The carbon monoxide prodrug oCOm-21 increases Ca2+ sensitivity of the cardiac myofilament.

Patients undergoing cardiopulmonary bypass procedures require inotropic support to improve hemodynamic function and cardiac output. Current inotropes such as dobutamine, can promote arrhythmias, prompting a demand for improved inotropes with little effect on intracellular Ca2+ flux. Low-dose carbon monoxide (CO) induces inotropic effects in perfused hearts. Using the CO-releasing pro-drug, oCOm-21, we investigated if this inotropic effect results from an increase in myofilament Ca2+ sensitivity. Male Sprague Dawley rat left ventricular cardiomyocytes were permeabilized, and myofilament force was measured as a function of -log [Ca2+ ] (pCa) in the range of 9.0-4.5 under five conditions: vehicle, oCOm-21, the oCOm-21 control BP-21, and levosimendan, (9 cells/group). Ca2+ sensitivity was assessed by the Ca2+ concentration at which 50% of maximal force is produced (pCa50 ). oCOm-21, but not BP-21 significantly increased pCa50 compared to vehicle, respectively (pCa50 5.52 vs. 5.47 vs. 5.44; p < 0.05). No change in myofilament phosphorylation was seen after oCOm-21 treatment. Pretreatment of cardiomyocytes with the heme scavenger hemopexin, abolished the Ca2+ sensitizing effect of oCOm-21. These results support the hypothesis that oCOm-21-derived CO increases myofilament Ca2+ sensitivity through a heme-dependent mechanism but not by phosphorylation. Further analyses will confirm if this Ca2+ sensitizing effect occurs in an intact heart.

Chemical Synthesis of the PAX Protein Inhibitor EG1 and Its Ability to Slow the Growth of Human Colorectal Carcinoma Cells.

Colorectal cancer is primarily a disease of the developed world. The incidence rate has continued to increase over time, reflecting both demographic and lifestyle changes, which have resulted in genomic and epigenomic modifications. Many of the epigenetic modifications occur in genes known to be closely associated with embryonic development and cellular growth. In particular, the paired box (PAX) transcription factors are crucial for correct tissue development during embryogenesis due to their role in regulating genes involved in proliferation and cellular maintenance. In a number of cancers, including colorectal cancer, the PAX transcription factors are aberrantly expressed, driving proliferation and thus increased tumour growth. Here we have synthesized and used a small molecule PAX inhibitor, EG1, to inhibit PAX transcription factors in HCT116 colorectal cell cultures which resulted in reduced proliferation after three days of treatment. These results highlight PAX transcription factors as playing an important role in the proliferation of HCT116 colorectal cancer cells, suggesting there may be a potential therapeutic role for inhibition of PAX in limiting cancer cell growth.

2-O-ß-d-Glucopyranosyl l-Ascorbic Acid, a Stable Form of Vitamin C, Is Widespread in Crop Plants.

2-O-ß-d-Glucopyranosyl l-ascorbic acid (AA-2ßG) is a stable, bioavailable vitamin C (AA) derivative. We report the distribution and seasonal variation of AA-2ßG in apples and its occurrence in other domesticated crops and in wild harvested Ma̅ori foods. Liquid chromatography-mass spectrometry analyses showed high AA-2ßG concentrations in crab apples (Malus sylvestris) but low concentrations in domesticated apples. Leaves of crab and domesticated apple cultivars contained similar intermediate AA-2ßG concentrations. Fruits and leaves of other crops were analyzed: mainly Rosaceae but also Actinidiaceae and Ericaceae. AA-2ßG was detected in all leaves (0.5-6.1 mg/100 g fr. wt.) but was at lower concentrations in most fruits (0.0-0.5 mg/100 g fr. wt.) except for crab apples (79.4 mg/100 g fr. wt.). Ma̅ori foods from Solanaceae, Piperaceae, Asteraceae, and a fern of Aspleniaceae also contained AA-2ßG. This extensive occurrence suggests a general role in AA metabolism for AA-2ßG.

Synthetic preparation and immunological evaluation of ß-mannosylceramide and related N-acyl analogues.

