Objective. This study investigated the chemical and structural changes in the mineral phase and collagen of dentin during application of a mild universal adhesive. Particular attention was paid to the role of isopropanol and changes in water molecules. Methods. In vitro application of the mild universal adhesive on dentin with two established etching modes (self-etch and etch-and-rinse) was studied using solid state nuclear magnetic resonance spectroscopy. Results. It was evidenced that the etch-and-rinse mode leads to a decrease of the inorganic apatite and a reorganization of the residual mineral phase with a low amount of adhesive phosphate monoesters calcium salt formed, compared to the self-etch mode. In contrast, the adhesive interacts very similarly to the level of dentin collagen in both protocols, with a strong decrease in the amount of the free water molecules induced by the presence of isopropanol as the adhesive solvent, but without significant changes in the initial collagen structure. For both modes, the adhesive acrylates monomers remain mobile and can infiltrate the collagen. Significance. Understanding the molecular interactions between dentin and adhesive solutions is a major challenge for designing products that lead to the formation of ideal dentin resin hybrid layer. Notably, one point considered essential is the presence of unbound water which, over time, is associated with a hydrolytic degradation of the organic matrix. Isopropanol, as an adhesive solvent, leads to a decrease in the amount of the less stable water molecules while the water molecules strongly attached to the collagen are retained, thus preserving the collagen structure. (c) 2021 The Academy of Dental Materials. Published by Elsevier Inc. All rights reserved.

Collecting information about molecular organisation on biological materials such as bone and dentine represents a major challenge in attaining a better understanding of their mechanical properties. To that end, solid state Nuclear Magnetic Resonance (ssNMR) spectroscopy is an appropriate strategy to provide atomic structural details on these amorphous composite materials. However, species like water molecules and hydroxyl groups are usually observed through 1H magic angle spinning (MAS) ssNMR that suffers from poor resolution due to strong signal overlapping, making their identification difficult. This paper proposes a set of ssNMR experiments for 1H characterization of the main components of human dentin, based on homo- and hetero-nuclear dipolar couplings and composed mostly of fast 1D experiments. The 1H assignment is assisted by straightforward sample modifications: vacuum drying, deuterium exchange and demineralization. These experiments allow the hydrogen signal edition of dentin species like water molecules, HPO42- and OH- groups, depending on their localization (bound to the organic phase, linked to apatite or at the interface) and their dynamic behaviour. This ssNMR toolbox has the potential to provide important structural and dynamic information on chemical and physical modifications of biomaterials.

A universal adhesive was applied to human dentin in both the etched and rinsed state and the normal non etched state, to compare the resulting properties and detect any significant differences. The study focused on observations of the hybrid layer by scanning electron microscopy and on fluid permeation measurements as a function of time. Spectroscopic characterizations included infrared and differential calorimetric curves of the samples. The results obtained show non-statistically significant fluid permeability between the two sample types. Both the etched and rinsed samples and the non-etched ones showed similar homogeneous hybrid layers that reduced the fluid flow, and corresponded to well spread polymer coatings. The infrared results illustrated the spectra obtained on going from the outside adhesive layer to the inside portion of the dentin-polymer interface and did not reveal any intermediate zone resembling demineralized collagen that would be water saturated and not infiltrated with adhesive. The Differential Scanning Calorimetry (DSC) curves corresponded to the curves obtained with ethanol wet bonding in that free water (melting at 0 degrees C) was removed by the universal adhesive, and that no collagen melting was observed for the non-etched samples. The Diffusion-Ordered Spectroscopy Nuclear Magnetic Resonance (DOSY NMR) spectrum of the virgin adhesive showed the presence of water and ethanol solvents and indicated that several monomer or prepolymer molecules were present with multiple acrylic functional groups with diffusion coefficients related to molecular weights.Overall, the results show that universal adhesive can be used in the milder self-etch mode and that more aggressive etch and rinse procedure can be reserved for the occasions with sclerotic dentin or enamel regions more difficult to treat. (C) 2016 Published by Elsevier B.V.

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The Stokes-Einstein expression of the diffusion coefficient as a function of the hydrodynamic radius of the diffusing object does not explicitly carry the mass dependency of the object. It is possible to correlate the translational self-diffusion coefficients D with the molecular weight M for an ensemble of cyclic or hollow clusters ranging from about 200 to 30,000 g x mol(-1). From this correlation, the mass of a cluster can be deduced from its diffusion coefficient. Consistency of diffusion as a power law of mass and Stokes-Einstein formulation is completely fulfilled with the selected compounds of this contribution.

