Picomolar inhibition of cholera toxin by a pentavalent ganglioside GM1os-calix[5]arene.

Cholera toxin (CT), the causative agent of cholera, displays a pentavalent binding domain that targets the oligosaccharide of ganglioside GM1 (GM1os) on the periphery of human abdominal epithelial cells. Here, we report the first GM1os-based CT inhibitor that matches the valency of the CT binding domain (CTB). This pentavalent inhibitor contains five GM1os moieties linked to a calix[5]arene scaffold. When evaluated by an inhibition assay, it achieved a picomolar inhibition potency (IC50 = 450 pM) for CTB. This represents a significant multivalency effect, with a relative inhibitory potency of 100,000 compared to a monovalent GM1os derivative, making GM1os-calix[5]arene one of the most potent known CTB inhibitors.

Copper-free click biofunctionalization of silicon nitride surfaces via strain-promoted alkyne-azide cycloaddition reactions.

Cu-free "click" chemistry is explored on silicon nitride (Si(3)N(4)) surfaces as an effective way for oriented immobilization of biomolecules. An ω-unsaturated ester was grafted onto Si(3)N(4) using UV irradiation. Hydrolysis followed by carbodiimide-mediated activation yielded surface-bound active succinimidyl and pentafluorophenyl ester groups. These reactive surfaces were employed for the attachment of bicyclononyne with an amine spacer, which subsequently enabled room temperature strain-promoted azide-alkyne cycloaddition (SPAAC). This stepwise approach was characterized by means of static water contact angle, X-ray photoelectron spectroscopy, and fluorescence microscopy. The surface-bound SPAAC reaction was studied with both a fluorine-tagged azide and an azide-linked lactose, yielding hydrophobic and bioactive surfaces for which the presence of trace amounts of Cu ions would have been problematic. Additionally, patterning of the Si(3)N(4) surface using this metal-free click reaction with a fluorescent azide is shown. These results demonstrate the ability of the SPAAC as a generic tool for anchoring complex molecules onto a surface under extremely mild, namely ambient and metal-free, conditions in a clean and relatively fast manner.

Organic modification and subsequent biofunctionalization of porous anodic alumina using terminal alkynes.

Porous anodic alumina (PAA) is a well-defined material that has found many applications. The range of applications toward sensing and recognition can be greatly expanded if the alumina surface is covalently modified with an organic monolayer. Here, we present a new method for the organic modification of PAA based on the reaction of terminal alkynes with the alumina surface. The reaction results in the the formation of a monolayer within several hours at 80 °C and is dependent on both oxygen and light. Characterization with X-ray photoelectron spectroscopy and infrared spectroscopy indicates formation of a well-defined monolayer in which the adsorbed species is an oxidation product of the 1-alkyne, namely, its α-hydroxy carboxylate. The obtained monolayers are fairly stable in water and at elevated temperatures, as was shown by monitoring the water contact angle. Modification with 1,15-hexadecadiyne resulted in a surface that has alkyne end groups available for further reaction, as was demonstrated by the subsequent reaction of N-(11-azido-3,6,9-trioxaundecyl)trifluoroacetamide with the modified surface. Biofunctionalization was explored by coupling 11-azidoundecyl lactoside to the surface and studying the subsequent adsorption of the lectin peanut agglutinin (PNA) and the yeast Candida albicans, respectively. Selective and reversible binding of PNA to the lactosylated surfaces was demonstrated. Moreover, PNA adsorption was higher on surfaces that exposed the ß-lactoside than on those that displayed the α anomer, which was attributed to surface-associated steric hindrance. Likewise, the lactosylated surfaces showed increased colonization of C. albicans compared to unmodified surfaces, presumably due to interactions involving the cell wall ß-glucan. Thus, this study provides a new modification method for PAA surfaces and shows that it can be used to induce selective adsorption of proteins and microorganisms.

The influence of ligand valency on aggregation mechanisms for inhibiting bacterial toxins.

Divalent and tetravalent analogues of ganglioside GM1 are potent inhibitors of cholera toxin and Escherichia coli heat-labile toxin. However, they show little increase in inherent affinity when compared to the corresponding monovalent carbohydrate ligand. Analytical ultracentrifugation and dynamic light scattering have been used to demonstrate that the multivalent inhibitors induce protein aggregation and the formation of space-filling networks. This aggregation process appears to arise when using ligands that do not match the valency of the protein receptor. While it is generally accepted that multivalency is an effective strategy for increasing the activity of inhibitors, here we show that the valency of the inhibitor also has a dramatic effect on the kinetics of aggregation and the stability of intermediate protein complexes. Structural studies employing atomic force microscopy have revealed that a divalent inhibitor induces head-to-head dimerization of the protein toxin en route to higher aggregates.

Glycosyltransferase-catalyzed synthesis of bioactive oligosaccharides.

