Enantioselective Intramolecular Michael Addition of Nitronates onto Conjugated Esters: Access to Cyclic γ-Amino Acids with up to Three Stereocenters

A highly stereoselective synthesis of conformationally constrained cyclic γ-amino acids has been devised. The key step involves an intramolecular cyclization of a nitronate onto a conjugated ester, promoted by a bifunctional thiourea catalyst. This methodology has been successfully applied to generate a variety of γ-amino acids, including some containing three contiguous stereocenters, with very high diastereoselectivity and excellent enantioselectivity. It is postulated that an interaction that is key to the success of the process is the simultaneous coordination of the thiourea functionality to both the conjugated ester and the nitronate. Finally, the synthetic utility of these compounds is demonstrated in the synthesis of two dipeptides derived from the C- and N-termini.

Rational conversion of substrate and product specificity in a Salvia monoterpene synthase: structural insights into the evolution of terpene synthase function

Terpene synthases are responsible for the biosynthesis of the complex chemical defense arsenal of plants and microorganisms. How do these enzymes, which all appear to share a common terpene synthase fold, specify the many different products made almost entirely from one of only three substrates? Elucidation of the structure of 1,8-cineole synthase from Salvia fruticosa (Sf-CinS1) combined with analysis of functional and phylogenetic relationships of enzymes within Salvia species identified active-site residues responsible for product specificity. Thus, Sf-CinS1 was successfully converted to a sabinene synthase with a minimum number of rationally predicted substitutions, while identification of the Asn side chain essential for water activation introduced 1,8-cineole and alpha-terpineol activity to Salvia pomifera sabinene synthase. A major contribution to product specificity in Sf-CinS1 appears to come from a local deformation within one of the helices forming the active site. This deformation is observed in all other mono- or sesquiterpene structures available, pointing to a conserved mechanism. Moreover, a single amino acid substitution enlarged the active-site cavity enough to accommodate the larger farnesyl pyrophosphate substrate and led to the efficient synthesis of sesquiterpenes, while alternate single substitutions of this critical amino acid yielded five additional terpene synthases.

S-nitrosylation of proteins at the leading edge of migrating trophoblasts by inducible nitric oxide synthase promotes trophoblast invasion

Nitric oxide regulates many important cellular processes including motility and invasion. Many of its effects are mediated through the modification of specific cysteine residues in target proteins, a process called S-nitrosylation. Here we show that S-nitrosylation of proteins occurs at the leading edge of migrating trophoblasts and can be attributed to the specific enrichment of inducible nitric oxide synthase (iNOS/NOS2) in this region. Localisation of iNOS to the leading edge is co-incidental with a site of extensive actin polymerisation and is only observed in actively migrating cells. In contrast endothelial nitric oxide synthase (eNOS/NOS3) shows distribution that is distinct and non-colocalised with iNOS, suggesting that the protein S-nitrosylation observed at the leading edge is caused only by iNOS and not eNOS. We have identified MMP-9 as a potential target for S-nitrosylation in these cells and demonstrate that it co-localises with iNOS at the leading edge of migrating cells. We further demonstrate that iNOS plays an important role in promoting trophoblast invasion, which is an essential process in the establishment of a successful pregnancy.

Platelet nitric oxide synthase is activated by tyrosine dephosphorylation: possible role for SHP-1 phosphatase

Background: Endothelial nitric oxide synthase (eNOS) activity in endothelial cells is regulated by post-translational phosphorylation of critical serine, threonine and tyrosine residues in response to a variety of stimuli. However, the post-translational regulation of eNOS in platelets is poorly defined. Objectives: We investigated the role of tyrosine phosphorylation in the regulation of platelet eNOS activity. Methods: Tyrosine phosphorylation of eNOS and interaction with the tyrosine phosphatase SHP-1 were investigated by coimmunoprecipitation and immunoblotting. An in vitro immunoassay was used to determine eNOS activity together with the contribution of protein tyrosine phosphorylation. Results: We found platelet eNOS was tyrosine phosphorylated under basal conditions. Thrombin induced a dose- and time-dependent increase in eNOS activity without altering overall level of tyrosine phosphorylation, although we did observe evidence of minor tyrosine dephosphorylation. In vitro tyrosine dephosphorylation of platelet eNOS using a recombinant protein tyrosine phosphatase enhanced thrombin-induced activity compared to thrombin alone, but had no effect on endothelial eNOS activity either at basal or after stimulation with bradykinin. Having shown that dephosphorylation could modulate platelet eNOS activity we examined the role of potential protein phosphatases important for platelet eNOS activity. We found SHP-1 protein tyrosine phosphatase, co-associated with platelet eNOS in resting platelets, but does not associate with eNOS in endothelial cells. Stimulation of platelets with thrombin increased SHP-1 association with eNOS, while inhibition of SHP-1 abolished the ability of thrombin to induce elevated eNOS activity. Conclusions: Our data suggest a novel role for tyrosine dephosphorylation in platelet eNOS activation, which may be mediated by SHP-1.

