Solid-Phase Synthesis and Combinatorial Technologies

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210.Kricka, L. J., Clin. Chem. 44, 2008–2014 (1998).

211.Campbell, D. P., Moore, J. L., Cobb, J. M., Hartman, N. F., Schneider, B. H. and Venugopal, M. G., Proc. SPIE-Int. Soc. Opt. Eng. 3540, 153–161 (1999).

212.Warren, M. E., Anex, D. S., Rakestraw, D. and Goudey, P. L., SANDIA Report UC-706, SAND98-0509. Sandia National Laboratories, Albuquerque, NM, and Livermore, CA, 1998.

213.Wilding, P. and Kricka, L. J., Trends Biotechnol. 17, 465–468 (1999).

214.Jacobson, S. C., McKnight, T. E. and Ramsey, J. M.,Anal. Chem. 71, 4455–4459 (1999).

215.Hosokawa, K., Fujii, T. and Endo, I., Anal. Chem. 71, 4781–4785 (1999).

216.Siebert, P., Petzold, G. and Muller, J., Proc. SPIE-Int. Soc. Opt. Eng. 3680, 562–571 (1999).

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218.MacBeath, G., Koehler, A. N. and Schreiber, S. L., J. Am. Chem. Soc. 121, 7967–7968 (1999).

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220.Fletcher, P. and Haswell, S., Chem. Brit., 38–41 (1999).

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5 mg-batches

single vessel/many beads:

1compound

a:resin portioning (1 to 100); b: coupling with amines; c: reduction; d: coupling with acids.

Figure 7.2 SP parallel synthesis: one compound per well.

extremely large discrete libraries (hundreds of thousands or even millions of components) cannot be made in a timely manner as of today.

The second approach, named many compounds per bead (Fig. 7.3), starts by coupling the solid support in a single reaction vessel with an equimolar mixture of the 100 amines (step a); then the mixture is reduced (step b) and the resin-bound amines are reacted with an equimolar mixture of the 100 acylating agents (step c). The 10,000-member library is obtained as a single 50-g pool of resin, and each bead contains similar quantities of each library individual. A bead has typically 1014–1015 reaction sites, so that each bead will contain an average of 1010–1011 copies of each library individual. The library synthesis could technically be considered successful if all the monomers react properly and the 10,000 compounds are actually present, but the identification of positives from this library for any specific application is not feasible. In fact, the cleavage of resin-bound materials produces an equimolar mixture of all the components, whose activity, if any, is the activity of a 10,000-member unresolved mixture. As a consequence of this major limitation, this SPS approach is not used for library synthesis.

The third approach can be considered as a compromise between parallel synthesis and mixtures on SP. It is named one compound per bead (13) and allows the synthesis

7.1 SYNTHESIS OF SOLID-PHASE POOL LIBRARIES 267

L

CHO

50 g-batch

a,b

50 g-batch

100 resin-bound amines

50 g-batch

10,000 resin-bound amides

single bead: 10,000 compounds

Figure 7.3 SP pool library synthesis: many compounds per bead.

of high-quality SP pool libraries from which structure determination of positives can be timely and effectively obtained. This method, usually called mix and split or divide and recombine, was reported at the very beginning of combinatorial technologies (2, 3) and has become extremely popular for the synthesis of oligomeric libraries. Even if the current trend for small organic molecule libraries is shifting toward discrete libraries, the synthesis of SP SOM pool libraries is still common and important, especially for encoded libraries (see Section 7.4), and has an ample margin left for quality improvements.

The synthesis of our hypothetical SP pool library of 10,000 amides by mix and split is reported in Fig. 7.4. Preparation of the 100 resin-bound amines follows an identical course to the one seen for parallel synthesis (steps a, b, and c), but then the 100 resin aliquots are mixed to give a single 50 g portion (step d). This portion is then split into 100 aliquots each containing all the 100 amines (step a), but each bead is loaded with

268 SYNTHETIC ORGANIC LIBRARIES: SOLID-PHASE POOL LIBRARIES

L

d

50 g-batch

100 resin-bound amines

1 compound per bead

1 compound per bead

a:resin portioning (1 to 100); b: coupling with amines;

c:reduction; d: mix in one pool; e: f: coupling with acids.

Figure 7.4 SP pool library synthesis: one compound per bead.

a single compound because the coupling of the amines to the solid support was performed in separate vessels. Each new aliquot is placed in a different vessel and the 100 acylating agents are added separately to each reactor (step e) to produce 100 pools of 100 compounds. Each pool contains only one acid monomer and the whole amine set, but each bead is loaded with a single individual.

Another hypothetical example of mix-and-split synthesis, involving a four-step process with five monomers for each of the four monomer sets, leads to a 5 × 5 × 5 ×

270 SYNTHETIC ORGANIC LIBRARIES: SOLID-PHASE POOL LIBRARIES

each aliquot but rather aliquots similar volumes of resin slurries (see next section), the library produced in such a case would contain several individuals that are represented more than once and others would be missing. Redundancy obviates this inconvenience: If 50 g of resin, that is, around 50,000,000 beads, is used for a 10,000-member library, a redundancy factor of 5000 ensures that the population of each library individual will vary roughly from double (10,000 beads) to half (2500 beads) of its theoretical amount at most but will probably be close to the theoretical 5000-bead representation. A redundancy of 5000 is generally too much, and typically lower redundancies (from 50 to 100) are suggested as the best compromise between fully represented libraries and cost savings. Redundancy can be safely decreased to 3–5 for a bead-based screening (vide infra) to ensure the presence of at least one bead loaded with each library component. If, as usual, the library will be screened on many assays, a redundancy of 5 × N, where N is the number of planned screenings, is needed.

