Solid-Phase Synthesis and Combinatorial Technologies
.pdf6.1 SYNTHESIS OF SOLID-PHASE DISCRETE LIBRARIES |
213 |
thesis of primary libraries, as realized by SP pool techniques and made of hundreds of thousands or even millions of individuals (see Chapter 7), is currently not possible using parallel synthesis. Even some recently designed multiblock synthesizers (28, 29) cannot prepare more than several hundred discretes in a given synthetic run. Innovative approaches that are beginning to address this crucial issue will be reported in Section 6.4.
These instruments are engineered to a high degree of complexity and allow the reliable control of the multiple operations and reaction conditions encountered in the automated synthesis protocol. This topic has been extensively covered in some recent reviews (30–40), and we will limit our discussion of automated SP discrete synthesizers to the implications of their use for the synthesis of small organic molecules on SP.
The most demanding requirement of a SP synthesizer is the necessity to perform a large variety of organic reactions. This means being able to deal with a wide temperature range (usually from –40 °C to +150°C) and the use of inert atmosphere for moisture-sensitive reaction conditions (the reaction blocks can be kept under an argon blanket or sealed with various materials) and also the ability to accommodate all types of reagents including acids, bases, and corrosive substances. Typically, glass and Teflon are used as inert construction materials for the reaction blocks, frits, tubing, and valves.
Complex hardware components are needed to constitute automated SP synthesizers, but their complexity should be reduced as much as possible, without slowing the synthetic protocols, to minimize the maintenance and the failures of the system. For example, a single valve or robotic pipette that fails to deliver or to withdraw a reagent compromises the whole automated synthesis, and so the minimum acceptable number of such devices should be used and overautomation should be avoided. The number of automated operations such as delivering and withdrawing solutions, washing, stirring, heating, and cooling should also be reduced to the minimum; additional manual steps such as the addition of solid reagents and the filtration of suspensions are only introduced in an automated SP library synthesis protocol if they are necessary to perform a high-quality synthesis.
Another important feature of an automated system is the software that controls the synthesizer. It must be very user friendly since engineers and programmers may be needed for maintenance, but their presence should not be required to run an automated SP protocol! Even though the program may have a complex architecture, the interface with the chemist should be straightforward. Usually the system is designed to automatically record any anomaly that may occur during the automated protocol such that an electronic logbook allows immediate troubleshooting. These instruments are quite expensive, and their use in nonspecialized chemical laboratories could be problematic. On the contrary, the use of several complementary SP synthesizers in a dedicated combinatorial laboratory allows the fast and reliable production of libraries. The production of libraries is often coupled with automated SP assessment studies that speed up the preparation of focused libraries of discretes.
214 SYNTHETIC ORGANIC LIBRARIES: SOLID-PHASE DISCRETE LIBRARIES
6.1.3 Analytical Methods for SP Discrete Library Synthesis
The synthesis of a library of discretes in SP makes extensive use of analytical 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 fully 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.
The exact nature of the monomer rehearsal of the library strongly depends on the size of the library. For example, if the planned library is small, the preparation of a few target compounds may suffice, while the number of compounds prepared during rehearsal may be larger when the discrete library is made by thousands of components. In both cases these compounds are prepared with the same procedure that will be used for the library synthesis, and thus the same steps will be present in both the monomer rehearsal and the final library synthesis. This step is crucial to rule out monomers that perform poorly and to determine the optimized reaction conditions.
A strong analytical support is required in this phase, and, ideally, all of the off-bead and on-bead techniques described in Chapter 1 should be used to determine the quality of the synthesis. Each reaction during the rehearsal must be carefully monitored to determine the ideal conditions for each representative monomer and to eventually define the most general conditions. Simple colorimetric techniques are used when possible, but on-bead monitoring methods based on IR spectroscopy (using either normal instruments by grinding a few milligrams of bead into a KBr pellet or single-bead techniques) or MAS-NMR spectroscopy (when this expensive and sophisticated instrumentation is available) are ideal techniques that not only allow the reaction to be followed but also allow the monitoring of side products and selection of the best conditions to minimize side reactions. The quantity of resin employed for the rehearsal of the synthesis may be relatively large so that off-bead analytical methods can also be used. The cleavage and the analytical characterization of each intermediate step should be performed, and the results compared with on-bead analysis. The use of several on-bead and off-bead techniques allows the selection of suitable monomers and reagents for the library as well as the determination of the most useful synthetic conditions for library production and of the most suitable cleavage conditions. In general, the more analytical information gathered in this phase, the higher the chance of success for the planned library.
