Multifunctional Catalysts in the Asymmetric Mannich Reaction of Malononitrile with N-Phosphinoylimines: Coactivation by Halogen Bonding versus Hydrogen Bonding.

A multifunctional (noncovalent) catalyst containing halogen-bond donor, hydrogen-bond donor, and Lewis basic sites was developed and applied in an enantioselective Mannich reaction between malononitrile and diphenylphosphinoyl-protected aldimine affording products in high yields (up to 98%) and moderate to high enantiomeric purities (ee up to 89%). Typically, noncovalent catalysts rely on several weak interactions to activate the substrate, with one or two of these giving the most notable contribution to activation. In this instance, instead of the initially proposed coactivation by halogen bonding, it was revealed that hydrogen bonding plays a key role in determining the enantioselectivity.

Asymmetric organocatalytic Michael addition of cyclopentane-1,2-dione to alkylidene oxindole.

An asymmetric Michael reaction between cyclopentane-1,2-dione and alkylidene oxindole was studied in the presence of a multifunctional squaramide catalyst. Michael adducts were obtained in high enantioselectivities and in moderate diastereoselectivities.

Designed whole-cell-catalysis-assisted synthesis of 9,11-secosterols.

A method for the synthesis of 9,11-secosteroids starting from the natural corticosteroid cortisol is described. There are two key steps in this approach, combining chemistry and synthetic biology. Stereo- and regioselective hydroxylation at C9 (steroid numbering) is carried out using whole-cell biocatalysis, followed by the chemical cleavage of the C-C bond of the vicinal diol. The two-step method features mild reaction conditions and completely excludes the use of toxic oxidants.

Gly188Arg substitution eliminates substrate inhibition in arachidonate 11R-lipoxygenase.

Lipoxygenases (LOXs) are dioxygenases that catalyze the oxygenation of polyunsaturated fatty acids to hydroperoxyl derivates. These products are precursors for different lipid mediators which are associated with pathogenesis of various diseases such as asthma, atherosclerosis and cancer. Several LOXs suffer from substrate inhibition, a potential regulatory mechanism, yet it is unclear what is the cause of this phenomenon. One such enzyme is the coral 11R-LOX which displays a significant decrease in turnover rate at arachidonic acid concentrations above 30 µM. In this report, site-directed mutagenesis and inhibition assays were employed to shed light on the mechanism of substrate inhibition in 11R-LOX. We found that introduction of a positive charge to the active site entrance with Gly188Arg substitution completely eliminates the slow-down at higher substrate concentrations. Inhibition of 11R-LOX by its catalysis product, 11(R)-hydroperoxyeicosatetraenoic acid, suggests an uncompetitive mechanism. We reason that substrate inhibition in 11R-LOX is due to additional fatty acid binding by the enzyme:substrate complex at an allosteric site situated in the very vicinity of the active site entrance.

Halo-1,2,3-triazolium Salts as Halogen Bond Donors for the Activation of Imines in Dihydropyridinone Synthesis.

In the past decade halogen bond (XB) catalysis has gained considerable attention. Halo-triazoles are known XB donors, yet few examples detail their use as catalysts. As a continuation of our previous work the catalytic properties of substituted enantiomerically pure halo-triazolium salts were explored in the reaction between an imine and Danishefsky's diene leading to the formation of dihydropyridinone. The catalytic activity of the XB donors was highly dependent on the choice of the halogen atom and on the counterion. Also, it was found that impurities in the diene affected the rate of the reaction.

Size-Control by Anion Templating in Mechanochemical Synthesis of Hemicucurbiturils in the Solid State.

Self-organization is one of the most intriguing phenomena of chemical matter. While the self-assembly of macrocycles and cages in dilute solutions has been extensively studied, it remains poorly understood in solvent-free environments. Provided here is the first example of using anionic templates to achieve selective assembly of differently-sized macrocycles in a solvent-free system. Using acid-catalyzed synthesis of cyclohexanohemicucurbiturils as a model, size-controlled, quantitative synthesis of 6- or 8-membered macrocycles by spontaneous anion-directed reorganization of mechanochemically-made oligomers in the solid state is demonstrated.

A PDZ-like domain mediates the dimerization of 11R-lipoxygenase.