Regulation of glycogen synthase kinase 3 in human platelets: a possible role in platelet function?

In this study we show that both glycogen synthase kinase 3 (GSK3) isoforms, GSK3alpha and GSK3beta, are present in human platelets and are phosphorylated on Ser(21) and Ser(9), respectively, in platelets stimulated with collagen, convulxin and thrombin. Phosphorylation of GSK3alpha/beta was dependent on phosphoinositide 3-kinase (PI3K) activity and independent of platelet aggregation, and correlated with a decrease in GSK3 activity that was preserved by pre-incubating platelets with PI3K inhibitor LY294002. Three structurally distinct GSK3 inhibitors, lithium, SB415286 and TDZD-8, were found to inhibit platelet aggregation. This implicates GSK3 as a potential regulator of platelet function. (C) 2003 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

A mechanistic study of cyclic siloxane pyrolyses at low pressures

Matrix isolation IR spectroscopy has been used to study the vacuum pyrolysis of hexamethylcyclotrisiloxane (D3), octamethylcyclotetrasiloxane (D4) and decamethyl cyclopentasiloxane (D5), and the results interpreted in the context of various kinetic models. In particular, it is shown that the significant pyrolysis products - which include CH3, CH4, C2H2, C2H4, C2H6 and SiO - may be satisfactorily accounted for by radical reactions involving dimethylsiloxane (D1), and estimates are made of the various chain lengths for the proposed reactions based on a range of ambient conditions.

Solvent-induced dynamic single-crystal-to-single-crystal transformation of a synthetic peptide-based cyclic compound

Solvent induced single-crystal-to-single-crystal transformation has been demonstrated indicating the dynamic behavior of one dimensional arrays obtained from a self-assembled new synthetic cyclic peptide.

Anharmonic vibrational levels of the two cyclic isomers of SiC3

Using coupled-cluster approach full six-dimensional analytic potential energy surfaces for two cyclic SiC3 isomers [C-C transannular bond (I) and Si-C transannular bond (II)] have been generated and used to calculate anharmonic vibrational wave functions. Several strong low-lying anharmonic resonances have been found. In both isomers already some of the fundamental transitions cannot be described within the harmonic approximation. Adiabatic electron affinities and ionization energies have been calculated as well. The Franck-Condon factors for the photodetachment processes c-SiC3-(I)-> c-SiC3(I) and c-SiC3-(II)-> c-SiC3(II) are reported. (c) 2006 American Institute of Physics.

Synthesis of fused cyclic systems containing medium-sized rings through tandem ROM-RCM of norbornene derivatives embedded in a carbohydrate template

A general approach for the synthesis of fused cyclic systems containing medium-sized rings (7-9) has been developed. The key steps involve a diastereoface-selective Diels-Alder reaction of the dienophiles 4a-d attached to a furanosugar with cyclopentadiene and ring opening (ROM)-ring closing metathesis (RCM) of the resulting norbornene derivatives 10a-d and 11a-d. Diels-Alder reaction of the dienophiles 4a-d with cyclopentadiene in the absence of a catalyst produced 10a-d as the major product arising through addition of the diene to the unhindered Si-face. The most interesting and new aspect of the Diels-Alder reaction of these dienophiles is the accessibility of the Re-face that was blocked by the alkenyl chains under Lewis acid catalysis producing the diastereoisomers 11a-d exclusively. The reversal of facial selectivity from an uncatalyzed reaction to a catalyzed one is unprecedented. The observed stereochemical dichotomy is attributed to rotation of the enone moiety along the or bond linking the sugar moiety during formation of the chelate 13. This makes the Re-face of the enone moiety in 4a-d unhindered. Diels-Alder reaction of the carbocyclic analogue 15 under Lewis acid catalysis produced a 1: 1 mixture of the adducts 16 and 17 confirming the participation of sugar ring oxygen in chelate formation. Finally ROM-RCM of 10a-d and 11a-d with Grubbs' catalyst afforded the cis-syn-cis and cis-anti-cis bicyclo-annulated sugars 21a-d and 23a-d, respectively, containing 7-9 membered rings.

Occurrence of ribbons of cyclic water pentamers in a metallo-organic framework formed by spontaneous fixation of CO2

In the reaction of equimolar amounts of copper(II) acetate with 2,2'-dipyridylamine (DPA) in aqueous tetrahydrofuran, in presence of KOH, aerial CO2 is spontaneously fixed to the carbonate anion yielding [Cu(DPA)(CO3)] . 3H(2)O (1). X-ray crystallography shows the presence of zigzag ribbons of cyclic water pentamers in the channels of a chain-like metallo-organic framework. The water ribbons are stabilised by hydrogen bonds to the metallo-organic backbone. Each (H2O)(5) pentamer is approximately planar.

