screening assay to identify lead compounds.
  • a preferred screening method involves contacting the library with an immobilized ligate and identifying the library molecules which bind to the ligate.
  • solution-phase competition assays can be used to screen combinatorial libraries (e.g., by contacting the library with an immobilized ligand in the presence of a soluble ligate for the ligand) and to assess the relative affinity of the library molecule for the ligate (Zucker ann, R. , et al., Proc. Natl. Acad. Sci.
  • assays are well known to those of ordinary skill in the art (see e.g., Lam, K. , et al. , su p ra . for a discussion of opiate peptide receptor assays) .
  • Other assays useful for identifying pharmaceutical and/or agricultural lead compounds include assaying for antimicrobial activity, assaying for antiviral activity (e.g., by measuring plaque inhibition) and assaying for anti-fungal activity.
  • iaat ⁇ refers to a molecule that has ah affinity for a ligand.
  • Ligate ⁇ may be naturally-occurring or non-naturally occurring and can be used in a soluble or an immobilized state, e.g., attached to a solid support.
  • Categories of ligate. for which the combinatorial librarie. of the invention are u.eful for identifying lead compound include, but are not limited to, receptor., antibodie., enzyme, and nucleic acid..
  • the preferred receptor ligate. of the invention include receptor, which modulate a humoral immune response, receptors which modulate a cellular immune response (e.g., T-c ⁇ ll receptors) and receptors which modulate a neurological response (e.g., glutamat ⁇ receptor, glycine receptor, gamma-amino butyric acid (GABA) receptor).
  • receptors which modulate a humoral immune response e.g., T-c ⁇ ll receptors
  • GABA gamma-amino butyric acid
  • cytokine receptors (implicated in arthritis, septic shock, transplant rejection, autoimmune disease and inflammatory diseases)
  • MHC major hi ⁇ tocompatibility
  • T-cell receptors and/or T- helper cell receptors
  • thrombin receptor implanticated in coagulation, cardiovascular disease
  • ⁇ орcerol ⁇ ионентs are u.eful for identifying pharmaceutical lead compound, include enzyme, implicated in arthriti., o.t.oporo.i., infla ⁇ atory di.ea.es, diabetes, allergies, organ transplant rejection, oncogene activation ( e.g., dihydrofolat. reducta.e ) , .ignal transduction, .elf-cycle regulation, transcription, DNA replication and repair.
  • enzyme implicated in arthriti., o.t.oporo.i., infla ⁇ atory di.ea.es, diabetes, allergies, organ transplant rejection, oncogene activation ( e.g., dihydrofolat. reducta.e ) , .ignal transduction, .elf-cycle regulation, transcription, DNA replication and repair.
  • the preferred nucleic acid ligate. of the instant invention include any .egment of DNA or RNA containing natural or non-naturally occurring nucleosides. Nucleic acid, are capable of .pecifically binding to other nucleic acid, or oligonucleotide. via complementary hydrogen- bonding and also are capable of binding to non-nucleic acid ligates. ( See, e.g.. Bock, L. , et al., Nature 355:564-566 (1992) which reports inhibition of the thrombin-catalyz ⁇ d conversion of fibrinogen to fibrin using aptamer DNA).
  • iaand refers to a molecule that is recognized by a ligate.
  • ead Molec le refers to a molecule which is capable of modulating the functional activity of a biological molecule. Screening assays are used to identify lead molecules in the combinatorial libraries of the invention. Examples of lead molecules that can be synthesized and selected in accordance with the invention include, but are not limited to, agonists an d antagonists for cell membrane receptors, toxins and venoms, viral epitopes, hormones, opiates, steroids, peptides, enzyme substrates, cofactors, drugs, lectins, sugars, oligonucleotide ⁇ , nucleic acids, oligosaccharides, lipids, proteins, and analogs of any of the foregoing molecules.
  • Analo g refers to a molecule which shares a common functional activity with the molecule to which it is deemed to be an analog and may share common structural features as well.
  • Pro-library refers to a type of combinatorial library of molecules which may be further processed into another state to form a second combinatorial library.
  • the pro-library molecules contain protecting groups which enhance the fat-solubility of the library molecules.
  • the pro-library i. purified by extracting the wat ⁇ r-.oluble core and tool molecule, from the fat-.olubl ⁇ pro-library molecule..
  • the pro-library can be further procea.ed by, for example, removing the protecting groups from the pro-library molecules, to yield a second combinatorial library which contains water-soluble molecules.
  • One method for forming a combinatorial library of the invention includes (a) admixing a plurality of core molecules, each having at least one reactive center with a plurality of different tool molecule., each having at least one functional group to form a reaction mixture; and (b) reacting the reactive center, of the core molecule, with the functional group, of the tool molecule, to orm a combinatorial library of molecules.
  • the phrase "combinatorial library” refer, to a collection of atructurally-diver.e molecule.. As will be explained in sore detail below, the extent of this diversity i. dictated by the number, the nature and the ratio of the core molecules and tool molecules from which the library molecule, are aynthe.ized. Although the core molecules and tool molecule, may have natural or .ynthetic origins, typically the product of the reaction between the core and tool molecules i. a non-naturally occurring molecule. In the preferred embodiments, the library molecules have structures that are not recognized by degradative enzymes in vivo, a feature which contributes to the metabolic stability and improved absorption properties of these molecules in vivo.
