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Our previously discovered monomeric aptamer for SARS-CoV-2 (MSA52) possesses a universal affinity for COVID-19 spike protein variants but is ultimately limited by its ability to bind only one subunit of the spike protein. The symmetrical shape of the homotrimeric SARS-CoV-2 spike protein presents the opportunity to create a matching homotrimeric molecular recognition element that is perfectly complementary to its structural scaffold, causing enhanced binding affinity. Here, we describe a branched homotrimeric aptamer with three-fold rotational symmetry, named TMSA52, that not only possesses excellent binding affinity but is also capable of binding several SARS-CoV-2 spike protein variants with picomolar affinity, as well as pseudotyped lentiviruses expressing SARS-CoV-2 spike protein variants with femtomolar affinity. Using Pd-Ir nanocubes as nanozymes in an enzyme-linked aptamer binding assay (ELABA), TMSA52 was capable of sensitively detecting diverse pseudotyped lentiviruses in pooled human saliva with a limit of detection as low as 6.3 × 10

Screening for a deficiency of glucose-6-phosphate dehydrogenase (G6PD) in red blood cells is vital for determining the potentially life-threatening presence of congenital, hereditary or induced hemolytic anemias. In this study, a "sample-to-readout" paper-based point-of-care (POC) colorimetric biosensor was developed for direct detection of G6PD in whole blood by simple visual comparison to a color card. The G6PD paper sensor was highly stable with no observable loss in performance after room temperature storage for at least 6 weeks, and worked equally well at room temperature and 37 °C. The simple printed paper format and the stability of the colorimetric reagents facilitates scalable manufacturing. The ability to utilize well established sample collection and preparation protocols along with a colorimetric visual readout should facilitate future transfer of this proof-of-concept POC biosensor to remote or resource-poor locations.

Polymorphonuclear leukocyte (PMN)-derived myeloperoxidase (MPO) contributes to the pathophysiology of numerous systemic inflammatory disorders through: (1) direct peroxidation of targets and (2) production of strong oxidizing compounds, e.g., hypohalous acids, particularly hypochlorous acid, which furthers oxidant damage and contributes to the propagation of inflammation and tissue injury/dysfunction. Carbon monoxide-releasing molecules (CORMs) offer potent anti-inflammatory effects; however, the mechanism(s) of action is not fully understood. This study assessed the potential of MPO activity inhibition by a water-soluble CORM, CORM-3. To this end, we used in vitro assays to study CORM-3-dependent modulation of MPO activity with respect to: (1) the inhibition of MPO's catalytic activity generally and (2) the specific inhibition of MPO's peroxidation and halogenation (i.e., production of hypochlorous acid) reactions. Further, we employed primary human umbilical vein endothelial cells (HUVECs) to investigate MPO-dependent cellular activation and dysfunction by measuring intracellular oxidant stress (DHR-123 oxidation) and HUVEC permeability (flux of Texas red-dextran), respectively. The results indicate that CORM-3 significantly inhibits MPO activity as well as MPO's peroxidation and hypohalous acid cycles specifically (p

Activity of the aminoglycoside phosphotransferase APH(3′)-Ia leads to resistance to aminoglycoside antibiotics in pathogenic Gram-negative bacteria, and contributes to the clinical obsolescence of this class of antibiotics. One strategy to rescue compromised antibiotics such as aminoglycosides is targeting the enzymes that confer resistance with small molecules. We demonstrated previously that ePK (eukaryotic protein kinase) inhibitors could inhibit APH enzymes, owing to the structural similarity between these two enzyme families. However, limited structural information of enzyme–inhibitor complexes hindered interpretation of the results. In addition, cross-reactivity of compounds between APHs and ePKs represents an obstacle to their use as aminoglycoside adjuvants to rescue aminoglycoside antibiotic activity. In the present study, we structurally and functionally characterize inhibition of APH(3′)-Ia by three diverse chemical scaffolds, anthrapyrazolone, 4-anilinoquinazoline and PP (pyrazolopyrimidine), and reveal distinctions in the binding mode of anthrapyrazolone and PP compounds to APH(3′)-Ia compared with ePKs. Using this observation, we identify PP derivatives that select against ePKs, attenuate APH(3′)-Ia activity and rescue aminoglycoside antibiotic activity against a resistant Escherichia coli strain. The structures described in the present paper and the inhibition studies provide an important opportunity for structure-based design of compounds to target aminoglycoside phosphotransferases for inhibition, potentially overcoming this form of antibiotic resistance.

