Analytical characterization of combinatorial libraries

A. General Strategy of Analytical Characterization of Combinatorial Libraries... 

In this chapter we will discuss current approaches for analytical characterization of combinatorial libraries in a pharmaceutical industry environment. Recently, several analytical groups have presented very similar strategies for analysis of libraries [7-9]. As will be shown later, the key to successful analytical characterization of a combinatorial library is to perform analytical and chemical work in parallel with the library development. The accumulation of data and analytical experience during this process results in an assessment of library quality with a high level of confidence, even if as little as 5-10% of the library components are analyzed. Utilization of the strategy will be demonstrated using two examples analysis of a library synthesized on a robotic station in spatially addressed format and analysis of a library synthesized in accordance with split-and-mix technology. 

Analytical characterization of OBOC library will be discussed for an example of a small-molecule combinatorial library with a complexity of 54,150 (57 x 25 x 38) compounds (Figure 10.9) [63]. In the result of an OBOC library analysis, identity, purity, and quantity of library components should be evaluated, but it is unknown which one out of thousands of compounds in a library is synthesized on the particular bead picked for analysis (Figure 10.10). General schemes of analytical characterization of combinatorial libraries (analysis of a small number of standard compounds, analysis of the model library, and analysis of the production-scale library) can be applied to analysis of OBOC libraries, but the issue of structure elucidation for OBOC libraries should be addressed through the scheme. 

Concluding this chapter, we would like to stress that the analytical characterization of combinatorial libraries is a very rapidly developing and changing area of analytical chemistry. The variety of synthetic strategies used in combinatorial chemistry continues to grow and requires development of new analytical methods. The authors would not be surprised if new ones replaced many of the approaches described in this chapter or radically... 

Any MS experiment begins with ionization of molecules of analyte. Numerous ionization techniques (electron ionization, fast atom bombardment, plasma desorption, electrospray ionization, etc.) allow MS analysis of a wide range of organic molecules. In most cases the characterization of combinatorial libraries means analysis of crude compounds i.e. one can expect not only the intended compound to be present in the analyte, but also products of side... 

Why A primary library can be planned for many purposes. Sometimes the project goal is purely academic, that is, simply to open a new combinatorial synthetic route and to report it. This respectable option allows complete freedom to prepare any primary library, with no constraints dictated by parameters such as application, cost, or resources. We will not comment further upon this approach in this section. More often, though, a primary library is prepared as a source of relevant active molecules on various biological targets. This obviously necessitates the availability of several robust and reliable HTS assays for these targets, as well as the synthetic resources, the analytical resources, and the instrumentation to ensure the successful synthesis and analytical characterization of the library. 

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 for all synthetic products to be tested in biological systems, a careful analytical characterization of peptide libraries is crucial in order to confirm their identity and establish their quality. Compared to individual peptides, however, the analysis of peptide libraries is complicated due to the fact that the peptides are either bound to a solid support or arranged in highly complex mixtures. This poses certain restrictions on which analytical methods can be used to characterize combinatorial libraries. For example, analytical methods that are based on the separation of product components, such as high performance liquid chromatography (HPLC) and capillary electrophoresis (CE), are only of limited use for the analysis of peptide libraries, in particular of those made up of complex nnixtures (>100 peptides per mixture). The analytical methods beneficially applicable to peptide libraries include amino acid analysis, mass spectrometry, and sequencing. 

Characterization of combinatorial libraries (product structure determination) can be performed by direct analytical methods, while compounds are still attached to the beads. Analytical techniques such as NMRandMAS-NMR,1314ESI-MSandHPLC-ESI-MS,15-17... 

The new field of molecular diversity raises three issues which need to be addressed by the organic analytical chemistry community (i) What tools can we use for following solid-phase reactions (ii) How can we analyze all these samples (iii) How much characterization of libraries is possible or appropriate This chapter deals with these problems and reviews the literature since a similar review written in June 1995 [2] (earlier seminal publications are described where appropriate). Other analytical issues such as decoding of combinatorial libraries or the applications of affinity separations and single-bead mass spectrometry for library deconvolution are dealt with in other chapters of this book. 

Sepetov N, Issakova O, Analytical characterization of synthetic organic libraries, in Combinatorial Chemistry and Combinatorial Technologies Methods and Applications (Eds. Fassina G, Miertus S), 237-268, 1998, 2005, Chapter 10, this volume. 

It is worth mentioning that a subject for analytical characterization is a library, but technically compounds are analyzed. What is the difference between analysis of a combinatorial library and analysis of a collection of many compounds There are at least two major differences related to quantitative and qualitative aspects of combinatorial libraries. The first is that combinatorial synthesis is a practically unlimited source of compounds. The number of compounds in a library may very easily reach the point at which none of the existing analytical methods will allow measurement of all compounds in a library. The second major difference between a combinatorial library and a... 

Issakova O, Nikolaev V, Ma N, Wade S, Sepetov N, Analytical characterization of one-bead-one-compound combinatorial libraries by LC/MS and LC/MS/ MS, Proc. 45th ASMS. Conf, p. 403, 1997. 

The term chemical assessment describes the process through which the reaction scheme to arrive at a target molecule is combinatorialized. This process may include the transfer of a reaction from solution onto SP and/or the adaptation of the reaction conditions to the use of many monomers with different reactivities and stabilities for library synthesis. Monomer rehearsal is an accurate check of the reactivity of a monomer set in the synthetic scheme for the buildup of the library so that the unreactive/difficult monomers are removed from the set. A model library is a small set of discretes, or a small pool, that is prepared using the planned synthetic route for the library and is fully characterized by the appropriate analytical tools Only if the results are satisfactory is the library synthesis carried out. Quality control determines the analytical profile of a library as a single entity, but data from each library individual, or a significant percentage of library individuals, are acquired. A library with 80% confirmed pure compounds is a good-quality library, but the 20% of samples that are... 

L. Zeng, and D. B. Kassel, Developments of a fully automated parallel HPLC/mass spectrometry system for the analytical characterization and preparative puriflca-tion of combinatorial libraries, Anal. Chem. 70 (1998), 4380-4388. 

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