7.1.2 Technical Aspects of SP Pool Libraries: Manual Versus Automated

Synthesis

The general aspects of SPS, which have been examined in Chapters 1–3, are common for all the SPS procedures leading to pool libraries. The aspects of automated SPS, which were covered during the description of SP discrete libraries in Section 6.1.2 (e.g., architecture of SP synthesizers, inertness of the system, complexity of hardware and software, and automated versus semiautomated devices), are also important for SP pool library synthesis. The most distinctive aspect of SP pool synthesis is represented by the mixing and splitting of resin aliquots, which can be performed by handling small aliquots of beads, or even single beads, either manually or by automated devices. We will briefly describe the principles and the techniques of this crucial operation.

Resin aliquots can be handled as solids after elimination of solvents and soluble reagents by filtration and repeated washings and drying of the resin. In this case, if a mixing step of ten 100-mg portions is involved, the aliquots are combined and mixed as dry solids; then ten new portions are created by weighing ten 100-mg aliquots of dry resin and partitioning them in different reaction vessels. It is more usual, though, to handle the resin as a slurry, which is easily mixed by pouring all the separate slurries in a common vessel and collecting all the residual beads from each vessel by repeated washings, possibly with heavy solvents such as DCM, in which the residual beads float. The splitting step is then performed by sampling ten equivalent volumes of the slurry, either stirring the slurry to have an equal density of beads in the liquid volume or, better, using an isopycnic solvent mixture with the same density as the beads. This allows the sampling of ten equivalent aliquots with a pipette and their partitioning into the reaction vessels.

This wet procedure is preferable because of the easier and faster splitting of slurries by pipetting volumes rather than weighing dry aliquots of resin. Moreover, its partial or even total automation is significantly easier. The use of robotic pipetting arms, which withdraw slurried aliquots and distribute them in different reaction vessels, is widespread. They may have one or more pipetting arms to speed the process, which

272 SYNTHETIC ORGANIC LIBRARIES: SOLID-PHASE POOL LIBRARIES

portion was split in 20 aliquots with a pipette (step h). The third monomer set was then coupled at 90 °C (20 amines and hydrazines, step i, Fig. 7.6) in 40 glass vials. No automation was used at any level, and the high-quality SP library L1, which was obtained as 40 pools of 300 compounds each, was used as a source of positives in many biological assays. When comparing this pool library to an equivalent 12,000-member SP discrete library, the ease of synthesis of the former should be underlined, especially when limited resources and budgets are available.

Another important technical problem for SP pool libraries is related to the handling of single beads. Often with encoded pool libraries or bead-based screening (vide infra), it is necessary at some stage to handle and deliver a certain number of single beads to single vessels/wells. This may well be done manually using a capillary tube and a microscope to check the pick of a single bead, but the process becomes tedious and time consuming when tens or hundreds of single beads must be processed. Automated instruments, called bead pickers, have been reported (21): they use microcapillaries mounted on a manifold (arrays of eight or more) that is switchable from vacuum (aspiration of a bead from a pool) to pressure (release of the single bead to a plate well) to pick and deliver up to 96 single beads in 5 min. The capillary ends are built so that the bead is retained by the vacuum but cannot enter because its diameter is bigger than the capillary end. The capillaries are rinsed in fresh solvents after delivering a bead and can then be used for another picking cycle. The percentage of errors, due either to the loss of bead during movement of the capillary to its plate destination or to multiple bead picking caused by bead lump formation, is reasonably low (<10% of total picking operations). These instruments can be built in a high-throughput version starting from commercially available 96-channel dispensing stations (22) and assembling around them a software/hardware system able to monitor the bead-picking operation, detecting the successful operations (one bead on one pipette tip), and discarding the unsuccessful ones (multiple beads or no beads on a tip).

7.1.3 Analytical Methods for the Synthesis, Quality Control, and Purification of Solid-Phase Pool Libraries

The synthesis of an SP pool library is more analytically challenging than the same library prepared as a discrete collection. The presence of many compounds in the same pool disturbs the qualitative and quantitative determination of purity and yields in all the synthetic steps, during the final quality control, and when necessary, during the off-bead purification of the pool. Several reviews (23–25) have dealt with the analytical chemistry aspects related to pool SP libraries; here we will review the analytical steps required for the synthesis, characterization, and purification of an SP pool library, while the structure determination of active components from a pool will be dealt with in the next three sections.

Monomer rehearsal is identical for SP discrete and pool libraries, but the generally larger dimensions of the latter require the use of more monomers and thus their qualification for the planned reaction scheme. The full analytical characterization of this step will be crucial. Discarding poorly performing monomers and identifying reliable monomers and optimized reaction conditions will guarantee the quality of the