Enhanced Gene Delivery Triggered by Dual pH/Redox Responsive Host-Guest Dimerization of Cyclooligosaccharide Star Polycations.

A robust strategy is reported to build perfectly monodisperse star polycations combining a trehalose-based cyclooligosaccharide (cyclotrehalan, CT) central core onto which oligoethyleneimine radial arms are installed. The architectural perfection of the compounds is demonstrated by a variety of physicochemical techniques, including NMR, MS, DLS, TEM, and GPC. Key to the strategy is the possibility of customizing the cavity size of the macrocyclic platform to enable/prevent the inclusion of adamantane motifs. These properties can be taken into advantage to implement sequential levels of stimuli responsiveness by combining computational design, precision chemistry and programmed host-guest interactions. Specifically, it is shown that supramolecular dimers implying a trimeric CT-tetraethyleneimine star polycation and purposely designed bis-adamantane guests are preorganized to efficiently complex plasmid DNA (pDNA) into transfection-competent nanocomplexes. The stability of the dimer species is responsive to the protonation state of the cationic clusters, resulting in dissociation at acidic pH. This process facilitates endosomal escape, but reassembling can take place in the cytosol then handicapping pDNA nuclear import. By equipping the ditopic guest with a redox-sensitive disulfide group, recapturing phenomena are prevented, resulting in drastically improved transfection efficiencies both in vivo and in vitro.

Investigation of action pattern of a novel chondroitin sulfate/dermatan sulfate 4-O-endosulfatase.

Recently, a novel CS/DS 4-O-endosulfatase was identified from a marine bacterium and its catalytic mechanism was investigated further (Wang, W., et. al (2015) J. Biol. Chem.290, 7823-7832; Wang, S., et. al (2019) Front. Microbiol.10, 1309). In the study herein, we provide new insight about the structural characteristics of the substrate which determine the activity of this enzyme. The substrate specificities of the 4-O-endosulfatase were probed by using libraries of structure-defined CS/DS oligosaccharides issued from synthetic and enzymatic sources. We found that this 4-O-endosulfatase effectively remove the 4-O-sulfate of disaccharide sequences GlcUAß1-3GalNAc(4S) or GlcUAß1-3GalNAc(4S,6S) in all tested hexasaccharides. The sulfated GalNac residue is resistant to the enzyme when adjacent uronic residues are sulfated as shown by the lack of enzymatic desulfation of GlcUAß1-3GalNAc(4S) connected to a disaccharide GlcUA(2S)ß1-3GalNAc(6S) in an octasaccharide. The 3-O-sulfation of GlcUA was also shown to hinder the action of this enzyme. The 4-O-endosulfatase exhibited an oriented action from the reducing to the non-reducing whatever the saturation or not of the non-reducing end. Finally, the activity of the 4-O-endosulfatase decreases with the increase in substrate size. With the deeper understanding of this novel 4-O-endosulfatase, such chondroitin sulfate (CS)/dermatan sulfate (DS) sulfatase is a useful tool for exploring the structure-function relationship of CS/DS.

Trifaceted Mickey Mouse Amphiphiles for Programmable Self-Assembly, DNA Complexation and Organ-Selective Gene Delivery.

Instilling segregated cationic and lipophilic domains with an angular disposition in a trehalose-based trifaceted macrocyclic scaffold allows engineering patchy molecular nanoparticles leveraging directional interactions that emulate those controlling self-assembling processes in viral capsids. The resulting trilobular amphiphilic derivatives, featuring a Mickey Mouse architecture, can electrostatically interact with plasmid DNA (pDNA) and further engage in hydrophobic contacts to promote condensation into transfectious nanocomplexes. Notably, the topology and internal structure of the cyclooligosaccharide/pDNA co-assemblies can be molded by fine-tuning the valency and characteristics of the cationic and lipophilic patches, which strongly impacts the transfection efficacy in vitro and in vivo. Outstanding organ selectivities can then be programmed with no need of incorporating a biorecognizable motif in the formulation. The results provide a versatile strategy for the construction of fully synthetic and perfectly monodisperse nonviral gene delivery systems uniquely suited for optimization schemes by making cyclooligosaccharide patchiness the focus.

