Structure Determination, Quality Control, and Purification of Solid-Phase Discrete Libraries

2 STRUCTURE DETERMINATION, QUALITY CONTROL, AND PURIFICATION OF SOLID-PHASE DISCRETE LIBRARIES 

After the final cleavage from the beads, the solutions containing the discrete library individuals are submitted to a work-up procedure and then the pure individuals are tested against one or a few selected targets. The results of the assays will, hopefully, create useful information that will allow further research to be focused on active structures. However, these results must be coupled with quality control, that is, the complete analytical characterization of the library. This allows the purity of each positive compound to be determined to check if the observed activity is due to the presence of impurities (false positives) and to locate the wells where the expected library individuals are absent (false negatives). Moreover, the analysis of the whole library will determine if a final purification of the compounds is required. 

In theory, it would be necessary to provide a profile of analyses for each member of the library to have a reliable and exhaustive quality control. However, this is feasible only for small libraries (a few tens of components), and when the numbers of components increase to hundreds or thousands, this process would be too time consuming. The best compromise is provided by a. fully automated technique that can 

Flow injection analysis mass spectrometry (FIA-MS) has been reported to be a fast method for the characterization of combinatorial libraries (55,56). The method verifies the presence of the molecular ions of the expected product and side products or impurities but does not provide information on the quality of the analyzed samples. Significant improvements related to the increased analytical throughput, obtained by reducing the time between each injection without increasing the intersample carry-over from each analysis, were recently reported (57, 58). When coupled with RP-HPLC, FIA-MS allows the separation and the determination of the molecular weight of the components of each sample. This is normally enough to unequivocally attribute the structure of the expected library component and of any side products from a library synthesis. 

As these detection methods are complementary rather than mutually exclusive, multiple detection is recommended for laboratories that routinely perform the synthesis of discrete libraries and can be realized by splitting the eluate of the HPLC/UV system in two or three aliquots that are sent to the mass spectrometer and the CLND and/or the ELSD instrument. Examples of multiple UV/MS/CLND (67) and UV/MS/ELSD (68) show how the different methods of detection are useful in 

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