Antioxidant capacity of an ethanolic extract of Elaeagnus x submacrophylla Servett. leaves.

In this study, we investigated the ethanolic extraction of the leaves of a very common but little studied plant species, Elaeagnus x submacrophylla Servett. and the opportunity of generating an antioxidant ingredient. The phytochemical profile of an ethanolic extract is also described here using gas chromatography and ultra-performance liquid chromatography, both combined with mass spectrometry (GC-MS and UPLC-MS), highlighting the presence of flavonoids, saponins, triterpenoids and a set of volatile compounds. Through in vitro assays (DPPH, ABTS, ORAC), the free radical scavenging capacity of the ingredient was then investigated (from 0.25 to 1.75 mmol TE/g) and compared with well-known standard antioxidants (BHT, gallic acid, quercetin, Trolox and vitamin C). In addition, in cellulo antioxidant capacity was performed using mice fibroblasts, revealing an activity equivalent to 50 mg/L of quercetin when tested the ethanolic extract in the concentration range of 50-300 mg/L, suggesting a synergistic combination effect of the identified phytochemicals. These results support the use of Elaeagnus x submacrophylla as a source of antioxidant ingredients.

Wound Healing Potential of an Oleoresin Essential Oil Chemotype from Canarium schweinfurthii Engl.

This study was conducted to investigate the chemical composition of essential oil (EO) extracted from an oleoresin of Canarium schweinfurthii widespread in the Gabonese tropical forest. A great variability in the chemical composition of EO was observed, among which a chemical profile rich in terpinolene and α-phellandrene (31.2 and 21.8%, respectively), was found and tested as a natural active ingredient for topical applications. After the evaluation of eye and skin irritancy and sensitization potentials of EO on in vitro and in chemico models, the in vitro modulating potential on a model of wound re-epithelialization was assessed. The terpinolene and α-phellandrene-rich chemotype have been proven to accelerate wound healing in a dose-dependent manner (concentration range from 1.8 to 9.0 µg/mL). In addition, the ability of this EO to modulate the pro-inflammatory response in human keratinocytes stimulated by UVB was observed in vitro by the reduction in levels of interleukin 6 (IL-6) and tumour necrosis factor-alpha (TNF-α), suggesting a possible implication during the inflammation phase of wound healing. Despite the high variability in EO composition, a method of solid-phase microextraction (SPME) of the oleoresin headspace is proposed for the in situ identification of the terpinolene and α-phellandrene-rich chemotype instead of conducting hydrodistillation. These results offer interesting perspectives for the development of innovative natural ingredients for the topical route, ingredients obtained in an eco-responsible and non-destructive way.

Xanthurenic Acid in the Shell Purple Patterns of Crassostrea gigas: First Evidence of an Ommochrome Metabolite in a Mollusk Shell.

Ommochromes are one of the least studied groups of natural pigments, frequently confused with melanin and, so far, exclusively found in invertebrates such as cephalopods and butterflies. In this study focused on the purple color of the shells of a mollusk, Crassostrea gigas, the first evidence of a metabolite of ommochromes, xanthurenic acid (XA), was obtained by liquid chromatography combined with mass spectrometry (UPLC-MS). In addition to XA and various porphyrins previously identified, a second group of high molecular weight acid-soluble pigments (HMASP) has been identified with physicochemical and structural characteristics similar to those of ommochromes. In addition, fragmentation of HMASP by tandem mass spectrometry (MS/MS) has revealed a substructure common to XA and ommochromes of the ommatin type. Furthermore, the presence of melanins was excluded by the absence of characteristic by-products among the oxidation residues of HMASP. Altogether, these results show that the purple color of the shells of Crassostrea gigas is a complex association of porphyrins and ommochromes of potentially ommatin or ommin type.

Volatile Compounds from Flowers of Elaeagnus x submacrophylla Servett.: Extraction, Identification of Flavonoids, and Antioxidant Capacity.

Beneficial to the ecosystem and with significant potential in permaculture, Elaeagnus x submacrophylla Servett. was studied here mainly for the identification of its floral odorants. After olfactory evaluation and determination of the volatile profile of freshly picked flowers by headspace/solid phase microextraction coupled with gas chromatography/mass spectrometry, an ethanolic extract was prepared and investigated for its antioxidant capacity. Unusual molecules were identified in the floral headspace, such as isochavicol or chrysanthemum acetate. The evaluation of the in vitro free radical scavenging capacity (from 0.4 to 1.3 mmol TE/g) and total phenolic content (65.1 mg GAE/g) of the extract pointed out a promising antioxidant activity, potentially related to the identification of several flavonoid glycosides. These results have to be considered in the context of the ever-increasing need to produce innovative natural extracts with notably interesting claims for the cosmetic field.

