Combinatorial libraries with mass spectrometry

For those readers who are not yet familiar with mass spectrometry, the introduction provides an explanation of the basics of mass spectrometry and its instrumentation as well as practical aspects and applications in bioanalysis. Next, a block of three chapters shows different affinity selection procedures suitable to identify hits from combinatorial compound libraries. This subject, being metaphorically speaking a search for a needle in a haystack, is of outstanding relevance for big pharma . The techniques described here offer real high throughput capabilities and are implemented already in the routine industrial screening... 

Mass spectrometry and isotope techniques can be used effectively for the encoding/decoding of pooled libraries. Two of these techniques are mass encoding [44] and stable isotope encoding [44,45]. Because stable isotope encoding is more direct and lends itself more readily to combinatorial library screening with mass spectrometry, it is discussed below. 

As with many areas of drug discovery, mass spectrometry has become intertwined with combinatorial chemistry, especially in the areas of library characterization and screening. Some future advances in mass spectrometry will be driven by the needs of combinatorial chemistry, and mass spectrometry will, to some extent, regulate the progress in combinatorial chemistry. 

Capillary electrophoresis (CE) is a powerful separation technique. It is especially useful for separation of ionic compounds and chiral mixtures. Mass spectrometry has been coupled with CE to provide a powerful platform for separation and detection of complex mixtures such as combinatorial libraries. However, the full potential of CE in the application of routine analysis of samples has yet to be realized. This is in part due to perceived difficulty in the use of the CE technique compared to the more mature techniques of HPLC and even SFC. Dunayevskiy et al. [136] analyzed a library of 171 theoretically disubstituted xanthene derivatives with a CE/ESI-MS system. The method allowed the purity and makeup of the library to be determined 160 of the expected compounds were found to be present, and 12 side products were also detected in the mixture. Due to the ability of CE to separate analytes on the basis of charge, most of the xanthene derivatives could be resolved by simple CE-MS procedures even though 124 of the 171 theoretical compounds were isobaric with at least one other molecule in the mixture. Any remaining unresolved peaks were resolved by MS/MS experiments. The method shows promise for the analysis of small combinatorial libraries with fewer than 1000 components. Boutin et al. [137] used CE-MS along with NMR and MS/MS to characterize combinatorial peptide libraries that contain 3 variable positions. The CE-MS method was used to provide a rapid and routine method for initial assessment of the construction of the library. Simms et al. [138] developed a micellar electrokinetic chromatography method for the analysis of combinatorial libraries with an open-tube capillary and UV detection. The quick analysis time of the method made it suitable for the analysis of combinatorial library samples. CE-MS was also used in the analysis... 

The purification of combinatorial libraries on a Berger system is deseribed by Farrell et al. at Pfizer for their parallel solution-phase syntheses. The overall process employs as well analytical SFC in combination with mass spectrometry and nitrogen chemiluminescence deteetion off-line of the preparative-scale SFC systems. Pre-purification analytical SFC/ MS/CLND allows the triage of samples for purification, and an in-house software package analyzes data for predicted quality based on an evaluation of UV and MS data for the potential of co-eluting peaks during purification. This same software package selects a collection time window for purification, which is necessary to limit the number of fractions per sample. This system accommodates the purification of samples up to 50 mg... 

ESI-MS has been used for the quantification of a number of substrates and products of enzymatic reactions [56,57]. Hsieh et al. report the use of ion spray mass spectrometry (a technical variation of electrospray ionization) coupled to HPLC for the kinetic analysis of enzymatic reactions in real time [58]. The hydrolysis of dinucleotides with bovine pancreatic ribonuclease A and the hydrolysis of lactose with 3-galactosidase were monitored and the resulting data were used for the estimation of and v x of these reactions. Another field of application of electrospray mass spectrometry is the screening of combinatorial libraries for potent inhibitors [31,59]. 

Another use for combinatorial libraries has been the screening of peptides for antimicrobrial properties. In this case, the design of the library is based on antimicrobial peptides found in nature. A combinatorial synthesis is used to find alternative unnatural amino acids expected to mimic the antimicrobial properties.23 Peptide libraries also have been used to find compounds that could bind the lytic peptide mellitin.24 The library was synthesized in solution phase, purified, and evaluated using time-of-flight mass spectrometry (TOF-MS). The sequences determined to bind to mellitin contained hydrophobic pairs. By binding to mellitin, they were able to prevent the cell-surface mellitin interaction. This is an example of a peptide library able to afford compounds that interact with other small peptides without having to find an interacting protein first. 

Wang, T. Zeng, L. Strader, T. Burton, L. Kassel, D. B. 1998. A new ultra-high throughput method for characterizing combinatorial libraries incorporating a multiple probe autosampler coupled with flow injection mass spectrometry analysis. Rapid Commun. Mass Spectrom., 12, 1123-1129. 

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. 

Using a GA for generation of diversity, one may chose a molecular property and select for molecules that are different with respect to that property. A dissimilar, unique molecular weight is such a property that would facilitate the deconvolution of combinatorial library mixtures by mass spectrometry. The optimal design of such mixtures was the target function in a recent application of a GA [26],... 

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. 

Because all library compounds must be monitored simultaneously, the compounds must be selected so that they have unique molecular weights. Also, one compound in the mixture should not suppress the ionization of another. Therefore, this approach is probably restricted to the screening of small combinatorial libraries that are similar in chemical structure and ionization efficiencies. Finally, the binding buffer used for affinity chromatography must be compatible with on-line APCI or electrospray mass spectrometry. This means that the mobile phase must be volatile and usually of low ionic strength (i.e., typically <40 mM for electrospray ionization). 

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