The Shiga toxin B subunit (STxB), which is involved in cell membrane attachment and trafficking of Shiga holotoxin, binds specifically to the glycosphingolipid Gb 3. In biological membranes, Gb 3 glycosphingolipids differ in their fatty acid composition and there is strong evidence that the fatty acid alters the binding behaviour of STxB as well as the intracellular routing of the Shiga toxin/Gb 3 complex. To analyse the binding of STxB to different Gb 3 s, we chemically synthesized saturated, unsaturated, a-hydroxylated Gb 3 s and a combination thereof, all based on a C 24-fatty acid chain starting from monosaccharide building blocks, sphingosine and the respective fatty acids. These chemically well-defined Gb 3 s were inserted into solid supported phase-separated lipid bilayers composed of DOPC/sphingomyelin/cholesterol as a simple mimetic of the outer leaflet of animal cell membranes. By fluorescenceand atomic force microscopy the phase behaviour of the bilayer as well as the lateral organization of bound STxB were analysed. The fatty acid of Gb 3 significantly alters the ratio between the ordered and disordered phase and induces a third intermediate phase in the presence of unsaturated Gb 3. The lateral organization of STxB on the membranes varies significantly. While STxB attached to membranes with Gb 3 s with saturated fatty acids forms protein clusters, it is more homogeneously bound to membranes containing unsaturated Gb 3 s. Large interphase lipid redistribution is observed for a-hydroxylated Gb 3 doped membranes. Our results clearly demonstrate that the fatty acid of Gb 3 strongly influences the lateral organization of STxB on the membrane and impacts the overall membrane organization of phase-separated lipid membranes.

A versatile and rapid access to various chain lengths of ethylene-bridged BODIPY motifs was discovered. Corresponding oligomers comprising up to eight monomeric units were studied with respect to their microstructures by photophysical, X-ray crystallographic, and computational means. The investigation of three different dipyrrin cores revealed a crucial dependence on the substitution pattern of the core, whereas the nature of the meso-periphery is less critical. The impact of substituent effects on the conformational space was investigated by Monte Carlo simulations and a set of DFT methods (B3LYP, PBEh-3c, TPSS/PWPB95), including dispersion effects. Cryptopyrrole-derived oligo-BODIPYs are characterized by a tight intramolecular arrangement triggering a dominant J-type excitonic coupling with red-shifts up to 45 nm, exceptionally small line widths of the absorption and emission event (up to 286 cm), outstandingly high attenuation coefficients (up to 1 042 000 M cm), and quantum yields of up to unity.

Shiga toxin subunit B (STxB) binding to its cellular receptor Gb3 leads to the formation of protein-lipid clusters and bending of the membrane. A newly developed synthetic route allowed synthesizing the biologically most relevant Gb3-C24:1 2OH species with both, the natural (Gb3-R) as well as the unnatural (Gb3-S) configuration of the 2OH group. The derivatives bind STxB with identical nanomolar affinity, while the propensity to induce membrane tubules in giant unilamellar vesicles is more pronounced for Gb3-S. Fluorescence and atomic force microscopy images of phase-separated supported membranes revealed differences in the lateral organization of the protein on the membrane. Gb3-R favorably induces large and tightly packed protein clusters, while a lower protein density is found on Gb3-S doped membranes.

A fundamental, highly fluorescent, and easily accessible scaffold derived from the BODIPY core is reported. The use of benzimidazole as a bridging ligand at the meso position enables the binding of two BF units to provide sufficient rigidity and enhanced electron-withdrawing strength. Absorption and emission events thus take place in the red (λ≈600 nm); the fluorescence quantum yields can reach unity (0.96) and show little dependence on solvent polarity. The synthetic route was shortened to two steps starting from commercially available precursors while the preparation is modular and tolerates various pyrrole and benzimidazole moieties. Fluoride replacement by propynyl groups, various halogenations, as well as Knoevenagel-type condensations were applied to extend the versatility of these new photostable fluorophores, which we termed BOIMPYs.

The use of photoactivatable dyes in STED microscopy has so far been limited by two-photon activation throught he STED beam and by the fact that photoactivatable dyesa re poorly solvable in water.H erein, we report ONB-2SiR, af luorophore that can be both photoactivated in the UV and specifically de-excited by STED at 775 nm. Likewise , we introduce ac onjugation and purification protocol to effectively label primary ands econdary antibodies with moderately water-soluble dyes. Greatly reducing dyea ggregation,o ur technique provides ad efineda nd tunable degree of labeling, and improves the imagingp erformance of dye conjugates in general.

Reactions of 2-arylcyclopropane dicarboxylates with naphthoquinones are reported. The key feature was the use of catalytic amounts of SnCl , which acts as both an electron donor and a Lewis acid. By an in situ umpolung of naphthoquinone the formerly electrophilic species is converted into a nucleophile that is able to trigger the ring-opening of the three-membered ring with formation of a new C-C bond. Treatment of these products with base under oxidative conditions resulted-through loss of methyl formate-in cyclopentannulated products with fully conjugated π systems exhibiting intensive absorptions in the visible range.

We present a straightforward, three‐step synthesis engaging an oligomerization and subsequent one‐pot oxidation step to form fully conjugated, benzene‐fused oligo‐BODIPYs from simple BODIPY precursors. FeCl3 serves as an efficient, bifunctional oxidant for a (multiple) cyclization/desaturation process, applied to ethylene‐bridged dimeric, trimeric and oligomeric species to transform linking ethano units into stiff benzene fusions between unsubstituted β‐positions of each BODIPY unit. The structural integrity was verified by X‐ray crystallography, and all target compounds were studied in detail by photophysical, electrochemical and computational means. The main S1 excited state gradually converges to a structure‐specific excitation limit, displaying a strong shift of the absorption event from about 500 nm (BODIPY monomer) to 955 nm (octamer) with attenuation coefficients up to ca. 500 000 M−1 cm−1.

The two lectins LecA from Pseudomonas aeruginosa and the B-subunit of Shiga toxin from Shigella dysenteriae (StxB) share the glycosphingolipid globotriaosylceramide (Gb3) as receptor. Counterintuitively, we found that LecA and StxB segregated into different domains after recognizing Gb3 at the plasma membrane of cells. We hypothesized that the orientation of the carbohydrate head group of Gb3 embedded in the lipid bilayer differentially influences LecA and StxB binding. To test this hypothesis, we reconstituted lectin-Gb3 interaction using giant unilamellar vesicles and were indeed able to rebuild LecA and StxB segregation. Both, the Gb3 fatty acyl chain structure and the local membrane environment, modulated Gb3 recognition by LecA and StxB. Specifically, StxB preferred more ordered membranes compared to LecA. Based on our findings, we propose comparing staining patterns of LecA and StxB as an alternative method to assess membrane order in cells. To verify this approach, we re-established that the apical plasma membrane of epithelial cells is more ordered than the basolateral plasma membrane. Additionally, we found that StxB recognized Gb3 at the primary cilium and the periciliary membrane, whereas LecA only bound periciliary Gb3. This suggests that the ciliary membrane is of higher order than the surrounding periciliary membrane.