DNA Encoded Libraries: Principle of DNA-Compatible Chemical Reactions for High-Throughput Small Molecule Discovery
DNA-encoded libraries (DELs) are powerful tools for drug discovery, uniquely combining features of genetically encoded libraries—via DNA barcoding—with the structural diversity of synthetic combinatorial libraries. In DEL synthesis, small molecules are covalently linked to unique DNA tags, enabling the pooling and parallel screening of vast compound libraries. Although it is theoretically possible to synthesize each small-molecule library member individually and attach a DNA tag, this process would undermine the efficiency advantages DELs offer over traditional high-throughput screening (HTS) methods. To preserve throughput, DEL synthesis relies on DNA-compatible chemical reactions performed in a split-and-pool format with the DNA barcode present throughout synthesis.
Key criteria for successful DNA-compatible chemistry in DEL construction include:
- Use of reagents and solvents that are physically compatible with DNA (e.g., mild conditions, water or polar solvents, non-acidic pH, limited temperature range).
- Maintenance of DNA integrity, particularly the preservation of sensitive functional groups such as deoxyribose sugars, phosphodiester backbones, and aniline-containing nucleobases.
- High-yielding reactions or robust purification strategies to eliminate side products in pooled mixtures.
- Broad substrate scope to maximize chemical diversity and expand DEL coverage.
These principles guide the design of effective and scalable DELs for identifying biologically active small molecules in early-stage drug development.
Common Synthetic Transformations for DEL Formation
Although multiple novel chemical transformations are reported every year, a few reactions are utilized in most synthesized DELs (Scheme 1) [Song and Hwang 2020, Shi 2021]:
- Amide formation
- Reductive amination
- Amine capping with electrophiles
- Nucleophilic aromatic substitution
- Transition metal-mediated cross coupling
- Cycloaddition
- Condensation reaction
This is both because these reactions are DNA-compatible across a broad range of similar building blocks and the availability of structurally diverse building blocks.
Scheme 1: Common synthetic steps utilized for the construction of DELs.
Transition metal-mediated cross coupling reactions are not only limited to the Suzuki, Sonogashira and Ullmann coupling shown in Scheme 1E, but also includes Heck coupling and other C-N coupling reactions. Similarly, a wide variety of condensation reactions have been reported for the formation of a broad range of heterocyclic moieties including:
- Benzimidazoles
- Imidazolidinones
- Quinazolinones
- Imidazopyridines
- Tetrahydroquinolines
- Polycyclic isoxazolidines
- Spiroheterocycles
- Thiazole fused dihydropyrans
- Isoquinolones
- Imidazoles
- Oxadiazoles
- Tetrazoles
Deprotection Reactions During DEL Synthesis
Albeit not introducing chemical diversity directly deprotection reactions are vital for DEL synthesis. Most often amines need to be liberated for subsequent diversification especially if bi- and tri-functional DEL building blocks are utilized. Besides, commonly employed deprotection strategies (removal of Boc and Fmoc, Scheme 2A) novel approaches have been developed. These include the reduction of azides to the corresponding amine (Scheme 2B) and (hetero)aromatic nitro groups to the corresponding anilines. The nitro reduction strategy is an elegant way to construct DELs by first utilizing the nitro to direct a SNAr reaction and secondly reduce it to an aniline which can be the next point of reaction (Scheme 2C). Besides the reactions shown in Scheme 2, examples of alloc deprotection has also been shown. Finally carboxylic acids can be accessed from the corresponding methyl, ethyl or tert-Butyl ester by hydrolysis.
Scheme 2: Common deprotection strategies to liberate free amines and anilines.
Multicomponent Reactions
Besides creating libraries by the stepwise addition of building blocks, in the past years multicomponent reactions have sparked interest as they allow for rapid diversification (Shi 2021). The reactions used for DEL synthesis include:
- Petasis reaction
- Ugi 4-component reaction (Scheme 3A) and variations of this
- Passerini reaction
- Povarov reaction
- Aza-Diels-Alder reaction (Scheme 3B)
Scheme 3: Examples of multicomponent reactions used for the synthesis of DNA-encoded libraries. * indicates that both enantiomers are formed.
The major issue with these reactions is the need for a diverse set of specialized building blocks such as isocyanides, aldehydes and olefins. As the commercially available building blocks of these types are limited the libraries which can be generated will be limited. Furthermore, at least one of the building blocks (typically an aldehyde) needs to be bifunctional to be attached to the DNA, which further limits the amount of available building blocks. Finally, e.g. the Ugi reaction has not been shown to be enantioselective.
Ring Closing Reactions
Scheme 4: Examples of synthesis of DNA encoded macrocyclic chemical libraries.
