Synthesis of N-acetylmannosamine-6-phosphate derivatives to investigate the mechanism of N-acetylmannosamine-6-phosphate 2-epimerase.

The synthesis of analogues of natural enzyme substrates can be used to help deduce enzymatic mechanisms. N-Acetylmannosamine-6-phosphate 2-epimerase is an enzyme in the bacterial sialic acid catabolic pathway. To investigate whether the mechanism of this enzyme involves a re-protonation mechanism by the same neighbouring lysine that performed the deprotonation or a unique substrate-assisted proton displacement mechanism involving the substrate C5 hydroxyl, the syntheses of two analogues of the natural substrate, N-acetylmannosamine-6-phosphate, are described. In these novel analogues, the C5 hydroxyl has been replaced with a proton and a methyl ether respectively. As recently reported, Staphylococcus aureus N-acetylmannosamine-6-phosphate 2-epimerase was co-crystallized with these two compounds. The 5-deoxy variant bound to the enzyme active site in a different orientation to the natural substrate, while the 5-methoxy variant did not bind, adding to the evidence that this enzyme uses a substrate-assisted proton displacement mechanism. This mechanistic information may help in the design of potential antibacterial drug candidates.

Glycan Array Evaluation of Synthetic Epitopes between the Capsular Polysaccharides from Streptococcus pneumoniae 19F and 19A.

Vaccination represents the most effective way to prevent invasive pneumococcal diseases. The glycoconjugate vaccines licensed so far are obtained from capsular polysaccharides (CPSs) of the most virulent serotypes. Protection is largely limited to the specific vaccine serotypes, and the continuous need for broader coverage to control the outbreak of emerging serotypes is pushing the development of new vaccine candidates. Indeed, the development of efficacious vaccine formulation is complicated by the high number of bacterial serotypes with different CPSs. In this context, to simplify vaccine composition, we propose the design of new saccharide fragments containing chemical structures shared by different serotypes as cross-reactive and potentially cross-protective common antigens. In particular, we focused on Streptococcus pneumoniae (Sp) 19A and 19F. The CPS repeating units of Sp 19F and 19A are very similar and share a common structure, the disaccharide ManNAc-ß-(1→4)-Glc (A-B). Herein, we describe the synthesis of a small library of compounds containing different combinations of the common 19F/19A disaccharide. The six new compounds were tested with a glycan array to evaluate their recognition by antibodies in reference group 19 antisera and factor reference antisera (reacting against 19F or 19A). The disaccharide A-B, phosphorylated at the upstream end, emerged as a hit from the glycan array screening because it is strongly recognized by the group 19 antisera and by the 19F and 19A factor antisera, with similar intensity compared with the CPSs used as controls. Our data give a strong indication that the phosphorylated disaccharide A-B can be considered a common epitope among different Sp 19 serotypes.

N-acetylmannosamine-6-phosphate 2-epimerase uses a novel substrate-assisted mechanism to catalyze amino sugar epimerization.

There are five known general catalytic mechanisms used by enzymes to catalyze carbohydrate epimerization. The amino sugar epimerase N-acetylmannosamine-6-phosphate 2-epimerase (NanE) has been proposed to use a deprotonation-reprotonation mechanism, with an essential catalytic lysine required for both steps. However, the structural determinants of this mechanism are not clearly established. We characterized NanE from Staphylococcus aureus using a new coupled assay to monitor NanE catalysis in real time and found that it has kinetic constants comparable with other species. The crystal structure of NanE from Staphylococcus aureus, which comprises a triosephosphate isomerase barrel fold with an unusual dimeric architecture, was solved with both natural and modified substrates. Using these substrate-bound structures, we identified the following active-site residues lining the cleft at the C-terminal end of the ß-strands: Gln11, Arg40, Lys63, Asp124, Glu180, and Arg208, which were individually substituted and assessed in relation to the mechanism. From this, we re-evaluated the central role of Glu180 in this mechanism alongside the catalytic lysine. We observed that the substrate is bound in a conformation that ideally positions the C5 hydroxyl group to be activated by Glu180 and donate a proton to the C2 carbon. Taken together, we propose that NanE uses a novel substrate-assisted proton displacement mechanism to invert the C2 stereocenter of N-acetylmannosamine-6-phosphate. Our data and mechanistic interpretation may be useful in the development of inhibitors of this enzyme or in enzyme engineering to produce biocatalysts capable of changing the stereochemistry of molecules that are not amenable to synthetic methods.

