Protecting carbohydrates with ethers, acetals and orthoesters under basic conditions.

Chlorinated ethyl and vinyl ethers are introduced at various positions of carbohydrates. Depending on the relative stereochemistry, vinylethers, acetals or orthoesters are formed under basic conditions. The products are stable, but are easily deprotected after dechlorination. The scope of the intramolecular protection is studied using common pentoses and hexoses.

Conformationally superarmed S-ethyl glycosyl donors as effective building blocks for chemoselective oligosaccharide synthesis in one pot.

A new series of superarmed glycosyl donors has been investigated. It was demonstrated that the S-ethyl leaving group allows for high reactivity, which is much higher than that of equally equipped S-phenyl glycosyl donors that were previously investigated by our groups. The superarmed S-ethyl glycosyl donors equipped with a 2-O-benzoyl group gave complete ß-stereoselectivity. Utility of the new glycosyl donors has been demonstrated in a one-pot one-addition oligosaccharide synthesis with all of the reaction components present from the beginning.

Rotigaptide protects the myocardium and arterial vasculature from ischaemia reperfusion injury.

AIM: Ischaemia-reperfusion injury (IRI) causes impaired endothelial function and is a major component of the adverse effects of reperfusion following myocardial infarction. Rotigaptide increases gap junction conductance via connexin-43. We tested the hypothesis that rotigaptide reduces experimental myocardial infarction size and ameliorates endothelial IRI in humans. METHODS: Myocardial infarction study: porcine myocardial infarction was achieved by catheter-induced occlusion of the left anterior descending artery. In a randomized double-blind study, rotigaptide (n = 9) or placebo (n = 10) was administered intravenously as a 10 min bolus prior to reperfusion and continuously during 2 h of reperfusion. Myocardial infarction size (IS) was assessed as proportion of the area at risk (AAR). Human translational study: forearm IRI was induced in the presence or absence of intra-arterial rotigaptide. In a randomized double-blind study, forearm arterial blood flow was measured at rest and during intra-arterial infusion of acetylcholine (5-20 µg min(-1) ; n = 11) or sodium nitroprusside (2-8 mg min(-1) ; n = 10) before and after intra-arterial infusion of placebo or rotigaptide, and again following IRI. RESULTS: Myocardial infarction study: Rotigaptide treatment was associated with a reduction of infarct size (IS/AAR[%]: 18.7 ± 4.1 [rotigaptide] vs. 43.6 ± 4.2 [placebo], P = 0.006). Human translational study: Endothelium-dependent vasodilatation to acetylcholine was attenuated after ischaemia-reperfusion in the presence of placebo (P = 0.007), but not in the presence of rotigaptide (P = NS). Endothelium-independent vasodilatation evoked by sodium nitroprusside was unaffected by IRI or rotigaptide (P = NS). CONCLUSIONS: Rotigaptide reduces myocardial infarction size in a porcine model and protects from IRI-related endothelial dysfunction in man. Rotigaptide may have therapeutic potential in the treatment of myocardial infarction.

Human L-ficolin recognizes phosphocholine moieties of pneumococcal teichoic acid.

Human L-ficolin is a soluble protein of the innate immune system able to sense pathogens through its fibrinogen (FBG) recognition domains and to trigger activation of the lectin complement pathway through associated serine proteases. L-Ficolin has been previously shown to recognize pneumococcal clinical isolates, but its ligands and especially its molecular specificity remain to be identified. Using solid-phase binding assays, serum and recombinant L-ficolins were shown to interact with serotype 2 pneumococcal strain D39 and its unencapsulated R6 derivative. Incubation of both strains with serum triggered complement activation, as measured by C4b and C3b deposition, which was decreased by using ficolin-depleted serum. Recombinant L-ficolin and its FBG-like recognition domain bound to isolated pneumococcal cell wall extracts, whereas binding to cell walls depleted of teichoic acid (TA) was decreased. Both proteins were also shown to interact with two synthetic TA compounds, each comprising part structures of the complete lipoteichoic acid molecule with two PCho residues. Competition studies and direct interaction measurements by surface plasmon resonance identified PCho as a novel L-ficolin ligand. Structural analysis of complexes of the FBG domain of L-ficolin and PCho revealed that the phosphate moiety interacts with amino acids previously shown to define an acetyl binding site. Consequently, binding of L-ficolin to immobilized acetylated BSA was inhibited by PCho and synthetic TA. Binding of serum L-ficolin to immobilized synthetic TA and PCho-conjugated BSA triggered activation of the lectin complement pathway, thus further supporting the hypothesis of L-ficolin involvement in host antipneumococcal defense.