Photoredox-Based Reactions
Photoredox based methods have emerged over the past few years in organic chemistry to generate radicals. These radicals have successfully been applied to react with radical acceptors (E.g. Michael acceptors and styrene’s) on-DNA for the successful DEL synthesis. Most radical precursors used for DEL synthesis are derived from α-amino acids, which can undergo decarboxylative conjugated additions as exemplified by the Giese reaction. Besides conjugative additions, Ni/Photoredox reactions have also been developed for various sp3-sp2 and sp3-sp3 cross-coupling reactions, defluorinative alkylations, and decarboxylative arylations. Utilizing N-Boc amino acids for the photoredox based decarboxylative reactions give easy access to an amine, which can be utilized for the subsequent rounds for DEL construction.
Scheme 5: Examples of Photoredox-based reaction for the construction of DNA Encoded Libraries.
Beyond aqueous conditions for on-DNA chemistry
Though not a new concept as it was initially described by Halpin and Harbury in 2004, DNA and hereby DELs can be reversibly absorbed to solid support, which allows for water-free reactions in organic solvents (Halpin and Harbury 2004). The solid support utilized is positively charged diethylammoniumethanol-sepharose or as reported by Baran and Dawson based
on quaternary ammonium. Allowing for near-anhydrous conditions opens op to broad range of reactions, and examples exist of Ni-mediated cross-couplings and electrochemical aminations (Flood 2019).
To overcome the challenges associated with DNA degradation when treated to acidic conditions a micellar system has been developed (Škopić 2019) Herein sulfonic acids were introduced in the lipophilic part of an amphiphilic copolymer. This allowed for the Brønsted acid to only be present within the micelle, where the small molecule can enter, but not the DNA-tag allowing for the diversification of aldehydes to tetrahydroquinolines and aminoimidazopyridines by Povarov and Groebke–Blackburn–Bienaymé reactions. Further the system was utilized for oxidation of alcohols to aldehydes as well as Boc deprotection under acidic conditions.
Conclusion
DNA-encoded libraries (DELs) have revolutionized early-stage drug discovery by enabling the rapid, high-throughput screening of vast chemical spaces through the fusion of combinatorial chemistry and DNA barcoding. Central to this platform is the ability to perform DNA-compatible chemical reactions that maintain the integrity of the DNA tag while allowing broad synthetic versatility. As the field has evolved, a robust toolbox of established reactions—including amine modifications, cross-couplings, and multicomponent transformations—has laid the groundwork for reliable DEL synthesis. Meanwhile, the emergence of advanced methodologies such as photoredox catalysis, ring-closing reactions, and solid-support-based approaches continues to expand the chemical space accessible through DELs. These innovations not only increase the structural diversity of libraries but also improve the functional group tolerance and synthetic efficiency necessary for navigating the complexity of drug-like molecules. Looking forward, the ongoing development of new DNA-compatible reactions, improved building block availability, and optimized reaction conditions will further enhance the scope and utility of DELs, cementing their role as a cornerstone technology in modern drug discovery.
References
- Flood, D. T. et. al. (2019), Expanding Reactivity in DNA-Encoded Library Synthesis via Reversible Binding of DNA to an Inert Quaternary Ammonium Support, 141, 25, 9998-10006. doi.org/10.1021/jacs.9b03774
- Flood, D. T. et. al. (2020), DNA Encoded Libraries: A Visitor’s Guide, Isr. J. Chem., 60, 268-280. doi.org/ 10.1002/ijch.201900133
- Halpin, D. R. and Harbury, R. B. (2004), DNA Display I. Sequence-Encoded Routing of DNA Populations, PLoS Biology, 2 (7), 1015-1021. doi.org/10.1371/journal.pbio.0020173
- Martí-Centelles, V. et. al. (2015), Macrocyclization Reactions: The Importance of Conformational, Configurational, and Template-Induced Preorganization, Chem. Rev., 115, 16, 8736-8834. doi.org/10.1021/acs.chemrev.5b00056
- Shi, Y. et. al. (2021), DNA-encoded libraries (DELs): a review of on-DNA chemistries and their output, RSC Adv., 11, 2359-2376. doi.org/ 10.1039/d0ra09889b
- Škopić, M. K. et. al. (2019), Micellar Brønsted Acid Mediated Synthesis of DNA-Tagged Heterocycles, J. Am. Chem. Soc., 141, 26, 10546-10555. doi.org/10.1021/jacs.9b05696
- Song, M., Hwang, G. T. (2020), DNA-Encoded Library Screening as Core Platform Technology in Drug Discovery: Its Synthetic Method Development and Applications in DEL Synthesis, J. Med. Chem., 63, 6578-6599. doi.org/10.1021/acs.jmedchem.9b01782a