United States Patent
`Cargill et al.
`
`[19]
`
`[54] METHODS AND APPARATUS FOR
`SYNTHESIZING LABELED
`COMBINATORIAL CHEMISTRY LIBRARIES
`
`[75] Inventors: John Cargill, San Diego; Robert W.
`Armstrong, Los Angeles, both of Calif.
`
`Assignee: Irori, La Jolla, Calif.
`
`Appl. No.: 08/383,766
`
`Filed:
`
`Feb. 2, 1995
`
`Related US. Application Data
`
`Continuation-in-part of application No. 08/180,863, Jan. 13,
`1994, abandoned, which is a continuation-in-part of appli
`cation No. 08/092,862, Jul. 16, 1993, abandoned.
`
`Int. Cl.7 ..................... .. G01N 33/543; G01N 33/544
`
`US. Cl. ........................ .. 436/518; 436/524; 436/525;
`436/526; 436/527; 436/528; 530/334; 435/4
`Field of Search ................................... .. 436/518, 523,
`436/524, 525, 526, 527, 528; 530/334,
`335
`
`US006087186A
`Patent Number:
`Date of Patent:
`
`[11]
`[45]
`
`6,087,186
`Jul. 11,2000
`
`Gallop et al., “Applications of Combinatorial Technologies
`to Drug Discovery. 1. Background and Peptide Combinato
`rial
`Libraries”, Journal of Medicinal Chemistry,
`37:1233—1251 (1994).
`Geysen et al., “Strategies for Epitome Analysis Using Pep
`tide Synthesis”, Journal of Immunological Methods,
`102:259—274 (1987).
`Gordon et al., “Applications of Combinatorial Technologies
`to Drug Discovery. 2. Combinatorial Organic Synthesis,
`Library Screening Strategies, and Future Directions”, J our
`nal ofMedicinal Chemistry, 37:1385—1401 (1994).
`Houghten, “General Method for the Rapid Solid—Phase
`Synthesis of Large Numbers of Peptides: Speci?city of
`Antigen—Antibody Interaction at the Level of Individual
`Amino Acids”, Proc. Natl. Acad. Sci. USA, 82:5131—5135
`(1985).
`Jung et al., “Multiple Peptide Synthesis Methods and Their
`Applications”, Angew. Chem. Int. Ed. Engl, 31:367—383
`(1992).
`Kerr et al., “Encoded Combinatorial Peptide Libraries Con
`taining Non—Natural Amino—Acids”, J. Am. Chem. Soc.,
`115:2529—2531 (1993).
`
`[56]
`
`References Cited
`
`(List continued on next page.)
`
`U.S. PATENT DOCUMENTS
`
`5,351,052
`5,525,962
`
`9/1994 D’Hont et al. .......................... .. 342/42
`6/1996 Urbas et a1. ..................... .. 340/870.17
`
`FOREIGN PATENT DOCUMENTS
`
`WO 93/06121
`WO 94/08051
`
`4/1993 WIPO.
`4/1994 WIPO.
`
`OTHER PUBLICATIONS
`
`Brenner et al., “Encoded Combinatorial Chemistry”, Proc.
`Natl. Acad. Sci. USA, 89:5381—5383 (1992).
`Fodor et al., “Light—Directed, Spatially Addressable Parallel
`Chemical Synthesis”, Science, 251:767—773 (1991).
`Furka et al., “General Method for Rapid Sythesis of Multi
`component Peptide Mixtures”, Int. J. Peptide Protein Res.,
`37:487—493 (1991).
`
`Primary Examiner—Stephanie Zitomer
`Attorney, Agent, or Firm—BroWn, Martin, Haller &
`McClain
`
`[57]
`
`ABSTRACT
`
`The present invention provides labeled synthetic libraries of
`random oligomers and methods and apparatus for generating
`labeled synthetic oligomer libraries. Each member of such a
`library is labeled With a unique identi?er tag that speci?es
`the structure or sequence of the oligomer. In a preferred
`embodiment of the present invention the identi?er tag is a
`microchip that is pre-encoded or encodable With information
`that is related back to a detector When the identi?er tag is
`pulsed With electromagnetic radiation.
`
`49 Claims, 13 Drawing Sheets
`
`1
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`
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`
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`@ is 0 5018505 support MLN is a linker
`
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`
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`