Experimental NMR diffusion measure on polymers and on globular proteins are presented. These results, complemented with results found in the literature, enable a general description of effective fractal dimension for objects such as small organic molecules, sugars, polymers, DNA, and proteins. Results are compared to computational simulations as well as to theoretical values. A global picture of the diffusion phenomenon emerges from this description. A power law relating molecular mass with diffusion coefficients is described and found to be valid over 4 orders of magnitude. From this law, the fractal dimension of the molecular family can be measured, with experimental values ranging from 1.41 to 2.56 in full agreement with theoretical approaches. Finally, a method for evaluating the molecular mass of unknown solutes is described and implemented as a Web page.

The Bipolar Pulse Pair Stimulated Echo NMR pulse sequence was modified to blend the original Excitation Sculpting water signal suppression. The sequence is a powerful tool to generate rapidly, with a good spectrum quality, bidimensional DOSY experiments without solvent signal, thus allowing the analysis of complex mixtures such as plant extracts or biofluids. The sequence has also been successfully implemented for a protein at very-low concentration in interaction with a small ligand, namely the salivary IB5 protein binding the polyphenol epigallocatechine gallate. The artifacts created by this sequence can be observed, checked and removed thanks to NPK and NMRnotebook softwares to give a perfect bidimensional DOSY spectrum.

La mesure de diffusion par RMN et les experiences de type DOSY sont de puissants outils analytiques qui ont ete longtemps sous exploites par les scientifiques. Les spectrometres RMN modernes permettent de realiser de telles experiences dans des conditions optimales. Toutefois, un traitement mathematique complexe des donnees recueillies - la transformee inverse de Laplace -est indispensable pour l'obtention de resultats pertinents et precis. Cette difficulte est aujourd'hui surmontee et le traitement des donnees RMN parfaitement maitrise grâce au logiciel Glfa©. Nous presentons ici l'experience DOSY/RMN appliquee a l'analyse de melanges complexes et a la detection de traces dans divers domaines industriels (agro-alimentaire, pharmaceutique, ecologie), et differents secteurs d'activite (recherche et developpement, analyse concurrentielle, controle qualite).

The solution structure of a new B-chain mutant of bovine insulin, in which the cysteines B7 and B19 are replaced by two serines, has been determined by circular dichroism, 2D-NMR and molecular modeling. This structure is compared with that of the oxidized B-chain of bovine insulin [Hawkins et al. (1995) Int. J. Peptide Protein Res.46, 424-433]. Circular dichroism spectroscopy showed in particular that a higher percentage of helical secondary structure for the B-chain mutant is estimated in trifluoroethanol solution in comparison with the oxidized B-chain. 2D-NMR experiments confirmed, among multiple conformations, that the B-chain mutant presents defined secondary structures such as a alpha-helix between residues B9 and B19, and a beta-turn between amino acids B20 and B23 in aqueous trifluoroethanol. The 3D structures, which are consistent with NMR data and were obtained using a simulated annealing protocol, showed that the tertiary structure of the B-chain mutant is better resolved and is more in agreement with the insulin crystal structure than the oxidized B-chain structure described by Hawkins et al. An explanation could be the presence of two sulfonate groups in the oxidized insulin B-chain. Either by their charges and/or their size, such chemical groups could play a destructuring effect and thus could favor peptide flexibility and conformational averaging. Thus, this study provides new insights on the folding of isolated B-chains.

Longibrachins are members of the class of natural Aib-containing peptides designated as peptaibols. Six longibrachins, LGA I-IV and LGB II and III, were purified from a Trichoderma longibrachiatum strain by a procedure employing several chromatography steps including reversed-phase HPLC. The amino acid sequence determination was based on a combination of liquid secondary ion mass spectrometry (LSIMS) and two-dimensional H-1 and C-13 NMR spectroscopy. Longibrachins are 20-residue peptaibols with a C-terminal phenylalaninol and either neutral (LGA; Gln(18)) or acidic (LGB; Glu(18)) character. Longibrachins LGB II and III have novel sequences. Both longibrachins LGA and LGB show significant bactericidal activity against mycoplasmas (Acholeplasma, Mycoplasma, and Spiroplasma), with minimal inhibitory concentrations in the range 1.56-12.5 muM (3-25 mug/mL), and also perturb the permeability of membrane bilayers. Longibrachin LGA IV is the most potent of the presently known 18-20-residue peptaibols. The antimicrobial and membrane-perturbing properties of longibrachins, which are described here for the first time, were shown to be correlated.