Chemical evidence of rare porphyrins in purple shells of Crassostrea gigas oyster.

The colour of oyster shells is a very diverse characteristic morphotype, forming intriguing vivid patterns both on the inside and outside of the shell. In the present study, we have identified for the first time, the presence of several porphyrins as constituents of the shell pigmentation of the Crassostrea gigas oyster consumed worldwide. The precise molecular structures of halochromic, fluorescent and acid-soluble porphyrins, such as uroporphyrin and turacin, are unambiguously determined by reverse phase liquid chromatography combined with high resolution mass spectrometry. Their presence account for the purple colouration of shells but also for the dark colouration of adductor muscle scars. We have also defined the endogenous origin of these porphyrins, specifically secreted or accumulated by the shell forming tissue. These findings are pioneering analytical proofs of the existence of the haem pathway in the edible oyster Crassostrea gigas, evidenced by the chemical identification of haem side-products and supported by the recent publication of the corresponding oyster genome.

Key Insights, Tools, and Future Prospects on Oyster Shell End-of-Life: A Critical Analysis of Sustainable Solutions.

Oyster farming represents one of the most developed aquaculture activities, producing delicacies unfortunately related to a direct accumulation of waste shells. Facing what is becoming an environmental issue, chemists are currently developing solutions to add value to this wild source of raw material in line with the principles of sustainable chemistry. An argumentative overview of this question is proposed here with a focus on recent data. Starting with a presentation of the environmental impact of oyster farming, existing and promising applications are then classified according to the type of raw materials derived from the oyster shell, namely the natural oyster shell (NOS), the calcined natural oyster shell (CNOS), and biomolecules of the organic matrix extracted from the oyster shell. Their relevance is discussed in regard to their scalability, originality, and sustainability. This review constitutes the first critical compilation on oyster shell applications, with the aim to provide essential elements to better comprehend the recycling of waste oyster shells.

A General Approach to the Aza-Diketomorpholine Scaffold.

A stereoconservative three-step synthesis to access to 1,2,4-oxadiazine-3,6-dione is presented. This underexplored platform could be considered as a constrained oxy-azapeptide or an aza-diketomorpholine, the methodology being then successfully applied to produce enantiopure aza-analogs of diketomorpholine natural products. Importantly, the first crystal structures were obtained and compared to diketomorpholine and diketopiperazine structures. Finally, a straightforward procedure concerning the coupling of this heterocyclic scaffold with various amino acids to afford original pseudodipeptide analogs was described.

MgI2 -chemoselective cleavage for removal of amino acid protecting groups: A fresh vision for peptide synthesis.

In the field of peptide synthesis, the key to a successful access to synthetic targets lies on a pertinent combination of protecting groups. Their choice is directed by their selective removal conditions. We present here the behavior of some of the most used protecting groups in peptide chemistry under experimental cleavage conditions, combining MgI2 with MW irradiation, using 2-Me-THF as a green solvent. In these experimental conditions, the benzyloxycarbonyl protecting group as well as the Merrifield resin can be re-considered in peptide chemistry.

Isoxazolidine: A Privileged Scaffold for Organic and Medicinal Chemistry.

The isoxazolidine ring represents one of the privileged structures in medicinal chemistry, and there have been an increasing number of studies on isoxazolidine and isoxazolidine-containing compounds. Optimization of the 1,3-dipolar cycloaddition (1,3-DC), original methods including electrophilic or palladium-mediated cyclization of unsaturated hydroxylamine, has been developed to obtain isoxazolidines. Novel reactions involving the isoxazolidine ring have been highlighted to accomplish total synthesis or to obtain bioactive compounds, one of the most significant examples being probably the thermic ring contraction applied to the total synthesis of (±)-Gelsemoxonine. The unique isoxazolidine scaffold also exhibits an impressive potential as a mimic of nucleosides, carbohydrates, PNA, amino acids, and steroid analogs. This review aims to be a comprehensive and general summary of the different isoxazolidine syntheses, their use as starting building blocks for the preparation of natural compounds, and their main biological activities.

MgI2 -mediated chemoselective cleavage of protecting groups: an alternative to conventional deprotection methodologies.

The scope of MgI2 as a valuable tool for quantitative and mild chemoselective cleavage of protecting groups is described here. This novel synthetic approach expands the use of protecting groups, widens the concept of orthogonality in synthetic processes, and offers a facile opportunity to release compounds from solid supports.

Stereoselective synthesis of original spirolactams displaying promising folded structures.

