Electrophilic Si-H Activation by Acetonitrilo Benzo[h]quinoline Iridacycles: Influence of Electronic Effects in Catalysis.

The performance of six newly synthesized benzo[h]quinoline-derived acetonitrilo pentamethylcyclopentadienyl iridium(III) tetrakis(3,5-bis-trifluoromethylphenyl)borate salts bearing different substituents -X (-OMe, -H, -Cl, -Br, -NO2 and -(NO2 )2 ) on the heterochelating ligand were evaluated in the dehydro-O-silylation of benzyl alcohol and the monohydrosilylation of 4-methoxybenzonitrile by Et3 SiH, two reactions involving the electrophilic activation of the Si-H bond. The benchmark shows a direct dependence of the catalytic efficiency with the electronic effect of -X, which is confirmed by theoretical assessment of the intrinsic silylicities Π of hydridoiridium(III)-silylium adducts and by the theoretical evaluation of the propensity of hydridospecies to transfer the hydrido ligand to the activated substrate. The revisited analysis of the Ir-Si-H interactions shows that the most cohesive bond in hydridoiridium(III)-silylium adducts is the Ir-H one, while the Ir-Si is a weak donor-acceptor dative bond. The Si…H interaction in all the cases is noncovalent in nature and dominated by electrostatics confirming the heterolytic cleavage of the hydrosilane's Si-H bond in this key catalytically relevant species.

Structural description of surfaces and interfaces in biominerals by DNP SENS.

Biological mineralized tissues are hybrid materials with complex hierarchical architecture composed of biominerals often embedded in an organic matrix. The atomic-scale comprehension of surfaces and organo-mineral interfaces of these biominerals is of paramount importance to understand the ultrastructure, the formation mechanisms as well as the biological functions of the related biomineralized tissue. In this communication we demonstrate the capability of DNP SENS to reveal the fine atomic structure of biominerals, and more specifically their surfaces and interfaces. For this purpose, we studied two key examples belonging to the most significant biominerals family in nature: apatite in bone and aragonite in nacreous shell. As a result, we demonstrate that DNP SENS is a powerful approach for the study of intact biomineralized tissues. Signal enhancement factors are found to be up to 40 and 100, for the organic and the inorganic fractions, respectively, as soon as impregnation time with the radical solution is long enough (between 12 and 24 h) to allow an efficient radical penetration into the calcified tissues. Moreover, ions located at the biomineral surface are readily detected and identified through 31P or 13C HETCOR DNP SENS experiments. Noticeably, we show that protonated anions are preponderant at the biomineral surfaces in the form of HPO42- for bone apatite and HCO32- for nacreous aragonite. Finally, we demonstrate that organo-mineral interactions can be probed at the atomic level with high sensitivity. In particular, reliable 13C-{31P} REDOR experiments are achieved in a few hours, leading to the determination of distances, molar proportion and binding mode of citrate bonded to bone mineral in native compact bone. According to our results, only 80% of the total amount of citrate in bone is directly interacting with bone apatite through two out of three carboxylic groups.

pH-Dependent Membrane Interactions of the Histidine-Rich Cell-Penetrating Peptide LAH4-L1.

The histidine-rich designer peptide LAH4-L1 exhibits antimicrobial and potent cell-penetrating activities for a wide variety of cargo including nucleic acids, polypeptides, adeno-associated viruses, and nanodots. The non-covalent complexes formed between the peptide and cargo enter the cell via an endosomal pathway where the pH changes from neutral to acidic. Here, we investigated the membrane interactions of the peptide with phospholipid bilayers and its membrane topology using static solid-state NMR spectroscopy. Oriented 15N solid-state NMR indicates that in membranes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS) 3:1 mol/mole and at neutral pH, the peptide adopts transmembrane topologies. Furthermore, 31P and 2H solid-state NMR spectra show that liquid crystalline 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and POPC/POPS 3:1 liposomes retain a bilayer macroscopic phase even at the highest peptide concentrations investigated, with an oblate orientational distribution of the phospholipids at a peptide/lipid ratio of 1:5. At pH 5, as it occurs in the endosome, the alignment of LAH4-L1 at a peptide/lipid ratio of 1:25 is predominantly parallel to POPC/POPS 3:1 bilayers (prolate deformation) when at the same time it induces a considerable decrease of the deuterium order parameter of POPC/2H31-POPS 3:1. In addition, when studied in mechanically supported lipid membranes, a pronounced disordering of the phospholipid alignment is observed. In the presence of even higher peptide concentrations, lipid spectra are observed that suggest the formation of magnetically oriented or isotropic bicelles. This membrane-disruptive effect is enhanced for gel phase DMPC membranes. By protonation of the four histidines in acidic environments, the overall charge and hydrophobic moment of LAH4-L1 considerably change, and much of the peptide is released from the cargo. Thus, the amphipathic peptide sequences become available to disrupt the endosomal membrane and to assure highly efficient release from this organelle.

