Mix and split procedure

Repeat this sequence of deprotecting and coupling as appropriate in the mix and split procedure... 

The nonchemical, robust, and automated RTF tag both for the encoding and the decoding is very appealing, and while this is not a single bead method and does not allow complete mix and split procedures, the preparation of... 

FIGURE 8.9 An outline of the standard library production scheme with four 96-well reaction blocks. Each of the 384 wells is preloaded with beads with a unique mass-encoded analytical constructed resin. Normally 16 monomer As are added across the rows and 24 monomer Bs down the columns. After a mix and split procedure, 96 monomer Cs are reacted in individual wells to generate 36,864 compounds. 

Iterative deconvolution, like many other methods presented in the next paragraphs and chapters, requires the use of the mix and split [36,37] technique, which is briefly described in Figure 2. A hypothetical three-step library of 3 x 3 x 3 = 27 members is prepared by first splitting the resin into three equivalent portions, then adding a different monomer of the subset A to each portion. The three portions are then mixed and split again into three portions, now containing similar amounts of the three A monomers but with a unique monomer on each resin bead. Repeating this procedure with monomers of the subset B, three pools where B is determined and A is randomized are obtained. Another mix and split ... 

This procedure marks the end of the spatial component of the library synthesis. The resin beads in the wells identified to proceed in the library synthesis are mixed and then split out into a 96-well reaction block. All 96 reaction wells are indistinguishable and consist of compounds that have all possible A-B combinations. Monomer set C typically consists of 96 unique monomers, where one unique monomer C is coupled in each well for the third point of diversity. QC is conducted after completion of the final synthesis step by the selection of a minimum of 12 beads from each well. The sampling rate does not permit the calculation of relative synthetic yields for all compounds in the library however, a global assessment on synthesis for each monomer C is produced. A narrow bandwidth of mass spectral-relative yields of the final product is selected for assay and assures a tight-Ugand concentration band. Those monomers that fail after the last synthetic step are not forwarded to biological assays. This synthetic scheme can produce a 36,864 compound library that is characterized by approximately 4200 mass-spectral data points. Normally, the final library size is between 20 and 30K after removal of the identified synthetic failures during the QC process. 

In the split-pool procedure, also known as the portioning-mixing method, the solid support is first divided into as many equal portions as the number of amino acids in the peptide s sequence [74,75,83], Each portion is coupled individually to only one amino acid. All portions of the resin are mixed, and the entire process of splitting and combining is repeated until all amino acids have been combined. An exorbitantly large number of derivatives can be synthesized at a time. 

Weigh out accurately 0.75 g pure DLTDP into a 100 ml beaker and dissolve in warm absolute ethyl alcohol. Transfer to a 100 ml volnmetric flask together with beaker washings and make up to the 100 ml mark with alcohol and mix well. Into five, 1 litre ground-glass stoppered extraction tnbes, measnre 800 ml of distilled water. Into three further sets of five tubes measure 800 ml of the ethanokwater extractant (1 1), 800 ml of the 5% sodium carbonate extractant and 800 ml of the 5% citric acid extractant. Into each of the four sets of five tubes, accurately pipette 0.0, 1.5, 3.5, 7.0 and 10.0 ml of 0.75% DLTDP in ethyl alcohol, stopper and mix well. Leave the tubes at 60 °C for 10 days. For the five ethyl alcohokwater extractants (1 1) remove alcohol by distillation and transfer the hot alcohol-free distillation residue to the liquid-liquid extractor as described previously. For the five distilled water, 5% sodium carbonate and 5% citric acid extractants, transfer the hot extractants to the liquid-liquid extractor as described previously. Carry out the ether extraction of the four sets of extractants and split each of the twenty 25 ml ether extracts into separate portions as described next. Use these solntions to calibrate the Schoniger procedure and the infrared procedures as described next. 

An important breakthrough in that respect was the use of soHd-phase organic synthesis (SPOS) where the attachment of the substrate to an insoluble support allowed for easy workup (filtration) and for rapid generation of products via split-mix procedures [1,2]. An important subsequent development consisted of the immobihzation of reagents, scavengers and catalysts. This technique, coined polymer-assisted solution phase chemistry (PASP), allowed solution phase synthesis of compoimds, yet still enjoying the bene-... 

Most important of all, however, is the possibility of running the Merrifield procedure on any number of resin beads (or other support systems) simultaneously in a number of reaction chambers. An example of this alternative is the so-called split and mix system of combinatorial chemistry. The first step in this kind of system is to prepare some number of monomer-support units (three in the example shown below), in which the monomer present differs from chamber to chamber. In the diagram below, the units are represented as -X, -Y, and -Z. These three units are washed and then mixed with each other in a single container. The mixture is then divided and placed into three separate containers. One of the most common containers used contains a number of wells in a plastic or glass dish that are miniature versions of the common petri dish used in biology experiments. 

Combinatorial synthesis on solid supports is usually carried out by using either the parallel synthesis (see section 6.2.1) or the Furka split and mix procedures (see section 6.2.2). The precise method and approach adopted when using these methods will depend on the nature of the combinatorial library being produced and also the objectives of the investigating team. However, in all cases it is necessary to determine the structures of the components of the library by either keeping a detailed record of the steps involved in the synthesis or giving beads a label that can be decoded to give the structure of the compound attached to that bead (see section 6.3). The method adopted to identify the components of the library will depend on the nature of the synthesis. 

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