The synthesis of the invariant natural killer (iNK) T cell agonist ß-mannosylceramide along with a series of fatty amide analogues is reported. Of the six ß-glycosylation protocols investigated, the sulfoxide methodology developed by Crich and co-workers proved to be the most effective where the reaction of a mannosyl sulfoxide and phytosphingosine derivative gave a key glycolipid intermediate as a 95 : 5 mixture of ß- to α-anomers in high yield. A series of mannosyl ceramides were evaluated for their ability to activate D32.D3 NKT cells and induce antitumour activity.

Discovery of a stable vitamin C glycoside in crab apples (Malus sylvestris).

Non-targeted LC-MS metabolomics on fruit of three wild and domesticated apple species (Malus sylvestris, M. sieversii and M. domestica) showed that two crab apple (M. sylvestris) accessions were distinguished by high concentrations of an ascorbic acid glycoside (AAG). This was partly purified, but key NMR signals were masked by inseparable sucrose. Reference samples of 2-O-ß-D-glucopyranosyl L-ascorbic acid and 2-O-ß-D-galactopyranosyl L-ascorbic acid were synthesised, but both coincided with the crab apple AAG on LC-MS. Peracetylation of the crab apple extract allowed both purification and characterisation, and the AAG was proven to be 2-O-ß-D-glucopyranosyl L-ascorbic acid by comparison of 1H NMR, HRMS and HPLC data with synthesised peracetylated ascorbyl glycoside standards. The stability of the natural AA 2-ß-glycoside was similar to synthetic 2-O-α-D-glucopyranosyl L-ascorbic acid, used widely in cosmetic and pharmaceutical products. This discovery in crab apples (Rosaceae) is only the fourth reported occurrence of any ascorbyl glycoside from plants, the others being from Cucurbitaceae, Solanaceae and Brassicaceae. It is hypothesised that AAGs may be more widespread in plants than currently realised.

Functionalisation of Virus-Like Particles Enhances Antitumour Immune Responses.

Virus-like particles (VLP) from the rabbit haemorrhagic disease virus (RHDV) can deliver tumour antigens to induce anticancer immune responses. In this study, we explored how RHDV VLP can be functionalised to enhance the immune response by increasing antigen loading, incorporating linkers to enhance epitope processing, and targeting receptor-mediated internalisation of VLP. RHDV VLP were developed to deliver up to three copies of gp10025-33 which contained proteasome cleavable linkers to target the correct processing of the epitope. Addition of mono- and dimannosides, conjugated to the surface of the gp100 VLP, would utilise a second pathway of internalisation, mannose receptor mediated, to further augment antigen internalised by phagocytosis/macropinocytosis. In vitro cell culture studies showed that a processing linker at the C-terminus of the epitope (gp100.1LC) induced enhanced T-cell activation (7.3 ng/ml interferon- (IFN-) γ release) compared to no linker (3.0 ng/ml IFN-γ) or the linker at the N-terminus (0.8 ng/ml IFN-γ). VLP delivering two (gp100.2L) or three (gp100.3L) gp100 epitopes induced similar high T-cell activation (7.6 ng/ml IFN-γ) compared to gp100.1LC. An in vivo cytotoxicity assay and a therapeutic tumour trial confirmed that mice vaccinated with either gp100.2L or gp100.3L induced a specific antitumour immune response. Mannosylation of the gp100.2L VLP further enhanced the generated immune response, demonstrated by prolonged survival of mice vaccinated with dimannosylated gp100.2L VLP (D-gp100.2L) by 22 days compared to gp100.2L-vaccinated mice. This study showed that functionalisation of RHDV VLP by addition of an epitope-processing linker and mannosylation of the surface facilitates the efficacy of VLP as vaccination vectors for tumour immunotherapy.

5-Bromo-norborn-2-en-7-one derivatives as a carbon monoxide source for palladium catalyzed carbonylation reactions.