`

`6,087,186
`Page 2
`
`OTHER PUBLICATIONS
`
`Needels et al., “Generation and Screening of an Oligonucle
`otide—Encoded Sythetic Peptide Library”, Proc. Natl. Acad.
`Sci. USA, 90:10700—10704 (1993).
`Nielsen et a1., “Synthetic Methods for the Implementation of
`Encoded Combinatorial Chemistry”, J. Am. Chem. Soc.,
`115:9812—9813 (1993).
`
`Ohlmeyer et a1., Complex Synthetic Chemical Libraries
`IndeXed With Molecular Tags, Proc. Natl. Acad. Sci. USA,
`90:10922—10926 (1993).
`
`E]. Moran et al. J. Amer. Chem. Soc., 1995, vol. 117, pp.
`10787—10788.
`
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`

`

`U.S. Patent
`
`Jul. 11,2000
`
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`
`6,087,186
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`

`

`U.S. Patent
`
`Jul. 11, 2000
`
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`

`

`U.S. Patent
`
`Jul. 11,2000
`
`Sheet 5 0f 13
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`6,087,186
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`Luminex/Irori - Page 7
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`Luminex/Irori - Page 8
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`

`

`U.S. Patent
`
`Jul. 11,2000
`
`Sheet 8 0f 13
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`6,087,186
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`
`

`

`U.S. Patent
`
`Jul. 11,2000
`
`Sheet 9 0f 13
`
`6,087,186
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`

`

`U.S. Patent
`
`Jul. 11,2000
`
`Sheet 10 0f 13
`
`6,087,186
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`

`

`U.S. Patent
`
`Jul. 11,2000
`
`Sheet 11 0f 13
`
`6,087,186
`
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`
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`

`

`U.S. Patent
`
`Jul. 11,2000
`
`Sheet 12 0f 13
`
`6,087,186
`
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`

`

`U.S. Patent
`
`Jul. 11,2000
`
`Sheet 13 0f 13
`
`6,087,186
`
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`
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`

