The conceptual and technical approaches that led to the explosive growth of combinatorial chemistry began approximately 20 years ago. In the past decade, combinatorial chemistry has continued to expand with new chemistries, technological improvements and, most importantly, a clear demonstration of its utility in the identification of active compounds for research and drug-discovery programs. This article describes the conceptual and practical breakthroughs that have been critical for the development of synthetic combinatorial methods and includes the most recent developments and applications of mixture-based combinatorial libraries.
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Merrifield, R.B. Peptide synthesis. I. The synthesis of a tetrapeptide. J. Am. Chem. Soc. 85, 2149–2154 (1963).
Merrifield, R.B. Solid-phase synthesis. Science 232, 341–347 (1986).
Frank, R. A new general approach for the simultaneous chemical synthesis of large numbers of oligonucleotides: segmental solid supports. Nucleic Acids Res. 11, 4365–4377 (1983).
Geysen, H.M., Meloen, R.H. & Barteling, S.J. Use of a peptide synthesis to probe viral antigens for epitopes to a resolution of a single amino acid. Proc. Natl. Acad. Sci. USA 81, 3998–4002 (1984).
Houghten, R.A. General method for the rapid solid-phase synthesis of large numbers of peptides: specificity of antigen–antibody interaction at the level of individual amino acids. Proc. Natl. Acad. Sci. USA 82, 5131–5135 (1985).
Leznoff, C.C. & Wong, J.Y. The use of polymer supports in organic synthesis III. Selective chemical reactions on one aldehyde group of symmetrical dialdehydes. Can. J. Chem. 51, 3756–3764 (1973).
Wong, J.Y. & Leznoff, C.C. The use of polymer supports in organic synthesis II. The syntheses of monoethers of symmetrical diols. Can. J. Chem. 51, 2452–2456 (1973).
Crowley, J.I. & Rapoport, H. Solid-phase organic synthesis: Novelty or fundamental concept? Acc. Chem. Res. 9, 135–144 (1976).
Bunin, B.A. & Ellman, J.A. A general and expedient method for the solid-phase synthesis of 1,4-benzodiazepine derivatives. J. Am. Chem. Soc. 114, 10997–10998 (1992).
Thompson, L.A. & Ellman, J.A. Synthesis and applications of small molecule libraries. Chem. Rev. 96, 555–600 (1996).
Nefzi, A., Ostresh, J.M. & Houghten, R.A. The current status of heterocyclic combinatorial libraries. Chem. Rev. 97, 449–472 (1997).
Dolle, R.E. Comprehensive survey of combinatorial library synthesis: 2000. J. Comb. Chem. 3, 477–517 (2001).
Bunin, B.A., Plunkett, M.J. & Ellman, J.A. The combinatorial synthesis and chemical and biological evaluation of a 1,4-benzodiazepine library. Proc. Natl. Acad. Sci. USA 91, 4708–4712 (1994).
Ostresh, J.M. et al. Solid-phase synthesis of trisubstituted bicyclic guanidines via cyclicization of reduced N-acylated dipeptides. J. Org. Chem. 63, 8622–8623 (1998).
Ostresh, J.M. et al. “Libraries from libraries”: chemical transformation of combinatorial libraries to extend the range and repertoire of chemical diversity. Proc. Natl. Acad. Sci. USA 91, 11138–11142 (1994).
Giannis, A. & Kolter, T. Peptidomimetics for receptor ligands—Discovery, development, and medical perspectives. Angew. Chem. Int. Ed. Engl. 32, 1244–1267 (1993).
Liskamp, R.M.J. Opportunities for new chemical libraries: Unnatural biopolymers and diversomers. Angew. Chem. Int. Ed. Engl. 33, 633–636 (1994).
Barkley, A. & Arya, P. Combinatorial chemistry toward understanding the function(s) of carbohydrates and carbohydrate conjugates. Chemistry 7, 555–563 (2001).
Lam, K.S. et al. A new type of synthetic peptide library for identifying ligand-binding activity. Nature 354, 82–84 (1991).
Houghten, R.A. et al. Generation and use of synthetic peptide combinatorial libraries for basic research and drug discovery. Nature 354, 84–86 (1991).
Hiemstra, H.S. et al. The identification of CD4+ T cell epitopes with dedicated synthetic peptide libraries. Proc. Natl. Acad. Sci. USA 94, 10313–10318 (1997).
Pinilla, C., Appel, J.R., Blanc, P. & Houghten, R.A. Rapid identification of high affinity peptide ligands using positional scanning synthetic peptide combinatorial libraries. Biotechniques 13, 901–905 (1992).
Houghten, R.A. et al. Mixture-based synthetic combinatorial libraries. J. Med. Chem. 42, 3743–3778 (1999).
Geysen, H.M., Rodda, S.J. & Mason, T.J. A priori delineation of a peptide which mimics a discontinuous antigenic determinant. Mol. Immunol. 23, 709–715 (1986).