Mammalian cell surfaces are all covered with bioactive oligosaccharides which play an important role in molecular recognition events such as immune recognition, cell-cell communication and initiation of microbial pathogenesis. Consequently, bioactive oligosaccharides have been recognized as a medicinally relevant class of biomolecules for which the interest is growing. For the preparation of complex and highly pure oligosaccharides, methods based on the application of glycosyltransferases are currently recognized as being the most effective. The present paper reviews the potential of glycosyltransferases as synthetic tools in oligosaccharide synthesis. Reaction mechanisms and selected characteristics of these enzymes are described in relation to the stereochemistry of the transfer reaction and the requirements of sugar nucleotide donors. For the application of glycosyltransferases, accepted substrate profiles are summarized and the whole-cell approach versus isolated enzyme methodology is compared. Sialyltransferase-catalyzed syntheses of gangliosides and other sialylated oligosaccharides are described in more detail in view of the prominent role of these compounds in biological recognition.

GM3, GM2 and GM1 mimics designed for biosensing: chemoenzymatic synthesis, target affinities and 900 MHz NMR analysis.

Undec-10-enyl, undec-10-ynyl and 11-azidoundecyl glycoside analogues corresponding to the oligosaccharides of human gangliosides GM3, GM2 and GM1 were synthesized in high yields using glycosyltransferases from Campylobacter jejuni. Due to poor water solubility of the substrates, the reactions were carried out in methanol-water media, which for the first time were shown to be compatible with the C. jejuni alpha-(2-->3)-sialyltransferase (CST-06) and beta-(1-->4)-N-acetylgalactosaminyltransferase (CJL-30). Bioequivalence of our synthetic analogues and natural gangliosides was examined by binding to Vibrio cholerae toxin and to the B subunit of Escherichia coli heat-labile enterotoxin. This bioequivalence was confirmed by binding mouse and human monoclonal antibodies to GM1 and acute phase sera containing IgM and IgG antibodies to GM1 from patients with the immune-mediated polyneuropathy Guillain-Barré syndrome. The synthesized compounds were analyzed by 1D and 2D 900 MHz NMR spectroscopy. TOCSY and DQF-COSY experiments in combination with 13C-1H correlation measurements (HSQC, HMBC) were carried out for primary structural characterization, and a complete assignment of all 1H and 13C chemical shifts is presented.

Selective depletion of neuropathy-related antibodies from human serum by monolithic affinity columns containing ganglioside mimics.

Monolithic columns containing ganglioside GM2 and GM3 mimics were prepared for selective removal of serum anti-ganglioside antibodies from patients with acute and chronic immune-mediated neuropathies. ELISA results demonstrated that anti-GM2 IgM antibodies in human sera and a mouse monoclonal anti-GM2 antibody were specifically and selectively adsorbed by monolithic GM2 mimic columns and not by blank monolithic columns or monolithic GM3 mimic columns. In control studies, serum antibodies against the ganglioside GQ1b from another neuropathy patient were not depleted by monolithic GM2 mimic columns. Fluorescence microscopy with FITC-conjugated anti-human immunoglobulin antibodies showed that the immobilized ganglioside mimics were evenly distributed along the column. The columns were able to capture ∼95% of the anti-GM2 antibodies of patients after only 2 min of incubation. A monolithic column of 4.4 µL can deplete 28.2 µL of undiluted serum. These columns are potential diagnostic and therapeutic tools for neuropathies related to anti-ganglioside antibodies.

Stereochemical preference of yeast epoxide hydrolase for the O-axial C3 epimers of 1-oxaspiro[2.5]octanes.

The 1-oxaspiro[2.5]octane moiety is a common motif in many biologically active spiroepoxide compounds. Stereochemistry plays an important role in the action of these spiroepoxides, since the O-axial C3 epimers are predominantly responsible for biological activity. In view of this, the reactivity of the yeast epoxide hydrolase (YEH) from Rhodotorula glutinis towards both O-axial and O-equatorial C3 epimers of various 1-oxaspiro[2.5]octanes was investigated. O-axial C3 Epimers were hydrolyzed faster than the O-equatorial C3 epimers. The stereochemical preference was greatly dependent on the type of substitution on the cyclohexane ring. The preference of YEH for O-axial C3 epimers, found throughout this study, illustrates the effectiveness of YEH in enzymatic detoxification of spiroepoxides.

Stereoselectivity and substrate specificity in the kinetic resolution of methyl-substituted 1-oxaspiro[2.5]octanes by Rhodotorula glutinis epoxide hydrolase.

The kinetic resolution of a range of methyl-substituted 1-oxaspiro[2.5]octanes by yeast epoxide hydrolase (YEH) from Rhodotorula glutinis has been investigated. The structural determinants of substrate specificity and stereoselectivity of YEH toward these substrates appeared to be the configuration of the epoxide ring and the substitution pattern of the cyclohexane ring. For all compounds tested, O-axial epoxides were hydrolyzed faster than the corresponding O-equatorial compounds. In concern of the ring substituents, YEH preferred methyl groups on the Re side of the ring. Placement of substituents close to the spiroepoxide carbon decreased the reaction rate but increased enantioselectivity. YEH-catalyzed kinetic resolutions of 4-methyl 1-oxaspiro[2.5]octane epimers were most enantioselective (E > 100).

Total synthesis and bioactivity of some naturally occurring pterulones.

The naturally occurring pterulones 1, 3, and 13 were synthesized in high overall yield from readily available methyl 3-(2-propenyl)-4-(2-propenyloxy)benzoate (6) by employing ring-closing metathesis (RCM) as a key step. The biological activities of the synthesized pterulones were tested using cells of Rhodotorulaglutinis and Saccharomyces cerevisiae.