Norbornenone (5b), obtained from the reaction of 2,5-dimethyl-3,4-diphenylcyclopentadienone dimer (3) with bromomaleic anhydride (4b), provides an excellent base-triggered source of carbon monoxide for palladium-catalysed carbonylation reactions. Aminocarbonylation, ketoamide synthesis, and Suzuki-Miyaura reactions of aryl iodides carried out in a two-chamber reaction vessel gave good to excellent yields of carbonylated products.

MIS416 as a siRNA Delivery System with the Ability to Target Antigen-Presenting Cells.

MIS416 is a microparticulate formulation derived from propionibacterium acnes cell wall skeletons with intrinsic adjuvant activity. Conjugates of MIS416-SS-peptide containing a disulfide linkage facilitate the cytoplasmic delivery and release of peptides in antigen-presenting cells (APCs). We hypothesized that MIS416-siRNA (small interfering RNA) conjugates, containing a disulfide linkage between MIS416 and the siRNA, would allow cytoplasmic release of siRNA in APCs. MIS416-SS-siStat3 conjugates added to cell culture medium of monolayers of DCs in culture flasks successfully targeted Stat3 mRNA in DCs in vitro without transfection, downregulating Stat3 mRNA and protein levels. These results suggest that MIS416-SS-siRNA conjugates can be used as a novel siRNA delivery system for the knockdown of mRNA levels in APCs.

Norborn-2-en-7-ones as physiologically-triggered carbon monoxide-releasing prodrugs.

A prodrug strategy for the release of the gasotransmitter CO at physiological pH, based upon 3a-bromo-norborn-2-en-7-one Diels-Alder cycloadducts of 2-bromomaleimides and 2,5-dimethyl-3,4-diphenylcyclopentadienone has been developed. Examples possessing protonated amine and diamine groups showed good water solubility and thermal stability. Half-lives for CO-release in TRIS-sucrose buffer at pH 7.4 ranged from 19 to 75 min at 37 °C and 31 to 32 h at 4 °C. Bioavailability in rats was demonstrated by oral gavage and oCOm-21 showed a dose dependent vasorelaxant effect in pre-contracted rat aortic rings with an EC50 of 1.6 ± 0.9 µM. Increased intracellular CO levels following oCOm-21 exposure were confirmed using a CO specific fluorescent probe.

Self-Assembly of Cyclohelicate [M3 L3 ] Triangles Over [M4 L4 ] Squares, Despite Near-Linear Bis-terdentate L and Octahedral M.

Self-assembly of 1:1:2 MII (BF4 )2 (M=Zn or Fe), pyrazine-2,5-dicarbaldehyde (1) and 2-(2-aminoethyl)pyridine gave trimetallic triangle architectures rather than the anticipated tetrametallic [2×2] squares. Options for the nontrivial synthesis of 1 are considered, and synthetic details provided for both preferred routes. Rare cyclohelicate triangle architectures are observed for the pair of structurally characterized yellow-brown [Zn3 L3 ](BF4 )6 and dark green [Fe3 L3 ](BF4 )6 complexes of the neutral bis-terdentate Schiff base L. In order to form these pyrazine-edged triangles, the octahedral metal ions-with all 6 N-donors provided by the terdentate binding pockets of two L-are located 0.4-0.5 Šout of the plane of the bridging pyrazines, towards the center of the triangle. Density functional theory calculations confirm that simple particle counting entropic arguments, which predict triangles over squares, are correct here, with the triangles shown to be energetically favored over the corresponding squares. However, importantly, DFT analysis of these and related triangle versus square systems also show that vibrational contributions to entropy dominate and may significantly influence the preferred architecture, such that simple particle counting cannot in general be reliably employed to predict the observed architecture.

Probing the Structure-Activity Relationship of the Natural Antifouling Agent Polygodial against both Micro- and Macrofoulers by Semisynthetic Modification.

