ß-Turn Induction by a Diastereopure Azepane-Derived Quaternary Amino Acid.

ß-Turns are one of the most common secondary structures found in proteins. In the interest of developing novel ß-turn inducers, a diastereopure azepane-derived quaternary amino acid has been incorporated into a library of simplified tetrapeptide models in order to assess the effect of the azepane position and peptide sequence on the stabilization of ß-turns. The conformational analysis of these peptides by molecular modeling, NMR spectroscopy, and X-ray crystallography showed that this azepane amino acid is an effective ß-turn inducer when incorporated at the i + 1 position. Moreover, the analysis of the supramolecular self-assembly of one of the ß-turn-containing peptide models in the solid state reveals that it forms a supramolecular helical arrangement while maintaining the ß-turn structure. The results here presented provide the basis for the use of this azepane quaternary amino acid as a strong ß-turn inducer in the search for novel peptide-based bioactive molecules, catalysts, and biomaterials.

Interference of the complex between NCS-1 and Ric8a with phenothiazines regulates synaptic function and is an approach for fragile X syndrome.

The protein complex formed by the Ca2+ sensor neuronal calcium sensor 1 (NCS-1) and the guanine exchange factor protein Ric8a coregulates synapse number and probability of neurotransmitter release, emerging as a potential therapeutic target for diseases affecting synapses, such as fragile X syndrome (FXS), the most common heritable autism disorder. Using crystallographic data and the virtual screening of a chemical library, we identified a set of heterocyclic small molecules as potential inhibitors of the NCS-1/Ric8a interaction. The aminophenothiazine FD44 interferes with NCS-1/Ric8a binding, and it restores normal synapse number and associative learning in a Drosophila FXS model. The synaptic effects elicited by FD44 feeding are consistent with the genetic manipulation of NCS-1. The crystal structure of NCS-1 bound to FD44 and the structure-function studies performed with structurally close analogs explain the FD44 specificity and the mechanism of inhibition, in which the small molecule stabilizes a mobile C-terminal helix inside a hydrophobic crevice of NCS-1 to impede Ric8a interaction. Our study shows the drugability of the NCS-1/Ric8a interface and uncovers a suitable region in NCS-1 for development of additional drugs of potential use on FXS and related synaptic disorders.

Chiral Microneedles from an Achiral Bis(boron dipyrromethene): Spontaneous Mirror Symmetry Breaking Leading to a Promising Photoluminescent Organic Material.

Supramolecular self-assembly of a highly flexible and achiral meso bis(boron dipyrromethene) [bis(BODIPY)] dye straightforwardly yields fluorescent microfibers, exhibiting an intriguing anisotropic photonic behavior. This performance includes the generation of chiroptical activity owing to spontaneous mirror symmetry breaking (SMSB). Repetition of several self-assembly experiments demonstrates that the involved SMSB is not stochastic but quasi deterministic in the direction of the induced chiral asymmetry. The origin of these intriguing (chiro)photonic properties is revealed by fluorescent microspectroscopy studies of individual micrometric objects, combined with X-ray diffraction elucidation of microcrystals. Such a study demonstrates that J-like excitonic coupling between bis(BODIPY) units plays a fundamental role in their supramolecular organization, leading to axial chirality. Interestingly, the photonic behavior of the obtained fibers is ruled by inherent nonradiative pathways from the involved push-pull chromophores, and mainly by the complex excitonic interactions induced by their anisotropic supramolecular organization.

An Example of Polynomial Expansion: The Reaction of 3(5)-Methyl-1H-Pyrazole with Chloroform and Characterization of the Four Isomers.

The reaction in phase-transfer catalyzed conditions of 3(5)-methyl-1H-pyrazole with chloroform affords four isomers 333, 335, 355 and 555 in proportions corresponding to the polynomial expansion (a + b)³, with a = 0.6 and b = 0.4, a and b being 3-methyl and 5-methyl proportions. The up (u) and down (d) conformation of the pyrazolyl rings with regard to the Csp³â»H atom was established by X-ray crystallography and by ¹H-, 13C- and 15N-NMR in solution combined with gauge-including atomic orbitals (GIAO)/B3LYP/6-311++G(d,p) calculations. A comparison with other X-ray structures of tris-pyrazolylmethanes was carried out.

