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1.  B4GAT1 is the priming enzyme for the LARGE-dependent functional glycosylation of α-dystroglycan 
eLife  2014;3:e03943.
Recent studies demonstrated that mutations in B3GNT1, an enzyme proposed to be involved in poly-N-acetyllactosamine synthesis, were causal for congenital muscular dystrophy with hypoglycosylation of α-dystroglycan (secondary dystroglycanopathies). Since defects in the O-mannosylation protein glycosylation pathway are primarily responsible for dystroglycanopathies and with no established O-mannose initiated structures containing a β3 linked GlcNAc known, we biochemically interrogated this human enzyme. Here we report this enzyme is not a β-1,3-N-acetylglucosaminyltransferase with catalytic activity towards β-galactose but rather a β-1,4-glucuronyltransferase, designated B4GAT1, towards both α- and β-anomers of xylose. The dual-activity LARGE enzyme is capable of extending products of B4GAT1 and we provide experimental evidence that B4GAT1 is the priming enzyme for LARGE. Our results further define the functional O-mannosylated glycan structure and indicate that B4GAT1 is involved in the initiation of the LARGE-dependent repeating disaccharide that is necessary for extracellular matrix protein binding to O-mannosylated α-dystroglycan that is lacking in secondary dystroglycanopathies.
eLife digest
Dystroglycan is a protein that is essential for muscles to function correctly, and helps to connect the interior framework of muscle cells with the external matrix of molecules that hold the cells together in the tissue. As is the case for many proteins, dystroglycan must have particular carbohydrate molecules joined to it in order to work correctly. Enzymes called glycosyltransferases assist with the reactions that build the carbohydrates on a protein.
Mutations in multiple glycosyltransferases that add carbohydrates to dystroglycan can cause a group of diseases that are characterized by a progressive loss of muscle function, known as congenital muscular dystrophies. Praissman et al. use biochemical experimentation to investigate the role of one of these enzymes, known as B3GNT1. The enzyme's name is based on a code that describes which carbohydrate it helps to bind to proteins. However, Praissman et al. (and independently, Willer et al.) discovered that this enzyme actually works with a different donor and acceptor than previously thought, and so should be called B4GAT1 instead.
Praissman et al. propose that the B4GAT1 enzyme starts the process of forming the carbohydrate structures that help muscle cells bind to the muscle tissue matrix. B4GAT1 forms short carbohydrates on the surface of the part of dystroglycan that sits on the surface of cells. These carbohydrates are then extended into longer chains by another glycosyltransferase called LARGE. The results of Praissman et al. suggest that another enzyme is also involved in this process, which will require further studies to identify. Understanding the role of B4GAT1 and other glycosyltransferases that build functionally glycosylated dystroglycan could help to develop treatments for diseases such as muscular dystrophies.
PMCID: PMC4227051  PMID: 25279697
congenital muscular dystrophy; O-mannosylation; glycosylation; alpha-dystroglycan; B4GAT1; B3GNT1; human
2.  Glycopeptide specific monoclonal antibodies suggest new roles for O-GlcNAc 
Nature chemical biology  2010;6(5):338-343.
Studies of post-translational modification by β-N-acetyl-D-glucosamine (O-GlcNAc) are hampered by a lack of efficient tools such as O-GlcNAc specific antibodies that can be employed for detection, isolation, and site localization. We have obtained a large panel of O-GlcNAc-specific IgG MAbs having a broad spectrum of binding partners by combining three-component immunogen methodology with hybridoma technology. Immunoprecipitation followed by large-scale shotgun proteomics led to the identification of more than 200 mammalian O-GlcNAc modified proteins, including a large number of novel glycoproteins. A substantial number of the glycoproteins were only enriched by one of the antibodies and this observation combined with results of inhibition ELISAs suggests that the antibodies in addition to their O-GlcNAc-dependence also appear to have different, but overlapping, local peptide determinants. The MAbs made it possible to delineate differentially modified proteins of liver in response to trauma-hemorrhage and resuscitation in a rat model.