`

`6,087,186
`
`1
`METHODS AND APPARATUS FOR
`SYNTHESIZING LABELED
`COMBINATORIAL CHEMISTRY LIBRARIES
`
`CROSS REFERENCE TO RELATED
`APPLICATIONS
`
`The present application is a continuation-in-part of appli
`cation Ser. No. 08/180,863 ?led Jan. 13, 1994, noW
`abandoned, Which is a continuation in part of application
`Ser. No. 08/092,862 ?led Jul. 16, 1993, noW abandoned.
`
`CLAIMS
`ABSTRACT
`
`FIELD OF THE INVENTION
`
`The present invention relates to labeled combinatorial
`synthesis libraries and methods and apparatus for labeling
`individual library members of a combinatorial synthesis
`library With unique identi?cation tags that facilitate eluci
`dation of the structures of the individual library members
`synthesiZed.
`
`BACKGROUND OF THE INVENTION
`
`The relationship betWeen structure and function of mol
`ecules is a fundamental issue in the study of biological
`systems. Structure-function relationships are important in
`understanding, for eXample, the function of enZymes, cel
`lular communication, and cellular control and feedback
`mechanisms. Certain macromolecules are knoWn to interact
`and bind to other molecules having a speci?c three
`dimensional spatial and electronic distribution. Any macro
`molecule having such speci?city can be considered a
`receptor, Whether the macromolecule is an enZyme, a
`protein, a glycoprotein, an antibody, an oligonucleotide
`sequence of DNA, RNA or the like. The various molecules
`that receptors bind are knoWn as ligands.
`Pharmaceutical drug discovery is one type of research that
`relies on the study of structure-function relationships. Most
`contemporary drug discovery involves discovering novel
`ligands With desirable patterns of speci?city for biologically
`important receptors. Thus, the time necessary to bring neW
`drugs to market could be greatly reduced by the discovery of
`novel methods Which alloW rapid screening of large num
`bers of potential ligands.
`Since the introduction of solid phase synthesis methods
`for peptides and polynucleotides neW methods employing
`solid phase strategies have been developed that are capable
`of generating thousands, and in some cases even millions, of
`individual peptide or nucleic acid polymers using automated
`or manual techniques. These synthesis strategies, Which
`generate families or libraries of compounds, are generally
`referred to as “combinatorial chemistry” or “combinatorial
`synthesis” strategies.
`Combinatorial chemistry strategies can be a poWerful tool
`for rapidly elucidating novel ligands to receptors of interest.
`These methods shoW particular promise for identifying neW
`therapeutics. See generally, Gorgon et al., “Applications of
`Combinatorial Technologies to Drug Discovery: II. Combi
`natorial Organic Synthesis, Library Screening Strategies,
`and Future Directions,” J. Med. Chem 37:1385—401 (1994)
`and Gallop et al., “Applications of Combinatorial Technolo
`gies to Drug Discovery: I. Background and Peptide Com
`binatorial Libraries,” J. Med. Chem 37:1233—51 (1994). For
`eXample, combinatorial libraries have been used to identify
`nucleic acid aptamers, Latham et al., “The Application of a
`Modi?ed Nucleotide in Aptamer Selection: Novel Thrombin
`Aptamers Containing 5-(1-Pentynyl)-2‘-DeoXy Uridine,”
`Nucl. Acids Res. 22:2817—2822 (1994); to identify RNA
`ligands to reverse transcriptase, Chen & Gold, “Selection of
`High-Affinity RNA Ligands to Reverse Transcriptase: Inhi
`bition of cDNA Synthesis and RNase H Activity,” Biochem
`istry 33:8746—56 (1994); and to identify catalytic antibodies
`speci?c to a particular reaction transition state, Posner et al.,
`“Catalytic Antibodies: Perusing Combinatorial Libraries,”
`Trends. Biochem. Sci. 19:145—50 (1994).
`The diversity of libraries generated using combinatorial
`strategies is impressive. For example, these methods have
`
`15
`
`20
`
`25
`
`30
`
`35
`
`TABLE OF CONTENTS
`CROSS REFERENCE TO RELATED APPLICATIONS
`FIELD OF THE INVENTION
`BACKGROUND OF THE INVENTION
`GLOSSARY
`SUMMARY OF THE INVENTION
`BRIEF DESCRIPTION OF THE FIGURES
`DETAILED DESCRIPTION OF THE INVENTION
`I. Labeled Oligomer Libraries
`II. Methods for Generating Labeled Oligomer Libraries
`III. Identifying the Sequence of Any Oligomer
`IV. Types of Identi?er Tags
`V. Linking the Oligomers to the Identi?er Tags
`VI. Encoding the Identi?er Tag Information
`VII. Recovering and Decoding the Identi?er Tag Infor
