Solid split-pool method

The HDAC inhibitors TSA and TPX (Fig. 2) have been utilized as structural leads in the early stages of the quest for new and more selective small molecule inhibitors of the HDAC enzyme family. In order to investigate the function of the individual HDAC members, Schreiber et al. synthesized a library of 7200 potential HDAC inhibitors based on the structural features of TSA and TPX [93]. The members of this library were prepared on solid support by means of split pool methods. The key characteristics of these compounds consist of a dioxane-containing capping region and a zinc binding motive, connected via an aliphatic chain. Three different zinc binders, i.e., carboxylic acid, o-aminoanilide and hydroxamic acid were used. 

Combinatorial libraries are prepared by the (1) parallel synthesis of arrays, (2) split-pool method, (3) biological method, or (4) spatially addressable parallel synthesis [74,78-80]. Parallel synthesis is carried out by the simultaneous synthesis of an array of different compounds. Several methods are available. In the multipin method, the peptide synthesis is carried out on polyethylene rods that have attached protected amino acids [81]. The amino acid sequence of a synthesized peptide on a particular pin depends on the order in which the amino acids are added. The number of products synthesized is the same as the number of pins. Another version of parallel synthesis, known as the teabag method, uses resin-filled bags in place of pins [74]. By pooling the resin portions from the appropriate bags, followed by redistribution and further coupling with a specific amino acid, a peptide library can be synthesized. The SPOT method uses a cellulose paper membrane as a solid support, which acts as an open reactor. Respective reagent solutions are pipetted onto several spots to synthesize as many peptides as the spots chosen [74,82]. 

Sun, Y., Chan, B.C., Ramnarayanan, R., Leventry, W.M., and Mallouk, T.E. Split-pool method for synthesis of solid state material combinatorial library. J. Comb. Chem. 2002, 4, 569-575. 

Dimeric carbohydrate scaffolds are important natural product mimics due to their abihty to activate cellular processes and to increase the binding affinity. One strategy for the dimerization of carbohydrates on solid support used cross-metathesis of two identical olefins, liao and coworkers [44] used a split-pool method to create polymer-boimd benzo[h]furans, which were subsequently treated with Grubbs catalyst, thus undergoing metathesis and forming dimeric molecules. 

The isorniinchnone cyclization/isocyanate cycloreversion process for substituted furan synthesis has been well studied, as exemplified by the conversion of 104 to 106 (Scheme 19.19). In a solid-phase adaptation of this transformation, two groups independently utilized this reaction to estabhsh a traceless self-cleaving method for the synthesis of substituted furans [176, 177]. Further investigation of the thermal requirements of this cycloreversion led to its application in the split-pool synthesis of a small library of amides [178]. 

The concept of reducing the number of reaction vessels and exponentially increasing the number of synthesized compounds was brought to a next level of simplicity by the split-and-pool method of Furka et al.5 The split-and-pool method was independently applied by Lam et al.6 in a one-bead-one-compound concept for the combinatorial synthesis of large compound arrays (libraries) and by Houghten et al.7 for the iterative libraries. Now several millions peptides could be synthesized in a few days. In Furka s method the resin beads receiving the same amino acid were contained in one reaction vessel—identical to Frank s method—however, the beads were pooled and then split randomly before each combinatorial step. Thus the method is referred to as the random split-and-pool method to differentiate it from Frank s method in which each solid-phase particle was directed into a particular reaction vessel (the directed split-and-pool method). 

The split-and-pool synthesis not only simplifies the complexity of the combinatorial synthetic process, but also offers additional important benefits. To undertake a full range of solid-phase chemical reactions, elaborate reaction conditions are needed for some chemical transformations. These include, but are not limited to, low temperature and inert atmosphere conditions. Parallel synthesis of a thousand compounds requires handling of a thousand reaction vessels. The timely addition of sensitive reagents (e.g., butyl lithium) at low temperature (—78°) under inert atmosphere during parallel synthesis is not a trivial task. It can be done if sophisticated automated synthesizer equipment is designed to handle and tolerate such reaction conditions. Such a synthesis can alternatively be performed easily in a manual fashion using a split-and-pool method that requires only a limited number of reaction vessels. Examples from Nicolaou s17 and Schrei-ber s18,19 laboratories have shown that the split-and-pool method is the methodology of choice for the synthesis of complex and diversity-oriented combinatorial libraries. 

Mixture analysis methods are of little value for interesting sized libraries. Single-bead analysis methods are being developed which can be used to assure the quality of larger solid-phase split pool libraries. 

Today s parallel methods remain limited in terms of their ability to produce the large numbers of compounds that many believe will be required by the large number of novel targets predicted to evolve from the Human Genome Project. These novel bioassays coupled with HTS will place an enormous burden on compound production and informatics capabilities. The techniques prepared to handle this need are the split synthesis and the direct divide solid phase synthesis methods. These methods involve the division and pooling of resin particles such that large libraries can be easily produced, usually with a minimum investment in automation. 

In the so-called tea-bag method, originated in 1984 by Houghten et al. [14] for multiple peptide synthesis, the split-pool protocol occurs batchwise on 15 x 22 mm polypropylene mesh packets with pm-sized pores known as tea bags, sealed with resin beads for solid-phase synthesis. This method offers the advantage that a greater quantity of each compound of the library is available at once (up to 500 pmol), which is sufficient for a complete biological and structural characterization. Furthermore, the structural identity of... 

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. 

A Directed Sorting Method intentionally prepares each selected compound from the combinatorial array only once, and the abundance of compounds in the split and pool method is driven by statistics due to the random distribution of pooled solid-phase particles. 

Combinatorial approaches have been applied to this chemistry. In a method amenable to split and pool, PAL, or Rink resin, 89 is modified with an acetoacetate to generate the solid supported aminocrotonate 90. Either a two- or three-component Hantzsch protocol is followed to produce 91. Treatment with TFA carries out the cleavage from the resin and the cyclization to dihydropyridine 92. 

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