PMCID: PMC2857662  PMID: 20305658
O-GlcNAc; Immunogen; Glycopeptide; Proteomics; Post-translational Modification
3.  Simple, clickable protocol for atomic force microscopy tip modification and its application for trace ricin detection by recognition imaging 
A simple two-step protocol for modification of atomic force microscopy tip and substrate by using a “click reaction” has been developed. The modified tip and substrate were applied to detect trace amounts of ricin by using single molecule recognition force microscopy (SMRFM1). A key feature of the approach is the use of a PEG derivative functionalized with a thiol and azide group end group. This compound could be attached to a gold coated AFM tip by a strong Au-thiol bond. The azide of the immobilized PEG was used for the attachment of an anti-ricin antibody modified with an alkyne group using a ‘click reaction’. The latter reaction is highly efficient, compatible with the presence of many functional groups and proceeds under mild reaction conditions. In a separate step, ricin was immobilized on the gold substrate surface that was modified by active esters. For this process, a novel bifunctional reagent was employed containing an active ester and a thioctic acid moiety. The SMRFM results showed a sub fg/mL level of detection sensitivity. The unbinding force between the anti-ricin antibody and ricin was studied by the force-distance curves (F-D). The unbinding force between the ricin and the antibody was determined to be 64.89±1.67 pN by constructing a force histogram.
PMCID: PMC2888661  PMID: 19437761
4.  Dissecting the Molecular Basis of the Role of the O-Mannosylation Pathway in Disease: α-Dystroglycan and Forms of Muscular Dystrophy 
Dystroglycanopathies are a subgroup of muscular dystrophies that arise from defects in the enzymes implicated in the recently elucidated O-mannosylation pathway, resulting in underglycosylation of α-dystroglycan. The emerging identification of additional brain proteins modified by O-mannosylation provides a broader context for interpreting the range of neurological consequences associated with dystroglycanopathies. This form of glycosylation is associated with protein mucin-like domains which present numerous serine and threonine residues as possible sites for modification. Further, the O-Man glycans coexist in this region with O-GalNAc glycans, conventionally associated with such protein sequences, resulting in a complex glycoconjugate landscape. Sorting out the relationships between the various molecular defects in glycosylation and the modes of disease presentation, as well as the regulatory interplay among the O-Man glycans, and the effects on other modes of glycosylation in the same domain is challenging. Here we provide a perspective on chemical biology approaches employing synthetic and analytical methods to address these questions.
PMCID: PMC3938021  PMID: 24318691
Glycopeptides; Carbohydrates; Muscular Dystrophy; α-Dystroglycan; Dystroglycanopathy; Protein O-Mannosylation
5.  Selective Exo-Enzymatic Labeling (SEEL) of N-Glycans of Living Cells by Recombinant ST6Gal I** 
Angewandte Chemie (International ed. in English)  2013;52(49):10.1002/anie.201307095.
SEEL cell surface glycans
N-Glycans of living cells could be selectively tagged by exogenously administered recombinant ST6Gal I sialyltransferase and azido-modified CMP-Neu5Ac followed by a strain promoted cycloaddition using biotin modified dibenzylcyclooctynol (DIBO). The methodology will make it possible to dissect the mechanisms that underlie altered glycoconjugate recycling and storage in disease and identify the glycoconjugates whose cell surface localization or secretion are affected.
PMCID: PMC3869382  PMID: 24129959
click chemistry; glycosyltransferase; N-glycans; sialic acid; trafficking
6.  Adaptive immune activation: glycosylation does matter 
Nature chemical biology  2013;9(12):776-784.
Major histocompatibility complex (MHC) class I and II are glycoproteins that can present antigenic peptides at the cell surface for recognition and activation of circulating T lymphocytes. Here, the importance of the modification of protein antigens by glycans on cellular uptake, proteolytic processing, presentation by MHC and subsequent T-cell priming is reviewed. Antigen glycosylation is important for a number of diseases and vaccine design. All of the key proteins involved in antigen recognition and the orchestration of downstream effector functions are glycosylated. The influence of protein glycosylation on immune function and disease is covered.
PMCID: PMC3966069  PMID: 24231619
7.  Multi-Functionalization of Polymers by Strain-Promoted Cycloadditions 
Macromolecules  2013;46(19):7759-7768.
