No abstract

The piperazine heterocycle is broadly exploited in FDA-approved drugs and biologically active compounds, but its chemical diversity is usually limited to ring nitrogen substitutions, leaving the four carbon atoms underutilized. Using an efficient four-step synthesis, chiral amino acids were transformed into 6-substituted piperazine-2-acetic acid esters as diastereomeric mixtures whose cis and trans products could be chromatographically separated. From six amino acids (both antipodes), a complete matrix of 24 monoprotected chiral 2,6-disubstituted piperazines was obtained, each as a single absolute stereoisomer in multigram quantities. These diverse and versatile piperazines can be functionalized on either nitrogen atom, allowing them to be used as scaffolds for parallel library synthesis or intermediates for the production of novel piperazine compounds.

Structured RNAs bind ligands and are attractive targets for small-molecule drugs. A wide variety of analytical methods have been used to characterize RNA−ligand interactions, but our experience is that most have significant limitations in terms of material requirements and applicability to complex RNAs. Surface plasmon resonance (SPR) potentially overcomes these limitations, but we find that the standard experimental framework measures notable nonspecific electrostatic-mediated interactions, frustrating analysis of weak RNA binders. SPR measurements are typically quantified relative to a non-target reference channel. Here, we show that referencing to a channel containing a non-binding control RNA enables subtraction of nonspecific binding contributions, allowing measurements of accurate and specific binding affinities. We validated this approach for small-molecule binders of two riboswitch RNAs with affinities ranging from nanomolar to millimolar, including low-molecular-mass fragment ligands. SPR implemented with reference subtraction reliably discriminates specific from nonspecific binding, uses RNA and ligand material efficiently, and enables rapid exploration of the ligand-binding landscape for RNA targets.

7SK is a highly conserved non-coding RNA that regulates eukaryotic transcription by sequestering positive transcription elongation factor b (P-TEFb). 7SK regulatory function likely entails changes in RNA structure, but characterizing dynamic RNA-protein complexes in cells has remained an unsolved challenge. We describe a new chemical probing strategy (DANCE-MaP) that uses maximum likelihood deconvolution and probabilistic read assignment to define simultaneously (i) per-nucleotide reactivity profiles, (ii) direct base pairing interactions, and (iii) tertiary and higher-order interactions for each conformation of multi-state RNA structural ensembles, all from a single experiment. We show that human 7SK RNA, despite significant heterogeneity, intrinsically codes for a large-scale structural switch that couples dissolution of the P-TEFb binding site to structural remodeling at distal release factor binding sites. The 7SK structural equilibrium is regulated by cell type, shifts dynamically in response to cell growth and stress, and can be exogenously targeted to modulate transcription in cells. Our data support that the 7SK structural ensemble functions as an integrator of diverse cellular signals to control transcription elongation in environment and cell specific ways, and establishes DANCE-MaP as a powerful strategy for comprehensively defining RNA structure and dynamics in cells.

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