First Steps to Rationalize Host-Guest Interaction between α-, ß-, and γ-Cyclodextrin and Divalent First-Row Transition and Post-transition Metals (Subgroups VIIB, VIIIB, and IIB).

Cyclodextrins (CDs) are cyclic oligosaccharides mainly composed of six, seven, and eight glucose units, so-called α-, ß-, and γ-CDs, respectively. They own a very particular molecular structure exhibiting hydrophilic features thanks to primary and secondary rims and delimiting a hydrophobic internal cavity. The latter can encapsulate organic compounds, but the former can form supramolecular complexes by hydrogen-bonding or electrostatic interactions. CDs have been used in catalytic processes to increase mass transfer in aqueous-organic two-phase systems or to prepare catalysts. In the last case, interaction between CDs and metal salts was considered to be a key point in obtaining highly active catalysts. Up to now, no work was reported on the investigation of factors affecting the binding of metal to CD. In the study herein, we present the favorable combination of electrospray ionization coupled to mass spectrometry [ESI-MS(/MS)] and density functional theory molecular modeling [B3LYP/Def2-SV(P)] to delineate some determinants governing the coordination of first-row divalent transition metals (Mn2+, Co2+, Ni2+, Cu2+, and Fe2+) and one post-transition metal (Zn2+) with α-, ß-, and γ-CDs. A large set of features concerning the metal itself (ionic radius, electron configuration, and spin state) as well as the complexes formed (the most stable conformer, relative abundance in MS, CE50 value in MS/MS, binding energy, effective coordination number, average bond lengths, binding site localization, bond dissociation energies, and natural bond orbital distribution) were screened. Taking into account all of these properties, various selectivity rankings have been delineated, portraying differential association/dissociation behaviors. Nonetheless, unique 3D topologies for each CD-metal complex were emphasized. The combination of these approaches brings a stone for building a compendium of molecular features to serve as a suitable descriptor or predictor for a better first round rationalization of catalytic activities.

Programmed Synthesis of Hepta-Differentiated ß-Cyclodextrin: 1 out of 117655 Arrangements.

Cyclodextrin poly-functionalization has fueled progress in their use in multiple applications such as enzyme mimicry, but also in the polymer sciences, luminescence, as sensors or for biomedical applications. However, regioselective access to a given pattern of functions on ß-cyclodextrin is still very limited. We uncover a new orienting group, the thioacetate, that expands the toolbox available for cyclodextrin poly-hetero-functionalization using diisobutylaluminum hydride (DIBAL-H) promoted debenzylation. The usefulness of this group is illustrated in the first synthesis of a precisely hepta-hetero-functionalized ß-cyclodextrin. By way of comparison, a random hepta-functionalization would give 117655 different molecules. This synthesis is not simply the vain quest for the Holy Grail of CD hetero-functionalization, but it illustrates the versatility of the DIBAL-H oriented hetero-functionalization strategy, opening the way to a multitude of useful functionalization patterns for new practical applications.

Click Synthesis of Size- and Shape-Tunable Star Polymers with Functional Macrocyclic Cores for Synergistic DNA Complexation and Delivery.