We report here a synthetic route to oxime, azide and nitrone-bearing copolymers via reversible addition-fragmentation chain transfer copolymerization of 4-vinylbenzaldehyde and 1-(chloromethyl)-4-vinylbenzene with styrene. The azide and nitrone moieties could be employed in strain-promoted 1,3-dipolar cycloadditions with various functionalized dibenzocyclooctynols (DIBO) for metal-free post-functionalization of the polymers. In situ oxidation of the oximes with hypervalent iodine gave nitrile oxides, which could also be employed as 1,3-dipoles for facile cycloadditions with DIBO derivatives. Kinetic measurements demonstrated that the pendant nitrile oxides reacted approximately twenty times faster compared to similar cycloadditions with azides. A block copolymer, containing azide and oxime groups in segregated blocks, served as a scaffold for attachment of hydrophobic and hydrophilic moieties by sequential strain-promoted alkyne-azide and strain-promoted alkyne-nitrile oxide cycloadditions. This sequential bi-functionalization approach made it possible to prepare in a controlled manner multi-functional polymers that could self-assemble into well-defined nanostructures.
PMCID: PMC3916133  PMID: 24511157
grafting; click chemistry; SPAAC; SPANOC; SPANC
8.  Microarray analysis of the human antibody response to synthetic Cryptosporidium glycopeptides 
Glycoproteins expressed by Cryptosporidium parvum are immunogenic in infected individuals but the nature of the epitopes recognised in C. parvum glycoproteins is poorly understood. Since a known immunodominant antigen of Cryptosporidium, the 17 kDa glycoprotein, has previously been shown to bind to lectins that recognise the Tn antigen (GalNAcα1-Ser/Thr-R), a large number of glycopeptides with different Tn valency and presentation were prepared. In addition, glycopeptides were synthesised based on a 40 kDa cryptosporidial antigen, a polymorphic surface glycoprotein with varying numbers of serine residues, to determine the reactivity with sera from C. parvum-infected humans. These glycopeptides and non-glycosylated peptides were used to generate a glycopeptide microarray to allow screening of sera from C. parvum-infected individuals for the presence of IgM and IgG antibodies. IgG but not IgM in sera from C. parvum-infected individuals bound to multivalent Tn antigen epitopes presented on glycopep-tides, suggesting that glycoproteins from C. parvum that contain the Tn antigen induce immune responses upon infection. In addition, molecular differences in glycosylated peptides (e.g. substituting Ser for Thr) as well as the site of glycosylation had a pronounced effect on reactivity. Lastly, pooled sera from individuals infected with either Toxoplasma or Plasmodium were also tested against the modified Cryptosporidium peptides and some sera showed specific binding to glycopeptide epitopes. These studies reveal that specific anti-glycopeptide antibodies that recognise the Tn antigen may be useful diagnostically and in defining the roles of parasite glycoconjugates in infections.
PMCID: PMC3937990  PMID: 23856596
Cryptosporidium parvum; Tn antigen; Glycopeptide microarrays; Toxoplasma; Plasmodium
9.  A General Strategy for the Chemoenzymatic Synthesis of Asymmetrically Branched N-Glycans 
Science (New York, N.Y.)  2013;341(6144):10.1126/science.1236231.
A systematic, efficient means of producing diverse libraries of asymmetrically branched N-glycans is needed to investigate the specificities and biology of glycan binding proteins. To that end, we describe a core pentasaccharide that at potential branching positions is modified by orthogonal protecting groups to allow selective attachment of unique saccharide moieties by chemical glycosylation. The appendages were selected in such a way that the antenna of the resulting deprotected compounds could be selectively extended by glycosyltransferases to give libraries of asymmetrical multi-antennary glycans. The power of the methodology was demonstrated by the preparation of a series of complex oligosaccharides that were printed as microarrays and screened for binding to lectins and influenza-virus hemagglutinins, which showed that recognition is modulated by presentation of minimal epitopes in the context of complex N-glycans.