  • the core molecules include at least one reactive center for reacting with the functional group of a tool molecule.
  • the core molecules serve a. a rigid or relatively rigid scaffold to which tool molecules can be linked in a fixed spatial orientation relative to one another.
  • core molecules that can be used in accordance with the methods of the invention, include, but are not limited to, the chemical compounds illustrated in figure 1 (e.g., monocyclic, polycyclic, heterocyclic and aromatic molecules) .
  • Figure 1 illustrates two classes of core molecules: (1) rigid core molecules, exemplified by the planar aromatic and planar heterocyclic molecules shown in the figure and (2) relatively rigid core molecules, exemplified by the non-aromatic monocyclic and polycyclic molecules shown in the figure.
  • the core molecules have a sufficiently rigid structure to maintain two or more tool molecules in a fixed spatial orientation relative to one another following covalent attachment of the tool molecules to the core molecule.
  • core molecules also include biochemical compounds (e.g., adenine, thymine, guanine, cytidine, uracil, ino.ine, a. well a. analog., nucleoside., and nucleotides of the foregoing nucleoba.e.) .
  • the core molecules are pharmaceutical molecules having a known biological activity.
  • the pharmaceutical core molecule are derivatized (e.g., by converting a reactive center of the pharmaceutical core molecule into a more potent nucleophile or electrophile (di.cua.ed below) to enhance reaction of the core molecule with a tool molecule containing a complementary functional group.
  • a pharmaceutical core molecule having an antibiotic activity can be reacted with a plurality of tool molecule, to form a library of derivatized antibiotic molecule.
  • the derivatized pharmaceutical molecule library can be screened using well-known colony inhibition assays to identify lead compounds which, for example, exhibit enhanced antibiotic potency and/or which confer aultidrug resistance.
  • Conventional and/or novel separation techniques and analytical methods e.g., HPLC and mass spectroscopy
  • HPLC and mass spectroscopy can be used to elucidate the structure of the novel lead molecules.
  • the invention also embrace, a method for forming a combinatorial library in which two or more different core molecules are reacted with a plurality of different tool molecules.
  • the inclusion of two or more different core molecule substantially increases the molecular diversity of the libraries formed in accordance with the methods of the invention.
  • the reactive centers of the core molecule form a covalent linkage with the functional groups of the tool molecules, although other types of linkages (e.g., a metal coordinate linkage in which a metal is used as the core molecule and the tool molecules form a coordinate bond to the metal, an ionic linkage) also fall within the scope of the invention.
  • linkages e.g., a metal coordinate linkage in which a metal is used as the core molecule and the tool molecules form a coordinate bond to the metal, an ionic linkage
  • different types of reaction mechanisms discussed below
  • the preferred reaction forms a covalent linkage between the reactive centers of the core molecule and the functional group of the tool molecule.. In general, almost any reaction for forming a covalent linkage can be used to link a tool molecule to a core molecule.
  • covalent linkages can be formed using a palladium or other transition metal catalyzed coupling reaction (see, e.g., N. Miyaura, H. Sugino e, and A. Suzuki, Tetrahedron irs ⁇ -Z-Z: 127-130 (1981)):
  • nucleophile/ ⁇ l ⁇ ctrophile for forming the covalent linkage between the core molecule and the tool molecule.
  • nucleophile and “electrophile” have their common meanings (see, e.g., March, J., ibid.).
  • electrophile such as a carbon atom of an activated carbonyl group
  • nucleophile such as an alcohol, an amine or a thiol
  • the core molecule can include one or more electrophilic reactive center( ⁇ ) which react with a nucleophilic functional group of a tool molecule (described in more detail below) to form a library molecule in which the core molecule and the tool molecule are linked via a covalent bond.
  • the core molecule can include an acid halide (e.g., an acid chloride) reactive center which is capable of reacting with a nucleophilic functional group, such as a primary amine or an alcohol, to form a library molecule in which the core molecule and the tool molecule are linked via an amide (figure 4) or an ester (figure 5) linkage, respectively.
  • the core molecule can include an alkyl halide (e.g., an alkyl chloride) reactive center which reacts with, for example, a primary amine or an alcohol, to form a library molecule including a secondary amine (figure 6) or ester linkage (figure 7) , respectively.
  • an alkyl halide e.g., an alkyl chloride
  • the core molecule can include one or more nucleophilic reactive center( ⁇ ) which are capable of reacting with an electrophilic functional group of a tool molecule to form a library molecule in which the core molecule and the tool molecule are coupled via a covalent bond.
  • the core molecule can include an alcohol reactive center which reacts with an electrophilic functional group, such as an acid halide or an alkyl halide on a tool molecule, to form a library molecule in which the core molecule and the tool molecule are linked via an e.ter (figure 8) or an ether linkage (figure 9), re ⁇ pectiv ⁇ ly. Lik ⁇ wi.
  • the core molecule can include a primary amine reactive center which, upon reaction with an acid chloride functional group or an alkyl halide, form, an amide linkage (figure 10) or a secondary amine linkage (figure 11) , respectively.
  • exemplary reactive canters include, but are not limited to, an acid halide, an amine, an alcohol and a thiol.
  • Other reactive center, and functional group will be apparent to the arti.an of ordinary .kill in the art in view of the broad categories of reactive groups disclosed herein.
