Reaction Monitoring in Solid-Phase Synthesis

Destructive methods, where the analytical sample is consumed by the analysis, and nondestructive methods will be presented and their qualitative or quantitative nature will be discussed. They will be divided into off-bead methods, where the resin-bound reaction product(s) are cleaved from the support with subsequent analysis of the cleavage solution, and on-bead methods, where single or multiple beads are analyzed directly. 

The cleavage of resin-bound materials and their full analytical characterization in solution are used as the most accurate way to monitor the outcome of a reaction carried out in the SP. The methods used are those of classical organic chemistry and will not be commented on further. The reaction products can be weighed and an accurate structure determination can be obtained. There are, however, some limitations to the usefulness of off-bead methods for reaction monitoring in SPS. 

the resin beads cleaved after each step of a multistep SPS are lost and the gravimetric yield determination requires a significant amount of compound. This may lead to a notable waste of precious materials and to a significant reduction of the target compound prepared. 

The use of fast, reliable, sensitive on-bead methods circumvents the drawbacks to off-bead analysis outlined above. The modification of common analytical techniques has provided the SP chemist with valuable and often preferred alternatives to off-bead methods for SPS reaction monitoring. 

Colored reagents to follow the appearance or the disappearance of a functional group have been widely used to monitor reactions in classical organic chemistry, particularly in TLC analysis. This technique has been successfully adapted to SPS for example, ninhydrin (118), bromophenol blue (119), nitrophenyl isothiocyanate-O-trityl (120), picric acid (121), and malachite green isothiocyanate (122) have all been used to show the presence or the absence of free resin-bound amines. The presence of free resin-bound thiol groups can also be detected (123). 

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P. Seneci, Chapter 1.3 Reaction monitoring in Solid-phase synthesis, in Solid-phase synthesis and combinatorial technologies, Wiley, New York, 2000. 

Infrared and Raman spectroscopy are nondestructive, quick and convenient techniques for monitoring the course of solid-phase reactions, and have therefore been widely used for the characterization of polymer supports and supported species [156-160]. In fact, the application of infrared spectroscopy in solid-phase synthesis has received much attention and has been the subject of several recent reviews [127, 128, 161-164]. Reactions involving either the appearance or disappearance of an IR-active functional group can be easily monitored using any of the IR techniques described in this section. Some beads are typically removed from the reaction mixture, then they are quickly washed and dried prior to IR analysis. Traditionally, polymer supports are diluted and ground with KBr, then conventional FT-IR analysis of the KBr disk is carried out Although this is a commonly used... 

T. Wehler and J. Westman, Magic angle spinning NMR A valuable tool for monitoring the progress of reactions in solid phase synthesis, Tet. Lett., 1996, 37, 4771-4774. 

Linkers are molecules which keep the intermediates in solid-phase synthesis bound to the support. Linkers should enable the easy attachment of the starting material to the support, be stable under a broad variety of reaction conditions, and yet enable selective cleavage at the end of a synthesis without damage to the product. Several types of linker have been developed, which meet these conflicting requirements to different extents [1]. Newer developments include linkers containing fluorine to facilitate the monitoring of solid-phase chemistry by NMR (see Section 1.3.5), and enantio-merically pure linkers that enable the synthesis on solid phase of enantiomerically enriched products [2],... 

The monitoring of solid-phase synthesis in a real time quantitative manner has always been a goal since invention of the process. Many qualitative color tests exist, principal amongst these being the ninhydrin-based Kaiser test.f 1 The Kaiser test is less sensitive when the N-terminal is a secondary amine and for this reason many other tests have been applied.t °l Although a quantitative Kaiser test has been proposed,t l it has never achieved prominence as a routinely applicable method and the results are not usually available in time to influence progress of the reaction. 

It would be of great utility if a better methodology could be provided to monitor polymer-bound reactions than gel phase heteronuclear NMR. However, as previously mentioned, the utility of gel phase proton NMR in solid phase synthesis monitoring is of little value, as the spectra obtained are generally quite broad. 

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. 

Although solid-phase synthesis revolutionized synthetic organic chemistry and triggered the development of combinatorial chemistry, it still exhibits several shortcomings originating from the nature of heterogeneous conditions, such as lower reaction rates and difficulties in reaction monitoring. 

Numerous resin supports are commercially available for solid-phase synthesis and some allow the acquisition of quite reasonable quality spectra of compounds bonded to them - and some don t. The resins to avoid (if you intend trying to monitor your reactions by MAS-NMR) are any that are based purely on cross-linked polystyrene. These are too rigid and afford little or no mobility to any bound compound. These resins are relatively cheap and have high specific loadings but will give very poor spectra even in a MAS probe. We see little point in running spectra of compounds on these resins as the quality of the spectra make them virtually useless - and perhaps worse - potentially misleading. 

FIGURE 5.4 Schematic representation of a continous-flow system for the solid-phase synthesis of peptides. Solvent is forced through the system by a pump. The support is in the form of a column that is stationary. A reaction is monitored by measuring the change in absorbance of the solvent stream. 

Solid-phase synthesis has unique advantages in accommodating purification at each individual reaction step without losing compound mass. However, to ensure reaction completion is a challenging task. Our capability to monitor reactions on... 

Monitoring reaction progress throughout a multistep synthesis is a relatively difficult task.22 Typical methods used for solution-phase synthesis, including thin-layer chromatography (TLC), GC, and most types of mass spectrometry (MS), are less informative for solid-phase methods. However, Fourier transform infrared (FTIR) spectroscopy and nuclear magnetic resonance (NMR) are particularly useful in solid-phase strategies. 

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