The development of selectively protected monosaccharide building blocks that can reliably be glycosylated with a wide variety of acceptors is expected to make oligosaccharide synthesis a more routine operation. In particular, there is an urgent need for the development of modular building blocks that can readily be converted into glycosyl donors for glycosylations that give reliably high 1,2-cis-anomeric selectivity. We report here that 1,2-oxathiane ethers are stable under acidic, basic, and reductive conditions making it possible to conduct a wide range of protecting group manipulations and install selectively removable protecting groups such as levulinoyl (Lev) ester, fluorenylmethyloxy- (Fmoc) and allyloxy- (Alloc) carbonates, and 2-methyl naphthyl ethers (Nap). The 1,2-oxathiane ethers could easily be converted into bicyclic anomeric sulfonium ions by oxidization to sulfoxides and arylated with 1,3,5-trimethoxybenzene. The resulting sulfonium ions gave high 1,2-cis anomeric selectivity when glycosylated with a wide variety of glycosyl acceptors including properly protected amino acids, primary and secondary sugar alcohols and partially protected thioglycosides. The selective protected 1,2-oxathianes were successfully employed in the preparation of a branched glucoside derived from a glycogen-like polysaccharide isolated form the fungus Pseudallescheria boydii, which is involved in fungal phagocytosis and activation of innate immune responses. The compound was assembled by a latent-active glycosylation strategy in which an oxathiane was employed as an acceptor in a glycosylation with a sulfoxide donor. The product of such a glycosylation was oxidized to a sulfoxide for a subsequent glycosylation. The use of Nap and Fmoc as temporary protecting groups made it possible to install branching points.

Methods to covalently conjugate Alexa Fluor dyes to cellulose nanocrystals, at limiting amounts that retain the overall structure of the nanocrystals as model cellulose materials, were developed using two approaches. In the first, aldehyde groups are created on the cellulose surfaces by reaction with limiting amounts of sodium periodate, a reaction well-known for oxidizing vicinal diols to create dialdehyde structures. Reductive amination reactions were then applied to bind Alexa Fluor dyes with terminal amino-groups on the linker section. In the absence of the reductive step, dye washes out of the nanocrystal suspension, whereas with the reductive step, a colored product is obtained with the characteristic spectral bands of the conjugated dye. In the second approach, Alexa Fluor dyes were modified to contain chloro-substituted triazine ring at the end of the linker section. These modified dyes then were reacted with cellulose nanocrystals in acetonitrile at elevated temperature, again isolating material with the characteristic spectral bands of the Alexa Fluor dye. Reactions with Alexa Fluor 546 are given as detailed examples, labeling on the order of 1% of the total glucopyranose rings of the cellulose nanocrystals at dye loadings of ca. 5 μg/mg cellulose. Fluorescent cellulose nanocrystals were deposited in pore network microfluidic structures (PDMS) and proof-of-principle bioimaging experiments showed that the spatial localization of the solid cellulose deposits could be determined, and their disappearance under the action of Celluclast enzymes or microbes could be observed over time. In addition, single molecule fluorescence microscopy was demonstrated as a method to follow the disappearance of solid cellulose deposits over time, following the decrease in the number of single blinking dye molecules with time instead of fluorescent intensity.

Aberrant glycosylation of α-dystroglycan (α-DG) results in loss of interactions with the extracellular matrix and is central to the pathogenesis of several disorders. To examine protein glycosylation of α-DG, a facile synthetic approach has been developed for the preparation of unusual phosphorylated O-mannosyl glycopeptides derived from α-DG by a strategy in which properly protected phospho-mannosides are coupled with a Fmoc protected threonine derivative, followed by the use of the resulting derivatives in automated solid phase glycopeptide synthesis using hyper-acid sensitive Sieber amide resin. Synthetic efforts also provided a reduced phospho-trisaccharide and the NMR data of this derivative confirmed the proper structural assignment of the unusual phospho-glycan structure. The glycopeptides made it possible to explore factors that regulate the elaboration of critical glycans. It was established that a glycopeptide having a 6-phospho-O-mannosyl residue is not an acceptor for action by the enzyme POMGnT1, which attaches β(1,2)-GlcNAc to O-mannosyl moietes, whereas the unphosphorylated derivate was readily extended by the enzyme. This finding implies a specific sequence of events in determining the structural fate of the O-glycan. It has also been found that the activity of POMGnT1 is dependent on the location of the acceptor site in the context of the underlying polypeptide/glycopeptide sequence. Conformational analysis by NMR has shown that the O-mannosyl modification does not exert major conformational effect on the peptide backbone. It is, however, proposed that these residues, introduced at the early stages of glycoprotein glycosylation, have an ability to regulate the loci of subsequent O-GalNAc additions, which do exert conformational effects. The studies show that through access to discrete glycopeptide structures, it is possible to reveal complex regulation of O-glycan processing on α-DG that has significant implications both for its normal post-translational maturation, and the mechanisms of the pathologies associated with hypoglycosylated α-DG.