Ten polyketide specialized metabolites, epoxynemanione A, nemanifuranones A-F, and nemanilactones A-C, were isolated from the culture filtrate of Nemania serpens (Pers.) Grey (1821), an endophytic fungus from a Riesling grapevine (Vitis vinifera) found in Canada's Niagara region. Additionally, four known metabolites 2-(hydroxymethyl)-3-methoxy-benzoic acid, phyllostine, 5-methylmellein and a nordammarane triterpenoid were isolated. A related known metabolite 2,3-dihydro-2-hydroxy-2,4-dimethyl-5-trans-propenylfuran-3-one has also been included for structural and biological comparison to the nemanifuranones. The latter was isolated from the culture filtrates of Mollisia nigrescens, an endophytic fungus from the leaves and stems of lowbush blueberry (Vaccinium angustifolium) found in the Acadian forest of Nova Scotia, Canada. Their structures were elucidated based on 1D and 2D NMR, HRESIMS measurements, X-ray crystallographic analysis of nemanifuranone A, the nordammarane triterpenoid and 2,3-dihydro-2-hydroxy-2,4-dimethyl-5-trans-propenylfuran-3-one compounds, and comparison of NOE and vicinal 1H-1H coupling constants to literature data for relative stereochemical assignments. Nemanifuranone A possesses a rare C2 hemiacetal and was active against both Gram-negative and Gram-positive bacteria.

Carbon monoxide-releasing molecules (CORMs) suppress inflammation by reducing polymorphonuclear leukocyte (PMN) recruitment to the affected organs. We investigated modulation of PMN-endothelial cell adhesive interactions by water-soluble CORM-401 using an experimental model of endotoxemia in vitro. Human umbilical vein endothelial cells (HUVEC) grown on laminar-flow perfusion channels were stimulated with 1 μg/mL lipopolysaccharide for 6 hours and perfused with 100 μmol/L CORM-401 (or inactive compound iCORM-401)-pretreated PMN for 5 minutes in the presence of 1.0 dyn/cm HUVEC: PMN co-cultures were perfused for additional 15 minutes with PMN-free medium containing CORM-401/inactive CORM-401. The experiments were videorecorded (phase-contrast microscopy), and PMN adhesion/migration were assessed off-line. In parallel, CORM-401-dependent modulation of PMN chemotaxis, F-actin expression/distribution, and actin-regulating pathways [eg, p21-activated protein kinases (PAK1/2) and extracellular signal-regulated kinase (ERK)/C-Jun N-terminal kinase (JNK) mitogen-activated protein kinases (MAPK)] were assessed in response to N-formyl-methionyl-leucyl-phenylalanine (fMLP) stimulation. Pretreating PMN with CORM-401 did not suppress PMN adhesion to HUVEC, but significantly reduced PMN transendothelial migration (P < 0.0001) and fMLP-induced PMN chemotaxis (ie, migration directionality and velocity). These changes were associated with CORM-401-dependent suppression of F-actin levels/cellular distribution and fMLP-induced phosphorylation of PAK1/2 and ERK/JNK MAPK (P < 0.05). CORM-401 had no effect on p38 MAPK activation. In summary, this study demonstrates, for the first time, CORM-401-dependent suppression of neutrophil migratory potential associated with modulation of PAK1/2 and ERK/JNK MAPK signaling and F-actin dynamics.