The anti-inflammatory action of the analgesic kyotorphin neuropeptide derivatives: insights of a lipid-mediated mechanism.

Recently, a designed class of efficient analgesic drugs derived from an endogenous neuropeptide, kyotorphin (KTP, Tyr-Arg) combining C-terminal amidation (KTP-NH2) and N-terminal conjugation to ibuprofen (Ib), IbKTP-NH2, was developed. The Ib moiety is an enhancer of KTP-NH2 analgesic action. In the present study, we have tested the hypothesis that KTP-NH2 is an enhancer of the Ib anti-inflammatory action. Moreover, the impact of the IbKTP-NH2 conjugation on microcirculation was also evaluated by a unified approach based on intravital microscopy in the murine cremasteric muscle. Our data show that KTP-NH2 and conjugates do not cause damage on microcirculatory environment and efficiently decrease the number of leukocyte rolling induced by lipopolysaccharide (LPS). Isothermal titration calorimetry showed that the drugs bind to LPS directly thus contributing to LPS aggregation and subsequent elimination. In a parallel study, molecular dynamics simulations and NMR data showed that the IbKTP-NH2 tandem adopts a preferential "stretched" conformation in lipid bilayers and micelles, with the simulations indicating that the Ib moiety is anchored in the hydrophobic core, which explains the improved partition of IbKTP-NH2 to membranes and the permeability of lipid bilayers to this conjugate relative to KTP-NH2. The ability to bind glycolipids concomitant to the anchoring in the lipid membranes through the Ib residue explains the analgesic potency of IbKTP-NH2 given the enriched glycocalyx of the blood-brain barrier cells. Accumulation of IbKTP-NH2 in the membrane favors both direct permeation and local interaction with putative receptors as the location of the KTP-NH2 residue of IbKTP-NH2 and free KTP-NH2 in lipid membranes is the same.

Membrane interactions of phylloseptin-1, -2, and -3 peptides by oriented solid-state NMR spectroscopy.

Phylloseptin-1, -2, and -3 are three members of the family of linear cationic antimicrobial peptides found in tree frogs. The highly homologous peptides encompass 19 amino acids, and only differ in the amino acid composition and charge at the six most carboxy-terminal residues. Here, we investigated how such subtle changes are reflected in their membrane interactions and how these can be correlated to their biological activities. To this end, the three peptides were labeled with stable isotopes, reconstituted into oriented phospholipid bilayers, and their detailed topology determined by a combined approach using (2)H and (15)N solid-state NMR spectroscopy. Although phylloseptin-2 and -3 adopt perfect in-plane alignments, the tilt angle of phylloseptin-1 deviates by 8° probably to assure a more water exposed localization of the lysine-17 side chain. Furthermore, different azimuthal angles are observed, positioning the amphipathic helices of all three peptides with the charged residues well exposed to the water phase. Interestingly, our studies also reveal that two orientation-dependent (2)H quadrupolar splittings from methyl-deuterated alanines and one (15)N amide chemical shift are sufficient to unambiguously determine the topology of phylloseptin-1, where quadrupolar splittings close to the maximum impose the most stringent angular restraints. As a result of these studies, a strategy is proposed where the topology of a peptide structure can be determined accurately from the labeling with (15)N and (2)H isotopes of only a few amino acid residues.

15N chemical shift referencing in solid state NMR.

