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WO1997029371A1 - Procede de marquage par taux de soufre pour bibliotheques combinatoires - Google Patents

Procede de marquage par taux de soufre pour bibliotheques combinatoires Download PDF

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Publication number
WO1997029371A1
WO1997029371A1 PCT/US1997/002109 US9702109W WO9729371A1 WO 1997029371 A1 WO1997029371 A1 WO 1997029371A1 US 9702109 W US9702109 W US 9702109W WO 9729371 A1 WO9729371 A1 WO 9729371A1
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WO
WIPO (PCT)
Prior art keywords
sulfur
beads
combinatorial
library
combinatorial library
Prior art date
Application number
PCT/US1997/002109
Other languages
English (en)
Inventor
Joseph Weinstock
Original Assignee
Smithkline Beecham Corporation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Smithkline Beecham Corporation filed Critical Smithkline Beecham Corporation
Priority to EP97906546A priority Critical patent/EP0891548A1/fr
Priority to JP09528733A priority patent/JP2000510819A/ja
Publication of WO1997029371A1 publication Critical patent/WO1997029371A1/fr

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Classifications

    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B20/00—Methods specially adapted for identifying library members
    • C40B20/04—Identifying library members by means of a tag, label, or other readable or detectable entity associated with the library members, e.g. decoding 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
    • B01J2219/00277—Apparatus
    • B01J2219/0054—Means for coding or tagging the apparatus or the reagents
    • B01J2219/00572—Chemical means
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N2030/022—Column chromatography characterised by the kind of separation mechanism
    • G01N2030/025—Gas chromatography