Lipoxygenases (LOXs), participating in inflammatory processes and cancer, are a family of enzymes with high potential as drug targets. Various allosteric effects have been observed with different LOX isozymes (e.g. lipid/ATP binding, phosphorylation), yet there is a lot of uncertainty concerning the regulation of these enzymes. It has been recently found that a number of LOXs form dimers, extending the list of possible allosteric mechanisms with oligomerization. Coral 11R-LOX is, unlike several mammalian counterparts, a stable dimer in solution facilitating quaternary structure studies that demand high sample homogeneity. By combining previous crystallographic data of 11R-LOX with small-angle X-ray scattering and chemical cross-linking, we were able to narrow down the possible dimerization interfaces, and subsequently determined the correct assembly by site-directed mutagenesis of potential contacting residues. The region of interest is located in the vicinity of an α+ß formation in the catalytic domain, also coined the PDZ-like domain. Being situated just between the active site and the dimer interface, our results further implicate this putative subdomain in the regulation of LOXs.

Synthesis and Characterisation of Chiral Triazole-Based Halogen-Bond Donors: Halogen Bonds in the Solid State and in Solution.

A general platform for the synthesis of various chiral halogen-bond (XB) donors based on the triazole core and the characterisation of factors that influence the strength of the halogen bond in the solid state and in solution are reported. The characterisation of XB donors in the solid state by X-ray crystallography and in solution by 1 H NMR titration can be used to aid the design of new XB donors. We describe the first example of a XB between iodotriazoles and thioureas in solution. In addition, the enantiodiscrimination of acceptors in solution through halogen-bond participation is described.

Two Catalytic Methods of an Asymmetric Wittig [2,3]-Rearrangement.

Two different approaches for asymmetric catalytic Wittig [2,3]-rearrangement were developed. Allyloxymalonate derivatives were converted into homoallyl alcohols via organocatalytic or Ca2+-catalyzed pathways in moderate to high enantioselectivities.

Two-step conversion of carboxylic esters into distally fluorinated ketones via ring cleavage of cyclopropanol intermediates: application of sulfinate salts as fluoroalkylating reagents.

Tertiary cyclopropanols easily available from carboxylic esters have been used in the synthesis of distally fluorinated ketones. Cyclopropane ring cleavage reactions in methanol with aqueous tert-butyl hydroperoxide in the presence of a copper(ii) acetate catalyst and sodium triflinate (Langlois reagent) afford ß-trifluoromethyl ketones in 16-74% isolated yields. Sodium triflinate serves as a precursor of reactive trifluoromethyl copper species, enabling ring-opening trifluoromethylation, as evidenced by mechanistic studies. We also demonstrate here that other sulfinate salts, such as sodium 1,1-difluoroethanesulfinate, sodium 2-(4-bromophenyl)-1,1-difluoroethanesulfinate and sodium 1-(trifluoromethyl)cyclopropanesulfinate, can be used as fluoroalkylation reagents, resulting in the corresponding fluorinated ketones.

Synthesis of γ-keto sulfones by copper-catalyzed oxidative sulfonylation of tertiary cyclopropanols.

Tertiary cyclopropanols undergo ring-opening oxidative sulfonylation to afford γ-keto sulfones when reacting with sulfinate salts in the presence of a copper(ii) acetate catalyst and an oxidant (tert-butyl hydroperoxide or atmospheric oxygen). Various fluoroalkyl, aryl and alkyl sulfinate salts are successfully employed as sulfonylation reagents, affording the corresponding sulfones in up to 94% yields. The experimental protocol is mild and tolerates a number of functionalities in the cyclopropanol substrate. The reaction proceeds via a one-pot oxidation-Michael addition mechanism and can serve as a useful addition to the existing methods for the preparation of γ-keto sulfones based on the sulfa-Michael reaction.

A conserved π-cation and an electrostatic bridge are essential for 11R-lipoxygenase catalysis and structural stability.

