Baiyang Mu, Yiwei Zhang, Xudong Wang, Rui Jin, Weiwei Lu, Nidhal Selmi, Sixiu Liu, Avinash Bhat, Sara Pahlén, Giulia Bergonzini, Zhiqiang Duan, Yinan Song, Xiaojie Lu

Bioconjugate Chemistry

DOI: 10.1021/acs.bioconjchem.4c00581

Abstract

The C­(sp²)–C­(sp³) cross-coupling reaction is an effective way to increase the C­(sp³) content in compound collections for drug discovery, enhancing molecular diversity and offering a unique chemistry starting point. In this study, we report a mild, DNA-compatible, and off-DNA-inert photochemical cross-coupling reaction inspired by the amino radical transfer strategy. This method demonstrates broad substrate scopes for DNA-encoded library (DEL) constructions, utilizing commonly available structures on DNA and diverse alkyl boronate ester building blocks, which have not been widely applied in the current DEL chemical space.

For years, a robust and general method for DNA compatible "reverse" Buchwald-Hartwig-type amination – coupling DNA-conjugated amines with external aryl halides – has been long sought, yet this transformation remained elusive due to the prohibitive challenge of DNA nucleobase interference. To overcome this long-standing technical bottleneck, the Ritter group at the Max-Planck-Institut für Kohlenforschung reported an air-stable ruthenium-mediated η6 π-arene activation strategy in the Journal of the American Chemical Society (JACS). This breakthrough finally enables the precise N-arylation of DNA-linked amines, unlocking a vast and previously inaccessible chemical space for drug discovery.

1. The Challenge: The DNA Nucleobase Interference

In DEL synthesis, C–N cross-coupling is a cornerstone for building structural diversity. While conventional Buchwald-Hartwig amination (DNA-linked aryl halides reacting with external amines) was well-established, the reverse approach has been hindered by the presence of multiple amino groups within DNA nucleobases (adenine, guanine, and cytosine).

1) Limitations of Traditional Catalysts: Conventional palladium-based methods often lead to undesired arylation of the DNA backbone rather than the intended terminal amine.

2) Chemoselectivity Hurdles: Because the amino groups on nucleobases are typically more acidic than aliphatic amines, palladium-catalyzed pathways (which involve deprotonated amine intermediates) preferentially target the DNA nucleobases.

2. Ruthenium-Mediated η6 π-Arene Activation

To overcome these limitations, the Ritter group developed a bench-stable, DNA-compatible acetyl-substituted cyclopentadienyl (CpAc) ruthenium complex (1).

Figure 1. N-arylation of DNA-conjugated amines in aqueous media. (a) SNAr of DNA-conjugated aliphatic amines with η6 arene complexes. (b) Electrophilicity of Ru π-arene complexes 5−7 and N-arylation of DNA-conjugate 2.

• Mechanism of Action: The ruthenium reagent activates commercially available haloarenes in situ through η6 π-arene coordination. This coordination significantly increases the electrophilicity of the arene ring, allowing for nucleophilic aromatic substitution (SNAr) directly with the DNA-conjugated amine.
• Operational Simplicity: Much like amide coupling reagents (e.g., HATU), the protocol involves simple activation of haloarenes by complex 1 followed by addition to the DNA conjugate. The final aniline product was released via photolysis at 390 nm, a wavelength safe for DNA integrity.

3. Superior Chemoselectivity and Validation

The core innovation lies in the complementary selectivity of ruthenium compared to traditional palladium catalysts.

1. Selective Amine Attack: Unlike palladium, the ruthenium-mediated pathway involves the attack of a neutral amine, which naturally favors the more nucleophilic aliphatic amine over the less nucleophilic DNA nucleobases.

Figure 2. Chemoselectivity of DNA N-arylation.

