Single bead reaction monitoring

The methods for monitoring reactions on-bead can also be performed with the help of IR microscopy [32-35]. A few resin beads are distributed on a KBr window, and flattened. The IR beam is focused on the resin bead for transmission measurement. 

The first use of this method for the monitoring of solid-phase reactions was described by Yan et al. [20]. Polystyrene hydroxymethyl resin as starting material was reacted with adipinic acid chloride and dipropargyle amine (Fig. 16.7). 

IR-microscopy allows the monitoring of solid-phase reactions during the time course analogous to FT-ATR-IR methods described above. Therefore, resin beads are removed from the reaction mixture at certain time intervals, washed and analyzed. The relation of 

The quality of the IR spectra depends on the size of the resin beads.The best results for sample measurement in transmission are received by smaller beads with a maximum diameter of 50 Bm. IR-measurements of bigger beads produces overloaded IR spectra.This can be explained by the longer IR beam passing through the bead and interfering with C02 and humidity. When those resin beads are flattened in order to reduce the path of the IR beam through the bead, the quality of the spectra is improved. 

Qualitative and quantitative analyzes of resin-bound molecules with any type of IR-spectroscopy is often limited by varying absorption coefficients and an overlapping of different vibrations.The use of deuterated reactants increases the selectivity of IR analysis, due to the fact that the region of C-D stretching vibration absorptions are in the range between 2300 and 2200 cm -. This region is free of other IR absorptions. An example of quantitative IR analysis is the benzoylation of amino methyl polystyrene resin [23], Fig- 

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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]. 

Scheme 1.2 Monitoring of a muiti-step reaction by Single-bead FT-iR spectoscopy.

A frequent complication in the use of an insoluble polymeric support lies in the on-bead characterization of intermediates. Although techniques such as MAS NMR, gel-phase NMR, and single bead IR have had a tremendous effect on the rapid characterization of solid-phase intermediates [27-30], the inherent heterogeneity of solid-phase systems precludes the use of many traditional analytical methods. Liquid-phase synthesis does not suffer from this drawback and permits product characterization on soluble polymer supports by routine analytical methods including UV/visible, IR, and NMR spectroscopies as well as high resolution mass spectrometry. Even traditional synthetic methods such as TLC may be used to monitor reactions without requiring preliminary cleavage from the polymer support [10, 18, 19]. Moreover, aliquots taken for characterization may be returned to the reaction flask upon recovery from these nondestructive... 

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]. 

Two acylation reactions as depicted in Schemes 12.7 and 12.8 had the same issue when monitored by single bead FTIR (Fig.s 12.11 and 12.12). The starting resins (13) and (15) did not have convenient signal to monitor by FTIR. A chloranil test [15] specific for the secondary amines was used to confirm the complete consumption of (13), therefore the reaction completion. An iron-chloride-pyridine test [16] was used to confirm the complete consumption of (15). In both color tests, a blue color would suggest the presence of the starting material. In both cases we observed the disappearance of the blue color that indicated the reaction completion. 

Yan, B. Kumaravel, G. Progression of Organic Reactions on Resin Supports Monitored by Single Bead FTIR Microspectroscopy, Tetrahedron 1996, 52, 843. 

Because the characterization of support-bound intermediates is difficult (see below), solid-phase reactions are most conveniently monitored by cleaving the intermediates from the support and analyzing them in solution. Depending on the loading, 5-20 mg of support will usually deliver sufficient material for analysis by HPLC, LC-MS, and NMR, and enable assessment of the outcome of a reaction. Analytical tools that are particularly well suited for the rapid analysis of small samples resulting from solid-phase synthesis include MALDI-TOF MS [3-5], ion-spray MS [6-8], and tandem MS [9]. MALDI-TOF MS can even be used to analyze the product cleaved from a single bead [5], and is therefore well suited to the identification of products synthesized by the mix-and-split method (Section 1.2). The analysis and quantification of small amounts of product can be further facilitated by using supports with two linkers, which enable either release of the desired product or release of the product covalently bound to a dye [10-13], to an isotopic label [11], or to a sensitizer for mass spectrometry [6,14,15] (e.g., product-linker-dye- analytical linker -Pol). 

The sensitivity requirements of HR-MAS NMR are determined to a certain extent by the chemistry used to produce a combinatorial library. In the most extreme case, each compound in the library is present only on a single bead of the solid-phase support and therefore it is necessary to obtain spectra from a single bead. Despite the practical difficulties associated with both the handling and data acquisition of single-bead spectra, it has been shown to be a suitable technique, both for analysis33 and reaction monitoring.34... 

In summary, the use of IR either as a simple (KBr pellets) or sophisticated (single-bead techniques) reaction monitoring system for SPS has become very important. The technology behind the methods outlined above is constantly evolving, and... 

Treatment of resin-bound a-arylazobenzhydryl 488 with a Lewis acid at room temperature followed by acidic cleavage furnished indazole 489 in quantitative yield, the reaction being monitored by single bead IR microspectroscopy (Equation 98) <1996TL8325>. Unsymmetrical azines 490 thermally cyclized to fused pyrazoles 491 (Equation 99) <2002TL6431>. Indazoles 493 were obtained from thermal cyclizations of (2-alkynylphenyl)triazenes 492 in the presence of methyl iodide as a solvent other solvents were tested where either no reaction or complex mixture of products was obtained (Equation 100) <2002JOC6395>. 

B Yan, JB Fell, G Kumaravel. Progression of organic reactions on resin supports monitored by single bead FTIR microspectroscopy. J Org Chem 61 7467-7472, 1996. 

Because of the simple sample preparation, FT-IR spectroscopy [12,13] has become an increasingly popular method in SPOS for reaction monitoring. For IR spectra in the transmission mode [14,15] or with ATR-[16],FT-Raman-[17,18] and DRIFTS-[19] methods, only 1-2 mg of resin beads are required for routine measurements. With FT-IR microscopy [20-24], it is even possible to obtain spectra from single resin beads or spatially resolved spectra [25]. 

KBr pellet or single bead samples are suitable for the purpose of solid-phase reaction monitoring and the study of reaction kinetics. For the KBr-embedded samples, a higher amount of resin material and longer sample preparation time is required, but these are compensated by the low costs for the measurement with KBr-pellets. The compromise here is the use of an ATR-instrumentation. With this technique the throughput of samples is much higher at low costs for additional equipment. ATR-IR spectra quality is at the same level than for the common methods mentioned before. 

The use of single bead IR spectroscopy is a valuable method for real-time monitoring of reaction kinetics or investigations of small amounts of resin beads. 

De Miguel, Y., Shearer, A.R. and Alison, S. (2001) Infrared spectroscopy in solid-phase synthesis. Biotechnol. Bioeng. 71 119-129. Yan, B., Fell, J. B. and Kumaravel, G (1996) Progression of organic reactions on resin supports monitored by single bead FTIR microscopy. J. Org. Chem. 61 7467-7472. 

THE APPLICATION OF SINGLE-BEAD FTIR AND COLOR TEST FOR REACTION MONITORING AND BUILDING BLOCK VALIDATION IN COMBINATORIAL LIBRARY SYNTHESIS... 

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