Aqueous synthetic methods and their applications in DNA-encoded chemical libraries

Water is the basis for all living organisms on Earth and most biochemical processes proceed in aqueous environments. To study biological systems, chemists have developed numerous procedures to perform chemical transformations in the presence of water. However, there is still a limited scope of reactions that proceed efficiently and reliably under physiological conditions. The search for new techniques to enable selective and reliable modifications of biomolecules and small molecules alike, has attracted the attention of many researchers in academia and industry. Such "water-friendly" reactions are highly desired for different areas of biochemical research, such as bioconjugation techniques and drug discovery procedures.
Schiff base formation is a workhorse in bioconjugation science, although the reactions proceed slowly under neutral conditions without catalysts. We investigated oxime and hydrazone formations of ortho-boronate carbonyl compounds with hydroxylamines and hydrazines. The boronic acid was found to strongly increase the Schiff base formation rate, which enabled the fluorescent labeling of antibodies. Hydrazones with an adjacent boronic acid group undergo a secondary cyclization reaction to form a stable, aromatic boron-heterocycle (BIQ, 4,3-borazaroisoquinoline). Upon modulation of the electronic properties of this boron-heterocycle with different substituents, we developed a blue fluorophore that formed upon cyclization to the BIQ product.
Based on the results from the bioconjugation techniques, we developed a library of macrocycles, in which every compound was attached to a DNA strand containing the information about the macrocycle structure. Despite initial difficulties, we obtained a 1.4 million member DNA-encoded library of natural product-like macrocycles with high scaffold diversity. After thorough analysis of the library properties, we screened the encoded macrocylce collection against three human proteins. Several hits were found and resynthesized without DNA tag. Binding affinities to the target proteins were evaluated by biophysical techniques. Differential scanning fluorimetry enabled the parallel screening of the hit compounds, giving a qualitative measurement for the protein affinities. Isothermal titration calorimetry yielded quantitative dissociation rate constants for the most promising compounds. We discovered a novel macrocyclic ligand for α-1-acid glycoprotein (AGP) with a low micromolar affinity, which holds promise for the development of new drug candidates. The developed encoded library methodology was shown to be well suited for early stage drug discovery.