Deciphering fucosylated protein-linked O-glycans in oral Tannerella serpentiformis: insights from NMR spectroscopy and glycoproteomics.

Tannerella serpentiformis is a health-associated Gram-negative oral anaerobe, while its closest phylogenetic relative is the periodontal pathogen Tannerella forsythia. The pathogen employs glycan mimicry through protein O-glycosylation, displaying a terminal nonulosonic acid aiding in evasion of host immune recognition. Like T. forsythia, T. serpentiformis cells are covered with a 2D-crystalline S-layer composed of two abundant S-layer glycoproteins-TssA and TssB. In this study, we elucidated the structure of the O-linked glycans of T. serpentiformis using 1D and 2D NMR spectroscopy analyzing S-layer glycopeptides and ß-eliminated glycans. We found that T. serpentiformis produces two highly fucosylated, branched glycoforms carrying non-carbohydrate modifications, with the structure [2-OMe-Fuc-(α1,2)]-4-OMe-Glc-(ß1,3)-[Fuc-(α1,4)]-2-NAc-GlcA-(ß1,4)-[3-NH2, 2,4-OMe-Fuc-(α1,3)]-Fuc-(α1,4)-Xyl-(ß1,4)-[3-OMe-Fuc-(α1,3)]-GlcA-(α1,2)-[Rha-(α1,4]-Gal, where the 3OMe-Fuc is variable; each glycoform contains a rare 2,4-methoxy, 3-amino-modified fucose. These glycoforms support the hypothesis that nonulosonic acid is a hallmark of pathogenic Tannerella species. A combined glycoproteomics and bioinformatics approach identified multiple sites within TssA (14 sites) and TssB (21 sites) to be O-glycosylated. LC-MS/MS confirmed the presence of the Bacteroidetes O-glycosylation motif (D)(S/T) (L/V/T/A/I) in Tannerella species, including the newly identified candidate "N" for the third position. Alphfold2 models of the S-layer glycoproteins were created revealing an almost uniform spatial distribution of the two glycoforms at the N-terminal two thirds of the proteins supported by glycoproteomics, with glycans facing outward. Glycoproteomics identified 921 unique glycopeptide sequences corresponding to 303 unique UniProt IDs. GO-term enrichment analysis versus the entire T. serpentiformis proteome classified these proteins as mainly membrane and cell periphery-associated glycoproteins, supporting a general protein O-glycosylation system in T. serpentiformis.

Decorating phenylalanine side-chains with triple labeled 13C/19F/2H isotope patterns.

We present an economic and straightforward method to introduce 13C-19F spin systems into the deuterated aromatic side chains of phenylalanine as reporters for various protein NMR applications. The method is based on the synthesis of [4-13C, 2,3,5,6-2H4] 4-fluorophenylalanine from the commercially available isotope sources [2-13C] acetone and deuterium oxide. This compound is readily metabolized by standard Escherichia coli overexpression in a glyphosate-containing minimal medium, which results in high incorporation rates in the corresponding target proteins.

Double Chalcogen Bonding Recognition Arrays in Solution.

N-substituted pyridino-based congeners of Ebselen, named here as Pyrselen, incorporating proximal Se and N atoms, undergo dimerization in solution and in the solid state through a dual donor-acceptor arrangement of chalcogen bonding sites. Dimerization constants were measured within the 15-50 M-1 range. Computational studies on the dimers depict a notable charge-transfer contribution to the association, validating Pyrselen as an effective scaffold for designing chalcogen-bonding-based recognition motifs. Insert abstract text here.

Constraining and Modifying Peptides Using Pd-Mediated Cysteine Allylation.

Over the past decades, several strategies for inducing and stabilizing secondary structure formation in peptides have been developed to increase their proteolytic stability and their binding affinity to specific interaction partners. Here, we report how our recently introduced chemoselective Pd-catalyzed cysteine allylation reaction can be extended to stapling and how the resulting alkene-containing staples themselves can be further modified to introduce additional probes into such stabilized peptides. The latter is demonstrated by introducing a fluorophore as well as a PEG moiety into different stapled peptides using bioorthogonal thiol-ene and Diels-Alder reactions. Furthermore, we investigated structural implications of our allyl staples when used to replace conformationally relevant disulfide bridges. To this end, we chose a selective binder of integrin α3 ß1 (LXY3), which is only active in its cyclic disulfide form. We replaced the disulfide bridge by different stapling reagents in order to increase stability and binding affinity towards integrin α3 ß1 .

