The ENCODE project revolutionized RNA biology by identifying thousands of non-coding RNAs that are essential in important cellular processes and are highly misregulated in numerous diseases. Despite this discovery, less than 100 chemical probes exist for a limited number of RNA targets. To date, the most successful probes were designed utilizing knowledge of small molecule specificity for the r(CUG) sequence of pathogenic repeat expansions in myotonic dystrophy. Although powerful, this RNA-design strategy is mostly limited to internal loop sequences, which represents only one of the five canonical RNA secondary structures. Recently, the oxazolidinone core was identified as privileged for binding to bulge motifs; however, the binding preferences among oxazolidinone derivatives are largely unknown with only limited knowledge of binding to the A-site of ribosomal RNA and a seven nucleotide bulge in the T-box riboswitch. Therefore, the goal of this work is to synthesize an RNA-targeted, oxazolidinone-based library and develop a repertoire of bulge-specific small molecules. Toward this aim, two synthetic routes have been explored. In one route, phenylalanine methyl ester was cyclized to an oxazolidinone scaffold in a stereocovergent and stereodivergent synthesis, allowing access to all four stereoisomers. The scaffold was diversified at the C(4) position to synthesize a library of bis-substituted small molecules. In a second route, mono-substituted oxazolidinones were synthesized using a commercially available scaffold, which was diversified at the C(5) position. Both diversification reactions utilized amine-based subunits that include known RNA-binding chemotypes or amino acid side chains enriched at RNA:protein interfaces. To date, the RNA-affinity and selectivity of the library has been assessed against a training set of sixteen RNA secondary structures. The results revealed unique bulge binding preferences with dissociation constants in the five to ten micromolar range. Future work will characterize the unique oxazolidinone:RNA interactions by NMR to develop a greater understanding for the basis of the specificity. Importantly, the bulge-specific small molecules identified in this work can be utilized to develop novel chemical probes for the many disease relevant RNAs containing this motif.

Synthesis and evaluation of oxazolidinone-based small molecule libraries for the selective recognition of RNA bulge motifs

254th National Meeting (2017)

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