Experimental and theoretical studies on the rearrangement of 2-oxoazepane α,α-amino acids into 2'-oxopiperidine ß(2,3,3) -amino acids: an example of intramolecular catalysis.

Enantiopure ß-amino acids represent interesting scaffolds for peptidomimetics, foldamers and bioactive compounds. However, the synthesis of highly substituted analogues is still a major challenge. Herein, we describe the spontaneous rearrangement of 4-carboxy-2-oxoazepane α,α-amino acids to lead to 2'-oxopiperidine-containing ß(2,3,3) -amino acids, upon basic or acid hydrolysis of the 2-oxoazepane α,α-amino acid ester. Under acidic conditions, a totally stereoselective synthetic route has been developed. The reordering process involved the spontaneous breakdown of an amide bond, which typically requires strong conditions, and the formation of a new bond leading to the six-membered heterocycle. A quantum mechanical study was carried out to obtain insight into the remarkable ease of this rearrangement, which occurs at room temperature, either in solution or upon storage of the 4-carboxylic acid substituted 2-oxoazepane derivatives. This theoretical study suggests that the rearrangement process occurs through a concerted mechanism, in which the energy of the transition states can be lowered by the participation of a catalytic water molecule. Interestingly, it also suggested a role for the carboxylic acid at position 4 of the 2-oxoazepane ring, which facilitates this rearrangement, participating directly in the intramolecular catalysis.

Synthesis of Enantiopure 3-Hydroxypiperidines from Sulfinyl Dienyl Amines by Diastereoselective Intramolecular Cyclization and [2,3]-Sigmatropic Rearrangement.

The highly diastereoselective base-promoted intramolecular cyclization of a variety of enantiopure sulfinyl dienyl amines provides novel sulfinyl tetrahydropyridines that are readily converted to 3-hydroxy tetrahydropyridines via sigmatropic rearrangement. The influence of N- and C- substituents on the process has been studied. Procedures to shorten the sequence such as the tandem cyclization followed by [2,3]-sigmatropic rearrangement, as well as cyclization of the free amine, under Boc- or ArSO- deprotection conditions have been examined. Good to excellent levels of selectivity are generally observed for the reported transformations (dr: 75/25 to >98/2). A novel protocol to access substituted amino dienyl sulfoxides is also reported.

The reaction of 2-amino-4H-pyrans with N-bromosuccinimide.

The reaction of racemic 2-amino-4H-pyrans, such as 3-amino-1-aryl-1H-benzo[f]chromene-2-carbonitriles, with N-bromosuccinimide (NBS), in CH2Cl2, at room temperature, is a very quick, regio, stereoselective, and high yielding process, affording major racemic (1S, 2S)-2-bromo-3-imino-benzo[f]chromene or racemic (1S, 2S)-2-bromo-3-(bromoimino)-benzo[f]chromene derivatives, when using 1.0 or 2.2 equivalents of NBS, respectively. This reaction, extended to isomeric 2-amino-4-aryl-4H-benzo[h]chromene-3-carbonitriles, showed an unexpected reactivity, affording racemic (3S,4S)-3-bromo-2-(bromoimino)-benzo[h]chromene-3-carbonitriles or 2-oxo-2H-benzo[h]chromene-3-carbonitriles, when using 2.2 or 1.0 equivalents of NBS, respectively. The reaction of alkyl 6-amino-5-cyano-2-methyl-4H-pyran-3-carboxylates has yielded unstable racemic (3S,4S)-alkyl 3-bromo-2-(bromoimino)-3-cyano-6-methyl-3,4-dihydro-2H-pyran-5-carboxylates. The mechanism of these reactions has been investigated by computational methods.

Substrate promiscuity of inositol 1,4,5-trisphosphate kinase driven by structurally-modified ligands and active site plasticity.