Synthesis of 4-Deoxy-4-Fluoro-d-Sedoheptulose: A Promising New Sugar to Apply the Principle of Metabolic Trapping.

Fluorinated carbohydrates are important tools for understanding the deregulation of metabolic fluxes and pathways. Fluorinating specific positions within the sugar scaffold can lead to enhanced metabolic stability and subsequent metabolic trapping in cells. This principle has, however, never been applied to study the metabolism of the rare sugars of the pentose phosphate pathway (PPP). In this study, two fluorinated derivatives of d-sedoheptulose were designed and synthesized: 4-deoxy-4-fluoro-d-sedoheptulose (4DFS) and 3-deoxy-3-fluoro-d-sedoheptulose (3DFS). Both sugars are taken up by human fibroblasts but only 4DFS is phosphorylated. Fluorination of d-sedoheptulose at C-4 effectively halts the enzymatic degradation by transaldolase and transketolase. 4DFS thus has a high potential as a new PPP imaging probe based on the principle of metabolic trapping. Therefore, the synthesis of potential radiolabeling precursors for 4DFS for future radiofluorinations with fluorine-18 is presented.

Supramolecular Chalcogen-Bonded Semiconducting Nanoribbons at Work in Lighting Devices.

This work describes the design and synthesis of a π-conjugated telluro[3,2-ß][1]-tellurophene-based synthon that, embodying pyridyl and haloaryl chalcogen-bonding acceptors, self-assembles into nanoribbons through chalcogen bonds. The ribbons π-stack in a multi-layered architecture both in single crystals and thin films. Theoretical studies of the electronic states of chalcogen-bonded material showed the presence of a local charge density between Te and N atoms. OTFT-based charge transport measurements showed hole-transport properties for this material. Its integration as a p-type semiconductor in multi-layered CuI -based light-emitting electrochemical cells (LECs) led to a 10-fold increase in stability (38 h vs. 3 h) compared to single-layered devices. Finally, using the reference tellurotellurophene congener bearing a C-H group instead of the pyridyl N atom, a herringbone solid-state assembly is formed without charge transport features, resulting in LECs with poor stabilities (<1 h).

Evaporation-Induced Self-Assembly of Small Peptide-Conjugated Silica Nanoparticles.

Self-assembly processes guide disordered molecules or particles into long-range organized structures due to specific supramolecular interactions among the building entities. Herein, we report a unique evaporation-induced self-assembly (EISA) strategy for four different silica nanoparticle systems obtained through peptide functionalization of the particle surface. First, covalent peptide-silica coupling was investigated in detail, starting with the grafting of a single amino acid (L-serine) and expanded to specific small peptides (up to four amino acids) and transferred to different particle types (MCM-48-type MSNs, solid nanoparticles, and newly developed virus-like nanoparticles). These materials were investigated regarding their ability to undergo EISA, which was shown to be independent of particle type and amount of peptide anchored to their surface. This EISA-based approach provides new possibilities for the design of future advanced drug delivery systems, engineered hierarchical sorbents, and nanocatalyst assemblies.

A Many-Faced Alkaloid: Polymorphism of (-)-Monophyllidin.

The synthesis of the alkaloid (-)-monophyllidin is described. The molecule is a hybrid of xanthoxyline and (S)-proline, accessible in one-step through a Mannich reaction. In the solid-state, defined structural arrangements with different physical properties are formed. Single crystal X-ray diffraction revealed structures of six distinct polymorphs. In the crystalline state, the alkaloid can host small polar molecules (preferably water), while the (S)-proline moiety is present in the zwitterionic state. Combined with the chelate, which is already present in the xanthoxyline substructure, an ideal disposition for multiple hydrogen bond networks evolve. Therefore, highly water-soluble polymorphs of monophyllidin can form. This structural flexibility explains the many faces of the molecule in terms of structure as well as analytical data. Furthermore, speculations about the biological role of the molecule, with regard to the manifold interactions with water, are presented.

Methylated Xanthones from the Rootlets of Metaxya rostrata Display Cytotoxic Activity in Colorectal Cancer Cells.