Resistance to β-lactam antibiotics is mediated primarily by enzymes that hydrolytically inactivate the drugs by one of two mechanisms: serine nucleophilic attack or metal-dependent activation of a water molecule. Serine β-lactamases are countered in the clinic by several codrugs that inhibit these enzymes, thereby rescuing antibiotic action. There are no equivalent inhibitors of metallo-β-lactamases in clinical use, but the fungal secondary metabolite aspergillomarasmine A has recently been identified as a potential candidate for such a codrug. Herein we report the synthesis of aspergillomarasmine A. The synthesis enabled confirmation of the stereochemical configuration of the compound and offers a route for the synthesis of derivatives in the future.

Summary Bacterially produced secondary metabolites are used as antibiotics, anticancer drugs, and for many other medicinal applications. The mechanisms that limit the production of these molecules in the laboratory are not well understood, and this has impeded the discovery of many important compounds. We have identified small molecules that remodel the yields of secondary metabolites in many actinomycetes and show that one set of these molecules does so by inhibiting fatty acid biosynthesis. This demonstrates a particularly intimate relationship between this primary metabolic pathway and secondary metabolism and suggests an approach to enhance the yields of metabolites for discovery and biochemical characterization.

Proton transfer to carbon represents a significant catalytic challenge because of the large intrinsic energetic barrier and the frequently unfavorable thermodynamics. Multiple kinetic isotope effects (KIEs) were measured for acid-catalyzed hydrolysis of the enol ether functionality of enolpyruvylshikimate 3-phosphate (EPSP) as a nonenzymatic analog of the EPSP synthase (AroA) reaction. The large solvent deuterium KIE demonstrated that protonating C3 was the rate-limiting step, and the lack of solvent hydron exchange into EPSP demonstrated that protonation was irreversible. The reaction mechanism was stepwise, with C3, the methylene carbon, being protonated to form a discrete oxacarbenium ion intermediate before water attack at the cationic center, that is, an AH(‡)*AN (or AH(‡) + AN) mechanism. The calculated 3-(14)C and 3,3-(2)H2 KIEs varied as a function of the extent of proton transfer at the transition state, as reflected in the C3-H(+) bond order, nC3-H+. The calculated 3-(14)C KIE was a function primarily of C3 coupling with the movement of the transferring proton, as reflected in the reaction coordinate contribution ((light)ν(‡)/(heavy)ν(‡)), rather than of changes in bonding. Coupling was strongest in early and late transition states, where the reaction coordinate frequency was lower. The other calculated (14)C and (18)O KIEs were more sensitive to interactions with counterions and solvation in the model structures than nC3-H+. The KIEs revealed a moderately late transition state with significant oxacarbenium ion character and with a C3-H(+) bond order ≈0.6.

Palladium complexes incorporating ligands based on a 1,3,5,7-tetramethyl-2,4,8-trioxa-6-phosphaadamantanyl scaffold were used to catalyze the arylation of ethyl cyanoacetate, malononitrile, and various ketones. The products from these reactions can be elaborated to substituted β-arylethylamines and used in microwave-assisted Pictet-Spengler reactions. The protocol developed is suitable for the synthesis of libraries of substituted isoquinolines.

The parallel synthesis of a library of toxoflavin derivatives is described. The microwave-assisted approach involves the de novo generation of the heterocyclic scaffold and allows for facile introduction of a variety of fragments.

The monodentate ligand 1,3,5,7-tetramethyl-2,4,8-trioxa-6-phenyl-6-phospha-adamantane (PA-Ph) is shown to be highly effective in palladium-catalyzed carbonylative cross-coupling. Aryl and vinyl halides were efficiently converted to carboxylic acids, amides and to primary, secondary, and tertiary esters, respectively. Application of the Pd(OAc)2/PA-Ph (1:1) catalyst system proved critical in the methoxycarbonylation of a functionalized nitroresorcinol halide, allowing convenient access to novel platensimycin sulfonamide analogs.

