Solid-Phase Synthesis and Combinatorial Technologies

Figure 4.4 Structure and deconvolution of the linear SP hexapeptide amide library L1.

appropriate for the preparation of large libraries of completely randomized peptides, antibodies, vaccines, or small proteins.

Many reports in which a structure (or some structural motif) has been identified from large synthetic pool libraries of peptides have appeared in the literature in the last 10–15 years, and some excellent reviews covering this field have appeared recently (24–26). These structures can be used to refine the binding mode hypothesis for an unknown receptor through biostructural studies (e.g., X-ray co-crystallization studies and NMR studies), or they can be used as assay tools to set up a HTS campaign for the specific receptor. Unfortunately, peptide ligands are generally not suitable as drug candidates because of their poor stability and pharmacokinetic profiles, among other reasons. Their use as starting points for a chemical modification program to give druglike analogues devoid of these drawbacks has not given generally good results as of today. Moreover, even a huge peptide library does not sample diversity in an appropriate way because the α-amino acidic–based oligomeric backbone makes each library component essentially similar to the others. Significantly smaller small organic molecule libraries can be much more diverse and are potentially more promising sources of valuable positives in a primary screen.

A cyclic pentapeptide SP library (L3, Fig. 4.5, top) was prepared by Spatola and Crozet (27) by randomizing 12 D- and L-α-amino acids in four positions and keeping D-Asp as a common fifth residue. The total number of individuals was 12 × 12 × 12 × 12 = 20,736, and they were prepared using the positional scanning technique (28), which produced four library copies in 48 pools containing 12 × 12 × 12 = 1728 individuals (Fig. 4.5, top). The structure of an endothelin antagonist obtained from this

D1-D4= determined amino acid; X1-X4=as above

Figure 4.5 Structure of a cyclic SP pentapeptide libraryL3 and of a trimeric SP peptide-related library L4.

library was identical to a known optimized structure, thus confirming the potential of pool screening to find active compounds.

A trimeric SP peptide-related library L4 was reported by Terrett et al. (29), where 32 × 31 × 31 = 30,752 compounds were produced using various amino acids as building blocks with final N-capping to produce the homopiperidine ureas (Fig. 4.5, bottom). The library was prepared as 31 library subsets (X1 fixed) of 992 individuals, and deconvolution produced a few peptidomimetic endothelin antagonists with nanomolar activity. Both these libraries show that the introduction of D-α-amino acids, exotic amino acidic building blocks, ureido bonds, and/or cyclization do not reduce the synthetic opportunities for library preparation (the availability of exotic building blocks may be the only limitation) while the diversity of the library components is enhanced and the poor profile of natural peptides as druglike compounds can be improved.

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Oligonucleotide libraries are commonly used either to encode biosynthetic display peptide libraries (Section 11.1) or to screen for novel nucleotide ligands (aptamer) or catalytic activities (ribozymes). They are produced using high-quality automated ON SP protocols seen in Section 2.2, but their amplification and selection are performed via biological protocols (see Sections 4.2.4 and 11.2).

The SP phosphoramidate library L5 (Fig. 4.6) reported by Fathi et al. (30) is an example of a oligonucleotide–peptide hybrid library consisting of 20 × 20 × 2 × 11 = 8800 products, including stereoisomers at each phosphoramidate P atom, prepared as 11 pools of 800 compounds. This ON-modified library was used as a generic source of lead compounds to be used on many biological targets. In this case the chemical assessment was more difficult because of differences between oligonucleotide and peptide synthesis and because of the stabilities of the products. Monomer rehearsal required the elimination of some building blocks that were unsuitable for incorporation into the final synthetic scheme. However, the final library was more diverse than a peptide, peptidelike, or oligonucleotide library. Such approaches have become popular and allow a more efficient coverage of diversity through the use of various oligomerrelated libraries.

Oligomeric libraries as sources of biological tools (ligands, catalytic antibodies, and metal coordination sequences, among others) were, and still are, very popular even if the development of SP and solution-phase techniques to generate SOM libraries has shifted the attention of combinatorial chemists toward these latter targets. A steady flow of reports concerning oligomeric libraries from various sources (Section 4.2.4) is nevertheless to be expected in the future, and major areas of interest will probably be the synthesis of new building blocks and the optimization of reaction conditions to produce new homogeneous or hybrid oligomeric structures.

prepared as 11 pools of 800 individuals (20x20=400 monomers, two phosphoramidate isomers)

Figure 4.6 Structure of an SP phosphoramidate library L5.

