Support-bound libraries, resin beads

Cyclic disulfide libraries have primarily been generated through sohd-phase oxidation of support-bound peptide libraries, either on resin beads l or on a cellulose membrane support.P A study comparing several methods for solid-phase and solution cyclizationt found oxidation with hydrogen peroxide (1.5 equiv at a peptide concentration of 0.5g L i) to be best suited for the generation of cyclic disulfide peptide hbraries in solution. 

The solid-bound library is treated with a labeled soluble biological target. For many studies a fluorescent label has been employed because of the high sensitivity of fluorescence detection. The labeled receptor binds to those resin beads that are derivatized with compounds that have the highest affinity to the biological receptor.The labeled beads are then selected followed by structural elucidation of the support-bound compound (see Section 1.4.1).The identity of the bioactive substance can be limited to a few alternative structures by mass spectrometric determination of the molecular mass. Highly efficient, automated methods have been developed to isolate the labeled beads, for example, by use of a fluorescence-activated cell sorting instrument [96]. 

Such a procedure is well established in the case of peptides, but solid-phase organic chemistry (SPOC) is more difficult. Optimization of the chemistry is required prior to library generation most of the time. Compound identification is complicated by the insolubility of the support. Release of the anchored structure in solution followed by standard spectroscopic analyses may impart delay and/or affect product integrity (9). A direct monitoring of supported organic reactions is thus preferable to the cleave and analyze methodology. Nevertheless, it presents several constraints. A common resin bead loaded at 0.8 mmol/g commonly produces nanomole quantities of the desired compound, and only 1% of the molecules are located at the outer surface of the bead (10). Very few materials, covalently bound to the insoluble support, are thus available for the analysis, which should ideally be nondestructive. 

Further cycloadditions used to prepare cycloalkenes on insoluble supports include the cyclopropanation of resin-bound alkynes and of polystyrene [165] (Figure 5.18). The latter reaction has been used to introduce tags onto polystyrene beads, which enable the recognition of a certain bead in compound libraries produced using the mix-and-split method (Section 1.2 [165-167]). The structure of polystyrene tagged in this way has not, however, been rigorously determined. 

The second approach, named many compounds per bead (Fig. 7.3), starts by coupling the solid support in a single reaction vessel with an equimolar mixture of the 100 amines (step a) then the mixture is reduced (step b) and the resin-bound amines are reacted with an equimolar mixture of the 100 acylating agents (step c). The 10,000-member library is obtained as a single 50-g pool of resin, and each bead contains similar quantities of each library individual. A bead has typically 10 " -10 reaction sites, so that each bead will contain an average of lO -lO copies of each library individual. The library synthesis could technically be considered successful if all the monomers react properly and the 10,000 compounds are acmally present, but the identification of positives from this library for any specific application is not feasible. In fact, the cleavage of resin-bound materials produces an equimolar mixture of all the components, whose activity, if any, is the activity of a 10,000-member unresolved mixmre. As a consequence of this major limitation, this SPS approach is not used for library synthesis. 

This on-bead screening is particularly useful for libraries of several thousand to a million compounds and the isolation of a few bioactive compounds from many inactive ones. By using an on-bead assay, the screening of a library of 107 resin-bound compounds can be accomplished routinely by one person in one day. One advantage of this method is that, once the library has been prepared and assayed, the remaining compounds may be reused for different biological assays. However, all of the difficulties of screening on the solid support remain. 

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