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

Comparison of boron-assisted oxime and hydrazone formations leads to the discovery of a fluorogenic variant

We use kinetic data, photophysical properties, and mechanistic analyses to compare recently developed high-rate constant oxime and hydrazone formations. We show that when Schiff base formation between aldehydes and arylhydrazines is carried out with an appropriately positioned boron atom, then aromatic B–N heterocycles form irreversibly. These consist of an extended aromatic structure amenable to the tailoring of specific properties such as reaction rate and fluorescence. The reactions work best in neutral aqueous buffer and can be designed to be fluorogenic – properties which are particularly interesting in bioconjugation

A DNA-Encoded Chemical Library Incorporating Elements of Natural Macrocycles

Abstract Here we show a seven-step chemical synthesis of a DNA-encoded macrocycle library (DEML) on DNA. Inspired by polyketide and mixed peptide-polyketide natural products, the library was designed to incorporate rich backbone diversity. Achieving this diversity, however, comes at the cost of the custom synthesis of bifunctional building block libraries. This study outlines the importance of careful retrosynthetic design in DNA-encoded libraries, while revealing areas where new DNA synthetic methods are needed

Boronic acids facilitate rapid oxime condensations at neutral pH

We report here the discovery and development of boron-assisted oxime formation as a powerful connective reaction for chemical biology. Oximes proximal to boronic acids form in neutral aqueous buffer with rate constants of more than 104 M−1 s−1, the largest to date for any oxime condensation. Boron's dynamic coordination chemistry confers an adaptability that seems to aid a number of elementary steps in the oxime condensation. In addition to applications in bioconjugation, the emerging importance of boronic acids in chemical biology as carbohydrate receptors or peroxide probes, and the growing list of drugs and drug candidates containing boronic acids suggest many potential applications

An assessment of the mutational load caused by various reactions used in DNA encoded libraries

DNA encoded libraries have become an essential hit-finding tool in early drug discovery. Recent advances in synthetic methods for DNA encoded libraries have expanded the available chemical space, but precisely how each type of chemistry affects the DNA is unstudied. Available assays to quantify the damage are limited to write efficiency, where the ability to ligate DNA onto a working encoded library strand is measured, or qPCR is performed to measure the amplifiability of the DNA. These measures read signal quantity and overall integrity, but do not report on specific damages in the encoded information. Herein, we use next generation sequencing (NGS) to measure the quality of the read signal in order to quantify the truthfulness of the retrieved information. We identify CuAAC to be the worst offender in terms of DNA damage amongst commonly used reactions in DELs, causing an increase of G → T transversions. Furthermore, we show that the analysis provides useful information even in fully elaborated DELs; indeed we see that vestiges of the synthetic history, both chemical and biochemical, are written into the mutational spectra of NGS datasets

Building Boron Heterocycles into DNA-Encoded Libraries

DNA-encoded library (DEL) technology uses DNA tags to track the synthetic history of individual members in a split-and-pool combinatorial synthesis scheme. DEL synthesis hinges on robust methodologies that tolerate combinatorial synthesis schemes while not destroying the information in DNA. We introduce here a DEL-compatible reaction that assembles a boron-containing pyridazine heterocycle. The heterocycle is unique because it can engage in reversible covalent interactions with alcohols─a feature that, until now, has not been deliberately engineered into DELs

A DNA-Encoded Macrocycle Library that Resembles Natural Macrocycles

Herein we perform a seven-step chemical synthesis of a DNA-encoded macrocycle library (DEML) on DNA. Inspired by polyketide and mixed peptide-polyketide natural products, the library was designed to incorporate rich backbone diversity. Achieving this diversity, however, comes at the cost of custom synthesis of bifunctional building block libraries. Our work outlines the importance of careful retrosynthetic design in DNA-encoded libraries, while revealing areas where new DNA synthetic methods are needed. </div