  • the reactive center is an acid chloride and the functional group of the tool molecule is an amine, an alcohol or a thiol group.
  • A.B COR, COOR. COOH. CN.
  • Exemplary tool molecules include, but are not limited to, the naturally-occurring nucleobases, nucleosides, nucleotides, oligonucleotide., amino acid., peptide. and carbohydrates, a. well as non- naturally occurring analogs of the foregoing molecules. Examples of amino acid, and other primary amine. that are suitable for use a. tool molecules in the generation of combinatorial libraries are shown in figure 2. Examples of nucleobases and modified nucleobases that are suitable as tool molecules are shown in figure 3.
  • the tool molecules can be entirely or partly of synthetic origin.
  • the tool molecule e.g., amino acid.
  • the tool molecule can be purchased in their naturally occurring state (e.g., not derivatized) or in a derivatized state.
  • a plurality of amino acid tool molecules are derivatized (e.g., to attach a fat-soluble protecting group to a second functional group of the tool molecule) prior to reacting the tool molecule with a core molecule reactive center. (See, e.g., the Examples. )
  • the diverse tool molecules of the invention have in common a functional group which is capable of reacting with a reactive center of the core molecule to form a linkage.
  • the same coupling reaction conditions can be used to react any electrophile (e.g., an acyl halide, an alkyl halide) with any nucleophile to covalently couple the ⁇ lectrophile- containing molecule (e.g., the core) to the nucleophile-containing molecule (e.g., the tool).
  • any electrophile e.g., an acyl halide, an alkyl halide
  • the incubation times for completing the coupling reaction will vary depending on the reactivity of the particular complementary nucleophil ⁇ /electrophile pair participating in the reaction.
  • a strong nucleophile e.g., an amine
  • a strong electrophile e.g., an acyl chloride
  • a longer incubation time or an increase in the incubation temperature e.g., from room temperature to 40 *C
  • the optimization of reaction conditions for each complementary pair of ⁇ l ⁇ ctrophile ⁇ and nucleophil ⁇ s is within the ordinary skill of the art.
  • the tool molecules that are used to form the combinatorial library include more than one functional group, e.g., a first functional group for reacting with the core molecule and a second functional group for reacting with a second molecule, such as a protecting group, to form a second linkage.
  • a method for forming a combinatorial library further includes the step of reacting the secondary functional group with a protecting group prior to admixing the plurality of core molecules with the plurality of tool molecules.
  • a particularly preferred procedure for synthesizing a protecting group-derivatized amino acid tool molecule is provided in the Examples.
  • a fatty acid also may serve as a removable protecting group, e.g., a fatty acid coupled to a tool molecule via an ester linkage may be cleaved from the tool molecule upon exposure to aqueous basic condition.
  • the protecting group primarily serve, to modulate the solubility properties of the protecting group-derivatized tool molecule and the library molecules formed therefrom.
  • a core molecule having a hydrophilic acid chloride reactive center
  • a lysine tool molecule in which a fat-soluble, protecting group is covalently linked to the epsilon amine group
  • a combinatorial library is generated by loading a plurality of core molecules, a plurality of different tool molecules and 5 ml of dichloromethane (or other solvent in which the core molecules and tool molecules are soluble) into a round bottom flask equipped with a magnetic stir bar.
  • the addition of base to the reaction mixture in the flask typically about 1 ml triethylamine
  • starts the coupling reaction which then is allowed to continue for 1 to 24 hours (depending on the reactivities of the particular electrophiles/nucleophiles present in the reaction) at room temperature under an argon atmosphere.
  • the amounts of core molecules and tool molecules used in the coupling reaction are selected so that the molar ratio of tool molecule functional groups to core molecule reactive centers is slightly greater than, or equal to, 1.
  • This selection criterion enhances the likelihood that each core reactive center will be linked to a tool molecule and that each tool molecule (regardless of whether it contains a strong or a weak electrophilic or nucleophilic functional group) will react with a core molecule reactive center.
  • the optimum total moles of a plurality of amino acid tool molecules (assuming each amino acid has a single functional group) that should be reacted with 10 moles of a core molecule having 4 reactive centers is slightly greater than, or equal to, 40 (because there are 40 moles reactive centers in the reaction mixture) .
  • the diver.ity of the combinatorial library may be limited b ⁇ cau.e only the most reactive functional group. v Hl react with the reactive centers. If the molar ratio of tool molecule functional groups to reactive center, i. l ⁇ . ⁇ than 1.0, the combinatorial library molecules may include unreacted reactive center, which will likely exhibit .imilar ⁇ olubility properties as the unreacted core molecule and extraction of the unreacted core molecules likely will be more difficult.
  • a particularly preferred procedure for separating the unreacted tool molecules and core molecules from the library molecules, based upon the ⁇ olubility differences between the unreacted molecules and the library molecules, is described herein.
  • the unreacted core molecules, unreacted tool molecules and library molecules containing core molecules coupled to one or more tool molecules are dissolved in approximately 50 ml of an organic solvent (preferably dichloromethane) .
  • an organic solvent preferably dichloromethane
  • a weak acid e.g. , 1 M citric acid
  • the unreacted tool molecule are extracted from the organic pha.e by washing the organic phase with two 75 ml washes of a 1 M citric acid solution.
  • Exposure to citric acid also results in converting the acid chloride reactive center of each core molecule into its corresponding free acid (which remains soluble in the organic phase) .
