Revising the structure of a new eicosanoid from human platelets to 8,9-11,12-diepoxy-13-hydroxyeicosadienoic acid.

Eicosanoids are critical mediators of fever, pain, and inflammation generated by immune and tissue cells. We recently described a new bioactive eicosanoid generated by cyclooxygenase-1 (COX-1) turnover during platelet activation that can stimulate human neutrophil integrin expression. On the basis of mass spectrometry (MS/MS and MS3), stable isotope labeling, and GC-MS analysis, we previously proposed a structure of 8-hydroxy-9,11-dioxolane eicosatetraenoic acid (DXA3). Here, we achieved enzymatic synthesis and 1H NMR characterization of this compound with results in conflict with the previously proposed structural assignment. Accordingly, by using LC-MS, we screened autoxidation reactions of 11-hydroperoxy-eicosatetraenoic acid (11-HpETE) and thereby identified a candidate sharing the precise reverse-phase chromatographic and MS characteristics of the platelet product. We optimized these methods to increase yield, allowing full structural analysis by 1H NMR. The revised assignment is presented here as 8,9-11,12-diepoxy-13-hydroxyeicosadienoic acid, abbreviated to 8,9-11,12-DiEp-13-HEDE or DiEpHEDE, substituted for the previous name DXA3 We found that in platelets, the lipid likely forms via dioxolane ring opening with rearrangement to the diepoxy moieties followed by oxygen insertion at C13. We present its enzymatic biosynthetic pathway and MS/MS fragmentation pattern and, using the synthetic compound, demonstrate that it has bioactivity. For the platelet lipid, we estimate 16 isomers based on our current knowledge (and four isomers for the synthetic lipid). Determining the exact isomeric structure of the platelet lipid remains to be undertaken.

Reoptimization of the Organocatalyzed Double Aldol Domino Process to a Key Enal Intermediate and Its Application to the Total Synthesis of Δ12 -Prostaglandin J3.

Re-investigation of the l-proline catalyzed double aldol cascade dimerization of succinaldehyde for the synthesis of a key bicyclic enal intermediate, pertinent in the field of stereoselective prostaglandin synthesis, is reported. The yield of this process has been more than doubled, from 14 % to a 29 % isolated yield on a multi-gram scale (32 % NMR yield), through conducting a detailed study of the reaction solvent, temperature, and concentration, as well as a catalyst screen. The synthetic utility of this enal intermediate has been further demonstrated through the total synthesis of Δ12 -prostaglandin J3 , a compound with known anti-leukemic properties.

A surface plasmon resonance assay for measurement of neuraminidase inhibition, sensitivity of wild-type influenza neuraminidase and its H274Y mutant to the antiviral drugs zanamivir and oseltamivir.

Antiviral resistance is currently monitored by a labelled enzymatic assay, which can give inconsistent results because of the short half-life of the labelled product, and variations in assay conditions. In this paper, we describe a competitive surface plasmon resonance (SPR) inhibition assay for measuring the sensitivities of wild-type neuraminidase (WT NA) and the H274Y (histidine 274 tyrosine) NA mutant to antiviral drugs. The two NA isoforms were expressed in High-five™ (Trichoplusia ni) insect cells. A spacer molecule (1,6-hexanediamine (HDA)) was conjugated to the 7-hydroxyl group of zanamivir, and the construct (HDA-zanamivir) was immobilized onto a SPR sensor chip to obtain a final immobilization response of 431 response units. The immobilized HDA-zanamivir comprised a bio-specific ligand for the WT and mutant proteins. The effects of the natural substrate (sialic acid) and two inhibitors (zanamivir and oseltamivir) on NA binding to the immobilized ligand were studied. The processed SPR data was analysed to determine 50% inhibitory concentrations (IC50-spr ), using a log dose-response curve fit. Although both NA isoforms had almost identical IC50-spr values for sialic acid (WT = 5.5 nM; H274Y mutant = 3.25 nM) and zanamivir (WT = 2.16 nM; H274Y mutant = 2.42 nM), there were significant differences between the IC50-spr values obtained for the WT (7.7 nM) and H274Y mutant (256 nM) NA in the presence of oseltamivir, indicating that oseltamivir has a reduced affinity for the H274Y mutant. The SPR inhibition assay strategy presented in this work could be applied for the rapid screening of newly emerging variants of NA for their sensitivity to antiviral drugs.