D-myo-inositol 1,4,5-trisphosphate (InsP3) is a fundamental second messenger in cellular Ca2+ mobilization. InsP3 3-kinase, a highly specific enzyme binding InsP3 in just one mode, phosphorylates InsP3 specifically at its secondary 3-hydroxyl group to generate a tetrakisphosphate. Using a chemical biology approach with both synthetised and established ligands, combining synthesis, crystallography, computational docking, HPLC and fluorescence polarization binding assays using fluorescently-tagged InsP3, we have surveyed the limits of InsP3 3-kinase ligand specificity and uncovered surprisingly unforeseen biosynthetic capacity. Structurally-modified ligands exploit active site plasticity generating a helix-tilt. These facilitated uncovering of unexpected substrates phosphorylated at a surrogate extended primary hydroxyl at the inositol pseudo 3-position, applicable even to carbohydrate-based substrates. Crystallization experiments designed to allow reactions to proceed in situ facilitated unequivocal characterization of the atypical tetrakisphosphate products. In summary, we define features of InsP3 3-kinase plasticity and substrate tolerance that may be more widely exploitable.

Structure-guided engineering of a receptor-agonist pair for inducible activation of the ABA adaptive response to drought.

Strategies to activate abscisic acid (ABA) receptors and boost ABA signaling by small molecules that act as ABA receptor agonists are promising biotechnological tools to enhance plant drought tolerance. Protein structures of crop ABA receptors might require modifications to improve recognition of chemical ligands, which in turn can be optimized by structural information. Through structure-based targeted design, we have combined chemical and genetic approaches to generate an ABA receptor agonist molecule (iSB09) and engineer a CsPYL1 ABA receptor, named CsPYL15m, which efficiently binds iSB09. This optimized receptor-agonist pair leads to activation of ABA signaling and marked drought tolerance. No constitutive activation of ABA signaling and hence growth penalty was observed in transformed Arabidopsis thaliana plants. Therefore, conditional and efficient activation of ABA signaling was achieved through a chemical-genetic orthogonal approach based on iterative cycles of ligand and receptor optimization driven by the structure of ternary receptor-ligand-phosphatase complexes.

Azepane quaternary amino acids as effective inducers of 3(10) helix conformations.

A simple method for the synthesis of an azepane quaternary amino acid in enantiopure form is described. Theoretical, NMR, and X-ray studies indicated that this azepane-derived amino acid is an effective stabilizer of 3(10) helical structures in short peptides.

Structure-Based Modulation of the Ligand Sensitivity of a Tomato Dimeric Abscisic Acid Receptor Through a Glu to Asp Mutation in the Latch Loop.

The binding of the plant phytohormone Abscisic acid (ABA) to the family of ABA receptors (PYR/PYL/RCAR) triggers plant responses to abiotic stress. Thus, the implementation of genetic or chemical strategies to modulate PYR/PYL activity might be biotechnologically relevant. We have employed the available structural information on the PYR/PYL receptors to design SlPYL1, a tomato receptor, harboring a single point mutation that displays enhanced ABA dependent and independent activity. Interestingly, crystallographic studies show that this mutation is not directly involved in ABA recognition or in the downstream phosphatase (PP2C) inhibitory interaction, rather, molecular dynamic based ensemble refinement restrained by crystallographic data indicates that it enhances the conformational variability required for receptor activation and it is involved in the stabilization of an active form of the receptor. Moreover, structural studies on this receptor have led to the identification of niacin as an ABA antagonist molecule in vivo. We have found that niacin blocks the ABA binding site by mimicking ABA receptor interactions, and the niacin interaction inhibits the biochemical activity of the receptor.

Quaternary α,α-2-oxoazepane α-amino acids: synthesis from ornithine-derived ß-lactams and incorporation into model dipeptides.

To explore further the chemistry of amino acid-derived ß-lactams, their conversion to α,α-heterocyclic quaternary amino acid derivatives is investigated. The latter derivatives, containing 2-oxoazepane as the α,α-substituent, are synthesized by a simple Pd-C-catalyzed hydrogenolysis of Orn(Z)-derived 2-azetidinones. The rearrangement from four- to seven-membered lactam ring is driven by the key intramolecular opening of the 1-Boc-ß-lactam, initiated by 7-exotrig ring closure from the NH(2) of the Orn side chain. The synthetic route is applied to the stereoselective preparation of enantiomerically pure 4-amino-3-methyl-2-oxoazepane-4-carboxylate derivatives, for which the structure and configuration is confirmed by X-ray diffraction. Molecular modeling and NMR experiments indicate that these quaternary amino acids are able to drive the adoption of ß-turn secondary structures when incorporated in model dipeptide derivatives.