The architectural perfection and multivalency of dendrimers have made them useful for biodelivery via peripheral functionalization and the adjustment of dendrimer generations. Modulation of the core-forming and internal matrix-forming structures offers virtually unlimited opportunities for further optimization, but only in a few cases this has been made compatible with strict diastereomeric purity over molecularly diverse series, low toxicity, and limited synthetic effort. Fully regular star polymers built on biocompatible macrocyclic platforms, such as hyperbranched cyclodextrins, offer advantages in terms of facile synthesis and flexible compositions, but core elaboration in terms of shape and function becomes problematic. Here we report the synthesis and characterization of star polymers consisting of functional trehalose-based macrocyclic cores (cyclotrehalans, CTs) and aminothiourea dendron arms, which can be efficiently synthesized from sequential click reactions of orthogonal monomers, display no cytotoxicity, and efficiently complex and deliver plasmid DNA in vitro and in vivo. When compared with some commercial cationic dendrimers or polymers, the new CT-scaffolded star polymers show better transfection efficiencies in several cell lines and structure-dependent cell selectivity patterns. Notably, the CT core could be predefined to exert Zn(II) complexing or molecular inclusion capabilities, which has been exploited to synergistically boost cell transfection by orders of magnitude and modulate the organ tropism in vivo.

Surface plasmon resonance imaging coupled to on-chip mass spectrometry: a new tool to probe protein-GAG interactions.

A biosensor device for the detection and characterization of protein-glycosaminoglycan interactions is being actively sought and constitutes the key to identifying specific carbohydrate ligands, an important issue in glycoscience. Mass spectrometry (MS) hyphenated methods are promising approaches for carbohydrate enrichment and subsequent structural characterization. In the study herein, we report the analysis of interactions between the glycosaminoglycans (GAGs) heparin (HP) and heparan sulfate (HS) and various cytokines by coupling surface plasmon resonance imaging (SPRi) for thermodynamic analysis method and MALDI-TOF MS for structural determination. To do so, we developed an SPR biochip in a microarray format and functionalized it with a self-assembled monolayer of short poly(ethylene oxide) chains for grafting the human cytokines stromal cell-derived factor-1 (SDF-1α), monocyte chemotactic protein-1 (MCP-1), and interferon-γ. The thermodynamic parameters of the interactions between these cytokines and unfractionated HP/HS and derived oligosaccharides were successively determined using SPRi monitoring, and the identification of the captured carbohydrates was carried out directly on the biochip surface using MALDI-TOF MS, revealing cytokine preferential affinity for GAGs. The MS identification was enhanced by on-chip digestion of the cytokine-bound GAGs with heparinase, leading to the detection of oligosaccharides likely involved in the binding sequence of GAG ligands. Although several carbohydrate array-based assays have been reported, this study is the first report of the successful analysis of protein-GAG interactions using SPRi-MS coupling.

Interactions of Calcium with Chlorogenic and Rosmarinic Acids: An Experimental and Theoretical Approach.

Chlorogenic (CA) and rosmarinic (RA) acids are two natural bioactive hydroxycinnamic acids whose antioxidant properties can be modulated by the chelation of metal ions. In this work, the interactions of these two carboxylic phenols with calcium ions and the impact of such interactions on their antioxidant activity were investigated. UV-Vis absorbance, mass spectroscopy and 1H and 13C liquid NMR were used to identify complexes formed by CA and RA with calcium. Antioxidant activities were measured by the Bois method. Density functional theory (DFT) calculations were performed to evaluate the most stable configurations and correlated with NMR data. Taken together, these data suggest that calcium ions mainly interact with the carboxylate groups of both molecules but that this interaction modifies the reactivity of the catechol groups, especially for RA. These results highlight the complex interplay between metal chelation and antioxidant properties of natural carboxylic phenols.

The uncommon strong inhibition of α-glucosidase by multivalent glycoclusters based on cyclodextrin scaffolds.