  • a weak base e.g., 100 ml of saturated sodium hydrogen carbonate solution
  • the organic phase deprotonates the unreacted core molecule acid moiety (i.e., the core molecule now carries a negative charge) , thereby rendering the unreacted core molecules soluble in the weak base but insoluble in the organic phase and allowing extraction of the unreacted core molecules.
  • the organic phase (containing the pro-library) is dried over MgSO, to remove trace amounts of aqueous phase and is concentrated to yield an oil.
  • the oil comprising the fat-soluble pro-library is dried under high vacuum to yield a foam which can be screened directly for the presence of a lead compound or which can be processed into a second, water-soluble library prior to screening.
  • the fat-soluble pro-library is converted into a water-soluble library by removing the protecting groups from the pro-library molecules (e.g., by chemically cleaving the protecting groups from the molecules under acidic or basic conditions, see Table 2). Because of its solubility properties, the fat-soluble pro-library is useful for identifying pharmaceutical lead compounds which modulate, for example, membrane-associated biological processes. Removal of the fat-soluble protecting group from the pro-library molecules yields a library of water- soluble molecules which is useful for identifying pharmaceutical lead compounds that modulate aqueous biological processes.
  • the method for forming a combinatorial library further includes the step of converting the fat-soluble pro-library into a biologically-active state, e.g. , by removing the fat-soluble protecting groups from the pro-library molecules (as described above) or by derivatizing the fat-soluble library molecules to render them water-soluble.
  • the methods of the invention further include the step of screening the combinatorial libraries for biologically-active molecules.
  • Numerous screening methods have been reported for identifying lead compounds which modulate the activity of a particular ligate. Such methods include selecting the library molecules which bind to an immobilized ligate (e.g., an antibody), selecting molecules which compete with a soluble ligand for binding to an immobilized or membrane-associated ligate (e.g., a receptor) and selecting molecule, which modulate the activity of the ligate (e.g., an enzyme, a microbe, a virus) in a dose- dependent manner.
  • an immobilized ligate e.g., an antibody
  • selecting molecules which compete with a soluble ligand for binding to an immobilized or membrane-associated ligate e.g., a receptor
  • selecting molecule, which modulate the activity of the ligate e.g., an enzyme, a microbe, a virus
  • molecules having antimicrobial activity and/or antiviral activity can be identified using well-known colony inhibition or plaque inhibition assay., respectively.
  • conventional and/or novel separation techniques and analytical method e.g. , HPLC and ma., .pectro.copy
  • HPLC and ma., .pectro.copy are used to elucidate the structure of the novel lead molecule.
  • the extant of molecular diversity of the libraries described herein is delineated by several variables: (1) the number of reactive centers per core molecule; (2) the number of different types of core molecules; (3) the number of functional groups per tool molecule; (4) the number of different tool molecules; and (5) the ratio of tool molecules to core molecules.
  • the dependence of molecular diversity upon these variables is illustrated in the following example.
  • the reaction yields a molecular library which has diversity, the extent of which is, at least in part, defined by the values of n and X.
  • a particular set of different tool molecule e.g., a .at of amino acids or peptides, a set of nucleoside. or oligonucleotide., a .et of tool molecules including a mixture of both amino acids and nucleosides
  • combinatorial libraries containing molecules having the potential to bind to a particular type of ligate e.g., an amino acid-binding ligate, nucleoside-binding ligate
  • an automated process for generating the combinatorial libraries of the invention as well as the equipment for performing the automated process, also are provided, in view of the simplicity of the reactions disclosed herein and the state of the art with respect to the automation of more complex methods for generating combinatorial libraries, automation of the processes disclosed herein is enabled by the instant disclosure (see, e.g.. Pavia, M. , et al., ______;
  • a fat-soluble combinatorial pro-library of non-naturally occurring molecules includes at least one tool molecule linked to a core molecule.
  • the tool molecules further include a removable, fat-soluble protecting group.
  • the fat-soluble pro-library is useful for identifying pharmaceutical and/or agricultural lead compounds which modulate organic phase-associated biological processes.
  • the pro-library can be further processed into a water-soluble library that is useful for identifying pharmaceutical and/or agricultural lead compounds which modulate aqueous-associated biological processes.
  • a molecular library containing a plurality of non-naturally occurring molecule, i. provided.
  • Each molecule includes a first tool molecule and a second tool molecule covalently coupled to a core molecule.
  • the first and second tool molecules are positioned at a fixed spatial orientation relative to one another.
  • Combinatorial libraries which differ solely in the spatial orientation of the first and second tool molecules are useful for identifying the optimal spatial orientation between tool molecules for binding to a particular ligate.
  • a first library can be generated by reacting a first core molecule (having reactive centers positioned at a first spatial orientation) with a plurality of tool molecules to form a first combinatorial library.
  • a second combinatorial library can be generated by reacting a second core molecule (having reactive centers positioned at a second spatial orientation) with the same plurality of tool molecules used to generate the first combinatorial library.
  • Each library is screened to identify a lead compound( ⁇ ) and the relative biological activities of the lead compound( ⁇ ) present in the first and second libraries are compared.
  • a comparison of the biological activities for the lead compounds found in each library makes it possible to optimize the spatial orientation of the lead molecules for optimum binding to a particular ligate.
