Synthesis of c‑di-GMP Analogs with Thiourea, Urea, Carbodiimide, and Guanidinium Linkages

The first syntheses of neutral thiourea, urea, and carbodiimide analogs, along with two guanidinium analogs, of the bacterial signaling molecule cyclic diguanosine monophosphate (c-di-GMP) are reported. The key intermediate, obtained in nine steps, is a 3′-amino-5′-azido-3′,5′-dideoxy derivative. The 5′-azide serves as a masked amine from which the amine is obtained by Staudinger reduction, while the 3′-amine is converted to an isothiocyanate that, while stable to chromatography, and Staudinger conditions, nevertheless reacts well with the 5′-amine

() The top view of the adenine triple (red) capping the top G-tetrad (cyan)

<p><b>Copyright information:</b></p><p>Taken from "Structure of the intramolecular human telomeric G-quadruplex in potassium solution: a novel adenine triple formation"</p><p></p><p>Nucleic Acids Research 2007;35(7):2440-2450.</p><p>Published online 29 Mar 2007</p><p>PMCID:PMC1874667.</p><p>© 2007 The Author(s)</p> The distance is 2.05 Å for A3H62/A21N1, and 2.27 Å for A21H2/A3N7, respectively. () The bottom view of the A25:T14 base pair (adenine in red and thymine in green) capping the bottom G-tetrad (blue). Figures are prepared using PyMOL

CD spectra of Tel26, WT-Tel26 and Tel22 in 100 mM Na or K solutions at 25°C

<p><b>Copyright information:</b></p><p>Taken from "Human telomeric sequence forms a hybrid-type intramolecular G-quadruplex structure with mixed parallel/antiparallel strands in potassium solution"</p><p>Nucleic Acids Research 2006;34(9):2723-2735.</p><p>Published online 19 May 2006</p><p>PMCID:PMC1464114.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> The same CD signatures are observed for Tel26 and WT-Tel26 in K solution, while similarly distinct CD signatures are observed for Tel22 and Tel26 in Na solution

() A G-tetrad with H1–H1 and H1–H8 connectivity patterns detectable in NOESY experiments

<p><b>Copyright information:</b></p><p>Taken from "Human telomeric sequence forms a hybrid-type intramolecular G-quadruplex structure with mixed parallel/antiparallel strands in potassium solution"</p><p>Nucleic Acids Research 2006;34(9):2723-2735.</p><p>Published online 19 May 2006</p><p>PMCID:PMC1464114.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> () Schematic diagram of the folding topology of the unimolecular human telomeric G-quadruplex in K solution. red ball, guanine; red box, () guanine; magenta box, () guanine; green ball, adenine; blue ball, thymine

The expanded H8/H6–H1′ region () and aromatic–aromatic region () of the non-exchangeable 2D-NOESY spectrum of Tel26 in K solution

<p><b>Copyright information:</b></p><p>Taken from "Structure of the intramolecular human telomeric G-quadruplex in potassium solution: a novel adenine triple formation"</p><p></p><p>Nucleic Acids Research 2007;35(7):2440-2450.</p><p>Published online 29 Mar 2007</p><p>PMCID:PMC1874667.</p><p>© 2007 The Author(s)</p> In (A), the sequential assignment pathway is shown. Missing connectivities are labeled with asterisks. The three peaks, A1H1′/A2H8, T19H1′/T20H8, T20H1′/A21H8, that appear to be missing but are not labeled are observable at a lower contour level, and are related with a broader linewidth. The H8-H1′ NOEs of the nucleotides in conformation are labeled in orange, while the H8-H1′ NOEs of the nucleotides in conformation are labeled in black. The characteristic G()H8/G( + 1)H1′ NOEs for the G()s are labeled by arrows. In (), the H2 protons are specified. () The expanded imino regions of the exchangeable proton 2D JR-NOESY spectrum of Tel26 in K solution. NOEs between loop residue protons and G-tetrad imino protons are labeled in red, intra-tetrad NOEs are labeled in green and inter-tetrad NOEs are labeled in blue. In the bottom panel, the aromatic H2 protons are specified. Conditions: 1°C, 25 mM K-phosphate pH 7.0, 70 mM KCl, 2.5 mM DNA

Nuclease-Resistant c‑di-AMP Derivatives That Differentially Recognize RNA and Protein Receptors

The ability of bacteria to sense environmental cues and adapt is essential for their survival. The use of second-messenger signaling molecules to translate these cues into a physiological response is a common mechanism employed by bacteria. The second messenger 3′–5′-cyclic diadenosine monophosphate (c-di-AMP) has been linked to a diverse set of biological processes involved in maintaining cell viability and homeostasis, as well as pathogenicity. A complex network of both protein and RNA receptors inside the cell activates specific pathways and mediates phenotypic outputs in response to c-di-AMP. Structural analysis of these RNA and protein receptors has revealed the different recognition elements employed by these effectors to bind the same small molecule. Herein, using a series of c-di-AMP analogues, we probed the interactions made with a riboswitch and a phosphodiesterase protein to identify the features important for c-di-AMP binding and recognition. We found that the <i>ydaO</i> riboswitch binds c-di-AMP in two discrete sites with near identical affinity and a Hill coefficient of 1.6. The <i>ydaO</i> riboswitch distinguishes between c-di-AMP and structurally related second messengers by discriminating against an amine at the C2 position more than a carbonyl at the C6 position. We also identified phosphate-modified analogues that bind both the <i>ydaO</i> RNA and GdpP protein with high affinity, whereas symmetrically modified ribose analogues exhibited a substantial decrease in ydaO affinity but retained high affinity for GdpP. These ligand modifications resulted in increased resistance to enzyme-catalyzed hydrolysis by the GdpP enzyme. Together, these data suggest that these c-di-AMP analogues could be useful as chemical tools to specifically target subsections of second-messenger signaling pathways

The expanded H1–H8/H2/H6 and H1–H1 regions of the exchangeable proton 2D JR-NOESY spectrum of Tel26 in K solution at 1°C

<p><b>Copyright information:</b></p><p>Taken from "Human telomeric sequence forms a hybrid-type intramolecular G-quadruplex structure with mixed parallel/antiparallel strands in potassium solution"</p><p>Nucleic Acids Research 2006;34(9):2723-2735.</p><p>Published online 19 May 2006</p><p>PMCID:PMC1464114.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> NOEs are labeled as follows: intra-tetrad NOEs are labeled in red, sequential NOEs are labeled in green, inter-tetrad NOEs are labeled in blue, NOEs between stacking adenine H2 protons and G-tetrad imino protons are labeled in black. Conditions: 1°C, 25 mM K-PO, 70 mM KCl, pH 7.0, 2.5 mM DNA