Diversity-oriented synthesis as a tool to expand the chemical space of DNA-encoded libraries

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Abstract

DNA-encoded libraries (DEL) represent a powerful technology for generating compound collections for drug discovery campaigns, that have allowed for the selection of many hit compounds over last three decades. However, the application of split-and-pool combinatorial methodologies, as well as the limitation imposed by DNA-compatible chemistry, has often brought to a limited exploration of the chemical space, with an over-representation of flat aromatic or peptide-like structures, whereas a higher scaffold complexity is generally associated with a more successful biological activity of the library. In this context, the application of Diversity-Oriented Synthesis, capable of creating sp3-rich molecular entities even starting from simple flat building blocks, can represent an efficient strategy to significantly broaden the chemical space explored by DELs. In this review, we present selected examples of DNA-compatible complexity-generating reactions that can be applied for the generation of DNA-encoded DOS libraries, including: (i) multicomponent reactions; (ii) C-H/C-X functionalization; (iii) tandem approaches; (iv) cycloadditions; (v) reactions introducing privileged elements. Also, selected case studies on the generation of DELs with high scaffold diversity are discussed, reporting their application in drug discovery programs.

Introduction

Since the pioneering work by Lerner and Brenner in 1992,1 DNA-encoded libraries (DELs) represent a powerful technology for generating compound collections for drug discovery campaigns.2, 3, 4, 5 DNA-encoded libraries are usually prepared following the principles of combinatorial chemistry, by exploiting the productive and efficient split-and-pool approach,6 which can rapidly give access to millions of library members in an exponential way.7, 8 Also, combinatorial chemistry allows for a convenient encoding technology, by covalently attaching in each synthetic step a unique DNA tag that specifically decodes for the identity of the building block being incorporated.9 To date, many hit compounds have been discovered by the application of DELs into different phenotypic or target-based screening assays,10, 11, 12 including the Receptor-Interacting serine/threonine-Protein Kinase 1 (RIPK1) inhibitor, developed by GSK, that recently entered phase II clinical trials for the treatment of psoriasis and arthritis.13 Very recently, a new method for the screening of DELs inside living cells under physiological conditions has been reported for the first time,14 thus enhancing the possibility of applying DELs in phenotypic approaches without the need of highly purified target proteins. However, the exploration of the chemical space of DNA-encoded combinatorial libraries is somewhat limited, as the diversity within the library is given only by the appendages around a common skeleton (Figure 1, top).15, 16, 17 Also, DNA-encoded combinatorial libraries are often enriched by flat aromatic or peptide-like structures, with a limited portion of three-dimensionally and stereochemically complex molecules.17 This is mainly due to the reaction toolbox for DNA-encoded libraries that is substantially small, as compared to classic medicinal chemistry synthesis.18 In fact, chemical reactions applicable to DEL platforms should be: (i) high-yielding and with minimal side-product formation, (ii) versatile, (iii) working in very dilute conditions to minimize the aggregation between DNA strands, (iv) working in water and under mild conditions, such as moderate temperature and pH, in order to avoid the degradation of DNA oligonucleotides and of the glycosidic and phosphodiester bonds.19 For these reasons, most of the reactions still frequently used in DEL synthesis are amidation,20, 21, 22 reductive amination,23, 24 cross-coupling reactions,25, 26, 27 and the Copper-Catalyzed Azide-Alkyne Cycloaddition (CuAAC).28, 29