  • kits for forming a combinatorial library contain a plurality of core molecules, each having at least one reactive center and instructions for reacting the core molecules with a plurality of tool molecules to form a combinatorial library.
  • the plurality of tool molecules are included in the kit.
  • the kits contain separate pluralities of core molecules which differ from one another in the nature and/or spatial orientation of the core reactive centers.
  • the kits are useful for generating more than one combinatorial library which differ in the type (e.g., amide, aldehyde) and/or spatial orientation of the linkage connecting the core and tool molecules.
  • a combinatorial library including a plurality of non-naturally occurring molecules.
  • the library molecules include at least one tool molecule that is covalently coupled to a xanthene molecule.
  • the tool molecules are selected from the group consisting of amino acids and nucleosides. Specific reactions for preparing the particularly preferred amino acid-derivatized xanthene molecules are disclosed in the Examples.
  • the utilities of the invention include the identification of novel pharmaceutical and/or agricultural lead compounds which antagonize, agonize or otherwise modulate the physiological activity of natural ligands.
  • the libraries disclosed herein contain compounds which have the potential to modulate the functional activity of biological processes implicated in (but not limited to) disease progression, immune system modulation, and neurological signal transmission.
  • the libraries of potential lead compounds include compounds which mimic the active determinants (i.e., the ligate-binding portion of the molecule) on hormones, cytokines, enzyme substrates, enzyme cofactors, virus, microbes and fungi.
  • the combinatorial libraries of the invention also are potentially rich sources of novel antimicrobial, anti-viral and anti-fungal agents.
  • the libraries are useful for identifying the epitopes of numerous types of Kenya and ligates (e.g., receptors, antibodies, enzymes, nucleic acids, carbohydrates, lipids), including both continuous epitopes (i.e., the epitope is formed of contiguous molecules) and discontinuous epitopes (i.e., the epitope is formed by the juxtaposition of non-adjacent ligand monomers as a result of, for example, secondary/tertiary structure folding) .
  • continuous epitopes i.e., the epitope is formed of contiguous molecules
  • discontinuous epitopes i.e., the epitope is formed by the juxtaposition of non-adjacent ligand monomers as a result of, for example, secondary/tertiary structure folding.
  • the majority of naturally-occurring ligates are believed to recognize discontinuous epitopes (Pavia, M. , et al., supra ...
  • the present invention provides methods for forming combinatorial libraries of molecules which are useful for identifying pharmaceutical and/or agricultural lead compounds.
  • the following Examples illustrate a particularly preferred embodiment of the method and representative utilities of the present invention.
  • the disclosure of the present invention illustrates the reaction of a number of different core molecule/tool molecules combinations.
  • a plurality of core molecules xanthene-di-acid chloride
  • n-2 two reactive centers
  • More complex combinatorial libraries also have been prepared by reacting a plurality of core molecules (xanthene-tetra-acid chloride) having four reactive centers (n-4) with 4 (figure 12), 7 (figure 13), 12 (figure 14) or 21 (figure 15) different tool molecules (each including a single amine functional group).
  • core molecules xanthene-tetra-acid chloride
  • n-4 four reactive centers
  • HPLC high pressure liquid chromatography
  • mass spectroscopy mass spectroscopy
  • Amino acids were purchased from Nova Biochem, La Jolla, CA.
  • Solvents, acids and bases were purchased from Fluka Chemie, Ag, Buchs, Switzerland, unless otherwise noted.
  • Reagent grade solvents were used throughout the procedure unless otherwise noted.
  • HPLC Chromatography was performed using a silica column (preparative or analytical, as described below) on a Waters 600E HPLC System with a Waters 490E UV detector (UV detection at 270 nm) and a Waters 717 autosampler (Millipore Corp., Waters Chromatography Division, Milford, MA).
  • the gradient used was threefold: 100% hexane to 100% ethyl acetate to 6% methanol/94% ethyl acetate.
  • All HPLC solvents were purchased from EM Science (Gibbstown, NJ) and were EM Science's Omni Solve • grade.
  • the analytical silica column was purchased from Beckman Instruments, Inc., Fullerton, CA (part #235341, ULTRASPHERE • SI column, 4.6 mm i.d. x 25 cm length, 80 angstrom, 5 micrometer).
  • the preparative silica column was purchased from Millipore, Waters Chromatography Division, Milford, MA (part #25823, NOVA PAR • HR silica, 19 mm i.d. x 30 cm length, 68 angstrom, 6 micrometer) .
  • This combinatorial library was generated by reacting the core molecule 9,9-dimethylxanthene-2,4,5,7-tetracarboxylic acid chloride with the following derivatized amino acid tool molecules:
  • a 10 ml one-neck, round bottomed flask equipped with a magnetic stir bar was charged with xanthene-2,4,5,7-tetracarboxylic acid chloride (100 ag, 0.217 mmol) (synthesized as described in Example 10), the mixture of derivatized amino acid tool molecules (shown above) and 5 ml dichloromethane.
  • the flask was stoppered with a rubber septum containing an argon inlet.
  • One ml of triethylamine was added to the reaction mixture with a syringe and the reaction mixture was stirred under an argon atmosphere for 3 hours.
