Design and synthesis of a chemically diverse, lead-like DNA-encoded library from sequential amide coupling

Abstract

DNA-encoded libraries (DELs) are established as an effective screening strategy to identify protein ligands and offer a cost-effective means of screening large numbers of compounds. However, the synthesis and utilisation of DELs is implemented by relatively few laboratories. Here, we describe the design and synthesis of a medium-sized DEL through simple amide coupling procedures. We provide details of chemistry and enzymatic steps and demonstrate their effectiveness by synthesising 300 thousand and 3 million-member DELs. We demonstrate their integrity through screening against carbonic anhydrase IX and show their chemical diversity through in silico comparison with an established high-throughput screening library. The DELs described can be used as a resource to accelerate hit identification for early-phase drug discovery and are available to the academic community for screening.

Scheme 1. DEL synthesis scheme comprising two rounds of amide-coupled N-Fmoc amino acids with a final capping carboxylic acid.

Fig. 1. Summary of building block validations: a) 14 nt DNA headpiece 1 used as a model in this work; b) amide coupling validation reaction; c) observed conversions for N-Fmoc amino acids (left) and carboxylic acids (right) determined by AUC integration of RP-LCMS BPC (1050–3000 Da) chromatogram.

Scheme 2. 300 thousand DEL synthesis comprising two sequential cycles of coupling of 55 N-Fmoc amino acids (with Fmoc deprotection) followed by capping with 96 carboxylic acids.

Fig. 2. a) Sequence counts for the cycle 3 monomer for selections of the 300 thousand-member library against CAIX, a) initial selection, b) selections after pre-clearing the library against the beads.

Fig. 3. Sequence counts for the cycle 3 monomer for selection of the 3 million-member DEL against CAIX.

Fig. 4. Calculated property distribution comparisons between the DEL (gold) and the enamine HTS set (teal).

Fig. 5. Chemical diversity principal components analysis of the DEL (gold) compared to the enamine HTS set (teal), left panel with DEL compounds on top, right panel with HTS compounds on top.

Fig. 6. Principal moments of inertia plots for the DEL (gold) and the enamine HTS set (teal).