However, it is now well clear how emphasizing the chemical diversity and complexity of the compounds representing a library can improve the success of drug discovery programs.30, 31, 32, 33, 34 Even though DNA-encoded technology allows to access very large compound collections, a challenge that still needs to be addressed is to increase the chemical space that can be covered by DELs, in particular enhancing the scaffold diversity of the library members. In this context, Martin and coworkers recently developed a chemoinformatics tool, called eDESIGNER, that can generate virtual DELs characterized by pre-defined molecular properties distribution and maximal diversity exploiting available on-DNA chemistry.35 This tool represents an interesting data-driven methodology to guide DEL library synthesis. Similarly, Diversity-Oriented Synthesis (DOS) can significantly broaden the chemical space explored by DELs,36 as this synthetic strategy has been conceived with the aim of generating highest diversity and complexity starting from simple structures.37 In DOS, synthetic pathways are planned using a forward-synthetic analysis, by exploiting efficient strategies composed of no more than 4–5 steps, that make use of complexity-generating reactions and cascade processes, capable of creating sp3-rich molecular entities. In the last few years, novel chemical methods resulted to interesting DNA-compatible complexity-generating reactions able to install rigidifying elements and multiple stereocenters on a DNA-tagged substrate. By elegantly designing the sequences of DNA-compatible complexity-generating reactions, it is possible to fold DNA-tagged intermediates into structurally complex compounds, that differ not only for the appendages but also for their molecular skeletons (Figure 1, bottom), following well-known DOS strategies, such as the Build/Couple/Pair,38 or the Relay Catalytic Branching Cascade (RCBC) approach.39 A great exploration of all DNA-compatible reactions has been recently reviewed in the literature.18, 40, 41 Herein we present selected examples of DNA-compatible complexity-generating reactions that can be applied for the generation of DELs following Diversity-Oriented Synthesis principles. In particular, reactions which rapidly create rings, stereocenters, carbon–carbon bonds, and functional groups are presented, including: (i) multicomponent reactions; (ii) C-H/C-X functionalization approaches; (iii) tandem reactions; (iv) cycloadditions; (v) reactions introducing privileged elements. Selected case studies on the generation of DNA-encoded DOS libraries are discussed, particularly reporting those examples in which the scaffold diversity is generated by a branching (or reagent-based) approach. Finally, a discussion on the off-DNA Diversity-Oriented Synthesis of different molecular scaffolds that are then conjugated to the DNA is presented, highlighting the great potential of this approach, still underdeveloped in the literature. Other strategies to expand DEL chemical space (including the use of quaternary ammonium cationic resins,42 copolymer micelles,43 solid-phase technology,44 or late-stage modification of natural products to attach DNA barcodes45) are not discussed in this review.

Section snippets

Multicomponent reactions

Multicomponent reactions (MCRs) represent a powerful tool in DOS, as they can form multiple bonds at a time, coupling simultaneously three or more starting molecules.46, 47 They allow to easily introduce diversity, just by varying the building blocks, and complexity, by combining multiple functional groups into a common skeleton, resulting in the synthesis of high-quality chemical libraries.48 MCRs have been exploited since the beginning even in the preparation of DELs and several research

Selected case studies on the generation of DNA-encoded DOS libraries

The development of modern complexity-generating DNA-compatible reactions, as seen in recent years, has opened the way to the application of Diversity-Oriented Synthesis principles for the preparation of DELs possessing high skeletal diversity. In DOS, skeletal diversity can be achieved with two main strategies: (1) reagent-based (or branching) approach and (2) substrate-based (or folding) approach.88 In the reagent-based approach, the application of different reaction conditions on the same

Generation of DELs by off-DNA synthesis of diverse scaffolds followed by on-DNA decoration

A complementary approach to increase the scaffold diversity of DELs is presented by the off-DNA Diversity-Oriented Synthesis of different molecular scaffolds, that are subsequently conjugated to the DNA and subjected to traditional split-and-pool combinatorial methodologies. This approach allows to create DELs able to explore wide area of the chemical space, being characterized by thousands of skeletally different compounds, without the limitations imposed by the use of DNA-compatible

Conclusions

Since their introduction three decades ago, DNA-encoded libraries (DEL) caught growing interest as a powerful technology for generating compound collections useful for drug discovery campaigns. Such increasing attention to this technology has been boosted by recent synthetic methods that take advantage of chemoselective and mild reaction conditions, making such chemistry amenable for the implementation to DNA-encoded chemical compounds. The major limitation of split-and-pool combinatorial

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

We acknowledge “Progetto Dipartimenti di Eccellenza 2018-2022” (allocated to Department of Chemistry “Ugo Schiff”) and Fondazione Cassa di Risparmio di Pistoia e Pescia (Bando Giovani@Ricerca scientifica 2018) for financial support.

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