Valproic (2-propylpentanoic) acid is a commonly used drug in the treatment of bipolar disorder and epilepsy. The molecular mechanism that underlies its clinical efficacy remains controversial and is complicated by the broad range of intracellular effects of valproic acid, including its ability to inhibit histone deacetylase (HDAC) and induce protein chaperone expression. Here we show that an established HDAC inhibitor, trichostatin A, promotes ER chaperone expression in HEK293 cells. Furthermore, we use chemical derivatives of valproic acid to show that the ability to promote GRP78 levels directly correlates with the induction of histone H4 hyperacetylation. These results suggest that exposure to valproic acid enhances chaperone expression by a mechanism that involves histone hyperacetylation.

Resistance to β-lactam antibiotics is mediated primarily by enzymes that hydrolytically inactivate the drugs by one of two mechanisms: serine nucleophilic attack or metal-dependent activation of a water molecule. Serine β-lactamases are countered in the clinic by several codrugs that inhibit these enzymes, thereby rescuing antibiotic action. There are no equivalent inhibitors of metallo-β-lactamases in clinical use, but the fungal secondary metabolite aspergillomarasmine A has recently been identified as a potential candidate for such a codrug. Herein we report the synthesis of aspergillomarasmine A. The synthesis enabled confirmation of the stereochemical configuration of the compound and offers a route for the synthesis of derivatives in the future.

Efficient synthetic routes for a number of deuterated analogues of 2-methoxy-3-isopropylpyrazines and 2-methoxy-3-isobutylpyrazines have been developed involving the condensation of glyoxal with an α-amino acid amide followed by methylation with iodomethane. In this way [2H3]2-methoxy-3-isopropylpyrazine, 2-methoxy-3-isopropyl-[2H2]pyrazine, [2H3]2-methoxy-3-isopropyl-[2H2]pyrazine, [2H3]2-methoxy-3-isobutylpyrazine; 2-methoxy-3-isobutyl-[2H2]pyrazine and [2H3]2-methoxy-3-isobutyl-[2H2]pyrazine were prepared and characterized by NMR and MS. Copyright © 2002 John Wiley & Sons, Ltd.

Substituted imidazoles can be prepared efficiently from cyclic or acyclic 1,2-aminoalcohols via a four-step procedure involving acylation of the amine, oxidation of the alcohol, imine formation and cyclization. Examples are presented and the methodology is applied in the generation of a library of compounds containing a fused imidazole-azepine motif.

The parallel synthesis of a library N1- and C3-substituted-pyrazolo[3,4-d]pyrimidines is described. The microwave-assisted approach involves the de novo generation of the heterocyclic scaffold, facile alkylation at N1 via either a Mitsunobu or a direct alkylation reaction and arylation at C3 via a Suzuki reaction.

Previous work has demonstrated that carbon monoxide releasing molecules (CORMs) suppress inflammation, however, the mechanisms are not well understood. Neutrophil (PMN)-derived myeloperoxidase (MPO...

The radical addition of PH3 to limonene results in the formation of 4,8-dimethyl-2-phosphabicyclo[3.3.1]nonane, which has been characterized by X-ray crystallographic analyses of its oxidation products, 2-(4-methylcyclohexyl)propylphosphonic acid and 4,8-dimethyl-2-phosphabicyclo[3.3.1]nonan-2-ol-oxide.

α-Diazoketones derived from pyrrolyl- and indolyl-carboxylic acids were prepared and their Rh2(OAc)4 catalyzed decomposition chemistry was studied. These reactions generally resulted in the alkylation of the heteroaromatic system by the ketocarbenoid and in some instances the systems underwent CH or NH insertions. Evidence that some of these reactions proceed via a cyclopropane intermediate is presented. The methodology described provides facile access to fused pyrrolyl– or indolyl–cycloalkanone systems wherein the carbonyl is beta to the heteroaromatic system.

