DNA-Encoded Solid-Phase Synthesis: Encoding LanguageDesign and Complex Oligomer Library Synthesis

摘要

The promise of exploiting combinatorial synthesis for small molecule discovery remains unfulfilled due primarily to the “structure elucidation problem”: the back-end mass spectrometric analysis that significantly restricts one-bead-one-compound (OBOC) library complexity. The very molecular features that confer binding potency and specificity, such as stereochemistry, regiochemistry, and scaffold rigidity, are conspicuously absent from most libraries because isomerism introduces mass redundancy and diverse scaffolds yield uninterpretable MS fragmentation. Here we present DNA-encoded solid-phase synthesis (DESPS), comprising parallel compound synthesis in organic solvent and aqueous enzymatic ligation of unprotected encoding dsDNA oligonucleotides. Computational encoding language design yielded 148 thermodynamically optimized sequences with Hamming string distance ≥ 3 and total read length <100 bases for facile sequencing. Ligation is efficient (70% yield), specific, and directional over 6 encoding positions. A series of isomers served as a testbed for DESPS’s utilityin split-and-pool diversification. Single-bead quantitative PCR detected9 × 10⁴ molecules/bead and sequencing allowed forelucidation of each compound’s synthetic history. We appliedDESPS to the combinatorial synthesis of a 75 645-member OBOClibrary containing scaffold, stereochemical and regiochemical diversityusing mixed-scale resin (160-μm quality control beads and 10-μmscreening beads). Tandem DNA sequencing/MALDI-TOF MS analysis of 19quality control beads showed excellent agreement (<1 ppt) betweenDNA sequence-predicted mass and the observed mass. DESPS synergisticallyunites the advantages of solid-phase synthesis and DNA encoding, enablingsingle-bead structural elucidation of complex compounds and synthesisusing reactions normally considered incompatible with unprotectedDNA. The widespread availability of inexpensive oligonucleotide synthesis,enzymes, DNA sequencing, and PCR make implementation of DESPS straightforward,and may prompt the chemistry community to revisit the synthesis ofmore complex and diverse libraries.