Self-Promoted Glycosylation of Carbamoylated Peptides on Solid Phase.

Self-promoted glycosylations with trichloroacetimidate glycosyl donors are demonstrated on solid-phase-anchored peptides orthogonally deprotected and tosylcarbamoylated on the side-chains of cysteine and serine, respectively. The donor scope included glucosyl as well as mannosyl trichloroacetimidates, carrying benzyl, acetyl, or isopropylidene protecting groups. Isopropylidene groups were found to be removed under the acidic conditions used for release of the neoglycopeptides from the solid support, yielding neoglycopeptides with unprotected hydroxyl groups. Glycosylation of a peptide containing a carbamoylated tyrosine was attempted as well, but the desired neoglycopeptide could not be synthesized due to thermal instability of the carbamate.

Ischaemia-reperfusion injury impairs tissue plasminogen activator release in man.

AIMS: Ischaemia-reperfusion (IR) injury causes endothelium-dependent vasomotor dysfunction that can be prevented by ischaemic preconditioning. The effects of IR injury and preconditioning on endothelium-dependent tissue plasminogen activator (t-PA) release, an important mediator of endogenous fibrinolysis, remain unknown. METHODS AND RESULTS: Ischaemia-reperfusion injury (limb occlusion at 200 mmHg for 20 min) was induced in 22 healthy subjects. In 12 subjects, IR injury was preceded by local or remote ischaemic preconditioning (three 5 min episodes of ipsilateral or contralateral limb occlusion, respectively) or sham in a randomized, cross-over trial. Forearm blood flow (FBF) and endothelial t-PA release were assessed using venous occlusion plethysmography and venous blood sampling during intra-arterial infusion of acetylcholine (5-20 µg/min) or substance P (2-8 pmol/min). Acetylcholine and substance P caused dose-dependent increases in FBF (P<0.05 for all). Substance P caused a dose-dependent increase in t-PA release (P<0.05 for all). Acetylcholine and substanceP-mediated vasodilatation and substanceP-mediated t-PA release were impaired following IR injury (P<0.05 for all). Neither local nor remote ischaemic preconditioning protected against the impairment of substance P-mediated vasodilatation or t-PA release. CONCLUSION: Ischaemia-reperfusion injury induced substanceP-mediated, endothelium-dependent vasomotor and fibrinolytic dysfunction in man that could not be prevented by ischaemic preconditioning. CLINICAL TRIAL REGISTRATION INFORMATION: Reference number: NCT00789243, URL: http://clinicaltrials.gov/ct2/show/NCT00789243?term=NCT00789243&rank=1.

Synthesis and thermotropic phase behavior of four glycoglycerolipids.

Four glycoglycerolipids with different head groups have been synthesized and their physicochemical properties studied. The lengths of the head groups from a mono-saccharide to a trisaccharide, in addition to the anomeric stereochemistry for the smaller glycoglycerolipids, have been modified. The synthesis has been optimized to avoid glycerol epimerization and to allow up-scaling. The physicochemical properties of the glycoglycerolipids were studied and a strong de-mixing of the gel-phase, depending on the head-group, was observed.

Quantifying the electronic effects of carbohydrate hydroxy groups by using aminosugar models.

Methyl amino-deoxy-glycosides with α- and ß-gluco, α-galacto, or α-manno stereochemistry with the amino functionality in each of the four possible non-anomeric positions have been synthesized and their pK(a) values determined by titration. These model compounds were chosen because they are the amino derivatives of the most common glycosyl acceptors. From this study it was possible to evaluate the electron density at each of the given positions in the carbohydrate and compare them. Some general trends were observed: The basicity of the amino groups decreases in the order 6-NH(2)>3-NH(2)>2-NH(2)>4-NH(2) (referring to the position). The basicity of a of an amino-deoxy-sugar generally increases when one or more substituents on the sugar ring are axial. The basicity decreases when the amine is antiperiplanar to an oxygen atom. These findings are in agreement with the observations obtained from glycosylation chemistry and the regioselective protection of sugars.

A study of anhydrocelluloses--is a cellulose structure with residues in a 1C4-conformation more prone to hydrolysis?