The reactions of ethyl diazoacetate with a series of 2-substituted thiophenes in the presence of catalytic rhodium (II) acetate were examined. In the cases of 2-methylthiophene and 2-(trimethylsilyl)thiophene, cyclopropane and thiopyran products predominated while thiophene-2-thiol and 2-(methylthio)thiophene gave rise to thiopyrans and ethyl 2-(2-thienylsulfanyl)acetate. No reaction was apparent when 2-pyrrolidinothiophene was used. 2-Alkoxythiophenes, however, allowed for the production of a series of ethyl 6-alkoxy-6-thioxo-2, 4-hexadienoates. The mechanistic implications are discussed. The reactions of EDA with 2-substituted thiophenes and catalytic Rh2(OAc)4 produced cyclopropanes, thiopyrans and polyenes. Download : Download full-size image

Treatment of 1-diazo-3-(2-thienyl)-2-propanone with catalytic rhodium (II) acetate results in cyclopropanation followed by acid-catalyzed ring opening and tautomerization to yield 5,6-dihydro-4H-cyclopenta[b]thiopen-5-one. Under the same conditions, however, the isomeric 1-diazo-3-(3-thienyl)-2-propanone generates a cyclopropane intermediate which undergoes [4+2] cycloreversion, isomerization and Diels-Alder dimerization to give a complex spiro-disulphide. While the 2-substituted thiophene behaves like other homologous members of the thienyl series, the isomeric 3-substituted thiophene undergoes chemistry seen previously with analogous furanyl compounds. The insight into the mechanistic underpinnings provided by preliminary molecular modeling at a PM3 level is discussed.

[structure: see text] Phosphorinanes are presented as a class of phosphine ligand suitable for organopalladium cross-coupling chemistry. Prepared via a direct double Michael addition of a monoalkyl- or arylphosphine to phorone followed by a Wolf-Kishner reduction, phosphorinanes are relatively inexpensive to manufacture and allow modification of one of the alkyl moieties permitting steric and electronic fine-tuning of the ligands. Library screening and applications of these ligands in the Suzuki, Sonogashira, ketone arylation, and aryl amination reactions are presented.

Aminoglycoside phosphotransferase 3'IIIa (APH3'IIIa) is a bacterial enzyme involved in antibiotic resistance through phosphorylation of aminoglycosides, which can potentially be overcome by co-administration of an APH3'IIIa inhibitor with the antibiotic. Current assay methods for discovery of APH3'IIIa inhibitors suffer from low specificity and high false positive/negative hit rates. Here, we describe a method for screening APH3'IIIa inhibitors based on direct detection of kanamycin A phosphorylation using MALDI-MS/MS, which is more rapid than conventional assays and does not require secondary assays or sample cleanup. The MALDI-MS/MS assay operates at an ionic strength of 45 mM and co-factors can be utilized at near-physiological levels for optimal enzyme activity. Detection via MALDI-MS/MS allowed for improved reproducibility when compared to ESI-MS/MS. Furthermore, the use of MS/MS provided better signal-to-noise ratios relative to MS alone on the MALDI instrument. The assay was validated via generation of Z'-factors, with values of 0.78 and 0.56 in the absence and presence of 0.2% DMSO, respectively. The assay was used to screen a kinase directed library of >200 compounds, assayed as 21 mixtures of 10 compounds each. Five novel synthetic inhibitors were identified following mixture deconvolution. Inhibition constants were obtained for the aforementioned inhibitors using the MALDI-MS/MS assay, revealing several low to mid micromolar "hits", and highlighting the quantitative nature of the assay.

The use of Pd 2 dba 3 ·CHCl 3 and 1,3,5,7-tetramethyl-2,4,8-trioxa-6-phenyl-6-phospha-adamantane has been shown to facilitate the effective amination of aryl halides with aromatic or aliphatic amines in high yields.

The use of a catalyst system based on Pd 2 dba 3 ·CHCl 3 and 1,3,5,7-tetramethyl-2,4,8-trioxa-6-phenyl-6-phospha-adamantane allows for Suzuki coupling aryl halides with an array of boronic acids on a solid-phase platform. The reactions can be carried out at room temperature with low palladium loadings in high yields.

