Spatially Addressed Libraries

Small molecule microarrays (SMMs) [162] can be used to identify small molecule-protein interactions. If the library is not covalently attached to the carrier material, this can simply be considered as another miniaturization in screening [163], where nanoliter volumes are handled [164]. 

Alternatively, each molecule can be attached to the carrier material (e.g., a PEG-based hydrogel or glass surfaces that carry thiol or olefin moieties). Synthesis is then performed via adding, for example, the components of an Ugi-reaction in a spatially defined fashion. The identity (or combination of diversity reagents) for each compound is thereby defined by its position in the microarray. Often, fluorescence is used for detection [165], but other technologies such as surfece plasmon resonance (SPR) have been used as well [166]. 

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The preferred mode of preparation for both mixture and split synthesis libraries is the solid phase. Both polymer matrices or pins and resin beads have been used. The solid-phase approach is preferred because of its simplicity and ease of purification and isolation of the reaction products. Unlike the spatially addressable library when structure is defined by position in a set of reaction vessels, the structure of interesting library members prepared by mixture or split synthesis must be defined by highly sensitive analytical methods or indirectly by encoding or by biological results combined with resynthesis, the so-called deconvolution method. 

The technology for materials discovery is still in the developmental stage, and future progress can still be influenced by theoretical considerations. In this spirit, I assume that the composition and noncomposition variables of each sample can be changed independently, as in spatially addressable libraries (Akporiaye et al., 1998 Pirrung, 1997). This is significant, because it allows great flexibility in how the space can be searched with a limited number of experimental samples. 

According to Blackwell [103] the application of microwave irradiation to expedite solid-phase reactions could be the tool that allows combinatorial chemistry to deliver on its promise - providing rapid access to large collections of diverse small molecules. Several different approaches to microwave-assisted solid-phase reactions and library synthesis are now available. These include the use of solid-supported reagents, multi-component coupling reactions, solvent-free parallel library synthesis, and spatially addressable library synthesis on planar solid support. 

AU combinatorial receptors in this account are created as spatially addressed libraries with each solution containing either one symmetric complex or mixtures of at most three symmetric and asymmetric complexes. These methods also permit the formation of large mixed libraries where a variety of monomers are mixed with one half an eqnivalent of the metal. 

Interest in solid phase synthesis continues to increase as articles which expand the scope of non-oligomer organic synthesis are published and combinatorial chemistry is applied to an increasing variety of problems. A special thematic issue of Chemical Reviews on combinatorial chemistry [1] has been published which includes reviews of solid-phase chemistry as applied to the one bead-one compound approach [2], heterocycle synthesis [3], spatially addressable libraries [4], as well as soluble resin-based synthesis [5]. This review focuses on work published from June 1996 through September 1997. A discussion of solid-phase based reagents is not included as this topic is covered elsewhere. 

Several combinatorial approaches to the discovery of transition metal based catalysts for olefin polymerization have been described. In one study Brookhart-type polymer-bound Ni- and Pd-(l,2-diimine) complexes were prepared and used in ethylene polymerization (Scheme 3).60,61 A resin-bound diketone was condensed with 48 commercially available aminoarenes having different steric properties. The library was then split into 48 nickel and 48 palladium complexes by reaction with [NiBr2(dme)] and [PdClMe(COD)], respectively, all 96 pre-catalysts being spatially addressable. 

An 8000-member library of trisamino- and aminooxy-l,3,5-triazines has been prepared by use of highly effective, microwave-assisted nucleophilic substitution of polypropylene (PP) or cellulose membrane-bound monochlorotriazines. The key step relied on the microwave-promoted substitution of the chlorine atom in monochlorotriazines (Scheme 12.7) [35]. Whereas the conventional procedure required relatively harsh conditions such as 80 °C for 5 h or very long reaction times (4 days), all substitution reactions were found to proceed within 6 min, with both amines and solutions of cesium salts of phenols, and use of microwave irradiation in a domestic oven under atmospheric reaction conditions. The reactions were conducted by applying a SPOT-synthesis technique [36] on 18 x 26 cm cellulose membranes leading to a spatially addressed parallel assembly of the desired triazines after cleavage with TFA vapor. This concept was later also extended to other halogenated heterocycles, such as 2,4,6-trichloropyrimidine, 4,6-dichloro-5-nitropyrimidine, and 2,6,8-trichloro-7-methylpurine, and applied to the synthesis of macrocyclic peptidomimetics [37]. 

As first practiced by Geysen and Houghton, the preparation of combinatorial libraries produced discrete compounds of known identity through a technique known as "spatial separation," which simply means that individual compounds in the library are produced discretely and are not mixtures. Such spatially addressable compound sets are produced in such a way as to keep separate the reaction flasks or resin beads containing the individual components of the library and perform bioassays on the discrete compounds, one at a time. Thus, if the "history" of the reaction conditions performed in each flask or on each solid support, the identity of the compounds produced is known, without resort to structure elucidation techniques. Initially, this technique, after typically an extensive reaction development stage, allowed the preparation of between 10 and 1000 discrete combinatorial products. 

Pirrung, M.C., Spatially addressable combinatorial libraries, Chem. Rev., 1997, 97, 473-488. 

The use of planar supports for presenting large arrays of spatially addressed molecules is one of the most powerful and versatile methods for creating combinatorial libraries.19,28-29... 

In this chapter we will discuss current approaches for analytical characterization of combinatorial libraries in a pharmaceutical industry environment. Recently, several analytical groups have presented very similar strategies for analysis of libraries [7-9]. As will be shown later, the key to successful analytical characterization of a combinatorial library is to perform analytical and chemical work in parallel with the library development. The accumulation of data and analytical experience during this process results in an assessment of library quality with a high level of confidence, even if as little as 5-10% of the library components are analyzed. Utilization of the strategy will be demonstrated using two examples analysis of a library synthesized on a robotic station in spatially addressed format and analysis of a library synthesized in accordance with split-and-mix technology. 

FIGURE 10.1 The scheme of analysis of a library synthesized in spatially addressed format (96-well plate). 

There are two approaches to analytical evaluation of OBOC libraries analysis of compounds from individuals bead [51,60] and analysis of mixtures of compounds detached from multiple beads [61, 62], It should be mentioned that analysis of mixtures from OBOC libraries is quite different from that described in section Analysis of Libraries Synthesized in the Format of Small Mixtures of 4-12 Compounds per Mixture. In the mixture of compounds cleaved from a few beads, which are randomly picked for analysis from the OBOC library, the structures of components of the mixture are unknown. Consequently, it is not possible to apply the strategy developed for the analysis of mixtures synthesized in spatially addressed format, which is based on information about the structures of components of each mixture. In the case of OBOC libraries, analysis of only very large mixtures is meaningful when the theoretical distribution of molecular weights within the whole library is compared with that experimentally observed [61, 62], The focus of this section is on characterization of OBOC libraries through analysis of individual beads. 

Figure 3 Spatially addressable combinatorial peptide library comprised of 125 peptides with the sequence of XCysYCysZCysGly, whereby X, Y, and Z are spacer residues of a-Glu, y-Glu, GABA, SerGly, and e-aminohexanoic acid (s-Ahx). (Reproduced...

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