Definitions

  • the field of this invention concerns combinatorial chemistry which involves the syntheses of one or more encoded combinatorial libraries where large numbers of products having varying compositions are obtained. This invention also relates to methods of encoding combinatorial libraries.
  • the standard method for conducting a search is to screen a variety of pre-existing chemical moieties, for example, naturally occurring compounds or compounds which exist in synthetic libraries or databanks.
  • the biological activity of the pre-existing chemical moieties is determined by applying the moieties to an assay which has been designed to test a particular property of the chemical moiety being screened, for example, a receptor binding assay which tests the ability of the moiety to bind to a particular receptor site.
  • Nonpeptidic organic compounds such as peptide mimetics
  • peptide mimetics can often surpass peptide ligands in affinity for a certain receptor of enzyme.
  • An effective strategy for rapidly identifying high affinity biological ligands, and ultimately new and important drugs, requires rapid construction and screening of diverse libraries of non-peptidic structures containing a variety of structural units capable of establishing one or more types of interactions with a biological acceptor (e.g., a receptor or enzyme), such as hydrogen bonds, salt bridges, pi-complexation, hydrophobic effects, etc.
  • a biological acceptor e.g., a receptor or enzyme
  • work on the generation and screening of synthetic test compound libraries containing nonpeptidic molecules is now in its infancy.
  • a key unsolved problem in the area of generation and use of nonpeptide libraries is the generation and use of nonpeptide libraries is the elucidation of the structure of molecules selected from a library that show promising biological activity.
  • An attempt to uncover the structures of peptides selected from a library using unique nucleotide sequence codes, which are synthesized in tandem with the peptide library, has been described by Brenner and Lerner (Brenner, S. and Lerner, R.A. Proc. Nat'l. Acad. Sci. USA. 1992 89 . 5381-5383).
  • the nucleotide sequence of the code attached to each peptide must be amplifiable via the polymerase chain reaction (PCR).
  • nucleotide synthesis techniques are not compatible with all of the synthetic techniques required for synthesis of many types of molecular libraries.
  • the close proximity of nucleotide and synthetic test compound in the library which can result in interactions between these molecules interfering with the binding of the ligand with a target receptor of enzyme during the biological assay, also limits this approach.
  • the nucleotide component of the library can also interfere during biological assays in a variety of other ways. Kerr et al. (J. Am. Chem. Soc. 1993, 115, 2520-2531) reported synthesizing solution phase libraries of peptides, containing non-natural amino acid residues, in parallel with peptide coding strands.
  • the peptide ligand and its coding strand in this library are covalently joined together, which allows isolation and sequence determination of pairs of synthetic test compound and corresponding code.
  • the coding peptide may interfere with the screening assay.
  • PCT/US93/09345 describes a method of identifying actives in a combinatorial library by attaching multiple tags in a predetermined binary coding system.
  • PCT/HU93/0030 describes fluorescently labeled sub-library peptide kits for use in peptide synthesis.
  • PCT/US94/06078 describes methods of encoding combinatorial libraries using polymeric sequences.
  • Shearer et al. describes the application of gas chromatography and flameless sulfur chemiluminescence detection to the analysis of petroleum products.
  • This invention relates to a method of encoding combinatorial libraries which comprises utilization of sulfur identifiers. This invention also relates to a method of encoding each choice of a combinatorial library with sulfur identifiers and combinatorial libraries encoded thereby. This invention also relates to beads with sulfur identifiers attached thereto. This invention also relates to beads with varying ratios of sulfur identifiers attached thereto.
  • beads means any solid support material capable of providing a base for combinatorial syntheses, such as 1 to 2% crosslinked polystyrene, polyacrylamide, polyethylene glycol polystyrene co-polymer, preferably polystyrene modified to inco ⁇ orate a polyethylene glycol side chain.
  • tag unless otherwise indicated, means an encoding characteristic of a bead or group of beads, such as differences in size, differences in material composition or differences in flow properties using varying ratios of sulfur identifiers.
  • sulfur identifier or "identifier” means a coding label attached to a bead or group of beads by adding varying ratios of sulfur moieties, preferably two, different sulfur moieties in varying ratios.
  • the term "intensity-differentiated” means an identifier (as used herein) in which varying ratios of different sulfur moieties are added to a bead or group of beads.
  • the term “choice” means the alternative variables for a given stage in a combinatorial synthesis (not limited to peptide chemistry), such as reactant, reagent, reaction conditions, and combinations thereof.
  • stage corresponds to a step in the sequential synthesis of a compound or ligand; the compound or ligand being the final product of a combinatorial synthesis.
  • registration as used herein, has the same meaning as the term "stage” as indicated above.
  • tags which reveal the reaction history of a particular bead either by themselves, or if not all reaction steps are labeled, in conjunction with MS are particularly useful.
  • Recently several convenient selective sulfur GC detectors flameless sulfur chemiluminescence detectors have been marketed at least one of which has a >5% accuracy starting at low picogram levels and extending over 3 to 4 orders of magnitude. These detectors do not respond to carbon or nitrogen thus the analyses are relatively free of interferences. Since the detector measures only the sulfur content of the peak, it is independent of the structure of the sulfur containing compounds.
  • the present invention provides for the identification of a large number of events on resin beads by the sulfur ratios and retention times with as few as 2 or 3 tags.
  • a typical large bead library will contain beads containing about 15 nmol of functionality for attaching ligands.
  • the amounts of 1 to 1000 pg of sulfur can currently be determined accurately enough so that factors of 2 can be distinguished.
  • the individual identifiers will have very similar reactivity for attachment and subsequent cleavage, thus, by varying two identifiers, 21 unique ratios of tags are available with a ratio range of 0.001 to 1000.
  • Using 3 identifiers in one step gives 126 identifiable ratios.
  • These sublibraries can be mixed and a third step carried out with numerous reagents to give very large sublibraries. For example, 100 third step reagents could give a tagged library of 1,586,600 (15,876 x 100) compounds using only 6 tags.
  • 3 tags including an allowance for wastage would require 1000 pg (0.032 nmole) of sulfur. This compares to about 15 nmol of functionality on a 250 micron bead and 0.15 nmol on a typical commercial polyethylene glycol (PEG) - polystyrene resin.
  • PEG polyethylene glycol
  • 6 tags could identify the reaction histories of 8100 sublibrary compounds, and for example, 81,000 compounds after a third step using only ten differentiating reagents.
  • the first tag may be attached to the resin by the same linker system as used by the ligand.
  • the tag will be in the ligand bioassay solution but at its low concentration will not interfere with the bioassay, and since the tag detector is sulfur selective and retention time selective, the ligand will generally not interfere with tag analysis.
  • the number of groups of beads will correspond to the number of choices in the first differentiating step.
  • the entirety of each group is entered into a separate container.
  • the beads will usually be divided up into groups of at least one bead each, usually a plurality of beads, generally 1000 or more, and may be 10 ⁇ or more depending on the total number of registries involved in the library and the number of beads desired for each final compound.
  • the procedure of dividing beads, followed by a synthetic stage (to form a registry) including a tagging step, and then recombining beads is iterated until the combinatorial library is completed.
  • the same reaction may be carried out in a manner to enhance the proportion of product having a particular substituent in a particular registry as compared to the other choices.
  • one or more of the registries may involve a portion of the beads being set aside and undergoing no reaction, so as to enhance the variability associated with the final product.
  • batches may be taken along entirely different synthetic pathways.
  • the library thus prepared will contain tagged beads which identify the reaction sequence of each choice therein.
  • intensity-differentiated sulfur-labeled beads can be prepared by the method outlined in Scheme 1 below.
  • a sample of beads preferably a polyethylene glycol modified polystyrene resin, 100 - 300 micron particles, is used.
  • the beads are divided into N pools.
  • the members of each pool are derivatized with an identifier unique to that pool.
  • the R and R groups of 2 are chosen such that the final 4- hydroxbenzenesulfonamides of 4 are separable by capillary GC either directly or after derivatization. Solutions of known concentrations of two or three different examples of 2 are prepared and aliquots are mixed to give solutions whose component ratios are precisely known.
  • tags are introduced into the GC by autosampler thus allowing unattended high throughput.
  • Another type of tag for use herein is indicated in scheme 1 as 5 (which can be used on place of 3) which releases the GC tag by oxidation with Ce(NH_i)2(NO3)6-
  • Scheme 1 Another type of tag for use herein is indicated in scheme 1 as 5 (which can be used on place of 3) which releases the GC tag by oxidation with Ce(NH_i)2(NO3)6-
  • linker strategies which could be used in the presently invented tagging methods are discussed in PCT US93/09345.
  • a combinatorial library is prepared, each choice therein being encoded by a tag using sulfur identifiers, and tested for biological activity.
  • An additionally preferred aspect of this invention relates to combinatorial libraries prepared using beads encoded by sulfur identifiers and to pharmaceutically active compounds identified by such combinatorial library.

