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A compact and negatively charged acceptor group, N‐(cyanamino)sulfonyl, is introduced for dye design and its influence on the absorption and emission spectra of the “push–pull” chromophores is demonstrated with 1,3,6‐tris[(cyanamino)sulfonyl]‐8‐aminopyrene. The new sulfonamides, including O‐phosphorylated (3‐hydroxyazetidine)‐N‐sulfonyl, are negatively charged electron acceptors and auxochromes. 1‐Aminopyrenes decorated with the new sulfonamides have three or six negative charges (pH ≥8), low m/z ratios, high mobilities in an electric field, and yellow to orange emission. We labeled maltodextrin oligomers by reductive amination, separated the products by electrophoresis, and demonstrated their high brightness in a commercial DNA analyzer and the distribution of the emission signal among the detection channels., Negatively charged electron acceptors and auxochromes? New sulfonamides make it possible! 1‐Aminopyrenes decorated with the new sulfonamides had low m/z ratios and high mobility in an electric field. Their glycoconjugates with reducing sugars were separated and detected at sub‐picomolar concentration by multicolor emission in a DNA analyzer (see picture).

The nanometer thickness of filaments and the dynamic behavior of actin – a protein playing crucial role in cellular function and motility – makes it attractive for observation with superresolution optical microscopy. We developed the solution-phase synthesis of des-bromo-des-methyl-jasplakinolide-lysine, used as the “recognition unit” (ligand) for F-actin in living cells. The first amino acid – Fmoc-O-TIPS-beta-tyrosine – was prepared in 78% yield (2 steps in one pot). The new solution-phase synthesis involves 2-phenylisopropyl protection of the carboxyl group and does not require excesses of commercially unavailable amino acids. The overall yield of the target intermediate obtained in 9 steps is about 8%. 2-Phenylisopropyl group can be cleaved from carboxyl with 2 – 3% (v/v) of TFA in acetonitrile (0-10°C), without affecting TIPS protection of the phenolic hydroxyl in beta-tyrosine and N-Boc protection in lysine. Des-bromo-des-methyl-jasplakinolide-lysine was coupled with red-emitting fluorescent dyes 580CP and 610CP (via 6-aminohexanoate linker). Actin in living cells was labeled with 580CP and 610CP probes, and the optical resolution measured as full width at half maximum of line profiles across actin fibers was found to be 300-400 nm and 100 nm under confocal and STED conditions, respectively. The solution-phase synthesis of des-bromo-des-methyl-jasplakinolide-lysine opens a way to better fluorescent probes perspective for actin imaging.

The capillary gel electrophoresis with laser-induced fluorescence detection (CGE-LIF) has become a key method in high-throughput glycan analysis. At present, CGE-LIF relies on 8-aminopyrene-1,3,6-trisulfonic acid (APTS). However, APTS has moderate reactivity in labeling of glycans, fixed selectivity profile and renders the detection to only green color. Here, we report synthesis of red-emitting and highly reactive fluo-rescent tags for glycan derivatization. The design is based on a 9-aminoacridine scaffold with various acceptor groups at C-2 (CN, SO2R) and a primary amino group at C-7 for conjugation via reductive amination. These reactive dyes exhibit absorption maxima close to 450 nm and emis-sion above 600 nm. They readily undergo conjugation with reducing sugars at the desired 1:1 stoichiometry. The red emission of conjugates with a maximum at 610-630 nm can be observed under excitation with 488 nm light and detected separately from the APTS-labeled oligosaccharides. Phosphorylated 7,9-diaminoacridine-2-SO2R derivatives with variable amounts of negative charges provide high mobilities of glycoconjugates on polyacrylamide gel electrophoresis (PAGE), as compared to those of APTS. We further demonstrate their utility by labeling and separating a maltodextrin ladder and sialyllactose isomers. The new dyes are expected to cross-validate and increase the glycan identification precision in CGE-LIF and help to reveal "heavy" glycans, yet undetectable with APTS label.

1‐Aminopyrenes with three ω‐hydroxylated N‐alkylsulfonamido or alkylsulfonyl residues in positions 3, 6, and 8 were prepared, O‐phosphorylated, and applied for reductive amination of oligosaccharides. The dyes (ϵ≈20 000 m −1 cm−1) with six negative charges (pH≥8) and low m/z ratios enable labeling and fluorescence detection of reducing sugars (glycans) related to the most structurally and functionally diverse class of natural products. Under excitation with a 488 nm laser, the new glycoconjugates emit yellow light of about 560 nm, outperforming (with respect to brightness and faster electrophoretic mobilities) the corresponding APTS derivatives (benchmark dye with green emission in conjugates)., Bright 1‐aminopyrenes (ϵ≈20.000 m −1 cm−1) with six negative charges (at pH≥8) and high mobility in the electric field are designed for labeling and fluorescence detection of glycans. The right and left lanes in the figure show the gel electrophoresis bands of new and reference dyes (lowest bands) and their conjugates.

