Reaction monitoring in SPS

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. 

We will now describe the main differences between SPS and organic synthesis in solution, focusing on (i) the types of solid supports available for SPS, (ii) the linkers used to anchor compounds to those supports, (iii) monitoring reactions in SPS, and (iv) estimation of the purity and yield of SP reactions. 

While specific applications of gel-phase NMR have been useful for SPS reaction monitoring, the great potential of SPS NMR is in the determination of structure and the measurement of purity and yields, especially through the use of magic angle spinning-high-resolution (MAS-HR) NMR techniques. This important topic will be addressed in Section 1.4.6. 

The suitability of this technique for acid-labile (142) and photocleavable linkers (143) has been demonstrated. Other cleavage conditions that do not produce residues (e.g., cleavage with gaseous ammonia) could also be used in theory. TOF-secondary ion mass spectrometry (TOF-SIMS) (144) has also been validated to monitor SP peptide synthesis (145) and could in future increase the versatility of MS monitoring of SP reactions. 

The use of infrared (IR) spectroscopy as a reaction monitoring technique for SPS has become more frequent over the last few years with the introduction of technologies specifically aimed toward SP reactions. Even so, a number of reports describing the use of classical IR by thorough mixing of a few milligrams of grounded resin beads in KBr pellets have appeared (150, 151). 

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... 

The determination of the yield of a reaction carried out in the SP and the structure and purity of the product is an essential component of the process of SPS. The same analytical methods that we examined in the previous section for reaction monitoring can be used, but their usefulness for qualitative analysis may vary, as we will describe in this section. 

The reaction conditions used in solution synthesis are generally adapted to the SPS the reaction times are usually increased, the reagents in solution are added in excess, and their concentration is increased to drive the SPS steps to completion. Typical experimental conditions are three- to fivefold excess of solution reagents and 0.2-1 M concentrations, while the optimal reaction time is evaluated via on-bead (FTIR) or off-bead reaction monitoring. If the reagents in solution are precious, a smdy to determine the minimum excess required to drive the reaction to completion will be necessary. 

The synthesis of a library of discretes in SP makes extensive use of anal54 ical tools to check the validity of the synthetic route and to monitor the reaction course for each library individual. Every reaction vessel contains a single entity with the possible presence of side products that can be fuUy characterized at any step in the synthesis. We will briefly review the most useful analytical techniques that can be employed for the SP discrete library synthesis. For a detailed description of each technique the reader is referred to Sections 1.3 and 1.4 and to the references cited therein. 

Potential representative monomers are then selected from commercially available compounds or from internal collections, possibly through the application of computational methods to select only the most significant examples, and the chemistry is rehearsed using these. The process is identical both in SP and in solution, but the support makes monitoring of the reactions, the precise determination of yields and purities of the reaction products, and the structure and the quantity of impurities more difficult and time consuming in the SP (Fig. 8.8). If the proper analytical equipment and expertise for working in SP are not available, the selection or rejection of a monomer candidate may be more difficult, less accurate, or even wrong. 

Solid supports for SP synthesis are an important option for the synthesis of libraries of both discretes and pools. We have extensively reviewed their properties, but we have also highlighted some critical issues such as the different reaction kinetics in heterogeneous reactions due to accessibility of inner reaction sites, the difficulty of monitoring reactions on SP, and the need for suitable expertise and instrumentation. 

The sp chains are metastable and have the tendency to undergo cross-linking reactions to form sp phase [7]. In order to characterize the sp metastable decay we have monitored the sp chain stability through the evolution of the C peak intensity, either keeping the sample under UHV for several days (at a pressure of about 2 x 10 Torr) or exposing it to different atmospheres (H2, He, N2 and dry air) [40]. In the case of UHV conditions, we have observed a slow decrease of the intensity of both the two components of the C peak and small changes in the shape of the G and D bands. The temporal evolution of is well described by an exponential decay plus a constant ... 

The Fmoc group has several properties that make it nearly ideal for use in SPS. It displays exceptional acid stability has a high ultraviolet absorption (A ax 267 nm), which permits monitoring of acylation and deprotection reactions and is fully compatible with ferf-butyl-based side-chain protection... 

