Hydrogermylation of 5‑Ethynyluracil Nucleosides: Formation of 5‑(2-Germylvinyl)uracil and 5‑(2-Germylacetyl)uracil Nucleosides

A stereoselective radical-mediated hydrogermylation of the protected 5-ethynyluracil nucleosides with trialkyl-, triaryl,- or tris­(trimethylsilyl)­germanes gave (<i>Z</i>)<i>-</i>5-(2-germylvinyl)­uridine, 2′-deoxyuridine, or <i>ara</i>-uridine as major products. Reaction of the β-triphenylgermyl vinyl radical intermediate with oxygen and fragmentation of the resulting peroxyradical provided also 5-[2-(triphenylgermyl)­acetyl]­pyrimidine nucleosides in low to moderate yields. Thermal isomerization of the latter in MeOH occurred via a four-centered activated complex, and subsequent hydrolysis of the resulting <i>O-</i>germyl substituted enol yielded 5-acetyluracil nucleosides in quantitative yield

Formation of Ternary Complexes with MgATP: Effects on the Detection of Mg²⁺ in Biological Samples by Bidentate Fluorescent Sensors

Fluorescent indicators based on β-keto-acid bidentate coordination motifs display superior metal selectivity profiles compared to current <i>o</i>-aminophenol-<i>N</i>,<i>N</i>,<i>O</i>-triacetic acid (APTRA) based chelators for the study of biological magnesium. These low denticity chelators, however, may allow for the formation of ternary complexes with Mg²⁺ and common ligands present in the cellular milieu. In this work, absorption, fluorescence, and NMR spectroscopy were employed to study the interaction of turn-on and ratiometric fluorescent indicators based on 4-oxo-4H-quinolizine-3-carboxylic acid with Mg²⁺ and ATP, the most abundant chelator of biological magnesium, thus revealing the formation of ternary complexes under conditions relevant to fluorescence imaging. The formation of ternary species elicits comparable or greater optical changes than those attributed to the formation of binary complexes alone. Dissociation of the fluorescent indicators from both ternary and binary species have apparent equilibrium constants in the low millimolar range at pH 7 and 25 °C. These results suggest that these bidentate sensors are incapable of distinguishing between free Mg²⁺ and MgATP based on ratio or intensity-based steady-state fluorescence measurements, thus posing challenges in the interpretation of results from fluorescence imaging of magnesium in nucleotide-rich biological samples

Thermodynamic Switch in Binding of Adhesion/Growth Regulatory Human Galectin‑3 to Tumor-Associated TF Antigen (CD176) and MUC1 Glycopeptides

A shift to short-chain glycans is an observed change in mucin-type O-glycosylation in premalignant and malignant epithelia. Given the evidence that human galectin-3 can interact with mucins and also weakly with free tumor-associated Thomsen-Friedenreich (TF) antigen (CD176), the study of its interaction with MUC1 (glyco)­peptides is of biomedical relevance. Glycosylated MUC1 fragments that carry the TF antigen attached through either Thr or Ser side chains were synthesized using standard Fmoc-based automated solid-phase peptide chemistry. The dissociation constants (<i>K</i>_d) for interaction of galectin-3 and the glycosylated MUC1 fragments measured by isothermal titration calorimetry decreased up to 10 times in comparison to that of the free TF disaccharide. No binding was observed for the nonglycosylated control version of the MUC1 peptide. The most notable feature of the binding of MUC1 glycopeptides to galectin-3 was a shift from a favorable enthalpy to an entropy-driven binding process. The comparatively diminished enthalpy contribution to the free energy (Δ<i>G</i>) was compensated by a considerable gain in the entropic term. ¹H–¹⁵N heteronuclear single-quantum coherence spectroscopy nuclear magnetic resonance data reveal contact at the canonical site mainly by the glycan moiety of the MUC1 glycopeptide. Ligand-dependent differences in binding affinities were also confirmed by a novel assay for screening of low-affinity glycan–lectin interactions based on AlphaScreen technology. Another key finding is that the glycosylated MUC1 peptides exhibited activity in a concentration-dependent manner in cell-based assays revealing selectivity among human galectins. Thus, the presentation of this tumor-associated carbohydrate ligand by the natural peptide scaffold enhances its affinity, highlighting the significance of model studies of human lectins with synthetic glycopeptides