Solid-Phase Synthesis and Combinatorial Technologies

4.2 COMBINATORIAL LIBRARIES 155

Quinic acid was decorated on the C1-carboxylic acid and on the C3, C4, C5-hydroxyls by Phoon and Abell (53) as in Fig. 4.16, producing two small arrays L24 (ten monoester-monoamide compounds) and L25 (six triester-monoamide compounds). The reliability and the robustness of both the scaffold and of the acylation/esterifications ensure an expansion of quinate-based libraries, which has already been reported for shikimate-based libraries (54).

The natural scaffold, or a scaffold resembling a natural product, can also be built during the synthesis of the library. An example presented by Nielsen and Lyngsoe (55) involved the synthesis of a library L26 inspired by the structure of balanol (Fig. 4.17). A disconnection study revealed α-amino alcohols Al, aromatic dicarboxylic acids Ar, and aromatic carboxylic acids R1 as monomer sets to mimic the natural constituents of balanol, and the small library L26 was prepared. It was made up of 2 × 4 × 4 = 32 compounds prepared as four pools of eight compounds that could be expanded by using enlarged monomer sets to produce primary balanol-biased combinatorial libraries. Introduction of modified building blocks (aliphatic diacids or monoacids, acyclic or aromatic amino alcohols, etc.) could lead, after substantial assessment of the chemistry, to a modular library of different balanol-inspired compounds with a high level of diversity.

A more complex combinatorial strategy based on a natural scaffold was reported by Nicolaou et al. (56) with the synthesis of an epothilone-based SP library L27 (Fig. 4.18). This biased library was prepared as a 3 × 3 × 5 = 45-member collection, but the final reaction vessels could, theoretically contain 4 different isomeric desoxyepothilones (Fig. 4.18), thus leading to a total number of 45 × 4 = 180 components. The library was prepared using a radiofrequency encoding technique (42), and the final

R1: 3 primary amines (n-propyl, benzyl and cyclohexyl)

R2: 5 carboxylic acids (acetic, benzoic, 4-pyridyl, phenylacetic and acrylic)

R3: 2 carboxylic acids (acetic and benzoic)

Figure 4.16 Structure of two quinicacid-inspired SP small arrays L24 and L25.

156 COMBINATORIAL TECHNOLOGIES: BASIC PRINCIPLES

prepared as four pools of 8 compounds

Figure 4.17 Structure of an SP balanol-inspired library L26.

cleavage solutions from each vessel were purified to give pure single isomers that were assayed to obtain a detailed SAR (56). The monomer sets consisted of three protected hydroxy aldehydes (R1), three protected hydroxy keto acids (R2), and five heterocyclic secondary alcohols (R3) prepared using published multistep routes, and the expansion of these sets would require significant synthetic effort. Once such an ambitious synthetic scheme has been set up, it would be desirable to move away from the initially used biased monomer sets and to introduce more diverse commercially available monomers to build larger, less epothilone-like libraries with the same synthetic strategy that could be used as primary libraries to search for other biological activities. This example shows that even complex natural scaffolds can be dissected and reconstructed using appropriate reactions and monomer sets. The balance between the appeal of the designed library and the resources/effort needed to set up the library synthesis should drive the strategic decision to go for the synthesis or not.

4.2.4 Biological Libraries

Libraries produced by microorganisms or through biochemical techniques have been frequently used to find either peptidic or oligonucleotidic sequences that bind certain receptors, or enzymes, and/or possess catalytic properties (catalytic antibodies and ribozymes, among others). Biological libraries can also be produced by combinatorial

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Figure 4.19 Structure of phage display S–S cyclic peptide libraries L28–L30.

electroporation. The libraries produced contained two terminal cysteines to provide S–S cyclization of the peptides: the CX3CX3CX3C library (L28), the CX7C library (L29), and the CX5C library (L30) (Fig. 4.19). Three distinct structural motifs were recovered after in vivo selection in tumor-bearing mice; NGR (in CNGRCVSGCAGRC from L28), RGD (in CDCRGDCFC from L29), and GSL (in CGSLVRC from L30). All three of them delivered peptidic sequences preferentially to the tumor and seemed to bind to different tumor receptors. Their use as drug delivery systems for the development of targeted chemotherapy looks extremely promising. The usefulness of phage display libraries in producing enormous numbers of peptidic sequences of various lengths combined with the possibility of tailoring the structures of the displayed population and the commercial availability of several phage systems makes this biological library source extremely appealing. Production of biochemical reagents (e.g., antibodies, diagnostics, and vaccines) or tools (e.g., to validate targets via their inhibition and to produce peptidic ligands) and gathering structural information about receptors by finding peptide agonists or antagonists have all been reported. Undoubtedly, the exploitation of phage libraries will increase and significant breakthroughs are to be expected in the future.

