Mix-and-split strategy

In addition, SPOS can easily be automated using appropriate robotics for both filtration and evaporation of the volatiles from the reaction mixture to obtain the cleaved product. Furthermore, SPOS can be applied to the powerful split-and-mix strategy, which has proved to be an important tool in combinatorial chemistry [7]. 

This dendritic support (1) was used to generate a library of indole derivatives with three centers of diversity following the Fischer protocol and the split and mix strategy. One example of this sequence is outlined in Scheme 7.1. 

The combinatorial approach that was pursued in search of an antiasthma drug based on a split-and-mix strategy [92] as a practical use of the operational principle of parsimony was to get the most with the least in this case, to get 343 different types of variants in only 21 reaction steps. Scheme 1-17 sketches... 

The split-and-mix strategy appears deceptively similar to parallel synthesis, but can produce libraries of several hundred thousand compounds in one mn. Although it can use the same style of apparatus as for parallel synthesis, there would normally be as many synthesis channels as one has monomers - 20 in the case of natural amino acids (although cysteine is often omitted). However, after the first coupling cycle has been completed, the batches of synthesis beads from each channel would all be combined, mixed well, and re-divided back into the 20 synthesis channels - the step from which the method takes its name. After this, another round of chain extension would commence. In this way all possible sequences are prepared at the same time, but each bead of solid support only contains one sequence. As the number of compounds rises exponentially with each chain extension cycle (hence it is combinatorial), large numbers of monomers limit the number of cycles that can be performed - usually to four rounds (i.e. a tetrapeptide library) with 20 different monomers, = 20" =160 000 compounds. This is because the number of possible sequences should be significantly less than the number of synthesis beads that can be contained in a reasonable volume. 

The first steps of solid-phase approaches in sensor development were linked to the emergence of solid-phase peptide synthesis. Split-and-mix strategies rendered one-bead one-peptide libraries that could be screened against any analyte of interest in a solid-phase assay. Colorimetric or fluorescence changes were generally used to report the interaction and enabled the discovery of sensors for a number of biomolecules (e.g., tripeptides" and ATP ). 

The solid-phase technique of split and mix synthesis relies on the efficiency of mixture-based synthesis to provide very large libraries (millions) of discrete compounds (Figure 4).[161 In this approach, each resin bead is treated with a single building block for each synthesis step. Thus any single resin bead possesses identical copies of one library member, but the identity of the library member on any bead is lost due to the mix step of the process. Elegant strategies have been developed to chemically encode the syn-... 

In Section II.C we will present novel tricyclic xanthene derived amino acid templates, which allow the construction of libraries of cyclic conformationally constrained peptide loop mimetics using the split-and-mix method without having to use tagging and deconvolution strategies. In Section III we will focus on parallel and combinatorial approaches devoted to the synthesis of small molecule, non-peptidic compound collections, which in addition offer the possibility to incorporate structural features derived from protein epitope mapping into conformationally constrained peptide mimetics. 

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. 

All the spectroscopic approaches applied for structural characterization of mixtures derive from methods originally developed for screening libraries for their biological activities. They include diffusion-ordered spectroscopy [15-18], relaxation-edited spectroscopy [19], isotope-filtered affinity NMR [20] and SAR-by-NMR [21]. These applications will be discussed in the last part of this chapter. As usually most of the components show very similar molecular weight, their spectroscopic parameters, such as relaxation rates or selfdiffusion coefficients, are not very different and application of these methodologies for chemical characterization is not straightforward. An exception is diffusion-edited spectroscopy, which can be a feasible way to analyze the structure of compounds within a mixture without the need of prior separation. This was the case for the analysis of a mixture of five esters (propyl acetate, butyl acetate, ethyl butyrate, isopropyl butyrate and butyl levulinate) [18]. By the combined use of diffusion-edited NMR and 2-D NMR methods such as Total Correlation Spectroscopy (TOCSY), it was possible to elucidate the structure of the components of this mixture. This strategy was called diffusion encoded spectroscopy DECODES. Another example of combination between diffusion-edited spectroscopy and traditional 2-D NMR experiment is the DOSY-NOESY experiment [22]. The use of these experiments have proven to be useful in the identification of compounds from small split and mix synthetic pools. 

The split and mix approach forms the basis of all solid support-based combinatorial synthetic strategies. 

Two distinct strategies of peptide lihrary preparation are commonly used parallel synthesis and so-called sptit-and-mix chemistry (also known as divide-couple- ecom-bine, portion-mixing and split-pool synthesis). Both are often described as combinatorial chemistry (combichem), although this is only strictly true for sptit-and-mix synthesis. Parallel synthesis is simply the preparation of many batches of peptide at the same time in separate parallel channels of one or more machines. In the case of library synthesis, the amino acid sequence is systematically varied between channels. Commonly this allows the synthesis of several hundred different sequences at the same time. It has the advantages that larger amounts are usually prepared than in split-and-mix, and that one can easily tell what sequence of residues was used in any potential hit. 

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