Synthetic Study toward Saccharomicin Based upon Asymmetric Metal Catalysis.

Here, we report a de novo metal-catalyzed approach toward the stereoselective glycosidic bond formation in saccharomicin. The signature step is highlighted by the Pd-catalyzed asymmetric coupling of ene-alkoxyallenes and highly functionalized alcohol substrates. The reaction showed high chemo-, regio-, and ligand-driven diastereoselectivity. In combination with the ring-closing metathesis and late-stage functionalization, this method led to highly efficient synthesis of saccharosamine-rhamnose and rhamnose-fucose fragments.

Understanding selectivity of metabolic labelling and click-targeting in multicellular environments as a route to tissue selective drug delivery.

Cancer cells generally exhibit higher metabolic demands relative to that of normal tissue cells. This offers great possibilities to exploit metabolic glycoengineering in combination with bio-orthogonal chemistry reactions to achieve tumour site-targeted therapeutic delivery. This work addresses the selectivity of metabolic glycan labelling in diseased (i.e., cancer) versus normal cells grown in a multicellular environment. Dibenzocylooctyne (DBCO)-bearing acetylated-d-mannosamine (Ac4ManNDBCO) was synthesised to metabolically label three different types of cell lines originating from the human lung tissues: A549 adenocarcinomic alveolar basal epithelial cells, MeT5A non-cancerous mesothelial cells, and MRC5 non-cancerous fibroblasts. These cell lines displayed different labelling sensitivity, which trended with their doubling time in the following order: A549 ≈ MeT5A > MRC5. The higher metabolic labelling efficiency inherently led to a higher extent of specific binding and accumulation of the clickable N3-conjugated gold nanoparticles (N3-AuNps, core diameter = 30 nm) in the DBCO-glycan modified A549 and MeT5A cells, but to a less prominent effect in MRC5 cells. These findings demonstrate that relative rates of cell metabolism can be exploited using metabolic labelling to recruit nanotherapeutics whilst minimising non-specific targeting of surrounding tissues.

Metabolic Glycoengineering in hMSC-TERT as a Model for Skeletal Precursors by Using Modified Azide/Alkyne Monosaccharides.

Metabolic glycoengineering enables a directed modification of cell surfaces by introducing target molecules to surface proteins displaying new features. Biochemical pathways involving glycans differ in dependence on the cell type; therefore, this technique should be tailored for the best results. We characterized metabolic glycoengineering in telomerase-immortalized human mesenchymal stromal cells (hMSC-TERT) as a model for primary hMSC, to investigate its applicability in TERT-modified cell lines. The metabolic incorporation of N-azidoacetylmannosamine (Ac4ManNAz) and N-alkyneacetylmannosamine (Ac4ManNAl) into the glycocalyx as a first step in the glycoengineering process revealed no adverse effects on cell viability or gene expression, and the in vitro multipotency (osteogenic and adipogenic differentiation potential) was maintained under these adapted culture conditions. In the second step, glycoengineered cells were modified with fluorescent dyes using Cu-mediated click chemistry. In these analyses, the two mannose derivatives showed superior incorporation efficiencies compared to glucose and galactose isomers. In time-dependent experiments, the incorporation of Ac4ManNAz was detectable for up to six days while Ac4ManNAl-derived metabolites were absent after two days. Taken together, these findings demonstrate the successful metabolic glycoengineering of immortalized hMSC resulting in transient cell surface modifications, and thus present a useful model to address different scientific questions regarding glycosylation processes in skeletal precursors.