`mation
`VIII. Screening Receptors With Labeled Synthetic Oligo
`mer Libraries
`EXAMPLE I. SYNTHESIS OF ONE-HUNDRED
`AMIDES
`EXAMPLE II. SYNTHESIS ON ELAMSTM OF FOUR
`PENTAPEPTIDES
`A. DerivatiZation of ELAMSTM
`B. Preparation of Boc-Gly-L-Phe-L-Leu-OH
`C. Preparation of Gly-L-Phe-L-Leu ELAMSTM
`D. Preparation of Gly-Gly-L-Phe-L-Leu (SEQ ID NO:5)
`ELAMSTM
`E. Preparation of L-Pro-Gly-L-Phe-L-Leu (SEQ ID
`NO:6) ELAMSTM
`F. Preparation of Tyr-Gly-Gly-L-Phe-L-Leu (SEQ ID
`NO:1) and Tyr-Pro-Gly-L-Phe-L-Leu (SEQ ID NO:2)
`ELAMSTM
`G. Preparation of Pro-L-Pro-Gly-L-Phe-L-Leu (SEQ ID
`NO:3) and Pro-Gly-Gly-L-Phe-L-Leu (SEQ ID NO:4)
`ELAMSTM
`H. Selection of ELAMSTM Containing Peptide Ligands
`for Monoclonal Antibody 3E7
`EXAMPLE III. PARALLEL SYNTHESIS OF PEPTIDES
`ON ELAMSTM
`A. DerivatiZing Amino ELAMSTM With a Linker
`B. Parallel Synthesis of Peptides
`EXAMPLE IV. PARALLEL SYNTHESIS OF OLIGO
`NUCLEOTIDE OCTAMERS
`A. Preparation of HydroXyl ELAMSTM
`B. Preparation of Linker
`C. Attachment of Synthesis Linker
`D. Preparation of Fluoresceinylated Probe
`E. Parallel Synthesis of Octanucleotides
`EXAMPLE V. SEQUENCE SPECIFIC TARGET
`65
`HYBRIDIZATION
`SEQUENCE LISTING
`
`40
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`45
`
`50
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`
`60
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`3
`been used to generate a library containing four trillion
`decapeptides, Pinilla et al., “Investigation of Antigen
`Antibody Interactions Using a Soluble, Non-Support-Bound
`Synthetic Decapeptide Library Composed of Four Trillion
`(4><1012) Sequences,” Biochem. J. 301:847—53 (1994); 1,4
`benZodiaZepines libraries, Bunin et al., “The Combinatorial
`Synthesis and Chemical and Biological Evaluation of a
`1,4-BenZodiaZepine Library,” Proc. Natl. Acad. Sci.
`91:4708—12 (1994) and US. Pat. No. 5,288,514, entitled
`“Solid Phase and Combinatorial Synthesis of BenZodiaZ
`epine Compounds on a Solid Support,” issued Feb. 22, 1994;
`libraries containing multiple small ligands tied together in
`the same molecules, Wallace et al., “A Multimeric Synthetic
`Peptide Combinatorial Library,” Pept. Res. 7:27—31 (1994);
`libraries of small organics, Chen et al., ‘“Analogous’
`Organic Synthesis of Compound Libraries: Validation of
`Combinatorial Chemistry in Small-Molecule Synthesis,” J.
`Am. Chem. Soc. 116:2661—2662 (1994); libraries of pepti
`dosteroidal receptors, Boyce & Nestler, “Peptidosteroidal
`Receptors for Opioid Peptides: Sequence-Selective Binding
`Using a Synthetic Receptor Library,” J. Am. Chem. Soc.
`116:7955—7956 (1994); and peptide libraries containing
`non-natural amino acids, Kerr et al., “Encoded Combinato
`rial Peptide Libraries Containing Non-Natural Amino
`Acids,” J. Am. Chem. Soc. 115:2529—31 (1993).
`To date, three general strategies for generating combina
`torial libraries have emerged: “spatially-addressable,” “split
`bead” and recombinant strategies. These methods differ in
`one or more of the folloWing aspects: reaction vessel design,
`polymer type and composition, control of physical constants
`such as time, temperature and atmosphere, isolation of
`products, solid-phase or solution-phase methods of assay,
`simple or complex mixtures, and method for elucidating the
`structure of the individual library members.
`Of these general strategies, several sub-strategies have
`been developed. One spatially-addressable strategy that has
`emerged involves the generation of peptide libraries on
`immobiliZed pins that ?t the dimensions of standard microti
`tre plates. See PCT Publication Nos. 91/17271 and
`91/19818, each of Which is incorporated herein by reference.
`This method has been used to identify peptides Which mimic
`discontinuous epitopes, Geysen et al., BioMed. Chem. Lett.
`3:391—404 (1993), and to generate benZodiaZepine libraries,
`US. Pat. No. 5,288,514, entitled “Solid Phase and Combi
`natorial Synthesis of BenZodiaZepine Compounds on a Solid
`Support,” issued Feb. 22, 1994 and Bunin et al., “The
`Combinatorial Synthesis and Chemical and Biological
`Evaluation of a 1,4-BenZodiaZepine Library,” Proc. Natl.
`Acad. Sci. 91:4708—12 (1994). The structures of the indi
`vidual library members can be decoded by analyZing the pin
`location in conjunction With the sequence of reaction steps
`used during the synthesis.
`A second, related spatially-addressable strategy that has
