Combinatorial Chemistry Review

Analytical Techniques

The resin bead mix and split method can be used to generate hundreds, thousands or even millions of different products. As an example, a four step synthesis employing 10 building blocks at each step would afford 10 000 different compounds in only 10*4 chemical steps. Although synthesis is rapid, the power of combinatorial libraries is only evident if structural information on active components may be easily obtained. The iterative resynthesis and rescreening offers a solution, but as it can be slow and requires a further dedication of synthetic and screening resource, there have been a number of new methods devised where information concerning the active compound may be carried on the bead in the form of a "tag".

The synthetic efficiency of the split synthesis technique can be contrasted with the technical difficulties encountered when analysing the resulting libraries. For example, the simple split synthesis scenario outline above results in a library consisting of 10 pools of 1 000 compounds each. These compounds can be cleaved into solution and screened as soluble pools, or the ligands can remain attached to the beads and screened in immobilised form. Neither scenario is ideal for several reasons. Because of limitations on solubility, the concentration of the individual compounds present in soluble pools must be correspondingly diminished as the pool size increase perhaps below a desirable threshold for screening. Biological screens performed on such large mixtures of soluble compounds can be ambiguous since the observed activity could be due to a single compound or due to a collection of compounds acting either collectively or synergistically. The subsequent identification of specific biologically active members is challenging, since the number of compounds present in the pools and their often-limited concentration deter their isolation and erase. Because of this, biologically active pools are often iteratively resynthesised and reassayed as increasingly smaller subsets until activity data are obtained on homogenous compounds.

This process of iterative resynthesis is time consuming, requires multiple bioassays, and the deconvolution of a single pool to its individual constituents typically require more synthetic step than were required to prepare the parent library. When multiple pool are active, the deconvolution process becomes additively complex if each active subset is chosen for resynthesis. In addition to begin inefficient, positive selection strategies such as iterative deconvolution ignore negative biological information, the knowledge of which is often important in the design of subsequent libraries.

In some instances, bead-based split synthesis libraries can be successfully assayed with the ligands still immobilised to the beads. In this process, a reporter system is employed in the biological assay such that beads displaying active ligands can be physically distinguished from those displaying inactive compounds. Suitable reporter system includes the use of fluorescently labelled receptors, or anti-receptors antibodies similarly labelled with a reporter molecule, that can be employed to "label" active beads. Beads thus marked are physically removed and analysed to identify the attached ligand. This technique is limited by the capacity of the biological screen to detect immobilised ligands, as well as the sensitivity of the analytical methods employed to unambiguously identify the attached compounds.