Efficient inhibition of O-glycan biosynthesis using the hexosamine analog Ac5GalNTGc.

There is a critical need to develop small-molecule inhibitors of mucin-type O-linked glycosylation. The best-known reagent currently is benzyl-GalNAc, but it is effective only at millimolar concentrations. This article demonstrates that Ac5GalNTGc, a peracetylated C-2 sulfhydryl-substituted GalNAc, fulfills this unmet need. When added to cultured leukocytes, breast cells, and prostate cells, Ac5GalNTGc increased cell-surface VVA binding by ∼10-fold, indicating truncation of O-glycan biosynthesis. Cytometry, mass spectrometry, and western blot analysis of HL-60 promyelocytes demonstrated that 50-80 µM Ac5GalNTGc prevented elaboration of 30%-60% of the O-glycans beyond the Tn-antigen (GalNAcα1-Ser/Thr) stage. The effect of the compound on N-glycans and glycosphingolipids was small. Glycan inhibition induced by Ac5GalNTGc resulted in 50%-80% reduction in leukocyte sialyl-Lewis X expression and L-/P-selectin-mediated rolling under flow conditions. Ac5GalNTGc was pharmacologically active in mouse. It reduced neutrophil infiltration to sites of inflammation by ∼60%. Overall, Ac5GalNTGc may find diverse applications as a potent inhibitor of O-glycosylation.

General Method for the Synthesis of α- or ß-Deoxyaminoglycosides Bearing Basic Nitrogen.

The introduction of glycosides bearing basic nitrogen is challenging using conventional Lewis acid-promoted pathways owing to competitive coordination of the amine to the Lewis acid promoter. Additionally, because many aminoglycosides lack a C2 substituent, diastereomeric mixtures of O-glycosides are often produced. Herein, we present a method for the synthesis of α- or ß- 2,3,6-trideoxy-3-amino- and 2,4,6-trideoxy-4-amino O-glycosides from a common precursor. Our strategy proceeds by the reductive lithiation of thiophenyl glycoside donors and trapping of the resulting anomeric anions with 2-methyltetrahydropyranyl peroxides. We apply this strategy to the synthesis of α- and ß-forosamine, pyrrolosamine, acosamine, and ristosamine derivatives using primary and secondary peroxides as electrophiles. α-Linked products are obtained in 60-96% yield and with >50:1 selectivity. ß-Linked products are obtained in 45-94% yield and with 1.7->50:1 stereoselectivity. Contrary to donors bearing an equatorial amine substituent, donors bearing an axial amine substituent favored ß-products at low temperatures. This work establishes a general strategy to synthesize O-glycosides bearing a basic nitrogen.

Metabolic Glycoengineering with Azide- and Alkene-Modified Hexosamines: Quantification of Sialic Acid Levels.

Metabolic glycoengineering (MGE) is an established method to incorporate chemical reporter groups into cellular glycans for subsequent bioorthogonal labeling. The method has found broad application for the visualization and isolation of glycans allowing their biological roles to be probed. Furthermore, targeting of drugs to cancer cells that present high concentrations of sialic acids on their surface is an attractive approach. We report the application of a labeling reaction using 1,2-diamino-4,5-methylenedioxybenzene for the quantification of sialic acid derivates after MGE with various azide- and alkene-modified ManNAc, GlcNAc, and GalNAc derivatives. We followed the time course of sialic acid production and were able to detect sialic acids modified with the chemical reporter group - not only after addition of ManNAc derivatives to the cell culture. A cyclopropane-modified ManNAc derivative, being a model for the corresponding cyclopropene analog, which undergoes fast inverse-electron-demand Diels-Alder reactions with 1,2,4,5-tetrazines, resulted in the highest incorporation efficiency. Furthermore, we investigated whether feeding the cells with natural and unnatural ManNAc derivative results in increased levels of sialic acids and found that this is strongly dependent on the investigated cell type and cell fraction. For HEK 293T cells, a strong increase in free sialic acids in the cell interior was found, whereas cell-surface sialic acid levels are only moderately increased.

