Screening and deconvolution

The success of a library depends not only on it containing the right compounds but also on the efficiency of the screening procedure. Furthermore, it is no use preparing a combinatorial library if there is not a suitable screening procedure 

The preparation of the first group of secondary libraries to find the first residue in the peptide. 

In order to be effective, deconvolution procedures require that both the synthesis and assay of the library be rapid. The procedure is complicated when there is more than one active component in the library. In this case it is necessary to prepare and test all the possible compounds indicated by deconvolution in order to identify the most active compound in the library. 

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Fig. 4.3 Primary screening and deconvolution stage mass spectra. (A) The region of the negative ion mass spectra containing the ions of interest is shown for two compounds, in two replicate primary screening experi-ments. Full-scale y-axis intensity values are normalized to 308 counts per second for compound 4 (m/z 436) and 162 counts per second for compound 5 (m/z 498).

A solid-supported reagent, 9.67, was used to activate the monomer set Mi (60 carboxylic acids, step a. Fig. 9.28) added to the solid support as 6 mixtures of 10 representatives. The activated ester pools 9.68 were reacted with the monomer set M2 (amines and primary alcohols, step b. Fig. 9.28) and the library L17 was recovered, after filtration, as a set of 10-member pools with yields varying from 60 to 90% (mass recovery). Screening and deconvolution produced 9.69, an active compound of interest (Fig. 9.28, bottom). Its structure made it suitable for further optimization via focused library efforts, while its similarity to known bleachers also pointed toward a specific mechanism of action (118). No further efforts were reported by the authors. 

This section provides an account of synthetic approaches and experimental procedures for the generation of peptide and peptidomimetic libraries with special emphasis on solid-phase methods, along with approaches for library screening and deconvolution. Other library techniques, such as phage display librariesf l and the generation of nonpeptide small-molecule libraries,are not covered here. 

Recently, Fmoc-N-protected (3-amino acid synthons have been prepared and used for the synthesis of (3-peptides on solid phase [103]. This methodology facilitates enormously the search for new bioactive compounds, above all through the generation of combinatorial (3-peptide libraries. In this context, (3-amino acids have been used as building blocks for RGD cyclic peptides and for the synthesis of an inhibitor of human cathepsin L, previously identified by screening and deconvolution of pentapeptide amide collections [104,105]. 

Various aspects of combinatorial methods and technology have been the subject of patent applications. These include screening and deconvolution... 

In the retesting and deconvolution phase of the procedure new compound mixtures were made based on the results of primary screening. These contained from nine to 14 compounds and no monoisotopic mass redundancy. Since most mass spectrometric peaks picked as hits in the primary screen contain more than one compound, and only one compound per peak is likely to be a binder, the nonmass redundant retest mixtures are unlikely to contain more than a few bona fide ligands, so once again target excess is maintained. Both the initial (round zero,... 

Fig. 4.3 (B) Deconvolution experiments for compound 4. The compound is screened in a much smaller mixture than in primary screening and with no mass redundancy.

Libraries prepared by application of real combinatorial synthetic methods are usually submitted to screening experiments, either as soluble mixtures or as unknown discrete compounds cleaved from, or tethered to individual beads of the solid support. The task in deconvolution is to identify the substance that has a desired property. The deconvolution methods can be classified into two groups deconvolution of mixtures, cleaved from support and deconvolution of tethered libraries. 

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