Library analysis

Since they are complementary, the two filters should be combined during chemical library analysis. 

Oprea, T.I. Rapid estimation ofhydro-phobicity for virtual combinatorial library analysis. SAR and QSAR Environ. Res. 2001, 12, 129-141. 

Selection-independent analysis In this case, library analysis occurs strictly after and apart from the library selection experiment. Typically, what this means is that the solution resulting from a library is analyzed by HPLC or HPLC-mass spectrometry (HPLC-MS), and compared with the chromatographic trace obtained for an identical library prepared in the absence of target. This provides an internal control for self-selection processes and (hopefully) allows direct identification library members undergoing enhancement through visual inspection. If selfselection is the goal, one simply compares HPLC traces of libraries at different time points. 

We have optimized an eight-way MUX coupled to a TOFMS analyzer to carry out eight-channel parallel LC/UV/MS analysis of combinatorial libraries14 in the past 2 years. This system has not only provided the capacity needed for library analysis, but also enabled simultaneous evaluation of experimental parameters to expedite the method development process. In this chapter, we discuss the optimization of this system and present a high-throughput protocol for combinatorial library analysis. We also compare the eight-channel parallel LC/UV/MS system to a conventional single channel LC/UV/MS system in terms of performance and operation. 

Without backpressure regulation for each channel, it is necessary to minimize the flow rate fluctuation over time. The relative standard deviation (RSD%) in retention time variation among the eight channels over 1 month for compounds A and B was less than 2% and for C and D it was less than 1%. The RSD% for all channels over a 1-month period for compounds A to D was 3.2, 2.4,1.6, and 1.5%, respectively. Therefore, this system is well suited for combinatorial library analysis. The UV chromatograms from channel 5 from different days are shown as an example in Fig. 2A. The retention times of the four compounds (compounds A to D) from all eight channels during a 1-month period are shown in Fig. 2B. 

To develop a method for combinatorial library analysis, we first analyzed six to eight representative compounds from each library under generic LC/UV/MS conditions. These conditions would be used for library analysis unless adjustments had to be made based on the study of these representative compounds. 

Figure 6.20 Computer screen capture of the Lib View library analysis program that illustrates the microtiter plate layout with the measured and predicted spectra shown in the foreground. (Reprinted with permission from Yates et al., 2001. Copyright 2001 American Chemical Society.)...

Zeng, L. Wang, X. Wang, T. Kassel, D. B. 1998a. New developments in automated prepLC/MS extends the robustness and utility of the method for compound library analysis and purification. Comb. Chem. High Throughput Screen, 1,101-111. 

There are two approaches to analytical evaluation of OBOC libraries analysis of compounds from individuals bead [51,60] and analysis of mixtures of compounds detached from multiple beads [61, 62], It should be mentioned that analysis of mixtures from OBOC libraries is quite different from that described in section Analysis of Libraries Synthesized in the Format of Small Mixtures of 4-12 Compounds per Mixture. In the mixture of compounds cleaved from a few beads, which are randomly picked for analysis from the OBOC library, the structures of components of the mixture are unknown. Consequently, it is not possible to apply the strategy developed for the analysis of mixtures synthesized in spatially addressed format, which is based on information about the structures of components of each mixture. In the case of OBOC libraries, analysis of only very large mixtures is meaningful when the theoretical distribution of molecular weights within the whole library is compared with that experimentally observed [61, 62], The focus of this section is on characterization of OBOC libraries through analysis of individual beads. 

Analytical characterization of OBOC library will be discussed for an example of a small-molecule combinatorial library with a complexity of 54,150 (57 x 25 x 38) compounds (Figure 10.9) [63]. In the result of an OBOC library analysis, identity, purity, and quantity of library components should be evaluated, but it is unknown which one out of thousands of compounds in a library is synthesized on the particular bead picked for analysis (Figure 10.10). General schemes of analytical characterization of combinatorial libraries (analysis of a small number of standard compounds, analysis of the model library, and analysis of the production-scale library) can be applied to analysis of OBOC libraries, but the issue of structure elucidation for OBOC libraries should be addressed through the scheme. 

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