Trihexyl(tetradecyl)phoshonium decanoate was shown to be an effective promoter for the Henry nitroaldol reaction of nitromethane with aromatic aldehydes. A mechanism is proposed involving Lewis acid activation of the carbonyl group proceeding through a trigonal-bipyramidal intermediate. Evidence in accord with the postulated pathway as opposed to a base or phase transfer-mediated route involving proton transfer from the nitroalkane is presented.

The syntheses of two cyclomaltoheptaose-based metal chelants, cyclomaltoheptaose-ethylenediaminetetraacetate (CD-EDTA) and cyclomaltoheptaose-diamide-disulfur (CD-DADS), are described. The chelant moieties are attached to the 6-position of a single pyranose in the cyclomaltoheptaose via a short diamine spacer chain. Characterization of these novel chelants has been achieved using NMR and MS techniques. The peculiar fluxional properties of the CD-EDTA molecules is also discussed.

[reaction: see text] A new class of sterically hindered phosphines based on a phospha-adamantane framework is described. Arylation or alkylation of the 1,3,5,7-tetramethyl-2,4,8-trioxa-6-phospha-adamantane system allows for the preparation of tertiary phosphines suitable for use in palladium-catalyzed cross-coupling reactions. For example, use of a catalytic system incorporating Pd(2)(dba)(3) and 1,3,5,7-tetramethyl-2,4,8-trioxa-6-phenyl-6-phospha-adamantane is shown to promote the Suzuki cross-coupling of aryl iodides, bromides, and activated chlorides with a variety of aryl boronic acids at room temperature in a few hours with high yields.

Proton transfer to carbon atoms is a significant catalytic challenge because of the large intrinsic energetic barrier and the frequently unfavorable thermodynamics. The main catalytic challenge for enolpyruvylshikimate 3-phosphate synthase (EPSP synthase, AroA) is protonating the methylene carbon atom of phosphoenolpyruvate, or EPSP, in the reverse reaction. We performed transition state analysis using kinetic isotope effects (KIEs) on AroA-catalyzed EPSP hydrolysis, which also begins with a methylene carbon (C3) protonation, as an analog of AroA's reverse reaction. As part of this analysis, an inorganic phosphate scavenging system was developed to remove phosphate which, though present in microscopic amounts in solution, is ubiquitous. The reaction was stepwise, with irreversible C3 protonation to form an EPSP cation intermediate; that is, an AH(‡)*AN mechanism. The large experimental 3-(14)C KIE, 1.032 ± 0.005, indicated strong coupling of C3 with the motion of the transferring proton. Calculated 3-(14)C KIEs for computational transition state models revealed that the transition state occurs early during C3-H(+) bond formation, with a C3-H(+) bond order of ≈0.24. The observed solvent deuterium KIE, 0.97 ± 0.04, was the lowest observed to date for this type of reaction, but consistent with a very early transition state. The large 2-(14)C KIE reflected an "electrostatic sandwich" formed by Asp313 and Glu341 to stabilize the positive charge at C2. In shifting the transition state earlier than the acid-catalyzed reaction, AroA effected a large Hammond shift, indicating that a significant part of AroA's catalytic strategy is to stabilize the positive charge in the EPSP cation. A computational model containing all the charged amino acid residues in the AroA active site close to the reactive center showed a similar Hammond shift relative to the small transition state models.