No abstract

Bacteriophages express endolysins which are the enzymes that hydrolyze peptidoglycan resulting in cell lysis and release of bacteriophages. Endolysins have acquired stringent substrate specificities, which have been attributed to cell wall binding domains (CBD). Although it has been realized that CBDs of bacteriophages that infect Gram-positive bacteria target cell wall carbohydrate structures, molecular mechanisms that confer selectivity are not understood. A range of oligosaccharides, derived from the secondary cell wall polysaccharides of Bacillus anthracis, has been chemically synthesized. The compounds contain an α-D-GlcNAc-(1→4)-β-D-ManNAc-(1→4)-β-D-GlcNAc backbone that is modified by various patterns of α-D-Gal and β-D-Gal branching points. The library of compounds could readily be prepared by employing a core trisaccharide modified by the orthogonal protecting groups Nα-9-fluorenylmethyloxycarbonate (Fmoc), 2-methylnaphthyl ether (Nap) and levulinoyl ester (Lev) and dimethylthexylsilyl ether (TDS) at key branching points. Dissociation constants for the binding the cell wall binding domains of the endolysins PlyL and PlyG were determined by surface plasmon resonance (SPR). It was found that the pattern of galactosylation greatly influenced binding affinities, and in particular a compound having a galactosyl moiety at C-4 of the non-reducing GlcNAc moiety bound in the low micromolar range. It is known that secondary cell wall polysaccharides of various bacilli may have both common and variable structural features and in particular differences in the pattern of galactosylation have been noted. Therefore, it is proposed that specificity of endolysins for specific bacilli is achieved by selective binding to a uniquely galactosylated core structure.

Pseudomonas aeruginosa is an opportunistic Gram‐negative bacterium that can cause life‐threatening infections in critically ill and cystic fibrosis patients. The Psl exopolysaccharide of P. aeruginosa offers an attractive serotype‐independent antigen for the development of immunotherapies. Here, the first chemical synthesis of a panel of oligosaccharides derived from the exopolysaccharide of P. aeruginosa by a synthetic strategy that efficiently deals with the stereoselective installation of several β‐mannosides and the formation of a mannoside that is extended by saccharide moieties at C‐1, C‐2, and C‐3 in a crowded 1,2,3‐cis configuration is described. The approach was employed to prepare tetra‐, penta‐, and hexa‐ and decasaccharide part structures. The compounds were employed to define the epitope requirements of several functionally active monoclonal antibodies (mAbs) that can bind three distinct epitopes of Psl (class I, II, and III). The class II mAb reacted potently with each oligosaccharide indicating its epitope resides within the tetrasaccharide and does not require the branched mannoside of Psl. The class III antibody did not bind the tetra‐ or pentasaccharide; however, it did react potently with the hexasaccharide and weakly with the decasaccharide, suggesting a terminal glucoside is required for optimal binding. Unexpectedly, the class I mAb did not bind any of the oligosaccharides indicating that Psl contains a yet to be elucidated sub‐stoichiometric isoform. This study demonstrates that functional activity of a mAb does not only depend on the avidity of binding but also on the location of an epitope within a bacterial polysaccharide. The results also provide a strong impetus to analyze further the structure of Psl to identify the class I epitope, that is expected to provide an attractive target for the development of a synthetic vaccine for P. aeruginosa.

