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.

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.

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.

Using exomarkers to assess mitochondrial reactive species in vivo.

BACKGROUND: The ability to measure the concentrations of small damaging and signalling molecules such as reactive oxygen species (ROS) in vivo is essential to understanding their biological roles. While a range of methods can be applied to in vitro systems, measuring the levels and relative changes in reactive species in vivo is challenging. SCOPE OF REVIEW: One approach towards achieving this goal is the use of exomarkers. In this, exogenous probe compounds are administered to the intact organism and are then transformed by the reactive molecules in vivo to produce a diagnostic exomarker. The exomarker and the precursor probe can be analysed ex vivo to infer the identity and amounts of the reactive species present in vivo. This is akin to the measurement of biomarkers produced by the interaction of reactive species with endogenous biomolecules. MAJOR CONCLUSIONS AND GENERAL SIGNIFICANCE: Our laboratories have developed mitochondria-targeted probes that generate exomarkers that can be analysed ex vivo by mass spectrometry to assess levels of reactive species within mitochondria in vivo. We have used one of these compounds, MitoB, to infer the levels of mitochondrial hydrogen peroxide within flies and mice. Here we describe the development of MitoB and expand on this example to discuss how better probes and exomarkers can be developed. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.

Physical characterization of synthetic phosphatidylinositol dimannosides and analogues in binary systems with phosphatidylcholine.

Native phosphatidylinositol mannosides (PIMs) from the cell wall of Mycobacterium bovis (M. bovis) and synthetic analogues have been identified to exert immunostimulatory activities. These activities have been investigated using particulate delivery systems containing native mannosylated lipids or total lipid extracts. Limited work has been carried out examining the incorporation of individual PIM lipids into suitable particulate formulations such as liposomes. The present study explored the possibility of constructing phosphatidylcholine (PC)-based liposomes containing synthetic PIM analogues. A series of six phosphatidylinositol dimannosides (PIM(2)s) and phosphatidylglycerol dimannosides (PGM(2)s) was characterized in this study. Binary Langmuir monolayers are a useful approach for establishing pharmaceutical properties, such as lipid-lipid interactions in mixed monolayers, to facilitate the design of liposome-based delivery systems. In mixed films the phosphoglycolipids were found to be miscible with PC based on evaluation of collapse pressures and deviations of experimental molecular areas from calculated ideal values. Concanavalin A (ConA) agglutination confirmed the presence of mannosylated lipids on the surface of the liposomes. Physicochemical properties of liposomes were affected by the presence of 2% (w/w) of phosphoglycolipids with liposome stability being increased by incorporation of long-chain PIM(2) and PGM2. Overall, while membrane stability of the liposomes was found to be dependent on incorporation of the phosphoglycolipids, all formulations retained proteins in amounts making them suitable for delivery.

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.

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.

Physicochemical and biological characterization of synthetic phosphatidylinositol dimannosides and analogues.

Native phosphatidylinositol mannosides (PIMs), isolated from the cell wall of Mycobacterium bovis, and synthetic PIM analogues have been reported to offer a variety of immunomodulating properties, including both suppressive and stimulatory activity. While numerous studies have examined the biological activity of these molecules, the aim of this research was to assess the physicochemical properties at a molecular level and correlate these characteristics with biological activity in a mouse model of airway eosinophilia. To accomplish this, we varied the flexibility and lipophilicity of synthetic PIMs by changing the polar headgroup (inositol- vs glycerol-based core) and the length of the acyl chains of the fatty acid residues (C0, C10, C16, and C18). A series of six phosphatidylinositol dimannosides (PIM2s) and phosphatidylglycerol dimannosides (PGM2s) were synthesized and characterized in this study. Langmuir monolayer studies showed that surface pressure-area (π-A) isotherms were greatly influenced by the length of the lipid acyl chains as well as the steric hindrance and volume of the headgroups. In aqueous solution, lipidated PIM2 and PGM2 compounds were observed to self-assemble into circular aggregates, as confirmed by dynamic light scattering and transmission electron microscopic investigations. Removal of the inositol ring but retention of the three-carbon glycerol unit maintained biological activity. We found that the deacylated PGM2, which did not show self-organization, had no effect on the eosinophil numbers but did have an impact on the expansion of OVA-specific CD4(+) Vα2Vß5 T cells.

The dimethoxyphenylbenzyl protecting group: an alternative to the p-methoxybenzyl group for protection of carbohydrates.

A reliable reagent system for the cleavage of 4-(3,4-dimethoxyphenyl)benzyl (DMPBn) ethers under acidic conditions has been established. Treatment of DMPBn-protected mono- and pseudodisaccharides with TFA in anhydrous CH2Cl2 and 3,4-(methylenedioxy)toluene as a cation scavenger resulted in the selective cleavage of the DMPBn ether giving the corresponding deprotected products in moderate to high yields. Examples are reported which show that allyl, benzyl, and p-bromobenzyl ethers, esters, and glycosidic linkages are stable to these reaction conditions. The selective cleavage of allyl, p-bromobenzyl, and PMB ethers in protected carbohydrates containing DMPBn ethers are also demonstrated. This work establishes the 4-(3,4-dimethoxyphenyl)benzyl ether as an effective and robust alternative to p-methoxybenzyl as a protecting group for alcohols.

Synthesis and mass spectral characterization of mycobacterial phosphatidylinositol and its dimannosides.