Access to diastereoisomeric forms of original spirolactam frameworks and investigation of their folded potentials are depicted here. Taking advantage of a stereoselective ring-contraction reaction, the Transannular Rearrangement of Activated Lactams (TRAL), followed by two unprecedented tandem reactions, we describe here an efficient access to elegant spirocyclic scaffolds. After dimerization, NMR analyses, circular dichroism, SEM and molecular modelling indicated the existence of an attractive edifice able to fold and behave as a PPII helix, a common yet neglected peptidic secondary structure.

Structural recognition mechanisms between human Src homology domain 3 (SH3) and ALG-2-interacting protein X (Alix).

The functions of Src family kinases are tightly regulated through Src homology (SH) domain-mediated protein-protein interactions. We previously reported the biophysical characteristics of the apoptosis-linked gene 2-interacting protein X (Alix) in complex with the haemopoietic cell kinase (Hck) SH3 domain. In the current study, we have combined ITC, NMR, SAXS and molecular modeling to determine a 3D model of the complex. We demonstrate that Hck SH3 recognizes an extended linear proline-rich region of Alix. This particular binding mode enables Hck SH3 to sense a specific non-canonical residue situated in the SH3 RT-loop of the kinase. The resulting model helps clarify the mechanistic insights of Alix-Hck interaction.

A specific protein disorder catalyzer of HIV-1 Nef.

The HIV-1 auxiliary protein Nef is required for the onset and progression of AIDS in HIV-1-infected persons. Here, we have deciphered the mode of action of a second-generation inhibitor of Nef, DLC27-14, presenting a competitive IC(50) of ∼16 µM measured by MALDI-TOF experiments. Thermal protein denaturation experiments revealed a negative effect on stability of Nef in the presence of a saturating concentration of the inhibitor. The destabilizing action of DLC27-14 was confirmed by a HIV protease-based experiment, in which the protease sensitivity of DLC27-14-bound Nef was three times as high as that of apo Nef. The only compatible docking modes of action for DLC27-14 suggest that DLC27-14 promotes an opening of two α-helices that would destabilize the Nef core domain. DLC27-14 thus acts as a specific protein disorder catalyzer that destabilizes the folded conformation of the protein. Our results open novel avenues toward the development of next-generation Nef inhibitors.

Stereoselective ring contraction of 2,5-diketopiperazines: An innovative approach to the synthesis of promising bioactive 5-membered scaffolds.

Ring contraction of 2,5-diketopiperazines by TRAL-alkylation led us to the stereoselective synthesis of original pyrrolidine-2,4-diones, a novel series of promising molecules with moderate anti-proliferative activity on breast cancer cells.

Identification and biophysical assessment of the molecular recognition mechanisms between the human haemopoietic cell kinase Src homology domain 3 and ALG-2-interacting protein X.

SFKs (Src family kinases) are central regulators of many signalling pathways. Their functions are tightly regulated through SH (Src homology) domain-mediated protein-protein interactions. A yeast two-hybrid screen using SH3 domains as bait identified Alix [ALG-2 (apoptosis-linked gene 2)-interacting protein X] as a novel Hck (haemopoietic cell kinase) SH3 domain interactor. The Alix-Hck-SH3 interaction was confirmed in vitro by a GST (glutathione transferase) pull-down assay and in intact cells by a mammalian two-hybrid assay. Furthermore, the interaction was demonstrated to be biologically relevant in cells. Through biophysical experiments, we then identified the PRR (proline-rich region) motif of Alix that binds Hck-SH3 and determined a dissociation constant of 34.5 µM. Heteronuclear NMR spectroscopy experiments were used to map the Hck-SH3 residues that interact with an ALIX construct containing the V and PRR domains or with the minimum identified interacting motif. Finally, SAXS (small-angle X-ray scattering) analysis showed that the N-terminal PRR of Alix is unfolded, at least before Hck-SH3 recognition. These results indicate that residues outside the canonical PxxP motif of Alix enhance its affinity and selectivity towards Hck-SH3. The structural framework of the Hck-Alix interaction will help to clarify how Hck and Alix assist during virus budding and cell-surface receptor regulation.

Transannular rearrangement of activated 2,5-diketopiperazines: a key route to original scaffolds.

An efficient and original stereocontrolled transannular rearrangement starting from activated 2,5-diketopiperazines has been developed, an opportunity for the medicinal chemistry field, which requests access to novel biological scaffolds. This powerful ring contraction, which can be related to a stereoselective aza-version of the Chan rearrangement, allows for example the one-step synthesis of various tetramic acids, access to 2-disubstituted statins, or the synthesis of relevant lactam-constrained dipeptide mimetics using a TRAL-RCM sequence.