  • This combinatorial library was generated by reacting the core molecule 9,9-dimethylxanthene-2,4,5,7-tetracarboxylic acid chloride with the following mixture of derivatized amino acid tool molecules:
  • This combinatorial library was generated by reacting the core molecule 9,9-dimethylxanthene-2,4,5,7-tetracarboxylic acid chloride with the following mixture of derivatized amino acid tool molecules:
  • H-L-Ala-OMe 11.1 mg H-L-Phe-OMe, 17.2 mg H-L-Met-OMe, 15.9 mg H-L-Pro-OMe, 13.2 mg H-L-Leu-OMe, 14.5 mg H-L-Lys(Boc)-OMe , 23.6 mg H-L-Ser(tBut)-OMe, 16.9 mg H-L-His(Trt)-NHR, 34.9 mg H-L-Asp(OtBut)- Me, 19.1 mg H-L-Glu(OtBut)-OMe, 20.2 mg H-L-Thr(tBut)-OMe, 18.0 mg Fur urylamine, 8.4 mg The reaction mixture contained 0.0785 mmol of each amine.
  • This combinatorial library was generated by reacting the core molecule 9,9-dimethylxanthene-2,4,5,7-tetracarboxylic acid chloride with the following mixture of derivatized amino acid tool molecules:
  • H-L-Trp-OMe 10.5 mg H-L-Val-OMe, 6.9 mg H-L-Ala-OMe, 5.7 mg H-L-Phe-OMe, 8.9 mg H-L-Met-OMe, 8.3 mg H-L-Pro-OMe, 6.9 mg H-L-L ⁇ u-OMe, 7.5 mg H-L-Ly ⁇ (Boc)-Me, 12.3 mg H-L-Ser(tBut)-OMe, 18.8 mg H-L-His(Trt)-NHR,, 18.1 mg H-L-Asp(OtBut)-OMe, 9.9 mg H-L-Glu(OtBut)-OM ⁇ , 10.5 mg H-L-Thr(tBut)-OMe, 9.3 mg H-L-Il ⁇ -OtBut, 9.3 mg H-L-Cys(Trt)-NHR, 18.7 mg H-L-Arg(Mtr)-NHR, 21.0 mg H-L-T
  • the FMOC-protected amino acid derivatives were activated with benzotriazol-l-yloxytris-(dimethylamine ) phosphonium hexafluoro-phosphate
  • Crude compound 35 (see figure 16) was purified by suspending the compound in an appropriate organic solvent (e.g., n-hexane/ethyl acetate (1:1).
  • Compounds 36, 37 and 38 were purified by chromatography on silica gel (described below). The chromatography fractions containing these compounds were concentrated in vacuo to yield a white foam. Synthesis of Compound *..
  • N"-Fluorenylmethoxycarbonyl-L-valine-cyclohexylamide (JJS w s synthesized by reaction of N"-fluorenylmethoxycarbonyl-L-valine (50 0 mg 1.5 mmol) with cyclohexylamine (150 mg, 1.5 mmol) and BOP (680 ag, 1.54 mmol) as the coupling reagent. Isolation of product was achieved by suspending the crude product in n-h ⁇ xane/ethyl acetate (1:1) and filtration. The residue was collected and concentrated to half of its volume. The slurry thus obtained was again filtered. The combined residues were dried under reduced pressure. The product was obtained as a white solid, 410 mg (66%).
  • N-Fluorenylmethoxycarbonyl-S-Trityl-cysteine-benzylamide (2&) was synthesized by reaction of N-fluorenylmethoxycarbonyl-S-trity ⁇ cysteine (500 ag, 0.85 mmol) with benzylamine (100 mg, 0.93 mmol) and BOP (500 ng, 1.1 mmol) as the coupling reagent.
  • the product was purified by chromatography on silica gel with n-hexane/ethyl acetate (2:l)eluant, R, -0.59. Yield - 442 mg (80%).
  • N*-Fluorenylmethoxycarbonyl-N*'"-trityl-histidine-prop-l-ylamide (38) was synthesized by reaction of N"-fluorenyaethoxycarbonyl-N*" i '-trityl- histidine (500 mg, 0.81 mmol) with n-propylamine (60 mg, 1.0 mmol) and BOP (500 mg, 0.9 mmol) as the coupling reagent.
  • the product was purified by chromatography on silica gel with ethyl acetate as eluant, R, - 0.80. Yield - 315 mg (59%).
  • the free amine (i.e., FMOC-deprotected) product was eluted with methanol/triethylamine (99:1) and was detectable on a kieselgel-precoated TLC plate (Merck * Co., Inc., Rahway, NJ) by staining with ninhydrin solution. Fractions containing the free amine product were concentrated in vacuo to afford a white foam with a yield greater than 90 %.
  • romnoun d 21 (Nx-( 4-Methoxy-2 , 3 , 6-trim ⁇ thyl-b ⁇ nz ⁇ n ⁇ - ⁇ ulfonyl )-L- arginine-4-methoxybenzylamid ⁇ (21)): ⁇ NMR (300 MHz, DMSO-d * 8.24 (br. ⁇ ,lH,NH) , 7.15 (d,2H,J - 8.5 Hz,ar-H), 6.85 (d,2H,J - 7.3 Hz,ar-H), 6.67 ( ⁇ ,lH,ar-H), 6.40 (br.