Solid-state NMR spectroscopy has much advanced during the last decade and provides a multitude of data that can be used for high-resolution structure determination of biomolecules, polymers, inorganic compounds or macromolecules. In some cases the chemical shift referencing has become a limiting factor to the precision of the structure calculations and we have therefore evaluated a number of methods used in proton-decoupled (15)N solid-state NMR spectroscopy. For (13)C solid-state NMR spectroscopy adamantane is generally accepted as an external standard, but to calibrate the (15)N chemical shift scale several standards are in use. As a consequence the published chemical shift values exhibit considerable differences (up to 22 ppm). In this paper we report the (15)N chemical shift of several commonly used references compounds in order to allow for comparison and recalibration of published data and future work. We show that (15)NH4Cl in its powdered form (at 39.3 ppm with respect to liquid NH3) is a suitable external reference as it produces narrow lines when compared to other reference compounds and at the same time allows for the set-up of cross-polarization NMR experiments. The compound is suitable to calibrate magic angle spinning and static NMR experiments. Finally the temperature variation of (15)NH4Cl chemical shift is reported.

Orientation and depth of surfactant protein B C-terminal helix in lung surfactant bilayers.

SP-B(CTERM) is a cationic amphipathic helical peptide and functional fragment composed of residues 63 to 78 of surfactant protein B (SP-B). Static oriented and magic angle spinning solid state NMR, along with molecular dynamics simulation was used to investigate its structure, orientation, and depth in lipid bilayers of several compositions, namely POPC, DPPC, DPPC/POPC/POPG, and bovine lung surfactant extract (BLES). In all lipid environments the peptide was oriented parallel to the membrane surface. While maintaining this approximately planar orientation, SP-B(CTERM) exhibited a flexible topology controlled by subtle variations in lipid composition. SP-B(CTERM)-induced lipid realignment and/or conformational changes at the level of the head group were observed using (31)P solid-state NMR spectroscopy. Measurements of the depth of SP-B(CTERM) indicated the peptide center positions ~8Å more deeply than the phosphate headgroups, a topology that may allow the peptide to promote functional lipid structures without causing micellization upon compression.

Membrane structure and conformational changes of the antibiotic heterodimeric peptide distinctin by solid-state NMR spectroscopy.

The heterodimeric antimicrobial peptide distinctin is composed of 2 linear peptide chains of 22- and 25-aa residues that are connected by a single intermolecular S-S bond. This heterodimer has been considered to be a unique example of a previously unrecorded class of bioactive peptides. Here the 2 distinctin chains were prepared by chemical peptide synthesis in quantitative amounts and labeled with (15)N, as well as (15)N and (2)H, at selected residues, respectively, and the heterodimer was formed by oxidation. CD spectroscopy indicates a high content of helical secondary structures when associated with POPC/POPG 3:1 vesicles or in membrane-mimetic environments. The propensity for helix formation follows the order heterodimer >chain 2 >chain 1, suggesting that peptide-peptide and peptide-lipid interactions both help in stabilizing this secondary structure. In a subsequent step the peptides were reconstituted into oriented phospholipid bilayers and investigated by (2)H and proton-decoupled (15)N solid-state NMR spectroscopy. Whereas chain 2 stably inserts into the membrane at orientations close to perfectly parallel to the membrane surface in the presence or absence of chain 1, the latter adopts a more tilted alignment, which further increases in the heterodimer. The data suggest that membrane interactions result in considerable conformational rearrangements of the heterodimer. Therefore, chain 2 stably anchors the heterodimer in the membrane, whereas chain 1 interacts more loosely with the bilayer. These structural observations are consistent with the antimicrobial activities when the individual chains are compared to the dimer.

Inorganic phosphate in growing calcium carbonate abalone shell suggests a shared mineral ancestral precursor.

The presence of phosphate from different origins (inorganic, bioorganic) is found more and more in calcium carbonate-based biominerals. Phosphate is often described as being responsible for the stabilization of the transient amorphous calcium carbonate phase. In order to specify the composition of the mineral phase deposited at the onset of carbonated shell formation, the present study investigates, down to the nanoscale, the growing shell from the European abalone Haliotis tuberculata, using a combination of solid state nuclear magnetic resonance, scanning transmission electron microscope and spatially-resolved electron energy loss spectroscopy techniques. We show the co-occurrence of inorganic phosphate with calcium and carbonate throughout the early stages of abalone shell formation. One possible hypothesis is that this first-formed mixed mineral phase represents the vestige of a shared ancestral mineral precursor that appeared early during Evolution. In addition, our findings strengthen the idea that the final crystalline phase (calcium carbonate or phosphate) depends strongly on the nature of the mineral-associated proteins in vivo.