A study of the effect of introduction of 3,6-anhydroglucose residues in the cellulose structure on glycoside hydrolysis rate was performed. A cellotetrose with an 3,6-anhydroglucose as the third residue was synthesised. Acidic hydrolysis of this tetrasaccharide showed that hydrolysis of the 3,6-anhydro-ß-D-glucoside linkage was 31.400 times faster than hydrolysis of cellobiose. A series of different 3,6-anhydrocelluloses with different degree of substitution were prepared by tosylation of cellulose with varying amounts of tosyl chloride in dimethylacetamide and subsequent treatment with sodium hydroxide. Anhydrocelluloses with degrees of substitution of 0.02, 0.07, 0.31 and 0.74 were obtained. The anhydrocelluloses were subjected to acidic hydrolysis in 2.0 M aqueous HCl and the rate of hydrolysis monitored by ion chromatography analysis of the amount of glucose and/or cellobiose formed. All 3,6-anhydrocelluloses hydrolyzed with a faster rate than cellulose, but the anhydrocellulose with a low degree of substitution (ds = 0.07) hydrolyzed fastest which was 90 times faster than cellulose.

Chemical synthesis of bacterial lipoteichoic acids: an insight on its biological significance.

During infections caused by Gram-negative bacteria, lipopolysaccharide (LPS, endotoxin) has a dominant role leading to fulminant pro-inflammatory reactions in the host. As there is no LPS in Gram-positive bacteria, other microbial cell wall components have been identified to be the causative agent for the pro-inflammatory activity since also Gram-positive bacterial infections lead to comparable clinical symptoms and reactions. On search for the "Gram-positive endotoxin" a widely accepted hypothesis has been raised in that the lipoteichoic acids (LTAs) serve as pathogen-associated molecular patterns (PAMPs) during Gram-positive sepsis, although the amount necessary for a pro-inflammatory in vitro response is several orders of magnitude higher than that for LPS. Therefore, LTA cannot be considered to be "the (endo)toxin of Gram-positive bacteria". Although LPS and LTA show structural relatedness (amphiphilic, negatively charged glycophospholipids), they are structurally quite different from each other and one might expect that they are also recognized by different receptors of the innate immune system, the so called toll-like receptors 4 and 2 (TLR4 and TLR2), respectively. Based on their chemical structure, the LTAs were classified into four types (type I-IV) of which we have carefully investigated the LTA of Staphylococcus aureus (type I), Lactococcus garvieae (type II) and Streptococcus pneumoniae (type IV). Hence, these LTAs have been synthesized in our group and biologically evaluated with respect to their potency to activate cytokines in transiently TLR2/CD14-transfected human endothelial kidney cells (HEK 293) or human macrophages and whole blood cells. Although LTA of type I and IV are structurally quite different, especially in their hydrophilic moiety, they originally were believed to interact with the same receptor (TLR2). Hence, the chemical syntheses leading to structurally defined, non-contaminated stimuli have a major impact on the outcome and interpretation of these biological studies of the innate immune system. With this material, it became evident that synthetic LTA from S. aureus and S. pneumoniae are not recognized by TLR2. Instead, another receptor of the innate immune system, the lectin pathway of the complement, known since many years to interact with LTA in quite a specific way, has gained increasing attractivity. With the help of synthetic LTA we obtained first evidences that this receptor is indeed the pathogen recognition receptor (PRR) for LTA.

d-Glucose Isomerization with PAMAM Dendrimers as Environmentally Friendly Catalysts.

The isomerization of d-glucose to d-fructose plays a key role in the biochemical and chemical conversion of biomass, and it is therefore desirable to develop and improve catalysts for this reaction. In this study, the environmentally friendly polymer poly(amidoamine) (PAMAM) dendrimer's properties as catalysts for this isomerization are investigated. The experimental results showed that the PAMAM dendrimers, which have basic terminal groups, can effectively promote the d-glucose isomerization reaction. Under the optimized reaction conditions, d-fructose was generated with a 20% maximum yield and above 90% selectivity. 13C and 2H isotope experiments by NMR were carried out to explore the reaction mechanism. When the reaction was performed in D2O, the C1 signal of d-fructose changed to a triplet, which confirmed that the C1 carbon binds to a deuterium atom, i.e., isotopic exchange. It was also found that the deuterium atom at the C2 position of d-glucose-2-d1 cannot transfer to d-fructose. These data indicate that PAMAM dendrimers catalyze d-glucose isomerization through a mechanism, which includes deprotonation, formation of ene-diol intermediate, and proton exchange with the solvent.

Quantifying electronic effects of common carbohydrate protecting groups in a piperidine model system.

