Split and mix

Mixtures containing up to several thousand distinct chemical entities are often synthesized and tested in mix-and-split combinatorial chemistry. The descriptor representation of a mixture may be approximated as the descriptor average of its individual component molecules, e.g., using atom-pair and topological torsion descriptors. 

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. 

The mix-and-split method was a combinatorial synthesis pioneered by Furka in 1988. The method is illustrated in figure 7.4 with three reaction vessels, each of which contains large quantities of small resin beads. The first vessel is treated with amino acid A to start a peptide, the second vessel with B, and the third vessel with C. Then the beads from the three vessels are mixed together and divided equally into three vessels. In the second generation of processing, the first vessel is treated with amino acid D, the second vessel with E, and the third vessel with F. Now we have a library... 

The synthetic strategy employed during the combinatorial syntheses can be used to assist in determining these pooling strategies. In random incorporation syntheses, a single bead could contain millions of different molecular species. In mix and split syntheses (also called pool and divide syntheses or one bead-one compound syntheses) only one compound is attached to any given solid-phase synthetic bead. 

The way in which the two excited configurations mix and split apart in energy is called configuration interaction. It is depicted in the energy level diagram of Figure 7.7 for the case at hand. 

Because the characterization of support-bound intermediates is difficult (see below), solid-phase reactions are most conveniently monitored by cleaving the intermediates from the support and analyzing them in solution. Depending on the loading, 5-20 mg of support will usually deliver sufficient material for analysis by HPLC, LC-MS, and NMR, and enable assessment of the outcome of a reaction. Analytical tools that are particularly well suited for the rapid analysis of small samples resulting from solid-phase synthesis include MALDI-TOF MS [3-5], ion-spray MS [6-8], and tandem MS [9]. MALDI-TOF MS can even be used to analyze the product cleaved from a single bead [5], and is therefore well suited to the identification of products synthesized by the mix-and-split method (Section 1.2). The analysis and quantification of small amounts of product can be further facilitated by using supports with two linkers, which enable either release of the desired product or release of the product covalently bound to a dye [10-13], to an isotopic label [11], or to a sensitizer for mass spectrometry [6,14,15] (e.g., product-linker-dye- analytical linker -Pol). 

Alternatively, the so-called mix-and-split method [17-22] can be used to prepare mixtures of support particles (beads, paper disks, etc.) or of small portions of support (e.g., tea bags ) with a well-defined quantity of one discrete compound linked to each portion of support. These compound libraries can be screened either directly on the support, or in solution after partial or total cleavage of the product from the support. 

The deconvolution of compound libraries prepared by the mix-and-split method can be greatly simplified by using polymeric supports that have been labelled with various dyes prior to library synthesis. In this case, the first monomer can be identified from the color or the UV spectrum of each bead. This type of labelling can, for instance, be achieved by partial derivatization of the support with different dyes [47], or by the use of polymers prepared from monomers showing characteristic IR or Raman spectra [48],... 

Some types of compound are also well suited to being screened as mixtures. This is, for instance, possible for peptides, which often either bind very strongly or not at all to a given receptor. In this instance, the most potent peptide can be identified by deconvolution, and tagging of the support particles is not required. As shown in Figure 1.2, oligomers prepared by the mix-and-split method can contain positions with... 

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]. 

For some applications it might be desirable to cleave the product from a support in two or more portions. This can be realized by derivatizing a functionalized support with a mixture of different linkers that enable a sequential cleavage [9]. The resulting support can, for instance, be used to prepare and screen combinatorial peptide libraries by the mix-and-split method ([10-12] one different peptide on each bead). The first portion of peptide released would be tested for biological activity, and, once an active peptide had been identified, the remaining peptide on the support could be used for structure elucidation. 

Further cycloadditions used to prepare cycloalkenes on insoluble supports include the cyclopropanation of resin-bound alkynes and of polystyrene [165] (Figure 5.18). The latter reaction has been used to introduce tags onto polystyrene beads, which enable the recognition of a certain bead in compound libraries produced using the mix-and-split method (Section 1.2 [165-167]). The structure of polystyrene tagged in this way has not, however, been rigorously determined. 

An important idea for split synthesis is that each well must contain a large number of individual resin beads. The large number of beads is important for two reasons. One, each well must include a representative sampling of beads from the previous reactions. Two, each reaction must form enough reacted beads to provide samples for each well in the next reaction. Since the mixing and splitting processes are not perfect, the use of large numbers of beads ensures that each desired library member will be prepared on at least one bead. 

Fig. 3. Solid-phase combinatorial synthesis of triazine-scaffolded library of ligands using a modified mix and split strategy.

A wide variety of encoding methods have been developed to record the history of a bead used in the Furka mix and split technique. This section outlines some of these methods. 

Mix and split into two aliquots and repeat the previous processes until the required library is obtained. 

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

Outline the parallel synthesis technique for carrying out a combinatorial synthesis. How does this method differ from Furka s mix and split method ... 

Outline a design for a combinatorial synthesis for the formation of a combinatorial library of nine compounds with the general formula B using the Furka mix and split method. Outline any essential practical details. Details of the chemistry of peptide link formation are not required it is sufficient to say that it is formed. 

Outline the range of encoding methods used to deduce the structures of compounds produced in a Furka mix and split combinatorial synthesis. 

If solid phase is selected, use parallel synthesis or Furka s mix and split ... 

The repetitive nature of oligomeric synthesis has enabled the rapid implementation of solid-phase and automated methods for DNA [20,21,85,86] and peptide combinatorial libraries. Using these systems for the synthesis of single compounds or mixtures of compounds, multiple reaction vessels numbering 8 [45], 15 [80], 20 [59], 24 [50], 25 [57,58], 36 [53-55,77-79], 48 [26,39-41], or 96 [42-44] can be manipulated. Only a few of these systems enable automated resin mixing and splitting within the instrument to generate mixtures of compounds [53,59,78,87,88],... 

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