Optimization of the Microwave Assisted Glycosylamines Synthesis Based on a Statistical Design of Experiments Approach.

Glycans carry a vast range of functions in nature. Utilizing their properties and functions in form of polymers, coatings or glycan derivatives for various applications makes the synthesis of modified glycans crucial. Since amines are easy to modify for subsequent reactions, we investigated regioselective amination conditions of different saccharides. Amination reactions were performed according to Kochetkov and Likhoshertov and accelerated by microwave irradiation. We optimized the synthesis of glycosylamines for N-acetyl-d-galactosamine, d-lactose, d-glucuronic acid and l-(-)-fucose using the design of experiments (DoE) approach. DoE enables efficient optimization with limited number of experimental data. A DoE software generated a set of experiments where reaction temperature, concentration of carbohydrate, nature of aminating agent and solvent were investigated. We found that the synthesis of glycosylamines significantly depends on the nature of the carbohydrate and on the reaction temperature. There is strong indication that high temperatures are favored for the amination reaction.

Aptamer-Directed Protein-Specific Multiple Modifications of Membrane Glycoproteins on Living Cells.

Understanding how a cell membrane protein functions on living cells remains a challenge for cell biology. Specific placement of functional molecules on specific proteins in their native environment would allow comprehensive study of proteins' dynamic functions. Existing methods cannot facilely achieve multiple modifications on specific membrane proteins. In this report, we describe an aptamer-induced, protein-specific bio-orthogonal modification technology for precise nongenetic immobilization of multiple small functional molecules on target membrane glycoproteins by combining metabolic technology and aptamer targeting. In brief, DNA probes were designed by modifying aptamers, which bind to target proteins on the surfaces of living cells pretreated with N-azidoacetylmannosamine-tetraacylated (Ac4ManNAz). The cyclooctynes tagged of DNA probes will approach the azide groups to trigger the bio-orthogonal reactions. After UV irradiation and hybridization with cDNA (complementary DNA), the aptamers can be removed, and the process can be repeated to achieve multiple modifications for multicolor imaging and cell surface nanoengineering on specific proteins.

Near-infrared light controlled fluorogenic labeling of glycoengineered sialic acids in vivo with upconverting photoclick nanoprobe.

Sialic acid serves as an important determinant for profiling cell activities in diverse biological and pathological processes. The precise control of sialic acid labeling to visualize its biological pathways under endogenous conditions is significant but still challenging due to the lack of reliable methods. Herein, we developed an effective strategy to spatiotemporally label thesialic acids with a near-infrared (NIR) light activated upconverting nanoprobe (Tz-UCNP). With this photoclickable nanoprobe and a stable N-alkene-d-mannosamine (Ac4ManNIPFA), metabolically synthesized alkene sialic acids on the cell surface were labeled and imaged in real time through fluorogenic cycloaddition. More importantly, we achieved spatially selective visualization of sialic acids in specific tumor tissues of the mice under NIR light activation in a spatially controlled manner. This in situ controllable labeling strategy thus enables the metabolic labeling of specific sialic acids in complex biological systems.

Conformational analysis by NMR and molecular dynamics of adamantane-doxorubicin prodrugs and their assemblies with ß-cyclodextrin: A focus on the design of platforms for controlled drug delivery.