PMCID: PMC3826785  PMID: 23888036
10.  Probing the Substrate Specificity of Golgi α-Mannosidase II by Use of Synthetic Oligosaccharides and a Catalytic Nucleophile Mutant 
Inhibition of Golgi α-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 α(1,3)- or α(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 α(1,3)-linked mannoside of the natural substrate. These studies also indicate that the conformational change of the bound mannoside to a high-energy B2,5 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 α(1,6)-linked Man of the natural substrate and the β(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 β(1,2)-linked GlcNAc. The structure is consistent with the ~80-fold preference of dGMII for the cleavage of substrates containing a nonreducing β(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.
PMCID: PMC3982601  PMID: 18558690
11.  A synthetic heparan sulfate oligosaccharide library reveals the novel enzymatic action of d-glucosaminyl 3-O-sulfotransferase-3a† 
Molecular bioSystems  2011;8(2):609-614.
Heparan sulfate (HS) glucosaminyl 3-O-sulfotranferases sulfate the C3-hydroxyl group of certain glucosamine residues on heparan sulfate. Six different 3-OST isoforms exist, each of which can sulfate very distinct glucosamine residues within the HS chain. Among these isoforms, 3-OST1 has been shown to play a role in generating ATIII-binding HS anticoagulants whereas 3-OST2, 3-OST3, 3-OST4 and 3OST-6 have been shown to play a vital role in generating gD-binding HS chains that permit the entry of herpes simplex virus type 1 into cells. 3-OST5 has been found to generate both ATIII- and gD-binding HS motifs. Previous studies have examined the substrate specificities of all the 3-OST isoforms using HS polysaccharides. However, very few studies have examined the contribution of the epimer configuration of neighboring uronic acid residues next to the target site to 3-OST action. In this study, we utilized a well-defined synthetic oligosaccharide library to examine the substrate specificity of 3-OST3a and compared it to 3-OST1. We found that both 3-OST1 and 3-OST3a preferentially sulfate the 6-O-sulfated, N-sulfoglucosamine when an adjacent iduronyl residue is located to its reducing side. On the other hand, 2-O-sulfation of this uronyl residue can inhibit the action of 3-OST3a on the target residue. The results reveal novel substrate sites for the enzyme actions of 3-OST3a. It is also evident that both these enzymes have promiscuous and overlapping actions that are differentially regulated by iduronyl 2-O-sulfation.
PMCID: PMC3970252  PMID: 22116385
12.  Spin-Labeled Analogs of CMP-NeuAc as NMR Probes of the α-2,6-Sialyltransferase ST6Gal I 
Chemistry & biology  2007;14(4):409-418.
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 15N phenylalanine-labeled version of the structurally uncharacterized, α-2,6-linked sialyltransferase, ST6Gal I.
PMCID: PMC3968682  PMID: 17462576
13.  The Molecular Basis of Inhibition of Golgi α-Mannosidase II by Mannostatin A 
Mannostatin A is a potent inhibitor of the mannose trimming enzyme Golgi α-mannosidase II (GMII), which acts late in the N-glycan processing pathway. Inhibition of this enzyme provides a route to blocking the transformation–associated changes in cancer cell surface oligosaccharide structures. In this paper, we report on the synthesis of new Mannostatin derivatives and analyze their binding in the active site of Drosophila GMII by X-ray crystallography. The results indicate that the interaction with the backbone carbonyl of Arg876 is crucial to the high potency of the inhibitor, an effect enhanced by the hydrophobic interaction between the thiomethyl group and an aromatic pocket vicinal to the cleavage site. The various structures indicate that differences in the hydration of protein-ligand complexes are also important determinant of plasticity as well as selectivity of inhibitor binding.
PMCID: PMC3956299  PMID: 19101978
14.  Fluorous Supported Modular Synthesis of Heparan Sulfate Oligosaccharides 
Organic letters  2013;15(2):342-345.
The modular synthesis of heparan sulfate fragments is greatly facilitated by employing an anomeric aminopentyl linker protected by a benzyloxycarbonyl group modified by a perfluorodecyl tag, which made it possible to purify highly polar intermediates by fluorous solid phase extraction. This tagging methodology made it also possible to perform repeated glycosylations to drive reactions to completion.
