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Combinatorial libraries employing the one-bead–one-compound technique are reviewed. Two distinguishing features characterize this technique. First, each compound is identified with a unique solid support, enabling facile segregation of active compounds. Second, the identity of a compound on a positively reacting bead is elucidated only after its biological relevance is established. Direct methods of structure identification (Edman degradation and mass spectroscopy) as well as indirect “coding” methods facilitating the synthesis and screening of nonpeptide libraries are discussed. Nonpeptide and “scaffold” libraries, together with a new approach for the discovery of a pentide binding motif using a “library of libraries,” are also discussed. In addition, the ability to use combinatorial libraries to optimize initially discovered leads is illustrated with examples using peptide libraries. © 1994 John Wiley & Sons, Inc.

Using a “split-synthesis” solid phase synthetic approach, bead libraries can be generated such that each bead displays only one chemical entity. This “one-bead one-compound” combinatorial library can then be assayed for specific biological properties using either a solid-phase on-bead binding or functional assay, or a releasable solution phase assay. Positive compound-beads can then be isolated for structure determination. Various assay systems to screen such a “one-bead one-compound” library are described. We have used this combinatorial library method to discover peptides that bind to the cell surface immunoglobulins of murine lymphoma cells. Such peptides, when presented in an oligomeric form to a lymphoma cell are able to induce signal transduction. Additionally, we have also applied the “one-bead onecompound” combinatory library approach to elucidate peptide substrate motifs for protein tyrosine kinases. Multiple distinct peptide motifs were identified for p60 c-src protein tyrosine kinase. Using the identified peptide substrates as templates, potent and highly specific pseudosubstrate-based peptide inhibitors were developed.

Combinatorial chemistry was first applied to the generation of peptide arrays in 1984. Since then, the field of combinatorial chemistry has evolved rapidly into a new discipline. There is a great need for the development of methods to examine the proteome functionally at a global level. Using many of the techniques and instruments developed for DNA microarrays, chemical microarray methods have advanced significantly in the past three years. High-density chemical microarrays can now be synthesized in situ on glass slides or be printed through covalent linkage or non-specific adsorption to the surface of the solid-support with fully automatic arrayers. Microfabrication methods enable one to generate arrays of microsensors at the end of optical fibers or arrays of microwells on a flat surface. In conjunction with the one-bead one-compound combinatorial library method, chemical microarrays have proven to be very useful in lead identification and optimization. High-throughput protein expression systems, robust high-density protein, peptide and small-molecule microarray systems, and automatic mass spectrometers are critical tools for the field of functional proteomics.

The applicability of polymer-supported diphenylprolinol derivatives in directing either asymmetric epoxidation or Michael addition of suitable α,β-unsaturated substrates has been assessed. Epoxidation of cinnamaldehyde in the solid state give poorer yields and stereoselectivities than in the solution-phase systems. In contrast Michael additions of several aldehyde enolates to 2-nitro-1-arylalkenes gave results that approached or surpassed those in solution, and could be extended successfully to a three-component Michael/Michael/aldol cascade process. Comparisons of the results of pendant- versus crosslinked functionalized resins in these applications were revealing of the benefits and limitations of each, as were attempts to reuse the polymer-bound auxiliaries.

Two methods that use chemoselective ligation chemistry to prepare peptide and small-molecule microarrays are described here. The first method involves the functionalization of a glass slide with a glyoxylyl group, followed by chemoselective ligation of small molecules or peptides to the functionalized surface via a covalent bond. In the second method, peptides or small molecules are first conjugated to a macromolecular scaffold. The final ligand-scaffold conjugates are then spotted and adsorbed onto the solid surface. Three different assay methods to screen such chemical microarrays are described.

Publisher Summary In the “one-bead one-compound” (OBOC) combinatorial library method, compound beads are prepared by a “split-mix” synthesis approach that results in the display of many copies of the same compound on one single bead. This chapter focuses on the methods used for preparing peptide libraries, and the various screening methods that can be applied to both peptide and small molecule libraries. TentaGel resin is a good choice for OBOC peptide libraries due to its uniformity in size as well as its nonstickiness. This resin can be swollen in a wide range of solvents from water to toluene. In principle, either the Fmoc/t-But (9-fluorenylmethoxycarbonyl/tertbutyl) or t-Boc/Bn (tert-butyloxycarbonyl/benzyl) amino acid protection strategies could be used for library synthesis on TentaGel. The chapter also describes the procedure that uses N,N’-diisopropylcarbodiimide (DIC) as an activating reagent and 1-hydroxybenzotriazole (HOBt) as an additive to synthesize peptide libraries. Regarding the OBOC combinatorial libraries screening, tens of thousands to millions of compound beads are first mixed with a target molecule. The beads that interact with the target molecule will be identified and then isolated for structure determination. The enzyme-linked colorimetric assay screening method is discussed that can be used to screen both peptide and small molecule OBOC libraries.

In the last few years, protein and chemical microarrays have emerged as two important tools in the field of proteomics. Specific proteins, antibodies, small molecule compounds, peptides, and carbohydrates can now be immobilized on solid surfaces to form high-density microarrays. Depending on their chemical nature, immobilization of these molecules on solid support is accomplished by in situ synthesis, nonspecific adsorption, specific binding, nonspecific chemical ligation, or chemoselective ligation. These arrays of molecules can then be probed with complex analytes such as serum, total cell extracts, and whole blood. Interactions between the analytes and the immobilized array of molecules are evaluated with a number of different detection systems. In this paper, various components, methods, and applications of the protein and chemical microarray systems are reviewed.