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Hematology (AREA)
  • Medicinal Chemistry (AREA)
  • Urology & Nephrology (AREA)
  • Organic Chemistry (AREA)
  • Biochemistry (AREA)
  • Biomedical Technology (AREA)
  • Analytical Chemistry (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Microbiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Food Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)

Abstract

L'invention concerne un procédé permettant de préparer des bibliothèques combinatoires, ainsi que des bibliothèques combinatoires ainsi préparées. Elle concerne également un procédé permettant d'identifier, dans une bibliothèque combinatoire, des composés ayant les caractéristiques désirées, ainsi qu'un procédé permettant de coder des bibliothèques combinatoires en utilisant des perles codées par des identificateurs soufrés.
PCT/US1997/002109 1996-02-12 1997-02-12 Procede de marquage par taux de soufre pour bibliotheques combinatoires WO1997029371A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP97906546A EP0891548A1 (fr) 1996-02-12 1997-02-12 Procede de marquage par taux de soufre pour bibliotheques combinatoires
JP09528733A JP2000510819A (ja) 1996-02-12 1997-02-12 コンビナトリアルライブラリーについての硫黄比率標識法

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US1149096P 1996-02-12 1996-02-12
US1745796P 1996-05-17 1996-05-17
US60/017,457 1996-05-17
US60/011,490 1996-05-17

Publications (1)

Publication Number Publication Date
WO1997029371A1 true WO1997029371A1 (fr) 1997-08-14

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Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1997/002109 WO1997029371A1 (fr) 1996-02-12 1997-02-12 Procede de marquage par taux de soufre pour bibliotheques combinatoires

Country Status (3)

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EP (1) EP0891548A1 (fr)
JP (1) JP2000510819A (fr)
WO (1) WO1997029371A1 (fr)

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
BIOPOLYMERS (PEPTIDE SCIENCE), 1995, Vol. 37, LEBL et al., "One-Bead-One-Structure Combinatorial Libaries", pages 177-198. *
EXP. OPIN. THER. PATENTS., September 1995, Vol. 5, No. 9, DEAN P.M., "Synthetic Peptide Combinatorial Libraries (SPCLs)", pages 887-899. *
JOURNAL OF IMMUNOLOGICAL METHODS, 1995, Vol. 180, LAM et al., "Application of a Dual Color Detection Scheme in the Screening of a Random Combinatorial Peptide Library", pages 219-223. *
PERSPECTIVES IN DRUG DISCOVERY AND DESIGN, April 1995, Vol. 2, No. 2, MADDEN et al., "Synthetic Combinatorial Libraries: Views on Techniques and Their Application", pages 269-285. *
PROC. NATL. ACAD. SCI. U.S.A., December 1993, Vol. 90, OHLMEYER et al., "Complex Synthetic Chemical Libraries Indexed with Molecular Tags", pages 10922-10926. *

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Publication number Publication date
JP2000510819A (ja) 2000-08-22
EP0891548A1 (fr) 1999-01-20

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