Irreversible Case. A heterogeneous electron transfer reaction can be represented as in Eq. (1). Assuming that all of the sample is initially present as O, the absorption signals of R may be monitored in the SPS/ CA experiment. If the magnitude of the potential applied to the system is sufficient to cause the forward reaction to proceed at a rate governed only by kf, the rate-dependent absorbance of R then is given by ... 

The kinetics of the formation of the porphyrin aggregate and its structure are sensitive of experimental conditions (Giovannetti et al., 2010). The monomer - aggregated sp>ecies is a system of multiple equilibria. Spectrophotometric monitoring in the time of the Uv- Vis absorbance permit to obtain information of intermediate species, of typ>e of the aggregate, and of their transformation. For this, for evaluated the polymerization kinetic constants, the concentrations of monomeric [M] and dimeric form [D], can be calculated from the relative absorption maxima at each time. If kpoi is the polymerisation kinetic constant. Cm and Co denoted the initial monomer and dimer concentrations, the reaction rate can be expressed as in equation (2) ... 

The progress of the reaction is conveniently monitored by gas chromatography on a 1/8" x 6 column packed with 6X SP-2100 on Supelcoport, 80-100 mesh, operated at 50°C for 4 min, then heated at 15°C/min to 250°C. The relative retention times are 4.0 min for trimethylvinyltin, 6.5 min for trimethyltin chloride, 13.1 min for 4-tert-butylcyclohexen-l-yl trifluoromethanesulfonate, and 14.7 min for l-(4-tert-butylcyclohexen-l-y1)-2-propen-l-one. Because of the extreme Volatility of trimethylvinyltin, it may be necessary to add additional small amounts of this reagent in order to drive the reaction to completion. 

This may be done using a simple boiling point column. We have employed either 10% UCW-98 on Chromosorb W or SP-2100 on 80/100 Supelcoport G2642. The checkers did not monitor the reaction except to extract a small sample after 30 hr in order to verify the absence of starting aldehyde by N NMR spectroscopy. 

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). 

The use of mass spectrometry (MS) techniques to monitor SP reactions has recently become possible through the use of matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) spectrometry (137) after in situ cleavage of a small number of resin beads (138-140). Although the compound is cleaved from the resin, the cleavage happens directly onto the center of the MALDI target and the method can be considered on-bead. 

Infrared spectroscopy may be considered to be one of the analytical techniques best suited to the rapid monitoring of the progress of chemical reactions in the SP, as has been discussed in Section 1.3.6. There are some intrinsic limitations to the use of this technique in the determination of purity and yields. The difficulty in quantifying reaction yields by following the simple appearance or disappearance of IR bands is worsened by the broader bands often obtained on SP, especially when using KBr pellets. Small quantities of side products cannot be detected easily due to the reduced intensity (or absence) of IR-specific bands. While some attempts have provided quite accurate estimations of the yield in a few cases (40, 154-156), other analytical techniques appear to be more suited for the quantitative determination of yield and purity of SP reactions. 

The MALDI-TOF monitored SP preparation of lysobactin, a natural cyclopeptide antibiotic, on PS resin bearing the Rink amine linker 1.6 has been reported (141). The SPS scheme is shown in Fig. 1.21. The classical peptide coupling steps, the allyl deprotection (see Section 1.3.5), and the macrocyclization step were all monitored by MALDI-TOF, and the purity of the products was also determined using this technique. The presence of small impurities in compounds 1.56-1.61 was easily detected, and the reaction conditions for the key deprotection of 1.59 and cyclization of 1.60 were rapidly optimized. A total yield of 15% was obtained after HPLC purification of released 1.62 (lysobactin). 

The use of gel-phase NMR for monitoring SPS reactions has been described previously. The application of this method to the determination of the purity and yield of a product is not recommended for the reasons already discussed in Section 1.3.4. Another SPS NMR technique, MAS-HR NMR, is more suited to this purpose. 

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