PMCID: PMC3563243  PMID: 23293947
15.  Stage-specific expression and antigenicity of glycoprotein glycans isolated from the human liver fluke, Opisthorchis viverrini 
Infection by Opisthorchis viverrini (liver fluke) is a major public health problem in southeastern Asia, resulting in hepatobiliary disease and cholangiocarcinoma. Fluke surface glycoconjugates are prominently presented to the host, thereby constituting a crucial immunological interface that can determine the parasite’s success in establishing infection. Therefore, N- and O-linked glycoprotein glycan profiles of the infective metacercarial stage and of the mature adult were investigated by nanospray ionization-linear ion trap mass spectrometry (NSI-MSn). Glycan immunogenicity was investigated by immunobloting with serum from infected humans. Metacercariae and adult parasites exhibit similar glycan diversity, although the prevalence of individual glycans and glycan classes varies by stage. The N-glycans of the metacercaria are mostly high mannose and monofucosylated, truncated-type oligosaccharides (62.7%), with the remainder processed to complex and hybrid type glycans (37.3%). The N-linked glycan profile of the adult is also dominated by high mannose and monofucosylated, truncated-type oligosaccharides (80.0%), with a smaller contribution from complex and hybrid type glycans (20.0%). At both stages, complex and hybrid type glycans are detected as mono-, bi-, tri-, or tetra-antennary structures. In metacercariae and adults, O-linked glycans are detected as mono- to pentasaccharides. The mucin type core 1 structure, Galβ1-3GalNAc, predominates in both stages but is less prevalent in the adult than in the metacercaria. Immunogenic recognition of liver fluke glycoproteins is reduced after deglycosylation but infected human serum was unable to recognize glycans released from peptides. Therefore, the most potent liver fluke antigenic epitopes are mixed determinants, comprised of glycan and polypeptide elements.
PMCID: PMC3547145  PMID: 23174105
Liver fluke; Opisthorchis viverrini glycoproteins; N-linked glycans; O-linked glycans; Antigenic epitopes
16.  Leishmania lipophosphoglycan: how to establish structure-activity relationships for this highly complex and multifunctional glycoconjugate? 
A key feature of many pathogenic microorganisms is the presence of a dense glycocalyx at their surface, composed of lipid-anchored glycoproteins and non-protein-bound polysaccharides. These surface glycolipids are important virulence factors for bacterial, fungal and protozoan pathogens. The highly complex glycoconjugate lipophosphoglycan (LPG) is one of the dominant surface macromolecules of the promastigote stage of all Leishmania parasitic species. LPG plays critical pleiotropic roles in parasite survival and infectivity in both the sandfly vector and the mammalian host. Here, we review the composition of the Leishmania glycocalyx, the chemical structure of LPG and what is currently known about its effects in the mammalian host, specifically. We will then discuss the current approaches employed to elucidate LPG functions. Finally, we will provide a viewpoint on future directions that this area of investigation could take to unravel in detail the biological activity of the specific molecular elements composing the structurally complex LPG.
PMCID: PMC4301024  PMID: 25653924
Leishmania glycoconjugates; lipophosphoglycan; LPG structure; LPG function; chemical synthesis; LPG structure-activity relationships
17.  A Fluorogenic Probe for the Catalyst-Free Detection of Azide-Tagged Molecules 
Journal of the American Chemical Society  2012;134(45):18809-18815.
Fluorogenic reactions in which non- or weakly-fluorescent reagents produce highly fluorescent products can be exploited to detect a broad range of compounds including biomolecules and materials. We describe a modified dibenzocyclooctyne that under catalyst-free conditions undergoes fast strain-promoted cycloadditions with azides to yield strongly fluorescent triazoles. The cycloaddition products are more than 1000-fold brighter compared to the starting cyclooctyne, exhibit large Stokes shift, and can be excited above 350 nm, which is required for many applications. Quantum mechanical calculations indicate that the fluorescence increase upon triazole formation is due to large differences in oscillator strengths of the S0 <–> S1 transitions in the planar C2v-symmetric starting material compared to the symmetry-broken and non-planar cycloaddition products. The new fluorogenic probe was successfully employed for labeling of proteins modified by an azide moiety.