146 COMBINATORIAL TECHNOLOGIES: BASIC PRINCIPLES

We have already examined the inherent differences and the higher degree of complexity embedded into oligosaccharides when compared to peptides and oligonucleotides (Section 2.3). Oligosaccharides are peculiar in the sense that their SP exploitation has still to happen, and major efforts leading to significant results are to be expected in the near future. Nevertheless, the extreme biological relevance of carbohydrate structures, or of sugar-decorated synthetic or natural products, has produced several significant combinatorial oligosaccharide libraries and will stimulate further efforts from leading groups.

prepared as an encoded library and on-bead screened (colorimetric assay)

Figure 4.7 Structure of an SP oligosaccharide encoded library L6.

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A list of recent papers (31, 32) and reviews (33–38) dealing with combinatorial libraries of oligosaccharides should give a flavor of the present and the future to the interested reader.

The first oligosaccharide SP-encoded (39) library L6 was reported by Liang et al. (40) and consisted of 1296 carbohydrates that were tested on-bead for their lectin binding activity using hydrophilic PS resin as a support. The structure of the library and selected building blocks are shown in Fig. 4.7. A high-quality diverse library of

Figure 4.8 Structure of a moenomycin-inspired SP discrete library L7.

148 COMBINATORIAL TECHNOLOGIES: BASIC PRINCIPLES

diand trisaccharides was assembled through the reaction of glycosyl acceptors, including the handle function, with glycosyl sulfoxide donors and acylating agents. It is clear that the degree of complexity of the chemistry involved is much greater when compared to either ON or peptide libraries and that the protected glycosidic building blocks must be built through complex syntheses. Another important point is that the reaction conditions must take into account the relative sensitivity of all the intermediates and of the final, resin-bound library components.

Sofia et al. (41) reported a SP library of disaccharides L7 made by 1300 individuals and inspired by the disaccharide core of moenomycin A, a bacterial cell wall inhibitor (Fig. 4.8). Both the structure of the library and its main features are shown in Fig. 4.8. The four major disaccharide scaffolds (X, Y, and W variations) were either built on resin via glycosylation or attached onto the photolabile linker, and subsequently decorated by introduction of R1, R2, and R3; radiofrequency encoding (42) was used to obtain a large number of discretes with an affordable number of reactions. The

Figure 4.9 Structure of an SP glycopeptide encoded library L8.

Figure 4.11 Structure of a nifedipine-inspired SP 1,4-dihydropyridine library L10.

scaffolds are often preferred because their decoration with meaningful monomers allows the creation of rigid structures that are able to explore the pharmacophoric space of various molecular targets. Many examples have been reported and among them scaffolds derived from natural products deserve a special mention (see next section).

A primary modular library was reported by Powers et al. (47), and its structure is shown in Fig. 4.13. The key chalcone intermediates were prepared as a discrete library L12 made up of 32 × 40 = 1280 individuals using acetophenones (R1) and aromatic aldehydes (R2) as monomer sets. This library was used to generate nine further libraries by addition of different reagents or monomer sets onto the α,β-unsaturated system (libraries L13–L15) or small monomer sets (L16–L21). The assembly of subsets L12–L21, composed of 1280 + 1280 + 1280 + 1280 + 7680 + 7680 + 12,800 + 7680 + 7680 + 25,600 = 74,240 discretes, can be considered as a modular library where the diversity of the chemical space is spanned by 10 different decorated scaffolds produced from the same intermediate library. This approach, which generates daughter libraries from a parent library, has an enormous potential to generate diverse primary libraries that can be focused on a specific scaffold during a second iteration of library synthesis after finding active structures from an HTS campaign.

Figure 4.12 Structure of a solution-phase triazine library L11.

4.2.3 Synthetic Organic Libraries: Natural Products

A specific class of libraries in which biologically active natural products are either built during the synthesis or used as scaffolds to be decorated has recently emerged and has gained considerable attention for pharmaceutical purposes. The biological information contained in the natural scaffold increases the chances of discovering novel active structures. Some examples depicted in Figs. 4.14–4.18 demonstrate the potential of natural product libraries.