The homeostasis disruption of d-glucose causes diabetes, a dramatic chronic disease worldwide. Type 1 diabetes is a successfully treatable form, where blood d-glucose is regulated by insulin treatment. In contrast type 2 diabetes, the non-insulin dependent kind, is problematic. The control of the d-glucose blood level via intestinal α-d-glucosidase inactivation can be achieved by using competitive inhibitors, such as iminosugars (e.g. acarbose) or sulfonium sugar derivatives (e.g. salacinol). Recently, an unprecedented result showed that multivalent diamond nanoparticles grafted with unmodified sugars displayed α-glucosidase inhibition at low micromolar concentrations. Herein we describe the synthesis of multivalent glycoclusters using cyclodextrins (CDs) as scaffolds and an assessment of their role as inhibitors of α-d-glucosidase. The glycoclusters were efficiently obtained from per-azido α, ß and γ-CD derivatives and propargyl glycosides using click-chemistry under microwave irradiation. The methodology was successfully applied to various protected and non-protected propargylated monosaccharides, including both O- and S-glycosides, giving clear evidence of its versatility. The targeted 6-per-glycosylated CDs were isolated in moderate to excellent yields (30-90%) by silica gel chromatography. The results showed inhibition of α-glucosidase from Saccharomyces cerevisiae with IC50 values in the 32-132 µM range, lower than that of acarbose (IC50 = ∼250 µM), a well-known competitive inhibitor used in the clinical treatment of type 2 diabetes. Preliminary experiments suggest a mixed-type non-competitive inhibition mode for these new glycoclusters.

Large-Ring Cyclodextrins as Chiral Selectors for Enantiomeric Pharmaceuticals.

Large-ring cyclodextrins (CD) are cyclic glucans composed of 9 or more α-1,4-linked glucose units. They are minor side products of bacterial glucanotransferases (CGTases, EC 2.4.1.19) and have previously been available only in very small amounts for studies of their properties in supramolecular complex formation reactions. We engineered a CGTase to synthesize mainly large-ring CD facilitating their preparation in larger amounts. By reversed phase chromatography, we obtained single CD samples composed of 10 to 12 glucose units (CD10, CD11, and CD12) with a purity of >90 %. Their identity was confirmed by high resolution mass spectrometry and fragmentation analysis. We demonstrated the non-toxicity of CD10-CD12 for human cell lines by a cell proliferation assay and impedimetric monitoring. We then showed that CD10 and CD11 are efficient chiral selectors for the capillary electrophoretic separation of the enantiomeric pharmaceuticals fluvastatin, mefloquine, carvedilol, and primaquine.

Biomimetic ion channels formation by emulsion based on chemically modified cyclodextrin nanotubes.

Biomimetic ion channels can be made to display the high sensitivity of natural protein nanopores and to develop new properties as a function of the material used. How to design the best future biomimetic channels? The main challenges are to control their sensitivity, as well as their syntheses, chemical modifications, insertion and lifetime in a lipid membrane. To address these challenges, we have recently designed short cyclodextrin nanotubes characterized by mass spectrometry and high-resolution transmission electron microscopy. They form non-permanent ion channels in lipid bilayers. Here we show how to improve the nanotube insertion in order to limit multiple insertions, how to stabilize biomimetic channels into the membrane, and how to understand the ion dynamics in confined medium scale.

Xylylene Clips for the Topology-Guided Control of the Inclusion and Self-Assembling Properties of Cyclodextrins.

The topology of ß-cyclodextrin can be molded, from toroidal to ovoid basket-shaped, by the installation of an o- or m-xylylene moiety connecting two consecutive d-glucopyranosyl units through the secondary O-2(I) and O-3(II) positions. This strategy can be exploited advantageously to precast the cavity for preferential inclusion of globular or planar guests as well as to privilege dimeric or monomeric species in water solution.

Embedding a Ruthenium-Based Structural Mimic of the [Fe]-Hydrogenase Cofactor into Papain.

We describe the synthesis of the ruthenacyclic carbamoyl complexes [Ru(2-NHC(O)C5H3NMe)(CO)2( o,o-Me2-C6H3S)(L)] (L = H2O or MeCN), which have a labile water or acetonitrile ligand at their sixth coordination sites. Steric bulk around the ruthenium center is essential in preventing isomerization and dimerization, and embedding within papain can be achieved via coordination of its sole free cysteine residue. The observed chemistry parallels that of the natural [Fe]-hydrogenase.