A recent report (41) has highlighted the usefulness of CE to determine the enantiomeric purity of individuals coming from parallel synthesis, either during chemical assessment to help revising the experimental protocols and as a library QC tool to determine the stereochemical quality and purity of chiral libraries. The authors studied the Pictet-Spengler reaction of chiral tryptophans with carbonyl compounds on SP to provide tetrahydro-β-carbolines, and clearly highlighted the reaction conditionsdependent racemization of the reaction products.
The same techniques can also be used during the library synthesis, and, depending on the size of the library, either all or only some of the intermediates may be analyzed
6.2 STRUCTURE DETERMINATION, QUALITY CONTROL, AND PURIFICATION |
215 |
at each step and any anomalous behavior recorded and double-checked during the quality control of the final library. The significant increase in the number of samples to be processed makes HPLC/MS, which is easily automated and fast, the technique of choice for off-bead characterization of the components of the final library after cleavage.
6.2 STRUCTURE DETERMINATION, QUALITY CONTROL, AND PURIFICATION OF SOLID-PHASE DISCRETE LIBRARIES
6.2.1 Structure Determination
The determination of the structure of a library member is not a problem for SP libraries of discretes as the structure of any given library component is positionally encoded, that is, by simply tracking the locations in which the different monomers are added, the structures of the expected final products are known. When dealing with automated syntheses and with large libraries, the software controlling the synthesizer determines the location of each monomer and delivers all the solutions of reagents accordingly. This information is provided to the chemist through spreadsheets or tables that represent the whole reaction block. When medium–large libraries are prepared manually, the tracking of monomer addition is made easier by segregating this monomer in specific areas of the plate or plates. A hypothetical example shown in Fig. 6.2 presents the synthesis of a 960-member SP discrete library in ten 96-well plates where the use of each monomer in the first set (10 compounds, A1–A10) is coupled to a different plate; then each monomer of the second set (8 compounds, B1–B8) is coupled with the same row of each plate; and finally the representatives of the third set (12 compounds, C1–C12) are coupled with the same column of each plate (Fig. 6.2).
6.2.2 Quality Control
After the final cleavage from the beads, the solutions containing the discrete library individuals are submitted to a work-up procedure and then the pure individuals are tested against one or a few selected targets. The results of the assays will, hopefully, create useful information that will allow further research to be focused on active structures. However, these results must be coupled with quality control, that is, the complete analytical characterization of the library. This allows the purity of each positive compound to be determined to check if the observed activity is due to the presence of impurities (false positives) and to locate the wells where the expected library individuals are absent (false negatives). Moreover, the analysis of the whole library will determine if a final purification of the compounds is required.
In theory, it would be necessary to provide a profile of analyses for each member of the library to have a reliable and exhaustive quality control. However, this is feasible only for small libraries (a few tens of components), and when the numbers of components increase to hundreds or thousands, this process would be too time consuming. The best compromise is provided by a fully automated technique that can
218 SYNTHETIC ORGANIC LIBRARIES: SOLID-PHASE DISCRETE LIBRARIES
commercially available instruments, and no further comment will be made here; however, it is worth mentioning that some autosamplers for automated HPLCs use the same 96-well format as used for the synthesis of discrete libraries, thus reducing the number of operations and transfers necessary for the analytical characterization of the library. Several recent reports have highlighted the use of capillary LC, in conjunction with MS, to accelerate the analysis throughput using the so-called rapid back-flush microseparation protocol (51); the use of supercritical fluid chromatography/mass spectrometry (SFC/MS) to successfully analyze and characterize a discrete library of thiohydantoins in high-throughput mode (52, 53); and the use of electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FT-ICR-MS), either alone or coupled with RP-HPLC, to analyze several hundreds of combinatorial discretes per day (54).
Flow injection analysis mass spectrometry (FIA-MS) has been reported to be a fast method for the characterization of combinatorial libraries (55, 56). The method verifies the presence of the molecular ions of the expected product and side products or impurities but does not provide information on the quality of the analyzed samples. Significant improvements related to the increased analytical throughput, obtained by reducing the time between each injection without increasing the intersample carry-over from each analysis, were recently reported (57, 58). When coupled with RP-HPLC, FIA-MS allows the separation and the determination of the molecular weight of the components of each sample. This is normally enough to unequivocally attribute the structure of the expected library component and of any side products from a library synthesis.