A study of the substituent effects of protecting groups in hydroxypiperidines was carried out and compared with the electronic effects in glycosylation chemistry. 1-Deoxynojirimycin, the aza-sugar analogue of 1-deoxy-D-glucose, was used as a carbohydrate model, and protected with the most common carbohydrate protecting groups. The different stabilization of positive charge on the ring heteroatom was determined by pK(a) measurements. The protecting groups could be ranked in the following way after their destabilization of the piperidinium ion: benzoyl ≥ acetyl ≫ 4,6-O-benzylidine >benzyl ≈ methyl > H > 3,6-anhydro > tert-butyldimethylsilyl. The observed effects of having protecting groups with different electronic characteristics were found to be in agreement with the "armed-disarmed" concept. Comparison of the pK(a) of benzylated and benzoylated epimers of 3-hydroxy-6-hydroxymethyl piperidines showed increased stabilization of the piperidinium ion in the axial epimer. The difference between axial and equatorial epimers was larger in the benzylated than in the benzoylated piperidines.

Catalytic and Atom-Economic Glycosylation using Glycosyl Formates and Cheap Metal Salts.

Benzylated glycosyl formates have been synthesized in one step from the corresponding hemiacetal or orthoester with formic acid as the sole reagent. The glycosyl formates are used as glycosyl donors under catalytic conditions with cheap metal catalysts based on iron or bismuth. A 13 C NMR spectroscopic method is developed and evaluated for screening reactions conditions, giving precise information on the selectivity, yield, and byproducts formed. The major side reaction is transesterification, which gives the formylated acceptor and regenerates the hemiacetal. By using this approach, catalyst loadings and solvents are optimized and the scope of the glycosylation is evaluated for a variety of glycosyl donors and acceptors. A proof of concept for a traceless glycosylation, utilizing a dual-purpose iron catalyst that catalyzes both glycosylation and dehydrogenation of formic acid, is also provided.

Development and Characterization of Mouse Monoclonal Antibodies Specific for Clostridiodes (Clostridium) difficile Lipoteichoic Acid.

Clostridiodes (Clostridium) difficile is an anaerobic Gram-positive, spore-forming nosocomial, gastrointestinal pathogen causing C. difficile-associated disease with symptoms ranging from mild cases of antibiotic-associated diarrhea to fatal pseudomembranous colitis. We developed murine monoclonal antibodies (mAbs) specific for a conserved cell surface antigen, lipoteichoic acid (LTA)of C. difficile. The mAbs were characterized in terms of their thermal stability, solubility, and their binding to LTA by surface plasmon resonance and competitive ELISA. Synthetic LTA molecules were prepared in order to better define the minimum epitope required to mimic the natural antigen, and three repeat units of the polymer were required for optimal recognition. One of the murine mAbs was chimerized with human constant region domains and was found to recognize the target antigen identically to the mouse version. These mAbs may be useful as therapeutics (standalone, in conjunction with known antitoxin approaches, or as delivery vehicles for antibody drug conjugates targeting the bacterium), as diagnostic agents, and in infection control applications.

An extended study of dimeric phenyl tropanes.

A series of dimeric phenyl tropanes consisting of two molecules of 4-chloro, 4-iodo or 4-(3-thiopheno)-phenyl tropane tethered together at the carboxylic acid moiety by a diamine or diol linker were prepared. The diamines used were a variety of linear, cyclic and aromatic diamines, while the diol tethered compounds were prepared by 'click' chemistry and contained a triazole in the linker. The new compounds were tested for binding to hDAT, hSERT and hNET. Amide linked chlorophenyl tropanes with an aromatic linker was found to be potent and selective DAT inhibitors with the best K(i) value for hDAT being 6nM. The ester linked halophenyl tropanes were more potent but displayed little selectivity in inhibition of monoamine transporter binding. Among the studied compounds an ester linker of 10 atoms between the tropane moieties gave the highest affinity. One monomeric phenyl tropane was made for comparison and was found to be less potent than the dimeric counterparts towards SERT and NET but remain highly active against DAT. Dimeric thiophenophenyl tropanes were in general found to be comparatively poor monoamine transporter binders, but significant gains of affinity of up to 45-fold could be achieved with selected dimeric chlorophenyl tropanes compared to the parent monomer. This observation implies that a secondary binding site that has affinity for phenyl tropanes, most likely the putative S2 site, is located within 13A of the primary central S1 binding site.

Preserved flow-mediated dilation in adults with cyanotic congenital heart disease.

