Characterization of combinatorial libraries

Fang, A. S. Vouros, P. Stacey, C. C. Kruppa, G. FL Laukien, F. FL Wintner, E. A. Carell, T. Rebek, J. Jr. 1998. Rapid characterization of combinatorial libraries using electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry. Comb. Chem. High Throughput Screen, 1,23-33. 

Kyranos, X N. Hogan, X C. 1998. High-throughput characterization of combinatorial libraries generated by parallel synthesis. Anal. Chem., 70, 389A-395A. 

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. 

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... 

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... 

Batt J, McDowall MA, Preece SW, Rontree JA, Automated LC/MS for the characterization of combinatorial libraries, Proc. 44th ASMS Conf, p. 1033, 1996. 

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. 

Mass spectrometry (MS) is playing an increasingly visible role in the molecular characterization of combinatorial libraries, natural products, drug metabolism and pharmacokinetics, toxicology and forensic investigations, and proteomics. Toward this end, electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), and atmospheric pressure photo-ionization (APPI) have proven valuable for both qualitative and quantitative screening of small molecules (e.g., pharmaceutical products) [9-14]. 

Initially, high-throughput characterization of combinatorial libraries was developed for and performed with single quadmpole instmments, providing unit-mass resolution and nominal molecular-mass determination. The application of high-resolution mass spectrometers, such as time-of-flight (TOF) and FT-ICR-MS, further enhances the power of the approach [22]. 

In the search for related substances, isolation of the impurities by preparative LC can be one of the steps to be performed. The characterization of combinatorial libraries by means of LC-MS reveals that the compounds generated in this way are often not sufficiently pure for successful biological screening. Therefore, there is a need for high-throughput preparative purification procedures. This led to the development of automated LC-MS controlled fractionation systems to be used in preparative LC. 

A.S. Fang, P. Vouros, C.C. Stacey, G.EI. Kruppa, F.EI. Laukien, E.A. Wintner, T. Carell, J. Rebek, Jr., Rapid characterization of combinatorial libraries using ESI-FT-ICR-MS, Comb. Chem. High Throughput Screen., 1 (1998) 23. 

This second updated, revised and expanded edition of this book on LC-MS was written and finished in a period when interface innovations somewhat calmed down. Electrospray and APCl have become the interfaces of choice. At present, no major developments in interface technology can be foreseen that will lead to another breaktluough in LC-MS. In terms of applications and versatility, innovations continue to appear, e.g., in the use of LC-MS in characterization of combinatorial libraries and in other phases of dmg development, in the advent of electrospray time-of-flight instramentation for impurity profiling, in applications in the field of biochemistiy and biotechnology. 

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... 

WL Fitch, A Lu, K Tsutsui, N Shah. Single bead mass spectrometry for characterization of combinatorial libraries. Proceedings of the 44th ASMS Conference on Mass Spectrometry and Allied Topics, Portlan, OR, 1996, p. 1043. 

JP Nawrocki, M Wigger, CH Watson, SA Benner, JR Eyler. Characterization of combinatorial libraries using ESI-FTICR mass spectrometry. Proceedings of the 45th ASMS Conference on Mass Spectrometry and Allied Topics, Palm Springs, CA, 1997, p. 1256. 

Kyranos, J.N. Hogan, J.C. Jr. High-Throughput Characterization of Combinatorial Libraries Generated by Parallel Synthesis, Anal. Chem. 70,389A-395A (1998). 

Drug discovery Purification and characterization of combinatorial libraries and lead compounds, ADME/PK screening HPLC MS, MS/MS, PDA, ELSD... 

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