A new family of peptide-nucleic acid nanostructures with potent transfection activities.

A family of His-rich peptides has been shown to complex DNA and efficiently deliver these nucleic acids into eukaryotic cells. Therefore, these nanoscale complexes have potential applications in gene therapy. Here, we review a number of spectroscopic and biophysical investigations aimed at characterizing the supramolecular interactions of the peptides with the nucleic acids and when overcoming the membrane barriers of the cell. Furthermore, solid-state NMR distance measurements for the first time reveal close interatomic distances between the amino acid side chains and the DNA phosphates within the transfection complex. A recent study indicates that the peptides are also potent transfectants of siRNAs and they could thereby be of potential interest for gene silencing therapies using these compounds. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.

Structure and alignment of the membrane-associated peptaibols ampullosporin A and alamethicin by oriented 15N and 31P solid-state NMR spectroscopy.

Ampullosporin A and alamethicin are two members of the peptaibol family of antimicrobial peptides. These compounds are produced by fungi and are characterized by a high content of hydrophobic amino acids, and in particular the alpha-tetrasubstituted amino acid residue ?-aminoisobutyric acid. Here ampullosporin A and alamethicin were uniformly labeled with (15)N, purified and reconstituted into oriented phophatidylcholine lipid bilayers and investigated by proton-decoupled (15)N and (31)P solid-state NMR spectroscopy. Whereas alamethicin (20 amino acid residues) adopts transmembrane alignments in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) or 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) membranes the much shorter ampullosporin A (15 residues) exhibits comparable configurations only in thin membranes. In contrast the latter compound is oriented parallel to the membrane surface in 1,2-dimyristoleoyl-sn-glycero-3-phosphocholine and POPC bilayers indicating that hydrophobic mismatch has a decisive effect on the membrane topology of these peptides. Two-dimensional (15)N chemical shift -(1)H-(15)N dipolar coupling solid-state NMR correlation spectroscopy suggests that in their transmembrane configuration both peptides adopt mixed alpha-/3(10)-helical structures which can be explained by the restraints imposed by the membranes and the bulky alpha-aminoisobutyric acid residues. The (15)N solid-state NMR spectra also provide detailed information on the helical tilt angles. The results are discussed with regard to the antimicrobial activities of the peptides.

Analysis of the amide (15)N chemical shift tensor of the C(alpha) tetrasubstituted constituent of membrane-active peptaibols, the alpha-aminoisobutyric acid residue, compared to those of di- and tri-substituted proteinogenic amino acid residues.

In protein NMR spectroscopy the chemical shift provides important information for the assignment of residues and a first structural evaluation of dihedral angles. Furthermore, angular restraints are obtained from oriented samples by solution and solid-state NMR spectroscopic approaches. Whereas the anisotropy of chemical shifts, quadrupolar couplings and dipolar interactions have been used to determine the structure, dynamics and topology of oriented membrane polypeptides using solid-state NMR spectroscopy similar concepts have been introduced to solution NMR through the measurements of residual dipolar couplings. The analysis of (15)N chemical shift spectra depends on the accuracy of the chemical shift tensors. When investigating alamethicin and other peptaibols, i.e. polypeptides rich in alpha-aminoisobutyric acid (Aib), the (15)N chemical shift tensor of this C(alpha)-tetrasubstituted amino acid exhibits pronounced differences when compared to glycine, alanine and other proteinogenic residues. Here we present an experimental investigation on the (15)N amide Aib tensor of N-acetyl-Aib-OH and for the Aib residues within peptaibols. Furthermore, a statistical analysis of the tensors published for di- (glycine) and tri-substituted residues has been performed, where for the first time the published data sets are compiled using a common reference. The size of the isotropic chemical shift and main tensor elements follows the order di- < tri- < tetra-substituted amino acids. A (15)N chemical shift-(1)H-(15)N dipolar coupling correlation NMR spectrum of alamethicin is used to evaluate the consequences of variations in the main tensor elements for the structural analysis of this membrane peptide.

