A practical synthesis of kifunensine analogues as inhibitors of endoplasmic reticulum alpha-mannosidase I

[reaction: see text] A practical synthesis of the potent class I alpha-mannosidase inhibitor kifunensine (1) beginning from the inexpensive and readily available starting material L-ascorbic acid (15) is described. The protected amino-alcohol ((2R,3R,4R,5R)-5-amino-2,3:4,6-diisopropylidenedioxyhexanol, 11) served as a key intermediate from which several N-1 substituted kifunensine analogues (including N-methyl, N-cyclohexyl, and N-bis(hydroxymethyl)methyl) and 2-desoxakifunensine analogues (including N-H and N-methyl) were prepared and screened for inhibition of human endoplasmic reticulum alpha-mannosidase I (ER Man I) and mouse Golgi alpha-mannosidase IA (Golgi Man IA). In addition, several pseudodisaccharide kifunensine analogues in which a mannose residue was tethered to N-1 of kifunensine via a two-, three-, or four-carbon linker and an affinity-bound kifunensine analogue were also prepared and evaluated for biological activity. While the synthesized N-1 kifunesine analogues were found to be less potent inhibitors of Class I alpha-mannosidases than kifuensine itself, the bis(hydroxymethyl)methylkifunensine analogue 6 was shown to selectively inhibit ER Man I over Golgi Man IA.     

A PEGylated Photocleavable Auxiliary Mediates the Sequential Enzymatic Glycosylation and Native Chemical Ligation of Peptides

Research aimed at understanding the specific role of glycosylation patterns in protein function would greatly benefit from additional approaches allowing direct access to homogeneous glycoproteins. Herein the development and application of an efficient approach for the synthesis of complex homogenously glycosylated peptides based on a multifunctional photocleavable auxiliary is described. The presence of a PEG polymer within the auxiliary enables sequential enzymatic glycosylation and straightforward isolation in excellent yields. The auxiliary‐modified peptides can be directly used in native chemical ligations with peptide thioesters easily obtained by direct hydrazinolysis of the respective glycosylated peptidyl resins and subsequent oxidation. The ligated glycopeptides can be smoothly deprotected by UV irradiation. We apply this approach to the preparation of variants of the epithelial tumor marker MUC1 carrying one or more Tn, T, or sialyl‐T antigens.     

Direct Visualization of Live Zebrafish Glycans via Single‐Step Metabolic Labeling with Fluorophore‐Tagged Nucleotide Sugars

Dynamic turnover of cell‐surface glycans is involved in a myriad of biological events, making this process an attractive target for in vivo molecular imaging. Metabolic glycan labeling coupled with bioorthogonal chemistry has paved the way for visualizing glycans in living organisms. However, a two‐step labeling sequence is required, which suffers from the tissue‐penetration difficulties of the imaging probes. Here, by exploring the substrate promiscuity of endogenous glycosyltransferases, we developed a single‐step fluorescent glycan labeling strategy by using fluorophore‐tagged analogues of the nucleotide sugars. Injecting fluorophore‐tagged sialic acid and fucose into the yolk of zebrafish embryos at the one‐cell stage enables systematic imaging of sialylation and fucosylation in live zebrafish embryos at distinct developmental stages. From these studies, we obtained insights into the role of sialylated and fucosylated glycans in zebrafish hematopoiesis.     

A Traveling Wave Ion Mobility Spectrometry (TWIMS) Study of the Robo1-Heparan Sulfate Interaction

Roundabout 1 (Robo1) interacts with its receptor Slit to regulate axon guidance, axon branching, and dendritic development in the nervous system and to regulate morphogenesis and many cell functions in the nonneuronal tissues. This interaction is known to be critically regulated by heparan sulfate (HS). Previous studies suggest that HS is required to promote the binding of Robo1 to Slit to form the minimal signaling complex, but the molecular details and the structural requirements of HS for this interaction are still unclear. Here, we describe the application of traveling wave ion mobility spectrometry (TWIMS) to study the conformational details of the Robo1-HS interaction. The results suggest that Robo1 exists in two conformations that differ by their compactness and capability to interact with HS. The results also suggest that the highly flexible interdomain hinge region connecting the Ig1 and Ig2 domains of Robo1 plays an important functional role in promoting the Robo1-Slit interaction. Moreover, variations in the sulfation pattern and size of HS were found to affect its binding affinity and selectivity to interact with different conformations of Robo1. Both MS measurements and CIU experiments show that the Robo1-HS interaction requires the presence of a specific size and pattern of modification of HS. Furthermore, the effect of N-glycosylation on the conformation of Robo1 and its binding modes with HS is reported.