Lipoxygenases (LOXs) are lipid-peroxidizing enzymes that consist of a regulatory calcium- and membrane-binding PLAT (polycystin-1, lipoxygenase, α-toxin) domain and a catalytic domain. In a previous study, the crystal structure of an 11R-LOX revealed a conserved π-cation bridge connecting these two domains which could mediate the regulatory effect of the PLAT domain to the active site. Here we analyzed the role of residues Trp107 and Lys172 that constitute the π-cation bridge in 11R-LOX along with Arg106 and Asp173-a potential salt bridge, which could also contribute to the inter-domain communication. According to our kinetic assays and protein unfolding experiments conducted using differential scanning fluorimetry and circular dichroism spectroscopy, mutants with a disrupted link display diminished catalytic activity alongside reduced stability of the protein fold. The results demonstrate that both these bridges contribute to the two-domain interface, and are important for proper enzyme activation.

CaCl2, Bisoxazoline, and Malonate: A Protocol for an Asymmetric Michael Reaction.

A mild protocol for the asymmetric Michael addition of dimethyl malonate to various α,ß-unsaturated carbonyl compounds was developed. The salient feature of this methodology is that a cheap and environmentally friendly Lewis acid, CaCl2, was used as a catalyst. An aminoindanol- and pyridine-derived ligand provided in the presence of CaCl2 Michael adducts in moderate to high enantioselectivities. The scope of the reaction was demonstrated.

Structural and catalytic insights into the algal prostaglandin H synthase reveal atypical features of the first non-animal cyclooxygenase.

Prostaglandin H synthases (PGHSs) have been identified in the majority of vertebrate and invertebrate animals, and most recently in the red alga Gracilaria vermiculophylla. Here we report on the cloning, expression and characterization of the algal PGHS, which shares only about 20% of the amino acid sequence identity with its animal counterparts, yet catalyzes the conversion of arachidonic acid into prostaglandin-endoperoxides, PGG2 and PGH2. The algal PGHS lacks structural elements identified in all known animal PGHSs, such as epidermal growth factor-like domain and helix B in the membrane binding domain. The key residues of animal PGHS, like catalytic Tyr-385 and heme liganding His-388 are conserved in the algal enzyme. However, the amino acid residues shown to be important for substrate binding and coordination, and the target residues for nonsteroidal anti-inflammatory drugs (Arg-120, Tyr-355, and Ser-530) are not found at the appropriate positions in the algal sequences. Differently from animal PGHSs the G. vermiculophylla PGHS easily expresses in Escherichia coli as a fully functional enzyme. The recombinant protein was identified as an oligomeric (evidently tetrameric) ferric heme protein. The preferred substrate for the algal PGHS is arachidonic acid with cyclooxygenase reaction rate remarkably higher than values reported for mammalian PGHS isoforms. Similarly to animal PGHS-2, the algal enzyme is capable of metabolizing ester and amide derivatives of arachidonic acid to corresponding prostaglandin products. Algal PGHS is not inhibited by non-steroidal anti-inflammatory drugs. A single copy of intron-free gene encoding for PGHS was identified in the red algae G. vermiculophylla and Coccotylus truncatus genomes.

Structure of a calcium-dependent 11R-lipoxygenase suggests a mechanism for Ca2+ regulation.

Lipoxygenases (LOXs) are a key part of several signaling pathways that lead to inflammation and cancer. Yet, the mechanisms of substrate binding and allosteric regulation by the various LOX isoforms remain speculative. Here we report the 2.47-Å resolution crystal structure of the arachidonate 11R-LOX from Gersemia fruticosa, which sheds new light on the mechanism of LOX catalysis. Our crystallographic and mutational studies suggest that the aliphatic tail of the fatty acid is bound in a hydrophobic pocket with two potential entrances. We speculate that LOXs share a common T-shaped substrate channel architecture that gives rise to the varying positional specificities. A general allosteric mechanism is proposed for transmitting the activity-inducing effect of calcium binding from the membrane-targeting PLAT (polycystin-1/lipoxygenase/α-toxin) domain to the active site via a conserved π-cation bridge.

Asymmetric synthesis of congested spiro-cyclopentaneoxindoles via an organocatalytic cascade reaction.