A family of naturally occurring mycobacterial phosphatidylinositol (PI) and its dimannosides (PIM(2), AcPIM(2), and Ac(2)PIM(2)) that all possess the predominant natural 19:0/16:0 phosphatidyl acylation pattern were prepared to study their mass spectral fragmentations. Among these, the first synthesis of a fully lipidated PIM (i.e., (16:0,18:0)(19:0/16:0)-PIM(2)) was achieved from (±)-1,2:4,5-diisopropylidene-D-myo-inositol in 16 steps in 3% overall yield. A key feature of the strategy was extending the utility of the p-(3,4-dimethoxyphenyl)benzyl protecting group for its use at the O-3 position of inositol to allow installation of the stearoyl residue at a late stage in the synthesis. Mass spectral studies were performed on the synthetic PIMs and compared to those reported for natural PIMs identified from a lipid extract of M. bovis BCG. These analyses confirm that fragmentation patterns can be used to identify the structures of specific PIMs from the cell wall lipid extract.

Chemical synthesis and immunosuppressive activity of dipalmitoyl phosphatidylinositol hexamannoside.

Phosphatidylinositol mannosides (PIMs) isolated from mycobacteria have been identified as an important class of phosphoglycolipids with significant immune-modulating properties. We present here the synthesis of dipalmitoyl phosphatidylinositol hexamannoside (PIM(6)) 1 and the first reported functional biology of a synthetic PIM(6). Key steps in the synthetic protocol included the selective glycosylation of an inositol 2,6-diol with a suitably protected mannosyl donor and construction of the glycan core utilizing a [3 + 4] thio-glycosylation strategy. The target 1 was purified by reverse phase chromatography and characterized by standard spectroscopic methods, HPLC, and chemical modification by deacylation to dPIM(6). The (1)H NMR spectrum of synthetic dPIM(6) obtained from 1 matched that of dPIM(6) obtained from nature. PIM(6) (1) exhibited dendritic cell-dependent suppression of CD8(+) T cell expansion in a human mixed lymphocyte reaction consistent with the well established immunosuppressive activity of whole mycobacteria.

A PIM2 analogue suppresses allergic airway disease.

Two approaches for the synthesis of a phosphatidylinositol dimannoside (PIM2) analogue 4 that mimics the suppressive activity of natural PIMs and also synthetic PIM2 have been developed. This analogue, where the inositol core was replaced by glycerol, was tested for its ability to suppress cellular inflammation in a mouse model of allergic asthma and shown to be effective in suppressing airway eosinophilia. Suppression of all inflammatory cells monitored was observed, indicating a general blockade of cellular activity. These data indicate that the inositol core is not essential for this suppressive activity.

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.

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.

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.

Role of phosphatidylinositol mannosides in the interaction between mycobacteria and DC-SIGN.

The C-type lectin dendritic cell (DC)-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) is the major receptor on DCs for mycobacteria of the Mycobacterium tuberculosis complex. Recently, we have shown that although the mannose caps of the mycobacterial surface glycolipid lipoarabinomannan (ManLAM) are essential for the binding to DC-SIGN, genetic removal of these caps did not diminish the interaction of whole mycobacteria with DC-SIGN and DCs. Here we investigated the role of the structurally related glycolipids phosphatidylinositol mannosides (PIMs) as possible ligands for DC-SIGN. In a binding assay with both synthetic and natural PIMs, DC-SIGN exhibited a high affinity for hexamannosylated PIM(6), which contains terminal alpha(1-->2)-linked mannosyl residues identical to the mannose cap on ManLAM, but not for di- and tetramannosylated PIM(2) and PIM(4), respectively. To determine the role of PIM(6) in the binding of whole mycobacteria to DC-SIGN, a mutant strain of M. bovis bacillus Calmette-Guérin deficient in the production of PIM(6) (Delta pimE) was created, as well as a double knockout deficient in the production of both PIM(6) and the mannose caps on LAM (Delta pimE Delta capA). Compared to the wild-type strain, both mutant strains bound similarly well to DC-SIGN and DCs. Furthermore, the wild-type and mutant strains induced comparable levels of interleukin-10 and interleukin-12p40 when used to stimulate DCs. Hence, we conclude that, like ManLAM, PIM(6) represents a bona fide DC-SIGN ligand but that other, as-yet-unknown, ligands dominate in the interaction between mycobacteria and DCs.

A synthetic analogue of phosphatidylinositol mannoside is an efficient adjuvant.

We recently described the synthesis of an ether linked analogue of phosphatidylinositol dimannoside (PIM(2)ME). In the current study, PIM(2)ME was found to significantly enhance the release of the key Th1 cytokine interleukin-12 (IL-12) by dendritic cells (DCs) of naive mice in vitro, but not interleukin-10 (IL-10). Based on this result, it was hypothesized that PIM(2)ME would be an effective adjuvant for cell-mediated immune responses. Injections of PIM(2)ME alone did not lead to weight loss and did not have toxic side effects, based on biomarkers of toxicity in serum,demonstrating that the compound induced no apparent adverse side effects. Mice were vaccinated with the core antigens of the hepatitis C virus by itself or with three different adjuvants, namely PIM(2)ME, a commercial preparation of monophosphoryl lipid A (MPL) or a preparation of aluminium hydroxide gel (alum). A control group of animals received the antigen only with no adjuvants. Immune responses to the Hepatitis C viral antigens were monitored by measuring antigen-specific production of interferon-gamma (IFN-gamma), the p40 subunit of interleukin-12 (IL-12) and interleukin-10 (IL-10) to assess cell-mediated immune responses. Vaccination of mice with Hepatitis C viral antigens with the adjuvant PIM(2)ME led to a significant increase in cell-mediated immune responses (IFN-gamma and IL-12). Injection of Hepatitis C viral antigens in alum led to no enhancement of the cell-mediated immune response. We conclude that PIM(2)ME is an efficacious adjuvant for enhancing cell-mediated immunity, and induces no observable adverse effects.

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.

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.