Libraries of compound mixtures

This issue highlights the characterization difference between parallel synthesis and combinatorial synthesis. Parallel synthesis is automated traditional organic chemistry. Each compound is made in a separate reactor, purified and characterized. There is no excuse for not fully characterizing compounds made by parallel synthesis. Jonathan Ellman s laboratory at UC Berkeley has been a pioneering academic center for solid-phase chemistry development. His philosophy is to synthesize libraries of discrete compounds in a spatially separate fashion, rather than libraries of compound mixtures, to allow for rigorous analytical characterization [48,49],... 

Depending on the synthetic strategy used the created combinatorial library can comprise compound mixtures, or separate, single substances. (The term combinatorial synthesis was strictly used only when different building blocks are treated in one reaction vessel, and compound mixtures are thus obtained. Since the boundary becomes blurred, herein syntheses of both libraries of compound mixtures and of separate, single substan-... 

Figure 1.4. Split-pool synthesis to prepare combinatorial libraries of compound mixtures. (Spheres represent resin beads, A, B, C represent the sets of building blocks, borders represent the reaction vessels.) In the case of three building blocks used, in each coupling step after three stages a total number of 27 different compounds, one on each resin bead, arc formed using nine individual reactions (ignoring deprotection).

Currently, the split-pool synthesis is the most popular method for the synthesis of large combinatorial libraries of compound mixtures comprising thousands to hundred thousands of compounds. A variety of methods have been developed to determine the structure of bioactive compounds within libraries of compound mixtures (see Section 1.4.2.1). 

In the so-called reagent mixture synthetic approach towards combinatorial libraries of compound mixtures, each reaction step of the combinatorial synthesis is carried out with... 

Combinatorial synthesis towards libraries of compound mixtures can be done either on a solid support or in solution. In both cases an effective decoding strategy is required to extract structural information from the results of the biological assay (see Section 1.4.2.1.2 Deconvolution by Orthogonal Libraries). 

If combinatorial libraries consist of compound mixtures (e.g., after cleavage from the resin beads), a reliable analytical characterization is difficult. In this case the utilization of mass spectrometry for analysis is based on the prediction of mass distribution of the library. Computer-generated distribution profiles can be compared with the actual profile obtained from the compound library [93,94]. Evaluation of mass distribution detects synthetic problems based on incomplete coupling (shift toward lower molecular masses), incomplete deprotection, or unwanted library modification, such as oxidation, acylation, or alkylation (shift toward higher molecular masses). However, for a larger library of compound mixtures many different compounds will have the same molecular mass. This greatly complicates structural determination and even makes it impossible at a certain mixture complexity. 

The discovery of oxazoline hydroxamates as potential inhibitors of LpxC was the result of high-throughput screening of large libraries of compounds at the Merck Research Laboratories in collaboration with the Department of Biochemistry, Duke University Medical Center [95]. The lead compound, L-573,655, was a racemic mixture of 4-carbohydroxamido-2-phenyl-2-oxazoline, which had been previously made by Stammer et al. [96] as a precursor in the chemical synthesis of cyclosporine. Namely, (R,S)-serine methyl ester hydrochloride (149) is converted into (R,S)-4-carbomethoxy-2-phenyl-2-oxazoline (150) via treatment with ethyl benzimidate using the Elliot procedure [97]. Treatment of this ester with one equivalent each of hydroxylamine and sodium methoxide in methanol at room temperature affords the desired (R,S)-4-carbohydroxamido-2-phenyl-2-oxazoline (151), as depicted in Scheme 30. 

The use of combinatorial chemistry to produce libraries of compounds is pivotal in drug discovery. Screen hits need to be analyzed to identify the structure of the individual active component. Large numbers of samples containing only small quantities of complex mixtures require... 

Some reactions afford mixtures of products. Mixtures include diastereomers, such as endo and exo products (10.1 and 10.2) of a Diels-Alder cycloaddition, and regioisomers, such as ortho and para products (10.3 and 10.4) from an electrophilic aromatic substitution (Scheme 10.1). Even a reaction that forms products as subtly similar as enantiomers is technically a mixture of products. Isomeric mixtures violate the spirit of one compound, one well in combinatorial chemistry. Isomeric mixtures, however, are often unavoidable and therefore tolerated in compound libraries. Mixtures are also tolerated in libraries of compounds that have been derived from natural sources. Examples include extracts from finely ground vegetation and microbial broths. 

Combinatorial chemistry is the production of libraries of compounds that represent permutations of a set of chemical variables. These variables include the nature of the substituent in a particular molecule, both in type and size, changes in the components in a mixture of materials, e.g. in ceramics and changes in process parameters, e.g. temperature, pH etc. Chemical libraries are usually created by one of two methods split and mix or parallel synthesis . Split and mix synthesis is used to produce small quantities of a relatively large number of compounds and requires assays to be performed on pools of compounds. Parallel synthesis is used to produce libraries... 

Figure 13.8. GPC followed by LC-MS-MS for screening mixtures of combinatorial libraries. After incubation of a receptor with a library of compounds, the ligand-receptor complexes (L-R) are separated from the low molecular weight unbound library compounds using GPC. Next, the L-R complexes are denatured during reversed phase HPLC to release the ligands for MS-MS identification.

Another example involves the reaction of Cu1 with the 2,2 2",6" 6",2" -quaterpyridine ligand (3) (Figure 3).15 A stoichiometric mixture of these molecules at ambient temperature and moderate concentration was found by ES-MS to contain a library of compounds which included the diastereomeric binuclear helicates, (4), and the oligonuclear circular helicates (5), (6), and (7). Concentration of the mixture resulted in an increase in the higher nuclearity species. At concentrations below 10 4 M, however, only the helicates were present. Crystallization transformed the mixture into (6) in the solid state. 

Validation of the compounds in HTOS libraries has become an area that is receiving more attention. Because of the movement away from pools of compounds (mixtures) to discrete compounds for SAR development, quality control and quality assurance of the samples has become an issue. The issue of quality vs. quantity in HTOS has been discussed in a recent paper by MacDonald et al. (71). Bauer has described an information management system that incorporates several quality checks on the data generated by HTOS systems (72). 

For library analysis of compound mixtures, reliable results are obtained by coupling mass spectrometry with HPLC or capillary electrophoresis (CE). In this arrangement, mixtures of several hundred compounds can be analyzed. A comparison of MS, CE, and NMR for library characterization has been made in [93]. It was concluded that existing analytical techniques can provide sufficient information about prepared libraries. 

Thus, synthetic combinatorial receptor libraries of high diversity are a prerequisite for further progress in the field of chemosensors. As yet, there are no specific receptors available for most of the analytes, but this can be overcome by the use of sensor arrays. A mathematical analysis by pattern recognition can lead to a successful analysis of compound mixtures which are exposed to an array of sensors with different selectivities ( bio-electronic nose ) [13,14]. 

An additional dimension to library analysis is introduced when mass spectrometry is coupled with common separation techniques, for example liquid chromatography (LC) and capillary electrophoresis (CE). While these couplings are compatible with spray ionization techniques such as ESI or APCI, they more or less exclude the use of the MALDI technique. Several contributions deal with LC-ES-MS [36, 37] and CE-ES-MS-coupling [38], For molecules with isobaric nominal masses, MS/MS-experiments are performed to confirm the identity of a library component. Separation and analysis of compound mixtures may also be performed by GC-MS [39, 40], As a supplement to the more common-... 

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