The tree fern Metaxya rostrata (Kunth) C. Presl is common in the rainforests of Central and South America, where suspensions of the dried rhizome are traditionally used to treat intestinal diseases. Two compounds from this plant, 2-deprenyl-rheediaxanthone B (XB) and 2-deprenyl-7-hydroxy-rheediaxanthone B (OH-XB), have been shown to be biologically highly active against colorectal cancer (CRC) cells in previous studies. The current investigation resulted in the isolation of the previously undescribed methylated xanthones 2-deprenyl-6-O-methyl-7-hydroxy-rheediaxanthone B, 2-deprenyl-5-O-methyl-7-methoxy-rheediaxanthone B, 2-deprenyl-5-O-methyl- 7-hydroxy-rheediaxanthone B and 2-deprenyl-7-methoxy-rheediaxanthone B. All compounds were isolated by column chromatography, structures were elucidated by one- and two-dimensional NMR-experiments and the identities of the compounds were confirmed by LC-HRMS. In logarithmically growing SW480 CRC cell cultures, cytotoxicity by neutral red uptake and MTT assays as well as caspase activation was analyzed. Cellular targets were examined by Western blot, and topoisomerase I (topo I) inhibition potential was tested. Comparing the structure-activity relationship with XB and OH-XB, the monomethylated derivatives showed qualitatively similar effects/mechanisms to their nonmethylated analogues, while dimethylation almost abolished the activity. Inhibition of topo I was dependent on the presence of an unmethylated 7-OH group.

Waste-Derived Low-Cost Mycelium Nanopapers with Tunable Mechanical and Surface Properties.

Mycelium, the vegetative growth of filamentous fungi, has attracted increasing commercial and academic interest in recent years because of its ability to upcycle agricultural and industrial wastes into low-cost, sustainable composite materials. However, mycelium composites typically exhibit foam-like mechanical properties, primarily originating from their weak organic filler constituents. Fungal growth can be alternatively utilized as a low-cost method for on-demand generation of natural nanofibrils, such as chitin and chitosan, which can be grown and isolated from liquid wastes and byproducts in the form of fungal microfilaments. This study characterized polymer extracts and nanopapers produced from a common mushroom reference and various species of fungal mycelium grown on sugarcane byproduct molasses. Polymer yields of ∼10-26% were achieved, which are comparable to those of crustacean-derived chitin, and the nanopapers produced exhibited much higher tensile strengths than the existing mycelium materials, with values of up to ∼25 MPa (mycelium) and ∼98 MPa (mushroom), in addition to useful hydrophobic surface properties resulting from the presence of organic lipid residues in the nanopapers. HCl or H2O2 treatments were used to remove these impurities facilitating tuning of mechanical, thermal, and surface properties of the nanopapers produced. This potentially enables their use in a wide range of applications including coatings, membranes, packaging, and paper.

Indium-mediated C-allylation of melibiose.

The indium-mediated allylation reaction has been applied to melibiose, a disaccharidic substrate. This elongation methodology allows for a short, efficient and diastereoselective approach towards complex glycosylated carbohydrate structures. The stereochemical outcome of the key intermediates, allylated disaccharides, has been determined by X-ray analysis. Ozonolysis of the introduced double bond yielded the unprotected elongated disaccharides in the equilibrium of the pyranoid as well as furanoid isomers in both anomeric forms, respectively. Per-O-acetylation has been performed to facilitate separation of the isomeric mixture for structural identification. The main product revealed to adopt a ß-pyranoid form of the elongated unit at the reducing end of the disaccharide.

Acetylated Furostene Glycosides from Solanum gilo Fruits.

In continuation of our work on a traditional mixture of spices called "Nkui", used in Cameroon for its influence on women's reproductive health, we investigated the chemical composition of Solanum gilo, one component of "Nkui". A methanolic extract was studied in detail. After dereplication of several known compounds, two furo-5-stene-derived saponin glycosides with acetylated sugar moieties were isolated. By extensive 1- and 2D NMR experiments and HR-MS and GC-MS methods, the structures were elucidated as 26-[(3‴,4‴,6‴-tri-O-acetyl)-ß-D-glucopyranosyloxy]-22-hydroxyfurost-5-en-3ß-yl-O-α-L-rhamnopyranosyl-(1″→2')-ß-D-glucopyranoside (A) and 26-[(3‴,4‴,6‴-tri-O-acetyl)-ß-D-glucopyranosyloxy]-22-hydroxyfurost-5-en-3ß-yl-[O-α-L-rhamnopyranosyl-(1''''→4')-O-α-L-rhamnopyranosyl-(1″→2')]-ß-D-glucopyranoside (B), both new natural compounds.

