HPLC report libraries

FIGURE 13 An HPLC report using mass detection and chemical structure libraries. 

Capillary HPLC-MS has been reported as a confirmatory tool for the analysis of synthetic dyes [585], but has not been considered as a general means for structural information (degradant identification, structural elucidation or unequivocal confirmation) positive identification of minor components (trace component MW, degradation products and by-products, structural information, thermolabile components) or identification of degradation components (MW even at 0.01 % level, simultaneous mass and retention time data, more specific and much higher resolution than PDA). Successful application of LC-MS for additive verification purposes relies heavily and depends greatly on the quality of a MS library. Meanwhile, MB, DLI, CF-FAB, and TSP interfaces belong to history [440]. 

The focus of this chapter has been on the synthesis of new catalysts by parallel and combinatorial methods. Another aspect important to the development of new catalysts by these methods is the screening of these large libraries. We will not attempt to cover this topic comprehensively but do feel it is necessary to summarize some of the approaches that have been taken. Methods for screening libraries can be divided into both serial and parallel methods. Generally, the serial methods are adaptations of standard methods that allow for rapid individual analysis of each member of a library. Serial approaches for the analysis of libraries can be as simple as use of an auto sampler on a GC or HPLC system or as advanced as laser-induced resonance-enhanced multiphoton ionization of reaction products above the head-space of a catalyst (16) or microprobe sampling MS (63). The determination of en-antioselectivity in catalysis is a particular problem. Reetz et al. (64) reported the use of pseudoenantiomers and MS in the screening of enantioselective catalysis while Finn and co-workers (65) used diastereoselective derivatization followed by MS to measure ee. 

This general procedure was applied to a variety of scaffolds to produce a small library of compounds. After the work-up and preparative HPLC, the final yields were in the range of 40-63%, which is comparable to the previously reported batch procedures. A typical 35 min run finally gave 0.5 mmol of the product that afforded 40-80 mg after the HPLC purification this accounts to approximately 80-160 mg/h productivity. 

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]. 

The Suzuki coupling reaction is a powerful tool for carbon-carbon bond formation in combinatorial library production.23 Many different reaction conditions and catalyst systems have been reported for the cross-coupling of aryl triflates and aromatic halides with boronic acids in solution. After some experimentation, we found that the Suzuki cleavage of the resin-bound perfluoroalkylsulfonates proceeded smoothly by using [l,l -bis (diphenylphosphino)ferrocene]dichloropalladium(II), triethylamine, and boronic acids in dimethylformamide at 80° within 8 h afforded the desired biaryl compounds in good yields.24 The desired products are easily isolated by a simple two-phase extraction process and purified by preparative TLC to give the biaryl compounds in high purity, as determined by HPLC, GC-MS, and LC-MS analysis. 

Stewart et al. have also reported the efforts at Molecular Nature Ltd. to generate a pure natural product library [41], Compounds for this library were isolated utilizing parallel, normal phase column chromatography followed by C-18 and/or ion exchange chromatography. To be accepted into the library, the compounds must be > 90% pure with structural verification via a combination of HPLC, NMR, MS and GC/MS. 

Among others, Wieboldt et al. [57] used a similar technique, immunoaffi-nity ultrafiltration, in conjunction with ion spray HPLC/MS, on a small benzodiazepine library tested for binding to a polyclonal sheep IgG antibody. Dunayevskiy et al. [58] used gel filtration together with CE/MS and LC/MS on a small library of drugs tested for binding to human serum albumin, Chu and coworkers et al. [59-61] reported an application of affinity capillary electrophoresis (ACE)-MS to some tri- and tetrpeptide libraries (>1000 compounds)... 

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. 

Fig. 6.10. The resin (15 g) was partitioned into 60 aliquots of 250 mg in 60 glass vials, and each was treated with an aniline and an olefin as 0.5 M solutions in acetonitrile in the presence of 2% TFA at room temperature for 24 h. The resin in each vial was filtered, washed, and dried and the product cleaved from the support with 15% TFA-DCM. The library individuals were obtained as solids (>80% by HPLC) after evaporation and trituration of the crude residue with Et20, with the yields reported in Table 6.1. The reactions proceeded well for alkenes 6.3-6.7 (60-84% yields), the only detectable impurities being the corresponding Schiff bases (5-10%). Alkene 6.8 gave poor yields of the desired product or failed to react completely in some cases (see Table 6.1). All of the tetrahydroquinolines were obtained as single diastereomers, as shown for compounds 6.14 and 6.15 (Fig. 6.10).

The dimensions of the library prevented its full analytical characterization, but enough data were acquired to judge its overall quality. Twelve percent of the individuals, that is, 1056 randomly selected compiounds, were analyzed by electrospray mass spectrometry, and 83% of the analyzed samples showed the expected molecular ion as the principal MS peak. Eighty-five randomly selected compounds (corresponding to 1% of the total) were analyzed by HPLC-MS and the average purity was determined as 73% area/area. Finally, six discretes (6.35-6.40) were randomly picked and fully characterized. An authentic sample of each was prepared in solution, purified, and used as a standard for the quantitative determination of the six library components. The structures of the six compounds and the overall satisfactory results from their analytical characterization are reported in Fig. 6.16. 

The majority of reports have used electrospray ionization mass spectroscopy (ESI-MS) as an analytical detection method because of its sensitivity and the soft namre of its ionization procedure, which generally only leads to the detection of the molecular ions of the positive library members. Many separation techniques have been coupled to ESI-MS, including affinity chromatography (49), size exclusion chromatography (50, 51), gel filtration (52), affinity capillary electrophoresis (53-58), capillary isoelectric focusing (59), immunoaffinity ultrafiltration (60), and immunoaffinity extraction (61). ESI-MS has also been used alone (62) to screen a small carbohydrate library. Other examples reported alternative analytical techniques such as MALDI MS, either alone (63, 64) or in conjunction with size exclusion methods (65), or HPLC coupled with immunoaffinity deletion (66). 

After library cleavage, screening, and selection of positives (steps a-c. Fig. 7.28), the positive beads, such as 7.46, are decoded simply by strong aqueous acid hydrolysis, neutralization, and dansylation of the residual amines to give the dansyl derivatives 7.47 (steps d-g), which are all distinct in an HPLC spectrum. Several recent reports have provided optimized HPLC/fluorescence decoding protocols (198), which shortened the average decoding procedure from 1 h to around 6 min, and also alternative... 

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