Split synthesis approach

The structure of the bioactive members of a combinatorial library prepared by split synthesis can be determined by several methods including bioactivity/deconvolution, microanalytical methods and encoding. In a generalized example of the biorecognition/ deconvolution approach, the final sublibraries are not pooled but tested as mixtures. The most active sublibrary then defines the synthon used in the last synthetic step. The synthesis is then repeated to the penultimate step these sublibraries are not mixed, but each is separately reacted with the preferred last-step synthon. These resulting sublibraries are tested the most active then defines the preferred synthon in the penultimate step, and hence the last two synthons present in the most active member of the family are defined. 

20 sublibraries, each with 160000 members and each with a different AA at position 1 

This iterative process is repeated until the full structure of the most active member is determined. The process is illustrated in Fig. 4 with an eight-membered library prepared in three steps using two synthons in each step. 

---

A library containing several million beads can be screened in a single afternoon. Furthermore, the library is reusable, as it may be washed in 8 M guanidine hydrochloride and then re-screened using a different probe. This split synthesis approach displays the ability to generate peptide libraries of incredible variety, variety that can be further expanded by incorporation of, for example, D-amino acids or rarely occurring amino acids. 

In a similar approach, a pretuned glass encased microchip set to emit a unique binary code is placed in a polypropylene tea bag loaded with polystyrene beads. Using a modified split synthesis approach, a 125-membered tripeptide library N-capped as the /r-carboxy-cinnamic acid amide was prepared on Rink resin. Each porous reactor contained a radio frequency transporter which successfully defined the structure of two inhibitors of protein tyrosine phosphatase [37],... 

Two main approaches to combinatorial chemistry are used—parallel synthesis and split synthesis. In parallel synthesis, each compound is prepared independently. Typically, a reactant is first linked to the surface of polymer beads, which are then placed into small wells on a 96-well glass plate. Programmable robotic instruments add different sequences of building blocks to tfie different wells, thereby making 96 different products. When the reaction sequences are complete, the polymer beads are washed and their products are released. 

Recently, Yoshida and coworkers demonstrated that the cation pool method can be used to make available Ai-acyliminium ion intermediates for parallel synthesis approaches to molecular libraries [106]. In this work, the cation pool was split into separate flasks following the electrolysis reaction. Different nucleophiles were then added to each flask in order to form a series of products (Scheme 49). 

Pibouleau et al. (1988) provided a more flexible representation for the synthesis problem by replacing the single reactor unit by a cascade of CSTRs. They also introduced parameters for defining the recovery rates of intermediate components into the distillate, the split fractions of top and bottom components that are recycled toward the reactor sequence, as well as parameters for the split fractions of the reactor outlet streams. A benzene chlorination process was studied as an example problem for this synthesis approach. In this example, the number of CSTRs in the cascade was treated as a parameter that ranged from one up to a maximum of four reactors. By repeatedly solving the synthesis problem, an optimum number of CSTRs was determined. 

Fig. 1. Split/mix synthesis approach to generate an OBOC combinatorial library.

Split synthesis is a completely different approach to library generation from parallel synthesis. Furthermore, split synthesis can be performed only on a resin solution phase is not an option. Split synthesis involves dividing, or splitting, a pile of loaded resin into batches for reaction with a set of building blocks. After the reaction, all the resin is pooled back together, thoroughly mixed, and then resplit for reaction with the next set of building blocks. Based on this protocol, split synthesis is often also called mix-split or pool-split synthesis. 

The word library is used to define a collection of compounds usually built around a common structural motif. There are three general approaches to library preparation parallel synthesis, mixture synthesis and split synthesis. One of these preferred strategies, parallel synthesis, is the approach where the compounds are made individually by automated or semi-automated methods. The library members may be made either in solution by classical methods, in solution attached to a polymeric carrier or on solid support. In parallel synthesis there must be linkage to a spatially defined position. The structure of the product is inferred from the position of the reactor and by the order of addition of the synthons and reagents at that position in space. Every possible member, resulting from the combinatorial mix of the synthons, need not be included in the library. 

The preferred mode of preparation for both mixture and split synthesis libraries is the solid phase. Both polymer matrices or pins and resin beads have been used. The solid-phase approach is preferred because of its simplicity and ease of purification and isolation of the reaction products. Unlike the spatially addressable library when structure is defined by position in a set of reaction vessels, the structure of interesting library members prepared by mixture or split synthesis must be defined by highly sensitive analytical methods or indirectly by encoding or by biological results combined with resynthesis, the so-called deconvolution method. 

With peptide and oligonucleotide libraries prepared by split synthesis, the structure of a bound ligand on an individual bead can be determined by microsequencing [ 17,18]. This approach has been applied to testing libraries where the bead bound compounds are evaluated for their ability to bind to monoclonal antibodies or other recognition macromolecules. In one such approach, fluorescence-based methods are used to select the most... 

Various sulhir-containing odorants, such as 2-thioalcanes, 3-acetylthio-2-alkyl aikanals, and trialklylated 1,3,5-dithiazines were prepared by conventional, parallel, and split-mix synthesis approaches. 2-Heptanethiol, newly identified in bell peppers, and 3-acetylthio-2-methylpentanal showed relatively low odor thresholds of 10 and 5 pg/kg water, respectively. Several odorants were found to develop interesting notes, which are compatible with both savory and sweet flavors. 

---