  • This combinatorial library was generated by reacting the core molecule xanthene-2,4,5,7-tetracarboxylic acid chloride with the following mixture of derivatized amino acid tool molecules: H-L-Ala-Ot-But, 15.7 mg; H-L-Tyr(t-But)-OH, 20.5 mg; H-L-Arg(Mtr)-OH, 33.3 mg; H-L-Trp-OMe, 22.0 mg; H-L-Ser(t-But)-OH, 18.3 mg; H-L-Glu(Ot-But)-Ot-But, 25.5 mg; H-L-Asn-Ot-But, 19.4 mg; H-L-Val-Ot-But, 18.1 mg; H-L-Asp(Ot-But), 24.3 mg; H-L-Pro-Ot-But, 18.0 mg; H-L-Thr(t-But)-OH, 15.1 mg; H-Hi ⁇ (Trt)-OH, 34.3 ag.
  • the reaction mixture contained 0.0865 mmol of each derivatized amino acid tool molecule.
  • This library was generated using the procedure disclosed in Example l.
  • the reaction mixture was concentrated to yield a tan oil which was dried under high vacuum conditions.
  • the dried oil was treated with cold diethylether to extract the cleaved protecting groups, leaving behind a white solid that was collected and dried in vacuo.
  • the white solid (50 mg) is dissolved in water (e.g., 0.5 ml) or a buffered aqueous solution for analysis.
  • an appropriate amount of the water-soluble combinatorial library is dissolved in a minimum amount of water or buffered solution for performing the screening assay.
  • "appropriate amount” refers to an amount which is within the detection limits of the screening assay. In general, the detection limits for an ELISA screening assay are between about 0.1 nM - 0.05 «. Thus, the "appropriate amount" of a library molecule for dissolution will be less for a screening method which employs a more sensitive detection method (e.g., a radiolabeled tag or amplification of the signal prior to detection) and will be more for a screening method which employs a less sensitive detection method.
  • a more sensitive detection method e.g., a radiolabeled tag or amplification of the signal prior to detection
  • An affinity matrix containing a plurality of ligate molecules immobilized to an insoluble support e.g., polyacrylamide, agarose, sepharose
  • an insoluble support e.g., polyacrylamide, agarose, sepharose
  • the affinity matrix is washed with buffer prior to applying the combinatorial library molecules.
  • An aliquot of serial dilutions of the combinatorial library (or a fraction thereof, such as a fraction eluted from an HPLC column) is added to the affinity matrix, followed by washing the matrix to remove unbound or non-specifically bound library molecules.
  • the matrix is contained in a column or is contained in, for example, a microcentrifuge tube for performing a batch separation.
  • the elution of unbound or non-specifically bound library molecules from the affinity matrix is determined by monitoring, for example, the absorbance at the wavelength at which the library molecules are known to absorb light, or by other detection methods (e.g., thin layer chromatography). Elution fractions containing a detectable amount of library molecules are collected, concentrated and analyzed.
  • the affinity matrix is rinsed with a concentrated solution of a ligand that is known to bind to a specific region of the ligate. Accordingly, washing the matrix with a solution of the ligand specifically elutes compounds that bind to the same region of ligate.
  • the eluted fraction(s) are subjected to the analysis procedure (described below).
  • the affinity matrix is rinsed with a solution of a denaturant which denatures the immobilized ligate, thereby releasing library molecules which have specifically bound to the immobilized ligate.
  • the eluted fraction(s) then are subjected to the analysis procedure.
  • the following example is intended to illustrate the above-disclosed general process for using an immobilized ligate to identify lead compounds in a combinatorial library.
  • the example is not intended to limit the invention to a particular embodiment.
  • the carbohydrate-binding protein concanavalin A (Con A) covalently bound to sepharose is purchased (e.g., Sigma Chemical Co., St. Louis, MO).
  • An appropriate amount of the library molecules are added to 20 ul of Con A- Sepharose contained in a aicrocentifuge tube, in a final volume of 200 ul Con A Buffer (50 mM NaCl/20 mM Mops, pH 6.8/2 mM MgClj/2 mM CaCi o.2 mM EOTA).
  • Con A Buffer 50 mM NaCl/20 mM Mops, pH 6.8/2 mM MgClj/2 mM CaCi o.2 mM EOTA.
  • the library molecules are allowed to bind to the Con A-S ⁇ pharose for 1 - 24 hours at room temperature with agitation. Unbound or non-specifically bound library molecules are removed by washing the Con A-Sepharose with Con A Buffer (e.g., three 5 minute washes) (described above). Specifically-bound library molecules are eluted with either 200 mM methyl alpha-D-mannopyranoside, 1 % mannan, or 100 mM citrate buffer (pH 3.0) for at least about 30 minutes at room temperature and are subjected to the analysis procedure (described below).
  • Con A Buffer e.g., three 5 minute washes
  • Fvamole 7 Screening procedures using a soluble ligate.
  • Combinatorial library molecules are assayed in a competition ELISA format over a concentration range of about o.l nM - 0.05 mM for each library molecule using procedures familiar to the artisan of ordinary skill in the art (see, e.g., Zuckermann, R. , et al., Proc. Nat. Acad. Sci. USA fl9_:4505-4509 (1992).
  • Microtiter plates are coated (e.g., about 0.2 ug ligand per well in 50 mM borate, pH 9.0, overnight at 4 *C) with a known ligand of the antibody ligate for which lead compounds are being screened.