Development of a surface plasmon resonance assay to measure the binding affinity of wild-type influenza neuraminidase and its H274Y mutant to the antiviral drug zanamivir.

Influenza is one of the most common infections of the upper respiratory tract. Antiviral drugs that are currently used to treat influenza, such as oseltamivir and zanamivir, are neuraminidase (NA) inhibitors. However, the virus may develop resistance through single-point mutations of NA. Antiviral resistance is currently monitored by a labelled enzymatic assay, which can be inconsistent because of the short half-life of the labelled product and variations in the assay conditions. In this paper, we describe a label-free surface plasmon resonance (SPR) assay for measuring the binding affinity of NA-drug interactions. Wild-type (WT) NA and a histidine 274 tyrosine (H274Y) mutant were expressed in High Five™ (Trichoplusia ni) insect cells. A spacer molecule (1,6-hexanediamine) was site-specifically conjugated to the 7-hydroxyl group of zanamivir, which is not involved in binding to NA, and the construct was immobilized onto a SPR sensor Chip to obtain a final immobilization response of 431 response units. Binding responses obtained for WT and H274Y mutant NAs were fitted to a simple Langmuir 1:1 model with drift to obtain the association (ka ) and dissociation (kd ) rate constants. The ratio between the binding affinities for the two isoforms was comparable to literature values obtained using labelled enzyme assays. Significant potential exists for an extension of this approach to test for drug resistance of further NA mutants against zanamivir and other antiviral drugs, perhaps paving the way for a reliable SPR biosensor assay that may replace labelled enzymatic assays.

Post-translational modification in the archaea: structural characterization of multi-enzyme complex lipoylation.

Lipoylation, the covalent attachment of lipoic acid to 2-oxoacid dehydrogenase multi-enzyme complexes, is essential for metabolism in aerobic bacteria and eukarya. In Escherichia coli, lipoylation is catalysed by LplA (lipoate protein ligase) or by LipA (lipoic acid synthetase) and LipB [lipoyl(octanoyl) transferase] combined. Whereas bacterial and eukaryotic LplAs comprise a single two-domain protein, archaeal LplA function typically involves two proteins, LplA-N and LplA-C. In the thermophilic archaeon Thermoplasma acidophilum, LplA-N and LplA-C are encoded by overlapping genes in inverted orientation (lpla-c is upstream of lpla-n). The T. acidophilum LplA-N structure is known, but the LplA-C structure is unknown and LplA-C's role in lipoylation is unclear. In the present study, we have determined the structures of the substrate-free LplA-N-LplA-C complex and E2lipD (dihydrolipoyl acyltransferase lipoyl domain) that is lipoylated by LplA-N-LplA-C, and carried out biochemical analyses of this archaeal lipoylation system. Our data reveal the following: (i) LplA-C is disordered but folds upon association with LplA-N; (ii) LplA-C induces a conformational change in LplA-N involving substantial shortening of a loop that could repress catalytic activity of isolated LplA-N; (iii) the adenylate-binding region of LplA-N-LplA-C includes two helices rather than the purely loop structure of varying order observed in other LplA structures; (iv) LplAN-LplA-C and E2lipD do not interact in the absence of substrate; (v) LplA-N-LplA-C undergoes a conformational change (the details of which are currently undetermined) during lipoylation; and (vi) LplA-N-LplA-C can utilize octanoic acid as well as lipoic acid as substrate. The elucidated functional inter-dependence of LplA-N and LplA-C is consistent with their evolutionary co-retention in archaeal genomes.

Protecting-group-free one-pot synthesis of glycoconjugates directly from reducing sugars.

