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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Superarming of glycosyl donors by combined neighboring and conformational effects.

A novel glycosyl donor that combines the concepts of both conformational and electronic superarming has been synthesized. The reactivity and selectivity of the donor have been tested in competition experiments.

Assessing and treating different suicidal states in a danish outpatient sample.

The studies presented compare two methodologies for categorizing suicidal patients based on clinical data. Discussion follows regarding implications for risk assessment and treatment. In these studies, 52 outpatient subjects were placed into different groups based on coding their "suicidal motivation" (Study 1) and their "internal struggle" ratings (Study 2) using data collected at intake. Self-report ratings of 6 Suicide Status Form (SSF) Core Constructs (Psychological Pain, Stress, Agitation, Hopelessness, Self-Hate, and Overall Risk of Suicide) recorded both at intake and at completion of treatment were then compared to determine differences in Core Construct ratings among groups at different time points. In Study 1, overall differences among motivation groups (Life-motivated, Ambivalent, and Death-motivated) were significant for ratings at treatment completion of Overall Risk of Suicide, Self-Hate, and Psychological Pain. In Study 2, overall differences among groups (Wish to live, Ambivalent, and Wish to die) were significant for ratings at intake of Overall Risk of Suicide. At completion of treatment, overall differences among groups were significant for ratings of Overall Risk of Suicide, Hopelessness, and Self-Hate. In addition, significant interactions were found between test time and group for Overall Risk of Suicide and Self-Hate. Results suggest that categorizing suicidal patients by motivation and by the nature of their internal struggle could be beneficial to differential risk assessment with implications for clinical treatment.

Sustained cardiovascular actions of APJ agonism during renin-angiotensin system activation and in patients with heart failure.

BACKGROUND: To assess cardiovascular actions of APJ agonism during prolonged (Pyr(1))apelin-13 infusion and renin-angiotensin system activation. METHODS AND RESULTS: Forty-eight volunteers and 12 patients with chronic stable heart failure attended a series of randomized placebo-controlled studies. Forearm blood flow, cardiac index, left ventricular dimensions, and mean arterial pressure were measured using bilateral venous occlusion plethysmography, bioimpedance cardiography, transthoracic echocardiography, and sphygmomanometry, respectively, during brief local (0.3-3.0 nmol/min) and systemic (30-300 nmol/min) or prolonged systemic (30 nmol/min) (Pyr(1))apelin-13 infusions in the presence or absence of renin-angiotensin system activation with sodium depletion or angiotensin II coinfusion. During sodium depletion and angiotensin II coinfusion, (Pyr(1))apelin-13-induced vasodilatation was preserved (P<0.02 for both). Systemic intravenous (Pyr(1))apelin-13 infusion increased cardiac index, whereas reducing mean arterial pressure and peripheral vascular resistance index (P<0.001 for all) irrespective of sodium depletion or angiotensin II (0.5 ng/kg per minute) coinfusion (P>0.05 for all). Prolonged 6-hour (Pyr(1))apelin-13 infusion caused a sustained increase in cardiac index with increased left ventricular ejection fraction in patients with chronic heart failure (ANOVA; P<0.001 for all). CONCLUSIONS: APJ agonism has sustained cardiovascular effects that are preserved in the presence of renin-angiotensin system activation or heart failure. APJ agonism may hold major promise to complement current optimal medical therapy in patients with chronic heart failure. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifiers: NCT00901719, NCT00901888, NCT01049646, NCT01179061.

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.

The influence of neighboring group participation on the hydrolysis of 2-O-substituted methyl glucopyranosides.

Does neighboring group participation actually enhance the reactivity of the anomeric center when the participating group is inherently disarming? To investigate the influence of the neighboring group effect from a 2-O protective group on acidic glycoside hydrolysis, 10 methyl glucosides having different protective groups on O2 have been synthesized and a clear trend between anomeric configuration, participation of the protective group, and the rate of hydrolysis could be observed.

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.

Bradykinin does not mediate remote ischaemic preconditioning or ischaemia-reperfusion injury in vivo in man.

OBJECTIVE: To examine whether endogenous bradykinin mediates the endothelium-dependent vasomotor dysfunction induced by ischaemia-reperfusion injury, or the protection afforded by remote ischaemic preconditioning in vivo in man. DESIGN: Randomised double-blind, cross-over study. SETTINGS: Royal Infirmary of Edinburgh, Wellcome Trust Clinical Research Facility. PATIENTS: Twenty healthy male volunteers. INTERVENTIONS: Subjects were randomised to intravenous infusion of the bradykinin B(2) receptor antagonist, HOE-140 (100 µg/kg), or saline placebo in a double-blind, crossover trial. Ischaemia-reperfusion injury was induced in the non-dominant arm by inflating a cuff to 200 mm Hg for 20 min in all subjects. Ischaemia-reperfusion injury was preceded by three cycles of remote ischaemic preconditioning in the dominant arm in 10 subjects. MAIN OUTCOME MEASURES: Bilateral forearm blood flow was assessed using venous occlusion plethysmography during intra-arterial infusion of acetylcholine (5-20 µg/min). RESULTS: Acetylcholine caused vasodilatation in all studies (p<0.05) that was attenuated by ischaemia-reperfusion injury, both in the presence (p=0.0002) and absence (p=0.04) of HOE-140. Remote ischaemic preconditioning abolished the impairment of endothelium-dependent vasomotor function induced by ischaemia-reperfusion injury. HOE-140 had no effect on the protection afforded by remote ischaemic preconditioning. CONCLUSIONS: These findings do not support a major role for endogenous bradykinin, acting via the B(2) kinin receptor, in the mechanism of ischaemia-reperfusion injury or the protective effects of remote ischaemic preconditioning in man. CLINICAL TRIAL REGISTRATION INFORMATION: NCT00965120 and NCT00965393.