  • ligand-coated microtiter wells To the ligand-coated microtiter wells is added a 50 ul aliquot of serial dilutions of the combinatorial library (or a fraction thereof, such as a fraction eluted from an HPLC column) with 50 ul of a diluted ligate antibody solution that is known to give a positive, detectable ELIS ⁇ signal when incubated with the ligand-coated microtiter well in the absence of a soluble competitor of the immobilized ligand. Wells to which no combinatorial library molecules are added are included as assay controls. Typically, the incubations are performed in a Trie-buffer for about one hour at 37 * C.
  • microtiter plates are washed to remove unbound or non- specifically bound antibody ligate, followed by incubating with a detection reagent (e.g., 100 ul of horseradish peroxidase-conjugated goat anti-mouse antibody (stock solution of 1 ag/ml diluted 1:1000, Boehringer Mannheim)) for about one hour at 37 *C and washed as above to remove unbound or non- specifically bound conjugated antibody.
  • a detection reagent e.g., 100 ul of horseradish peroxidase-conjugated goat anti-mouse antibody (stock solution of 1 ag/ml diluted 1:1000, Boehringer Mannheim)
  • the amount of bound conjugated antibody is quantitated by color development with 100 ul of o- phenylenediamine at 5 mg/ml in 50 mM sodium citrate/0.02% H.O., pH 5.1 and measurement of the absorbance at 450 nm.
  • the presence of a library molecule which specifically inhibits the binding of the antibody ligate to its known (immobilized) ligand is indicated by a reduction in the absorbance at 450 nm.
  • the library molecules which specifically inhibit the binding are subjected to the analysis procedure described below.
  • a known number of tool molecules (X) are used to generate a first combinatorial library.
  • the library is screened to identify library molecules which modulate the biological activity of an immobilized ligate (see, e.g., Example 6) or a soluble ligate (see, e.g., Example 7).
  • an immobilized ligate see, e.g., Example 6
  • a soluble ligate see, e.g., Example 7
  • the ability of a library molecule to modulate the biological activity of an enzyme is determined by observing a change in enzyme activity as increasing amounts of library molecules are included in the enzyme assay reaction mixture.
  • a number of different screening assays can be used to quantitate the effect of the first combinatorial library on the biological activity of the ligate.
  • a second combinatorial library is prepared using the same mixture of tool molecules as used to generate the first combinatorial library with one exception: one of the tool molecules included in the generation of the first library is absent from the mixture of tool molecules used to generate the second library.
  • a fraction containing the library molecules of interest (e.g., a fraction which has exhibited binding to an immobilized ligate) is subjected to mass spectroscopy and the empirically obtained mass values are compared to the theoretical mass values calculated for each possible compound in the combinatorial library. By matching the empirically obtained mass values and calculated mass values, the molecular weights of library molecules that are able to bind to the ligate are deduced.
  • the structures of the library molecules of interest are deduced from their molecular weights.
  • an iterative process is used to identify the structure of the biologically active library molecule.
  • the iterative process involves generating a second combinatorial library using only those tool molecules having molecular weights which the mass spectroscopy data indicates are present in the biologically active fraction.
  • the second library contains a higher concentration of the biologically-relevant library molecules, a factor which facilitates mass spectroscopy analysis.
  • the second library is screened, subjected to mass spectroscopy and the empirical mass values are compared to the theoretic mass values (as described above) to deduce the structures of the library molecules of interest. This process is repeated until there is sufficient molecular weight data to determine the structure of the biologically active library molecule.
  • EXAMPLE 10- Synthesis of 9. -dimethvlxanthene diacvl- and tetraacyl- chloride.
  • the xanthene tetranitrile (5.00 g, 1 eq) was suspended in H,0 (20 mL). Sodium hydroxide (3.21 g) in 20 mL water was added, and the brown mixture was stirred at reflux for 14 h.

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Abstract

On décrit des procédés permettant de former des banques combinatoires, et les banques produites de cette manière. Selon un aspect préféré de l'invention on fait réagir une pluralité de molécules nucléaires avec une pluralité de molécules outils différentes pour former une banque de molécules ayant une diversité moléculaire n'existant pas naturellement. Ces banques sont utiles pour identifier des composés 'chefs de file' qui modulent l'activité fonctionnelle d'une molécule biologique.
PCT/US1998/002812 1997-02-14 1998-02-13 Procede permettant de creer de la diversite moleculaire WO1998035923A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001002355A2 (fr) * 1999-07-01 2001-01-11 Axys Pharmaceuticals, Inc. Syntheses d'indoles
WO2016033095A1 (fr) * 2014-08-26 2016-03-03 Drexel University Nouveaux procédés de préparation de protéoglycanes biomimétiques

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995019359A1 (fr) * 1994-01-12 1995-07-20 Massachusetts Institute Of Technology Procede de production de composes a base de xanthene ou de cubane et inhibitors de protease

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995019359A1 (fr) * 1994-01-12 1995-07-20 Massachusetts Institute Of Technology Procede de production de composes a base de xanthene ou de cubane et inhibitors de protease

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001002355A2 (fr) * 1999-07-01 2001-01-11 Axys Pharmaceuticals, Inc. Syntheses d'indoles
WO2001002355A3 (fr) * 1999-07-01 2001-06-28 Axys Pharm Inc Syntheses d'indoles
WO2016033095A1 (fr) * 2014-08-26 2016-03-03 Drexel University Nouveaux procédés de préparation de protéoglycanes biomimétiques

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Publication number Publication date
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