Lam, K.S., Lebl, M. & Krchnak, V. The “one-bead-one-compound” combinatorial library method. Chem. Rev. 97, 411–448 (1997).
Ohlmeyer, M.H.J. et al. Complex synthetic chemical libraries indexed with molecular tags. Proc. Natl. Acad. Sci. USA 90, 10922–10926 (1993).
Houghten, R.A. Soluble combinatorial libraries: extending the range and repertoire of chemical diversity. Methods: Companion Methods Enzymol. 6, 354–360 (1994).
Berman, J., Halm, K., Adkison, K. & Shaffer, J. Simultaneous pharmacokinetic screening of a mixture of compounds in the dog using API LC/MS/MS analysis for increased throughput. J. Med. Chem. 40, 827–829 (1997).
Cheng, Y. et al. A combinatorial library of indinavir analogues and its in vitro and in vivo studies. Bioorg. Med. Chem. Lett. 12, 529–532 (2002).
Thornberry, N.A. et al. A combinatorial approach defines specificities of members of the caspase family and granzyme B—Functional relationships established for key mediators of apoptosis. J. Biol. Chem. 272, 17907–17911 (1997).
Rano, T.A. et al. A combinatorial approach for determining protease specificities: application to interleukin-1β converting enzyme (ICE). Chem. Biol. 4, 149–155 (1997).
Backes, B.J., Harris, J.L., Leonetti, F., Craik, C.S. & Ellman, J.A. Synthesis of positional-scanning libraries of fluorogenic peptide substrates to define the extended substrate specificity of plasmin and thrombin. Nature Biotechnol. 18, 187–193 (2000).
Harris, J.L. et al. Rapid and general profiling of protease specificity by using combinatorial fluorogenic substrate libraries. Proc. Natl. Acad. Sci. USA 97, 7754–7759 (2000).
Harris, J.L. et al. Definition of the extended substrate specificity determinants for β-tryptases I and II. J. Biol. Chem. 276, 34941–34947 (2001).
Salter, J.P. et al. Cercarial elastase is encoded by a functionally conserved gene family across multiple species of schistosomes. J. Biol. Chem. 277, 24618–24624 (2002).
Mathieu, M.A. et al. Substrate specificity of schistosome versus human legumain determined by P1-P3 peptide libraries. Mol. Biochem. Parasitol. 121, 99–105 (2002).
Dauber, D.S. et al. Altered substrate specificity of drug-resistant human immunodeficiency virus type 1 protease. J. Virol. 76, 1359–1368 (2002).
Nazif, T. & Bogyo, M. Global analysis of proteasomal substrate specificity using positional-scanning libraries of covalent inhibitors. Proc. Natl. Acad. Sci. USA 98, 2967–2972 (2001).
Harris, J.L., Alper, P.B., Li, J., Rechsteiner, M. & Backes, B.J. Substrate specificity of the human proteasome. Chem. Biol. 8, 1131–1141 (2001).
Nefzi, A., Giulianotti, M.A. & Houghten, R.A. Solid-phase synthesis of substituted 2,3-diketopiperazines from reduced polyamides. Tetrahedron 56, 3319–3326 (2000).
Acharya, A.N., Nefzi, A., Ostresh, J.M. & Houghten, R.A. Tethered libraries: solid-phase synthesis of substituted urea-linked bicyclic guanidines. J. Comb. Chem. 3, 189–195 (2001).
Pinilla, C., Appel, J.R. & Houghten, R.A. Investigation of antigen-antibody interactions using a soluble nonsupport-bound synthetic decapeptide library composed of four trillion sequences. Biochem. J. 301, 847–853 (1994).
Udaka, K., Wiesmüller, K.-H., Kienle, S., Jung, G. & Walden, P. Self-MHC-restricted peptides recognized by an alloreactive T lymphocyte clone. J. Immunol. 157, 670–678 (1996).
Hemmer, B. et al. Identification of high potency microbial and self ligands for a human autoreactive class II-restricted T cell clone. J. Exp. Med. 185, 1651–1659 (1997).
Pinilla, C. et al. Exploring immunological specificity using synthetic peptide combinatorial libraries. Curr. Opin. Immunol. 11, 193–202 (1999).
Hemmer, B. et al. Identification of candidate T cell epitopes and molecular mimics in chronic Lyme disease. Nature Med. 5, 1375–1382 (1999).
Zhao, Y. et al. Combinatorial peptide libraries and biometric score matrices permit the quantitative analysis of specific and degenerate interactions between clonotypic TCR and MHC peptide ligands. J. Immunol. 167, 2130–2141 (2001).
La Rosa, C. et al. Enhanced immune activity of cytotoxic T-lymphocyte epitope analogs derived from positional scanning synthetic combinatorial libraries. Blood 97, 1776–1786 (2001).