Nanoscale design and construction of affinity-based drug delivery systems (ADDS) is an active research area with enormous potential for the improvement of cancer treatment. For the therapeutic load of these ADDS, a promising strategy is the design of pH-sensitive prodrugs based on the construction of conjugates between adamantane and doxorubicin (Ad-Dox), which stands out as an excellent model system to obtain novel supramolecular materials. Construction of these prodrugs involves a modification of three zones of doxorubicin which in principle does not affect the action mechanism: the carbonyl group C13 (hydrazone linker), the primary alcohol neighboring the carbonyl (ester linker) and the 3' amino group of daunosamine sugar (amide linker). These modifications are aimed to improve the efficacy and reduce the systemic toxicity of the drug chemotherapy by controlling its release in cancer cells. In this work, we performed 2D NMR experiments and molecular dynamics simulations to characterize the conformational changes of three constructed prodrugs. Our results demonstrated that ring A and the daunsamine sugar of the hydrazone and amide linkers conserve the half-chair state 9H8, while the ester linker disrupts this conformation. Our study also showed that the hydrazone-linked compound (Ad-h-Dox) does not modify the conformation of the original drug and maintains cytotoxic activity. Moreover, the inclusion complex (IC) of Ad-h-Dox with ß-cyclodextrin (ßCD) generated a highly soluble platform in water, whereas the ester-linked compound (Ad-e-Dox) causes the loss of biological activity. This study proves that Ad-h-Dox prodrug can be an optimum prodrug and act as a building block for a more complex drug transport system.

Identifying Sialylation Linkages at the Glycopeptide Level by Glycosyltransferase Labeling Assisted Mass Spectrometry (GLAMS).

Precise assignment of sialylation linkages at the glycopeptide level is of importance in bottom-up glycoproteomics and an indispensable step to understand the function of glycoproteins in pathogen-host interactions and cancer progression. Even though some efforts have been dedicated to the discrimination of α2,3/α2,6-sialylated isomers, unambiguous identification of sialoglycopeptide isomers is still needed. Herein, we developed an innovative glycosyltransferase labeling assisted mass spectrometry (GLAMS) strategy. After specific enzymatic labeling, oxonium ions from higher-energy C-trap dissociation (HCD) fragmentation of α2,3-sailoglycopeptides then generate unique reporters to distinctly differentiate those of α2,6-sailoglycopeptide isomers. With this strategy, a total of 1236 linkage-specific sialoglycopeptides were successfully identified from 161 glycoproteins in human serum.

Synthesis of orthogonally protected and functionalized bacillosamines.

2,4-Diamino-2,4,6-trideoxyglucose (bacillosamine) is a monosaccharide found in many pathogenic bacteria, variation in the functionalities appended to the amino groups occurs depending on the species the sugar is derived from. We here report the first synthesis of bacillosamine synthons that allow for the incorporation of two different functionalities at the C-2-N-acetyl and C-4-amines. We have developed chemistry to assemble a set of conjugation ready Neisseria meningitidis C-2-N-acetyl bacillosamine saccharides, carrying either an acetyl or (R)- or (S)-glyceroyl at the C-4 amine. The glyceroyl bacillosamines have been further extended at the C-3-OH with an α-d-galactopyranose to provide structures that occur as post-translational modifications of N. meningitidis PilE proteins, which make up the bacterial pili.

Loss of GFAT-1 feedback regulation activates the hexosamine pathway that modulates protein homeostasis.

Glutamine fructose-6-phosphate amidotransferase (GFAT) is the key enzyme in the hexosamine pathway (HP) that produces uridine 5'-diphospho-N-acetyl-D-glucosamine (UDP-GlcNAc), linking energy metabolism with posttranslational protein glycosylation. In Caenorhabditis elegans, we previously identified gfat-1 gain-of-function mutations that elevate UDP-GlcNAc levels, improve protein homeostasis, and extend lifespan. GFAT is highly conserved, but the gain-of-function mechanism and its relevance in mammalian cells remained unclear. Here, we present the full-length crystal structure of human GFAT-1 in complex with various ligands and with important mutations. UDP-GlcNAc directly interacts with GFAT-1, inhibiting catalytic activity. The longevity-associated G451E variant shows drastically reduced sensitivity to UDP-GlcNAc inhibition in enzyme activity assays. Our structural and functional data point to a critical role of the interdomain linker in UDP-GlcNAc inhibition. In mammalian cells, the G451E variant potently activates the HP. Therefore, GFAT-1 gain-of-function through loss of feedback inhibition constitutes a potential target for the treatment of age-related proteinopathies.