Protein protein interaction inhibition (2P2I) combining high throughput and virtual screening: Application to the HIV-1 Nef protein.

Protein-protein recognition is the cornerstone of multiple cellular and pathological functions. Therefore, protein-protein interaction inhibition (2P2I) is endowed with great therapeutic potential despite the initial belief that 2P2I was refractory to small-molecule intervention. Improved knowledge of complex molecular binding surfaces has recently stimulated renewed interest for 2P2I, especially after identification of "hot spots" and first inhibitory compounds. However, the combination of target complexity and lack of starting compound has thwarted experimental results and created intellectual barriers. Here we combined virtual and experimental screening when no previously known inhibitors can be used as starting point in a structure-based research program that targets an SH3 binding surface of the HIV type I Nef protein. High-throughput docking and application of a pharmacophoric filter on one hand and search for analogy on the other hand identified drug-like compounds that were further confirmed to bind Nef in the micromolar range (isothermal titration calorimetry), to target the Nef SH3 binding surface (NMR experiments), and to efficiently compete for Nef-SH3 interactions (cell-based assay, GST pull-down). Initial identification of these compounds by virtual screening was validated by screening of the very same library of compounds in the cell-based assay, demonstrating that a significant enrichment factor was attained by the in silico screening. To our knowledge, our results identify the first set of drug-like compounds that functionally target the HIV-1 Nef SH3 binding surface and provide the basis for a powerful discovery process that should help to speed up 2P2I strategies and open avenues for new class of antiviral molecules.

Fluorescent pirenzepine derivatives as potential bitopic ligands of the human M1 muscarinic receptor.

Following a recent description of fluorescence resonance energy transfer between enhanced green fluorescent protein (EGFP)-fused human muscarinic M1 receptors and Bodipy-labeled pirenzepine, we synthesized seven fluorescent derivatives of this antagonist in order to further characterize ligand-receptor interactions. These compounds carry Bodipy [558/568], Rhodamine Red-X [560/580], or Fluorolink Cy3 [550/570] fluorophores connected to pirenzepine through various linkers. All molecules reversibly bind with high affinity to M1 receptors (radioligand and energy transfer binding experiments) provided that the linker contains more than six atoms. The energy transfer efficiency exhibits modest variations among ligands, indicating that the distance separating EGFP from the fluorophores remains almost constant. This also supports the notion that the fluorophores may bind to the receptor protein. Kinetic analyses reveal that the dissociation of two Bodipy derivatives (10 or 12 atom long linkers) is sensitive to the presence of the allosteric modulator brucine, while that of all other molecules (15-24 atom long linkers) is not. The data favor the idea that these analogues might interact with both the acetylcholine and the brucine binding domains.

Circular dichroism and nuclear magnetic resonance spectroscopic analysis of immunogenic gluten peptides and their analogs.

Celiac Sprue, or gluten-sensitive enteropathy, is an inheritable human disease of the small intestine that is triggered by the dietary intake of gluten. Recently, several Pro- and Gln-rich peptide sequences (most notably PQPQLPY and analogs) have been identified from gluten with potent immunogenic activity toward CD4(+) T cells from small intestinal biopsies of Celiac Sprue patients. These peptides have three unusual properties. First, they are relatively stable toward further proteolysis by gastric, pancreatic, and intestinal enzymes. Second, they are recognized and deamidated by human tissue transglutaminase (tTGase) with high selectivity. Third, tTGase-catalyzed deamidation enhances their affinity for HLA-DQ2, the disease-specific class II major histocompatibility complex heterodimer. In an attempt to seek a mechanistic explanation for these properties, we undertook secondary structural studies on PQPQLPY and its analogs. Circular dichroism studies on a series of monomeric and dimeric analogs revealed a strong polyproline II helical propensity in a subset of them. Two-dimensional nuclear magnetic resonance spectroscopic analysis confirmed a polyproline II conformation of PQPQLPY, and was also used to elucidate the secondary structure of the most helical variant, (D-P)QPQLPY. Remarkably, a strong correlation was observed between polyproline II content of naturally occurring gluten peptides and the specificity of human tTGase toward these substrates. Analogs with up to two D-amino acid residues retained both polyproline II helical content and transglutaminase affinity. Since the Michaelis constant (K(m)) is the principal determinant of tTGase specificity for naturally occurring gluten peptides and their analogs, our results suggest that the tTGase binding site may have a preference for polyproline II helical substrates. If so, these insights could be exploited for the design of selective small molecule inhibitors of this pharmacologically important enzyme.