Arginine side-chain modification that occurs during copper-catalysed azide-alkyne click reactions resembles an advanced glycation end product.

Dehydroascorbate is a by-product of copper-catalysed azide-alkyne click (CuAAC) reactions and also forms advanced glycation end products (AGEs) in tissues undergoing oxidative stress. Here we isolate and characterize an arginine-dehydroascorbate adduct formed during CuAAC reactions, investigate strategies for preventing its formation, and propose its biological relevance as an AGE.

Stereoselective gold(I) domino catalysis of allylic isomerization and olefin cyclopropanation: mechanistic studies.

Gold(I) catalysis of olefin activation is still a rare feature in the repertoire of that metal. Mechanistic studies on the gold(I)-catalyzed cyclopropanation of allyl-substituted sulfonium ylides, including kinetic analysis as well as detailed computational studies, reveal that the reaction proceeds through activation of the alkene moiety. Furthermore, a novel competitive allylic isomerization pathway that interconverts "linear" and "branched" allylic isomers is uncovered. The subtle interplay of cyclopropanation and olefin isomerization results in an intriguing domino process where two independent catalytic transformations combine with near-perfect regio- and stereoselectivities.

Intermolecular Reactions of a Foiled Carbene with Carbonyl Compounds: The Effects of Trishomocyclopropyl Stabilization.

endo-Tricyclo[3.2.1.0(2,4)]oct-8-ylidene is a foiled carbene reaction intermediate. It was generated by thermolyzing Δ(3)-1,3,4-oxadiazoline precursors dissolved in benzaldehyde and acetophenone. The products appear to stem from direct insertion of the carbene's divalent C atom into the α-bonds of the carbonyl compounds; however, this is only superficial. The strict stereochemistry observed is due to the topologies of the reaction intermediates of the proposed two-step mechanism. Bimolecular nucleophilic addition generates bent 1,3-zwitterions. The neutral reaction intermediates undergo pinacolic rearrangements to form the observed adducts. Product ratios reflect the migratory aptitudes of the carbonyl compounds' α-substituents. The carbene reaction was modeled using DFT. The singlet carbene's bicoordinate C atom bends 31° toward the endo-fused cyclopropane bond, elongating it to r = 1.69 Å. The resulting trishomocyclopropyl HOMO{-1} is a three-center two-electron bond responsible for the electron-deficient carbene's nucleophilicity. Its calculated properties are consistent with this assertion: (1) singlet-triplet (ΔE(S-T)) energy gap of -25 kcal/mol, (2) gas-phase proton affinity (PA) value of 272 kcal/mol, (3) hard and soft acid and base (HSAB) ΔN value of -0.2 in its initial reaction with the carbonyl compounds, and (4) negative frontier orbital interaction values ΔΔE(PhC(O)H) = -4.38 eV and ΔΔE(PhC(O)Me) = -3.97 eV.

Hybrids of salicylalkylamides and Mannich bases: control of the amide conformation by hydrogen bonding in solution and in the solid state.

3-Aminomethylation of salicylalkylamides afforded hybrids with a Mannich base. In addition, it triggered the rotation of the amide bond. The observed conformational switch is driven by strong intramolecular hydrogen bonding between the Mannich base and phenolic group. Crystal structure analysis reveals the stabilization of the hybrid molecules by double hydrogen bonding of the phenolic OH, which acts as an acceptor and donor simultaneously. The molecules contain an amide site and a Mannich base site in an orthogonal spatial arrangement. The intramolecular hydrogen bonds are persistent in a nonpolar solvent (e.g., chloroform). The conformational change can be reversed upon protection or protonation of the Mannich base nitrogen.

Indium-mediated allylation in carbohydrate synthesis: A short and efficient approach towards higher 2-acetamido-2-deoxy sugars.

Higher aminosugars are interesting targets in carbohydrate synthesis since these compounds play important roles in biological systems. However, their availability from natural sources is limited. Thus, in order to investigate their biological function, the development of facile and adaptable routes to this class of compounds is of fundamental importance. Our synthetic route towards these target molecules makes use of readily accessible pentoses and hexoses, which are subjected to indium-mediated two-carbon chain elongation. Subsequent ozonolysis and treatment with base yields α,ß-unsaturated aldehydes, which are stereoselectively epoxidized using Jørgenson's protocol. After Wittig chain elongation the obtained allylic epoxides were regio- and stereoselectively opened with trimethylsilyl azide under palladium catalysis. Finally, a suitable deprotection protocol, starting with acidic acetate cleavage and ozonolysis was established. Peracetylation of the products simplifies purification and subsequent azide reduction followed by final deacetylation using methanolic sodium methoxide furnishes the title compounds.

