Split and pool synthesis

The procedure makes it possible to prepare millions or even trillions of compounds as mixtures that can be used in drug research.

According to traditional methods, most organic compounds are synthesized one by one from building blocks coupling them together one after the other in a stepwise manner.

So the productivity of the split and pool method invented by Prof. Á. Furka (Eötvös Loránd University Budapest Hungary), in 1982 seemed incredible at first sight.

The document is written in Hungarian and translated to English[1] Motivations that led to the invention are found in a 2002 paper[2] and the method was first published in international congresses in 1988[3] then in print in 1991.

Choosing the solid phase method in the S&P synthesis is reasonable since otherwise removal of the by-products from the mixture of compounds would be very difficult.

In fact, the theoretical maximum number of components depends on the quantity of the library expressed in moles.

As far as the chemistry of the couplings makes it possible the components of the libraries form in nearly equal molar quantity.

The solid phase method makes it possible to use the reagents in excess to drive the reactions close to completion since the surplus can easily be removed by filtration.

Although a considerable amount of labor could be saved by using the two mixtures approach when a high number of BBs are coupled in each position, it is advisable to stick to the normally used S&P procedure.

It depends on the decision of the chemist to use the library in the tethered (OBOC) form or cleave down the compounds from the beads and use it as a solution.

In the early years of combinatorial chemistry, an automatic machine was constructed and commercialized at AdvancedChemTech (Louisville KY USA).

For this reason, encoding methods had been introduced to help to determine the identity of the compound contained in a selected bead.

[7] They used mixtures of 18 tagging molecules that after cleaving them from the beads could be identified by Electron Capture Gas Chromatography.

The method was implemented by Nielsen, Brenner, and Janda[12] using the bifunctional linker of Kerr et al. to attach the encoding DNA oligomers.

Han et al. described a method that made it possible to keep the advantages of both the high efficiency of S&P synthesis and that of a homogeneous media in the chemical reactions.

So, the theoretical maximum number of compounds depends on the quantity of the solid support and the size of the beads.

An important modification was introduced in the synthesis of DNA encoded combinatorial libraries by Harbury and Halpin.

This makes it possible to synthesize libraries containing even trillions of components and screen them using affinity binding methods.

By attaching Rf groups to the substrate the synthesis can be carried out in solution and the product can be separated from the reaction mixture by liquid extraction using a fluorous solvent like perfluoromethylcyclohexane or perfluorohexane.

[17] One of the best examples of the special features caused by DNA encoding is the synthesis of the self-assembling library introduced by Mlecco et al.[18] First, two sublibraries are synthesized.

The two sublibraries are mixed in equimolar quantities, heated to 70 °C then allowed to cool to room temperature, heterodimerize and form the self-assembling combinatorial library.

In affinity screening, the two BBs of the pharmacophore may interact with the two adjacent binding sites of the target protein.

The yoctoreactor method introduced by Hansen et al.[21] is based on the geometry and stability of a three-dimensional DNA structure that creates a yoctoliter (10−24 L) size chemical reactor in which proximity of BBs brings about reactions among them.

The DNA oligomers comprise the DNA-barcode for the attached BBs and form the structural elements of the reactor.

The figure shows one member of a simple ssDNA template library (A) containing the codes of three BBs (2, 4, 6) that planned to be successively attached.

The sequence directed procedure uses a series of columns of resin beads each coated with the anticodon of one of the BBs (B).

The final library contains all of the synthesized organic compounds attached to their encoding DNA oligomers.

Modifications had been developed enabling the split and pool synthesis to produce known compounds in larger quantities than the content of a bead of solid support and retain the high efficiency of the original method.