Systematic Evaluation and Optimization of Modification Reactions of Oligonucleotides with Amines and Carboxylic Acids for the Synthesis of DNA-Encoded Chemical Libraries

Abstract

DNA-encoded chemical libraries are collections of small molecules, attached to DNA fragments serving as identification barcodes, which can be screened against multiple protein targets, thus facilitating the drug discovery process. The preparation of large DNA-encoded chemical libraries crucially depends on the availability of robust synthetic methods, which enable the efficient conjugation to oligonucleotides of structurally diverse building blocks, sharing a common reactive group. Reactions of DNA derivatives with amines and/or carboxylic acids are particularly attractive for the synthesis of encoded libraries, in view of the very large number of building blocks that are commercially available. However, systematic studies on these reactions in the presence of DNA have not been reported so far. We first investigated conditions for the coupling of primary amines to oligonucleotides, using either a nucleophilic attack on chloroacetamide derivatives or a reductive amination on aldehyde-modified DNA. While both methods could be used for the production of secondary amines, the reductive amination approach was generally associated with higher yields and better purity. In a second endeavor, we optimized conditions for the coupling of a diverse set of 501 carboxylic acids to DNA derivatives, carrying primary and secondary amine functions. The coupling efficiency was generally higher for primary amines, compared to secondary amine substituents, but varied considerably depending on the structure of the acids and on the synthetic methods used. Optimal reaction conditions could be found for certain sets of compounds (with conversions >80%), but multiple reaction schemes are needed when assembling large libraries with highly diverse building blocks. The reactions and experimental conditions presented in this article should facilitate the synthesis of future DNA-encoded chemical libraries, while outlining the synthetic challenges that remain to be overcome.

What is a “DNA-Compatible” Reaction?

“…A handful of studies now exist describing the preparation and screening of solution-phase libraries that incorporate diverse reactions—including those rehearsed above—though only with conversion yield data. 20–23,32–34 We sought to determine whether our assay could be extensible to the investigation of solution-phase DNA-encoded reaction development, and whether and how those data could apply to solution-phase library planning and synthesis.…”

“…The impact of synthesis conditions on DNA’s informational integrity, and consequently its viability in PCR to generate templates for sequencing-based decoding, is currently not a consideration in the development of new DNA-encoded reactions. 32–35 …”

DNA Compatible Multistep Synthesis and Applications to DNA Encoded Libraries

“…Table 1 Entry 20 provides a method for reacting amines with a bromoacetamide functionalized DNA-conjugate. Franzinin et al 53 have recently reported a similar reaction using chloroacetamide functionalized DNA. Table 1 Entry 21 provides a method for a DNA-conjugated HWE reaction which may be applied to split-and-pool or high-throughput DEL synthesis.…”

“Cap-and-Catch” Purification for Enhancing the Quality of Libraries of DNA Conjugates

“…19 However, yields for acylation reactions of secondary amines tend to be lower than for primary amine oligonucleotide conjugates. 19 Test biotinylations with two DNA conjugates bearing secondary amines (DNA-DMBG and DNA-AzPro, Scheme 1) provided lower modification yields than for DNA-NH 2 . In a test reaction on pseudosolid phase (200 mM B-NHS, 20 mM DMAP, 2 h, room temperature) biotinylation yields were 33% and 73% for DNA-DMBG and DNA-AzPro, respectively (Supporting Information Figure S3).…”

“…27 We demonstrate the concept of "cap-and-catch" purification for the attachment of carboxylic acids to amino-modified DNAs, a frequently used reaction for the preparation of DECLs. 19 In this protocol, following the synthesis step, electrophilic biotin reagents cap unmodified amino-DNAs and the resulting biotinylated DNAs can be removed by affinity capture on streptavidin-coated sepharose ( Figure 1). Several tested reaction protocols provided excellent biotinylation yields and enabled the straightforward purification of DNA-conjugates from unreacted amino-DNA.…”