`emerged involves solid-phase synthesis of polymers in indi
`vidual reaction vessels, Where the individual vessels are
`arranged into a single reaction unit. An illustrative example
`of such a reaction unit is a standard 96-Well microtitre plate;
`the entire plate comprises the reaction unit and each Well
`corresponds to a single reaction vessel. This approach is an
`extrapolation of traditional single-column solid-phase syn
`thesis.
`As is exempli?ed by the 96-Well plate reaction unit, each
`reaction vessel is spatially de?ned by a tWo-dimensional
`matrix. Thus, the structures of individual library members
`can be decoded by analyZing the sequence of reactions to
`Which each Well Was subjected.
`Another spatially-addressable strategy employs “tea
`bags” to hold the synthesis resin. The reaction sequence to
`
`10
`
`25
`
`35
`
`45
`
`55
`
`65
`
`6,087,186
`
`4
`Which each tea bag is subject is recorded, Which determines
`the structure of the oligomer synthesiZed in each tea bag. See
`for example, Lam et al., “A NeW Type of Synthetic Peptide
`Library for Identifying Ligand-Binding Activity,” Nature
`354:82—84 (1991); Houghten et al., “Generation and Use of
`Synthetic Peptide Combinatorial Libraries for Basic
`Research and Drug Discovery,” Nature 354:84—86 (1991);
`Houghten, “General Method for the Rapid Solid-Phase
`Synthesis of Large Numbers of Peptides: Speci?city of
`Antigen-Antibody Interaction at the Level of Individual
`Amino Acids,” Proc. Natl.Acad. Sci. 82:5131—5135 (1985);
`and Jung et al., Agnew. Chem. Int. Ed. Engl. 91:367—383
`(1992), each of Which is incorporated herein by reference.
`In another recent development, scientists combined the
`techniques of photolithography, chemistry and biology to
`create large collections of oligomers and other compounds
`on the surface of a substrate (this method is called
`“VLSIPSTM”). See, for example, US. Pat. No. 5,143,854;
`PCT Publication No. 90/15070; PCT Publication No.
`92/ 10092 entitled “Very Large Scale ImmobiliZed Polymer
`Synthesis,” Jun. 25, 1992; Fodor et al., “Light-Directed
`Spatially Addressable Parallel Chemical Synthesis,” Science
`251:767—773 (1991); Pease et al., “Light-Directed Oligo
`nucleotide Arrays for Rapid DNA Sequence Analysis,”
`Proc. Natl. Acad. Sci. 91:5022—5026 (1994); and Jacobs &
`Fodor, “Combinatorial Chemistry: Applications of Light
`Directed Chemical Synthesis,” Trends. Biotechnology 12(1)
`:19—26 (1994), each of Which is incorporated herein by
`reference.
`Others have developed recombinant methods for prepar
`ing collections of oligomers. See, for example, PCT Publi
`cation No. 91/17271; PCT Publication No. 91/19818; Scott,
`“Discovering Peptide Ligands Using Epitope Libraries,”
`TIBS 17:241—245 (1992); CWirla et al., “Peptides on Phage:
`A Vast Library of Peptides for Identifying Ligands,” Proc.
`Natl. Acad. Sci. 87:6378—6382 (1990); Devlin et al., “Ran
`dom Peptide Libraries: ASource of Speci?c Protein Binding
`Molecules,” Science 249:404—406 (1990); and Scott &
`Smith, “Searching for Peptide Ligands With an Epitope
`Library,” Science 249:386—390 (1990). Using these
`methods, one can identify each oligomer in the library by
`determining the coding sequences in the recombinant organ
`ism or phage. HoWever, since the library members are
`generated in vivo, recombinant methods are limited to
`polymers Whose synthesis is mediated in the cell. Thus,
`these methods typically have been restricted to constructing
`peptide libraries.
`A third general strategy that has emerged involves the use
`of “split-bead” combinatorial synthesis strategies. See, for
`example, Furka et al., Int. J. Pept. Protein Res. 37:487—493
`(1991), Which is incorporated herein by reference. In this
`method synthesis supports are apportioned into aliquots,
`each aliquot exposed to a monomer, and the beads pooled.
`The beads are then mixed, reapportioned into aliquots, and
`exposed to a second monomer. The process is repeated until
`the desired library is generated.
`Since the polymer libraries generated With the split-bead
`method are not spatially-addressable, the structures of the
`individual library members cannot be elucidated by analyZ
`ing the reaction histogram. Rather, structures must be deter
`mined by analyZing the polymers directly. Thus, one limi
`tation of the split-bead approach is the requisite for an