Coating of polydopamine on polyurethane open cell foams to design soft structured supports for molecular catalysts.

Polydopamine-coated polyurethane open cell foams are used as structured supports for molecular catalysts through the covalent anchoring of alkoxysilyl arms by the catechol groups of the mussel-inspired layer. This strong bonding prevents their leaching. No alteration of the mechanical properties of the flexible support is observed after repeated uses of the catalytic materials.

Helix orientations in membrane-associated Bcl-X(L) determined by 15N-solid-state NMR spectroscopy.

Controlled cell death is fundamental to tissue hemostasis and apoptosis malfunctions can lead to a wide range of diseases. Bcl-x(L) is an anti-apoptotic protein the function of which is linked to its reversible interaction with mitochondrial outer membranes. Its interfacial and intermittent bilayer association makes prediction of its bound structure difficult without using methods able to extract data from dynamic systems. Here we investigate Bcl-x(L) associated with oriented lipid bilayers at physiological pH using solid-state NMR spectroscopy. The data are consistent with a C-terminal transmembrane anchoring sequence and an average alignment of the remaining helices, i.e. including helices 5 and 6, approximately parallel to the membrane surface. Data from several biophysical approaches confirm that after removal of the C-terminus from Bcl-x(L) its membrane interactions are weak. In the presence of membranes Bcl-x(L) can still interact with a Bak BH3 domain peptide suggesting a model where the hydrophobic C-terminus of the protein unfolds and inserts into the membrane. During this conformational change the Bcl-x(L) hydrophobic binding pocket becomes accessible for protein-protein interactions whilst the structure of the N-terminal region remains intact.

Proton-decoupled 15N and 31P solid-state NMR investigations of the Pf3 coat protein in oriented phospholipid bilayers.

The coat proteins of filamentous phage are first synthesized as transmembrane proteins and then assembled onto the extruding viral particles. We investigated the transmembrane conformation of the Pseudomonas aeruginosa Pf3 phage coat protein using proton-decoupled 15N and 31P solid-state NMR spectroscopy. The protein was either biochemically purified and uniformly labelled with 15N or synthesized chemically and labelled at specific sites. The proteins were then reconstituted into oriented phospholipid bilayers and the resulting samples analysed. The data suggest a model in which the protein adopts a tilted helix with an angle of approximately 30 degrees and an N-terminal 'swinging arm' at the membrane surface.

Polyelectrolytes to produce nanosized polydopamine.

"Polydopamine" (PDA) is the oxidation product of dopamine and can be obtained as thin films covering the surface of all kinds of known materials and simultaneously as insoluble and useless precipitates from dopamine solutions in the presence of appropriate oxidants. The valorization of such precipitates to obtain stable suspensions of functional nanomaterials is highly desirable owing to the chemical and optical properties of PDA. We show that a vast repertoire of polyelectrolytes polycations as well as polyanions, allow to control the size of PDA particles in the 10-100 nm size range. Simultaneously to the production of smaller nanoparticles, a progressive inhibition of PDA deposition on the surface of quartz plates (as well as on the surface of the reaction vessel) is found as the concentration of the polyelectrolytes is increased in the dopamine solution. The mechanism of size control-inhibition of film deposition is investigated in the particular case of poly(allylamine) but remains not understood in the case of polyanions.

The alignment, structure and dynamics of membrane-associated polypeptides by solid-state NMR spectroscopy.

Solid-state NMR spectroscopy is being developed at a fast pace for the structural investigation of immobilized and non-crystalline biomolecules. These include proteins and peptides associated with phospholipid bilayers. In contrast to solution NMR spectroscopy, where complete or almost complete averaging leads to isotropic values, the anisotropic character of nuclear interactions is apparent in solid-state NMR spectra. In static samples the orientation dependence of chemical shift, dipolar or quadrupolar interactions, therefore, provides angular constraints when the polypeptides have been reconstituted into oriented membranes. Furthermore, solid-state NMR spectroscopy of aligned samples offers distinct advantages in allowing access to dynamic processes such as topological equilibria or rotational diffusion in membrane environments. Alternatively, magic angle sample spinning (MAS) results in highly resolved NMR spectra, provided that the sample is sufficiently homogenous. MAS spinning solid-state NMR spectra allow to measure distances and dihedral angles with high accuracy. The technique has recently been developed to selectively establish through-space and through-bond correlations between nuclei, similar to the approaches well-established in solution-NMR spectroscopy.