The structure and flexibility analysis of the Arabidopsis synaptotagmin 1 reveal the basis of its regulation at membrane contact sites.

Non-vesicular lipid transfer at ER and plasma membrane (PM) contact sites (CS) is crucial for the maintenance of membrane lipid homeostasis. Extended synaptotagmins (E-Syts) play a central role in this process as they act as molecular tethers of ER and PM and as lipid transfer proteins between these organelles. E-Syts are proteins constitutively anchored to the ER through an N-terminal hydrophobic segment and bind the PM via a variable number of C-terminal C2 domains. Synaptotagmins (SYTs) are the plant orthologous of E-Syts and regulate the ER-PM communication in response to abiotic stress. Combining different structural and biochemical techniques, we demonstrate that the binding of SYT1 to lipids occurs through a Ca2+-dependent lipid-binding site and by a site for phosphorylated forms of phosphatidylinositol, thus integrating two different molecular signals in response to stress. In addition, we show that SYT1 displays three highly flexible hinge points that provide conformational freedom to facilitate lipid extraction, protein loading, and subsequent transfer between PM and ER.

Controlling optical properties and function of BODIPY by using asymmetric substitution effects.

Asymmetrically substituted BODIPY analogues of the dye PM567 have been synthesised from 2-acylpyrroles and pyrroles that bear indene, fluorene or difluorene units. The type of linkage between the fluorene and the BODIPY core plays an important role in the photophysics of the BODIPY chromophore. Indeed, an aliphatic bridge gives rise to an energy-transfer process between the chromophores, whereas a vinyl spacer allows an electronic interaction between them, leading to a large red shift of the spectral bands. The laser action of the new dyes has been analysed under transversal pumping at 10 Hz repetition rate, in both liquid phase and incorporated into solid polymeric matrices. Lasing efficiencies of up to 40% were reached with high photostabilities with the laser output remaining at the initial level after 1×10(5) pump pulses in the same position of the sample. The laser action of the new dyes outperforms the laser behaviour of commercial dyes that emit in the same spectral region. The replacement of fluorene by indene quenches the fluorescence and laser emission, but allows the development of an iron cation fluorescent sensor.

Smart lanthanide antennas for sensing water.

Two new families of lanthanide antennas are described. 8-Methoxy-4,5-dihydrocyclopenta[de]quinolin-2(1H)-one phosphonates or carboxylates behave as selective antennas exhibiting Eu3+ luminescence in organic solvents, while quinolin-2(1H)-one analogues selectively sensitize the Tb3+ emission. These emissions are quenched by H2O addition. Based on this behaviour, the new lanthanide antennas can be used as highly sensitive water sensors.

Environment-Sensitive Probes for Illuminating Amyloid Aggregation In Vitro and in Zebrafish.

The aberrant aggregation of certain peptides and proteins, forming extracellular plaques of fibrillar material, is one of the hallmarks of amyloid diseases, such as Alzheimer's and Parkinson's. Herein, we have designed a new family of solvatochromic dyes based on the 9-amino-quinolimide moiety capable of reporting during the early stages of amyloid fibrillization. We have rationally improved the photophysical properties of quinolimides by placing diverse amino groups at the 9-position of the quinolimide core, leading to higher solvatochromic and fluorogenic character and higher lifetime dependence on the hydrophobicity of the environment, which represent excellent properties for the sensitive detection of prefibrillar aggregates. Among the different probes prepared, the 9-azetidinyl-quinolimide derivative showed striking performance in the following ß-amyloid peptide (Aß) aggregation in solution in real time and identifying the formation of different types of early oligomers of Aß, the most important species linked to cytotoxicity, using novel, multidimensional fluorescence microscopy, with one- or two-photon excitation. Interestingly, the new dye allowed the visualization of proteinaceous inclusion bodies in a zebrafish model with neuronal damage induced by the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Our results support the potential of the novel fluorophores as powerful tools to follow amyloid aggregation using fluorescence microscopy in vivo, revealing heterogeneous populations of different types of aggregates and, more broadly, to study protein interactions.