The conversion of sugars into glycomimetics typically involves multiple protecting-group manipulations. The development of methodology allowing the direct aqueous conversion of free sugars into glycosides, and mimics of oligosaccharides and glycoconjugates in a high-yielding and stereoselective process is highly desirable. The combined use of 2-azido-1,3-dimethylimidazolinium hexafluorophosphate and the Cu-catalyzed Huisgen cycloaddition allowed the synthesis of a range of glycoconjugates in a one-step reaction directly from reducing sugars under aqueous conditions. The reaction, which is completely stereoselective, may be applied to the convergent synthesis of triazole-linked glycosides, oligosaccharides, and glycopeptides. The procedure provides a method for the one-pot aqueous ligation of oligosaccharides and peptides bearing alkyne side chains.

Cloning, expression, purification, crystallization and preliminary X-ray diffraction studies of N-acetylneuraminate lyase from methicillin-resistant Staphylococcus aureus.

The enzyme N-acetylneuraminate lyase (EC 4.1.3.3) is involved in the metabolism of sialic acids. Specifically, the enzyme catalyzes the retro-aldol cleavage of N-acetylneuraminic acid to form N-acetyl-D-mannosamine and pyruvate. Sialic acids comprise a large family of nine-carbon amino sugars, all of which are derived from the parent compound N-acetylneuraminic acid. In recent years, N-acetylneuraminate lyase has received considerable attention from both mechanistic and structural viewpoints and has been recognized as a potential antimicrobial drug target. The N-acetylneuraminate lyase gene was cloned from methicillin-resistant Staphylococcus aureus genomic DNA, and recombinant protein was expressed and purified from Escherichia coli BL21 (DE3). The enzyme crystallized in a number of crystal forms, predominantly from PEG precipitants, with the best crystal diffracting to beyond 1.70 Šresolution in space group P21. Molecular replacement indicates the presence of eight monomers per asymmetric unit. Understanding the structural biology of N-acetylneuraminate lyase in pathogenic bacteria, such as methicillin-resistant S. aureus, will provide insights for the development of future antimicrobials.

Ruthenium-catalysed oxidative synthesis of heterocycles from alcohols.

Ruthenium-catalysed hydrogen transfer has been successfully used for the conversion of alcohols into either 2,3-dihydroquinazolines or quinazolines. The choice of reaction conditions allows for the selective formation of either heterocycle and the methodology can also be applied to the sulfonamide analogue.

Borrowing hydrogen methodology for amine synthesis under solvent-free microwave conditions.

Application of microwave heating to the Borrowing Hydrogen strategy to form C-N bonds from alcohols and amines is presented, removing the need for solvent and reducing the reaction times while still yielding results comparable with those using thermal heating.

Ruthenium-catalyzed N-alkylation of amines and sulfonamides using borrowing hydrogen methodology.

The alkylation of amines by alcohols has been achieved using 0.5 mol % [Ru(p-cymene)Cl(2)](2) with the bidentate phosphines dppf or DPEphos as the catalyst. Primary amines have been converted into secondary amines, and secondary amines into tertiary amines, including the syntheses of Piribedil, Tripelennamine, and Chlorpheniramine. N-Heterocyclization reactions of primary amines are reported, as well as alkylation reactions of primary sulfonamides. Secondary alcohols require more forcing conditions than primary alcohols but are still effective alkylating agents in the presence of this catalyst.

Exploring Tandem Ruthenium-Catalyzed Hydrogen Transfer and SNAr Chemistry.

A hydrogen-transfer strategy for the catalytic functionalization of benzylic alcohols via electronic arene activation, accessing a diverse range of bespoke diaryl ethers and aryl amines in excellent isolated yields (38 examples, 70% average yield), is reported. Taking advantage of the hydrogen-transfer approach, the oxidation level of the functionalized products can be selected by judicious choice of simple and inexpensive additives.

Structure and inhibition of N-acetylneuraminate lyase from methicillin-resistant Staphylococcus aureus.