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Probing the substrate specificity of Golgi alpha-mannosidase II by use of synthetic oligosaccharides and a catalytic nucleophile mutant

Inhibition of Golgi alpha-mannosidase II (GMII), which acts late in the N-glycan processing pathway, provides a route to blocking cancer-induced changes in cell surface oligosaccharide structures. To probe the substrate requirements of GMII, oligosaccharides were synthesized that contained an alpha(1,3)- or alpha(1,6)-linked 1-thiomannoside. Surprisingly, these oligosaccharides were not observed in X-ray crystal structures of native Drosophila GMII (dGMII). However, a mutant enzyme in which the catalytic nucleophilic aspartate was changed to alanine (D204A) allowed visualization of soaked oligosaccharides and led to the identification of the binding site for the alpha(1,3)-linked mannoside of the natural substrate. These studies also indicate that the conformational change of the bound mannoside to a high-energy B 2,5 conformation is facilitated by steric hindrance from, and the formation of strong hydrogen bonds to, Asp204. The observation that 1-thio-linked mannosides are not well tolerated by the catalytic site of dGMII led to the synthesis of a pentasaccharide containing the alpha(1,6)-linked Man of the natural substrate and the beta(1,2)-linked GlcNAc moiety proposed to be accommodated by the extended binding site of the enzyme. A cocrystal structure of this compound with the D204A enzyme revealed the molecular interactions with the beta(1,2)-linked GlcNAc. The structure is consistent with the approximately 80-fold preference of dGMII for the cleavage of substrates containing a nonreducing beta(1,2)-linked GlcNAc. By contrast, the lysosomal mannosidase lacks an equivalent GlcNAc binding site and kinetic analysis indicates oligomannoside substrates without non-reducing-terminal GlcNAc modifications are preferred, suggesting that selective inhibitors for GMII could exploit the additional binding specificity of the GlcNAc binding site.     

Spin-labeled analogs of CMP-NeuAc as NMR probes of the alpha-2,6-sialyltransferase ST6Gal I

Structural data on mammalian proteins are often difficult to obtain by conventional NMR approaches because of an inability to produce samples with uniform isotope labeling in bacterial expression hosts. Proteins with sparse isotope labels can be produced in eukaryotic hosts by using isotope-labeled forms of specific amino acids, but structural analysis then requires information from experiments other than nuclear Overhauser effects. One source of alternate structural information is distance-dependent perturbation of spin relaxation times by nitroxide spin-labeled analogs of natural protein ligands. Here, we introduce spin-labeled analogs of sugar nucleotide donors for sialyltransferases, specifically, CMP-TEMPO (CMP-4-O-[2,2,6,6-tetramethylpiperidine-1-oxyl]) and CMP-4carboxyTEMPO (CMP-4-O-[4-carboxy-2,2,6,6-tetramethylpiperidinine-1-oxyl]). An ability to identify resonances from active site residues and produce distance constraints is illustrated on a (15)N phenylalanine-labeled version of the structurally uncharacterized, alpha-2,6-linked sialyltransferase, ST6Gal I.     

A Glycan Array‐Based Assay for the Identification and Characterization of Plant Glycosyltransferases

Growing plants with modified cell wall compositions is a promising strategy to improve resistance to pathogens, increase biomass digestibility, and tune other important properties. In order to alter biomass architecture, a detailed knowledge of cell wall structure and biosynthesis is a prerequisite. We report here a glycan array‐based assay for the high‐throughput identification and characterization of plant cell wall biosynthetic glycosyltransferases (GTs). We demonstrate that different heterologously expressed galactosyl‐, fucosyl‐, and xylosyltransferases can transfer azido‐functionalized sugar nucleotide donors to selected synthetic plant cell wall oligosaccharides on the array and that the transferred monosaccharides can be visualized “on chip” by a 1,3‐dipolar cycloaddition reaction with an alkynyl‐modified dye. The opportunity to simultaneously screen thousands of combinations of putative GTs, nucleotide sugar donors, and oligosaccharide acceptors will dramatically accelerate plant cell wall biosynthesis research.     

Modulating Cell‐Surface Receptor Signaling and Ion Channel Functions by In Situ Glycan Editing

Glycans anchored on cell‐surface receptors are active modulators of receptor signaling. A strategy is presented that enforces transient changes to cell‐surface glycosylation patterns to tune receptor signaling. This approach, termed in situ glycan editing, exploits recombinant glycosyltransferases to incorporate monosaccharides with linkage specificity onto receptors in situ. α2,3‐linked sialic acid or α1,3‐linked fucose added in situ suppresses signaling through epidermal growth factor receptor and fibroblast growth factor receptor. We also applied the same strategy to regulate the electrical signaling of a potassium ion channel–human ether‐à‐go‐go‐related gene channel. Compared to gene editing, no long‐term perturbations are introduced to the treated cells. In situ glycan editing therefore offers a promising approach for studying the dynamic role of specific glycans in membrane receptor signaling and ion channel functions.