Compounds on Beads

FIGURE 2.4 Formation of a single compound on each bead. Large circle resin bead smaller black, gray and white circles monomers (e.g. amino acids). 

In the split-mix synthesis, like in other solid phase procedures, the beads behave very much like tiny reaction vessels, which do not interchange their contents with the other ones. Each of the millions of these reaction vessels preserves its content until the end of the synthesis, when they become containers of a single substance. If peptides are produced, their identity can be determined by automatic sequencing [9], It is sufficient to sacrifice a fraction of the total quantity for this purpose. All this means that the split-mix synthesis is, in fact, a parallel procedure, with unprecedented efficiency, however, leading to individual compounds. This feature of the split and mix synthesis allows screening the products in three different ways  

Doing binding experiments with the individual compounds uncleaved from the beads 

Cleaving the product from a single bead and test it as an individual 

Pooling the beads before cleavage, then carrying out screening with a solution of a mixture. 

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Combinatorial Chemistry. Figure 2 Chemical libraries are prepared either by parallel synthesis or by the split-and-recombine method. In the latter case, coupling m building blocks in m separated reaction flasks through n synthetic cycles on a beaded polymer carrier generates a combinatorial library with nf individual compounds and one compound per bead. 

Because of their ease of synthesis and their structural similarity to peptides, many laboratories have used peptoids as the basis for combinatorial drug discovery. Peptoids were among the first non-natural compounds used to establish the basic principles and practical methods of combinatorial discovery [17]. Typically, diverse libraries of relatively short peptoids (< 10 residues) are synthesized by the mix-and-split method and then screened for biological activity. Individual active compounds can then be identified by iterative re-synthesis, sequencing of compounds on individual beads, or indirect deduction by the preparation of positional scanning libraries. 

IR spectroscopy is not a very sensitive analytical tool and is, therefore, not well suited to the detection of small amounts of material. If, however, intermediates have intense and well-resolved IR absorptions, the progress of their chemical transformation can be followed by IR spectroscopy [83,88,91-93], Near-infrared spectroscopy, in combination with an acousto-optic tunable filter, can be sufficiently sensitive to enable the on-bead identification of polystyrene-bound di- and tripeptides, even if the peptides have very similar structures (e.g., Leu-Ala-Gly-PS and Val-Ala-Gly-PS) or differ only in their amino acid sequence (e.g., Leu-Val-Gly-PS and Val-Leu-Gly-PS) [94]. Special resins displaying an IR and Raman barcode have been developed, which may facilitate the deconvolution of combinatorial compound libraries prepared by the mix-and-split method [48]. 

In 1991, we first introduced the one-bead one-compound (OBOC ) combinatorial library method.1 Since then, it has been successfully applied to the identification of ligands for a large number of biological targets.2,3 Using well-established on-bead binding or functional assays, the OBOC method is highly efficient and practical. A random library of millions of beads can be rapidly screened in parallel for a specific acceptor molecule (receptor, antibody, enzyme, virus, etc.). The amount of acceptor needed is minute compared to solution phase assay in microtiter plates. The positive beads with active compounds are easily isolated and subjected to structural determination. For peptides that contain natural amino acids and have a free N-terminus, we routinely use an automatic protein sequencer with Edman chemistry, which converts each a-amino acid sequentially to its phenylthiohydantoin (PTH) derivatives, to determine the structure of peptide on the positive beads. 

We first reported the one-bead one-compound (OBOC) combinatorial library method in 1991.1 In this method, compound beads are prepared by a split-mix synthesis approach1 3 (Fig. 1) that results in the display of many copies of the same compound on one single bead.1,4 Tens of thousands to millions of these compound beads can easily be prepared. The bead library is then mixed with a target molecule, such as a protein, an... 

The expense of screening depends very much on the number of samples tested. Consequently, the density format of titer-plates has increased in recent years from 96-well up to the 9600-well format. The next big step towards miniaturization would be the complete avoidance of any container, which then results in the smallest well possible and a well-less, so-called lawn-format assay develops. This is exactly what is proposed by a number of authors (see review [47]). Screening in a lawn format does not mean avoiding any structure or arrangements. Samples are still prepared on beads, which are produced by split-mix synthesis, but the beads are arrayed directly on the well-less assay. A typical matrix applied for such biological screening is the agarose lawn. Active beads are then picked from the assay matrix and decoded for compound identification. 

In parallel synthesis the compounds are prepared in separate reaction vessels but at the same time, that is, in parallel. The array of individual reaction vessels often takes the form of either a grid of wells in a plastic plate or a grid of plastic rods called pins attached to a plastic base plate (Figure 6.6) that fits into a corresponding set of wells. In the former case the synthesis is carried out on beads placed in the wells whilst in the latter case it takes place on so called... 

The Furka method produces the library of compounds on resin beads. It may be used to make both large (thousands) and small (hundreds) combinatorial libraries. Large libraries are possible because the technique produces one type of compound on each bead, that is, all the molecules formed on one bead are the same but different from those formed on all the other beads. Each bead will yield up to 6 x 1013 product molecules, which is sufficient to carry out high throughput screening procedures. The technique has the advantage that it reduces the number of reactions required to produce a large library. 

In a trial to identify hydrolytically active members in a metal-complexed un-decapeptide library, Berkessel and coworkers used the phosphate ester 8 as a substrate [14]. On hydrolysis the insoluble blue indigo dye 9 is formed and precipitates on beads that carry compounds with hydrolytic activity (Figure 5.4.5). 

One bead one sequence" approach allows the preparation of simultaneously millions of physically separate individual compounds on solid microcarriers (beads)... 

The second group can be represented by single bead methods, and relies on either bioanalytical methods to select the active compounds or on-bead screening to determine the beads carrying active compounds. It is limited to solid-phase chemistry and does not require chemical steps after library synthesis but does require sophisticated analytical methods to determine the structure of the active compounds. A recent hybrid deconvolution-single beaddecoding method named DRED (dual recursive deconvolution) requires both deconvolutive techniques and sophisticated analytical capacities. 

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