Library-On-Bead

The rapid increase in the separation factors observed for the individual series of columns reflected not only the improvement in the intrinsic selectivities of the individual selectors but also the effect of increased loading with the most potent selector. Although the overall loading determined from nitrogen content remained virtually constant at about 0.7 mmol g for all CSPs, the fractional loading of each selector increased as the number of selectors in the mixture decreased. Thus, the whole method of building block selection and sublibrary synthesis can be also viewed as an amplification process. 

In contrast, there are fewer limitations from the chemical point of view. The preparation of large, well-defined, libraries that involve amino acid building blocks has been demonstrated many times. Carefully optimized reaction conditions for the preparation of other mixed libraries can also ensure that each desired compound is present in sufficient amount. However, the reaction rates of some individual selectors with the activated solid support may be lower than that of others. As a result, the more reactive selectors would occupy a majority of the sites within the beads. Since the most reactive selectors may not be the most selective, testing of a slightly larger number of specifically designed CSPs may be required to reduce the effect of falsenegative results. 

Chiral separation media are quite complex systems. Therefore, neither combinatorial methods nor even the identification of the best selector can ensure that an outstanding chiral separation medium will be prepared. This is because some other variables of the system such as the support, spacer, and the chemistry used for their con- 

Support of this research by a grant of the National Institute of General Medical Sciences, National Institutes of Health (GM-44885) is gratefully acknowledged. This work was also partly supported by the Division of Materials Sciences of the U.S. Department of Energy under Contract No. DE-AC03-76SF00098. 

Chiral Separation Techniques A Practical Approach, Second, completely revised and updated edition 

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Fig. 3-17. Schematic of the deconvolution process used in the library-on-bead approach.

The availability of several dye tags with several distinguishable excitation and emission has already been exploited for purposes of identifying combinatorial chemistry libraries on beads, but the broad nature of molecular absorption and emission spectra puts a bandwidth limit on the number of dyes that can be practically applied. The logic approach mentioned in the previous paragraph can increase the diversity available with a single color combination manifold times. 

Scheme 7.34 Generation of pyrazole and oxazole libraries on cellulose beads.

To ensure complete conversion for all examples of a 21-member library, irradiation times of 30-60 min were used (Scheme 7.39), employing a multi-vessel rotor system for parallel microwave-assisted synthesis (see Fig. 3.7). The results were confirmed by on-bead FTIR analysis, accurate weight-gain measurements of washed and dried resins, and post-cleavage analysis of the prepared enones. 

There is one more report on the synthesis of a library of phosphorus ligands on solid phase. Waldmann et al. prepared a library of phosphoramidites on beads (Fig. 36.5), but these were only applied in enantioselective C-C-bond formation. In fact, as two ligands need to be bound to the catalyst, the use of an immobilized monodentate ligands should most likely be avoided unless the proximity between the ligands is sufficiently close. In addition, crosslinking by the metal may have a negative impact on the permeability of the polymer for the substrate. 

M. Halkes, . H. Gotfredsen, M. Grotli, L. P. Miranda, J. O. Duus and M. Meldal, Solid-phase glycosylation of peptide templates and on-bead MAS-NMR analysis Perspectives for glycopeptide libraries, Chemistry-A European Journal, 2001, 7, 3584-3591. 

IR spectroscopy is not a very sensitive analytical tool and is, therefore, not well suited to the detection of small amounts of material. If, however, intermediates have intense and well-resolved IR absorptions, the progress of their chemical transformation can be followed by IR spectroscopy [83,88,91-93], Near-infrared spectroscopy, in combination with an acousto-optic tunable filter, can be sufficiently sensitive to enable the on-bead identification of polystyrene-bound di- and tripeptides, even if the peptides have very similar structures (e.g., Leu-Ala-Gly-PS and Val-Ala-Gly-PS) or differ only in their amino acid sequence (e.g., Leu-Val-Gly-PS and Val-Leu-Gly-PS) [94]. Special resins displaying an IR and Raman barcode have been developed, which may facilitate the deconvolution of combinatorial compound libraries prepared by the mix-and-split method [48]. 

In 1991, we first introduced the one-bead one-compound (OBOC ) combinatorial library method.1 Since then, it has been successfully applied to the identification of ligands for a large number of biological targets.2,3 Using well-established on-bead binding or functional assays, the OBOC method is highly efficient and practical. A random library of millions of beads can be rapidly screened in parallel for a specific acceptor molecule (receptor, antibody, enzyme, virus, etc.). The amount of acceptor needed is minute compared to solution phase assay in microtiter plates. The positive beads with active compounds are easily isolated and subjected to structural determination. For peptides that contain natural amino acids and have a free N-terminus, we routinely use an automatic protein sequencer with Edman chemistry, which converts each a-amino acid sequentially to its phenylthiohydantoin (PTH) derivatives, to determine the structure of peptide on the positive beads. 

On-Bead Screening of Encoded Peptidomimetic and Small Molecule Libraries... 

Various on-bead binding or functional screening methods for OBOC combinatorial libraries are described in Chapter 17 in this volume and will not be repeated here. The positive beads are physically isolated under a microscope for structure determination. 

The modified mix-and-splif combinatorial method is used for the synthesis of the ligand library yields n<->n members, where n represents the number of different amines chosen. Usually 5 g of each immobilized ligand are synthesized however, this amount depends on the screening strategy preferred (for example, the FITC-based system or the ELISA on beads require less resin than the conventional affinity chromatography). 

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