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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

The structures of two scorpionates: thallium tetrakis(3-phenyl-1H-pyrazol-1-yl)borate and potassium tetrakis(3-cyclopropyl-1H-pyrazol-1-yl)borate.

The introduction of poly(1H-pyrazolyl)borate anions, better known as scorpionates, as negatively charged ligands for a great diversity of metal cations has had a tremendous influence in coordination chemistry. The structures of two salts of tetrakispyrazolylborate, namely [tetrakis(3-phenyl-1H-pyrazol-1-yl)borato]thallium(I), [Tl(C36H28BN8)], and catena-poly[potassium-[µ2-tetrakis(3-cyclopropyl-1H-pyrazol-1-yl)borato]], [K(C24H28BN8)]n, have been determined at 296 K in the monoclinic P21/c and C2/c space groups, respectively. In their crystal structures, the thallium salt presents discrete molecular motifs, while the potassium salt shows infinite polymeric chains. The 13C and 15N CPMAS (cross polarization magic angle spinning) NMR spectra of these compounds were recorded and the chemical shifts compared with theoretically calculated ones at the GIAO/B3LYP/6-311++G(d,p) level. Both techniques are complementary and mutually consistent.

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.

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.

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.

Sulfoxide-directed enantioselective synthesis of functionalized tetrahydropyridines.

The highly selective base-promoted cyclization of enantiopure sulfinyl dienamines provides stereodefined sulfinyl 1,2,3,6-tetrahydropyridines (dr up to 99:1). Subsequent sigmatropic rearrangement affords tetrahydropyridin-3-ols in good yields and selectivities.

Efficient light harvesters based on the 10-(1,3-dithiol-2-ylidene)anthracene core.

Three new push-pull chromophores based on the 10-(1,3-dithiol-2-ylidene)anthracene core were synthesized and fully characterized. The new chromophores display broad absorption spectra, nearly covering the whole visible region, with high extinction coefficients. Electrochemistry and theoretical calculations allowed the understanding of these singular electronic properties. The molecular structures were unambiguously confirmed by X-ray diffraction.

Highly functionalized 1,2-diamino compounds through reductive amination of amino acid-derived ß-keto esters.

1,2-Diamine derivatives are valuable building blocks to heterocyclic compounds and important precursors of biologically relevant compounds. In this respect, amino acid-derived ß-keto esters are a suitable starting point for the synthesis of ß,γ-diamino ester derivatives through a two-step reductive amination procedure with either simple amines or α-amino esters. AcOH and NaBH(3)CN are the additive and reducing agents of choice. The stereoselectivity of the reaction is still an issue, due to the slow imine-enamine equilibria through which the reaction occurs, affording mixtures of diastereoisomers that can be chromatographically separated. Transformation of the ß,γ-diamino esters into pyrrolidinone derivatives allows the configuration assignment of the linear compounds, and constitutes an example of their potential application in the generation of molecular diversity.