The aim of this work was to explore the synthesis of a tosylated derivative of diprenorphine (DPN) able to be radiolabeled with either fluorine-18 or iodine-123, making it suitable for PET or SPECT imaging studies of central opioid receptors, respectively. The strategy was based on the reactivity of the C-19 alcohol tertiary function. As an unexpected deacetylation of the phenolic function of the diprenorphine occurred, the prosthetic group reacted with the deprotected C-3 phenolic function instead of the C-19 alcohol group. UV spectroscopy and H-1 and C-13 NMR studies provided good evidence for the 3-phenolic substituted diprenorphine structure. Thorough 2D NMR experiments such as H-1-H-1 COSY, H-1-H-1 TOCSY, H-1-C-13 HMQC, H-1-C-13 HMBC and H-1-H-1 NOESY allowed us to assign fully 3-O-[(Z)-4-fluorobut-2-enyl] diprenorphine (10a) and gave us an unambiguous proof of the C-3 prosthetic group position. In vitro binding studies showed low affinity for both fluoro and iodo derivatives of diprenorphine, K-i = 0.31 +/- 0.05 and 0.09 +/- 0.03 mum, respectively, for mouse brain membranes, these inhibition constants also being in agreement with a 3-phenolic substituted structure. Copyright (C) 2001 John Wiley & Sons, Ltd.

Mono-2 and mono-6-O-pentenyl-beta-cyclodextrin (mono-2-pent-beta-CD and mono-6-pent-beta-CD), covalently linked to mercaptopropylsilica gel (thiol-Si) through thioether or sulfone linkage, reveal differentiated enantioselectivities in the separation of piperidine-2,6-dione-related drugs, namely aminoglutethimide and thalidomide, in supercritical fluid conditions. Supercritical fluid chromatographic resolution on completely defined mono-cyclodextrin derivative-based chiral stationary phases (CSP) is a method of choice for the separation of aminoglutethimide but not effective for thalidomide. For both high performance liquid chromatography (HPLC) and supercritical fluid chromatography (SFC) conditions, the impact of the position, imposed to be 2 or 6 in our synthetic pathway, of the pentenyl moiety on one of the glucopyranosidics of the CD cage is of crucial importance in the chiral discrimination phenomenon. Additionally, the nature of the heteroatom present in the spacer arm between the CD and the silica gel, in this case thioether or sulfone functionality, is also essential for the chiral recognition mechanism(s) for the solute enantiomer.

Various NADH models with the following characteristics were studied and compared with previously reported models: (1) use of (S)-phenylalaninol as chiral auxiliary; (2) orientation in or out of the plane of the amide carbonyl. Despite the occurrence of apparently similar characteristics, they gave very different results in the asymmetric reduction of methyl benzoylformate. A detailed NMR study was performed in order to explain the behaviour of these models.

Microcin J25 (MccJ25) is the single representative of the immunity group J of the microcin group of peptide antibiotics produced by Enterobacteriaceae. It induces bacterial filamentation in susceptible cells in a non‐SOS‐dependent pathway [R. A. Salomon and R. Farias (1992) J. Bacteriol.174, 7428–7435]. MccJ25 was purified to homogeneity from the growth medium of a microcin‐overproducing Escherichia coli strain by reverse‐phase HPLC. Based on amino acid composition and absolute configuration determination, liquid secondary ion and electrospray mass spectrometry, extensive two‐dimensional NMR, enzymatic and chemical degradations studies, the structure of MccJ25 was elucidated as a 21‐residue peptide, cyclo(‐Val1‐Gly‐Ile‐Gly‐Thr‐Pro‐Ile‐Ser‐Phe‐Tyr‐Gly‐Gly‐Gly‐Ala‐Gly‐His‐Val‐Pro‐Glu‐Tyr‐Phe21‐). Although MccJ25 showed high resistance to most of endoproteases, linearization by thermolysin occurred from cleavage at the Phe21‐Val1 bond and led to a single peptide, MccJ25‐L. While MccJ25 exhibited remarkable antibiotic activity towards Salmonella newport and several E. coli strains (minimal inhibitory concentrations ranging between 0.01 and 0.2 µg·mL–1), the thermolysin‐linearized microcin showed a dramatic decrease of the activity, indicating that the cyclic structure is essential for the MccJ25 biological properties. As MccJ25 is ribosomally synthesized as a larger peptide precursor endowed with an N‐terminal extremity, the present study shows that removal of this extension and head–tail cyclization of the resulting propeptide are the only post‐translational modifications involved in the maturation of MccJ25, that appears as the first cyclic microcin.

The solution structure of the R2 repeat of the DNA binding domain of the protooncogene c-Myb contains a N-terminal structural motif comprising two antiparallel helices. The motif is stabilized by interactions involving conserved residues. The recognition region in C-terminal position is flexible. This structure differs from that of R2 of another c-Myb protein.