(-)Epicatechin stimulates ERK-dependent cyclic AMP response element activity and up-regulates GluR2 in cortical neurons

Emerging evidence suggests that the cellular actions of flavonoids relate not simply to their antioxidant potential but also to the modulation of protein kinase signalling pathways. We investigated in primary cortical neurons, the ability of the flavan-3-ol, (-)epicatechin, and its human metabolites at physiologically relevant concentrations, to stimulate phosphorylation of the transcription factor cAMP-response element binding protein (CREB), a regulator of neuronal viability and synaptic plasticity. (-)Epicatechin at 100-300 nmol/L stimulated a rapid, extracellular signal-regulated kinase (ERK)- and PI3K-dependent, increase in CREB phosphorylation. At micromolar concentrations, stimulation was no longer apparent and at the highest concentration tested (30 mu mol/L) (-)epicatechin was inhibitory. (-)Epicatechin also stimulated ERK and Akt phosphorylation with similar bell-shaped concentration-response characteristics. The human metabolite 3 '-O-methyl-(-)epicatechin was as effective as (-)epicatechin at stimulating ERK phosphorylation, but (-)epicatechin glucuronide was inactive. (-)Epicatechin and 3 '-O-methyl-(-)epicatechin treatments (100 nmol/L) increased CRE-luciferase activity in cortical neurons in a partially ERK-dependent manner, suggesting the potential to increase CREB-mediated gene expression. mRNA levels of the glutamate receptor subunit GluR2 increased by 60%, measured 18 h after a 15 min exposure to (-)epicatechin and this translated into an increase in GluR2 protein. Thus, (-)epicatechin has the potential to increase CREB-regulated gene expression and increase GluR2 levels and thus modulate neurotransmission, plasticity and synaptogenesis.

Rat brain serotonin neurones that express neuronal nitric oxide synthase have increased sensitivity to the substituted amphetamine serotonin toxins 3,4-methylenedioxymethamphetamine and p-chloroamphetamine

Substituted amphetamines such as p-chloroamphetamine and the abused drug methylenedioxymethamphetamine cause selective destruction of serotonin axons in rats, by unknown mechanisms. Since some serotonin neurones also express neuronal nitric oxide synthase, which has been implicated in neurotoxicity, the present study was undertaken to determine whether nitric oxide synthase expressing serotonin neurones are selectively vulnerable to methylenedioxymethamphetamine or p-chloroamphetamine. Using double-labeling immunocytochemistry and double in situ hybridization for nitric oxide synthase and the serotonin transporter, it was confirmed that about two thirds of serotonergic cell bodies in the dorsal raphe nucleus expressed nitric oxide synthase, however few if any serotonin transporter immunoreactive axons in striatum expressed nitric oxide synthase at detectable levels. Methylenedioxymethamphetamine (30 mg/kg) or p-chloroamphetamine (2 x 10 mg/kg) was administered to Sprague-Dawley rats, and 7 days after drug administration there were modest decreases in the levels of serotonin transporter protein in frontal cortex, and striatum using Western blotting, even though axonal loss could be clearly seen by immunostaining. p-Chloroamphetamine or methylenedioxymethamphetamine administration did not alter the level of nitric oxide synthase in striatum or frontal cortex, determined by Western blotting. Analysis of serotonin neuronal cell bodies 7 days after p-chloroamphetamine treatment, revealed a net down-regulation of serotonin transporter mRNA levels, and a profound change in expression of nitric oxide synthase, with 33% of serotonin transporter mRNA positive cells containing nitric oxide synthase mRNA, compared with 65% in control animals. Altogether these results support the hypothesis that serotonin neurones which express nitric oxide synthase are most vulnerable to substituted amphetamine toxicity, supporting the concept that the selective vulnerability of serotonin neurones has a molecular basis.

Asymmetric phase-transfer-catalyzed synthesis of five-membered cyclic gamma-Amino acid precursors

The first example of an intramolecular enantioselective Michael addition of nitronates onto conjugated systems utilizing a chiral phase-transfer catalyst is described. A range of five-membered gamma-nitro esters with up to three stereocentres have been prepared and the relative and absolute configurations proven by chemical and crystallographic methods. The products are rapidly obtained and are precursors to five-membered cyclic gamma-amino acids.

Probing the effect of allylic substitution on cyclic ammonium ylid rearrangements

The [2,3]-sigmatropic rearrangement of tetrahydropyridine-derived ammonium ylids is a valuable method for the preparation of substituted pyrrolidine carboxylates. The presence of an allylic substituent does not intrinsically reduce the yield of rearrangements, and the diastereoselectivity of rearrangement is related to the structure of the diazo reactant. The method represents a very rapid means of accessing complex pyrrolidines, as shown by preparation of a precursor to the core of lactacystin.