Adults with cyanotic congenital heart disease (CCHD) have been shown to have endothelial dysfunction in the forearm resistance vessels as assessed with venous occlusion plethysmography. Whether these abnormalities are confined to the microvasculature or reflect generalized endothelial dysfunction remain unknown. We used high-resolution ultrasound to compare flow responses and endothelial-dependent flow-mediated dilation (FMD) in the brachial artery of 13 adult patients with CCHD and 14 healthy controls. High-dose vitamin C was infused to evaluate the possible role of reactive oxygen species on endothelial vasomotor function. FMD was measured both prior to and after vitamin C infusion. Sublingual glyceryl nitrate was given to assess endothelium-independent responses. FMD did not differ among patients with CCHD and controls either before (6.2 +/- 4.1, 5.1 +/- 2.6%, p = 0.44) or after (5.1 +/- 2.8, 5.2 +/- 3.1%, p = 0.90) vitamin C infusion. Endothelium-independent vasodilatation was similar in both groups (14.3 +/- 3.7, 13.2 +/- 4.4%). There were no differences in baseline flow or in measures of reactive hyperemia. Adults with CCHD appear to have preserved endothelial function in their conduit arteries. This suggests that these patients are not at an increased risk of premature atherosclerotic cardiovascular events.

Testing an App-Assisted Treatment for Suicide Prevention in a Randomized Controlled Trial: Effects on Suicide Risk and Depression.

Suicide is a global public health problem and effective psychological interventions are needed. The objective of the present study was to evaluate the effect of an app-assisted suicide prevention treatment on suicide risk and depression. One hundred twenty-nine participants were randomized to treatment as usual (TAU), consisting of psychotherapy adhering to the framework of Collaborative Assessment and Management of Suicidality (CAMS), with (TAU+APP, N = 60) or without (TAU, N = 69) access to a mobile application (i.e., LifeApp'tite). Suicide risk and symptoms of depression were assessed pre- and posttherapy, and at 4-month follow-up. The TAU+APP group showed a smaller decrease on self-reported suicide risk at the end of treatment, corresponding to a medium between-group effect size (p = .008, d = 0.46). At the 4-month follow-up this was the case only at the trend level, where the effect size was also of a smaller magnitude (p = .057, d = 0.30). No differences between the treatment groups were observed on self-reported depressive symptoms, either immediately following treatment (p = .732, d = 0.05) or at follow-up (p = .467, d = 0.11). The unexpected negative effect concerning suicide risk points to crucial consideration of issues pertaining to timing, dosing, and content when adding new technology to existing treatments both in this and other populations.

Glycosylation intermediates studied using low temperature 1H- and 19F-DOSY NMR: new insight into the activation of trichloroacetimidates.

Low temperature 1H- and 19F-DOSY have been used for analyzing reactive intermediates in glycosylation reactions, where a glycosyl trichloroacetimidate donor has been activated using different catalysts. The DOSY protocols have been optimized for low temperature experiments and provided new insight into acid catalyzed glycosylation chemistry. From the study a new glycosylation intermediate was characterized.

ß-Selective mannosylation with a 4,6-silylene-tethered thiomannosyl donor.

Mannosylations using the new conformationally restricted donor phenyl 2,3-di-O-benzyl-4,6-O-(di-tert-butylsilylene)-1-thio-α-D-mannopyranoside (6) have been found to be ß-selective with a variety of activation conditions. The simplest activation conditions were NIS/TfOH, in which case it is proposed that the ß-mannoside is formed from ß-selective glycosylation of the oxocarbenium ion 25 in a B(2,5) conformation.

Remote ischemic preconditioning with--but not without--metabolic support protects the neonatal porcine heart against ischemia-reperfusion injury.

BACKGROUND: While remote ischemic preconditioning (rIPC) protects the mature heart against ischemia-reperfusion (IR) injury, the effect on the neonatal heart is not known. The neonatal heart relies almost solely on carbohydrate metabolism, which is modified by rIPC in the mature heart. We hypothesized that rIPC combined with metabolic support with glucose-insulin (GI) infusion improves cardiac function and reduces infarct size after IR injury in neonatal piglets in-vivo. METHODS AND RESULTS: 32 newborn piglets were randomized into 4 groups: control, GI, GI+rIPC and rIPC. GI and GI+rIPC groups received GI infusion continuously from 40 min prior to ischemia. rIPC and GI+rIPC groups underwent four cycles of 5 min limb ischemia. Myocardial IR injury was induced by 40 min occlusion of the left anterior descending artery followed by 2 h reperfusion. Myocardial lactate concentrations were assessed in microdialysis samples analyzed by mass spectrometry. Infarct size was measured using triphenyltetrazolium chloride staining. Systolic recovery (dP/dt(max) as % of baseline) after 2 h reperfusion was 68.5±13.8% in control, 53.7±11.2% in rIPC (p<0.05), and improved in GI (83.6±18.8%, p<0.05) and GI+rIPC (87.0±15.7%, p<0.01). CONCLUSION: rIPC+GI protects the neonatal porcine heart against IR injury in-vivo. rIPC alone has detrimental metabolic and functional effects that are abrogated by simultaneous GI infusion.