N-Acetylneuraminate lyase is the first committed enzyme in the degradation of sialic acid by bacterial pathogens. In this study, we analyzed the kinetic parameters of N-acetylneuraminate lyase from methicillin-resistant Staphylococcus aureus (MRSA). We determined that the enzyme has a relatively high KM of 3.2 mm, suggesting that flux through the catabolic pathway is likely to be controlled by this enzyme. Our data indicate that sialic acid alditol, a known inhibitor of N-acetylneuraminate lyase enzymes, is a stronger inhibitor of MRSA N-acetylneuraminate lyase than of Clostridium perfringens N-acetylneuraminate lyase. Our analysis of the crystal structure of ligand-free and 2R-sialic acid alditol-bound MRSA N-acetylneuraminate lyase suggests that subtle dynamic differences in solution and/or altered binding interactions within the active site may account for species-specific inhibition.

Achiral 2-Hydroxy Protecting Group for the Stereocontrolled Synthesis of 1,2-cis-α-Glycosides by Six-Ring Neighboring Group Participation.

Glycosylation of a fully armed donor bearing a 2-O-(trimethoxybenzenethiol) ethyl ether protecting group is completely α-selective with a range of carbohydrate alcohol acceptors. Low-temperature NMR studies confirm the intermediacy of cyclic sulfonium ion intermediates arising from six-membered ß-sulfonium ring neighboring group participation. Selective protecting group removal is achieved in high yield in a single operation by S-methylation and base-induced ß-elimination.

Direct aqueous synthesis of non-protected glycosyl sulfoxides; weak inhibitory activity against glycosidases.

A flavinium catalyst, in conjunction with hydrogen peroxide as stoichiometric oxidant, allowed the aqueous conversion of non-protected thioglycosides into the corresponding glycosyl sulfoxides. These glycosyl sulfoxides displayed only very weak inhibitory activity against corresponding glycosidases.

Synthesis of arabinose glycosyl sulfamides as potential inhibitors of mycobacterial cell wall biosynthesis.

A series of arabinose glycosyl sulfamides with varying alkyl chain types and lengths were synthesised as mimics of decaprenolphosphoarabinose (DPA), and as potential inhibitors of mycobacterial cell wall biosynthesis. Unprecedented conversion of the desired furanose to the thermodynamically more stable pyranose form occurred during final de-protection. Biological testing against Mycobacterium smegmatis revealed low to moderate anti-mycobacterial activity with marked dependence on alkyl chain length, which in the case of mono-substituted sulfamides was maximal for a C-10 chain.

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of N-acetylmannosamine-6-phosphate 2-epimerase from methicillin-resistant Staphylococcus aureus.

Sialic acids are one of the most important carbohydrate classes in biology. Some bacterial pathogens can scavenge sialic acids from their surrounding environment and degrade them as a source of carbon, nitrogen and energy. This sequestration and subsequent catabolism of sialic acid require a cluster of genes known as the `Nan-Nag' cluster. The enzymes coded by these genes are important for pathogen colonization and persistence. Importantly, the Nan-Nag genes have proven to be essential for Staphylococcus aureus growth on sialic acids, suggesting that the pathway is a viable antibiotic drug target. The enzyme N-acetylmannosamine-6-phosphate 2-epimerase is involved in the catabolism of sialic acid; specifically, the enzyme converts N-acetylmannosamine-6-phosphate into N-acetylglucosamine-6-phosphate. The gene was cloned into an appropriate expression vector, and recombinant protein was expressed in Escherichia coli BL21 (DE3) cells and purified via a three-step procedure. Purified N-acetylmannosamine-6-phosphate 2-epimerase was screened for crystallization. The best crystal diffracted to a resolution of beyond 1.84 Å in space group P21212. Understanding the structural nature of this enzyme from methicillin-resistant S. aureus will provide us with the insights necessary for the development of future antibiotics.

Ruthenium-catalyzed aromatic C-H activation of benzylic alcohols via remote electronic activation.

Remote electronic activation of benzylic alcohols via temporary oxidation facilitates ruthenium-catalyzed arene C-H activation for a range of aromatic alcohols.

Ruthenium-catalyzed oxidation of alcohols into amides.

The synthesis of secondary amides from primary alcohols and amines has been developed using commercially available [Ru(p-cymene)Cl(2)](2) with bis(diphenylphosphino)butane (dppb) as the catalyst.