2. Substrate Scope: The method demonstrates exceptional tolerance for diverse functional groups, including carbonyls, sulfonamides, and even Lewis-basic heterocycles when treated with HBF4⋅Et2O to prevent metal coordination. It successfully arylated a wide array of primary and secondary amine-DNA conjugates, including natural and non-natural amino acid.

Figure 3. Substrate scope of N-arylation for various amine-DNA conjugates. Aryl halide (c = 10 mM) and Ru complex 1 (c = 1.0 mM) in DMC, 80oC, 2 h; then amine-DNA conjugate (c = 0.10 mM), in sodium borate buffer (c = 0.50 M, pH 9.4): DMSO (1:9), 40°C, 2−16 h; then 390 nm irradiation in water (c = 0.10 mM). aHBF4·Et2O was used for the complexation step.

3. Preservation of DNA Integrity: High-throughput compatibility was confirmed through qPCR analysis and ligation tests, proving that the DNA barcode remains stable and readable throughout the reaction sequence.

4. Paradigm Shift in DEL Synthesis

This ruthenium-mediated strategy reconfigures the drug discovery workflow for DELs in three key ways:

1) Unlocking "Reverse" Disconnections: Researchers can now use DNA-linked amines as a starting point for C–N coupling with a vast library of commercially available aryl halides, drastically expanding structural diversity.

2) Orthogonal Reactivity: By providing a method that is chemoselective for aliphatic amines over DNA nucleobases, it offers a "surgical" precision that palladium-based methods lack for this specific disconnection.

3) Establishing a Bench-Stable Standard: Complex 1 provides a practical, scalable, and single-reagent tool for both academic research and industrial DEL production.

5. Conclusion

This study marks the first successful "reverse" Buchwald-Hartwig-type amination on DNA. While "normal" pathways (DNA-halides + external amines) are well-established, the reverse route (DNA-amines + external halides) was historically hindered by nucleobase interference. Due to the lower pKa of nucleobase amines compared to aliphatic amines, traditional palladium catalysis often leads to undesired DNA backbone modification. Ruthenium reagent 1 overcomes this via η6 π-arene activation, targeting neutral amines with "surgical" precision to bypass nucleobase competition. This paradigm shift significantly expands the accessible chemical space for DEL-driven drug discovery while ensuring rigorous DNA integrity

References

1. Kanoo, S., de Pedro Beato, E., et al. (2025). Ruthenium-Mediated N-Arylation for DNA-Encoded Libraries. J. Am. Chem. Soc. https://doi.org/10.1021/jacs.5c11842

Raphael M. Franzini

Molecular Informatics

DOI: 10.1002/minf.70045

Abstract

Linkers play a central role in many areas of medicinal chemistry, including proximity inducers, small‐molecule conjugates, and DNA‐encoded libraries. However, little is known about the accessibility of molecules to linker attachment when bound to proteins. Here, we analyze linker accessibility across protein–ligand complexes in cocrystal structures. A computational workflow was developed to evaluate the linkerability of modifiable positions on molecules based on solvent accessibility, local steric space for introduction of a linker atom, and the geometry of solvent‐directed escape paths approximated as conical frustums. Analysis of 8,228 protein–ligand cocrystal structures with 131 431 modifiable positions shows that approximately 22% of positions can accommodate linkers without significant geometric restriction. Limited linkerability of positions influences DEL data and may confound efforts to use such data for lead prediction.

Slavko Rast, Marie Morgan-Fisher, Sarah D. Blomquist, Jorge Peiró Cadahía, Sanne Cowland, Thomas Franch, Emil Glibstrup, Alex Gouliaev, Margit Haahr Hansen, Aleksejs Kontijevskis, Titi Kronborg, Loris Moretti, Anna Nadali, Søren Nielsen, Sebastian Leth-Petersen, Michael Rabe, Adili Alafate, Jennifer R. Allen, Abhisek Banerjee, Shon K. Booker, John R. Butler, Imelda Hot, David Huang, Matthew R. Kaller, Rajiv Kapoor, Qingyian Liu, Patricia Lopez, Vu Ma, Francesco Manoni, Jose M. Medina, Alexander J. Pickrell, Hui-Ling Wang, Jingjing Xie, Wenhan Zhang, Christopher Mohr, Kui Chen, Anne Y. Saiki, Paul Wang, Monica Leavitt, Karen Rex, Guo Zhong, Ling Zou, Julie Lade, Upendra P. Dahal, Nashid Farhan, Prashant Agarwal, Borna Zandkarimi, Kai Zhu, Gitte Husemoen, Nuria A. Tamayo, Brian A. Lanman

