Mass spectrometry combinatorial compounds

Enjalbal, C. Manx, D. Martinez, J. Combarieu, R. Aubagnac, J.-L. Mass Spectrometry and Combinatorial Chemistry New Approaches for Direct Support-Bound Compound Identification. Comb. Chem. High Throughput Screening 2001, 4,363-373. 

Kaur, S., McGuire, L., Tang, D., Dollinger, G., Heubner, V. Affinity selection and mass spectrometry-based strategies to identify lead compounds in combinatorial libraries. J. Prot. Chem. 1997, 16, 505-511. 

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... 

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. 

Most of the mass spectrometry applications for combinatorial chemistry will be described in the following sections of this chapter. Here we will give a short overview of MS techniques utilized for the characterization of resin-bound molecules. The majority of publications in this field describe applications of matrix-assisted laser desorption ionization (MALDI), combined with time-of-flight (TOF) detection. The major difference of MS application for analysis of resin-bound molecules from the above-described NMR and IR applications is that analyte should not be covalently bound to solid support prior to mass measurement. Detachment of compound molecules from resin can be done chemically (for example, by bead exposure to TFA vapors) [30,31] or photochemically, such that cleavage, desorption, and ionization of molecules occur simultaneously upon stimulation by laser radiation [32], Since the... 

Some particular features of the analysis of products obtained by combinatorial methods have impaired the use of NMR spectroscopy in the initial phase of the development of this technique. Combinatorial chemistry produces large number of compounds in a very short period of time, in small quantities and instead of using traditional glassware for synthesis employs 96-well microtiter plates to store, transport and sometimes even to synthesize the compounds of interest. Another issue is the need to characterize solution and solid samples, since solid phase synthesis is extensively used in combichem. In this context, the need of an efficient and universal sample analysis remains a challenge. Actually, most combichem programs obtain mass spectrometry and UV (photodiode-array detection) data on their samples but clearly the use of NMR spectroscopy provides a structural characterization unparalleled by the aforementioned techniques. In the last years an increasing number of new NMR methods opened the possibility for the utilization of this analytical technique for monitoring combinatorial chemistry reactions. The first part of this chapter will focus on the recent developments introduced in NMR spectroscopy to overcome these difficulties. 

LC-NMR data may be acquired in either onflow or stopped-flow modes. In the onflow mode, effluent flows directly from the column through the probe, where spectra are collected at fixed time intervals. Figure 4 shows a typical onflow LC-NMR spectrum.6 In the stopped-flow mode, several fractions are collected and analysed individually at a later time. Although mass spectrometry continues to be the favoured technique for the analysis of combinatorial libraries, the value of LC-NMR was demonstrated in a study by Chin et al.A1 in a stopped-flow study of four positional isomers of dimethoxybenzoylglycine. Iso-molecular-weight mixtures such as this can be problematic to analyse using mass spectrometry however, in this case assignment of the compounds was readily accomplished from the LC-NMR data (Fig. 5). 

LC/MS emerged as the method of choice for the quality control assessment to support parallel synthesis because the technique, unlike flow injection mass spectrometry, provides the added measure of purity (and quantity) of the compound under investigation. In addition, universal-like HPLC gradients (e.g., 10% to 90% acetonitrile in water in 5 minutes) have been found to satisfy the separation requirements for the vast majority of combinatorial and parallel synthesis libraries. Fast HPLC/MS has been found to serve as good surrogate to conventional HPLC for assessing library quantity and purity [34-37]. Fast HPLC/MS is simple in concept. It involves the use of short columns (typically 4.6 mm i.d. x 30 mm in length) operated at elevated flow rates (typically 3-5mL/min). 

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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