`available means to analyZe the polymer composition. While
`sequencing techniques are available for peptides and nucleic
`acids, sequencing reactions for polymers of other
`composition, such as for example carbohydrates, organics,
`peptide nucleic acids or mixed polymers may not be readily
`knoWn.
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`5
`Variations on the “split-bead” scheme have emerged that
`obviate the need to sequence the library member directly.
`These methods utilize chemicals to tag the growing poly
`mers With a unique identi?cation tag (“co-synthesis”
`strategies). See, for example, PCT Publication No. WO
`94/08051 entitled “Complex Combinatorial Chemical
`Libraries Encoded With Tags,” Apr. 14, 1994; Nestler et al.,
`“A General Method for Molecular Tagging of Encoded
`Combinatorial Chemistry Libraries,” J. Org. Chem.
`59:4723—4724 (1994); PCT Publication No. WO 93/06121
`entitled “Method of SynthesiZing Diverse Collections of
`Oligomers,” Apr. 1, 1993; Needels et al., Proc. Natl. Acad.
`Sci. 90:10700—10704 (1993); Kerr et al., “Encoded Combi
`natorial Peptide Libraries Containing Non-Natural Amino
`Acids,” J. Amer. Chem. Soc. 115:2529—2531 (1993); and
`Brenner & Lerner, “Encoded Combinatorial Chemistry,”
`Proc. Natl. Acad. Sci. 89:5381—5383 (1992), each of Which
`is incorporated herein by reference.
`Encoding library members With chemical tags occurs in
`such a fashion that unique identi?ers of the chemical struc
`tures of the individual library members are constructed in
`parallel, or are co-synthesiZed, With the library members.
`Typically, in a linear three component synthesis containing
`building blocks A, B and C in the process of generating
`library member ABC, an encoding tag is introduced at each
`stage such that the tags TA, TB and TC Would encode for
`individual inputs in the library. The synthesis Would proceed
`as folloWs: (a) Chemical Ais coupled onto a synthesis bead,
`immediately folloWed by coupling tag TA to the bead; (b)
`The bead is subject to deprotection conditions Which remove
`the protecting group selectively from A, leaving TA pro
`tected. Chemical B is coupled to the bead, generating the
`sequence AB. The bead is then subject to deprotection Which
`selectively removes the protecting group from TA, and TB is
`coupled to the bead, generating tag sequence TATE; (c) The
`third component C and concomitant tag TC is added to the
`bead in the manner described above, generating library
`sequence ABC and tag sequence TATBTC.
`For large libraries containing three chemical inputs, the
`chemical tagging sequence is the same. Thus, to generate a
`large library containing the complete set of three-input, one
`hundred unit length polymers, or 1003=106 library members,
`unique identifying tags are introduced such that there is a
`unique identi?er tag for each different chemical structure.
`Theoretically, this method is applicable to libraries of any
`complexity as long as tagging sequences can be developed
`that have at least the same number of identi?cation tags as
`there are numbers of unique chemical structures in the
`library.
`While combinatorial synthesis strategies provide a poW
`erful means for rapidly identifying target molecules, sub
`stantial problems remain. For example, since members of
`spatially addressable libraries must be synthesiZed in spa
`tially segregated arrays, only relatively small libraries can be
`constructed. The position of each reaction vessel in a
`spatially-addressable library is de?ned by an XY coordinate
`pair such that the entire library is de?ned by a tWo
`dimensional matrix. As the siZe of the library increases the
`dimensions of the tWo-dimensional matrix increases. In
`addition, as the number of different transformation events
`used to construct the library increases linearly, the library
`siZe increases exponentially. Thus, While generating the
`complete set of linear tetramers comprised of four different
`inputs requires only a 16x16 matrix (44=256 library
`members), generating the complete set of linear octamers
`composed of four different inputs requires a 25 6x256 matrix
`(48=65,536 library members), and generating the complete
`
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`GLOSSARY
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`The folloWing terms are intended to have the folloWing
`general meanings as they are used herein:
`
`6,087,186