The role of CO in the modulation of antioxidant enzyme function has not been investigated, yet. In this study we assessed the effects and potential mechanisms of the ruthenium-based water-soluble CO-releasing molecule CORM-3 in the modulation of superoxide dismutase (SOD) activity/binding in vascular endothelial cells (HUVECs). To this end, HUVECs were treated with CORM-3 (100 μM) and assessed for total SOD activity in cell lysates (cell-associated SOD activity) and cell culture supernatants (soluble SOD). In parallel, release/binding of extracellular SOD (SOD-3) in the absence or presence of heparin (1-10 IU/ml), a key factor regulating SOD-3 cell-surface binding, was investigated. In addition, the effects of CORM-3 on the modulation of purified SOD-1 and SOD-2 activity in a cell-free system were also assessed. The results obtained indicate that CORM-3 effectively suppresses the activity of both purified SOD-1 and SOD-2. These findings were accompanied by CORM-3-dependent attenuation of total cell-associated SOD activity (without affecting SOD-1/SOD-2 protein expression) and a subsequent increase in ROS production (DHR123 oxidation) in HUVECs. In parallel, a concomitant increase in soluble-SOD activity (due to increased SOD-3 release from the cell surface) was observed in the cell culture supernatants. However, in the presence of heparin, total cell-associated SOD activity was significantly increased by CORM-3, because of increased binding of SOD-3 to HUVECs. Taken together these findings indicate for the first time that CORM-3 modulates both the activity of intracellular SOD (i.e., SOD-1 and SOD-2) and the binding of extracellular SOD (SOD-3) to the cell surface.

Palladium complexes of 1,3,5,7-tetramethyl-2,4,8-trioxa-6-phenyl-6-phosphaadamantane are shown to be effective catalytic systems facilitating the sequential application of a microwave-assisted Sonogashira and carbonylative annulation reaction for the preparation of substituted flavones.

Recently, it has been shown that carbon monoxide (CO)-releasing molecule (CORM)-released CO can suppress inflammation. In this study, we assessed the effects and potential mechanisms of a ruthenium-based water-soluble CO carrier [tricarbonylchloroglycinate-ruthenium(II) (CORM-3)] in the modulation of polymorphonuclear leukocyte (PMN) inflammatory responses in an experimental model of sepsis. Sepsis in mice was induced by cecal ligation and puncture. CORM-3 (3 mg/kg iv) was administered 15 min after the induction of cecal ligation and puncture. PMN accumulation in the lung (myeloperoxidase assay), bronchoalveolar lavage (BAL) fluid, and lung vascular permeability (protein content in BAL fluid) were assessed 6 h later. In in vitro experiments, human PMNs were primed with LPS (10 ng/ml) and subsequently stimulated with formyl-methionyl-leucylphenylalanine (fMLP; 100 nM). PMN production of ROS (L-012/dihydrorhodamine-123 oxidation), degranulation (release of elastase), and PMN rolling, adhesion, and migration to/across human umbilical vein endothelial cells (HUVECs) were assessed in the presence or absence of CORM-3 (1–100 μM). The obtained results indicated that systemically administered CORM-3 attenuates PMN accumulation and vascular permeability in the septic lung. Surprisingly, in in vitro experiments, treatment of PMNs with CORM-3 further augmented LPS/fMLP-induced ROS production and the release of elastase. The latter effects, however, were accompanied by an inability of PMNs to mobilize elastase to the cell surface (plasma membrane), an event required for efficient PMN transendothelial migration. The CORM-3-induced decrease in cell surface levels of elastase was followed by decreased PMN rolling/adhesion to HUVECs and complete prevention of PMN migration across HUVECs. In contrast, treatment of HUVECs with CORM-3 had no effect on PMN transendothelial migration. Taken together, these findings indicate that, in sepsis, CORM3-released CO, while further amplifying ROS production and degranulation of PMNs, concurrently reduces the levels of cell surface-bound elastase, which contributes to suppressed PMN transendothelial migration.

Glycogen synthase kinase-3beta (GSK-3beta) is involved in the hyperphosphorylation of previously phosphorylated (primed) substrates, and is currently assayed using an approach based on the incorporation of gamma-(32)P-radiolabelled isotopes into substrate peptides. The requirement to detect hyperphosphorylation of a primed substrate poses a particular challenge for development of a high-throughput screening assay, as many current kinase assays are designed to produce a signal in the presence of any phosphorylation site, and thus are only suitable for beta-unphosphorylated substrates. Herein, we have developed an electrospray-ionization tandem mass spectrometry (ESI-MS/MS) assay to allow for direct detection of a hyperphosphorylated product which is formed in a solution reaction involving a primed peptide substrate (GSM peptide) and GSK-3beta. Optimum reaction conditions (level of Mg(2+), buffer type, ionic strength, pH, enzyme concentration, and reaction time) were established to both maintain the activity of GSK-3beta and allow for substrate and product quantification through ESI/MS/MS. We show that the MS-based assay allows for rapid determination of GSK-3beta activity from reaction volumes of approximately 40 microL and that it can be used to assess IC(50) values and the site of action of known inhibitors. It also can be used for automated screening of small-molecules mixtures to identify inhibitors of GSK-3beta.

