Obtain a compound library

To conduct a chemical genetic phenotypic screen there is an absolute requirement for a large selection of bioactive molecules. The primary limits on these molecules availability are the synthetic routes to them. For this reason there are three main classes of such compounds natural products, synthetic peptides and pharmaceutical-like molecules. 

A particularly convenient feature of this technique, by contrast with solution-phase synthesis, is that there is no need for multistep purification of the product because the by-products and debris are easily washed away from the solid support before proceeding to the next coupling cycle with a new amino acid. The solid support can be reacted with reagents in various types of apparatus it can be simply held in a tube sandwiched between porous frits whilst reagents are pumped past, or reagents can be injected into and sucked out of vials sealed with a rubber septum, or it can be treated in 96-well plate format blocks of miniature reactors. There are also several proprietory styles of solid support designed for convenience of use. 

Peptides The most obvious feature that makes synthetic peptides good candidates for hhrary synthesis is that, since natnre is full of them, they are clearly bioactive. However, the delivery properties of peptides vary widely, and they are subject to enzymatic degradation, giving them potentially short half-lives in vivo. In spite of these limitations, they lend themselves to library synthesis because, being a linear hetero-polymer, they are amenable to automated synthesis (Box 1), and therefore for the preparation of hbraries. 

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. 

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. 

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