The methods of detection most commonly used are based on the UV and MS properties of the compounds. The acquisition of a UV chromatogram using a diode array detector with a wide wavelength window and of the total ion current (TIC) usually allows detection of all the components of a sample and allows an approximate but reliable quantitation. As an example, the UV and MS spectra of two compounds from an SP discrete library are reported in Fig. 6.4 (59); the different properties of the desired product and of an impurity are highlighted by their different UV and ionization behavior. Some classes of compounds, though, have poor or no chromophoric groups present in their structures (and hence no UV absorption) or ionize only poorly, thus giving no MS signal. For this reason alternative detection methods that can be coupled with HPLC/MS/UV are also used. Evaporative light-scattering detection (ELSD; 60–63) is based on the light-scattering properties of nonvolatile compounds. In this technique, the recorded signal is roughly proportional to the molecular weight of the molecule and does not significantly vary in intensity between members of the same chemical class; however, it has the major drawbacks of being relatively insensitive and being readily influenced by sudden changes in the gradient of the mobile phase. Chemiluminescent nitrogen detection (CLND; 64–66) is based on the total combustion of nitrogen and thus is able to detect organic compounds containing at least one nitrogen atom producing a reliable chromatogram with a high sensitivity and without any influence from other structural factors. ELSDand CLND-based detectors are commercially available and can be easily coupled to automated RP-HPLC systems.
6.2 STRUCTURE DETERMINATION, QUALITY CONTROL, AND PURIFICATION |
219 |
Figure 6.4 UV (top) and MS (bottom) detection for an analytical sample.
As these detection methods are complementary rather than mutually exclusive, multiple detection is recommended for laboratories that routinely perform the synthesis of discrete libraries and can be realized by splitting the eluate of the HPLC/UV system in two or three aliquots that are sent to the mass spectrometer and the CLND and/or the ELSD instrument. Examples of multiple UV/MS/CLND (67) and UV/MS/ELSD (68) show how the different methods of detection are useful in
220 SYNTHETIC ORGANIC LIBRARIES: SOLID-PHASE DISCRETE LIBRARIES
determining and quantifying the composition of an injected sample. Two examples of different sensitivities for the multiple system UV/MS/ELSD are reported in Figs. 6.5 and 6.6 (68). The former consists of a well from a discrete carbohydrate library containing two major components, the more hydrophilic of which (Rt = 9.85) does not contain a chromophore and is not detected by UV (Fig. 6.5b) but is clearly visible using ELSD or MS detection (respectively Figs. 6.5a and c). The latter consists of a well from a benzoic acid–inspired library containing two major components, the more lipophilic of which (Rt = 2.20) requires a detection method with high sensitivity and thus is not spotted by ELSD (Fig. 6.6a) but is clearly visible using UV or MS detection (respectively Figs. 6.6b and c).
The application of the powerful and information-rich combination of RP-HPLC and NMR, which has previously been used above all for the determination of drug metabolites (69, 70), to the problem of library analysis has been reviewed recently (71). NMR as a detection method is able to provide more detailed structural information than any other technique. Integrated HPLC-NMR systems can be created using commercially available flow NMR probes (72), and small libraries have been successfully analyzed with this method (73, 74). Quality control may be carried out either on-line, recording the NMR spectra during the elution, or by stopping the HPLC flow when a chromatographic peak elutes. Both methods are relatively insensitive and require significantly more substance and time than HPLC-MS, but the structural information provided can be unique, especially when isomeric compounds or compounds with the same molecular weight are present in the library. A recent example (74) reported the separation and structure determination of four isomers by analysis of their 1H-NMR spectra (two of the peaks are partially overlapping, but determination of the structures was still possible), while a discrete 96-member library of methylene malonic acids has been fully characterized by HPLC-NMR (75). This method will become increasingly important for the quality control of libraries, and a multicomponent detection system that includes an NMR spectrometer is the method of choice for discrete library quality control in dedicated combinatorial laboratories.
The use of “ultrafast” HPLC gradients and of automated integrated instruments allows the processing of up to several hundred samples per day, which is satisfactory for most of the SP discrete libraries produced. If a large library of discretes made up of several thousand members is being considered, a significant percentage (typically 10–25% of the samples) is processed and the quality control obtained on this sample is assumed to reflect the quality of the library as a whole. If one or more positives from the screening of the library are among the nonprocessed samples, their characterization will follow immediately. Another useful procedure is to first run a fast FIA-MS analysis and discard the library wells/samples where the expected components are absent and only afterward analyze the confirmed library samples by HPLC with UV detection.
The processing of several hundreds of samples per day using a multidetection system means the acquisition of more than a thousand chromatograms, the evaluation of which by the chemist would require substantial time and effort. Software that is able to discriminate between “good” and “bad” library components without human intervention is desirable in order to speed up the process of quality control. Some software of this type has been developed and is commercially available (76, 77) or, alternatively,