Efficient recovery of glycosaminoglycan oligosaccharides from polyacrylamide gel electrophoresis combined with mass spectrometry analysis.

To promote efficient separation and structural analysis of glycosaminoglycan oligosaccharides, we developed a straightforward method that combined gel electrophoresis and mass spectrometry (MS). Potential limitations of this approach (e.g., low extraction yields and weak compatibility with MS) were resolved by developing an active extraction procedure that yielded a quantitative amount of sulfated oligosaccharides from excised gel bands. The compatibility of obtained oligosaccharides for subsequent MS analysis was ensured using a single, simple clean-up step on a mixed C18/graphite carbon solid-phase column that was fully effective for polymerization degrees ranging from di- to dodecasaccharides. The reported combination of carbohydrates-polyacrylamide gel electrophoresis with MS was successfully applied to glucosamino- (heparin) and galactosamino- (dermantan sulfate) glycans, demonstrating the potential of our method for structural analysis of bioactive sulfated carbohydrates extracted from biological matrices. Graphical Abstract ᅟ.

Interactions between shape-persistent macromolecules as probed by AFM.

Water-soluble shape-persistent cyclodextrin (CD) polymers with amino-functionalized end groups were prepared starting from diacetylene-modified cyclodextrin monomers by a combined Glaser coupling/click chemistry approach. Structural perfection of the neutral CD polymers and inclusion complex formation with ditopic and monotopic guest molecules were proven by MALDI-TOF and UV-vis measurements. Small-angle neutron and X-ray (SANS/SAXS) scattering experiments confirm the stiffness of the polymer chains with an apparent contour length of about 130 Å. Surface modification of planar silicon wafers as well as AFM tips was realized by covalent bound formation between the terminal amino groups of the CD polymer and a reactive isothiocyanate-silane monolayer. Atomic force measurements of CD polymer decorated surfaces show enhanced supramolecular interaction energies which can be attributed to multiple inclusion complexes based on the rigidity of the polymer backbone and the regular configuration of the CD moieties. Depending on the geometrical configuration of attachment anisotropic adhesion characteristics of the polymer system can be distinguished between a peeling and a shearing mechanism.

Biomimetic Nanotubes Based on Cyclodextrins for Ion-Channel Applications.

Biomimetic membrane channels offer a great potential for fundamental studies and applications. Here, we report the fabrication and characterization of short cyclodextrin nanotubes, their insertion into membranes, and cytotoxicity assay. Mass spectrometry and high-resolution transmission electron microscopy were used to confirm the synthesis pathway leading to the formation of short nanotubes and to describe their structural parameters in terms of length, diameter, and number of cyclodextrins. Our results show the control of the number of cyclodextrins threaded on the polyrotaxane leading to nanotube synthesis. Structural parameters obtained by electron microscopy are consistent with the distribution of the number of cyclodextrins evaluated by mass spectrometry from the initial polymer distribution. An electrophysiological study at single molecule level demonstrates the ion channel formation into lipid bilayers, and the energy penalty for the entry of ions into the confined nanotube. In the presence of nanotubes, the cell physiology is not altered.

On-line capillary isoelectric focusing hyphenated to native electrospray ionization mass spectrometry for the characterization of interferon-γ and variants.

The on-line hyphenation of Capillary IsoElectric Focusing (CIEF) with ElectroSpray Ionization Mass Spectrometry (ESI/MS) has been carried out in a non-denaturing detection mode at the CIEF-MS interface. This CIEF-MS coupling methodology relied on the use of 40% glycerol-water medium as anti-convective agent in the CE capillary and the addition of 10 mM ammonium acetate buffer, pH 5, as a volatile aqueous sheath liquid. These CIEF-MS coupling conditions allowed the characterization of the highly basic cytokine human interferon-gamma (IFN-γ) and its detection as a non-covalent homodimer (33,814.3 g mol(-1)) corresponding to the active form of this immune-regulatory protein. An experimental pI value of 9.95 was determined for the human IFN-γ homodimer in these conditions. The CIEF-MS analysis of several variants bearing punctual or deletion mutations within the two D1 and D2 basic clusters at the C-terminal end of IFN-γ revealed the different contribution of these domains to the charge properties of this heparan sulfate-binding protein.