The current study represents the first comprehensive investigation into the general antifouling activities of the natural drimane sesquiterpene polygodial. Previous studies have highlighted a high antifouling effect toward macrofoulers, such as ascidians, tubeworms, and mussels, but no reports about the general antifouling effect of polygodial have been communicated before. To probe the structural and chemical basis for antifouling activity, a library of 11 polygodial analogues was prepared by semisynthesis. The library was designed to yield derivatives with ranging polarities and the ability to engage in both covalent and noncovalent interactions, while still remaining within the drimane sesquiterpene scaffold. The prepared compounds were screened against 14 relevant marine micro- and macrofouling species. Several of the polygodial analogues displayed inhibitory activities at sub-microgram/mL concentrations. These antifouling effects were most pronounced against the macrofouling ascidian Ciona savignyi and the barnacle Balanus improvisus, with inhibitory activities observed for selected compounds comparable or superior to several commercial antifouling products. The inhibitory activity against the microfouling bacteria and microalgae was reversible and significantly less pronounced than for the macrofoulers. This study illustrates that the macro- and microfoulers are targeted by the compounds via different mechanisms.

Carbazole-based N4-donor Schiff base macrocycles: obtained metal free and as Cu(ii) and Ni(ii) complexes.

The very different multi-step routes to the closely related pair of diformyl-carbazole head units, 1,8-diformyl-3,6-di-tert-butyl-9H-carbazole (1tBu) and 1,8-diformyl-9H-carbazole (1H), are detailed and compared. The first examples of Schiff base macrocycles derived from diformyl-carbazole head units are reported. Specifically, the direct cyclisation of 1tBu or 1H with diethylenetriamine gives the two metal-free [1 + 1] Schiff base macrocycles HLH and HLtBu in high yields. Four carbazole-based macrocyclic complexes, [CuIIL(OH2)]OAc and [NiIIL]OAc, where L = LH or LtBu, were accessed either by metallation of these macrocycles, or by metal templated reaction of the macrocycle components. [CuIILtBu(OH2)]OAc·0.5(Ether) and [NiLH]OAc·EtOH, were structurally characterised, confirming the nickel(ii) complexes are square planar (both show diamagnetic NMR spectra) and that the copper(ii) complexes are square pyramidal with a water molecule bound in the axial site. Like porphyrins, both of these N4-donor macrocycles, which differ only in the R group present at the 3 and 6 positions (H or tBu), impose a strong ligand field.

Bridging Small Molecules to Modified Bacterial Microparticles Using a Disulphide Linkage: MIS416 as a Cargo Delivery System.

MIS416 is an intact minimal cell wall skeleton derived from Proprionibacterium acnes that is phagocytosed by antigen presenting cells, including dendritic cells (DCs). This property allows MIS416 to be exploited as a vehicle for the delivery of peptide antigens or other molecules (for example, nucleic acids) to DCs. We previously showed that covalent (non-cleavable) conjugation of OVA, a model antigen derived from ovalbumin, to MIS416 enhanced immune responses in DCs in vivo, compared to unconjugated MIS416 and OVA. Intracellular trafficking promotes the lysosomal degradation of MIS416, leading to the destruction of MIS416 plus the associated cargos conjugated to MIS416. However, lysosomal degradation of cargo may not be desired for some MIS416 conjugates. Here we have investigated whether a cleavable linkage could facilitate release of the cargo in the cytoplasm of DCs to avoid lysosomal degradation. DCs were treated in vitro with disulfide-containing conjugates, and as hypothesised faster release of SIINFEKL peptide in the cytoplasm of DCs was observed with the inclusion of a disulfide bond between MIS416 and cargo. The inclusion of a cleavable disulfide bond in the conjugates did not significantly alter the amount of SIINFEKL antigens presented on MHC I molecules on DCs as compared with conjugates without a disulfide bond. However, the conjugates containing disulfide-linkages performed either slightly better (p<0.05) than, or the same as conjugates without a disulfide bond with respect to in vitro OT-1 T-cell proliferation induced by the presentation of SIINFEKL antigens on DCs, or DC activation studies, respectively. However, disulfide-containing conjugates were less effective than conjugates without a disulfide bond in in vivo cytotoxicity assays. In conclusion, inclusion of a disulfide bond in MIS416-peptide conjugates was associated with efficient release of peptides in the cytoplasm of DCs, an important consideration for MIS416-mediated delivery of degradation-sensitive cargoes. However, treatment of DCs with disulfide-containing conjugates did not significantly alter the presentation of peptide antigens on MHC class I molecules to T-cells, or greatly enhance antigen-associated T-cell proliferation in vitro.

