On-bead synthesis

Methods of Conducting Flow Chemistry 4.2.3.1 On-Bead Synthesis 

The dehydration of diethylcarbinol in 1932 was the first example of synthetic chemistry attempted in flow mode using a heterogeneous reagent [67] although no experimental details were given, it was reported that the use of acidic silica gel at high temperature promoted the dehydration of the compound to various alkenes. 

However, it was not until Merrifield developed a polystyrene matrix for peptide synthesis that a broad range of functionalized solid supports became available [91], leading to solid phase organic synthesis (SPOS) and eventually to polymer-assisted solution synthesis (PASS). Both of these techniques are heavily utilized by industry, especially for rapid library synthesis. 

Despite the breakthrough associated with Merrifield s approach, there are several limitations such as the discontinuous nature of the reaction, the need for large excesses of reagent and the mechanical instability of the polymer matrix. An early solution to the restrictions imposed by Merrifield s polystyrene supported batch process was the use of commercially available benzyl alcohol-functionalized silica (used for H PLC columns). This was initially derivatized with the first member of the peptide chain to be propagated. The synthesis of a tetrapeptide in flow was completed in half the time required for the equivalent batch mode assembly and required significantly smaller excesses of the solution-phase reagent [92], 

A flow synthesis of peptides using a macroporous polymer resin soon followed additionally incorporating an in-line UV detector, which constantly monitored the progress of the reaction by analyzing the output of the column the reaction was recirculated until it reached completion. However, backpressures of500-1000 psi were encountered, making these systems only suitable for metal-encased columns [93], 

---

Although these examples illustrate opportunities for using a variety of immobilized reagents for individual transformations, a more imaginative use of these methods is in multistep syntheses. In this respect our group s approach has been to minimize the use of conventional workup procedures, yet be able to prepare complex natural products and related compounds simply and efficiently. In this chapter we refrain from commenting on the use of on-bead synthesis of natural compounds or on the diversity-oriented procedures. 

On-Bead Solid-Phase Synthesis of Chiral Dipeptides... 

To ensure complete conversion for all examples of a 21-member library, irradiation times of 30-60 min were used (Scheme 7.39), employing a multi-vessel rotor system for parallel microwave-assisted synthesis (see Fig. 3.7). The results were confirmed by on-bead FTIR analysis, accurate weight-gain measurements of washed and dried resins, and post-cleavage analysis of the prepared enones. 

With the following examples, we will investigate and discuss the following (a) Scale-up phenomena of different solid phase reactions and the corresponding on-bead analytics (b) the effect of loading (an equivalent to concentration in solution-phase chemistry) (c) comparison with the solution-phase alternative and (d) the synthesis and use of new trityl linkers. 

There is one more report on the synthesis of a library of phosphorus ligands on solid phase. Waldmann et al. prepared a library of phosphoramidites on beads (Fig. 36.5), but these were only applied in enantioselective C-C-bond formation. In fact, as two ligands need to be bound to the catalyst, the use of an immobilized monodentate ligands should most likely be avoided unless the proximity between the ligands is sufficiently close. In addition, crosslinking by the metal may have a negative impact on the permeability of the polymer for the substrate. 

Tools for On-Bead Monitoring and Analysis in Solid-Phase Oligosaccharide Synthesis... 

In order to meet these demands, a number of on-bead analytical techniques2 were adopted. The methods briefly outlined below have an immense impact on the development of new methods for solid-phase oligosaccharide synthesis by allowing direct monitoring of the reactions as they unfold. 

The requirements for solid-phase synthesis are diverse. The support must be insoluble, in the form of beads of sufficient size to allow quick removal of solvent by filtration, and stable to agitation and inert to all the chemistry and solvents employed. For continuous-flow systems, the beads also must be noncompressible. Reactions with functional groups on beads imply reaction on the inside of the beads as well as on the surface. Thus, it is imperative that there be easy diffusion of reagents inside the swollen beads and that the reaction sites be accessible. Accessibility is facilitated by a polymer matrix that is not dense and not highly functionalized. A matrix of defined constitution allows for better control of the chemistry. Easier reaction is favored by a spacer that separates the matrix from the reaction sites. Coupling requires an environment of intermediate polarity such as that provided by dichloromethane or dimethylformamide benzene is unsuitable as solvent. 

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... 

Meldal and coworkers developed polyacrylamides cross-linked with poly(ethylene glycol), referred to as PEGA, as supports for solid-phase synthesis and on-bead enzymatic assays [181-183]. Functionalization of the polymer was performed in a similar fashion as in the case of other polyacrylamides, i.e. either by copolymerization with N-acryloylsarcosine ethyl ester followed by aminolysis with ethylenediamine, or by copolymerization with an amino group containing monomer. The monomers used for a high-capacity (0.4-0.8 mmol/g [182]) and a low-capacity (0.2-0.4 mmol/g [181]) PEGA support are sketched in Figure 2.7. 

The modified mix-and-splif combinatorial method is used for the synthesis of the ligand library yields n<->n members, where n represents the number of different amines chosen. Usually 5 g of each immobilized ligand are synthesized however, this amount depends on the screening strategy preferred (for example, the FITC-based system or the ELISA on beads require less resin than the conventional affinity chromatography). 

The expense of screening depends very much on the number of samples tested. Consequently, the density format of titer-plates has increased in recent years from 96-well up to the 9600-well format. The next big step towards miniaturization would be the complete avoidance of any container, which then results in the smallest well possible and a well-less, so-called lawn-format assay develops. This is exactly what is proposed by a number of authors (see review [47]). Screening in a lawn format does not mean avoiding any structure or arrangements. Samples are still prepared on beads, which are produced by split-mix synthesis, but the beads are arrayed directly on the well-less assay. A typical matrix applied for such biological screening is the agarose lawn. Active beads are then picked from the assay matrix and decoded for compound identification. 

In parallel synthesis the compounds are prepared in separate reaction vessels but at the same time, that is, in parallel. The array of individual reaction vessels often takes the form of either a grid of wells in a plastic plate or a grid of plastic rods called pins attached to a plastic base plate (Figure 6.6) that fits into a corresponding set of wells. In the former case the synthesis is carried out on beads placed in the wells whilst in the latter case it takes place on so called... 

---