PMCID: PMC3525324  PMID: 23095037
click chemistry; cycloaddition; bioorthogonal; fluorogenic probe; cyclooctyne
18.  Endolysins of Bacillus anthracis Bacteriophages Recognize Unique Carbohydrate Epitopes of Vegetative Cell Wall Polysaccharides with High Affinity and Selectivity 
Journal of the American Chemical Society  2012;134(37):15556-15562.
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.
PMCID: PMC3489029  PMID: 22935003
19.  Chemical Synthesis and Immunological Evaluation of the Inner-Core Oligosaccharide of Francisella tularensis 
Journal of the American Chemical Society  2012;134(34):14255-14262.
Francisella tularensis, which is a gram negative bacterium that causes tularemia, has been classified by the Center for Disease Control and Prevention (CDC) as a category A bioweapon. The development of vaccines, immunotherapeutics and diagnostics for F. tularensis requires a detailed knowledge of the saccharide structures that can be recognized by protective antibodies. We have synthesized the inner core region of the lipopolysaccharide (LPS) of F. tularensis to probe antigenic responses elicited by a live and subunit vaccine. The successful preparation of the target compound relied on the use of a disaccharide which was modified by the orthogonal protecting groups diethylisopropylsilyl (DEIPS), 2-naphthylmethyl (Nap), allyl ether (All) and levulinoyl (Lev) ester. The ability to remove the protecting groups in different orders made it possible to establish the optimal glycosylations sequence to prepare a highly crowded 1,2,3-cis configured branching point. A variety of different methods were exploited to control anomeric selectivities of the glycosylations. A comparison of the 1H NMR spectra of isolated material and the synthetic derivative confirmed the reported structural assignment of the inner core oligosaccharide of F. tularensis. The observation that immunizations with LPS lead to antibody responses to the inner core saccharides provides an impetus to further explore this compound as a vaccine candidate.
PMCID: PMC3458509  PMID: 22867268
20.  Stereoselective Assembly of Complex Oligosaccharides Using Anomeric Sulfonium Ions as Glycosyl Donors 
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.
PMCID: PMC3353542  PMID: 22475263
stereoselective glycosylations; modular building blocks; auxiliary; sulfonium ions; glucosides
21.  A Chemo-Mechanical Tweezer for Single Molecular Characterization of Soft Materials 
A new Atomic Force Microscopy (AFM)-based chemo-mechanical tweezer has been developed that can measure mechanical properties of individual macromolecules in supramolecular assembly and reveal positions of azide-containing polymers. A key feature of the new technology is the use of an AFM tip densely modified with 4-dibenzocyclooctynols (chemo-mechanical tweezer) that can react with multiple azide containing macromolecules of micelles to give triazole “clicked” compounds, which during retracting phases of AFM imaging are removed from the macromolecular assembly thereby providing a surface topographical image and positions of azide-containing polymers. The force-distance curves gave mechanical properties of removal of individual molecules from a supramolecular assembly. The new chemo-mechanical tweezer will make it possible to characterize molecular details of macromolecular assemblies thereby offering new avenues to tailor properties of such assemblies.
PMCID: PMC3319842  PMID: 22374886
22.  Polar Dibenzocyclooctynes for Selective Labeling of Extracellular Glycoconjugates of Living Cells 
Although strain-promoted alkyne-azide cycloadditions (SPAAC) have found wide utility in biological and material sciences, the low polarity and limited water solubility of commonly used cyclooctynes represents a serious shortcoming. To address this problem, an efficient synthetic route has been developed for highly polar sulfated dibenzocyclooctynylamides (S-DIBO) by a Friedel-Crafts alkylation of 1,2-bis(3-methoxyphenyl)ethylamides with trichlorocyclopropenium cation followed by a controlled hydrolysis of the resulting dichlorocyclopropenes to give bis(3-methoxyphenyl)cyclooctacyclopropenones, which were subjected to methoxy group removal of the phenols, O-sulfation, and photochemical unmasking of the cyclopropenone moiety. Accurate rate measurements of the reaction of benzyl azide with various dibenzylcyclooctyne derivatives demonstrated that aromatic substitution and the presence of the amide function had only a marginal impact on the rate constants. Biotinylated S-DIBO 8 was successfully used for labeling azido-containing glycoconjugates of living cells. Furthermore, it was found that the substitution pattern of the dibenzylcyclooctynes influences subcellular location and in particular it has been shown that DIBO derivative 4 can enter cells thereby labeling intra- and extracellular azido-modified glycoconjugates, whereas S-DIBO 8 cannot pass the cell membrane and therefore is ideally suited for selective labeling of cell surface molecules. The ability to selectively label cell surface molecules will yield unique opportunities for glycomic analysis and the study of glycoprotein trafficking.