Synthesis and Activity of 6″-Deoxy-6″-thio-α-GalCer and Peptide Conjugates.

A major challenge in the development of highly defined synthetic vaccines is the codelivery of vaccine components (i.e., antigen and adjuvant) to secondary lymphoid tissue to induce optimal immune responses. This problem can be addressed by synthesizing vaccines that comprise peptide antigens covalently attached to glycolipid adjuvants through biologically cleavable linkers. Toward this, a strategy utilizing previously unreported 6″-deoxy-6″-thio analogues of α-GalCer that can undergo chemoselective conjugation with peptide antigens is described. Administration of these conjugate vaccines leads to enhanced priming of antigen specific T cells. This simple vaccine design is broadly applicable to multiple disease indications such as cancer and infectious disease.

A mitochondria-targeted derivative of ascorbate: MitoC.

Mitochondrial oxidative damage contributes to a wide range of pathologies. One therapeutic strategy to treat these disorders is targeting antioxidants to mitochondria by conjugation to the lipophilic triphenylphosphonium (TPP) cation. To date only hydrophobic antioxidants have been targeted to mitochondria; however, extending this approach to hydrophilic antioxidants offers new therapeutic and research opportunities. Here we report the development and characterization of MitoC, a mitochondria-targeted version of the hydrophilic antioxidant ascorbate. We show that MitoC can be taken up by mitochondria, despite the polarity and acidity of ascorbate, by using a sufficiently hydrophobic link to the TPP moiety. MitoC reacts with a range of reactive species, and within mitochondria is rapidly recycled back to the active ascorbate moiety by the glutathione and thioredoxin systems. Because of this accumulation and recycling MitoC is an effective antioxidant against mitochondrial lipid peroxidation and also decreases aconitase inactivation by superoxide. These findings show that the incorporation of TPP function can be used to target polar and acidic compounds to mitochondria, opening up the delivery of a wide range of bioactive compounds. Furthermore, MitoC has therapeutic potential as a new mitochondria-targeted antioxidant, and is a useful tool to explore the role(s) of ascorbate within mitochondria.

NKT cell-dependent glycolipid-peptide vaccines with potent anti-tumour activity.

It is known that T cells can eliminate tumour cells through recognition of unique or aberrantly expressed antigens presented as peptide epitopes by major histocompatibility complex (MHC) molecules on the tumour cell surface. With recent advances in defining tumour-associated antigens, it should now be possible to devise therapeutic vaccines that expand specific populations of anti-tumour T cells. However there remains a need to develop simpler efficacious synthetic vaccines that possess clinical utility. We present here the synthesis and analysis of vaccines based on conjugation of MHC-binding peptide epitopes to α-galactosylceramide, a glycolipid presented by the nonpolymorphic antigen-presenting molecule CD1d to provoke the stimulatory activity of type I natural killer T (NKT) cells. The chemical design incorporates an enzymatically cleavable linker that effects controlled release of the active components in vivo. Chemical and biological analysis of different linkages with different enzymatic targets enabled selection of a synthetic vaccine construct with potent therapeutic anti-tumour activity in mice, and marked in vitro activity in human blood.

Soluble human TLR2 ectodomain binds diacylglycerol from microbial lipopeptides and glycolipids.

