On September 28, 2025, a pioneering study by Miyachi et al. published in the International Journal of Molecular Sciences introduced a transformative diazide platform molecule (DAP) designed to overcome critical limitations in DNA-encoded library (DEL) technology. This research addresses the constrained chemical space of traditional DELs, which often rely on limited reaction sets like CuAAC, hindering exploration of complex targets such as protein-protein interactions. The innovative DAP features orthogonal aromatic and aliphatic azide groups, enabling a stepwise "double-click" strategy: first, an organocatalyzed [3+2] cycloaddition for 1,4,5-trisubstituted triazoles, followed by CuAAC or reduction for further diversification. Validation via mock DEL synthesis confirmed high fidelity, minimal DNA damage, and access to unprecedented chemical regions, expanding opportunities for novel therapeutic discovery against challenging biological targets.

The Challenge: Overcoming Chemical Space Limitations in DELs

Traditional DEL synthesis often relies on a limited set of highly efficient reactions, such as the copper-catalyzed azide-alkyne cycloaddition (CuAAC), which predominantly generates 1,4-disubstituted triazoles. While powerful, this approach can restrict the structural complexity and diversity of the resulting library members. Exploring uncharted chemical space is crucial for discovering hits against challenging targets, such as those involved in protein-protein interactions (PPIs), which require compounds with greater molecular complexity and specific three-dimensional architectures. The strategic use of platform molecules—core scaffolds with multiple orthogonal reactive handles—offers a pathway to more diverse and innovative libraries.

The Innovative Solution: A Smart Diazide Platform with Orthogonal Reactivity

The research team designed and synthesized a novel on-DNA platform molecule featuring both an aromatic and an aliphatic azide group within a single scaffold. The key innovation lies in the orthogonal reactivity of these two azides:

• Aromatic azide: Readily undergoes an organocatalyzed [3+2] cycloaddition with active methylene compounds to form structurally complex 1,4,5-trisubstituted 1,2,3-triazoles.
• Aliphatic azide: Remains inert during the first reaction but can be utilized in a subsequent CuAAC "click" reaction with terminal alkynes or reduced to a primary amine for further diversification (e.g., amidation).

This stepwise "double-click" strategy allows for the modular and efficient construction of highly diverse compounds from a single, versatile starting point. The team meticulously optimized the platform using a proline-based scaffold (4N₃-BA-(2S,4S)-N₃-Pro–HP) to prevent undesirable side reactions, ensuring high fidelity during library synthesis.

Fig.1 Validation of a mock DEL synthesis utilizing double click chemistry. (Miyachi, et al., 2025)

Key Advancements and Validation

• Synthetic Versatility

The platform successfully underwent sequential transformations with a wide range of building blocks, including β-ketoanilides, ketosulfones, and various alkynes, achieving high yields and demonstrating excellent compatibility with DNA.

• Practical Application

A mock DEL synthesis was performed, creating a 12-compound mixture to simulate real-world conditions. Analysis confirmed the successful and specific formation of all target compounds, proving the feasibility of this approach for large-scale library production.

• DNA Integrity

A critical concern in DEL technology is maintaining DNA integrity throughout chemical synthesis. Quantitative PCR (qPCR) analysis of a full-length DEL construct confirmed that the multi-step synthetic process induced negligible DNA damage, ensuring the reliability of the encoded information.

• Uncharted Chemical Space

Perhaps the most compelling finding came from a computational analysis comparing a virtual 6-million-compound library built from this DAP with known bioactive molecules in the ChEMBL database. The DAP-based library was shown to occupy distinct, previously unexplored regions of chemical space, highlighting its potential to generate truly novel chemotypes for drug discovery. The 1,4,5-trisubstituted triazole scaffold (Scaffold A) covered a chemical space over 60% larger than the simpler 1,4-disubstituted analog.

Synergy with CD BioGlyco's Expertise

This pioneering work resonates deeply with the core capabilities at CD BioGlyco. While this study focuses on a general diazide platform, the principles of orthogonal reactivity, modular synthesis, and chemical space exploration are fundamental to our advanced service platforms. Our expertise in custom carbohydrate and glycoconjugate synthesis is ideally suited to incorporate sophisticated sugar-based building blocks into such innovative DEGL platforms, creating libraries with physicochemical properties and targeting capabilities.

Conclusion: A New Era for Library Design

The development of this diazide platform molecule represents a significant leap forward in DEL technology. By enabling a stepwise, divergent synthesis strategy, it provides access to complex and diverse chemical structures that were previously difficult to obtain. This approach holds immense promise for interrogating challenging biological targets and discovering first-in-class therapeutics.

At CD BioGlyco, we are at the forefront of integrating such advancements. We provide the tools and expertise to leverage these novel strategies for your specific research needs, from custom library design based on innovative chemistries to comprehensive screening and analysis.