The three-dimensional solution structure of harzianin HC IX, a peptaibol antibiotic isolated from the fungus Trichoderma harzianum, was determined using CD, homonuclear, and heteronuclear two-dimensional nmr spectroscopy combined with molecular modeling. This 14-residue peptide, Ac Aib1 Asn2 Leu3 Aib4 Pro5 Ala6 Ile7 Aib8 Pro9 Iva10 Leu11 Aib12 Pro13 Leuol14 (Aib, α-aminoisobutyric acid; Iva, isovaline; Leuol, leucinol), is a main representative of a short-sequence peptaibol class characterized by an acetylated N-terminus, a C-terminal amino alcohol, and the presence of three Aib-L-Pro motifs at positions 4–5, 8–9, and 12–13, separated by two dipeptide units. In spite of a lower number of residues, compared to the 18/20-residue peptaibols such as alamethicin, harzianin HC IX exhibits remarkable membrane-perturbing properties. It interacts with phospholipid bilayers, increasing their permeability and forming voltage-gated ion channels through a mechanism slightly differing from that proposed for alamethicin. Sequence-specific 1H- and 13C-nmr assignments and conformational nmr parameters (3JNHCαH coupling constants, quantitative nuclear Overhauser enhancement data, temperature coefficients of amide and carbonyl groups, NH–ND exchange rates) were obtained in methanol solution. Sixty structures were calculated based on 98 interproton distance restraints and 6 Φ dihedral angle restraints, using high temperature restrained molecular dynamics and energy minimization. Thirty-seven out of the sixty generated structures were consistent with the nmr data and were convergent. The peptide backbone consists in a ribbon of overlapping β-turns twisted into a continuous spiral from Asn2 to Leuol14 and forming a 26 Å long helix-like structure. This structure is slightly amphipathic, with the three Aib–Pro motifs aligned on the less hydrophobic face of the spiral where the Asn2 side chain is also present, while the more hydrophobic bulky side chains of leucines, isoleucine, isovaline, and leucinol are located on the concave side. The repetitive (Xaa–Yaa–Aib–Pro) tetrapeptide subunit, making up the peptide sequence, is characterized by four sets of (Φ,Ψ) torsional angles, with the following mean values: Φi = −90°, Ψi = −27°; Φi+1 = −98°, Ψi+1 = −17°; Φi+2 = −49°, Ψi+2 = −50°; Φi+3 = −78°, Ψi+3 = +3°. We term this particular structure, specifically occurring in the case of (Xaa–Yaa–Aib–Pro)n sequences, the (Xaa–Yaa–Aib–Pro)-β-bend ribbon spiral. It is stabilized by 4 → 1 intramolecular hydrogen bonds and differs from both the canonical 310-helix made of a succession of type III β-turns and from the β-bend ribbon spiral that has been described in the case of (Aib–Pro)n peptide segments. © 1999 John Wiley & Sons, Inc. Biopoly 50: 71–85, 1999

Trichorzianin TA VII, Ac0 U1 A2 A3 U4 J5 Q6 U7 U8 U9 S10 L11 U12 P13 V14 U15 I16 Q17 Q18 Fol19, is a nonadecapeptide member of the peptaibol antibiotics biosynthesized by Trichoderma soil fungi, which is characterized by a high proportion of the alpha, alpha-dialkylated amino acids, alpha-aminoisobutyric acid (Aib, U) and isovaline (Iva, J), an acetylated N-terminus and a C-terminal phenylalaninol (Pheol, Fol). The main interest in such peptides stems from their ability to interact with phospholipid bilayers and form voltage-dependent transmembrane channels in planar lipid bilayers. In order to provide insights into the lipid-peptide interaction promoting the voltage gating, the conformational study of TA VII in the presence of perdeuterated sodium dodecyl sulfate (SDS-d25) micelles has been carried out. 1H sequential assignment have been performed with the use of two-dimensional homo- and -heteronuclear nmr techniques including double quantum filtered correlated spectroscopy, homonuclear Hartmann-Hahn, nuclear Overhauser effect spectroscopy, 1H-13C heteronuclear single quantum correlation, and heteronuclear multiple bond correlation. Conformational parameters, such as 3JNHC alpha H coupling constants, temperature coefficients of amide protons (delta gamma/delta TNH) and quantitative nuclear Overhauser enhancement data, lead to detailed structural information. Ninety-eight three-dimensional structures consistent with the nmr data were generated from 231 interproton distances six phi dihedral angle restraints, using restrained molecular dynamics and energy minimization calculations. The average rms deviation between the 98 refined structures and the energy-minimized average structure is 0.59 A for the backbone atoms. The structure of trichorzianin TA VII associated with SDS micelles, as determined by these methods, is characterized by two right-handed helical segments involving residues 1-8 and 11-19, linked by a beta-turn that leads to an angle about 90 degrees-100 degrees between the two helix axes; residues 18 and 19 at the end of the C-terminal helix exhibit multiple conformations.