Silica nanoparticle conjugation with gallic acid towards enhanced free radical scavenging capacity and activity on osteosarcoma cells in vitro.

Gallic acid (GA), derived from land plants, possesses diverse physiological benefits, including anti-inflammatory and anticancer effects, making it valuable for biomedical applications. In this study, GA was used to modify the surface of dendritic mesoporous silica nanoparticles (DMSNs) via carbamate (DMSN-NCO-GA) or amide (DMSN-NH-GA) bonds, using a post-grafting technique. To explore GA-conjugated materials' potential in modulating cancer cell redox status, three variants of osteosarcoma cells (U2-OS) were used. These variants comprised the wild-type cells (NEO), the cells overexpressing the wild-type human Golgi anti-apoptotic protein (hGAAP), and the null mutant of hGAAP (Ct-mut), as this protein was previously demonstrated to play a role in intracellular reactive oxygen species (ROS) accumulation and cell migration. In the absence of external ROS triggers, non-modified DMSNs increased intracellular ROS in Ct-mut and NEO cells, while GA-conjugated materials, particularly DMSN-NH-GA, significantly reduced ROS levels, especially pronounced with higher GA concentrations and notably in hGAAP cells with inherently higher ROS levels. Additionaly, NH-GA conjugates were less cytotoxic, more effective in reducing cell migration, and had higher ROS buffering capacity compared to DMSN-NCO-GA materials. However, in the presence of the external stressor tert-butyl-hydroperoxide (TBHP), NCO-GA conjugates showed more efficient reduction of intracellular ROS. These findings suggest that varying chemical decoration strategies of nanomaterials, along with the accessibility of functional groups to the cellular environment, significantly influence the biological response in osteosarcoma cells. Highlighting this, GA-conjugation is a promising method for implementing antioxidant properties and inhibiting cancer cell migration, warranting further research in anticancer treatment and drug development.

MANNosylation of Mesoporous Silica Nanoparticles Modifies TLR4 Localization and NF-κB Translocation in T24 Bladder Cancer Cells.

D-mannose is widely used as non-antibiotic treatment for bacterial urinary tract infections. This application is based on a well-studied mechanism of binding to the type 1 bacterial pili and, therefore, blocking bacteria adhesion to the uroepithelial cells. To implement D-mannose into carrier systems, the mechanism of action of the sugar in the bladder environment is also relevant and requires investigation. Herein, two different MANNosylation strategies using mesoporous silica nanoparticles (MSNs) are described. The impact of different chemical linkers on bacterial adhesion and bladder cell response is studied via confocal microscopy imaging of the MSN interactions with the respective organisms. Cytotoxicity is assessed and the expression of Toll-like receptor 4 (TLR4) and caveolin-1 (CAV-1), in the presence or absence of simulated infection with bacterial lipopolysaccharide (LPS), is evaluated using the human urinary bladder cancer cell line T24. Further, localisation of the transcription factor NF-κB due to the MANNosylated materials is examined over time. The results show that MANNosylation modifies bacterial adhesion to the nanomaterials and significantly affects TLR4, caveolin-1, and NF-κB in bladder cells. These elements are essential components of the inflammatory cascade/pathogens response during urinary tract infections. These findings demonstrate that MANNosylation is a versatile tool to design hybrid nanocarriers for targeted biomedical applications.

Probing the nature and extent of stabilization within foiled carbenes: homoallylic participation by a neighboring cyclopropane ring.

Oxadiazoline 6 was synthesized to generate endo-tricyclo[3.2.1.0(2,4)]octan-8-ylidene (3) by either photolysis or thermolysis. Diastereomer 6a thermally decomposed twice as fast as 6b. Carbene 3 was trapped stereoselectively by acrylonitrile and diethylamine in high yields. It behaved as a nucleophilic carbene with electron-poor alkenes, like acrylonitrile, but as an electrophile with very electron-rich species, such as diethylamine. However, when the reactions were performed in cyclohexane and cyclohexene, isomerization of 3 was favored. Replacement of the double bond in 7-norbornenylidene (1) by the single bond in the endo-fused cyclopropane unit of carbene 3 led to similar outcomes. Carbene 3 rightfully belongs to the family of foiled carbenes.