5-Formylcytosin (5fC) und 5-Formyluracil (5fU) sind naturliche modifizierte Nucleobasen, die durch Oxidation aus 5-Methylcytosin und Thymin (oder 5-Methyluracil) entstehen. Hier beschreiben wir die chemoselektive Markierung von 5-Formylpyrimidinnucleotiden in DNA und RNA mithilfe fluorogener Aldolkondensationsreaktionen mit 2,3,3-Trimethylindolderivaten. Unter milden und spezifischen Reaktionsbedingungen entstehen aus 5fU und 5fC Hemicyanin-artige Chromophore mit charakteristischen photophysikalischen Eigenschaften. Die Reaktionsbedingungen wurden an DNA-Oligonucleotiden optimiert und konnten direkt auf RNA ubertragen werden. Der positionsspezifische Nachweis gelang durch Fluoreszenzmessung und Primerverlangerungsexperimente. Diese Methode der direkten Markierung von 5-Formylpyrimidinen wird zur Untersuchung von Vorkommen, enzymatischen Transformationen und biologischen Funktionen von epigenetischen/epitranskriptomischen Modifikationen von Nucleobasen in DNA und RNA beitragen.

5-Formylcytosine (5fC) and 5-formyluracil (5fU) are natural nucleobase modifications that are generated by oxidative modification of 5-methylcytosine and thymine (or 5-methyluracil). Herein, we describe chemoselective labeling of 5-formylpyrimidine nucleotides in DNA and RNA by fluorogenic aldol-type condensation reactions with 2,3,3-trimethylindole derivatives. Mild and specific reaction conditions were developed for 5fU and 5fC to produce hemicyanine-like chromophores with distinct photophysical properties. Residue-specific detection was established by fluorescence readout as well as primer-extension assays. The reactions were optimized on DNA oligonucleotides and were equally suitable for the modification of 5fU- and 5fC-modified RNA. This direct labeling approach of 5-formylpyrimidines is expected to help in elucidating the occurrence, enzymatic transformations, and functional roles of these epigenetic/epitranscriptomic nucleobase modifications in DNA and RNA.

Nucleolytic ribozymes catalyze site-specific cleavage of their phosphodiester backbones. A minimal version of the twister ribozyme is reported that lacks the phylogenetically conserved stem P1 while retaining wild-type activity. Atomic mutagenesis revealed that nitrogen atoms N1 and N3 of the adenine-6 at the cleavage site are indispensable for cleavage. By NMR spectroscopy, a pKa value of 5.1 was determined for a (13) C2-labeled adenine at this position in the twister ribozyme, which is significantly shifted compared to the pKa of the same adenine in the substrate alone. This finding pinpoints at a potential role for adenine-6 in the catalytic mechanism besides the previously identified invariant guanine-48 and a Mg(2+) ion, both of which are directly coordinated to the non-bridging oxygen atoms of the scissile phosphate; for the latter, additional evidence stems from the observation that Mn(2+) or Cd(2+) accelerated cleavage of phosphorothioate substrates. The relevance of this metal ion binding site is further emphasized by a new 2.6 Å X-ray structure of a 2'-OCH3 -U5 modified twister ribozyme.

Distance measurements in RNAs by pulse EPR with TEMPO-labeled nucleotides allow for model free conversion of distances into base-pair separation., Structural information at atomic resolution of biomolecular assemblies, such as RNA and RNA protein complexes, is fundamental to comprehend biological function. Modern spectroscopic methods offer exceptional opportunities in this direction. Here we present the capability of pulse EPR to report high-resolution long-range distances in RNAs by means of a recently developed spin labeled nucleotide, which carries the TEMPO group directly attached to the nucleobase and preserves Watson–Crick base-pairing. In a representative RNA duplex with spin-label separations up to 28 base pairs (≈8 nm) we demonstrate that the label allows for a model-free conversion of inter-spin distances into base-pair separation (Δbp) if broad-band pulse excitation at Q band frequencies (34 GHz) is applied. The observed distance distribution increases from ±0.2 nm for Δbp = 10 to only ±0.5 nm for Δbp = 28, consistent with only small deviations from the “ideal” A-form RNA structure. Molecular dynamics (MD) simulations conducted at 20 °C show restricted conformational freedom of the label. MD-generated structural deviations from an “ideal” A-RNA geometry help disentangle the contributions of local flexibility of the label and its neighboring nucleobases and global deformations of the RNA double helix to the experimental distance distributions. The study demonstrates that our simple but strategic spin labeling procedure can access detailed structural information on RNAs at atomic resolution over distances that match the size of macromolecular RNA complexes.