Split-and-mix technique

An important difference between the split-and-mix technique and parallel synthesis is that the former method affords small quantities of a large number of peptides, whereas the latter method yields larger quantities of a smaller number of peptides (37). A discussion of the many novel techniques and advances in combinatorial peptide synthesis is beyond the scope of this chapter the interested reader may want to check recent current reviews on this topic (13,41). 

On-bead sensors have also been developed on the basis of the fluorescence energy transfer (FRET) mechanism (Figure 14.2b). FRET signaling is less stringent than PET in terms of binding domain structure but requires large changes in the distance between donor and acceptor, which results in their main application within peptide or nucleic acid structures. On-bead FRET sensors constructed by split-and-mix techniques were initially... 

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-... 

There are two basic combinatorial chemistry techniques (1) parallel synthesis and (2) split and mix methods. They are illustrated next. 

An example of a 3x3x3 combinatorial split and mix combinatorial synthesis is shown in Figure 15.2. The technique involves three initial batches of resin beads to which are initially coupled, for example, a different amino acid. These batches are then combined, mixed and split again into three batches each batch now containing a mixture of beads containing different amino acids. A different amino acid is then coupled to each of these batches of beads, the beads mixed, split and the process repeated a third time. This simple... 

Almost all of the analytical characterization tools (e.g., HPLC, NMR, FTIR, and LC/MS) are serial-based techniques, and parallel synthesis is inherently parallel. Consequently, this led rapidly to a new bottleneck in the discovery process (i.e., the analysis and purification of compound libraries). Parallel synthesis suffers from some of the same shortcomings of split and mix synthesis (e.g., the expected compound may not be pure, or even synthesized in suffi-... 

Figure 1 The split and mix combinatorial technique for three subunits, A, B, and C.

Combinatorial syntheses can be performed in two ways (i) split-and-pool (also referred to as the split-and-mix or the split-and-recombine method)and its variant directed sorting" and (ii) parallel synthesis. A hybrid approach is the split-and-split technique. " ... 

Synthesis of combinatorial arrays of compounds can, in principle, be performed three different ways. The Split and Mix (split and pool/recombine) concept introduced by Furka (4-6) is the most efficient method for preparation of sizable libraries (tens of thousands of compounds). However, the technique requires tracking of the chemical history of the resin beads and limits the quantity of synthesized material to the loading per solid phase unit (one unit can be represented by one particle, bead, lantern, etc., or one container, T-bag, Kan, etc.). Parallel synthesis, on the other hand, can supply any quantity per compound, but it requires handling large numbers of reaction vessels at one time. 

Diversity Sciences developed a library synthesis strategy that combines the simplicity of parallel synthesis and the power of resin-mixing techniques. The general format is four 96-well plates that give rise to 384 synthetic wells, as shown in Figure 8.9. The layout of the synthesis blocks enables 16 unique monomers in monomer position A (across rows) and 24 unique monomers in monomer position B (down the columns). All of the 384 wells are preloaded with off-the-shelf resin where each well has a unique binary code embedded in the analytical construct. The first two points of diversity (monomer A and monomer B) is added in all possible combinations by parallel synthesis. Each spatial location has a unique binary-mass code that encodes for a particular combination of monomer A and monomer B. For example, binary code number 8 represents monomer Al and monomer B8. After the addition of monomer B, the resin from all 384 wells is mixed together and split into 96 identical pools, to which monomer C is added. The third monomer, monomer C, is spatially encoded, since the 96 pools are not mixed after the last step and screened as pools. Upon decoding, the identification of the binary code reveals the combination of monomer A and monomer B on each bead. 

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