Tracking glycosylation in live cells using FTIR spectroscopy.

This is the first demonstration of the study of glycan protein turnover in living cells by FTIR with commercially available tetraacetylated N-Azidoacetyl-D-Mannosamine (Ac4ManNAz) label. The FTIR analysis has shown to be able to monitor the metabolism of glycans in living cells in real time. The method is simple, quantitative and requires equipment that are available in many laboratories. It can be used in a wide range of applications such as the study of glycosylation and cell-signalling.

The basis for non-canonical ROK family function in the N-acetylmannosamine kinase from the pathogen Staphylococcus aureus.

In environments where glucose is limited, some pathogenic bacteria metabolize host-derived sialic acid as a nutrient source. N-Acetylmannosamine kinase (NanK) is the second enzyme of the bacterial sialic acid import and degradation pathway and adds phosphate to N-acetylmannosamine using ATP to prime the molecule for future pathway reactions. Sequence alignments reveal that Gram-positive NanK enzymes belong to the Repressor, ORF, Kinase (ROK) family, but many lack the canonical Zn-binding motif expected for this function, and the sugar-binding EXGH motif is altered to EXGY. As a result, it is unclear how they perform this important reaction. Here, we study the Staphylococcus aureus NanK (SaNanK), which is the first characterization of a Gram-positive NanK. We report the kinetic activity of SaNanK along with the ligand-free, N-acetylmannosamine-bound and substrate analog GlcNAc-bound crystal structures (2.33, 2.20, and 2.20 Å resolution, respectively). These demonstrate, in combination with small-angle X-ray scattering, that SaNanK is a dimer that adopts a closed conformation upon substrate binding. Analysis of the EXGY motif reveals that the tyrosine binds to the N-acetyl group to select for the "boat" conformation of N-acetylmannosamine. Moreover, SaNanK has a stacked arginine pair coordinated by negative residues critical for thermal stability and catalysis. These combined elements serve to constrain the active site and orient the substrate in lieu of Zn binding, representing a significant departure from canonical NanK binding. This characterization provides insight into differences in the ROK family and highlights a novel area for antimicrobial discovery to fight Gram-positive and S. aureus infections.

A Validamycin Shunt Pathway for Valienamine Synthesis in Engineered Streptomyces hygroscopicus 5008.

Valienamine is the key functional component of many natural glycosidase inhibitors, including the crop protectant validamycin A and the clinical antidiabetic agent acarbose. Due to its important biomedical activity, it is also the prominent lead compound for the exploration of therapeutic agents, such as the stronger α-glucosidase inhibitor voglibose. Currently, the main route for obtaining valienamine is a multistep biosynthetic process involving the synthesis and degradation of validamycin A. Here, we established an alternative, vastly simplified shunt pathway for the direct synthesis of valienamine based on an envisioned non-natural transamination in the validamycin A producer Streptomyces hygroscopicus 5008. We first identified candidate aminotransferases for the non-natural ketone substrate valienone and conducted molecular evolution in vitro. The WecE enzyme from Escherichia coli was verified to complete the envisioned step with >99.9% enantiomeric excess and was further engineered to produce a 32.6-fold more active mutant, VarB, through protein evolution. Subsequently, two copies of VarB were introduced into the host, and the new shunt pathway produced 0.52 mg/L valienamine after a 96-h fermentation. Our study thus illustrates a dramatically simplified alternative shunt pathway for valienamine production and introduces a promising foundational platform for increasing the production of valienamine and its valuable N-modified derivatives for use in pharmaceutical applications.

Synthesis of 2-azido-2-deoxy- and 2-acetamido-2-deoxy-D-manno derivatives as versatile building blocks.

Reported herein is the synthesis of a number of building blocks of 2-amino-2-deoxy-d-mannose from common d-glucose precursors.