Journal of Medicinal Chemistry

DOI: 10.1021/acs.jmedchem.6c01357

Abstract

Activating mutations in the Kirsten rat sarcoma (KRAS) gene are prevalent oncogenic drivers in nonsmall cell lung cancer (NSCLC). Patients harboring KRAS-mutant lung cancers frequently develop central nervous system (CNS) metastases. Although approved KRAS G12C inhibitors (i.e., sotorasib and adagrasib) show promising clinical CNS activity, these agents demonstrate low preclinical brain-to-plasma ratios, raising the question of whether compounds with elevated preclinical Kp,uu,brain values might show enhanced clinical performance. Here, we report the first successful application of DNA-encoded library (DEL) screening technology to the identification of CNS-penetrant covalent inhibitors of KRAS G12C. In this effort, a property-biased covalent DEL-screening approach enabled the discovery of a structurally novel series of hydrogen bond donor-free KRAS G12C inhibitors with improved CNS exposure. Leveraging structure-based design, we refined this hit series to deliver lead compound AM-8719, a CNS-penetrant, orally efficacious KRAS G12C inhibitor exhibiting 200-fold improved potency with respect to initial screening hits.

Xudong Wang, Xuanjing Shen, Zhiqiang Duan, Xiaojie Lu

Bioconjugate Chemistry

DOI: 10.1021/acs.bioconjchem.6c00344

Abstract

DNA-encoded library (DEL) technology has emerged as a powerful platform for small-molecule discovery, in which on-DNA reaction development plays a central role in determining accessible chemical space. Early on-DNA chemistry mainly focused on establishing robust DNA-compatible transformations under mild aqueous conditions but often generated structurally limited libraries. Recent advances in photochemistry, electrochemistry, biocatalysis, and complexity-generating reactions have substantially expanded the scope of accessible on-DNA transformations and enabled the incorporation of increasingly diverse and medicinally relevant scaffolds into DELs. In this Viewpoint, we discuss the recent progress and emerging trends in on-DNA reaction development, with particular emphasis on the transition from compatibility-driven chemistry toward function-oriented DEL synthesis. We further highlight current challenges and future opportunities for developing precision on-DNA chemistry to support next-generation ligand discovery.

John P Burdick, Erika M Cerna Arroyo, Samantha R Levine, Brian M Paegel

Journal of Medicinal Chemistry

DOI: 10.1021/acs.jmedchem.6c01339

Abstract

One-bead-one-compound DNA-encoded library (OBOC-DEL) technology enables synthesis and activity-based screening of diverse compound collections but requires complex microfluidic instrumentation and laborious manual bead handling. Here, we execute DEL synthesis on 2.8-μm-dia magnetic beads and encapsulate them in a functionalized polyacrylamide hydrogel to support in-gel activity assays. Magnetic-bead loading capacity was ∼10 amol of small molecule and ∼104 DNA encoding tags per bead. We developed a fully automated magnetic solid-phase synthesis using a KingFisher instrument. Bulk emulsification of DEL beads with assay reagents eliminated the need for microfluidics. Activity-based screening against Factor Xa identified enzyme inhibitors, including the positive control (rivaroxaban, incorporated into the library), which was recovered as the most abundant hit (replicate k class = 14). This ultraminiaturized platform uses commercially available automation and flow cytometry, making OBOC-DEL technology broadly accessible and distributable.