The objective of this project is development of new mass spectral techniques in order to provide innovative and/or more rapid solutions to problems involving (1) chemical structure determination, (2) complex mixture analysis and (3) measurement of trace components in biological systems. Matrix-assisted laser desorption ionization (MALDI) mass spectrometry, electrospray ionization mass spectrometry (ESI/MS), tandem mass spectrometry (MS/MS), combined liquid chromatography-mass spectrometry (LC/MS), combined capillary electrophoresis-mass spectrometry (CE/MS) and accurate mass measurement are the techniques of current interest. On-line CE sample concentration techniques with subsequent peak collection are being investigated as a preparative-scale method for the off-line combination of CE with high resolution MALDI/MS and post-source decay fragmentation analysis. The off-line combination of high-performance liquid chromatography (HPLC) with MALDI/MS is also being investigated for the analysis of complex synthetic mixtures.? ? A number of synthetic compounds built on a constrained glycerol scaffold (disubstituted DAG-lactone) have been identified as potent agonists of protein kinase C (PK-C). Depending on the structure of the substituents comprising R1 and R2, these DAG-lactones appear to have some degree of PK-C isozyme specificity. A solid-phase combinatorial approach is being applied in the LMC to investigate chemical diversity at R1 and R2 in order to produce more specific C1 domain ligands. It is important that these synthetic libraries be rapidly characterized and their structures established before biological evaluation. Fast atom bombardment mass spectrometry (FAB/MS), flow-injection ESI/MS, flow-injection APCI/MS and MALDI/MS have been investigated as rapid mass spectral approaches for the initial characterization of these synthetic DAG-lactone libraries. One strategy for the rapid, qualitative structural characterization of these libraries employs FAB/MS mixture analysis. Because the FAB mass spectrum of a typical DAG-lactone derivative is both predictable and structurally informative, it can be used to identify the DAG-lactone derivatives present in simple mixtures. Initially, selected library components are examined individually to evaluate the efficiency of the chemistry and to confirm that mass spectra behave as predicted. Simple 4- to 6-component mixtures encompassing the entire chemical space are then assembled, analyzed and interpreted. Library components not detected during mixture analysis are subsequently analyzed individually. Mixture analysis correlates directly with individual analysis, substantially reduces the number of samples to be examined, results in enhanced analytical turn-around and is amenable to accurate mass measurement. Flow-injection ESI/MS is not suitable for analysis of these DAG-lactones because of their high lipophilicity and limited basicity. Flow injection-APCI/MS produces spectra that consist of MH+ as well as various fragment and solvent-adduct ions. This later approach appears to be complementary to FAB/MS in terms of spectral information. MALDI/MS also appears useful as a tool for the rapid characterization of these small molecule libraries although variable alkali metal ion cationization complicates spectral interpretation. Matrix-less MALDI is currently under investigation as an approach to resolve this problem and provide an even more rapid analysis approach. Our ultimate goal is the structural characterization of all 96 library components in a one day. Aliquots of each library component are being archived for possible future analysis should later biological evaluation warrant additional characterization.? ? FAB/MS is routinely employed to support the LMC synthetic effort through structural characterization of new compounds and synthetic intermediates.?
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