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`set of linear tetramers composed of tWenty different inputs
`requires a 400x400 matrix (204=160,000 library members).
`Therefore, not only does the physical siZe of the library
`matrix quickly become unWieldy (constructing the complete
`set of linear tetramers composed of tWenty different inputs
`using spatially-addressable techniques requires 1667
`microtitre plates), delivering reagents to each reaction vessel
`in the matrix requires either tedious, time-consuming
`manual manipulations, or complex, expensive automated
`equipment.
`While the VLSIPSTM method attempts to overcome this
`limitation through miniaturiZation, VLSIPSTM requires spe
`cialiZed photoblocking chemistry, expensive, specialiZed
`synthesis equipment and expensive, specialiZed assay equip
`ment. Thus, the VLSIPSTM method is not readily and eco
`nomically adaptable to emerging solid phase chemistries and
`assay methodologies.
`Split bead methods also suffer severe limitations.
`Although large libraries can theoretically be constructed
`using split-bead methods, the identity of library members
`displaying a desirable property must be determined by
`analytical chemistry. Accordingly, split-bead methods can
`only be employed to synthesiZe compounds that can be
`readily elucidated by microscale sequencing, such as
`polypeptides and polynucleotides.
`Co-synthesis strategies have attempted to solve this struc
`ture elucidation problem. HoWever, these methods also
`suffer limitations. For example, the tagging structures may
`be incompatible With synthetic organic chemistry reagents
`and conditions. Additional limitations folloW from the
`necessity for compatible protecting groups Which alloW the
`alternating co-synthesis of tag and library member, and
`assay confusion that may arise from the tags selectively
`binding to the assay receptor.
`Finally, since methods such as the preceding typically
`require the addition of like moieties, there is substantial
`interest in discovering methods for producing labeled librar
`ies of compounds Which are not limited to sequential addi
`tion of like moieties, and Which are amenable to any
`chemistries noW knoWn or that Will be later developed to
`generate chemical libraries. Such methods Would ?nd
`application, for example, in the modi?cation of steroids,
`sugars, co-enZymes, enZyme inhibitors, ligands and the like,
`Which frequently involve a multi-stage synthesis in Which
`one Would Wish to vary the reagents and/or conditions to
`provide a variety of compounds.
`In such methods the reagents may be organic or inorganic
`reagents, Where functionalities or side groups may be
`introduced, removed or modi?ed, rings opened or closed,
`stereochemistry changed, and the like.
`From the above, one can recogniZe that there is substantial
`interest in developing improved methods and apparatus for
`the synthesis of complex labeled combinatorial chemical
`libraries Which readily permit the construction of libraries of
`virtually any composition and Which readily permit accurate
`structural determination of individual compounds Within the
`library that are identi?ed as being of interest. Many of the
`disadvantages of the previously-described methods as Well
`as many of the needs not met by them are addressed by the
`present invention, Which as described more fully hereinafter,
`provides myriad advantages over these previously-described
`methods.
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`7
`Labeled Synthetic Oligomer Library: A“labeled synthetic
`oligomer library” is a collection of random synthetic oligo
`mers Wherein each member of such a library is labeled With
`a unique identi?er tag from Which the structure or sequence
`of each oligomer can be deduced.
`Identi?er Tag: An “identi?er tag” is any detectable
`attribute that provides a means Whereby one can elucidate
`the structure of an individual oligomer in a labeled synthetic
`oligomer library. Thus, an identi?er tag identi?es Which
`transformation events an individual oligomer has experi
`enced in the synthesis of a labeled synthetic oligomer
`library, and at Which reaction cycle in a series of synthesis
`cycles each transformation event Was experienced.
`An identi?er tag may be any detectable feature, including,
`for example: a differential absorbance or emission of light;
`magnetic or electronically pre-encoded information; or any