PMCID: PMC3319718  PMID: 22376061
click chemistry; cycloaddition; carbohydrates; bioorthogonal; O-sulfated cyclooctynes
Glycosaminoglycan (GAG) carbohydrates provide a challenging analytical target for structural determination due to their polydisperse nature, non-template biosynthesis, and labile sulfate modifications. The resultant structures, although heterogeneous, contain domains which indicate a sulfation pattern or code that correlates to specific function. Mass spectrometry, in particular electron detachment dissociation Fourier transform ion cyclotron resonance (EDD FT-ICR MS), provides a highly sensitive platform for GAG structural analysis by providing cross-ring cleavages for sulfation location and product ions specific to hexuronic acid stereochemistry. To investigate the effect of sulfation pattern and variations in stereochemistry on EDD spectra, a series of synthetic heparan sulfate (HS) tetrasaccharides are examined. Whereas previous studies have focused on lowly sulfated compounds (0.5–1 sulfate groups per disaccharide), the current work extends the application of EDD to more highly sulfated tetrasaccharides (1–2 sulfate groups per disaccharide) and presents the first EDD of a tetrasaccharide containing a sulfated hexuronic acid. For these more highly sulfated HS oligomers, alternative strategies are shown to be effective for extracting full structural details. These strategies inlcude sodium cation replacement of protons, for determining the sites of sulfation, and desulfation of the oligosaccharides for the generation of product ions for assigning uronic acid stereochemistry.
PMCID: PMC3517180  PMID: 23230388
Glycosaminoglycan; electron detachment dissociation; EDD; FT-ICR; desulfation; heparan sulfate
24.  Synthetic, Structural and Biosynthetic Studies of an Unusual Phospho-Glycopeptide Derived from α-Dystroglycan 
Journal of the American Chemical Society  2011;133(36):14418-14430.
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.
PMCID: PMC3176502  PMID: 21812486
25.  Combining High-energy C-trap Dissociation and Electron Transfer Dissociation for Protein O-GlcNAc Modification Site Assignment 
Journal of proteome research  2011;10(9):4088-4104.
Mass spectrometry-based studies of proteins that are post-translationally modified by O-linked β-N-acetylglucosamine (O-GlcNAc) are challenged in effectively identifying the sites of modification while simultaneously sequencing the peptides. Here we tested the hypothesis that a combination of high-energy C-trap dissociation (HCD) and electron transfer dissociation (ETD) could specifically target the O-GlcNAc modified peptides and elucidate the amino acid sequence while preserving the attached GlcNAc residue for accurate site assignment. By taking advantage of the recently characterized O-GlcNAc-specific IgG monoclonal antibodies and the combination of HCD and ETD fragmentation techniques, O-GlcNAc modified proteins were enriched from HEK293T cells and subsequently characterized using the LTQ Orbitrap Velos™ ETD (Thermo Fisher Scientific) mass spectrometer. In our dataset, 83 sites of O-GlcNAc modification are reported with high confidence confirming that the HCD/ETD combined approach is amenable to the detection and site assignment of O-GlcNAc modified peptides. Realizing HCD triggered ETD fragmentation on a linear ion trap/Orbitrap platform for more in-depth analysis and application of this technique to other post-translationally modified proteins are currently underway. Furthermore, this report illustrates that the O-GlcNAc transferase appears to demonstrate promiscuity with regards to the hydroxyl-containing amino acid modified in short stretches of primary sequence of the glycosylated polypeptides.
PMCID: PMC3172619  PMID: 21740066
O-GlcNAc; HCD; ETD; tandem mass spectrometry; site assignment; post-translational modification; glycosylation

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