TLRs are key innate immune receptors that recognize conserved features of biological molecules that are found in microbes. In particular, TLR2 has been reported to be activated by different kinds of microbial ligands. To advance our understanding of the interaction of TLR2 with its ligands, the recombinant human TLR2 ectodomain (hTLR2ED) was expressed using a baculovirus/insect cell expression system and its biochemical, as well as ligand binding, properties were investigated. The hTLR2ED binds synthetic bacterial and mycoplasmal lipopeptides, lipoteichoic acid from Staphylococcus aureus, and synthetic lipoarabinomannan precursors from Mycobacterium at extracellular physiological conditions, in the absence of its co-receptors TLR1 and TLR6. We also determined that lipopeptides and glycolipids cannot bind simultaneously to hTLR2ED and that the phosphatidyl inositol mannoside 2 (Pim2) is the minimal lipoarabinomannan structure for binding to hTLR2ED. Binding of hTLR2ED to Pim4, which contains a diacylglycerol group with one of its acyl chains containing 19 carbon atoms, indicates that hTLR2ED can bind ligands with acyl chains longer than 16 carbon atoms. In summary, our data indicate that diacylglycerol is the ligand moiety of microbial glycolipids and lipoproteins that bind to hTLR2ED and that both types of ligands bind to the same binding site of hTLR2ED.

Resolution of orthogonally protected myo-inositols with novozym 435 providing an enantioconvergent pathway to Ac2PIM1.

Orthogonally protected chiral myo-inositol derivatives are important intermediates for higher order myo-inositol-containing compounds. Here, the use of the immobilized enzyme Novozym 435 to efficiently catalyze the acetylation of the 5R configured enantiomer of racemic 1,2-O-isopropylidene-myo-inositols possessing chemically and sterically diverse protecting groups at O-3 and O-6 is described. The resolutions were successful with allyl, benzyl, 4-bromo-, 4-methoxy-, 4-nitro-, and 4-(3,4-dimethoxyphenyl)benzyl, propyl, and propargyl protection at O-6 in combination with either allyl or benzyl groups at O-3. Bulky protecting groups slow the rate of acetylation. No reaction was observed for 3,6-di-O-triisopropylsilyl-1,2-O-isopropylidene-myo-inositol. The utility of this methodology was demonstrated by the first reported synthesis of an Ac2PIM1 (9), which used both enantiomers of the resolved 3-O-allyl-6-O-benzyl-1,2-O-isopropylidene-myo-inositol in a convergent synthesis.

A self-adjuvanting vaccine induces cytotoxic T lymphocytes that suppress allergy.

Epitope-based peptide vaccines encompass minimal immunogenic regions of protein antigens to allow stimulation of precisely targeted adaptive immune responses. However, because efficacy is largely determined by the functional status of antigen-presenting cells (APCs) that acquire and present peptides to cells of the adaptive immune system, adjuvant compounds are needed to enhance immunogenicity. We present here a vaccine consisting of an allergen-derived peptide conjugated to a prodrug of the natural killer-like T (NKT) cell agonist α-galactosylceramide, which is highly effective in reducing inflammation in a mouse model of allergic airway inflammation. Unlike other peptide-adjuvant conjugates that directly activate APCs through pattern recognition pathways, this vaccine encourages third-party interactions with NKT cells to enhance APC function. Therapeutic efficacy was correlated with marked increases in the number and functional activity of allergen-specific cytotoxic T lymphocytes (CTLs), leading to suppression of immune infiltration into the lungs after allergen challenge in sensitized hosts.

Mannosylation of virus-like particles enhances internalization by antigen presenting cells.

Internalization of peptides by antigen presenting cells is crucial for the initiation of the adaptive immune response. Mannosylation has been demonstrated to enhance antigen uptake through mannose receptors, leading to improved immune responses. In this study we test the effect of surface mannosylation of protein-based virus-like particles (VLP) derived from Rabbit hemorrhagic disease virus (RHDV) on uptake by murine and human antigen presenting cells. A monomannoside and a novel dimannoside were synthesized and successfully conjugated to RHDV VLP capsid protein, providing approximately 270 mannose groups on the surface of each virus particle. VLP conjugated to the mannoside or dimannoside exhibited significantly enhanced binding and internalization by murine dendritic cells, macrophages and B cells as well as human dendritic cells and macrophages. This uptake was inhibited by the inclusion of mannan as a specific inhibitor of mannose specific uptake, demonstrating that mannosylation of VLP targets mannose receptor-based uptake. Consistent with mannose receptor-based uptake, partial retargeting of the intracellular processing of RHDV VLP was observed, confirming that mannosylation of VLP provides both enhanced uptake and modified processing of associated antigens.