An example of deoxyribozyme (deoxyoligonucleotidic enzymatic activity) selection applied to the isolation of a broad specificity RNA-cleaving activity was reported by Santoro and Joyce (58). A specific construct was built up by assembling a 5′-biotin moiety (A), attached by a short ODN spacer (B), to a target 12-mer ribonucleotide containing the target cleavage sequence (C) and a 50-mer random ODN (E). This latter sequence was surrounded by fixed-sequence ODN spacers (D) on the 3′ and 5′ ends (Fig. 4.20). The corresponding library L31 (1014 ON–ODN components) was applied to a streptavidin-coated support and bound tightly to it via the biotin–streptavidin recognition. The few RNA-cleaving DNA sequences released only the cleaved constructs, which, after 10 rounds of polymerase chain reaction (PCR) amplification, eventually produced two catalytic motifs (DNA 1 and 2, Fig. 4.20) which were thoroughly studied and refined. Many other reports of ribozymes and deoxyribozymes have been reported, as the use of PCR amplification to select ON–ODN aptamers that bind to receptors/small molecules.

in solution

PCR amplification

50-member ODNs

DNA 1, DNA 2

Figure 4.20 Structure of an SP ON/ODN library L31.

The generation of biased libraries from synthetic or natural precursors using either purified enzymes or whole microorganisms was reported by Khmelnitsky et al. (59) adopting the so-called combinatorial biocatalysis approach. The library L32, based on (±)-(2-endo,3-exo)-bicyclo[2.2.2]oct-5-ene-2,3-dimethanol (BOD), was obtained by submitting BOD to a panel of multistep enzymatic transformations. A total of 1222 compounds were obtained and some of the representative structures are shown in Fig. 4.21. This approach allows the generation of very diverse and complex compounds from a common scaffold employing mild enzymatic transformations that can also accommodate sensitive substrates. These transformations are often stereoselective and regioselective, so that complex derivatives may be obtained without protection/deprotection steps. A lot of effort is required to process the reaction samples and to isolate, purify, and structurally characterize the final library components. However, this strategy is particularly useful when a lead compound with a complex structure, which has already proven its value for a specific application, must be optimized for progression as a drug candidate.

Combinatorial biosynthesis is an approach aimed at the modification of the cellular machinery involved in the biosynthetic pathways that produce natural products. There are several groups active in the field, and an example reported by McDaniel et al. (60)

4.2 COMBINATORIAL LIBRARIES 161

Engineered polyketide biosynthesis

Manipulation of the following steps currently feasible:

CHAIN LENGTH of the product

KETOREDUCTION (yes or no)

1st RING STEREOSPECIFICITY

1st RING AROMATIZATION

2nd RING CYCLIZATION (yes or no)

RATIONALLY ENGINEERED POLYKETIDES

Figure 4.22 Combinatorial biosynthesis: manipulation of the aromatic polyketide pathway.

reported in Fig. 4.23, which will be used to briefly define the properties and opportunities provided by these libraries.

A library L33 made up of different compositions and stoichiometries of LnxMy- CoOz (Ln = La or Y, M = Ba, Sr, Ca, or Pb) was reported by Briceno et al. (61) as a source of new magnetoresistant materials. It was composed of 128 discretes (Fig. 4.23) and was prepared in duplicate using a combination of thin-film deposition and physical masking to vary the individual components. Each library copy was analyzed for magnetoresistance using a different procedure. A significant number of individuals showed a meaningful (>5%) magnetoresistant effect, and a few of them were characterized in detail. Automated synthesis of such discrete material libraries, their analysis for different applications in material sciences, and the automated and reliable readout of the results make this class of libraries extremely useful, and in fact similar approaches are appearing more and more frequently in the literature.