Role of surfactants in the control of dopamine-eumelanin particle size and in the inhibition of film deposition at solid-liquid interfaces.

Anionic and cationic surfactants such as sodium dodecylsulfate (SDS) and hexadecyltrimethylammonium bromide (HTAB) are able to control the size of "polydopamine" particles produced from dopamine solutions and to simultaneously strongly inhibit the deposition of "polydopamine" on surfaces. Indeed, dynamic light scattering experiments allowed to show that the hydrodynamic radius of polydopamine progressively decreases from about 1 µm to a few nanometer upon an increase in the SDS and CTAB concentration. At the highest surfactant concentration used (50 mM) the size of the aggregates is only slightly larger than the size of the surfactant micelles. On the other hand, the non-ionic Triton X-100 surfactant has no significant influence on both phenomena. It is suggested that the observed effect originates from the anionic and cationic surfactants acting as a template in which the growth of "polydopamine" is confined.

One-step synthesis of a highly homogeneous SBA-NHC hybrid material: en route to single-site NHC-metal heterogeneous catalysts with high loadings.

The one-step synthesis of a mesoporous silica of SBA type, functionalized with a 1-(2,6-diisopropylphenyl)-3-propyl-imidazolium (iPr2Ar-NHC-propyl) cation located in the pore channels, is described. This material was obtained by the direct hydrolysis and co-condensation of tetraethylorthosilicate (TEOS) and 1-(2,6-diisopropylphenyl)-3-[3-(triethoxysilyl)propyl]-imidazolium iodide in the presence of Pluronic P123 as a non-ionic structure-directing agent and aqueous HCl (37%) as an acid catalyst. Small-angle X-ray diffraction measurements, scanning and transmission electron microscopies, as well as dinitrogen sorption analyses revealed that the synthesized material is highly mesoporous with a 2D hexagonal arrangement of the porous network. (13)C and (29)Si CP-MAS NMR spectroscopy confirmed that the material contains intact iPr2Ar-NHC-propyl cations, which are covalently anchored via silicon atoms fused into the silica matrix. Moreover, comparison of the latter data with those of an analogous post-synthetic grafted SBA-NHC material allowed us to establish that, as expected, (i) it is most probably more homogeneous and (ii) it shows a more robust anchoring of the organic units. Finally, elemental mapping by energy dispersive X-ray spectroscopy in the scanning electron microscope demonstrated a very homogeneous distribution of the imidazolium units within the one-pot material, moreover with a high content. This study thus demonstrates that a relatively bulky and hydrophilic imidazolium unit can be directly co-condensed with TEOS in the presence of a structure-directing agent to provide in a single step a highly ordered and homogeneous mesoporous hybrid SBA-NHC material, possessing a significant number of cationic NHC sites.

One step preparation of plasma based polymer films for drug release.

Among various atmospheric pressure plasma deposition techniques, the so-called "Atmospheric Pressure Plasma Dielectric Barrier Discharges" (APDBD) has recently received a lot of attention due to the easy ignition of a stable discharge and its scalability. In the present work we aim at designing plasma polymer based films for biomedical applications, in which the drug to be released will be directly incorporated in the film during its deposition. Plasma polymer films made of methacrylic acid (MAA) and of ethylene glycol dimethacrylate (EGDMA) were prepared, allowing to obtain smart coatings able to release the molecule of interest, acetaminophen. A combination of different analytic tools shows that the functional groups of the film are well preserved with respect to the monomer structure and that the drug initially put in the gas phase is embedded in the plasma film with its structure being preserved. The physical and chemical characteristics of the elaborated film allowed for the progressive release of acetaminophen by simply dipping the film into a deionized water solution. However only 8% of the acetaminophen present in the monomer mixture could be released slowly in the presence of water. The significance of this result will be discussed.