Single bead analysis

For on-bead analysis vibrational spectroscopy (IR-spectroscopy) can be employed attenuated total reflection is a method allowing fast and nondestructive on-bead analysis of small samples (single bead analysis) without significant sample preparation. Solid phase NMR is the method of choice if complex structural analysis is intended on the support. Spatially resolved analysis on the resin is possible with microscopic techniques. 

Mixture analysis methods are of little value for interesting sized libraries. Single-bead analysis methods are being developed which can be used to assure the quality of larger solid-phase split pool libraries. 

A broad variety of particle sizes are available, from microspheres with less than 5-pm particle size up to resins of 700-pm particle size. The large beads can be used as polymeric microreactors.P l As the separation between resin and liquid is still performed by filtration, different glass filters have to be used the porosity and nominal pore size of these filters are listed in Table 5. In those applications where single beads are used (split/mix, single-bead analysis, bead-based assays) it is essential to know the capacity of the single beads. For practical purposes a correlation between bead size and capacity per bead is reported in Table 6. 

It has been shown by Egner et al. (64,65) that it is possible to analyze compounds directly from prepared polystyrene beads. This approach has been useful to verify and improve the synthetic method for the generation of combinatorial libraries. Assuming that there are approximately 106 beads in 1 gram of polystyrene resin, and the substitution factor for the solid phase chemistry is 0.4 mmol/g or more, there is about 400 pmol of compound attached to each single bead. MALDI-TOF analysis has detection limits in the femtomole region, which makes it compatible with single-bead analysis (76). 

While IR transmission spectroscopy is a general analytical method for resin samples, internal reflection spectroscopy is especially suited for solid polymer substrates known as pins or crowns. Single-bead analysis is best done by IR microspectroscopy, whereas photoacoustic spectroscopy allows totally nondestructive analysis of resin samples. 

A special applicability of MALDI is the single bead analysis of peptides (— 1000 Da) with a TOF-analyzer [25] also using different resin linkers [26]. Improved resolution of mass spectra was achieved with the development of reflectron devices and post source decay for a direct structural readout of single compounds [27]. For direct ionization and desorption in the MALDI source, photosensitive linkers were developed [28,29]. 

First an introduction to the physical parameters of the FT-ICR method is presented, which has been used seldomly for routine analysis.This shows the possibilities but also the limits of the mass detection method. Then the immense potentials of 1CR-MS for analysis in combinatorial chemistry will be demonstrated by examples of measurements of synthetic compound mixtures and simple compound collections. The application of the coupling of micro-HPLC and ES-FT-ICR mass spectrometer underlines the universal applicability of this powerful detection method for single bead analysis. 

In summary the advantages of single bead analysis are combined with those of library synthesis without creating new disadvantages. 

Yan, B. Single bead analysis in combinatorial chemistry. Curr. Opin. Chem. Biol. 2002, 6, 328-332. 

An important tool for the fast characterization of intermediates and products in solution-phase synthesis are vibrational spectroscopic techniques such as Fourier transform infrared (FTIR) or Raman spectroscopy. These concepts have also been successfully applied to solid-phase organic chemistry. A single bead often suffices to acquire vibrational spectra that allow for qualitative and quantitative analysis of reaction products,3 reaction kinetics,4 or for decoding combinatorial libraries.5... 

Structural Analysis of Compounds Linked to Single Beads by MAS-NMR... 

While parallel synthesis of arrays of glycopeptides is readily achieved by implementation of the building-block approach (Scheme 14.1, Strategy 2),101 glycopeptide library synthesis in a combinatorial manner via the split-mix method has yet to prove routine. The difficulty lies in the structural analysis of the vast number of compounds generated in picomolar quantities on a single bead. Whereas peptides on... 

FT-IR microspectroscopy is a new nondestructive, fast and rehable technique for solid-phase reaction monitoring. It is the most powerful of the currently available IR methods as it usually requires only a single bead for analysis, thus it is referred to as single bead FT-IR [166]. (See also Chapter 12 for further details). The high sensitivity of the FT-IR microscope is achieved thanks to the use of an expensive liquid nitrogen-cooled mercury cadmium telluride (MCT) detector. Despite the high cost of the instrument, this technique should become more widely used in the future as it represents the most convenient real-time reaction monitoring tool in SPOS [166, 167]. 

ATR FT-IR spectroscopy allows for analysis of the polymer surface, rather than the bulk of the sample. Whereas the spectra obtained from the FT-IR microscope and BCA are recorded in transmittance mode and are used to analyze the entire bead, an ATR objective can be brought into direct contact with the sample in order to yield information about the chemistry taking place mainly on the periphery of the bead. Some FT-IR microscopes are equipped with an ATR crystal, and ATR analysis can be achieved on a single bead [172], but there have been no reports of automated ATR instruments. This technique has been used in kinetic studies, to prove that the esterification of Wang resin (4) (Scheme 1.4) to give the corre-... 

The bromination reaction (Scheme 12.3) was also carried out on resins (1) of three different sizes (Fig. 12.5). Single bead FTIR study and the kinetics analysis were carried out as in the esterification reaction studies. Rate constants are hsted in Tab. 12.2. The relationship between the rate constants and the bead size is shown in Fig. 12.7b. 

To check the completion of the reaction in Scheme 12.6, the single bead FTIR measurement (Fig. 12.10) alone was not conclusive because there was no IR band from the starting resin (11) to monitor. Resin elemental analysis (Cl) of (11) could conclusively show if the reaction was complete. The accuracy and the reproducibility of the resin elemental analysis methods have been evaluated before [14]. 

Resins (19) ( 30 mg each) reacted with 5% TFA in DCM. Droplet of suspension was taken at various time intervals for single bead FTIR (Fig. 12.15) and kinetics analysis (Fig. 12.16). The data was also fitted to a first order reaction rate equation and rate constants were determined to be 4.8x10 (5% TFA). Cleavage of carbamides (18), (20), (21), ureas (22-25), amides (26-29), and sulfonamides (30-33) were studied in the same way. 

The rate of a chemical reaction depends on temperature. A rule of thumb for many organic reactions in solution is that a 10 °C change in temperature causes a two- to three-fold change in rate of reaction [25]. To study the temperature dependence of solid-phase reactions, the cleavage reaction of resin (35) with n-butyla-mine at 20, 40 and 60 °C were carried out. The cleavage time courses and pseudo-first-order rate fits at these three temperatures are shown in Fig. 12.20. The rate constants from single bead FTIR analysis are Hsted in Tab. 12.4. Compared with the reaction at 20 °C, the solid-phase cleavage reaction of resin 3b was two times faster at 40 °C and four times faster at 60 °C. 

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