Direct Structure Determination of Positives from Solid-Phase Pool Libraries

2 DIRECT STRUCTURE DETERMINATION OF POSITIVES FROM SOLID-PHASE POOL LIBRARIES 

Direct structure determination methods, where positives are characterized directly via off-bead or on-bead identification of their chemical structure, will be described in detail in this section. Indirect methods that determine the structure of positives from the library architecture will be covered later they use either deconvolutive methods (Section 7.3), where the iterative synthesis of library pools with decreasing complexity via sequential determination of the best monomers leads to the identification of a positive structure, or encoding methods (Section 7.4), where, during the library synthesis, the structure of each component is coupled to a tag that can be read from a single bead after the library screening. 

We will describe these methods illustrating their complementarity with respect to the skills and the equipment available in the laboratory where the SP pool library must be prepared, but most of all with respect to the specific synthetic scheme, format, and size that are planned for each library. 

The majority of reports have used electrospray ionization mass spectroscopy (ESI-MS) as an analytical detection method because of its sensitivity and the soft namre of its ionization procedure, which generally only leads to the detection of the molecular ions of the positive library members. Many separation techniques have been coupled to ESI-MS, including affinity chromatography (49), size exclusion chromatography (50, 51), gel filtration (52), affinity capillary electrophoresis (53-58), capillary isoelectric focusing (59), immunoaffinity ultrafiltration (60), and immunoaffinity extraction (61). ESI-MS has also been used alone (62) to screen a small carbohydrate library. Other examples reported alternative analytical techniques such as MALDI MS, either alone (63, 64) or in conjunction with size exclusion methods (65), or HPLC coupled with immunoaffinity deletion (66). 

As a case example we will consider the approach of van Breemen and co-workers (67-69), who applied pulsed ultrafiltration ESI-MS (70) to the screening for inhibitors of adenosine deaminase (67, 68) and dehydrofolate reductase (69). A schematic representation of the whole process is repiorted in Fig. 7.12. The authors used an HPLC/MS apparatus where the column was substituted with an ultrafiltration chamber (around 100 pL volume) equipped with a 10,000-MW cutoff membrane. Aqueous buffer solutions of the library components and the receptor were incubated, then loaded 

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