Probing the common alkali metal affinity of native and variously methylated ß-cyclodextrins by combining electrospray-tandem mass spectrometry and molecular modeling.

In the study herein, we investigated the solution and gas phase affinity of native and variously methylated ß-cyclodextrins (CDs) as hosts towards three common alkali metals as guests namely lithium, sodium and potassium. For this purpose, two complementary approaches have been employed: electrospray-tandem mass spectrometry (ESI-MS/MS) with two energetic regimes: Collision Induced Dissociation (CID) and Higher Collision Dissociation (HCD), respectively, and DFT molecular modeling. These approaches have been achieved by taking into account the interaction of either one or two alkali metals with the host molecules. The results showed a good agreement between experimental and theoretical data. It was demonstrated that increasing the methylation degree strengthened the gas phase affinity towards all studied alkali metals. Furthermore, it was established that the cation selectivity was Na(+) > Li(+) > K(+) and Li(+) > Na(+) > K(+) for the solution and gas phase, respectively.

Identification of novel PAMP-triggered phosphorylation and dephosphorylation events in Arabidopsis thaliana by quantitative phosphoproteomic analysis.

Signaling cascades rely strongly on protein kinase-mediated substrate phosphorylation. Currently a major challenge in signal transduction research is to obtain high confidence substrate phosphorylation sites and assign them to specific kinases. In response to bacterial flagellin, a pathogen-associated molecular pattern (PAMP), we searched for rapidly phosphorylated proteins in Arabidopsis thaliana by combining multistage activation (MSA) and electron transfer dissociation (ETD) fragmentation modes, which generate complementary spectra and identify phosphopeptide sites with increased reliability. Of a total of 825 phosphopeptides, we identified 58 to be differentially phosphorylated. These peptides harbor kinase motifs of mitogen-activated protein kinases (MAPKs) and calcium-dependent protein kinases (CDPKs), as well as yet unknown protein kinases. Importantly, 12 of the phosphopeptides show reduced phosphorylation upon flagellin treatment. Since protein abundance levels did not change, these results indicate that flagellin induces not only various protein kinases but also protein phosphatases, even though a scenario of inhibited kinase activity may also be possible.

Derivatization strategies for CE-LIF analysis of biomarkers: Toward a clinical diagnostic of familial transthyretin amyloidosis.

We report three derivatization strategies for CE analysis with LIF detection (CE-LIF) of two synthetic peptides mimicking the wild and mutated fragments of interest for the diagnosis of familial transthyretin amyloidosis. The precapillary derivatization of the peptides with three optical tags, 5-carboxytetramethylrhodamin succinimidyl ester (TAMRA-SE), naphtalene-2,3-dicarboxyaldehyde (NDA), and 3-(2-furoyl)quinoline-2-carboxyaldehyde (FQ) has been investigated by CE-LIF detection and MS. Results provide evidence that high reaction yields have been reached whereas the multitagging phenomenon has occurred for both NDA and TAMRA-SE labeling procedures. The derivatization and electrokinetic separation of a mixture of the two peptides of interest for the pathology diagnosis (22-aa peptides that differ only from one amino acid) were achieved using both approaches. The highest resolution with a value of 2.5 was obtained with TAMRA-SE labeled derivatives whereas NDA gave the best detection sensitivity (LOD of 2.5 µM). The validation of the developed methods showed a good linearity (R ≥ 0.997) between the peak area of the labeled derivatives and the peptide concentration for both NDA and FQ labeling procedures. The intraday RSDs of A and the migration times were less than 3.8 and 2.2%, respectively.