High chemical yields and high levels of stereochemical inversion are demonstrated in the phosphorane-mediated esterification reaction of chiral alcohols with non-hindered carboxylic acids through the incorporation of sterically non-hindered alkyl groups of phosphorus.

The Heck cross-coupling of aryl iodides and bromides with olefins proceeds in the phosphonium salt ionic liquid trihexyl(tetradecyl)phosphonium chloride (THP-Cl) in excellent yields. Furthermore, it is shown that the counter anion matched to the phosphonium cation exerts a measurable effect on the overall yield.

A family of compounds based upon the chemical structure of valproate were synthesized and assayed for their ability to inhibit glycogen synthase kinase (GSK)-3 alpha and beta activity in vitro. This data is correlated to the known anti-convulsant properties of these compounds in order to determine the potential role of GSK-3 inhibition in the therapeutic efficacy of these drugs.

The sensitization of Eu(III) and Tb(III) by ethylenediaminetetraaceticacid (EDTA)-derivatized tryptophan (Trp), 7-azatryptophan (7AW) and 5-hydroxytryptophan (5HW) has been examined. These Trp analogs were utilized in the present study because they can be incorporated into proteins in place of native Trp residues and because they absorb strongly beyond 305 nm (where Trp absorbance goes to zero), allowing selective excitation of such species in the presence of other Trp-containing proteins. All three indole derivatives were able to sensitize Tb(III) luminescence, with the relative sensitization being in the order Trp5HW7AW. On the other hand, only the 7AW-EDTA complex was able to sensitize Eu(III) luminescence, likely owing to a better spectral overlap between 7AW emission and Eu(III) absorbance. The sensitized emission of Tb(III) and Eu(II) displayed the expected long emission lifetimes at 545 nm [for Tb(III)] and 617 nm [for Eu(III)], indicating that long-lifetime lanthanide emission could be produced using nonnatural amino-acid donors. Thus, 7AW- and 5HW-sensitized lanthanide emissions should prove to be useful in biophysical studies, such as the use of fluorescence energy transfer to probe biomolecular interactions in vivo.

The poly-methylene interrupted fatty acid 5(Z),11(Z)-eicosadienoic acid and its tetradeuterated analogue were prepared via a convergent synthetic sequence.

Uroporphyrinogen III synthase, the product of the hemD gene, is the enzyme responsible for the cyclisation of the linear tetrapyrrole, hydroxymethylbilane. The hemD gene isolated from Bacillus subtilis was manipulated by PCR to enable direct cloning behind a synthetic ribosome-binding site downstream of tandem bacteriophage lambda PR and PL promoters in a pCE30-derived vector. Following thermal induction of transcription, the resulting plasmid (pPS21) directed the synthesis of uroporphyrinogen III synthase. The protein produced was soluble and was readily purified. Pure uroporphyrinogen III synthase is monomeric with an isoelectric point of 4.1 and an optimum pH for activity of 8.3. Its specific activity by assay using synthetic hydroxymethylbilane as substrate is 565 units mg-1 and the Km for this substrate is 330 +/- 30 nM. The N-terminal sequence of the enzyme is Met-Glu-Asn-Asp-Phe-Pro-Leu, in agreement with the gene-derived sequence. Studies based on amino acid modifications suggest that arginine, lysine and probably histidine residues are essential for the activity of uroporphyrinogen III synthase. Significantly, this synthase from B. subtilis is substantially more thermostable than the enzymes from previously studied sources.