Hemmer, B. et al. Contribution of individual amino acids within MHC molecule or antigenic peptide to TCR ligand potency. J. Immunol. 164, 861–871 (2000).
Anderson, B., Park, B.J., Verdaguer, J., Amrani, A. & Santamaria, P. Prevalent CD8+ T cell response against one peptide/MHC complex in autoimmune diabetes. Proc. Natl. Acad. Sci. USA 96, 9311–9316 (1999).
Judkowski, V. et al. Identification of MHC class II-restricted peptide ligands, including a glutamic acid decarboxylase 65 sequence, that stimulate diabetogenic T cells from transgenic BDC2.5 nonobese diabetic mice. J. Immunol. 166, 908–917 (2001).
Pinilla, C. et al. Combinatorial peptide libraries as an alternative approach to the identification of ligands for tumor reactive cytolytic T lymphocytes. Cancer Res. 61, 5153–5160 (2001).
Rubio-Godoy, V. et al. Combinatorial peptide library based identification of peptide ligands for tumor-reactive cytolytic T lymphocytes of unknown specificity. Eur. J. Immunol. 32, 2292–2299 (2002).
Linnemann, T. et al. Mimotopes for tumor-specific T lymphocytes in human cancer determined with combinatorial peptide libraries. Eur. J. Immunol. 31, 156–165 (2001).
Rubio-Godoy, V. et al. Towards synthetic combinatorial peptide libraries in positional scanning format (PS-SCL)-based identification of CD8+ tumor-reactive T-cell ligands: A comparative analysis of PS-SCL recognition by a single tumor-reactive CD8+ CTL. Cancer Res. 62, 2058–2063 (2002).
Borras, E. et al. Findings on T cell specificity revealed by synthetic combinatorial libraries. J. Immunol. Methods 267, 79–97 (2002).
Dooley, C.T. & Houghten, R.A. The use of positional scanning synthetic peptide combinatorial libraries for the rapid determination of opioid receptor ligands. Life Sci. 52, 1509–1517 (1993).
Reixach, N., Crooks, E., Ostresh, J.M., Houghten, R.A. & Blondelle, S.E. Inhibition of β-amyloid-induced neurotoxicity by imidazopyridoindoles derived from a synthetic combinatorial library. J. Struct. Biol. 130, 247–258 (2000).
Blondelle, S.E., Crooks, E., Ostresh, J.M. & Houghten, R.A. Mixture-based heterocyclic combinatorial positional scanning libraries: discovery of bicyclic guanidines having potent antifungal activities against Candida albicans and Cryptococcus neoformans. Antimicrob. Agents Chemother. 43, 106–114 (1999).
Boger, D.L., Fink, B.E. & Hedrick, M.P. Total synthesis of distamycin A and 2640 analogs: A solution-phase combinatorial approach to the discovery of new, bioactive DNA binding agents and development of a rapid, high-throughput screen for determining relative DNA binding affinity or DNA binding sequence selectivity. J. Am. Chem. Soc. 122, 6382–6394 (2000).
Willoughby, C.A. et al. Combinatorial synthesis of 3-(amidoalkyl) and 3-(aminoalkyl)-2-arylindole derivatives: discovery of potent ligands for a variety of G-protein coupled receptors. Bioorg. Med. Chem. Lett. 12, 93–96 (2002).
Appel, J.R., Johnson, J., Narayanan, V.L. & Houghten, R.A. Identification of novel antitumor agents from mixture-based synthetic combinatorial libraries using cell-based assays. Mol. Divers. 4, 91–102 (1999).
Sternson, S.M., Wong, J.C., Grozinger, C.M. & Schreiber, S.L. Synthesis of 7200 small molecules based on a substructural analysis of the histone deacetylase inhibitors trichostatin and trapoxin. Org. Lett. 3, 4239–4242 (2001).
McMillan, K. et al. Allosteric inhibitors of inducible nitric oxide synthase dimerization discovered via combinatorial chemistry. Proc. Natl. Acad. Sci. USA 97, 1506–1511 (2000).
We thank R. Martin and R. Simon for their contributions to the development of the biometrical analysis first used in T-cell studies; C. Dooley for the opioid receptor research; D. Wilson and S. Blondelle for their involvement in the use of combinatorial libraries in many different biological assays; and J. Ostresh, A. Nefzi and the chemistry group at Torrey Pines Institute for Molecular Studies for the continuing development of synthetic chemistry for the preparation of mixture-based combinatorial libraries. Supported by NCI grant PO1 CA78040, NIDA grant RO1 DA09410 and MSNRI funding.
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Pinilla, C., Appel, J., Borràs, E. et al. Advances in the use of synthetic combinatorial chemistry: Mixture-based libraries. Nat Med 9, 118–122 (2003). https://doi.org/10.1038/nm0103-118
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