Further evidence for 2-alkyl-2-carboxyazetidines as gamma-turn inducers.

Reverse turns, a common motif in proteins and peptides, have attracted attention due to their relevance in a wide variety of biological processes. In an attempt to artificially imitate and stabilize these turns in short peptides, we have developed versatile synthetic methodologies for the preparation of 2-alkyl-2-carboxyazetidines and incorporated them into the i + 1 position of model tetrapeptides, where they have shown a tendency to induce gamma-turns. However, to ascertain the general utility of these restricted amino acids as gamma-type reverse turn inducers, it was then required to study the conformational preferences when located at other positions. To this end, model tetrapeptides R-CO-Ala-Xaa-NHMe, containing differently substituted azetidine moieties (Xaa = Aze, 2-MeAze, 2-BnAze) at the i + 2 position, were synthesized and subjected to a thorough conformational analysis. The theoretical and experimental results obtained, including the X-ray diffraction structure of a dipeptide derivative containing this skeleton, provide evidence that the 2-alkyl-2-carboxyazetidine scaffold is able to efficiently induce gamma-turns when incorporated into these short peptides, irrespective of their localization in the peptide chain.

New Quinolylnitrones for Stroke Therapy: Antioxidant and Neuroprotective ( Z)- N- tert-Butyl-1-(2-chloro-6-methoxyquinolin-3-yl)methanimine Oxide as a New Lead-Compound for Ischemic Stroke Treatment.

We describe herein the synthesis and neuroprotective capacity of an array of 31 compounds comprising quinolyloximes, quinolylhydrazones, quinolylimines, QNs, and related heterocyclic azolylnitrones. Neuronal cultures subjected to oxygen-glucose deprivation (OGD), as experimental model for ischemic conditions, were treated with our molecules at the onset of recovery period after OGD and showed that most of these QNs, but not the azo molecules, improved neuronal viability 24 h after recovery. Especially, QN ( Z)- N-tert-butyl-1-(2-chloro-6-methoxyquinolin-3-yl)methanimine oxide (23) was shown as a very potent neuroprotective agent. Antioxidant analysis based on the ability of QN 23 to trap different types of toxic radical oxygenated species supported and confirmed its strong neuroprotective capacity. Finally, QN 23 showed also neuroprotection induction in two in vivo models of cerebral ischemia, decreasing neuronal death and reducing infarct size, allowing us to conclude that QN 23 can be considered as new lead-compound for ischemic stroke treatment.

Deciphering the Inhibition of the Neuronal Calcium Sensor 1 and the Guanine Exchange Factor Ric8a with a Small Phenothiazine Molecule for the Rational Generation of Therapeutic Synapse Function Regulators.

Protein-protein interactions (PPIs) are known to play an essential role between the neuronal calcium sensor 1 (NCS-1) and the guanine exchange factor Ric8a to regulate synapse function, emerging as a druggable interface for synaptopathies such as the fragile X syndrome (FXS). Recently, the phenothiazine FD44 has been identified as an inhibitor of this PPI, decreasing the abnormally high synapse number and enhancing associative learning in a FXS animal model. Here, we have integrated advanced experimental and computational studies to obtain important structural insights into Drosophila NCS-1/FD44 recognition to understand the basis of its affinity and specificity and generate improved PPI regulators. This has allowed the identification of a new small drug-like molecule, IGS-1.76, which efficiently inhibits the human NCS-1/Ric8a complex with improved binding potency. The crystal structure of the Drosophila NCS-1/IGS-1.76 complex demonstrates that the new inhibitor, although chemically different from FD44, shares the same mechanism of action and constitutes a new hit candidate for FXS.

Synthesis, photophysical properties, and laser behavior of 3-amino and 3-acetamido BODIPY dyes.

The asymmetrically substituted BODIPY dyes 9a and 9b have been synthesized through a key redox step involving the alpha-nitroso derivative of the starting pyrrol. Both dyes emit fluorescence with quantum yields of ca. 0.7, but only 8b behaves as a good laser dye, with an efficiency of 48% in ethanol solution.