Starting from simple alkylidene oxindoles and nitroketones, a highly stereoselective methodology was developed for the synthesis of spiro-cyclopentaneoxindoles with four consecutive stereogenic centers. Using an organocatalytic cascade of Michael and aldol reactions in the presence of a chiral thiourea catalyst products were obtained in moderate to high yields and excellent enantioselectivities. Nitro, ester, and hydroxyl groups were introduced to the spiro ring, which could be used to facilitate further functionalization of the products.

Identification of glycolytic proteins as binding partners of Bri2 BRICHOS domain.

Human integral membrane protein 2B (ITM2B or Bri2) is a member of the BRICHOS family, that can attenuate Aß pathology in the brain. As a result, the identification of novel Bri2 BRICHOS client proteins has been sought to help elucidate signaling pathways and the potential identification of novel therapeutic targets. To identify Bri2 BRICHOS interacting partners, we carried out a 'protein fishing' experiment using recombinant human (rh) Bri2 BRICHOS-coated magnetic particles, in combination with proteomic analysis on cytosolic and membrane fractions of cortical homogenates from C57BL/6 J WT mouse. We identified 4 proteins from the cytosolic fractions and 44 proteins from the membrane fractions that had significant interactions (p < 0.05) with Bri2 BRICHOS domain, of which 11 proteins were previously identified as proteins that interacted with Bri2 BRICHOS domain. Enrichment analysis of the retained proteins identified glycolysis/gluconeogenesis as the most enriched pathway, with several proteins identified playing roles in carbon metabolism, amino acid synthesis. The data suggested that Bri2 BRICHOS may have a role in cellular energy demands in the brain via glycolysis and mitochondrial oxidative phosphorylation and may play a role in mitochondrial homeostasis.

Activation of 11R-lipoxygenase is fully Ca(2+)-dependent and controlled by the phospholipid composition of the target membrane.

Activation of some lipoxygenases (LOX) is found to be related to the selective membrane binding upon cell stimulation. In this study, a systematic analysis of the effect of the lipid composition on the membrane binding efficiency, Ca(2+) affinity, and enzymatic activity of 11R-LOX was performed. The analysis of the membrane targeting by fluorometric and surface plasmon resonance measurements in the absence of Ca(2+) showed an exclusive binding of 11R-LOX to the anionic phospholipids (phosphatidylinositol < phosphatidylglycerol ≈ phosphatidylserine) containing model membranes. The presence of Ca(2+) enhanced the rate of interaction and influenced its mode. The modulation of the activity of 11R-LOX indicated that (i) Ca(2+) binding is a prerequisite for productive membrane association, (ii) the reaction of 11R-LOX with arachidonic acid coincided with and was driven by its Ca(2+)-mediated membrane association, and (iii) phosphatidylethanolamine and anionic phospholipids had a synergistic effect on the Ca(2+) affinity, in line with a target-activated messenger affinity mechanism [Corbin, J. A., et al. (2007) Biochemistry 46, 4322-4336]. According to the mechanism proposed in this report, 11R-LOX can bind to the membranes in two different modes and the efficiency of productive membrane binding is determined by a concerted association of Ca(2+) and lipid headgroups.

Diastereoselective multicomponent cascade reaction leading to [3.2.0]-heterobicyclic compounds.

A general three-component triple cascade reaction through an iminium-enamine-iminium sequential activation initiated by a hetero-Michael addition to α,ß-unsaturated aldehydes affords [3.2.0]heterobicycles in high diastereoselectivity. The rate and diastereoselectivity of the reaction depended on the (E)-4-heterocrotonate and size of the secondary amine. The enantiomers of the major diastereoisomer of oxa- and azabicyclo[3.2.0]heptane derivatives were separated by enzymatic kinetic resolution with immobilized Candida antarctica Lipase B (CALB), with E values up to 153. The absolute configuration of the nonacylated enantiomer of oxabicyclo[3.2.0]heptane was determined by single crystal X-ray analysis.

Organocatalytic asymmetric addition of malonates to unsaturated 1,4-diketones.

The organocatalytic Michael addition of malonates to symmetric unsaturated 1,4-diketones catalyzed by thiourea and squaramide derivatives with Cinchona alkaloids afforded the formation of a new C-C bond in high yields (up to 98%) and enantiomeric purities (up to 93%). The absolute configuration of the product was suggested from comparison of the experimental and calculated VCD spectra of the reaction product 3a.