Siderophores are iron (Fe)-binding secondary metabolites that have been investigated for their uranium-binding properties. Previous work has focused on characterizing hydroxamate types of siderophores, such as desferrioxamine B, for their uranyl (UO2)-binding affinity. Carboxylate forms of these metabolites hold potential to be more efficient chelators of UO2, yet they have not been widely studied. Desmalonichrome is a carboxylate siderophore that is not commercially available and so was obtained from the fungus Fusarium oxysporum cultivated under Fe-depleted conditions. The relative affinity for UO2 binding of desmalonichrome was investigated using a competitive analysis of binding affinities between UO2 acetate and different concentrations of Fe(III) chloride using electrospray ionization mass spectrometry. In addition to desmalonichrome, three other siderophores, including two hydroxamates (desferrioxamine B and desferrichrome) and one carboxylate (desferrichrome A), were studied to understand their relative affinities for the UO2(2+) ion at two pH values. The binding affinities of hydroxamate siderophores to UO2(2+) ions were observed to decrease with increasing Fe(III)Cl3 concentration at the lower pH. On the other hand, decreasing the pH has a smaller impact on the binding affinities between carboxylate siderophores and the UO2(2+) ion. Desmalonichrome in particular was shown to have the greatest relative affinity for UO2 at all pH and Fe(III) concentrations examined. These results suggest that acidic functional groups in the ligands are important for strong chelation with UO2 at lower pH.

Sequence control in polymers,well-known in nature, encodes structure and functionality.H ere we introduce an ew architecture,b ased on the nucleophilic aromatic substitution chemistry of cyanuric chloride,t hat creates an ew class of sequence-defined polymers dubbed TZPs.Proof of concept is demonstrated with two synthesized hexamers,h aving neutral and ionizable side chains.M olecular dynamics simulations show backbone-backbone interactions,i ncluding H-bonding motifs and pi-pi interactions.T his architecture is arguably biomimetic while differing from sequence-defined polymers having peptide bonds.The synthetic methodology supports the structural diversity of side chains knowninpeptides,aswell as backbone-backbone hydrogen-bonding motifs,a nd will thus enable new macromolecules and materials with useful functions.Sequence control in synthetic polymers has gained renewed interest, [1] because sequence leads to structure and function. Sequence-controlled polymers have repeated sequence motifs (e.g.,( ABC)n)w hereas sequence-defined polymers have monomers in any predetermined order (e.g.ABCADC). Thel atter polymers are epitomized by natural biopolymers such as polypeptides and poly(nucleic acids), where pendant side chains distinguish one monomer from another. In polypeptides,t he sequencing leads to diverse structures and functions that are vital to life,i ncluding material architectures,b iocatalysis,m olecular recognition, and transport across membranes.S equence in polymers was discussed in acritical review by Lutz et al. in 2013, including discussions of relevance to materials science.[1b] Some sequence-defined oligomers and polymers fold into conformational structures such as helices,and hence are called "foldamers", which have potential applications in materials science,c atalysis,a nd molecular recognition. [2] Construction of synthetic poly(peptides) using biomolecular machinery,solution synthesis,orsolid-phase synthesis,is well-established.[1b] Synthetic sequence-defined polymers typically have monomers selected from natural and unnatural alpha-amino acids,non-alpha-amino acids,and pseudo-amino acids.Peptoids have amide bonds in the backbone where the side chains are attached to an itrogen atom instead of ac arbon atom, prepared by the solid-phase submonomer synthesis approach without using amino acid monomers or protecting groups.[3] Until recently,m ost synthetic sequencedefined polymers have as imilarity to polypeptides by virtue of having amide bonds.With resurgent interest in sequence-controlled and sequence-defined polymers,a dditional bond-forming reactions are being implemented to create new polymer architectures [1g-r] Lutz et al. described alternating cycloaddition and amidation reactions in 2009 to create (AB) n sequencecontrolled polymer segments.[1h] This group employed the same or similar strategies 2014 and 2105 to design sequencedefined oligomers that encode digital information.[1i-l] The sequencing could be decoded with tandem mass spectrometry. [1k, l] In 2013, Madder et al. ...