`other distinctive mark With the required information. An
`identi?er tag may be pre-encoded With unique identi?er
`information prior to synthesis of a labeled synthetic oligo
`mer library, or may be encoded With a identi?er information
`concomitantly With a labeled synthetic oligomer library.
`In this latter embodiment, the identi?er information added
`at each synthesis cycle is preferably added in a sequential
`fashion, such as, for example digital information, With the
`identi?er information identifying the transformation event
`of synthesis cycle tWo being appended onto the identi?er
`information identifying the transformation event of synthe
`sis cycle one, and so forth.
`Preferably, an identi?er tag is impervious to the reaction
`conditions used to construct the labeled synthetic oligomer
`library.
`Apreferred example of an identi?er tag is a microchip that
`is pre-encoded or encodable With information, Which infor
`mation is related back to a detector When the microchip is
`pulsed With electromagnetic radiation.
`Pre-Encoded Identi?er Tag: A “pre-encoded identi?er
`tag” is an identi?er tag that is pre-encoded With unique
`identi?er information prior to synthesis of a labeled syn
`thetic oligomer library. A preferred example of such a
`pre-encoded identi?er tag is a microchip that is pre-encoded
`With information, Which information is related back to a
`detector When the microchip is pulsed With electromagnetic
`radiation.
`Encodable Identi?er Tag: An “encodable identi?er tag” is
`an identi?er tag that is capable of receiving identi?er infor
`mation from time to time. An encodable identi?er tag may
`or may not be pre-encoded With partial or complete identi?er
`information prior to synthesis of a labeled synthetic oligo
`mer library. A preferred example of such an encodable
`identi?er tag is a microchip that is capable of receiving and
`storing information from time to time, Which information is
`related back to a detector When the microchip is pulsed With
`electromagnetic radiation.
`Transformation Event: As used herein, a “transformation
`event” is any event that results in a change of chemical
`structure of an oligomer or polymer. A “transformation
`event” may be mediated by physical, chemical, enZymatic,
`biological or other means, or a combination of means,
`including but not limited to, photo, chemical, enZymatic or
`biologically mediated isomeriZation or cleavage; photo,
`chemical, enZymatic or biologically mediated side group or
`functional group addition, removal or modi?cation; changes
`in temperature; changes in pressure; and the like. Thus,
`“transformation event” includes, but is not limited to, events
`that result in an increase in molecular Weight of an oligomer
`or polymer, such as, for example, addition of one or a
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`plurality of monomers, addition of solvent or gas, or coor
`dination of metal or other inorganic substrates such as, for
`example, Zeolities; events that result in a decrease in
`molecular Weight of an oligomer or polymer, such as, for
`example, de-hydrogenation of an alcohol to form an alkene
`or enZymatic hydrolysis of an ester or amide; events that
`result in no net change in molecular Weight of an oligomer
`or polymer, such as, for example, stereochemistry changes at
`one or a plurality of a chiral centers, Claissen rearrangement,
`or Cope rearrangement; and other events as Will become
`apparent to those skilled in the art upon revieW of this
`disclosure. See, for example, application Ser. No. 08/180,
`863 ?led Jan. 13, 1994, Which is assigned to the assignee of
`the present invention and PCT Publication WO 94/08051
`entitled “Complex Combinatorial Libraries Encoded With
`Tags,” Apr. 14 (1994), each of Which is incorporated herein
`by reference.
`Monomer: As used herein, a “monomer” has the same
`meaning as de?ned beloW.
`Oligomer or Polymer: As used herein, an “oligomer” or
`“polymer” is any chemical structure that can be synthesiZed
`using the combinatorial library methods of this invention,
`including, for example, amides, esters, thioethers, ketones,
`ethers, sulfoxides, sulfonamides, sulfones, phosphates,
`alcohols, aldehydes, alkenes, alkynes, aromatics,
`polyaromatics, heterocyclic compounds containing one or
`more of the atoms of: nitrogen, sulfur, oxygen, and
`phosphorous, and the like;